Synthesis, β-glucuronidase inhibition and molecular docking studies of hybrid bisindole-thiosemicarbazides analogs

Article abstract of DOI:10.1016/j.bioorg.2016.07.008

Hybrid bisindole-thiosemicarbazides analogs (1–18) were synthesized and screened for β-glucuronidase activity. All compounds showed varied degree of β-glucuronidase inhibitory potential when compared with standard D-saccharic acid 1,4-lactone (IC₅₀

Full text of DOI:10.1016/j.bioorg.2016.07.008

Bioorganic Chemistry 68 (2016) 56–63
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, b-glucuronidase inhibition and molecular docking studies
of hybrid bisindole-thiosemicarbazides analogs
a,b,
⇑
, Nor Hadiani Ismail a,b, Syahrul Imran a,b, Fazal Rahim , Abdul Wadood ,
d
c
Muhammad Taha
c
d
e
e
Huma Khan , Hayat Ullah , Uzma Salar , Khalid Mohammed Khan
a
Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA, Puncak Alam 42300, Malaysia
Faculty of Applied Science, Universiti Teknologi MARA, Shah Alam 40450, Selangor D.E., Malaysia
Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali Khan University Mardan, Pakistan
Department of Chemistry, Hazara University, Mansehra-21300, Khyber Pakhtunkhwa, Pakistan
b
c
d
e
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:
Hybrid bisindole-thiosemicarbazides analogs (1–18) were synthesized and screened for b-glucuronidase
activity. All compounds showed varied degree of b-glucuronidase inhibitory potential when compared
Received 28 April 2016
Revised 30 June 2016
Accepted 17 July 2016
Available online 18 July 2016
with standard
8 showed exceptional b-glucuronidase inhibition with IC50 values ranging from 0.1 to 5.7
Compounds 1, 3, 8, 16, 13, 2 and 14 also showed better activities than standard with IC50 values ranging
from 7.12 to 15.0 M. The remaining compounds 10, 11, and 15 showed good inhibitory potential with
IC50 values 33.2 ± 0.75, 21.4 ± 0.30 and 28.12 ± 0.25 M respectively. Molecular docking studies were
carried out to confirm the binding interaction of the compounds.
Ó 2016 Elsevier Inc. All rights reserved.
D
-saccharic acid 1,4-lactone (IC50 = 48.4 ± 1.25
l
M). Compounds 4, 7, 9, 6, 5, 12, 17 and
1
l
M.
l
Keywords:
Synthesis
Bisindole
b-glucuronidase
Docking studies
l
1
. Introduction
responsible for the promotion of beneficial estrogen metabolism
and produce apoptosis in human cancer cells [21]. Bis(indolyl)
methanes and its derivatives are also used as dietary supplements
for humans [22–24]. It has inhibitory activity against bladder can-
cer growth [25], renal cell carcinoma growth [26], inhibit lung can-
cer [27] and colon cancer [28,29]. Some derivatives also show
inhibitory activity against mammary tumor growth [30,31] and
few analogs have potent antitumor activity [32]. They are applied
as chemotherapeutic agents against tumors [33].
In this study, a novel series of hybrid analogs of bis(indolyl)
methane and thiosemicarbazide had been synthesized and tested
for their ability to inhibit b-glucuronidase activity. In our previous
study, simple bis(indolyl)methane and thiourea derivatives had
shown good inhibitory activity against b-glucuronidase [34,35].
The binding modes of active compounds were also identified to
further understand how the molecules bind with b-glucuronidase
protein.
b-Glucuronidase (EC 3.2.1.31) is an inducible enzyme found in
an aerobic Escherichia, Bacteroides, Clostridia and Peptostreptococcus
genera which catalyzes the cleavage of b-glucuronosyl-O-bonds [1].
In human body, this enzyme is present in fluids and organs such as
spleen, serum, bile, urine and kidney [2,3]. b-Glucuronidase
improves activity in diverse pathological circumstances such as
renal diseases [4], epilepsy [5], breast, larynx, testes [6] and in uri-
nary tract infections [7–10]. Moreover, b-glucuronidase has been
reported to be released in the synovial fluid in the inflammatory
joint diseases, for instance, rheumatoid arthritis [11,12]. The
involvement of b-glucuronidase in the colon cancer, and higher
intestinal levels of the enzyme is correlated to the higher incidence
of colon carcinoma [13]. Therefore, inhibition of b-glucuronidase
enzyme is effective in preventing various diseases.
Bis(indolyl)methanes form an important class of heterocyclic
compounds [14–18] that show a variety of pharmacological activ-
ities and play an important role in the treatment of chronic fatigue,
fibromyalgia and irritable bowel syndrome [19,20]. They are also
2
. Results and discussion
2
.1. Chemistry
⇑
Corresponding author at: Atta-ur-Rahman Institute for Natural Product
Discovery, Universiti Teknologi MARA, Puncak Alam 42300, Malaysia.
E-mail addresses: taha_hej@yahoo.com, muhamm9000@puncakalam.uitm.edu.
my (M. Taha).
In the continuation working on active molecules based on
indole [36–41]; we synthesized of bisindole-thiosemicarbazides
http://dx.doi.org/10.1016/j.bioorg.2016.07.008
0
045-2068/Ó 2016 Elsevier Inc. All rights reserved.

M. Taha et al. / Bioorganic Chemistry 68 (2016) 56–63
57
analogs 1–18 started with the synthesis of methyl 4-(di(1H-indol-
-yl)methyl)benzoate I. The indole (double molar equivalent) was
remaining compounds 7, 13, and 15 also showed remarkable inhi-
bitory potential with IC50 values 33.2 ± 0.75, 21.4 ± 0.30 and
3
treated to methyl 4-formylbenzoate in the presence of acetic acid
28.12 ± 0.25 lM respectively.
to form methyl 4-(di(1H-indol-3-yl)methyl)benzoate I. The methyl
Structure-activity relationship suggested that the activity of a
specific molecule is superficially directed by the substitution pre-
sent at aromatic residues attach to the thiosemicarbazides. The
4
-(di(1H-indol-3-yl)methyl)benzoate I was treated with methano-
lic hydrazine hydrate to form 4-(di(1H-indol-3-yl)methyl)benzohy
drazide II. The 4-(di(1H-indol-3-yl)methyl)benzohydrazide II was
treated with different isothiocyanates to form bisindole-
thiosemicarbazides derivatives 1–18.
ortho-fluoro analog 2 (IC50 = 0.1 ± 0.001
3 (IC50 = 0.5 ± 0.001 M), ortho-bromo analog 4 (IC50 = 2.9 ± 0.01
M), para-fluoro analog (IC50 = 3.1 ± 0.04 M), meta-fluoro
analog 12 (IC50 = 2.3 ± 0.01 M), meta-nitro analog 14
(IC50 = 5.7 ± 0.01 M), 3,4-dichloro analog 16 (IC50 = 0.12 ± 0.001
M), and para-trifluoromethane analog 17 (IC50 = 0.20 ± 0.001 M)
lM), ortho-chloro analog
l
l
6
l
l
2.2. In vitro b-glucuronidase inhibitory potential
l
l
l
Compounds 1–18 were screened for b-glucuronidase activity.
