Design, synthesis and biological evaluation of 8-substituted-6-hydrazonoindolo[2,1-b]quinazolin-12(6H)-one scaffolds as potential cytotoxic agents: IDO-1 targeting molecular docking studies

Article abstract of DOI:10.1016/j.bmcl.2017.08.064

Herein, we have reported the synthesis of 18 novel 8-substituted tryptanthrin analogues based on our earlier work. All these tryptanthrin analogues were well characterized by ¹H & ¹³C NMR, FT-IR, Mass Spectrometry and Elemental Analy

Full text of DOI:10.1016/j.bmcl.2017.08.064

Accepted Manuscript
Design, synthesis and biological evaluation of 8-substituted-6-hydrazonoindolo
[2, 1-b] quinazolin-12(6H)-one scaffolds as potential cytotoxic agents: IDO-1
targeting molecular docking studies
Ramu Guda, Rajashekar Korra, Siripireddy Balaji, Rambabu Palabindela,
Rakesh Eerla, Harikiran Lingabathula, Narsimha Reddy Yellu, Girijesh Kumar,
Mamatha Kasula
PII:
S0960-894X(17)30858-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.08.064
BMCL 25258
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 May 2017
20 August 2017
25 August 2017
Please cite this article as: Guda, R., Korra, R., Balaji, S., Palabindela, R., Eerla, R., Lingabathula, H., Yellu, N.R.,
Kumar, G., Kasula, M., Design, synthesis and biological evaluation of 8-substituted-6-hydrazonoindolo [2, 1-b]
quinazolin-12(6H)-one scaffolds as potential cytotoxic agents: IDO-1 targeting molecular docking studies,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.08.064
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Design, synthesis and biological evaluation of 8-substituted-6-hydrazonoindolo
[2, 1-b] quinazolin-12(6H)-one scaffolds as potential cytotoxic agents: IDO-1
targeting molecular docking studies
Ramu Guda,a Rajashekar Korra,a Siripireddy Balaji,b Rambabu Palabindela,a
Rakesh Eerlac, Harikiran Lingabathula,d Narsimha Reddy Yellud Girijesh Kumar,e*
Mamatha Kasulaa*
aDepartment of Chemistry, Kakatiya University, Warangal, India –506009.
bDepartment of Chemistry Vellore Institute of Technology, Tamilnadu –500046, India.
cDepartment of Microbiology, Kakatiya University, Warangal, India –506009.
dUniversity College of Pharmaceutical Sciences, Kakatiya University, Warangal, Telangana –
506009, India.
eDepartment of Chemistry and Centre for Advanced Studies in Chemistry, Panjab University,
Chandigarh –160014, India.
*Corresponding Authors
E-mail address: mamathakasula2016@gmail.com (K. Mamatha)
E-mail address: girijeshchem@gmail.com (Girijesh Kumar)
1

ABSTRACT
Herein, we have reported the synthesis of 18 novel 8-substituted tryptanthrin analogues
based on our earlier work. All these tryptanthrin analogues were well characterized by 1H & 13C
NMR, FT-IR, Mass Spectrometry and Elemental Analysis. All these 8-substituted analogues
were screened for their anti-oxidant activity by DPPH radical scavenging assay. Out of all the
tested compounds, T11, T12, T17 and T18 showed potent anti-oxidant activity. The anti-cancer
activity have been performed by using MTT assay protocol and their results depicts that
compounds having the 4-pyridyl or 4-carboxyphenyl substituents at the 8th position of the
tryptanthrin framework are found to be the most promising cytotoxic agent against A549, MCF-7
and HeLa human cancer cell lines compared to others as well as with the standard drug cisplatin.
Moreover, the comparative molecular docking studies against the three protein receptors IDO1,
EGFR and HER2 strongly suggested that IDO1 is the best target protein, which exhibits lowest
binding energies of –11.73 and –11.61 kcal mol–1 for T11 and T12 scaffolds, respectively towards
the in vitro anti-cancer activity.
Keywords: tryptanthrin, cytotoxicity, anti-oxidant activity, MTT assay, DPPH, molecular
docking, IDO1.
2

