Synthesis and antitumor activity of pyrido [2,3-d]pyrimidine and pyrido[2,3-d] [1,2,4]triazolo[4,3-a]pyrimidine derivatives that induce apoptosis through G1 cell-cycle arrest

Article abstract of DOI:10.1016/j.ejmech.2014.06.027

New series of 2-(2-arylidenehydrazinyl)pyrido[2,3-d]pyrimidines 5a-e and pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidines 6-15 were synthesized and evaluated for their cytotoxic activity against two cancer cell lines, namely PC-3 prostate cancer and A-549 lung cancer. Some of the tested compounds displayed high growth inhibitory activity against PC-3 cells. Whereas, compounds 5b and 15f showed relatively potent antitumor activity against PC-3 and A-549 cell lines. In particular, 4-(3-acetyl-5-oxo-6-phenyl-8-(thiophen-2-yl)pyrido[2, 3-d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide 15f exhibited superior antitumor activity against both cell lines at submicromolar level (IC₅₀ = 0.36, 0.41 μM, respectively). Moreover, the potential mechanisms of the cytotoxic activity of the promising compound 15f on the more sensitive cell line PC-3 were studied. The data indicated that 15f was able to cause cell cycle arrest at least partly through enhancing the expression level of the cell cycle inhibitor p21 and induced cancer cell apoptosis via caspase-3 dependent pathway.

Full text of DOI:10.1016/j.ejmech.2014.06.027

Accepted Manuscript
Synthesis and antitumor activity of pyrido [2,3-d]pyrimidine and pyrido[2,3-d]
[1,2,4]triazolo[4,3-a]pyrimidine derivatives that induce apoptosis through G cell-cycle
1
arrest
Mohamed Fares, Sahar Mahmoud Abou-Seri, Hatem A. Abdel-Aziz, Safinaz E-S.
Abbas, Mohieldin Magdy Youssef, Radwa Ahmed Eladwy
PII:
S0223-5234(14)00546-7
10.1016/j.ejmech.2014.06.027
EJMECH 7070
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 December 2013
Revised Date: 11 June 2014
Accepted Date: 12 June 2014
Please cite this article as: M. Fares, S.M. Abou-Seri, H.A. Abdel-Aziz, S.E.-S Abbas, M.M.
Youssef, R.A. Eladwy, Synthesis and antitumor activity of pyrido [2,3-d]pyrimidine and pyrido[2,3-d]
[1,2,4]triazolo[4,3-a]pyrimidine derivatives that induce apoptosis through G cell-cycle arrest, European
1
Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.027.
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ACCEPTED MANUSCRIPT
Graphical abstract
Synthesis and antitumor activity of pyrido[2,3-d]pyrimidine and pyrido[2,3-d]
[1,2,4]triazolo[4,3-a]pyrimidine derivatives that induce apoptosis through G1 cell-cycle
arrest
Mohamed Fares, Sahar Mahmoud Abou-Seri, Hatem A. Abdel-Aziz, Safinaz E-S Abbas, Mohi
Magdy Yosseff, Radwa Ahmed Eladwy
New series of pyrido[2,3-d]pyrimidine and pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine
derivatives were synthesized as antitumor agents. The most potent compound 15f caused cell
cycle arrest and induced apoptosis in PC-3 cancer cell line.

ACCEPTED MANUSCRIPT
Highlights
ꢀ New pyridopyrimidine and pyridotriazolopyrimidine derivatives were synthesized.
ꢀ The new compounds displayed significant antitumor activity against PC-3 cancer cells.
ꢀ The most potent compound 15f was able to cause G1 cell cycle arrest at PC-3 cells.
ꢀ Also, 15f demonstrated activity as caspase-3 activator and apoptosis inducer.

ACCEPTED MANUSCRIPT
Synthesis and antitumor activity of pyrido [2,3-d]pyrimidine and pyrido[2,3-d]
[1,2,4]triazolo[4,3-a]pyrimidine derivatives that induce apoptosis through G1 cell-cycle
arrest
Mohamed Faresa, Sahar Mahmoud Abou-Serib,*, Hatem A. Abdel-Azizc,d,*, Safinaz E-S Abbasb,
Mohieldin Magdy Youssefe,f, Radwa Ahmed Eladwye
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, Egyptian Russian University,
Badr City, Cairo, P.O. Box 11829, Egypt.
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini
Street, Cairo, P.O. Box 11562, Egypt.
cDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box
2457, Riyadh 11451, Saudi Arabia.
dDepartment of Applied Organic Chemistry, National Research Center, Dokki, Giza, P.O. Box
12622, Egypt.
eDepartment of Pharmacology and Toxicology, College of Pharmacy, Egyptian Russian
University, Badr City, Cairo, P.O. Box 11829, Egypt.
fDepartment of Biology, School of Science and Engineering (SSE), American University in Cairo,
New Cairo, P.O. Box 11835, Egypt.
* Corresponding author: Sahar Mahmoud Abou-Seri; Associate Professor of Pharmaceutical
Chemistry, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University,
Kasr El-Aini street, Cairo, P.O. Box, 11562, Egypt.
Tel.: +2-02-33144107, +2-1227059742; fax: +2-02-2362 8426.
E-mail address: saharshaarawy_69@yahoo.com
* Corresponding author: Hatem A. Abdel Aziz; Associate Professor, Department of
Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box. 2457 - Riyadh
11451, Saudi Arabia.
Tel: -+966-146-77341; Fax : -+966-146-76220.
E-mail address: hatem_741@yahoo.com
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Abstract
New series of 2-(2-arylidenehydrazinyl)pyrido[2,3-d]pyrimidines 5a-e and pyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidines 6-15 were synthesized and evaluated for their cytotoxic
activity against two cancer cell lines, namely PC-3 prostate cancer and A-549 lung cancer. Some
of the tested compounds displayed high growth inhibitory activity against PC-3 cells. Whereas,
compounds 5b and 15f showed relatively potent antitumor activity against PC-3 and A-549 cell
lines. In particular, 4-(3-acetyl-5-oxo-6-phenyl-8-(thiophen-2-yl)pyrido[2,3-d][1,2,4]triazolo[4,3-
a]pyrimidin-1(5H)-yl)benzenesulfonamide 15f exhibited superior antitumor activity against both
cell lines at submicromolar level (IC50 = 0.36, 0.41 µM, respectively). Moreover, the potential
mechanisms of the cytotoxic activity of the promising compound 15f on the more sensitive cell
line PC-3 were studied. The data indicated that 15f was able to cause cell cycle arrest at least
partly through enhancing the expression level of the cell cycle inhibitor p21 and induced cancer
cell apoptosis via caspase-3 dependent pathway.
Key words: Pyrido[2,3-d]pyrimidine; pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine; antitumor
activity; apoptosis; cell cycle arrest.
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1. Introduction:
Regardless of the immense advances in the field of basic and clinical research related to cancer
therapy which have resulted in higher cure rates for a number of malignancies, cancer remains the
second leading cause of death in developing as well as developed countries [1]. Chemotherapy is
still one of the primary modalities for the treatment of cancer. However, the use of available
chemotherapeutics is often limited mainly due to toxicities and drug-resistance [2]. This clearly
underlies the urgent need of developing novel chemotherapeutic agents with safe potent antitumor
activities.
Apoptosis or programmed cell death is a normal process that ensures equilibrium between cell
proliferation and cell death and plays a regulatory role in controlling the size of cell populations as
well as in tissues homeostasis [3]. Inadequate or abnormal inhibition of apoptosis leads to
unchecked cell proliferation resulting in cell accumulation and is considered as a hallmark of
cancer [4]. It has been reported that, drugs which restore the normal apoptotic pathway have the
potential for effectively treating cancer that depend on aberration of apoptotic pathway to stay
alive. This has encouraged a change in anticancer therapy trends, from classical cytotoxic
strategies to the development of new non-harmful therapies which target apoptosis [5]. This
process allows for the selective apoptotic destruction of oncogenic cells without causing vicinal
inflammation in normal body tissues [6]. In addition, by inducing apoptosis, these new agents may
overcome tumor resistance to conventional anticancer agents [7]. Therefore, the identification of
apoptosis inducers has become an attractive approach for the discovery and development of
potential anticancer agents.
Among the wide range of compounds tested as potential anticancer agents, pyrido[2,3-
d]pyrimidines were reported to exhibit antitumor activity which may be attributed to inhibition of
cyclin dependent kinase [8, 9], check point kinase [10] or mammalian target of rapamycin [11].
Moreover, several derivatives having pyrido[2,3-d]pyrimidine core were found to induce
apoptosis and/or reduce cell proliferation in different solid tumors and leukemia cell lines [5, 12-
14]. For example, a series of 2,4-bis substituted pyrido[2,3-d]pyrimidine nucleosides I exhibited
dose dependent cytostatic effects against HT-29 colon cancer through activation of signaling
pathways leading to cell cycle arrest and rapid apoptosis [5]. Later, 2-(alkylsulfanyl)-N-
alkylarylpyrido[2,3-d]pyrimidine derivatives showed good profile as caspase-3 activator and
apoptosis inducers in breast, colon and bladder cancer cells lines [12,13]. Furthermore, the novel
analog;
2-[(3-chloro-4-fluorophenyl)amino]-6-(2,6-dichlorophenyl)-8-methylpyrido[2,3-
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d]pyrimidin-7(8H)-one II potently inhibited p210Bcr-Abl tyrosine kinase and induced apoptosis of
K562 leukemic cell line [14].
