Dicationic phenyl-2,2′-bichalcophenes and analogues as antiprotozoal agents

Article abstract of DOI:10.1016/j.bmc.2010.11.047

A series of phenyl-2,2'-bichalcophene diamidines 1a-h were synthesized from the corresponding dinitriles either via a direct reaction with LiN(TMS) ₂, followed by deprotection with ethanolic HCl or through the bis-O-acetoxyamidoxime followed by

Full text of DOI:10.1016/j.bmc.2010.11.047

Bioorganic & Medicinal Chemistry 19 (2011) 978–984
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dicationic phenyl-2,20-bichalcophenes and analogues as antiprotozoal agents
Mohamed A. Ismail a,e, Serry A. El Bialy b,e, Reto Brun c,d, Tanja Wenzler c,d, Rupesh Nanjunda e,
W. David Wilson e, David W. Boykin e,
⇑
a Department of Chemistry, Faculty of Science, Mansoura University, Mansoura 35516, Egypt
b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
c Swiss Tropical and Public Health Institute, Basel CH4002, Switzerland
d University of Basel, Basel CH4002, Switzerland
e Department of Chemistry and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303-3083, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of phenyl-2,2’-bichalcophene diamidines 1a–h were synthesized from the corresponding dinitr-
iles either via a direct reaction with LiN(TMS)2, followed by deprotection with ethanolic HCl or through
the bis-O-acetoxyamidoxime followed by hydrogenation in acetic acid and EtOH over Pd–C. These diami-
Received 9 October 2010
Revised 16 November 2010
Accepted 22 November 2010
Available online 5 December 2010
dines show a wide range of DNA affinities as judged from their
DTm values which are remarkably sensi-
tive to replacement of a furan unit with a thiophene one. These differences are explained in terms of the
effect of subtle changes in geometry of the diamidines on binding efficacy. Five of the eight compounds
were highly active (below 6 nM IC50) in vitro against Trypanosoma brucei rhodesiense (T. b. r.) and four
gave IC50values less than 7 nM against Plasmodium falciparum (P. f.). Only one of the compounds was
as effective as reference compounds in the T. b. r. mouse model for the acute phase of African
trypanosomiasis.
Keywords:
Diamidines
Bichalcophenes
Antiprotozoal agents
Stille coupling
Heck coupling
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
we have made compounds in which the phenyl group(s) of
furamidine have been replaced with pyridyl group(s) (IIb). Several
Aromatic diamidines exhibit broad-spectrum anti-microbial
activity including effectiveness against the protozoan diseases
caused by Trypanosoma sp. and Plasmodium sp.1 The broad activity
of the aromatic diamidines notwithstanding, pentamidine (I) is the
only compound of this class to be used extensively in the clinic
(Fig. 1).1 Furamidine (IIa), a diamidino diphenylfuran, has been
shown to be more potent and less toxic than pentamidine in mur-
ine models of trypanosomiasis.2 The oral prodrug of furamidine,
2,5-bis[4-(methoxyamidino)phenyl]furan (pafuramidine), showed
promising results in Phase I and II clinical trials against both human
African trypanosomiasis (HAT) and malaria.1 Unfortunately, in an
additional safety study of pafuramidine paralleling the Phase III tri-
als, liver and kidney toxicities in some volunteers were found and
the development of pafuramidine was terminated.2 Many of the ac-
tive aromatic diamidines for various structural classes have been
shown to bind to the minor groove of DNA at AT rich sites.3–9 It
has been assumed that the minor groove binding of these type of
compounds leads to inhibition of one or more DNA dependant
enzymes which gives rise to the anti-microbial effect.10–12 Recently,
of these aza-analogues show in vivo activity which is superior to
that of furamidine.13 More recently, one of these aza analogs was
found to be effective in a mouse model for second stage African
sleeping sickness.14 Furan units are often key structural elements
in the aromatic frame work for the more effective diamidines.15–17
The furan analogue III has been found to bind to DNA in a unique
stacked dimer array which has potential for development of new
gene regulation molecules.18–21 Compound III has shown some
activity in an immunosuppressed rat model for Pneumocystis carinii
pneumonia18 and in the STIB900 mouse model for acute stage Afri-
can trypanosomiasis.22 The bichalcophene diamidine IV from our
laboratory has been shown to recognize G-quadruplex DNA.23 More
recently, the diverse modes of the interaction of IV with multi-
stranded DNA structures has been reported.24
As part of a research program directed to drug discovery of
antiprotozoal agents and due to the unusual DNA binding proper-
ties of III and IV we decided to prepare additional analogues of
this series of bichalcophene diamidines in order to investigate
structure–activity relationships (SAR) and their DNA binding
profiles. We report here the synthesis of novel diamidino
phenyl-bifuran derivatives and structural isosteres and their
evaluation versus Trypanosoma brucei rhodesiense (T. b. r.) and
Plasmodium falciparum (P. f.).
⇑
Corresponding author. Tel.: +1 404 413 5498; fax: +1 404 413 5505.
E-mail address: dboykin@gsu.edu (D.W. Boykin).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.11.047

M. A. Ismail et al. / Bioorg. Med. Chem. 19 (2011) 978–984
979
O
O
NH
NH2
O
NH
HN
NH
NH2
X
NH2
NH2
IIa; X = CH
IIb; X = N
I
N
O
HN
H2N
HN
O
NH
O
NH
NH
NH2
IV
H2N
III
NH2
Figure 1.
2. Results and discussion
2.1. Chemistry
4a–d. Suzuki coupling of 4a–d with p-cyanophenylboronic acid
was accomplished in the presence of Pd (PPh3)4 as a catalyst,
Na2CO3 as base and toluene as solvent.
Compound 2h was obtained in three steps starting with a Stille
coupling reaction of the readily available 6-(5-bromofuran-2-
Our strategy for the synthesis of diamidines 1 is based upon
the conversion of dinitriles either by direct reaction using
lithium bis(trimethylsilyl)amide LiN(TMS)2 or through the bis-O-
acetoxyamidoxime followed by hydrogenation in acetic acid.
