Synthesis, biological evaluation and DFT calculation of novel pyrazole and pyrimidine derivatives

Article abstract of DOI:10.1016/j.molstruc.2018.11.008

The utility of the enaminonitriles 3a and 3b for the synthesis of the pyrazole derivatives 5a,b, 7a,b, diaminopyrimidine derivatives 9, 11, pyrazolo[1,5-a]pyrimidines 12, 15, triazolo[4,3-a]pyrimidines 13, 16, imidazo[1,2-a]pyrimidine derivatives 14 and 17, was explored. Most of the synthesized compounds showed excellent in vitro antitumor activity against MCF-7 cell line. They also exhibted high antimicrobial and antioxidant activities. Density functional theory (DFT) calculations at the B3LYP/6-31G level of theory have been carried out to investigate the equilibrium geometry of the novel phenylpyrazolo[1,5-a]pyrimidin-6-yl)methanone derivative 12 and phenylpyrazolo[1,5-a]pyrimidine derivative 15. The structure-activity relationship (SAR) has been used to correlate the biological activity with the appropriate quantum such as total energy. The energy of the HOMO and LUMO and Mulliken atomic charges were also calculated.

Full text of DOI:10.1016/j.molstruc.2018.11.008

Accepted Manuscript
Synthesis, Biological Evaluation and DFT Calculation of Novel Pyrazole and
Pyrimidine Derivatives
Ahmad M. Farag, Asmaa M. Fahim
PII:
S0022-2860(18)31312-7
10.1016/j.molstruc.2018.11.008
MOLSTR 25837
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Accepted Date:
16 September 2018
05 November 2018
Please cite this article as: Ahmad M. Farag, Asmaa M. Fahim, Synthesis, Biological Evaluation and
DFT Calculation of Novel Pyrazole and Pyrimidine Derivatives, Journal of Molecular Structure
(2018), doi: 10.1016/j.molstruc.2018.11.008
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Graphical Abstract:

ACCEPTED MANUSCRIPT
Synthesis, Biological Evaluation and DFT Calculation of Novel Pyrazole
and Pyrimidine Derivatives
Ahmad M. Faraga and Asmaa M. Fahimb
a Department of Chemistry, Faculty of Science, Cairo University, Giza 12613 Egypt
bDepartment of Green Chemistry, National Research Center Dokki, P.O. Box.12622 Cairo, Egypt
Abstract:
The utility of the enaminonitriles 3a and 3b for the synthesis of the pyrazole derivatives
5a,b, 7a,b, diaminopyrimidine derivatives 9, 11, pyrazolo[1,5-a]pyrimidines 12, 15,
triazolo[4,3-a]pyrimidines 13, 16, imidazo[1,2-a]pyrimidine derivatives 14 and 17, was
explored. Most of the synthesized compounds showed excellent in vitro antitumor activity
against MCF-7 cell line. They also exhibted high antimicrobial and antioxidant activities.
Density functional theory (DFT) calculations at the B3LYP/6-31G level of theory have
been carried out to investigate the equilibrium geometry of the novel phenylpyrazolo[1,5-
a]pyrimidin-6-yl)methanone derivative 12 and phenylpyrazolo[1,5-a]pyrimidine
derivative 15 . The structure-activity relationship (SAR) has been used to correlate the
biological activity with the appropriate quantum such as total energy. The energy of the
HOMO and LUMO and Mulliken atomic charges were also calculated.
Keywords: Enaminone, Pyrazole, Pyrimidine, MCF-7 cells, Antimicrobial, Antioxidant,
DFT Calculations.
1. Introduction:
Enaminonitriles are versatile reagents for the synthesis of heteroaromatics [1-9]. On the
other hand, pyrazole derivatives showed broad spectrum of biological activities, such as
oxidase inhibitor [10], anticonvulsant [11], antibacterial [12], hypotensive [13], antipyretic
[14] anti-inflammatory [15], antimicrobialand anthelmintic activities [16]. Moreover, the
nucleus of pyrazole represent the central unit in a variety of drugs [17] such as celecobix
(Celebrex) I, and sildenafil (Viagra) II (Figure 1). Also, pyrimidine nucleus exhibited a
wide range of pharmacological activities including antifungal, anti-inflammatory [18],
antihypertensive antiviral, antidiabetic, antioxidant, anticancer activities [20]. Several
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antibacterial drugs containing pyrimidine derivatives III-IV [20-22] are shown in Figure
1.
Figure 1. Some drugs incorporating pyrazole and pyrimidine heterocycles
The physicochemical and pharmacological properties of bis-heterocyclic compounds [23]
such as WMC-26 and LU 79553, both showing high effectiveness against in vivo
antitumor xenografts [24-26] (Figure 2).
O
O
O
N
NH
N
NH
N
O
N
NH
NH
N
N
O
O
N
(LU 79553)
VI
(WMC-26)
V
Figure 2. Some bis-heterocycles with antitumor activity.
The main goal of the research reported here was to synthesize and characterize mono- and
bis-heterocyclic compounds and to estimate of the energies of these molecules which is
very important both in theoretical studies and their chemical reactivity [27-29]. In this
context, we report the synthesis of pyrazolopyrimidine derivatives starting from dimethyl
terephthalate 1a and 4-(methoxycarbonyl)benzoic acid 1b and their conversion to the
corresponding enaminonitrile derivatives [30-32]. DFT calculations of selected examples
the synthesized pyrazolo[1,5-a]pyrimidin derivatives have also been carried out [33].
2. Results and Discussion:
2.1. Chemistry:
The adopted route for the reaction of dimethyl terephthalate 1a and 4-(methoxycarbonyl)
benzoic acid 1b with acetonitrile, in the presence of Sodium hydride (NaH), furnished the
corresponding 3,3'-(1,4-phenylene)bis(3-oxopropanenitrile) (2a) and 4-(2-cyanoacetyl)
benzoic acid (2b),[34] respectively (Scheme 1). Treatment of each of compound 2a and
2b with N,N-dimethylformamide dimethyl acetal (DMF-DMA) afforded high yields of the
corresponding enaminonitriles 3a and 3b, respectively.
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The IR spectrum of the product 3b exhibited a strong C=O absorption bands at 1723 cm-1
and 1677; respectively, a nitrile absorption band at 2359 cm-1 and OH band at 3406 cm-1
Its 1H NMR revealed signals at δ 3.37 and at δ 7.89 due to N,N-dimethylamino and CH
proton, respectively, in addition to an aromatic protons in the region 7.93-8.12 ppm. Its
mass spectrum revealed a peak at m/z 244 corresponding to its molecular ion. The reactivity
of the enaminonitriles 3a and 3b towards some nitrogen nucleophiles was investigated.
Thus, when the enaminonitrile 3a was treated with hydrazine and with phenyl hydrazine,
it afforded the corresponding pyrazole products 5a and 5b, respectively as shown in
Scheme 2. The 1H NMR spectrum of compound 5a taken as an example, revealed a singlet
signal at δ 8.08 (pyrazole-CH-2) and two singlet signals at δ 7.9-8.07 due to aromatic
protons, respectively The IR spectrum of the same compound revealed the presence of a
strong absorption band at 1720 cm-1 due to carbonyl and a broad absorption band in the
region 3262-3211cm-1 due to amino group and signal at 3365 due to NH group.
Scheme1. Synthesis of the oxopropanenitriles 2a,b and the enaminonitrile derivatives
3a,b.
