Synthesis of non-toxic anticancer active forskolin-indole-triazole conjugates along with their in silico succinate dehydrogenase inhibition studies

Article abstract of DOI:10.1002/jhet.4332

Biologically important three different pharmacophores, forskolin, indole and 1,2,3-triazoles are coupled to obtain a hybrid molecule. Here, we described the synthesis of novel series of forskolin-indole-triazole conjugates 5a-5l by using the Cu(I) catalyzed 1,3-dipolar cycloaddition reaction. Furthermore, the biological significance of the synthesized molecules was assessed by in silico and in vitro modes. All the synthesized compounds were evaluated for in vitro anticancer activity against PC-3, MCF-7, MDA-MB-231, COLO-205, HeLa, WRL-68, RAJI, CHANG and RAW-264.7 cell lines. Compound 5g was found to be the most potent in all the tested cell lines (IC₅₀ range 9.6–21.66 μg/ml, except COLO-205), 5a, 5b and 5k were observed to exert its effect only against WRL-68 (IC₅₀ range 27.69–48.18 μg/ml), when compared to parent 3 (IC₅₀ > 100 μg/ml, tested concentrations 10–50 μg/ml) and standard Doxorubicin (IC₅₀ range 0.42–3.16 μg/ml). The most potent compound 5g (MEF₅₀ 0.57) was found non-toxic to human erythrocytes as compared to control (MEF₅₀ 0.60) at tested concentration (50 μg/ml). In silico-based succinate dehydrogenase inhibition showed that the synthesized compounds were having potent binding affinity compared to forskolin. Predictive ADMET and toxicity risk assessment analysis revealed that most of the compounds were complying with the standard limit of Lipinski's rule of five for oral bioavailability.

Full text of DOI:10.1002/jhet.4332

Received: 31 March 2021
DOI: 10.1002/jhet.4332
Revised: 20 June 2021
Accepted: 24 June 2021
A R T I C L E
Synthesis of non-toxic anticancer active forskolin-indole-
triazole conjugates along with their in silico succinate
dehydrogenase inhibition studies
Devendar Ponnam1 | Niranjana Kumar Arigari1
|
Satya Srinivas Kalvagunta Venkata Naga1 | Kotesh Kumar Jonnala1
Shilpi Singh2 | Abha Meena3 | Pallavi Misra2 | Suaib Luqman2
|
1Natural Product Chemistry Division,
Abstract
CSIR-Central Institute of Medicinal and
Aromatic Plants, Research Centre,
Boduppal, Hyderabad, India
2Molecular Bio-Prospection Department,
CSIR-Central Institute of Medicinal and
Aromatic Plants, Lucknow, India
3Metabolic and Structural Biology
Department, CSIR-Central Institute of
Medicinal and Aromatic Plants, Lucknow,
India
Biologically important three different pharmacophores, forskolin, indole and
1,2,3-triazoles are coupled to obtain a hybrid molecule. Here, we described the
synthesis of novel series of forskolin-indole-triazole conjugates 5a-5l by using
the Cu(I) catalyzed 1,3-dipolar cycloaddition reaction. Furthermore, the bio-
logical significance of the synthesized molecules was assessed by in silico
and in vitro modes. All the synthesized compounds were evaluated for in vitro
anticancer activity against PC-3, MCF-7, MDA-MB-231, COLO-205, HeLa,
WRL-68, RAJI, CHANG and RAW-264.7 cell lines. Compound 5g was found
to be the most potent in all the tested cell lines (IC50 range 9.6–21.66 μg/ml,
except COLO-205), 5a, 5b and 5k were observed to exert its effect only against
Correspondence
Kotesh Kumar Jonnala and Satya Srinivas
Kalvagunta Venkata Naga, Natural
Product Chemistry Division, CSIR-Central
Institute of Medicinal and Aromatic
Plants, Research Centre, Boduppal,
Hyderabad 500 092, India.
WRL-68 (IC50 range 27.69–48.18 μg/ml), when compared to parent 3 (IC50
>
100 μg/ml, tested concentrations 10–50 μg/ml) and standard Doxorubicin (IC50
range 0.42–3.16 μg/ml). The most potent compound 5g (MEF50 0.57) was
found non-toxic to human erythrocytes as compared to control (MEF50 0.60) at
tested concentration (50 μg/ml). In silico-based succinate dehydrogenase inhi-
bition showed that the synthesized compounds were having potent binding
affinity compared to forskolin. Predictive ADMET and toxicity risk assessment
analysis revealed that most of the compounds were complying with the stan-
dard limit of Lipinski's rule of five for oral bioavailability.
Email: koteshkumarj@yahoo.com (JKK);
kvn.satyasrinivas@cimap.res.in (KVNSS).
K E Y W O R D S
anti-cancer activity, forskolin, indole, tsriazole
1 | INTRODUCTION
have increasingly attracted the interests of researchers
involved in cancer therapy [1,2]. In fact, most anticancer
Natural products (NPs) and the molecules based on NP
scaffolds, being the proven source of therapeutic agents,
drugs available today are either originated from nature or
synthesized as natural identical. One of such important
class of secondary metabolites is terpenoid which has
pharmacologically active scaffold and provides privileged
motifs for further modifications and structure activity
Devendar Ponnam and Niranjana Kumar Arigari have equal
contribution and considered as first authors.
J Heterocyclic Chem. 2021;1–12.
wileyonlinelibrary.com/journal/jhet
© 2021 Wiley Periodicals LLC.
1

2
PONNAM ET AL.
relationship analysis, etc. To date, several reports are
available wherein simple or advanced modifications have
been performed for bringing out the desired biological
properties [3,4].
Our earlier studies on the cyclic acetal protection of
hydroxyls of forskolins and andrographolides with suit-
able functional groups, significantly increased its cytotox-
icity [41–43]. In view of the above finding, it is envisaged
that synthesis of a hybrid of forskolin-indole-1,2,3-
triazole (FIT) and its anlogues may result in high value,
potent, selective and less toxic bioactive molecules. A
series of novel FITs were generated by employing Cu(I)-
catalyzed Huisgen [3 + 2] cycloaddition (click chemis-
try). The resulting derivatives were screened for their
anticancer activity in in vitro mode using MTT assay
against selected human cancer cell lines. MTT assay, cat-
alyzed by succinate dehydrogenase, is an indicator of cell
viability in terms of mitochondrial respiration. Assess-
ment of molecular interactions shown by these analogues
for succinate dehydrogenase inhibition (SDHI) and
ADMET analysis in silico mode was also carried out to
support the drawn in vitro inferences. The compounds
showing potent anticancer activity were further tested for
toxicity to human erythrocytes by performing osmotic
fragility test.
Although, several plant-derived anticancer drugs, such
as Podophyllotoxin, Vincristine, Vinblastine, Ellipticine,
Paclitaxel, Camptothecin, and Combretastatin A4, are
used clinically, they failed to achieve the desired therapeu-
tic effect due to multi-drug resistance, toxicity, or poor bio-
availability. Therefore, it is necessary to identify and
develop more effective and safe anticancer drugs [5,6].
Forskolin 3 is a labdane diterpenoid isolated from
Coleus forskohlii. Forskolin increases intracellular levels
of important signaling agent cyclic AMP (for biological
response of cells to hormones and other extracellular sig-
nals) by activating adenylyl cyclase enzyme. Forskolin
and its analogues have shown various biological activi-
ties, such as antihypertensive, positive inotropic [6–8],
antiobesity [9,10], antidiabetic [11], and anticancer activi-
ties [12,13]. Experimental studies on different cancer cell
lines have shown synergistic effects of forskolin in
upregulating cellular responses [14–17]. A protective
effect against ionizing radiation [18] and UBV-induced
apoptosis [19] has been shown in in vitro. Forskolin has
been demonstrated to have an angiogenic effect via
cAMP-dependent protein kinase pathways [20]. How-
ever, the water-insoluble nature of forskolin limits its
clinical usefulness.
