Design, synthesis and antitubercular activity of certain nicotinic acid hydrazides

Article abstract of DOI:10.3390/molecules20058800

Three series of 6-Aryl-2-methylnicotinohydrazides 4a-i, N'-Arylidene-6-(4-bromophenyl)-2-methylnicotino hydrazides 7a-f, and N'-(un/substituted 2-oxoindolin-3-ylidene)-6-(4-fluorophenyl)-2-methylnicotinohydrazides 8a-c were synthesized and evaluated for their potential in vitro antimycobacterial activity against M.Tuberculosis. The results showed that isatin hydrazides 8a-c are remarkably more active than the parent hydrazide 4c. Hydrazides 8b and 8c exhibited the highest activity among all the tested compounds (MIC = 12.5 and 6.25 μg/mL, respectively). Compounds 8b and 8c were also devoid of apparent cytotoxicity to HT-29, PC-3, A549, HepG2 and MCF-7 cancer cell lines. Besides, 8b and 8c showed good drug-likeness scores of 0.62 and 0.41, respectively. Those two isatin hydrazides could offer an excellent framework for future development to obtain more potent antitubercular agents. The SAR study suggested that lipophilicity of the synthesized derivatives is a crucial element that accounts for their antimycobacterial activity. Finally, a theoretical kinetic study was established to predict the ADME of the active derivatives.

Full text of DOI:10.3390/molecules20058800

Molecules 2015, 20, 8800-8815; doi:10.3390/molecules20058800
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design, Synthesis and Antitubercular Activity of Certain
Nicotinic Acid Hydrazides
Wagdy M. Eldehna 1,*, Mohamed Fares 1, Marwa M. Abdel-Aziz 2 and Hatem A. Abdel-Aziz 3,4,
*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,
Badr City, Cairo 11829, Egypt; E-Mail: ph.fares@yahoo.com
2
The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo 11759, Egypt;
E-Mail: marwa2rcmb@yahoo.com
3
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
P.O. Box 2457, Riyadh 11451, Saudi Arabia
4
Department of Applied Organic Chemistry, National Research Center, Dokki, Cairo 12622, Egypt
* Authors to whom correspondence should be addressed; E-Mails: wagdy2000@gmail.com (W.M.E.);
hatem_741@yahoo.com (H.A.A.-A.); Tel.: +20-228-609-114 (W.M.E.); +966-146-77341 (H.A.A.-A.);
Fax: +20-228-609-117 (W.M.E.); +966-146-76220 (H.A.A.-A.).
Academic Editor: Derek J. McPhee
Received: 17 April 2015 / Accepted: 12 May 2015 / Published: 15 May 2015
Abstract: Three series of 6-aryl-2-methylnicotinohydrazides 4a–i, N′-arylidene-6-(4-
bromophenyl)-2-methylnicotino hydrazides 7a–f, and N′-(un/substituted 2-oxoindolin-3-
ylidene)-6-(4-fluorophenyl)-2-methylnicotinohydrazides 8a–c were synthesized and evaluated
for their potential in vitro antimycobacterial activity against M. tuberculosis. The results
showed that isatin hydrazides 8a–c are remarkably more active than the parent hydrazide
4c. Hydrazides 8b and 8c exhibited the highest activity among all the tested compounds
(MIC = 12.5 and 6.25 µg/mL, respectively). Compounds 8b and 8c were also devoid of
apparent cytotoxicity to HT-29, PC-3, A549, HepG2 and MCF-7 cancer cell lines. Besides,
8b and 8c showed good drug-likeness scores of 0.62 and 0.41, respectively. Those two
isatin hydrazides could offer an excellent framework for future development to obtain
more potent antitubercular agents. The SAR study suggested that lipophilicity of the
synthesized derivatives is a crucial element that accounts for their antimycobacterial
activity. Finally, a theoretical kinetic study was established to predict the ADME of the
active derivatives.

Molecules 2015, 20
Keywords: synthesis; nicotinic acid; hydrazides; antimycobacterial; ADME
8801
1. Introduction
The Nineteenth World Health Organization (WHO) Tuberculosis Report indicates that TB is one of
the world’s deadliest communicable diseases [1]. It estimates that in 2013 there were 9.0 million new
cases and 1.5 million deaths from TB, including 400,000 deaths associated with co-infection with HIV.
The highest rates per capita occurred in the African Region (25%), while South-East Asia, the Western
Pacific and African Regions account for around 81% of all the total cases [1]. The disease is
aggravated by the worldwide continuous emergence of multidrug-resistant strains of M. tuberculosis
(MDR-TB), extensively drug-resistant tuberculosis (XDR-TB) and totally drug-resistant tuberculosis
(TDR-TB) [2,3]. The magnitude and extent of drug-resistant strains have increased concern that TB
may once again become an incurable disease [4,5]. Moreover, the increasing incidence of the disease
in immunocompromised patients along with the longer durations of therapy emphasize the need for
new drugs to extend the range of effective TB treatment options [6–8].
TB treatment is tedious, challenging and time-consuming. It requires the administration of
appropriate treatment regimens for at least six months via directly observed therapy (DOT) and
follow-up support [2]. Treatment regimens require a minimum six months in two separate phases. The
duration of phase one is about two months and involves four drugs (isoniazid, rifampicin,
pyrazinamide and ethambutol), followed by four months of phase two (using isoniazid plus
rifampicin) [9]. However, the present treatment regimen has some limitations such as drug toxicity and
intolerance, drug–drug interactions and poor patient adherence due to the lengthy treatment
duration [10]. Therefore, there is an urgent need for the development and more efficient evaluation of
new TB drugs and shorter treatment regimens. Isoniazid (INH, Figure 1), a critical frontline drug in TB
treatment discovered by Dogmagk, is a prodrug that requires activation in vivo by mycobacterial
catalase peroxidase (KatG) [11,12]. INH exerts its anti-tubercular activity via interference with the
synthesis of mycolic acid, one of the essential chemical pathways responsible for the formation of cell
walls in M. tuberculosis [13].
The enzymatic acetylation of isoniazid by N-acetyltransferase (NAT) represents a major metabolic
pathway for isoniazid in humans [14], so blocking acetylation via chemical modification of the
hydrazine unit with a functional group, while preserving potent antimycobacterial action, has the
potential to counterbalance the known side effects of INH, improve clinical outcomes and reduce the
emergence of acquired isoniazid resistance in patients. Subsequently, numerous studies have
pointed out the importance of developing novel INH hydrazides as promising anti-tubercular agents
(Figure 1) [15–22].
Recently, Narang et al. [23] developed a novel series of nicotinic acid hydrazide derivatives as
potential antimycobacterial agents with a general structure represented by VI (Figure 1). The results
showed that the presence of lipophilic electron-withdrawing halogen groups at the para position of the
phenyl ring improved the antimycobacterial activity. Concerning the related fused pyridine
heterocycles, Adhikari and co-workers reported two studies on the design, synthesis and biological
evaluation of two different series of new quinoline-3-carbohydrazone derivatives VII, (Figure 1), as

