Efficient one-pot preparation of novel fused chromeno[2,3-d]pyrimidine and pyrano[2,3-d]pyrimidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.09.040

Some novel chromeno[2,3-d]pyrimidinone, pyrano[2,3-d]pyrimidine, dihydropyrimidine, pyridopyranopyrimidine and pyrimidopyranopyrimidine have been synthesized. The structures of target compounds were confirmed by elemental analyses and spectral data. The a

Full text of DOI:10.1016/j.ejmech.2011.09.040

European Journal of Medicinal Chemistry 47 (2012) 18e23
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Original article
Efficient one-pot preparation of novel fused chromeno[2,3-d]pyrimidine and
pyrano[2,3-d]pyrimidine derivatives
,
b
Hala M. Aly a , Mona M. Kamal
*
a Department of Chemistry, Faculty of Science (Girl’s), Al-Azhar University, PO box 11754, Nasr City, Cairo, Egypt
b Department of Drug Radiation Research, National Center for Radiation Research and Technology, Atomic Energy Authority, Nasr City, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Some novel chromeno[2,3-d]pyrimidinone, pyrano[2,3-d]pyrimidine, dihydropyrimidine, pyridopy-
ranopyrimidine and pyrimidopyranopyrimidine have been synthesized. The structures of target
compounds were confirmed by elemental analyses and spectral data. The antimicrobial activity of all the
target synthesized compounds were tested against various microorganismst such as Pseudomonas aer-
uginosa; Staphylococcus aureus (Bacteria), Aspergillus flavus (Fungus) and Candida albicans (Yeast fungus)
by the disc diffusion method. In general, the novel synthesized compounds showed a good antimicrobial
activity against the previously mentioned microorganisms.
Received 11 December 2010
Received in revised form
22 September 2011
Accepted 22 September 2011
Available online 1 October 2011
Keywords:
Pyrimidine
Ó 2011 Elsevier Masson SAS. All rights reserved.
Antibacterial activity
Antifungal activity
1. Introduction
The significance of pyranopyrimidine derivatives is recognized
as a result of their occurrence in the structure of various natural
Pyrane and fused 4H-pyrane derivatives have attracted a great
interest owing to their antimicrobial activity [1e3], inhibition of
influenza, virus sialidases [4], mutagenic activity [5], antiviral [6]
antiproliferaction agents [7], sex-pheromones [8] antitumor [9]
and anti-inflammatory agents [10]. Moreover, pyrane derivatives
are well known for their antihistaminic activity [11]. Also, pyrim-
idines and its fused derivatives play an essential role in several
biological processes and chemical and pharmacological impor-
tance. In particular, pyrimidine nucleus can be found in a broad
variety of antibacterial and antitumor agents as well as in agro-
chemical and veterinary products [12e15]. Based on the above
information’s and due to our interest in developed the synthetic
strategies for the construction of novel fused pyrimidinones as
a biologically active pharmacophore [16e18], we have reported
a facile methodology for the synthesis and transformation of
pyrimidindione to pyrimidinones fused at C-5 and C-6 position.
Herein, a detailed account of our focused attention toward
construction of the pyrimidinones was reported, having latent
functionalities at C-5 position, which could form useful building
blocks for the synthesis of various C-5, C-6 heterocyclic ring
fused pyrimidinones via a Knoevenagel condensation and other
reactions.
products, their biological activity, and their synthetic potential
[19e21]. We thought it would be of interest to combine the above
mentioned heterocyclic compounds in a molecular framework to
investigate a possible additive effect of these rings regarding bio-
logical activity.
2. Results and discussion
2.1. Chemistry
A Knoevenagel condensation of 2-thioxo-dihydropyrimidine-4,
6(1H,5H)-dione 1 with aromatic aldehyde in ethanol under reflux
in the presence of piperidine yielded the chromeno[2,3-d]pyrim-
idine derivative 3. Assignment of chromeno[2,3-d]pyrimidine
derivative 3 was based on the basis of elemental and spectral data.
Its infrared spectrum exhibited the absence of one carbonyl group
absorption band while it showed the presence of broad band for
OH group stretching at 3432 cmꢀ1. Its mass spectrum showed
a molecular ion peak at m/z 246 (Mþ) (10.98) together with a base
peak at m/z 86 (100). The formation of compound 3 is assumed to
proceed through the in situ intramolecular cyclization of the non-
isolable intermediate 2 via nucleophilic addition of the hydroxyl
group to the carbonyl group followed by elimination of water
(Scheme 1).
* Corresponding author. Tel.: þ20 105356623.