Second pharmacophore, the indole and its various con-
jugates present in many natural and synthetic molecules
exhibit a wide range of significant biological activities,
such as antidiabetic [21], anti-inflammatory [22],
antimalarial [23], antibacterial [24], antifungal [25], ant-
iviral [26] and anticancer [27]. This moiety is very impor-
tant for its medicinal and biological aspects, thus
attracting a lot of scientific attention.
The third pharmacophore, 1,2,3-triazole has been
shown to possess several desirable structural features in
the context of medicinal and click chemistry. The syn-
thetically prepared 1,2,3-triazole derivatives have shown
diverse biological activities, such as anticancer [28],
antibiotic [29], antifungal, antibacterial, antitubercular
and antiviral [30–32]. Triazoles are stable to acid/base
hydrolysis and reductive/oxidative conditions, indicative
of a high aromatic stabilization and, also relatively resis-
tant to metabolic degradation. At the same time, they
readily associate with biological targets, through hydro-
gen bonding and dipole interactions [33,34]. NPs hooked
with the 1,2,3-triazole moiety have also shown excellent
anticancer activities, such as Podophyllotoxin (A) [35],
Oleanolic acid (B) [36], betulinic acid (C) [37],
dehydroabietic acid (D) [38], imbricatolic acid (E) [39]
and 10β-artemisinin (F) derivatives [40] (Figure 1).
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
A three-step synthesis to obtain the target molecules is
depicted in Schemes 1 and 2. In the first step, the precur-
sor N-propargyl-indole-3-carbaldehyde 2 was synthesized
by treating indole-3-carbaldehyde 1 with propargyl bro-
mide/K2CO3 in DMF (92% yield). In the second step, alde-
hyde 2 was subjected to acetalization with forskolin 3 in
presence of Amberlist-15 (wet) in CH2Cl2 at room temper-
ature for 24 h, to give novel 3,19-O-(N-propargyl-3-indo-
lylidene)forskolin 4 in 75% yield (Scheme 1). In the third
step, terminal alkyne 4 and various substituted azides a-l
(synthesized in situ from their halides/amine precursors)
[44] in the presence of catalytic Cu(I) (CuSO4/sodium
ascorbate), resulted in a series of 12 1,4-disubstituted
triazoles 5a-5l in high regioselectivity (Scheme 2). Aqueous
conditions (H2O/t-BuOH, 2:1) facilitated precipitation of
products, which were isolated in high purity [45].
The structures of all the hybrids were elucidated by
NMR, IR and mass spectral data. For example, structures
of 4 and 5l were assigned as follows. The compound
4 showed characteristic stretching in IR spectra at
2123 cmÀ1 (C C), confirmed the new triple bond. A
molecular ion peak [M + H]+ at m/z 576.3 was observed
1
in ESI-MS spectra. In H NMR spectrum, the presence of
characteristic singlet at δH 5.74 (1H) corresponding to the
newly formed acetal ring (O-CH-O). A triplet in up field
at δ 2.41 (3JH,H = 2.4 Hz, 1H) coupled with a doublet at δ

PONNAM ET AL.
3
O
N
H
O
N
N
N
N
N
N
O
COOH
NO2
O
O
O
O
N
N
N
O
HO
O
NC
(B)
MeO
OMe
(C)
OH
(A)
O
N
N
N
C5H11
N
N
N
O
H
OH
H
O
O
O
O
H
O
O
N
N
N
(F)
(D)
(E)
FIGURE 1 Semisynthetic cytotoxic 1,2,3-triazole conjugates of 10β-Podophyllotoxin (A), Oleanolic acid (B), Betulinic acid (C),
Dehydroabietic acid (D), Imbricatolic acid (E) and 10β-Artemisinin (F)
Step 1: Propargylation of Indole 3-carbaldehyde
δH 5.65 (1H) confirmed the new cyclic indolylidene acetal
ring (O-CH-O). The bridged indole N-CH2 protons were
showed as a singlet at δH 5.44 (2H). A characteristic dou-
blet δH 4.96 (2H, J = 6 Hz, overlapped), multiplet δH
6.03–5.94 (1H) and two overlapped doublet of doublet at
δH 5.22 (1H), 4.87 (1H) confirm the end allyl group
((N-CH2-CH═CH2) on triazole ring. Presence of two dou-
blets (J = 8 Hz) at δH 7.75 (1H), 7.61 (1H) and two multi-
plets at δH 7.16 (1H), 7.04 (1H) confirmed the four
phenyl ring protons of Indole. Also, a molecular ion peak
[M + H]+ observed at m/z 659.33 in ESI-MS spectrum,
confirmed the assigned structure 5l.
O
H
O
H
K2CO3
Br
N
DMF
N
H
RT, 12 h
1
2 (92 %)
Propargyl
bromide
Indole-3-carbaldehyde
N-Propargyl-indole-
3-carbaldehyde
Step 2: Acetalization of Forskolin
N
O
O
O
H
O
O
OH
Amberlist-15 (Wet)
O
OH
OH
OH
1,2-Dichloroethane
RT, 24 h
N
OAc
OAc
OH
4
2
3
(75 %)
1,9-O-(N-propargyl-3-
Forskolin
N-Propargyl-indole-
3-carbaldehyde
2.2 | In silico succinate dehydrogenase
inhibition
indolylidene) forskolin
SCHEME 1 Synthesis of 1,9-O-(N-Propargyl-3-indolylidene)
forskolin 4
Molecular docking of succinate dehydrogenase (1YQ3), a
NAD (P) H-dependent cellular oxido-reductase enzyme
known to catalyze the conversion of tetrazolium dye into
its insoluble purple colored formazan crystals, is des-
cribed here. Under the defined condition it reflects the
number of viable cells, therefore, considered as a front-
line target for cytotoxicity evaluation. Docking study was
performed to understand the molecular interaction of
forskolin derivatives with SDH and the obtained results
are expressed in terms of binding energy (BE) and inhibi-
tion constant (Ki). All the compounds showed strong
binding interaction with the SDH in terms of lowest
4.84 (3JH,H = 2.4 Hz, 2H), confirms the presence of
N-propargyl group (N–CH2–C CH). In aromatic region,
a characteristic singlet at δH 7.33 (1H, pyrrole ring H)
along with two doubles (J = 8 Hz) at δH 7.35 (1H, Ar-H),
7.36 (1H, Ar-H) and two multiplets in the region of δH
7.11–7.25 (2H, Ar-H), due to the presence of 3-substituted
indole. In 1H NMR spectrum of 5l, two characteristic sin-
glets at δH 7.99 (1H), 7.60 (1H) corresponding to protons
of triazole ring and pyrrole ring, respectively. A singlet at

4
PONNAM ET AL.
Step 3: Azide-Alkyne cycloaddition/Click reaction
SCHEME 2 Synthesis of forskolin-indole-
triazole conjugates 5a-5l
R
N
N
N
N
N
O
O
O
O
CuSO4.5H2O
Sodium ascorbate
O
O
R
O
O
N
N
N
H2O/t-BuOH, 2:1
OAc
RT, 12 h.
