Synthesis and biological activity of substituted urea and thiourea derivatives containing 1,2,4-triazole moieties

Article abstract of DOI:10.3390/molecules18033562

A series of novel thiourea and urea derivatives containing 1,2,4-triazole moieties were synthesized and evaluated for their antifungal and larvicidal activity. Triazole derivatives 3a-e and 4a-e were synthesized by reacting thiocarbohydrazide with thiourea and urea compounds 1a-e and 2a-e, respectively, in a 130-140 °C oil bath. The proposed structures of all the synthesized compounds were confirmed using elemental analysis, UV, IR, 1H-NMR and mass spectroscopy. All compounds were evaluated for antifungal activity against plant pathogens, larvicidal and biting deterrent activity against the mosquito Aedes aegypti L. and in vitro cytotoxicity and anti-inflammatory activity against some human cell lines. Phomopis species were the most sensitive fungi to these compounds. Compounds 1b, 1c, 3a and 4e demonstrated selectively good activity against Phomopis obscurans and only 1b and 4e showed a similar level of activity against P. viticola. Compound 3d, with a LD₅₀ value of 67.9 ppm, followed by 1c (LD₅₀ = 118.8 ppm) and 3e (LD₅₀ = 165.6 ppm), showed the highest toxicity against Aedes aegypti larvae. Four of these compounds showed biting deterrent activity greater than solvent control, with the highest activity being seen for 1c, with a proportion not biting (PNB) value of 0.75, followed by 1e, 2b and 1a. No cytotoxicity was observed against the tested human cancer cell lines. No anti-inflammatory activity was observed against NF-κB dependent transcription induced by phorbol myristate acetate (PMA) in human chondrosarcoma cells.

Full text of DOI:10.3390/molecules18033562

Molecules 2013, 18, 3562-3576; doi:10.3390/molecules18033562
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Activity of Substituted Urea and
Thiourea Derivatives Containing 1,2,4-Triazole Moieties
Bedia Kocyigit-Kaymakcioglu 1,*, Ahmet Ozgur Celen 1, Nurhayat Tabanca 2, Abbas Ali 2,
Shabana I. Khan 2,3, Ikhlas A. Khan 2,3 and David E. Wedge 4
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University,
Haydarpasa 34668, Istanbul, Turkey; E-Mail: ahmozgcelen@hotmail.com
2
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences,
The University of Mississippi, University, MS 38677, USA;
E-Mails: ntabanca@olemiss.edu (N.T.); aali@olemiss.edu (A.A.); ikhan@olemiss.edu (S.I.K.);
skhan@olemiss.edu (I.A.K.)
3
Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, University,
MS 38677, USA
4
Natural Products Utilization Research Unit, Agricultural Research Service,
United States Department of Agriculture, University, MS 38667, USA;
E-Mail: dwedge@olemiss.edu
* Author to whom correspondence should be addressed; E-Mail: bkaymakcioglu@marmara.edu.tr.
Received: 26 December 2012; in revised form: 27 February 2013 / Accepted: 11 March 2013 /
Published: 19 March 2013
Abstract: A series of novel thiourea and urea derivatives containing 1,2,4-triazole moieties
were synthesized and evaluated for their antifungal and larvicidal activity. Triazole
derivatives 3a–e and 4a–e were synthesized by reacting thiocarbohydrazide with thiourea
and urea compounds 1a–e and 2a–e, respectively, in a 130–140 °C oil bath. The proposed
structures of all the synthesized compounds were confirmed using elemental analysis, UV,
IR, 1H-NMR and mass spectroscopy. All compounds were evaluated for antifungal activity
against plant pathogens, larvicidal and biting deterrent activity against the mosquito
Aedes aegypti L. and in vitro cytotoxicity and anti-inflammatory activity against some
human cell lines. Phomopis species were the most sensitive fungi to these compounds.
Compounds 1b, 1c, 3a and 4e demonstrated selectively good activity against
Phomopis obscurans and only 1b and 4e showed a similar level of activity against
P. viticola. Compound 3d, with a LD50 value of 67.9 ppm, followed by 1c
(LD50 = 118.8 ppm) and 3e (LD50 = 165.6 ppm), showed the highest toxicity against

