Synthesis, biological evaluation, and docking studies of novel thiourea derivatives of bisindolylmethane as carbonic anhydrase II inhibitor

Article abstract of DOI:10.1016/j.bioorg.2015.08.001

Abstract This article describes discovery of 29 novel bisindolylmethanes consisting of thiourea moiety, which had been synthesized through three steps. These novel bisindolylmethane derivatives evaluated for their potential inhibitory activity against car

Full text of DOI:10.1016/j.bioorg.2015.08.001

Bioorganic Chemistry 62 (2015) 83–93
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, biological evaluation, and docking studies of novel thiourea
derivatives of bisindolylmethane as carbonic anhydrase II inhibitor
a,b,
c
Syahrul Imran a,b, Muhammad Taha a,b, , Nor Hadiani Ismail
, Sharmeen Fayyaz ,
⇑
⇑
Khalid Mohammed Khan d, Muhammad Iqbal Choudhary c,d
a Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia
b Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia
c Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
d H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan
a r t i c l e i n f o
a b s t r a c t
Article history:
This article describes discovery of 29 novel bisindolylmethanes consisting of thiourea moiety, which had
been synthesized through three steps. These novel bisindolylmethane derivatives evaluated for their
potential inhibitory activity against carbonic anhydrase (CA) II. The results for in vitro assay of carbonic
anhydrase II inhibition activity showed that some of the compounds are capable of suppressing the
activity of carbonic anhydrase II. Bisindoles having halogen at fifth position showed better inhibitory
activity as compared to unsubstituted bisindoles. Derivatives showing inhibition activity docked to
further, understand the binding behavior of these compounds with carbonic anhydrase II. Docking
studies for the active compound 3j showed that nitro substituent at para position fits into the core of
the active site. The nitro substituent of compound 3j is capable of interacting with Zn ion. This interaction
believed to be the main factor causing inhibition activity to take place.
Received 16 June 2015
Revised 29 July 2015
Accepted 5 August 2015
Available online 5 August 2015
Keywords:
Bisindolylmethane
Thiourea
Carbonic anhydrase II
Enzyme inhibition
Docking study
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
Studies showed that human carbonic anhydrases play impor-
tant role in various physiological processes. It was also found that
Carbonic anhydrases (CA), which form a family of zinc metal-
loenzymes, are present in almost all living organism [1]. Carbonic
anhydrases function by catalyzing conversion between carbon
dioxide and bicarbonate in eukaryotes, archaea, and bacteria
[2,3]. In human, carbonic anhydrase catalyzes reaction involving
carbon dioxide, hydrogen carbonate, and proton, which is essential
for pathological and physiological processes such as electrolyte
secretion, pH and CO2 homeostasis, lipogenesis, and respiration
[4]. Studies done on carbonic anhydrase manage to identify 16 dif-
ferent isozymes in mammal so far. These isozymes are categorized
into several forms like mitochondrial, membrane bound, and
cytosolic form. Two major carbonic anhydrase isozymes that are
present at high concentrations in the cytosol in erythrocytes are
CA II and I. Among all the carbonic anhydrase identified in human,
carbonic anhydrase isozyme II is found to be most active [5,6].
abnormal activities of these enzymes are often linked to various
diseases [7], which include epileptic [8,9], leukemia [10], and glau-
coma [11]. For the past few years, several human carbonic anhy-
drases were targeted to design new inhibitors and activators for
carbonic anhydrase [12–14]. Various studies originally employ car-
bonic anhydrase inhibitors as diuretic [15], and anticonvulsant
[16]. The use of carbonic anhydrase inhibitors as drugs for antiobe-
sity [17] and management of hypoxic tumors were validated
recently [18]. Some examples of human carbonic anhydrase
inhibitors used clinically include acetazolamide, brinzolamide,
dorzolamide, and indisulam. However, due to large number of
human carbonic anhydrase isozymes [19], there is a need to
improve the selectivity and inhibition activity for carbonic anhy-
drase inhibitor. This is to avoid side effects from inhibition of iso-
zymes, which do not participate in a certain pathological processes.
Thiourea compounds work as building block in the synthesis of
heterocyclic compounds [20]. Thioureas are the subject of signifi-
cant interest due their contribution in medicinal chemistry. They
often found to be biologically active and function as fungicides,
herbicides [21], rodenticides [22], and phenoloxidase enzymatic
inhibitors [23]. Substituted thioureas have recently gained much
interest in the preparation of wide variety of biologically active
⇑
Corresponding authors at: Atta-ur-Rahman Institute for Natural Product
Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar
Puncak Alam, Selangor, Malaysia.
E-mail addresses: taha_hej@yahoo.com, muhamm9000@puncakalam.uitm.edu.
my (M. Taha), norhadiani@salam.uitm.edu.my (N.H. Ismail).
http://dx.doi.org/10.1016/j.bioorg.2015.08.001
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

84
S. Imran et al. / Bioorganic Chemistry 62 (2015) 83–93
compounds [24,25]. Thiourea derivatives and their metal com-
plexes exhibit analgesic, anti-inflammatory, antimicrobial, and
anticancer activities [26]. Thiourea derivatives are valuable build-
ing blocks for the synthesis of amides, guanidines, and varieties
of heterocycles [27]. Among the numerous methods for the synthe-
sis of thioureas, condensation of primary and secondary amine
with isothiocyanate, thiophosgene or its derivatives constitutes
the most widely accepted general methods [28].
discovering new biological activity for novel thiourea derivatives
of bisindolylmethane by taking into consideration that compounds
having indole and bisindole moieties do show some significance
effect in suppressing activity of carbonic anhydrase [56,57].
2. Results and discussion
2.1. Chemistry
Besides thiourea, indole derivatives had also received substan-
tial attention due of their broad biological activity range [29].
Indole moiety found in various natural products and synthetic
compounds. Some of them are physiologically relevant and thera-
peutically used [30–32]. Bisindolylmethane derivatives, which are
prevalent compounds containing two indole rings, have attracted
interest of researchers due to their biological activities in recent
years [33–37]. Bisindolylmethane is an important class of hetero-
cyclic compound with a variety of pharmacological activities.
They played an important role in the treatment of chronic fatigue,
fibromyalgia, and irritable bowel syndrome [38,39]. Some of them
are responsible for beneficial estrogen metabolism and induce
apoptosis in cancer cells of human [40]. Bisindolylalkanes from
marine and terrestrial sources found to possess diverse activities,
like coronary dilatory properties, antibacterial activity, and geno-
toxicity [41]. Some bisindole alkaloids, such as macrocarpamine,
macralstonine acetate and villastonine possess significant in vitro
antiprotozoal activity against Plasmodium falciparum and
Entamoeba histolytica [42,43]. Some bisindolylmethanes employed
as dietary supplements for humans [44]. They exhibit inhibitory
activity against bladder cancer [45], renal cell carcinoma growth
[46], lung [47], and colon cancers [48,49]. Bisindolylmethanes
showed inhibitory activity against phenobarbital-induced hepatic
CYP mRNA expression [50] and act as cytodifferentiating agents
[51]. Some bisindolylmethanes derivatives have DPPH radical scav-
enging [52], antimetastatic [53,54], and analgesic activities [55].
In the present study, we synthesize novel thiourea derivatives
of bisindolylmethane and evaluate them for in vitro carbonic anhy-
drase II inhibition activity. This study focuses on designing and
In continuation of our work on enzyme inhibition [58] we syn-
thesized novel derivatives of bisindolylmethanes. Synthesis
involves initially forming various bisindolylmethanes having nitro
group (1a–1e). In this study, we use five different indole deriva-
tives. These indoles undergo electrophilic substitution reaction
with nitro substituted benzaldehydes in glacial acetic acid for 8 h
to produce bisindolylmethanes 1a–1e. The nitro group was being
reduced into amine using sodium borohydride/nickel acetate in
acetonitrile: water (3:1) (Scheme 1) [36].
Bisindolylmethanes 2a–2e were used to synthesize derivatives
of thiourea by reacting with various isothiocyanates (Scheme 2).
The products yield between 63% and 89%. Structures of the final
products (3a–4f) were confirmed by using NMR and CHN analysis.
Results for mass analysis found to be in good agreement with the
calculated values.
2.2. Carbonic anhydrase II inhibitory activity
Carbonic Anhydrase isozyme II from bovine erythrocytes was
used to evaluate inhibitory activity for Carbonic Anhydrase II
(Table 1). Observation on the results in Fig. 1 showed that most com-
pounds having nitro and chloro substitution on the R group inhibit
more than 50% of the CA II activity. The results for inhibition activity
at 200 lM (Fig. 1) showed that all compounds having nitro substitu-
tion at para position are capable of inhibiting carbonic anhydrase II.
Thissuggeststhatnitroisresponsiblefortheactivity. Bisindoleshav-
ing halogensat fifth positionshowed competitiveinhibitoryactivity.
Scheme 1. Reaction scheme for synthesis of compounds 1a–1e and compounds 2a–2e. Reagents and condition: (a) Glacial acetic acid, reflux, 8 h; (b) sodium borohydride,
nickel acetate, acetonitrile:water (3:1), room temperature, 6 h.

S. Imran et al. / Bioorganic Chemistry 62 (2015) 83–93
85
Scheme 2. Reaction of compound 2a–2e with isothiocyanates to produce thiourea derivatives 3a–4f.
The results showed that compounds having bisindole with
bromo substitution at fifth position inhibit more than 50% of the
enzyme’s activity. None of the compounds having unsubstituted
indole is showing inhibition more than 50%. The trend of this result
signifies the importance of halide substituted indole for inhibition
activity to take place.
Fig. 2 represents the IC50 values for thiourea derivatives that
inhibit more than 50% at 200 lM. Analysis shows that compounds
bearing bromo at fifth position on indole ring (3g–3l) recorded the
best inhibition activity with IC50 value ranging between 30.10 and
anchoring of the inhibitor to the Zn-bound solvent molecule. This
mechanism is important since the active site cavity conserved in
all carbonic anhydrase isozymes identified so far in mammals [59].
