Synthesis, DNA binding, fluorescence measurements and antiparasitic activity of DAPI related diamidines

Article abstract of DOI:10.1016/j.bmc.2009.12.011

A novel series of extended DAPI analogues were prepared by insertion of either a carbon-carbon triple bond (16a-d) or a phenyl group (21a,b and 24) at position-2. The new amidines were evaluated in vitro against both Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.). Five compounds (16a, 16b, 16d, 21a, 21b) exhibited IC₅₀ values against T. b. r. of 9 nM or less which is two to nine folds more effective than DAPI. The same five compounds exhibited IC₅₀ values against P. f. of 5.9 nM or less which is comparable to that of DAPI. The fluorescence properties of these new molecules were recorded, however; they do not offer any advantage over those of DAPI.

Full text of DOI:10.1016/j.bmc.2009.12.011

Bioorganic & Medicinal Chemistry 18 (2010) 557–566
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, DNA binding, fluorescence measurements and antiparasitic
activity of DAPI related diamidines
Abdelbasset A. Farahat a,b, Arvind Kumar a, Martial Say a, Alaa El-Din M. Barghash b, Fatma E. Goda b,
Hassan M. Eisa b, Tanja Wenzler c, Reto Brun c, Yang Liu a, Leah Mickelson a, W. David Wilson a,
David W. Boykin a,
*
a Department of Chemistry, Georgia State University, Atlanta, GA 30303, United States
b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
c Department of Medical Parasitology and Infection Biology, Swiss Tropical Institute, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of extended DAPI analogues were prepared by insertion of either a carbon–carbon triple
bond (16a–d) or a phenyl group (21a,b and 24) at position-2. The new amidines were evaluated
in vitro against both Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.). Five com-
pounds (16a, 16b, 16d, 21a, 21b) exhibited IC50 values against T. b. r. of 9 nM or less which is two to nine
folds more effective than DAPI. The same five compounds exhibited IC50 values against P. f. of 5.9 nM or
less which is comparable to that of DAPI. The fluorescence properties of these new molecules were
recorded, however; they do not offer any advantage over those of DAPI.
Received 3 November 2009
Revised 2 December 2009
Accepted 3 December 2009
Available online 11 December 2009
Keywords:
DAPI
Diamidines
Ó 2009 Elsevier Ltd. All rights reserved.
Indole acetylenes
Biphenyl indole
DNA binding
Fluorescence and antiprotozoal activity
1. Introduction
variety of biological effects, including antifungal, antibacterial,
antitrypanosomal and antiviral activities.5,6 Also, DAPI is a fluores-
Eukaryotic parasitic diseases, such as sleeping sickness and ma-
laria are caused by ancient, unusual, and often quite deadly micro-
organisms.1 Trypanosoma brucei, which causes sleeping sickness,
limits the health and economic hopes of 50,000–70,000 people/
year, and if not effectively treated, is fatal.2 Dicationic systems that
bind in the DNA minor groove are promising agents against these
diseases. Pentamidine (Fig. 1) is the only one of this class which
has seen significant human clinical use.3 Furamidine (Fig. 1), a
diphenyl furan diamidine analogue, has been shown to be more
potent and less toxic than pentamidine in murine models of
trypanosomiasis.4 The orally effective prodrug of furamidine
(pafuramidine) (Fig. 1), showed promising results in Phase I and
II clinical trials against both sleeping sickness and malaria.3,4
Unfortunately, in an additional safety study of pafuramidine
parallelling the Phase III trials, liver and kidney toxicities in some
volunteers were found and the development of pafuramidine was
suspended.4 DAPI (40,6-diamidino-2-phenylindole) (Fig. 1), was
developed as a compound related to diminazene and stilbamidine,
to be used as an antitrypanosomal agent.5 DAPI, however showed a
cent dye which exhibits several binding modes to DNA7 and so it
has been frequently utilized as a DNA specific probe for flow
cytometry, chromosome staining, DNA visualization and quantita-
tion,8 and it is now an important tool in molecular biology. The
antiparasitic mode of action of these diamidines is thought to in-
volve binding to the minor groove of DNA at AT rich regions in
the nucleus or kinetoplast.9 For decades, strong minor groove bind-
ing was thought to require a curved molecule (crescent shape) that
complemented the shape of the DNA minor groove. Furamidine ap-
pears to work in this way as it binds strongly to the parasite DNA
and causes rapid destruction of the mitochondrial kinetoplast.10 In
contrast to curved compounds, linear molecules have not been sig-
nificantly investigated. The classical model for minor groove bind-
ing emphasizes complementary curvature between DNA and the
binding compound.11 This model indicates that compounds, which
are linear or have a large radius of curvature, should bind weakly to
the minor groove. DB921 (Fig. 1), a near linear heterocyclic diami-
dine, however, binds quite strongly to the DNA minor groove, even
more strongly than related curved compounds.12 DB921 was found
to simulate the curved structure of DNA minor groove by incorpo-
ration of a water molecule into the recognition complex with
DNA.12,13 Based on both the discovery of this new binding mode
* Corresponding author. Tel.: +1 404 651 3798; fax: +1 404 651 1416.
E-mail address: dboykin@gsu.edu (D.W. Boykin).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.011

558
A. A. Farahat et al. / Bioorg. Med. Chem. 18 (2010) 557–566
O
O
RN
NH
HN
HN
O
NR
NH2
H2N
NH2
NH2
R
H
Pentamidine
Furamidine
Pafuramidine OMe
NH
NH
N
HN
N
N
H
NH2
NH2
H
NH2
NH2
DAPI
DB921
Figure 1. Active antiparasitic agents.
and the favorable biological activities and fluorescence of DAPI, we
prepared new series of near linear cationic indole derivatives (16a–
d, 21a–b, 24) which retain many of the features of both DAPI and
DB921.
higher yields.18 The reduction of the nitro compound 8 is a poten-
tial challenge due to practical drawbacks regarding the use of tra-
ditional reducing agents such as tin or iron metal, including large
amount of metal oxide waste and difficulties with workup.19 Also
palladium catalyzed hydrogenation could affect the bromo groups
or the double bond. Due to these potential problems, we sought a
more promising method for reduction. Vanadium-doped palladium
on carbon catalyst reported by Baumister appeared to be useful.20
The presence of vanadium is thought to minimize contamination
by undesired hydroxylamines formed in the reduction process.
Using this approach, the amine 9 was obtained in excellent yield
using a mixture of toluene and ethyl acetate (3:1) as the hydroge-
nation solvent. This amine was allowed to undergo cross coupling
with the terminal acetylene 4a according to the method of Lautens
and co-workers19 This process involves Ullman cyclization to form
2-bromoindoles which undergoes Sonogashira coupling with ter-
minal alkynes giving the desired 2-alkynylindole 10. In our hands,
a yield of 15% was obtained. The relatively poor yield lead us to
search for another method for preparation of the indole acetylenes.
We decided to attempt Sonogashira coupling between the unre-
ported 2-iodoindole 14 and the ethynyl benzonitrile derivatives
4a–d.
Scheme 3 outlines the preparation of the indole acetylenes 15a–
d in high yield. The enamine 6 undergoes catalytic reductive cycli-
zation using Pd/C in tetrahydrofuran to form 1H-indole-6-carboni-
trile.21 The Boc-protected indole 12 was prepared in high yield by
employing a procedure we have used previously22 employing di-
tert-butyldicarbonate (Boc2O) and 4-(dimethylamino) pyridine
(DMAP) in dichloromethane as the solvent. We tried a number of
iodination methods to prepare the 2-iodoindole derivative 14,
including the use of diiodoethane in presence of butyl lithium,23
2. Results and discussion
2.1. Chemistry
Scheme 1 outlines our approach to the synthesis of new ethynyl
benzonitrile derivatives which will be used in the synthesis of the
target compounds 10, 15a–d. We employed Sonogashira-type14
cross coupling reactions of bromo benzonitrile derivatives with tri-
methylsilylacetylene to give the acetylenes 3a–d which were desi-
lylated14 by stirring with tetrabutylammonium fluoride in
tetrahydrofuran-methylene chloride mixture to afford the ethynyl
benzonitrile derivatives 4a–d.
