Design and synthesis of thiadiazolo-carboxamide bridged β-carboline-indole hybrids: DNA intercalative topo-IIα inhibition with promising antiproliferative activity

Article abstract of DOI:10.1016/j.bioorg.2020.104357

The conjoining of salient pharmacophoric properties directing the development of prominent cytotoxic agents was executed by constructing thiadiazolo-carboxamide bridged β-carboline-indole hybrids. On the evaluation of in vitro cytotoxic potential, 12c exhibited prodigious cytotoxicity among the synthesized new molecules 12a–k, with an IC₅₀ < 5 μM in all the tested cancer cell lines (A549, MDA-MB-231, BT-474, HCT-116, THP-1) and the best cytotoxic potential was expressed in lung cancer cell line (A549) with an IC₅₀ value of 2.82 ± 0.10 μM. Besides, another compound 12a also displayed impressive cytotoxicity against A549 cell line (IC₅₀: 3.00 ± 1.40 μM). Further target-based assay of these two compounds 12c and 12a revealed their potential as DNA intercalative topoisomerase-IIα inhibitors. Additionally, the antiproliferative activity of compound 12c was measured in A549 cells by traditional apoptosis assays revealing the nuclear, morphological alterations, and depolarization of membrane potential in mitochondria and externalization of phosphatidylserine in a concentration-dependent manner. Cell cycle analysis unveiled the G0/G1 phase inhibition and wound healing assay inferred the inhibition of in vitro cell migration by compound 12c in lung cancer cells. Remarkably, the safety profile of compound 12c was disclosed by screening against normal human lung epithelial cell line (BEAS-2B: IC₅₀: 71.2 ± 7.95 μM) with a selectivity index range of 14.9–25.26. Moreover, Molecular modeling studies affirm the intercalative binding of compound 12c and 12a in the active pocket of topo-IIα. Furthermore, in silico prediction of physico-chemical parameters divulged the propitious drug-like properties of the synthesized derivatives.

Full text of DOI:10.1016/j.bioorg.2020.104357

Journal Pre-proofs
Design and Synthesis of Thiadiazolo-Carboxamide Bridged β-Carboline-In-
dole Hybrids: DNA Intercalative Topo-IIα Inhibition with Promising Antipro-
liferative Activity
Ramya Tokala, Sravani Sana, Uppu Jaya Lakshmi, Prasanthi Sankarana,
Dilep Kumar Sigalapalli, Nikhil Gadewal, Jyoti Kode, Nagula Shankaraiah
PII:
S0045-2068(20)31655-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.104357
YBIOO 104357
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
29 June 2020
3 September 2020
5 October 2020
Please cite this article as: R. Tokala, S. Sana, U. Jaya Lakshmi, P. Sankarana, D. Kumar Sigalapalli, N. Gadewal,
J. Kode, N. Shankaraiah, Design and Synthesis of Thiadiazolo-Carboxamide Bridged β-Carboline-Indole
Hybrids: DNA Intercalative Topo-IIα Inhibition with Promising Antiproliferative Activity, Bioorganic Chemistry
(2020), doi: https://doi.org/10.1016/j.bioorg.2020.104357
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Design and Synthesis of Thiadiazolo-Carboxamide Bridged β-Carboline-Indole
Hybrids: DNA Intercalative Topo-IIα Inhibition with Promising Antiproliferative
Activity
Ramya Tokalaa, Sravani Sanaa, Uppu Jaya Lakshmia, Prasanthi Sankaranaa, Dilep Kumar
Sigalapallia, Nikhil Gadewal,b Jyoti Kode,c,d Nagula Shankaraiaha*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad - 500 037, India
bBioinformatics Centre, cTumor Immunology & Immunotherapy Group, Advanced Centre
for Treatment, Research & Education in Cancer, Tata Memorial Centre, Kharghar, Navi
Mumbai-410210; dHomiBhabha National Institute (HBNI), Training School Complex,
Anushakti Nagar, Mumbai 400094, India
*Corresponding author: Dr. Nagula Shankaraiah, E-mail: shankar@niperhyd.ac.in,
shankar.niperhyd@gov.in
Research Highlights
 Compound 12c displayed superlative cytotoxicity in the cancer cell lines
 Selectivity index of 12c is in the range of 14.90–25.26
 Compound 12c along with 12a inhibited topoisomerase IIα and interacted with DNA
via intercalative mode of binding
 Compound 12c induced apoptosis in A549 lung cancer cell line
 In silico and in vitro evaluation established the cytotoxic potential of compound 12c
Abstract: The conjoining of salient pharmacophoric properties directing the development of
prominent cytotoxic agents was executed by constructing thiadiazolo-carboxamide bridged
β-carboline-indole hybrids. On the evaluation of in vitro cytotoxic potential, 12c exhibited
prodigious cytotoxicity among the synthesized new molecules 12a–k, with an IC50 < 5 μM
in all the tested cancer cell lines (A549, MDA-MB-231, BT-474, HCT-116, THP-1) and the
best cytotoxic potential was expressed in lung cancer cell line (A549) with an IC50 value of
2.82 ± 0.10 μM. Besides, another compound 12a also displayed impressive cytotoxicity
against A549 cell line (IC50: 3.00 ± 1.40 μM). Further target-based assay of these two
compounds 12c and 12a revealed their potential as DNA intercalative topoisomerase-IIα
inhibitors. Additionally, the antiproliferative activity of compound 12c was measured in
A549 cells by traditional apoptosis assays revealing the nuclear, morphological alterations,
and depolarization of membrane potential in mitochondria and externalization of
1

phosphatidylserine in a concentration-dependent manner. Cell cycle analysis unveiled the
G0/G1 phase inhibition and wound healing assay inferred the inhibition of in vitro cell
migration by compound 12c in lung cancer cells. Remarkably, the safety profile of
compound 12c was disclosed by screening against normal human lung epithelial cell line
(BEAS-2B: IC50: 71.2 ± 7.25 μM) with a selectivity index range of 14.9–25.26. Moreover,
Molecular modeling studies affirm the intercalative binding of compound 12c and 12a in the
active pocket of topo-IIα. Furthermore, in silico prediction of physico-chemical parameters
divulged the propitious drug-like properties of the synthesized derivatives.
Keywords: β-Carboline, Thiadiazole, DNA Intercalation, Topo-IIα inhibitors, Apoptosis,
Cytotoxicity.
1. Introduction
Promising anticancer agents should be necessarily developed as cancer remains as
public health concern worldwide [1] along with the additional origin of a newer pandemic
like COVID-19 [2]. The drug repurposing strategy has been employed to reduce COVID-19
induced inflammation, coagulopathy and dysfunction by using anticancer agents like
interleukin inhibitors, corticosteroids, kinase inhibitors, etc [2d]. The types, mutations [3],
metastasis [4], resistance [5], and after-effects of clinical treatment, make it a gruesome
disease [6] without ideal treatment. Consequently, there lies a lacuna in the progression of
novel agents to combat cancer. Inhibition of more than one target with a key role in cell
replication and proliferation set forth magnificent directions in the generation of new
chemical entities (NCEs) towards chemical research.
The discovery and comprehension of the structure and function of DNA are
paramount of all the discoveries in the last century [7]. Hitherto, DNA is a major
pharmacological target for small molecules which are effective in various ailments which
originated from virus [8], bacteria [9] and also in cancer [10]. Based on the interaction of the
small molecules with DNA, they have been broadly divided into (i) Covalent inhibitors,
which are irreversible binders that form covalent adducts, thereby cause serious damage to
the DNA process and induce cell death. e.g., cisplatin (ii) Non-covalent inhibitors are
reversible binders; further classified based on modes of binding with duplex DNA, (a)
Surface binders (exhibits electrostatic interactions) (b) Intercalators (between DNA base
pairs) (c) Groove binders (major and minor groove binding) [11]. Usually, the small
molecules bind with DNA with more than one binding mode. Also, enzymes acting on DNA
play a promising role in replication and repair. One such enzyme is DNA Topoisomerase II
2

