Novel chromenes and benzochromenes bearing arylazo moiety: molecular docking, in-silico admet, in-vitro antimicrobial and anticancer screening

Article abstract of DOI:10.1007/s00044-019-02387-5

Novel derivatives of benzochromene 9a–e and chromene 10a–e were synthesized via multi components reaction that utilizes azo dyes, malononitrile, and arylaldehydes as starting materials. The structures of the newly synthesized compounds were elucidated by the IR, ¹H-NMR, ¹³C-NMR and mass spectral data. The new synthesized compounds were explored for their in-silico ADMET properties. The docking investigations verified good docking outcomes in terms of score and binding affinities of the examined compounds on the desired proteins, namely the GlcN-6-P synthase and PI3Ks. The in-vitro studies, which encompassed the antibacterial activity against two types of Gm +ve and two of Gm –ve, and the antifungal activity against four fungi micro-organisms were evaluated by the well diffusion method for the synthesized compounds. Moreover, the anticancer activity was tested against three human carcinoma cell lines [human colon carcinoma (HCT-116), human breast adenocarcinoma (MCF-7), and liver carcinoma (HEPG-2)], exhibiting promising anticancer activity in comparison to Vinblastine, Colchicine and Doxorubicin as standard drugs. The data suggests that some of the new derivatives of chromene scaffold which emerged promising in the in-silico molecular docking studies displayed good in-vitro antimicrobial and anticancer activities and could be exploited as leads for further optimization.

Full text of DOI:10.1007/s00044-019-02387-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02387-5
ORIGINAL RESEARCH
Novel chromenes and benzochromenes bearing arylazo moiety:
molecular docking, in-silico admet, in-vitro antimicrobial and
anticancer screening
Tarek H. Afifi1 Sayed M. Riyadh1,2 Anwar A. Deawaly1 Arshi Naqvi1
●
●
●
Received: 30 March 2019 / Accepted: 19 June 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Novel derivatives of benzochromene 9a–e and chromene 10a–e were synthesized via multi components reaction that utilizes
azo dyes, malononitrile, and arylaldehydes as starting materials. The structures of the newly synthesized compounds were
elucidated by the IR, 1H-NMR, 13C-NMR and mass spectral data. The new synthesized compounds were explored for their
in-silico ADMET properties. The docking investigations verified good docking outcomes in terms of score and binding
affinities of the examined compounds on the desired proteins, namely the GlcN-6-P synthase and PI3Ks. The in-vitro
studies, which encompassed the antibacterial activity against two types of Gm +ve and two of Gm –ve, and the antifungal
activity against four fungi micro-organisms were evaluated by the well diffusion method for the synthesized compounds.
Moreover, the anticancer activity was tested against three human carcinoma cell lines [human colon carcinoma (HCT-116),
human breast adenocarcinoma (MCF-7), and liver carcinoma (HEPG-2)], exhibiting promising anticancer activity in
comparison to Vinblastine, Colchicine and Doxorubicin as standard drugs. The data suggests that some of the new
derivatives of chromene scaffold which emerged promising in the in-silico molecular docking studies displayed good in-
vitro antimicrobial and anticancer activities and could be exploited as leads for further optimization.
●
●
●
Keywords Arylazochromene Anticancer activity Antimicrobial Activity In-silico screenings
Introduction
chromene demonstrates several other incredible applica-
tions, including: antileishmanial (Foroumadi et al. 2010;
Tanaka et al. 2007), antioxidant (Fadda et al. 2012
; Nareshkumar et al. 2009), vascular-disrupting (Kasibhatla
et al. 2004), blood platelet antiaggregating (Lee et al. 2006),
analgesic and hypolipidemic effects (Sashidhara et al.
2011). Furthermore, the appliance of these moieties for
disease therapy and for the enhancement of potent antitumor
agents has presented efficacious models in literature. For
instance, Crolibulin™ (A) is currently in phase I/II of
clinical trials for the treatment of advanced solid tumors
(Tiana et al. 2008; Hermanson et al. 2009). 2-Amino-4-(3-
bromo-4,5-dimethoxyphenyl)-7-(dimethylamino)-4H-chro-
mene-3-carbonitrile (B) has been known as a tubulin inhi-
bitor (El-Agrody et al. 2014) (Fig. 1).
The synthetic methodology of chromenes has been
accomplished over the years, employing various pathways,
such as multicomponent reactions (MCRs) (Kirilmis et al.
2008; Elinson et al. 2010; Hosseini-Sarvari and Shafiei
Haghighi 2011; Boominathan et al. 2011), heterogeneous
catalytic methods (Mehrabi and Kazemi-Mireki 2011; Yadav
Chromene has been explored as a privileged structure,
which can be identified in synthetic medicinal chemistry as
a single molecular framework able to provide ligands for
diverse receptors (Atta-ur-Rahman and Thomsen 2001).
The majority of researches have been centered on the
development of 2-amino-4H-chromene and their deriva-
tives. These compounds have been recognized as ideal
medicinal scaffolds because of their unique pharmacologi-
cal and biological activities, as antibacterial (Ali et al. 2015)
and/or anticancer agents (Cai et al. 2006). In addition,
* Tarek H. Afifi
afifith@yahoo.com
1
Department of Chemistry, Faculty of Science, Taibah University,
30002 Al-Madinah Al-Munawarah, Saudi Arabia
2
Department of Chemistry, Faculty of Science, Cairo University,
Giza 12613, Egypt

Medicinal Chemistry Research
OCH
substitutedphenyl) (phenyl)methanone] (4a, b) in the pre-
sence of nitrous acid at 0–5 °C afforded the corresponding
diazonium salts. The coupling reaction of these diazonium
salts with the phenolic components, containing reactive
hydrogen atom(s) such as, 2-naphthol or resorcinol, led to
the formation of the azo dyes 3a, b or 6a, b, respectively
(Scheme 1). The structure identities of the obtained azo
dyes were deduced from their spectral analysis. For exam-
ple, the infrared spectra of these products showed the
3
H CO
Br
3
Ar
O
CN
NH
CN
NH
Me N
2
2
H N
2
O
2
NH
2
appearance of the absorption band at 1450–1446 cm−1
,
(A)
Crolibulin
(B)
MX58151
Ar = 3-Br-4,5-MeOC H
which is a characteristic of the azo group (N = N). Mean-
while, the carbonyl absorption band of compound 3b
appeared at 1685 cm1, and the absorption of the hydroxyl
6
2
1
group occurred at 3460–3257 cm−1. The representative H
Fig. 1 Chromene compounds with promising biological activities
NMR spectrum of compound 6a showed singlet signals at
10.69 and 11.36 ppm, assigned to the OH and NH protons,
respectively.
The multicomponent reaction of 3a or 3b, malononitrile
(8), and the aromatic aldehyde derivatives (7a–d) in
refluxing ethanol, and in the presence of the catalytic
amount of piperidine, afforded the corresponding azo dyes,
containing chromene moieties 9a–e (Scheme 2).
et al. 2007), electro catalytic procedures (Makarem et al.
2008), microwave and ultrasound techniques (Hamelin et al.
2002; Zbancioc and Mangalagiu 2006; Bejan and Mantu
2012; Zbancioc and Zbancioc 2014; Safari and Javadian
2015). Among these strategies, multicomponent reactions
(MCR) are deemed as one of the most effective routes due to
the simplicity of usage, low cost, and the production of a
sufficient yield of the desired product. The combination of the
chromene derivatives with the azo dyes hadn’t gained the
attentiveness of scientists even though the chromene scaffold
appears to be an attractive precursor for the formation of
novel azo chromophores. Recently, we reported the first
model of chromene, containing azo chromophores, and
investigated their biological properties. As a continuation to
our preceding work (Afifi et al. 2017), herein we introduce
the synthesis and characterization of a novel series of azo
chromophores with chromene moieties. The synthesized
compounds were investigated for their pharmacodynamic
and pharmacokinetic properties via in-vivo ADMET
(absorption, distribution, metabolism, excretion and toxicity)
predictions. Moreover, the chromene derivatives have been
reported as glucosamine-6-phosphate synthase (GlcN-6-P
synthase) and Phosphoinositide 3-kinases (PI3Ks) inhibitors
(Govori et al. 2017; Chen-Chen M and Zhao-Peng 2016;
Cheng et al. 2003; Mao et al. 2011). In this regard, the in-
silico molecular docking screenings are employed mainly to
understand the drug-receptor interaction. Along with these
in-silico studies, this work also incorporates the screening of
the biological features of the targeted compounds.
The sequence of the reaction was initiated by the con-
densation of malononitrile (8) and the aldehyde derivatives
(7a–d) to generate arylidinemalononitrile, which is
manipulated as intermediates. These intermediates have
been captured by the aromatic (CH-) of 2-naphthol to give a
non-isolable product. The intermolecular Michael type
addition of the latter intermediate furnished the respective
benzo[g]chromene-3-carbonitriles (9a–e) as the end pro-
duct. The characterization of the isolated products has been
1
performed, using the spectroscopic techniques (FTIR, H-
NMR, and 13C-NMR). For example, the IR spectrum of
compound 9e revealed stretching vibration bands at 2195
and 1450 cm−1 for the (CN) and the (N = N), respectively.
In addition, a pair of bands at 3460 and 3420 cm−1 was
1
assigned to the (NH2) group. The H NMR of 9e exhibited
singlet signals at 5.74 and 5.97 ppm for the CH-pyran at
position-4 and the NH2-protons, respectively (Widelski
et al. 2009). Also, the 13C NMR spectrum revealed a
characteristic signal at 43.57 ppm, which was assigned to
the C-4 of the pyran ring (Widelski et al. 2009).
To explore the scope of the multicomponent reactions,
we extended the procedure to various arylazo dyes. Thus,
refluxing a mixture of [5-substituted-2-((2,4-dihydrox-
yphenyl) azo) phenyl] [phenyl]methanone (6a, b), mal-
ononitrile (8), and arylaldehydes (7a–d) under similar
conditions led to the formation of the corresponding 2-
amino-6-[(2-benzoyl-4-substitutedphenyl) azo]-7-hydroxy-
4-aryl-4H-chromene-3- carbonitriles (10a–e) (Scheme 3).
The elucidation of the structure of the isolated products
was confirmed by the spectral data. The IR spectra showed
stretching vibration bands at 3465–3460, 2220–2190, and
Results and discussion
Chemistry
The diazotization of aniline derivatives [4-toludine
(1a) and 4-aminoacetophenone (1b)] or [(2-amino-5-

