Synthesis and antimicrobial activity of new functionalized derivatives of [1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one

Article abstract of DOI:10.1002/hc.20311

New functionalized 1,7-diaryl-6-cyano-1,2,4-triazolo[4,3-a]pyrimidin-5(1H)- one derivatives (5a-j) were synthesized via reaction of 5-cyano-6-phenyl-2- thiouracil 1 with the respective hydrazonoyl halides 2a-j and their biological activity was evaluated.

Full text of DOI:10.1002/hc.20311

Heteroatom Chemistry
Volume 18, Number 4, 2007
Synthesis and Antimicrobial Activity
of New Functionalized Derivatives
of [1,2,4]Triazolo[4,3-a]pyrimidin-5(1H)-one
Ahmad S. Shawali,1 Asma M. Mahran,2 and Afaf A. Nada2
1Department of Chemistry, Faculty of Science, University of Cairo, Giza, Egypt
2Photochemistry Department, National Research Center, Dokki, Giza, Egypt
Received 17 January 2006; revised 25 June 2006
with carbon-inserting agents such as ortho esters
ABSTRACT: New functionalized 1,7-diaryl-6-cyano-
[6]. The required hydrazine derivatives are usu-
1,2,4-triazolo[4,3-a]pyrimidin-5(1H)-one derivatives
ally prepared by hydrazinolysis of the respective
(5a–j) were synthesized via reaction of 5-cyano-
2-thiouracils [7,8]. This general route is, however,
6-phenyl-2-thiouracil 1 with the respective hydra-
limited for synthesis of only 1-unsubstituted-
zonoyl halides 2a–j and their biological activity
1,2,4-triazolo[4,3-a]pyrimidin-5(1H)-ones. In the
was evaluated. The mechanism and the regios-
present paper, we wish to report one-step synthesis
electivity of the studied reactions are discussed.
of functionalized 1,2,4-triazolo[4,3-a]pyrimidin-
ꢀ
C
2007 Wiley Periodicals, Inc. Heteroatom Chem 18:393–
5(1H)-ones directly from 2-thiouracils 1 (Scheme 1)
398, 2007; Published online in Wiley InterScience
and the biological activity of the compounds
(www.interscience.wiley.com). DOI 10.1002/hc.20311
prepared.
INTRODUCTION
RESULTS AND DISCUSSION
Various
derivatives
of
1,2,4-triazolo[4,3-
The
starting
5-cyano-6-phenyl-3,4-dihydro-2-
a]pyrimidin-5(1H)-one were reported to be useful
as calcium-channel–blocking vasodilators, some
have antihypertensive [1], cardiovascular [2,3]
and anxiolytic activities [4] and other are used as
components in photographic materials [5]. These
pharmacological activities have prompted us to
develop a new synthetic strategy for functionalized
derivatives of such a ring system. This is because
at present the main synthetic strategy reported
in literature for synthesis of 1,2,4-triazolo[4,3-
a]pyrimidin-5(1H)-ones involves cyclization of the
2-hydrazino-3,4-dihydropyrimidin-4-one derivatives
thiouracil 1 [9] and the hydrazonoyl halides 2a–j
[10] were prepared by literature methods. Reaction
of 1 with each of 2a–j in refluxing ethanol in the
presence of sodium ethoxide gave, in each case,
one isolable product as evidenced by TLC analysis
of the crude product. This finding indicates that
the studied reactions are regioselective. On the
basis of elemental analyses and IR and 1H and
13C NMR spectra, which showed all the expected
signals (see Experimental), the isolated products
were assigned the structure of 1,2,4-triazolo[4,3-
a]pyrimidin-5(1H)-one 5 rather than the isomeric
structure of 1,3,4-triazolo[4,3-a]pyrimidin-7(1H)-
one 7 (Scheme 1). Formation of compound 5
could be accounted for by one of the two pathways
indicated in Scheme 1. As depicted in the latter,
Correspondence to: Asma M. Mahran; e-mail: dr asma chem@
yahoo.com.