E-mail address: hala_mali@yahoo.com (H.M. Aly).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.040

H.M. Aly, M.M. Kamal / European Journal of Medicinal Chemistry 47 (2012) 18e23
19
The infrared spectrum of compound 10 indicated the presence of
characteristic absorption bands for NH2 and ester functional groups.
In addition, the molecular structure of compound 10 was established
by 1H NMR spectrum which exhibited lack of the characteristic
O
CHO
O
OH
OH
HN
HN
O
HO
O
S
N
H
S
N
H
signal of methylene group and the presence of triplet at
quartet at 4.4 ppm assigned for ethoxycarhonyl moiety in addition
to amino protons at 5.8 ppm. The molecular ion peak of compound
d 1.2 ppm,
OH
d
1
d
O
O
N
10 was found in the mass spectrum at m/z 379 (MþeOC2H5) (9.5)
corresponding to the molecular formula C16H14ClN3O4S with a base
peak at m/z 186 (100). Thionation reaction of the o-aminoester of
pyrano[2,3-d]pyrimidine derivative 10 was occurred by the inter-
actionwith phosphorus pentasulphide afforded the ethyl -7-amino-
5-(4-chlorophenyl)-2,4-dithioxo-2,3,4,5-tetrahydro-1H-thiopyr-
ano[2,3-d]pyrimidine-6-carboxylate 11. The infrared spectrum of
compound 11 indicated the presence of one carbonyl group
absorption band and other characteristic absorption bands at 1567
and 1355 cmꢀ1 for the two C]S functional group. The molecular ion
peak of compound 11 was found in the mass spectrum at m/z
411(MþeNH2) (2.13) corresponding to the molecular formula
C16H14ClN3O2S3. 5-Ethoxymethylene derivative 12 was obtained in
87% yield bythe alkylation reaction of startingmaterial 1 with excess
amount of triethyl orthoformate at 200 ꢁC under solvent free
condition (Scheme 3). The reactivityof compound 12 which contains
chalcone system, toward hydrazine hydrate was investigated. Thus,
hydrazinolysis of compound 12 with hydrazine hydrate furnished
hydrazinyl methylene derivative 13 and attempts to cyclize the
hydrazide derivative 13 by refluxing in ethanol containing piperi-
dine and/or pyridine to afford 6-thioxo-6,7-dihydropyrazolo[3,4-d]
pyrimidinone derivative 14 were unsuccessful on the basis of
analytical and spectral data. Furthermore, o-aminoester of pyrano
[2,3-d]pyrimidine derivative 6 gave characteristic reaction for
enaminonitriles so it used as a key precursor for the synthesis of
condensed heterocyclic compounds of expected biological activity.
The interaction of pyrano[2,3-d]pyrimidine 6 with malononitrile in
the presence of piperidine as basic condition afforded the corre-
sponding pyridopyranopyrimidine 15. 2-arylsulfonylamino pyrano
[2,3-d]pyrimidine derivative 16 was obtained by refluxing of
compound 6 with benzenesulfonyl chloride in dry benzene. The
structure of compound 16 was demonstrated by spectral data. IR
spectrum revealed band for (C^N). Mass spectrum of 16 showed
a molecular ion peak m/z at 528 [Mþ] (2.1), with a base peak at 222
(100). Finally, the behavior of compound 6 toward acid derivative
was also investigated. Thus, heating compound 6 with formic acid
caused cyclization to give the corresponding pyrimidopyranopyr-
imidine derivative 17 with a new ring system. Compound 17 was
formed via Dimruth rearrangement illustrated in Scheme 4. The
-H2O
HN
HN
S
N
H
O
OH
S
O
OH
OH
2
3
Scheme 1.
The active methylene group in pyrimidindione derivative 1
was exploited to synthesize novel pyranopyrimidine and hydrazi-
nyl-methylene-2-thioxo-dihydropyrimidine derivatives through its
reactions with some electrophiles. Thus, the 7-aminopyrano[2,3-d]
pyrimidine-6-carbonitrile derivative 6 was synthesized in a good
yield, as stable crystalline solids and easily recrystallized from dioxane,
when treatment of compound 1 and 2-(3,4,5-trimethoxybenzylidene)
malononitrile 4 under reflux in absolute ethanol, in the presence of
catalytic amounts of piperidine. The structure of compound 6 was
characterized by elemental and spectroscopic analysis. Thus, IR spec-
trum of compound 6 revealed the presence of characteristic bands for
amino, cyano and one carbonyl functional groups. In addition, the 1H
NMR spectrum of compound 6 in DMSO-d6 revealed the absence of
methylene moiety. The structure of compound 6 was supported by its
mass spectrum which revealed a molecular ion peak at m/z 388 (Mþ)
(2.86). Pyrano[2,3-d]pyrimidine derivative 6 was assumed to be
formed via Michael addition of the active methylene group in pyr-
imidindione derivative 1 to the activated double bond in cyanoar-
ylidine derivative 4 to form the non-isolable intermediate 5 followed
by the intramolecular cyclization to furnish o-aminocarbonitrile of
pyrano[2,3-d]pyrimidinone derivative 6 (Scheme 2).