Molecules 2015, 20
8802
potential antimycobacterial agents [24,25]. Aboul-Fadl et al. [26] also explored the anti-tubercular
activity of Schiff bases VIII of nalidixic acid-3-carbohydrazides and isatins. On the other hand,
isatin-based compounds are known to exhibit excellent anti-TB properties [27–30].
Figure 1. Structures of antitubercular drugs I–VIII and the target derivatives 4a–i, 7a–f and 8a–c.
In this work, the aforementioned findings motivated us to synthesize three series of nicotinic acid
hydrazide derivatives 4a–i, 7a–f and 8a–c with the aim of obtaining new antimycobacterial agents.
The first series comprises different nine 6-aryl-2-methylnicotinohydrazides with free hydrazine units in
a similar fashion to INH. Subsequently, two derivatives (compounds 4c and 4e) with considerable
lipophilicity (LogP = 1.53 and 2.17, respectively) were chosen for further chemical modification. The
non-classical ring opening bioisosterism for structures VI and VIII was adopted to develop a series of
aldehyde hydrazides 7a–f and a series of isatin hydrazides 8a–c.
The in vitro cytotoxic activity of subset of compounds was evaluated against HT-29, PC-3, A549,
HepG2 and MCF-7 cancer cell lines to determine the toxicity of these agents. In addition, we describe
an ADME study and SAR description in order to explore the structural requirements controlling these
observed antitubercular activities.

Molecules 2015, 20
8803
2. Results and Discussion
2.1. Chemistry
The synthetic pathways employed to prepare the new targeted derivatives are depicted in Schemes 1
and 2. In a one-pot three-component heterocyclocondensation process, ethyl 2-methyl-6-arylnicotinates
3a–i was obtained via the reaction of enaminones 2a–i with ethyl acetoacetate and ammonium acetate
in refluxing acetic acid. Preparation of the nicotinic acid hydrazides 4a–i in 79%–90% yield was
achieved via the hydrazinolysis of ester derivatives 3a–i with refluxing hydrazine hydrate (Scheme 1).
Reagents and conditions: (i) xylene, reflux 8h; (ii) NH4OAc/AcOH/reflux 5 h; (iii)
NH2NH2·H 2O/reflux 3 h.
Scheme 1. Synthesis of nicotinic acid hydrazides 4a–i.
The IR spectra of hydrazides 4a–i showed absorption bands due to the carbonyl group in the
1635–1664 cm−1 region, in addition to peaks in the region from 3187 to 3414 cm−1, assigned to the NH
1
and NH2 groups. The H-NMR spectra of 4a–i showed two singlet D2O-exchangeable signals
attributable to NH and NH2 protons in the δ 9.58–9.62 and 4.52–4.60 ppm region, respectively, while
13
the methyl (-CH3) protons appeared as singlets around δ 2.59–2.61 ppm. Furthermore, the C-NMR
spectra of 4f–h showed signals resonating around δ 167 ppm attributable to the carbons of carbonyl
groups, while the carbons of the methyl groups appeared in the δ 23.53–23.59 ppm range.
While the hydrazide 4e was reacted with different aldehydes 5a–f, the hydrazide 4c was reacted
with three isatins 6a–c in ethanol in the presence of a catalytic amount of glacial acetic acid to furnish
the target derivatives 7a–f and 8a–c, respectively (Scheme 2).
The IR spectra of hydrazides 7a–f revealed the presence of two peaks in the 3412–3413 and
1635–1654 cm−1 regions, assigned to the NH and carbonyl groups, respectively. The 1H-NMR spectra
of these compounds revealed D2O-exchangeable signals in the δ 11.80–12.05 ppm region which were
assigned to NH protons, in addition to the signal of the methine proton (-CH=N-) in the δ 8.06–8.96 ppm
region. Furthermore, the 13C-NMR spectra of 7a–f showed two signals resonating at δ 23.41–23.47 and
δ 162.00–170.39 ppm attributable to the methyl (CH3) and carbonyl (=C-C=O) carbons, respectively.

Molecules 2015, 20
8804
Reagents and conditions: i, EtOH/AcOH (catalytic)/reflux 4 h.
Scheme 2. Synthesis of nicotinic acid hydrazides 7a–f and 8a–c.
2.2. Biological Evaluation
2.2.1. In Vitro Antimycobacterial Activity
Biological assays were performed at the Regional Center for Mycology and Biotechnology
(RCMB), Al-Azhar University (Cairo, Egypt). Target compounds 4a–i, 7a–f and 8a–c were evaluated
in vitro for their anti-tubercular activity against M. tuberculosis (RCMB 010126) using the microplate
Alamar blue assay (MABA) [31]. Isoniazide and pyrazinamide were used as reference drugs. The
results as percent inhibition and minimum inhibitory concentration (MIC) are presented in Table 1.
With regard to the issue of activity, valuable information about cell wall structure, host-pathogen
interactions and drug targets provide opportunities for rational drug design strategies focused on drug
lipophilicity. Increases in lipophilic character, however, result in changes in pathways of diffusion
across the cell wall, enhancing the contribution of diffusion through the lipid domain, so increasing the
lipophilicity of an antimycobacterial agent enhances its efficacy [32–35]. The calculated lipophilicity
(miLogP) values are listed in Table 1.
Drug-likeness model scores were computed for all the compounds using the MolSoft software and
are presented in Table 1. Drug-likeness models help to optimize the pharmacokinetic and
pharmaceutical properties, for example, solubility, chemical stability, bioavailability and distribution
profile of compounds [36]. Counterparts having zero or negative values should not be considered as
drug-like candidates. Compounds 7f and 8a–c possessed the maximum drug-likeness score ranging
from 0.41 to 0.88.

