Synthesis and biological evaluation of some novel thiazole compounds as potential anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2013.04.005

In the present investigation, furo[2,3-d]thiazol-5(2H)-one 5 was obtained from reaction of thiosemicarbazone derivative 2 with diethyl acetylene dicarboxylate. A series of newly synthesized 2-(hydrazinyl)thiazol-4(5H)-one 6, 7 & 8 and 2-(4-(substituted)-thiazol-2-yl)hydrazono derivatives 9a, b & 10 were synthesized from treatment of thiosemicarbazone derivative 2 with appropriate α-halogenated compounds. Also, a one pot synthesis of thiazole derivatives 13 & 15 was achieved from three components reaction of hydrazone derivative 11 with phenyl isothiocyanate and α-halogenated compounds catalyzed by DMF/KOH. 4-(4-Morpholino phenyl) thiazol-2-amino 17 was obtained via the reaction of acetophenone derivative 1 with thiourea in presence of iodine. The reactivity of 2-aminothiazole 17 toward some electrophilic reagents was investigated. The structure of the newly compounds was confirmed on the basis of elemental analysis and spectral data. The antibacterial activity towards two Gram negative (Proteus mirabilis & Serratia marcesens) and two Gram positive (Staphylococcus aureus & Bacillus cereus) bacteria was investigated. The anti-inflammatory activity was also investigated and the inhibition of the carrageenin-induced oedema by these compounds was established.

Full text of DOI:10.1016/j.ejmech.2013.04.005

European Journal of Medicinal Chemistry 65 (2013) 517e526
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of some novel thiazole compounds
as potential anti-inflammatory agents
,
b
,
c
,
,
,g
M.H.M. Helal a , M.A. Salem b, M.S.A. El-Gaby d e, M. Aljahdali f
*
a Department of Chemistry, Faculty of Arts and Science, Northern Border University, Rafha, Saudi Arabia
b Department of Chemistry, Faculty of Science, Al-Azhar University, 11284 Nasr City, Cairo, Egypt
c Department of Chemistry, Faculty of Science and Arts, King Khalid University, Mohail Assir, Saudi Arabia
d Department of Chemistry, Faculty of Science and Arts (Girl’s), King Khalid University, El-nams Al-Nmas, Saudi Arabia
e Department of Chemistry, Faculty of Science, Al-Azhar University, 71524 Assiut, Egypt
f Department of Chemistry, Faculty of Pharmacy, Northern Border University, Rafha, Saudi Arabia
g Department of Chemistry, Faculty of Science, King Abdulaziz University, Jeddah 21589, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
In the present investigation, furo[2,3-d]thiazol-5(2H)-one 5 was obtained from reaction of thio-
semicarbazone derivative 2 with diethyl acetylene dicarboxylate. A series of newly synthesized 2-
(hydrazinyl)thiazol-4(5H)-one 6, 7 & 8 and 2-(4-(substituted)-thiazol-2-yl)hydrazono derivatives 9a, b &
Received 6 October 2012
Received in revised form
1 April 2013
Accepted 2 April 2013
Available online 30 April 2013
10 were synthesized from treatment of thiosemicarbazone derivative 2 with appropriate
compounds. Also, a one pot synthesis of thiazole derivatives 13 & 15 was achieved from three compo-
nents reaction of hydrazone derivative 11 with phenyl isothiocyanate and -halogenated compounds
a-halogenated
a
catalyzed by DMF/KOH. 4-(4-Morpholino phenyl) thiazol-2-amino 17 was obtained via the reaction of
acetophenone derivative 1 with thiourea in presence of iodine. The reactivity of 2-aminothiazole 17
toward some electrophilic reagents was investigated. The structure of the newly compounds was
confirmed on the basis of elemental analysis and spectral data. The antibacterial activity towards two
Gram negative (Proteus mirabilis & Serratia marcesens) and two Gram positive (Staphylococcus aureus &
Bacillus cereus) bacteria was investigated. The anti-inflammatory activity was also investigated and the
inhibition of the carrageenin-induced oedema by these compounds was established.
Keywords:
Thiazole
Furo[2,3-d]thiazole
Thiosemecarbazone
Antibacterial
Anti-inflammatory activity
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
as antidibetic [14], antihyperlipo-proteinemics [15], antiemetic
[16], platelet aggregation inhibitors, bronchodilators, growth
Thiazolidinone has an important role as a widely exploited
pharmacophore in medicinal chemistry [1] having varied biological
activity such as antifungal [2], antibacterial [3,4], antimycobacterial
[5], antipsychotic [6], anti-inflammatory [7]. Also, substituted
thiazolidine derivatives represent important key intermediates for
the synthesis of pharmacologically active drugs. It is well known
that thiazole compounds have recently been grown up due to their
biological activity [8]. They can be used as anticonvulsants [9],
antibacterial [10,11] and antifungal agents [12]. Recently, some new
thiazole compounds are used as anti-inflammatory [13]. In addi-
tion, morpholine is a simple heterocyclic compound with a great
industrial importance. Many N-functionalized morpholines have
found to possess diverse pharmacological activities. They are re-
ported to exert a number of important physiological activities such
stimulates [17] and antidepressants [18]. These were also used in
the treatment of inflammatory diseases, pain, migraine and asthma
[19]. In view of the above facts and in continuation of our research
program directed towards the development of a new, simple and
efficient procedure for the synthesis of heterocyclic compounds
[20e31]. It seems of considerable interest to synthesize newly the
thiazole derivatives containing morpholine moiety. Additionally,
our objective is also to study the antibacterial and anti-
inflammatory activities of the synthesized compounds.
2. Results and discussion
2.1. Chemistry
The starting 1-(4-morpholinophenyl)ethanone 1 was obtained
by nucleophilic substitution of 4-fluroacetophenone with appro-
priate morpholine in dimethyl sulphoxide (DMSO) in the presence
of potassium carbonate as a base under reflux [32] (Scheme 1).
* Corresponding author. Department of Chemistry, Faculty of Science, Al-Azhar
University, 11284 Nasr City, Cairo, Egypt. Tel.: þ90 567 227 904.
