Synthesis of 1,4-dihydropyrazolo[4,3-b]indoles via intramolecular C(sp2)-N bond formation involving nitrene insertion, DFT study and their anticancer assessment

Article abstract of DOI:10.1016/j.bioorg.2021.105114

We herein report a new synthetic route for a series of unreported 1,4-dihydropyrazolo[4,3-b]indoles (6–8) via deoxygenation of o-nitrophenyl-substituted N-aryl pyrazoles and subsequent intramolecular (sp²)-N bond formation under microwave irradiation expedite modified Cadogan condition. This method allows access to NH-free as well as N-substituted fused indoles. DFT study and controlled experiments highlighted the role of nitrene insertion as one of the plausible reaction mechanisms. Furthermore, the target compounds exhibited cytotoxicity at low micromolar concentration against lung (A549), colon (HCT-116), and breast (MDA-MB-231, and MCF-7) cancer cell lines, induced the ROS generation and altered the mitochondrial membrane potential of highly aggressive MDA-MB-231 cells. Further investigations revealed that these compounds were selective Topo I (6h) or Topo II (7a, 7b) inhibitors.

Full text of DOI:10.1016/j.bioorg.2021.105114

Bioorganic Chemistry 114 (2021) 105114
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis of 1,4-dihydropyrazolo[4,3-b]indoles via intramolecular C(sp2)-N
bond formation involving nitrene insertion, DFT study and their
anticancer assessment
Manpreet Kaura, Vikrant Mehtab, Aabid Abdullah Wanic, Sahil Aroraa, Prasad V. Bharatamc,
,
,*
Ashoke Sharond, Sandeep Singhb *, Raj Kumara
a Laboratory for Drug Design and Synthesis, Department of Pharmaceutical Sciences and Natural Products, School of Pharmaceutical Sciences, Central University of
Punjab, Bathinda-151401, Punjab, India
b Department of Human Genetics and Molecular Medicine, Central University of Punjab, Bathinda-151401, Punjab, India
c Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), SAS. Nagar, India
d Department of Chemistry, Birla Institute of Technology, Mesra, Ranchi, Jharkhand, 835215, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
We herein report a new synthetic route for a series of unreported 1,4-dihydropyrazolo[4,3-b]indoles (6–8) via
deoxygenation of o-nitrophenyl-substituted N-aryl pyrazoles and subsequent intramolecular (sp2)-N bond for-
mation under microwave irradiation expedite modified Cadogan condition. This method allows access to NH-free
as well as N-substituted fused indoles. DFT study and controlled experiments highlighted the role of nitrene
insertion as one of the plausible reaction mechanisms. Furthermore, the target compounds exhibited cytotoxicity
at low micromolar concentration against lung (A549), colon (HCT-116), and breast (MDA-MB-231, and MCF-7)
cancer cell lines, induced the ROS generation and altered the mitochondrial membrane potential of highly
aggressive MDA-MB-231 cells. Further investigations revealed that these compounds were selective Topo I (6h)
or Topo II (7a, 7b) inhibitors.
1,4-Dihydropyrazolo[4,3-b]indoles
–
C
N bond formation
Cadogan Cyclization
DFT study
Cancer
Topoisomerase inhibitors
1. Introduction
[12] indazoles,[13] and indoles[14] are gaining significant importance
as they involve the direct conversion of a nitro group into reactive ni-
–
C
N bond formation [1–3] offers opportunities to construct diverse
trogen species. The Cadogan/Cadogan-Sundberg cyclization[15–16] is
one of the well-studied reductive cyclization reactions for synthesizing
these skeletons, and the literature is witnessed on their advancements.
[1]
N-heterocycles with broader applications in pharmaceuticals,[4] su-
pramolecular chemistry,[5] crop protecting agents,[6] etc. The majority
–
of the C N bond formation methods such as Buchwald-Hartwig ami-
nation reaction,[7] Ugi reaction,[8] and Eschweiler-Clarke methylation
reaction[9] either utilize amines, imines or amides as one of the starting
materials (nitrogen source) in the presence or absence of metal and/or
ligand to access heterocyclic skeletons. The admittance to molecular
diversity has amplified inspiring enthusiasm among synthetic chemists
from the past decade because of its vivacious role in drug discovery. It is,
therefore, of high importance to introduce efficient methods to synthe-
size new compounds of pharmaceutical interests. Reactions involving
intramolecular reductive cyclization of o-functionalized nitroarenes
using a reductant to construct heterocycles[10–11] such as carbazoles,
Cancer has been recognized as one of the major diseases which is
causing mortality worldwide, thus remains a major health problem to
resolve. From the last five years, almost 29% of the drugs have been
approved by the US to treat various types of cancers.[17] Nowadays, to
combat cancer, the anticancer treatment approach has been advanced to
target-based drug discovery[18] like targeting topoisomerases (Topos),
[19] epidermal growth factor receptor,[20] HDACs,[21] etc. as their
overexpression mediate growth of a variety of cancers, especially lung,
breast, ovary, colon, etc. Among these targets, topoisomerases, due to
their role in maintaining the topology of DNA, are identified as one of
Abbreviations: CPT, Camptothecin; IC50, Inhibitory Concentration at half-maximal; Topo, Topoisomerase; MTT, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium Bromide; ROS, Reactive Oxygen Species; MMP, Mitochondrial Membrane Potential.
* Corresponding authors.
E-mail addresses: sandeepsingh82@cup.edu.in, sandeepsingh82@gmail.com (S. Singh), raj.khunger@gmail.com, rajcps@cup.edu.in (R. Kumar).
https://doi.org/10.1016/j.bioorg.2021.105114
Received 27 April 2021; Received in revised form 17 June 2021; Accepted 18 June 2021
Available online 29 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