In a similar manner, the reaction of starting material 1 with
equimolar amount of ethyl 3-(4-chlorophenyl)-2-cyanoacrylate 7
under refluxing ethanol in the presence of piperidine afforded ethyl
7-amino-5-(4-chlorophenyl)-4-oxo-2-thioxo-2,3,4,5-tetrahydro-
1H-pyrano[2,3-d]pyrimidine-6-carboxylate 10. Both elemental and
spectral data supported the proposed structure 10 and the forma-
tion of Michael adduct 8 followed by intramolecular cyclization
through elimination of ethanol to afforded 9 was ruled out
(Scheme 3).
O
O
O
Ar
O
H
O
Ar
H
H
CN
H
Ar
C
H
O
CN
B
HN
HN
HN
BH
HN
NH
C
1,4-addition
S
N
H
O
S
N
H
NH
S
N
H
C
NC
N
S
N
H
O
H
4
1
Ar=C6H2(OCH3)-3,4,5
O
Ar
O
Ar
CN
CN
NH
1,3-proton shift
HN
HN
S
N
H
O
NH2
S
N
H
O
H
6
5
Scheme 2.

20
H.M. Aly, M.M. Kamal / European Journal of Medicinal Chemistry 47 (2012) 18e23
Scheme 3.
structure of compound 17 was supported by its analytical and
spectral data. The infrared spectrum of compound 17 showed the
absence of (C^N) band and the presence bands at 3293,3224 cmꢀ1
(br, NH), 1690, 1676 cmꢀ1 (2C]O), 1620 cmꢀ1 (C]N). The mass
spectrum of compound 17 revealed a molecular ion peak m/z at 416
[Mþ] (39.71), with a base peak at 180 (100).
2.2. Antimicrobial activity
All of the target synthesized compounds were screened against
cultures of two fungal species, namely Aspergillus flavus and
Candida albicans as well as two bacteria species, namely Pseudo-
monas aeruginosa and Staphylococcus aureus to detect their
6
CN
HCOOH
CN
O
Ar NH
HN
O
O
Ar NH2
PhSO2Cl
CN
S
N
H
O
N
HN
O
Ar
S
N
H
O
N
NH2
Dimurth rearrangment
CN
15
HN
O
O
Ar
O
N
S
N
H
O
N S Ph
H
O
HN
NH
16
S
N
O
H
Ar = C6H2(OCH3)-3,4,5
17
O
N
O
N
NH
NH
O
NH
NH
O
Nu
N
Nu
N
Nu
Mechanism of Dimurth rearrangment
Scheme 4.

H.M. Aly, M.M. Kamal / European Journal of Medicinal Chemistry 47 (2012) 18e23
21
Table 1
moiety and biologically active groups like 3-hydoxy, eOCH3,
4-chloro groups attached to phenyl ring of the pyrane ring and
eOC2H5,eNHNH2 groups attached to pyrimidine moiety and 2,4-
diamino-3-cyano-pyridyl, benzenesulfonamide and pyrano[2,3-d]
pyrimidine moiety attached to the pyrano[2,3-d]pyrimidine of the
title compounds are responsible for the good antimicrobial activity.
From the obtained antifungal and antibacterial results, we can
conclude that the entire tested compounds are more active toward
bacteria than fungi.
Antimicrobial activity of chemical substances tested.
Compound No.