E-mail address: m.h.helal.chem@gmail.com (M.H.M. Helal).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.04.005

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M.H.M. Helal et al. / European Journal of Medicinal Chemistry 65 (2013) 517e526
The behavior of thiosemicarbazone derivative 2 toward some a-
halogenated compounds was investigated to synthesize versatile
hitherto unreported thiazole derivative. Thus, the reaction of thi-
osemicarbazone derivative 2 with ethyl chloroacetate in glacial
acetic acid containing a catalytic amount of fused sodium acetate
afforded the corresponding 4-thiazolidinone derivative 6. The
molecular structure of compound 6 was established on the basis of
its elemental analysis and spectral data. The infrared spectrum of 6
revealed characteristic absorption bands at 3140 and 1708 cmꢀ1 for
NH and carbonyl group, respectively. A molecular ion peak at m/
z ¼ 318 (35%) was observed in the mass spectrum of compound 6
with base peak at m/z ¼ 317. The 1HNMR spectrum of 7 showed
Scheme 1.
Thiosemicarbazone derivative 2 was obtained via condensation
of ethanone derivative 1 with thiosemicarbazide in presence of a
catalytic amount of conc. HCl in ethanol as the solvent affording in
satisfactory yield (70%). IR spectrum of compound 2 revealed
characteristic absorption bands at ʋ ¼ 3418, 3288 and 3167 cmꢀ1
assignable for (NH2 & NH), while its 1HNMR spectrum (DMSO-d6)
indicated singlet signals at 2.23 ppm for CH3, two triplet at 3.13,
3.73 ppm for morphonyl protons with two singlet at 8.17 and
10.07 ppm for NH and NH2 respectively. Also, 13CNMR (DMSO-d6) of
signals at
(s, 2H, SCH2) and 9.42 ppm (s, 1H, NH). Similarly, reaction of thio-
semicarbazone 3 with ethyl- -chloropropionate resulted in the
d 2.37 (s, 2H, CH3), 3.24, 3.71 (2t, 8H, morphonyl-H), 3.88
a
formation of 5-methyl-4-thiazolinone derivative 7 according to the
spectral data of the isolated product (Scheme 3). The IR spectrum of
compound 7 revealed intense absorption bands at 3179 (NH) and
1718 cmꢀ1 (C]O). 1HNMR spectrum (DMSO-d6) of isolated product
7 showed signals at d 1.70 (d, 3H, CH3), 4.16 (q, 1H, CH) with singlet
compound 2 revealed signals at
d
17.00 (CH3), 39.98 (C3, C5 of
at 9.70 ppm for NH proton. The formation of compound 7 may be
assumed to proceed through initial alkylation followed by intra-
molecular cyclization with elimination of ethanol. On the other
hand reaction of thiosemicarbazone derivative 2 with chloro acetyl
chloride afforded the corresponding 5-thiazolidinone derivative 8
(Scheme 3). 1HNMR spectrum (DMSO-d6) of 8 revealed singlet
signal at 3.82 ppm for CH2 thiazole. Also, the structure of compound
8 was confirmed on the basis of 13CNMR which revealed signals at
morpholine), 77.62 (C2, C6 of morpholine), 147.55 (C]N) and
178.28 (C]S) ppm. Treatment of thiosemicarbazone derivative 2
with diethyl acetylene dicarboxylate afforded furo[2,3-d]thiazole
derivative 5 and other expected structure 3 was ruled out on the
basis of elemental analysis and spectral data. IR spectrum of com-
pound 5 showed characteristic absorption band at 1703 cmꢀ1 cor-
responding to carbonyl group. 1HNMR spectrum (CDCl3) of
compound 5 revealed singlet signals at 6.80 ppm for CH furan with
two triplets at 3.26 and 3.88 ppm for the morphonyl protons. Also,
mass spectrum of 5 showed a molecular ion peak at m/z 356 (55%)
with base peak at m/z ¼ 162. The formation of compound 5 was
assumed to proceed via nucleophilic attack of SH group on the
activated triple bond of diethyl acetylene dicarboxylate followed by
in situ heterocyclization with elimination of two ethanol molecule
d
14.35 for CH3, 32.75 (C4 of thiazole), 47.62 (C3, C5 of morpholine
moiety) and 173.91 ppm (thazole-C5). Cyclocondensation of thio-
semicarbazone 2 with chloroacetone and phenacyl bromide in
refluxing ethanol containing catalytic amount of fused sodium ac-
etate resulted in the formation of 1,3-thiazoles 9a, b. The 1HNMR
spectrum (DMSO-d6) of the isolated products revealed in each case
a singlet signal at 6.97 ppm for thiazole-H5. 13CNMR spectrum
to afford compound
(Scheme 2).
5
through non-isolable intermediate
4
(DMSO-d6) of 9b revealed signals at
morpholine), 65.02 (C2, C6 of morpholine), 103.77 (thiazole-C5)
d 14.35 (CH3), 47.62 (C3, C5 of
Scheme 2.

M.H.M. Helal et al. / European Journal of Medicinal Chemistry 65 (2013) 517e526
519
Scheme 3.
and 170.05 (thiazole-C2). In addition, interaction of thio-
semicarbazone 2 with chloroacetonitrile afforded 4-aminothiazole
derivative 10 (Scheme 3). The structure of 10 was confirmed on the
basis of elemental analysis and spectral data. IR spectrum of com-
pound 10 showed absorption bands at ʋ ¼ 3316, 3104 cmꢀ1 for
NH2/NH groups. 1HNMR spectrum (DMSO-d6) of 10 indicated that,
the reaction product exist in the imino form 10a rather than the
amino form 10b.