respectively exhibited marvelous inhibitory potential among the
series. Although the whole series of compounds is active, but it
was observed that bringing about variation in substitution pattern
of aromatic ring attach to thiosemicarbazides resulted in activities
difference. Compound 2 (ortho-fluoro analog) is found to be the
most active analog among the series with IC50 value
0.1 ± 0.001 lM. The most potent activity of this compound might
be due to fluoro group an electronegative group which might be
involved in hydrogen bonding. Compound 16 got the second posi-
All compounds showed varied degree of b-glucuronidase inhibi-
tory potential when compared with standard -saccharic acid
M). Compounds 2, 3, 4, 6, 12,
4, 16 and 17 showed exceptional b-glucuronidase inhibition with
D
1
1
,4-lactone (IC50 value 48.4 ± 1.25 l
IC50 values 0.1 ± 0.001, 0.5 ± 0.001, 2.9 ± 0.01, 3.1 ± 0.04, 2.3 ± 0.01,
.7 ± 0.01, 0.12 ± 0.001 and 0.20 ± 0.001 M, respectively, which
are many fold better than the standard inhibitor. Compound 1, 5,
, 9, 10, 11 and 18 showed potential 2–6 times better the standard
with IC50 values 7.12 ± 0.04, 15.0 ± 0.38, 8.5 ± 0.06, 11.6 ± 0.15,
2.01 ± 0.11, 14.7 ± 0.16, and 9.40 ± 0.01 M respectively. The
5
l
8
tion in series with IC50 value 0.12 ± 0.001
lM. The compound has
1
l
3,4-dichloro group substituent an electronegative group which
Table 1
The b-glucuronidase activity of bisindole analogs (1–18).
S. no.
R
IC50
(l
M ± SEMa)
S. no.
R
IC50 (l
M ± SEMa)
1
7.12 ± 0.04
10
12.01 ± 0.11
2
3
0.1 ± 0.001
0.5 ± 0.001
11
12
14.7 ± 0.16
2.3 ± 0.01
4
5
2.9 ± 0.01
13
14
21.4 ± 0.30
5.7 ± 0.01
15.0 ± 0.38
6
7
8
3.1 ± 0.04
33.2 ± 0.75
8.5 ± 0.06
15
16
17
28.12 ± 0.25
0.12 ± 0.001
0.20 ± 0.001
9
11.6 ± 0.15
18
9.40 ± 0.01
D-saccharic acid 1,4-lactone
48.4 ± 1.25 lM
a
IC50 values are expressed as mean ± standard error of mean.

5
8
M. Taha et al. / Bioorganic Chemistry 68 (2016) 56–63
Table 2
2.3. Docking study
Predicted docking scores of bisindole analogs (1–18).
Compound
Docking score
Compound
Docking score
From the docking results a good correlation between the dock-
ing scores and biological activities of these newly synthesized
compounds was observed (Tables 1 and 2). For example the dock-
ing scores of the most active compounds, compound 2, 3, 4, 12, 14,
1
2
3
4
5
6
7
8
9
À12.3022
À16.4534
À14.0556
À13.9201
À10.9523
À13.8253
À09.716
10
11
12
13
14
15
16
17
18
À11.0304
À11.1638
À13.6479
À10.2442
À12.9388
À10.3289
À15.0909
À15.0664
À12.6581
1
6 and 17 were found to be more negative as compare to the less
active compounds, compound 5, 7 and 15 (Tables 1 and 2). The
docking conformations of all the compound showed that all the
active derivatives showed significant binding interactions with
the important catalytic site residues of the target protein. Com-
pounds 2, 3, 4, 12, 14, 16 and 17 give good interactions with the
important active site residues of the enzyme. The docking confor-
mation of the most active compound, compound 2 (ortho-fluoro
analog) showed eight interactions with the important active site
residues of b-glucuronidase. The ortho-fluoro analog makes
arene-arene interaction with Tyr 508, the hydrogen atom of
thiourea forms hydrogen bond with the Glu 451 while the indole
and benzamide moiety of the compound forms week interactions
with the His 509 residue of the protein (Fig. 1a). The second most
active compound, compound 16 formed two arene-cation interac-
tions. The meta and para-chloro analog forms arene-cation interac-
tion with His 509 and the indole moiety of the compound forms
arene-cation interaction with Arg 600 residues of the enzyme
À11.9401
À10.2003
might be responsible for this potent inhibition. Compound 17 got
the third position among the series with IC50 value
.20 ± 0.001 lM. This compound has trifluoromethane group at
para position of aromatic ring which might be helpful in this inhibi-
tion. The compounds 6 and 12 having para-fluoro and meta-fluoro
group with IC50 values 3.1 ± 0.04 and 2.3 ± 0.01 lM, respectively
0
also showed potent inhibition. The potential of these analogs are
less than compound 2. The slight activity difference among these
analogs might be due to differences in position of fluoro group on
the aromatic ring. Compound 3 (ortho-chloro analog) is also the
most active analog among the series. The activity of compound
might be due to chloro group an electronegative group. If we com-
pared compound 3 with compound 8 a para-chloro analog with IC50
value 8.5 ± 0.06
ference in position of chloro group as well. Compound 1 ortho-
methyl analog with IC50 value 8.5 ± 0.06 M, 5 para-methyl analog
with IC50 value 15.0 ± 0.38 M and 11 a meta-methyl analog with
IC50 value 14.7 ± 0.16 M. The great activity difference is mainly
because of the position difference of substituent. Similarly if we
see to the compound 9 a para-methoxy analog with IC50 value
1.6 ± 0.15
8.12 ± 0.25
.40 ± 0.01
(Fig. 1b).
lM. The changes in activity might be due to the dif-
The third most active compound, compound 17 showed four
interactions with the active site residues. Tyr 205 form hydrogen
bond with the hydrogen of indole moiety and His 509 interact with
l
l
the
p electron system of trifluoromethyl benzene group of the
l
compound (Fig. 2a). These three compounds have halogens atoms
at the ortho, meta, and para positions of R-group and showed
interactions with important residues of the binding pocket of
b-glucuronidase. Fluorine which due to its smaller size and high
electronegativity produces enough polarizability in the molecule
and thus molecules having F at proper position showed good
results.
For example, compound 12 having a meta-fluoro group, also
showed good interactions with the active pocket of protein. In
compound 12 the aromatic ring of meta-fluoro group forms
1
2
9
l
l
l
M, 15 a meta- methoxy analog with IC50 value
M and 18 a ortho-methoxy analog with IC50 value
M. We have found here that either electron withdraw-
ing group or electron donating group at ortho position showed great
inhibition, but the electron withdrawing group are superior to elec-
tron donating group overall. In case of bromo substituted analogs 4,
7
and 13 and nitro-substituted analogs 10 and 14 we have observed
similar results. The binding interactions of the most active com-
pounds were confirmed through molecular docking studies.
p
-interaction with the Arg 600 residue and the indole group made
two arene-arene interactions with the His 509 (Fig. 2b). Bromo and
Fig. 1. Docking conformation of compound 2 (a) and compound 16 (b) in the active site of b-D-glucuronidase.

