Synthesis of new bicarbazole-linked triazoles as non-cytotoxic reactive oxygen species (ROS) inhibitors

Article abstract of DOI:10.1080/00397911.2019.1620281

Carbazole analogs 3 and 4 and a new library of bicarbazole-linked triazoles 6–11 were prepared via new synthetic methodology. Metal-catalyzed oxidative coupling reaction was utilized for the synthesis of bicarbazole acetylene 4 and different metals (Zn

Full text of DOI:10.1080/00397911.2019.1620281

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Synthesis of new bicarbazole-linked triazoles
as non-cytotoxic reactive oxygen species (ROS)
inhibitors
Shazia Iqbal, Maria A. Khan, Zulqarnain Ansari, Almas Jabeen, Aisha Faheem,
Saba Fazal-Ur-Rehman & Fatima Z. Basha
To cite this article: Shazia Iqbal, Maria A. Khan, Zulqarnain Ansari, Almas Jabeen, Aisha Faheem,
Saba Fazal-Ur-Rehman & Fatima Z. Basha (2019) Synthesis of new bicarbazole-linked triazoles
as non-cytotoxic reactive oxygen species (ROS) inhibitors, Synthetic Communications, 49:18,
2330-2341, DOI: 10.1080/00397911.2019.1620281
To link to this article: https://doi.org/10.1080/00397911.2019.1620281
View supplementary material
Published online: 28 Jun 2019.
Submit your article to this journal
Article views: 22
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=lsyc20

R
SYNTHETIC COMMUNICATIONSV
2019, VOL. 49, NO. 18, 2330–2341
https://doi.org/10.1080/00397911.2019.1620281
Synthesis of new bicarbazole-linked triazoles as non-
cytotoxic reactive oxygen species (ROS) inhibitors
Shazia Iqbala, Maria A. Khana, Zulqarnain Ansarib, Almas Jabeenc, Aisha Faheemc,
Saba Fazal-Ur-Rehmanb, and Fatima Z. Bashaa
aH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
b
University of Karachi, Karachi, Pakistan; Department of Chemistry, University of Karachi, Karachi,
c
Pakistan; PCMD, International Center for Chemical and Biological Sciences, University of Karachi,
Karachi, Pakistan
ABSTRACT
ARTICLE HISTORY
Received 28 November 2018
Carbazole analogs 3 and 4 and a new library of bicarbazole-linked
triazoles 6–11 were prepared via new synthetic methodology. Metal-
catalyzed oxidative coupling reaction was utilized for the synthesis
KEYWORDS
of bicarbazole acetylene 4 and different metals (Znþ2, Coþ2, Feþ3
,
Carbazole; bicarbazole;
triazoles; reactive oxygen
species (ROS);
Niþ2, Cuþ2, Mnþ2) as catalyst were screened. Only Fe-catalyzed reac-
tion was found to be excellent and gave homocoupled product 4.
Cu-catalyzed 1,3-dipolar cycloaddition was also utilized to install tri-
azole moiety for the synthesis of hybrid molecules 6–11. Based on
reported anti-inflammatory activity of carbazole and triazole scaf-
folds, all compounds were screened for their reactive oxygen species
(ROS) inhibitory potential. Results from these studies revealed tri-
azole 9 as most active compound (IC50 value of 7.6 0.1 mg/mL on
human whole blood and 2.7 0.09 mg/mL on isolated neutrophils)
using ibuprofen as a standard. Therefore, class described herein can
serve as attractive structural element for further studies on ROS
inhibition.
oxidative stress
GRAPHICAL ABSTRACT
CONTACT Fatima Z. Basha
bashafz@gmail.com
H. E. J. Research Institute of Chemistry, International Center for
Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lsyc.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2019 Taylor & Francis Group, LLC

R
SYNTHETIC COMMUNICATIONSV
2331
Introduction
Reactive oxygen species (ROS) are oxygen-containing molecules with an unpaired elec-
tron. They are produced during cellular respiration in mitochondria or during metabolic
activity of certain enzymes.[1] However, overproduction of ROS or imbalance in the
metabolism of oxidative defense mechanism results in cell damage, and ultimately leads
to cancer, neurodegenerative diseases, atherosclerosis, autoimmune diseases, and
aging.[2a,2b,3,4] Therefore, controlling or inhibiting the production of ROS can serve as
an alternate to control associated problems. Currently several immunosuppressants, and
anti-inflammatory agents, such as naproxen, asprin, ibuprofen, etc. are available, but
their non-selectivity, and side effects decrease their utility[5] as shown in Figure 1.
Carbazole analogs have gained significant attention owing to a large number of
interesting biological properties. Among them, a number of reports were available
describing anti-inflammatory activities of carbazole analogs, mainly as COX-1, COX-
2, TNF-a, and IL-6 inhibitors.[6] These compounds have carbazole linked with differ-
ent moieties, such as N-phenylacetamide, carbamates, indoles, pyran, benzoxazepine,
pyrazoline, etc.[7] Encouraged by these reports, and interest in ROS inhibition, our
attention is drawn to select carbazole as main skeleton, and linked it with other bio-
logically active heterocycles, such as triazoles, which is already known to display a
number of pharmacological activities including anti-tubercular, antifungal, anti-
inflammatory, antioxidant, antiepileptic, and antibacterial activities (Figure 2).[8]
Fe-catalysis have emerged as a powerful procedure for oxidation reactions and con-
sidered as a low cost, and efficient alternate to noble transition metals (Pd, Pt, etc.).
This strategy is well known for the formation of C–X (X ¼ C, N, P, O, and S) bonds via
oxidation coupling or functionalization reactions. Among wide applications, oxidative
homocoupling of heterocycles can be easily achieved by this method to obtained dimeric
heterocycles. Cu-catalyzed 1,3-dipolar cycloaddition is one of the feasible method to
install triazole moiety. In addition, Cu-catalysis adds efficiency, and regioselectivity to
the reaction.
Based on the fact that carbazole and triazole scaffolds have been reported to exhibit
good anti-inflammatory activity, both were integrated to form a hybrid molecule, which
believed to demonstrate excellent ROS inhibition (Figure 2). Herein, carbazole analogs,
and a new library of bicarbazole-linked triazole analogs were synthesized using
Figure 1. Structures of common anti-inflammatory agents.