Molecules 2015, 20
8805
Table 1. Antitubercular activities, LogP measurements and drug-likeness model scores of
nicotinic acid hydrazide derivatives.
Mean of
MIC
Drug-Likeness
Model Score b
−0.1
Compound
Ar
R1 R2
R3
LogP a
Inhibition % (µg/mL)
4a
C6H5
4-CH3C6H4
4-FC6H4
42.52 ± 0.63
36.33 ± 0.58
NA
25
25
1.36
1.81
1.53
2.04
2.17
1.42
1.01
0.99
1.15
4.95
5.11
5.63
6.23
5.01
6.13
3.54
4.19
4.32
4b
−0.36
−0.06
0.05
4c
NA
100
50
4d
4-ClC6H4
12.45 ± 0.58
20.63 ± 0.63
42.63 ± 0.16
22.63 ± 0.16
18.32 ± 0.72
22.63 ± 0.20
NA
4e
4-BrC6H4
−0.25
−0.25
0.13
4f
4-CH3OC6H4
3,4(CH3O)2C6H3
3,4,5(CH3O)3C6H2
thiophen-2-yl
25
4g
50
4h
50
0.33
4i
50
−0.10
0.12
7a
H
H
H
H
H
H
H
H
F
NA
NA
NA
100
NA
100
25
7b
NA
0.00
7c
H
Cl
NA
0.09
7d
Cl
H
Cl
13.57 ± 0.72
NA
0.13
7e
MeO
0.09
7f
H
-(CH)4-
14.32 ± 0.58
31.44 ± 0.58
52.63 ± 0.58
77.42 ± 0.93
93.25 ± 0.63
-
0.46
8a
8b
H
0.88
Cl
Br
12.5
6.25
3.21
0.75
0.62
8c
0.41
Pyrazinamid
Isoniazide
a: Calculated by [37]; b: Calculated by [38]; NA: No Activity (>100 µg/mL).
As shown in Table 1, compound 8c emerged as the most potent analog with good antimycobacterial
activity (MIC = 6.25 µg/mL). Compound 8b also possessed reasonable activity with a MIC value of
12.5 µg/mL. Besides, derivatives 4a, 4b, 4f and 8a displayed moderate activity (MIC = 25 µg/mL). On
the other hand, compounds 4d, 4e, 4g–i, 7d and 7f exhibited modest antimycobacterial activity with
MIC values ranging from 50 to 100 µg/mL.
2.2.2. Structure Activity Relationships (SAR)
Observing the results, we could deduce valuable data about the structure activity correlations of the
tested compounds. Firstly, we explored the impact of substitution of the 4-position of the phenyl group
in the first series compounds 4a–i. Incorporation of an unsubstituted phenyl group led to compound 4a
with good activity against M. tuberculosis (MIC = 25 µg/mL). Introduction of a fluorine atom in the

Molecules 2015, 20
8806
4-position (compound 4c) led to complete loss of activity, suggesting that the presence of a strongly
electron-withdrawing group is not favorable to the activity. Meanwhile, compounds 4d and 4e bearing
more lipophilic chlorine and bromine substituents at the same position, elicited better activity
(MIC = 50 and 100 µg/mL, respectively) than that of analog 4c. Thence, the order of activities of the
halogenated members in the first series, decreased in the order of Br > Cl > F, indicating that the
increased lipophilicity is a crucial element for the antitubercular activity. Conversely, substitution at
the 4-position with electron-donating groups as methyl and methoxy groups (compounds 4b and 4f)
retained the activity at MIC = 25 µg/mL. Interestingly, di- and trimethoxy substitutions (compounds 4g
and 4h) led to decrease in the lipophilicity with subsequent decrease in the antimycobacterial activity
(MIC = 50 µg/mL). Furthermore, the bioisosteric replacement of the phenyl group with a 2-thienyl group
(compound 4i) led to a decrease in the activity.
Considering the aldehyde hydrazides of the second series 7a–f, antimycobacterial activity was only
observed in counterparts 7d and 7f (MIC = 100 µg/mL). The remaining hydrazides exhibited no
activity. Noteworthily, compounds 7d and 7f are the most lipophilic analogs in this series with LogP
values of 6.23 and 6.13, respectively.
Finally, the effect of substitution of the 5-position of the incorporated isatin moiety in hydrazides
8a–c was investigated. Compound 8a with an unsubstituted isatin moiety showed good anti-TB
activity (MIC = 25 µg/mL). The introduction of a chlorine substituent at the 5-position (compound 8b)
resulted in increased activity with MIC = 12.50 µg/mL. Moreover, incorporation of a bromine atom
(compound 8c) caused a remarkable increase of activity against M. tuberculosis (MIC = 6.25 µg/mL).
To summarize, the order of activities of the isatin hydrazides of the third series decreased in the order
Br > Cl > H, confirming the importance of lipophilicity for the antimycobacterial activity.
In general, the active members in the second series, compounds 7d and 7f, are less active than their
parent compound 4e, indicating that condensation of free hydrazides with aldehydes results in a sharp
decrease in activity, whilst, the compounds of the third series are remarkably more potent than their
parent compound 4c, hinting that condensation with isatins greatly enhances the antitubercular activity.
2.2.3. In Vitro Cytotoxicity
In vitro cytotoxicity of the most active antitubercular compounds 8a–c was examined against HT-29
colon cancer and PC-3 prostate cancer cell lines using a sulforhodamine B (SRB) colorimetric assay as
described by Skehan et al. [39]. Besides, these compounds were evaluated in a previous study for their
cytotoxicity against HepG-2 liver cancer, A-549 human lung cancer and MCF-7 breast cancer cell
lines [40]. Doxorubicin was included in the experiment as a reference cytotoxic compound. The results
were expressed as growth inhibitory concentration (IC50) values which represent the compound
concentration required to produce a 50% inhibition of cell growth after 72 h of incubation compared to
untreated control (Table 2). Interestingly, none of the tested compounds displayed any significant
cytotoxicity, thereby providing a high therapeutic index.