M. Kaur et al.
Bioorganic Chemistry 114 (2021) 105114
the most important cellular targets for clinically potent anticancer
agents.[22]
phosphorus) and with or without an electrophile (BnCl) under micro-
wave irradiations (Scheme 1d) along with DFT studies and their anti-
cancer assessment.
Indoles are attractive molecules due to their ubiquity in nature.
[5,23–24] Heteroaryl-substituted or fused-indoles epitomize privileged
architectural units frequently found in pharmaceuticals and bioactive
molecules.[25] Sunitinib, indomethacin, pindolol, physostigmine,
delavirdine, metralindole, vincristine, and sumatriptan are some of the
FDA approved indole containing drugs for the treatment of cancer,
cardiovascular and neurologic disorders (Fig. 1).[26]
2. Results and discussions
2.1. Chemistry
2.1.1. Optimisation of reaction conditions
Recently carbazoles, pyrazolocarbazoles and N-acetyl pyrazolines
(Fig. 2) have emerged as potent anticancer agents via topoisomerase
inhibition as one of their primary targets.[27–29] Considering the
structural scaffolds of the compounds as mentioned above, we designed
new 1,4-dihydropyrazolo[4,3-b]indoles as Topo I/II inhibitors based on
merging of active pharmacophores and their preliminary docking (6 h; a
representative compound) into the proteins, which rationalized their
proposed inhibitory activity.
To begin with, nitro-substituted precursors (5a-5j) were synthesized
(Scheme S1; see supplementary material file) as per the methodology
reported by our research group.[33] To determine the standard reaction
conditions for the synthesis of target compounds, 5d was chosen as the
model substrate. The standard Cadogan reaction conditions were
selected that employ reductant, i.e. PPh3 (3 equiv; method A) or P(OEt)3
(3 equiv; method B) to carry out exhaustive deoxygenation of 5d (1
equiv) in different solvents at high temperature (Table 1).
It was surprising to know that only a few synthetic methods to access
pyrazolo[4,3-b] or [3,4-b]indole derivatives have been developed dur-
ing the past decade (Scheme 1a-c). These involve either intramolecular
palladium-catalyzed Heck-type heteroarylation of o-bromoanilinopyr-
azoles (Scheme 1a),[30] gold-catalyzed three-component annulation
reaction of alkynes, hydrazines and ketone/aldehydes (Scheme 1b),[31]
or a three-step one-pot process consisting of the iodination of indole-2-
carbaldehyde under basic conditions, followed by hydrazone formation
and intramolecular cross-coupling using copper iodide (Scheme 1c).[32]
Besides, none of the methods offers an inexpensive methodology with
diverse substitutions at N-1, C-3 and N-4 positions of 1,4-dihydropyr-
azolo[4,3-b]indoles.
The initial screening revealed that 6d was solely obtained (61%;
entry 3, Table 1) under method A in carrying out the reaction of 5d in
toluene at 180 ◦C for 20 min under MW, whereas method B yielded 6d
(52%) along with 7d (18%) when 5d was heated under MW in toluene at
210 ◦C for 30 min (entry 12, Table 1). Reactions under solvent-free
conditions failed to afford the desired products in method A or B (en-
tries 1, 9 and 10, Table 1). Microwave heating of reaction[34] was found
to accelerate the reaction rate and increase the yield in both the methods
(compare entry 2 with 3; compare entry 11 with 12, Table 1) except
under neat conditions. Further, non-polar solvent, particularly toluene,
emerged as the best choice for both the methods amidst solvents such as
1,4-dioxane, DMF, CH3CN, MeOH and water. Unexpectedly, N-ethoxy
product (9d) was not obtained with method B, which is generally
observed along with NH product in P(OEt)3-mediated reductive cycli-
zation.[1] Interestingly possible formation of compounds with dibenzo
[b,f]pyrazolo[1,5-d][1,4]diazepine ring system (10d-12d, Table 1) was
We report herein the synthesis of unreported 1,4-dihydropyrazolo
[4,3-b] indoles (6) and their derivatives (7 and 8) via intramolecular
reductive cyclization of 5-(o-nitroaryl)-1,3-diaryl-1H-pyrazole (5) under
modified-Cadogan conditions in the presence of a reductant (trivalent
Fig. 1. Indole based FDA approved drugs.
2

M. Kaur et al.
Bioorganic Chemistry 114 (2021) 105114
Fig. 2. Design of target compounds (6–8) as topoisomerase I/II inhibitors.
Scheme 1. Synthetic approaches for pyrazolo[4,3-b] or [3,4-b]indoles.
3

M. Kaur et al.
Bioorganic Chemistry 114 (2021) 105114
Table 1
Optimization of reaction conditionsa.
Entry
Solvent
Method
Reaction Conditions
Yield (%)b
6d
Tracec
22
61d
49
17
15
11
–
7d
1
–
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
B
B
180 ◦C, 24 h
–
2
Toluene
Toluene
1,4-Dioxane
DMF
Reflux, 24 h
–
3
MW, 180 ◦C, 20 min
MW, 180 ◦C, 20 min
MW, 180 ◦C, 20 min
MW, 180 ◦C, 20 min
MW, 180 ◦C, 20 min
MW, 180 ◦C, 20 min
Reflux, 24 h
–
4
–
5
–
6
CH3CN
MeOH
H2O
–
7
–
8
–
9
–
–
3
Trace
10
11
12
13
14
15
16
17
MW, 210 ◦C, 30 min
Reflux, 24 h
8
3
Toluene
Toluene
1,4-Dioxane
DMF
16
52d
29
19
11
7
9
MW, 210 ◦C, 30 min
MW, 210 ◦C, 30 min
MW, 210 ◦C, 30 min
MW, 210 ◦C, 30 min
MW, 210 ◦C, 30 min
MW, 210 ◦C, 30 min
18d
10
9
CH3CN
MeOH
H2O
6
2
–
–
a
Substrate 5d (1 mmol, 1 equiv) was treated either with PPh3 (3 equiv; method A) or P(OEt)3 (3 equiv; method B), bIsolated yield, creaction under MW (sealed tube)
at 180 ◦C did not improve the yield. dIncrease in time upto 2 h did not have much effect on the % yield.
also not detected.
limiting step for the formation of the product (6k). Two products are
possible from 5kb_C2 along two different pathways (Pathway A and
Pathway B; Fig. 3). Pathway A leads to the formation of N-OH (6kb)
product, which is exergonic by 9.15 kcal/mol with an activation barrier
of 30.39 kcal/mol, whereas Pathway B would lead to the formation of
To further explore the scope and limitations of reductive cyclization,
nitro-substituted precursors were treated using method A or B under
optimized conditions (Scheme 2a) to afford the target compounds (6 and
7). The reaction was found to be compatible with diverse functional
groups on both the phenyl rings. For unknown reasons, corresponding
products 6i and 6j of 5i and 5j were not obtained in method B.
Furthermore, the methodology was found to help construct N4
alkylated pyrazolo[4,3-b]indole in situ. For instance, when a mixture of
5d (1 equiv), PPh3 (3 equiv) and benzyl chloride (1.5 equiv) was heated
under MW irradiations (sealed tube) at 180 ◦C for 20 min, it resulted in
the formation of N4-benzylated product (8d; 61%; Scheme 2b). This
further extends the scope of methodology.
–
N
H (6k) product, which is exergonic by 78.85 kcal/mol with an
activation barrier of 14.91 kcal/mol. The energetics of the two pathways
and the experimental results motivated us to explore Pathway B
(Fig. 3B), which involves the nitrene formation and its insertion to give
the product. The formation of nitrene intermediate 5kc from 5kb_C2 is
exergonic by 5.72 kcal/mol and hence is a thermodynamically favour-
able process.
The cyclization process involves the insertion of nitrene into the
–
pyrazole C H bond, resulting in the formation of a cyclized product
2.1.2. Mechanism of reaction (DFT study)
which is exergonic by 69.05 kcal/mol. This insertion process involves an
activation barrier of 14.91 kcal/mol. The role of triethyl phosphate of
To understand the mechanism of reaction and formation of 6 and 7
under Cadogan conditions particularly using P(OEt)3 as reductant,
quantum chemical studies on 5k (R1 = R2 = R3 = R4 = R5 = R6 = H; a
model substrate) were performed using Density Functional B3LYP and
6–31 + G(d) basis set (see Supplementary material).[35–37] The overall
cyclization process was found to be thermodynamically favorable
(Fig. 3) and exergonic by 70.59 kcal/mol, which can be seen from the
free energy profile diagram (Fig. 4). The formation of products firstly
involves the nucleophilic attack of the lone pair of P(OEt)3 on the oxygen
atom of the nitro of 5 k followed by removal of PO(OEt)3, resulting in the
formation of nitroso intermediate 5 ka.[16,38–42] Further, 5ka changes
into product 6 k via nitrene pathway, which involves deoxygenation of
nitroso intermediate by P(OEt)3 followed by the release of PO(OEt)3 and
formation of nitrene intermediate 5 kb (Fig. 4).
–
5kb is to activate the pyrazole C H bond, facilitating the nitrene
–
insertion into the C H bond of the pyrazole ring. Hence, triethyl
phosphate here is analogous to iron which has been found to catalyze a
–
similar kind of C H activation followed by nitrene insertion and
cyclization.[43].
Further, detection of a phosphorimidate byproduct in LC-MS (15d;
Scheme S2) along with 6d, 7d and absence of N-OEt[44] product (9d)
during a controlled experiment of 5d with in excess of P(OEt)3
confirmed that present reaction occurs possibly via a nitrene mechanism
and not by [13,16] non-nitrene pathway. The formation of N-substituted
products like N-ethyl (ΔG = -1.71, Ea = 68.39) (7) and N-benzyl (ΔG =
6.08, Ea = 55.10) (8) can be justified as the products of nucleophilic
substitution reactions of (6k) with the P(OEt)3 and benzyl chloride,
respectively under given reaction conditions. All the final products were
new and fully characterized (mp, NMR, HRMS and X-ray; see SI).
The conformational analysis of 5kb revealed the possibility of
another conformer, 5kbꢀ C2. The formation of 5kb_C2 has been found
to be endergonic by 4.18 kcal/mol (Fig. 4). The activation barrier
–
required for the formation of the O P bond in 5kb_C2 is 70.55 kcal/
mol. The quantum chemical studies suggest the activation energy
–
needed for the formation of the O P bond in 5kb_C2 can serve as a rate-
4