Inhibition zone diameter (mm)
Antibacterial
Antifungal
Pseudomonas
aeruginosa
Staphylococcus
aureus
Aspergillus
flavus
Candida
albicans
3
6
10
11
12
13
15
16
14
13
19
18
13
18
12
13
13
28
e
15
13
16
16
13
18
13
14
15
26
e
0
0
0
14
0
36
0
0
10
13
14
12
0
10
12
11
e
4. Experimental
4.1. Chemistry
0
0
17
Tetracyclin
Amphotericin B
e
All melting points are uncorrected and were determined on
a Stuart melting point apparatus. IR spectra were recorded on
a Shimadzu-440 IR spectrophotometer using the KBr technique
(Shimadzu, Japan). 1H NMR spectra were measured on a Varian
Mercury VX-300 NMR spectrometer in DMSO-d6 as a solvent and
were run at 300 MHz, using tetramethylsilane (TMS) as an internal
standard. The mass spectra were recorded on Shimadzu GCMS-QP-
1000EX mass spectrometers at 70 eV. The purity of the synthesized
compounds was monitored by TLC. Elemental analyses were
carried out by the Microanalytical Research Center, Faculty of
Science, Cairo University. Analytical results for C, H, N and S were
within ꢂ0.4 of the calculated values.
16
19
Tetracyclin used as a standard (antibacterial agent).
Amphotericin B used as a standard (antifungal agent).
antimicrobial activities. The antimicrobial activity was biologi-
cally assayed using the diffusion technique. The investigation of
antibacterial and antifungal screening data revealed that all the
tested compounds 3, 6, 10, 11, 12, 13, 16 and 17 showed compar-
atively good activity against all the bacterial strains. The organ-
isms were tested against the activity of solutions with
concentration; 1.0 mg/mL of each compound; and using inhibition
zone diameter (IZD) in centimeter as the criterion for antimicro-
bial activity. Amphotericin B as an antifungal agent and Tetracyclin
as an antibacterial agent were used as references to evaluate the
potency of the tested compounds under the same conditions. The
minimum inhibitory concentration (MIC) of the biologically active
compounds was measured by a two-fold serial dilution method.
The results are depicted in Table 1. The good activity is attributed
to the presence of pharmacologically active 3-hydoxy, eOCH3,
4-chloro groups attached to phenyl ring on the pyrane ring and
eOC2H5,eNHNH2 groups attached to pyrimidine moiety and 2,4-
diamino-3-cyanopyridyl, benzenesulfonamide and pyrano[2,3-d]
pyrimidine moiety attached to the pyrano[2,3-d]pyrimidine. In
conclusion, we reported herein a simple and convenient route for
the synthesis of some new heterocyclic compounds based on
2-thiobarbituric acid for antimicrobial evaluation. Most of the
compounds were effective against C. albicans. In case of Ampho-
tericin B; compounds 6, 10, 11,12,15,16 and 17 are mostly effective,
while 3, 13 have no activity. The pyrano[2,3-d]pyrimidine-6-
carboxylate 11 nearly as active as the standard fungicide Ampho-
tericin B. Among these nine active compounds, the most and the
only more effective compound against A. flavus than the standard
fungicide Amphotericin B was 13, which includes hydrazide
subsistent on pyrimidine moiety while other effective compounds
does not have. However, it was also observed that the substations
on the pyrane derivatives had no determining influence on the
4.1.1. 8-Hydroxy-2-thioxo-2,3-dihydrochromeno[2,3-d]pyrimidin-
4-one (3)
A mixture of compound 1 (0.72 g, 0.005 mol), 2,4-dihydroxy
benzaldehyde (0.69 g, 0.005 mol), and piperidine (0.005 mol) in
ethanol (60 mL) was heated under reflux for 3 h, The solid product
was filtered on hot, dried, and crystallized from dioxane to give
orange powders 3: Yield, 80%; m.p. 185e186 ꢁC; IR, cmꢀ1: 3432
(br,OH), 3325 (NH), 1694 (C]O), 1563 (C]S). 1H NMR (DMSO-d6)
d:
6.9e7.4 (m, 4H, AreH), 10.0 (s, H, OH), 11.6 (s, H, NH pyrimidine).
MS, m/z (%): 246 (M) (10.98), 86 (100). Anal. Calcd. For C11H6N2O3S:
C, 53.65; H, 2.46; N,11.38; S,13.02. Found: C, 53.55; H, 2.06; N,11.18;
S, 13.00.
4.1.2. General procedure for the reaction of barbituric acid with
arylidine derivatives
A
mixture of
trimethoxy- benzylidene)malononitrile 4 (4.64 g, 0.02 mol) or
ethyl 3-(4-chlorophenyl)-2-cyanoacrylate and triethylamine
1 (2.88 g, 0.02 mol), and either 2-(3,4,5-
7
(0.02 mol) in ethanol (10 mL) was refluxed for 3 h. After cooling, the
resulting solid product was collected by filtration, washed with
water, and the crude product recrystallized from ethanol to give 6
and 10, respectively.