Retrosynthetic studies (Fig. 2) for this strategy suggested that the
triaryl structure 2 could be obtained by the application of palla-
dium catalyzed reactions which permit the formation of C–C bond
with full regioselectivity. The bichalcophenes 3 could be converted
into dinitriles 2 using the Heck coupling reaction. The compounds
4 could be converted into the dinitriles 2 using a Suzuki coupling
reaction. For SAR study, a two-carbon spacer could be inserted into
dinitriles 2 using Sonogashira coupling27 by the reaction of 4a with
trimethyl silyl acetylene derivative. Furthermore, Stille coupling is
another Pd catalyzed reaction which is an alternative pathway to
furnish certain analogues using compound 5.
We initiated our study by the synthesis of dinitrile 2a which
was obtained from 2,20-bifuran-5-carbonitrile (3a), prepared
according to our published procedure,25 via a Heck coupling reac-
tion with the commercially available 5-bromopyridine-2-carboni-
trile as shown in Scheme 1. The dinitrile 2b was prepared
employing similar procedures used for 2a using 2,20-bithiophene-
5-carbonitrile (3b). The synthesis of dinitriles 2c–f was accom-
plished in two steps. The regioselective bromination of 3a–d using
N-bromosuccinamide (NBS) in DMF furnished bromo derivatives
yl)nicotinonitrile
5 and 2-tributylstannylfuran to form the
corresponding 6-(2,20-bifuran-5-yl)-nicotinonitrile (6) (Scheme
2). Bromination of 6 with N-bromosuccinimide in DMF, furnished
bromo-bifuran derivative 7 in a 58% yield. A subsequent cyanation
reaction of compound 7 with Cu(I)CN in DMF gave the dinitrile 2h.
As outlined in Scheme 3, a series of diamidines1a–h was obtained
from the dinitriles 2a–h either by direct reaction using LiN(TMS)2 (cf.
1a–g) or through the bis-O-acetoxyamidoxime followed by hydroge-
nation in glacial acetic acid as in case of 1c, h. Thus, 50-(2-amidino-
pyridin-5-yl)-2,20-bifuran-5-carboxamidine (1a) was synthesized
fromthe corresponding dinitrile 2a by treatment with LiN(TMS)2 fol-
lowed by deprotection with ethanolic HCl(g). 6-(50-Amidino-2,20-
bifuran-5-yl)-nicotinamidine acetate salt (1h) was synthesized from
6-(50-cyano-2,20bifuran-5-yl)-nicotinonitrile (2h), through the bis-
O-acetoxyamidoxime followed by hydrogenation. The hydrochlo-
ride salts of the diamidines 1a–g were obtained by passing hydrogen
chloride gas into ethanolic solutions of their free bases.
2.2. Biology
Aromatic diamidines are well known cationic molecules that
have a strong and reversible interaction with DNA. Recent work
L
X
HN
Y
M
Z
NH
1
H2N
H2N
Bu3Sn
O
Br
NC
Z
L
X
X
Br
O
Heck
Y
Stille
Y
NC
NC
M
M
Reaction
Z
Coupling
CN
CN
2
3
5
OH
Suzuki
Coupling
Sonogashira
coupling
B
SiMe3
NC
OH
NC
Br
X
Y
O
O
Br
CN
CN
4
4a
Figure 2. Retrosynthetic study of diamidines.

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M. A. Ismail et al. / Bioorg. Med. Chem. 19 (2011) 978–984
(ii)
2
X
Y
Yield (%)
( 55 )
X
X
Y
Y
Br
O
O
CN
CN
Br
CN
O
O
a
3a-d
4a-d
4a
( 60 )
( 74 )
S
O
S
O
b
c
Br
B(OH)2
(iii)
(iv)
SiMe3
(i)
NC
( 76 )
( 70 )
O
S
S
S
d
e
NC
N
NC
( 75 )
( 25 )
S
O
O
f
O
X
X
O
g
Y
Y
O
NC
CN NC
CN NC
N
CN
2a,b
2g
2c-f
Scheme 1. Reagents and conditions: (i) DMF, KOAc, Pd(PPh3)4, 125–135 °C; (ii) NBS, DMF; (iii) toluene, Pd(PPh3)4, Na2CO3, 80 °C; (iv) Pd(OAc)2, NaOAc, Bu4NCl, DMF, 70 °C.
by 1c including a more curved structure, a shorter distance be-
tween the two amidine units and a smaller stacking surface pro-
vided by the three aryl rings. The first two differences seem
likely to be the more important contributors to the reduction in
Bu3Sn
O
Br
(ii)
(i)
O
O
O
O
N
N
N
NC
NC
NC
5
6
D
Tm value since the more curved structure would be expected to
prevent optimal fit in the groove by pushing the central portion
of the molecule away from the floor of the groove and the shorter
distance between the amidine units could lead to a disadvanta-
geous offset in H-bond indexing with the minor groove base pairs.
Replacement of one of the furan rings with a thiophene ring (1d or
(iii)
O
O
O
N
NC
Br
CN
7
2h
1f) results in an increase in the
DTm by approximately 5 °C. This re-
sult is consistent with the increased affinity of other thiophene
based diamidines in comparison to furan counterparts.28 There is
a small increase in bond angle for C–S–C in thiophene compared
to that of C–O–C in furan attributable to the differences in van
der Waals radii between S and O. This small angle difference when
amplified to the terminal amidine units leads to a significant differ-
ence in the positions of the amidines in the two different molecules
and likely leads to the increase in binding affinities for the thio-
phene analogues.28 The bithiophene analog 1e gives the highest
Scheme 2. Reagents and conditions: (i) Pd(PPh3)4, 1,4-dioxan 100–110 °C; (ii) NBS,
DMF; (iii) Cu(I)CN, DMF.
