Microwave-assisted synthesis of some new pyrazolopyridines and their antioxidant, antitumor and antimicrobial activities

Article abstract of DOI:10.1016/j.ejmech.2013.04.043

The chemical behavior of 4-(dimethylaminomethylene)-1-phenyl-3-(pyridin-3- yl)-1 H-pyrazol-5(4H)-one (enaminone) (2) toward some active methylene reagents has been reported to give pyrazolopyridine derivatives. All the reactions were carried out by conventional heating and microwave irradiation technique. The antioxidant activity of the prepared compounds was studied using 1,1-phenyl-2-picrylhydrazyl (DPPH) assay. Compounds (4c) and (4d) showed the highest activity. The antitumor activity against liver and breast cell lines was tested. Compounds (6), (9) and (11) showed the highest activity for liver cell line while compounds (6) and (9) showed the highest activity for breast cell line. Compounds (4a-d) were screened for their antibacterial activity against Gram-positive, Gram-negative bacteria and antifungal activity.

Full text of DOI:10.1016/j.ejmech.2013.04.043

Accepted Manuscript
Microwave- assisted synthesis of some new pyrazolopyridines and their antioxidant,
antitumor and antimicrobial activities
M.A. El-Borai, H.F. Rizk, D.M. Beltagy, I.Y. El-Deeb
PII:
S0223-5234(13)00275-4
10.1016/j.ejmech.2013.04.043
EJMECH 6153
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 November 2012
Revised Date: 17 March 2013
Accepted Date: 21 April 2013
Please cite this article as: M.A. El-Borai, H.F. Rizk, D.M. Beltagy, I.Y. El-Deeb, Microwave- assisted
synthesis of some new pyrazolopyridines and their antioxidant, antitumor and antimicrobial activities,
European Journal of Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.04.043.
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ACCEPTED MANUSCRIPT
New pyrazolopyridine compounds were prepared by conventional heating and
microwave irradiation technique. The synthesized compounds were screened for
antioxidant, antitumor and antimicrobial activities.
R
N(Me)2
O
N
N
N
N
N
N
NH2
H
N
N
N
N
N
N
O
N
DMFDMA
reflux
Acetic acid
N
R
(13)
O
O
O
(2)
N
(1)
CN
CN
g
Piperidine
EtOH
R
X
l
a
c
OEt
i
a
H2C
l
a
CN
c
e
t
i
c
or
a
c
i
d
O
N
CN
N
H2C
COOC2H5
R
CNH2
X
N
N
N
O
X
N
H
N
N
N
CN
N
N
N
N
H
X
(4)
(11)
X=O,S
(6,9)

ACCEPTED MANUSCRIPT
1
Microwave- assisted synthesis of some new pyrazolopyridines and
their antioxidant, antitumor and antimicrobial activities.
M.A. El-Boraia*, H.F. Rizka, D.M. Beltagyb, I.Y. El-Deeba
a Department of Chemistry, Faculty of Science, Tanta University, 31527 Tanta, Egypt
b
Biochemistry Division, Department of Chemistry, Faculty of Science, Damanhour University,
Damanhour, Egypt
ABSTRACT
The chemical behavior of 4-(dimethylaminomethylene)-1-phenyl-3-(pyridin-3-yl)-
1H-pyrazol-5(4H)-one (enaminone) (2) towards some active methylene reagents has
been reported to give pyrazolopyridine derivatives. All the reactions were carried out
by conventional heating and microwave irradiation technique. The antioxidant activity
of the prepared compounds was studied using 1, 1-phenyl-2-picrylhydrazyl (DPPH)
assay. Compounds (4c) and (4d) showed the highest activity. The antitumor activity
against liver and breast cell lines was tested. Compounds (6), (9) and (11) showed the
highest activity for liver cell line while compounds (6) and (9) showed the highest
activity for breast cell line. Compounds (4a-d) were screened for their antibacterial
activity against Gram-positive, Gram-negative bacteria and antifungal activity.
Keywords: enaminones, microwave technology, antioxidant, antitumor, antimicrobial
activities.

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1. Introduction
The chemistry of the enaminones is potentially an area of considerable scope and has
recently received a very important attention. They have multiple electrophilic and
nucleophilic centers and undergo a variety of cycloaddition and self condensation
reactions. For these reasons they considered versatile synthetic intermediates [1-5].
Pyrazolo[3,4-b]pyridine skeleton had proven to be interesting class of heterocycles
due to diverse biological properties including antitubercular, antibacterial and
antioxidant activities [6- 15]. Recently, the pyrazolo pyridine compounds found a
great importance in the synthesis of some fluorescence dyes [16] and as anti corrosion
protection of stainless steel in aggressive media [17].
Microwave irradiation has broken new grounds in synthetic organic chemistry not
only in term of the reduction of reaction time, but also simplicity of reaction work up,
besides energy saving, high efficiency and environmental benign procedures [18-20].
In this paper, we have continued seeking the development of new and simple methods
for the synthesis of pyrazolopyridine derivatives.
2. Results and Discussion
2.1. Chemistry
In regards to our previous work [21] targeting to explore the synthesis of new
pyrazolopyridine derivatives because of their role as pharmacophores of considerable
importance [8]. We like to report here the condensation of dimethylformamide-
dimethylacetal (DMFDMA) with the active methylene group in the pyrazolone
system in dry condensation to give 4-(dimethylamino methylene)-1-phenyl-3-
(pyridin-3-yl)-1H-pyrazol-5(4H)-one (enaminone) (2) (Scheme 1). This interesting
compound (2) enables us to continue our research for the synthesis of new series of
pyrazolopyridine derivatives.
This reaction normally producing two stereoisomers (2E) and (2Z), the formation of
isomer (2Z) was not observed (TLC and 1H NMR spectrum), which revealed a singlet
at 7.15 ppm characteristic of the exocyclic (C=CH) proton for isomer (E), while this
proton was reported to appear at 6.90 ppm for isomer (Z) [22].
Of noteworthy observation the reaction of pyrazolone with aromatic aldehydes always
giving the corresponding arylidenes in the isomer (E) [23].

