Exploration of DAPI analogues: Synthesis, antitrypanosomal activity, DNA binding and fluorescence properties

Article abstract of DOI:10.1016/j.ejmech.2017.01.037

The DAPI structure has been modified by replacing the phenyl group with substituted phenyl or heteroaryl rings. Twelve amidines were synthesized and their DNA binding, fluorescence properties, in?vitro and in?vivo activities were evaluated. These compounds are shown to bind in the DNA minor groove with high affinity, and exhibit superior in?vitro antitrypanosomal activity to that of DAPI. Six new diamidines (5b, 5c, 5d, 5e, 5f and 5j) exhibit superior in?vivo activity to that of DAPI and four of these compounds provide 100% animal cure at a low dose of 4?×?5?mg/kg i.p. in T.?b. rhodesiense infected mice. Generally, the fluorescence properties of the new analogues are inferior to that of DAPI with the exception of compound 5i which shows a moderate increase in efficacy while compound 5k is comparable to DAPI.

Full text of DOI:10.1016/j.ejmech.2017.01.037

European Journal of Medicinal Chemistry 128 (2017) 70e78
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Exploration of DAPI analogues: Synthesis, antitrypanosomal activity,
DNA binding and fluorescence properties
,
b
,
, d
Abdelbasset A. Farahat a *, Arvind Kumar a, Martial Say a, Tanja Wenzler c
,
Reto Brun c d, Ananya Paul 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, Mansoura University, Mansoura 35516, Egypt
c Swiss Tropical and Public Health Institute, Basel 4002, Switzerland
d University of Basel, Basel, 4003, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The DAPI structure has been modified by replacing the phenyl group with substituted phenyl or het-
eroaryl rings. Twelve amidines were synthesized and their DNA binding, fluorescence properties, in vitro
and in vivo activities were evaluated. These compounds are shown to bind in the DNA minor groove with
high affinity, and exhibit superior in vitro antitrypanosomal activity to that of DAPI. Six new diamidines
(5b, 5c, 5d, 5e, 5f and 5j) exhibit superior in vivo activity to that of DAPI and four of these compounds
provide 100% animal cure at a low dose of 4 ꢀ 5 mg/kg i.p. in T. b. rhodesiense infected mice. Generally,
the fluorescence properties of the new analogues are inferior to that of DAPI with the exception of
compound 5i which shows a moderate increase in efficacy while compound 5k is comparable to DAPI.
Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
Received 12 December 2016
Received in revised form
17 January 2017
Accepted 23 January 2017
Available online 24 January 2017
Keywords:
Diamidines
Stille coupling
Pinner reaction
DAPI
DNA minor groove binders
Antitrypanosomal activity
Fluorescence properties
1. Introduction
eflornithine (NECT). The treatment selection is based on the sub-
species of T. brucei and on the disease stage. All drugs have disad-
Human African trypanosomiasis also known as sleeping sick-
ness is a neglected tropical disease in Sub-Saharan Africa caused by
two subspecies of Trypanosoma brucei (T. b. gambiense and T. b.
rhodesiense). It threatens over 65 million people among the poorest
in the world in 36 countries [1a]. The trypanosomes are transmitted
mainly by blood-feeding tsetse flies. Once infected, the disease
generally progresses through two stages which are defined by the
location of the parasites in the patient. In the first hemolymphatic
stage the parasites reside in the blood and lymph system whereas
in the second meningoencephalitic stage the parasites invade
additionally the central nervous system [1b]. It is generally
accepted that without an effective treatment the disease is lethal.
Drugs currently in use for sleeping sickness are pentamidine, sur-
vantages such as difficult administration, serious side effects or
resistance issues. Better drugs are needed to control or eliminate
sleeping sickness. There are two new molecules, fexinidazole and
the oxaborole SCYX-7158 at the stage of clinical development [1c].
However, due to the high attrition rate in drug development it is
essential to continue with drug discovery to find new drugs to cure
sleeping sickness.
Dicationic amidines that bind in the DNA minor groove are
promising agents against African trypanosomiasis. So far, pent-
amidine (Fig. 1) is the only one of this class which has seen sig-
nificant human clinical use [2]. However, it is the most frequently
used drug for the treatment of first stage sleeping sickness since
almost 8 decades and it is still effective by a parenteral route of
application.
amin, melarsoprol, and
a combination of nifurtimox and
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 [3]. DAPI has been subsequently found
to exhibit a variety of other biological effects, including antifungal,
antibacterial, and antiviral activities [3,4]. Also, DAPI is a fluorescent
* Corresponding author. Department of Chemistry, Georgia State University,
Atlanta, GA 30303, United States.
