Microwave-assisted synthesis of antimicrobial dihydropyridines and tetrahydropyrimidin-2-ones: Novel compounds against aspergillosis

Article abstract of DOI:10.1016/j.bmc.2005.09.014

Ten 4-aryl-1,4-dihydropyridine and three 4-aryl-1,2,3,4- tetrahydropyrimidin-2-one derivatives have been synthesized and examined for their activity against pathogenic strains of Aspergillus fumigatus and Candida albicans. Although none of the three compo

Full text of DOI:10.1016/j.bmc.2005.09.014

Bioorganic & Medicinal Chemistry 14 (2006) 973–981
Microwave-assisted synthesis of antimicrobial dihydropyridines
and tetrahydropyrimidin-2-ones: Novel compounds
against aspergillosisq
Anil K. Chhillar,a Pragya Arya,b Chandrani Mukherjee,b Pankaj Kumar,b,c
Yogesh Yadav,b,c Ajendra K. Sharma,b,d Vibha Yadav,a Jyotsana Gupta,a
Rajesh Dabur,a Hirday N. Jha,c Arthur C. Watterson,d Virinder S. Parmar,b,d,
*
Ashok K. Prasadb,* and Gainda L. Sharmaa,*
aInstitute of Genomics & Integrative Biology, Mall Road, Delhi 110 007, India
bBioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
cDepartment of Chemistry, MMH College, CCS University, Meerut 250 002, India
dInstitute for Nano Science and Engineering Technology, Department of Chemistry, University of Massachusetts,
Lowell, MA 01854, USA
Received 2 August 2005; revised 3 September 2005; accepted 6 September 2005
Available online 7 October 2005
Abstract—Ten 4-aryl-1,4-dihydropyridine and three 4-aryl-1,2,3,4-tetrahydropyrimidin-2-one derivatives have been synthesized and
examined for their activity against pathogenic strains of Aspergillus fumigatus and Candida albicans. Although none of the three
compounds belonging to pyrimidin-2-one series showed any activity against two pathogens, two of the compounds of the dihydro-
pyridine series, that is, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate and dimethyl 4-(4-methoxy-
phenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate, exhibited significant activity against A. fumigatus in disc diffusion,
microbroth dilution and percent spore germination inhibition assays. The most active diethyl dihydropyridine derivative exhibited
a MIC value of 2.92 lg/disc in disc diffusion and 15.62 lg/ml in microbroth dilution assays. The MIC90 value of the most active
compound by percent germination inhibition assay was found to be 15.62 lg/ml. The diethyl dicarboxylate derivative of dihydro-
pyridine also exhibited appreciable activity against C. albicans. The in vitro toxicity of the most active diethyl dihydropyridine deriv-
ative was evaluated using haemolytic assay, in which the compound was found to be non-toxic to human erythrocytes even at a
concentration of 625 lg/ml. The standard drug amphotericin B exhibited 100% lysis of erythrocytes at a concentration almost 16
times less than the safer concentration of the most active dihydropyridine derivative.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
risk of developing fatal mycosis, predominantly asper-
gillosis and candidiasis. Aspergillus and Candida species
are the most frequent isolates from blood cultures in
hospitals with large populations of immunocompro-
mised patients.2,3 Infections by these pathogenic fungal
species are being recognized as emerging threat to public
health.4,5 It is, therefore, imperative to diagnose the
mycotic infections at an early stage and treat them effec-
tively. The drugs derived from natural sources or pre-
pared synthetically are available for treating the fungal
infections; however, none of these drugs is ideal.6
Fungal infections are reported to cause lots of morbidity
and mortality despite recent advances in antifungal che-
motherapeutic regimen.1 Immunocompromised individ-
uals and patients with malignancies remain at higher
Keywords: Antifungal agent; Aspergillosis; 4-Aryl-1,4-dihydropyri-
dine; 4-Aryl-1,2,3,4-tetrahydropyrimidinone; Microwave-assisted
reaction.
q
A part of the work has been presented at ÔIUPAC International
Conference on Biodiversity and Natural Products: Chemistry and
Amphotericin B, which is considered to be the drug of
choice at present and remains effective therapeutic agent
in severe conditions of invasive mycosis, has been found
Medical ApplicationsÕ, held in Delhi, India, on 26–31 January 2004.
Corresponding authors. Tel.: +1 978 934 3659; fax: +1 978 458
9571; e-mail: virinder_parmar@uml.edu
*
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.014

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A. K. Chhillar et al. / Bioorg. Med. Chem. 14 (2006) 973–981
to be highly toxic and immunosuppressive.7,8 The non-
availability of an absorbable oral form of amphotericin
B for long maintenance therapy in immunocompro-
mised patients is another important drawback of this
drug. Several synthetic drugs, such as ketoconazole, itr-
aconazole and fluconazole, have been found to be very
effective in treating the fungal diseases. Variconazole is
a recent addition to the list of synthetic antifungals.
