Novel polyfunctional pyridines as anticancer and antioxidant agents. Synthesis, biological evaluation and in silico ADME-T Study

Article abstract of DOI:10.1248/cpb.c16-00761

Two series of novel alkoxylated 2-oxo(imino)-3-pyridinecarbonitriles (structurally-relevant to some reported anticancer pyridines with phosphodiesterase 3A (PDE3A) inhibitory activity) were synthesized and evaluated for their in vitro differential tumor c

Full text of DOI:10.1248/cpb.c16-00761

442
Chem. Pharm. Bull. 65, 442–454 (2017)
Vol. 65, No. 5
Regular Article
Novel Polyfunctional Pyridines as Anticancer and Antioxidant Agents.
Synthesis, Biological Evaluation and in Silico ADME-T Study
,a,b
Mona Hany Badr,* Sherif Ahmed Fawzi Rostom,a,b and Mohammed Fouad Radwana,c
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University; P.O. Box 80260, Jeddah
b
21589, Saudi Arabia: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University;
c
Alexandria 21521, Egypt: and Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University; Minia
61519, Egypt.
Received September 23, 2016; accepted February 20, 2017
Two series of novel alkoxylated 2-oxo(imino)-3-pyridinecarbonitriles (structurally-relevant to some re-
ported anticancer pyridines with phosphodiesterase 3A (PDE3A) inhibitory activity) were synthesized and
evaluated for their in vitro differential tumor cell growth inhibitory potential against the breast MCF7,
hepatocellular Hep-G2, colon CACO-2 cell lines, and a normal human foreskin fibroblast Hs27 cell line.
Compounds 8, 16 and 19 displayed recognizable growth inhibitory ability and selectivity towards the breast
MCF7 (LC50 19.15, 17.34 and 14.70µM, respectively) as compared with doxorubicin (LC50 3.94µM). Mean-
while, compounds 8, 15, 16, and 19 revealed a marginal inhibitory effect on the growth of the normal human
foreskin fibroblast Hs27 cell line, beside a distinctive antioxidant potential in the 2,2-diphenyl-1-picrylhy-
drazyl (DPPH) assay. These four compounds were further assessed for their in vitro inhibition of PDE3A (a
current antitumor therapeutic target), where 16 and 19 showed moderate to weak PDE3A inhibitory as com-
pared with milrinone, the positive control. No clear straightforward liaison between the anticancer potential
and PDE3A inhibitory activity could be deduced. Computations of the predicted pharmacokinetic properties,
toxicity effects (ADME-T), drug-likeness and drug scores for the newly developed compounds showed non-
violations of Lipinski’s RO5 and Veber’s criteria for good bioavailability, with a predicted high safety profile.
Key words pyridine; anticancer; antioxidant; phosphodiesterase-3A (PDE3A); pharmacokinetic property and
effect (ADME-T)
The success of the conventional chemotherapeutic agents was reported to suppress cell proliferation of certain leukemia,
in the management of cancer was hampered by their inherent breast, cervical and prostate cancer cell lines.12–14) Subse-
toxicity, harmful side effects together with the evolution of quently, finding out novel specific PDE3 inhibitors became a
drug resistance by tumor cells. Therefore, along the past two prominent selective target for anticancer therapy, which offers
decades there has been a noticeable movement towards the an unprecedented opportunity to rescue healthy cells from the
recognition of the biomolecular aspects of malignancy for the destructive harmful effects of conventional cytotoxic agents.
development of more safe and selective anticancer agents.1,2)
On the other hand, oxidative stress generated by the pre-
Among the various cellular and molecular processes recent- dominance of extravagant reactive oxygen species (ROS) that
ly studied, the impairment of intracellular cyclic nucleotides forcibly offence and destroy different body cells is known to
cAMP and/or guanosine 5′-cyclic monophosphate (cGMP) lead to oxidative damage of DNA; a leading cause of car-
production by excessive expression of phosphodiesterases cinogenesis.15) Moreover, ROS are known to provoke tumor
(PDEs) has been linked to diverse cancer etiologies.3) In other growth and metastasis through induction of angiogenesis,16)
words, modulation of the levels of such cyclic nucleotides therefore, antioxidants capable of scavenging free radicals
through inhibiting their breakdown by PDEs has received par- proved to contribute efficiently in the treatment of many types
ticular attention as a promising approach in tumor therapy.4) of cancer.17)
Eleven families of PDEs (PDE1–11), including many isoforms,
As far as the chemotherapeutic potential is concerned,
have been identified, which vary in their amino acid sequenc- pyridine derivatives have come into view as heterocycles with
es, tissue distribution, substrate affinities, type of inhibitors versatile antimicrobial,18) antitubercular19) and anticancer20–22)
and their bioregulatory pathways.5) Thorough literature survey activities. Most notably, several functionalized pyridine-
revealed that concomitant inhibition of certain PDE isoforms carbonitriles have received much interest for their peculiar
(e.g. PDE3, PDE4 and PDE5) led to elevation in the intracellu- antineoplastic23–25) as well as antioxidant26,27) activities. In
lar cAMP and/or cGMP levels which was reported to retrieve this context, various 2-oxopyridine-3-carbonitriles and their
regular intracellular signaling, inhibit tumor cell growth, 2-amino isosteres have revealed promising molecular-based
retard angiogenesis, induce apoptosis and cell cycle arrest in anticancer activities being able to interfere with Pim-1 proto-
malignant rather than normal cells.6–8) In particular, beside oncogene, serine/threonine kinase (PIM1) Kinase and survivin
the documented cardiovascular contributions of the PDE3 protein which played articular roles in suppressing cancer-
isozymes (PDE3A and B),9) overexpression of PDE3s has been ous cell survival and proliferation, in addition to induction
accounted for the induction of several malignancies includ- of apoptosis.28,29) Several in silico molecular docking and in
ing osteosarcoma, squamous cell carcinoma, melanoma, and vitro testing studies have emphasized the importance of the
malignant salivary gland cells.10,11) Moreover, PDE3 inhibition pyridinecarbonitrile scaffold as novel PDE3 inhibitors and
*To whom correspondence should be addressed. e-mail: badrmona@hotmail.com
© 2017 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
443
Fig. 1. 2-Oxo(imino)pyridine-3-carbonitrile-Based PDE3 Inhibitors
Milrinone, I30), II34), III35) and the proposed structure modifications in the scaffold to obtain the target compounds A–C.
anticancer agents.30–33) Among these, compound I (Fig. 1) linkage based on literature reports revealing the remarkable
stemmed as the most potent PDE3A inhibitor with significant contribution of this functionality in diverse anticancer activi-
cytotoxic potential.30) Furthermore, another relevant series be- ties.45–47) Such substitution pattern was thought to be useful in
longing to the previously-descibed skeleton with a C4 2-alkyl- assisting the interaction of the target molecules with different
thio-1-benzyl-5-imidazolyl moiety as a basic counterpart was cellular targets through various intermolecular forces.
reported,34) where in silico, in vitro PDE3A and anticancer
The differential in vitro anticancer potential of the target
studies suggested the existence of a clear correlation between compounds was investigated against a panel of three human
their biological activities and PDE3A’s molecular binding tumor cell lines including the Caucasian breast adenocarcino-
mode. Among which compound II exhibited the strongest ma MCF7, hepatocellular carcinoma Hep-G2, colon carcinoma
PDE3A inhibition with IC50=3.76 nM (Fig. 1). Additionally, a CACO-2, and normal human foreskin fibroblast Hs27 cell
molecular study of a series of 2-pyridones (III; Fig. 1) with lines. In a trial to search for a plausible mechanism behind the
a substitution pattern relevant to the PDE3 inhibitor milri- anticancer efficacy of the most active compounds, their anti-
none (Fig. 1), revealed a considerable affinity of five analogs oxidant activity was determined using the 2,2-diphenyl-1-pic-
to the PDE3A isozyme.35) In previous publications, during rylhydrazyl (DPPH) assay, and the in vitro inhibitory activity
our continuous efforts to find out novel pyridine-containing against PDE3A isozyme was measured. Additionally, an in
lead structures with diverse chemotherapeutic activities, the silico computation of the molecular properties, physicochemi-
substituted 2-oxo(imino)pyridine-3-carbonitrile scaffold have cal profile, drug score and drug-likeness of the biologically
emerged as a corner stone for building potential broad-spec- active compounds was performed to predict their pharmaco-
trum antitumor and/or antimicrobial agents.36–41)
kinetic and toxicity properties (ADME-T), and to assess their
In this investigation, we report the synthesis of some novel suitability to serve as possible orally-active dug candidates.
polyfunctionalized pyridines with the general structures A–C
(Fig. 1). The targeted compounds were designed so as to pre-
serve the 2-oxopyridine-3-carbonitrile scaffold, while intro-
Results and Discussion
Chemistry The synthetic strategies adopted for the
ducing additional cyano and/or carboxylate functionalities as synthesis of the intermediates and target compounds
H-bond acceptors, and OH and/or NH2 groups as H-bond do- are depicted in Charts 1, 2 and 3. In Chart 1, the key
nors. Alkoxylated aryl rings (either directly hocked to the pyr- intermediates (E)-N′-(3,4-disubstituted benzylidene)-2-cya-
idinecarbonitrile moiety at C1, C4 and/or C6, or separated by noacetohydrazide 1 and 2 were prepared by reacting cya-
an azavinyl linkage at N1) were selected as basic counterparts noacetic acid hydrazide with the appropriate substituted
owing to their reported augmentation of several chemothera- benzaldehyde.48) Refluxing 1 and 2 with acetyl acetone, ethyl
peutic activities through glorification of the overall lipophilic- acetoacetate, ethyl cyanoacetate or malononitrile in absolute
ity of the molecules, beside their documented role in exert- ethanol containing catalytic amount of piperidine afforded
ing antioxidant potential.42–44) The selection of the azavinyl the targeted (E)-1-((3,4-disubstituted benzylidene)amino)-4,6-

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Reagents and conditions: a) Ethanol, r.t., 1h; b) Acetyl acetone, piperidine, ethanol, reflux, 5h.; c) Ethyl acetoacetate, piperidine, ethanol, reflux, 4h; d) Ethyl cyanoac-
etate, piperidine, ethanol, reflux, 3h; e) Malononitrile, piperidine, ethanol, reflux, 3h.