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< Scheme 1 >
The reaction of enaminone (2) with some β- ketonitriles (3a-d) in ethanolic medium
in the presence of piperidine as catalyst gave 5-substituted aroyl-1-phenyl-3-(pyridin-
3-yl)-1H-pyrazolo[3,4-b] pyridin-6(7H)-one derivatives (4a-d) (Scheme 2). The IR
spectra of compounds (4a-d) showed absorption at ν 1597-1663 cm-1 for exocyclic
carbonyl group and carbonyl of pyridinone nucleus and ν 3389-3432 cm-1 for imine or
hydroxyl group.
< Scheme 2 >
The reaction of enaminone (2) with malononitrile in refluxing ethanolic sodium
ethoxide solution may give two possible products: 6-imino-1-phenyl-3-(pyridin-3-yl)-
1,6-dihydropyrano[2,3-c]pyrazol-5-carbonitrile (5) or 6-oxo-1-phenyl-3-(pyridin-3-
yl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-5-carbonitrile (6) (Scheme 3). Structure of
compound (6) was ruled out based on IR and 1H NMR spectral data. The IR spectrum
revealed two absorption bands at ν 1651 and 2198 cm-1 for the amidic carbonyl and
C≡N groups respectively. The 1H NMR spectrum of compound 6, showed the absence
of imine signal. It is worth noting that the reaction of enaminone (2) with
cyanoacetamide gave the same structure (6) obtained by the reaction of (2) with
malononitrile. This is clear by (TLC, m.p., mixed m.p. and spectral data) (see
Experimental). In a similar behavior
the reaction of enaminone (2) with
cyanothioacetamide gave 1-phenyl-3-(pyridin-3-yl)-6-thioxo-6,7-dihydro-1H-
pyrazolo[3,4-b]pyridin-5-carbonitrile (9) not compound (8) ruled out by IR spectral
data which revealed two absorption bands at ν 1373 and 2202 cm-1 corresponding to
amidic thione and cyano groups respectively. These results are in accordance with that
previously reported work [15]. The reaction of pyrazolopyridone (6) with
phosphorous pentasulphide in boiling pyridine gave compound (9) (TLC, m.p., mixed
m.p and spectral data).
< Scheme 3 >

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The reaction of enaminone (2) with ethyl-3-oxo-3-(pyridin-3-yl)propanoate (10) in boiling acetic
acid and ammonium acetate gave ethyl -1-phenyl-3,6-di(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridin-
5-carboxylate (11) (Scheme 4). The IR spectrum of compound (11) showed absorption
band at ν 1615 cm-1 for the carbonyl of ester group.
< Scheme 4 >
The reaction of enaminone (2) with 3-substituted-5-amino-1H-pyrazoles (12 a-b) in boiling
acetic acid gave 7- aryl-3-(pyridin-3-yl)-1H-bispyrazolo [3,4-e:2',3'-a] pyrimidine derivatives
(13a-c) (Scheme 5). This reaction was accomplished by the nucleophilic attack of amino
group to the double bond via Michael addition reaction beginning of elimination of the
dimethylamino group followed by dehydration to give (13).
< Scheme 5 >
All the prepared compounds (Schemes 2, 3, 4 and 5) were done by conventional
heating and microwave irradiation technique. From the comparison of the results cited
in (Table 1), it is clear that the reaction time of is reduced from (8-15 hr) to (15-25
min) and the yield is increased from (60-80%) to (82-93%).
< Table 1>
3. Pharmacology
3.1 Antioxidant evaluation
Considerable progress has been made in recent years in relating ageing to oxidation in
biological cells. The reactive oxygen species (ROS), cause of oxidation in biological
cells are basically involved in detoxification of invading organism and chemicals,
ROS also initiate lipid peroxidation in healthy cells leading to diverse pathologies
such as Alzheimer’s disease, atherosclerosis, diabetes, Parkinson’s disease, etc. [24].
Thus reduction of the rate of these life-linacting metabolic processes by use of
chemicals [25, 26] has been a subject of current research.