OAc
azides (a-l)
OH
OH
5a-5l
up to 91 % yields
4
Where R=
Cl
Cl
Cl
O2N
F
F
(5f)
(5e)
CN
(5c)
(5d)
(5j)
(5b)
(5h)
(5a)
Ac
HOOC
NC
(5g)
(5k)
(5l)
(5i)
either by superimposing one or more atoms in one struc-
ture on corresponding atom (s) of the other, or by
ordered group in one structure over the other. The dis-
tance (error) RMS value in molecular geometry between
two structures was calculated and showed (Table S1).
The results indicate that RMSD between the compound
3 (forskolin) and the synthesized compounds were not
significantly different, which indicated that perhaps
most of the compounds are having potential cytotoxic
activity.
In addition to calculate RMSD, the predictive
ADMET, drug-likeness and toxicity levels (Tables S2–
S4) were also evaluated. ADMET is helpful in deciding
the acceptance of forskolin derivatives as potential anti-
cancer compounds. The toxicity screening data can be
used for (a) deciding safe concentration level to be used
for animal assays, (b) in the reduction of animal usage
during screening out of potentially toxic molecules and
(c) in optimizing the therapeutic ratios of the potential
anticancer forskolin derivatives. All derivatives showed
compliance with the standard range. As per in silico
studies, in comparison to forskolin 3 most of the synthe-
size compounds showed good binding affinity, low
RMSD, low hepatotoxicity, genotoxicity, carcinogenicity
and mutagenicity, and were following Lipinski's rule of
five for oral bioavailability. Hence these results were
validated using in vitro MTT assay.
TABLE 1 Binding energy and inhibition constants for forskolin
and its derivatives
Succinate dehydrogenase
(PDB ID: 1YQ3) Docking
Entry score (kcal/mole)
Inhibitory
constant (Ki)
3
À6.97
À6.37
À7.49
À8.22
À8.51
À9.70
À9.14
À7.08
À9.02
À8.56
À8.04
À9.17
À6.75
À7.51
7.76 μM
4
21.36 μM
3.24 μM
5a
5b
5c
5d
5e
5f
5g
5h
5i
940.37 nM
581.01 nM
77.60 nM
198.06 nM
6.42 μM
245.32 nM
528.08 nM
1.27 μM
5j
190.91 nM
11.29 μM
3.12 μM
5k
5l
binding energy ranged from À6.37 to À9.70 Kcal/mol
and Ki from 77.60 nM to 21.36 μM (Table 1), when com-
pared to forskolin and standard reference compound
Malonate (À5.64 kcal/mol and 73.96 μM). Hence, it may
be interpreted that these compounds have strong inhibi-
tory activity for SDH and thus modulate the activity of
this enzyme and affect the viability of the cells.
2.3 | In vitro cytotoxic activity
Further, to find out root mean square distance
(RMSD), molecular geometry comparison was done
All the compounds evaluated for in vitro cytotoxic activi-
ties against 10 cancer cell lines MCF-7 (human breast

PONNAM ET AL.
5
adenocarcinoma), MDA-MB-231 (human breast adeno-
carcinoma), CHANG (human liver cell line), WRL-68
(human hepatocellular carcinoma), COLO-205 (human
colon carcinoma), PC-3 (human prostrate carcinoma),
K562 (human leukemia carcinoma), Hela (human cervi-
cal carcinoma), RAW-264.7 (mouse macrophages cell
line) and RAJI (human Burrkit's lymphoma cell line)
using MTT assay. Doxorubicin was used as a standard
(Table 2). Among all the synthesized derivatives, 5g
(p-COOH on phenyl) possessed excellent cytotoxicity
(IC50 9.6–21.66 μg/ml) in most of the tested cell lines
(except COLO-205) and 5a, 5b and 5k displayed moder-
ate cytotoxicity against WRL-68 cell line (IC50 27.69–
48.18 μg/ml) and remaining compounds 5c-5f, 5h-5j and
5l were found inactive (IC50 > 100 μg/ml), when com-
pared to parent (in all the cell lines IC50 > 100 μg/ml,
tested concentrations 10–50 μg/ml) and standard Doxoru-
bicin (IC50 range 0.42–3.16 μg/ml). From Table 2, it
clearly indicates that FIT hybrids having p-flurobenzyl,
2.4 | Erythrocyte osmotic fragility assay
Erythrocyte tensile strength is responsible for normal
function of erythrocytes. Determination of osmotic fragil-
ity patterns is a good index of membrane integrity, so it is
frequently used for the measurement of erythrocyte ten-
sile strength and resistance. Compound 5g possessed sig-
nificant cytotoxicity was further evaluated for osmotic
fragility. Compound 5g has exerted a pronounced protec-
tive effect on erythrocyte osmotic fragility which is evi-
dent by a significant shift of the curve to the right and a
decrease in mean erythrocyte fragility (MEF), rep-
resenting a resistance to hemolysis (Figure 3), when
exposed to an environment of hypotonic solutions. At
higher tested concentration (50 μg/ml) protective effect
of both compounds on osmotic fragility of erythrocyte is
shown (Table S5) which gives the MEF of erythrocyte
incubated for 30 min at 370ꢀC.
A significant (p < 0.01) decrease in MEF, compared
with control was observed when erythrocytes are incu-
bated with respective compounds. Hence, the tested con-
centration of 5g may be considered as non-toxic toward
human erythrocytes.
p-nitrobenzyl
o,p-dichlorobenzyl,
m-chlorobenzyl,
p-acetylphenyl, p-cyanophenyl, n-butyl, allyl group sub-
stitutions were found non-toxic against all the tested cell
lines at tested concentration. Whereas substitutions like
n-octyl, biphenyl, o-flurobenzyl substitutions moderate
active against only WRL-68 cell line, and with a suitable
polar p-carboxyphenyl on triazole ring was found to be
highly potential against eight cell lines.
3 | CONCLUSION
Since compound 5g showed better cytotoxicity pro-
files, its interaction with the SDH receptor was further
studied in depth and found that the interacting amino
acids residues at the binding site were ARG 464, SER
57, GLU 46, GLU 34, LYS 52,THR 475, GLY 217, GLN
477, TYR 220, ASP 92, LYS 87, THR 86, GLY 256, THR
367, THR 228 for compound 5g with four H-bonds
(Figure 2).
In conclusion, synthesis of molecular hybrids with multi-
ple pharmacophores is an emerging structural modifica-
tion tool toward designing potential medicinal drugs. In
the present synthesis, the introduced N-propargyl group
on 1,9-O-(3-indolylidene) forskolin provided a uniquely
reactive handle within the structure and was subse-
quently reacted with a series of aryl, benzyl, aliphatic and
allylic azides to afford 12 forskolin-indole-triazole (FIT)
TABLE 2 Cytotoxic potential of forskolin and their conjugates against various human cancer cell lines by MTT assay
Cytotoxic activity (IC50 μg/ml)a
Entry
3
5a
5b
5g
5k
Doxo
3.16
1.65
1.97
0.49
0.51
1.92
3.06
0.42
2.96
PC-3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
46.46
>100
>100
>100
>100
>100
>100
>100
>100
27.69
>100
>100
>100
9.89
9.93
6.00
>100
12.86
9.39
21.66
15.30
9.20
>100
>100
>100
>100
>100
48.18
>100
>100
>100
MCF-7
MDA-MB-231
COLO-205
HeLa
WRL-68
RAJI
CHANG
RAW-264.7
aIC50 values depicted are mean of three experiments (n = 3) in duplicate.

6
PONNAM ET AL.
FIGURE 2 Binding pocket for
compound 5g within the succinate
dehydrogenase (PDB ID: 1YQ3).