On the other hand, several studies have been devoted to the antiproliferative activity of
hydrazones, where a variety of hydrazone derivatives -like the hydrazinopyrimidines III-have
been reported to inhibit the growth and/or induce apoptosis in a panel of human tumor cells
including leukemia, lung, colon and breast cancer cell lines [15-17]. Stimulated by the successful
applications of such class of compounds as apoptotic inducers, a new series of 2-
(arylidenehydrazinyl)pyrido[2,3-d]pyrimidines 5a-e was synthesized to explore the influence of
incorporating hydrazonyl moiety on the antitumor activity of pyridopyrimidines (Scheme1, Fig.
1).
Furthermore, 1,2,4-triazolo[4,3-a]pyrimidine ring system has been well acknowledged to
possess anticancer activity [18-20]. This was exemplified by a series of triazolo[4,3-a]pyrimidin-
6-sulfonamide derivatives IV that demonstrated potent inhibitory effects on the growth of a wide
range of cancer cell lines including leukemia, prostate and lung cancer at low dose levels [18].
Accordingly, it seemed of interest to synthesize some fused pyrido[2,3-d][1,2,4]triazolo[4,3-
a]pyrimidin-5-ones 6-15, hoping that the hybridization of the pharmacophoric features of the
triazolopyrimidine and pyridopyrimidine scaffolds would produce enhanced antitumor effect(Fig.
1). Surveying literatures revealed that no studies dealt with the anticancer activity of this tricyclic
ring system concerning substitution at N-1 and C-3 positions. Therefore, structural modifications
on pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine core involved monosubstition on the fused
triazole ring with 3-oxo 7 or 3-amino 9 functionality as well as their isosteres 3-thioxo 8 and 3-
methyl 10 derivatives (Scheme 2), in addition to 1,3-disubstitution as 3-acetyl-1-un/substituted
phenyl 15a-e and their ethyl carboxylate analogs 15g-j (Scheme 3). The proposed structural
modifications were aimed at gaining insight into the influence of some parameters like electronic
nature, lipophilicity and steric effect on cytotoxic activity.
Herein, we report the synthesis, cytotoxic activity and structure activity relationship of new
series
of
2-(2-arylidenehydrazinyl)pyrido[2,3-d]pyrimidines
5a-e
and
pyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidines 6-15. In addition, the most potent compound 15f was selected
to investigate its mechanism of action. Results showed that, it was able to cause cell cycle arrest
and induced cancer cell apoptosis in PC-3 cell line via caspase-3 dependent pathway.
Figure 1
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2. Results and discussion :
2.1 Chemistry
The reaction between heterocyclic amines and aromatic α-β unsaturated ketones is a very
convenient and versatile method for the fusion of the pyridine ring to the preexisting heterocycle
[21]. The starting compound, 5-phenyl-7-(thiophen-2-yl)-2-thioxo-2,3-dihydropyrido[2,3-
d]pyrimidin-4(1H)-one 3 was prepared via reacting 6-amino-2-thiouracil 2 and 3-phenyl-1-
(thiophen-2-yl)prop-2-en-1-one 1 [22] in DMF according to the procedure reported by Quiroga et
al [23]. Reacting 2-thioxopyridopyrimidine 3 with hydrazine hydrate in absolute ethanol afforded
2-hydrazinylpyrido[2,3-d]pyrimidin-4(3H)-one 4. Condensation of the latter with appropriate
aldehyde furnished the required 2-(2-arylidenehydrazinyl)-5-phenyl-7-(thiophen-2-yl)pyrido[2,3-
d]pyrimidin-4(3H)-ones 5a-e (Scheme 1).
Scheme 1
2-Hydrazinylpyrido[2,3-d]pyrimidin-4(3H)-one 4 is considered the key intermediate for the
synthesis of a variety of 3-substituted pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidines 6-10.
Reacting the 2-hydrazinyl derivative 4 with triethyl orthoformate resulted in the formation of 6-
phenyl-8-(thiophen-2-yl)pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one
6.
While,
cyclocondensation of the 2-hydrazinyl derivative 4 with ethyl chloroformate in dry pyridine or
carbon disulphide in ethanolic KOH solution produced 3-oxo(thioxo)pyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidines 7 and 8, respectively. Alternatively, the 3-amino derivative 9
was obtained by refluxing the 2-hydrazinyl derivative 4 with ammonium isothiocyanate in acetic
acid. Meanwhile, the preparation of the 3-methyl derivative 10 was achieved via the reaction of
the 2-hydrazinyl derivative 4 with acetyl chloride in dry pyridine (Scheme 2).
Scheme 2
Reaction of 2-thioxopyridopyrimidine 3 with hydrazonoyl chlorides 11a-j in dioxane in the
presence of triethylamine furnished one isolable product. As depicted in Scheme 3, the reaction
proceeded through S-alkylation to give S-alkylated products 12 followed by Smiles rearrangement
to afford intermediates 13 which were consumed in situ via elimination of hydrogen sulfide gas to
give one of the isomeric fused triazole derivatives 15A or 15B. Both spectroscopic data (IR, 1H
NMR and Ms) and elemental analyses were consistent with either structure. The IR spectra of
15a-f exhibited characteristic absorption band at1710-1728 cm-1 due to acetyl C=O, while that of
the ethyl carboxylate functionality in 15g-j was observed at 1737-1755 cm-1. 1H NMR spectra of
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compounds 15a-f displayed singlet signal resonating at
δ
2.43-2.89 ppm representing acetyl CH3
protons. Meanwhile, 1H NMR of 15g-j showed a typical triplet-quartet pattern of the ethyl
protons at δ 1.30-1.31 and 4.42-4.44 ppm. Distinction between the two structures (15A or 15B)
was reached by comparing the 13C NMR spectra with those of similar annulated pyrimidinones.
Literature report [24] has shown that the chemical shift for the carbonyl carbon in pyrimidin-4-one
derivatives is markedly affected by the nature of the adjacent nitrogen (pyrrole type as in 15A or
pyridine type as in 15B). For example, 13C NMR spectral data of compounds 15b and 15f revealed
carbonyl carbon signals of the pyrimidinone at 162.44 and 162.62 ppm, respectively, suggesting
that N-4 near to C=O is sp3-hybridized (pyrrole type) which is different from C=O adjacent to a
sp2-hybridized nitrogen (pyridine type) that usually appears at 170-175 ppm [24]. Accordingly, the
isolated products 15a-j existed in one form namely, A rather than B. This result is in agreement
with other reported cyclocondensation reactions of hydrazonoyl chloride with similar condensed
2-thioxopyridopyrimidine derivatives [25-27].
Furthermore, single-crystal X-ray analysis of compound 15g gave an absolute confirmation for
the structure of 15a-j, in addition to a unique view for this system (Fig. 2). The X-ray analysis of
compound 15g showed the planarity of the tricyclic fused system as approximately planar system.
It also revealed the presence of thiophene and phenyl of the 1,2,4-triazole in the same plane of the
fused tricyclic system, while the phenyl ring on the pyridine is almost perpendicular to the main
plane of the fused system. The X-ray analysis of compound 15g displayed the binding resonance
of ester function which is common in X-ray measurements at room temperature.
Scheme 3
Figure 2
2.2 Biological activity
2.2.1 In vitro cytotoxic activity.
Based on the reported cytotoxic activity of a large array of bioactive cores incorporating
sulfonamide moiety, compound 15f was selected to carry out a preliminary screening for its
cytotoxic effects against four metastatic human cancer cell lines, including MCF-7 breast cancer,
HepG2 liver cancer, A-549 lung cancer and PC-3 prostate cancer. The selection of such cell lines
was inspired by the declared anticancer activity of a number of hydrazones, triazolo[4,3-
a]pyrimidines and pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine derivatives against the mentioned
cell lines [15-18]. The cytotoxic activity was evaluated using the Sulfo-rhodamine B (SRB)
colorimetric assay [28].The results revealed potent growth inhibitory activity against A-459 and
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PC-3 cell lines and fair activity against MCF-7 and HepG2 cancer cells (Table 1).Therefore, the
cytotoxic activity of all the newly synthesized compounds was evaluated against the two sensitive
cell lines, namely PC-3 and A-549. The conventional anticancer drug in clinical use, 5-
fluorouracil was used as positive control. 5-FU is a pharmacologically nontoxic compound, which
has been widely used in chemotherapy for a wide range of metastatic tumors including androgen-
independent or hormone-refractory prostate cancer [29,30]. The cytotoxic activities are expressed
as the median growth inhibitory concentration (IC50) and are provided in Table 2. From the
results, it is evident that some of the tested compounds displayed significant growth inhibitory
activity. Compounds 5b, 5d and 15f (IC50 = 1.54, 0.63 and 0.36 µM, respectively) were found to
be more potent and efficacious than 5-FU (IC50 = 12.00 µM) against PC-3 cell line. Moreover,
compounds 6, 7 and 9 were almost equipotent to the reference drug against the same cell line. In
addition, compound 15f was about 10 folds more potent than 5-FU against A-549 cell line (IC50
=0.41, 4.21 µM, respectively), while compound 5b (IC50 = 3.36 µM) exhibited slightly higher
cytotoxic effect than that expressed by 5-FU.