c
ꢀ
2007 Wiley Periodicals, Inc.
393

394 Shawali, Mahran, and Nada
O
NC
Ph
H
S
N
Route A
Route B
+
R
C(Cl) NNHAr
2
N
H
− HX
− HX
1
O
N
O
R
H
NC
NC
N
R
N
NNHAr
S
NNHAr
Ph
Ph
N
H
S
3
6
O
O
R
NC
Ph
NC
H
N
N
N
NHCSR
− H2S
− H2S
N
N
Ph
N
N
Ar
4
5
Ar
Ar = XC6H4
R / X : a, CH 3CO / H; b, CH3CO / 4-Me;
c, CH3CO / 4-Cl; d, EtOCO / H;
O
NC
e, EtO CO / 4-Me; f, EtO CO / 4-Cl;
g, PhNHCO / H; h, PhNHCO / 4-Me;
i, PhNHCO / 4-Cl; j, Ph / H
N
Ar
Ph
N
N
N
R
7
SCHEME 1
it is suggested that the studied reactions started
with the hydrazonoylation of 1 to give the thiohy-
drazonate esters 3. This is followed by Smiles-type
rearrangement [11] of the latter esters to form the
respective thiohydrazides 4, which in turn under-
went cyclization to give 5 as end products (Route A,
Scheme 1). It seems that both intermediates 3
and 4 are consumed, under the employed reaction
conditions, as soon as they are formed since all
attempts to isolate them failed. However, by varying
the reaction conditions, we succeeded in isolation
of one of these intermediates. For example, when
1 was reacted with 2i in ethanol in the presence
of triethylamine at room temperature, it gave 4i.
The structure of the latter was established on the
basis of its microanalysis and spectra (IR, MS, and
1H NMR) (see Experimental) and its conversion
into 5i upon refluxing it with ethanolic sodium
ethoxide. Alternatively, the formation of 5 can also
be accomplished via cyclization of the amidrazone
intermediates 6 (Route B, Scheme 1). This latter
alternative pathway has been ruled out, however,
because alkylation and acylation of 2-thiouracil
derivatives have been known to give S-alkyl and
S-acyl derivatives, respectively [12–16].
The involvement of 3 and 4 as intermediates in
the formation of 5 was further evidenced by alternate
synthesis of 5g (Scheme 2). Thus, treatment of 1 with
α-chloroacetoacetanilide in ethanol in the presence
of sodium ethoxide afforded the substituted product
8. Coupling of 8 with benzenediazonium chloride in
ethanol in the presence of sodium acetate yielded
the coupling product 9 (Scheme 2). Treatment of
the latter with sodium ethoxide in refluxing ethanol,
in an attempt to effect Japp-Klingemann cleavage
of the acetyl group in 9 [17] to give the respective
thiohydrazonate (3g), was found to give 5g directly
(Scheme 2). This finding indicates that 3 and 4 are
Heteroatom Chemistry DOI 10.1002/hc

Antimicrobial Activity of [1,2,4]Triazolo[4,3-a]pyrimidin-5(1H)-ones 395
O
O
NC
Ph
H
S
NC
Ph
H
S
N
CONHPh
N
CH3COCH(Cl)CONHPh
− HCl
COCH3
H
N
N
H
1
8
O
NC
Ph
H
S
N
CONHPh
COCH3
PhN2Cl
EtONa / EtOH
EtOH /AcONa
N
N
N
Ph
9
O
O
NC
H
S
NC
Ph
H
N
N
CONHPh
NNHPh
Ph
N
N
Ph
N
− H2S
NHCSCONHPh
3g
4g
O
CONHPh
N
NC
N
N
Ph
N
Ph
5g
SCHEME 2
intermediates in the studied reactions and they are
consumed as soon as they are formed.
each compound and using inhibition zone diameter
(IZD) in centimeter as the criterion for antimicro-
bial activity. Terbinafin as an antifungal agent and
chloramphenicol as an antibacterial agent were used
as references to evaluate the potency of the tested
compounds under the same conditions. The results
are depicted in Table 1. The results reveal that com-
pounds 5a and 5e exhibited the highest degree of
inhibition against the tested organisms SA and BS,
respectively, whereas compounds 5h and 5j exhib-
ited maximum inhibition against AF. Their activity
is similar to that of the standard antifungal and an-
tibacterial agents used. All other compounds either
exhibit no activity or are less active against the tested
species (Table 1).
