A convenient one-pot synthesis and antimicrobial activity of pyrimido[1,2-b][ 1,2,4,5]tetrazines

Article abstract of DOI:10.1002/jhet.5570390105

The synthesis of the hitherto unreported 3-amino-2,3-dihydro-6-phenyl-2-thioxo-4(1H)-pyrimidine 2 and 3-amino-2-methylthio-6-phenyl-4(3H)-pyrimidinone 3 is described. Reactions of hydrazonoyl halides 1 with either 2 or 3 afforded 6H-pyrimido[1,2-b][1,2,4,

Full text of DOI:10.1002/jhet.5570390105

Jan-Feb 2002
A Convenient One-Pot Synthesis and Antimicrobial
Activity of Pyrimido[1,2-b][1,2,4,5]tetrazines
Ahmad S. Shawali*, Magda A. Abdallah and Mohie M. Zayed
45
Department of Chemistry, Faculty of Science, University of Cairo, Giza, Egypt
Received March 12, 2001
The synthesis of the hitherto unreported 3-amino-2,3-dihydro-6-phenyl-2-thioxo-4(1H)-pyrimidine 2 and
3-amino-2-methylthio-6-phenyl-4(3H)-pyrimidinone 3 is described. Reactions of hydrazonoyl halides 1
with either 2 or 3 afforded 6H-pyrimido[1,2-b][1,2,4,5]tetrazin-6-ones 6. The latter products were screened
for their antifungal and antibacterial properties. The mechanism of the studied reactions is discussed.
J. Heterocyclic Chem., 39, 45 (2002).
Introduction.
NMR) and microanalyses. For example, their IR spectra
reveal the NH and CO bands in the regions 3300-3100
and 1680-1650 cm . The H NMR spectrum of 2 shows,
2
Recently, we have shown that reactions of hydrazonoyl
halides 1 with heterocyclic thiones, having a thiourea
residue in their structure, provide direct, efficient and
regioselective routes for synthesis of various azoloazole
derivatives [1-4]. In continuation of such studies in this area,
we felt it would be interesting to extend our investigations to
reactions of 1 with heterocyclic systems having a thiosemi-
carbazide residue in their structures such as 3-amino-2,3-
dihydro-6-phenyl-2-thioxo-4(1H)-pyrimidinone 2 in an
attempt to develop a new simple strategy for synthesis of
functionalized pyrimido[1,2-b][1,2,4,5]tetrazines. At
present, the synthesis of the latter ring system from
3-amino-6-substituted-2-thiouracils comprises three steps
viz. (i) conversion of the latter into the respective
2-methylthio derivatives, (ii) hydrazinolysis of such
2-methylthiouracils to get 3-amino-2-hydrazino-4(3H)-
pyrimidinones and (iii) condensation of the latter with one
carbon cyclizing reagents such as ortho esters, dimethyl-
formamide dimethylacetal, or dimethoxymethyl acetate in
glacial acetic acid to get the respective pyrimido[1,2-b]-
[1,2,4,5]tetrazines [5]. The interest in the latter derivatives is
due to the recent finding that derivatives of such ring system
were reported to be strong inhibitors of human
cytomegalovirus protease [6]. In the present paper, we wish
to report the synthesis of both 2 and 3, which have been
unreported hitherto, and the results of the study of their
reactions with 1. In addition, the results of screening of the
products, namely pyrimido[1,2-b][1,2,4,5]tetrazines 6a-i,
isolated from the studied reactions, for their potential
antifungal and antibacterial properties are reported.
-1
1
in addition to the aromatic proton multiplet, three
characteristic signals at δ 4.72, 6.14 and 11.00 assignable
1
to NH , ring C5-H and NH protons, respectively. The H
2
NMR spectrum of 3 shows the presence of the S-CH ,
3
NH and C5-H protons as three singlets at δ 3.13, 5.72 and
2
6.71, respectively.
Refluxing of 1 with 2 in ethanol in the presence of
triethylamine until hydrogen sulfide ceased to evolve and
working up the reaction mixture gave, in each case, one
product as evidenced by tlc analysis. At first, it was
anticipated that such reactions would yield the respective
pyrimido[4,3-b][1,3,4]thiadiazine derivatives by analogy to
the reactions of 1 with 2-aminothiophenols which were
reported to give benzothiadiazine derivatives [7].
Surprisingly, however, the products isolated from the
reactions of 1a-i with 2 were found to be free of sulfur. All
such products display in their IR spectra absorption bands in
Results and Discussion.
