Design, synthesis, molecular modelling, and biological evaluation of novel substituted pyrimidine derivatives as potential anticancer agents for hepatocellular carcinoma

Article abstract of DOI:10.1080/14756366.2019.1612889

New anticancer agents are highly needed to overcome cancer cell resistance. A novel series of pyrimidine pyrazoline-anthracene derivatives (PPADs) (4a-t) were designed and synthesised. The anti-liver cancer activity of all compounds was screened in vitro against two hepatocellular carcinoma (HCC) cell lines (HepG2 and Huh-7) as well as normal fibroblast cells by resazurin assay. The designed compounds 4a-t showed a broad-spectrum anticancer activity against the two cell lines and their activity was more prominent on cancer compared to normal cells. Compound 4e showed high potency against HepG2 and Huh-7 cell lines ((IC₅₀=5.34 and 6.13 μg/mL, respectively) comparable to that of doxorubicin (DOX) activities. A structure activity relationship (SAR) has been investigated and compounds 4e, 4i, 4m, and 4q were the most promising anticancer agents against tested cell lines. These compounds induced apoptosis in HepG2 and Huh-7 cells through significant activation of caspase 3/7 at all tested concentrations. In conclusion, 4e could be a potent anticancer drug.

Full text of DOI:10.1080/14756366.2019.1612889

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis, molecular modelling, and
biological evaluation of novel substituted
pyrimidine derivatives as potential anticancer
agents for hepatocellular carcinoma
Naglaa Mohamed Ahmed, Mahmoud Youns, Moustafa Khames Soltan &
Ahmed Mohammed Said
To cite this article: Naglaa Mohamed Ahmed, Mahmoud Youns, Moustafa Khames Soltan &
Ahmed Mohammed Said (2019) Design, synthesis, molecular modelling, and biological evaluation
of novel substituted pyrimidine derivatives as potential anticancer agents for hepatocellular
carcinoma, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 1110-1120, DOI:
10.1080/14756366.2019.1612889
To link to this article: https://doi.org/10.1080/14756366.2019.1612889
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UK Limited, trading as Taylor & Francis
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2019, VOL. 34, NO. 1, 1110–1120
https://doi.org/10.1080/14756366.2019.1612889
RESEARCH PAPER
Design, synthesis, molecular modelling, and biological evaluation of novel
substituted pyrimidine derivatives as potential anticancer agents for
hepatocellular carcinoma
Naglaa Mohamed Ahmeda, Mahmoud Younsb,c, Moustafa Khames Soltand,e and Ahmed Mohammed Saida,f
b
aPharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University, Cairo, Egypt; Biochemistry Department, Faculty of
c
Pharmacy, Helwan University, Cairo, Egypt; Department of Functional Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg,
d
e
Germany; Medicinal Chemistry Department, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt; Oman College of Health Sciences,
f
Muscat, Sultanate of Oman; Department of Chemistry, University at Buffalo, The State University of New York, Buffalo, NY, USA
ABSTRACT
ARTICLE HISTORY
Received 18 March 2019
Revised 21 April 2019
Accepted 23 April 2019
New anticancer agents are highly needed to overcome cancer cell resistance. A novel series of pyrimidine
pyrazoline-anthracene derivatives (PPADs) (4a-t) were designed and synthesised. The anti-liver cancer
activity of all compounds was screened in vitro against two hepatocellular carcinoma (HCC) cell lines
(HepG2 and Huh-7) as well as normal fibroblast cells by resazurin assay. The designed compounds 4a-t
showed a broad-spectrum anticancer activity against the two cell lines and their activity was more prom-
inent on cancer compared to normal cells. Compound 4e showed high potency against HepG2 and Huh-7
cell lines ((IC50¼5.34 and 6.13 lg/mL, respectively) comparable to that of doxorubicin (DOX) activities. A
structure activity relationship (SAR) has been investigated and compounds 4e, 4i, 4m, and 4q were the
most promising anticancer agents against tested cell lines. These compounds induced apoptosis in HepG2
and Huh-7 cells through significant activation of caspase 3/7 at all tested concentrations. In conclusion, 4e
could be a potent anticancer drug.
KEYWORDS
Pyrimidine; pyrazoline;
anthracene; liver cancer;
apoptosis; caspase 3/7
GRAPHICAL ABSTRACT
Introduction
chemotherapy, radiotherapy, and immunotherapy. These types of
treatment are often accompanied by high systemic toxicity and
sometimes drug resistance. Therefore, new therapeutic agents are
highly needed to overcome this tumour drug resistance4.
Pyrimidine ring is one of the mostly used heterocyclic scaffolds
in medicinal chemistry. Pyrimidine derivatives have been well rec-
ognised for their therapeutic applications i.e. antiviral5,6, antibac-
Cancer (called tumour) is the result of uncontrolled growth and
spread of abnormal cells1. Cancer is considered the leading cause
of death worldwide based on the world health organisation
(WHO) statistics. The most common types of cancer are lung,
stomach, liver, colorectal, and female breast cancers.
Hepatocellular carcinoma (HCC), one of the most common cancer
types, is a primary malignancy of the liver and occurs predomin-
antly in patients with underlying chronic liver disease. The mortal-
ity rate of HCC has significantly increased worldwide. In 2018, the
American Cancer Society estimated that new liver cancer patients
in USA to be over 40,000 cases2. One of the most common risk
factors for developing HCC is the chronic viral infection with hepa-
titis C virus (HCV) or hepatitis B virus (HBV) or both3. There are
terial7,8
,
antifungal9, anti-inflammatory10, COX-2 inhibitors11,
antioxidant12, antithyroid13, anticonvulsant14, and anti-diabetic15.
Pyrimidine ring is also an integral part of DNA nucleic acid com-
position. Various drugs containing pyrimidine nucleus are being
used as potent anticancer agents through different mechanisms
R
of action i.e. 5-FluorouracilV (5-FU) I as thymidylate synthase
R
inhibitor16, MerbaroneV II as DNA topoisomerase II (topoII) cata-
many therapeutic strategies for targeting HCC. This includes lytic inhibitor17, Ceritinib (LDK378) III as anaplastic lymphoma
CONTACT Naglaa Mohamed Ahmed
nogamoon2005@yahoo.com
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Helwan University, Ein
Helwan, P.O. Code11795, Cairo, Egypt
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1111
kinase (ALK) inhibitor18, Dasatinib IV as multi-targeted of Bcr-Abl anticancer agents in hepatic HepG2 cancer cell line and induced
and Src family kinases inhibitor apoptosis inducer19, Imatinib V as HepG2 cells apoptosis
(Figure 2).
31,32
a receptor tyrosine kinase (TKI) inhibitor20, Ibrutinib (IBR) VI as
Bruton’s tyrosine kinase (BTK) inhibitor21, Ruxolitinib (INC424) VII
as Janus kinase (JAK) inhibitor22 and Nilotinib VIII as tyrosine kin-
ase inhibitor and apoptosis inducer23 (Figure 1).
Another organic moiety of high interest as anticancer pharma-
cophore is the anthracene nucleus. Several anthracene derivatives
showed significant anticancer activity against a wide range of
human tumour cell lines33–35 i.e. the well-known Doxorubicin
Pyrazoline ring is another widely used heterocyclic nucleus in (DOX) XV, is a Topoisomerase II inhibitor36 and anthracene-9-
medicinal chemistry because of its wide spectrum of pharmaco-
logical activities i.e. anti-inflamatory24, antibacterial25, and antioxi-
dant26 activities. Many pyrazoline derivatives was found to have
ylmethylene-[2-methoxyethoxymethylsulfanyl]-5-pyridin-3-yl- [1, 2,
4] triazole-4-amine (HL-37) XVI37 (Figure 3).
