Sulphamethazine derivatives as immunomodulating agents: New therapeutic strategies for inflammatory diseases

Article abstract of DOI:10.1371/journal.pone.0208933

Sulfamethazine (SMZ) (1) is an antibacterial sulfa drug which suppresses the synthesis of dihydrofolic acid. It is used for the treatment of infections in livestock; such as gastrointestinal, and respiratory tract infections. During the current study, syn

Full text of DOI:10.1371/journal.pone.0208933

RESEARCH ARTICLE
Sulphamethazine derivatives as
immunomodulating agents: New therapeutic
strategies for inflammatory diseases
Hina Siddiqui 1*, Haroon M. Haniffa1,2, Almas Jabeen3, Atta-ur -Rahman1,3, M.
ID
Iqbal Choudhary1,3,4
*
1 H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi, Pakistan, 2 Department of Physical Sciences, Faculty of Applied Sciences,
South Eastern University, Oluvil, Sri Lanka, 3 Dr. Panjwani Center for Molecular Medicine and Drug
Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi,
Pakistan, 4 Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
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* hej@cyber.net.pk (MIC); hinahej@gmail.com (HS)
Abstract
Sulfamethazine (SMZ) (1) is an antibacterial sulfa drug which suppresses the synthesis of
dihydrofolic acid. It is used for the treatment of infections in livestock; such as gastrointesti-
nal, and respiratory tract infections. During the current study, synthesis, characterization,
and evaluation of immunomodulatory activities of derivatives of sulfamethazine (SMZ) (3–
39) was carried out. These derivatives were synthesized by the reaction of sulfamethazine
with a range of acid chlorides. All the compounds were characterized by using modern spec-
troscopic techniques, such as 1H-, and 13C-NMR, EI-MS, and HRFAB-MS. Compounds 3–
10, 14, and 15 were identified as new compounds. Immunomodulatory effect of compounds
3–39 on different parameters of innate immune response was evaluated, including the pro-
duction of Reactive Oxygen Species (ROS) from human whole blood and isolated polymor-
phonuclear neutrophils (PMNs), nitric oxide (NO), and pro-inflammatory cytokine TNF-α. All
the new compounds, except 14 and 15, showed a significant anti-inflammatory activity.
Compounds 3–39 were also evaluated for their anti-bacterial activity and cytotoxicity (3T3
mouse fibroblast cell lines). All the compounds were found to be non-cytotoxic against nor-
mal cell lines.
OPEN ACCESS
Citation: Siddiqui H, Haniffa HM, Jabeen A,
-Rahman A-u, Choudhary MI (2018)
Sulphamethazine derivatives as
immunomodulating agents: New therapeutic
strategies for inflammatory diseases. PLoS ONE 13
(12): e0208933. https://doi.org/10.1371/journal.
pone.0208933
Editor: Mohammad Shahid, Aligarh Muslim
University, INDIA
Received: August 27, 2018
Accepted: November 26, 2018
Published: December 19, 2018
Copyright: © 2018 Siddiqui et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files.
Introduction
Funding: HMH is thankful to the ICCBS for the
post-doctoral fellowship for one year. HMH is also
thankful to the COMSATS (The Commission on
Science and Technology for Sustainable
Chronic inflammation is a persistent inflammatory condition which can cause tissue destruc-
tion. Macrophages are critical players in the initiation of inflammatory process, which trans-
forms into chronic inflammation in some cases [1]. The white blood cells move in bulk, and
begin to attack internal organs or other important tissues and cells [2]. Chronic inflammation
is linked to a variety of ailments, including osteoarthritis, asthma, atherosclerosis, inflamma-
tory bowel disease, psoriasis, cancers, Crohn’s disease, and ankylosing spondylitis [3].
Development in the South) for the travel support.
Competing interests: The authors have declared
that no competing interests exist.
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Sulphamethazine derivatives as immunomodulating agents
Oxidative stress has a crucial role in the pathogenesis of chronic inflammatory diseases.
During inflammation, activated phagocytes including neutrophils, monocytes and macro-
phages, utilize NADPH oxidase system to generate highly toxic free radicals, called oxidative
burst. The deamination of L-arginine through an enzyme nitric oxide synthase (NOS) gener-
ates nitric oxide (NO) which reacts with superoxide to produces highly reactive peroxynitrite
radicals. Physiological concentrations of ROS and reactive nitrogen species (RNS) have various
roles, such as regulation of cellular metabolism, intracellular signaling and maintain cellular
redox status, etc. ROS also defend against invading pathogens. The overexpression of these
molecules during chronic inflammation is the key factor in pathogenesis of various metabolic,
and autoimmune diseases [4].
Cytokines play a major role in immune system by facilitating interactions between various
immune cells. TNF-α is among the key promoters of chronic inflammation, and thus play a
pivotal role in the pathogenesis of inflammatory, and autoimmune diseases. It is known to
enhance the production of pro-inflammatory cytokines, leukocyte adhesion molecules, prosta-
glandin E-2 (PGE-2), collagenase, nitric oxide (NO), etc. TNF-α inhibitor reduces the inflam-
mation, and joint damage in rat model of inflammatory arthritis. TNF and its various
superfamily members are among the most important therapeutic targets against the chronic
inflammation [5].
Certain natural and synthetic drugs have been developed for the treatment of chronic
inflammation and associated diseases. These drugs are divided in two groups; steroidal anti-
inflammatory drugs, such as corticosteroids, and non-steroidal anti-inflammatory drugs
(NSAIDs) (e.g. asprin, naproxen, and Ibuprofen) [6]. Short-term use of corticosteroids is
linked with mild side effects, including cutaneous effects, electrolyte abnormalities, hyperten-
sion, hyperglycemia, and neuropsychologic effects. While, long-term use of corticosteroid is
associated with more serious consequences, such as osteoporosis, aseptic joint necrosis, adre-
nal insufficiency, hyperlipidemia, growth suppression, and possible congenital malformations
[7]. Because of the above mentioned adverse effects, there is an urgent need for developing
new, safe and effective anti-inflammatory drugs.
The process of finding new uses of existing drugs is termed drug repositioning or drug
repurposing, which has gained major importance in recent years. The libraries of drug mole-
cules and rapid advances in disease biology, genomics and bioinformatics has enhanced the
pace of activity-based, as well as in silico drug repositioning. It is a promising, fast, and cost
effective method that can meet the challenges of traditional de novo drug discovery and devel-
opment [8, 9].
Sulfonamides are well known antimicrobial agents. They have been reported to possess var-
ious biological activities such as anti-cancer, anti-angiogenesis, anti-convulsant, anti-diuretics,
anti-inflammatory, hypoglycemic, carbonic anhydrase, histone deacetylase, HIV protease, and
cholinestrases inhibitors.
Sulfamethazine (SMZ) is an antibacterial sulfa drug. It is known to inhibit the synthesis of
dihydrofolic acid. Mostly this drug have been used for the treatment of various infections in
livestock, such as gastrointestinal and respiratory tract infections [10]. Since the amide is an
important functional group present in a number of drugs, the synthesis of amide linkages has
special significance in organic synthesis, and in pharmaceuticals industries [11]. Amide deriva-
tives of sulfamethazine have also been reported for other biological activities, such as anti-bac-
terial [12–15] and anti-cancer [16] properties.
A total of 37 amide derivatives of sulfamethazine were synthesized by using conventional
protocols being used in synthetic chemistry. Among them, 3–10, 14, and 15 were identified as
new analogues. Interestingly all these compounds were not been reported for their anti-
inflammatory activities. Present study describes the effects of test compounds 3–39 on various
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Sulphamethazine derivatives as immunomodulating agents
inflammatory mediators of innate immune responses, including effect on the production of
intracellular ROS fromzymosan-activated whole blood phagocytes, and isolated PMNs, nitric
oxide (NO) production from lippopolysaccharide (LPS) activated J774.2 mouse macrophages,
and the production of pro-inflammatory cytokine TNF-α from THP-1 human monocytic leu-
kemia cell line. All the compounds were also evaluated for their anti-bacterial, and cytotoxicity
activities.
Materials and methods
General experimental conditions
Melting points were determined on a Bu¨chi 535 melting point apparatus. IR spectra were
recorded on a FTIR-8900 spectrophotometer. UV Spectra were obtained on a Hitachi UV
3200 spectrophotometer. Electron impact mass spectra (EI-MS) were recorded on a JEOL
JMS-600H mass spectrometer with a MASPEC data system. FAB- and HRFAB-MS were per-
formed on a Jeol JMS HX 110 mass spectrometer using glycerol as the matrix. The 1H-NMR
spectra were recorded in deuterated solvents on Bruker Avance 300 and 400, MHz NMR spec-
trometers, while the 13C-NMR experiments were conducted on the same instrument at 75.45,
100, and 125 MHz respectively. The chemical shifts (δ) were in ppm, relative to the chemical
shift of tetramethylsilane (TMS), as internal standard and coupling constants J in Hz. Thin-
layer chromatography (TLC) analyses were performed on precoated ALUGRAM, SIL G/
UV254 aluminum plates (Kieselgel 60, 20 x 20, 0.5mm thick, E. Merk, Germany). The reagents
and solvents were obtained from Aldrich (St. Louis, Missouri, (USA)), E. Merck Darmstadt,
(Germany), and Fluka (Buchs, Switzerland). Reagents and solvents were used without purifica-
tion. Chromatograms, developed on TLC plates, were visualized under ultraviolet light at 254
nm for fluorescence quenching spots and 365 nm for florescent spots. Chromatograms, devel-
oped on TLC plates, were visualized under the ultraviolet light at 254 nm for fluorescence
quenching spots, and 365 nm for florescent spots. General reaction for the synthesis of com-
pounds 3–39 can be seen in the supporting information S39 Fig.
A solution of sulfamethazine drug (0.5 mmol, 0.14 g) in acetonitrile (3.0 mL) was stirred at
room temperature for 15–20 minutes. Then the corresponding acid chloride (0.5 mmol) was
added slowly, and the reaction mixture was refluxed for 3–4 hours. Progress of the reaction
was monitored by TLC (Silica gel, hexane: ethyl acetate, 4: 6). After complete consumption of
substrate, the reaction mixture was cooled down, and the precipitate was filtered off and
washed with hexane, ethyl acetate, and acetone to remove impurities. Structures of all synthetic
compounds 3–39 were characterized by different spectroscopic techniques; such as 1H-, and
13C NMR, EI-MS, FAB-MS, HR-FAB-MS, UV and IR spectroscopy.
2,3-Dibromo-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)propanamide
(3). Slightly brown solid; yield: 50%; m. p. 185–187 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf =
0.52; IR (KBr): νmax cm-1 3276, 3185, 3039, 2996, 1704, 1598, 1434, 1361, 1320, 1162, 1083, 853,
585; UV/Vis (MeOH): λmax nm 207, 272; 1H-NMR (300 MHz, DMSO-d6): δ 11.20 (1H, s,
0
0
0
0
0
0
0
0
SO2NH), 10.90 (1H, s, CONH), 7.97 (2H, d, J2 , 3 /6 , 5 = 8.4 Hz, H-2 /H-6 ), 7.74 (2H, d, J3 , 2 /
50, 60
= 8.4 Hz, H-30/H-50), 6.75 (1H, s, pyrimidine-H), 4.88 (1H, dd, J = 5.1 Hz, J = 10.2 Hz, H-
2), 3.97 (2H, m, H-2a, H-2b), 2.25 (6H, s, pyrimidine-CH3); 13C-NMR (100 MHz, DMSO-d6):
δ 167.2 (CONH), 165.3 (C-3", C-5"), 156.0 (C-1"), 142.1 (C-1’), 135.6 (C-4’), 129.4 (C-3’, C-5’),
118.4 (C-2’, C-4’), 113.4 (C-4"), 44.2 (C-2), 30.7 (C-3), 22.7 (CH3-3", CH3-5"); EI-MS m/z (%
rel. abund.): 350.0 [M+4]+-SO2 -Br, 1.3], 348.0 [M+2]+-SO2 -Br, 65], 346.0 [M+-SO2-Br, 30],
267.1 (16), 239.1 (8), 213.2 (86), 186.0 (2.2); HR-FAB-MS (positive mode) m/z: [M+H]+calcd.
for C15H17Br2N4O3S, 490.9388; found, 490.9388.