It was observed that in order for inhibition to take place, the
right conformation and sufficient energy comes together as the
main factor addressing successful inhibition activity. In this study,
which focuses on inhibiting activity of carbonic anhydrase, there
are several factors being considered such as formation of ligand-
receptor complex and ligand’s interaction with important residues
like zinc (cofactor) and amino acids (Thr199, His94, His96 and
His119). This is proven by acetazolamide, which is a primary sul-
fonamide and well-known inhibitor for carbonic anhydrase. The
primary sulfonamide moiety of acetazolamide forms a tetrahedral
adduct with Thr199 and Zn ion (Fig. 3b) and this is most probably
one of the reason supporting its potent activity toward carbonic
anhydrase. Observing the way acetazolamide binds to the active
site (Fig. 3b) points out several factors contributing toward the
low activity observed for bisindolylmethane with thiourea
derivatives.
The result in Table 1 shows that generally the best derivatives
that are capable of inhibiting carbonic anhydrase are having nitro
substituent of the extended ring. Docking studies for the active
compound 3j (Fig. 4) showed that nitro substituent at para position
fits into the core of the active site. Fig. 4 shows that nitro sub-
stituent of compound 3j is capable of interacting with Zn ion.
The nitro substituent of compound 3j forms a coordination and
hydrogen bond with catalytic zinc ion and backbone (HG1) of
Thr199 (2.70 Å), respectively (Fig. 4). This interaction is believed
to be the main factor causing inhibition activity to take place.
This interaction partially resembles the interaction between sul-
fonamide of acetazolamide with zinc atom as in Fig. 3b.
Observation also shows that the other oxygen of nitro group forms
bonding with Thr199. These interactions prevent zinc-coordinated
hydroxide ion from forming hydrogen bond with Thr199 and
results in inhibition activity. Thr199, which is the key residue of
the second coordination sphere, is essential for enzyme activity;
together with Thr200, they are involved in a finely tuned network
70.36 lM.
The results in Fig. 2 clearly show the important role played by
nitro substitution at para position. For compounds from two other
series 3r–3w and 4a–4f, those with nitro substitution at para posi-
tion were found to be the only compound having inhibition
activity.
Compounds with unsubstituted phenyl ring are showing inhibi-
tion less than 50%, which clearly indicates that suitable substituent
on phenyl ring is important for inhibition activity to take place.
Compounds that are less bulky and possess electron-withdrawing
substituents showed good inhibitory activity. The results suggest
that a bulky group such as nitro or chloro in the ortho position
introduces unfavorable steric effect to the binding pocket on car-
bonic anhydrase and thus reduces the binding affinity relative to
the para-substituted inhibitor. On the other hand, hydrogen bond
acceptor like nitro in the para position could interact well with
the binding pocket on CA II and thus inhibit the enzyme’s activity.
2.3. Docking studies
Carbonic anhydrase inhibition takes place through some
mechanisms, which mainly involves interaction with Zn2+ that is
the core of the enzyme’s activity. One of the mechanisms involves
inhibitor coordination to Zn2+ ion, which results in replacement of
zinc-bound water/hydroxide ion. This leads to a tetrahedral geom-
etry of Zn through addition of inhibitor to the metal coordination
sphere, when the Zn2+ ion is in a trigonal bipyramidal geometry,

86
S. Imran et al. / Bioorganic Chemistry 62 (2015) 83–93
Table 1
could resulting in a big different for inhibition activity of both com-
pounds. This had left us with the only structural difference, which is
the presence of different halides at fifth position for both com-
pounds. A valid reason for the observed activity is that it might be
due to different halide groups of both compounds, which could pos-
sibly change lipophilicity of these compounds. In this case com-
pound 3p which has chloro at fifth position on indole ring is
claimed to be having lower solubility toward lipid as compared to
compound 3k, which possesses bromo at fifth position. Further
analysis carried out using logP values showed that predicted logP
value for compound 3p is 9.31 which is lower that compound 3k,
predicted to be 9.85. Estimated free energy of binding for both com-
pounds were also compared to identify any possible factor leading
to the low inhibition activity of compound 3p. Binding energy for
compound 3p and 3k was found to be ꢀ8.42 kcal/mol and
ꢀ8.58 kcal/mol respectively. The higher binding energy further
confirms the important role played by bromo substituent in stabi-
lizing the ligand-enzyme complex. Compound 3q docking shows
that the compound adopts similar interaction as compound 3p.
Compound 3q only interacts with Thr200 (3.59 Å) and Glu69
(2.91 Å), which not sufficient enough to inhibit the activity.
Carbonic anhydrase II inhibition activity for thiourea derivatives (3a–4f).
Compound
R
% Inhibitiona
IC50 (l
M) SEMb
NAc
NAc
3a
3b
Phenyl
44.9
31.2
3c
3d
3e
3f
3g
3h
3-Bromophenyl
2,5-Dichlorophenyl
3-Chlorophenyl
4-Nitrophenyl
Phenyl
59.0
60.6
63.4
65.8
49.4
69.2
131 2.06
68.3 1.14
112.0 2.4
45.9 2.86
NAc
70.36 0.49
3i
3j
3k
3l
3m
3n
3o
3p
3q
3-Bromophenyl
3-Chlorophenyl
4-Nitrophenyl
2,5-Dichlorophenyl
3-Bromophenyl
4-Nitrophenyl
70.7
76.0
75.3
100.0
11.1
100
71.3
62.6
64.3
30.1 1.31
42.9 0.69
32.3 0.61
33.0 0.65
NAc
39.67 0.43
54.36 3.08
138.81 2.37
136.77 2.24
3-Chlorophenyl
2,5-Dichlorophenyl
Despite being able to form hydrogen bonding with the active site,
the ligand-enzyme complexes remain unstable. It was observed that
there is very little interaction involving Pi–Pi and Pi-cation interac-
tion between the ligands and enzyme which leads to incapability of
compounds to form stable ligand-enzyme complexes.
3r
3s
3t
3u
3v
3w
Phenyl
32.8
5.07
35.1
68.8
37.08
30.8
NAc
2,5-Dichlorophenyl
3-Bromophenyl
4-Nitrophenyl
3-Chlorophenyl
NAc
NAc
44.09 1.37
3. Conclusion
NAc
NAc
This study manages to identify important factors influencing the
inhibition activity of CA II by thiourea derivatives of bisindolyl-
methane. The CA II inhibition activity subjected through docking
studies to evaluate properly structural contribution toward the
activity. Molecular docking study carried out to understand further
on the effect substituents. Docking study showed that presence of
nitro substituent at para position is highly important for the inhibi-
tion activity to take place. Compounds with nitro substituent were
found to be able interact closely with the Zn ion, Thr199, His94,
His96 and His119 and therefore interfere in the Zn-OH-Thr199-
Glu106 hydrogen bond network. Even though indole ring was not
able to interact with the active site due to its bulky molecule, NH
of indole observed to form hydrogen bonding with Glu69 to stabi-
lize ligand-receptor complex. These novel compounds possess inhi-
bition activity that can be considered for future development.
4a
4b
4c
4d
4e
4f
3-Bromophenyl
3-Chlorophenyl
2,5-Dichlorophenyl
4-Nitrophenyl
Phenyl
31.0
34.3
49.3
60.8
47.6
14.4
NAc
NAc
NAc
32.88 1.4
NAc
NAc
Standard
Acetazolamide
97.0
0.18
a
b
c
% inhibition at 200 lM.
Standard error mean.
No activity.
of hydrogen bonds leading toward the solvent-exposed His64,
which is located at the entrance of the active-site channel. The
ligand’s ability to occupy the active site partially deactivates the
enzyme from catalyzing hydration process of carbon dioxide into
bicarbonate, which then undergo further dehydration in acidic
medium to regenerate carbon dioxide.
The most non-active compounds come from the same class of
bisindole bearing chloro at fifth position on the indole ring.
Docking of some thiourea derivatives which are least active
showed that compound 3k interacts with zinc ion through a direct
ring-metal linkage (Fig. 5). Side interaction includes interaction
between thiourea linkage with Thr200 (2.24 Å) and NH of indole
interaction with Glu69 (2.04 Å). Docking for two compounds hav-
ing highest IC50 in thiourea series provides some relevant reasons
for their inability to inhibit the activity of carbonic anhydrase II.
2D interaction diagram for 3p (Fig. 6) showed that it typically
adopts the same binding mode as observed for compound 3k
(Fig. 5). Compound 3p forms hydrogen bonding with Thr200
(2.24 Å) and Glu69 (2.12 Å).
4. Experimental
4.1. Material and methods
Carbonic anhydrase was purchased from Sigma Aldrich (Cat. No.
C-3666), 4-nitrophenyl acetate was purchased from MP Bio (Cat.
No. 160888). HEPES buffer was purchase from DOJINDO (Cat. No.
346-01373), tris-(hydroxymethyl)-aminomethane (reagent grade)
was purchase from Scharlau (Cat. No. TR0423). DMSO and ethanol
of reagent grade were used in the experiment. The experiment
was performed in triplicate. The IC50 values were calculated by
using EZ-fit enzyme kinetics software (USA). NMR experiments
were performed on Avance Bruker AV 300 MHz, 400 MHz,
500 MHz, and 600 MHz. IR experiments were performed on
Perkin Elmer FT-IR. CHN analysis was performed on a Carlo Erba
Strumentazion-Mod-1106, Italy. Electron impact mass spectra (EI
MS) were recorded on a Finnigan MAT-311A, Germany. Thin layer
chromatography (TLC) was performed on pre-coated silica gel alu-
minum plates (Kieselgel 60, 254, E. Merck, Germany).