However, all these triazole drugs also induce serious side
effects in human beings.9–11 Further, the development of
resistance in fungi to most available drugs has been
reported.12–14 Therefore, there is an urgent need to iden-
tify new lead molecules from other classes of compounds
to develop antifungal drugs with high therapeutic index.
fused potassium carbonate to afford diethyl 2,6-dimeth-
yl-4-(3-epoxymethoxyphenyl)-1,4-dihydropyridin-3,5-
dicarboxylate (2h) in 78% yield (Scheme 2). Treatment
of compound 2h with dilute hydrochloric acid in
methanol led to the formation of diethyl 4-[3-(3-chloro-
2-hydroxypropanoxy)phenyl]-1,4-dihydropyridin-3,5-
dicarboxylate (2i) in 72% yield (Scheme 2). The
structures of 4-aryl-1,4-dihydropyridine derivatives 2a–
2i, 3a and 3b were unambiguously established from the
1
analysis of their spectral data (IR, H-, 13C NMR and
mass). The structures of known compounds 2a–2g, 3a
and 3b were further confirmed by comparison of their
melting points and/or spectral data with those reported
in the literature.34–39
Dihydropyridine derivatives, such as nifedipine, nitren-
dipine and nimodipine, have been found to be commer-
cially useful molecules as calcium channel blockers.15–17
A number of dihydropyridine calcium antagonists have
been introduced as potential drugs for the treatment of
congestive heart failure.18,19 Further, cerebrocrast, a
dihydropyridine derivative, has been introduced as a
neuroprotective agent.20 Together with calcium channel
blocker and neuroprotective activity, a number of dihy-
dropyridine derivatives have been found as vasodilators,
antihypertensive, bronchodilators, antiatherosclerotic,
hepatoprotective, antitumour, antimutagenic, geropro-
tective, antidiabetic and antiplatelet aggregation
agents.21–25 However, the potential of dihydropyridines
as antifungal agents has not been studied. Therefore,
the current study was undertaken to synthesize different
dihydropyridine derivatives and investigate their anti-
fungal potential using Aspergilli and Candida as model
pathogens. Further, the interest to synthesize tetrahy-
dropyrimidin-2-ones for their antifungal studies arises
from their structural similarities to 1,4-dihydropyri-
dines26 and also because of interesting biological proper-
ties of several marine alkaloids27–29 based upon
dihydropyrimidine system, viz crambine, batzelladine
(potent HIV gp-120-CD4 inhibitor), and ptilomycalin
A. The derivatization of dihydropyrimidinones, special-
ly at C-4, has led to the recognition of several lead com-
pounds that show a very similar pharmacological profile
to that of 1,4-dihydropyridine based drugs.30–33
2.2. Synthesis of 4-aryl-1,2,3,4-tetrahydropyrimidin-2-one
derivatives 5–7
Ethyl 4-(4-hydroxyphenyl)-6-methyl-1,2,3,4-tetrahydro-
pyrimidin-2-one-5-carboxylate (5) was synthesized by
Biginelli cyclocondensation reaction of ethyl acetoace-
tate, urea and 4-hydroxybenzaldehyde (4) in the pres-
ence of ferric chloride under microwave conditions in
85% yield following the modified literature procedure.40
The compound 5 was acetylated to afford ethyl 4-(4-
acetoxyphenyl)-6-methyl-1,2,3,4-tetrahydropyrimidin-2-
one-5-carboxylate (6) in quantitative yield using acetic
anhydride-pyridine and a catalytic amount of dimeth-
ylaminopyridine (DMAP) (Scheme 3). Further, epox-
ymethylation of tetrahydropyrimidin-2-one
5 was
carried out by refluxing the compound with epichloro-
hydrin in dry ethanol in the presence of fused potassium
carbonate to afford ethyl 4-(4-epoxymethoxy)phenyl-6-
methyl-1,2,3,4-tetrahydropyrimidin-2-one-5-carboxylate
(7) in 77% yield (Scheme 3). All the three hydroxyphe-
nyl-, acetoxyphenyl- and epoxymethoxyphenyl-1,2,3,4-
tetrahydropyrimidin-2-ones 5–7 were unambiguously
characterized on the basis of their spectral data (IR,
1H, 13C NMR and mass). The structure of known tetra-
hydropyrimidine 5 was further confirmed by compari-
son of its melting point and spectral data with those
reported in the literature.40
3. Antifungal activity
2. Results and discussion
Ten 4-aryl-1,4-dihydropyridines 2a–2g, 2i, 3a and 3b
and three 4-aryl-1,2,3,4-tetrahydropyrimidin-2-ones 5–
7 have been examined for activity against pathogenic
strains of Aspergillus fumigatus and Candida albicans.