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Sulphamethazine derivatives as immunomodulating agents
3-(Chloromethyl)-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)benzamide
(4). White solid; yield:46%; m. p. 201–203 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.53; IR
(KBr): νmax cm-1 3405, 3344, 3062, 2948, 1663, 1594, 1529, 1319, 1158,1078, 709, 582;UV/Vis
(MeOH): λmax nm224, 272; 1H-NMR (400 MHz, DMSO-d6): δ 11.46 (1H, s, SO2NH), 10.60
0
0
0
0
0
0
0
0
0
0
(1H, s, CONH), 7.97 (2H, d, J2 , 3 /6 , 5 = 8.8 Hz, H-2 /H-6 ), 7.92 (2H, d, J3 ,2 /5 , 6 = 8.8 Hz, H-
30/H-50), 7.96 (1H, d, J6,5 = 7.6 Hz, H-6), 7.94 (1H, s, H-2), 7.66 (1H, d, J4,5 = 7.6 Hz, H-4), 7.55
(1H, t, J = 7.6 Hz, H-5), 6.75 (1H, s, pyrimidine-H), 4.84 (2H, s, CH2Cl), 2.25 (6H, s, CH3);
13C-NMR (100 MHz, DMSO-d6): δ 167.5 (CONH), 161.7 (C-3", C-5"), 156.1 (C-1"), 142.6 (C-
1’), 138.0 (C-3), 136.2 (C-4’), 134.8 (C-1), 132.2 (C-4), 129.0 (C-5), 128.8 (C-6), 128.1 (C-3’, C-
5’), 127.6 (C-2), 119.2 (C-2’, C-4’), 113.5 (C-4"), 45.5 (CH2Cl-3), 22.9 (CH3-3", CH3-5");
FAB-MS (positive mode) m/z 433.0 [M+2]+, 431.2 [M+H]+; HRFAB-MS (positive mode) m/z:
[M+H]+calcd. for C20H20ClN4O3S, 431.0945; found, 431.0945.
4-(Chloromethyl)-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)benzamide
(5). White solid; yield: 46%; m. p. 246–248 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.52; IR
(KBr): νmax cm-1 3301, 2855, 1656, 1595, 1518, 1397,1320, 1160,1083, 883, 585; UV/Vis
(MeOH): λmax nm 213, 273; 1H-NMR (400 MHz, DMSO-d6): δ 10.58 (1H, s, CONH), 7.96
0
0
0
0
0
0
0
0
0
0
0
0
(2H, d, J2 , 3 /6 , 5 = 8.8 Hz, H-2 /H-6 ), 7.92 (2H, d, J3 , 2 /5 , 6 = 8.8 Hz, H-3 /H-5 ), 7.95 (2H, d,
2, 3/6, 5 = 8.0 Hz, H-2/H-6), 7.58 (2H, d, J3, 2/5, 6 = 8.0 Hz, H-3/H-5), 6.75 (1H, s, pyrimidine-
J
H), 4.83 (2H, s, CH2Cl), 3.65 (1H, s, SO2NH), 2.25 (6H, s, CH3); 13C-NMR (100 MHz, DMSO-
d6): δ 166.4 (CONH), 165.4 (C-3", C-5"), 156.1 (C-1"), 142.7 (C-4), 141.3 (C-1’), 134.9 (C-4’),
134.2 (C-1), 129.0 (C-3’, C-5’), 128.7 (C-3, C-5), 128.1 (C-2, C-6), 119.6 (C-2’, C-4’), 113.4 (C-
4"), 45.3 (CH2Cl-4), 22.5 (CH3-3", CH3-5"); EI-MS m/z (% rel. abund.): 368.1 [M+2]+-SO2
(64), 23], 366.1 [M+-SO2 (64), 68], 348.1 (29), 332.2 (63), 213.1 (72), 153.1 (40), 119.1 (100);
HRFAB-MS (positive mode) m/z: [M+H]+calcd. for C20H20ClN4O3S, 431.0945; found,
431.0945.
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3,5-bis(trifluoromethyl)ben-
zamide (6). White solid; yield: 53%; m. p. 257–259 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf =
0.53; IR (KBr): νmax cm-1 3419, 3312, 2925, 1684, 1598, 1539, 1281, 1136, 676, 585; UV/Vis
(MeOH): λmax nm 225, 277; 1H-NMR (400 MHz, DMSO-d6): δ 11.44 (1H, s, SO2NH), 10.90 0
0
0
0
0
(1H, s, CONH), 8.58 (2H, s, H-2/H-6), 8.38 (1H, s, H-4), 8.01 (2H, d, J2 , 3 /6 , 5 = 8.4 Hz, H-2 /
0
0
H-60), 7.92 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz, H-3 /H-5 ), 6.75 (1H, s, pyrimidine-H), 2.25 (6H, s,
CH3); 13C NMR (100 MHz, DMSO-d6): δ 167.4 (CONH), 162.9 (C-3", C-5"), 156.1 (C-1"),
142.0 (C-1’), 136.7 (C-1), 135.4 (C-4’), 130.2 (C-3, C-5), 129.1 (C-3’, C-5’), 128.6 (C-2, C-6),
125.3 (C-4), 124.3 (CF3-4), 119.5 (C-2’, C-4’), 113.6 (C-4"), 22.8 (CH3-3", CH3-5"); EI-MS m/z
(% rel. abund.): 455.2 (14), 454.2 [M+-SO2 (64), 70], 453.2 (100), 365.1 (2), 332.1 (3), 241.1
(33), 213.1 (55); HRFAB-MS (positive mode) m/z: [M+H]+calcd. for C21H17F6N4O3S,
519.0926; found, 519.0926.
0
0
0
0
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3,5-difluorobenzamide (7).
White solid; yield: 62%; m. p. 237–240 ˚C; TLC (Hexane: EtOAc, 8:2 v/v): Rf = 0.58; IR (KBr):
ν
max cm-1 3387, 3311, 3122, 2924, 1673, 1596, 1532, 1326, 1159, 589; UV/Vis (MeOH): λmax
nm 208, 277; 1H-NMR (400 MHz, DMSO-d6): δ 10.66 (1H, s, CONH), 7.97 (2H, d, J2 , 3 /6 , 5
0
0
0
0
=
8.8 Hz, H-20/H-60), 7.91 (2H, d, J3 , 2 /5 , 6 = 8.8 Hz, H-3 /H-5 ), 7.68 (2H, d, J ,
2 3F/6,5F = 6.4 Hz,
0
0
0
0
0
0
H-2/ H-6), 7.53 (1H, t, J3F,5F = 6.4 Hz, H-4), 6.75 (1H, s, pyrimidine-H), 3.66 (1H, s, SO2NH),
2.24 (6H, s, CH3); 13C-NMR (100 MHz, DMSO-d6): δ 167.2 (CONH), 163.2 (C-3", C-5"), 160.8
(C-3, C-5), 156.1 (C-1"), 142.2 (C-1’), 137.7 (C-1), 135.4 (C-4’), 129.0 (C-3’, C-5’), 119.4 (C-2’,
C-4’), 113.4 (C-4"), 111.4 (C-2, C-6), 107.2 (C-4), 22.7 (CH3-3", CH3-5"); EI-MS m/z (% rel.
abund.): 355.1 (20), 354.1 (95), 353.1 [M+-SO2 (64), 100], 213.1 (66), 141.1 (33), 113.1 (18);
HRFAB-MS (positive mode) m/z: [M+H]+calcd. for C19H17F2N4O3S, 419.0989; found,
419.0989.
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Sulphamethazine derivatives as immunomodulating agents
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3-fluoro-5-(trifluoromethyl)
benzamide (8). White solid; yield: 50%; m. p. 210–212 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf
= 0.48; IR (KBr): νmax cm-1 3386, 3306, 3200, 2924, 1633, 1594, 1528, 1358,1315, 1166, 1132,
836, 574; UV/Vis (MeOH): λmax nm 207, 282; 1H-NMR (400 MHz, DMSO-d6): δ 10.81 (1H, s,
0
0
0
0
0
CONH), 8.16 (1H, s, H-6), 8.12 (1H, J2, 3F = 9.2 Hz, H-2), 7.99 (2H, d, J2 , 3 /6 , 5 = 8.8 Hz, H-2 /
H-60), 7.97 (H, d, J4, 3F = 7.6 Hz, H-4), 7.92 (2H, d, J3 , 2 /5 , 6 = 8.8 Hz, H-3 /H-5 ), 6.75 (1H, s,
pyrimidine-H), 3.81 (1H, s, SO2NH), 2.24 (6H, s, CH3); 13C-NMR (100 MHz, DMSO-d6): δ
163.1 (CONH), 163.0 (C-3", C-5"), 160.5 (C-3), 156.1 (C-1"), 142.2 (C-1’), 137.9 (C-1), 135.6
(C-4’), 130.9 (C-5), 129.0 (C-3’, C-5’), 123.1 (CF3-5), 120.7 (C-6), 119.4 (C-2’, C-4’), 119.1 (C-
2), 116.1 (C-4), 113.5 (C-4"), 22.7 (CH3-3", CH3-5"); EI-MS m/z (% rel. abund.): 406.2 (8),
405.2 (63), 404.1 (98), 403.2 [M+-SO2 (64), 100], 385.2 (2), 282.1 (9), 213.1 (96), 191.1 (86),
163.1 (73); HRFAB-MS (positive mode) m/z: [M+H]+ calcd. for C20H17F4N4O3S, 469.0957;
found, 469.0957.
0
0
0
0
0
0
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3-fluoro-4-(trifluoromethyl)
benzamide(9). White solid; yield: 89%; m. p. 204–206 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf
= 0.56; IR (KBr):νmax cm-1 3405, 3129, 2949, 1685, 1593, 1530, 1313, 1315, 1170, 1134, 839,
579;UV/Vis (MeOH): λmax nm 209, 274; 1H-NMR (400 MHz, DMSO-d6): δ 10.83 (01H, s,
0
0
0
0
0
CONH), 8.06 (1H, d, J5,6 = 11.6 Hz, H-5), 7.98 (2H, d, J2 , 3 /6 , 5 = 8.8 Hz, H-2 /H-6 ), 7.97
0
0
0
0
0
0
(2H, m, H-2, H-6), 7.93 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz, H-3 /H-5 ), 6.75 (1H, s, pyrimidine-H),
4.10 (1H, s, SO2NH), 2.25 (6H, s, CH3); 13C-NMR (100 MHz, DMSO-d6): δ 167.3 (CONH),
163.3 (C-3", C-5"), 159.8 (C-3), 156.1 (C-1"), 142.1 (C-1’), 140.1 (C-1), 135.5 (C-4’), 129.0 (C-
3’, C-5’), 127.7 (C-5), 124.4 (C-6), 123.6 (CF3-4), 120.9 (C-4), 116.4 (C-2), 119.4 (C-2’, C-4’),
113.4 (C-4"), 22.7 (CH3-3", CH3-5"); EI-MS m/z (% rel. abund.): 406.4 (1.), 405.4 (16.4), 404.1
(72.1), 403.2 [M+-SO2 (64), 100], 333.2 (2), 282.1 (3), 213.1 (68), 191.1 (37), 163.1 (33);
HR-FAB-MS (positive mode) m/z: [M+H]+calcd. for C20H17F4N4O3S, 469.0957; found,
469.0957.