E-mail address: afarahat@gsu.edu (A.A. Farahat).
http://dx.doi.org/10.1016/j.ejmech.2017.01.037
0223-5234/Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

A.A. Farahat et al. / European Journal of Medicinal Chemistry 128 (2017) 70e78
71
Fig. 1. Antitrypanosomal diamidines.
dye which exhibits several binding modes to DNA [5] and it has
been widely utilized as a DNA specific probe for flow cytometry,
chromosome staining, DNA visualization and quantitation [6], and
has become now an important tool in molecular biology. We pre-
viously have reported some phenyl acetylene derivatives of DAPI
(compound I, Fig.1), that showed strong DNA binding and exhibited
potent in vitro activity superior to that of DAPI [7]. In a related
investigation, we extended the overall length of DAPI by adding one
phenyl group at the 5 or 6 position of indole and replacing the
phenyl group of DAPI with different aryl and heteroaryls (com-
pound II, Fig. 1), these diamidines showed strong DNA binding
comparable to that of DAPI and showed stronger in vitro anti-
trypanosomal activity with a comparable or stronger in vivo activity
than that of DAPI [8]. Moreover, we recently studied the replace-
ment of the indole ring of DAPI with a benzimidazole ring (com-
pound III, Fig. 1), these benzimidazole derivatives showed good
in vitro activity but exhibited very limited in vivo activity [9], which
demonstrated the importance of the indole ring of DAPI for the
antitrypanosomal activity. Here we report the replacement of the
phenyl ring of DAPI with different substituted phenyl and hetero-
aryl rings in anticipation of finding enhanced in vivo activity and
physicochemical properties.
furnish the dihydrochloride salts of the diamidines 5a-k.
Scheme 2 illustrates the synthesis of the monoamidine 8. The
commercially available 1-bromo-4-methoxybenzene was coupled
with the indole stannane 2a utilizing Stille coupling conditions to
afford the mononitrile 7 in high yield. This mononitrile was con-
verted to the monoamidine hydrochloride 8 by utilizing the Pinner
methodology [12] by stirring in dry ethanolic HCl to yield the
imidate ester hydrochloride which was separated, dried and sus-
pended in ethanolic ammonia to furnish the monoamidine 8.
2.2. Biology
Table 1 summarizes the DNA binding affinities for the twelve
new amidino analogues of DAPI as well as both the in vitro and
in vivo activities of these compounds against Trypanosoma brucei
rhodensiense (T. b. r.). For comparative purposes, data for DAPI are
included. The thermal melting increase
DTm (Tm of DNA-ligand
complex e Tm of free DNA) is a rapid and reliable method for
ranking binding affinities for large numbers of aryldiamidines [13].
The
DTm values for the complexes between poly (dA-dT) and the
new analogues vary with the change in the substitution of the 2-
aryl group and ranges from 10 to >26 ꢂC. The methyl 5b, methoxy
5d and the o-pyridyl 5e analogues exhibit high D
Tm values (>26 ꢂC)
2. Results and discussion
comparable to that of DAPI. These three compounds have the
amidine group at the 6-position of indole, while their isomers with
the amidine group at 5-position (5a, 5c, 5f) of indole exhibit lower
2.1. Chemistry
DTm values. We hypothize that this decrease in DNA affinity is likely
Scheme 1 describes the preparation of the diamidines 5a-k in
good yield. 5 (6)-Cyanoindoles 1a,b were protected by employing
di-tert-butyldicarbonate in dichloromethane using 4-(dimethyla-
mino)pyridine as a catalyst. Lithiation of the Boc-protected indoles
2a,b was readily achieved with lithium diisopropylamide (LDA) in
anhydrous tetrahydrofuran at ꢁ20 ꢂC. Subsequent reaction of the
lithioindole intermediate with trimethyltin chloride gave the
indole stannanes 3a,b in good yield [10]. The stannane in-
termediates were allowed to react with different bromoaryl and
heteroaryl benzonitriles under Stille coupling conditions in the
presence of 5 mol % Pd(PPh3)4 using dioxane as a solvent to afford
the bisnitriles 4a-k in good yield. The bisnitriles 4a-k were allowed
to react with lithium bis(trimethylsilyl)amide [11] in THF, followed
by deprotection of the silylated amidines with ethanolic HCl to
due to the fact that in the complex 6- amidino indole can form a
strong hydrogen bond involving the indole NH to a thymine
carbonyl as has been previously reported [7]. While in the analo-
gous complex for the 5-amidino indole such is not possible as the 3-
position CeH is pointed into the groove. Removal of the amidine
groups from DAPI as in the monoamidine 8 results in a big reduc-
tion of binding affinity consistent with the loss of one key cationic
binding center.
Generally, heterocyclic diamidines have been found to bind in
the DNA minor groove in AT base pair sequences of DNA [14].Cir-
cular dichroism (CD) spectroscopy is 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 complex formation. Fig. 2 shows the CD data

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A.A. Farahat et al. / European Journal of Medicinal Chemistry 128 (2017) 70e78
Scheme 1. Reagents and conditions. (a) Boc2O, DMAP, CH2Cl2; (b) ClSn(CH3)3, LDA/THF; (c) Br-Ar-CN, Pd(PPh3)4, Dioxane (d) 1-LiN(TMS)2/THF,2-HCl gas/EtOH.
Scheme 2. Reagents and conditions: (a) Pd(PPh3)4, Dioxane (b) i-HCl gas/EtOH.ii-NH3/EtOH.
for the interaction between DNA and 5d, 5e and 5j along with that
of DAPI as a reference compound. Minor groove binders yield a
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
Fig. 2(a). The expected positive signal in the compound absorption
region with relatively small changes in the DNA region are
observed. The results shown in Fig. 2 shows that the CD data are
compound 8 (SI ¼ 6), which was in vitro inactive versus T. b. r.
