Synthesis and biological evaluation of some novel polysubstituted pyrimidine derivatives as potential antimicrobial and anticancer agents

Article abstract of DOI:10.1002/ardp.200800223

Synthesis and evaluation of the antimicrobial and cytotoxic activity of two series of polysubstituted pyrimidines comprising the thioether functionality and other pharmacophores, reported to contribute to various chemotherapeutic activities are described.

Full text of DOI:10.1002/ardp.200800223

Arch. Pharm. Chem. Life Sci. 2009, 342, 299–310
S. A. F. Rostom et al.
299
Full Paper
Synthesis and Biological Evaluation of Some Novel
Polysubstituted Pyrimidine Derivatives as Potential
Antimicrobial and Anticancer Agents*
Sherif A. F. Rostom1, Hayam M. A. Ashour2, and Heba A. Abd El Razik2
1 Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
Synthesis and evaluation of the antimicrobial and cytotoxic activity of two series of polysubsti-
tuted pyrimidines comprising the thioether functionality and other pharmacophores, reported
to contribute to various chemotherapeutic activities are described. All newly synthesized com-
pounds were subjected to in-vitro antibacterial and antifungal screening. Out of the compounds
tested, 18 derivatives displayed an obvious inhibitory effect on the growth of the tested Gram-
positive and Gram-negative bacterial strains, with special effectiveness against the Gram-positive
strains. Compounds 1, 2, 6, 7, 9, 10, 11, 21, and 24 revealed remarkable broad antibacterial spec-
trum profiles. Among those, compounds 1, 2, 6, 7, 9, and 24 exhibited an appreciable antifungal
activity against C. albicans. Compound 2 proved to be the most active antimicrobial member
identified here as it showed twice the activity of ampicillin against B. subtilis and the same activ-
ity of ampicillin against M. Luteus and P. aeruginosa together with a moderate antifungal activity.
Further, eleven analogs were evaluated for their in-vitro cytotoxic potential utilizing the stand-
ard MTT assay against a panel of three human cell lines: breast adenocarcinoma MCF7, hepato-
cellular carcinoma HePG2, and colon carcinoma HT29. The obtained data revealed that six of
the tested compounds 1, 3, 7, 12, 13, and 15 showed a variable degree of cytotoxic activity
against the tested cell lines at both the LC50 and LC90 levels. Compound 7 proved to be the most
active cytotoxic member in this study with special effectiveness against the colon carcinoma
HT29 and breast cancer MCF7 human cell lines for LC50 and LC90. Thus, compounds 1 and 7 could
be considered as possible dual antimicrobial-anticancer agents.
Keywords: Antibacterial / Anticancer activity / Antifungal / Pyrimidines / Thioether /
Received: December 7, 2008; accepted: February 5, 2009
DOI 10.1002/ardp.200800223
resistant Staphylococcus aureus (MRSA) and vancomycin-
resistant Enterococcus faecium (VRE) [1, 2]. Moreover, there
has been a rapid spread in primary and opportunistic
fungal infections particularly Candida albicans, because
of the increased number of immuno-compromised
patients suffering from AIDS, cancer, and organ trans-
plantation [3, 4]. Consequently, such types of infections
continue to provide impetus for the search and discovery
of novel, more potent, and selective non-traditional anti-
Introduction
Over the past two decades, the health benefits ascribed to
commercially available antimicrobials became doubtful,
since many commonly used antibiotics have become less
effective against certain bacterial infections, not only
because of the toxic reactions they produce, but also due
to emergence of drug resistant bacteria like methicillin-
Correspondence: Sherif A. F. Rostom, Ph.D., Department of Chemistry,
Faculty of Science, King Abdulaziz University, PO Box 80203, Jeddah
21589, Saudi Arabia.
E-mail: sherifrostom@yahoo.com
Fax: +9662-6400000-22327
* This work was presented in the 11th Ibn-Sina International Conference
on Pure and Applied Heterocyclic Chemistry, in Cairo, Egypt, December
13–16, 2008.
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S. A. F. Rostom et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 299–310
microbial agents so that no cross-resistance with the
present therapeuticals can take place.
A literature survey revealed that many pyrimidine-5-
carbonitriles [28, 29] and pyrimidinethione derivatives
On the other hand, the increasing number of neoplas- [30] proved to exhibit potent anticancer as well as antimi-
tic diseases together with the accompanied high mortal- crobial activities. Moreover, it has been well documented
ity rates [5] has stimulated an unprecedented level of that incorporation of alkoxy substituents (methoxy
research directed towards the search for new structure and / or benzyloxy moieties) results in significant
leads that might be of use in designing novel antitumor enhancement of several biological activities due to the
drugs.
magnification of compounds' lipophilicity [31–34]. Con-
Patients with neoplastic disorders that are subjected to sequently, the newly synthesized compounds were
chemotherapeutic treatment are highly susceptible to designed so as to constitute essentially a pyrimidine-
microbial infections due to subsequent lack of immun- thione ring system substituted basically with a cyano
ity. Co-administration of multiple drugs for treating group and a highly lipophilic alkoxylated aryl moiety.
patients suffering from cancer disease accompanied with The thrust in derivatization of such scaffold was focussed
microbial infections might inflect some added health on the thione function that was linked to a variety of
problems especially in patients with impaired liver and /
pharmacologically active substituents and functional-
or kidney functions. Therefore, the concept of monother- ities through -CH2- or -CH2CO- bridges, or directly hooked
apy by a single drug which possesses dual utility might to the sulfur atom to produce the target thioethers. Thio-
be advantageous from both therapeutic and cost-effective ethers were found to show enhanced antimicrobial and
stand points.
antitumor activities [35–37], beside being analogs of S-
Among the pharmacologically most interesting chemi- DABOs (dihydro-alkylthio-benzyl-oxopyrimidines) which
cal scaffolds, are pyrimidines, widely spread in many nat- possess antiproliferative as well as antiviral activities
ural products and in several interesting nucleoside and [38, 39]. Moreover, at a certain stage of this investigation,
non-nucleoside compounds. Being a building unit of the synthesis of chloropyrimidines was planned owing to
DNA and RNA, pyrimidine derivatives were found to be their reported potential antimicrobial and antitumor
associated with a variety of chemotherapeutic effects activities [40, 41]. The fact that hydrazino derivatives are
including antimicrobial [6–10], antitubercular [11], anti- capable of exerting anticancer activity by alkylating DNA
fungal [12], antimalarial [13], and antiviral [14, 15] activ- through a free radical intermediate [42], prompted the
ities. Furthermore, pyrimidine-containing compounds synthesis of some substituted amino- and hydrazinopyri-
were reported to contribute to a variety of anticancer midines in order to study the influence of such structure
potentials including antitumor [16], antineoplastic [17], variation on the anticipated biological activities.
