Exploration of larger central ring linkers in furamidine analogues: Synthesis and evaluation of their DNA binding, antiparasitic and fluorescence properties

Article abstract of DOI:10.1016/j.bmc.2011.02.045

The effects of replacing the central furan ring of furamidine with indole and benzimidazole on their DNA binding affinity, antiparasitic activity and fluorescence are reported. The bis-cyanophenylindoles required to make the corresponding amidines were prepared by sequential Stille and/or Suzuki coupling reactions. The bis-cyanophenylbenzimidazoles were obtained by coupling 4-cyanobenzaldehydes with the appropriate cyano substituted phenylenediamine. The bis-nitriles were converted to the diamidines by reaction with LiN[Si(CH₃)₃]₂ or by Pinner methodology. Specifically, we have prepared new series of 2,6- and 2,5-diaryl indoles (6a,b, 12 and 17a-d) and the related benzimidazoles (24, 30 and 35). The new compounds bind in the DNA minor groove in DNA AT base pair sequences and eight of the ten new analogues exhibit ΔT_m values comparable to or higher than that of furamidine. Six of ten of the new compounds exhibit lower IC ₅₀ values against Trypanosoma brucei rhodesiense (T. b. r.) and eight of ten exhibit lower IC₅₀ values against Plasmodium falciparum (P. f.) than furamidine. Four of the ten show greater efficacy than furamidine in the rigorous T. b. r. STIB900 mouse model for African trypanosomiasis. Generally, the fluorescence properties of the new analogues are similar to that of DAPI.

Full text of DOI:10.1016/j.bmc.2011.02.045

Bioorganic & Medicinal Chemistry 19 (2011) 2156–2167
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploration of larger central ring linkers in furamidine analogues:
Synthesis and evaluation of their DNA binding, antiparasitic and fluorescence
properties
Abdelbasset A. Farahat a,b, Ekaterina Paliakov a, Arvind Kumar a, Alaa-Eldin M. Barghash b, Fatma E. Goda b,
Hassan M. Eisa b, Tanja Wenzler c,d, Reto Brun c,d, Yang Liu a, W. David Wilson a, David W. Boykin a,
⇑
a Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA
b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
c Swiss Tropical and Public Health Institute, Basel CH-4002, Switzerland
d University of Basel, Basel CH-4002, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The effects of replacing the central furan ring of furamidine with indole and benzimidazole on their DNA
binding affinity, antiparasitic activity and fluorescence are reported. The bis-cyanophenylindoles
required to make the corresponding amidines were prepared by sequential Stille and/or Suzuki coupling
reactions. The bis-cyanophenylbenzimidazoles were obtained by coupling 4-cyanobenzaldehydes with
the appropriate cyano substituted phenylenediamine. The bis-nitriles were converted to the diamidines
by reaction with LiN[Si(CH3)3]2 or by Pinner methodology. Specifically, we have prepared new series of
2,6- and 2,5-diaryl indoles (6a,b, 12 and 17a–d) and the related benzimidazoles (24, 30 and 35). The
new compounds bind in the DNA minor groove in DNA AT base pair sequences and eight of the ten
Received 28 December 2010
Revised 18 February 2011
Accepted 23 February 2011
Available online 1 March 2011
Keywords:
Furamidine
Indole diamidines
Benzimidazole diamidines
DNA binding
new analogues exhibit
DTm values comparable to or higher than that of furamidine. Six of ten of the
new compounds exhibit lower IC50 values against Trypanosoma brucei rhodesiense (T. b. r.) and eight of
ten exhibit lower IC50 values against Plasmodium falciparum (P. f.) than furamidine. Four of the ten show
greater efficacy than furamidine in the rigorous T. b. r. STIB900 mouse model for African trypanosomiasis.
Generally, the fluorescence properties of the new analogues are similar to that of DAPI.
Ó 2011 Elsevier Ltd. All rights reserved.
Fluorescence and antiprotozoal activity
1. Introduction
and thiazole.10 Only the triazole spacer was found to be more effec-
tive than the furan linker. DAPI (III, 40,6-diamidino-2-phenylindole,
The antimicrobial activity of aromatic diamidines was first re-
ported in the 1930s.1 Numerous dicationic systems have been sub-
sequently investigated, with the primary objective of discovering
useful therapeutic agents, however pentamidine (I, Fig. 1) is the
only compound of this class which has seen significant human
use.2 Furamidine (IIa, Fig. 1), was found to be more potent and less
toxic than pentamidine in murine models of Trypanosomiasis.3 An
orally effective prodrug of furamidine (IIb, pafuramidine, Fig. 1),
showed promising results in Phase II clinical trials against both
African sleeping sickness and malaria.2,3 Unfortunately, in an addi-
tional safety study of pafuramidine paralleling Phase III trials, liver
and kidney toxicities in some volunteers were found and the devel-
opment of pafuramidine was terminated.3 A number of spacers be-
tween the amidinophenyl moieties have been investigated in
search for effective compounds including; thiophene,4 pyrrole,5
N-methyl pyrrole,4 phenyl,6 selenophene,7 isoxazole8 triazole9
Fig. 1), was developed as an antitrypanosomal agent related to
diminazene and stilbamidine.10 DAPI showed a variety of biological
effects, including antifungal, antibacterial, antitrypanosomal and
antiviral activities.11,12 DAPI, an important tool in molecular biol-
ogy, is a fluorescent dye which exhibits several binding modes to
DNA13 and it has been frequently utilized as a DNA specific probe
for flow cytometry, chromosome staining, DNA visualization and
quantitation.14 The near linear molecules, DB921 (IV)15 and
DB1883 (V)16 (Fig. 1) are active antiparasitic agents, that have a
new sequence of aryl rings, a biphenyl at 2-position of benzimid-
azole or indole, respectively. Here we report the replacement of
the furan linker of IIa with the larger indole and benizimidazole
rings (new curved isomers of IV and V) in anticipation of finding
enhanced DNA binding and improved physiochemical properties.
The new compounds may be viewed as DAPI analogues with in-
creased length and the effects of these modifications on DNA bind-
ing and antiprotozoal activity are evaluated. Specifically, we have
prepared new series of 2,6 and 2,5-diaryl indoles (6a,b, 12 and
17a–d) and the related benzimidazoles (24, 30 and 35) all as
hydrochloride salts.
⇑
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 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.045

A. A. Farahat et al. / Bioorg. Med. Chem. 19 (2011) 2156–2167
2157
O
O
RN
NH
O
HN
HN
NR
NH2
H2N
NH2
NH2
R
H
I Pentamidine
IIa Furamidine
IIb Pafuramidine OMe
NH
NH
N
HN
N
H
N
H
NH2
NH2
NH2
NH2
III DAPI
IV
NH
HN
N
NH2
H
NH2
V
Figure 1.
Br
Br
Br
(a)
(b)
Sn
N
N
N
76%
93%
H
O
O
O
O
1
2
3
NC
Br
(c)
79%
(d)
81-85%
Ar CN
Ar CN
N
N
O
O
O
O
4a,b
5a,b
NH
HCl
H2N
NH
(e)
Ar
HCl
60-65%
N
NH2
H
6a,b
5,6
Ar =
a
b
S
substituted at 5 position
with cyano or amidine group
substituted at 4 position
with cyano or amidine group
Scheme 1. Reagents and conditions: (a) (BOC)2O, DMAP,CH2Cl2; (b) ClSn(CH3)3, LDA/THF; (c) Br-Ar-CN, Pd(PPh3)4, dioxane; (d) p-cyanophenylboronic acid, Pd(PPh3)4,
Na2CO3, toluene; (e) (i) LiN(TMS)2/THF; (ii) HCl gas/EtOH.
coupling reaction18 between the commercially available haloaryl
2. Results and discussion
nitriles and the indole stannane 3 using 5 mol % Pd(PPh3)4 and
1,4-dioxane as the solvent afforded 4a,b in good yield. The dinitr-
iles 5a,b were prepared in good yield by employing a Suzuki
coupling reaction19 between p-cyanophenylboronic acid and the
5-bromoindole derivatives 4a,b in the presence of 5 mol %
Pd(PPh3)4. The dinitriles 5a,b were allowed to react with lithium
bis(trimethylsilyl)amide in THF,20 followed by deprotection of
2.1. Chemistry
Scheme 1 outlines our approach to the synthesis of the hydro-
chloride salts of 2,5-diaryl indole diamidines 6a,b. The Boc-pro-
tected indole 2 and the indole stannane 3 were prepared in high
yield by employing a procedure we have used previously.17 A Stille

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A. A. Farahat et al. / Bioorg. Med. Chem. 19 (2011) 2156–2167
(a)
(b)
Sn
98%
N
N
H
N
Br
Br
Br
70%
O
O
O
O
7
9
8
(d)
(c)
I
CN
83%
N
79%
N
Br
O
O
O
NC
O
11
10
NH
(e)
HCl
N
H
NH2
46%
HN
HCl
NH2
12
Scheme 2. Reagents and conditions: (a) (Boc)2O, DMAP,CH2Cl2; (b) ClSn(CH3)3, LDA/THF; (c) I2/THF; (d) p-cyanophenylboronic acid, Pd(PPh3)4, Na2CO3, toluene; (e) (i)
LiN(TMS)2/THF; (ii) HCl gas/EtOH.
the silylated amidines with ethanolic HCl to furnish the hydrochlo-
ride salts of 6a,b.
derivative 19 in good yield.22 Compound 19 was protected by acet-
ylation after heating in acetic anhydride at 100 °C to yield 20.23
Compound 20 was allowed to react with p-cyanophenylboronic
acid according to a published procedure23 in the presence of DAP-
CY (diacetylpalladium (II)bis(dicyclohexylamine)) and potassium
phosphate to afford 21. The nitro compound 21 was hydrogenated
at 50 psi using 10% Pd/C to afford the diamine 22.24 A solution of
the diamine 22 in DMF and p-cyanobenzaldehyde in the presence
of a catalytic amount of nitrobenzene was heated at reflux to afford
the diphenyl benzimidazole 23.15 Stirring the dinitrile 23 in etha-
nolic HCl according to Pinner methodology25 afforded the corre-
sponding bis-imidate ester hydrochloride which was converted
to the hydrochloride salt of the diamidine 24 by stirring with eth-
anol saturated with ammonia gas.