Chart 1. Synthesis of Compounds 1–10
Reagents and conditions: a) Ethyl ethoxymethylenecyanoacetate, potassium carbonate, ethanol, reflux, 3h; b) 2-Arylidenemalononitrile, piperidine, ethanol, reflux, 3h.; c)
2-Arylidenecyanoacetae, sodium ethoxide, ethanol, reflux, 2h.
Chart 2. Synthesis of Compounds 11–20
disubstituted-2-oxo-1,2-dihydropyridine-3-carbonitriles 3–8.
Shifting to Chart 3, (E)-1-aryl-3-(3,4,5-trimethoxyphenyl)-
In Chart 2, reacting 1 and 2 with ethyl ethoxymethylene- prop-2-en-1-ones 21–23 were served as key intermedi-
cyanoacetate in the presence of anhydrous K2CO3 produced ates. They were prepared in excellent yields by condensing
the respective (E)-1-((3,4-disubstituted benzylidene)amino)-6- 3,4,5-trimethoxybenzaldehyde with the appropriate substi-
hydroxy-2-oxo-1,2-dihydropyridine-3,5-dicarbonitriles 11 and tuted acetophenone in the presence of potassium hydroxide
12. On the other hand, cyclocondensation of 1 and 2 with according to a reported literature procedure.49) Refluxing
the appropriate 2-arylidenemalononitrile in the presence of 21–23 with either ethyl cyanoacetate or malononitrile in the
piperidine resulted in the formation of 2-oxo-1,2-dihydropyr- presence of ammonium acetate yielded the corresponding
idine-3,5-dicarbonitrile derivatives 13–16. Whereas, the ethyl 2-oxo(imino)-1,2-dihydropyridines 24–28.
carboxylate analogs 17–20 were obtained by refluxing 1 and
2 with 2-arylidenecyanoacetate in the presence of sodium
ethoxide.

Vol. 65, No. 5 (2017)
Chem. Pharm. Bull.
445
Reagents and conditions: a) 6% ethanolic KOH, r.t., 24h; b) Ethyl cyanoacetate, ammonium acetate, ethanol, reflux, 12h; c) Malononitrile, ammonium acetate, ethanol,
reflux, 24h.
Chart 3. Synthesis of Compounds 21–28
Table 1. Cytotoxic Effects (LC50; µM)a) of the Active Compounds on
Three Human Tumor and One Normal Non-transformed Cell Lines Using
the MTT Assay
of the tested tumor cell lines particularly the breast MCF7,
whereas the rest of the investigated compounds were totally
inactive. Moreover, the three tested human tumor cell lines
exhibited a variable degree of sensitivity profiles towards the
Human normal non-
transformed cell line
Human tumor cell lines
active compounds. For instance, the MCF7 cell line showed a
recognizable sensitivity against compounds 8, 16 and 19 (LC50
19.15, 17.34 and 14.70µM, respectively) which represented
nearly 20–27% of the activity of doxorubicin (LC50 3.94 µM).
A moderate cytotoxic potential was displayed by compounds
10, 15 and 25 (LC50 41.04, 30.67 and 35.42µM, respectively),
whereas the rest of the active compounds showed weak ef-
ficiencies against the same cell line. On the other hand, the
growth of Hep-G2 cell line was found to be mildly inhibited
by five compounds (8, 10, 15, 16 and 25) with LC50 range
45.78–125.55 µM, among which the analogs 8 and 16 were the
most active (LC50 48.95 and 45.78µM, respectively). Regard-
ing the CACO-2 cell line, it was proved to be the least sensi-
tive tumor cell line as its growth was affected only by four
analogs (8, 10, 15 and 16), with LC50 range 111.63–226.73µM,
representing nearly 10–20% of the activity of doxorubicin
(LC50 22.81 µM) against the same cell line. Taking both the
growth inhibitory potential and the spectrum of cytotoxic
activity as preference parameters, compounds 8 and 16 have
Cpd. No.
MCF7b) Hep-G2c) CACO-2d)
Hs27e)
f)
6
116.73
19.15
41.04
30.67
17.34
14.70
35.42
70.23
3.94
—
—
NDg)
>500
ND
8
48.95
109.33
125.55
45.78
—
121.71
226.73
142.00
111.63
—
10
15
16
19
25
28
Dox.h)
>500
>500
>500
ND
111.64
—
—
—
ND
3.11
22.81
ND
a) LC50: Lethal concentration of the compound which causes death of 50% of
cells in 24h (µM). b) MCF7 (Caucasian breast adenocarcinoma cell line). c) Hep-G2
(Human hepatocellular carcinoma cell line). d) CACO-2 (Human colon carcinoma
cell line). e) Hs27 (normal non-transformed human foreskin fibroblast cell line). f)
Totally inactive against this cell line. g) ND: not determined. h) Doxorubicin: positive
control cytotoxic agent.
Biology
In Vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium stemmed as the most distinguished members in this study,
Bromide (MTT) Cytotoxicity Assay with special effectiveness against the breast MCF7 tumor cell
The newly synthesized target compounds 3–20 and 24–28 line, as compared with doxorubicin. Additionally, a marginal
were subjected to evaluation of their in vitro cytotoxic effect growth inhibitory effect on the Hs27 normal cell line (LC50
utilizing the standard MTT method50,51) against a panel of values >500µM) was displayed by the analogs 8, 15, 16 and
three human tumor cell lines namely; Caucasian breast ad- 19 (which showed distinctive growth inhibitory potential on
enocarcinoma MCF7, hepatocellular carcinoma Hep-G2, colon the breast MCF7 tumor cell line), confirming their differential
carcinoma CACO-2, and a normal human foreskin fibroblast cytotoxicity towards tumor rather than normal cell lines.
Hs27 cell line. The results are presented in Table 1 as LC50
(µM) which is the lethal concentration of the compound that
causes death of 50% of the cells in 24h.
Structure–Activity Correlation
A close examination of the structures of the active com-
pounds revealed that they belong to two series: 1-((3,4-di-
The obtained data revealed that, eight compounds namely; substituted benzylidene)amino)-4,5,6-trisubstituted-2-oxo-1,2-
6, 8, 10, 15, 16, 19, 25 and 28 were able to affect the viability dihydropyridine-3-carbonitriles 3–20 (Charts 1, 2) and the

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Table 2. DPPH Radical-Scavenging Activity of the Cytotoxic-Active
Compounds 6, 8, 10, 15, 16, 19, 25 and 28
4,6-disubstituted-2-oxo(imino)-1,2-dihydropyridines
24–28
(Chart 3). In general, compounds belonging to the first series
comprising the N-azavinylpyridine scaffold displayed better
cytotoxic effects than the second one. Besides, the biological
activity proved to be governed with the type and electronic
nature of the substituents. For instance, compounds compris-
ing the benzodioxole moiety (R1, R2=OCH2O), displayed
better cytotoxic effects when compared with the 3,4-dime-
thoxyphenyl congeners. In addition, the introduction of H-
bond donors (e.g., hydroxyl and amino groups) and H-bond ac-
ceptors (cyano and/or carboxylate functions), together with the
pivotal 2-oxo and 3-carbonitrile groups on the pyridine ring,
seems to modulate the targeted cytotoxic activity.
