Synthesis and antimicrobial activity of some new 1,3,4-thiadiazole and 1,2,4-triazole compounds having a D,L-methionine moiety

Article abstract of DOI:10.3390/12010103

New 1,3,4-thiadiazole, 5a-e, and 1,2,4-triazolecompounds 6a-c, containing a D,L-methionine moiety were synthesized by intramolecular cyclization of 1,4-disubstituted thiosemicarbazides 4a-e in acid and alkaline media, respectively. The potential antimicro

Full text of DOI:10.3390/12010103

Molecules 2007, 12, 103-113
molecules
ISSN 1420-3049
http://www.mdpi.org
Full Paper
Synthesis and Antimicrobial Activity of Some New
1,3,4-Thiadiazole and 1,2,4-Triazole Compounds Having a
D,L-Methionine Moiety
Otilia Pintilie 1, Lenuta Profire 2,*, Valeriu Sunel 1, Marcel Popa 3 and Aurel Pui 4
1
Department of Organic Chemistry and Biochemistry and 4 Department of Inorganic Chemistry;
Faculty of Chemistry, “Al. I. Cuza” University, Bd. Carol I, no. 11, 700506 Iasi, Romania; e-mails:
povpintilie@yahoo.com, vsunel@uaic.ro
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Gr. T. Popa” Medicine and
Pharmacy University, Universitatii Street, no. 16, 700115 Iasi, Romania
3
Department of Natural and Synthetic Polymers, Faculty of Chemical Engineering, “Gh. Asachi”
Technical University, Bd. Mangeron, no. 71A, 700050, Iasi, Romania; e-mail: marpopa@ch.tuiasi.ro
* Author to whom correspondence should be addressed; E-mail: nprofire@yahoo.com, Phone (+40)
232 267801; Fax: (+40) 232 211802.
Received: 4 December 2006; in revised form: 25 January 2007 / Accepted: 26 January 2007 /
Published: 29 January 2007
Abstract: New 1,3,4-thiadiazole, 5a-e, and 1,2,4-triazolecompounds 6a-c, containing a
D,L-methionine moiety were synthesized by intramolecular cyclization of 1,4-disubstituted
thiosemicarbazides 4a-e in acid and alkaline media, respectively. The potential
antimicrobial effects of the synthesized compounds were investigated using the
Staphylococcus aureus ATCC 25923, Bacillus antracis ATCC 8705, Bacillus cereus ATCC
10987, Sarcina lutea ATCC 9341 and Escherichia coli ATCC 25922 strains. The newly
synthesized compounds exhibited promising activities against Bacillus antracis and Bacillus
cereus.
Keywords: 1,3,4-Thiadiazole; 1,2,4-triazole; thiosemicarbazide; D,L-methionine;
antimicrobial activity.

Molecules 2007, 12
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Introduction
The therapeutic effects of compounds containing 1,3,4-thiadiazole and 1,2,4-triazole rings have
been well studied for a number of pathological conditions including inflammation [1, 2], pain [3-5] or
hypertension [6]. Moreover, synthesis of thiadiazoles and triazoles has attracted widespread attention
due to their diverse applications as antibacterial [7], antimycobacterial [8, 9], antimycotic [10, 11],
antifungal [12, 13] and antidepressant agents [14]. Meanwhile, N-acylated aminoacids are known for
their hepatoprotective and antimicrobial effects [15, 16].
Taking these observations into account in the present study, some new 1,3,4-thiadiazoles and
1,2,4-triazoles having a D,L-methionine moiety have been synthesized and their structures were
1
characterized by H-NMR, IR spectroscopy and elemental analysis. The potential antimicrobial
activity and degree of toxicity of the synthesized compounds were also investigated.