Presently, cancer is one of the most challenging diseases found worldwide and also the most
stimulating factor for the human morbidity and mortality. Therefore, the design and development
of an anticancer drug is still one of the most challenging task for the people working in the
domain of medicinal chemistry. As we know that the drug currently available in the market have
several side effects on the normal cells and thus causing several abnormalities in the body
despite of their enormous utilizations. The cost and availability are the another major concern of
the present anti-cancer drugs.1,2 Hence, developing the new therapeutic drugs having the advance
proficiency and efficacy on the cancer cells is one of the foremost goal for the researchers.3 In
addition, to understand the molecular biology and pharmacology of cancer at a molecular level
the target based drug-design and discovery is another emerging and challenging filed for the
researchers.4–6 In this regards, the highly developed technology in molecular biology advantages
to identify many cancer targets in human body.7 So, considerable attention and effort have been
needed for the discovery and development of new and potential anti-cancer drugs.
In this regards, indoloquinazolines received considerable curiosity in the field of
therapeutic and diagnostic medicines. The natural product tryptanthrin (Indolo [2,1-
b]quinazoline-6,12-dione) is obtained from the Chinese medicinal plants Polygonum tinctorium
and Isatis tinctoria (Chinese wood) and shows significant biological activities due to the
presence of indoloquinazolines moiety (Fig. 1).8 Furthermore, as tryptanthrin framework is
derived from the isatin, which is well known inhibitors for anti-bacterial, anti-viral and anti-
fungal agents and thereby also capable to influence their therapeutic effect of the concerned
drugs.9-15 These important classes of heterocycles have attracted remarkable attention16 and
possession of the researchers to investigate their synthesis, pharmacological and biological
screening such as anti-leishmanial17 anti-microbial18,19 anti-tuberculosis20 anti-inflammatory21–23
,
3

anti-oxidant agents24 and their cytotoxicity against many cancer cell lines in mammalian cells.25
This compound can control the activity of COX-226,27 and inhibits the expression of nitric oxide
synthase and prostaglandin E(2) in cells.28–30 Even though tryptanthrin has wide range of
applications it has some small drawback such as poor aqueous solubility as well. Inspiring with
the aforementioned fascinating importance of the tryptanthrin scaffolds, we are interested to
develop some new tryptanthrin based anti-cancer agents and IDO1 inhibitor.
In literature 8-floro substituted tryptanthrin derivatives were successfully demonstrated as
potential anti-cancer agent and IDO1-inhibitor.31 wherein, 8-bromo substituted hydrazono
tryptanthrins were found to be an excellent cytotoxic agents as well as a good inhibitors for IDO-
1 enzyme. The in vitro anti-cancer activity and molecular docking studies were also well
exploited in our previous report.32 Herein, we have disclosed the design and synthesis of a series
of 18 novel 8-substituted (F, Cl, I, NO2, 4-pyridyl or 4-carboxyphenyl) tryptanthrin analogues T1
– T18 (Scheme 1 and Fig. 2) and investigated their anti-cancer evaluation by MTT assay using
human cancer cell lines MCF-7, A549, HeLa and anti-oxidant activity screening by using DPPH
radical scavenging method. The molecular docking studies were also performed using the three
target proteins IDO1, EGFR and HER2. The tryptanthrin analogues T1 –T4 were prepared by
adapting the literature protocol and using the respective substituted (F, Cl, Br, I and NO2)
indoline-2,3-dione and 2H-benzo[d][1,3]oxazine-2,4(1H)-dione followed by the facile
condensation using the hydrazine hydrate in presence of acid.33 Compounds T1–T4 afforded T7–
T10 and T13–T16 after the reaction with benzoyl acetone and o-vanillin, respectively in presence
of acetic acid and following the literature protocol.34,35 Moreover, Suzuki coupling reaction of
respective boronic acid and bromo substituted tryptanthrin analogue (see Scheme 1) gave T5 and
T6. However, T11 and T12 were prepared by refluxing T5 and T6 with benzoyl acetone. In similar
4