Among the first series, in spite of the inactivity shown
by the 4,6-dimethyl derivatives (3 and 4), yet the 6-hy-
droxy analog 6 (R1, R2=OCH2O) displayed a weak cyto-
toxic effect against MCF7 cell line. Introducing an additional
amino group at the pyridine C4 furnished compound 8 (R1,
R2=OCH2O), with a noticeable enhancement in both cytotoxic
potency and spectrum as shown from its capability of inhib-
iting the growth of the three tested cell lines, with activity
percentages lying between 6–20% of that of doxorubicin. This
improvement might be ascribed to the increase in H-bond
forming ability with certain tumor cell targets caused by both
the hydroxyl and amino group. However, isosteric replacement
of the 6-hydroxyl functionality with another amino group as
shown in the 4,6-diamino derivative 10 (R1, R2=OCH2O),
DPPH radical scavenging activity
Compound Concentration (M)
(% of control)a)
Controlb)
BHTc)
—
100 0.05
10−4
10−3
10−4
10−3
10−4
10−3
10−4
10−3
10−4
10−3
10−4
10−3
10−4
10−3
10−4
10−3
10−4
10−3
20
36
14
26
17
31
18
32
15
28
19
34
12
23
10
21
9
0
1
1
2
1
2
1
1
0
2
1
2
2
1
2
1
0
2
6
8
10
15
16
19
25
28
19
a) Values are recorded as the mean of three independent experiments S.D. b)
Control: DPPH radical solution in methanol. c) BHT: Butylated hydroxyl toluene
(reference standard antioxidant).
resulted in about two-fold reduction in the cytotoxic potential (SA%) in relation to a control and butylated hydroxyl toluene
against MCF7 and Hep-G2 cell lines, when compared with the (BHT) was utilized as a reference standard antioxidant agent
analog 8. This could be attributed to the reduction in the elec- (Table 2).
tronegativity of the amino group when compared with that of
At the 10−3 M concentration level, the substituted 2-oxopyri-
the hydroxyl one. On the other hand, while the 3,5-dicarboni- dine-3-carbonitriles 8, 10 and 16 (SA 31, 32 and 34%, respec-
trile derivatives 11 and 12 were deprived of any cytotoxic effi- tively) were nearly equiactive with BHT; the standard antioxi-
cacy, yet increasing the lipophilic character by introducing an dant utilized in this assay (SA 36%). Whereas, the analogs 6,
alkoxylated phenyl substituent at the pyridine C4, yielded two 15 and 19 displayed an appreciable antioxidant potential (SA
active compounds 15 (R1, R2=OCH2O; R3=OCH3, R4=H) and 26, 28 and 23%, respectively). Nevertheless, the structure
16 (R1, R2=OCH2O; R3=R4=OCH3), among which the ana- variants 25 and 28 displayed a relatively weak antioxidant ac-
log 16 displayed acceptable cytotoxic spectrum and potency tivity (scavenging activity 21 and 19%), when compared with
profiles, showing 23 and 25% of the activity of doxorubicin compounds 8, 10 and 16, the most active antioxidants in this
against MCF7 and CACO-2 cell lines, respectively. Surpris- study. In a similar pattern, the same order of DPPH radical
ingly, exchanging the essential C3 carbonitrile functionality in scavenging potential was expressed at the 10−4 M concentration
15 with a carboxylate as in 19 (R1, R2=OCH2O; R3=OCH3; level, with a scavenging activity range 9–19%, when com-
R4=H), resulted in a two-fold improvement in the activ- pared with BHT (scavenging activity 20%).
ity and selectivity towards the breast MCF7 cell line (14.7 vs
30.67µM, respectively), however, with loss of activity against
the other tested tumor cell lines.
Determination of the in Vitro PDE3A Inhibitory Activity
The most active anticancer compounds 8, 15, 16 and 19
were assessed for their in vitro inhibition of PDE3A (a pos-
In the second series (Chart 3), structure modifications in- sible molecular target for the antitumor activity) using fluores-
volved the exchange of the benzylidineamino counterpart at cence polarization (FP). The basic principle for this assay is
position-1 with a lipophilic phenyl moiety at position-6, while to cleave the fluorescently labeled c-AMP (substrate) into its
conserving the essential 2-oxo (or 2-amino) and the 3-carbo- respective nucleotide by PDE3A. Binding agent is then added
nitrile functionalities, together with the 4-substituted phenyl to produce a change in FP that can be measured using a fluo-
moiety. Such transformation furnished two mildly active com- rescence reader. Milrinone was employed as a positive control
pounds 25 and 28, with an obvious reduction in both cytotoxic and the IC50 values were determined by testing a range of 10
potential and spectrum (Table 1).
DPPH Radical Scavenging Antioxidant Assay
Compounds 6, 8, 10, 15, 16, 19, 25 and 28 which exhibited
concentrations (0.01–100µM), each in duplicate and the results
were shown in Table 3.
The results revealed that all the investigated four com-
cytotoxic activity, were further evaluated for their antioxidant pounds showed moderate to weak PDE3A inhibitory activity
potential through their ability to scavenge the DPPH radical, (IC50 range 34.78–85.33µM), as compared with milrinone (IC50
according to a modified procedure from that described by 12.03 µM), the reference positive control utilized in this assay.
Blois.52) The antioxidant potential of the tested compounds Although compounds 8, 16 and 19 displayed nearly equal
was calculated as the percentage DPPH scavenging activity LC50 values against the breast MCF7 cancer cell line (19.15,

Vol. 65, No. 5 (2017)
Chem. Pharm. Bull.
447
Table 3. Anticancer Activity against the MCF7 Breast Cancer Cell Linea) and in Vitro Inhibitory Effect on PDE3A Enzymeb) of the Most Active Com-
pounds 8, 15, 16 and 19
17.34 and 14.7µM, respectively), yet their ability to inhibit the properties, affinity to proteins, reactivity, toxicity and meta-
PDE3A enzyme is variable and not going in hand with the bolic stability.
anticancer results. In particular, although compound 19 proved
In this context, a computational study for the biologically
to be the most active anticancer agent against the MCF7 cell active compounds (5–10, 12–17, 19, 20, 24–26 and 28) utiliz-
line (LC50 14.7 µM), yet it did not express the highest PDE3A ing the Molinspiration online property calculation toolkit,55)
inhibitory activity (IC50 49.15 µM) which represented about was carried out to determine the Lipinski’s molecular proper-
25% of the activity of milrinone. On the contrary, the analog ties and the number of rotatable bonds (nROTB), together with
16 with a relatively lesser anticancer potential (LC50 17.34 µM), the topographical polar surface area (TPSA; a sum of polar
displayed an obviously higher PDE3A inhibitory potential atoms’ surfaces: a descriptor for drug absorption, penetrabil-
(IC50 34.78 µM; about 35% of the activity of milrinone).
ity and bioavailability), the percentage of absorption (ABS%)
Since the PDE3A and the tumor growth inhibitory activi- calculated as (ABS%=109−0.345×TPSA)56) and the molecular
ties are not going in hand, such enzymatic inhibition would volume (a determinant of the transport characteristics).
be considered as a possible (but not the principle) mechanism
The results presented in Table 4 revealed that all the tested
of action behind their anticancer potential, therefore other compounds comply with Lipinski’s rule of 5, where LogP val-
phosphodiesterases and/or different targets might participate ues ranged between 0.45–4.15 (<5), molecular weight (MW)
in the anticancer activity. This finding could be reinforced by range 297–490 (<500), HBA range 5–10 (≤10) and HBD
the inactivity of the positive control milrinone to inhibit the range 1–4 (<5), suggesting that these compounds would not
growth of some tumor cell line despite its inhibition of PDE3 be expected to cause problems with oral bioavailability. The
as reported by Abadi et al.30)
In Silico Evaluation of the ADME-T and Drug-Likeness
only exception was noted for compound 20 which showed
one violation to the RO5 (HBD >10). Moreover, all the tested
Drug-likeness is a term that describes an integrated equi- compounds showed nROTB values of 2–9 (<10) indicating ac-
librium between multiple molecular properties and structure ceptable molecular flexibility with consequent expected good
features that define whether a particular compound is compa- permeability and oral bioavailability. Additionally, all the
rable to already known drugs. Among the common principles evaluated compounds showed TPSA range 84.36–138.19Å2
applied to evaluate of the drug-like properties of a compound, (<140 Å2), indicating good permeability and transport of the
stemmed prominently the Lipinski’s rule of 5 (RO5)53) and compounds in the cellular plasma membrane; except for the
Veber’s criteria.54) These properties comprise hydrophobicity, analogs 14, 16 and 20 which were not so far from the ideal
electronic distribution, hydrogen-bonding capability, molecule value (PSA 144.91, 144.91 and 147.43Å2, respectively). Fur-
size and flexibility that would affect the behavior of a mol- thermore, all the tested compounds exhibited a considerable
ecule in a living system including bioavailability, transport % ABS range 58.14–79.9%, which is a designation of good

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Chem. Pharm. Bull.
Vol. 65, No. 5 (2017)
Table 4. In Silico ADME-T Calculations, Lipinski’s Parameters Drug-Likeness, and Drug Scorea, b) of the Synthesized Compounds
Lipinski’s parameters
Cpd.
nROTBh)
TPSAi) % ABS j) Volumek) Log S l) Drug-likeness Drug-score
LogPc)
MWd)
HBAe)
HBD f)
Violationsg)
5
6
1.72
1.96
0.75
0.99
0.45
0.70
1.27
2.50
2.10
2.75
2.34
2.96
3.20
2.79
3.31
3.99
2.96
4.15
313.31
297.27
314.30
298.26
313.32
297.27
308.25
429.44
459.46
413.39
443.42
476.49
460.45
490.47
362.38
396.83
422.44
395.85
7
7
1
1
3
3
4
4
1
2
2
2
2
2
2
2
1
1
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
4
2
4
2
4
2
2
6
7
4
5
9
7
8
5
5
7
5
96.86
95.15
75.58
76.17
67.22
66.61
64.61
64.61
67.38
62.19
59.01
62.19
59.01
61.32
61.32
58.14
79.90
79.90
73.53
77.81
275.59
248.43
270.32
243.16
273.59
246.43
248.73
376.12
401.66
348.95
374.5
−4.12
−4.80
−4.00
−4.68
−4.08
−4.75
−4.94
−5.32
−5.34
−6.00
−6.01
−5.07
−5.75
−5.77
−3.47
−4.21
−3.51
−3.39
2.72
0.93
0.77
0.62
0.73
0.52
0.72
0.51
0.61
0.35
0.33
0.29
0.29
0.17
0.16
0.15
0.47
0.41
0.45
0.43
7
8
121.1
1.33
8
8
122.88
128.68
128.68
120.65
135.68
144.91
135.68
144.91
138.19
138.19
147.43
84.36
0.47
9
8
1.23
10
12
13
14
15
16
17
19
20
24
25
26
28
8
−0.56
0.88
8
9
2.99
10
9
2.59
1.26
10
10
10
11
6
1.84
420.59
393.43
418.97
324.33
337.86
375.42
341.02
−5.72
−7.46
−6.92
2.44
5
84.36
3.03
8
102.82
90.41
3.99
6
1.60
a) Molinspiration chemoinformatics property calculator (2014). b) OSIRIS property explorer (2014). c) Partition coefficient. d) Molecular weight. e) Number of H-Bond
acceptors (O and N atoms). f) Number of H-Bond donors (OH and NH groups). g) number of Rule of 5 violations. h) number of rotatable bonds. i) Topological polar surface
area. j) Absorption %. k) Molecular volume. l) Aqueous solubility prediction.