Hydrogen bonds formed between the
compound 5g and various amino acid
residues within the pockets are
represented by dashed lines (magenta
color)
FIGURE 3 Osmotic fragility curve
of compound 5g
library via Cu (I)-catalyzed 1,3-dipolar cycloaddition.
Novel derivatives, clean reaction profiles, greater selectiv-
ity, simple experimental/product isolation procedures,
makes it a useful and attractive strategy for the synthesis
of forskolin-indole-triazole hybrids.
The derivatives were evaluated for anticancer activity
against PC-3, MCF-7, K562, MDA-MB-231, COLO-
205, HeLa, WRL-68, RAJI, CHANG and RAW-264.7 cell
lines. Compound 5g has shown significant cytotoxicity
IC50 < 21.66 μg/ml against all the tested cell lines except
COLO-205. To validate the above MTT assay results, an
in silico-based molecular docking of the derivatives was
performed against SDH to understand binding interac-
tions. Most of the compounds showed strong inhibitory
activity for SDH when compared to parent forskolin.
Mean erythrocyte fragility (MEF50) assay revealed
compound 5g was nontoxic to human erythrocytes at
50 μg/ml. All the experimental results prompt us to

PONNAM ET AL.
7
consider the designed scaffold as a starting point for the
development of the novel and more potent forskolin anti-
cancer agents in the future.
7.34 (m, 2H), 4.94 (d, J = 2.4 Hz, 2H), 2.56 (t, J = 2.4 Hz,
1H); ESI-MS: positive ion mode: m/z = 183.96 [M + H]+,
calculated mass (M) for C12H9NO is 183.07.
4 | EXPERIMENTAL SECTION
4.1 | Chemistry
4.2.2 | Synthesis of 1,9-O-(N-propargyl-
3-indolylidene)forskolin 4 (Step 2)
To a solution of forskolin 3 (0.5 g, 1.22 mmol) and
N-propargyl-indole-3-carbaldehyde 2 (0.33 g, 1.83 mmol)
in 1,2-dichloroethane (DCE), Amberlist-15 wet (1 g) was
added and the reaction mixture was further stirred at rt
for 24 h. After completion of reaction, reaction mixture
was filtered and washed with DCE and evaporated off
under reduced pressure. The obtained crude product was
subjected to neutral Al2O3 column chromatography using
50% ethyl acetate in n-hexane as mobile phase to give
pure 4. Yield: 0.53 g (75%), white solid, m.p. 190–192ꢀC;
IR (KBr): 3443, 2931, 2123, 1737, 1697, 1370, 1238,
All other chemicals and reagents purchased from Aldrich
(India), AVRA Chemicals Pvt. Ltd. (India) and were used
without further purification. TLC silica gel 60 F254
(Merck, Germany) was used for TLC. Visualization of the
developed TLC was performed by UV light or 5% H2SO4
in MeOH stain. Melting points were measured using
A. KRUSS OPTRONIC (Germany) melting point appara-
tus and are uncorrected. IR spectra were recorded on a
Bruker Optics (Tensor 27 model, Germany) FT-IR spec-
trophotometer (using KBr pellets) and reported in wave
1
1
number (cmÀ1). H NMR (300/400 MHz) and 13C NMR
1053 cmÀ1; H NMR (400 MHz, CDCl3): δ (ppm) 7.75 (d,
(75/100 MHz) spectra were measured in CDCl3 at room
J = 8 Hz, 1H), 7.37 (d, J = 8 Hz, 1H), 7.33 (s, 1H), 7.25–
7.22 (m, 1H), 7.16–7.12 (m, 1H), 5.75 (dd, J = 17.2 and
10.8 Hz, 1H), 5.74 (s, 1H), 5.51 (d, J = 3.6 Hz, 1 H), 5.18
(dd, J = 17.2 and 1.2 Hz, 1H), 4.84 (d, J = 2.8 Hz, 2H),
4.74 (dd, J = 10.8 and 1.2 Hz, 1H), 4.51 (brs, 1H), 4.27
(brs, 1H), 2.93 (d, J = 16.4 Hz, 1H), 2.54 (d, J = 2 Hz,
1H), 2.45 (d, J = 16.4 Hz, 1H), 2.41 (t, J = 2.4 Hz, 1H),
2.16 (m, 1H), 2.15 (s, 3H), 1.83–1.70 (m, 2H), 1.75 (s, 3H),
1.64–1.61 (m, 1H), 1.60 (s, 3H), 1.34 (s, 3H), 1.29
(s, 3H), 1.08–1.05 (m, 1H), 1.03 (s, 3H); 13C NMR
(100 MHz, CDCl3): δ (ppm) 206.20, 169.62, 146.10, 136.05,
126.18, 122.17, 120.60, 120.14, 113.96, 110.30, 109.41,
94.37, 85.48, 80.86, 77.50, 76.61, 76.48, 75.42, 73.76, 70.21,
50.20, 43.11, 39.39, 36.53, 35.88, 34.53, 33.05, 31.26, 24.81,
23.73, 23.38, 21.20, 17.58; ESI-MS: positive ion mode:
m/z = 576.32 [M + H]+, 598.27 [M + Na]+, 614.24
[M + K]+, calculated mass (M) for C34H41NO7 is 575.29.
temperature on
a Bruker Avance-III 300/400 MHz
(Switzerland) instruments. The chemical shifts are re-
ported as δ parts per million (ppm) in CDCl3 (δH = 7.26;
δC = 77.0) using tetramethylsilane as an internal stan-
dard. Data are reported as follows: chemical shift, multi-
plicity (s: singlet, d: doublet, t: triplet, dd: doublet of
doublet, q: quartet, br: broad, m: multiplet), coupling
constants (J in Hz) and integration.
4.2 | General procedure and spectral
data of synthesized compounds
4.2.1 | Synthesis of N-propargyl-indole-
3-carbaldehyde 2 (Step 1)
To a solution of indole-3-carbaldehyde 1 (0.73 g, 5 mmol)
and potassium carbonate (1.04 g, 7.5 mmol) in DMF
(20 ml), propargyl bromide (0.7 ml g, 7.5 mmol) was
added drop wise and the resulting mixture was allowed
to stir overnight at temperature. After completion of reac-
tion monitored by TLC, the reaction mixture was par-
titioned between CH2Cl2 and water, and the CH2Cl2
layer was collected. The aqueous layer was extracted two
times with CH2Cl2, the combined organic extracts was
dried over anhydrous Na2SO4 and concentrated under
vacuum. It was crystallized from i-propanol-water (1:9) to
obtain the desired N-propargyl-Indole-3-carbaldehyde
2 in pure form. Yield: 0.85 g (92%), white solid, m.p. 104–
4.2.3 | Synthesis of forskolin-indole-triazole
conjugates 5a-5l (Step 3)
To a solution of 4 (100 mg, 0.174 mmol), aryl azide (a-l,
0.34 mmol) were suspended in 2:1 mixture of water and
tert-butyl alcohol (10 ml total volume). Sodium ascorbate
(10 mol%, 1 M) and CuSO4.5H2O (5 mol%, 0.1M) were
added sequentially to the reaction mixture and the mix-
ture was allowed to room temperature for next 12 h.
