New 2H-tetrahydro-1,3,5-thiadiazine-2-thiones incorporating glycine and glycinamide as potential antifungal agents

Article abstract of DOI:10.1002/1521-4184(200212)335:9<438::AID-ARDP438>3.0.CO;2-E

The new title derivatives (4 b-h and 5 a-i) were synthesized by reaction of the appropriate primary amine, carbon disulphide, and formaldehyde. These derivatives were prepared in order to study the effects of introducing polar groups at N3 or N5 or at both positions on the biological activity. The 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 compounds exhibited varied inhibitory effects on growth or sporulation of some tested fungal species.

Full text of DOI:10.1002/1521-4184(200212)335:9<438::AID-ARDP438>3.0.CO;2-E

438 Aboul-Fadl et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 9, 438–442
Tarek Aboul-Fadla,
New 2H-Tetrahydro-1,3,5-thiadiazine-2-thiones
Incorporating Glycine and Glycinamide as Potential
Antifungal Agents
Mostafa A. Husseina,
Abdel-Nasser El-Shorbagia,
Abdel-Raouf Khallilb
a
Faculty of Pharmacy,
Assiut University,
Assiut-71526, Egypt
Faculty of Science,
Assiut University,
Assiut, Egypt
The new title derivatives (4 b–h and 5 a–i) were synthesized by reaction of the ap-
propriate primary amine, carbon disulphide, and formaldehyde. These derivatives
were prepared in order to study the effects of introducing polar groups at N3 or N5 or
at both positions on the biological activity.The compounds were tested for their anti-
fungal activity in vitro against pathogenic (Trichophyton rubrum and Candida albi-
cans), phytopathogenic (Penicillum expansum, Trichoderma hazianum, and Fasa-
rium oxysporum), and aflatoxin-producing (Aspergillus flavus) fungi. These com-
pounds exhibited varied inhibitory effects on growth or sporulation of some tested
fungal species.
b
Keywords: 3,5-Disubstituted 2H-tetrahydro-1,3,5-thiadiazine-2-thiones; Glycine;
Glycinamide; Antifungal activity
Received: April 11, 2002 [FP690]
Introduction
Systemic mycosis in patients suffering from debilitating
diseases such as neopliasis and in persons on long term
total parentral nutrition [1] is creating a critical need for
more and better antifungal agents. It is well established
that the tetrahydro-1,3,5-thiadiazine-2-thione (THTT)
moiety possesses a significant antifungal activity, possi-
bly through the production of isothiocyanate and/or di-
thiocarbamic acids [2–11]. Recently, we reported ethyl-
enebis-THTT derivatives [12, 13].
In order to focus our efforts on the structural require-
ments for antifungal activity of theTHTT nucleus, several
new derivatives were prepared through incorporation of
glycine and glycinamide at N3 or N5 or at both positions
of this ring structure. The compounds were screened,
and a variable antifungal activity was established.
Results and discussion
Chemistry
amate salts (2 a–i) were then treated with formaldehyde
to give (3 a–i).The latter derivatives were then allowed to
react with glycinamide to give (4 b–h). Compound 3 i
was reacted with 1 i or with the appropriate alkyl, cy-
cloalkyl, or aralkyl amine (1 a–h) to provide the desired
derivatives (5 a–i), as shown in Charts 1 and 2.
The target compounds were synthesized by reaction of
the appropriate alkyl, cycloalkyl, aralkyl amine or glycine
(1 a–i) with carbon disulphide and potassium hydroxide.
The amount of KOH was doubled in the case of glycine to
provide potassium glycinate. The resulting dithiocarb-
It was suggested that the reaction proceeds through the
formation of the intermediates (3 a–i) in situ [11–16].
One of these intermediates, 3 c, was isolated by using
40 % KOH (instead of 20 % KOH, see Experimental
Correspondence: Abdel-Nasser El-Shorbagi, Faculty of
Pharmacy, Assiut University, Assiut-71526, Egypt. e-mail:
el-shorbagi@lycos.com.
© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/09/0438 $ 17.50+.50/0