Medicinal Chemistry Research
O
C
NH2
HO
R
NH2
+
4a,b
4a. R = Cl
4b. R = NO2
R
1
2
i) HCl / NaNO2
ii) NaOH
HO
iii) 0-5oC
OH
HO
5
i) HCl / NaNO2
ii) NaOH
N
N
R
iii) 0-5oC
HO
3a,b
N
OH
3a. R = CH3
3b. R = COCH3
R
N
C
O
6a,b
6a. R = Cl
6b. R = NO2
Scheme 1 The synthesis of azo chromophores 3a, b and 6a, b
1450 cm−1 for the (OH), (CN), and (N = N), respectively.
In addition, pair of bands at 3455–3450 and
chromophores has no effect on their wavelength values.
This behavior is due to the non-conjugated system of the
chromene motifs and is consistent with our previous report
(Afifi et al. 2017). Table 1 shows the UV-Vis of compounds
3a, b, 6a, b and the isolated products 10a–d as repre-
sentative examples of the target molecules. Moreover, the
structures of the azo dyes 3a, b and 6a, b and the isolated
products 10a–d could have more than one tautomeric form,
namely as hydroxyl-azo (A) or keto-hydrazone (B) (Fig. 2).
The electronic absorption spectral data of the investi-
gated azo dyes (3a, b and 6a, b) and the arylazo chromenes
10a–d can be used to differentiate between the hydroxyl-
azo (A) and the keto-hydrazone one (B), which exhibited a
split peak for the latter form. As shown in Table 1, com-
pounds 3a, 10a, and 10c exhibited two absorption bands at
(354–398 and 404–444 nm) in the DMF solution while 3b,
6a, 6b, 10b, and 10d gave a single absorption peak at
a
3260–3220 cm−1 were ascribed to the (NH2) group. The 1H
NMR exhibited singlet signals at 4.65–4.70, 8.54–9.88, and
11.33–11.59 ppm for the CH-pyran at the 4 position, the
(OH) group, and the NH-hydrazone, respectively, while the
broad signal at 6.88–7.05 ppm was assigned to the amino
protons. Furthermore, the 13C NMR spectra of compounds
10a–e revealed characteristic signals between 35.63-
36.85 ppm, which corresponds to the C-4 of pyran ring
(Widelski et al. 2009).
UV-Vis Study
The UV-Vis analysis of the azo dyes 3a, b and 6a, b and
arylazo chromene 10a–e compounds revealed that the
incorporation of the chromene moieties into the azo

Medicinal Chemistry Research
HO
CHO
CN
CN
N
H2C
8
+
+
N
R
EtOH / Pip.
Reflux
3a,b
R1
7a-d
3a. R = CH3
3b. R = COCH3
H2N
O
CN
R1
9a, R = CH3,
9b, R = CH3,
9c, R = CH3,
R1 = OCH3
R1 = F
R1 = Cl
N
9d, R = COCH3, R1 = F
9e, R = COCH3, R1 = Br
R
N
9a-e
Scheme 2 Synthesis of benzo[g]chromenes containing azo chromophore moieties 9a–e
HO
CHO
N
OH
CN
N
O
R
+
+
H C
2
CN
EtOH / Pip.
Reflux
C
1
R
8
7a-d
6a,b
6a. R = Cl
6b. R = NO
HO
2
N
O
R
N
NH
2
1
10a; R = Cl,
10b; R = Cl,
10c; R = NO ,
R
R
= OCH
= F
= OCH
3
1
1
R
R
2
3
C
O
CN
1
10d; R = NO ,
= Br
2
1
10e; R = NO , R = Cl
2
R
1
10a-e
Scheme 3 Synthesis of azo chromene derivatives 10a–e
(405–444 nm). The probable reason for this behavior is that
the dyes 3a, 10a, and 10c are present predominantly in a
mixture of the hydroxyl-azo (A) and keto-hydrazone (B)
forms while the other dyes 3b, 6a, 6b, 10b, and 10d are
presented in a single tautomeric form [keto-hydrazone (B)]
(Wainwright 2008). This behavior was in a good agreement
with the explanation for the 1H NMR spectra for these dyes
due to the presence of the NH signal in the regions of
11.33–16.15 ppm. A bathochromic shift was also observed
in the DMF solution for the azo dye 3b (λmax = 440 nm) as
compared to 3a (λmax = 409 nm), due to the effect of the
electron withdrawing character of the acetyl group.