In view of the high in vitro activity and selectivity of these new
amidines, they were evaluated for efficacy in the acute stage of
infection in the rigorous T. b. r. STIB900 mouse model (Table 1). The
compounds were tested by daily intraperitoneal dosage of 5 mg/kg
for four consecutive days. Four of the twelve new compounds (5d,
5e, 5f and 5j) cured all 4 infected mice and gave 100% cure rate. Two
compounds (5b, 5c) cured 3 out of 4 mice and gave 75% cure rate,
while 5g and 5h gave 25% cure rate. Compound 5i was toxic killing
2 out of 4 mice and did not cure any of the two remaining mice. The
in vivo results for six compounds (5b, 5c, 5d, 5e, 5f and 5j) are
superior to that of DAPI and pentamidine that is in clinical use for
1st stage HAT since 80 years (both cure 2 out of 4 mice at 4 ꢀ 5 mg/
kg i.p.) in the STIB900 acute mouse model and therefore the six new
compounds merit further evaluation as potential drug candidates
for HAT.
consistent with the
D Tm results for 5d, 5e and 5j. Reflecting strong
binding to DNA, compound 5d exhibits large induced CD spectra in
its absorption regions that are higher than that for DAPI while 5e
and 5j exhibit comparable CD spectra to that of DAPI. This data
demonstrates the DNA minor groove binding of these compounds.
DAPI exhibits excellent in vitro activity against T. b. r (18 nM). All
of the newly synthesized compounds, except 5e and 8, showed an
in vitro activity stronger than that of DAPI ranging from a 2e6 fold
increase in activity. All the newly synthetized compounds showed
good selectivity indices ranging from 133 to 7833 indicating that
they are clearly less cytotoxic than DAPI (SI ¼ 48) with exception of
The widespread use of DAPI in molecular biology is in large part
due to its fluorescence properties. During the study of these new
indole amidines we observed that several of the compounds were

Table 1
DNA binding, in vitro and in vivo antitrypanosomal activity of the DAPI analogues.
Compound
Code
DAPI
D
Tma (ꢂC)
Cytotoxb
0.86
(
m
M)
T. b. rc (nM)
18
SId
48
In vivof(cures)
>27
2/4
NH
HN
NH2
N
H
NH2
5a
5b
19.2
1.7
3
5
566
0/4
3/4
H
N
NH2
NH
H2N
NH
>26
15.3
>26
15.7
3140
H3C
NH
HN
NH2
N
H
NH2
5c
23.5
29.6
3
6
7833
4876
3/4
4/4
H3C
H
N
NH2
NH
H2N
NH
5d
5e
5f
22.1
>26
15.3
12.1
3.2
24
4
133
4/4
4/4
1/4
1/4
NH
HN
NH2
N
N
H
NH2
21.3
2.0
5325
666.7
394
5g
5h
3
NH
HN
NH2
N
H
N
NH2
1.8
5
5i
5j
11
7.2
2.7
9
3
800
900
0/2
4/4
HN
NH
NH2
N
H
O
NH2
21.4
H
N
H2N
NH
NH2
O
HN
5k
8
10
4.5
29
11
409
6
0/4
HN
NH
NH2
N
S
H
NH2
12.6
4869
NDe
a
Increase in thermal melting of poly (dA-dT)n.
b
c
d
e
f
Cytotoxicity was evaluated using cultured L6 rat myoblast cells.
The T. b. r. (Trypanosoma brucei rhodesiense) strain was STIB900. The values are the average of duplicate determinations.
Selectivity Index for T. b. r. expressed as the ratio: IC50 (L6)/IC50 (T.b.r.).
Not determined as in vitro inactive.
In vivo efficacy was determined in T. b. rhodesiense (STIB900) infected mice at 4 ꢀ 5 mg/kg i.p. dose.

74
A.A. Farahat et al. / European Journal of Medicinal Chemistry 128 (2017) 70e78
fluorescent in the visible region. Therefore we decided to record the
fluorescence data for the new compounds and compare the results
with that of DAPI. These data are presented in Table 2.
compounds that bind in the DNA minor groove with high affinity
and exhibit superior in vitro antitrypanosomal activity to that of
DAPI. In vivo efficacy of six new diamidines (5b, 5c, 5d, 5e, 5f and 5j)
was further improved compared to that of DAPI, four of them gave
100% animal cures at the low dose of 4 ꢀ 5 mg/kg i.p. Generally,
there is a decline in the fluorescence properties of the new ana-
logues compared to that of DAPI with the exception of compound 5i
that shows a moderate increase in efficacy whereas compound 5k
is comparable to DAPI.
The absorption maximum for the new amidines ranges from 312
to 370 nm; that for DAPI is 343 nm. The emission maximum ranges
from 418 to 493 nm in comparison with a value of 458 nm for DAPI.
A qualitative comparison of quantum yield of emission for the in-
doles shows that comp 5i shows a moderate increase in efficacy
compared to that of DAPI while compound 5k is comparable to
DAPI. Both new diamidines have the advantage of being less cyto-
toxic than DAPI (Table 1). Compounds 5b, 5j and 8 shows 11e18%
decrease from that of DAPI, the rest of the compounds shows
significantly lower quantum yield by a factor of 2e25.
4. Experimental
4.1. Biology
3. Conclusion
4.1.1. Efficacy studies
The in vitro viability assays [15] with the T. b. r. strain STIB 900 as
well as the efficacy studies in the STIB900 acute mouse model for
T. b. r. at a 4 ꢀ 5 mg/kg i.p. dosage were carried out as previously
reported [16].
