Synthesis and biological activity of some new 1,3,4-thiadiazole and 1,2,4-triazole compounds containing a phenylalanine moiety

Article abstract of DOI:10.3390/molecules14072621

New 1,3,4-thiadiazole, 6, 7 and 1,2,4-triazole derivatives, 8, 9 containing a phenylalanine moiety have been synthesized by intramolecular cyclization of 1,4- disubstituted thiosemicarbazides, 4, 5, in acid and alkaline media, respectively; the thiosemica

Full text of DOI:10.3390/molecules14072621

Molecules 2009, 14, 2621-2631; doi:10.3390/molecules14072621
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Activity of Some New 1,3,4-Thiadiazole
and 1,2,4-Triazole Compounds Containing a Phenylalanine
Moiety
Mihaela Moise 1, Valeriu Sunel 1, Lenuta Profire 2,*, Marcel Popa 3, Jacques Desbrieres 4 and
Cristian Peptu 5
1
Department of Organic Chemistry and Biochemistry, Faculty of Chemistry, “Al. I. Cuza”
University, Iasi, 11 B-dul Carol I, 700506, Romania;
E-mails: moise_mihaela82@yahoo.com (M.M.), vsunel@uaic.ro (V.S.)
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Gr. T. Popa” Medicine and
Pharmacy University, 16 Universitatii Street, 700115, Romania
3
Department of Natural and Synthetic Polymers, Faculty of Chemical Engineering and
Environmental Protection, “Gh. Asachi” Technical University, 71A B-dul Mangeron, Iasi, 700050,
Romania; E-mail: marpopa@ch.tuiasi.ro (M.P.)
4
Pau et Pays de l’Adour University, Institut Pluridisciplinaire de Recherche sur l’Environnement et
les Matériaux – Equipe de Physique et Chimie des Polymèrs (IPREM/EPCP), UMR 5254 CNRS,
Hélioparc Pau Pyrénées 2, av. President Angot, 64053 Pau Cedex 09, France;
E-mail: jacques_desbrieres@univ-pau.fr (J.D.)
5
“Petru Poni" Institute of Macromolecular Chemistry, 41A Grigore Ghica Voda Alley 700487 Iasi,
Romania
* Author to whom correspondence should be addressed; E-mail: nprofire@yahoo.com
Received: 11 May 2009; in revised form: 24 June 2009 / Accepted: 8 July 2009 /
Published: 16 July 2009
Abstract: New 1,3,4-thiadiazole, 6, 7 and 1,2,4-triazole derivatives, 8, 9 containing a
phenylalanine moiety have been synthesized by intramolecular cyclization of 1,4-
disubstituted thiosemicarbazides, 4, 5, in acid and alkaline media, respectively; the
thiosemicarbazides were obtained by reaction of hydrazide 3 with appropriate aromatic
isothiocyanates. The toxicity of the synthesized compounds was evaluated and the anti-
inflammatory study of the triazole compound 9 established an appreciable anti-
inflammatory activity that is comparable with that of other nonsteroidal anti-inflammatory
agents.

Molecules 2009, 14
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Keywords: phenylalanine; thiosemicarbazide; 1,3,4-thiadiazole; 1,2,4-triazole;
anti-inflammatory activity
1. Introduction
The progress achieved in the synthesis of heterocyclic compounds with biological potential is due
to improvement of the methodological study of tested substances too. It is known that many 1,3,4-
thiadiazole and 1,2,4-triazole derivatives have biological activity, with their antibacterial [1-3],
antimycobacterial [4,5], antimycotic [6], antifungal [7,8], antidepressive [9], and cardiotonic [10]
action being notable. Recent research has also established for these heterocycles an analgesic [11] and
anti-inflammatory [12,13] activity. Meanwhile, N-acylated amino acids are known for their
hepatoprotective [14], antimicrobial [15,16] and antitumoral [17,18] action. Taking these data into
account, in the present study, some new 1,3,4-thiadiazole and 1,2,4-triazole derivatives having a
phenylalanine moiety have been synthesized and their structure confirmed by elemental and spectral
1
(FT-IR, H-NMR, MS) analyses. The degree of toxicity of the compounds was established and the
potential anti-inflammatory activity of the triazole compound 9 was also investigated.
2. Results and Discussion
2.1. Chemistry
The synthesis of new 1,3,4-thiadiazole and 1,2,4-triazole compounds was performed in several
steps. In the first step, 2-(p-nitrophenyl)-4-benzyl-Δ2-oxazolin-5-one (2) was obtained by heating N-(p-
nitrobenzoyl)-L-phenylalanine (1) at 70-75°C for 60 minutes under the dehydrating action of excess
acetic anhydride (1:10 molar rate) (Scheme 1).
Scheme 1. Synthesis of oxazolone 2.
H
C
O
H
C
C6H5 H2C
COOH
C6H5 CH2
NH
O
(CH3CO)2O
- 2 CH3COOH
N C
C O
NO2
2
NO2
1
1
The structure of oxazolone 2 was established through by spectroscopic (IR, H-NMR, MS) as well
as elemental analyses data. In the IR spectra the absorption bands characteristic for the >C=O lactonic
group and >C=N bond were identified at 1,825 cm-1 and 1,620 cm-1, respectively, and in the 1H-NMR
spectrum the proton signals due to the heterocyclic proton appeared at 4.85 ppm. In the MS spectra the

