Synthesis 1-acyl-3-(2'-aminophenyl) thioureas as anti-intestinal nematode prodrugs

Article abstract of DOI:10.3390/molecules15106941

A series of 1-acyl-3-(2'-aminophenyl) thiourea derivatives were designed and synthesized. The structures of all the newly synthesized compounds were identified by IR, elemental analysis, ¹H-NMR and ¹³C-NMR. Their anti-intestinal nema

Full text of DOI:10.3390/molecules15106941

Molecules 2010, 15, 6941-6947; doi:10.3390/molecules15106941
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis 1-Acyl-3-(2'-aminophenyl) thioureas as Anti-Intestinal
Nematode Prodrugs
Li-Ping Duan, Jia Xue, Li-Li Xu and Hao-Bing Zhang *
National Institute for Parasitic Diseases, Chinese Center for Disease Control and Prevention, Shanghai,
200025, China
* Author to whom correspondence should be addressed; E-Mail: zhanghaobing@hotmail.com.
Received: 4 August 2010; in revised form: 6 September 2010/ Accepted: 25 September 2010 /
Published: 8 October 2010
Abstract: A series of 1-acyl-3-(2'-aminophenyl) thiourea derivatives were designed and
synthesized. The structures of all the newly synthesized compounds were identified by IR,
1
elemental analysis, H-NMR and 13C-NMR. Their anti-intestinal nematode activities
against Nippostrongylus brazilliensis were evaluated in rats by an oral route. Among these
compounds, at concentrations of 10 mg/kg of rat, compound (1-(2'-furanyl)acyl-3- (2'-
aminophenyl) thiourea) (5h) produced the highest activity with 89.4% deparasitization.
The present work suggests that 1-acyl-3-(2'-aminophenyl) thiourea derivatives may
become useful lead compounds for anti-intestinal nematode treatment.
Keywords: acyl; thiourea; deparasitization; Nippostrongylus brazilliensis
1. Introduction
Mebendazole and albendazole have been used against human and animal helminth parasites for
more than two decades [1-5]. They are derived from benzimidazole which has a broad spectrum of
activity and is used to treat nematode and trematode infections in domestic animals. The limited
solubility of benzimidazoles may have a major influence on their absorption and clinical efficacy [6,7].
Furthermore, when used in lengthy therapies, they can produce side-effects, such as severe headaches,
fever, fatigue, hair loss, and liver degeneration [8] and hence are not recommended for patients with
hepatic problems. A way to overcome these problems is to use prodrugs [9-11], such as 4-amino-3-(3'-
methoxycarbonyl-2'-thioureido)benzophenone [12]. It is a soluble prodrug, which is enzymatically

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cyclized to mebendazole in vitro. On the other hand, thiourea derivatives also exhibit potent antiviral,
antibacterial and cytotoxic activities [13,14]. Several works demonstrate their activity against parasites
such as Plasmodium falciparum, Trichomonas vaginalis and Trypanosoma. cruzi [15,16]. Based on
these reports, we report herein the synthesis, characterization, and in vitro evaluation of anti-intestinal
nematode activity of eight different novel thiourea derivatives bearing the o-aminobenzene moiety.
2. Results and Discussion
2.1. Synthesis and Characterization of 1-Acyl-3-(2-aminophenyl) thiourea Derivatives 5a-5h
The synthetic route to the target compounds 5a-5h is shown in Scheme 1. Firstly, acids 1a-1h were
acylated by SOCl2 followed by isothiocyanation and coupling reactions with 2-nitrobenzenamine to
give 1-acyl-3-(2'-nitrophenyl) thioureas 4a-4h in moderate yield. Then the title compounds 5a-5h were
successfully obtained in 50-60% overall yield using SnCl2 as reducing agent. Compounds 5a-5h were
characterized by 1H-NMR, 13C-NMR and elemental analysis. All results are in full agreement with the
1
proposed structures. For example, the H-NMR spectrum of compound 5b showed a singlet at 2.40
ppm (CH3), singlets at 12.9 and 11.6 ppm (NHCSNH) and a multiplet from δ = 7.91 to δ = 6.75 for
aromatic hydrogens. Moreover, the 13C-NMR spectrum showed δ 29.6 (CH3), 154.19, 141.62, 133.95,
130.41, 125.70, 124.63, 122.27, 119.28, 115.78, 114.44, 113.15, 109.34 (benzene C), 165.1 (C=O),
180.2 (C=S), all consistent with its proposed structure. The elemental analyses results were in good
agreement with those calculated for the suggested formulae. The melting points are sharp, indicating
the purity of these compounds.
Scheme 1. Synthesis of compounds 5a-5h.
SOCl2
KSCN
R
CONCS
3a-h
R
COOH
R
COCl
1a-h
2a-h
O
S
O
S
R
CNHCNH
R
CNHCNH
SnCl2
H2N
O2N
H2N
O2N
5a-h
4a-h
F
Cl
H3C
d
a
b
c
O
Br
N
N
h
f
g
e
2.2. Anti-intestinal Nematode Activity
From Table 1, we can see that some of compounds showed significant anti-intestinal nematode
activity in a two-day in vivo test in rats. At concentrations of 10 mg/kg of rat, compound 5h produced
the highest activity against Nippostrongylus brazilliensis with 89.4 % deparasitization. For anti-
intestinal nematode activity, it appears that a variety of substituents can be introduced on the phenyl
ring without significantly altering the activity relative to the unsubstituted phenyl analogue 5a. For

