Synthesis, in?vitro β-glucuronidase inhibitory potential and molecular docking studies of quinolines

Article abstract of DOI:10.1016/j.ejmech.2017.08.052

In this study synthesis and β-glucuronidase inhibitory potential of 3/5/8 sulfonamide and 8-sulfonate derivatives of quinoline (1–40) are discussed. Studies reveal that all the synthetic compounds were found to have good inhibitory activity against β-gluc

Full text of DOI:10.1016/j.ejmech.2017.08.052

Accepted Manuscript
Synthesis, in vitro β-glucuronidase inhibitory potential and molecular docking studies
of quinolines
Bilquees Bano, Arshia, Khalid Mohammed Khan, Kanwal, Bibi Fatima, Muhammad
Taha, Nor Hadiani Ismail, Abdul Wadood, MehreenGhufran, Shahnaz Perveen
PII:
S0223-5234(17)30663-3
10.1016/j.ejmech.2017.08.052
EJMECH 9696
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 March 2017
Revised Date: 22 August 2017
Accepted Date: 23 August 2017
Please cite this article as: B. Bano, Arshia, K.M. Khan, Kanwal, B. Fatima, M. Taha, N.H. Ismail, A.
Wadood, MehreenGhufran, S. Perveen, Synthesis, in vitro β-glucuronidase inhibitory potential and
molecular docking studies of quinolines, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.08.052.
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ACCEPTED MANUSCRIPT
Synthesis, in vitro
glucuronidase inhibitory potential and molecular docking studies of quinolines
a
a
a
a
a
b,c
Bilquees Bano, Arshia, Khalid Mohammed Khan, Kanwal, Bibi Fatima , Muhammad Taha,
b
d
d
e
Nor Hadiani Ismail, Abdul Wadood, Mehreen Ghufran, Shahnaz Perveen
a
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
b
Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300
Bandar Puncak Alam, Selangor D. E. Malaysia
c
Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), University of Dammam, Dammam
3
1441, Saudi Arabia
d
Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali Khan University, Mardan,
Pakistan
e
PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280, Pakistan
O
NH2
S Ar/Alkyl
O
HN
O
S
O
O
O
S
O
N
Pyridine
Pyridine
S
+ Cl
Ar/Alkyl
Refluxing, 40-45 min
NH
+
Cl
Ar/Alkyl
Alkyl/Ar
Refluxing, 40-45 min
N
O
9-20
N
N
NH2
1-7
O
H
O
NH2
N
S
Pyridine
N
Ar/Alkyl
O
S
O
Cl
Ar/Alkyl
S
O
O
+
O
Pyridine
Refluxing, 40-45 min
S
Refluxing, 40-45 min
O
N
+ Cl
Ar/Alkyl
Alkyl/Ar
N
O
N
2
1-32
3
3-40
OH
Cl
Cl
Cl
O
O
S
S
NH
NH
Cl O
O
N
N
2
1
(IC50 = 2.10 ± 0.10 M) (IC50 = 4.50 ± 0.01 M)
Cl
Cl
O
O
Cl
S
NH
O
S
NH
OHO
Cl
N
N
5
24
(IC50 = 5.50 ± 0.10 M)
(
IC50 = 1.60 ± 0.10 M)
Synthetic compounds
Docking conformation of (a) compound 5, (b) compound
, (c) compound 1 and (d) compound 24 in the active site
of -glucuronidase.
2

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Synthesis, in vitro
β-glucuronidase inhibitory potential and molecular docking
studies of quinolines
a
a
a∗
a
a
b,c
Bilquees Bano, Arshia, Khalid Mohammed Khan, Kanwal, Bibi Fatima, Muhammad Taha,
b
d
d
e
Nor Hadiani Ismail, Abdul Wadood, MehreenGhufran, Shahnaz Perveen
a
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
b
Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM),
Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia
c
Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
University of Dammam, Dammam 31441, Saudi Arabia
d
Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul
Wali Khan University, Mardan, Pakistan
e
PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi-75280,
Pakistan
Abstract: In this study synthesis and β-glucuronidase inhibitory potential of 3/5/8 sulfonamide
and 8-sulfonate derivatives of quinoline (1-40) are discussed. Studies reveal that all the synthetic
compounds were found to have good inhibitory activity against β-glucuronidase. Nonetheless,
compounds 1, 2, 5, 13, and 22-24 having IC50 values in the range of 1.60 – 8.40
µM showed
superior activity than the standard saccharic acid 1,4-lactone (IC = 48.4 ± 1.25 M). Moreover,
µ
5
0
molecular docking studies of selected compounds were also performed to see interactions
between active compounds and binding sites. Structures of all the synthetic compounds were
1
confirmed through H-NMR, EI-MS and HREI-MS spectroscopic techniques.
Keywords: β-Glucuronidase; quinoline; sulfonamide; sulfonate; molecular docking studies.
1. Introduction:
Quinoline is a aza heterocyclic compound and consists of a structurally fused benzene and
pyridine nucleus [1]. It is found in many natural products and there are many methods reported
for its synthesis [2]. Many drugs are based on a quinoline scaffold such as captothecine
anticancer [3] and cryptolepine antimalarial [4] drugs. Moreover, this skeleton also showed a
broad spectrum of biological activities including antidiabetic [5], antiasthmatic [6],
antitoxoplasma [7], antibacterial [8], anti-HIV [9] and antifungal [10] activities. The quonoline
*Corresponding Authors: khalid.khan@iccs.edu, drkhalidhej@gmail.com, Tel.: +922134824910, Fax:
+
922134819018
1

