ω-Conotoxin GVIA mimetics based on an anthranilamide core: Effect of variation in ammonium side chain lengths and incorporation of fluorine

Article abstract of DOI:10.1016/j.bmc.2009.07.063

A number of ω-conotoxin GVIA mimetics based on an anthranilamide core were prepared and tested for their affinity for rat brain Ca_v2.2 channels. Features such as the presence of hydroxyl and fluoro substituents on the tyrosine side chain mimic,

Full text of DOI:10.1016/j.bmc.2009.07.063

Bioorganic & Medicinal Chemistry 17 (2009) 6659–6670
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
x
-Conotoxin GVIA mimetics based on an anthranilamide core: Effect of
variation in ammonium side chain lengths and incorporation of fluorine
c,d,
Asa Andersson a, Jonathan B. Baell b, Peter J. Duggan c, , Janease E. Graham
, Richard J. Lewis a,
*
Natalie G. Lumsden a, C. Elisabet Tranberg c, Kellie L. Tuck d, Aijun Yang a
a Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia
b The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia
c CSIRO Molecular and Health Technologies, Bag 10, Clayton South, Victoria 3169, Australia
d School of Chemistry, Monash University, Clayton, Victoria 3800, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of
x-conotoxin GVIA mimetics based on an anthranilamide core were prepared and tested for
Received 12 June 2009
Revised 23 July 2009
Accepted 26 July 2009
Available online 30 July 2009
their affinity for rat brain Cav2.2 channels. Features such as the presence of hydroxyl and fluoro substit-
uents on the tyrosine side chain mimic, the length of the chains on the lysine/arginine side chain mimics
and the use of diguanidino and diamino substituents rather than mono-guanidine/mono-amine substitu-
tion were examined. The diguanidinylated compounds proved to be the most active and deletion of the
hydroxyl substituent had a limited influence on activity. The SAR associated with variation in the lysine/
arginine side chain mimics was not strong. The introduction of a fluoro substituent into the tyrosine
mimic produced the most active compound prepared in this study (2g), with an EC50 at rat brain
Keywords:
Conotoxin
N-type calcium channels
Cav2.2
Channel blockers
Pain
Cav2.2 channels of 6 lM.
Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
1. Introduction
pathic pain, primarily due to the success of Prialt (Ziconotide), a
synthetic peptide identical to
x-conotoxin MVIIA, a component
Neuropathic pain is a hypersensitive pain response that is often
dysregulated and self-perpetuating. It results from nerve injury
associated with limb amputation, certain types of viral infections,
cancer, stroke, spinal cord injury and lower back pain caused by
disk compression or herniation. It is estimated that 26 million peo-
ple worldwide are affected by this condition,1a with the quality of
life of sufferers being greatly impaired. Neuropathic pain is often
unresponsive to existing therapies. Typically, a combination of
drugs such as opioids, antidepressants and anticonvulsants are
prescribed, but these generally only provide relief in 50% of cases,
of the venom of the marine cone snail, Conus magnus. This peptide
is known to bind to mammalian Cav2.2 channels. Prialt shows non-
addictive properties and is used clinically for the treatment of
chronic pain where opioids are not effective. This drug however,
has a narrow therapeutic window and must be administered intra-
thecally.2 The GABA analogues Gabapentin (Neurontin) and Pre-
gabalin (Lyrica) are known to bind to the a2–d sub-unit of Cav2.2
and are considered by some physicians to be the best available
treatment for neuropathic pain,1a further validating the Cav2.2
channels as targets for therapeutic intervention.
and even in these cases only
a
30–50% pain reduction is
A number of industry and academic groups have investigated
selective, low molecular weight binders of Cav2.2 as potential drug
leads for the effective treatment of neuropathic pain, either for oral
or intravenous administration. This includes a large campaign by
the Parke–Davis laboratories3 and concerted efforts by Ono Phar-
maceutical Co.,5 Ajinomoto Co.,6 Ionix Pharmaceuticals,7 the Bei-
jing Institute of Pharmacology8 and a team at the University of
Firenze.9 The most successful to date is Neuromed Technologies,
who produced NMED-160 which reached Phase II clinical trials.10
Although no adverse effects were observed with NMED-160, fur-
ther development of this compound was halted in 2007 as it did
not demonstrate ‘ideal pharmaceutical characteristics’.11 With
few exceptions,12 the initial hit structures for these investigations
have been identified through the screening of peptide or proprie-
experienced.1
The transmission and regulation of pain is a complicated pro-
cess involving ascending and descending spinal cord signals. Neu-
ronal voltage-gated N-type calcium channels (Cav2.2) concentrated
in presynaptic terminals are an important component in the regu-
lation of nociceptive neurons and pain signal transmission. They
play a critical role in synaptic neurotransmitter release. These cal-
cium channels are validated targets for the treatment of neuro-
* Corresponding author. Tel.: +61 3 9545 2560; fax: +61 3 9545 2446.
E-mail address: peter.duggan@csiro.au (P.J. Duggan).
J. E. Graham performed all of the organic synthesis described in this article.
0968-0896/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.063

6660
A. Andersson et al. / Bioorg. Med. Chem. 17 (2009) 6659–6670
tary compound libraries, or through the modification of existing
CNS-active drugs.
O
O
NH2
5
O
HN
O
Given the high selectivity of some conotoxins for Cav2.2 chan-
nels,13 and in particular
x-conotoxin GVIA, which is present in
HO
N
H
the venom of Conus geographus, we have developed Cav2.2 binders
which mimic important features of these peptides. The constrained
nature of these conotoxins makes them highly amenable to the
development of small molecule mimetics. The method used fol-
lows the approach of Bartlett and Lauri,14 and involves matching
X
3
1a
1b
X = N=C(NH2)2
X = NH2
the a,b-bond vectors of important amino acid residues on x-cono-
toxin GVIA, with bond projections on synthetic scaffolds.15,16 Two
mimetic classes were prepared in this way and the focus of the
present study are the mimetics based on an anthranilamide core
1a and 1b (Fig. 1).17 While the mono-guanidinylated compound
O
O
N
NH2HCl
O
HN
O
n
NH2
1a shows impressive binding affinity (3.5 lM), its large polar sur-
R
N
H
face area (190 Å2)à and number of hydrogen bond donors means
that it is unlikely to penetrate the CNS.18 Given the narrow therapeu-
tic window of Prialt, however, a small molecule drug with greater
selectivity for Cav2.2, even if it required intrathecal administration,
would still be a major advance. Thus, before addressing the issue
of CNS penetration, we are using relatively simple changes to the
hit structure to better define the optimum chemical properties that
promote strong association of these anthranilamide-based com-
pounds with the Cav2.2 channel. Along these lines, we have recently
shown that activity is retained if one of the ether links in 1b is chan-
ged to an amide.19 Encouraged by the calcium channel affinity
shown by long-chain aliphatic amines like dodecylamine,20 here
we describe the preparation and Cav2.2 affinity of a series of ana-
logues of 1a and 1b where variation occurs in the length of ammo-
nium side chains. This has been extended to the preparation and
testing of an analogue that bears a fluorine substituent on the
peripheral aromatic moiety.
N
m
NH2HCl
NH2
2a
2b
2c
2d
2e
2f
m = 3, n = 4, R = H
m = 3, n = 5, R = H
m = 3, n = 7, R = H
m = 4, n = 5, R = H
m = 4, n = 7, R = H
m = 3, n = 7, R = OH
m = 3, n = 7, R = F
2g
Figure 1. Previously tested anthranilamide-based
x
-conotoxin GVIA mimetics 1a
and 1b (EC50 3.5 and 13.1
l
M, respectively),17 and analogues 2a–g prepared and
tested in this study.
O
NO2
O
NO2
OH
a
HO
tBuO
2. Chemistry
Previous results obtained with a
x-conotoxin GVIA mimic
OH
O
3
4
based on a benzothiazole scaffold suggested that Cav2.2 affinity,
although reduced, could be retained when the hydroxyl group on
the peripheral aromatic moiety was deleted.21 Thus, most of the
analogues of 1a and 1b described here also lack the p-hydroxyl
substituent on the peripheral aromatic group. One example was
prepared with the p-hydroxyl substituent present (2f) and another
with a p-fluoro substituent (2g). In addition, while the original mi-
mic (1a) possessed both a primary amine and a guanidinium side
chain, here the diamino and diguanidinylated compounds were
prepared. Our initial target compounds were therefore 2a–g with
the corresponding diamino analogues being precursors to these
compounds.
NO2
O
O
NO2
O
tBuO
HO
b
c
Cl
m
Cl
m
5a
6a
m=3
m=3
5b m=4
6b m=4
Scheme 1. Reagents and yields: (a) MgSO4, H2SO4, t-BuOH, DCM, 70%; (b) K2CO3,
DMF, 1-bromo-3-chloropropane; 5a 67%; 1-bromo-4-chlorobutane; 5a 84%; (c) TFA,
DCM; 6a 78%; 6b 66%.
To allow the rapid preparation of 2a–g a new synthetic route
was developed beginning with 5-hydroxy-2-nitrobenzoic acid (3,
Scheme 1). Protection of the acid as a tert-butyl ester (4), followed
by alkylation of the phenol with 1-bromo-3-chloropropane or 1-
bromo-4-chlorobutane gave the chloro compounds 5a and 5b.
Cleavage of the tert-butyl esters gave the acids 6a–b, which were
coupled with the p-phenoxyanilines21,22 (7a–c, Scheme 2) using
EDC activation furnishing the nitro-amides 8a–d. The nitro groups
in 8a,b and d were then reduced by hydrogenation over palladium
on carbon, whereas the compound bearing the benzyloxy substitu-
ent 8c was reduced with sodium dithionite. The subsequent ani-
lines 9a–d were then acylated with either an appropriate
bromocarboxylic acid using carbodiimide activation or with 6-
bromohexanoyl chloride to furnish the corresponding dihalides
10a–g. These dihalides were then converted to diamines 12a–g
in two steps involving initial conversion to the diazides 11a–g with
tetra-n-butylammonium azide, generated in situ from trimethyl-
silyl azide and tetra-n-butylammonium fluoride,23 then hydroge-
nation over palladium on carbon. Guanidinylation to afford 2a–g
was achieved by treatment with 1H-pyrazole-1-carboximidamide
hydrochloride.24 Hydrogenation for an extended period (1.5–
2 days) was used to remove the benzyl protecting groups from
2h and 12f to furnish 2f and 12h, respectively (Scheme 3).
3. Results and discussion
The synthesised compounds, diamino (12a–g) and diguanidini-
um (2a–g), were evaluated for their ability to bind to rat brain
Cav2.2 channels using a previously described radio-ligand displace-
ment assay.17,19,21,28 The results are summarised in Table 1.
à
tPSA; ChemBioDraw Ultra 11.0.

A. Andersson et al. / Bioorg. Med. Chem. 17 (2009) 6659–6670
6661
O
O
O
NO2
O
a
R
N
H
6a
6b
or
R
NH2
7a
7b
8a
R = H
m = 3, R = H
Cl
m
R = OBn
8b m = 4, R = H
7c R = F
8c
m = 3, R = OBn
8d m = 3, R = F
O
O
O
Br
n
O
HN
O
O
NH2
O
c
b
R
N
H
R
N
H
9a m=3, R = H
10a m = 3, n = 4, R = H
Cl
m
Cl
m
9b
10b
m = 3, n = 5, R = H
m=4, R = H
9c m=3, R = OBn
9d
10c m = 3, n = 7, R = H
10d m = 4, n = 5, R = H
m=3, R = F
10e
m = 4, n = 7, R = H
10f m = 3, n = 7, R = OBn
10g
m = 3, n = 7, R = F
O
O
O
N3
O
NH2
O
HN
O
n
O
HN
O
n
d
e
R
N
R
N
H
H
N3
12a m = 3, n = 4, R = H
12b m = 3, n = 5, R = H
12c m = 3, n = 7, R = H
12d m = 4, n = 5, R = H
12e m = 4, n = 7, R = H
NH2
11a m = 3, n = 4, R = H
m
11b
m = 3, n = 5, R = H
m
11c m = 3, n = 7, R = H
11d m = 4, n = 5, R = H
11e
m = 4, n = 7, R = H
12f m = 3, n = 7, R = OBn
12g m = 3, n = 7, R = F
11f m = 3, n = 7, R = OBn
11g
m = 3, n = 7, R = F
O
O
N
NH2HCl
NH2
O
HN
O
n
R
N
f
H
N
m
NH2HCl
NH2
2a m = 3, n = 4, R = H
2b m = 3, n = 5, R = H
2c m = 3, n = 7, R = H
2d m = 4, n = 5, R = H
2e m = 4, n = 7, R = H
2g m = 3, n = 7, R = F
2h m = 3, n = 7, R = OBn
Scheme 2. Reagents and yields: (a) TEA, DMAP, EDCIꢀHCl, DCM; 8a 40%, 8b 87%: or TEA, DCC, DMAP, DCM, 8c 58%, 8d 47%; (b) H2, Pd/C, THF; 9a 48%, 9b 95%, 9d 90%: or
Na2S2O4, EtOH, THF; 9c 97%; (c) TEA, DMAP, EDCIꢀHCl, DCM, bromocarboxylic acid; 10a 91%, 10b 89%, 10c 96%, 10e 96%: or 6-bromohexanoyl chloride, TEA, DMAP, DCM; 10d
70%: or TEA, DMAP, DCC, DCM, bromocarboxylic acid; 10f 20%, 10g 11%; (d) TMS azide, TBAF, THF; 11a 68%, 11b 94%, 11c 95%, 11d 89%, 11e 98%, 11f 47%, 11g 28%; (e) Pd/C,
H2(g), THF, MeOH; 12a 98%, 12b 80%, 12c 96%, 12d 83%, 12e 81%, 12f 97%, 12g 87%; (f) 1H-pyrazole-1-carboximidamide hydrochloride, DIEA, DMF; 2a 33%, 2b 69%, 2c 46%, 2d
96%, 2e 52%, 2g 63%, 2h 47%.
O
O
X
7
O
HN
O
a
12f
2h
or
HO
N
H
X
3
12h
X = NH2
2f X = N=C(NH2)2.HCl
Scheme 3. Reagents and yields: (a) Pd/C, H2(g), MeOH, DMF; 12h 36 h 74%; 2f 48 h 84%.

