Synthesis of some dihydropyrimidine-based compounds bearing pyrazoline moiety and evaluation of their antiproliferative activity

Article abstract of DOI:10.1016/j.ejmech.2013.10.003

Two series of 2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-1-methyl-6-oxo-4- phenyl-1,6-dihydropyrimidine-5-carbonitriles 5a-h and 4-(4-chlorophenyl)-2-(3,5- diaryl-4,5-dihydropyrazol-1-yl)-1-methyl-6-oxo-1,6-dihydropyrimidine-5- carbonitriles 6a-h were synthesized via a cyclocondensation reaction of the corresponding 2-hydrazinopyrimidines 3a,b with the appropriate 2-propen-1-ones 4a-h. The target compounds were screened for their antiproliferative activity against A 549 (lung), HT 29 (colon), MCF 7 and MDA-MB 231 (breast) cell lines. The two most susceptible cell lines were the colon (HT 29) and breast (MDA-MB 231). Generally, the 4-unsubstitutedphenylpyrimidine derivatives 5a-h were more active than their 4-chlorophenylpyrimidine analogs 6a-h. Compounds 5e and 5g, showed high activity against three of the cell lines. The most active compound 5c possessed IC₅₀ = 1.76 μM against A 549 cell line.

Full text of DOI:10.1016/j.ejmech.2013.10.003

Accepted Manuscript
Synthesis of some dihydropyrimidine-based compounds bearing pyrazoline moiety
and evaluation of their antiproliferative activity
Fadi M. Awadallah, Gary A. Piazza, Bernard D. Gary, Adam B. Keeton, Joshua C.
Canzoneri
PII:
S0223-5234(13)00644-2
10.1016/j.ejmech.2013.10.003
EJMECH 6466
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 June 2013
Revised Date: 24 September 2013
Accepted Date: 3 October 2013
Please cite this article as: F.M. Awadallah, G.A. Piazza, B.D. Gary, A.B. Keeton, J.C. Canzoneri,
Synthesis of some dihydropyrimidine-based compounds bearing pyrazoline moiety and evaluation
of their antiproliferative activity, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.10.003.
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ACCEPTED MANUSCRIPT
Graphical abstract:
Some pyrimidine-pyrazoline hybrids were synthesized via cyclocondensation of the
precursor 2-hydrazonepyrimidines 3a,b and the appropriate propenones 4a-h. The
compounds were screened for their antiproliferative activity against four human cell
lines.
O
NC
CH3
N
N
R2
N
N
R
R1
5a-h, 6a-h

ACCEPTED MANUSCRIPT
Synthesis of some dihydropyrimidine-based compounds bearing pyrazoline moiety and
evaluation of their antiproliferative activity
Fadi M. Awadallah,a,1Gary A. Piazza,b Bernard D. Gary,b Adam B. Keetonb and Joshua C.
Canzonerib
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Eini 11562, Cairo Egypt.
bDepartment of Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute, University of
South Alabama, 1660 Springhill Avenue, Mobile, AL 36604-1405, USA
Abstract.
Two series of 2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-1-methyl-6-oxo-4-phenyl-1,6-
dihydropyrimidine-5-carbonitriles 5a-h and 4-(4-chlorophenyl)-2-(3,5-diaryl-4,5-dihydropyrazol-
1-yl)-1-methyl-6-oxo-1,6-dihydropyrimidine-5-carbonitriles 6a-h were synthesized via
a
cyclocondensation reaction of the corresponding 2-hydrazinopyrimidines 3a,b with the
appropriate 2-propen-1-ones 4a-h. The target compounds were screened for their antiproliferative
activity against A 549 (lung), HT 29 (colon), MCF 7 and MDA-MB 231 (breast) cell lines. The
two most susceptible cell lines were the colon (HT 29) and breast (MDA-MB 231). Generally, the
4-unsubstitutedphenylpyrimidine derivatives 5a-h were more active than their 4-
chlorophenylpyrimidine analogs 6a-h. Compounds 5e and 5g, showed high activity against three
of the cell lines. The most active compound 5c possessed IC50= 1.76 µM against A 549 cell line.
Keywords: Pyrimidine; Pyrazoline; Antiproliferative activity; Human colon (HT 29) cell line,
Human breast (MDA-MB 231) cell line.
1. Introduction.
Cancer is a major worldwide health problem. Although there has been a progress in the
development of treatment and prevention of cancer, this disease remains the second major cause of
death in the world. Still, the successful treatment of cancer remains a challenge in the 21st century,
and there is a need to search for newer and safer anticancer agents that have a broader spectrum of
cytotoxicity to tumor cells [1]. With the discovery of multicomponent reaction, the
dihydropyrimidinones (DHPM’s) were reported for the first time by Biginelli over a century ago
[2]. The multifunctionalized dihydropyrimidinones scaffold represents a class of heterocyclic
compounds with significant pharmacological efficiency and are receiving considerable amount of
interest. They exhibit a diverse pharmacological profile like calcium channel blockade, α1a-
adrenoreceptor antagonism, antibacterial, antifungal and other related properties [3-6]. From
natural marine sources, several alkaloids containing the dihydropyrimidine core unit were isolated
such as batzelladine alkaloids, which were found to be potent HIV gp-120-CD4 inhibitors [7].