Scheme 2 outlines the synthesis of indole acetylene 10, starting
with 5 which contains a methyl group ortho to the strong electron
withdrawing nitro group that can be easily functionalized by con-
densation with N,N-dimethylformamide dimethyl acetal to provide
the corresponding N,N-dimethylamino alkene 6.15 Oxidative cleav-
age of 6 using sodium periodate in tetrahydrofuran-water mixture
afforded the aldehyde 715,16 Since the Ramirez olefination is sensi-
tive to amine or amide functional groups, so we decided to start
with the nitro compound 7 which we planned to reduce later.
The dibromoalkene 8 was prepared efficiently with the Ramirez
(Wittig-type) dibromoolefination of the corresponding aldehyde
7 using carbon tetrabromide as a reagent and triphenylphosphine
as a catalyst.17 Recently, it was discovered that performing this
reaction in the presence of zinc powder, a bromine scavenger, gave
(a)
X2 X1
X2 X1
+
NC
NC
Br
Si
Si
X3
X3
1 a-d
3 a-d
2
(b)
X1
X2
X3
a
b
C
C
C
N
H
H
X2 X1
NC
c
N
C
C
C
H
F
X3
4 a-d
d
Scheme 1. Reagents: (a) PdCl2(PPh3)2, CuI, TEA, THF; (b) TBAF, THF, CH2Cl2.

A. A. Farahat et al. / Bioorg. Med. Chem. 18 (2010) 557–566
559
N
(a)
CHO
NO2
(b)
NC
NC
NO2
NC
NO2
(d)
5
6
7
Br
(c)
Br
(e)
Br
NH2
Br
NO2
NC
NC
8
9
CN
N
NC
H
10
Scheme 2. Reagents: (a)DMF–DMA, DMF; (b) NaIO4, THF/H2O;(c)CBr4, P(Ph)3, CH2Cl2;(d)H2/Pd(C), toluene/EtOAc;(e) Pd(C), P(4-OCH3Ph)3, CuI, toluene, 4-ethynylbenzonitrile.
N
(a)
(b)
N
H
N
NC
NC
NC
NO2
O
O
6
12
11
(d)
(c)
Sn
I
N
N
NC
NC
O
O
O
O
14
13
X1 X2
(e)
(f)
CN
N
NC
X3
O
O
15 a-d
X1
X2
X3
a
b
c
C
C
N
C
C
N
C
C
H
H
H
F
X1 X2
NH
NH2
HN
N
H
X3
NH2
16 a-d
d
Scheme 3. Reagents: (a) H2/Pd(C), THF; (b) BOC2O, DMAP, CH2Cl2; (c) ClSn(CH3)3, LDA/THF; (d) I2/THF; (e) PdCl2(PPh3)2, CuI, TEA, THF, ethynylbenzonitrile derivatives;
(f) (i) LiN(TMS)2/THF; (ii) HCl gas/EtOH.
iodine monochloride-pyridine complex in acetic acid,24 molecular
iodine with butyl lithium in tetrahydrofuran,25 N-iodosuccinin-
mide with butyl lithium in tetrahydrofuran.26 However we were
unable to obtain 14 in a reasonable yield using these methods,
therefore we decided to introduce a trimethylstannyl group at
the 2-position of indole which has been reported in other systems
to react with iodine to form the corresponding iodo compound.27
Lithiation of the Boc-protected indole 12 was readily achieved with
lithium diisopropylamide (LDA) in anhydrous tetrahydrofuran at
ꢀ20 °C. Subsequent reaction of the lithioindole intermediate with
trimethyltin chloride gave the indole stannane 13 in good yield.22
Stirring the indole stannane 13 with molecular iodine in dry tetra-
hydrofuran at room temperature give the iodoindole 14 in high
yield (88%). We note that the preparation of the 2-iodoindole 14
was recently reported also employing an indirect route starting
with the analogous boronic acid.28 The alkynylindole derivatives
15a–d were prepared through Sonogashira coupling of iodoindole
14 with the terminal alkynes 4a–d using PdCl2(PPh3)2 as the

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A. A. Farahat et al. / Bioorg. Med. Chem. 18 (2010) 557–566
catalyst, in anhydrous tetrahydrofuran as the solvent under nitro-
gen atmosphere. The dinitriles 15a–d were allowed to react with
lithium bis(trimethylsilyl)amide29 in THF, followed by deprotec-
tion of the silylated amidines with ethanolic HCl to furnish hydro-
chloride salts of the diamidines 16a–d.
Scheme 4 outlines the synthesis of the linear diphenyl indole
diamidines 21a,b. 4,40-Bromobiphenyl benzonitrile 19 was pre-
pared in good yield by employing a Suzuki coupling reaction30 be-
tween the boronic acid 17 and the iodobenzonitrile 18 in the
presence of 5 mol % Pd(PPh3)4. A Stille coupling reaction31 between
19 and 1-(tert-butoxycarbonyl)-2-(trimethylstannyl)-1H-indole-5
or 6-carbonitrile22 using 5 mol % Pd(PPh3) and 1,4-dioxane as the
solvent afforded the dinitriles 20a,b in good yield. These dinitriles
were converted to the diamidines 21a,b utilizing the lithium
bis(trimethylsilyl)amide method.
analogues are presented in Table 1. For comparative purposes re-
lated data for pentamidine, furamidine, DAPI and DB921 are also
included in Table 1. The DNA binding affinity as deduced from
D
Tm values varies with structure and ranges from quite strong to
moderate. For a relatively small molecule the affinity of DAPI is
quite strong as previously reported32 and in the current work a
D
Tm value of greater than 27 °C is found. Extension of the length
of DAPI by insertion of a carbon–carbon triple bond at position-2
(16a) results in a reduction in Tm value of about 30% of that of
D
DAPI. In contrast, an insertion of a phenyl group at position-2
(21b) results in a molecule with comparable DNA affinity to that
of DAPI.
Since the overall dimensions of 16a and 21b are roughly similar,
it seems likely that the lower affinity of 16a is attributable, in large
part, to weaker stacking interactions of 16a with the groove. The
two isomers 21b and 21a show significantly different affinities.
Scheme 5 outlines the synthesis of the monoamidine 24. The
Stille coupling reaction between 13 and the commercially available
22 gave the mononitrile 23 which was converted to the amidine 24
utilizing the lithium bis(trimethylsilyl)amide method.
Compound 21b shows a larger
DTm value than 21a. Molecular
modeling suggests that the 20% difference in affinity is likely due
to the fact that in the complex 21b can form a strong hydrogen
bond involving the indole N–H to a thymine carbonyl. Whereas
in the analogous complex for 21a such is not possible as only the
3-postion C–H is pointed into the groove. Removal of the amidine
on the terminal phenyl ring to form 24 results in about 40% reduc-
tion of affinity consistent with the loss of one key cationic binding
2.2. Biology
The results for assessment of DNA binding affinity and the
activity against P. f. K1 and T. b. r. STIB900 for seven new indole
OH
(a)
Br
B
I
CN
Br
CN
OH
17
18
19
(b)
NH
NH2
HN
(c)
CN
NC
N
N
H
H2N
O
O
21 a: 5 amidin
21 b: 6 amidin
20 a: 5 CN
20 b: 6 CN
Scheme 4. Reagents: (a) Pd(PPh3)4, Na2CO3, toluene; (b) 1-(tert-butoxycarbonyl)-2-(trimethylstannyl)-1H-indole-5 or 6-carbonitrile, Pd(PPh3)4, dioxane; (c) (i) LiN(TMS)2/
THF; (ii) HCl gas/EtOH.