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-4-fluoro-3-(trifluoromethyl)
benzamide (10). White solid; yield: 64%; m. p. 218–220 ˚C; TLC (Hexane: EtOAc, 6:4 v/v):
Rf = 0.50; IR (KBr): νmax cm-1, 3316, 3097, 2931, 1692, 1632, 1591, 1498, 1313, 1164,1135, 836,
581; UV/Vis (MeOH): λmax nm 207, 274; 1H-NMR (400 MHz, DMSO-d6): δ 10.71 (1H, s,
0
0
0
0
0
CONH), 8.35 (1H, J6,5 = 6.0 Hz, H-6), 8.33 (1H, s, H-2), 7.98 (2H, d, J2 , 3 /6 , 5 = 8.8 Hz, H-2 /
0
0
H-60), 7.91 (2H, d, J3 , 2 /5 , 6 = 8.8 Hz, H-3 /H-5 ), 7.35 (1H, J5,6 = 9.2 Hz, H-5), 6.75 (1H, s,
pyrimidine-H), 3.66 (1H, s, SO2NH), 2.24 (6H, s, CH3); 13C-NMR (100 MHz, DMSO-d6): δ
167.3 (CONH), 163.4 (C-3", C-5"), 162.0 (C-4), 156.1 (C-1"), 142.3 (C-1’), 135.3 (C-4’), 135.2
(C-6), 130.1 (C-1), 129.0 (C-3’, C-5’), 127.6 (C-2), 123.6 (CF3-3), 119.4 (C-2’, C-4’), 117.4 (C-
5), 113.4 (C-4"), 22.7 (CH3-3", CH3-5"); EI-MS m/z (% rel. abund.): 406.2 (8), 405.3 (54), 404.2
(97), 403.2 [M+-SO2 (64), 100], 335.3 (3), 282.1 (7), 213.1 (88), 191.1 (83), 163.1 (53);
HRFAB-MS (positive mode) m/z: [M+H]+calcd. for C20H17F4N4O3S, 469.0957; found,
469.0957.
0
0
0
0
2-Chloro-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)propanamide (11).
White solid; yield:55%;m. p. 256–258 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.53; IR (KBr):
ν
max cm-13276, 3188, 3047, 2997, 1706, 1600, 1544, 1430, 1350, 1161,1081, 855, 583;UV/Vis
(MeOH): λmax nm212, 261; 1H-NMR (400 MHz, DMSO-d6): δ 11.38 (1H, s, SO2NH), 10.63
0
0
0
0
0
0
0
0
0
0
(1H, s, CONH), 7.94 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz, H-3 /H-5 ), 7.73 (2H, d, J2 , 3 /6 , 5 = 8.4 Hz, H-
20/H-60), 6.75 (1H, s, pyrimidine-H), 4.66 (1H, q, J = 6.8 Hz, H-2), 2.25 (6H, s, pyrimidine-
CH3). 1.59(1H, d, J = 6.8 Hz, CH3-2); FAB-MS (positive mode) m/z 371.2 [M + H + 2]+, 369.1
[M+ H]+.
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-4-fluorobenzamide (12).
White solid; yield:50%; m. p. 190–192 ˚C; TLC (Hexane: EtOAc, 8:2 v/v): Rf = 0.62; IR (KBr):
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Sulphamethazine derivatives as immunomodulating agents
ν
λ
max cm-1 3453, 3243, 3066, 2928, 1625, 1531, 1502, 1317,1160, 851, a1572; UV/Vis (MeOH):
max nm 213, 274; 1H-NMR (300 MHz, DMSO-d6): δ10.59 (1H, s, CONH), 8.04 (2H, dd, J2,
0
0
0
0
0
0
(3,4F)/6, (5, 4F) = 9.0 Hz, 2.1 0Hz, H-2/H-6), 7.96 (2H, d, J2 , 3 /6 , 5 = 9.0 Hz, H-2 /H-6 ), 7.91 (2H,
0
0
0
0
0
d, J3 , 2 /5 , 6 = 9.0 Hz, H-3 /H-5 ), 7.36 (2H, t, J3, (2,4F)/5, (6, 4F) = 9.0 Hz, H-3/H-5), 6.75 (1H, s,
pyrimidine-H), 3.99 (1H, s, SO2NH), 2.24 (6H, s, CH3); EI-MS m/z (% rel. abund.): 338.3 (7),
337.3 (96), 336.2 [M+-SO2 (64), 100], 335.2 (96), 213.2 (86), 185.1 (3), 123.0 (100), 95.0 (55).
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3-nitrobenzamide (13).
Slightly yellow solid; yield:80%; m. p. 251–253 ˚C; TLC (Hexane:EtOAc, 8:2 v/v): Rf = 0.54;IR
(KBr): νmax cm-13381, 3072, 2925, 1668, 1592, 1524, 1348, 1319, 1157, 1084; UV/Vis (MeOH):
λ
max nm 216, 272; 1H-NMR (400 MHz, DMSO-d6): δ 11.52 (s, 1H, SO2NH), 10.85 (1H, s,
CONH), 8.78 (1H, s, H-2), 8.44(1H,0d, J4, 5 = 8.4 Hz, H-4), 8.38 (1H, d, J6,5 = 8.0 Hz, H-6), 7.99
0
0
0
0
0
0
0
0
0
0
0
(2H, d, J2 , 3 /6 , 5 = 8.0 Hz, H-2 /H-6 ), 7.93 (2H, d, J3 , 2 /5 , 6 = 8.0 Hz, H-3 /H-5 ),7.84 (1H, t,
J = 8.0 Hz, H-5), 6.75 (1H, s, pyrimidine-H), 2.25 (6H, s, CH3), FAB-MS (positive mode) m/z
428 [M+H]+.
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-2-fluoro-3-(trifluoromethyl)
benzamide (14). White solid; yield:57%; m. p. 215–217 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf
= 0.53; IR (KBr): νmax cm-1 3405, 3129, 2949, 1685, 1593, 1530, 1313, 1315, 1170, 1134, 839,
579; UV/Vis (MeOH): λmax nm 211, 272; 1H-NMR (400 MHz, DMSO-d6): δ, 11.47 (1H, s,
0
0
0
0
SO2NH), 10.95 (1H, s, CONH), 8.01 (1H, d, J6, 5 = 6.4 Hz, H-6), 7.98 (2H, d, J2 , 3 /6 , 5 = 8.4
0
0
Hz, H-20/H-60), 7.93 (1H, d, J4,5 = 6.4 Hz, H-4), 7.84 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz, H-3 /H-5 ),
7.54 (1H, t, J = 7.6 Hz, H-5), 6.75 (1H, s, pyrimidine-H), 2.25 (6H, s, CH3);); 13C-NMR (100
MHz, DMSO-d6): δ 167.5 (CONH), 161.7 (C-3", C-5"), 157.0 (C-2), 156.1 (C-1"), 141.9 (C-1’),
135.5 (C-4’), 134.6 (C-4), 129.4 (C-6), 129.2 (C-3’, C-5’), 126.2 (C-1), 125.2 (C-5), 123.7 (CF3-
3), 121.0 (C-3), 118.8 (C-2’, C-4’), 113.5 (C-4"), 22.7 (CH3-3", CH3-5"); EI-MS m/z (% rel.
abund.): 406.2 (2), 405.3 (21), 404.2 (94), 403.2 [M+-SO2(64), 100], 282.1(4), 213.1(51), 191.1
(50), 163.1(15); HR-FAB-MS (positive mode) m/z: [M+H]+calcd. for C20H17F4N4O3S,
469.0957; found, 469.0957.
0
0
0
0
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-4,7,7-trimethyl-3-oxo-2-oxa-
bicyclo[2.2.1]heptane-1-carboxamide (15). White solid; yield:50%;m. p. 254–256 ˚C; TLC
(Hexane: EtOAc, 6:4 v/v): Rf = 0.48; IR (KBr): νmax cm-13368, 3234, 2967, 1791, 1692, 1597,
1528, 1434, 1321, 1159,1095, 875, 583; UV/Vis (MeOH): λmax nm211, 261; 1H-NMR (400
0
0
0
0
MHz, DMSO-d6): δ 11.55 (1H, s, SO2NH),10.13 (1H, s, CONH), 7.93 (2H, d, J3 , 2 /5 , 6 = 8.8
0
0
Hz, H-30/H-50), 7.87 (2H, d, J2 , 3 /6 , 5 = 8.8 Hz, H-2 /H-6 ), 6.75 (1H, s, pyrimidine-H), 2.45
(1H, m, H-6a), 2.25 (6H, s, pyrimidine-CH3), 1.98 (1H, m, H-6b), 1.97 (1H, m, H-5a), 1.58
(1H, m, H-5b), 1.02 (6H, s, CH3-7), 0.88 (3H, s, CH3-4); 13C-NMR (100 MHz, DMSO-d6): δ
177.7 (COO), 167.1 (CONH), 165.8 (C-3", C-5"), 156.1 (C-1"), 141.3 (C-1’), 135.6 (C-4’), 128.8
(C-3’, C-5’), 120.0 (C-2’, C-4’), 113.5 C-4"), 91.0 (C-2), 54.5 (C-4), 53.6 (C-7), 30.0 (C-6), 28.3
(C-5), 22.9 (CH3-3", CH3-5"), 16.4 (CH3-7), 16.2 (CH3-7), 9.4 (CH3-4); FAB-MS (positive
mode) m/z 459.1 [M+H]+; HRFAB-MS (positive mode) m/z: [M+H]+calcd. for C22H27N4O5S,
459.1702; found, 459.1702.
0
0
0
0
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3-fluorobenzamide (16).
White solid; yield: 35%; m. p. 236–238 ˚C; TLC (Hexane: EtOAc, 8:2 v/v): Rf = 0.48; IR (KBr):
ν
λ
max cm-1 3383, 3121, 2924, 1671, 1591, 1529, 1467, 1432, 1320, 1160, 560; UV/Vis (MeOH):
max nm 207, 278; 1H-NMR (400 MHz, DMSO-d6): δ 11.39 (1H, s,SO2NH), 10.58 (1H, s,
0
0
0
0
0
0
0
0
0
0
0
CONH), 7.97 (2H, d, J2 , 3 /6 , 5 = 8.4 Hz, H-2 /H-6 ), 7.91 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz, H-3 /H-
50), 7.79 (1H, d, J6,5 = 7.6 Hz, H-6), 7.74 (1H, s, H-2), 7.58 (1H, q, J = 8.0 Hz, H-4), 7.46 (1H, t,
J = 8.4 Hz, H-5), 6.75 (1H, s, pyrimidine-H), 2.25 (6H, s, CH3); EI-MS m/z (% rel. abund.):
338.3 (3), 337.2 (20), 336.1 [M+-SO2 (64), 98], 335.1 (100), 213.1(65), 185.1(2), 123.0 (50).
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Sulphamethazine derivatives as immunomodulating agents
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-2-fluorobenzamide (17).