The anti-Aspergillus activity of all the 13 compounds
has been evaluated by the disc diffusion (DDA), micro-
broth dilution (MDA) and percentage spore germina-
tion inhibition (PSGIA) assays;41 the results are given
in Table 1 (results of PSGIA are also depicted in
Fig. 1). The anti-Candida activity of all the 13 com-
pounds 2a–2g, 2i, 3a, 3b, and 5–7 has been evaluated
by counting colony-forming units42 (Fig. 2, inactive
compounds have not been displayed in the figure). The
anti-Candida activity of most active compound 2e, iden-
tified on the basis of counting colony-forming units as-
say, was further evaluated by microbroth dilution
assay (Fig. 3).
2.1. Synthesis of 4-aryl-1,4-dihydropyridine derivatives
2a–2i, 3a and 3b
Diethyl 4-aryl-2,6-dimethyl-1,4-dihydropyridin-3,5-di-
carboxylates 2a–2g and dimethyl 4-aryl-2,6-dimethyl-
1,4-dihydropyridin-3,5-dicarboxylates 3a and 3b were
prepared by Biginelli cyclocondensation of ethyl aceto-
acetate/methyl acetoacetate, urea, and corresponding
aromatic aldehydes 1a–1g under microwave condition
following the procedure of Yadav et al.25 in 65–80%
yield (Scheme 1). Epoxymethylation of one of the
4-aryl-1,4-dihydropyridines, that is, diethyl 2,6-dimeth-
yl-4-(3-hydroxyphenyl)-1,4-dihydropyridin-3,5-dicarb-
oxylate (2d), was carried out by refluxing the compound
with epichlorohydrin in dry ethanol in the presence of

A. K. Chhillar et al. / Bioorg. Med. Chem. 14 (2006) 973–981
975
R1
R1
R2
R2
R1
R2
R3
R3
R3
O
O
O
silica gel
+
+
MeOOC
H3C
COOMe
EtOOC
H3C
COOEt
H2N
NH2
microwave
OEt/OMe
H3C
CHO
N
H
CH3
N
H
CH3
1
3
2
a
b
c
d
e
f
g
a
b
1 & 2
R1
3
R1
R2
R3
H
Br
H
H
OH
H
OMe
H
H
H
OMe
H
OMe
OH
H
OH
OMe
H
R2
Cl
H
H
OH
H
R3
H
H
Br
H
Scheme 1.
OH
O
2"
4'
OH
Cl
O
O
3"
1"
2'
6'
(i)
(ii)
EtOOC
H3C
COOEt
CH3
5
3
EtOOC
H3C
COOEt
CH3
EtOOC
COOEt
CH3
N
H
N
H3C
N
H
1
H
2d
2i
2h
Scheme 2. Reagents and conditions: (i) epichlorohydrin, dry EtOH, fused K2 CO3, reflux; (ii) MeOH, dilute HCl, stirring at 25–28 ꢁC.
OH
OCOCH3
OH
O
ii
O
O
O
O
i
+
+
H2N
NH2
H5C2O
NH
OC2H5
H5C2O
NH
CHO
N
H
O
N
O
4
H
5
6
iii
O
2"
1"
O
4'
3"
2'
6'
5
O
3
H5C2O
NH
N
O
1
H
7
Scheme 3. Reagents and conditions: (i) microwave 1–2 min, FeCl3, SiO2; (ii) acetic anhydride, DMAP, 60 min, 25–30 ꢁC; (iii) epichlorohydrin, dry
EtOH, fused K2CO3, reflux.
3.0.1. Anti-Aspergillus activity. The results of anti-
Aspergillus activity evaluation revealed that one of the
dihydropyridines, that is, diethyl 4-(4-methoxyphenyl)-
2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (2e), is
a potential inhibitor of the growth of A. fumigatus.
The compound 2e exhibited appreciable activity even
at a concentration of 2.92 lg/disc in disc diffusion and
15.62 lg/ml in microbroth dilution assays (Table 1).