(a)
Br
N
NC
O
O
23
22
(b)
HN
N
H
NH2
24
Scheme 5. Reagents: (a) 1-(tert-butoxycarbonyl)-2-(trimethylstannyl)-1H-indole-6-carbonitrile, Pd(PPh3)4, dioxane; (b) (i) LiN(TMS)2/THF; (ii) HCl gas/EtOH.

A. A. Farahat et al. / Bioorg. Med. Chem. 18 (2010) 557–566
561
Table 1
The IC50 values of the new diamidino indoles against T. b. r.
range from 2 to 9 nM (with the exception of 16c; IC50 = 215 nM),
which are in the range of those for furamidine and pentamidine,
but 2–9-folds more active than DAPI. Consistent with other
monoamidines of active diamidines, 24 is essentially inactive
DNA affinities and antiprotozoal activity for DAPI analogues
Cyctoxicityc
M)
T. b. r.b
(nM)
SIT
P.f.b
(nM)
SIP
a
d
e
Compd
D
(°C)
Tm
(
l
Pentamidine
Furamidine
DB921
DAPI
16a
16b
16c
16d
21a
21b
12.6
25
46
6.7
17
0.86
8.7
1.6
13.0
4.5
38.9
10.5
8.4
2.2
4.5
7.7
18
6
3
215
9
15,533
2093
2207
51
1450
533
46.4
15.5
0.5
3
3.8
3.1
14.1
5.9
0.7
3.3
858
991
486
34,000
287
2289
516
933
763
55,571
3182
9.8
(IC50 = 8.7 l
M).3a The activity of the diamidines against P. f.
>27
>27
19.0
19.1
12.2
16.0
20.9
>27
16.0
followed a similar trend, 16c was the least active and the other
diamidines gave IC50 values ranging from 0.7 to 5.9 nM. The activ-
ity of the monoamidine 24 is greatly reduced (IC50 = 0.86 lM) in
comparison to 21a and 21b. The antiplasmodial activity of the
indoles is superior to that of furamidine and pentamidine and com-
parable to that of DAPI. The potency of 21a (IC50 = 0.7 nM) places it
in a small group of highly active diamidines in the STI screen.35 The
selectivity indices for both T. b. r.(SIT) and P. f. (SIP) are listed in Ta-
ble 1. The SIT values for the five most active compounds (16a, 16b,
16d, 21a, 21b) range from 500 to 9725, a clear improvement over
the value of 51 for DAPI. The SIP values for the same five
compounds ranges from 516 to 55571which are all superior to
the DAPI value of 287. Clearly these compounds merit in vivo eval-
uation against both organisms.
60
500
4
2
8723
9725
5250
0.96
24
a
Increase in thermal melting of poly(dA-dT)n.38
The T. b. r. (Trypanosoma brucei rhodesiense) strain was STIB900 and the P. f.
b
(Plasmodium falciparum) strain was K1. The values are the average of duplicate
determinations.37
c
Cyctotoxicity was evaluated using cultered L6 rat myoblast cells.37
Selectivity index for T. b. r. expressed as the ratio: IC50(L6)/IC50(T.b.r).
Selectivity index for P. f. expressed as the ratio: IC50(L6)/IC50(P.f.)
d
e
Given the general use of DAPI in molecular biology, in large part
due to its fluorescence properties, we have recorded fluorescence
data for the new indoles which is found in Table 2. For comparative
evaluation, also included in Table 2 are results for DAPI. The
absorption maximum for the new indoles ranges from 340 to
368 nm (with the exception of the fluoro analogue 16d which are
comparable to that of DAPI (341 nm). The emission maximum
ranges from 425 to 514 nm in comparison of a value of 458 nm
for DAPI. With two exceptions, a qualitative comparison of quan-
tum yield to that of DAPI shows a loss of efficacy by 13–43-fold.
One exception, 16a shows only a twofold decline and interestingly
the monoamidine 24 shows a moderate increase compared to that
of DAPI. An important feature for the molecular biology use of DAPI
fluorescence is that on binding to DNA, its fluorescence is enhanced
by about 20-folds.36 Therefore, we compared the fluorescence of
the new analogues to DAPI on binding to DNA. Figure 3 shows
the expected enhancement of DAPI fluorescence on interaction
with DNA, however none of the new analogues cause a similar
enhancement; illustrated in Figure 3 with compound 21b. We con-
clude that the fluorescence properties of these new molecules do
not offer any advantage over those of DAPI.
center. A limited study of the effect of substitution at the benzam-
idine portion of 16a shows that DNA affinity is sensitive to such
variations. Introduction of nitrogen ortho to the amidine 16b was
well tolerated, however when nitrogen is placed meta to the ami-
dine 16c the
DTm value is reduced by approximately one-third.
Similar trends have been noted for other aza-analogues of minor
groove binders and are believed to arise from differences in hydra-
tion of the small molecules.33 Introduction of a fluorine atom ortho
to the benzamidine 16d results in about 15% reduction in affinity,
perhaps due to twisting of the amidine group relative to the phenyl
ring. In order to determine if the new analogues 16a and 21b are
binding in the DNA minor groove and to compare them to DAPI,
we acquired CD spectra of these compounds on binding to DNA
(see Fig. 2). Minor groove binding agents give a large positive in-
duced CD signal on binding to AT DNA sequences and cause only
small changes in the shape of the DNA CD spectrum.34 Such a result
is observed on adding DAPI to poly(dA)ꢁpoly (dT) (Fig. 2) with a
strong, positive induced signal between 350 and 400 nm and little
change in the DNA signal near 260 nm. As can be seen in Figure 2,
both 16a and 21b have similar CD spectra with poly(dA)ꢁpoly (dT).
The biphenyl indole, 21b has a particularly strong induced CD sig-
nal at around 390 nm indicating a strong interaction with the AT
base pairs. These results clearly support a minor groove binding
mode for 16a and 21b which is the case for DAPI.34 A detailed anal-
ysis of the effect of structure induced variation in DAPI analogues
DNA affinities awaits the results of the quantitative biophysical
studies which are in progress.
2.3. Conclusion
The new indoles show significant DNA binding affinities which
vary with structure consistent with that noted for other diami-
dines. The diamidines are highly active in vitro against both T. b.
r. and P. f. and are scheduled for in vivo evaluation. The fluorescent
Figure 2. Comparison of CD spectra of poly(dA)ꢁpoly(dT) in complex with different compounds at various mixing ratios. (A) CD titration spectra of poly(dA)ꢁpoly(dT) binding
with DAPI. (B) CD titration spectra of poly(dA)ꢁpoly(dT) binding with 16a. (C) CD titration spectra of poly(dA)ꢁpoly(dT) binding with 21b.

562
A. A. Farahat et al. / Bioorg. Med. Chem. 18 (2010) 557–566
Table 2
tained under computer control and are increased at 0.5 °C/min.
Fluorescence data for DAPI analogues
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 add 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.38
a,b
a,b
Compd
kex (nm)
kem (nm) Sw/ExSw 5/20
DAPI
16a
16b
16c
16d
21a
21b
24
341
343
348
368
290
341
348
340
458
465
461
514
425
511
461
454c
821
378
27
42
19
32
63
721
3.1.3. Circular dichroism (CD)
CD spectra were collected with 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 a de-
sired wavelength range. Compounds 16a and 21b at increasing ra-
tios were then titrated into the same cuvette and the complexes
rescanned under same conditions.39
a
b
c
Wavelenths(k) indicated are for excitation and emission.
