Design, synthesis and pharmacophoric model building of novel substituted nicotinic acid hydrazones with potential antiproliferative activity

Article abstract of DOI:10.1007/s12272-012-0904-2

Novel 6-aryl-2-methylnicotinic acid hydrazides 4a-c and their corresponding hydrazones 5a-c and 6a-i were synthesized. X-ray single crystal diffraction of 6h confirmed the chemical structure of hydrazones 6a-i. Antiproliferative activity of the synthetic compounds was investigated against K562 leukemia cell lines. Variable cell growth inhibitory activities were obtained with IC ₅₀ range from 24.99 to 66.78 μM where the compound 6c exhibited the maximum activity. Structure activity relationship analysis has been performed and a common pharmacophore model for the synthesized derivatives has been obtained by using the pharmacophore elucidation module of the software MOE. The best model obtained is characterized by two projected locations of potential H-bond donors (F3 and F4) and two Aromatic annotations (F1 and F2).

Full text of DOI:10.1007/s12272-012-0904-2

Arch Pharm Res Vol 35, No 9, 1543-1552, 2012
DOI 10.1007/s12272-012-0904-2
Design, Synthesis and Pharmacophoric Model Building of Novel
Substituted Nicotinic Acid Hydrazones with Potential Antiprolifera-
tive Activity
Hatem A. Abdel-Aziz1,5, Tarek Aboul-Fadl1,§, Abdul-Rahman M. Al-Obaid1, Mohamed Ghazzali2,
Abdullah Al-Dhfyan3, and Alessandro Contini4
1Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia,
3
2Department of Chemistry, College of Science, King Saud University, Riyadh 11451, Saudi Arabia, Stem Cell Therapy Pro-
4
gram, King Faisal Specialized Hospital and Research Center, Riyadh 11211, Saudi Arabia, Dipartimento di Scienze Farma-
ceutiche - Sezione di Chimica Generale e Organica “A. Marchesini”, Università degli Studi di Milano, via Venezian 21,
5
20133 Milano, Italy, and Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt
(Received December 20, 2011/Revised March 2, 2012/Accepted May 1, 2012)
Novel 6-aryl-2-methylnicotinic acid hydrazides 4a
-c and their corresponding hydrazones 5a-c
and 6a-i were synthesized. X-ray single crystal diffraction of 6h confirmed the chemical struc-
ture of hydrazones 6a-i. Antiproliferative activity of the synthetic compounds was investigated
against K562 leukemia cell lines. Variable cell growth inhibitory activities were obtained with
IC50 range from 24.99 to 66.78 µM where the compound 6c exhibited the maximum activity.
Structure activity relationship analysis has been performed and a common pharmacophore
model for the synthesized derivatives has been obtained by using the pharmacophore elucida-
tion module of the software MOE. The best model obtained is characterized by two projected
locations of potential H-bond donors (F3 and F4) and two Aromatic annotations (F1 and F2).
Key words: Nicotinic acid hydrazones, X-ray single crystal diffraction, K562 Leukemia cell
line, Antiproliferative, Molecular modeling, Pharmacophore model building
apy, succeed only when malignant cells are confined
to the treated area. The use of chemotherapy is there-
INTRODUCTION
Cancer is one of the most dreaded diseases of man- fore essential for the systemic treatment of metastases
kind; it is a leading cause of death throughout the world accompanying the local and regional growth of tumors
(Ahmedin et al., 2008). More than 10 million new cancer (Skeel et al., 2007). Metastases are the primary cause
cases occur annually, roughly half of which is in the of death from cancer. The anticancer drugs used in
developed countries, and the disease causes over 6 chemotherapy are systemic antiproliferative agents
million deaths a year (Cozzi et al., 2004; Sinha et al., that preferentially kill those cells that are dividing.
2004). Unlimited and uncontrolled cell proliferation is These cytotoxic agents are antimetabolites, alkylating
an obvious characteristic of tumor cells (Rew et al., agents, DNA-complexing agents, mitosis inhibitors or
2000). Current cancer treatment strategies employ a hormones, and they exert their activity by interfering
combination of surgery, radiation, and chemotherapy. with some aspect of DNA replication, repair, transla-
The localized therapies, such as surgery and radiother- tion or cell division. They mainly rely on enhanced
proliferative rate of cancer cells and therefore, are not
truly selective for cancer cells. The prolonged use of
chemotherapy results in lethal damage to proliferat-
ing non-cancerous cells and this is particularly true in
the treatment of solid tumors, where the majority of the
tumor cells do not divide rapidly (Skeel et al., 2007). It
has been observed that the use of cytotoxins in patients
§Address on leave: Faculty of Pharmacy, Assiut University,
Assiut 71526, Egypt
Correspondence to: Tarek Aboul-Fadl, Department of Pharma-
ceutical Chemistry, College of Pharmacy, King Saud University,
P.O. Box 2457, Riyadh 11451, Saudi Arabia
Tel: 966-1-467-7341, Fax: 966-1-467-6220
E-mail: fadl@ksu.edu.sa
1543

1544
H. A. Abdel-Aziz et al.
with appreciable tumor burdens leads to remissions of electrothermal melting point apparatus (Stuart Scien-
limited duration and varying degrees, followed by re- tific), and are uncorrected. Precoated silica gel plates
growth and spread of more malignant and multi-drug (kiesel gel 0.25 mm, 60G F254, Merck) were used for
resistant forms of cancer (Gottesman et al., 2002).
thin layer chromatography. Developing solvent system
Despite several decades of intensive research, the of chloroform-methanol (10:3) was used and the spots
long-term outlook for patients with aggressive cancer were detected by ultraviolet light. IR spectra (KBr disc)
remains discouraging, and there is a need for innovative were recorded on a Perkin Elmer FT-IR spectrophoto-
approaches towards the design of anticancer drugs with meter at the research center, college of Pharmacy, King
reduced toxicity and improved therapeutic indices Saud University, Saudi Arabia. 1H-NMR Spectra were
(Chabner et al., 2005; Kamb et al., 2007). Due to its scanned in DMSO-d6 on a Jeol NMR spectrophotometer
synthetic and biological versatility, in recent years operating at 400 MHz for 1H and 100 MHz for 13C, at
investigations on compounds containing hydrazide or the College of Science, King Saud University, Saudi
hydrazone moieties are attractive target compounds Arabia. Chemical shifts are expressed in d-values (ppm)
for new drug development, because of their potentially relative to TMS as an internal standard. Exchangeable
biological activities involving antiproliferative activities protons were confirmed by an addition of drops of D2O.