bioavailability upon oral administration. It is to be noted that a panel of three human tumor cell lines. Eight compounds
TPSA is inversely proportional to %ABS e.g. the analog 25 (6, 8, 10, 15, 16, 19, 25 and 28) displayed growth inhibitory
possesses the maximum absorption (79.9%) whereas its cor- capability towards the tested cell lines, among which 8, 16
responding TPSA was least among the series (84.36Å2).
and 19 displayed recognizable growth inhibitory ability and
On the other hand, the OSIRIS Property Explorer (2014: selectivity towards the breast MCF7 (LC50 19.15, 17.34 and
Version 2) software57) was utilized to calculate the aqueous 14.70 µM, respectively) as compared with doxorubicin (LC50
solubility (LogS) of the tested compounds (being significantly 3.94 µM). Meanwhile, compounds 8, 15, 16, and 19 revealed a
affecting absorption as well as distribution characteristics), marginal inhibitory effect on the growth of the normal human
where they gave moderate LogS values ranging between foreskin fibroblast Hs27 cell line, beside a distinctive anti-
−3.39 and −6.01mol/L. Finally, the same OSIRIS software oxidant potential in the DPPH assay. These four compounds
was employed to determine the overall and drug score val- were further assessed for their in vitro inhibition of PDE3A (a
ues which is an expression representing integration of drug- current antitumor therapeutic target), where 16 and 19 showed
likeness, several physicochemical parameters and toxicity moderate to weak PDE3A inhibitory (IC50 34.78 and 49.15µM,
probabilities in one numerical value that can be utilized to respectively) as compared with milrinone (IC50 12.03 µM), the
foresee compound’s ability to act as a drug candidate. A posi- positive control. In general, the antioxidant potential showed a
tive value for drug-likeness and drug score indicates that the considerable alignment with the cytotoxic experimental data
compound contains fragments that are often present in most suggesting a possible role of free radical scavenging in the
of currently used drugs. The results revealed that about 80% antitumor effect of the active compounds. On the other hand,
of the evaluated compounds gave positive values for drug- the PDE3A and the tumor cell growth inhibitory activities
likeness lying between 0.47 and 3.99, excluding four com- are not in parallel, indicating that inhibition of PDE3A would
pounds (10, 17, 19 and 20) which displayed negative values not be the principle mechanism, but other molecular targets
of drug-likeness (between −7.46 and −0.56). Additionally, all might participate in the anticancer activity. Collectively, the
the evaluated compounds (including those compounds with observed in vitro cytotoxic and antioxidant potentials of the
negative drug-likeness scores) have displayed positive values active compounds were engaged with the compounds compris-
ranged from 0.15 to 0.77 in the drug score calculation (Table ing the N-azavinylpyridine scaffold, and were modulated by
4). According to OSIRIS findings, prediction of the expected the H-bond forming functionalities and/or substitution of the
toxicity risks pointed out that none of the twenty three investi- pyridine ring with benzodioxole moieties. These findings are
gated compounds would exert tumorigenic, mutagenic, irritant concordant with previous studies that reported the important
or reproductive toxicity.
roles displayed by the lipophilicity and the excellent bioavail-
ability of the benzodioxole fragment in relevant biological ac-
tivities.58–61) Moreover, in silico computation of the predicted
Conclusion
The main objective of this research work was to synthesize ADME-T of the newly developed compounds showed non-
two series of novel alkoxylated 2-oxo(imino)-3-pyridinecarbo- violations of Lipinski’s RO5 and Veber’s criteria, suggesting
nitriles supported with different pharmacophores, to be evalu- their liability to act as new orally-active drug candidates with
ated for their in vitro differential anticancer potential against a predicted high safety profile. Finally, the selective anticancer

Vol. 65, No. 5 (2017)
Chem. Pharm. Bull.
449
potential of the active compounds against the breast MCF7 (m, 3H, benzodioxole-H), 7.80 (s, 1H, N=CH). 13C-NMR
tumor cell line and their reliable antioxidant activity, together (600MHz, [D6]DMSO) δ: 18.9 (CH3), 25.9 (CH3), 89.9 (O–
with their in vitro PDE3A inhibitory activity, make such type CH2–O), 119.8 (CN), 104.8, 110.5, 114.0, 123.5, 128.9, 132.4,
of polyfunctional pyridines the appropriate matrix for future 135.2, 136.8, 150.5, 156.9 (Ar C), 165.0 (C=N), 168.6 (CO). IR
derivatization and optimization hoping to determine the scope (KBr) cm−1: 2223 (CN), 1675 (C=O). MS m/z (%): 295 (21)
and limitations of their bioactivities, and to find out more [M]+. Anal. Calcd for C16H13N3O3: C, 65.08; H, 4.44; N, 14.23.
active and selective lead pyridine derivatives as anticancer Found: C, 65.19; H, 4.17; N, 13.97.
agents with possible antioxidant and/or PDE3A inhibitory
activities.
General Procedure for the Synthesis of (E)-1-((3,4-Di-
methoxybenzylidene)amino)-6-hydroxy-4-methyl-2-oxo-1,2-
dihydropyridine-3-carbonitrile (5) and (E)-1-((Benzo[1,3]-
dioxol-5-ylmethylene)amino)-6-hydroxy-4-methyl-2-oxo-1,2-
Experimental
Chemistry Melting points (mps) were determined in open dihydropyridine-3-carbonitrile (6) To a solution of 1 or
glass capillaries on a Gallenkamp melting point apparatus and 2 (2mmol) in absolute ethanol (10mL) containing four drops
were uncorrected. The IR spectra were recorded on Shimadzu of piperidine, ethyl acetoacetate (0.26g, 2mmol) was added.
FT-IR 8400S infrared spectrophotometer using the KBr pellet The reaction mixture was heated under reflux for 4h then al-
1
technique. H-NMR and 13C-NMR spectra were recorded on lowed to attain room temperature. Working up of the reaction
a Bruker WM-600 FT NMR spectrometer using tetramethyl- mixture was carried out as described under 3 and 4. The sepa-
silane (TMS) as an internal standard and dimethyl sulfoxide rated solid product was filtered, washed with water, dried and
(DMSO)-d6 as a solvent (Chemical shifts in δ, ppm). Splitting recrystallized from ethanol.
patterns were designated as follows: s: singlet; d: doublet; t
triplet; q: quartet; m: multiplet. Mass spectra were recorded methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile (5)
on Agilent LC-MS 6120 single quad. Elemental analyses were
Yield: 68%. mp: 160–161°C. 1H-NMR (600MHz,
(E)-1-((3,4-Dimethoxybenzylidene)amino)-6-hydroxy-4-
performed on a 2400 PerkinElmer, Inc. Series 2 analyser and [D6]DMSO) δ: 2.18 (s, 3H, CH3), 3.76, 3.81 (2s, 6H, 2 OCH3),
the found values were within 0.4% of the theoretical values. 5.68 (s, 1H, pyridine-C5-H), 6.92–7.15 (m, 3H, dimethoxyphe-
Follow up of the reactions and checking the homogeneity of nyl–H), 8.01 (s, 1H, N=CH). 13C-NMR (600MHz, [D6]DMSO)
the compounds were made by TLC on silica gel-protected alu- δ: 19.3 (CH3), 57.8 (OCH3), 58.6 (OCH3), 118.9 (CN), 104.1,
minium sheets (Type 60 F254, Merck) and the spots were de- 111.6, 115.2, 122.8, 127.8, 133.4, 135.6, 137.2, 155.6, 157.2 (Ar
tected by exposure to UV-lamp at λ 254. The synthesis of (E)- C), 164.8 (C=N), 168.5 (CO). IR (KBr) cm−1: 3430 (OH), 2220
N′-(3,4-disubstituted benzylidene)-2-cyanoacetohydrazide 1, (CN), 1685 (C=O). MS m/z (%): 314 (48) [M+H]+. Anal. Calcd
248) and (E)-1-aryl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one for C16H15N3O4: C, 61.34; H, 4.83; N, 13.41. Found: C, 61.15;
21–2349) were performed according to reported literature pro- H, 4.65; N, 13.15.
cedures.