Also, it was observed that PC-3 cell line was more susceptible to the influence of most of the
tested compounds than A-549 cell line. With the exception of the sulfonamido derivative 15f and
4-fluorobenzylidene derivative 5b, the new compounds displayed poor antitumor activity against
A-549 cell line, especially in comparison with 5-FU. Accordingly, the SAR of the target
compounds will be discussed in relation to their activity toward PC-3 cell line. Analysis of the
data in Table 2 showed that, 2-(2-arylidenehydrazinyl)pyridopyrimidines 5a-e exhibited potent to
moderate potency (IC50 = 0.63 - 26.80 µM). The highest growth inhibitory effect was associated
with 4-methylbenzylidene 5d and 4-fluorobenzylidene 5b congeners (IC50 =0.63, 1.54 µM,
respectively), which displayed excellent activity relative to 5-FU (IC50 = 12.00 µM). Generally,
the order of antitumor activity was found to be 4-methylbenzylidene 5d > 4-fluorobenzylidene 5b
> 4-methoxybenzylidene 5e > 4-chlorobenzylidene 5c > unsubstituted benzylidene 5a, indicating
that substitution at the 4-position of benzylidene moiety with small electron donating (CH3) or
electron withdrawing (F) group of considerable lipophilicity greatly enhanced the activity (c.f. 5a,
5b and 5d). Conversely, substitution with the more bulky chloro or methoxy substituent produced
compounds with reduced cytotoxic activity, suggesting that the steric effect rather than the
electronic nature of substituent may be the main factor affecting the potency of these compounds.
The pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5-ones could be classified according to the
substitution on the triazole ring into: 3-un/substituted 1,2,4-triazolo derivatives 6-10 and 1,3-
disubstituted ones 15a-j. Examination of the data concerning the 3-un/substituted derivatives 6–10
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revealed that their anticancer activities were influenced by the C-3 substituent on the 1,2,4-triazole
ring. The unsubstituted derivative 6 had potent growth inhibitory activity (IC50 = 11.71 µM) that is
nearly equivalent to 5-FU (IC50 = 12.00 µM). Meanwhile, the 3-oxo derivative 7 and the 3-amino
substituted counterpart 10 elicited similar cytotoxic activities (IC50 = 12.66 and 12.29 µM,
respectively), which were however, slightly lower than the parent compound 6, suggesting that
substitution with a small hydrophilic group had little effect on potency. On the other hand,
substitution with lipophilic group like the 3-thioxo derivative 8 and the 3-methylated analog 10
resulted in partial or complete loss of activity (IC50 = 32.39 and 83.88 µM, respectively).
Considering 1,3-disubstituted triazolo derivatives 15a-j, the substituent on the N-1 phenyl ring
appeared to be determinal for activity of 3-acetyl-1-un/substituted phenyl derivatives 15a-f which
exhibited a wide range of cytotoxic activity (IC50 = 0.36 - 35.95 µM). Compound 15a having
unsubstituted phenyl demonstrated fair cytotoxic effect (IC50 = 32.33 µM). Substitution on the
phenyl ring with 4-fluoro resulted in compound 15b with diminished activity (IC50 = 35.95 µM).
Conversely, grafting 4-chloro, 4-methyl or 4-methoxy group to the phenyl ring contributed to an
increase in potency (IC50 = 22.90, 16.92, 19.47µM, respectively). Moreover, the introduction of 4-
sulfonamido functionality afforded the most potent analog 15f with superior antitumor activity
(IC50 = 0.36 µM), highlighting the importance of sulfonamido substituent as a potential antitumor
pharmacophore that is reported to play a vital role in the proper binding of several antitumor
agents to their biotargets [31-33]. Finally, replacement of the 3-acetyl moiety in 15a, 15c, 15d and
15f with 3-ethyl carboxylate produced compounds 15g-j with reduced cytotoxic efficacy, probably
due to the low stability of ester function [34]. The best growth inhibitory activity among the 3-
ethyl carboxylate derivatives 15g-j was observed with the benzenesulfonamide derivative 15j
(IC50 = 7.15 µM).
2.2.2 Morphological investigation:
Chromatin condensation and fragmented nuclei are known as the classic characteristics of
apoptosis [35]. Therefore, to determine whether the observed cell death induced by the most
potent anti-proliferative agent 15f was due to apoptosis or necrosis, PC-3 treated cells were
examined using acridine orange (AO) and ethidium bromide (EB) double staining under
fluorescence microscopy after 24h and 48h of treatment [36].
AO permeates into living cells, emitting green fluorescence after intercalation into DNA. EB is
only taken up by cells when cytoplasmic membrane integrity is lost, and stains the nucleus red.
Thus live cells have normal green nuclei; early apoptotic cells have bright green nuclei and display
condensed or fragmented chromatin; late apoptotic cells have orange stained nuclei with
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condensed and fragmented chromatin. Cells that have died from direct necrosis have a structurally
normal red nucleus [37].
As shown in Figure 3, it was found that the untreated control cells were morphologically
normal, mostly green with intact nuclei (Fig. 3A). On the other hand, cells treated with 15f at its
IC50 displayed marked morphological changes. After 24h of treatment, the cells were wrinkled,
and the chromatin was condensed; some proportion of cells took only acridine orange and stained
bright green with fragmented chromatin showing early apoptosis (Fig. 3B). While, after 48h it was
observed that yellow to orange fluorescence has been enhanced in some cells which indicated
latter stage of apoptosis (Fig. 3C).
Figure 3
2.2.3 Caspase-3 activity (Key executor of apoptosis):
It is well known that caspases, a family of proteolytic enzymes plays a pivotal role in the
apoptotic process. Activation of these proteases -which are normally present inside cells as
inactive zymogens- results in the cleavage of many protein substrates within the cell leading to
irreversible apoptotic cell death. Among these caspases, caspase-3 is one of the most important
downstream caspases and is called an effector caspase [38]. Therefore, the activation of caspase-3
in PC-3 cells treated with compound 15f was investigated. The level of active caspase-3 was
measured in ng/g protein using colorimetric assay that apply sandwich enzyme immunoassay
technique. The assay uses monoclonal antibody and biotin conjugated antibodies, both of which
are specific to caspase-3. As shown in Figure 4, treatment of PC-3 cells with 15f for 24 h and 48h
caused a significant increase in caspase-3 level by about 1.5 and 2 folds respectively, compared to
control. These results supported the observation of chromatin condensation and DNA
fragmentation during morphological investigations and suggested that 15f induced apoptosis
through activation of caspase-3.
Figure 4
2.2.4. Cell-cycle analysis:
Cell cycle is the series of events that take place in a cell leading to its division and duplication
(replication). The cell cycle consists of four distinct phases: G1 phase, S phase (synthesis), G2
phase (collectively known as interphase) and M phase (mitosis). During G1, preparation of energy
and material for DNA replication occurs. The S phase is the stage when DNA replicates. During
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G2, the new DNA is checked and any error is usually repaired. The M stage is “mitosis” when
nuclear and cytoplasmic division occur [39].
The apoptosis inducing activity of 15f was also characterized by flow cytometric analysis of the
DNA profile in PC-3 cells. Figure 5 showed that exposure of PC-3 cells to 15f (0.36 µM) for 24 h
induced a significant cell cycle arrest at G0/G1 phase with concurrent reduction in the percentage
of cells at S and G2/M phases compared to control. Meanwhile, exposure of PC-3 cells to 15f for
48 h resulted in significant increase in the percentage of cells at the pre-G phase (cells with
subdiploid DNA), a marker of apoptotic cells. These results were consistent with morphological
observations and caspase-3 activation assay. The data also indicated that compound 15f arrested
cells in G1 phase, with subsequent induction of apoptosis.
Figure 5
2.2.5 CDK4/Cyclin D1 and CDK6/Cyclin D1profiling:
Cyclin-dependent kinases (CDKs) are a family of protein kinases that is involved in regulating
the cell cycle. They control the cell cycle progression from one phase to the next. Activation of
CDKs is achieved via complexation with regulatory proteins called cyclins. Different types of
cyclins and CDKs play their roles at various stages of the cell cycle. For instance, in the G1 phase,
CDK4 and CDK6 are activated upon binding with cyclin D1 leading to phosphorylation of the
tumor suppressor protein retinoblastoma (pRb)[40]. Phosphorylation of Rb early in the G1 phase
indicates changes in gene transcription that carry cells through G1/S transition and to DNA
replication. Therefore, CDK4 or CDK6 inhibitors will inhibit Rb phosphorylation and prevent
tumor cell from entering the S phase causing cell cycle arrest at G1 phase resulting in suppression
of DNA replication and decrease tumor cell proliferation [41].