Medicinal Chemistry Research
Table 1 UV absorption bands of compounds 3a, b, 6a, b, and 10a–d
candidates. All the tested compounds can be categorized as
well absorbed compounds as they displayed good human
intestinal absorption with HIA% from 87.39%–100%
including the standard drugs namely ampicillin and vin-
blastine with predicted HIA% of 81.48 and 96.06 respec-
tively. The tested synthesized compounds also appeared as
potent plasma protein binding candidates with PPB more
than 90%. The predictions of PPB in standard drugs
revealed to be lower ranging from 31.16%–71.67%. Com-
pounds 4b and 6b likewise ampicillin and gentamicin
exhibited low Caco-2 cell permeability while all the other
compounds and standard drugs showed moderate Caco-2
cell permeability, ranging from 11.00% to 39.36% with
highest predicted Caco-2 permeability of 39.36% for the
standard drug, vinblastine. Most of the compounds along
with the standard drugs were predicted as low permeable
compounds for the MDCK cells (0.03%–21.12%). 3a, 4a,
and 4b have displayed moderate MDCK cell permeability
(29.92%–76.92%). All the predicted compounds are P-gp
inhibitors except for 4a. Amphotericin B and vinblastine
were forecasted as P-gp inhibitors while rest of the drugs
were predicted as non-inhibitors. The toxicity in terms of
mutagenicity (Ames Test) and carcinogenicity (mouse and
rat) for the tested compounds/drugs is reported in Table 3.
All the focused compounds and some standard drugs are
predicted as mutagenic by the Ames test except for 10a,
10d, 10e, Amphotericin B, vinblastine and doxorubicin.
The prediction of carcinogenicity revealed that most of the
tested compounds and all the predicted standard drugs
except amphotericin B, possess carcinogenic activity in
mouse. The forecast of rat carcinogenicity discloses that
most of the tested compounds and standard drugs are car-
cinogenic in nature.
Compound No. Substituents
λmax (nm) Absorbance
3a
R = CH3
R = COCH3
X = Cl
398, 409 1.33, 1.32
3b
6a
440
405
407
1.5
1.34
1.36
6b
10a
X = NO2
X = Cl; Ar = 4-
362, 404 1.62, 1.49
CH3OC6H4
10b
10c
X = Cl; Ar = 4-FC6H4
442
1.4
X = NO2; Ar = 4-
354, 444 1.48, 0.67
CH3OC6H4
10d
X = NO2; Ar = 4-ClC6H4 444
1.55
ADMET predictions
In the current biomedical framework, the in-silico compu-
ter-aided drug designing (CADD) concept expedites virtual
screenings from a vast quantity of compound libraries into
miniature clusters of projected bioactive candidates, thus,
optimizing the ADMET (absorption, distribution, metabo-
lism, excretion, and toxicity) outline and bypassing the
hurdles affiliated to safety. The prediction of the
Blood–Brain Barrier (BBB) penetration plays a key role in
the pharmaceutical sphere by determining whether the
selected drug candidates would be able to pass across the
blood – brain barrier or not. Compounds, which are Central
Nervous System (CNS) active, are proficient in passing
across the BBB (Ma et al. 2005). The venture of a drug’s
action in terms of its efficacy and disposition is governed by
its binding to the plasma proteins (PPB), therefore, making
the PPB% a major pharmacokinetic factor (Mannhold et al.
2008). The forecasting of the human intestinal absorption
(HIA%) is equally appraised as a critical feature in the drug
development phase. The ratio of excretion/cumulative
excretion in urine, bile, and feces is applied to gauge the
sum of absorption and bioavailability as HIA% (Zhao et al.
2010). In order to predict the oral absorption of drugs,
models viz., the Madin–Darby canine kidney (MDCK) and
Caco-2 cells are judged to be well-grounded in-vitro models
(Hou et al. 2004; Irvine et al. 1999). The Ames test is a
straight forward method to foretell the mutagenicity and
carcinogenicity of the targeted compound (Bruce et al.
1973).
In-silico molecular docking studies
While designing a candidate drug, interaction between drug
candidates and the targeted receptor is mostly understood
by employing the molecular docking studies. This not only
predicts the activity but also foretells the affinity of the
tested drug candidates on the chosen receptor. Enzymes,
which are associated with the biosynthetic procedure of the
microbes’ cell wall, are estimated to be the supreme targets
for docking while designing novel compounds as anti-
microbial drug candidates. The enzyme glucosamine-6-
phosphate synthase (GlmS, GlcN-6-P synthase, L-gluta-
mine: D-fructose-6P amido-transferase, EC 2.6.1.16) is
materialized to be an attractive target for performing
docking studies while designing both antibacterial and
antifungal drug candidates (Chmara et al. 1984). This par-
ticular enzyme is involved in the genesis of the chief
building block, namely N-acetyl Glucosamine (the core
amino sugar) of the fungal and bacterial cell wall (Marshall
PreADMET was used to forecast the in-vivo ADMET
properties of all the synthesized compounds i.e. information
of rates for the BBB penetration, PPB%, HIA%, Caco-2,
MDCK cell permeability and P-glycoprotein substrate
inhibition, Table 2. All the investigated compounds can be
classified as CNS candidates specially the compounds 3a
and 6a, with highest absorption to CNS. All the standard
drugs except vinblastine were predicted as non-CNS

Medicinal Chemistry Research
Fig. 2 Tautomeric forms of the azo dyes 3a, b and 6a, b and the arylazo chromenes 10a–d
et al. 2003; Lather et al. 2018). The molecular docking was
considered to study the binding interactions of the synthe-
sized compounds with the target protein GlcN-6-P synthase
(PDB ID: 2VF5).
Phosphoinositide 3-kinases (PI3Ks) is a member of the
lipid kinases family and plays a crucial role in boosting the
survival and cellular growth of several types of cancer. In
cancer cells, the PI3Ks are the mandatory regulators of
apoptosis. The PI3Ks catalyzes the phosphorylation of
phosphatidylinositol of the inositol ring at the 3′-OH posi-
tion, procuring the secondary messenger lipid, namely
phosphatidylinositol-3,4,5-trisphosphate. This trisphosphate
activates the serine/threonine kinase AKT, which in turn
regulates several signaling pathways, thus, governing the
survival of cells, the apoptosis process, the proliferation of
cells, and the motility (Engelman 2009; Zhao and Vogt
2008). PI3Ks are categorized into three classes on basis of
their structural traits and substrate specificity. Class I PI3Ks
are the most assessed class of PI3Ks and it is grouped into 4
isoforms: p110α, p110β, p110δ, and p110γ. The PI3KCA
gene, encoding p110α, is the most routinely mutated gene
in human tumors, thus, inculcating to be the most striking

Medicinal Chemistry Research
Table 2 In-silico ADME
properties of the selected
Compound No.
BBBa
PPBb
HIAc
Caco-2d
MDCKe
P-gpf
compounds/some standard drugs
3a
2.82
0.97
0.20
0.25
0.24
0.97
0.61
0.91
0.85
2.55
0.19
0.10
0.17
0.57
0.94
0.93
0.06
0.03
0.03
0.87
0.02
0.04
92.41
99.25
90.47
99.66
100
96.05
97.07
97.00
97.03
97.36
97.07
97.35
96.18
93.54
94.74
90.03
96.23
96.10
93.48
97.68
96.93
81.48
5.28
29.50
22.04
29.58
24.39
22.25
22.04
21.16
11.00
0.83
76.92
0.10
4.21
8.49
21.12
0.10
0.03
29.92
48.16
0.16
0.08
0.04
0.04
0.04
0.04
0.04
0.94
0.47
0.04
0.04
119.16
1.02
Inhibitor
3b
Inhibitor
9a
Inhibitor
9b
Inhibitor
9c
Inhibitor
9d
99.26
100
Inhibitor
9e
Inhibitor
4a
87.39
88.76
97.59
100
Non-inhibitor
Inhibitor
4b
6a
19.23
6.99
Inhibitor
6b
Inhibitor
10a
100
20.42
20.33
15.23
18.74
18.68
0.63
Inhibitor
10b
100
Inhibitor
10c
100
Inhibitor
10d
100
Inhibitor
10e
100
Inhibitor
Ampicillin
Gentamicin
Amphotericin B
Vinblastine
Colchicine
Doxorubicin
36.16
46.93
39.01
71.67
65.42
31.16
Non-inhibitor
Non-inhibitor
Inhibitor
4.47
4.68
14.14
39.36
37.45
17.73
96.06
96.90
56.84
Inhibitor
Non-inhibitor
Non-inhibitor
aBlood–brain barrier penetration, bPlasma protein binding, cHuman intestinal absorption, dCaco-2 cell
e
f
permeability, MDCK cell permeability, P-glycoprotein substrate
target for tumor therapy (Mukohara 2015; Liu et al. 2014;
Yuan and Cantley 2008). Molecular docking screenings
were performed to gain a better understanding of the
binding mode of the synthesized compounds with the PI3K
p110α domain (PDB ID: 2RD0).
Biological study
Antimicrobial activity
The compounds 3a, b; 4a, b; 6a, b; 9a–e and 10a–e were
screened for their antimicrobial activity against two gram
positive bacteria [S.P (Streptococcus pneumoniae) and B.S
(Bacillus subtilis)], two gram negative bacteria [P.A
(Pseudomonas aeruginosa) and E.C (Escherichia coli)],
and four Fungi [A.F (Aspergillus fumigatus), S.R (Synce-
phalastrum racemosum), G.C (Geotricum candidum), and
C.A (Candida albicans)], using the diffusion agar technique
(Widelski et al. 2009) at different concentrations. Ampi-
cillin, Gentamicin, and Amphotericin B were used as con-
trol drugs (Yadav et al. 2007). The observed inhibition zone
(IZ) is given in Table 5 and presented in Figs 4 and 5 while
Table 6 illustrates the MIC values of the most active
molecules.
Evidently, some of the investigated compounds exhibited
moderate to good activity against gram positive bacteria
(Streptococcus pneumonia, Bacillis subtilis) and gram
negative bacteria (Escherichia coli). However, compounds
3b, 9a, 9d, 9e, and 4b didn't demonstrate activity against
the gram negative bacteria (Pseudomonas aeruginosan).
In order to establish the best in-silico conformation, the
molecular docking screenings of the newly synthesized
compounds on the Glucosamine-6-phosphate of the GlcN-
6-P synthase and the PI3K as the target proteins was exe-
cuted. To please this motive, a Lamarckian genetic algo-
rithm docking program, AutoDock 4.0, was employed. All
the synthesized compounds were docked on these two target
receptors. The docking of some selected ligands (with the
highest docking score among the tested compounds) is
shown in Fig. 3a, b. The minimum binding energy of all the
docked compounds on the respective receptors is docu-
mented in Table 4. The ligands (synthesized compounds
and the standard drugs) exhibited good in-silico docking
scores towards the target receptors viz., the GlcN-6-P syn-
thase, and PIK3, ranging from −8.7 to −6.37 and −10.2 to
−7.0 Kcal/mol, respectively. Compound 9e possess the
highest docking score of −8.7 towards the GlcN-6-P syn-
thase, and compounds 4a and 6a displayed the highest
docking score of −9.8 towards PIK3.

