Synthesis and bioactivity evaluation of new 6-aryl-5-cyano thiouracils as potential antimicrobial and anticancer agents

Article abstract of DOI:10.3390/molecules17089868

Several novel 6-aryl-5-cyano thiouracil derivatives were synthesized and explored for their activities as antibacterial, antifungal and anticancer agents. The antimicrobial evaluation revealed that compounds 7b and 7c possessed superior antibacterial activity against the Gram positive bacteria S. aureus and B. subtilis compared to the reference drug amoxicillin. Moreover, compound 4i was found to be a broad spectrum antimicrobial agent and it also exhibited the highest antifungal activity against C. albicans, even higher than the reference drug amphotericin B (MIC = 2.34, 3.00 μg/mL respectively). Selected compounds were tested for in vitro cytotoxicity at a single 10^-5 M concentration in accordance to the NCI (USA) protocol. The preliminary screening results showed that most of the compounds had limited cytotoxic activity against renal cancer UO-31 and/or A498 cell lines. Nevertheless, compounds 6d and 6i displayed potent growth inhibitory effect toward non-small cell lung cancer HOP-92 and leukemia MOLT-4 cell lines, respectively.

Full text of DOI:10.3390/molecules17089868

Molecules 2012, 17, 9868-9886; doi:10.3390/molecules17089868
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Bioactivity Evaluation of New 6-Aryl-5-cyano
Thiouracils as Potential Antimicrobial and Anticancer Agents
Azza Taher Taher 1 and Sahar Mahmoud Abou-Seri 2,*
1
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Cairo University,
Kasr El-Aini Street, Cairo, P.O. Box, 11562, Egypt; E-Mail: azzataher2005@yahoo.com
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University,
Kasr El-Aini Street, Cairo, P.O. Box, 11562, Egypt
* Author to whom correspondence should be addressed; E-Mail: saharshaarawy_69@yahoo.com;
Tel.: +20-2-2559-1683; Fax: +20-2-2362-8426.
Received: 16 July 2012; in revised form: 25 July 2012 / Accepted: 10 August 2012 /
Published: 17 August 2012
Abstract: Several novel 6-aryl-5-cyano thiouracil derivatives were synthesized and
explored for their activities as antibacterial, antifungal and anticancer agents. The
antimicrobial evaluation revealed that compounds 7b and 7c possessed superior
antibacterial activity against the Gram positive bacteria S. aureus and B. subtilis compared
to the reference drug amoxicillin. Moreover, compound 4i was found to be a broad
spectrum antimicrobial agent and it also exhibited the highest antifungal activity against
C. albicans, even higher than the reference drug amphotericin B (MIC = 2.34, 3.00 μg/mL
respectively). Selected compounds were tested for in vitro cytotoxicity at a single 10−5 M
concentration in accordance to the NCI (USA) protocol. The preliminary screening results
showed that most of the compounds had limited cytotoxic activity against renal cancer UO-31
and/or A498 cell lines. Nevertheless, compounds 6d and 6i displayed potent growth
inhibitory effect toward non-small cell lung cancer HOP-92 and leukemia MOLT-4 cell
lines, respectively.
Keywords: 6-aryl-5-cyano thiouracils; antibacterial; antifungal; anticancer

Molecules 2012, 17
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1. Introduction
Pyrimidines are an important component of nucleic acids and they have been used as building
blocks in pharmaceuticals for the synthesis of antiviral [1], antineoplastic [2] antibacterial and
antifungal [3] agents. Similarly, the related thiouracil derivatives are potential therapeutics as antiviral,
anticancer and antimicrobial agents [4–6]. For example, S-alkylation and N-alkylation products have
been recently reported as novel antibacterial, cytotoxic agents [7,8] and unique HIV reverse
transcriptase inhibitors [9,10]. Moreover, a literature survey revealed that the thiouracilcarbonitrile
ring system has occupied a marked position in the design and synthesis of novel chemotherapeutic
agents with remarkable antitumor and antimicrobial activities (Figure 1). In particular, 2-[(1H-
benzoimidazol-2-yl)methylthio]-4-hydroxy-6-phenylpyrimidine-5-carbonitrile (I) possessed significant
broad spectrum antiproliferative activity in vitro [11]. In addition, thiouracil quinoxaline hybrids II
demonstrated strong inhibitory effects on the EBV-EA activation with chemopreventive effect against
carcinogenesis on Raji cells [12]. On the other hand, the nitrofuran analog III displayed a distinctive
inhibitory activity against a panel of Gram positive bacteria [13]. Meanwhile, an array of 4-anilino-
and 4-hydrazinothiopyrimidine-5-carbonitriles and their condensed heterocycles exerted promising
chemotherapeutic activity as antimicrobial and anticancer agents [14–16]. Also, it was of great interest
that specifically functionalized S-aralkylated 6-aryl-5-cyano-2-thiouracils may possess specific
biological properties, including inhibition of bacterial protein translocase SecA (compound IV) [17],
hepatitis C viral NS5B RNA dependent RNA polymerase (compound V) [18] and potent antagonist of
Epac protein—a therapeutic target of cancer—(compound VI) [19].
Figure 1. Structures of some potent antimicrobial and anticancer 6-aryl-5-cyano-2-thiouracil
lead compounds.
OH
O
N
O
N
NC
NC
NC
N
NH
NH
N
H
N
O
N
N
S
S
NO2
S
N
Cl
R
III
I
II
O
N
O
O
N
NC
NC
NC
NH
NH
NH
CH3
S
N
S
S
Br
R
IV
Sec A inhibiror
V
VI
Epac antagonist
CH3
NS5B RNA-dependent
RNA polymerase inhibitor
In view of the biological significance of the above mentioned thiouracils, we herein report the
synthesis and biological evaluation of novel 6-aryl-5-cyano-2-thiouracil derivatives 6a–i and 7a–c as
potential antimicrobial and cytotoxic agents. The design of the target compounds 6a–i was based on
previous report that, for a series of 5-substituted-2-anilinopyrimidinones, a systematic increase in
antimicrobial potency was observed upon elongation of the alkyl spacer between the phenyl ring and
pyrimidinone pharmacophore from one to three carbons [20]. In analogy to this strategy, a new series