has shown that both curved and linear molecules exhibit excellent
biological activity against trypanosomes.22,26 The mechanism of
action for anti-trypanosomal activity has been suggested to involve
the inhibition of DNA dependent enzymes or direct inhibition of
transcription.1,9,10
D
Tm value (18.1 °C) for the bichalcophenes studied, consistent with
Table 1 contains the DNA binding affinities for the new bichal-
cophene diamidines as well as the in vitro activities for these com-
pounds against T. b. r. and P. f. For comparative purposes, similar
data for furamidine and pentamidine are included. The use of the
the previous observations. Introduction of nitrogen into the phenyl
ring in this system (1a, 1b, 1h) results in a 1.4–3.0 °C decrease in
D
Tm values compared to that of their carbocycle analogues. This
type decline has been noted previously for other diamidines and
has been attributed to differences in hydration of the phenyl and
pyridyl ring systems.13,29–31 The bifuran 1g in which a carbon–car-
bon triple bond has been inserted between the furan and the phe-
thermal melting increase
D
Tm (Tm of complex ꢀ Tm of free DNA)
is a rapid and reliable method for comparing binding affinities
for a large number of diverse classes of diamidines. The complexes
between poly (dA–dT) and the bichalcophene diamidines give an
nyl rings yields the bichalcophene compound with the lowest
DTm
unexpected wide range (from 4 to 18 °C) of
DTm values. The DTm
value of those investigated. A similar reduction in binding affinity
was noted in DAPI analogues in which a carbon–carbon triple bond
had been inserted.30 The origin of the decline seems likely to be
associated with poor stacking interactions of the triple bond and
possible changes in H-bond indexing as a result of further separa-
tion of the amidine groups.
value (8.5 °C) for the bifuran 1c is significantly lower than that of
both pentamidine (12.6 °C) and furamidine (25 °C). Due to struc-
tural similarities this analysis will focus on comparisons between
furamidine and the bichalcophene analogues. The large drop in
D
Tm value on replacing one of the furamidine phenyl groups with
a furan ring is probably due to a combination of factors presented
(i)
L
1
L
M
X
2
X
Y
Z
Yield (%)
Y
NC
M
Z
65
71
73
70
81
77
-
-
-
-
-
-
CH
CH
CH
CH
CH
CH
CN
a
b
c
d
e
O
S
O
O
S
S
O
S
O
S
S
O
N
L
X
HN
N
Y
(ii)
M
Z
NH
1
CH
CH
CH
CH
H2N
H2N
(iii)
HON
O
M
8a;
O
f
g
NOH
H2N
M=CH
b; M=N
78
59
C
C
CH
N
O
O
O
O
CH
CH
H2N
-
h
Scheme 3. Reagents and conditions: (i) (a) LiN(TMS)2, THF, rt, 12 h; (b) HCl(g), dry EtOH, rt, 12 h; (ii) NH2OHꢁHCl/KO-t-Bu, DMSO; (iii) (a) AcOH/Ac2O; (b) H2/Pd–C, AcOH.

M. A. Ismail et al. / Bioorg. Med. Chem. 19 (2011) 978–984
981
The IC50 values for the bichalcophene diamidines against T. b. r.
range from 2 to 97 nM. Five (1a, 1b, 1d, 1e, 1f) of the eight com-
pounds studied give IC50 values of 6 nM or less which are all in
the range of the values of pentamidine and furamidine. These com-
pounds were quite effective against P. f. with IC50 values ranging
from of 0.9 to 41 nM. Against this organism four compounds (1a,
1b, 1f, 1h) gave IC50 values less than 7 nM. On comparison of the
T. b. r. activity of non-pyridyl containing analogues (1c–g) the thio-
phene compounds (1b, 1e) are the most effective. A similar trend is
also seen for the pyridyl analogues (1a, 1b, 1h); however, the differ-
ence in IC50 values for 1a and 1b is within experimental error. There
is not an obvious SAR pattern for the P. f. activity. As we have noted
previously, there is no direct correlation between DNA binding
affinity and antiparasitic activity.1 While some DNA affinity seems
essential, transport of diamidines into the parasite plays and impor-
tant role.1 There does appear to be a rough correlation between
DNA affinity and cytotoxicity.31 Nevertheless, the selectivity of
these compounds for the parasitic organisms is generally quite high
as judged from their cytotoxicity to cultured L6 rat myoblast cells
shown in Table 1. The selectivity indices range from 277 to 38,500.
Given the in vitro selectivity and activity of the bichalcophenes
they were evaluated in the stringent T. b. r. mouse model for the
acute phase of African trypanosomiasis.14 The compounds were
evaluated by daily intraperitoneal dosage of 5 mg/kg (except for
1c which was tested in an earlier protocol at 20 mg/kg) for four
consecutive days. The results for these studies are included in
Table 1. At the low dosage of 5 mg/kg only 1f provided cures
(2/4) which represents activity comparable to that of the reference
compounds pentamidine and furamidine. Compound 1f merits
further evaluation in other models for HAT.
2.3. Conclusion
A new series of bichalcophene diamidines has been prepared in
good yields. These analogues show a wide range of DNA affinities as
judged from their
DTm values which are remarkably sensitive to
replacement of a furan unit with a thiophene one. These differences
are explained in terms of the effect of subtle changes in geometry of
the diamidines on binding efficacy. Five of the eight compounds
were highly active (below 6 nM IC50) in vitro against T. b. r. and four
gave IC50values less than 7 nM against P. f. Only one of the com-
pounds was as effective as reference compounds in the T. b. r. mouse
model for the acute phase of African trypanosomiasis.
3. Experimental section
3.1. Biology
3.1.1.
DTm measurements
Thermal melting experiments were conducted with a Cary 300
spectrophotometer. Cuvettes for the experiment are mounted in a
thermal block and the solution temperatures are monitored by a
thermistor in the reference cuvette. Temperatures were main-
tained under computer control and are increased at 0.5 °C/min.
The experiments were conducted in 1 cm path length quartz cuv-
ettes in CAC 10 buffer (cacodylic acid 10 mM, EDTA 1 mM, NaCl
100 mM with NaOH added to give pH 7.0). The concentrations of
DNA were determined by measuring the absorbance at 260 nm.
A ratio of 0.3 mol compound per mole of DNA was used for the
complex and DNA with no compound was used as a control.26,32
3.1.2. In vitro activity determination
In vitro assays with T. b. rhodesiense STIB 900 and P. falciparum
K1 strain and cell toxicity assays using cultured L6 rat myoblast
cells were carried out as previously described.33

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M. A. Ismail et al. / Bioorg. Med. Chem. 19 (2011) 978–984
3.1.3. STIB900 acute mouse model of trypanosomiasis
(300 mL) and aqueous solution containing 15 mL of concentrated
ammonia. The organic layer was dried (Na2SO4), and then concen-
trated to dryness under reduced pressure.