M. Kaur et al.
Bioorganic Chemistry 114 (2021) 105114
Scheme 2. a and b. Synthesis of target compounds.
2.2. Biology
Compounds 6h, 7a, and 7b were tested for Topo I mediated DNA
relaxation assay to assess Topo I inhibitory potential. The results for
Topo I inhibition assay showed that these chosen compounds exerted
Topo I inhibitory activity. In the presence of Topo I inhibitor, the
enzyme is unable to remove the supercoils. The inhibition of relaxation
was measured by densitometry showing that compound 6h showed
better Topo I inhibition compared to camptothecin (Fig. 6A; Lane 7). 7a
(Lane 5) and 7b (Lane 6) also displayed inhibition of Topo I mediated
relaxation of supercoiled DNA though they were less effective than
camptothecin (Fig. 6B).
2.2.1. Evaluation of anticancer activity, alteration in mitochondrial
membrane potential and redox parameter, and topoisomerase inhibition by
the target compounds
All the target compounds were tested for cytotoxicity potential
against A549 (lung), HCT-116 (colon), and MDA-MB-231 (breast), and
MCF-7 (breast) cancer cell lines. All the compounds showed good anti-
proliferative activities (Table 2) with IC50 values in the low micromolar
range. The results were then represented from three independent ex-
periments with the absorbance of the formazan formed in control (media
only) cells taken as 100% viability, and the results of the MTT assay were
plotted in graphical form (Fig. 5). Camptothecin (CPT) and etoposide
were taken as reference drugs. Three different concentrations (1 µM, 5
µM, 25 µM) of the compounds were used, and treatments were given for
24 h. Although all the tested compounds exhibited low µM IC50s, 7a, 7b,
and 6h emerged as broad-spectrum antiproliferative compounds (IC50
range 0.58ꢀ 2.41 µM; Table 2 and Fig. 5) comparable to standard drugs.
Further, to determine the toxicity of these lead compounds on normal
cells, we isolated human peripheral blood mononuclear cells (hPBMCs),
and an MTT assay was performed. The results did not show any signif-
icant toxicity towards hPBMC tested at 10 and 25 µM concentration of
the compounds for 48 h (Figure S2). After that, compounds were
examined further for their various other impacts on cancer cells.
Furthermore, for the Topo II inhibition assay, kDNA was used as a
substrate for Topo II. Gel image shows that in the presence of Topo II,
kDNA gets decatenated. However, in the presence of a Topo II inhibitor
like etoposide, the decatenation gets reduced (Fig. 6 C; Lane 3). Inter-
estingly, compounds 7a and 7b emerged as better Topo II inhibitors than
etoposide. In contrast to these, compound 6h (Lane 6) displayed weak
Topo II inhibitory activity, and thus, we deduce that compound 7a (Lane
4) and 7b (Lane 5) are strong Topo II inhibitors (Fig. 6 D). Furthermore,
topoisomerase binding assays revealed that 6h inhibited Topo I,
whereas 7a and 7b inhibited Topo II to a greater extent than the stan-
dard drugs.
This observation was further supported by in silico studies
(Figure S3), which revealed some critical interactions (Fig. 6E and F),
such as when 7a docked at the Topo I binding site, it intercalates with
5

M. Kaur et al.
Bioorganic Chemistry 114 (2021) 105114
Fig. 3. A plausible mechanism of the reaction.
DNA and interacts with DG-12, DC-111, DC-112 residues. In addition,
pyrazole ring nitrogen of 7a showed hydrogen bonding with ARG364,
and the aromatic hydrogens of two phenyl rings showed weak hydrogen
bonding with ASP533. Furthermore, the docking of 7a into Topo II
alter mitochondrial membrane potential (MMP) and ROS level in MDA-
MB-231 cells. Interestingly, the studies revealed that these compounds
modulated the MMP as indicated by an increase in the Red Green ratio
leading to hyperpolarization of the membrane, as shown in Fig. 7.
Additionally, the intracellular ROS was measured by treating the
washed cells with H2DCFDA (2′, 7′–dichlorofluorescein diacetate) for
30 min at 37˚C and then analyzed using a flow cytometer. This investi-
gation revealed that compounds 7a, 7b, and 6h induced oxidative stress,
as evidenced by an upsurge in ROS generation compared to the control
(Fig. 8).
disclosed that chlorine substituted phenyl ring displayed π-cation in-
teractions with ARG98, weak hydrogen bonding with SER149. Inter-
estingly, the 1,4-dihydropyrazolo[4,3-b]indole nucleus interacted with
conserved Walker A motif lined by ARG162, ASN163, GLY164, TYR165,
GLY166 and ALA167, responsible for Topo II inhibition.
Further, the tested compounds were investigated for their ability to
6