M. Taha et al. / Bioorganic Chemistry 68 (2016) 56–63
59
Fig. 2. Docking conformation of compound 17 (a) and compound 12 (b) in the active site of b-D-glucuronidase.
Fig. 3. Docking conformation of compound 4 (a) and compound 14 (b) in the active site of b-D-glucuronidase.
nitro-substituted analogs (compound 4 and 14) also showed inter-
4. Experimental
actions with the binding site residues of the enzyme. In case of
compound 4, the bromine substituted moiety showed interactions
with His 509 whereas the indole moiety makes interactions with
the Tyr 508 residues of the enzyme (Fig. 3a). In case of compound
4.1. Chemistry
NMR experiments were performed on Ultra Shield Bruker FT
NMR 500 MHz; CHN analysis was performed on a Carlo Erba
Strumentazion-Mod-1106, Italy. Electron impact mass spectra
(EI-MS) were recorded on a Finnegan MAT-311A, Germany. Thin
layer chromatography (TLC) was performed on pre-coated silica
gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany).
Chromatograms were visualized by UV at 254 and 365 nm.
1
4, the docking conformation showed that the nitro-substituted
moiety interacts with His 509 and the aromatic ring of benzamide
moiety makes arene-arene interaction with Tyr 508 residues of the
enzyme (Fig. 3b).
3
. Conclusion
The use of b-glucuronidase for glucuronide hydrolysis is a
4.2. Synthesis of methyl 4-(di(1H-indol-3-yl)methyl)benzoate (I)
common practice in bio-analytical techniques. The present study
reports the synthesis and biological evaluation of a series of new
hybrids bisindole-thiosemicarbazide derivatives (1–18). All syn-
thesized derivatives showed excellent b-glucuronidase inhibitory
Methyl 2-hydroxybenzoate (4.03 g, 27 mmol) and indole
(6.32 g, 53 mmol) were mixed in glacial acetic acid (100 mL). The
mixture was refluxed for 8 h. Upon completion, reaction mixture
was treated with sodium carbonate and extracted using ethyl
acetate (50 mL Â 4). The solvent was evaporated under reduced
pressure and the product was crystallized with methanol. Yield is
potential if compared with the standard
D-saccharic acid 1,
4
-lactone. The results of our synthesized derivatives encouraged
us to further develop potential b-glucuronidase inhibitors based
on bisindole in future.
1
9.51 g (92.7%). White solid, m.p. 219–221 °C; H NMR (500 MHz,

6
0
M. Taha et al. / Bioorganic Chemistry 68 (2016) 56–63
DMSO-d
6
): d d 7.95 (d, J = 7.4 Hz, 2H), 7.72 (dd, J = 7.5, 1.6 Hz, 2H),
.54 (dd, J = 7.5, 1.5 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 7.39 (td, J = 8.0,
.5 Hz, 2H), 7.27 (td, J = 7.4, 1.5 Hz, 2H), 6.96 (s, 2H), 5.72 (s, 1H),
H = 4.51, N = 13.14; HR MS Calcd for C31
533.1675.
5
H24FN OS: 533.1686 found
7
1
3
1
1
1
1
3
6
.95 (s, 3H); C NMR (DMSO-d , 125 MHz): d 167.38, 1402,
38.42, 138.42, 130.81, 128.61, 128.61, 128.19, 128.19, 126.97,
26.97, 123.48, 123.48, 121.67, 121.67, 121.42, 121.42, 118.42,
18.42, 113.39, 113.39, 110.18, 110.18, 52.13, 37.27; HREI-MS:
4.4.3. N-(2-Chlorophenyl)-2-(4-(di(1H-indol-3-yl)methyl)benzoyl)
hydrazinecarbothioamide (3)
Yield: 0.40 g (73%); 1H NMR (500 MHz, DMSO-d
6
): d 12.02 (s,
m/z calcd for C25H20N2O2 [M]+ 380.1525; Found 380.1532; Anal.
Calcd for C25 , C, 78.93; H, 5.30; N, 7.36 found C, 78.94; H,
2H), 10.48 (s, 2H), 8.82 (s, 1H), 8.02 (s, 1H), 7.82 (d, J = 7.9 Hz,
2H), 7.50 (t, J = 7.0 Hz, 2H), 7.43 (d, J = 7.0 Hz, 2H), 7.38 (d,
J = 8.0 Hz, 2H), 7.31 (d, J = 7.8 Hz, 2H), 7.12 (t, J = 7.5 Hz, 2H),
20 2 2
H N O
5
.28; N, 7.35.
1
3
7
.04–6.96 (m, 3H), 6.89 (s, 2H), 5.95 (s, 1H); C NMR (125 MHz,
DMSO-d ):d 180.87, 166.97, 142.39, 138.52, 138.52, 134.64,
34.42, 129.85, 129.36, 129.36, 129.28, 127.73, 127.73, 127.23,
26.84, 126.84, 125.34, 125.08, 123.53, 123.53, 121.48, 121.48,
21.35, 121.35, 118.12, 118.12, 113.47, 113.47, 110.19, 110.19,
4.3. Synthesis of 4-(di(1H-indol-3-yl)methyl)benzohydrazide (II)
6
1
1
1
3
A mixture of compound I (7.00 g, 18.4 mmol) and hydrazine
hydrate (20 mL) were refluxed for 6 h in 100 mL methanol. When
reaction has completed, methanol was evaporated and the residue
was washed with diethyl ether, filtered, dried, and crystallized
7.40; Anal. Calcd for
31 5
C H24ClN OS, C = 67.69, H = 4.40,
N = 12.73; Found C = 67.67, H = 4.42, N = 12.75; HR MS Calcd for
24ClN OS: 549.1390 found 549.1398.
31
C H
5
from ethanol and to give a brownish solid, (5.85 g, 83.7%). m.p.