The required starting materials namely 3-amino-6-
phenyl-2-thiouracil 2 and its 2-methylthio derivative
3 have not been reported hitherto. They were prepared in
this work as depicted in Scheme 1. Thus, reaction of
thiosemicarbazide with ethyl benzoylacetate in ethanol in
the presence of sodium ethoxide yielded the 2-thiouracil
derivative 2. Methylation of the latter with methyl iodide
in ethanol in the presence of sodium ethoxide afforded the
2-methylthio derivative 3 in 74% yield. The structures of
-1
the regions 3260-3200 and 1700-1670 cm due to the NH
1
and C=O groups, respectively. Their H NMR spectra, while
they show a common characteristic singlet signal in the
region δ 8.9-9.4 assignable to the NH proton, they reveal the
absence of N-NH proton signal present in the spectrum of 2
2
at δ 4.72. Their mass spectra show, in addition to the
expected molecular ions, peaks at m/z corresponding to
+
+
+
1
[M - R] , [M - RC(NH):NNHAr], [M -RC(:N)NH),
both 2 and 3 were evidenced by their spectra (mass, IR, H

46
A. S. Shawali, M. A. Abdallah and M. M. Zayed
Vol. 39
[ArN] and [Ar] fragments (see Experimental). On the basis
of these spectral characteristics together with their microana-
lytical data, the products isolated from the reactions of 2 with
1a-i were assigned the pyrimido[1,2-b][1,2,4,5]tetrazine
structures 6a-i, respectively (Scheme 2). Such structure
were reported to give the corresponding hydrazidines [8].
Alternatively, reaction of 1 with 2 may start with the
formation of the thiohydrazonate esters 5 which undergo in
situ Smiles rearrangement [9,10] under the reaction condi-
tions employed to afford the corresponding thiohydrazides
7. Then the thiohydrazides 7 undergo cyclization as soon
as they are formed with concurrent elimination of
hydrogen sulfide to give 6 as the end products (Route B,
Scheme 2). All attempts to isolate any of the aforemen-
tioned intermediates 4, 5 and 7 were unsuccessful,
however. Presumably, such intermediates are converted
under the employed reaction conditions to the final
products 6 as soon as they are formed.
13
assignment was substantiated by C nmr spectra. For exam-
13
ple, the C nmr spectrum of 6c in CDCl reveals, as
3
expected, fifteen signals at δ 24.2, 103.5, 123.9, 126.8, 126.9,
128.6, 128.7, 130.9, 135.5, 139.7, 143.2, 143.6, 157.1, 160.1,
189.2.
To account for the formation of 6, the two possible path-
ways A and B depicted in Scheme 2 were considered.
Thus, it is suggested that reactions of 1 with 2 or 3
presumably proceed through initial formation of the
respective hydrazidine derivatives 4 which subsequently
undergo cyclization with concurrent elimination of
hydrogen sulfide to give 6 (Route-A). The formation of 4
is analogous to the reactions of 1 with hydrazines which
To distinguish between these two alternative pathways,
the reactions of 1a-i each with 3-amino-2-methylthio-6-
phenyl-4(3H)-one derivative 3 were investigated. Thus,
refluxing a mixture of 1 and 3 in pyridine afforded, in each
1
case, one product whose H NMR spectrum showed the
R/Z/X: a, EtOCO/H/Cl; b, PhNHCO/H/Cl; c, CH CO/H/Cl; d, PhCO/H/Br; e, 2-Naphthoyl/H/Br; f, 2-Thenoyl/4-Me/Br; g, Ph/H/Cl;
3
h, PhCH=CH/H/Cl; i, CH /4-NO /Br.
3
2

Jan-Feb 2002
A Convenient One-Pot Synthesis and Antimicrobial Activity
conditions. The results are depicted in Table 1.
47
absence of both the methylthio and amino proton signals
present at δ 3.13 and 5.72, respectively in the spectrum of
the respective 3-amino-2-methylthiopyrimidinone 3.
Instead, the spectra of the products isolated revealed in each
case, a characteristic NH proton singlet signal in the region
δ 9.0 - 9.4. Furthermore, such products proved identical in
all respects (m.p., mixed m.p., IR) with those obtained
above from 1 and 2. As compound 3 cannot form thio-
hydrazonates with 1, it is not unreasonable to conclude that
route-A in Scheme 2 seems to be the most plausible
mechanism for the studied reactions of 1 with 3 leading to 6.