With the advent of combinatorial synthesis, which is particularly useful for multicomponent
reactions like Biginelli condensation, diverse DHMPs libraries have been synthesized and
subjected to high throughput screening for biological activity [8]. By the end of the last century a
structurally simple compound, monastrol I has been identified on screening a large library of
diverse small molecules, as a novel cell permeable molecule that causes mitotic arrest by blocking
bipolar mitotic spindle in mammalian cells [9]. Moreover, many diversely substituted
1 Corresponding author: Tel.: 2 01223483941; fax: 20 2 2362 8426.
E-mail address: fadi2mae@gmail.com (F.M. Awadallah).
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dihydropyrimidinones have been synthesized showing promising antitumor activity among which
the ethanehydrazonoylpyrimidinone II is an example [10] (Fig. 1).
On the other hand, many pyrazoline derivatives are acknowledged to possess a wide range
of bioactivities. The pyrazoline motif makes up the core structure of numerous biologically active
compounds. Thus, some representatives of this heterocycle exhibit antiviral/antitumor [11-13],
antibacterial [14,15], anti-inflammatory [16], analgesic [17], fungistatic [18], and anti-
hyperglycemic activities [19]. A series of novel 3,5-diarylpyrazolines III [20] and
thiazolylpyrazoline derivatives IV were recently reported as potent anticancer agents [21] (Fig. 1).
Figure 1
In view of these observations it was thought of interest to study the influence of pyrazoline
moiety and dihydropyrimidinone scaffold combination, so that the two combined substructures,
might exhibit synergistic antitumor effect. Therefore, two series of new pyrazolinyl-
dihydropyrimidine derivatives, 5a-h and 6a-h, were synthesized and screened for their antitumor
activity. On the molecular design level, the substitutions on the two phenyl rings on the pyrazoline
moiety (R1 and R2) were subject to modifications regarding their electronic and lipophilic nature
(Fig. 2). The impact of these molecular manipulations was studied from the results obtained from
the cytotoxic biological assessment of all the synthesized compounds against the human lung cell
line A 549, colon cancer cell line HT 29 and the breast cancer cell lines MCF 7 and MDA-
MB231.
Figure 2
2. Results and discussion.
2.1. Chemistry
The
starting
1-methyl-2-(methylthio)-6-oxo-4-pheny/(4-chlorophenyl)-1,6-
dihydropyrimidine-5-carbonitriles 2a,b were synthesized in a one pot reaction from the
corresponding aldehyde, ethyl cyanoacetate and thiourea followed by methylation. The
corresponding 2-hydrazino-1-methyl-6-oxo-4-pheny/(4-chlorophenyl)-1,6-dihydropyrimidine-5-
carbonitriles 3a,b were obtained through hydrazinolysis of the precursor methylthio derivatives
(Scheme 1). The propen-1-one derivatives 4a-h were synthesized via a base-catalyzed Claisen-
Schmidt condensation of the appropriate benzaldehyde and acetophenone derivatives (Scheme 2).
Scheme1, Scheme 2
The target compounds 5a-h and 6a-h were obtained through a cyclocondensation reaction
of the corresponding hydrazinopyrimidine derivatives 3a,b and the appropriate propenones 4a-h
in absolute ethanol in the presence of sodium hydroxide (Scheme 3). Postulated structures of the
1
newly synthesized compounds were in good agreement with their IR, H NMR, 13C NMR, mass
1
spectral and elemental analyses data. The H NMR spectra of the target compounds showed a
prominent AMX system for the protons at C-4 and C-5 of the pyrazoline ring. Proton at C-4 (HA)
appeared as doublet of doublet at δ 2.65-3.23 ppm, proton at C-4 (HM) appeared as doublet of
doublet at δ 3.11-3.92 ppm, and proton at C-5 (HX) appeared as doublet of doublet at δ 4.10-5.30
ppm. There are three coupling constants: JAX = 6.3-6.9 Hz, JAM = 17.1-18.2 Hz, and JMX = 11.6-
12.3 Hz. Other protons (N-CH3, OCH3, aromatic Hs) were shown in their usual range. 13C NMR
2

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showed the characteristic C-4 and C-5 carbon signals of the pyrazoline ring at δ 40.3-44.3 and
52.5-60.6 ppm, respectively, in addition to the other signals attributed to the carbon skeleton of the
target compounds (c.f. the experimental section).
Scheme 3
2.2. Antiproliferative activity
The antiproliferative activity of all synthesized compounds were investigated against four
human cell lines, namely, lung (A 549), colon (HT 29) and breast (MCF 7 and MDA-MB 231)
using doxorubicin (Dox) as positive control. Compounds were first evaluated in triplicate for their
percent proliferation inhibition. All the tested compounds revealed percentage inhibition above
60% and subsequently, their IC50 values were calculated in µM from a graph displaying the dose-
survival percentage curve obtained after testing 8 concentrations for each tested compound with
four replicates per concentration (Table 1). Generally, results showed that the two most
susceptible cell lines were the colon (HT 29) and the breast (MDA-MB 231) cell lines that were
inhibited by the tested compounds at IC50 values ranging from 2.49-19.51 µM and 3.99-29.14 µM,
respectively. Apart from some exceptions, inhibition of the other two cell lines required higher
concentrations of the tested compounds. Also, it could be noticed that the 4-
chlorophenylpyrimidine derivatives 6a-h were generally less potent than their corresponding 4-
unsubstitutedphenylpyrimidine analogs 5a-h. Regarding the activity of the tested compounds
against HT 29 and MDA-MB 231 cell lines, the most active compound against both cell lines was
5a which had no substitutions on any of the phenyl rings (IC50= 2.49 and 3.99 µM, respectively).