M. Kaur et al.
Bioorganic Chemistry 114 (2021) 105114
Fig. 4. Potential Energy Surface Diagram representing the cyclization process via nitrene pathway.
Therefore, from the biological evaluation, the investigation provided
4. Experimental section
significant findings in the context of the anti-cancer potential of the
synthesized compounds. Interestingly, all the compounds exhibited
antiproliferative activity at a low micro-molar range, among which 6h,
7a and 7b were the most effective compounds. The further elucidation
of the anticancer mechanism revealed that 6h inhibited topoisomerase I
while 7a and 7b were potent topoisomerase II inhibitors. It is found from
the literature that the cancer cells usually have fine-tuned ROS ho-
meostasis, which allows them to operate pro-tumorigenic signaling
without undergoing ROS induced cell death[45], and the cancer treat-
ments like chemotherapy rely on generating ROS levels which activate
intrinsic apoptosis[46]. Thus, to know about the mechanism of cell
death, the compounds were further investigated, and it was found that
our compounds were also able to induce cell death via ROS burst and
hyperpolarisation of the mitochondrial membrane.
4.1. Chemistry
General Methods and Materials. Biotage® Initiator microwave
synthesizer (Company: Biotage® Model No. 355,301 (Initiator EXPEU))
(used for sealed reactions) and Discover System; Company: CEM; Model
No. 908010; Serial No. DU9671) (used for open reflux reactions) were
used for carrying out microwave reactions at 200 W power. 1H NMR and
13C NMR spectra were obtained in CDCl3/d6-DMSO on 400/500 MHz
and 100/125 MHz Bruker Advance II NMR spectrometer/Jeol, respec-
tively TMS (δ = 0) as an internal standard. Data were reported as fol-
lows: Chemical shifts (δ) are reported in ppm, coupling constants (J) are
in Hertz (Hz). Abbreviations used to explain the multiplicities: s =
singlet, d = doublet, t = triplet, q = quartet, m = multiplet. Column
chromatography was performed with silica gel (60–120 and 100–200
mesh ASTM) to purify compounds. Melting points were measured with a
melting point instrument and were uncorrected.
3. Conclusions
To encapsulate, we have described a new synthetic protocol for the
construction of undocumented 1,4-dihydropyrazolo[4,3-b]indoles
involving intramolecular C(sp2)-N bond formation under modified
Cadogan reaction. DFT calculations rationalized the formation of
products via the nitrene mechanism. The biological studies revealed that
7a, 7b, and 6h were cytotoxic towards cancer cells, induced ROS gen-
eration and altered the mitochondrial membrane potential of highly
aggressive MDA-MB-231 cells. Further investigations revealed that these
compounds were selective Topo I (6h) or Topo II (7a, 7b) inhibitors.
Thus, the work offers the opportunity to synthesize products with sub-
stitution at N-1, C-3 and N-4 positions of 1,4-dihydropyrazolo[4,3-b]
indoles required for further structure–activity relationship (SAR) study
for pharmaceuticals as well as supramolecular chemistry perspective.
All the reagents were purchased from commercial suppliers and used
without further purification. Reactions were monitored by TLC (detec-
tion with UV light).
General procedure for the synthesis of nitro precursors. (a) To a
methanol solution containing acetophenones 1 (1.0 equiv) and 2-nitro-
benzaldehyde 2 (1 equiv) was added NaOH (10%). The reaction mixture
was stirred at room temperature for 2–3 h. After the completion of the
reaction (TLC), the reaction mixture was poured into the water and
filtered. The solid compounds (3) were washed with methanol and
dried.
Analytical data of reference compound, 3a (precursor of 4a).
(E)-1-(4-chlorophenyl)-3-(2-nitrophenyl)prop-2-en-1-one (3a). Cream
yellow solid; 75% yield; 1H NMR (500 MHz, DMSO‑d6): δ 8.22 (d, J =
7.8 Hz, 1H), 8.13 – 8.12 (m, 3H), 8.04 – 8.00 (m, 1H), 7.92 – 7.82 (m,
4H), 7.75–7.72 (m, 1H); 13C NMR (125 MHz, DMSO‑d6) δ 188.75,
7