When an ethanolic solution of the enaminonitrile 3b was treated with hydrazine
derivatives, it afforded the corresponding pyrazole derivatives 7a and 7b, respectively. The
IR spectrum of compound 7b, taken as a typical example, showed a characteristic
absorption band at 3423 cm-1 due to OH function, at 3262-3216 cm-1 due to amino group
and two strong absorption bands of carbonyl groups at 1722 cm-1 and 1698 cm-1. The 1H
NMR spectrum of the same compound revealed singlet signal at δ 9.80 due to pyrazole
proton and δ 12.30 (D2O-exchangable) due to OH protons. Its mass spectrum revealed a
peak at m/z 307 corresponding to its molecular ion (Scheme 2).
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O
O
N
N
H3C
H
N
CH3
N
O
CH3
N
CH3
CH3
CH3
N
O
O
3b
3a
RNHNH2
RNHNH2
N
CH3
H3C
N
O
O
H
N
N
NHR
H
O
NHR
NHR
N
H
N
N
H
O
H3C
N
CH3
O
N
H3C
6a,b
CH3
4a,b
-(CH3)2NH
-(CH3)2NH
O
O
H2N
R N
H
N
N
N
N R
O
R
N
R
a
b
NH2
O
NH2
O
H
Ph
7a,b
5a,b
Scheme 2. Synthesis of the pyrazole derivatives 5 and 7.
Treatment of the enaminonitriles 3a and 3b with guanidine, in basic medium, afforded an
excellent yield of a products identified as 1,4-phenylenebis((2,4-diaminopyrimidin-5-
yl)methanone) (9) and 4-(2,4-diaminopyrimidine-5-carbonyl) benzoic acid (11),
respectively. The formation of the latter products was assumed to take place via addition
of the N-nuclophile to the activated ethylenic double bond of the enaminonitriles 3a and
3b followed by cyclization and elimination of dimethylamine molecule as depicted in
Scheme 3.
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O
O
N
N
H
CH3
CH3
N
N
CH3
N
O
CH3
H3C
H3C
N
O
O 3b
3a
NH
NH
K2CO3/
isopropyl alcohol
K2CO3/
isopropyl alcohol
H2N NH2
O
H2N NH2
O
N
N
N(CH3)2
H2N
NH
H
NH
H
N
NH
O
H
NH
N
(H3C)2N
10
(H3C)2N
N
O
NH2
O
NH2
8
O
NH2
O
NH2
N
N
H2N
N
O
H
N
N
NH2
N
NH2
O
11
9
NH2
O
Scheme 3. Synthesis of diaminopyrimidine derivatives 9 and 11.
The IR spectrum of compound 9 showed strong absorption bands at 3366-3300 cm-1 and
3206-3039 cm-1 due to two amino group, in addition of carbonyl band at 1710 cm-1. The
1H NMR spectrum of the same compound revealed singlet signal at δ 7.87 due to
pyrimidine proton and Its mass spectrum revealed a peak at m/z 350 corresponding to its
molecular ion
In the same manner, the enaminonitrile 3a reacts regioselectively with heterocyclic amines
such as methyl-aminopyrazole, 1,2,4-aminotraizole and 2-aminobenzimidazole, in
refluxing pyridine, to afford the corresponding bis-heterocyclic systems 12, 13, and 14
(Scheme 4). The spectral data of the isolated compounds are in complete agreement with
the assigned structure. The 1H NMR spectrum of compound 13, taken as a typical example,
revealed a singlet signal at δ 8.33 (triazole-CH-2) and two singlet signals at δ 8.83, 8.97
due to pyrimidine protons (CH-6, CH-5) respectively, in addition to aromatic protons as a
multiplet in the region 8.02-8.12 ppm. The IR spectrum of the same compound revealed a
presence of strong absorption band at 1716 cm-1 corresponding to a carbonyl group.
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The IR spectrum of compound 14 revealed bands due to amino function at 3250-3100
1
(NH2) and carbonyl groups at 1733 and 1699 cm-1, respectively. Its H NMR spectrum
revealed an aromatic a multiplet in region δ 7.96, in addition to triplet at δ 7.43 and doublet
8.66 due to imdazole protons [cf. Experimental part].
CH3
N
O
N
H3C
N
CH3
N
3a
O
CH3
Ph
N
CH3
NH2
N
NH2
N
N
H
H
N
N
NH2
N
O
NH2
O
NH2
N
H
Ph
N
N
N
N
N
H3C
CH3
N
N
N
N
N
N
Ph
H2N
NH2
O
O
14
NH2
O
12
N
N
N
N
N
N
N
N
O
NH2
13
Scheme 4. Synthesis of bis(pyrimidine) derivatives 12, 13 and 14.
The enaminonitrile 3b reacts also with heterocyclic amines to afford the corresponding
pyrazolo[1,5-a] pyrimidine derivatives 15, 4-(5-amino-[1,2,4]triazolo[4,3-a]pyrimidine-6-
carbonyl)benzoic acid (16) and 4-(4-aminobenzo[4,5]imidazo[1,2-a]pyrimidine-3-
carbonyl)benzoic acid (17), respectively (Scheme 5). The elemental analyses and spectral
data are in complete agreement with the assigned structures (cf. Experimental part).
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O
N
H
CH3
N
O
Ph
H3C
CH3
NH2
H
N
3b
N
O
HN
NH2
N
H
N
H2N
O
NH2
N
N
N
O
NH2
N
N
N
O
N
Ph
H
O
17
O
H
N
O
CH3
O
H
N
N
15
O
N
N
O
NH2
16
Scheme 5. Synthesis of the pyrimidine derivatives 15, 16 and 17
3. Biological evaluation:
3.1 Anticancer screening studies:
Selected examples of the newly synthesized heterocyclic compounds (2a, 2b, 3a, 3b, 5a, 5b,
7a, 7b, 9, 11, 12, 13, 14, 15, 16 and 17) were screened for their anticancer activity on breast
cell line by one dose assay at the National Institute of Cancer (Cairo, Egypt) by SRB assay.
[35] Most of compounds showed potential cytotoxicity effect at four concentrations. The
cell line used in the present investigation was MCF-7 (breast carcinoma). The IC50 was
calculated with respect to the saline control group and the potency was calculated with
respect to the percentage of change for Tamoxifen and tested compounds, as presented in
Table 1 and illustrated in Figure. 3. In comparison to standard drugs, the tested compounds
exhibited high to a low antitumor activity toward the one cell line, with IC50 values ranging
from 3-50 µg/ml for MCF-7 breast cell line.
Table 1. In vitro anti-proliferative activities of the newly prepared derivatives against
MCF-7cell line.
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Compound
MCF-7
IC50(µg/ml)a
Compound
IC50(µg/ml)a
MCF-7
2a
28±0.13
7b
45.6±0.14
2b
3a
36.1±0.19
11.8±0.03
9
6.3±0.01
11
9.97±0.03
3b
5a
16.9±0.06
38.6±0.11
29.5±0.12
7.6±0.01
12
13
3.0±0.0051
5.4±0.01
5b
14
18.2±0.03
50±0.13
7a
15
16
16.2±0.05
8.31±0.0102
17
41±0.119
8.31±0.0102
(TAM)a
(TAM)a
TAM (tamoxifen, standard drug for breast cancer a IC50 values are the mean ± S.D. of three separate experiments.
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Figure 3. Survival Plot of MCF7 cells grown for 48 h in the presence of increasing concentrations of
compounds 3a,7b,14,7a,6b, 12, 17,6a,16,15,13, 2a, 9 and 11 in comparison to tamoxifen.
