Synthesis of β-hydroxypropanoic acid derivatives as potential anti-inflammatory, analgesic and antimicrobial agents

Article abstract of DOI:10.1002/ardp.200600016

A series of new 3-(substituted) 3-hydroxy-propanoic acid ethyl esters 1a-c, hydrazides 2a-c, thiosemicarbazides 3a-f, and semicarbazides 3g, 3h has been synthesized. Cyclization of compounds 3a-d in basic medium yielded 1,2,4-triazole-5-thiones 4a-d. On t

Full text of DOI:10.1002/ardp.200600016

378
Arch. Pharm. Chem. Life Sci. 2006, 339, 378–387
Full Paper
Synthesis of b-Hydroxypropanoic Acid Derivatives as Potential
Anti-inflammatory, Analgesic and Antimicrobial Agents
Hamdy M. Abdel-Rahman1 and Mostafa A. Hussein2
1 Pharmaceutical Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt
2 Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt
A series of new 3-(substituted) 3-hydroxy-propanoic acid ethyl esters 1a–c, hydrazides 2a–c, thio-
semicarbazides 3a–f, and semicarbazides 3g, 3h has been synthesized. Cyclization of com-
pounds 3a–d in basic medium yielded 1,2,4-triazole-5-thiones 4a–d. On the other hand, reaction
of hydrazides 2a–c with CS2 in basic medium afforded 1,3,4-oxadiazole-5-thiones 5a–c. All the
synthesized compounds were characterized by their physical and spectral analyses data. The
newly synthesized compounds were evaluated for their anti-inflammatory, analgesic, and anti-
microbial activities. Compounds 1c, 3g, 4a, 4b, 4c, and 5c exhibited comparable anti-inflamma-
tory activity to that of indomethacin and compounds 1c, 4c, and 5c were more analgesics than
acetyl salicylic acid. Compounds 4b, 4c, and 5c showed superior GI safety profile (33.3%, 33.3%
and 50.0% ulceration) than that of indomethacin (100% ulceration) at 100 mg/kg oral dose. Com-
pounds 4b, 4c, and 5c were also non-toxic with a median lethal dose (LD50) up to 200 mg/kg. The
antibacterial and antifungal screenings identified compounds 3c, 4b, 4d, 5a, and 5b as the most
effective against a variety of tested microorganisms.
Keywords: b-Hydroxypropanoic acid derivatives / Anti-inflammatory / Antimicrobial / 1,2,4-Triazoles / 1,3,4-Oxadia-
zoles /
Received: January 23, 2006; accepted: March 16, 2006
DOI 10.1002/ardp.200600016
Introduction
Bacterial infections often produce pain and inflamma-
tion. In normal practice, two groups of agents (che-
motherapeutic, analgesic, and anti-inflammatory) are
prescribed simultaneously. Compounds possessing all
three activities are not common. Among the many
reported classes of anti-inflammatory agents, aryl and
heteroaryl (alkyl) carboxylic acids possessed wide clinical
Figure 1. Chemical structure of F-776 (I).
applications but with the incidence of gastrointestinal
damage and ulceration [1, 2]. b-Hydroxy carboxylic acid
derivatives as salicylic acid and its derivatives are well-
known drugs for their anti-inflammatory activity [1].
Furthermore, other b-hydroxy carboxylic acid derivatives
as F-776 (I) (Fig. 1) and compound 2c (Scheme 1) are
reported to possess a promising anti-inflammatory activ-
ity [3, 4]. On the other hand, 1,2,4-triazoles and 1,3,4-oxa-
diazole derivatives were also reported to possess a variety
of pharmacological activities such as anti-inflammatory
[5–12], antitubercular [13], antifungal [14], and antican-
cer activities [15].
Correspondence: Hamdy M. Abdel-Rahman, Pharmaceutical Medicinal
Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut
71526, Egypt.
E-mail: hamdym@acc.aun.edu.eg
Fax: +20 88 233-2776
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Arch. Pharm. Chem. Life Sci. 2006, 339, 378–387
Anti-inflammatory b-Hydroxypropanoic acid Derivatives
379
Results and discussion
Chemistry
Ethyl 3-(substituted) 3-hydroxypropionates 1a–c were
synthesized starting from the readily available p-bromo
and p-chlorobenzaldehydes or benzophenone via Refor-
matsky reaction [4, 18, 19]. Hydrazinolysis of the esters
1a–c afforded the corresponding key intermediate
hydrazides 2a–c (Scheme 1). Reaction of the hydrazides
2a–c with substituted isothiocyanate and phenyl isocya-
nate, yielded thiosemicarbazides 3a–f and semicarba-
zides 3g and 3h, respectively. Cyclization of 3a–d was car-
ried out through reflux in aqueous NaOH solution afford-
ing substituted 1,2,4-triazole-5-thiones 4a–d in good
yields. On the other hand, reaction of the hydrazides 2a–
c with CS2 in the presence of aqueous NaOH afforded the
1,3,4-oxadiazole-2-thiones 5a–c (Scheme 2). The struc-
tures of the synthesized compounds were verified by IR,
1H-NMR, mass spectra as well as the elemental analyses.
Briefly, IR spectra of compounds 2a–c and 3a-h showed a
strong absorption bands at 3545–3140 cm– 1 (OH, NH, or
NH2 stretching), in addition to a strong absorption band
at 1686–1638 cm– 1 for the amidic carbonyl group. Disap-
pearance of the later band upon formation of com-
Scheme 1. Synthesis route for compounds 2a–c.