(Topo II), with a potential role in cell division and demarcation by causing nicking and
relegation of DNA and is an imperative target in drug discovery [12]. There are two
isoforms of Topo II with gene codes Topo IIα and Topo IIβ which are analogous in structure
and are uniform in catalytic properties. However, topo IIα has positive coordination on the
mitotic process of cells such as rate of cell proliferation, expression of mRNA, and also on
different phases of the cell cycle nevertheless, cellular functions of topo IIβ are yet unclear
[13]. Topo II, in general, modifies DNA topology and induces double-strand breaks through
multiple steps that require ATP hydrolysis and Mg2+ for activity [14a] and agents inhibiting
it are categorized into interfacial poisons (IFPs) and catalytic inhibitory compounds (CICs).
Anticancer agents like doxorubicin and etoposide are renowned Topo II poisons that cause
accretion of deplorable linear DNA by inhibition of the relegation of cleaved DNA strands
through the stabilization of the Topo-DNA complex. Catalytic inhibitors such as novobiocin
and merbarone, block binding and nicking of DNA respectively. On the contrary, agents
such as suramin, a potent catalytic inhibitor block topo II’s ATP binding site [14]. In
addition to the above features, agents that induce apoptosis are considered as remarkable
anticancer agents as they target an important hallmark of cancer [15]. Thus the discovery of
new chemical entities targeting topo II through DNA intercalation is an important area of
cancer research. Based on the literature, the rationale to exhibit intercalative topo II
inhibition by NCE’s is to possess a chromophore (head part) linked to a side chain (tail part)
through a cationic species. The mechanistic details indicate that the chromophore is a
polyaromatic ring system which intercalates between DNA base pairs. The linker and
extending heterocycles impart stabilization by interacting with phosphate groups and by
orienting into the groove of DNA respectively [16].
The structural features of naturally occurring β-carboline alkaloids make it a potential
chromophore to intercalate with DNA in between the base pairs. The tricyclic pyrido [3,4-b]
ring system (norharmane A) possesses an affinity to stack between GC residues of DNA
[17]. Furthermore, these Peganum harmala derived agents (harmine B) followed up by their
chemical functionalization were reported as DNA damaging and photocleavage agents [18].
In synergism, inhibitions of other enzymes like topoisomerases [12, 19], kinases [20] make
β-carboline a promising entity in cancer research along with indications in several diseases
[21].
1,3,4-Thiadiazole is another privileged heterocyclic scaffold with excellent therapeutic
potential against diverse health problems. The potential of this bioactive scaffold was far
3

recognized as an antiepileptic (acetazolamide C), antibacterial, antiprotozoal (megazol D),
and to treat glaucoma [22]. Farooqi et. al., revealed the affinity of 1,3,4-thiadiazole towards
DNA interaction by hybridization of 1,3,4-thiadiazole to the clinical drugs (Ibuprofen and
Ciprofloxacin)[23]. Plech et. al., proved the affinity of 1, 3, 4-thiadiazole towards topo II by
disubstitution on the heterocycles [24]. Indeed, this entity reveals wide pharmacological
activities such as antimicrobial, antitubercular, anti-inflammatory, antiviral, anticonvulsant,
and activity against central nervous system (CNS) disorders, cardiovascular system (CVS)
diseases and cancer [22].
Indole is a versatile and privileged scaffold with an extensive range of biological
actions principally in cancer by targeting diverse enzymes (Topoisomerases, Tubulin
polymerases, Aromatase) pathways (NF-kB/mTOR/PI3K/AKT), and mechanisms like DNA
binding and apoptosis [25]. Naturally occurring antitumor antibiotic like duocarmycin has a
strong affinity for DNA interaction, nonetheless unable to reach the clinic. However,
synthetic analogs like adozelesin with substitution on 2nd position (E) reached clinical trials
in the treatment of cancer. Highly effective D-64131(F) and D 68144 (G) were recognized
as potent anticancer agents even in paclitaxel-resistant tumors [26] (Figure 1).
<Insert Figure 1 here>
2. Rationale
In maintaining our persistence to achieve the goal of reaching successful clinical
candidates towards cancer [27], by considering the advantage of structural features of the
chemically diverse scaffolds like β-carboline, 1,3,4-thiadiazole, indole and exploiting
pharmacophore hybridization to carboxamide formation, we have methodologically
designed a new series (12a–k) of DNA intercalative topo II inhibitors with classical
apoptosis-inducing features. The rationale for this design has been established based on
literature and structural functionalities as illustrated in Figure 2. Foremost requirements for
intercalative topoisomerase II inhibition are the presence of a chromophore (intercalation
into DNA), adjoining cationic center (electrostatic interaction with base pairs), and a groove
extending side-chain/heterocycles (stabilization). DNA intercalative topoisomerase II
inhibitors such as doxorubicin and PD-115934 also possess this kind of arrangement
(chromophore + cationic center + groove extending side chain) [16] and support the
rationale to this design. The chemical synthesis of β-carboline acid from amino acid L-
tryptophan satisfies the utmost feature as a chromophore for dual activity. Further, the
4

cationic center of 1,3,4-thiadiazole on the 2nd position of indole (extending heterocycles) has
been constructed by utilizing chemical functionalities of indole-2-acid and
thiosemicarbazide followed by cyclization resulting in the free amino moiety. The
chromophore has been adjoined with the rest of the scaffold by pharmacophore
hybridization [28] by carboxamide formation through acid amine coupling.
<Insert Figure 2 here>
3. Chemistry
The desired hybrids 1,3,4-thiadiazolo carboxamide bridged β-carboline-indole hybrids
12a–k were synthesized via amide formation between β-carboline acid and indol-2-yl-1,3,4-
thiadiazol-2-amine as depicted in Scheme 2. 1-Aryl-9H-pyrido[3,4-b] indole-3-carboxylic
acids 6a–f were synthesized by earlier reported procedures, [12] from commercially
available L-tryptophan (Spectrochem; CAS NO: 73-22-3) 1 which was esterified 2 and
subjected to Pictet-Spengler condensation using acid catalyst trifluoroacetic acid with
substituted aromatic benzaldehydes 3a–f to obtain tetrahydro-β-carboline esters 4a–f. The β-
carboline esters 5a–f were obtained by further aromatization of 4a–f using KMnO4 and were
subjected to basic hydrolysis to yield the respective β-carboline acids 6a–f (Scheme 1).
<Insert Scheme 1 here>
The amine components 11a–c were attained by esterification of indole-2-acid 7, which
(8) was further alkylated using different alkyl halides to yield alkylated indole-2-esters 9a–
c. Thus obtained esters, on basic hydrolysis result in respective alkylated indole-2-acids
10a–c. This acid component undergoes cyclization with thiosemicarbazide in POCl3 to
succumb amine counterpart. Further, the title compounds 12a–k was achieved by acid-
amine coupling between 6a–f and 11a–c in good yields (Scheme 2). All these compounds
were purified by using column chromatography.
<Insert Scheme 2 here>
The representative compound 12a of synthesized derivatives was thus characterized by
1H NMR spectroscopy. The appearance of singlet at δ = 12.14 corresponds to the amidic N-
H proton and confirms the formation of titled compounds. The two singlets at δ = 12.58 and
5

12.19 ppm account for the NH proton of indole and 9H-indolic proton of β-carboline ring
respectively. The proton at the C4 position of β-carboline appeared as a singlet at δ = 9.13
ppm. All the other 14 aromatic protons of both β-carboline and indole are accounted for in
the range of δ = 8.55 to 7.13 ppm. Further analysis by 13C NMR spectroscopy disclosed that
amidic carbonyl carbon resonated at δ = 164.1 ppm. The signals at δ = 158.2 and 158.0 ppm
account for amide attached C2 carbon and indole attached C5 carbon of 1,3,4-thiadiazole
respectively. All the remaining carbons of 12a appeared in the region of δ = 142.1-104.5
ppm. Next, IR spectra show the band at 1673 cm-1 which indicates the C=O stretching of the
amide of the compound 12a. The HRMS exhibited specific [M + H]+ peaks that matched
13
with their molecular formula. Similarly, spectral data (1H, C, FT-IR) of all the remaining
compounds 12b–k matched accurately with their respective structures.
4. Biological Evaluation
4.1 In vitro cytotoxicity assay
Thus, the synthesized compounds 12a–k were evaluated for their cytotoxic potential
against a panel of human cancer cell lines and compared with the standard harmine and
doxorubicin, VP-16 (etoposide) and represented in Table 1. The MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [29a] was performed for all
the derivatives on different cancer cell lines such as (i) adherent cell lines viz lung cancer
(A549), breast cancer (MDA-MB-231, BT-474), colon cancer (HCT-116), and (ii)
suspension cell lines viz leukemia (HL-60) and acute monocytic leukemia (THP-1) (Figure
S1 and S2 of ESI) and compared to normal human cell line (BEAS-2B). Notably, all the
tested compounds exhibited potent cytotoxicity against all the tested cell lines, particularly
12c with the highest cytotoxic potential of IC50 of 2.82 ± 0.10 µM in the A549 cell line.
Further, it has leading cytotoxicity in all the cell lines tested with an IC50 of 3.18 ± 0.49 µM
and 3.83 ± 0.94 µM in THP-1 and HL-60 respectively. Also, in both breast cancer cell lines,
12c expressed an IC50 of 3.28 ± 0.43 µM and 4.09 ± 0.59 µM in MDA-MB-231 (triple-
negative) and BT-474. Also, the compound 12c has exhibited in vitro cytotoxicity in HCT-
116 with an IC50 value of 4.78 ± 1.29 µM and the order of cytotoxicity for compound 12c in
different cell lines is A549 > THP-1 > MDA-MB-231 > HL-60 > BT-474 > HCT-116.
<Insert Table 1 here>
6