manner, T5 and T6 also afforded T17 and T18 after the reaction with o-vanillin. The synthetic
route adapted for the preparation of all the aforementioned 8-substituted-6-hydrazonoindolo [2,1-
b]quinazolin-12-(6H)-one and its derivatives were illustrated in Scheme 1. Whereas, details of
the synthesis and characterization of these tryptanthrin analogues T1 –T18 were depicted in
Supporting Information (Fig. S1 – S14).
The 1H NMR spectra of the compounds T1 – T6 showed two signals in the range of δ 11.02 –
11.35 ppm and δ 10.20 – 10.55 ppm, which is assigned as NH2 protons. However, compounds T7
– T12 exhibits a signal in the region of δ 13.65 – 16.25 ppm, which corresponds to the enolic
proton (-OH). The singlet appered in the region of δ 5.45 – 6.35 ppm attributed to the alkene
(=CH-) proton. For compounds T13 – T18 singlet related to the =CH proton appread in the region
δ 8.57 – 8.45 ppm. The 13C NMR spectra showed the signals in the region of δ 198.2 – 187.8 and
δ 167.8 –155.7 ppm related to the enolic carbon and carbonyl group of quinazoline ring and thus
strengthens the aforementioned statement. The FT–IR spectrum of T12 also displays peaks in the
region of 3332 – 3375 cm–1 and at 1738 cm–1, which are assigned for the ν(OH) and ν(C=O)
,
respectively. The LCMS spectrum of the compounds T11 and T12 shows peaks at m/z 485 and
425, which is related to the [M+H]+ peak of the T11 and T12, respectively.
Fig. 1. Some biologically potent 8-substituted-6-hydrazonoindolo [2, 1-b] quinazolin-12-(6H)-one derivatives.
5

Scheme 1. (i) Et3N, 2–4 h, toluene, reflux. (ii) NH2-NH2.H2O, 12 h, THF, reflux. (iii) Acetic acid, benzoyl acetone,
reflux. (iv) o-vanillin, CH3OH, reflux 12 h. (v) Na2CO3 (20 mL, PdCl2(PPh3)2, DMF, 9 h, 110 °C, 4-pyridineboronic
acid or 4-carboxyphenyl boronic acid. (vi) Acetic acid, benzoyl acetone, reflux.
6

NH2
N
NH2
NH2
N
NH2
N
N
N
N
N
N
O
N
O
N
O
N
N
O2N
NH2
F
I
Cl
O
T1
T2
T3
T4
NH2
N
N
N
N
N
O
N
O
N
O
HO
T6
T5
H3C
H3C
H3C
H3C
OH
OH
OH
OH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
O2N
I
Cl
F
O
O
O
O
T7
T8
T9
T10
H3C
H3C
OH
OH
N
N
N
N
N
N
N
O
N
N
O
O
HO
T12
T11
CH3
CH3
CH3
CH3
O
O
O
O
OH
OH
OH
OH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
F
Cl
I
O2N
O
O
O
O
T13
T14
T15
T16
CH3
CH3
O
O
OH
OH
N
N
N
N
N
N
N
N
N
HOOC
T17
O
T18
O
Fig. 2. Chemical structures of all the 8-substituted-6-hydrazonoindolo [2, 1-b] quinazolin-12-(6H)-one derivatives.
7