Scheme 5 outlines the synthesis of the hydrochloride salt of
1-methyl-2.5-diamidinobenzimidazole 30 starting with the nitro
activated fluoro derivative 25 which on reaction with methylamine
in dichloromethane in the presence of potassium carbonate affor-
ded the N-methylaniline analogue 26.26 The bromo derivative 26
was allowed to react with p-cyanophenylboronic acid under Suzuki
coupling conditions in the presence of DAPCy and potassium phos-
phate to afford 27.27 The nitro compound 27 was hydrogenated un-
der 50 psi using 10% Pd/C to afford 28 in high yield (90%). Heating a
DMF solution of the diamine 28 with p-cyanobenzaldehyde in the
presence of sodium bisulphite afforded the diphenyl benzimid-
azole 29.15 The hydrochloride salt of the diamidine 30 was ob-
tained by the lithium bis(trimethylsilyl)amide method.20
Scheme 6 outlines the synthesis of the hydrochloride salt of
1-hydroxy-2,5-diamidinobenzimidazole 35 starting with the nitro
activated fluoro derivative 25 which on reaction with p-cyanoben-
zylamine 31 in dioxane in the presence of potassium carbonate
gave 32. The bromo derivative 32 was allowed to react with p-cya-
nophenylboronic acid under Suzuki coupling conditions in the
presence of DAPCy and potassium phosphate to give 33.27 The
dicyano derivative 33 was cyclized by heating in a sodium methox-
ide/methanol solution to afford the N-hydroxybenzimidazole 34.
The hydrochloride salt of the diamidine 35 was obtained by apply-
ing Pinner methodology as discussed above.25
Scheme 2 outlines the synthesis of the hydrochloride salt of the
diamidine 12. Compound 8 was prepared by the same approach as
the indole 2.17 As previously reported,16 we tried a number of
iodination methods to prepare the 2-iodoindole derivative 10,
however we were unable to obtain 10 in a reasonable yield until
we reacted the indole stannane 917 with molecular iodine at room
temperature to give the iodoindole 10 in good yield (83%). We note
that the introduction of the iodo group at 2-position of indole was
recently reported also by employing an indirect route starting with
the analogous boronic acid.21 The dinitrile 11 was prepared in good
yield by employing a Suzuki coupling reaction19 between two
equivalents of p-cyanophenylboronic acid and the dihaloindole
10 in the presence of 5 mol % Pd(PPh3)4. The diamidine 12, as a
hydrochloride salt, was obtained by the lithium bis(trimethyl-
silyl)amide method discussed before.20
After preparation of the symmetric 2,6-diphenylindole
diamidine 12, we decided to prepare dissymmetric analogues by
introduction of heterocyclic rings at position 2 of the indole. The
synthesis of the diamidines 17a–d was achieved as outlined in
Scheme 3. 6-Bromoindole
7 was allowed to react with
p-cyanophenylboronic acid under Suzuki coupling conditions19 in
the presence of 5 mol % Pd(PPh3)4, however this reaction gave 13
in low yield (<20%). Therefore, we decided to attempt the reaction
with the Boc-protected bromoindole 8, which was more soluble
than 7. This approach provided compound 14 in good yield
(87%), by using the standard Suzuki coupling reaction.19 As before,
the indole stannane 15 was obtained in a similar manner as 3. A
Stille coupling reaction18 between the commercially available
haloaryl nitriles and the indole stannane 15 using 5 mol %
Pd(PPh3)4 and 1,4-dioxane as the solvent afforded the dinitriles
16a–d in good yield. The hydrochloride salts of the diamidines
17a–d were obtained by the lithium bis(trimethylsilyl)amide
method.20
Scheme 4 outlines the synthesis of the hydrochloride salt of 2,5-
diamidinobenzimidazole 24. Bromination of the starting material
18 with hydrogen peroxide mediated HBr afforded the bromo

A. A. Farahat et al. / Bioorg. Med. Chem. 19 (2011) 2156–2167
2159
(a)
N
N
H
30%
Br
H
NC
13
7
87%
(b)
(b)
(a)
N
H
N
N
Br
Br
98%
78%
O
O
O
NC
O
7
8
14
(c)
74%
(d)
Ar CN
Sn
N
N
62-70%
O
O
O
NC
NC
O
16a-d
15
NH
(e)
33-47%
HCl
Ar
N
H
NH2
HN
HCl
NH2
17a-d
16,17
a
b
c
d
N
Ar =
N
S
O
substituted at 5 position
with cyano or amidine group
substituted at 4 position
with cyano or amidine group
Scheme 3. Reagents and conditions: (a) p-cyanophenylboronic acid, Pd(PPh3)4, Na2CO3, toluene; (b) (Boc)2O, DMAP, CH2Cl2; (c) ClSn(CH3)3, LDA/THF; (d) Br-Ar-CN, Pd(PPh3)4,
dioxane; (e) (i) LiN(TMS)2/THF; (ii) HCl gas/EtOH.
(a)
(b)
Br
Br
NHCOCH3
NO2
20
NH2
NO2
19
NH2
NO2
18
(c)
(d)
(e)
NC
NH2
NH2
NC
NH2
NO2
80%
60%
22
21
HCl NH
H2N
NC
N
NH
N
(f)
HCl
CN
N
H
N
H
NH2
68%
24
23
Scheme 4. Reagents and conditions: (a) 49% HBr, MeOH, 30% H2O2; (b) Ac2O, 100 °C; (c) p-cyanophenylboronic acid, DAPCy, K3PO4; (i) PrOH; (d) EtOAc, Pd/C, H2; (e) 4-
cyanobenzaldehyde, p-nitrobenzene, DMF, 145 °C; (f) (i) HCl gas/EtOH; (ii) NH3 gas/EtOH.

2160
A. A. Farahat et al. / Bioorg. Med. Chem. 19 (2011) 2156–2167
F
NHMe
(b)
(a)
NC
NHMe
NO2
60%
Br
NO2
Br
NO2
25
27
26
NC
N
N
(d)
73%
(c)
90%
NC
NHMe
CN
NH2
Me
28
29
NH
HCl
H2N
NH
N
N
(e)
HCl
89%
NH2
Me
30
Scheme 5. Reagents and conditions: (a) 40% MeNH2, K2CO3, CH2Cl2; (b) 4-cyanophenylboronic acid, DAPCy, K3PO4, EtOH; (c) EtOH, Pd/C, H2; (d) (i) 4-cyanobenzaldehyde,
NaHSO3, EtOH; (ii) DMF; (e) (i) LiN(TMS)2/THF; (ii) HCl gas/EtOH.
F
CN
(a)
(b)
+
H2N
Br
NH
NO2
CN
98%
Br
NO2
80%
32
31
25
N
N
CN
CN
(c)
NC
NH
NO2
OH
93%
NC
34
33
N
N
NH
(d)
HCl
78%
NH2
HN
OH
HCl
35
H2N
Scheme 6. Reagents and conditions: (a) K2CO3, dioxane; (b) p-cyanophenylboronic acid, DAPCy, K3PO4, EtOH; (c) (i) MeOH, NaOMe; (ii) HCl; (d) (i) HCl gas/EtOH; (ii) NH3 gas/
EtOH.
2.2. Biology
Table 1 contains the DNA binding affinities for the new diami-
dines with central ring indoles and benzimidazoles as well as the
in vitro activities for these compounds against T. b. r. and P. f. For
comparative purposes, similar data for DAPI, furamidine, pentam-
idine and the two near linear analogues IV and V are included.
not melt under the conditions of the DTm measurements, ranking
and analysis of structural effects must await detailed biophysical
studies performed under conditions for which affinities can be di-
rectly compared. Nevertheless, it is clear that, in general, replace-
ment of the central furan ring of furamidine with an indole or
benzimidazole ring (but not N-substituted benzimidazole 30 and
35) leads to compounds with quite high affinity for AT rich DNA
minor grooves.