General Procedure for the Synthesis of (E)-1-((3,4- methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile (6)
Dimethoxybenzylidene)amino)-4,6-dimethyl-2-oxo-1,2-
Yield: 65%. mp: 240–242°C. 1H-NMR (600MHz,
(E)-1-((Benzo[1,3]dioxol-5-ylmethylene)amino)-6-hydroxy-4-
dihydropyridine-3-carbonitrile (3) and (E)-1-((Benzo[1,3]- [D6]DMSO) δ: 2.39 (s, 3H, CH3), 5.79 (s, 1H, pyridine-C5–H),
dioxol-5-ylmethylene)amino)-4,6-dimethyl-2-oxo-1,2-di- 6.08 (s, 2H, O–CH2–O), 7.09–7.34 (m, 3H, benzodioxole–H),
hydropyridine-3-carbonitrile (4) A solution of 1 or 2 8.06 (s, 1H, N=CH), 11.39 (s, 1H, OH, D2O exchangeable).
(2mmol) and acetylacetone (0.20g, 2mmol) in absolute etha- 13C-NMR (600MHz, [D6]DMSO) δ: 19.5 (CH3), 89.7 (O–
nol (10mL) containing four drops of piperidine, was heated CH2–O), 119.6 (CN), 105.2, 110.5, 114.0, 123.5, 128.9, 132.4,
under reflux for 5h. After cooling to room temperature, the 135.2, 136.8, 154.9, 156.1 (Ar C), 165.5 (C=N), 167.0 (CO). IR
reaction mixture was poured onto crushed ice containing few (KBr) cm−1: 3343 (OH), 2226 (CN), 1673 (C=O). MS m/z (%):
drops of dilute hydrochloric acid. The separated solid product 298 (33) [M+H]+. Anal. Calcd for C15H11N3O4: C, 60.61; H,
was filtered, washed with water, dried and recrystallized from 3.73; N, 14.14. Found: C, 60.43; H, 3.89, N; 14.10.
dioxane.
(E)-1-((3,4-Dimethoxybenzylidene)amino)-4,6-dimethyl-2- 1-((3,4-dimethoxybenzylidene)amino)-6-hydroxy-2-oxo-
oxo-1,2-dihydropyridine-3-carbonitrile (3)
1,2-dihydropyridine-3-carbonitrile (7) and (E)-4-Amino-1-
General Procedure for the Synthesis of (E)-4-Amino-
Yield: 76%. mp: 150–152°C. 1H-NMR (600MHz, ((benzo[1,3]dioxol-5-ylmethylene)amino)-6-hydroxy-
[D6]DMSO) δ: 2.01 (s, 3H, CH3), 2.38 (s, 3H, CH3), 3.71, 3.82 2-oxo-1,2-dihydropyridine-3-carbonitrile (8) To a solution
(2s, 6H, 2 OCH3), 5.69 (s, 1H, pyridine-C5–H), 6.92–7.23 (m, of 1 or 2 (2mmol) in absolute ethanol (10mL) containing four
3H, dimethoxyphenyl-H), 8.02 (s, 1H, N=CH). 13C-NMR drops of piperidine, ethyl cyanoacetate (0.23g, 2mmol) was
(600MHz, [D6]DMSO) δ: 19.9 (CH3), 27.4 (CH3), 56.9 (OCH3), added. The reaction mixture was heated under reflux for 3h
57.9 (OCH3), 118.8 (CN), 106.8, 110.8, 114.0, 123.5, 129.2, then allowed to attain room temperature. Working up of the
132.4, 135.2, 137.2, 152.5, 157.2 (Ar C), 164.0 (C=N), 168.9 reaction mixture was carried out as described under 3 and 4.
(CO). IR (KBr) cm−1: 2227 (CN), 1687 (C=O). MS m/z (%): The separated solid product was filtered, washed with water,
311 (28) [M]+. Anal. Calcd for C17H17N3O3: C, 65.58; H, 5.50; dried and recrystallized from the proper solvent.
N, 13.50. Found: C, 65.43; H, 5.32; N, 13.61.
(E)-1-((Benzo[1,3]dioxol-5-ylmethylene)amino)-4,6- hydroxy-2-oxo-1,2-dihydropyridine-3-carbonitrile (7)
(E)-4-Amino-1-((3,4-dimethoxybenzylidene)amino)-6-
1
dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile (4)
Yield: 70%. mp: 192–194°C (MeOH). H-NMR (600MHz,
Yield: 82%. mp: 190–192°C. 1H-NMR (600MHz, [D6]DMSO) δ: 3.73, 3.79 (2s, 6H, 2 OCH3), 5.66 (s, 1H,
[D6]DMSO) δ: 2.09 (s, 3H, CH3), 2.39 (s, 3H, CH3), 5.82 pyridine-C5–H), 6.73–7.02 (m, 3H, dimethoxyphenyl–H),
(s, 1H, pyridine-C5–H), 6.13 (s, 2H, O–CH2–O), 7.09–7.34 7.92 (s, 1H, N=CH), 8.39 (brs, 2H, NH2, D2O exchangeable).

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Chem. Pharm. Bull.
Vol. 65, No. 5 (2017)
11.68 (brs, 1H, OH, D2O exchangeable). 13C-NMR (600MHz, (2mmol), ethyl ethoxymethylenecyanoacetate (0.34g, 2mmol)
[D6]DMSO) δ: 57.9 (OCH3), 59.9 (OCH3), 118.8 (CN), 106.8, and anhydrous potassium carbonate (0.28g, 2mmol) in ab-
110.5, 114.0, 123.5, 129.2, 132.4, 135.2, 137.2, 152.2, 157.2 (Ar solute ethanol (10mL), was heated under reflux for 3h. The
C), 165.0 (C=N), 169.2 (CO). IR (KBr) cm−1: 3465–3239 (OH, solvent was evaporated under reduced pressure and the ob-
NH2), 2225 (CN), 1670 (C=O). MS m/z (%): 315 (34) [M+H]+. tained residue was dissolved in water then acidified with di-
Anal. Calcd for C15H14N4O4: C, 57.32; H, 4.49; N, 17.83. lute hydrochloric acid to pH 3–4. The precipitate thus formed
Found: C, 57.14; H, 4.61; N, 17.59.
was filtered, washed with water, dried and recrystallized from
(E)-4-Amino-1-((benzo[1,3]dioxol-5-ylmethylene)amino)-6- dioxane.
hydroxy-2-oxo-1,2-dihydropyridine-3-carbonitrile (8)
(E)-1-((3,4-Dimethoxybenzylidene)amino)-6-hydroxy-2-
Yield: 58%. mp: 171–173°C (EtOH). 1H-NMR (600MHz, oxo-1,2-dihydropyridine-3,5-dicarbonitrile (11)
[D6]DMSO) δ: 5.98 (s, 1H, pyridine-C5–H), 6.05 (s, 2H, O–
Yield: 48%. mp: 260–262°C. 1H-NMR (600MHz,
CH2–O), 6.94–7.16 (m, 3H, benzodioxole-H), 7.91 (s, 1H, [D6]DMSO) δ: 3.89 (s, 6H, 2 OCH3), 6.75–7.32 (m, 3H, di-
N=CH), 11.62 (s, 1H, OH, D2O exchangeable). 13C-NMR methoxyphenyl–H), 7.79 (s, 1H, pyridine-C4–H), 8.27 (s, 1H,
(600MHz, [D6]DMSO) δ: 88.9 (O–CH2–O), 119.2 (CN), 104.9, N=CH), 10.84 (brs, 1H, OH, D2O exchangeable). 13C-NMR
111.5, 115.2, 123.5, 128.3, 132.6, 135.7, 137.5, 153.4, 156.8 (Ar (600MHz, [D6]DMSO) δ: 56.1 (OCH3), 57.2 (OCH3), 116.4
C), 164.3 (C=N), 168.3 (CO). IR (KBr) cm−1: 3451–3241 (OH, (CN), 118.3 (CN), 102.9, 106.5, 111.9, 127.9, 128.9, 132.5,
NH2), 2212 (CN), 1682 (C=O). MS m/z (%): 299 (28) [M+H]+. 132.9, 133.4, 152.2, 156.4 (Ar C), 165.8 (C=N), 169.8 (CO). IR
Anal. Calcd for C14H10N4O4: C, 56.38; H, 3.38; N, 18.78. (KBr) cm−1: 3400 (OH), 2210 (CN), 1664 (C=O). MS m/z (%):
Found: C, 56.56; H, 3.34; N, 18.91.
General Procedure for the Synthesis of (E)-4,6- 3.73; N, 17.28. Found: C, 58.98; H, 3.85; N, 17.13.
Diamino-1-((3,4-dimethoxybenzylidene)amino)-2-oxo- (E)-1-((Benzo[1,3]dioxol-5-ylmethylene)amino)-6-
1,2-dihydropyridine-3-carbonitrile (9) and (E)-4,6-Diami- hydroxy-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (12)
no-1-((benzo[1,3]dioxol-5-ylmethylene)amino)-2-oxo-1,2-
Yield: 53%. mp: 181–183°C. 1H-NMR (600MHz,
325 (17) [M+H]+. Anal. Calcd for C16H12N4O4: C, 59.26; H,
dihydropyridine-3-carbonitrile (10) To a solution of 1 or 2 [D6]DMSO) δ: 6.11 (s, 2H, O–CH2–O), 6.72–7.32 (m, 3H,
(2mmol) in absolute ethanol (10mL) containing four drops of benzodioxole–H), 7.87 (s, 1H, pyridine-C4-H), 8.07 (s, 1H,
piperidine, malononitrile (0.13g, 2mmol) was added. The re- N=CH), 10.87 (brs, 1H, OH, D2O exchangeable). 13C-NMR
action mixture was heated under reflux for 3h then allowed to (600MHz, [D6]DMSO) δ: 87.4 (O–CH2–O), 117.1 (CN), 118.6
attain room temperature. Working up of the reaction mixture (CN), 103.9, 106.2, 110.8, 127.8, 128.5, 132.2, 132.9, 133.5,
was carried out as described under 3 and 4. The separated 153.1, 155.9 (Ar C), 164.9 (C=N), 170.2 (CO). IR (KBr) cm−1:
solid product was filtered, washed with water, dried and re- 3465 (OH), 2216 (CN), 1678 (C=O). MS m/z (%): 309 (12)
crystallized from the proper solvent.