In view of these facts and in continuation of our search
for novel anti-inflammatory agents [16, 17], the present
paper involves the design, synthesis, and biological eva-
luation of a series of 3-(substituted) 3-hydroxypropanoic
acid derivatives and their cyclized products.
Scheme 2. Synthesis route of compounds
4a–d and 5a–c.
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H. M. Abdel-Rahman et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 378–387
Table 1. Physical data of 3-substituted 3-hydroxypropanoic acid derivatives (2, 3).
No
R1
R2
R
M. p.
(8C)
Yield
(%)
Mol. Formula
(Mol. Wt.)
2a
2b
2c
3a
3b
3c
3d
3e
3f
Cl
Br
H
Cl
Br
H
Cl
Br
H
Cl
Br
H
H
C6H5
H
H
C6H5
H
H
C6H5
H
H
H
H
H
211-3
212
80
80
95
82
85
77
80
84
75
78
80
C9H11ClN2O2 (214.65)
C9H11BrN2O2 (259.10)
C15H16N2O2 (265.30)
C12H16ClN3O2S (301.79)
C12H16BrN3O2S (346.24)
C18H21N3O2S (301.79)
C16H16ClN3O2S (349.84)
C16H16BrN3O2S (394.29)
C22H21N3O2S (391.49)
C16H16N3O3 (333.77)
C16H16BrN3O3 (378.22)
114-6
170-2
164-5
160-2
158-9
183-5
178-80
200-2
200
C(S)NHC2H5
C(S)NHC2H5
C(S)NHC2H5
C(S)NHC6H5
C(S)NHC6H5
C(S)NHC6H5
C(O)NHC6H5
C(O)NHC6H5
3g
3h
Table 2. Physical data of 1,2,4-triazoles 4a–d and 1,3,4-oxadiazoles 5a–c.
No
R1
R2
R3
Y
M. p.
(8C)
Yield
(%)
Mol. Formula
(Mol. Wt.)
4a
4b
4c
4d
5a
5b
5c
Cl
Br
H
Cl
Cl
Br
H
H
H
C6H5
H
H
C2H5
C2H5
C2H5
C6H5
–
N
N
N
N
O
O
O
170-2
70-2
155-8
94-6
175-7
175-6
155-6
78
80
70
72
75
87
70
C12H14ClN3OS (283.78)
C12H14BrN3OS (328.23)
C18H19N3OS (325.43)
C16H14ClN3OS (331.82)
C10H9ClN2O2S (256.71)
C10H9BrN2O2S (301.16)
C16H14N2O2S (298.63)
H
C6H5
–
–
pounds 4a–d or 5a–c is a preliminary indication for the tion of the carboxylic acid hydrazides or thiosemicaba-
1
cyclization process. Moreover, H-NMR spectra were in zides to azoles such as 1,2,4-triazoles and 1,3,4-oxadia-
accordance with the proposed structures of the synthe- zoles were also reported to yield potent nonulcerogenic
sized compounds. (Details of structures, yields, melting derivatives [6, 7].
points, and spectral data are shown in Tables 1 and 2 and
in the experimental section, part 3.)
In the present study, twelve compounds namely 1c, 2c,
3f, 3g, 3h, 4a–d, and 5a–c were selected to test their anti-
inflammatory activity using the carragenan-induced rat
paw edema method [23]. Indomethacin was used as a
reference drug. All compounds and the reference drug
Biological screening
Anti-inflammatory activity
Previously reported results indicated that the conversion were tested at a dose level of 10 mg/kg and the results are
of the free acid moiety of NSAIDs into amides resulted in presented in Table 3. After 3 h of administration, com-
more potent derivatives [20, 21] and some of them pounds 1c, 3g, 4a, 4b, 4c, and 5c exhibited anti-inflamma-
showed reduced ulcerogenic effects [22]. Further cycliza- tory activities similar to that of indomethacin while the
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Arch. Pharm. Chem. Life Sci. 2006, 339, 378–387
Anti-inflammatory b-Hydroxypropanoic acid Derivatives
381
Table 3. Effect of compounds 1, 2, 3, 4, 5, and indomethacin on carrageenan-induced rat paw edema test (10 mg/kg i.p. dose).
No
Edema inhibition (%)*
0.5 h
1 h
2 h
3 h
5 h
Indomethacin
6.7 l 0.99
3.0 l 0.81
3.0 l 0.78
1.0 l 0.35
0.0
2.2 l 0.44
3.4 l 0.83
3.4 l 0.56
0.0
6.7 l 0.79
0.0
3.4 l 0.86
3.4 l 0.92
12.9 l 0.79
9.7 l 0.59
12.9 l 0.91
6.0 l 0.49
9.6 l 1.27
6.5 l 0.85
9.7 l 0.95
16.1 l 1.37
9.7 l 0.31
6.5 l 0.42
9.7 l 1.27
12.9 l 0.93
12.9 l 1.36
21.2 l 0.95
30.3 l 0.75
24.2 l 1.02
12.1 l 0.59
24.2 l 1.68
15.2 l 0.66
18.2 l 0.61
24.2 l 0.96
24.2 l 0.92
12.1 l 0.98
24.2 l 1.33
27.3 l 0.85
30.3 l 1.27
36.4 l 1.08
36.4 l 0.65
24.2 l 0.82
21.1 l 0.91
30.3 l 1.27
18.2 l 1.39
30.3 l 0.86
30.3 l 1.16
27.3 l 1.29
21.2 l 0.28
24.2 l 1.70
24.2 l 1.02
33.4 l 0.88
44.0 l 0.83
38.2 l 0.97
35.3 l 0.69
17.6 l 0.87
35.3 l 0.90
26.5 l 0.65
32.4 l 0.93
38.2 l 1.31
41.2 l 0.78
20.6 l 0.41
23.5 l 1.69
26.3 l 0.65
38.2 l 0.84
1c
2c
3f
3g
3h
4a
4b
4c
4d
5a
5b
5c
* Each value represents the mean l S.E. and all showed at least significant difference at P a 0.05 in comparison with control group.