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An imbalance between antioxidants and ROS result in oxidative stress, leading to
cellular damage. Several authors demonstrated that antioxidant intake is inversely
related to mortality from coronary heart disease and to incidence of heart attack [27-
29]. Antioxidant defense system of our body, which includes vitamin C (ascorbic
acid), vitamin E (α - tocopherol, γ tocopherol), carotenoids (β- carotene, α- carotene,
β- cryptoxathin, butein, zeaxanthin, lycopene and several polyphenolic compounds
including flavonoids (catechins, flavonals, flavones and iso flavonoids) [30- 32].
Dietary can also impede carcinogenesis by scavenging oxygen radicals as interfering
with the binding of carcinogens to DNA.
3. 1. 2 Materials and methods
DPPH radical scavenging assay
The antioxidant activities of prepared compounds were assessed using the 1,1-
diphenyl-2-picrylhydrazyl (DPPH) according to Ardestani and Yazdanparast with
ascorbic acid as standard [33]. Each tested sample and ascorbic acid (reference
compound) (50 µg) was dissolved in 1 ml DMSO. The dissolved sample (250 ꢀl) was
added to 1 ml DPPH/DMSO solution (6 mg/50 ml) and the total volume was adjusted
to 3 ml with DMSO. An equal amount of DMSO was used as a control. After
vortexing the mixture was incubated for 30 min in dark at room temperature.
Absorbance was measured using a spectrophotometer at 517 nm.
DPPH radical scavenging % = [1 – (A sample/ A control)] x 100.
Serial dilutions (5-50 µg/ml) of each compound were measured by the same assay to
obtain EC50 (the amount of compound that gives half-maximal scavenging response)
according to Brand-Williams et al [34].
3.1.2 Results and discussion
Radical scavenging activities of the ten compounds were measured. The
results were summarized in Table 2, Figure 1 and Figure 2. Compound (4c) has the
most potent antioxidant activity that was found to be very close to the values of
standard ascorbic acid due to the presence of fluorine group in the aromatic ring.
Compound (11) has no antioxidant activity. The other eight compounds were
considerably less effective radical scavengers in the order 4d > 13c > 4b > 4a > 13b >
9 > 13a > 6.

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< Table 2>
<Figure 1>
<Figure 2>
3.2 Antitumor evaluation
The search for novel drugs is still a priority goal for cancer therapy, due to the rapid
development of resistance to multiple chemotherapeutic drugs. In addition, the high
toxicity usually associated with cancer chemotherapy drugs and their undesirable side
effects increase the demand for novel antitumor drugs active against untreatable
tumors with fewer side effects and/or with greater therapeutic efficiency [35]. Breast
cancer is a major health problem worldwide [36]. Breast cancer is the most frequently
diagnosed cancer and the leading cause of cancer death in females worldwide,
accounting for 23% (1.38 million) of the total new cancer cases and 14% (458,400) of
the total cancer deaths in 2008 [37]. Liver cancer rapidly reduces quality of life and
typically causes death six months - one year from diagnosis [38]. Globally, it is the
fifth leading cause of cancer and the third leading cause of cancer death. This cancer
varies widely in incidence throughout the world, with rising incidence in Egypt [39].
3. 2. 1 Antitumor activity
The anticancer activities of prepared compounds were investigated on two human
cancer cell lines, HepG-2 (liver cancer) and MCF7 (breast cancer) according to SRB
assay [40].
3.3.2 Results and discussion
Antitumor activities of the tested compounds were measured used SRB assay for
breast cancer (Figure 3) and liver cancer (Figure 4). These results were tabulated in
Table 3. The overall results indicated that, some compounds have antitumor activity
through induction of apoptosis in MCF7 breast cancer cell line, suggesting that they
might be a potential alternative agent for human breast cancer therapy. The ten

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compounds can be ordered according to their anti-breast cancer as 9> 6> 4d> 11>
4c>4b> 4a> 13c> 13b> 13a. Some compounds have antitumor activity through
induction of apoptosis in HEPG2 cancer cell line, suggesting that they might be a
potential alternative agent for human hepatic cancer therapy. The ten compounds can
be ordered according to their anti-hepatic cancer as 6> 11> 9> 4c> 4b> 4d> 4a> 13c>
13b> 13a.
< Table 3>
<Figure 3>
<Figure 4>
3.3 Antimicrobial evaluation
Antimicrobial compounds could be used as antibacterial, antifungal and antiviral
agents. Random screening as tool in discovering new biologically active molecules
has been most productive in the area of antibiotics [41, 42].
3. 3. 1 Antibacterial assay
The microorganisms used in this study included Gram-positive bacteria (Bacillus),
Gram-negative bacteria (E. coli, E. cloaca, serratia) in addition to some fungal
species (F. oxysporum, P. expansum). The strains under study were obtained from the
culture collection of Microbiology Unit (Faculty of Science, Tanta University, Tanta,
Egypt). Bacteria were cultured on nutrient agar and the fungus was cultured on
sabaroud agar slopes. Antimicrobial activities of the synthesized compounds were
tested in vitro on nutrient agar at 30ºC after 24 h by the cut-plug method according to
Pridham et al. [43].
3.3.2. Results and discussion
The results of the antimicrobial activities are summarized in (Table 4). Antimicrobial
activities of compounds (4a-d) showed negative results for the Bacillus and F.
oxysporum strains. Only compound (4b) showed a good antimicrobial activity for P.
expansum while other three compounds gave negative results. On the other hand, the
four compounds gave relatively good antibacterial activities for Serratia in the order