Anti-proliferative activity of the newly heterocyclic derivatives 2a, 2b, 3a, 3b, 5a, 5b, 7a,
7b, 9, 11, 12, 13, 14, 15, 16 and 17 was examined in the MCF-7 breast cancer using
sulforhodamine B (SRB) colorimetric assay as described by Skehan et al.[36] From the
obtained results. Compound 12 exhibited the highest antitumor activity compared to
standard drugs for the breast cancer cell lines with (IC50 = 3.0 µg/ml for MCF-7) compared
to the mono-substituent of pyrazolo[1,5-a]pyrimidine 15 (IC50 = 50.0 µg/ml for MCF-7)
which exhibited low activity as shown in Table 1. The benzoic acid derivatives 5a, 5b, 7b,
15 and 17 decreased the anticancer activity against MCF-7(IC50 =38.6, 29.5, 45.6, 50 and
41 µg/ml), whereas compounds 7a, 9, 11 and 13 possessed excellent anti-proliferative
activities against MCF-7 cells (IC50= 7.6, 6.3, 9.97 and 5.4 µg/ml, respectively).
Moreover, compounds 3a, 3b, 14 and 16 displayed good activities against MCF-7 cell line
with IC50 of 11.8, 16.9, 18.2 and 16.2 µg/ml; respectively. It seems that the benzoic acid
derivatives decrease the anticancer activity rather than the novel bis-heterocyclic
derivatives which enhanced biodegradation and thus affected drug availability to the target
sites. The obtained results are in good agreement with previous studies.[37,38]
3.2. Antimicrobial activity and structure–activity relationship (SAR):
The synthesized compounds 5a, 5b, 9, 12, 13, 14, 7a, 7b, 14, 11, 15 and 17 were examined
for antimicrobial strains. For the optimization purpose, the most active agent is compound
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17 to all strains and was selected for further modification, hoping to increase the the
antimicrobial activities due to carboxyl group. On the other hand, compounds 5b and 11
are almost having the same activity. It is worth mentioning that incorporation of carboxyl
group in compounds 16, 5b, 11 and 15 showed a high antimicrobial activity than the bis-
derivatives. Compounds 14 and 12 show moderate activities against all strains. These
results indicate that additional bulky substituents decrease the antibacterial activity.
Geotricum candidum is largely responsible for increased the incidence of invasive
aspergillosis (IA) in immune compromised patients[39]. Moreover, the mortality rate due
to invasive fungal diseases is still unacceptable high, because of a limited number of
antifungal agents.[40] Data in Tables 2 revealed that compounds 5b, 16 showed remarkable
activity.
Table 2. Antimicrobial activities of the synthesized compounds against the pathological
organisms expressed as inhibition diameter zonesin millimeters
Compound
Gram Positive Bacteria
Gram Negative Bacteria
Fungi
Bs
Sp
Ec
Pa
Af
Ca
Ss
Gc
5a
11.3±0.25
19.3±0.19
12.0±0.25
18.9±0.25
16.9±0.44
18.7±0.25
15.2±0.37
16.3±.37
20.3±0.58
14.9±0.25
23.4±0.37
16.2±0.44
25.4±0.1
-
NA*
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
19.7±0.2
-
9.3±0.58
16.7±0.58
11.8±0.58
18.3±0.25
16.8±0.58
17.3±0.44
14.3±0.58
14.6±0.58
18.7±0.19
13.4±0.37
20.9±0.58
15.8±0.19
23.7±0.1
-
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
-
NA
11.3±0.25
18.7±0.44
11.4±0.34
17.2±0.37
13.4±0.19
16.3±0.25
12.4±0.44
13.4±.44
17.8±0.63
11.7±0.19
21.9±0.25
14.7±0.58
-
12.5±0.19
20.6±0.19
12.6±0.37
18.7±0.37
14.8±0.58
18.8±0.44
14.6±0.37
15.8±0.44
19.3±0.44
12.9±0.19
29.8±0.19
16.2±0.44
-
5b
17.8±0.44
10.6±0.37
16.8±0.19
14.2±0.44
16.2±0.58
13.6±0.58
12.3±0.44
17.9±0.63
11.7±0.37
19.1±0.25
13.9±0.19
28.7±0.2
-
16.8±0.58
10.3±0.37
14.9±0.58
11.9±0.25
14.8±0.37
11.2±0.25
12.4±0.25
14.8±0.19
11.4±0.25
17.8±0.25
13.3±0.25
-
9
12
13
14
7a
7b
11
15
16
17
Amphotericin B
Ampicilline
Gentamicin
-
-
23.8±0.2
-
32.4±0.3
-
-
-
-
-
17.3±0.1
19.9±0.3
NA*: No Activity; The screening organisms, Mould: Gram positive bacteria: B. subtilis (RCMB 010069,Bs) and S. pneumonia
(RCMB 010010,Sp), two Gram-negative bacteria: P. aeruginosa (RCMB 010043, Pa), and E. coli (RCMB 010052,Ec),four
fungi A. fumigatus (RCMB 02568,Af), , Candida albicans (RCMB 05036,Ca), Syncephala strumracemosum(RCMB 05922,Sa)
and Geotricum candidum(RCMB 05097,Gc).
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The preliminary SAR study has focused on the effect of mono-carboxyl subsituent is
essential for the activity against Fungi and Bacteria. Amongst the dozens of
phenylpyrazoles found in recent few years, Compound 16 showed one of the highest
observed activities against B. subtilis and Geotricum candidum. It can be considered as a
lead compound in this field. Further studies are in progress on the same compound to
increaseits efficacy and understand its QSAR. Also, benzoic acid moiety is important to
increase antipseudomonal activity as observed in compound 11. The overall results of the
present study can be considered very promising in the perspective of new drugs discovery,
with respect to the medical importance of the tested microorganisms. P. aeruginosa has
emerged as one of the most problematic Gram-negative pathogens, with the alarmingly
high antibiotics resistance rates [41]. Even with the most effective antibiotics against this
pathogen, namely carbapenems (imipenem and meropenem), the resistance rates were
detected as 15-20.4% amongst 152 P. aeruginosa strains [42b]. This pathogen was found
to be sensitive to compounds 16, 11,14,18 and 5b (Table 2). Candida albicans and other
Candida species causing candidiasis are increasingly important diseases that are distributed
worldwide since they are frequent opportunistic pathogens in AIDS patients. This fungus
was found to be sensitive to most of the synthesized compounds especially triazolo[4,3-
a]pyrimidine 16 (Table 2).
3.3 DPPH radical scavenging:
The aim of this study is to identify the potential heterocyclic compound for antioxidant
activity. Amongst the tested compounds 1,4-phenylenebis-((5-amino-[1,2,4]triazolo[4,3-
a]pyrimidin-6-yl)methanone) (13) (27.7 μM ±2.4) was found to be potential antioxidant
agents. This may be due to effective conjugation. On the other hand, 4-(5-amino-1H-
pyrazole-4-carbonyl)benzoic acid (5b) (10.3 μM ±6.3) exhibited comparatively higher
antioxidant activity than 1,4-phenylene-bis((5-amino-1H-pyrazol-4-yl)methanone (5a)
(20.6 μM ±11.3). The presence of similarity substituent on the aromatic ring enhanced the
activity which may be due to +M effect. This evidenced that compound 3a showed
excellent radical scavenging activity when compared with the standard ascorbic acid. It
was also perceived that compounds 2a exhibited good activity. However, compound 14
displayed the least activity, The IC50 value of the standard drug ascorbic acid in DPPH
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method was found to be (34μ M ±6.17), respectively. The data presented in Table 3 showed
that all new synthesized compounds showed good to moderate anti-oxidative activity
[42,43].