6662
A. Andersson et al. / Bioorg. Med. Chem. 17 (2009) 6659–6670
Table 1
the original mimetic design and the synthetic simplifications intro-
duced here. In addition, the deletion of the hydroxyl substituent
had a limited effect on activity. In one case, a fluorine substituent
was incorporated in place of the hydroxyl group (2g) and this led
to the most active compound prepared in this study. The effect
of chain length variation on activity was initially a major focus of
this study however this did not lead to strong a SAR and demon-
strated that such structural variation is well tolerated in the
Cav2.2 channel. Only the compound with the shortest combined
length of the two flexible chains (2a) was strongly non-preferred.
Given that the contact surface between the conotoxins and the
Cav2.2 channel is likely to be quite large, it would be interesting
to study the binding of mimics with even longer side chains than
those investigated here. The use of correctly oriented rigidification
in these side chains will also be critical in the achievement of sub-
micromolar binding.
Cav2.2 binding affinities of diamino and diguanidinium anthranilamides (95%
confidence intervals are shown in parentheses)
Diamino
compound
EC50 Cav2.2
M)
Diguanidinium
compound
EC50 Cav2.2
(lM)
(l
12a
12b
12c
12d
12e
12h
12g
77 (56–106)
107 (85–134)
72 (62–85)
99 (80–122)
90 (70–115)
Weak
2a
2b
2c
2d
2e
2f
116 (95–141)
16 (12–21)
13 (9–17)
17 (13–22)
15 (13–17)
16 (12–21)
6 (4–11)
Weak
2g
In the original investigation of the use of the anthranilamide
scaffold in -conotoxin GVIA mimicry, the design strategy was
tested with two compounds (1a and 1b).17 The observation now
of several analogues of 1a with Cav2.2 affinities of <20 M supports
the ,b-bond vector approach to N-type channel blockers and con-
x
l
a
5. Experimental
firms the suitability of the anthranilamide core as a scaffold with
which to develop lead structures.
Starting materials and reagents were purchased from Sigma–Al-
drich and were used without purification. Tetrahydrofuran (THF)
was distilled under a nitrogen atmosphere from sodium benzophe-
none ketal. Dry DCM, methanol and DMF were obtained by passage
through two alumina columns on the Solvent Dispensing System
built by J. C. Meyer and based on the original design by Grubbs
and co-workers.26 Solutions were dried over anhydrous magne-
sium sulfate (MgSO4) or sodium sulfate (Na2SO4). Flash chromatog-
raphy was performed on Merck silica gel No. 9385. Melting points
were recorded on a Stuart melting point apparatus and are uncor-
rected. Infrared spectra were recorded on a Perkin Elmer 1600 FTIR
spectrometer or a Bruker IFS 55 Equinox FTIR fitted with a Golden
Gate single bounce diamond micro-ATR (Attenuated Total Reflec-
tance) cell and were recorded as Nujol mulls, solutions in chloro-
form applied as a thin film on NaCl plates or neat samples, as
indicated. Proton (1H) NMR spectra were acquired at 400 MHz
and carbon (13C) NMR spectra at 100 MHz on a Bruker Av400 spec-
trometer. NMR spectra were referenced to residual solvent peak
[chloroform (dH 7.26, dC 77.0), acetone (dH 2.05, dC 29.8, 205.9),
methanol (dH 4.87, 3.30, dC 49.86)]. To aid the assignment of
NMR spectra COSY and HMQC spectroscopy were used. The units
for all coupling constants (J) are hertz (Hz). Low resolution mass
spectra were recorded on a Micromass Platform spectrometer or
VG Platform spectrometer. Accurate mass determinations were
carried out at high resolution on an Agilent G1969A LC-TOF system
with reference and mass correction at 4000 V capillary voltage for
ESI. Microanalysis was performed at the Campbell Microanalytical
Laboratory, University of Otago, Dunedin, New Zealand.
The majority of synthesised compounds lack the hydroxyl
group on the peripheral aromatic moiety. The effect of this simpli-
fication can be gauged by comparison of the activities of direct ana-
logues 12b and 1b,17 and 2f and 2c. With the diamines (12b and
1b) a sixfold loss in activity was observed while the diguanidines
(2f and 2c) showed essentially identical activity. Taken together,
these results do not represent large differences and validate the
deletion of the peripheral hydroxyl group from the target
compounds.
The diguanidines generally showed the strongest activities,
with all but one (2a), having a Cav2.2 affinity of <20 lM. The same
compound (2a) was also the only diguanidine not to have signifi-
cantly stronger affinity than the corresponding diamine.
The SAR uncovered here demonstrates that chain length varia-
tion in these anthranilamide based compounds is well tolerated,
with the diguanidine with the shortest chains (2a) being the only
strongly non-preferred analogue. There is only a weak correlation
between side chain lengths and binding affinity but as the propoxy
octanamides (12c and 2c) gave the lowest measured IC50 values for
the diamino and diguanidino compounds, respectively, it was
decided to make and test their hydroxylated analogues (12h and
2f). As discussed above, this did not lead to a significant improve-
ment in activity. Fluorine is widely used as a hydroxyl surrogate in
bioactive discovery both for electronic and metabolic reasons. It is
also known that phenoxy groups are very quickly metabolized in
the liver and this pathway is delayed if the phenoxy group is fluo-
rinated at the 4 position.25 This substitution to give 2g led to the
lowest measured IC50 value in this study, although the 95% confi-
dence interval for this number does overlap slightly with that
determined for 2c. This compound has comparable activity to the
5.1. tert-Butyl-5-hydroxy-2-nitrobenzoate (4)
most active
x
-conotoxin GVIA mimics reported to date.17,21
The title compound (4) was prepared following the method for
the synthesis of tert-butyl esters reported by Wright et al.4 Magne-
sium sulfate (33.1 g, 0.275 mol) vigorously stirring was extensively
dried in vacuo with a heat gun for 30 min. Dry DCM (220 mL) was
then added to the flask under a dry nitrogen atmosphere and the
mixture stirred for 5 min before the addition of the benzoic acid
(3) (10.0 g, 0.055 mol) and tert-butyl alcohol (26.0 mL,
0.275 mol). Concentrated sulfuric acid (5.50 mL, 0.0550 mol) was
then carefully added and the RBF tightly sealed. The reaction was
stirred at room temperature for 36 h after which magnesium sul-
fate was removed from the reaction mixture by filtration and the
residues washed with DCM. The reaction mixture was cooled to
0 °C and then carefully neutralised by addition of satd NaHCO3(aq)
(300 mL). The product was then extracted into DCM (3 ꢁ 50 mL),
the combined extracts dried, and the solvent removed in vacuo
4. Conclusions
In summary, we have developed a new synthetic route to
anthranilamide-based
x-conotoxin GVIA mimics which has al-
lowed the ready production of an expanded set of analogues. There
was evidence from previous work21 that deletion of the hydroxyl
group from the original mimic15,17 may not lead to a significant
drop in activity. It was also unclear from the original work15,17 if
a single guanidinium group was necessary for good activity or if
diguanidinylated compounds would be suitable. Thus, diguanid-
inylated compounds were the targets here. This approach also al-
lowed for a shortened the synthetic approach.
Six of the synthesised compounds, the diguanidinylated com-
pounds (2b–g,) had Cav2.2 affinities of <20 lM, strongly validating