Molecules 2013, 18
Aedes aegypti larvae. Four of these compounds showed biting deterrent activity greater
3563
than solvent control, with the highest activity being seen for 1c, with a proportion not
biting (PNB) value of 0.75, followed by 1e, 2b and 1a. No cytotoxicity was observed
against the tested human cancer cell lines. No anti-inflammatory activity was observed
against NF-B dependent transcription induced by phorbol myristate acetate (PMA) in
human chondrosarcoma cells.
Keywords: thiourea; urea; 1,2,4-triazole; synthesis; fungicide; plant pathogens; larvicides;
mosquitoes; anti-inflammatory; cytotoxicity
1. Introduction
Thioureas are important sulphur and nitrogen-containing compounds that have proved to be useful
substances in drug research in recent years [1–6]. Some urea derivatives possess valuable
antituberculosis, antibacterial and anticonvulsant properties [7–10]. Most of these compounds include
heterocyclic rings such as oxadiazoles, thiadiazoles, triazoles, and pyrazoles. It is well known that the
1,2,4-triazole-derived N-bridged heterocycles find applications in the field of medicine, agriculture and
industry [11–14]. The 1,2,4-triazole nucleus has also been incorporated into a wide variety of
therapeutically important molecules to transform them into better drugs. Drugs such as fluconazole,
itraconazole, and the new generation of triazoles posaconazole, voriconazole, and ravuconazole are the
best examples of potent antifungal molecules possessing triazole nuclei [15–17].
Thioureas can be used in the control of plant pathogens like Penicillum expansum and
Fusarium oxysporum [18]. 1,3-Dialkyl or diaryl thioureas exhibited significant antifungal activity
against Pyricularia oryzae and Drechslera oryzae [19]. Cao et al. reported that γ-aryl-1H-1,2,4-triazole
derivatives showed potent antifungal activity against Fusarium oxysporium, Rhizoctonia solani,
Botrytis cinerea, Gibberella zeae, Dothiorella gregaria, and Colletotrichum gossypii species [20,21].
Crank et al. [22] reported for the first time the antifungal activity of 2-aminooxazole thiourea
derivatives against Botrytis cinerea.
Data on the biological activity of new thiourea and urea derivatives bearing 1,2,4-triazole moieties
is scanty. Considering the biological activities of these compounds, in this work a series of new thiourea
and urea derivatives bearing 1,2,4-triazole moieties were prepared and evaluated for their antifungal
activity against several filamentous fungal plant pathogens, mosquito (Aedes aegypti) larvicidal and
biting deterrent activity and cytotoxicity and anti-inflammatory activity in some human cell lines.
2. Results and Discussion
2.1. Synthesis
A series of new thiourea and urea derivatives bearing 1,2,4-triazole rings were prepared
according to Scheme 1. The thiourea derivatives 1a–e were previously synthesized and reported
by Celen et al. [23]. Compounds 2a–e were prepared by refluxing equimolar amounts of
4-(aminophenyl)acetic acid and various isocyanates in acetone. The reactions of compounds 1a–e and

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2a–e with thio-carbohydrazide in an oil bath afforded the corresponding 1,2,4-triazoles 3a–e and 4a–e.
All compounds were isolated in satisfactory yields (45–83%) and readily purified by recrystallization
from acetonitrile. The purity of the compounds was checked by TLC and elemental analyses. The
chemical structures of all compounds were characterized by various spectroscopic methods, and both
the analytical and spectral data of all the synthesized compounds were in full agreement with the
proposed structures. Physical and chemical properties of all compounds are presented in Table 1.
Scheme 1. General synthetic route for title compounds 1a–e, 2a–e, 3a–e and 4a–e.
COOH
H2N
b
a
O
COOH
S
COOH
Ar
Ar
NH
NH
NH
NH
2a-e
d
1a-e
c
N
N
O
S
NH
NH
Ar
N
Ar
N
NH
NH
NH
NH
H2N
H2N
S
S
4a-e
3a-e
Reagents and Conditions: (a) Ar-NCS, 100 °C; (b) Ar-NCO, 100 °C; (c) NH2NHCSNHNH2, 130–140 °C;
(d) NH2NHCSNHNH2, 130–140 °C.
Table 1. Structure and physical data of compounds 1a–e, 2a–e, 3a–e and 4a–e.
Compd.
1a
Ar
M.P. (°C)
214–215
206–207
193–194
240–241
200–201
276–277
258–259
234–235
250–251
245–246
227–228
206–207
228–230
240–241
234–236
237–238
M.F.
M.W. Yield (%)
2,4,6-Cl-C6H2
2,6-Cl2-C6H4
4-CH3S-C6H4
4-CF3-C6H5
4-NO2-C6H5
2,4,6-Cl-C6H2
2,6-Cl2-C6H4
4-CH3S-C6H4
4-CF3-C6H5
4-NO2-C6H5
2,4,6-Cl-C6H2
2,6-Cl2-C6H4
4-CH3S-C6H4
4-CF3-C6H5
4-NO2-C6H5
2,4,6-Cl-C6H2
C15H11Cl3N2O2S
C15H13ClN2O2S
C16H16N2O3S
C15H13N3O4S
C15H13FN2O2S
C15H11Cl3N2O3
C15H13ClN2O3
C16H16N2O4
389.68
320.79
316.37
331.34
304.34
373.62
304.73
300.31
315.28
288.27
459.80
390.85
386.56
401.47
374.46
443.74
50.5
48.3
61.1
45.5
67.2
77.3
78.0
83.5
67.4
80.9
46.4
50.8
59.3
55.4
52.2
58.0
1b
1c
1d
1e
2a
2b
2c
2d
2e
C15H13N3O5
C15H13FN2O3
C16H13Cl3N6S2
C16H15ClN6S2
C17H18N6OS2
C15H17N7O2S2
C16H15FN6S2
C16H13Cl3N6OS
3a
3b
3c
3d
3e
4a