To verify if the G1 phase arrest caused by compound 15f is mediated through CDK
inhibition, the kinase inhibitory effect of 15f was evaluated against CDK4 and CDK6 at
concentrations of 1, 10 and 100 ꢀM using radioisotope assay [42]. The broad spectrum CDK
inhibitor staurosporine was used as a positive control. The profiling data in Table 3 showed that
15f had weak inhibition at the highest tested concentration of the compound against both enzymes.
At 100 ꢀM, the CDK4/cyclin D1 and CDK6/cyclin D1 activities were inhibited by 21% and 17%
respectively compared to control. On the other hand, staurosporine showed potent inhibition of
both enzymes at 1 ꢀM concentration, the CDK4/cyclin D1 and CDK6/cyclin D1 activities were
inhibited by 93% and 90% respectively compared to control.
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2.2.6. Expression levels of cyclin-dependent kinase inhibitor proteins p21 and p27
Cyclin-dependent kinase inhibitors p21and p27 are proteins that bind to and inhibit the
activity of CDK2/cyclin E, CDK4/cyclin D1 and/or CDK6/cyclin D1 complexes, and thus control
the cell cycle progression at G1phase [43]. The up regulated expression of these proteins is
reported to mediate cell cycle G1 phase arrest in response to a variety of stress stimuli [44].
Therefore, the expression of p21 and p27 were examined in PC-3 cells treated with 15f at IC50
(0.36 µM) by immunocytochemistry staining. Exposure to compound 15f resulted in an elevation
in the p21 positively stained PC-3 cells compared to control group (Fig. 6A). However, treatment
with 15f did not induce changes in the p27 expression in PC-3 cells (Fig. 6B). These results were
further supported by Western blot analysis. The obtained data revealed that the protein level of
p21 in PC-3 treated cells was increased by about 1.7 folds compared to control (Fig. 6C).
In summary, the sulfonamido derivative 15f was able to cause G1cell cycle arrest through
enhancing the expression of cyclin-dependent kinase inhibitors p21 not by direct inhibition of
CDK /cyclin activity.
Figure 6
3. Conclusion:
New series of 2-(2-arylidenehydrazinyl)pyrido[2,3-d]pyrimidines 5a-e and pyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidines 6-15 were synthesized and evaluated for their cytotoxic
activity against two cancer cell lines, namely PC-3 prostate cancer and A-549 lung cancer. The
results revealed that compounds 5d, 6, 7 and 9 displayed high growth inhibitory activity against
PC-3 cells. Whereas, compounds 5b and 15f showed relatively potent antitumor activity against
both PC-3 and A-549 cell lines. In particular, 4-(3-acetyl-5-oxo-6-phenyl-8-(thiophen-2-
yl)pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-1(5H)-yl)benzenesulfonamide
15f
exhibited
superior antitumor activity against both cell lines at submicromolar level (IC50 = 0.36, 0.41 µM,
respectively). Moreover, the potential mechanisms of the cytotoxic activity of the promising
compound 15f on the more sensitive cell line PC-3 were investigated. The data indicated that 15f
was able to cause cell cycle arrest at least partly through boosting the expression levels of the cell
cycle inhibitor p21 as shown by immune-staining and western blotting. Also, 15f exhibited pro-
apoptotic activity as evidenced by its ability to induce nuclear fragmentation, in addition to
augmenting caspase-3 activation. Hence, it could be considered as good lead-candidate for further
optimization of new potent antitumor agents.
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4. Experimental:
4.1. Chemistry
4.1.1. General
Melting points were measured with a Gallenkamp apparatus and were uncorrected. IR spectra
were recorded on Shimadzu FT-IR 8101 PC infrared spectrophotometer. The NMR spectra were
recorded on a Bruker spectrophotometer at 300 or 400 MHz. 1H NMR spectra were run at 300 or
13
400 MHz and C NMR spectra were run at 75 or 100 MHz in deuterated dimethylsulphoxide
(DMSO-d6). Chemical shifts (δH) are reported relative to TMS as internal standard. All coupling
constant (J) values are given in hertz. Chemical shifts (δC) are reported relative to DMSO-d6 as
internal standards. The abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. Mass
spectra were measured on a GCMS-QP1000 EX spectrometer at 70 e.V. Elemental analyses was
carried out at the Regional Center for Microbiology and Biotechnology, Al-Azhar University,
Cairo, Egypt and the results were within ±0.4%. Analytical thin layer chromatography (TLC) on
silica gel plates containing UV indicator was routinely employed to follow the course of reactions
and to check the purity of the products. All reagents and solvents were purified and dried by
standard techniques.
4.1.2. 5-Phenyl-7-(thiophen-2-yl)-2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one (3). A
solution of 3-phenyl-1-(thiophen-2-yl) prop-2-en-1-one 1 (2.14 g, 0.01 mol) and 6-amino-2-
thiouracil 2 (1.43 g, 0.01 mol) in dry DMF (30 mL) was refluxed for 15 h. The reaction mixture
was cooled and the solid formed was filtered, dried and crystallized from DMF. Yield: 70 %, m.p
1
>300oC; IR (KBr) ν: 3402 (NH), 1705 (C=O) cm-1; H NMR (DMSO-d6, 300 MHz) δ: 7.18 (m,
1H, H4 thiophene), 7.38-7.52 (m, 5H, Ar-H), 7.59 (m, 1H , H5 thiophene), 7.92 (s, 1H, pyridine
H), 8.07 (m, 1H, H3 thiophene ), 12.29 (br.s, 1H, NH, D2O exchangeable), 13.05 (br.s, 1H, NH ,
D2O exchangeable); MS m/z [%]: 339 [(M+2)+, 23.70], 337 [M+, 63.38], 83.05 [100]; Anal. Calcd
C17H11N3OS2: C, 60.51; H, 3.29; N, 12.45; S, 19.01. Found: C, 60.30; H, 3.26; N, 12.59; S, 19.08.
4.1.3. 2-Hydrazinyl-5-phenyl-7-(thiophen-2-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (4). A mixture
of 2-thioxopyrido[2,3-d]pyrimidine 3 (1.36 g, 4 mmol) and hydrazine hydrate (3 mL, 99 %, 60
mmol) in absolute ethanol (20 mL) was heated under reflux for 15 h. The reaction mixture was
cooled and the solid formed was filtered, dried and crystallized from DMF. Yield: 70 %; m.p 285-
290°C; IR (KBr) ν: 3317, 3336 (NH, NH2), 1681 (C=O) cm-1; 1H NMR (DMSO-d6, 300 MHz) δ
2.49 (br.s, 2H, NHNH2, D2O exchangeable), 7.16 (m, 1H, H4 thiophene), 7.38-7.42 (m, 5H, Ar-
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H), 7.59 (s, 1H, pyridine H), 7.62 (m, 1H, H5 thiophene), 7.96 (m, 1H, H3 thiophene), 8.25 (br.s,
1H, NHNH2, D2O exchangeable), 12.15 (br s, 1H, NH pyrimidine, D2O exchangeable); MS m/z
[%]: 337 [(M+2)+, 83.58], 335 [M+, 14.08], 304 [100]. Anal. Calcd for C17H13N5OS: C, 60.88; H,
3.91; N, 20.88. Found: C, 60.92; H, 3.97; N, 21.04.
4.1.4. 2-(2-Arylidenehydrazinyl)-5-phenyl-7-(thiophen-2-yl)pyrido[2,3-d]pyrimidin-4(3H)-ones
5a-e. A mixture of 2-hydrazinylpyrido[2,3-d]pyrimidine 4 (0.34 g, 1 mmol) and the appropriate
aldehyde (benzaldehyde, 4-fluorobenzaldehyde, 4-chlorobenzaldehyde, 4-tolylaldehyde or 4-
anisaldehyde) (2 mmol) in glacial acetic acid (15 mL) was heated under reflux for 4 h. The
reaction mixture was cooled and the solid formed was filtered, dried and crystallized from
DMF/Ethanol [v:v, 1:1]
4.1.4.1. 2-(2-Benzylidenehydrazinyl)-5-phenyl-7-(thiophen-2-yl)pyrido[2,3-d]pyrimidin-4(3H)-one
(5a). Yield: 92 %; m.p 293- 295°C; IR (KBr) ν: 3365 (NH), 1647 (C=O) cm-1; 1H NMR (DMSO-
d6, 300 MHz) δ: 7.21 (m, 1H, H4 thiophene), 7.40-7.45 (m ,9H, 8 Ar-H + pyridine H), 7.77 (m, 1H,
H5 thiophene), 7.95-8.03 (m, 3H, 2 Ar-H+ H3 thiophene, 8.12 (s, 1H, -N=CH), 11.29 (s, 1H, -NH-
N=, D2O exchangeable), 11.89 (s, 1H, NH pyrimidine, D2O exchangeable); MS m/z [%]: 425
[(M+2)+, 7.84], 423 [M+, 92.08], 346 [100]. Anal. Calcd for C24H17N5OS: C, 68.07; H, 4.05; N,
16.54. Found: C, 68.14; H, 4.11; N, 16.73.
4.1.4.2. 2-(2-(4-Fluorobenzylidene)hydrazinyl)-5-phenyl-7-(thiophen-2-yl)pyrido[2,3-d]pyrimidin-
4(3H)-one (5b): Yield: 88%; m.p >300°C; IR (KBr) ν: 3381 (NH), 1645 (C=O) cm-1; 1H NMR
(DMSO-d6, 300 MHz) δ: 7.18-7.28 (m, 3H, H4 thiophene, 2 Ar-H), 7.42-7.45 (m, 6H, 5 Ar-H +
pyridine H), 7.77 (m, 1H , H5 thiophene), 8.03-8.1 ( m, 3H, 2 Ar-H + H3 thiophene), 8.12 (s, 1H, -
N=CH), 11.37 (s, 1H, -NH-N=, D2O exchangeable), 11.87 (s, 1H, NH pyrimidine, D2O
exchangeable); 13C NMR (DMSO-d6, 100 MHz) δ: 115.82, 116.04, 116.26, 127.82, 128.16,
128.36, 128.94, 129.19, 130.22, 130.30, 131.18, 131.59, 140.03, 144.29, 151.65, 153.75, 161.22,
162.18, 164.64, 172.50. MS m/z [%]: 443 [(M+2)+, 7.84], 441 [M+, 92.08], 346 [100]. Anal. Calcd
for C24H16FN5OS: C, 65.29; H, 3.65; N, 15.86. Found: C, 65.32; H, 3.71; N: 16.02.