Experiments were performed as previously reported14 with
minor modifications. Briefly, female NMRI mice were infected
intraperitoneally (ip) with 2 ꢂ 104 STIB900 bloodstream forms.
Groups of four mice were treated ip with the tested dications on
4 consecutive days from day 3 through 6 post infection. A control
group was infected but was not treated. The tail blood of all mice
was checked for parasitemia until 60 days post infection. Surviving
and parasite free mice at day 60 were considered cured and then
euthanized. Death of animals (including the aparasitemic mice,
>60) was recorded to calculate the mean survival time in days.
3.2.2.1. 50-(4-Cyanophenyl)-2,20-bifuran-5-carbonitrile (2c).
Yellow solid, yield 74%, mp 194–195 °C (DMF).25 1H NMR
(DMSO-d6) d 7.17 (d, J = 3.6 Hz, 1H), 7.21 (d, J = 3.6 Hz, 1H), 7.43
(d, J = 3.6 Hz, 1H), 7.76 (d, J = 3.6 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H),
7.99 (d, J = 8.4 Hz, 2H).
3.2.2.2. 5-[5-(4-Cyanophenyl)-furan-2-yl]-thiophene-2-car-
bonitrile (2d). Yellowish green solid, yield 76%, mp 187–
188 °C.25 1H NMR (DMSO-d6); d 7.22 (d, J = 3.6 Hz, 1H), 7.35 (d,
J = 3.6 Hz, 1H), 7.62 (d, J = 3.9 Hz, 1H), 7.85–7.94 (m, 5H).
3.2. Chemistry
Melting points were recorded using a Thomas-Hoover (Uni-Melt)
capillary melting point apparatus and are uncorrected. TLC analysis
was carried out on Silica Gel 60 F254 precoated aluminum sheets and
detected under UV light. 1H and 13C NMR spectra were recorded
employing a Varian Unity Plus 300 spectrometer and a Bruker
Avance 400 MHz spectrometer, and chemical shifts (d) are in ppm
relative to TMS as internal standard. Mass spectra were recorded
on a VG analytical 70-SE spectrometer. Elemental analyses were ob-
tained from Atlantic Microlab Inc. (Norcross, GA) and are within 0.4
of the theoretical values. The compounds reported as salts
frequently analyzed correctly for fractional moles by water and/or
ethanol of solvation. In each case proton NMR showed the presence
of indicated solvent (s). All chemicals and solvents were purchased
from Aldrich Chemical Co., Fisher Scientific or Frontier.
3.2.2.3. 50-(4-Cyanophenyl)-2,20-bithiophene-5-carbonitrile
(2e). Yellow solid, yield 70%, mp 231–233 °C.25 1H NMR (DMSO-
d6) d 7.50 (d, J = 3.9 Hz, 1H), 7.59 (d, J = 3.9 Hz, 1H), 7.73 (d,
J = 3.9 Hz, 1H), 7.84–7.87 (m, 4H), 7.91 (d, J = 3.9 Hz, 1H).
3.2.2.4. 5-[5-(4-Cyanophenyl)-thiophen-2-yl]-furan-2-car-
bonitrile (2f). Yellow solid, yield 75%, mp 216.5–218 °C.25 1H
NMR (DMSO-d6) d 7.09 (d, J = 3.9 Hz, 1H), 7.64–7.66 (m, 2H), 7.75
(d, J = 3.9 Hz, 1H), 7.84–7.88 (m, 4H).
3.2.3. 50-[(4-Cyanophenyl)ethynyl]-2,20-bifuran-5-carboni-
trile (2g). A mixture of 4-[(trimethylsilyl)ethynyl]benzonitrile
(1.99 g, 10 mmol), 50-bromo-2,20-bifuran-5-carbonitrile (4a)
(1.19 g, 5 mmol), palladium acetate (56 mg, 0.25 mmol), tri(o-
tolyl)phosphine (152 mg, 0.5 mmol), Bu4NCl (1.39 g, 5 mmol),
and sodium acetate (1.64 g, 20 mmol) in DMF (20 mL) was heated
at 70 °C for 3 h. The reaction mixture was poured onto water and
extracted with ethyl acetate 200 mL (3ꢂ). The extract was evapo-
rated, and the residue was chromatographed on silica gel using
hexanes/EtOAc (95:5) as an eluent to furnish compound 2g as yel-
low solid in 25% yield, mp 172–173 °C. 1H NMR (DMSO-d6); d 7.16
(d, J = 3.6 Hz, 1H), 7.21 (d, J = 3.6 Hz, 1H), 7.23 (d, J = 3.6 Hz, 1H),
7.76–7.79 (m, 3H), 7.93 (d, J = 8.0 Hz, 2H). 13C NMR (DMSO-d6); d
148.7, 145.1, 136.1, 132.7, 131.9, 125.6, 125.5, 124.5, 119.8,
118.3, 111.7, 111.2, 108.6, 93.8, 82.5. MS (EI) m/e (rel. int.); 284
(M+, 10), 228 (35), 201 (40), 164 (60), 64 (100). Anal. Calcd for
3.2.1. General procedure of Heck reaction (2a, b)
A mixture of 2,20-bifuran-5-carbonitrile 3a or 2,20-bithiophene-
5-carbonitrile 3b (10 mmol), 5-bromopyridine-2-carbonitrile
(1.82 g, 10 mmol), Pd(PPh3)4(0) (400 mg), and KOAc (5 g, 50 mmol)
in dry DMF (10 mL) was heated under nitrogen at 130–135 °C for
overnight. The reaction mixture then poured onto cold-water.
The precipitate which formed was collected, recrystallized from
DMF.