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Table 1. Cont.
Compd.
Ar
M.P. (°C)
M.F.
M.W. Yield (%)
4b
4c
4d
4e
2,6-Cl2-C6H4
4-CH3S-C6H4
4-CF3-C6H5
4-NO2-C6H5
258–259
180–182
214–216
240–241
C16H15ClN6OS
C17H19N6O2S
C16H15N7O3S
C16H15FN6OS
374.85
371.43
385.40
358.39
55.9
51.8
50.2
48.8
In the IR spectra of compounds 3a–e, two C=S stretching bands due to the thiourea and thioxo
1
groups were seen at 1320–1350 cm−1 and 1234–1294 cm−1. In the H-NMR spectrum, the carboxylic
acid O-H peaks were observed as singlets at 11.98–12.58 ppm for compounds 1a–e [23], but were
absent in the spectra of the 1,2,4-triazole derivatives 3a–e due to the ring closure. The N-H peaks of
the triazole rings were seen at 11.70–12.30 ppm as singlets and the thiourea N-H peaks were observed
at 7.83–9.79 ppm as two singlets. In the IR spectra of compounds 2a–e, two C=O stretching bands due
to the carboxylic acid and urea groups were detected at 1629–1697 cm−1. Urea N-H peaks were seen at
8.22–9.33 ppm as two singlets. The carboxylic acid O-H peaks were observed at 12.13–12.24 ppm as
singlets, but these peaks disappeared in the spectra of 1,2,4-triazole derivatives 4a–e due to the ring
closure. The N-H peaks of the triazole rings were seen at 12.40–13.48 ppm as a singlet but some
compounds (e.g., 4a, 4b) have no triazole ring-related N-H peaks due to deuterium exchange with the
NMR solvent. The other aliphatic and aromatic protons were detected in the expected regions for all
compounds. Mass spectra (MS-ES) of compounds showed [MH]+ peaks in agreement with their
molecular formulae.
2.2. Biological Activity
All compounds were evaluated for growth inhibition of filamentous plant pathogenic fungi
belonging to genera Colletotrichum, Botrytis, Fusarium and Phomopsis using 96-well micro-dilution
broth assays. Microbioassay results indicated that the thiourea derivatives 1b, 1c, 3a and 3d bearing
2,6-dichloro-, 2,4,6-trichloro-, 4-methysulfanyl- and 4-trifluoromethyl- groups on the phenylthiourea
ring and the urea derivative 4e having a 4-nitro group were the most active against Phomopsis obscurans
and P. viticola. Compounds 1b, 3a, and 1c and 4e at 30 M inhibited the growth of P. obscurans by
100% and 80%, respectively, after 120 h exposure, whereas 1b, 3d and 4e showed 80%, 60% and 80%
growth inhibition, respectively, at 30 M, respectively (Figure 1). Compound 4e showed 100% growth
inhibition of P. viticola at 30 M, whereas 1b and 1c inhibited its growth by 80% and 60%,
respectively (Figure 2). Compounds 1b, 3a and 4e having 2,6-dichloro-, 2,4,6-trichloro- and
4-nitro-groups on the phenylthiourea ring showed fungal growth inhibition that was similar to that of
30 M of the positive control captan, a well known multisite inhibitor fungicide with no systemic
activity, used as a commercial protectant fungicide to prevent anthracnose diseases in fruits and
ornamentals [24]. Tested compounds showed a very weak activity against Colletotrichum, Botrytis and
Fusarium species.

Molecules 2013, 18
Figure 1. Growth inhibition of Phomposis obscurans and P. viticola after 120 h using a 96
3566
well microdilution broth assay in a dose response with the commercial fungicide standard
captan as reference.
In biting deterrent bioassays with A. aegypti, compounds 1a, 2b, 1e and 1c showed significantly
higher biting deterrent activity (Figure 2) than the control, while the activity in the other compounds
was similar to that of the ethanol control. Compound 1c bearing a 2,4,6-trichloro group had the highest
activity, with a portion not biting (PNB) value of 0.75, followed by 1e, 2b and 1a having 4-nitro-,
2,6-dichloro- and 2,4,6-trichloro-groups, with PNB values of 0.71, 0.68 and 0.55, respectively, at
25 nmol/cm2. However, biting deterrent activity in all these compounds was significantly lower than
that of the reference compound DEET (PNB = 0.89).
In order to discover new mosquito toxicants, synthesized compounds were also screened against 1 d
old larvae. The three compounds 1c, 3d and 3e which showed larvicidal activity in preliminary
screening were selected for further study in dose response bioassays. Dose mortality data of these
thiourea compounds are presented in Table 2.