Encouragingly, the compound 12c displayed the lowest in vitro cytotoxicity with an
IC50 value of 71.25 ± 7.95 µM in normal human lung epithelial cells (BEAS-2B).
Remarkably, the compounds 12a, 12f, 12h, and 12j exhibited notable cytotoxicity potential
in all the cell lines tested with overall IC50 ≤ 10 µM. Moreover, compounds 12b, 12d, 12i,
and 12k also expressed a reasonable in vitro cytotoxicity on A549 and MDA-MB-231 cell
lines with an IC50 value < 10 µM. From the detailed analysis of the in vitro cytotoxicity
table, it was comprehended that 12c is a potent compound and possesses the least inhibition
in BEAS-2B and the cytospecificity profile on all the human cancer cell lines along with
normal human cell line was represented in graphical form in Figure 3a.
<Insert Figure 3a here>
4.2. Selectivity Index
After contemplating the specificity exhibited by potent compound 12c in different
cancer cell lines and normal human cell line (Figure 3a), the selectivity index (SI) was
calculated and portrayed in Figure 3b. The selectivity index is the ratio of IC50 in the non-
tumor cell line to IC50 in the tumor cell line [29b] and a compound is said to be selective
only if the value of SI ≥ 10 [29c]. It was observed that compound 12c exhibited selectivity
in all the cell lines tested with selectivity range of 25.26–14.90 in the following order A549
> THP-1 > MDA-MB-231 > HL-60> BT-474 > HCT-116.
<Insert Figure 3b here>
4.3. Structure-Activity Relationship (SAR)
Based on the established in vitro cytotoxicity data against human cancer cell lines, the
structure-activity relationship (SAR) was constructed to provide insights regarding the
pattern of efficiency of the synthesized NCEs as illustrated in Figure 4. Two-point diverse
modifications have been performed on the C1-phenyl of the β-carboline (R1) and nitrogen of
indole (R2). Noticeably, the compounds 12a, 12c, 12h, and 12j without substitution on
indole (R2 = H) were found to be potent than substituted derivatives 12b, 12d–g, 12i, and
12k. Additionally, increase in length of alky chain (R2 = H > Me > Et) on indole resulted in
derivatives with lower cytotoxicity potential (12c > 12d > 12e & 12a > 12b). The better
cytotoxicity was observed when R1 is substituted by electron-donating group (EDG) (12c–g,
12k: R1 = 4-Me, 4-OMe & 3,4,5-triOMe) than no substitution (12a, b: R1 = H) and followed
by electron-withdrawing group (EWG) (12h–j: R1 = 4-F & 4-Cl). The in vitro cytotoxic
7

potential was compared with EDG and EWG in the following order: (a) EDG: (i) In
derivatives where R2 = methyl; and R1 is 3,4,5-trimethoxy substituted, such derivative
exhibited superior inhibition than 4-methoxy containing corresponding derivative (12f >
12d); (ii) In compounds with R2 = ethyl; the compound with 4-methyl substitution on R1
displayed better inhibition than derivative with 3,4,5-trimethoxy followed by 4-methoxy
substituted compound (12k > 12g > 12e); (b) EWG: In compounds with R2 = H; the
derivative with 4-chloro substitution at R1 position exhibited higher inhibition than 4-fluoro
substituted derivative (12j > 12h). Interestingly, a similar pattern of inhibition was observed
for all the compounds in the tested cell lines.
<Insert Figure 4 here>
5. Target-based assays
5.1. Topoisomerase IIα inhibition
Topoisomerase IIα is located in the nucleus and functions as a DNA nick inducing
enzyme with established importance in DNA replication. It induces nicks on both the DNA
strands, religates the phosphodiester bonds, and thereby alters the DNA topology. Thus,
inhibition of topoisomerase IIα results in either blockage of the enzyme to induce nicks
which thereby interferes the cell replication process (CICs) or prevent the relegation of
nicks caused by the enzyme by stabilization of DNA-ternary complex which results in the
initiation of recombination pathways (IFPs) and ultimately results in cell death [30]. Topo-
IIα inhibitory assay has been performed using Topo-IIα drug screening kit (TG1019-1,
TopoGEN, USA) with enzyme human topoisomerase IIα (TG2000H-2, TopoGEN, USA).
In this assay, the two most potent compounds 12c and 12a from IC50 data were evaluated for
their topo-IIα inhibitory activity. The catenated kDNA and topo II (5 units) along with
compound 12c (10 μM) and 12a (10 μM) (lane 1 and 2 respectively) didn’t result in the
formation of any linear DNA (a & b). Further, decatenated kDNA and linear DNA in lines 3
and 4 were the reference markers provided with the screening kit. From Figure 5, it is
evident that the compound 12c and 12a inhibited topo IIα to induce nicks and linearize
kDNA and fall into the category of catalytic inhibitory compounds.
<Insert Figure 5 here>
5.2. DNA Intercalation Assays
8

Intercalation of the ligand into DNA doesn’t always cause alterations in hydrogen
bonds between base pairs, but the ligand-DNA complex is stabilized by van der Waals,
charge transfer, π-π stacking with the planar scaffold, hydrophobic and hydrogen-bonding
interactions [31]. Intercalation stiffens, lengthens, reduces helical twist, and unwinds the
DNA double helix [32], thereby resulting in inhibition of cell proliferation and triggers cell
death. [33] Among various classes of DNA binding agents, intercalating agents were widely
studied by considering different biophysical assays. By relating absorbance and viscosity
measurements using CT-DNA with a concentration of the tested compound, these assays
suggest the mode of binding of the novel synthesized compounds.
5.2.1. Relative viscosity experiment
To understand the binding mode of newly synthesized series, a relative viscosity
experiment has been performed for potent compounds 12c and 12a using CT-DNA as per
the reported protocol [34]. The different modes of binding of ligand result in alterations in
the viscosity of DNA. The increase in viscosity is a result of an increase in the axial length
of DNA to accommodate the ligand in between the base pairs, which indicates the
intercalative mode of binding. However, no or minimal alteration in viscosity is observed
with compounds possessing minor groove binding affinity. Conversely, covalent binders
such as cisplatin result in a decrease of viscosity due to absolute functional inhibition of
DNA through irreversible binding [35]. Ethidium bromide (EtBr) and Hoechst33258
generally cause an increase and comparative alteration in relative viscosity of DNA
indicating their mode of binding as intercalator and minor groove binder respectively [34].
In the current study, different concentrations of compound 12c and 12a along with
harmine, Hoechst 33258, and EtBr are incubated with CT-DNA, and changes in viscosity
were recorded. The controls EtBr and harmine displayed an increase in viscosity whereas;
Hoechst33248 exhibited a minimal change in viscosity with the increase in concentration.
The tested compounds exhibited an increase in viscosity in a concentration-dependent
manner indicating intercalation as their mode of DNA binding. Therefore, a graph is plotted
with the concentration of compound [compound/CT-DNA] on X-axis and viscosity [η/η0]1/3
on Y-axis and is represented in Figure 6.
<Insert Figure 6 here>
5.2.2. Absorbance Spectroscopy
9