Table 4. Analgesic activity of test compounds in the hot-plate test (10 mg/kg i.p dose).
No
Reaction time (s)a)
3 h
0.5 h
1 h
2 h
4 h
5 h
Control
19.8 l 0.45
20.5 l 0.92
55.1 l 1.11
37.6 l 0.73
33.3 l 0.46
32.3 l 0,98
39.1 l 1.15
38.5 l 0.91
38.9 l 0.94
21.1 l 1.02
45.4 l 1.22
31.6 l 0.88
24.2 l 1.02
23.1 l 0.78
41.1 l 1.36
39.5 l 0.13
39.0 l 0.83
22.4 l 0.61
36.0 l 1.02
30.1 l 0.72
26.0 l 0.94
24.5 l 0.42
34.9 l 1.08
24.1 l 1.46
28.2 l 1.32
25.0 l 0.33
35.9 l 1.44
26.2 l 0.96
20.7 l 0.56
25.0 l 1.11b)
27.3 l 0.87
21.6 l 1.16
25.9 l 0.28
21.1 l 0.47
28.6 l 1.36
21.3 l 0.44b)
20.1 l 0.32
23.3 l 0.99
25.8 l 0.45
22.1 l 0.95
25.7 l 0.86
Acetyl salicylic acid 36.1 l 0.92
1c
2c
3g
4b
4c
5c
37.6 l 0.81
23.0 l 0.88
30.3 l 1.28
33.9 l 0.86
39.2 l 1.17
37.7 l 1.26
a)
Each value represents the mean l S.E. and all showed at least significant difference at P a 0.05 in comparison with control group.
not significant.
b)
rest of the test compounds gave around half of the activ- 104.3%, respectively) while compound 4b showed nearly
ity of indomethacin. After 5 h, compounds 1c, 2c, 3g, 4b, the same activity as that of acetyl salicylic acid (93.8%).
4c, and 5c exhibited 79–93% anti-inflammatory activity
compared to that of indomethacin while the rest of the Ulcerogenic effects
test compounds gave 38–72%. As a general result, all The administration of most nonstereoidal anti-inflam-
cyclized compounds such as 5c showed an improved anti- matory drugs (NSAIDs) has been limited because of the
inflammatory activity compared to their open analogs as incidence of gastrointestinal damage (bleeding, ulcera-
2c in accordance with the reported results [6, 7].
tion). Compounds 4b, 4c, and 5c, which exhibited both
potent an anti-inflammatory and analgesic profile in the
pre-mentioned animal models, were evaluated for their
Analgesic activity
The most active anti-inflammatory compounds 1c, 2c, 3g, ulcerogenic effect in rats [25] and the results are pre-
4b, 4c, and 5c were tested for their analgesic activity rela- sented in Table 5. The test compounds showed superior
tive to acetyl salicylic acid at a dose level of 10 mg/kg [24] GI safety profile (33.3%, 33.3%, and 50.0%) at an oral dose
and the results are presented in Table 4. At the 3 h inter- of 100 mg/kg, respectively when compared to indo-
val, the results revealed that the analgesic activity of the methacin, which was found to cause 100% ulceration.
test compounds 4b, 4c, and 5c is about 90.4%, 86.9%, and
85.9% of acetyl salicylic acid, respectively. However, com- Acute toxicity
pounds 1c, 4c, and 5c were more potent than acetyl sal- The median lethal dose (LD50) of the most effective com-
icylic acid at the 5 h interval (104.2%, 108.5%, and pounds 4b, 4c, and 5c was determined in mice [26] that
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382
H. M. Abdel-Rahman et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 378–387
Table 5. Gastric ulcerogenic effects of compound 4b, 4c, 5c,
and indomethacin at 100 mg/kg.
were injected (i.p.) with graded doses of the test com-
pounds and the LD50 was determined. Compounds 4b, 4c,
and 5c were also nontoxic with a median lethal dose
(LD50) up to 200 mg/kg.
Compound
Ratio of ulcer- % Ulceration Ulcer index
ated animals
(mean lS.E.)
Indomethacin 6/6
100
33
33
1.01 l 0.1
1.1 l 0.2
1.8 l 0.1
1.0 l 0.1
Antimicrobial activities
4b
4c
5c
2/6
2/6
3/6
Antibacterial activity
50
The newly synthesized compounds (2a–b, 3a–g, 4a–d,
and 5a–c) were tested for their in vitro antibacterial activ-
ity against Bacillus cereus, Micrococcus roseus, and M. loteus
as representatives of Gram-positive strains and Escherichia
coli and Serratia rodenii, as representatives of Gram-nega-
tive ones, using a reported method as described in Experi-
mental (part 3) [27]. The results are cited in Table 6 and
expressed as inhibition zones in mm in comparison to
penicillin G as a reference drug.
