Synthesis and antitumor activity evaluation of some N-heterocycles derived from pyrazolyl-substituted 2(3H)-furanone

Article abstract of DOI:10.1080/00397911.2016.1193755

Pyrazolyl-substituted 2(3H)-furanone was allowed to react with different nitrogen nucleophiles such as hydrazine hydrate, ethylenediamine, ethanolamine, and anthranilic acid to give pyrrolone and benzoxazinone derivatives. The acid hydrazide 3 was reacted with some carbonyl compounds such as 4-chlorobenzaldehyde, chloroacetyl chloride, and acetic anhydride to give thiazolidinone, oxadiazole, and pyridazinone derivatives. Selected examples of the synthesized compounds were evaluated as anticancer agents against three types of carcinoma cell lines (HePG 2, HCT116, and PC3), using Doxorubicin as a reference drug. The result revealed that some of the new compounds showed high activities. Compound 6a was more potent than the standard drug. A docking study using MOE 2008.10 program was performed.

Full text of DOI:10.1080/00397911.2016.1193755

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: http://www.tandfonline.com/loi/lsyc20
Synthesis and Antitumor Activity Evaluation of
Some N-Heterocycles Derived From Pyrazolyl-
Substituted 2(3H)-furanone
Wael S. I. Abou-Elmagd, Ahmed K. EL-Ziaty, Magdy I. El-Zahar, Sayed K.
Ramadan & Ahmed I. Hashem
To cite this article: Wael S. I. Abou-Elmagd, Ahmed K. EL-Ziaty, Magdy I. El-Zahar, Sayed K.
Ramadan & Ahmed I. Hashem (2016): Synthesis and Antitumor Activity Evaluation of Some N-
Heterocycles Derived From Pyrazolyl-Substituted 2(3H)-furanone, Synthetic Communications,
DOI: 10.1080/00397911.2016.1193755
To link to this article: http://dx.doi.org/10.1080/00397911.2016.1193755
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Date: 07 June 2016, At: 01:28

Synthesis and antitumor activity evaluation of some N-heterocycles derived from
pyrazolyl-substituted 2(3H)-furanone
1
1
2
1
Wael S. I. Abou-Elmagd , Ahmed K. EL-Ziaty , Magdy I. Elzahar , Sayed K. Ramadan ,
1
Ahmed I. Hashem
1
Chemistry Department, Faculty of Science, Ain Shams University, Abassia, Cairo,
2
Egypt Therapeutic chemistry department, National Research Center, Dokki, Cairo,
Egypt
Corresponding should be addressed E-mail: waelmagd97@yahoo.com
Abstract
Pyrazolyl-substituted 2(3H)-furanone was allowed to react with different nitrogen
nucleophiles such as hydrazine hydrate, ethylenediamine, ethanolamine and anthranilic
acid to give pyrrolone and benzoxazinone derivatives. The acid hydrazide 3 was reacted
with some carbonyl compounds such as 4-chlorobenzaldehyde, chloroacetyl chloride and
acetic anhydride to give thiazolidinone, oxadiazole, pyridazinone derivatives. Selected
examples of the synthesized compounds were evaluatedas anticancer agents against three
types of carcinoma cell lines (HePG 2, HCT116 and PC3), using Doxorubicin as a
reference drug. The result revealed that some of the new compounds showed high
activities. Compound 6a was more potent than the standard drug. Docking study using
MOE 2008.10 program was performed.
Graphical Abstract
1

KEYWORDS: 2(3H)-furanone, pyrazole, pyrrolones, thiazolidinones, Antitumor
activities
INTRODUCTION
There is a variety of N-heterocycles bearing pyrazole derivatives developed as a new
scaffold of antitumor agents. All are effective against lungs cell carcinoma (A549) and
also showed anti-proliferative activity against human lung cancer cell lines and inhibited
[1-14]
tubular polymerization.
Also, it has been documented that different newly designed
and synthesized pyrazolyl derivatives showed anticancer activity due to their B-Raf
[
15-18]
inhibitory activities with IC50 values in the nano levels as compounds I, II, III, IV.
The utilization of 2(3H)-furanones for the construction of a wide variety of nitrogenous
heterocyclic ring systems of synthetic and biological importance had been a subject of
[
1,19-32]
research concern by our research group.
These diverse biological activities initiated
our interest in utilizing the 2(3H)-furanone 1 bearing a pyrazolyl moiety as side chain for
the synthesis of nitrogen containing heterocycles of anticipated antitumor activities.
RESULTS AND DISCUSSION
2