Results and Discussion
The synthesis of new 1,3,4-thiadiazole and 1,2,4-triazole compounds was performed in several
steps. In the first step, 2-(3-nitrophenyl)-4-(2-methylthioethyl)-∆2-5-oxazolinone (2) was obtained by
cyclization of N-(3-nitrobenzoyl)-D,L-methionine (1) in the presence of acetic anhydride. This
intermediate, through reaction with 98% hydrazine hydrate solution in a dioxane medium, gave the
hydrazide of N-(3-nitrobenzoyl)-D,L-methionine (3), which, in the next step was reacted with different
isothiocyanates whereupon new 1,4-disubstituted thiosemicarbazides 4a-e were obtained (Scheme 1).
Scheme 1. Synthesis of the intermediates 4a-e.
O
(CH3CO)2O
- 2CH3COOH
NH2 NH2
CH COOH
CH2
CH2
CH3S CH2
HC
N C
CH3S CH2
O
NH
CO
NO2
CH CO NH NH C NH R
CH2
NO2
2
1
R N C
S
CH CO NH
CH2
CH3S CH2
CH3S CH2
NH
CO
S
NH
CO
NH2
NO2
NO2
4a-e
3
In the last step new 1,3,4-thiadiazole, 5a-e, and 1,2,4-triazole compounds 6a-c were obtained by
intramolecular cyclization of the thiosemicarbazides 4a-e in acid and alkaline media, respectively
(Scheme 2).The structures of all the synthesized compounds were confirmed by IR, 1H-NMR spectral
measurements and elemental analysis.

Molecules 2007, 12
105
Scheme 2. Synthesis of the compounds 5a-e and 6a-c.
N
N
H2SO4
CH2 CH
CH3S CH2
CH2 CH CO NH NH C NH R
CH3S CH2
S
NHR
NH
CO
NH
CO
S
NO2
NO2
5a-e
4a-e
NaOH
N
N
R
CH3S CH2 CH2 CH
a
CH3
C6H5
N
R
SH
NH
CO
b
c
C6H4 CH3 (4)
d
e
C6H4 Br (4)
CH2 CH CH2
NO2
6a-c
The IR spectra of compounds 4a-e showed intense absorption bands within the 3181-3367 cm-1
range that were attributed to NH and NH2 function vibrations. The absorption band of the –C=O
function appears in the 1641-1644 cm-1 region and the bands which appear at 1171-1173 cm-1 were
attributed to the −C=S function. In the 1H-NMR spectra, the proton signals due to the NH group were
recorded between 9.07-10.80 ppm and the aromatic protons signals appear at 7.19-8.74 ppm.
In the IR spectra of compounds 5a-e the absorption band of the –NH-CO function appears at 2916-
1
3302 cm-1 and the absorption band of the C=N group was identified at 1431-1433 cm-1. In the H-
NMR spectra the proton signal due to the –NH-CO function was observed as a singlet between 9.06-
9.07 ppm.
The IR spectra of compounds 6a-c showed an intense absorption band between 2589-2592 cm-1 that
was attributed to the −SH function and in the 1H-NMR spectra, the proton signal due to the SH group
appeared as a singlet at 12.60-12.80 ppm.
Moreover, the elemental analysis results were all in a good agreement with the structures proposed
for compounds 2, 3, 4a-e, 5a-e and 6a-c.
Biological activity
The potential antimicrobial activity of compounds 4a-e, 5a-e and 6a-c towards five standard
bacterial strains was investigated. From the date presented in Table 1 it may be seen that
thiosemicarbazides 4a-e exhibit relatively good activity against Staphylococcus aureus and
Escherichia coli, but are only slightly active against Bacillus antracis, Bacillus cereus and Sarcina
lutea strains. Upon cyclization of thiosemicarbazides to the corresponding thiadiazoles 5a-e and
triazoles 6a-c the activity towards Staphylococcus aureus and Escherichia coli decreases, while the

Molecules 2007, 12
106
activity against Bacillus antracis and Bacillus cereus increases. Concerning the activity towards the
Sarcina lutea strain, it was noted that the activity of the triazoles is comparable to that of the
thiosemicarbazideas, but the thiadiazoles are inactive. The most active compounds against Bacillus
antracis and Bacillus cereus are 5c (a thiadiazole compound) and 6c (a triazole compound), both with
a 4-methylphenyl moiety on the heterocyclic ring. This could be explained by electropositive effect of
methyl group attached to the phenyl moiety because of the known favorable influence of electron
donating groups on the potency of the heterocyclic nuclei. Further investigations are in progress.