ACCEPTED MANUSCRIPT
nitrogen atom can act as a chelating agent as well as a weak base [11]. The quinoline moiety can
undergo both nucleophilic and electrophilic substitution reactions.
β-Glucuronidase (EC 3.2.1.31) found in unicellular microorganisms including Escherichia coli
and Pepto streptococcus genera to multicellular organisms including plants and humans [12,15].
Moreover, this enzyme is found in many parts of human body including kidney, lung, liver
serum, spleen, and urine blood cells [16]. β-Glucuronidase is a glycosidase that catalyses the
hydrolysis of β-linked glucuronides to yield derivatives and free glucuronic acid. It is involved in
degradation of glucuronic acid containing glycosaminoglycans such as heparin, haparan,
dermatan, and chondroitin sulfates [17]. β-Glucuronidase has enhanced activity in a variety of
pathological conditions such as urinary tract infection [18-21], epilepsy [22], neoplasm of
bladder [23], renal diseases [24], breast, larynx testes, and alimentary tract cancer [25]. In
addition, β-glucuronidase has been reported to be released in the synovial fluid in inflammatory
joint diseases, for instance, rheumatoid arthritis [26,27]. The involvement of β-glucuronidase in
colon cancer and the higher intestinal levels of the enzyme can be correlated to the higher
incidence of colon carcinoma [28]. Liver damage results in increased β-glucuronidase levels in
blood which can cause liver cancer [29,30]. Therefore, β-glucuronidase inhibitors can function to
minimize this carcinogenic potential [31]. Consequently, inhibition of β-glucuronidase enzyme is
effective in preventing various diseases. Our research group is working for many years in this
field to discover new lead molecules as β-glucuronidase inhibitors [32-34]. Recently, our
research group published work on the quinazoline nucleus which has structural similarity to
quionline and showed good β-glucuronidase inhibition. Therefore, to extend our research, we
synthesized several quinoline derivatives and screened them against β-glucuronidase enzyme
activity and got encouraging results (Figure-1) [35]. To the best of our knowledge compounds 4,
1
6-20, 26, 27 [36-40] 10, 11, and 13 [41] are previously known, while the remaining compounds
are being reported for the first time.
2

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Figure-1: Rationale of present study
2
2
.
Results and discussions:
.1. Chemistry:
Sulfonamides of 5/3/8 aminoquinoline and sulphonyl ester of 8-hydroxyquinoline were
synthesized 1-40 in the presence of pyridine. The reaction was performed using 5-amino, 3-
amino, 8-amino and 8-hydroxy quinoline and benzene sulphonyl chloride. Pyridine acts as a base
where it abstracts the proton from respective quinolines and converts into anions which then
attack the benzene sulphonyl chloride by replacing the chloride ion to afford the desired product.
3

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Schemes-1: Synthesis of quinoline derivatives
2
.2. Spectroscopic studies of new and most active compound 3,5-dichloro-2-hydroxy-N-
(quinolin-5-yl)benzene sulfonamide (5):
Structure of 3,5-dichloro-2-hydroxy-N-(quinolin-5-yl) benzene sulfonamide (5) was elucidated
1
13
by H- and C-NMR in deuterated methanol and DMSO and recorded on Bruker AM 400 MHz
1
and 300 MHz instruments, respectively. In H-NMR spectrum the most downfield signal
appeared at δH 8.83 for H-2 due to the presence of electronegative nitrogen adjacent to C-2. H-6
also appeared in downfield region at δH 8.71 (J(6,7) = 8.8 Hz) as doublet, due to attachment of a
sulfonamide nitrogen at the adjacent carbon-5. H-3 resonated as triplet at δH 7.68 (J3(4,2)= 8.0
′
′
,
Hz). However, two singlets resonate at δH 7.41 and 7.43 for H-4 and H-6 respectively.
4

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However, others aromatic protons resonate at their resonace frequency along with their
respective J values (Figure-2).
Cl
7
.41 (s)
5'
7.43 (s)
6
'
4
3
'
O
'
1'
2'
Cl
S
NH
O
OH
7.92 (d, J = 8,4 Hz)
5
4
7.68 (t, J = 8,0 Hz)
8
.71 (d, J = 8,8 Hz)
6
3
2
7
.56 (ovp) 7 8
1
8.83 (ovp )
N
7
.56 (ovp)
1
Figure-2: H-NMR Chemical shift values for compound 5
1
3
In broad band decoupled C-NMR spectra, total 15 signals resonated, out of which 8 signals
were for methines, and 7 signals for quarternary carbons. The most deshielded signal resonated
′
at
δ
C
151.6 and was due to phenolic carbon-2 . Signal at
δ
C
149.3 which in turn was due to
′
aromatic carbon-2 directly attached to a cyclic nitrogen C-4. Adjacent to sulfonamide two
′
carbon C-1 and C-5 resonated at δC 132.9, and 139.8, respectively. Rest of carbons in the
compound resonated in the normal aromatic range (Figure-3).
Cl
1
35.4
1
33.8
5' 131.0
6'
4
3
'
O
'
1' 132.9
2'
Cl 134.0
S
151.6
NH
O
OH
1
30.4
1
39.8 125.8
5
4
137.8
149.3
1
37.7
6
3
2
7
8
1
N
1
28.4
1
27.2
22.5
1
1
3
Figure-3: C-NMR Chemical shift values for compound 5
5

ACCEPTED MANUSCRIPT
Sulfonamide part
Alkyl/Aryl
O
S
5-Amino quinoline part
NH
5
O
4
6
3
2
7
N
8
1
Figure-4: General structure of 5-amino quinoline sulfonamide
Table-1: β-Glucuronidsase inhibitory activity of 5-amino quinoline derivatives 1-8
a
Compound No.
Category
Ar/Alkyl
IC ± SEM [µM]
50
1
A
4.50 ± 0.01
2
A
2.10 ± 0.10
3
4
5
6
7
A
A
A
A
A
23.50 ± 0.20
12.20 ± 0.20
1.60 ± 0.10
50.20 ± 1.30
43.20 ± 0.85
6

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8
A
75.10 ± 1.60
a
SEM is the standard error of mean
5
-Amino quinoline part
5
4
6
7
3
2
8
N 1
O
NH
S
Alkyl/Aryl
O
Sulfonamide part
Figure-5: General structure of 5-amino quinoline sulfonamide
Table-2: β-Glucuronidase inhibitory activity of 8-amino quinoline derivatives 9-20
a
Compound No.
9
Category
Ar/Alkyl
IC ± SEM [µM]
50
B
42.60 ± 0.85
1
0
1
B
B
49.20 ± 1.10
31.30 ± 0.40
1
12
13
14
B
B
B
68.50 ± 1.25
6.10 ± 0.10
24.50 ± 0.25
CH3
Cl
CH3
7