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example, the substituted F, CH3, Cl, and Br acyl derivatives all have the almost same activity as 5a.
Moreover, the 3'-pyridylacyl derivative 5g is slightly more active than the corresponding 2'-isomer 5f.
On the other hand, the structural variation between compounds 5b and 5f results in different activity.
Compound 5a, 5b, 5c, 5d and 5e contains benzene moieties, while 5f and 5g have a pyridyl group
moiety. This pyridyl group appears to be particularly responsible for anti-intestinal nematode activity.
Compounds 5f and 5g, which contain a pyridyl moiety, and 5h that contains a furanyl moiety all seemed
to be much more effective in terms of anti-intestinal nematode activity. Because compound 5h displayed
anti-intestinal nematode potency that is comparable to albendazole (10 mg/kg), further anti-intestinal
nematode activity assay was carried out for compound 5h. It was found that at concentrations of 18
mg/kg of rat, 5h produced the highest activity against Nippostrongylus brazilliensis with 100%,
effectiveness, which implies further possibilities for lead compound development.
Table 1. Results of the chemotherapeutic trials of thiourea derivatives bearing
O-aminobenzene moieties in rats.
Number of worms recovered from rats (including worms in rats at post mortem)
Dose(mg/kg)
1
2
3
Average Deparasitization(%)
Control
Albendazole
0
200
0
198
0
199
0
199
0
10
10
10
10
10
10
10
10
10
100.00
13.7
10.5
10.5
7.0
13.5
45.2
48.2
89.4
5a
5b
5c
5d
5e
5f
180
178
183
188
177
109
108
22
170
180
177
183
170
110
107
23
176
178
174
184
170
108
96
175
178
178
185
172
109
103
21
5g
5h
19
3. Conclusions
In summary, various types of 1-acyl-3-(2-aminophenyl) thioureas were synthesized and their
varying biological activities towards the N. brazilliensis was demonstrated. Among these compounds,
5h produced the highest activity against N. brazilliensis with 89.4% deparasitization. The present work
suggest that 5h may be a useful lead compound for anti-intestinal nematode medicine development.
Further studies of the structure-biology activity relationships around the designed compounds
are underway.
4. Experimental
4.1. General
All the reagents and solvents were of the commercial quality and were used without purification.
Elemental analysis was performed on a PE-2400 elemental analyzer, the C, H and N analysis were
1
13
repeated twice. H-NMR and C-NMR spectra were obtained in DMSO-d6 with TMS as internal