2332
S. IQBAL ET AL.
Figure 2. Rationale for the synthesis of bicarbazole-linked triazole analogs.
Fe-catalyzed oxidative coupling, propargylation, and Cu-catalyzed 1,3-dipolar cycloadd-
ition reactions. Compounds were screened for their ROS inhibitory potential. To our
knowledge, this is the first report in literature on ROS inhibitory activity of triazole ana-
logs linked with bicarbazole-type molecules.
Results and discussion
Chemistry
A small library of bicarbazole-linked triazoles (6–11) was prepared starting from com-
mercially available carbazole (1), as presented in Scheme 1. At first, carbazole acetylene
(3) was prepared via nucleophilic substitution of 1 with propargyl bromide, according
to our previous report.[8b] Bicarbazole acetylene (4) was synthesized via metal-catalyzed
oxidative coupling strategy. Number of metal catalysts were screened for the synthesis
of bicarbazole acetylene (4). Only FeCl3 anhydrous was considered as an excellent cata-
lyst among the other metal catalysts, as it gave oxidatively homocoupled product (4)
and rest of the metals were not catalyzed the reaction (Table 1).
Later on, bicarbazole acetylene (4) was synthesized via two alternate strategies. In the
first strategy, carbazole (1) was first subjected to iron-catalyzed oxidative coupling to
afford bicarbazole (2) (71% yield), which in the second step underwent alkylation with
propargyl bromide to afford 4 in 52% yield (Method 1). An alternate strategy can also
be used to prepare bicarbazole acetylene (4) in 72% yield, where acetylene 3 is directly

R
SYNTHETIC COMMUNICATIONSV
2333
Scheme 1. Synthetic route towards bicarbazole-linked triazoles (6–11).
Table 1. Optimization of metal-catalyzed oxidative coupling reaction for the synthesis of bicarbazole
acetylene (4).
ꢀ
Entry
Metal catalyst
Temperature
Time (h)
Yield (%)
1
2
3
4
5
6
ZnCl2
FeCl3
NiCl2
MnCl2
CoCl2
CuSO4
0 ꢁC! rt
0ꢁC! rt
0 ꢁC! rt
0 ꢁC! rt
0 ꢁC! rt
0 ꢁC! rt
1
1
1
1
1
1
0a
27
0a
0a
0a
0a
ꢀThe crude product was monitored by 1H NMR.
aCarbazole acetylene (4) did not react with respective metal catalyst. Reaction conditions for all were same: solvent was chloroform, 2 equivalents of each
metal catalyst used.
subjected to iron-catalyzed oxidative coupling (Method 2). Second route is considered
as a feasible method, as it gave comparatively better yields (Scheme 1).
Later, several conditions were screened for better yield of bicarbazole acetylene (4)
via second route (Method 2) in Scheme 1, an increase in % yield was observed when
equivalences of iron chloride (FeCl3) anhydrous were switched from 2.0, 4.5, 5.0, to 5.5,
and a decrease in % yield was observed due to the degradation of product when reac-
tion time increased from 1 h to 2 h, as shown in Table 2.
Dipolar partner azides 5a–5f were prepared according to literature,[8a] in which ace-
tophenone was subjected to a-bromination using N-bromosuccinimide (NBS), followed
by substitution of bromide group with azide. In the end, copper-catalyzed 1,3-dipolar
cycloaddition of acetylene 4 (dipolarophiles) with acetophenone azides (dipolar mole-
cules) (5a–5f) was done to afford subsequent bicarbazole-linked triazoles in 84–87%
yields (Scheme 2). The reaction regioselectively gave 1,4-disubstituted-1,2,3-triazoles,
and to the best of our knowledge, compounds 4 and 6–11 were newly identified.