Chromatograms were visualized by UV at 254 and 365 nm.
Small difference in bond distance and binding mode between
compound 3p and 3k does not signify any possible reason which

S. Imran et al. / Bioorganic Chemistry 62 (2015) 83–93
87
Fig. 1. Inhibition activity for thiourea derivatives at 200 lM.
Fig. 2. IC50 (lM) values for derivatives having percent inhibition more than 50% at 200 lM.
Fig. 3. (a) Superimpose of acetazolamide with its crystal native ligand in 3HS4 and (b) interaction of acetazolamide with important residues.
4.2. General procedure for synthesis of bisindolylmethane derivatives
(1a–1e)
monitored using TLC on the following day. Deionized water was
added to allow product to precipitate. Reaction mixture was
being transferred to
a
container containing crushed ice.
Nitro-substituted benzaldehyde (18 mmol) and indole
(36 mmol) were added to a 250-ml round bottom flask containing
15 ml of acetic acid and refluxed overnight. Reaction progress
Precipitate formed was filtered and rinsed using diethyl ether to
afford pure product. The pure product was left to dry at room
temperature.

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S. Imran et al. / Bioorganic Chemistry 62 (2015) 83–93
Fig. 4. Docking and 2D interaction diagram of 3j.
Fig. 5. Docking for compound 3k.
Fig. 6. Docking for compound 3p and 3q.
4.2.1. 3,30-((4-Nitrophenyl)methylene)bis(1H-indole) (1a)
C = 75.19; H = 4.66; N = 11.44; Found: C = 75.20; H = 4.64;
Light yellow solid; Yield 92.3%; m.p. 219–221 °C; IR (ATR):
N = 11.43; EI MS m/z (% rel. abund.): 367.40 (M+, 78.2%).
3420, 1543, 1457, 1342, 1226 cmꢀ1 1H NMR (600 MHz, DMSO-
;
4.2.2. 3,30-((4-Nitrophenyl)methylene)bis(5-bromo-1H-indole) (1b)
White solid; Yield 83.6%; m.p. 274–276 °C; IR (ATR): 3417,
d6) d 11.05 (s, 2H), 8.16 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 8.8 Hz, 2H),
7.37 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 7.9 Hz, 2H), 7.06 (t, J = 7.6 Hz,
2H), 6.92–6.86 (m, 4H), 6.03 (s, 1H).; Anal. Calcd for C23H17N3O2:
1636, 1452, 1414, 1336, 1094, 743 cmꢀ1 1H NMR (500 MHz,
;

S. Imran et al. / Bioorganic Chemistry 62 (2015) 83–93
89
DMSO-d6) d 11.18 (d, J = 1.7 Hz, 2H), 8.21–8.15 (m, 2H), 7.60 (d,
J = 8.7 Hz, 2H), 7.49 (d, J = 1.8 Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H),
7.18 (dd, J = 1.9 Hz, 8.6 Hz, 2H), 6.97 (d, J = 2.2 Hz, 2H), 6.11 (s,
1H); Anal. Calcd for C23H15Br2N3O2: C = 52.62; H = 2.90; N = 8.00;
Found: C = 52.61; H = 2.92; N = 7.98; EI MS m/z (% rel. abund.):
525.27 (M+, 53.7%).
4.3.3. 4-(Bis(5-chloro-1H-indol-3-yl)methyl)aniline (2c)
Brown solid; Yield 95.2%; m.p. 241–243 °C; IR (ATR): 3426,
3384, 1638, 1531, 1456, 1417, 1332, 1094, 743 cmꢀ1 1H NMR
(500 MHz, DMSO-d6): d 10.98 (s, 2H), 7.36 (d, J = 9.0 Hz, 2H), 7.23
(d, J = 2.0 Hz, 2H), 7.03 (dd, J = 8.5 Hz, 2.0 Hz, 2H), 6.96–6.98 (m,
2H), 6.85 (d, J = 2.0 Hz, 2H), 6.49 (d, J = 8.5 Hz, 2H), 5.62 (s, 1H),
4.86 (s, 2H); Anal. Calcd for C23H17Cl2N3: C = 67.99; H = 4.22;
N = 10.34; Found: C = 68.01; H = 4.23; N = 10.32; EI MS m/z (% rel.
abund.): 495.21 (M+, 69.2%), 497.2 (M + 2, 21.6%).
;
4.2.3. 3,30-((4-Nitrophenyl)methylene)bis(5-chloro-1H-indole) (1c)
Orange solid; Yield 79.6%; m.p. 235–237 °C; IR (ATR): 3458,
1637, 1175, 1452, 1416, 1095, 748 cmꢀ1 1H NMR (500 MHz,
;
4.3.4. 4-(Bis(7-methyl-1H-indol-3-yl)methyl)aniline (2d)
DMSO-d6): d 11.18 (s, 2H), 8.18 (d, J = 9.0 Hz, 2.0 Hz, 2H), 7.61 (d,
J = 9.0 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 2.0 Hz, 2H),
7.07 (dd, J = 8.5 Hz, 2.0 Hz, 2H), 6.09 (s, 1H), 7.00 (d, J = 2.0 Hz,
2H); Anal. Calcd for C23H15Cl2N3O2: C = 63.32; H = 3.47; N = 9.63;
Found: C = 63.33; H = 3.45; N = 9.62; EI MS m/z (% rel. abund.):
435.05 (M+, 49.2%), 437.3 (M + 2, 18.1%).
Orange solid; Yield 81%; m.p. 257–258 °C; IR (ATR): 3419, 3287,
1636, 1452, 1414, 1336, 1094, 743 cmꢀ1 1H NMR (500 MHz,
;
DMSO-d6): d 2.43 (s, 6H), 4.80 (s, 2H), 6.46 (d, J = 8.5 Hz, 2H),
6.76 (d, J = 2 Hz, 2H), 6.84 (t, J = 7.5 Hz, 2H), 6.98–7.03 (m, 4H),
7.26 (d, J = 8 Hz, 2H), 7.32 (d, J = 8 Hz, 2H), 10.72 (s, 2H); Anal.
Calcd for
C25H23N3: C = 82.16; H = 6.34; N = 11.50; Found:
C = 82.17; H = 6.36; N = 11.48; EI MS m/z (% rel. abund.): 365.15
(M+, 63.4%).
4.2.4. 3,30-((4-Nitrophenyl)methylene)bis(7-methyl-1H-indole) (1d)
White solid; Yield 85%; m.p. 234–236 °C; IR (ATR): 3416, 1637,
1528, 1457, 1417, 1091, 743 cmꢀ1 1H NMR (DMSO-d6, 500 MHz):
;
4.3.5. 3-(Di(1H-indol-3-yl)methyl)aniline (2e)
d 6.00 (s, 1H), 6.80 (t, J = 7.5 Hz, 2H), 6.86–6.87 (m, 4H), 7.12 (d,
J = 8.0 Hz, 2H), 7.60 (d, J = 7.5 Hz, 2H), 8.15 (d, J = 8.5 Hz, 2H),
10.89 (s, 2H); Anal. Calcd for C25H21N3O2: C = 75.93; H = 5.35;
N = 10.63, Found: C = 75.91; H = 5.33; N = 10.64; EI MS m/z (% rel.
abund.): 395.67 (M+, 52.4%).
Dark orange solid; Yield 95%; m.p. 229–231 °C; IR (ATR): 3437,
3372, 1638, 1529, 1456, 1418, 1127, 744 cmꢀ1; 1H NMR (500 MHz,
DMSO-d6): d 4.87 (s, 2H), 5.63 (s, 1H), 6.37 (dd, J = 7.9, 1.2 Hz, 1H),
6.54 (d, J = 7.6 Hz, 1H), 6.58 (s, 1H), 6.81 (d, J = 1.6 Hz, 2H), 6.86 (t,
J = 7.5 Hz, 2H), 6.91 (t, J = 7.7 Hz, 1H), 7.03 (dd, J = 11.1, 4.0 Hz, 2H),
7.30 (d, J = 7.9 Hz, 2H), 7.34 (d, J = 8.1 Hz, 2H), 10.76 (s, 2H); Anal.
4.2.5. 3,30-((3-Nitrophenyl)methylene)bis(1H-indole) (1e)
Calcd for C23H19N3: C = 81.87; H = 5.68; N = 12.45; Found:
Brownish solid; Yield 87%; m.p. 231–233 °C; IR (ATR): 3440,
C = 81.89; H = 5.70; N = 12.43; EI MS m/z (% rel. abund.): 337.15
(M+, 49.1%).
1637, 1455, 1416, 1341, 1101, 741 cmꢀ1
;
1H NMR (DMSO-d6,
500 MHz): d 6.06 (s, 1H), 6.87–6.90 (m, 4H), 7.06 (t, J = 7.5 Hz,
2H), 7.30 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 7.58 (t,
J = 8.0 Hz, 1H), 7.84 (d, J = 7.5 Hz, 1H), 8.07 (d, J = 8.0 Hz, 2H),
10.91 (s, 2H); Anal. Calcd for C23H17N3O2: C = 75.19; H = 4.66;
N = 11.44; Found: C = 75.19; H = 4.66; N = 11.44; EI MS m/z (% rel.
abund.): 367.24 (M+, 71.5%).
4.4. General procedure for synthesis of bisindolylmethane thiourea
derivatives (3a–4f)
Thiourea derivatives were synthesized by mixing equal mole of
key intermediate and phenylisothiocyanates in 5 ml of acetone.
The mixture stirred at room temperature to yield various thiourea
derivatives. Thiourea forms precipitate upon addition of hexane.
The products filtered and washed with plenty of hexane to afford
pure product.