Molecules 2015, 20
Table 2. Levels of cytotoxicity induced by hydrazides 8a–c on different cell lines.
8807
IC50 (µM)
A549
Compound
HT-29
>200
>200
>200
PC-3
>200
>200
>200
HepG2
>200
MCF-7
>200
8a
8b
8c
>200
>200
>200
>200
>200
>200
>200
Doxorubicin 7.3 ± 1.11 6.5 ± 1.07 7.6 ± 1.37
6.9 ± 2.05
6.1 ± 1.95
2.3. ADME Study
The ADME of the biologically active counterparts 4a, 4b, 4d–i, 7d, 7f and 8a–c was predicted via a
theoretical kinetic study performed by means of the Discovery Studio software (Table 3). Both
AlogP98 and PSA_2D descriptors were calculated to evaluate the lipophilicity and polar surface area.
Also, solubility, absorption and CYP2D inhibition levels were predicted. Active members of the first
series were expected to have good solubility, while compounds 7d and 7f showed very low solubility
levels and compounds 8a–c showed low solubility levels. All the examined compounds showed good
absorption levels, except compound 7d that displayed a moderate absorption level. Finally, with
exception to compounds 4b–f, all members were predicted to be CYP2D non-inhibitors.
Table 3. Computer aided ADME study of the active derivatives.
Solubility Absorption
CYP2D6
Probability g
0.306
Compound AlogP98 a PSA_2D b Solubility c
CYP2D6 f
Level d
Level e
4a
4b
4d
4e
4f
1.419
1.905
2.083
2.167
1.402
1.386
1.37
67.912
67.912
67.912
67.912
76.842
85.772
94.702
67.912
52.695
52.695
82.806
82.806
82.806
−2.578
−3.087
−3.496
−3.57
3
3
3
3
3
3
3
3
1
1
2
2
2
0
0
0
0
0
0
0
0
1
0
0
0
0
0
1
1
1
1
0
0
0
0
0
0
0
0
0.772
0.831
0.712
−2.838
−3.101
−3.353
−2.546
−6.834
−6.647
−4.835
−5.608
−5.682
0.722
4g
4h
4i
0.366
0.316
1.145
5.839
5.419
3.048
3.713
3.797
0.158
7d
7f
0.287
0.306
8a
8b
8c
0.485
0.415
0.495
b
c
a: Lipophilicity descriptor; : Polar surface area; : Solubility parameter.(0~−2 = optimal, −2~−4 = good,
−4~−6 = low, −6~−8 = very low); d: Solubility level. (0 = extremely low, 1 = very low but possible, 2 = low,
e
f
3 = good, 4 = optimal); : Absorption level. (0 = good, 1 = moderate, 2 = low, 3 = very low); : CYP2D
inhibition. (0 = non inhibitor, 1 = inhibitor); g: CYP2D6 Probability: 0–0.5 = non inhibitor; 0.5–1 = inhibitor.

Molecules 2015, 20
8808
3. Experimental Section
3.1. General Information
Melting points were measured with a Stuart melting point apparatus and are uncorrected. Infrared
(IR) spectra were recorded as KBr disks using a Perkin Elmer FT-IR Spectrum BX apparatus (Akron,
OH, USA). NMR Spectra were recorded on a Bruker AV-500 MHz NMR spectrometer (Billerica,
MA, USA). 1H-NMR spectra were run at 500 MHz and 13C spectra was run at 125 MHz in deuterated
dimethylsulfoxide (DMSO-d6). Chemical shifts are expressed in δ values (ppm) using the solvent peak
as internal standard. All coupling constants (J) values are given in Hertz. The abbreviations used are as
follows: s, singlet; d, doublet; m, multiplet. Microanalyses were carried out using Perkin Elmer PE 2400
CHN Elemental Analyzer and the results were within ±0.4%. Reaction courses and product mixtures
were routinely monitored by thin layer chromatography (TLC) on silica gel precoated F254 plates
Merck (Merck KGaA, Darmstadt, Germany). Unless otherwise noted, all solvents and reagents were
commercially available and used without further purification.
3.2. Synthesis
3.2.1. Ethyl 2-methyl-6-arylnicotinates 3a–i
To a solution of the appropriate enaminone 2a–i (5 mmol) in glacial acetic acid (15 mL), ethyl
acetoacetate (5.5 mmol) and ammonium acetate (40 mmol) were added. The reaction mixture was
heated under reflux for 5 h. After cooling and pouring into ice-water, the residue obtained was filtered
and washed with petroleum ether then with water and finally crystallized from ethanol [41]. The yields
of compounds 3a–i were 80%, 77%, 81%, 84%, 86%, 80%, 785, 76%, 83%, respectively.
3.2.2. 6-Aryl-2-methylnicotinohydrazides 4a–i
A mixture of the appropriate ester 4a–i (5 mmol) and 99% hydrazine hydrate (5 mL) was refluxed
for 3 h. The solid product obtained upon cooling was filtered off and recrystallized from dioxan to
afford the corresponding 6-aryl-2-methylnicotinohydrazides 4a–i, respectively. The physical properties
and spectral data of 4a–d and 4i were identical with those reported [40,42]. The yields of 4a–d and 4i
were 83, 78, 84, 86 and 84%, respectively.
6-(4-Bromophenyl)-2-methylnicotinohydrazide (4e). White crystals (yield 90%), m.p. 213–215 °C; IR
1
(KBr, ν cm−1): 3194, 3294 (NH, NH2) and 1643 (C=O); H-NMR δ ppm: 2.61 (s, 3H, CH3), 4.60 (s,
2H, NH2, D2O exchangeable), 7.56 (d, J = 8.5 Hz, 2H, Ar-H), 7.79 (d, J = 8.1 Hz, 1H, H-4 pyridine,),
7.87 (d, J = 8.1 Hz, 1H, H-5 pyridine,), 8.14 (d, J = 8.5 Hz, 2H, Ar-H), 9.62 (s, 1H, NH, D2O
exchangeable); Anal. calcd. for C13H12BrN3O (306.16): C, 51.00; H, 3.95; N, 13.72. Found C, 51.13;
H, 4.10; N, 13.78.
6-(4-Methoxyphenyl)-2-methylnicotinohydrazide (4f). White crystals (yield 81%), m.p. 195–197 °C; IR
1
(KBr, ν cm−1): 3292, 3350 (NH, NH2) and 1635 (C=O); H-NMR δ ppm: 2.59 (s, 3H, CH3), 3.83 (s,
3H, OCH3), 4.52 (s, 2H, NH2, D2O exchangeable), 7.05 (d, J = 8.5 Hz, 2H, Ar-H), 7.70 (d, J = 8.1 Hz,