The medicinal chemistry importance of the three pharmaco-
anticancer activity against various human cancer types with differ- phores, i.e. pyrimidine, pyrazoline and anthracene, encouraged us
ent mode of action i.e. Pyrazoloacridine (PZA) (IX)27, Axitinib to design novel series of compounds hybridised with the three
(AG013736) (X)28, Pazopanib (GW786034) (XI)29 and 3–(50- moieties. The hybridisation of these pharmacophores in a single
Hydroxymethyl-20-furyl)-1-benzyl indazole (YC-1) (XII)30. New pyra- molecule (Figure 4) is expected to result in a hybrid molecule that
zoline derivatives XIII and XIV were reported as potential possesses anticancer activity. In this study, we report the design,
Figure 1. Anticancer drugs have pyrimidine ring in their structure.
Figure 2. Biologically active anticancer compounds have pyrazoline ring.

1112
N. M. AHMED ET AL.
synthesis and anticancer activity of a novel hybrids of pyrimidine 6-Aryl-2-hydrazinyl-4-oxo-1,4-dihydropyrimidine-5-carbonitriles
derivatives (4a-t) bearing pyrazoline and anthracene pharmaco-
phores against both human liver cancer cell lines (HepG2 and
Huh-7) and normal fibroblast cells by resazurin assay. In addition,
apoptosis and effects on caspase-3 and -7 activation against
human liver cancer cell lines HepG2 and Huh-7 were also per-
formed for the most active derivatives. Structure and activity rela-
tionship (SAR) and possible mechanisms of action of these
compounds were also investigated.
(2a-e)
The titled compounds 2a39, 2b40,41, 2c42, 2d43, and 2e were syn-
thesised according to the reported methods.
A mixture of 1a-d (0.005 mol) and hydrazine hydrate (0.005 mol,
99%) in 30 mL ethanol was refluxed for 30 h, then cooled and
poured on ice/water. The produced precipitate was filtered off, dried
and crystallised from ethanol to give compounds 2a-e.
4-[4-(Dimethylamino)phenyl]-2-hydrazino-6-oxo-1,6-dihydropyrimi-
dine-5-carbonitrile (2e). Yellow solid, yield 60%, m.p. 214–216 ꢀC.
IR (KBr) v max (cmꢁ1): 3366, 3295 (NH, NH2), 3069 (CH-Ar), 2959
(CH-sp3), 2209 (CN), 1689 (C¼O).1H NMR (300 MHz, DMSO-d6) d:
2.5 (s, 6H, CH3), 5.2 (s, 2H, NH2, D2O exchangeable), 7.38–7.57 (m,
4H, Ar-H), 10, 11 (s, 2H, 2NH, D2O exchangeable). 13C NMR
Materials and methods
Instruments
All melting points were determined with Electro-thermal IA 9100
apparatus (Shimadzu, Tokyo, Japan) and were uncorrected. FT-IR
spectra were recorded as potassium bromide pellets on a Perkin-
Elmer 1650 spectrophotometer (PerkinElmer, Waltham, MA),
(300 MHz, DMSO-d6):
d 43.6 (N-CH3), 90.4 (C-5 pyrimidine),
114.1–157.9 (aromatic Cs), 115.3 (CN), 159.5 (C-2 pyrimidine),
161.9(C¼O), 175.7(C-6 pyrimidine). MS(EI): m/z: 270[Mþ] (12.3%),77
(100%). Anal. Calcd for C13H14N6O (270.29): C, 57.77; H, 5.22; N,
31.09; Found: C, 57.78; H, 5.32; N, 31.10.
Faculty of Science, Cairo University, Cairo, Egypt. HNMR and 13C-
1
NMR spectra were recorded in DMSO-d6 on a Varian Mercury
(300 MHz) spectrometer (Varian, Oxford, UK) and chemical shifts
were given as ppm from TMS as internal reference (Faculty of
Science, Cairo University, Cairo, Egypt). Mass spectra were
recorded on 70 eV EI Ms-QP 1000 EX (Shimadzu, Tokyo, Japan),
Faculty of Science, Cairo University, Cairo, Egypt. Microanalyses
were performed using Vario, Elementar apparatus (Shimadzu,
Tokyo, Japan), Organic Microanalysis Unit, Faculty of Science, Cairo
University, Cairo, Egypt and the results were within the accepted
range (0.40) of the calculated values. Column chromatography
was performed on (Merck & Co., Kenilworth, NJ) silica gel 60 (par-
ticle size 0.06–0.20 mm).
(2E)-1–(9-anthryl)-3- substituted prop-2-en-1-ones (3a-d)
Compounds 3a, b, and c44 were synthesised according to the lit-
erature procedure.
General procedure for the preparation of compounds (3a-d). A
mixture of 9-acetylanthracene (0.01mol) and the appropriate alde-
hyde (0.01 mol) in 50 mL 10% ethanolic KOH solution was stirred at
room temperature for 24 h. The solution was cooled, poured on ice/
water acidified with dil. HCl. The produced precipitate was filtered
off, dried and crystallised from ethanol to give compounds 3a-d.
Chemistry
(2E)-1–(9-anthryl)-3-(1H-indol-3-yl)prop-2-en-1-one (3d). Brown
solid, yield 85%, m.p. 170–172 ꢀC. IR (KBr) v max (cmꢁ1): 3177 (CH-
Ar), 1665 (C¼O), 1604 (C¼C).1H NMR (300 MHz, DMSO-d6) d:
6-Aryl-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitriles
(1a-e)
The titled compounds 1a-e was synthesised according to the 6.6–8.7(m, 14H, Ar-H), 7.3 (d, 1H, J ¼ 6.9 Hz,ACH¼CHA), 7.5 (d, 1H,
J ¼ 19.2 Hz, ACH¼CHA) 11.2 (s, 1H, NH, D2O exchangeable). 13C
reported methods38.
NMR (300 MHz, DMSO-d6) d: 112.7–144.1 (aromatic Cs), 131.26,
140.8 (CH¼CH), 189.7 (C¼O). MS (EI): m/z: 347 [Mþ] (20%), 206
(100%). Anal. Calcd for C25H17NO (347.408): C, 86.43; H, 4.93; N,
4.03; O, 4.61; Found: C, 86.52; H, 5.03; N, 4.12; O, 4.70.
General procedure for the preparation of compounds (4a-t). A
mixture of compound 2a-e (4 mmol), the appropriate propenone
3a-d (4 mmol) and potassium hydroxide (0.2 g, 5 mmol) in abso-
lute ethanol (30 mL) was refluxed for 72 h. The reaction mixture
was poured on water, neutralised with 2 N hydrochloric acid and
the residue was filtered off. The crude product obtained was crys-
tallised from ethanol.
Figure 3. Examples of some anthracene-based anticancer agents.