(Kamal et al., 2007; Onnis et al., 2009). Several studies Mass spectra were taken on a Varian 320 - MS spectro-
have been devoted to the antiproliferative activity of meter at the research center, College of Pharmacy, King
aroylhydrazone derivatives (Easmon et al., 2006; Putt Saud University, Saudi Arabia. Mass spectral data
et al 2006; Vogel et al., 2008; Xia et al., 2008; Onnis et al., are given as m/z (Intensity %). Diffraction data were
2009). It has been suggested that the antiproliferative collected using a Bruker SMART APEX diffractometer
activity of these hydrazones may be attributed to in- equipped by CCD detector utilizing Mo-Kα radiation (
hibition of kinases (Hellmuth et al., 2008; Xu et al., = 0.71073 Å) with graphite monochromator. The data
2008; Horiuchi et al 2009), generation of radicals, and were collected using
-scans at a temperature of 294 ±
dissipation of the mitochondrial membrane poten- 2 K to a maximum 2
of 55.0o. Antiproliferative activity
tial (Hofmann et al., 2009).
was examined at the Stem Cell Therapy Program, King
λ
ω
θ
On the other hand, synthesis of the pyridine ring Faisal Specialized Hospital and Research Center, Riyadh,
system and its derivatives occupy an important place Saudi Arabia.
in the realm of natural and synthetic organic chemistry;
due to their therapeutic and pharmacological properties,
they have emerged as integral backbones of over 7000
existing drugs (Henry et al., 2004).
Chemistry
General procedure for synthesis of 6-aryl-2-
methylnicotinohydrazide 4a-c
In the light of previous information and in continu-
A mixture of the appropriate ester 3a-c (10 mmol)
ation of our interest in the biologically active hydrazides and hydrazine hydrate (1.5 mL, 99%) was refluxed for
and hydrazones (Aboul-Fadl et al., 2003, 2010, 2011a, 6 h, then left to cool at room temperature. The separated
2011b, 2011c; Dawood et al., 2006; Abdel-Aziz et al., white solid was filtered off, and recrystallized from
2007, 2009a, 2009b, 2010; Abdel-Wahab et al., 2008, EtOH to afford the corresponding 6-aryl-2-methylnico-
2009; Abdel-Aziz and Gamal-Eldeen, 2009), the current tinohydrazides 4a
-c, respectively.
work report the synthesis and antiproliferative activity
of hydrazone derivatives of some novel 6-aryl-2-methyl- 2-Methyl-6-phenylnicotinohydrazide (4a)
nicotinic acid hydrazides. In addition, a pharmacophore White powder; yield (82%); mp 138-140oC. IR (KBr):
model for the synthesized hydrazones was derived from 3296-3193 (NH, NH2), 1639 (C=O), 1585 (C=N). 1H-NMR
their structural features and their evaluated antipro- (DMSO-d6): 2.62 (s, 3H, CH3), 4.56 (s, 2H, NH2, D2O-
liferative activity using the Molecular Operating En- exchangeable), 7.45-7.52 (m, 3H, ArH), 7.77 (d, 1H,
J =
vironment (MOE) software package.
MATERIALS AND METHODS
General
8.04 Hz, H of C4 in pyridine), 7.83 (d, 1H, J = 8.08 Hz, H
of C5 in pyridine), 8.90-8.12 (m, 2H, ArH), 9.64 (s, 1H,
NH, D2O-exchangeable). 13C-NMR (DMSO-d6): 23.58
(-CH3), 117.58, 127.22, 129.34, 129.72, 129.90, 13698,
138.59, 156.17, 156.36, 167.79 (-C=O); MS m/z [%]: 227
Acetophenones, ethyl 3-oxobutanoate, pentane-2,4- [M+, 9.44], 196 [100], 168 [27.45], 141 [36.08], 115 [15.39].
dione, hydrazine hydrate, DMF-DMA, isatins, and piper-
onaldehyde were purchased from Sigma-Aldrich Com- 6-(4-Chlorophenyl)-2-methylnicotinohydrazide (4b)
pany. All other chemicals were of commercially available White powder; yield (86%); mp 220-222oC. IR (KBr):
1
research-grade. Melting points were determined on an 3322-3195 (NH, NH2), 1643 (C=O), 1587 (C=N). H-

Antiproliferative Activity of Nicotinic Acid Hydrazones
1545
NMR (DMSO-d6): 2.60 (s, 3H, CH3), 4.54 (s, 2H, NH2, (C=N). 1H-NMR (DMSO-d6): 2.65 (s, 3H, CH3), 6.00 and
D2O-exchangeable), 7.56 (d, 2H, = 8.80 Hz, ArH), 6.11 (2s, -OCH2O-, cis and trans isomers); 6.85-8.23 (m,
7.78 (d, 1H, = 8.08 Hz, H of C4 in pyridine), 7.86 (d, 9H, ArH and 1H, -CH=N-), 11.84 and 11.86 (2br. s, 1H,
1H, = 8.08 Hz, H of C5 in pyridine), 8.14 (d, 2H,
NH, D2O-exchangeable, cis and trans isomers). 13C-
J
J
J
J =
8.80 Hz, ArH), 9.63 (s, 1H, NH, D2O-exchangeable). NMR (DMSO-d6): 23.56 (-CH3), 102.18, 105.33, 105.72,
13C-NMR (DMSO-d6): 23.56 (-CH3), 117.62, 128.97, 109.06, 117.68, 124.07, 129.01, 129.07, 129.40, 134.92,
129.37, 130.02, 134.77, 137.10, 137.34, 155.01, 156.27, 137.28, 137.43, 144.54, 148.26, 148.58, 149.80, 155.42,
167.63 (-C=O). MS m/z [%]: 261 [M+, 7.30], 230 [69.16], 156.52, 164.30. MS m/z [%]: 394 [M+, 0.16], 207 [100],
207 [68.47], 167 [60.04], 140 [60.09], 78 [68.98], 73 73 [32.21].