Condensation of acetophenone derivative 1 with cyanoacetic
acid hydrazide afforded hydrazone derivative 11 on the basis of
spectral data which indicated the presence of characteristic ab-
sorption bands at ʋ ¼ 3200 and 2257 cmꢀ1 assignable for NH and
C^N groups in the infrared spectrum. 1HNMR spectrum (DMSO-d6)
be assumed to proceed through initial alkylation followed by
intramolecular cyclization and elimination of ethanol molecule to
afford the two isomeric structures 14 and 15. In addition, ternary
condensation of hydrazone derivative 11, aryl isothiocyanate and
sulfur metal in refluxing ethanol and in the presence of a catalytic
amount of triethyl amine resulted in the formation of 4-
aminothiazol-2-thione derivative 16a, b. Infrared spectrum of iso-
lated product 16a revealed absorption bands at ʋ ¼ 3327, 3310,
3200 and 1672 cmꢀ1 corresponding to (NH2/NH) and (C]O)
groups, respectively. 1H NMR spectrum (DMSO-d6) of 16a revealed
singlet at
d 2.52 for CH3, multiplet aromatic protons with NH2 at
d
6.99e7.61 ppm with singlet signal at 9.00 ppm for imino group.
Mass spectrum of compound 16b showed a molecular ion peak at
m/z ¼ 503 (25%) with base peak at m/z ¼ 388 (Scheme 4).
of compound 11 revealed singlet signals at d 3.99 and 10.70 ppm for
active methylene protons and NH proton. Also, the structure of
Furthermore, 4-(4-morpholinophenyl) thiazol-2-amino 17 was
obtained via the reaction of acetophenone derivative 1 with thio-
urea in presence of iodine [33]. Compound 17 was characterized by
its elemental analysis and spectral data. IR spectrum showed
characteristic absorption bands at 3303, 3117 cmꢀ1 assignable for
amino group. 1H NMR spectrum (CDCl3) of 17 showed singlet signal
at 6.58 assignable for thiazole-H5 with singlet signal at 7.94 ppm
assignable for NH2. Also, 13CNMR spectrum of 17 revealed signals at
compound 11 was confirmed on the basis of 13CNMR (DMSO-d6)
which revealed signals at
d 13.14, 24.79 for CH3 and CH2, 66.21,
77.05 for morpholine moiety and 149.90, 196.64 for C^N and C]O
groups (Scheme 4). The non-isolable potassium sulphide salt 12
was achieved by the nucleophilic addition of active methylene
group in compound 11 to phenyl isothiocyanate in dry dime-
thylformamide at room temperature in the presence of potassium
hydroxide (Scheme 4). The potassium salt 12 was exploited to
synthesize some new thiazolidine derivatives. Cyclocondensation
of intermediate 12 with phenacyl bromide gave 4-phenylthiazole
derivative 13 (Scheme 4). Infrared spectrum of compound 13
indicated the presence of NH, C^N and C]O functional groups.
1HNMR spectrum of compound 13 revealed singlet signals at
d
102.00 (thiazole-C5), 150.30 (thiazole-C4), and 168.90 (thiazole-
C2) (Scheme 5). The reactivity of 2-aminothiazole derivative 17
toward some electrophilic reagents was investigated. Thus,
condensation of compound 17 with 4-methylbenzaldehyde in
refluxing ethanol and in the presence of a catalytic amount of
piperidine resulted in the formation of the imino derivative 18.
1HNMR spectrum (DMSO-d6) of the isolated product 18 showed
singlet signal at 2.17, 5.95 and 7.55 ppm assignable for CH3, thia-
zole-H5 and CH-benzylidine, respectively. Also, acetylation of 2-
aminothiazole 17 with acetic anhydride on refluxing afforded
N,N-(diacetyl)aminothiazole derivative 19 on the basis of spectral
data. IR spectrum showed the absence of NH2 group with the
presence of a characteristic absorption band at 1691 cmꢀ1 assign-
able for carbonyl group. Also, 1HNMR spectrum (DMSO-d6) of 19
revealed two singlet signals 2.23, 2.40 ppm for two methyl with
singlet at 6.92 ppm for thiazole-H5. Finally, condensation of 2-
amino-4-phenyl thiazole with ethanone derivative 1 afforded the
d
6.88 ppm for thiazole-H5. Also, the mass spectrum of compound
13 revealed a molecular ion peak at m/z ¼ 521 (36%) which is
characteristic for the molecular formula C30H27N5O2S. Treatment of
intermediate 12 with ethyl-a-chloropropionate at room tempera-
ture gave 4-hydroxythiazole derivative 15. The structure of com-
pound 15 was preferred rather than the compound 14 according to
the spectral data. IR spectrum showed abroad absorption band at
3420 cmꢀ1 corresponding to hydroxyl group. 1HNMR spectrum
(DMSO-d6) of compound 15 revealed singlet signals at
(2s, 6H, 2CH3), 3.13, 3.72 (2t, 8H, morpholine moiety), 10.07 (s, 1H,
NH) with singlet at 11.88 for OH group. The formation of 15 may
d 1.89, 2.29
d

520
M.H.M. Helal et al. / European Journal of Medicinal Chemistry 65 (2013) 517e526
Scheme 4.
corresponding thiazole derivative 20. 1H NMR spectrum (CDCl3) of
the isolated product 20 showed singlet signal at 2.53 for CH3 with
signal at 5.22 ppm for thiazole-H5 (Scheme 5).
out on the basis of the semi-empirical PM3 level provided by
HyperChem 7.5 software. The calculated bond length and bond
angles after geometrical optimization of compound 17 structure as
a representative example of thiazole compounds are given in
Table 1. The molecular structure of compound 17 structure along
with the atom numbering scheme are given in Fig. 1.
2.2. Molecular modeling calculations
In order to throw light on the molecular conformation of the
synthesized compounds, energy minimization studies were carried
2.3. Biological activity
2.3.1. Antibacterial activity
The synthesized compounds were tested for their inhibitory
effects on the growth of two Gram negative (Proteus mirabilis &
Serratia marcesens) and two Gram positive bacteria (Staphylococcus
aureus & Bacillus cereus) bacteria in DMSO as solvent using Ampi-
cillin as standard material because such organisms can achieve
resistance to antibiotics through biochemical and morphological
modification [34]. The antibacterial activity of the new compounds
is listed in Table 2. The antibacterial activity was tested by using the
disc diffusion method. The antimicrobial results showed that:
1. Only compound 11 were found to be the more active com-
pounds against S. aureus (NCTC-7447) (Fig. 2).