Intercalation of ligands with DNA results in a slight bathochromic and significant
hypochromic shift in absorbance at the specific wavelength [36]. This principle has been
applied to find the efficacy of compounds 12c and 12a using CT-DNA.
5.2.2.1. Nanodrop method
Nanodrop is a kind of UV-spectrophotometer which uses a small quantity (nano
quantity) to measure the quality of the nucleic acids [37]. The specific absorbance was
measured at 260/230 nm wavelength with the different concentrations of compound 12c and
12a along with control CT-DNA. Harmine, Dox, Hoechst-33258, and EtBr were used as
standards. It is evident from Figures 7A and 7B, that compound 12c and 12a caused
hypochromic shift at specific wavelength respectively, and are potent intercalators.
<Insert Figure 7 here>
5.2.2.2. UV-Visible spectroscopy
In addition to the nanodrop method, the absorbance alterations induced by compounds
12a and 12c on CT-DNA were evaluated at a specific concentration along with potent
intercalator EtBr as a positive control. The plain CT-DNA without the addition of any
ligand was used as a control, and from Figure 8, it is evident that the order of absorption is
EtBr > 12c > 12a. The results of the absorbance spectroscopic methods are consistent with
relative viscosity measurements and establish the intercalative mode of binding of
compounds tested with CT-DNA. [38]
<Insert Figure 8 here>
6. Determination of Apoptosis
Programmed cell death is evaded by cancer cells and prevails in the different organs;
however, new cancer agents that induce apoptosis are always the prime choice of cancer
research [39]. To understand the ability of compound 12c in apoptosis induction, various
qualitative (morphological) and quantitative assays were performed on the A549 lung
cancer cell line.
6.1. Determination of morphological changes
10

The morphological assays reveal the formation of characteristic apoptotic features in
the cells with the aid of different nuclear affinity dyes. Henceforth, these are the elemental
line of apoptosis identification in diverse cancer cells.
6.1.1. Phase-contrast microscopy
The changes in the morphological features of A549 cells were observed after the
treatment with compound 12c at different concentrations along with the untreated control
cells. Further, images reported in Figure 9A were captured using phase-contrast microscopy
after 72 h, reveal the characteristic apoptotic features like changes in morphology (shape,
shrinkage) of the cell, reduction in the number of live cells.
6.1.2. Acridine orange staining
Acridine orange is selective toward nucleic acid and penetrates live cells and stains the
nuclei green [40]. In the present study, A549 cells after treatment with compound 12c for 72
h exhibited the formation of apoptotic features such as the appearance of membrane blebs
and inverse proportion in the number of cells with a concentration of compound tested. In
addition, the cationic dye has the potential to bind lysosomes and thereby also exhibiting the
vacuoles as represented in Figure 9B.
6.1.3. DAPI staining
The initial discovery of 4,6-diamidino-2-phenylindole (DAPI) in the treatment of
protozoal diseases retarded due to a strong binding affinity towards DNA base pairs. Later,
this ability of passage through cell membranes made it an efficient stain to detect nuclear
changes in the apoptosis cells [41]. The DAPI staining was executed according to the
reported procedure [40] to detect apoptosis induction. Compound 12c treated A549 cells
after 72 h, on staining with DAPI visualized the chromatin condensation, pyknotic (inset of
1.25 μM), and condensed (bright colored: inset of 2.5 μM) nuclei formation as depicted in
Figure 9C.
<Insert Figure 9 here>
6.2. Flow cytometry analysis
6.2.1. Quantification of apoptosis by dual staining assay
11

To quantify apoptotic cell death and determine the phase of apoptosis, dual staining
assay with annexin V-FITC/PI was employed by using flow cytometry. The effect of
different concentrations of compound 12c on A549 cells was studied using the principle of
phosphatidylserine externalization [42] and compared with control (untreated) cells. The
percentage of apoptotic cells at different phases are represented on a four quadrant graph.
From Figure 10, it is clear that induction of slight early (+ annexin V/– PI) and significant
late apoptosis (+ annexin V/+ PI) after 72 h of incubation with compound 12c.
<Insert Figure 10 here>
6.2.2. Depolarization of mitochondrial membrane potential (Δψm)
Mitochondria are the source of energy and also the crucial targets for oxidative stress
in a cell [43a-b]. JC-1 staining was performed by standard protocol [43c] to assess the
impact of 12c on the mitochondrial membrane potential of A549 lung cancer cells. After the
treatment period of 72 h, JC-1 dye was added and normal polarized healthy mitochondria
labeled as J aggregates (red color) in the control remained intact, whereas 12c treated
resulted in the formation of depolarized mitochondria categorized as J monomers. However,
the concentration of compound 12c and the formation of J monomers increased
proportionately (Figure 11).
<Insert Figure 11 here>
6.2.3. Cell cycle analysis
Cell cycle analysis was conducted on A549 cells using different concentrations (1.25,
2.5, 5 μM) of compound 12c to figure out the phase where cells are arrested. Further, after
the treatment for 48 h, cells are interpreted through the flow analyzer after staining with
propidium iodide (PI). The number of cells viz., 55.68 in the G0/G1 phase in control
increased exponentially to 73.27 in compound 12c (5 μM) treated cells. It is apparent from
Figure 12A & B that the treatment arrested the G0/G1 phase of the cell cycle in a
concentration-dependent manner.
<Insert Figure 12 here>
7. Metastatic assay
12

Cancer cells detach from the site of origin, migrate, and develop the disease at the
secondary site is known as metastasis, which makes the disease perilous and abating the
survival rate of the patient [4,44].
7.1. Cell migration assay
To understand the capability of 12c on cell migration, wound healing assay has been
performed on A549 cells. This assay is based on the ability of the healing of an artificial
wound called “scratch” created using a sterile pipette tip (200 μL).[45] The cells were
treated with a range of concentrations varying from 1.25-5 μM of 12c, along with control,
and images were captured at 0 and 72 h and compared. It is inferred from Figure 13, that
the wound in control was practically healed, whereas, the cell migration is inhibited in 12c
treated cells after the period of treatment.
<Insert Figure 13 here>
8. Computational Analysis
8.1. Molecular docking
Further affirmation of intercalative topo-IIα inhibition by synthesized derivatives,
molecular docking [46] was performed against active site of human topoisomerase II alpha
complexed with DNA [47] using Glide (Schrödinger 2017-1) [48] and the best-scored
compounds were detailed along with their interactive pattern. Structural accommodation of
the ligand 12c and 12a revealed that β-carboline moiety exhibited intercalation into the
DNA in the active site whereas, indole chain extended into the hydrophobic region via
thiadiazolo carboxamide as illustrated in A, C of Figure 14.
<Insert Figure 14 here>
The analysis of docking interactions indicated that all the structural fragments (β-carboline,
thiadiazole, and indole) of ligand 12c established key interactions with residues in the active
pocket such as the formation of two hydrogen bonds between amide attached thiadiazole’s
nitrogen and indolic nitrogen with dC8 (d = 3.65 Å) and dT9 (d = 3.51 Å) residues
respectively as represented in B of Figure 14. Further, π-cation interaction was exhibited by
tricyclic pyridine NH with dA12 residue and π- π interaction was observed with phenyl ring
of β-carboline with dA12 and dG13 residues. Notably, several hydrophobic interactions
13

were observed with different amino acid residues such as Leu486, Met762, Met766, Ile769,
Ala801, and Pro803. Additionally, compound 12a exhibited another crucial interaction on
the β-carboline viz hydrogen bond between indolic N-H and Arg487 (d = 2.87 Å), along
with other interactions as displayed in D of Figure 14. The docking results reveal the topo II
inhibition is a collective contribution by all the structural fragments (Figure S3, ESI) of the
compounds and are compatible with the in vitro biological evaluation and encourage the
structural diversification on the ligands in the near future.
8.2. Binding energy and Lipinski’s rules
Binding energies for the molecules were calculated to identify the affinity and complex
forming energy of the ligands in the active sites of the human topo-IIα complex with DNA
and topo I using the prime MM/GBSA method. The lead molecules exhibited superlative
binding energy values of -58.91(12c) and -53.67 (12a) kcal/mol in the active domain of
5GWK (topoisomerase IIα) in comparison to binding energy values of -46.87 (12c) and -
40.52 (12a) in the active pocket of 1SC7 (topoisomerase I) (Table S1, ESI). The binding
energy values of harmine and co-crystal (etoposide) are -40.94 and -72.21 kcal/mol
respectively.
The drug likeliness of the potent molecules and standards (Harmine, Etoposide) was
analyzed by the evaluation of the Lipinski rule of five and is incorporated in Table 2.
Although the compounds other than 12a violate the Lipinski rule in terms of molecular
weight, the optimal range [49] obtained by evaluating the 95% of known drugs recommends
the acceptable range up to 725 Daltons reveals the compounds tested satisfies the entire
parameters and possess good drug-like properties [50, 51].
<Insert Table 2 here>
8.3. In silico ADME/T profile
The prediction of pharmacokinetic parameters provides an idea regarding the
acceptable range for human use which was obtained by analyzing 95% of known drugs [51,
52]. The Physico-chemical parameters of the five best performers in the cytotoxicity table
and standards (Harmine, Etoposide) were predicted by using the QikProp module [53] of
Schrödinger and some key pharmacokinetic descriptors along with the recommended range
are tabulated. The evaluation of computed parameters such as QPlogKhsa predicts the
binding of a ligand to serum albumin which determines the availability of free drug and
14