Table 6. The antibacterial zones of inhibition (mm) of the test
compounds.
No
Bacteria
Bacillus Micrococcus M. loteus E. coli
Serrati
rodenii
cereus
roseus
As shown in Table 6, compounds 2a–c are either inac-
tive or possessed slight activity against one or more of
the used microorganisms compared to the reference
drug. It is also noted that compound 3c showed higher
activity than penicillin G against all microorganisms. On
the other hand, the antibacterial activity was greatly
improved when compounds 2a–c and 3a–c were
cyclized to their corresponding 1,2,4-triazole and 1,3,4-
oxadiazole derivatives. Compounds 4a, 4c, 4d, 5a, and 5b
are more effective than the reference drug against most
of the tested microorganisms.
2a
2b
2c
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
5a
5b
5c
–
–
–
–
–
8
13
8
–
–
14
–
15
–
–
14
–
–
12
–
–
–
–
–
8
–
16
–
8
–
–
15
–
14
–
12
8
–
–
–
–
–
–
14
–
7
–
15
–
17
–
–
12
8
–
12
12
–
–
10
16
–
–
–
–
12
–
14
13
12
15
–
8
14
15
–
12
–
–
–
16
19
15
14
16
10
Antifungal activity
Compounds (2a–b, 3a–g, 4a–d, and 5a–c) were tested
for their in vitro antifungal activity using the standard
Penicillin G 15
–
11
11
Table 7. The antifungal zones of inhibition (mm) of the test compounds.
No
Aspergillus
Candida al-
bicans
Trichophyton
Microsporium
gypseum
A. umigatus A. terreus A. flavus A. niger
A. ochraceous
T. rubrum T. tonsurans T. georgi
2a
2b
2c
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
5a
5b
5c
–
–
–
–
–
7
–
–
–
–
–
10
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12
–
–
–
–
–
–
–
–
14
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
9
–
–
–
–
–
–
–
–
14
–
–
–
–
–
–
–
15
12
8
–
30
–
–
–
–
–
14
8
–
8
–
10
–
–
12
12
12
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12
20
–
–
25
–
–
–
–
–
–
–
–
–
–
–
–
–
13
18
–
–
25
–
–
20
–
–
20
20
–
–
–
–
13
–
18
25
20
–
8
8
8
7
8
–
–
7
–
–
–
–
–
–
–
–
–
–
–
10
9
7
12
10
–
10
13
8
–
Fluconazole –
–
–
–
–
15
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Arch. Pharm. Chem. Life Sci. 2006, 339, 378–387
Anti-inflammatory b-Hydroxypropanoic acid Derivatives
383
IR spectra (KBr discs) were recorded on a Shimadzu IR-470
1
spectrometer (Shimadzu, Japan). H-NMR spectra were scanned
on a Varian EM-360 L NMR spectrometer (60 MHz), (Varian, USA).
Chemical shifts are expressed in d values (ppm) relative to tetra-
methylsilane (TMS) as an internal standard, using DMSO-d6 as
solvent. Coupling constants (J values) are expressed in Hertz (Hz).
Elemental analyses were performed at the Unit of Microanalysis,
Assiut University, Assiut, Egypt. MS spectra were recorded on a
JEOL JMS 600 mass spectrometer (JEOL, Japan) at the Microanaly-
tical Center, Faculty of Science, Cairo University, Egypt.
Most of the chemicals used are of commercial grade: p-bromo-
benzaldehyde, p-chlorobenzaldehyde, benzophenone, ethyl bro-
moacetate (Aldrich, Germany), hydrazine hydrate and carboxy
methyl cellulose (El Nasr Pharm. Co., Egypt), carrageenan
(Sigma, USA), carbon disulphide (Riedel-de Haꢀn), indomethacin
(Nile Co., Egypt) and acetyl salicylic acid (ADCO Co., Egypt).
Potato dextrose agar (PDA) or sabouraud agar (SA) media are pre-
pared in the Department of Botany, Faculty of Science, Assiut
University.
Figure 2. Chemical structure of fluconazole (II).
agar disc diffusion method [27] against Aspergillus fumiga-
tus, A. terreus, A. flavus, A. nige, and A. ochraceous thom, Can-
dida albicans (Robin) Berkhout, Trichophyton rubrum (Cas-
tellani) Sabouraud, T. tonsurans, T. georgi, and Microspor-
ium gypseum gruby. The results of the antifungal activity
are given in Table 7 and expressed as inhibition zones in
mm using fluconazole as a reference drug.
The starting 3-(substituted) 3-hydroxy-propanoic acid ethyl
esters (1a–c) were prepared via Reformatsky reaction of ethyl
bromoacetate with aldehydes or ketones following previously
reported literature methods [4, 18, 19].
Again, compound 2a–c, 3a–g (except 3c) showed slight
antifungal activity against most of the used fungi. On the
other hand, the antifungal activity was greatly improved
with the cyclized 1,2,4-triazole-5-thione and 1,3,4-oxadia-
zole-2-thione derivatives, which might be attributed to
their structural similarity of azoles antifungal agents as
fluconazole (II) (Fig. 2). Compounds 4d, 5a, and 5b are the
most active in this series with compound 5a showing a
broad-spectrum antifungal activity.