Medicinal Chemistry Research
Table 3 In-silico toxicity prediction of the selected compounds/some
standard drugs
Cytotoxic screening
Compound No. Ames Test
Mouse
Rat
The in-vitro cytotoxic activity of the studied molecules was
performed by the MTT assay (Alley et al. 1988) against
three human carcinoma cell lines: human colon carcinoma
(HCT-116), human hepatocellular carcinoma (HEPG-2),
and human breast adenocarcinoma (MCF-7). Doxorubicin,
Vinblastine, and Colchicine were used as positive controls.
The inhibitory effects of compounds 3a, b, 4a, b, 6a, b,
9a–e, and 10a–e on the growth of the three cell lines are
presented in Table 7.
As can be seen from Table 7 and Fig. 7, the values of
compounds 3b, 4a, 6a, 9d and 9e from the investigated
compounds in comparison to Colchicine as reference drug
exhibited good IC50s, ranging from 5.4 to 19.2 µM, against
the HCT-116 cell line. While compounds 3a, 3b, 4a, 6a,
6b, 9d and 9e displayed inhibitory effects against the MCF-
7 cell line with IC50 (8.6–44.3 µM), excluding 9a and 9b,
which accounts for the same value of Colchicine as standard
drug. Moreover, the compounds 3b, 6a, 9d and 9e showed
slightly high activity in the case of the HepG-2 cell line by
an IC50 range of (10.6 to 15.0 µM). All the investigated
compounds displayed less activity against the three cancer
cell lines as compared to both Vinblastine and Doxorubicin
reference drugs.
Mutagenicity Carcinogenicity Carcinogenicity
3a
3b
9a
9b
9c
Mutagenic
Mutagenic
Mutagenic
Mutagenic
Mutagenic
Mutagenic
Mutagenic
Mutagenic
Mutagenic
Mutagenic
Mutagenic
Negative
Negative
Positive
Positive
Positive
Negative
Negative
Positive
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Negative
Negative
Positive
Negative
Negative
Positive
Negative
Positive
Negative
9d
9e
4a
4b
6a
6b
10a
Non-
Mutagenic
10b
10c
10d
Mutagenic
Mutagenic
Negative
Negative
Negative
Negative
Positive
Negative
Non-
Mutagenic
10e
Non-
Negative
Negative
Mutagenic
Ampicillin
Gentamicin
Mutagenic
Mutagenic
Negative
Negative
Positive
Negative
Negative
Positive
Amphotericin B Non-
Mutagenic
Non-
Conclusion
Vinblastine
Negative
Positive
Mutagenic
A new series of azo dyes, containing chromene moieties,
has been synthesized, using the multicomponent reaction
(MCRs) strategy containing azo dye, malononitrile, and the
corresponding aldehydes. The structure identity of the
desired compounds was confirmed, using the spectroscopic
techniques. These molecules have been employed for sev-
eral theoretical studies in order to predict their performance
as a new drug system. ADMET properties were predicted in
order to gauge the pharmacokinetic and pharmacodynamic
features of the synthesised compounds. As per predictive
results, the analyzed compounds can be classified as CNS
and skillfully absorbed candidates as compared to the pre-
dicted standard drugs, as the synthesized compounds
flaunted good BBB permeation (≥0.1) and HIA%
(87.39%–100%) along with PPB more than 90%. Most of
the predicted compounds emerged as P-gp inhibitors along
with Amphotericin B and vinblastine. The predicted com-
pounds possess low to moderate Caco-2 and MDCK cells
permeability with the highest predicted Caco-2 permeability
for vinblastine (39.36%). The toxicity prediction reveals
that most of the compounds are mutagenic in nature when
compared with the predicted non-mutagenic nature of some
of the standard drugs. Most the tested compounds and
standard drugs were forecasted to be carcinogenic both in
Colchicine
Mutagenic
Negative
Negative
Positive
Doxorubicin
Non-
Mutagenic
Negative
Among the azo dye derivatives, 6a displayed good activity
against (Streptococcus pneumonia) and (Bacillis subtilis)
with respect to the standard drug Ampicillin as shown in
Table 5. In addition, 3a and 9b showed higher activity
against (Pseudomonas aeruginosa) and (Escherichia coli)
in comparison to the reference drug Gentamicin, Table 5.
Moreover, the obtained results imply that compounds 3a,
9b, 9e; 6a and 10b could be attractive new
antibacterial drugs.
As shown in (Table 5) and (Fig. 6), the antifungal per-
formance of the target compounds, which displays moderate
to good activity against the studied fungi (Aspergillus
fumigates, Syncephalastrum racemosum, Geotricum can-
didum and Candida albicans). Based on the obtained data,
compounds 3a, 9c, 9d, 4a, 4b, 6a, 6b, 10a, 10d, and 10e
did not exhibit any activity against (Syncephalastrum
racemosum). However, 3b, 6a, 6b, and 10b derivatives
portrayed very good antifungal activity against most of the
fungi as compared to the reference drug Amphotericin-B.

Medicinal Chemistry Research
Fig. 3 a Docking of compound 9e into active site of glucosamine-6-phosphate (GlcN-6-P) synthase (PDB ID: 2VF5). b Docking of compound 4a
into active site of Phosphoinositide 3-kinases (PI3K, PDB ID: 2RD0)
rat and mouse except amphotericin B. In-silico molecular
docking study unveiled the interaction between the syn-
thesized ligands and the targeted protein receptor to be
favorable with good docking scores. The highest docking
score was obtained by compound 9d i.e −8.7 Kcal/mol
towards the GlcN-6-P synthase, and compounds 4a and 6a
i.e −9.8 Kcal/mol towards PIK3.
In order to endorse these in-silico results, the new
compounds were then screened for their in-vitro anti-
microbial and antitumor behaviour, sequentially, investigate
their potential as new drug candidates. The antibacterial
study stated that compounds 3a, 6a, 6b, 9e, and 10b have
the most antibacterial effect while 3b, 6a, 6b, and 10b
derivatives displayed very good antifungal activity against
most of the tested fungi. The in-vitro results were validated
as these compounds emerged as promising candidates in the
molecular docking studies with energetically favorable
binding poses and good docking score as compared to other
compounds which are neither promising candidates in in-
silico nor in in-vitro studies. Meanwhile, the in-vitro
anticancer examination showed that most of the target
molecules displayed unexpected comparable cytotoxicity to