White solid; yield:65%; m. p. 233–235 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.61; IR (KBr):
ν
max cm-1 3405, 3129, 2949, 1685, 1593, 1530, 1313,1315, 1170, 1134, 839, 579; UV/Vis
(MeOH): λmax nm 212, 278; 1H-NMR (400 MHz, DMSO-d6): δ 11.41 (1H, s, SO2NH), 10.74
0
0
0
0
0
0
0
0
0
0
(1H, s, CONH), 7.96 (2H, d, J2 , 3 /6 , 5 = 8.4 Hz, H-2 /H-6 ), 7.85 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz, H-
30/H-50), 7.60 (1H, t, J = 7.2 Hz, H-5), 7.57 (1H, q, J = 7.2 Hz, H-3), 7.35 (1H, t, J = 8.0 Hz, H-
4), 7.31 (1H, t, J = 7.2 Hz, H-5), 6.75 (1H, s, pyrimidine-H), 2.25 (6H, s, CH3); 13C-NMR (100
MHz, DMSO-d6): δ 167.9 (CONH), 163.1 (C-3", C-5"), 157.6 (C-2), 156.1 (C-1"), 142.3 (C-1’),
135.2 (C-4’), 132.7 (C-4), 129.8 (C-5), 129.2 (C-3’, C-5’),), 124.4 (C-1), 124.5 (C-6), 118.7 (C-2’,
C-4’), 116.0 (C-3), 113.5 (C-4"), 22.8 (CH3-3", CH3-5") EI-MS m/z (% rel. abund.): 338.3 (3),
337.2 (20), 336.3 [M+-SO2 (64), 100], 335.1 (95), 213.1(70), 123.0 (83), 95.0 (22); HRFAB-MS
(positive mode) m/z: [M+H]+calcd. for C19H18FN4O3S, 401.1084; found, 401.1084.
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3,4-difluorobenzamide (18).
Slightly yellow solid; yield:74%; TLC (Hexane: EtOAc, 8:2 v/v): Rf = 0.53; m.p. 212–218 ˚C; IR
(KBr): νmax cm-1 3245, 3062, 2926, 1672, 1624, 1513, 1316, 1284, 1159, 843, 579; UV/Vis
(MeOH): λmax nm 207, 277;1H-NMR (300 MHz, DMSO-d6): δ 10.68 (1H, s, CONH), 8.06
0
0
0
0
0
0
(1H, m, H-6), 7.97 (2H, d, J2 , 3 /6 , 5 = 8.8 Hz, H-2 /H-6 ), 7.93 (1H, d, J = 7.2 Hz, H-6), 7.92
0
0
0
0
0
0
(2H, d, J3 , 2 /5 , 6 = 8.8 Hz, H-3 /H-5 ), 7.86 (1H, m, H-2), 7.61 (1H, m, H-5), 6.74 (1H, s,
pyrimidine-H), 4.96 (1H, s, SO2NH), 2.24 (6H, s, CH3); EI-MS m/z (% rel. abund.): 355.2 (17),
356.2 (2.6), 354.1 [M+-SO2(64), 95], 353.1 (100), 232.1(30), 213.2 (65), 141.0 (53), 113.0 (20).
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-4-(trifluoromethyl)benzamide
(19). White solid; yield: 64%; m. p. 225–228 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.64; IR
(KBr): νmax cm-1 3444, 3239, 2925, 1626, 1591, 1528, 1319, 1167, 1065, 835, 580; UV/Vis
(MeOH): λmax nm 223, 273; 1H-NMR (400 MHz, DMSO-d6): δ 10.78 (1H, s, CONH), 7.98
0
0
0
0
0
0
0
0
0
0
0
0
(2H, d, J2 , 3 /6 , 5 = 8.8 Hz, H-2 /H-6 ), 7.91 (2H, d, J3 , 2 /5 , 6 = 8.8 Hz, H-3 /H-5 ), 8.14 (2H, d,
J
2, 3/6, 5 = 8.0 Hz, H-2/H-6), 7.93 (2H, d, J3, 2/5, 6 = 8.0 Hz, H-3/H-5), 6.75 (1H, s, pyrimidine-
H), 3.59 (1H, s, SO2NH), 2.25 (6H, s, CH3); EI-MS m/z (% rel. abund.): 387.3 (19), 386.2 [M+-
SO2 (64), 97], 385.3 (100), 297.2 (4), 264.1 (3), 213.1(63), 173.1 (42), 145.1 (31).
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3- trifluoromethyl)benzamide
(20). White solid; yield:65%;m. p. 237–239 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.63; IR
(KBr): νmax cm-13386, 3115, 2921, 1669, 1593, 1532, 1314,1260, 1165,1124, 694, 558;UV/Vis
(MeOH): λmax nm 211, 277; 1H-NMR (400 MHz, DMSO-d6): δ11.51 (1H, s, SO2NH), 10.74
0
0
0
0
(1H, s, CONH), 8.28 (1H, s, H-2), 8.24 (1H, d, J6,5 = 8.0 Hz, H-6), 7.99 (2H, d, J2 , 3 /6 , 5 = 8.8
0
0
Hz, H-20/H-60), 7.96 (H, d, J4,5 = 8.0 Hz, H-4), 7.92 (2H, d, J3 , 2 /5 , 6 = 8.8 Hz, H-3 /H-5 , 7.78
(H, t, J = 7.6 Hz, H-5), 6.75 (1H, s, pyrimidine-H), 2.25 (6H, s, CH3); EI-MS m/z (% rel.
abund.): 387.2 (20.5), 386.2 [M+-SO2, 88.4], 385.2 (100), 297.2 (2), 264.1 (3), 213.1(57), 173.1
(45), 145.0(29).
0
0
0
0
3-chloro-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)benzamide (21).
White solid; yield:65%; m. p. 237–246 ˚C; TLC (Hexane: EtOAc, 8:2 v/v): Rf = 0.48; IR (KBr):
ν
max cm-1 3384, 3063, 2927, 1665, 1592, 1521, 1316, 113, 959, 552; UV/Vis (MeOH): λmax nm
212, 278; 1H-NMR (400 MHz, DMSO-d6): δ 11.44 (1H, s, SO2NH), 10.62 (1H, s, CONH), 7.99
0
0
0
0
0
0
(1H, s, H-2), 7.97 (2H, d, J2 , 3 /6 , 5 = 8.0 Hz, H-2 /H-6 ), 7.89 (1H, d, J6,5 = 6.8 Hz, H-6), 7.87
0
0
0
0
0
0
(2H, d, J3 , 2 /5 , 6 = 8.0 Hz, H-3 /H-5 ), 7.67 (1H, d, J4,5 = 6.8 Hz, H-4), 7.56 (1H, t, J = 8.0 Hz,
H-5), 6.75 (1H, s, pyrimidine-H), 2.25 (6H, s, CH3); EI-MS m/z (% rel. abund.): 354.0 [M
+ 2]+-SO2 (64), 37], 353.0 (52), 352.0 [M+-SO2 (64), 100], 351.0 (88), 230.0 (3), 213.1 (73),
139.0 (43), 111.0 (21).
4-chloro-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)benzamide (22).
White solid yield: 94%; m. p. 223–226 ˚C; TLC (Hexane: EtOAc, 8:2 v/v): Rf = 0.66;IR (KBr):
ν
max cm-1 3308, 3200, 2924, 1658, 1595, 1520, 1312, 1159, 581; UV/Vis (MeOH): λmax nm 208,
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Sulphamethazine derivatives as immunomodulating agents
276; 1H-NMR (400 MHz, DMSO-d6): δ 11.40 (s, 1H, SO2NH), 10.59 (1H, s, CONH), 7.97 (2H,
0
0
0
0
0
0
0
0
0
0
d, J2 ,3 /6 ,5 = 8.4 Hz, H-2 /H-6 ), 7.97 (2H, d, J2, 3/6, 5 = 8.4 Hz, H-2/H-6), 7.91 (2H, d, J3 , 2 /5 , 6
= 8.4 Hz, H-30/H-50), 7.61 (2H, d, J3,2/5,6 = 8.4 Hz, H-3/H-5), 6.75 (1H, s, pyrimidine-H), 2.24
(6H, s, CH3); EI-MS m/z (% rel. abund.): 354.0 [M+2]+-SO2 (64), 34], 353.0 (4), 352.0 [M+-
SO2 (64), 100], 351.0 (86), 230.0 (3), 213.0 (71), 139.0 (78), 111.0 (24).
2,5-dichloro-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)benzamide (23).
White solid; yield: 88%; m. p. 230–232 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.53; IR (KBr):
ν
max cm-1 3239, 3061, 2966, 1657, 1598, 1540, 1399, 1263, 138, 1080, 830, 585; UV/Vis
(MeOH): λmax nm 210, 228, 268; 1H-NMR (400 MHz, DMSO-d6): δ 11.43 (1H, s, SO2NH),
0
0
0
0
0
0
0
0
0
0
10.90 (1H, s, CONH), 7.97 (2H, d, J2 , 3 /6 , 5 = 8.4 Hz, H-2 /H-6 ), 7.82 (2H, d, J3 , 2 /5 , 6 = 8.4
Hz, H-30/H-50), 7.75 (1H, s, H-6), 7.59 (2H, s, H-3, H-4), 6.75 (1H, s, pyrimidine-H), 2.25 (6H,
s, CH3); EI-MS m/z (% rel. abund.): 390.1[M + 4]+-SO2 (64), 10], 389.1 (22), 388.1[M+2]+-SO2
(64), 59], 387.0 (74), 386.1[M+-SO2 (64), 99], 385.1 (86), 213.1 (100), 173.0 (56), 145.0 (22).
2,4-dichloro-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)benzamide (24).
White solid; yield:62%; m. p. 233–236 ˚C; TLC (Hexane: EtOAc, 8:2 v/v): Rf = 0.43; IR (KBr):
ν
max cm-13600, 3064, 2923, 1660, 1592, 1540, 1140, 849, 706, 585; UV/Vis (MeOH): λmax
nm213, 272; 1H-NMR (400 MHz, DMSO-d6): δ 11.49 (1H, s, SO2NH), 10.87 (1H, s,0CONH),
0
0
0
0
0
0
0
0
0
0
0
7.97 (2H, d,J2 , 3 /6 , 5 = 8.4 Hz, H-2 /H-6 ), 7.82 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz, H-3 /H-5 ), 7.76
(1H, s, H-3), 7.64 (1H, d, J5,6 = 8.0 Hz, H-6), 7.55 (1H, dd, J = 8.0 Hz, J = 1.6 Hz, H-5), 6.75
(1H, s, pyrimidine-H), 2.25 (6H, s, CH3); FAB-MS (positive mode) m/z 455.0 [M+H +4]+,
453.0 [M+H+2]+, 451.1 [M+H]+.
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-4-nitrobenzamide (25).
Slightly yellow solid; yield: 51%; m. p. 237–239 ˚C; TLC (Hexane: EtOAc, 8:2 v/v): Rf = 0.42;
IR (KBr): νmax cm-1 3300, 3079, 2924, 1632, 1593, 1528, 1342, 1317, 1164, 842; UV/Vis
(MeOH): λmax nm 207, 258; 1H-NMR (400 MHz, DMSO-d6): δ 10.87 (1H, s, CONH),
8.36 (2H, d, J3, 2/5, 6 = 8.8 Hz, H-3/H-5), 8.17 (2H, d, J2, 3/6, 5 = 8.8 Hz, H-2/H-6), 7.95 (2H, d,
0
0
0
0
0
0
0
0
0
0
0
0
J2 , 3 /6 , 5 = 8.8 Hz, H-2 /H-6 ), 7.93 (2H, d, J3 , 2 /5 , 6 = 8.8 Hz, H-3 /H-5 ), 6.75 (1H, s, pyrimi-
dine-H), 3.50 (s, 1H, SO2NH), 2.25 (6H, s, CH3); EI-MS m/z (% rel. abund.): 364.2 (22), 363.1
[M+-SO2 (64), 93], 362.1 (100), 333.2 (8), 316.1 (9), 213.1 (59), 150.0 (20).
