Synthesis and antifungal activity of 3,3′-ethylenebis(5-alkyl-1,3,5-thiadiazine-2-thiones)

Article abstract of DOI:10.1002/1521-4184(200110)334:10<305::AID-ARDP305>3.0.CO;2-O

In a search for promising antifungal compounds, nine new 3,3′-ethylenebis(5-alkyl-1,3,5-thiadiazine-2-thiones) were synthesized by the reaction of ethylene diamine, carbon disulfide, formaldehyde, and the appropriate alkyl amine. The title compounds were

Full text of DOI:10.1002/1521-4184(200110)334:10<305::AID-ARDP305>3.0.CO;2-O

Full Papers
Mostafa A. Husseina),
Abdel-Nasser El-Shorbagia),
Abdel-Raouf Khallilb)
Synthesis and antifungal activity of
3,3′-ethylenebis(5-alkyl-1,3,5-thiadiazine-
2-thiones)
a)Faculty of Pharmacy and b)Faculty
of Science, Assiut University, Assiut-
71526, Egypt
In a search for promising antifungal compounds, nine new 3,3′-ethylenebis(5-alkyl-1,3,5-
thiadiazine-2-thiones) were synthesized by the reaction of ethylene diamine, carbon
disulfide, formaldehyde, and the appropriate alkyl amine. The title compounds were
tested for their antifungal activity in vitro against pathogenic (Trichophyton rubrum and
Candida albicans), phytopathogenic (Penicillum expansum, Trichoderma hazianum, and
Fasarium oxysporum), and aflatoxin producing (Aspergillus flavus) fungi. These com-
pounds exhibited varied inhibitory effects on growth or sporulation of some tested fungal
species.
Key Words: 3,3′-Ethylenebis(5-alkyl-1,3,5-thiadiazine-2-thiones); Ethylenediamine;
Antifungal activity
Received: June 11, 2001 [FP588]
S
H
Introduction
K S
N
NH2
+ 2CS2
+
2KOH
N
H2N
The need for more and better antifungal agents is becoming
more critical because of the increasing detection of sys-
temic mycosis in patients suffering from debilitating dis-
eases such as neoplasia and in persons on long term total
parenteral nutrition[1]. It is well established that the 1,3,5-
thiadiazine-2-thione moiety possesses a significant antifun-
gal activity through the production of isothiocyanate and
dithiocarbamic acids[2–10]. Recently, a variety of 1,3,5-
thiadiazine-2-thione derivatives prepared through incorpo-
ration of glycine and glycinamide at N3 and N5 positions of
the 1,3,5-thiadiazine-2-thione structure were screened,
and a variable antifungal activity was established[11]. In our
effort to shed light on the structural requirements for the
antifungal activity of the 1,3,5-thiadiazine-2-thione nucleus,
we prepared several new derivatives of the 1,3,5-
thiadiazine-2-thione nucleus. The study was based on in-
troduction of two moieties of 1,3,5-thiadiazine-2-thione
through an ethylene bridge and investigation of the antifun-
gal activity of these structural analogues.
S K
H
S
[1]
S
N
CH2OH
R-NH2
[3a-i]
4 HCHO
S
N
CH2OH
HOH2C
S
HOH2C
S
[2]
R
S
S
N
N
N
N
S
S
R
[4a-i]
Scheme 1. R = CH3 (a), C2H5 (b), C3H7 (c), i-C3H7 (d), C4H9 (e),
i-C4H9 (f), cyclo-C6H11 (g), C6H5-CH2 (h), C6H5-(CH2)2 (i).
amine (3a–i) to provide the desired derivatives (4a–i),
Scheme 1.
Results and Discussion
It was suggested that the reaction proceeds through the
formation of the intermediate (2) in situ [12–15]. Structures
of the synthesized compounds were verified on the basis
of spectral and elemental methods of analyses. Table 1
shows the physicochemical constants of the newly synthe-
sized derivatives (4a–i).
Chemistry
The target compounds were synthesized by the reaction of
ethylenediamine with carbon disulfide and potassium hy-
droxide. The resulting ethylene bisthiocarbamate salt (1)
was subjected to cyclocondensation reaction with formal-
dehyde and the appropriate alkyl-, cycloalkyl-, or aralkyl
All spectral data are in accordance with the assumed
structures. The IR spectra of compounds (4a–i) show
bands around 2930–2950 cm–1 (C-H stretch aliphatic),
3030–3050 (C-H stretch aromatic) and at about 1450 cm–1
(C=S stretch).
———
Correspondence: Prof. Mostafa A. Hussein, Faculty of Phar-
macy, Assiut University, Assiut-71526, Egypt.
E-mail: mostafa17@hotmail.com
Fax: +20 88 332776
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2001
0365-6233/01/1010/0305 $17.50 +.50/0