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Figure 2. Biting deterrent effects of DEET 1a, 1c and 1e and 2b at 25 nmol/cm2 against A. aegypti.
Table 2. Toxicity of substituted urea and thiourea 1,2,4-triazole derivatives 1c, 3d and 3e
against 1-d old larvae of Aedes aegypti at 24-h post treatment.
Compounds
LD50 (95% CI) *
118.8 (105.3–135.2)
67.4 (59.0–77.0)
LD90 (95% CI) *
216.4 (182.4–280.4)
139.4 (116.4–180.6)
370.95 (278.5–619.3)
Chi square
61.7
DF
38
38
38
1c
3d
3e
75.0
165.6 (141.7–205.2)
40.3
* LD values are in ppm; 95% CI = 95% confidential intervals.
Compound 3d carrying a 4-trifluorophenyl group (LD50 value of 67.9 ppm) showed the highest
mortality, followed by 1c having a 4-methylsulfanylphenyl group on the thiourea moiety
(LD50 = 118.8 ppm) and 3e with a 4-nitrophenyl moiety (LD50 = 165.6). The comparison of larvicidal
activity of thiourea and urea derivatives showed that thiourea derivatives were much more potent than
the corresponding urea derivatives. There was not not much increase in mortality at 48 h post treatment.
All synthesized compounds were tested for their cytotoxicity and anti-inflammatory activity in
cellular assays. No anti-inflammatory activity was observed against NF-B mediated transcription
induced by phorbol myristate acetate (PMA) in human chondrosarcoma cells up to the highest tested
concentration of 25 μg/mL and no cytotoxicity was observed against mammalian kidney cells (Vero
and LLC-PK11) or human solid tumor cells (SK-MEL, SK-OV-3, BT-549 and KB) up to a highest
concentration of 25 μg/mL, except for compound 3e having 4-nitro group, which showed mild
cytotoxicity with IC50 values in the range of 11.0–25.0 μg/mL towards most of the tested cell lines.

Molecules 2013, 18
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3. Experimental
3.1. General
Reactions were monitored by thin layer chromatography (TLC) performed on 60 F-254 silica gel
plates with visualization by UV-light using chloroform and methanol as solvent system and purity of
the products was checked by High Performance Liquid Chromatography (HPLC, Agilent Technologies,
Palo Alto, CA, USA). Melting points were determined on a SMP II apparatus (Schorpp Geaetetechnik
Gehrden, Germany). The UV spectra were measured with a Shimadzu UV–2100 S (Shimadzu, Kyoto,
Japan).The IR spectra were recorded on a Shimadzu FTIR 8400S spectrometer (Shimadzu, Kyoto,
Japan). 1H-NMR spectra were recorded at 400 MHz on Bruker Avance-DPX-400 spectrometer (Bruker
Spectrospin, Billerica, MA, USA) in DMSO-d6. Chemical shifts were recorded in parts per million
(ppm) downfield from TMS. The splitting patterns of 1H-NMR were designated as follows: s: singlet,
d: doublet, t: triplet, q: quartet, m: multiplet. Mass spectra were recorded on an Agilent 1100 LC-MS
series system (Agilent Technologies, Palo Alto, CA, USA) in the electrospray mode. Elemental
analysis was performed on a Leco CHNS-932 analyzer (Leco, Michigan, MI, USA). All chemicals and
solvents were procured from Merck or Aldrich (Darmstad and Steinheim, Germany). The thiourea
derivatives 1a–e were prepared by reacting 4-(aminophenyl)acetic acid with the corresponding
isothiocyanate. Spectroscopic characterisation of 1a–e has been previously reported [23].
3.1.1. General Procedure for the Preparation of 2a–e
4-(Aminophenyl)acetic acid (0.500 g, 3.3 mmol) was dissolved in acetone (5 mL) at 100 °C. Then,
a solution of the corresponding isocyanate (3.3 mmol) in acetone (5 mL) was added in three portions
separated by 30 min. After 6–8 h, the reaction was finalized according to TLC monitoring. Solid
material was filtered off and recrystallized from a suitable solvent.
(4-{[(2,4,6-Trichlorophenyl)carbamoyl]amino}phenyl)acetic acid (2a). UV λmax. (EtOH) nm (log ):
251 (4.45). IR (max, cm−1): 3286 (N-H, O-H), 3286 (C-H), 1687, 1651 (C=O), 1543, 1510 (N-H).
1H-NMR : 3.48 (2H, s, -CH2), 6.99–7.83 (8H, m, Ar-H), 8.22 (1H, s, -NH-), 8.94 (1H, s, -NH-), 12.24
(1H, s, OH). Anal. Calcd. for C15H11Cl3N2O3; C: % 48.22; H: % 2.97; N: % 7.50 Found: C: % 47.95;
H: % 3.00; N: % 7.30. MS-ES (m/z): 374.62 (MH+).
(4-{[(2,6-Dichlorophenyl)carbamoyl]amino}phenyl)acetic acid (2b). UV λmax. (EtOH) nm (log ): 251
(4.41). IR (max, cm−1): 3252 (N-H, O-H), 3016 (C-H), 1697, 1651 (C=O), 1573, 1545 (N-H). 1H-NMR
: 3.49 (2H, s, -CH2), 7.09–7.60 (7H, m, Ar-H), 8.18 (1H, s, -NH-), 8.91 (1H, s, -NH-), 12.24 (1H, s,
OH). Anal. Calcd. for C15H12Cl2N2O3; C: % 53.12; H: % 3.57; N: % 8.26 Found: C: % 52.84;
H: % 3.55; N: % 8.15. MS-ES (m/z): 340.18 (MH+).
4-{[(4-(Methylsulfanyl)phenyl)carbamoyl]amino}phenyl)acetic acid (2c). UV λmax. (EtOH) nm (log ):
279 (3.12). IR (max, cm−1): 3290 (N-H, O-H), 3007 (C-H), 1695,1629 (C=O), 1550 (N-H). 1H-NMR :
3.35 (3H, s, CH3), 3.54 (2H, s, -CH2), 7.02–7.62 (8H, m, Ar-H), 8.63 (1H, s, -NH-), 8.70 (1H,
s, -NH-), 12.24 (1H, s, OH). Anal. Calcd. for C16H16N2O3S; C: % 60.74; H: % 5.10; N: % 8.85, S:
10.14 Found: C: % 60.54; H: % 5.09; N: % 8.73, S: 10.00. MS-ES (m/z): 317.37 (MH+).