2334
S. IQBAL ET AL.
Table 2. Optimization of reaction condition of Fe-catalyzed oxidative coupling reaction using carba-
zole acetylene for the synthesis of bicarbazole acetylene (4).
ꢀ
ꢀ
Entry
Equivalence of FeCl3 (anhydrous) used
Temperature
Time (h)
Yield (%)
Time (h)
Yield (%)
1
2
3
4
2.0 equiv.
4.5 equiv.
5.0 equiv.
5.5 equiv.
0 ꢁC
1a
1b
1c
1c
27
72
75
78
2c
2c,d
2d
27
69
52
65
0 ꢁC! rt
0 ꢁC! rt
0 ꢁC! rt
2d
ꢀThe product was monitored by 1H NMR.
a50% SM was consumed.
b90% SM was consumed.
c100% SM was consumed.
dMultiple spots was appeared/product was degrading upon prolonged reaction time.
Scheme 2. Library of bicarbazole-linked triazole analogs (6–11).
All the synthesized compounds were evaluated using spectroscopic techniques.
Bicarbazole (2) formation was confirmed by the disappearance of singlet for H-3 in car-
1
bazole due to Fe-catalyzed oxidative coupling reaction in H-NMR, and reappearance of
two H-2 (d 7.81 ppm) and two H-4 (d 8.49 ppm) protons, respectively, as doublet and
singlet instead of triplet and doublet. Two protons for amino groups appeared at
11.24 ppm, while rest of the aromatic protons appeared at d 8.23, 7.58, 7.50, 7.39 and
7.17 ppm for two sets of H-5, H-1, H-8, H-7, and H-6 protons, respectively. Structure
of bicarbazole acetylene (4) was deduced by the disappearance of NH, and appearance
of two acetylenic protons at d 1.36 ppm as a triplet, and four methylene protons at d
5.35 ppm as a doublet in addition to aromatic protons of bicarbazole moiety (d
8.58–7.26 ppm). For spectral data discussion of bicarbazole-linked triazoles, compound
1
10 was taken as a representative example. In the H-NMR, appearance of singlet for
two triazolyl protons H-500 (d 7.24 ppm) confirmed the triazole formation via cycloadd-
ition reaction. Its formation was also supported by the disappearance of triplet for two
acetylenic protons (d 1.36 ppm). In addition to aromatic protons of bicarbazole moiety
(d 8.56–7.80 ppm), two singlets at d 6.03 and 5.77 ppm correspond to two sets of methy-
lene signals OCH2 and NCH2, respectively. Methoxy protons at d 3.84 ppm appeared as
a singlet for six protons. 13C-NMR of compound 10 showed characteristic peak for tria-
zolyl methine carbons at 125.1 ppm in addition to two C ¼ O, OCH3, CH2O, and CH2N

R
SYNTHETIC COMMUNICATIONSV
2335
Table 3. ROS inhibitory activities (IC50, lM) of carbazole analogs 1–4 and 6–11.
ROS inhibitory activity IC50 SDa (mg/mL)
Compound
Whole blood Neutrophils
Cytotoxicity IC50 SDa (mM)
1
2
3
4
6
7
8
9
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
7.6 0.1
15.6 0.7
inactive
11.2 1.9
–
–
–
–
–
–
–
–
Inactive[8b]
Inactive
Inactive[8b]
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
–
2.7 0.09
10
11
–
–
2.5 0.6
–
Ibuprofenb
Cycloheximidec
0.26 0.12
aSD: Standard deviation;
bStandard for ROS inhibitory activity;
cStandard for Cytotoxicity.
signals, at 190.4, 55.7, 55.4, and 37.8 ppm, respectively. Rest of the aromatic methines
and quarternary carbon signals appeared in the region between 163.9 and 109.8 ppm.
Moreover, mass and IR spectral data also supported the formation of target products.
Spectral data of carbazole acetylene (3), and azide analogs 5a–5f was not included in
the report as it was already mentioned in our previous literature.[8a,8b]
Reactive oxygen species (ROS) inhibitory activity
ROS inhibition is considered to play an important role in controlling inflammation and
related problems where phagocytes are the main producers of ROS. Carbazole (1) and
all the synthesized analogs 2–4 and 6–11 have been evaluated for their phagocyte oxida-
tive burst inhibitory activity using ibuprofen as a standard (IC50 ¼ 11.2 1.9 lg/mL).
Chemiluminescence technique was applied to evaluate the inhibitory activity of synthe-
sized compounds against ROS. The generation of ROS from human peripheral blood
phagocytes was stimulated by serum opsonized zymogen (SOZ). Luminol was used as a
probe in this technique, which can be easily penetrated in phagocyte and reacted with
intracellular oxidative species to emit light. Inhibition of ROS by test compounds results
in decrease emission of light and can be monitored by this technique.
Structure-activity relationship (SAR) investigation showed no inhibition for carbazole
(1), and its acetylenic analog 3, when tested for ROS inhibition from whole blood.
Interestingly, oxidative coupled products bicarbazole (2) and bicarbazole acetylene (4)
were also found inactive in this assay, as shown in Table 3. To expand the scope of
study, length of these analogs was increased by converting bicarbazole acetylenes (4)
into acetophenone-linked triazole moieties (in compounds 6–11). This functionalization
resulted in increased ROS inhibitory activity for two triazoles with IC50 values ranging
from 7.6 0.1 to 15.6 0.7 lg/mL. SAR results revealed that bicarbazole-linked triazole
(6) with no substitution was found inactive; however, changing electronics on acetophe-
none ring have substantial effect on the inhibitory activity. Substitution of electron
withdrawing groups at 40-position of acetophenone ring in compounds 7, 8, and 11 did
not show any appreciable results and were found to be inactive. In contrast,