Cytotoxic Activity. All these 8-substituted-6-hydrazonoindolo [2,1-b] quinazolin-12-(6H)-one
derivatives T1 – T18 were evaluated for their in vitro anti-cancer activity against the human
breast cancer MCF-7, Lung cancer A549 and HeLa cervical cancer cell lines according to
procedure described in literature and taking cisplatin as the reference drug.36,37 The structure of
most potent tryptanthrin derivative is shown in Fig.3. The relationship between surviving
fraction and drug concentration was plotted to obtain the survival curves of MCF-7, A549 and
HeLa (Fig. 4). The response parameter calculated was the IC50 value, which corresponds to the
concentration required for 50% inhibition of cell viability. Among all the 8-substituted-6-
hydrazonoindolo [2, 1-b] quinazolin-12-(6H)-one derivatives used in this studies compounds T8,
T9 T11 and T12 shows better potent behavior than others. In particular T11 and T12 are showing
,
most potent cytotoxicity against three tumor cell lines with their IC50 values of 11.60 1.821
µM, 9.42 1.239 µM against MCF-7, 6.01 1.116 µM, 7.19 0.991µM against A549 and
12.20 0.239 µM, 9.42 1.594 µM against HeLa, respectively, compared to the standard drug
cisplatin. Compounds T8 and T9 shown moderate IC50 values of 19.71 0.937 µM, 19.89 1.44
µM for MCF-7, 18.07 0.375 µM, 20.36 1.832 µM for A549 and 23.64 1.629 µM, 18.82
1.168 µM for HeLa, respectively. These results indicates that compounds T8 – T12 shows good
cytotoxic activity towards the three cell lines MCF-7, A549 and HeLa. Under identical
conditions the remaining compounds exhibits poor anti-cancer activity with the IC50 values
ranging from 20.368 0.190 µM to 67.68 0.862 µM for the three cell lines. We have also
screened cytotoxic activity of T8 – T12 against HEK-293 (Human Embryonic Kidney-293) using
MTT-micro cultured tetrazolium assay and noticed IC50 values of 76.09 0.901µM,
64.12 1.482µM, 71.04 1.034µM and 82.54 0.694 µM for T11, T12, T8 and T9, respectively and
8

the detail results are presented in Table 1. Notably and importantly, the results summarized in
Table 1 advocated that there are no adverse effects noticed on the normal cell lines.
Table 1. Cytotoxic activities of newly synthesized 8-substituted-6-hydrazonoindolo [2,1-b] quinazolin-
12-(6H)-one derivatives T1 – T18 on human cancer cell lines MCF-7, A549 and HeLa [in vitro (IC50
µM)]a.
S.No.
1
Compound
T1
MCF-7
A549
HeLa
HEK293
54.55 0.871
67.51 1.453
44.94 0.841
53.56 0.745
50.38 0.977
61.10 1.004
20.368 0.190
19.71 0.937
19.89 1.44
31.52 1.234
11.60 1.821
9.42 1.239
35.51 1.11
35.38 0.982
30.38 1.021
34.25 0.237
24.77 1.568
37.07 1.471
4.28 0.355
44.59 0.974
62.12 1.190
43.44 1.454
45.55 0.896
47.76 0.511
62.16 0.540
32.00 1.230
18.07 0.375
20.36 1.832
28.39 0.708
6.01 1.116
7.19 0.991
31.20 1.075
40.84 1.234
32.29 1.964
40.06 0.873
32.87 1.342
39.25 1.803
5.14 0.421
67.68 0.862
55.67 1.122
51.05 1.091
50.29 0.540
44.12 0.801
54.26 1.538
24.46 0.836
23.64 1.629
18.82 1.168
33.10 0.300
12.20 0.239
9.42 1.594
34.28 0.886
36.56 1.547
29.22 1.237
38.03 0.966
24.77 1.452
32.29 1.053
3.88 0.354
ND
ND
2
T2
T3
T4
T5
T6
T7
T8
3
ND
4
ND
5
ND
6
ND
7
ND
8
71.04 1.034
82.54 0.694
ND
9
T9
10
11
12
13
14
15
16
17
18
19
T10
T11
T12
T13
T14
T15
T16
T17
T18
76.09 0.901
64.12 1.482
ND
ND
ND
ND
ND
ND
Cisplatin
ND
aValues are expressed as mean SEM. Cytotoxicity as IC50 for each cell line, is the concentration of compound
which is reduced by 50% the optical density of treated cell with respect to untreated cell using the MTT assay.
9