The two diamidines, DAPI and pentamidine are highly active
against African trypanosomes. Substitutions on the phenyl ring of
DAPI or its replacement with heterocyclic rings results in
Fig. 2. Circular dichroism spectral profiles on the addition of representative compounds, with Poly (dA).Poly (dT) duplex DNA sequences in buffer (10 mM MES, 100 mM NaCl, 1 mM
EDTA, at pH, 7.4).

A.A. Farahat et al. / European Journal of Medicinal Chemistry 128 (2017) 70e78
75
mixture was heated at 90e100 ꢂC for 24 h. The solvent was evap-
orated under reduced pressure, the resulting solid was partitioned
between ethyl acetate (200 mL) and 5 mL of concentrated ammonia
to remove the palladium complex, washed with water, passed
through celite to remove the catalyst, dried over sodium sulfate and
evaporated. Purification by column chromatography on silica gel,
using hexanes/ethyl acetate (75/25, v/v).
Table 2
Fluorescence data for the new amidines.
Compound
l
exa,b (nm)
l
ema,b (nm)
Em. intensityc
DAPI
5a
5b
5c
5d
5e
5f
5g
5h
5i
343
322
351
341
351
348
339
342
358
322
348
370
312
458
481
463
461
493
470
462
480
475
418
460
481
433
821
176
611
33
452
461
260
53
200
875
680
810
690
5.1. 1-(tert-Butoxycarbonyl)-2-(2-methyl-4-cyanophenyl)-1H-
indole-6-carbonitrile (4b)
White solid, yield (1.98 gm, 75%). mp 164e164.5 ꢂC; 1HNMR
5j
5k
8
(CDCl3, 400 MHz)
2H), 7.55 (br d, 1H, J ¼ 8 Hz), 7.41 (d, 1H, J ¼ 8 Hz), 6.57 (s, 1H), 2.23
(s, 3H), 1.31 (s, 9H); 13CNMR (CDCl3, 100 MHz)
156.4, 151.1, 149.2,
d
8.66 (s, 1H), 7.67 (d, 1H, J ¼ 8 Hz)760e7.58 (m,
d
a
Wavelengths (l)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
b
140.4, 137, 136.1, 132.5, 131.2, 130.0, 129.8, 127.6, 126.2, 125.1, 121.2,
c
121.1, 119.2, 112.8109.1, 104.2, 27.5, 21.5; ESI-MS: m/z calculated for
22H19N3O2: 357.41, found: 358.2 (Mþþ1); Anal. Calcd. For
slit width of 20.
C
C
22H19N3O2: C, 73.93; H, 5.36; N, 11.76. Found: C, 73.69; H, 5.32; N,
11.99.
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 maintained
under computer control and increased at 0.5 ꢂC/min. The experi-
ments were conducted in 1 cm path length quartz cuvettes in CAC
10 buffer (cacodylic acid 10 mM, EDTA 1 mM, NaCl 100 mM with
NaOH added to give pH ¼ 7.0). The concentrations of DNA were
determined by measuring its absorbance at 260 nm. A ratio of
0.3 mol compound per mole of DNA was used for the complex and
5.2. 1-(tert-Butoxycarbonyl)-2-(2-methyl-4-cyanophenyl)-1H-
indole-5-carbonitrile (4c)
White solid, yield (0.85 gm, 66%), mp 191e193 ꢂC; 1HNMR
(DMSO-d6, 400 MHz)
d
8.11 (d, 1H, J ¼ 1.5 Hz), 7.83 (s, 1H), 7.79 (dd,
1H, J ¼ 1.5 Hz, J ¼ 8.1 Hz), 7.74 (d, 1H, J ¼ 8.1 Hz), 7.58 (d, 1H,
J ¼ 8.4 Hz), 7.47 (dd, 1H, J ¼ 1.5 Hz, J ¼ 8.4 Hz), 6.88 (d, 1H,
J ¼ 1.5 Hz), 2.52 (s, 3H), 1.29 (s, 9H); 13CNMR (CDCl3, 100 MHz)
d
138.2, 137.7, 137.1, 135.9, 134.2, 129.6, 129.4, 127.7, 125.8, 124.5,
120.2, 118.4, 112.4, 110.4, 103.9, 101.5, 27.1, 21.3; ESI-MS: m/z
DNA alone was used as a control [13]. DTm values were determined
by the peak in first derivative curves (dA/dT).
calculated for C17H11N3: 257 (Mþ).
5.3. 1-(tert-Butoxycarbonyl)-2-(5-cyanopyridin-2-yl)-1H-indole-6-
carbonitrile (4e)
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 ꢂC in MES 10 buffer
(10 mM MES, 100 mM NaCl, I mM EDTA). A DNA solution in a 1-cm
quartz cuvette was first scanned over the desired wavelength
range. DAPI, 5d, 5e and 5j at increasing ratios, were titrated into the
same cuvette and the complexes rescanned under the same con-
ditions [17].