Molecules 2015, 20
8809
1H, H-4 pyridine), 7.73 (d, J = 8.1 Hz, 1H, H-5 pyridine), 8.07 (d, J = 8.5 Hz, 2H, Ar-H), 9.58 (s, 1H,
NH, D2O exchangeable); 13C-NMR δ ppm: 23.53, 55.72, 114.61, 116.55, 128.54, 129.00, 130.96,
136.78, 155.89, 156.03, 160.85, 167.78; Anal. calcd. for C14H15N3O2 (257.12): C, 65.35; H, 5.88; N,
16.33. Found C, 65.54; H, 5.93; N, 16.53.
6-(3,4-Dimethoxyphenyl)-2-methylnicotinohydrazide (4g). White crystals (yield 85%), m.p. 204–205 °C;
IR (KBr, ν cm−1): 3187, 3414 (NH, NH2) and 1648 (C=O); 1H-NMR δ ppm: 2.60 (s, 3H, CH3), 3.83 (s,
3H, OCH3), 3.86 (s, 3H, OCH3), 4.53 (s, 2H, NH2, D2O exchangeable), 7.06 (d, J = 8.5 Hz, 1H, Ar-H),
7.68–7.74 (m, 3H, Ar-H), 7.81 (d, J = 8.5 Hz, 1H, Ar-H), 9.59 (s, 1H, NH, D2O exchangeable);
13C-NMR δ ppm: 23.59, 56.03, 110.41, 112.19, 116.81, 119.95, 128.76, 131.15, 136.71, 149.37,
150.57, 155.83, 156.09, 167.77; Anal. calcd. for C15H17N3O3 (287.13): C, 62.71; H, 5.96; N, 14.63; O,
16.71, Found C, 62.84; H, 6.11; N, 14.89.
2-Methyl-6-(3,4,5-trimethoxyphenyl)nicotinohydrazide (4h). White crystals (yield 85%), m.p. 185–187 °C;
IR (KBr, ν cm−1): 3282, 3310 (NH, NH2) and 1664 (C=O); 1H-NMR δ ppm: 2.61 (s, 3H, CH3), 3.73 (s,
3H, OCH3), 3.87 (s, 6H, OCH3), 4.55 (s, 2H, NH2, D2O exchangeable), 7.41 (s, 2H, Ar-H), 7.75 (d,
J = 8.0 Hz, 1H, H-4 pyridine), 7.89 (d, J = 8.0 Hz, 1H, H-5 pyridine), 9.61 (s, 1H, NH, D2O
exchangeable); 13C-NMR δ ppm: 23.58, 56.46, 60.57, 104.53, 117.51, 129.31, 134.02, 136.73, 139.23,
153.61, 155.84, 155.92, 167.66; Anal. calcd. for C16H23N3O4 (321.17): C, 59.80; H, 7.21; N, 13.08.
Found C, 60.09; H, 7.43; N, 13.22.
3.2.3. General Procedure for Synthesis of N′-arylidene-6-(4-bromophenyl)-2-methylnicotinyl
Hydrazides 7a–f
To a stirred solution of the hydrazide 4e (5 mmol) in hot ethanol (20 mL), aldehydes 5a–f (5 mmol)
and catalytic amount of glacial acetic acid were added. The reaction mixture was heated under reflux
for 4 h. The precipitate formed was collected by filtration while hot, washed with hot ethanol, dried
and crystallized from ethanol/DMF to afford compounds 7a–f in 65%–80% yield.
N′-Benzylidene-6-(4-bromophenyl)-2-methylnicotinohydrazide (7a). White crystals (yield 75%), m.p.
270–272 °C; IR (KBr, ν cm−1): 3410 (NH), 1635 (C=O); 1H-NMR δ ppm: 2.67 (s, 3H, CH3), 7.31–7.49
(m, 4H, Ar-H), 7.72-7.75 (m, 3H, Ar-H), 7.93-8.13 (m, 4H, Ar-H), 8.33 (s, 1H, CH=N), 11.94 (s, D2O
exch., 1H, –CONH–); 13C-NMR δ ppm: 23.47, 117.59, 123.67, 127.11, 127.65, 129.27, 130.73,
132.22, 132.26, 134.58, 137.39, 137.50, 148.39, 155.48, 156.47, 164.38; Anal. calcd. for C20H16BrN3O
(393.05): C, 60.93; H, 4.09; N, 10.66. Found C, 61.07; H, 4.30; N, 10.93.
6-(4-Bromophenyl)-N'-(4-fluorobenzylidene)-2-methylnicotinohydrazide (7b). White crystals (yield
65%), m.p. 252–254 °C; IR (KBr, ν cm−1): 3413 (NH), 1647 (C=O); 1H-NMR δ ppm:
2.66 (s, 3H, CH3), 7.31 (t, J = 8.25 Hz, 2H, Ar-H), 7.72 (d, J = 8.5 Hz, 2H, Ar-H), 7.80–7.97 (m, 4H,
Ar-H), 8.11-813 (m, 2H, Ar-H), 8.33 (s, 1H, CH=N), 11.95 (s, D2O exch., 1H, –CONH–); 13C-NMR δ
ppm: 23.41, 116.34 (2JF-C = 22.5 Hz), 117.25, 117.58, 123.68, 129.21 (3JF-C = 7.5 Hz), 129.64, 130.48,
131.21, 132.21, 137.38, 143.79, 147.26, 154.75, 162.70 (1JF-C = 211.3 Hz), 170.39; Anal. calcd. for
C20H15BrFN3O (411.04): C, 58.27; H, 3.67; N, 10.19. Found C, 58.38; H, 3.83; N, 10.25.