Molecules 2012, 17
9870
of thiouracil-5-carbonitile derivatives 6a–i was prepared in which structure modification was focused on
changing the aryl methyl moiety in lead compounds I, II and IV to a bromobenzoylmethyl moiety and
simultaneously introducing various substituted aryl groups at the 6 position of the thiouracil ring. The
substituent on the aryl group was selected so as to confer different lipophilic and electronic environments
on the molecules.
Furthermore, utilizing the active methylene site in 6 for incorporation of additional pharmacophoric
group, the 4-hydroxyphenylhydrazono derivatives 7a–c were prepared. Hydrazones represents an
important class of compounds that show, besides broad spectrum antitumor activity, distinguished
antifungal and antibacterial effects [21,22].
2. Results and Discussion
2.1. Chemistry
The synthetic approaches adopted to obtain the target compounds 6–9 are depicted in Schemes 1 and 2.
The structures of the newly synthesized compounds were established on the basis of their elemental
analyses and spectral data.
Scheme 1. Synthetic pathway for target compounds 4a–i and 6a–i.
O
O
N
Br
NC
Ar
NH
O
i
1
+
Br
S
N
H
S
O
Ar-CHO
+
4 a-i
H2N
NH2
5
ii
2
3
O
4a, 6a
4b, 6b
4d, 6d
4f, 6f
4h, 6h
Ar=ph,
Ar=4-Br-ph,
Ar=4-CH3-ph,
Ar=3,4,5-triOCH3-ph,
Ar=5-CH3-thiophen
Ar=4-Cl-ph
NC
Ar
Br
4c, 6c
4e, 6e
4g, 6g
4i, 6i
Ar=2,6-diCl-ph
Ar=4-OCH3-ph
Ar=5-CH3-furan
NH
N
S
O
6 a-i
Reagents and conditions: (i) anhydrous K2CO3, absolute ethanol, reflux 12 h; (ii) anhydrous
K2CO3, dry benzen, reflux, 24 h.
Scheme 2. Synthetic pathways for compounds 7a–c and 9.
OH
O
N
O
N
O
N
NH
O
NC
NC
Br
NC
Br
N
NH
N
NH
ii
i
S
S
S
O
Br
R
R
Br
6 a,c,e
7 a-c
9
O
ii
7a R= H
7b
R= Br
7c R= CH3
O
OC2H5
NC
Br
NH
N
S
O
Br
8
Reagents and conditions: (i) p-aminophenol, glacial acetic acid, NaNO2, stir at −5 °C, 30 min.
(ii) (C2H5O)3CH, acetic anhydride, reflux, 8 h.

Molecules 2012, 17
9871
Ternary condensation of ethyl cyanoacetate 1 with the appropriate aldehyde 2 and thiourea 3 in the
presence of anhydrous potassium carbonate afforded the 6-aryl-4-oxo-2-thioxo-1,2,3,4-tetrahydro-
pyrimidine-5-carbonitrile (thiouracils) 4a–i. The IR spectra of these compounds were characterized by
the presence of NH stretching bands at 3410–3124 cm−1, C≡N bands at 2214–2152 cm−1 along with
C=O bands at 1652–1625 cm−1 and C=S bands at 1253–1222 cm−1.
Selective S-alkylation of 4a–i with bromophenacyl bromide 5 to produce compounds 6a–i was
carried out in refluxing dry benzene utilizing potassium carbonate as base catalyst (Scheme 1). The IR
spectral data of compounds 6a–i displayed no absorption bands for C=S, while an additional benzoyl
1
C=O band was observed at 1735–1693 cm−1. Their H-NMR spectra revealed a singlet signal
resonating at 5.98–4.51 ppm assignable to SCH2. Compounds 6a–i may exist in one of two tautomeric
13
forms A and B (Figure 2). To distinguish between these forms, C-NMR of compounds 6b–f were
recorded. The spectra showed two carbonyl signals corresponding to the pyrimidinone C=O at
166.61–160.36 and the benzoyl C=O at 194.50–194.09 ppm. Based on literature reports [23–27], the
chemical shift of the pyrimidinone carbonyl is markedly affected by the nature of the adjacent
nitrogen. The δ values of the pyrimidinone C=O in compounds 6b–f suggest that N-(3) near to C=O is
sp3-hybridized (pyrrole type) as it is similar to that found in the methyl derivative 10 and different
from the C=O adjacent to sp2-hybridized nitrogen (pyridine type), which appears at 175–170 ppm
(compound 11) [26,27] (Figure 2). Accordingly, compounds 6a–i are found as one tautomeric form
namely, A rather B.
Figure 2. Possible tautomeric structures of compounds 6a–i and 13C-NMR chemical shifts
of reported thiouracils.
166.61-160.36
O
N
O
NC
Ar
Br
NC
Ar
Br
NH
N
S
N
H
S
O
O
B
A
194.50-194.09
175-170
164-161
CH3
O
O
N
N
N
R'
R'
R
N
S
R
S
CH3
10
11
Reaction of compound 6a, 6c or 6e with diazotized p-aminophenol in aqueous sodium hydroxide
solution at −5 °C produced the corresponding arylhydrazono derivatives 7a–c (Scheme 2). Compounds
7a–c can exist in one or more of four tautomeric structures C–F (Figure 3). Their IR spectral data seem
to be consistent more with the hydrazone structures (C or D) rather than the enolazo tautomeric forms
(E or F). For example, all compounds exhibited two carbonyl bands in the regions 1654–1632 and
1670–1660 cm−1 corresponding to the stretching vibrations of the pyrimidinone and the benzoyl

Molecules 2012, 17
9872
carbonyl groups, respectively. The low value of the wave number assigned for the latter C=O
stretching band appears to result from chelation with NH and conjugation with the C=N double bond
1
as required by hydrazone form C or D [28,29]. The H-NMR showed three exchangeable singlet
signals at the range of 9.41–13.20 ppm due to the phenolic OH, the hydrazone NH and the
pyrimidinone NH, while those derived from SCH2 were not detected. Finally, to differentiate between
the C and D tautomers (Figure 3), the 13C-NMR spectrum of 7c was recorded and compared with those
of 6b–f. The pyrimidinone C=O was detected at 161.04 ppm similar to those of 6b–f and 10. This
finding indicates that the hydrazone derivatives 7a–c exist predominantly in form C.
Figure 3. Possible tautomeric structures of compounds 7a–c.
OH
OH
O
O
NC
Ar
N
NC
Ar
N
H
NH
N
H
N
N
O
O
N
S
N
H
S
C
D
Br
Br
OH
OH
O
O
NC
Ar
N
NC
Ar
N
N
N
H
NH
N
H
O
O
N
H
S
N
S
E
F
Br
Br
Refluxing 6c with triethyl orthoformate in acetic anhydride gave the thiazolo[3,2-a]pyrimidine
derivative 9 instead of the expected ethoxy methene derivative 8, as a result of intramolecular
cyclization of the latter (Scheme 2). The 1H-NMR spectrum of the product revealed the disappearance
of SCH2 signal and the appearance of a singlet signal at 7.65 ppm assigned for the thiazole CH. A
notable feature in the 1H-NMR spectrum was the absence of the triplet-quartet pattern of the ethoxy group
and D2O exchangeable (NH) signal, which asserted the production of the cyclic thiazolo[3,2-a]pyrimidine
derivative 9.
2.2. Biological Evaluation
2.2.1. Antimicrobial Activity
The newly synthesized compounds were evaluated for their in vitro antibacterial activity against
Staphylococcus aureus ATCC 6538P, Bacillus subtilis ATCC CC33, Escherichia coli ATCC 5087 and