4.3. General procedure for synthesis of bisindolylmethane derivatives
(2a–2e)
Purified bisindolylmethanes (1a–1e) were dissolved in acetoni-
trile:water (3:1) and reduced using mixture of sodium borohydride
(4 equiv) and nickel acetate tetrahydrate (0.2 equiv). Reaction pro-
gress was monitored using TLC. Upon completion, product was
extracted using dichloromethane and rinsed using diethyl ether
to afford pure product.
4.4.1. 1-(4-(Di(1H-indol-3-yl)methyl)phenyl)-3-phenylthiourea (3a)
Brown solid; Yield 72.9%; m.p. 212–214 °C; IR (ATR): 3426,
3342, 1581, 1515, 1463, 1339, 1093, 785 cmꢀ1
;
1H NMR
(300 MHz, DMSO-d6): d 10.78 (s, 2H), 9.68 (s, 1H), 9.67 (s, 1H),
7.44 (d, J = 10.4 Hz, 2H), 7.27–7.37 (m, 10H), 7.10 (t, J = 9.6 Hz,
1H), 7.02 (t, J = 9.6 Hz, 2H), 6.82–6.88 (m, 4H), 5.80 (s, 1H); Anal.
Calcd for C30H24N4S: C = 76.24; H = 5.12; N = 11.85; Found:
C = 76.26; H = 5.13; N = 11.84; EI MS m/z (% rel. abund.): 473.14
(M+, 68.3%).
4.3.1. 4-(Di(1H-indol-3-yl)methyl)aniline (2a)
Dark orange solid; Yield 95.8%; m.p. 230–232 °C; IR (ATR):
3425, 3387, 1629, 1276 cmꢀ1 1H NMR (500 MHz, DMSO-d6) d
;
10.73 (s, 2H), 7.34 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 7.9 Hz, 2H),
7.07–6.95 (m, 4H), 6.85 (t, J = 7.5 Hz, 2H), 6.78 (d, J = 1.8 Hz, 2H),
6.48 (d, J = 8.3 Hz, 2H), 5.63 (s, 1H), 4.82 (s, 2H); Anal. Calcd for
4.4.2. Ethyl (4-(bis(1H-indol-3-yl)methyl)phenyl) thioureaoate (3b)
Dark red solid; Yield 83.7%; m.p. 255–257 °C; IR (ATR): 3422,
3339, 1594, 1504, 1461, 1412, 1332, 1110, 794 cmꢀ1 1H NMR
;
C23H19N3: C = 81.87; H = 5.68; N = 12.45; Found: C = 81.86;
(300 MHz, DMSO-d6): d 11.49 (s, 1H), 11.13 (s, 1H), 10.79 (s, 2H),
7.50 (d, J = 8.4 Hz, 2H), 7.27–7.37 (m, 7H), 7.02 (t, J = 7.2 Hz, 1H),
6.83–6.88 (m, 4H), 5.83 (s, 1H), 4.18 (q, J = 7.2 Hz, 2H), 1.24 (t,
J = 7.2 Hz, 3H); Anal. Calcd for C27H24N4O2S: C = 69.21; H = 5.16;
N = 11.96; Found: C = 69.22; H = 5.17; N = 11.94; EI MS m/z (% rel.
abund.): 468.27 (M+, 45.8%).
H = 5.69; N = 12.44. EI MS m/z (% rel. abund.): 337.42 (M+, 43.7%).
4.3.2. 4-(Bis(5-bromo-1H-indol-3-yl)methyl)aniline (2b)
White solid; Yield 84.6%; m.p. 258–259 °C; IR (ATR): 3414,
3354, 1628, 1456, 1417, 1352, 1096, 1017, 744 cmꢀ1 1H NMR
;
(500 MHz, DMSO-d6): d 11.00 (s, 2H), 7.40 (s, 2H), 7.33 (d,
J = 8.5 Hz, 2H), 7.15 (dd, J = 8.5 Hz, 1.5 Hz, 2H), 6.98 (d, J = 8.5 Hz,
2H), 6.83 (s, 2H), 6.50 (d, J = 5.0 Hz, 2H), 4.87 (s, 2H), 5.64 (s,
1H); Anal. Calcd for C23H17Br2N3: C = 55.78; H = 3.46; N = 8.49;
Found: C = 55.79; H = 3.45; N = 8.47; EI MS m/z (% rel. abund.):
495.21 (M+, 69.2%).
4.4.3. 1-(3-Bromophenyl)-3-(4-(di(1H-indol-3-yl)methyl)phenyl)
thiourea (3c)
Light red solid; Yield 79.2%; m.p. 239–241 °C; IR (ATR): 3406,
3325, 1669, 1582, 1493, 1456, 1341, 738 cmꢀ1
;
1H NMR
(400 MHz, DMSO-d6): d 10.80 (s, 2H), 9.87 (s, 1H), 9.79 (s, 1H),

90
S. Imran et al. / Bioorganic Chemistry 62 (2015) 83–93
7.77 (s, 1H), 7.26–7.34 (m, 11H), 7.02 (t, J = 7.6 Hz, 2H), 6.86 (t,
J = 7.2 Hz, 2H), 6.83 (t, J = 7.6 Hz, 2H), 5.80 (s, 1H); Anal. Calcd for
C = 50.80; H = 2.98; N = 7.90; Found: C = 50.82; H = 2.97;
N = 7.88; EI MS m/z (% rel. abund.): 710.15 (M+, 67.2%).
C30H23BrN4S: C = 65.33; H = 4.20; N = 10.16; Found: C = 65.34;
H = 4.21; N = 10.14; EI MS m/z (% rel. abund.): 552.17 (M+, 61.3%).
4.4.10. 1-(4-(Bis(5-bromo-1H-indol-3-yl)methyl)phenyl)-3-(3-
chlorophenyl)thiourea (3j)
Dark orange solid; Yield 81.9%; m.p. 205–207 °C; IR (ATR):
4.4.4. 1-(2,5-Dichlorophenyl)-3-(4-(di(1H-indol-3-yl)methyl)phenyl)
thiourea (3d)
3420, 3346, 1594, 1456, 1413, 1331, 1110, 792 cmꢀ1 1H NMR
;
(300 MHz, DMSO-d6): d 11.05 (s, 2H), 9.87 (s, 1H), 9.80 (s, 1H),
7.66 (s, 1H), 7.44 (d, J = 2.0 Hz, 2H), 7.28–7.38 (m, 8H), 7.12–7.16
(m, 3H), 6.90 (d, J = 2.4 Hz, 2H), 5.83 (s, 1H); Anal. Calcd for
Orange solid; Yield 69.7%; m.p. 225–226 °C; IR (ATR): 3431,
3319, 1582, 1508, 1460, 1092, 794 cmꢀ1 1H NMR (300 MHz,
;
DMSO-d6,): d 10.78 (s, 2H), 10.07 (s, 1H), 9.35 (s, 1H), 7.77 (d,
J = 3.2 Hz, 1H), 7.50 (d, J = 11.6 Hz, 1H), 7.27–7.41 (m, 9H), 7.02
(t, J = 10.0 Hz, 2H), 6.83–6.88 (m, 4H), 5.81 (s, 1H); Anal. Calcd
C30H21Br2ClN4S: C, 54.20; H, 3.18; N, 8.43; Found: C, 54.21; H,
3.17; N, 8.45; EI MS m/z (% rel. abund.): 663.58 (M+, 51.9%).
for
C30H22Cl2N4S: C = 66.54; H = 4.10; N = 10.35; Found:
4.4.11. 1-(4-(Bis(5-bromo-1H-indol-3-yl)methyl)phenyl)-3-(4-
nitrophenyl)thiourea (3k)
C = 66.53; H = 4.09; N = 10.33; EI MS m/z (% rel. abund.): 540.13
(M+, 64.1%).
Dark brown solid; Yield 79.3%; m.p. 221–223 °C; IR (ATR): 3421,
3334, 1585, 1509, 1457, 1410, 1092, 792 cmꢀ1; 1H NMR (300 MHz,
DMSO-d6): d 11.05 (s, 2H), 10.28 (s, 1H), 10.17 (s, 1H), 8.18 (d,
J = 12.4 Hz, 2H), 7.82 (d, J = 12.4 Hz, 2H), 7.40–7.46 (m, 4H), 7.31–
7.34 (m, 4H), 7.14 (dd, J = 11.6 Hz, 2.4 Hz, 2H), 6.92 (d, J = 2.4 Hz,
2H), 5.85 (s, 1H); Anal. Calcd for C30H21Br2N5O2S: C = 53.35;
H = 3.13; N = 10.37; Found: C = 53.34; H = 3.12; N = 10.35; EI MS
m/z (% rel. abund.): 675.17 (M+, 35.0%).
4.4.5. 1-(3-Chlorophenyl)-3-(4-(di(1H-indol-3-yl)methyl)phenyl)
thiourea (3e)
Brown solid; Yield 74.9%; m.p. 249–251 °C; IR (ATR): 3421,
3345, 1592, 1454, 1411, 1332, 1097, 794 cmꢀ1
;
1H NMR
(400 MHz, DMSO-d6): d 10.80 (s, 2H), 9.87 (s, 1H), 9.81 (s, 1H),
7.65 (s, 1H), 7.28–7.34 (m, 11H), 7.02 (t, J = 7.6 Hz, 2H), 6.83–
6.87 (m, 4H), 5.80 (s, 1H); Anal. Calcd for C30H23ClN4S: C = 71.06;
H = 4.57; N = 11.05; Found: C = 71.05; H = 4.56; N = 11.06; EI MS
m/z (% rel. abund.): 507.26 (M+, 63.0%).