Synthesis
[
19]
The 2(3H)-furanone 1 was previously prepared by our research group. Fusion of the
0
furanone 1 with ammonium acetate in a sand bath at 150 C gave the pyrrolone derivative
2
. The structure of 2 is substantiated from its spectral data. The IR spectrum shows
disappearance of absorption of the lactone C=O group and the appearance of absorption
-1
band of C=O group for the cyclic amide at 1699cm (cf. experimental part). Treatment of
1
with other nitrogen nucleophiles such as hydrazine hydrate, benzyl amine, anthranilic
acid ethanolamine and ethylenediamine led to the formation of a wide variety of N-
heterocycles (Scheme 1). Thus, refluxing 2 with hydrazine hydrate and benzylamine in
ethanol gave the acid hydrazide and N-benzylamide derivatives 3 and 4 respectively. The
structures of 3 and 4 were illustrated from their spectral data and supported by
comparison with an authentic sample prepared by treatment of furanone 1 with hydrazine
[
16]
hydrate and benzylamine respectively.
Treatment of the 2(3H)-furanone 1 with
anthranilic acid in acetic acid in the presence of anhydrous sodium acetate gave the
benzoxazinone derivative 5. The structure of 5 was elucidated from its analytical and
spectral data (cf. experimental part). The IR spectrum showed disappearance of
absorption of C=O group of the 2(3H)-furanone and the appearance of absorption peaks
-1
for C=O group of benzoxazinone at 1744 cm (cf. experimental part). The assigned
1
structure is supported by H-NMR and mass spectra. The MS spectrum showed the
correct molecular ion peak beside some of abundant peaks (cf. experimental part).
Stirring of 1 with ethanolamine or ethylenediamine in ethanol at room temperature
afforded the amide derivative 6a or 6b respectively. Heating the amide derivative 6a in
3

n-butanol gave the pyrrolone derivative 7. The structures of 6 and 7 were confirmed from
their analytical and spectral data (cf. experimental part). On the other hand, refluxing 1
was ethylenediamine in ethanol/ acetic mixture gave the dipyrrolonyl derivatives 8.
Compound 8 was also obtained by refluxing the pyrrolone 7 with the furanone 1 under
the same reaction conditions. The structures of compounds 6-8 were substantiated from
their spectral and analytical data (cf. experimental part).
The acid hydrazide derivative 3 was treated with some carbonyl compounds to give
oxadiazole, pyridazinone and thiazolidinone derivatives. Thus, when 3 was heated with
4
-chlorobenzaldehyde in ethanol, it gave the Schiff base 9. However, the oxadiazole
derivative 10 was obtained when the reaction was carried in dioxane. The mechanistic
pathway for the formation of the oxadiazole derivative 10 is represented in (Scheme 2).
The Schiff base 9 was easily converted into the thiazolidinone derivative 11 when treated
with thioglycolic acid. On the other hand, chloroacetyl chloride reacted with the acid
hydrazide derivative 3 to give the corresponding pyrrolone derivative 12. Acetic
anhydride was allowed to react with the acid hydrazide 3 under two reaction conditions.
Stirring 3 with acetic anhydride at room temperature gave the diamide derivative 13.
Ring closure of 13 using HCl/AcOH mixture gave the pyridazinone derivative 15.
However, the oxadiazole derivative 14 was obtained by refluxing 3 with acetic anhydride.
The thiosemicarbazide derivative 16 was obtained by treating 3 with phenyl
isothiocyanate. Treatment of 16 with ethyl bromoacetate and maleic anhydride gave the
4

thiazolidinone derivatives 17 and 18 respectively. The mechanistic pathway for the
formation of the thiazolidinone derivatives 18 is represented in (Scheme 3).
The structures of compounds 9–18 were confirmed by the analytical and spectroscopic
1
data. The assigned structures were strongly confirmed from H-NMR and mass spectra
(cf. experimental part). All the foregoing reactions are illustrated by (Scheme 4).
Evaluation Of The Antitumor Activity
Cytotoxic Effect On Human Cell Lines (Hepg 2, HCT116 And PC3)
The pyrazolyl derivatives were examined as anticancer agents against three types of
carcinoma cell lines, Human liver carcinoma (HePG 2), Human colon carcinoma
(
HCT116) and Human prostate cancer (PC3) cell lines, using Doxorubicin as a reference
drug. As a trial to get more effective and less toxic agents, only four compounds (3, 6a,
2, 13) exhibited the desired activity. The biological results showed that the most potent
1
activity was gained upon conjugation of the parent pyrazole heterocycle with amidic long
chain functionality (compound 6a). The obtained activity was higher than that obtained
by the reference drug against the three examined carcinoma cell lines (IC50; 6.25-12.40
µg/ml, IC50Dox; 21.6-37.6 µg/ml) (cf. Tables part).
The reduction in the length of the amidic side chain to form the pyrazolohydrazide
derivative 3 retains the anticancer potency against HCT116 and PC3, that is higher than
that of Doxorubicin (IC50; 14.1, 11.6 µg/ml), while the activity decreased against HePG
2
cell lines to be less than the reference drug (IC50; 49.0 µg/ml).
5