A. Andersson et al. / Bioorg. Med. Chem. 17 (2009) 6659–6670
6663
to yield the product as a brown solid. Recrystallization from DCM/
hexanes yielded the product (9.1 g, 70%) as a pale yellow crystal-
line solid. Mp 100–103 °C; mmax (Nujol)/cmꢂ1 3336br (OH), 2928s
and 2854s (CH), 1702s (CO), 1598s (Ar), 1522s and 1360w (NO2);
dH (400 MHz, CDCl3) 1.61 (s, 9H, t-Bu), 6.91 (dd, J = 2.7 and 8.9,
1H, H4), 6.97 (d, J = 2.7, 1H, H6), 7.93 (d, J = 8.9, 1H, H3); dC
(100 MHz, CDCl3) 27.7 (C30), 85.0 (C20), 115.5 and 117.1 (C4 and
C6), 126.9 (C3), 132.4 (C1), 139.1(C2), 161.2 (C5), 166.8 (C10);
HRMS (ESI+) [M+Na]+ calcd for C11H13NO5Na 262.0691 found
262.0691. Anal. Calcd for C11H13NO5: C, 55.23; H, 5.48; N, 5.86.
Found: C, 55.30; H, 5.48; N, 5.94.
J = 2.7, 1H, H6), 8.12 (d, J = 8.7, 1H, H3); dC (100 MHz, acetone-d6)
32.6 (C20), 42.4 (C30), 64.2 (C10), 116.3, 118.6, 132.6, 133.6, 137.7,
164.0, 167.7; HRMS (ESI+) [M+Na]+ calcd for C10H16ClNNaO5
282.0145 found 282.0140. Anal. Calcd for C10H10ClNO5: C, 46.26;
H, 3.88; N, 5.39. Found: C, 46.61; H, 3.91; N, 5.49.
5.5. 5-(4-Chlorobutoxy)-2-nitrobenzoic acid (6b)
Following the same procedure described for the preparation of
6a, the benzoic acid (6b) (3.2 g, 66%) was isolated as a white pow-
der. Mp 66–69 °C; m
max (Nujol)/cmꢂ1 3382b (OH), 2954s and 2924s
(CH), 1707s (CO), 1591w and 1490w (Ar), 1515s and 1378s (NO2),
836w (CCl); dH (400 MHz, methanol-d4) 1.82–1.85 (m, 4H, H20 and
H30), 3.52 (t, J = 6.0, 2H, H40), 4.02 (t, J = 5.7, 2H, H10), 7.01 (dd,
J = 2.7 and 8.9, 1H, H4), 7.03 (d, J = 2.7, 1H, H6), 7.87 (d, J = 8.9,
1H, H3); dC (100 MHz, methanol-d4) 26.1, 28.9, 44.0, 68.2, 114.4
(C6), 115.5, 126.2, 131.8, 140.0, 162.7, 167.7; HRMS (ESI+)
[M+Na]+ calcd for C10H16ClNNaO5 296.0302 found 296.0305. Anal.
Calcd for C10H10ClNO5: C, 48.28; H, 4.42; N, 5.12. Found: C, 48.00;
H, 4.34; N, 5.12.
5.2. tert-Butyl-5-(3-chloropropoxy)-2-nitrobenzoate (5a)
A solution of 1-bromo-3-chloropropane (1.97 g, 12.5 mmol) in
dry DMF (10 mL) was added dropwise under an atmosphere of
nitrogen to a flask containing nitrophenol (4) (3.00 g, 12.5 mmol),
potassium carbonate (5.10 g, 37.6 mmol) and DMF (120 mL). The
mixture was stirred at room temperature until no starting material
remained (typically 36 h) after which the reaction mixture was
poured into 1 M HCl(aq) (200 mL) on ice and the product extracted
into ether (5 ꢁ 100 mL). The combined ether extracts were then
washed with water (3 ꢁ 150 mL), dried, and the solvent removed
in vacuo to yield a pale yellow oil which, after purification by flash
chromatography (4:1, hexanes/ethyl acetate) gave the phenyl
ether (5a) (2.51 g, 67%) as a colourless oil. mmax (ATR)/cmꢂ1
2979w (CH), 1723vs (CO), 1584s and 1477w (Ar), 1525vs and
1370s (NO2), 845w (CCl); dH (400 MHz, CDCl3) 1.57 (s, 9H, t-Bu),
2.24–2.31 (m, 2H, H20), 3.74 (t, J = 6.2, 2H, H30), 4.22 (t, J = 5.8,
2H, H10), 6.99 (dd, J = 2.8 and 9.0, 1H, H4), 7.05 (d, J = 2.7, 1H,
H6), 7.97 (d, J = 9.0, 1H, H3); dC (100 MHz, CDCl3) 28.4 (C300), 33.2
(C20), 42.5 (C30), 67.1 (C10), 84.2 (C200), 116.3 (C6), 117.3 (C4),
127.9 (C3), 133.7, 141.7, 164.1, 165.7; HRMS (ESI+) [M+Na]+ calcd
for C14H18NO5Na 338.0777 found 338.0763.
5.6. 5-(3-Chloropropoxy)-2-nitro-N-(4-phenoxyphenyl)-
benzamide (8a)
The anthranilamide 8a was prepared following a procedure
similar to that used by Banwell, Easton and co-workers.27 A solu-
tion of nitrobenzoic acid (6a) (3.2 g, 12.4 mmol) in DCM (300 mL)
was maintained at 0 °C under a nitrogen atmosphere and Et3N
(2.4 mL, 17.3 mmol) and 4-DMAP (0.61 g, 4.94 mmol) were added
and stirred for 15 min before addition of EDCꢀHCl (2.84 g,
14.8 mmol). After stirring for a further 45 min a solution of 4-phe-
noxyaniline (7a) (2.74 g, 14.8 mmol) in DCM (20 mL) was added
dropwise. The resulting mixture was stirred for 1 h at 0 °C before
being allowed to warm to room temperature. Stirring was contin-
ued until TLC analysis showed no starting material remained (typ-
ically 16 h). The reaction mixture was then washed with water
(3 ꢁ 200 mL), dried, and the solvent removed in vacuo to yield a
brown oil. Purification by flash chromatography (2:1, hexanes/
ethyl acetate) yielded the title compound (8a) (3.1 g, 40%) as a
white powder. Mp 82–84 °C; mmax (Nujol)/cmꢂ1 3390 m and
3222 m (NH), 2925s and 2854s (CH), 1660w (CO), 1636w and
1505 (NO2), 1589 m and 1485s (Ar), 870 m (CCl); dH (400 MHz,
CDCl3) 2.31–2.25 (m, 2H, H20), 3.73 (t, J = 6.1, 2H, H30), 4.23 (t,
J = 5.9, 2H, H10), 6.93–7.01 (m, 4H, H4, H6, H400 and H700), 7.10 (t,
J = 7.4, 1H, H90), 7.33 (t, J = 8.4, 2H, H800), 7.46 (d, J = 8.88, 2H,
H300), 7.90 (br s, 1H, NH), 8.05, (d, J = 9.10, 1H, H3); dC (100 MHz,
CDCl3) 31.7, 40.9, 65.5, 114.1, 115.6, 118.6, 119.5, 122.3, 123.3,
127.2, 129.8, 132.7, 135.2, 138.6, 154.2, 157.3, 163.0, 164.6. HRMS
(ESI+) [M+Na]+ calcd for C22H19ClN2O5Na 449.0880 found
449.0878. Anal. Calcd for C22H19ClN2O5: C, 61.90; H, 4.49; N,
6.56. Found: C, 61.92; H, 4.60; N, 6.60.
5.3. tert-Butyl 5-(4-chlorobutoxy)-2-nitrobenzoate (5b)
Following the same procedure used to prepare 5a, the phenyl
ether (5b) was isolated as a colourless oil after purification by flash
chromatography (7:1, hexanes/ethyl acetate) (6.7 g, 84%). mmax
(ATR)/cmꢂ1 2979s (CH), 1728vs (CO), 1584s and 1487w (Ar),
1524vs and 1370s (NO2), 844 m (CCl); dH (400 MHz, CDCl3) 1.56
(s, 9H, t-Bu), 1.96–1.99 (m, 4H, H20 and H30), 3.60 (t, J = 6.1, 2H,
H40), 4.08 (t, J = 5.9, 2H, H10), 6.95 (dd, J = 2.7 and 9.0, 1H, H4),
7.00 (d, J = 2.7, 1H, H6), 7.95 (d, J = 9.0, 1H, H3); dC (100 MHz,
CDCl3) 26.3, 27.7, 29.0, 44.4, 68.1, 83.6, 114.5, 115.5, 126.4,
132.6, 139.9, 162.6, 164.9; m/z (ESI+, 50 eV): m/z (%): 352 (20)
[M+Na]+.
5.4. 5-(3-Chloropropoxy)-2-nitrobenzoic acid (6a)
The cleavage of the tert-butyl ester (5a) was achieved using a
27
procedure similar to that used by Banwell, Easton and co-workers
5.7. 5-(4-Chlorobutoxy)-2-nitro-N-(4-phenoxyphenyl)-
TFA (4 mL, 52.6 mmol) was added dropwise to a flask maintained
at 0 °C containing a stirred solution of ester 5a (2.5 g, 7.92 mmol)
in DCM (3 mL). The reaction was maintained at 0 °C for 1 h then
stirred at room temperature until TLC analysis showed no starting
material remained (typically 4 h). The reaction mixture was then
diluted with DCM (20 mL), washed with 1 M HCl(aq) and dried be-
fore removal of the solvent in vacuo to yield a white solid.
Recrystallisation from DCM/hexanes gave the title compound
(6a) (2.0 g, 78%) as a white powder. Mp 93–94 °C; mmax (Nujol)/
cmꢂ1 3381b (OH), 2957s and 2926s (CH), 1732s (CO), 1657w and
1487w (Ar), 1524 s and 1342s (NO2), 847 (CCl); dH (400 MHz,
methanol-d4) 2.29–2.23 (m, 2H, H20), 3.76 (t, J = 6.4, 2H, H30),
4.26 (t, J = 5.9, 2H, H10), 7.16 (dd, J = 2.7 and 8.7, 1H, H4), 7.18 (d,
benzamide (8b)
Following the procedure used to prepare 8a the title compound
(8b) was prepared from 6b and 7a. The product was isolated as a
brown oil and further purified by flash chromatography (4:1, hex-
anes/ethyl acetate) to yield 8b (4.5 g, 89%) as a white powder. Mp
127–128 °C; mmax (Nujol)/cmꢂ1 3390 m and 3222 m (NH), 3019s
(CH), 1666 m (CO), 1589w and 1506 (Ar), 1486s and 1324w
(NO2); dH (400 MHz, CDCl3) 1.93–1.95 (m, 4H, H20 and H30), 3.59
(t, J = 5.9, 2H, H40), 4.03 (t, J = 5.3, 2H, H10), 6.86 (dd, J = 9.1 and
2.2, 1H, H4), 6.92–6.89 (m, 3H, H400 and H6), 6.96 (d, J = 7.7, 2H,
H700), 7.08 (t, J = 7.4, 1H, H900), 7.29–7.33 (m, 2H, H800), 7.42 (d,
J = 9.0, 2H, H300), 7.95 (d, J = 9.1, 1H, H3), 8.45 (br s, 1H, NH); dC

6664
A. Andersson et al. / Bioorg. Med. Chem. 17 (2009) 6659–6670
(100 MHz, CDCl3) 26.3, 28.9, 44.5, 68.3, 113.9, 115.5, 118.5, 119.4,
122.2, 123.2, 127.0, 129.8, 133.0, 135.2, 138.3, 153.9, 157.4, 163.2,
164.8. m/z (ESIꢂ) 439.1 ([MꢂH]ꢂ). Anal. Calcd for C23H21ClN2O5: C,
62.66; H, 4.80; N, 6.35. Found: C, 62.66; H, 4.82; N, 6.35.
119.6, 120.4 (C4), 121.7, 124.4 (C900), 124.5 (C300), 131.0 (C800),
135.6, 144.4, 152.0, 155.3, 159.3, 170.1.(C100); HRMS (ESI+)
[M+H]+ calcd for C22H22ClN2O3 397.1319 found 397.1330.
5.11. 2-Amino-5-(4-chlorobutoxy)-N-(4-phenoxyphenyl)
5.8. N-[4-(4-Benzyloxy-phenoxy)-phenyl]-5-(3-chloro-pro-
benzamide (9b)
poxy)-2-nitro-benzamide (8c)
The title compound (9b) was prepared from 8b according to the
procedure used to prepare 9a. One batch of the aniline (9b) (1.4 g,
95%) was isolated as a white solid. mmax (ATR)/cmꢂ1 3421w and
3414w and 3278br (NH), 2986w and 2857w (CH), 1642w (CO),
1592s and 1500s and 1478vs (Ar), 800 (CCl); dH (400 MHz, CDCl3)
1.90–1.99 (m, 4H, H20 and H30), 3.62 (t, J = 6.1, 2H, H40), 3.96 (t,
J = 6.0, 2H, H10), 6.71 (d, J = 8.8, 1H, H3), 6.90 (d, J = 8.8 and 2.6,
1H, H4), 6.99–7.08 (m, 5H, H6, H400 and H700), 7.09–7.11 (m, 1H,
H900), 7.33 (t, J = 7.7, 2H, H800), 7.54 (d, J = 8.9, 2H, H300), 8.08 (br s,
1H, NH); d (100 MHz, CDCl3) 26.7, 29.3, 44.7, 68.1, 113.2, 118.1,
118.5, 119.5, 119.6, 120.3, 122.3, 123.1, 129.7, 133.2, 141.8,
151.0, 153.7, 157.5, 166.9; HRMS (ESI+) [M+H]+ calcd for
C23H24ClN2O3 411.1476 found 411.1472.
The title compound (8c) was prepared from 6a and 4-(4-benzyl-
oxyphenoxy)phenylamine (7b)21 by application of the procedure
used for the preparation of 8a, with the substitution of DCC for
EDCꢀHCl and THF as the solvent. The product was isolated as a
brown oil and further purified by flash chromatography (6:1, hex-
anes/ethyl acetate) to yield 8c (0.6 g, 58%) as a white powder. Mp
122–124 °C; mmax (ATR)/cmꢂ1 3282s (NH), 2927w and 2850w
(CH), 1658s (CO), 1607w and 1588w and 1542s (Ar) 1494s and
1335s (NO2); dH (400 MHz, CDCl3) 2.24 (m, 2H, H20), 3.72 (t,
J = 5.9, 2H, H30), 4.18 (t, J = 5.6, 2H, H10), 5.04 (s, 2H, H1000), 6.88
(d, J = 8.8, 2H, H800), 6.90–7.00 (m, 6H, H4, H6, H400 and H700), 7.34
(d, J = 6.9, 1H, H5000), 7.37–7.43 (m, 6H, H300, H3000 and H4000), 8.00
(d, J = 9.2, 1H, H3), 8.28 (br s, 1H, NH); dC (100 MHz, CDCl3) 33.7,
41.0, 65.5, 70.6, 114.1, 115.5, 116.0, 118.3, 120.4, 122.4, 127.1,
127.5, 128.0, 128.6, 132.2, 135.3, 136.9, 138.6, 150.6, 155.0,
155.3, 162.9, 164.7; m/z (ESIꢂ, 50 eV): m/z (%): 531 (100) [MꢂH]ꢂ,
567 (82) [M+Cl]ꢂ, 613 (5) [M+EtOH+Cl]ꢂ.
5.12. 2-Amino-N-(4-(4-(benzyloxy)phenoxy)phenyl)-5-(3-
chloropropoxy)benzamide (9c)
The aniline (9c) was prepared following our previously reported
procedure.17 To a solution of the nitrobenzene (8c) (250 mg,
0.464 mmol) in THF (15 mL) and EtOH (20 mL) at reflux was added
Na2S2O4 (326 mg, 1.88 mmol) and H2O (4 mL). The mixture was
stirred at reflux until no starting material was evident by TLC (typ-
ically 2 h) at which time the solvent was removed, the residues dis-
solved in ethyl acetate (150 mL) and washed with satd NaHCO3(aq)
(3 ꢁ 100 mL). The organic layer was then dried and the solvent re-
moved in vacuo to yield the product 9c (230 mg, 97%) as a beige
solid which was used for subsequent steps without further purifi-
cation. m/z (ESI+, 50 eV): m/z (%): 525 (50) [M+Na]+, 541 (11)
[M+K]+.
5.9. 5-(3-Chloro-propoxy)-N-[4-(4-fluoro-phenoxy)-phenyl]-2-
nitro-benzamide (8d)
Following the same procedure as that used to prepare 8c the ti-
tle compound (8d) was prepared from 6a and 4-(4-fluoro-phen-
oxy)-phenylamine (7c).22 The product was isolated as a brown
oil and further purified by flash chromatography (6:1, hexanes/
ethyl acetate) to yield 8d (0.41 g, 47%) as a white powder. Mp
137–140 °C; mmax (ATR)/cmꢂ1 3263s (NH), 3069w and 2959w
(CH), 1648s (CO), 1607w and 1588w and 1533s (Ar) 1494s and
1343s (NO2); dH (400 MHz, CDCl3) 2.29 (m, 2H, H20), 3.75 (t,
J = 6.0, 2H, H30), 4.24 (t, J = 5.7, 2H, H10), 6.96–7.04 (m, 7H, H4,
H300, H400 and H700), 7.52 (d, 2H, J = 8.7, 2H, H800), 7.57 (s, 1H, H6),
8.13 (d, J = 8.9, 1H, H3); dC (100 MHz, CDCl3) 31.7, 40.9, 65.4,
114.2, 115.5, 116.4 (d, J = 23.4, C700), 119.0, 120.2 (d, J = 8.2, C600),
122.4, 127.1, 132.7, 135.2, 138.6, 153.0, 154.5, 158.8 (d, J = 241.6,
C800), 160.0, 163.0, 164.6. m/z (ESIꢂ, 50 eV): m/z (%): 443 (100)
[MꢂH]ꢂ.
5.13. 2-Amino-5-(3-chloropropoxy)-N-(4-(4-fluorophenoxy)
phenyl)benzamide (9d)
The title compound (9d) was prepared from 8d according to the
procedure used to prepare 9a. After filtration through a plug of Cel-
ite the solvent was removed and the aniline (9d) (0.34 g, 90%) was
used in subsequent transformations without further purification.
HRMS (ESI+) [M+H]+ calcd for C22H21ClFN2O3 415.1224 found
415.12143.
5.10. 2-Amino-5-(3-chloropropoxy)-N-(4-phenoxyphenyl)
benzamide (9a)
5.14. 2-(5-Bromopentanamido)-5-(3-chloropropoxy)-N-(4-
The title compound was prepared following a procedure used
by Banwell, Easton and co-workers.27 A vigorously stirred flask
phenoxyphenyl)benzamide (10a)
containing
a
mixture of the nitro compound (8a) (0.67 g,
To a stirred solution of 5-bromovaleric acid (0.22 g, 1.33 mmol)
in DCM (50 mL) maintained at 0 °C under a nitrogen atmosphere
was added TEA (0.24 mL, 1.4 mmol) and 4-DMAP (0.138 g,
0.20 mmol). After stirring for 5 min EDCꢀHCl (0.231 g, 1.21 mmol)
was added and the mixture stirred for a further 15 min after which
a solution of the aniline (9a) (0.218 g, 1.21 mmol) in THF (10 mL)
was added. When TLC analysis showed no starting material re-
mained (typically 36 h) the reaction mixture was washed with
brine (3 ꢁ 50 mL), satd NaHCO3(aq) (3 ꢁ 50 mL) and 5% citric acid(aq)
(3 ꢁ 50 mL). The organic solution was dried and the solvent re-
moved in vacuo to yield a brown oil which was purified by flash
chromatography (3:1 hexanes/ethyl acetate). The title compound
(10a) (2.3 g, 91%) was obtained as a white powder. Mp 131–
133 °C; mmax (CHCl3)/cmꢂ1 3284 m and 3044 m (NH), 2936w
(CH), 1731s and 1660s (CO), 1588s and 1505s and 1489s (Ar),
844w (CCl), 752s (CBr); dH (400 MHz, CDCl3) 1.84–1.92 (m, 2H),
1.57 mmol) and 10% wt palladium on carbon (15 mg, 0.45 mmol)
in THF (5 mL) was degassed, flushed with hydrogen and stirred un-
der an atmosphere of hydrogen for 3 h. The flask was then flushed
with nitrogen and filtered though Celite. This solution was used
immediately for subsequent transformations without further puri-
fication. One batch of the aniline (9a) (0.30 g, 48%) after solvent re-
moval in vacuo was obtained as a white solid. Mp 116–118 °C; mmax
(Nujol)/cmꢂ1 3427 w and 3323w (NH), 2824s and 2853s (CH),
1643w (CO), 1591s and 1490s (Ar), 1516s and 1460 (NO2); dH
(400 MHz, CDCl3) 2.15 (m, 2H, H20), 3.10 (br s, 2H, NH2) 3.69 (t,
J = 6.3, 2H, H30), 4.09 (t, J = 5.9, 2H, H10), 6.66 (d, J = 8.8, 1H, H3),
6.86 (dd, J = 2.8 and 8.8, 1H, H4), 6.93–6.97 (m, 4H, H400 and
H700), 7.03 (t, J = 7.4, 1H, H900), 7.07 (d, J = 2.8, 1H, H6), 7.50 (m,
2H, H800), 7.50 (d, J = 8.9, 2H, H300); d (100 MHz, methanol-d4)
33.8 (C20), 42.5 (C30), 66.7 (C10), 115.1 (C6), 119.4, 119.5 (C3),