M. Kaur et al.
Bioorganic Chemistry 114 (2021) 105114
(5a). Yellow solid; 89% yield; 1H NMR (400 MHz, DMSO‑d6): δ 8.07 (d,
J = 8.1 Hz, 1H), 7.97 (d, J = 8.5 Hz, 2H), 7.84 (t, J = 7.5 Hz, 1H), 7.74 (t,
J = 7.3 Hz, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.54 (d, J = 8.5 Hz, 2H),
7.40–7.33 (m, 3H), 7.26 (d, J = 7.2 Hz, 2H), 7.20 (s, 1H), 13C NMR (100
MHz, DMSO‑d6): δ 150.40, 148.63, 140.12, 139.23, 134.36, 133.31,
133.18, 131.75, 131.42, 129.66, 129.36, 128.28, 127.57, 125.23,
125.11, 124.56, 106.65; MS (ESI): m/z = 375.8
Table 2
IC50 values of synthesized compounds against various cancer cell lines.
Antiproliferative potential IC50 (µM)a
Compound Code
MDA-MB-231
(Breast)
MCF7
A549
(Lung)
3.35 ±
0.32
HCT116
(Colon)
(Breast)
2.6 ± 0.12
6a
2.55 ± 0.21
2.8 ± 0.26
6b
6c
3.22 ± 0.11
3.41 ± 0.51
2.3 ± 0.23
2.97 ± 0.32
2.65 ± 0.24
3.39 ± 0.18
2.6 ± 0.13
3.1 ± 0.69
2.89 ± 0.31
1.69 ± 0.26
1.29 ± 0.28
0.58 ± 0.13
3.28 ± 0.47
3.35 ± 0.41
2.53 ± 0.38
2.78 ± 0.36
2.84 ± 0.47
3.1 ± 0.24
3.25 ± 0.46
2.49 ± 0.23
3.47 ± 0.41
0.97 ± 0.27
2.84 ±
0.47
1.84 ± 0.1
2.7 ± 0.36
3.01 ± 0.3
2.87 ± 0.36
6.47 ± 0.11
4.14 ± 0.4
1.69 ± 0.2
1.47 ± 0.42
2.41 ± 0.44
2.99 ± 0.11
3.01 ± 0.27
3.01 ± 0.21
2.97 ± 0.39
3.03 ± 0.19
2.62 ± 0.17
3.76 ± 0.26
3.1 ± 0.32
3.58 ± 0.28
0.95 ± 0.3
General Procedure for the synthesis of 1,4-Dihydropyrazolo
[4,3-b]indoles (6 and 7). To an oven-dried Microwave vial was added
pyrazole-based nitro compound 5 (1 mmol, 1 equiv), PPh3/P(OEt)3 (3
equiv), and toluene (5 mL). The mixture was stirred at 180/210 ◦C
(method A and B) in the CEM (sealed tube). After TLC indicated that 5
was consumed entirely, toluene was evaporated under vacuum, and the
mixture was poured in water and extracted with ethyl acetate (25 mL).
Then the solvent was evaporated under vacuum. Finally, the crude
products were purified using flash column chromatography (eluent:
Petroleum ether/Acetone) on silica gel to afford the desired products 6
and 7.
2.3 ±
0.29
6d
6e
2.78 ±
0.42
2.84 ± 0.27
2.34 ± 0.74
3.53 ± 0.42
1.58 ± 0.56
0.99 ± 0.24
0.84 ± 0.19
2.38 ± 0.21
2.49 ± 0.25
1.51 ± 0.29
2.34 ± 0.15
3.01 ± 0.14
2.43 ± 0.41
3.1 ± 0.1
3.56 ±
0.3
6f
4.72 ±
0.37
6g
4.67 ±
0.59
6h
7a
1.87 ±
0.47
1.21 ±
0.50
Analytical data of products (6 and 7)
3-(4-chlorophenyl)-1-phenyl-1,4-dihydropyrazolo[4,3-b]indole (6a)
Compound 6a was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 19:1) afforded 6a (60%) as a cream solid; mp:
191–193 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 11.58 (s, 1H), 8.09 (d, J =
8.5 Hz, 2H), 7.91 (d, J = 7.8 Hz, 2H), 7.85 (d, J = 8.0 Hz, 1H), 7.65 –
7.53 (m, 5H), 7.40–7.33 (m, 2H), 7.13 (t, J = 7.5 Hz, 1H). 13C NMR (100
MHz, DMSO‑d6): δ 144.98, 140.76, 132.93, 132.67, 132.12, 131.54,
130.36, 130.26, 129.51, 127.73, 126.88, 125.35, 120.80, 119.51,
119.50, 113.70, 112.75; HRMS: for C21H14ClN3, Exact mass: 343.0876;
observed [M + H]+: 344.0952.
7b
7c
1.63 ±
0.24
3.43 ±
0.51
7d
7e
3.24 ±
0.31
2.24 ±
0.45
7f
3.24 ±
0.29
7g
2.24 ±
0.16
7i
3.28 ±
0.21
3-(4-bromophenyl)-1-phenyl-1,4-dihydropyrazolo[4,3-b]indole (6b)
Compound 6b was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 19:1) afforded 6b (57%) as a yellow white solid; mp:
212–214 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 11.58 (s, 1H), 8.02 (d, J =
8.5 Hz, 2H), 7.91 (d, J = 7.8 Hz, 2H), 7.85 (d, J = 8.0 Hz, 1H), 7.71 (d, J
= 8.4 Hz, 2H), 7.63 (t, J = 7.8 Hz, 2H), 7.54 (d, J = 8.3 Hz, 1H),
7.40–7.33 (m, 2H), 7.13 (t, J = 7.5 Hz, 1H); 13C NMR (100 MHz,
DMSO‑d6): δ 144.97, 140.73, 132.97, 132.39, 132.14, 131.86, 130.36,
130.24, 128.00, 126.91, 125.37, 121.23, 120.80, 119.54, 119.48,
113.70, 112.73; HRMS: for C21H14BrN3, Exact mass: 387.0371; observed
[M + H]+: 388.0436.
7j
7.46 ±
0.16
8d
Etoposide
3.22 ± 0.62
2.05 ± 0.33
0.21 ± 0.11
3.41 ±
0.21
3.14 ±
0.29
Camptothecin
(CPT)
1.65 ±
0.37
a
Data was represented as mean ± S.E. from three independent experiments.;
Camptothecin and Etoposide were taken as positive controls.
149.30, 139.51, 136.54, 134.27, 132.45, 131.68, 131.23, 130.14,
130.04, 128.24, 126.56, 125.23; MS (ESI): m/z = 287.7
1-phenyl-3-(p-tolyl)-1,4-dihydropyrazolo[4,3-b]indole (6c)
Compound 6c was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 19:1) afforded 6c (56%) as a light yellow solid; mp:
218–220 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 11.50 (s, 1H), 7.97 (d, J =
8.1 Hz, 2H), 7.90 (d, J = 7.9 Hz, 2H), 7.85 (d, J = 8.0 Hz, 1H), 7.62 (t, J
= 7.9 Hz, 2H), 7.54 (d, J = 8.3 Hz, 1H), 7.31–7.38 (m, 4H), 7.12 (t, J =
7.5 Hz, 1H), 2.35 (s, 3H); 13C NMR (100 MHz, DMSO‑d6): δ 144.92,
140.92, 137.66, 134.22, 131.84, 130.31, 130.03, 129.85, 126.55,
126.04, 125.13, 122.50, 120.61, 119.45, 119.37, 113.68, 112.81, 21.46;
HRMS: for C22H17N3, Exact Mass: 323.1422; observed [M + H]+:
324.1485.
(b) Further, 3 (1 mmol) were mixed with various substituted phenyl
hydrazines (2–3 mmol) in methanol and refluxed for 3 h. The reaction
was monitored by TLC, and the solvent was evaporated under vacuum to
get 4.
Analytical data of reference compound, 4a (precursor of 5a). 3-
(4-chlorophenyl)-5-(2-nitrophenyl)-1-phenyl-4,5-dihydro-1H-pyrazole
(4a). Peach solid; 93% yield; 1H NMR (500 MHz, DMSO‑d6): δ 8.16 (d, J
= 8.1 Hz, 1H), 7.77 (d, J = 8.5 Hz, 2H), 7.66 (t, J = 7.6 Hz, 1H), 7.56 (t,
J = 7.7 Hz, 1H), 7.50 (m, 2H), 7.26 (t, J = 11.2 Hz, 1H), 7.18 (t, J = 7.9
Hz, 2H), 6.96 (d, J = 8.1 Hz, 2H), 6.76 (t, J = 7.3 Hz, 1H), 5.95–5.91 (m,
1H), 4.08 (m, 1H), 3.32 – 3.29 (m, 1H); 13C NMR (125 MHz, DMSO‑d6) δ
147.85, 147.12, 143.87, 136.72, 134.94, 133.79, 131.42, 129.57,
129.53, 129.18, 128.08, 127.96, 125.93, 119.60, 113.21, 60.53, 42.82;
MS (ESI): m/z = 377.8
3-(4-methoxyphenyl)-1-phenyl-1,4-dihydropyrazolo[4,3-b]indole
(6d)
Compound 6d was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 19:1) afforded 6d (61%) as a cream solid; mp:
221–223 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 11.48 (s, 1H), 8.00 (d, J =
8.6 Hz, 2H), 7.90–7.84 (m, 3H), 7.61 (t, J = 7.0 Hz, 2H), 7.53 (d, J = 8.4
Hz, 1H), 7.37–7.31 (m, 2H), 7.14 – 7.06 (m, 3H), 3.81 (s, 3H); 13C NMR
(100 MHz, DMSO‑d6): δ 159.53, 144.91, 140.96, 134.16, 131.79,
130.30, 130.11, 127.51, 126.41, 125.22, 125.09, 120.52, 119.47,
119.33, 114.89, 113.67, 112.85, 55.77; HRMS: for C22H17N3O, Exact
Mass: 339.1372; observed [M + H]+: 340.1452.
(c) Catalytic amount of molecular iodine was added to 4 in DMSO
and refluxed for 4 h. TLC was done for reaction confirmation. The
mixture was poured in ice cold water and ethyl acetate was used for
extraction of 5. The organic layer was washed with sodium thiosulphate
solution, so that the traces of iodine get removed. Then the organic layer
was filtered via drying it over sodium sulphate. The filtrate was evapo-
rated under vacuum to get 5.
Analytical data of representative nitro substrate, 5a (precursors
of 6a). 3-(4-chlorophenyl)-5-(2-nitrophenyl)-1-phenyl-1H-pyrazole
8