Table 3. DPPH radical scavenging activity of new synthesized compounds
Compound
Inhibition%
90.20%
80.9%
DPPH IC50 (µM)a
34±6.17b
Ascorbic acid
2a
3a
5a
5b
9
47.0±7.8
88.9%
10.4±1.1
84.8%
20.6±11.3
10.3±6.3
99.5%
82.25%
91.3%
37.5±10.08
27.7±2.4
13
14
73.16%
66.4±10.4
bIC50 values represent as mean±SD of three determinations bReported IC50 = 15.3µMb
The overview of the results presented in Table 1, 2 and 3 revealed that, in general, most of
the bis-heterocyclic compounds were more active than those containing aromatic rings.
More studies are needed to be carried out to find the correlation between IC50 of the
evaluated noval pyrazole and pyrimidine derivatives and their molecular descripts, such as
electronic, lipophilic, and steric parameters.
4. Molecular Orbital Calculations:
Goal studies of structure-activity relationship (SAR) to correlate the biological activity of
a series of compounds with some appropriate descriptors such as the electronic properties
of molecules and the quantum chemical descriptors based on DFT have been found useful
in several SAR studies.[44] From the results of DFT calculations, different descriptors such
as some of optimized geometries (bond lengths and bond angles) as well as ground state
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energies (total energy ET, energy of highest occupied MO EHOMO, energy of lowest
unoccupied MO ELUMO, energy gap (Eg), dipole moment (µ), and net charge utilizing
Gaussian 09 [45] on fused dihydropyrazolo[1,5-a]pyrimidin (12) and phenylpyrazolo[1,5-
a]pyrimidine (15) using B3LYP/6-31G were selected for SAR analysis. The computed
quantum chemical descriptors based upon DFT calculations are presented in Tables 4 and
5. The energies of frontier molecular orbitals are important parameters in several chemical
and pharmacological processes. EHOMO measures the electron donating character of a
compound, while ELUMO measures its electron accepting character. Also, DFT
calculation was employed to study the stability of the (7-amino-2-methyl-3-phenyl-3,7-
dihydropyrazolo[1,5-a]pyrimidin-6-yl)(4-(7-amino-2-methyl-3-phenylpyrazolo[1,5-
a]pyrimidine-6-carbonyl)phenyl)methanone
(12)
and
4-(7-amino-2-methyl-3-
phenylpyrazolo[1,5-a]pyrimidine-6-carbonyl)benzoic acid (15) which they are both have
pyrazolo[1,5-a]pyrimidine moiety, but they differ in the arrangement of the rings.
From the results listed in Tables 4,5 and Figures. 4,5; the following conclusions were
inferred:
(1) For both optimized structures DHP (12) of fused dihydropyrazolo[1,5-a]pyrimidine
derivative is shown in Figure 4, and the PCA (15) of phenylpyrazolo[1,5-a]pyrimidine
derivative (Figure 5), the two compounds are non-planar, where both the pyrazolo[1,5-
a]pyrimidine are out of the molecular plane .
(2) The optimized bond length of both dihydropyrazolo[1,5-a]pyrimidine derivatives 12
fall in range 1.050 to 1.394 the same agreement with experimental [46] also phenyl-
pyrazolo[1,5-a]pyrimidine 15 alls in the range of 0.971to 1.531 Å which are in good
agreement with the experimental data 0.930-1.47 Å, for C=O bonds the optimized length
obtained by B3LYP/6-31G is 1.247-1.240 Å were slightly shorter than the experimental
value.[47,48]
(3) For dihydropyrazolo[1,5-a]pyrimidine derivatives 12, the pyrazolo[1,5-a]pyrimidine
are out of the molecular plane by -0.00227 (N29-N25-C26-N30) and 0.00259 (N12-N8-
C9-N13), respectively, which allow the interaction with the appropriate
dihydropyrazolo[1,5-a]pyrimidine 12 to afford the corresponding target molecule.
(4)- For phenylpyrazolo[1,5-a]pyrimidine PCA (15), the the pyrazolo[1,5-a]pyrimidine
was out of the molecular plane by -179.968 (N8-N7-N24-C25) and -0.25288 (H41-O40-
13

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C38-O39), respectively which may be due the stability of 4-(7-amino-2-methyl-3-phenyl
pyrazolo[1,5-a]pyrimidine-6-carbonyl)benzoic acid (15) between pyrazolo[1,5-
a]pyrimidine and benzoic acid ring.
Table4: Optimized bond length Å, and bond angle degrees dihedral angle degrees of
Compound DHP (12) and PCA(15) using DFT/B3LYP/6-31G(d)
DHP (12)
B3LYP/6-31G(d)
Parameters of
bond angles
PCA(15)
B3LYP/6-31G(d)
Parameters of bond
angles
Parameters of
bond lengths
Å
Degrees(o) Parameter
s of bond
Å
Degrees(o)
lengths
N12 – N8
N8-C7
N13-C9
N13-H46
N6-C7
C9-C4
C2-O3
C1-C2
N12-C11
N25-C26
N30-H54
N25-N29
C19-O20
1.3428
1.3795
1.2660
1.0500
1.3497
1.3948
1.2081
1.3509
1.3406
1.3888
<C21-C19-C20
<C21-C26-N30
<N25-C26-N30
<C28-N29-N25
<C27-C24-N 25
<C21-C19-O20
<N29-N25-C24
<N12-N8-C9
<N12-N8-C9
<N13-C9-C4
119.998
27.262
N24-N7
N7-N8
C5-N8
C26-O27
C38-O39
C38-O40
N7-C1
C4-C1
C5-C4
C26-C28
O40-H41
C25-N24
C26-C2
1.3665
1.4303
1.3826
1.2473
1.2421
1.4033
1.4264
1.4167
1.4411
1.5317
0.9718
1.3442
1.5299
<C2-C26-C27
<N24-C25-C2
<N8-N7-N24
<C5-N8-N7
<C5-C4-C1
<C2-C26-C27
<O40-C38-O39
<H41-O40-C38
<C5-C4-C20
118.532
124.008
128.326
106.282
109.012
118.532
120.936
108.942
27.8105
122.307
-179.968
-0.25288
0.21265
122.194
102.803
105.485
119.998
33.440
123.520
122.453
112.178
-0.00227
0.00259
179.997
<C10-C9-C5
1.0500 <N29-N25-C26-N30
1.3395
1.2081
<N8-N7-N24-C25
<H41-O40-C38-O39
<N7-N8-C5-C4
<N12-N8-C9-N13
<N6-C5-C4-C2
From the above results (Tables 1-4), it is clear that as the dihydropyrazolo[1,5-
a]pyrimidine (DHP) 12 and phenylpyrazolo[1,5-a]pyrimidine (PCA) 15, come close to the
molecular plane of the pyrazolo[1,5-a]pyrimidine moiety. On the other hand, compounds
12 and 15 moieties go far from the molecular plane of the pyrazolo[1,5-a]pyrimidine ring,
by increasing its dihedral angles.
14

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(5)
The
two
p-isoelectronic
structures
12
and
15
are
though dihydropyrazolo[1,5-a]pyrimidine 12 seems
a
more stable than
phenylpyrazolo[1,5-a]pyrimidine 15 by 3.0717 eV (≈70.835kcal) (Table 5).[49]
Table 5: Quantum-chemical descriptors based on DFT calculations used for SAR studies at DFT/
B3LYP/6-31G(d).