4.1.2.1. 7-Amino-4-oxo-2-thioxo-5-(3,4,5-trimethoxyphenyl)-2,3,4,5-
tetrahydro-1H-pyrano[2,3-d]pyrimidine-6-carbonitrile (6). Yellow
crystals: Yield, 85%; m.p. 221e223 ꢁC. IR, cmꢀ1: 3369, 3330,
3251(NH2,NH), 3189 (CH arom.), 2986 (CH aliph.), 2203(C^N),
antifungal activity (MIC values <50
compounds exhibited no activity against the tested species
(Table 1).
mg/mL). All of the other
1689(C]O), 1567(C]S), 1H NMR (DMSO-d6)
d: 3.7(s,9H, 3OCH3),
4.16 (s, 1H, H-5), 7.19 (s, 2H, NH2), 7.20e7.35 (m,2H, AreH), 11.5,
12.01 ppm (2s, 2H, 2NH pyrimidine). MS, m/z (%): 388 (Mþ) (2.86),
244 (100). Anal. Calcd. For C17H16N4O5S: C, 52.57; H, 4.15; N, 14.43;
S, 8.26. Found: C, 52.87; H, 4.35; N, 14.53; S, 8.46.
3. Conclusions
The research study reports the successful synthesis and anti-
microbial activity of new chromene, pyrane and pyranopyridine
derivatives bearing 2-thiobarbituric acid moiety. The antimicrobial
activity study revealed that all the tested compounds showed
moderate to good antibacterial and antifungal activities against
pathogenic strains. Structure and biological activity relationship of
title compounds showed that the presence of thiopyrane
4.1.2.2. Ethyl-7-amino-5-(4-chlorophenyl)-4-oxo-2-thioxo-2,3,4,5-
tetrahydro-1H-pyrano[2,3-d]pyrimidine-6-carboxylate
(10). Off
white crystals: yield, 88%; m.p. 210e212 ꢁC; IR, cmꢀ1: 3372, 3334,
3245 (NH2, NH), 3132 (CH arom.), 3091 (CH aliph.), 1756,1694 (2C]
O), 1562 (C]S). 1H NMR (DMSO-d6)
d: 1.2 (t, 3H, CH3), 3.4 (s, 1H, H-

22
H.M. Aly, M.M. Kamal / European Journal of Medicinal Chemistry 47 (2012) 18e23
5), 4.4 (q, 2H, CH2), 5.8 (s, 2H, NH2), 7.4e7.6 (m,4H, AreH), 11.6,
12.1 ppm (2s, 2H, 2NH pyrimidine).MS, m/z (%): 379 (MþeOC2H5)
(9.5), 186 (100). Anal. Calcd. For C16H14ClN3O4S: C, 50.60; H, 3.72; N,
11.06; S, 8.44. Found: C, 50.40; H, 3.42; N, 11.04; S, 8.04.
ice water, then acidified with dilute HCl, the obtained solid was
crystallized from dioxane to give pale yellow powder 16: Yield,
69%; m.p. 228e230 ꢁC; IR, cmꢀ1: 3270, 3251 (3NH), 3093 (CH
arom.), 2940, 2886 (CH aliph.), 2185 (CN), 1677(C]O), 1578 (C]S).
1H NMR (DMSO-d6)
d: 3.8 (s, 9H, 3OCH3), 4.5 (s, 1H, CH-5 pyrane),
4.1.3. Ethyl-7-amino-5-(4-chlorophenyl)-2,4-dithioxo-2,3,4,5-
tetrahydro-1H-thiopyrano[2,3-d]pyrimidine-6-carboxylate (11)
A mixture of 10 (1.8 g, 0.005 mol,) and phosphorus pentasul-
phide (1.1 g, 0.005 mol) in dry xylene (50 mL) was heated under
reflux for 1 h then the solution was concentrated. The product was
obtained by filtration and recrystallized from xylene to give yellow
crystals 11: Yield, 75%; m.p. 220e222 ꢁC; IR, cmꢀ1: 3361, 3339, 3219
(NH2, NH), 3176 (CH arom.), 3101 (CH aliph.), 1769 (C]O), 1567,
7.0e7.5 (m, 7H, AreH), 11.5, 12.2 ppm (2s, 2H, 2NH pyrimidine). MS,
m/z (%): 528 (M) (2.1), 222 (100). Anal. Calcd. For C23H20N4O7S2: C,
52.26; H, 3.81; N, 10.60; S, 12.13. Found: C, 52.36; H, 3.91; N, 10.90;
S, 12.23.