ACCEPTED MANUSCRIPT
15
B
16.10 ± 0.30
1
6
7
B
B
18.20 ± 0.20
48.60 ± 1.10
1
18
B
33.40 ± 0.60
1
9
0
B
B
70.50 ± 1.40
66.40 ± 1.40
2
a
SEM is the standard error of mean
8

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Sulfonamide part
3
-Amino quinoline part
H O
N
5
4
Alkyl/Aryl
6
7
S
3
2
O
N
8
1
Figure-6:General structure of 3-amino quinoline sulfonamide
Table-3: β-Glucuronidase inhibitory activity of 3-amino quinoline derivatives 21-32
a
Compound No
Category
Ar/Alkyl
50
IC ± SEM [µM]
21
22
23
C
33.50 ± 0.60
8.10 ± 0.20
8.40 ± 0.1
C
C
2
4
5
C
C
5.50 ± 0.10
2
34.40 ± 0.60
26
C
31.60 ± 0.45
9

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27
28
29
C
C
C
46.20 ± 0.90
53.40 ± 1.20
66.50 ± 1.40
30
C
53.60 ± 1.30
3
1
2
C
C
27.20 ± 0.35
75.10 ± 1.60
3
n-Octyl
a
SEM is the standard error of mean
5
8-Amino quinoline part
4
6
7
3
2
N
8
O
1
Alkyl/Aryl
O
S
O
Sulfonate part
Figure-7: General structure of 8-amino quinoline sulfonate
Table-4:
Compound No.
β
-Glucuronidase inhibitory activity of 8-sulfonatequinoline derivatives 21-32
a
Category
Ar/Alkyl
IC ± SEM [
µM]
5
0
33
D
49.50 ± 1.20
10

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34
35
36
D
D
D
49.50 ± 1.20
30.10 ± 0.50
9.70 ± 0.10
3
7
8
D
D
15.40 ± 0.30
6.40 ± 0.10
3
3
9
0
D
D
84.40 ± 1.50
4
41.60 ± 0.85
48.4 ± 1.25
Standard
Saccharic acid 1,4-lactone
a
SEM is the standard error of mean
2
.2
Enzyme inhibitory study
Structure-activity relationship (SAR) studies suggested that the
β-glucuronidase inhibitory
activities of these class of compounds depend mainly on the substitutions on the phenyl ring and
different quinoline substituents. In order to understand SAR, all synthetic compounds were
divided into four categories i.e. A, B, C, and D. Categories A, B, and C correspond to
compounds in which the quinoline ring is substituted with an amino moiety at position 5, 8, and
3
, respectively, while in category D, the quinoline ring is substituted with a hydroxyl moiety at
11

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position 8. Several synthetic analogs showed better activity than the standard saccharic acid 1,4-
lactone.(IC50 = 48.4 ± 1.25 µM).
Category A:
It is worth noting that in category A, seven compounds 1-7 are more active than the standard
saccharic acid 1,4-lactone (IC50 = 48.4 ± 1.25 M). 2-Hydroxy-3,5-dichloro substituted
compound 5 (IC50 = 1.60 ± 0.10) was found to be thirty-fold more active than the standard in
category A. Comparison of 5 with compound 2 (IC50 = 2.10 ± 0.10 M) suggested that the
hydroxyl substitution in compound 5 plays an important role in -glucuronidase inhibition and
µ
µ
β
absence of hydroxyl group in compound 2 showed decreased activity as compared to 5 but was
still twenty-four time more active than the standard. Similarly, when the chloro group was
shifted from position ortho, and para to ortho and meta as in compound 1 (IC50 = 4.50 ± 0.01
µ
M), decreased activity was observed as compared to compound 2. Replacement of hydroxyl
and chloro group with methyl as in compound 3 (IC50 = 23.50 ± 0.20 M), also decreases its
activity. Replacement of chloro group with a nitro group as in compound 4 (IC = 12.20 ± 0.20
µ
5
0
µ
M) results in increased activity. Compounds 7, 8 showed IC values of 43.20 ± 0.85 and 75.1 ±
50
1
.60 µM, respectively (Figure-8) (Table-1).
12

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Figure-8: SAR studies of category A
Category B:
This category consists of eleven compounds of 8-substituted sulfonamide analogs of quinoline.
Compound 13 (IC50 = 6.10 ± 0.10 M) was found to be the most active compound in this
category having an eight-fold better inhibitory activity than the standard saccharic acid 1,4-
lactone (IC50 = 48.4 ± 1.25 M). If we compare it’s activity with the closely related compound 5
µ
µ
13

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(
IC = 1.60 ± 0.10
µM), it was observed that the 5-amino substitution is more effective for its
5
0
activity than its 8-amino counterpart.
The single meta-nitro group containing compound 15 (IC50 = 16.10 ± 0.30
µM) showed a
decreased activity as compared to compound 5 which is the most active analog of the seires.
When the nitro group was shifted from meta to ortho, a comparable activity was observed as in
1
6 (IC50 = 18.20 ± 0.20
31.30 ± 0.40 M) lost half of its activity as compared to compounds 15 and 16. Nevertheless,
still these compounds were about three-fold more active than the standard saccharic acid 1,4-
µM). Surprisingly, when it was switched to para then compound 11 (IC50
=
µ
lactone.(IC = 48.4 ± 1.25
µ
M). The non-substituted benzene containing compound 20 (IC =
5
0
50
6
6.40 ±1.40
µ
M) and methoxy and bromo substituted 12 (IC = 68.50 ± 1.25
µ
M) were found to
M) and
M) showed good inhibitory
potential. Mono-methoxy and dimethoxy compounds 10 (IC50 = 49.20 ± 1.10 M) and 9 (IC50
2.60 ± 0.85 M) displayed weak activity, respectively.When aryl was replaced by alkyl groups
5
0
be weakly active. Whilst, mono-chloro substituted compound 18 (IC = 33.40 ± 0.60
µ
5
0
chloro and methyl substituted compound 14 (IC50 = 24.50 ± 0.25
µ
µ
=
4
µ
as in compound 17 (48.60 ± 1.10
µM) and 19 (70.50 ± 1.40 µM), a decreased activity was
observed (Figure-9) (Table-2).
14