Molecules 2010, 15
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standard on a Bruker AM-400 spectrometer. Chemical shifts are reported as ppm. Melting points were
determined by an X-6 micro-melting point apparatus and are uncorrected.
4.2. General Procedure for the Preparation of 1-Acyl-3-(2-aminophenyl)thioureas 5a-5h
According to our reported procedure [17] different acids 1a-1h were treated with SOCl2 and KSCN,
respectively, affording moderate yields of around 75% of the intermediates 3a-3h, which were used
directly without further purification. The subsequent nucleophilic reactions of 3a-3h with 2-nitro-
benzenamine led to the key intermediates 4a-4h, respectively. Then reduction of 4a-4h with SnCl2 in
CH3COOH afforded the target compounds 5a-5h, which were recrystallized twice from DMF/H2O.
1-Phenylacyl-3-(2'-aminophenyl) thiourea (5a): Yield 60%, mp 154~156oC. IR (KBr) : 3165, 1628,
1
1255 cm−1; H-NMR δ: 12.52 (s, 1H, NH), 11.44 (s, 1H, NH), 8.03-6.80 (m, 9H, Ar-H), 5.30 (s, 2H,
Ar-NH); 13C-NMR δ: 180.34, 165.48, 154.39, 133.62, 132.95, 130.41, 130.10, 128.63, 128.27, 127.28,
125.78, 124.05, 119.32, 118.44; Anal. Calcd. for C14
C 62.00, H 4.83, N 15.60.
H13
N3OS (271.2): C 61.97, H 4.83, N 15.49; found
1-(4'-Methylphenyl)acyl-3-(2'-aminophenyl) thiourea (5b): Yield 58%, mp 164~165oC. IR (KBr) :
1
3208, 1645, 1240 cm−1; H-NMR δ: 12.91 (s, 1H, NH), 11.64 (s, 1H, NH), 7.91-6.75 (m, 8H, Ar-H),
13
5.32 (s, 2H, Ar-NH), 2.40 (s, 3H, CH3); C-NMR δ: 180.24, 165.18, 154.19, 141.62, 133.95, 130.41,
125.70, 124.63, 122.27, 119.28, 115.78, 114.44, 113.15, 109.34, 29.64; Anal. calcd. for C15
(285.1): C 63.13, H 5.30, N 14.73; found C 63.56, H 5.41, N 14.73.
H15
N3OS
1-(4'-Fluorophenyl)acyl-3-(2'-aminophenyl) thiourea (5c): Yield 53%, mp 173~174oC. IR (KBr): 3235,
1650, 1235 cm−1; 1H-NMR δ: 12.93 (s, 1H, NH), 11.67 (s, 1H, NH), 8.12-6.80 (m, 8H, Ar-H), 5.35 (s,
13
2H, Ar-NH); C-NMR δ: 180.27, 166.18, 165.27, 155.29, 140.18, 139.19, 134.62, 133.95, 126.41,
124.70, 123.68, 122.58, 121.20, 118.54; Anal. calcd. for C14
H
12FN OS (289.2): C 58.12, H 4.18, N 14.52;
3
found C 58.56, H 4.14, N 14.68.
1-(4'-Chlorophenyl)acyl-3-(2'-aminophenyl) thiourea (5d): Yield 58%, mp 182~184oC. IR (KBr): 3225,
1630, 1240 cm−1; 1H-NMR δ: 12.90 (s, 1H, NH), 11.34 (s, 1H, NH), 7.97-6.80 (m, 8H, Ar-H), 5.23(s,
13
2H, Ar-NH); C-NMR δ: 180.23, 165.19, 154.67, 141.13, 139.37, 136.89, 135.05, 130.78, 128.95,
126.73, 125.47, 121.40, 120.85, 119.44; Anal. calcd. for C14
13.74; found C 56.00, H 3.94, N 13.80.
H
12ClN OS (305.7): C 54.99, H 3.96, N
3
1-(4'-Bromophenyl)acyl-3-(2'-aminophenyl) thiourea(5e): Yield 50%, mp 156~158oC. IR (KBr): 3230,
1
1680, 1245 cm−1; H-NMR δ: 12.70 (s, 1H, NH), 11.44(s, 1H, NH), 7.92-6.75 (m, 8H, Ar-H), 5.19(S,
13
2H, Ar-NH); C-NMR δ: 180.30, 165.21, 154.89, 141.19, 139.17, 136.69, 135.75, 130.80, 128.99,
126.80, 125.87, 122.67, 121.78, 120.56; Anal. calcd. for C14
12.00; found C 48.06, H 3.54, N 12.08.
H
12BrN OS (350.2): C 48.01, H 3.45, N
3