Medicinal Chemistry Research
Table 4 Docking scores as minimum binding energy (Kcal/mol) of the
synthesized compounds/standard drugs on the target receptors i.e
GlcN-6-P synthase and PIK3 respectively
Table 5 Mean zone of inhibition in mm produced on a range of
environmental clinically pathogenic microorganisms using (5 mg/mL)
concentration of tested samples
Compound no.
GlcN-6-P synthase
PIK3
Compounds
Tested microorganisms
Gm +ve Gm −ve
Fungi
3a
−7.3
−7.5
−8.1
−8.2
−8.5
−8.6
−8.7
−6.6
−7.1
−8.1
−8.4
−7.5
−8.1
−5.7
−6.4
−6.5
−7.8
−7.4
−8.1
–
−8.5
−9.7
−8.3
−8.1
−8.4
−9.6
−9.6
−9.8
−8.4
−9.8
−8.4
−7.6
−7.6
−7.0
−7.4
−7.8
–
3b
S.P B.S P.A E.C A.F S.R G.C C.A
9a
3a
16.4 17.3 17.5 19.3 16.2 NA 17.6 17.2
16.2 18.1 NA 15.6 19.3 17.2 21.3 18.3
17.5 19.3 NA 16.6 18.5 17.8 19.4 17.5
18.6 18.7 19.2 17.6 17.7 18.3 19.7 18.5
17.6 18.2 16.5 17.8 18.6 NA 18.8 19.3
18.2 17.5 NA 18.2 17.5 NA 19.2 18.8
18.5 19.2 NA 18.2 17.8 19.6 19.6 18.5
17.3 16.8 16.8 17.5 16.5 NA 17.4 16.8
16.8 17.4 NA 17.2 17.6 NA 16.8 17.6
20.2 23.8 16.3 17.5 19.8 NA 22.3 21.1
19.2 20.1 17.9 16.2 18.2 NA 21.4 20.6
16.7 17.8 14.6 16.5 15.7 NA 16.1 15.2
17.8 18.2 16.5 15.4 19.2 16.7 20.5 19.8
15.9 16.1 15.8 15.2 17.8 16.3 18.7 19.6
17.5 16.8 16.2 15.3 16.5 NA 17.9 18.5
18.1 17.7 16.5 16.2 17.3 NA 17.2 18.6
9b
3b
9c
9a
9d
9b
9e
9c
4a
9d
4b
9e
6a
4a
6b
4b
10a
6a
10b
6b
10c
10a
10d
10b
10e
10c
Ampicillin
Gentamicin
Amphotericin B
Vinblastine
Colchicine
Doxorubicin
10d
–
10e
–
Ampicillin
Gentamicin
Amphotericin B
23.8 32.4
–
–
–
–
–
–
–
–
–
–
−9.5
−9.3
−10.2
–
–
–
–
17.3 19.9
–
–
–
23.7 19.7 28.7 25.4
–
Mean Zone of Inhibiꢀon in mm for the tested
compounds for Gm+ve microorganisms
the reference drug, in particular, compounds 3b, 4a, 6a, 9e
and 9d which demonstrated good IC50 values against the
HCT-116 cell line, as compared to Colchicine. While,
compounds 3a, 3b, 4a and 6a exhibited excellent IC50
values against the MCF-7, as compared to Colchicine.
These results were found to be consistent with the in-silico
molecular docking studies as these compounds 3b, 4a, 6a,
and 9d had shown the highest binding energy toward the
target protein PIK3 ranging from −9.8 to −9.6 kJ mol−1
and have good affinity toward the protein. Thus, they may
be considered as good inhibitors of PIK3 by in-silico studies
which was supported by good in-vitro anticancer results.
This alliance between in-silico and in-vitro studies may
facilitate structural based design of more potent and selec-
tive compounds in the future.
35
30
25
20
15
10
5
0
S.P B.S
Fig. 4 Gm +ve microorganism antibacterial activity of the investi-
gated compounds
Shimadzu IR – 8400S, using the KBr pellets (ν in cm−1).
The NMR spectra were recorded on a Bruker Avance
400 MHz, spectrometer, using the TMS as an internal
standard and the CDCl3 (DMSO) as solvents, [Chemical
shifts in δ (ppm)]. The MS were measured on a Shimadzu
GC/MS-QP5050A spectrometer. The UV-Vis spectra were
recorded on a Jasco UV-Vis spectrophotometer double-
beam spectrophotometer (range 350 to 700 nm) with the
scan speed at 200 nm min−1, using a 1.0 cm quartz cell. A
cell containing the pure solvent was used as reference. All
Experimental section
Materials & instrumentation
All the melting points are measured by a MEL-TEMP
Electrothermal Melting point apparatus and were uncor-
rected. The IR absorption spectra were recorded by a

Medicinal Chemistry Research
Mean Zone of Inhibiꢀon in mm for the tested
Mean Zone of Inhibiꢀon in mm for the tested
compounds for Fungi's
compounds for Gm-ve microorganisms
25
20
15
10
5
35
30
25
20
15
10
5
0
0
P.A E.C
A.F S.R G.C C.A
Fig. 5 Gm −ve microorganism antibacterial activity of the investi-
gated compounds
Fig. 6 Antifungal activity of the investigated compounds
Table 6 MIC (μg/mL) of tested
samples against tested
microorganisms
Compounds
Tested microorganisms
Gm +ve
Gm –ve
Fungi
S.P
B.S
3.9
P.A
E.C
A.F
S.R
G.C
1.95
C.A
3b
7.81
7.81
7.81
31.25
7.81
7.81
7.81
3.9
NA
31.25
31.25
7.81
7.81
7.81
3.9
3.9
3.9
7.81
7.81
3.9
3.9
31.25
7.81
1.95
3.9
9a
7.81
3.9
NA
3.9
1.95
3.9
9b
1.95
31.25
NA
7.81
3.9
9c
7.81
7.81
3.9
NA
NA
1.95
NA
NA
NA
NA
16.7
31.25
NA
NA
–
9d
7.81
7.81
31.25
3.9
1.95
1.95
7.81
1.95
3.9
9e
NA
7.81
31.25
3.9
4a
31.25
1.95
3.9
31.25
31.25
31.25
31.25
7.81
31.25
31.25
31.25
–
7.81
7.81
31.25
15.63
31.25
31.25
31.25
31.25
–
6a
6b
7.81
7.81
7.81
31.25
15.63
7.81
0.98
–
7.81
31.25
3.9
1.95
31.25
3.9
10a
7.81
3.9
15.63
1.95
7.81
7.81
15.63
–
10b
10c
31.25
31.25
7.81
0.24
–
7.81
15.63
15.63
–
7.81
3.9
10d
10e
7.81
–
Ampicillin
Gentamicin
Amphotericin B
15.63
–
3.9
–
–
–
–
–
–
–
0.98
3.9
0.49
0.49
the measurements were made at room temperature. All
reactions have been followed by the TLC. The biological
evaluation of the products was carried out in the Medical
Mycology Laboratory of the Regional Center for Mycology
and Biotechnology of Al-Azhar University, Cairo, Egypt.
was stirred gently to ensure complete dissolution of aniline
at 0–5 °C. Sodium nitrite (10 mmol), dissolved in water
(10 mL), was used to diazotize the aniline derivatives at
0–5 °C. The nitrite solution was intermittently added to the
amine solution with stirring, and the temperature was
monitored to ensure that it did not exceed 5 °C. The dia-
zonium salt solution was left in the freezing mixture and
was stirred occasionally until the temperature came down to
0 °C for 2 hrs. The coupling component, 2-naphthol (2) or
resorcinol (5) (10 mmol), was dissolved in a 10% sodium
hydroxide solution and then cooled to 0 °C by adding ice.
The previously prepared diazonium solution was added
drop wise over 30 min with stirring. The mixture was stirred
for a further 2 hrs. at 5 °C, and the pH of this solution was
Chemistry
General procedure for the preparation of 1-arylazo-
naphthalen-2-ol (3a,b) and [5-substituted-2-(2,4-
dihydroxyphenylazo)-phenyl] [phenyl]methanone (6a, b)
Aniline derivatives 1a, b or 4a, b (10 mmol) were dissolved
in a solution of hydrochloric acid and water. The mixture