[64.84], 63 [100].
N'-(Benzo[d][1,3]dioxol-5-ylmethylene)-2-methyl-
2-Methyl-6-(4-tolyl)nicotinohydrazide (4c)
6-(4-tolyl)nicotinohydrazide (5c)
White powder; yield (75%); mp 178-180oC. IR (KBr): White fibers; yield (72%); the ratio of cis/trans: 65/35;
3297-3195 (NH, NH2), 1637 (C=O), 1587 (C=N). H- mp 221-223oC. IR (KBr): 3219 (NH), 1648 (C=O), 1586
1
NMR (DMSO-d6): 2.36 (s, 3H, CH3), 2.60 (s, 3H, CH3), (C=N). 1H-NMR (DMSO-d6): 2.37 (s, 3H, CH3), 2.65 (s,
4.54 (s, 2H, NH2, D2O-exchangeable), 7.30 (d, 2H,
8.04 Hz, ArH), 7.74 (d, 1H, = 8.08 Hz, H of C4 in conformers); 6.86-8.24 (m,9H, ArH and 1H, -CH=N-),
pyridine), 7.89 (d, 1H, = 8.08 Hz, H of C5 in pyridine), 11.82 and 11.88 (2br. s, 1H, NH, D2O-exchangeable,
8.00 (d, 2H,
J
=
3H, CH3), 6.00 and 6.11 (2s, -OCH2O-, cis and trans
J
J
J
= 8.08 Hz, ArH), 9.60 (s, 1H, NH, D2O- cis and trans conformers); 13C-NMR (DMSO-d6): 21.42
exchangeable). 13C-NMR (DMSO-d6): 21.41 (-CH3), 23.61 (-CH3), 23.62 (-CH3), 102.03, 102.17, 105.37, 105.71,
(-CH3), 117.15, 127.12, 129.37, 129.93, 135.83, 136.91, 109.05, 116.81, 117.21, 123.23, 124.05, 127.15, 127.22,
139.50, 156.05, 156.33, 167.82 (-C=O). MS m/z [%]: 129.11, 129.15, 129.96, 135.77, 135.90, 137.22, 137.36,
241 [M+, 10.80], 210 [100], 207 [40.73], 167 [27.81], 139.45, 139.68, 144.41, 148.15, 148.35, 148.58, 149.78,
155 [19.96], 73 [24.57].
154.94, 156.04, 156.33, 156.76, 164.49, 170.47. MS m/
z [%]: 373 [M+, 0.90], 207 [100], 91 [95.09], 73 [48.19].
General procedure for synthesis of N'-(benzo
[d][1,3]dioxol-5-ylmethylene)-2-methyl-6-arylni- General procedure for synthesis of (
cotinohydrazide 5a-c N'-(2-oxoindolin-5-substituted-3-ylidene)-6-aryl-
A mixture of the appropriate hydrazide 4a
and piperonaldehyde (0.15 g, 1 mmol) in ethanol (50
Z)-2-methyl-
-c (1 mmol)
ylnicotinohydrazide 6a-i
A mixture of the appropriate isatin derivative and
mL) was refluxed for 6 h. After cooling, the solid product the hydrazides 4a-c in ethanol in presence of catalytic
was collected by filtration, washed with ethanol and amount of glacial acetic acid was refluxed for 8 h.
dried. Recrystallization from the ethanol afforded the After cooling, the resulting solid was recrystallized
corresponding hydrazones 5a
-
c
, respectively.
from EtOH-DMF to afford the corresponding isatin
hydrazones 6a
-i.
N'-(Benzo[d][1,3]dioxol-5-ylmethylene)-2-methyl-
6-phenylnicotinohydrazide (5a)
(Z)-2-Methyl-N'-(2-oxoindolin-3-ylidene)-6-phenyl-
White fibers; yield (76%); the ratio of cis/trans: 72/28; nicotinohydrazide (6a)
mp 220-222oC. IR (KBr): 3221 (NH), 1647 (C=O), 1591 Yellow crystalls; yield (77%); mp 288-290oC. IR (KBr):
(C=N). 1H-NMR (DMSO-d6): 2.66 (s, 3H, CH3), 6.00 and 3169 (2NH), 1705, 1677 (2C=O), 1618 (C=N). 1H-NMR
6.12 (2s, -OCH2O-, cis and trans isomers); 6.86-8.24 (m, (DMSO-d6): 2.70 (s, 3H, CH3), 6.95-8.18 (m, 11H, ArH
10H, ArH and 1H, -CH=N-), 11.84 and 11.88 (2br. s, 1H, and 1H, NH), 11.45 (br. s, 1H, NH, D2O-exchangeable).