Molecules 2012, 17
9873
Pseudomonas aeruginosa ATCC 9027, as well as for their antifungal activity against Candida albicans
ATCC 60193 and Aspergillus niger ATCC 1718109 using the microbroth dilution method [30]. The
minimum inhibitory concentration (MIC) and concentration that inhibit 50% of microorganisms (IC50)
as measures of the microbial inhibitory activity as well as minimum bactericidal concentration (MBC)
that reflects the bactericidal activity of the tested compounds were calculated at μg/mL (Table1). The
data presented in Table 1 revealed that compounds 6a and 6c had good to fair broad spectrum
antibacterial activity, other compounds 4d, 4h, 6f, 6g, 7a–c were only active against Gram positive
strains. As for antifungal activity, compound 4g exhibited moderate activity against C. albicans and
A. niger, while 9 elicited weak antifungal activity against A. niger with Gram positive antibacterial
activity. Among the tested compounds, only compound 4i displayed pronounced broad spectrum
antibacterial and antifungal activities. On the other hand, the remaining compounds 6b, 6d, 6e, 6h and
6i had no significant activity against any of the tested strains at concentration up to 50 μg/mL (Figure 4).
Table 1. Antimicrobial activity of the synthesized compounds expressed as minimum
inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and
concentration that inhibit 50% of microorganisms (IC50) in µg /mL against the pathological
strains based on two fold serial dilution technique.
Gram positive bacteria
Gram negative bacteria
Fungi
Compound
4d
S. aureus
9.38
9.30
4.20
>50
B. subtlis
9.38
9.30
6.25
>50
E. coli
>50
P. aeruginosa C. albicans
A. niger
>50
MIC
MBC
IC50
>50
>50
>50
>50
>50
9.38
9.30
6.25
>50
>50
>50
2.34
2.30
1.17
>50
>50
>50
>50
>50
>50
> 50
> 50
> 50
>50
>50
>50
>50
>50
>50
>50
>50
4g
4h
4i
MIC
MBC
IC50
>50
>50
18.75
18.75
12.50
>50
>50
>50
>50
>50
>50
>50
>50
>50
MIC
MBC
IC50
1.17
1.56
0.40
2.34
2.30
1.17
4.69
3.80
2.30
>50
1.17
1.56
0.78
9.38
9.30
6.25
9.38
9.00
6.25
>50
>50
>50
>50
>50
>50
>50
>50
>50
MIC
MBC
IC50
9.38
9.30
3.13
18.75
18.75
12.50
>50
9.38
9.30
3.13
37.50
37.50
25.00
>50
4.69
6.25
3.13
>50
6a
6b
6c
6d
MIC
MBC
IC50
>50
>50
MIC
MBC
IC50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
MIC
MBC
IC50
37.50
37.50
12.50
>50
37.50
37.50
25.00
>50
37.50
37.50
25.00
>50
37.50
37.50
25.00
>50
> 50
> 50
> 50
>50
MIC
MBC
IC50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50

Molecules 2012, 17
9874
Table 1. Cont.
Compound
6e
Gram positive bacteria
Gram negative bacteria
Fungi
S. aureus
>50
B. subtlis
>50
E. Coli
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
NA
P. aeruginosa C. albicans
A. niger
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
37.50
50.00
37. 50
NA
MIC
MBC
IC50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
NA
8.00
NA
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
NA
NA
3.00
>50
>50
>50
>50
6f
6g
6h
6i
MIC
MBC
IC50
2.34
3.13
1.56
4.69
4.70
1.56
>50
4.69
6.25
3.13
4.69
4.70
1.56
>50
MIC
MBC
IC50
MIC
MBC
IC50
>50
>50
>50
>50
MIC
MBC
IC50
>50
>50
>50
>50
>50
>50
7a
7b
7c
9
MIC
MBC
IC50
18.75
18.75
12.50
1.17
1.17
0.78
0.19
0.20
0.15
37.50
37.50
12.50
1.25
NA
18.75
18.75
12.50
2.34
2.30
1.17
1.17
1.17
0.40
37.50
50.00
37.50
150
MIC
MBC
IC50
MIC
MBC
IC50
MIC
MBC
IC50
Amoxicillin
Gentamicin
MIC
MIC
NA
1.00
NA
NA
Amphotericin B MIC
NA
NA
1.25
Figure 4. The antimicrobial spectrum of tested compounds.

Molecules 2012, 17
9875
The structure activity correlation of the tested compounds showed that, the starting 6-aryl-5-cyano-
2-thiouracil derivatives 4d, 4g–i had moderate to potent antimicrobial activity. Both the antimicrobial
spectrum and potency of such compounds seem to be dependent on the the aryl substituent at the 6
position of the thiouracil ring. Compound 4d with a 2,6-dichlorophenyl was moderately active against
Gram positive bacteria only. An eight-fold increase in inhibitory activity against the same bacterial
isolates was observed with methylfuran analog 4h. Appending a methylthiophene to the thiouracil ring
as in 4i changed the antimicrobial spectrum from narrow to broad. S. aureus was the most sensitive
bacteria to 4i followed by B. subtilis, E. coli and P. aeruginosa. Moreover, it exhibited the highest
antifungal activity against C. albicans, even stronger than the reference drug amphotericin B (MIC = 2.34,
3.00 μg/mL respectively) and fairly potent activity against A. niger. On the other hand, modest
antifungal activity was observed for the trimethoxyphenyl congener 4g.
Considering the S-bromobenzoylmethyl thiouracil derivatives 6a–i, they proved to have no
antifungal activity. This suggested that S-alkylation has a negative impact on the antifungal activity of
the synthesized thiouracils (4g versus 6g and 4i versus 6i). On the other hand, the antibacterial activity
of such compounds appears to be affected by the nature of aryl substituent on the thiouracil scaffold.
Compound 6a with an unsubstituted phenyl group displayed good activity against S. aureus and
B. subtilis, in addition to moderate to low activity against the Gram-negative E. coli and P. aeruginosa.
Meanwhile, the presence of a phenyl-bearing mesomeric electron-donating methoxy group resulted in
compounds 6f and 6g with enhanced activity against Gram positive bacteria only. Conversely, analogs
with heteroaryl substituent 6h and 6i or phenyl attached to inductive electron-donating or electron
withdrawing atoms as in 6b–e were either inactive or weakly active against the investigated bacteria,
assuming that the electronic nature of the substituents is of major importance.
The incorporation of a 4-hydroxyphenylhydrazono moiety to the active methylene in 6a, 6c and 6e
produced 7a–c with remarkable inhibition effect against Gram positive S. aureus and B. subtilis.
Compound 7a with phenyl substituent at the 6-postion of the thiouracil ring possessed moderate
activity. However, the more lipophilic p-bromophenyl 7b and p-tolyl 7c analogs had superior
antibacterial activity compared to the reference drug amoxicillin. In particular, 7c being the most
potent antibacterial agent in this study with MIC 0.19 and 1.17 μg/mL against S. aureus and B. subtilis,
respectively, indicating that, the presence of an electron-donating methyl group is more favorable for
the antibacterial effect of this class of compounds than electron-withdrawing bromine atom. Also, the
excellent antibacterial activity of 7a–c might be attributed to the presence of 4-hydroxyphenylhydrazono
moiety. It has been reported [31–33], that phenolic compounds can sensitize the phospholipid bilayer
of the microbial cytoplasmic membrane causing increased permeability, unavailability of vital
intracellular constituents and/or impairment of bacterial enzyme systems related to energy production.
The cyclization of 6c into the corresponding thiazolo[3,2-a]pyrimidine derivative 9 affected the
microbial spectrum rather than potency. While, 6c proved to have mild broad spectrum antibacterial
activity with no antifungal activity, 9 demonstrated weak antimicrobial activities toward Gram positive
bacterial strains and the fungus A. niger. Finally, the narrow concentration difference between the
MIC and MBC of active compounds indicated that they have bactericidal effects rather than
bacteriostatic ones.