Activity of 5a against colon HT 29 cell line was slightly higher than doxorubicin (IC50 Dox= 2.75
µM). Substitution on the phenyl groups located on positions 3 and 5 of the pyrazoline ring led to
decrease in activity. Compounds substituted with (R1) only such as compound 5f or having
another substitution (R2) such as 5e, 5g and 5h were more effective against both HT 29 and MDA-
MB 231 cell lines than those unsubstituted with R1 and which had only R2 substituent as in 5b-5d.
The pattern of activity of compounds 6a-h against HT 29 and MDA-MB 231 cell lines was
different from that of compounds 5a-h. The promising compounds emerging in this series were
those substituted with R1 such as 6e and 6f, in addition to the derivatives substituted with R2 only
as in 6c and 6d.
Regarding activity against lung (A 549) and breast (MCF 7) cell lines, the 4-
unsubstitutedphenylpyrimidine analogs 5a-h were more effective than their 4-chlorophenyl
analogs. Regarding the activity against the lung (A 549) cell line, the most active compound was
5c (IC50= 1.76 µM). Compounds 5b, 5c and 5e showed activity against lung A 549 cell line
comparable to or higher than doxorubicin (IC50= 3.25, 1.76 and 2.00 µM, respectively, c.f. IC50
Dox= 3.13 µM). Good activity was also demonstrated by compound 5g (IC50= 6.07 µM,
respectively). Lower activity was observed with the other tested compounds. Considering the
activity against the breast (MCF 7) cell line, only two compounds exhibited promising activity;
namely 5b and 5g (IC50= 3.28 and 4.80 µM, respectively).
Concerning the nature of substituent groups R1 and R2, there was no consistent relation
that could be established between the lipophilicity and/or electronic property of these groups and
the antiproliferative activity.
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From this SAR analysis, it was noteworthy that some compounds exhibited promising
activity against three of the cell lines such as compounds 5e (IC50= 2.00, 4.62 and 5.10 µM against
A 549, HT 29 and MDA-MB 231, respectively) and 5g (IC50= 6.07, 9.12 and 4.80 µM against A
549, HT 29 and MCF 7, respectively). These compounds may be candidates for future lead
optimization.
Table 1
3. Conclusion
Two series of pyrimidine-pyrazoline hybrids, 5a-h and 6a-h, were synthesized via a
cyclocondensation reaction of the precursor 2-hydrazonepyrimidines 3a,b and the appropriate
propenones 4a-h. The target compounds were screened for their antiproliferative activity against
four human cell lines: lung (A 549), colon (HT 29), breast (MCF 7 and MDA-MB 231) cell lines.
The two most susceptible cell lines were the colon (HT 29) and breast (MDA-MB 231). Generally
the 4-unsubstitutedphenylpyrimidine analogs 5a-h were more effective than their chloro analogs
6a-h. Compounds 5e and 5g, showing high activity against three of the cell lines, were considered
promising lead compounds for future optimization.
4. Experimental.
4.1. Chemistry
Melting points were determined with Stuart SMP3 version 5 apparatus and were uncorrected. FT-
IR spectra were recorded on Bruker FT-IR 8400S spectrophotometer using KBr cell. Unless
1
otherwise noted, H NMR spectra were recorded in DMSO-d6 on Varian mercury 300BB at 300
MHz. 13C NMR spectra were run at 75.46 MHz. Chemical Shifts were given in δ as parts per
million (ppm) downfield from tetramethylsilane (TMS) as internal standard. The electron impact
(EI) mass spectra were recorded on Finnigan Mat SSQ 7000 (70 ev) mass spectrometer. Elemental
microanalysis was performed at the Regional Center for Mycology and Biotechnology, Azhar
University. TLC were monitored on FLUKA silica gel TLC aluminum cards (0.2 mm thickness)
with fluorescent indicator 254 nm using chloroform/methanol (9.5:0.5) as eluent to follow the
course of the reactions and to check the purity of the products. All reagents and solvents were
purified and dried by standard techniques.
4.1.1. 6-oxo-4-pheny/(4-chlorophenyl)-2-sulfanyl-1,6-dihydropyrimidine-5-carbonitriles (1a,b):
The titled compounds 1a and 1b were synthesized according to the reported methods [22].
4.1.2.
carbonitriles
1-Methyl-2-(methylthio)-6-oxo-4-pheny/(4-chlorophenyl)-1,6-dihydropyrimidine-5-
(2a,b) and 2-hydrazino-1-methyl-6-oxo-4-pheny/(4-chlorophenyl)-1,6-
dihydropyrimidine-5-carbonitriles (3a,b):
The titled compounds 2a, 2b, 3a and 3b were synthesized according to the reported methods [23].
4.1.3. (E)-1-(un)substitutedphenyl-3-(un)substitutedphenylprop-2-en-1-ones (4a-h):
4

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Compounds 4a, 4c, 4f, 4g [24], 4b [25], 4d, 4e [26] and 4h [27] were synthesized according to the
literature procedures.
4.1.4. General procedure for the preparation of compounds (5a-h) and (6a-h):
A mixture of compound 3a/3b (4 mmol), the appropriate propenone 4a-h (4 mmol) and
sodium hydroxide (0.2g, 5mmol) in absolute ethanol (30 ml) was refluxed for 72 h. The reaction
mixture was poured on water, neutralized with 2N hydrochloric acid and the residue was filtered
off. The crude product obtained was crystallized from isopropanol.
4.1.4.1.