We continue to use the thermal melting increase
DTm (Tm of com-
plex ꢀ Tm of free DNA) as a rapid and reliable method for ranking
Generally, heterocyclic diamidines have been found to bind in
the DNA minor groove in AT base pair sequences of DNA.28 Circular
dichroism (CD) spectroscopy has proven to be a valuable technique
for characterizing minor groove interactions. In order to determine
if these new analogues bind in the DNA minor groove, we observed
the CD spectroscopy of the complex formation. Figure 2 shows the
CD data for the interaction between DNA and 12 and 24 along with
that of DAPI as a reference compound. Minor groove binders yield a
binding affinities for a large number of diverse classes of aryldiami-
dines.16 The
DTm values for the complexes between poly(dA-dT)
and the linker modified bis-benzamidines are quite high ranging
from 23 to >27 °C for all the diamidines except for 30 and 35.
The compounds with high DNA affinity (6a, 6b, 12, 17a, 17b, 17c,
17d and 24) exhibit
DTm values comparable to or higher than that
of furamidine. Since the complexes of five of these compounds do

A. A. Farahat et al. / Bioorg. Med. Chem. 19 (2011) 2156–2167
2161
Table 1
activity of the six indoles compares favorably with that of the ref-
erence compounds. The indole analogues are somewhat less active
against P. f. compared to that against T. b. r., with only three com-
pounds (6a, 6b and 12) giving IC50 values ranging between 5 and
7 nM and the remaining four gave values between 9 and 18 nM.
None of the indole compounds achieve better antimalarial activity
than DAPI, IV and V, several show higher activity than furamidine
and all are superior to that of pentamidine. Generally, the indoles
show acceptable selectivity for both parasites, SI values range from
541 to 4950 against T. b. r. and from 198 to 1394 versus P. f. The
activity against T. b. r. for two of the three benzimidazole com-
pounds (24, 30; IC50 values 4 and 15 nM, respectively) was compa-
rable to that of the indoles. The same two compounds were
moderately more active against P. f. (IC50 values 3.7 and 1.3 nM,
respectively) than the indoles. The benzimidazoles generally show
greater selectivity than the indoles; SI values against T. b. r range
from 256 to 15,600 and against P. f. from 6880 to >130,000.
The in vitro activity and selectivity of these new diamidines
lead us to evaluate them in the rigorous T. b. r. STIB900 mouse
model for the acute phase of African trypanosomiasis and the re-
sults are presented in Table 2. The compounds were screened by
daily intraperitoneal dosage of 5 mg/kg for four consecutive days.
Four of the seven indole compounds (6a, 17b, 17c and 17d) gave
at least 2 of 4 cures. No cures were obtained with the three benz-
imidazoles (24, 30 and 35) which may reflect differences in PK
properties from that of the indoles. The in vivo results for four
compounds (6a, 17b, 17c and 17d) compare favorably with that
for pentamidine and is superior to that of furamidine in this mouse
model and merit further evaluation.
The widespread use of DAPI in molecular biology is largely due
to its fluorescence properties. During the study of these new indole
diamidines we observed that several of the compounds were fluo-
rescent in the visible region. Therefore, we decided to record the
fluorescence data for the new indole and benzimidazole com-
pounds and compare the results with that of DAPI. These data
are presented in Table 3. The absorption maximum for the new in-
doles ranges from 341 to 380 nm; that for DAPI is 343 nm. The
absorption maximum for the new benzimidazoles is a small range
of 280–286 nm. The emission maximum ranges from 508 to
579 nm for the indoles and from 468 to 503 nm for the benzimi-
dazoles; that for DAPI is 458 nm. A qualitative comparison of quan-
tum yield of emission for the indoles shows a 2.5–30-fold decline
from that of DAPI; whereas the benzimidazole 24 shows a modest
20% increase in emission intensity over that of DAPI. The other two
benzimidazoles showed a decline in emission intensity similar to
the decline noted for the indoles.
DNA binding and in vitro antiparasitic data for centrally linked indoles and
benzimidazoles
a
d
e
f
D
Tm (°C) Cytoxicityb T. b. rc SIT
P. f
SIP
(
lM)
(nM)
(nM)
DAPI (III)
Furamidine (IIa) 25
Pentamidine (I) 12.6
V
IV
6a
6b
12
17a
17b
17c
17d
24
>27
0.86
6.4
46
10.5
17
9.9
18
4.5
2.2
2
7.7
2
48
3
287
1422
20,909
5250
2207
4950
2333
1133
541
550
2320
650
15,600
>11,266 1.3
256 18
15.5 413
46.4 991
>27
>27
25.5
23.7
>27
24.9
>27
>27
>27
>27
11.2
12.2
3.3
0.5
7.1
5.7
5.2
3182
34,000
1394
1175
653
6.7
3
3.4
3
9.2
2.2
17
4
12.5 736
11.1 198
11.6
2.6
5
4
18
9.8
3.7
644
265
16,864
>130,000
6,888
62.4
>169
124
4
15
484
30
35
a
b
c
Increase in thermal melting of poly(dA-dT)n in °C.32
Cytotoxicity (IC50) was evaluated using cultured L6 rat myoblast cells.9
IC50 values obtained against the STIB900 strain of T. b. r. The IC50 values are the
mean of two independent assays. Individual values differed by less than 50% of the
mean value.9
d
Selectivity index for T. b. r. expressed as the ratio: IC50 (L6)/IC50 (T. b. r.).
IC50 values obtained against the K1 strain of P. f. The IC50 values are the mean of
e
two independent assays. Individual values differed by less than 50% of the mean
value.9
f
Selectivity index for P. f. expressed as the ratio: IC50 (L6)/IC50 (P. f.).
large positive induced CD signal on binding to AT sequences and
only exhibit small changes in the CD pattern of the DNA spectrum.
The induced CD spectrum of DAPI is shown as a reference in Figure
2a. The expected positive signal in the compound absorption re-
gion with relatively small changes in the DNA region are observed.
Figure 2 also shows the CD results for the interaction between DNA
and 12 and 24. In agreement with their strong binding to DNA,
both of these compounds exhibit large induced CD spectra in their
absorption regions that is approximately twice the DAPI value.
They are clearly binding in the DNA minor groove. Interestingly,
both of these compounds cause larger changes in the DNA CD spec-
tral region than DAPI. It seems likely that their shapes and H-bond
donor groups do not initially match the DNA groove shape and H-
bond acceptors as well as DAPI. In this case they induce comple-
mentary changes in DNA that yield a very favorable final complex
with high Tm values.
DAPI and a number of analogues have exhibited excellent
in vitro activity against T. b. r. and P. f.16 Six of the seven new indole
linked analogues (6a, 6b, 12, 17b, 17c and 17d) give IC50 values
against T. b. r. ranging between 2 and 5 nM. The remaining indole
17a give an IC50 value of 17 nM. The in vitro antitrypanosomal
DAPI on binding to DNA exhibits an approximately 20-fold
enhancement in its fluorescence, an attribute which significantly
Figure 2. The concentration of polydAꢁpolydT is 30
lM. The ratio of compound to DNA is shown in the inset.

2162
A. A. Farahat et al. / Bioorg. Med. Chem. 19 (2011) 2156–2167
Table 2
3. Conclusions
Antitrypanosomal evaluation of indole and benzimidazole analogues in the acute
STIB900 mouse modela
Replacement of the central furan ring of furamidine with indole
or benzimidazole (but not N-substituted benzimidazoles) yields
compounds that bind in the DNA minor groove with high affinity
and exhibit high in vitro antiparasitic activity. In general, the
DNA affinities and the in vitro antiparasitic activities are compara-
ble to or better than that of furamidine. The DNA affinities of the
indole analogues are also comparable to those of the structurally
related bis-benzamdinoimidazo[1,2-a]pyridines, however the
in vitro activity of the indoles is generally superior to that of the
imidazo[1,2-a]pyridines.30 In vivo efficacies of four indoles (6a,
17b, 17c and 17d) are comparable to that of pentamidine and
superior to that of furamidine. Generally, the fluorescence proper-
ties of the new analogues are similar to that of DAPI. The two com-
pounds 12 and 24 show emission enhancement on binding to DNA,
albeit 4-fold less than DAPI, however their longer emission wave-
length maybe beneficial in certain circumstances.
b
Compound
In vitro IC50 (nM)
Curesc
MRDd
DAPI (III)
Pentamidine(I)
Furamidine (IIa)
6a
6b
12
17a
17b
17c
17d
24
18
2.2
4.5
2
3
3
17
4
5
4
4
2/4
2/4
1/4
2/4
1/4
0/4
0/4
2/4
3/4
2/4
0/4
0/4
0/4
17
20
24
17
19.7
20
17.75
17
24
24
18
11
7.75
30
35
15
484
a
See Ref. 31 for details of STIB900 model; dosage at 5 mg/kg/day for four con-
secutive days by intraperitoneal administration.
b
IC50 values obtained against the STIB900 strain of T. b. r. The values are the
average of duplicate determinations.9
c
Number of mice that survive and are parasite free on day 60.
d
Mean relapse day of parasitemia.