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 438–442
New 2H-Tetrahydro-1,3,5-thiadiazine-2-thiones 439
to provide a concentrated solution inducing crystalliza-
tion of 3 c after addition of the required amount of formal-
dehyde. Structures of the synthesized compounds were
verified on the bases of spectral and elemental methods
of analyses. Table 1 shows the physicochemical con-
stants of the newly synthesized derivatives.
Antifungal activity
The synthesized compounds (4 b–h and 5 a–i) were
tested for their antifungal activity in vitro against patho-
genic (Trichophyton rubrum and Candida albicans),
phytopathogenic (Penicillum expansum, Trichoderma
hazianum, and Fasarium oxysporum), and aflatoxin-pro-
ducing (Aspergillus flavus) fungi using the standard agar
diffusion method [17]. Table 3 shows the results of the
antifungal activity of the tested compounds expressed
as the inhibition zone in mm.
All spectral data are in accordance with the assumed
structures. The IR spectra of compounds (4 b–h)
showed the characteristic stretching absorption of the
amide at the range 3350–3150 cm–1 (NH) and at 1660–
1680 cm–1 (C=O). Compounds (5 a–i) showed the char-
acteristic stretching absorption of the carboxylic acid at
the range 3500–3200 cm–1 (OH) and at 1705–1715 cm–1
(for the carboxylic C=O).All derivatives showed the other
common stretching bands around 2900–2950 cm–1 (C-H
stretch aliphatic), 3000–3050 (C-H stretch aromatic) and
at about 1400–1450 cm–1 (C=S stretch).
The study was carried out at a concentration of
100 µmol/mL in DMSO for each compound.The results
clearly indicate that the introduction of a polar group
(acetamide derivatives 4 b–h and 5 i) at N5 of the THTT
system abolished the activity.However, introducing a po-
lar group (acetic acid derivatives) at N3 gave good to
moderate activity. Compound 5 d (R = CH(CH3)2)
100 µmol/mL shows the highest activity against the
growth and also against the sporulation of most of the
tested species.The sporulation of T. rubrum and C. albi-
cans, P. expansum, T. hazianum, and F. oxysporum was
conspicuously inhibited in the presence of compound
5 d. Meanwhile, increasing the length or the bulkiness of
the alkyl side chain (N5–R) in derivatives 5 a–i resulted
in various effects, depending upon the tested fungus.
Aralkyl derivatives (5 g and 5 h) were inactive or weakly
active towards most organisms.
In the 1H-NMR spectra, a common pattern for the
methylenes of C4 and C6 of all derivatives was found.The
differences in sets and patterns were attributed to the N3
and N5-alkyls or aralkyls (Table 2).
Table 1. Physicochemical data of the newly synthesized
We can conclude that the antifungal activity of the newly
synthesized derivatives is greatly affected by the posi-
tion carrying a polar group. The derivatives bearing the
polar group at N3 are active.The highest activity was ob-
tained with less bulky groups.Preparation of other deriv-
atives with various types of side chain and testing of their
antifungal activity is currently being carried out in our
laboratory.
derivatives.
Compd.
No.
R
Formulaa
Yield Mp
[%] [°C]
3 c
4 b
4 c
4 d
4 e
4 f
4 g
4 h
5 a
5 b
5 c
5 d
5 e
5 f
C3H7
C2H5
C3H7
i-C3H7
C4H9
C6H13NO2S2 · H2O 45 215–6
C7H13N3OS2
C8H15N3OS2
C8H15N3OS2
C9H17N3OS2
60 184
57 89
65 170
62 165
50 196
55 194
58 198
50 126
55 128
86 128
87 123
80 247
78 121
88 173
85 141
62 212
Experimental
Cyclo-C6H11 C11H19N3OS2
C6H5-CH2 C12H15N3OS2
C6H5-(CH2)2 C13H17N3OS2
Materials and equipment
Melting points were determined on an electrothermal melting
point apparatus [Stuart Scientific, UK] and were uncorrected.
Precoated silica gel plates (kiesel gel 0.25 mm, 60G F254,
Merk) were used for thin layer chromatography. A chloroform/
methanol (10 : 3) developing solvent system was used and the
spots were detected by ultraviolet light.
CH3
C12H15N3OS2
C7H12N2O2S2
C8H14N2O2S2
C8H14N2O2S2
C9H16N2O2S2
C2H5
C3H7
i-C3H7
C4H9
IR spectra (KBr disc) were recorded on a Shimadzu (Japan) IR-
470 spectrometer. 1H-NMR spectra were scanned on a Varian
(USA) EM-360 L NMR spectrometer (60 MHz). 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 measured at the Department of Botany, Faculty of
Science, Assiut University, Assiut, Egypt.
Cyclo-C6H11 C11H18N2O2S2
C6H5-CH2 C12H14N2O2S2
C6H5-(CH2)2 C13H16N2O2S2
C7H10N2O4S2
5 g
5 h
5 i
–
a)
Elemental analyses were satisfactory within ±0.5 % of
the calculated values.