2. None of the other tested compounds showed superior activity
over the reference.
3. The activity index was calculated and the results are given in
Table 3. The activity index of compound 11 reaches 75, sug-
gesting that this compound be considered as the most prom-
ising potent broad spectrum antimicrobial compound (Table 4)
4. The tested heterocyclic compounds were more active against
gram-positive than Gram-negative bacteria, it may be
concluded that the antimicrobial activity of the compounds is
related to cell wall structure of the bacteria. It is possible
because the cell wall is essential to the survival of bacteria and
some antibiotics are able to kill bacteria by inhibiting a step in
Scheme 5.

M.H.M. Helal et al. / European Journal of Medicinal Chemistry 65 (2013) 517e526
521
Table 1
Bond distances (Å) and angles (ꢁ) for compound 17.
Atoms
Bond distances (Å)
Atoms
Angle (ꢁ)
Atoms
Angle (o)
C(17)eH(33)
C(17)eH(32)
C(16)eH(31)
C(16)eH(30)
C(15)eH(29)
C(15)eH(28)
C(14)eH(27)
C(14)eH(26)
N(12)eH(25)
N(12)eH(24)
C(11)eH(23)
C(5)eH(22)
C(4)eH(21)
C(2)eH(20)
C(1)eH(19)
N(13)eC(15)
C(16)eC(15)
O(18)eC(16)
C(17)eO(18)
C(14)eC(17)
N(13)eC(14)
C(1)eC(6)
C(5)eC(6)
C(4)eC(5)
C(3)eC(4)
C(2)eC(3)
C(1)eC(2)
C(7)eN(8)
C(9)eN(8)
S(10)eC(9)
C(11)eS(10)
C(7)eC(11)
C(3)eN(13)
C(9)eN(12)
C(6)eC(7)
1.113
1.113
1.113
1.113
1.113
1.113
1.113
1.113
1.05
1.05
1.1
1.1
1.1
C(17)eO(18)eC(16)
H(33)eC(17)eH(32)
H(33)eC(17)eO(18)
H(33)eC(17)eC(14)
H(32)eC(17)eO(18)
H(32)eC(17)eC(14)
O(18)eC(17)eC(14)
H(31)eC(16)eH(30)
H(31)eC(16)eO(18)
H(31)eC(16)eC(15)
H(30)eC(16)eO(18)
H(30)eC(16)eC(15)
O(18)eC(16)eC(15)
H(29)eC(15)eH(28)
H(29)eC(15)eC(16)
H(29)eC(15)eN(13)
H(28)eC(15)eC(16)
H(28)eC(15)eN(13)
C(16)eC(15)eN(13)
H(27)eC(14)eH(26)
H(27)eC(14)eC(17)
H(27)eC(14)eN(13)
H(26)eC(14)eC(17)
H(26)eC(14)eN(13)
C(17)eC(14)eN(13)
C(15)eN(13)eC(14)
C(15)eN(13)eC(3)
C(14)eN(13)eC(3)
H(25)eN(12)eH(24)
H(25)eN(12)eC(9)
H(24)eN(12)eC(9)
H(23)eC(11)eS(10)
H(23)eC(11)eC(7)
S(10)eC(11)eC(7)
C(11)eS(10)eC(9)
N(12)eC(9)eS(10)
N(12)eC(9)eN(8)
110.6935
110.7607
108.3961
108.3961
108.9845
108.9845
111.3236
110.7596
108.3971
108.3971
108.9849
108.9849
111.322
111.4295
107.8376
107.8376
108.744
108.744
112.2766
111.4286
107.8384
107.8384
108.7444
108.7444
112.2753
108.2826
125.8587
125.8587
120
C(11)eC(7)eN(8)
C(11)eC(7)eC(6)
N(8)eC(7)eC(6)
C(7)eC(6)eC(5)
C(7)eC(6)eC(1)
C(5)eC(6)eC(1)
H(22)eC(5)eC(6)
H(22)eC(5)eC(4)
C(6)eC(5)eC(4)
H(21)eC(4)eC(5)
H(21)eC(4)eC(3)
C(5)eC(4)eC(3)
N(13)eC(3)eC(4)
N(13)eC(3)eC(2)
C(4)eC(3)eC(2)
H(20)eC(2)eC(3)
H(20)eC(2)eC(1)
C(3)eC(2)eC(1)
H(19)eC(1)eC(6)
H(19)eC(1)eC(2)
C(6)eC(1)eC(2)
115.6691
122.1654
122.1654
120.0002
120.0002
119.9996
120.0014
120.0014
119.9972
119.9984
119.9984
120.0033
119.9999
119.9999
120.0002
120.0015
120.0015
119.9969
119.9986
119.9986
120.0029
1.1
1.1
1.4756
1.5364
1.4334
1.4333
1.5364
1.4756
1.3948
1.3949
1.3948
1.3948
1.3949
1.3948
1.3813
1.3152
1.7161
1.7154
1.3788
1.266
1.266
1.337
120
120
125.1462
125.1462
109.7076
89.2503
121.8996
121.8996
116.2008
109.1722
S(10)eC(9)eN(8)
C(9)eN(8)eC(7)
the synthesis of peptidoglycan. Gram-positive bacteria possess
a thick cell wall containing many layers of peptidoglycan and
teichoic acids, but in contrast, Gram negative bacteria have a
relatively thin cell wall consisting of a few layers of peptido-
glycan surrounded by a second lipid membrane containing li-
popolysaccharides and lipoproteins. These differences in cell
Table 2
Antibacterial activity of the synthesized compounds and inhibition zones (mm).
Compd. no.
Gþ
Gꢀ
Staphylococcus Bacillus cereus Proteus mirabilis Serratia
aureus
(NCTC-14579) (NCTC-289)
marcesens
(NCTC-7447)
(IMRU-70)
2
5
4
5
4
4
2
2
3
3
6
4
4
3
3
7
5
5
2
2
8
5
4
2
3
9a
5
3
2
2
9b
4
4
3
2
10
5
4
2
3
11
13
18
5
4
4
3
2
2
3
15
5
3
3
3
16a
4
3
2
2
16b
5
3
2
2
17
5
4
2
3
18
4
4
2
3
19
5
4
3
2
20
5
4
3
2
Standard
(ampicillin)
DMSO
(control)
24
23
21
21
0
0
0
0
Fig. 1. Molecular modelling of compound 17.