A closer look of the structure-activity relationship demonstrates that usually the potency of
heterocyclic compounds towards biological activity mostly depends on the position and type of
substituents present on the core scaffold. Substituents present at C2 and C8 position of indole and
quinazolone rings in tryptanthrin changes the cytotoxic activity against different cell lines.31,32
However, substituents which can influence the core moiety either by donating electrons or by
withdrawing the electron density of indole ring of the tryptanthrin also effects the cytotoxicity
proficiency against the different human cancer cell lines. In this work we have noticed enhance
cytotoxic activity in case of 4-pyridyl and 4-carbaxyphenyl substituted (at 8th position)
tryptanthrin derivatives against three human cancer cell lines MCF-7, A549 and HeLa compared
with non-substituted tryptanthrin derivatives and less cytotoxicity against HEK293 (normal
human cancer cell line).
Fig. 3. Structure of most potent 8-substituted-6-hydrazonoindolo [2,1-b] quinazolin-12-(6H)-one analogues T11 and
T12.
10

(a)
(b)
(c)
Fig. 4a–c. Survival curves of MCF-7, A549 and HeLa cell lines.
In Vitro Anti-Oxidant Assay. In order to investigate the potential application of T1 – T18 towards
the in vitro anti-oxidant activity, DPPH radical scavenging assay was performed. In this
experiment, 10–6 molar methanolic solution of the concerned compounds was used followed by
the seriel dilution. In small test tubes 1 mL (in each tube) mixture of scaffold (100µL) and DPPH
radical (10–4 molar, 900µL) were taken and incubated at 37 °C under dark conditions for 30 min
(French et al. 1994). The absorbance of the DPPH solution and mixture was recorded using the
11

UV-visible spectrophotometer. The DPPH solution exhibits λmax of 517 nm (typical range of
DPPH radical, Blois et al. 1958; Lu and Foo, 2000; Zhu et al. 2002). Notably, the absorbance is
decreases in presence of the concerned compounds with the colour change from the blue to
yellow. In principle, DPPH has an odd electron and thus can accept an electron or hydrogen free
radical and or also able to liberate this free radical, which can be scavenges by synthesized
compounds. The shifting of absorbance value towards the lower wavelength side indicates the
increasing radical scavenging activity of the test compounds (Gulçin et al. 2004). The percentage
of inhibition values were compared by using absorbance values of standard ascorbic acid under
identical conditions. The anti-oxidant activity was considered in IC50 in µM (the effective
concentration at which 50% of the radicals were scavenged) and shown in Table 2.38,39
Moreover, all the samples were tested in triplicate to maintain the accuracy and to find out the
standard deviation.40 From Table 2, it is clear that these synthesized compounds showed
remarkable anti-oxidant activity compared to the standard drug. Importantly, compounds T11,
T12, T17 and T18 were found to be the most potent anti-oxidants with the least IC50 values of 6.23
µM (for T11), 5.02 µM (for T12), 4.98 µM (for T17) and 5.31 µM (for T18), respectively, among
all the synthesized compounds.
We argued that, this better in vitro anti-oxidant activity of compounds T11, T12, T17 and T18
are might be due to the presence of electron-rich functionality such as 4-pyridyl or 4-
carboxypheny in the tryptanthrin scaffolds. The proton exchange capability between carbonyl
and –NH group of compounds T11 and T12 may possibly be another aspect for enhanced radical
scavenging activity. Moreover, T5, T8, T9, and T15 displaying moderate anti-oxidant activity
compared with standard ascorbic acid having the IC50 values of 11.23 µM (for T5), 12.45 µM
(T8), 11.60 µM (for T9) and 10.90 µM (for T15), respectively. The remaining compounds
12