Table 1. Antimicrobial activity of compounds 4a-e, 5a-e and 6a-c.
Minimum Inhibitory Concentration (MIC) (µg/mL)
Comp. no.
Sa
Ba
618
620
587
613
636
143
137
119
131
130
142
154
122
Bc
714
774
898
883
716
186
194
128
147
173
168
205
131
Sl
638
Ec
193
185
201
198
228
734
836
869
765
892
759
629
858
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
171
113
105
126
138
732
489
642
529
725
852
748
456
782
761
791
829
1264
1137
1142
1153
1215
615
549
521
(Sa): Staphylococcus aureus ATCC 25923; (Ba): Bacillus antracis ATCC 8705; (Bc): Bacillus
cereus ATCC 10987; (Sl): Sarcina lutea ATCC 9341; (Ec) Escherichia coli ATCC 25922.
The synthesized compounds were also investigated for their toxicity (Table 2) and it was observed
that all tested compounds had a low toxicity.
Table 2. The DL50 values of the tested compounds.
Comp. DL50 mg/Kg body Comp. DL50 mg/Kg body
4a
4b
4c
4d
4e
5a
1465
1275
1625
1315
1260
1825
5c
5d
5e
6a
6b
3100
2025
2816
4620
5010
6c
4920
5b
2110

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Conclusions
New 1,3,4-thiadiazole, 5a-e, and 1,2,4-triazole compounds 6a-c possessing a D,L-methionine
moiety were synthesized by intramolecular cyclization of 1,4-disubstituted thiosemicarbazides in acid
and alkaline media, respectively. The potential antimicrobial effects of the synthesized compounds
were investigated using the Staphylococcus aureus ATCC 25923, Bacillus antracis ATCC 8705,
Bacillus cereus ATCC 10987, Sarcina lutea ATCC 9341 and Escherichia coli ATCC 25922 strains.
The most active compounds were 5c and 6c containing a 4-methylphenyl susbtituent on the
heterocyclic ring, which exhibited promising activities against Bacillus antracis and Bacillus cereus.
Experimental Section
General
All melting points were determined on a Melt-Temp R apparatus equipped with a digital
thermometer and are uncorrected. The combustion analysis was performed on an Elemental Exeter
Analytical CE 440 Apparatus. The IR spectra were measured as potassium bromide pellets on a
Digilab Scimitar Series spectrophotometer; the wave numbers are given in cm-1. The 1H-NMR spectra
were recorded in DMSO-d6 solutions on Brucker ARX-300 spectrometer (1H: 300 MHz) at ambient
temperature. Chemical shifts were recorded as δ values in parts per millions (ppm) and were indirectly
1
referenced to tetramethylsilane via the residual solvent signal (2.49 for H). All chemical reagents
were obtained from the Aldrich Chemical Company.
Synthesis of 2-(3-nitrophenyl)-4-(2-methylthioethyl)-∆2-5-oxazolinone (2).
N-(3-Nitrobenzoyl)-D,L-methionine (5.6 g, 0.018 mol) was dissolved in acetic anhydride (15 mL)
and heated at 65-70 ºC for half an hour. After cooling the solution was added under stirring to a
mixture of petroleum ether (50 mL) and dried ethyl ether (10 mL). The reaction mixture was stirred for
30 minutes, the ethereal layer was removed and the oily product obtained was washed with petroleum
ether. The crude product was dissolved in dioxane and crystallized from anhydrous ethyl ether –
anhydrous petroleum ether (1:1). The solid compound obtained was dried under vacuum at 40-45 ºC.