General method for preparation of 3-(substituted)-3-
hydroxy propanoic acid hydrazides (2)
3-(Substituted) 3-hydroxy-propanoic acid ethyl esters 1a–c
(0.01 mol) and hydrazine hydrate (0.02 mol) were refluxed in
absolute ethanol (20 mL) for 3 h. The reaction mixture was con-
centrated, cooled, and poured onto ice-cooled water. The solid
thus separated out was filtered, dried, and crystallized from
ethanol (Scheme 1, Table 1).
Aspergillosis is one of the most serious fungal infec-
tions where fluconazole does not appear to be effective in
the prevention or treatment of such infections [28]. As
shown in Table 7, fluconazole was inactive against all
Aspergillus spp., while compounds 3b, 3c, 3e, 4a, 4b, 4d,
5a, 5b, and 5c were active against one or more of such spe-
cies. These results are in accordance with the reported
data about the antifungal activity of azoles, which indi-
cate that they are active against dermatophytes such as
Trichophyton, Epidermophyton, Microsporum spp., and yeast-
like fungi such as Candida albicans but are inactive
against Aspergillus spp. [14, 28]. The difference in the
obtained antifungal activity between the tested com-
pounds and fluconazole might be attributed to their
different behavior towards the fungal cytochrome P-450,
which is responsible for the growth of fungi [14].
3-(4-Chlorophenyl)-3-hydroxy propanoic acid hydrazide
(2a)
1H-NMR (DMSO-d6) d: 2.5 (d, 2H, CH2, J = 8), 3.6 (brs, 2H, NH2), 5.0
(t, 1H, CH, J = 8 and 8), 7.2 (s, 4H, Ar-H) 8.5 (s, 1H, NH). IR (KBr)
cm– 1: 3470, 3300, 3165, 1640, 1610. Anal. calcd. for C9H11ClN2O2:
C, 50.36; H, 5.17; N, 13.05 Found: C, 50.28; H, 5.46; N, 12.99.
3-(4-Bromophenyl)-3-hydroxypropanoic acid hydrazide
(2b)
1H-NMR (DMSO-d6) d: 2.4 (d, 2H, CH2, J = 8), 3.7 (brs, 2H, NH2), 5.0
(t, 1H, CH, J = 8 and 7), 7.5 (dd, 4H, Ar-H, J = 10 and 8). IR (KBr) cm–
1: 3470, 3295, 3135, 2910, 1638, 1486, 1073, 1010, 820, 734. Anal.
calcd. for C9H11BrN2O2: C, 41.72; H, 4.28; N, 10.81 Found: C,
41.90; H, 4.47; N, 10.96.
3-Hydroxy-3,3-diphenylpropanoic acid hydrazide (2c) [4]
1H-NMR (DMSO-d6) d: 2.5 (s, 2H, CH2), 3.4 (brs, 2H, NH2), 6.8 (s, 1H,
OH), 7.2-7.8 (m, 10H, Ar-H), 9.5 (brs, 1H, NH). IR (KBr) cm-1: 3470,
3300, 3165, 1640, 161.
Experimental
General method for preparation of 3-(substituted)
3-hydroxypropanoic acid thiosemicarbazides (3a–f)
and semicarbazides (3g, h)
A mixture of the corresponding hydrazide 2a–c (0.01 mol) and
the appropriate isothiocyanate or isocyanate derivative
(0.01 mol) in ethanol (20 mL) was heated under reflux for 2–3 h.
Melting points were determined on an electrothermal melting
point apparatus (Stuart Scientific, Model SMM, UK) and were
uncorrected. TLC was carried out using silica gel 60 F254 pre-
coated sheets (E. Merck, Darmstadt, Germany) and was visua-
lized using UV lamp (Spectroline Model CM 10, USA), and/or
iodine stain.
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The formed solid was filtered, washed with ethanol, and crystal-
lized from ethanol (Scheme 2, Tables 1).
each of 1H, 3H). IR (KBr) cm– 1: 3470, 3340, 3215, 1639, 1605,
1590. Anal. calcd. for C16H16ClN3O3: C, 57.58; H, 4.83; N, 12.59.
Found: C, 55.88; H, 5.12; N, 12.67.
1-[3-(4-Chlorophenyl)-3-hydroxypropanoyl]-4-
ethylthiosemicarbazide (3a)
1-[3-(4-Bromophenyl)-3-hydroxypropanoyl]-4-
1H-NMR (DMSO-d6) d: 1.1 (t, 3H, CH2CH3, J = 8 and 8), 2.5 (d, 2H, CH2,
J = 7), 3.3–3.8 (m, 2H, CH2CH3), 4.9–5.3 (m, 1H, CH), 5.9 (d, 1H,
OH, J = 8), 7.6 (s, 4H, Ar-H), 7.7 (t, 1H, NH), 9.5 (s, 1H, NH), 10.0 (s,
1H, NH). IR (KBr) cm– 1: 3250, 2975, 1686, 1555, 828. Anal. calcd.
for C12H16ClN3O2S: C, 47.76; H, 5.34; N, 13.92. Found: C, 47.89; H,
5.40; N, 13.98.
phenylsemicarbazide (3h)
1H-NMR (DMSO-d6) d: 2.5 (d, 2H, CH2, J = 8), 4.3 (brs, 1H, OH), 5.2 (t,
1H, CH, J = 7 and 7), 7.4–8.0 (m, 9H, Ar-H), 8.4, 8.8, 10.1 (3 brss
each of 1H, 3NH). IR (KBr) cm– 1: 3415, 3225, 3055, 2930, 1674,
1610, 1587, 1483, 1061, 746, 617. Anal. calcd. for C16H16BrN3O3:
C, 50.81; H, 4.26; N, 11.11. Found: C, 51.04; H, 3.68; N, 11.16.