Gþ: Gram positive bacteria and Gꢀ: Gram negative bacteria.

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M.H.M. Helal et al. / European Journal of Medicinal Chemistry 65 (2013) 517e526
30
25
20
15
10
5
Results are presented in Table 3, Fig.3, as percent edema increase at
the right hind paw and percent inhibition.
Carrageenin-induced edema is nonspecific inflammation
Serratia Marcesens (IMRU-70)
Proteus mirabilis (NCTC-289)
Bacillus cereus (NCTC-14579)
Staphylococcus aureus (NCTC-7447)
a
resulting from a complex of diverse mediators (Shen) [40]. Since
edemas of this type are highly sensitive to nonsteroidal anti-
inflammatory drugs (NSAIDs), carrageenin has been accepted as a
useful agent for studying new anti-inflammatory drugs (Winter
et al.). This model reliably predicts anti-inflammatory efficacy of
the NSAIDs, and during the second phase it detects compounds
which are anti-inflammatory agents as a result of inhibition of
prostaglandin amplification.
Significant anti-inflammatory activity was observed with inhi-
bition in edema in the range of 35e87% after 4 h. The standard drug
indomethacin has shown 91% inhibition after 4 h. Among all the
screened compounds (5 and 20) were found to be potent in the
series with 85 and 87% inhibition after 4 h, respectively, while the
least potent one was compound (6) in the series with 35% inhibition
after 4 h. The inflammatory compounds (5 and 17) have a high
degree of inhibition reaches 85 and 87%, respectively. Thus these
compounds can be considered as the most promising potent broad
spectrum inflammatory compounds among the synthesized
compounds.
0
(5)
(7)
(11)
(17)
Ampicillin
Fig. 2. Antibacterial activity of synthesized compounds.
wall structure can produce differences in antibacterial sus-
ceptibility and some antibiotics can kill only Gram-positive
bacteria and is infective against Gram-negative pathogens
[35e37].
5. The MIC value of compound 11 is 40 mg/ml.
6. The mode of action of the compounds may involve the for-
mation of a hydrogen bond through the azomethine nitrogen
atom (>C]N) with the active centers of cell constituents,
resulting in interference with the normal cell process [34,36e
38].
7. The variation in the effectiveness of different compounds
against different organisms depends on either the imperme-
ability of the cells of the microbes or on differences in ribosome
of microbial cells [39].
3. Experimental
All melting points are uncorrected. IR spectra (KBr) were
measured on Shimadzu 440 spectrometer, 1H NMR spectra were
obtained in DMSO on a Varian Gemini 600 MHz spectrometer using
TMS as internal standard; chemical shifts are reported as (ppm).
Mass spectra were obtained on GCMS/QP 1000 Ex mass spec-
trometer at 70 eV. Elemental analyses were carried out at the
Department of Chemistry, Faculty of Science, King Abdul-Aziz
University, Jeddah21589, KSA. Microbiology screening was carried
out in Microbiology Department, Faculty of Pharmacy, Al-Mansoura
University and Pharmacology Department, National Research
Center, Cairo, Egypt BOX: 12622.
2.3.2. Anti-inflammatory activity
The synthesized thiazole compounds were tested for their anti-
inflammatory activity but only seven compounds (2, 5, 6, 8, 9b, 17
and 20) were found biologically active with inhibition percent
ranges from 35% to 87%. These seven compounds were screened for
in vivo anti-inflammatory activity by inhibition of carrageenan
induced rat paw edema method at the dose of 50 mg/kg orally.
3.1. Chemistry
3.1.1. 2-(1-(4-Morpholinophenyl)ethylidene)
hydrazinecarbimidothioic acid 2
Table 3
Activity index percent of the synthesized compounds and inhibition zones (mm).
Equimolar amounts of compound
1 (0.01 mol), thio-
Compound no.
Gþ
Gꢀ
semicarbazide (0.01 mol) and a few drops of conc. HCl in ethanol
(30 ml) was refluxed for 3 h. The solid product which produced on
heating was collected and recrystallized from acetic acid as yellow
crystals. Yield (70%); m.p. 215 ꢁC; IR (KBr, cmꢀ1): 3418, 3288, 3167
(NH2, NH), 2966, 2829 (aliph. CH) and 1205, 1050 (C]S). MS: 278
Staphylococcus
aureus
(NCTC-7447)
Bacillus cereus
(NCTC-14579)
Proteus
mirabilis
(NCTC-289)
Serratia
marcesens
(IMRU-70)
2
5
6
7
8
9a
9b
10
11
13
15
16a
16b
17
18
19
16.67
20.83
16.67
20.83
20.83
20.83
16.67
20.83
75
20.83
20.83
16.67
20.83
20.83
16.67
20.83
20.83
24
17.39
17.39
17.39
21.74
16.67
13.04
17.39
17.39
17.39
17.39
13.04
13.04
13.04
17.39
17.39
17.39
17.39
23
9.52
9.52
14.28
9.52
9.52
9.52
14.28
9.52
14.28
9.52
14.28
9.52
9.52
9.52
9.52
14.28
14.28
21
15.72
15.72
15.72
10.52
15.72
15.72
15.72
15.72
15.72
15.72
15.72
15.72
15.72
15.72
15.72
15.72
15.72
19
(Mþ, 29%), 204 (100%). 1H NMR (600 MHz, DMSO-d6):
d
¼ 2.22 (s,
3H, CH3), 3.13, 3.73 (2t, 8H, morphonyl-H), 6.88e7.82 (2d, 4H, Ar-H)
and 10.07 (s, 1H, NH) ppm. 13CNMR (600 MHz, DMSO-d6):
d
¼ 17.00
(CH3), 39.98 (C3, C5 of morpholine), 77.62 (C2, C6 of morpholine),
114.46, 127.32, 129.78, 153.71 (phenyl-C), 147.55 (C]N) and 178.28
(C]S) ppm. Elemental analysis for C13H18N4OS. Calcd: C, 56.09; H,
6.52; N, 20.13; Found: C, 56.00; H, 6.30; N, 19.90.