Molecules 2013, 18
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(4-{[(4-Trifluoromethylphenyl)carbamoyl]amino}phenyl)acetic acid (2d). UV λmax. (EtOH) nm (log ):
261 (4.06). IR (max, cm−1): 3304 (N-H, O-H), 3304 (C-H), 1697,1630 (C=O), 1552, 1512 (N-H).
1H-NMR : 3.48 (2H, s, -CH2), 6.81–7.42 (8H, m, Ar-H), 8.43 (1H, s, -NH-), 8.53 (1H, s, -NH-), 12.21
(1H, s, OH). Anal. Calcd. for C16H13F3N2O3; C: % 56.81; H: % 3.87; N: % 8.28 Found: C: % 56.60; H:
% 3.72; N: % 8.16. MS-ES (m/z): 339.28 (MH+).
(4-{[(4-Nitrophenyl)carbamoyl]amino}phenyl)acetic acid (2e). Compound 2e was previously
synthesized by Deny et al. [25]. UV λmax. (EtOH) nm (log ): 249 (4.08). IR (max, cm−1): 3367 (N-H,
1
O-H), 3308 (C-H), 1672, 1640 (C=O), 1556,1496 (N-H). H-NMR : 3.25 (2H, s, -CH2), 7.11–8.30
(8H, m, Ar-H), 8.70 (1H, s, -NH-), 9.33 (1H, s, -NH-), 12.13 (1H, s, OH). Anal. Calcd. for
C15H13N3O5: C: % 57.14; H: % 4.16; N: % 13.33 Found: C: % 57.27; H: % 4.10; N: % 13.38. MS-ES
(m/z): 316.28 (MH+).
3.1.2. General Procedure for the Preparation of 3a–e and 4a–e
Equimolar mixtures of thiocarbohydrazide (0.1 mol) and thiourea or urea derivatives 1a–e, 2a–e
were heated in an oil-bath at 130–140 °C for 2–3 h. The fused mass obtained was dispersed with hot
water to obtain the new thiourea and urea derivatives 3a–e, 4a–e containing 1,2,4-triazole moieties.
The products were recrystallized from methanol.
1-{4-[(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]phenyl}-3-(2,4,6-trichlorophenyl)-
thiourea (3a). UV λmax. (EtOH) nm (log ): 258 (3.97). IR (max, cm−1): 3252 (N-H), 3020 (C-H) 1651
(C=N), 1600, 1510 (N-H), 1345, 1246 (C=S), 1182 (C-N). 1H-NMR : 3.20 (2H, s, -CH2), 5.48 (2H, s,
NH2), 7.20–7.46 (6H, m, Ar-H), 7.83 (1H, s, -NH-), 8.55 (1H, s, -NH-), 11.70 (1H, s, triazole NH).
Anal. Calcd. for C16H13Cl3N6S2; C: % 41.79; H: % 2.85; N: % 18.28; S: % 13.95 Found: C: % 41.64;
H: % 3.09; N: % 18.25; S: % 13.72. MS-ES (m/z): 460.80 (MH+).
1-{4-[(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]phenyl}-3-(2,6-dichlorophenyl)-
thiourea (3b). UV λmax. (EtOH) nm (log ): 278 (3.51). IR (max, cm−1): 3194 (N-H), 3012 (C-H), 1650
(C=N), 1541 (N-H), 1350, 1240 (C=S), 1180 (C-N). 1H-NMR : 3.47 (2H, s, -CH2), 5.50 (2H, s, NH2),
7.15–7.59 (7H, m, Ar-H), 9.68 (1H, s, -NH-), 9.79 (1H, s, -NH-), 11.80 (1H, s, triazole NH). Anal.
Calcd. for C16H14Cl2N6S2; C: % 45.18; H: % 3.32; N: % 19.76; S: % 15.08. Found: C: % 45.10; H:
% 3.30; N: % 19.66; S: % 15.00. MS-ES (m/z): 426.36 (MH+).
1-{4-[(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]phenyl}-3-(4-(methylsulfanyl)-
phenyl)thiourea (3c). UV λmax. (EtOH) nm (log ): 282 (3,30). IR (max, cm−1): 3220 (N-H), 3026
(C-H), 1537 (N-H), 1345, 1234 (C=S), 1178 (C-N). 1H-NMR : 3.30 (3H, s, CH3), 3.49 (2H, s, -CH2),
5.10 (2H, s, NH2), 6.90–7.40 (8H, m, Ar-H), 9.40 (1H, s, -NH-), 9.46 (1H, s, -NH-), 12.00 (1H, s,
triazole NH). Anal. Calcd. for C17H18N6OS2; C: % 50.72; H: % 4.51; N: % 20.88; S: % 23.90. Found:
C: % 50.27; H: % 4.70; N: % 20.69; S: % 23.92. MS-ES (m/z): 403.56 (MH+).
1-{4-[(4-amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]phenyl}-3-(4-(trifluoromethyl)-
phenyl)thiourea (3d). UV λmax. (EtOH) nm (log ): 257 (4.62). IR (max, cm−1): 3288 (N-H, C-H),
1697 (C=N), 1627, 1552 (N-H), 1346, 1294 (C=S), 1188 (C-N). 1H-NMR : 3.41 (4H, d, -NH2, -CH2),