NH, D2O-exchangeable, cis and trans conformers). 13C- 13C-NMR (DMSO-d6): 23.97 (-CH3), 111.80, 120.25,
NMR (DMSO-d6): 23.59 (-CH3), 102.04, 102.17, 105.71, 123.32, 127.48, 127.61, 129.41, 130.32, 130.50, 132.51,
109.06, 117.65, 124.07, 127.26, 127.33, 129.08, 129.37, 138.27, 143.14, 178.94. MS m/z [%]: 356 [M+, 1.38], 281
129.50, 137.30, 137.39, 138.53, 148.20, 148.58, 149.42, [29.05], 207 [100], 73 [45.72].
149.80, 156.40, 156.76, 164.45. MS m/z [%]: 359 [M+,
0.30], 207 [100], 73 [44.09].
(Z)-2-Methyl-N'-(2-oxoindolin-5-chloro-3-ylidene)-
6-phenylnicotinohydrazide (6b)
N'-(Benzo[d][1,3]dioxol-5-ylmethylene)-2-methyl- Yellow powder; yield (79%); mp 302-304oC. IR (KBr):
6-(4-chlorophenyl)nicotinohydrazide (5b)
3265 (2NH), 1701, 1672 (2C=O), 1624 (C=N). 1H-NMR
White fibers; yield (78%); the ratio of cis/trans: 70/30; (DMSO-d6): 2.70 (s, 3H, CH3), 6.96-8.18 (m, 10H, ArH
mp 240-242oC. IR (KBr): 3160 (NH), 1684 (C=O), 1589 and 1H, NH), 11.36 (br. s, 1H, NH, D2O-exchangeable);

1546
H. A. Abdel-Aziz et al.
1
13C-NMR (DMSO-d6): 24.00 (-CH3), 113.36, 122.00, Yellow powder; yield (72%); mp 310-312oC. H-NMR
127.35, 127.50, 129.41, 130.34, 131.85, 141.84, 179.00. (DMSO-d6): 2.38 (s, 3H, CH3), 2.68 (s, 3H, CH3), 6.96-
MS m/z [%]: 390 [M+, 0.45], 207 [100], 91 [83.26], 63 8.09 (m, 9H, ArH and 1H, NH), 11.35 (br. s, 1H, NH,
[22.31].
D2O-exchangeable). 13C-NMR (DMSO-d6): 21.47 (-CH3),
24.02 (-CH3), 113.38, 117.45, 122.04, 126.00, 127.141,
130.02, 131.84, 135.45, 140.06, 141.95. MS m/z [%]:
404 [M+, 1.23], 281 [36.48], 207 [100], 73 [45.08]. The
(Z)-2-Methyl-N'-(2-oxoindolin-5-bromo-3-ylidene)-
6-phenylnicotinohydrazide (6c)
Yellow powder; yield (74%); mp 301-303oC. IR (KBr): bright yellow single crystal of 6 h was cultured from
3215 (2NH), 1703, 1680 (2C=O), 1617 (C=N). 1H-NMR EtOH-DMF (V/V = 4:1) by slow evaporation at room
(DMSO-d6): 2.70 (s, 3H, CH3), 6.91-8.18 (m, 10H, ArH temperature. Crystal data for compound 6h: Molecular
and 1H, NH), 11.42 (br. s, 1H, NH, D2O-exchangeable). formula C22H17ClN4O2, Formula weight: 404.85, Mono-
13C-NMR (DMSO-d6): 24.00 (-CH3), 113.81, 114.97, clinic, P21/a,
122.40, 127.35, 127.50, 129.41, 130.34, 134.64, 138.22, (11) Å,
142.21, 162.00. MS m/z [%]: 435 [M+, 1.29], 281 [23.60], cm−3, yellow block with 0.2
207 [100], 73 [36.74]. 35428 reflections were measured, of which 4500 were
independent. Rint = 0.086, Dataset (h; k; l) = 13; 13;
17:17;
18:18. Refinement of F2, against all reflections,
led to R( ) [ > 2
)] = 0.0655, Rw(F2) = 0.1734, S =
a
= 10.3096(9),
= 97.538(2)o, V = 1969.0(3)Å3, Dcalc = 1.366 g
0.2 0.1 mm. A total of
b = 13.4072(11), c = 14.3696
β
×
×
−
(
Z
)-6-(4-Chlorophenyl)-2-methyl-N'-(2-oxoindolin-
−
−
F
3-ylidene)nicotinohydrazide (6d)
I
σ (I
Yellow powder; yield (79%); mp 303-305oC. H-NMR 1.05, min & max Residual density (e/Å3) = 0.31, 0.36.
(DMSO-d6): 2.70 (s, 3H, CH3), 6.95-8.22 (m, 10H, ArH Crystallographic data for the structure 6h has been
and 1H, NH), 11.35 (br. s, 1H, NH, D2O-exchangeable). deposited with the Cambridge Crystallographic Data
MS m/z [%]: 390 [M+, 0.88], 281 [6.05], 207 [100], 73 Centre (CCDC) under the number 823366. Copies of
1
[39.61].
the data can be obtained, free of charge, on application
to CCDC 12 Union Road, Cambridge CB2 1EZ, UK.
(Z)-6-(4-Chlorophenyl)-2-methyl-N'-(2-oxoindolin-
5-chloro-3-ylidene)nicotinohydrazide (6e)
(
Z
)-2-Methyl-N'-(2-oxoindolin-5-bromo-3-ylidene)-
Yellow powder; yield (82%); mp 338-340oC. H-NMR 6-(4-tolyl)nicotinohydrazide (6i)
(DMSO-d6):
2.70 (s, 3H, CH3), 6.97-8.22 (m, 9H, ArH Yellow powder; yield (75%); mp 323-325oC. H-NMR
1
1
δ
and 1H, NH), 11.38 (br. s, 1H, NH, D2O-exchangeable). (DMSO-d6): 2.38 (s, 3H, CH3), 2.69 (s, 3H, CH3), 6.92-
MS m/z [%]: 425 [M+, 0.28], 281 [23.74], 207 [100], 73 8.09 (m, 9H, ArH and 1H, NH), 11.37 (br. s, 1H, NH,
[49.72].