Molecules 2012, 17
9876
2.2.2. Anticancer Activity
Thirteen compounds 4d, 4g–i and 6a–i were selected by the National Cancer Institute (NCI)
Developmental Therapeutic Program (www.dtp.nci.nih.gov) to be screened for their anticancer activity
in vitro. The anticancer assays were performed in accordance with the protocol of the Drug Evaluation
Branch, NCI, Bethesda [34–36]. The compounds were first evaluated at one dose primary anticancer
assay towards a panel of approximately 60 cancer lines (concentration 10−5 M). The human tumor cell
lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, central nervous
system (CNS), ovarian, renal, prostate and breast cancers. A 48 h drug exposure protocol was used and
sulforhodamine B (SRB) protein assay was applied to estimate the cell viability and growth [37].
Results for each tested agent were reported as the percentage growth of the treated cells compared to
the untreated control cells and also, presented as mean graph of the growth present. The preliminary
screening results (Table 2) showed that most of the compounds had limited cytotoxic activity against
the tested cell lines with percentage growth ranging from 73.09 to 133.71%. Nevertheless, compounds
6d and 6i displayed potent growth inhibitory potency toward non-small cell lung cancer HOP-92 and
leukemia MOLT-4 with growth % 41.03 and 42.38, respectively.
Regarding sensitivity of individual cell lines, all the evaluated compounds exhibited a distinguished
sensitivity profile toward one or more of the renal cancer cell lines in comparison with other cell lines,
namely UO-31, A498 or 786-0. Moreover, compounds 4i and 6i restrained the growth of melanoma
LOX IMVI cancer cell line. It is noteworthy that both compounds have highly lipophilic
methylthiophen motif as substituent on thiouracil moiety, which might increase their availability and
penetration to melanoma cells. In addition, compounds 6d and 6i were found especially effective
against non-small cell lung cancer HOP-92 and leukemia MOLT-4, respectively.
Structurally, elaborating the thiouracils 4g–i with bromobenzoylmethyl moiety in 6g–i led to
compounds that had comparable or slightly better inhibitory efficacy with almost similar antitumor
spectrum. For instance, the analogue 6i exhibited a marginal activity increase toward the same
melanoma and renal cancer cell lines as 4i, in addition to remarkable high activity against MOLT-4
leukemia cell line (growth % 42.38). Interestingly, applying the same structure modification to 4d
afforded 6d with a unique broad anticancer spectrum. The latter, elicited the highest growth inhibitory
potential against seven tumor sub-panels; leukemia, colon cancer, CNS cancer, melanoma, renal
cancer and breast cancer with noticeable antiproliferative effect on non small lung cancer HOP-92
(growth % 41.03).
Eventually, it seems that the position rather than electronic nature of substituents on 6-aryl group of
thiouracil ring had a prominent effect on the activity profile of compounds 6a–i. The steric hindrance
generated by the ortho-ortho dichloro substituents on the 6-aryl in 6d can create constrain and impose
particular orientation of the aryl group relative to the rest of the molecule. Such conformation might be
favorable to cytotoxicity and could explain the observed activity of 6d. In brief, the coexistence of
methylthiophene or 2,6-dichlophenyl at position 6 and bromobezoylmethyl moiety at position 2 of the
thiouracil ring increases the cytotoxic activity against specific cell lines.

Molecules 2012, 17
9877
Table 2. Cytotoxic activity of the tested compounds against a panel of 60 cancer cell lines
at 10 µM concentration.
Compound
Mean
growth, %
102.90
Range of
Panel
Most sensitive cell lines
growth, %
growth, %
4d
4g
4h
4i
76.85–126.64
81.19–121.32
82.37–133.71
76.99–127.08
Renal Cancer
Renal Cancer
Renal Cancer
Melanoma
UO-31 (76.85)
102.53
A498 (81.19)
104.95
UO-31 (82.37)
105.24
L OX IMVI (76.99)
Renal Cancer
Renal Cancer
Renal Cancer
Renal Cancer
Leukemia
786-0 (83.54) , UO-31 (84.31)
A498 (85.49) , UO-31 (80.39)
A498 (80.27)
6a
6b
6c
6d
102.14
103.26
102.26
89.03
80.39–119.48
80.27–121.43
80.07–117.90
41.03–118.60
A498 (80.07)
HL-60(TB) (75.34), K-562
(73.85), MOLT-4 (73.33),
RPMI-8226 (72.35), SR (79.59).
EKVX (79.94), HOP-92 (41.03),
NCI-H522 (75.25).
Non Small Cell Lung
Cancer
HCT-116 (74.54),
HTC-15 (80.89).
Colon Cancer
CNS Cancer
Melanoma
SF-295 (81.77).
SK-MEL-2 (82.18),
UACC-62 (80.12)
Renal Cancer
780-0 (73.64), A498 (75.93),
CAKI-1 (71.34), UO-31 (80.25).
MCF-7 (83.81), T-47D (74.23),
MDA-MB-468 (73.27)
UO-31 (82.81)
Breast Cancer
6e
6f
101.92
102.30
82.81–119.49
78.50–121.80
Renal Cancer
Non-Small Cell Lung
Cancer
HOP-92 (85.86)
Renal Cancer
Renal Cancer
Renal Cancer
Leukemia
A498 (78.50), UO-31 (86.29)
A498 (84.19), UO-31 (83.81)
UO-31 (73.09)
6g
6h
6i
102.98
101.72
102.41
83.81–124.78
73.09–114.65
42.38–119.33
MOLT-4 (42.38)
Melanoma
L OX IMVI (75.06)
Renal Cancer
786-0 (81.16), UO-31 (80.26)
3. Experimental
3.1. Chemistry
Melting points are uncorrected and determined in one end open capillary tubes using Gallenkamp
melting point apparatus MFB-595-010M (Gallenkamp, London, England). Microanalysis was carried
out at Micro-analytical Unit, Regional Centre for Microbiology and Biotechnology, Al-Azhar
University. Infrared spectra were recorded on a Shimadzu FT-IR 8400S spectrophotometer (Shimadzu,
Kyoto, Japan), using potassium bromide discs and results are expressed in wave numbers (cm−1). The
NMR spectra were recorded on a Varian Mercury VX-300 NMR spectrometer. 1H- spectra were run at