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3,5-dinitrobenzamide (26).
Slightly yellow solid; yield: 26% m. p. 265–267 ˚C; TLC (Hexane: EtOAc, 8:2 v/v): Rf = 0.64; IR
(KBr):νmax cm-13392, 3121, 2925, 1686, 1598, 1542, 1342, 1155;UV/Vis (MeOH): λmax nm2 12,
252; 1H-NMR (300 MHz, DMSO-d6): δ 11.72 (s, 1H, SO2NH), 11.10 (1H, s, CONH), 9.15 (2H,
0
0
0
0
0
0
d, J5,3 = 2.1 Hz, H-2), 9.00 (1H, t, J = 2.1 Hz, H-3), 8.01 (2H, d, J2 , 3 /6 , 5 = 8.7 Hz, H-2 /H-6 ),
0
0
0
0
0
0
7.94 (2H, d, J3 , 2 /5 , 6 = 9.0 Hz, H-3 /H-5 ), 6.74 (1H, s, pyrimidine-H), 2.24 (6H, s, CH3);
EI-MS m/z (% rel. abund.): 409.2 (20), 408.2 [M+-SO2 (64), 86], 407.1 (100), 378.2 (22), 239.1
(30), 213.2 (84), 195.1 (22).
2-chloro-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-4-nitrobenzamide
(27). White solid; yield: 47%; m. p. 216–219 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.48; IR
(KBr): νmax cm-1 3258, 3104, 2927, 1700, 1594, 1532, 1323, 1157, 584; UV/Vis (MeOH): λmax
nm 214, 261; 1H-NMR (300 MHz, DMSO-d6): δ 11.67 (s, 1H, SO2NH), 11.08 (1H, s, CONH),
0
0
0
0
8.41 (1H, d, J3,5 = 2.0 Hz, H-3), 8.29 (1H, dd, J = 8.4 Hz, J = 1.6 Hz, H-5), 7.99 (2H, d, J2 , 3 /6 , 5
0
= 8.4 Hz, H-20/H-60), 7.91 (1H, d, J6,5 = 8.4.0 Hz, H-6), 7.83 (2H, d, J3 , 2 /5 , 6 = 8.4.0 Hz, H-3 /
0
0
0
0
H-50), 6.75 (1H, s, pyrimidine-H), 2.25 (6H, s, CH3); EI-MS m/z (% rel. abund.): 399.3[M+2]+-
SO2 (64), 29], 397.3 [M+-SO2 (64), 88], 396.3 (98), 362.3 (18), 350.3 (10), 213.2 (97.4), 184.1
(16).
2-Bromo-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)benzamide (28).
White solid; yield:47%; m. p. 235–237 ˚C; TLC (Hexane: EtOAc, 8:2 v/v): Rf = 0.52; IR (KBr):
ν
max cm-1 3237, 3059, 2854, 1659, 1595, 1535, 1395, 1139, 845, 585; UV/Vis (MeOH): λmax nm
PLOS ONE | https://doi.org/10.1371/journal.pone.0208933 December 19, 2018
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Sulphamethazine derivatives as immunomodulating agents
213, 264; 1H-NMR (400 MHz, 0DMSO-d6): δ 11.46 (s, 1H, SO2NH), 10.83 (1H, s, CONH), 7.97
0
0
0
0
0
0
0
0
0
0
0
(2H, d, J2 , 3 /6 , 5 = 8.4 Hz, H-2 /H-6 ), 7.84 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz, H-3 /H-5 ), 7.71 (1H, d,
J3,4 = 7.6 Hz, H-3), 7.55 (1H, d, J6,5 = 7.2 Hz, H-6), 7.49 (1H, t, J = 7.2 Hz, H-5), 7.42 (1H, t,
J = 8.0 Hz, H-4), 6.76 (1H, s, pyrimidine-H), 2.25 (6H, s, CH3); EI-MS m/z (% rel. abund.):
398.0 [M +2]+-SO2 (64), 94], 397.0 (81), 396.0 [M+-SO2 (64), 100], 395.0 (70), 317.1 (4), 213.0
(100), 184.9 (76), 155.0 (13), 157.0 (14).
4-Bromo-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)benzamide (29).
White solid; yield: 82%; m. p. 227–228 ˚C; TLC (Hexane: EtOAc, 4:6 v/v): Rf = 0.44; IR (KBr):
ν
max cm-1 3276, 3059, 2854, 1655, 1594, 1521, 1397, 1313, 1158, 581; UV/Vis (MeOH): λmax
nm 208, 274; 1H-NMR (400 MHz, DMSO-d6): δ 10.64 (1H, s, CONH), 7.96 (2H, d, J2 , 3 /6 , 5
0
0
0
0
=
0
8.8 Hz, H-20/H-60), 7.92 (2H, d, J2,3/6,5 = 7.6 Hz, H-2/H-6), 7.90 (2H, d, J3 , 2 /5 , 6 = 8 Hz, H-3 /
H-50), 7.74 (2H, d, J3,2/5,6 = 8 Hz, H-3/H-5), 6.75 (1H, s, pyrimidine-H), 4.2 (1H, s, SO2NH),
2.24 (6H, s, CH3); EI-MS m/z (% rel. abund.): 398.9 (15.9), 397.9 [M+2]+-SO2 (64), 68], 396.9
(80.7), 395.9 [M+-SO2 (64), 71], 394.9 (60), 213.0 (100), 184.9 (63), 154.9 (19), 156.9 (19).
3-Bromo-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)benzamide (30).
White solid; yield:76%; m. p 228–230 ˚C; TLC (Hexane: EtOAc, 8:2 v/v): Rf = 0.52IR (KBr):
0
0
0
0
ν
max cm-1 3385, 3062, 2945, 1665, 1592, 1523, 1316, 1159, 582, 553; UV/Vis (MeOH): λmax nm
213, 278; 1H-NMR (300 MHz, DMSO-d6): δ 11.56 (s, 1H, C0ONH), 10.62 (1H, s, SO2NH), 8.13
0
0
0
0
0
(1H, t, J = 1.8 Hz, H-2), 7.98 (2H, d, J2 , 3 /6 , 5 = 9.0 Hz, H-2 /H-6 ), 7.93 (1H, d, J6,5 = 7.2 Hz,
0
0
0
0
0
0
H-6), 7.90 (2H, d, J3 , 2 /5 , 6 = 8.7 Hz, H-3 /H-5 ), 7.80 (1H, dd, J = 7.2 Hz, J = 1.0 Hz, H-4),
7.50 (1H, t, J = 7.8 Hz, H-5), 6.75 (1H, s, pyrimidine-H), 2.24 (6H, s, CH3); EI-MS m/z (% rel.
abund.): 398.2 [M+2]+-SO2 (64), 87.6], 397.1 (100), 396.2 [M+-SO2 (64), 82], 395.1 (76), 213.2
(67), 183.0 (33), 185.0 (32).
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3-iodobenzamide (31).
White solid; yield:55%; m. p. 208–210 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.44; IR (KBr):
ν
max cm-1 3388, 3074, 2923, 1664, 1592, 1525, 1316, 1160, 1081, 556;UV/Vis (MeOH): λmax nm
217, 275; 1H-NMR (400 MHz, DMSO-d6): δ 11.39 (1H, s, SO2NH), 10.59 (1H, s, CONH), 8.28
0
0
0
0
0
0
(1H, s, H-2), 7.94 (2H, d, J2 , 3 /6 , 5 = 8.8 Hz, H-2 /H-6 ), 7.97 (2H, d, J6,5 = 7.6 Hz, H-4, H-6),
0
0
0
0
0
0
7.90 (2H, d, J3 , 2 /5 , 6 = 8.8 Hz, H-3 /H-5 ), 7.33 (H, t, J = 7.6 Hz, H-5), 6.75 (1H, s, pyrimi-
dine-H), 2.25 (6H, s, CH3); EI-MS m/z (% rel. abund.): 446.1 (3), 445.1 (18), 444.1 [M+-SO2
(64), 100], 443.1 (85), 316.2 (4), 291.2 (2), 231.0 (50), 213.2 (48), 203.0 (20), 76.0 (13).
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3-methoxybenzamide (32).
White crystalline solid; yield: 62%; m. p. 188–190 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.49;
IR (KBr): νmax cm-1 3346, 3158, 3062, 2925, 1665, 1592, 1530, 1319, 1163, 1068, 842, 587; UV/
Vis (MeOH): λmax nm 212, 277; 1H-NMR (400 MHz, DMSO-d6): δ 10.51 (1H, s0, CONH), 7.96
0
0
0
0
0
0
0
0
0
0
0
(2H, d, J2 , 3 /6 , 5 = 8.8 Hz, H-2 /H-6 ), 7.92 (2H, d, J3 , 2 /5 , 6 = 8.8 Hz, H-3 /H-5 ), 7.52 (1H, d,
J6,5 = 7.6 Hz, H-6), 7.47 (1H, s, H-2), 7.44 (1H, t, J = 7.6 Hz, H-5), 7.16 (1H, d, J4,5 = 8.0 Hz, H-
4), 6.75 (1H, s, pyrimidine-H), 3.82 (3H, s, CH3O-), 3.47 (1H, s, SO2NH), 2.25 (6H, s, CH3);
EI-MS m/z (% rel. abund.): 349.3 (20), 348.3 [M+-SO2 (64), 100], 347.1 (58), 259.2 (3), 213.2
(31), 135.1 (77), 107.1 (20).
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3,5-dimethoxybenzamide
(33). White solid; yield:41%; m. p. 226–227 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.43; IR
(KBr): νmax cm-1 3306, 2932, 2949, 1653, 1593, 1510, 1313, 1315, 1162, 1049, 945; UV/Vis
(MeOH): λmax nm 214, 278; 1H-NMR (400 MHz, DMSO-d6): δ 11.44 (1H, s, SO2NH), 10.45
0
0
0
0
0
0
0
0
0
0
(1H, s, CONH), 7.96 (2H, d, J2 , 3 /6 , 5 = 8.4 Hz, H-2 /H-6 ), 7.90 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz, H-
30/H-50), 7.82 (2H, d, J2,4/6,4 = 2 Hz, H-2/H-6), 6.75 (1H, s, pyrimidine-H), 6.72 (1H, s, H-4),
3.80 (6H, s, CH3O-) 2.25 (6H, s, CH3); EI-MS m/z (% rel. abund.): 379.3 (27), 378.3 [M+-SO2
(64), 100], 377.1 (65), 256.2 (4), 213.1 (17), 165.1 (80), 122.1 (15)
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Sulphamethazine derivatives as immunomodulating agents
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-4-methylbenzamide (34).
White solid; yield: 83%; m. p. 216–218 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.63; IR (KBr):
ν
λ
max cm-1 3371, 3135, 2794, 1667, 1593, 1534, 1320, 1161, 1083, 831, 558; UV/Vis (MeOH):
max nm 210, 274;01H-NMR (400 MHz, DMSO-d6): δ10.46 (1H, s, CONH), 7.96 (2H, d, J2 , 3 /
0
0
0
0
0
60, 50
0
0
0
0
= 9.2 Hz, H-2 /H-6 ), 7.93 (2H, d, J3 , 2 /5 , 6 = 9.2 Hz, H-3 /H-5 ), 7.86 (2H, d, J2,3/6,5 = 8.0
Hz, H-2/H-6), 7.33 (2H, d, J3,2/5,6 = 8.0 Hz, H-3/H-5), 3.95 (1H, s, SO2NH), 6.75 (1H, s, pyrim-
idine-H), 2.37 (3H, s, CH3-4). 2.25 (6H, s, pyrimidine-CH3); EI-MS m/z (% rel. abund.): 333.0
(28.4), 332.0 [M+-SO2 (64), 100], 331.0 (79.9), 213.2 (26), 119.0 (100), 91.0 (46).