Yield 76.20 %; m.p. 114-115 ºC; Anal. Calc. for C12H12N2O4S (280.05): 51.42% C, 4.28% H, 10.00%
N, 11.42% S; found 51.44% C, 4.01% H, 9.81% N, 11.26% S; IR (ν, cm-1): 725 (CH3S), 819 (aromatic
CH), 1350 (symmetric vibrations of NO2), 1433 (-C=N), 1529 (asymmetric vibrations of NO2), 1707
(CO). 1H-NMR δ: 2.09 (s, 3H, CH3,), 2.52 (m, 2H, CH2), 2.65 (m, 2H, CH2) 4.50 (t, 1H, CH), 7.80 (s,
1H, ArH), 8.30 (d, 1H, ArH), 8.40 (d, 1H, ArH), 8.70 (s, 1H, ArH).
N-(3-nitrobenzoyl)-D,L-methionyl-hydrazide (3).
The 2-(3-nitrophenyl)-4-(2-methylthioethyl)-∆2-5-oxazolinone (2, 1 g, 0.035 mol) was dissolved in
dioxane (10 mL) and aqueous hydrazine hydrate solution (98%, 0.172 mL, 0.0035 mol) was added.
The reaction mixture was heated at 65-70 ºC for one hour. After cooling the solvent was removed and

Molecules 2007, 12
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the obtained oily product was dissolved in absolute ethanol and precipitated with water. The solid
compound was dried under vacuum at 40-45 ºC. The crude product was crystallized from ethanol-
water. Yield 54.78 %; m.p. 102-104 ºC; Anal. Calc. for C12H16N4O4S (327.11): 46.15% C, 5.12% H,
17.94% N, 11.42% S; found 46.02% C, 4.98% H, 17.89% N, 11.26% S; IR (ν, cm-1): 716 (CH3S); 818
(aromatic CH); 1352 (symmetric vibrations of NO2); 1527 (asymmetric vibrations of NO2); 1638
1
(CO); 2916, 3103, 3368 (NH); H-NMR δ: 2.10 (s, 3H, CH3); 2.50 (m, 4H, CH2); 4.50 (t, 1H, CH);
7.80 (t, 1H, Ar); 8.34 (d, 1H, Ar); 8.42 (d, 1H, Ar); 8.74 (s, 1H, Ar); 9.05 (d, 1H, NH); 10.80 (s, 1H,
NH); 11.20 (s, 2H, NH2).
General procedure for the synthesis of 1,4-disubstituted thiosemicarbazides 4a-e.
N-(3-nitrobenzoyl)-D,L-methionylhydrazide (3, 0.78 g, 0.0025 mol) was dissolved in methanol (10
mL) and a solution of the appropriate isothiocyanate (0.0025 mol) in methanol (10 mL) was added.
The reaction mixture was heated at 70-80 ºC for two hours. After cooling the solvent was evaporated
under reduced pressure and the solid was dried under vacuum at room temperature. The crude product
was purified by crystallization from ethanol.
1-[N-(3-nitrobenzoyl)-D,L-methionyl]-4-methyl-thiosemicarbazide (4a). Yield 58.33 %; m.p. 121-122
ºC; Anal. Calc. for C14H19N5O4S2 (385.09): 43.63% C, 4.93% H, 18.18% N, 16.62% S; found 43.59%
C, 4.75% H, 18.03% N, 16.41% S; IR (ν, cm-1): 720 (CH3S); 819 (aromatic CH); 1173 (C=S); 1351
(symmetric vibrations of NO2); 1528 (asymmetric vibrations of NO2); 1643 (CO); 3302-3367 (NH-
CO); 1H-NMR δ: 2.11 (s, 3H, CH3); 2.51 (d, 3H, CH3 ); 2.54 (m, 4H, CH2); 4.50 (t, 1H, CH); 7.80 (t,
1H, Ar); 8.30 (d, 1H, Ar); 8.40 (d, 1H, Ar); 8.70 (s, 1H, Ar); 9.07 (s, 1H, NH); 9.80 (m, 1H, NH);
10.10 (s, 1H, NH).