After completion of reaction (TLC monitoring), the reac-
tion mixture was diluted with water (10 ml) and cooled
on ice. The precipitate was isolated by vacuum filtration
and washed with cold water (3 Â 5 ml) and cold
n-hexane (3 Â 5 ml) to afford crude compound. It was
1
106ꢀC; IR (KBr): 3196, 2121, 1740, 1641 cmÀ1; H NMR
(400 MHz, CDCl3): δH (ppm) 10.02 (s, 1H), 8.32 (dd,
J = 6.8 and 2.0 Hz, 1H), 7.90 (s, 1H), 7.42 (m, 1H), 7.39–

8
PONNAM ET AL.
purified using flash column chromatography to afford
mode: m/z = 727.31 [M + H]+, 749.33 [M + Na]+, calcu-
pure compounds 5a-5l.
lated mass (M) for C41H47FN4O7 is 726.34.
4.2.4 | 1,9-O-{N-[(1-Biphenyl-1H-
1,2,3-triazol-4-yl)methyl]-3 indolylidene}
forskolin (5a)
4.2.6 | 1,9-O-{N-((1-[p-flurobenzyl]-1H-
1,2,3-triazol-4-yl)methyl)-3-indolylidene}
forskolin (5c)
Yield: 102 mg (72%), brown solid, m.p. 108–110ꢀC; IR
Yield: 115 mg (91%), yellow solid, m.p. 120–122ꢀC; IR
1
1
(KBr): 3463, 2965, 2226, 1739, 1370, 1223, 1023 cmÀ1; H
(KBr): 3461, 2964, 1738, 1370, 1224, 1021 cmÀ1; H NMR
NMR (400 MHz, CDCl3): δ (ppm) 7.76 (d, J = 8 Hz, 2H),
7.63 (td, J = 8 and 1.2 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H),
7.46 (d, J = 7.6 Hz, 2H), 7.29 (m, 3H), 7.29 (d, J = 7.6 Hz,
2H), 7.21–7.09 (m, 2H), 5.72 (s, 1H), 5.67 (dd, J = 17.2 and
10.4 Hz, 1H), 5.52 (s, 2H), 5.48 (d, J = 3.6 Hz, 1H), 5.42 (d,
J = 1.6 Hz, 2H), 5.07 (dd, J = 17.2 and 1.6 Hz, 1H), 4.53
(dd, J = 10.4 and 1.2 Hz, 1H), 4.49 (brs, 1H), 4.25 (brs, 1H),
2.90 (d, J = 16.4 Hz, 1H), 2.52 (brs, 1H), 2.44
(d, J = 16.4 Hz, 1H), 2.14 (m, 3H), 2.13 (s, 3H), 1.86–1.76
(m, 2H), 1.74 (s, 3H), 1.70–1.63 (m, 1H), 1.58 (s, 3H), 1.32
(s, 3H), 1.29 (s, 3H), 1.09–1.06 (m, 1H), 1.02 (s, 3H); 13C
NMR (100 MHz, CDCl3): δH (ppm) 206.11, 169.70, 146.15,
144.99, 145.53, 138.53, 136.18, 135.11, 133.80, 132.92,
130.04, 129.48, 129.48, 128.35, 128.35, 127.90, 126.64, 126.0,
122.23, 122.15, 120.51, 120.06, 118.51, 113.95, 111.20,
110.09, 109.74, 94.36, 85.40, 80.85, 77.3, 76.5, 75.37, 70.17,
53.74, 50.13, 43.12, 42.32, 39.34, 36.55, 34.53, 33.06, 31.24,
24.79, 23.71, 23.34, 21.24, 17.57; ESI-MS: positive ion mode:
m/z = 810 [M + H]+, 832.34 [M + Na]+, 848.31 [M + K]+,
calculated mass (M) for C48H51N5O7 is 809.38.
(400 MHz, CDCl3): δ (ppm) 7.75 (d, J = 7.6 Hz, 1H), 7.30
(d, J = 8 Hz, 1H), 7.25 (s, 1H), 7.23–7.18 (m, 4H), 7.13–
7.09 (m, 1H), 7.00 (t, J = 8.4 Hz, 2H), 5.71 (s, 1H), 5.64
(dd, J = 17.2 and 10.8 Hz, 1H), 5.47 (d, J = 4 Hz, 1H), 5.41
(s, 2H), 5.39 (d, J = 2.8 Hz, 1H), 5.05 (dd, J = 17.2 and
1.6 Hz, 1H), 4.49 (brs, 1H), 4.48 (dd, J = 10.8 and 1.2 Hz,
1H), 4.51 (brs, 1H), 4.26 (brs, 1H), 2.88 (d, J = 16.4 Hz,
1H), 2.51 (d, J = 2 Hz, 1H), 2.44 (d, J = 16.4 Hz, 1H),
2.17–2.10 (m, 1H), 2.14 (s, 3H), 1.84–1.79 (m, 2H), 1.74
(s, 3H), 1.68–1.63 (m, 1H), 1.59 (s, 3H), 1.33 (s, 3H), 1.30
(s, 3H), 1.10–1.06 (m, 1H), 1.03 (s, 3H); 13C NMR
(100 MHz, CDCl3): δ (ppm) 205.97, 169.69, 164.07, 146.17,
145.04, 136.16, 130.38, 130.02, 129.94, 126.58, 126.01,
122.21, 121.77, 120.50, 120.07, 116.19, 115.97, 113.98,
110.0, 109.68, 94.34, 85.42, 80.83, 77.3, 76.5, 75.33, 70.20,
53.41, 50.11, 43.14, 42.33, 39.36, 36.54, 34.52, 33.06, 31.27,
24.78, 23.71, 23.33, 21.21, 17.57; ESI-MS: positive ion
mode: m/z = 727.38 [M + H]+, 749.33 [M + Na]+, calcu-
lated mass (M) for C41H47FN4O7 is 726.34.
4.2.7 | 1,9-O-{N-((1-[p-nitrobenzyl]-1H-
1,2,3-triazol-4-yl)methyl)-3-indolylidene}
forskolin (5d)
4.2.5 | 1,9-O-{N-[(1-[o-flurobenzyl]-1H-
1,2,3-triazol-4-yl)methyl]-3-indolylidene}
forskolin (5b)
Yield: 118 mg (90%), yellow solid, m.p. 108–110ꢀC; IR
1
Yield: 105 mg (83%), yellow solid, m.p. 118–120ꢀC; IR
(KBr): 3464, 2963, 1738, 1350, 1224, 1020 cmÀ1; H NMR
1
(KBr): 3463, 2964, 1739, 1370, 1226, 1099 cmÀ1; H NMR
(400 MHz, CDCl3): δ (ppm) 8.18 (d, J = 8.4 Hz, 1H), 7.75
(d, J = 7.8 Hz, 1H), 7.38–7.28 (m, 3H), 7.24–7.09 (m, 4H),
5.72 (s, 1H), 5.65 (dd, J = 16.8 and 10.4 Hz, 1H), 5.56
(s, 2H), 5.49–5.36 (m, 2H), 5.04 (d, J = 16.8 Hz, 1H), 4.45
(m, 2H), 4.26 (brs, 1H), 2.88 (d, J = 16.4 Hz, 1H), 2.49–
2.44 (m, 2H), 2.14 (s, 3H), 2.04 (m, 1H), 1.78–1.68
(m, 2H), 1.74 (s, 3H), 1.58 (s, 3H), 1.32 (s, 3H), 1.30
(s, 3H), 1.10–1.06 (m, 1H), 1.01 (s, 3H); 13C NMR
(75 MHz, CDCl3): δ (ppm) 205.92, 169.87, 148.02, 146.23,
145.52, 141.47, 136.07, 128.74, 128.74, 126.54, 126.01,
124.30, 124.30, 122.30, 122.22, 120.54, 120.17, 114.14,
109.92, 109.63, 94.26, 85.36, 80.83, 76.52, 76.44, 75.31,
70.18, 53.10, 50.04, 43.13, 42.32, 39.32, 36.54, 34.53, 33.04,
31.24, 24.76, 23.71, 23.32, 21.27, 17.59; ESI-MS: positive
ion mode: m/z = 754.32 [M + H]+, 776.27 [M + Na]+,
calculated mass (M) for C41H47N5O9 is 753.34.