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of 4c > 4b, 4d > 4a and an excellent activities for E. coli and E. cloaca in the order of
4d > 4b > 4c > 4a and 4c > 4d > 4b > 4a, respectively.
<Table 4 >
4. Experimental
4.1. General
Melting points were recorded on a Gallenkamp melting point apparatus and are
reported uncorrected. The infrared spectra were recorded on Perkin–Elmer FTIR
1430 spectrophotometer using KBr disk technique. The 1H NMR spectra were
recorded at 250 C in DMSO-d6 with TMS as an internal standard. Chemical shifts
are reported in ppm and results are expressed as δ values on a Bruker AC
spectrometer (300 MHz). Reactions were conducted under microwave irradiation
in closed vessels under magnetic stirring by using Synthos 3000 dual magnetrons
system and with maximum power of 1000 W. Reaction progress was monitored by
thin layer chromatography (TLC) using benzene/acetone (2/1 by volume) as eluent.
Mass spectra were measured on a Finnigan MAT 8222 EX mass spectrometer at 70
eV. Absorbance was measured using a Jenway 6305 spectrophotometer.
Microanalyses were preformed on Perkin.Elemer 2400 Elemental Analyzer at
Microanalytical center at Cairo University.
Compounds (3a-d) were prepared according literature procedures [44, 45],
compound (10) [46] and compounds (12a-c) [47].
4.2.
4-(Dimethylaminomethylene)-1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-5(4H)-one
(2).
A mixture of 1 (2.99 g, 12.6 mmol) and DMFDMA (2.519 ml) was heated under reflux for 10
h. The reaction mixture was triturated with ethanol to give a solid product. Filtration and
recrystallized from ethanol to give faint green crystals in yield (69%); m.p. 220-222o C; IR
(KBr) ν max / cm_1= 1400 (C-N), 1602 (C=N pyrazole ring), 1671 (C=O), 2918 (Aliph.-H),
3013 (Arom.-H). 1HNMR (DMSO- d6): δppm = 3.35 (s, 6H, 2CH3), 7.13 (s, 1H, CH),
7.33- 7.74 (m, 5H, Ar-H), 8.12-8.94 (m, 4H, pyridine ring). MS m/z (%): M = 292
(74%), 248 (77), 222(11), 194(24), 159 (35), 116 (89), 77 (100), 63 (24), 51 (44).

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Anal.; For C17H16N4O (292.34) Calcd.: C 69.85 ; H 5.52; N 19.17% Found: C 70.27 ;
H 5.79; N 19.42%.
4.3. General procedure for the synthesis of 5-aroyl-1-phenyl-3-(pyridin-3-yl)-1H-
pyrazolo[3,4-b]pyridin-6(7H)-one (4a-d).
4.3.1. Method (A):
To a solution of 2 (0.49 g, 1.7 mmol) in ethanol (20 ml) and piperidine (0.5 ml) as a catalyst
was added substituted β- ketonitriles 3a-d (1.7 mmol). The reaction mixture was heated under
reflux for (8- 10) h and the progress of the reaction was monitored by TLC using
benzene/acetone (2:1) as eluent. The solvent was evaporated under reduced pressure; the oil
residue was treated with petroleum ether 40-600 C (3 x 10 ml) and recrystallized from
ethanol to give (4a-d).
4.3.2. Method (B):
The procedure was similar to that described in Method (A) except that the mixture
was capped in closed vessels and irradiated in a microwave oven at 130 0C for 20 min
using Synthos 3000 (500 W). The reaction was worked as usual and the obtained solid
was recrystallized from ethanol to give (4a-d).
4.3.3. 5-Nicotinoyl-1-phenyl-3-(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one
4a
Brown crystals in yield (75%); m.p. 290-292o C; IR (KBr) ν max / cm_1= 1663 (C=O),
2195 (Aliph.-H), 3074 (Arom.-H), 3389 (NH or OH). 1HNMR (DMSO- d6): δppm =
7.33- 7.74 (m, 5H, Ar-H), 8.15-8.77 (m, 8H, pyridine ring), 8.92 (s, 1H, broad NH),
8.97 (s, 1H, pyridone ring). MS m/z (%): M = 392 (29%), 332(98), 289(31), 277(91),
264(100), 242(31), 218(38), 207(42), 181(38), 152(40), 95(64), 50(38). Anal.; For
C23H15N5O2 (393.44) Calcd.: C 70.22; H 3.84; N 17.80 Found: C 70.44 H 4.01; N
17.99%.
4.3.4.
5-(4-Methylbenzoyl)-1-phenyl-3-(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridin-
6(7H)-one 4b