The MIC90 value of the most active compound 2e by
percentage spore germination inhibition assay was
found to be 15.62 lg/ml (Table 1 and Fig. 1). The next
most active compound among dihydropyridines is
dimethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihy-
dropyridine-3,5-dicarboxylate (3a), which exhibited

976
A. K. Chhillar et al. / Bioorg. Med. Chem. 14 (2006) 973–981
Normal germinating
Spores
Spores treated with 15.62 µg/ml
of compound 2e
Figure 1. Inhibition of spore germination by compound 2e.
control
101
136
Amphotericin B
2a
2c
2e
2f
74
12
128
94
2g
2i
96
91
3a
3b
59
88
Figure 2. Inhibition of colonies of Candida albicans by different 1,4-
dihydropyridines at a concentration of 31.25 lg/ml (inactive com-
pounds 2b, 2d and 5–7 are not displayed in the figure).
activity at 11.68 lg/disc in DDA and 31.25 lg/ml, both
in MDA and PSGIA. The only difference in the struc-
ture of compounds 2e and 3a is the presence of carbome-
thoxy group at C-3 and C-5 positions in compound 3a
instead of carboethoxy group at the same positions in
compound 2e; both these compounds have 4-methoxy-
phenyl substituents at C-4 position of dihydropyridine
ring. The dihydropyridine derivatives, that is, 2c, 2f,
2g and 2i having carboethoxy groups at C-3 and C-5
positions, do not show any appreciable activity and
the other three compounds of this series, 2a, 2b and
2d, are completely inactive. In all these dihydropyridine
derivatives having carboethoxy group at C-3 and C-5
positions, the C-4 aryl group is different than the 4-
methoxyphenyl groups in active compounds 2e and 3a.
This indicated that 4-methoxyphenyl group is one of
the essential features for a dihydropyridine to exhibit
inhibition of growth of A. fumigatus. Both compounds
2e and 3a have 4-methoxyphenyl substituent at C-4 po-
sition, but the activity of latter compound is less than
the activity of the former indicating that C-3 and C-5-
carboalkoxy groups also affect the antifungal activity
of dihydropyridines, and carboethoxy groups at these
positions are better than the carbomethoxy groups. It
is interesting to note that compound 2f is much less ac-
tive than compound 2e, although it has methoxy group
at C-4 position in the phenyl ring of the compound
together with carboethoxy groups at C-3 and C-5-posi-
tions. This may be because of the presence of a hydroxyl
group at C-3 position in the C-4 phenyl moiety in com-
pound 2f. This again indicates the importance of substit-

A. K. Chhillar et al. / Bioorg. Med. Chem. 14 (2006) 973–981
977
MIC 90
100
90
80
70
60
50
40
30
20
10
0
0
0.5
1
1.5
2
2.5
3
ln C (µg/ml)
Figure 3. MIC90 of compound 2e against Candida albicans.
uents present on the C-4 phenyl group of the dihydro-
pyridines. On the basis of structure–activity relationship
drawn above compound 3b should not exhibit apprecia-
ble inhibition of A. fumigatus growth, which is also re-
vealed by experiment. Finally to examine the
importance of dihydropyridine ring, we investigated
the activity of three tetrahydropyrimidin-2-one deriva-
tives 5–7, which have similar structures as the dihydro-
pyrimidines under study; none of the compounds 5–7
showed inhibition of A. fumigatus.
substituent at C-3 and C-5 positions exhibited better
therapeutic index than the other with carbomethoxy
group at same positions. The anti-Candida activity of
compound 2e was further evaluated by microbroth dilu-
tion assay (Fig. 3). It was observed that there was more
than 90% inhibition of growth of C. albicans cells in
wells treated with 125.0 lg/ml of compound 2e
(Fig. 3). Thus, anti-Candida activity testing of 4-aryl-
1,4-dihydropyridines and 4-aryl-1,2,3,4-tetrahydropyr-
imidin-2-ones by counting colony-forming units showed
that compound 2e is a potential anticandidal agent,
which is further confirmed by 90% inhibition of growth
of C. albicans cells by the compound in microbroth dilu-
tion assay.43
3.0.2. Anti-C. albicans activity. The preliminary anti-
Candida activity of ten 4-aryl-1,4-dihydropyridines 2a–
2g, 2i, 3a and 3b and three 4-aryl-1,2,3,4-tetrahydropyri-
din-2-ones 5–7 have been evaluated by counting colony-
forming units42 (CFU/ml). The control experiment
exhibited the growth of 136 colonies of C. albicans,
out of which 124 colonies were inhibited by standard
drug amphotericin B at a concentration of 31.25 lg/
ml, that is, amphotericin B exhibited 91.2% inhibition
(Fig. 2). As in the case of antifungal activity against
A. fumigatus, compound 2e exhibited maximum activity
against C. albicans by inhibiting 77 colonies (56.6% inhi-
bition) out of 136 colonies followed by compound 3a
(45.6%) at a concentration of 31.25 lg/ml (Fig. 2). This
may again be due to the similar structures of compounds
2e and 3a, which differ only in the substitution at C-3
and C-5 positions of dihydropyridine ring. The antican-
didal activity of compounds 2c, 2f, 2g and 2i is low and
of the same order, that is, 33.1, 35.3, 29.4 and 30.9%,
respectively (Fig. 2). Compounds 2a and 3b did not
show any appreciable activity. The other two 4-aryl-
1,4-dihydropyridine derivatives 2b and 2d, and three 4-
aryl-1,2,3,4-tetrahydropyrimidin-2-ones 5–7 evaluated
for their anti-Candida potential under this study were
found inactive. These results again revealed that carbo-
ethoxy substituent at C-3 and C-5 positions and 4-meth-
oxyphenyl substituent at C-4 position of phenyl ring in
4-aryl-1,4-dihydropyridine make the compound a better
anticandidal agent. Among the two 4-methoxyphenyl-
1,4-dihydropyridines 2e and 3a, one with carboethoxy
3.1. Cytotoxicity study on diethyl 4-(4-methoxyphenyl)-
2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate (2e)
The in vitro cell cytotoxicity of 4-aryl-1,4-dihydropyri-
dine 2e was investigated using haemolytic assay44
(Fig. 4). In a dose-dependent study, compound 2e was
Amphotericin B
Compound 2e
120
100
80
60
40
20
0
0
1
2
3
4
5
log concentration (µg/ml)
Figure 4. Cytotoxicity of compound 2e against erythrocytes using
haemolytic assay.