Compound 12
Compound 15
-1254.570
-5.1598
ET (au)
-1883.705
-5.4423
-2.3706
3.0717
ET (au)
EHOMO (eV)
ELOMO (eV)
Eg (eV)
EHOMO (eV)
ELOMO (eV)
Eg (eV)
-2.3399
3.1799
µ (D)
6.4786
µ (D)
4.8810
N30
N13
N6
N25
O20
O3
C19
C24
C10
-0.884
-0.812
-0.502
-0.455
-0.461
-0.452
0.313
0.415
-0.125
a Eg= ELOMO - EHOMO.
N13
N8
N6
N12
O3
O20
O43
C10
C7
-0.817
-0.443
-0.503
-0.430
-0.461
-0.465
-0.562
-0.125
0.549
Net charges
Net charges
(6) From the calculations of the energy gap, Eg, which measure the chemical activity, the
dihydropyrazolo[1,5-a]pyrimidine (DHP) 12 was found to be more reactive than
phenylpyrazolo[1,5-a]pyrimidine (PCA) 15 by -2.4951 kcal as shown in Figures 4 and 5.
(7) The polarity or charge separation over the molecule, which is measured by the dipole
moment µ, showed that µ of dihydropyrazolo[1,5-a]pyrimidine (DHP) 12 > µ of amino
phenylpyrazolo[1,5-a]pyrimidine (PCA) 15 by 1.5976 D.
15

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From the above results (5–7), one can conclude that although the total energy (ET) of
dihydropyrazolo[1,5-a]pyrimidine (DHP) 12 is less than that of phenylpyrazolo[1,5-
a]pyrimidine (PCA) 15 by a factor of -394788.257 kcal together with the fact that polarity,
as well as the reactivity of compound 12, are more than that of 15, experimental results
showed that dihydropyrazolo[1,5-a]pyrimidine (DHP) 12 give excellent biological
activity rather than compound 15 which its energy is more so give less activity
Figure 4. The optimized geometry, numbering system, and and the vector of the dipole
moment of DHP (12) using B3LYP/6-31G.
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Figure 5. The optimized geometry, numbering system, and and the vector of the dipole
moment PCA (15) using B3LYP/6-31G.
4.1 Frontier Molecular Orbitals (FMO):
The importance of the frontier molecular orbital (FMO) contribution is lying in
determining the charge-separated states and reflecting the chemical activity of the studied
molecules. General, hybrid funcionals were used to improve the description of the ground
state energies for small molecules,[50] One of the most commonly hybrid functional used
is B3LYP which leads to improved band gaps as good as that obtained from sophisticated
correlated calculation. Therefore, in order to rationalized this increased accuracy by
comparison with results from time-dependent DFT (TD-DFT) calculations so we found the
HOMO-LUMO gap energy for compound 12 was (0.16109 eV) rather than compound 15
which has energy gap (1.0753 eV). The small energy gap in compound 12 expose a numeral
of unique properties such as infrequent electronic properties, for instance thermo-excited
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intermolecular electron transfer (in solution) and metal conductivity (in the solid state),[51]
as demonstrated in Figure 6.
Figure 6. illustrated the electronic structures of HOMO and LUMO of compounds 12 and
15. As can be seen from that figure, strong delocalization of the HOMO for compound 12
occurs on the pyrazolopyrimidine donor moiety while strong delocalization of the LUMO
occurs on the benzene bridge (acceptor moiety) between the pyrazolopyrimidine moieties
demonstrating the flow of electron density along compound 12. Furthermore, strong
delocalization of the HOMO for compound 15 occurs on the pyrazolopyrimidine donor
moiety while strong delocalization of the LUMO occurs on the benzoic acid as an acceptor
moiety. Consequently, the lowering of the HOMO-LUMO energy gap is essentially a
consequence of the large stabilization of the LUMO due to the strong electron-accepting
capability of the electron-acceptor group and influences the biological activity of the
molecule.
Figure 6. Gap energy (HOMO–LUMO) (eV) are calculated for compounds 12 and 15
using TD-DFT B3LYP/6-31G (d).
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5. Conclusion
In this investigation, we have developed an efficient and simple method for the synthesis
of a series of novel heterocycles of 5-amino-1H-pyrazol-4-yl derivatives 5 a,b and 7 a,b,
2,4-diaminopyrimidin 9 and 11, pyrazole[1,5-a] pyrimidine 12 and 15, triazolo [4,3-
a]pyrimidine 13 and 16 and dihydropyrimido[1,2-b]indazole 15 and 18 from the ester
derivatives 1a,b. (7-Amino-2-methyl-3-phenyl-3,7-dihydropyrazolo[1,5-a] pyrimidin-6-
yl)(4-(7-amino-2-methyl-3-phenylpyrazolo[1,5-a]pyrimidine-6-carbonyl)phenyl)
methanone (12) showed a higher antitumor activity than tamoxifen standard drug, while 4-
(5-amino-[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonyl)benzoic acid (16) showed high
antimicrobial activity in all strain media comparable with commercial compounds. Also,
1,4-phenylenebis((5-amino-1-phenyl-1H-pyrazol-4-yl)methanone) (5b) showed high
activity against DPPH as antioxidant activity. DFT calculations at B3LYP/6-31G
predicted that the dihedral angles of fused pyrazolo[1,5-a]pyrimidine rings of both p-
isoelectronic structures 12 and 15 are a new building block heterocyclic compounds
responsible for that chemical and biological activities and therefore, become lead
molecules for further synthetic and biological evaluation. It can be concluded that this class
of compounds certainly holds great promise towards pursuit to discover a novel class of
antitumor, antimicrobial and antioxidant agents. Further studies are being conducted to
acquire more information about quantitative structure-activity relations.
6. Experimental:
6.1. General
All melting points were measured on a Gallenkamp melting point apparatus and were
uncorrected. The infrared spectra were recorded in potassium bromide disks on a
PyeUnicam SP-3-300 and Shimadzu FT-IR 8101 PC infrared spectrophotometers. The
NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer. 1HNMR
spectra were run at 300 MHz and 13CNMR spectra were run at 75.46 MHz in dimethyl
sulphoxide (DMSO-d6). Chemical shifts were related that of the solvents. Mass spectra
were recorded on a Shimadzu GCMS-QP-1000EX mass spectrometer at 70 e.V.
19

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6.1.1 Material and reagents:
Dimethyl terephthalate, 4-(methoxycarbonyl)benzoic acid, acetonitrile, sodium hydride,
dimethylformamide dimethyl acetal (DMF-DMA), hydrazine hydrate, phenylhydrazine,
triethylamine, 2-aminobenzimidazole, aminopyrazole and aminotriazole were purchased
from Aldrich Chemical CO. Ethanol, pyridine, toluene, THF and piperidine purchased
from Aldrich Chemical CO. Methanol, petroleum ether; chloroform where BDH
reagents.
6.1.2. Preparation of 3-oxopropanenitrile derivatives 2a, b:
General procedure:
A three-necked, round bottomed flask (500-ml), equipped with a magnetic stirrer,
thermometer, decanter and condenser was charged with the proper ester 1a or 1b (10g),
acetonitrile (10 ml, 10 mmole), in dry THF and stirred well, then an equivalent weight of
NaH and DMF were added then refluxed to 100oC for 2 h then left to cool, and filtered
washed with petroleum ether. The formed salt was dissolve in ice-cold water and
acidified HCl to afford the corresponding 3-oxo-propanenitrile derivatives 2a and 2b;
respectively.