306 Antifungal agents
Arch. Pharm. Pharm. Med. Chem. 2001, 334, 305–308
Table 1. 3,3′-Ethylenebis(5-alkyl-1,3,5-thiadiazine-2-thiones).
Antifungal Activity
————————————————————————————–
The newly synthesized compounds (4a–i) were tested for
their antifungal activity in vitro against pathogenic (Tricho-
phyton rubrum and Candida albicans), phytopathogenic
(Penicillum expansum, Trichoderma hazianum, and Fasar-
ium oxysporum), and aflatoxin producing (Aspergillus fla-
Compd.R
No.
Moleculara
formula
Yield Mp
(%)
————————————————————————————–
4a
4b
4c
4d
4e
4f
CH3
C10H18N4S4
C12H22N4S4
C14H26N4S4
C14H26N4S4
C16H30N4S4
C16H30N4S4
C20H34N4S4
C22H26N4S4
C24H30N4S4
60
65
80
85
86
85
80
86
87
183
vus) fungi using the standard agar diffusion method[16]
.
C2H5
178
Table 3 shows the results of the antifungal activity of the
tested compounds expressed as the inhibition zone in mm.
C3H7
180
i-C3H7
172–173
163–165
100–101
130–132
186–187
180–182
The study was carried out at concentrations (100, 50, and
25 µmol/ml in DMSO) for each compound. The results
clearly indicate that compound 4a (R = CH3) at 100 µmol/ml
is the most active one against the growth and also against
the sporulation of the most tested species. The sporulation
of P. expansum, F. oxysporum, and A. flavus was conspicu-
ously inhibited in the presence of compound (4a). Mean-
while, increasing the length or the bulkiness of the alkyl side
chain (N5′-R) resulted in varied effects depending upon the
tested fungus and the employed dose. Branching of the
alkyl side chain (4c–f) gave comparable results. Almost all
alkyl and cyclohexyl derivatives (4a–g) showed good ac-
tivities against P. expansum and F. oxysporum. Aralkyl
derivatives (4h and 4i) were inactive at the concentrations
used on all organisms; as an exception 4i provided excel-
lent activity against T. rubrum.
C4H9
i-C4H9
4g
4h
4i
cyclo-C6H11
C6H5-CH2
C6H5-(CH2)2
————————————————————————————–
a) Elemental analyses were satisfactory within ± 0.5% of the calcu-
lated values.
In the 1H-NMR spectra, a common pattern for the ethylene
and the methylenes of C4&C4′ and C6&C6′ of all deriva-
tives was found. The differences in sets and patterns were
only attributed to the N5&N5′-alkyls or aralkyls where they
give patterns in accordance with the structure of the target
compounds (Table 2).
Table 2. The 1H-NMR chemical shifts of compounds (4a–i) in DMSO-d6.
R
S
4/
S
N
5/
/
6/
N
3
N
2/
1/
N
S
S
R
————————————————————————————————————————————————————————————
No CH2CH2 4&4′-CH2 6&6′-CH2
————————————————————————————————————————————————————————————
R
4a
4b
CH3
2.55 (s, 6H, 2CH3)
4.15, s, 4H
4.00, s, 4H
4.35, s, 4H
4.25, s, 4H
4.40, s, 4H
4.30, s, 4H
C2H5
1.20 (t, 6H, 2CH3), and
2.70 (q, 4H, 2CH2)
4c
C3H7
0.8 (t, 6H, 2CH3), 1.40 –1.65
(m, 4H, 2CH2), and 2.75
(t, 4H, 2CH2)
4.25, s, 4H
4.50, s, 4H
4.60, s, 4H
4d
4e
i-C3H7
C4H9
1.15 (d, 12H, 4CH3), and
3.55–3.70 (m, 2H, 2CH)
4.15, s, 4H
4.25, s, 4H
4.50, s, 4H
4.50, s, 4H
4.60, s, 4H
4.55, s, 4H
0.85 (t, 6H, 2CH3), 1.35–1.50
(m, 8H, 2CH2CH2), and
2.65 (t, 4H, 2CH2),
4f
i-C4H9
0.85 (d, 12H, 4CH3), and
2.55–2.70 (m, 2H, 2CH),
and 3.25 (d, 4H, 2CH2)
4.15, s, 4H
4.10, s, 4H
4.45, s, 4H
4.45, s, 4H
4.55, s, 4H
4.50, s, 4H
4g
cyclo-C6H11
1.15–2.20 (m, 20H, 2C6H11),
and 3.00–3.15 (m, 2H, H1 of
C6H11
)
4h
4i
C6H5-CH2
4.00 (s, 4H, 2CH2C6H5), and
7.50 (s, 10H, 2C6H5)
4.25, s, 4H
4.25, s, 4H
4.45, s, 4H
4.55, s, 4H
4.65, s, 4H
4.60, s, 4H
C6H5-(CH2)2
3.00 (s, 8H, 2CH2CH2), and
7.50 (s, 10H, 2C6H5)
————————————————————————————————————————————————————————————

