Cytotoxic activity assessment, QSAR and docking study of novel bis-carboxamide derivatives of 4-pyrones synthesized by Ugi four-component reaction

Article abstract of DOI:10.1016/j.ejmech.2013.05.049

Fourteen novel bis-carboxamide derivatives of 4-pyrones were designed and synthesized via Ugi four-component reactions of 4-pyrone carbaldehydes, aromatic amines, isocyanides and carboxylic acids. The cytotoxic activity of synthesized derivatives was eval

Full text of DOI:10.1016/j.ejmech.2013.05.049

Accepted Manuscript
Cytotoxic Activity Assessment, QSAR and Docking Study of Novel bis-Carboxamide
Derivatives of 4-Pyrones Synthesized by Ugi Four-Component Reaction
Aziz Shahrisa, Somayeh Esmati, Ramin Miri, Omidreza Firuzi, Najmeh Edraki,
Maryam Nejati
PII:
S0223-5234(13)00372-3
10.1016/j.ejmech.2013.05.049
EJMECH 6232
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 December 2012
Revised Date: 14 May 2013
Accepted Date: 16 May 2013
Please cite this article as: A. Shahrisa, S. Esmati, R. Miri, O. Firuzi, N. Edraki, M. Nejati, Cytotoxic
Activity Assessment, QSAR and Docking Study of Novel bis-Carboxamide Derivatives of 4-Pyrones
Synthesized by Ugi Four-Component Reaction, European Journal of Medicinal Chemistry (2013), doi:
10.1016/j.ejmech.2013.05.049.
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ACCEPTED MANUSCRIPT
Cytotoxic Activity Assessment, QSAR and Docking Study of Novel bis-Carboxamide
Derivatives of 4-Pyrones Synthesized by Ugi Four-Component Reaction
Aziz Shahrisa*a, Somayeh Esmatia, Ramin Miri*b, Omidreza Firuzib, Najmeh Edrakib and
Maryam Nejatib
a Department of Organic Chemistry and Biochemistry , Faculty of Chemistry, University of Tabriz, Tabriz
5166614766, Iran
b Medicinal & Natural Products Chemistry Research Centre, Shiraz University of Medical Sciences, Shiraz, Iran
(phone.: +98 411 339 3118; fax: +98 411 334 0191; e-mail: ashahrisa@yahoo.com (A. Shahrisa); mirir@sums.ac.ir (R. Miri) )
Graphical Abstract
A series of novel bis-carboxamide derivatives of 4-pyrones were synthesized via Ugi four
component reaction and their cytotoxic activities were evaluated against three different cell lines
using MTT reduction assay.
High Lights
- New bis-amide derivatives of 4-pyrones were synthesized via Ugi 4-component reaction.
- For the first time cytotoxic activity of Ugi adducts was evaluated.
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- Some of the synthesized compounds have strong cytotoxic potential in HL-60 cell line.
- The QSAR study indicated that topological properties influence cytotoxic activity.
- Docking studies of these compounds were conducted into Src tyrosine kinase.
Abstract
Fourteen novel bis-carboxamide derivatives of 4-pyrones were designed and synthesized via Ugi
four-component reactions of 4-pyronecarbaldehydes, aromatic amines, isocyanides and
carboxylic acids. The cytotoxic activity of synthesized derivatives was evaluated against LS180,
MCF-7 and HL-60 cell lines using MTT reduction assay. Synthesized compounds demonstrated
strong cytotoxic potential in HL-60 cell line. Compound 12n was the most potent derivative with
IC50 values of 16.1, 9.1 and 13.8 µM in LS180, MCF-7 and HL-60 cells, respectively. The
results of MLR-QSAR study indicated that topological property of these derivatives directly
influenced the cytotoxic potential in HL-60 cell line. Docking study of compounds, conducted
for ATP binding site of Src tyrosine kinase, demonstrated the key H-bond interaction with Met
347 of the hinge region.
Key words: Ugi reaction, 4-pyrone derivatives, QSAR, Docking, Src tyrosine kinase
1. Introduction
4H-pyran-4-one (4-pyrone) derivatives constitute a promising versatile class of heterocyclic
compounds, which are widely distributed in a variety of natural and synthetic biologically active
compounds [1-4]. The results of different studies have indicated that amide containing 4-pyrones
exhibit valuable biological activities; e.g. amidopyrones 1 are inhibitors of matrix
metalloproteinases [5]. Some of amide containing 4-pyrones has been found in a variety of
natural products. For example, pestalamide A 2 has been isolated from cultures of the plant
pathogenic fungus Pestalotiopsis theae [6]. Furthermore, the anticancer and cytotoxic potential of
natural and synthetic 4H-pyran-4-one derivatives have been well established [7-9]. Bransova and
colleagues have reported the in vitro cytotoxic properties of different derivatives of a naturally
occurring 4H-pyran-4-one, Kojic acid 3 (5-hydroxy-2-hydroxymethyl-4-pyrone), in leukemia
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cell lines [10]. Moreover, the anticancer property of 5-benzyloxy-2-thiocyanatomethyl-4- pyrone
4 in rat pituitary carcinoma cells (GH4C1), human leukemia and murine leukemia cells has been
well characterized [10,11]. In this regard, 5-benzyloxy-4-oxo-4H-pyran-2-carboxamides 5 (Fig.
1) have been reported as good ligands for ATP-binding site of Src tyrosine kinase [12] which
plays important roles in cell growth, proliferation and invasion in many types of cancer.
Therefore, Src is an attractive target for cancer chemotherapy and different types of Src
inhibitors have been developed [13-15].
< Figure 1 >
On the other hand, Multicomponent reactions (MCRs) have recently become popular for
preparation of pharmacologically active compounds [16]. One of the most important classes of
MCRs is the isocyanide based multicomponent reaction (IMCR) [17]. Passerini developed one of
the first IMCRs in 1921 [18]. This reaction was later expanded by Ugi in 1959 to afford di-amide
derivatives [19] by a four-component coupling reaction of aldehyde, amine, isocyanide and
carboxylic acid according to the mechanism shown in Scheme 1. Ugi reaction has widely used
not only to prepare peptide-like molecules, but also have achieved profound attention for the
synthesis and discovery of new lead structures and different types of biologically active scaffolds
[20-28]
< Scheme 1 >
Based on the above evidences and as a part of our ongoing research on synthesis of
biologically active derivatives of 4-pyrones [29-32], here we report Ugi four-component
reactions for the synthesis of novel anticancer bis-carboxamide derivatives of 4H-pyran-4-one
along with cytotoxic activity evaluation of these compounds in three different cancer cell lines
including LS180, MCF-7 and HL-60 cells. Furthermore, synthesized compounds were subjected
to QSAR study in order to find important structural features that influence anticancer activity of
these novel derivatives. Finally, we performed a molecular docking simulation study in order to
find out the binding potential of these compounds with the Src tyrosine kinase enzyme as a
probable molecular target that could explain the experimental results on the cytotoxic activity.
2. Results and discussion
2.1. Chemistry
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In view of interesting biological activities of pyrone motif containing carboxamide groups, we
designed and synthesized new bis-amide derivatives of 4-pyrones via Ugi four component
reactions. This is the first time that 4-pyrone carbaldeydes are used in Ugi reaction. As
mentioned in Scheme 2 aldehyde 7 was obtained from commercially available Kojic acid 3 by
benzylation of phenolic OH [33], followed by oxidation of the hydroxymethyl group with active
MnO2. Aldehyde 11 was synthesized from the corresponding ethoxycarbonyl derivative 9
according to the reported procedure [34]. The ethoxycarbonyl derivative itself was prepared
through cyclization of the related 1,3,5-triketone 8 under acidic conditions, which is an important
method for the synthesis of a variety of 4-pyrone derivatives [35-37].
< Scheme 2 >
As shown in the Scheme 3, novel Ugi products 12a-n were synthesized by the reaction of 4-
pyrone carbaldehydes, isocyanides, carboxylic acids and amines in the absence of any catalyst in
methanol. The reaction mixtures were stirred at room temperature for 24 hours and the final
products were obtained with good yields. The results are summarized in Table 1. This method is
simple and convenient and would be applicable for the synthesis of different types of 4-pyrone
bis-amides, since operates at room temperature under catalyst-free condition.
< Scheme 3 >
< Table 1>
1
13
The structures of final products were confirmed by H NMR, C NMR, FT-IR and elemental
analysis.
2.2. Cytotoxic activity
The in vitro anti-cancer activity of target compounds 12a-n was determined against LS 180
(intestinal human colon adenocarcinoma cells), MCF-7 (breast cancer human cell lines) and
HL60 (human promyelocytic leukemia cell line) cells using MTT 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide method [38]. Briefly, cell viability was assessed by MTT
colorimetric assay in which the viable cell number is directly proportional to the production of
purple formazan. This color product can be measured spectrophotometrically at 570 nm by an
ELISA plate reader. The percent inhibition of viability for each concentration of compound was
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calculated compared to the control wells and IC50 values (concentration of the compound that
induces 50% inhibition of cell viability) were calculated by linear regression and expressed in
Mean ± SD. The results are represented in Table 2.
< Table 2>
The results of cytotoxic data indicate that most of synthesized compounds showed moderate
to strong cytotoxic potential especially in HL-60 and MCF-7 cell lines. Although all synthesized
bis-carboxamide derivatives of 4-pyrones possess lower cytotoxic potential than doxorubicin in
all three cell lines, some of them showed superior potency to cisplatin especially in MCF-7 and
HL-60 cell lines (compounds 12e in MCF-7 and 12n in HL-60 cells).
According to the R1, R2, R3 and R4 substituents shown in Fig. 2 and based on the cytotoxic data
in Table 2, the following structure-activity relationship may be developed:
< Figure 2 >
The compounds could be classified into two groups according to the R1 substituent:
benzyloxy-4-pyrones (12a-i) and phenyl-4-pyrone derivatives (12j-n). The benzyloxy
derivatives of 4-pyrones demonstrated more cytotoxic potential than phenyl-4-pyrones in some
cases. For instance, the benzyloxy derivative 12c is more potent than its phenyl-4-pyrone
counterpart 12k in all three cell lines; IC50 values for 12c against HL-60, LS180 and MCF-7
were 12.1, 63.0 and 56.3 μM, respectively, while 12k was inactive in two first lines and its IC50
value against HL-60 cells was 22.2 μM.
R2 moiety in the synthesized compounds could be cyclohexyl or t-butyl. There was no
significant difference in the cytotoxic potency of cyclohexyl and t-butyl bearing derivatives.
However, by considering the substitution pattern on R3 group, some differences were observed
between cyclohexyl and t-butyl derivatives: The introduction of 2-chlorophenyl in the R3 moiety
enhanced cytotoxic potential of t-butyl bearing derivatives, while it reduced the potency of
cyclohexyl bearing derivatives. For instance, it is noteworthy to compare activity of compounds
12c and 12e which possessing cyclohexyl at R2 (R1 and R4 groups are also the same in 12c and
12e ). Compound 12e with phenyl group as R3, showed significant cytotoxic potential against all
three cell lines (IC50 values in HL-60, LS180 and MCF-7 cell lines were 11.3, 20.2 and 11.2 μM,
respectively), while compound 12c with 2-chlorophenyl as R3 showed lower potency especially
against LS180 and MCF-7 cell line (IC50= 63.0 and 56.3 μM respectively). This observation may
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be described as a result of geometrical hindrance imposed in cyclohexyl derivatives by
introduction of chlorine atom into R3-phenyl moiety, which can hinder the ligand-receptor
access. However, this substitution pattern in t-butyl containing derivatives caused the invers
outcome, as exemplified in the comparison of the activity of compounds 12l and 12n. Compound
12l containing phenyl moiety as R3 showed lower activity than its 2-chlorophenyl analog 12n in
all three cell lines. Thus compound 12n is one of the promising synthesized derivatives in all
three cell lines (IC50 values in HL-60, LS180 and MCF-7 cell lines were 13.8, 16.1 and 9.1,
respectively).
On the other hand, the results indicated that the introduction of different substituents such as
methyl, n-butyl, methoxy and chlorine in para position of phenyl in R4 moiety resulted in
enhanced cytotoxic potential of compounds in both series of pyrone derivatives (benzyloxy and
phenylpyrones) especially in HL-60 cell line; e.g. the corresponding IC50 value for compound
12a containing phenyl group at R4 (Y=H) in HL-60 cell lines is 66.2 μM. Introduction of methyl
group in para position of phenyl ring (Z=methyl, 12c) considerably enhances the cytotoxic
potency of this compound in this cells (IC50=12.1 μM).
2.3. QSAR study
QSAR analysis of synthetic compounds (12a-n) was carried out by MLR analysis. The
correlation coefficient (R2), standard error of regression (SE), correlation coefficient for cross-
validation significance (Q2) and root mean square error (RMS) were employed to judge the
validity of regression equation. As collinearity degrades the performances of the MLR based
QSAR equation, correlation analysis was performed to detect the collinear descriptors (Mager,
1983). Therefore, the correlation of descriptors with each other and with activity data was
evaluated and among the collinear descriptors one of them that represented the highest
correlation with activity was retained and the rest were omitted.
Due to limited available cytotoxic data in LS180 and MCF-7 cell lines (8 and 10 respectively),
no valid QSAR model is obtained for quantitative structure-activity elucidation of bis-amide
derivatives of pyrone in these two cell lines.