1-[3-(4-Bromophenyl)-3-hydroxypropanoyl]-4-
ethylthiosemicarbazide (3b)
1H-NMR (DMSO-d6) d: 1.2 (t, 3H, CH2CH3, J = 8 and 8), 2.5 (d, 2H, CH2,
J = 7), 3.3–3.8 (m, 2H, CH2CH3), 4.5 (brs, 1H, NH), 5.2 (t, 1H, CH, J =
6 and 6), 5.7 (brs, 1H, OH), 7.4–8.0 (m, 4H, Ar-H), 9.3 (brs, 2H,
2NH). IR (KBr) cm– 1: 3435, 3295, 3056, 2945, 1655, 1410, 1180,
1010, 845, 715. Anal. calcd. for C12H16BrN3O2S: C, 41.63; H, 4.66;
N, 12.14. Found: C, 41.90; H, 4.80; N, 11.95.
General method for preparation of 3-[2-(substituted)-
2-hydroxyethyl]-4-alkyl/aryl-4,5-dihydro-1H-1,2,4-
triazole-5-thiones (4a–d)
The appropriate thiosemicarbazide, 3a–d (0.01 mol) was dis-
solved in 6% aqueous NaOH solution (20 mL) and refluxed for
4 h. The resulting solution was cooled, filtered, and acidified
with dilute HCl to pH 5. The solid formed was filtered off,
washed with water, dried, and crystallized from aqueous etha-
nol (Scheme 2, Table 2).
1-[3-Hydroxy-3,3-diphenylpropanoyl]-4-
ethylthiosemicarbazide (3c)
1H-NMR (DMSO-d6) d: 1.1 (t, 3H, CH3, J = 7 and 7), 3.3–4.0 (m, 4H,
2CH2), 6.7 (brs, 1H, OH), 7.8–8.4 (m, 11H, Ar-H and NH), 9.9 (brs,
IH, NH), 10.6 (brs, 1H, NH). IR (KBr) cm– 1: 3545, 3410, 3190, 2935,
1661, 1636, 1560, 1527, 1215. Anal. calcd. For C18H21N3O2S: N,
12.23; S, 9.34. Found: N, 12.11; S, 9.23.
3-[2-(4-Chlorophenyl)-2-hydroxyethyl]-4-ethyl-4,5-
dihydro-1H-1,2,4-triazole-5-thione (4a)
1H-NMR (DMSO-d6) d: 1.4 (t, 3H, CH2CH3, J = 7 and 7), 3.2 (d, 2H, CH2,
J = 8), 3.6 (brs, 1H, NH), 4.0–4.4 (q, 2H, CH2CH3), 5.1 (t, 1H, CH, J =
7 and 8), 6.1 (brs, 1H, OH), 7.7–7.8 (m, 4H, Ar-H). IR (KBr) cm– 1
:
1-[3-(4-Chlorophenyl)-3-hydroxypropanoyl]-4-
3555, 3415, 3245, 3160, 2970, 1566, 1488. Anal. calcd. for
C12H14ClN3OS.H2O: C, 47.76; H, 5.34; N, 13.92; S, 10.62. Found: C,
48.16; H, 4.81; N, 14.21; S, 10.31.
phenylthiosemicarbazide (3d)
1H-NMR (DMSO-d6) d: 2.6 (d, 2H, CH2, J = 7), 5.0 (t, 1H, CH, J = 8 and
8), 6.1 (brs, 1H, OH), 7.2–7.7 (m, 9H, Ar-H), 9.4, 9.7, 10.1 (3 brss,
each of 1H, 3H). IR (KBr) cm– 1: 3420, 3265, 1659, 1541, 1472.
Anal. calcd. for C16H16ClN3O2S: C, 54.93; H, 4.61; N, 12.01. Found:
C, 55.84; H, 4.89; N, 11.69
3-[2-(4-Chlorophenyl)-2-hydroxyethyl]-4-phenyl-4,5-
dihydro-1H-1,2,4-triazole-5-thione (4b)
1H-NMR (DMSO-d6) d: 2.9 (d, 2H, CH2, J = 8), 3.6 (brs, 1H, NH), 4.9 (t,
1H, CH, J = 7 and 7), 5.8 (brs, 1H, OH), and 7.3–7.8 (m, 9H, Ar-H).
IR (KBr) cm– 1: 3415, 3300, 3190, 2930, 1595, 1494. Anal. calcd.
for C16H14ClN3OS: C, 57.91; H, 4.25; N, 12.66; S, 9.66. Found: C,
57.40; H, 4.29; N, 12.26; S, 9.39
1-[3-(4-Bromophenyl)-3-hydroxypropanoyl]-4-
phenylthiosemicarbazide (3e)
1H-NMR (DMSO-d6) d: 2.7 (d, 2H, CH2, J = 7), 5.3 (t, 1H, CH, J = 7 and
7), 6.0 (brs, 1H, OH), 7.3–8.0 (m, 9H, m, Ar-H), 9.7, 10.1, 10.5 (3
brss each of 1H, 3NH). IR (KBr) cm– 1: 3420, 3295, 3020, 2910,
1639, 1544, 1487, 1073, 1010, 821, 734. Anal. calcd. for
C16H16BrN3O2S: C, 48.74; H, 4.09; N, 10.66. Found: C, 47.82; H,
4.31; N, 10.24.