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Figure-9: SAR studies of category B
Category C:
This category consists of twelve compounds of 3-substituted sulfonamide analogs of quinoline.
In category C, compound 24 (IC = 5.5 ± 0.10 M) having ortho and meta dichloro substituents
µ
5
0
15

ACCEPTED MANUSCRIPT
showed superior activity than the standard saccharic acid 1,4-lactone (IC = 48.4 ± 1.25 µM). It
5
0
also confirmed the pronounced effect of 5-amino over 8- and 3-amino quinoline with reference
to -glucuronidase inhibitory activity of this series. Compound 23 (IC = 8.4 ± 0.10 M) having
ortho and para dichloro substituent displayed a six-fold more activity than the standard. The
compound 25 (IC = 34.40 ± 0.60 M) having para chloro along with two methyls at ortho and
meta, displayed a decreased activity as compared to compounds 23 and 24. Compound 29 (IC50
66.50 ± 1.40 M) having ortho methoxy and meta bromo substituents demonstrated a weak
activity.
β
µ
5
0
µ
5
0
=
µ
In nitro substituted derivatives, ortho substituted compound 22 (IC50 = 8.10 ± 0.20
substituted 26 (IC50 = 31.60 ± 0.45 M), and meta substituted 27 (IC50 = 46.20 ± 0.90
showed different activities due to the different positions of the substituents. Alkyl substituted
compounds 31 (IC50 = 27.20 ± 0.35 M) and 32 (IC50 = 43.20 ± 0.85 M) showed less activity.
However, methoxy containing compound 30 (IC = 53.60 ± 1.30 M), showed sharp decrease in
µM), para
µ
µM)
µ
µ
µ
5
0
activity. Compound 29 having methoxy and bromo substituent at aryl part and non-substituted
aryl part containing compound 28 exhibited weak activities with IC50 values of 66.50 ± 1.40 and
5
3.40 ± 1.20 µM, respectively (Figure-10) (Table-3).
16

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Figure-10: SAR studies of category C
Category D:
This category has eight compounds of 8-substituted sulfonates of quinoline and compound 38
was found to be the most active with an IC50 value of 6.4 ± 0.10 M and superior than the
standard, whilst, its positional isomers 35 (IC = 30.10 ± 0.50 M) and 37 (IC = 15.54 ± 0.45
µ
µ
5
0
50
µ
M) displayed less activity than 38. However, dichloro compound 36 (IC50 = 9.70 ±0.10
was found to be more active than the standard. Placement of bromo group at the aryl part, as in
4 (IC50= 49.50 ± 1.20 M) resulted in decreased activity. Non-substituted and methyl
substituted aryl part compounds 40 and 33 exhibited IC50values of 41.60 ± 0.85 and 49.50 ± 1.20
M, respectively (Figure-11) (Table-4).
µM)
3
µ
µ
17

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Figure-11: SAR studies of category D
2
.4 Molecular docking studies:
To find the binding interactions of these synthetic compounds in the active site of
glucuronidase, molecular docking was performed using MOE-Dock program
www.chemcomp.com). The x-ray crystallographic structure of human -D-glucuronidase
having 80% sequence similarity with bovine -D-glucuronidase [42] was retrieved from the
β-D-
(
β
β
18

ACCEPTED MANUSCRIPT
protein data bank (PDB Code 1BHG) [43] as the 3D structure of bovine β-D-glucuronidase is not
reported yet. The β-chain of protein and hetero-atoms including co-factors were removed from
the original pdb file. The energy of the protein molecule was minimized after the 3D protonation
using the default parameters of MOE (Molecular Operating Environment) software
(
www.chemcomp.com) energy minimization algorithm (gradient: 0.05, Force Field: Amber99).
The three dimensional coordinates of the synthetic compounds were derived by using MOE
www.chemcomp.com). These molecules were than energy minimized using the default
(
parameters of MOE energy minimization algorithm (gradient: 0.05, Force Field: Amber99). All
the minimized molecules were saved in the mdb file format as an input file.
Prior to docking of the synthetic compounds, p-nitrophenyl β-D-glucuronide which is a known
substrate molecule [46], was docked first into the active site of the enzyme by utilizing docking
program MOE-Dock with default docking parameters. Similar to our previous results [44, 45],
the docked conformation of the substrate displayed that the glycoside bond of p-nitrophenyl β-D-
glucuronide was appropriately oriented towards the catalytic residues such as Glu540, Glu451,
and Tyr504. Selected compounds were then docked into the binding pocket of β-glucuronidase
enzyme using the same docking parameters. The catalytic site of β-D-glucuronidase [41] (PDB
Code 1BHG) [42] is composed of Glu540, Glu451, and Tyr504 residues. The molecular docking
studies have also disclosed some other important residues like Asp207, Arg600, Trp507, Tyr508,
and Tyr511 in the β-D-glucuronidase inhibition in addition to the catalytic site residues of the
enzyme. Thus the docking study of the sulfonamide and sulfonate derivatives of quinoline (1-40)
and the esteemed variations of their binding site residues in addition to the catalytic site of the
enzyme may lead to further investigation of these interesting interactions.
2
.5 Molecular docking interactions:
From the docking studies, it was observed that selected compounds showed significant binding
interactions with the catalytic site residues. In the case of the most active compound 5 (IC50
.60 ± 0.10, docking score = -15.9876) form five hydrogen bonds and two arene-cation linkages
=
1
with the active site residues of the enzyme. Asp207 and Arg600 made hydrogen bonds with the
nitrogen atom of the quinoline ring, Trp507 and Arg600 formed hydrogen bond with the oxygen
atom of the sulfonamide moiety, whereas the Tyr511 established hydrogen bond with hydroxyl
moiety of the compound, respectively (Figure-14a). This compound also formed two arene-
19