1
2
53–255 °C; H NMR (500 MHz, DMSO-d
6
): d 8.45 (s, 2H), 7.89
(
d, J = 7.5 Hz, 2H), 7.72 (dd, J = 7.3, 1.6 Hz, 2H), 7.54 (dd, J = 7.4,
.5 Hz, 2H), 7.41 (d, J = 7.5 Hz, 2H), 7.39 (dd, J = 7.3, 1.6 Hz, 2H),
4
.4.4. N-(2-Bromophenyl)-2-(4-(di(1H-indol-3-yl)methyl)benzoyl)
1
7
2
1
1
1
hydrazinecarbothioamide (4)
.28 (dd, J = 7.3, 1.6 Hz, 2H), 7.08 (s, 2H), 5.66 (s, 1H), 3.82 (s,
1
1
3
Yield: 0.42 g (71%); H NMR (500 MHz, DMSO-d ): d 12.05 (s,
H); C NMR (150 MHz, DMSO-d
6
): d 167.54, 142.19, 138.56,
6
2
H), 11.20 (s, 2H), 8.70 (s, 1H), 7.83 (d, J = 8.0 Hz, 2H), 7.56 (d,
38.56, 134.94, 129.49, 129.49, 126.93, 126.93, 126.87, 126.87,
23.43, 121.49, 121.49, 121.35, 121.35, 118.12, 118.12, 113.39,
J = 8.0 Hz, 2H), 7.50 (t, J = 7.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.31
(
(
d, J = 8.0 Hz, 2H), 7.15 (t, J = 7.5 Hz, 2H), 6.93–6.88 (m, 3H), 6.89
s, 2H), 5.96 (s, 1H); C NMR (125 MHz, DMSO-d
13.39, 110.09, 110.09, 37.36; HREI-MS: m/z calcd for C24
H
N
20
N
4
O, C,
4
O
1
3
6
):d 181.86,
[
M]+ 380.1637; Found 380.1645; Anal. Calcd for C24
H
20
1
1
1
1
67.92, 142.39, 138.63, 138.63, 136.56, 134.523, 132.69, 129.41,
29.41, 128.72, 127.73, 127.73, 126.84, 126.84, 125.07, 124.09,
23.42, 123.42, 121.67, 121.67, 121.35, 121.35, 118.28, 118.28,
7
5.77; H, 5.30; N, 14.73; found C, 75.77; H, 5.30; N, 14.73;
4
.4. Synthesis of 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-phenylhy-
17.44, 113.30, 113.30, 110.17, 110.17, 37.40; Anal. Calcd for C31
-
drazinecarbothioamides (1–18)
H
24BrN OS, C = 62.63, H = 4.07, N = 11.78; Found C = 62.61,
5
H = 4.09, N = 11.76; HR MS Calcd for C31
found 593.0879.
5
H24BrN OS: 593.0885
Equal amount of bisindole hydrazide II (1 mmole) and isothio-
cyanates (1 mmole) were mixed in the presence of ethanol
(
6
20 mL) and catalytic amount of triethylamine and refluxed for
h. The completion of reaction was confirmed by TLC. The crude
4
.4.5. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(p-tolyl)
hydrazinecarbothioamide (5)
Yield: 0.47 g (89%); 1H NMR (500 MHz, DMSO-d
H), 10.07 (s, 2H), 8.46 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.63 (d,
J = 7.5 Hz, 2H), 7.52 (d, J = 7.9 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H),
products were further recrystallized from methanol to afford pure
compounds with the yield between 72% and 92%.
6
): d 11.70 (s,
2
4
.4.1. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(o-tolyl)
hydrazinecarbothioamide (1)
7
.33 (d, J = 7.5 Hz, 2H), 7.26 (d, J = 7.5 Hz, 2H), 7.12 (t, J = 7.6 Hz,
H), 6.95 (t, J = 7.5 Hz, 2H), 6.87 (s, 2H), 5.94 (s, 1H), 2.35 (s, 3H);
C NMR (125 MHz, DMSO-d ): d 179.39, 166.80, 142.39, 138.67,
6
Yield: 0.43 g (81%); 1H NMR (500 MHz, DMSO-d
): d 10.30 (s,
6
2
2
H), 9.85 (s, 2H), 9.70 (s, 1H), 7.80 (t, J = 7.8 Hz, 2H), 7.50 (d,
13
J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 7.9 Hz, 2H),
7
6
1
1
1
1
38.67, 137.62, 134.50, 132.72, 130.36, 130.36, 129.48, 129.48,
27.54, 127.54, 126.97, 126.97, 123.62, 123.62, 121.37, 121.37,
21.25, 121.25, 120.97, 120.97, 118.12, 118.12, 113.39, 113.39,
.30–7.25 (m, 4H), 7.11 (t, J = 7.8 Hz, 2H), 6.92 (t, J = 7.4 Hz, 2H),
13
.85 (s, 2H), 5.93 (s, 1H), 2.45 (s, 3H);
):d 180.87, 166.91, 142.37, 138.72, 138.72, 137.96,
34.51, 132.74, 129.92, 129.56, 129.56, 127.76, 127.50, 127.50,
C NMR (125 MHz,
DMSO-d
6
10.18, 110.18, 37.27, 21.02; Anal. Calcd for
32 27 5
C H N OS,
1
1
1
3
C = 72.56, H = 5.14, N = 13.22; Found C = 72.57, H = 5.11,
N = 13.26; HR MS Calcd for C32 OS: 529.1936 found 529.1945.
26.98, 126.98, 124.63, 123.51, 123.51, 123.08, 121.45, 121.45,
21.41, 121.41, 118.28, 118.28, 113.39, 113.39, 110.05, 110.05,
7.35, 17.31; Anal. Calcd for C32H N OS, C = 72.56, H = 5.14,
27 5
27 5
H N
N = 13.22; Found C = 72.58, H = 5.13, N = 13.24; HR MS Calcd for
OS: 529.1936 found 529.1941.
4
.4.6. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(4-fluorophenyl)
hydrazinecarbothioamide (6)
Yield: 0.51 g (96%); 1H NMR (500 MHz, DMSO-d
32 27 5
C H N
6
): d 11.90 (s,
4
.4.2. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(2-fluorophenyl)
2H), 10.20(s, 2H), 8.44 (s, 1H), 7.84–7.76 (m, 4H), 7.52 (d,
J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H),
7.32–7.26 (m, 2H), 7.11 (t, J = 7.6 Hz, 2H), 6.92 (t, J = 7.5 Hz, 2H),
hydrazinecarbothioamide (2)
Yield: 0.46 g (86%); 1H NMR (500 MHz, DMSO-d
6
): d 11.91 (s,
1
3
2
H), 10.85 (s, 2H), 8.70 (s, 1H), 7.81 (d, J = 8.0 Hz, 2H), 7.50 (t,
6
6.86 (s, 2H), 5.93 (s, 1H); C NMR (125 MHz, DMSO-d ):d 180.38,
J = 8.5 Hz, 4H), 7.41 (d, J = 8.2 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H),
165.90, 161.53 (d, J = 262.3 Hz), 159.43, 141.38, 137.62, 137.62,
134.51, 129.36, 129.36, 127.51, 127.51, 126.97, 126.97, 123.42,
123.42, 122.49, 122.49, 121.87, 121.87, 121.35, 121.35, 118.28,
118.28, 115.35, 115.35, 113.38, 113.38, 110.09, 110.09, 36.30; Anal.
7
6
1
1
1
.30–7.25 (m, 2H), 7.11 (t, J = 7.5 Hz, 2H), 6.96 (t, J = 7.5 Hz, 2H),
.91 (s, 2H), 5.97 (s, 1H); 13C NMR (125 MHz, DMSO-d
6
):d 180.87,
66.90, 158.99 (d, J = 262.3 Hz), 142.38, 138.62, 134.52, 129.46,
27.82, 127.61, 127.53, 126.94, 125.70, 124.43, 123.96, 123.52,
21.47, 118.22, 117.25, 117.04, 113.37, 110.07, 37.30; Anal. Calcd
Calcd for
31 5
C H24FN OS, C = 69.77, H = 4.53, N = 13.12; Found
C = 69.77, H = 4.52, N = 13.14; HR MS Calcd for C31
533.1686 found 533.1679.