antiproliferative [18, 19], thymidylate synthase and dihy-
drofolate reductase inhibitory [20], angiogenesis inhibit-
ing [21], and cyclin-dependent kinase inhibitory [22]
activities. Interest in the chemotherapeutic activity of
Results and discussion
pyrimidines stemmed from the early success of some pyr- Chemistry
imidine-based antimetabolites such as 5-flurouracil, car- Synthesis of the intermediate and target compounds was
mofur, 5-azauracil, cytarabine, and gemcitabine as accomplished according to the steps depicted in
potential antineoplastic agents [23]. Diverse mechanisms Schemes 1, 2, and 3. In Scheme 1, the key intermediate 6-
of actions were reported to be encountered with the che- (4-benzyloxy-3-methoxyphenyl)-4-oxo-2-thioxo-1,2,3,4-tet-
motherapeutic bioactivity of pyrimidines including rahydropyrimidine-5-carbonitrile 1 was prepared accord-
inhibition of kinases, inhibition of enzymes involved in ing to previously reported reaction conditions [43]. Stir-
pyrimidine biosynthesis, incorporation into RNA and ring 1 with an equivalent amount of either methyl iodide
DNA which subsequently cause misreading and inhibi- or benzyl chloride in dry DMF containing anhydrous
tion of DNA polymerase [23].
potassium carbonate gave rise to the S-alkyl derivatives 2
In view of the above mentioned facts, and inspired by and 3; respectively. However, treating 1 with three equiv-
the promising chemotherapeutic activities encountered alents of methyl iodide under the same reaction condi-
with some pyrimidine derivatives reported in our pre- tions yielded the dimethyl analog 4. Moreover, treating
vious publications [24–27], we considered it worthwhile the thione 1 with chloroacetonitrile afforded the corre-
to synthesize some novel polysubstituted pyrimidines sponding 2-cyanomethylsulfanyl derivative 5. Further-
with the hope of discovering more active and selective more, alkylation of the same starting compound 1 either
antimicrobial and / or anticancer agents.
with 4-substituted phenacyl bromide or chloroacetone
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Reagents and reaction conditions: i) RX, K2CO3, DMF, rt.; ii) CH3I (3 eq.), K2CO3, DMF, rt.; iii) ClCH2CN, K2CO3, DMF, rt.; iv) R-C6H4COCH2Br,
K2CO3, DMF, rt.; v) ClCH2COCH3, K2CO3, DMF, rt.
Scheme 1. Synthesis of compounds 1–9.
Reagents and reaction conditions: i) ClCH2COOC2H5, K2CO3, DMF, rt.; ii) NH2NH2, EtOH, rt.; iii) NH2NH2, EtOH, reflux; iv) 4-Cl-C6H4-CHO,
EtOH, reflux.
Scheme 2. Synthesis of compounds 10–13.
yielded the targeted substituted phenylcarbonylmethyl-
sulfanyl 6–8 and 2-oxoprop-1-ylsulfanyl 9 analogs.
At this stage, it was planned to synthesize some 4-(4-
benzyloxy-3-methoxyphenyl)-2-substituted amino-6-oxo-
On the other hand, alkylation of the thione 1 with 1,6-dihydropyrimidine-5-carbonitriles 14–16 (Scheme 3).
ethyl chloroacetate afforded the substituted ethyl acetate This was achieved by fusion of the acetate ester 10 with
derivative 10. Stirring of 10 with excess hydrazine four equivalents of the appropriate amine in an oil bath
hydrate in absolute ethanol at room temperature at 160–1708C. Furthermore, the 2-benzylamino deriv-
afforded the acid hydrazide 11 in a moderate yield. How- ative 14 was utilized for the synthesis of the chloro deriv-
ever, refluxing 10 with excess hydrazine hydrate resulted ative 17 through reflux in POCl3 for 15 min, since increas-
in the corresponding hydrazino derivative 12. Addition- ing the reaction time led to an increase in the percentage
ally, the azomethine 13 was prepared by condensing the of by-products probably due to cleavage of the ether link-
acid hydrazide 11 with 4-chlorobenzaldehyde in ethanol age. Refluxing 17 with aniline or benzyl amine in abso-
(Scheme 2).
lute ethanol yielded the corresponding amino deriv-
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Reagents and reaction conditions: i) RNH2, heating 160 – 1708C; ii) POCl3, reflux, 15 min; iii) RNH2, EtOH, reflux, 6 h; iv) 4-R-C6H4NHNH2 N HCl,
anhydrous NaOAc, EtOH, reflux, 12 h; v) C6H5CH2CONHNH2, EtOH, reflux, 18 h; vi) NH2CSNHNH2, EtOH, reflux, 18 h; vii) C6H5SH, K2CO3, DMF, rt.
Scheme 3. Synthesis of compounds 14–24.
atives 18 and 19, respectively. However, refluxing 17 with potential against Candida albicans (ATCC 10231) and
the selected arylhydrazine hydrochloride in absolute Aspergillus niger (recultured) fungal strains.
ethanol containing anhydrous sodium acetate resulted
The agar-diffusion method was used for determination
in the formation of the pyrazolo[3,4-d]pyrimidines 20, 21. of preliminary antibacterial and antifungal activity.
IR spectra of the latter compounds revealed the absence Ampicillin trihydrate (antibiotic) and clotrimazole (anti-
of the characteristic cyano group, whereas, their 1H-NMR fungal) were used as reference drugs. The results were
spectra displayed a D2O-exchangeable singlet attribut- recorded for each tested compound as the average diame-
able to a NH2 group. Moreover, the mass spectrum of com- ter of inhibition zones (IZ) of bacterial or fungal growth
pound 20 was in favor of the unexpected structure as it around the discs in mm. The minimum inhibitory con-
showed a molecular ion peak at m/z 528. On the other centration (MIC) measurement was determined for com-
hand, refluxing 17 with phenyl acetic acid hydrazide or pounds that showed significant growth inhibition zones
thiosemicarbazide in absolute ethanol yielded the requi- (F14 mm) using the two-fold serial dilution method [44].
site derivatives 22 and 23, respectively. Finally, stirring The MIC values (in lg/mL) of the active compounds
17 with an equivalent amount of thiophenol in dry DMF against the tested microbial strains are recorded in
containing anhydrous potassium carbonate afforded the Table 1.
target 6-phenylsulfanyl derivative 24.
Concerning the antibacterial activity, the results
revealed that 18 out of the tested 24 compounds dis-
played variable inhibitory effects on the growth of the
In-vitro antibacterial and antifungal activities
All the newly synthesized compounds were evaluated for tested Gram-positive and Gram-negative bacterial strains.
their in-vitro antibacterial activity against Staphylococcus In general, most of the tested compounds revealed better
aureus (ATCC 6538), Bacillus subtilis (NRRL B-14819), and antibacterial activity against the Gram-positive rather
Micrococcus luteus (ATCC 21881) as examples of Gram-posi- than the Gram-negative bacteria. Among the Gram-posi-
tive bacteria and Escherichia coli (ATCC 25922), Pseudomo- tive bacteria tested, two strains namely, S. aureus and B.
nas aeruginosa (ATCC 27853), and Klebsiella pneumonia subtilis, showed relatively high sensitivity towards the
(clinical isolate) as examples of Gram-negative bacteria. tested compounds. In this view, compounds 1, 2, 9, 17,
They were also evaluated for their in-vitro antifungal 19, and 21 (MIC = 12.5 lg/mL) were 50% less active than
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Pyrimidines as In-vitro Antimicrobial / Anticancer Agents
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Table 1. Minimal inhibitory concentrations (MIC, in lg/mL) of the active newly synthesized compounds.