Molecules 2015, 20
8810
6-(4-Bromophenyl)-N'-(4-chlorobenzylidene)-2-methylnicotinohydrazide (7c). White crystals (yield
1
80%), m.p. 265–267 °C; IR (KBr, ν cm−1): 3413 (NH),1654 (C=O); H-NMR δ ppm: 2.66 (s, 3H,
CH3), 7.20 (d, J = 8.50 Hz, 1H, Ar-H), 7.54 (d, J = 8.50 Hz, 1H, Ar-H), 7.72 (d, J = 8.50 Hz, 2H,
Ar-H), 7.77 (d, J = 8.50 Hz, 2H, Ar-H), 7.98 (d, J = 4.5 Hz, 2H, Ar-H), 8.11 (d, J = 8.5 Hz, 2H,
Ar-H), 8.32 (s, 1H, CH=N), 12.01 (s, D2O exch., 1H, –CONH–); 13C-NMR δ ppm: 23.47, 114.00,
117.59, 129.28, 129.46, 132.26 (2C), 135.16 (2C), 136.50, 137.41, 137.52, 148.00, 152.00, 155.52,
168.00; Anal. calcd. for C20H15BrClN3O (427.01): C, 56.03; H, 3.53; N, 9.80. Found C, 56.09; H, 3.76;
N, 9.92.
6-(4-Bromophenyl)-N'-(2,4-dichlorobenzylidene)-2-methylnicotinohydrazide (7d). White crystals
1
(yield 70%), m.p. 269–271 °C; IR (KBr, ν cm−1): 3413 (NH), 1650 (C=O); H-NMR δ ppm: 2.67 (s,
3H, CH3), 7.55 (d, J = 8.50 Hz, 1H, Ar-H), 7.54 (d, J = 8.50 Hz, 1H, Ar-H), 7.72 (d, J = 8.50 Hz, 2H,
Ar-H), 7.96 (d, J = 6.50 Hz, 1H, Ar-H), 8.02 (d, J = 8.5 Hz, 1H, Ar-H), 8.05 (d, J = 8.5 Hz, 1H,
Ar-H), 8.11 (d, J = 8.5 Hz, 2H, Ar-H), 7.68 and 8.45 (s, 1H, Ar-H), 8.67 (s, 1H, CH=N), 12.01 (s, D2O
exch., 1H, –CONH); Anal. calcd. For C20H14BrCl2N3O (463.15): C, 51.86; H, 3.05; N, 9.07. Found C,
51.95; H, 3.12; N, 9.17.
6-(4-Bromophenyl)-N'-(4-methoxybenzylidene)-2-methylnicotinohydrazide (7e). White crystals (yield
1
75%), m.p. 251–253 °C; IR (KBr, ν cm−1): 3413 (NH), 1652 (C=O); H-NMR δ ppm: 2.66 (s, 3H,
CH3), 3.74 and 3.83 (s, 3H, -OCH3), 7.04 (d, J = 8.50 Hz, 2H, Ar-H), 7.69 (d, J = 8.50 Hz, 2H, Ar-H),
7.72 (d, J = 8.50 Hz, 2H, Ar-H), 7.92–7.98 (m, 2H, Ar-H), 8.11 (d, J = 8.50 Hz, 2H, Ar-H), 8.26 (s,
1H, CH=N), 11.80 (s, D2O exch., 1H, –CONH–); 13C-NMR δ ppm: 23.44, 55.79, 114.85, 117.58,
127.00, 129.00, 129.26 (2C), 132.25 (2C), 135.50, 137.00, 137.34, 138.00, 148.26, 156.41, 162.00;
Anal. calcd. for C21H18BrN3O2 (423.06): C, 59.45; H, 4.28; N, 9.90; Found C, 59.69; H, 4.41;
N, 10.12.
6-(4-Bromophenyl)-2-methyl-N'-(naphthalen-2-ylmethylene)nicotinohydrazide (7f). White crystals
1
(yield 73%), m.p. 273–275 °C; IR (KBr, ν cm−1): 3413 (NH), 1648 (C=O); H-NMR δ ppm: 2.71 (s,
3H, CH3), 7.20–8.95 (m, 14H, Ar-H), 8.96 (s, 1H, CH=N), 12.03 (s, D2O exch., 1H, –CONH–); Anal.
calcd. for C24H18BrN3O (443.06): C, 64.88; H, 4.08; N, 9.46. Found C, 65.07; H, 4.37; N, 9.65.
3.2.4. General Procedure for Preparation of Target Compounds 8a–c
Indoline-2,3-dione derivative 6a–c (1 mmol) was added to a suspension of 6-(4-fluorophenyl)-2-
methylnicotinohydrazide (4c, 1 mmol) in ethanol (10 mL) and a catalytic amount of glacial acetic acid.
The reaction mixture was refluxed for 4 h. The precipitate formed was collected by filtration while hot,
washed with hot ethanol, dried and crystallized from ethanol/DMF to furnish compounds 8a–c. The
physical properties and spectral data of 8a–c were identical with those reported [40]. The yields of
compounds 4a–c ranged from 75%–80%.

Molecules 2015, 20
8811
3.3. Biological Evaluation
3.3.1. Antimycobacterial Activity
The M. tuberculosis (RCMB 010126) strain was provided from culture collection of the Regional
Center for Mycology and Biotechnology (RCMB), Al-Azhar University (Cairo, Egypt). Isoniazide and
pyrazinamide were used as reference drugs. Antimycobacterial activity of the synthesized compounds
was evaluated using the microplate Alamar blue assay (MABA) which was performed in black,
clear-bottomed, 96 well microplates (in order to minimize background effects). Outer perimeter wells
were filled with sterile water to prevent dehydration in experimental wells. Initial compounds dilutions
were prepared in dimethyl sulfoxide and subsequent twofold dilutions were performed in the
microplates. 0.1 ml of 105 CFU/mL Mycobacterium tuberculosis inoculum was added to wells,
additional control wells consisted of bacteria only (B). Plates were incubated at 37 °C. Starting at day 4
of incubation, 20 µL of alamarBlue solution (Alamar Biosciences/Accumed, Westlake, OH, USA) and
12.5 µL of 20% Tween 80 were added added to the entire plate. Plates were then incubated at 37 °C,
and results were recorded at 24 h post-reagent addition at 590 nm. Percent inhibition was defined as:
1 − (mean of test well/mean of B wells) × 100. Visual MICs were defined as the lowest concentration
of drug that prevented a color change.
3.3.2. In Vitro Cytotoxic Activity
HT-29 colon cancer and PC-3 prostate cancer cell lines were obtained from the National Cancer
Institute (Cairo, Egypt). HT-29 and PC-3, cells were grown in RPMI-1640. Media were supplemented
with 10% heat-inactivated FBS, 50 units/mL of penicillin and 50 g/mL of streptomycin and maintained
at 37 °C in a humidified atmosphere containing 5% CO2. The cells were maintained as a “monolayer
culture” by serial subculturing. Cytotoxicity was determined using the SRB method as previously
described by Skehan et al. [39]. Exponentially growing cells were collected using 0.25% trypsin-EDTA
and seeded in 96-well plates at 1000–2000 cells/well in supplemented DMEM medium. After 24 h,
cells were incubated for 72 h with various concentrations of the tested compounds as well as
doxorubicin as the reference compound. Following 72 h of treatment, the cells were fixed with 10%
trichloroacetic acid for 1 h at 4 °C. Wells were stained for 10 min at room temperature with 0.4% SRB
dissolved in 1% acetic acid. The plates were air dried for 24 h, and the dye was solubilized with
Tris-HCl for 5 min on a shaker at 1600 rpm. The optical density (OD) of each well was measured
spectrophotometrically at 564 nm with an ELISA microplate reader (ChroMate-4300, Palm City, FL,
USA). The IC50 values were calculated according to the equation for Boltzmann sigmoidal
concentration-response curve using the nonlinear regression models (Graph Pad, Prism Version 5).
The results reported are means of at least three separate experiments. Significant differences were
analyzed by one-way ANOVA wherein the differences were considered to be significant at p < 0.05.
4. Conclusions
In summary, we have synthesized eighteen derivatives based on the nicotinic acid hydrazide
scaffold and evaluated their antimycobacterial activity. From the obtained results, it was obvious that