4.1.4.3. (2-(4-Chlorobenzylidene)hydrazinyl)-5-phenyl-7-(thiophen-2-yl)pyrido[2,3-d]pyrimidin-
4(3H)-one (5c). Yield: 90%; m.p >300°C; IR (KBr) ν: 3381 (NH), 1658 (C=O) cm-1; 1H NMR
(DMSO-d6, 300 MHz) δ: 7.19 (m, 1H , H4 thiophene), 7.42-7.51 (m, 8H, 7 Ar-H + pyridine H),
7.72 (m, 1H, H5 thiophene), 7.89-7.92 (m, 3H, 2 Ar-H+ H3 thiophene), 8.08 (s, 1H, -N=CH),
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11.40 (s, 1H, -NH-N=, D2O exchangeable), 11.85 (s, 1H, NH pyrimidine, D2O exchangeable); MS
m/z [%]: 459 [(M+2)+, 24 ], 457 [M+, 72.45], 171 [100]. Anal. Calcd for C24H16ClN5OS: C, 62.95;
H, 3.52; N, 15.29. Found: C, 62.97; H, 3.57; N: 15.36.
4.1.4.4. 2-(2-(4-Methylbenzylidene)hydrazinyl)-5-phenyl-7-(thiophen-2-yl)pyrido[2,3-d]pyrimidin-
4(3H)-one (5d). Yield: 90%; m.p > 300°C; IR (KBr) ν: 3427 (NH), 1728 (C=O) cm-1; 1H NMR
(DMSO-d6, 300 MHz) δ: 2.34 (s ,3H ,CH3), 7.18 -7.24 (m, 3H, H4 thiophene + 2Ar-H ), 7.43-7.49
(m, 6H, 5 Ar-H + pyridine H), 7.76 ( m, 1H, H5 thiophene), 7.84 (d, J = 7.8 Hz, 2H, Ar-H), 8.01
(m, 1H, H3 thiophene), 8.08 (s, 1H, -N=CH), 11.21 (s, 1H, -NH-N=, D2O exchangeable), 11.76(s,
1H, NH pyrimidine, D2O exchangeable); MS m/z [%]: 439 [(M+2)+, 8.72], 437 [M+, 100]. Anal.
Calcd for C25H19N5OS: C, 68.63; H, 4.38; N, 16.01. Found: C, 68.67; H, 4.41; N: 16.14.
4.1.4.5.
2-(2-(4-Methoxybenzylidene)hydrazinyl)-5-phenyl-7-(thiophen-2-yl)pyrido[2,3-d]
pyrimidin-4(3H)-one (5e). Yield: 89%; m.p 298-300°C; IR (KBr) ν 3385 (NH), 1676 (C=O) cm-1;
1H NMR (DMSO-d6, 300 MHz) δ: 3.82 (s, 3H, OCH3), 6.98 (d, J = 9 Hz, 2H, Ar-H ),7.20 (m, 1H,
H4 thiophene), 7.42 (m, 6H, 5 Ar-H + pyridine H ), 7.77 (m , 1H , H5 thiophene), 7.88 (d, J = 9
Hz, 2H , Ar-H), 8.01 (m, 1H, H3 thiophene), 8.09 (s, 1H, -N=CH), 11.16 (s, 1H, -NH-N=, D2O
exchangeable), 11.85 (s, 1H, NH pyrimidine, D2O exchangeable). MS m/z [%]: 455 [(M+2)+, 30],
453.1 [M+, 100]. Anal. Calcd for C25H19N5O2S: C, 66.21; H, 4.22; N, 15.44. Found: C, 66.24; H,
4.27; N: 15.58.
4.1.5. 6-Phenyl-8-(thiophen-2-yl)pyrido[2,3-d] [1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one (6). A
mixture of 2-hydrazinylpyrido[2,3-d]pyrimidine 4 (0.34 g, 1 mmol) and triethyl orthoformate (8
mL) was heated under reflux for 3 h. The formed solid was filtered, dried and crystallized from
DMF:EtOH mixture [v:v, 1:1]. Yield: 72%; m.p >300°C; IR (KBr) ν: 3415 (NH), 1693 (C=O) cm-
1
1; H NMR (DMSO-d6, 300 MHz) δ: 7.26 (m, 1H , H4 thiophene), 7.44 (m, 5H, 5 Ar-H), 7.87 (s,
1H, pyridine H), 7.91 (m, 1H, H5 thiophene), 8.22 (m , 1H , H3 thiophene ), 9.20 (s, 1H, H3
triazole), 12.60 (br s, 1H, NH, D2O exchangeable); MS m/z [%]: 347 [(M+2)+, 51.89], 345 [M+,
66.04], 75 [100]. Anal. Calcd for C18H11N5OS: C, 62.60; H, 3.21; N, 20.28. Found: C, 62.63; H,
3.27; N: 20.42.
4.1.6. 6-Phenyl-8-(thiophen-2-yl)-1,2-dihydropyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidine-3,5-
dione (7). A mixture of 2-hydrazinylpyrido[2,3-d]pyrimidine 4 (0.34 g, 1 mmol) and ethyl
chloroformate (0.22 g, 2 mmol) in dry pyridine (10 mL) was heated under reflux for 9 h. The
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reaction mixture was cooled and the obtained solid was filtered, washed with ethanol, dried and
crystallized from DMF:EtOH [v:v, 1:1]. Yield: 86%, mp: > 300°C; IR (KBr) ν: 3371 (NH), 1687,
1651 (2C=O) cm-1; 1H NMR (DMSO-d6, 300 MHz) δ: 7.2 (m, 1H, H4 thiophene), 7.41 (m, 6H, 5
Ar-H + pyridine H), 7.79 (m, 1H, H5 thiophene), 8.06 (m, 1H, H3 thiophene), 12.23 (br s ,1H, NH,
D2O exchangeable), 12.96 ( br s, 1H, NH, D2O exchangeable); MS m/z [%]: 363 [(M+2)+, 9.25],
361 [M+, 36.87], 79 [100]. Anal. Calcd for C18H11N5O2S: C, 59.82; H, 3.07; N, 19.38. Found: C,
59.91; H, 3.05; N: 19.46.
4.1.7.
6-Phenyl-8-(thiophen-2-yl)-3-thioxo-2,3-dihydropyrido[2,3-d][1,2,4]triazolo[4,3-a]
pyrimidin-5(1H)-one (8). A mixture of 2-hydrazinylpyrido[2,3-d]pyrimidine 4 (0.68 g, 2 mmol),
potassium hydroxide (0.23 g, 4 mmol) and carbon disulphide (4 mL) in absolute ethanol (40 mL)
was heated under reflux for 5 h. The reaction mixture was evaporated to dryness and water (200
mL) was added then, the alkaline solution was filtered. The filtrate was acidified with conc. HCl
(10 mL) and the separated solid was filtered, dried and crystallized from DMF:EtOH [v:v, 1:1].
Yield: 62%, m.p >300°C; IR (KBr) ν: 3421 (NH), 1668 (C=O) cm-1; 1H NMR (DMSO-d6, 300
MHz) δ: 7.17 (m, 1H, H4 thiophene), 7.37 (m, 5H, Ar-H), 7.64 (s, 1H, pyridine H), 7.74 (m, 1H,
H5 thiophene), 8.04 (m, 1H, H3 thiophene), 12.45 (br s, 1H, NH, D2O exchangeable), 13.52 (br s,
1H, NH, D2O exchangeable); MS m/z [%]: 379 [(M+2)+, 12.7], 377 [M+, 100]. Anal. Calcd for
C18H11N5OS2: C, 57.28; H, 2.94; N, 18.55. Found: C, 57.34; H, 3.02; N: 18.68.