5
H24FN OS:
5
for C31H24FN OS, C = 69.77, H = 4.53, N = 13.12; Found C = 69.79,

M. Taha et al. / Bioorganic Chemistry 68 (2016) 56–63
61
4
.4.7. N-(4-Bromophenyl)-2-(4-(di(1H-indol-3-yl)methyl)benzoyl)
7.29 (s, 2H), 7.11 (t, J = 7.6 Hz, 2H), 6.97 (t, J = 7.5 Hz, 2H), 6.88 (s,
2H), 5.94 (s, 1H), 3.40 (s, 3H); C NMR (125 MHz, DMSO-d ): d
6
183.38, 171.90, 152.38, 140.26, 138.52, 138.52, 138.37, 134.32,
129.47, 129.47, 128.50, 127.52, 127.52, 126.97, 126.97, 126.59,
123.59, 123.59, 121.73, 121.57, 121.57, 121.35, 121.35, 120.17,
118.28, 118.28, 113.27, 113.27, 110.08, 110.08, 37.31, 21.23; Anal.
1
3
hydrazinecarbothioamide (7)
Yield: 0.48 g (81%); 1H NMR (500 MHz, DMSO-d
6
): d 11.90 (s,
2H), 9.88 (s, 2H), 8.33 (s, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.56 (d,
J = 8.3 Hz, 2H), 7.46 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H),
7
2
.32 (d, J = 8.0 Hz, 2H), 7.12 (t, J = 7.6 Hz, 2H), 6.92 (t, J = 7.4 Hz,
1
3
H), 6.87 (s, 2H), 6.83 (d, J = 8.4 Hz, 2H), 5.96 (s, 1H); C NMR
): d 179.47, 166.80, 142.28, 138.32, 138.32,
32 27 5
Calcd for C H N OS, C = 72.56, H = 5.14, N = 13.22; Found
(
125 MHz, DMSO-d
6
C = 72.58, H = 5.10, N = 13.24; HR MS Calcd for C32
529.1936 found 529.1929.
27 5
H N OS:
1
1
1
1
37.92, 134.58, 131.26, 129.36, 129.36, 127.51, 127.51, 126.97,
26.97, 123.42, 123.42, 123.18, 123.18, 121.46, 121.46, 121.25,
21.25, 118.27, 118.27, 114.51, 114.51, 113.38, 113.38, 110.09,
4.4.12. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(3-fluorophenyl)
10.09, 35.32; Anal. Calcd for C31
H24BrN
5
OS, C = 62.63, H = 4.07,
hydrazinecarbothioamide (12)
N = 11.78; Found C = 62.63, H = 4.10, N = 11.75; HR MS Calcd for
24BrN OS: 593.0885 found 593.0871.
Yield: 0.44 g (83%); 1H NMR (500 MHz, DMSO-d
6
): d 11.88 (s,
C
31
H
5
2H), 10.23 (s, 2H), 8.45 (s, 1H), 7.80 (d, J = 7.5 Hz, 2H), 7.50 (t,
J = 8.5 Hz, 3H), 7.42 (d, J = 8.0 Hz, 2H), 7.31(d, J = 7.5 Hz, 2H), 7.12
(t, J = 7.5 Hz, 2H), 7.04–6.96 (m, 5H), 6.86 (s, 2H), 5.93 (s, 1H);
4.4.8. N-(4-Chlorophenyl)-2-(4-(di(1H-indol-3-yl)methyl)benzoyl)
1
3
hydrazinecarbothioamide (8)
6
C NMR (125 MHz, DMSO-d ): d 179.38, 166.90, 160.02 (d,
Yield: 0.43 g (78%); 1H NMR (500 MHz, DMSO-d
J = 262.3 Hz), 162.38, 150.11, 137.62, 137.62, 134.51, 129.81,
129.81, 129.36, 127.52, 127.52, 126.91, 126.91, 123.82, 123.82,
121.67, 121.67, 121.25, 121.25, 118.45, 118.28, 118.28, 113.47,
6
): d 11.92 (s,
2H), 10.03 (s, 2H), 8.41 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.76 (d,
J = 7.0 Hz, 2H), 7.50 (t, J = 8.0 Hz, 4H), 7.42 (d, J = 8.0 Hz, 2H), 7.31
(
d, J = 8.0 Hz, 2H), 7.12 (t, J = 7.5 Hz, 2H), 6.95 (t, J = 7.5 Hz, 2H),
113.47, 112.29, 111.21, 110.09, 110.09, 37.20; Anal. Calcd for C31
-
13
6
1
1
1
1
.89 (s, 2H), 5.92 (s, 1H);
C NMR (125 MHz, DMSO-d
6
): d
5
H24FN OS, C = 69.77, H = 4.53, N = 13.12; Found C = 69.77,
82.38, 166.91, 142.39, 139.82, 138.65, 138.65, 134.49, 129.56,
29.56, 129.17, 129.17, 128.17, 127.56, 127.56, 126.98, 126.98,
23.42, 123.42, 121.85, 121.85, 121.42, 121.42, 121.17, 121.17,
18.12, 118.12, 113.38, 113.38, 110.17, 110.17, 37.36; Anal. Calcd
H = 4.51, N = 13.13; HR MS Calcd for C31
533.1690.
5
H24FN OS: 533.1686 found
4.4.13. N-(3-Bromophenyl)-2-(4-(di(1H-indol-3-yl)methyl)benzoyl)
for C31
H
24ClN
5
OS, C = 67.69, H = 4.40, N = 12.73; Found C = 67.68,
hydrazinecarbothioamide (13)
H = 4.41, N = 12.77; HR MS Calcd for C31
found 549.1384.
H24ClN
5
OS: 549.1390
Yield: 0.47 g (79%); 1H NMR (500 MHz, DMSO-d
6
): d 11.82 (s,
2H), 10.20 (s, 2H), 8.33 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.52 (d,
J = 8.1 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H),
7.26 (t, J = 7.5 Hz, 2H), 7.20 (s, 1H), 7.15–7.10 (m, 3H), 6.92 (t,
4
.4.9. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(4-methoxyphenyl)
hydrazinecarbothioamide (9)
Yield: 0.46 g (84%); 1H NMR (500 MHz, DMSO-d
H), 9.90 (s, 2H), 8.36 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.68(d,
J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H),
1
3
J = 7.5 Hz, 2H), 6.88 (s, 2H), 5.94 (s, 1H); C NMR (125 MHz,
DMSO-d ): d 176.38, 169.90, 142.28, 140.92, 138.52, 138.52,
6
): d 11.60 (s,
6
2
134.59, 129.76, 129.76, 129.49, 127.43, 127.43, 126.34, 126.34,
125.71, 123.62, 123.62, 122.50, 122.02, 121.46, 121.46, 121.35,
121.35, 120.77, 118.32, 118.32, 113.38, 113.38, 110.17, 110.17,
7
.32 (d, J = 8.0 Hz, 2H), 7.12 (t, J = 7.6 Hz, 2H), 7.04 (d, J = 8.0 Hz,
H), 6.91 (t, J = 7.5 Hz, 2H), 6.87 (s, 2H), 5.94 (s, 1H), 3.81 (s, 3H);
C NMR (125 MHz, DMSO-d ): d 179.12, 165.87, 154.87, 142.28,
6
2
37.35; Anal. Calcd for
N = 11.78; Found C = 62.61, H = 4.08, N = 11.76; HR MS Calcd for
24BrN OS: 593.0885 found 593.0877.