A. Andersson et al. / Bioorg. Med. Chem. 17 (2009) 6659–6670
6665
1.92–1.97 (m, 2H), 2.20–2.26 (m, 2H, H20), 2.40 (t, J = 7.2, 2H, H2000),
3.42 (t, J = 6.5, 2H, H5000), 3.74 (t, J = 6.2, 2H, H30), 4.13 (t, J = 5.9, 2H,
H10), 6.99 (dd, J = 2.8 and 9.1, 1H, H4), 7.01–7.14 (m, 6H, H6, H400,
H700 and H900), 7.32–7.36 (m, 2H, H800), 7.58 (d, J = 8.8, 2H, H300),
8.21 (br s, 1H, NH), 8.35 (d, J = 9.1, 1H, H3), 10.30 (br s, 1H, NH);
dC (100 MHz, CDCl3) 23.8, 24.1, 32.1, 33.0, 36.9, 41.3, 64.9, 113.3,
117.8, 118.7, 119.5, 122.5, 123.1, 123.3, 123.8, 129.8, 132.4,
132.5, 154.2, 154.3, 157.2, 166.8, 171.2; HRMS (ESI+) [M+Na]+ calcd
for C27H28BrClN2NaO4 581.0819 found 581.0820.
solution was then dried and the solvent removed in vacuo to
yield a brown oil. After purification by flash chromatography
(3:1 hexanes/ethyl acetate) and recrystallisation from DCM/hex-
anes the title compound (10d) (0.40 g, 70%) was isolated as
white crystals. Mp 145–146 °C; mmax (CHCl3)/cmꢂ1 3270br (NH),
3017w and 2937w (CH), 1656vs and 1603s (CO), 151506s and
1490s (Ar); dH (400 MHz, CDCl3) 1.51 (m, 2H, H4000), 1.68–1.75
(m, 4H, H3000 and H5000), 1.87–1.93 (m, 4H, H20 and H30), 2.36
(t, J = 7.36, 2H, H2000), 3.39 (t, J = 6.7, 2H, H6000), 3.60 (t, J = 5.7,
2H, H40), 3.97 (t, J = 5.5, 2H, H10), 6.90 (dd, J = 2.3 and 9.0, 1H,
H4), 7.02–7.07 (m, 5H, H6, H400 and H700), 7.11 (t, J = 7.5, 1H,
H900), 7.33 (t, J = 7.5, 2H, H800), 7.62 (d, J = 8.7, 2H, H300), 8.26
(d, J = 9.0, 1H, H3), 8.55 (br s, 1H, NH), 10.24 (br s, 1H, NH);
dC (100 MHz, CDCl3) 24.6, 26.6, 27.8, 29.2, 32.4, 33.5, 37.7,
44.6, 67.6, 113.7, 117.3, 118.7, 119.5, 122.3, 123.3, 123.4,
123.7, 129.7, 132.0, 132.9, 154.2, 154.3, 157.4, 166.1, 171.7;
HRMS (ESI+) [M+Na]+ calcd for C29H32BrClN2O4Na 611.1111
found 611.1107. Anal. Calcd for C30H34BrClN2O4: C, 59.24; H,
5.49; N, 4.76. Found: C, 59.19; H, 5.42; N, 4.75.
5.15. 2-(6-Bromohexanamido)-5-(3-chloropropoxy)-N-(4-
phenoxyphenyl)benzamide (10b)
The title compound (10b) was prepared from 9a using 6-
bromohexanoic acid according to the procedure used to prepare
10a. The crude product was obtained as a pale yellow solid and
purified by flash chromatography (2:1 hexanes/ethyl acetate) then
recrystallised from DCM/hexanes to yield the product (10b)
(0.49 g, 89%) as white fluffy crystals. Mp 128–131 °C; mmax
(CHCl3)/cmꢂ1 3280s (NH), 3016 m and 2940 m (CH), 1660s (CO),
1600s and 1504vs (Ar); dH (400 MHz, CDCl3) 1.49–1.55 (m, 2H,
H4000), 1.70–1.78 (m, 2H, H3000), 1.85–1.92 (m, 2H, H5000), 2.18–2.24
(m, 2H, H20), 2.37 (t, J = 7.6, 2H, H2000), 3.40 (t, J = 6.7, 2H, H6000),
3.73 (t, J = 6.2, 2H, H30), 4.11 (t, J = 5.8, 2H, H10), 6.96 (dd, J = 2.7
and 9.1, 1H, H4), 7.02–7.13 (m, 6H, H6, H400, H700 and H900), 7.35–
7.37 (m, 2H, H800), 7.61 (d, J = 8.9, 1H, H300), 8.28 (d, J = 9.1, 1H,
H3), 8.50 (br s, 1H, NH), 10.25 (br s, 1H, NH); dC (100 MHz, CDCl3)
24.6, 27.7, 32.1, 32.5, 33.5, 37.7, 41.3, 64.9, 113.6, 117.6, 118.7,
119.5, 122.4, 123.3, 123.4, 123.7, 129.8, 132.1, 132.8, 154.2,
154.3, 166.8, 171.7; HRMS (ESI+) [M+Na]+ calcd for
C28H30BrClN2NaO4 595.0975 found 595.0975.
5.18. 2-(8-Bromooctanamido)-5-(4-chlorobutoxy)-N-(4-
phenoxyphenyl)benzamide(10e)
The title compound (10e) was prepared from 9b using 8-
bromooctanoic acid according to the procedure used to prepare
10a. The product (10e) (0.6 g, 96%) was obtained as white crystals
after purification by flash chromatography (2:1 hexanes/ethyl ace-
tate) then recrystallisation from DCM/hexanes. Mp 133–134 °C;
mmax (CHCl3)/cmꢂ1 3299br (NH), 3017 and 2935 and 2858 (CH),
1652 and 1591 (CO), 1595 and 1487 (Ar); dH (400 MHz, CDCl3)
1.32–1.43 (m, 6H, H4000, H5000and H6000), 1.71 (m, 2H, H3000), 1.83
(m, 2H, H7000), 1.93–1.97 (m, 4H, H20 and H30), 2.35 (t, J = 7.6, 2H,
H2000), 3.38 (t, J = 6.9, 2H, H5000), 3.62 (t, J = 5.9, 2H, H40), 3.97 (t,
J = 5.83, 2H, H10), 6.90 (dd, J = 2.9 and 9.1, 1H, H4), 7.01–7.04 (m,
4H, H400 and H700), 7.06–7.11 (m, 1H, H900), 7.33–7.37 (m, 3H, H6
and H800), 7.59 (d, J = 9.0, 2H, H300), 8.26 (br s, 1H, NH), 8.33 (d,
J = 9.1, 1H, H, H3), 10.23 (br s, 1H, NH); dC (100 MHz, CDCl3) 25.4,
26.6, 28.0, 28.5, 29.0, 29.2, 32.7, 33.9, 38.1, 44.6, 67.6, 113.5,
117.7, 118.6, 118.8, 119.5, 122.5, 123.4, 123.9, 129.8, 132.3,
132.8, 154.4, 157.3, 166.9, 172.1; m/z (ESI+, 50 eV): m/z (%): 615
(100) [M+H]+ Anal. Calcd for C31H36BrClN2O4: C, 60.44; H, 5.89;
N, 4.55. Found: C, 60.50; H, 5.61; N, 4.71.
5.16. 2-(8-Bromooctanamido)-5-(3-chloropropoxy)-N-(4-
phenoxyphenyl)benzamide (10c)
The title compound (10c) was prepared from 9a using 8-
bromooctanoic acid according to the procedure used to prepare
10a. The product 10c (0.60 g, 96%) was obtained as white crys-
tals after purification by flash chromatography (2:1 hexanes/
ethyl acetate) and recrystallisation from DCM/hexanes. Mp
133–134 °C; mmax (CHCl3)/cmꢂ1 3314b (NH), 3019w and 2933w
(CH), 1654w and 1604w (CO), 1506s and 1490s (Ar); dH
(400 MHz, CDCl3) 1.31–1.46 (m, 6H, H4000, H5000 and H6000), 1.67–
1.75 (m, 2H, H3000) 1.79–1.89 (m, 2H, H7000), 2.30–2.23 (m, 2H,
H20), 2.35 (t, J = 7.4, 2H, H2000), 3.38 (t, J = 6.8, 2H, H8000), 3.73 (t,
J = 6.2, 2H, H30), 4.13–4.10 (m, 2H, H10), 6.97 (dd, J = 2.8 and
9.1, 1H, H4), 7.02–7.07 (m, 4H, H400 and H700), 7.10 (d, J = 2.66,
1H, H6), 7.11–7.14 (m, 1H, H900), 7.34–7.38 (m, 2H, H800), 7.61
(d, J = 8.9, 2H, H300), 8.34 (d, J = 9.1, 1H, H3), 8.37 (br s, 1H,
NH), 10.24 (br s, 1H, NH); dC (100 MHz, CDCl3) 25.4, 28.0, 28.4,
29.0, 32.1, 32.7, 33.9, 38.0, 41.3, 64.9, 113.4, 117.3, 118.5,
119.5, 122.4, 123.3, 122.7, 123.8, 129.8, 132.3, 132.7, 154.3,
154.1, 157.2, 166.8, 172.1; m/z (ESIꢂ, 50 eV): m/z (%): 599
(100) [MꢂH]ꢂꢂ. Anal. Calcd for C30H34BrClN2O4: C, 59.86; H,
5.69; N, 4.65. Found: C, 59.60; H, 5.80; N, 4.55.
5.19. N-(4-(4-(Benzyloxy)phenoxy)phenyl)-2-(8-
bromooctanamido)-5-(3-chloropropoxy)benzamide (10f)
The title compound (10f) was prepared from 9c using 8-bromo-
octanoic acid according to the procedure used to prepare 8c. The
product (10f) (44 mg, 20%) was obtained as white crystals after
purification by flash chromatography (2:1 hexanes/ethyl acetate)
then recrystallisation from DCM/hexanes. Mp 111–113 °C; mmax
(ATR)/cmꢂ1 3396w and 3323w (NH), 2927w and 2851w (CH),
1686w and 1623w (C@O), 1586w, 1511w, 1493s (Ar); dH
(400 MHz, CDCl3) 1.35–1.38 (m, 4H, H4000 and H5000), 1.42 (m, 2H,
H6000), 1.71 (m, 2H, H6000), 1.83 (m, 2H, H7000), 2.20 (m, 2H, H20),
2.34 (t, J = 7.5, 2H, H2000), 3.38 (t, J = 6.9, 2H, H8000), 3.71 (t, J = 6.2,
2H, H30), 4.08 (t, J = 5.9, 2H, H10), 5.06 (s, 2H, H1000), 6.92 (dd,
J = 2.8 and 9.1, 1H, H4), 6.94–7.00 (m, 6H, H400 and H700 and H800),
7.33 (d, J = 7.1, 1H, H50000), 7.40 (m, 2H, H40000), 7.44 (m, 2H, H30000),
7.57 (d, J = 8.9, 2H, H300), 8.29 (d, J = 9.1, 1H, H3), 8.52 (br s, 1H,
NH), 10.25 (br s, 1H, NH); dC (100 MHz, CDCl3) 25.4, 28.0, 28.5,
29.0, 32.1, 32.7, 33.9, 38.0, 41.3, 64.9, 70.6, 113.6, 116.0, 117.6,
118.3, 120.5, 121.6, 122.4, 123.4, 123.7, 127.5, 128.0, 128.6,
132.2, 136.9, 150.5, 154.1, 155.1, 155.5, 166.8, 172.1; HRMS
(ESI+) [M+Na]+ calcd for C37H41BrClN2O5 707.18874 found
707.18743.
5.17. 2-(6-Bromohexanamido)-5-(4-chlorobutoxy)-N-(4-
phenoxyphenyl)benzamide (10d)
A solution of 6-bromohexanoyl chloride (0.231 g, 1.17 mmol)
in DCM (20 mL) was added dropwise to a stirring solution of
the aniline (9b) (400 mg, 0.976 mmol) in DCM (70 mL) at 0 °C
under a nitrogen atmosphere. When TLC analysis indicated the
absence of starting material (typically 10 min) the reaction mix-
ture was washed with brine (3 ꢁ 50 mL), satd NaHCO3(aq)
(3 ꢁ 50 mL) and 5% citric acid(aq) (3 ꢁ 50 mL). The organic