3.1.2. 2-(2-(1-(4-Morpholinophenyl)ethylidene)hydrazinyl)furo
[2,3-d]thiazo-l-5(2H)-one 5
A mixture of compound 2 (0.01 mol) and acetylene dicarbox-
ylate (0.01 mol) in ethanol (30 ml) was stirred under reflux for 24 h
at 160 ꢁC. The solid product which produced on heating was
collected and recrystallized from acetic acid as yellow crystals.
Yield (50%); m.p. 246 ꢁC; IR (KBr, cmꢀ1): 2963 (aliph. CH), 1703 (C]
O) and 1630 (C]N). MS: 356 (Mþ, 55%), 162 (100%). 1H NMR
20
Standard
(ampicillin)
DMSO
(control)
0
0
0
0
(600 MHz, CDCl3):
morphonyl-H), 6.80 (s, 1H, Furan-H) and 6.89, 7.85 (2d, 4H, Ar-H)
d
¼ 2.42 (s, 3H, CH3), 3.26, 3.88 (2t, 8H,
Gþ: Gram positive bacteria and Gꢀ: Gram negative bacteria.

M.H.M. Helal et al. / European Journal of Medicinal Chemistry 65 (2013) 517e526
523
Table 4
Oedema inhibiting activity of synthesized compounds.
% Edema
% Inhibition
1st
Time (h)
1st
2nd
3rd
4th
83 ꢂ 4.7
2nd
3rd
4th
Control
54.8 ꢂ 2.9
49.1 ꢂ 4.2
74 ꢂ 6.7
67.1 ꢂ 4.4
36.8 ꢂ 4.1*
51.7 ꢂ 6.4
30 ꢂ 1.6*
79.5 ꢂ 4.1
19.5 ꢂ 2.9*
35 ꢂ 3.5*a
18 ꢂ 0.9*
56 ꢂ 4.0*a
29.1 ꢂ 3.0*
30 ꢂ 1.7*
19.7 ꢂ 2.2*
32 ꢂ 1.9*
e
e
e
e
Indomethacin
Compound 2
Compound 5
Compound 6
Compound 8
Compound 9b
Compound 17
Compound 20
7.2 ꢂ 0.11
13.2 ꢂ 2.6*
11.8 ꢂ 1.0*
53 ꢂ 2.9*a
18.1 ꢂ 1.8*
17.4 ꢂ 1.6*
11 ꢂ 1.3*
10.5
ꢀ35
23.3
ꢀ58.6
29.5
ꢀ1.6
ꢀ17
27.2
45.1
23
75.5
55.7
75.7
29
63.4
62.2
75.3
59.8
91.3
83.6
85
35.7
78.2
79.1
86.8
81.6
38 ꢂ 3.6*a
87.0 ꢂ 1.57
38.7 ꢂ 3.9
55 ꢂ 4.4
50.6
ꢀ8
72.5 ꢂ 1.4
47.9 ꢂ 2.7*
43.6 ꢂ 4.2*
45.7 ꢂ 4.3*
51.8 ꢂ 4.8
28.6
35
64.1 ꢂ 5
31.7
22.9
40 ꢂ 2.5
15 ꢂ 1.5*
e Values are expressed as means ꢂ SEM (n ¼ 6).
* Significantly different from control group at P < 0.05.
a
Significantly different from indomethacin group at P < 0.05.
ppm. Elemental analysis for C17H18N4O3S. Calcd: C, 56.97; H, 5.06;
N, 15.63; Found: C, 56.60; H, 4.95; N, 15.46.
42%), 303 (100%). 1H NMR (600 MHz, DMSO-d6): 1.70 (d, 3H, CH3),
3.22, 3.88 (2t, 8H, morphonyl-H), 4.16 (q, 1H, CH), 6.88, 7.90 (2d, 4H,
Ar-H), 9.80 (br, 1H, NH) ppm. Elemental analysis for C16H20N4O2S.
Calcd: C, 57.81; H, 6.06; N, 16.85; Found: C, 57.50; H, 9.80; N, 16.40.
3.1.3. Preparation of compounds (6, 7, 8, 9a, b & 10): general
procedure
A mixture of compound 1 (0.01 mol), appropriate
a
-halo com-
3.1.6. 2-(2-(1-(4-Morpholinophenyl)ethylidene)hydrazinyl)thiazol-
5(4H)-one 8
pounds namely (ethylchloroacetate, ethyl -chloropropionate,
a
chloro-acetylchloride, chloroacetone, phenacyl bromide, chlor-
oacetonitrile) (0.01 mol) and sodium acetate (0.01 mol) in acetic
acid (30 mL) was refluxed for 4 h. The solid product which pro-
duced on heating was collected and recrystallized from the proper
solvents.
Yield (45%); white solid (ethanol); m.p. 267e68 ꢁC; IR (KBr,
cmꢀ1): 3100 (NH), 2983, 2825 (aliph. CH), 1715 (C]O). 1H NMR
(600 MHz, DMSO-d6):
morphonyl-H), 3.82 (s, 2H, SCH2), 6.94, 7.70 (2d, 4H, Ar-H) and 11.86
(s, 1H, NH) ppm. 13CNMR (600 MHz, DMSO-d6):
d
¼ 2.28 (s, 3H, CH3), 3.18, 3.73 (2t, 8H,
d
¼ 14.35 (CH3),
39.08 (SCH2), 47.62 (C3, C5 of morpholine), 65.99 (C2, C6 of mor-
pholine), 113.47, 114.04, 127.47, 127.98, 130.22 (phenyl-C), 152.09
(thiazole-C2), 159.92 (C]N) and 173.91(thiazole-C5) ppm.
Elemental analysis for C15H18N4O2S. Calcd. C, 56.58; H, 5.70; N,
17.60; Found: C, 56.20; H, 5.50; N, 17.30.