Arch. Pharm. Pharm. Med. Chem. 2001, 334, 305–308
Antifungal agents 307
Table 3. Antifungal activity of the tested compounds (expressed as the diameter of the inhibition zonea).
————————————————————————————————————————
Compd No.
T.
C.
P.
T.
F.
A.
µmol/ml
rubrum
albicans
expansum harzianum oxysporum flavus
————————————————————————————————————————
4a
100
50
28
–
22
19
–
12
16
14
10
10
–
–
–
–
7
–
–
–
–
7
–
16
–
–
–
–
19
–
16
10
–
25
–
–
4b
100
50
–
–
13
–
7
–
–
7
25
–
–
–
9
4c
4d
4e
100
100
100
50
–
–
8
9
–
–
–
7
8
–
–
–
13
17
–
10
–
–
–
–
–
–
–
–
–
4f
100
100
100
100
–
–
12
13
–
4g
9
–
10
–
4h
–
–
4i
23
–
–
–
–
Control
–
–
–
————————————————————————————————————————
a)Average of three observations.
Inhibition zone in mm. Disc diameter is 6 mm.
”–” no inhibition zone.
Generally, we can conclude that the antifungal activity of
the newly synthesized derivatives (4a–i) is greatly affected
by the bulkiness of the side chain. The highest activity was
obtained with the least bulky groups (R = CH3). Preparation
of other derivatives depending upon the type of the side
chain simultaneously with testing of their antifungal activity
is currently being carried out in our laboratory.
At concentrations of 50 µmol/ml, compounds 4a, 4b, and
4e still show antifungal activity against three types of the
used fungi. Moreover, compounds 4a and 4b revealed
promising antifungal activity against P. expansum and A.
flavus, respectively, at concentrations of 25 µmol/ml.
Similar inhibitory effects were obtained by several authors
who dealt with the antifungal activities of 1,3,5-thiadiazine-
2-thione derivatives. In this respect, Ertan et al.[17] prepared
ten derivatives of 3-(2-phenethyl)-5-substituted-1,3,5-
thiadiazine-2-thione and tested their antifungal activities
against four species of Candida (C. albicans, C. parap-
silosis, C. stellatoidea, and C. pseudotropicalis). These
derivatives exerted variable inhibitory effects depending
upon the structure and the tested species. In addition, it is
reported that growth, sporulation, and nucleic acid contents
of Fasarium oxysporum f. sp. Lycopersici and Helmin-
thosporium oryzae were retarded in the presence of
2-aminopyrimidine derivatives[18]. Moreover, thiazol[2,3-c]-
1,2,4-triazole derivatives are reported to be toxic against
Experimental
Materials and equipment
Melting points were determined on an electrothermal melting
point apparatus [Sturat Scientific, UK], and were uncorrected.
Precoated silica gel plates (kiesel gel 0.25 mm, 60G F254,
Merck) were used for thin layer chromatography. A developing
solvent system of chloroform/methanol (10:3) was used and the
spots were detected by ultraviolet light.
IR spectra (KBr disc) were recorded on a Shimadzu IR-470
spectrometer, Japan. 1H-NMR Spectra were scanned on a
Varian EM-360 L NMR spectrometer (60 MHz), USA. Chemical
shifts are expressed in δ-values (ppm) relative to TMS as an
internal standard, using DMSO-d6 as a solvent. Elemental
analyses were performed at the Department of Chemistry,
Faculty of Science, Assiut University, Assiut, Egypt. Antifungal
activity was performed at the Department of Botany, Faculty of
Science, Assiut University, Assiut, Egypt.
Aspergillus flavus and Fasarium solani[19]
.
The rest of the derivatives were devoid of activity at 50 and
25 µmol/ml. Conversely, some tested compounds not only
showed no inhibitory effects on growth or sporulation of the
tested fungal species, but also allowed a dense mycelial
growth. This indicates that these fungi can utilize such
compounds as carbon or nitrogen sources. In this connec-
tion it is reported that some pyrimidine, uracil, uridine,
cytosine, cytidine, deoxycytidine, and dihydrouracil deriva-
tives allowed mycelial growth, sporulation, and nucleic acid
synthesis[18] or could be utilized by some fungal species as
Synthesis of new 3,3′-ethylenebis(5-alkyl-1,3,5-thia-
diazine-2-thiones)
Carbon disulfide (80 mmol) was added portionwise to a stirred
mixture of ethylene diamine (10 mmol) and potassium hydrox-
ide (40%, 40 mmol) in water (5 ml), stirring was continued at
ambient temperature for 2 h. Formaldehyde solution (35%,
a sole nitrogen source[20]
.