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7.05–7.52 (8H, m, Ar-H), 8.45 (1H, s, -NH-), 8.69 (1H, s, -NH-), 12.30 (1H, s, triazole NH). Anal.
Calcd. for C17H15F3 N6S2; C: % 48.10; H: % 3.56; N: % 19.80; S: % 15.11 Found: C: % 50.29; H:
% 4.12; N: % 22.50; S: % 17.17. MS-ES (m/z): 425.47 (MH+).
1-{4-[(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]phenyl}-3-(4-nitrophenyl)thiourea
(3e). UV λmax. (EtOH) nm (log ): 246 (4.06). IR (max, cm−1): 3280 (N-H), 3023 (C-H), 1593 (C-NO2
1
assymetrical stretching), 1494 (N-H), 1320, 1298 (C=S), 1170 (C-N). H-NMR : 3.55 (2H, s, -CH2),
5.25 (2H, s, NH2), 7.11–8.36 (8H, m, Ar-H), 9.20 (1H, s, -NH-), 9.40 (1H, s, -NH-), 12.20 (1H, s,
triazole NH). Anal. Calcd. for C16H15N7O2S2; C: % 47.87; H: % 3.77; N: % 24.42; S: % 15.97. Found:
C: % 47.80; H: % 3.74; N: % 24.40; S: % 15.96. MS-ES (m/z): 402.47 (MH+).
1-{4-[(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]phenyl}-3-(2,4,6-trichlorophenyl)-
urea (4a). UV λmax. (EtOH) nm (log ): 253 (3.24). IR (max, cm−1): 3271 (N-H, C-H), 1651 (C=O),
1595 (C=N), 1539, 1508 (N-H), 1284–1253 (C=S), 1182 (C-N). 1H-NMR : 4.96 (4H, s, -NH2, -CH2),
7.15–7.79 (6H, m, Ar-H), 9.12 (1H, s, -NH-), 9.63 (1H, s, -NH-). Anal. Calcd. for C16H13Cl3N6OS; C:
% 43.31; H: % 2.95; N: % 18.94; S: % 7.23 Found: C: % 42.95; H: % 2.91; N: % 19.03; S: % 7.24.
MS-ES (m/z): 444.74 (MH+).
1-{4-[(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]phenyl}-3-(2,6-dichlorophenyl)-
urea (4b). UV λmax. (EtOH) nm (log ): 263 (4.60). IR (max, cm−1): 3300 (N-H, C-H), 1699 (C=O),
1
1633 (C=N), 1591, 1541 (N-H), 1228 (C=S), 1089 (C-N). H-NMR : 3.47 (4H, s, -NH2, -CH2),
7.10–7.58 (7H, m, Ar-H), 9.29 (1H, s, -NH-), 9.48 (1H, s, -NH-). Anal. Calcd. for C16H14Cl2N6OS2; C:
% 45.18; H: % 3.45; N: % 20.53; S: % 7.83 Found: C: % 47.00; H: % 3.42; N: % 20.50; S: % 7.92.
MS-ES (m/z): 410.29 (MH+).
1-{4-[(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]phenyl}-3-(4-methylsulfanyl-
phenyl)urea (4c). UV λmax. (EtOH) nm (log ): 261 (4.06). IR (max, cm−1): 3304 (N-H, O-H), 3304
1
(C-H), 1697, 1633 (C=O), 1552,1512 (N-H). H-NMR : 3.32 (3H, s, CH3), 3.48 (2H, s, -CH2), 5.20
(2H, s, -NH2), 6.81–7.42 (8H, m, Ar-H), 8.43 (1H, s, -NH-), 8.53 (1H, s, -NH-), 12.40 (1H, s, triazole
NH). Anal. Calcd. for C17H18N6OS; C: % 52.83; H: % 4.69; N: % 21.70; S: 16.59. Found: C: % 52.70;
H: % 4.49; N: % 21.67; S: 16.50. MS-ES (m/z): 387.49 (MH+).
1-{4-[(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]phenyl}-3-(4-trifluoromethyl-
phenyl)urea (4d). UV λmax. (EtOH) nm (log ): 258 (4.33). IR (max, cm−1): 3290 (N-H), 1699 (C=O),
1
1556 (C=N), 1510, 1494 (N-H), 1211-1186 (C=S), 1151, 1095 (C-N). H-NMR : 3.42 (4H, d, -NH2,
-CH2), 7.02–7.66 (8H, m, Ar-H), 8.65 (2H, d, -NH-, -NH-), 12.50 (1H, s, triazole NH). Anal. Calcd.
for C17H15F3 N6OS; C: % 50.00; H: % 3.70; N: % 20.58; S: % 7.85 Found: C: % 50.10; H: % 3.67;
N: % 20.50; S: % 7.89. MS-ES (m/z): 409.40 (MH+).
1-{4-[(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]phenyl}-3-(4-nitrophenyl)urea
(4e). UV λmax. (EtOH) nm (log ): 253 (3,36). IR (max, cm−1): 3290 (N-H, C-H), 1695 (C=O), 1629
(C=N), 1589, 1545 (N-H), 1296 (C=S), 1111 (C-N). 1H-NMR : 3.48 (2H, s, -CH2), 5.40 (2H, s, -NH2),
7.01–7.62 (8H, m, Ar-H), 8.52 (1H, s, -NH-), 9.93 (1H, s, -NH-), 13.48 (1H, s, triazole NH). Anal.