Molecules 2009, 14
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base peak was observed at m/z = 297.09 and it was assigned as protonated compound having the
monoisotopic mass 296.09. The [M+Na]+ species at m/z = 319.07 and Na adducts of two oxazolone
molecules were also detected. The accurate mass measurements allowed the evaluation of the isotopic
profile for the peaks situated at 297.09, 298.09 and 299.09.
By reaction of Δ2-oxazolin-5-one 2 with hydrazine hydrate (98% solution) in anhydrous dioxane
under reflux for 3 hours, N-(p-nitrobenzoyl)-D,L-phenylalanine hydrazide (3) was obtained
(Scheme 2).
Scheme 2. Synthesis of hydrazide 3.
H
O
C6H5 CH2
CO NH NH2
C
H
C
C6H5 CH2
NH
CO
NH2 NH2
98%
O
N C
NO2
3
NO2
2
1
The structure of hydrazide 3 was established through by spectroscopic (IR, H-NMR) as well as
elemental analyses data. The IR spectra showed two characteristic bands for NH groups at 2,857 cm-1
and 3,269 cm-1, respectively, and the absorption band for CO amide group appeared at 1,661 cm-1. In
1
the H-NMR spectrum the NH groups appeared as two signals at 9.0 ppm and 10.40 ppm, while the
proton signals of the NH2 group were identified at 5.37 ppm.
The new 1,4-disubstituted thiosemicarbazides 4, 5 were obtained through reaction of hydrazide 3
with p-bromophenyl- and p-tolylisothiocyanates in methanol media under reflux (Scheme 3).
Scheme 3. Synthesis of thiosemicarbazides 4, 5.
H
OC
HN C NH R
S
NH
C6H5 CH2 CH
NH
C6H5 CH2 C CO NH NH2
NH
CO
R N C S
CO
4: R = -C6H4-Br (p)
5: R= -C6H4-CH3 (p)
NO2
NO2
4, 5
3
The formation of thiosemicarbazides 4, 5 was indicated in the IR spectra by a shift in the CO amide
group absorption of hydrazide 3 from 1,661 cm-1 to 1,654-1,683 cm-1 in the thiosemicarbazide

