Facile and expedient access to bis-coumarin-iminothiazole hybrids by molecular hybridization approach: synthesis, molecular modelling and assessment of alkaline phosphatase inhibition, anticancer and antileishmanial potential

Article abstract of DOI:10.1039/c5ra14900b

In the design of new drugs, the hybridization approach might allow obtaining molecules with improved biological activity with respect to the corresponding lead compounds. Thus, adopting this approach, a new series of novel bis-coumarin-iminothiazole hybrids was designed, synthesized and tested for their biological action against alkaline phosphatase, leishmaniasis and cancerous cells. The structures of the synthesized hybrid compounds (5a-m) were characterized and established by using spectro-analytical data. The synthesized analogues were evaluated against alkaline phosphatase where compound 5j was emerged as a potent inhibitor with IC₅₀ value of 1.38 ± 0.42 μM. This inhibitory efficacy is two-fold higher as compared to the standard inhibitor. The synthesized compounds were also assayed for their anti-leishmanial potential against Leishmania major and compound 5i was observed as the lead candidate with 70.4% inhibition. The prepared compounds also showed cytotoxic behavior against kidney fibroblast (BHK-21) and lung carcinoma (H-157) cancer cell lines. Molecular docking of the synthesized library of iminothiazole derivatives against ALP was performed to delineate ligand-protein interactions at molecular level which suggested that the major interacting residues in the binding sites of the proteins might have an instrumental role in the inhibition of enzyme's function. Our results inferred that compounds 5j-m may serve as potential surrogates for the development of potent inhibitors of ALP.

Full text of DOI:10.1039/c5ra14900b

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Facile and expedient access to bis-coumarin–
iminothiazole hybrids by molecular hybridization
approach: synthesis, molecular modelling and
assessment of alkaline phosphatase inhibition,
anticancer and antileishmanial potential†
b
Cite this: RSC Adv., 2015, 5, 89919
Aliya Ibrar,a Sumera Zaib,b Imtiaz Khan,a Farukh Jabeen,cd Jamshed Iqbal
*
a
*
and Aamer Saeed
In the design of new drugs, the hybridization approach might allow obtaining molecules with improved
biological activity with respect to the corresponding lead compounds. Thus, adopting this approach,
a new series of novel bis-coumarin–iminothiazole hybrids was designed, synthesized and tested for their
biological action against alkaline phosphatase, leishmaniasis and cancerous cells. The structures of the
synthesized hybrid compounds (5a–m) were characterized and established by using spectro-analytical
data. The synthesized analogues were evaluated against alkaline phosphatase where compound 5j was
emerged as a potent inhibitor with IC50 value of 1.38 ꢀ 0.42 mM. This inhibitory efficacy is two-fold
higher as compared to the standard inhibitor. The synthesized compounds were also assayed for their
anti-leishmanial potential against Leishmania major and compound 5i was observed as the lead
candidate with 70.4% inhibition. The prepared compounds also showed cytotoxic behavior against
kidney fibroblast (BHK-21) and lung carcinoma (H-157) cancer cell lines. Molecular docking of the
synthesized library of iminothiazole derivatives against ALP was performed to delineate ligand–protein
interactions at molecular level which suggested that the major interacting residues in the binding sites of
the proteins might have an instrumental role in the inhibition of enzyme's function. Our results inferred
that compounds 5j–m may serve as potential surrogates for the development of potent inhibitors of ALP.
Received 27th July 2015
Accepted 14th October 2015
DOI: 10.1039/c5ra14900b
www.rsc.org/advances
transport, modulation of organic cation transport, and
involvement in cell proliferation.4,5 The biological action of the
Introduction
alkaline phosphatase in serum is associated with metabolic
bone (hypophosphatasia) and liver diseases and also is used as
a marker of osteoblastic differentiation.6 Alkaline phosphatases
may potentially be employed as therapeutic agents and show
several uses in clinical medicine and in biotechnology. The
most common human cancers like breast, kidney, lung and
prostate have a great potential to metastasize to bone, directing
towards painful and fatal consequences.7 Bisphosphonates are
widely used to reduce symptoms of osteolytic bone metastasis
commonly seen in breast, kidney and lung cancer.8 High serum
level of tissue non-specic alkaline phosphatases represents
elevated osteoblast activity in patients with metastatic prostate
cancer.9,10 The chemical agent that inhibit alkaline phosphatase
enzyme may be useful to prevent increased osteoblast activity,
commonly seen in bone metastasis.
Alkaline phosphatase (ALP, E.C. 3.1.3.1.) is a non-specic
phosphomonoester hydrolase that catalyzes the hydrolysis
and transphosphorylation of a wide variety of organic mono-
phosphates and regulates the functions of many biological
systems.1–3 The most common occurrence of ALP in nature
suggests its involvement in fundamental biochemical
processes, however, there is no positive evidence regarding its
physiological functions. The possible roles of ALP have been
suggested as hydrolysis of phosphoesters, phosphate trans-
ferase activity, protein phosphatase activity, phosphate
aDepartment of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan.
E-mail: aamersaeed@yahoo.com; Fax: +92-51-9064-2241; Tel: +92-51-9064-2128
bCentre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad-22060, Pakistan. E-mail: drjamshed@ciit.net.pk
cFlorida Center for Heterocyclic Compounds, Department of Chemistry, University of
Florida, Fl. 32611, USA
Cancer is a notably complex, widespread and lethal disease
and at present one of the most leading causes of death
accounting for 7.6 million (around 13% of all deaths) in 2008,
that are projected to continue rising, with an estimated 13.1
million deaths in 2030.11 Cancer can affect almost every tissue
dCenter for Computationally Assisted Science and Technology, North Dakota State
University, Fargo, ND, 58102, USA
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra14900b
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 89919–89931 | 89919

View Article Online
RSC Advances
Paper
lineage in the human body and poses great challenges to are major causative agents of VL, while L. major, L. tropica, L.
medical science. Most cancers are characterized by uncon- aethiopica, L. braziliensis, L. panamensis, L. amazonensis and L.
trolled cell proliferation, lack of cell differentiation and loss of mexicana cause CL.19 L. braziliensis and L. panamensis are also
contact inhibition, which confers upon the tumor cell a capa- agents of muco-cutaneous leishmaniasis. Therapy of patients
bility to invade local tissues and metastasize.12,13 Nearly all with leishmaniasis is still a serious problem and urgent and
cancers are caused by abnormalities in the genetic material of sustained efforts are needed for the development of new, effi-
the transformed cells and involve deregulation of essential cient, and safe drugs.
enzymes and other proteins controlling cell division and
proliferation.14 Many efforts have been made to the discovery of istry20–22 and are an integral part of the chemical and life
new anticancer agents which is of a paramount importance. sciences. Heterocyclic ring systems have emerged as powerful
Heterocycles occupy a central position in organic chem-
Over three billion people are estimated to be at risk for the scaffolds for many biological evaluations playing an important
parasitic diseases such as malaria, trypanosomiasis and leish- role in the design and discovery of new physiological/
maniasis.15 Leishmaniasis is a widespread parasitic disease that pharmacologically active molecules.23 Among them, 1,3-thia-
is caused by protozoan parasites of the genus Leishmania in zole is an important and versatile structural analogue possess-
tropical and subtropical areas in both the old and new worlds. ing a diverse spectrum of biological actions.24–30 On the other
According to recent World Health Organization reports, 88 hand, as a fragrant compound, coumarin derivatives are known
countries are affected, comprehending 12 million infected to possess multiple biological functions, including anti-HIV,
people worldwide, with approximately 350 million people at anti-acetylcholinesterase,
antifungal,
antioxidant,
anti-
risk. The incidence is increasing worldwide, with 1–2 million helmintic, antitumor, antibacterial, antiviral, anticancer, and
new cases registered annually, despite all efforts being made to anticlotting activities, and nd extensive application in phar-
ght the disease.15–18 It occurs in three major forms; cutaneous maceuticals and agrochemicals.31–35
(CL), muco-cutaneous (MCL) and visceral leishmaniasis (VL),
The molecular hybridization strategy involving the integra-
depending upon parasite species. L. donovani and L. infantum tion of two or more pharmacophoric units in the same
Fig. 1 Examples of bioactive coumarin-hybrid motifs and our designed hybrid molecules.
89920 | RSC Adv., 2015, 5, 89919–89931
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
molecule, is a rationally attractive approach for the design and characteristic coumarin proton (H-4) at 8.66 ppm along with
development of new bioactive agents.36,37 These combined other aromatic protons at appropriate chemical shi values.
pharmacophores probably offer some advantages such as in Methyl protons were also observed as singlet at 2.10 ppm. 3-
overcoming drug resistance38 as well as improving their bio- Acetyl-2H-chromen-2-one was also conrmed by 13C NMR data
logical potency.39 The literature survey revealed the biological where characteristic carbonyl carbon (ketone) appeared at 197.3
importance of coumarin-hybrid compounds associated with an ppm in addition to lactonic carbon resonating at 156.6 ppm.
array of biological functions. These molecules possess
3-Acetyl-2H-chromen-2-one was brominated by reuxing
coumarin and other heterocyclic and non-heterocyclic skeletons with Br2 solution in chloroform.40 FTIR spectral data indicated
(Fig. 1).40 In view of literature ndings and our continued the formation of compound (3) where stretching absorptions
interest aiming at developing new molecules as potent biolog- appeared at 1743 and 1716 cmꢁ1 for ester and ketone carbonyls.
ical candidates,31,41,42 we designed and synthesized a new series 1H NMR exhibited characteristic signal for methylene carbon as
of bis-coumarin–iminothiazole hybrids using molecular singlet at 4.90 ppm. Furthermore, 13C NMR also conrmed the
hybridization approach. The synthesized compounds were bromination of acetyl coumarin as methylene carbon resonated
evaluated for their alkaline phosphatase inhibition, anticancer at 55.4 ppm.
and antileishmanial potential. In addition, molecular docking
Reaction of 3-(2-bromoacetyl)-2H-chromen-2-one (3) and
studies of these compounds have also been carried out to gain substituted thiosemicarbazides (4a–m), synthesized previ-
further insights into the biological properties. The results of ously,31 in methanol under basic conditions afforded title
this study are presented in this paper.
products (5a–m) in pleasing yields.44 The structural diversity on
this scaffold was ensured by using a range of electron-rich and
electron-decient functional groups, attached to the thiazole
core. The formation of thiazole derivatives (5a–m) was evi-
denced by IR spectroscopy where characteristic stretching
absorptions observed in the range of 3410–3293 and 1629–1607
cmꢁ1 were due to N–H and C]N groups, respectively. In
1H NMR spectra, these compounds showed dominant signals in
the range of 11.90–10.98 ppm, assigned to N–H moiety. 13C
NMR spectra also conrmed the presence of C]N group in the
range of 159.7–157.2 ppm. The purity of the synthesized prod-
ucts (5a–m) was ascertained by elemental analysis.