3,3'-(1,4-Phenylene)bis(3-oxopropanenitrile) (2a) [33] pale brown powder (EtOH/DMF),
m.p:175-177oC, yield:75% C12H8N2O2, IR (KBr, cm-1): ν= 2549 (C≡N), 1717 (C=O) ;
1HNMR (DMSO-d6) 3.875 (s, 4H, H2C), 8.03-8.07 (s, 4H, H-Ar), 13C NMR (DMSO-d6):
29.3 (CH2), 129.9 (CH), 134.4(C≡N), 135.4 (CH), 167.10 (C=O); MS, (m/z): 149 (M+,
100.0%),163(38.46%), 212 (25.23%), 104(60.52%); Anal calcd C12H8N2O2 (212.20); C,
67.92; H,3.80; N, 13.20%; Found: C, 67.88; H, 3.77; N, 13.24%.
4-(2-Cyanoacetyl)benzoic acid (2b), white crystals (EtOH), m.p:170-172oC, yield: 75%,
C10H7NO3 IR (KBr, cm-1) ν=3374(OH), 2237(C≡N), 1715 (C=O) , 1689 (C=O); 1HNMR
(DMSO-d6): 3.480 (s, 2H, H2C), 7.93-8.00(d, 2H, HC), 8.01-8.15(d, 2H, HC), 12.30(s, 1H,
13
HOOC D2O exchangeable); C NMR (DMSO-d6):δ 29.77 (CH2), 129-130 (CH), 134
(CN), 135 (CH),139 (CH), 165 (C=O), 170(C=O); MS (m/z): 149 (M+, 100.0%), 189
(94.3%),179 (83.8%), 120 (70.4%); Anal calcd C10H7NO3 (189.17) C, 63.49; H, 3.73; N,
7.40%; Found C, 63.53 %; H, 3.77%; N, 7.45%;
20

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6.3. Synthesis of Enaminonitrile derivatives 3a,b:
General procedure:
A solution of the 3-oxopropanenitrile derivatives 2a or 2b (10 mmol) in dry THF (50 ml)
and dimethylformamide-dimethyl acetal (DMF-DMA) (10mmol) was refluxed for 2 h and
then left to cool. The white precipitated was filtered off, washed with dry petroleum ether
(40-60 oC) and dried.
(Z)-2-(4-((E)-2-cyano-3-(dimethylamino)acryloyl)benzoyl)-3-(dimethylamino)
acrylonitrile (3a) brown crystals (EtOH), m.p:197-199oC, yield: 80%: C18H18N4O2; IR
(KBr, cm-1) ν=: 2200(C≡N), 1712(C=O); 1HNMR (DMSO-d6): 3.9 (s, 12H, H3C), 7.9 (s,
4H, H-Ar), 13C NMR (DMSO-d6): 41 (CH3), 85.2 (CH), 115.8 (C≡N), 129.3 (CH), 137
(CH), 141 (CH), 168 (C=O); MS (m/z): 322.36 (M+, 100%), 228(14.3%) Anal calcd
C18H18N4O2 (322.36) C, 67.07; H, 5.63; N, 17.38%; Found: C, 67.03; H, 5.67; N, 17.42%
(E)-4-(2-cyano-3-(dimethylamino)acryloyl)benzoic acid (3b) pale white crystals (EtOH),
m.p: 182-184oC, yield: 88%; C13H12N2O3; IR (KBr, cm-1), ν=3406 (OH), 2359 (C≡N),
1723(C=O), 1677 (C=O); 1HNMR (DMSO-d6): 3.37(s, 6H, H3C), 7.89 (s, 1H, HC), 7.93-
8.03(d, 2H, HC aromatic), 8.06-8.12(d, 2H, HC aromatic), 12.30(s, 1H, 1H, HOOC D2O
13
exchangeable); CNMR (DMSO-d6): δ 42.7 (CH3), 85.2 (CH), 115.8 (CN), 129.8 (CH),
136 (CH), 141 (CH), 157.2 (CH), 169.3 (C=O), 186.4 (C=O); (m/z): 244 (M+, 100.0%),
55(74%), 57 (6.86%); Analysis for C13H12N2O3 (244.25); C, 63.93;%; H, 4.95;% N,
11.47%; Found C, 63.98;%; H, 5.00;%; N, 11.43%.
6.4. Reaction of the enaminonitrile derivatives 3 with hydrazine derivatives:
General procedure:
To an ethanolic solution of the appropriate enaminonitrile 3 (0.44 g, 2 mmol), (20 ml),
hydrazine hydrate (80 %, 0.2 ml), phenylhydrazine (0.2 ml, 2 mmol) was added and the
resulting mixture was refluxed for 6h then left to cool. The solid formed was filtered off,
and washed with ethanol and dried, to afford the corresponding pyrazoles derivatives 5a,
b and 7a, b.
21

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1,4-phenylenebis((5-amino-1H-pyrazol-4-yl)methanone) (5a) pale brown crystals:
(EtOH), m.p: 211-213oC yield: 66%, C14H12N6O2; IR (KBr, cm-1) ν= 3365(NH), 3262-
3211(NH2), 1720 (C=O); 1HNMR (DMSO-d6): 7.34 (s, 4H, H2N D2O-exchangeable),
7.9-8.07(s, 4H, HC aromatic), 8.08 (s, 2H, H2C pyrazole), 13.26(s, 2H, HN D2O-
exchangeable); 13C NMR (DMSO-d6): δ 96.3 (CH), 128.3(CH), 129.6 (CH), 135.1 (CH),
138.2 (CH), 145.2 (CH), 196.3 (C=O), MS (m/z): 296 (M+, 100.0%), 187 (25%),
110(12.3%); Anal calcd C14H12N6O2 (296.28); C, 56.75; H, 4.08; N, 28.36% Found: C,
56.79; H, 4.03; N, 28.40%.
1,4-Phenylenebis((5-amino-1-phenyl-1H-pyrazol-4-yl)methanone) (5b), dark brown
crystals (EtOH), m.p: 231-233oC yield:64%, C26H20N6O2, IR (KBr, cm-1) ν=3362-3330
(NH2), 1646 (C=O): 1HNMR (DMSO-d6): 6.43-6.49 (t, 2H, HC, J=3.3Hz), 6.7-7.3(m,
8H, HC), 7.4-7.8(s, 4H, HC), 7.93(s, 4H, H2N D2O-exchangeable), 8.41(s, 2H, HC-
pyrazole); MS (m/z): 448 (M+, 100%), 290(36%), 158(12.3%) Anal calcd C26H20N6O2
(448.16): C, 69.63; H, 4.49; N, 18.74, Found C, 69.68; H, 4.52; N, 18.79.
4-(5-Amino-1H-pyrazole-4-carbonyl)benzoic acid (7a) white crystals (EtOH), m.p:198-
199oC , yield: 69%,C11H9N3O3; IR (KBr, cm-1) ν=: 3423 (OH), 3330 (NH), 3262-3216
(NH2), 1723(C=O), 1693(C=O); 1HNMR (DMSO-d6): 7.43 (s, 2H, H2N D2O-
exchangeable), 7.62-7.96 (d, 2H, HC aromatic, J=12Hz), 7.9-8.1 (d, 2H, HC aromatic,
J=12Hz), 9.80 (s, 1H, HC pyrazole), 12.30 (s, 1H, HO D2O-exchangeable), 13.09(s, 1H,
HND2O- exchangeable); MS (m/z): 233(M+, 100.0%), 205 (47.39%), 64 (35.98%); Anal
calcd C11H9N3O3 (231.21) C, 57.14; H, 3.92 ; N, 18.17%, Found: C, 57.17; H, 3.96; N,
18.20%.