Molecules 2009, 14
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1
derivatives and by the band of >C=S bond that appears at 1,234-1,299 cm-1. In the H-NMR spectra
the protons linked to nitrogen appear at 9.00-10.48 ppm. In the MS spectra of thiosemicarbazide 4 the
base peak was observed at m/z = 564.02 and it was assigned as its Na adduct (564 = 541 +23). Beside
the Na adduct there were observed (M+H)+ and (M+K)+ adducts at m/z = 542 and 580, respectively.
Two cluster molecules charged with Na were also observed at m/z = 1105 (541×2 + 23). For the MS
spectra of thiosemicarbazide 5 the base peak was observed at m/z = 500.13 and it is associated with the
presence of the Na adduct of this thiosemicarbazide (477+23). The peaks accompanying the base peak
are (M+H)+ at m/z = 478 and (M+K)+ at 516. An additional peak at m/z = 977 representing the Na
adduct of two thiosemicarbazide molecules (977=477×2+23) was also observed.
In the last step by intramolecular cyclization of thiosemicarbazides 4, 5 in acid and alkaline media,
respectively, new 1,3,4-thiadiazole and 1,2,4-triazole compounds 6, 7 and 8, 9 were obtained (Scheme
4). The 1,2,4-triazole derivatives can exist in two tautomeric forms – thiole (I) and thione (II), the
reaction conditions leading to the chemical stabilization of the tautomeric thione form [19, 20].
Scheme 4. Synthesis of 1,3,4-thiadiazole (6, 7) and 1,2,4-triazole (8, 9) compounds.
N
N
OC
HN C NH R
S
NH
C6H5 CH2 CH
NH
CO
C6H5 CH2 CH
NH
NH R
S
H2SO4
- H2O
CO
C6H4 NO2 (p)
NO2 (p)
6, 7
C6H4
4, 5
- + NaOH
H2O
N
NH
N
N
C6H5 CH2 CH
S
C6H5 CH2 CH
N
SH
N
R
NH
CO
NH
CO
R
C6H4 NO2 (p)
(II)
C6H4 NO2 (p)
(I)
8, 9
6, 8: R = -C6H4-Br (p)
7, 9: R= -C6H4-CH3 (p)
1
The structure of the final compounds was confirmed by elemental and spectral (FT-IR, H-NMR
and MS) analyses. In the IR spectra of the 1,3,4-thiadiazoles 6, 7 the >C=N group appears as an
absorption band at 1,665-1,672 cm-1, and the C-S bond was identified at 640-652 cm-1. In the 1H-NMR
spectra of 1,3,4-thiadiazoles 6, 7 the proton signal for the amine group appears as singlet at
6.00-6.05 ppm and that for the amide group appears at 9.08-9.10 ppm. The CH3 group is present as a
singlet at 2.34 ppm. In the mass spectrum of thiadiazole 6 the base peak was observed at
m/z = 524, corresponding to the [M-H]+ ions of the product. The base peak for thiadiazole 7 is situated
at m/z = 460.13 corresponding to the [M-H]+ ions of the product.

Molecules 2009, 14
2625
In the IR spectra of 1,2,4-triazoles 8, 9 the >C=N group was identified at 1,566-1,595 cm-1 and the
1
characteristic C=S function absorption band appeared at 1,300-1,320 cm-1 region. In the H-NMR
spectra the proton signal for the NH from the triazole ring was observed as a singlet at 12.80 ppm. The
mass spectra of triazole 8 showed the base peak at m/z = 524.03 corresponding to the [M-H]+ ions of
the product with monoisotopic mass of 523.03 g/mol. The smaller peaks are assigned as Na and K
adducts of the same molecule or species adducts formed by physical association of two molecules (e.g.
1069.01 = 2×523+23). The base peak observed in the case of triazole 9 is situated at m/z = 460.13
corresponding to the [M-H]+ ion of the product.
2.2. Biological Activities
The synthesized compounds were investigated for their toxicity (Table 1). It was ascertained that all
tested compounds have low toxicity, the lowest toxic compounds being 1,3,4-thiadiazole 7 and 1,2,4-
thiazole 9, both having a p-tolyl moiety in the structure.
Table 1. DL50 values of the tested compounds.
DL50 (mg/kg body weight)
Comp.
24 hours
1,110
1,100
1,650
2,050
2,350
5,200
2,250
5,150
48 hours
1,110
1,100
1,650
2,050
2,350
5,200
2,250
5,150
7 days
975
Average
1,065
1,025
1,620
1,970
2,270
4,620
2,150
5,010
2
3
4
5
6
7
8
9
875
1,560
1,810
2,110
3,460
1,950
4,730
The potential anti-inflammatory activity of the 1,2,4-triazole (9) comparative with standard
nonsteroidal anti-inflammatory drugs (indometacine, phenylbutazone, acetylsalicylic acid) was also
investigated (Table 2).
The substances were administered in two concentrations, 1/10 of DL50 and 1/5 of DL50,
respectively, relative to body weight of the animal tested. From the results presented in Table 2 it is
observed that at small doses (1/10 DL50) only indometacine has an appreciable anti-inflammatory
activity (60.30% inhibition of oedema) while at higher doses (1/5 DL50) all tested compounds reduced
the nystatin-induced oedema (45.33-61.20% inhibition of oedema). The activity of the 1,2,4-triazole
(9) is similar to the phenylbutazone’s activity (49.03% inhibition of oedema). This effect could be
explained through both the lysosomal stabilization and the inhibition of prostaglandin biosynthesis
[21,22,23].