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-2-methylbenzamide (35).
White solid; yield:63%; m. p. 216–218 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.62; IR (KBr):
ν
max cm-1 3330, 3240, 3162, 2924, 1664, 1595, 1526, 1315, 1157, 1091, 878, 581; UV/Vis
(MeOH): λmax nm 211, 272; 1H-NMR (400 MHz, DMSO-d6): δ 11.41 (1H, s, SO2NH), 10.62
0
0
0
0
0
0
0
0
0
0
(1H, s, CONH), 7.95 (2H, d, J2 , 3 /6 , 5 = 8.4 Hz, H-2 /H-6 ), 7.87 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz, H-
30/H-50), 7.45 (1H, d, J6,5 = 7.2, H-6), 7.39 (1H, t, J = 6.8 Hz, H4), 7.30 (1H, d, J3,4 = 7.6 Hz, H-
3), 7.29 (1H, t, J = 7.6 Hz, H-5), 6.75 (1H, s, pyrimidine-H), 2.49 (3H, s, CH3-2), 2.25 (6H, s,
pyrimidine-CH3); EI-MS m/z (% rel. abund.): 333.0 (24), 332.0 [M+-SO2 (64), 99], 331.0 (65),
213.2 (18), 119.0 (100), 91.0 (40).
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3-methylbenzamide (36).
White solid; yield: 61%; m. p. 219–220 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf. = 0.58; IR (KBr):
ν
λ
max cm-13349, 3062, 2924, 1659, 1594, 1527, 1318, 1157, 1082, 857, 580; UV/Vis (MeOH):
max nm 213, 273; 1H-NMR (400 MHz, DMSO-d6): δ 11.53 (1H, s, SO2NH), 10.50 (1H, s,
0
0
0
0
0
0
0
0
0
0
0
CONH), 7.96 (2H, d, J2 , 3 /6 , 5 = 8.4 Hz, H-2 /H-6 ), 7.92 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz, H-3 /H-
50), 6.75 (1H, s, pyrimidine-H), 2.38 (3H, s, CH3-3), 2.25 (6H, s, pyrimidine-CH3); EI-MS m/z
(% rel. abund.): 333.0 (24), 332.1 [M+-SO2 (64), 100], 331.0 (65), 213.2 (29), 119.0 (84), 91.0
(33).
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3-phenylpropanamide (37).
White solid; yield: 50%; m. p. 195–197 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.67; IR (KBr):
ν
max cm-1 3287, 3176, 3034, 2928, 1697, 1596, 1535, 1429, 1160, 1083, 858, 582; UV/Vis
(MeOH): λmax nm 211, 266; 1H-NMR (400 MHz, DMSO-d6): δ 10.27 (s, 1H, CONH), 7.89
0
0
0
0
0
0
0
0
0
0
0
0
(2H, d, J3 , 2 /5 , 6 = 9.0 Hz, H-3 /H-5 ), 7.70 (2H, d, J2 , 3 /6 , 5 = 9.0 Hz, H-2 /H-6 ), 7.13–7.28
(5H, m, C6H5-), 6.75 (1H, s, Pyrimidine-H), 2.88 (2H, t, J = 7.5 Hz, CH2-3), 2.64 (2H, t, J = 7.5
Hz, CH2-2), 2.25 (6H, s, CH3); EI-MS m/z (% rel. abund.): 347.2 (23), 346.1 [M+-SO2 (64),
100], 345.1.0 (88), 214.2 (92), 105.1 (13), 91.0 (22).
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)-3,5-dimethylbenzamide
(38). White solid; yield:55%;m. p. 216–218 ˚C; TLC (Hexane: EtOAc, 6:4 v/v): Rf = 0.55; IR
(KBr):νmax cm-13332, 3092, 2921, 1659, 1595, 1522, 1312, 1164, 1086, 836, 582; UV/Vis
(MeOH): λmax nm 213, 278; 1H-NMR (400 MHz, DMSO-d6): δ10.46 (1H, 0s, CONH), 7.95
0
0
0
0
0
0
0
0
0
0
0
(2H, d, J2 , 3 /6 , 5 = 9.2 Hz, H-2 /H-6 ), 7.91 (2H, d, J3 , 2 /5 , 6 = 9.2 Hz, H-3 /H-5 ), 7.54 (2H, s,
H-2/H-6), 7.22 (1H, s, H-4), 6.75 (1H, s, pyrimidine-H), 3.52 (1H, s, SO2NH), 2.34 (6H, s,
CH3-3), 2.25 (6H, s, pyrimidine-CH3); FAB-MS (positive mode) m/z 411.3 [M+ H]+.
N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)phenyl)benzamide (39). White solid;
yield:79%; m. p. 233–236 ˚C; TLC (Hexane: EtOAc, 4:6 v/v): Rf = 0.45; IR (KBr): νmax cm-1
3344, 3108, 2925, 1660, 1595, 1528, 1157; UV/Vis (MeOH): λmax nm 209, 207; 1H-NMR (400
0
0
0
0
MHz, DMSO-d6): δ 11.49 (s, 1H, SO2NH), 10.54 (1H, s, CONH), 7.97 (2H, d, J2 , 3 /6 , 5 = 9.2
0
0
Hz, H-20/H-60), 7.95 (2H, d, J3 , 2 /5 , 6 = 9.2 Hz, H-3 /H-5 ), 7.94 (2H, d, J2, 3/6, 5 = 8.4 Hz, H-2/
H-6), 7.60 (1H, t, J = 7.2 Hz, H-4), 7.50 (2H, t, J = 7.6 Hz, H-3/H-5), 6.75 (1H, s, pyrimidine-
H), 2.25 (6H, s, CH3). EI-MS m/z (% rel. abund.): 213.0 (85.9), 214.1 [M+-SO2 (64), 100], 215.1
(10.9), 228.2 (1.4), 213.1 (100), 199.1 (7.2).
0
0
0
0
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Sulphamethazine derivatives as immunomodulating agents
Immunomodulatory studies. All studies on human blood cells was carried out after an
approval from independent ethics committee (Prof. Dr. Ghazala H. Rizwani Chair, IEC, Prof.
Qamar Amin, Prof, Dr. Muddasir Uddin, Dr. Shahnaz Ghazi, Dr. Samiuz Zaman, Prof. Dr.
Ahsana Dar Farooq, Dr. M. Raza Shah member IEC), International Center for Chemical and
Biological Sciences, University of Karachi, No: ICCBS/IEC-008-BC-2015/Protocol/1.0.
Informed consents were obtained from the volunteers before drawing the blood. All the exper-
iments were performed in accordance with relevant guidelines and regulations. The approval
of institutional ethical committee approval can be seen from supporting information.
LSM (50494, MP Biomedicals, lllkirch, France); HBSS++ and HBSS—(14025, Gibco, Cali-
fornia, USA); Ethanol (1070172511, Merck, Darmstadt, Germany); FBS (A11-104, PAA,
Pasching, Austria); RPMI-1640 (R8758, Sigma Aldrich, St. Louis, USA); 10 mL sterile syringes
(305559, BD Biosciences, NJ, USA); Luminol (A14597, Alfa Aesar, Karlsrushe, Germany);
96-well white half area plates (3693,Costar, NY, USA); Zymosan (26701494, Wako, Shanghai,
China).
Isolation of human polymorphonuclear cells (PMNs). Heparinized blood was obtained
by vein puncture aseptically from healthy volunteers. The PMNs were isolated by ficoll-Hypa-
que density gradient method. The cells were isolated after centrifugation from the tube base.
Cells were washed twice and suspended in Hank’s Balance salt solution (HBSS--) (Ca2+ and
Mg2+ free), RBCs were lysed using hypotonic solution. Cells were adjusted to their required
concentration (1x106 cells/mL) using HBSS++containing Ca2+ and Mg2+.
All studies on human blood cells was carried out after an approval from independent ethics
committee (Prof. Dr. Ghazala H. Rizwani Chair, IEC, Prof. Qamar Amin, Prof, Dr. Muddasir
Uddin, Dr. Shahnaz Ghazi, Dr. Samiuz Zaman, Prof. Dr. Ahsana Dar Farooq, Dr. M. Raza
Shah member IEC), International Center for Chemical and Biological Sciences, University of
Karachi, No: ICCBS/IEC-008-BC-2015/Protocol/1.0. Informed consents were obtained from
the volunteers before drawing the blood. All the experiments were performed in accordance
with relevant guidelines and regulations.
Oxidative burst assay. Luminol-enhanced chemiluminescence assay was performed, as
described by Helfandet al., 1982 [17], with some modifications. Briefly 25 μL of diluted whole
blood in HBSS++ (Hanks Balanced Salt Solution, containing calcium chloride and magnesium
chloride) [Sigma, St. Louis, USA] was incubated with 25 μL of three different concentrations
of compounds (1, 10, and 100 μg/mL), each in triplicate. Control wells received HBSS++ and
cells, but no compounds. Test was performed in white half area 96-well plates [Costar, NY,
USA], which was incubated at 37 ˚C for 15 minutes in the thermostat chamber of luminometer
[Labsystems, Helsinki, Finland]. After incubation, 25 μL of serum opsonized zymosan (SOZ)
[Fluka, Buchs, Switzerland] and 25 μL of intracellular reactive oxygen species detecting probe,
luminol [Research Organics, Cleveland, OH, USA] were added into each well, except blank
wells (containing only HBSS++). The level of the ROS was recorded in luminometer in term of
relative light units (RLU).
Measurement of nitric oxide. The mouse macrophage cell line J774.2 (European Collec-
tion of Cell Cultures, UK) was cultured in 75 cc flasks IWAKI (Asahi Techno Glass, Japan) in
DMEM Sigma-Aldrich (Steinheim, Germany) supplemented with 10% fetal bovine serum
GIBCO (New York, USA)1% streptomycin/penicillin. Flasks were kept at 37 ˚C in atmosphere
of humidified air containing 5% CO2, cells were seeded in 96-well plate (106 cells/mL) and
were induced by 30 μg/mL e. coli lipopolysaccharide (LPS) (DIFCO Laboratories michigon,
USA). The test compounds were added at concentrations of (1, 10 and 100 μg/mL) and plate
was incubated at 37 ˚C in 5% CO2. Nitrite accumulation in culture supernatant was measured
using the Griess reagents as described previously [18].