4.4.12. 1-(4-(Bis(5-bromo-1H-indol-3-yl)methyl)phenyl)-3-(2,5-
dichlorophenyl)thiourea (3l)
Brown solid; Yield 89.1%; m.p. 218–220 °C; IR (ATR): 3408,
4.4.6. 1-(4-Nitrophenyl)-3-(4-(di(1H-indol-3-yl)methyl)phenyl)
thiourea (3f)
1594, 1455, 1416, 741 cmꢀ1 1H NMR (300 MHz, DMSO-d6): d
;
11.05 (s, 2H), 10.09 (s, 1H), 9.38 (s, 1H), 7.76 (d, J = 3.2 Hz, 1H),
7.51 (d, J = 11.6 Hz, 1H), 7.41–7.45 (m, 4H), 7.28–7.34 (m, 5H),
7.14 (dd, J = 11.2 Hz, 2.4 Hz, 2H), 6.92 (d, J = 2.4 Hz, 2H), 5.85 (s,
Red solid; Yield 75.9%; m.p. 239–241 °C; IR (ATR): 3420, 3331,
1696, 1508, 1445, 1316, 1095, 792 cmꢀ1 1H NMR (400 MHz,
;
DMSO-d6): d 10.80 (s, 2H), 10.30 (s, 1H), 10.19 (s, 1H), 8.17 (d,
J = 8.8 Hz, 2H), 7.79 (d, J = 8.8 Hz, 2H), 7.28–7.39 (m, 8H), 7.02 (t,
J = 7.6 Hz, 2H), 6.84–6.87 (m, 4H), 5.81 (s, 1H); Anal. Calcd for
1H); Anal. Calcd for
C30H20Br2Cl2N4S: C = 51.53; H = 2.88;
N = 8.01; Found: C = 51.54; H = 2.89; N = 7.99; EI MS m/z (% rel.
abund.): 699.27 (M+, 74.8%).
C30H23N5O2S: C = 69.61; H = 4.48; N = 13.53; Found: C = 69.60;
H = 4.46; N = 13.52; EI MS m/z (% rel. abund.): 517.17 (M+, 48.8%).
4.4.13. 1-(4-(Bis(5-chloro-1H-indol-3-yl)methyl)phenyl)-3-(3-
bromophenyl)thiourea (3m)
4.4.7. 1-(4-(Bis(5-bromo-1H-indol-3-yl)methyl)phenyl)-3-
phenylthiourea (3g)
Orange solid; Yield 84.7%; m.p. 219–221 °C; IR (ATR): 3408,
3323, 1672, 1581, 1494, 1455, 1339, 737 cmꢀ1
;
1H NMR
Brown solid; Yield 65.1%; m.p. 244–245 °C; IR (ATR): 3422,
(500 MHz, DMSO-d6,): d 11.06 (s, 2H), 9.92 (s, 1H), 9.85 (s, 1H),
7.79 (s, 1H), 7.40 (d, J = 7.0 Hz, 1H), 7.35–7.37 (m, 4H), 7.29–7.31
(m, 4H), 7.23–7.26 (m, 2H), 7.02 (dd, J = 8.5 Hz, 2.0 Hz, 2H), 6.93
(d, J = 1.6 Hz, 2H), 5.83 (s, 1H). Anal. Calcd for C30H21BrCl2N4S,
C = 58.08; H = 3.41; N = 9.03; Found: C = 58.07; H = 3.40;
N = 9.01; EI MS m/z (% rel. abund.): 620.2 (M+, 64.7%), 622.1
(M + 2, 24.5%).
3211, 1718, 1507, 1456, 1341, 1035, 792 cmꢀ1
;
1H NMR
(300 MHz, DMSO-d6): d 11.04 (s, 2H), 9.70 (s, 1H), 9.68 (s, 1H),
7.44–7.46 (m, 4H), 7.37 (d, J = 8.4 Hz, 2H), 7.26–7.33 (m, 6H),
7.05–7.16 (m, 3H), 6.89 (d, J = 1.5 Hz, 2H), 5.83 (s, 1H); Anal.
Calcd for
C30H22Br2N4S: C = 57.16; H = 3.52; N = 8.89; Found:
C = 57.17; H = 3.53; N = 8.91; EI MS m/z (% rel. abund.): 630.11
(M+, 41.5%).
4.4.14. 1-(4-(Bis(5-chloro-1H-indol-3-yl)methyl)phenyl)-3-(4-
nitrophenyl)thiourea (3n)
4.4.8. Ethyl (4-(bis(5-bromo-1H-indol-3-yl)methyl)phenyl)
thioureaoate (3h)
Light orange solid; Yield 62.6%; m.p. 236–238 °C; IR (ATR):
3408, 3350, 1586, 1511, 1455, 1416, 1338, 1092, 742 cmꢀ1 1H
;
Light orange solid; Yield 73.8%; m.p. 222–224 °C; IR (ATR):
3402, 3346, 1598, 1503, 1335, 1098, 743 cmꢀ1
;
1H NMR
NMR (500 MHz, DMSO-d6): d 11.06 (s, 2H), 10.36 (s, 1H), 10.241
(s, 1H), 8.17 (d, J = 9.0 Hz, 2H), 7.82 (d, J = 9.0 Hz, 2H), 7.41 (d,
J = 8.5 Hz, 2H), 7.36 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.5 Hz, 2H),
7.30 (d, J = 1.5 Hz, 2H), 7.03 (dd, J = 8.5 Hz, 2.0 Hz, 2H), 6.95 (d,
J = 2.0 Hz, 2H), 5.85 (s, 1H); Anal. Calcd for C30H21Cl2N5O2S:
C = 61.44; H = 3.61; N = 11.94; Found: C = 61.45; H = 3.63;
N = 11.95; EI MS m/z (% rel. abund.): 586.1 (M+, 56.1%), 588.2
(M + 2, 19.1%).
(300 MHz, DMSO-d6): d 11.50 (s, 1H), 11.14 (s, 1H), 11.06, (s, 2H),
7.53 (d, J = 11.2 Hz, 2H), 7.45 (s, 2H), 7.31–7.34 (m, 4H), 7.15 (dd,
J = 11.6 Hz, 2.0 Hz, 2H), 6.90 (d, J = 2.4 Hz, 2H), 5.87 (s, 1H), 4.19
(q, J = 9.2 Hz, 2H), 1.24 (t, J = 9.6 Hz, 3H); Anal. Calcd for
C27H22Br2N4O2S: C = 51.77; H = 3.54; N = 8.94; Found: C = 51.78;
H = 3.53; N = 8.97; EI MS m/z (% rel. abund.): 626.34 (M+, 74.1%).
4.4.9. 1-(4-(Bis(5-bromo-1H-indol-3-yl)methyl)phenyl)-3-(3-
bromophenyl)thiourea (3i)
4.4.15. 1-(4-(Bis(5-chloro-1H-indol-3-yl)methyl)phenyl)-3-(3-
chlorophenyl)thiourea (3o)
White solid; Yield 75.7%; m.p. 212–214 °C; IR (ATR): 3419,
3337, 1594, 1457, 1333, 1110, 742 cmꢀ1
;
1H NMR (300 MHz,
Brown solid; Yield 78.2%; m.p. 197–199 °C; IR (ATR): 3409,
DMSO-d6): d 11.05 (s, 2H), 9.87 (s, 1H), 9.79 (s, 1H), 7.78 (s, 1H),
7.25–7.45 (m, 11H), 7.13 (dd, J = 11.6 Hz, 2.4 Hz, 2H), 6.90 (d,
3194, 1715, 1507, 1339, 1035, 740 cmꢀ1 1H NMR (300 MHz,
;
DMSO-d6): d 11.06 (s, 2H), 9.92 (s, 1H), 9.86 (s, 1H), 7.66 (s, 1H),
7.35–7.45 (m, 5H), 7.29–7.31 (m, 5H), 7.14 (d, J = 10.0 Hz, 1H),
J = 2.4 Hz, 2H), 5.83 (s, 1H); Anal. Calcd for
C30H21Br3N4S:

S. Imran et al. / Bioorganic Chemistry 62 (2015) 83–93
91
7.03 (dd, J = 11.2 Hz, 2.0 Hz, 2H), 6.93 (d, J = 1.6 Hz, 2H), 5.83 (s,
1H); Anal. Calcd for C30H21Cl3N4S: C = 62.56; H = 3.68; N = 9.73;
Found: C = 62.57; H = 3.69; N = 9.71; EI MS m/z (% rel. abund.):
575.2 (M+, 62.9%), 577.2 (M + 2, 23.7%).
(d, J = 8.8 Hz, 2H), 7.80 (d, J = 9.2 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H),
7.31 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 7.6 Hz, 2H), 6.82 (t, J = 6.8 Hz,
2H), 6.81 (d, J = 2.0 Hz, 2H), 6.79 (d, J = 8.0 Hz, 2H), 5.76 (s, 1H),
2.43 (s, 6H); Anal. Calcd for C32H27N5O2S: C = 70.44; H = 4.99;
N = 12.83; Found: C = 70.46; H = 5.01; N = 12.82; EI MS m/z (% rel.
abund.): 545.17 (M+, 57.5%).
4.4.16. 1-(4-(Bis(5-chloro-1H-indol-3-yl)methyl)phenyl)-3-(2,5-
dichlorophenyl)thiourea (3p)
Red solid; Yield 82.9%; m.p. 263–265 °C; IR (ATR): 3409, 3221,
4.4.22. 1-(4-(Bis(7-methyl-1H-indol-3-yl)methyl)phenyl)-3-(3-
chlorophenyl)thiourea (3v)
1589, 1510, 1479, 1415, 1337, 1094, 742 cmꢀ1
;
1H NMR
(300 MHz, DMSO-d6): d 11.06 (s, 2H), 10.12 (s, 1H), 9.40 (s, 1H),
7.76 (d, J = 3.2 Hz, 1H), 7.51 (d, J = 11.6 Hz, 1H), 7.40–7.38 (m,
3H), 7.33–7.35 (m, 2H), 7.29–7.32 (m, 4H), 7.02 (dd, J = 11.2 Hz,
1.6 Hz, 2H), 6.93 (d, J = 1.6 Hz, 2H), 5.84 (s, 1H); Anal. Calcd for
Dark red solid; Yield 74.7%; m.p. 204–206 °C; IR (ATR): 3421,
3321, 1606, 1586, 1536, 1454, 1339, 1095, 741 cmꢀ1 1H NMR
;
(400 MHz, DMSO-d6,): d 10.75 (s, 2H), 9.86 (s, 1H), 9.81 (s, 1H),
7.65 (s, 1H), 7.28–7.34 (m, 6H), 7.15 (d, J = 7.6 Hz, 1H), 7.10 (d,
J = 8.0 Hz, 2H), 6.82 (t, J = 6.8 Hz, 2H), 6.78 (d, J = 2.0 Hz, 2H), 6.76
(d, J = 7.2 Hz, 2H), 5.76 (s, 1H), 2.43 (s, 6H); Anal. Calcd for
C30H20Cl4N4S: C = 59.03; H = 3.30; N = 9.18; Found: C = 59.02;
H = 3.29; N = 9.16; EI MS m/z (% rel. abund.): 610.1 (M+, 55.3%),
612.1 (M + 2, 19.3%).