Medicinal Chemistry Research
d, J = 8 Hz, Ar–CH), 16.15 (1H, s, NH hydrazo form); 13C
NMR (CDCl3, ppm) δ: 21.30 (CH3), 119.19, 121.64,
123.98, 125.32, 128.21, 128.50, 128.59, 129.91, 130.20,
133.71, 138.38, 138.85, 143.90 (Ar-C), 168.50 (C = O of
hydrazo form).
Table 7 Inhibitory activity against HCT-116, MCF-7 and HepG-2
cell lines
Compounds
IC50 (μM)
HCT-116
MCF-7
HepG-2
3a
69.0
14.9
50.4
50.4
59.2
19.2
17.5
11.4
167.2
5.4
15.1
9.5
115.5
10.6
32.0
26.7
28.1
14.2
14.6
43.1
152.3
15.0
74.6
307.3
302.9
144.3
149.2
126.6
5.7
3b
1-[4-(2-Hydroxynaphthalene-1-ylazo)phenyl]ethanone
(3b) Red solid (85%), m.p. 144 °C; IR (KBr) cm−1: IR
vmax (cm−1, KBr): 3257(OH), 1446 (N = N), 1685 (C = O);
1H NMR (CDCl3, ppm) δ: 2.61 (3H, s, COCH3), 6.72 (1H,
d, J = 9.6 Hz, Ar–CH), 7.40 (1H, t, J = 6.8 Hz, Ar–CH),
7.52-7.56 (2H, m, Ar–CH), 7.66–7.68 (3H, m, Ar–CH),
8.04 (2H, d, J = 8.8 Hz, Ar–CH), 8.43 (1H, d, J = 8.4 Hz,
Ar–CH), 16.13 (1H, s, NH hydrazo form); 13C NMR
(CDCl3, ppm) δ: 26.55 (CH3), 116.91, 122.20, 126.17,
126.89, 128.35, 128.96, 129.49, 130.24, 131.21, 133.33,
134.29, 142.51, 146.76 (Ar-C); 178.44 (C = O of hydrazo
form), 19676 (COCH3).
9a
95.4
96.9
103.1
44.3
29.2
12.1
185.4
8.6
9b
9c
9d
9e
4a
4b
6a
6b
252.7
374.3
278.1
253.9
368.9
298.9
3.2
40.7
159.4
139.4
170.8
210.3
207.6
7.5
10a
10b
10c
[5-Chloro-2-(2,4-dihydroxyphenylazo)-phenyl]
[phenyl]
10d
methanone (6a) Reddish orange solid (84%), m.p. 118 °
C; IR (KBr) cm−1: 3460, (OH), 1685 (CO), 1450 (N = N);
1H NMR (DMSO-d6, ppm) δ: 6.25 (1H, s, Ar–CH), 6.32-
6.34 (1H, dd, Ar–CH), 7.14 (1H, d, Ar–CH), 7.50 (2H, t,
Ar–CH), 7.63 (1H, t, Ar–CH), 7.69 (1H, d, Ar–CH), 7.71
(1H, d, Ar–CH), 7.72 (1H, s, Ar–CH), 7.78 (1H, d, Ar–CH),
8.02(1H, d, Ar–CH), 10.69 (1, s, OH), 11.36 (1H, s, NH
hydrazo form); 13C NMR (DMSO-d6, ppm) δ: 103.29,
109.85, 120.61, 128.57, 128.66, 129.32, 129.67, 131.54,
132.87, 134,17, 134.71, 137.41, 137.79, 147.91, 157.09,
164.42 (Ar-C), 195.35 (C = O of carbonyl group).
10e
Vinblastine
Colchicine
Doxorubicin
44.3
1.6
107.2
1.9
26.5
2.2
Inhibitory Acꢀvity against HCT-116, MCF-7 and
HepG-2 cell lines
400
350
300
250
200
150
100
50
[2-(2,4-Dihydroxyphenylazo)-5-nitrophenyl]
[phenyl]
methanone (6b) Red solid (76%), m.p. 154 °C; IR (KBr)
0
1
cm−1: 3460 (OH), 1685 (CO), 1450 (N = N); H NMR
(DMSO-d6, ppm) δ: 6.28 (1H, s, Ar–CH), 6.35–6.50 (1H,
dd, Ar–CH), 7.14 (1H, d, Ar–CH), 7.51 (2H, t, Ar–CH),
7.65 (1H, t, Ar–CH), 7.74 (1H, d, Ar–CH), 7.82 (1H, d,
Ar–CH), 8.18 (1H, s, Ar–CH), 8.41 (1H, d, Ar–CH), 8.49
(1H, d, Ar–CH), 11.70 (1, s, OH), 13.78 (1H, s, NH
hydrazo form).
IC50 (μM)
IC50 (μM)
IC50 (μM)
Fig. 7 Antitumor activity of the investigated compounds
adjusted to 4–5 by adding the hydrochloric acid slowly,
followed by the filtration and washing until neutral and the
drying and then crystallizing from ethanol afforded azo dyes
3a, b or 6a, b, respectively.
General procedure for synthesis of 2-amino-4H-benzo[g]
chromene-3-carbonitriles (9a–e) and 2-amino-4H-
chromene-3-carbonitriles (10a–e)
1-[(4-Methylphenyl)azo]naphthalene–2-ol (3a) Red solid
(94%), m.p. 119 °C; IR (KBr) cm−1: IR vmax (cm−1, KBr):
3436 (OH), 1446 (N = N); H NMR (CDCl3, ppm) δ: 2.41
(3H, s, CH3), 6.92 (1H, d, J = 9.2 Hz, Ar–CH), 7.28 (2H, d,
J = 8 Hz, Ar–CH), 7.39 (1H, t, Ar–CH), 7.55 (1H, t,
Ar–CH), 7.62 (1H, d, J = 8 Hz, Ar–CH), 7.67 (2H, d, J =
8 Hz, Ar–CH), 7.72 (1H, d, J = 9.2 Hz, Ar–CH), 8.61 (1H,
A mixture of azodyes 3a, b or 6a, b (2.3 mmol), mal-
ononitrile (2.3 mmol), and aryl aldehyde (2.3 mmol) was
dissolved in ethanol (10 mL) and few drops of pipredine
were added. The reaction mixture was refluxed (4–6 h.).
After the completion of the reaction (monitored by TLC),
the mixture was cooled, filtered, and washed with ethanol
and hexane to afford 9a–e or 10a–e, respectively.
1