White solid, yield (2.21 gm, 87%). mp 171e171.5 ꢂC; 1HNMR
(CDCl3, 400 MHz)
d
8.96 (d, 1H, J ¼ 1.2 Hz), 8.51 (s, 1H), 8.08 (dd, 1H,
J ¼ 2.0 Hz, J ¼ 8.0 Hz), 7.71 (d, 1H, J ¼ 8 Hz), 7.69 (d, 1H, J ¼ 8 Hz),
7.53 (dd, 1H, J ¼ 1.2 Hz, J ¼ 8 Hz), 6.95 (s, 1H), 1.44 (s, 9H); 13CNMR
(CDCl3, 400 MHz)
d 155.2, 151.7, 148.8, 140.6, 139.4, 136.9, 131.6,
126.2, 123.1, 122.2, 119.8, 116.4, 112.3, 108.6, 109, 85.6, 27.6; ESI-MS:
m/z calculated for C20H16N4O2: 344.37, found: 345.2 (Mþþ1); Anal.
Calcd. For C20H16N4O2: C, 69.76; H, 4.68; N, 16.27. Found: C, 69.49;
H, 4.58; N, 16.08.
4.2. Chemistry
All commercial reagents were used without purification.
Melting 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 sol-
vents. Mass spectra were obtained from the Georgia State Univer-
sity Mass Spectrometry Laboratory, Atlanta, GA. If the compounds
reported as salts contain a fraction of water and/or solvents, these
fractions are seen in HNMR spectra. Elemental analysis were per-
formed by Atlantic Microlab Inc., Norcross, GA. Compounds 5a [18]
and 5d [19] were previously reported.
5.4. 1-(tert-Butoxycarbonyl)-2-(5-cyanopyridin-2-yl)-1H-indole-5-
carbonitrile (4f)
White solid, yield (2.06 gm, 81%). mp 180e181 ꢂC; 1HNMR
(CDCl3, 400 MHz)
2H), 7.55 (dd, 1H, J ¼ 1.6 Hz, J ¼ 8.0 Hz), 7.41 (d, 1H, J ¼ 8.0 Hz), 6.57
(s, 1H), 1.31 (s, 9H); 13CNMR (CDCl3, 100 MHz)
150.7, 148.9, 139.4,
d
8.65 (s,1H), 7.67 (s,1H, J ¼ 8.0 Hz), 7.61e7.58 (m,
d
137.0, 136.5, 133.3, 128.7, 128.5, 127.6, 125.9, 119.3, 117.0, 116.7112.0,
107.2, 86.2, 27.7; ESI-MS: m/z calculated for C20H16N4O2: 344.37,
found: 345.2 (Mþþ1); Anal. Calcd. For C20H16N4O2: C, 69.76; H,
4.68; N, 16.27. Found: C, 69.69; H, 4.81; N, 15.99.
5. General procedure for the synthesis of the bisnitriles 4a-k
and 7
5.5. 1-(tert-Butoxycarbonyl)-2-(6-cyanopyridin-3-yl)-1H-indole-6-
carbonitrile (4g)
Tetrakistriphenylphosphine palladium (0.288 gm, 0.25 mmol)
was added to a stirred mixture of the indole stannane (3a or 3b)
(5 mmol) and the aryl halide (5 mmol) in de-aireated dioxane
(15 mL) under a nitrogen atmosphere. The vigorously stirred
White solid, yield (2.14 gm, 84%). mp 168e168.5 ꢂC; 1HNMR
(CDCl3, 400 MHz)
7.92 (br d, 1H, J ¼ 8.0 Hz), 7.81 (d, 1H, J ¼ 8.0 Hz), 7.66 (d,1H,
d
8.81 (s, 1H), 8.35 (d, 1H, J ¼ 8.8 Hz), 7.97 (s, 1H),
J ¼ 8.8 Hz), 6.79 (s, 1H), 1.45 (s, 9H); 13CNMR (CDCl3, 100 MHz)

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A.A. Farahat et al. / European Journal of Medicinal Chemistry 128 (2017) 70e78
d
155.0, 151.8, 148.9, 140.7, 139.5, 137.0, 131.2, 126.7, 122.9, 122.4,
for C21H20N2O3: 348.4, found: 349.3 (Mþþ1); Anal. Calcd. For
119.3, 116.5, 112.4, 108.7, 108.7, 108.4, 85.6, 27.7; ESI-MS: m/z
calculated for C20H16N4O2: 344.37, found: 345.2 (Mþþ1); Anal.
Calcd. For C20H16N4O2: C, 69.76; H, 4.68; N, 16.27. Found: C, 69.66;
H, 4.62; N, 16.21.
C21H20N2O3: C, 72.40; H, 5.79; N, 8.04. Found: C, 72.35; H, 5.68; N,
8.12.
6. General procedure for the synthesis of the diamidines 5a-k
5.6. 1-(tert-Butoxycarbonyl)-2-(6-cyanopyridin-3-yl)-1H-indole-5-
carbonitrile (4h)
The dinitriles (4a-k) (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 mixture
was then cooled to zero oC 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 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 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 diamidines salt.
White solid, yield (2.11 gm, 83%). mp 197e197.5 ꢂC; 1HNMR
(CDCl3, 400 MHz)
d
8.81 (s, 1H), 8.34 (d, 1H, J ¼ 8.8 Hz), 7.97 (s, 1H),
7.93 (br d, 1H, J ¼ 8.0 Hz), 7.81 (d, 1H, J ¼ 8.8 Hz), 7.66 (d, 1H,
J ¼ 8.0 Hz), 6.79 (s, 1H), 1.44 (s, 9H); 13CNMR (CDCl3, 100 MHz)
d
150.8, 149.1, 139.7, 137.4, 136.3, 132.9, 128.9, 128.3, 127.5, 126.3,
119.2, 116.9, 116.8, 112.0, 107.2, 86.3, 27.7; ESI-MS: m/z calculated for
C
C
20H16N4O2: 344.37, found: 345.2 (Mþþ1); Anal. Calcd. For
20H16N4O2: C, 69.76; H, 4.68; N, 16.27. Found: C, 69.72; H, 4.66; N,
16.17.