ACCEPTED MANUSCRIPT
cation interactions with the Tyr504 and Arg600 residues. The higher inhibitory potential of
compound 5 might be due to the availability of the electron donating-OH group and the electron
withdrawing group (-Cl) which creates an electron flow in the compound, making it more
polarizable and a more potent inhibitor compared to the compounds having only electron
withdrawing moiety (-Cl) e.g., compound 2, 13, 1 etc. The docking conformation of the second
most active compound 2 (IC50= 2.10 ± 0.10, docking score = -15.8651) in the series, showed
three hydrogen bonds and two arene-arene interactions where the active site residues of the
enzyme.Asp207, Tyr508 and Arg600 were found to be involved in hydrogen bonding with the
oxygen atoms of the sulfonamide moiety of the compound (Figure-12b). The arene-arene
interaction was observed between the central phenyl ring of the compound and the hydrophobic
residue Trp507 and Tyr511 of
β-glucouronidase enzyme (Figure-12b). The slightly lower
biological activity and poor binding interactions of compound 2 as compared to compound 5
might be due to the absence of the electron donating moiety. In case of compound 1 the third
most active compound (IC50 = 4.50 ± 0.01) of the series, the docking results showed that this
compound formed four hydrogen bonding with the active site residues (Tyr205, Asp207, Tyr508
and Arg600) of the enzyme (Figure-13c). Tyr205 made hydrogen bonds with the nitrogen atom
of the methanamine moiety while Asp207, Tyr508, and Arg600 formed hydrogen bonds with the
oxygen atoms of the sulfonamide moiety of the compound. If, we compare the structures of
compounds 1 and 2, they have almost similar structures but the only difference is the positions
of the -Cl moiety. The different attachment positions of chlorine might play a role in the
activities as well as binding interaction of these compounds (Table-5 and Figure-13). The
docking conformation of the forth most active compound, compound 24 (IC = 5.50 ± 0.01)
5
0
showed two hydrogen bonds, one arene-cation and onearene-arene linkage with the Phe206,
Tyr508, and Arg600 residues of the enzyme (Figure-12d). Phe206 showed hydrogen bond with
the nitrogen atom of the quinoline ring and Arg600 established hydrogen bonding with the
nitrogen atom of the sulfonamide moiety of the compound. Tyr508 and Arg600 showed arene-
arene and arene-cation interactions with this compound, respectively. If, we compare the
structures of compound 24 and 1, both compounds have about similar structures the only
difference is the attachment positions of the 2,5-dichloro-benzenesulfonamide substituent to the
quinoline ring. This attachment position might play a role in the biological activities as well as
binding modes of these compounds (Table-5, and Figure-12).
20

ACCEPTED MANUSCRIPT
For the nitro phenyl containing compounds (22, 26, 27), it was observed that the position of the
nitro moiety plays a role in the biological activity as well as binding modes of these compounds.
For example, when the nitro group is at ortho position of the phenyl ring, then the compound
(22) showed comparatively good inhibitory activity and interaction as compared to the
compounds (26 and 27) in which the nitro group present at meta and para position of phenyl ring
of the compounds (Figures 13a and 13b, compounds 25 and 26). Comparable results were
observed for compounds 39 and 38 (Table-5 and Figure-14c and-14d).
The methoxy benzene containing compounds (9, 10, 21) showed very low biological activities
and poor interactions. The poor inhibitory activities and interactions of these compounds might
be due to the presence of methoxy groups.
Overall the docking results showed that electron donating moiety is favorable for biological
activities as compared to electron withdrawing moiety. Furthermore, the position of attachment
at the aryl part as well as the quinoline ring played a role in the activities of these synthetic
compounds.
Generally, a good correlation was observed between the docking stabilizing energies and
biological activities of the active compounds showing a correlation coefficient of 0.114 as given
in (Figure-14).
21

ACCEPTED MANUSCRIPT
Figure-12: Docking conformation of (a)compound 5, (b)compound 2, (c) compound 1and (d)
compound 24 in the active site of β-glucuronidase
22

ACCEPTED MANUSCRIPT
Figure-13: Docking conformation of (a)compound 22, (b) compound28, (c) compound 38 and
(d) compound 39 in the active site of β-glucuronidase
Figure-14: A correlation graph for docking predicted activity and IC values
5
0
23

ACCEPTED MANUSCRIPT
3
.
Conclusion:
This study deals with synthesis of quinoline analogs and their
β-glucuronidase inhibitory
potential. Amongst synthetic compounds most of the compounds that have electron donating
substituents showed more inhibitory activity. However, some nitro substituted compounds also
showed good inhibitory activity. These results were further supported by molecular docking
studies which show efficient binding interaction between the active compound and
glucuronidase enzyme proteins. Conclusively, some new lead molecules are identified as
glucuronidase inhibitors and further studies on these molecules may direct to better inhibitors.
β
-
-
β
4
4
.
Experimental:
.1.
Material and methods:
Different sulfonyl chloride, 3-, 5-, 8-amino quinoline, 8-hydroxy quinoline and pyridine were
purchased from TCI (Japan) and used as received without purification. UV light of wavelengths
of 254 and 365 nm was used for visualization of chromatograms.
1
13
H- and C-NMR spectra were recorded on Bruker NMR 300, 400 and 500 MHz machines
(
Wissembourg, France). EI-MS, HREI-MS and FAB-MS spectroscopic analysis have been
obtained on a Finnigan-MAT-311-A instrument (Bremen, Germany). Thin layer chromatography
TLC) was performed using pre-coated silica gel plates (Merck, Kieselgel 60 F-254, 0.20 mm).
(
4
.2.
β-Glucuronidase inhibition study:
β
-Glucuronidase (E.C. 3.2.1.31, from bovine liver, G-0251) was purchased from Sigma
-Glucuronidase activity was determined by measuring the absorbance at
-nitrophenol formed from the substrate by the spectrophotometric method. The total
reaction volume was 250 L. DMSO (100%) was used to dissolve the compound (5 L) which
become 2% in the final assay (250 L) and the same conditions were used for standard (D-
saccharic acid 1,4-lactone). The reaction mixture contained 185
of test compound solution, 10
Chemical Co. (USA).
05 nm of
β
4
p
µ
µ
µ
µ
L of 0.1 M acetate buffer, 5 µL
°
µ
L of enzyme solution was incubated at 37 C for 30 min. The
plates were read on a multi-plate reader (SpectraMax plus 384) at 405 nm after the addition of 50
-nitrophenyl- -D-glucuronide. All assays were run in triplicate [47].
µ
L
p
β
24