Molecules 2010, 15
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1-(2'-Pyridyl)acyl-3-(2'-aminophenyl) thiourea (5f): Yield 78%, mp 180~181oC. IR (KBr): 3190, 1675,
1250 cm−1; 1H-NMR δ: 12.90 (s, 1H, NH), 11.68 (s, 1H, NH), 8.02-7.83 (m, 4H, Py-H), 7.03-6.75 (m,
4H, Ar-H), 5.20 (s, 2H, Ar-NH); 13C-NMR δ: 180.36, 166.90, 154.48, 151.23, 147.67, 137.50, 130.48,
126.76, 125.51, 124.56, 124.00, 119.96,114.90; Anal. calcd for C13
N 20.57; found C 57.36, H 4.44, N 20.68.
H12
N4OS (272.3): C 57.34, H 4.44,
1-(3'-Pyridyl)acyl-3-(2'-aminophenyl) thiourea (5g): Yield 49%, mp 180~181oC. IR (KBr): 3215, 1665,
1242 cm−1; 1H-NMR δ: 12.91 (s, 1H, NH), 11.64 (s, 1H, NH), 8.02-7.83 (m, 4H, Py-H), 7.03-6.75 (m,
4H, Ar-H), 5.21 (s, 2H, Ar-NH); 13C-NMR δ: 180.36, 166.92, 154.48, 151.23, 147.57, 137.51, 130.48,
126.76, 125.55, 124.56, 124.02, 119.99, 114.90; Anal. calcd. for C13
N 20.57; found C 57.37, H 4.44, N 20.66.
H12
N4OS (272.3): C 57.34, H 4.44,
1-(2'-Furanyl)acyl-3-(2'-aminophenyl) thiourea (5h): Yield 48 %, mp 121~122oC. IR (KBr): 3220,
1640, 1250 cm−1; 1H-NMR δ: 12.91 (s, 1H, NH), 11.64 (s, 1H, NH), 8.09-6.83 (m, 3H, furyl-H), 7.03-
6.75 (m, 4H, Ar-H), 5.20 (s, 2H, Ar-NH); 13C-NMR δ: 180.30, 166.78, 154.45, 147.23, 143.65, 130.48,
125.53, 124.56, 119.63, 115.36, 109.71; Anal. calcd. for C12H11N3O2S (261.3): C 55.16, H 4.24, N
16.08; found C 55.20, H 4.24, N 16.09.
4.3. Biological Assays
All analogues were tested against N. brazilliensis to evaluate their anti-intestinal nematode
activities using the screening method described by Cavier [18].. The compounds were dissolved in
dimethyl formamide (DMF) and serially diluted with water containing Triton X-80 (0.1 mg/L) to get
the required test concentrations. Each rat in the respective group received 10 mg/kg body weight using
oral candle. These compounds were tested on ten groups of rats, each containing three rats.
Evaluations were based on a percentage scale of 0-100, in which 100 was total kill and 0 was no
activity. All results are shown in Table 1. The reference compound was albendazole, and water
containing DMF (0.5 mg/L) and Triton X-80 (0.1 mg/L) was used as a negative control. The trials
commenced on the 10th day after infecting each of 30 rats with 250 N. brazilliensis larvae. The
percentage deparasitization was calculated using the following formula:
N n
X 100
N
where N = average number of worms found in the control animals and n = average number of worms
found in the groups of treated animals (including worms in rats at post mortem).
Acknowledgements
The authors wish to acknowledge that this project is supported by National Institute for Diseases
(2010A102) and Shanghai Health Bureau (2009Y109).

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Sample Availability: Samples of the compounds 5a-5h are available from the authors.
© 2010 by the authors; licensee MDPI, 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/).