3.2.1.1. 50-(2-Cyanopyridin-5-yl)-2,20-bifuran-5-carbonitrile
(2a). Yellow solid, yield 55%, mp 213–214 °C. 1H NMR (DMSO-d6);
d 7.25 (d, J = 3.6 Hz, 1H), 7.27 (d, J = 3.6 Hz, 1H), 7.59 (d, J = 3.6 Hz,
1H), 7.80 (d, J = 3.6 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.41 (dd, J = 8.0,
2.4 Hz, 1H), 9.22 (d, J = 2.4 Hz, 1H). 13C NMR (DMSO-d6); d 149.9,
149.1, 146.5, 145.1, 131.7, 130.7, 129.3, 128.3, 125.6, 124.4, 117.6,
113.1, 112.1, 111.8, 108.5. MS (EI) m/e (rel. int.); 262 (M++1, 100),
207 (18), 179 (24), 148 (80). Anal. Calcd for C15H7N3O2: C, 68.96;
H, 2.70. Found: C, 68.72; H, 2.79.
C18H8N2O2: C, 76.05; H, 2.83. Found: C, 75.80; H, 2.96.
3.2.4. 6-(2,20-Bifuran-5-yl)-nicotinonitrile (6). A mixture of
6-(5-bromofuran-2-yl)nicotinonitrile13 5 (2.48 g, 10 mmol), 2-n-
tributyltin furan (3.58 g, 10 mmol), and tetrakis(triphenylphos-
phine) palladium (300 mg) in dry dioxane (20 mL) was heated un-
der nitrogen at 100–110 °C for 24 h. The solvent was evaporated
under reduced pressure and the resulting residue was dissolved
in ethyl acetate. This solution was passed through celite to remove
Pd. The solution was evaporated, and the residue was chromato-
graphed on silica gel using hexanes/EtOAc (70:30) as an eluent to
furnish compound 6 as a golden yellow solid in 78% yield, mp
169–170 °C. 1H NMR (CDCl3); d 6.52 (dd, J = 3.6, 1.8 Hz, 1H),
6.72–6.76 (m, 2H), 7.31 (d, J = 3.6 Hz, 1H), 7.49 (d, J = 1.8 Hz, 1H),
7.79 (d, J = 8.4 Hz, 1H), 7.95 (dd, J = 8.4, 2.1 Hz, 1H), 8.80 (d,
J = 2.1 Hz, 1H). 13C NMR (CDCl3); d 152.5, 151.3, 151.0, 148.7,
145.5, 142.9, 139.6, 117.6, 117.0, 114.3, 111.7, 108.1, 107.1,
106.6. Anal. Calcd for C14H8N2O2: C, 71.18; H, 3.41; N, 11.85.
Found: C, 70.83; H, 3.61; N, 11.84.
3.2.1.2. 50-(2-Cyanopyridin-5-yl)-2,20-bithiophene-5-carbo-
nitrile (2b). Yellow solid, yield 60%, mp 257–258.5 °C. 1H NMR
(DMSO-d6); d 7.59 (d, J = 4.0 Hz, 1H), 7.72 (d, J = 4.0 Hz, 1H), 7.95
(d, J = 4.0 Hz, 1H), 8.00 (d, J = 4.0 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H),
8.34 (dd, J = 8.0, 2.4 Hz, 1H), 9.14 (d, J = 2.4 Hz, 1H). 13C NMR
(DMSO-d6); d 147.6, 143.0, 140.4, 138.9, 136.3, 133.6, 132.2,
131.0, 129.3, 129.0, 128.5, 125.4, 117.5, 114.1, 107.0. MS (EI) m/e
(rel. int.); 293 (M+, 10), 152 (12), 133 (15), 69 (100). Anal. Calcd
for C15H7N3S2: C, 61.41; H, 2.41. Found: C, 61.54; H, 2.47.
3.2.2. General procedure of Suzuki coupling (2c–f)
To a stirred solution of 50-bromo-2,20-bifuran-5-carbonitrile25
4a (4.74 g, 20 mmol), and Pd(PPh3)4(0) (600 mg) in toluene
(40 mL) under a nitrogen atmosphere was added 20 mL of a
1.5 M aqueous solution of Na2CO3 followed by 4-cyanophenylbo-
ronic acid (3.21 g, 22 mmol) in 5 mL of methanol. The vigorously
stirred mixture was warmed to 80 °C for 16 h. The solvent was
evaporated, the precipitate was partitioned between CH2Cl2
3.2.5. 6-(50-Bromo-2,20-bifuran-5-yl)-nicotinonitrile (7). To
a solution of 6 (1.41 g, 6 mmol) in DMF (20 mL) was added
portionwise N-bromosuccinimide (1.07 g, 6 mmol) with stirring.
The reaction mixture was stirred overnight, then poured onto
cold-water. The precipitate which formed was collected, washed

M. A. Ismail et al. / Bioorg. Med. Chem. 19 (2011) 978–984
983
with water and dried to give 7 as a golden yellow solid in 58%
(EtOH), mp 143–145 °C. 1H NMR (CDCl3); d 6.44 (d, J = 3.6 Hz,
1H), 6.70 (d, J = 3.6 Hz, 1H), 6.75 (d, J = 3.6 Hz, 1H), 7.30 (d,
J = 3.6 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.95 (dd, J = 8.4, 2.1 Hz,
1H), 8.80 (d, J = 2.1 Hz, 1H). 13C NMR (CDCl3); d 152.5, 151.2,
151.1, 147.5, 147.3, 139.7, 122.8, 117.7, 117.0, 114.3, 113.5,
109.3, 108.6, 106.9. MS (EI) m/e (rel. int.); 314 (M+, 60), 285 (10),
235 (20), 207 (100), 179 (10). HRMS calcd for C14H7BrN2O2:
313.9690. Observed 313.9661.
J = 3.6 Hz, 1H), 7.23 (d, J = 3.6 Hz, 1H), 7.36 (d, J = 3.6 Hz, 1H),
7.75 (d, J = 3.6 Hz, 1H), 7.85 (d, J = 7.8 Hz, 2H), 8.01 (d, J = 7.8 Hz,
2H). Anal. Calcd for C16H14N4O2ꢁ2.0HClꢁ1.0H2O: C, 49.88; H, 4.70;
N, 14.54. Found: C, 49.92; H, 4.50; N, 14.41.