Results and discussion
Synthesis and characterization of bis-coumarin–
iminothiazole hybrids
The reaction sequence employed for the synthesis of bis-
coumarin–iminothiazole compounds (5a–m) is illustrated in
Scheme 1. To a cold mixture of salicylaldehyde (1) and ethyl
acetoacetate (2) was added catalytic amount of piperidine. The
reaction mixture was stirred for 30 min at room temperature.
The precipitated solid was ltered off and recrystallized (chlo-
roform) to afford 3-acetyl-2H-chromen-2-one in good yield.43
The formation of 3-acetyl-2H-chromen-2-one was indicated by
its IR spectrum where absorption bands observed at 1759 and
Pharmacology
Alkaline phosphatase inhibition. The newly synthesized
1705 cmꢁ1 were attributed to the C]O of ketone and lactone bis-coumarin–iminothiazole hybrid compounds (5a–m) were
1
functionalities. H NMR spectral data exhibited resonance for assayed for their alkaline phosphatase inhibition using
Scheme 1 Synthesis of bis-coumarin–iminothiazole hybrid compounds (5a–m).
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 89919–89931 | 89921

View Article Online
RSC Advances
Paper
Table 1 Alkaline phosphatase inhibition of bis-coumarin–iminothia-
zole hybrids
other compounds in the series with mono substitution at
various positions of aryl ring showed diminished activities.
These substitutions include electron-withdrawing as well as
electron-donating groups. Overall, within the synthesized
series, derivatives bearing two methyl groups at variable posi-
tions of the aromatic ring attached to iminothiazole skeleton
showed remarkable inhibitory potential and indicated the
inuence of steric factors on the modulation of alkaline phos-
phatase potential.
ALP inhibition
Entry
Substituent (R)
IC50 ꢀ SEM (mM)
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
4-Cl
5.78 ꢀ 0.92
3.92 ꢀ 0.79
4.36 ꢀ 0.83
3.62 ꢀ 0.65
4.38 ꢀ 0.72
3.47 ꢀ 0.67
2.59 ꢀ 0.33
1.38 ꢀ 0.42
1.79 ꢀ 0.27
1.39 ꢀ 0.21
1.41 ꢀ 0.19
2.43 ꢀ 0.04
2-OMe
3-OMe
2-Me
3-Me
4-Me
2,3-diMe
2,4-diMe
2,6-diMe
3,4-diMe
3,5-diMe
—
Anti-leishmanial activity. The antileishmanial activity of
novel bis-coumarin–thiazole hybrids was measured by MTT
method,46,47 and %inhibition of all the synthesized compounds
at different concentrations is reported in Table 2. Amphotericin
B was used as a standard drug for anti-leishmanial activity.
The careful examination of the anti-leishmanial activity
results revealed that most of the compounds showed >50%
inhibition whereas some of the tested iminothiazoles showed
good inhibition of Leishmania major in vitro. Among all the
compounds screened in the series, 5i showed highest inhibition
of 70.4 ꢀ 2.2% at 100 mM, whereas amphotericin B showed
79.8% inhibition. Compound 5i incorporates two methyl
substituents on the aryl ring attached directly to thiazole ring.
This compound also showed highest potential at variable
concentrations. The activity was reduced when these dimethyl
substituents were present at variable positions as depicted by
compound 5j. Also, compound 5g with mono methyl substit-
uent at meta-position also exhibited signicant inhibition
(62.7 ꢀ 1.6 mM). Apart from these substituents, electron-rich
(–OMe) as well as electron-poor (–Cl) groups were tolerated
which showed moderate inhibition of Leishmania major. Least
active compound among the series was 5c displaying 40.6 ꢀ
2.2% inhibition at 100 mM.
5m
KH2PO4
potassium dihydrogen phosphate as standard drug (IC50
¼
2.43 ꢀ 0.04 mM).45 The activity results are summarized in Table
1. The careful observation of the bioactivity results revealed that
several compounds possess potent alkaline phosphatase
inhibitory activity, more than the standard inhibitor.
Among the tested thiazole analogues, compound 5j with an
IC50 value of 1.38 ꢀ 0.42 mM emerged as the most active
compound of the series. This two-fold strong inhibitory activity
may be attributed to the two methyl groups present at 3- and 5-
position of aryl ring directly attached to the thiazole core. A
slight modication in substituent pattern on aryl ring gave
compound 5l where substituents were present at 3- and 4-
position. This compound also exhibits strong inhibitory activity
(IC50 ¼ 1.39 ꢀ 0.21 mM) as compared to the standard drug.
Moreover, a similar inhibitory potential was observed when
methyl groups shi to 3- and 5-position at the aromatic ring as
depicted by compound 5m (IC50 ¼ 1.41 ꢀ 0.19 mM). Compound
5k with methyl groups at 2- and 6-position also afforded potent
inhibition, higher than the standard reference. Compound with
methyl groups at 2- and 3-position of the aromatic ring main-
tained inhibitory potential comparable to standard drug. The
Cytotoxic activity. Antiproliferative activity of the newly
synthesized bis-coumarin–thiazole compounds was measured
in vitro at four different concentrations (100, 10, 1 and 0.1 mM)
by the cell growth inhibition against kidney broblast (BHK-21)
and lung carcinoma (H-157) cell lines.48 Vincristine was used as
a standard drug for comparison to get percent inhibition of
each compound.
Table 2 Anti-leishmanial activity of bis-coumarin–iminothiazole hybrids
Leishmaniasis (%inhibition)
100 mM 10 mM
Entry
Substituent (R)
1 mM
0.1 mM
5c
5d
5e
5f
5g
5h
5i
5j
4-Cl
40.6 ꢀ 2.2
58.3 ꢀ 2.1
59.4 ꢀ 1.5
52.2 ꢀ 1.7
62.7 ꢀ 1.6
59.0 ꢀ 1.4
70.4 ꢀ 2.2
60.6 ꢀ 1.5
58.4 ꢀ 1.4
53.2 ꢀ 1.2
53.8 ꢀ 2.1
79.8 ꢀ 1.8
34.6 ꢀ 1.4
56.8 ꢀ 1.7
56.3 ꢀ 1.3
51.9 ꢀ 2.3
59.4 ꢀ 1.2
57.2 ꢀ 2.1
66.2 ꢀ 1.4
58.7 ꢀ 1.5
56.2 ꢀ 1.4
47.9 ꢀ 3.7
49.7 ꢀ 1.2
76.3 ꢀ 1.4
31.7 ꢀ 1.6
50.4 ꢀ 2.2
52.9 ꢀ 1.5
48.6 ꢀ 1.7
56.3 ꢀ 1.4
54.1 ꢀ 1.6
60.7 ꢀ 1.5
54.4 ꢀ 1.1
49.1 ꢀ 1.7
43.2 ꢀ 2.2
45.3 ꢀ 1.3
74.8 ꢀ 2.7
22.4 ꢀ 1.4
45.5 ꢀ 1.4
49.3 ꢀ 1.7
41.7 ꢀ 2.2
53.7 ꢀ 1.2
38.2 ꢀ 1.3
53.9 ꢀ 1.6
52.8 ꢀ 1.7
43.2 ꢀ 1.1
38.6 ꢀ 1.3
36.4 ꢀ 1.2
69.9 ꢀ 2.3
2-OMe
3-OMe
2-Me
3-Me
4-Me
2,3-diMe
2,4-diMe
2,6-diMe
3,4-diMe
3,5-diMe
—
5k
5l
5m
Amphotericin B
89922 | RSC Adv., 2015, 5, 89919–89931
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Most of the compounds among thiazole series exhibited 3- and 4-position of aryl ring attached to thiazole core. A slight
(>50%) inhibition for both cell lines with slightly different downward shi in activity was observed when position of
capacity due to their structure diversity in terms of attached substituents is moved to 2- and 4-position or 3- and 5-position,
functional groups with thiazole skeleton (Tables 3 and 4). respectively. Compounds 5j and 5m with dimethyl substituents
Among the tested compounds, 5m showed the highest inhibi- at 2- and 4-position and 3- and 5-position showed 61.4 ꢀ 1.6 and
tion of 66.8 ꢀ 1.1 and 64.2 ꢀ 2.3% at 100 and 10 mM, respec- 61.1 ꢀ 1.2% inhibitions at 10 mM, respectively as compared to
tively, against BHK-21 cells. This compound possesses dimethyl vincristine (72.6 ꢀ 3.1% at 10 mM). Least activity was observed in
substituent at 3- and 5-position of the aryl ring at thiazole case of 5g which incorporates only one methyl group at meta-
skeleton. A slight decrease in inhibition was observed when position.
substitution pattern is changed to 2,4- or 3,4-positions as
In general, among the tested bis-coumarin–iminothiazole
depicted by compound 5j and 5l, respectively. Several other hybrids, the compounds with dual substitutions were found to
substitutions including electron-donating and electron- possess highest activities and emerged as lead candidates. This
withdrawing were also tolerated but afforded reduced inhibi- pattern also suggests that the steric factors are generally more
tion. Compound 5k bearing dimethyl substitution at 2- and operative in modulating the activity prole as compared to the
6-position gave least inhibition of 37.8 ꢀ 1.2% at 0.1 mM electronic properties. These results also speculate some further
concentration. Compounds with only one methyl group at research to optimize bis-coumarin–iminothiazole template against
ortho- or para-position exhibited inhibition slightly greater than a range of biological targets with promising inhibitory potential.
50% at 100 and 10 mM.