D2O-exchangeable). MS m/z [%]: 449 [M+, 1.67], 207
[100], 73 [46.57].
(Z)-6-(4-Chlorophenyl)-2-methyl-N'-(2-oxoindolin-
5-bromo-3-ylidene)nicotinohydrazide (6f)
(E)-3-(Benzo[d][1,3]dioxol-5-yl)-1-(2-methyl-6-
phenylpyridin-3-yl)prop-2-en-1-one (8)
1
Yellow powder; yield (85%); mp 323-325oC. H-NMR
(DMSO-d6): 2.69 (s, 3H, CH3), 6.91-8.22 (m, 9H, ArH
To a stirred solution of pyridine 7 (0.22 g, 1 mmol)
and 1H, NH), 11.37 (br. s, 1H, NH, D2O-exchangeable). and piperonaldehyde (0.15 g, 1 mmol) in ethanol (30
MS m/z [%]: 469 [M+, 1.19], 281 [50.32], 207 [100], 78 mL), 10% aqueous sodium hydroxide (5 mL) was added
[72.43], 73 [78.24].
)-2-Methyl-N'-(2-oxoindolin-3-ylidene)-6-(4-tolyl)
portion-wise at room temperature for 10 min, the reac-
tion mixture was stirred further for 12 h. The result-
ing solid was filtered off, washed with water, dried,
(Z
nicotinohydrazide (6g)
and crystallized from EtOH to afford chalcone 8, as
1
Yellow powder; yield (70%); mp 280-282oC. H-NMR pale yellow powder, in 78% yield; mp 113-115oC; IR
1
(DMSO-d6): 2.38 (s, 3H, CH3), 2.69 (s, 3H, CH3), 6.94- (KBr) 1661 (C=O), 1635 (C=N). H-NMR (DMSO-d6):
8.11 (m, 10H, ArH and 1H, NH), 11.41 (br. s, 1H, NH, 2.59 (s, 3H, CH3), 6.11 (s, 2H, CH2), 6.99-8.18 (m, 12H,
D2O-exchangeable). 13C-NMR (DMSO-d6): 21.47 (-CH3), ArH). 13C-NMR (DMSO-d6): 24.42 (-CH3), 102.28, 107.56,
24.01 (-CH3), 111.79, 117.52, 120.27, 121.45, 121.57, 109.13, 117.74, 124.33, 126.65, 127.44, 129.38, 130.19,
123.30, 127.11, 127.22, 127.38, 130.01, 132.45, 134.46, 133.04, 138.14, 138.39, 145.87, 148.70, 150.36, 156.90,
135.48, 137.23, 139.70, 140.02, 143.13, 157.54, 163.31. 157.02, 193.90. MS m/z [%]: 343 [M+, 1.69], 281 [40.42],
MS m/z [%]: 370 [M+, 0.12], 207 [100], 73 [42.26].
207 [100], 73 [42.42].
(Z)-2-Methyl-N'-(2-oxoindolin-5-chloro-3-ylidene)-
Cell proliferation analysis
6-(4-tolyl)nicotinohydrazide (6h)
K562 chronic myelogenous leukemia cells were pur-

Antiproliferative Activity of Nicotinic Acid Hydrazones
1547
chased from the American Type Culture Collection.
Cells were maintained in RPMI 1640 (Sigma), supple-
mented with 10% FCS (Cambrex Bio Science), 100 IU/
mL penicillin, 100 mg/mL streptomycin and 2 mmol/l
L-glutamine (Sigma). Cells were seeded into 96-well
plates at 0.4
×
104/well and incubated overnight. The
medium was replaced with fresh one containing the
desired concentrations of the compounds. After 48 h,
10 µL of the WST-1 reagent was added to each well
and the plates were reincubated for 4 h at 37oC. The
amount of formazan was quantified using ELISA reader
at 450 nm.
RESULTS
Chemistry
6-Aryl-2-methylnicotinohydrazides 4a
not been reported hitherto, were prepared by the
reaction of esters 3a (Kantevari et al., 2007) with
-c, which had
-c
Scheme 2. Reagents and conditions: a) piperonaldehyde,
EtOH, reflux 6 h, 72-78%; b) Isatin or 5-chloro/bromo isatin,
EtOH-AcOH, reflux 8 h, 70-85%.
hydrazine hydrate (Scheme 1). Their IR spectra showed,
in each case, the appearance of absorption bands due
do NH2 and NH functions in the region 3297-3193 cm−1
in addition to the carbonyl absorption bands in the
1
region 1643-1637 cm− . Their mass spectra showed the region 3221-3160 cm−1 and the band of carbonyl
peaks corresponding to their molecular ions.
Next, treatment of compounds 4a with piperonal-
dehyde in refluxing ethanol yielded the corresponding priate isatin, in the presence of catalytic amount of
function in the region 1684-1647 cm−1.
The treatment of hydrazides 4a with the appro-
-c
-c
hydrazones 5a-c (Scheme 2). The compounds contain- acetic acid, afforded the hydrazones 6a-i, respectively.
ing arylidene–hydrazide structure may exist as E/Z The 1H-NMR spectra of the latter hydrazones showed
geometrical isomers about –C=N– double bond and two D2O-exchangeable signals and their mass spectra
cis/trans amide conformers (Demirbas et al., 2004). revealed, in each case, a peak corresponding to the mole-
Compounds 5a
form of geometrical
with cis/trans amide conformers. H NMR spectra of
hydrazones 5a
due to -CH=N- proton and D2O-exchangeable NH signal with the 6h ellipsoidal presentation are displayed in
cf. Experimental part). The IR spectra of hydrazones Fig. 1. Hydrogen bonding scheme and geometries of
5a showed the appearance of NH absorption band in hydrazone 6h are given in Fig. 2.