3.2.7.4. 5-[5-(4-Amidinophenyl)-furan-2-yl]-thiophene-2-
carboxamidine hydrochloride (1d). The same procedure de-
scribed for preparation of 1a was used starting with the dinitrile
2d. Brown solid, yield 70%, mp 250–252 °C. 1H NMR (D2O/DMSO-
d6); d 7.14 (d, J = 3.6 Hz, 1H), 7.25 (d, J = 3.6 Hz, 1H), 7.58 (d,
J = 3.6 Hz, 1H), 7.79 (d, J = 7.8 Hz, 2H), 7.85–7.93 (m, 3H). 13C
NMR (D2O/DMSO-d6); d 165.9, 159.2, 153.2, 148.8, 140.2, 136.1,
134.8, 129.8, 127.6, 127.5, 125.5, 125.0, 112.9, 112.7. MS (ESI) m/
e (rel. int.); 311 (M+ +1, 92), 156 (100). HRMS calcd for C16H15N4OS:
311.0967. Observed: 311.0977. Anal. Calcd for C16H14N4OSꢁ2.0HClꢁ
1.5H2Oꢁ0.25EtOH: C, 46.97; H, 4.89; N, 13.28. Found: C, 46.82; H,
4.69; N, 13.13.
3.2.6. 6-(50-Cyano-2,20-bifuran-5-yl)-nicotinonitrile (2h).
A
mixture of 7 (942 mg, 3 mmol) and Cu(1)CN (540 mg, 6 mmol) in
dry DMF (25 mL) was refluxed for 48 h. The reaction mixture was
poured onto water/ammonia and extracted with methylene chlo-
ride. The extract was washed with water and brine, dried over
Na2SO4, then passed on silica gel to give compound 2h as yellow
solid in 27% yield, mp 209–210.5 °C. 1H NMR (DMSO-d6); d 7.23
(d, J = 3.6 Hz, 1H), 7.27 (d, J = 3.6 Hz, 1H), 7.52 (d, J = 3.6 Hz, 1H),
7.79 (d, J = 3.6 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 8.39 (dd, J = 8.4,
2.4 Hz, 1H), 9.03 (d, J = 2.4 Hz, 1H). Anal. Calcd for C15H7N3O2: C,
68.96; H, 2.70. Found: C, 68.81; H, 2.64.
3.2.7.5. 50-(4-Amidinophenyl)-2,20-bithiophene-5-carbox-
amidine hydrochloride (1e). The same procedure described
for preparation of 1a was used starting with the dinitrile 2e. Redish
brown solid, yield 81%, mp 271–273 °C. 1H NMR (D2O/DMSO-d6); d
7.49 (d, J = 3.6 Hz, 1H), 7.53 (d, J = 3.6 Hz, 1H), 7.67 (d, J = 3.6 Hz, 1H),
7.84 (s, 4H), 7.94 (d, J = 3.6 Hz, 1H). 13C NMR (D2O/DMSO-d6); d
165.5, 158.7, 144.1, 143.3, 138.4, 136.1, 136.0, 129.8, 129.0, 128.1,
127.5, 127.4, 126.4, 126.2. MS (ESI) m/e (rel. int.); 327 (M+ +1,
100), 208 (17), 164 (90). HRMS calcd. for C16H15N4S2: 327.0738. Ob-
served: 327.0739. Anal. Calcd for C16H14N4S2ꢁ2.0HClꢁ1.5H2O: C,
45.06; H, 4.49; N, 13.13. Found: C, 45.00; H, 4.38; N, 13.03.
3.2.7. General procedure of diamidines synthesis (Method A)
The dinitrile 2a (0.87 g, 3.34 mmol), suspended in freshly dis-
tilled THF (10 mL), was treated with LiN(TMS)2 (1 M solution in
THF, 8 mL, 8 mmol) and the reaction was allowed to stir overnight.
The reaction mixture was then cooled to 0 °C to which was added
HCl saturated ethanol (40 mL) whereupon a precipitate started
forming. The mixture was left to run overnight whereafter it was
diluted with ether and the resultant solid was collected by filtra-
tion. The diamidine was purified by neutralization with 1 N NaOH
followed by filtration of the resultant solid and washing with water
(3ꢂ 10 mL). Finally, the free base was stirred with ethanolic HCl
overnight, diluted with ether, and the solid formed was filtered
and dried to give the diamidine 1a hydrochloride salt.
3.2.7.6.
5-[5-(4-Amidinophenyl)-thiophen-2-yl]-furan-2-
carboxamidine hydrochloride (1f). The same procedure de-
scribed for preparation of 1a was used starting with the dinitrile
2f. Yellow solid, yield 77%, mp 248–249.5 °C. 1H NMR (D2O/
DMSO-d6); d 7.19 (d, J = 3.6 Hz, 1H), 7.78–7.82 (m, 3H), 7.91–7.96
(m, 4H). 13C NMR (D2O/DMSO-d6); d 165.5, 153.8, 153.4, 143.7,
140.2, 138.5, 131.9, 129.6, 128.9, 127.4, 126.4, 126.0, 123.8,
121.8. MS (ESI) m/e (rel. int.); 311 (M+ +1, 100), 156 (87). HRMS
calcd. for C16H15N4OS: 311.0967. Observed: 311.0966. Anal. Calcd
for C16H14N4OSꢁ2.0HClꢁ1.5H2O: C, 46.83; H, 4.67; N, 13.65. Found:
C, 46.90; H, 4.66; N, 13.30.
3.2.7.1. 50-(2-Amidinopyridin-5-yl)-2,20-bifuran-5-carbox-
amidine hydrochloride (1a). Yellow solid, yield 65%, mp
>320 °C. 1H NMR (DMSO-d6); d 7.30 (d, J = 2.4 Hz, 1H), 7.38 (d,
J = 2.4 Hz, 1H), 7.67 (d, J = 2.4 Hz, 1H), 8.05 (d, J = 2.4 Hz, 1H),
8.51–8.57 (m, 2H), 9.26 (s, 2H), 9.29 (s, 1H), 9.53 (s, 2H), 9.60 (s,
2H), 9.69 (s, 2H). 13C NMR (DMSO-d6); d 161.4, 153.3, 150.3,
148.8, 145.4, 145.0, 142.2, 140.5, 132.1, 129.1, 123.8, 120.9,
113.0, 112.4, 109.5. MS (ESI) m/e (rel. int.); 296 (M++1, 100), 208
(7), 148 (52). HRMS calcd for C15H14N5O2: 296.1147. Observed:
296.1144. Anal. Calcd for C15H13N5O2ꢁ2.0HClꢁ1.25H2O: C, 46.10;
H, 4.51; N, 17.92. Found: C, 46.01; H, 4.44; N, 17.63.