4.1.8. 3-Amino-6-phenyl-8-(thiophen-2-yl)pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-
one (9). A mixture of 2-hydrazinylpyrido[2,3-d]pyrimidine 4 (0.68 g, 2 mmol) and ammonium
thiocyanate (2.38 g, 30 mmol) in glacial acetic acid (20 mL) was heated under reflux for 10 h. The
reaction mixture was cooled, poured onto water (50 mL) and the formed solid was filtered, dried
and crystallized from acetic acid. Yield: 68% , m.p >300°C; IR (KBr) ν: 3444, 3419 (NH + NH2),
1699 (C=O) cm-1; 1H NMR (DMSO-d6, 300 MHz) δ: 6.9 (s, 2H, NH2, D2O exchangeable), 7.25
(m, 1H, H4 thiophene), 7.44 (m, 5H, Ar-H), 7.69 (s, 1H, pyridine H), 7.87 ( m, 1H, H5 thiophene),
8.16 (m, 1H, H3 thiophene), 12.48 (br s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6, 100
MHz) δ: 105.2, 108.94, 117.15, 119.88, 127.91, 128.43, 129.03, 129.61, 129.8, 132.28, 139.47,
142.95, 148.17, 153.47, 155.47, 160.18; MS m/z [%]: 362 [(M+2)+, 6.3], 360 [M+, 16.02], 80
[100]. Anal. Calcd for C18H12N5OS: C, 59.99; H, 3.36; N, 23.32. Found: C, 60.08; H, 3.43; N:
23.50.
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4.1.9.
3-
Methyl-6-phenyl-8-(thiophen-2-yl)pyrido[2,3-d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-
one (10). A Suspension of 2-hydrazinylpyrido[2,3-d]pyrimidine 4 (0.34 g, 1 mmol) and acetyl
chloride (0.1mL, 1.5 mmol) in dry pyridine (8 mL) was heated under reflux for 20 h. The reaction
mixture was cooled and the formed solid was filtered, dried and crystallized from DMF:EtOH
[v:v, 1:1]. Yield: 55%, m.p > 300°C; IR (KBr) ν: 3445 (NH), 1705 (C=O) cm-1; 1H NMR (DMSO-
d6, 300 MHz) δ: 2.95 (s, 3H, -CH3), 7.24 (m, 1H, H4 thiophene), 7.44 (m, 5H, Ar-H ), 7.73 (s, 1H,
pyridine H), 7.88 (m, 1H, H5 thiophene), 8.15 (m, 1H, H3 thiophene), 12.56 (br s, 1H, NH, D2O
exchangeable); MS m/z [%]: 361 [(M+2)+, 7.68], 359 [M+, 100]. Anal. Calcd for C19H13N5OS: C,
63.49; H, 3.65; N, 19.49. Found: C, 63.52; H, 3.71; N: 19.62.
4.1.10.
3-Substituted-1-(aryl)-6-phenyl-8-(thiophen-2-yl)pyrido[2,3-d][1,2,4]triazolo[4,3-a]
pyrimidin-5(1H)-ones 15a-i. To a mixture of 5-phenyl-7-(thiophen-2-yl)-2-thioxo-2,3-
dihydropyrido[2,3-d] pyrimidin-4(1H)-one 3 (0.34 g, 1 mmol) and the appropriate hydrazonoyl
chloride 11a-i (1 mmol) in dioxane (50 mL), triethylamine (0.14 mL, 1 mmol) was added. The
reaction mixture was refluxed for 6-10 h till the disappearance of starting materials (monitored by
TLC) and hydrogen sulfide gas ceased to liberate. The solvent was removed under vacuum and the
residue was triturated with methanol. The formed solid was filtered and crystallized from DMF:
EtOH [v;v, 1:1].
4.1.10.1. 3-Acetyl-1,6-diphenyl-8-(thiophen-2-yl)pyrido[2,3-d][1,2,4]triazolo[4,3-a] pyrimidin-
1
5(1H)-one (15a): Yield: 68%; m.p:300 °C; IR (KBr) ν: 1710, 1697 (2C=O) cm-1; H NMR
(DMSO-d6, 300 MHz) δ: 2.64 (s, 3H, COCH3), 7.23 (m, 1H, H4 thiophene), 7.46 (m, 6H, Ar-H),
7.67-7.73 (m, 3H, 2 Ar-H + pyridine H), 7.85 (m, 1H, H5 thiophene), 8.16 (m, 1H, H3
thiophene), 8.23 (d , J = 7.5 Hz, 2H, Ar-H); MS m/z [%]: 464 [(M+1)+, 0.43], 463 [M+, 2.99], 73
[100]. Anal. Calcd for C26H17N5O2S: C, 67.37; H, 3.70; N, 15.11. Found: C, 67.35; H, 3.74; N:
15.30.
4.1.10.2. 3-Acetyl-1-(4-fluorophenyl)-6-phenyl-8-(thiophen-2-yl)pyrido[2,3-d][1,2,4]triazolo [4,3-
a] pyrimidin-5(1H)-one (15b). Yield: 75%; m.p> 300°C; IR (KBr) ν: 1726, 1691 (2C=O) cm-1; 1H
NMR (DMSO-d6, 300 MHz) δ: 2.62 (s, 3H, COCH3), 7.24 (m, 1H, H4 thiophene), 7.47-7.59 (m,
7H, Ar-H), 7.73 (s, 1H, pyridine H), 7.85 (m, 1H, H5 thiophene), 8.16 (m, 1H, H3 thiophene),
13
8.21-8.24 ( m, 2H, Ar-H); C NMR (DMSO-d6, 75 MHz) δ: 29.4 (COCH3), 106.58, 116.16,
116.47, 117.03, 123.53, 127.60, 127.89, 128.19, 128.77, 128.99, 131.53, 139.22, 141.39, 143.31,
146.93, 154.26, 154.74, 156.57, 160.08, 162.44, 187.07; MS m/z [%]: 483 [(M+2)+, 5.78], 481
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[M+, 43.72], 105 [100]. Anal. Calcd for C26H16FN5O2S: C, 64.86; H, 3.35; N, 14.54. Found: C,
64.92; H, 3.37; N: 14.72%.
4.1.10.3. 3-Acetyl-1-(4-chlorophenyl)-6-phenyl-8-(thiophen-2-yl)pyrido[2,3-d][1,2,4]triazolo [4,3-
1
a]pyrimidin-5(1H)-one (15c). Yield: 71%; m.p >300°C; IR (KBr) ν: br, 1676 (2C=O) cm-1; H
NMR (DMSO-d6, 300 MHz) δ: 2.65 (s, 3H, COCH3), 7.24 (m, 1H, H4 thiophene), 7.45 (m, 5H, Ar-
H), 7.71 (s, 1H, pyridine H), 7.73 (d, J = 6.9 Hz, 2H, Ar-H), 7.85 (m, 1H, H5 thiophene), 8.13 (m,
1H , H3 thiophene), 8.27 (d, J = 6.9 Hz , 2H, Ar-H); MS m/z [%]: 499 [(M+2)+, 41.24], 497 [M+,
100]. Anal. Calcd for C26H16ClN5O2S: C, 62.71; H, 3.24; N, 14.06. Found: C, 62.76; H, 3.27; N:
14.15.
4.1.10.4.
3-Acetyl-6-phenyl-8-(thiophen-2-yl)-1-p-tolylpyrido[2,3-d][1,2,4]triazolo[4,3-
a]pyrimidin-5(1H)-one (15d). Yield: 70%; m.p > 300°C; IR (KBr) ν: 1715, 1697 (C=O) cm-1; 1H
NMR (DMSO-d6, 300 MHz) δ: 2.43 (s, 3H, CH3), 2.64 (s, 3H, COCH3), 7.24 (m, 1H, H4
thiophene), 7.47 (m, 7H, Ar-H), 7.72 (s, 1H, pyridine H), 7.86 (m, 1H, H5 thiophene), 8.07 (d, J
= 8.4 Hz, 2H, Ar-H), 8.16 (m, 1H, H3 thiophene); MS m/z [%]: 479 [(M+2)+, 10.19], 477 [M+,
100]. Anal. Calcd for C27H19N5O2S: C, 67.91; H, 4.01; N, 14.67. Found: C, 67.98; H, 3.97; N:
14.79.
4.1.10.5. 3-Acetyl-1-(4-methoxyphenyl)-6-phenyl-8-(thiophen-2-yl)pyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one (15e). Yield: 75%; m.p >300°C; IR (KBr) ν: 1728,
1695 (2C=O) cm-1; 1H NMR (DMSO-d6, 300 MHz) δ: 2.65 (s, 3H, COCH3), 3.31 (s, 3H, OCH3),
7.25 (m, 1H, H4 thiophene), 7.45 (m, 5H, Ar-H), 7.74 (s, 1H, pyridine H), 7.77 (d, J = 6.9 Hz, 2H,
Ar-H), 7.86 (m, 1H, H5 thiophene), 8.17 (m, 1H, H3 thiophene), 8.27 (d, J = 6.9 Hz, 2H , Ar-H);
MS m/z [%]: 493 [M+, 60], 66 [100]. Anal. Calcd for C27H19N5O3S: C, 65.71; H, 3.88; N, 14.19.
Found: C, 65.74; H, 3.93; N: 14.32.
4.1.10.6.