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Buff crystals in yield (67%); m.p. 276-277o C; IR (KBr) ν max / cm_1= 1639 (C=O), 2193
(Aliph.-H), 3076 (Arom.-H), 3432 (NH or OH). 1HNMR (DMSO- d6): δppm = 2.25
(s, 3H, CH3), 7.31- 7.66 (m, 9H, Ar-H), 8.22-8.83 (m, 4H, pyridine ring), 8,93 (s, 1H,
broad NH), 8.98 (s, 1H, pyridone ring). MS m/z (%): M = 406 (99%), 392(35),
379(49), 351(75), 324(51), 230(44), 207(58), 195(100), 169(61), 124(43), 110(74), 92(76),
68(59). Anal.; For C25H18N4O2 (406.44) Calcd.: C 73.88; H 4.46; N 13.78% Found: C
74.11; H 4.65; N 13.95%.
4.3.5
5-(4-Fluorobenzoyl)-1-phenyl-3-(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridin-
6(7H)-one 4c
Faint brown crystals in yield (70%); m.p. 300-302o C; IR (KBr) ν max / cm_1= 1597 (C=O),
2196 (Aliph.-H), 3046 (Arom.-H), 3430 (NH or OH). 1HNMR (DMSO- d6): δppm =
7.33- 7.68 (m, 9H, Ar-H), 8.15-8.77 (m, 4H, pyridine ring), 8.90 (s, 1H, broad NH),
8.98 (s, 1H, pyridone ring). MS m/z (%): M = 410(68%), 394(43), 380(46), 363(73),
308(60), 260(77), 233(92), 207(46), 183(78), 127(46), 95(100), 77(95), 65(55), 53(51).
Anal.; For C24H15FN4O2 (410.40) Calcd.: C 70.24; H 3.68; F 4.63; N 13.65, % Found:
C 70.63; H 3.89; F 4.73 N 13.89%.
4.3.6.
5-(4-Chlorobenzoyl)-1-phenyl-3-(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridin-
6(7H)-one 4d
Faint brown crystals in yield (62%); m.p. 304-306o C; IR (KBr) ν max / cm_1= 1659 (C=O),
2188 (Aliph.-H), 3036 (Arom.-H), 3423 (NH or OH). 1HNMR (DMSO- d6): δppm =
7.33- 7.66 (m, 9H, Ar-H), 8.15-8.77 (m, 4H, pyridine ring), 8.93 (s, 1H, broad NH),
8.96 (s, 1H, pyridone ring). MS m/z (%): M+2 = 428 (20%), 426 (60), 362(52), 350(76),
324(58), 320(67), 289(80), 236(72), 206(78), 193(60), 170(67), 138(67), 107(74), 97(79),
60(100). Anal.; For C24H15ClN4O2 (426.85) Calcd.: C 67.53; H 3.54; Cl 8.31; N 13.13%
Found: C 67.85; H 3.75; Cl 8.63; N 13.46%.
4.4 Synthesis of 6-oxo-1-phenyl-3-(pyridin-3-yl)-6,7-dihydro-1H-pyrazolo[3,4-
b]pyridin-5-carbonitrile (6)
4.4.1. Method (A):
To a solution of 2 (0.49 g, 1.7 mmol) in ethanolic sodium ethoxide solution (0.1 g of sodium
metal in 10 ml absolute ethanol) malononitrile or cyanoactamide (1.7 mmol) was added. The

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reaction mixture was heated under reflux for 12 h. The solvent was evaporated under reduced
pressure, then washed with diethyl ether (2 x 10 ml) and recrystallized from ethanol to give
compound (6).
4.4.2. Method (B)
The procedure was similar to that described in Method (A) except that the mixture
was capped in closed vessels and irradiated in a microwave oven at 120 oC for 25 min
using Synthos 3000 (500 W). The reaction was worked as usual and the obtained solid
was recrystallized from ethanol to give (6) as Buff crystals in yield (63%) m.p. 290-293o
1
C; IR (KBr) ν
/ cm_1= 1651 (C=O), 2198 (C≡N), 3077 (Arom.-H). HNMR
max
(DMSO- d6): δppm = 7.33- 7.66 (m, 5H, Ar-H), 8.15-8.77 (m, 4H, pyridine ring &
1H, pyridone ring), 8.90 (s, 1H, broad NH). MS m/z (%): M = 313 (50%), 301(48),
279(73), 227(73), 209(60), 165(55), 127(68), 105(100), 80(61), 63(85). Anal.; For
C18H11N5O (313.31) Calcd.: C 69.00; H 3.54; N 22.35% Found: C 69.40; H 3.69; N
22.68%.
4.5. Synthesis of 1-phenyl-3-(pyridin-3-yl)-6-thioxo-6,7-dihydro-1H-pyrazolo[3,4-
b]pyridin-5-carbonitrile (9)
4.5.1. Method (A):
To a solution of 2 (0.49 g, 1.7 mmol) in ethanolic sodium ethoxide solution (0.1 g of
sodium metal in 10 ml absolute ethanol) was added cyanothioactamide (0.17 g, 1.7
mmol). The reaction mixture was heated under reflux for 12 h. The solvent was
evaporated under reduced pressure, then washed with diethyl ether (2 x 10 ml) and
recrystallized from ethanol to give (9).
4.5.2. Method (B):
A mixture of 6 (0.51 g, 1.5 mmol) and phosphorous pentasulphide (1.02 g, 2.2 mmol)
in 20 ml pyridine was refluxed for 10 h. The solvent was evaporated under reduced
pressure, washed with diethyl ether (2 x 10 ml) and crystallized from ethanol to give
(9) with the same m.p. and mixed m.p. as mentioned in method (A)
4.5.3. Method (C):