exhibits reasonable to poor anti-oxidant activity with IC50 value ranging from 16.45 µM to 29.71
µM. Notably, T10 and T16 shows poorest anti-oxidant activity among all the synthesized
compounds with IC50 values of 29.71 µM and 28.68 µM, respectively (Table 2) attributed to the
presence of electron withdrawing -NO2 group at 8-position of the tryptanthrin framework.
Table 2. Anti-oxidant activity of 8-substituted-6-hydrazonoindolo [2, 1-b] quinazolin-12-(6H)-
one derivatives (T1 – T18) by DPPH assay.
S. No
Compounds
IC50 (µm)
1
T1
16.45
19.87
24.87
27.32
11.23
21.96
20.14
12.45
11.60
29.71
6.23
2
T2
3
T3
4
T4
5
T5
6
T6
7
T7
8
T8
9
10
T9
T10
11
T11
12
T12
5.02
13
T13
21.07
22.21
10.90
28.68
4.98
14
T14
15
T15
T16
T17
16
17
18
T18
5.31
Standard
Ascorbic acid
3.48
Molecular docking studies. Docking investigation was performed to compounds T1 – T18 with
pharmacological target proteins, indoleamine 2,3-dioxygenase (IDO1), epidermal growth factor
receptor (EFGR) and human epidermal growth factor receptor 2 (HER2) to compare their
13

potential as drug candidates. IDO1, which is an attractive target in design of anti-cancer drugs as
it catalyzes essential amino acid L-tryptophan to N-formylkynurenine.41 In principle, IDO1 is the
primary and rate-limiting enzyme of tryptophan catabolism and responsible for the depletion of
tryptophan leading to halted growth of microbes as well as T-cells.42 The EGFR is well known
receptor for the members of epidermal growth factor family of extracellular protein.43 It induces
receptor dimerization and tyrosine autophoshorylation and leads to cell proliferation,
differentiation, motility and cell servival 44–46 Over expression of EGFR is up regulated in colon
cancers and most neoplasms. Whereas, mutations comprising EGFR lead to its constant
activation and produces uncontrolled cell devision.47,48 Moreover, HER2 is a member of human
epidermal growth factor receptor (HER/EGFR/ERBB) family. The overexpression of oncogene
play a vital role in the development and propagation of certain types of cancers such as breast,
lung, stomach, ovarian, adenocarcinoma and uterine cancer (uterine serous epidermal
carcinoma).49–52 For the reason that we have chosen IDO1, EGFR and HER2 proteins as the
target receptors for the docking studies and their comparative binding energy are compiled in
Table 3 (readers are advised to refer the Supporting Information for the details of docking
protocol). Whereas, hydrogen bonding profile for the compounds T1 – T18 are shown in Table 4
along with residues. The comparative molecular docking demonstrations of the organic scaffolds
T1 – T18 with the target receptors IDO1, EGFR and HER2 (as shown in Table 3) clearly indicates
that the affinity of these drug candidates with receptor IDO1 is better than that of receptors
EGFR and HER2 and that’s why we have chooses IDO1 as the target receptor for the details
discussion. Out of these series of drug candidates exploited for the doc score, scaffolds T12, T11,
T9 and T8 are found to be the best inhibitors and their probable reasons are discussed below.
14

From Table 3 it is clear that T8 is a potential candidate for the anticancer drug with the
binding energy of -11.20 kcal mol–1. Notably, T8 exhibits strong hydrogen bonding interactions
between amino acid His346 and N-atom of hydrazine tryptanthrin as well as between Ala264 and
carbonyl group of benzoyl acetone with bond length of 2.7678 Å and 3.1517 Å, respectively
(Fig. 5). All the other interactions are hydrophobic in nature and found between the amino acid
of target receptor IDO1 and phenyl ring of tryptanthrin core with the bond distance of 3.6445 Å,
which is off course larger than the drug – receptor hydrogen bonding interaction.
Table 3. Comparative binding energies of 8-substituted-6-hydrazonoindolo [2, 1-b] quinazolin-12-(6H)-
one compounds T1 – T18 against IDO1, EGFR and HER2.
Binding Energy
(kcal mol–1)
HER2 (PDB id: 3ppo)
Binding Energy
(kcal mol–1)
IDO1 (PDB id: 2dU0)
Binding Energy
(kcal mol–1)
EGFR (PDB id: 4hjo)
S.No. Compound
1
2
T1
T2
-8.22
-8.88
-7.24
-7.69
-8.09
-7.86
-8.94
-9.08
-8.91
-10.43
-10.76
-11.00
-10.06
-10.49
-9.53
-9.05
-9.83
-9.59
-8.89
-9.41
-7.80
-7.80
3
T3
-7.68
-9.47
-9.04
4
T4
-7.92
5
T5
-9.92
-9.07
6
T6
-8.10
-9.29
7
T7
-10.79
-11.20
-11.71
-10.91
-11.73
-11.61
-10.52
-9.83
-10.79
-10.63
-9.59
T8
T9
8
9
10
11
12
13
14
15
16
17
18
T10
T11
T12
T13
T14
T15
T16
T17
T18
-10.47
-10.20
-9.84
-9.84
-10.92
-10.88
-11.29
-10.84
-10.12
-10.31
-10.00
-10.80
-9.96
15