440 Aboul-Fadl et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 438–442
1
Table 2. The H-NMR chemical shifts of synthesized compounds in DMSO-d6.
No. R
5 or 3
4 and 6 CH2
R and others
CH2CO
3 c C3H7
–
4.10 (2 H, br. s),
4.55 (2 H, br. s)
0.85 (3 H, t, N3-CH2CH2CH3), 1.60 (2 H, m, N3-
CH2CH2CH3), 3.05–4.50 (4 H, m, N3-CH2CH2CH3,
2 OH (exchangeable).
4 b C2H5
4 c C3H7
3.30 (2 H, s)
3.21 (2 H, s)
4.50 (4 H, br. s)
4.50 (4 H, br. s)
1.22 (3 H, t, CH2CH3), 4.00 (2 H, q, CH2CH3),
7.10–7.3 (2 H, br. s, CONH2)
0.88 (3 H, t, N3-CH2CH2-CH3), 1.75 (2 H, m, N3-
CH2CH2CH3), 3.65 (2 H, t, N3-CH2CH2CH3), 7.00–7.3
(2 H, br. s, CONH2)
4 d i-C3H7
4 e C4H9
3.28 (2 H, s)
3.25 (2 H, s)
4.58 (4 H, br. s)
4.48 (4 H, br. s)
1.25 (6 H, br. d, N3-CH(CH3)2), 5.96 (1 H, m, N3-
CH(CH3)2), 6.90–7.25 (2 H, br. s, CONH2)
0.90 (3 H, t, butyl CH3), 1.10–1.90 (4 H, br.m, N3-
CH2CH2CH2CH3), 3.96 (2 H, t, N3-CH2CH2CH2CH3),
7.10–7.35 (2 H, br. s, CONH2)
4 f Cyclo-C6H11 3.30 (2 H, s)
4.56 (4 H, br. s)
0.90–2.35 (10 H, br.m, the cyclohexyl five
methylenes), 5.68 (1 H, br.m, N3-CH methine of
cyclohexyl), 7.10–7.30 (2 H, br. s, CONH2)
4 g C6H5-CH2
3.30 (2 H, s)
4.40 (4 H, br. s)
4.50 (4 H, br. s)
5.37 (2 H, s, N3-CH2C6H5), 7.45 (5 H, s, benzyl C6H5),
6.70–7.15 (2 H, br. s, CONH2)
4 h C6H5-(CH2)2 3.30 (2 H, s)
4.56 (4 H, br. s)
3.01 (2 H, t, N3-β-CH2), 4.16 (2 H, t, N3-α-CH2), 7.43
(5 H, s, C6H5), 7.00–7.30 (2 H, br. s, CONH2)
5 a CH3
5 b C2H5
5 c C3H7
4.75 (2 H, s)
4.75 (2 H, s)
4.80 (2 H, s)
4.50 (4 H, br. s)
4.55 (4 H, br. s)
4.60 (4 H, br. s)
2.65 (s, 6 H, 2 CH3)
1.15 (3 H, t, CH2CH3), 2.90 (2 H, q, CH2CH3)
0.82 (3 H, t, propyl CH3), 1.70 (2 H, m, N5-
CH2CH2CH3), 2.95 (2 H, t, N5-CH2CH2CH3
5 d i-C3H7
5 e C4H9
4.55 (2 H, s)
4.56 (2 H, s)
4.45 (4 H, br. s)
4.50 (4 H, br. s)
1.15 (6 H, d, CH(CH3)2, 3.25 (1 H, m, CH(CH3)2)
0.85 (3 H, t, butyl CH3), 1.20–1.75 (4 H, br. m, N5-
CH2CH2CH2CH3), 2.86 (2 H, t, N5-CH2CH2CH2CH3)
5 f Cyclo-C6H11 4.60 (2 H, s)
4.65 (4 H, br. s)
1.10–2.25 (10 H, br. m, the cyclohexyl five methylene
groups), 3.15 (1 H, br. m, N3-CH methine of
cyclohexyl)
5 g C6H5-CH2
4.55 (2 H, s)
4.05 (2 H, br. s)
4.20 (2 H, br. s)
3.42 (2 H, s, N5-CH2C6H5), 7.30 (5 H, s, benzyl C6H5)
5 h C6H5-(CH2)2 4.70 (2 H, s)
4.50 (4 H, br. s)
2.60–3.30 (4 H, m, N5-CH2CH2), 7.30 (5 H, s, C6H5)
5 i –
3.66 (2 H, s, N5-CH2), 4.58 (4 H, br. s)
4.72 (2 H, s, N3-CH2)