ACCEPTED MANUSCRIPT
4
.3
General procedure for synthesis of compounds 1-40
Quinoline analogs were synthesized by refluxing 1 mmol of sulfonyl chloride with 1 mmol of
different quinoline in 10 mL of pyridine for 6 hours. Succession of reaction was studied by thin
layer chromatography (TLC) analysis. On completion of reactions, the mixture was poured into
cool water which resulted in precipitation. The precipitates were filtered, washed with hexane
and dried to afford target compounds.
2
,5-Dichloro-N-(quinolin-5-yl)benzenesulfonamide (1)
°
1
Yield: 55%; m.p. 176-180 C; H-NMR (400 MHz, CD
3
OD),
4,2 = 2.4 Hz, 1H, H-4), 7.72 (d,
.59 (d, J4’,3’ = 8.4 Hz, 1H, H-4’), 7.54 (overlapping multiplet, 4H, H-6’/H-3/H-6/ H-5); C-
): c 149.5, 138.8, 138.1, 136.6, 134.3, 133.6, 132.6, 132.0, 130.5,
29.5, 128.1, 126.7, 123.8, 122.5, 117.6; EI-MS: m/z (rel. abund. %) 354 [M+2] (4.6), 352 [M]
7.0), 253 (100), 143 (87.8), 116 (62.6); HREI-MS Calcd for C H Cl N O S , 351.9840.Found,
δ
H
8.84 (d,
J
2,4 = 2.8 Hz, 1H, H-2),
8
7
.26 (d,
J
7,6 = 8.0Hz, 1H, H-7), 8.14 (d,
J
J3’,4’ = 7.2 Hz, 1H, H-3’),
1
3
NMR (125 MHz, DMSO-d6 δ
+
+
1
(
1
5
10
2
2
2
3
2
51.9822.
,4-Dichloro-N-(quinolin-5-yl)benzenesulfonamide (2)
°
1
Yield: 65%; m.p. 155-159 C; H-NMR (400 MHz, CD OD), δH 8.83 (overlapping multiplet, 1H,
3
H-2), 8.71 (d, J6,5 = 8.4 Hz 1H, H-6), 7.90 (d, J4,3 = 8.4 Hz 1H, H-4), 7.79 (d, J7,6 = 8.8 Hz 1H,
,
,
,
H-7), 7.69 (d,
J
3’,5’ = 1.6 Hz
,
1H, H-3’), 7.64 (t,
J
3,2
=
3,4 = 8.0 Hz
,
1H, H-3) ,7.55 (overlapping
1
3
multiplet, 1H, H-8), 7.37 (overlapping multiplet, 2H, H-5’/H-6’); C-NMR (125 MHz, DMSO-
d6): c 150.7, 148.1, 137.5, 136.5, 136.1, 133.6, 132.4, 132.2, 131.4, 131.3, 129.0, 127.7, 124.7,
δ
+
+
1
3
23. 2 , 121.3., 19.1, 18.8; EI-MS: m/z (rel. abund. %) [M+4] 356 (1.2), [M+2] (3.7) (1.3), [M]
52 (8.6), 253 (23.5), 209 (8.4), 143 (71.1), 116 (100), 109 (27.6), 89 (56.4); HREI-MS Calcd
for C H Cl N O S , 351.9840.Found, 351.9815.
1
5
10
2
2
2
4
-Chloro-2,5-dimethyl-N-(quinolin-5-yl)benzene sulfonamide (3)
°
1
Yield: 75%; m.p. 197-200 C; H-NMR (400 MHz, CD OD), δH 8.83 (overlapping multiplet 1H,
3
,
H-4), 8.55 (d,
J5,6 =8.4 Hz
,
1H, H-6), 7.92 (d,
J4,3 = 8.4 Hz
,
1H, H-4), 7.64 (t,
J
3,2
=
3,4
=
J6,’7’ = 8.0
Hz 1H, H-3), 7.52 (s, 1H, H-6’), 7.52 (overlapping multiplet, 1H, H-8), 7.34 (s, 1H, H-3’), 7.21
,
1
3
(
3 3
d, J7,6 = 7.6 Hz, 1H, H-7), 2.45 (s, 3H, CH ), 2.24 (s, 3H, CH ); C-NMR (125 MHz, DMSO-
25