4. Experimental
4.1. Biology
Table 3
Fluorescence data for new indole and benzimidazole diamidines
4.1.1. Efficacy studies
The in vitro assays9 with T. b. r. STIB 900 and P. f. K1 strain as
well as the efficacy studies in an acute mouse model for T. b. r. STIB
90031 were carried out, using the hydrochloride salts, as previously
reported.
a,b
a,b
Compound
kex (nm)
kem (nm)
Em intensityc
DAPI
6a
6b
343
368
341
358
370
380
369
368
280
286
284
458
519
517
508
508
532
515
579
496
503
468
821
177
42
12
32
17a
17b
17c
17d
24
146
358
55
4.1.2. Tm measurements
Thermal melting experiments were conducted with a Cary 300
spectrophotometer. Cuvettes for the experiment were mounted in
a thermal block and the solution temperatures monitored by a
thermistor in the reference cuvette. Temperatures were main-
tained under computer control and increased at 0.5 °C/min. The
experiments were conducted in 1 cm path length quartz curvettes
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 its absorbance at 260 nm. A ratio of 0.3
moles compound per mole of DNA was used for the complex and
27
986
106
51
30
35
a
b
c
Wavelengths (k) are indicated for excitation (ex) and emission (em).
Measurements were made with 0.001 M solutions in distilled water.
Emission intensities were measured at excitation slit width of 5 and emission
slit width of 20.
contributes to its utility for molecular biology studies.29 To further
access the possible utility of the new compounds we have exam-
ined the effect of DNA binding on the fluorescence of 12 and 24
as shown in Figure 2. While the emission intensity of 24 is approx-
imately 30 fold greater than that of 12, both show an approxi-
mately 5-fold enhancement on binding to DNA (Fig. 3). DAPI
shows a 4-fold greater enhancement of emission on binding to
DNA. Nevertheless, in special circumstances 12 or 24 may be of
utility since their emission maxima are approximately 50 nm
greater than that of DAPI.
DNA alone was used as a control.32
DTm values were determined
by the peak in first derivative curves (dA/dT).
4.1.3. Circular dichroism (CD)
CD spectra were collected employing a Jasco J-810 spectrometer
at different ratios of compound to DNA at 25 °C in MES 10 buffer. A
DNA solution in a 1-cm quartz cuvette was first scanned over the
desired wavelength range. DAPI, 12 and 24, at increasing ratios,
were titrated into the same cuvette and the complexes rescanned
under the same conditions.33
4.2. Chemistry
Fluorescence comparison
All commercial reagents were used without purification. Melt-
ing points were determined on a Mel-Temp 3.0 melting point
apparatus, and are uncorrected. TLC analysis was carried out on
Silica Gel 60 F254 precoated aluminum sheets using UV light for
detection. 1H and 13C NMR spectra were recorded on a Bruker
400 MHz spectrometer (except as noted) using the indicated
solvents. Mass spectra were obtained from the Georgia State
University Mass Spectrometry Laboratory, Atlanta, GA. Compounds
reported as salts often analyzed correctly for solvates of water and/
or other solvents, in each case the solvates were detected by 1H
NMR. Elemental analysis was performed by Atlantic Microlab
Inc., Norcross, GA.
24
12
Wavelength (nm)
Figure 3. The free compound concentrations were 0.5
l
M, polydAꢁpolydT was
titrated into the cell at 0.025 lM.

A. A. Farahat et al. / Bioorg. Med. Chem. 19 (2011) 2156–2167
2163
4.2.1. General procedure for 4a,b
111.4, 110.7, 84.7, 27.8; ESI-MS: m/z calcd for
C
27H21N3O2:
Tetrakistriphenylphosphine palladium (0.288 g, 0.25 mmol)
was added to a stirred mixture of the indole stannane 3 (2.29 g,
5 mmol) and the halo arylnitriles (5-bromothiophene-2-carboni-
trile and 4-bromobenzonitrile) (5 mmol) in deaireated dioxane
(20 mL) under a nitrogen atmosphere. The mixture was warmed
to 90–100 °C and stirred for 24 h. The solvent was evaporated un-
der reduced pressure. The resultant solid was partitioned between
ethyl acetate (200 mL) containing 5 mL of concentrated ammonia
to destroy the palladium complex, washed with water, passed
through celite, dried over sodium sulfate and evaporated. Purifica-
tion was by column chromatography on silica gel, using hexanes/
ethyl acetate (80:20, v/v) as the eluent.
419.47, found: 420.3 (M++1). Anal. Calcd for C27H21N3O2: C,
77.31; H, 5.05; N, 10.02. Found: C, 77.68; H, 5.22; N, 9.97.
4.2.3. General procedure for 6a,b
The dinitriles 5a,b (0.66 mmol), were suspended in freshly dis-
tilled tetrahydrofuran (5 mL), and treated with lithium bis(tri-
methylsilyl)amide 1 M solution in tetrahydrofuran (4 mL,
3.98 mmol), and stirred for three days at room temperature. The
reaction mixture was cooled to 0 °C to which was added HCl satu-
rated ethanol (2 mL). The mixture was stirred for two days, diluted
with ether and the resultant solid was collected by filtration. The
diamidine was purified by neutralization with 1 N sodium hydrox-
ide followed by filtration of the resultant solid, washed with water
and dried. Finally, the free base was stirred with ethanolic HCl for
one week to ensure that the Boc groups were completely removed,
diluted with ether, and the solid formed was filtered and dried to
give the diamidine salt.
4.2.1.1.
5-Bromo-1-(tert-butoxycarbonyl)-2-(5-cyanothien-2-
yl)-1H-indole (4a). White solid (1.56 g, 79%). Mp 152–153 °C;
1H NMR (CDCl3): d 8.08 (d, 1H, J = 8.2 Hz), 7.70 (s, 1H), 7.60 (d,
1H, J = 3.6 Hz), 7.47 (d, 1H, J = 8.2 Hz), 7.15 (d, 1H, J = 3.6 Hz),
6.71 (s, 1H), 1.49 (s, 9H); 13C NMR (CDCl3): d 149.2, 142.3, 136.9,
136.3, 130.9, 130, 128.6, 128.1, 123.5, 117.1, 116.7, 113.9, 112.7,
110, 85.1, 27.8. Anal. Calcd for C18H15BrN2O2S: C, 53.61; H, 3.75;
N, 6.95. Found: C, 53.58; H, 3.84; N, 6.63.
4.2.3.1. Dihydrochloride salt of 5-(4-amidinophenyl)-2-(5-
amidinothien-2-yl)-1H-indole (6a). Yellow solid (0.179 g, 60%),
mp >300 °C; 1H NMR (DMSO-d6) d 12.40 (s, 1H), 9.49 (s, 2H),
9.46 (s, 2H), 9.24 (s, 2H), 9.21 (s, 2H), 8.17 (d, 1H, J = 4 Hz), 8.07
(d, 1H, J = 4 Hz), 7.96 (br s, 4H), 7.82 (d, 1H, J = 4 Hz), 7.64 (dd,
1H, J = 1.6 Hz, J = 8.4 Hz), 7.57 (d, 1H, J = 8.4 Hz), 7.01 (d, 1H,
J = 1.6 Hz); 13C NMR (DMSO-d6) d 165.8, 163.9, 147, 143.2, 138.2,
135.9, 132, 131, 129.2, 127.2, 127, 126, 125.3, 123, 119.7, 112.8,
102.4; ESI-MS: m/z calcd for C20H17N5S: 359.45, found: 360.50
(amidine base M++1). Anal. Calcd for C20H17N5Sꢁ2HClꢁ1.25H2O: C,
52.80; H, 4.76; N, 15.39. Found: C, 52.80; H, 4.72; N, 15.22.
4.2.1.2. 5-Bromo-1-(tert-butoxycarbonyl)-2-(4-cyanophenyl)-
1H-indole (4b). White solid (1.59 g, 79%). Mp 188–188.5 °C ; 1H
NMR (DMSO-d6): d 8.07 (d, 1H, J = 9.2 Hz), 7.94 (d, 2H, J = 8 Hz),
7.86 (d, 1H, J = 1.2 Hz), 7.7 (d, 2H, J = 8 Hz), 7.53 (dd, 1H, J = 9.2,
1.2 Hz), 6.71 (s, 1H), 1.49 (s, 9H); 13C NMR (DMSO-d6): d 149.4,
140.1, 138.8, 136.6, 132.2, 131.1, 130.1, 127.9, 123.8, 118.9,
117.2, 116.1, 111.2, 110.7, 85.2, 27.7. Anal. Calcd for C20H17BrN2O2:
C, 60.47; H, 4.31; N, 7.05. Found: C, 60.55; H, 4.33; N, 6.92.
4.2.3.2. Dihydrochloride salt of 2,5-bis(4-amidinophenyl)-1H-
indole (6b). Yellow solid (0.19 g, 65%), mp >300 °C; 1H NMR
(DMSO-d6) d 12.31 (s, 1H), 9.55 (s, 2H), 9.52 (s, 2H), 9.35 (s, 2H),
9.33 (s, 2H), 8.24 (d, 2H, J = 8.8 Hz), 8.06 (d, 2H, J = 8.4 Hz), 7.99–
7.92 (m, 4H), 7.67 (d, 2H, J = 8.4 Hz), 7.61 (s, 1H), 7.19 (s, 1H). 13C
NMR (DMSO-d6) d 165.8, 165.5, 147.2, 138.4, 137.5, 130.7, 129.4,
129.3, 129.2, 127, 126.5, 125.8, 125.3, 122.4, 119.8, 112.8, 102.2;
ESI-MS: m/z calcd for C22H19N5: 353.42, found: 354.30 (amidine
base M++1). Anal. Calcd for C22H19N5. 2HClꢁ0.90H2O: C, 59.70; H,
5.19; N, 15.82. Found: C, 60.05; H, 5.12; N, 15.57.