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Calcd. for C16H15FN7O3S; C: % 49.86; H: % 3.92; N: % 25.44; S: % 8.32 Found: C: % 50.08; H:
% 4.08; N: % 25.37; S: % 8.26. MS-ES (m/z): 386.40 (MH+).
3.2. Biological Activity
3.2.1. Antifungal Activity
A standardized 96-well micro-dilution broth assay as developed by Wedge and Kuhajek [26] was
used to evaluate the antifungal activity of the compounds towards Botrytis cinerea, Colletotrichum
acutatum, C. fragariae, C. gloeosporioides, P. viticola, P. obscurans and Fusarium oxysporum. The
fungicide captan was used as an internal fungicide standard in all assays. Each fungus was challenged
in duplicate in a dose-response format using test compounds where the final treatment concentrations
were 0.3, 3.0 and 30.0 M. Microtiter plates (Nunc MicroWell, untreated, Roskilde, Denmark) were
covered with a plastic lid and incubated in a growth chamber as described previously [27]. Fungal
growth was then evaluated by measuring absorbance of each well at 620 nm using a microplate
photometer (Packard Spectra Count, Packard Instrument Co., Downers Grove, IL, USA). Sixteen wells
containing broth and inoculum served as positive growth controls, eight wells containing solvent at the
appropriate concentration and broth without inoculum were used as negative growth controls. The
experiments were repeated at three times over a period of time. Mean absorbance values and standard
errors were used to evaluate fungal growth at 48 h and 72 h except for P. obscurans and P. viticola
where the data were recorded at 144 h. Means for percent inhibition of each fungus at each dose of test
compound relative to the untreated positive growth controls were used to evaluate fungal growth. The
SAS Proc ANOVA module [28] was used to identify significant factors, and Fisher’s protected LSD
was used to separate means [29].
3.2.2. Mosquito Larval Bioassay
Aedes aegypti (L.) used in these studies were from a laboratory colony maintained since 1952 at the
Mosquito and Fly Research Unit at Center for Medical, Agricultural and Veterinary Entomology,
USDA-ARS, Gainesville, FL, USA. This colony is maintained using standard procedures [30].
Bioassays to determine the larvicidal activity of urea and thiourea derivatives against A. aegypti were
conducted by using the system described by Pridgeon et al. [31]. In brief, the eggs were hatched under
vacuum (about 1 h) by placing a piece of a paper towel with eggs in a cup filled with 100 mL of
deionized water containing small quantity of larval diet. Larvae were removed from vacuum and held
overnight in the cup in a temperature-controlled room maintained at a temperature of 27 ± 2 °C and
60 ± 10% RH at a photoperiod regimen of 12:12 (L:D) h. Five 1-d-old first instar A. aegypti were
added to each well of 24-well plates in a droplet of water using a disposable 22.5-cm Pasteur pipette by
placing on an illuminated light box. One mL deionized water was added to each well using a
Finnpipette stepper (Thermo Fisher, Vantaa, Finland). Fifty µL of larval diet (2% slurry of 3:2 beef
liver powder (Now Foods, Bloomingdale, IL, USA) and brewer’s yeast (Lewis Laboratories Ltd.,
Westport, CT, USA) were then added to each well. All chemicals to be tested were diluted in dimethyl
sulfoxide (DMSO). Eleven microliters of the test chemical was added to the labeled wells, and in
control treatments 11 µL of DMSO alone was added. After the treatment, the plates were swirled in