Molecules 2009, 14
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Table 2. The anti-inflammatory effects of tested compounds.
Dose (mg/kg
body, p.o.)
5*
Inflammatory oedema (mL/100 g body) Inhibition
Comp.
(%)
Control
Comp.
Indometacine
0.280±0.110
0.310±0.005
0.280±0.110
0.310±0.005
0.310±0.005
0.300±0.100
0.280±0.110
0.310±0.095
0.111±0.008
0.124±0.070
0.190±0.062
0.158±0.025
0.250±0.057
0.164±0.072
0.186± 0.060
0.158±0.024
60.30
61.20
32.10
49.03
19.35
45.33
33.57
49.03
10**
50*
100**
150 *
250**
80*
Phenylbutazone
Acetylsalicylic
Acid
1,2,4-Triazole
(9)
160**
* = 1/10 DL50, **1/5DL50
3. Experimental Section
3.1. Chemistry
3.1.1. 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 FT-IR Spectrophotometer; the wave numbers are given in cm-1. The 1H-NMR
spectra were recorded in DMSO-d6 or CD3COCD3 solutions on Bruker ARX-300 spectrometer at
ambient temperature. Chemical shifts were recorded as δ values in parts per millions (ppm) and were
1
indirectly referenced to tetramethylsilane via residual solvent signal (2.49 for H). MS spectra were
obtained using an instrument produced by Agilent Technologies, Wilmington, DE, USA. The
instrument, Accurate Mass Q-TOF LC/MS 6520 was operated via the manufacture’s software, Mass
Hunter. Samples were dissolved in acetonitrile/water mixture (95/5 v/v) to obtain a concentration of
10 μg/mL and 0.05 mL were directly injected into the electrospray source using the autosampler at a
rate of 0.05 mL/min. The instrument was operated in High resolution mode with an acquisition rate of
4 GHz. The source voltage was set at 4,000 V, the spray gas flow at 5 L/min, heating gas temperature
at 325 °C and the fragmentor potential at 215 V. All chemical reagents were obtained from the Aldrich
Chemical Company.
3.1.2. Synthesis of 2-(p-nitrophenyl)-4-benzyl-Δ2-oxazolin-5-one (2)
N-(p-nitrobenzoyl)-L-phenylalanine (5.32 g, 0.018 mol) was dissolved in acetic anhydride (18 mL)
and heated under reflux at 70-75ºC for 45-60 minutes. After cooling, the solution was added under
stirring over a mixture of dried petroleum ether (55 mL) and dried ethyl ether (35 mL). The reaction
mixture was stirred for 15 minutes, the ether layer was removed and the oily product obtained was
washed many times with dried ethyl ether until a solid product was obtained that was filtered and dried
for 8-10 hours under vacuum at 40-45 °C. Crystallization from anhydrous dioxane gave a pure yellow
solid (383 mg, 72% yield), mp 120-123 °C; IR (cm-1) ν: 1,825 (CO), 1,620 (C=N), 1,520 (asymmetric