3.1.4. 2-(2-(1-(4-Morpholinophenyl)ethylidene)hydrazinyl)thiazol-
4(5H)-one 6
Yield (75%); white solid (dioxane); m.p.200 ꢁC; IR (KBr, cmꢀ1):
3140 (NH), 2949, 2860 (aliph. CH), 1708 (C]O). MS: 318 (Mþ, 35%),
317 (100%). 1H NMR (600 MHz, CDCl3):
d
¼ 2.37 (s, 2H, CH3), 3.24,
3.71 (2t, 8H, morphonyl-H), 3.88 (s, 2H, SCH2), 6.89, 7.82 (2d, 4H,
3.1.7. 4-(4-(1-(2-(4-Methylthiazol-2-yl) hydrazono) ethyl) phenyl)
morpholine 9a
Ar-H) and 9.42 (br, 1H, OH) ppm. 13CNMR (600 MHz, CDCl3):
d
¼ 14.87 (CH3), 33.04 (thiazole-C5), 66.20 (C3, C5 of morpholine),
Yield (55%); white crystals (ethanol); m.p. 291 ꢁC; IR (KBr,
77.05 (C2, C6 of morpholine), 111.46, 114.02, 127.32, 128.21 (phenyl-
C), 150.00 (C]N), 161.99 (thiazole-C2) and 172.48 (thiazole-C4)
ppm. Elemental analysis for C15H18N4O2S. Calcd: C, 56.58; H, 5.70;
N, 17.60; Found: C, 56.10; H, 5.40; N, 17.50.
cmꢀ1): 3278, 3166 (NH), 2965 (aliph. CH.) and 1600 (C]N). 1HNMR
(600 MHz, DMSO-d6):
d
¼ 2.29 (s, 6H, 2CH3), 3.20, 3.85 (2t, 8H,
morphonyl-H), 6.97e7.79 (m, 5H, Ar-H þ thiazole-H5) and 9.55 (s,
1H, NH) ppm. Elemental analysis for C16H20N4OS. Calcd. C, 60.73; H,
6.37; N, 17.71; Found: C, 60.60; H, 6.20; N, 17.40.
3.1.5. 5-Methyl-2-(2-(1-(4-morpholinophenyl)ethylidene)
hydrazinyl)thiazol-4(5H)-one) 7
3.1.8. 4-(4-(1-(2-(4-Phenylthiazol-2-yl) hydrazono) ethyl) phenyl)
morpholine 9b
Yield (65%); white solid (dioxane); m.p.205e207 ꢁC; IR (KBr,
cmꢀ1): 3179 (NH), 2962, 2831 (aliph. CH),1718 (C]O). MS: 332 (Mþ,
Yield (40%); brown solid (ethanol); m.p.280e82 ꢁC; IR (KBr,
cmꢀ1):3109 (NH), 2958, 2854 (aliph. CH) and 1607 (C]N). 1H NMR
(600 MHz, DMSO-d6):
morphonyl-H), 6.94e7.87 (m, 10H, Ar-H þ thiazole-H5) and 11.07 (s,
1H, NH) ppm. 13CNMR (600 MHz, DMSO-d6):
d
¼ 2.25 (s, 3H, CH3), 3.14, 3.74 (2t, 8H,
d
¼ 13.65 (CH3), 66.60
(C3, C5 of morpholine), 77.05 (C2, C6 of morpholine), 102.64
(thiazole-C5), 114.20, 125.81, 127.36, 127.48, 128.11, 130.31 (2
phenyl-C), 152.06 (thiazole-C2) and 169.40 (C]N)ppm. Elemental
analysis for C21H24N4OS. Calcd. C, 66.29; H, 6.36; N, 14.72; Found: C,
66.00; H, 6.10; N, 14.50.
3.1.9. 2-(2-(1-(4-Morpholinophenyl) ethylidene) hydrazinyl)
thiazol-4(5H)-imine 10
Yield (45%); brown solid (ethanol); m.p.136 ꢁC; IR (KBr, cmꢀ1):
3316, 3104 (NH2/NH), 2967, 2866 and (aliph. CH). MS: 317 (Mþ,
39%), 301 (100%). 1H NMR (600 MHz, DMSO-d6):
d
¼ 2.25 (s, 3H,
CH3), 3.22, 3.70 (2t, 8H, morphonyl-H), 3.87 (s, 2H, SCH2), 6.40, 8.60
(2s, 2H, 2NH; cancelled with D2O) and 6.86e7.90 (m, 4H, Ar-H)
ppm. Elemental analysis for C15H19N5OS. Calcd. C, 56.76; H, 6.03;
N, 22.06; Found: C, 56.50; H, 5.80; N, 21.90.
Fig. 3. Anti-inflammatory of synthesized compounds.

524
M.H.M. Helal et al. / European Journal of Medicinal Chemistry 65 (2013) 517e526
3.1.10. 2-Cyano-N0-(1-(4-morpholinophenyl) ethylidene)
acetohydrazide 11
3.32, 3.86 (2t, 8H, 4CH2), 6.98e7.60 (m, 11H, Ar-H þ NH2), 9.00 (s,
1H, NH) ppm. Elemental analysis for C22H23N5O2S2. Calcd. C, 58.26;
H, 5.11; N, 15.44; Found: C, 58.00; H, 5.00; N, 15.20.
Equimolar amounts of compound 1 (0.01 mol), cyanoacetic acid
hydrazide (0.01 mol) and a few drops of conc. HCl in ethanol (30 ml)
was refluxed for 3 h. The solid product which produced on heating
was collected and recrystallized from acetic acid as white solid.
Yield (65%); m.p. 205 ꢁC; IR (KBr, cmꢀ1): 3200 (NH), 2971, 2867
(aliph. CH), 2257 (C^N) and 1671 (C]O). 1HNMR (600 MHz,
3.1.16. 4-Amino-N0-(1-(4-morpholinophenyl)ethylidene)-3-
(naphthalen-2-yl)-2-thioxo-2,3-dihydrothiazole-5-carbohydrazide
16b
Yield (55%); brown solid (acetic acid); m.p. 269e70 ꢁC; IR (KBr,
DMSO-d6):
3.96 (s, 2H, CH2), 6.87, 7.86 (2d, 4H, Ar-H) and 10.70 (s,1H, NH) ppm.