Finally, the suggestion that cyclization of
the thiohydrazide intermediates 4 to give 1,2,4-
triazolo[4,3-a]pyrimidin-5(1H)-ones 5 rather than
the isomeric 1,2,4-triazolo[4,3-a]pyrimidin-7(1H)-
ones 7 (Scheme 1) is consistent with literature re-
ports. For example, it has been reported that cycliza-
tion of 2-substituted uracil derivatives having no
substituent at N-3 proceeds regioselectively to give
the respective 1,2,4-triazolo[4,3-a]pyrimidin-5(1H)-
ones [18–21].
Antimicrobial Activity
The compounds 5a–j were tested for their antimi-
crobial activities against four fungal species, namely
Aspergillus fumigatus (AF), Penicillium italicum (PI),
Syncephalastrum racemosum (SR), and Candida al-
bicans (CA) as well as four bacteria species, namely
Staphylococcus aureus (SA), Pseudomonas aerugi-
nosa (PA), Bacillus subtilis (BS), and Escherichia coli
(EC). The organisms were tested against the activ-
ity of solutions of concentration of 1.0 mg/mL of
EXPERIMENTAL
Melting points were determined on a Gallenkamp
apparatus and are uncorrected. IR spectra were
recorded in potassium bromide using PU 9712
spectrophotometer. H and 13C NMR spectra were
1
recorded in deuterated chloroform using a Varian
Gemini 300 NMR spectrometer. Mass spectra were
Heteroatom Chemistry DOI 10.1002/hc

396 Shawali, Mahran, and Nada
TABLE 1 Antimicrobial Activity of the Products 5a–j∗
Microorganism IZD (cm)†
Aspergillus Penicillium Syncephalastrum Candida Staphylococcus Pseudomonas Bacillus Escherichia
Compound Fumigatus
Italicum
Racemosum
Albicans
Aureus
Aeruginosa
Subtilis
Coli
5a
5b
5c
5d
5e
5f
+
+
+
0
0
0
0
0
0
0
0
0
0
+
0
0
0
0
0
+
0
0
0
0
0
0
+
+
0
++
+
+
0
0
0
+
+
+
0
0
0
0
+
+
0
0
0
0
+
0
0
0
+
+
++
+
+
0
0
0
5g
5h
5i
+
0
0
0
+
0
0
0
0
0
++
+
+
+
0
+
5j
++
+
+
+
CA‡
++
++
++
§
TE
++
++
++
++
++
∗Note: IZD, inhibition zone diameter; ++, inhibition value 0.6–1.0 cm; +, inhibition value 0.1–0.5 cm beyond control; 0, no inhibition detected.
†50 mL of solution in DMF, whose concentration of 1.0 mg/mL was tested.
‡Chloramphenicol as standard antibacterial agent.
§
Terbinafin as standard antifungal agent.
recorded on a 75 Kratos spectrometer. Elemental
analyses were carried out at the Microanalytical Lab-
oratory of National Research Center, Giza, Egypt. 5-
Cyano-6-phenyl-3,4-dihydro-2-thiouracil (1) [9] and
hydrazonoyl halides (2a–j) [10] were prepared as
previously described.
3-Acetyl-6-cyano-7-phenyl-1-(4-methylphenyl)-[1,2,
4]triazolo[4,3-a]-pyrimidin-5(1H)-one (5b). White
crystals, yield 2.65 g (72%), mp 221–222◦C (EtOH).