Compound
S. aureus
B. subtilis
M. luteus
E. coli
P. aeruginosa
K. pneumonia
C. albicans
1
2
3
5
6
12.5
12.5
50
100
25
50
12.5
25
25
100
25
12.5
50
12.5
50
25
6.25
50
100
12.5
12.5
25
25
12.5
100
100
25
25
50
50
25
50
12.5
100
100
12.5
25
50
50
50
100
50
100
50
50
100
25
–
12.5
6.25
–
25
12.5
100
100
25
50
25
50
25
–
100
50
100
–
25
50
50
50
50
–
–
–
–
–
–
–
–
12.5
50
–
a)
–
12.5
25
25
–
–
–
–
–
–
–
–
–
–
50
–
7
9
10
11
13
14
17
18
19
20
21
22
24
Ab)
Cc)
25
25
100
50
12.5
100
50
50
12.5
50
–
100
50
50
–
–
-
100
100
50
–
100
50
–
50
12.5
–
12.5
50
25
6.25
–
–
25
12.5
–
100
12.5
–
100
12.5
–
6.25
a)
(–): totally inactive (MIC F 200 lg/mL).
A: Ampicillin trihydrate (standard broad-spectrum antibiotic).
C: Clotrimazole (standard broad-spectrum antifungal agent).
b)
c)
ampicillin (MIC = 6.25 lg/mL) against S. aureus. Moreover, ited moderate activity against the same organism when
the analogs 6, 10, 11, 14, and 24 (MIC = 25 lg/mL) showed compared with ampicillin (MIC = 6.25 lg/mL). Addition-
25% of the activity of ampicillin against the same organ- ally, the tested P. aeruginosa and K. pneumonia strains
ism. The rest of the active compounds revealed moderate revealed moderate to weak sensitivity to most of the
activity against S. aureus (MIC range: 50–100 lg/mL). active compounds (MIC range: 25–100 lg/mL). An excep-
With regard to the activity against B. subtilis, an outstand- tion was shown by compound 2 which was proved to be
ing growth inhibitory activity was displayed by com- equipotent to ampicillin against P. aeruginosa (MIC =
pound 2 (MIC = 6.25 lg/mL) which was twice as active as 12.5 lg/mL).
ampicillin (MIC = 12.5 lg/mL) against this organism. Fur-
Concerning the antifungal activity of the tested com-
thermore, compounds 6, 7, 17, and 21 were equipotent to pounds, the results revealed that only six compounds
ampicillin against the same organism (MIC = 12.5 lg/mL), namely; 1, 2, 6, 7, 9, and 24 were able to produce appreci-
whereas the analogs 1, 9, 10, 11, and 24 (MIC = 25 lg/mL) able growth inhibitory activity against C. albicans (MIC
exhibited half the potency of ampicillin (MIC = 12.5 lg/ values: 12.5–100 lg/mL, respectively) comparable to that
mL) against the same species. M. luteus proved to be the of clotrimazole (MIC = 6.25 lg/mL), the standard antifun-
least sensitive Gram-positive microorganism to most of gal agent utilized in this assay. Among these, the analogs
the tested compounds, however, compound 2 was shown 1 and 6 exhibited the best antifungal activity (MIC =
to act equipotent to ampicillin against this organism 12.5 lg/mL) that is 50% of the standard. It should be
(MIC = 12.5 lg/mL). Four compounds namely, 1, 6, 7, and noted that all of the tested compounds lacked antifungal
11 (MIC 25 lg/mL) exhibited appreciable growth inhibi- activity against Asp. niger.
tory effect towards M. luteus which was 50% of the activity
A deep insight into the structures of the active com-
of ampicillin (MIC = 12.5 lg/mL). On the other hand, pounds revealed that the tested compounds belong to
investigation of the antibacterial potency of the active two main series namely; 4-(4-benzyloxy-3-methoxy-
compounds against the three tested Gram-negative phenyl)-2-substituted sulfanyl-6-oxo-1,6-dihydropyrimi-
strains revealed that three analogs namely 2, 6, and 24 dine-5-carbonitriles 1–13 (Schemes 1 and 2) and 4-(4-ben-
were able to produce noticeable growth inhibitory activ- zyloxy-3-methoxy-phenyl)-2,6-disubstitutedaminopyrimi-
ity against E. coli (MIC = 12.5 lg/mL) which represents dine-5-carbonitriles 14–24 (Scheme 3).
half the activity of ampicillin (MIC = 6.25 lg/mL).
Within the first series (Scheme 1), the results revealed
Whereas, compounds 7 and 21 (MIC = 25 lg/mL) exhib- that the parent thione 1 exhibited potential activity
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Arch. Pharm. Chem. Life Sci. 2009, 342, 299–310
against all the tested microorganisms. It showed 50% of = CH2-C6H5) was the sole analog with moderate activity
the activity of ampicillin against the Gram-positive against Gram-positive bacteria (MIC = 25–100 lg/mL).
organisms S. aureus, B. subtilis, M. Luteus and the Gram- Chlorination of the benzylamino derivative 14 led to the
negative strain P. aeruginosa. However, the antimicrobial formation of an obviously active chloropyrimidine ana-
potential of the derived compounds is obviously depend- log 17, with moderate activity against Gram-positive bac-
ant on the nature of the substituent of the thio function teria but with a specially high activity against B. subtilis
at position 2. In this respect, alkylation of the 2-thione where it exerted the same level of activity as ampicillin.
functionality resulted in two active compounds (2: R = Conversion of the chloro substituent to a secondary
CH3 and 3: R = CH2-C6H5), of which the methyl analog 2 amino moiety as in 18 and 19, resulted in a noticeable
showed significant enhancement in activity especially reduction in activity. Only one analog namely, 19 (R =
against B. subtilis as the compound displayed double the C6H5), retained the activity against S. aureus. It is worth
activity of ampicillin against this organism. It revealed mentioning that, although addition of substituted phe-
also a two-fold improvement in the antibacterial poten- nylhydrazines led unexpectedly to cyclized pyrazolopyri-
tial against M. Luteus, P. Aeruginosa, and K. pneumonia and midines 20 and 21, the latter analog showed considerable
a four-fold improvement in activity against E. coli. Mean- antimicrobial activity. Among these, analog 21 (R =
while, it is shown to be equipotent to ampicillin against SO2NH2) exhibited better activity against both Gram-posi-
M. Luteus and P. aeruginosa. However, the benzyl analog 3 tive and -negative bacteria that was comparable to or
showed a dramatic reduction in the overall antimicro- even better than that of the parent chloro analog 17.
bial spectrum when compared with the parent thione 1.
On the contrary, introduction of a phenylacetohydra-
Dimethylation at positions 2 and 3 resulted in complete zide moiety as in 22 resulted in a dramatic reduction in
abolishment of the antimicrobial activity. Introduction activity, whereas the introduction of thiosemicarbazido
of a cyanomethyl moiety as in compound 5, did not offer moiety as in 23 led to a total loss of antimicrobial poten-
any advantage over the parent compound 1, on the con- tial. Finally, increasing compound's lipophilicity
trary, it resulted in a dramatic reduction in the activity through the incorporation of a phenyl thioether counter-
against all the tested organisms. On the other hand, part, led to a remarkable change in both the antimicro-
introducing a 4-substituted phenylcarbonylmethyl frag- bial potential and spectrum. When compared with the
ment at position 2 resulted in two active compounds (6: R parent chloroderivative 17, the analog 24 showed about
= H and 7: R = Cl). Both were proved to be equipotent to two-fold reduction in the overall activity against the
ampicillin against B. subtilis, which was even better than three tested Gram-positive strains, whereas it exhibited a
the parent thione 1. It is worth mentioning that replace- better anti-Gram-negative potential especially towards E.
ment of the chlorine atom with bromine one compound, coli (50% of the activity of ampicillin).