M. Kaur et al.
Bioorganic Chemistry 114 (2021) 105114
Fig. 5. In vitro evaluation of the antiproliferative potential of the indicated compounds in different cell lines at indicated concentrations after 24 h of treatment.
Camptothecin (CPT) and etoposide were used as positive controls.
1-phenyl-3-(3,4,5-trimethoxyphenyl)-1,4-dihydropyrazolo[4,3-b]
indole (6e)
silica gel (PE/A = 19:1) afforded 6 g (54%) as a yellow solid; mp:
177–178 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 11.42 (s, 1H), 7.98–7-75
(m, 3H), 7.74 – 7.63 (m, 2H), 7.49 (d, J = 8.3 Hz, 1H), 7.34 – 7.26 (m,
2H), 7.07 – 7.0 (m, 3H), 3.80 (s, 3H). 13C NMR (100 MHz, DMSO‑d6) δ
159.60, 144.76, 137.53, 134.80, 134.51, 133.79, 130.77, 130.21,
129.18, 129.00, 127.58, 125.10, 125.02, 119.27, 119.18, 114.88,
113.47, 112.93, 55.77; HRMS: for C22H15Cl2N3O, Exact Mass: 407.0592;
observed [M + H]+: 408.0662
Compound 6e was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 19:1) afforded 6e (51%) as a cream solid; mp:
225–227 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 11.5 (s, 1H), 7.9 (m, 3H),
7.7 (m, 2H), 7.6 (m, 1H), 7.4 (m, 2H), 7.3 (m, 2H), 7.2 (m, 1H), 4 (s, 6H),
3.8 (s, 3H). 13C NMR (100 MHz, DMSO‑d6): δ 153.96, 145.01, 140.84,
139.01, 137.99, 134.23, 132.08, 130.32, 128.37, 126.70, 125.25,
122.67, 120.81, 119.46, 113.67, 112.95, 103.77, 60.70, 56.76; HRMS:
for C24H21N3O3, Exact Mass: 399.1583; observed [M + H]+: 400.1670.
1-(4-fluorophenyl)-3-(4-methoxyphenyl)-1,4-dihydropyrazolo[4,3-
b]indole (6f)
3-(2,4-dimethoxyphenyl)-1-phenyl-1,4-dihydropyrazolo[4,3-b]
indole (6h)
Compound 6 h was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 19:1) afforded 6 h (52%) as a light yellowish solid;
mp: 158–160 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 10.35 (s, 1H), 7.88 (d,
J = 7.9 Hz, 2H), 7.82 (d, J = 8.5 Hz, 2H), 7.62–7.56 (m, 3H), 7.36–7.27
(m, 2H), 7.07 (t, J = 7.6 Hz, 1H), 6.72 (d, J = 2.2 Hz, 1H), 6.66–6.63 (m,
1H), 3.94 (s, 3H), 3.81 (s, 3H). 13C NMR (100 MHz, DMSO‑d6): δ 161.31,
158.08, 144.06, 140.96, 132.14, 131.68, 130.84, 130.25, 129.90,
126.29, 124.71, 120.68, 119.22, 118.82, 114.26, 113.78, 112.50,
106.18, 99.05, 56.66, 55.90; HRMS: for C23H19N3O2, Exact Mass:
369.1477; observed [M + H]+: 370.1558.
Compound 6f was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 19:1) afforded 6f (59%) as a light yellowish solid; mp:
175–176 ◦C; 1H NMR (400 MHz, DMSO‑d6) δ 11.44 (s, 1H), 7.99 (d, J =
8.3 Hz, 2H), 7.92–7.89 (m, 2H), 7.81 (d, J = 8.0 Hz, 1H), 7.53 (d, J = 8.3
Hz, 1H), 7.45 (t, J = 8.7 Hz, 2H), 7.35–7.31 (m, 1H), 7.13 – 7.06 (m,
3H), 3.81 (s, 3H). 13C NMR (100 MHz, DMSO- d6) δ 159.54, 144.91,
137.52, 134.18, 131.88, 130.04, 127.51, 125.12, 122.55, 122.47,
119.35, 119.31, 117.21, 116.98, 114.88, 113.67, 112.71, 55.77; HRMS:
for C22H16FN3O, Exact Mass: 357.1277; observed [M + H]+: 358.1371.
1-(2,4-dichlorophenyl)-3-(4-methoxyphenyl)-1,4-dihydropyrazolo
[4,3-b]indole (6g)
3-(4-chlorophenyl)-4-ethyl-1-phenyl-1,4-dihydropyrazolo[4,3-b]
indole (7a)
Compound 7a was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 99:1) afforded 7a (21%) as a cream solid; mp:
170–172 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 7.89 – 7.81 (m, 5H), 7.67 –
Compound 6 g was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
9

M. Kaur et al.
Bioorganic Chemistry 114 (2021) 105114
Fig. 6. A. Agarose gel image depicting the inhibition of Topo I activity B. Graph showing relative inhibition of relaxation of supercoiled DNA by compounds (100
µM). M: Marker; C: CPT. C. Gel image showing decatenation of kDNA when treated with Topo II and its inhibition by compounds (100 µM). D. Quantification of
relative decatenation showing Topo II inhibition. E. Binding pose of 7a with Topo I and F. Topo II active site residues.
7.58 (m, 5H), 7.39 (t, J = 7.7 Hz, 2H), 7.14 (t, J = 7.5 Hz, 1H), 4.33 (q, J
= 7.0 Hz, 2H), 1.07 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, DMSO‑d6): δ
144.32, 140.58, 134.24, 133.38, 131.91, 131.53, 131.06, 130.49,
130.35, 129.38, 127.11, 125.46, 121.15, 119.61, 119.42, 112.60,
111.78, 40.61, 14.98; HRMS: for C23H18ClN3, Exact Mass: 371.1189;
observed [M + H]+: 372.1277.
Compound 7b was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 99:1) afforded 7b (22%) as a cream solid; mp:
174–176 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 7.89–7.606 (m, 10H),
7.39–7.37 (m, 2H), 7.16–7.11 (m, 1H), 4.308 (m, 2H), 1.07 (m, 3H); 13
C
NMR (100 MHz, DMSO‑d6): δ 144.45, 140.58, 134.28, 132.29, 131.57,
31.03, 130.77, 130.35, 127.12, 125.48, 122.00, 121.16, 119.61, 119.43,
112.60, 111.79, 40.20, 14.98; HRMS: for C23H18BrN3, Exact Mass:
3-(4-bromophenyl)-4-ethyl-1-phenyl-1,4-dihydropyrazolo[4,3-b]
indole (7b)
10