2-(3,5-Diphenyl-4,5-dihydropyrazol-1-yl)-1-methyl-6-oxo-4-phenyl-1,6-
dihydropyrimidine-5-carbonitrile (5a):
M.p. 160-162oC, yield: 75%. IR υmax/cm-1: 3057 (CH aromatic), 2950 (CH aliphatic), 2222 (CN),
1
1681 (C=O), 1614 (C=N). H NMR (CDCl3): δ 2.55 (s, 3H, N-CH3), 3.23 (dd, 1H, C4-HA
pyrazoline, JAX= 6.6 Hz, JAM= 17.9 Hz), 3.58 (dd, 1H, C4-HM pyrazoline, JMX= 11.8 Hz, JMA=
17.9 Hz), 4.75 (dd, 1H, C5-HX pyrazoline, JXA= 6.6 Hz, JXM= 11.9 Hz), 7.27-7.80 (m, 15H,
aromatic H). Anal. Calcd. for C27H21N5O (431.39): C, 75.16; H, 4.91; N, 16.23. Found: C, 75.22;
H, 4.95; N, 16.35. correct in all
4.1.4.2. 2-[5-(4-Chlorophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl]-1-methyl-6-oxo-4-phenyl-1,6-
dihydropyrimidine-5-carbonitrile (5b):
M.p. 173-174oC, yield: 71%. IR υmax/cm-1: 3061 (CH aromatic), 2920, 2850 (CH aliphatic), 2223
(CN), 1681 (C=O), 1622 (C=N). 1H NMR: δ 2.51 (s, 3H, N-CH3), 2.65 (dd, 1H, C4-HA pyrazoline,
JAX= 6.4 Hz, JAM= 17.8 Hz), 3.11 (dd, 1H, C4-HM pyrazoline, JMX= 11.9 Hz, JMA= 17.8 Hz), 4.38
(dd, 1H, C5-HX pyrazoline, JXA= 6.4 Hz, JXM= 11.9 Hz), 7.40-7.67 (m, 14H, aromatic H). MS, m/z
(%): 465.40 (M+, 0.28); 80.00 (100). Anal. Calcd. for C27H20ClN5O (465.93): C, 69.60; H, 4.33;
N, 15.03. Found: C, 69.58; H, 4.37; N, 15.11.
4.1.4.3. 2-[5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-yl]-1-methyl-6-oxo-4-phenyl-1,6-
dihydropyrimidine-5-carbonitrile (5c):
M.p. 92-94oC, yield: 65%. IR υmax/cm-1: 3059 (CH aromatic), 2922, 2848 (CH aliphatic), 2222
1
(CN), 1681 (C=O), 1608 (C=N). H NMR: δ 2.47 (s, 3H, N-CH3), 2.95 (dd, 1H, C4-HA
pyrazoline, JAX= 6.5 Hz, JAM= 17.9 Hz), 3.28 (dd, 1H, C4-HM pyrazoline, JMX= 11.9 Hz, JMA=
17.9 Hz), 3.78 (s, 3H, OCH3), 4.24 (dd, 1H, C5-HX pyrazoline, JXA= 6.5 Hz, JXM= 11.9 Hz), 7.33-
7.87 (m, 14H, aromatic H). 13C NMR: δ 27.9 (N-CH3), 40.3 (C-4 pyrazoline), 54.7 (C-5
pyrazoline), 55.9 (OCH3), 112.9-143.8 (aromatic Cs), 150.5 (C-2 pyrazoline), 160.2 (C-OCH3),
161.0 (C-2 pyrimidine), 161.3 (C=O). Anal. Calcd. for C28H23N5O2 (461.51): C, 72.87; H, 5.02; N,
15.17. Found: C, 72.89; H, 5.11; N, 15.26.
4.1.4.4. 2-[5-(4-Fluorophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl]-1-methyl-6-oxo-4-phenyl-1,6-
dihydropyrimidine-5-carbonitrile (5d):
5

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M.p. 130-131oC, yield: 68%. IR υmax/cm-1: 3061 (CH aromatic), 2920, 2848 (CH aliphatic), 2222
1
(CN), 1681 (C=O), 1602 (C=N). H NMR: δ 2.57 (s, 3H, N-CH3), 2.95 (dd, 1H, C4-HA
pyrazoline, JAX= 6.4 Hz, JAM= 17.9 Hz), 3.20 (dd, 1H, C4-HM pyrazoline, JMX= 11.9 Hz, JMA=
17.9 Hz), 4.25 (dd, 1H, C5-HX pyrazoline, JXA= 6.4 Hz, JXM= 11.9 Hz), 7.15-7.95 (m, 14H,
aromatic H). 13C NMR: δ 26.5 (N-CH3), 44.3 (C-4 pyrazoline), 60.6 (C-5 pyrazoline), 114.5-
142.6 (aromatic Cs), 150.3 (C-2 pyrazoline), 158.3 (C-2 pyrimidine), 160.2 (C=O). Anal. Calcd.
for C27H20FN5O (449.48): C, 72.15; H, 4.48; N, 15.58. Found: C, 72.21; H, 4.53; N, 15.69.