3.2.7.7. 50-[(4-Amidinophenyl)ethynyl]-2,20-bifuran-5-ami-
dine hydrochloride (1g). The same procedure described for
preparation of 1a was used starting with the dinitrile 2g. Yellow
solid, yield 78%, mp >320 °C. 1H NMR (DMSO-d6); d 7.19 (d,
J = 3.6 Hz, 1H), 7.25 (d, J = 3.6 Hz, 1H), 7.32 (d, J = 3.6 Hz, 1H),
7.83 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 3.6 Hz,
1H), 9.26 (s, 2H), 9.40 (s, 2H), 9.58, 9.61 (2s, 4H). 13C NMR
(DMSO-d6); d 164.9, 153.2, 148.3, 145.4, 140.5, 136.2, 131.4,
128.7, 128.4, 125.9, 120.9, 119.7, 111.5, 109.6, 94.1, 81.95. MS
(ESI) m/e (rel. int.); 319 (M++1, 100), 160 (75). HRMS calcd for
3.2.7.2. 50-(2-Amidinopyridin-5-yl)-2,20-bithiophene-5-car-
boxamidine hydrochloride (1b). The same procedure de-
scribed for preparation of 1a was used starting with the dinitrile
2b. Orange solid, yield 71%, mp 318–321 °C. 1H NMR (D2O/
DMSO-d6); d 7.55 (d, J = 4.0 Hz, 1H), 7.60 (d, J = 4.0 Hz, 1H), 7.82
(d, J = 4.0 Hz, 1H), 7.94 (d, J = 4.0 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H),
8.30 (dd, J = 8.4, 2.0 Hz, 1H), 9.06 (d, J = 2.0 Hz, 1H). 13C NMR
(D2O/DMSO-d6); d 161.5, 158.4, 146.6, 143.7, 142.4, 139.4, 137.0,
135.8, 134.3, 133.4, 129.2, 128.8, 127.2, 126.3, 123.9. MS (ESI) m/
e (rel. int.); 328 (M+ + 1, 75), 224 (10), 203 (7), 164 (100). HRMS
calcd. for C15H14N5S2: 328.0691. Observed: 328.0696. Anal. Calcd
for C15H13N5S2ꢁ2.0HClꢁ0.5H2Oꢁ0.25EtOH: C, 44.23; H, 4.19; N,
16.64. Found: C, 44.18; H, 4.13; N, 16.61.
C18H15N4O2: 319.1195. Observed 319.1206. Anal. Calcd for
C
18H14N4O2ꢁ2.0HClꢁ1.25H2O: C, 52.24; H, 4.51; N, 13.54. Found: C,
52.50; H, 4.54; N, 13.23.
3.2.8. General procedure of diamidines synthesis (Method B)
3.2.8.1. N-Hydroxy-50-[4-(N-hydroxyamidino)-phenyl]-2,20-
bifuran-5-carboxamidine (8a). A mixture of hydroxylamine
hydrochloride (695 mg, 10 mmol, 10 equiv) in anhydrous DMSO
(8 mL) was cooled to 5 °C under nitrogen and potassium t-butoxide
(1.12 g, 10 mmol, 10 equiv) was added in portions. The mixture
was stirred for 30 min. This mixture was added to the dinitrile
derivative 2c (260 mg, 1 mmol, 1 equiv). The reaction mixture
was stirred overnight at room temperature. The reaction mixture
was then poured slowly onto ice-water. The precipitate was
3.2.7.3.
50-(4-Amidinophenyl)-2,20-bifuran-5-carboxami-
dine hydrochloride (1c). The same procedure described for
preparation of 1a was used starting with the dinitrile 2c. Yellow
solid, yield 73%, mp >300 °C. 1H NMR (D2O/DMSO-d6); d 7.16 (d,

984
M. A. Ismail et al. / Bioorg. Med. Chem. 19 (2011) 978–984
filtered and washed with water and then ethanol to afford 8a (free
base) in 93%, mp 205–206 °C. 1H NMR (DMSO-d6); d 5.83 (br s, 4H),
6.86–6.89 (m, 3H), 7.14 (s, 1H), 7.75 (s, 4H), 9.70 (s, 1H), 9.75 (s,
1H). 13C NMR (DMSO-d6); d 152.3, 150.3, 146.7, 145.0, 144.9,
144.1, 132.3, 129.9, 125.8, 123.1, 109.7, 108.5, 107.2. MS (FAB)
m/e (rel. int.); 327 (M++1, 40), 307 (100), 299 (60), 273 (10),
220 (30). HRMS calcd for C16H15N4O4 ms 327.1093. Observed
327.1137.
by a Fulbright Fellowship awarded by the Binational Fulbright
Commission of Egypt.
References and notes
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Antimicrobial Agents In Small Molecule DNA and RNA Binders: From Synthesis
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Tanious, F. A.; Mathis, A.; Hall, J. E.; Stephens, C. E.; Boykin, D. W. Curr. Med.
Chem. Anti-Cancer Agents 2005, 5, 389; (c) Soeiro, M. N. C.; de Souza, E. M.;
Stephens, C. E.; Boykin, D. W. Expert Opin. Invest. Drugs 2005, 14, 957; (d)
Dardonville, C. Expert Opin Ther. Patents 2005, 15, 1241; (e) Werbovetz, K. A.
Curr. Opin. Invest. Drugs 2006, 7, 147; (f) Paine, M. F.; Wang, M. Z.; Generaux, C.
N.; Boykin, D. W.; Wilson, W. D.; De Koning, H. P.; Olson, C. A.; Pohling, G.;
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Opin. Invest. Drugs 2010, 11, 876.