In a similar fashion drug T9 (binding energy, -11.71 kcal mol–1, Tables 3 and 4) also exhibit
hydrogen bonding interaction with receptor IDO1 (Fig. 6). Which takes place between His346
and nitrogen atom of hydrazono group of the tryptanthrin core with bond distance of 2.6800 Å.
Whereas, the hydrophobic interaction observed between the phenyl ring of quinazoline fused
tryptanthrin core and Ser167 with bond distance 3.9972 Å. Furthermore, T11 also shows drug –
receptor hydrogen bonding interaction (binding energy, -11.73 kcal mol–1, Tables 3 and 4)
analogous to T9 (-11.71 kcal mol–1). In this case hydrogen bonding interaction took place in
between His346 and nitrogen atom of hydrazono group appended to tryptanthrin moiety with
bond distance of 2.7190 Å (Fig. 7). Drug T11 also features hydrophobic interactions between
Ala264 and carbonyl group of benzoylactone attached with tryptanthrins core in addition to
interactions between Ser167 and phenyl ring of quinazoline induced tryptanthrin framework
having the distance of 3.2152 Å and 3.6072 Å, respectively. The structure of complex derived
from the interaction of T12 and receptor IDO1 also displays hydrogen bonding interaction
(binding energy, -11.61 kcal mol–1, Tables 3 and 4) between His346 and nitrogen atom of
hydrazono tryptanthrin core with bond distance of 3.0734 Å (Fig. 8). This drug-receptor complex
also features hydrophobic interaction between Ala264 and carbonyl group of benzoylacetone
group of tryptanthrin core with bond distance of 3.1209 Å. Notably, all these cases the
synthesized organic scaffolds (T12, T11, T9 and T8) are interacted with the target protein (receptor
IDO1) through the hydrogen bonding between His346, Ala264 and/or Ser167 and different
substituted functionality of the tryptanthrin core. Whereas, the affinity between the target
receptor and drug candidates are enhanced via the hydrophobic interactions.53 Notably,
compounds other than T12, T11, T9 and T8 display moderate dock score and binding affinity
against all the three protein receptors IDO1, EGFR and HER2 (see Table 3 for details).
16

Table 4. Binding energies, number of hydrogen bonds and residues involved in hydrogen bonding of
compounds 8-substituted-6-hydrazonoindolo [2, 1-b] quinazolin-12-(6H)-one T1– T18 against IDO1.
Binding Energy
(kcal mol–1)
Number of
hydrogen bonds
S.No. Compound
Residues
1
2
T1
T2
-8.22
-8.88
2
2
2
2
2
2
1
2
1
2
1
1
3
2
2
2
1
1
Ser167
Ser267, Glu171
Ser267, Glu171
Ser167
3
T3
-9.47
-9.04
4
T4
5
T5
-9.92
Glu171
6
T6
-8.10
Arg343
7
T7
-10.79
-11.20
-11.71
-10.91
-11.73
-11.61
-10.52
-9.83
His346
T8
T9
His346, Ala264
His346
8
9
10
11
12
13
14
15
16
17
18
T10
T11
T12
T13
T14
T15
T16
T17
T18
His346, Ser263
His346
His346
Ser267, Ser167, His346
His346
-10.31
-10.00
-10.80
-9.96
His346
Ser267, His346
Ala234
Arg343
17