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 438–442
New 2H-Tetrahydro-1,3,5-thiadiazine-2-thiones 441
Table 3. Antifungal activity of the tested compounds (expressed as the diameter of the inhibition zonea).
Compd No.
T.
C.
P. expansum
T. harzianum
F. oxysporum
A.
4 f
4 g
5 a
5 b
5 c
5 d
5 g
5 h
–
9
8
–
15
10
–
22
–
11
–
–
–
–
13
10
10
–
–
–
–
–
–
7
–
–
–
–
–
7
–
–
19
–
7
–
–
–
–
–
–
8
–
–
–
16
13
15
–
–
–
–
–
Control
–
a)
Average of three observations.
Inhibition zone in mm. Disc diameter is 6 mm.
“–” no inhibition zone.
Compounds 4 b–e, 4 h, 5 e, 5 f, and 5 i are inactive.
General procedure for synthesis of 3,5-disubstituted tetrahy-
dro-2H-1,3,5-thiadiazine-2-thione 4 b–h
solid collected was recrystallized from ethanol-chloroform
(1 : 1) to afford 5 a–i.Yields, melting points, physical and spec-
tral data are given in Tables 1 and 2.
Carbon disulfide (60 mmol) was added portionwise to a stirred
mixture of the appropriate alkyl-, cycloalkyl-, or aralkylamine
1 b–h (10 mmol) and potassium hydroxide (20 %, 10 mmol) in
ethanol (10 mL) [note 1]. Stirring was continued for 3 h at ambi-
ent temperature. To the reaction mixture, which contains the
dithiocarbamates 2 b–h, formaldehyde solution (35 %, 22
mmol) was added, and stirring was continued for a further 1 h.
The resulting clear solution of 3 b–h was added portionwise
during 15 min to a stirred solution of glycinamide (10 mmol) in
phosphate buffer (pH 7.8, 20 mL). After stirring for 4 h at ambi-
ent temperature, the reaction mixture was acidified with dilute
hydrochloric acid (5 %, ف 15–18 mL) to pH 2 and stirring was
continued for a further 30 min.The resulting precipitate was col-
lected by filtration, washed with 0.5 % hydrochloric acid, and
dried. The crude solid collected was recrystallized from etha-
nol-chloroform (1 : 1) to afford 4 b–h. Yields, melting points,
physical and spectral data are given in Tables 1 and 2.
Organisms, culture conditions, and antifungal activity
Six pathogenic or phytopathogenic fungal species were used in
the present study:
Trichoderma rubrum (Castellani) Sabouraud (a dermatophyte
causing tinea corporis, tinea pedis, tinea manuum, and ony-
chomycosis), Candida albicans (Robin) Berkhout (a cause of
candidiasis) [18], Penicillium expansum Link (a cause of apple
rot), Trichoderma harzianum Rifai (a cause of soft rot of some
fruits), Fusarium oxysporum Schlechtendal (a cause of tomato
fruit rot), and. Aspergillus flavus Link (aflotoxin-producing spe-
cies).
T. rubrum and C. albicans were isolated from clinical cases in
the Assiut University hospitals [19]. The other fungal species
were isolated from rotted fruits (Penicillium expansum, Tri-
choderma harzianum, and Fusarium oxysporum) and soil (As-
pergillus flavus). These species were maintained on Sabour-
aud agar dextrose (SAD) in the collection herbarium of the
Botany Department.
Note 1: Potassium hydroxide (40 %, 10 mmol) was used to
enable separation of compound 3 d.
General procedure for synthesis of 3,5-disubstituted tetra-
hydro-2H-1,3,5-thiadiazine-2-thione 5 a–i
Spore suspension in sterile distilled water was prepared from
3–5-day old cultures 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.
The plates were shaken gently to homogenize the inoculum.
Carbon disulfide (60 mmol) was added portionwise to a stirred
mixture of glycine 1 i (10 mmol) and potassium hydroxide
(40 %, 20 mmol).Stirring was continued for 3 h at ambient tem-
perature.To the reaction mixture, which contains the dithiocar-
bamate 2 d, formaldehyde solution (35 %, 22 mmol) was added
and stirring was continued for a further 1 h. The reaction mix-
ture was filtered through a filter paper (to separate the excess
carbon disulfide).The resulting clear solution of 3 d was added
portionwise during 15 min to a stirred solution of the appropri-
ate alkyl-, cycloalkyl-, aralkylamine or glycine 1 a–i (10 mmol)
in phosphate buffer (pH 7.8, 20 mL). After stirring for 4 h at am-
bient temperature, the reaction mixture was acidified with dilute
hydrochloric acid (5 %) to pH 2 and stirring was continued for a
further 30 min.The resulting precipitate was collected by filtra-
tion, washed with 0.5 % hydrochloric acid, and dried.The crude
Antifungal activity of the tested compounds was performed by
the standard agar disk diffusion method [17] as follows:
Sterile 6 mm filter paper disks (Whatman) were impregnated
with solutions of the tested compound (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 at the center of each plate. The
seeded plates were incubated at 30 ± 2 °C for 7 days.The radii
of the inhibition zones (in mm) were measured at successive in-

442 Aboul-Fadl et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 438–442
[10] A. El-Shorbagi, Arch. Pharm. Pharm. Med. Chem., 2000,
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2000, Cairo, Egypt.
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