In case of H-157 cell lines, among the tested thiazole hybrids,
Molecular modelling
compound 5l was the most potent analogue with inhibition
value of 65.0 ꢀ 1.8% at 100 mM while 57.3 ꢀ 0.7% at 10 mM. This
activity may be attributed to the presence of dimethyl groups at
Druglikeness, Lipinski's rule of ve. Molecular descriptors
were calculated for all the synthesized compounds by the ligand
Table 3 Anticancer activity of bis-coumarin–iminothiazole hybrids against kidney fibroblast (BHK-21) cell lines
BHK-21 cell lines (%inhibition)
Entry
Substituent (R)
100 mM
10 mM
1 mM
0.1 mM
5c
5d
5e
5f
5g
5h
5i
5j
4-Cl
50.5 ꢀ 1.4
59.4 ꢀ 1.3
53.2 ꢀ 1.7
54.7 ꢀ 1.2
52.9 ꢀ 1.3
57.3 ꢀ 1.5
57.2 ꢀ 1.2
62.9 ꢀ 3.2
54.7 ꢀ 2.1
62.1 ꢀ 1.8
66.8 ꢀ 1.1
74.5 ꢀ 2.9
48.3 ꢀ 1.2
55.3 ꢀ 1.7
50.2 ꢀ 2.1
51.8 ꢀ 2.4
50.7 ꢀ 1.5
54.8 ꢀ 2.8
51.6 ꢀ 2.5
61.1 ꢀ 2.6
51.8 ꢀ 2.4
61.8 ꢀ 1.2
64.2 ꢀ 2.3
72.6 ꢀ 3.1
47.2 ꢀ 1.8
52.8 ꢀ 1.2
46.3 ꢀ 1.8
49.9 ꢀ 1.6
47.3 ꢀ 2.3
50.5 ꢀ 1.7
47.2 ꢀ 2.6
60.7 ꢀ 1.8
44.3 ꢀ 2.7
56.4 ꢀ 2.9
59.4 ꢀ 1.2
70.9 ꢀ 2.4
41.8 ꢀ 2.5
47.0 ꢀ 1.7
45.5 ꢀ 1.2
47.2 ꢀ 1.6
45.1 ꢀ 1.8
48.7 ꢀ 1.2
41.9 ꢀ 3.3
56.4 ꢀ 2.4
37.8 ꢀ 1.2
52.9 ꢀ 1.9
55.5 ꢀ 2.7
69.8 ꢀ 1.9
2-OMe
3-OMe
2-Me
3-Me
4-Me
2,3-diMe
2,4-diMe
2,6-diMe
3,4-diMe
3,5-diMe
—
5k
5l
5m
Vincristine
Table 4 Anticancer activity of bis-coumarin–iminothiazole hybrids against lung carcinoma (H-157) cell lines
H-157 cell lines (%inhibition)
Entry
Substituent (R)
100 mM
10 mM
1 mM
0.1 mM
5c
5d
5e
5f
5g
5h
5i
5j
4-Cl
53.7 ꢀ 1.6
54.4 ꢀ 1.5
52.5 ꢀ 0.7
56.6 ꢀ 1.4
51.0 ꢀ 1.3
54.6 ꢀ 1.8
54.4 ꢀ 3.9
63.6 ꢀ 2.0
57.9 ꢀ 1.1
65.0 ꢀ 1.8
63.0 ꢀ 3.1
74.5 ꢀ 2.9
50.3 ꢀ 0.8
52.3 ꢀ 1.3
49.8 ꢀ 1.1
52.4 ꢀ 1.9
44.2 ꢀ 2.5
51.8 ꢀ 2.7
51.7 ꢀ 1.7
61.4 ꢀ 1.6
55.2 ꢀ 0.4
57.3 ꢀ 0.7
61.1 ꢀ 1.2
72.6 ꢀ 3.1
47.2 ꢀ 2.7
49.4 ꢀ 1.7
44.9 ꢀ 1.2
47.5 ꢀ 2.8
38.1 ꢀ 2.4
50.9 ꢀ 1.1
47.2 ꢀ 1.2
56.7 ꢀ 2.4
51.6 ꢀ 1.1
53.8 ꢀ 2.6
57.4 ꢀ 2.1
70.9 ꢀ 2.4
44.7 ꢀ 1.2
48.4 ꢀ 1.2
35.2 ꢀ 1.6
44.3 ꢀ 2.7
32.8 ꢀ 1.3
47.6 ꢀ 1.5
43.6 ꢀ 1.3
52.1 ꢀ 1.4
44.4 ꢀ 1.6
45.5 ꢀ 1.7
54.3 ꢀ 2.5
69.8 ꢀ 1.9
2-OMe
3-OMe
2-Me
3-Me
4-Me
2,3-diMe
2,4-diMe
2,6-diMe
3,4-diMe
3,5-diMe
—
5k
5l
5m
Vincristine
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 89919–89931 | 89923

View Article Online
RSC Advances
Paper
while phenyl ring of the same moiety established H–arene inter-
action in all the ligands except 5d and 5k. In addition to these,
tremendous polar interactions also played important role for
strong binding of the ligands inside the active site. Compounds
5j–m are found to be the most active compounds of the series and
these compounds were also found to exhibit strong interaction
with the amino acids residues inside the active site. Compound
5m inhibited the catalytic activities of the ALP by anchoring over
active site through multiple polar interactions, a hydrophobic
interaction and Pi–H interaction although it didn't establish
hydrogen bonding like other three most active compounds 5j, 5k
and 5l. Fig. 3 shows plausible binding modes for three most active
representative compounds 5j, 5k and 5l of the series. While
images of 2D interaction for all the ligands along with the detailed
legend are shown in Fig. S1.† The binding free energies derived
from docking (London dG ranging from ꢁ11.4376 to ꢁ7.4100 kcal
molꢁ1) indicate that almost all the synthesized imino-thiazole
derivatives exhibited stronger binding affinity for alkaline phos-
phatase (Table S2†). The poses are ranked by the scores from the
GBVI/WSA binding free energy calculation in the “S” eld. It is
quite interesting to note that the binding free energy in “S” eld is
in quite narrow range from ꢁ6.0532 to ꢁ6.8090 kcal molꢁ1 (Table
S2†). According to the computations performed, all the docked
ligands are located in the active site of alkaline phosphatase (PDB
code: 4KJG).51 Investigation of the binding mode analysis of the
adopted conformations revealed that both the phenyl rings
(coumarin moiety) are oriented almost in parallel to the main
plane. This orientation of phenyl rings may lead to its interaction
(H–arene) with the amino acid residues in the active site of
alkaline phosphatase.
The calculated geometries of the inhibitor 5l in the enzyme's
active site was analyzed to study the orientation of its docking
pose and binding mode as shown in Fig. 4. As a result the
positions of oxygen, it formed a strong H-bonding with the
Asp167, His153 and Lys108 in addition to H–arene interaction
of the phenyl ring with His432. Several strong polar interactions
may reect additional xation of inhibitor 5l which occupied
the enzyme's active site. Again binding of two phenyl rings
through Pi–H interaction with key residues could be the strong
reason of low energy and good activity of the inhibitor 5l (Fig. 3c
and 4). Examination of the most preferred docked pose of
inhibitor 5k (Fig. 3b) revealed that the oxygen atom of the
carbonyl group established a strong hydrogen bond with
Lys108. In addition to these, several polar interactions also
played a signicant role for strong binding, low binding energy
and in turn good activity of the entire imino-thiazole library
against ALP. The synthesized library 5a–m is composed basi-
cally of three phenyl rings, causing the whole structure to be
quite rigid and bulky. The rigidity imparted the expanded
orientation to the compounds which favored the maximum
exposure of the ligand to active site residues leading to
remarkable interactions.
Fig. 2 Superimposition of the ligands inside the active site; (a) over-
laying of all the minimum energy docked poses inside the pocket.
Ligands are shown in cyan color in stick mode while key residues are
shown in green color in stick mode. Backbone receptor is shown in
cartoon and ribbon form. (b) Close view of the active site, pocket is
marked with surfaces.
property calculation function of MOE (Table S1†) and all of
them were found to obey Lipinski's Ro5 and Veber's Ro3, cut-off
limits,49,50 revealing the fact that these could be potent alkaline
phosphatase inhibitors.
According to Lipinski's Ro5, most drug like molecules have
molecular weight # 500, logarithm of the octanol/water parti-
tion coefficient (log P) # 5, total polar surface area (TPSA) <
2
˚
140 A , number of hydrogen bond donors (HBD) # 5 and
hydrogen bond acceptor (HBA) # 10.50 Further modications in
the Ro5 were made by Veber et al.49 who suggested the number
of rotatable bond (NOR) of drug like molecule must be fewer or
equal to 10.50 Molecules violating more than one of these rules
may have problem with bioavailability.
Docking studies. For elucidation of the molecular mecha-
nism of inhibition of the synthesized library of imino-thiazole
derivatives, compounds were docked computationally to the
active site of alkaline phosphatase (ALP) from rat (PDB code:
4KJG).51
Structural analyses of the 3D generated model of the ALP
showed that enzyme is constructed with two chains with the co-
crystallized ligand in each chain, pocket was found around the
co-crystallized ligand. Aer docking the standard drug in the
pocket of both chains, the active site of the chain B was selected
for further studies. Pocket was found to be a small cavity, mostly
constructed with hydrophilic residues some of these are Tyr107,
Lys108, Arg166, His153, Val89, His 320, Ser429, Thr431 and
Gln317. Molecular docking studies were carried out to deter-
mine the probable binding modes of these compounds in the
active sites of the enzyme, rat Intestinal Alkaline Phosphate
(IALP) (PDB code: 4KJG). All the docked conformations of each
ligands were analyzed. From the results of calculations, we
chose variants with the minimal energy of the enzyme–inhibitor
complex. All the preferred docked conformations formed one
cluster inside the pocket as shown in Fig. 2.
It can be concluded from the docking computations that
quite low binding free energy (ꢁ6.0532 to ꢁ6.8090 kcal molꢁ1
)
Almost all the compounds have similar binding interaction
pattern. In all the ligands except 5c, 5f, 5h, 5i and 5m, the carbonyl
oxygen present on the coumarin moiety formulated strong
hydrogen bonding with the amino acid residues in active site,
coupled with the multiple interactions with amino acids resi-
dues provided the rational for good activities of these inhibitors
specially 5j–m (IC50 ¼ 1.38 to 1.79 mM) which were noticed to be
89924 | RSC Adv., 2015, 5, 89919–89931
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Fig. 3 Interaction of ligands with the active site residues of receptor in 2D space; dotted green lines present hydrogen bonding and other
interactions. Detailed description is given in the Fig. S1 in ESI† under the caption legend (a) compound 5j (b) compound 5k and (c) compound 5l.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 89919–89931 | 89925

View Article Online
RSC Advances
Paper
operative in modulating the activity prole as compared to the
electronic properties. The detailed binding mode analysis with
docking simulation showed that the inhibitors could be stabi-
lized in the active site through the formation of multiple
interactions with catalytic residues and the establishment of
hydrophobic contacts in a cooperative fashion. Finally, based
on the activity ndings and docking analysis, it could be pro-
claimed that the synthesized inhibitors could be further devel-
oped as novel and potent inhibitors of alkaline phosphatase.