4.1.4.5. 2-[5-(4-Fluorophenyl)-3-(4-tolyl)-4,5-dihydropyrazol-1-yl]-1-methyl-6-oxo-4-phenyl-1,6-
dihydropyrimidine-5-carbonitrile (5e):
M.p. 145-147oC, yield: 62%. IR υmax/cm-1: 3059 (CH aromatic), 2920, 2850 (CH aliphatic), 2222
(CN), 1674 (C=O), 1622 (C=N). 1H NMR: δ 2.31 (s, 3H, CH3), 2.52 (s, 3H, N-CH3), 3.06 (dd, 1H,
C4-HA pyrazoline, JAX= 6.5 Hz, JAM= 17.9 Hz), 3.34 (dd, 1H, C4-HM pyrazoline, JMX= 11.9 Hz,
JMA= 17.9 Hz), 4.23 (dd, 1H, C5-HX pyrazoline, JXA= 6.5 Hz, JXM= 11.9 Hz), 7.22-7.85 (m, 13H,
aromatic H). 13C NMR: δ 21.1 (CH3), 26.4 (N-CH3), 40.3 (C-4 pyrazoline), 57.6 (C-5 pyrazoline),
115.5-143.4 (aromatic Cs), 150.3 (C-2 pyrazoline), 159.6 (C-2 pyrimidine), 161.6 (C=O). Anal.
Calcd. for C28H22FN5O (463.51): C, 72.56; H, 4.78; N, 15.11. Found: C, 72.58; H, 4.83; N, 15.25.
4.1.4.6. 2-[3-(4-Chlorophenyl)-5-phenyl-4,5-dihydropyrazol-1-yl]-1-methyl-6-oxo-4-phenyl-1,6-
dihydropyrimidine-5-carbonitrile (5f):
M.p. 133-135oC, yield: 77%. IR υmax/cm-1: 3061 (CH aromatic), 2920, 2848 (CH aliphatic), 2223
(CN), 1681 (C=O), 1616 (C=N). 1H NMR: δ 2.51 (s, 3H, N-CH3), 3.05 (dd, 1H, C4-HA pyrazoline,
JAX= 6.6 Hz, JAM= 18.0 Hz), 3.42 (dd, 1H, C4-HM pyrazoline, JMX= 11.9 Hz, JMA= 18.0 Hz), 4.25
(dd, 1H, C5-HX pyrazoline, JXA= 6.6 Hz, JXM= 11.9 Hz), 7.10-7.95 (m, 14H, aromatic H). Anal.
Calcd. for C27H20ClN5O (465.93): C, 69.60; H, 4.33; N, 15.03. Found: C, 69.66; H, 4.37; N, 15.14.
4.1.4.7. 2-[3-(4-Chlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-1-methyl-6-oxo-4-
phenyl-1,6-dihydropyrimidine-5-carbonitrile (5g):
M.p. 124-126oC, yield: 65%. IR υmax/cm-1: 3061 (CH aromatic), 2922, 2872 (CH aliphatic), 2223
(CN), 1681 (C=O), 1621 (C=N). 1H NMR (CDCl3): δ 2.30 (s, 3H, N-CH3), 3.09 (dd, 1H, C4-HA
pyrazoline, JAX= 6.4 Hz, JAM= 18.2 Hz), 3.84 (s, 3H, OCH3), 3.87 (dd, 1H, C4-HM pyrazoline,
JMX= 11.9 Hz, JMA= 18.2 Hz), 4.80 (dd, 1H, C5-HX pyrazoline, JXA= 6.4 Hz, JXM= 11.9 Hz), 6.84-
7.59 (m, 13H, aromatic H). 13C NMR: δ 27.5 (N-CH3), 40.4 (C-4 pyrazoline), 52.5 (C-5
pyrazoline), 55.8 (OCH3), 120.7-142.4 (aromatic Cs), 151.5 (C-2 pyrazoline), 159.3 (C-OCH3),
160.0 (C-2 pyrimidine), 162.3 (C=O). MS, m/z (%): 495.95 (M+, 2.81); 105.00 (100). Anal. Calcd.
for C28H22ClN5O2 (495.96): C, 67.81; H, 4.47; N, 14.12. Found: C, 68.02; H, 4.53; N, 14.31.
4.1.4.8.
2-[3,5-Bis-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-1-methyl-6-oxo-4-phenyl-1,6-
dihydropyrimidine-5-carbonitrile (5h):
M.p. 196-198oC, yield: 58%. IR υmax/cm-1: 3061 (CH aromatic), 2916, 2848 (CH aliphatic), 2222
(CN), 1681 (C=O), 1600 (C=N). 1H NMR (CDCl3): δ 2.33 (s, 3H, N-CH3), 3.15 (dd, 1H, C4-HA
pyrazoline, JAX= 6.4 Hz, JAM= 17.9 Hz), 3.89 (s, 6H, 2 OCH3), 3.92 (dd, 1H, C4-HM pyrazoline,
JMX= 11.8 Hz, JMA= 17.9 Hz), 5.30 (dd, 1H, C5-HX pyrazoline, JXA= 6.4 Hz, JXM= 11.8 Hz), 6.91-
7.67 (m, 13H, aromatic H). MS, m/z (%): 491.05 (M+, 23.97); 343.00 (100). Anal. Calcd. for
C29H25N5O3 (491.54): C, 70.86; H, 5.13; N, 14.25. Found: C, 70.89; H, 5.17; N, 14.33.
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4.1.4.9.