When we used pIC50 of bis-carboxamide derivatives of pyrone against HL-60 cell line as
dependent variable the three parametric equation E1 with good statistical quality was obtained
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from the pool of calculated descriptors (R2=0.93, S.E.= 0.09 and Q2=0.83) (Table 3). Selected
variables indicated that topological property (PJI2: 2D petitjean shape index) of bis-amide 4-
pyrones directly affect the cytotoxic potential of compounds against HL-60 cell line. Whereas,
GETAWAY parameters including HGM (geometric mean on the leverage magnitude) and ISH
(standardized information content on the leverage equality) demonstrated negative relation with
cytotoxic potency of compounds in these cells.
< Table 3 >
The data of selected descriptors used in this study together with the experimental and
corresponding predicted value of activity of compounds such as pIC50 (-Log IC50) are listed in
Table 4. The respective predicted values of activity, refined from the calibration and cross-
validation, by using equation 1 is plotted against the experimental values. As it could be seen,
there is a close agreement between the experimental and predicted activities obtained by QSAR
model in HL-60 cell line (Fig. 3.)
< Table 4 >
< Figure 3 >
2.4. Docking study
Based on the crystal structure of active Src tyrosine kinase inhibitor 1-{4-[4-amino-5-(3-
methoxyphenyl)-7H-pyrrolo[2,3-D]pyrimidin-7-yl]benzyl}piperidin-4-ol (S03) (PDB ID: 1YOL)
[39] we docked the bis-carboxamide derivatives of 4-pyrone into ATP site of this enzyme using
Autodock 4.2 software by flexible ligand/rigid macromolecule protocol. Docking optimization
and validation was performed according to the cited method based on the RMSD (root mean
square deviation) of the best-docked conformation of cognate ligand from experimental one
(internal validation) [40]. Docking performance examined by re-docking the co-crystallized
conformation of a native ligand (S03) to Src tyrosine kinase ATP site. The best-docked and
actual conformation of inhibitor superimposed quite well with an RMSD of 1.463 Aº (The
RMSD value of below 2 Aº is considered as an index of successful prediction [41]. The results of
internal validation along with important binding interactions are reported in Table 5. This
inhibitor produces a key hydrogen bonding interaction with Met 343 of hinge region of the active
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site, which is important for directing the ligand for other hydrogen bonding interaction with Src
gate keeper residue Thr340 and also with Glu342. Moreover the 3-methoxyphenyl moiety of the
ligand is mainly oriented towards the hydrophobic backpocket of the enzyme and produces
important hydrophobic interactions with this pocket.
< Table 5 >
As described by Farard and co-workers [12], the 4-pyrone moiety could be served as a
hydrogen-bond acceptor moiety for interaction with the hinge region of Src tyrosine kinase.
Therefore, we performed a docking study on the synthesized bis-amide derivatives of 4-pyrones
into the active site of Src tyrosine kinase to further evaluate the binding affinity of these
compounds into ATP site of this enzyme. Docking results including free binding energies (ΔGb),
inhibition constants (Ki) and hydrogen bonds are demonstrated in Table 6. Top ranked binding
energies (kcal/mol) in AutoDock dlg output file were considered as response in each run.
< Table 6 >
Some of these derivatives (12a, 12g, 12j, 12k and 12n) demonstrated key hydrogen-bonding
interaction with Met 343 of hinge region. The carbonyl group of 4-pyrone moiety is mainly
involved in this interaction in cyclohexyl containing derivatives (12a, 12j and 12k), while in t-
butyl containing derivatives the different orientation was observed, since other part of molecule
participated in this key H-bond interaction. The binding interaction of the most potent cytotoxic
derivative 12n containing t-butyl moiety as R2 group is depicted in Fig. 4A. The R4 substituent is
oriented towards the hinge region; since the methoxy group substituted on the para position of
phenyl ring produced H-bond interaction with Met343. These parts of ligand (R4) along with 2-
chlorophenyl group (R3) are involved in hydrophobic interaction with the backpocket of active
site (composed of Lys 297 and Leu275) as observed in S03 binding pose. The other important
interactions are depicted in Fig. 4A. The attached phenyl moiety on the C2 position of 4-pyrone
ring oriented toward Thr340. Accordingly, substitution of suitable groups in this part could
provide the other important H-bonding with this residue. On the other hand, as it is depicted in
Fig. 4B, the R4 (p-methylphenyl) and R3 (2-chlorophenyl) substitute of cyclohexyl containing
derivative 12k also oriented toward the backpocket and involved in important hydrophobic
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interaction with this part of active site. As mentioned previously, the pyrone moiety of 12k is
directed toward hinge part to provide the key H-bond interaction with this part.
Overall, the results of the docking study indicate that the binding orientation of bis-
carboxamide derivatives towards the hinge region is mainly dependent on the substituted moiety
on the R4 position of this scaffold. Regarding this substitution, cyclohexyl derivatives
demonstrated different binding orientation than t-butyl ones. This effect could be interpreted
with the results of QSAR study that topological descriptors influence the cytotoxic activity of
bis-carboxamide derivatives of 4-pyrones. It could be concluded that the R4 substitute might
topologically affects ligand-protein interaction. Furthermore, new H-bond interactions with Leu
275, Ser 347, Asp 350, Gln 377 and Ala 390 residues were detected for these new 4-pyrone
derivatives. The results indicate that attachment of suitable groups with H-bond interacting
potential into C-2 position of the 4-pyrone ring would result in the other important H-bond
interaction with Thr340 residue of active site and should be noticed in development of potent Src
tyrosine kinase inhibitors of this type.
< Figure 4 >
3. Conclusions
In conclusion, we have described an efficient Ugi four-component reaction for the synthesis
of novel bis-amide derivatives of 4-pyrones under mild and neutral reaction conditions. This
method for synthesis of bis-amide derivatives has the advantages of simple operation in one-pot
and room temperature reaction conditions, simple experimental work up procedures and good
yields. These derivatives demonstrated considerable cytotoxic potential especially in HL-60 cell
line, which was mainly dependent on the topological properties of the molecule as revealed by
QSAR study. Inhibition of Src tyrosine kinase enzyme might be considered as one of the
probable cytotoxic mechanisms of these compounds. However; different cytotoxic profiles of
these compounds indicate that other molecular targets might be involved for anticancer activity
of these derivatives.
4. Experimental protocols
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4.1. Chemistry
Melting points were determined with a MEL-TEMP model 1202D and are uncorrected. FT-IR
1
spectra were recorded on a Bruker Tensor 27 spectrometer as KBr disks. The H NMR spectra
were recorded with a Bruker Spectrospin Avance 400 spectrometer with CDCl3 as solvent and
13
TMS as internal standard. C NMR spectra were determined on the same instrument at 400
MHz. All chemical shifts were reported as  (ppm) and coupling constants (J) are given in Hz.
Elementray analyses (C, H, N) were performed on a Vario EL III analyzer. Thin-layer
chromatography was done with prepared glass-backed plates (20 20 cm2, 500 µ) using silica
gel (Merk Kieselgel 60 HF254, Art. 7739). The chemical reagents used in synthesis were
purchased from Merck and Sigma-Aldrich.
4.1.1. Procedure for the synthesis of 4-pyrone carbaldehydes 7 and 11
Alcohol derivatives (6) or (10) (2.4 mmol) was dissolved in dichloromethane (20 cm3) and
activated manganese (IV) oxide (21 mmol) was added. The reaction mixture was stirred
efficiently for 3 days at room temperature then the black suspension was filtered through a pad of
silica, which was washed with warm ethyl acetate (50 cm3). The resulting solution was
evaporated under reduced pressure and the crude product was purified by recrystallization from
chloroform/ethanol to give the pure aldehyde.
4.1.1.1. 5-Benzyloxy-4-Oxo-4H-pyran-2-carbaldehyde (7)
Pale yellow solid; Yield: 65%; mp: 118-120°C; FT-IR (KBr): ν 3087, 2923 , 2860 (aldehyde
1
CH), 1713 (aldehyde C=O), 1647 (pyrone C=O), 1615, 1211, 1139 cm-1; H NMR (400 MHz,
CDCl3):  5.16 (s, 2H, OCH2Ph), 7.02 (s, 1H, H-3 pyrone), 7.36–7.43 (m, 5H, Ph-H), 7.69 (s,
1H, H-6 pyrone), 9.67 (s, 1H, CHO) ppm.
4.1.1.2. 4-Oxo-6-phenyl-4H-pyran-2-carbaldehyde (11)
Pale yellow solid; Yield: 62%; mp: 115-116°C; FT-IR (KBr): ν 3046, 2858 (aldehyde C–H),
1
2733 (aldehyde C–H), 1714 (aldehyde C=O), 1647 (pyrone C=O) cm-1; H NMR (400 MHz,
CDCl3):  6.89 (d, 1H, J = 2 Hz, pyrone-H), 6.93 (d, 1H, J = 2 Hz, pyrone-H), 7.49-7.58 (m, 3H,
Ph-H), 7.84-7.87 (m, 2H, Ph-H), 9.76 (s,1H,–CHO) ppm; 13C NMR (100 MHz, CDCl3):  112.1,
120.6, 125.2, 128.2, 129.2, 131.2, 155.6, 162.8, 178.4, 183.3 ppm.
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4.1.2. General procedure for the synthesis of Ugi products 12a-n
A mixture of pyrone carbaldehydes 7 or 11 (0.5 mmol) and suitable amine (R4-NH2) (0.5
mmol) in MeOH (2 ml) was stirred for 30 min at room temperature and then carboxylic acid (R3-
COOH) (0.5 mmol) and isocyanide (R2-NC) (0.6 mmol) was added and reaction mixture was
stirred at room temperature for 24h (The progress of reaction was monitored by TLC). After
completion of reaction, the residue was purified by preparative thin layer chromatography PTLC
(silica gel, acetone- n-hexane, 1:3) to give corresponding products 12a-n.
4.1.2.1.
N-[(5-(Benzyloxy)-4-oxo-4H-pyran-2-yl)-(cyclohexylcarbamoyl)-methyl]-2-chloro-N-
phenylbenzamide (12a)
Pale yellow solid; Yield: 84%; mp: 65-67°C; FT-IR (KBr):  3294 (N-H), 3068 (aromatic C-
H), 2929 (aliphatic C-H), 2854 (aliphatic C-H), 1695 (amide C=O), 1647 (pyrone C=O) cm-1; 1H
NMR (400 MHz, CDCl3):  1.09–1.94 (m, 10H, 5CH2 of cyclohexyl), 3.83 (m, 1H, CH–N of
cyclohexyl), 4.96 (d, 1H, J= 12 Hz, benzylic-H), 5.01 (d, 1H, J= 12 Hz, benzylic-H), 5.97 (s, 1H,
C-H), 6.62 (s, 1H, pyrone-H), 6.93 (bd, 1H, J= 8 Hz, N-H), 7.03-7.08 (m, 9H, Ar-H), 7.27-7.35
13
(m, 5H, Ar-H), 7.43 (s, 1H, pyrone-H) ppm; C NMR (100 MHz, CDCl3):  23.6, 23.7, 24.3,
31.4, 31.5, 48.1, 62.8, 70.6, 116.1, 125.2, 126.7, 127.3, 127.5, 127.6, 127.6, 127.7, 127.8, 128.3,
129.2, 129.2, 133.9, 134.3, 137.9, 140.3, 145.9, 159.0, 163.3, 167.9, 173.1 ppm; Anal. Calcd. For
C33H31ClN2O5: C, 69.41; H, 5.47; N, 4.91; Found: C, 69.23; H, 5.38; N, 4.86%
4.1.2.2.
N-[(5-(Benzyloxy)-4-oxo-4H-pyran-2-yl)-(tert-butylcarbamoyl)-methyl]-N-
phenylbenzamide (12b)
Pale yellow solid; Yield: 78%; mp: 62-63°C; FT-IR (KBr):  3308 (N-H), 3069 (aromatic C-
H), 2963 (aliphatic C-H), 2924 (aliphatic C-H), 2858 (aliphatic C-H), 1692 (amide C=O), 1644
(pyrone C=O) cm-1; 1H NMR (400 MHz, CDCl3):  1.35 (s, 9H, 3 CH3), 4.97 (d, 1H, J= 12 Hz,
benzylic-H), 5.02 (d, 1H, J= 12 Hz, benzylic-H), 5.87 (s, 1H, C-H), 6.53 (bs, 1H, N-H), 6.65 (s,
13
1H, pyrone-H), 7.12-7.34 (m, 15H, Ar-H), 7.44 (s, 1H, pyrone-H) ppm; C NMR (100 MHz,
CDCl3):  27.5, 51.1, 64.6, 70.9, 115.7, 126.7, 126.8, 126.9, 127.3, 127.4, 127.6, 127.7, 127.8,
128.1, 129.3, 133.5, 134.5, 139.9, 140.6, 146.0, 159.7, 163.6, 170.2, 173.2 ppm; Anal. Calcd. For
C31H30N2O5: C, 72.92; H, 5.92; N, 5.49; Found: 72.75; H, 5.95; N, 5.41%
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4.1.2.3. N-[(5-(Benzyloxy)-4-oxo-4H-pyran-2-yl)-(cyclohexylcarbamoyl)-methyl]-2-chloro-N-p-
tolylbenzamide (12c)
Pale yellow solid; Yield: 88%; mp: 67-68°C; FT-IR (KBr):  3295 (N-H), 3069 (aromatic C-
H), 2930 (aliphatic C-H), 2855 (aliphatic C-H), 1682 (amide C=O), 1649 (pyrone C=O) cm-1; 1H
NMR (400 MHz, CDCl3):  1.11-1.92 (m, 10H, 5CH2 of cyclohexyl), 2.14 (s, 3H, CH3), 3.80 (m,
1H, CH–N of cyclohexyl), 4.96 (d, 1H, J= 12 Hz, benzylic-H), 5.00 (d, 1H, J= 12 Hz, benzylic-
H), 5.99 (s, 1H, C-H), 6.58 (s, 1H, pyrone-H), 6.84 (d, 2H, J= 8 Hz, Ar-H), 7.04-7.18 (m, 7H,
13
Ar-H, N-H), 7.31-7.35 (m, 5H, Ar-H), 7.46 (s, 1H, pyrone-H) ppm; C NMR (100 MHz,
CDCl3):  19.9, 23.6, 23.7, 24.3, 31.5, 31.6, 48.1, 62.8, 70.7, 116.2, 125.3, 126.7, 126.9, 127.3,
127.4, 127.5, 127.6, 128.3, 128.5, 129.1, 134.1, 134.4, 135.3, 137.5, 140.3, 145.9, 158.9, 163.3,
168.1, 173.0 ppm; Anal. Calcd. For C34H33ClN2O5: C, 69.80; H, 5.68; N, 4.79; Found: C, 69.62;
H, 5.71; N, 4.70%
4.1.2.4. N-[(5-(Benzyloxy)-4-oxo-4H-pyran-2-yl)-(tert-butylcarbamoyl)-methyl]-2-chloro-N-p-
tolylbenzamide (12d)
Pale yellow solid; Yield: 85%; mp: 60-62°C; FT-IR (KBr):  3303 (N-H), 3075 (aromatic C-
H), 2966 (aliphatic C-H), 2924 (aliphatic C-H), 2865 (aliphatic C-H), 1687 (amide C=O), 1646
(pyrone C=O) cm-1; 1H NMR (400 MHz, CDCl3):  1.39 (s, 9H, 3 CH3), 2.16 (s, 3H, CH3), 4.97
(d, 1H, J= 12 Hz, benzylic-H), 5.02 (d, 1H, J= 12 Hz, benzylic-H), 5.82 (s, 1H, C-H), 6.61 (s,
1H, pyrone-H), 6.73 (bs, 1H, N-H), 6.86 (d, 2H, J= 8Hz, Ar-H), 7.02-7.19 (m, 6H, Ar-H), 7.30-
13
7.38 (m, 5H, Ar-H), 7.46 (s, 1H, pyrone-H) ppm; C NMR (100 MHz, CDCl3):  19.9, 27.4,
51.0, 63.5, 70.7, 116.0, 125.2, 126.7, 127.2, 127.3, 127.5, 128.3, 128.4, 129.0, 129.1, 134.1,
134.4, 135.4, 137.4, 140.3, 145.9, 159.2, 163.3, 168.0, 173.0 ppm; Anal. Calcd. For
C32H31ClN2O5: C, 68.75; H, 5.59; N, 5.01; Found: C, 68.67; H, 5.61; N, 4.97%
4.1.2.5.
N-[(5-(Benzyloxy)-4-oxo-4H-pyran-2-yl)-(cyclohexylcarbamoyl)-methyl]-N-p-
tolylbenzamide (12e)
Pale yellow solid; Yield: 78%; mp: 95-97°C; FT-IR (KBr):  3240 (N-H), 3051 (aromatic C-
H), 2989 (aliphatic C-H), 2927 (aliphatic C-H), 2854 (aliphatic C-H), 1677 (amide C=O), 1643
12