assessment of QPPCaco, where Caco cells act as a model for gut-blood barrier and
QPPMDCK, where MDCK (Madin-Darby Canine Kidney) cells are a mimic for blood-brain
barrier assist in the estimation of bioavailability. From the in silico data delineated in Table
3, all the newly synthesized compounds assessed through QikProp fall in the optimal range
and exhibit pharmaceutical significance.
<Insert Table 3 here>
8.4. In Silico toxicity profiling
In order to understand the toxicity profile of the synthesized compounds and harmine,
TOPKAT (TOxicity Prediction by Komputer Assisted Technology) tool from Discovery
Studio v19.1.0.18287 was utilized. TOPKAT employs robust and cross-validated
Quantitative Structure Toxicity Relationship (QSTR) models to predict toxicological
endpoints of given molecules chemical structure. Toxicity model development involves the
use of 2D molecular, spatial and electronic descriptors. The optimal Predictive Space
validation method of TOPKAT gives assurance to the prediction [54, 55].
In silico toxicity profiling results for some of the synthesized compounds and harmine was
depicted in Table 4. The toxicity profile assessment has been predicted using different
animal models. The toxicity of synthesized compounds was assessed and compared with
harmine in different animal models. Broadly, the probability values of the synthesized
compounds showed a lower toxicity profile than harmine. All the analyzed compounds
including harmine have shown higher toxicity probabilities values for Ames mutagenicity.
Compounds 12a, 12c, 12f, 12h and 12j did not show carcinogenicity call in National
Toxicology Program (NTP) carcinogenicity (male and female mouse) model and found to
be safe, while harmine has shown positive carcinogenicity signs in the NTP model. All the
analyzed molecules had shown a positive sign for carcinogenicity in NTP (male rat) and
weight of evidence rodent carcinogenicity (except 12j) model. Except for 12f and harmine,
all the molecules had shown a negative sign for carcinogenicity in NTP (female rat) model.
Moreover, all the compounds are non-degradable and negative for skin sensitization, skin
irritancy and developmental toxicity potential. These compounds had shown higher
probability for various animal models which include Rat Male FDA None vs Carcinogen,
Rat Male FDA Single vs Multiple, Rat Female FDA None vs Carcinogen and Rat Female
FDA Single vs Multiple. However, in silico toxicity studies indicated that the synthesized
compounds were found to be safer in many models with a minimal toxicological profile.
15

<Insert Table 4 here>
9. Conclusion
In summary, new molecules 12a–k were rationally designed by taking into
consideration of structural fragments and pharmacophoric hybridization approach to build
intercalative topoisomerase IIα inhibitors targeting cancer. Based on the earlier presented
pharmacophoric profile of these heterocycles (β-carboline, indolo-thiadiazole), the synthesis
was performed and adjoined by carboxamide formation which resulted in the target
molecules with good yields. From the preliminary in vitro screening, it can be observed that
the compounds 12c and 12a remarkably expressed its cytotoxicity among all the derivatives
tested in all the cell lines (solid and liquid tumors) with magnificent IC50 values of 2.82 ±
0.10 and 3.00 ± 1.40 μM, respectively in A549 cell line. Also, cytospecificity and selectivity
index of potent compound 12c towards cancer cells was revealed by calculating cytotoxicity
(IC50: 71.25 ± 7.95) against normal human lung epithelial cell line (BEAS-2B). Catalytic
inhibition of topo-IIα by compounds 12c and 12a was evinced through gel-based
electrophoresis and intercalation into DNA was revealed by evaluating alterations in
parameters such as absorbance, viscosity, and correlation with standard intercalators.
Additionally, deeper insights into mechanistic features by different flow cytometric assays
(Annexin V/FITC, JC-1) indicated that compound 12c is a potent inducer of apoptosis. Cell
cycle progression in A549 cells was arrested at the G0/G1 phase by compound 12c in a
dose-dependent manner. Furthermore, cellular and nucleo-morphological changes caused by
12c were also visualized using diverse staining assays (Acridine-orange and DAPI) revealed
the induction of abundant apoptotic features in A549 cells. Scratch wound assay by
compound 12c indicated the inhibition of in vitro cell migration and deterrence of wound
closure in A549 cells. Further, computational studies supported the in vitro target-based
assays as compounds 12c and 12a accommodated well in the catalytic region of topo-IIα
with residual amino acid and DNA base pairs interactions (PDB code: 5GWK). Further, in
silico evaluation of ADME/T profile and drug-likeliness indicated that all the molecules fall
under the specified range deduced from known drugs. As a concluding remark, the current
study primarily emphasizes on assembling varied pharmacological aspects of designed
compounds and can be suitably derivatized to mark their progression as promising
anticancer agents.
10.0. Experimental section
16

10.1. Chemistry
Materials and methods: All the reagents and solvents were obtained from commercial
suppliers and were used without further purification. Analytical thin-layer chromatography
(TLC) was performed on MERCK precoated silica gel 60-F254 (0.5 mm) aluminium plates.
Visualization of the spots on TLC plates was achieved by UV light. Wherever required,
column chromatography was performed using silica gel (60-120). The reactions wherever
anhydrous conditions are required were carried under nitrogen positive pressure using
freshly distilled solvents. All evaporation of solvents was carried out under reduced pressure
o
1
13
using a rotary evaporator below 45 C. H and C NMR spectra were recorded on Bruker
500 MHz by making a solution of samples in the DMSO using tetramethylsilane (TMS) as
the internal standard. Chemical shifts for 1H and 13C are reported in parts per million (ppm)
downfield from tetramethylsilane. Spin multiplicities are described as s (singlet), d
(doublet), t (triplet), q (quartet), and m (multiplet). Coupling constant (J) values are reported
in hertz (Hz). HRMS was determined with Agilent QTOF mass spectrometer 6540 series
instrument. Melting points were determined with an electrothermal digital melting point
apparatus IA9100 and are uncorrected. The names of all the compounds given in the
experimental section were taken from Chem Bio Draw Ultra, Version 12.0.
10.1.1. General procedure for N-(5-(1-alkyl-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)-1-aryl-
9H-pyrido[3,4-b]indole-3-carboxamide (12a–k)
To a mixture of 1-aryl-9H-pyrido[3,4-b]indole-3-carboxylic acid (6a–f, 1.1 equiv.),
EDC (1.5 equiv.), HOBt (1.5 equiv.) under nitrogen conditions add dichloromethane and
allowed to stir at room temperature for 15 min. Later, the amino component (5-(1-alkyl-
indol-2-yl)-1,3,4-thiadiazol-2-amine 11a–c, 1 equiv.) dissolved in dichloromethane, was
added and the reaction was allowed to stir at room temperature till complete consumption of
the starting materials as determined by TLC. The reaction mixture was extracted with
dichloromethane and water (3 × 50 mL) and purified by column chromatography to yield N-
(5-(1-alkyl-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)-1-phenyl-9H-pyrido[3,4-b]indole-3-
carboxamide 12a–k in moderate to good yields (72-85%).
10.1.1.1. N-(5-(1H-Indol-2-yl)-1,3,4-thiadiazol-2-yl)-1-phenyl-9H-pyrido[3,4-b]indole-3-
carboxamide (12a)
Pale yellow solid; 72% yield; mp: 233-237 oC; FT-IR (cm-1): 3472, 3253, 3058, 2920, 1673,
1296; 1H NMR (500 MHz, DMSO-d6): δ 12.58 (s, 1H), 12.19 (s, 1H), 12.14 (s, 1H), 9.13 (s,
1H), 8.55 (d, J = 7.9 Hz, 1H), 8.39 (d, J = 7.1 Hz, 2H), 7.84–7.62 (m, 6H), 7.52 (d, J = 8.1
17