C32H27ClN4S: C = 71.83; H = 5.09; N = 10.47; Found: C = 71.82;
H = 5.08; N = 10.49; EI MS m/z (% rel. abund.): 534. 32 (M+, 72.5%).
4.4.17. Ethyl (4-(bis(5-chloro-1H-indol-3-yl)methyl)phenyl)
thioureaoate (3q)
4.4.23. Ethyl (4-(bis(7-methyl-1H-indol-3-yl)methyl)phenyl)
thioureaoate (3w)
Red solid; Yield 72.9%; m.p. 193–195 °C; IR (ATR): 3408, 3338,
1594, 1504, 1455, 1414, 1333, 742 cmꢀ1
;
1H NMR (300 MHz,
Orange solid; Yield 76.3%; m.p. 218–220 °C; IR (ATR): 3393,
DMSO-d6,): d 11.50 (s, 1H), 11.18 (s, 1H), 11.07 (s, 2H), 7.52 (d,
J = 10.8 Hz, 2H), 7.30–7.38 (m, 6H), 7.02 (dd, J = 11.2 Hz, 2.0 Hz,
2H), 6.93 (s, 2H), 5.86 (s, 1H), 4.2 (q, J = 9.2 Hz, 2H), 1.21 (t,
3170, 1701, 1526, 1450, 1344, 1038, 739 cmꢀ1 1H NMR
;
(400 MHz, DMSO-d6,): d 11.49 (s, 2H), 11.16 (s, 1H), 10.77 (s, 1H),
7.49 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 8.4 Hz,
2H), 6.84 (d, J = 6.8 Hz, 2H), 6.78 (s, 2H), 6.75 (d, J = 7.2 Hz, 2H),
5.80 (s, 1H), 4.20 (q, J = 7.2 Hz, 2H), 2.43 (s, 6H), 1.24 (t,
J = 7.2 Hz, 3H); Anal. Calcd for C29H28N4O2S: C, 70.14; H, 5.68; N,
11.28; Found: C, 70.15; H, 5.66; N, 11.27; EI MS m/z (% rel. abund.):
496.11 (M+, 62.4%).
J = 9.2 Hz, 3H); Anal. Calcd for
C27H22Cl2N4O2S: C = 60.34;
H = 4.13; N = 10.42; Found: C = 60.35; H = 4.12; N = 10.40; EI MS
m/z (% rel. abund.): 537.12 (M+, 55.3%), 539.12 (M+, 18.5%).
4.4.18. 1-(4-(Bis(7-methyl-1H-indol-3-yl)methyl)phenyl)-3-
phenylthiourea (3r)
Light red solid; Yield 85.8%; m.p. 238–239 °C; IR (ATR): 3427,
4.4.24. 1-(3-Bromophenyl)-3-(3-(di(1H-indol-3-yl)methyl)phenyl)
thiourea (4a)
3340, 1582, 1515, 1463, 1339, 1093, 785 cmꢀ1
;
1H NMR
(400 MHz, DMSO-d6,): d 10.74 (s, 2H), 9.69 (s, 2H), 7.44 (d,
J = 7.6 Hz, 1H), 7.43 (s, 1H), 7.26–7.35 (m, 6H), 7.10 (d, J = 7.6 Hz,
3H), 6.82 (d, J = 7.2 Hz, 2H), 6.78 (d, J = 1.6 Hz, 2H), 6.76 (t,
J = 7.2 Hz, 2H), 5.76 (s, 1H), 2.43 (s, 6H); Anal. Calcd for
Orange solid; Yield 84.5%; m.p. 235–237 °C; IR (ATR): 3412,
3323, 1593, 1510, 1446, 1344, 1063, 1019, 744 cmꢀ1 1H NMR
;
(400 MHz, DMSO-d6): d 10.80 (s, 2H), 9.85 (s, 1H), 9.74 (s, 1H),
7.79 (s, 1H), 7.19–7.38 (m, 11H), 7.02 (t, J = 7.6 Hz, 2H), 6.82–
6.88 (m, 4H), 5.79 (s, 1H); Anal. Calcd for C30H23BrN4S: C = 65.33;
H = 4.20; N = 10.16; Found: C = 65.34; H = 4.21; N = 10.14; EI MS
m/z (% rel. abund.): 552.13 (M+, 82.6%).
C32H28N4S: C = 76.77; H = 5.64; N = 11.19; Found: C = 76.75;
H = 5.65; N = 11.17; EI MS m/z (% rel. abund.): 500.23 (M+, 63.2%).
4.4.19. 1-(4-(Bis(7-methyl-1H-indol-3-yl)methyl)phenyl)-3-(2,5-
dichlorophenyl)thiourea (3s)
4.4.25. 1-(3-Chlorophenyl)-3-(3-(di(1H-indol-3-yl)methyl)phenyl)
thiourea (4b)
Light red solid; Yield 71%; m.p. 197–199 °C; IR (ATR): 3408,
3323, 1590, 1526, 1455, 1419, 1338, 1093, 742 cmꢀ1
;
1H NMR
Light orange solid; Yield 75.2%; m.p. 268–270 °C; IR (ATR):
(400 MHz, DMSO-d6,): d 10.75 (s, 2H), 10.08 (s, 1H), 9.37 (s, 1H),
10.75 (s, 1H), 7.76 (d, J = 4.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.38
(d, J = 8.0 Hz, 2H), 7.29–7.32 (m, J = 4.0 Hz, 3H), 7.11 (d, J = 8.0 Hz,
2H), 6.82–6.76 (m, 6H), 5.77 (s, 1H), 2.43 (s, 6H); Anal. Calcd for
3413, 3328, 1587, 1522, 1441, 1342, 1091, 781, 745 cmꢀ1 1H
;
NMR (400 MHz, DMSO-d6): d 10.80 (s, 2H), 9.85 (s, 1H), 9.75 (s,
1H), 7.67 (s, 1H), 7.29–7.39 (m, 8H), 7.22 (t, J = 7.6 Hz, 1H), 7.22
(d, J = 9.6 Hz, 1H), 7.13 (dd, J = 7.6 Hz, 2.0 Hz, 1H), 7.02 (t,
J = 7.6 Hz, 2H), 6.82–6.88 (m, 4H), 5.80 (s, 1H); Anal. Calcd for
C32H26Cl2N4S: C = 67.48; H = 4.60; N = 9.84; Found: C = 67.47;
H = 4.61; N = 9.83; EI MS m/z (% rel. abund.): 568.19 (M+, 55.8%).
C30H23ClN4S: C = 71.06; H = 4.57; N = 11.05; Found: C = 71.04;
H = 4.56; N = 11.04; EI MS m/z (% rel. abund.): 506.31 (M+, 43.3%).
4.4.20. 1-(4-(Bis(7-methyl-1H-indol-3-yl)methyl)phenyl)-3-(3-
bromophenyl)thiourea (3t)
4.4.26. 1-(2,5-Dichlorophenyl)-3-(3-(di(1H-indol-3-yl)methyl)phenyl)
thiourea (4c)
Brown solid; Yield 69.3%; m.p. 227–229 °C; IR (ATR): 3408,
3327, 1586, 1529, 1456, 1412, 1339, 1095, 743 cmꢀ1
;
1H NMR
Orange solid; Yield 89.6%; m.p. 251–253 °C; IR (ATR): 3412,
(400 MHz, DMSO-d6): d 10.75 (s, 2H), 9.86 (s, 1H), 9.79 (s, 1H),
7.78 (s, 1H), 7.40 (d, J = 7.2 Hz, 1H), 7.25–7.34 (m, 6H), 7.10 (d,
J = 7.6 Hz, 2H), 6.82 (d, J = 6.8 Hz, 2H), 6.79 (d, J = 2.0 Hz, 2H),
6.76 (d, J = 6.8 Hz, 2H), 5.76 (s, 1H), 2.43 (s, 6H); Anal. Calcd for
3323, 1587, 1508, 1429, 1342, 781, 746 cmꢀ1 1H NMR (400 MHz,
;
DMSO-d6): d 10.80 (s, 2H), 10.08 (s, 1H), 9.31 (s, 1H), 7.78 (d,
J = 2.0 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.42 (s, 2H), 7.19–7.33 (m,
7H), 7.02 (t, J = 7.2 Hz, 2H), 6.87 (d, J = 1.6 Hz, 2H), 6.84 (t,
C32H27BrN4S: C, 66.32; H, 4.70; N, 9.67; Found: C, 66.30; H, 4.68;
J = 8.0 Hz, 2H), 5.81 (s, 1H); Anal. Calcd for C30H22Cl2N4S:
N, 9.68; EI MS m/z (% rel. abund.): 580.04 (M+, 36.1%).
C = 66.54; H = 4.10; N = 10.35; Found: C = 66.53; H = 4.09;
N = 10.36; EI MS m/z (% rel. abund.): 540.18 (M+, 57.8%).