On the other hand, the attachment of two amide groups at pyrazole-C4 (compound 13)
produced a noticeable reduction in the activity against HCT116 carcinoma cell lines
(
IC50; 70.5 µg/ml) and abolishes the activity completely against PC3 cell lines, but
retains the potent anticancer activity against HePG 2 (IC50; 13.9 µg/ml). Cyclization of
the side chain at pyrazole-C4 to form the pyrrole-acetamide nucleus (compound 12) led
to further decrease in the potency against the examined cancer cell lines (IC50; 46.3-76.1
µg/ml).
Molecular Modeling Studies
All the molecular modeling calculations and docking simulation studies were performed
®
[33]
using Molecular Operating Environment (MOE ) 2008.10. All the interaction energies
and different calculations were automatically calculated.
The target compounds were constructed into a 3D model using the builder interface of the
MOE program. After checking their structures and the formal charges on atoms by 2D
depiction, the following steps were carried out: the target compounds were subjected to a
conformational search. All conformers were subjected to energy minimization, all the
minimizations were performed with MOE until a RMSD gradient of 0.01 Kcal/mole and
RMS distance of 0.1 Å with MMFF94X force-field and the partial charges were
automatically calculated. The obtained data base was then saved as MDB file to be used
in the docking calculations.
6

The origin of the selectivity of (1E)-5-(1-piperidin-4-yl-3-pyridin-4-yl-1H- pyrazol-4-yl)-
,3-dihydro-1H-inden-1-one oxime),SM5 (I) for B-Raf seems to be due to interactions
2
with several B-Raf amino acids, as well as the presence of the indane-oxime. In
particular, a Phe583 in the COOH-terminal lobe of B-Raf forms favorable π-stacking
interactions with the pyrazole and pyridine rings of SM5 (I). Furthermore, a Trp531 is
located above the opposing face of the pyridine, forming favorable π-stacking
interactions. Additionally, the hydroxyl group of indane-oxime forms two strong
hydrogen bonds with Lys 483 and Glu501, thus likely contributing to the selectivity and
potency of SM5 (I). (Fig. 1).
The X-ray crystallographic structure of B-Raf receptor complexes with SM5 (PDB ID:
3
D4Q) was obtained from the Protein Data Bank through the internet. The enzyme was
prepared for docking studies by removing the ligand molecule, SM5 from B-Raf receptor
active sites. Hydrogen atoms were added to the system with their standard geometry. The
atoms connection and type were checked for any errors with automatic correction.
Selection of the receptor and its atoms potential were fixed. MOE Alpha Site Finder was
used for the active site search in the enzyme structure using all default items. Dummy
atoms were created from the obtained alpha Spheres. Re-docking of co-crystalline ligand
to the receptor active sites to insure the docking method was efficient and the active
pocket was saved as MOE file to be used for docking simulation of the selected
compounds.
7

Docking of the conformation database of the target compounds was done using MOE-
Dock software. The following methodology was generally applied via loading of the
enzyme active site file and the Dock tool was initiated. The MDB file of the ligand to be
docked was loaded and Dock calculations were run automatically. The obtained poses
were studied and the poses showed best ligand-enzyme interactions were selected and
stored for energy calculations. The 2D interaction and stereo view for compounds 3, 6a
and 13 inside the active site of B-Raf enzyme were obtained and saved as both MOE and
photo files. (cf. Figures. part (Fig. 2, 3 and 4)).
CONCLUSIONS
In this work we designed and synthesized some nitrogen containing heterocycles such as
pyrrolone, benzoxazinone, thiazolidinone, oxadiazole and pyridazinone derivatives, to
use them as anticancer agents against three types of carcinoma cell lines (HePG 2,
HCT116 and PC3), using Doxorubicin as a reference drug. The binding modes and
orientation of the most promising anticancer compounds 3, 6a and 13 at the active site of
the ATP binding site of B-Raf kinase were studied. The result revealed that some of the
new compounds showed high activities. Compound 6a was more potent than the standard
drug.
EXPERIMENTAL PART
Chemistry
Melting points were measured on a Gallen Kamp electric melting point apparatus and are
uncorrected. The infrared spectra were recorded using potassium bromide disks on FTIR
8