-c were present in DMSO-d6 in the cular ion (Scheme 2).
E
isomer about –C=N– bond and
X-Ray crystallography of hydrazone 6h showed the
configuration of –C=N- group in solid state. The
1
Z
-c revealed the appearance of signal important bond distances, angles and torsion angles
(
-c
Scheme 1. Reagents and conditions: (
free, NH2NH2 H2O, reflux 6 h, 75-86%.
A) AcNH4, EAA, reflux 2.5 h, 84-95%; (B) AcNH4, EAA, reflux 4 h, 75%; (C) solvent
·

1548
H. A. Abdel-Aziz et al.
Fig. 1. The thermal ellipsoids (50% probability level) and labelling scheme of 3e. Hydrogen atoms are drawn as spheres of
arbitrary radius. Selected bond distances: Cl1-C2: 1.746(3) Å; O1-C8: 1.232(3) Å; O2-C9: 1.220(3) Å; N1-C5: 1.412(3) Å; N1-
C8:1.358(4) Å; N2-N3: 1.375(3) Å; N2-C7: 1.292(3) Å; N3-C9:1.362(4) Å; N4-C11: 1.335(4) Å. Selected bond angles: C5-N1-C8:
110.9(2)o, N3-N2-C7: 115.2(2)o, N2-N3-C9: 118.6(2)o, C11-N4-C13: 119.9(2)o, Cl1-C2-C1:118.6(2)o, Cl1-C2-C3:119.4(2)o, N1-C5-
C4: 128.2(2)o, N1-C5-C6: 110.1(2)o. Selected torsion angle; C7-N2-N3-C9: -168.15(2)o, C8-C7-N2-N3; -1.910(4)o. The molecule is
inclined by the N2-N3-C9 spacer, with best fit planes dihedral angle of 55.101(3)o.
In an attempt to understanding the potentiality of the cell growth inhibition assay against K562 chronic
5a
light on their activity, propenone
Thus, the reaction of 1-(2-methyl-6-phenylpyridin-3- was conducted by the use of WST-1 reagent for the de-
yl)ethanone ( ) with piperonaldehyde in ethanol con- termination of IC50 values, results are given in Table I.
taining 10% aqueous sodium hydroxide affording the Variable cell growth inhibitory activities were obtained
propenone (Scheme 3). with IC50 range from 24.99 to 66.78 M where com-
-c
in antiproliferative investigation and to shed some myelogenous leukemia cells. The general in vitro anti-
8
was synthesized. proliferative evaluation of the synthesized compounds
7
8
µ
pound 6c revealed the maximum activity.
It is hard to find a constant activity pattern that
Antiproliferative activity
Antiproliferative activity was measured in vitro by distinguishes between the synthesized compounds. In
addition, the effect of the variations of -substituent
N
Table I. Antiproliferative activity of the synthesized
compounds against K562 chronic myelogenous leukemia
cells
a
a
Compd.
IC50
(µM)
Compd.
IC50 (µM)
4a
4b
4c
5a
5b
5c
6a
6b
29.58 ± .060
25.54 ± .007
> 100
36.74 ± .020
> 100
50.16 ± .030
> 100
66.78 ± .100
6c
6d
6e
6f
6g
6h
6i
24.99 ± .050
> 100
26.97 ± .080
> 100
> 100
51.36 ± .030
79.54 ± .017
> 100
8
Fig. 2. Presentation of the structure in 6h in the a-axis,
showing the intermolecular N ···O and C ···O hydrogen
bonding. Hydrogen bonds are represented as dotted lines.
Hydrogen bonding geometry: D ···A; H···A Å: D A Å: D
···Ao: N1 H1A···O2ii; 1.95, 2.786(3), 139: C1 H1···O1i; 2.44,
3.360(3), 173: C4—H4···O2ii; 2.59, 3.230(3), 127.
aIC50: concentration of the compound (
µ
M) producing 50%
−
H
−H
cell growth inhibition after 48 h of compound exposure, as
determined by the WST-1 assay. Each experiment was run
a minimum of two times, and the results are presented as
average values ± S.D.
−H
−
−
H
−
−
Scheme 3. Reagents and conditions: a) piperonaldehyde, EtOH, 10% NaOH, r.t., stirring 12 h, 78%.

Antiproliferative Activity of Nicotinic Acid Hydrazones
1549
Fig. 3. Different scaffolds identified from the SAR analysis
on activity was not clear; however, the hydrazide moiety trend also confirmed when R1=CH3, where compound
has a significant role in antiproliferative activity, as 6h (R2=Cl; IC50 = 51.36
M) resulted more active than
propenone M). Those results suggest
showed no activity. Consequently, the ob- 6i (R2=Br; IC50 = 79.54
µ
8
µ
tained biological activity was subjected to a molecular that a strict balance between over whole dimensions
modeling study to generate a pharmacophoric model and lipophilicity should be maintained.
for the active compounds and shed light on their
Structure-Activity Relationship (SAR).
A Quantitative SAR analysis was attempted as well.