(300 MHz, CDCl3): δ (ppm) 7.74 (d, J = 7.5 Hz, 1H), 7.33
(d, J = 8.1 Hz, 1H), 7.29 (s, 1H), 7.26–7.03 (m, 7H), 5.71
(s, 1H), 5.67 (dd, J = 17.1 and 10.5 Hz, 1H), 5.51 (s, 2H),
5.48 (d, J = 3.9 Hz, 1H), 5.39 (brs, 1H), 5.07 (d, J =
17.1 Hz, 1H), 4.53 (d, J = 11.1 Hz, 1H), 4.50 (brs, 1H),
4.25 (brs, 1H), 2.89 (d, J = 16.2 Hz, 1H), 2.52 (brs, 1H),
2.44 (d, J = 16.2 Hz, 1H), 2.14 (m, 4H), 1.84–1.79
(m, 2H), 1.74 (s, 3H), 1.63 (m, 1H), 1.59 (s, 3H), 1.33
(s, 3H), 1.29 (s, 3H), 1.09 (m, 1H), 1.03 (s, 3H); 13C NMR
(75 MHz, CDCl3): δ (ppm) 206.47, 170.00, 146.53, 145.22,
136.57, 134.5, 131.16, 130.85, 126.99, 126.42, 125.16,
122.58, 122.50, 120.89, 120.42, 116.34, 116.06, 114.33,
110.45, 110.10, 94.78, 85.85, 81.24, 77.8, 76.9, 75.76, 70.60,
50.54, 48.04, 43.53, 42.68, 39.77, 36.93, 34.91, 33.43, 31.67,
25.18, 24.10, 23.72, 21.56, 17.96; ESI-MS: positive ion

PONNAM ET AL.
9
4.2.8 | 1,9-O-{N-((1-[o,p-dichlorobenzyl]-1H-
1,2,3-triazol-4-yl)methyl)-3-indolylidene}
forskolin (5e)
(d, J = 8.0 Hz, 1H), 7.95 (dd, J = 8.0 and 1.2 Hz, 1H),
7.78 (d, J = 8.0 Hz, 1H), 7.56 (t, J = 8 Hz, 1H), 7.40
(d, J = 8.0 Hz, 1H), 7.37 (s, 1H), 7.22 (t, J = 7.2 Hz, 1H),
7.13 (t, J = 7.2 Hz, 1H), 5.76 (s, 1H), 5.75 (dd, J = 17.2
and 10.4 Hz, 1H), 5.48 (d, J = 4.0 Hz, 1H), 5.42 (s, 2H),
5.41 (d, J = 2.4 Hz, 2H), 5.07 (dd, J = 17.2 and 1.2 Hz,
1H), 4.53–4.48 (m, 2H), 4.25 (brs, 1H), 2.88
(d, J = 16.4 Hz, 1H), 2.52 (d, J = 2.0 Hz, 1H), 2.44
(d, J = 16.4 Hz, 1H), 2.18 (m, 1H), 2.15 (s, 1H), 1.84–1.79
(m, 2H), 1.74 (s, 3H), 1.67–1.61 (m, 1H), 1.59 (s, 3H), 1.33
(s, 3H), 1.30 (s, 3H), 1.15–1.10 (m, 1H), 1.02 (s, 3H); ESI-
MS: positive ion mode: m/z = 761.30 [M + Na]+, calcu-
lated mass (M) for C41H46N4O9 is 738.32.
Yield: 98 mg (72%), white solid, m.p. 98–100ꢀC; IR (KBr):
1
3435, 2970, 1741, 1369, 1222 cmÀ1; H NMR (400 MHz,
CDCl3): δ (ppm) 7.74 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 2 Hz,
1H), 7.32 (d, J = 8 Hz, 1H), 7.29 (s, 1H), 7.26 (s, 1H), 7.22–
7.16 (m, 2H), 7.13–7.09 (m, 2H), 7.05 (d, J = 8 Hz, 1H), 5.72
(s, 1H), 5.66 (dd, J = 17.2 and 10.8 Hz, 1H), 5.54 (s, 2H),
5.48 (d, J = 3.6 Hz, 1H), 5.41 (d, J = 2 Hz, 2H), 5.08 (dd, J =
17.2 and 1.6 Hz, 1H), 4.53 (d, J = 10.8 and 1.6 Hz, 1H), 4.49
(t, J = 3.2 Hz, 1H), 4.25 (brs, 1H), 2.88 (d, J = 16.4 Hz, 1H),
2.52 (d, J = 2.4 Hz, 1H), 2.44 (d, J = 16.4 Hz, 1H), 2.16 (m,
1H), 2.14 (s, 1H), 1.82–1.79 (m, 2H), 1.74 (s, 3H), 1.71–1.63
(m, 1H), 1.59 (s, 3H), 1.33 (s, 3H), 1.29 (s, 3H), 1.09–1.05 (m,
1H), 1.03 (s, 3H); 13C NMR (100 MHz, CDCl3): δ (ppm)
206.06, 169.69, 146.16, 144.93, 136.15, 135.49, 134.07, 130.99,
130.96, 129.75, 127.93, 126.58, 126.02, 122.32, 122.23, 120.50,
120.09, 113.99, 110.04, 109.70, 94.32, 85.42, 80.84, 77.3,
76.50, 75.35, 70.19, 50.85, 50.11, 43.13, 42.30, 39.35, 36.54,
34.53, 33.07, 31.25, 24.78, 23.71, 23.33, 21.20, 17.57; ESI-MS:
positive ion mode: m/z = 777.68 [M + H]+, 779.22 [M +
Na]+, calculated mass (M) for C41H46Cl2N4O7 is 776.27.
4.2.11 | 1,9-O-{N-((1-[p-acetylphenyl]-1H-
1,2,3-triazol-4-yl)methyl)-3-indolylidene}
forskolin (5h)
Yield: 111 mg (87%), white solid, m.p. 144–146ꢀC; IR
1
(KBr): 3466, 2963, 1738, 1685, 1367, 1228, 1020 cmÀ1; H
NMR (400 MHz, CDCl3): δ (ppm) 8.02 (d, J = 8.4 Hz,
1H), 7.34 (d, J = 8.1 Hz, 1H), 7.78–7.69 (m, 3H), 7.20–
7.07 (m, 3H), 5.72 (s, 1H), 5.67 (dd, J = 17.1 and 10.5 Hz,
1H), 5.48–5.46(m, 3H), 5.07 (d, J = 17.1 Hz, 1H), 4.53
(d, J = 17.1 Hz, 1H), 4.46 (brs, 1H), 4.23 (brs, 1H), 2.88
(d, J = 16.5 Hz, 1H), 2.58 (s, 3H), 2.44 (m, 2H), 2.10
(s, 3H), 2.0 (m, 1H), 1.71–1.66 (m, 2H), 1.71 (s, 3H), 1.67–
1.61 (m, 1H), 1.55 (s, 3H), 1.26 (s, 3H), 1.29 (s, 3H), 1.10
(m, 1H), 1.0 (s, 3H); ESI-MS: positive ion mode:
m/z = 759.39 [M + Na]+, calculated mass (M) for
C42H48N4O8 is 736.34.