Molecules 2009, 14
2627
1
NO2 vibrations), 1,342 (symmetric NO2 vibrations), 786 (aromatic CH); H-NMR (CD3COCD3) δ:
3.15 (m, 1H, CH2), 3.25 (m, 1H, CH2), 4.85 (t, 1H, CH), 7.20 (m, 5H, Ph), 8.15 (d, 2H, C6H4), 8.35 (d,
2H, C6H4); MS (acetonitrile/water 95/5, v/v) m/z: 297.09 (base peak), 319.07 (14.5%), 615.11 (21%);
Anal. calcd. for C16H12N2O4 (%): C, 64.86; H, 4.05; N, 9.45; found: C, 65.32; H, 4.26; N, 9.91.
3.1.3. N-(p-nitrobenzoyl)-D,L-phenylalanine hydrazide (3)
2-(p-Nitrophenyl)-4-benzyl-Δ2-oxazolin-5-one (2, 5.94 g, 0.02 mol) was dissolved in dioxane
(50 mL) and hydrazine hydrate solution (98%, 0.5 mL, 0.02 mol) was added. The reaction mixture was
heated at 65-70ºC for three hours, under reflux on a thermostated silicone oil bath. The solvent was
removed by distillation under reduced pressure till 10-15 mL, and then water was added until a yellow
semisolid product was separated. Crystallization from ethanol-water mixture gave a pure yellow solid
(514 mg, 78.35% yield), mp 210-212 °C; IR (cm-1) ν: 3,269, 2,857 (NH), 1,661 (CO), 1,526
1
(asymmetric NO2 vibrations), 1,346 (symmetric NO2 vibrations), 723, 868 (aromatic CH); H-NMR
(DMSO-d6, 300 MHz) δ: 3.30 (m, 2H, CH2), 4.80 (m, 1H, CH), 5.37 (s, 2H, NH2), 7.40 (m, 5H, Ph),
8.05 (d, 2H, C6H4), 8.30 (d, 2H, C6H4), 9.0 (m, 1H, NH), 10.40 (s, 1H, NH); Anal. calcd. for
C16H16N4O4 (%): C, 58.53; H, 4.87; N, 17.07; found: C, 58.75; H, 5.33; N, 17.46.
3.1.4. General procedure for synthesis of 1,4-disubstituted thiosemicarbazides 4, 5
N-(p-nitrobenzoyl)-D,L-phenylalanine hydrazide (3, 1.64 g, 0.005 mol) was dissolved in dried
methanol (10 mL) and a solution of the corresponding isothiocyanate (0.005 mol) in dried methanol
(10 mL) was added. The reaction mixture was heated under reflux at 70-80ºC for three hours. After
cooling the solvent was evaporated under reduced pressure and the solid was dried under vacuum at
room temperature. The rough product was purified by crystallization from ethanol.
1-[N-(p-nitrobenzoyl)-D,L-phenylalanyl-4-(p-bromophenyl)-thiosemicarbazide (4): white solid, 184
mg (68.33% yield); mp 188-190°C; IR (cm-1) ν: 3,541, 3,460, 3,169 (NH), 1,683 (CO), 1,573
(asymmetric NO2 vibrations), 1,379 (symmetric NO2 vibrations), 1,299 (C=S), 754, 796 (aromatic
1
CH), 565 (C-Br); H-NMR (DMSO-d6, 300 MHz) δ: 3.04 (m, 1H, CH2), 3.10 (m, 1H, CH2), 4.70 (m,
1H, CH), 7.17-7.33 (m, 6H, Ar CH), 7.56 (m, 3H, Ar CH), 8.01 (d, 2H, Ar CH), 8.31 (m, 2H, Ar CH),
9.0 (d, 1H, NH), 9.91 (s, 1H, NH), 10.48 (s, 1H, NH); MS (acetonitrile/water 95/5, v/v) m/z: 564 (base
peak) (541+23), 542 (541+1) (30%), 580 (541+39) (19%), 1105 (541×2+23) (4%); Anal. calcd. for
C23H20N5O4BrS (%): C, 51.01; H, 3.69; N, 12.93; Br, 14.78; S, 5.91; found: C, 51.37; H, 3.88; N,
13.35; Br, 15.19; S, 6.27.
1-[N-(p-nitrobenzoyl)-D,L-phenylalanyl-4-(p-tolyl)-thiosemicarbazide (5): yellow solid, 186 mg
(78.35% yield), mp 179-180°C; IR (cm-1) ν: 3,321, 3,211 (NH), 1,654 (CO), 1558 (asymmetric NO2
vibrations), 1,357 (symmetric NO2 vibrations), 1,234 (C=S), 873 (aromatic CH); 1H-NMR (DMSO-d6,
300 MHz) δ: 2.29 (s, 3H, CH3), 3.40 (m, 1H, CH2), 4.70 (m, 1H, CH), 7.14-7.50 (m, 8H, Ar CH), 7.95
(s, 1H, Ar CH), 8.01 (d, 2H, Ar CH), 8.30 (d, 2H, Ar CH), 9.19 (s, 2H, NH), 9.71 (s, 1H, NH), 10.44
(s, 1H, NH); MS (acetonitrile/water 95/5, v/v) m/z: 500.13 (base peak) (477+23), 478.15 (477+1)