Measurements were made in 0.001 M solution in distilled water.
kem obtained at Ex. sw/Em. sw 2.5/20.
3.2. Chemistry
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 sil-
ica gel 60 F254 precoated aluminum sheets using UV light for
detection. 1H and 13C NMR spectra were recorded on a Bruker
400 MHz spectrometer using the indicated solvents. Mass spectra
were obtained from the Georgia State University Mass Spectrome-
try Laboratory, Atlanta, GA. Elemental analysis were performed by
Atlantic Microlab Inc., Norcross, GA.
3.2.1. General procedure for the synthesis of4-[(trimethylsilyl)-
ethynyl] arylnitrile (3a–d)
A
dry flask was charged with 4-bromoarylnitrile (1a–d)
(55 mmol), PdCl2(PPh3)2 (2 g, 2.75 mmol), and CuI (1.3 g, 5.5 mmol).
After three successive nitrogen/vacuum cycles, degassed anhydrous
tetrahydrofuran (200 mL), trimethylsilylacetylene (2) (16.05 mL,
110 mmol), and degassed anhydrous triethylamine (250 mL) were
introduced via a syringe. Once triethylamine was added, the reaction
mixture turned black. The reaction mixture was stirred under a nitro-
gen atmosphere at room temperature for 24 h, passed through celite
to remove the catalyst, concentrated under vacuum, extracted with
ethyl acetate (3 ꢂ 100 mL), washed with ammonium chloride (10%
solution) to remove copper salts and then with water, dried over
magnesium sulfate and concentrated. Purification by column
chromatography on silica gel using hexanes/ethyl acetate (95/5, v/v).
Figure 3. Excitation spectra for DAPI and 21b are plotted as intensity (arbitary
units) versus wavelength. The free compound concentrations were 0.5 M,
poly(dA)ꢁpoly(dT) DNA was titrated into the cell at 0.1 increments. Both
l
lM
excitation and emission slit widths were 5 nm. The buffer was 10 mM cacodylic
acid, 1 mM EDTA and 0.1 M NaCl with the pH adjusted to 6.2 with NaOH prior to
dilution to the final volume. Excitation wavelength is the same as listed in Table 1.
DAPI gave the expected fluorescence enhancement but none of the DAPI analogues
gave any significant enhancement of fluorescence when bound to the AT DNA
polymer. All were similar to 21b in the Figure.
3.2.1.1. 4-[(Trimethylsilyl)ethynyl]benzonitrile (3a). Yellowish
brown solid (8.4 g, 83%), mp 109–111 °C, literature14 mp 109–
111 °C; spectroscopic data are consistent with those reported pre-
viously for this compound.
properties of the new molecules do not vary greatly from that of
DAPI.
3.2.1.2. 5-[(Trimethylsilyl)ethynyl]pyridine-2-carbonitrile (3b).
Yellow solid (9.47 g, 86%), mp 91 °C; 1H NMR (CDCl3, 400 MHz) d
8.39 (br s, 1H), 7.85 (d, 1H, J = 8 Hz), 7.64 (d, 1H, J = 8 Hz), 0.30 (s,
9H); 13C NMR (CDCl3, 400 MHz) d 153.6, 139.4, 132.1, 127.6,
123.9, 116.8, 103.7, 99.7, ꢀ0.4; ESI-MS: m/z calcd for C11H12N2Si:
200.31, found: 201.1 (M++1). Anal. Calcd for C11H12N2Si: C, 65.96;
H, 6.04; N, 13.98. Found: C, 66.22; H, 6.14; N, 13.82.
3. Experimental
3.1. Biology
3.1.1. In vitro activity determination
In vitro assays with T. b. r. STIB900 and P. f. K1 strain and cell
toxicity assays using cultured L6 rat myoblast cells were carried
out as previously reported.37
3.2.1.3. 6-[(Trimethylsilyl)ethynyl] pyridine-3-carbonitrile (3c).
White solid (10 g, 91%), mp 127 °C; 1H NMR (CDCl3, 400 MHz) d
8.82 (br s, 1H), 7.91 (dd, 1H, J = 2 Hz, J = 8 Hz), 7.53 (d, 1H,
J = 8 Hz), 0.23 (s, 9H); 13C NMR (CDCl3, 400 MHz) d 152.4, 146.2,
139.3, 126.9, 116.2, 108.6, 102, 100.7, ꢀ0.5; ESI-MS: m/z calcd for
C11H12N2Si: 200.31, found: 201.1 (M++1). Anal. Calcd for
C11H12N2Si: C, 65.96; H, 6.04; N, 13.98. Found: C, 66.12; H, 5.96;
N, 13.91.
3.1.2.
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-

A. A. Farahat et al. / Bioorg. Med. Chem. 18 (2010) 557–566
563
3.2.1.4. 2-Fluoro-4-[(trimethylsilyl)ethynyl]benzonitrile (3d).
Brown solid (9.32 g, 78%), mp 112 °C; 1H NMR (CDCl3, 400 MHz)
d 7.57–7.54 (m, 1H,), 7.33 (m, 2H), 0.27 (s, 9H); ESI-MS: m/z calcd
for C12H12FNSi: 217.31, found: 218.1 (M++1). Anal. Calcd for
C12H12FNSi: C, 66.32; H, 5.57; N, 6.45. Found: C, 66.51; H, 5.72;
N, 6.12.
sure. Purification by column chromatography on silica gel using
hexanes/ethyl acetate (60/40, v/v) gave yellow crystals (4.18 g,
92%), mp 142–142.5 °C; 1H NMR (CDCl3, 400 MHz) d 7.38 (d, 1H,
J = 8 Hz), 7.07 (dd, 1H, J = 1.6 Hz, J = 8 Hz), 6.97 (d,1H, J = 8 Hz),
3.95 (br s, 2H); 13C NMR (CDCl3, 400 MHz) d 137.1, 135, 131.2,
130.3, 126.5, 119.3, 112.5, 99, 95.1; ESI-MS: m/z calcd for
C9H6Br2N2: 301.97, found: 303 (M++1). Anal. Calcd for C9H6Br2N2:
C, 35.80; H, 2.00; N, 9.28. Found: C, 35.99; H, 2.27; N, 9.32.
3.2.2. General procedure for the synthesis of 4-ethynylarylni-
triles (4a–d)
A solution of tetrabutyl ammonium fluoride trihydrate (12.1 g,
35.2 mmol) in tetrahydrofuran (50 mL) was added to a solution
of the silylated compounds (3a–d) (32 mmol) in dichloromethane
(100 mL). The reaction mixture was stirred at 0 °C for 2 h, washed
twice with water, dried over magnesium sulfate and concentrated.
Purification by column chromatography on silica gel using hex-
anes/ethyl acetate (97/3, v/v).