31 5
C H24BrN OS, C = 62.63, H = 4.07,
1
3
1
1
1
1
38.67, 138.67, 134.42, 133.06, 129.36, 129.36, 127.57, 127.57,
26.95, 126.95, 123.53, 123.53, 122.12, 122.12, 121.48, 121.48,
21.15, 121.15, 118.24, 118.24, 114.23, 114.23, 113.38, 113.38,
C
31
H
5
4.4.14. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(3-nitrophenyl)
10.17, 110.17, 56.02, 37.27; Anal. Calcd for
C
32
H
27
N
5
O
2
S,
hydrazinecarbothioamide (14)
C = 70.44, H = 4.99, N = 12.83; Found C = 70.46, H = 5.01,
N = 12.85; HR MS Calcd for S: 545.1885 found
Yield: 0.54 g (96%); 1H NMR (500 MHz, DMSO-d
6
): d 12.05 (s,
C
H
32 27
N
5
O
2
2H), 10.65 (s, 2H), 8.55 (s, 1H), 8.24 (d, J = 7.5 Hz, 1H), 8.13 (d,
J = 7.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.74 (t, J = 8.0 Hz, 1H), 7.51
5
45.1874.
(
d, J = 8.0 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H),
4
.4.10. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(4-nitrophenyl)
7.13 (t, J = 7.5 Hz, 2H), 7.02–6.96 (m, 3H), 6.89 (s, 2H), 5.94 (s,
1H); C NMR (125 MHz, DMSO-d ): d 178.38, 166.91, 150.52,
6
142.39, 141.37, 138.42, 138.42, 134.57, 129.56, 129.33, 129.33,
127.58, 127.58, 126.97, 126.97, 126.64, 123.58, 123.58, 121.87,
121.87, 121.55, 121.55, 118.28, 118.28, 118.12, 115.95, 113.38,
1
3
hydrazinecarbothioamide (10)
Yield: 0.51 g (91%); 1H NMR (500 MHz, DMSO-d
6
): d 12.08 (s,
2H), 10.70 (s, 2H), 8.52 (s, 1H), 8.32 (d, J = 8.0 Hz, 2H), 8.01 (d,
J = 7.0 Hz, 2H), 7.82 (d, J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H),
7
2
.42 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.12 (t, J = 7.5 Hz,
113.38, 110.19, 110.19, 32.35; Anal. Calcd for
C = 66.41, H = 4.31, N = 14.99; Found C = 66.42, H = 4.32,
31 24 6 3
C H N O S,
1
3
H), 6.93 (t, J = 7.5 Hz, 2H), 6.89 (s, 2H), 5.96 (s, 1H); C NMR
): d 179.32, 166.91, 143.57, 143.34, 142.48,
(
125 MHz, DMSO-d
6
N = 15.00; HR MS Calcd for
560.1624.
31 24 6 3
C H N O S: 560.1631 found
1
1
1
1
38.65, 138.65, 134.51, 129.56, 129.56, 127.52, 127.52, 126.91,
26.91, 125.07, 125.07, 123.53, 123.53, 121.68, 121.68, 121.35,
21.35, 120.95, 120.95, 118.12, 118.12, 113.38, 113.38, 110.17,
4.4.15. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(3-methoxyphenyl)
10.17, 37.35; Anal. Calcd for C31
24
H N
6
O
3
S, C = 66.41, H = 4.31,
hydrazinecarbothioamide (15)
N = 14.99; Found C = 66.43, H = 4.34, N = 15.01; HR MS Calcd for
S: 560.1631 found 560.1638.
Yield: 0.43 g (79%); 1H NMR (500 MHz, DMSO-d
6
): d 11.70 (s,
2H), 9.91(s, 1H), 9.62 (s, 1H), 8.35 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H),
.52 (d, J = 80 Hz, 2H), 7.41(d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz,
31 24 6 3
C H N O
7
4
.4.11. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(m-tolyl)
2H), 7.25 (t, J = 8.0 Hz, 2H), 7.18 (s, 1H), 7.14–7.08 (m, 3H), 6.92
hydrazinecarbothioamide (11)
(t, J = 7.5 Hz, 2H), 6.89 (s, 2H), 5.94 (s, 1H) 3.89 (s, 3H); 13C NMR
Yield: 0.38 g (72%); 1H NMR (500 MHz, DMSO-d
6
): d 11.46 (s,
6
(125 MHz, DMSO-d ): d 179.49, 166.80, 158.49, 142.48, 140.44,
2
H), 10.20 (s, 2H), 8.43 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.53 (d,
138.72, 138.72, 134.51, 129.45, 129.45, 128.97, 127.52, 127.52,
126.92, 126.92, 123.50, 123.50, 121.67, 121.67, 121.35, 121.35,
J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 7.5 Hz, 2H),

6
2
M. Taha et al. / Bioorganic Chemistry 68 (2016) 56–63
1
1
18.42, 118.42, 113.57, 113.57, 112.96, 110.47, 110.47, 110.47,
09.16, 56.13, 32.57; Anal. Calcd for C32 S, C = 70.44,
4.6. Molecular docking
27 5 2
H N O
H = 4.99, N = 12.83; Found C = 70.45, H = 4.98, N = 12.81; HR MS
Calcd for C32 S: 545.1885 found 545.1878.
Molecular docking studies were performed in order to get more
insight into the binding mode of Bis(indolyl)methanes derivatives
H
27
N
5
O
2
within the active site of b-D-glucuronidase and to obtain further
4
.4.16. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(3,4-dichlorophenyl)-
validations for our experimental results. All the docking calcula-
tions were performed on Intel (R) xenon (R) CPU
E5620@2.40 GHz system having 3.8 GB RAM with the open 11.4
(X 86_64) operating platform. The software package MOE (Molec-
ular Operating Environment) (www.Chemcomp.comwas) used for
docking. LigPlot implemented in MOE was used to examine the
interactions between the enzyme and Ligands.
hydrazinecarbothioamide (16)
Yield: 0.50 g (85%); 1H NMR (500 MHz, DMSO-d
6
): d 11.52 (s,
2
7
2
H), 9.37 (s, 1H), 9.24 (s, 1H), 8.25 (s, 1H), 7.82 (d, J = 8.0 Hz, 2H),
.48 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 7.5 Hz,
H), 7.23 (s, 1H), 7.13 (t, J = 7.5 Hz, 2H), 6.93–6.98 (m, 2H), 6.88
13
(
s, 2H), 6.76 (d, J = 8.0 Hz, 2H), 5.94 (s, 1H); C NMR (125 MHz,
DMSO-d ): d 180.76, 166.80, 142.39, 138.52, 138.52, 136.47,
34.51, 132.94, 129.45, 129.45, 127.72, 127.55, 127.38, 127.38,
6
The crystal structure of b-D-glucuronidase (PDB code 1BHG)
1
1
1
3
was retrieved from protein data bank for docking studies. The
structure was checked for missing atoms, bonds and contacts.