4.2.9 | 1,9-O-{N-((1-[m-chlorobenzyl]-1H-
1,2,3-triazol-4-yl)methyl)-3-indolylidene}
forskolin (5f)
Yield: 102 mg (79%), white solid, m.p. 118–120ꢀC; IR
1
(KBr): 3464, 2959, 1738, 1370, 1222, 1021 cmÀ1; H NMR
(400 MHz, CDCl3): δ (ppm) 7.75 (d, J = 8.0 Hz, 1H), 7.31
(d, J = 8.4 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H), 7.26 (s, 1H),
7.24–7.09 (m, 5H), 5.72 (s, 1H), 5.65 (dd, J = 17.2 and
10.8 Hz, 1H), 5.48 (d, J = 4.0 Hz, 1H), 5.42 (s, 2H), 5.41
(d, J = 2.4 Hz, 2H), 5.07 (dd, J = 17.2 and 1.2 Hz, 1H),
4.53–4.48 (m, 2H), 4.25 (brs, 1H), 2.88 (d, J = 16.4 Hz,
1H), 2.52 (d, J = 2.0 Hz, 1H), 2.44 (d, J = 16.4 Hz, 1H),
2.18 (m, 1H), 2.15 (s, 1H), 1.84–1.79 (m, 2H), 1.74 (s, 3H),
1.67–1.61 (m, 1H), 1.59 (s, 3H), 1.33 (s, 3H), 1.30
(s, 3H), 1.15–1.10 (m, 1H), 1.02 (s, 3H); ESI-MS: positive
ion mode: m/z = 765.36 [M + Na]+ calculated mass (M)
for C41H47ClN4O7 is 742.31.
4.2.12 | 1,9-O-{N-((1-[p-cyanophenyl]-1H-
1,2,3-triazol-4-yl)methyl)-3-indolylidene}
forskolin (5i)
Yield: 114 mg (91%), yellow solid, m.p. 140–142ꢀC; IR
(KBr): 3463, 2959, 2231, 1737, 1370, 1235, 1042 cmÀ1; H
1
NMR (400 MHz, CDCl3): δ (ppm) 7.75 (d, J = 8.0 Hz,
1H), 7.31 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 2.4 Hz, 1H),
7.26 (s, 1H), 7.24–7.09 (m, 5H), 5.72 (s, 1H), 5.65 (dd,
J = 17.2 and 10.8 Hz, 1H), 5.48 (d, J = 4.0 Hz, 1H), 5.42
(s, 2H), 5.41 (d, J = 2.4 Hz, 2H), 5.07 (dd, J = 17.2 and
1.2 Hz, 1H), 4.53–4.48 (m, 2H), 4.25 (brs, 1H), 2.88
(d, J = 16.4 Hz, 1H), 2.52 (d, J = 2.0 Hz, 1H), 2.44
(d, J = 16.4 Hz, 1H), 2.18 (m, 1H), 2.15 (s, 1H), 1.84–1.79
(m, 2H), 1.74 (s, 3H), 1.67–1.61 (m, 1H), 1.59 (s, 3H), 1.33
(s, 3H), 1.30 (s, 3H), 1.15–1.10 (m, 1H), 1.02 (s, 3H); ESI-
MS: positive ion mode: m/z = 720.33 [M + H]+, 742.35
[M + Na]+, 758.31 [M + K]+, calculated mass (M) for
C41H45N5O7 is 719.33.
4.2.10 | 1,9-O-{N-((1-[p-carboxyphenyl]-1H-
1,2,3-triazol-4-yl)methyl)-3-indolylidene}
forskolin (5g)
Yield: 107 mg (83%), white solid, m.p. 139–141ꢀC; IR
1
(KBr): 2967, 2929, 1739, 1370, 1223, 1047 cmÀ1; H NMR
(400 MHz, CDCl3):
δ
(ppm) 8.33 (s, 1H), 8.11

10
PONNAM ET AL.
4.2.13 | 1,9-O-{N-((1-[n-butyl]-1H-
1,2,3-triazol-4-yl)methyl)-3-indolylidene}
forskolin (5j)
(400 MHz, CDCl3): δ (ppm) 7.99 (s, 1H), 7.75 (d,
J = 8 Hz, 1H), 7.61 (d, J = 8 Hz, 1H), 7.51 (s, 1H), 7.16
(m, 1H), 7.04 (m, 1H), 6.03–5.94 (m, 1H), 5.70 (dd,
J = 17.2, 10.4 Hz, 1H), 5.65 (s, 1H), 5.44 (s, 2H), 5.22 (dd,
J = 10.4 and 1.2 Hz, 1H), 5.15 (dd, J = 17.2 and 1.2 Hz,
1H), 5.02–4.92 (m, 4H), 4.54 (dd, J = 10.8 and 0.8 Hz,
1H), 4.41 (brs, 1H), 4.16 (s, 1H), 2.93 (d, J = 17.2 Hz, 1H),
2.55 (d, J = 17.2 Hz, 1H), 2.43 (s, 1H), 2.10–2.06 (m,
1H), 2.02 (s, 3H), 1.76–1.73 (m, 1H), 1.69 (s, 3H), 1.49 (s,
3H), 1.47–1.39 (m, 2H), 1.27 (s, 6H), 1.13–1.11 (m, 1H),
1.04 (s, 3H); ESI-MS: positive ion mode: m/z = 659.33
[M + H]+, 681.28 [M + Na]+, calculated mass (M) for
C37H46N4O9 is 658.34.
Yield: 93 mg (79%), white solid, m.p. 100–102ꢀC; IR (KBr):
;
3461, 2928, 1739, 1370, 1223, 1021 cmÀ1 1H NMR
(300 MHz, CDCl3): δ (ppm) 7.77 (d, J = 7.8 Hz, 1H), 7.34–
7.26 (m, 2H), 7.20–7.09 (m, 3H), 5.73 (s, 1H), 5.72 (dd,
J = 17.1 and 10.5 Hz, 1H), 5.48 (d, J = 3.9 Hz, 1H), 5.42
(brs, 2H), 5.10 (d, J = 16.8 Hz, 1H), 4.58 (d, J = 10.5 Hz,
1H), 4.50 (brs, 1H), 4.28–4.23 (m, 3H), 2.91 (d, J = 16.5 Hz,
1H), 2.54 (brs, 1H), 2.46 (d, J = 16.5 Hz, 1H), 2.17 (m, 1H),
2.14 (s, 3H), 1.83–1.78 (m, 5H), 1.75 (s, 3H), 1.59 (s, 3H),
1.33 (m, 4H), 1.30 (s, 3H), 1.10 (m, 1H), 1.04 (s, 3H), 0.91 (t,
J = 7.5 Hz, 2H); 13C NMR (100 MHz, CDCl3): δ (ppm)
206.40, 170.02, 146.64, 144.85, 136.60, 127.07, 126.44, 122.58,
122.08, 120.91, 120.42, 114.31, 110.46, 110.13, 94.80, 85.84,
81.23, 77.8, 76.9, 75.75, 70.58, 50.54, 50.54, 43.54, 42.84,
39.76, 36.95, 34.92, 33.46, 32.53, 31.63, 25.18, 24.11, 23.74,
21.57, 20.07, 17.96, 13.78; ESI-MS: positive ion mode: m/
z = 675.36 [M + H]+, 697.31 [M + Na]+, 713.28 [M + K]+,
calculated mass (M) for C38H50N4O7 is 674.37.