Molecules 2012, 17
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300 MHz and 13C- spectra were run at 75.46 MHz in dimethylsulphoxide (DMSO-d6). Chemical shifts
are quoted in δ and were related to that of the solvents. Mass spectra were recorded using a Hewlett
Packard Varian (Varian, Palo Alto, CA, USA) and Shimadzu Gas Chromatograph Mass Spectrometer-
QP 1000 EX (Shimadzu, Kyoto, Japan). TLC were carried out using Art.DC-Plastikfolien, Kieselgel
60 F254 sheets (Merck, Darmstadt, Germany), the developing solvents were chloroform/methanol
(9:1) and the spots were visualized at 366 and 254 nm by UV Vilber Lourmat 77,202 (Vilber, Marne
La Vallee, France). Compounds 4a–c and 4e–g were obtained according to the reported procedures
[15,18,38–40], while compound 6a is commercially available.
6-Aryl-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitriles 4a–i.
A
mixture of ethyl
cyanoacetate 1 (1.13 g, 1.07 mL, 0.01 mol), thiourea 3 (0.76 g, 0.01mol), anhydrous potassium
carbonate (2.07 g, 0.015 mol) and the appropriate aromatic aldehyde 2 (0.01 mol) in absolute ethanol
(25 mL) was heated under reflux for 12 h. The reaction mixture was allowed to cool and the formed
precipitate was filtered. The residue was triturated with water and neutralized with acetic acid. The
precipitate was filtered, washed twice with cold water (20 mL), dried, and crystallized from ethanol.
6-(2,6-Dichlorophenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4d): Yield 55%,
m.p: 261–263 °C. 1H-NMR: δ: 7.48–7.60 (3H, m), 7.63 (1H, d, J = 7.8), 7.86 (1H, d, J = 6.9). 13C-NMR
δ: 125.03, 128.18, 128.37, 129.09, 130.97, 137.35, 159.94, 160.00, 176.13. IR cm−1: 3344 and 3124
(NH), 3012 (CH aromatic), 2939 and 2900 (CH aliphatic), 2206 (C≡N), 1631 (C=O), 1253 (C=S).
Anal. Calcd. for C11H5Cl2N3OS (298.15): C, 44.31; H, 1.69; N, 14.09. Found: C, 44.61; H, 1.77; N, 14.28.
4-Oxo-2-thioxo-6-(3,4,5-trimethoxyphenyl)-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4g) [15]:
Yield 62%, m.p: 250–252 °C, (reported m.p: 245–247 °C). 1H-NMR δ: 3.73 (3H, s), 3.82 (6H, s), 6.61
13
(2H, s), 11.53 (2H, br s). C-NMR δ: 55.69, 60.05, 84.84, 106.27, 119.05, 132.64, 138.95, 152.13,
152.54, 162.54, 166.65, 182.56. IR cm−1: 3344 and 3294 (NH), 3136 (CH aromatic), 2954 and 2839
(CH aliphatic), 2152 (C≡N), 1627 (C=O), 1246 (C=S). Anal. Calcd. for C14H13N3O4S (319.34): C,
52.66; H, 4.10; N, 13.16. Found: C, 52.86; H, 4.23; N, 13.52.
6-(5-Methylfuran-2-yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4h): Yield 48%,
m.p: 242 °C decomp. 1H-NMR δ: 2.36 (3H, s), 6.33 (1H, d, J = 2.2), 7.20 (1H, d, J = 2.2), 11.47 (2H, br
s, D2O exchangeable). IR cm−1: 3394, br (NH), 3039 (CH aromatic), 2924 and 2854 (CH aliphatic),
2214 (C≡N), 1652 (C=O), 1222 (C=S). Anal. Calcd. for C10H7N3O2S (233.03): C, 51.49; H, 3.02; N,
18.02. Found: C, 51.62; H, 3.14; N, 18.09.
6-(5-Methylthiophen-2-yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (4i): Yield 45%,
m.p: 201–203 °C. 1H-NMR δ: 2.48 (3H, s), 6.89 (1H, d, J = 3.6), 7.44 (1H, d, J = 3.9), 11.45 (2H, br s,
D2O exchangeable). IR cm−1: 3410, br (NH), 3093 (CH aromatic), 2920 and 2854 (CH aliphatic), 2210
(C≡N), 1625 (C=O), 1242 (C=S). Anal. Calcd. for C10H7N3OS2 (249.31): C, 48.18; H, 2.83; N, 16.85.
Found: C, 48.60; H, 2.90; N, 17.31. MS m/z: 249 [M]+.
6-Aryl-2-(4-bromobenzoylmethylthio)-4-oxo-3,4-dihydropyrimidine-5-carbonitriles 6a–i. To a solution
of thiouracil derivatives 4a–i (0.01 mol) in dry benzene (10 mL) was added anhydrous potassium