ACCEPTED MANUSCRIPT
1
(pyrone C=O) cm-1; H NMR (400 MHz, CDCl3):  1.11-1.90 (m, 10H, 5CH2 of cyclohexyl),
2.21 (s, 3H, CH3), 3.80 (m, 1H, CH–N of cyclohexyl), 4.97 (d, 1H, J= 12 Hz, benzylic-H), 5.02
(d, 1H, J= 12 Hz, benzylic-H), 5.92 (s, 1H, C-H), 6.61 (s, 1H, pyrone-H), 6.77 (bd, 1H, J= 8Hz,
N-H), 6.91 (d, 2H, J= 8 Hz, Ar-H), 6.96 (d, 2H, J= 8 Hz, Ar-H), 7.14-7.17 (m, 2H, Ar-H), 7.23-
7.35 (m, 8H, Ar-H), 7.45 (s, 1H, pyrone-H) ppm; 13C NMR (100 MHz, CDCl3):  19.9, 23.6,
24.3, 31.5, 31.6, 47.9, 64.0, 70.7, 115.7, 126.7, 126.8, 127.4, 127.5, 127.6, 128.7, 129.2, 131.6,
133.6, 134.5, 136.9, 137.2, 140.3, 145.9, 159.5, 163.7, 170.4, 173.1 ppm; Anal. Calcd. For
C34H34N2O5: C, 74.16; H, 6.22; N, 5.09; Found: C, 74.01; H, 6.25; N, 5.01%
4.1.2.6.
N-[(5-(Benzyloxy)-4-oxo-4H-pyran-2-yl)-(tert-butylcarbamoyl)-methyl]-N-(4-butyl-
phenyl)-2-chlorobenzamide (12f)
Pale yellow solid; Yield: 82%; mp: 70-72°C; FT-IR (KBr):  3296 (N-H), 3081 (aromatic C-
H), 2960 (aliphatic C-H), 2927 (aliphatic C-H), 2861 (aliphatic C-H), 1679 (amide C=O), 1647
(pyrone C=O) cm-1; 1H NMR (400 MHz, CDCl3):  0.84 (t, 3H, J= 8Hz, CH3), 1.14-1.23 (m, 2H,
CH2), 1.39-1.46 (m, 11H, 3×CH3, CH2), 2.43 (t, 2H, J= 8Hz, CH2), 4.97 (d, 1H, J= 12 Hz,
benzylic-H), 5.03 (d, 1H, J= 12 Hz, benzylic-H), 5.78 (s, 1H, C-H), 6.62 (s, 1H, pyrone-H), 6.75
(bs, 1H, N-H), 6.87 (d, 2H, J= 8Hz, Ar-H), 6.99-7.19 (m, 6H, Ar-H), 7.27-7.36 (m, 5H, Ar-H),
13
7.46 (s, 1H, pyrone-H) ppm; C NMR (100 MHz, CDCl3):  12.8, 20.9, 27.4, 31.9, 33.9, 51.1,
63.9, 70.7, 115.9, 125.2, 126.7, 127.2, 127.3, 127.5, 127.6, 127.8, 128.3, 129.1, 129.2, 134.0,
134.5, 135.7, 140.3, 142.4, 146.0, 159.2, 163.4, 168.1, 173.1 ppm; Anal. Calcd. For
C35H37ClN2O5: C, 69.93; H, 6.20; N, 4.66; Found: C, 69.74; H, 6.23; N, 4.59%
4.1.2.7. N-[(5-(Benzyloxy)-4-oxo-4H-pyran-2-yl)-(tert-butylcarbamoyl)-methyl]-2-chloro-N-(4-
methoxyphenyl) benzamide (12g)
Pale yellow solid; Yield: 72%; mp: 90-92°C; FT-IR (KBr):  3304 (N-H), 3073 (aromatic C-
H), 2966 (aliphatic C-H), 2927 (aliphatic C-H), 1682 (amide C=O), 1647 (pyrone C=O) cm-1; 1H
NMR (400 MHz, CDCl3):  1.41 (s, 9H, 3 CH3), 3.67 (s, 3H, CH3), 4.98 (d, 1H, J= 12 Hz,
benzylic-H), 5.04 (d, 1H, J= 12 Hz, benzylic-H), 5.82 (s, 1H, C-H), 6.56 (bs, 1H, N-H), 6.58 (s,
1H, pyrone-H), 6.60 (d, 2H, J= 4Hz, Ar-H), 7.07-7.20 (m, 6H, Ar-H), 7.31-7.34 (m, 5H, Ar-H),
7.45 (s, 1H, pyrone-H) ppm; 13C NMR (100 MHz, CDCl3):  27.5, 51.1, 54.2, 63.3, 70.8, 112.9,
116.3, 125.3, 126.7, 127.4, 127.5, 127.6, 128.3, 128.8, 129.1, 130.5, 134.2, 134.5, 140.5, 145.9,
13