4.3 | In silico studies
The 2D chemical structures of synthesized compounds
were drown, modeled and later optimized through
molecular mechanics (MM2/MM3) force field for conver-
sion into 3D structure using Chem Bio Office Software v6
(Cambridge-Soft Inc.) [46]. X-ray crystallographic 3D
structures of succinate dehydrogenase (PDB ID: 1YQ3)
[47] was retrieved from Brookhaven Protein Data Bank
(http://www.rcsb.org). Binding energy (BE) and inhibi-
tion constant (Ki) values were calculated by using Auto
dock version 4.2 (autodock.scripps.edu) [48]. Polar hydro-
gen atoms were added to the macromolecules, and all
rotatable bonds present in the ligands were set as rotat-
able bond for experiments. A grid box was created for
macromolecule (dimensions of the grid box was
126 Â 126 Â 126 with grid spacing of 0.381). A GA was
used to run Auto dock. Population sizes of 150 and 10 mil-
lion energy were used for the 100 times search, with
126 Â 126 Â 126 dimensions of grid box size and 0.381 Å
grid spacing around the catalytic triad. The best confor-
mation was selected from the lowest docking energy
identified among the different pockets of the macromole-
cule for each ligand, with not more than 1.5 Å RMSD.
The formula for calculation is as follows: Ki = exp (ΔG/
RT; http:// www.autodock.scripps.edu.org). The RMSD
values were calculated using the PyMOL Molecular
Graphics System, Version 1.level.
4.2.14 | 1,9-O-{N-((1-[n-octyl]-1H-
1,2,3-triazol-4-yl)methyl)-3-indolylidene}
forskolin (5k)
Yield: 102 mg (80%), white solid, m.p. 75–77ꢀC; IR (KBr):
2968, 1740, 1370, 1223 cmÀ1; 1H NMR (400 MHz, CDCl3):
δ (ppm) 7.77 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8 Hz, 1H),
7.28 (s, 1H), 7.30–7.10 (m, 3H), 5.73 (s, 1H), 5.72 (dd,
J = 17.2 and 10.8 Hz, 1H), 5.49 (d, J = 4 Hz, 1H), 5.42 (d,
J = 3.2 Hz, 2H), 5.11 (dd, J = 17.2 and 1.6 Hz, 1H), 4.59
(dd, J = 10.8 and 1.2 Hz, 1H), 4.51 (brs, 1H), 4.26–4.22 (m,
3H), 2.91 (d, J = 16.4 Hz, 1H), 2.55 (d, J = 2.4 Hz, 1H),
2.46 (d, J = 16.4 Hz, 1H), 2.19–2.11 (m, 1H), 2.14 (s, 3H),
1.84–1.80 (m, 4H), 1.75 (s, 3H), 1.70–1.64 (m, 1H), 1.59 (s,
3H), 1.34 (s, 3H), 1.30 (s, 3H), 1.26–1.23 (m, 10H), 1.10–
1.06 (m, 1H), 1.04 (s, 3H), 0.86 (t, J = 7.2 Hz, 3H); 13C
NMR (100 MHz, CDCl3): δ (ppm) 206.03, 169.63, 146.23,
144.45, 136.20, 126.71, 126.04, 122.19, 121.67, 120.52,
120.04, 113.88, 110.08, 109.75, 94.41, 85.45, 80.83, 77.0,
76.54, 75.35, 70.18, 50.45, 50.15, 43.14, 42.49, 39.36, 36.56,
34.53, 33.08, 31.67, 31.25, 30.20, 29.0, 28.9, 26.46, 24.79,
23.72, 23.36, 22.57, 21.19, 17.57, 14.04; ESI-MS: positive
ion mode: m/z = 731.44 [M + H]+, 753.45 [M + Na]+,
calculated mass (M) for C42H58N4O7 is 730.43.
4.4 | ADMET screening
Various pharmacokinetics parameters related with
absorption, distribution, metabolism, excretion and toxic-
ity (ADMET) were calculated by using Discovery Studio
v3.5 [49]. The ADMET properties calculated were blood
brain barrier penetration, hepatotoxicity, cytochrome
P450 2D6 binding, aqueous solubility, plasma protein
4.2.15 | 1,9-O-{N-[(1-allyl-1H-1,2,3-triazol-
4-yl)methyl]-3-indolylidene}forskolin (5l)
Yield: 86 mg (75%), white solid, m.p. 95–97ꢀC; IR (KBr):
3462, 2948, 1737, 1370, 1224, 1021 cmÀ1 1H NMR
;

PONNAM ET AL.
11
binding and intestinal absorption. For toxicity levels, also
TOPKAT protocols of Discovery studio v3.5 was
used [50].
Spectro photo metrically at 540 nm. Likewise, heparin-
ized blood was incubated with the effective concentration
of compound 50 and 100 mg/ml at 37ꢀC for 60 min and
then transferred to the tubes containing decreasing con-
centrations of PBS. The cell suspensions were left to
equilibrate for 30 min and then centrifuged. The absor-
bance of the supernatants was read at 540 nm and the
percent lysis was calculated by dividing the optical den-
sity (OD) of the supernatant obtained from a particular
saline concentration by the OD of the standard rep-
resenting 100% hemolysis. Osmotic fragility curves were
constructed by plotting the lysis percentage against the
concentration of the saline solution. The results are
expressed in terms of MEF50, which is the level of hemo-
lysis at 50% PBS. The MEF50 values at standard pH and
temperature were then obtained from the standard curve.
Curcumin, podophyllotoxin and DMSO were used as a
positive control, standard drug control and vehicle con-
trol, respectively.
4.5 | In vitro anticancer activity
4.5.1 | Chemicals
Dulbecco's modified essential eagle medium (DMEM) and
fetal bovine serum (FBS) were taken from Gibco, India.
3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bro-
mide (MTT) dye, HEPES, trypsin–EDTA, antibiotic/anti-
mitotic (Ab/Am), phosphate buffer saline (PBS) were
purchased from Sigma Aldrich. Sodium bicarbonate
(NaHCO3) was purchased from Himedia, India while
dimethyl sulphoxide (DMSO), ethanol and isopropanol
were purchased from Merck India, Ltd.
4.5.2 | Cytotoxicity
ACKNOWLEDGMENTS
The authors thank the director, CSIR-CIMAP, Lucknow,
for his constant encouragement and support. Author
Devendar is thankful to UGC, New Delhi, for providing
financial assistance in the form of a Senior Research
Fellowship.
MCF-7 (human breast adenocarcinoma), MDA-MB-231
(human breast adenocarcinoma), CHANG (human liver
cell line), WRL-68 (human hepatocellular carcinoma),
COLO-205 (human colon carcinoma), PC-3 (human pros-
trate carcinoma), HeLa (human cervical carcinoma),
RAW-264.7 (mouse macrophages cell line) and RAJI
(human Burrkit's lymphoma cell line) were grown in
DMEM supplemented with 10% FBS and Ab/Am, under
5% CO2, 95% humidity and 37ꢀC temperature.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bro-
mide (MTT) assay was done by Mosmann method
reported previously [51,52]. Seeding in 96 well plates was
done with approximately 2 Â 103cells/well and treatment
of the compound was given for 24 h then MTT dye
(500 μg/ml) was added to the media in each well and
plate was incubated at 37ꢀC for 4 h. After the incubation
period, media was discarded and DMSO was added to
dissolve the formazan crystal and absorbance was taken
at 570 nm.
DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are avail-
able in the supplementary material of this article
ORCID
Kotesh Kumar Jonnala https://orcid.org/0000-0001-
8294-2234
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SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
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How to cite this article: D. Ponnam, N.
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