Figure 4. Design strategy of new pyrimidine hybrid compounds as anticancer agent.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1
1113
2-[3-Anthracene-9-yl-5–(4-fluoro-phenyl)-4,5-dihydro-pyrazol-1-yl]-
6-oxo-4-phenyl-1,6-dihydro-pyrimidine-5-carbonitrile (4a). Yellow
solid, yield 51%, m.p. 150–151 ꢀC. IR (KBr) v max (cmꢁ1): 3077 (CH-
Ar), 2828 (CH-sp3), 2228 (CN), 1685 (C¼O), 1603 (C¼N). 1HNMR
(300 MHz, DMSO-d6) d: 2.5–3.38 (dd, 2H, C4-Hpyrazoline), 4.50 (dd,
1H, J ¼ 7.5 Hz, J ¼ 13.8 Hz, C5-Hpyrazoline), 7–8.8 (m, 18H, Ar-H),
10.2 (s, 1H, NH, D2O exchangeable). 13C NMR (300 MHz, DMSO-d6):
d 41.7 (C-4 pyrazoline), 42.0 (C-5 pyrazoline), 91.7 (C-5 pyrimidine),
111.6–147.3 (aromatic Cs), 117.8 (CN), 152.4 (C-2 pyrazoline), 163.0
(C-2 pyrimidine), 168.1 (C¼O), 175.7 (C, C-6 pyrimidine). MS (EI):
m/z: 535 [Mþ] (8%), 178 (100). Anal. Calcd for C34H22FN5O
(535.569): C, 76.25; H, 4.14; N, 13.08; Found: C, 76.29; H, 4.18;
N, 13.17.
(C¼O), 1621 (C ¼ N). H NMR (300 MHz, DMSO-d6) d: 2.50–3.38 (dd,
2H, C4-H pyrazoline), 5.5 (dd, 1H, J ¼ 3.6 Hz, J ¼ 12.6 Hz, C5-H pyra-
zoline), 7.30–8.25 (m, 17H, Ar-H), 10.1 (s, 1H, NH, D2O exchange-
able). 13C NMR (300 MHz, DMSO-d6): d 40.2(C-4 pyrazoline), 42.2
(C-5 pyrazoline), 93.0 (C-5 pyrimidine), 103.0–150.0 (aromatic Cs),
115.0 (CN), 158.2 (C-2 pyrazoline), 164.1 (C-2 pyrimidine),
86.2(C¼O), 172.1 (C, C-6 pyrimidine). MS (EI): m/z: 553 [Mþ] (8%),
95 (100%). Anal. Calcd for C34H21F2N5 O (553.56): C, 73.77; H, 3.82;
N, 12.65; Found: C, 73.80; H, 3.90; N, 12.70.
2-[3-Anthracene-9-yl-5–(4-dimethylamino-phenyl)-4,5-dihydro-pyr-
azol-1-yl]-4–(4-fluoro-phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-car-
bonitrile (4f). Brown crystals, yield 54%, m.p. 130–132 ꢀC. IR (KBr) v
max (cmꢁ1): 3059 (CH-Ar), 2922, 2872 (CH-sp3), 2220 (CN), 1680
1
2-[3-Anthracene-9-yl-5–(4-dimethylamino-phenyl)-4,5-dihydro-pyr-
azol-1-yl]-6-oxo-4-phenyl-1,6-dihydro-pyrimidine-5-carbonitrile
(4b). Yellowish brown crystals, yield 58%, m.p. 120–122 ꢀC. IR (KBr)
v max (cmꢁ1): 3062 (CH-Ar), 2920 (CH-sp3), 2223(CN), 1681 (C¼O),
(C¼O), 1614 (C¼N). H NMR (300 MHz, DMSO-d6) d: 2.3, 2.5 (s, 6H,
N-CH3), 2.65–3.1 (dd, 2H, C4-H pyrazoline), 5.6 (dd, 1H, J ¼ 11.8 Hz,
J ¼ 17.8 Hz, C5-H pyrazoline), 7–8.5 (m, 17 H, Ar-H), 10.0 (s, 1H, NH,
D2O exchangeable). 13C NMR (300 MHz, DMSO-d6): d 40.4 (C-4 pyr-
azoline), 43.0 (N-CH3), 42.4 (C-5 pyrazoline), 93.3 (C-5 pyrimidine),
104.0–147.0 (aromatic Cs), 115.0 (CN), 158.0 (C-2 pyrazoline), 164.0
(C-2 pyrimidine), 168.2 (C¼O), 172.0 (C, C-6 pyrimidine). MS (EI):
m/z: 578 [Mþ] (12%), 482 (100%). Anal. Calcd for C36H27FN6O
(578.637): C, 74.72; H, 4.70; N, 14.52; Found: C, 74.83; H, 4.80;
N, 14.60
1
1620 (C¼N). H NMR (300 MHz, DMSO-d6) d: 2.3, 2.4 (s, 6H, N-CH3),
2.5–3.5 (dd, 2H, C4-H pyrazoline), 5.2 (dd, 1H, J ¼ 6.6 Hz,
J ¼ 17.8 Hz, C5-H pyrazoline), 7.1–8.9 (m, 18H, Ar-H), 10.1(s, 1H, NH,
13
D2O exchangeable).
C NMR (300 MHz, DMSO-d6): d 41.0 (C-4
pyrazoline), 42.1 (C-5 pyrazoline), 43.0 (N-CH3), 91.2 (C-5 pyrimi-
dine), 110.0–140.0 (aromatic Cs), 117.0 (CN), 152.0 (C-2 pyrazoline),
163.0 (C-2 pyrimidine), 168.0 (C¼O), 175.0 (C, C-6 pyrimidine). MS
(EI): m/z: 560 [Mþ] (4%), 439 (100%). Anal. Calcd for C36H28 N6O
(560.647): C, 77.12; H, 5.03; N, 14.99; Found: C, 77.18; H, 5.14;
N, 15.02.
2-[3-Anthracene-9-yl-5–(3,4,5-trimethoxy-phenyl)-4,5-dihydro-pyra-
zol-1-yl]-4–(4-fluoro-phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-car-
bonitrile (4g). Brown crystals, yield 58%, m.p. 154–156 ꢀC. IR (KBr)
v max (cmꢁ1): 3062 (CH-Ar), 2930, 2850 (CH-sp3), 2223 (CN), 1681
(C¼O), 1620 (C¼N). 1HNMR (300 MHz, DMSO-d6) d: 2.62–3.2 (dd,
2-[3-Anthracene-9-yl-5–(3,4,5-trimethoxy-phenyl)-4,5-dihydro-pyra-
zol-1-yl]-6-oxo-4-phenyl-1,6-dihydro-pyrimidine-5-carbonitrile (4c). 2H, C4-H pyrazoline), 5.7 (dd, 1H, J ¼ 6.4 Hz, J ¼ 11.8 Hz, C5-H pyra-
Brown crystals, yield 60%, m.p. 110–112 ꢀC. IR (KBr) v max (cmꢁ1): zoline), 3.4–3.6 (s, 9H, OCH3), 7.1–8.6 (m, 15H, Ar-H), 10.1 (s, 1H,
3060 (CH-Ar), 2922, 2850 (CH-sp3), 2222 (CN),1680 (C¼O), 1618 NH, D2O exchangeable). 13C NMR (300 MHz, DMSO-d6): d 40.2 (C-4
1
(C¼N). H NMR (300 MHz, DMSO-d6) d: 2.6–3.2 (dd, 2H, C4-H pyra- pyrazoline), 42.3 (C-5 pyrazoline), 56.2, 56.4 (OCH3), 93.2 (C-5 pyr-
zoline), 5.4 (dd, 1H, J ¼ 6.4 Hz, J ¼ 17.5 Hz, C5-H pyrazoline), 3.3–3.5 imidine), 105.0–145.0 (aromatic Cs), 115.0 (CN), 158.4 (C-2 pyrazo-
(s, 9H, OCH3), 6.9–8.5 (m, 16H, Ar-H), 10.2 (s, 1H, NH, D2O line), 164.2 (C-2 pyrimidine), 168.1 (C¼O), 172.4 (C, C-6
exchangeable). 13C NMR (300 MHz, DMSO-d6): d 41.2 (C-4 pyrazo- pyrimidine). MS (EI): m/z: 625 [Mþ] (12%), 108 (100%). Anal. Calcd
line), 42.0 (C-5 pyrazoline), 56.2, 56.4 (OCH3),92.0 (C-5 pyrimidine), for C37H28FN5O4 (625.647): C, 71.03; H, 4.51; N, 11.19; Found: C,
111.0–142.0 (aromatic Cs), 117.0 (CN), 152.2 (C-2 pyrazoline), 163.0 71.13; H, 4.60; N, 11.22.