1-[N-(3-nitrobenzoyl)-D,L-methionyl]-4-phenyl-thiosemicarbazide (4b). Yield 68 %; m.p. 110-111 ºC;
Anal. Calc. for C19H21N5O4S2 (447.10): 51.00% C, 4.69% H, 15.65% N, 14.31% S; found 49.72% C,
4.48% H, 15.37% N, 14.25% S; IR (ν, cm-1): 721 (CH3S); 823 (aromatic CH); 1173 (C=S); 1349
(symmetric vibrations of NO2); 1532 (asymmetric vibrations of NO2); 1643 (CO); 3181- 3301 (NH-
CO); 1H-NMR δ: 2.09 (s, 3H, CH3); 2.50 (m, 4H, CH2); 4.50 (t, 1H, CH); 7.19 (t, 1H, Ar); 7.29 (t, 2H,
Ar); 7.38 (d, 2H, Ar); 7.81 (t, 1H, Ar); 8.41 (d, 1H, Ar); 8.47 (d, 1H, Ar); 8.70 (s, 1H, Ar); 9.07 (s, 1H,
NH); 9.81 (s, 1H, NH); 10.10 (s, 1H, NH).
1-[N-(3-nitrobenzoyl)-D,L-methionyl]-4-(4-methylphenyl)-thiosemicarbazide (4c). Yield 78.35 %;
m.p. 112-113 ºC; Anal. Calc. for C20H23N5O4S2 (461.12): 52.06% C, 4.98% H, 15.18% N, 13.88% S;
found 51.92% C, 4.75% H, 15.01% N, 13.58% S; IR (ν, cm-1): 721 (CH3S); 819 (aromatic CH); 1173
(C=S); 1350 (symmetric vibrations of NO2); 1528 (asymmetric vibrations of NO2); 1644 (CO); 3301-
1
3366 (NH-CO); H-NMR δ: 2.11 (s, 3H, CH3); 2.43 (s, 3H, CH3); 2.50 (m, 4H, CH2); 4.50 (t, 1H,
CH); 7.80 (t, 1H, Ar); 8.12 (d, 2H, Ar); 8.32 (d, 2H, Ar); 8.39 (d, 1H, Ar); 8.41 (d, 1H, Ar); 8.74 (s,
1H, Ar); 9.07 (d, 1H, NH); 9.80 (s, 1H, NH); 10.10 (s, 1H, NH).

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1-[N-(3-nitrobenzoyl)-D,L-methionyl]-4-(4-bromophenyl)-thiosemicarbazide (4d). Yield 73.05 %;
m.p. 95-96 ºC; Anal. Calc. for C19H20N5O4S2Br (525.01): 43.34% C, 3.80% H, 13.30% N, 12.16% S;
found 43.18% C, 3.72% H, 13.05% N, 12.11% S; IR (ν, cm-1): 718 (CH3S); 819 (aromatic CH); 1171
(C=S); 1350 (symmetric vibrations of NO2); 1528 (asymmetric vibrations of NO2); 1641 (CO); 3101-
3300 (NH-CO); 1H-NMR δ: 2.11 (s, 3H, CH3); 2.50 (m, 4H, CH2); 4.50 (t, 1H, CH); 7.80 (t, 1H, Ar);
8.17 (d, 2H, Ar); 8.30 (d, 2H, Ar); 8.39 (d, 1H, Ar); 8.45 (d, 1H, Ar); 8.70 (s, 1H, Ar); 9.07 (d, 1H,
NH); 9.81 (s, 1H, NH); 10.10 (s, 1H, NH).