(E)-4,6-Diamino-1-((3,4-dimethoxybenzylidene)amino)-2- 18.18. Found: C, 58.73; H, 2.65; N, 18.02.
oxo-1,2-dihydropyridine-3-carbonitrile (9)
General Procedure for the Synthesis of (E)-6-Amino-
[M+H]+. Anal. Calcd for C15H8N4O4: C, 58.45; H, 2.62; N,
1
Yield: 78%. mp: 252–254°C (MeOH). H-NMR (600MHz, 4-aryl-1-((3,4-dimethoxybenzylidene)amino)-2-oxo-1,2-
[D6]DMSO) δ: 3.79, 3.84 (2s, 6H, 2 OCH3), 5.65 (s, 1H, dihydropyridine-3,5-dicarbonitriles (13, 14) and (E)-6-
pyridine-C5–H), 7.02–7.13 (m, 3H, dimethoxyphenyl–H), 8.15 Amino-4-aryl-1-((benzo[1,3]dioxol-5-ylmethylene)amino)-
(s, 1H, N=CH), 8.39, 9.81 (2 brs, 4H, 2 NH2, D2O exchange- 2-oxo-1,2-dihydropyridine-3,5-dicarbonitriles (15, 16) To
able). 13C-NMR (600MHz, [D6]DMSO) δ: 57.6 (OCH3), 59.8 a solution of 1 or 2 (2mmol) in absolute ethanol (10mL), were
(OCH3), 119.5 (CN), 105.9, 110.8, 117.8, 128.2, 129.7, 131.5, added the appropriate 2-arylidenemalononitrile (2mmol) and
132.9, 133.4, 154.2, 156.2 (Ar C), 165.4 (C=N), 169.7 (CO). IR four drops of piperidine. The reaction mixture was heated
(KBr) cm−1: 3387–3258 (NH2), 2220 (CN), 1678 (C=O). MS under reflux for 3h. After cooling, the separated product was
m/z (%): 313 (38) [M]+. Anal. Calcd for C15H15N5O3: C, 57.50; filtered, washed with ethanol, dried, and recrystallized from
H, 4.83; N, 22.35. Found: C, 57.82; H, 4.79; N, 22.46.
the proper solvent.
(E)-4,6-Diamino-1-((benzo[1,3]dioxol-5-ylmethylene)-
(E)-6-Amino-1-((3,4-dimethoxybenzylidene)amino)-4-(4-
methoxyphenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile
amino)-2-oxo-1,2-dihydropyridine-3-carbonitrile (10)
1
Yield: 85%. mp: 238–239°C (EtOH). H-NMR (600MHz, (13)
[D6]DMSO) δ: 5.64 (s, 1H, pyridine-C5–H), 6.14 (s, 2H,
Yield: 58%. mp: 257–259°C (DMF). 1H-NMR (600MHz,
O–CH2–O), 6.98 (d, J=8.1Hz, 1H, benzodioxole-C7–H), [D6]DMSO) δ: 3.84, 3.85, 3.87 (3s, 9H, 3 OCH3), 6.99–7.71
7.06–7.09 (m, 2H, benzodioxole-C4,6–H), 8.11 (s, 1H, N=CH), (m, 7H, Ar-H), 8.36 (brs, 2H, NH2, D2O exchangeable), 8.77
8.40 (brs, 2H, NH2, D2O exchangeable). 13C-NMR (600MHz, (s, 1H, N=CH). 13C-NMR (600MHz, [D6]DMSO) δ: 57.3
[D6]DMSO) δ: 89.7 (O–CH2–O), 120.3 (CN), 106.5, 111.9, (OCH3), 58.4 (OCH3), 63.1 (OCH3), 119.6 (CN), 120.4 (CN),
116.8, 127.5, 128.9, 132.5, 132.9, 133.4, 153.1, 157.2 (Ar C), 104.2, 109.9, 111.9, 123.1, 128.2, 128.9, 131.7, 132.9, 137.2,
165.8 (C=N), 170.1 (CO). IR (KBr) cm−1: 3366–3250 (NH2), 138.5, 139.1, 139.7, 139.7, 140.3, 141.5, 153.5 (Ar C), 167.4
2213 (CN), 1667 (C=O). MS m/z (%): 297 (29) [M]+. Anal. (C=N), 170.2 (CO). IR (KBr) cm−1: 3388–3239 (NH2), 2221
Calcd for C14H11N5O3: C, 56.56; H, 3.73; N, 23.56. Found: C, (CN), 1663 (C=O). MS m/z (%): 430 (18) [M+H]+. Anal. Calcd
56.38; H, 3.71; N, 23.59.
for C23H19N5O4: C, 64.33; H, 4.46; N, 16.31. Found: C, 64.21;
General Procedure for the Synthesis of (E)-1-((3,4- H, 4.32; N, 16.54.
Dimethoxybenzylidene)amino)-6-hydroxy-2-oxo-1,2-dihy-
(E)-6-Amino-1-((3,4-dimethoxybenzylidene)amino)-4-
dropyridine-3,5-dicarbonitrile (11) and (E)-1-((Benzo[1,3]- (3,4-dimethoxyphenyl)-2-oxo-1,2-dihydropyridine-3,5-
dioxol-5-ylmethylene)amino)-6-hydroxy-2-oxo-1,2-dihydro- dicarbonitrile (14)
1
pyridine-3,5-dicarbonitrile (12) A mixture of 1 or 2
Yield: 66%. mp: 245–247°C (dioxane). H-NMR (600MHz,

Vol. 65, No. 5 (2017)
Chem. Pharm. Bull.
451
[D6]DMSO) δ: 3.80, 3.82, 3.85, 3.87 (4s, 12H, 4 OCH3), exchangeable), 8.26 (s, 1H, N=CH). 13C-NMR (600MHz,
7.00–7.19 (m, 4H, Ar-H), 7.48 (d, J=8.4Hz, 1H, dimethoxy- [D6]DMSO) δ: 15.9 (CH3), 57.9 (OCH3), 59.2 (OCH3), 65.7
benzylidene-C6–H), 7.71 (s, 1H, dimethoxybenzylidene-C2–H), (OCH3), 80.1 (CH2–O), 87.3 (O–CH2–O), 118.7 (CN), 104.2,
8.35 (brs, 2H, NH2, D2O exchangeable), 8.76 (s, 1H, N=CH). 110.3, 120.3, 122.7, 127.9, 128.7, 132.4, 133.2, 134.6, 137.4,
13C-NMR (600MHz, [D6]DMSO) δ: 57.1 (OCH3), 58.2 138.7, 138.9, 139.6, 139.9, 141.2, 152.1 (Ar C), 164.2 (C=N),
(OCH3), 62.4 (OCH3), 64.0 (OCH3), 119.9 (CN), 120.9 (CN), 168.2 (CO), 170.7 (CO). IR (KBr) cm−1: 3360–3274 (NH2),
104.6, 109.9, 111.8, 122.3, 127.4, 128.7, 131.2, 132.9, 137.5, 2227 (CN), 1730 (C=O ester), 1675 (C=O). MS m/z (%): 477
138.3, 139.4, 139.6, 139.7, 139.9, 140.1, 152.5 (Ar C), 168.9 (18) [M+H]+. Anal. Calcd for C25H24N4O6: C, 63.02; H, 5.08;
(C=N), 170.7 (CO). IR (KBr) cm−1: 3370–3232 (NH2), 2230 N, 11.76. Found: C, 63.21; H, 5.16; N, 11.56.
(CN), 1675 (C=O). MS m/z (%): 459 (15) [M]+. Anal. Calcd
for C24H21N5O5: C, 62.74; H, 4.61; N, 15.24. Found: C, 62.85; amino)-4-(3,4-dimethoxyphenyl)-2-oxo-1,2-dihydropyridine-3-
(E)-Ethyl 6-Amino-5-cyano-1-((3,4-dimethoxybenzylidene)-
H, 4.66; N, 15.18.