6666
A. Andersson et al. / Bioorg. Med. Chem. 17 (2009) 6659–6670
5.20. 2-(8-Bromooctanamido)-5-(3-chloropropoxy)-N-(4-(4-
fluorophenoxy)phenyl)benzamide (10g)
26.3, 28.6, 37.8, 48.1, 51.2, 53.8, 65.2, 113.6, 117.6, 118.7, 119.5,
119.6, 122.4, 123.3, 123.7, 129.8, 132.2, 132.8, 154.1, 154.2,
157.2, 166.8, 171.7 (C100); HRMS (ESI+) [M+Na]+ calcd for
C28H30N8O4Na 565.2288 found 565.2282.
The title compound (10g) was prepared from 9d using 8-
bromooctanoic acid according to the procedure used to prepare
8c. The product (10g) (62 mg, 11%) was obtained as a pale brown
solid after purification by flash chromatography (2:1 hexanes/ethyl
acetate) then recrystallisation from DCM/hexanes. Mp 101–105 °C;
5.23. 2-(8-Azidooctanamido)-5-(3-azidopropoxy)-N-(4-
phenoxyphenyl)benzamide (11c)
m
max (ATR)/cmꢂ1 3427w and 3307w (NH), 2928w and 2855w (CH),
The diazide (11c) was prepared from the dihalide (10c)
according to the method used to prepare 11a. The product
(11c) (28 mg, 95%) was isolated as white crystals. mmax (CHCl3)/
cmꢂ1 3300br (NH), 2933s (CH), 2097vs (N3), 1653w and 1591s
(CO), 1521s and 1506vs and 1489s (Ar); dH (400 MHz, CDCl3)
1.35 (m, 4H, H4000 and H5000), 1.44 (m, 2H, H6000), 1.58 (m, 2H,
H3000), 1.69 (m, 2H, H7000), 2.02 (m, 2H, H20), 2.46 (t, J = 7.5, 2H,
H2000), 3.23 (t, J = 6.9, 2H, H8000), 3.50 (t, J = 6.6, 2H, H30), 4.03 (t,
J = 5.9, 2H, H10), 6.93 (dd, J = 2.7 and 9.1, 1H, H4), 7.01–7.14
(m, 6H, H6, H400, H700 and H900), 7.35 (m, 2H, H800), 7.55 (d,
J = 8.9, 2H, H300), 8.42 (d, J = 9.1, 1H, H3), 8.54 (br s, 1H, NH),
10.27 (br s, 1H, NH); dC (100 MHz, CDCl3) 25.4, 26.5, 28.71,
28.74, 28.8, 29.0, 38.1, 48.1, 51.4, 65.1, 113.5, 117.6, 118.4,
118.7, 119.5, 122.4, 123.3, 123.7, 129.8, 132.3, 132.8, 154.0,
154.2, 157.3, 166.8, 172.0; HRMS (ESI+) [M+Na]+ calcd for
C30H34N8O4Na 593.2601 found 593.2593.
1687w and 1641w (C@O), 1590w, 1511w and 1493s (Ar); dH
(400 MHz, CDCl3) 1.25–1.43 (m, 4H, H4000, H5000 and H6000), 1.68–
1.71 (m, 2H, H3000), 1.81–1.85 (m, 2H, H7000), 2.17–2.20 (m, 2H,
H20), 2.33 (t, J = 7.6, 2H, H2000), 3.37 (t, 2H, J = 6.8, 2H, H8000), 3.70
(t, J = 6.2, 2H, H30), 4.08 (t, J = 5.8, 2H, H10), 6.9 (dd, J = 2.6 and
9.1, 1H, H4), 6.97–7.04 (m, 8H, H400, H600, H700 and H800), 7.07 (d,
J = 2.6, 1H, H6), 7.61 (d, J = 8.8, 2H, H300), 8.23 (d, J = 8.5, 1H, H3),
8.71 (br s, 1H, NH), 10.22 (br s, 1H, NH); dC (100 MHz, CDCl3)
25.4, 28.0, 28.5, 29.0, 32.1, 32.7, 33.9, 38.0, 41.3, 64.7, 113.7,
116.3 (J 23.3, C800), 117.5, 118.9, 120.3 (J 8.3, C700), 122.4, 123.4,
123.7, 132.1, 132.9, 152.9, 154.1, 154.6, 158.8 (J 241.8, C900),
166.8, 172.1; HRMS (ESI+) [M+Na]+ calcd for C30H33BrClFN2O4Na
641.1194 found 641.1187.
5.21. 2-(5-Azidopentanamido)-5-(3-azidopropoxy)-N-(4-
phenoxyphenyl)benzamide (11a)
5.24. 5-(4-Azidobutoxy)-2-(6-azidohexanamido)-N-(4-
The diazide (11a) was prepared from the dihalide (10a) fol-
lowing the method of Takaya.23 To a stirred solution of the
dihalide (100 mg, 0.17 mmol) in THF (5 mL), maintained at 0 °C
under an atmosphere of nitrogen was added trimethylsilylazide
(60 mg, 0.52 mmol) then tetra-butylammonium fluoride (1 M)
in THF (0.52 mL, 0.52 mmol). The reaction mixture was then al-
lowed to warm slowly to room temperature then heated at 45 °C
until NMR analysis showed no starting material remained (typi-
cally 50 h). The solvent was then removed in vacuo to give a
brown oil which was purified by flash chromatography (4:1 hex-
anes/ethyl acetate) then recrystallised from DCM/hexanes to
yield the title compound (11a) (62 mg, 68%) as white crystals.
Care was taken to ensure the dry diazide was not subjected to
shock or heat. mmax (CHCl3)/cmꢂ1 3322br (NH), 3017s and
2960s (CH), 2099vs (N3), 1656s and 1591s (CO), 1505vs and
1489vs (Ar); dH (400 MHz, CDCl3) 1.66 (m, 2H, H3000), 1.80 (m,
2H, H4000), 2.06 (m, 2H, H20), 2.42 (t, J = 7.2, 2H, H2000), 3.32 (t,
J = 6.6, 2H, H5000), 3.52 (t, J = 6.5, 2H, H30), 4.06 (t, J = 6.1, 2H,
H10), 6.99–7.14 (m, 7H, H4, H6, H400, H700 and H900) 7.35 (m,
2H, H800), 7.78 (d, J = 8.9, 2H, H300), 8.18 (br s, 1H, NH), 8.42 (d,
J = 9.1, 1H, H3), 10.47 (br s, 1H, NH); dC (100 MHz, CDCl3) 22.6,
28.4, 28.7, 37.3, 48.1, 51.1, 65.2, 113.5, 117.7, 118.7, 119.5,
122.5, 123.1, 123.4, 123.7, 129.8, 132.4, 132.6, 154.1, 154.4,
157.2, 166.8, 171.2; HRMS (ESI+) [M+H]+ calcd for C27H29N8O4
529.2312 found 529.2339.
phenoxyphenyl)benzamide(11d)
The diazide (11d) was prepared from the dihalide (10d)
according to the method used to prepare 11a. The product
(11d) (0.34 g, 89%) was isolated as white crystals. mmax (CHCl3)/
cmꢂ1 3280br (NH), 3014w and 2933s (CH), 2098vs (N3), 1655s
and 1591s (CO), 1521s and 1506vs and 1488s (Ar); dH
(400 MHz, CDCl3) 1.45 (m, 2H, H4000), 1.63 (m, 2H, H3000), 1.71–
1.83 (m, 4H, H30 and H5000), 1.84–1.89 (m, 2H, H20), 2.38 (t,
J = 7.5, 2H, H2000), 3.26 (t, J = 6.9, 2H, H6000), 3.37 (t, J = 6.5, 2H,
H40), 4.00 (t, J = 6.0, 2H, H10), 6.98 (dd, J = 2.8 and 9.1, 1H, H4),
7.02–7.07 (m, 4H, H400 and H700), 7.09 (d, J = 2.8, 1H, H6), 7.12
(m, 1H, H900), 7.33–7.37 (m, 2H, H800), 7.56 (d, J = 8.9, 2H, H300),
8.16 (br s, 1H, NH), 8.37 (d, J = 9.1, 1H, H3), 10.28 (br s, 1H,
NH); dC (100 MHz, CDCl3) 25.0, 25.6, 26.3, 26.5, 28.6, 37.8,
51.2, 53.9, 65.2, 113.7, 117.4, 118.7, 119.5, 119.6, 122.4, 123.4,
123.7, 129.8, 132.0, 132.8, 154.29, 154.31, 157.3, 166.8, 171.6;
HRMS (ESI+) [M+Na]+ calcd for C29H32N8O4Na 579.2444 found
579.2444.
5.25. 5-(4-Azidobutoxy)-2-(8-azidooctanamido)-N-(4-
phenoxyphenyl)benzamide (11e)
The diazide (11e) was prepared from the dihalide (10e) accord-
ing to the method used to prepare 11a. The product (11e) (0.14 mg,
98%) which was isolated as white crystals. mmax (CHCl3)/cmꢂ1
3320br (NH), 3028s and 2933s (CH), 2100vs (N3), 1652s and
1591s (CO), 1521s and 1505vs and 1489s (Ar); dH (400 MHz, CDCl3)
1.32–1.41 (m, 6H, H4000and H5000 and H6000), 1.54–1.59 (m, 2H, H3000),
1.67–1.72 (m, 2H, H7000), 1.74–1.80 (m, 2H, H30) 1.82–1.89 (m, 2H,
H20), 2.35 (t, J = 7.5, 2H, H2000), 3.26 (t, J = 6.9, 2H, H8000), 3.37 (t,
J = 6.6, 2H, H40), 4.00 (t, J = 5.9, 2H, H10), 6.98 (dd, J = 2.7 and 9.1,
1H, H4), 7.07–7.02 (m, 4H, H400 and H700), 7.09 (d, J = 2.7, 1H, H6),
7.12 (t, J = 7.5, 1H, H900), 7.35 (m, 2H, H800), 7.61 (d, J = 8.8, 2H,
H300), 8.30 (d, J = 9.1, 1H, H3), 8.48 (br s, 1H, NH), 10.22 (br s, 1H,
NH); dC (100 MHz, CDCl3) 25.4, 25.6, 26.4, 26.5, 28.7, 28.9, 29.0,
38.0, 51.1, 51.4, 67.8, 113.7, 117.2, 118.7, 119.5, 119.6, 122.3,
123.3, 123.6, 129.8, 131.7, 132.8, 154.21, 154.24, 157.3, 166.9,
172.1; HRMS (ESI+) [M+Na]+ calcd for C31H36N8O4Na 607.2757
found 607.2756.
5.22. 2-(6-Azidohexanamido)-5-(3-azidopropoxy)-N-(4-
phenoxyphenyl)benzamide (11b)
The diazide (11b) was prepared from the dihalide (10b) accord-
ing to the method used to prepare 11a. The product (11b) (280 mg,
94%) was isolated as white crystals. mmax (CHCl3)/cmꢂ1 3310br
(NH), 3019s and 2934s (CH), 2100vs (N3), 1655w and 1592w
(CO), 1521s and 1506vs (Ar); dH (400 MHz, CDCl3) 1.54 (m, 2H,
H4000), 1.71 (m, 2H, H3000), 1.81 (m, 2H, H5000), 2.15 (m, 2H, H20),
2.48 (t, J = 7.40, 2H, H2000), 3.41 (t, J = 6.9, 2H, H6000), 3.66 (t, J = 6.7,
2H, H30), 4.23 (t, J = 6.1, 2H, H10), 7.09–7.15 (m, 4H, H400and H700),
7.19–7.23 (m, 2H, H4 and H900), 7.47 (m, 2H, H800), 7.51 (d, J = 2.9,
1H, H6), 7.88 (d, J = 9.0, 2H, H300), 8.50 (d, J = 9.1, 1H, H3), 9.84
(br s, 1H, NH), 10.56 (br s, 1H, NH); dC (100 MHz, CDCl3) 25.0,