4.2.2. General procedure for 5a,b
An aqueous solution of Na2CO3 (5 mL 2 M, deaireated) and
4-cyanophenyl boronic acid (0.73 g, 5 mmol) in 5 mL deaireated
methanol were added to a stirred solution of the bromoindole
derivatives 4a,b (5 mmol), and tetrakistriphenylphosphine palla-
dium (0.288 g, 0.25 mmol) in deaireated toluene (20 mL) under a
nitrogen atmosphere. The vigorously stirred mixture was warmed
to 80 °C for 24 h. The solvent was evaporated under reduced pres-
sure, the solid was partitioned between ethyl acetate (200 mL) and
2 M aqueous Na2CO3 (25 mL) containing 5 mL of concentrated
ammonia, to destroy the palladium complex, washed with water,
passed through celite to remove the catalyst, dried over sodium
sulfate and evaporated. Purification was by column chromatogra-
phy on silica gel, using hexanes/ethyl acetate (80:20, v/v) as the
eluent.
4.2.4. 6-Bromo-1-(tert-butoxycarbonyl)-2-(iodo)-1H-indole (10)
A molecular iodine (25 g, 98.76 mmol) solution in dry tetra-
hydrofuran (100 mL) was added slowly to a solution of the indole
stannane 9 (22.61 g, 49.38 mmol) in dry tetrahydrofuran (100 mL).
The mixture was stirred at room temperature for 6 h, the solvent
was removed under reduced pressure, the residue dissolved in
ethyl acetate (200 mL), washed with sodium thiosulfate solution
(10%) to remove any excess iodine, with water the organic phase
was dried over sodium sulfate and evaporated. Purification was
by column chromatography on silica gel, using hexanes/ethyl ace-
tate (97:3, v/v) as the eluent. White solid (17.21 g, 83%). Mp 48–
48.5 °C; 1H NMR (CDCl3) d 8.33 (s, 1H), 7.34–7.30 (m, 2H), 6.96
(s, 1H), 1.74 (s, 9H); 13C NMR (CDCl3) d 148.8, 138, 129.8, 126.1,
121.5, 120.2, 118.6, 85.9, 75.3, 28.2; ESI-MS: m/z calcd for
4.2.2.1. 1-(tert-Butoxycarbonyl)-5-(4-cyanophenyl)-2-(5-cyan-
othien-2-yl)-1H-indole (5a). Yellow solid (1.72 g, 81%). Mp
237–237.5 °C ; 1H NMR (CDCl3): d 8.30 (d, 1H, J = 8.8 Hz), 7.81 (br
s, 1H), 7.76 (br s, 4H), 7.63 (d, 1H, J = 8.8 Hz), 7.52 (d, 1H,
J = 4 Hz), 7.18 (d, 1H, J = 4 Hz), 6.86 (s, 1H), 1.52 (s, 9H); 13C NMR
(CDCl3) d 149.3, 145.6, 142.5, 137.7, 136.8, 134.7, 132.6, 130.9,
129, 128, 127.8, 125, 119.6, 118.9, 116.3, 113.9, 113.8, 110.7,
109.2, 85.1, 27.8; ESI-MS: m/z calcd for C25H19N3O2S: 425.50,
found: 426.2 (M++1). Anal. Calcd for C25H19N3O2S: C, 70.57; H,
4.50; N, 9.88. Found: C, 70.24; H, 4.51; N, 9.72.
C
C
13H13BrINO2: 420.92, found: 422.2 (M++1). Anal. Calcd for
13H13BrINO2: C, 36.99; H, 3.10; N, 3.32. Found: C, 37.21; H,
3.08; N, 3.30.
4.2.2.2.
1-(tert-Butoxycarbonyl)-2,5-bis(4-cyanophenyl)-1H-
indole (5b). White solid (1.78 g, 85%). Mp 222–222.5 °C; 1H
NMR (CDCl3): d 8.32 (d, 1H, J = 8.8 Hz)), 7.82 (d, 1H, J = 1.6 Hz),
7.76 (br s, 4H), 7.75 (d, 2H, J = 8.4 Hz), 7.64 (dd, 1H, J = 1.6 Hz,
J = 8.8 Hz), 7.58 (d, 2H, J = 8.4 Hz), 6.72 (s, 1H), 1.41 (s, 9H); 13C
NMR (CDCl3) d 149.2, 145.9, 139.4, 139.1, 137.8, 134.6, 132.6,
131.7, 129.6, 129.3, 127.8, 124, 119.4, 119, 118.7, 116.2, 111.6,
4.2.5. 1-(tert-Butoxycarbonyl)-2,6-bis(4-cyanophenyl)-1H-
indole (11)
An aqueous solution of Na2CO3 (10 mL 2 M, deaireated) and
4-cyanophenyl boronic acid (1.46 g, 10 mmol) in 10 mL deaireated
methanol were added to a stirred solution of the dihalo indole 10
(2.11 g, 5 mmol), and tetrakistriphenylphosphine palladium

2164
A. A. Farahat et al. / Bioorg. Med. Chem. 19 (2011) 2156–2167
(0.57 g, 0.5 mmol) in deaireated toluene (30 mL) under a nitrogen
atmosphere. The vigorously stirred mixture was warmed to 80 °C
for 24 h. The solvent was evaporated under reduced pressure, the
solid was partitioned between ethyl acetate (200 mL) and 2 M
aqueous Na2CO3 (25 mL) containing 5 mL of concentrated
ammonia, to destroy the palladium complex, washed with water,
passed through celite to remove the catalyst, dried over sodium
sulfate and evaporated. Purification was by column chromatogra-
phy on silica gel, using hexanes/ethyl acetate (85:15, v/v) as the
eluent. White solid (1.65 g, 79%). Mp 230–230.5 °C ; 1H NMR
(DMSO-d6): d 8.47 (s, 1H), 7.98–7.90 (m, 6H), 7.79 (d, 1H,
J = 8 Hz), 7.73 (d, 2H, J = 8 Hz), 7.70 (d, 1H, J = 8 Hz), 6.96 (s, 1H),
1.17 (s, 9H); 13C NMR (DMSO-d6) d 154.6, 145.8, 139.4, 139.1,
137.8, 134.5, 132.6, 131.6, 129.6, 129.3, 127.8, 124.4, 119.5, 119,
118.6, 116.1, 111.8, 111.4, 110.6, 84.7, 27.7; ESI-MS: m/z calcd
for C27H21N3O2: 419.47, found: 420.3 (M++1). Anal. Calcd for
C27H21N3O2: C, 77.31; H, 5.05; N, 10.02. Found: C, 77.45; H, 5.39;
N, 9.89.
extracted with ether (2 ꢂ 200 ml), the organic phase was washed
with potassium fluoride solution (10%) to remove any excess
stannane, with water and the combined extract was dried over so-
dium sulfate and the solvent was evaporated. Crystallization from
hexanes/ethanol (4:1) mixture gave a give yellow solid (13.43 g,
74%), mp 118–118.5 °C. 1H NMR (CDCl3): d 8.29 (s, 1H), 7.72–
7.58 (m, 4H), 7.63 (d, 1H, J = 8 Hz), 7.47 (d, 1H, J = 8 Hz), 6.80 (s,
1H), 1.71 (s, 9H), 0.39 (s, 9H); 13C NMR (CDCl3) d 151.9, 146.9,
145.5, 138, 134.5, 132.5, 127.8, 121.6, 120.6, 118.9, 117.8, 114.3,
110.4, 108.3, 84.4, 28.2, ꢀ0.1; ESI-MS: m/z calcd for C23H26N2O2Sn:
481.17, found: 482.1 (M++1). Anal. Calcd for C23H26N2O2Sn: C,
57.41; H, 5.45; N, 5.82. Found: C, 57.72; H, 5.63; N, 5.53.
4.2.9. General procedure for 16a–d
These series were prepared by following the experimental pro-
cedure used for compounds 4a,b.
4.2.9.1. 1-(tert-Butoxycarbonyl)-6-(4-cyanophenyl)-2-(5-cyano-
fur-2-yl)-1H-indole (16a). White solid (1.38 g, 68%), mp 178–
179 °C; 1H NMR (CDCl3): d 8.52 (d, 1H, J = 1.6 Hz), 7.81 (d, 2H,
J = 6.8 Hz), 7.77 (d, 2H, J = 6.8 Hz), 7.71 (d, 1H, J = 8 Hz), 7.55 (dd,
1H, J = 1.6 Hz, J = 8 Hz), 7.22 (d, 1H, J = 3.6 Hz), 6.93 (s, 1H), 6.73
(d, 1H, J = 3.6 Hz), 1.55 (s, 9H); 13C NMR (CDCl3) d 150, 145.7,
142.6, 137.7, 136.9, 134.7, 132.6, 131.2, 129, 128.2, 127.8, 125,
119.6, 119, 116.6, 113.6, 113.5, 110.7, 110, 85.2, 28.2; ESI-MS: m/
z calcd for C25H19N3O3: 409.44, found: 410.20 (M++1). Anal. Calcd
for C25H19N3O3: C, 73.34; H, 4.68; N, 10.26. Found: C, 73.17; H,
4.67; N, 10.22.