4.4.21. 1-(4-(Bis(7-methyl-1H-indol-3-yl)methyl)phenyl)-3-(4-
nitrophenyl)thiourea (3u)
4.4.27. 1-(4-Nitrophenyl)-3-(3-(di(1H-indol-3-yl)methyl)phenyl)
thiourea (4d)
Dark red solid; Yield 83.9%; m.p. 251–252 °C; IR (ATR): 3403,
3348, 1596, 1506, 1334, 1111, 744 cmꢀ1
;
1H NMR (400 MHz,
Dark brown solid; Yield 88.4%; m.p. 224–226 °C; IR (ATR): 3407,
DMSO-d6): d 10.76 (s, 2H), 10.29 (s, 1H), 10.18 (s, 1H), 8.17
3334, 1595, 1507, 1335, 787, 746 cmꢀ1 1H NMR (400 MHz,
;

92
S. Imran et al. / Bioorganic Chemistry 62 (2015) 83–93
DMSO-d6): d 10.80 (s, 2H), 10.23 (s, 1H), 10.17 (s, 1H), 8.15 (d,
J = 9.2 Hz, 2H), 7.76 (d, J = 9.2 Hz, 2H), 7.41 (s, 1H), 7.22–7.37 (m,
7H), 7.02 (t, J = 7.6 Hz, 2H), 6.87 (d, J = 1.6 Hz, 2H), 6.84 (d,
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2015.08.
001.
J = 8.0 Hz, 2H), 5.80 (s, 1H); Anal. Calcd for
C30H23N5O2S:
C = 69.61; H = 4.48; N = 13.53; Found: C = 69.62; H = 4.46;
N = 13.51; EI MS m/z (% rel. abund.): 517.26 (M+, 70.6%).
References
4.4.28. 1-(3-(Di(1H-indol-3-yl)methyl)phenyl)-3-phenylthiourea (4e)
Brown solid; Yield 80.9%; m.p. 187–189 °C; IR (ATR): 3410,
[1] C.T. Supuran, Bioorg. Med. Chem. Lett. 20 (2010) 3467.
[2] C.T. Supuran, J.Y. Winum (Eds.), Drug Design of Zinc-Enzyme Inhibitors:
Functional, Structural, and Disease Applications, Wiley, Hoboken, NJ, 2009. 15.
[3] S. Elleuche, S. Pöggeler, Curr. Gen. 55 (2009) 211.
3319, 1588, 1519, 1428, 1342, 784, 743 cmꢀ1 1H NMR (400 MHz,
;
DMSO-d6): d 10.80 (s, 2H), 9.70 (s, 1H), 9.63 (s, 1H), 7.43 (d,
J = 8.0 Hz, 2H), 7.40 (s, 1H), 7.27–7.37 (m, 7H), 7.22 (t, J = 8.0 Hz,
1H), 7.17 (d, J = 8.0 Hz, 1H), 7.09 (t, J = 8.0 Hz, 1H), 7.02 (t,
J = 8.0 Hz, 2H), 6.87 (d, J = 1.6 Hz, 2H), 6.84 (t, J = 7.6 Hz, 2H), 5.79
(s, 1H); Anal. Calcd for C30H24N4S: C = 76.24; H = 5.12; N = 11.85;
Found: C = 76.25; H = 5.14; N = 11.83; EI MS m/z (% rel. abund.):
472.15 (M+, 36.8%).
[4] V. Alterio, A. Di Fiore, K. D’Ambrosio, C.T. Supuran, G. De Simone, Chem. Rev.
112 (2012) 4421.
[5] G. De Simone, V. Alterio, C.T. Supuran, Expert Opin. Drug Disc. 8 (2013) 793.
[6] C.T. Supuran, J. Enzyme Inhib. Med. Chem. 27 (2012) 759.
[7] C.T. Supuran, Nat. Rev. Drug Disc. 7 (2008) 168.
[8] J.G. Millichap, D.M. Woodbury, L.S. Goodman, J. Pharm. Exp. Ther. 115 (1955)
251.
[9] M. Aggarwal, B. Kondeti, R. McKenna, Expert Opin. Ther. Pat. 23 (2013) 717.
[10] M. Leppilampi, P. Koistinen, E.R. Savolainen, J. Hannuksela, A.K. Parkkila, O.
Niemelä, S. Pastoreková, J. Pastorek, A. Waheed, W.S. Sly, S. Parkkila, H.
Rajaniemi, Clin. Cancer Res. 8 (2002) 2240.
4.4.29. Ethyl (4-(bis(1H-indol-3-yl)methyl)phenyl) thioureaoate (4f)
Light red solid; Yield 78.4%; m.p. 224–226 °C; IR (ATR): 3410,
[11] F. Mincione, A. Scozzafava, C.T. Supuran, Curr. Pharm. Des. 14 (2008) 649.
[12] J.Y. Winum, D. Vullo, A. Casini, J.L. Montero, A. Scozzafava, C.T. Supuran, J. Med.
Chem. 46 (2003) 2197.
[13] F. Abbate, J.Y. Winum, B.V. Potter, A. Casini, J.L. Montero, A. Scozzafava, C.T.
Supuran, Bioorg. Med. Chem. Lett. 14 (2004) 231.
[14] J.Y. Winum, A. Scozzafava, J.L. Montero, C.T. Supuran, Curr. Pharm. Des. 14
(2008) 615.
[15] C.T. Supuran, Curr. Pharm. Des. 14 (2008) 641.
3208, 1717, 1510, 1334, 1036, 784, 744 cmꢀ1 1H NMR (400 MHz,
;
DMSO-d6): d 11.45 (s, 2H), 11.15 (s, 1H), 10.81 (s, 1H), 7.54 (s,
1H), 7.33 (d, J = 8.0 Hz, 2H), 7.27–7.33 (m, 5H), 7.02 (t, J = 7.2 Hz,
2H), 6.85 (d, J = 1.6 Hz, 2H), 6.84 (t, J = 7.6 Hz, 2H), 5.82 (s, 1H),
4.17 (q, J = 6.8 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H); Anal. Calcd for
[16] A. Thiry, J.M. Dogne, C.T. Supuran, B. Masereel, Curr. Top. Med. Chem. 7 (2007)
855.
C27H24N4O2S: C = 69.21; H = 5.16; N = 11.96; Found: C = 69.22;
[17] G.D. Simone, A.D. Fiore, C.T. Supuran, Curr. Pharm. Des. 14 (2008) 655.
[18] D. Neri, C.T. Supuran, Nat. Rev. Drug Disc. 10 (2011) 767.
[19] E.T. Mack, P.W. Snyder, R. Perez-Castillejos, B. Bilgiçer, D.T. Moustakas, M.J.
Butte, G.M. Whitesides, J. Am. Chem. Soc. 134 (2011) 333.
[20] M. Kodomari, M. Suzuki, K. Tanigawa, T. Aoyama, Tetrahedron Lett. 46 (2005)
5841.
H = 5.17; N = 11.94; EI MS m/z (% rel. abund.): 468.15 (M+, 42.7%).
4.5. Assay for carbonic anhydrase (CA) II inhibition activity
[21] C. Walpole, S.Y. Ko, M. Brown, D. Beattie, E. Campbell, F. Dickenson, S. Ewan, G.
A. Hughes, M. Lemaira, J. Lerpiniere, S. Patel, L. Urban, J. Med. Chem. 41 (1998)
3159.
This was assay performed using buffer containing Tris and
HEPES at the pH of 7.2–7.9 and a total concentration of 20 mM.
[22] (a) D.C. Schroeder, Chem. Rev. 55 (1955) 181–228;
(b) G.Y. Sarkis, E.D. Faisal, J. Heterocycl. Chem. 22 (1985) 137.
[23] A.G. Makhsumov, A.S. Safaev, S.V. Abidova, Chem. Abstr. 71 (1969) 101668.
[24] M. Taha, N.H. Ismail, W. Jamil, K.M. Khan, U. Salar, S.M. Kashif, F. Rahim, Y.
Latif, Med. Chem. Res. 24 (24) (2015) 3166–3173.
[25] K.M. Khan, F. Naz, M. Taha, A. Khan, S. Perveen, M.I. Choudhary, W. Voelter,
Eur. J. Med. Chem. 74 (2014) 314–323.
[26] F.T. Elmali, U. Avciata, N. Demirhan, Main Group Chem. 10 (2011) 17.
[27] C. Levallet, J. Lerpiniere, S.Y. Ko, Tetrahedron 53 (1997) 5291.
[28] (a) M. Taha, N.H. Ismail, M. Ali, K.M. Khan, W. Jamil, S.M. Kashif, M. Asraf, Med.
Chem. Res. 23 (2014) 5282–5293;
(b) F. Rahim, F. Malik, H. Ullah, A. Wadood, F. Khan, M.T. Javid, M. Taha, W.
Rehman, A.U. Rehman, K.M. Khan, Bioorg. Chem. 60 (2015) 42–48.
[29] (a) F. Rahim, H. Ullah, M.T. Javid, A. Wadood, M. Taha, M. Ashraf, A. Shaukat, M.
Junaid, S. Hussain, W. Rehman, R. Mehmood, M. Sajid, M.N. Khan, K.M. Khan,
Bioorg. Chem. 62 (2015) 15–21;
96-well plates were filled with 140
20 L of bovine erythrocyte carbonic anhydrase II solution (0.1–
0.2 mg/2000 mL), and 20 L of 0.7 mM para-nitrophenyl acetate
(diluted in ethanol). Test compounds were incubated at 25–28 °C
for 15 min prior to addition of para-nitrophenyl acetate, which
starts the reaction. The reaction was allowed to taking place for
30 min at 25–28 °C. All compounds tested in triplicate at different
concentration. Amount of product that forms during the reaction
monitored at 400 nm after 30 min using SPECTRA-max 340 spec-
trophotometer, Molecular Devices (USA) [60].
lL of Tris-HEPES solution,
l
l
4.6. Molecular docking
(b) M.A. Mesaik, K.M. Khan, F. Rahim, M. Taha, S.M. Haider, S. Perveen, A.S.