ACCEPTED MANUSCRIPT
d6):
δ
c 150.7, 148.1, 137.5, 136.5, 136.1, 133.6, 132.4, 132.2, 131.4, 131.3, 129.0, 127.7, 124.7,
+
+
1
23. 2 , 121.3., 19.1, 18.8; EI-MS: m/z (rel. abund. %), 348 [M+2] (18.2), [M] 346 (45.5), 282
(
31.1), 143.1 (100), 116.2 (93.2); HREI-MS Calcd for C H ClN O S, 346.0543, Found,
1
7
15
2
2
3
46.0562.
-Nitro-
3
N-(quinolin-5-yl)benzenesulfonamide (4)
°
1
Yield: 65%; m.p. 252-256 C; H-NMR (300 MHz, CD
3 H
OD), δ 8.82 (overlapping multiplet, 1H,
H-2), 8.47 (overlapping multiplet 3H, H-6’/H-4’/H-6), 7.99 (overlapping multiplet 1H, H-4),
,
,
7
8
1
.93 (s, H, H-2’), 7.72 (overlapping multiplet 2H, H-3/H-7), 7.49 (overlapping multiplet, 1H, H-
,
1
3
6 C
), 7.27 (dd, J5,’6’ = 7.5 Hz, J5’,7’ = 0.9 Hz, 1H, H-5’); C-NMR (75 MHz, DMSO-d ): δ 150.8,
48.1, 147.8, 140.9, 132.6, 131.3, 129.0, 128.2, 127.5, 124.8, 124.0, 121.4; EI-MS: m/z (rel.
+
abund. %), [M] 329 (13.5), 218 (0.7), 143 (98.2), 116 (100), 89 (45.1). HREI-MS Calcd for
3
29.0470, Found 329.0472.
3
,5-Dichloro-2-hydroxy-
N-(quinolin-5-yl)benzene sulfonamide (5)
°
1
Yield: 45%; m.p. 174-178 C; H-NMR (400 MHz, CD
3
OD),
δ
H
8.83 (overlapping multiplet, 1H,
J3,4 = 8.0 Hz,
H-2), 8.71 (d, J6,5 = 8.8 Hz 1H, H-6), 7.92 (d, J4,3 = 8.4 Hz 1H, H-4), 7.68 (t, J3,2
=
,
,
1
3
1
H, H-3), 7.56 (overlapping multiplet, 2H, H-7/H-8), 7.43 (s, 1H, H-6’), 7.41 (s, 1H, H-4’); C-
NMR (75 MHz, DMSO- ): 151.6, 149.3, 139.8, 137.8, 137.7, 135.4, 134.0, 133.8, 132.9,
31.0, 130.4, 128.4, 127.2, 125.8, 122.5; EI-MS: m/z (rel. abund. %), [M+4] 372 (0.70), [M+2]
d
6
δ
C
+
+
1
3
+
70 (10), [M] 368 (12), 304 (4.7), 162 (10.5), 144 (100), 11 6(45.4); HREI-MS Calcd for
S, 367.9789, .Found, 367.9794.
15 2 2 3
C H10Cl N O
5
-Bromo-2-methoxy-N-(quinolin-5-yl)benzene sulfonamide (6)
°
1
Yield: 45%; m.p. 203-208 C; H-NMR (300 MHz, CD OD), δH 8.82 (overlapping multiplet, 1H,
3
H-2), 8.68 (d,
J6,5 = 8.7 Hz, 1H, H-6), 7.90 (d, J4,5, = 8.7 Hz, 1H, H-4), 7.69 (overlapping
multiplet, 3H ,H-3’/H-4’/H-6’), 7.55 (overlapping multiplet, 1H, H-3), 7.39 (d, J7,6, = 7.2 Hz, 1H,
13
H-7), 7.09 (d, J8,7 = 9.0 Hz, 1H, H-8), 3.86 (s, 3H, OCH ); C-NMR (75 MHz, DMSO-d6): δC
3
1
5
55.6, 150.6, 148.0, 137.4, 132.4, 131.5, 129.0, 128.7, 127.5, 124.5, 122.9, 121.2, 115.3, 110.8,
+
+
6.2; EI-MS: m/z (rel. abund. %), [M+2] 394 (36.6) [M] , 392.0 (34.1), 327.9 (6.5), 312.8 (6.5),
26

ACCEPTED MANUSCRIPT
2
2 3
98.9 (25.3), 218 (43.4), 157 (26.6), 143 (100); HREI-MS Calcd for C16H13BrN O S, 391.9830,
Found 391.9818.
3
,4-Dimethoxy-N-(quinolin-5-yl)benzenesulfonamide (7)
°
1
Yield: 55%; m.p.165-169 C; (ethyl acetate/hexanes 3:7), H NMR (400 MHz, CD OD), δH 8.80
3
(
overlapping multiplet, 1H, H-2), 8.48 (d, J6,7 = 8.4.Hz, 1H, H-6), 7.91(d, J4,3 = 8.4 Hz, 1H, H-4),
.66 (t, J3(2,4) 8.0 Hz, 1H, H-3), 7.46 (overlapping multiplet, 1H, H-8), 7.27 (overlapping
multiplet, 2H, H-7/H-6’), 7.09 (d, J2’,6’ = 2.0 Hz, 1H, H-2’), 6.95 (d, J5’,6’ = 8.4 Hz, 1H, H-5’),
7
=
1
3
3
1
.82 (s, 3H, OCH ), 3.66 (s, 3H, OCH ); C-NMR (75 MHz, DMSO-d6): δc 151.9, 150.3, 148.2,
3 3
32.6, 131.3, 130.5, 128.6, 127.2, 124.4, 123.0, 120.8, 120.3, 110.7, 109.1, 55.5, 55.3; EI-MS:
+
m/z (rel. abund. %), [M] 344 (100), 280 (90.2), 265 (11.0), 201 (35.5),143 (43.8) ,116 (72.7), 89
(24.3); HREI-MS Calcd for C17
16 2 4
H N O S 344.0831, Found 344.0844
4
-Propyl-N-(quinolin-5-yl) sulfonamide (8)
°
1
Yield: 55%; m.p 145-150 C; H-NMR (400 MHz, CD
H-2), 8.42 (d, J4,2 = 8.4.Hz, 1H, H-4), 7.90 (d, J8,7 = 8.8Hz, 1H, H-8), 7.64 (d,
H-6), 7.54 (d, J2.’,3’ 6’,5’ = 8.4Hz, 2H, H-2’/H-6’), 7.42 (overlapping multiplet, 1H, H-7), 7.27
overlapping multiplet, 3H , H-3’/H-5’/H-3), 2.62 (t, 2H, CH ), 1.62 (overlapping multiplet, 2H,
c 151.9, 150.3, 148.2, 132.6, 131.3,
3
OD),
δ
H
8.78 (overlapping multiplet, 1H,
J6,4 = 8.0 Hz, 1H,
=
J
(
2
1
3
CH ), 0.89 (s, 3H, CH ); C-NMR (75 MHz, DMSO-d6):
δ
2
3
1
30.5, 128.6, 127.2, 124.4, 123.0, 120.8, 120.3, 110.7, 109.1, 55.5, 55.3; EI-MS m/z (rel. abund.
+
%
), 326 [M] , 262 (6.9), 233 (8.4), 219 (7.4). 183 (15.4), 143 (100.0), 116 (41.1); HREI-MS
Calcd for C H N O S 326.1089, Found 326.1085
1
8
18
2
2
3
,4-Dimethoxy-
N-(quinolin-8-yl)benzene sulfonamide (9)
°
1
Yield: 50%; m.p 180-184 C; H-NMR (300 MHz, CD OD), δH 8.80 (overlapping multiplet, 1H,
3
H-2), 8.23 (dd,
,
J7,6 = 8.4, J7,5= 1.5 Hz, 1H, H-7), 7.84 (d, J4,3 = 6.9 Hz 1H, H-4), 7.58 (d, J6,5 =7.2
Hz 1H, H-6), 7.49 (overlapping multiplet, 3H, H-3/H-5/H-6’), 7.31 (d, J2’,4‘ = 2.1 Hz 1H, H-2’),
,
,
13
6
.89 (d, J5’,6’ = 9.0 Hz 1H, H-5’), 3.74 (s, 3H, OCH ), 3.67 (s, 3H , OCH ); C-NMR (75 MHz,
, 3 3
DMSO-
d
6
):
δ
C
151.9,150.3, 148.2, 132.6, 131.3, 130.5, 128.6, 127.2, 124.4, 123.0, 120.8, 120.3,
+
1
10.7, 109.1, 55.5, 55.3; EI-MS: m/z (rel. abund. %), [M] 344 (11.3), 280 (94.2), 265 (100), 143
(
49.3), 116 (29); HREI-MS Calcd for C H N O S, 344.0831, Found 344.0846
17 16 2 4
27