2336
S. IQBAL ET AL.
bicarbazole-linked triazoles (9 and 10), bearing electron donating substituents showed
remarkable increase in activity. Comparison of IC50 values revealed that 40-methyl sub-
stituted triazole (9) (7.6 0.1 lg/mL) was found to be slightly better in activity than
standard drug (IC50 ¼ 11.2 1.9 lg/mL), and ꢂ2-fold better than its methoxy substi-
tuted congener 10 (IC50 ¼ 15.6 0.7 lg/mL). The change in activity of two congeners
was attributed by intervening oxygen atom in the methoxy group, which may be altered
the hydrophobicity, and electronics of the molecule. Most potent triazole 9 with methyl
substitution at 40-position of acetophenone ring was further tested for its potency to
inhibit ROS on isolated neutrophils, and was found to exhibit potent inhibition with
IC50 value of 2.7 0.09 lM. The results obtained from limited SAR, therefore, revealed
that increasing length of the molecule (triazole formation), and changing electronics (by
varying substituents on acetophenone ring linked to triazole moiety) played an import-
ant structural element for attenuating potency of the molecules.
Cytotoxicity
Using MTT assay, cytotoxicity of compounds 1–4 and 6–11 against 3T3 cell lines were
evaluated. Cytotoxicity of carbazole (1) and its analog 3 was already reported in our
previous literature[8b] and showed non-cytotoxic profile. Compounds 2 and 4 and bicar-
bazole-linked triazoles (6–11) showed cytotoxicity less than 30 mM as compared to the
standard cycloheximide (IC50 ¼ 0.26 0.12 lM) and were found to be safe (Table 3).
Conclusion
Propargylated and Fe-catalyzed oxidative coupled analogs of carbazole, and a small
library of bicarbazole-linked triazoles (6–11) were synthesized and studied using differ-
ent spectroscopic techniques. Carbazole (1) and all the synthesized analogs were eval-
uated for ROS inhibition using ibuprofen (IC50 ¼ 11.2 1.9 lg/mL) as the standard
inhibitor. The two compounds 9 (IC50 ¼ 7.6 0.1 lg/mL), and 10 (IC50 ¼ 15.6 0.7 lg/
mL) were found to be active among the series, with compound 9 as the most active
analog. Limited SAR of carbazole and its analogs revealed that conversion to triazole
linked with acetophenone moiety (bearing electron donating substituents) resulted in
potent inhibitory activities. In summary, these results suggested that linking triazoles
with bicarbazole could be an important structural element for tuning biological proper-
ties and for controlling inflammation and associated degenerative problems.
Experimental
Material and methods
All chemicals were bought from Alfa aesar and Sigma Aldrich and were used without
1
purification. H-NMR (400 MHz) and 13C-NMR (100 MHz) spectra were obtained on
Avance Bruker spectrometer in appropriate solvents. JEOL MS Route 600 Hz instrument
was used to record the Mass spectra, while MALDI spectra were recorded on Ultraflex
TOF/TOF mass spectrometer. IR spectra were taken on a Shimadzu FT-IR 8900

R
SYNTHETIC COMMUNICATIONSV
2337
spectrometer. UV spectra were obtained on Hitachi U-3200 ND Thermoscientic
Evolution 310 spectrophotometer.
Procedure for synthesis of 9 H,9H-3,3-bicarbazole (2)
To a suspended solution of ferric chloride (FeCl3) (12.88 g, 79.85 mmol) in 100 mL of
dry chloroform (CHCl3) under nitrogen atmosphere, a solution of carbazole (1) (5.55 g,
17.89 mmol) in CHCl3 (50 mL) was slowly added at 25 ꢁC. After 3 h of stirring, metha-
nol (MeOH) (300 mL), and dichloromethane (DCM) (100 mL) were added, and the
mixture extracted using distilled water (2 ꢃ 200 mL). Brine solution was used to wash
the combined organic layers which were then dried using anhydrous sodium sulfate
(Na2SO4), filtered, and the solvent was removed to dryness. Column chromatography
was carried out to purify the desired product, using a mixture of ethyl acetate and n-
hexanes. White solid; Yield: 71%; m.p 163–167 ꢁC; IR (KBr, cmꢄ1) tmax: 3396, 3044,
1
2961, 1602, 1459, 1329, 1237, 749; UV (DMSO) nm: ømax 303 nm; H-NMR: (400 MHz,
DMSO-d6): d 11.24 (s, 2ꢃNH, 2H), 8.49 (s, 2ꢃH-4, 2H), 8.23 (d, 2ꢃH-5, J5,6 ¼ 7.6 Hz,
2H), 7.81 (d, 2ꢃH-2, J2,1 ¼ 8.8 Hz, 2H), 7.58 (d, 2ꢃH-1, J1,2 ¼ 8.8 Hz, 2H), 7.50 (d,
2ꢃH-8, J8,7 ¼ 8.0 Hz, 2H), 7.39 (t, 2ꢃH-7, J7,(6,8) ¼ 7.6 Hz, 2H), 7.17 (t, 2ꢃH-6, J6,(5,7)
¼ 7.6 Hz, 2H); MS (EI) m/z (%): 332 (Mþ, 100), 167 (6), 166 (27); HREI-MS: calcd for
C24H16N2 (M): m/z 332.1301, found: 332.1313.
Procedure for synthesis of 9,9-di(prop-2-yn-1-yl)-9H,9H-3,3-bicarbazole (4)
To carbazole acetylene (18.7 mmol) in CHCl3 (40 mL) at 0 ꢁC, FeCl3 (78.3 mmol) was
added and stirred at 25 ꢁC for 1 h. Ice cold water was used to precipitate out the prod-
uct. Column chromatography was carried out to purify the desired product using mix-
ture of ethyl acetate and n-hexanes. White solid; Yield: 72%; IR (KBr, cmꢄ1) tmax: 3398,
3045, 2933, 1604, 1459, 1331, 1238, 1169, 1062, 802, 749; UV (DMSO) nm: ømax
1
303 nm; H-NMR: (400 MHz, DMSO-d6): d 8.58 (s, 2ꢃH-4, 2H), 8.30 (d, 2ꢃH-5, J5,6
¼
7.6 Hz, 2H), 7.93 (d, 2ꢃH-2, J2,1 ¼ 8.8 Hz, 2H), 7.80 (d, 2ꢃH-1, J1,2 ¼ 8.8 Hz, 2H), 7.70
(d, 2ꢃH-8, J8,7 ¼ 8.0 Hz, 2H), 7.50 (t, 2ꢃH-7, J7,(6,8) ¼ 7.6 Hz, 2H), 7.26 (t, 2ꢃH-6,
J6,(5,7) ¼ 7.6 Hz, 2H), 5.35 (s, 2 ꢃ CH2, 4H), 1.36 (s, 2ꢃꢅCH, 2H); MS (EI) m/z (%):
408 (Mþ, 100), 369 (61), 330 (48), 204 (13), 167 (4), 166 (5), 140 (2); HREI-MS: calcd
for C30H20N2 (M): m/z 408.1672, found: 408.1626.
General procedure for synthesis of bicarbazole–linked triazoles (6–11)
To 0.5 mmol of bicarbazole acetylene (4) in DMF: water mixture (3: 2 v/v), 0.9 mmol of
acetophenone azide (5a–5f) was added at room temperature. After that, 0.5 mmol of
copper(II) sulfate pentahydrate (CuSO4.5H2O) and 0.7 mmol of sodium L-ascorbate
(0.7 mmol) was added and stirred for 2 h. Upon completion, ice cold water was added
to the mixture and was kept for 2 h until precipitates were formed. Precipitates were
then filtered, washed with 25% ethyl acetate in n-hexanes, and purified using column
chromatography (eluent: 20–90% ethyl acetate in n-hexanes) to get pure products in
82–87% yields (See supplementary material).