4.2.6. Dihydrochloride salt of 2,6-bis(4-amidinophenyl)-1H-
indole (12)
This compound was prepared by following the experimental
procedure used for compounds 6a,b. Yellow solid, yield (0.140 g,
46%), mp >300 °C; 1H NMR (DMSO-d6): 12.28 (s, 1H), 9.46 (s, 4H),
9.22 (s, 4H), 8.19 (d, 2H, J = 8.4 Hz), 7.98 (d, 2H, J = 8.4 Hz), 7.97
(br s, 4H), 7.81 (s, 1H), 7.73 (d, 1H, J = 8.4 Hz), 7.48 (dd, 1H,
J = 1.2 Hz, J = 8.4 Hz), 7.25 (s, 1H); 13C NMR (DMSO-d6) d 165.7,
165.4, 147, 138.6, 137.8, 137.3, 133.3, 129.3, 129.4, 127.9, 126.6,
126.3, 125.6, 125.2, 121.7, 119.8, 110.5, 101.7; ESI-MS: m/z calcd
for C22H19N5: 353.42, found: 354.3 (amidine base M++1). Anal.
Calcd for C22H19N5ꢁ2HClꢁ2H2O: C, 57.14; H, 5.44; N, 15.14. Found:
C, 57.44; H, 5.62; N, 14.88.
4.2.9.2. 1-(tert-Butoxycarbonyl)-6-(4-cyanophenyl)-2-(5-cyan-
othien-2-yl)-1H-indole (16b). Yellow solid (1.48 g, 70%), mp
186–187 °C; 1H NMR (CDCl3): d 8.52 (d, 1H, J = 1.2 Hz), 7.80 (d,
2H, J = 8.4 Hz), 7.77 (d, 2H, J = 8.4 Hz), 7.69 (d, 1H, J = 8 Hz), 7.63
(d, 1H, J = 4 Hz), 7.55 (dd, 1H, J = 2 Hz, J = 8 Hz), 7.17 (d, 1H,
J = 4 Hz), 6.83 (s, 1H), 1.49 (s, 9H); 13C NMR (CDCl3) d 149.3,
145.7, 142.5, 137.7, 136.9, 134.7, 132.6, 131, 129, 128, 127.8,
125, 119.6, 119, 116.3, 114, 113.8, 110.7, 110, 85.1, 27.8; ESI-MS:
m/z calcd for C25H19N3O2S: 425.12, found: 426.2 (M++1). Anal.
Calcd for C25H19N3O2S: C, 70.56; H, 4.50; N, 9.88. Found: C,
70.61; H, 4.52; N, 9.66.
4.2.7. 1-(tert-Butoxycarbonyl)-6-(4-cyanophenyl)-1H-indole
(14)
An aqueous solution of Na2CO3 (50 mL 2 M, deaireated) and 4-
cyanophenyl boronic acid (7.3 g, 50 mmol) in 50 ml deaireated
methanol were added to a stirred solution of indole derivative 8
(14.75 g, 50 mmol), and tetrakistriphenyl-phosphine palladium
(2.31 g, 2 mmol) in deaireated toluene (150 mL) under a nitrogen
atmosphere. The vigorously stirred mixture was warmed to 80 °C
for 24 h. After evaporation of the solvent under reduced pressure,
the solid was partitioned between ethyl acetate (250 mL) and
2 M aqueous Na2CO3 (50 mL) containing 20 mL of concentrated
ammonia, to destroy the palladium complex, then washing with
water, passed through celite to remove the catalyst, dried over so-
dium sulfate and evaporated. Purification was by column chroma-
tography on silica gel, using hexanes/ethyl acetate (90:10, v/v) as
the eluent. White solid (12.4 g, 78%). Mp 123–124 °C; 1H NMR
(CDCl3): d 8.51 (s, 1H), 7.79 (d, 2H, J = 7.2 Hz), 7.74 (d, 2H,
J = 7.2 Hz), 7.69–7.62 (m, 2H), 7.49 (d, 1H, J = 8 Hz), 6.63 (s, 1H),
1.71 (s, 9H); 13C NMR (CDCl3) d 149.6, 146.4, 135.9, 135.4, 128.7,
127.9, 127.2, 122, 121.5, 119.1, 114.2, 110.4, 107, 84, 28.2; ESI-
MS: m/z calcd for C20H18N2O2: 318.37, found: 319.2 (M++1). Anal.
Calcd for C20H18N2O2: C, 75.45; H, 5.70; N, 8.80. Found: C, 75.49;
H, 5.74; N, 8.80.
4.2.9.3. 1-(tert-Butoxycarbonyl)-6-(4-cyanophenyl)-2-(5-cyano-
pyridin-2-yl)-1H-indole (16c). White solid (1.36 g, 65%), mp
193–195 °C; 1H NMR (CDCl3): d 8.96 (d, 1H, J = 2 Hz), 8.48 (d, 1H,
J = 1.6 Hz), 8.05 (dd, 1H, J = 2 Hz, J = 8 Hz), 7.81 (d, 2H, J = 6.8 Hz),
7.78 (d, 2H, J = 6.8 Hz), 7.69 (d, 2H, J = 8 Hz), 7.56 (dd, 1H,
J = 1.6 Hz, J = 8 Hz), 6.97 (s, 1H), 1.38 (s, 9H); 13C NMR (CDCl3) d
150.8, 149.5, 146, 138.4, 136.9, 136.3, 136, 134, 132.7, 132.2,
129, 128, 127.6, 123, 121.7, 119, 117.2, 114.8, 112.7, 110.8, 85.4,
27.8; ESI-MS: m/z calcd for C26H20N4O2: 420.16, found: 421.2
(M++1). Anal. Calcd for C26H20N4O2: C, 74.27; H, 4.79; N, 13.33.
Found: C, 74.13; H, 5.03; N, 13.19.
4.2.9.4. 1-(tert-Butoxycarbonyl)-6-(4-cyanophenyl)-2-(6-cyano-
pyridin-3-yl)-1H-indole (16d). Yellowish white solid (1.29 g,
62%), mp 172–174 °C. 1H NMR (CDCl3): d 8.83 (d, 1H, J = 2 Hz),
8.55 (s, 1H), 7.92 (dd, 1H, J = 2 Hz, J = 8 Hz), 7.82 (d, 2H,
J = 6.8 Hz), 7.80 (d, 2H, J = 6.8 Hz), 7.79 (d, 1H, J = 8 Hz), 7.72 (d,
1H, J = 8 Hz), 7.58 (dd, 1H, J = 2 Hz, J = 8 Hz), 6.78 (s, 1H), 1.44 (s,
9H); 13C NMR (CDCl3) d 150.8, 149.5, 146, 138.4, 136.8, 136,
136.1, 134, 132.6, 132.2, 129, 128, 127.6, 123.5, 121.7, 118,
117.3, 114.7, 113, 110.4, 85.4, 27.2; ESI-MS: m/z calcd for
4.2.8. 1-(tert-Butoxycarbonyl)-6-(4-cyanophenyl)-2-
(trimethylstannyl)-1H-indole (15)
A 2.0 M solution of LDA in heptane-tetrahydrofuran-ethylben-
zene (22.63 mL, 45.27 mmol) was added slowly to a solution of
the indole derivative 14 (12 g, 37.73 mmol) and trimethyltin chlo-
ride (14.5 g, 42.76 mmol) in dry tetrahydrofuran (100 mL) at
ꢀ20 °C. The reaction mixture was left to warm to room tempera-
ture with stirring for 48 h, quenched with water (50 mL), the sol-
vent was removed under reduced pressure, the residue was
C
C
26H20N4O2: 420.1, found: 421.2 (M++1). Anal. Calcd for
26H20N4O2: C, 74.27; H, 4.79; N, 13.33. Found: C, 74.21; H, 4.94;
N, 13.07.

A. A. Farahat et al. / Bioorg. Med. Chem. 19 (2011) 2156–2167
2165
4.2.10. General procedure for 17a–d
These series was prepared by following the experimental proce-
dure used for compounds 6a,b.
4.2.12. Trihydrochloride salt of 2,5-bis(4-amidinophenyl)-1H-
benzimidazole (24)
p-Nitrobenzene (0.5 mL, 4.5 mmol) was added to a stirred mix-
ture of 22 (310 mg, 1.5 mmol) and 4-cyanobenzaldehyde (195 mg,
1.5 mmol), in dry dimethylformamide. The vigorously stirred mix-
ture was warmed to 145 °C for 48 h under a nitrogen atmosphere.