3.2.5. Synthesis of 2-[(4-(cyanophenyl)ethynyl]-1H-indole-6-
carbonitrile (10)
A dry flask was charged with 4-ethynylbenzonitrile (4a) (0.63 g,
4.95 mmol), dibromoolefin (9) (1 g, 3.3 mmol), Pd/C (0.176 g,
0.165 mmol), CuI (0.063 g, 0.33 mmol) and 4-trimethoxytriphenyl-
phosphine (0.11 g, 0.33 mmol). After three successive nitrogen/
vacuum cycles, degassed anhydrous toluene (20 mL) and degassed
anhydrous isopropyl amine (0.72 mL, 8.25 mmol), were introduced
via a syringe. The reaction mixture was stirred at 100 °C under a
nitrogen atmosphere for 24 h, passed through celite to remove
the catalyst, concentrated under vacuum, extracted with ethyl ace-
tate (3 ꢂ 50 mL), washed with ammonium chloride (10% solution)
to remove copper salts and then with water, dried over magnesium
sulfate and concentrated. Purification by column chromatography
on silica gel using hexanes/dichloromethane (50/50, v/v) gave
brownish white crystals (0.13 g, 15%), mp 205–206 °C; 1H NMR
(DMSO-d6, 400 MHz) d 12.5 (s, NH); 7.94 (d, 2H, J = 8 Hz), 7.89
(br s, 1H), 7.79 (d, 2H, J = 8 Hz), 7.76 (d, 1H, J = 8 Hz), 7.40 (dd,
1H, J = 1.2 Hz, J = 8 Hz), 7.06 (s, 1H); 13C NMR (CDCl3, 400 MHz) d
135.8, 133.2, 132.2, 130.7, 126.6, 122.9, 122.2, 121.9, 120.6,
118.8, 116.7, 111.9, 109.6, 105.1, 92.4, 85.9; ESI-MS: m/z calcd
for C18H9N3: 267.28, found: 268.1 (M++1). Anal. Calcd for
C18H9N3: C, 80.88; H, 3.39; N, 15.72. Found: C, 80.64; H, 3.48; N,
15.49.
3.2.2.1. 4-Ethynylbenzonitrile (4a). Yellow solid (3.19 g, 78%),
mp 156–158 °C, literature14 mp 156–158 °C; spectroscopic data
are consistent with those reported previously for this compound.
3.2.2.2. 5-Ethynyl pyridine-2-carbonitrile (4b). Yellow solid
(3.36 g, 82%), mp 126–126.5 °C; 1H NMR (CDCl3, 400 MHz) d 8.78
(dd, 1H, J = 0.8 Hz, J = 2 Hz), 7.92 (dd, 1H, J = 2 Hz, J = 8 Hz), 7.69
(dd, 1H, J = 0.8 Hz, J = 8 Hz), 3.51 (s, 1H); 13C NMR (CDCl3,
400 MHz) d 153.7, 139.9, 132.6, 127.7, 122.9, 116.8, 84.9, 78.9;
ESI-MS: m/z calcd for C8H4N2: 128.13, found: 129.1 (M++1). Anal.
Calcd for C8H4N2: C, 74.99; H, 3.15; N, 21.86. Found: C, 75.12; H,
3.19; N, 21.92.
3.2.2.3. 6-Ethynyl pyridine-3-carbonitrile (4c). Greenish white
solid (3.03 g, 74%), mp 158 °C; 1H NMR (CDCl3, 400 MHz) d 8.56
(d, 1H, J = 2 Hz), 7.97 (dd, 1H, J = 2 Hz, J = 8 Hz), 7.59 (d, 1H,
J = 8 Hz), 3.44 (s, 1H); 13C NMR (CDCl3, 400 MHz) d 153.7, 139.9,
132.6, 127.7, 122.9, 116.7, 84.9, 78.9; ESI-MS: m/z calcd for
C8H4N2: 128.13, found: 129.0 (M++1). Anal. Calcd for C8H4N2: C,
74.99; H, 3.15; N, 21.86. Found: C, 74.77; H, 3.11; N, 21.89.
3.2.6. Synthesis of 1-(tert-butoxycarbonyl)-2-iodo-1H-indole-6-
carbonitrile (14)
A molecular iodine (25 g, 98.76 mmol) solution in dry tetrahydro-
furan (100 mL) was added slowly to a solution of the indole stann-
ane22 (13) (20 g, 49.38 mmol) in dry tetrahydrofuran (100 mL). The
mixture was stirred at room temperature for 6 h; the solvent was re-
moved under reduced pressure, the residue dissolved in ethyl ace-
tate (200 mL), washed with sodium thiosulfate solution (10%) to
remove any excess iodine, then with water, passed through sodium
sulfate and evaporated. Purification by column chromatography on
silica gel, using hexanes/ethyl acetate (97/3, v/v) gave white solid
(16 g, 88%), mp 72 °C; 1H NMR (CDCl3, 400 MHz) d 8.47 (d, 1H,
J = 1.2 Hz), 7.54 (dd, 1H, J = 0.8 Hz, J = 8.4 Hz), 7.46 (dd, 1H,
J = 1.2 Hz, J = 8.4 Hz), 7.07 (d, 1H, 0.8 Hz), 1.79 (s, 9H); 13C NMR
(CDCl3, 400 MHz) d 148.4, 136.4, 134, 126, 121.6, 120, 119.9, 119.7,
107.1, 86.8, 80, 28.2; ESI-MS: m/z calcd for C14H13IN2O2: 368.17,
found: 369.1 (M++1). Anal. Calcd for C14H13IN2O2: C, 45.67; H,
3.55; N, 7.60. Found: C, 45.91; H, 3.71; N, 7.35.
3.2.2.4. 4-Ethynyl-2-fluorobenzonitrile
(4d). Yellow
solid
(3.24 g, 70%), mp 120 °C; 1H NMR (CDCl3, 400 MHz) d 7.70–7.15
(br m, 3H), 3.4 (s, 1H); ESI-MS: m/z calcd for C9H4FN: 145.13,
found: 146.2 (M++1). Anal. Calcd for C9H4FN: C, 74.48; H, 2.78; N,
9.65. Found: C, 74.61; H, 2.89; N, 9.42.
3.2.3. Synthesis of 4-(2,2-dibromovinyl)-3-nitrobenzonitrile (8)
Triphenylphosphine (7.9 g, 30 mmol) was added portionwise to
an ice cold solution of 4-cyano-2-nitro-benzaldehyde15,16 (7)
(1.76 g, 10 mmol), carbon tetrabromide (5.0 g, 15 mmol) and zinc
powder (0.65 g, 10 mmol) in anhydrous methylene chloride
(100 mL), stirred overnight, filtered to remove triphenylphosphine
oxide and zinc bromide, concentrated, purification by column
chromatography on silica gel using hexanes/dichloromethane
(70/30, v/v) to give yellow crystals (2.95 g, 89%), mp 101–102 °C;
1H NMR (CDCl3, 400 MHz) d 8.43 (d, 1H, J = 1.6 Hz), 7.96 (dd, 1H,
J = 1.6 Hz, J = 8 Hz), 7.82 (d, 1H, J = 8 Hz), 7.81 (s, 1H); 13C NMR
(CDCl3, 400 MHz) d 136.1, 135.5, 132.9, 132.1, 128.6, 116.2,
113.7, 100, 95.9. Anal. Calcd for C9H4Br2N2O2: C, 32.56; H, 1.21;
N, 8.44. Found: C, 32.63; H, 1.32; N, 8.22.
3.2.7. General procedure for the synthesis of 1-(tert-butoxy-
carbonyl)-2-[4-(ethynylarylnitrile)]-1H-indole-6-carbonitrile
(15a–d)
A
dry flask was charged with 4-ethynylarylnitrile (4a–d)
(10.1 mmol), iodoindole derivative (14) (2.5 g, 6.79 mmol),
PdCl2(PPh3)2 (0.246 g, 0.33 mmol), CuI (0.12 g, 0.67 mmol), after
three successive nitrogen/vacuum cycles, degassed anhydrous tet-
rahydrofuran (30 mL) and degassed anhydrous triethylamine
(10 mL), were introduced via a syringe. The reaction mixture was
stirred at room temperature under a nitrogen atmosphere for
24 h. The reaction mixture was passed through celite to remove
the catalyst, concentrated under vacuum, extracted with ethyl
acetate (3 ꢂ 100 mL), washed with ammonium chloride (10%
3.2.4. Synthesis of 3-amino-4-(2,2-dibromovinyl)benzonitrile (9)
Pd/C (10%) (0.3 g) and ammonium metavanadate (0.017 g,
1 mol %) was added to a solution of the nitro compound (8) (5 g,
15 mmol) in toluene: ethyl acetate (30 mL: 10 mL) mixture. Shak-
ing in a Parr hydrogenator under 50 psi was continued until the
uptake of hydrogen ceased; the solution was passed through celite
to remove the catalyst, and then concentrated under reduced pres-

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A. A. Farahat et al. / Bioorg. Med. Chem. 18 (2010) 557–566
solution) to remove copper salts and then with water, dried over
magnesium sulfate and concentrated. Purification by column chro-
matography on silica gel using hexanes/ethyl acetate (75/25, v/v)
and then crystallization by hexanes/ethyl acetate mixture.