2338
S. IQBAL ET AL.
2,2-((9000H,9000H-[3000,3000-Bicarbazole]-9000,9000-diylbis(methylene))bis(100H-100,200,300-triazoleꢄ- 1
400,100-diyl))bis(1-phenylethan-1-one) (6). Off white solid; Yield: 84%; IR (KBr, cm
)
tmax: 3408, 3058 (C-HAr), 2936 (C-HAl), 1791, 1686 (C ¼ O), 1597 (C ¼ N), 1457 (tri-
azole), 1328 (C-NAr), 1229 (C-NAl), 1051 (C–O), 799, 749; UV (DMSO) nm: ømax
1
303 nm; H-NMR: (400 MHz, DMSO-d6): d 8.56 (s, 2H, 2ꢃH-4000), 8.29 (d, 2H, J5
¼
000,6000
7.8 Hz, 2ꢃH-5000), 7.99-7.89 (m, 2ꢃH-20, 2ꢃH-60, 2ꢃH-500, 2ꢃH-1000, 2ꢃH-2000, 10H),
7.82 (d, 2ꢃH-8000, J8
¼ 8.1 Hz, 2H), 7.69 (t, 2ꢃH-40, J4 ,(5 ,3 ) ¼ 7.5 Hz, 2H), 7.55 (t,
0
0
0
000,7000
2ꢃH-30, 2ꢃH-50, J4 ,(3 ,5 )/5 ,(4 ,6 ) ¼ 7.5 Hz, 4H), 7.48 (t, 2ꢃH-7000, J7
¼ 7.8 Hz,
0
0
0
0
0
0
000,(6000,8000)
2H), 7.24 (t, 2ꢃH-6000, J6
¼ 7.2 Hz, 2H), 6.09 (s, 2 ꢃ CH2CO, 4H), 5.79 (s,
000,(5000,7000)
13
2 ꢃ CH2N, 4H); C-NMR (100 MHz, DMSO-d6): d 192.1 (2ꢃC ¼ O), 143.1 (2ꢃC-10),
140.3 (2ꢃC-400), 138.9 (2ꢃC-8a000), 134.1 (2ꢃC-40), 132.6 (2ꢃC-10), 128.9 (2ꢃC-30,
2ꢃC-50), 128.1 (2ꢃC-20, 2ꢃC-60), 125.8 (2ꢃC-7000), 125.1 (2ꢃC-500), 124.7 (2ꢃC-2000),
122.9 (2ꢃC-3000), 122.5 (2ꢃC-4a000, 2ꢃC-4b000), 120.5 (2ꢃC-5000), 119.0 (2ꢃC-6000), 118.3
(2ꢃC-4000), 110.0 (2ꢃC-1000), 109.7 (2ꢃC-8000), 55.7 (2ꢃCH2CO), 37.7 (2ꢃCH2N); MS
(MALDI-TOF): m/z: 730.28 (Mþ), HRMS Calcd for [C46H34N8O2]þ: 730.2805,
Found: 730.2832.
2,2-((9000H,9000H-[3000,3000-Bicarbazole]-9000,9000-diylbis(methylene))bis(100H-100,200,300-triazole-
400,100-diyl))bis(1-(40-methoxyphenyl)ethan-1-one) (10). Cream colored solid; Yield: 85%;
IR (KBr, cmꢄ1) tmax: 3416, 3054 (C-HAr), 2935 (C-HAl), 2839, 1686 (C ¼ O), 1599
(C ¼ N), 1461 (triazole), 1326 (C-NAr), 1239 (C-NAl), 1173 (C–N), 1023 (C–O), 834,
1
802, 747; UV (DMSO) nm: ømax 296 nm; H-NMR: (400 MHz, DMSO-d6): d 8.56 (s,
2ꢃH-4000, 2H), 8.28 (d, 2ꢃH-5000, J5
¼ 7.6 Hz, 2H), 7.97–7.89 (m, 2ꢃH-30, 2ꢃH-50,
000,6000
2ꢃH-500, 2ꢃH-1000, 2ꢃH-2000, 10H), 7.81 (d, 2ꢃH-8000, J8
¼ 8.4 Hz, 2H), 7.50 (t,
¼ 7.6 Hz, 2H), 7.08 (d,
000,7000
2ꢃH-7000, J7
¼ 7.6 Hz, 2H), 7.24 (t, 2ꢃH-6000, J6
000,(6000,8000
)
000,(5000,7000
)
2ꢃH-20, 2ꢃH-60, J2 ,3 /6 ,5 ¼ 8.8 Hz, 4H), 6.00 (s, 2 ꢃ CH2CO, 4H), 5.77 (s, 2 ꢃ CH2N,
4H), 3.84 (s, 2 ꢃ OCH3, 6 H); 13C-NMR (100 MHz, DMSO-d6): d 190.4 (2ꢃC¼O), 163.9
(2ꢃC-40), 140.3 (2ꢃC-400), 138.9 (2ꢃC-8a000), 132.7 (2ꢃC-10), 130.5 (2ꢃC-20, 2ꢃC-60),
126.9 (2ꢃC-9a000), 125.9 (2ꢃC-7000), 125.1 (2ꢃC-500), 123.0 (2ꢃC-3000), 122.6 (2ꢃC-4a000,
2ꢃC-4b000), 120.5 (2ꢃC-5000), 119.0 (2ꢃC-6000), 118.3 (2ꢃC-4000), 114.1 (2ꢃC-30, 2ꢃC-50),
110.0 (2ꢃC-1000, 2ꢃC-2000), 109.8 (2ꢃC-8000), 55.7 (2 ꢃ OCH3), 55.4 (2ꢃCH2CO), 37.8
(2ꢃCH2N); MS (MALDI-TOF): m/z: 790.30 (Mþ), HRMS Calcd for [C48H38N8O4]þ:
790.3016, Found: 790.3062.
0
0
0
0
Oxidative burst assay for immunomodulatory activity
The assay was performed using Helfand protocol with some modifications as described
in literature.[9]
In vitro cytotoxicity assay
The MTT assay was performed according to the procedure given in our previous report
using Masson protocol.[8b,10]