A. Andersson et al. / Bioorg. Med. Chem. 17 (2009) 6659–6670
6667
5.26. 2-(8-Azidooctanamido)-5-(3-azidopropoxy)-N-(4-(4-
5.29. 2-(6-Aminohexanamido)-5-(3-aminopropoxy)-N-(4-
(benzyloxy)phenoxy)phenyl)benzamide (11f)
phenoxyphenyl)benzamide (12b)
The diazide (11f) was prepared from the dihalide (10f)
according to the method used to prepare 11a (53 mg, 47%).
mmax (ATR)/cmꢂ1 3244w and 3123w and 3039w (NH), 2930s
and 2857w (CH), 2091s (N3), 1656s and 1639s (CO), 1600vs
and 1547s and 1493s (Ar); dH (400 MHz, CDCl3) 1.35 (m, 6H,
H4000, H5000 and H6000), 1.58 (m, 2H, H3000), 1.71 (m, 2H, H7000),
1.02 (m, 2H, H20), 2.34 (t, J = 7.6, 2H, H2000), 3.23 (t, J = 7.0,
2H, H8000), 3.49 (t, J = 6.6, 2H, H30), 4.02 (t, J = 6.1, 2H, H10),
5.06 (s, 2H, H10000), 6.92 (dd, J = 2.7 and 9.4, 2H, H4), 6.78–
7.00 (m, 6H, H400, H700 and H800), 7.08 (d, J = 2.7, 1H, H6),
7.33–7.38 (m, 1H, H50000), 7.39–7.45 (m, 4H, H30000 and H4000),
7.56 (d, J = 8.9, 2H, H300), 8.30 (d, J = 9.1, 1H, H3), 8.49 (br s,
1H, NH), 10.26 (br s, 1H, NH); dC (100 MHz, CDCl3) 25.4,
26.6, 28.7, 28.8, 28.9, 29.1, 38.0, 48.1, 51.4, 65.2, 70.6, 113.6,
115.0, 117.6, 118.4, 120.5, 121.5, 123.7, 127.5, 128.0, 128.6,
132.2, 132.3, 136.9, 150.5, 154.1, 155.1, 155.5, 166.8, 172.1;
HRMS (ESI+) [M+Na]+ calcd for C37H41N8O5Na 699.3019 found
699.3013.
The diamine (12b) was prepared from the diazide (11b) accord-
ing to the method used to prepare 12a. The product (12b) (66 mg,
80%) was isolated as a white powder. Mp 147 °C (decomposed);
mmax (ATR)/cmꢂ1 3258b and 3150b and 3057b (NH), 2928s and
2860s (CH), 1649s and 1587s (CO), 1503vs and 1485vs (Ar); dH
(400 MHz, methanol-d4) 1.41 (m, 2H, H4000), 1.53 (m, 2H, H3000),
1.69 (m, 2H, H5000), 1.98 (m, 2H, H20), 2.38 (t, J = 7.2, 2H, H2000),
2.69 (t, J = 7.2, 2H, H6000), 2.89 (t, J = 6.9, 2H, H30), 4.13 (t, J = 6.1,
2H, H10), 6.97–7.01 (m, 4H, H400 and H700), 7.08–7.11 (m, 2H, H4
and H900), 7.30 (d, J = 2.6, 1H, H6), 7.35 (m, 2H, H800), 7.64 (d,
J = 8.9, 2H, H300), 7.91 (d, J = 9.0, 1H, H3); dC (100 MHz, methanol-
d4) 25.2, 26.0, 31.5, 31.7, 36.7, 38.2, 40.7, 66.1, 113.7, 117.2,
118.2, 118.8, 122.6, 122.9, 124.6, 127.3, 129.5, 131.1, 133.7,
154.0, 155.6, 157.5, 167.4, 173.0; HRMS (ESI+) [M+H]+ calcd for
C28H35N4O4 491.2658 found 491.2658.
5.30. 2-(8-Aminooctanamido)-5-(3-aminopropoxy)-N-(4-
phenoxyphenyl)benzamide (12c)
The diamine (12c) was prepared from the diazide (11c) accord-
ing to the method used to prepare 12a. The product (12c) (156 mg,
96%) was isolated as a white powder. Mp 132 °C (decomposed);
mmax (ATR)/cmꢂ1 3262b and 3043b (NH), 2927s and 2854s (CH),
1728s and 1589s (CO), 1503vs and 1486vs (Ar); dH (400 MHz,
methanol-d4) 1.29–1.33 (m, 6H, H4000 and H5000 and H6000), 1.39
(m, 2H, H7000), 1.64 (m, 2H, H3000), 1.94 (m, 2H, H20), 2.36 (t,
J = 7.4, 2H, H2000), 2.66 (t, J = 7.2, 2H, H8000), 2.88 (t, J = 7.0, 2H,
H30), 4.13 (t, J = 6.0, 2H, H10), 6.97–7.01 (m, 4H, H400 and H700),
7.08–7.11 (m, 2H, H4 and H900), 7.29 (d, J = 2.7, 1H, H6), 7.33 (m,
2H, H800), 7.64 (d, J = 8.6, 2H, H300), 7.90 (d, J = 9.0, 1H, H3); dC
(100 MHz, methanol-d4) 27.0 (C3000), 28.0, 30.4, 30.5, 33.5 (C20),
34.0 (C7000), 38.4 (C2000), 39.8 (C30), 42.7 (C8000), 67.7 (C10), 115.1
(C4), 118.6 (C6), 119.6, 120.4, 123.9 (C900), 124.1 (C3), 124.4
(C300), 126.2, 131.1 (C800), 131.2, 135.5, 155.3, 157.2, 159.1, 169.0,
174.8; HRMS (ESI+) [M+Na]+ calcd for C30H39N4O4Na 519.2971
found 519.2970.
5.27. 2-(8-Azidooctanamido)-5-(3-azidopropoxy)-N-(4-(4-
fluorophenoxy)phenyl)benzamide (11g)
The diazide (11g) was prepared from the dihalide (10g) accord-
ing to the method used to prepare 11a. The product (11g) (50 mg,
28%) which was isolated as white crystals. m
max (ATR)/cmꢂ1 3309br
and 3241s (NH), 2928s (CH), 2096s (N3), 1645s and 1589s (CO),
1520s and 1593vs and 1466s (Ar); dH (400 MHz, CDCl3) 1.35 (m,
6H, H4000, H5000 and H6000), 1.58 (m, 2H, H3000), 1.71 (m, 2H, H7000),
2.04 (m, 2H, H20), 2.35 (t, J = 7.5, 2H, H20), 3.23 (t, J = 6.9, 2H,
H8000), 3.51 (t, J = 6.3, 2H, H30), 4.05 (t, J = 5.9, 2H, H10), 6.97–7.04
(m, 7H, H4, H400, H700 and H800), 7.09 (d, J = 2.5, 1H, H6), 7.57 (d,
J = 8.8, 2H, H300), 8.25 (br s, 1H, NH), 8.35 (d, J = 9.1, 1H, H3),
10.23 (br s, 1H, NH); dC (100 MHz, CDCl3) 25.4, 26.5, 28.7, 28.7,
28.9, 29.0, 38.0, 48.1, 51.4, 65.2, 113.4, 116.4 (J = 23.4, C800),
117.8, 118.9, 120.4 (J = 8.3, C700), 122.5, 123.1, 123.8, 132.5, 132.6,
151.0, 154.1, 154.8, 158.6 (J = 241.4, C900), 166.8, 171.6; HRMS
(ESI+) [M+Na]+ calcd for C30H33N8O4Na 611.25065 found
611.24969.
5.31. 5-(4-Aminobutoxy)-2-(6-aminohexanamido)-N-(4-
phenoxyphenyl)benzamide (12d)
5.28. 2-(5-Aminopentanamido)-5-(3-aminopropoxy)-N-(4-
phenoxyphenyl)benzamide (12a)
The diamine (12d) was prepared from the diazide (11d) accord-
ing to the method used to prepare 12a. The product (12d) (101 mg,
83%) was isolated as a white powder. Mp 106 °C (decomposed);
mmax (ATR)/cmꢂ1 3270b and 3047b (NH), 2925s and 2852s (CH),
1708s and 1624s (CO), 1503vs and 1486vs (Ar); dH (400 MHz,
methanol-d4) 1.31 (m, 2H, H4000), 1.40 (m, 2H, H3000), 1.55–1.62
(m, 4H, H30 and H5000), 1.76 (m, 2H, H20), 2.23 (t, J = 7.4, 2H, H2000),
2.53 (t, J = 7.1, 2H, H6000), 2.66 (t, J = 7.2, 2H, H40), 4.00 (t, J = 6.3,
2H, H10), 6.93–6.91 (m, 4H, H400 and H700), 7.02 (dd, J = 2.7 and
9.0, 1H, H4), 7.06 (t, J = 7.4, 1H, H900), 7.20–7.25 (m, 2H, H6),
7.26–7.30 (m, 2H, H800), 7.58–7.60 (d, J = 7.85, 2H, H300), 7.89 (d,
J = 8.49, 1H, H3); dC (100 MHz, methanol-d4) 26.7 (C3000), 27.5
(C4000), 27.8 (C20), 30.6 (C30), 33.7 (C5000), 38.3 (C2000), 42.38 (C600),
42.43 (C40), 69.4 (C10), 115.2 (C6), 118.6 (C4), 119.6 (C400), 120.3
(C700), 124.0 (C300), 124.3, 125.8 (C3), 128.4, 130.9 (C800), 131.5
(C300), 135.3, 155.3, 156.9, 158.9, 168.8, 174.3; HRMS (ESI+)
[M+H]+ calcd for C29H37N4O4 505.2815 found 505.2815.
A flask containing a solution of the diazide (11a) (48 mg,
0.10 mmol) and 10% wt palladium on carbon (16 mg,
0.071 mmol) in THF/methanol (1:1) (4 mL) was degassed, flushed
with hydrogen and stirred with an atmosphere of hydrogen for
3 h. After this time the reaction mixture was filtered through
Celite and the solvent removed in vacuo to give a pale yellow
oil. After recrystallisation from methanol/ethyl acetate the title
compound (12a) (43 mg, 98%) was isolated as a white powder.
Mp 152 °C (decomposed); mmax (ATR)/cmꢂ1 3228b (NH), 2930s
and 2857s (CH), 1636s and 1600s (CO), 1528vs and 1501vs
and 1485vs (Ar). dH (400 MHz, methanol-d4) 1.50 (m, 2H, H3000),
1.66 (m, 2H, H4000), 1.95 (m, 2H, H20), 2.38 (t, J = 7.4, 2H, H2000),
2.62 (t, J = 7.1, 2H, H5000), 2.85 (t, J = 6.8, 2H, H30), 4.11 (t,
J = 6.1, 2H, H10), 6.96–6.99 (m, 4H, H400 and H700), 7.06–7.10
(m, 1H, H900), 7.28 (d, J = 2.7, 1H, H6), 7.34 (m, 2H, H800), 7.63
(d, J = 8.9, 2H, H300), 7.90 (d, J = 9.0, 1H, H3); dC (100 MHz, meth-
anol-d4) 22.5, 23.9, 31.9, 37.8, 39.5, 41.7, 67.5, 115.2, 118.7,
119.6, 120.2, 124.1, 124.4, 126.0, 128.6, 130.9, 131.4, 135.1,
155.4, 156.9, 158.9, 168.8, 174.0; HRMS (ESI+) [M+H]+ calcd for
C27H33N4O4 477.2502 found 477.2498.
5.32. 5-(4-Aminobutoxy)-2-(8-aminooctanamido)-N-(4-
phenoxyphenyl)benzamide (12e)
The diamine (12e) was prepared from the diazide (11e) accord-
ing to the method used to prepare 12a. The product (12e) (59 mg,