4-(4-Chlorophenyl)-2-(3,5-diphenyl-4,5-dihydropyrazol-1-yl)-1-methyl-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile (6a):
M.p. 270-272oC, yield: 68%. IR υmax/cm-1: 3061 (CH aromatic), 2918, 2848 (CH aliphatic), 2193
(CN), 1681 (C=O), 1614 (C=N). 1H NMR (CDCl3): δ 2.34 (s, 3H, N-CH3), 3.10 (dd, 1H, C4-HA
pyrazoline, JAX= 6.9 Hz, JAM= 17.1 Hz), 3.91 (dd, 1H, C4-HM pyrazoline, JMX= 12.3 Hz, JMA=
17.1 Hz), 5.23 (dd, 1H, C5-HX pyrazoline, JXA= 6.9 Hz, JXM= 12.3 Hz), 6.77-7.65 (m, 14H,
aromatic H). Anal. Calcd. for C27H20ClN5O (465.93): C, 69.60; H, 4.33; N, 15.03. Found: C,
68.68; H, 4.34; N, 15.12.
4.1.4.10. 4-(4-Chlorophenyl)-2-[5-(4-chlorophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl]-1-methyl-
6-oxo-1,6-dihydropyrimidine-5-carbonitrile (6b):
M.p. 265-267oC IR υmax/cm-1: 3062 (CH aromatic), 2920, 2848 (CH aliphatic), 2193 (CN), 1662
(C=O), 1601 (C=N). 1H NMR (CDCl3): δ 2.50 (s, 3H, N-CH3), 3.05 (dd, 1H, C4-HA pyrazoline,
JAX= 6.3 Hz, JAM= 17.7 Hz), 3.17 (dd, 1H, C4-HM pyrazoline, JMX= 11.9 Hz, JMA= 17.7 Hz), 3.86
(dd, 1H, C5-HX pyrazoline, JXA= 6.3 Hz, JXM= 11.9 Hz), 6.84-7.67 (m, 13H, aromatic H). 13C
NMR: δ 27.4 (N-CH3), 40.4 (C-4 pyrazoline), 56.6 (C-5 pyrazoline), 114.4-142.2 (aromatic Cs),
153.3 (C-2 pyrazoline), 158.9 (C-2 pyrimidine), 164.2 (C=O). MS, m/z (%): 500.95 (M+, 16.43);
120.00 (100). Anal. Calcd. for C27H19Cl2N5O (500.38): C, 64.81; H, 3.83; N, 14.00. Found: C,
64.86; H, 3.81; N, 14.13.
4.1.4.11.
4-(4-Chlorophenyl)-2-[5-(4-methoxyphenyl)-3-phenyl-4,5-dihydropyrazol-1-yl]-1-
methyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (6c):
M.p. 275-277oC, yield: 64%. IR υmax/cm-1: 3064 (CH aromatic), 2920, 2848 (CH aliphatic), 2193
(CN), 1666 (C=O), 1597 (C=N). 1H NMR (CDCl3): δ 2.40 (s, 3H, N-CH3), 3.15 (dd, 1H, C4-HA
pyrazoline, JAX= 6.6 Hz, JAM= 17.9 Hz), 3.89 (dd, 1H, C4-HM pyrazoline, JMX= 11.6 Hz, JMA=
17.9 Hz), 4.80 (s, 3H, OCH3), 4.23 (dd, 1H, C5-HX pyrazoline, JXA= 6.6 Hz, JXM= 11.6 Hz), 6.80-
7.58 (m, 13H, aromatic H). 13C NMR: δ 26.5 (N-CH3), 44.0 (C-4 pyrazoline), 54.8 (C-5
pyrazoline), 55.3 (OCH3), 113.5-136.7 (aromatic Cs), 150.7 (C-2 pyrazoline), 159.2 (C-OCH3),
162.8 (C-2 pyrimidine), 168.25 (C=O). Anal. Calcd. for C28H22ClN5O2 (495.96): C, 67.81; H,
4.47; N, 14.12. Found: C, 67.79; H, 4.51; N, 14.23.
4.1.4.12. 4-(4-Chlorophenyl)-2-[5-(4-fluorophenyl)-3-phenyl-4,5-dihydropyrazol-1-yl]-1-methyl-
6-oxo-1,6-dihydropyrimidine-5-carbonitrile (6d):
M.p. 281-282oC, yield: 76%. IR υmax/cm-1: 3064 (CH aromatic), 2920, 2850 (CH aliphatic), 2194
(CN), 1662 (C=O), 1621 (C=N). 1H NMR: δ 2.50 (s, 3H, N-CH3), 3.18 (dd, 1H, C4-HA pyrazoline,
JAX= 6.5 Hz, JAM= 17.9 Hz), 3.41 (dd, 1H, C4-HM pyrazoline, JMX= 11.9 Hz, JMA= 17.9 Hz), 4.10
(dd, 1H, C5-HX pyrazoline, JXA= 6.5 Hz, JXM= 11.9 Hz), 7.18-7.88 (m, 13H, aromatic H). MS, m/z
(%): 483.10 (M+, 0.64); 105.00 (100). Anal. Calcd. for C27H19ClFN5O (483.92): C, 67.01; H, 3.96;
N, 14.47. Found: C, 67.08; H, 4.01; N, 14.58.
4.1.4.13. 4-(4-Chlorophenyl)-2-[5-(4-fluorophenyl)-3-(4-tolyl)-4,5-dihydropyrazol-1-yl]-1-methyl-
6-oxo-1,6-dihydropyrimidine-5-carbonitrile (6e):
M.p. 269-271oC, yield: 66%. IR υmax/cm-1: 3061 (CH aromatic), 2922, 2850 (CH aliphatic), 2194
(CN), 1661 (C=O), 1597 (C=N). 1H NMR: δ 2.26 (s, 3H, CH3), 2.54 (s, 3H, N-CH3), 2.95 (dd, 1H,
7

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C4-HA pyrazoline, JAX= 6.3 Hz, JAM= 17.8 Hz), 3.41 (dd, 1H, C4-HM pyrazoline, JMX= 11.8 Hz,
JMA= 17.8 Hz), 4.23 (dd, 1H, C5-HX pyrazoline, JXA= 6.3 Hz, JXM= 11.8 Hz), 7.13-7.88 (m, 12H,
aromatic H). Anal. Calcd. for C28H21ClFN5O (497.95): C, 67.54; H, 4.25; N, 14.06. Found: C,
67.57; H, 4.26; N, 14.13.