(C-2 pyrimidine), 168.0 (C¼O), 176.0 (C, C-6 pyrimidine). MS (EI):
m/z: 607 [Mþ] (6%), 437 (100%). Anal. Calcd for C37H29N5O4
2-[3-Anthracene-9-yl-5-(1H-indol-3-yl)-4,5-dihydro-pyrazol-1-yl]-
(607.657): C, 73.13; H, 4.81; N, 11.53; Found: C, 73.18; H, 4.90;
N, 11.59.
4–(4-fluoro-phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-carbonitrile
(4h). Brown crystals, yield 59%, m.p. 143–145 ꢀC. IR (KBr) v max
(cmꢁ1): 3177 (CH-Ar), 2893 (CH-sp3), 2226 (CN), 1680 (C¼O), 1604
(C¼N). 1H NMR (300 MHz, DMSO-d6) d: 2.51–3.38 (dd, 2H, C4-H
2-[3-Anthracene-9-yl-5-(1H-indol-3-yl)-4,5-dihydro-pyrazol-1-yl]-6-
oxo-4-phenyl-1,6-dihydro-pyrimidine-5-carbonitrile(4d).
Yellow
pyrazoline), 4.51 (dd, 1H, J ¼ 7.5 Hz, J ¼ 10.8 Hz, C5-H pyrazoline),
7.0–8.38 (m, 18H, Ar-H), 9.58, 10.20 (s, 2H, NH, D2O exchangeable).
13C NMR (300 MHz, DMSO-d6): d 40.2 (C-4 pyrazoline), 42 0.0 (C-5
pyrazoline), 93.7 (C-5 pyrimidine), 103.6–152.0 (aromatic Cs), 115.2
(CN), 158.0 (C-2 pyrazoline), 164.0 (C-2 pyrimidine), 168.0 (C¼O),
172.0 (C, C-6 pyrimidine). MS (EI): m/z: 574 [Mþ] (10%), 178
(100%). Anal. Calcd for C36H23FN6 O (574.605): C, 75.25; H, 4.03; N,
14.63; Found: C, 75.31; H, 4.15; N, 14.71.
crystals, yield 59%, m.p. 135–137 ꢀC. IR(KBr) v max (cmꢁ1): 3062
(CH-Ar), 2852 (CH-sp3), 2220 (CN), 1682 (C¼O), 1608 (C¼N).
1HNMR (300 MHz, DMSO-d6) d: 2.51–3.42 (dd, 2H, C4-H pyrazoline),
4.51 (dd, 1H, J ¼ 3.3 Hz, J ¼ 14.7 Hz, C5-H pyrazoline), 7.4–8.40 (m,
19 H, Ar-H), 10.2, 11.8 (s, 2H, NH, D2O exchangeable). 13C NMR
(300 MHz, DMSO-d6): d 41.5 (C-4 pyrazoline), 42.2 (C-5 pyrazoline),
91.2 (C-5 pyrimidine), 112.0–145.0 (aromatic Cs), 117 (CN), 152.0
(C-2 pyrazoline), 162.0 (C-2 pyrimidine), 168.0 (C¼O), 170.0 (C, C-6
pyrimidine). MS (EI): m/z: 556 [Mþ] (3%), 419 (100%). Anal. Calcd
for C36H24N6O (556.61536): C, 77.68; H, 4.35; N, 15.10; Found: C,
77.71; H, 4.41; N, 15.22.
2-[3-Anthracene-9-yl-5–(4-fluoro-phenyl)-4,5-dihydro-pyrazol-1-yl]-
4-(1H-indol-3-yl)-6-oxo-1,6-dihydro-pyrimidine-5-carbonitrile (4i). A
brown solid, yield 55%, m.p. 184–185 ꢀC. IR (KBr) v max (cmꢁ1):
3062 (CH-Ar), 2940, 2850 (CH-sp3), 2219 (CN), 1682 (C¼O), 1601
1
2-[3-Anthracene-9-yl-5–(4-fluoro-phenyl)-4,5-dihydro-pyrazol-1-yl]-
4–(4-fluoro-phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-carbonitrile
(4e). Yellowish brown crystals, yield 50%, m.p. 138–140 ꢀC. IR (KBr) (m, 18H, Ar-H), 10.1, 10.3 (s, 2H, NH, D2O exchangeable). 13C NMR
v max (cmꢁ1): 3058 (CH-Ar), 2920, 2848 (CH-sp3), 2222(CN), 1672 (300 MHz, DMSO-d6): d 41.4 (C-4 pyrazoline), 43.0 (C-5 pyrazoline),
(C¼N). HNMR (300 MHz, DMSO-d6) d: 2.95–4.1 (dd, 2H, C4-H pyra-
zoline), 4.9 (dd, 1H, J ¼ 6.5 Hz, J ¼ 17.7 Hz, C5-H pyrazoline), 7–8.5

1114
N. M. AHMED ET AL.
93.1 (C-5 pyrimidine), 102.1–148.1 (aromatic Cs), 114.2 (CN), 152.4 C37H28FN5O4 (625.647): C, 71.03; H, 4.51; N, 11.19; Found: C, 71.14;
(C-2 pyrazoline), 162.0 (C-2 pyrimidine), 165.0 (C¼O), 178.0 (C, C-6
H, 4.60; N, 11.20.
pyrimidine). MS (EI): m/z: 574 [Mþ] (3%), 117 (100%). Anal. Calcd
for C36H23FN6O (574.605): C, 75.25; H, 4.03; N, 14.63; Found: C,
75.29; H, 4.15; N, 14.68.
2-[3-Anthracene-9-yl-5–(4-dimethylamino-phenyl)-4,5-dihydro-pyr-
azol-1-yl]-6-oxo-4–(3,4,5-trimethoxy-phenyl)-1,6-dihydro-pyrimi-
dine-5-carbonitrile (4n). Dark brown crystals, yield 60%, m.p.