3-[2-(4-Bromophenyl)-2-hydroxyethyl]-4-ethyl-4,5-
dihydro-1H-1,2,4-triazole-5-thione (4c)
1H-NMR (DMSO-d6) d: 1.2 (t, 3H, CH2CH3, J = 7 and 7), 3.1 (d, 2H, CH2,
J = 7), 3.5 (brs, 1H, NH), 4.2 (q, 2H, CH2CH3), 5.1 (t, 1H, CH, J = 7 and
7), 5.9 (brs, 1H, OH), 7.6 (dd, 4H, Ar-H, J = 9 and 10). IR (KBr) cm– 1
:
1-[3-Hydroxy-3,3-diphenylpropanoyl]-4-
3465, 3045, 2980, 1638, 1490, 1187, 823, and 765. EI-MS: m/z 325
[M]9+, 326 [M+1]+, 182. Anal. calcd. for C12H14BrN3OS: C, 43.91; H,
4.30; N, 12.80; S, 9.77. Found: C, 44.04; H, 4.40; N, 12.82; S, 9.63.
phenylthiosemicarbazide (3f)
1H-NMR (DMSO-d6) d: 3.6 (s, 2H, CH2), 6.8 (brs, 1H, OH), 7.5–8.4
(m, 15H, Ar-H), 10.0 (brs, 1H, NH), 10.5 (brs, 1H, NH), 10.9 (brs,
1H, NH). IR (KBr) cm– 1: 3455, 3370, 3285, 3200, 1664, 1636, 1535.
Anal. calcd. for C22H21N3O2S: C, 67.50; H, 5.41; N, 10.73; S, 8.19.
Found: C, 67.58; H, 5.53; N, 10.71; S, 8.55.
3-[2-(4-Bromophenyl)-2-hydroxyethyl]-4-phenyl-4,5-
dihydro-1H-1,2,4-triazole-5-thione (4d)
1H-NMR (DMSO-d6) d: 2.8 (d, 2H, CH2, J = 7), 4.8 (t, 1H, CH, J = 7 and
7), 4.2 (brs, 1H, NH), 5.5 (brs, 1H, OH), 7.1–7.9 (m, 9H, Ar-H). IR
(KBr) cm– 1: 3415, 3070, 2960, 1564, 1440, 1068, 827, 741. Anal.
calcd. for C16H14BrN3OS: C, 51.07; H, 3.75; N, 11.17; S, 8.52. Found:
C, 51.04; H, 3.67; N, 11.16; S, 8.53.
1-[3-(4-Chlorophenyl)-3-hydroxypropanoyl]-4-
phenylsemicarbazide (3g)
1H-NMR (DMSO-d6) d: 2.6 (d, 2H, CH2, J = 7), 5.0–5.3 (m, 1H, CH),
6.1 (brs, 1H, OH), 7.2–7.7 (m, 9H, Ar-H), 8.3, 8.7, and 9.9 (3 brss,
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Arch. Pharm. Chem. Life Sci. 2006, 339, 378–387
Anti-inflammatory b-Hydroxypropanoic acid Derivatives
385
1 h of carrageenan injection to detect the inflammation induced
by carrageenan. Test compounds were injected i.p. at doses of
10 mg/kg to the rats 1 h after injection of carageenan. The con-
trol group received the vehicle (0.5% NaCMC in water) while the
reference group received indomethacin at 10 mg/kg.
The difference between the thicknesses of the two paws was
taken as a measure of edema. The measurement was carried out
at 0.5, 1, 2, 3, and 5 h after injection of the test compounds, refer-
ence drug, and the vehicle. The results of anti-inflammatory
activity of the test compounds and the reference drug are listed
in Table 3. The percentage edema inhibition was calculated as
follows:
General method for preparation of 5-(substituted)
1,3,4-oxadiazole-2(3H)-thiones (5a–c)
To a solution of the hydrazides 2a–c (0.01 mol) in ethanol
(15 mL) at 08C, carbon disulphide (2 mL) and potassium hydro-
xide (0.6 g) were added. After addition, the reaction mixture was
refluxed until the evolution of H2S ceased. Excess solvents were
evaporated under reduced pressure and the residue was dis-
solved in water and acidified with dilute HCl to pH l 5. The pre-
cipitate was filtered off, dried, and crystallized from ethanol
(Scheme 2, Table 2).
5-[2-(4-Chlorophenyl)-2-hydroxyethyl]-1,3,4-oxadiazole-
%edema inhibition = (Va–Vb/Va)6100
2(3H)-thione (5a)
1H-NMR (DMSO-d6) d: 3.1 (d, 2H, CH2, J = 7), 3.5 (brs, 1H, NH), 5.2 (t,
1H, CH, J = 7 and 7), 6.0 (brs, 1H, OH), and 7.6 (s, 4H, Ar-H). IR (KBr)
cm– 1: 3465, 3370, 1600, 1494. Anal. calcd. For C10H9ClN2O2S: C,
46.79; H, 3.53; N, 10.91; S, 12.49. Found: C, 47.77; H, 3.51; N,
10.46; S, 12.97.
where Va is the increase in paw size in the absence of the test
compounds or indomethacin and Vb is the increase in paw size
after injection of the test compounds or indomethacin
Analgesic activity
The analgesic activity of the most effective anti-inflammatory
derivatives, compounds 1c, 2c, 3g, 4b, 4c, and 5c was determined
in mice using the hot plate method [24] using acetyl salicylic
acid as reference drug. In this method, the latency time taken
(seconds) by the mouse to lick its feet or to jump within a Plexi-
glas cylinder placed on a hot plate surface (558C) was deter-
mined. This reaction time was taken as the end point and the
increase in hot plate latency time was taken as a measure of the
analgesic activity.