Fig. 5. Shows the binding poses and interactions of 8-substituted-6-hydrazonoindolo [2,1-b] quinazolin-12-(6H)-one
analogue T8 to the binding sites of target protein IDO1 (PDB ID: 2d0U).
Fig. 6. Shows the binding poses and interactions of 8-substituted-6-hydrazonoindolo [2,1-b] Quinazolin-12-(6H)-
one analogue T9 to the binding sites of target protein IDO1 (PDB ID: 2d0U).
18

Fig. 7. Shows the binding poses and interactions of 8-substituted-6-hydrazonoindolo [2,1-b] quinazolin-12-(6H)-one
analogue T11 to the binding sites of target protein IDO1 (PDB ID: 2d0U).
Fig. 8. Shows the binding poses and interactions of 8-substituted-6-hydrazonoindolo [2,1-b] quinazolin-12-(6H)-one
analogue T12 to the binding sites of target protein IDO1 (PDB ID: 2d0U).
In summary, 18 novel, 8-substituted-6-hydrazonoindolo [2,1-b] quinazolin-12-(6H)-ones T1 – T18
were synthesized by well-established synthetic protocol followed by the Suzuki coupling
19

reaction and screened for their anti-oxidant, anti-cancer and molecular docking studies. The
compounds T12, T11, T17 and T18 were found to be the most potent anti-oxidant agents by DPPH
radical scavenging assay. Whereas, T12, T11, T8 and T9 are showing a promising anti-cancer
activity against the MCF-7, A549, HeLa human cancer cell lines using the MTT assay protocol
and using cisplatin as a reference drug. We reasoned that the presence of 4-pyridyl or 4-
carboxyphenyl substituents at 8th position of tryptanthrin framework might be a cause for their
potent anti-cancer and anti-oxidant activity. The comparative molecular docking studies have
been also performed against the three protein receptors IDO1, EGFR and HER2 and found IDO1
is the best one with binding energies of -11.73 kcal mol–1, -11.61 kcal mol–1, -11.20 kcal mol–1
and -11.20 kcal mol–1 for compounds T12, T11, T8, and T9, respectively. Importantly, molecules
having the lowest binding energies are also displaying the highest cytotoxicity against the tested
MCF-7, A549, HeLa human cancer cell lines. In a similar fashion, compounds with the better
binding energies also showing excellent anti-oxidant. Moreover, this results will provide
advantage to the medicinal chemist in the design and development of new tryptanthrin based
anti-cancer drugs in near future.
Acknowledgements
We gratefully acknowledge Prof. Samar K. Das and UGC-Networking Resource Center of
the School of Chemistry, University of Hyderabad for the analytical data collection. The
financial assistance from the University Grant Commission (UGC), New Delhi is also
acknowledged.
20

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Research Highlights
ꢀ Designed and synthesized 18-novel tryptanthrin analogues as potential cytotoxic agents
ꢀ Compounds T11 and T12 were found to be best cytotoxic agents with lowest binding
energies in docking
ꢀ Compounds T11, T12, T17and T18 were found to be promising anti-oxidants
ꢀ IDO-1 protein is the best target for 8-substitued tryptanthrin analogues.
26

Graphical Abstract
Leave this area blank for abstract info.
Design, synthesis and biological evaluation of
8-substituted-6-hydrazonoindolo [2,1-b]quinazolin-
12(6H)-one scaffolds as potential cytotoxic agents:
IDO-1 targeting molecular docking studies
Ramu Guda,a Rajashekar Korra,a Siripireddy Balaji,b Rambabu Palabindela,a Rakesh Eerlac, Harikiran
Lingabathula,d Narsimha Reddy Yellud Girijesh Kumar,e* Mamatha Kasula a*
Novel tryptanthrin derivatives have exhibited excellent anti-cancer activity against three
human cancer cell lines MCF-7, A549 and HeLa.