Molecules 2009, 14
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(39.5%), 516.11 (477+39) (13%), 977 (477×2+23) (30%). Anal. calcd. for C24H23N5O4S (%): C,
60.37; H, 4.82; N, 14.67; S, 6.70; found: C, 60.81; H, 5.01; N, 15.10; S, 7.19.
3.1.5. General procedure for synthesis of 1,3,4-thiadiazoles 6, 7
To the corresponding thiosemicarbazide 4, 5 (0.006 mol), concentrated H2SO4 (1 mL) was added
under stirring. The reaction mixture was stirred at room temperature for one hour and then was added
dropwise in cold water and stirred again till a solid product was obtained that was separated and dried
under vacuum at 45-50ºC. The rough product was purified by crystallization from ethanol.
2-[1-(p-Nitrobenzoylamino)-2-phenyl]-ethyl-5-(p-bromophenylamino)-1,3,4-thiadiazole (6): light
yellow solid, 223 mg (71.50% yield), mp 151-153°C; IR (cm-1) ν: 3,263, 3,066 (NH), 1,672 (C=N),
1,620 (CO), 1,520 (asymmetric NO2 vibrations), 1,341 (symmetric NO2 vibrations), 812 (aromatic
1
CH), 750 (C-Br), 640 (C-S); H-NMR (DMSO-d6, 300 MHz) δ: 2.60 – 2.65 (m, 2 H, CH2), 4.76 (m,
1H, NH), 6.00 (m, 1H, CH), 6.56 (d, 2H, ArCH), 7.63 – 7.78 (m, 5H, ArCH), 8.30 (d, 2H, ArCH), 8.40
(d, 2H, ArCH), 8.80 (d, 2H, ArCH), 9.10 (s, 1H, -NH-CO-); MS (acetonitrile/water 95/5, v/v) m/z: 524
(base peak), 1047 (1.5%); Anal. calcd. for C23H18N5O3BrS (%): C, 52.67; H, 3.43; N, 13.35; Br, 15.26;
S, 6.10; found: C, 53.02; H, 3.89; N, 13.84; Br, 15.67; S, 6.58.
2-[1-(p-Nitrobenzoylamino)-2-phenyl]-ethyl-5-(p-tolylamino)-1,3,4-thiadiazole (7): yellow solid, 195
mg (71.27% yield), mp 180-182°C; IR (cm-1) ν: 3,259, 3,192 (NH), 1,665 (C=N), 1,620 (CO), 1,552
1
(asymmetric NO2 vibrations), 1,384 (symmetric NO2 vibrations), 815 (aromatic CH), 652 (C-S); H-
NMR (DMSO-d6, 300 MHz) δ: 2.34 (s, 3H, CH3), 2.60 – 2.63 (m, 2H, CH2), 4.76 (m, 1H, NH), 6.05
(m, 1H, CH), 6.58 (d, 2H, ArCH), 7.63-7.80 (m, 5H, ArCH), 8.33 (d, 2H, ArCH), 8.45 (d, 2H, ArCH),
8.75 (d, 2H, ArCH), 9.08 (s, 1H, -NH-CO-); MS (acetonitrile/water 95/5, v/v) m/z: 460.13 (base peak),
482.02 (1%), 919.14 (2%), 941.12 (1%); Anal. calcd. for C24H21N5O3S (%): C, 62.74; H, 4.57; N,
15.25; S, 6.97; found: C, 63.22; H, 4.91; N, 15.70; S, 7.47.
3.1.6. General procedure for the synthesis of 1,2,4-triazoles 8, 9
To corresponding thiosemicarbazide 6, 7 (0.0014 mol), a solution of NaOH 2N (10 mL) was added.
The reaction mixture was heated under reflux at 80-90 °C for four hours and then a solution of HCl 1N
was added until it reached pH 4.5 when a solid product was formed. The rough product was separated
and dried under vacuum at 55-60ºC and then it was recrystallized from ethanol.
4-(p-bromophenyl)-5-[1-(p-nitrobenzoylamino)-2-phenyl-ethyl]-3-thio-1,2,4-triazole (8): yellow-
orange solid, 46 mg (64.45% yield), mp 147-149°C; IR (cm-1) ν: 3,062 (NH), 1,664 (CO), 1,595
(C=N), 1,546 (asymmetric NO2 vibrations), 1,300 (C=S), 1,332 (symmetric NO2 vibrations), 925, 873
(aromatic CH), 752 (C-Br); 1H-NMR (DMSO-d6, 300 MHz) δ: 2.62 – 2.66 (m, 2H, CH2), 4.86 (m, 1H,
CH), 7.25 – 7.30 (m, 5H, ArCH), 7.80 (d, 2H, ArCH), 8.39 ( d, 2H, ArCH), 8.50 (d, 2H, ArCH), 8.75
(d, 2H, ArCH), 9.05 (m, 1H, -NH-CO-), 12.80 (s, 1H, NH); MS (acetonitrile/water 95/5, v/v) m/z:
524.03 (base peak), 541.94 (33%), 563.92 (25%), 1069.01 (9%); Anal. calcd. for C23H18N5O3BrS (%):
C, 52.67; H, 3.43; N, 13.35; Br, 15.26; S, 6.10; found: C, 53.08; H, 3.85; N, 13.85; Br, 15.26; S, 6.49.