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clockwise and counter clockwise motions and front and back and side to side five times to ensure even
mixing of the chemicals. Larval mortality was recorded 24- and 48-h after treatment. Larvae that
showed no movement in the well after manual disturbance of water by a pipette tip were recorded as
dead. A series of concentrations were used in each treatment to get a range of mortality. Treatments
were replicated 15 times for each compound. LD50 values for larvicidal data were calculated by using
the SAS Proc Probit module [28]. Control mortality was corrected by using Abbott’s formula.
3.2.3. Mosquito Biting Bioassays
Experiments were conducted using a six-celled in vitro Klun & Debboun (K & D) module bioassay
system as developed by Klun et al. [32] for quantitative evaluation of bite deterrent properties of
candidate compounds. The term deterrent refers to a chemical that inhibits feeding when present in a
place where the insects feed in its absence and the repellent is a chemical that causes insects to make
oriented movement away from its source (Dethier [33]). Briefly, the assay system consists of a six well
reservoir with each of the 3 cm  4 cm wells containing 6 mL of blood. As described by Ali et al. [34],
a feeding solution consisting of CPDA-1 and ATP was used instead of blood. N,N-diethyl-meta-
toluamide (DEET) was obtained from Sigma Aldrich (St. Louis, MO, USA) and used as a positive
control. Molecular biology grade ethanol was obtained from Fisher Scientific Chemical Co. (Fairlawn,
NJ, USA). All synthesized compounds (Table 1) were screened in this study and DEET at 25 nmol/cm2
was used as positive control. All the treatments were freshly prepared in ethanol at the time of bioassay.
The temperature of the solution in the reservoirs was maintained at 37.5 °C by continuously passing
the warm water through the reservoir using a circulatory bath. The reservoirs were covered with a layer
of collagen membrane. The test compounds were randomly applied to six 4 cm × 5 cm areas of
organdy cloth and positioned over the membrane-covered CPDA-1+ATP solution with a separator
placed between the treated cloth and the six-celled module. A six celled K & D module containing five
females per cell was positioned over cloth treatments covering the six CPDA-1+ATP solution
membrane wells, and trap doors were opened to expose the treatments to these females. The number of
mosquitoes biting through cloth treatments in each cell was recorded after a 3 min exposure and
mosquitoes were prodded back into the cells. These mosquitoes were then squashed to determine the
number which has actually engorged the solution. A replicate consisted of six treatments: four test
compounds, DEET (a standard biting deterrent) and ethanol treated cloth as solvent control. A set of
five replications was conducted on different days using a new batch of females in each replication.
Treatments were replicated 15 times.
3.2.4. Cytotoxicity Activity
In vitro cytotoxicity was determined against a panel of human cancer cell lines (SK-MEL, KB,
BT-549, SK-OV-3) as well as noncancerous pig kidney epithelial (LLC-PK11) and monkey kidney
fibroblast (VERO) cells. The assay was performed in 96-well tissue culture-treated microplates. Cells
were seeded to the wells of the plate (25,000 cells/well) and incubated for 24 hours. Samples were
added and again incubated for 48 h. The number of viable cells was determined using the supravital
dye Neutral Red according to the procedure described earlier [35]. Doxorubicin was used as control
drug for cytotoxicity.

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3.2.5. Anti-Inflammatory Activity
Inhibition of NF-κB mediated transcription was determined in human chondrosarcoma (SW1353)
cells by a reporter gene assay as described earlier [36,37]. In brief, at about 75% confluency, cells were
harvested and transfected with NF-κB reporter luciferase plasmid construct at 160 V and one
70-ms pulse in a BTX Electro Square Porator T 820. Transfected cells were plated in 96-well plates
(1 × 105 cells/well) and incubated for 24 h. After 24 h, cells were treated with test samples and after
30 min treated with PMA (70 ng/mL) for the activation of NF-κB and incubated for 8 h. After
removing medium, cells were lysed by adding 40 µL of a 1:1 mixture of LucLite reagent and PBS
containing 1 mM calcium and magnesium. Luciferase activity was measured as light output on a
SpectraMax plate reader. Sp-1 was used as a control transcription factor to evaluate the toxicity of
tested compounds in the same assay and parthenolide was used as a positive control as described
earlier [37].
4. Conclusions
The molecules described in this study demonstrated no cytotoxicity and no anti-inflammatory
activity, but some of them showed good antifungal activity against Phomopsis species. Based on the
biological activity evaluation these compounds may not be useful as therapeutic or pharmaceutical
agents. However, selective compounds exhibiting antifungal activity could have a potential to be
developed as agrochemicals, and further work should be performed to modify their structure with an
aim to increase their activity. Phomopsis appeared to be the most sensitive fungal species to the tested
compounds. Phomopsis cane and leaf spot (P. viticola) causes serious economic losses to the vine
grape production in the United States of America and Europe, while P. obscurans causes Phomopsis
leaf blight and fruit rot of strawberry. Compound 3d was the most active derivative among the tested
compounds in a larvicidal assay against A. aegypti. The comparison of antifungal and insecticidal
activities of thiourea and urea derivatives showed that thiourea derivatives bearing 2,6-dichlorophenyl,
2,4,6-trichlorophenyl, 4-nitophenyl, 4-trifluoromethylphenyl substituents were the active compounds.
Thiourea compounds, 1b, 1c, 3a, 3d, 3e, and urea compound 4e show sufficient potential to warrant
further modification to discover new antifungal and insecticidal molecules.
Acknowledgments
The authors wish to thank Marmara University Scientific Research Projects Commission (BAPKO,
SAG-C-YLP-270109-0013, 2009 and SAG-D-200611-0205 2011) for financial support. This study
was supported in part by USDA/ARS grant No. 56-6402-1-612 and Deployed War-Fighter Protection
Research Program Grant funded by the U.S. Department of Defense through the Armed Forces Pest
Management Board. We thank Jessie Linda Robertson, Ramona Pace, John Trott, and Katherine
Martin for the bioassays. We thank James J. Becnel, Mosquito and Fly Research Unit, Center for
Medical, Agricultural and Veterinary Entomology (USDA-ARS, Gainesville, FL, USA) for supplying
A. aegypti eggs.

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