308 Antifungal agents
Arch. Pharm. Pharm. Med. Chem. 2001, 334, 305–308
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44 mmol) was added to the mixture and stirring was continued
for a further 3 h. To the resulting clear solution obtained, a
solution of the appropriate alkyl or aralkyl amine (20 mmol) in
phosphate buffer ( pH 7.8, 5 ml) was added portionwise during
15 min. After stirring for 5 h at ambient temperature, the pre-
cipitate formed was collected by filtration, washed with aqueous
methanol, dried, and crystallized from chloroform/ethanol.
Yields, melting points, and physical data are given in Table 1.
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Six pathogenic or phytopathogenic fungal species were used
in the present study: Trichoderma rubrum (Castellani)
Sabouraud (a dermatophyte causing of tinea corporis, tinea
pedis, tinea manuum, and onychomycosis), Candida albicans
(Robin) Berkhout (a cause of candidiasis)[21], Penicillium ex-
pansum Link (a cause of apple rot), Trichoderma harzianum
Rifai (a cause of soft rot of some fruits), Fusarium oxysporum
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Martinez, Arzneim-Forsch./Drug Res. 1999, 49, 764–769.
T. rubrum and C. albicans were isolated from clinical cases in
the Assiut University hospitals[22]. The other fungal species
were isolated from rotted fruits (Penicillium expansum,
Trichoderma harzianum, and Fusarium oxysporum) and soil
(Aspergillus flavus). These species were maintained on
Sabouraud agar dextrose (SAD) in the herbarium collection of
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[11] T. Aboul-Fadl, M. A. Hussein, A. El-Shorbagi, presented at
the 27th Int. Conf. of Pharm. Sciences, Jan. 2000, Cairo,
Egypt.
Spore suspension in sterile distilled water was prepared from
3–5 days old culture of the test fungi growing on SAD medium.
The final spore concentration was 5×104 spores/ml. About
20 ml of growth medium was introduced on sterilized plates of
9 cm diameter and inoculated with 1 ml of spore suspension.
Plates were shaken gently to homogenize the inoculum.
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Antifungal activity of the tested compounds was determined by
the standard agar disk diffusion method[16] as follows:
Sterile 6 mm filter paper disks (Whatman) were impregnated
with solutions of various concentrations of the tested compound
(25, 50, and 100 µM/ml in DMSO). In addition, other disks were
impregnated with the solvent (DMSO) and served as control.
The impregnated disks were then dried for 1 h and placed in
the center of each plate. The seeded plates were incubated at
30±2 °C for 7 days. The radius of the inhibition zones (in mm)
were measured at successive intervals during the incubation
period. Triplicate set were applied for each treatment. Results
are given in Table 3.
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[20] T. P. West, F. Microbios 1988, 56, 226, 27–36.
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