Experimental
General
Unless otherwise noted, all materials were obtained from
commercial suppliers (Aldrich and Merck companies) and used
without further purication. Thin layer chromatography (TLC)
was performed on Merck DF-Alufoilien 60F254 0.2 mm pre-
coated plates. Product spots were visualized under UV light at
254 and 365 nm. Melting points were recorded on a Stuart
melting point apparatus (SMP3) and are uncorrected. Infra-red
(IR) spectra were recorded on FTS 3000 MX, Bio-Rad Merlin
(Excalibur model) spectrophotometer. 1H NMR spectra were
recorded on a Bruker Avance (300 MHz) spectrometer. Chemical
shis (d) are quoted in parts per million (ppm) downeld of
tetramethylsilane, using residual solvent as internal standard
(DMSO-d6 at 2.50 ppm). Abbreviations used in the description
of resonances are: s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet), Ar (aromatic). Proton-decoupled 13C NMR spectra
were recorded on a Bruker Avance (75 MHz) spectrometer using
deuterated solvent as internal standard (DMSO-d6 at 39.52
ppm). The elemental analysis was performed on Leco CHNS-932
Elemental Analyzer, Leco Corporation (USA).
Fig. 4 Docking pose of the ligand 5l inside the active site. Interaction
of ligand 5l with the active site residues of receptor in 3D space;
dashed blue lines represent hydrogen bonding while dotted yellow line
represents H–arene interaction.
more potent as compared to standard drug (IC50 ¼ 2.43 mM)
against IALP. This is in accordance with ndings that intro-
ducing coumarin moieties at the both terminal of the structure
of thiazoles lead to greater strength in addition to imparting the
at, expanded orientation to the structures which consequently
helped in strong binding. This can provide the rational for more
effective binding through hydrogen bonding and arene–H
interaction in the enzyme active site. These results proclaimed
compounds 5j–m to be further developed as ALP inhibitors.
Synthesis of 3-acetyl-2H-chromen-2-one
Conclusions
To a cold mixture of salicylaldehyde 1 (1.0 mol) and ethyl ace-
toacetate 2 (1.0 mol), catalytic amount of piperidine was added
and reaction mixture was stirred for 30 min at room tempera-
ture. The solid separated was ltered and washed with ethanol.
Recrystallization of the solid from chloroform afforded 3-acetyl-
2H-chromene-2-one.43 Yield (93%): m.p.: 119–121 ꢂC; Rf: 0.61
(20% EtOAc/hexane). The data for 3-acetyl-2H-chromene-2-one
was consistent with literature.52
In summary, a series of novel bis-coumarin–iminothiazole
hybrids (5a–m) has been prepared by the reaction of bromoa-
cetyl coumarin and a range of thiosemicarbazide derivatives in
methanol. The structural diversity of the synthesized derivatives
was ensured by using a diverse range of substituents including
electron-donating and electron-withdrawing groups on aryl part
of thiosemicarbazide. The synthesized analogues were screened
against alkaline phosphatase where 5j emerged as a potent
inhibitor with an IC50 value of 1.38 ꢀ 0.42 mM which is two-fold
strong inhibition as compared to the standard inhibitor. The
Synthesis of 3-(2-bromoacetyl)-2H-chromen-2-one (3)
synthesized compounds were also assayed for their anti- 3-Acetyl-2H-chromene-2-one (10.0 mmol) was dissolved in dry
leishmanial potential and compound 5i was found to be the chloroform and a solution of Br2 (10.0 mmol) in chloroform was
lead candidate with 70.4 ꢀ 2.2% inhibition at 100 mM. The added dropwise with continuous stirring and mixture was
prepared compounds also showed cytotoxic behavior against heated at reux for 6–7 h. The progress of the reaction was
kidney broblast (BHK-21) and lung carcinoma (H-157) cell monitored by thin layer chromatography (TLC). Aer comple-
lines. Compound 5l showed dual inhibition (66.8 ꢀ 1.1 and tion of the reaction, the mixture was concentrated under
65.0 ꢀ 1.8%) against BHK-21 and H-157 cell lines, respectively. reduced pressure, and washed with diethyl ether and recrys-
In general, among the tested bis-coumarin–iminothiazole tallized (70% CHCl3/EtOH) to afford 3-(2-bromoacetyl)-2H-
hybrids, the compounds with dual substitutions were found to chromen-2-one (3).40 Yield: (69%); m.p.: 161–163 ꢂC; Rf: 0.26
possess highest activities and emerged as lead candidates. This (20% EtOAc/hexane). The data for 3 was consistent with those
pattern also suggests that the steric factors are generally more observed in the literature.52
89926 | RSC Adv., 2015, 5, 89919–89931
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
144.0, 139.8, 135.7, 133.8, 131.0, 129.3, 127.8, 125.5, 122.4,
119.1, 117.6, 116.6, 114.7, 22.3, 19.3; anal. calcd for
Synthesis of 1,4-disubstituted thiosemicarbazides (4a–m)
The carbohydrazide (6.8 mmol) was dissolved in methanol
(30 mL), and a solution of substituted isothiocyanate (6.6 mmol)
separately dissolved in methanol (10 mL) was added dropwise
with continuous stirring. The reaction mixture was heated at
reux for 10–12 h and monitored by TLC. Aer consumption of
the starting materials, the mixture was cooled to room
temperature. The methanol was evaporated under reduced
pressure leaving behind the crude product as oil that became
solidied on cooling and was recrystallized (20% EtOAc/hexane)
to yield thiosemicarbazides (4a–m).31 The spectroscopic data for
thiosemicarbazides (4a–h) has previously been reported in our
papers31 (Rf ¼ 20% EtOAc/hexane).
C
19H17N3O3S: C, 62.11; H, 4.62; N, 11.40; S, 8.67; found: C,
62.00; H, 4.52; N, 11.37; S, 8.61.
N-(3,5-Dimethylphenyl)-2-(2-oxo-2H-chromene-3-carbonyl)-
hydrazinecarbothioamide (4m). Yield: (63%); m.p.: 160–162 ꢂC;
Rf: 0.52; IR (ATR, cmꢁ1): 3423–3281 (NH), 1708 (C]O), 1562,
1529 (C]C), 1259 (C]S); 1H NMR (300 MHz, DMSO-d6): d 11.48
(s, 1H, NH–C]O), 11.09 (s, 2H, NH–C]S), 9.09 (s, 1H, H-4),
7.79–7.66 (m, 2H, ArH), 7.42–7.39 (m, 2H, ArH), 6.69–6.66
(m, 3H, ArH), 2.27 (s, 6H, CH3); 13C NMR (75 MHz, DMSO-d6):
d 175.6, 163.2, 157.4, 151.2, 140.8, 137.0, 131.2, 129.6, 127.8,
124.5, 123.1, 120.2, 118.8, 115.0, 113.5, 19.9; anal calcd for
C
19H17N3O3S: C, 62.08; H, 4.59; N, 11.38; S, 8.69; found: C,
N-(2,3-Dimethylphenyl)-2-(2-oxo-2H-chromene-3-carbonyl)-
hydrazinecarbothioamide (4i). Yield: (59%); m.p.: 171–173 C;
62.03; H, 4.50; N, 11.33; S, 8.59.
ꢂ
Rf: 0.51; IR (ATR, cmꢁ1): 3464–3223 (NH), 1717 (C]O), 1572,
1540 (C]C), 1271 (C]S); 1H NMR (300 MHz, DMSO-d6): d 11.51
(s, 1H, NH–C]O), 11.13 (s, 2H, NH–C]S), 9.01 (s, 1H, H-4),
7.71–7.68 (m, 2H, ArH), 7.43–7.38 (m, 1H, ArH), 7.27–7.17
(m, 1H, ArH), 7.00–6.95 (m, 2H, ArH), 6.16–6.13 (m, 1H, ArH),
2.27 (s, 3H, CH3), 2.09 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-
d6): d 177.3, 164.4, 159.8, 152.7, 140.1, 136.9, 133.3, 130.8, 127.4,
125.1, 123.5, 120.7, 119.0, 117.3, 116.3, 114.1, 112.7, 22.2, 17.3;
anal. calcd for C19H17N3O3S: C, 62.10; H, 4.64; N, 11.43; S, 8.71;
found: C, 62.02; H, 4.58; N, 11.39; S, 8.65.
Synthesis of (E)-N0-(3-(substituted)-4-(2-oxo-2H-chromen-3-yl)-
thiazol-2(3H)-ylidene)-2-oxo-2H-chromene-3-carbohydrazide
(5a–m)
To a mixture of substituted thiosemicarbazide (4a–m) (1.0
mmol) and 3-(2-bromoacetyl)-2H-chromen-2-one (3) (1.0 mmol)
in methanol, anhydrous sodium acetate (4.0 mmol) was added
and heated at reux for 5–6 h. The reaction mixture was then
cooled to room temperature and the solid product formed was
ltered off, washed with water, dried (MgSO4), and recrystal-
lized (EtOH) to afford title products (5a–m) in good yields44 (Rf ¼
20% EtOAc/hexane).
N-(2,4-Dimethylphenyl)-2-(2-oxo-2H-chromene-3-carbonyl)-
ꢂ
hydrazinecarbothioamide (4j). Yield: (68%); m.p.: 166–168 C;
Rf: 0.53; IR (ATR, cmꢁ1): 3392–3263 (NH), 1716 (C]O), 1569,
1528 (C]C), 1269 (C]S); 1H NMR (300 MHz, DMSO-d6): d 11.54
(s, 1H, NH–C]O), 11.10 (s, 2H, NH–C]S), 9.13 (s, 1H, H-4),
7.73–7.69 (m, 2H, ArH), 7.43–7.40 (m, 2H, ArH), 6.97–6.95
(m, 1H, ArH), 6.67–6.63 (m, 1H, ArH), 6.13–6.11 (m, 1H, ArH),
2.27 (s, 3H, CH3), 2.02 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-
d6): d 178.0, 163.1, 159.8, 153.1, 143.3, 139.0, 132.3, 130.7, 129.1,
127.3, 126.1, 122.6, 120.3, 119.6, 118.2, 116.1, 114.3, 21.3, 17.8;
anal. calcd for C19H17N3O3S: C, 62.10; H, 4.59; N, 11.40; S, 8.69;
found: C, 62.03; H, 4.51; N, 11.31; S, 8.63.