170–172 ꢀC. IR (KBr) v max (cmꢁ1): 3061 (CH-Ar), 2922, 2850 (CH-
2-[3-Anthracene-9-yl-5–(4-dimethylamino-phenyl)-4,5-dihydro-pyr-
sp3), 2222 (CN), 1686 (C¼O), 1620(C¼N). 1H NMR (300 MHz,
azol-1-yl]-4-(1H-indol-3-yl)-6-oxo-1,6-dihydro-pyrimidine-5-carboni-
trile (4j). A dark brown solid, yield 59%, m.p. 240–242 ꢀC. IR (KBr) v
DMSO-d6) d: 2.5, 2.8 (s, 6H, N-CH3), 2.95–3.4 (dd, 2H, C4-H pyrazo-
max (cmꢁ1): 3064 (CH-Ar), 2920, 2850 (CH-sp3), 2218 (CN), 1686
line), 3.5 (s, 9H, OCH3), 5.3 (dd, 1H, J ¼ 6.5 Hz, J ¼ 17.9 Hz, C5-H pyr-
(C¼O), 1598 (C¼N). 1HNMR (300 MHz, DMSO-d6) d: 2.3, 2.5 (s, 6H,
azoline), 6.7–8.8 (m, 15H, Ar-H), 11.1 (s, 1H, NH, D2O
exchangeable). 13C NMR (300 MHz, DMSO-d6): d 41.2 (C-4pyrazo-
line), 42.2 (C-5 pyrazoline), 43.0 (N-CH3), 56.3, 56.4 (OCH3), 95.7 (C-
5 pyrimidine), 102.0–148.0 (aromatic Cs), 115.0 (CN), 152.2 (C-2
pyrazoline), 160.1 (C-2 pyrimidine), 168.5 (C¼O), 175.4 (C, C-6 pyr-
imidine). MS (EI): m/z: 725 [Mþ] (4%), 108 (100%). Anal. Calcd for
C39H34N6O4 (650.725.06): C, 71.98; H, 5.27; N, 12.91; Found: C,
72.01; H, 5.31; N,13.02.
N-CH3), 2.95–4.2 (dd, 2H, C4-H pyrazoline), 4.80 (dd, 1H, J ¼ 6.3 Hz,
J ¼ 17.8 Hz, C5-H pyrazoline), 6.8–8.6 (m, 18H, Ar-H), 10, 10.2 (s,
2H, NH, D2O exchangeable). 13CNMR (300 MHz, DMSO-d6): d 41.6
(C-4 pyrazoline), 43.0 (N-CH3), 43.2 (C-5 pyrazoline), 93.0 (C-5 pyr-
imidine), 105.0–145.0(aromatic Cs), 114.0 (CN), 152.4 (C-2 pyrazo-
line), 162.0 (C-2 pyrimidine), 164.5 (C¼O), 177.0 (C, C-6
pyrimidine). MS (EI): m/z: 599 [Mþ] (6%), 478 (100%). Anal. Calcd
for C38H29N7O (599.683): C, 76.11; H, 4.87; N, 16.35; Found: C,
76.18; H, 4.91; N, 16.40.
2-[3-Anthracene-9-yl-5–(3,4,5-trimethoxy-phenyl)-4,5-dihydro-pyra-
zol-1-yl]-6-oxo-4–(3,4,5-trimethoxy-phenyl)-1,6-dihydro-pyrimidine-
5-carbonitrile (4o). Brown crystals, yield 62%, m.p. 165–166 ꢀC. IR
(KBr) v max (cmꢁ1): 3061 (CH-Ar), 2920, 2850 (CH-sp3), 2223(CN),
1680 (C¼O), 1618 (C¼N). 1HNMR (300 MHz, DMSO-d6) d: 2.6–3.1
(dd, 2H, C4-H pyrazoline), 4.85(dd, 1H, J ¼ 6.6 Hz, J ¼ 18.2 Hz, C5-H
pyrazoline), 3.3, 3.5 (s, 18H, OCH3), 6.5–8.5 (m, 13H, Ar-H), 10.3
(s,1H, NH, D2O exchangeable). 13C NMR (300 MHz, DMSO-d6): d
41.2 (C-4 pyrazoline), 42.7 (C-5 pyrazoline), 56.2, 56.4, 56.5 (OCH3),
95.2 (C-5 pyrimidine), 102.0–147.0 (aromatic Cs), 115.0 (CN), 152.4
(C-2 pyrazoline), 160.2 (C-2 pyrimidine), 168.2 (C¼O), 175.7 (C, C-6
pyrimidine). MS (EI): m/z: 697 [Mþ] (5%), 457 (100%). Anal. Calcd
for C40H35N5O7 (697.7352): C, 68.86; H, 5.06; N, 10.04; Found: C,
68.91; H, 5.17; N, 10.15.
2-[3-Anthracene-9-yl-5–(3,4,5-trimethoxy-phenyl)-4,5-dihydro-pyra-
zol-1-yl]-4-(1H-indol-3-yl)-6-oxo-1,6-dihydro-pyrimidine-5-carboni-
trile (4k). A brown solid, yield 61%, m.p. 228–230 ꢀC. IR (KBr) v
max (cmꢁ1): 3344 (CH-Ar), 2959 (CH-sp3), 2213 (CN), 1686 (C¼O),
1606 (C¼N). 1H NMR (300 MHz, DMSO-d6) d: 2.5–3.03 (dd, 2H, C4-
H pyrazoline), 5.5 (dd, 1H, J ¼ 6 Hz, J ¼ 15.6 Hz, C5-H pyrazoline),
3.3 (s, 9H, OCH3), 6.76–8.12 (m, 16H, Ar-H), 9.6, 10.6 (s, 2H, NH,
D2O exchangeable). 13C NMR (300 MHz, DMSO-d6): d 41.6 (C-4 pyr-
azoline), 43.7 (C-5 pyrazoline), 55.5, 55.7 (OCH3), 93.0 (C-5 pyrimi-
dine), 102.2–146.7 (aromatic Cs), 114.0 (CN), 152.0 (C-2 pyrazoline),
162.9 (C-2 pyrimidine), 164.7 (C ¼ O), 178.0 (C, C-6 pyrimidine). MS
(EI): m/z: 646 [Mþ] (9%), 77 (100%). Anal. Calcd for C39H30N6O4
(646.6933): C, 72.43; H, 4.68; N, 13.00; Found: C, 72.48; H, 4.70;
N, 13.04.
2-[3-Anthracene-9-yl-5-(1H-indol-3-yl)-4,5-dihydro-pyrazol-1-yl]-6-
oxo-4–(3,4,5-trimethoxy-phenyl)-1,6-dihydro-pyrimidine-5-carboni-
trile (4p). Brown crystals, yield 63%, m.p. 157–158 ꢀC. IR
(KBr) v max (cmꢁ1): 3064 (CH-Ar), 2920, 2850 (CH-sp3), 2220 (CN),
1686 (C¼O), 1618 (C¼N). 1H NMR (300 MHz, DMSO-d6) d:
2.51–4.03 (dd, 2H, C4-H pyrazoline), 5.51 (dd, 1H, J ¼ 6.5 Hz,
J ¼ 17.2 Hz, C5-H pyrazoline), 3.5 (s, 9H, OCH3), 6.5–8.7 (m, 16H, Ar-
H), 11.2, 11.3 (s, 2H, NH, D2O exchangeable). 13CNMR (300 MHz,
DMSO-d6): d 41.3 (C-4 pyrazoline), 42.4 (C-5 pyrazoline), 56.2, 56.7
(OCH3), 95.1 (C-5 pyrimidine), 102.0–146.0 (aromatic Cs), 115.0
(CN), 152.0 (C-2pyrazoline), 160.1 (C-2 pyrimi dine), 168.5 (C¼O),
175.4 (C, C-6 pyrimidine). MS (EI): m/z: 644 [Mþ] (9%), 77 (100%).
Anal. Calcd for C40H32N6O3 (644.720): C, 74.52; H, 5.00; N, 13.04;
Found: C, 74.61; H, 5.12; N, 13.15.