Ethyl acetate was added, and the precipitated crystals were filtered
and recrystalized from ethanol to give 2,5 (6)-bis(4-cyanophenyl)-
1H-benzimidazole 23 (0.288 g, 60%), mp >300 °C. 1H NMR (DMSO-
d6) d 13.22 (s, 1H), 8.36 (d, 2H, J = 8.1 Hz), 8.02 (d, 2H, J = 8.1 Hz),
7.91 (m, 5H), 7.74 (d, 1H, J = 8.1 Hz), 7.63 (d, 1H, J = 8.4 Hz); HRMS
(ESI): calcd for C21H13N4 m/z: 321.1140 (M++1); Found m/z:
321.1154. Compound 23 was used in the next step without further
characterization. The dinitrile 23 (0.16 g, 0.5 mmol) was dissolved
in saturated ethanolic HCl (5 mL) and stirred at room temperature
for 1 week, isolated from air and moisture. Dry ether was added,
the crystals were filtered, dried under vacuum for 1 h and dis-
solved in absolute ethanol (10 mL), ammonia gas was passed for
30 min while cooling and the resulting solution was stirred for
4 days. Dry ether was added, the precipitated crystals (HCl salt)
were filtered. The diamidine salt was purified by neutralization
with 1 N sodium hydroxide solution followed by filtration of the
resultant solid and washing with water and dried. Finally, the free
base was stirred with ethanolic HCl (5 mL) for 2 days, diluted with
ether, and the solid formed was filtered and dried to give the
diamidine HCl salt (0.12 g, 68%), mp >300 °C dec.; 1H NMR
(DMSO-d6) d 9.50 (s, 2H), 9.39 (s, 2H), 9.31 (s, 2H), 9.20 (s, 2H),
8.54 (d, 2H, J = 8.4 Hz), 8.08 (d, 2H, J = 8.4 Hz), 8.06 (s, 1H), 7.99
(s, 4H), 7.84 (d, 1H, J = 8.4 Hz), 7.74 (dd, 1H, J = 1.5 Hz, J = 8.4).
13C NMR (DMSO-d6) d 165.2, 164.8, 149.7, 145.5, 137.0, 136.6,
135.0, 131.6, 130.1, 129.2, 129.0, 127.7, 127.5, 126.4, 124.0,
115.7, 113.4. ESI-MS: m/z calcd for C21H18N6: 354.4, found: 355.1
(amidine base M++1). Anal. Calcd for C21H18N6ꢁ3HClꢁ2H2O: C,
50.46; H, 5.04; N, 16.81. Found: C, 50.53; H, 4.76; N, 16.49.
4.2.10.1. Dihydrochloride salt of 6-(4-amidinophenyl)-2-(5-ami-
dinofur-2-yl)-1H-indole (17a). Brown solid (0.138 g, 47%), mp
>300 °C; 1H NMR (DMSO-d6) d 12.58 (s, 1H), 9.72 (s, 2H), 9.58 (s,
2H), 9.25 (s, 2H), 9.17 (s, 2H), 7.99–7.95 (m, 5H), 7.77 (s, 1H),
7.74 (s, 1H), 7.49 (d, 1H, J = 8.4 Hz), 7.30 (d, 1H, J = 3.2 Hz), 7.15
(s, 1H).; 13C NMR (DMSO-d6) d 165.3, 160.4, 151.8, 146.2, 139.3,
137.5, 133.2, 128.6, 128.5, 128.2, 126.8, 125.7, 121.4, 121, 119.4,
109.8, 108.6, 101.2; ESI-MS: m/z calcd for C20H17N5O: 343.38,
found: 344.30 (amidine base M++1). Anal. Calcd for
C20H17N5Oꢁ2HClꢁ1.75H2O: C, 53.63; H, 5.06; N, 15.63. Found: C,
53.81; H, 5.17; N, 15.31.
4.2.10.2. Dihydrochloride salt of 6-(4-amidinophenyl)-2-(5-
amidinothien-2-yl)-1H-indole (17b). Yellow solid (0.136 g,
45%), mp >300 °C; 1H NMR (DMSO-d6) d 12.38 (s, 1H), 9.44 (br s,
4H), 9.18 (s, 2H), 9.13 (s, 2H), 8.14 (d, 1H, J = 3.6 Hz), 7.99 (br s,
4H), 7.83 (d, 1H, J = 3.6 Hz), 7.78 (s, 1H), 7.71 (d, 1H, J = 8.4 Hz),
7.49 (d, 1H, J = 8.4 Hz), 6.99 (s, 1H); 13C NMR (DMSO-d6) d 165.6,
160.9, 147.5, 144, 138.2, 135.9, 132, 130.8, 129.2, 127, 126.9,
125.9, 125.1, 123.1, 119, 112.2, 101.8; ESI-MS: m/z calcd for
C
20H17N5S: 359.45, found: 361.50 (amidine base M++2). Anal. Calcd
for C20H17N5Sꢁ2HClꢁ0.75H2Oꢁ0.2Et2O: C, 54.22; H, 4.93; N, 15.20.
Found: C, 54.57; H, 4.53; N, 15.30.
4.2.10.3. Trihydrochloride salt of 6-(4-amidinophenyl)-2-(5-
amidinopyridin-2-yl)-1H-indole (17c). Yellow solid (0.103 g,
33%), mp >300 °C; 1H NMR (DMSO-d6) d 12.18 (s, 1H), 9.61 (s,
2H), 9.43 (s, 2H), 9.30 (s, 2H), 9.15 (br s, 2H), 9.08 (s, 1H), 8.32–
8.30 (m, 2H), 7.97 (d, 2H, J = 8.4 Hz), 7.94 (d, 2H, J = 8.4 Hz), 7.85
(s, 1H), 7.78 (d, 1H, J = 8.4 Hz), 7.49 (d, 1H, J = 8.4 Hz), 7.47 (s,
1H); 13C NMR (DMSO-d6) d 165.8, 164, 149.4, 146.9, 138.8, 137.6,
137.5, 134.2, 129.3, 129, 127.4, 126.5, 122.5, 122.4, 120, 119.9,
111.2, 104, 103.7; ESI-MS: m/z calcd for C21H18N6: 354.16, found:
355.3 (amidine base M++1). Anal. Calcd for C21H18N6ꢁ3HClꢁ0.75H2O:
C, 52.84; H, 4.75; N, 17.60. Found: C, 52.96; H, 5.01; N, 17.31.
4.2.13. 2,5-Bis(4-cyanophenyl)-1-N-methyl-1H-benzimidazole
(29)
DAPCy (0.3 g, 0.44 mmol %) and K3PO4 (3.8 g, 17.8 mmol) were
added to a solution of 26 (2.5 g, 8.9 mmol) and p-cyanophenylbo-
ronic acid (1.55 g, 10.6 mmol) in deaireated absolute ethanol
(60 mL). The vigorously stirred mixture was warmed to 80 °C for
24 h under a nitrogen atmosphere. The solvent was concentrated
under reduced pressure; the crystals which formed were filtered,
dissolved in dichloromethane and passed through celite. Purifica-
tion by column chromatography on silica gel, using hexanes/ethyl
acetate (70:30, v/v) as the eluent gave 4-(4-N-methylamino-3-
nitrophenyl)-benzonitrile (27) (1.35 g, 60%). Mp 194.4–195.4 °C.
1H NMR (DMSO-d6, 300 MHz) d 8.39 (d, 1H, J = 2.4 Hz), 8.14 (br s,
1H), 7.95 (dd, 1H, J = 2.4, J = 9.0 Hz), 7.83 (m, 4H), 7.12 (d, 1H,
J = 9.3 Hz), 3.01 (s, 3H); 13C NMR (DMSO-d6, 75 MHz) d 145.9,
142.7, 134.8, 132.9, 131.5, 126.6, 124.7, 124.2, 119.0, 115.4,
109.4, 29.7. Compound 27 was used in the next step without fur-
ther characterization. Pd/C (10%) (0.5 g) was added to a solution
of the nitro compound 27 (4 g, 15.6 mmol) in ethanol: ethyl acetate
(60 mL: 20 mL) mixture. Shaking in a Parr hydrogenator at 50 psi
was continued until the uptake of hydrogen ceased; the solution
was passed through celite to remove the catalyst, and concentrated
under reduced pressure. Purification by crystallization from ace-
tone gave 4-(3-amino-4-N-methylaminophenyl)-benzonitrile (28)
(3.2 g, 90%). Mp 215–216 °C. 1H NMR (DMSO-d6, 300 MHz) d 7.63
(m, 4H), 7.13 (dd, 1H, J = 2.1, J = 8.1 Hz), 6.98 (d, 1H, J = 1.8 Hz),
6.72 (d, 1H, J = 8.4 Hz), 3.40 (br s, 2H), 2.92 (s, 3H). ESI-MS: m/z
calcd for C14H13N3: 223.27, found: 224.0 (M++1). Compound 28
was used in the next step without further characterization. 4-Cya-
nobenzaldehyde (0.7 g, 5.6 mmol) and 28 (1.4 g, 5.4 mmol) were
stirred in anhydrous dimethylformamide for 1 h, then sodium
4.2.10.4. Dihydrochloride salt of 6-(4-amidinophenyl)-2-(6-ami-
dinopyridin-3-yl)-1H-indole (17d). Yellow solid (0.118 g, 37%),
mp >300 °C; 1H NMR (DMSO-d6) d 12.49 (s, 1H)), 9.73 (s, 2H),
9.43 (s, 2H), 9.41 (s, 2H), 9.17 (s, 2H), 8.68 (d, 1H, J = 8.8 Hz), 8.45
(d, 1H, J = 8.8 Hz), 7.99 (s, 1H), 7.98 (br s, 4H), 7.84 (s, 1H), 7.77
(d, 1H, J = 8 Hz), 7.50 (d, 1H, J = 8 Hz), 7.39 (s, 1H); 13C NMR
(DMSO-d6); d 165.8, 164.1, 149.4, 146.1, 138.7, 137.9, 137, 134.5,
129.3, 129, 127.5, 126.5, 122.5, 122.4, 120, 119.9, 111.3, 104.4,
102.3; ESI-MS: m/z calcd for C21H18N6: 354.16, found: 355.3 (ami-
dine base M++1). Anal. Calcd for C21H18N6ꢁ2HClꢁ2H2O: C, 54.43; H,
5.22; N, 18.13. Found: C, 54.52; H, 5.11; N, 17.98.