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The procedure was similar to that described in Method (A) and Method (B) except
that the mixture was capped in closed vessels and irradiated in a microwave oven at
120 0C for 25 min using Synthos 3000 (500 W). The reaction was worked as usual
and the obtained solid was recrystallized from ethanol to give (9) as faint brown crystals
in yield (64%); m.p. 283-286o C; IR (KBr) ν max / cm_1= 1373 (C=S), 2202 (C≡N), 3085
(Arom.-H). 1HNMR (DMSO- d6): δppm = 7.30- 7.56 (m, 5H, Ar-H), 8.25-8.74 (m,
4H, pyridine ring & 1H, pyridone ring), 8.88 (s, 1H, broad NH). MS m/z (%): M =
329(56%), 220(61), 203(83), 193(68), 169(61), 143(62), 130(69), 115(100), 96(73), 71(86),
64(52), 53(62). Anal.; For C18H11N5S (329.38) Calcd.: C 65.64; H 3.37; N 21.26; S
9.74% Found: C 65.96; H 3.59; N 21.60; S 9.97%.
4.6. Synthesis of ethyl-1-phenyl-3,6-di(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridin-5-
carboxylate (11)
4.6.1. Method (A):
To a solution of 2 (0.49 g, 1.7 mmol) in 20 ml glacial acetic acid in the presence of
ammonium acetate (0.15 g) was added ethyl-3-oxo-3-(pyridin-3-yl)propanoate (10) (0.32
g, 1.7 mmol). The reaction mixture was heated under reflux for 8 h. The reaction
mixture was monitored by TLC using benzene/acetone (2:1) as eluent. The solvent
was evaporated under reduced pressure, washed with diethyl ether (2 x 10 ml) and
recrystallized from methanol to give (11).
4.6.2. Method (B):
The procedure was similar to that described in Method (A)except that the mixture was
capped in closed vessels and irradiated in a microwave oven at 120 oC for 15 min
using Synthos 3000 (500 W). The reaction was worked as usual and the obtained solid
was recrystallized from ethanol to give (11) as faint yellow crystals in yield (71%); m.p.
273-275o C; IR (KBr) ν max / cm_1= 1615 (C=O), 3017 (Arom.-H). 1HNMR (DMSO-
d6): δppm = 1.32 (t, J䟞7.0 Hz, 3H, CH3), 4.40 (q, J䟞7.1 Hz, 2H, CH2), 7.33- 7.74
(m, 5H, Ar-H), 8.32-8.94 (m, 9H, pyridine ring). MS m/z (%): M = 421 (54%),
392(50), 376(53), 348(49), 322(49), 259(100), 242(73), 206(50), 164(58), 127(87), 105(71),

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13
77(96). Anal.; For C25H19N5O2 (421.45) Calcd.: C 71.25; H 4.54; N 16.62 Found: C
71.60; H 4.78; N 16.98%.
4.7. General procedure for the synthesis of 7-aryl-3-(pyridin-3-yl)-1H-dipyrazolo
[4,3-e:2',3'-a] pyrimidine (13a-c)
4.7.1. Method (A):
To a solution of 2 (0.49 g, 1.7 mmol) in acetic acid (10 ml) was added 3-substituted-5-
amino-1H-pyrazole (1.6 mmol). The mixture was heated under reflux for (8-10) h.
The mixture was poured into ice cold water; the formed solid was collected by
filtration, dried and crystallized from DMF to give (13a-c).
4.7.2. Method (B):
The procedure was similar to that described in Method (A) except that the mixture
was capped in closed vessels and irradiated in a microwave oven at 130 0C for 20 min
using Synthos 3000 (500 W). The reaction was worked as usual and the obtained solid
was recrystallized from DMF to give (13a-c).
4.7.3. 7-Phenyl-3-(pyridin-3-yl)-1H-dipyrazolo[4,3-e:2',3'-a]pyrimidine (13a)
Yellow crystals in yield (80 %) ; m.p. 304-305 0 C; IR (KBr) ν max / cm_1= 1617 (C=N),
3062 (Arom.-H). 1HNMR (DMSO- d6): δppm = 7.24 (s, 1H, CH of pyrazole), 7.25-7.79
(m, 10H, Ar-H), 8.22-9.13(m, 4H, pyridine ring & 1H, pyrimidine ring). MS m/z (%): M =
388 (17%), 329(37), 312(59), 248(100), 234(18), 220(22), 191(13), 115(12), 100(15),
77(52). Anal.; For C24H16N6 (388.42) Calcd.: C 74.21; H 4.15; N 21.64% Found: C
74.62; H 4.32; N 21.99%.
4.7.4.
7-(4-Chlorophenyl)-3-(pyridin-3-yl)-1H-dipyrazolo[4,3-e:2',3'-a]pyrimidine
(13b)
Yellow crystals in yield (74 %); m.p. 310-312 0 C; IR (KBr) ν max / cm_1= 1659 (C=N),
3065 (Arom.-H). 1HNMR (DMSO- d6): δppm = 7.24 (s, 1H, CH of pyrazole), 7.22-
7.78(m, 9H, Ar-H), 8.22-9.11(m, 4H, pyridine ring & 1H, pyrimidine ring). MS m/z (%): M
= 423 (45%), 335(63), 315(40), 296(40), 273(48), 237(54), 193(54), 164(58), 146(55), 127(64),