4.1.4.14. 4-(4-Chlorophenyl)-2-[3-(4-chlorophenyl)-5-phenyl-4,5-dihydropyrazol-1-yl]-1-methyl-
6-oxo-1,6-dihydropyrimidine-5-carbonitrile (6f):
M.p. 245-247 oC, yield: 75%. IR υmax/cm-1: 3061 (CH aromatic), 2920, 2850 (CH aliphatic), 2194
(CN), 1660 (C=O), 1614 (C=N). 1H NMR: δ 2.50 (s, 3H, N-CH3), 2.99 (dd, 1H, C4-HA pyrazoline,
JAX= 6.5 Hz, JAM= 17.7 Hz), 3.38 (dd, 1H, C4-HM pyrazoline, JMX= 11.9 Hz, JMA= 17.7 Hz), 4.26
(dd, 1H, C5-HX pyrazoline, JXA= 6.5 Hz, JXM= 11.9 Hz), 7.23-7.95 (m, 13H, aromatic H). MS, m/z
(%): 500.20 (M+, 3.46); 138.95 (100). Anal. Calcd. for C27H19Cl2N5O (500.38): C, 64.81; H, 3.83;
N, 14.00. Found: C, 64.86; H, 3.88; N, 14.05.
4.1.4.15. 4-(4-Chlorophenyl)-2-[3-(4-chlorophenyl)-5-(4-methoxyphenyl)-4,5-dihydropyrazol-1-
yl]-1-methyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (6g):
M.p. 262-263oC, yield: 72%. IR υmax/cm-1: 3061 (CH aromatic), 2922, 2848 (CH aliphatic), 2193
(CN), 1662 (C=O), 1595 (C=N). 1H NMR: δ 2.51 (s, 3H, N-CH3), 3.07 (dd, 1H, C4-HA pyrazoline,
JAX= 6.3 Hz, JAM= 18.2 Hz), 3.51 (dd, 1H, C4-HM pyrazoline, JMX= 11.6 Hz, JMA= 18.2 Hz), 3.80
(s, 3H, OCH3), 4.31 (dd, 1H, C5-HX pyrazoline, JXA= 6.3 Hz, JXM= 11.6 Hz), 7.15-7.95 (m, 12H,
aromatic H). 13C NMR: δ 26.5 (N-CH3), 41.2 (C-4 pyrazoline), 53.5 (C-5 pyrazoline), 55.9
(OCH3), 120.7-138.4 (aromatic Cs), 151.5 (C-2 pyrazoline), 159.8 (C-OCH3), 160.5 (C-2
pyrimidine), 162.3 (C=O). Anal. Calcd. for C28H21Cl2N5O2 (530.40): C, 63.40; H, 3.99; N, 13.20.
Found: C, 63.44; H, 4.02; N, 13.31.
4.1.4.16. 4-(4-Chlorophenyl)-2-[3,5-bis-(4-methoxyphenyl)-4,5-dihydropyrazol-1-yl]-1-methyl-6-
oxo-1,6-dihydropyrimidine-5-carbonitrile (6h):
M.p. >300oC, yield: 74%. IR υmax/cm-1: 3061 (CH aromatic), 2922, 2850 (CH aliphatic), 2193
(CN), 1654 (C=O), 1598 (C=N). 1H NMR (CDCl3): δ 2.34 (s, 3H, N-CH3), 3.05 (dd, 1H, C4-HA
pyrazoline, JAX= 6.8 Hz, JAM= 17.9 Hz), 3.85 (dd, 1H, C4-HM pyrazoline, JMX= 11.8 Hz, JMA=
17.9 Hz), 3.88 (s, 6H, 2 OCH3), 5.80 (dd, 1H, C5-HX pyrazoline, JXA= 6.7 Hz, JXM= 11.8 Hz),
7.10-7.91 (m, 12H, aromatic H). Anal. Calcd. for C29H24ClN5O3 (525.99): C, 66.22; H, 4.60; N,
13.31. Found: C, 66.21; H, 4.67; N, 13.35.
4.2. Antiproliferative activity
4.2.1. Cell culture:
The tumor cells were obtained from ATCC. Cells were incubated under standard cell
culture conditions at 37 oC in a humidified atmosphere with 5% CO2. Cells were grown in RPMI
1640 supplemented with 5% fetal bovine serum, 2 mM glutamine, 100 units/mL streptomycin, and
0.25 mg/mL amphotericin. Cellswere harvested at 70 and 90% confluence with trypsin/EDTA and
used immediately. Cell count and viability was determined by Trypan blue staining followed by
hemocytometry. Only cultures displaying >95% cell viability were used for performing the
experiments [28].
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4.2.2. Growth assay:
Tissue culture treated microtiter 96-well plates were seeded at a density of 5000 cells/well.