In conclusion, the foregoing results indicate collectively
that the studied reactions of 1 with either 3-amino-6-
phenyl-2-thioxopyrimidin-4(1H)-one 2 or 3-amino-2-
methylthio-6-phenylpyrimidin-4(3H)-one 3 provide easy
access to pyrimido[1,2-b][1,2,4,5]tetrazine derivatives 6.
The latter compounds represent important extensions in
the chemistry of ring-fused 4H-1,2,4,5-tetrazines. The
availability of the 4-nitrogen atom for further substitution
offers the potential for novel biologically active materials
or dyestuffs.
The results revealed that compound 6b exhibited the
highest degree of inhibition against all tested
organisms. Furthermore, while the data showed that all
other compounds 6c-f,h,i are active against the two
species PA and PV, they exhibit no inhibition of the two
bacteria species EC and SS and the fungi species SR.
Compounds 6a and 6g showed activity only against the
two micro-organisms PA and PV. However, the
activities of the tested compounds are much less than
that of the standard antifungal and antibacterial agents
used.
EXPERIMENTAL
All melting points were determined in capillary tubes on a
1
13
Gallenkamp apparatus and are uncorrected. H and C NMR
spectra were recorded using a Varian Gemini 200 spectrometer in
deuterated chloroform with tetramethylsilane (TMS) as an
internal standard. IR spectra were determined with a Fourier
Transform and Pye Unicam Infrared spectrophotometer using a
potassium bromide wafer. Mass spectra were recorded on a
GCMS-QP 1000 EX spectrometer at an ionizing potential of
70 eV. Elemental analyses were carried out at the Microanalytical
Laboratory of Cairo University, Giza, Egypt.
Antimicrobial Activity.
The compounds 6a-i were tested for their antimicrobial
activities using four fungi species namely Rhizopus spp.
RS, Aspergillus fumigatus AF, Syncephalastrum racemo-
sum SR and Candida spp. CS. Also, five bacteria species
namely Pseudomonas aeruginosa PA, Proteus vulgaris
PV, Bacillussubtilis BS, Escherichia coli EC and
Salmonella spp. SS were tested. The organisms were tested
against the activity of solutions of concentration of
1.0 µg/ml of each compound and using inhibition zone
diameter in cm (IZD) as criterion for the antimicrobial
activity. The fungicide Terbinafin and the bactericide
Chloramphenicol were used as references to evaluate the
potency of the tested compounds under the same
The biological evaluations of the products 6a-i were carried
out at The Regional Center for Mycology and Biotechnology of
Al-Azhar University, Cairo, Egypt. The hydrazonoyl halides 1a-i
were prepared by following the procedures reported earlier
[11-17].
3-Amino-2,3-dihydro-6-phenyl-2-thioxo-4(1H)pyrimidinone (2).
To an ethanolic sodium ethoxide solution, prepared from
sodium metal (4.6 g, 0.2 mol) and absolute ethanol (100 ml),
were added thiosemicarbazide (9.1 g, 0.1 mole) and ethyl
benzoylacetate (19.2 g, 0.1 mole) with stirring. The reaction
mixture was refluxed for 4 hours and then cooled. The solid that
Table 1
Antimicrobial Activity of the Products 6a-I [a]
Compound No.
Micro-organism/IZD (cm)
RS
0
1.9
0
1.3
1.3
0
0
0
0
4.7
AF
0
SR
0
1.8
0
0
0
0
0
0
0
CS
0
PA
1.2
1.8
1.2
1.2
1.1
1.3
1.2
1.1
1.3
3.6
PV
1.3
2.0
1.2
1.2
1.4
1.3
1.2
1.2
1.3
BS
1.4
1.8
0
1.5
1.4
1.3
0
EC
0
1.5
0
0
0
0
0
0
0
SS
0
1.4
0
0
0
0
0
0
0
6a
6b
6c
6d
6e
6f
6g
6h
2.0
1.1
1.7
1.5
0
0
0
1.4
5.0
1.9
1.4
1.2
1.2
1.3
0
1.3
1.3
3.0
1.4
1.2
6i
TE [b]
CA [c]
3.6
2.6
2.6
2.8
2.4
[a] 50 ml of solution whose concentration 1.0 µg/ml was tested; [b] Terbinafin; [c] Chloramphenicol.

48
A. S. Shawali, M. A. Abdallah and M. M. Zayed
Vol. 39
precipitated was isolated by filtration and was dissolved in water
(100 ml). The resulting solution was acidified with acetic acid,
and crystallized from ethanol to give pure 2: Yield 15 %, m.p.