R
SYNTHETIC COMMUNICATIONSV
2339
Funding
Authors are thankful to the Higher Education Commission, Pakistan for providing finan-
cial assistance.
References
[1] (a) Mittal, M.; Siddiqui, M. R.; Tran, K.; Reddy, S. P.; Malik, A. B. Reactive Oxygen
Species in Inflammation and Tissue Injury. Antioxid. Redox Signal. 2014, 20, 1126–1167.
DOI: 10.1089/ars.2012.5149. (b)Yousuf, S.; Khan, K. M.; Salar, U.; Jabeen, A.; Ahmed, S.;
Muhammad, M. T.; Faheem, A.; Perveen, S. New Bis-Pyrazolones as Potential Leads for
Ros Inhibition; Environment Friendly Green Synthesis, Structural Characterization, and in
Vitro Studies. Mc 2018, 14, 536–548. DOI: 10.2174/1573406414666180112122001.
[2] (a) Beckhauser, T. F.; Francis-Oliveira, J.; De Pasquale, R. Reactive Oxygen Species:
Physiological and Physiopathological Effects on Synaptic Plasticity. J. Exp. Neurosci. 2016,
10, 23–48. (b) Kim, G. H.; Kim, J. E.; Rhie, S. J.; Yoon, S. The Role of Oxidative Stress in
Neurodegenerative Diseases. Exp. Neurobiol. 2015, 24, 325–340. DOI: 10.5607/
en.2015.24.4.325.
[3] (a) Hidalgo, C.; Arias-Cavieres, A. Calcium, Reactive Oxygen Species, and Synaptic
Plasticity. Physiology (Bethesda, Md.) 2016, 31, 201–215. DOI: 10.1152/physiol.00038.2015.
(b) Hsu, H.-T.; Tseng, Y.-T.; Wong, W.-J.; Liu, C.-M.; Lo, Y.-C. Resveratrol Prevents
Nanoparticles-Induced Inflammation and Oxidative Stress via Downregulation of PKC-a
and NADPH Oxidase in Lung Epithelial A549 Cells. BMC Complement. Altern. Med.
2018, 18, 211. DOI: 10.1186/s12906-018-2278-6.
[4] (a) Molatlhegi, R. P.; Phulukdaree, A.; Anand, K.; Gengan, R. M.; Tiloke, C.; Chuturgoon,
A. A. Cytotoxic Effect of a Novel Synthesized Carbazole Compound on a549 Lung Cancer
Cell Line. PloS One 2015, 10, e0129874. DOI: 10.1371/journal.pone.0129874. (b) Salar, U.;
Khan, K.; Jabeen, A.; Faheem, A.; Fakhri, M. I.; Saad, S.; Perveen, S.; Taha, M.; Hameed,
A. Coumarin Sulfonates: As Potential Leads for ROS Inhibition. Bioorg. Chem. 2016, 69,
37–47. DOI: 10.1016/j.bioorg.2016.09.006. (c) Kattoor, A. J.; Pothineni, N. V. K.; Palagiri,
D.; Mehta, J. L. Oxidative Stress in Atherosclerosis. Curr. Atheroscler. Rep. 2017, 19, 42.
DOI: 10.1007/s11883-017-0678-6. (d) Goncharov, N. V.; Avdonin, P. V.; Nadeev, A. D.;
Zharkikh, I. L.; Jenkins, R. O. Reactive Oxygen Species in Pathogenesis of atherosclerosis.
Curr. Pharm. Des. 2015, 21, 1134–1146. DOI: 10.2174/1381612820666141014142557. (e)
Davalli, P.; Mitic, T.; Caporali, A.; Lauriola, A.; D’Arca, D. ROS, Cell Senescence, and
Novel Molecular Mechanisms in Aging and Age-Related Diseases. Oxid. Med. Cell Longev.
2016, 2016, 1. DOI: 10.1155/2016/3565127.
[5] (a) Yuan, J.; Kashiwagi, S.; Reeves, P.; Nezivar, J.; Yang, Y.; Arrifin, N. H.; Nguyen, M.;
Jean-Mary, G.; Tong, X.; Uppal, P.; et al. A Novel Mycobacterial Hsp70-Containing
Fusion Protein Targeting Mesothelin Augments Antitumor Immunity and Prolongs
Survival in Murine Models of Ovarian Cancer and Mesothelioma. J. Hematol. Oncol.
2014, 7, 15. DOI: 10.1186/1756-8722-7-15. (b) Matsui, H.; Shimokawa, O.; Kaneko, T.;
Nagano, Y.; Rai, K.; Hyodo, I. The Pathophysiology of Non-Steroidal anti-Inflammatory
Drug (NSAID)-Induced Mucosal Injuries in Stomach and Small Intestine. J. Clin.
Biochem. Nutr. 2011, 48, 107–111. DOI: 10.3164/jcbn.10-79. (c) Salar, U.; Khan, K.;
Jabeen, A.; Faheem, A.; Ali, F.; Syed, S.; Ahmed, K.; Perveen, S. Anti-Inflammatory
Activity of 3-Thiazolyl Coumarins. J. Chem. Soc. Pak. 2017, 39, 578–585.
[6] (a) Nooron, N.; Athipornchai, A.; Suksamrarn, A.; Chiabchalard, A. Mahanine Enhances
the Glucose-Lowering Mechanisms in Skeletal Muscle and Adipocyte Cells. Biochem.
Biophys. Res. Commun. 2017, 494, 101–106. DOI: 10.1016/j.bbrc.2017.10.075. (b)
Mounika, K. N.; Jyothi, A. N.; Raju, G. N.; Rao, R. Carbazole Derivatives in Cancer
Treatment-a Review. WJPPS 2015, 4, 420–428.