IR (KBr) ν cm−1: 2221, 1685, 1660. MS m/z (%): 370
(M+ + 1, 68), 369 (M+, 9), 341 (4), 327 (100), 299
1
(32), 272 (21), 127 (3), 104 (11), 77 (10). H NMR
(CDCl3) δH: 2.30 (s, 3H, CH3), 2.36 (s, 3H, COCH3),
7.52–8.10 (m, 9H, ArH). Anal. Calcd. for C21H15N5O2
(369): C, 68.35%; H, 4.09%; N, 18.97%. Found: C,
68.34%; H, 4.10%; N, 18.96%.
Synthesis of Compounds 5a–j
General Procedure: To a stirred ethanolic sodium
ethoxide solution, prepared by dissolving sodium
metal (0.01 g.atom) in ethanol (30 mL), was added
5-cyano-6-phenyl-2-thiouracil (1) (2.30 g, 0.01 mol).
To the resulting mixture was added the appropriate
hydrazonoyl halide (2) (0.01 mol) portionwise. Af-
ter the addition was complete, the reaction mixture
was refluxed until hydrogen sulfide ceased to evolve.
The reaction mixture was then evaporated in a rota-
tory evaporator and the residue was triturated with
methanol. The solid that precipitated was filtered,
washed with water and dried, and finally crystallized
from the proper solvent to give the respective 1,2,4-
triazolo[4,3-a]pyrimidin-5(1H)-one derivative (5).
3-Acetyl-6-cyano-7-phenyl-1-(4-chlorophenyl)-[1,2,
4]triazolo[4,3-a]-pyrimidin-5(1H)-one (5c). White
crystals, yield 2.80 g (72%), mp 238–240◦C (EtOH).
IR (KBr) ν cm−1: 2219, 1685, 1660. MS m/z (%): 392
(M+ + 2, 2), 390 (M+, 2), 363 (2), 347 (41), 321 (10),
293 (6), 127 (58), 104 (15), 77 (42). 1H NMR (CDCl3)
δH: 2.36 (s, 3H, COCH3), 7.20–8.10 (m, 9H, ArH).
Anal. Calcd. for C20H12ClN5O2 (390): C, 61.15%; H,
3.09%; N, 17.95%. Found: C, 61.00%; H, 3.11%; N,
17.80%.
3-Acetyl-6-cyano-1,7-diphenyl[1,2,4]triazolo[4,3-
a]pyrimidin-5(1H)-one (5a). White crystals, yield
2.48 g (70%), mp 148–149◦C (EtOH). IR (KBr)
ν cm−1: 2225, 1685, 1660. MS m/z (%): 356 (M+ + 1,
72), 355 (M+, 2), 327 (4), 313 (100), 285 (62), 258
Ethyl-6-cyano-1,7-diphenyl-[1,2,4]triazolo[4,3-a]-
pyrimidin-5(1H)-one-3-carboxylate
(5d). White
crystals, yield 2.77 g (72%), mp 209–210◦C (EtOH).
IR (KBr) ν cm−1: 2225, 1685. MS m/z (%): 385 (M+,
100), 357 (5), 312 (14), 259 (6), 258 (6), 127 (10), 103
1
1
(28), 127 (3), 103 (10), 77 (13). H NMR (CDCl3) δH:
(10), 77 (9). H NMR (CDCl3) δH: 1.5 (t, 3H, CH3),
2.36 (s, 3H, COCH3), 7.20–8.10 (m, 10H, ArH). Anal.
Calcd. for C20H13N5O2 (355): C, 67.66%; H, 3.68%;
N, 19.72%. Found: C, 67.65%; H, 3.67%; N, 19.71%.
4.62 (q, 2H, CH2) 7.20–8.10 (m, 10H, ArH). Anal.
Calcd. for C21H15N5O3 (385): C, 65.51%; H, 3.92%;
N, 18.18%. Found C, 65.32%; H, 3.90%; N, 18.20%.
Heteroatom Chemistry DOI 10.1002/hc

Antimicrobial Activity of [1,2,4]Triazolo[4,3-a]pyrimidin-5(1H)-ones 397
Ethyl-6-cyano-7-phenyl-1-(4-methylphenyl)-[1,2,4]-
triazolo[4,3-a]pyrimidin-5(1H)-one-3-carboxylate (5e).