Molecules 2012, 17
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carbonate (4.14 g, 0.03 mol) and bromophenacyl bromide 5 (2.78 g, 0.01 mol). The mixture was
heated under reflux for 24 h. Then, the solvent was reduced under vacuum and the reaction mixture was
cooled. The precipitate was filtered, washed twice with cold water, dried and crystallized from methanol.
2-(4-Bromobenzoylmethylthio)-4-oxo-6-phenyl-3,4-dihydropyrimidine-5-carbonitrile (6a): Yield 68%,
m.p: 242–244 °C. IR cm−1: 3429, br (NH), 3086 (CH aromatic), 2920 and 2854 (CH aliphatic), 2194
(C≡N), 1701 and 1631 (C=Os). Anal. Calcd. for C19H12BrN3O2S (426.29): C, 53.53; H, 2.84; N, 9.86.
Found: C, 53.42; H, 2.64; N, 10.14. MS m/z: 425 [M−1]+.
2-(4-Bromobenzoylmethylthio)-6-(4-chlorophenyl)-4-oxo-3,4-dihydropyrimidine-5-carbonitrile (6b):
1
Yield 62%, m.p: 248–250 °C. H-NMR δ: 4.55 (2H, s), 7.40 (2H, d, J = 8.1), 7.63 (2H, d, J = 8.1),
13
7.71 (2H, d, J = 8.4), 7.94 (2H, d, J = 8.4), 11.75 (1H, br s, D2O exchangeable). C-NMR δ: 37.26,
89.06, 119.69, 127.21, 127.92, 129.73, 130.24, 131.60, 134.32, 135.25, 136.12, 165.49, 169.91,
170.69, 194.15. IR cm−1: 3371, br (NH), 3105 (CH aromatic), 2916 (CH aliphatic), 2194 (C≡N), 1697
and 1650 (C=Os). Anal. Calcd. for C19H11BrClN3O2S (460.73): C, 49.53; H, 2.41; N, 9.12. Found: C,
49.46; H, 2.61; N, 9.42. MS m/z: 459 [M]+, 461 [M+2]+, 463 [M+4]+.
6-(4-Bromophenyl)-2-(4-bromobenzoylmethylthio)-4-oxo-3,4-dihydropyrimidine-5-carbonitrile (6c):
Yield 68%, m.p: 250–252 °C. 1H-NMR δ: 4.55 (2H, s), 7.52 (2H, d, J = 8.1), 7.59–7.82 (4H, m), 7.93
(2H, d, J = 8.4), 11.70 (1H, br s). 13C-NMR δ: 37.26, 88.97, 119.71, 123.085, 123.54, 127.23,
128.41, 129.77, 130.19, 130.85, 131.59, 135.20, 162.37, 169.86, 170.72, 194.09. IR cm−1: 3421, br
(NH), 3050 (CH aromatic), 2916 (CH aliphatic), 2194 (C≡N), 1693-1628 (C=Os). Anal. Calcd. for
C19H11Br2N3O2S (505.18): C, 45.17; H, 2.19; N, 8.32. Found: C, 45.19; H, 2.34; N, 8.47. MS m/z: 504
[M+1]+, 505 [M+2]+, 506 [M+3]+, 507 [M+4]+.
2-(4-Bromobenzoylmethylthio)-6-(2,6-dichlorophenyl)-4-oxo-3,4-dihydropyrimidine-5-carbonitrile (6d):
1
Yield 65%, m.p: > 300 °C. H-NMR δ: 5.98 (2H, s), 7.23 (3H, m), 7.37 (4H, d, J = 7.8), 8.74 (1H, s,
13
D2O exchangeable). C-NMR δ: 107.49, 116.46, 119.40, 123.17, 127.45, 128.39, 131.11, 132.54,
133.15, 135.26, 141.72, 147.03, 163.17, 169.64, 194.50. IR cm−1: 3197, br (NH), 3128 (CH aromatic),
2974 (CH aliphatic), 2160 (C≡N), 1735 and 1619 (C=Os). Anal. Calcd. for C19H10BrCl2N3O2S
(495.18): C, 46.09; H, 2.04; N, 8.49. Found: C, 46.13; H, 2.33; N, 8.86. MS m/z: 493 [M]+, 494
[M+1]+, 495 [M+2]+, 496 [M+3]+.
2-(4-Bromobenzoylmethylthio)-4-oxo-6-p-tolyl-3,4-dihydropyrimidine-5-carbonitrile (6e): Yield 70%,
1
m.p: 262–264 °C. H-NMR δ: 2.32 (3H, s), 4.89 (2H, s), 7.07 (2H, d, J = 8.1), 7.50 (2H, d, J = 8.1),
13
7.75 (2H, d, J = 8.4), 7.95 (2H, d, J = 8.4), 11.30 (1H, br s, D2O exchangeable). C-NMR δ: 20.85,
37.23, 88.78, 120.06, 127.19, 128.13, 128.43, 130.25, 131.59, 134.57, 135.25, 139.22, 166.61, 170.27,
170.43, 194.23. IR cm−1: 3387, br (NH), 3012 (CH aromatic), 2916 (CH aliphatic), 2194 (C≡N), 1701
and 1631 (C=Os). Anal. Calcd. for C20H14BrN3O2S (440.31): C, 54.56; H, 3.20; N, 9.54. Found: C,
54.83; H, 3.29; N, 9.82. MS m/z: 439 [M]+, 441 [M+2]+.
2-(4-Bromobenzoylmethylthio)-6-(4-methoxyphenyl)-4-oxo-3,4-dihydropyrimidine-5-carbonitrile (6f):
Yield 75%, m.p: 285–287 °C. 1H-NMR δ: 3.82 (3H, s), 4.56 (2H, s), 6.87 (2H, d, J = 6.9), 7.63 (2H, d,