Thermo Electron Nicolet 7600 (USA) infrared spectrometer at the Central laboratory of
1
Faculty of Science, Ain shams University. The H-NMR spectra were run at 300 MHz on
a GEMINI 300 BB NMR spectrometer using tetramethyl silane (TMS) as internal
standard in deuterated dimethylsulphoxide (DMSO-d ) at the Main Defense Chemical
6
laboratory. The mass spectra were recorded on a shimadzu GC-MS QP- 1000EX mass
spectrometer operating at 70 ev at the Micro analytical Center of Cairo university. The
reactions were monitored by the thin layer chromatography using Merck Kiesel gel 60
F254 aluminum backed plates. The biological activity was performed at Central
Laboratory for Genetic Engineering, National Research Center, Dokki, Cairo, Egypt.
Action Of Ammonium Acetate On Furanone 1
A mixture of furanone 1 (10 mmol) and ammonium acetate (40 mmol) was heated in a
o
sand bath at 150-160 C for 2 hrs. The reaction mixture was cooled then poured onto
water. The obtained solid was filtered off and dried, recrystallized from ethanol/dioxane
mixture (1:1) to give the pyrrolone 2.
3
-((1,3-Diphenyl-1H-pyrazol-4-yl)methylene)-5-phenyl-1H-pyrrol-2(3H)-one 2
o
Orange crystals; mp.294-296 C. yield 83%. Anal. Calcd.for C H N O (389.15): C,
2
6
19
3
-
8
0.18; H, 4.92; N, 10.79. Found: C, 80.22; H, 4.85; N, 10.71. FTIR (KBr) ( max, cm
1
1
)
:3142 (NH),3059 (aryl-H), 1699 (C=O), 1617 (C=N),755, 686 (δ5H). H-NMR
(
DMSO-d6): δH(ppm) 10.49 (s, 1H, NH, D O-exchangeable), 9.17 (s, 1H, C-
2
9

HPyrazole),8.09-7.39 (m, 15H, Ar-H),7.08 (s, 1H, C-HPyrrolone), 7.00 (s, 1H, =CH). MS (m/z,
+
.
%
): 389 (M ,15), 345 (31), 289 (23), 256 (24), 181 (32), 105 (100), 77 (41), 64(54).
General Procedure For The Reaction Of Furanone 1 With Ethylenediamine And
Ethanolamine
To a solution of furanone 1 (5 mmol) in absolute ethanol or dioxane (20 ml),
ethylenediamine or ethanolamine (5 mmol) was added with stirring. The reaction mixture
was stirred for 10 hrs. at room temperature. The solvent was evaporated under reduced
pressure. The residue was triturated with light petroleum recrystallized from benzene to
give the amide derivatives 6a and 6b, respectively.
N-(2-Aminoethyl)-2-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)-4-oxo-4-
phenylbutanamide 6a
o
Pale yellow crystals, mp. 115-117 C (Light Petroleum), yield 68%.Anal. Calcd.for
C H N O (450.21): C, 74.65; H, 5.82; N, 12.44. Found: C, 74.69; H, 5.76; N, 12.40.
28
26
4
2
-1
FTIR, (KBr) ( max, cm ): 3359, 3290 (NH, NH ), 3058, 3028 (Aryl-H), 2953, 2924
2
1
(
Alkyl-H), 1684,1653 (C=O), 762, 702 (δ5H). H-NMR (DMSO-d6): δH(ppm) 8.70 (s,
1
H, NH, D O-Exchangeable), 8.28 (s, 1H, C-HPyrazole),8.15-7.27 (m, 15H, Ar-H),7.21
2
(
s, 1H, =CH), 3.82 (t, 2H, CH NH, J= 6.5 Hz ), 3.77 (s, 2H, CH CO), 3.45 (t, 2H,
2
2
CH NH , J= 6.3 Hz), 5.21 (br.s, 2H, NH , D O-Exchangeable). MS (m/z, %): 450
2
2
2
2
+
.
(
M ,11), 432 (31), 391 (15), 258 (100), 229 (12), 105 (27), 77 (100), 64(24).
-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)-N-(2-hydroxyethyl)-4-oxo-4-
phenylbutanamide 6b
2
1
0