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158.1, 159.0, 163.4, 168.3, 173.0 ppm; Anal. Calcd. For C32H31ClN2O6: C, 66.84; H, 5.43; N,
4.87; Found: C, 66.60; H, 5.39; N, 4.71%
4.1.2.8. N-[(5-(Benzyloxy)-4-oxo-4H-pyran-2-yl)-(cyclohexylcarbamoyl)-methyl]-2-chloro-N-(4-
chlorophenyl)benzamide (12h)
Pale yellow solid; Yield: 74%; mp: 60-62°C; FT-IR (KBr):  3297 (N-H), 3072 (aromatic C-
H), 2929 (aliphatic C-H), 2855 (aliphatic C-H), 1682 (amide C=O), 1649 (pyrone C=O) cm-1; 1H
NMR (400 MHz, CDCl3):  1.12–1.91 (m, 10H, 5CH2 of cyclohexyl), 3.80 (m, 1H, CH–N of
cyclohexyl), 4.97 (d, 1H, J= 12 Hz, benzylic-H), 5.02 (d, 1H, J= 12 Hz, benzylic-H), 6.01 (s, 1H,
C-H), 6.58 (s, 1H, pyrone-H), 6.75 (bd, 1H, J= 8 Hz, N-H), 7.00-7.21 (m, 8H, Ar-H), 7.32-7.39
13
(m, 5H, Ar-H), 7.41 (s, 1H, pyrone-H) ppm; C NMR (100 MHz, CDCl3):  23.6, 23.7, 24.3,
31.4, 31.5, 48.3, 62.1, 70.7, 116.5, 125.4, 126.7, 127.4, 127.5, 127.6, 127.7, 127.9, 128.4, 129.1,
129.3, 129.4, 133.4, 133.8, 134.2, 136.2, 140.5, 145.9, 158.7, 163.1, 167.7, 172.9 ppm; Anal.
Calcd. For C33H30Cl2N2O5: C, 65.46; H, 4.99; N, 4.63; Found: C, 65.39; H, 5.02; N, 4.58%
4.1.2.9.
N-[(5-(Benzyloxy)-4-oxo-4H-pyran-2-yl)-(cyclohexylcarbamoyl)-methyl)]-N-(4-
chlorophenyl)benzamide (12i)
Pale yellow solid; Yield: 71%; mp: 64-66°C; FT-IR (KBr):  3301 (N-H), 3067 (aromatic C-
H), 2928 (aliphatic C-H), 2854 (aliphatic C-H), 1677 (amide C=O), 1645 (pyrone C=O) cm-1; 1H
NMR (400 MHz, CDCl3):  1.12–1.90 (m, 10H, 5CH2 of cyclohexyl), 3.78 (m, 1H, CH–N of
cyclohexyl), 4.97 (d, 1H, J= 12 Hz, benzylic-H), 5.02 (d, 1H, J= 12 Hz, benzylic-H), 6.02 (s, 1H,
C-H), 6.58 (d, 1H, J= 2Hz, pyrone-H), 6.66 (bd, 1H, J= 8 Hz, N-H), 6.99-7.37 (m, 14H, Ar-H),
13
7.39 (s, 1H, pyrone-H) ppm; C NMR (100 MHz, CDCl3):  23.6, 23.7, 24.3, 31.5, 31.6, 48.1,
62.9, 70.7, 116.1, 126.7, 126.8, 127.4, 127.5, 127.6, 127.7, 127.8, 128.1, 128.9, 129.4, 132.7,
134.3, 138.0, 140.4, 145.9, 159.1, 163.5, 170.1, 172.9 ppm; Anal. Calcd. For C33H31ClN2O5: C,
69.41; H, 5.47; N, 4.91; Found: C, 69.28; H, 5.57; N, 4.75%
4.1.2.10.
2-Chloro-N-[(cyclohexylcarbamoyl)-(4-oxo-6-phenyl-4H-pyran-2-yl)-methyl)]-N-
phenylbenzamide (12j)
14