5.7. 1-(tert-Butoxycarbonyl)-2-(5-cyanofuran-2yl)-1H-indole-6-
carbonitrile (4i)
6.1. 2-(4-Amidino-3-methylphenyl)-1H-indole-6-amidine (5b)
Yellow solid, yield (0.149 gm, 61%), mp 277e279 ꢂC; 1HNMR
Yellow solid, yield (1.58 gm, 64%). mp 154e155 ꢂC; 1HNMR
(CDCl3, 400 MHz)
d
8.3e8.15 (m, 2H), 7.81e7.74 (m, 2H), 7.20 (s, 1H),
150.7, 148.8,
(DMSO-d6, 400 MHz) d 12.32 (s, 1H), 9.47 (s,2H), 9.32 (s,2H), 9.20
7.14 (br s, 1H), 1.45 (s, 9H); 13CNMR (CDCl3, 100 MHz)
d
(s,2H), 8.96 (s,2H), 8.01 (s, 1H), 7.92 (s, 1H), 7.83e7.78 (m, 3H), 7.49
148.6, 139.0, 129.3, 129.2, 128.3, 127.4, 125.4, 119.6, 116.3, 113.3,
(d, 1H, J ¼ 8.4 Hz), 6.93 (s, 1H), 2.59 (s, 3H);; 13CNMR (DMSO-d6,
112.7, 112.0, 106.4, 85.9, 27.5; ESI-MS: m/z calculated for
100 MHz) d 167.0, 165.6, 140.4, 137.1, 136.2, 132.6, 131.2, 130.0, 127.6,
C
C
19H15N3O3: 333.34, found: 334.2 (Mþþ1); Anal. Calcd. For
126.2, 121.2, 121.1, 119.2, 112.8, 104.2, 21.6; ESI-MS: m/z calculated
for C17H17N5: 291.35, found: 292 (amidine base Mþþ1); Anal. Calcd.
For C17H17N5þ2HCl þ H2O: C, 53.41; H, 5.53; N, 18.31. Found: C,
53.15; H, 5.67; N, 18.12.
19H15N3O3: C, 68.46; H, 4.54; N, 12.61. Found: C, 68.32; H, 4.61; N,
12.53.
5.8. 1-(tert-Butoxycarbonyl)-2-(5-cyanofuran-2yl)-1H-indole-5-
carbonitrile (4j)
6.2. 2-(4-Amidino-3-methylphenyl)-1H-indole-5-amidine (5c)
pale solid, yield (0.130 gm, 53%), mp 285e287 ꢂC dec.; 1HNMR
Yellow solid, yield (1.7 gm, 69%). mp 162e162.5 ꢂC; 1HNMR
(CDCl3, 400 MHz)
d
8.32 (d, 1H, J ¼ 8.4 Hz), 7.94 (s, 1H), 7.66e7.62
(DMSO-d6, 400 MHz) d 12.39 (s, 1H), 9.55 (s, 2H), 9.35 (s, 2H), 9.32
(m, 2H), 7.18 (d, 1H, J ¼ 3.6 Hz), 6.84 (s, 1H), 1.5 (s, 9H); 13CNMR
(s, 2H), 9.12 (s, 2H), 8.23 (s, 1H), 7.93 (s, 1H), 7.87e7.82 (br s, 2H),
(CDCl3, 100 MHz)
d 148.8, 141.5, 139.2, 136.9, 132.0, 128.5, 128.4,
7.64 (br s, 2H), 6.95 (s, 1H), 2.59 (s, 1H); 13CNMR (DMSO-d6,
128.3,125.8,119.3,116.6,113.7,110.5,112.9,107.0, 86.0, 27.7; ESI-MS:
m/z calculated for C19H15N3O3: 333.34, found: 334.2 (Mþþ1); Anal.
Calcd. For C19H15N3O3: C, 68.46; H, 4.54; N, 12.61. Found: C, 68.45;
H, 4.56; N, 12.6.
100 MHz) d 166.5, 165.1, 139.5138.1, 136.5, 136.4, 130.8, 129.3, 127.7,
126.9, 125.8, 121.7, 121.4, 118.7, 112.0, 104.5, 21.3; ESI-MS: m/z
calculated for C17H17N5: 291.35, found: 292 (amidine free base
Mþþ1); Anal. Calcd. For C17H17N5þ2HClþ0.25H2O: C, 55.37; H,
5.39; N, 18.99. Found: C, 55.46; H, 5.43; N, 18.72.