M. Kaur et al.
Bioorganic Chemistry 114 (2021) 105114
Fig. 7. A. Measurement of alteration of Mitochondrial Membrane Potential by flow cytometer. B. Ratiometric values showing the alteration in the ratio of Red (J-
aggregates) to green (J-Monomer) fluorescence intensity.
Fig. 8. Measurement of ROS alteration by flow cytometry. Compared to control, all the selected compounds could enhance ROS production at IC50 values in MDA-
MB-231 cells.
415.0684; observed [M + H]+: 416.0760.
silica gel (PE/A = 99:1) afforded 7e (20%) as a cream yellow; mp:
138–140 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 8 (d, J = 8.7 Hz, 1H),
7.93–7.87 (m, 2H), 7.74(d, J = 8.8 Hz, 1H), 7.69–7.64 (m, 2H),
7.44–7.42 (m, 2H), 7.17 (m, 1H), 7.09 (d, J = 4 Hz, 2H), 4.42–4.40 (m,
2H), 3.8 (s, 6H), 3.75 (s, 3H), 1.25–1.20 (m, 3H); 13C NMR (100 MHz,
DMSO‑d6): δ 153.60, 144.13, 140.69, 139.03, 135.55, 131.21, 130.33,
128.58, 126.93, 125.32, 122.97, 121.12, 119.59, 119.29, 112.55,
111.73, 106.07, 60.69, 56.51, 40.62, 15.12; HRMS: for C26H25N3O3,
Exact Mass: 427.1896; observed [M + H]+: 428.1967.
4-ethyl-1-phenyl-3-(p-tolyl)-1,4-dihydropyrazolo[4,3-b]indole (7c)
Compound 7c was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 99:1) afforded 7c (21%) as a cream solid; mp:
171–173 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 7.88–7.83 (m, 3H), 7.69 –
7.59 (m, 5H), 7.39 – 7.32 (m, 4H), 7.13 (t, J = 7.5 Hz, 1H), 4.35–4.30
(m, 2H), 2.37 (s, 3H)1.06 (t, J = 7.0 Hz, 3H); 13C NMR (100 MHz,
DMSO‑d6): δ 144.22, 140.73, 138.05, 135.57, 131.25, 131.19, 130.31,
130.14, 129.87, 128.72, 126.83, 125.28, 120.99, 119.60, 119.29,
112.69, 111.75, 40.63, 21.44, 14.99; HRMS: for C24H21N3, Exact Mass:
351.1735; observed [M + H]+: 352.1831.
4-ethyl-1-(4-fluorophenyl)-3-(4-methoxyphenyl)-1,4-dihydropyr-
azolo[4,3-b]indole (7f)
Compound 7f was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 99:1) afforded 7f (22%) as a cream solid; mp:
158–159 ◦C; 1H NMR (400 MHz, DMSO‑d6) δ 7.90–7.87 (m, 2H), 7.80 (d,
J = 8.1 Hz, 1H), 7.71 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.4 Hz, 1H), 7.44
(t, J = 8.7 Hz, 3H), 7.14 – 7.07 (m, 3H), 4.34–4.29 (m, J = 7.0 Hz, 2H),
3.81 (s, 3H), 1.07 (m, 3H). 13C NMR (100 MHz, DMSO‑d6) δ 161.91,
159.78, 144.20, 137.32, 135.43, 131.28, 131.07, 130.10, 125.25,
122.99, 122.91, 119.50, 119.25, 117.22, 116.99, 114.71, 112.57,
111.72, 55.73, 40.65, 15.00.
4-ethyl-3-(4-methoxyphenyl)-1-phenyl-1,4-dihydropyrazolo[4,3-b]
indole (7d)
Compound 7d was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 99:1) afforded 7d (18%) as a cream solid; mp:
152–154 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 7.88 – 7.83 (m, 3H), 7.74 –
7.70 (m, 2H), 7.64–7.59 (m, 3H), 7.40–7.35 (m, 2H), 7.15 – 7.07 (m,
3H), 4.35–4.30 (m, 2H), 3.81 (s, 3H), 1.07 (t, J = 7.1 Hz, 3H); 13C NMR
(100 MHz, DMSO‑d6): δ 159.76, 144.18, 140.76, 135.43, 131.16,
130.30, 130.12, 126.73, 125.31, 125.24, 120.91, 119.61, 119.26,
114.70, 112.70, 111.72, 55.73, 40.67, 15.01; HRMS: for C24H21N3O,
Exact Mass: 367.1685; observed [M + H]+: 368.1761.
1-(2,4-dichlorophenyl)-4-ethyl-3-(4-methoxyphenyl)-1,4-dihy-
dropyrazolo[4,3-b]indole (7 g)
Compound 7 g was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 99:1) afforded 7 g (22%) as a cream solid; mp:
124–125 ◦C; 1H NMR (400 MHz, DMSO‑d6) δ 7.94 (d, J = 2.2 Hz, 1H),
7.76 – 7.58 (m, 5H), 7.34–7.31 (m, 2H), 7.09 – 7.02 (m, 3H), 4.35–4.30
4-ethyl-1-phenyl-3-(3,4,5-trimethoxyphenyl)-1,4-dihydropyrazolo
[4,3-b]indole (7e)
Compound 7e was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
11