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Yellow solid; Yield: 86%; mp: 57-59°C; FT-IR (KBr):  3292 (N-H), 3063 (aromatic C-H),
1
2927 (aliphatic C-H), 2854 (aliphatic C-H), 1694 (amide C=O), 1653 (pyrone C=O) cm-1; H
NMR (400 MHz, CDCl3):  1.12–2.03 (m, 10H, 5CH2 of cyclohexyl), 3.90 (m, 1H, CH–N of
cyclohexyl), 6.18 (s, 1H, C-H), 6.57 (s, 1H, pyrone-H), 6.62 (s, 1H, pyrone-H), 6.82 (bd, 1H, J=
8 Hz, N-H), 7.03-7.19 (m, 7H, Ar-H), 7.27-7.29 (m, 2H, Ar-H), 7.41-7.54 (m, 3H, Ar-H), 7.59
13
(d, 2H, J= 8Hz, Ar-H) ppm; C NMR (100 MHz, CDCl3):  23.6, 23.7, 24.3, 31.6, 31.7, 48.2,
62.5, 109.8, 116.8, 124.7, 125.3, 127.6, 127.8, 127.9, 127.9, 128.3, 129.2, 129.3, 129.4, 130.5,
134.1, 137.9, 159.4, 162.5, 163.2, 167.9, 178.4 ppm; Anal. Calcd. For C32H29ClN2O4: C, 71.04;
H, 5.40; N, 5.18; Found: C, 70.95; H, 5.43; N, 5.12%
4.1.2.11.
2-Chloro-N-[(cyclohexylcarbamoyl)-(4-oxo-6-phenyl-4H-pyran-2-yl)methyl)]-N-p-
tolylbenzamide (12k)
Yellow solid; Yield: 89%; mp: 102-104°C; FT-IR (KBr):  3297 (N-H), 3064 (aromatic C-
H), 2928 (aliphatic C-H), 2854 (aliphatic C-H), 1693 (amide C=O), 1653 (pyrone C=O) cm-1; 1H
NMR (400 MHz, CDCl3):  1.18–2.03 (m, 10H, 5CH2 of cyclohexyl), 2.07 (s, 3H, CH3), 3.90
(m, 1H, CH–N of cyclohexyl), 6.17 (s, 1H, C-H), 6.59 (d, 1H, J= 2Hz, pyrone-H), 6.65 (d, 1H,
J= 2Hz, pyrone-H), 6.78-6.83 (m, 3H, Ar-H, N-H), 7.06-7.19 (m, 6H, Ar-H), 7.41-7.51 (m, 3H,
13
Ar-H), 7.59 (d, 2H, J= 8Hz, Ar-H) ppm; C NMR (100 MHz, CDCl3):  19.9, 23.6, 23.7, 24.4,
31.7, 31.8, 48.2, 62.6, 109.9, 116.9, 124.8, 125.3, 127.5, 127.6, 127.9, 128.4, 128.5, 129.1, 129.2,
129.5, 130.6, 134.2, 135.3, 137.7, 159.5, 162.7, 163.3, 168.2, 178.6 ppm; Anal. Calcd. For
C33H31ClN2O4: C, 71.41; H, 5.63; N, 5.05; Found: C, 71.25; H, 5.66; N, 5.01%
4.1.2.12. N-[(tert-Butylcarbamoyl)-(4-oxo-6-phenyl-4H-pyran-2-yl)methyl)]-N-p-tolylbenzamide
(12l)
Yellow solid; Yield: 82%; mp: 78-80°C; FT-IR (KBr):  3301 (N-H), 3062 (aromatic C-H),
2962 (aliphatic C-H), 2923 (aliphatic C-H), 2858 (aliphatic C-H), 1696 (amide C=O), 1650
1
(pyrone C=O) cm-1; H NMR (400 MHz, CDCl3):  1.39 (s, 9H, 3CH3), 2.14 (s, 3H, CH3), 6.00
(s, 1H, C-H), 6.56 (d, 1H, J= 2Hz, pyrone-H), 6.63 (bs, 1H, N-H), 6.65 (d, 1H, J= 2Hz, pyrone-
H), 6.89 (d, 2H, J= 8Hz, Ar-H), 7.05 (d, 2H, J= 8 Hz, Ar-H), 7.15-7.50 (m, 8H, Ar-H), 7.61 (d,
13
2H, J= 8Hz, Ar-H) ppm; C NMR (100 MHz, CDCl3):  19.9, 27.5, 51.0, 64.9, 110.1, 116.0,
15