8, experienced a total loss of activity. Furthermore,
replacement of the aryl group in compounds 6–8 with Preliminary in-vitro anticancer screening
an aliphatic one as in the analog 9, almost maintained In-vitro MTT cytotoxicity assay
the activity which was about 50% that of ampicillin Out of the newly synthesized compounds, eleven analogs
against S. aureus, B. Subtilis, and P. aeruginosa, in addition namely, 1, 3, 5, 7, 12, 13, 15, 19, 21, 22, and 24 were
to a moderate antifungal activity against C. albicans.
selected to be evaluated for their in-vitro cytotoxic effect
Regarding the thioether derivatives presented in via the standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
Scheme 2, the key intermediate ester derivative 10 exhib- diphenyltetrazolium bromide) method [45–47], against
ited appreciable activity against all the tested microor- a panel of three human tumor cell lines. Caucasian
ganisms with MIC range: 25–50 lg/mL. Conversion of breast adenocarcinoma MCF7, hepatocellular carcinoma
the ester group to an acid hydrazide functionality (com- HePG2, and colon carcinoma HT29. The results are pre-
pound 11) did not result in a significant change in the sented in Table 2 as LC50 and LC90 (lg/mL) which are the
antimicrobial profile, whereas synthesis of the azome- lethal concentrations of the compound which cause
thine 13 led to an obvious reduction in both antimicro- death of 50% or 90% of the cells in 24 h, respectively.
bial spectrum and potency. Furthermore, replacement of
the thioether moiety with a hydrazine counterpart as in human tumor cell lines exhibited variable degrees of sen-
13, led to a total loss of activity. sitivity profiles towards six of the tested compounds
The obtained data revealed that the three tested
Concerning the antimicrobial activity of the second namely; 1, 3, 7, 12, 13, and 15, at both the LC50 and LC90
series (Scheme 3), replacement of the thioether moiety levels. Among these, the human colon carcinoma HT29
with a substituted amino group gave rise to three com- cell line showed pronounced sensitivity towards com-
pounds 14–16, among which the benzyl derivative 14 (R pound 15 with LC50 value of 3.4 lg/mL. Such high activity
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Table 2. Cytotoxic effects (LC50 and LC90, in lg/mL) of the active compounds on some human tumor cell lines using the MTT assay.
Compound
Human colon carcinoma HT29
Human hepatocellular carcinoma HePG2
Human breast cancer MCF7
a)
b)
LC50
LC90
LC50
LC90
LC50
LC90
1
3
7
12
13
15
41.4
55.9
10.1
19.4
21.2
3.4
65.2
89.7
34.6
67.7
91.3
24.9
16.9
14.3
20.9
26.7
17.7
19.1
28.3
37.3
40.6
79.4
47.6
71.5
46.4
–
1.9
48.7
–
101
–
30.4
93.8
–
156
c)
63.4
a)
LC50: lethal concentration of the compound which causes death of 50% of cells in 24 h (lg/mL).
LC90: lethal concentration of the compound which causes death of 90% of cells in 24 h (lg/mL).
Totally inactive against this cell line.
b)
c)
was maintained at the LC90 level which showed also the
greatest activity (24.9 lg/mL). Moreover, a remarkable
cytotoxic potential was displayed by compound 7 against
the same cell line at both the LC50 and LC90 levels (10.1
and 34.6 lg/mL, respectively). Meanwhile, compounds 12
and 13 revealed an obvious cytotoxicity profile against
colon carcinoma HT29 at the LC50 level (19.4 and 21.2 lg/
mL, respectively). Whereas, mild activity was displayed
by compounds 1 and 3 at the LC50 level (41.4 and 55.9 lg/
mL, respectively). Furthermore, the growth of the human
hepatocellular carcinoma HePG2 cell line was found to
be inhibited by all the six active compounds. It showed
nearly the same sensitivity profile towards the six active
compounds with LC50 values range of 14.3–26.7 lg/mL.
Among these, the highest cytotoxic activity was dis-
played by compounds 1 and 3 which were almost equipo-
tent (LC50 = 16.9 and 14.3 lg/mL, respectively). However,
their LC90 pattern was reversed (LC90 = 28.3 and 37.3 lg/
mL, respectively). On the other hand, human breast can-
cer MCF7 was proved to be the least sensitive among the
cell lines tested as it was affected by only four test com-
pounds. However, an outstanding growth inhibition
potential was shown by compound 7 as evidenced from
its LC50 and LC90 values (1.9 and 30.4 lg/mL, respectively).
The remaining three active compounds, namely 1, 12,
and 15 showed mild activity against the same cell line
with LC50 values of 46.4, 63.4, and 48.7 lg/mL, respec-
tively.
Conclusion
In conclusion, the present study focussed on the synthe-
sis and investigation of the antimicrobial and cytotoxic
activity of two series of polysubstituted pyrimidines with
the hope of discovering new structure leads serving as
potential broad spectrum antimicrobial and / or anti-
cancer agents. The obtained results revealed that 18 com-
pounds were able to display variable growth inhibitory
effects on the tested Gram-positive and Gram-negative
bacterial strains, among them, six compounds exhibited
some moderate antifungal activity against C. albicans.
Globally, most of the tested compounds showed better
activity against the Gram-positive rather than the Gram-
negative bacteria, particularly S. aureus and B. subtilis.
Structurally, compounds belonging to the first series (thi-
oethers) displayed better overall antimicrobial activity
than that of the second one (substituted amines). Among
these, compounds 1, 2, 6, 7, 9, 10, 11, 21, and 24 revealed
a considerable broad spectrum of antibacterial activity
against both Gram-positive and negative bacteria. More-
over, compounds 1, 2, 6, 7, 9, and 24 exhibited an appreci-
able antifungal activity against C. albicans comparable to
that of clotrimazole. An outstanding broad spectrum
antibacterial profile was displayed by compound 2 which
was proved to be twice as active as ampicillin against B.
subtilis and equipotent to ampicillin against M. Luteus,
and P. aeruginosa, together with a moderate antifungal
activity. Consequently, it is considered to be the most
active antimicrobial member identified in this study.
On the other hand, eleven analogs were evaluated for
their in-vitro cytotoxic effect utilizing the standard MTT
assay, against a panel of three human cell lines: breast
adenocarcinoma MCF7, hepatocellular carcinoma
HePG2, and colon carcinoma HT29. The obtained data
revealed that six of the tested compounds namely; 1, 3, 7,
12, 13, and 15 showed a variable degree of cytotoxic activ-
ity against the tested cell lines at both the LC50 and LC90
Further interpretation of the results revealed that com-
pounds 7 and 15 showed a considerable broad spectrum
of cytotoxic activity against the three tested human
tumor cell lines at both the LC50 and LC90 levels. In partic-
ular, compound 7 proved to be the most active member
in this study with a broad spectrum of activity against
the tested cell lines, with special effectiveness against the
human colon carcinoma HT29 and human breast cancer
MCF7 cell lines at both the LC50 and LC90 levels (LC50
=
10.1, 1.9 and LC90 = 34.6, 30.4 lg/mL, respectively).