2-[3-Anthracene-9-yl-5-(1H-indol-3-yl)-4,5-dihydro-pyrazol-1-yl]-
4-(1H-indol-3-yl)-6-oxo–1,6-dihydro-pyrimidine-5-carbonitrile
(4l). Reddish brown crystals, yield 62%, m.p. 220–222 ꢀC. IR (KBr) v
max (cmꢁ1): 3070 (CH-Ar), 2920, 2850 (CH-sp3), 2222 (CN), 1686
1
(C¼O), 1608 (C¼N). H NMR (300 MHz, DMSO-d6) d: 2.62–3.03 (dd,
2H, C4-H pyrazoline), 5.6 (dd, 1H, J ¼ 3.6 Hz, J ¼ 13.2 Hz, C5-H pyra-
zoline), 6.7–7.9 (m, 19H, Ar-H), 10, 11.6, 11.7 (s, 3H, NH, D2O
exchangeable). 13CNMR (300 MHz, DMSO-d6): d 41.4 (C-4 pyrazo-
line), 43.0 (C-5 pyrazoline), 93.0 (C-5 pyrimidine), 104.0 –144.2 (aro-
matic Cs), 114.0 (CN), 152.2 (C-2 pyrazoline), 162.4 (C-2
pyrimidine), 164.0 (C¼O), 178.0 (C, C-6 pyrimidine). MS (EI): m/z:
595 [Mþ] (12%), 178 (100%). Anal. Calcd for C38H25N7O (595.6514):
C, 76.62; H, 4.23; N, 16.46; Found: C, 76.68; H, 4.33; N, 16.51.
2-[3-Anthracene-9-yl-5–(4-fluoro-phenyl)-4,5-dihydro-pyrazol-1-yl]-
4–(4-dimethylamino-phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-car-
bonitrile (4q). Reddish brown solid, yield 57%, m.p. 182–184 ꢀC.
2-[3-Anthracene-9-yl-5–(4-fluoro-phenyl)-4,5-dihydro-pyrazol-1-yl]-
6-oxo-4–(3,4,5-trimethoxy-phenyl)-1,6-dihydro-pyrimidine-5-car-
bonitrile (4m). Dark brown crystals, yield 58%, m.p. 148–150 ꢀC. IR
(KBr) v max (cmꢁ1): 3348 (CH-Ar), 2953, 2850 (CH-sp3), 2212 (CN),
1684 (C¼O), 1601 (C¼N). 1H NMR (300 MHz, DMSO-d6) d: 2.9–3.1
(dd, 2H, C4-H pyrazoline), 4.9 (dd, 1H, J ¼ 6.6 Hz, J ¼ 16.5 Hz, C5-H
pyrazoline), 3.45 (9H, s, OCH3), 6.8–8.7 (m, 15H, Ar-H), 11.4 (s, 1H,
NH, D2O exchangeable). 13C NMR (300 MHz, DMSO-d6): d 41.5 (C-4
pyrazoline), 43.7 (C-5 pyrazoline), 56.2, 56.7 (OCH3), 95.7 (C-5 pyr-
imidine), 102.0–146.4 (aromatic Cs), 115.5 (CN), 152.4 (C-2 pyrazo-
line), 159.6 (C-2 pyrimidine), 168.5(C¼O), 175.7 (C, C-6 pyrimidine).
IR (KBr)
v
max (cmꢁ1): 3066 (CH-Ar), 2923 (CH-sp3),
2222 (CN), 1680 (C¼O), 1602 (C¼N). 1H NMR (300 MHz, DMSO-d6)
d: 2.5 (s, 6H, N-CH3), 2.9–3.36 (dd, 2H, C4-H pyrazoline), 5.50 (dd,
1H, J ¼ 6.4 Hz, J ¼ 18.2 Hz, C5-H pyrazoline), 6.7–8.8 (m, 17H, Ar-H),
10.2 (s, 1H, NH, D2O exchangeable). 13C NMR (300 MHz, DMSO-d6):
d 40.2 (C-4 pyrazoline), 43.2 (N-CH3), 55.2 (C-5 pyrazoline), 93.2 (C-
5 pyrimidine), 104.0–142.0 (aromatic Cs), 114.0 (CN), 152.1(C-2 pyr-
azoline), 164.0 (C-2 pyrimidine), 160.0 (C¼O), 170.0 (C, C-6 pyrimi-
MS (EI): m/z: 625 [Mþ] (12%), 77 (100%). Anal. Calcd for dine). MS (EI): m/z: 578 [Mþ] (12%), 178 (100%). Anal. Calcd for

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1115
C36H27FN6O (578.637): C, 74.72; H, 4.70; N, 14.52; Found: C, 74.80; Caspase-Glo 3/7 assay
H, 4.78; N, 14.60.
The influence of our test samples on caspase 3/7 activity in liver
cancer cells (HepG2 cells) was detected using Caspase-Glo 3/7
Assay kit (Promega, Madison, WI) as previously described45 (see
Supplementary).
2-[3-Anthracene-9-yl-5–(4-dimethylamino-phenyl)-4,5-dihydro-pyr-
azol-1-yl]-4–(4-dimethylamino-phenyl)-6-oxo-1,6-dihydro-pyrimi-
dine-5-carbonitrile (4r). Brown crystals, yield 59%, m.p. 174–175 ꢀC.
IR (KBr) v max (cmꢁ1): 3061 (CH-Ar), 2920, 2850 (CH-sp3), 2222
1
(CN), 1682 (C¼O), 1602 (C¼N). H NMR (300 MHz, DMSO-d6) d: 2.3,
Data analysis
2.5 (s, 12H, N-CH3), 2.95–3.23 (dd, 2H, C4-H pyrazoline), 5.70 (dd,
1H, J ¼ 6.3 Hz, J ¼ 17.6 Hz, C5-H pyrazoline), 6.7–8.5 (m, 17H, Ar-H),
10.1 (s, 1H, NH, D2O exchangeable). 13C NMR (300 MHz, DMSO-d6):
d 40.1 (C-4 pyrazoline), 43.1 (N-CH3), 55.4 (C-5 pyrazoline), 93.2 (C-
5 pyrimidine), 104.0–144.0 (aromatic Cs), 114.0 (CN), 152.0 (C-2
pyrazoline), 164.0 (C-2 pyrimidine), 160.0 (C¼O), 170.2 (C, C-6 pyr-
Results are represented as M SEM of at least three independent
experiments. Data analysis has been done using graph pad prism
and Sigma Plot 11 software.
imidine). MS (EI): m/z: 603 [Mþ] (8%), 77 (100%). Anal. Calcd for Molecular modelling procedure
C38H33N7O (603.714): C, 75.60; H, 5.51; N, 16.24; Found: C, 75.67; H,
The modelling experiment described in his study was performed
5.61; N, 16.30.
by using the Discovery Studio version 4.5 (Accelrys Inc., San
Diego, CA) software as previously described13. The required pdb
coordinates were downloaded from the Brookhaven website
(www.rcsb.org). The hydrogen atoms were then added to both
the small molecule and the DNA structure. The atom and bond
types as well as the protonation state for the small molecule and
the binding site were checked and corrected when needed. Water
molecules were deleted. This was followed by minimising the
complex with the Discovery Studio (DS) force field by using the
default parameters. The binding mode of the designed compound
and that of the DOX in bound to part of the DNA structure will
be discussed later.
2-[3-Anthracene-9-yl-5–(3,4,5-trimethoxy-phenyl)-4,5-dihydro-pyra-
zol-1-yl]-4–(4-dimethylamino-phenyl)-6-oxo-1,6-dihydro-pyrimi-
dine-5-carbonitrile (4s). A brown solid, yield 61%, m.p. 160–162 ꢀC.