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124.8, 126.8, 127.7, 127.9, 128.7, 129.2, 129.5, 129.7, 130.5, 133.7, 136.9, 137.3, 160.1, 162.6,
163.7, 170.3, 178.5 ppm; Anal. Calcd. For C31H30N2O4: C, 75.28; H, 6.11; N, 5.66; Found: C,
75.12; H, 6.17; N, 5.62%
4.1.2.13.
2-Chloro-N-[(cyclohexylcarbamoyl)-(4-oxo-6-phenyl-4H-pyran-2-yl)methyl]-N-(4-
methoxyphenyl)benzamide (12m)
Yellow solid; Yield: 76%; mp: 105-107°C; FT-IR (KBr):  3298 (N-H), 3065 (aromatic C-
H), 2929 (aliphatic C-H), 2852 (aliphatic C-H), 1694 (amide C=O), 1653 (pyrone C=O) cm-1; 1H
NMR (400 MHz, CDCl3):  1.12–2.04 (m, 10H, 5CH2 of cyclohexyl), 3.57 (s, 3H, CH3), 3.90
(m, 1H, CH–N of cyclohexyl), 6.21 (s, 1H, C-H), 6.51 (d, 2H, J= 8Hz, Ar-H), 6.59 (s, 1H,
pyrone-H), 6.65 (s, 1H, pyrone-H), 6.74 (bd, 1H, J= 8Hz, N-H), 7.05-7.20 (m, 6H, Ar-H), 7.42-
13
7.55 (m, 3H, Ar-H), 7.61 (d, 2H, J= 8Hz, Ar-H) ppm; C NMR (100 MHz, CDCl3):  23.7,
23.8, 24.3, 31.6, 31.7, 48.2, 54.1, 62.2, 109.9, 112.9, 117.0, 124.7, 125.4, 127.4, 127.9, 128.3,
129.0, 129.1, 129.3, 130.2, 130.6, 130.8, 134.2, 158.1, 159.4, 162.6, 163.3, 168.4, 178.6 ppm;
Anal. Calcd. For C33H31ClN2O5: C, 69.41; H, 5.47; N, 4.91; Found: C, 69.25; H, 5.51; N, 4.88 %
4.1.2.14.
N-[(tert-Butylcarbamoyl)-(4-oxo-6-phenyl-4H-pyran-2-yl)methyl]-2-chloro-N-(4-
methoxyphenyl)benzamide (12n)
Yellow solid; Yield: 74%; mp: 88-90°C; FT-IR (KBr):  3307 (N-H), 3066 (aromatic C-H),
2966 (aliphatic C-H), 2926 (aliphatic C-H), 2855 (aliphatic C-H), 1694 (amide C=O), 1653
1
(pyrone C=O) cm-1; H NMR (400 MHz, CDCl3):  1.43 (s, 9H, 3CH3), 3.56 (s, 3H, CH3), 6.12
(s, 1H, C-H), 6.51 (d, 2H, J= 8Hz, Ar-H), 6.57 (d, 1H, J= 2Hz, pyrone-H), 6.64 (d, 1H, J= 2Hz,
pyrone-H), 6.74 (bs, 1H, N-H), 7.04-7.19 (m, 6H, Ar-H), 7.42-7.50 (m, 3H, Ar-H), 7.63 (d, 2H,
J= 8Hz, Ar-H) ppm; 13C NMR (100 MHz, CDCl3):  27.5, 51.2, 54.1, 62.9, 109.9, 112.8, 116.9,
124.7, 125.3, 127.5, 127.9, 128.3, 129.0, 129.1, 129.5, 130.3, 130.5, 130.8, 134.3, 158.1, 159.5,
162.5, 163.4, 168.2, 178.5 ppm; Anal. Calcd. For C31H29ClN2O5: C, 68.31; H, 5.36; N, 5.14;
Found: C, 68.14; H, 5.41; N, 5.10 %
4.2. MTT assay
16

ACCEPTED MANUSCRIPT
RPMI 1640, fetal bovine serum (FBS), trypsin and phosphate buffered saline (PBS) were
purchased from Biosera (Ringmer, UK). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) was obtained from Sigma-Aldrich (Saint Louis, MO, USA) and
penicillin/streptomycin was purchased from Invitrogen (San Diego, CA, USA). Doxorubicin and
dimethyl sulphoxide were obtained from EBEWE Pharma (Unterach, Austria) and Merck
(Darmstadt, Germany), respectively. HL-60 (human promyelocytic leukemia), LS180 (human
colon adenocarcinoma) and MCF-7 (human breast adenocarcinoma) cells were obtained from the
National Cell Bank of Iran, Pasteur Institute, Tehran, Iran. Cell lines were maintained in RPMI
1640 supplemented with 100 units/ml penicillin-G and 100 g/ml streptomycin as well as 10%
FBS (except for HL-60 cells that needed 20% FBS). Cells were maintained at 37°C in
humidified air containing 5% CO2. LS180 and MCF-7 cells were grown in monolayer cultures,
while HL-60 cells were grown in suspension.
Cell viability following exposure to synthetic compounds was estimated by using the MTT
reduction assay [38,42]. HL-60, LS180 and MCF-7 cells were plated in 96-well flat bottomed
microplates at densities of 40,000, 50,000 and 30,000 cells/ml (100 μl per well), respectively.
Control wells contained the same number of cells without any drugs, while blank wells contained
only growth medium for background correction. After overnight incubation at 37 °C, half of the
growth medium was removed and 50 μl of medium supplemented with 3-4 different
concentrations of synthetic compounds were added in duplicate. Plates with HL-60 cells were
centrifuged before this procedure. Compounds were all first dissolved in DMSO and then diluted
in the growth medium. The maximum concentration of DMSO in the wells was 0.5%. Cells were
further incubated for 72 hr and at the end of the incubation time fresh medium containing 0.5
mg/ml of MTT was added. Plates were incubated for another 4 hr at 37 °C. Then the formazan
crystals formed inside viable cells were solubilized in 200 μl DMSO. The optical density was
measured at 570 nm with background correction at 655 nm using a Bio-Rad microplate reader
(Model 680). The percent inhibition of viability for each concentration of compound was
calculated compared to the control wells and IC50 values (concentration of the compound that
induces 50% inhibition of cell viability) were calculated with the CurveExpert software version
1.34 for Windows. Each experiment was repeated 3-5 times. Data are presented as mean  S.D.
17

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4.3. QSAR analysis
4.3.1. Descriptor generation
The chemical structure of molecules was constructed using HyperChem (Version 7, Hypercube
Inc., http://www.hyper.com, USA). The Z-matrices of the structures were provided by the
software and were then transferred to the Gaussian 98 program [43]. Complete geometry
optimization was performed taking the most extended conformations as starting geometries.
Semi-empirical molecular orbital calculations (AM1) of the structures were performed using
Gaussian 98 program.
A large number of molecular descriptors were calculated using HyperChem (HyperCube Inc.),
Gaussian 98 and Dragon (Todeschini, http://www.disat.unimib.it/vhm/) Packages. Gaussian 98
were employed for calculation of different quantum chemical descriptors including dipole
moment (DM), local charges, HOMO and LUMO energies, hardness (η); softness (S);
electronegativity (χ); and electrophilicity (ω). Dragon software was used to calculate different
descriptors including functional groups, topological, geometrical, constitutional, charge
descriptors and aromaticity indices for each molecule. Some chemical parameters including
molecular volume (MV), molecular surface area (SA), hydrophobicity (logP) and hydration
energy (HE) were calculated using HyperChem software.
4.3.2. Data processing and modeling
The calculated descriptors were collected in a data matrix, D. First the descriptors were checked
for constant or near constant values and those detected were removed from the original data
matrix. Then, the correlation of descriptors with each other and with the activity data was
determined. Among the collinear descriptors detected (r >0.9), one of them that had the highest
correlation with activity was retained and the rest were omitted. MLR analysis with stepwise
selection and elimination of variables was applied for developing QSAR models using SPSS
software (SPSS Inc., version 17). The resulted models were validated by leave-one out cross-
validation procedure (using MATLAB software) to check their predictive ability and robustness.
4.4. Molecular docking study
18

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The docking studies were carried out using AutoDock 4.2 (The Scripps Research Institute, La
Jolla, CA, http://autodock.scripps.edu/). Lamarckian Genetic Algorithm of the AutoDock 4.2
program was used to perform the flexible-ligand docking studies (Morris et al. 2009). Receptor
X-ray crystal structure in complex with the inhibitor, 1-{4-[4-amino-5-(3-methoxyphenyl)-7H-
pyrrolo[2,3- D]pyrimidin-7-yl]benzylpiperidin-4-ol (S03) (PDB ID 1YOL; http://www.pdb.org/)
[39] was obtained from the Brookhaven protein data bank. All pre-processing procedures of
target protein and ligand files were performed within Accelrys ViewerLite 5.0 software and
AutoDock Tools 1.5.4 program (ADT) which has been released as an extension suite to the
Python Molecular Viewer [44]. The three-dimensional structures of the ligands were constructed
using Chem3D Ultra 8.0 software [Chemical Structure Drawing Standard; Cambridge Soft
Corporation, USA (2003)] to obtain standard 3D structures (pdb format). The constructed
compounds were energetically minimized (100 steepest descent steps using MM+ force field with
a gradient convergence value of 0.1 kcal/mol ) using HyperChem (HyperCube Inc.).
For docked ligands, non-polar hydrogens were added; Gasteiger charges and torsions degrees of
freedom were also allocated by ADT program. One-hundred independent genetic algorithm (GA)
runs were considered for each ligand under study. For Lamarckian GA method, a maximum
number of 2,500,000 energy evaluations; 27,000 maximum generations; a gene mutation rate of
0.02; and a crossover rate of 0.8 were used. A grid of 40×40× 40 points in x, y, and z direction
with grid spacing of 0.375 Ao was built centered on the center of mass of the catalytic site.
Cluster analysis was performed on the docked results using a root mean square (RMS) tolerance
of 2 Ao. Ranking of the clusters was carried out according to the obtained average lowest binding
energy of members of the cluster to highest.
Acknowledgments
The authors wish to thank the Research Concil of University of Tabriz and Shiraz University of
Medical Sciences for their financial support.
19