Being aware that the available dataset is not suffici-
ently wide to derive a true predictive QSAR model, we
were interested in determining if the observed activity
Pharmacophoric model building
Aiming to a better rationalization of the obtained was correlated with one or more particular chemical
results, an SAR analysis has been attempted by taking physical property. In order to do that, all compounds
advantage of the Structure-Activity Report application were flexibly aligned using the default options of MOE
implemented in the MOE software package (MOE, software. The obtained alignment (see Fig. 1, Supporting
2010). This application, starting from a small collec- information), obtained independently and without any
tion of structures each of which features an activity bias coming from the crystalline structure, perfectly
value, generates a web page providing an interactive matches with the experimental X-ray geometry of 6h
,
view onto the collection of molecules, thus allowing a giving support to the adopted computational method.
clear interpretation of the role of substituents in affect- All the molecular descriptors available in the MOE
ing the activity of a small number of related common software were then calculated for the aligned geometries
scaffolds, automatically identified by the program and systematically analyzed by correlating the computed
(Clark et al., 2009). Consequently, compounds were descriptor value to the experimental IC50 through a linear
divided into three classes as showed in Fig. 3.
Compounds belonging to scaffolds and
regression analysis. By considering all compounds, no
are limit- descriptors showing a significant correlation with the
4
5
ed to only three structures and thus, for those com- experimental activity were actually found (only a very
pounds, an exhaustive SAR analysis cannot be derived. modest correlation with the adjacency and distance
Indeed, for compounds
atom at R1 seems not to be mandatory, as compounds 1999) was observed with a correlation coefficient r =
4a (R1=H, IC50 = 29.58 M) and 4b (R1=Cl, IC50 = 25.54 0.34). Nevertheless by excluding compounds
, an evi-
M) showed similar activities, while the presence of a dent inverse correlation was found between the exper-
methyl group (4c, R1=CH3, IC50 > 100) completely sup- imental IC50 and MNDO computed dipole moment
presses activity. On the other hand, when scaffold
is (Dewar et al., 1977), with a r = 0.78 and a r2 = 0.61
considered, the presence of a substituent at the R1 (Fig. 4).
4, the presence of a halogen matrix descriptor GCUT_SLOGP_3 (Wildman et al.,
µ
4
µ
5
position seems to negatively affect the activity, where
By only considering compounds 6, the linear regres-
the IC50 drops from 36.74 (5a, R1=H) to 50.16 (5c, R1= sion between IC50 and MNDO dipole provide an r2 =
CH3), to above 100 when R1=Cl (5b). Interestingly, in 0.52, thus about 0.1 lower than the one observed when
compound 5a, the replacement of both exocyclic nitro- all compounds, with the exception of
gen atoms with carbons completely suppresses this By also considering those latter derivatives, the corre-
activity. Concerning the most promising scaffold , it lation coefficient drops to 0 because of the high dipole
can be shown that the presence of a halogen atom at computed for compounds . This findings suggest that
the R2 position is essential for activity. When R1=H, compounds
and probably share a common target
the best results were obtained for R2=Br (IC50 = 24.99), and binding mode, while compounds
cannot be con-
followed by R2=Cl (IC50 = 66.78). However, when sidered as belonging to the same class, as they either
R1=Cl the highest activity was shown for R2=Cl (IC50
bind to a different target or to the same target, but
26.99
M), while no activity was obtained for R2=Br, a with a substantially different binding mode. From this
4, were included.
6
4
5
6
4
=
µ

1550
H. A. Abdel-Aziz et al.
Fig. 4. Correlation plot between experimental IC50 and MNDO computed dipole
analysis, it appears that a MNDO computed dipole mo- ware MOE (Dewar et al., 1977), which exhaustively
ment of 3.5 D or above, positively affects the compound generates ph4 queries for a set of flexibly aligned mole-
activity. Compound 6f behaves as an outlier, as it is cules, scoring them on the basis of a combined meas-
the only inactive compound of series
5 and 6 showing ure of how well they overlay the ligands (overlap) and
an MNDO dipole above 3.5, but no activity. Interest- how well they classify the data set into actives and
ingly, despite that all compounds satisfy the Lipinski’s inactives (accuracy) (Labute et al., 2001). Accuracy =
rule of five (Lipinski et al., 1997), compound 6f is the m/N where N is the number of molecules in the input
only one not satisfying Oprea’s test for drug-likeness collection and m is the sum of the number of actives
(see Table I, Supporting Information) (Oprea et al., that match the query and the number of inactive that
2000). Indeed, in previous works, we found Oprea’s test do not match the query. The best model obtained (over-
for drug-likeness being more effective in discriminating lap = 6.68, accuracy = 0.54) is represented in Fig. 5.
drug-like molecules in the virtual screening of large
databases (Ferri et al., 2009).
The model is characterized by two projected locations
of potential H-bond donors (F2 and F3), corresponding
A common pharmacophore (ph4) model was finally to the sp2 nitrogen and amidic carbonyl moiety, respec-
derived by considering compounds belonging to both tively, and two aromatic annotations (F1 and F4)
scaffolds
5
and
6
,
as, from both SAR and QSAR analyses, corresponding to the bicyclic aromatic ring and to the
are characteriz- phenyl group at the 5 position of the pyridine moiety.
we can safely assume that compounds
4
ed by different binding modes. The ph4 model was ob- This model, together with qualitative and quantitative
tained by using the ph4 Elucidator module of the soft- observations reported above, will help developing new
Fig. 5. Pharmacophore model. Features F1 and F4 are aromatic rings, features F2 and F3 are projected hydrogen bond
acceptor.

Antiproliferative Activity of Nicotinic Acid Hydrazones
1551
thesis and antimicrobial activity of benzofuran-based (1
E)-
,
compounds. For instance, by observing Fig. 5, it appears
that the substitution by a halogen atom at the 3-posi-
tion of the methylendioxyaryl group characterizing com-
1-(piperidin-1-yl)-N2-arylamidrazones. Eur. J. Med. Chem.
44, 3985-4997 (2009).