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3.2.8.2. N-Hydroxy-6-[50-(N-hydroxyamidino)-2,20-bifuran-
5-yl]-nicotinamidine (8b). The same procedure described for
8a was used starting with 2h. Yield 89%, mp 248–250 °C. 1H
NMR (DMSO-d6); d 5.88 (s, 2H), 6.04 (s, 2H), 6.92–6.96 (m, 3H),
7.29 (d, J = 3.6 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 8.11 (dd, J = 8.4,
2.1 Hz, 1H), 8.88 (d, J = 2.1 Hz, 1H), 9.80 (s, 1H), 9.92 (s, 1H). 13C
NMR (DMSO-d6); d 152.2, 148.7, 147.8, 147.0, 146.6, 146.0, 144.6,
144.0, 133.6, 127.3, 117.7, 111.3, 109.7, 108.5, 107.9. MS (EI) m/e
(rel. int.); 327 (M+, 15), 311 (5), 295 (10), 278 (85), 261 (100).
HRMS calcd for C15H13N5O4: 327.0967. Observed 327.0974.
3.2.8.3.
dine acetate (1c). To
50-(4-Amidinophenyl)-2,20-bifuran-5-carboxami-
solution of the diamidoxime 8a
a
(326 mg, 1 mmol) in glacial acetic acid (10 mL) was slowly added
acetic anhydride (0.35 mL). After stirring for overnight TLC indi-
cated complete acylation of the starting material, ethanol (10 mL)
and 10% palladium on carbon (80 mg) were then added. The mix-
ture was placed on Parr hydrogenation apparatus at 50 psi for
4 h at room temperature. The mixture was filtered through hyflo
and the filter pad washed with water. The filtrate was evaporated
under reduced pressure and the precipitate was collected and
washed with ether to give 1c acetate salt in 67% yield, mp 240–
242 °C. 1H NMR (D2O/DMSO-d6); d 1.80 (s, 2 x CH3), 7.06 (d,
J = 3.6 Hz, 1H), 7.11 (d, J = 3.6 Hz, 1H), 7.36 (d, J = 3.6 Hz, 1H),
7.39 (d, J = 3.6 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.4 Hz,
2H). Anal. Calcd for C16H14N4O2ꢁ2.0AcOHꢁ2.4H2O: C, 52.48; H,
5.87; N, 12.23. Found: C, 52.28; H, 5.49; N, 11.81.
16. Del Poeta, M.; Schell, W. A.; Dykstra, C. C.; Jones, S. K.; Tidwell, R. R.; Czarny, A.;
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Wilson, W. D. Biochemistry 2001, 40, 2511.
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3.2.8.4.
50-(4-Amidinophenyl)-2,20-bifuran-5-carboxami-
dine (1c). It was prepared by dissolving the acetate salt 1c
(50 mg) in water (5 mL) and by neutralization with 1 N NaOH.
The precipitate was filtered, dried to afford free amidine of 1c.
Mp 202–203.5 °C. 1H NMR (D2O/DMSO-d6); d 6.93 (d, J = 3.6 Hz,
1H), 7.01 (d, J = 3.6 Hz, 1H), 7.11 (d, J = 3.6 Hz, 1H), 7.22 (d,
J = 3.6 Hz, 1H), 7.83 (s, 4H). 13C NMR (D2O/DMSO-d6); d 162.2,
153.9, 152.4, 147.4, 145.7, 145.0, 134.1, 131.1, 127.3, 123.1,
111.9, 109.4, 109.2, 107.7. MS (EI) m/e (rel. int.); 294 (M+, 50),
277 (100), 261 (25). HRMS calcd for C16H14N4O2: 294.1116. Ob-
served: 294.1101.
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27. Sørensen, U. S.; Pombo-Villar, E. Tetrahedron 2005, 61, 2697.
28. Mallena, S.; Lee, M. P. H.; Bailly, C.; Neidle, S.; Kumar, A.; Boykin, D. W.; Wilson,
W. D. J. Am. Chem. Soc. 2004, 126, 13659.
29. Wilson, W. D.; Tanious, F. A.; Mathis, A.; Tevis, D.; Hall, J. E.; Boykin, D. W.
Biochimie 2008, 90, 999.
30. Farahat, A. A.; Kumar, A.; Say, M.; Barghash, A. E. M.; Goda, F. E.; Eisa, H. M.;
Wenzler, T.; Brun, R.; Liu, Y.; Mickelson, L.; Wilson, W. D.; Boykin, D. W. Bioorg.
Med. Chem. 2010, 18, 557.
3.2.8.5.
6-(50-Amidino-2,20-bifuran-5-yl)-nicotinamidine
acetate (1h). The same procedure described for preparation of
1c was used starting with 8b. Yellow solid, yield 59%, mp 269–
271 °C. 1H NMR (D2O/DMSO-d6); d 7.15 (d, J = 3.6 Hz, 1H), 7.23
(d, J = 3.6 Hz, 1H), 7.47 (d, J = 3.6 Hz, 1H), 7.57 (d, J = 3.6 Hz, 1H),
8.04 (d, J = 8.4 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.99 (s, 1H). MS
(EI) m/e (rel. int.); 296 (M++1, 100), 273 (12), 239 (40). HRMS calcd
for C15H14N5O2: 296.1147. Observed 296.1189. Anal. Calcd for
C
15H13N5O2ꢁ2.0AcOHꢁ2.65H2Oꢁ0.5EtOH: C, 49.41; H, 6.07; N,
14.40. Found: C, 49.72; H, 5.96; N, 14.02.
31. We appreciate a reviewer making this observation.
32. Branowska, D.; Farahat, A. A.; Kumar, A.; Wenzler, T.; Brun, R.; Liu, Y.; Wilson,
W. D.; Boykin, D. W. Bioorg. Med. Chem. 2010, 18, 3551.
Acknowledgments
33. Bakunova, S. M.; Bakunov, S. A.; Patrick, D. A.; Suresh Kumar, E. V. K.; Ohemeng,
K. A.; Bridges, A. S.; Wenzler, T.; Barszcz, T.; Susan Jones, S. K.; Werbovetz, K. A.;
Brun, R.; Tidwell, R. R. J. Med. Chem. 2009, 52, 2016.
This work was supported by NIH Grant AI64200, and an award
from the Bill and Melinda Gates Foundation. S.A.E.B. was supported