(E)-6-Amino-1-((benzo[1,3]dioxol-5-ylmethylene)-
carboxylate (18)
Yield: 65%. mp: 165–167°C (MeOH). H-NMR (600MHz,
1
amino)-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3,5- [D6]DMSO) δ: 1.24 (t, J=6.9Hz, 3H, ester–CH3), 3.81 (s,
dicarbonitrile (15) 6H, 2 OCH3), 3.84 (s, 6H, 2 OCH3), 4.26 (q, J=6.9Hz, 2H,
1
Yield: 58%. mp: 265–267°C (DMF). H-NMR (600MHz, ester–CH2), 6.79–7.57 (m, 6H, Ar-H), 7.99 (s, 2H, NH2, D2O
[D6]DMSO) δ: 3.85 (s, 3H, OCH3), 6.17 (s, 2H, O–CH2–O), exchangeable), 8.32 (s, 1H, N=CH). 13C-NMR (600MHz,
6.98–7.99 (m, 7H, Ar-H), 8.37 (brs, 2H, NH2, D2O exchange- [D6]DMSO) δ: 16.1 (CH3), 58.2 (OCH3), 59.4 (OCH3), 64.9
able), 8.80 (s, 1H, N=CH). 13C-NMR (600MHz, [D6]DMSO) (OCH3), 66.2 (OCH3), 80.4 (CH2-O), 87.8 (O–CH2–O), 119.2
δ: 62.0 (OCH3), 88.9 (O–CH2–O), 118.7 (CN), 119.6 (CN), (CN), 103.9, 110.5, 121.1, 123.3, 127.0, 128.9, 132.6, 133.5,
103.9, 110.1, 112.4, 122.3, 127.4, 129.2, 131.4, 132.8, 137.5, 134.7, 137.4, 138.2, 138.7, 139.6, 140.2, 142.9, 153.5 (Ar
138.3, 139.1, 139.7, 140.1, 140.9, 141.6, 152.9 (Ar C), 169.1 C), 165.1 (C=N), 169.1 (CO), 171.3 (CO). IR (KBr) cm−1:
(C=N), 170.8 (CO). IR (KBr) cm−1: 3367–3240 (NH2), 2219 3357–3271 (NH2), 2215 (CN), 1722 (C=O ester), 1663 (C=O).
(CN), 1670 (C=O). MS m/z (%): 413 (11) [M]+. Anal. Calcd for MS m/z (%): 507 (21) [M+H]+. Anal. Calcd for C26H26N4O7:
C22H15N5O4: C, 63.92; H, 3.66; N, 16.94. Found: C, 64.13; H, C, 61.65; H, 5.17; N, 11.06. Found: C, 61.39; H, 5.12; N, 10.86.
3.78; N, 16.81.
(E)-Ethyl
6-Amino-1-((benzo[1,3]dioxol-5-ylmethylene)-
(E)-6-Amino-1-((benzo[1,3]dioxol-5-ylmethylene)amino)-4- amino)-5-cyano-4-(4-methoxyphenyl)-2-oxo-1,2-dihydro-
(3,4-dimethoxyphenyl)-2-oxo-1,2-dihydropyridine-3,5- pyridine-3-carboxylate (19)
1
dicarbonitrile (16)
Yield: 62%. mp: 208–209°C (EtOH). H-NMR (600MHz,
1
Yield: 53%. mp: 260–262°C (DMF). H-NMR (600MHz, [D6]DMSO) δ: 0.95 (t, J=6.9Hz, 3H, ester–CH3), 3.81 (s,
[D6]DMSO) δ: 3.81, 3.87 (2s, 6H, 2 OCH3), 6.17 (s, 2H, O– 3H, OCH3), 3.91 (q, J=6.9Hz, 2H, ester–CH2), 6.09 (s, 2H,
CH2–O), 6.98–7.72 (m, 6H, Ar-H), 8.37 (brs, 2H, NH2, D2O O–CH2–O), 6.71–7.74 (m, 7H, Ar-H), 8.12 (s, 1H, N=CH).
exchangeable), 8.79 (s, 1H, N=CH). 13C-NMR (600MHz, 13C-NMR (600MHz, [D6]DMSO) δ: 15.8 (CH3), 65.9 (OCH3),
[D6]DMSO) δ: 62.6 (OCH3), 63.9 (OCH3), 87.8 (O–CH2–O), 79.5 (CH2-O), 87.4 (O–CH2–O), 118.7 (CN), 103.9, 110.1,
119.2 (CN), 120.4 (CN), 104.3, 111.1, 113.3, 122.3, 127.5, 120.4, 123.1, 127.4, 128.6, 131.3, 132.9, 134.6, 137.2, 138.1,
128.5, 131.2, 133.4, 136.6, 138.1, 138.9, 139.6, 140.2, 140.9, 138.9, 139.6, 139.9, 141.3, 152.3 (Ar C), 169.3 (C=O), 171.4
141.8, 153.5 (Ar C), 164.7 (CN), 168.4 (C=N), 169.9 (CO). IR (CO). IR (KBr) cm−1: 3355–3266 (NH2), 2221 (CN), 1725
(KBr) cm−1: 3380–3245 (NH2), 2225 (CN), 1665 (C=O). MS (C=O ester), 1670 (C=O). MS m/z (%): 461 (23) [M+H]+.
m/z (%): 443 (10) [M]+. Anal. Calcd for C23H17N5O5: C, 62.30; Anal. Calcd for C24H20N4O6: C, 62.60; H, 4.38; N, 12.17.
H, 3.86; N, 15.79. Found: C, 62.54; H, 3.94; N, 16.00.
General Procedure for the Synthesis of (E)-Ethyl
Found: C, 62.83; H, 4.37; N, 12.18.
(E)-Ethyl 6-Amino-1-((benzo[1,3]dioxol-5-ylmethylene)-
6-Amino-4-aryl-5-cyano-1-((3,4-dimethoxybenzylidene)- amino)-5-cyano-4-(3,4-dimethoxyphenyl)-2-oxo-1,2-
amino)-2-oxo-1,2-dihydropyridine-3-carboxylates (17, dihydropyridine-3-carboxylate (20)
18) and (E)-Ethyl 6-Amino-4-aryl-1-((benzo[1,3]dioxol- Yield: 67%. mp: 182–184°C (MeOH). H-NMR (600MHz,
1
5-ylmethylene)amino)-5-cyano-2-oxo-1,2-dihydropyridine- [D6]DMSO) δ: 1.23 (t, J=6.9Hz, 3H, ester–CH3), 3.87 (s,
3-carboxylates (19, 20) To a solution of 1 or 2 (2mmol) in 3H, OCH3), 4.21 (q, J=6.9Hz, 2H, ester–CH2), 6.22 (s, 2H,
absolute ethanol (10mL) containing sodium metal (0.046g, O–CH2–O), 6.94–7.56 (m, 6H, Ar-H), 8.00 (s, 2H, NH2, D2O
2mmol), was added the appropriate 2-arylidenecyanoacetate exchangeable), 8.34 (s, 1H, N=CH). 13C-NMR (600MHz,
(2mmol). The reaction mixture was heated under reflux for [D6]DMSO) δ: 15.2 (CH3), 66.2 (OCH3), 68.2 (OCH3), 79.2
2h, then the solvent was evaporated under reduced pressure. (CH2-O), 87.1 (O–CH2–O), 118.4 (CN), 104.6, 109.8, 120.1,
The obtained residue was dissolved in water and neutralized 122.2, 127.4, 128.7, 131.2, 132.9, 134.4, 137.4, 138.4, 138.6,
with dilute hydrochloric acid to pH 7. The precipitate thus 139.7, 139.9, 140.4, 150.9 (Ar C), 169.9 (C=O), 171.7 (CO). IR
formed was filtered, washed with water, dried and recrystal- (KBr) cm−1: 3357–3271 (NH2), 2219 (CN), 1727 (C=O ester),
lized from the proper solvent.
1668 (C=O). MS m/z (%): 491 (19) ([M+H]+. Anal. Calcd for
(E)-Ethyl 6-Amino-5-cyano-1-((3,4-dimethoxybenzylidene)- C25H22N4O7: C, 61.22; H, 4.52; N, 11.42. Found: C, 61.51; H,
amino)-4-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3- 4.61; N, 11.26.
carboxylate (17)
General Procedure for the Synthesis of 6-Aryl-
Yield: 41%. mp: 183–185°C (EtOH). 1H-NMR (600MHz, 2-oxo-4-(3,4,5-trimethoxyphenyl)-1,2-dihydropyridine-
[D6]DMSO) δ: 1.24 (t, J=6.9Hz, 3H, ester–CH3), 3.80 (s, 3-carbonitriles (24–26) A mixture of chalcone 21–23 (2.5
3H, OCH3), 3.82 (s, 6H, 2 OCH3), 4.26 (q, J=6.9Hz, 2H, mmole), ammonium acetate (1.54g, 20 mmole) and ethyl
ester–CH2), 6.75–7.56 (m, 7H, Ar-H), 7.79 (s, 2H, NH2, D2O cyanoacetate (0.28g, 0.28mL, 2.5 mmole) in absolute ethanol

452
Chem. Pharm. Bull.
Vol. 65, No. 5 (2017)
(50mL) was heated under reflux for 12h during which yellow [D6]DMSO) δ: 56.2 (OCH3), 57.4 (OCH3), 60.1 (OCH3), 121.3
crystals started to form. After cooling, the separated solid was (CN), 102.3, 103.7, 112.1, 128.5, 129.1, 133.2, 137.4, 148.7,
filtered, washed with ethanol, dried and recrystallized from 153.1, 157.3, 163.1, 164.2, 169.0, 169.7 (Ar C), 176.8 (C=N).
the proper solvent.
IR (KBr) cm−1: 3266 (NH), 2212 (CN). MS m/z (%): 361 (36)
2-Oxo-6-phenyl-4-(3,4,5-trimethoxyphenyl)-1,2-dihydro- [M]+. Anal. Calcd for C21H19N3O3: C, 69.79; H, 5.30; N, 11.63.
pyridine-3-carbonitrile (24)
Yield: 90%. mp: 274–276°C (dioxane). H-NMR (600MHz,
Found: C, 69.58; H, 5.41; N, 11.61.