Abdel-Aziz, H. A., Gamal-Eldeen, A. M., Hamdy, N. A., and
Fakhr, I. M. I., Immunomodulatory and anti-cancer activity
of some novel 2-substituted-6-bromo-3-methylthiazolo[3,2-
a]benzimidazole derivatives. Arch. Pharm., 342, 230-237
(2009a).
Abdel-Aziz, H. A.,Mekawey, A. A. I., and Dawood, K. M., Con-
venient synthesis and antimicrobial evaluation of some
novel 2-substituted-3-methylbenzofuran derivatives. Eur.
J. Med. Chem., 44, 3637-3644 (2009b).
pounds
5 could improve the activity, and designing
and preparing such compounds will be the object of
future investigations.
In conclusion novel 6-aryl-2-methylnicotinic acid hy-
drazides 4a-c, their corresponding hydrazones 5a-c
and 6a-i were synthesized in addition to the propenone
derivative 8, structural analog of hydrazone 5a. Anti-
proliferative activity of the synthesized compounds was
investigated against K562 leukemia cell lines. Variable
cell growth inhibitory activities were obtained with
Abdel-Aziz, H. A., Abdel-Wahab, B. F., and Badria, F. A.,
Stereoselective synthesis and antiviral activity of (1E,2Z,
3E)-1-(piperidin-1-yl)-1-(arylhydrazono)-2-[(benzoyl/benzo-
IC50 range from 24.99 to 66.78 µM, where compound
6c revealed the maximum activity. SAR analysis has
been attempted and compounds were divided into
three scaffolds; 4, 5 and 6. Concerning the most pro-
mising scaffold 6, it could be shown that the presence
of a halogen atom at the fifth position of isatin moiety
(R2) is essential for activity. When R1=H, the best
results were obtained for R2=Br (IC50 = 24.99), followed
by R2=Cl (IC50 = 66.78). However, when R1=Cl the
highest activity was shown for R2=Cl (IC50 = 26.99),
while no activity was obtained for R2=Br, a trend also
confirmed when R1=CH3, where compound 6h (R2=Cl;
thiazol-2-oyl)hydrazono]-4-(aryl1)but-3-enes. Arch. Pharm.
343, 152-159 (2010).
,
Abdel-Wahab, B. F., Abdel-Aziz, H. A., and Ahmed, E. M., Con-
venient synthesis and antimicrobial activity of some new
3-substituted-5-(benzofuran-2-yl)-pyrazole derivatives. Arch.
Pharm., 341, 734-739 (2008).
Abdel-Wahab, B. F., Abdel-Aziz, H. A., and Ahmed, E. M., Syn-
thesis and antimicrobial evaluation of some new 1,3-thiazole,
1,3,4-thiadiazole, 1,2,4-triazole and [1,2,4]triazolo[3,4-b][1,3,
4]thiadiazine derivatives including 5-(benzofuran-2-yl)-1-
phenyl-pyrazole moiety. Monatsh. Chem., 140, 601-605
(2009).
IC50 = 51.36) were more active than 6i (R2=Br; IC50
=
Aboul-Fadl, T., Mohammed, F. A., andHassan, E. A., Synthe-
sis, antitubercular activity and pharmacokinetic studies of
some schiff bases derived from 1-alkylisatin and isonicotinic
acid hydrazide (INH). Arch. Pharm. Res., 26, 778-784 (2003).
Aboul-Fadl, T., Bin-Jubair, F. A. S., and Aboul-Wafa, O.,
Schiff bases of indoline-2,3-dione (isatin) derivatives and
nalidixic acid carbohydrazide, synthesis, antitubercular
activity and pharmacophoric model building. Eur. J. Med.
Chem., 45, 4578-4586 (2010).
Aboul-Fadl, T., Abdel-Aziz, H. A., Kadi, A., Bari, A., Ahmad, P.,
Al-Samani, T., and Ng, S. W., Microwave-assisted one-step
synthesis of fenamic acid hydrazides from the correspond-
ing acids. Molecules, 16, 3544-3551 (2011a).
79.54). Those results suggest that a strict balance
between over whole dimensions and lipophilicity should
be observed. Furthermore, hydrazide moiety has a
significant role in the antiproliferative activity as pro-
penone 8 showed no activity. A common ph4 model for
derivatives 4, 5 and 6 has been obtained by using the
ph4 elucidation module of the software MOE. The best
model obtained is characterized by two projected
locations of potential H-bond donors (F3 and F4) and
two Aromatic annotations (F1 and F2).
ACKNOWLEDGEMENTS
Aboul-Fadl, T., Abdel-Aziz, H. A., Kadi, A., Ahmad, P., Elsaman,
T., Attwa, M. W., and Darwish, I. A., Microwave-assisted
solution-phase synthesis and DART-Mass spectrometric
monitoring of combinatorial library of indolin-2,3-dione
Schiff bases with potential antimycobacterial activity.
Molecules, 16, 5194-5206 (2011b).
Aboul-Fadl, T., Abdel-Aziz, H. A., Elsaman, T., Abdel-Hamid,
M. K., Thanassi, J., and Pucci, M. J., Schiff bases of indoline-
2,3-dione: Potential novel inhibitors of Mycobacterium
tuberculosis (Mtb) DNA Gyrase, Molecules, 16, 7864-7879
(2011c).
Ahmedin, J., Siegel, R., Ward, E., Hao, Y., Xu J., Murray, T.,
and Thun, M. J., Cancer statistics. AC Cancer J. Clin., 58,
71-96 (2008).
Chabner, B. A. and Roberts, T. G., Chemotherapy and the war
on cancer. Nat. Rev. Cancer, 5, 65-72 (2005).
This work is supported by the research center, College
of Pharmacy and Deanship of Scientific Research, King
Saud University, project no. 080130.
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