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14
119(53), 90(52), 71(42), 54(100).. Anal.; For C24H15ClN6 (422.87) Calcd.: C 68.17; H 3.58;
Cl 8.38; N 19.87% Found: C 68.60; H 3.72; Cl 8.60; N 20.26%.
4.7.5. 7-(4-Toloyl -3-(pyridin-3-yl)-1H-dipyrazolo[4,3-e:2',3'-a]pyrimidine (13c)
Deep yellow crystals in yield (60 %); m.p. 320-322 0 C; IR (KBr) ν max / cm_1= 1615
(C=O), 2991 (Aliph.-H), 3060 (Arom.-H). 1HNMR (DMSO- d6): δppm = 2.42 (s, 3H,
CH3), 7.22 (s, 1H, CH of pyrazole), 7.23-7.73(m, 9H, Ar-H), 8.00-9.11(m, 4H, pyridine ring
& 1H, pyrimidine ring). MS m/z (%): M = 402 (25%), 396(67), 342(24), 314(100), 286(48),
248(13), 199(29), 173(62), 149(37), 119(62), 97(51), 77(78). Anal.; For C25H18N6 (402.45)
Calcd.: C 74.61; H 4.51; N 20.88% Found: C 74.89; H 4.69; N 21.22%.
5. Conclusion
The objective of this work was established by testing antioxidant, anticancer and
antimicrobial activities for the new synthesized compounds. The results demonstrate
that compounds (4c) and (4d) showed the highest antioxidant activity, compounds (6)
and (9) showed the highest anticancer activity while (4d) exhibited the strong activity
against most of the tested organisms.
Acknowledgement
The authors express their deep thanks to Dr. M. E. H. Osman, Professor of plant
physiology, Botany Department, Faculty of science, Tanta University, Tanta, for
carrying out the biological activities at his research laboratory.

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15
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Figure 1: Assessment of the antioxidant activities of the compounds in comparison
with the ascorbic acid.
Figure 2: Scavenging antioxidant percentage of the ten compounds tested
Figure 3: IC50 of the ten compounds tested on MCF7 for breast cancer
Figure 4: IC50 of the ten compounds tested on HEPG2 for liver cancer.
Scheme 1. Synthesis of 4-(dimethylaminomethylene)-1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-5(4H)-one (2).
Scheme 2. Synthesis of 5-aroyl-1-phenyl-3-(pyridin-3-yl)-1H-pyrazolo [3,4-b]pyridin-6(7H)-one (4a-d).
Scheme 3. Synthesis of 6-oxo-1-phenyl-3-(pyridin-3-yl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-5-carbonitrile (6)
and 1-Phenyl-3-(pyridin-3-yl)-6-thioxo-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-5-carbonitrile (9)
Scheme 4. Synthesis of ethyl-1-phenyl-3,6-di(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylate
(11)
Scheme 5. Synthesis of 7-aryl-3-(pyridin-3-yl)-1H-dipyrazolo [4,3-e:2',3'-a] pyrimidine (13a-c)
Table 1: Comparison between conventional heating and microwave irradiation
Table 2: Decrease of DPPH absorbance (%) by the synthesized compounds
Table3: IC50 of the compounds tested on breast and liver cancer
Table 4: Diameters of inhibition zones (mm) of the compounds against different
tested microorganisms

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19
Table 1: Comparison between conventional heating and microwave irradiation
methods.
Compound no.
X
R
Time
Yield (%)
Microwave irradiation Conventional heating Microwave
irradiation
Conventional
heating
4a
4b
4c
N
H
130 o C, 20 minutes
130 o C, 20 minutes
130 o C, 20 minutes
130 o C, 20 minutes
120 o C, 25 minutes
120 o C, 25 minutes
120 o C, 15 minutes
130 o C, 20 minutes
130 o C, 20 minutes
130 o C, 20 minutes
Refluxing 8 hours
Refluxing 10 hours
Refluxing 8 hours
Refluxing 10 hours
Refluxing 12 hours
Refluxing 12 hours
Refluxing 8 hours
Refluxing 10 hours
Refluxing 8 hours
Refluxing 10 hours
90
83
82
87
90
92
90
93
88
85
75
67
70
62
63
64
71
80
74
60
CH CH3
CH
F
Cl
-
4d
6
CH
-
-
-
-
-
-
9
-
11
13a
13b
13c
-
H
Cl
CH3