The plates were incubated for 18-24 h prior to any treatment. All test compounds were solubilized
in 100% DMSO and diluted with media to obtain final DMSO concentration of 0.1%. Cells were
dosed. Cell viability was measured 72 h after treatment by Cell Titer Glo Assay (Promega), which
is a luminescent assay that is an indicator of live cells as a function of metabolic activity and ATP
content. The assay was performed according to the manufacturer’s specifications. Luminescence
was measured by a Perkin Elmer Victor® multi-label plate reader.
Effects of the synthesized compounds on tumor cell growth were assessed and potency
was expressed in terms of the compounds IC50 values. After testing 8 concentrations for each
compound with four replicates per concentration, dose-response curves were constructed and
analyzed using PrismTM 4 (Graph-Pad software, San Diego, CA). IC50 values were calculated in
µM from those graphs displaying the dose-survival percentage curve for each compound using a
four parameter logistic equation.
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Scheme captions:
- Scheme 1. Preparation of compounds 3a and 3b.
- Scheme 2. Preaparation of compounds 4a-h.
- Scheme 3. Synthesis of compounds 5a-h and 6a-h.
Figure captions:
- Figure 1. Structures of some DHPM's and pyrazolines with antitumor activity.
- Figure 2. General structure of target compounds.
Table caption:
Table 1. IC50 values (µM) of the in vitro antiproliferative activity of the tested compounds against
A 549 (lung), HT 29 (colon), MCF 7 and MDA-MB 231(breast) cell lines.
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Highlights:
- Pyrimidine-pyrazoline hybrids were synthesized.
- Antiproliferative activity against four human cell lines was performed.
- Compounds 5e and 5g, showed high activity against three of the cell lines.
- IC50 of compound 5c against A 549 (lung) cell line was 1.76 µM.

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Table 1. IC50 values (µM) of the in vitro antiproliferative activity of the tested
compounds against A 549 (lung), HT 29 (colon), MCF 7 and MDA-MB 231(breast)
cell lines.
O
NC
CH3
N
N
R2
N
N
R
R1
5a-h, 6a-h
Cpd R
R1
R2
A 549
HT 29 MCF 7
MDA-MB
231
H
H
H
52.86
3.25
2.49
35.35
3.28
3.99
5a
5b
5c
5d
5e
5f
H
H
Cl
19.17
14.67
8.15
28.02
12.83
11.82
5.10
H
H
OCH3
1.76
17.97
38.14
23.11
12.25
4.80
H
H
F
39.17
2.00
H
CH3
Cl
F
4.62
H
H
26.93
6.07
6.60
10.64
11.09
16.23
29.14
25.12
19.53
20.89
19.30
12.42
24.84
28.29
0.11
H
Cl
OCH3
OCH3
H
9.12
5g
5h
6a
6b
6c
6d
6e
6f
H
OCH3
H
26.52
28.09
24.74
23.81
27.84
35.59
43.86
35.65
39.69
3.13
9.42
126.76
34.37
46.32
52.38
36.83
115.97
25.28
27.27
99.81
1.20
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
-
19.51
15.00
10.95
13.70
10.52
8.72
H
Cl
H
OCH3
F
H
CH3
Cl
F
H
Cl
OCH3
OCH3
-
15.10
18.21
2.75
6g
6h
Dox
OCH3
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O
NH
O
O
CH3
NH
H3C
N
O
N
NH
Cl
N
S
H
OH
II
COCH3
I (Monastrol)
CH3
Cl
S
O
CH3
CH3
N
N
N
N
N
OH
CH3
F
IV
Cl
III
Figure 1. Structures of some DHPM's and pyrazolines with antitumor activity.
O
NC
CH3
N
N
R2
N
N
R
R1
5a-h, 6a-h
Figure 2. General structure of target compounds.

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O
NC
C2H5
O
N
O
NC
NH
SH
a
NH2
S
CHO
H2N
R
R=H
R
1a
1b
R=Cl
b
O
N
O
NC
CH3
c
NC
CH3
N
N
NH2
CH3
N
N
S
H
R
R
R=H
R=H
3a
3b
R=Cl
2a
2b
R=Cl
Scheme 1. Preparation of compounds 3a and 3b.
Reagents and conditions. a: potassium carbonate/ absolute ethanol, reflux, 12h; b:
methyl iodide, potassium carbonate, dry DMF, RT., 3h; c: hydrazine hydrate/ absolute
ethanol, reflux, 12h.
O
O
CHO
a
H3C
+
R1
R2
R2
R1
1
2
1
2
R = H,
R = H
R = F,
R = CH
3
4a
4b
4c
4d
4e
1
2
1
2
R = Cl,
R =H
R = H,
R = Cl
4f
4g
4h
1
2
1
2
R = OCH , R = H
R = OCH , R = Cl
3
3
1
2
1
2
R = F,
R = H
R = OCH , R = OCH
3 3
Scheme 2. Preparation of compounds 4a-h.
Reagents and conditions. a: sodium hydroxide/ absolute ethanol, RT., 24h.

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O
N
O
N
NC
CH3
N
O
NC
CH3
N
N
a
R2
N
NH2
N
+
H
R1
R2
R
R
R=H
R=Cl
3a
3b
4a-h
R1
5a-h R=H
6a-h R=Cl
a R1= H,
R2= H
b R1= H,
c R1= H,
d R1= H,
R2=Cl
R2=OCH3
R2= F
e R1= CH3, R2= F
f R1= Cl,
g R1= Cl,
R2= H
R2= OCH3
h R1= OCH3, R2= OCH3
Scheme 3. Synthesis of compounds 5a-h and 6a-h.
Reagents and conditions. a: sodium hydroxide/ absolute ethanol, reflux, 72h.

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