(E)-N0-(3-(2-Chlorophenyl)-4-(2-oxo-2H-chromen-3-yl)thiazol-
2(3H)-ylidene)-2-oxo-2H-chromene-3-carbohydrazide
(5a).
Yield: (58%); m.p.: 190–192 C; Rf: 0.42; IR (ATR, cmꢁ1): 3329
(NH), 1717, 1703 (C]O), 1629 (C]N); 1H NMR (300 MHz,
DMSO-d6): d 11.23 (s, 1H, NH), 9.67 (s, 1H, H-4), 8.98 (s, 1H, H-
40), 7.96–7.79 (m, 4H, ArH), 7.43–7.40 (m, 5H, ArH), 7.23–7.21
(m, 1H, ArH), 7.01–6.86 (m, 2H, ArH), 6.66–6.60 (m, 1H, ArH);
13C NMR (75 MHz, DMSO-d6): d 168.3, 159.1, 154.0, 151.2, 148.6,
143.2, 133.9, 131.6, 130.4, 129.1, 128.6, 127.3, 126.7, 125.3,
123.5, 121.2, 119.8, 118.0, 116.3, 112.5, 98.6; anal. calcd for
ꢂ
N-(2,6-Dimethylphenyl)-2-(2-oxo-2H-chromene-3-carbonyl)-
C
28H16ClN3O5S: C, 62.04; H, 2.96; N, 7.73; S, 5.90; found: C,
ꢂ
hydrazinecarbothioamide (4k). Yield: (66%); m.p.: 161–163 C;
62.00; H, 2.90; N, 7.68; S, 5.87.
Rf: 0.51; IR (ATR, cmꢁ1): 3442–3293 (NH), 1712 (C]O), 1572,
1523 (C]C), 1269 (C]S); 1H NMR (300 MHz, DMSO-d6): d 11.43
(s, 1H, NH–C]O), 11.02 (s, 2H, NH–C]S), 9.09 (s, 1H, H-4),
7.78–7.70 (m, 2H, ArH), 7.45–7.43 (m, 2H, ArH), 6.95–6.92
(m, 2H, ArH), 6.63–6.61 (m, 1H, ArH), 2.19 (s, 6H, CH3); 13C
NMR (75 MHz, DMSO-d6): d 171.1, 161.9, 158.2, 153.0, 141.1,
138.4, 133.0, 128.1, 127.0, 125.1, 123.7, 121.0, 119.3, 116.7,
114.2, 23.9; anal. calcd for C19H17N3O3S: C, 62.09; H, 4.60; N,
11.39; S, 8.68; found: C, 62.01; H, 4.54; N, 11.35; S, 8.61.
N-(3,4-Dimethylphenyl)-2-(2-oxo-2H-chromene-3-carbonyl)-
(E)-N0-(3-(3-Chlorophenyl)-4-(2-oxo-2H-chromen-3-yl)thiazol-
2(3H)-ylidene)-2-oxo-2H-chroꢂmene-3-carbohydrazide
(5b).
Yield: (60%); m.p.: 182–184 C; Rf: 0.46; IR (ATR, cmꢁ1): 3298
(NH), 1732, 1712 (C]O), 1619 (C]N); 1H NMR (300 MHz,
DMSO-d6): d 11.48 (s, 1H, NH), 9.89 (s, 1H, H-4), 9.01 (s, 1H, H-
40), 8.01–7.93 (m, 4H, ArH), 7.42–7.09 (m, 5H, ArH), 7.03–6.95
(m, 2H, ArH), 6.90–6.86 (m, 1H, ArH), 6.65–6.63 (m, 1H, ArH);
13C NMR (75 MHz, DMSO-d6): d 169.1, 158.3, 153.4, 150.1, 149.6,
142.0, 135.7, 133.0, 131.8, 130.6, 128.5, 127.0, 126.4, 125.1,
122.7, 121.3, 119.6, 118.1, 116.6, 114.8, 99.1; anal. calcd for
ꢂ
hydrazinecarbothioamide (4l). Yield: (55%); m.p.: 164–166 C;
C
28H16ClN3O5S: C, 62.04; H, 2.96; N, 7.73; S, 5.90; found: C,
Rf: 0.56; IR (ATR, cmꢁ1): 3453–3343 (NH), 1720 (C]O), 1565,
1549 (C]C), 1263 (C]S); 1H NMR (300 MHz, DMSO-d6): d 11.63
(s, 1H, NH–C]O), 11.11 (s, 2H, NH–C]S), 8.86 (s, 1H, H-4),
7.73–7.65 (m, 2H, ArH), 7.42–7.41 (m, 2H, ArH), 6.86 (m, 1H,
ArH), 6.23–6.21 (m, 2H, ArH), 2.36 (s, 3H, CH3), 2.27 (s, 3H,
CH3); 13C NMR (75 MHz, DMSO-d6): d 179.2, 163.9, 158.4, 152.2,
62.02; H, 2.88; N, 7.70; S, 5.85.
(E)-N0-(3-(4-Chlorophenyl)-4-(2-oxo-2H-chromen-3-yl)thiazol-
2(3H)-ylidene)-2-oxo-2H-chromene-3-carbohydrazide
(5c).
Yield: (66%); m.p.: 196–198 C; Rf: 0.48; IR (ATR, cmꢁ1): 3311
(NH), 1725, 1705 (C]O), 1612 (C]N); 1H NMR (300 MHz,
DMSO-d6): d 11.48 (s, 1H, NH), 8.59 (s, 1H, H-4), 8.19 (s, 1H, H-
ꢂ
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 89919–89931 | 89927

View Article Online
RSC Advances
Paper
40), 7.89–7.80 (m, 4H, ArH), 7.68–7.61 (m, 4H, ArH), 7.48–7.36 (m, 2H, ArH), 6.36–6.29 (m, 2H, ArH), 2.20 (s, 3H, CH3); 13C NMR
(m, 4H, ArH); 13C NMR (75 MHz, DMSO-d6): d 158.8, 158.7, (75 MHz, DMSO-d6): d 170.0, 159.1, 154.2, 150.2, 147.2, 139.0,
155.1, 154.9, 149.2, 149.1, 147.8, 135.6, 135.1, 131.5, 131.3, 132.2, 131.5, 130.6, 128.4, 127.5, 125.9, 124.3, 123.3, 120.1, 118.8,
125.6, 125.5, 123.2, 122.6, 118.6, 118.5, 118.3, 116.7; anal. calcd 116.2, 114.6, 96.3, 23.9; anal. calcd for C29H19N3O5S: C, 66.77; H,
for C28H16ClN3O5S: C, 62.04; H, 2.96; N, 7.73; S, 5.90; found: C, 3.67; N, 8.05; S, 6.13; found: C, 66.69; H, 3.60; N, 8.01; S, 6.09.
62.00; H, 2.90; N, 7.69; S, 5.87.
(E)-N0-(3-(2,3-Dimethylphenyl)-4-(2-oxo-2H-chromen-3-yl)-thia-
(E)-N0-(3-(2-Methoxyphenyl)-4-(2-oxo-2H-chromen-3-yl)thiazol- zol-2(3H)-ylidene)-2-oxo-2H-chromene-3-carbohydrazide
(5i).
2(3H)-ylidene)-2-oxo-2H-chromene-3-carbohydrazide (5d). Yield: Yield: (65%); m.p.: 128–130 ꢂC; Rf: 0.42; IR (ATR, cmꢁ1): 3312
(69%); m.p.: 174–176 C; Rf: 0.42; IR (ATR, cmꢁ1): 3312 (NH), (NH), 1717 (C]O), 1615 (C]N); 1H NMR (300 MHz, DMSO-d6):
ꢂ
1717, 1698 (C]O), 1615 (C]N); 1H NMR (300 MHz, DMSO-d6): d 11.143 (s, 1H, NH), 9.10 (s, 1H, H-4), 8.69 (s, 1H, H-40), 7.96–7.84
d 11.90 (s, 1H, NH), 8.48 (s, 1H, H-4), 8.33 (s, 1H, H-40), 8.32–8.25 (m, 4H, ArH), 7.69–7.49 (m, 4H, ArH), 7.43–7.40 (m, 1H, ArH),
(m, 2H, ArH), 7.90 (t, J ¼ 8.7 Hz, 1H, ArH), 7.28–7.00 (m, 3H, 6.99–6.96 (m, 2H, ArH), 6.65–6.59 (m, 1H, ArH), 2.23 (s, 3H, CH3),
ArH), 6.98–6.96 (m, 3H, ArH), 6.94–6.92 (m, 2H, ArH), 6.89–6.86 2.01 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): d 169.3, 159.7,
(m, 3H, ArH), 3.88 (s, 3H, OCH3); 13C NMR (75 MHz, DMSO-d6): 152.8, 150.3, 147.7, 141.1, 136.8, 131.3, 129.1, 128.4, 127.5, 126.6,
d 169.7, 159.2, 152.7, 147.3, 144.4, 138.4, 138.2, 136.6, 134.4, 125.2, 124.7, 121.0, 120.0, 119.3, 118.3, 116.5, 114.7, 100.2, 23.5,
132.1, 129.2, 128.9126.1, 125.2, 121.1, 119.6, 116.3, 113.2, 111.9, 19.3; anal. calcd for C30H21N3O5S: C, 67.28; H, 3.94; N, 7.84; S,
111.5, 64.5; anal. calcd for C29H19N3O6S: C, 64.79; H, 3.54; N, 5.98; found: C, 67.23; H, 3.90; N, 7.80; S, 5.93.
7.80; S, 5.96; found: C, 64.73; H, 3.49; N, 7.73; S, 5.91.
(E)-N0-(3-(3-Methoxyphenyl)-4-(2-oxo-2H-chromen-3-yl)thiazol- zol-2(3H)-ylidene)-2-oxo-2H-chromene-3-carbohydrazide
(E)-N0-(3-(2,4-Dimethylphenyl)-4-(2-oxo-2H-chromen-3-yl)thia-
(5j).