978
A. K. Chhillar et al. / Bioorg. Med. Chem. 14 (2006) 973–981
found to be non-toxic up to the concentration of
625.0 lg/ml and it lysed only 17.95% of human erythro-
cytes at the highest dose tested, that is, at a concentra-
tion of 10,000 lg/ml (Fig. 4). The standard drug
amphotericin B lysed 100% erythrocytes at a concentra-
tion of 39.05 lg/ml. This result is further confirmed by
the study of Cybulska et al.45, who have reported that
1.70 lg/ml of amphotericin B caused 50% haemoglobin
loss from erythrocytes.
5.3. Pathogens
Pathogenic strain of A. fumigatus was obtained from
Microbiology Department of Vallabhbhai Patel Chest
Institute, Delhi, India, and C. albicans was obtained
from IARI, New Delhi, India. A. fumigatus and C. albi-
cans were grown on SabouraudÕs dextrose agar at 37 ꢁC.
5.4. General method of synthesis of 4-aryl-1,4-dihydro-
pyridine derivatives 2a–2g, 3a and 3b
4. Conclusion
A mixture of aromatic aldehyde (1a–1g, 4 mmol), ethyl
acetoacetate/methyl acetoacetate (8 mmol) and urea
(4 mmol) was thoroughly mixed with silica gel (100–
200 mesh, 2 g). The mixture was taken in a beaker,
placed in an alumina bath and subjected to microwave
irradiation for 1.5–2 min at 850 W (Scheme 1).25 After
completion of the reaction as indicated by TLC, the
reaction-mass was directly loaded on a silica gel column,
and compounds 2a–2g, 3a and 3b were eluted with 4-7%
acetone in chloroform as colourless/pale yellow solids in
65–80% yields. The structures of 2a–2g, 3a and 3b were
unambiguously established on the basis of their spectral
analysis (IR, 1H-, 13C and mass) and comparison of
their melting points and/or spectral data with those
reported in the literature.34–39
Eleven 4-aryl-1,4-dihydropyridines, including the inter-
mediate epoxide 2h and three 4-aryl-1,2,3,4-tetrahydro-
pyrimidine derivatives, have been synthesized out of
which four compounds are novel and have not been
synthesized earlier. The antifungal activity evaluation
studies on 4-aryl-1,4-dihydropyridines 2a–2g, 2i, 3a
and 3b, and 4-aryl-1,2,3,4-tetrahydropyrimidin-2-ones
5–7 have revealed that diethyl 4-(4-methoxyphenyl)-
2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate 2e is
a potent antifungal agent. Although the potency of
compound 2e is less than one of the most active anti-
fungal compounds amphotericin B, the toxicity of this
active dihydropyridine derivative is about 16 times less
than the toxicity of amphotericin B. This has revealed
that the active dihydropyridine derivative is a much
safer drug candidate and can be taken up for the devel-
opment of a suitable antifungal drug through genera-
tion of a library of analogues of 2e and for further
studies.
5.5. Synthesis of diethyl 4-[3-(3-chloro-2-hydroxyprop-
oxy)phenyl]-2,6,dimethyl-1,4-dihydropyridine-3,5-dicarb-
oxylate (2i)
The title compound 2i was synthesized in two steps
starting from the refluxing of a mixture of compound
2d (2 mmol) and epichlorohydrin (2.2 mmol) in dry eth-
anol (20 ml) in the presence of fused potassium carbon-
ate (1 g) (Scheme 2). On completion of the reaction,
potassium carbonate was filtered off and solvent was re-
moved under reduced pressure. The crude product, thus
obtained, was purified over silica gel column using
petroleum ether–ethyl acetate as mobile phase to afford
pure 2h as a colourless solid (650 mg) in 78% yield. Mp.