Molecules 2015, 20
8812
condensation of free hydrazides with aldehydes sharply decreased the activity, while isatin hydrazides
enhanced the activity. Compounds 8b and 8c emerged as the most potent counterparts among all the
tested compounds (MIC = 12.5 and 6.25 µg/mL, respectively). They also displayed good drug-likeness
scores of 0.62 and 0.41, respectively. The cytotoxicity of hydrazides 8a–c was evaluated against
HT-29, PC-3, A549, HepG2 and MCF-7 cancer cell lines. None of the tested hydrazides exhibited
cytotoxicity up to 200 µM. The importance of lipophilicity for the antimycobacterial activity was
explored via a SAR study. Finally, a theoretical kinetic study was established to predict the ADME of
the active derivatives.
Acknowledgments
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,
Cairo, Egypt, is highly appreciated for supporting this research. The authors would like to extend their
sincere appreciation to the Deanship of Scientific Research at King Saud University for its funding of
this research through the Research Group Project no. RGP-VPP-321.
Author Contributions
Hatem A. Abdel-Aziz and Wagdy M. Eldehna formulated the research idea and participated in the
preparation of manuscript; Wagdy M. Eldehna and Mohamed Fares carried out the experimental,
interpreted the data and prepared the manuscript; Marwa M. Abdel-Aziz performed the biological
screening. All authors have read and approved the final manuscript.
Conflicts of Interes
The authors declare no conflict of interest.
References
1. World Health Organization. Global Tuberculosis Report; WHO: Geneva, Switzerland, 2013.
2. Zumla, A.; Nahid, P.; Cole, S.T. Advances in the development of new tuberculosis drugs and
treatment regimens, Nat. Rev. Drug Discov. 2013, 12, 388–404.
3. Goldman, R.C.; Plumley, K.V.; Laughon, B.E. The evolution of extensively drug resistant
tuberculosis (XDR-TB): History, status and issues for global control. Infect. Disord. Drug Targets
2007, 7, 73–91.
4. Benatar, S.R. Extensively drug resistant tuberculosis—Problem will get worse in South Africa
unless poverty is alleviated. Br. Med. J. 2006, 333, doi:10.1136/bmj.333.7570.705-a.
5. Lawn, S.D.; Wilkinson, R. Extensively drug resistant tuberculosis—A serious wake-up call for
global health. Br. Med. J. 2006, 333, 559–560.
6. Dahle, U.R. Extensively drug resistant tuberculosis—Beware patients lost to follow-up. Br. Med. J.
2006, 333, doi:10.1136/bmj.333.7570.705.
7. Gandhi, N.R.; Moll, A.; Sturm, A.W.; Pawinski, R.; Govender, T.; Lalloo, U.; Zeller, K.; Andrews, J.;
Friedland, G. Extensively drug-resistant tuberculosis as a cause of death in patients co-infected
with tuberculosis and HIV in a rural area of South Africa. Lancet 2006, 368, 1575–1580.

Molecules 2015, 20
8813
8. Manissero, D.; Fernandez, K. Extensive drug-resistant TB: A threat for Europe? Euro Surveill.
2006, 11, E060928.
9. World Health Organization. Treatment of Tuberculosis Guidelines, 4th ed.; WHO: Geneva,
Switzerland, 2010.
10. Mitnick, C.D.; McGee, B.; Peloquin, C.A. Tuberculosis pharmacotherapy: Strategies to optimize
patient Care. Expert. Opin. Pharmacother. 2009, 10, 381–401.
11. Domagk, G.; Offe, H.A.; Siefken, W. Ein weiterer Beitrag zur experimentellen Chemotherapie der
Tuberkulose (Neoteben). Dtsch. Med. Wochenschr. 1952, 77, 573–578.
12. Janin, Y.L. Antituberculosis drugs: Ten years of research. Bioorg. Med. Chem. 2007, 15, 2479–2513.
13. Boechat, N.; Ferreira, V.F.; Ferreira, S.B.; Ferreira, M.G.; de C. da Silva, F.; Bastos, M.M.;
dos S. Costa, M.; Lourenc, M.C.S.; Pinto, A.C.; Krettli, A.U.; et al. Novel 1,2,3-triazole derivatives
for use against Mycobacterium tuberculosis H37Rv (ATCC 27294) strain. J. Med. Chem. 2011,
54, 5988–5999.
14. Sandy, J.; Mushtaq, A.; Kawamura, A.; Sinclair, J.; Sim, E.; Noble, M. The Structure of
Arylamine N-acetyltransferase from Mycobacterium smegmatis—An Enzyme which Inactivates the
Antitubercular Drug, Isoniazid. J. Mol. Biol. 2002, 318, 1071–1083.
15. Lourençoa, M.C.S.; Ferreirab, M.L.; Souza, M.V.N.; Peralta, M.A.; Vasconcelos, T.R.A.;
das Graças, M.; Henriques, M.O. Synthesis and antimycobacterial activity of (E)-N'
(monosubstituted-benzylidene)isonicotinohydrazide derivatives. Eur. J. Med. Chem. 2008, 43,
1344–1347.
16. Cardoso, S.H.; de Assis, J.V.; de Almeida, M.V.; Lourenço, M.C.S.; Vicente, F.R.C.;
de Souza, M.V.N. Synthesis and antitubercular activity of isoniazid condensed with carbohydrate
derivatives. Quim. Nova 2009, 32, 1557–1560.
17. Bottari, B.; Maccari, R.; Monforte, F.; Ottana, R.; Rotondo, E.; Vigorita, M.G. Isoniazid-related
copper(II) and nickel(II) complexes with antimycobacterial in vitro activity. part 9. Bioorg. Med.
Chem. Lett. 2000, 10, 657–660.
18. Roye, W.E.; Ewart, G.E. A Preliminary Report on the Safety and Therapeutic Activity of a Salizid
INH Derivative. Dis. Chest 1958, 33, 261–267.
19. Pershin, G.N.; Makeeva, O.O. Chemotherapeutic effect of phthivazide in experimental
tuberculosis. Probl. Tuberk. 1953, 2, 16–20.
20. Rubbo, S.D.; Edgar, J.; Vaughan, G. Chemotherapy of tuberculosis. I. Antituberculous activity of
verazide and related hydrazones. Am. Rev. Tuberc. 1957, 76, 331–345.
21. Soldatov, V.E. Saluzide in the treatment of tuberculous meningitis in adults. Probl. Tuberk. 1955,
6, 16–21.
22. Judge, V.; Narasimhan, B.; Ahuja, M.; Sriram, D.; Yogeeswari, P.; Clercq, E.D.; Pannecouque, C.;
Balzarini, J. Synthesis, antimycobacterial, antiviral, antimicrobial activities, and QSAR studies of
isonicotinic acid-1-(substituted phenyl)-ethylidene/cycloheptylidene hydrazides. Med. Chem. Res.
2012, 21, 1935–1952.
23. Narang, R.; Narasimhan, B.; Sharma, S.; Sriram, D.; Yogeeswari, P.; Clercq, E.D.; Pannecouque, C.;
Balzarini, J. Synthesis, antimycobacterial, antiviral, antimicrobial activities, and QSAR studies of
nicotinic acid benzylidene hydrazide derivatives. Med. Chem. Res. 2012, 21, 1557–1576.