Found: C, 63.7; H, 4.7; N, 18.6 %.
N-Phenyl-(1,8-diphenyl-6H-pyrimido[1,2-b][1,2,4,5]tetrazin-6-
one)-3-carboxamide (6b).
-1
1
238-40 °C, IR: ν (cm ) 3286, 3132 (NH ), 1658 (CO); H NMR:
2
δ 4.72 (s, 2H), 6.14 (s, 1H), 7.49 - 7.67 (m, 5H), 11.0 (s, 1H); MS
m/z (%) 220 (14), 219 (100), 191 (9), 188 (19), 146 (44), 129
(10), 117 (8), 103 (27), 77 (31).
Compound 6b was obtained in 42% yield; m.p. 200-202 °C
(EtOH); IR: ν (cm ) 3217, 1700, 1660 (cm ); H NMR: δ 6.64
-1
-1
1
(s, 1H), 7.34-7.70 (m, 15H), 8.6 (s, 1H), 9.4 (s, 1H); MS m/z (%)
+
Anal. Calcd. for C H N OS: C, 54.78; H, 4.14; N, 19.16.
422 (M , 88), 357 (17), 302 (38), 262 (16), 247 (12), 171 (9), 145
10
9 3
Found: C, 54.9; H, 4.2; N, 19.0 %.
(9), 105 (23), 91 (12), 77 (100).
Anal. Calcd. for C H N O : C, 68.24; H, 4.29; N, 19.89.
Found: C, 67.4; H, 4.1; N, 19.9 %.
24 18
6 2
3-Amino-6-phenyl-2-methylthio-4(3H)-pyrimidinone (3).
To an ethanolic sodium ethoxide solution, prepared from
sodium metal (0.23 g, 0.01 mol) and absolute ethanol (100 ml),
was added compound 2 (2.19 g, 0.01 mole) with stirring. To the
resulting solution was added methyl iodide (1.42 g, 0.01 mol),
and the mixture was refluxed on a water bath for 1 hour and then
left at room temperature overnight. The precipitated solid was
collected by filtration and crystallized from ethanol to give pure
3-Acetyl-1,8-diphenyl-6H-pyrimido[1,2-b][1,2,4,5]tetrazin-6-
one (6c).
Compound 6c was obtained in 66% yield; m.p. 214-216 °C
-1
1
(EtOH); IR: ν (cm ) 3217, 1700, 1666; H NMR: δ 2.54 (s, 3H),
6.64 (s, 1H), 7.34-7.71 (m, 10H), 9.09 (s, 1H); MS m/z (%) 345
+
(M , 89), 302 (100), 262 (10), 171 (11), 145 (8), 129 (13), 103
-1
3: Yield 74 %, m.p. 176°C, ir: ν (cm ) 3302, 3201 (NH ), 1680
(28), 91 (9), 77 (69).
2
1
(CO); H nmr: δ 3.13 (s, 3H), 5.72 (s, 2H), 6.71 (s, 1H),
Anal. Calcd. for C H N O : C, 66.08; H, 4.38; N, 20.28.
19 15
5 2
7.44 - 8.03 (m, 5H); MS m/z (%) 234 (74), 233 (79), 217 (100),
204 (69), 192 (37), 160 (17), 129 (39), 119 (43), 116 (35), 102
(68), 89 (41), 77 (90).
Found: C, 66.1; H, 4.6; N, 20.0 %.
3-Benzoyl-1,8-diphenyl-6H-pyrimido[1,2-b][1,2,4,5]tetrazin-6-
one (6d).
Anal. Calcd. for C H N OS: C, 56.63; H, 4.75; N, 18.01.
11 11
3
Found: C, 56.8; H, 4.7; N, 18.0 %.
Compound 6d was obtained in 35% yield; m.p. 224-226 °C
(EtOH); IR: ν (cm ) 3247, 1674, 1650; H NMR: δ 6.70
-1
1
1,3-Disubstituted-8-phenyl-6H-pyrimido[1,2-b][1,2,4,5]tetrazin-
6-ones (6).
(s, 1H), 7.25-8.26 (m, 15H), 9.42 (s, 1H); MS m/z (%) 407
+
(M , 34), 302 (3), 262 (1), 171 (2), 145 (4), 105 (100), 91 (3),
To a mixture of equimolecular quantities of the appropriate
hydrazonoyl halide 1 and 2-thiouracil derivative 2 (0.005 mole
each) in absolute ethanol (40 ml) was added triethylamine
(0.7 ml, 0.005 mole). The resulting mixture was refluxed until
hydrogen sulfide ceased to evolve (4 - 6 hours) and then cooled.