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20
Table 2: Decrease of DPPH absorbance (%) by the synthesized compounds
Compound no.
Decrease of DPPH absorbance % EC50 (µg/ml)
mean ± SD (n = 3)
35.98 ± 1.60
38.89 ± 1.15
72.34 ± 1.61
63.63 ± 2.14
1.14 ± 0.53
17.04 ± 2.67
-ve effect
38.64
54.76
4.21
4a
4b
4c
8.97
4d
> 100
92.85
-ve effect
> 100
80.50
53.76
3.22
6
9
11
9.47 ± 1.60
29.35 ± 0.79
39.59 ± 1.17
82.77 ± 1.37
13a
13b
13c
Ascorbic acid (standard)
Table3: IC50 of the compounds tested on breast and liver cancer
Compound no
IC50 (µg/ml)
breast cancer
liver cancer
11.00
8.85
10.10
4a
9.90
4b
8.70
7.80
4c
6.45
9.00
4d
3.00
3.30
6
2.70
6.90
3.90
9
3.75
11
15.90
17.30
16.20
12.90
13a
13b
14.70
10.50
13c
MCF7-DOX (4.40)
HEPG2-DOX (3.10)
Standard-DOX

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21
Table 4: Diameters of inhibition zones (mm) of the compounds against different
tested microorganisms
Compound Bacillus Echerechia Enterobacter Serratia
Fusarium
oxysporum
Penicillium
expansum
no.
4a
ab
4c
coli
10.5
17
cloaca
11.5
12.5
21
-ve
-ve
-ve
-ve
21
12.5
17
-ve
-ve
-ve
-ve
12
-ve
10
15
17.5
17
-ve
-ve
10
19
19
4d
Chloram-
phenicol
18
17
25.8
The concentration used was 10 mg/ml. Control discs were performed in DMSO
(dimethylsulfoxide) and no an inhibitions zones of were observed (-ve resistance).
90
80
70
60
50
40
30
20
10
0
Asc.
4a
4b
4c
4d
6
9
11
13a
13b
13c
Compound
Figure 1: Assessment of the antioxidant activities of the compounds in comparison with the
ascorbic acid.

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22
4 a
4 b
4 c
4 d
6
9
1 1
1 3 a
1 3 b
1 3 c
A sco rb ic
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
C onc. (µg/m l)
Figure 2: Scavenging antioxidant percentage of the tested compounds
4 a
4 b
4 c
1 . 0
4 d
6
9
1 1
0 . 8
1 3 a
1 3 b
1 3 c
0 . 6
0 . 4
0 . 2
0 . 0
0
1 0
2 0
3 0
4 0
5 0
C o n c ; µ g /m l
Figure 3: IC50 of the ten compounds tested on MCF7 for breast cancer
4 a
4 b
4 c
4 d
6
591
1 . 3
1 . 2
1 . 1
1 . 0
0 . 9
0 . 8
0 . 7
0 . 6
0 . 5
0 . 4
0 . 3
0 . 2
9
1 1
1 3 a
1 3 b
1 3 c
0
1 0
2 0
3 0
4 0
5 0
C o n c ; µ g / m l

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23
Figure 4: IC50 of the ten compounds tested on HEPG2 for liver cancer.
N(Me)2
N
N
N
N(Me)2
N
N
N
N
O
N
O
N
O
DMFDMA
reflux
+
(2Z)
(2E)
(1)
Scheme 1. Synthesis of 4-(dimethylaminomethylene)-1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-5(4H)-one (2).
N(Me)2
O
O
N
N
O
CN
R
EtOH
+
X
N
N
N
N
H
N
O
Piperidine
R
X
X = N or CH
(4a-d)
(3a-d)
(2)
Scheme 2. Synthesis of 5-aroyl-1-phenyl-3-(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridin-6(7H)-one (4a-d).

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24
N
CN
NH
N
CN
N
O
H2C
CN
EtOH/ EtONa
(5)
N
CN
O
N
CN
H2C
N
N
N(Me)2
O
H
CONH2
N
EtOH/ EtONa
N
(6)
N
N
CN
N
N
O
NH2
(2)
(7)
N
CN
CN
N
H2C
NH2
N
S
CSNH2
EtOH/ EtONa
(8)
N
CN
S
N
N
N
H
(9)
Scheme 3. Synthesis of 6-oxo-1-phenyl-3-(pyridin-3-yl)-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-5-carbonitrile (6)
and 1-Phenyl-3-(pyridin-3-yl)-6-thioxo-6,7-dihydro-1H-pyrazolo[3,4-b]pyridin-5-carbonitrile (9)

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25
N(Me)2
O
O
N
N
COOC2H5
OEt
glacial acetic acid
amm.acetate
+
N
N
N
O
N
N
N
N
(10)
(2)
(11)
Scheme 4. Synthesis of ethyl-1-phenyl-3,6-di(pyridin-3-yl)-1H-pyrazolo[3,4-b]pyridin-5-carboxylate
(11)
R
N(Me)2
N
N
N
+
Acetic acid
N
N
N
O
N
N
N
N
H
NH2
N
R
(12 a-c)
(13 a-c)
(2)
Scheme 5. Synthesis of 7-aryl-3-(pyridin-3-yl)-1H-dipyrazolo [4,3-e:2',3'-a] pyrimidine (13a-c)

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> 4-(dimethylaminomethylene)-1-phenyl-3-(pyridin-3-yl)-1H-pyrazol-5(4H)-one
(enaminone) was reported.> The chemical behavior of enaminone with some active
methylene reagent was examined.> The reactions were carried out by conventional
heating and microwave irradiation.> Most of the synthesized compounds were
screened for antioxidant, antitumor and antimicrobial activities