Synthesis of some pyrazolines and pyrimidines derived from polymethoxy chalcones as anticancer and antimicrobial agents

Article abstract of DOI:10.1002/ardp.201100077

The synthesis of a series of certain polymethoxy chalcones and some derived pyrazole, pyrimidine, and thiazolopyrimidine ring structures is reported. Eleven compounds 4, 6, 9, 11, 14-17, 22, 24, and 25 were selected by the National Cancer Institute (NCI) to be screened for their in-vitro anticancer activity, whereas all the synthesized compounds were evaluated for their in-vitro antimicrobial activity. Compounds 4, 6, and 11 were found to possess a significant broad spectrum antitumor potential against most of the tested subpanel tumor cell lines. The pyrazolines 4 and 6 displayed remarkable growth inhibitory activities (GI₅₀ MG-MID values of 2.10 and 1.38 μM, respectively), together with moderate cytostatic effects (TGI MG-MID values of 47.9 and 42.7 μM, respectively). Meanwhile, the pyrimidin-2-one 11 showed a noticeable overall tumor growth inhibitory activity, together with high cytostatic and cytotoxic efficacies (GI₅₀, TGI and LC₅₀ MG-MID values of 3.39, 17.4, and 61.7 μM, respectively). On the other hand, compounds 3, 4, 13, 15, 19, 20, and 23 were found to be the most active antimicrobial members in this investigation with a broad spectrum of activity. Compound 23 was four times superior to ampicillin against Pseudomonas aeruginosa. The best antifungal activity was demonstrated by compounds 4, 5, and 11 which possessed almost half the activity of clotrimazole against Candida albicans. Collectively, the obtained biological results suggest that compound 4 could be considered as a possible dual antimicrobial-anticancer agent. 22 novel compounds derived from polymethoxylated chalcones including dihydropyrazole, pyrimidine, and thiazolopyrimidine cyclized ring structures were synthesized, characterized, and evaluated for their biological activity as anticancer and/or antimicrobial agents. The results revealed that compounds 4, 6, and 11 were found to be the most active anticancer members with a significant broad spectrum antitumor potential against most of the tested subpanel tumor cell lines. Copyright

Full text of DOI:10.1002/ardp.201100077

572
Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
Full Paper
Synthesis of Some Pyrazolines and Pyrimidines Derived
from Polymethoxy Chalcones as Anticancer and
Antimicrobial Agents*
Sherif A.F. Rostom1,2, Mona H. Badr2, Heba A. Abd El Razik2, Hayam M.A. Ashour2, and
Abeer E. Abdel Wahab3
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi
Arabia
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
3 Genetic Engineering and Biotechnology Research Institute (GEBRI), Mubarak City for Scientific Research
and Technology Application, Borg El-Arab, Alexandria, Egypt
The synthesis of a series of certain polymethoxy chalcones and some derived pyrazole, pyrimidine,
and thiazolopyrimidine ring structures is reported. Eleven compounds 4, 6, 9, 11, 14–17, 22, 24, and 25
were selected by the National Cancer Institute (NCI) to be screened for their in-vitro anticancer activity,
whereas all the synthesized compounds were evaluated for their in-vitro antimicrobial activity.
Compounds 4, 6, and 11 were found to possess a significant broad spectrum antitumor potential
against most of the tested subpanel tumor cell lines. The pyrazolines 4 and 6 displayed remarkable
growth inhibitory activities (GI50 MG-MID values of 2.10 and 1.38 mM, respectively), together with
moderate cytostatic effects (TGI MG-MID values of 47.9 and 42.7 mM, respectively). Meanwhile, the
pyrimidin-2-one 11 showed a noticeable overall tumor growth inhibitory activity, together with high
cytostatic and cytotoxic efficacies (GI50, TGI and LC50 MG-MID values of 3.39, 17.4, and 61.7 mM,
respectively). On the other hand, compounds 3, 4, 13, 15, 19, 20, and 23 were found to be the most
active antimicrobial members in this investigation with a broad spectrum of activity. Compound 23
was four times superior to ampicillin against Pseudomonas aeruginosa. The best antifungal activity was
demonstrated by compounds 4, 5, and 11 which possessed almost half the activity of clotrimazole
against Candida albicans. Collectively, the obtained biological results suggest that compound 4 could be
considered as a possible dual antimicrobial-anticancer agent.
Keywords: Antibacterial / Anticancer activity / Antifungal / Chalcones / Pyrazoles / Pyrimidines
Received: February 27, 2011; Revised: March 15, 2011; Accepted: April 6, 2011
DOI 10.1002/ardp.201100077
Introduction
significant toxicity exerted by most anticancer agents and
the development of cellular drug resistance remain the
primary hurdle for effective chemotherapy [2]. Meanwhile,
patients with neoplastic disorders that are subjected to
chemotherapeutic treatment are mostly susceptible to
microbial infections due to subsequent lack of immunity.
Additionally, the emergence of some pathogenic bacteria and
opportunistic fungi resistant to currently marketed antimi-
crobial agents poses a serious challenge to the medical com-
munity and highlights the importance and urgent need for
Cancer is considered to be the second major cause of deaths
after cardio and cerebrovascular diseases accounting for
about 12% of deaths worldwide [1]. Despite the important
efforts and investment in research, the management of
malignancies in humans still constitutes a major challenge
for contemporary medicinal chemistry. Unfortunately, the
Correspondence: Dr. Sherif A.F. Rostom, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, King Abdulaziz University, P.O. Box 80260,
Jeddah 21589, Saudi Arabia.
E-mail: sherifrostom@yahoo.com
Fax: þ9662-6408404
*This work was presented in the 3rd International IUPAC Conference on
Green Chemistry, Ottawa, Canada, 15–18 August, 2010, as a Poster.
P 209.
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Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
Anticancer/antimicrobial polymethoxy chalcone derivatives
573
new, more potent and selective non-traditional antimicrobial
agents [3]. Therefore, the concept of monotherapy by a single
drug which would possess dual utility for those patients
suffering from cancer disease accompanied with microbial
infections might be advantageous from both therapeutic and
cost-effective stand points, with the hope of better and more
complete response, together with minimal side effects and
more improvement of patients’ life.
tion of alkoxy substituents would result in significant
enhancement of several biological activities due to the mag-
nification of compounds’ lipophilicity [44–46]. The newly
synthesized compounds were designed so as to comprise
two polymethoxylated aryl rings attached to either a pyra-
zole ring substituted with formyl, acetyl or substituted aryl
moieties, or a pyrimidine counterpart with different ketone,
thione, and thioether functionalities. Moreover, it was con-
sidered of interest to annulate the pyrimidine ring in a
thiazolopyrimidine structure in order to investigate the
effect of such structure modification on the expected bio-
logical activities. The aim of the present study is the develop-
ment of novel chemical entities that might serve as lead
molecules in the anticancer and/or antimicrobial drug dis-
covery paradigm.
Chalcones (1,3-diarylprop-2-en-1-ones) are important
precursors of many biologically-active compounds. This
class of compounds has been reported to possess diverse
chemotherapeutic effects including antituberculosis [4],
antifungal [5] in addition to antitumor activities for which
different mechanisms of action have been proposed [6–8].
A literature survey revealed that Nobiletin, Tangeretin and
Combretastatin A-4 (CA-4) are typical polymethoxy chalcone
derivatives in which the double bond is locked in the cis- Results and Discussion
configuration, were found to be highly cytotoxic against a
variety of human cancer cell lines [9–11].
Chemistry
On the other hand, pyrazole-containing compounds have
received considerable attention owing to their diverse che-
motherapeutic potentials including versatile antineoplastic
activities. It has been well-documented that some pyrazoles
have been implemented as antileukemic [12, 13], antitumor
[14, 15], and antiproliferative [16, 17] agents, beside their
capability to exert remarkable anticancer effects through
inhibiting different types of enzymes that play important
role in cell division [18, 19]. In addition, it was found that the
introduction of the pyrazole nucleus between two aryl rings
of chalcones played an integral role for the increase in cyto-
toxic potential [20].
The synthetic strategies adopted to obtain the intermediate
and target compounds are depicted in Schemes 1–3. In
Scheme 1, the key intermediate chalcones 1 and 2 were
synthesized in excellent yields by condensing 3,4,5-tri-
methoxybenzaldehyde with the appropriate substituted
acetophenone in the presence of potassium hydroxide under
Claisen–Schmidt reaction conditions. These chalcones were
subjected to cycloaddition condensation reactions using
hydrazine hydrate in boiling formic or acetic acid to give
the corresponding dihydropyrazole-1-carbaldehydes 3, 4, and
1-acetyldihydropyrazoles 5, 6, respectively, in good yields.
Analogously, refluxing the same chalcones 1 and 2 with
phenylhydrazine or 4-nitrophenylhydrazine in ethanol con-
taining few drops of acetic acid furnished the dihydropyra-
zoles 7-10. It is to be noted that the pyrazoline derivatives 3, 5,
and 7 were prepared following previously published reaction
conditions [47].
Moreover, pyrimidines are among the pharmacologically
most interesting chemical scaffolds that are widely spread in
many natural products and in several interesting nucleoside
and non-nucleoside compounds. Being a building unit of
DNA and RNA, pyrimidine derivatives were found to be
associated with a variety of chemotherapeutic effects includ-
ing antimicrobial [21, 22], antitubercular [23], antifungal [24],
antiviral [25], and antitumor activities [26, 27]. Furthermore,
many pyrimidinethiones and their thioether derivatives
were proved to exhibit potent anticancer as well as antimi-
crobial activities [28, 29], besides being analogs of S-DABOs
(dihydro-alkylthio-benzyl-oxopyrimidines) which possess
antiproliferative as well as antiviral activities [30].