1-[N-(3-nitrobenzoyl)-D,L-methionyl]-4-allyl-thiosemicarbazide (4e). Yield 76.27 %; m.p. 117-118
ºC; Anal. Calc. for C16H21N5O4S2 (411.10): 46.71% C, 5.10% H, 17.03% N, 15.57% S; found 46.48%
C, 5.02% H, 16.98% N, 15.23% S; IR (ν, cm-1): 768 (CH3S); 823 (aromatic CH); 1172 (C=S); 1348
(symmetric vibrations of NO2); 1529 (asymmetric vibrations of NO2); 1644 (CO); 3082-3302 (NH-
CO); 1H-NMR δ: 1.80 (d, 2H, CH2); 2.11 (s, 3H, CH3); 2.50 (m, 4H, CH2); 4.50 (t, 1H, CH); 5.90 (t,
2H, CH2); 6.32 (m, 1H, CH); 7.80 (t, 1H, Ar); 8.34 (d, 1H, Ar); 8.47 (d, 1H, Ar); 8.74 (s, 1H, Ar); 9.07
(d, 1H, NH); 9.82 (t, 1H, NH); 10.10, 10.30 (s, 1H, NH).
General procedure for the synthesis of 1,3,4-thiadiazole compounds 5a-e.
To corresponding thiosemicarbazide 4a-e (0.006 mol) concentrated H2SO4 (1 mL) was added
under stirring. The reaction mixture was stirred at room temperature for one hour and then added
dropwise to cold water. The obtained solid was dried under vacuum at 45 ºC. The crude product was
purified by crystallization from ethanol.
2-[1-(3-nitrobenzoylamino)-3-(methylthio)]-propyl-5-(methylamino)-1,3,4-thiadiazole (5a). Yield
69.76 %; m.p. 116-117 ºC; Anal. Calc. for C14H17N5O3S2 (367.08): 45.77% C, 4.63% H, 19.07% N,
17.43% S; found 45.55% C, 4.58% H, 18.92% N, 17.21% S; IR (ν, cm-1): 725 (CH3S); 821 (aromatic
CH); 1350 (symmetric vibrations of NO2); 1433 (C=N); 1527 (asymmetric vibrations of NO2); 1645
1
(CO); 3302 (NH-CO); H-NMR δ: 2.06 (s, 3H, CH3); 2.50 (m, 4H, CH2); 2.80 (d, 3H, CH3); 4.50 (t,
1H, CH); 7.06 (m, 1H, NH); 7.80 (t, 1H, Ar); 8.30 (d, 1H, Ar); 8.40 (d, 1H, Ar); 8.70 (s, 1H, Ar); 9.07
(d, 1H, NH).
2-[1-(3-nitrobenzoylamino)-3-(methylthio)]-propyl-5-(phenylamino)-1,3,4-thiadiazole (5b). Yield
68.72 %; m.p. 105-106 ºC; Anal. Calc. for C19H19N5O3S2 (429.09): 53.11% C, 4.42% H, 16.31% N,
14.91% S; found 52.98% C, 4.38% H, 16.22% N, 14.81% S; IR (ν, cm-1): 725 (CH3S); 821 (aromatic
CH); 1350 (symmetric vibrations of NO2); 1433 (C=N); 1527 (asymmetric vibrations of NO2); 1645
(CO); 2916-3302 (NH-CO); 1H-NMR δ: 2.09 (s, 3H, CH3); 2.50 (m, 4H, CH2); 4.50 (t, 1H, CH); 7.10
(t, 1H, Ar); 7.20 (t, 2H, Ar); 7.30 (d, 2H, Ar); 7.80 (t, 1H, Ar); 8.41 (d, 1H, Ar); 8.47 (d, 1H, Ar); 8.74
(s, 1H, Ar); 9.06 (d, 1H, NH); 10.43 (s, 1H, NH).
2-[1-(3-nitrobenzoylamino)-3-(methylthio)]-propyl-5-(4-methylphenylamino)-1,3,4-thiadiazole (5c).
Yield 62.43 %; m.p. 98-99 ºC; Anal. Calc. for C20H21N5O3S2 (443.11): 54.17% C, 4.74% H, 15.80%
N, 14.44% S; found 54.12% C, 4.38% H, 15.72% N, 14.28% S; IR (ν, cm-1): 725 (CH3S); 819

Molecules 2007, 12
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(aromatic CH); 1350 (symmetric vibrations of NO2); 1433 (C=N); 1527 (asymmetric vibrations of
NO2); 1645 (CO); 3084-3302 (NH-CO); 1H-NMR δ: 2.09 (s, 3H, CH3); 2.50 (m, 4H, CH2); 4.50 (t, 1H,
CH); 7.10 (s, 3H, CH3); 7.78 (t, 1H, Ar); 7.82 (d, 2H, Ar); 7.94 (d, 2H, Ar); 8.30 (d, 1H, Ar); 8.40 (d,
1H, Ar); 8.00 (s, 1H, Ar); 9.07 (d, 1H, NH); 10.43 (s, 1H, NH).