White crystals, yield 2.87 g (72%), mp 155–156◦C
(EtOH). IR (KBr) ν cm−1: 2225, 1685. MS m/z (%):
399 (M+, 3), 371 (9), 326 (100), 299 (33), 272 (18),
1664. MS m/z (%): 468 (M+ + 2, 17), 466 (M+, 70),
446 (2), 361 (100), 347 (25), 319 (13), 127 (9), 114
(4), 77 (16). 1H NMR (CDCl3) δH: 7.20–8.10 (m, 14H,
ArH), 8.52 (s, 1H, NH). 13C NMR (CDCl3) δ: 86.99,
116.61, 120.71, 124.31, 125.80, 129.45, 129.52,
129.86, 130.51, 132.61, 133.93, 134.80, 136.32,
138.13, 139.55, 147.84, 153.28, 155.77, 170.63. Anal.
Calcd. for C25H15ClN5O2 (466): C, 64.43%; H, 3.24%;
N, 18.03%. Found: C, 64.50%; H, 3.24%; N, 18.00%.
1
127 (2), 104 (9), 77 (4). H NMR (CDCl3) δH: 1.51
(t, 3H, CH3), 2.36 (s, 3H, CH3), 4.62 (q, 2H, CH2),
7.20–8.10 (m, 9H, ArH). Anal. Calcd. for C22H17N5O3
(399): C, 66.22%; H, 4.29%; N, 17.55%. Found: C,
66.01%; H, 4.10%; N, 17.60%.
1,3,7-Triphenyl-6-cyano-[1,2,4]triazolo[4,3-a]-
pyrimidin-5(1H)-one (5j). White crystals, yield 2.8
g (72%), mp 302–303◦C (EtOH). IR (KBr) ν cm−1:
2225, 1685. MS m/z (%): 399 (M+ + 1, 5), 389 (M+,
Ethyl-6-cyano-7-phenyl-1-(4-chlorophenyl)-[1,2,4]-
triazolo[4,3-a]pyrimidin-5(1H)-one-3-carboxylate (5f).
Brown crystals, yield 2.89 g (72%), mp 239–240◦C
(EtOH). IR (KBr) ν cm−1: 2225, 1700, 1685. MS
m/z (%): 421 (M+ + 2, 31), 419 (M+, 100), 391 (2),
346 (14), 319 (8), 292 (2), 127 (9), 103 (1), 77
(5). 1H NMR (CDCl3) δH: 1.5 (t, 3H, CH3), 4.62
(q, 2H, CH2), 7.20–8.10 (m, 9H, ArH). 13C NMR
(CDCl3) δ: 13.89, 64.70, 114.91, 122.41, 128.75,
129.15, 129.82, 132.24, 134.11, 134.78, 135.37,
136.45, 146.47, 154.00, 155.43, 170.69. Anal. Calcd.
for C21H14ClN5O3 (419): C, 60.19%; H, 3.36%; N,
16.71%. Found: C, 60.20%; H, 3.40%; N, 16.80%.
1
100), 360 (24), 258 (10), 127 (2), 103 (5), 77 (9). H
NMR (DMSO-d6) δH: 7.20–8.16 (m, 15H, ArH). 13C
NMR (DMSO-d6) δ: 67.90, 87.23, 117.86, 123.52,
126.84, 129.27, 129.95, 103.12, 131.01, 132.06,
132.47, 132.98, 137.27, 137.57, 146.62, 149.75,
157.57, 170.58; Anal. Calcd. for C24H15N5O2 (389): C,
74.10%; H, 3.88%; N, 18.00%. Found: C, 74.10%; H,
3.90%; N, 18.01%.
Synthesis of the Thiohydrazide (4i)
N-Phenyl-6-cyano-1,7-diphenyl-[1,2,4]triazolo[4,3-
a]pyrimidin-5(1H)-one-3-carboxamide (5g). Yellow
crystals, yield 3.09 g (72%), mp 218–220◦C (EtOH).