4-(3-Acetyl-5-oxo-6-phenyl-8-(thiophen-2-yl)pyrido[2,3-d][1,2,4]triazolo[4,3-a]
pyrimidin-1(5H)-yl)benzenesulfonamide (15f). Yield: 65%; m.p >300°C; IR (KBr) ν: 3402, 3358
(NH2), br, 1710 (2C=O), 1348, 1166 (SO2) cm-1; 1H NMR (DMSO-d6, 300 MHz) δ: 2.88 (s, 3H,
COCH3), 7.25 (m, 1H, H4 thiophene), 7.47-7.48 (m, 5H, Ar-H), 7.53 (br.s, 2H, NH2, D2O
exchangeable), 7.77 (s, 1H, pyridine H), 7.88 (m, 1H, H5 thiophene), 8.13 (d, J = 8.7 Hz, 2H, Ar-
H), 8.16 (m, 1H, H3 thiophene), 8.46 (d, J = 8.7 Hz, 2H, Ar-H ); 13C NMR (DMSO-d6, 75 MHz)
δ: 29.38 (COCH3), 106.78, 117.28, 120.67, 127.06, 127.69, 128.06, 128.16, 128.97, 129.08,
131.66, 138.69, 138.92, 141.65, 142.01, 142.92, 146.84, 154.32, 154.59, 156.51, 159.82, 162.62,
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187.37; MS m/z [%]: 543 [(M+1)+, 0.46], 542 [M+, 0.47], 73.05 [100]. Anal. Calcd for
C26H18N6O4S2: C, 57.55; H, 3.34; N, 15.49. Found: C, 57.54; H, 3.42; N: 15.63.
4.1.10.7. Ethyl 5-oxo-1,6-diphenyl-8-(thiophen-2-yl)-1,5-dihydropyrido[2,3-d][1,2,4]triazolo [4,3-
a]pyrimidine-3-carboxylate (15g). Yield: 70%; m.p 290-292°C; IR (KBr) ν: 1749, 1705 (2C=O)
cm-1; 1H NMR (DMSO-d6, 300 MHz) δ: 1.30 (t, J = 6.9 Hz, 3H, CH2CH3), 4.43 (q, J = 6.9 Hz, 2H,
CH2CH3), 7.24 (m, 1H, H4 thiophene), 7.46-7.52 (m, 6H, Ar-H), 7.66-7.71 (m, 2H, Ar-H), 7.73 (s,
1H, pyridine H), 7.85 (m, 1H, H5 thiophene), 8.15-8.18 (m, 3H, 2Ar-H + H3 thiophene); MS m/z
[%]: 495 [(M+2)+, 3.44], 493 [M+, 39.34], 71.05 [100]. Anal. Calcd for C27H19N5O3S: C, 65.71;
H, 3.88; N, 14.19. Found: C, 65.74; H, 3.90; N: 14.35. The purified product 15g was dissolved in
ethanol/DMF (v/v = 3/1) and yellow single crystals were separated after 3 days. Crystal data for
compound 15g: Molecular formula C27H19N5O3S, Formula weight: 493.54, Orthorhombic, Pbca, a
= 19.6064 (6) Å, b = 10.9685 (4) Å, c = 21.7093 (8) Å, V = 4668.7 (3) Å3, Dcalc = 1.404 Mg m−3,
colorless block with 0.32 × 0.19 × 0.15 mm. A total of 29344 reflections were measured, of which
3939 were independent. Rint = 0.078, Dataset (h; k; l) = −23,23; -12,12; -23,25. Refinement of F2,
against all reflections ,led to R [F2 > 2σ(F2)] = 0.090, wR(F2) = 0.296, S = 1.06.
4.1.10.8.
Ethyl1-(4-chlorophenyl)-5-oxo-6-phenyl-8-(thiophen-2-yl)-1,5-dihydropyrido[2,3-d]
[1,2,4] triazolo[4,3-a]pyrimidine-3-carboxylate (15h). Yield: 75%; m.p >300°C; IR (KBr) ν:
1743, 1699 (2C=O) cm-1; 1H NMR (DMSO-d6, 300 MHz) δ: 1.3 (t, J = 7.5 Hz, 3H, CH2CH3), 4.42
(q, J = 7.5 Hz, 2H, CH2CH3), 7.23 (m, 1H, H4 thiophene), 7.45 (m, 5H, Ar-H), 7.74 (s, 1H,
pyridine H), 7.75 (d, J = 8.7 Hz, 2H, Ar-H), 7.86 (m, 1H, H5 thiophene), 8.18 (m, 1H, H3
13
thiophene), 8.22 (d, J =8.7 Hz ,2H , Ar-H); C NMR (DMSO-d6, 75 MHz) δ: 13.61(CH2CH3),
63.42 (CH2CH3), 106.48, 117.09, 122.45, 127.61, 127.97, 128.23, 128.81, 129.09, 129.44, 131.59,
131.66, 135.13, 135.57, 139.07, 143.27, 146.5, 154.13, 154.44, 156.07, 156.60, 160.06; MS m/z
[%]: 529 [(M+2)+, 1.38], 527 [M+, 3.85], 80 [100]. Anal. Calcd for C27H18ClN5O3S: C, 61.42; H,
3.44; N, 13.26. Found: C, 61.48; H, 3.42; N: 13.39.
4.1.10.9.
Ethyl
5-oxo-6-phenyl-8-(thiophen-2-yl)-1-p-tolyl-1,5-dihydropyrido[2,3-d][1,2,4]
triazolo [4,3-a]pyrimidine-3-carboxylate (15i). Yield: 68%; m.p 283-285°C; IR (KBr) ν: 1755,
1716 (2C=O) cm-1; 1H NMR (DMSO-d6, 300 MHz) δ: 1.3 (t, J = 6.9 Hz, 3H, CH2CH3), 2.43 (s,
3H, CH3 ), 4.42 (q, J = 6.9 Hz, 2H, CH2CH3), 7.24 (m, 1H, H4 thiophene), 7.46-7.50 (m, 7H, Ar-
H), 7.71 (s, 1H, pyridine H), 7.86 (m, 1H, H5 thiophene), 8.03 (d, J = 8.1 Hz , 2H, Ar-H), 8.16
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13
(m, 1H, H3 thiophene); C NMR (DMSO-d6, 75 MHz) δ: 13.62(CH2CH3), 20.6 (CH3), 63.32
(CH2CH3), 106.3, 116.84, 121.27, 127.59, 127.93, 128.24, 128.79, 128.98, 129.77, 131.55,
133.82, 135.23, 137.22, 139.17, 143.38, 146.53, 154.11, 154.53, 156.19, 156.53, 160.22; MS m/z
[%]: 508 [(M+1)+ ,1.25], 507 [M+,5.26], 73[100]. Anal. Calcd for C28H21N5O3S: C, 66.26; H, 4.17;
N, 13.80. Found: C, 66.34; H, 4.21; N: 13.96.
4.1.10.10. ethyl 5-oxo-6-phenyl-1-(4-sulfamoylphenyl)-8-(thiophen-2-yl)-1,5-dihydropyrido[2,3-
d][1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate (15j). Yield: 71%; m.p >300 °C; IR (KBr) ν:
3325, 3286 (NH2), br, 1737,1707 (2C=O), 1346, 1159 (SO2) cm-1; 1H NMR (DMSO-d6,400 MHz)
δ: 1.31 (t, J = 7.12 Hz, 3H, CH2CH3), 4.44 (q, J = 7.12 Hz, 2H, CH2CH3), 7.25 (t, J = 4.64 Hz, 1H,
H4 thiophene), 7.43-7.48 (m, 5H, Ar-H),7. 53 (br.s, 2H, NH2, D2O exchangeable), 7.77 (s, 1H,
pyridine H), 7.87 (d, J = 4.92 Hz ,1H, H5 thiophene), 8.12 (d, J = 8.76 Hz , 2H, Ar-H), 8.19 (d, J =
3.68 Hz, 1H, H3 thiophene), 8.43 (d, J = 8.76 Hz, 2H, Ar-H). 13C NMR (DMSO-d6, 100 MHz) δ:
13.74 (CH2CH3), 63.40 (CH2CH3), 106.4, 116.87, 117.04, 121.11, 127.64, 128.16, 128.78, 129.10,
129.49, 131.64, 131.77, 135.38, 136.25, 139.16, 143.37, 146.59, 154.1, 154.57, 156.23, 156.55,
160.19. Anal. Calcd for C27H20N6O5S2: C, 56.63; H, 3.52; N, 14.68; O, 13.97; S, 11.20. Found: C,
56.42; H, 3.43; N: 14.86.
4.2 Biological evaluation
4.2.1. In vitro cytotoxic activity [28]:
A-549 human lung cancer cells and PC-3 human prostate cancer cells were grown in DMEM
and RPMI-1640 respectively. Both were supplemented with 10% heat inactivated FBS, 50
units/mL of penicillin and 50 g/mL of streptomycin and maintained at 37°C in a humidified
atmosphere containing 5% CO2. The cells were maintained as “monolayer culture” by serial
subculturing. Cytotoxicity was determined using SRB method as previously described by Skehan
et al. [28]. Exponentially growing cells were collected using 0.25% Trypsin-EDTA and seeded in
96-well plates at 1000-2000 cells/well in DMEM supplemented medium. After 24 h, cells were
incubated for 48 h with various concentrations of the tested compounds as well as 5-fluorouracil
as reference compound. Following 48 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,
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FL, USA). The IC50 values were calculated according to the equation for Boltzman 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. Significant
differences were analyzed according by way ANOVA wherein the differences were considered to
be significant at p < 0.05.