105 ꢁC; IR (KBr): 3336 (NH), 1693 (CO), 1489, 1300,
5. Experimental
5.1. General
Melting points were determined on a sulfuric acid bath
and are uncorrected. The IR spectra were recorded
either on a Perkin-Elmer model 2000 FT-IR or RXI
FT-IR spectrophotometer. The 1H NMR and 13C
NMR spectra were recorded on a Bruker Avance 300
spectrometer at 300 and 75.5 MHz, respectively, using
TMS as internal standard. The chemical shift values
are on d scale and the coupling constant values (J) are
in Hz. The HRMS were recorded on a JMS-AX 505W
instrument at 70 eV in FAB (positive or negative ion
mode) using bis-hydroxyethyldisulphide (HEDS) doped
with sodium ions and 3-nitrobenzyl alcohol (NBA) as
matrix. Microwave reactions were performed in a
domestic microwave oven of 850 W and 1.2 Cft (33L,
Infodisplay, Sharp Carosel). Analytical TLCs were per-
formed on pre-coated Merck silica gel 60F254 plates; the
spots were detected either by viewing under UV light or
by charring with 10% alcoholic H2SO4.
1271, 1202 and 1097 cmꢀ1
;
1H NMR (300 MHz,
CDCl3): d 1.22 (6H, t, J = 7.1 Hz, 2· COOCH2CH3),
2.31 (6H, s, C-2 and C-6 CH3), 2.72 (1H, dd, J = 2.6
and 4.8 Hz, C-300Ha) and 2.90 (1H, t, J = 4.6 Hz, C-
300Hb), 3.33 (1H, m, C-200H), 3.90 (1H, dd, J = 5.6 and
10.9 Hz, C-100Ha), 4.07 (4 H, m, 2· COOCH2CH3),
4.15 (1H, dd, J = 3.8 and 11.0 Hz, C-100Hb), 4.98 (1H,
s, C-4H), 5.98 (1H, br s, NH), 6.66 (1H, dd, J = 1.9
and 7.9 Hz, C-60H), 6.85 (1H, br s, C-20H), 6.90 (1H,
d, J = 7.6 Hz, C-40H) and 7.11 (1H, t, J = 7.7 Hz, C-
50H); 13C NMR (75.5 MHz, CDCl3):
d
14.68
(2· COOCH2CH3), 19.96 (2· CH3), 39.95 (C-300), 45.28
(C-200), 50.61 (C-4), 60.12 (2· COOCH2CH3), 68.99 (C-
100), 104.30 (C-2 and C-6), 111.99, 115.28, 121.55,
129.04 (C-20, C-40, C-50 and C-60), 144.37 (C-3 and C-
5), 149.85 (C-10), 158.67 (C-30) and 167.98 (2· CO);
HRMS: calculated for C22H27NO6 [M]+ 401.1736, ob-
served [M]+ 401.1750. The epoxide 2h (1.2 mmol) was
dissolved in methanolic–HCl (15 ml) and stirred at 25–
28 ꢁC until complete conversion of the starting material
into a slow moving product on TLC was seen. The
5.2. Materials
SabouraudÕs dextrose agar and SabouraudÕs dextrose
broth were purchased from Hi Media, Mumbai, India.
Amphotericin B and DMSO were purchased from Sig-
ma Chemical Company, USA.

A. K. Chhillar et al. / Bioorg. Med. Chem. 14 (2006) 973–981
979
solvent of the reaction was removed, ice-cold water
(25 ml) was added and the product was extracted with
chloroform (2 · 25 ml). Combined organic layer was
dried over Na2SO4, solvent was removed and the crude
product was purified over silica gel column using ethyl
acetate petroleum ether as eluent to afford compound
2i as a colourless solid (378 mg) in 72% yield. Mp.
141–142 ꢁC; IR (KBr): 3400 (OH), 2979, 2366, 1669
J = 1.9 Hz, C-4H), 5.85 (1H, br s, N-1H), 7.02 (2H, d,
J = 8.5 Hz, C-30H and C-50H), 7.32 (2H, d, J = 8.5 Hz,
C-20H and C-60H) and 8.19 (1H, s, N-3H); 13C NMR
(75.5 MHz, CDCl3): d 13.86 (COOCH2CH3), 18.39
and 20.79 (OCOCH3 and C-6CH3), 54.86 (C-4), 59.79
(COOCH2CH3), 101.03 (C-5), 121.46 (C-30 and C-50),
127.44 (C-20 and C-60), 140.99 (C-10), 146.10 (C-6),
149.97 (C-40), 153.01 (C-2), 165.24 (COOCH2CH3) and
169.01(OCOCH3); HRMS: calculated for C16H18N2O5
[M]+ 318.1216, observed [M]+ 318.1201.