(s, 2H), 9.26 (s, 2H), 9.17 (s, 2H), 7.93 (br s, 1H), 7.92 (d, 2H,
J = 7.2 Hz), 7.78 (d, 2H, J = 7.2 Hz), 7.73 (d, 1H, J = 8 Hz), 7.5 (d,
1H, J = 8 Hz), 6.96 (s, 1H); 13C NMR (DMSO-d6, 400 MHz) d 166.8,
165.4, 136, 132.5, 131.4, 129.2, 128.6, 127.1, 122.4, 122.3, 121.4,
119.5, 112.7, 109.2, 92.4, 85.5; ESI-MS: m/z calcd for C18H15N5:
301.35, found: 302.20 (amidine base M++1). Anal. Calcd for
C18H15N5ꢁ2HClꢁ3.1H2O: C, 50.35; H, 5.45; N, 16.32. Found: C,
50.00; H, 5.22; N, 16.69.
3.2.7.1. 1-(tert-Butoxycarbonyl)-2-[(4-cyanophenyl)ethynyl]-1-H-
indole-6-carbonitrile (15a). White solid (1.74 g, 70%), mp 175–
176 °C; 1H NMR (CDCl3, 400 MHz) d 8.54 (d, 1H, J = 1.6 Hz), 7.71
(d, 2H, J = 6.8 Hz), 7.67 (d, 2H, J = 6.8 Hz), 7.64 (d, 1H, J = 8 Hz),
7.52 (dd, 1H, J = 1.6 Hz, J = 8 Hz), 7.07 (s, 1H), 1.74 (s, 9H); 13C
NMR (CDCl3, 400 MHz) d 148.6, 135.2, 132.2, 132, 131.6, 127.2,
126.3, 123.1, 121.6, 120.3, 119.7, 118.2, 116.5, 112.3, 108.6, 94.9,
85.9, 85, 28.2; ESI-MS: m/z calcd for C23H17N3O2: 367.40, found:
368.2 (M++1). Anal. Calcd for C23H17N3O2: C, 75.18; H, 4.66; N,
11.43. Found: C, 74.98; H, 4.79; N, 11.12.
3.2.8.2. 2-[(6-Amidino-3-pyridyl)ethynyl]-6-amidino-1H-indole
dihydrochloride (16b). Yellow solid (0.115 g, 40%), mp 273–
276 °C 1H NMR (DMSO-d6, 400 MHz) d 12.75 (s, 1H), 9.78 (br,
2H), 9.62 (br, 2H), 9.40 (br, 2H), 9.20 (br, 2H), 9.03 (br s, 1H),
8.48 (d, 1H, J = 8.4 Hz), 8.39 (d, 1H, J = 8.4 Hz),, 8.03 (br s, 1H),
7.81 (d, 1H, J = 8.4 Hz),, 7.51 (d, 1H, J = 8.4 Hz),, 6.99 (s, 1H); 13C
NMR (DMSO-d6, 400 MHz) d 166.7, 161.8, 151.8, 143.5, 140.7,
136.2, 131.3, 123.7, 123.5, 122.8, 121.6, 121.4, 119.6, 112.9,
109.8, 89.7, 88.7; ESI-MS: m/z calcd for C17H14N6: 302.33, found:
303.20 (amidine base M++1). Anal. Calcd for C17H14N6ꢁ-
2HClꢁ1.65H2O: C, 50.41; H, 4.80; N, 20.75. Found: C, 50.55; H,
4.75; N, 20.37.
3.2.7.2. 1-(tert-Butoxycarbonyl)-2-[(6-cyano-3-pyridyl)ethynyl]-
1H-indole-6-carbonitrile (15b). Yellow solid (1.8 g, 72%), mp
175 °C; 1H NMR (CDCl3, 400 MHz) d 8.89 (br s, 1H), 8.53 (br s,
1H), 7.99 (brd, 1H, J = 8 Hz), 7.74 (d, 1H, J = 8 Hz), 7.67 (d, 1H,
J = 8 Hz), 7.53 (d, 1H, J = 8 Hz), 7.13 (s, 1H), 1.74 (s, 9H); 13C NMR
(CDCl3, 400 MHz) d 153, 148.4, 139, 135.2, 132.4, 131.5, 127.8,
126.4, 123.3, 122.4, 121.8, 120.4, 119.6, 117.2, 116.8, 109, 91.8,
88.4, 86.2, 28.2; ESI-MS: m/z calcd for C22H16N4O2: 368.38, found:
369.2 (M++1). Anal. Calcd for C22H16N4O2: C, 71.73; H, 4.38; N,
15.21. Found: C, 71.65; H, 4.77; N, 14.86.
3.2.8.3. 2-[(5-Amidino-2-pyridyl)ethynyl]-6-amidino-1H-indole
(16c) dihydrochloride. Yellow solid (0.111 g, 39%), mp 249–
252 °C 1H NMR (DMSO-d6, 400 MHz) d 12.7 (br s, 1H), 9.64 (br s,
2H), 9.37 (br s,4H), 9.09 (d, 1H, 2.4 Hz), 9.06 (br, 2H), 8.34 (dd,
1H, J = 2.4 Hz, J = 8.4 Hz), 8.19 (d, 1H, J = 8.4 Hz), 8.0 (s, 1H), 7.96
(br s, 1H), 7.83 (d, 1H, J = 8.4), 7.48 (dd, 1H, J = 1.6, J = 8.4 Hz),
7.19 (s, 1H); 13C NMR (DMSO-d6, 400 MHz) d 166.7, 160.3, 151.3,
143.5, 140, 135.2, 131.3, 124.2, 123.5, 122.8, 122.2, 120.3, 119,
111.9, 109.8, 90.2, 88.1; ESI-MS: m/z calcd for C17H14N6: 302.33,
found: 303.22 (amidine base M++1). Anal. Calcd for C17H14N6ꢁ-
3HClꢁ1.75H2O: C, 46.19; H, 4.67; N, 19.02. Found: C, 46.29; H,
4.71; N, 18.76.
3.2.7.3. 1-(tert-Butoxycarbonyl)-2-[(5-cyano-2-pyridyl)ethynyl]-
1H-indole-6-carbonitrile (15c). Yellow solid (1.72 g, 69%), mp
186 °C; 1H NMR (CDCl3, 400 MHz) d 8.92 (d, 1H, J = 2.4 Hz), 8.55 (s,
1H), 8.01 (dd, 1H, J = 2.4 Hz, J = 8.4 Hz), 7.69 (d, 1H, J = 8.4 Hz), 7.67
(d, 1H, J = 8.8 Hz), 7.53 (dd, 1H, J = 1.2 Hz, J = 8.8 Hz), 7.18 (s, 1H),
1.74 (s, 9H); 13C NMR (CDCl3, 400 MHz) d 152.6, 147.5, 139, 137.1,
132.4, 132, 127.8, 126.9, 123.3, 123.1, 121.2, 120.4, 118.9, 117, 116,
108.8, 91.8, 89.1, 86.7, 28.1; ESI-MS: m/z calcd for C22H16N4O2:
368.38, found: 369.2 (M++1). Anal. Calcd for C22H16N4O2: C, 71.73;
H, 4.38; N, 15.21. Found: C, 71.65; H, 4.29; N, 15.17.