2(3H)-ylidene)-2-oxo-2H-chromene-3-carbohydrazide (5e). Yield: Yield: (71%); m.p.: 135–137 ꢂC; Rf: 0.43; IR (ATR, cmꢁ1): 3296
(62%); m.p.: 167–169 C; Rf: 0.40; IR (ATR, cmꢁ1): 3332 (NH), (NH), 1717 (C]O), 1612 (C]N); 1H NMR (300 MHz, DMSO-d6):
ꢂ
1721, 1702 (C]O), 1623 (C]N); 1H NMR (300 MHz, DMSO-d6): d 11.02 (s, 1H, NH), 9.09 (s, 1H, H-4), 8.58 (s, 1H, H-40), 7.93–7.87
d 11.67 (s, 1H, NH), 9.15 (s, 1H, H-4), 8.68 (s, 1H, H-40), 8.38–8.27 (m, 2H, ArH), 7.83–7.74 (m, 2H, ArH), 7.61–7.59 (m, 1H, ArH),
(m, 2H, ArH), 7.73–7.62 (m, 4H, ArH), 7.30–7.26 (m, 1H, ArH), 7.37–7.19 (m, 4H, ArH), 6.96–6.91 (m, 1H, ArH), 6.73–6.65 (m,
7.10–6.99 (m, 4H, ArH), 6.38–6.31 (m, 2H, ArH), 3.65 (s, 3H, 2H, ArH), 2.34 (s, 3H, CH3), 2.02 (s, 3H, CH3); 13C NMR (75 MHz,
OCH3); 13C NMR (75 MHz, DMSO-d6): d 169.7, 159.3, 157.1, DMSO-d6): d 168.1, 158.5, 152.0, 151.6, 148.3, 143.8, 139.1, 137.4,
151.6, 150.7, 148.1, 143.9, 132.0, 130.7, 128.1, 126.1, 125.2, 136.3, 133.2, 131.6, 130.4, 129.1, 127.3, 126.7, 125.5, 121.2, 119.3,
124.6, 123.3, 121.7, 120.3, 119.9, 116.6, 111.6, 110.7, 95.8, 56.3; 117.1, 114.7, 100.1, 26.3, 15.8; anal. calcd for C30H21N3O5S: C,
anal. calcd for C29H19N3O6S: C, 64.79; H, 3.54; N, 7.80; S, 5.96; 67.27; H, 3.95; N, 7.83; S, 5.97; found: C, 67.19; H, 3.84; N, 7.76; S,
found: C, 64.71; H, 3.50; N, 7.70; S, 5.89.
5.83.
(E)-N0-(3-(2,6-Dimethylphenyl)-4-(2-oxo-2H-chromen-3-yl)-
(E)-2-Oxo-N0-(4-(2-oxo-2H-chromen-3-yl)-3-o-tolylthiazol-2(3H)-
ylidene)-2H-chromene-3-carbohydrazide (5f). Yield: (67%); m.p.: thiazol-2(3H)-ylidene)-2-oxo-2H-chromene-3-carbohydrazide
109–111 ꢂC; Rf: 0.48; IR (ATR, cmꢁ1): 3381 (NH), 1719, 1699 (C] (5k). Yield: (69%); m.p.: 152–154 ꢂC; Rf: 0.46; IR (ATR, cmꢁ1):
1
O), 1609 (C]N); H NMR (300 MHz, DMSO-d6): d 11.70 (s, 1H, 3316 (NH), 1717 (C]O), 1607 (C]N); 1H NMR (300 MHz,
NH), 8.47 (s, 1H, H-4), 8.10 (s, 1H, H-40), 7.43–7.41 (m, 4H, ArH), DMSO-d6): d 11.11 (s, 1H, NH), 9.39 (s, 1H, H-4), 9.00 (s, 1H,
7.26–7.14 (m, 4H, ArH), 7.00–6.81 (m, 4H, ArH), 2.30 (s, 3H, CH3); H-40), 7.83–7.69 (m, 4H, ArH), 7.54–7.51 (m, 2H, ArH), 7.39–
13C NMR (75 MHz, DMSO-d6): d 167.2, 153.0, 149.6, 146.2, 144.8, 7.34 (m, 3H, ArH), 6.99–6.96 (m, 2H, ArH), 6.67–6.63 (m, 1H,
138.9, 137.0, 134.8, 131.7, 128.9, 127.5, 126.2, 120.7, 119.6, 116.4, ArH), 2.09 (s, 6H, 2 ꢃ CH3); 13C NMR (75 MHz, DMSO-d6):
115.7, 114.6, 111.4, 21.0; anal. calcd for C29H19N3O5S: C, 66.77; H, d 169.9, 157.2, 151.4, 150.3, 149.0, 147.4, 133.5, 130.6, 129.1,
3.67; N, 8.05; S, 6.13; found: C, 66.68; H, 3.60; N, 8.01; S, 6.08.
128.6, 126.9, 125.4, 123.2, 121.7, 119.1, 118.8, 116.3, 114.5,
(E)-2-Oxo-N0-(4-(2-oxo-2H-chromen-3-yl)-3-m-tolylthiazol-2(3H)- 99.0, 17.6; anal. calcd for C30H21N3O5S: C, 67.27; H, 3.95; N,
ylidene)-2H-chromene-3-carbohydrazide (5g). Yield: (63%); m.p.: 7.83; S, 5.97; found: C, 67.20; H, 3.89; N, 7.79; S, 5.86.
113–115 ꢂC; Rf: 0.47; IR (ATR, cmꢁ1): 3398 (NH), 1716, 1705 (C]
(E)-N0-(3-(3,4-Dimethylphenyl)-4-(2-oxo-2H-chromen-3-yl)thia-
(5l).
1
O), 1616 (C]N); H NMR (300 MHz, DMSO-d6): d 11.21 (s, 1H, zol-2(3H)-ylidene)-2-oxo-2H-chromene-3-carbohydrazide
NH), 9.43 (s, 1H, H-4), 8.86 (s, 1H, H-40), 7.86–7.79 (m, 4H, ArH), Yield: (67%); m.p.: 109–111 ꢂC; Rf: 0.42; IR (ATR, cmꢁ1): 3329
7.53–7.35 (m, 5H, ArH), 7.03–6.98 (m, 2H, ArH), 6.66–6.60 (m, 1H, (NH), 1723 (C]O), 1629 (C]N); 1H NMR (300 MHz, DMSO-d6):
ArH), 6.53–6.49 (m, 1H, ArH), 2.12 (s, 3H, CH3); 13C NMR (75 MHz, d 11.14 (s, 1H, NH), 9.01 (s, 1H, H-4), 8.67 (s, 1H, H-40), 7.97–7.92
DMSO-d6): d 169.1, 158.3, 153.6, 151.2, 148.5, 137.2, 132.9, 131.2, (m, 1H, ArH), 7.81–7.69 (m, 2H, ArH), 7.63–7.57 (m, 1H, ArH),
129.1, 128.6, 127.1, 126.0, 124.7, 122.5, 121.7, 119.2, 117.6, 116.1, 7.48–7.20 (m, 4H, ArH), 7.12–7.02 (m, 1H, ArH), 6.99–6.93 (m,
114.3, 111.5, 98.7, 20.1; anal. calcd for C29H19N3O5S: C, 66.77; H, 1H, ArH), 6.89–6.80 (m, 2H, ArH), 2.22 (s, 3H, CH3), 2.10 (s, 3H,
3.67; N, 8.05; S, 6.13; found: C, 66.70; H, 3.62; N, 8.00; S, 6.06.
CH3); 13C NMR (75 MHz, DMSO-d6): d 168.1, 158.2, 151.1, 149.0,
(E)-2-Oxo-N0-(4-(2-oxo-2H-chromen-3-yl)-3-p-tolylthiazol-2(3H)- 147.7, 140.1, 137.8, 131.5, 130.2, 129.6, 127.3, 126.8, 125.2, 123.7,
ylidene)-2H-chromene-3-carbohydrazide (5h). Yield: (68%); m.p.: 121.0, 119.7, 118.4, 116.3, 115.1, 114.8, 99.0, 20.8, 18.1; anal.
108–110 ꢂC; Rf: 0.49; IR (ATR, cmꢁ1): 3410 (NH), 1725, 1707 calcd for C30H21N3O5S: C, 67.26; H, 3.93; N, 7.81; S, 5.96; found:
1
(C]O), 1607 (C]N); H NMR (300 MHz, DMSO-d6): d 11.09 (s, C, 67.20; H, 3.87; N, 7.75; S, 5.89.
1H, NH), 9.68 (s, 1H, H-4), 8.94 (s, 1H, H-40), 7.98–7.84 (m, 4H,
(E)-N0-(3-(3,5-Dimethylphenyl)-4-(2-oxo-2H-chromen-3-yl)thia-
ArH), 7.65–7.62 (m, 2H, ArH), 7.46–7.39 (m, 3H, ArH), 6.99–6.93 zol-2(3H)-ylidene)-2-oxo-2H-chromene-3-carbohydrazide (5m).
89928 | RSC Adv., 2015, 5, 89919–89931
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Yield: (66%); m.p.: 125–128 ꢂC; Rf: 0.40; IR (ATR, cmꢁ1): 3293 10% heat inactivated fetal bovine serum (FBS), 100 IU mLꢁ1
(NH), 1715 (C]O), 1618 (C]N); 1H NMR (300 MHz, DMSO-d6): penicillin and 100 mg mLꢁ1 streptomycin, then washed 3 times
d 11.13 (s, 1H, NH), 9.10 (s, 1H, H-4), 8.89 (s, 1H, H-40), 7.83–7.71 with PBS by centrifugation at 1500 rpm for 10 min at room
(m, 2H, ArH), 7.68–7.63 (m, 1H, ArH), 7.42–7.31 (m, 4H, ArH), temperature and resuspended at a concentration of 2.5 ꢃ 106
7.07–7.00 (m, 2H, ArH), 6.95–6.89 (m, 1H, ArH), 6.65–6.53 (m, parasites per mL in medium.