M. Kaur et al.
Bioorganic Chemistry 114 (2021) 105114
(m, 2H), 3.8(s, 3H), 1.12–1.08 (m, 3H). 13C NMR (100 MHz, DMSO‑d6) δ
159.81, 144.04, 137.26, 135.93, 133.96, 133.77, 130.82, 130.23,
130.08, 129.99, 129.20, 125.19, 125.14, 119.38, 119.23, 114.74,
112.65, 111.54, 55.73, 39.49, 15.20.
experimental setup.
Human peripheral blood mononuclear cells (hPBMCs) were isolated
from healthy individuals. RBCs were lysed in lysis buffer, and the
hPBMCs were cultured in RPMI media and treated accordingly with the
compounds as discussed below to assess the selectivity of investigational
compounds toward cancer cells. The assay was performed strictly as per
protocol no. CUPB/cc/14/IEC/4483 approved by Institutional Ethics
Committee of Central University of Punjab, Bathinda, and according to
the Indian Council of Medical Research (ICMR) guidelines Govt. of
India.
4-ethyl-3-(3-iodo-4-methoxyphenyl)-1-phenyl-1,4-dihydropyrazolo
[4,3-b]indole (7i)
Compound 7i was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 99:1) afforded 7i (41%) as a cream solid; mp:
173–175 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 8.16–8.18 (m, 1H), 7.94
(d, J = 8.6 Hz, 1H), 7.88 – 7.77 (m, 3H), 7.69 (d, J = 8.6 Hz, 1H), 7.64 –
7.59 (m, 2H), 7.38 (t, J = 7.0 Hz, 2H), 7.16–7.11 (m, 2H), 4.32–4.28 (m,
2H), 3.88 (s, 3H), 1.13–1.08 (m, 3H); 13C NMR (100 MHz, DMSO‑d6): δ
158.36, 144.22, 140.30, 139.01, 138.93, 134.34, 130.33, 130.15,
127.03, 125.48, 122.94, 121.06, 119.79, 119.62, 112.53, 112.11,
111.73, 91.54, 86.95, 57.06, 40.67, 15.05; HRMS: for C24H20IN3O,
Exact Mass: 493.0651; observed [M + H]+: 494.0714.
5.2. Evaluation of antiproliferative potential of synthesized compounds by
3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay
The antiproliferative potential of the investigational compounds was
assessed using MTT assay (Invitrogen) in different cells. 7 × 103 cells
were seeded in a 96-well plate. The following day, cells were serum-
starved, and synthetic compounds were treated and incubated for 24 h
following which 10 µL of MTT dye (5 mg/mL of 1X PBS) was added to
each well and incubated in CO2 atmosphere in the dark for 4 h to allow
formazan crystals formation. 100 µL DMSO (100%) was added to each
well, and absorbance was read using a microplate reader at 570 nm. %
cell viability was calculated using the formula given below, and IC50
values were calculated using ORIGIN software.
4-ethyl-3-(naphthalen-2-yl)-1-phenyl-1,4-dihydropyrazolo[4,3-b]
indole (7j)
Compound 7j was synthesized in accordance with the typical pro-
cedure discussed above. Purification by column chromatography on
silica gel (PE/A = 99:1) afforded 7j (48%) as a white solid; mp:
177–179 ◦C; 1H NMR (400 MHz, DMSO‑d6): δ 8.34 (s, 1H), 8.07–8.04
(m, 2H), 7.99 – 7.92 (m, 4H), 7.90–7.86 (m, 1H), 7.75 (d, J = 8.7 Hz,
1H), 7.69–7.62 (m, 2H), 7.58 – 7.53 (m, 2H), 7.42–7.38 (m, 2H),
7.18–7.14 (m, 1H), 4.44 – 4.41 (m, 2H), 1.10–1.05 (m, 3H). 13C NMR
(100 MHz, DMSO‑d6): δ 144.40, 140.70, 139.06, 133.51, 133.04,
131.53, 130.49, 130.36, 128.83, 128.70, 128.25, 127.60, 127.50,
127.20, 127.01, 126.79, 125.41, 122.99, 121.13, 119.64, 119.41,
112.72, 111.82, 40.62, 15.02; HRMS: for C22H21N3, Exact Mass:
388.1805; observed [M + H]+: 388.1801.
% Cell Viability = OD Test Compound/ OD Media (Control) * 100
5.3. Topoisomerase assay
Topoisomerase I (Catalogue No TG2005H-RC) and Topoisomerase II
(Catalogue No TG2000H-1) assay kits were procured from Topogen
(Topogen, Inc. Buena Vista, CO, USA). DNA relaxation and DNA deca-
tenation assays were performed as per the manufacturer’s instructions.
Negatively supercoiled DNA (SC DNA) and camptothecin (CPT) were
used as substrate and positive control. The total reaction mixture was
incubated at 37 ◦ C for 30 min and stopped by the addition of 2 µL 10%
SDS. The samples were run on 0.8% agarose gel for 1–2 h at 50 V and
then stained with the help of EtBr for 15 min followed by destaining in
water, and images were recorded in Chemi-Doc™ XRS+ (Bio-Rad). For
topoisomerase-II, kinetoplast DNA (kDNA) acted as substrate and eto-
poside, an inhibitor of Topo II, was used as a positive control as
described previously.[47]
General Procedure for the synthesis of 8d in-situ benzylation. To
an oven-dried Microwave vial was added 5d (1 mmol, 1 equiv), PPh3 (3
equiv), benzyl chloride (1.5 equiv) and toluene (5 mL). The mixture was
stirred at 180 ◦C in the CEM (sealed tube) for 20 min. After TLC indi-
cated that 5d was completely consumed. Toluene was evaporated under
vaccum, and the mixture was poured in water and extracted with ethyl
acetate (25 mL). Then the solvent was evaporated under vacuum. The
crude product was purified using flash column chromatography (eluent:
Petroleum ether/Acetone) on silica gel to afford the desired product 8d.
Cream solid; Purification by column chromatography on silica gel (PE/
A = 19:1): 61% yield; mp: 140–142 ◦C: 1H NMR (500 MHz, DMSO‑d6): δ
7.92–7.95 (m, 3H), 7.69–7.64 (m, 5H), 7.45 – 7.38 (m, 2H), 7.20 (d, J =
8.6 Hz, 4H), 7.04 (d, J = 10.4 Hz, 2H), 6.94 (d, J = 8.5 Hz, 2H), 5.56 (s,
2H), 3.82 (s, 3H); 13C NMR (125 MHz, DMSO‑d6): δ 159.74, 144.71,
140.66, 138.12, 135.43, 131.62, 131.24, 130.27, 130.15, 129.07,
127.78, 126.78, 126.76, 125.38, 125.05, 120.91, 119.63, 119.62,
114.62, 112.87, 112.17, 55.69, 47.75; HRMS: for C29H23N3O, Exact
Mass: 429.1841; observed [M + H]+: 430.1920.
5.4. Molecular docking studies
The ligand-bound crystal structures of human topoisomerase I and II
(PDB ID’s: 1T8I [48] and 4R1F [49]) were imported from Protein Data
Bank (www.rcsb.org) in Schrodinger (2020–4). Both the proteins were
prepared using protein preparation wizard in Maestro 12.6. The binding
cavity was defined using receptor grid generation, and the grid was
generated around the already bound ligand. All the synthesized ligands
were drawn using ChemDraw Professional in 2D format and saved in
‘sdf’ format. All the ligands were prepared using the LIGPREP module in
a 3D format using the OPLS3 force field. All the prepared ligands were
docked at the topoisomerase I and II binding sites using the GLIDE
module in Maestro 12.6. The result revealed the affinity and binding
interactions of ligands with the proteins. The docking scores were
generated.[50]
Procedure for controlled experiments. To gather more pieces of
evidence in favor of the nitrene pathway, we set a controlled experiment
(Scheme S2) where a reaction of 5d (1 equiv) with triethyl phosphite (3
equiv) was carried out in toluene as solvent. The reaction mixture was
refluxed for 4 h, and the sample was collected from the reaction mixture
for LCMS analysis.
5. Biology
5.1. Cell lines and cell culture
5.5. Mitochondrial membrane potential (MMP) and reactive oxygen
species (ROS) assay
Cell lines (MDA-MB-231, MCF-7, A549, and HCT-116) were pro-
cured from National Centre for Cell Science (NCCS) Pune, India and
maintained per instructions in DMEM media mixed with 10% FBS, 1%
Penicillin-Streptomycin solution in a CO2 incubator. Upon reaching 80%
confluency, they were passaged with the help of 0.25% Trypsin-EDTA
solution (Gibco) and were further utilized according to the
To assess MMP and ROS level alteration induced by compounds, cells
were plated in 100 mm culture dishes and treated with chosen com-
pounds at their respective IC50 values for 24 h. The pellets were washed
twice with the help of sterile 1X PBS (Gibco). The MMP in MDA-MB-231
12

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Declaration of Competing Interest
[21] C. Zhao, H. Dong, Q. Xu, Y. Zhang, Histone deacetylase (HDAC) inhibitors in
cancer: a patent review (2017-present), Expert Opin. Ther. Pat. 30 (4) (2020)
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MK thanks the DST-Inspire, New Delhi (IF 180460), for providing the
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The Supplementary data includes copies of 1H and 13C NMR spectra,
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and other figures (such as docking images and drug response curves)
(S2-S7) and Schemes. Supplementary data to this article can be found
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