3.2.8.4. 2-[(4-Amidino-3-fluorophenyl)ethynyl]-6-amidino-1H-
indole dihydrochloride (16d). Brown solid (0.144 g, 51%), mp
258–260 °C 1H NMR (DMSO-d6, 400 MHz) d 12.55 (s, 1H), 9.55 (br
s, 2H), 9.37 (br s, 2H), 9.29 (br s, 2H), 8.96 (br s, 2H), 7.9 (br s, 1H),
7.79–7.73 (m, 2H), 7.72 (d, 1H, J = 1.2 Hz), 7.61 (dd, 1H, J = 1.6 Hz,
J = 8 Hz), 7.45 (dd, 1H, J = 1.6 Hz, J = 8 Hz), 6.03 (d, 1H, J = 1.2 Hz);
ESI-MS: m/z calcd for C18H14FN5: 319.34, found: 320.20 (amidine
base M++1). Anal. Calcd for C18H14FN5ꢁ2HClꢁ2.5H2O: C, 49.43; H,
4.84; N, 16.01. Found: C, 49.51; H, 4.93; N, 15.77.
3.2.7.4. 1-(tert-Butoxycarbonyl)-2-[(4-cyano-3-fluorophenyl)ethy-
nyl]-1H-indole-6-carbonitrile (15d). Brown solid (2 g, 80%), mp
185 °C; 1H NMR (CDCl3, 400 MHz) d 8.52 (s, 1H), 7.72–7.63 (m,
2H), 7.52 (dd, 1H, J = 1.2 Hz, J = 8 Hz), 7.46 (dd, 1H, J = 1.2 Hz,
J = 8 Hz), 7.41 (dd, 1H, J = 1.2 Hz, J = 8 Hz), 7.10 (s, 1H), 1.73 (s,
9H); ESI-MS: m/z calcd for C23H16FN3O2: 385.39, found: 385.00
(M+). Anal. Calcd for C23H16FN3O2: C, 71.68; H, 4.18; N, 10.90.
Found: C, 71.39; H, 4.15; N, 10.82.
3.2.9. Synthesis of 40-bromobiphenyl-4-carbonitrile (19)
5 mL deaireated 2 M aqueous solution of Na2CO3 and 4-bromo-
phenyl boronic acid (17) (1 g, 5 mmol) in 5 mL deaireated metha-
nol were added to a stirred solution of 4-iodobenzonitrile (18)
(1.14 g, 5 mmol), and Tetrakistriphenylphosphine palladium
(0.288 g, 0.25 mmol) in deaireated toluene (20 mL) under a nitro-
gen atmosphere. The vigorously stirred mixture was warmed to
80 °C for 24 h. Evaporation of the solvent under reduced pressure,
the solid was partitioned between ethyl acetate (200 mL) and 2 M
aqueous Na2CO3 solution (25 mL) containing 5 mL of concentrated
ammonia, to destroy the palladium complex, then washed with
water, passed through celite to remove the catalyst, and finally
passed through sodium sulfate, and evaporated. Purification by
column chromatography on silica gel, using hexanes/ethyl acetate
(85/15, v/v) gave white solid (1 g, 79%), mp 155–155.5 °C 1H NMR
(DMSO-d6, 400 MHz) d 7.9 (m, 4H), 7.7 (br s, 4H); 13C NMR (DMSO-
d6, 400 MHz) d 143.3, 137.4, 132.9, 132, 129.1, 127.5, 122.4, 118.7,
110.4. Anal. Calcd for C13H8BrN: C, 60.49; H, 3.12; N, 5.43. Found: C,
60.33; H, 2.99; N, 5.15.
3.2.8. General procedure for the synthesis of 2-((4-amidino-
aryl)ethynyl)-6-amidino-1H-indole dihydrochlorides (16a–d)
The dinitriles (15a–d) (0.66 mmol) were suspended in freshly
distilled THF (5 mL), and treated with lithium trimethylsilylamide
1 M solution in tetrahydrofuran (4 mL, 3.98 mmol), the mixture
was stirred for three days at room temperature. The reaction mix-
ture was then cooled to 0 °C and HCl saturated ethanol (2 mL) was
added. 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 1N sodium hydroxide solution
followed by filtration of the resultant solid and washing with water
and drying. Finally, the free base was stirred with ethanolic HCl for
one week to make sure that the (Boc)2O group was completely re-
moved, diluted with ether, and the solid formed was filtered and
dried to give the diamidine salt.
3.2.8.1. 2-((4-Amidinophenyl)ethynyl)-6-amidino-1H-indole di-
hydrochloride (16a). Yellow solid (0.113 g, 40%), mp 260–262 °C
1H NMR (DMSO-d6, 400 MHz) d 12.65 (s, 1H), 9.58 (s, 2H), 9.34

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565
3.2.10. General procedure for the synthesis of 1-(tert-butoxycar-
bonyl)-2-(40-cyanobiphenyl-4-yl)-1H-indole-5 or-6-carbonitrile
(20a,b)
3.2.11.2. 2-(40-Amidinobiphenyl-4-yl)-6-amidino-1H-indole dihy-
drochloride (21b). Yellow solid (0.154 g, 51%), mp >300 °C 1H
NMR (DMSO-d6, 400 MHz) d 12.65 (s, 1H), 9.56 (s,2H), 9.36
(s,4H), 9.13 (s, 2H), 8.17 (d, 2H, J = 7.2 Hz), 8.07–7.96 (m, 5H),
7.88 (d, 2H, J = 7.2 Hz), 7.76 (d, 1H, 8.4 Hz), 7.48 (d, 1H,
J = 8.4 Hz), 7.19 (s, 1H); 13C NMR (DMSO-d6, 400 MHz) d 165.7,
165.4, 147, 138.6, 137.8, 137.3, 133.3, 129.4, 129.3, 127.3, 126.6,
126.3, 125.6, 121.7, 119.8, 110.5, 101.7; ESI-MS: m/z calcd for
C22H19N5: 353.4, found: 354.2 (amidine base M++1). Anal. Calcd
for C22H19N5ꢁ2HClꢁ1.85H2O: C, 57.48; H, 5.41; N, 15.23. Found: C,
57.12; H, 5.63; N, 14.96.
Tetrakistriphenylphosphine palladium (0.288 g, 0.25 mmol)
were added to a stirred mixture of the N-BOC-5 or 6-cyanoindole
stannane (2.02 g, 5 mmol) and the 40-bromobiphenyl-4-carboni-
trile (19) (1.29 g, 5 mmol) in deaireated dioxane (20 mL) under a
nitrogen atmosphere. The vigorously stirred mixture was warmed
to 90–100 °C for 24 h. Evaporation of the solvent under reduced
pressure, the solid was partitioned between ethyl acetate
(200 mL) containing 5 mL of concentrated ammonia to destroy
the palladium complex, then washed with water, passed through
celite to remove the catalyst, and finally passed through sodium
sulfate, and evaporated. Purification by column chromatography
on silica gel, using hexanes/ethyl acetate (80/20, v/v).