Hz, 1H), 7.44 (t, J = 7.4 Hz, 1H), 7.27 (t, J = 7.1 Hz, 1H), 7.22 (d, J = 1.4 Hz, 1H), 7.13 (t, J
= 7.4 Hz, 1H).; 13C NMR (125 MHz, DMSO-d6): δ 164.1, 158.2, 156.0, 142.1, 142.1, 137.9,
137.5, 137.3, 135.5, 130.3, 129.7, 129.5, 129.4, 129.2, 128.4, 128.2, 123.9, 122.6, 121.6,
121.4, 121.1, 120.4, 115.6, 113.3, 112.4, 104.5; HRMS (ESI): m/z calcd for C28H18N6OS
487.1336 found 487.1334 [M+H]+.
10.1.1.2.
N-(5-(1-Methyl-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)-1-phenyl-9H-pyrido[3,4-
b]indole-3-carboxamide (12b)
Pale yellow solid; 75% yield; mp: 287-291oC; FT-IR (cm-1): 3622, 3207, 1686, 1592, 1407,
1244; 1H NMR (500 MHz, DMSO-d6): δ 12.56 (s, 1H), 12.10 (s, 1H), 9.08 (s, 1H), 8.50 (d,
J = 7.9 Hz, 1H), 8.33 (d, J = 7.1 Hz, 2H), 7.76–7.59 (m, 7H), 7.38 (t, J = 7.6 Hz, 1H), 7.32
(t, J = 7.6 Hz, 1H), 7.19 (s, 1H), 7.15 (t, J = 7.3 Hz, 1H), 4.18 (s, 3H); 13C NMR (125 MHz,
DMSO-d6): δ 164.2, 158.3, 156.3, 142.1, 139.4, 137.5, 137.2, 135.5, 130.3, 129.7, 129.5,
129.4, 129.2, 129.0, 127.3, 124.1, 122.6, 121.6, 121.5, 121.1, 120.8, 115.6, 113.3, 111.0,
106.6, 32.6; HRMS (ESI): m/z calcd for C29H20N6OS 501.1492 found 501.1489 [M+H]+.
10.1.1.3. N-(5-(1H-Indol-2-yl)-1,3,4-thiadiazol-2-yl)-1-(4-methoxyphenyl)-9H-pyrido[3,4-
b]indole-3-carboxamide (12c)
Pale yellow solid; 73% yield; mp: 265-270 oC; FT-IR (cm-1): 3430, 3528, 3054, 1691, 1025,
893; 1H NMR (500 MHz, DMSO-d6): δ 12.50 (s, 1H), 12.15 (s, 1H), 12.04 (s, 1H), 9.02 (s,
1H), 8.48 (d, J = 7.9 Hz, 1H), 8.31 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.2 Hz, 1H), 7.69–7.60
(m, 2H), 7.47 (d, J = 8.2 Hz, 1H), 7.37 (t, J = 7.1 Hz, 1H), 7.23 (dd, J = 17.3, 8.0 Hz, 3H),
13
7.17 (d, J = 1.4 Hz, 1H), 7.08 (t, J = 7.1 Hz, 1H), 3.93 (s, 3H); C NMR (125 MHz,
DMSO-d6): δ 164.2, 160.6, 158.2, 156.0, 142.1, 142.0, 137.9, 137.1, 135.3, 131.0, 130.1,
130.0, 129.3, 128.4, 128.2, 123.9, 122.6, 121.6, 121.4, 121.0, 120.4, 115.0, 114.6, 113.3,
112.4, 104.5, 55.8; HRMS (ESI): m/z calcd for C29H20N6O2S 517.1441 found 517.1436
[M+H]+.
10.1.1.4.
1-(4-Methoxyphenyl)-N-(5-(1-methyl-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)-9H-
pyrido[3,4-b]indole-3-carboxamide (12d)
Pale yellow solid; 72% yield; mp: 287-291 oC; FT-IR (cm-1): 3277, 3055, 1671, 1407, 1042,
1026; 1H NMR (500 MHz, DMSO-d6): δ 12.46 (s, 1H), 12.02 (s, 1H), 9.00 (s, 1H), 8.45 (d,
J = 7.9 Hz, 1H), 8.37–8.13 (m, 2H), 7.73 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.62
(t, J = 8.1 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.36 (t, J = 7.2 Hz, 1H), 7.31 (t, J = 7.6 Hz,
1H), 7.24 (d, J = 8.8 Hz, 2H), 7.20–7.03 (m, 2H), 4.17 (s, 3H), 3.93 (s, 3H); 13C NMR (125
MHz, DMSO-d6): δ 164.2, 160.7, 158.3, 156.2, 142.1, 139.4, 137.1, 135.3, 130.9, 130.1,
130.0, 129.3, 129.0, 127.3, 124.1, 122.5, 121.6, 121.5, 121.0, 120.8, 115.1, 114.7, 113.3,
18

110.9, 106.6, 55.9, 32.6; HRMS (ESI): m/z calcd for C30H22N6O2S 531.1598 found
531.1595 [M+H]+.
10.1.1.5.
N-(5-(1-Ethyl-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)-1-(4-methoxyphenyl)-9H-
pyrido[3,4-b]indole-3-carboxamide (12e)
o
Pale yellow solid; 74% yield; mp: 278-282 C; FT-IR (cm-1): 3370, 3283, 2974, 1674,
1
1495,1031; H NMR (500 MHz, DMSO-d6): δ 12.48 (s, 1H), 12.05 (s, 1H), 9.03 (s, 1H),
8.47 (d, J = 7.9 Hz, 1H), 8.29 (d, J = 8.8 Hz, 2H), 7.75 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 7.9
Hz, 1H), 7.66–7.58 (m, 2H), 7.37 (t, J = 7.1 Hz, 1H), 7.32 (t, J = 7.1 Hz, 1H), 7.26 (d, J =
8.8 Hz, 2H), 7.19 (d, J = 0.5 Hz, 1H), 7.15 (t, J = 7.1 Hz, 1H), 4.77 (q, J = 7.0 Hz, 2H), 3.93
13
(s, 3H), 1.36 (t, J = 7.1 Hz, 3H); C NMR (125 MHz, DMSO-d6): δ 164.2, 160.6, 158.2,
156.1, 142.0, 138.3, 137.0, 135.3, 130.9, 130.0, 129.9, 129.3, 128.1, 127.5, 124.1, 122.6,
121.7, 121.6, 121.0, 120.8, 115.1, 114.6, 113.3, 110.9, 107.0, 55.9, 27.3, 15.8; HRMS (ESI):
m/z calcd for C31H24N6O2S 545.1754 found 545.1747 [M+H]+.
10.1.1.6. N-(5-(1-Methyl-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)-1-(3,4,5-trimethoxyphenyl)-
9H-pyrido[3,4-b]indole-3-carboxamide (12f)
Pale yellow solid; 85% yield; mp: 225-230 oC; FT-IR (cm-1): 2942, 1736, 1683, 1001, 905,
782; 1H NMR (500 MHz, DMSO-d6): δ 12.50 (s, 1H), 12.09 (s, 1H), 9.07 (s, 1H), 8.50 (d, J
= 7.9 Hz, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.69 – 7.66 (m, 1H), 7.65-7.54(m, 2H), 7.47–7.42
(m, 2H), 7.38 (t, J = 6.5 Hz, 1H), 7.33 (t, J = 10.5 Hz, 1H), 7.20 (s, 1H), 7.15 (t, J = 7.1 Hz,
13
1H), 4.19 (s, 3H), 4.00 (s, 6H), 3.83 (s, 3H); C NMR (125 MHz, DMSO-d6): δ 164.3,
158.2, 156.3, 153.6, 142.4, 142.07, 139.4, 138.9, 137.0, 135.6, 132.9, 130.0, 129.4, 129.0,
127.3, 124.1, 122.7, 121.6, 121.5, 121.0, 120.8, 115.6, 113.3, 111.0, 107.1, 106.6, 60.5,
56.6, 32.6; HRMS (ESI): m/z calcd for C32H26N6O4S 591.1809 found 591.1805 [M+H]+.
10.1.1.7.
N-(5-(1-Ethyl-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)-1-(3,4,5-trimethoxyphenyl)-
9H-pyrido[3,4-b]indole-3-carboxamide (12g)
Pale yellow solid; 78% yield; mp: 283-286 oC; FT-IR (cm-1): 3280, 2971, 1706, 1507, 1353,
1246; 1H NMR (500 MHz, DMSO-d6): δ 12.46 (s, 1H), 12.10 (s, 1H), 9.08 (s, 1H), 8.50 (d,
J = 7.9 Hz, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.70–7.56 (m, 3H), 7.47 (s, 2H), 7.38 (t, J = 7.2
Hz, 1H), 7.32 (t, J = 7.2 Hz, 1H), 7.20 (s, 1H), 7.15 (t, J = 7.4 Hz, 1H), 4.77 (dd, J = 14.0,
13
6.9 Hz, 2H), 4.00 (s, 6H), 3.83 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H); C NMR (125 MHz,
DMSO-d6): δ 164.1, 158.2, 156.2, 153.6, 142.4, 142.0, 138.9, 138.3, 136.9, 135.5, 132.9,
130.0, 129.4, 128.0, 127.5, 124.2, 122.6, 121.7, 121.6, 121.1, 120.8, 115.5, 113.3, 110.8,
19