The solid that precipitated upon cooling was isolated by filtra-
tion, washed with water, dried and finally crystallized from
ethanol to give the respective pure pyrimidotetrazine derivative 6
in 45-67% yield.
77 (58).
Anal. Calcd. for C H N O : C, 70.75; H, 4.21; N, 17.19.
Found: C, 70.0; H, 4.2; N, 17.3 %.
24 17
5 2
3-(2-Naphthoyl)-1,8-diphenyl-6H-pyrimido[1,2-b][1,2,4,5]-
tetrazin-6-one (6e).
Compound 6e was obtained in 50% yield; m.p. 232-234°C
(EtOH); IR: ν (cm ) 3294, 1681, 1650; H NMR: δ 6.70 (s, 1H),
7.34-8.94 (m, 17H), 9.48 (s, 1H); MS m/z (%) 458 (M , 42), 302
-1
1
+
Alternatively, the latter products 6 were also prepared as
follows. A mixture of equimolar quantities of the 2-methylthio
derivative 3 and the appropriate hydrazonoyl halide 1 (5 mmol
each) were refluxed in pyridine for 10 hours and cooled. The
cold reaction mixture was then poured onto ice-cold
hydrochloric acid with stirring. The solid that precipitated was
collected, washed with water and finally crystallized from
ethanol to give the corresponding pyrimidotetrazines 6 which
were found identical in all respects with that obtained above
from 1 and 2. The various pyrimido[1,2-b][1,2,4,5]tetrazines
6a-i that were prepared, together with their physical constants,
are listed subsequently.
(3), 262 (3), 171 (2), 155 (100), 127(69), 103 (4), 91 (3), 77 (34).
Anal. Calcd. for C H N O : C, 73.51; H, 4.19; N, 15.31.
28 19
5 2
Found: C, 73.1; H, 4.2; N, 15.1 %.
3-(2-Thenoyl)-1-(4-methylphenyl)-8-phenyl-6H-pyrimido-
[1,2-b][1,2,4,5]tetrazin-6-one (6f).
Compound 6f was obtained in 52% yield; m.p. 228-230 °C
-1
1
(EtOH); IR: ν (cm ) 3232, 1689, 1670; H NMR: δ 2.45 (s, 3H),
6.66 (s, 1H), 7.25-8.3 (m, 12H), 9.49 (s, 1H); MS m/z (%) 427
+
(M , 30), 316 (7), 261 (26), 171 (3), 129 (3), 111 (100), 105 (2),
91 (24), 77 (6).
Anal. Calcd. for C H N O S: C, 64.62; H, 4.01; N, 16.38.
23 17
5 2
Found: C, 64.9; H, 4.1; N, 16.4 %.
Ethyl(1,8-diphenyl-6H-pyrimido[1,2-b][1,2,4,5]tetrazin-6-one)-
3-carboxylate (6a).
1,3,8-Triphenyl-6H-pyrimido[1,2-b][1,2,4,5]tetrazin-6-one (6g).
Compound 6g was obtained in 50% yield; m.p. 200-202 °C
Compound 6a was obtained in 45 % Yield; m.p. 138-140 °C
-1
-1 1
-1
1
(EtOH); IR: ν (cm ) 3286, 1720, 1681 cm ; H NMR: δ 1.41
(EtOH); IR: ν (cm ) 3201, 1681; H NMR: δ 6.66 (s, 1H),
+
(t, 3H), 4.44 (q, 2H), 6.65 (s, 1H), 7.27-7.70 (m, 10H), 9.03
7.25-7.87 (m, 15H), 9.00 (s, 1H); MS m/z (%) 379 (M , 86),
262 (3), 247 (8), 220 (3), 171 (6), 117 (3), 105 (27), 91 (5), 77
(100).
+
(s, 1H); MS m/z (%) 376 (M , 79), 348 (10), 303 (7), 262 (6), 248
(22), 247 (19), 171 (9), 145 (14), 129 (9), 116 (8), 103 (15), 91
(6), 77 (100).
Anal. Calcd. for C H N O: C, 72.81; H, 4.52; N, 18.48.
23 17
5
Anal. Calcd. for C H N O : C, 63.99; H, 4.56; N, 18.66.
Found: C, 73.0; H, 4.4; N, 18.1 %.