2-[1-(3-nitrobenzoylamino)-3-(methylthio)]-propyl-5-(4-bromophenylamino)-1,3,4-thiadiazole (5d).
Yield 61.18 %; m.p. 106-107 ºC; Anal. Calc. for C19H18N5O3S2Br (507.00): 44.88% C, 3.54% H,
13.77% N, 12.59% S; found 44.81% C, 3.25% H, 13.51% N, 12.42% S; IR (ν, cm-1): 769 (CH3S); 825
(aromatic CH); 1349 (symmetric vibrations of NO2); 1432 (C=N); 1529 (asymmetric vibrations of
NO2); 1644 (CO); 3082-3302 (NH-CO); 1H-NMR δ: 2.11 (s, 3H, CH3); 2.50 (m, 4H, CH2); 4.50 (t, 1H,
CH); 7.90 (t, 1H, Ar); 8.05 (d, 2H, Ar); 8.08 (d, 2H, Ar); 8.41 (d, 1H, Ar); 8.47 (d, 1H, Ar); 8.74 (s,
1H, Ar); 9.07 (d, 1H, NH); 10.43 (s, 1H, NH).
2-[1-(3-nitrobenzoylamino)-3-(methylthio)]-propyl-5-(allylamino)-1,3,4-thiadiazole (5e). Yield 71.03
%; m.p. 112-113 ºC; Anal. Calc. for C16H19N5O3S2 (393.09): 48.85% C, 4.83% H, 17.81% N, 16.28%
S; found 48.69% C, 4.78% H, 17.62% N, 16.01% S; IR (ν, cm-1): 723 (CH3S); 819 (aromatic CH);
1350 symmetric vibrations of (NO2); 1431 (C=N); 1527 (asymmetric vibrations of NO2); 1645 (CO);
1
3082-3300 (NH-CO); H-NMR δ: 2.06 (s, 3H, CH3); 2.50 (m, 4H, CH2); 3.70 (d, 2H, CH2); 4.50 (t,
1H, CH); 5.90 (t, 2H, CH2); 6.30 (m, 1H, CH); 7.80 (t, 1H, Ar); 8.30 (d, 1H, Ar); 8.40 (d, 1H, Ar);
8.70 (s, 1H, Ar); 9.07 (d, 1H, NH); 10.42 (t, 1H, NH).
General procedure for the synthesis of 1,2,4-triazole compounds 6a-c.
To corresponding thiosemicarbazide 4a-e (0.0014 mol) a solution of NaOH 2N (10 mL) was
added. The reaction mixture was heated at 80 °C for one hour, diluted with water (1:1) and then a
solution of HCl 1N was added till pH 4.5. The obtained solids were dried at 45 ºC and then
recristalized from ethanol.
3-mercapto-4-methyl-5-[1-(3-nitrobenzoylamino)-3-methylthio]-propyl-1,2,4-triazole (6a). Yield
62.43 %; m.p. 280-281 ºC; Anal. Calc. for C14H17N5O3S2 (367.08): 45.77% C, 4.63% H, 19.07% N,
17.43% S; found 45.62% C, 4.48% H, 18.93% N, 17.21% S; IR (ν, cm-1): 686 (CH3S); 866 (aromatic
CH); 1350 (symmetric vibrations of NO2); 1436 (C=N); 1580 (asymmetric vibrations of NO2); 1648
(CO); 2592 (SH); 2974-3271 (NH-CO); 1H-NMR δ: 2.11 (s, 3H, CH3); 2.50 (m, 4H, CH2); 2.80 (s, 3H,
CH3); 4.50 (t, 1H, CH); 7.80 (t, 1H, Ar); 8.41 (d, 1H, Ar); 8.47 (d, 1H, Ar); 8.74 (s, 1H, Ar); 9.05 (d,
1H, NH); 12.60 (s, 1H, SH).