On the other hand, the same chalcones 1 and 2 were
employed as precursors for the synthesis of the targeted
pyrimidine derivatives 11-23 (Scheme 2). In this view, the
pyrimidin-2-one 11 was achieved by reaction of the chalcone
1 with urea in absolute ethanol containing conc. hydro-
chloric acid. Analogously, the 3,4-dihydro-1H-pyrimidine-2-
thiones 12-15 were obtained by refluxing 1 or 2 with thiourea
or substituted thiourea in the presence of a basic catalyst.
Alkylation of the thiones 14 and 15 either with 2-chloro-N-
arylacetamides or chloroacetone in dry DMF containing
anhydrous potassium carbonate at room temperature
afforded the pyrimidine thioethers 16-20. Unexpectedly, stir-
ring a suspension of 14 with ethyl bromoacetate in dry DMF
containing anhydrous potassium carbonate in an attempt to
prepare the target acetic acid ethyl ester derivative 21 gave
directly the thiazolopyrimidinone 22. The structure of
In view of the aforementioned facts, and as a continuation
of an on-going research program devoted to the synthesis
and characterization of different heterocyclic ring systems
endowed with potential chemotherapeutic activities [31–43],
it was sought of interest to design and synthesize certain
chalcones and some derived pyrazole, pyrimidine, and thia-
zolopyrimidine cyclized ring structures substituted basically
with polymethoxy groups based on the fact that incorpora-
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S. A. F. Rostom et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
R
R1
O
O
O
OCH3
OCH3
OCH3
OCH3
ii
i
OCH3
OCH3
H
R
N
N
R
+
R1
1: R = R1 = OCH3
OCH3
OCH3
H
R1
OCH3
OCH3
O
2: R,R1 = -OCH2O-
3: R = R1 = OCH3
4: R,R1= -OCH2O-
R
R
R1
R1
OCH3
OCH3
OCH3
OCH3
iii
N
iv
N
N
N
OCH3
O
R2
5: R = R1 = OCH3
6: R,R1 = -OCH2O-
7: R = R1 = OCH3, R2 = H
8: R = R1 = OCH3, R2 = NO2
9: R,R1 = -OCH2O-, R2 = H
10: R,R1 = -OCH2O-, R2 = NO2
R
e
agents and rea
ction conditions: i) 6% ethanolic KOH, r.t..; ii) NH2NH2.H2O, formic acid, reflux, 12 h; iii) NH2NH2.H2O, acetic acid, reflux, 12 h;
i
v) R-C6H4NHNH2, EtOH, reflux, 6 h.
Scheme 1. Synthesis of compounds 1–10.
22 was substantiated by elementary analyses and 1H-NMR
spectral data which showed the absence of both the triplet
and quartet signals characteristic for the ethyl group and
the NH signal. Refluxing the latter compound with hydrazine
hydrate in absolute ethanol yielded the hydrazino derivative
23. In Scheme 3, the thiazolopyrimidinone 22 could be pre-
pared using the method described by Holla et al. [48] which
involves a reaction of pyrimidinethione 14 with chloroacetic
acid in the presence of anhydrous sodium acetate, acetic
acid, and acetic anhydride. Finally, condensing the latter
compound with the appropriate methoxylated aldehyde in
the presence of piperidine afforded the corresponding aryli-
dene derivatives 24 and 25.
compounds [49–51]. All compounds submitted to the NCI-60
cell screen are tested initially at a single high dose (10 mM) in
the full NCI-60 cell panel including leukemia, non-small cell
lung, colon, CNS, melanoma, ovarian, renal, prostate, and
breast cancer cell lines. Only compounds which satisfy pre-
determined threshold inhibition criteria would proceed to
the five-dose screen. The threshold inhibition criteria for
proceeding to the five-dose screen was designed to efficiently
capture compounds with anti-proliferative activity, and it
is based on careful analysis of historical Development
Therapeutic Program (DTP) screening data. Data are reported
as a mean graph of the percent growth of treated cells, and
presented as percentage growth inhibition (GI %) caused by
the test compounds (Table 1).
Biology
In-vitro anticancer screening
The obtained results revealed that compounds 9, 15, 16, 17,
24, and 25 showed moderate to weak sensitivity profiles
against some of the tested subpanel tumor cell lines (GI %
range of 17.89–69.29). Compound 14 was proved to be the
weakest anticancer member in this screen owing to its low
potency and narrow margin of activity, which was against
only four cell lines and with GI % range of 12.39–28.04.
Compound 22 was able to exhibit promising broad spectrum
anticancer activity particularly against the non-small cell
lung cancer NCI-H522 and melanoma MDA-MB-435 cell lines
(GI % values of 71.72 and 83.89, respectively). However, its
overall antitumor profile did not meet the pre-determined
Preliminary in-vitro one-dose antitumor screening
Out of the newly synthesized compounds, eleven derivatives
namely: 4, 6, 9, 11, 14–17, 22, 24, and 25 were selected by the
National Cancer Institute (NCI) in-vitro disease-oriented
human cells screening panel assay to be evaluated for their
in-vitro antitumor activity. An effective one-dose assay has
been added to the NCI-60 cell screen in order to increase
compound throughput and reduce data-turnaround time to
suppliers while maintaining efficient identification of active
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Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
Anticancer/antimicrobial polymethoxy chalcone derivatives
575
OCH3
OCH3
H3CO
OCH3
OCH3
H3CO
OCH3
H3CO
OCH3
N
H
N
H
S
R
N
N
S
H
O
N
H3CO
H3CO
O
N
R1
R2
H3CO
H3CO
N
H
O
16: R = R1 = OCH3; R2 = Cl
17: R = R1 = OCH3; R2 = OCH3
18: R,R1 = -OCH2O-; R2 = Cl
20
11
19: R,R1 = -OCH2O-; R2 = OCH3
iv
v
14
i
1
OCH3
OCH3
OCH3
H3CO
H3CO
OCH3
vi
iii
O
H
S
H
S
OCH3
N
N
R
14
H3CO
H3CO
O
N
H
N
R
OCH3
OCH3
O
R1
R1
14: R = R1 = OCH3
15: R,R1 = -OCH2O-
1: R = R1 = OCH3
2: R,R1 = -OCH2O-
21
ii
vi
14
1
OCH3
OCH3
OCH3
OCH3
H3CO
H3CO
OCH3
H3CO
OCH3
vii
O
N
H
S
N
N
H3CO
H3CO
NH2
N
N
H
H3CO
H3CO
H3CO
H3CO
S
N
R
N
22
23
12: R = CH3
13: R = C6H5
Reagents and reaction conditions: i) NH CONH , conc. HCl, EtOH, reflux, 6 h.; ii) RNHCSNH , NaOH, EtOH,
`
2
2
reflux, 8 h; iii) NH2CSNH2, K2CO3, EtOH, r2eflux 12 h.; iv) R-C6H4NHCOCH2Cl, K2CO3, DMF, r.t.; v) ClCH2COCH3,
K2CO3, DMF, r.t.; vi) BrCH2COOC2H5, K2CO3, DMF, r.t.; vii) N2H4.H2O, EtOH, reflux, 3 h.
Scheme 2. Synthesis of compounds 11–23.
threshold inhibition criteria and therefore was not sufficient
to proceed to the five-dose screen. On the other hand, com-
pounds 4, 6, and 11 were found to be the most active mem-
bers in this preliminary screen as evidenced by their ability to
exert potential growth inhibitory activity against most of the
tested subpanel tumor cell lines in addition to some lethal
activities (GI % > 100) against some other cell lines.
Consequently, they passed successfully this assay and were
carried over to the five-dose screen against a panel of about 60
different tumor cell lines.
Full in-vitro five-dose antitumor assay
About 60 cell lines of nine tumor subpanels, including leu-
kemia, non-small cell lung, colon, CNS, melanoma, ovarian,
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S. A. F. Rostom et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
OCH3
OCH3
OCH3
H3CO
OCH3
OCH3
H3CO
H3CO
OCH3
O
O
N
ii
H
S
i
N
H3CO
H3CO
N
S
N
H3CO
H3CO
H3CO
H3CO
S
N
R
N
H
OCH3
24: R = H
25: R = OCH3
22
14
Reagents and reaction conditions: i) Ac2O, HOAc, ClCH2COOH, CH3COONa reflux, 8 h; ii) RCHO, piperidine, EtOH, reflux, 6 h.
Scheme 3. Synthesis of compounds 24 and 25.
renal, prostate, and breast cancer cell lines, were incubated
with five concentrations (0.01-100 mM) for each compound
and were used to create log concentration % growth inhi-
0.286, 0.198, and 0.297 mM, respectively). It also showed a
significant sensitivity against most of the tested subpanel
tumor cell lines with GI50 range of 0.318–18.8 mM. On the
other hand, compound 11 was able to display a reasonable
activity against all the tested tumor cell lines (GI50 range of
1.57–12.5 mM), with particular effectiveness against the leu-
kemia HL-60 (TB), K-562, non-small cell lung NCI-H460, renal
A498, and prostate DU-145 cell lines (GI50 values of 1.57, 1.96,
1.92, 1.83, and 1.90 mM, respectively).
bition curves. Three response parameters (GI50, TGI, and LC50
)
were calculated for each cell line. The GI50 value (growth
inhibitory activity) corresponds to the concentration of the
compounds causing 50% decrease in net cell growth, the TGI
value (cytostatic activity) is the concentration of the com-
pounds resulting in total growth inhibition and the LC50
value (cytotoxic activity) is the concentration of the com-
pounds causing net 50% loss of initial cells at the end of
the incubation period (48 h). Subpanel and full panel mean-
graph midpoint values (MG-MID) for certain agents are the
average of individual real and default GI50, TGI, or LC50 values
of all cell lines in the subpanel or the full panel, respectively
[49–51].