3.2.12. 1-(tert-Butoxycarbonyl)-2-(biphenyl-4-yl))-1H-indole-
6-carbonitrile (23)
Tetrakistriphenylphosphine palladium (0.288 g, 0.25 mmol)
were added to a stirred mixture of the N-BOC-6-cyanoindole
stannane (13) (2.02 g, 5 mmol) and the 4-bromobiphenyl (22)
(1.16 g, 5 mmol) in deaireated dioxane (20 mL) under a nitrogen
atmosphere. The vigorously stirred mixture was warmed to 90–
100 °C for 24 h. Evaporation of the solvent under reduced pressure,
the solid was partitioned between ethyl acetate (200 mL) contain-
ing 5 mL of concentrated ammonia to destroy the palladium com-
plex, then washing with water, passed through celite to remove the
catalyst, and finally passed through sodium sulfate, and evapo-
rated. Purification by column chromatography on silica gel, using
hexanes/ethyl acetate (90/10, v/v). Afforded white solid (1.13 g,
58%), mp 192–193 °C 1H NMR (CDCl3, 400 MHz) d 8.62 (s, 1H),
7.17–7.64 (m, 5H), 7.54–7.49 (m, 5H), 7.27 (dd, 1H, J = 7.2 Hz),
6.68 (s, 1H), 1.38 (s, 9H).; 13C NMR (CDCl3, 400 MHz) d 149.5,
143.8, 141.3, 140.4, 136.5, 132.7, 132.5, 129.2, 128.9, 127.7127.1,
126.1, 121.2, 120.2, 119.9, 109.7, 106.9, 84.9, 27.5; ESI-MS: m/z
calcd for C26H22N2O2: 394.47, found: 395.3 (M++1). Anal. Calcd
for C26H22N2O2: C, 79.16; H, 5.62; N, 7.10. Found: C, 79.1; H,
5.65; N, 7.18.
3.2.10.1. 1-(tert-Butoxycarbonyl)-2-(40-cyanobiphenyl-4-yl)-1H-
indole-5-carbonitrile (20a). White solid (0.91 g, 45%), mp 191–
192 °C 1H NMR (DMSO-d6, 400 MHz) d 8.09 (br s, 1H), 8.06–7.87
(m, 8H), 7.58 (d, 1H, J = 8.1 Hz), 7.47 (d, 1H, J = 8.1 Hz), 7.16 (s,
1H), 1.51 (s, 9H); 13C NMR (DMSO-d6, 400 MHz) d 148.7, 144.2,
139.9, 139.4, 138.1, 133.3, 131.9, 128.8, 128.1, 127.8, 126.4,
126.1, 125, 121.1, 119.3, 113, 110.6, 102.1, 100.5, 84.3, 27.2; ESI-
MS: m/z calcd for C27H21N3O2: 419.47, found: 420.2 (M++1). Anal.
Calcd for C27H21N3O2: C, 77.31; H, 5.05; N, 10.02. Found: C,
77.35; H, 5.31; N, 9.72.
3.2.10.2. 1-(tert-Butoxycarbonyl)-2-(40-cyanobiphenyl-4-yl)-1H-
indole-6-carbonitrile (20b). White solid (1 g, 49%), mp 202–
202.5 °C 1H NMR (DMSO-d6, 400 MHz) d 8.59 (s, 1H), 7.82 (m,
4H), 7.70 (d, 2H, J = 8.0 Hz), 7.66 (d, 1H, J = 8.4 Hz), 7.58 (d, 2H,
J = 8.0 Hz), 7.54 (d, 1H, J = 8.0 Hz), 6.70 (s, 1H), 1.40 (s, 9H).; 13C
NMR (DMSO-d6, 400 MHz) d 149.3, 144.8, 143.1, 139.1, 136.4,
134.1, 132.7, 132.4, 129.5, 127.6, 126.8, 126.2, 121.3, 120, 119.9,
118.8, 111.3, 110.1, 107.2, 85, 27.6; ESI-MS: m/z calcd for
C27H21N3O2: 419.47, found: 420.2 (M++1). Anal. Calcd for
C27H21N3O2: C, 77.31; H, 5.05; N, 10.02. Found: C, 77.29; H, 5.15;
N, 9.89.
3.2.13. 2-(Biphenyl-4-yl)-6-amidino-1H-indole hydrochloride
(24)
The mononitrile (23) (0.26 g, 0.66 mmol) was suspended in
freshly distilled THF (5 mL), and treated with lithium trim-
ethylsilylamide 1 M solution in tetrahydrofuran (2 mL, 1.99 mmol),
the mixture was stirred for three days at room temperature. The
reaction mixture was then cooled to 0 °C and HCl saturated ethanol
(1 mL) was added. 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 solution followed by filtration of the resultant solid and wash-
ing with water and drying. Finally, the free base was stirred with
ethanolic HCl for one week to make sure that the (Boc)2O group
was completely removed, diluted with ether, and the solid formed
was filtered and dried to give the diamidine salt as yellow solid
(0.133 g, 55%), mp >280 dec °C 1H NMR (DMSO-d6, 400 MHz) d
12.40 (s, 1H), 9.30 (s, 2H), 8.98 (s, 2H), 8.08 (d, 2H, J = 8.4 Hz),
7.96 (s, 1H), 7.84 (d, 2H, J = 8.4 Hz), 7.79–740 (m, 3H), 7.55–7.38
(m, 4H), 7.15 (s, 1H); 13C NMR (DMSO-d6, 400 MHz) d 166, 144.8,
140.5, 137.1, 136.9, 132.8, 129.1, 128.7, 127.6, 126.6, 125.8,
125.4, 121.3, 120.1, 109.4, 100.1; ESI-MS: m/z calcd for C21H17N3:
311.38, found: 312.2 (amidine base M++1). Anal. Calcd for
C21H17N3ꢁ1HClꢁ1.15H2O: C, 68.43; H, 5.55; N, 11.4. Found: C,
68.11; H, 5.4; N, 11.13.
3.2.11. General procedure for the synthesis of 2-(40-amidinobi-
phenyl-4-yl)-5 or 6-amidino-1H-indole dihydrochlorides (21a,b)
The dinitriles (20a,b) (0.276 g, 0.66 mmol) were suspended in
freshly distilled THF (5 mL), and treated with lithium trim-
ethylsilylamide 1 M solution in tetrahydrofuran (4 mL, 3.98 mmol),
the mixture was stirred for three days at room temperature. The
reaction mixture was then cooled to 0 °C and HCl saturated ethanol
(2 mL) was added. 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 1N sodium hydrox-
ide solution followed by filtration of the resultant solid and wash-
ing with water and drying. Finally, the free base was stirred with
ethanolic HCl for one week to make sure that the (Boc)2O group
was completely removed, diluted with ether, and the solid formed
was filtered and dried to give the diamidine salt.
3.2.11.1. 2-(40-Amidinobiphenyl-4-yl)-5-amidino-1H-indole dihy-
drochloride (21a). Yellow solid (0.126 g, 42%), mp >300 °C 1H
NMR (DMSO-d6, 400 MHz) d 12.55 (s, 1H),), 9.55 (s, 2H), 9.30
(br s, 4H), 9.06 (s, 2H), 8.22–7.81 (m, 8H), 7.63 (br s, 2H), 7.23
(s, 1H); 13C NMR (DMSO-d6, 400 MHz) d 166.9, 165.6, 144.8,
140.6, 138, 132, 129.3, 128.7, 128, 127.3, 126.7, 126.2, 121.8,
121.6, 119.1, 112.3, 100.8, 100.5; ESI-MS: m/z calcd for C22H19N5:
353.4, found: 354.2 (amidine base M++1). Anal. Calcd for
C22H19N5ꢁ2HClꢁ2.45H2O: C, 56.16; H, 5.54; N, 14.88; Found: C,
56.33; H, 5.67; N, 14.62.
Acknowledgments
This work was supported by the Egyptian government through
the channel program (AAF) and by an award from the Bill and Me-
linda Gates Foundation (RB, WDW, DWB).

566
A. A. Farahat et al. / Bioorg. Med. Chem. 18 (2010) 557–566
C.; Colson, J.; Stenek, J.; Brun, R.; Neidle, S.; Wilson, W. D. Biophys. J. 2004, 86,
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