4.1.8. 4,6-Dioxo-2-thioxo-5-(3,4,5-trimethoxyphenyl)-2,3,4,5,6,7-
sexahydro-1H-pyrimido [20,30:4,5]pyrano[2,3-d]pyrimidine (17)
A solution of compound 6 (1.94 g, 0.005 mol) in formic acid
(10 mL) was heated under reflux for 12 h. The reaction mixture was
concentrated in vacuum and the obtained solid was crystallized
from ethanol to give yellow powder 17: Yield, 73%; m.p.110e112 ꢁC;
IR, cmꢀ1: 3293, 3224 (br, NH), 3098 (CH aliph.), 3098 (CH arom.),
1355 (2C]S). 1H NMR (DMSO-d6)
d: 1.4 (t, 3H, CH3), 4.2 (q, 2H, CH2),
6.2 (s, 2H, NH2), 7.3e7.8 (m, 4H, AreH), 11.8, 12.0 (2s, 2H, NH). MS,
m/z (%): 411 (MþeNH2) (2.13), 144 (100). Anal. Calcd. For
C16H14ClN3O2S3: C, 46.65; H, 3.43; N, 10.20; S, 23.35. Found: C,
47.05; H, 3.63; N, 10.03; S, 23.65.
1690,1676 (2C]O),1620 (C]N),1562 (C]S). 1H NMR (DMSO-d6)
d:
3.6 (s, 9H, 3OCH3), 4.1 (s,1H, CH benzylic), 6.9e7.3 (d, 2H, AreH), 7.5
(s, 1H, CH]N), 8.0 (s, 1H, NH exchangeable), 11.8, 12.3 ppm (2s, 2H,
2NH pyrimidine). MS, m/z (%): 416(M) (39.71), 180 (100). Anal.
Calcd. For C18H16N4O6S: C, 51.92; H, 3.87; N, 13.45; S, 7.70. Found: C,
51.72; H, 3.67; N, 13.65; S, 7.90.
4.1.4. 5-(Ethoxymethylene)-2-thioxo-dihydropyrimidine-
4,6(1H,5H) dione (12)
A mixture of compound 1 (0.7 g, 0.005 mol) and triethyl
orthoformate (8 mL) was heated under reflux for 2 h. The obtained
solid was crystallized from ethanol to give yellow crystals 12: Yield,
87%; m.p. 230e232 ꢁC; IR, cmꢀ1: 3322 (NH), 3091, 3008 (CH aliph.),
4.3. Microbiological studies
1690, 1678 (2C]O), 1569 (C]S). 1H NMR (DMSO-d6)
d: 1.3 (t, 3H,
CH3), 4.0 (q, 2H, CH2), 8.0 (s, 1H, C]CHeOC2H5), 11.4, 12.3 ppm (2s,
2H, 2NH pyrimidine). Anal. Calcd. For C7H8N2O3S: C, 41.99; H, 4.03;
N, 13.99; S, 16.02. Found: C, 41.79; H, 3.83; N, 13.69; S, 15.72.
4.3.1. Antibacterial
Antibacterial activities were investigated using agar well diffu-
sion method. The activity of tested samples was studied against the
S. aureus (as gram positive bacteria) while P. aeruginosa (as gram
negative bacteria). Centrifuged pelletes of bacteria from a 24 h old
culture containing approximately 104e106 CFU (colony forming
unit) per ml were spread on the surface of Nutrient agar (typetone
1%, yeast extract 0.5%, NaCl 0.5%, agar 1%, 1000 mL of distilled water,
PH 7.0) which was autoclaved under 121 ꢁC for at least 20 min. Wells
were created in medium with the help of a sterile metallic bores and
then cooled down to 45 ꢁC. The activity was determined by
4.1.5. 5-(Hydrazinylmethylene)-2-thioxo-dihydropyrimidine-
4,6(1H,5H)dione (13)
A mixture of 12 (1 g, 0.005 mol, 5 mmol) and hydrazine hydrate
(0.25 g, 0.005 mol) in EtOH (60 mL) was heated under reflux for 3 h.
The solid product was filtered on hot, dried, and crystallized from
proper solvent to give yellow crystals 13: Yield, 78%; m.p. >260 ꢁC;
IR, cmꢀ1: 3372, 3380,3205 (NH2,NH), 3077 (CH aliph.), 1731, 1688
(2C]O), 1575 (C]S). 1H NMR (DMSO-d6)
d: 6.5 (s, 2H, NH2
measuring the diameter of the inhibition zone (in mm).100 ml of the
exchangeable), 7.9 (s, 1H, CH-NHNH2), 8.9 (s, 1H, NH exchangeable),
11.4, 12.3 ppm (2s, 2H, 2NH pyrimidine). MS, m/z (%): 186 (Mþ)
(100). Anal. Calcd. For C5H6N4O2S: C, 32.25; H, 3.25; N, 30.09; S,
17.22. Found: C, 32.05; H, 3.55; N, 30.39; S, 17.02.
tested samples (10 mg/mL) were loaded into the wells of the plates.