With regard to the broad spectrum antitumor activity,
the results revealed that all of the three active compounds;
4, 6, and 11; revealed effective growth inhibition GI50 (MG-
MID) values of 2.10, 1.38, and 3.39 mM, respectively, beside
cytostatic activity TGI (MG-MID) values of 47.9, 42.7, and
17.4 mM, respectively (Tables 3 and 4). In addition, com-
pounds 6 and 11 possessed some cytotoxic efficacy with
LC50 (MG-MID) values of 97.7 and 61.7 mM, respectively
(Table 4).
In the present study, the active three compounds; 4, 6, and
11 exhibited potential antitumor activities against most of
the tested subpanel tumor cell lines (GI50, TGI, and LC50
values <100 mM). These compounds showed a distinctive
pattern of sensitivity against some individual cell lines
(Table 2), as well as a broad spectrum of antitumor activity
(Tables 3 and 4).
Further interpretation of the data presented in Tables 3
and 4 revealed that, compound 6 displayed the highest
growth inhibitory potential (GI50 MG-MID of 1.38 mM),
together with a reasonable cytostatic (TGI MG-MID of
42.7 mM) and weak cytotoxic (LC50 MG-MID of 97.7 mM) activi-
ties. Moreover, it exhibited a pronounced activity on the
leukemia and breast cancer cell lines (GI50 MG-MID values
of 1.89 and 1.14 mM, respectively). Compound 4 showed
almost half of the growth inhibitory potency (GI50 MG-MID
2.10 mM) of the analog 6. However, it exhibited nearly the
same cytostatic activity (TGI MG-MID of 47.9 mM) and lacked
any cytotoxic effect (LC50 values > 100 mM). It should be
pointed out that, both analogs 4 and 6 proved to be highly
active with a significant potential against all the tested sub-
panel tumor cell lines, with distinctive effectiveness on the
colon cancer subpanel at the GI50 level (0.95 and 0.67 mM,
respectively). Despite the fact that compound 11 displayed a
relatively lower growth inhibitory activity (GI50 MG-MID of
3.39 mM) when compared with compounds 4 and 6 (2.10 and
1.38 mM, respectively), yet it showed a significantly better
Concerning the sensitivity against some individual cell
lines (Table 2), compound 4 exhibited a remarkable activity
against colon cancer cell lines with GI50 range of 0.467–
3.19 mM. It also showed remarkable high activity against
CNS SF-295, melanoma MDA-MB-435, and renal A498 cancer
cell lines (GI50 values of 0.255, 0.202, and 0.218 mM, respec-
tively) in addition to an appreciable growth inhibitory
activity against most of the tested subpanel tumor cell lines
with GI50 range of 0.363–9.46 mM. The analog 6 displayed the
ever highest growth inhibitory potential against colon cell
lines with GI50 range of 0.378–2.10 mM, beside a remarkably
high activity against leukemia HL-60 (TB), K-562, non-small
cell lung NCI-H522, melanoma MDA-MB-435, ovarian OVCAR-
3, NCI/ADR-RES, renal A498, and breast MDA-MB-468 cancer
cell lines (GI50 values of 0.289, 0.302, 0.272, 0.184, 0.274,
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Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
Anticancer/antimicrobial polymethoxy chalcone derivatives
577
Table 1. In vitro percentage growth inhibition (GI %) caused by the test compounds against some selected tumor cell lines at the single-dose
assay.a
Cpd. No. NSC No.
4 754197
Panel
Leukemia
Subpanel tumor cell lines (% growth inhibitory activity)
CCRF-CEM (75.49), HL-60 (TB) (99.43), K-562 (82.36), MOLT-4 (55.04), RPMI-8226 (47.23),
SR (66.54)
Non small cell lung
Colon cancer
A549/ATCC (53.79), EKVX (62.12), HOP-62 (63.28), HOP-92 (25.44), NCI-H226 (41.71),
NCI-H23 (40.07), NCI-H322M (85.88), NCI-H460 (84.73), NCI-H522 (103.85)
COLO 205 (87.44), HCC-2998 (35.84), HCT-116 (79.36), HCT-15 (66.48), KM12 (76.67),
SW-620 (72.97)
CNS cancer
Melanoma
SF-268 (55.2), SF-295 (101.84), SNB-19 (57.24), SNB-75 (113.52), U251 (67.99)
LOX IMVI (65.54), MALME-3M (103.87), M14 (73.35), MDA-MB-435 (125.87), SK-MEL-2 (68.75),
SK-MEL-28 (49.36), SK-MEL-5 (82.82), UACC-257 (34.67), UACC-62 (59.13)
OVCAR-3 (128.99), OVCAR-4 (42.31), OVCAR-8 (46.14), NCI/ADR-RES (84.52), SK-OV-3 (75.26)
786-0 (62.56), A498 (68.78), CAKI-1 (93.58), RXF 393 (54.3), SN12C (53.71), TK-10 (46.77)
PC-3 (52.07), DU-145 (68.34)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
MCF7 (83.23), MDA-MB-231/ATCC (32.0), HS 578T (91.49), BT549 (59.72), MDA-MB-468
(112.66)
6
754198
Leukemia
Non-small cell lung
CCRF-CEM (83.99), K-562 (81.84), MOLT-4 (63.96), RPMI-8226 (60.76), SR (80.06)
A549/ATCC (63.97), EKVX (61.31), HOP-62 (64.54), HOP-92 (25.36), NCI-H226 (47.58),
NCI-H23 (55.7), NCI-H322M (112.91), NCI-H460 (89.31), NCI-H522 (129.83)
COLO 205 (112.80), HCC-2998 (52.24), HCT-116 (69.13), HCT-15 (59.57), KM12 (69.42),
SW-620 (73.39)
Colon cancer
CNS cancer
Melanoma
SF-268 (47.31), SF-295 (107.31), SNB-19 (52.38), SNB-75 (112.15), U251(71.75)
LOX IMVI (64.3), MALME-3M (76.7), Ml4 (70.67), MDA-MB-435 (148.97), SK-MEL-2 (71.51),
SK-MEL-28 (47.34), SK-MEL-5 (88.27), UACC-257 (33.46), UACC-62 (53.83)
OVCAR-3 (105.71), OVCAR 4 (45.48), OVCAR-8 (57.97), NCI/ADR-RES (96.0), SK-OV-3 (89.41)
786-0 (57.93), A498 (71.2), CAKI-1 (97.6), RXF 393 (92.78), SN12C (53.68), TK-10 (61.79)
PC-3 (56.23), DU-145 (86.27)
MCF7 (82.55), MDA-MB-231/ATCC (38.72), HS 578T (107.69), BT-549 (39.34), MDA-MB-468
(126.82)
K-562 (51.19), RPM1-8226 (24.35), SR (28.13)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
9
754199
754204
Leukemia
Non-small cell lung
Colon cancer
CNS cancer
Melanoma
Breast cancer
Leukemia
NCI-H522 (26.64)
KM12 (39.12)
SNB-75 (41.21), U251 (61.06)
MDA-MB-435 (69.29), UACC-62 (23.27)
MDA-MB-231/ATCC (17.83), BT-549 (28.09), MDA-MB-468 (24.29)
CCRF-CEM (93.41), HL-60(TB) (97.05), K-562 (89.4), MOLT-4(82.12), RPMI-8226 (95.2),
SR (90.43)
11
Non-small cell lung
Colon cancer
A549/ATCC (80.95), EKVX (64.5), HOP-62 (67.44), HOP-92 (93.76), NCI-H226 (35.75), NCI-H23
(72.7), NCI-H322M (109.67), NCI-460 (98.75), NCI-H522 (108.71)
COLO 205 (124.09), HCC-2998 (81.26), HCT-116 (86.55), HCT-15 (72.49), KM12 (126.84),
SW-620 (82.68)
CNS cancer
Melanoma
SF-268 (67.77), SF-295 (93.47), SNB-19 (61.87), SNB-75 (97.83), U251 (78.89)
LOX IMVI (92.96), MALME-3M (88.83), M l4 (82.79), MDA-MB-435 (92.69), SK-MEL-2 (79.31),
SK-MEL-28 (60.0), SK-MEL-5 (81.53), UACC-257 (42.82), UACC-62 (55.53)
IGROV l (l17.70), OVCAR-3 (124.05), OVCAR-4 (81.23), OVCAR-5 (65.35), OVCAR-8 (81.25),
NCI/ADR-RES (86.34), SK-OV-3 (76.69)
786-0 (72.14), A498 (80.67), CAK1-1 (94.18), RXF 393 (99.38), SN12C (76.25), TK-10 (87.16),
UO-31 (77.21)
PC-3 (49.03), DU-145 (117.07)
Ovarian cancer
Renal cancer
Prostate cancer
Breast cancer
MCF7 (85.0), MDA-MB-231/ATCC (78.24), HS 578T (92.38), BT-549 (91.28), MDA-MB-468
(112.63)
14
15
754200
754201
Melanoma
Renal cancer
Leukemia
Non-small cell lung
Colon cancer
CNS cancer
Melanoma
Renal cancer
Prostate cancer
LOX IMVI (12.39), MDA-MB-435 (28.04), UACC-62 (12.67).