5.9. 1-(tert-Butoxycarbonyl)-2-(5-cyanothiophen-2yl)-1H-indole-
6-carbonitrile (4k)
6.3. 2-(5-Amidinopyridine-2-yl)-1H-indole-6-amidine (5e)
Dark yellow solid, yield (1.83 gm, 71%). mp 181e181.3 ꢂC;
Yellow solid, yield (0.171 gm, 71%), mp > 300 ꢂC; 1HNMR
(DMSO-d6, 400 MHz) d 12.65 (s, 1H), 9.70 (s,2H), 9.39 (s,2H), 9.13
1HNMR (CDCl3, 400 MHz)
d
8.31 (d, 1H, J ¼ 8.8 Hz), 7.95 (s, 1H), 7.64
(d, 1H, J ¼ 8.8 Hz), 7.23 (d, 1H, J ¼ 3.6HZ), 6.95 (s, 1H), 6.76 (d, 1H,
(s,1H), 9.08 (s, 2H), 8.38 (br s,2H), 8.01 (s, 1H), 7.85 (d, 1H,
J ¼ 3.6 Hz), 1.55 (s, 9H); 13CNMR (CDCl3, 100 MHz)
d
158.8, 150.1,
J ¼ 8.4 Hz), 7.51 (s, 1H), 7.46 (d, 1H, J ¼ 8.4 Hz); 13CNMR (DMSO-d6,
148.6, 139.2, 129.3, 128.7, 128.1, 126.1, 123.1, 123.1, 119.2, 116.5, 112.6,
100 MHz) d 166.9, 162.0, 146.1, 143.1, 141.3, 136.1, 134.0, 132.3, 127.3,
112.4, 111.2, 107.0, 85.8, 27.8; ESI-MS: m/z calculated for
124.5,122.1,118.9,113.2,104.3; ESI-MS: m/z calculated for C15H14N6:
C
C
19H15N3O2S: 349.41, found: 349.3 (Mþþ1); Anal. Calcd. For
278.31, found: 279.2 (amidine base Mþþ1); Anal. Calcd. For
19H15N3O2S: C, 65.31; H, 4.33; N, 12.03. Found: C, 65.28; H, 4.41; N,
C15H14N6þ2HClþ1.35H2Oþ 0.2 Et2O: C, 48.61; H, 5.34; N, 21.52.
11.99.
Found: C, 48.29; H, 4.95; N, 21.24.
5.10. 1-(tert-Butoxycarbonyl)-2-(4-methoxyphenyl)-1H-indole-6-
carbonitrile (7)
6.4. 2-(5-Amidinopyridine-2-yl)-1H-indole-5-amidine (5f)
Yellow solid, yield (0.157 gm, 65%), mp > 300 ꢂC; 1HNMR
White solid, yield (1.42 gm, 82%). mp 206e207 ꢂC as reported
(DMSO-d6, 400 MHz) d 13.0 (s, 1H), 9.70 (s, 2H), 9.50 (s, 2H), 9.48(s,
[20]; 1HNMR (CDCl3, 400 MHz)
d
8.56 (s,1H), 7.61 (dd,1H, J ¼ 2.0 Hz,
H), 9.33 (s, 2H), 9.09 (s, 2H), 8.76 (dd,1H, J ¼ 2.0 Hz, J ¼ 8.0 Hz), 7.51
J ¼ 8.0 Hz), 7.51 (d, 1H, J ¼ 8 Hz), 7.36 (d, 2H, J ¼ 8.8HZ), 6.99 (d, 2H,
(d, 1H, J ¼ 8.0 Hz), 8.26 (s, 1H), 7.69 (d, 1H, J ¼ 8.8 Hz), 7.67 (d, 1H,
J ¼ 8.8HZ), 6.58 (d, 1H, J ¼ 2 Hz), 3.89 (s, 3H), 1.38 (s, 9H); 13CNMR
J ¼ 8.8 Hz), 7.55 (s, 1H); 13CNMR (DMSO-d6, 100 MHz)
d 166.7, 161.9,
(CDCl3, 100 MHz)
d
159.9, 149.6, 144, 136.3, 132.6, 130, 126.2, 126.1,
146.9, 142.6, 140.9, 136.0, 134.7, 131.9, 128.1, 124.0,122.6, 119.7, 112.7,
121, 119.9, 113.5, 109.2, 106, 84.7, 55.4, 27.6; ESI-MS: m/z calculated
104.0; ESI-MS: m/z calculated for C15H14N6: 278.31, found: 279.4

A.A. Farahat et al. / European Journal of Medicinal Chemistry 128 (2017) 70e78
77
(amidine base Mþþ1); Anal. Calcd. For C15H14N6þ2HClþ1.65H2O-
6.10. 2-(4-methoxyphenyl)-1H-indole-6-amidine (8)
þ0.45Et2O: C, 48.7; H, 5.79; N, 20.28. Found: C, 48.81; H, 5.93; N,
19.99.
The mononitrile 7 (0.23 g, 0.66 mmol) was dissolved in satu-
rated ethanolic HCl (5 mL) and stirred at room temperature for 1
week, isolated from air and moisture. Dry ether was added, the
crystals which formed were filtered, dried under vacuum for 1 h,
dissolved in absolute ethanol, ammonia gas was passed into the
solution for 30 min while cooling and the resulting solution was
stirred for 4 days at room temperature. Dry ether was added, the
precipitated crystals (HCl salt) were filtered. The diamidine 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 at room temperature, diluted with ether, and the
formed solid was filtered and dried.