107.0, 106.9, 60.6, 56.5, 15.7; HRMS (ESI): m/z calcd for C33H28N6O4S 605.1996 found
605.1991 [M+H]+.
10.1.1.8.
N-(5-(1H-Indol-2-yl)-1,3,4-thiadiazol-2-yl)-1-(4-fluorophenyl)-9H-pyrido[3,4-
b]indole-3-carboxamide (12h)
Pale yellow solid; 76% yield; mp: 260-265 oC; FT-IR (cm-1): 3463, 3335, 1456, 1042, 892,
861; 1H NMR (500 MHz, DMSO-d6): δ 12.64 (s, 1H), 12.16 (s, 1H), 12.11 (s, 1H), 9.08 (s,
1H), 8.50 (d, J = 7.9 Hz, 1H), 8.45–8.41 (m, 2H), 7.75 (d, J = 8.2 Hz, 1H), 7.68–7.63 (m,
2H), 7.53 (t, J = 8.9 Hz, 2H), 7.47 (d, J = 8.2 Hz, 1H), 7.39 (t, J = 7.4 Hz, 1H), 7.22 (t, J =
13
7.1 Hz, 1H), 7.17 (d, J = 1.4 Hz, 1H), 7.08 (t, J = 7.1 Hz, 1H); C NMR (125 MHz,
DMSO-d6): δ 164.2, 162.3, 158.3, 156.0, 142.1, 141.1, 137.9, 137.3, 135.4, 133.9, 131.9 (d,
J = 8.4 Hz), 130.4, 129.4, 128.4, 128.2, 123.9, 122.6, 121.6, 121.4, 121.1, 120.4, 116.2,
116.1, 115.9, 115.6, 113.3, 112.4, 104.5; HRMS (ESI): m/z calcd for C28H17FN6OS
505.1241 found 505.1236 [M+H]+.
10.1.1.9.
1-(4-Fluorophenyl)-N-(5-(1-methyl-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)-9H-
pyrido[3,4-b]indole-3-carboxamide (12i)
Pale yellow solid; 73% yield; mp: 270-275 oC; FT-IR (cm-1): 3299, 3064, 1673, 1605, 1408,
843; 1H NMR (500 MHz, DMSO-d6): δ 12.64 (s, 1H), 12.10 (s, 1H), 9.07 (s, 1H), 8.49 (d, J
= 7.9 Hz, 1H), 8.46–8.32 (m, 2H), 7.74 (d, J = 8.2 Hz, 1H), 7.66 (dd, J = 15.3, 7.5 Hz, 2H),
7.60 (d, J = 8.1 Hz, 1H), 7.53 (t, J = 8.9 Hz, 2H), 7.38 (t, J = 7.4 Hz, 1H), 7.32 (t, J = 7.1
13
Hz, 1H), 7.19 (s, 1H), 7.15 (t, J = 7.1 Hz, 1H), 4.19 (s, 3H); C NMR (125 MHz, DMSO-
d6):δ 164.3, 158.3, 156.3, 142.1, 141.1, 139.4, 137.2, 135.4, 134.0, 131.9, 131.9, 130.4,
129.4, 129.0, 127.3, 124.1, 121.6, 121.5, 121.1, 120.8, 116.1, 116.0, 115.7, 113.3, 111.0,
106.6, 32.6; HRMS (ESI): m/z calcd for C29H19FN6OS 519.1398 found 519.1397 [M+H]+.
10.1.1.10. N-(5-(1H-Indol-2-yl)-1,3,4-thiadiazol-2-yl)-1-(4-chlorophenyl)-9H-pyrido[3,4-
b]indole-3-carboxamide (12j)
Pale yellow solid; 82% yield; mp: 285-290 oC; FT-IR (cm-1): 3293, 2919, 2855, 1624, 1093,
1013; 1H NMR (500 MHz, DMSO-d6): δ 12.65 (s, 1H), 12.14 (d, J = 17.3 Hz, 2H), 9.09 (s,
1H), 8.50 (d, J = 7.9 Hz, 1H), 8.41 (t, J = 7.6 Hz, 2H), 8.12 (d, J = 8.5 Hz, 1H), 7.77–7.73
(m, 2H), 7.70 (d, J = 8.5 Hz, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.47 (d, J = 8.2 Hz, 1H), 7.39 (t,
J = 7.5 Hz, 1H), 7.22 (t, J = 7.3 Hz, 1H), 7.15 (t, J = 10.9 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H);
13C NMR (125 MHz, DMSO-d6): δ 164.2, 158.3, 156.0, 142.2, 140.7, 137.9, 137.4, 136.7,
136.3, 135.5, 134.5, 131.5, 131.0, 130.5, 129.5, 129.3, 129.1, 128.4, 128.2, 123.9, 122.7,
20

121.6, 121.4, 121.1, 120.4, 113.3, 112.4, 104.5; HRMS (ESI): m/z calcd for C28H17ClN6OS
521.0946 found 521.0939 [M+H]+.
10.1.1.11.
N-(5-(1-Ethyl-1H-indol-2-yl)-1,3,4-thiadiazol-2-yl)-1-(p-tolyl)-9H-pyrido[3,4-
b]indole-3-carboxamide (12k)
o
Pale yellow solid; 76% yield; mp: 228-232 C; FT-IR (cm-1): 3283, 3068, 1739, 1625,
1245,823; 1H NMR (500 MHz, DMSO-d6): δ 12.45 (s, 1H), 12.04 (s, 1H), 9.05 (s, 1H), 8.48
(d, J = 7.9 Hz, 1H), 8.21 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.2 Hz, 1H), 7.68 (d, J = 7.9 Hz,
1H), 7.66–7.57 (m, 2H), 7.52 (d, J = 7.9 Hz, 2H), 7.38 (t, J = 7.5 Hz, 1H), 7.32 (t, J = 7.6
Hz, 1H), 7.19 (s, 1H), 7.15 (t, J = 7.5 Hz, 1H), 4.77 (q, J = 7.0 Hz, 2H), 2.50 (s, 3H), 1.36 (t,
13
J = 7.1 Hz, 3H); C NMR (125 MHz, DMSO-d6): δ 164.2, 158.2, 156.1, 142.2, 142.1,
139.3, 138.4, 137.1, 135.5, 134.7, 130.2, 129.8, 129.4, 128.1, 127.5, 124.1, 122.6, 121.7,
121.0, 120.8, 115.4, 113.3, 110.9, 107.0, 21.4, 15.8; HRMS (ESI): m/z calcd for
C31H24N6OS 529.1805 found 529.1800 [M+H]+.
10.2. Biological evaluation
10.2.1. Cell culture
Normal human bronchial epithelial cell line (BEAS-2B) and cancer cell lines like lung
(A549), breast (MDA-MB-231, BT-474), colon (HCT-116), Leukemia (HL-60, THP-1),
were maintained in appropriate media supplemented with 10% fetal bovine serum (FBS)
stabilized with 1% antibiotic-antimycotic solution (Sigma Aldrich) cells were maintained at
o
37 C with 5% CO2 and 98% relative humidity in the incubator. When the cells reached up
to 80-90% of confluency, sub-culturing was performed using a 0.25% trypsin/1 mM EDTA
solution for further passage. The stock solution of 10 mM was prepared by dissolving test
compounds and standards in DMSO. Required concentrations were further obtained by
suitable dilutions with respective media.
10.2.2. MTT assay
MTT (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide) is a dye that
converts into insoluble formazan by mitochondrial succinate dehydrogenase enzyme, where
MTT assay is a colorimetric assay, thereby measures the reduction of MTT. Metabolically
active cells can reduce the MTT and the level of activity indicates the viability of cells.
Briefly, cells were seeded in 96-well plates at a density of 1000 to 4000 cells per well in 100
µl of complete medium and allowed to grow overnight for attachment onto the wells. After
the media was replaced with fresh media, the cells were treated with various concentrations
21