UO-31 (15.05)
CCRF-CEM (30.74), K-562 (40.84), MOLT-4 (23.85), RPMI-8226 (41.55), SR (31.75)
HOP-92 (36.68), NCI-H522 (35.75)
HCT-116 (44.46), HCT-15 (27.37)
SNB-75 (33.37)
MDA-MB-435 (41.89)
UO-31 (44.04)
PC-3 (37.05)
continued
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Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
Table 1. (continued )
Cpd. No. NSC No.
Panel
Subpanel tumor cell lines (% growth inhibitory activity)
Breast cancer
Leukemia
Renal cancer
Prostate cancer
Leukemia
MCF7 (41.89)
16
17
754202
754203
MOLT-4 (28.78), RPMI-8226 (34.04)
CAKI-1(20.18), UO-31(68.75)
PC-3 (23.52)
RPMI-8226 (21.78)
Non-small cell lung
CNS cancer
Renal cancer
Leukemia
Non-small cell lung
Colon cancer
CNS cancer
HOP-92 (23.21), NCI-H522 (20.44)
SNB-75 (23.96)
UO-31(64.64)
CCRF-CEM (40.48), K-562 (47.36), MOLT-4 (22.78), RPMI-8226 (38.0), SR (39.07)
A549/ATCC (21.68), HOP-92 (49.04), NCI-H322M (19.73), NCI-H522 (71.72)
HCT-116 (25.64), HCT-15 (21.78), KM12 (30.73)
SF-295 (26.17), SNB-75 (29.31)
22
754205
Melanoma
MDA-MB-435 (83.89), SK-MEL-5 (55.51), UACC-62 (36.32)
OVCAR-3 (28.5), NCI/ADR-RES (33.93)
786-0 (26.6), A498 (38.09), CAKI-1 (30.43)
MCF7 (49.37), BT-549 (32.78), MDA-MB-468 (31.01)
NCI-H522 (32.07)
SNB-75 (26.98)
OVCAR-4 (25.71)
786-0 (21.42)
Ovarian cancer
Renal cancer
Breast cancer
Non-small cell lung
CNS cancer
Ovarian cancer
Renal cancer
Leukemia
24
25
754206
754207
CCRF-CEM (35.59), RPMI-8226 (36.26)
HOP-92 (36.65), NCI-H522 (41.93)
SNB-75 (23.79)
Non-small cell lung
CNS cancer
Renal cancer
786-0 (21.93)
a
Data obtained from NCI in-vitro disease-oriented human tumor cell screen at 10 mM concentration
broad spectrum of cytostatic (TGI MG-MID of 17.4 mM) and
cytotoxic (LC50 MG-MID of 61.7 mM) potentials.
42.7 mM), whereas a weak cytotoxic effect was observed (LC50
MG-MID of 97.7 mM). Here, it should be pointed out that
introduction of an aromatic moiety at position 1 of the
dihydropyrazole ring (as in 9) led to a dramatic reduction
in the anticancer activity. On the other hand, enclosure of the
polymethoxylated chalcones in a pyrimidine-2-one ring
structure (11) led to a relative reduction in the overall growth
inhibitory activity (GI50 MG-MID of 3.39 mM) and a remark-
able increase in both cytostatic and cytotoxic efficacies (TGI50
and LC50 MG-MID of 17.4 and 61.7 mM, respectively).
However, replacement of the 2-one group with a thione
(14 and 15) or thioether (16 and 17) functionalities led to a
dramatic loss of the anticancer activity. Finally, annulation of
the pyrimidine moiety into a thiazolopyrimidine structure
(22) led to a compound with a promising broad spectrum
anticancer activity that deserves further derivatization and
modification in order to explore the scope and limitations of
its activity.
The ratio obtained by dividing the compound full panel
MG-MID (mM) by its individual subpanel MG-MID (mM) is
considered as a measure of compound selectivity. Ratios
between 3 and 6 refer to moderate selectivity, ratios > 6
indicate high selectivity towards the corresponding cell line,
while compounds meeting neither of these criteria are rated
non-selective [52]. In this context, the active compounds in
the present study were found to be non-selective with broad
spectrum antitumor activity against the nine tumor subpa-
nels tested with selectivity ratios ranging between 0.55–2.35
at the GI50 MG-MID level.
Structurally, compounds 4 and 6 are 4,5-dihydropyrazole
derivatives comprising trimethoxyphenyl and 1,3-benzo-
dioxol-5-yl moieties together either with a formyl group at
position 1 (compound 4) or an acetyl one at the same position
(compound 6). In this view, the N-formyl derivative 4 showed
an overall remarkable growth inhibitory (GI50 MG-MID of
2.10 mM) and mild cytostatic (TGI MG-MID of 47.9 mM) activi-
ties, while it lacked any cytotoxic effect. Replacement of the
formyl functionality with an acetyl group (as in 6) resulted in
about twofold increase in the overall growth inhibitory
activity (GI50 MG-MID of 1.38 mM), in addition to some
noticeable improvement in activity against some individual
subpanels including leukemia, colon and breast cell lines. Its
cytostatic potential was marginally affected (TGI MG-MID of
In-vitro antibacterial and antifungal activities
All of the newly synthesized compounds were evaluated for
their in-vitro antibacterial activity against Staphylococcus aureus
(DSM 1104) and Bacillus subtilis (ATCC 6633) as Gram-positive
bacteria; Escherichia coli (ATCC 11775) and Pseudomonas aerugi-
nosa (ATCC 10145) as Gram-negative bacteria. They were also
investigated for their in-vitro antifungal potential against
Candida albicans (DSM 70014). Agar cup-diffusion method
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Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
Anticancer/antimicrobial polymethoxy chalcone derivatives
579
Table 2. (continued )
Table 2. Growth inhibitory concentrations (GI50, mM) of
compounds 4, 6, and 11.a
Cell lines
4
6
11
Cell lines
4
6
11
A498
0.218
3.53
0.509
NT
13.6
8.97
4.33
0.198
0.695
0.53
1.52
7.17
7.99
7.88
1.83
3.50
2.26
3.04
2.42
4.48
2.54
ACHN
CAKI-1
RXF 393
SN12C
TK-10
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
2.48
1.18
2.65
1.57
1.96
2.41
3.03
2.11
0.371
0.419
4.23
7.31
0.409
0.289
0.302
1.49
7.73
0.355
UO-31
Prostate cancer
PC-3
DU-145
4.92
1.43
4.39
0.744
10.2
1.90
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
NAb
1.67
1.35
NA
4.02
2.43
0.78
NA
18.8
1.69
3.30
4.74
2.85
3.99
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
T-47D
0.581
6.41
1.60
1.72
NT
0.397
3.77
0.888
0.358
0.297
4.26
2.84
2.15
2.63
3.21
12.5
NTc
8.40
3.48
4.19
1.92
2.78
3.14
6.04
0.501
0.456
MDA-MB-468
0.407
0.272
a
Data obtained from NCI’s in-vitro disease-oriented human
b
tumor cell screen. NA: Not active (GI50 value > 100 mM).
NT: Not tested.
c
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
0.521
3.19
0.401
2.10
2.65
3.77
3.17
2.90
4.05
2.25
2.96
was used for determination of preliminary antibacterial and
antifungal activity [53]. Ampicillin trihydrate (antibiotic) and
clotrimazole (antifungal) were used as reference drugs.
Dimethylsulfoxide (DMSO) was used as a blank and showed
no antimicrobial activity. The results were recorded for each
tested compound as the average diameter of inhibition zones
(IZ) of bacterial or fungal growth around the discs in mm. The
minimal inhibitory concentration (MIC) and minimal bac-
tericidal concentration (MBC) measurements were deter-
mined for all compounds using the two-fold serial dilution
method [54].
0.664
0.686
0.467
0.614
0.528
0.509
0.475
0.378
0.435
0.407
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
7.61
0.255
1.42
5.0
0.682
4.75
11.4
3.46
2.34
2.28
4.73
2.42
3.82
0.318
0.578
1.12
0.354
1.17
As revealed from Tables 5 and 6, all of the tested com-
pounds displayed variable inhibitory effects on the growth of
the tested Gram-positive and Gram-negative microorganisms
with special pronounced activity against P. aeruginosa.
Moreover, some members exhibited moderate antifungal
activity against C. albicans. Concerning the antibacterial
potency of the active compounds against S. aureus, com-
pounds 3, 19, 20, and 22 (MIC ¼ 25 mg/mL) showed mild
activity, whereas the remaining compounds showed weak
activity. With regard to the activity against B. subtilis, com-
pounds 3, 7, and 20 (MIC ¼ 12.5 mg/mL) were found to be
equipotent to ampicillin, while compounds 4, 13, and 19
showed half the activity of ampicillin against the same organ-
ism. Interestingly, the Gram-negative P. aeruginosa was found
to be the most sensitive microorganism to most of the tested
compounds. Compound 23 (MIC ¼ 12.5 mg/mL) was four
times as active as ampicillin, whereas compounds 3, 4, 17,
20 and 25 showed double the activity of ampicillin against
the same organism (MIC ¼ 25 mg/mL). Furthermore, com-
pounds 3, 15, and 23 (MIC ¼ 12.5 mg/mL) were nearly equiv-
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
1.27
1.06
0.616
0.202
9.46
8.29
NTc
0.66
3.05
5.06
3.59
3.10
4.96
6.97
4.41
0.838
0.471
0.184
NA
16.3
0.488
NA
0.449
NA
0.755
10.8
4.31
Ovarian cancer
IGROV1
0.981
0.363
9.12
6.53
NA
1.87
0.274
15.0
4.38
7.56
0.286
0.63
3.80
2.13
3.61
2.28
4.25
3.02
3.67
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
0.442
1.24
Renal cancer
786-0
5.26
2.06
4.65
continued
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580
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Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
Table 3. Median growth inhibitory concentrations (GI50, mM) of in-vitro subpanel tumor cell lines.a
Compd. No.