4.2.11. 4-(4-Amino-3-nitrophenyl)-benzonitrile (21)
DAPCy (0.435 g, 0.6 mmol %) and K3PO4 (6.3 g, 30 mmol) were
added to a solution of 20 (3.88 g, 15 mmol) and p-cyanophenylbo-
ronic acid (2.64 g, 18 mmol) in deaireated iso-propanol (60 mL).
The vigorously stirred mixture was warmed to 80 °C for 24 h under
a nitrogen atmosphere. The solvent was concentrated under re-
duced pressure, the crystals which formed were filtered, dissolved
in dichloromethane and passed through celite and evaporated un-
der reduced pressure to give 21 (2.9 g, 80%). Mp 204.4–205.4 °C.;
1H NMR (DMSO-d6, 300 MHz):
d 9.66 (s, 1H), 7.73 (d, 2H,
J = 7.2 Hz), 7.57 (d, 2H, J = 7.2 Hz), 7.43 (d, 2H, J = 9.0 Hz), 5.77 (s,
2H). 13C NMR (DMSO-d6, 75 MHz) d 145.9, 142.5, 133.7, 132.5,
130.6, 126.2, 125.0, 123.2, 119.9, 118.6, 109.1; HRMS (ESI): Calcd
for C13H8N3O2 m/z: 238.0617 (M++1); Found m/z: 238.0612.

2166
A. A. Farahat et al. / Bioorg. Med. Chem. 19 (2011) 2156–2167
bisulfite (0.6 g, 5.6 mmol) was added and the resulting mixture
was stirred at 130 °C for 48 h, cooled to room temperature, poured
into water and the resulting crystals were filtered and recrystalized
from ethanol to give 29 (1.4 g, 73% yield). Mp 257–258 °C. 1H NMR
(DMSO-d6, 300 MHz) d 8.10 (m, 3H), 8.03 (d, 2H, J = 8.7 Hz), 7.95 (d,
2H, J = 8.7 Hz), 7.89 (d, 2H, J = 8.7 Hz), 7.80 (d, 1H, J = 8.7 Hz), 7.72
(dd, 1H, J = 1.5, J = 8.4 Hz), 3.95 (s, 3H). 13C NMR (DMSO-d6,
75 MHz) d 152.2, 145.2, 142.9, 137, 134.1, 132.7, 132.5, 132.3,
129.8, 127.4, 122.2, 118.6, 118.1, 117.7, 112.1, 111.2, 109.2, 31.7.
ESI-MS: m/z calcd for C22H14N4: 334.12, found: 335.1 (M++1). Anal.
Calcd for C22H14N4ꢁ0.2H2O: C, 78.18; H, 4.29; N, 16.58. Found: C,
78.28; H, 4.15; N, 16.88.
4.2.16. Trihydrochloride salt of 2,5-bis(4-amidinophenyl)-1-N-
hydroxy-1H-benzimidazole (35)
This compound was prepared by following the experimental
procedure used for compound 24. Yellow solid (0.118 g, 78%), mp
>300 °C. 1H NMR (DMSO-d6) d 9.63 (br s, 2H), 9.52 (br s, 2H),
9.39 (br s, 2H), 9.28 (br s, 2H), 8.54 (d, 2H, J = 8.0 Hz), 8.05 (t, 4H,
J = 7.2 Hz), 7.98 (d, 3H, J = 8.8 Hz), 7.87 (d, 1H, J = 8.4 Hz), 7.80 (d,
1H, J = 8.4 Hz); 13C NMR (DMSO-d6, 75 MHz) d 165.1, 146.3,
145.4, 137.7, 134.0, 133.8, 132.8, 129.0, 128.6, 128.3, 127.0,
126.2, 122.1, 119.7, 119.7, 108.0. ESI-MS: m/z calcd for
C
21H18N6O: 370.40, found: 370.4 (amidine base M+). Anal. Calcd
for C21H18N6Oꢁ3HClꢁ0.5H2Oꢁ0.4EtOH: C, 51.62; H, 4.84; N, 16.56.
Found: C, 51.62; H, 4.61; N, 16.24.
4.2.14. Trihydrochloride salt of 2,5-bis(4-amidinophenyl)-1-N-
methyl-1H-benzimidazole (30)
Acknowledgments
This compound was prepared by following the experimental
procedure used for compounds 6a,b. Yellow solid (0.183 g, 89%).
Mp >300 °C dec. 1H NMR (DMSO-d6) d 9.68 (s, 2H), 9.52 (s, 2H),
9.45 (s, 2H), 9.29 (s, 2H), 8.18 (d, 2H, J = 9.0 Hz), 8.13 (d, 2H,
J = 8.4 Hz), 8.02 (s, 3H), 7.95 (d, 2H, J = 8.7 Hz), 7.89 (d, 2H,
J = 8.4 Hz), 4.02 (s, 3H); 13C NMR (DMSO-d6) d 167.4, 165.9, 141,
138.7, 136.7, 136.6, 136.1, 133.2, 131.2, 130.3, 129.6, 127.5,
126.5, 126.3, 123.5, 121, 21.4; ESI-MS: m/z calcd for C22H20N6:
368.43, found: 368.40 (amidine base M+). Anal. Calcd for
This work was supported by the Egyptian government through
the channel program (A.A.F.) and by an award from the Bill and
Melinda Gates Foundation (R.B., W.D.W., D.W.B.).
References and notes
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50.13; H, 5.30; N, 15.44.
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4.2.15. 2,5-Bis(4-cyanophenyl)-1-N-hydroxy-1H-benzimidazole
(34)
3-Bromo-6-fluoronitrobenzene 25 (1.7 mL, 13.6 mmol) was
added to a solution of 4-cyanobenzylamine 31 (3.4 g, 20.3 mmol)
and K2CO3 (5.6 g, 40.5 mmol) in dioxane (150 mL). The resulting
solution was heated to reflux under nitrogen for 24 h. The inorgan-
ics were filtered from the mixture, the solution was concentrated,
hexane was added and the product, orange crystals were filtered
and recrystalized from ethyl acetate to give 4.4 g of 4-(N-4-cya-
nobenzylamino)-3-nitro-bromobenzene 32 in 98% yield, mp 153–
154 °C. Compound 32 was used in the next step without further
characterization. DAPCy (0.2 g, 0.37 mmol %) and K3PO4 (3.1 g,
15 mmol) were added to a solution of 32 (2.65 g, 7.5 mmol) and
p-cyanophenylboronic acid (1.1 g, 7.5 mmol) in deaireated abso-
lute ethanol (60 mL). The vigorously stirred mixture was warmed
to 80 °C for 72 h under a nitrogen atmosphere. The precipitated
product was filtered. The solid was dissolved in acetone, filtered
through celite and recrystalized from EtOAc/MeOH to give 4-(4-
cyanobenzylamino-3-nitrophenyl)-benzonitrile (33) (2.1 g, 80%
yield), mp 181–182 °C. 1H NMR (DMSO-d6, 300 MHz) d 8.86 (br s,
1H), 8.43 (d, 1H, J = 2.1 Hz), 7.85 (m, 7 H), 7.57 (d, 2H, J = 8.1 Hz),
6.96 (d, 1H, J = 9.0 Hz), 4.80 (d, 2H, J = 6.3 Hz). 13C NMR (DMSO-
d6, 75 MHz) d 144.4, 144.3, 142.4, 134.5, 132.8, 132.0, 132.1,
127.8, 126.5, 125.5, 124.3, 118.7, 118.6, 115.6, 109.9, 109.5, 45.4.
ESI-MS: m/z calcd for C21H14N4O2: 354.36, found: 355.1 (M++1).
Compound 33 was used in the next step without further character-
ization. A solution of sodium methoxide (0.3 g, 5.6 mmol) and 33
(1 g, 2.8 mmol) in 30 mL dry methanol was heated at reflux for
24 h under nitrogen atmosphere. The reaction mixture was cooled
to room temperature, brought to pH 7 with 20% HCl, the resulting
yellow crystals were filtered and dried to give 34 (0.9 g, 93%). Mp
244.4–244.5 °C; 1H NMR (DMSO-d6, 300 MHz) d 8.46 (d, 2H,
J = 8.7 Hz), 7.94 (d, 2H, J = 8.1 Hz), 7.90 (d, 2H, J = 5.7 Hz), 7.86
(m, 2H), 7.81 (m, 1H), 7.75 (d, 1H, J = 8.7 Hz), 7.61 (dd, 1H, J = 1.5,
J = 8.1 Hz); 13C NMR (DMSO-d6, 75 MHz) d 144.9, 138.4, 134.2,
132.8, 132.7, 132.6, 128.7, 128.5, 127.8, 122.3, 120.3, 118.8,
118.4, 112.3, 109.7, 109.9, 107.9. HRMS (ESI) calcd for C21H13N4O
m/z: 337.1090 (M++1); found m/z: 337.1173.
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