All compounds was prepared in Dimethyl Sulfoxide (DMSO), DMSO
was loaded as control. The plates were kept for incubation at 37 ꢁC
for 24 h and then the plates were examined for the formation of zone
of inhibition. Each inhibition zone was measured three times by
caliper to get an average value. The test was performed three times
for each bacterium culture. Tetracyclin was used as antibacterial
standard drugs [22]. Zones of inhibitionwere determined for 3, 6,10,
11, 12, 13, 15, 16 and 17 and the results are summarized in Table 1.
4.1.6. 6,8-Diamino-4-oxo-2-thioxo-5-(3,4,5-trimethoxyphenyl)-
2,3,4,5-tetrahydro-1H-pyrido [30,20:4,5]pyrano[2,3-d]pyrimidine-7-
carbonitrile (15)
A suspension of 6 (1.94 g, 0.005 mol) in C2H5OH (50 mL) con-
taining a catalytic amount of piperidine was treated with malo-
nonitrile (0.35 g, 0.005 mol). The reaction mixture was refluxed
for 3 h. The separated solid was filtered off on hot and crystallized
from dioxane to give off white crystals 15: Yield, 70%; m.p.
188e190 ꢁC; IR, cmꢀ1: 3378, 3299 (br, NH2þNH), 3166 (CH arom.),
2906 (CH aliph.), 2198(CN), 1674 (C]O), 1561 (C]S). 1H NMR
4.3.2. Antifungal
Newly prepared compounds were screened separately in vitro
for their antifungal activity against cultures of two fungal species,
namely, Fungus, A. flavus and one yeast fungus C. albicans on Sab-
ouraud dextrose agar plates. The culture of fungi was purified by
single spore isolation technique. The antifungal activity was
detected by agar well diffusion method [23] by the following
procedure Sabouraud dextrose agar plates: A homogeneous mixture
of glucoseepeptoneeagar (40:10:15) was sterilized by autoclaving
at 121 ꢁC for 20 min. The sterilized solution (25 mL) was poured in
each sterilized petridish in laminar flow and left for 20 min to form
the solidified sabouraud dextrose agar plate. These plates were
inverted and kept at 30 ꢁC in incubator to remove the moisture and
to check for any contamination.
(DMSO-d6) d: 3.7(s, 9H, 3OCH3), 4.2(s,1H, CH-5 pyrane), 5.8, 6.4 (2s,
4H, NH2), 6.9e7.8 (m, 2H, AreH), 11.3, 12.3 ppm (2s, 2H, 2NH
pyrimidine). MS, m/z (%): 456 (M) (7.25), 144 (100). Anal. Calcd. For
C20H18N6O5S: C, 52.86; H, 3.99; N, 18.49; S, 7.06. Found: C, 52.46; H,
3.79; N, 18.09; S, 7.36.
4.1.7. N-(6-Cyano-4-oxo-2-thioxo-5-(3,4,5-trimethoxyphenyl)-
2,3,4,5-tetrahydro-1H-pyrano[2,3-d]pyrimidin-7-yl)
benzenesulfonamide (16)
A mixture of compound 6 (1.94 g, 0.005 mol) and benzene
sulfonylchloride (0.8 g, 0.005 mol) in dry benzene (30 mL) was
refluxed for 2 h, the reaction mixture was cooled and poured on to
Antifungal assay: Fungal strain was grown in 5 mL sabouraud
dextrose broth (glucose: peptone; 40:10) for 3e4 days to achieve
105 CFU/mL cells. The fungal culture (0.1 mL) was spread out

H.M. Aly, M.M. Kamal / European Journal of Medicinal Chemistry 47 (2012) 18e23
23
[4] R.N. Taylor, A. Cleasby, O. Singh, T. Skarzynski, J. Med. Chem. 41 (1998)
798e807.
[5] K. Hirmoto, A. Nasuhara, K. Michiloshi, T. Kato, K. Kikugawa, Mutat. Res. 395
(1997) 47e56.
[6] A.G. Martinez, L.J. Marco, Bioorg. Med. Chem. Lett. 7 (1997) 3165e3170.