Subpanel tumor cell linesb
MG-MIDc
I
II
III
IV
V
VI
VII
VIII
IX
4
6
11
2.54
1.89
2.29
2.19
3.96
4.98
0.95
0.67
3.11
3.29
2.49
3.18
3.09
2.77
5.14
3.11
4.29
3.25
5.20
3.50
3.09
3.18
2.57
6.05
2.58
1.14
3.02
2.10
1.38
3.39
a
Median values calculated according to the data obtained from NCI’s in-vitro disease-oriented human tumor cell screen. b I, Leukemia;
II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian cancer; VII, renal cancer; VIII, prostate cancer;
IX, breast cancer. c GI50 (mM) full panel mean-graph midpoint (MG-MID) ¼ the average sensitivity of all cell lines towards the test agent.
Table 4. Median total growth inhibitory concentrations (TGI, mM) and lethal concentration LC50, mM) of in-vitro subpanel tumor cell lines.a
Compd. No.
Subpanel tumor cell linesb
MG-MIDc
I
II
III
IV
V
VI
VII
VIII
IX
4
83.9
–
88.4
–
86.1
–
52.1
–
87.6
–
72.2
–
82.3
–
53.4
77.1
–
47.9
–
6
83.5
–
14.0
–
89.2
–
15.4
(71.3)
79.9
(90.2)d
11.4
(39.7)
62.5
–
11.2
(41.4)
71.1
–
18.1
(46.9)
63.6
–
10.6
(32.4)
87.6
–
19.0
(41.4)
52.3
–
4.45
(11.1)
60.0
–
16.7
(69.5)
42.7
(97.7)
17.4
(61.7)
11
a
Median values calculated according to the data obtained from NCI’s in-vitro disease-oriented human tumor cell screen. b For subpanel
tumor cell lines, see footnote (b) of Table 3. c TGI (mM) full panel mean-graph midpoint (MG-MID) ¼ the average sensitivity of all cell
lines towards the test agent. d LC50 (mM) full panel mean-graph midpoint (MG-MID) ¼ the average sensitivity of all cell lines towards
the test agent.
Table 5. Inhibition zones (IZ) in mm diameter of the tested compounds.
Cpd. No. S. aureus
B. subtilis
P. aeruginosa
E. coli
C. albicans
1
2
3
4
5
6
7
8
11
14
13
13
14
14
15
12
11
16
12
12
16
16
17
14
14
12
14
12
15
16
16
17
13
12
12
14
14
13
13
13
12
12
14
15
12
14
12
14
14
12
11
12
11
12
14
15
12
12
12
13
14
11
12
12
12
14
14
12
11
12
11
12
14
14
15
16
14
17
15
16
12
14
15
14
15
12
14
16
14
14
15
16
11
10
12
14
15
16
16
14
15
14
15
15
14
16
16
14
12
15
14
14
14
15
15
16
16
14
17
15
14
14
16
14
14
16
14
17
9
10
11
12
13
14
15
16
17
18
19
20
22
23
24
25
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Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
Anticancer/antimicrobial polymethoxy chalcone derivatives
581
Table 6. Minimal inhibitory concentrations (MIC, mg/mL) and minimal bactericidal concentrations (MBC, mg/mL) of the tested compounds.
Cpd. No.
S. aureus
B. subtilis
P. aeruginosa
E. coli
C. albicans
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
22
23
24
25
100
100
25
50
100
50
100
200
50
50
100
50
100
100
50
200
100
100
200
200
100
100
100
100
25
50
50
200
100
50
50
100
12.5
25
100
50
12.5
50
50
100
100
100
25
100
50
100
100
100
25
12.5
50
100
100
25
100
100
25
100
100
25
50
25
12.5
50
100
50
50
50
50
25
50
100
25
100
12.5
100
25
100
50
50
100
12.5
25
50
10
50
50
25
50
100
50
100
50
50
50
50
100
50
100
25
100
50
100
50
50
100
25
25
100
–
100
25
50
12.5
12.5
25
50
100
50
25
12.5
50
100
50
100
25
100
50
100
50
100
50
50
25
–
5
200
50
100
25
25
50
100
200
50
50
25
50
100
100
25
100
50
100
200
100
50
200
50
100
100
200
50
25
50
100
100
100
50
100
100
50
50
50
100
100
50
25
50
100
25
100
12.5
50
25
50
100
100
100
100
100
100
100
50
100
100
100
50
50
50
100
50
200
25
50
50
100
100
50
100
100
100
100
100
100
50
100
100
25
25
25
50
25
50
200
100
200
50
100
100
100
50
100
50
100
50
100
50
50
5
50
50
100
100
12.5
–
100
100
–
Ampicillin
Clotrimazole
–
–
–
–
–
–
–
–
–
–
alent to ampicillin against E. coli, meanwhile, compounds 2,
10, 13, 17, and 24 possessed nearly half the potency of ampi-
cillin against the same organism. On the other hand, inves-
tigation of the antifungal activity of the tested compounds
revealed that only three analogs 4, 5, and 11 were able
to display a noticeable growth inhibitory activity (MIC ¼
12.5 mg/mL) against C. albicans when compared with clotri-
mazole. According to the MIC and MBC specifications of the
latest National Committee on Clinical Laboratory Standards
(NCCLS), it could be deduced whether the test compound is
bactericidal or bacteriostatic to the test organism [55].
Accordingly, concerning the Gram-positive bacteria, com-
pound 19 was bactericidal against S. aureus, whereas com-
pounds 9, 15, 22, and 23 were bactericidal against B. subtilis.
Meanwhile, most of the active compounds showed bacteri-
cidal activity against the Gram-negative P. aeruginosa among
which compound 3 was the most active, whereas compound
24 showed the highest bactericidal potential against E. coli
(Table 6).
pyrazolin-1-carbaldehydes (3 and 4) resulted in an obvious
enhancement in both the antimicrobial potential and
spectrum. Accordingly, compound 3 displayed a fourfold
increase in antimicrobial activity against S. aureus, B. subtilis,
P. aeruginosa, and E. coli (MIC ¼ 25, 12.5, 25, and 12.5 mg/mL,
respectively) and exhibited a twofold increase in activity
against C. albicans (MIC ¼ 50 mg/mL). On the other
hand, compound 4 demonstrated a fourfold increase in anti-
microbial activity against B. subtilis and P. aeruginosa
(MIC ¼ 25 mg/mL for both organisms), and a twofold increase
in activity against S. aureus and C. albicans (MIC ¼ 50 and
12.5 mg/mL, respectively). Replacement of the formyl func-
tion at N-1 of the pyrazoline ring by an acetyl group markedly
reduced the activity against all the test organisms except for
compound 5 that showed a fourfold increase in antimicrobial
activity against C. albicans (MIC ¼ 12.5 mg/mL). Introduction
of an aryl moiety at position-1 of the pyrazoline ring resulted
in four compounds (7–10), among which compounds 7 and 9
stemmed as the most active members in this series, however,
with limited potential when compared with the formyl and
acetyl congeners. On the other hand, cyclization of chalcone
1 to the corresponding pyrimidinone counterpart 11 did not
significantly affect the antimicrobial potential, with the
exception of a twofold increase and eightfold increase in
The obtained antimicrobial activity of the tested com-
pounds could be correlated to the structural variations
and modifications. The key precursors, chalcones 1 and 2,
showed moderate to mild overall antimicrobial activity.
Cyclocondensation of these chalcones to the corresponding
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582
S. A. F. Rostom et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
activity against P. aeruginosa and C. albicans (MIC ¼ 50 and
12.5 mg/mL, respectively). Condensation of the chalcone 1
with N-substituted thiourea afforded two moderately active
derivatives (12 and 13), of which the N-phenyl analog 13
exhibited significant activity against B. subtilis, P. aeruginosa,
and E. coli (MIC ¼ 25, 50, 25 mg/mL). Meanwhile, conden-
sation of 1 and 2 with thiourea yielded two compounds
(14 and 15) of which compound 15 demonstrated the highest
activity against E. coli (MIC 12.5 mg/mL). Alkylation of the
thione group furnished five thioethers 16–20 with altered
antimicrobial activity, among which compound 20 possessed
the best antimicrobial profile and spectrum. Introduction of
a hydrazino group at position 2 of the pyrimidine ring (23)
led to a noticeable improvement in the antimicrobial activity
marked by an eightfold increase in growth inhibitory activity
against P. aeruginosa and E. coli (MIC ¼ 12.5 mg/mL for both
organisms). Finally, incorporating the pyrimidine ring into a
thiazolo[3,2-a]pyrimidine structure as in 22 led to a fourfold
and twofold increase in activity against S. aureus and B. subtilis,
respectively. Condensation of the latter with aromatic alde-
hydes furnished two compounds 24 and 25 with a decreased
antimicrobial activity against Gram positive organisms and
enhanced activity against Gram negative organisms and
C. albicans. Collectively, compounds 3, 4, 13, 15, 19, 20, and
23 could be considered as the most active broad spectrum
antimicrobial members identified in this study.
with special high activity against the Gram-negative
P. aeruginosa, in addition to a variable degree of antifungal
activity against C. albicans. Compounds 3, 4, 13, 15, 19, 20, and
23 could be considered as the most active broad spectrum
antimicrobial members identified in this study. Among
these, compound 23 emerged with the highest potential
against P. aeruginosa as it was found to be four times superior
to ampicillin against this organism. The best antifungal
activity was demonstrated by compounds 4, 5, and 11 which
possessed almost half the activity of clotrimazole against
C. albicans.
Finally, the obtained antimicrobial and anticancer data
makes such type of compounds a fruitful matrix for further
development of more potent and selective anticancer and/or
antimicrobial agents. In particular, compound 4 could be
considered as a possible dual antimicrobial-anticancer can-
didate that deserves further investigation and derivatization
in order to explore the scope and limitation of its biological
activities.