20 17
5 3

Jan-Feb 2002
A Convenient One-Pot Synthesis and Antimicrobial Activity
49
1,8-Diphenyl-3-(2-phenylethenyl)-6H-pyrimido[1,2-b][1,2,4,5]-
tetrazin-6-one (6h).
inhibitory action of the compound 6. The fungicide Terbinafin
and the bactericide Chloramphenicol were used as standards
under the same conditions. Measurements were considered after
72 hours for fungi and 24 hours for bacteria. The results are
summarized in Table 1.
Comound 6h was obtained in 54% yield; m.p. 250-251°C
-1
1
(EtOH); IR: ν (cm ) 3209, 1674; H NMR: ν 6.65 (s, 1H), 6.70
(d, 1H), 6.76 (d, 1H), 7.15-7.77 (m, 15H), 8.80 (s, 1H); MS m/z
+
(%) 405 (M , 100), 314 (3), 302 (2), 262 (3), 247 (7), 220 (3),
REFERENCES AND NOTES
171 (6), 145 (6), 129 (14), 105 (23), 91 (5), 77 (87).
Anal. Calcd. for C H N O: C, 74.06; H, 4.72 ; N, 17.27.
25 19
5
Found: C, 73.9; H, 4.7; N, 17.2%.
[*] Reprint requests to Dr. Ahmad Sami Shawali, e-mail:
shawali@chem-sci.cairo.eun.eg
[1] A. S. Shawali; M. A. Abdallah, Adv. Heterocyclic Chem., 63,
277 (1995).
3-Methyl-1-(4-nitrophenyl)-8-phenyl-6H-pyrimido[1,2-b]-
[1,2,4,5]tetrazin-6-one (6i).
Compound 6i was obtained in 58% yield ; m.p. > 300 °C
[2] A. S. Shawali, Chem. Rev., 93, 2731 (1993).
[3] A. S. Shawali, Heterocycles, 20, 2239 (1983).
[4] A. S. Shawali; C. Parkanyi, J. Heterocyclic Chem. , 17, 833
(1980).
+
-1
(EtOH); IR: ν (cm ) 3193, 1651; MS m/z (%) 362 (M , 100),
347 (2), 293 (10), 266 (3), 247 (4), 171 (11), 169 (4), 136 (4), 122
(40), 116 (8), 105 (4), 90 (4), 77 (14).
Anal. Calcd. for C H N O : C, 59.67; H, 3.89; N, 23.19.
Found: C, 60.1; H, 4.1; N, 23.4 %.
[5] P. Bitha; J. J. Hlavka; Y. Lin, J. Org. Chem., 52, 2220 (1987).
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Can. J. Chem., 53, 1484 (1975).
18 14
6 3
Antimicrobial Assay.
Cultures of four fungi species namely Rhizopus spp. RS,
Aspergillus fumigatus AF, Syncephalastrum racemosum SR and
Candida spp. CS as well as five bacteria species namely
Pseudomonas aeruginosa PA, Proteus vulgaris PV,
Bacillussubtilis BS, Escherichia coli EC and Salmonella spp. SS
were used to investigate the antimicrobial activity of the com-
pounds 6a-i. The antimicrobial activity was assayed biologically
using diffusion plate technique. The latter technique was carried
out by pouring a spore suspension of the fungal species (one ml
[10] A. J. Elliott, M. S. Gibson, M. M. Kayser and G. A. Pawelchak,
Can. J. Chem., 51, 4115 (1973).
8
of sterile water contains approximately 10 conidia) or spreading
[11] G. Favrel, Bull. Chim. Soc. Fr. , 31, 150 (1904).
[12] W. Dieckmann and O. Platz, Chem. Ber., 38, 2989 (1906).
[13] P. Wolkoff, Can. J. Chem., 53, 1333 (1975).
[14] A. S. Shawali and A. O. Abdelhamid, Bull. Chem. Soc. Jpn.,
49, 321 (1976).
[15] H. M. Hassaneen, A. S. Shawali and N. M. Abunada,
Org. Prep. Proc. Int., 24, 171 (1992).
[16] A. M. Farag and M. S. Algharib, Org. Prep. Proc. Int., 20, 521
(1988).
bacterial suspension over a solidified malt agar medium. The
layer is allowed to set for 30 minutes. A solution of the test
compound 6 (1.0 µg/ml) was placed onto sterile 5 mm filter paper
discs and allowed to dry, then the discs were placed on the centre
of the malt agar plate and incubated at optimum incubation tem-
perature 28 ± 2 °C. Test organism growth 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
[17] T. Curtius, J. Prakt. Chem., 51, 168 (1899).