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References
[1] F. Caturla, M. Amat, R.F. Reinoso, M. Cordoba, G. Warrellow, Racemic and chiral
sulfoxides as potential prodrugs of the COX-2 inhibitors Vioxx and Arcoxia, Bioorg. Med.
Chem. Lett. 16 (2006) 3209-3212.
[2] P.W. Groundwater, K.R.H. Solomons, The synthesis of pyranoacridinone inhibitors of
protein tyrosine kinases, J. Chem. Soc. Perkin Trans. 1 (1994) 173-177.
[3] T. Kamino, K. Kuramochi, S. Kobayashi, A concise approach to 5-substituted-pyrones from
kojic acid, Tetrahedron Lett. 44 (2003) 7349-7351.
[4] D.T. Puerta, J. Mongan, B.L. Tran, J.A. McCammon, S.M. Cohen, Potent, selective pyrone-
based inhibitors of stromelysin-1, J. Am. Chem. Soc. 127 (2005) 14148-14149.
[5] Y.L. Yan, S.M. Cohen, Efficient synthesis of 5-amido-3-hydroxy-4-pyrones as inhibitors of
matrix metalloproteinases, Org. Lett. 9 (2007) 2517-2520.
[6] G. Ding, L. Jiang, L. Guo, X. Chen, H. Zhang, Y. Che, Pestalazines and pestalamides,
bioactive metabolites from the plant pathogenic fungus Pestalotiopsis theae, J. Nat. Prod. 71
(2008) 1861-1865.
[7] J.C. Henrikson, T.K. Ellis, J.B. King, R.H. Cichewicz, Reappraising the structures and
distribution of metabolites from black aspergilli containing uncommon 2-benzyl-4H-pyran-4-one
and 2-benzylpyridin-4(1H)-one systems, J. Nat. Prod. 74 (2011) 1959-1964.
[8] D.H. Li, S.X. Cai, L. Tian, Z.J. Lin, T.J. Zhu, Y.C. Fang, P.P. Liu, Q.Q. Gu, W.M. Zhu, Two
new metabolites with cytotoxicities from deep-sea fungus, Aspergillus sydowi YH11-2, Arch.
Pharmacal Res. 30 (2007) 1051-1054.
[9] R.C. Barcelos, J.C. Pastre, V. Caixeta, D.B. Vendramini-Costa, J.E. de Carvalho, R.A. Pilli,
Synthesis of methoxylated goniothalamin, aza-goniothalamin and gamma-pyrones and their in
vitro evaluation against human cancer cells, Bioorg. Med. Chem. 20 (2012) 3635-3651.
[10] J. Bransova, M. Uher, L. Novotny, J. Brtko, 5-benzyloxy-2-thiocyanatomethyl-4-pyrone, a
novel heterocyclic compound: synthesis, structure determination and effects on neoplastic cell
growth, Anticancer Res. 17 (1997) 1175-1178.
[11] J. Brtko, L. Rondahl, M. Fickova, D. Hudecova, V. Eybl, M. Uher, Kojic acid and its
derivatives: history and present state of art, Cent. Eur. J. Public health. 12 Suppl (2004) S16-18.
20

ACCEPTED MANUSCRIPT
[12] J. Farard, G. Lanceart, C. Loge, M.R. Nourrisson, F. Cruzalegui, B. Pfeiffer, M. Duflos,
Design, synthesis and evaluation of new 6-substituted-5-benzyloxy-4-oxo-4H-pyran-2-
carboxamides as potential Src inhibitors, J. Enzym. Inhib. Med. Chem. 23 (2008) 629-640.
[13] M. Susva, M. Missbach, J. Green, Src inhibitors: drugs for the treatment of osteoporosis,
cancer or both?, Trends Pharmacol. Sci. 21 (2000) 489-495.
[14] J.M. Summy, G.E. Gallick, Src family kinases in tumor progression and metastasis, Cancer
Metastasis Rev. 22 (2003) 337-358.
[15] A.P. Belsches-Jablonski, M.L. Demory, J.T. Parsons, S.J. Parsons, The Srcpathway as
atherapeuticstrategy, Drug. Discov. Today Ther. Strat. 2 (2005) 313-321.
[16] I. Ugi, A. Domling, B. Werner, Since 1995 the new chemistry of multicomponent reactions
and their libraries, including their heterocyclic chemistry , J. Heterocyclic Chem. 37 (2000) 647-
658.
[17] A. Domling, I. Ugi, Multicomponent reactions with isocyanides, Angew. Chem. Int. Ed.
Engl. 39 (2000) 3168-3210.
[18] M. Passerini, L. Simone, Sopra gli isonitrili (I). Composto del p-isonitrilazobenzolo con.
Acetone ed acido acetico, Gazz. Chim. Ital. 52 (1921) 126-129.
[19] C.C. Musonda, D. Taylor, J. Lehman, J. Gut, P.J. Rosenthal, K. Chibale, Application of
multi-component reactions to antimalarial drug discovery. Part 1: Parallel synthesis and
antiplasmodial activity of new 4-aminoquinoline Ugi adducts, Bioorg. Med. Chem. Lett. 14
(2004) 3901-3905.
[20] C.M. Haney, C. Schneider, B. Beck, J.L. Brodsky, A. Domling, Identification of Hsp70
modulators through modeling of the substrate binding domain, Bioorg. Med. Chem. Lett. 19
(2009) 3828-3831.
[21] P.G. Wyatt, M.J. Allen, A.D. Borthwick, D.E. Davies, A.M. Exall, R.J. Hatley, W.R. Irving,
D.G. Livermore, N.D. Miller, F. Nerozzi, S.L. Sollis, A.K. Szardenings, 2,5-Diketopiperazines
as potent and selective oxytocin antagonists 1: Identification, stereochemistry and initial SAR,
Bioorg. Med. Chem. Lett. 15 (2005) 2579-2582.
[22] H.J. Sanganee, A. Baxter, S. Barber, A.J. Brown, D. Grice, F. Hunt, S. King, D. Laughton,
G. Pairaudeau, B. Thong, R. Weaver, J. Unitt, Discovery of small molecule human C5a receptor
antagonists, Bioorg. Med. Chem. Lett. 19 (2009) 1143-1147.
21

ACCEPTED MANUSCRIPT
[23] I.W. Ku, S. Cho, M.R. Doddareddy, M.S. Jang, G. Keum, J.H. Lee, B.Y. Chung, Y. Kim, H.
Rhim, S.B. Kang, Morpholin-2-one derivatives as novel selective T-type Ca2+ channel blockers,
Bioorg. Med. Chem. Lett. 16 (2006) 5244-5248.
[24] D.J. Parks, L.V. Lafrance, R.R. Calvo, K.L. Milkiewicz, V. Gupta, J. Lattanze, K.
Ramachandren, T.E. Carver, E.C. Petrella, M.D. Cummings, D. Maguire, B.L. Grasberger, T.
Lu, 1,4-Benzodiazepine-2,5-diones as small molecule antagonists of the HDM2-p53 interaction:
discovery and SAR, Bioorg. Med. Chem. Lett. 15 (2005) 765-770.
[25] K. Groebke Zbinden, D.W. Banner, J. Ackermann, A. D'Arcy, D. Kirchhofer, Y.H. Ji, T.B.
Tschopp, S. Wallbaum, L. Weber, Design of selective phenylglycine amide tissue factor/factor
VIIa inhibitors, Bioorg. Med. Chem. Lett. 15 (2005) 817-822.
[26] N.A. Yehia, W. Antuch, B. Beck, S. Hess, V. Schauer-Vukasinovic, M. Almstetter, P.
Furer, E. Herdtweck, A. Domling, Novel nonpeptidic inhibitors of HIV-1 protease obtained via a
new multicomponent chemistry strategy, Bioorg. Med. Chem. Lett. 14 (2004) 3121-3125.
[27] X. Fan, X. Zhang, C. Bories, P.M. Loiseau, P.F. Torrence, The Ugi reaction in the
generation of new nucleosides as potential antiviral and antileishmanial agents, Bioorg. Chem.
35 (2007) 121-136.
[28] A. Arabanian, M. Mohammadnejad, S. Balalaie, J.H. Gross, Synthesis of novel Gn-RH
analogues using Ugi-4MCR, Bioorg. Med. Chem. Lett. 19 (2009) 887-890.
[29] A. Shahrisa, M. Saraei, Synthesis of pyrone carbaldehydes, pyrone sulfonium ylides and
related epoxides, J. Heterocyclic Chem. 46 (2009) 268-272.
[30] A. Shahrisa, R. Miri, S. Esmati, M. Saraei, A.R. Mehdipour, M. Sharifi, Synthesis and
calcium channel antagonist activity of novel 1,4-dihydropyridine derivatives possessing 4-pyrone
moieties, Med. Chem. Res. 21 (2012) 284-292.
[31] A. Shahrisa, Z. Ghasemi, Synthesis of fused pyrimidone derivatives of 4-pyrones from the
acetates of Baylis–Hillman adducts, Chem. Heterocyclic Compd. 46 (2010) 30-36.
[32] A. Shahrisa, Z. Ghasemi, M. Saraei, Synthesis of 2,6-bis(1H-indole-6-yl)-4H-pyran-4-ones
via Leimgruber–Batcho indole synthesis, J. Heterocyclic Chem. 46 (2009) 273-277.
[33] A.F. Thomas, A. Marxer, Heilmittelchemische Studien in der heterocyclischen Reihe. 30.
Mitteilung. Die Reaktion von Kojis ure mit Hydrazin. 2. Teil. Umsetzung von Kojis ure thern
mit Hydrazin, Helv. Chim. Acta. 43 (1960) 469-477.
22