Experimental
Chemistry
Melting points were determined in open glass capillaries using
Stuart Capillary melting point apparatus (Stuart Scientific Stone,
Staffordshire, UK) and are uncorrected. Infrared (IR) spectra were
recorded on Perkin-Elmer 1430 infrared spectrophotometer
(Perkin Elmer, Beaconsfield, UK). 1H-NMR spectra were scanned
on Jeol-500 MHz spectrometer (Jeol, Tokyo, Japan) using tetra-
methylsilane (TMS) as internal standard and DMSO-d6 as the
solvent (chemical shifts are given in d, ppm). Splitting patterns
were designated as follows: s: singlet; d: doublet; dd: doublet of
doublet; t: triplet; m: multiplet. Elemental analyses were per-
formed at the microanalytical unit, Faculty of Science, Cairo
University, and at the central lab, Faculty of Pharmacy,
Alexandria University, Egypt and were found within 0.4% of
the theoretical values. Follow up of the reactions and checking
the purity of the compounds was made by thin layer chroma-
tography (TLC) on silica gel-precoated aluminum sheets (Type 60
GF254; Merck, Germany) and the spots were detected by
Conclusion
Twenty two novel compounds derived from polymethoxy-
lated chalcones including dihydropyrazole, pyrimidine,
and thiazolopyrimidine cyclized ring structures, were suc-
cessfully synthesized and characterized by IR and 1H-NMR
spectral data, in addition to elementary analyses. They were
also evaluated for their biological activity as anticancer and/
or antimicrobial agents.
The results obtained from the NCI’s in-vitro anticancer
screening revealed that compounds 4, 6, and 11 were found
to be the most active anticancer members in this study with a
significant broad spectrum antitumor potential against most
of the tested subpanel tumor cell lines. The pyrazolines 4 and
6 displayed a remarkable growth inhibitory activities (GI50
MG-MID values of 2.10 and 1.38 mM, respectively), together
with moderate cytostatic effects (TGI MG-MID values of 47.9
and 42.7 mM, respectively). The pyrimidine-2-one analog 11
possessed a relatively lower growth inhibitory activity (GI50
MG-MID of 3.39 mM) than compounds 4 and 6, however it
showed a pronounced cytostatic and cytotoxic efficacy (TGI
and LC50 MG-MID values of 17.4 and 61.7 mM, respectively).
On the other hand, most of the newly synthesized com-
pounds displayed promising antibacterial activity against
the tested Gram-positive and Gram-negative bacterial strains,
exposure to UV lamp at l 254 nm for few seconds.
Compounds 3, 5, and 7 were prepared following previously
published reaction conditions [47].
3-(Benzo[1,3]dioxol-5-yl)-5-(3,4,5-trimethoxyphenyl)-4,5-
dihydropyrazole-1-carbaldehyde 4
A mixture of the chalcone 2 (0.34 g, 1.0 mmol), hydrazine
hydrate 99% (0.2 g, 0.19 mL, 4.0 mmol), and formic acid
(5 mL) was heated under reflux for 12 h, then allowed to cool
to room temperature. The obtained precipitate was filtered,
washed with cold ethanol and crystallized. Physicochemical
and analytical data are recorded in Table 7.
IR (KBr, cmꢀ1): 1658 (C O), 1593 (C N), 1249, 1037 (C–O–C).
–
–
–
–
1H-NMR (d, ppm): 3.09 (dd, J ¼ 18.0, 5.0 Hz, 1H, pyrazoline C4-H),
3.59 (s, 3H, OCH3), 3.70 (s, 6H, 2 OCH3), 3.79 (dd, J ¼ 18.0, 11.7 Hz,
1H, pyrazoline C4-H), 5.40 (dd, J ¼ 11.7, 5.0 Hz, 1H, pyrazoline
C5-H), 6.06 (s, 2H, O–CH2–O), 6.46 (s, 2H, trimethoxyphenyl
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Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
Anticancer/antimicrobial polymethoxy chalcone derivatives
583
Table 7. Physicochemical and analytical data of compounds 4–25.
Cpd.
4
R, R1
R2
Yield (%)
72
M.p. (8C) Cryst. solventꢁ
Mol. Formula (Mol. wt.) ꢁꢁ
OCH2O
–
204-6
D/E
C20H20N2O6
(384.38)
6
OCH2O
–
70
122-4
E
C21H22N2O6
(398.41)
8
R ¼ R1 ¼ OCH3
NO2
73
68-70
D/E
C26H27N3O7
(493.51)
9
OCH2O
H
78
134-6
D/E
C25H24N2O5
(432.47)
10
11
12
13
14
15
16
17
18
19
20
22
23
24
25
OCH2O
NO2
70
114-6
D/E
118-20
E
84-6
A/W
122-4
A/W
225-7
D/E
180-82
D/E
194-6
D/E
185-7
D/E
204-6
D/E
179-81
E
142-4
EA
159-61
E
108-10
E
137-9
D/W
126-28
D/W
C25H23N3O7
(477.47)
C21H24N2O6
(400.43)
C22H26N2O5S
(430.52)
C27H28N2O5S
(492.59)
C21H24N2O5S
(416.49)
C20H20N2O5S
(400.45)
C29H28ClN3O6S
(582.07)
C30H31N3O7S
(577.65)
C28H24ClN3O6S
(566.02)
C29H27N3O7S
(561.61)
C24H28N2O6S
(472.56)
C23H24N2O6S
(456.51)
C21H24N4O5.2H2O
(448.47)
C31H30N2O7S
(574.64)
C32H32N2O8S
(604.67)
–
–
42
CH3
–
35
C6H5
–
40
R ¼ R1 ¼ OCH3
–
45
OCH2O
–
55
R ¼ R1 ¼ OCH3
Cl
65
R ¼ R1 ¼ OCH3
OCH3
60
OCH2O
Cl
63
OCH2O
OCH3
51
–
–
–
–
–
–
43
–
–
45
40
H
48
OCH3
36
ꢁ
Crystallization solvent (s): A: acetic acid, D: 1,4-dioxane, E: ethanol, EA: ethyl acetate, W: water. **Found values are within ꢂ0.4% of
the calculated values.
C2,6-H), 6.97 (d, J ¼ 8.0 Hz, 1H, benzodioxole C7-H), 7.22 (d,
J ¼ 8.0 Hz, 1H, benzodioxole C6-H), 7.31 (s, 1H, benzodioxole
C4-H), 8.84 (s, 1H, HCO).
1H, pyrazoline C5-H), 6.09 (s, 2H, O-CH2-O), 6.44 (s, 2H, trimethoxy-
phenyl C2,6-H), 6.99 (d, J ¼ 8.0 Hz, 1H, benzodioxole C7-H), 7.23
(d, J ¼ 8.0 Hz, 1H, benzodioxole C6-H), 7.36 (s, 1H, benzodioxole
C4-H).
1-(3-(Benzo[1,3]dioxol-5-yl)-5-(3,4,5-trimethoxyphenyl)-
4,5-dihydropyrazol-1-yl)ethanone 6
General procedure for the preparation of 3-(3,4-
dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1-(4-
nitrophenyl)-4,5-dihydro-1H-pyrazole 8 and 1-aryl-3-
(benzo[1,3]dioxol-5-yl)-5-(3,4,5-trimethoxyphenyl)-4,5-
A mixture of the chalcone 2 (0.34 g, 1.0 mmol), hydrazine
hydrate 99% (0.2 g, 0.19 mL, 4.0 mmol), and acetic acid (5 mL)
was heated under reflux for 12 h, then allowed to cool to room
temperature. The obtained precipitate was filtered, washed with
EtOH, and crystallized. Physicochemical and analytical data are
recorded in Table 7.
dihydro-1H-pyrazoles 9, 10
To a solution of the appropriate chalcone 1 or 2 (1.0 mmol) in
absolute ethanol (5 mL) containing 2 drops of glacial acetic acid,
the appropriate aryl hydrazine (1.0 mmol) was added. The reac-
tion mixture was heated under reflux for 6 h, then allowed to
cool to room temperature. The separated crystalline solid was
IR (KBr, cmꢀ1): 1660 (C O), 1590 (C N), 1247, 1028 (C–O–C).
–
–
–
–
1H-NMR (d, ppm): 2.28 (s, 3H, CH3), 3.11 (dd, J ¼ 18.0, 4.8 Hz, 1H,
pyrazoline C4-H), 3.63 (s, 3H, OCH3), 3.73 (s, 6H, 2 OCH3), 3.76 (dd,
J ¼ 18.0, 11.6 Hz, 1H, pyrazoline C4-H), 5.45 (dd, J ¼ 11.6, 4.8 Hz,
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584
S. A. F. Rostom et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
filtered, washed with ethanol, dried, and recrystallized.
Physicochemical and analytical data are recorded in Table 7.
for 12 h, then allowed to cool to room temperature. The obtained
precipitate was filtered and washed with ethanol. The product
was then suspended in water and neutralized with conc. hydro-
chloric acid, whereupon the desired product was obtained. It
was filtered, washed with water, dried, and recrystallized.
Physicochemical and analytical data are recorded in Table 7.