Molecules 2012, 17
9880
J = 6.9), 7.72 (2H, d, J = 6.9), 7.95 (2H, d, J = 6.6), 11.55 (1H, br s). 13C-NMR δ: 37.27, 55.20, 88.25,
113.18, 120.35, 127.24, 129.56, 129.64, 129.66, 130.33, 131.66, 135.25, 160.36, 166.02, 170.36,
194.22. IR cm−1: 3394, br (NH), 3012 (CH aromatic), 2916 (CH aliphatic), 2194 (C≡N), 1697 and
1635 (C=Os). Anal. Calcd. for C20H14BrN3O3S (456.31): C, 52.64; H, 3.09; N, 9.21. Found: C, 52.28;
H, 3.19; N, 9.53. MS m/z: 455 [M]+, 457 [M+2]+.
2-(4-Bromobenzoylmethylthio)-4-oxo-6-(3,4,5-trimethoxyphenyl)-3,4-dihydropyrimidine-5-carbonitrile
(6g): Yield 55%, m.p: > 300 °C. 1H-NMR δ: 3.80 (3H, s), 3.82 (3H, s), 3.84 (3H, s), 5.23 (2H, s), 7.06
(1H, s), 7.19 (1H, s), 7.39 (2H, d, J = 8.7), 7.85 (2H, d, J = 8.7), 10.10 (1H, br s, D2O exchangeable).
IR cm−1: 3425, br (NH), 3012 (CH aromatic), 2924 (CH aliphatic), 2200 (C≡N), 1701 and 1635
(C=Os). Anal. Calcd. for C22H18BrN3O5S (516.36): C, 51.17; H, 3.51; N, 8.14. Found: C, 51.29; H,
3.22; N, 8.54. MS m/z: 515 [M]+.
2-(4-Bromobenzoylmethylthio)-6-(5-methylfuran-2-yl)-4-oxo-3,4-dihydropyrimidine-5-carbonitrile (6h):
1
Yield 40%, m.p: 192 °C decomp. H-NMR δ: 2.33 (3H, s), 4.55 (2H, s), 7.15 (1H, d, J = 3.6), 7.42
(2H, d, J = 8.1), 7.54 (1H, d, J = 3.6), 7.77 (2H, d, J = 8.1), 11.50 (1H, br s, D2O exchangeable). IR
cm−1: 3441, br (NH), 3089 (CH aromatic), 2912 (CH aliphatic), 2218 (C≡N), 1693 and 1658 (C=Os).
Anal. Calcd. for C18H12BrN3O3S (430.28): C, 50.25; H, 2.81; N, 9.77. Found: C, 50.18; H, 2.76; N,
9.97. MS m/z: 429 [M]+, 431 [M+2]+.
2-(4-Bromobenzoylmethylthio)-6-(5-methylthiophen-2-yl)-4-oxo-3,4-dihydropyrimidine-5-carbonitrile
1
(6i): Yield 45%, m.p: 196–198 °C. H-NMR δ: 2.37 (3H, s), 4.51 (2H, s), 6.88 (1H, d, J = 3.9), 7.39
(2H, d, J = 8.1), 7.69 (1H, d, J = 3.9), 7.97 (2H, d, J = 8.1), 11.40 (1H, br s, D2O exchangeable).
IR cm−1: 3421, br (NH), 3043 (CH aromatic), 2916 (CH aliphatic), 2210 (C≡N), 1700 and 1616
(C=Os). Anal. Calcd. for C18H12BrN3O2S2 (446.34): C, 48.44; H, 2.71; N, 9.41. Found: C, 48.43; H,
2.61; N, 9.73. MS m/z: 445 [M]+, 447 [M+2]+.
6-Aryl-5-cyano-4-oxo-3,4-dihydropyrimidin-2-yl
2-(4-bromophenyl)-N'-(4-hydroxyphenyl)-2-oxo-
ethanehydrazonothioates 7a–c. An ice cold diazonium salt solution of p-aminophenol (prepared
from p-aminophenol (1.09 g, 0.01 mol), glacial acetic acid (4 mL) and sodium nitrite (0.69 g,
0.01 mol) in water (15 mL)) was added to a chilled solution of the appropriate 6-Aryl-2-(4-
bromobenzoylmethylthio)-4-oxo-3,4-dihydropyrimidine-5-carbonitrile 6a, 6c or 6e (0.01 mol) and
sodium hydroxide (1.6 g, 0.04 mol) in water (25 mL). The reaction mixture was maintained at −5 °C
with continuous stirring for 30 min, and then acidified with glacial acetic acid till pH 5-5.5. The
resulting solid was filtered, washed twice with water, dried and crystallized from methanol.
5-Cyano-4-oxo-6-phenyl-3,4-dihydropyrimidin-2-yl 2-(4-bromophenyl)-N'-(4-hydroxyphenyl)-2-oxo-
1
ethanehydrazonothioate (7a): Yield 65%, m.p: 188–190 °C. H-NMR δ: 6.86 (2H, d, J = 8.4), 7.43
(2H, d, J = 6.9), 7.54–7.64 (5H, m), 7.66 (4H, d, J = 8.1), 10.11 (1H, br s, D2O exchangeable), 11.85
(1H, s, D2O exchangeable), 13.20 (1H, s, D2O exchangeable). IR cm−1: 3417–3252 (NH and OH),
3086 (CH aromatic), 2210 (C≡N), 1660 and 1640 (C=Os). Anal. Calcd. for C25H16BrN5O3S (546.40):
C, 54.95; H, 2.95; N, 12.82. Found: C, 54.93; H, 3.03; N, 12.97. MS m/z: 547 [M+2]+, 549 [M+4]+.

Molecules 2012, 17
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6-(4-Bromophenyl)-5-cyano-4-oxo-3,4-dihydropyrimidin-2-yl 2-(4-bromophenyl)-N'-(4-hydroxyphenyl)-
2-oxoethanehydrazonothioate (7b): Yield 60%, m.p: 240 °C decompose. 1H-NMR δ: 7.41 (2H, d, J = 8.4),
7.63–7.75 (8H, m), 7.92 (2H, d, J = 8.7), 10.00 (1H, br s, D2O exchangeable), 10.70 (1H, br s, D2O
exchangeable), 11.85 (1H, s, D2O exchangeable). IR cm−1: 3417–3300 (NH and OH), 3089 (CH
aromatic), 2214 (C≡N), 1670 (C=O), 1654 (C=O). Anal. Calcd. for C25H15Br2N5O3S (625.29): C,
48.02; H, 2.42; N, 11.20. Found: C, 48.11; H, 2.45; N, 11.37. MS m/z: 623 [M]+, 625 [M+2] +.
Cyano-4-oxo-6-p-tolyl-3,4-dihydropyrimidin-2-yl
2-(4-bromophenyl)-N'-(4-hydroxyphenyl)-2-oxo-
1
ethanehydrazonothioate (7c): Yield 58%, m.p: 231 °C decompose. H-NMR δ: 2.32 (3H, s), 7.07 (2H,
d, J = 8.1), 7.34 (2H, d, J = 8.4), 7.51 (2H, d, J = 8.1), 7.72 (2H, d, J = 8.7), 7.85 (2H, d, J = 8.4), 7.92
13
(2H, d, J = 8.7), 9.41 (1H, br s), 10.05 (1H, s), 11.80 (1H, s). C-NMR δ: 21.01, 92.45, 115.79,
127.82, 128.42, 128.72, 129.12, 130.23, 131.23, 131.64, 131.74, 131.80, 134.52, 134.81, 141.85,
161.04, 165.03, 166.81, 192.15. IR cm−1: 3417–3124 (NH and OH), 3066 (CH aromatic), 2214 (C≡N),
1660 (C=O), 1632 (C=O). Anal. Calcd. for C26H18BrN5O3S (560.42): C, 55.72; H, 3.24; N, 12.50.
Found: C, 55.78; H, 3.31; N, 12.66. MS m/z: 561 [M+2]+.
2-(4-Bromobenzoyl)-5-(4-bromophenyl)-7-oxo-7H-thiazolo[3,2-a]pyrimidine-6-carbonitrile 9. A mixture
of 6-(4-bromophenyl)-2-(4-bromobenzoylmethylthio)-4-oxo-3,4-dihydropyrimidine-5-carbonitrile 6c
(0.01 mol) and triethyl orthoformate (1.48 g, 1.3 mL, 0.01 mol) in acetic anhydride (10 mL) was
heated under reflux with stirring for 8 h. The solvent was concentrated under reduced pressure and the
reaction mixture was left overnight. The formed solid was collected, dried and crystallized from
aqueous methanol. Yield 40%, m.p: 170–172 °C. 1H-NMR δ: 7.60 (4H, d, J = 8.4), 7.65 (1H, s), 7.82
(4H, d, J = 8.4). IR cm−1: 3086 (CH aromatic), 2210 (C≡N), 1660 (C=O), 1640 (C=O). Anal. Calcd.
for C20H9Br2N3O2S (515.18): C, 46.63; H, 1.76; N, 8.16. Found: C, 46.71; H, 1.82; N, 8.28. MS m/z:
513 [M]+, 515 [M+2]+.
3.2. Biological Evaluation
3.2.1. Determination of the Antimicrobial Activitiess
The antimicrobial activity expressed as MIC, MBC and IC50 of tested compounds were determined
against four reference bacterial strains; Staphylococcus aureus ATCC 6538P, Bacillus subtilis ATCC
CC33, Escherichia coli ATCC 5087, Pseudomonas aeruginosa ATCC 9027 as well as against two
fungi strains; Candida albicans ATCC 60193 and Aspergillus niger ATCC 1718109. Amoxicillin,
gentamicin and amphotericin B were used as positive control. All assays were conducted in triplicate
under strict aseptic conditions.
3.2.1.1. Determination of the Minimum Inhibitory Concentration (MIC)
The preliminary MICs were firstly determined by the microbroth dilution method [30]. Briefly, 100 µL
of double strength DMSO (Sigma-Aldrich, Munich, Germany) were placed in each well of a 96-well
microtiter plate. Aliquot of 100 µL of the solutions to be tested were added to the first column. Then
2-fold dilutions were carried out from one well to the next up to final well in each row for each
tested compound.