From the original protein data bank file the B-chain of protein
and hetero-atoms including cofactors were removed. After 3D pro-
tonation of the enzyme the energy of the retrieved protein mole-
cule was minimized using the default parameters of MOE energy
minimization algorithm (gradient: 0.05, Force Field: MMFF94X).
Energy minimization was terminated when the root mean square
gradient falls below the 0.05.
The 3D structures of these newly synthesized Bis(indolyl)
methanes derivatives were constructed using MOE-Builder tool
and hydrogens were added. Then, these molecules were energy
minimized using the default parameters of MOE energy minimiza-
tion algorithm (gradient: 0.05, Force Field: MMFF94X). All the min-
imized molecules were saved in the mdb file format. The prepared
ligands were used as input file for MOE-Dock in the next step.
MOE docking program was used to analyze the binding modes
of the ligands with the protein molecule. To find the correct confor-
mations of the ligands and to obtain minimum energy structures,
ligands were allowed to be flexible. The default parameters of
MOE-Dock program were used for the molecular docking of the
ligands. The top ranked pose of each compound was selected on
the basis of docking score (S) for further analysis. At the end of
docking, the best conformations on the basis of score were ana-
26.97, 126.97, 126.39, 123.62, 123.62, 122.38, 121.67, 121.67,
21.41, 121.41, 118.12, 118.12, 113.33, 113.33, 110.13, 110.13,
7.15; Anal. Calcd for
31 23 l2 5
C H C N OS, C = 63.70, H = 3.97,
N = 11.98; Found C = 63.71, H = 3.99, N = 11.99; HR MS Calcd for
OS: 583.1000 found 583.1010.
31 23 l2 5
C H C N
4
.4.17. 2-(4-(di(1H-indol-3-yl)methyl)benzoyl)-N-(4(trifluoromethyl)
phenyl)hydrazinecarbothio amide (17)
Yield: 0.45 g (78%); 1H NMR (500 MHz, DMSO-d
6
): d 12.04 (s,
2
H), 11.90 (s, 2H), 8.63 (d, J = 4.2 Hz, 2H), 8.42 (s, 1H), 7.81 (d,
J = 8.0 Hz, 2H), 7.68 (s, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.42 (d,
J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H), 7.12 (t, J = 7.5 Hz, 2H), 6.96
(
t, J = 7.5 Hz, 2H), 6.89 (s, 2H), 5.96 (s, 1H); 13C NMR (125 MHz,
DMSO-d
6
): d 179.48, 167.50, 143.53, 142.28, 138.72, 138.72,
1
1
1
1
34.42, 129.45, 129.45, 127.52, 127.52, 126.84, 126.84, 126.33,
26.33, 126.02, 124.53 (d, J = 248.9 Hz), 123.40, 123.40, 121.48,
21.48, 121.15, 121.15, 120.62, 120.62, 118.12, 118.12, 113.38,
24 3 5
13.38, 110.17, 110.17, 37.35; Anal. Calcd for C32H F N OS,
C = 65.85, H = 4.14, N = 12.00; Found C = 65.81, H = 4.15,
N = 12.01; HR MS Calcd for OS: 583.1654 found
5
32 24 3 5
C H F N
83.1662.
4
.4.18. 2-(4-(Di(1H-indol-3-yl)methyl)benzoyl)-N-(2-methoxyphenyl)
hydrazinecarbothioamide (18)
lyzed for hydrogen bonding/p-p interactions.
Yield: 0.44 g (81%); 1H NMR (500 MHz, DMSO-d
6
): d 11.80 (s,
2
H), 9.95 (s, 2H), 8.85 (s, 1H), 8.16 (d, J = 2.0 Hz, 1H), 8.06 (d,
Acknowledgment
J = 8.0 Hz, 1H), 7.82 (d, J = 7.5 Hz, 2H), 7.70–7.66 (m, 1H), 7.50 (d,
J = 8.0 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H),
The authors would like to acknowledge Universiti Teknologi
MARA, Malaysia, for the financial support under the Research
Intensive Faculty grant scheme with reference number UiTM
7
1
1
1
1
1
.11 (t, J = 7.5 Hz, 2H), 6.94 (t, J = 7.5 Hz, 2H), 6.89 (s, 2H), 5.96 (s,
1
3
H), 3.90 (s, 3H);
6
C NMR (125 MHz, DMSO-d ): d 181.85,
67.96, 151.17, 140.39, 136.68, 136.68, 135.52, 132.16, 129.48,
29.48, 127.43, 127.43, 126.96, 126.96, 124.92, 123.47, 123.47,
22.52, 122.29, 121.48, 121.48, 121.35, 121.35, 118.21, 118.21,
13.38, 113.38, 111.21, 110.09, 110.09, 56.58, 37.25; Anal. Calcd
6
00-RMI/DANA 5/3/LESTARI (54/2015).
Appendix A. Supplementary material
for C32
H
27
N
5
O
2
S, C = 70.44, H = 4.99, N = 12.83; Found C = 70.43,
S: 545.1885 found
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2016.07.
H = 4.97, N = 12.84; HR MS Calcd for C32
27 5 2
H N O
5
45.1874.
0
08.
4.5. Bioassay
References
b-Glucuronidase activity was determined by measuring the
[
1] B. Sperker, J.T. Backman, K. Kromer, Clin. Pharmacokinet. 33 (1997) 18–31.
absorbance at 405 nm of p-nitrophenol formed from the substrate
by the spectrophotometric method. The total reaction volume was
[2] W.H. Fishman, Academic Press, New York, 1970, p. 519.
[3] K. Paigen, Prog. Nucleic Acid Res. Mol. Biol. 37 (1989) 155–205.
[4] H.C. Gonick, H.J. Kramer, A.E. Schapiro, Arch. Intern. Med. 132 (1973) 63–69.
[5] C.M. Plum, Enzymol. Biol. Clin. 8 (1967) 97–112.
2
50
lL. The reaction mixture contained 185
lL of 0.1 M acetate
buffer, 5
l
L of test compound solution, 10 L of enzyme solution
l
[6] E. Boyland, J.E. Gasson, D.C. Williams, Br. J. Cancer 11 (1957) 120–129.
[7] N. Bank, S.H. Bailine, N. Engl. J. Med. 272 (1965) 70–75.
was incubated at 37 °C for 30 min. The plates were read on a mul-
tiplate reader (SpectraMax plus 384) at 405 nm after the addition
of 50 lL of 0.4 mM p-nitrophenyl-b-D-glucuronide. All assays were
run in triplicate [42–45]. Furthermore to avoid precipitation, the
[
8] A.P. Roberts, J. Frampton, S.M.M. Karim, R.W. Beard, N. Engl. J. Med. 276 (1967)
468–1470.