Medicinal Chemistry Research
2-Amino-4-(4-methoxyphenyl)-10-[(4-methylphenyl)azo]-
4H-benzo[g]chromene- 3-carbonitrile (9a) Dark red solid
(75%), m.p. 118 °C; IR (KBr) cm−1: 3450, 3386 (NH2),
2979, 2935, 2869 (CH), 2210 (CN), 1430 (N = N); 1H
NMR (DMSO-d6, ppm) δ: 2.29 (3H, s, CH3), 3.65 (3H, s,
OCH3), 4.95 (1H, s, CH pyran), 5.97 (2H, s, NH2), 6.80-
7.15 (4H, m, ArCH), 7.55–7.70 (4H, m, ArCH), 8.15–8.26
(2H, m, ArCH), 8.36 (1H, s, ArCH), 8.46 (2H, d, ArCH);
13C NMR (DMSO-d6, ppm) δ: 20.96 (CH3), 40.57 (CH
pyran), 52.8 (OCH3), 60.83 (CN), 112.5, 115.5, 121.5,
122.6, 123.5, 125.8, 127.7, 129.2, 129.5, 130.08, 131.5,
132.8, 133.01, 134.6, 135.12, 136.9, 143.4, 148.6, 158.7,
177.7 (Ar-C). MS, m/z (%): 446 (M+, 51.36) with a base
peak at 259 (100).
132.08, 135.5, 138.0, 139.01, 140.5, 142.8, 146.6, 148.4,
157.6, 161.7, 178.6 (Ar-C), 197.39 (C = O). MS, m/z (%):
462 (M+, 40.31) with a base peak at 367 (100).
10-[(4-Acetylphenyl)azo]-2-amino-4-(4-bromophenyl)-4H-
benzo[g]chromene-3-carbonitrile (9e) Red solid (74%), m.
p. 218 °C; IR (KBr) cm−1: 3460, 3420 (NH2), 2979, 2935,
1
2869 (CH), 2195 (CN), 1450 (N = N); H NMR (DMSO-
d6, ppm) δ: 2.69 (3H, s, COCH3), 5.74 (1H, s, CH pyran),
5.97 (2H, s, NH2), 7.36 (2H, d, J = 8.4 Hz, ArCH), 7.56
(2H, d, J = 8.4 Hz, ArCH), 7.72-7.76 (2H, m, ArCH), 7.85
(1H, t, ArCH), 8.03 (1H, d, ArCH), 8.12 (1H, t, ArCH),
8.38 (2H, d, J = 8.0 Hz, ArCH), 8.82 (2H, d, J = 8.0 Hz,
ArCH); 13C NMR (DMSO-d6, ppm) δ: 26.96 (COCH3),
43.57 (CH pyran), 60.83 (CN), 102.1, 103.2, 121.3, 122.8,
127.4, 128.6, 129.9, 130.0, 130.08, 130.5, 131.0, 133.01,
135.5, 137.8, 138.6, 138.9, 143.4, 150.6, 151.7, 178.6, (Ar-
C), 197.39 (C = O). MS, m/z (%): 522 (M+, 51.36) with a
base peak at 367 (100).
2-Amino-4-(4-fluorophenyl)-10-[(4-methylphenyl)azo]-4H-
benzo[g]chromene-3-carbonitrile
(9b) Reddish-brown
solid (70%), m.p. 126 °C; IR (KBr) cm−1: 3266, 3258
(NH2), 2979, 2935, 2869 (CH), 2200 (CN), 1430 (N = N);
1H NMR (DMSO-d6, ppm) δ: 2.31 (3H, s, CH3), 5.45 (1H,
s, CH pyran), 6.52 (2H, s, NH2), 7.15–7.35 (4H, m, ArCH),
7.65–7.85 (4H, m, ArCH); 8.12–8.30 (2H, m, ArCH), 8.40
(1H, s, ArCH), 8.46 (2H, m, ArCH); 13C NMR (DMSO-d6,
ppm) δ: 21.25 (CH3), 39.95 (CH pyran), 60.83 (CN), 114.5,
118.5, 124.5, 126.6, 127.5, 128.8, 129.6, 129.9, 130.21,
131.87, 132.08, 133.5, 134.08, 135.8, 136.01, 138.6, 139.9,
145.4, 148.2, 161.7, 179.7 (Ar-C). MS, m/z (%): 434 (M+,
45.62) with a base peak at 339 (100).
2-Amino-6-[(2-benzoyl-4-chlorophenyl)azo]-7-hydroxy-4-(4-
methoxyphenyl)-4H- chromene-3-carbonitrile (10a) Red-
dish orange solid (84%), m.p. 250 °C; IR (KBr) cm−1: 3460
(OH), 3450, 3220 (NH2), 2220 (CN), 1685 (CO), 1450 (N
1
= N); H NMR (DMSO-d6, ppm) δ: 3.85 (3H, s, OCH3),
4.69 (1H, s, CH pyran), 6.65 (1H, s, Ar–CH), 6.88 (2H, s,
NH2), 6.95 (2H, d, ArCH), 7.05 (2H, t, Ar–CH), 7.15 (2H,
d, Ar–CH), 7.45 (2H, t, Ar–CH), 7.65 (1H, t, Ar–CH), 7.69
(2H, d, Ar–CH), 8.19 (1H, s, Ar–CH), 9.81 (1, s, OH),
11.36 (1H, s, NH hydrazo form); 13C NMR (DMSO-d6,
ppm) δ: 36.05 (CH pyran), 44.26 (OCH3), 57.77 (CN),
108.9, 113.5, 114.2, 114.9, 114.22, 114.93. 122.43, 123.54,
124.23, 124.92, 126.11, 127.21, 128.65, 129.36, 129.67,
131.88, 132.39, 134.10, 136.51, 139.12, 147.83, 154.74,
159.95, 163.16, (Ar-C), 195.35 (C = O). MS, m/z (%): 536
(M+, 35.64) with a base peak at 244 (100).
2-Amino-4-(4-chlorophenyl)-10-[(4-methylphenyl)azo]-4H-
benzo[g]chromene-3-carbonitrile (9c) Dark red solid
(72%), m.p. 118 °C; IR (KBr) cm−1: 3450, 3278 (NH2),
2979, 2935, 2869 (CH), 2220 (CN), 1435 (N = N); 1H
NMR (DMSO-d6, ppm) δ: 2.29 (3H, s, CH3), 5.45 (1H, s,
CH pyran), 6.52 (2H, s, NH2), 7.19–7.39 (4H, m, ArCH),
7.43–7.49 (4H, m, ArCH), 8.15–8.30 (2H, m, ArCH), 8.47
(1H, s, ArCH), 8.66 (2H, m, ArCH); 13C NMR (DMSO-d6,
ppm) δ: 23.25 (CH3), 40.95 (CH pyran), 62.73 (CN), 116.5,
117.5, 123.5, 125.6, 126.5, 127.9, 128.04, 129.6, 130.9,
131.25, 132.08, 133.5, 134.8, 135.69, 136.01, 137.6, 139.9,
145.4, 148.2, 160.7, 177.7 (Ar-C). MS, m/z (%): 450 (M+,
32.46) with a base peak at 339 (100).
2-Amino-6-[(2-benzoyl-4-chlorophenyl)azo]-7-hydroxy-4-(4-
fluorophenyl)-4H- chromene-3-carbonitrile (10b) Reddish
orange solid (84%), m.p. 154 °C; IR (KBr) cm−1: 3465 (OH),
3455, 3260 (NH2), 2190 (CN), 1685 (CO), 1450 (N = N); 1H
NMR (DMSO-d6, ppm) δ: 4.70 (1H, s, CH pyran), 6.15 (1H,
s, ArCH), 6.25 (1H, s, ArCH), 6.33 (1H, t, ArCH), 6.60 (1H,
d, J = 9.2 Hz, ArCH), 6.95 (2H, t, ArCH), 7.00 (1H, d,
ArCH), 7.03 (1H, t, ArCH), 7.05 (2H, s, NH2), 7.10–7.81
(2H, m, ArCH), 8.04 (2H, d, ArCH), 8.18 (1H, d, ArCH),
8.54 (1H, s, OH), 11.33 (1H, s, NH hydrazo form); 13C NMR
(DMSO-d6, ppm) δ: 35.76 (CH pyran), 56.93 (CN), 108.44,
112.36, 114.13, 114.99, 115.21, 116.80, 117.02, 119.87,
128.10, 128.70, 128.82, 129.04, 129.18, 131.10, 133.36,
133.65, 135.65, 135.58, 137.19, 147.40, 153.41, 159.43
(ArCH), 194.83 (C = O). MS, m/z (%): 524 (M+, 29.56) with
a base peak at 324 (100).
10-[(4-Acetylphenyl)azo]-2-amino-4-(4-fluorophenyl)-4H-
benzo[g]chromene-3-carbonitrile (9d) Red solid (78%), m.
p. 208 °C; IR (KBr) cm−1: 3400, 3350 NH2), 2979, 2935,
1
2869 (CH), 2210 (CN), 1445 (N = N); H NMR (DMSO-
d6, ppm) δ: 2.67 (3H, s, COCH3), 5.25 (1H, s, CH pyran),
6.59 (2H, s, NH2), 7.15–7.35 (4H, m, ArCH), 7.65–7.85
(4H, m, ArCH), 8.19-8.35 (2H, m, ArCH), 8.40 (1H, s,
ArCH), 8.46 (2H, m, ArCH); 13C NMR (DMSO-d6, ppm) δ:
26.96 (COCH3), 41.57 (CH pyran), 60.83 (CN), 114.2,
116.1, 120.2, 121.3, 123.8, 127.4, 128.6, 129.9, 131.0,