ACCEPTED MANUSCRIPT
[34] M. Rudas, I. Fejes, M. Nyerges, A. Szöllõsy, L. Tõke, P.W. Groundwater, Substituent
effects on the 4π + 2π cycloadditions of 4H-pyran-4-one derivatives J. Chem. Soc., Perkin Trans.
1 (1999) 1167-1172.
[35] D. Hobuss, S. Laschat, A. Baro, Concise two-step synthesis of γ-pyrone from acetone,
synlett (2005) 123-124.
[36] V.I. Tyvorskii, D.N. Bobrov, O.G. Kulinkovich, N. De Kimpe, K.A. Tehrani, New
synthetic approaches to 2-perfluoroalkyl-4H-pyran-4-ones, Tetrahedron, 54 (1998) 2819-2826.
[37] H. Arimoto, S. Nishiyama, S. Yamamum, Mild conditions for cyclization of β-triketides to
the corresponding γ-pyronescarryingadjacentchiralcenterstowardbiomimeticsynthesis of fully
substituted γ-pyrone-containing natural products, Tetrahedron Lett. 31 (1990) 5619-5620.
[38] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55-63.
[39] C.B. Breitenlechner, N.A. Kairies, K. Honold, S. Scheiblich, H. Koll, E. Greiter, S. Koch,
W. Schafer, R. Huber, R.A. Engh, Crystal structures of active SRC kinase domain complexes, J.
Mol. Biol. 353 (2005) 222-231.
[40] Y. Fukunishi, Y. Mikami, H. Nakamura, Similarities among receptor pockets and among
compounds: analysis and application to in silico ligand screening, J. Mol. Graph. Model. 24
(2005) 34-45.
[41] J. Pan, G.Y. Liu, J. Cheng, X.J. Chen, X.L. Ju, CoMFA and molecular docking studies of
benzoxazoles and benzothiazoles as CYP450 1A1 inhibitors, Eur. J. Med. Chem. 45 (2010) 967-
972.
[42] R. Miri, O. Firuzi, P. Peymani, M. Zamani, A.R. Mehdipour, Z. Heydari, M.M. Farahani, A.
Shafiee, Synthesis, cytotoxicity, and QSAR study of new aza-cyclopenta[b]fluorene-1,9-dione
derivatives, Chem. Biol. Drug Des. 79 (2012) 68-75.
[43] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, V.G.
Zakrzewski, J.A. Montgomery Jr., R.E. Stratmann, J.C. Burant, S. Dapprich, J.M. Millam, A.D.
Daniels, K.N. Kudin, M.C. Strain, Ö. Farkas, J. Tomasi, V. Barone, M. Cossi, R. Cammi, B.
Mennucci, C. Pomelli, C. Adamo, S. Clifford, J. Ochterski, G.A. Petersson, P.Y. Ayala, Q. Cui,
K. Morokuma, P. Salvador, J.J. Dannenberg, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B.
Foresman, J. Cioslowski, J.V. Ortiz, A.G. Baboul, B.B. Stefanov, G. Liu, A. Liashenko, P.
23

ACCEPTED MANUSCRIPT
Piskorz, I. Komáromi, R. Gomperts, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y.
Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, J.L.
Andres, C. Gonzalez, M. Head-Gordon, E.S. Replogle, J.A. Pople, Gaussian 98 (Gaussian, Inc.,
Pittsburgh, PA, 1998).
[44] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson,
AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility, J.
Comput. Chem. 30 (2009) 2785-2791.
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Captions:
Fig. 1. Chemical structure of some of biologically active pyrones.
Fig. 2. Substitution pattern of bis-carboxamide derivatives of 4-pyrone
Fig. 3. Plot of the predicted activity against the experimental activity for the QSAR model
obtained by Equation 1 in HL-60 cell line.
Fig. 4. Best docked pose of compound 12n (A) and 12k (B) docked into Src tyrosine kinase
ATP-binding site.
Scheme 1. Mechanism of the Ugi reaction.
Scheme 2. Synthesis of 4-pyrone carbaldehyde derivatives. Reagents and conditions: i) PhCH2Br
(1 eq), NaOH (1eq), MeOH/H2O: 10/1, 60oC, 4h, 75%; ii) MnO2 (8eq), CH2Cl2, rt., 3days, 65%;
iii) (2eq), DME, reflux, 4h, 67%; iv)H2SO4, rt., 4h, 62%; v) NaBH4 (4eq), MeOH, reflux, 3h,
42%; vi) MnO2 (8eq), CH2Cl2, rt., 3days, 62%.
Scheme 3. General description of Ugi adducts.
Table 1. Chemical structures of bis-carboxamide derivatives of 4-pyrones 12a-n.
Table 2. Cytotoxic activity of bis-carboxamide derivatives of pyrone 12a-n.
Table 3. Statistical equations obtained by QSAR analysis.
Table 4. Data of the selected descriptors used in this study and the experimental and predicted
values of pIC50 in HL-60 cell line.
Table 5. The results of docking validation study obtained by re-docking of cognate ligand ″1-{4-
[4-amino-5-(3-methoxyphenyl)-7H-pyrrolo[2,3- D]pyrimidin-7-yl]benzyl}piperidin-4-ol (S03)″
into Src tyrosine kinase.
Table 6. Results of docking study of synthesized bis-amide derivatives of 4-pyrones docked into
Src tyrosine kinase active site.
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Table 1. Chemical structures of bis-carboxamide derivatives of 4-pyrones 12a-n
m.p.
(oC)
Yield
(%)
Entry
R1
R2
R3
R4
Product
Cyclohexyl
65-67
84
1
t-Butyl
62-63
78
2
Cyclohexyl
67-68
60-62
95-97
88
85
78
3
4
5
t-Butyl
Cyclohexyl
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t-Butyl
70-72
82
6
t-Butyl
90-92
72
7
Cyclohexyl
60-62
74
8
Cyclohexyl
64-66
71
9
Cyclohexyl
57-59
86
10
Cyclohexyl
102-104
89
11
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t-Butyl
78-80
82
12
Cyclohexyl
105-107
76
13
t-Butyl
88-90
74
14
Table 2. Cytotoxic activity of bis-carboxamide derivatives of pyrone 12a-n.
IC50 (μM)
Compound
LS180 cells
MCF-7 cells
HL-60 cells
12a
12b
12c
12d
12e
12f
12g
12h
12i
97.3(±5.1)
Inactive
84.2(±14.6)
63.1(±9.6)
56.3(±11.6)
53.8(±8.9)
11.2(±2.1)
28.6(±4.2)
47.2(±6.6)
168.5(±24.1)
Inactive
66.2(±4.5)
57.3(±0.5)
12.1(±0.9)
42.0(±3.6)
11.3(±1.9)
16.2(±2.1)
52.9(±4.2)
30.5(±5.2)
14.7(±0.9)
72.0(±8.8)
22.2(±4.9)
33.0(±1.8)
Inactive
63.0(±7.2)
88.3(±5.9)
20.2(±2.0)
88.2(±13.3)
77.3(±7.5)
Inactive
Inactive
12j
Inactive
Inactive
12k
12l
Inactive
Inactive
121.8(±18.0)
Inactive
54.9(±7.1)
Inactive
12m
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12n
16.1(±1.2)
10.0(±1.7)
0.04(±4.7)
9.1(±0.9)
26.2(±8.4)
0.2 (±22.1)
13.8(±5.2)
3.2(±0.8)
0.01(±1.8)
Cisplatin
Doxorubicin
Values in parentheses represent the average of 3-4 experiments ± S.E.M.
Table 3. Statistical equations obtained by QSAR analysis.
Cell
Equation
R2
S.Ea
Q2
RMScv
nb
line
HL-60
E1: Y= -1.35(±0.16) HGM -15.24 (±3.2) ISH + 1.34(±0.40)
PJI2+23.0 (±3.05)
0.93 0.09 0.83
0.13
13
a Standard error of regression
b Number of compounds used for QSAR analysis
Table 4. Data of the selected descriptors used in this study and the experimental and predicted values of pIC50 in
HL-60 cell line.
Descriptors
ISH
pIC50
Experimental
Compound
12a
HGM
3.59
3.60
3.40
3.48
3.14
3.10
3.52
3.51
3.31
3.61
3.40
3.47
3.33
PJI2
1.00
1.00
1.00
1.00
1.00
0.90
0.89
1.00
1.00
0.86
0.86
0.86
1.00
Predicted
4.31
4.27
4.75
4.43
5.06
4.82
4.37
4.51
4.78
3.99
4.94
4.37
4.76
1.00
4.18
4.24
4.92
4.38
4.95
4.79
4.28
4.52
4.83
4.14
4.65
4.48
4.86
12b
12c
1.00
0.98
12d
12e
1.00
0.99
12f
1.00
12g
12h
12i
0.99
0.99
0.99
12j
1.00
12k
12l
0.97
0.99
12n
0.99
29