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306
S. A. F. Rostom et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 299–310
Table 3. Physicochemical and analytical data for compounds 1–24.
Compound
R
Yield (%)
Mp. (8C) Cryst. Solventa)
Molecular formula (Mol. wt.)b)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
–
CH3
C6H5CH2
–
–
H
Cl
Br
–
–
–
–
65
32
70
75
78
80
80
82
75
81
60
82
68
75
67
31
52
76
76
67
81
52
53
70
250–252 (D)
C19H15N3O3S (365.41)
C20H17N3O3S (379.43)
C26H21N3O3S (455.53)
C21H19N3O3S (393.46)
C21H16N4O3S (404.44)
C27H21N3O4S (483.55)
C27H20ClN3O4S (517.98)
C27H20BrN3O2 (562.44)
C22H19N3O4S (421.47)
C23H21N3O5S (451.50)
C21H19N5O4S (437.47)
C19H17N5O3 (363.37)
C28H22ClN5O4S (560.02)
C26H22N4O3 (438.48)
C25H20N4O3 (424.45)
C25H26N4O3 N 1.5H2O (457.53)
C26H21ClN4O2 (456.92)
C33H29N5O2 N 0.5H2O (536.64)
C32H27N5O2 N 0.5H2O (522.61)
C32H28N6O2 (528.60)
174–176 (D / E)
234–35 (D / E)
206–207 (D / E)
188–190 (D / E)
208–210 (D)
220–222 (DMF / E)
A 300 (DMF / E)
178–179 (EA)
223–224 (D)
292–293 (E)
246–247 (DMF / W)
294–296 (D)
246–248 (E)
218–219 (D)
–
C6H5CH2
C6H5
cyclo-C6H11
–
C6H5CH2
C6H5
H
213–214 (E)
133–135 (E / PE.)
144–145 (D / E)
122–124 (E)
216–217 (E)
210–211 (DMF / W)
168–169 (E)
SO2NH2
C32H29N7O4S (607.68)
C34H30N6O3 (570.64)
C27H25N7O2S N H2O (529.62)
C32H26N4O2S N H2O (548.67)
–
–
–
230–231 (D)
170–171 (D / E)
a)
Crystallization solvent(s): D: dioxane, DMF: N,N-dimethylformamide, E: ethanol, EA: ethyl acetate, PE.: petroleum ether (60 : 80),
W: water.
b)
Found values are within l 0.4% of the calculated values.
levels. Among these, compounds 7 and 15 showed consid-
erable broad spectrum of cytotoxic activity against the
three tested human tumor cell lines. Compound 7 was
proved to be the most active member in this study with a
broad spectrum of activity against the tested cell lines,
with special effectiveness against the human colon carci-
noma HT29 and human breast cancer MCF7 cell lines at
both the LC50 and LC90 levels. Collectively, the antimicro-
bial and cytotoxic results suggest that compounds 1 and
7 could be considered as possible dual antimicrobial-anti-
cancer candidates that deserve further investigation and
derivatization in order to explore the scope and limita-
tion of their biological activities. Physiochemical and
analytical data are recorded in Table 3.
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) spec-
tra were recorded on Perkin-Elmer 1430 infrared spectropho-
tometer (Perkin-Elmer, Norwalk, CT, USA). 1H-NMR spectra were
scanned on Jeol-500 MHz spectrometer (Jeol, Japan) using tetra-
methylsilane (TMS) as internal standard and DMSO-d6 as the sol-
vent (chemical shifts are given in d ppm). Splitting patterns were
designated as follows; s: singlet; br s: broad singlet d: doublet;
dd: double doublet; t: triplet; m: multiplet. Mass spectra were
carried out using a Schimadzu GCMS-QP-1000EX mass spectrom-
eter (Shimadzu, Tokyo, Japan) at 70 ev, Faculty of Science, Cairo
University. Elemental analyses were performed at the microana-
lytical unit, Faculty of Science, Cairo University and were within
l0.4% of the theoretical values. Follow-up of the reactions and
checking the purity of the compounds was made by thin layer
chromatography (TLC) on silica gel-precoated aluminium sheets
(Type 60 GF254; Merck, Germany) and the spots were detected by
exposure to UV lamp at k254 for a few seconds.
Sincere gratitude is conveyed to Dr. Y. R. Abdel-Fattah, Genetic
Engineering and Biotechnology Research Institute (GEBRI),
Mubarak City for Scientific Research and Technology Applica-
tions, Bourg El-Arab, Alexandria, Egypt, for carrying out the
in-vitro antimicrobial assay. Extendable thanks are expressed
to the authorities of the Bioassay-Cell Culture Laboratory, in-
vitro bioassays unit on human tumor cell lines for drug discov-
ery, National Research Center (NRC), Cairo, Egypt, for their
efforts in performing the MTT cytotoxicity assay.
6-(4-Benzyloxy-3-methoxyphenyl)-4-oxo-2-thioxo-
1,2,3,4-tetrahydropyrimidine-5-carbonitrile 1
A mixture of thiourea (1.9 g, 25 mmol), ethyl cyanoacetate
(2.83 g, 2.66 mL, 25 mmol), 4-benzyloxy-3-methoxybenzalde-
hyde (6 g, 25 mmol), and anhydrous K2CO3 (3.45 g, 25 mmol) in
ethanol (150 mL) was heated under reflux for 12 h. After being
The authors have declared no conflict of interest.
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Arch. Pharm. Chem. Life Sci. 2009, 342, 299–310
Pyrimidines as In-vitro Antimicrobial / Anticancer Agents
307
cooled to room temperature, the obtained precipitate was fil-
tered and washed with ethanol. The product was then dissolved
in hot water, filtered while hot and neutralized with conc. HCl
whereupon the desired thione was obtained. It was filtered,
washed with water, dried, and crystallized. Physicochemical and
analytical data are recorded in Table 3. IR KBr (cm– 1): 3311, 3212
(NH), 2234 (CN), 1694 (C=O), 1530, 1306, 1153, 1048 (N-C=S),
1235, 1025 (C-O-C).1H-NMR d (ppm): 3.78 (s, 3H, OCH3), 5.15 (s, 2H,
OCH2), 7.17 (d, J = 8.6 Hz, 2H, alkoxyphenyl C5-H), 7.27-7.47 (m,
7H, benzyloxy-H & alkoxyphenyl C2,6-H), 9.8, 12.77 (2s, 2H, 2NH,
D2O exchangeable).