2340
S. IQBAL ET AL.
[7] (a) Pattanashetty, S. H.; Hosamani, K.; Shettar, A. K.; Mohammed Shafeeulla, R. Design,
Synthesis and Computational Studies of Novel Carbazole N-Phenylacetamide Hybrids as
Potent Antibacterial, anti-Inflammatory, and Antioxidant Agents. J. Heterocyclic Chem.
2018, 55, 1765–1774. DOI: 10.1002/jhet.3214. (b) Ma, Y.; Wang, Q.; Yu, K.; Fan, X.; Xiao,
W.; Cai, Y.; Xu, P.; Yu, M.; Yang, H. 6-Formylindolo(3,2-b)Carbazole Induced Aryl
Hydrocarbon Receptor Activation Prevents Intestinal Barrier Dysfunction through
Regulation of Claudin-2 Expression. Chem.-Biol. Interact. 2018, 288, 83–90. DOI: 10.1016/
j.cbi.2018.04.020. (c) Nalli, Y.; Khajuria, V.; Gupta, S.; Arora, P.; Riyaz-Ul-Hassan, S.;
Ahmed, Z.; Ali, A. Correction: Four New Carbazole Alkaloids from Murraya Koenigii
That Display anti-Inflammatory and anti-Microbial Activities. Org. Biomol. Chem. 2018,
16, 1994. 1994; DOI: 10.1039/C8OB90030B. (d) Nalli, Y.; Khajuria, V.; Gupta, S.; Arora,
P.; Riyaz-Ul-Hassan, S.; Ahmed, Z.; Ali, A. Four New Carbazole Alkaloids from Murraya
Koenigii That Display anti-Inflammatory and anti-Microbial Activities. Org. Biomol.
Chem. 2016, 14, 3322–3332. DOI: 10.1039/C6OB00267F. (e) Muniyappan, G.;
Kathvarayan, S.; Kella, C. R.; Kalliyappan, E.; Ponnusamy, S.; Thirumalai, P. P. Synthesis
of Novel 4-Hydroxycarbazole Derivatives and Evaluation of Their in Vitro anti-
Inflammatory, anti-Oxidant Activities and Molecular Docking. Res. Chem. Intermed. 2017,
43, 3377–3393. DOI: 10.1007/s11164-016-2831-1. (f) Prabhakar, V.; Babu, K. S.;
Ravindranath, L.; SwarnaKumari, M.; Latha, J. Synthesis, Characterisation and Biological
Evaluation of 1,5-Benzoxazepine Derivatives Containing Carbazole Ring. Heterocycl. Lett.
2015, 5, 579–594. (g) Shen, D.-Y.; Chan, Y.-Y.; Hwang, T.-L.; Juang, S.-H.; Huang, S.-C.;
Kuo, P.-C.; Thang, T. D.; Lee, E.-J.; Damu, A. G.; Wu, T.-S. Constituents of the Roots of
Clausena Lansium and Their Potential anti-Inflammatory Activity. J. Nat. Prod. 2014, 77,
1215–1223. DOI: 10.1021/np500088u.
[8] (a) Khan, M. A.; Saleem, A.; Ghouri, N.; Hameed, A.; Choudhary, M. I.; Z Basha, F.
Synthesis and in Vitro Evaluation of Dibenzoazepine Triazole Derivatives: A Novel Class
of
Antileishmanial
Agents.
Lddd.
2015,
12,
597–606.
DOI:
10.2174/
1570180812999150225111959. (b) Iqbal, S.; Khan, M. A.; Javaid, K.; Sadiq, R.; Fazal-Ur-
Rehman, S.; Choudhary, M. I.; Basha, F. Z. New Carbazole Linked 1, 2, 3-Triazoles as
Highly Potent Non-Sugar a-Glucosidase Inhibitors. Bioorg. Chem. 2017, 74, 72–81. DOI:
10.1016/j.bioorg.2017.07.006. (c) Khan, M. A.; Javaid, K.; Wadood, A.; Jamal, A.; Batool,
F.; Fazal-Ur-Rehman, S.; Basha, F. Z.; Choudhary, M. I. In Vitro Alpha-Glucosidase
Inhibition by Non-Sugar Based Triazoles of Dibenzoazepine, Their Structure-Activity
Relationship, and Molecular Docking. Med. Chem. 2017, 13, 698–704. (d) Zhang, S.; Xu,
Z.; Gao, C.; Ren, Q.-C.; Chang, L.; Lv, Z.-S.; Feng, L.-S. Triazole Derivatives and Their
anti-Tubercular Activity. Eur. J. Med. Chem. 2017, 138, 501–513. DOI: 10.1016/
j.ejmech.2017.06.051. (e) Sadeghpour, H.; Khabnadideh, S.; Zomorodian, K.; Pakshir, K.;
Hoseinpour, K.; Javid, N.; Faghih-Mirzaei, E.; Rezaei, Z. Design, Synthesis, and Biological
Activity of New Triazole and Nitro-Triazole Derivatives as Antifungal Agents. Molecules
ꢀ
ꢀ
2017, 22, 1150. DOI: 10.3390/molecules22071150. (f) Ramırez-Villalva, A.; Gonzalez-
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Calderon, D.; Rojas-Garcıa, R. I.; Gonzalez-Romero, C.; Tamarız-Mascarua, J.; Morales-
ꢀ
ꢀ
Rodrıguez, M.; Zavala-Segovia, N.; Fuentes-Benıtes, A. Synthesis and Antifungal Activity
of Novel Oxazolidin-2-One-Linked 1,2,3-Triazole Derivatives. Med. Chem. Commun.
2017, 8, 2258–2262. 8, DOI: 10.1039/C7MD00442G. (g) Ansari, K.; Lal, C.; Khitoliya, R.
Synthesis and Biological Activity of Some Triazole-Bearing Benzimidazole Derivatives. J.
Serb. Chem. Soc. 2011, 76, 341–352. DOI: 10.2298/JSC100301029A. (h) Kharb, R.; Sharma,
P. C.; Yar, M. S. Pharmacological Significance of Triazole Scaffold. J. Enzym. Inh. Med.
Chem. 2011, 26, 1–21. DOI: 10.3109/14756360903524304. (i) Singhal, N.; Sharma, P.;
Dudhe, R.; Kumar, N. Recent Advancement of Triazole Derivatives and Their Biological
Significance. J. Chem. Pharm. Res. 2011, 3, 126–133. (j) Anandhan, R.; Kannan, A.;
Rajakumar, P. Synthesis and anti-Inflammatory Activity of Triazole-Based Macrocyclic
Amides through Click Chemistry. Syn. Comm. 2017, 47, 671–679. DOI: 10.1080/
00397911.2016.1254800. (k) Song, M.-X.; Deng, X.-Q. Recent Developments on Triazole