1
(CO), 1492, 1373, 1215, 1125, 1047 and 747 cmꢀ1; H
NMR (300 MHz, CDCl3): d 1.25 (6H, t, J = 7.1 Hz,
2· COOCH2CH3), 2.31 (6H, s, C-2 and C-6 CH3),
2.60 (1H, br s, C-200 OH), 3.71 (2H, m, C-300H), 4.10
(7H, m, 2· COOCH2CH3, C-100H and C-200H), 4.98
(1H, s, C-4H), 5.84 (1H, br s, NH), 6.67 (1H, dd,
J = 1.9 and 7.9 Hz, C-60H), 6.84 (1H, br s, C-20H),
6.90 (1H, d, J = 7.6 Hz, C-40H) and 7.11 (1H, t,
J = 7.9 Hz, C-50H); 13C NMR (75.5 MHz, CDCl3): d
14.66 (2· COOCH2CH3), 19.81 (2· CH3), 40.02 (C-300),
46.43 (C-4), 60.19 (2· COOCH2CH3), 68.86 and 70.34
(C-100 and C-200), 104.14 (C-2 and C-6), 112.16, 115.12,
121.64, 129.16 (C-20, C-40, C-50 and C-60), 144.64 (C-3
and C-5), 149.97 (C-10), 158.45 (C-30) and 168.16
(2· CO); HRMS: calculated for C22H28NO6Cl [M]+
437.1509, observed [M]+ 437.1503.
5.7. Synthesis of ethyl 4-(4-epoxymethoxyphenyl)-6-
methyl-1,2,3,4-tetrahydropyrimidin-2-one-5- carboxylate
(7)
A mixture of compound 5 (2 mmol) and epichlorohy-
drin (2.2 mmol) in dry ethanol (20 ml) was refluxed in
the presence of fused potassium carbonate (2 mmol).
On completion of the reaction, potassium carbonate
was filtered off, solvent was removed under reduced
pressure and the crude product, thus obtained, was puri-
fied over silica gel column using CHCl3 as mobile phase
to afford pure 7 as a colourless solid (511 mg) in 77%
yield. Mp. 196–198 ꢁC; IR (KBr): 3246 (NH), 3119,
2927, 1721 (CO), 1648 (CO), 1513, 1457, 1235, 1099,
1
5.6. Synthesis of ethyl 4-(4-acetoxyphenyl)-6-methyl-
1,2,3,4-tetrahydropyrimidin-2-one-5- carboxylate (6)
1033, 765 cmꢀ1; H NMR (300 MHz, CDCl3): d 1.16
(3H, t, J = 6.0 Hz, COOCH2CH3), 2.34 (3H, s, C-
6CH3), 2.74 (1H, br s, C-300Ha), 2.89 (1H, t,
J = 6.03 Hz, C-300Hb), 3.33 (1H, br s, C-200H), 3.94
(1H, dd, J = 5.4 and 10.8 Hz, C-100Ha), 4.07 (2H, q,
J = 6.0 Hz, COOCH2CH3), 4.20 (1H, dd, J = 1.5 and
10.8 Hz, C-100Hb), 5.35 (1H, br s, C-4H), 5.41 (1H, br
s, N-1H), 6.85 (2H, d, J = 6.8 Hz, C-30H and C-50H),
7.24 (2H, d, J = 6.8 Hz, C-20H and C-60H) and 7.28
(1H, br s, N-3H); 13C NMR (75.5 MHz, CDCl3 + few
drops of DMSO-d6): d 14.15 (COOCH2CH3), 18.17
(C-6CH3), 44.17 (C-300), 49.96 (C-200), 54.25 (C-4),
59.39 (COOCH2CH3), 68.82 (C-100), 100.36 (C-5),
114.35 (C-30 and C-50), 127.79 (C-20 and C-60), 137.71
(C-10), 147.53 (C-6), 152.85 (C-40), 157.69 (C-2) and
165.79 (COOCH2CH3); HRMS: calculated for
C17H20N2O5 [M]+ 332.1372, observed [M]+ 332.1372.