Molecules 2009, 14
2629
5-[1-(p-nitrobenzoylamino)-2-phenyl-ethyl]-3-thio-4-(p-tolyl)-1,2,4-triazole (9): brown solid, 43 mg
(69% yield), mp 172-174°C; IR (cm-1) ν: 3,437 (NH), 1,670 (CO), 1,566 (C=N), 1,520 (asymmetric
1
NO2 vibrations), 1,320 (C=S), 1,338 (symmetric NO2 vibrations), 962, 785 (aromatic CH); H-NMR
(DMSO-d6, 300 MHz) δ: 2,32 (t, 3H, CH3), 2.57 – 3.35 (m, 2H, CH2), 7.14 – 7.19 (m, 4H, ArCH),
7.78 – 7.82 (m, 5 H, ArCH), 8.45 (d, 2H, ArCH), 8.75 (d, 2H, ArCH), 9.08 (s, 1H, -NH-CO-), 12.80 (s,
1H, NH); MS (acetonitrile/water 95/5, v/v) m/z: 460.13 (base peak), 482.04 (18%), 941.13 (11%).
Anal. calcd. for C24H21N5O3S (%): C, 62.74; H, 4.57; N, 15.25; S, 6.97; found: C, 63.16; H, 5.06; N,
15.62; S, 7.41.
3.2. Toxicity study
The acute toxicity was estimated by intraperitoneal administration of the compounds 2-9 as a
suspension in Tween 80 to groups of six male mice, each weighing 20-22 g, according to the classical
laboratory methodology [24]. The animals were monitored and the death rate ascertained after
24 hours, 48 hours and 7 days. The DL50 was established using the Spearman-Karber method [25].
3.3. Anti-inflammatoty study
The nystatin-induced paw oedema in rats was realized according to the Niemegeers et al. method
[24]. A group of six rats weighing 120-160 g housed at 21.5 °C with free-food and water for 24 hours
was used. After the measurement of the initial volume of paw according to the Whitehouse method
[19], the animals were injected in their left posterior paw with 0.1 mL nystatin suspension
(3,500 U/mg) with concentration of 65 mg/mL in 0.9% NaCl solution. Two hours after injection, the
triazole compound 9 and the standard anti-inflammatory drugs (indometacine, phenylbutazone and
acetylsalicylic acid) were administered through tube-feeding, in 1/10 and 1/5 of DL50 dose as a
suspension in CMC 0.5%, 20 ml/kg body. The control groups received only vehicle. The final volume
of each paw was measured four hours later, and the difference between the initial and the final paw
volume was established. This difference, expressed in mL/100 g body, represents the inflammatory
oedema; the mean paw volume in the drug-treated and control groups respectively was also
established. The percentage oedema was calculated, using student’s – t test for statistic interpretation.
4. Conclusions
New 1,3,4-thiadiazole (6-7) and 1,2,4-triazole (8-9) compounds having a D,L-phenylalanine moiety
were synthesized by intramolecular cyclization of 1,4-disubstituted thiosemicarbazide in acid and
alkaline medium, respectively. The corresponding thiosemicarbazides (4, 5) were obtained by addition
of N-(p-nitrobenzoyl)-D,L-phenylalanine hydrazide to the aromatic isothiocyanates. The DL50 values
for all synthesized compounds were established, all compounds having a low toxicity. The potential
anti-inflammatory effects of the thiazole 9 by using the nystatin-induced paw oedema in rats was also
studied. The triazole 9, at a dose of 160 mg/kg body (1/5 DL50), reduced the inflammatory oedema
considerably, this action being comparable with that of the tested standard anti-inflammatory drugs.

Molecules 2009, 14
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Sample Availability: Samples of the compounds 2-9 are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).