Khalid, O.M. Abdalla, S. Soomro, W. Voelter, Bioorg. Chem. 60 (2015) 118–122;
(c) F. Rahim, K. Ullah, H. Ullah, A. Wadood, M. Taha, A.U. Rehman, I. uddin, M.
Ashraf, A. Shaukat, W. Rehman, S. Hussain, K.M. Khan, Bioorg. Chem. 58 (58)
(2015) 81–87.
In docking studies, AutoDock 4.2 [61] was used to dock the
small molecules into the protein active site. Lamarckian-Genetic
Algorithm (LGA) was employed for this study. Human carbonic
anhydrase containing native crystal structure of acetazolamide
(PDB ID: 3HS4) [62], at resolution of 1.10 Å, which was used
throughout the docking study, was being selected using
SwissModel [63]. All the water molecules removed from the pro-
tein and hydrogens were added. Protein–ligand interactions ana-
lyzed using Discovery Studio Visualizer 3.5 [64] and PyMOL [65].
[30] N. Saracoglu, Functionalization of indole and pyrrole cores via Michael-type
additions, in: Bioactive Heterocycles V, Springer, Berlin Heidelberg, 2007, pp.
1–61.
[31] S. Sakemi, H.H. Sun, J. Org. Chem. 56 (1991) 4304.
[32] K. Wang, Z. Liu, Syn. Commun. 40 (2009) 144.
[33] C. Pal, S. Dey, S.K. Mahato, J. Vinayagam, P.K. Pradhan, V.S. Giri, P. Jaisankara, T.
Hossaina, S. Baruria, D. Raya, S.M. Biswas, Bioorg. Med. Chem. Lett. 17 (2007)
4924.
[34] G. Sivaprasad, P.T. Perumal, V.R. Prabavathy, N. Mathivanan, Bioorg. Med.
Chem. Lett. 16 (2006) 6302.
[35] X. Chang, J.C. Tou, C. Hong, H.A. Kim, J.E. Riby, G.L. Firestone, L.F. Bjeldanes,
Carcinogenesis 26 (2005) 771.
Acknowledgments
[36] S. Imran, M. Taha, N.H. Ismail, K.M. Khan, F. Naz, M. Hussain, S. Tauseef,
Molecules 19 (2014) 11722.
[37] (a) K.M. Khan, F. Rahim, A. Wadood, M. Taha, M. Khan, S. Naureen, N. Ambreen,
S. Hussain, S. Perveen, M.I. Choudhary, Bioorg. Med. Chem. Lett. 24 (2014)
1825;
Authors would like to acknowledge Universiti Teknologi MARA
for the financial support under grant file No. UiTM 600-RMI/DANA
5/3/CIFI (142/2013). The first author would like to thank UiTM and
Ministry of Higher Education Malaysia for the financial funding
through Dana Kecemerlangan Pendidikan UiTM and MyBrain15
scholarship.
(b) K.M. Khan, F. Rahim, S.A.A. Shah, M. Taha, N.H. Ismail, M. Manzoor, G.A.
Miana, S. Perveen, J. Chem. Soc. Pak. 36 (2014) 1158–1161;
(c) K.M. Khan, M. Taha, F. Rahim, W. Jamil, S. Perveen, M.I. Choudhary, J. Iran
Chem. Soc. 9 (2012) 81–83.

S. Imran et al. / Bioorganic Chemistry 62 (2015) 83–93
93
[38] C.A. Bradfield, L.F. Bjeldanes, J. Toxicol. Environ. Health, Part A: Current Issues
21 (1987) 311.
[57] O. Talaz, H. Çavdar, S. Durdagi, H. Azak, D. Ekinci, Bioorg. Med. Chem. 21 (2013)
1477.
[39] R.H. Dashwood, L. Uyetake, A.T. Fong, J.D. Hendricks, G.S. Bailey, Food Chem.
Toxicol. 27 (1989) 385.
[40] M.A. Zeligs, J. Med. Food 1 (1998) 67.
[58] (a) M. Taha, N.H. Ismail, W. Jamil, H. Rashwan, S.M. Kashif, A.A. Sain, M.I.
Adenan, E.H. Anouar, M. Ali, F. Rahim, K.M. Khan, Eur. J. Med. Chem. 84 (2014)
731;
[41] M. Chakrabarty, R. Basak, Y. Harigaya, Heterocycles 55 (2001) 2431.
[42] C.W. Wright, J.D. Phillipson, Phytother. Res. 4 (1990) 127.
[43] C.W. Wright, D. Allen, Y. Cai, J.D. Phillipson, I.M. Said, G.C. Kirby, D.C. Warhurst,
Phytother. Res. 6 (1992) 121.
[44] C. Bonnesen, I.M. Eggleston, J.D. Hayes, Cancer Res. 61 (2001) 6120.
[45] T. Inamoto, S. Papineni, S. Chintharlapalli, S.D. Cho, S. Safe, A.M. Kamat, Mol.
Cancer Ther. 7 (2008) 3825.
(b) Z.S. Saify, A. Kamil, S. Akhtar, M. Taha, A. Khan, K.M. Khan, S. Jahan, F.
Rahim, S. Perveen, M.I. Choudhary, Med. Chem. Res. 23 (2014) 4447;
(c) M. Taha, N.H. Ismail, S. Lalani, M.Q. Fatmi, Atia-tul-Wahab, S. Siddiqui, K.M.
Khan, S. Imran, M.I. Choudhary, Eur. J. Med. Chem. 92 (2015) 387;
(d) M. Taha, N.H. Ismail, M.S. Baharudin, S. Lalani, S. Mehboob, K.M. Khan, S.
Yousuf, S. Siddiqui, F. Rahim, M.I. Choudhary, Med. Chem. Res. 24 (2015) 1310;
(e) K.M. Khan, F. Rahim, A. Wadood, N. Kosar, M. Taha, S. Lalani, A. Khan, M.I.
Fakhri, M. Junaid, W. Rehman, M. Khan, S. Perveen, M. Sajid, M.I. Choudhary,
Eur. J. Med. Chem. 81 (2014) 245.
[46] M. York, M. Abdelrahim, S. Chintharlapalli, S.D. Lucero, S. Safe, Clin. Cancer Res.
13 (2007) 6743.
[47] N. Ichite, M.B. Chougule, T. Jackson, S.V. Fulzele, S. Safe, M. Singh, Clin. Cancer
Res. 15 (2009) 543.
[48] S.D. Cho, K. Yoon, S. Chintharlapalli, M. Abdelrahim, P. Lei, S. Hamilton, S. Khan,
S.K. Ramaiah, S. Safe, Cancer Res. 67 (2007) 674.
[49] S. Chintharlapalli, S. Papineni, S.J. Baek, S. Liu, S. Safe, Mol. Pharmacol. 68
(2005) 1782.
[50] D.R. Parkin, Y. Lu, R.L. Bliss, D. Malejka-Giganti, Food Chem. Toxicol. 46 (2008)
2451.
[59] C.T. Supuran, A. Scozzafava, Bioorg. Med. Chem. 15 (2007) 4336.
[60] (a) K.M. Khan, M. Khan, M. Saleem, M. Taha, S. Perveen, M.I. Choudhary, J. Pak.
Chem. Soc. 35 (2013) 901–904;
(b) P.S. Richard, R.D. Dennis, J.S. Anthony, B. Meir, Epilepsy Res. 63 (2005) 103;
(c) Y. Pocker, J.T. Stone, Biochemistry 6 (1967) 668.
[61] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J.
Olson, J. Comp. Chem. 16 (2009) 2785.
[62] Katherine H. Sippel, Arthur H. Robbins, John Domsic, Caroli Genis, Mavis
Agbandje-McKenna, Robert McKenna, Acta Cryst. Sect. F: Struct. Biol. Cryst.
Commun. 65 (2009) 992.
[63] (a) F. Kiefer, K. Arnold, M. Kunzli, L. Bordoli, T. Schwede, Nucleic Acids Res. 37
(2009) D387–D392;
(b) K. Arnold, L. Bordoli, J. Kopp, T. Schwede, Bioinformatics 22 (2006) 195;
(c) N. Guek, M.C. Peitsch, T. Schwede, Electrophoresis 30 (2009) S162.
[64] Accelrys Software Inc., Discovery Studio Modeling Environment, Release 3.5,
Accelrys Software Inc., San Diego, 2012.
[51] C. Pisano, P. Kollar, M. Giannı, Y. Kalac, V. Giordano, F.F. Ferrara, R. Tancredi, A.
´
Devoto, A. Rinaldi, A. Rambaldi, S. Penco, M. Marzi, G. Moretti, L. Vesci, O. Tinti,
P. Carminati, M. Terao, E. Garattini, Blood 100 (2002) 3719.
[52] S.H. Benabadji, R. Wen, J.B. Zheng, X.C. Dong, S.G. Yuan, Acta Pharmacol. Sin. 25
(2004) 666.
[53] L. Valeria, M. Ernesto, EP0887348, 1998.
[54] L. Valeria, M. Ernesto, O. Ambrogio, G. Frank, K. Hans-Willi, EP0991645, 2000.
[55] K. Sujatha, P.T. Perumal, D. Muralidharan, M. Rajendran, Indian J. Chem. Sect. B
48 (2009) 267.
[65] The PyMol Molecular Graphics System, Version 1.1 Schrödinger LLC.
[56] Ö. Güzel, C. Temperini, A. Innocenti, A. Scozzafava, A. Salman, C.T. Supuran,
Bioorg. Med. Chem. Lett. 18 (2008) 152.