6-(4-Chlorophenyl)-2-imino-4-(3,4,5-trimethoxyphenyl)-
1
[D6]DMSO) δ: 3.75 (s, 3H, OCH3), 3.87 (s, 6H, 2 OCH3), 1,2-dihydropyridine-3-carbonitrile (28)
6.89 (s, 1H, pyridine–C5–H), 7.06 (s, 2H, trimethoxyphe- Yield: 91%. mp: 206–208°C (DMF). H-NMR (600MHz,
1
nyl–C2,6–H), 7.52–7.56 (m, 3H, phenyl–C3,4,5–H), 7.90 (d, [D6]DMSO) δ: 3.80 (s, 3H, OCH3), 3.82 (s, 6H, 2OCH3), 6.98
J=7.2Hz, 2H, phenyl–C2,6–H), 12.74 (s, 1H, NH, D2O ex- (s, 1H, pyridine-C5–H), 7.03 (s, 2H, trimethoxyphenyl-C2,6–H),
changeable). 13C-NMR (600MHz, [D6]DMSO) δ: 56.2 (OCH3), 7.59 (d, J=8.4Hz, 2H, p-chlorophenyl-C2,6–H), 7.83 (d,
57.3 (OCH3), 60.4 (OCH3), 120.3 (CN), 101.3, 103.9, 112.5, J=8.4Hz, 2H, p-chlorophenyl-C3,5–H). 13C-NMR (600MHz,
128.3, 129.5, 132.3, 137.6, 149.2, 153.4, 157.1, 163.2, 164.7, [D6]DMSO) δ: 56.1 (OCH3), 57.4 (OCH3), 60.2 (OCH3), 121.4
168.9, 170.1 (Ar C), 176.8 (CO). IR (KBr) cm−1: 3348 (NH), (CN), 101.9, 103.7, 112.0, 128.7, 129.3, 132.3, 137.1, 148.9,
2217 (CN), 1688 (C=O). MS m/z (%): 362 (48) [M]+. Anal. 153.2, 157.4, 163.2, 164.4, 169.3, 170.3 (Ar C), 177.2 (C=N).
Calcd for C21H18N2O4: C, 69.60; H, 5.01; N, 7.73. Found: C, IR (KBr) cm−1: 3372 (NH), 2219 (CN). MS m/z (%): 396 (29)
69.73; H, 5.16; N, 7.79.
6-(4-Chlorophenyl)-2-oxo-4-(3,4,5-trimethoxyphenyl)-1,2- 63.72; H, 4.58; N, 10.62. Found: C, 63.88; H, 4.62; N, 10.81.
[M]+, 398 (10) [M+2]+. Anal. Calcd for C21H18ClN3O3: C,
dihydropyridine-3-carbonitrile (25)
Biology
Yield: 93%. mp: 280–281°C (DMF). 1H-NMR (600MHz,
[D6]DMSO) δ: 3.74 (s, 3H, OCH3), 3.86 (s, 6H, 2 OCH3),
In Vitro MTT Cytotoxicity Assay
The synthesized compounds were investigated for their
6.95 (s, 1H, pyridine–C5–H), 7.06 (s, 2H, trimethoxyphenyl– in vitro cytotoxic effect via the standard method MTT50,51)
C2,6–H), 7.61 (d, J=8.4Hz, 2H, p-chlorophenyl–C2,6–H), 7.94 against a panel of three human tumor cell lines (ATCC®)
(d, J=8.4Hz, 2H, p-chlorophenyl–C3,5–H), 12.78 (s, 1H, NH, namely; Caucasian breast adenocarcinoma MCF7 (HTB-22™),
D2O exchangeable). 13C-NMR (600MHz, [D6]DMSO) δ: 56.1 hepatocellular carcinoma HepG2 (HB-8065™), colon car-
(OCH3), 57.4 (OCH3), 60.2 (OCH3), 121.4 (CN), 101.9, 103.7, cinoma CACO-2 (HTB-37™), and normal human foreskin
112.0, 128.7, 129.3, 132.3, 137.1, 148.9, 153.2, 157.4, 163.2, fibroblast Hs27 normal cell line (ATCC® CRL-1634™). The
164.4, 169.3, 170.3 (Ar C), 177.2 (CO). IR (KBr) cm−1: 3291 procedures were done in a sterile area using a Laminar flow
(NH), 2210 (CN), 1671 (C=O). MS m/z (%): 397 (41) [M]+, cabinet biosafety class II level (Baker, SG403INT, Stanford,
399 (14) (M+2]+. Anal. Calcd for C21H17ClN2O4: C, 63.56; H, ME, U.S.A.). Cells were batch cultured for 10d, then seeded
4.32; N, 7.06. Found: C, 63.47; H, 4.30; N, 6.99.
6-(3,4-Dimethoxyphenyl)-2-oxo-4-(3,4,5-trimethoxy- medium in 96-well microtiter plastic plates at 37°C for 24h
phenyl)-1,2-dihydropyridine-3-carbonitrile (26) under 5% CO2 using a water jacketed carbon dioxide incuba-
at concentration of 10×103 cells/well in fresh complete growth
1
Yield: 87%. mp: 250–252°C (DMF). H-NMR (600MHz, tor (Sheldon, TC2323, Cornelius, OR, U.S.A.). Media was
[D6]DMSO) δ: 3.74 (s, 3H, OCH3), 3.87 (s, 2H, 4 OCH3), aspirated, fresh medium (without serum) was added and cells
6.88 (s, 1H, pyridine–C5–H), 7.04 (s, 2H, trimethoxyphenyl- were incubated either alone (negative control) or with different
C2,6–H), 7.10 (d, J=8.4Hz, 1H, dimethoxyphenyl-C5–H),7.48 concentrations of the test compounds. DMSO was employed
(s, 1H, dimethoxyphenyl-C2–H), 7.54 (d, J=8.4Hz, 1H, as a vehicle for dissolution of the tested compounds and its
dimethoxyphenyl-C6–H), 12.62 (s, 1H, NH, D2O exchange- final concentration on the cells was less than 0.2%. Cells were
able). 13C-NMR (600MHz, [D6]DMSO) δ: 56.2 (OCH3), 57.3 suspended in RPMI 1640 medium (for HepG2 and CACO-2
(OCH3), 60.4 (OCH3), 60.4 (OCH3), 60.4 (OCH3), 120.3 (CN), cell lines) and Dulbecco’s modified Eagle’s medium (DMEM)
101.3, 103.9, 112.5, 128.3, 129.5, 132.3, 137.6, 149.2, 153.4, (for MCF7 cell line), 1% antibiotic-antimycotic mixture
157.1, 163.2, 164.7, 168.9, 170.1 (Ar C), 176.8 (CO). IR (KBr) (10000IU/mL penicillin potassium, 10000µg/mL streptomy-
cm−1: 3324 (NH), 2215 (CN), 1676 (C=O). MS m/z (%): 422 cin sulphate and 25µg/mL amphotericin B), and 1% L-gluta-
(39) [M]+. Anal. Calcd for C23H22N2O6: C, 65.39; H, 5.25; N, mine in 96-well flat bottom microplate at 37°C under 5% CO2.
6.63. Found: C, 65.42; H, 5.29; N, 6.62.
After 24h of incubation, the medium was aspirated, 40µL of
General Procedure for the Synthesis of 6-Aryl-2-imino- MTT salt (2.5µg/mL) were added to each well and incubated
4-(3,4,5-trimethoxyphenyl)- 1,2-dihydropyridine-3-carbo- for further 4h at 37°C under 5% CO2. To stop the reaction and
nitriles (27, 28) A mixture of chalcone 21, 22 (2.5 mmole), dissolve the formed crystals, 200µL of 10% sodium dodecyl
ammonium acetate (1.54g, 20 mmole) and malononitrile sulphate (SDS) in deionized water was added to each well and
(0.33g, 2.5 mmol) in absolute ethanol (50mL) was heated incubated overnight at 37°C. The absorbance was then mea-
under reflux for 24h then left to cool to room temperature. sured using a microplate multi-well reader (Bio-Rad Labora-
The separated solid was filtered, washed with water, dried and tories Inc., model 3350, Hercules, CA, U.S.A.) at λ 595nm and
recrystallized from the proper solvent.
2-Imino-6-phenyl-4-(3,4,5-trimethoxyphenyl)-1,2- was tested between samples and negative control (cells with
dihydropyridine-3-carbonitrile (27) vehicle) using independent t-test by SPSS 11 program. The re-
a reference wavelength of λ 620nm. A statistical significance
1
Yield: 82%. mp: 175–177°C (dioxane). H-NMR (600MHz, sults are presented in Table 1 as LC50 (µM) which is the lethal
[D6]DMSO) δ: 3.83 (s, 3H, OCH3), 3.93 (s, 6H, 2 OCH3), concentration of the compound which causes death of 50% of
7.20 (s, 1H, pyridine–C5–H), 7.51 (s, 2H, trimethoxyphenyl– the cells in 24h.
C2,6–H), 7.52–7.86 (m, 5H, phenyl–H). 13C-NMR (600MHz,

Vol. 65, No. 5 (2017)
Chem. Pharm. Bull.
453
DPPH Radical Scavenging Antioxidant Assay
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Acknowledgements This paper was funded by the Dean-
ship of Scientific Research (DSR), King Abdulaziz Univer-
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