R
SYNTHETIC COMMUNICATIONSV
2341
Nucleus in Anticonvulsant Compounds: A Review. J. Enzym. Inh. Med. Chem. 2018, 33,
453–478. DOI: 10.1080/14756366.2017.1423068.
[9] (a) Helfand, S. L.; Werkmeister, J.; Roder, J. C. Chemiluminescence Response of Human
Natural Killer Cells. I. The Relationship between Target Cell Binding, Chemiluminescence,
and Cytolysis. J. Exp. Med. 1982, 156, 492–505. DOI: 10.1084/jem.156.2.492. (b)
Adhikaria, A.; Vohra, M. I.; Jabeen, A.; Dastagir, N.; Choudhary, M. I. Antiinflammatory
Steroidal Alkaloids from Sarcococca Wallichii of Nepalese Origin. Nat. Prod. Comm. 2015,
10, 1533–1536.
[10] Masson, P.; Froment, M.-T.; Fortier, P.-L.; Visicchio, J.-E.; Bartels, C. F.; Lockridge, O.
Butyrylcholinesterase-Catalysed Hydrolysis of Aspirin, a Negatively Charged Ester, and
Aspirin-Related Neutral Esters. Biochim. Biophys. Acta Protein Struct. Molec. Enzym. 1998,
1387, 41–52. DOI: 10.1016/S0167-4838(98)00104-6.