Molecules 2015, 20
8814
24. Eswaran, S.; Adhikari, A.V.; Pal, N.K.; Chowdhury, I.H. Design and synthesis of some new
quinoline-3-carbohydrazone derivatives as potential antimycobacterial agents. Bioorg. Med.
Chem. Lett. 2010, 20, 1040–1044.
25. Thomas, K.D.; Adhikari, A.V.; Telkar, S.; Chowdhury, I.H.; Mahmoode, R.; Pal, N.K.; Rowd, G.;
Sumesh, E. Design, synthesis and docking studies of new quinoline-3-carbohydrazide derivatives
as antitubercular agents, Eur. J. Med. Chem. 2011, 46, 5283–5292.
26. Aboul-Fadl, T.; Bin-Jubair, F.A.S.; 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. 2010, 45, 4578–4586.
27. Feng, L.; Liu, M.; Zhang, S.; Chai, Y.; Wang, B.; Zhang, Y.; Lv, K.; Guan, Y.; Guo, H.; Xiao, C.
Synthesis and in vitro antimycobacterial activity of 8-OCH3 ciprofloxacin methylene and ethylene
isatin derivatives. Eur. J. Med. Chem. 2011, 46, 341–348.
28. Feng, L.; Liu, M.; Wang, B.; Chai, Y.; Hao, X.; Meng, S.; Guo, H. Synthesis and in vitro
antimycobacterial activity of balofloxacin ethylene isatin derivatives. Eur. J. Med. Chem. 2010,
45, 3407–3412.
29. Sriram, D.; Yogeeswari, P.; Basha, J.S.; Radhab, D.R.; Nagaraja, V. Synthesis and antimycobacterial
evaluation of various 7-substituted ciprofloxacin derivatives. Bioorg. Med. Chem. 2005, 13,
5774–5778.
30. AbouI-Fadl, T.; Mohammed, F.A.; Hassan, E.A. Synthesis, antitubercular activity and
pharmacokinetic studies of some schiff bases derived from 1-alkylisatin and isonicotinic acid
hydrazide (INH). Arch. Pharm. Res. 2003, 26, 778–784.
31. Collins, L.A.; Franzblau, S.G. Microplate Alamar Blue Assay versus BACTEC 460
System for High-Throughput Screening of Compounds against Mycobacterium tuberculosis and
Mycobacterium avium. Antimicrob. Agents Chemother. 1997, 41, 1004–1009.
32. Christensen, H.; Garton, N.; Harobin, R.; Minnikin, D.E.; Barer, M.R. Lipid domains of
mycobacteria studied with fluorescent molecular probes. Mol. Microbiol. 1999, 31, 1561–1572.
33. Maccari, R.; Ottana, R.; Vigorita, M.G. In vitro advanced antimycobacterial screening of
isoniazid-related hydrazones; hydrazides and cyanoboranes: Part 14. Bioorg. Med. Chem. Lett.
2005, 15, 2509–2513.
34. Hearn, M.J.; Cynamon, M.H. Design and synthesis of antituberculars: Preparation and evaluation
against Mycobacterium tuberculosis of an isoniazid Schiff base. J. Antimicrob. Chemother. 2004,
53, 185–191.
35. Rodrigues, M.O.; Cantos, J.B.; D’Oca, C.R.; Soares, K.L.; Coelho, T.S.; Piovesan, L.A.;
Russowsky, D.; da Silva, P.A.; D’Oca, M.G. Synthesis and antimycobacterial activity of isoniazid
derivatives from renewable fatty acids. Bioorg. Med. Chem. 2005, 21, 6910–6914.
36. Vistoli, G.; Pedretti, A.; Testa, B. Assessing drug-likeness—What are we missing? Drug Discov.
Today 2008, 13, 285–294.
37. Molinspiration Cheminformatics. Available online: http://www.molinspiration.com (accessed on
15 May 2015).
38. Drug-Likeness and molecular property prediction. Available online: http://www.molsoft.com/mprop/
(accessed on 15 May 2015).

Molecules 2015, 20
8815
39. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.T.;
Bokesch, H.; Kenney, S.; Boyd, M.R. New colorimetric cytotoxicity assay for anticancer-drug
screening. J. Natl. Cancer Inst. 1990, 82, 1107–1112.
40. Eldehna, W.M.; Altoukhy, A.; Mahrous, H.; Abdel-Aziz, H.A. Design, synthesis and QSAR study
of certain isatin-pyridine hybrids as potential anti-proliferative agents. Eur. J. Med. Chem. 2015,
90, 684–694.
41. Al-Saleh, B.; Abdel-Khalik, M.M.; Eltoukhy, A.M.; Elnagdi, M.H. Enaminones in Heterocyclic
Synthesis: A New Regioselective Synthesis of 2,3,6-Trisubstituted Pyridines, 6-Substituted-3-
Aroylpyridines and 1,3,5-Triaroylbenzenes. J. Heterocycl. Chem. 2002, 39, 1035–1038.
42. Abdel-Aziz, H.A.; Aboul-Fadl, T.; Al-Obaid, A.M.; Ghazzali, M.; Al-Dhfyan, A.; Contini, A.
Design, Synthesis and Pharmacophoric Model Building of Novel Substituted Nicotinic Acid
Hydrazones with Potential Antiproliferative Activity. Arch. Pharm. Res. 2012, 35, 1543–1552.
Sample Availability: Not available.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).