o
6
b: Yellow crystals, mp. 129-131 C (Light Petroleum), yield 72%.Anal. Calcd.for
C H N O (451.19):C, 74.48; H, 5.58; N, 9.31. Found: C, 74.42; H, 5.63; N, 9.25.
28
25
3
3
-1
FTIR,(KBr) ( max, cm ): 3278 (br. OH), 3060, 3030 (Aryl-H), 2950, 2927 (Alkyl-H),
1
1
677,1648 (C=O), 762, 701 (δ5H). H-NMR (DMSO-d6): δH(ppm) 10.01 (s, 1H, NH,
D O-Exchangeable), 8.32 (s, 1H, C-HPyrazole), 8.01-7.10 (m,16H, Ar-H),4.43 (t, 2H,
2
CH O, J=5.4 Hz), 3.99 (t, 2H, CH N, J= 5.4 Hz), 3.72 (s, 2H, CH CO), 4.87 (br.s, 1H,
2
2
2
+
.
OH, D O-Exchangeable).MS (m/z, %): 452 (M+1, 0.9),451 (M ,10), 433 (M-.OH, 70),
2
4
02 (22), 286 (6), 231 (30), 167 (9), 104 (40), 77 (71), 64(100), 55 (17).
SUPPLEMENTAL MATERIAL
(Full experimental detail, tables, figures and results of biological testing) This material
can be found as supplemental data on the publisher’s website.
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3
1
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1
6

Table 1 Cytotoxic effect on human cell lines (HePG 2, HCT116and PC3)
Sample Code
HePG 2
HCT116
LC50
PC3
LC50
LC90 (µg/ml)
LC90 (µg/ml) LC50
LC90
(µg/ml)
21.0
(
µg/ml)
(µg/ml)
14.1
(µg/ml)
11.6
3
5
49.0
86.1
22.5
--------
--------
--------
7
--------
12.4
6
a
100% up to
100% up to
6.5 ppm
6
.25ppm
7
8
1
1
1
1
1
1
1
--------
--------
--------
46.3
--------
--------
--------
74.5
79.6
132.8
--------
--------
70.6
--------
--------
119.2
0
2
3
4
5
6
8
76.1
70.5
125.4
114.9
13.9
25.2
--------
--------
--------
--------
--------
--------
--------
23.8
--------
--------
--------
--------
--------
--------
--------
--------
--------
--------
--------
--------
--------
--------
--------
--------
--------
--------
DMSO
--------
--------
37.6
--------
--------
Negative control --------
Doxorubicin 21.6
LC : Lethal concentration of the sample which causes the death of 50% of cells in 48 hrs
5
0
LC : Lethal concentration of the sample which causes the death of 90% of cells in 48
9
0
hrs)
1
7

Table 2 Docking results of the compounds 3, 6a and 13 with B-Raf kinase in comparison
with the ligand SM5 using MOE software version 2008.10.
Compd.
NO.
Docking score Amino acid residues
Atoms of compound
Type of bond
o
(Kcal/mol)
-6.38
(bond length A )
Cys532(2.7);
Glu501(2.1);
Lys483(2.4);
Trp531;
SM5
N(pyridine);
OH;
H-don
H-don
OH;
H-don
Pyridine;
Pyridine, pyrazole
Arene-Arene
Arene-Arene
H-acc, H-don
H-acc
Phe583
3
-7.24
-6.32
-8.43
Ser465(2.7), (2.9);
Ser535(2.7);
Ser536(3);
Trp531;
N(NH );
2
O(COPh);
O(COPh);
3-Phenyl;
3-Phenyl
O(COPh);
O(COPh);
H(NH2);
H-acc
Arene-Arene
Arene-Arene
H-acc
Phe583
6
a
Ser535(2.7);
Ser536(2.9);
Asn581(3.2),
Asp594(1.7);
Phe583
H-acc
H-don
H(NH2);
H-don
3-Phenyl
O(COPh);
O(COCH3);
O(COPh);
3-Phenyl
Arene-Arene
H-acc
1
3
Ser535(3);
Ser535(2.8);
Ser536(3);
Phe583
H-acc
H-acc
Arene-Arene
1
8

Scheme 1.
1
9

Scheme 2. (Formation of oxadiazole 10)
2
0

Scheme 3. (Formation of thiazolidinone derivative 18)
2
1

Scheme 4.
2
2

Fig. 1. The proposed binding mode of compound 3 docked in the active site of B-Raf; A
and B showing 2D and 3D ligand-receptor interactions (hydrogen bonds are illustrated as
arrows; C atoms are colored gray, N blue and O red).
2
3

Fig. 2. The proposed binding mode of compound 6a docked in the active site of B-Raf; A
and B showing 2D and 3D ligand-receptor interactions (hydrogen bonds are illustrated as
arrows; C atoms are colored gray, N blue and O red).
2
4

Fig. 3. The proposed binding mode of compound 13 docked in the active site of B-Raf; A
and B showing 2D and 3D ligand-receptor interactions (hydrogen bonds are illustrated as
arrows; C atoms are colored gray, N blue and O red).
2
5