General procedure for the synthesis of 4-(4-benzyloxy-3-
methoxyphenyl)-6-oxo-2-(substituted
phenylcarbonylmethyl- or 2-oxoprop-1-ylsulfanyl)-1,6-
dihydropyrimidine-5-carbonitriles 6–9
A mixture of 1 (0.37 g, 1 mmol), the appropriate phenacyl bro-
mide (for 6–8) (1 mmol) or, chloroacetone (for 9) (0.09 g,
0.08 mL, 1 mmol), and anhydrous K2CO3 (0.14 g, 1 mmol) in dry
DMF (3 mL) was stirred at room temperature overnight. Working
up of the reaction mixture was carried out as described under
compound 5. Physicochemical and analytical data are recorded
in Table 3. IR KBr (cm– 1): 3415–3109 (NH), 2214–2212 (CN),
1692–1650 (C=O), 1612–1585 (C=N). 1H-NMR d (ppm) for 7 (R =
Cl): 3.61 (s, 3H, OCH3), 4.87 (s, 2H, SCH2), 5.08 (s, 2H, OCH2), 6.92
(d, J = 8.4 Hz, 1H, alkoxyphenyl C5-H), 7.26–7.43 (m, 7H, benzy-
loxy-H and alkoxyphenyl C2,6-H), 7.55, 7.99 (2d, J = 8.4 Hz, 4H,
chlorophenyl C3,5-H and C2,6-H). 1H-NMR d (ppm) for 9: 2.61 (s, 3H,
O=C-CH3), 3.03 (s, 2H, SCH2), 3.81 (s, 3H, OCH3), 5.17 (s, 2H, OCH2),
7.23 (d, J = 8.4 Hz, 1H, alkoxyphenyl C5-H), 7.33 (t, J = 7.65 Hz, 1H,
benzyloxy C4-H), 7.39 (t, J = 7.65 Hz, 2H, benzyloxy C3,5-H), 7.45 (d,
J = 7.65 Hz, 2H, benzyloxy C2,6-H), 7.57 (s, 1H, alkoxyphenyl C2-H),
7.62, 7.64 (dd, J = 8.4, 2.3 Hz, 1H, alkoxyphenyl C6-H).
4-(4-Benzyloxy-3-methoxyphenyl)-2-(methyl or
benzyl)sulfanyl-6-oxo-1,6-dihydro-pyrimidine-5-
carbonitriles 2, 3
To a suspension of 1 (0.7 g, 1.9 mmol) in DMF (5 mL) containing
anhydrous K2CO3 (0.26 g, 1.9 mmol), methyl iodide (compound
2) or benzyl chloride (compound 3) (1.9 mmol) was added. The
reaction mixture was stirred at room temperature for 30 min
(for 2) or for overnight (for 3), then poured onto cold water and
the formed precipitate was filtered, washed with water, dried,
and crystallized. Physicochemical and analytical data are
recorded in Table 3. IR KBr (cm– 1): 3196–3148 (NH), 2214–2204
(CN), 1649–1648 (C=O), 1598–1595 (C=N).1H-NMR d (ppm) for 3:
3.73 (s, 3H, OCH3), 4.52 (s, 2H, SCH2), 5.16 (s, 2H, OCH2), 7.19–7.45
(m, 11H, alkoxyphenyl C5-H, benzylsulfanyl-H & benzyloxy-H),
7.57 (s, 1H, alkoxyphenyl C2-H), 7.61 (d, J = 8.4 Hz, 1H, alkoxy-
phenyl C6-H).
Ethyl 2-[4-(4-Benzyloxy-3-methoxyphenyl)-5-cyano-6-
oxo-1,6-dihydropyrimidin-2-yl-sulfanyl]acetate 10
To a suspension of 1 (0.37 g, 1 mmol) in dry DMF (3 mL) contain-
ing anhydrous K2CO3 (0.14 g, 1 mmol), ethyl chloroacetate
(0.12 g, 1 mmol) was added. The reaction mixture was stirred at
room temperature for 6 h, poured onto crushed ice. The
obtained precipitate was filtered, washed with H2O, dried, and
crystallized. Physicochemical and analytical data are recorded
in Table 3. IR KBr (cm– 1): 3296 (NH), 2218 (CN), 1736, 1654 (C=O),
1596 (C=N).1H-NMR d (ppm): 1.01 (t, J = 6.9 Hz, 3H, CH2CH3), 3.82
(s, 3H, OCH3), 3.99 (q, J = 6.9 Hz, 2H, CH2CH3), 4.12 (s, 2H, SCH2),
5.18 (s, 2H, OCH2), 7.18 (d, J = 8.4 Hz, 1H, alkoxyphenyl C5-H), 7.32
(t, J = 7.02 Hz, 1H, benzyloxy C4-H), 7.37 (t, J = 7.02 Hz, 2H, benzy-
loxy C3,5-H), 7.43 (d, J = 7.02 Hz, 2H, benzyloxy C2,6-H), 7.53–7.58
(m, 2H, alkoxyphenyl C2,6-H), 12.4 (s, 1H, NH, D2O exchangeable).
4-(4-Benzyloxy-3-methoxyphenyl)-1-methyl-2-methyl-
sulfanyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 4
To a solution of equimolar amounts of the thione 1 (0.7 g,
1.9 mmol) and anhydrous K2CO3 (0.26 g, 1.9 mmol) in dry DMF
(5 mL), methyl iodide (0.82 g, 0.36 mL, 5.7 mmol) was added and
the reaction mixture was stirred at room temperature 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 3. IR KBr (cm– 1): 2214 (CN), 1665 (C=O), 1593
(C=N). 1H-NMR d(ppm): 2.62 (s, 3H, N-CH3), 3.39 (s, 3H, SCH3), 3.79
(s, 3H, OCH3), 5.13 (s, 2H, OCH2), 7.20 (d, J = 8.98 Hz, 1H, alkoxy-
phenyl C5-H), 7.32 (t, J = 7.65 Hz, 1H, benzyloxy C4-H), 7.38 (t, J =
7.65 Hz, 2H, benzyloxy C3,5-H), 7.43 (d, J = 7.65 Hz, 2H, benzyloxy
C2,6-H), 7.65 (s, 1H, alkoxyphenyl C2-H), 7.67 (dd, J = 8.98, 2.68 Hz,
1H, alkoxyphenyl C6-H).
2-[4-(4-Benzyloxy-3-methoxyphenyl)-5-cyano-6-oxo-1,6-
dihydropyrimidin-2-ylsulfanyl]acetic acid hydrazide 11
To a suspension of 10 (1 g, 2.2 mmol) in absolute ethanol (10 mL)
hydrazine hydrate 98% (0.55 g, 0.53 mL, 11 mmol) was added
and the mixture was stirred at room temperature for 3 h. The
reaction mixture cleared after 1 h, then a white precipitate sepa-
rated out. After 3 h, the precipitate was filtered, washed with
ethanol, dried, and crystallized. Physicochemical and analytical
data are recorded in Table 3. IR KBr (cm– 1): 3347, 3279, 3186
(NH2, NH), 2205 (CꢀN), 1647 (C=O), 1604 (C=N). 1H-NMR d (ppm):
3.71 (s, 3H, OCH3), 3.78 (s, 2H, NH2, D2O exchangeable), 4.80 (s,
2H, SCH2), 5.16 (s, 2H, OCH2), 7.12 (d, J = 8.4 Hz, 1H, alkoxyphenyl
C5-H), 7.30-7.60 (m, 7H, benzyloxy-H & alkoxyphenyl C2,6-H), 9.4,
12.4 (2s, 2H, 2NH, D2O exchangeable).
4-(4-Benzyloxy-3-methoxyphenyl)-2-cyanomethyl-
sulfanyl-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 5
To a suspension of 1 (0.37 g, 1 mmol) in dry DMF (3 mL) contain-
ing anhydrous K2CO3 (0.14 g, 1 mmol), chloroacetonitrile (0.08 g,
0.06 mL, 1 mmol) was added. The reaction mixture was stirred
at room temperature overnight, poured onto crushed ice, then
acidified with dilute HCl. The obtained precipitate was filtered,
washed with H2O, dried, and crystallized. Physicochemical and
analytical data are recorded in Table 3. IR KBr (cm– 1): 3144 (NH),
2219 (CN), 1650 (C=O), 1615 (C=N). 1H-NMR d (ppm): 3.75 (s, 3H,
OCH3), 4.12 (s, 2H, SCH2), 5.13 (s, 2H, OCH2), 7.20 (d, J = 8.98 Hz,
1H, alkoxyphenyl C5-H), 7.30–7.40 (m, 5H, benzyloxy-H), 7.66 (s,
1H, alkoxyphenyl C2-H), 7.67 (dd, J = 8.98, 2.68 Hz, 1H, alkoxy-
phenyl C6-H).