2H, ArH), 2.31 (s, 6H, 2 ꢃ CH3); 13C NMR (75 MHz, DMSO-d6):
Antileishmanial activity assay (MTT assay). The anti-
d 169.2, 158.1, 154.0, 151.6, 147.2, 141.3, 137.7, 131.5, 129.0, leishmanial activity of the newly synthesized compounds was
128.1, 126.7, 125.2, 123.1, 121.8, 120.5, 118.7, 116.9, 114.3, 96.9, evaluated in vitro against the promastigote forms of Leishmania
24.3; anal. calcd for C30H21N3O5S: C, 67.26; H, 3.93; N, 7.81; S, major
using
a
MTT
(3-(4,5-dimethylthiazol-2yl)-2,5-
5.96; found: C, 67.23; H, 3.85; N, 7.77; S, 5.83.
diphenyltetrazoliumbromide)-based microassay as a marker of
cell viability. The MTT assay used was based on that originally
described by Mosmann46 modied by Niks and Otto.47 A stock
solution of MTT (Sigma Chemical Co., St. Louis, Mo.) was
prepared by dissolving in PBS at 5 mg mLꢁ1 and storing in the
dark at 4 ꢂC for up to 2 weeks before use. For the anti-
leishmanial activity assays, 100 mL per well of the culture which
contained 2.5 ꢃ 106 cells per mL promastigotes was seeded in
96-well at-bottom plates. Then, 10 mL per well from various
concentrations of synthesized compounds were added to trip-
licate wells and plates were incubated for 72 h at 25 ꢀ 1 ꢂC. The
rst well of 96 wells was as a blank well which only contained of
100 mL culture medium without any compound, drug or para-
site. Amphotericin B was used as standard drug. At the end of
incubation, 10 mL of MTT was added to each well and plates
were incubated for 3 h at 25 ꢀ 1 ꢂC. Enzyme reaction was then
stopped by the addition of 100 mL of 50% isopropanol and 10%
sodium dodecyl sulfate. The plates were incubated for an
additional 30 min under agitation at room temperature. Rela-
tive optical density (OD) was then measured at a wavelength of
570 nm using a 96-well microplate reader (Bio-Tek ELx 800 TM,
Instruments, Inc. USA). The background absorbance of plates
was measured at 690 nm and subtract from 570 nm measure-
ment. The absorbance of the formazan produced by the action
of mitochondrial dehydrogenases of metabolically active cells is
shown to correlate with the number of viable cells. All experi-
ments were repeated at least three times. Results reported are
mean of three independent experiments (ꢀSEM) and expressed
as percent inhibitions calculated by the formula:
Pharmacological protocols
Alkaline phosphatase inhibition. Newly synthesized bis-
coumarin–thiazole compounds were tested against calf intes-
tinal alkaline phosphatase (CIAP). The compounds were
initially tested against these enzymes at 10 mM concentration to
further make a dose-response curve of potential inhibitors of
synthesized compounds by using previously described method
by J. Iqbal.45 The solution was made in assay buffer (pH 9.5)
comprises of tris-hydrochloride (50 mM), MgCl2 (5 mM), ZnCl2
(0.1 mM) and glycerol 50% in the solution. The solution of
enzyme substrate p-nitrophenyl phosphate (p-NPP) and test
compounds was also prepared in the same buffer without
glycerol. The assay was started in 96 wells plate by adding 70 mL
of assay buffer, 10 mL of test compound (from 100 mM
compound stock solution), 10 mL of optimized enzyme (0.5 U
mLꢁ1) and then incubated at 37 ꢂC for 10 minutes. To initialize
the reaction a 10 mL of substrate (5 mM p-NPP) was added into
each well and again incubated for additional 30 minutes. The
change in absorbance of released p-nitrophenolate was moni-
tored at 405 nm, using a 96-well microplate reader (Bio-Tek ELx
800 TM, Instruments, Inc. USA). The percent inhibition of each
compound was calculated by comparing the results with the
control wells having no inhibitor at all. The compounds which
showed $50% inhibition were selected for further determina-
tion of dose-response curves against all compounds. For this
purpose 7–9 serial dilutions of each compound spanning three
orders of magnitude were prepared in enzyme assay buffer and
their dose response curves were obtained by adopting the same
methods used for initial screening. All experiments were
repeated three times in triplicate. Results reported are mean of
Inhibition (%) ¼ [100 ꢁ (abs of test compound/abs of control) ꢃ 100]
three independent experiments (ꢀSEM) and expressed as Cytotoxicity assay
percent inhibitions calculated by the formula;
Cell lines and cultures. Lung carcinoma (H-157), (ATCC CRL-
5802) and kidney broblast (BHK-21), (ATCC CCL-10) cell lines
were kept in RPMI-1640 [having heat-inactivated fetal bovine
serum (10%) glutamine (2 mM), pyruvate (1 mM), 100 U mLꢁ1
penicillin and 100 mg mLꢁ1 streptomycin] in T-75 cm2 sterile
Inhibition (%) ¼ [100 ꢁ (abs of test compound/abs of control)
ꢃ 100]
tissue culture asks in a 5% CO2 incubator at 37 C.53 96-well
ꢂ
IC50 values of selected compounds exhibiting >50% activity
at 0.5 mM were calculated aer dilutions using computer
program GraphPad, San Diego, California, USA.
plates were used for growing H-157 and BHK-21 cells by inoc-
ulating 104 cells per 100 mL per well and plates were incubated
in CO2 incubator. Within 24 h, a uniform monolayer was
formed which was used for experiments.
Leishmanicidal assay
Cytotoxicity analysis by sulforhodamine B (SRB) assay. To
Parasite and culture. Leishmania major promastigotes were perform cytotoxicity assay with H-157 and BHK-21 cells,
cultured at 25 ꢀ 1 ꢂC to logarithmic phase in D-MEM/F-12 a previously described method by Skehan et al.,48 was adopted
medium (Gibco BRL) without phenol red, supplemented by with some modications. Briey, cells were cultured in
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 89919–89931 | 89929

View Article Online
RSC Advances
Paper
different 96 well plates for 24 h. The compounds in different
concentrations (100, 10, 1 and 0.1 mM) were inoculated in test
wells while control and blank wells were also prepared con-
taining standard drug (vincristine) and culture media with
cells, respectively. The plates were then incubated for 48 h.
Aer that cells were xed with 50 mL of 50% ice cold tri-
chloroacetic acid solution (TCA) at 4 ꢂC for 1 h. The plates were
washed 5 times with phosphate-buffered saline (PBS) and air
dried. Fixed cells were further treated with 0.4% w/v sulfo-
rhodamine B dye prepared in 1% acetic acid solution and le
at room temperature for 30 min. Aer that the plates were
rinsed with 1% acetic acid solution and allowed to dry. In
order to solubilize the dye, the dried plates were treated with
10 mM Tris base solution for 10 min at room temperature.
Absorbance was measured at 490 nm subtracting the back-
ground measurement at 630 nm.54 All experiments were
repeated at least three times. Results reported are mean of
three independent experiments (ꢀSEM) and expressed as
percent inhibitions calculated by the formula:
References
1 M. H. le Du and J. L. Millan, J. Biol. Chem., 2002, 277, 49808–
49814.
2 L. Zhang, M. Balcerzak, J. Radisson, C. Thouverey, S. Pikula,
G. Azzar and R. Buchet, J. Biol. Chem., 2005, 280, 37289–
37296.
3 S. P. Coburn, J. D. Mahuren, M. Jain, Y. Zubovic and
J. Wortsman, J. Clin. Endocrinol. Metab., 1998, 83, 3951–3957.
4 D. Sarrouilhe, P. Lalegerie and M. Baudry, Biochim. Biophys.
Acta, 1992, 1118, 116–122.
5 M. Muda, N. N. Rao and A. Torriani, J. Bacteriol., 1992, 174,
8057–8064.
6 M. Li, W. Ding, B. Baruah, D. C. Crans and R. Wang, J. Inorg.
Biochem., 2008, 102, 1846–1853.
7 G. R. Mundy, Nat. Rev. Cancer, 2002, 2, 584–593.
8 G. D. Roodman, N. Engl. J. Med., 2004, 350, 1655–1664.
9 C. J. Logothetis and S.-H. Lin, Nat. Rev. Cancer, 2005, 5, 21–
28.
¨
10 K. Jung, M. Lein, C. Stephan, K. von Hosslin, A. Semjonow,
Inhibition (%) ¼ [100 ꢁ (abs of test compound/abs of control) ꢃ 100]
P. Sinha, S. A. Loening and D. Schnorr, Int. J. Cancer, 2004,
111, 783–791.
11 D. Sloane, Methods Mol. Biol., 2009, 471, 65–83.
12 M. Mareel and A. Leroy, Physiol. Rev., 2003, 83, 337–376.
13 J. Weschel, K. Haglund and E. M. Haugsten, Biochem. J.,
2011, 437, 199–213.
14 J. Cummings, T. H. Ward, M. Ranson and C. Dive, Biochim.
Biophys. Acta, 2004, 1705, 53–66.
Molecular docking
Preparation of receptor. The crystal structure of alkaline
phosphate (PDB code: 4KJG)51 was retrieved from protein data
bank with its cognate ligand and active site was found around
the cognate ligand by using Lig X module of MOE.
15 WHO, Research Priorities for Chagas Disease, Human African
Trypanosomiasis and Leishmaniasis, WHO Technical Report
Series No. 975, 2012, p. 116.
16 C. R. Davies, R. P. Kaye, S. L. Cro and S. Sundar, BMJ [Br.
Med. J.], 2003, 15, 377–382.
Preparation of ligands. The ligand les for molecular dock-
ing studies were prepared in molecular operating environment
(MOE) by chemical computing group (CCG).55 Drawn geome-
tries were followed by energy optimization at standard MMFF94
force eld level, with a 0.0001 kcal molꢁ1 energy gradient
convergence criterion.49 These ligands were saved in molecular
data base le for docking studies.
17 P. Trouiller, E. Torreele, P. Olliaro, N. White, S. Foster,
´
D. Wirth and B. Pecoul, Trop. Med. Int. Health, 2001, 6,
945–951.
Druglikeness and molecular docking studies. Physical
descriptors were calculated by making use of the “Descriptor
module” of MOE program to gauge the druglikeness of all the
compounds. The optimized ligands were docked with the IALP
(PDB code: 4KJG) protein using the MOE-Dock program. A
total of 20 independent docking runs were performed using
MOE docking simulation program. The best scored pose
(minimum energy) for each compound was retained for
further studies of interaction evaluation. The 2D ligand–
protein interactions were visualized using the MOE ligand
interactions program. Detailed docking results are recorded in
Table S2 given in ESI.†
´
18 J. Alvar, I. D. Velez, C. Bern, M. Herrero, P. Desjeux, J. Cano,
J. Jannin and M. den Boer, PLoS One, 2012, 7, e35671.
19 Working to Overcome the Global Impact of Neglected Tropical
Diseases, First WHO Report on Neglected Tropical Diseases,
ed. D. W. T. Compton and P. Peters, World Health
Organization, Geneva, 2010.