IR (KBr) v max (cmꢁ1): 3064 (CH-Ar), 2922, 2850 (CH-sp3), 2222
(CN), 1684 (C¼O), 1604 (CN). 1H NMR (300 MHz, DMSO-d6) d: 2.5
(s, 6H, N-CH3), 2.85–3.3 (dd, 2H, C4-H pyrazoline), 5.60 (dd, 1H,
J ¼ 6.6 Hz, J ¼ 17.6 Hz, C5-H pyrazoline), 3.5 (s, 9H, OCH3), 6.6–8.4
(m, 15H, Ar-H), 10.3(s, 1H, NH, D2O exchangeable). 13C NMR
(300 MHz, DMSO-d6): d 40.0 (C-4 pyrazoline), 43.3 (N-CH3), 55.2 (C-
5 pyrazoline), 56.2, 56.7 (OCH3), 93.2 (C-5 pyrimidine), 104.0–143.0
(aromatic Cs), 114.0 (CN), 152.2 (C-2 pyrazoline), 164.0 (C-2 pyrimi-
dine), 160.0 (C¼O), 170.0 (C, C-6 pyrimidine). MS (EI): m/z: 650
[Mþ] (9%), 480 (100%). Anal. Calcd for C39H34N6O4 (650.725): C,
71.98; H, 5.27; N, 12.91; Found: C, 72.01; H, 5.32; N, 13.00.
Results and discussion
Chemistry
2-[3-Anthracene-9-yl-5-(1H-indol-3-yl)-4,5-dihydro-pyrazol-1-yl]-
4–(4-dimethylamino-phenyl)-6-oxo-1,6-dihydro-pyrimidine-5-car-
bonitrile (4t). A brown solid, yield 62%, m.p. 190–191 ꢀC. IR (KBr) v
max (cmꢁ1): 3333 (CH-Ar), 2944 (CH-sp3), 2211(CN), 1681 (C¼O),
1600 (C¼N). 1H NMR (300 MHz, DMSO-d6) d: 2.49 (s, 6H, N-CH3),
2.56–3.33 (dd, 2H, C4-H pyrazoline), 5.49 (dd, 1H, J ¼ 1.5 Hz,
J ¼ 18 Hz, C5-H pyrazoline), 7.4–8.5 (m, 19H, Ar-H), 9.3, 10.09 (s,
2H, NH, D2O exchangeable). 13C NMR (300 MHz, DMSO-d6): d 40.2
(C-4 pyrazoline), 43.8 (N-CH3), 55.4 (C-5 pyrazoline), 93.9 (C-5 pyr-
imidine), 104.2–141.0 (aromatic Cs), 114.0 (CN), 152.0 (C-2 pyrazo-
line), 164.0 (C-2 pyrimidine), 159.7.0 (C¼O), 170.0 (C, C-6
pyrimidine). MS (EI): m/z: 599 [Mþ] (8%), 117 (100%). Anal. Calcd
for C38H29N7O (599.68316): C, 76.11; H, 4.87; N, 16.35; Found: C,
76.14; H, 4.94; N,16.50.
The synthetic pathways adopted for the synthesis of the desired
new compounds are illustrated in Scheme 1A–C. The starting 6-
aryl-4-oxo-2-thioxo-1,2,3,4-tetrahydro
pyrimidine-5-carbonitriles
(1a-e) were synthesised from thiourea, ethyl cyanoacetate and the
corresponding aldehydes in sodium ethoxide38 in a one pot reac-
tion. The corresponding 2-hydrazino-6-oxo-4-phenyl/(4-substitu-
tedphenyl)-1,6-dihydropyrimi-dine-5-carbonitriles
2a-e
were
obtained through hydrazinolysis of the precursor thio derivatives40
(Scheme 1A). The (2E)-1–(9-anthryl)-3- substituted prop-2-en-1-
ones derivatives 3a-d were synthesised via a base-catalysed
Claisen Schmidt condensation of the appropriate aldehyde and
9-acetylanthracene in 10% ethanolic KOH (Scheme 1B).
Cyclocondensation reaction of the corresponding 2-hydrazinopyr-
imdine derivatives 2a-e and the appropriate propenones 3a-d in
absolute ethanol in the presence of potassium hydroxide afforded
the target compounds 4a-t (Scheme 1C). The structures of the
newly synthesised compounds were in good agreement with their
IR, MS, 1H-NMR, 13C-NMR and elemental analyses data. The 1H
NMR spectra of compounds 4a-t showed the protons at C-4 and
C-5 of the pyrazoline ring. Proton at C-4 appeared as doublet of
In vitro cytotoxicity activity
All compounds 2a-e, 3a-d, and PPAD’s 4a-t were subjected to a doublet at d 2.50–4.3 ppm and proton at C-5 appeared as doublet
screening system for evaluation of their anticancer activity against of doublet at d 4.5–5.70 ppm. Other protons (N-CH3, OCH3, and
cell line of human cancer, namely liver (HepG2 and Huh-7) cancer aromatic Hs) were shown in their usual range. 13C-NMR showed
and normal fibroblast cells obtained from the German Cancer the characteristic C-4 and C-5 carbon signals of the pyrazoline
Research Center (DKFZ), following the Resazurin Cell Growth ring at d 40– 41.7 and 42– 55.4 ppm, respectively, in addition to
Inhibition Assay45 in comparison to the standard treatment using the other signals for the carbons of the target compounds (c.f.
DOX (see Supplementary).
Experimental section).

1116
N. M. AHMED ET AL.
Scheme 1. Synthesis of the designed compounds (4a-t).
Anticancer evaluation
-F and N, N(CH3)2 such as 4f and 4q. Compounds substituted with
both (R) and (R’) were more effective than those having only R’
substituent as in 4b-d against HepG2 cell line. On the other hand,
testing the compounds against Huh-7 cell line, all the tested
PPAD’s 4a-s showed anticancer activities with IC50 values ranging
from 4.78 to 16.75 lg/mL. Interestingly, compounds 4e and 4g
(IC50¼6.13 which is equivalent to 11.1 lM and 4.78 lg/mL which is
equivalent to 7.6 lM, respectively) showed activity against Huh-7
cell line higher than DOX. Compound 4i (IC50¼6.47 lg/mL which
is equivalent to 11.25 lM) showed activity comparable to DOX
(Figure 5). However, compounds 4m, 4r, 4t, 4f, 4p, and 4q had
significant activity (IC50¼7.50, 7.61, 7.42, 8.45, 8.14, and 8.66 lg/
mL, respectively). The activity of the tested compounds against
Huh-7cell line had the following descending order: (4g > 4e >4i >
4t > 4m > 4r > 4p> 4d >4f > 4q > 4n> 4h >4b > 4s> 4c > 4l >
4j > 4k > 4o> 4a).
This study presents the synthesis and antiproliferative activity of
compounds having pyrimidine, pyrazoline, and anthracene phar-
macophores. All compounds 2a-e, 3a-d, and PPAD’s 4a-t were
investigated for their in vitro anti-HCC activity against HepG2,
Huh-7 cell lines, and normal fibroblast cells and the anticancer
drug DOX was used as standard treatment. The cytotoxic activities
of tested compounds were measured as IC50 (in lg/mL) value (the
dose that reduces cell growth to 50%) (Table 1). The results
showed that the tested compounds exhibited good to moderate
anti-proliferative activities against the tested cell lines compared
to normal cells. Compounds 2a-e, 3a-d were found to show mod-
erate activity against the two cell lines. Regarding the activity of
PPAD’s 4a-t against HepG2 cell line, the results showed that com-
pound 4f (IC50¼4.2 lg/mL which is equivalent to 7.2 lM) pos-
sessed the highest degree of cytotoxicity, 4e (IC50¼5.34 lg/mL
which is equivalent to 9.6 lM) was almost equipotent to DOX
(IC50¼5.43 lg/mL which is equivalent to 10.1 lM) and 4q
(IC50¼6.85 lg/mL which is equivalent to 11.8 lM) was quite less
potent than DOX (Figure 5). The anticancer activity of the tested
PPAD’s against HepG2 cell line had the following descending
order: (4f > 4e > 4q > 4g > 4h > 4m > 4r > 4s> 4t > 4i > 4n >
4o > 4p >4a> 4j > 4k > 4l > 4b > 4c >4d).