Medicinal Chemistry Research
2-Amino-6-[(2-benzoyl-4-nitrophenyl)azo]-7-hydroxy-4-(4-
methoxyphenyl)-4H- chromene-3-carbonitrile (10c) Red-
dish orange solid (84%), m.p. 208 °C; IR (KBr) cm−1: 3460
(OH), 3450, 3220 (NH2), 2220 (CN), 1690 (CO), 1450 (N =
pharmacokinetic etiquette. The ADMET predictions were
done by exploiting the PreADMET software.
In-silico molecular docking studies
1
N); H NMR (DMSO-d6, ppm) δ: 3.72 (3H, s, OCH3), 4.65
(1H, s, CH pyran), 6.59 (1H, d, J = 8.8 Hz, Ar–CH), 7.01
(2H, s, NH2), 7.07 (2H, d, J = 8.8 Hz, Ar–CH), 7.51 (2H, t,
Ar–CH), 7.66 (1H, t, Ar–CH), 8.38 (1H, d, J = 8.8 Hz,
Ar–CH), 8.46 (1H, s, Ar–CH), 8.51 (1H, d, J = 8.8 Hz,
Ar–CH), 9.88 (1, s, OH), 11.59 (1H, s, NH hydrazo form);
13C NMR (DMSO-d6, ppm) δ: 35.63 (CH pyran), 55.06
(OCH3), 57.47 (CN), 109.00, 113.16, 113.80, 119.34,
120.03, 121.15, 124.17, 126.26, 128.24, 128.85, 129.33,
133.78, 135.75, 136.94, 137.05, 137.46, 147.67, 152.18,
153.67, 155.49, 158.02, 159.25 (Ar-C), 194.35 (C = O). MS,
m/z (%): 547 (M+, 25.61) with a base peak at 334 (100).
The In-silico docking strategy was recruited to perform
structure based drug designing in order to procure the more
effective inhibitors of GlcN-6-P and PI3K from the syn-
thesized drug candidates. The ACD/Labs-Chemsketch pro-
gram was utilized to create the 3D atomic coordinates of the
ligands. 2VF5 and 2RD0 (PDB ID’s), respectively, were
reviewed as the most appropriate pure 3D crystal structure of
the Glucosamine-6-phosphate of GlcN-6-P synthase and the
p110α subunit of PI3K as fetched from the protein data bank
(PDB) (Source: www.rcsb.org/pdb/). Energy of the mole-
cules was minimized, using a Dundee PRODRG2 server,
and was exploited to minimize the energy of the targeted
2-Amino-6-[(2-benzoyl-4-nitrophenyl)azo]-7-hydroxy-4-(4-
bromophenyl)-4H- chromene-3-carbonitrile (10d) Reddish
orange solid (84%), m.p. 250 °C; IR (KBr) cm−1: 3460
(OH), 3450, 3220 (NH2), 2220 (CN), 1655 (CO), 1445 (N
= N); 1H NMR (DMSO-d6, ppm) δ: 4.64 (1H, s, CH pyran),
6.59 (1H, d, J = 8.8 Hz, Ar–CH), 6.87 (2H, d, J = 8.8 Hz,
ArCH), 6.96 (2H, d, J = 8.8 Hz, ArCH), 7.05 (2H, s, NH2),
7.07 (2H, d, J = 8.8 Hz, Ar–CH), 7.51 (2H, t, Ar–CH), 7.66
(1H, t, Ar–CH), 7.72 (2H, d, J = 8.4 Hz, Ar–CH), 8.46 (1H,
s, Ar–CH), 9.70 (1, s, OH), 11.45 (1H, s, NH hydrazo form);
13C NMR (DMSO-d6, ppm) δ: 36.83 (CH pyran), 55.06
(OCH3), 57.47 (CN), 109.00, 113.16, 113.80, 119.34,
120.03, 121.15, 124.17, 126.26, 128.24, 128.85, 129.33,
133.78, 135.75, 136.94, 137.05, 137.46, 147.67, 152.18,
153.67, 155.49, 159.25 (Ar-C), 194.35 (C = O). MS, m/z
(%): 595 (M+, 31.5) with a base peak at 334 (100).
ligands (Schuttelkopf and Aalten 2004). The docking
̈
simulations were prepared, ran, and analyzed by the means
of the Graphical User Interface program, namely Autodock4
from “Auto-Dock Tools (ADT, 1.5.6)” (Morris et al. 1998).
The assignment of the Kollman united-atom charges
along with the addition of the only polar hydrogens to the
receptor protein was achieved. Ligands were assigned with
the Gasteiger charge, and the torsions were set. The grids
were made and adjusted in an X, Y, Z-axis so that it
comprises the whole active site of the targeted proteins with
a grid spacing of 0.375 Å. The Lamarckian Genetic Algo-
rithm (LGA) was preferred over the Monte Carlo method,
which is present in earlier versions of AutoDock. The
docking specifications were stationed to the software's
default values and a standard decorum was maintained and
followed throughout the docking screenings. The result
outcomes were elucidated on the basis of a pdf file gener-
ated by the software by ranking the different ligands with
respect to the forecasted binding energy.
A cluster analysis, based on values of the root mean
square deviation, was conducted. The minimum energy co-
ordinates were ascertained, using the same rmsd table cre-
ated in the dlg file. The cluster, securing the lowest energy,
was appraised as the most reliable solution.
For 3D visualization of ligand protein interaction, we
employed the UCSF Chimera 1.11.2 and mcule, a web
interface, which uses the WebGL/Javascript based molecule
viewer of GLmol.
2-Amino-6-[(2-benzoyl-4-nitrophenyl)azo]-7-hydroxy-4-(4-
chlorophenyl)-4H- chromene-3-carbonitrile (10e) Reddish
orange solid (84%), m.p. 250 °C; IR (KBr) cm−1: 3460
(OH), 3450, 3220 (NH2), 2220 (CN), 1655 (CO), 1450 (N
= N); 1H NMR (DMSO-d6, ppm) δ: 4.65 (1H, s, CH
pyran), 7.05 (2H, s, NH2), 6.25–8.85 (13H, m, ArCH), 9.85
(1H, s, OH), 11.59 (1H, s, NH hydrazo form): 13C NMR
(DMSO-d6, ppm) δ: 36.83 (CH pyran), 57.47 (CN), 109.00,
113.16, 113.80, 119.34, 120.03, 121.15, 124.17, 126.26,
128.24, 128.85, 129.33, 133.78, 135.75, 136.94, 137.05,
137.46, 147.67, 152.18, 153.67, 155.49, 158.02, 159.25
(Ar-C), 194.35 (C = O). MS, m/z (%): 551 (M+, 34.6) with
a base peak at 335 (100).
Biological studies
Absorption, Distribution, Metabolism, Excretion, and
Toxicity (ADMET) Predictions
Antimicrobial screening
The microorganism inoculums were uniformly spread using
sterile cotton swabs on a sterile Petri dish malt extract agar
(for fungi) and nutrient agar (for bacteria). One hundred
The molecular structure of the examined candidates played
a crucial task in deciding the ADMET properties and the

Medicinal Chemistry Research
milliliters of each sample (at 10 mg/ml concentrations dis-
solved in Dimethyl Sulfoxide) was added to each well (6-
mm-diameter holes were cut in the agar gel, 30 mm apart
from one another). The samples were compared to the
standard drug as positive control and DMSO solvent con-
trol. The cultures were incubated for 24 h at 37 °C (for
bacteria) and for 48 h at 28 °C (for fungi). After incubation,
the microorganism growth was observed. Inhibition zones
of the bacterial and fungal growth were measured in milli-
meters. Tests were performed in triplicate (Cappuccino and
Sherman 1999; Vanden-Berghe and Vlientinck 1991).
(IC50), the concentration required to cause toxic effects in
50% of intact cells, was estimated from graphic plots.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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