[7] C.P. Dell, C.W. Smith, Eur. Pat. 537, 94, 9, 21 Apr 1993; ref. Chem. Abstr. 119
(1993) 139102d.
[8] G. Bianchi, A. Tava, Agric. Biol. Chem. 51 (1987) 2001e2002.
[9] F. Eiden, F. Denk, Arch. Pharm. Weinheim Ger. 324 (1991) 353e354.
[10] C.J. Shishoo, M.B. Devani, G.V. Ullas, S. Ananthan, V.S. Bahadit, J. Heterocyclic
Chem. 18 (1981) 43e46.
[11] K. Noda, A. Nakagawa, Y. Nakajima, H. Ide, Japan. Kokai 7785, 194, 15 Jul
1977;ref. Chem. Abstr. 88 (1978) P50908q.
[12 ] Z.H. Ismail, M.M. Ghorab, E.M.A. Mohamed, H.M. Aly, M.S.A. El-Gaby, Phos-
phorus, Sulfur and Silicon 183 (2008) 2541.
[13] M.S.A. El-Gaby, S.M. Abdel-Gawad, M.M. Ghorab, H.I. Heiba, H.M. Aly, Phos-
phorus, Sulfur and Silicon 181 (2006) 279.
[14] L.P. Prikazchikova, B.M. Khutova, I.F. Vladimirtsev, I.V. Boldyrev,
uniformly on the Sabouraud dextrose agar plates by sterilized
triangular folded glass rod. Plates were left for 5e10 min so that
culture is properly adsorbed on the surface of sabouraud dextrose
agar plates. Now small wells of size (4 mm ꢃ 2 mm) were cut into
the plates with the help of well cutter and bottom of the wells were
sealed with 0.8% soft agar to prevent the flow of test sample at the
bottom of the well. 100 ml of the tested samples (10 mg/mL) were
loaded into the wells of the plates. All compounds was prepared in
dimethyl sulfoxide (DMSO), DMSO was loaded as control. The
plates were kept for incubation at 30 ꢁC for 3e4 days and then the
plates were examined for the formation of zone of inhibition. Each
inhibition zone was measured three times by caliper to get an
average value. The test was performed three times for each fungus.
Amphotericin B was used as references to evaluate the potency of
the tested compounds under the same conditions. Zones of inhi-
bition were determined for 3, 6, 10, 11, 12, 13, 15, 16 and 17 and the
results are summarized in Table 1.
N.I. Zhuravskaya, Fiziol. Akt. Veshchestva
83:127346m.
7 (1975) 84 ref. Chem. Abstr.
[15] D. Brown, in: A.R. Katritzky, C.W. Rees (Eds.), J. Comprehensive Heterocyclic
Chemistry, 3, Pergamon Press, Oxford, 1984, p. 443.
[16] H.M. Aly, Phosphorus, Sulfur and Silicon 185 (2010) 211e221.
[17] H.M. Aly, Monatsch. Chem. 142 (2011) 935e941.
[18] H.M. Aly, N.M. Saleh, H.A. Elhady, Eur. J. Med. Chem. 46 (2011) 4566e4572.
[19] J. Hren, F. Pozgan, A. Bunic, V.I. Parvulescu, S. Polanc, M. Kocevar, Tetrahedron
65 (2009) 8216e8221.
References
ꢀ
ꢀ
ꢀ
[20] H.M.F. Madkour, M.R. Mahmoud, M.H. Nassar, M.M. Habashy, Molecules 5
(2000) 746e755.
[1] A.M. El-Agrody, M.S. Abd El-Latif, N.A. El-Hady, A.H. Fakery, A.H. Bedair,
Molecules 6 (2001) 519e527.
[21] Y. Jing, W. Hanqing, Synth. Commun. 35 (2005) 3133e3140.
[22] A. Rahman, M.I. Choudhary, W.J. Thomsen, Bioassay Techniques for Drug
Development. Harwood Academic Publishers, the Netherlands, 2001, pp. 16e26.
[23] H.S. Rathore, S. Mittal, S. Kumar, Pestic. Res. J. 12 (2000) 103.
[2] A.H. Bedair, Nagwa A. El-Haddy, M.S. Abd El-Latif, A.H. Fakery, A.M. El-Agrody,
lL Farmaco 55 (2000) 708e714.
[3] A.M. El-Agrody, M.H. El-Hakim, M.S. Abd El-Latif, A.H. Fakery, E.M. El-Sayed,
K.A. El-Ghareab, Acta Pharm. 50 (2000) 111e120.