IR (KBr, cmꢀ1): 1597–1594 (C N), 1238–1236, 1033–1029
–
–
(C–O–C). 1H-NMR (d, ppm) for compound 9: 3.05 (dd, J ¼ 17.2,
7.3 Hz, 1H, pyrazoline C4-H), 3.59 (s, 3H, OCH3), 3.66 (s, 6H,
2 OCH3), 3.79 (dd, J ¼ 17.2, 12.0 Hz, 1H, pyrazoline C4-H), 5.41
(dd, J ¼ 12.0, 7.3 Hz, 1H, pyrazoline C5-H), 6.03 (s, 2H, O-CH2-O),
6.57 (s, 2H, trimethoxyphenyl C2,6-H), 6.69 (t, J ¼ 7.8 Hz, 1H,
phenyl C4-H), 6.92 (d, J ¼ 7.7 Hz, 1H, benzodioxole C7-H), 6.98
(d, J ¼ 7.8 Hz, 2H, phenyl C2,6-H), 7.10–7.50 (m, 3H, phenyl C3,5-H
and benzodioxole C6-H), 7.34 (s, 1H, benzodioxole C4-H).
IR (KBr, cmꢀ1): 3198–3164 (NH), 1669–1664 (C N), 1598–1578
–
–
–
(C C), 1236–1235, 1035–1023 (C–O–C), 1123–1119, 968–965
–
(C–S). 1H-NMR (d, ppm) for compound 14: 3.61 and 3.72 (2 ꢃ s,
each 3H, 2 OCH3), 3.73 (s, 6H, 2 OCH3), 3.77 (s, 3H, OCH3), 5.03 and
5.32 (2 ꢃ d, J ¼ 4.6 Hz, each 1H, pyrimidine C4,5-H), 6.62 (s, 2H,
trimethoxyphenyl C2,6-H), 6.90 (d, J ¼ 9.2 Hz, 1H, dimethoxy-
phenyl C6-H), 7.02–7.06 (m, 2H, dimethoxyphenyl C2,5-H), 9.0,
9.80 (2 ꢃ s, each 1H, 2 NH, D2O exchangeable).
6-(3,4-Dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-3,4-
dihydropyrimidin-2(1H)-one 11
To a solution of urea (0.6 g, 10 mmol) in ethanol (20 mL) and
conc. hydrochloric acid (5 mL) was added the chalcone 1 (1.8 g,
5.0 mmol) and the reaction mixture was heated under reflux for
6 h, during which the solution turns red. After being cooled to
room temperature, the mixture was neutralized with dil. ammo-
nia solution (10%) and set aside in the refrigerator for 2 h. The
obtained fine yellow solid product was filtered, thoroughly
washed with water, dried, and recrystallized. Physicochemical
General procedure for the preparation of 2-(4-(3,4,5-
trimethoxyphenyl)-6-(3,4-dimethoxyphenyl)pyrimidin-2-
ylthio)-N-arylacetamides 16, 17 and 2-(4-
(benzo[1,3]dioxol-5-yl)-6-(3,4,5-trimethoxyphenyl)-
pyrimidin-2-ylthio)-N-arylacetamides 18, 19
To a solution of thione 14 or 15 (1.0 mmol) in dry DMF (5 mL)
containing anhydrous potassium carbonate (0.14 g, 1.0 mmol),
the appropriate 2-chloro-N-arylacetamide (1.0 mmol) was added
and the reaction mixture was stirred at room temperature for an
overnight. The mixture was poured onto crushed ice and the
obtained solid product was filtered, washed with water, dried,
and crystallized. Physicochemical and analytical data are
recorded in Table 7.
and analytical data are recorded in Table 7.
IR (KBr, cmꢀ1): 3450–3080 (NH), 1680 (C O). H-NMR (d, ppm):
1
–
–
3.86 (s, 6H, 2 OCH3), 3.89 (s, 9H, 3 OCH3), 5.46 and 6.77 (2 ꢃ d,
J ¼ 4.6 Hz, each 1H, pyrimidine C4,5-H), 6.83–7.57 (m, 5H, Ar-H),
8.14 and 8.36 (2 ꢃ s, each 1H, 2 NH, D2O exchangeable).
IR (KBr, cmꢀ1): 3254–3249 (NH), 1669–1662 (C O), 1570–1569
General procedure for the preparation of 6-(3,4-
dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1-methyl-
3,4-dihydropyrimidine-2(1H)-thione 12 and 6-(3,4-
dimethoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-1-phenyl-
–
–
–
(C N), 1300–1295, 1128–1126 (C-S-C), 1241–1237, 1034–1033
–
(C–O–C). 1H-NMR (d, ppm) for compound 17: 3.66, 3.70, 3.79
and 3.80 (4 ꢃ s, each 3H, 4 OCH3), 3.83 (s, 6H, 2 OCH3), 4.13 (s,
2H, SCH2), 6.82 (d, J ¼ 8.8 Hz, 2H, methoxyphenyl C3,5-H), 7.0 (d,
J ¼ 8.4 Hz, 1H, dimethoxyphenyl C6-H), 7.44 (d, J ¼ 8.8 Hz, 2H,
methoxyphenyl C2,6-H), 7.54 (s, 2H, trimethoxyphenyl C2,6-H),
7.82 (s, 1H, dimethoxyphenyl C2-H), 7.94 (d, J ¼ 8.4 Hz, 1H,
dimethoxyphenyl C5-H), 8.16 (s, 1H, pyrimidine C5-H), 10.09 (s,
1H, NH, D2O exchangeable).
3,4-dihydropyrimidine-2(1H)-thione 13
A mixture of the chalcone 1 (1.8 g, 5.0 mmol) and the appro-
priate substituted thiourea (10 mmol) in ethanolic sodium
hydroxide (30%, 20 mL) was heated under reflux for 8 h. The
reaction mixture was reduced to half of its original volume,
diluted with ice cold water, then acidified with dilute hydro-
chloric acid (10%) and kept in the refrigerator for 3 h. The
precipitate thus formed was filtered and washed with cold etha-
nol and recrystallized. Physicochemical and analytical data are
1H-NMR (d, ppm) for compound 18: 3.70 (s, 3H, OCH3), 3.84 (s,
6H, 2 OCH3), 4.15 (s, 2H, SCH2), 6.10 (s, 2H, O-CH2-O), 6.91 (d,
J ¼ 8.4 Hz, 1H, benzodioxole C6-H), 7.31 (d, J ¼ 8.4 Hz, 2H, chlor-
ophenyl C3,5-H), 7.53 (s, 2H, trimethoxyphenyl C2,6-H), 7.59 (d,
J ¼ 8.4 Hz, 2H, chlorophenyl C2,6-H), 7.90–7.94 (m, 2H, benzo-
dioxole C4,7-H), 8.13 (s, 1H, pyrimidine C5-H), 10.47 (s, 1H, NH,
D2O exchangeable).
recorded in Table 7.
1
IR (KBr, cmꢀ1): 3220–2980 (NH), 1190–1180 (C S). H-NMR (d,
–
–
ppm) for compound 12: 2.56 (s, 3H, CH3), 3.87 (s, 6H, 2 OCH3), 3.92
(s, 9H, 3 OCH3), 4.94 and 5.17 (2 ꢃ d, J ¼ 4.6 Hz, each 1H,
pyrimidine C4,5-H), 7.19–7.83 (m, 5H, Ar-H), 8.08 (s, 1H, NH,
D2O exchangeable).
1-[4-(3,4-Dimethoxyphenyl)-6-(3,4,5-trimethoxyphenyl)-
1H-NMR (d, ppm) for compound 13: 3.85 (s, 6H, 2 OCH3), 3.89
(s, 9H, 3 OCH3), 4.89 and 5.26 (2 ꢃ d, J ¼ 4.6 Hz, each 1H,
pyrimidine C4,5-H), 7.21–7.94 (m, 10H, Ar-H), 8.19 (s, 1H, NH,
D2O exchangeable).
1,6-dihydropyrimidin-2-yl-sulfanyl]propan-2-one 20
To a solution of 14 (0.42 g, 1.0 mmol) in dry DMF (5 mL) con-
taining anhydrous potassium carbonate (0.14 g, 1.0 mmol),
chloroacetone (0.09 g, 0.08 mL, 1.0 mmol) was added and the
reaction mixture was stirred at room temperature for an over-
night. The mixture was poured onto crushed ice and the
obtained solid product was filtered, washed with water, dried,
and crystallized. Physicochemical and analytical data are
recorded in Table 7.
4-(3,4,5-Trimethoxyphenyl)-6-(3,4-dimethoxyphenyl)-3,4-
dihydropyrimidine-2(1H)-thione 14 and 6-benzo[1,3]dioxol-
5-yl-4-(3,4,5-trimethoxyphenyl)-3,4-dihydro-1H-
IR (KBr, cmꢀ1): 3470 (NH), 1631 (C O), 1593 (C N), 1326, 1128
–
–
pyrimidine-2-thione 15
–
–
A mixture of the appropriate chalcone 1 or 2 (1.0 mmol), thio-
urea (0.08 g, 1.0 mmol), and anhydrous potassium carbonate
(0.14 g, 1.0 mmol) in ethanol (10 mL) was heated under reflux
(C-S-C), 1238, 1018 (C–O–C). 1H-NMR (d, ppm): 2.10 (s, 3H, COCH3),
3.59, 3.69, 3.70 and 3.71 (4 ꢃ s, each 3H, 4 OCH3), 3.72 (s, 5H,
OCH3 and SCH2), 5.43 and 5.47 (2 ꢃ d, J ¼ 4.55 Hz, each 1H,
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Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
Anticancer/antimicrobial polymethoxy chalcone derivatives
585
pyrimidine C4,5-H), 6.45 (s, 1H, NH, D2O exchangeable), 6.56 (s,
2H, trimethoxyphenyl C2,6-H), 6.83 (d, J ¼ 8.4 Hz, 1H, dimethoxy-
phenyl C6-H), 7.18 (s, 1H, dimethoxyphenyl C2-H), 7.20 (d,
J ¼ 8.4 Hz, 1H, dimethoxyphenyl C5-H).