PLOS ONE | https://doi.org/10.1371/journal.pone.0208933 December 19, 2018
11 / 22

Sulphamethazine derivatives as immunomodulating agents
Cytokine production and their quantification. RPMI-1640 (R8758, Sigma Aldrich,
St. Louis, USA); L-glutamine (25030081, Gibco, California, USA); D-glucose (G8270, Sigma
Aldrich, St. Louis, USA); 2-mercaptoethanol (190242, MP Biomedicals, lllkirch, France);
sodium pyruvate (11360–070, Gibco, California, USA); FBS (A11-104, PAA Laboratories,
Pasching, Austria); HEPES (H3375, Sigma Aldrich, St. Louis, USA); PMA (151864, MP Biome-
dicals, lllkirch, France); lipopolysaccharide B of E. coli 0111:B4 (LPS) (3922–25, DIFCO Labo-
ratories, USA); 24- well tissue culture plates (CLS3524, Corning, Dublin, USA); Flat bottom
96-well tissue culture plates (CLS3300, Corning, New York, USA). BSA (151429, MP Biomedi-
cals, lllkirch, France); PBS tablets (41620016–4, Bioworld, Dublin, Ireland); Tween 20 (822184,
Merck, Damstadt, Germany); Human TNF-α DuoSet ELISA Kit (DY210, R&D Systems, Min-
neapolis, USA); Human IL-1β DuoSet ELISA Kit (DY201, R&D Systems, Minneapolis, USA);
and Human IL-2 DuoSet ELISA Kit (DY202, R&D Systems, Minneapolis, USA); ELISA plate
sealers (DY992, R&D Systems, Minneapolis, USA); Clear flat bottom 96-well microplates
(DY990, R&D Systems, Minneapolis, USA). Human monocytic leukemia cells THP-1 were
obtained from (European Collection of Cell Cultures, UK). The cells were maintained in
RPMI-1640 containing 5.5 mmol/L glucose (BioM Laboratories, Chemical Division, Malay-
sia), 50 μmol/L mercaptoethanol (Merck Damstadt, Germany), 10% FBS (fetal bovine serum),
2 mmol/L; L-glutamine (PAA Laboratories, GmbH, Pasching, Austria). Cells were grown in 75
cc flasks, harvested and 2.5×105 cells/mL was then plated in 24-well tissue culture plates. 20
ng/mL of phorbol-12-myristate-13-acetate (PMA), (SERVA, Heidelberg, Germany) was added
followed by incubation for 24 hours at 37 ˚C in 5% CO2to convert them into macrophage like
cell. Cells were then stimulated with E. coli lipopolysacchride B, (DIFCO Laboratories, michi-
gon, USA) at a final concentration of 50 ng/mL and treated with compounds at three different
concentration (1, 10 and 100 μg/mL) and then incubated for 4 hours at 37 ˚C in 5% CO2. The
supernatants collected were analyzed for the level of TNF -α using the human TNF-α Duo Set
ELISA (R&D Systems, Minneapolis, USA), and according to manufacturer’s instructions [18].
Cytotoxicity assay. Cytotoxic activity of compounds was evaluated in 96-well flat-bottom
micro titer plates by using the standard MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tet-
razolium bromide) colorimetric assay [19]. In this assay, mouse fibroblast 3T3 cells were
grown in Dulbecco’s Modified Eagle Medium (DMEM), along with 5% Fetal Bovine Serum
(FBS), 100 μg/mL of streptomycin, and 100 IU/ mL of penicillin in 75 cm2 flasks. The flasks
were then incubated at 37 ˚C in 5% CO2. Cells were harvested, followed by counting with hae-
mocytometer, and dilution with a medium. Cell culture was prepared with the concentration
of 5 x 104 cells/mL, followed by introduction (100 μL/ well) into 96-well plates. Medium was
removed after overnight incubation. Freshly prepared medium (200 μL) was added with vari-
ous concentrations of test compounds (1–30 μM). After 48 hours, 200 μL MTT dye (0.5 mg/
mL) was added into all wells, and again incubated for 4 hours. DMSO (100 μL) was added to
each well. The formation of formazan dye within the cells was calculated by measuring the
absorbance at 540 nm using a using a micro plate reader. The cytotoxicity was recorded as
concentration causing 50% growth inhibition (IC50).
Anti-bacterial activity. The anti-bacterial activity of synthesized compound were evalu-
ated by the Microplate Alamar Blue Assay [20, 21]. Staphylococcus aureus (ATCC 25923),
Pseudomonas aeruginosa(POA 286), Salmonella typhi (ATCC 14028), Bacillus subtilis ATCC
6633) and Escherichia coli (ATCC 12022) were used in this study as a standard bacterial strains
and they were obtained from microbial bank of PCMD, International Center for Chemical
and Biological Sciences, University of Karachi, Karachi-75270, Pakistan. The bacterial colonies
grown in Trypton Soya Agar (TSA) (Oxoid Limited, UK) were inoculated in Mueller Hinton
Broth (MHB) (Oxoid Limited, UK) and incubated overnight at 37 ˚C. Fully grown bacterial
cultures were then diluted to adjust with 0.5 McFarland Turbidity Index (equivalent to
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Sulphamethazine derivatives as immunomodulating agents
1.5 × 108 CFU/mL). Stock solutions (4 mg/mL) of different test compounds were prepared in
DMSO and 10μL each of these stock solutions were placed in well of flat bottom, polystyrene,
sterile 96-wells plate except the positive control wells (media + bacteria). This gives 200μg/mL
concentrations of compounds in final 200 μL solution. Each compound was run in triplicates.
Finally, bacterial suspension (5 x 106 CFU/mL) was added in each well. Plates were sealed by
parafilm and incubated at 37 ˚C for 18–22 hrs. Next day, 20 μL of 0.02% resazurin sodium salt
dye (Chem-Impex-Int’LInc) was added in each well and incubated in shaking incubator at 80
rpm, 37 ˚C for 2–3 hrs. The color change from blue to reddish pink indicates the growth of
bacteria. For quantitative analysis, plates were read at 570 nm and 600 nm in Multiskan GOmi-
croplate spectrophotometer, (Thermo Scientific, USA). The % inhibition of bacterial growth
was calculated using below mentioned formula.
For the calculation of the % difference in reduction between treated and control cells in
cytotoxicity/proliferation assays:
ðεOXÞl2 Al1 À ðεOXÞl1 Al2 of test agent
¼
� 100
ðεOXÞl2 A�l1 À ðεOXÞl1 A�l2 of positive growth control
For the % inhibition of test compound as compared to positive control cell:
¼ 100 À ð%ddifference in reduction between treated and positive control cellsÞ
Statistical analysis. Results are expressed as mean values SD (Standard Deviation). The
statistical significance of the difference between control and treated samples was calculated by
the Student’s t-test. The differences were considered to be highly significant at P � 0.05.
Results and discussion
During the current study 37 membered library of amide derivatives of sulfamethazine was syn-
thesized by using conventional methods being employed in organic synthesis. The purified
yields of resulting amides ware between 42–96%. Progress of the reaction was monitored by
thin layer chromatography (TLC). After complete consumption of substrate, the reaction mix-
ture was cooled, precipitates were filtered, washed with mixture of hexane, and ethyl acetate,
followed by washing with acetone. Structures of all synthetic compounds 3–39 were elucidated
by modern spectroscopic technique; such as 1H- and 13C-NMR, EI-MS, FAB-MS, HRFAB-MS,
UV, and IR spectroscopy.
General structure elucidation of a representative compound 6
1H- and 13C-NMR spectra of one of the most bioactive members of the series (compounds 6)
is presented here. In 1H-NMR spectrum, recorded in DMSO-d6, downfield broad singlets of
the acidic SO2NH proton, and CONH appeared at δH11.44 and10.90, respectively. The para-
disubstituted aromatic H-20/H-60 resonated at δH 8.01 (2H, d, J2 , 3 /6 , 5 = 8.4 Hz) with ortho
0
0
0
0
couplings with H-30/H-50. Similarly, H-30/H-50 resonated at δH 7.92 (2H, d, J3 , 2 /5 , 6 = 8.4 Hz)
showed ortho couplings with H-20/H-60. The other downfield set of protons H-2/H-6, and H-4
of 3,5-bis(trifluoromethyl)benzoyl substituent appeared as singlets at δH 8.58 and 8.38, respec-
tively. This downfield signal was due to the electron withdrawing effect attachedCF3 groups.
The two CH3 protons attached to pyrimidine ring appeared as singlet at δH 2.25. The charac-
teristic signal of methine H-4@ of pyrimidine ring appeared as a singlet at δH 6.75 (Fig 1).
In 13C-NMR broad-band decoupled DEPT spectrum of 6, in DMSO-d6 showed 21 carbon
signals, including 2 methyl, 8 methine, and 11 quarternary carbons. The downfield signal of
amide carbonyl carbon appeared at δC 167.4. The second most downfield signal of C-5’’
0
0
0
0
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Sulphamethazine derivatives as immunomodulating agents
1
Fig 1. H-NMR chemical shifts of compound 6.
https://doi.org/10.1371/journal.pone.0208933.g001
appeared at δC162.9 due to the quaternary carbon directly, attached to nitrogen of the pyrimi-
dine ring. Another quaternary carbon (C-1@) which is in proximity three nitrogen atoms
appeared at δ156.1. Two quaternary carbons (C-1´ and C-4´) that are directly attached to NH
of amide and SO2 of sulphonamide appeared at δC 142.0, and 135.4, respectively. Four aro-
matic methine carbons of the para-disubstituted aromatic ring appeared at δC 119.5, and 129.1
assigned to C-2’/C-6’, and C-3’/ C-5’, respectively. Three aromatic methine carbons of 3, 5-bis
(trifluoromethyl)benzoyl substituent appeared at δC125.3, and 128.6 assigned to C-2/C-6 and
C-4, respectively. C-4’’ of pyrimidine ring appeared at δC113.6. CF3 carbon appeared at
δC124.3 while aromatic quaternary C-3/5, ipso to CF3 group resonated at δC130.2. Aromatic
quaternary carbon attached to CO of amide bond resonated at δC 136.7. The most up field
methyl carbons attached to C-3’’and C-5’’ appeared at δC 22.8 (Fig 2).
The EI-MS of compound 6 did not show the molecular ion peak [M]+ at 518.1but showed
the peak at 454.2 [M+-SO2(64)] arose from the elimination SO2 group. Loss of COPh (CF3)2
from m/z 454.2 [M+-SO2(64)] yielded a peak at m/z 320, while a radical cation for COPh(CF3)2
appeared at m/z 241.
HRFAB-MS (positive mode) displayed the [M+H]+ at m/z 519.0926, supporting the com-
position of C21H17F6N4O3S(Calcd.519.0926).
Fig 2. 13C-NMR chemical shifts of compound 6.
https://doi.org/10.1371/journal.pone.0208933.g002
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Sulphamethazine derivatives as immunomodulating agents
Anti-inflammatory activities in vitro
All sulfamethazine amide derivatives 3–39 were evaluated for their in vitro protein anti-
inflammatory activity, as well as on various parameters of inflammation. Among them com-
pounds 3–10, 14, and 15 were identified as new compounds. All the compounds have not
been reported for their anti-inflammatory activities earlier. During the current study, com-
pounds 3–39 and parent drug sulfamethazine (1) were initially tested for their inhibitory
potential on the production of intracellular ROS from zymosan activated whole blood phago-
cytes. Compounds 1, and 3–13 inhibited the production of ROS from human whole blood
cells. Compound 1, and its derivatives 3–13 were further evaluated for the inhibition of the
ROS production from neutrophils, isolated from human whole blood. All tested compounds
were found to be the potent inhibitors of ROS, except compounds 5 and 11 which showed a
moderate level of inhibition (IC50 = 20.5 2.2, and 34.6 1.2 μg/mL, respectively) as com-
pared to standard drug ibuprofen (IC50 = 11.2 1.9 μg/mL). Fig 3 represents the effect of com-
pounds 1 and 3–13 on oxidative burst in the graphical form. Among all tested derivatives, 8
was found to be the most potent inhibitor of ROS from whole blood, and isolated PMNs with
an IC50 values of 4.2 0.2 and 2.4 0.07, respectively. Fig 4 represents the effect of compounds
1 and 3–13 on oxidative burst activity of polymorphonuclear neutrophils (PMNs).
The parent drug 1 along with synthesized derivatives were tested for their NO (nitric oxide)
scavenging activity, produced from LPS stimulated mouse macrophage cell lines. All com-
pounds showed a promising inhibitory effect (Table 1), except 11 which showed relatively
moderate level of inhibition (IC50 = 26.3 1.8 μg/mL) compared to standard LNMMA (IC50
=
24.2 0.6 μg/mL). Compounds 3 and 4 were found to be the most potent inhibitor of NO with
an IC50 value of <1 μg/mL.