4-(4-Benzyloxy-3-methoxyphenyl)-2-hydrazino-6-oxo-
1,6-dihydropyrimidine-5-carbonitrile 12
A mixture of 10 (1 g, 2.2 mmol) and hydrazine hydrate 98%
(0.22 g, 0.21 mL, 4.4 mmol) in absolute ethanol (20 mL) was
heated under reflux for 2 h during which a crystalline white pre-
cipitate separated out. It was filtered, washed with water, dried,
and crystallized. Physicochemical and analytical data are
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308
S. A. F. Rostom et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 299–310
recorded in Table 3. IR KBr (cm– 1): 3439, 3275 (NH2, NH), 2205
(CN), 1667 (C=O), 1591 (C=N).1H-NMR d (ppm): 3.43 (br s, 2H, NH2,
D2O exchangeable), 3.77 (s, 3H, OCH3), 5.13 (s, 2H, OCH2), 7.12 (d,
J = 8.4 Hz, 1H, alkoxyphenyl C5-H), 7.31 (t, J = 7.46 Hz, 1H, benzy-
loxy C4-H), 7.37 (t, J = 7.46 Hz, 2H, benzyloxy C3,5-H), 7.41–7.50
(m, 4H, benzyloxy C2,6-H & alkoxyphenyl C2,6-H), 9.40, 12.5 (2s,
2H, 2NH, D2O exchangeable).
2-Benzylamino-4-(4-benzyloxy-3-methoxyphenyl)-6-
substituted aminopyrimidine-5-carbonitriles 18, 19
A suspension of 17 (0.5 g, 1.1 mmol) and the appropriate amine
(2.2 mmol) in ethanol (15 mL) was heated under reflux for 6 h.
The reaction mixture was left overnight whereupon a crystalline
white solid separated out. It was filtered, washed with ether,
dried, and recrystallized. Physicochemical and analytical data
are recorded in Table 3. IR KBr (cm– 1): 3365–3242 (NH), 2198–
2197 (CN), 1604–1590 (C=N).1H-NMR d (ppm) for 18: 3.77 (s, 3H,
OCH3), 4.41 (d, J = 6.1 Hz, 2H, pyrimidine-C2-NHCH2), 4.53 (d, J =
5.9 Hz, 2H, pyrimidine-C4-NHCH2), 5.12 (s, 2H, OCH2), 7.09–7.46
(m, 18H, Ar-H), 7.99 (t, J = 6.1 Hz, 1H, pyrimidine-C2-NH), 8.23 (t,
J = 5.9 Hz, 1H, pyrimidine-C4-NH).
N1-(4-chlorobenzylidene)-2-[4-(4-benzyloxy-3-
methoxyphenyl)-5-cyano-6-oxo-1,6-dihydropyrimidin-2-
ylsulfanyl]acetic acid hydrazide 13
A suspension of the acid hydrazide 11 (0.6 g, 1.37 mmol) in etha-
nol (10 mL) was heated under reflux with an equimolar amount
of 4-chlorobenzaldehyde (0.19 g, 1.37 mmol) for 2 h. The reac-
tion mixture was left to attain room temperature and the sepa-
rated solid product was filtered, washed with cold ethanol,
dried, and crystallized. Physicochemical and analytical data are
recorded in Table 3. IR KBr (cm– 1): 3155 (NH), 2209 (CN), 1652
(C=O), 1600 & 1585 (C=N). 1H-NMR d (ppm): 3.51 (s, 2H, SCH2), 3.79
(s, 3H, OCH3), 5.11 (s, 2H, OCH2), 7.19 (d, J = 8.4 Hz, 1H, alkoxy-
phenyl C5-H), 7.33 (t, J = 7.65 Hz, 1H, benzyloxy C4-H), 7.38 (t, J =
7.65 Hz, 2H, benzyloxy C3,5-H), 7.44 (d, J = 7.65 Hz, 2H, benzyloxy
3-Amino-6-benzylamino-4-(4-benzyloxy-3-
methoxyphenyl)-1-phenylpyrazolo[3,4-d]pyrimidine 20
and 4-[3-amino-6-benzylamino-4-(4-benzyloxy-3-
methoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-
yl]benzenesulfonamide 21
A suspension of 17 (0.5 g, 1.1 mmol), the appropriate aryl hydra-
zine hydrochloride (1.1 mmol) and anhydrous sodium acetate
(0.09 g, 1.1 mmol) in absolute ethanol (15 mL) was heated under
reflux for 12 h. The reaction mixture was left overnight and the
separated solid product was filtered, washed with ethanol,
dried, and crystallized. Physicochemical and analytical data are
recorded in Table 3. IR KBr (cm– 1): 3398–3350, 3289–3256 &
3155–3125 (NH2 & NH), 1636–1589 (C=N).1H-NMR d (ppm) for
20: 3.60 (br s, 2H, NH2, D2O exchangeable), 3.83 (s, 3H, OCH3),
4.58 (s, 2H, NHCH2), 5.16 (s, 2H, OCH2), 7.15–7.58 (m, 17H, Ar-H),
7.98 (d, J = 8.4 Hz, 1H, alkoxyphenyl C6-H), 8.53 (br s, 1H, NH, D2O
exchangeable). MS m/z (relative abundance%) for compound 20:
529 [M+ + 1] (4.5), 528 [M+] (7.6), 438 (6.4), 437 (4.7), 91 (100), 90
(4.7), 89 (2.9), 78 (2.5), 77 (10.7), 66 (3.7), 65 (11.9).
C2,6-H), 7.47 (d, J = 8.8 Hz, 2H, chlorophenyl C3,5-H), 7.49 (s, 1H,
alkoxyphenyl C2-H), 7.51 (dd, J = 8.4, 2.3 Hz, 1H, alkoxyphenyl C6-
H), 8.05 (d, J = 8.8 Hz, 2H, chlorophenyl C2,6-H), 8.13 (s, 1H, N=CH),
12.5 (br s, 2H, 2NH, D2O exchangeable).
4-(4-Benzyloxy-3-methoxyphenyl)-2-substituted amino-6-
oxo-1,6-dihydropyrimidine-5-carbo-nitriles 14–16
A mixture of 10 (1 g, 2.2 mmol) and the selected amine
(8.8 mmol) was fused in an oil bath at 160–1708C for 30 min.