6668
A. Andersson et al. / Bioorg. Med. Chem. 17 (2009) 6659–6670
81%) was isolated as a white powder. Mp 122 °C (decomposed);
mmax (ATR)/cmꢂ1 3325b and 3230b and 3125b (NH), 2929s and
2851s (CH), 1644s and 1602s (CO), 1511vs and 1488vs (Ar); dH
(400 MHz, methanol-d4) 1.35–1.37 (m, 6H, H4000, H5000 and H6000),
1.45 (m, 2H, H7000), 1.69–1.71 (m, 4H, H30 and H3000), 1.89 (m, 2H,
H20), 2.39 (t, J = 6.3, 2H, H2000), 2.60 (t, J = 7.2, 2H, H8000), 2.76 (t,
J = 6.2, 2H, H40), 4.13 (m, 2H, H10), 7.03 (m, 4H, H400 and H700),
7.09–7.15 (m, 2H, H4 and H900), 7.36–7.40 (m, 3H, H6 and H800),
7.66 (d, J = 8.0, 2H, H300), 7.93 (d, J = 8.7, 1H, H3); dC (100 MHz,
methanol-d4) 26.9, 27.8, 27.9, 30.2, 30.3, 30.6, 34.0, 38.5, 42.4,
42.6, 69.4, 115.2, 118.6, 119.5, 120.3, 124.0, 124.8, 125.9, 129.5,
130.1, 130.9, 135.9, 155.1, 157.0, 159.1, 168.9, 174.5; HRMS
(ESI+) [M+H]+ calcd for C31H41N4O4 533.3128 found 533.3119.
65.9, 114.1, 116.2, 117.2, 120.2, 120.5, 122.2, 127.6, 128.3, 133.8,
137.0, 148.2, 154.2, 160.7, 166.5, 166.5, 175.1; HRMS (ESI+)
[M+H]+ calcd for C30H39N4O5 535.2921 found 535.2906.
5.36. 2-(5-Guanidinopentanamido)-5-(3-guanidinopropoxy)-N-
(4-phenoxyphenyl)benzamide dihydrochloride (2a)
The diamine (12a) (43 mg, 0.091 mmol), DIEA (0.031 mL,
0.18 mmol), 1H-pyrazole-1-carboximidamide hydrochloride (26 mg,
0.18 mmol) and DMF (3 mL) were combined and stirred under a
nitrogen atmosphere overnight. The solvent was then removed in
vacuo and the residues recrystallised twice from ether/methanol
to yield the title compound (2a) (20 mg, 33%) as colourless crystals.
Mp 76–80 °C; mmax (ATR)/cmꢂ1 3242b and 3290b and 3159b (NH),
2950s and 2862s (CH), 1631s (CO), 1504 and 1487 and 1471 (Ar);
dH (500 MHz, methanol-d4) 1.60 (m, 2H, H4000), 1.69 (m, 2H, H3000),
2.06 (m, 2H, H20), 2.39 (t, J = 7.3, 2H, H2000), 3.12 (t, J = 7.1, 2H,
H5000), 3.40 (t, J = 6.8, 2H, H30), 4.16 (t, J = 5.8, 2H, H10), 6.95–6.98
(m, 4H, H400 and H700), 7.08 (m, 1H, H900), 7.14 (dd, J = 2.7 and 9.0,
1H, H4), 7.34–7.37 (m, 3H, H6 and H800), 7.65 (d, J = 8.7, 2H, H300),
7.87 (d, J = 9.0, 1H, H3); dC (100 MHz, methanol-d4) 23.8 (C4000),
29.4 (C3000), 29.8 (C20), 37.5 (C2000), 39.7 (C5000), 42.3 (C30), 66.7
(C10), 115.3 (C6), 118.8 (C4), 119.7 (C400), 119.8 (C700), 120.4 (C300),
124.2 (C900), 126.4 (C3), 129.5 (C1), 131.1 (C800), 131.4 (C200),
135.2 (C2), 155.6, 157.0 (C600), 158.7 (C6000), 158.9 (C40), 159.1,
169.0, 174.4; HRMS (ESI+) [M+H]+ calcd for C29H37N8O4 561.2938
found 561.2941.
5.33. 2-(8-Aminooctanamido)-5-(3-aminopropoxy)-N-(4-(4-
(benzyloxy)phenoxy)phenyl)benzamide (12f)
The diamine (12f) was prepared from the diazide (11f) accord-
ing to the method used to prepare 12a. The product (12f) (49 mg,
97%) was isolated as a colourless oil. dH (400 MHz, methanol-d4)
1.28–1.38 (m, 6H, H4000, H5000 and H6000), 1.45 (m, 2H, H7000), 1.65
(m, 2H, H3000), 1.96 (m, 2H, H20), 2.34 (t, J = 7.4, 2H, H2000), 2.63 (t,
J = 7.3, 2H, H8000), 2.86 (t, J = 7.0, 2H, H30), 4.11 (t, J = 6.6, 2H, H10),
5.05 (s, 2H, H10000), 6.87–7.00 (m, 6H, H400, H700 and H800), 7.08
(dd, J = 2.7 and 8.9, 1H, H4), 7.27 (m, 1H, H6), 7.30 (d, J = 7.2, 1H,
H50000), 7.34–7.44 (m, 4H, H30000 and H40000), 7.59 (d, J = 9.0, 2H,
H300), 7.90 (d, J = 8.9, 1H, H3); dC (100 MHz, methanol-d4) 25.4,
26.4, 28.8, 29.0, 31.8, 31.9, 36.8, 38.2, 41.0, 66.2, 70.1, 114.2,
115.8, 116.1, 117.6, 119.8, 121.6, 122.5, 124.7, 127.2, 127.5,
128.1, 129.8, 133.1, 137.3, 148.9, 150.7, 155.2, 155.6, 167.4,
173.2; m/z (ESI+, 50 eV): m/z (%): 625 (65) [M+H]+.
5.37. 2-(5-Guanidinohexanamido)-5-(3-guanidinopropoxy)-N-
(4-phenoxyphenyl)benzamide dihydrochloride (2b)
The diguanidinium compound (2b) was prepared from the
diamine (12b) according to the method used to prepare 2a. The
product (2b) (30 mg, 69%) was isolated as colourless crystals. Mp
106–111 °C; mmax (ATR)/cmꢂ1 3316b (@N–H), 3241b and 3159b
(NH), 2950s and 2863s (CH), 1632s (CO), 1562w (C@N), 1504s
and 1486s and 1471s (Ar); dH (400 MHz, methanol-d4) 1.43 (m,
2H, H4000), 1.59 (m, 2H, H5000), 1.61 (m, 2H, H3000), 2.10 (m, 2H,
H20), 2.41 (t, J = 7.3, 2H, H2000), 3.14 (t, J = 7.1, 2H, H6000), 3.45 (t,
J = 6.7, 2H, H30), 4.18 (t, J = 5.8, 2H, H10), 6.99–7.03 (m, 4H, H400
and H700), 7.13 (m, 1H, H900), 7.15 (dd, J = 2.8 and 9.0, 1H, H4),
7.35–7.39 (m, 3H, H6 and H800), 7.69 (d, J = 8.9, 2H, H300), 7.89 (d,
J = 9.0, 1H, H3); dC (100 MHz, methanol-d4) 26.3, 27.2, 29.5, 29.7,
37.9, 39.6, 42.3, 66.7, 115.3, 118.8, 119.6, 120.2, 124.1, 124.4,
126.3, 129.3, 130.9, 131.3, 135.1, 155.5, 156.9, 158.6, 158.8,
158.9, 168.9, 174.4; HRMS (ESI+) [M+2H]2+ calcd for C30H39N8O4
288.1586 found 288.1577.
5.34. 2-(8-Aminooctanamido)-5-(3-aminopropoxy)-N-(4-(4-
fluorophenoxy)phenyl)benzamide (12g)
The diamine (12g) was prepared from the diazide (11g) accord-
ing to the method used to prepare 12a, using a 4 h reaction time.
The product (12g) (46 mg, 87%) was isolated as a white powder.
Mp 84 °C (decomposed); mmax (ATR)/cmꢂ1 3282b (NH), 2952s and
2864w (CH), 1730s and 1629s (CO), 1503w and 1459s (Ar); dH
(400 MHz, methanol-d4) 1.25–1.37 (m, 6H, H4000, H5000 and H6000),
1.41 (m, 2H, H3000), 1.65 (m, 2H, H7000), 1.94 (m, 2H, H20), 2.35 (t,
J = 7.4, 2H, H2000), 2.58 (t, J = 7.0, 2H, H8000), 2.89 (t, J = 7.0, 2H,
H30), 4.11 (t, J = 6.6, 2H, H10), 6.96–7.02 (m, 5H, H4, H400 and
H700), 7.09 (J = 8.4, 2H, H800), 7.29 (d, J = 2.3, 1H, H6), 7.63 (d,
J = 8.8, 2H, H300), 7.84 (d, J = 8.9, 1H, H3); dC (100 MHz, methanol-
d4) 26.8, 27.8, 30.2, 30.3, 33.4, 33.8, 38.4, 39.7, 42.6, 67.6, 115.1,
117.3 (J = 8.3, C800), 118.7, 119.8, 121.2 (J = 8.3, C700), 124.0, 126.0,
128.7, 131.4, 135.2, 151.0, 154.9, 160.1 (J = 240.0, C900), 162.6,
166.9, 174.6; HRMS (ESI+) [M+H]+ calcd for C30H38FN4O4
537.2877 found 537.2876.
5.38. 2-(5-Guanidino-octanamido)-5-(3-guanidinopropoxy)-N-
(4-phenoxyphenyl)benzamide dihydrochloride (2c)
The diguanidinium compound (2c) was prepared from the dia-
mine (12c) according to the method used to prepare 2a. The product
(2c) (26 mg, 46%) was isolated as colourless crystals. Mp 117–
119 °C; mmax (ATR)/cmꢂ1 3316b (@N–H), 3241b and 3159b (NH),
2950s and 2863s (CH), 1632s (CO), 1562w (C@N), 1504s and 1486s
and 1471s (Ar); dH (400 MHz, methanol-d4) 1.43 (m, 2H, H4000),
1.59 (m, 2H, H5000), 1.61 (m, 2H, H3000), 2.10 (m, 2H, H20), 2.41 (t,
J = 7.3, 2H, H2000), 3.14 (t, J = 7.1, 2H, H6000), 3.45 (t, J = 6.7, 2H, H30),
4.18 (t, J = 5.8, 2H, H10), 6.99–7.03 (m, 4H, H400 and H700), 7.13 (m,
1H, H900), 7.15 (dd, J = 2.8 and 9.0, 1H, H4), 7.35–7.39 (m, 3H, H6
and H800), 7.69 (d, J = 8.9, 2H, H300), 7.89 (d, J = 9.0, 1H, H3); dC
(100 MHz, methanol-d4) 26.3, 27.2, 29.5, 29.7, 37.9, 39.6, 42.3,
66.7, 115.3, 118.8, 119.6, 120.2, 124.1, 124.4, 126.3, 129.3, 130.9,
131.3, 135.1, 155.5, 156.9, 158.6, 158.8, 158.9, 168.9, 174.4; HRMS
(ESI+) [M+2H]2+ calcd for C30H39N8O4 288.1586 found 288.1577.
5.35. 2-(8-Aminooctanamido)-5-(3-aminopropoxy)-N-(4-(4-
hydroxyphenoxy)phenyl)benzamide (12h)
The phenol (12h) was prepared from the benzyl ether (12f)
according to the method used to prepare 12a, using a 36 h reaction
time. The product (12h) (17 mg, 74%) was isolated as a white pow-
der. Mp 100 °C (decomposed); m
max (ATR)/cmꢂ1 3310b (OH), 3278b
(NH), 2929s and 2855s (CH), 1654s (CO), 1598s and 1503w and
1498vs (Ar); dH (400 MHz, methanol-d4) 1.09–1.19 (m, 10H, H3000,
H4000, H5000, H6000 and H7000), 1.48 (m, 2H, H20), 1.78 (m, 2H, H2000),
2.16–2.25 (m, 2H, H30 and H8000), 4.00 (m, 2H, H10), 6.71–6.75 (m,
2H, H400), 6.79–6.87 (m, 4H, H700 and H800), 7.01 (m, 1H, H4), 7.24
(m, 1H, H6), 7.61 (m, 2H, H300), 7.93 (m, 1H, H3); dC (100 MHz,
methanol-d4) 26.7, 28.5, 28.8, 29.0, 32.6, 33.3, 36.9, 38.5, 41.6,