5-[2-(4-Bromophenyl)-2-hydroxyethyl]-1,3,4-oxadiazole-
2(3H)-thione (5b)
1H-NMR (DMSO-d6) d: 3.1 (d, 2H, CH2, J = 7), 5.1 (t, 1H, CH, J = 7 and
7), 6.3 (brs, 2H, OH and NH), 7.6 (dd, 4H, Ar-H, J = 13 and 10). IR
(KBr) cm– 1: 3485, 3050, 2975, 1494, 1180, 824, 768. Anal. calcd.
For C10H9BrN2O2S: C, 39.88; H, 3.01; N, 9.30. Found: C, 39.35; H,
2.85; N, 9.07.
Male adult albino mice (20–25 g) were divided into eight
groups, each of five animals. Six test compounds and the refer-
ence drug were injected i. p. at a dose level of 10 mg/kg into mice.
The control group of animals was similarly treated with 0.5%
NaCMC. The reaction time was evaluated directly after 0.5, 1, 2, 3,
4, and 5 h of injection. The results of the analgesic activity of the
test compoundsand acetylsalicylic acidare listed in Table 4.
5-(2-Hydroxy-2,2-diphenylethyl)-1,3,4-oxadiazole-2(3H)-
thione (5c)
1H-NMR (DMSO-d6) d: 3.4 (brs, 1H, NH), 3.9 (s, 2H, CH2), 6.5 (brs,
1H, OH), 7.3–7.8 (m, 10H, Ar-H). IR (KBr) cm– 1: 3390, 3140, 2995,
2790, 1486. EI-MS: m/z 280 [M-H2O]9+, 208, 180, 179, 102. Anal.
calcd. For C16H14N2O2S: C, 64.41; H, 4.73; N, 9.39; S, 10.75. Found:
C, 65.92; H, 4.71; N, 8.72; S, 10.28.
Ulcerogenic effects
Biological screening
Observation of the gastrointestinal mucosa for the presence of
lesions following oral administration of a dose of 100 mg kg– 1 of
the test compounds (4b, 4c, and 5c) as well as indomethacin has
been taken as an indication for the ulcerogenic effects. Both the
frequency of ulceration (expressed as ratio of ulcerated animals)
and the severity of ulceration (expressed as ulcer index) were
used for comparison of the tested compound and indomethacin
[25]. Four groups each of six male adult albino mice were fasted
for 24 h. The tested compounds and indomethacin were admi-
nistered orally in doses of 100 mg/kg, as suspensions in 0.5%
NaCMC aqueous solution. After 6 h, the animals were sacrified,
the stomachs were removed and gastric lesions on the mucosa
were determined by using stereoscopic microscope. Ulcer was
defined as at least one lesion that was 0.5 mm or more in length.
All lesions of more than 0.1 mm in length were summed to
obtain the ulcer index and results were cited in Table 5. The per-
centage ulceration for each group was calculated as follows:
Animals were housed in separate cages, six animals each, in tem-
perature-controlled rooms at 258C. Animals were allowed free
access to food and water and maintained at 12 h light/dark cycle.
The experiments were conducted in accordance with the inter-
nationally accepted principles for laboratory animal use and
care as found in the European Community guide lines.
Analgesic and anti-inflammatory activities were performed at
the Department of Pharmacology, Faculty of Medicine, Assiut
University, Assiut, Egypt.
Bacterial and fungal cultures were obtained from Assiut Uni-
versity Mycological Center (AUMC), and the screening tests were
carried out at the Department of Botany, Faculty of Science,
Assiut University, Assiut, Egypt.
Anti-inflammatory activity
Compounds 1c, 2c, 3f, 3g, 3h, 4a–d, and 5a–c were evaluated for
their anti-inflammatory activities using the carageenan-induced
rat paw edema as previously described [23]. In this procedure, a
pedal inflammation in rat paws was induced by sub-plantar
injection of 0.2 mL carrageenan (0.2%) suspension into their
right hind. Male adult albino rats (100–120 g) were divided into
14 groups, of five animals each. The thickness of rat paw was
measured by a Veriner caliper (SMIEC, China) before and after
number of ulcerated animals in a group
%Ulceration ¼
6100
total number of animals in the same group
Determination of acute toxicity (LD50)
The median lethal dose (LD50) of the most effective compounds
4b, 4c, and 5c was determined in mice. Groups of male adult
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H. M. Abdel-Rahman et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 378–387
albino mice, each of five animals (25–30 g) were injected i. p.
with graded doses of the test compound. The percentage of mor-
tality in each group of animals was determined 72 h after injec-
tion.
The authors are greatly indebted to Dr. Mahran Shaker, Department
of Pharmacology, Faculty of Medicine, Assiut University for his kind
assistance in performing the anti-inflammatory and analgesic screen-
ings. Also, the authors greatly thank Dr. Mohammed Hashem Depart-
ment of Botany, Faculty of Science, Assiut University for carrying out
the antimicrobial activity screenings.
Antimicrobial screening
Antibacterial activity
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