NMR (d, ppm) for compound 24: 3.72 (s, 3H, OCH3), 3.86 (s, 6H,
2 OCH3), 3.91 (s, 9H, 3 OCH3), 5.82 and 5.97 (2 ꢃ d, J ¼ 4.6 Hz,
each 1H, thiazolopyrimidine C5,6-H), 6.93–7.74 (m, 9H, Ar-H), 8.77
–
(s, 1H, CH C).
–
1H-NMR (d, ppm) for compound 25: 3.67 (s, 6H, 2 OCH3), 3.79 (s,
6H, 2 OCH3), 3.86 (s, 9H, 3 OCH3), 5.79 and 6.01 (2 ꢃ d, J ¼ 4.6 Hz,
each 1H, thiazolopyrimidine C5,6-H), 7.02–7.79 (m, 8H, Ar-H), 8.80
7-(3,4-Dimethoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-2H-
thiazolo[3,2-a]pyrimidin-3(5H)-one 22
Method A:
–
(s, 1H, CH C).
–
To a solution of 14 (0.42 g, 1.0 mmol) in dry DMF (5 mL)
containing anhydrous potassium carbonate (0.14 g, 1.0 mmol),
ethyl bromoacetate (0.17 g, 0.11 mL, 1.0 mmol) was added The
reaction mixture was stirred at room temperature for an over-
night, then poured onto crushed ice. The obtained precipitate
was filtered, washed with water, dried, and crystallized.
Method B:
In-vitro anticancer activity
Preliminary in-vitro one-dose antitumor screening
Out of the newly synthesized compounds, eleven derivatives
namely 4, 6, 9, 11, 14–17, 22, 24, and 25 were selected by the
National Cancer Institute (NCI) in-vitro disease-oriented human
cells screening panel assay to be evaluated for their in-vitro anti-
cancer activity. Primary in vitro one dose anticancer assay was
performed using the full NCI 60 cell panel in accordance with the
current protocol of the Drug Evaluation Branch, NCI, Bethesda
[49–51]. These cell lines were incubated with one concentration
(10 mM) for each tested compound. A 48 h continuous drug
exposure protocol was used, and a sulphorhodamine B (SRB)
protein assay was employed to estimate cell viability or growth.
Three compounds 4, 6, and 11 passed this primary anticancer
assay and were carried over to the 5-dose screen against a panel of
about 60 different tumor cell lines. The results are presented as %
growth inhibition in Table 1.
A mixture of compound 14 (1.25 g, 3.0 mmol), chloroacetic
acid (0.43 g, 4.5 mmol), anhydrous sodium acetate (0.37 g,
4.5 mmol), and acetic anhydride (5 mL) was heated under reflux
in glacial acetic acid (10 mL) for 8 h. After being cooled to room
temperature, the reaction mixture was poured onto ice cold
water and the precipitated solid was filtered, thoroughly washed
with water, dried, and recrystallized from glacial acetic acid
containing few drops of water. Physicochemical and analytical
data are recorded in Table 7.
IR (KBr, cmꢀ1): 1723 (C O), 1593 (C N), 1254, 1127 (C-S-C),
–
–
–
–
1230, 1017 (C–O–C). 1H-NMR (d, ppm): 3.60 (s, 3H, OCH3), 3.71
(s, 6H, 2 OCH3), 3.72 and 3.73 (2 ꢃ s, each 3H, 2 OCH3), 4.06 and
4.14 (2 ꢃ d, J ¼ 16.8 Hz, each 1H, SCH2), 5.69 and 5.90 (2 ꢃ d,
J ¼ 4.6 Hz, each 1H, thiazolopyrimidine C5,6-H), 6.53 (s, 2H, tri-
methoxyphenyl C2,6-H), 6.90 (d, J ¼ 8.8 Hz, 1H, dimethoxyphenyl
C6-H), 7.26 (s, 1H, dimethoxyphenyl C2-H), 7.32 (d, J ¼ 8.8 Hz, 1H,
dimethoxyphenyl C5-H).
Full in-vitro five-dose antitumor assay
According to the protocol of the NCI in-vitro disease-oriented
human cells screening panel assay [49–51], about 60 cell lines
of nine tumor subpanels, including leukemia, non-small cell
lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast
cancer cell lines, were incubated with five concentrations (0.01–
100 mM) for each of the preliminary active compounds (4, 6, and
11), and were used to create log(concentration–% growth inhi-
[4-(3,4-Dimethoxyphenyl)-6-(3,4,5-
trimethoxyphenyl)pyrimidin-2-yl]hydrazine 23
bition) curves. Three response parameters (GI50, TGI, and LC50
)
A mixture of 22 (0.46 g, 1.0 mmol) and hydrazine hydrate 99%
(0.1 g, 0.1 mL, 2.0 mmol) in absolute ethanol (5 mL) was heated
under reflux for 3 h. After cooling, the separated crystalline
white precipitate was filtered, washed with water, dried, and
recrystallized. Physicochemical and analytical data are recorded
were calculated for each cell line. The results are recorded in
Tables 2 to 4.
In-vitro antibacterial and antifungal activities
Inhibition-zone (IZ) measurements
in Table 7. IR (KBr, cmꢀ1): 3410, 3287 (NH), 1661 (C N), 1231,
–
–
All the synthesized compounds were evaluated by the agar cup
diffusion technique [53] using a 1-mg/mL solution in DMSO. The
test organisms utilized were Staphylococcus aureus (DSM 1104) and
Bacillus subtilis (ATCC 6633) as Gram-positive bacteria; Escherichia
coli (ATCC 11775) and Pseudomonas aeruginosa (ATCC 10145) as
Gram-negative bacteria. Candida albicans (DSM 70014) was also
used as a representative for fungi. Each 100 mL of sterile molten
agar (at 458C) received 1 mL of 6 h-broth culture and then the
seeded agar was poured into sterile Petri dishes. Cups (8 mm in
diameter) were cut in the agar. Each cup received 0.1 mL of the 1-
mg/mL solution of the test compounds. The plates were then
incubated at 378C for 24 h or, in case of C. albicans, for 48 h. A
control using DMSO without the test compound was included for
each organism. Ampicillin was used as standard antibacterial,
while clotrimazole was used as antifungal reference. The result-
ing inhibition zones are recorded in Table 5.
1017 (C–O–C). 1H-NMR (d ppm): 3.72, 3.83, 3.88, 3.91 and 3.92
(5 ꢃ s, each 3H, 5 OCH3), 4.26 (br s, 2H, NH2, D2O exchangeable),
7.08 (d, J ¼ 8.4 Hz, 1H, dimethoxyphenyl C6-H), 7.55 (s, 2H, tri-
methoxyphenyl C2,6-H), 7.83 (s, 1H, dimethoxyphenyl C2-H), 7.94
(d, J ¼ 8.4 Hz, 1H, dimethoxyphenyl C5-H), 8.16 (s, 1H, pyrimi-
dine C5-H), 9.27 (s, 1H, NH, D2O exchangeable).
2-(Substituted benzylidene)-5-(3,4,5-trimethoxyphenyl)-7-
(3,4-dimethoxyphenyl)-5H-thiazolo[3,2-a]pyrimidin-3-ones
24, 25
To a solution of 22 (0.91 g, 2.0 mmol) and piperidine (3 drops) in
absolute ethanol (10 mL) was added the appropriate aldehyde
(2.0 mmol). The mixture was heated under reflux for 6 h where a
solid product partially crystallized. The reaction mixture was left
to cool and the separated solid product was filtered, washed with
cold ethanol, dried, and recrystallized. Physicochemical and
analytical data are recorded in Table 7.
Minimal inhibitory concentration (MIC) measurement
The minimal inhibitory concentrations (MIC) of the most active
compounds were measured using the twofold serial broth
IR (KBr, cmꢀ1): 1715–1712 (C O), 1585–1580 (C N), 1245–
–
–
–
–
1240, 1121–1117 (C-S-C), 1235–1232, 1015–1013 (C–O–C). 1H-
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

586
S. A. F. Rostom et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 572–587
dilution method [54]. The test organisms were grown in their
suitable broth: 24 h for bacteria and 48 h for fungi at 378C.
Twofold serial dilutions of solutions of the test compounds were
prepared using 200, 100, 50, 25, and 12.5 mg/mL. The tubes were
then inoculated with the test organisms; each 5 mL received
0.1 mL of the above inoculum and were incubated at 378C for
48 h. Then, the tubes were observed for the presence or absence
of microbial growth. The MIC values of the prepared compounds
are listed in Table 6.
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Minimal bactericidal concentration (MBC) measurement
MIC tests were always extended to measure the MBC as follows: A
loop-full from the tube not showing visible growth (MIC) was
spread over a quarter of Mu¨ller–Hinton agar plate. After 18 h of
incubation, the plates were examined for growth. Again, the
tube containing the lowest concentration of the test compound
that failed to yield growth on subculture plates was judged to
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The authors are deeply thankful to the staff members of the Department of
Health and Human Services, National Cancer Institute (NCI), Bethesda,
Maryland, U.S.A. for carrying out the anticancer screening of the newly
synthesized compounds.
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