ACCEPTED MANUSCRIPT
4
-Methoxy-N-(quinolin-8-yl)benzenesulfonamide (10)
°
1
Yield: 65%; m.p 162-167 C; H-NMR (300 MHz, CD
3 H
OD), δ 8.79 (overlapping multiplet, 1H,
H-2), 8.22 (dd, J7,5 = 1.5, J7,6 = 8.4 Hz, 1H, H-7), 7.81 (overlapping multiplet, 3H, H-4/H-3/H-5),
7
3
1
2
.57 (overlapping multiplet, 3H, H-2’/H-6’/H-6), 6.88 (overlapping multiplet, 2H, H-5’/H-3’),
1
3
.72 (s, 3H, OCH ); C-NMR (75 MHz, DMSO-d6):
δ
c 162.4, 150.3, 148.2, 132.6, 131.3, 128.6,
3
+
27.2, 124.4, 123.0, 121.1, 114.2, 55.5; EI-MS: m/z (rel. abund. %), [M] 314 (11.0) 250 (100)
35 (76.1), 143 (91.9), 116(64.7); HREI-MS Calcd forC H N O S. 314.0725, Found 314.0728.
1
6
14
2
3
4
-Nitro-N-(quinolin-8-yl)benzenesulfonamide (11)
°
1
Yield: 45%; m.p 168-172 C; H-NMR (400 MHz, CD OD), δH 8.76 (overlapping multiplet, 1H,
3
H-2), 8.22 (overlapping multiplet, 3H, H-7/H-5’/H-3’), 8.09 (d,
2’,3’
J = J6’,5’= 8.8 Hz, 2H, H-2’/H-
6
’), 7.89 (d, J4,3, = 7.6 Hz, 1H, H-4), 7.63 (d, J5,6, = 8.4 Hz, 1H, H-5), 7.52 (d, J6,5, = 8.0 Hz, 1H,
1
3
H-6 ), 7.47 (overlapping multiplet, 1H, H-3); C-NMR (75 MHz, DMSO- 6
d ): δc 149.7, 149.4,
1
45.3, 139.5, 136.4, 133.0, 128.4, 128.2, 126.6, 124.2, 124.1, 122.2, 119.1, 40.3; EI-MS: m/z
+
(
rel. abund. %), [M] 329 (19) 265 (65.6), 235 (8), 219 (14.7), 143 (100), 116(69.8); HREI-MS
Calcd for C H N O S 329.0470, Found 329.0485.
1
5
11
3
4
5
-Bromo-2-methoxy-N-(quinolin-8-yl)benzene sulfonamide (12)
°
1
Yield: 65%; m.p 203-208 C; H-NMR (400 MHz, CD OD), δH 8.88 (overlapping multiplet, 1H,
3
H-2), 8.24 (overlapping multiplet, 1H, H-7), 7.95 (overlapping multiplet, 1H, H-4), 7.76 (d,
7.6 Hz, 1H, H-4’), 7.56 (overlapping multiplet 3H, H-5/H-3’/H-6’), 7.45 (t, J3,2 J3,4 = 8.0 Hz,
); C-NMR (75 MHz, DMSO- ):
c 154.9, 148.9, 144.0, 137.6, 137.2, 136.0, 132.5, 132.3, 131.1, 126.2, 122.4, 121.9, 115.0,
J
4’,3’
=
=
,
1
3
1
δ
1
H, H-3), 6.90 (d,
J
6,5 = 8.8 Hz, 1H, H-6), 3.78 (s, 3H, OCH
3
d
6
+
+
14.8, 110.3, 56.1; EI-MS: m/z (rel. abund.%), [M+2] 394 (2.4), 392 [M] (1.8), 330 (7.5), 297
(
100) 218.2 (10.6), 143 (72.2), 116 (75.5); HREI-MS Calcd for C H BrN O S, 391.9830,
16 13 2 3
Found 391.9809.
2
,4-Dichloro-N-(quinolin-8-yl)benzenesulfonamide (13)
°
1
Yield: 54%; m.p 165-169 C; H-NMR (400 MHz, CD OD), δH 8.84 (overlapping multiplet, 1H,
3
,
H-2), 8.25 (overlapping multiplet, 1H, H-7), 8.14 (d, J4,5 = 8.4 Hz 1H, H-4), 7.69 (d, J6,7 = 7.2
,
28