The data presented in this study revealed that all the tested
PPAD’s 4a-t had broad spectrum anticancer activity against all
screened HCC cell lines compared to normal cells. Some of our
PPAD’s exhibited potent antiproliferative activity against HepG2
and Huh-7 cell lines. The cytotoxicity of all compounds was more
prominent on cancer cells compared to normal ones (Table 1).
These results suggest that pyrimidine pyrazoline-anthracene back-
bone is an interesting anticancer pharmacophore. Moreover, some
PPAD’s were even more potent than the standard drug Dox. The
To shed the light on the SAR of these series of compound. The
data suggest that the promising compounds emerging were those
substituted with fluorine atom (-F) in both R and R’ such as 4e or most potent compound in this study was 4e. This could be

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1117
explained by the electronegative effect of the fluorine atom on Regarding the structure SAR of compounds (Figure 7), there was a
the pyrimidine and pyrazoline -anthracene backbone. PPAD’s 4e,
4i, 4m, and 4q were the most promising anticancer agents
against all the tested cell lines (Figure 6). Interestingly, PPAD’s 4e
showed high potency against HepG2 and Huh-7 cell lines.
consistent relation between the lipophilicity and/or electronic
property of the substituent groups R and R’ groups and the anti-
proliferative activity. Concerning the nature of substituent groups
R and R’, the order of activity of tested compounds against tested
cell lines was the following: (F > N(CH3)2 >3,4,5-(OCH3)3 > 3-
Indolyl > H). The introduction of a more electronegative and a
lipophilic substituent to phenyl rings of the pyrimidine and pyra-
zoline enhance potency against HepG2 and Huh-7 cancer cell lines
as shown in the fluorine substituted PPAD’s 4e. However, a drop
in potency against the same cell lines was observed with unsub-
stituted phenyl ring or 3-indolyl substituted PPAD’s as in 4a-d.
The sensitivity of the tested cell lines to PPAD’s 4a-t was in the
following descending order: (HepG2 > Huh-7).
Table 1. IC50 values in lg/mL for cytotoxic activity of the compounds against
hepatocellular carcinoma cell lines and fibroblast cells.
ꢂ
Cytotoxic activity IC50 (lg/mL)
Compounds
HepG2 cell line
Huh-7 cell line
Fibroblast
2a
2b
2c
2d
2e
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
20.8 1.6
22.2 2.20
20.2 1.88
20.6 2.14
20.1 3.00
22.2 2.45
21.4 2.76
21.5 2.54
22.5 2.80
11.34 2.11
15.31 4.21
15.32 4.22
17.24 4.04
5.34 0.21
4.22 0.94
7.22 1.85
7.45 1.11
9.54 1.24
12.18 2.53
14.13 3.01
14.63 2.12
8.12 1.82
9.66 1.31
10.19 1.02
11.21 1.2
6.85 0.85
8.44 1.10
8.66 1.44
9.21 1.05
5.43 0.24
21.7 1.18
21.6 2.19
21.3 2.37
21.8 3.18
21 4.12
40.01 1.24
40.58 2.45
40.23 2.13
40.13 1.37
40.42 1.89
40.56 2.48
40.71 2.34
40.82 4.51
40.81 4.72
32.31 5.22
33.02 6.51
29.32 3.51
21.51 4.07
24.12 4.63
33.14 6.52
25.32 4.87
23.52 4.21
31.42 2.07
34.14 5.17
27.04 4.12
20.23 3.18
24.13 4.34
18.57 3.17
28.05 3.31
18.24 4.02
34.31 6.42
25.24 5.24
20.31 4.15
23.51 3.82
24.11 3.53
21.8 1.24
21.00 1.12
21.5 4.13
22.6 3.30
16.75 3.45
10.95 2.14
11.33 1.09
8.42 1.04
6.13 1.01
8.45 0.75
4.78 0.24
9.77 1.48
6.47 0.41
13.24 3.05
13.52 2.32
12.44 1.75
7.50 0.98
9.54 2.16
14.52 1.24
8.14 1.65
8.66 1.52
7.61 0.21
11.28 0.61
7.42 1.48
6.40 0.43
Caspase 3/7 assay
The caspases are a family of endoproteases that provide critical
links in cell regulatory networks through controlling inflammation
and cell death (apoptosis). Caspase-3 and caspase-7 cleave pro-
teins involved in programmed cell death events46. It is well estab-
lished that the induction of the apoptotic cascade is one of the
main mechanisms of most of the currently available anticancer
agents45,47,48. To determine the mechanism involved in the antitu-
mor activity of our PPADs demonstrated above whether is a result
of apoptosis or not, Caspase-Glo 3/7 assay was performed47.
HepG-2 (Figure 8) and Huh-7 cell lines (Figure 9) were treated
with the target PPAD’s samples or DMSO (solvent control). Figures
8 and 9 show that PPADs 4e, 4i, 4m, and 4q stimulated caspase
activity in both cell lines at all tested concentrations and caused
significant increase in activation of caspase 3/7 in a dose-depend-
ent manner. These results suggest that our compounds induced
apoptosis is, in part, due to activation of caspses3/7 and apoptosis
is may be the main mechanism of action. Moreover, the apoptosis
induced by the tested target compounds on Huh-7 cell line was
greater than its effect on HepG2 cell line.
4t
Doxorubicin
ꢂ
Three independent experiments were performed for each concentration.
Figure 5. The most potent pyrimidine derivatives against HepG2 cell line and Huh-7 cell line, respectively.

1118
N. M. AHMED ET AL.
Figure 6. The most potent PPADs as anticancer agents.
Figure 7. Structure activity relationship of the pyrimidine derivatives.
Figure 9. Caspase 3/7 assay results of PPADs 4e, 4i, 4m, and 4q against Huh-7
cell line (24h incubation). The results were significant; p < .05, n ¼ 3.
Figure 8. Caspase 3/7 assay results of PPADs 4e, 4i, 4m, and 4q against HepG2
cell line (24h incubation). The results were significant; p < .05, n ¼ 3.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1119
Acknowledgements
We like to convey our grateful thanks for Mossad Sayed Mohamed
(Professor of Pharmaceutical Organic Chemistry) for his inspiration
and support. The authors are also thankful to Dr. Joerg Hoheisel,
head of functional genome analysis department, German Cancer
Research Center, Germany for his generous help and support
throughout the whole work.
Disclosure statement
No conflict of interest was reported by the authors.
Figure 10. (A) The modeled structure of compound 4e predicting that com-
pound 4e will form a covalent crosslink to DNA using the anthracene ring while
the rest of the compound is in the minor groove like the crystal structure of DOX
(pdb:1D12); (B) 3 D structure of compound 4e.
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A novel series of pyrimidine pyrazoline-anthracene hybrids 4a-t
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