4.2.2. Morphological investigation:
PC-3 cells were cultured in cell culture flask 25 CM² (3x106 cells / flask) and treated with a
fixed concentration IC50 (0.356 µM) of the tested compound 15f for 24 or 48 h. Then the cells
were stained using the nucleic acid-binding dye mixture of 100 µg/ml acridine orange and 100
µg/ml ethidium bromide in PBS, and examined by fluorescence microscopy.
4.2.3. Caspase-3 activity determination:
The level of caspase-3 was measured using the colorimetric assay kit (Uscn Life Science
E90626Mu, China) according to the manufacturer’s instructions. Briefly, PC-3 cells were treated
with 15f (0.356 µM) for 24 or 48 h. The cell were harvested by trypsinization and rinsed with
PBS. After centrifugation, the pellet (105- 106 cells) was suspended in 1 mL of PBS. Cells were
freezed at < -20oC and thawed with gentle mixing. Freeze/thaw cycle repeated for 3 times, then
centrifuged at 1500×g for 10 minutes at 2- 8oC to remove cellular debris.
The test principle applied in this kit is sandwich enzyme immunoassay. The microtiter plate
provided in this kit has been pre-coated with an antibody specific to active caspase-3. The cell
lysates were then added to the appropriate microtiter plate wells with a biotin-conjugated antibody
specific to caspase-3. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each
microplate well and incubated. After TMB substrate solution is added, only those wells that
contain caspase-3, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a
change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid
solution and the color change is measured spectrophotometrically at a wavelength of 450nm ±
10nm. The concentration of caspase-3 in the samples is then determined in ng/g protein.
4.2.4. Cell cycle analysis:
The PC-3 cells were treated with 0.356 µM of 15f for 24 or 48 h. After treatment, the cells
were washed twice with ice-cold PBS, collected by centrifugation, and fixed in ice-cold 70% (v/v)
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ethanol, washed with PBS, re-suspended with 0.1 mg/ml RNase, stained with 40 mg/ml PI, and
analyzed by flow cytometry using FACScalibur (Becton Dickinson). The cell cycle distributions
were calculated using Cell Quest software (Becton Dickinson).
4.2.5. CDK4/Cyclin D1 and CDK6/Cyclin D1profiling:
A radioisotope assay format was used for profiling evaluation of protein kinase targets and all
assays are performed in a designated radioactive working area. Protein kinase assays (in duplicate)
were performed at ambient temperature for 20-30 minutes in a final volume of 25 ꢀL according to
[5 ꢀL of diluted active CDK4/Cyclin D1 or CDK6/Cyclin D (~10-50 nM final concentration in the
assay)], 5 ꢀL of stock solution of substrate (1-5 ꢀg of peptide substrate), 5 ꢀL of kinase assay
33
buffer, 5 ꢀL of compound (various concentration) or 10% DMSO and 5 ꢀL of P-ATP (250 ꢀM
stock solution, 0.8 ꢀCi).
33
The assay was initiated by the addition of P-ATP and the reaction mixture was incubated at
ambient temperature for 20-30 minutes. After the incubation period, the assay was terminated by
spotting 10 ꢀL of the reaction mixture onto Multiscreen phosphocellulose P81 plate. The
Multiscreen phosphocellulose P81 plate was washed 3 times for approximately 15 minutes each in
a 1% phosphoric acid solution. The radioactivity on the P81 plate was counted in the presence of
scintillation fluid in a Trilux scintillation counter.
Blank control was set up that included all the assay components except the addition of the
appropriate substrate (replaced with equal volume of assay dilution buffer). The corrected activity
for CDK4/Cyclin D1 and CDK6/Cyclin D1 was determined by subtracting the blank control
value.
4.2.6. Immunocytochemistry staining for p21 and p27 [45]:
PC-3 cells were seeded on ibidi® µ-Chamber 12-well slide (Munich, Germany) at a
density of 2 x 105 cells/mL. After exposure to the tested compound 15f at a concentration
equivalent to its IC50 (0.36 µM) for 48h, cells were fixed with 70% of ethanol. Then, they were
washed in phosphate-buffered saline (PBS) and incubated with 0.01% Triton X-100 in PBS for 1
min to permeabilize the cell membranes. Cells were afterwards incubated with 0.3% of H2O2 in
PBS for 20 min to quench endogenous peroxidase activity and then in 5% of normal horse serum
in Tris-buffered saline plus Tween-20 (TBST) for 30 min to block the nonspecific binding of the
secondary antibody. Thereafter, cells were incubated overnight with primary anti-p21 antibody or
anti-p27 antiboby (Abcam plc, Cambridge, MA). In the following day, the slides were incubated
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with the corresponding conjugated anti-rabbit IgG (dilution, 1:2,000, Santa Cruz Biotechnology,
Dallas, TX). Cells were treated afterwards with streptavidin horseradish peroxidase complex
(dilution, 1:100; ABC/HRP; Vector Laboratories, Burlingame, CA, USA) in TBST for 50 min.
The color reaction was developed for 5 min in 3,30-diaminobenzidine (DAB) solution (Santa Cruz
Biotechnology, Dallas, TX).
4.2.8. Western Blot Analysis [45]:
PC-3 cells were seeded, cultured and exposed to IC50 of the tested compound 15f (0.36
µM) for 48h. Whole-cell protein lysates were prepared according to standard protocol using RIPA
buffer (Cell Signaling, Danvers, MA). Protein (50 mg) was loaded per well of a 10% SDS-PAGE
gel using electrophoresis buffer (0.192 M glycine, 25 mM Tris and 0.1% SDS). After
electrophoresis, the gel was transferred onto a PVDF membrane (Bio-Rad Laboratorie, Hercules,
CA) using transfer buffer (0.192 M glycine, 25 mM Tris, 0.025% SDS and 10% methanol).
Membranes were blocked in TBS-T with 5% BSA and incubated overnight with the primary
antibody anti-p21 antibody (1:1,000; Abcam plc, Cambridge, MA) then incubated with secondary
HRP-linked antibody (1:5,000). Development was done using Pierce ECL 2 chemiluminescent
and chemifluorescent substrate (Thermo Fisher Scientific, Waltham, MA). Anti-β-tubulin
antibody (Abcam plc, Cambridge, MA) was used for loading correction. Band densities were
quantified using the free software “ImageJ”.
Acknowledgments
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Egypt, is
highly appreciated for funding this research work, and for X-ray research, the authors would like
to extend their sincere appreciation to the Deanship of Scientific Research at King Saud
University for its funding of this research through the Research Group Project no. RGP-VPP-321.
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FIGURE CAPTIONS
Figure 1: Structure of some anticancer pyrido[2,3-d]pyrimidines I,II, hydrazinopyrimidines III,
1,2,4-triazolo[4,3-a]pyrimidines IV and the targeted compounds 5(a-e), 6-10 and 15(a-j).
Figure 2. ORTEP diagram of compound 15g.
Figure 3: Fluorescence photomicrographs of PC-3 cells stained using acridine orange/ethidium
bromide (AO/EB). (A) Represents the control cells with intact nuclei stained uniformly green with
AO. (B) and (C) show the apoptotic cells with chromatin condensation or nuclear fragmentation
after treatment with compound 15f at its IC50 for 24 h and 48 h respectively. The experiment was
done in triplicate.
Figure 4: Levels of active caspase-3 in PC-3 cells treated with IC50 of compound 15f (0.36 µM)
for 24h and 48h respectively. The experiment was done in triplicate. Data are mean ±SEM. *
significantly different from control at p<0.05.
Figure 5: DNA-flow cytometry analysis for PC-3 cells treated with compound 15f for 24 h and
48h at its IC50. The experiment was done in triplicate. Data are mean ±SEM. * significantly
different from control at p<0.05.
Figure 6: Effect of compound 15f on cell-cycle regulatory proteins in PC-3 cells treated with a
fixed concentration (IC50, 0.36 µM) of the tested compound for 48h. (A) Immunocytochemistry
staining for cyclin-dependent kinase inhibitor p21CIP. (B) Immunocytochemistry staining for
cyclin-dependent kinase inhibitor p27kip. (C) Effect on the expression of p21 measured by Western
blot. One of three repeated experiment is shown. * Significantly different from control at P<0.05.
TABLE CAPTIONS
Table 1. Cytotoxic activity of compound 15f against MCF-7, HepG2, A-549 and PC-3 cells
cancer cell lines.
Table 2. Cytotoxic activity of the new compounds against A-549 and PC-3 cells cancer cell lines.
Table 3. The % inhibition of CDK4/Cyclin D1 and CDK6/Cyclin D1 in the presence of
compound 15f (1-100 µM) or staurosporine (1 µM).
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Table 1. Cytotoxic activity of compound 15f against MCF-7, HepG2, A-549 and PC-
3 cancer cell lines.
IC50 (µM)a
HepG2
Compound
MCF-7
A-549
PC-3
37.96±1.97 56.65±3.65 0.41±0.03 0.36±0.02
a IC50 values are mean of three separate experiments ± S.D.
15f