1
[
9] A.R. Ronald, F. Silverblatt, H. Clark, R.E. Cutler, M. Turck, Appl. Microbiol. 21
(1971) 990–992.
[
[
[
10] H.A. Kallet, L. Lapco, J. Urol. 97 (1967) 352–356.
11] J.C. Caygill, D.A. Pitkeathly, Ann. Rheum. Dis. 25 (1966) 137–144.
12] G. Weissmann, R.B. Zurier, P.J. Spieler, I.M. Goldstein, J. Exp. Med. 134 (1971)
149–165.
concentration of the compounds was decreased and the reaction
volume was high (200
lL) so the chance of precipitation was less
hence the addition of detergents was not needed.

M. Taha et al. / Bioorganic Chemistry 68 (2016) 56–63
63
[
[
13] B.R. Goldin, S.L. Gorbach, J. Natl. Cancer Inst. 57 (1976) 371–375.
14] K.M. Khan, M. Taha, F. Rahim, W. Jamil, S. Perveen, M.I. Choudhary, J. Iran.
Chem. Soc. 9 (2012) 81–83.
[32] I. Chen, A. McDougal, F. Wang, S. Safe, Carcinogenesis 19 (1998) 1631–1639.
[33] D. Maciejewska, I. Szpakowska, I. Wolska, M. Niemyjska, M. Mascini, M. Maj-
Zurawska, Bioelectrochemistry 69 (2006) 1–9.
[
15] S. Imran, M. Taha, N.H. Ismail, S. Fayyaz, K.M. Khan, M.I. Choudhary, Bioorg.
Chem. 62 (62) (2015) 83–93.
[34] M. Taha, N.H. Ismail, W. Jamil, K.M. Khan, U. Salar, S.M. Kashif, F. Rahim, Y.
Latif, Med. Chem. Res. 24 (2015) 3166–3173.
[
[
16] S. Imran, M. Taha, N.H. Ismail, Curr. Med. Chem. 22 (2015) 4412–4433.
17] S. Imran, M. Taha, N.H. Ismail, K.M. Khan, F. Naz, M. Hussain, S. Tauseef,
Molecules 19 (2014) 11722–11740.
[35] (a) M. Taha, N.H. Ismail, S. Imran, E.H. Anouar, M. Ali, W. Jamil, N. Uddin, S.M.
Kashif, RSC Adv. 6 (2016) 3003–3012;
(b) 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)
1825–1829.
[
18] 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.
[
[
19] C.A. Bradfield, L.F. Bjeldanes, J. Toxicol. Environ. Health 21 (1987) 311–323.
20] R.H. Dashwood, L. Uyetake, A.T. Fong, J.D. Hendricks, G.S. Bailey, Food Chem.
Toxicol. 27 (1989) 385–392.
[36] K.M. Khan, F. Rahim, N. Ambreen, M. Taha, S. Iqbal, S.M. Haider, S. Perveen, J.
Chem. Soc. Pak. 34 (2012) 748–757.
[37] M. Taha, N.H. Ismail, K. Javaid, S. Imran, A. Wadood, M. Ali, K.M. Khan, S.M.
Saad, F. Rahim, M.I. Choudhary, Bioorg. Chem. 63 (2015) 24–35.
[38] 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 (2015)
81–87.
[39] 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, Synthetic indole Mannich bases: their
ability to modulate in vitro cellular immunity, Bioorg. Chem. 60 (2015)
118–122.
[
[
[
21] M.A. Zeligs, J. Med. Food 1 (1998) 67–82.
22] C. Bonnesen, I.M. Eggleston, J.D. Hayes, Cancer Res. 61 (2001) 6120–6130.
23] M.J. Anderton, M.M. Manson, R. Verschoyle, A. Gescher, W.P. Steward, M.L.
Williams, D.E. Mager, Drug Metabol. Dispos. 32 (2004) 632–638.
24] T.H. Carter, K. Liu, W. Ralph Jr., D.Z. Chen, M. Qi, S. Fan, F. Yuan, E.M. Rosen, K.J.
Auborn, J. Nutrit. 132 (2002) 3314–3324.
[
[
[
[
[
[
[
[
25] T. Inamoto, S. Papineni, S. Chintharlapalli, S.D. Cho, S. Safe, A.M. Kamat, Mol.
Cancer Ther. 7 (2008) 3825–3833.
26] M. York, M. Abdelrahim, S. Chintharlapalli, S.D. Lucero, S. Safe, Clin. Cancer Res.
3 (2007) 6743–6752.
27] N. Ichite, M.B. Chougule, T. Jackson, S.V. Fulzele, S. Safe, Clin. Cancer Res. 15
2009) 543–552.
28] S.D. Cho, K. Yoon, S. Chintharlapalli, M. Abdelrahim, P. Lei, S. Hamilton, S. Khan,
S.K. Ramaiah, S. Safe, Cancer Res. 67 (2007) 674–683.
[40] M. Taha, N.H. Ismail, M. Ali, K.M. Khan, W. Jamil, S.M. Kashif, M. Asraf, Med.
Chem. Res. 23 (2014) 5282–5293.
1
[41] M. Taha, N.H. Ismail, A. Khan, S.A.A. Shah, A. Anwar, S.A. Halim, M.Q. Fatmi, S.
Imran, F. Rahim, K.M. Khan, Bioorg. Med. Chem. Lett. 25 (2015) 3285–3289.
[42] W. Jamil, S. Perveen, S.A.A. Shah, M. Taha, N.H. Ismail, S. Perveen, N. Ambreen,
K.M. Khan, M.I. Choudhary, Molecules 19 (2014) 8788–8802.
[43] M. Taha, N.H. Ismail, S. Imran, M. Selvaraj, A. Rahim, M. Ali, S. Siddiqui, F.
Rahim, K.M. Khan, Bioorg. Med. Chem. 23 (2015) 7394–7404.
[44] K.M. Khan, S.M. Saad, N.N. Shaikh, S. Hussain, M.I. Fakhri, S. Perveen, M. Taha,
M.I. Choudhary, Bioorg. Med. Chem. 22 (2014) 3449–3454.
[45] K.M. Khan, N. Ambreen, M. Taha, S.A. Halim, S. Naureen, S. Rasheed, S. Perveen,
S. Ali, M.I. Choudhary, J. Comput. Aided Mol. Des. 28 (2014) 577.
(
29] S. Chintharlapalli, S. Papineni, S.J. Baek, S. Liu, S. Safe, Mol. Pharm. 68 (2005)
1
782–1792.
30] A. McDougal, M.S. Gupta, D. Morrow, K. Ramamoorthy, J.E. Lee, S.H. Safe, Breast
Cancer Res. 66 (2001) 147–157.
31] A. McDougal, M. Sethi Gupta, K. Ramamoorthy, G. Sun, S.H. Safe, Cancer Lett.
1
51 (2000) 169–179.