A. Andersson et al. / Bioorg. Med. Chem. 17 (2009) 6659–6670
6669
5.39. 2-(5-Guanidinohexanamido)-5-(3-guanidinobutoxy)-N-
5.42. N-(4-(4-(Benzyloxy)phenoxy)phenyl)-2-(8-guanidinooctana
(4-phenoxyphenyl)benzamide dihydrochloride (2d)
mido)-5-(3-guanidinopropoxy)benzamide dihydrochloride (2h)
The diguanidinium compound (2d) was prepared from the
diamine (12d) according to the method used to prepare 2a. The
product (2d) (81 mg, 96%) was isolated as colourless crystals. Mp
106–113 °C; mmax (ATR)/cmꢂ1 3320b (@N–H), 3247b and 3150b
and 3065b (NH), 2938s and 2869s (CH), 1634s (CO), 1543s
(C@N), 1597vs, 1502vs and 1489vs (Ar); dH (400 MHz, methanol-
d4) 1.39 (m, 2H, H4000), 1.56 (m, 2H, H5000), 1.67 (m, 2H, H3000), 1.78
(m, 2H, H30), 1.86 (m, 2H, H20), 2.37 (t, J = 7.3, 2H, H2000), 3.11 (t,
J = 7.1, 2H, H6000), 3.27 (t, J = 7.0, 2H, H40), 4.05 (t, J = 6.1, 2H, H10),
6.96–7.00 (m, 4H, H400 and H700), 7.07–7.11 (m, 2H, H4 and H900),
7.26 (d, J = 2.7, 1H, H6), 7.34 (m, 2H, H800), 7.64 (d, J = 8.9, 2H,
H300), 7.83 (d, J = 8.9, 1H, H3); dC (100 MHz, methanol-d4) 26.3
(C3000), 26.7 (C4000), 27.2 (C30), 27.5 (C20), 29.5 (C5000), 37.9 (C2000),
42.30 (C6000), 42.38 (C40), 69.0 (C10), 115.2 (C6), 118.8 (C4), 119.6
(C400), 120.2 (C700), 124.2 (C900), 124.4 (C300), 126.3 (C3), 129.3
(C1), 130.9 (C800), 131.0 (C200), 135.1 (C2), 155.4 (C500), 157.1
(C600), 158.6 (C7000), 158.7 (C50), 158.9 (C5), 168.9 (C1000), 174.7
(C100); HRMS (ESI+) [M+H]+ calcd for C31H41N8O4 589.3251 found
589.3247.
The diguanidinium compound (2h) was prepared from the
diamine (12f) according to the method used to prepare 2a. The
product (2h) (25 mg, 47%) was isolated as colourless crystals. Mp
75–81 °C; mmax (ATR)/cmꢂ1 3363b (@N–H), 3248b and 3150b
(NH), 2931s and 2858s (CH), 1643s (CO), 1603 (C@N), 1493vs
(Ar); dH (400 MHz, methanol-d4) 1.19–1.26 (m, 6H, H4000, H5000
and H6000), 1.40 (m, 2H, H3000), 1.52 (m, 2H, H7000), 1.92 (m, 2H,
H20), 2.25 (t, J = 6.5, 2H, H2000), 3.04 (m, 2H, H8000), 3.29 (m, 2H,
H30), 4.11 (t, J = 4.9, 2H, H10), 5.06 (s, 2H, H10000), 6.80–7.03 (m,
8H, H400, H700 and H800), 7.10 (d, J = 8.9 1H, H4), 7.29–7.33 (m, 1H,
H50000), 7.33–7.45 (m, 5H, H6, H30000 and H40000), 7.90 (br s, 1H, NH),
7.94 (d, J = 8.8, 1H, H3), 8.10 (br s, 1H, NH); dC (100 MHz, DMSO-
d6) 24.9 (C5000), 25.8 (C4000), 28.1 (C6000), 28.2 (C3000), 28.3 (C7000),
36.6 (C20), 37.6 (C2000), 38.8 (C30), 40.5 (C8000), 65.2 (C10), 69.6 (C10000),
114.1 (C6), 115.9 (C400), 116.1 (C4), 117.8 (C700), 119.8 (C800), 122.4
(C300), 126.2 (C3), 127.5 (C30000), 127.7 (C50000), 128.3 (C40000), 130.7
(C1), 133.4 (C20000), 133.8 (C200), 137.0 (C2), 148.3 (C900), 150.2
(C500), 153.6 (C600), 154.3 (C5), 157.1 (C9000), 157.2 (C40), 166.1
(C1000), 170.8 (C100); HRMS (ESI+) [M+H]+ calcd for C33H45N8O4
709.3826 found 709.3805.
5.40. 2-(5-Guanidino-octanamido)-5-(3-guanidinobutoxy)-N-
(4-phenoxyphenyl)benzamide dihydrochloride (2e)
5.43. 2-(8-Guanidinooctanamido)-5-(3-guanidinopropoxy)-N-
(4-(4-hydroxyphenoxy)phenyl)benzamide dihydrochloride (2f)
The diguanidinium compound (2e) was prepared from the
diamine (12e) according to the method used to prepare 2a.
The product (2e) (41 mg, 52%) was isolated as colourless crys-
tals. Mp 61–65 °C; mmax (ATR)/cmꢂ1 3320b (@N–H), 3251b and
3150b and 3065b (NH), 2934s and 2899s (CH), 1645s (CO),
1605 (C@N), 1597vs, 1504vs and 1487vs (Ar); dH (400 MHz,
methanol-d4) 1.31–1.40 (m, 6H, H4000, H5000, H6000 and H7000),
1.55 (m, 2H, H3000), 1.67 (m, 2H, H7000), 1.80 (m, 2H, H30), 1.88
(m, 2H, H20), 2.36 (t, J = 7.4, 2H, H2000), 3.12 (t, J = 7.1, 2H,
H8000), 3.30 (t, J = 6.9, 2H, H40), 4.10 (t, J = 6.0, 2H, H10), 6.96–
7.00 (m, 4H, H400 and H700), 7.11 (m, 2H, H900), 7.09 (dd,
J = 2.6 and 9.0, 1H, H4), 7.28 (d, J = 2.6, 1H, H6), 7.35 (m, 2H,
H800), 7.64 (d, J = 8.9, 2H, H300), 7.85 (d, J = 9.0, 1H, H3); dC
(100 MHz, methanol-d4) 25.2, 25.3, 25.99, 26.02, 28.3, 28.5,
28.6, 36.7, 40.8, 41.0, 67.6, 115.2, 118.7, 119.5, 120.3, 124.0,
124.4, 126.3, 129.4, 130.9, 131.1, 135.2, 155.4, 157.2, 158.6,
158.7, 159.0, 168.8, 174.7; HRMS (ESI+) [M+H]+ calcd for
C33H45N8O4 617.3564 found 617.3559.
The phenol (2f) was prepared from the benzyl ether (2h)
according to the method used to prepare 12a, using DMF/methanol
(1:2) (1.5 mL) as the reaction solvent and a 48 h reaction time. The
product (2f) (20 mg, 84%) was isolated as a white powder. Mp
169 °C (decomposed); mmax (ATR)/cmꢂ1 3351b (@N–H), 3260b
and 3169b and 3065b (NH), 2933s and 2859s (CH), 1650s (CO),
1603 (C@N), 1496vs and (Ar). dH (400 MHz, DMSO-d6) 1.19–1.28
(m, 6H, H4000, H5000 and H6000), 1.37 (m, 2H, H3000), 1.56 (m, 2H,
H7000), 1.90 (m, 2H, H20), 2.26 (t, J = 7.1, 2H, H2000), 3.00 (t, J = 6.8,
2H, H8000), 3.25 (t, J = 6.3, 2H, H30), 4.03 (t, J = 5.9, 2H, H10), 6.75
(d, J = 8.8, 2H, H800), 6.83–6.86 (m, 4H, H400 and H700), 7.02 (dd,
J = 2.8 and 8.7, 1H, H4), 7.48 (d, J = 2.4, 1H, H6), 7.61 (d, J = 8.8,
2H, H300), 8.01 (d, J = 8.8, 1H, H3). dC (100 MHz, DMSO-d6) 26.6,
28.3, 28.5, 28.5, 28.6, 29.7, 32.0, 40.5, 45.0, 69.5, 114.5, 115.8,
116.3, 117.2, 118.8, 122.8, 123.7, 127.7, 128.3, 134.3, 137.0,
154.0, 157.1, 157.8, 158.0, 160.7, 161.4, 170.6; HRMS (ESI+)
[M+H]+ calcd for C32H43N8O5 619.3356 found 619.3332.
5.44. Cav2.2 binding assay
5.41. N-(4-(4-Fluorophenoxy)phenyl)-2-(8-guanidinooctanamido)-
5-(3-guanidinopropoxy)benzamide dihydrochloride (2g)
Radioligand binding assays were run in triplicate in 96-well
plates at room temperature as previously described.28 Each assay
contained the test compound, radiolabelled peptide (125I-GVIA)
The diguanidinium compound (2g) was prepared from the
diamine (12g) according to the method used to prepare 2a.
The product (2g) (33 mg, 63%) was isolated as colourless crystals.
Mp 73–79 °C; mmax (ATR)/cmꢂ1 3320b (@N–H), 3253b and 3150b
(NH), 2931s and 2857s (CH), 1648s (CO), 1604s (C@N), 1508s
and 1492vs (Ar); dH (400 MHz, methanol-d4) 1.32–1.38 (m, 6H,
H4000, H5000 and H6000), 1.55 (m, 2H, H3000), 1.66 (m, 2H, H7000),
2.09 (m, 2H, H20), 2.36 (t, J = 7.4, 2H, H2000), 3.13 (t, J = 7.0, 2H,
H8000), 3.42 (t, J = 6.8, 2H, H30), 4.15 (t, J = 5.8, 2H, H10), 6.97–
7.02 (m, 4H, H400 and H800), 7.08–7.14 (m, 3H, H4 and H700),
7.32 (d, J = 2.3, 1H, H6), 7.64 (d, J = 8.6, 2H, H300), 7.87 (d,
J = 8.7, 1H, H3); dC (100 MHz, methanol-d4) 26.7, 27.5, 29.7,
29.8, 29.9, 30.0, 38.1, 39.6, 42.4, 66.7, 115.3 (d, J = 23.7, H800),
117.3, 118.6, 119.8 (d, J = 8.3, C700), 121.3, 124.1, 126.2, 129.2,
131.4, 135.1, 154.8, 156.3, 158.6, 158.8, 160.2 (d, J = 240.3,
C900), 161.4, 168.8, 174.7; HRMS (ESI+) [M+H]+ calcd for
C32H42N8O4 621.3313 found 621.3308.
and 8
were made in assay buffer (20 mM HEPES, 75 mM NaCl, 0.2 mM
EDTA, 0.2 mM EGTA, 2 M leupeptin, 2 L apoprotinin (to 30 mL
assay buffer) and 0.1% BSA, pH 7.4). The final volume in each well
was 150 L. After shaking for 1 h, the membrane was filtered
lg of crude rat brain membrane, added last. All dilutions
l
l
l
(Wallac, Finland glass fibre filters pre-soaked in 0.6% polyethylene-
imine) and washed with 20 mM HEPES, 125 mM NaCl, pH 7.4 on a
Tomtec harvester. After addition of scintillant, radioactivity bound
to the filter was counted using a 1450 MicroBeta (Wallac, Finland).
The data were analysed using GraphPad Prism 2.0 (GraphPad Soft-
ware, Inc, San Diego, USA).
Acknowledgements
The Monash-CSIRO Collaborative Research Support Scheme is
acknowledged for funding, as well as Monash University for

6670
A. Andersson et al. / Bioorg. Med. Chem. 17 (2009) 6659–6670
6. (a) Yamamoto, T.; Niwa, S.; Ohno, S.; Onishi, T.; Matsueda, H.; Koganei, H.;
Uneyama, H.; Fujita, S. I.; Takeda, T.; Kito, M.; Ono, Y.; Saitou, Y.; Takahara, A.;
Iwata, S.; Shoji, M. Bioorg. Med. Chem. Lett. 2006, 16, 798; (b) Yamamoto, T.;
Niwa, S.; Iwayama, S.; Koganei, H.; Fujita, S.; Takeda, T.; Kito, M.; Ono, Y.;
Saitou, Y.; Takahara, A.; Iwata, S.; Yamamoto, H.; Shoji, M. Bioorg. Med. Chem.
2006, 14, 5333; (c) Yamamoto, T.; Niwa, S.; Ohno, S.; Tokumasu, M.; Masuzawa,
Y.; Nakanishi, C.; Nakajo, A.; Onishi, T.; Koganei, H.; Fujita, S.; Takeda, T.; Kito,
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providing J.E.G. with a Monash Graduate Scholarship and CSIRO for
providing a scholarship top-up. Meagan Clark and Zelong Lim are
thanked for technical assistance provided during their CSIRO Vaca-
tion Scholarships.
Supplementary data
7. Knutsen, L. J. S.; Hobbs, C. J.; Earnshaw, C. G.; Fiumana, A.; Gilbert, J.; Mellor, S.
L.; Radford, F.; Smith, N. J.; Birch, P. J.; Burley, J. R.; Ward, S. D. C.; James, I. F.
Bioorg. Med. Chem. Lett. 2007, 17, 662.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2009.07.063.
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