Molecules 2012, 17
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MICs were then determined using agar streaking technique as per Clinical Laboratory Standard
Institute guidelines [30]. A total of 15 mL molten (45 °C) Neutrient agar (Sigma-Aldrich) were
supplemented with the required concentration then were added into sterilized Petri dishes, allowed to
solidify. Then 10 µL of each bacterial or fungal suspension (105 CFU mL−1) were streaked onto the
surface. Finally all plates were incubated at 37 °C for 24 h for bacterial strains and 25 °C for 48 h
fungal strains under aerobic conditions. MIC was determined as the average between the last plate had
growth and the first plate with no growth.
3.2.1.2. Determination of the MBC and IC50
MBC and IC50 were determined in 96 well microtiter plate where a 100 µL of trypcase soya broth
(Oxoid, Lenexa, KS, USA) for bacterial isolates or sabaroud's dextrose broth for fungal strains were
placed in each well. A proper amount of the stock solution of the tested compounds was added to reach
the desired concentration. All columns were then inoculated with 20 µL of bacterial suspension
(106 CFU mL−1) and incubated for 5–6 h. An aliquot of 100 µL from each well was transferred into
another pre-supplemented with 100 µL f Dey-engly broth medium (Fluka, St. Louis, MO, USA) and
allowed to stand for 10–20 min to neutralized any antimicrobial activities. Then these neutralized
solutions were subjected to proper dilutions and streaked onto trypcase soya agar or sabaroud's
dextrose agar plates to determine the viable count [41]. Controls were done for sterility and growth and
subjected to the same regimen of treatment. MBC was determined as the lowest concentration which
decreased the number of viable bacteria by 3 log units. IC50 was determined as the lowest
concentration reduced the viable count by about 50%.
3.2.2. Anticancer Activity [37]
The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium
containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells are
inoculated into 96 well microtiter plates in 100 µL at plating densities ranging from 5,000 to 40,000
cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter
plates are incubated at 37 °C, 5% CO2, 95% air and 100% relative humidity for 24 h prior to addition
of experimental drugs. After 24 h, two plates of each cell line are fixed in situ with trichloroacetic acid
(TCA), to represent a measurement of the cell population for each cell line at the time of drug addition
(Tz). Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum
test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen
concentrate is thawed and diluted to twice the desired final maximum test concentration with complete
medium containing 50 µg/mL gentamicin. Aliquots of 100 µL of the compound dilution is added to the
appropriate microtiter wells already containing 100 µL of medium, resulting in the required final
compound concentrations. Following compound addition, the plates are incubated for an additional 48 h
at 37 °C, 5% CO2, 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by
the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 µL of cold 50% (w/v)
TCA (final concentration, 10% TCA) and incubated for 60 min at 4 °C. The supernatant is discarded,
and the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution
(100 µL) at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 minutes

Molecules 2012, 17
9883
at room temperature. After staining, unbound dye is removed by washing five times with 1% acetic
acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and
the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells,
the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of
the wells by gently adding 50 µL of 80% TCA (final concentration, 16% TCA). Using the seven
absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug
at the five concentration levels (Ti)], the percentage growth is calculated at each of the drug
concentrations levels. Percentage growth inhibition is calculated as: [(Ti-Tz)/(C-Tz)] × 100 for
concentrations for which Ti >/= Tz or [(Ti-Tz)/Tz] × 100 for concentrations for which Ti<Tz.
4. Conclusions
This study reports the synthesis of 6-aryl-5-cyanothiouracil based compounds 4a–i, 6a–i, 7a–c and
9 as potential antimicrobial and antitumor agents. Several newly synthesized derivatives displayed
promising antimicrobial activity compared to the reference drugs, amoxicillin, gentamicin and
amphotericin B. The activity against gram positive bacteria S. aureus and to a lesser extent B. subtilis
was a characteristic of the majority of active compounds. It can be stated that final compounds 6f and
6g with mesomeric electron donating methoxy substituents on the phenyl at the 6 position of the
thiouracil scaffold were found more active than the other analogs against both Gram positive strains. In
addition, the introduction of 4-hydroxyphenylhdrazono moiety in 7b and 7c contributed to excellent
potency toward the same bacterial strains. On the other hand, in vitro cytotoxicity screening of selected
compounds 4d, 4g–i and 6a–i at a single concentration of 10−5 M—revealed that most of the
compounds had limited cytotoxic activity against renal cancer UO-31 or A498 cell lines. However,
compounds 6d and 6i exhibited potent growth inhibitory effect toward non-small cell lung cancer
HOP-92 and leukemia MOLT-4 cell lines, respectively.
Acknowledgments
The authors are grateful to the staff members of Biotechnology Center, Faculty of Pharmacy,
Cairo University for running the antimicrobial screening. The authors would like to express their
sincere thanks to the NCI staff, Bethesda, MD, USA for performing the antitumor testing of the
synthesized compounds.
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Sample Availability: Samples of the compounds 6a–i, 7a–c are available from the authors.
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