4-(5-Amino-1-phenyl-1H-pyrazole-4-carbonyl)benzoic acid (7b) brown solid crystals
(EtOH), m.p: 235-237oC, yield:60%,C17H13N3O3, IR (KBr, cm-1) ν=: 3423 (OH), 3262-
3216 (NH2), 1722 (C=O), 1698 (C=O); 1HNMR (DMSO-d6): 7.43-7.83(m, 5H, H-Ar),
7.93 (s, 2H, H2N D2O-exchangeable), 8.07-8.12 (m, 4H, H-Ar), 9.80 (s,1H, HC
pyrazole), 12.30 (s, 1H, HO D2O-exchangeable); MS (m/z): 307(M+, 100%), 311
22

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(7.57%), 68 (33.2%); Anal calcd C17H13N3O3 (307.30); C, 66.44; H, 4.26;N, 13.67%;
Found :C, 66.48; H, 4.31; N, 13.63%.
6.5. Reaction of the enaminonitriles 3 with guanidine:
General procedure:
To a mixture of the appropiate enaminonitrile 3a or 3b (0.44 g, 2 mmol) and guanidine
nitrate (2.3 mmol), in isopropyl alcohol (30 ml), anhydrous potassium carbonate (0.552 g,
4 mmol) was added. The resulting mixture was refluxed for 6h and then allowed to cool to
room temperature and diluted with water (20ml). The solid product was collected by
filtration, washed with water and dried. Recrystallization from ethanol afforded the
corresponding (2,4-diaminopyrimidin-5-yl) derivatives 9 and 11; respectively.
1,4-Phenylenebis((2,4-diaminopyrimidin-5-yl)methanone) (9) yellow crystals (EtOH),
m.p: 221-223oC yield: 65%, C16H14N8O2; IR (KBr, cm-1) ν = 3366-3300 (NH2), 3206-3039
(NH2), 1710 (C=O) ;1HNMR (DMSO-d6): 6.98 (s, 4H, H2N D2Oexchangeable),7.63(s,
4H,H2N-D2Oexchangable) 7.87 (s, 2H, HC-pyrimidine), 8.07 (s, 4H, H-Ar); MS (m/z): 350
(M+, 100.0%),213(29.3%), 137.2(9.6%); Anal calcd C16H14N8O2 (350.33); C, 54.85; H,
4.03; N, 31.98 % Found: C, 54.88; H, 4.00; N, 32.02%.
4-(2,4-Diaminopyrimidine-5-carbonyl)benzoic acid (11) white crystals (EtOH), m.p:
>300oC, yield: 66%, C12H10N4O3, IR (KBr, cm-1) ν=: 3699(OH), 3411-3396(NH2), 3256-
1
3102 (NH2), 1723 (C=O), 1688 (C=O); HNMR (DMSO-d6): 6.96(s, 2H, H2N D2O-
exchangeable), 7.085 (s, 1H, HC pyrimidine7.83(s, 2H, H2N D2O-exchangeable), 7.9-8.02
(d, 2H, HC aromatic, J=1.2Hz ), 8.02-8.06 (d, 2H, HC aromatic, J=3Hz), 12.36(s, 1H, HO
D2O-exchangeable),; MS (m/z): 258 (M+, 100.0%), 149 (50.02%), 104(77.3%); Anal calcd
C12H10N4O3 (258.23); C, 55.81;H, 3.90; N, 21.70% Found: C, 55.85; H, 3.94; N, 21.74%.
6.6. Reaction of the enaminonitrile 3a, b with heterocyclic amines:
General procedure:
A mixture of the enaminonitrile 3 (2 mmol) and the corresponding heterocyclic amine
[aminoyrazole or 2-aminobenzimidazole or 1,2,4- aminotriazole] (2 mmol) in pyridine (15
23

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ml) was refluxed for 6h, then left to cool to room temperature. The precipitated product
was filtered off, washed with ethanol and recrystallized from the proper solvent to afford
the products 12, 13, 14, 15, 16 and 17.
1,4-phenylenebis((7-amino-2-methyl-3-phenylpyrazolo[1,5-a]pyrimidin-6-yl)methanone)
(12) brown powder (EtOH/DMF), m.p: 263-265oC yield: 71%; C34H26N8O2, IR (KBr, cm-
1) ν=: 3421-3319 (NH2), 3309-3260 (NH2), 1714(C=O); 1HNMR (DMSO-d6): 2.23 (s, 6H,
H3C), 7.23-7.44 (m, 10H, H-Ar), 7.75(s, 4H, H2ND2O-exchangeable), 8.11-8.17 (s, 4H, H-
Ar), 8.34 (s, 2H, HC pyrimidine); 13C NMR (DMSO-d6): δ 11.0 (CH3), 115.3 (CH), 126.5
(CH), 128.7 (CH), 129 (CH), 132.6 (CH), 137.3 (CH), 160 (CH), 164 (CH), 169 (CH), 190
(C=O), 196.3 (C=O) MS (m/z): 578(M+, 100.0%), 357(14.3%), 255 (25.15%), 166
(13.55%), 149(63.2%), 121(54.92%); Anal calcd. C34H26N8O2 (578.62); C, 70.58; H, 4.53;
N, 19.37% Found: C, 70.62; H, 4.57; N, 19.41%
1,4-phenylenebis((5-amino-[1,2,4]triazolo[4,3-a]pyrimidin-6-yl)methanone) (13) pale
yellow crystals (EtOH/DMF), m.p: 268-270oC, yield:72%, C18H12N10O2, IR (KBr, cm-1)
1
ν=3320-3214(NH2), 3125-3065 (NH2), 1716 (C=O); HNMR (DMSO-d6): 7.95 (s, 4H,
H2N, D2O exchangeable), 8.02-8.12 (m,4H, H-Ar), 8.33 (s, 2H, triazole- HC-2, J = 7.2Hz),
8.83(s,1H, HC, pyrimidine), 8.97(s,1H, HC, pyrimidine), MS (m/z): 400 (M+, 100.0%),
340 (47.68%), 166 (4512%), 149 (10.2%), 79.20 (99.34%); 13C NMR (DMSO-d6); 111.7
(CH), 121(CH), 129 (CH), 136 (CH), 146 (CH), 154 (CH), 166 (CH), 168 (CH), 169
(C=O); Anal calcd C18H12N10O2 (400.35); C,54.00; H, 3.02; N, 34.99% Found: C, 54.04;
H, 3.06; N 34.95%.
1,4-phenylenebis((4-aminobenzo[4,5]imidazo[1,2-a]pyrimidin-3-yl)methanone) (14)
dark yellow crystals (EtOH/DMF), m.p: 298-299o C; yield: 69%; C28H18N8O2, IR (KBr,
cm-1) ν=3250-3100 (NH2), 1733 (C=O), 1699 (C=O); 1HNMR (DMSO-d6): 7.21-7.29 (d,
4H, HC), 7.43(t, 2H, HC, J=6.3Hz), 7.71(s, 4H, H2N D2O exchangeable),7.96 (s, 4H, H-
Ar), 8.12 (s, 2H, HC), 8.66 (d, 2H, HC, J =7.8 Hz), MS (m/z): 149 (M+, 100.0%), 166
(23.08%); Anal calcd C28H18N8O2 (498.49); C, 67.46; H, 3.64; N, 22.48%; Found: C,
67.50; H,3.60; N, 22.42%.