IR (KBr) ν cm−1: 3080, 2225, 1685. MS m/z (%): 433
(M+ + 1, 33), 432 (M+, 65), 404 (2), 327 (100), 312
To a mixture of the thione 1 and 2i (0.01 mol each)
in ethanol (50 mL) was added triethylamine (1.4 mL,
0.01 mol) and the reaction mixture was stirred for 3 h
at room temperature. The solid that was precipitated
was filtered and crystallized from ethanol to give 4i:
yellow crystals, yield 3.21 g (76%), mp 195–196◦C
(EtOH). IR (KBr) ν cm−1: 3160, 2219, 1698, 1664,
1465. MS m/z (%): 425 (M+ + 2, 12), 423 (M+, 50).
1H NMR (CDCl3) δH: 1.53 (s, 3H, CH3), 7.00–7.60 (m,
9H, ArH), 8.50 (s, 1H, NH), 11.83 (s, 1H, NH). Anal.
Calcd. for C20H14ClN5O2S (423): C, 56.73%; H, 3.30%;
N, 16.54%; Found C, 56.68%; H, 3.23%; N, 16.60%.
1
(25), 286 (4), 358 (3), 127 (3), 104 (21), 77 (29). H
NMR (CDCl3) δH: 7.20–8.10 (m, 15H, ArH), 8.51 (s,
1H, NH). 13C NMR (CDCl3) δ: 13.89, 29.67, 64.71,
87.95, 114.9, 122.41, 128.75, 129.15, 129.82, 132.24,
134.11, 134.76, 135.37, 136.45, 146.47, 154.00,
155.43, 170.69. Anal. Calcd. for C25H16N6O2 (432): C,
69.50%; H, 3.72%; N, 19.45%. Found: C, 69.40%; H,
3.80%; N, 19.41%.
2-(5-Cyano-6-phenyl-3H-4-oxo-2-
pyrimidinylthio)acetoacetanilide (8)
N-Phenyl-6-cyano-7-phenyl-1-(4-methylphenyl)-[1,
2,4]triazolo[4,3-a]pyrimidin-5(1H)-one-3-carboxa-
mide (5h). Yellow crystals, yield 3.2 g (72%), mp
211–212◦C (EtOH). IR (KBr) ν cm−1: 3072, 2225,
1685, 1660. MS m/z (%): 447 (M+ + 1, 18), 446 (M+,
80), 419 (2), 341 (100), 327 (40), 300 (15), 272 (10),
To
a
mixture of equimolar amounts of 2-
chloroacetoacetanilide and 1 (0.01 mol each) in ab-
solute ethanol (25 mL) was added triethylamine
(1.4 mL, 0.01 mol). The resulting mixture was stirred
at room temperature for 10 h. The solid that precip-
itated was filtered and crystallized from ethanol to
give 8: white crystals, yield 3.0 g (75%), mp 197–
198◦C (EtOH). IR (KBr) ν cm−1: 3430, 3124, 2223,
1700, 1685, 1650. MS m/z (%): 406 (M+ + 1, 1), 405
1
127 (6), 103 (18), 77 (18). H NMR (CDCl3) δH: 2.30
(s, 3H, CH3), 7.20–8.10 (m, 14H, ArH) 8.52 (s, 1H,
NH). Anal. Calcd. for C26H18N6O2 (446): C, 69.95%;
H, 4.03%; N, 18.83%; Found: C, 69.89%; H, 4.00%;
N, 18.79%.
1
(M+, 2). H NMR (CDCl3) δH: 1.53 (s, 3H, CH3), 5.9
N-Phenyl-6-cyano-7-phenyl-1-(4-chlorophenyl)-[1,
2,4]triazolo[4,3-a]-pyrimidin-5(1H)-one-3-carboxa-
mide (5i). Yellow crystals, yield 3.2 g (70%), mp
250–251◦C (EtOH). IR (KBr) ν cm−1: 2225, 1685,
(s, 1H, CH), 7.00–7.60 (m, 10H, ArH), 8.50 (s, 1H,
NH), 11.83 (s, 1H, NH). Anal. Calcd. for C21H16N4O3S
(404): C, 62.22%; H, 3.95%; N, 13.82%; Found C,
62.13%; H, 4.00%; N, 13.19%.