6.5. 2-(6-Amidinopyridine-3-yl)-1H-indole-6-amidine (5g)
Yellow solid, yield (0.171 gm, 71%), mp > 300 ꢂC; 1HNMR
(DMSO-d6, 400 MHz) d 12.85 (s,1H), 9.68 (s,2H), 9.48 (br s, 3H), 9.32
(s, 2H), 9.07 (s,2H), 8.74 (d,1H, J ¼ 8.4 Hz), 7.49 (d, 1H, J ¼ 8.4 Hz),
8.25 (s, 1H), 7.70 (d, 1H, J ¼ 8.4 Hz), 7.66 (d, 1H, J ¼ 8.4 Hz), 7.56 (s,
1H); 13CNMR (DMSO-d6,100 MHz)
d 166.4,162.9,146.9,142.7,140.9,
136.0, 134.4, 131.9, 128.2, 124.0, 122.7, 119.7, 112.7, 104.3; ESI-MS: m/
z calculated for C15H14N6: 278.31, found: 279.2 (amidine base
Mþþ1); Anal. Calcd. For C15H14N6þ2HClþ0.35H2O: C, 50.4; H, 4.7;
N, 23.5. Found: C, 50.72; H, 4.65; N, 23.16.
White solid, yield (0.11 gm, 54%), mp 281e283 ꢂC; 1HNMR
(DMSO-d6, 400 MHz)
d 12.03 (s, 1H), 9.26 (s, 2H), 8.97 (s, 2H), 7.92
6.6. 2-(6-Amidinopyridine-3-yl)-1H-indole-5-amidine (5h)
(m, 3H), 7.69 (d, 1H, J ¼ 8.4 Hz), 7.44 (d, 1H, J ¼ 8.4 Hz), 7.08 (d, 2H,
J ¼ 8.4 Hz), 6.95 (s, 1H); 13CNMR (DMSO-d6, 100 MHz)
d 167, 142.8,
Yellow solid, yield (0.166 gm, 69%), mp > 300 ꢂC; 1HNMR
137.2, 136.4, 133.5, 129.7, 127.7, 124.3, 120.3, 120, 119.2, 115, 112.2,
55.8; ESI-MS: m/z calculated for C16H15N3O: 265.31, found: 266.1
(amidine base Mþþ1); Anal. Calcd. For C16H15N3O þ1HClþ1.15H2O:
C, 59.59; H, 5.71; N, 13.03. Found: C, 59.35; H, 5.78; N, 13.01.
(DMSO-d6, 400 MHz)
d 13.12 (s, 1H), 9.76 (s,2H), 9.51 (s,2H), 9.48
(s,1H), 9.33 (s, 2H), 9.10 (s,2H), 8.75 (d,1H, J ¼ 8.4 Hz), 7.51 (d, 1H,
J ¼ 8.4 Hz), 8.26 (s, 1H), 7.71 (d, 1H, J ¼ 8.8 Hz), 7.6 (d, 1H, J ¼ 8.8 Hz),
7.56 (s, 1H); 13CNMR (DMSO-d6, 100 MHz)
d 166.9, 162.1, 147.0,
142.7, 141.0, 136.0, 134.2, 132.0, 127.7, 124.1, 122.7, 119.9, 112.8, 104.3;
ESI-MS: m/z calculated for C15H14N6: 278.31, found: 279.2 (amidine
base Mþþ1); Anal. Calcd. For C15H14N6þ2HClþ0.3H2Oþ 0.2 Et2O: C,
51.08; H, 5.04; N, 22.62. Found: C, 50.94; H, 4.73; N, 22.94.
Acknowledgements
This work was supported by an award from the Bill and Melinda
Gates Foundation through the Consortium for Parasitic Drug
Development (RB, WDW, DWB).
6.7. 2-(5-Amidinofuran-2-yl)-1H-indole-6-amidine (5i)
Appendix A. Supplementary data
Yellow solid, yield (0.173 gm, 49%), mp 277e279 ꢂC; 1HNMR
(DMSO-d6, 400 MHz)
d 12.98 (s, 1H), 9.70 (s,2H), 9.39 (s,2H), 9.30
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.01.037.
(s,2H), 9.07 (s,2H), 8.25 (s, 1H), 7.99 (d, 1H, J ¼ 3.6 Hz), 7.73 (d, 1H,
J ¼ 8.8 Hz), 7.64(b, 1H, J ¼ 8.8 Hz), 7.36 (d, 1H, J ¼ 3.6 Hz), 7.32 (s,
1H); 13CNMR (DMSO-d6, 100 MHz)
d 166.7, 153.7, 151.6, 140.3, 140.1,
References
139.1, 130.0, 127.9, 122.7, 121.4, 119.7, 112.4, 110.0, 102.6,; ESI-MS: m/
z calculated for C14H13N5O: 267.29, found: 268.4 (amidine base
Mþþ1); Anal. Calcd. For C14H13N5O þ2HCl þ H2Oþ0.23Et2O: C,
47.75; H, 5.18; N, 18.66. Found: C, 47.62; H, 5.48; N, 18.28.
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Yellow solid, yield (0.159 gm, 45%), mp 263e265 ꢂC; 1HNMR
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d 12.85 (s, 1H), 9.54 (s,2H), 9.42 (s,2H), 9.31
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(DMSO-d6, 400 MHz)
d 13.0 (s, 1H), 9.61 (s,2H), 9.30 (s,2H), 9.20
(s,2H), 9.03 (s,2H), 8.25 (s,1H), 8.02 (d, 1H, J ¼ 4 Hz), 7.76 (dd, 1H,
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d 166.8, 153.2, 151.9, 140.4,
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