20 (a) V. Polshettiwar and R. S. Varma, Curr. Opin. Drug
Discovery Dev., 2007, 10, 723–737; (b) I. Khan, A. Ibrar,
N. Abbas and A. Saeed, Eur. J. Med. Chem., 2014, 76, 193–244.
21 (a) A. Padwa and S. K. Bur, Tetrahedron, 2007, 63, 5341–5378;
(b) I. Khan, A. Ibrar, W. Ahmed and A. Saeed, Eur. J. Med.
Chem., 2015, 90, 124–169.
22 D. M. D'Souza and T. J. Muller, Chem. Soc. Rev., 2007, 36,
1095–1108.
Acknowledgements
A. I is grateful to the Higher Education Commission of Pakistan
for the nancial support under Indigenous 5000 PhD Fellow-
ship Program. We are thankful to Dr Charles Dennis Hall and
Prof. Alan R. Katritzky (late) (University of Florida) for the MOE
soware license. The Department of Energy, United State of
America, is gratefully acknowledged for support through Grant
No. DE-SC0001717.
23 G. Eren, S. Unlu, M. T. Nunez, L. Labeaga, F. Ledo,
A. Entrena, E. B. Lu, G. Costantino and M. F. Sahin, Bioorg.
Med. Chem., 2010, 18, 6367–6376.
24 R. J. Gillespie, A. I. Cliffe, C. E. Dawson, C. T. Dourish,
S. Gaur, P. R. Giles, A. M. Jordan, A. R. Knight,
A. Lawrence, J. Lerpiniere, A. Misra, R. M. Pratt,
89930 | RSC Adv., 2015, 5, 89919–89931
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
R. S. Todd, R. Upton, S. M. Weiss and D. S. Williamson,
Bioorg. Med. Chem. Lett., 2008, 18, 2920–2923.
25 V. Jaishree, N. Ramdas, J. Sachin and B. Ramesh, J. Saudi
Chem. Soc., 2012, 16, 371–376.
26 A. T. Ricardo, A. Luz and D. P. Carlos, Bioorg. Med. Chem.,
2003, 11, 2175–2182.
27 A. Zablotskaya, I. Segal, A. Geronikaki, T. Eremkina,
RSC Advances
C. Giommarelli and F. Zunino, Bioorg. Med. Chem., 2010,
18, 3543–3550; (e) Q. Sun, D.-Y. Peng, S.-G. Yang, X.-L. Zhu,
W.-C. Yang and G.-F. Yang, Bioorg. Med. Chem., 2014, 22,
4784–4791; (f) K. Paul, S. Bindal and V. Luxami, Bioorg.
Med. Chem. Lett., 2013, 23, 3667–3672; (g) R. Aggarwal,
S. Kumar, P. Kaushik, D. Kaushik and G. K. Gupta, Eur. J.
Med. Chem., 2013, 62, 508–514.
S. Belyakov, M. Petrova, I. Shestakova, L. Zvejniece and 41 (a) I. Khan, S. Zaib, A. Ibrar, N. H. Rama, J. Simpson and
V. Nikolajeva, Eur. J. Med. Chem., 2013, 70, 846–856.
28 M. H. M. Helal, M. A. Salem, M. S. A. El-Gaby and
M. Aljahdali, Eur. J. Med. Chem., 2013, 65, 517–526.
J. Iqbal, Eur. J. Med. Chem., 2014, 78, 167–177; (b) I. Khan,
S. J. A. Shah, S. A. Ejaz, A. Ibrar, S. Hameed, J. Lecka,
´
J. L. Millan, J. Sevigny and J. Iqbal, RSC Adv., 2015, 5,
29 R. Romagnoli, P. G. Baraldi, M. K. Salvador, M. E. Camacho,
64404–64413.
D. Preti, M. A. Tabrizi, M. Bassetto, A. Brancale, E. Hamel, 42 (a) I. Khan, A. Ibrar, S. Zaib, S. Ahmad, N. Furtmann,
R. Bortolozzi, G. Basso and G. Viola, Bioorg. Med. Chem.,
2012, 20, 7083–7094.
30 K. N. Venugopala, M. Krishnappa, S. K. Nayak,
B. K. Subrahmanya, J. P. Vaderapura, R. K. Chalannavar,
R. M. Gleiser and B. Odhav, Eur. J. Med. Chem., 2013, 65,
295–303.
S. Hameed, J. Simpson, J. Bajorath and J. Iqbal, Bioorg. Med.
Chem., 2014, 22, 6163–6173; (b) I. Khan, S. M. Bakht, A. Ibrar,
S. Abbas, S. Hameed, J. M. White, U. A. Rana, S. Zaib,
M. Shahid and J. Iqbal, RSC Adv., 2015, 5, 21249–21267; (c)
A. Saeed, P. A. Channar, Q. Iqbal and J. Mahar, Chin. Chem.
Lett., DOI: 10.1016/j.cclet.2015.09.011.
31 (a) A. Saeed and A. Ibrar, Phosphorus, Sulfur Silicon Relat. 43 T. Somerai, L. Szilagyi and S. Horvath, Arch. Pharm., 1986,
Elem., 2011, 186, 1801–1810; (b) A. Raza, A. Saeed, A. Ibrar, 139, 238–241.
M. Muddassar, A. A. Khan and J. Iqbal, ISRN Pharmacol., 44 K. M. Dawood, H. Abdel-Gawad, H. A. Mohamed and
2012, 2012, 1–11; (c) A. Saeed, P. A. Channar, Q. Iqbal and F. A. Badria, Med. Chem. Res., 2011, 20, 912–919.
J. Mahar, Chin. Chem. Lett., 2015, DOI: 10.1016/ 45 J. Iqbal, Anal. Biochem., 2011, 414, 226–231.
j.cclet.2015.09.011. 46 T. Mosmann, J. Immunol. Methods, 1983, 65, 55–63.
¨ ¨
¨
32 O. Domotor, T. Tuccinardi, D. Karcz, M. Walsh, B. S. Creaven 47 M. Niks and M. Otto, J. Immunol. Methods, 1990, 130, 149–151.
´
and E. A. Enyedy, Bioorg. Chem., 2014, 52, 16–23.
48 P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon,
D. Vistica, J. T. Warren, H. Bokesch, S. Kenney and
M. R. Boyd, J. Natl. Cancer Inst., 1990, 82, 1107–1112.
33 Y. Zhao, Q. Zheng, K. Dakin, K. Xu, M. L. Martinez and
W.-H. Li, J. Am. Chem. Soc., 2004, 126, 4653–4663.
34 J. R. Hwu, S.-Y. Lin, S.-C. Tsay, E. de Clercq, P. Leyssen and 49 D. F. Veber, S. R. Johnson, H.-Y. Cheng, B. R. Smith,
J. Neyts, J. Med. Chem., 2011, 54, 2114–2126.
35 S.-C. Tsay, J. R. Hwua, R. Singha, W.-C. Huang, Y. H. Chang,
K. W. Ward and K. D. Kopple, J. Med. Chem., 2002, 45,
2615–2623.
M. H. Hsu, F.-K. Shieh, C.-C. Lin, K. C. Hwang, J. C. Horng, 50 C. A. Lipinski, F. Lombardo, B. W. Dominy and P. Feeney,
E. de Clercq, I. Vliegen and J. Neyts, Eur. J. Med. Chem., 2013,
63, 290–298.
36 K. Nepali, S. Sharma, M. Sharma, P. M. S. Bedi and
K. L. Dhar, Eur. J. Med. Chem., 2014, 77, 422–487.
37 F. W. Muregi and A. Ishih, Drug Dev. Res., 2010, 71, 20–32.
Adv. Drug Delivery Rev., 1997, 23, 3–25.
51 K. Ghosh, T. D. Mazumde, R. Anumula, B. Lakshmaiah,
P. P. Kumar, S. Singaram, T. Matan, S. Kallipatti,
S. Selyam, P. Krishnamurthy and M. Ramarao, J. Struct.
Biol., 2013, 184, 182–192.
38 C. Hubschwerlen, J. L. Specklin, C. Sigwalt, S. Schroeder and 52 N. Ingale, V. Maddi, M. Palkar, P. Ronad, S. Mamledesai,
H. H. Locher, Bioorg. Med. Chem., 2003, 11, 2313–2319.
39 R. Pingaew, S. Prachayasittikul, S. Ruchirawat and
V. Prachayasittikul, Med. Chem. Res., 2014, 23, 1768–1780.
40 (a) K. V. Sashidhara, R. K. Modukuri, S. Singh, K. B. Rao,
G. A. Teja, S. Gupta and S. Shukla, Bioorg. Med. Chem. Lett.,
A. H. M. Vishwanathswamy and D. Satyanarayana, Med.
Chem. Res., 2012, 21, 16–26.
53 K. Araki-Sasaki, S. Aizawa, M. Hiramoto, M. Nakamura,
O. Iwase, K. Nakata, Y. Sasaki, T. Mano, H. Handa and
Y. Tano, J. Cell. Physiol., 2000, 182, 189–195.
2015, 25, 337–341; (b) K. V. Sashidhara, S. R. Avula, 54 G. S. Longo-Sorbello, G. m. Say, D. Banerjee and J. R. Bertino,
K. Sharma, G. R. Palnati and S. R. Bathula, Eur. J. Med.
Chem., 2013, 60, 120–127; (c) K. V. Sashidhara,
Cytotoxicity and cell growth assays, Cell Biology, Four-Volume
Set: A Laboratory Handbook, 2005, vol. 1, pp. 315–324.
R. K. Modukuri, D. Choudhary, K. B. Rao, M. Kumar, 55 Molecular Operating Environment (MOE), 2013.08, Chemical
V. Khedgikar and R. Trivedi, Eur. J. Med. Chem., 2013, 70,
802–810; (d) F. Belluti, G. Fontana, L. D. Bo, N. Carenini,
Computing Group Inc., 1010, Sherbooke St. West, Suite
#910, Montreal, QC, Canada, H3A 2R7, 2015.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 89919–89931 | 89931