When tested for their effect on production of pro-inflammatory cytokine TNF-α, all com-
pounds were found to be the potent inhibitors of this cytokine except parent compound 1 and
derivative 11. Compounds 3–6 were found to be the most potent inhibitors of TNF-α with an
Fig 3. The graph represents the effect of compounds 1 and 3–13 on oxidative burst. Compounds were tested on three different concentrations. Results are
presented in relative light units (RLU) and oxidative burst activity of whole blood using luminol as a probe. Each vertical bar represents a mean of triplicate. Error bars
represent mean Standard deviation of three determinations. Significance difference was compared to the control = no drug.
https://doi.org/10.1371/journal.pone.0208933.g003
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Sulphamethazine derivatives as immunomodulating agents
Fig 4. The graph represents the effect of compounds 1 and 3–13 on oxidative burst. Compounds were tested on three different concentrations. Results are
presented in relative light units (RLU) and oxidative burst activity of polymorphonuclear neutrophils (PMNs) using luminol as a probe. Each vertical bar represents a
mean of triplicate. Error bars represent mean Standard deviation of three determinations. Significance difference was compared to the control = no drug.
https://doi.org/10.1371/journal.pone.0208933.g004
IC50 value of >1μg/mL. Among 37 synthetic derivatives of sulfamethazine 11 derivatives
showed a significant anti-inflammatory potential at a varying levels. Compounds 3, 4, 6, 7, 8,
9, and 13 were found to have a better anti-inflammatory activity than parent compound 1.
All the new compounds, except 14 and 15, showed anti-inflammatory activities. The cyto-
toxicity of compounds 3–39 was also evaluated against the mouse fibroblast NIH-3T3 cell line.
All the compounds were found to be non-toxic (Table 1). These results showed that modifica-
tion of sulfonamide’s primary amino group brings promising improvement in the anti-inflam-
matory profile of parent drug 1. Limited Structure-Activity-Relationship shows that there is
no particular trend for the potent activity which compounds 3–39 follows, however, it was
observed that compounds with alkyl and/or halogen substituents at meta or para positions of
benzamide ring showed potent activity.
Antibacterial activity
The synthesized compounds 3–39 were evaluated for their antibacterial activity by Microplate
Alamar Blue Assay [20, 21] in comparison to ofloxacin (standard antibiotic) against Staphylo-
coccus aureus (ATCC 25923), Pseudomonas aeruginosa (POA 286), Salmonella typhi (ATCC
14028), Bacillus subtilis (ATCC 6633) and Escherichia coli (ATCC 12022). The results indicated
that most of the compounds, including 1, showed moderate activity against Staphylococcus
aureus. The compounds were found to be in active against above mentioned bacteria
(Table 2).
Conclusion
During the current study we have synthesized new derivatives of sulfamethazine, and evalu-
ated their immunomodulating effect. Results showed that these compounds possess a promis-
ing anti-inflammatory activity which can be used further as a candidate for the treatment of
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Sulphamethazine derivatives as immunomodulating agents
Table 1. Effect of compounds (1, and 3–39) on oxidative burst from whole blood phagocytes and isolated PMNs, nitric oxide, TNF-α production, and cytotoxicity
on 3T3 cells. The IC50 (μg/mL) was calculated using three doses (1, 10, and 100 μg/mL) of each compound. Values are expressed as mean SD of three determinations.
S. No.
Oxidative Burst Inhibition
(IC50μg/mL)
NO Inhibition
(IC50 μg/mL)
TNF-α Inhibition
(IC50 μg/mL)
Cytotoxicity NIH-3T3 cell line
(IC50 μg/mL)
WB
8.9 1.3
6.5 1.3
7.3 0.4
20.5 2.2
5.7 0.8
5.8 0.6
4.2 0.2
5.7 0.2
10.6 0.5
34.6 1.2
10.6 0.5
9.2 0.6
NA
PMNs
Sulfamethazine (1)
5.2 0.05
3.6 0.05
6.1 0.08
3.0 0.06
3.4 0.1
2.9 0.3
2.4 0.07
2.8 0.05
3.4 0.4
6.2 0.13
3.3 0.05
4.9 0.2
NT
18.9 1.7
<1
>100
<1
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
-
3
4
<1
<1
5
6.9 1.9
7.8 0.3
8.0 0.3
7.3 0.3
7.3 0.4
7.3 0.2
26.3 1.8
10.8 2.1
10.9 0.5
NT
<1
6
<1
7
38.9 0.7
9.5 0.4
2.1 0.2
34.5 4.1
>100
9.9 0.2
19.5 1.0
NT
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
NA
NT
NT
NT
40
(Ibuprofen)
11.2 1.9
2.5 0.6
-
-
41
(L-NMMA)
-
-
-
-
-
-
24.2 0.6
-
-
42
(Pentoxifillin)
-
-
94.8 2.1
-
-
43
(Cyclohexamide)
0.26 0.1
WB = Whole Blood, PMNs = Polymorphonuclear neutrophils, NT = Not tested, NA = Not active
L-NMMA = NG monomethyl L-arginine acetate
https://doi.org/10.1371/journal.pone.0208933.t001
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Sulphamethazine derivatives as immunomodulating agents
Table 2. Antimicrobial activity of the synthesized compounds determined by Microplate Alamar Blue Assay.
Percentage (%) Inhibition of compound
Type
Gram positive
Gram negative
Pseudomonas
aerugenosa
Compounds
(200 μg/mL)
Bacillus
subtilis
Staphylococcus
aureus
Escherichia
coli
Slamonella
typhi
1
3
NI
NI
NI
NI
NI
NI
NI
NI
NI
35.07
NI
NI
NI
38.37
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
93.77
42.17
60.05
3.21
NI
NI
NI
NI
NI
NI
NI
NI
NI
8.92
5.93
NI
NI
NI
NI
NI
NI
4
5
34.98
32.66
54.33
47.26
27.94
1.09
NI
6
NI
7
NI
13.10
29.44
0.94
NI
8
4.31
NI
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
Ofloxacin
NI
NI
3.93
NI
26.5
NI
29.58
18.97
NI
49.69
34.30
27.35
14.12
47.23
24.33
49.73
49.37
30.78
54.62
44.33
5.76
NI
6.58
1.04
29.36
NI
NI
25.43
29.34
19.36
11..01
22.46
0.99
9.30
14.61
11.59
NI
NI
NI
NI
NI
NI
NI
NI
2.37
1.66
27.99
9.78
1.54
NI
NI
NI
NI
NI
48.40
17.48
34.74
50.12
58.46
52.33
34.21
13.01
32.92
31.34
NI
7.45
1.21
NI
18.41
4.64
NI
6.35
NI
NI
NI
3.72
11.11
14.10
4.26
NI
4.65
NI
3.99
14.97
NI
NI
NI
NI
NI
NI
19
NI
NI
NI
NI
15.42
16.48
20.54
NI
0.65
0.20
NI
NI
NI
NI
NI
10.19
NI
NI
NI
NI
18.96
NI
NI
41.09
94.08
1.84
94.43
12.49
89.75
94.14
NI: No inhibition
https://doi.org/10.1371/journal.pone.0208933.t002
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Sulphamethazine derivatives as immunomodulating agents
inflammation and related disorder. Further optimization of the synthesized compounds is
required in order to obtain enhanced biological activities.
Supporting information
S1 Fig. Spectral data of compound 3.
(PDF)
S2 Fig. Spectral data of compound 4.
(PDF)
S3 Fig. Spectral data of compound 5.
(PDF)
S4 Fig. Spectral data of compound 6.
(PDF)
S5 Fig. Spectral data of compound 7.
(PDF)
S6 Fig. Spectral data of compound 8.
(PDF)
S7 Fig. Spectral data of compound 9.
(PDF)
S8 Fig. Spectral data of compound 10.
(PDF)
S9 Fig. Spectral data of compound 11.
(PDF)
S10 Fig. Spectral data of compound 12.
(PDF)
S11 Fig. Spectral data of compound 13.
(PDF)
S12 Fig. Spectral data of compound 14.
(PDF)
S13 Fig. Spectral data of compound 15.
(PDF)
S14 Fig. Spectral data of compound 16.
(PDF)
S15 Fig. Spectral data of compound 17.
(PDF)
S16 Fig. Spectral data of compound 18.
(PDF)
S17 Fig. Spectral data of compound 19.
(PDF)
S18 Fig. Spectral data of compound 20.
(PDF)
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Sulphamethazine derivatives as immunomodulating agents
S19 Fig. Spectral data of compound 21.
(PDF)
S20 Fig. Spectral data of compound 22.
(PDF)
S21 Fig. Spectral data of compound 23.
(PDF)
S22 Fig. Spectral data of compound 24.
(PDF)
S23 Fig. Spectral data of compound 25.
(PDF)
S24 Fig. Spectral data of compound 26.
(PDF)
S25 Fig. Spectral data of compound 27.
(PDF)
S26 Fig. Spectral data of compound 28.
(PDF)
S27 Fig. Spectral data of compound 29.
(PDF)
S28 Fig. Spectral data of compound 30.
(PDF)
S29 Fig. Spectral data of compound 31.
(PDF)
S30 Fig. Spectral data of compound 32.
(PDF)
S31 Fig. Spectral data of compound 33.
(PDF)
S32 Fig. Spectral data of compound 34.
(PDF)
S33 Fig. Spectral data of compound 35.
(PDF)
S34 Fig. Spectral data of compound 36.
(PDF)
S35 Fig. Spectral data of compound 37.
(PDF)
S36 Fig. Spectral data of compound 38.
(PDF)
S37 Fig. Spectral data of compound 39.
(PDF)
S38 Fig. Institutional ethical committee approval.
(PDF)
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Sulphamethazine derivatives as immunomodulating agents
S39 Fig. General reaction for the synthesis of compounds 3–39.
(TIF)
Acknowledgments
We are thankful to Ms. Narjis Fatima, Research Assistant at the PCMD, ICCBS for conducting
the cytotoxicity, Ms. Tooba, Research Assistant at ICCBS for conducting anti-bacterial assays,
and Ms. Ayesha Fahim, Research Assistant at ICCBS for conducting the anti-inflammatory
assays.
Author Contributions
Conceptualization: Hina Siddiqui, M. Iqbal Choudhary.
Data curation: Hina Siddiqui, Haroon M. Haniffa, Almas Jabeen, M. Iqbal Choudhary.
Formal analysis: Hina Siddiqui, Haroon M. Haniffa, Almas Jabeen, M. Iqbal Choudhary.
Funding acquisition: Hina Siddiqui, M. Iqbal Choudhary.
Investigation: Hina Siddiqui, Haroon M. Haniffa, Almas Jabeen, M. Iqbal Choudhary.
Methodology: Hina Siddiqui, Haroon M. Haniffa, Almas Jabeen, M. Iqbal Choudhary.
Project administration: Hina Siddiqui, Atta-ur -Rahman, M. Iqbal Choudhary.
Resources: Hina Siddiqui, Atta-ur -Rahman, M. Iqbal Choudhary.
Software: Hina Siddiqui, M. Iqbal Choudhary.
Supervision: Hina Siddiqui, Atta-ur -Rahman, M. Iqbal Choudhary.
Validation: Hina Siddiqui, Haroon M. Haniffa, Almas Jabeen, Atta-ur -Rahman, M. Iqbal
Choudhary.
Visualization: Hina Siddiqui, M. Iqbal Choudhary.
Writing – original draft: Hina Siddiqui, Haroon M. Haniffa, Almas Jabeen, Atta-ur -Rahman,
M. Iqbal Choudhary.
Writing – review & editing: Hina Siddiqui, Haroon M. Haniffa, Almas Jabeen, Atta-ur -Rah-
man, M. Iqbal Choudhary.
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