3-mercapto-4-phenyl-5-[1-(3-nitrobenzoylamino)-3-methylthio]-propyl-1,2,4-triazole (6b). Yield
60.31 %; m.p. 233-234 ºC; Anal. Calc. for C19H19N5O3S2 (429.09): 53.14% C, 4.42% H, 16.31% N,
14.91% S; found 53.02% C, 4.16% H, 16.28% N, 14.85% S; IR (ν, cm-1): 684 (CH3S); 866 (aromatic
CH); 1348 (symmetric vibrations of NO2); 1440 (C=N); 1550 (asymmetric vibrations of NO2); 1691
(CO); 2589 (SH); 2981-3462 (NH-CO); 1H-NMR δ: 2.08 (s, 3H, CH3); 2.50 (m, 4H, CH2); 4.50 (t, 1H,

Molecules 2007, 12
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CH); 7.18 (t, 1H, Ar); 7.27 (t, 2H, Ar); 7.38 (d, 2H, Ar); 7.80 (t, 1H, Ar); 8.30 (d, 1H, Ar); 8.40 (d, 1H,
Ar); 8.70 (s, 1H, Ar); 9.07 (d, 1H, NH); 12.80 (s, 1H, SH).
3-mercapto-4-(4-methylphenyl)-5-[1-(3-nitrobenzoylamino)-3-methylthio]-propyl-1,2,4-triazole (6c).
Yield 68.38 %; m.p. 285-286 ºC; Anal. Calc. for C20H19N5O3S2 (443.11): 54.17% C, 4.74% H, 15.80%
N, 14.44% S; found 54.03% C, 4.58% H, 15.52% N, 14.39% S; IR (ν, cm-1): 686 (CH3S); 866
(aromatic CH); 1350 (symmetric vibrations of NO2); 1450 (C=N); 1580 (asymmetric vibrations of
NO2); 1698 (CO); 259 (SH); 2976-3486 (NH-CO); 1H-NMR δ: 2.06 (s, 3H, CH3); 2.50 (m, 4H, CH2);
4.50 (t, 1H, CH); 7.10 (s, 3H, CH3); 7.80 (t, 1H, Ar); 7.90 (d, 2H, Ar); 8.08 (d, 2H, Ar); 8.41 (d, 1H,
Ar); 8.47 (d, 1H, Ar); 8.74 (s, 1H, Ar); 9.07 (d, 1H, NH); 12.70 (s, 1H, SH).
Antimicrobial activity assessment
The test microorganisms used to evaluate the potential antimicrobial activity of the new
synthesized compounds were: Staphylococcus aureus ATCC 25923, Bacillus antracis ATCC 8705,
Bacillus cereus ATCC 10987, Sarcina lutea ATCC 9341 and Escherichia coli ATCC 25922. All the
new compounds were weighed and dissolved in dimethylsulphoxide (DMSO) to prepare an extract
stock solution of 100 mg/mL. The antimicrobial effects of the substances were quantitatively tested in
the respective broth media by using double dilution and the Minimal Inhibitory Concentration (MIC)
values (µg/mL) were determined [17]. The antibacterial assays were performed in Mueller-Hinton
broth (MH) at pH 7.3. The MIC was defined as the lowest concentration that showed no growth.
Dimethylsulfoxide (DMSO) with dilution of 1:10 was used as solvent control.
Toxicity study
The acute toxicity was estimated by intraperitoneal administration of the compounds as a
suspension in Tween 80 to groups of fourteen mice, each weighting 20-25 g, according to the classical
laboratory methodology [18]. The animals were observed and the death rate ascertained after 7 days.
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Sample Availability: Samples of the compounds 5a-e and 6a-c are available from authors.
© 2007 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.