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4-(7-amino-2-methyl-3-phenylpyrazolo[1,5-a]pyrimidine-6-carbonyl)benzoic acid (15)
white crystals (EtOH), m.p: >300o C yield: 61%,(C21H16N4O3), IR (KBr, cm-1) ν =3419
(OH), 3329-3201(NH2), 1721(C=O), 1689 (C=O);1HNMR (DMSO-d6): 1.04 (s, 3H, H3C),
7.63-7.77 (s, 5H, H-Ar), 7.88(s, 2H, H2N D2O-exchangeable), 8.29-8.30 (dd, 4H, H-Ar, J
= 3.2 Hz), 8.33(d, 1H, HC, J =1.2Hz), 12.32(s, 1H, HO D2O- exchangeable), MS(m/z):
372(M+, 100.0%), 75(85.37%); Anal calcd C21H16N4O3 (372.38); C,67.73; H,4.33;
N,15.05% Found: C,67.77; H, 4.38; N, 15.01%
4-(5-Amino-[1,2,4]triazolo[4,3-a]pyrimidine-6-carbonyl)benzoic acid (16) white crystals
o
-1
(EtOH), m.p:> 300 C yield: 68%; C13H9N5O3; IR (KBr, cm ): 3423(OH), 3210-3065
1
(NH2), 1722 (C=O), 1685 (C=O); HNMR (DMSO-d6): 7.89 (s, 2H, H2N D2O-
exchangeable) 7.83- 7.86 (m, 4H, H-Ar), 7.99 (s, 1H, HC pyrimidine), 8.08 (d, 1H,
13
triazole-HC-2, J = 3.2Hz), 12.28 (s, 1H, HO D2O-exchangeable), C NMR (DMSO-d6),
115 (CH), 130 (CH), 134 (CH), 139 (CH), 152 (CH), 160 (CH), 168 (CH), 169 (C=O), 196
(C=O); MS (m/z): 283(M+, 100.0%), 84(43%), 149 (28.67%); Anal calcd C13H9N5O3
(283.24); C, 55.13; H,3.02; N,24.73% Found: C, 55.10; H, 3.06; N,24.77%.
4-(4-aminobenzo[4,5]imidazo[1,2-a]pyrimidine-3-carbonyl)benzoic acid (17) yellow
crystals (EtOH), m.p: 305-308 yield: 67%; C18H12N4O3; IR (KBr, cm-1): 3237 (OH), 3128-
3087(NH2), 1712(C=O), 1682(C=O);1HNMR (DMSO-d6): 7.20-7.31 (d, 2H, HC), 7.47 (d,
1H, HC), 7.89 (s, 2H, H2ND2O-exchangeable), 8.13-8.25(m, 4H, H-Ar), 8.31 (s, 1H, HC),
8.9 (d, 1H, HC-pyrimidine, J = 7.8 Hz), 12.32(s, 1H, HO D2O-exchangeable), 13C NMR
(DMSO-d6), 111.9 (CH), 119 (CH), 123 (CH), 129(CH), 134(CH), 140 (CH), 155 (CH),
166 (CH), 169(C=O), 196(C=O); MS (m/z): 332 (M+, 100.0%), 166 (23.08%), 149(23.6%);
Anal calcd C18H12N4O3 (332.31); C, 65.06; H, 3.64; N, 16.86% Found: C,65.10; H,3.70;
16.82%.
6.7. In vitro Cytotoxic Activity:
MCF-7 breast cancer cell lines, obtained from the National Cancer Institute (Cairo,
Egypt), were grown in RPMI-1640. Media were supplemented with 10% heat-inactivated
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FBS, 50 units/ml of penicillin and 50 g/mL of streptomycin and maintained at 37°C in a
humidified atmosphere containing 5% CO2. Cytotoxicity was determined using SRB
method as previously described via Skehan et al [35]. Exponentially growing cells were
collected using 0.25% Trypsin-EDTA and seeded in 96-well plates at 1000-2000
cells/well in RPMI-1640 supplemented medium. After 24 h, cells were incubated for 72 h
with various concentrations of the tested compounds as well as tamoxifen as reference
drug. Following 72 h of treatment, the cells will be fixed with 10% trichloroacetic acid
for 1 h at 4 ºC. Wells were stained for 10 min at room temperature with 0.4% SRB
dissolved in 1% acetic acid. The plates were air dried for 24 h and the dye was
solubilized with Tris-HCl for 5 min on a shaker at 1600 rpm. The optical density (OD) of
each well was measured spectrophotometrically at 564 nm with an ELISA microplate
reader (ChroMate-4300, FL, USA) The IC50 values were calculated according to the
equation for Boltzmann sigmoidal concentration response curve using the nonlinear
regression fitting models (Graph Pad, Prism Version 5). The results reported are means of
at least three separate experiments. Statistical differences were analyzed according to one
way ANOVA test wherein the differences were considered to be significant at p< 0.05.
6.8 Antimicrobial Screening:
Antibacterial and antifungal activities were performed at the Regional Center for Mycology
and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. Initially, target
compounds 2a-17 and reference drugs were evaluated in vitro for their antibacterial and
antifungal activity, by inhibition zone technique and minimum inhibitory concentration
(MIC), using four fungi: A. fumigates (RCMB 02568), Candida albicans (RCMB 05036),
Syncephala strumracemosum (RCMB 05922) and Geotricum candidum (RCMB 05097),
two Gram-positive bacteria: S. pneumonia (RCMB 010010) and B. subtitles (RCMB
010069), two Gram-negative bacteria: P. aeruginosa (RCMB 010043), and E. coli (RCMB
010052) and the results were compared with respect to those of Amphotericin B,
Ampicillin and Gentamicin as a standard drugs. Suspension of the above-mentioned micro-
organisms was prepared by inoculating fresh stock cultures into separate broth tubes, each
containing 7 ml of nutrient broth (pepton, 0.3%) beef extract (0.3%). The inoculated tubes
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were incubated at 37 oC for 24 h. Solutions of the tested compounds and reference drugs
were prepared by dissolving 0.5 g of the compound in 10 ml DMF.[39,40]
6.9. Antioxidant Activity:
Organic acids and esters and O-N-heterocycles, in particular, have been recognized as
antioxidant activity . Furthermore, their mechanism of action and the structure-activity
relationships (SAR) were extensively studied the antioxidant activity of synthesized
heterocyclic was evaluated using DPPH radical scavenging assays. This widely used
method determines antioxidant activity by measuring the hydrogen donating ability of the
compound being studied. IC50 values are displayed in Table 3 Vitamin C was used as a
positive standard for the antioxidant activity in all experiments. Antioxidant assay by
ABST method % Inhibition = {[A sample – Atest] / A control} × 100. Compounds 2a, 3a,
5a, 5, 9, 13 and 14 were evaluated for antioxidant property by 2,2'-diphenyl-1-
picrylhydrazyl (DPPH). [42] The observed data on the antioxidant activity of the
compounds and control drug are displayed in Table 3 .
6.10. Computational method:
Calculations have been performed using Khon–Sham's DFT method subjected to the
gradient-corrected hybrid density functional B3LYP. This function is a combination of
the Becke's three parameters non-local exchange potential with the non-local correlation
functional of Lee et al. [52] For each structure, a full geometry optimization was
performed using this function and the 6-31G bases set as implemented by Gaussian 09
package.[45] All geometries were visualized either using Gauss View5.0.9 [53] and
chemcraft1.6 53 [54]. No symmetry constrains were applied during the geometry
optimization.
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