of the compounds for a period of 72 h. After incubation, the media was aspirated and 100 µl
of MTT (0.5 mg/mL) was added and incubated at 37 °C for 4 h. Then MTT reagent was
aspirated and the formazan crystals formed were dissolved by the addition of 200 µL of
DMSO for 20 min. at 37 °C. The formazan product quantity was measured by using a
spectrophotometric microtiter plate reader (Spectra Max, M4 Molecular Devices, USA) at
570 nm wavelength.
10.3. Target-based assays
10.3.1. Topoisomerase IIα inhibition
Topo IIα inhibition of potent compounds 12c and 12a at a concentration of 10 μM was
evaluated by performing ATP dependent decatenation of kDNA by the protocol described in
the Drug Screening Kit of Topo II (TG1019-1, TopoGEN, USA). Reactions were carried out
in 20 μL and contained two different types of buffer in equal ratio and 200–300 ng of
kDNA, and topoisomerase II (TG2000H-2, TopoGEN, USA). The quantity of
topoisomerase II (5 units) was attuned in pilot experiments to decatenate around 100% of
the kDNA under our assay conditions. Authentic decatenated DNA and linear DNA was
used as a control to identify the changes. The samples were made with buffer, kDNA, test
compounds, topo-IIα and incubated at 37 °C for 30 min. and terminated by the addition of 2
μL of a stop buffer containing 10% (w/v) SDS and 2 μL of 0.5 mg/mL proteinase-K and
incubated for 10 min at 37 °C. After completion of the reaction, the products in the reaction
mixture were separated by 1% agarose gel. The products in the agarose gel were visualized
after staining with ethidium bromide (0.2 mg/mL). The gels were run at 100 V for about 30
min. and visualized under UV transillumination (BIO-RAD gel doc XR+, USA).
10.3.2. DNA Intercalation assays
10.3.2.1. Relative viscosity studies
The viscosities of the DNA-ligand complexes were determined by the Lovis 2000
M/ME Rolling-ball viscometer (Anton Paar GmbH, Graz, Austria), based on the falling ball
principle. The temperature was controlled at ± 0.005 K through an internal Peltier
thermostat. A calibrated 1.59 mm glass capillary containing a steel ball was filled with the
sample for measuring the ball falling time at angles in the range from 20° to 70°. The
kinematic, as well as dynamic viscosities at 25 °C, were estimated based on the ball falling
time and densities DNA solution was prepared in 100 mM Tris-HCl (pH 7.4) and viscosity
was measured while each derivative 12c and 12a (5 µM) was added to CT-DNA solution
22

(50 µM). Ethidium bromide, Harmine and Hoechst 33258 at different concentrations were
used as controls. Data was represented graphically as (ŋ/ŋ0)1/3 vs. the ratio of the
concentration of the hybrid to CT-DNA, where ŋ is the viscosity of CT-DNA in the
presence of the various concentrations of derivatives and ŋ0 is the viscosity of CT-DNA
solution.
10.3.2.1. Absorbance spectroscopy
10.3.2.1.1. DNA Nanodrop method
DNA intercalation is determined by nanodrop spectrophotometric analysis. The
intercalating agents decrease the absorption and increase the wavelengths. In our
experiment, we incubated the 50 μM of calf thymus DNA (Sigma-Aldrich, USA) with
compound 12c and 12a, Ethidium bromide (EtBr), Doxorubicin (DOX) and Harmine for 10
min at 1 μM concentration. Later, the TAE buffer used as blank and absorbance and
concentrations were determined by NanoDrop™ 2000/2000c Spectrophotometer (Thermo
Fisher Scientific, USA).
10.3.2.1.2. UV-Visible spectroscopy
DNA intercalation is determined by UV-Visible spectroscopic analysis. In our
experiment, we incubated the 50 μM of calf thymus DNA (Sigma-Aldrich, USA) with
compound 12c and 12a, and Ethidium bromide (EtBr), for 10 min at 1 μM concentration
and compared with control (i.e. CT-DNA alone). Later, the TAE buffer used as blank and
absorbance and concentrations were determined by UV-visible Spectrophotometer (Jasco;
V-650).
10.4. Apoptosis detection studies
10.4.1. Morphological observations
10.4.1.1. Phase-contrast microscopy
A549 cells were plated in 12 well culture plates with a cell density of 1 × 106 cells/mL
and allowed to adhere overnight. The cells were incubated with different (1.25, 2.5, and 5
µM) concentrations of the compound 12c for 72 h treatment. Later cells were checked for
the morphological changes, and images were taken using a phase-contrast microscope
(Nikon, Inc. Japan).
23

10.4.1.2. Acridine Orange (AO) staining
A549 cells were plated at a concentration of 1 × 106 cells/mL and treated with various
concentrations of compound 12c and the plates were incubated for 72 h. 10 µL of
fluorescent dye, Acridine Orange (AO) was added into each well in equal volumes (10
µg/ml) respectively then the cells were visualized immediately under a fluorescence
microscope (Nikon, Inc. Japan) with excitation (488 nm) and emission (550 nm) at 200X
magnification.
10.4.1.3. DAPI nucleic acid staining
DAPI staining aids in the observation of morphological changes in the nucleus. After
treatment with compound 12c for 72 h, lung cancer A549 cells were washed with PBS and
permeabilized with 0.1% Triton X for 10 min followed by staining with 1 µM DAPI.
Control and treated cells were observed with a fluorescence microscope with excitation at
359 nm and emission at 461 nm using DAPI filter at 200X magnification.
10.4.2. Flow cytometric analysis
10.4.2.1. Annexin V assay
This assay was performed by the method [56] given by Rieger et al. with slight
modifications. Briefly, 1 × 105 cells were seeded in a 12-well plate and treated with different
concentrations of compound 12c for 72 h. The collected cells were washed twice with ice-
cold PBS, then incubated with 200 μL of 1 × binding buffer containing 1 μL propidium
iodide (PI) for 15 min. at room temperature in the dark. After incubation, cells were
analyzed for apoptosis using a flow cytometer (BD FACSVerseTM, USA). Apoptosis and
necrosis were analyzed with quadrant statistics on propidium iodide-negative cells,
fluorescein positive cells, and propidium iodide (PI)-positive cells, respectively.
10.4.2.2. Measurement of mitochondrial membrane potential
A549 cells (1 × 106 cells/ml) were seeded in 12 well plates and allowed to adhere
overnight. The cells were incubated with compound 12c at 1.25, 2.5, and 5 µM
concentrations for 72 h. Cells were collected and washed with PBS and resuspended in a
solution of JC-1 (1 µM) and incubated for 30 min. in an incubator at 37 °C. The cells were
washed twice with PBS and analyzed by flow cytometer (BD FACSVerse™, USA).
10.4.2.3. Cell cycle analysis
In general, the novel compounds exert their cytotoxic or growth inhibitory effect by
arresting the specific checkpoint in the cell cycle. Flow cytometric analysis (FACS) was
performed to calculate the distribution of the cell population in various cell cycle phases.
Here A549 cancer cells were incubated with compound 12c at various concentrations from
24

1.25 to 5 µM for 48 h. Untreated and treated cells were harvested, washed, and fixed
overnight in 70% ethanol in PBS at -20 °C. Fixed cells were pelleted and stained with cell
cycle analysis reagent propidium iodide (50 μg/ml) with RNase A for 20 min at 37 °C in
dark according to the manual instructions and about 10,000 events were acquired and
analyzed on a flow cytometer BD FACSVerseTM (BD Biosciences, USA).
10.5. Metastatic assays
10.5.1. Cell migration assay/wound healing assay
A549 cells were plated at a cell density of 3.5 × 105 cells/well into 12-well plates in
RPMI medium and allowed to form a confluent monolayer, and then the scratch is made
with 200 μL sterile pipette tip and then treated with 1.25, 2.5 and 5 μM of the compound
12c. The migration of lung cancer cells were captured by microscopic observations at 0 h
and 72 h.
10.3. Computational studies
The DNA crystal structure has been retrieved from Protein Data Bank (PDB ID:
5GWK) [46]. The protein preparation tool was used for the preparation of the human
topoisomerase II alpha with DNA. The missing atoms will be added up and peripheral water
molecules will be removed with a distance of more than 5 Å from the pocket through the
protein preparation wizard. The grid is generated by picking the active site where the co-
crystal is located and grid box of 10 × 10 × 10 Å (Schrödinger 2017-1) [47]. The potent
hybrids were sketched (12c & a) by using 2D sketcher and energy minimized, and prepared
using Ligprep for the generation of different conformers (Schrödinger 2017-1). The
different conformers thus obtained were subjected to molecular docking with SP Glide
(Schrödinger 2017-1). The poses generated were evaluated and the best one was described.
Five potent compounds and standards were subjected to ligand preparation and the diverse
conformers thus generated were envisaged for their Physico-chemical parameters using
QikProp [52]. The different descriptors were thus reported.
Acknowledgments
Authors are thankful to DoP, Ministry of Chemicals & Fertilizers, Govt. of India, New
Delhi, for the award of the NIPER fellowship. We acknowledge Dr. Chandraiah Godugu
and his lab members, Department of Regulatory Toxicology, NIPER, Hyderabad for all time
support to carry out the biological studies.
25

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