A mixture of 4-hydroxybenzaldehyde (4, 5 mmol), ethyl
acetoacetate (5.5 mmol), urea (15 mmol) and ferric chlo-
ride hexahydrate (5 mmol) was thoroughly mixed with
silica gel (100–200 mesh, 2 g). The mixture was taken
in a beaker, which was placed in an alumina bath and
subjected to microwave irradiation of four shots of
15 s each at an interval of 1 min at 850 W (Scheme
3).40 After completion of the reaction as indicated by
TLC, the reaction-mass was directly loaded on a silica
gel column and eluted with 1% methanol in chloroform
to afford compound 5 as a yellow solid. For further puri-
fication, compound 5 was dissolved in methanol (50 ml),
the solution passed through a pad of Celite (5 g) and the
solvent was removed to afford 5 as a colourless solid
(1.17 g, mp 140–141 ꢁC; literature40 mp 141–142 ꢁC) in
85% yield. The structure of compound 5 was unambigu-
ously established on the basis of its spectral analysis (IR,
6. Antifungal activity assay
1
UV, H-, 13C and mass) and comparison of the data
with those reported in the literature.40 To a solution of
compound 5 (1 mmol) in acetic anhydride (1.1 equiv)
and pyridine (2 equiv) was added a catalytic amount
of 4-N,N-dimethylaminopyridine and the reaction mix-
ture was stirred at 25–28 ꢁC for an hour when TLC
examination showed complete conversion of the starting
material compound into a product of higher Rf value.
Ice-cold water (30 ml) was added to the reaction under
vigorous stirring, and solid separated was filtered off
and washed with water and petroleum ether to afford
6 as a white solid (280 mg) in 88% yield. Mp. 141–
142 ꢁC; IR (KBr): 3249, 3117, 2981, 1723 (CO of ethyl
ester), 1704 (CO of acetate), 1651 (CO of amide),
The anti-A. fumigatus activity of all the compounds was
studied by disc diffusion, microbroth dilution and per-
centage spore germination inhibition assays.41 The
activity against C. albicans was carried out by the meth-
od of Riffel et al.42 and Iijima et al.43
7. Anti-Aspergillus activity assay
7.1. Disc diffusion
The disc diffusion assay was performed in radiation ster-
ilized petri plates of 10.0 cm diameter (Tarsons) as
described.41 Different concentrations in the range of
750–1.46 lg of the test compounds were impregnated
on the sterilized discs (5.0 mm in diameter) of Whatman
filter paper No 1. The discs were placed on the surface of
1508, 1225, 1094, 785 cmꢀ1
;
1H NMR (300 MHz,
CDCl3): d 1.17 (3H, t, J = 6.0 Hz, COOCH2CH3), 2.28
and 2.33(6H, 2s, 3H each, OCOCH3 and C-6CH3),
4.08 (2H, q, J = 6.0 Hz, COOCH2CH3), 5.40 (1H, d,

980
A. K. Chhillar et al. / Bioorg. Med. Chem. 14 (2006) 973–981
the agar plates already inoculated with A. fumigatus
spores. The plates were incubated at 37 ꢁC and exam-
ined at 24, 48 and 96 h for zone of inhibition, if any,
around the discs. The concentration, which developed
the zone of inhibition of at least 6.0 mm diameter, was
considered as minimum inhibitory concentration (MIC).
assay.44 A slight modification was employed to deter-
mine the haemolytic effect of antifungal dihydropyridine
derivative 2e. Human erythrocytes collected from appar-
ently healthy volunteers were washed thrice with PBS
and 2.0% (v/v) suspension of erythrocytes was prepared
in phosphate buffer saline at pH 7.2. Half millilitre of
(2.0%) human erythrocyte suspension in 16 duplicate
sets of tubes was treated with compound 2e at a concen-
tration of 1.22 lg/ml for 1 h at 37 ꢁC. After incubation,
tubes were centrifuged at 5000 rpm for 10 min. The
supernatant was collected and the OD was measured
at A415 nm using a spectrophotometer (UV–Vis Spect
Lambda Bio 20 Perkin-Elmer). Results were expressed
as % haemolysis by the compound. Only buffer of pH
7.2 was used for background lysis in negative control
sets, whereas in positive controls, lysis buffer was used
for completely lysing the erythrocytes.
7.2. Microbroth dilution
The test was performed in 96-well culture plates (Nunc,
Nunclon). Various concentrations of synthetic com-
pounds in the range of 1000–7.81 lg/ml were prepared
in the wells by twofold dilution method. Assay was per-
formed as per standard method described earlier.41
7.3. Percentage spore germination inhibition
Various concentrations of the test compounds in 90.0 ll
of culture medium were prepared in 96-well flat-bottomed
micro-culture plates (Nunc, Nunclon) by double dilution
method. The wells were prepared in triplicates for each
concentration. Each well was then inoculated with
10.0 ll of spore suspension containing 100 5 spores.
The plates were incubated at 37 ꢁC for 8 h and then exam-
ined for spore germination with an inverted microscope
(Nikon, Diphot). The number of germinated and non-
germinated spores was counted. The percentage spore
germination inhibition was calculated.41 All the tests were
repeated at least three times. The lowest concentration of
the compound, which resulted in >90% inhibition of ger-
mination of spores in the wells was considered as MIC90.
Acknowledgment
A.K.C. is thankful to the Department of Science and
Technology (DST, Government of India, New Delhi)
for a grant of Young Scientist Fellowship.
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