The reaction mixture was left to cool and then triturated with
diethyl ether (2615 mL). The obtained precipitate was filtered,
washed with ether, dried, and crystallized. Physicochemical and
analytical data are recorded in Table 3. IR (KBr, cm– 1): 3395–
3211 (NH), 2212–2210 (CN), 1665–1652 (C=O), 1616–1613
(C=N). 1H-NMR d(ppm) for 14 (R = CH2C6H5): 3.7 (s, 3H, OCH3), 4.58
(d, J = 5.35 Hz, 2H, NH-CH2), 5.13 (s, 2H, OCH2), 7.13 (d, J = 8.4 Hz,
1H, alkoxyphenyl C5-H), 7.22 (t, J = 7.25 Hz, 1H, benzylamino C4-
H), 7.28–7.35 (m, 5H, benzylamino C2,3,5,6-H & benzyloxy C4-H),
7.36–7.40 (m, 3H, benzyloxy C3,5-H and alkoxyphenyl C2-H), 7.43
(d, J = 6.9 Hz, 2H, benzyloxy C2,6-H), 7.48 (d, J = 8.4 Hz, 1H, alkoxy-
phenyl C6-H), 7.87, 11.82 (2s, 2H, 2NH, D2O exchangeable).
General procedure for the synthesis of 2-benzylamino-4-
(4-benzyloxy-3-methoxyphenyl)-6-substituted pyrimidine-
5-carbonitriles 22, 23
A mixture of 17 (0.5 g, 1.1 mmol) and phenyl acetic acid hydra-
zide (for 22) (0.17 g, 1.1 mmol); or thiosemicarbazide (for 23)
(0.12 g, 1.3 mmol) in ethanol (20 mL) was heated under reflux
for 18 h. The reaction mixture was left overnight and the pre-
cipitated product was filtered, washed with ethanol, dried, and
crystallized. Physicochemical and analytical data are recorded
in Table 3. IR KBr (cm– 1): 3436–3418 & 3335–3283 (NH2 & NH),
1
2211–2205 (CN), 1669 (C=O), 1630–1603 (C=N). H-NMR d(ppm)
for 22: 3.77 (s, 3H, OCH3), 4.35 (s, 2H, O=C-CH2), 4.52 (d, J = 6.5 Hz,
2H, NHCH2), 5.12 (s, 2H, OCH2), 7.11–7.45 (m, 18H, Ar-H), 8.31 (t, J
= 6.5 Hz, 1H, NHCH2, D2O exchangeable), 9.25, 10.15 (2s, 2H,
2NH, D2O exchangeable). 1H-NMR d(ppm) for 23: 3.54 (s, 2H, NH2,
D2O exchangeable), 3.83 (s, 3H, OCH3), 4.61 (s, 2H, NHCH2), 5.17
(s, 2H, OCH2), 7.19–7.50 (m, 13H, Ar-H), 8.09, 8.48, 12.96 (3s, 3H,
3NH, D2O exchangeable).
2-Benzylamino-4-(4-benzyloxy-3-methoxyphenyl)-6-
chloropyrimidine-5-carbonitrile 17
A solution of 14 (5 g, 11.4 mmol) in POCl3 (20 mL) was heated
under reflux for 15 min. The reaction mixture was left to attain
room temperature, then poured onto crushed ice. The precipi-
tated product was filtered, washed with water, dried, and puri-
fied by column chromatography using CH2Cl2 / petroleum ether
60/80 (120 : 10) as eluting solvent. Physicochemical and analyti-
cal data are recorded in Table 3. IR KBr (cm– 1): 3240 (NH), 2198
2-Benzylamino-4-(4-benzyloxy-3-methoxyphenyl)-6-
phenylsulfanylpyrimidine-5-carbonitrile 24
To a well stirred suspension of 17 (0.5 g, 1.1 mmol) and anhy-
drous K2CO3 (0.15 g, 1.1 mmol) in dry DMF (5 mL), an equimolar
amount of thiophenol (0.12 g, 0.11 mL) was added and the mix-
ture was left stirred at room temperature overnight. The reac-
1
(CN), 1597 (C=N). H-NMR d (ppm): 3.45, 3.69 (2d, J = 18 Hz, 2H,
NHCH2), 4.03 (s, 3H, OCH3), 5.08 (s, 2H, OCH2), 7.01–7.40 (m, 13H,
Ar-H), 7.8 (s, 1H, NH, D2O exchangeable).
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Arch. Pharm. Chem. Life Sci. 2009, 342, 299–310
Pyrimidines as In-vitro Antimicrobial / Anticancer Agents
309
tion mixture was poured onto crushed ice and the precipitated
product was filtered, washed with water, dried, and crystallized.
Physicochemical and analytical data are recorded in Table 3. IR
lines) and DMEM (for MCF7 cell line), 1% antibiotic-antimycotic
mixture (10000 IU/mL penicillin potassium, 10000 lg/mL strep-
tomycin sulphate, and 25 lg/mL amphotericin B), and 1% L-glu-
tamine in 96-well flat bottom microplate at 378C under 5% CO2.
After 48 h of incubation, the medium was aspirated, 40 lL of
MTT salt (2.5 lg/mL) were added to each well and incubated for
further 4 h at 378C under 5% CO2. To stop the reaction and dis-
solve the formed crystals, 200 lL of 10% sodium dodecyl sul-
phate (SDS) in de-ionized water was added to each well and incu-
bated overnight at 378C. A positive control which composed of
100 lg/mL of Annona cherimolia extract was used as known
cytotoxic natural agent that gives 100% lethality under the same
experimental conditions. The absorbance was then measured
using a microplate multi-well reader (model 3350, Bio-Rad Labo-
ratories Inc., Hercules, CA, USA) at 595 nm and a reference wave-
length of 620 nm.
1
KBr (cm– 1): 3403 (NH), 2206 (CN), 1595 (C=N). H-NMR d (ppm):
3.78 (s, 3H, OCH3), 4.06 (d, J = 6.47 Hz, 2H, NHCH2), 5.14 (s, 2H,
OCH2), 6.76–7.62 (m, 18H, Ar-H), 8.83 (t, J = 6.47 Hz, 1H, NH, D2O
exchangeable).
Biology
In-vitro antibacterial and antifungal activities
Standard sterilized filter paper discs (5 mm diameter) impreg-
nated with a solution of the test compound in DMSO (1 mg/mL)
were placed on an agar plate seeded with the appropriate test
organism in triplicates. The utilized test organisms were: Staph-
ylococcus aureus (ATCC 6538), Bacillus subtilis (NRRL B-14819), and
Micrococcus luteus (ATCC 21881) as examples of Gram-positive
bacteria and Escherichia coli (ATCC 25922), Pseudomonas aerugi-
nosa (ATCC 27853), and Klebsiella pneumonia (clinical isolate) as
examples of Gram-negative bacteria. They were also evaluated
for their in-vitro antifungal potential against Candida albicans
(ATCC 10231) and Aspergillus niger (recultured) fungal strains
were utilized as representatives for fungi. Ampicillin trihydrate
and clotrimazole were used as standard antibacterial and anti-
fungal agents, respectively. DMSO alone was used as control at
the same above-mentioned concentration. The plates were incu-
bated at 378C for 24 h for bacteria and for seven days for fungi.
Compounds that showed significant growth inhibition zones
(F14 mm), using the two-fold serial dilution technique, were fur-
ther evaluated for their minimal inhibitory concentrations
(MICs).
Statistical significance was tested between samples and nega-
tive control (cells with vehicle) using independent t-test by SPSS
11 program. The percentage change in viability was calculated
according to the formula:
[Reading of the sample/
(Reading of the negative control–1)]6100
(1)
A probit analysis was carried for LC50 and LC90 determinations
using SPSS 11 program.
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were inoculated to the corresponding wells. Plates were incu-
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