Heteroatom Chemistry DOI 10.1002/hc

398 Shawali, Mahran, and Nada
dry; then the discs were placed on the centre of the
malt agar plate and incubated at optimum incuba-
tion temperature 28
2-Phenylazo-2-(5-cyano-6-phenyl-3H-4-oxo-2-
pyrimidinylthio)acetoacetanilide (9)
2◦C. Test organism growth
To a stirring solution of 8 (10 m moles) in ethanol
(50 mL) was added sodium acetate trihydrate (3 g)
and the mixture was cooled in an ice bath to 0–
5◦C. To the resulting cold solution was added por-
tionwise a cold solution of benzenediazonium chlo-
ride, prepared by diazotizing aniline (10 mmol) in
hydrochloric acid (6 mL, 6M) with sodium nitrite
(0.7 g, 10 mmol) in a low amount of water. After all
of the diazonium salt solution was added, the reac-
tion mixture was stirred for 1 h in ice bath and left
in the refrigerator. The solid that precipitated was
filtered off, washed with water, air dried, and finally
crystallized from ethanol to give 9: brown powder,
yield 3.45 g (68%), mp 161–162◦C. (EtOH). IR (KBr)
ν cm−1: 3421, 3100, 2217, 1700, 1687, 1650, 1567,
may be affected by the inhibitory action of the test
compound, so a clear zone around the disc appears
as an indication of the inhibition of test organism
growth. The size of the clearing zone is proportional
to the inhibitory action of the compound. The fungi-
cide Terbinafin and the bactericide chloramphenicol
were used as standards under the same conditions.
Measurements were considered after 72 h for fungi
and 24 h for bacteria. The results are summarized in
Table 1.
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1508. MS m/z (%): 509 (M+ + 1, 2), 508 (M+, 5). H
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NMR (DMSO-d6) δH : 1.50 (s, 3H, CH3), 7.3–8.1 (m,
15H, ArH), 8.5 (s, 1H, NH), 10.9 (s, 1H, NH); Anal.
Calcd. for C27H20N6O3S (508): C, 63.77%; H, 3.93%;
N, 16.53%; Found C, 63.69%; H, 3.92%; N, 16.50%.
Cyclization of 2-phenylazo-2-(5-cyano-6-phenyl-
3H-4-oxo-2-pyrimidinylthio)acetoacetanilide (9)
To a stirred sodium ethoxide solution, prepared from
sodium metal (0.69 g, 0.003 g.atom) and absolute
ethanol (20 ml), compound 9 (1.524 g, 0.003 mol)
was added and the mixture was refluxed, while be-
ing stirred, till all hydrogen sulfide ceased to evolve
(3 h), then cooled. The solid that precipitated was
filtered off, washed with water, air dried, and finally
crystallized from ethanol to give a yellow crystaline
product that proved identical in all respects with 5g
(mp 220◦C, yield 65%) obtained above from 1 and 2g.
Antimicrobial Assay
Cultures of four fungal species, namely, Aspergillus
fumigatus, Penicillium italicum, Syncephalastrum
racemosum, and Candida albicans, as well as four
bacterial species, namely, Staphylococcus aureus,
Pseudomonas aeruginosa, Bacillus subtilis, and
Escherichia coli were used to investigate the antimi-
crobial activity of compounds 5a–j. The antimicro-
bial activity was assayed biologically using the diffu-
sion plate technique. The latter technique involved
pouring a spore suspension of the fungal species
(1 mL of sterile water contains approximately 108
conidia) or spreading bacterial suspension over a
solidified malt agar medium. The layer is allowed
to set for 30 min. A solution of the test compound
5 (1.0 mg/mL) in dimethylformamide was placed
onto sterile 5-mm filter paper discs and allowed to
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Heteroatom Chemistry DOI 10.1002/hc