13CNMR (600 MHz, DMSO-d6):
d
¼ 2.24 (s, 3H, CH3), 3.20, 3.83 (2t, 8H, morphonyl-H),
cmꢀ1): 3300, 3215, 3110 (NH2/NH), 1650 (C]O). MS: 503 (Mþ, 25%),
388 (100%). 1H NMR (600 MHz, DMSO-d6):
3.37, 3.86 (2t, 8H, 4CH2), 6.99e7.61 (m, 13H, Ar-H þ NH2), 9.00 (s,
1H, NH) ppm. Elemental analysis for C26H25N5O2S2. Calcd. C, 62.00;
H, 5.00; N, 13.91; Found: C, 61.80; H, 4.90; N, 13.70.
d
¼ 2.52 (s, 3H, CH3),
d
¼ 13.14 (CH3), 24.79 (CH2), 66.21
(C3, C5 of morpholine), 77.05 (C2, C6 of morpholine), 114.01, 115.78,
127.78, 133.28 (phenyl-C), 149.90 (C^N), 164.63 (C]N) and 196.64
(C]O). Elemental analysis for C15H18N4O2. Calcd. C, 62.92; H, 6.34;
N, 19.57; Found: C, 62.60; H, 6.00; N, 19.20.
3.1.17. 4-(4-Morpholinophenyl)thiazol-2-amine 17
A mixture of acetophenone derivative 1 (0.1 mol), thiourea
(0.2 mol) and Iodine (0.1 mol) was heated on a steam bath for 4 h. The
hydroiodide separated, was filtered, washed with ether and dried. It
was dissolved in hot water, filtered while hot and the clear solution
neutralized with a strong solution of ammonia. The solid separated
was filtered, washed with water and recrystallized from benzene as
yellow crystals. Yield (65%); m.p. 227 ꢁC; IR (KBr, cmꢀ1): 3303, 3117
(NH2) 2970, 2838 (aliph. CH), 1606 (C]N). MS: 261 (Mþ, 83%), 99
3.1.11. Preparation of compounds (13 & 15); general procedure
To suspension of finally powdered potassium hydroxide
(0.01 mol) in dry dimethylformamide (20 ml) the active methylene
compound (11, 0.01 mol) and then the phenyl isothiocyanate
(0.01 mol) were added in portions. The reaction mixture was stirred
at room temperature for 1 h and then treated with a-halogenated
compound (0.01 mol) and left at room temperature for 2 h; then it
was poured into ice/water and acidified with 0.1 N HCl at pH 3e4.
The resulting precipitate was filtered off, dried, and recrystallized
from the proper solvent.
(100%).1H NMR (600 MHz, CDCl3):
8H, morphonyl-H), 6.58 (s, 1H, thiazole-H5), 6.87, 7.66 (2d, 4H, Ar-H),
7.94 (s, 2H, NH2) ppm.13CNMR (600 MHz, CDCl3):
d
¼ 2.57 (s, 2H, CH3), 3.15, 3.82 (2t,
d
¼ 65.04 (C3, C5 of
morpholine), 75.05 (C2, C6 of morpholine), 102.00 (thiazole-C5),
112.80,122.50,128.30,149.50(phenyl-C),150.30 (thiazole-C4),168.90
(thiazole-C2) ppm. Elemental analysis for C13H15N3OS. Calcd. C,
59.74; H, 5.79; N, 16.08; Found: C, 59.50; H, 5.40; N, 16.00.
3.1.12. 2-Cyano-2-(3,4-diphenylthiazol-2(3H)-ylidene)-N0-(1-(4-
morpholinoph-enyl) ethyl-idene) acetohydrazide 13
Yield (55%); yellow solid (ethanol); m.p. 180 ꢁC; IR (KBr, cmꢀ1):
3100 (NH), 2210 (C^N) and 1650 (C]O). MS: 521 (Mþ, 36%), 237
(100%). 1H NMR (600 MHz, CDCl3):
(2t, 8H, morphonyl-H), 6.86 (s, 1H, thiazole-H5), 6.88e7.90 (m, 15H,
Ar-H þ NH) ppm. Elemental analysis for C30H27N5O2S. Calcd. C,
69.08; H, 5.22; N, 13.43; Found: C, 68.80; H, 5.10; N, 13.10.
d
¼ 2.53 (s, 2H, CH3), 3.20, 3.83
3.1.18. N-(4-methylbenzylidene)-4-(4-morpholinophenyl)thiazol-2-
amine 18
Equimolar amounts of compound 17 (0.01 mol), 4-
methylbenzaldehyde (0.01 mol) and a few drops of piperidine in
ethanol (30 ml) were refluxed for 3 h. The solid product which
produced on heating was collected and recrystallized from the
acetic acid as white solid. Yield (65%); m.p. 251 ꢁC; IR (KBr, cmꢀ1):
2900, 2851 (aliph. CH), 1620(C]N). MS: 363 (Mþ, 17%), 86 (100%).
3.1.13. 2-Cyano-2-(4-hydroxy-5-methyl-3-phenylthiazol-2(3H)-
ylidene)-N0-(1-(4-morph-olinophenyl) ethylidene) acetohydrazide
15
Yield (53%); brown solid (ethanol); m.p. 239e40 ꢁC; IR (KBr,
1HNMR (600 MHz, DMSO-d6):
8H, 4CH2), 5.95 (s,1H, thiazole-H5), 6.68e7.27 (m, 8H, Ar-H), 7.55 (s,
1H, benzyldine-CH) ppm. Elemental analysis for C21H21N3OS. Calcd.
C, 69.39; H, 5.82; N, 11.56; Found: C, 69.10; H, 5.60; N, 11.30.
d
¼ 2.17 (s, 3H, CH3), 3. 14, 3.83 (2t,
cmꢀ1): 3420 (OH), 3288, 3166 (NH), 1593 (C]N). MS: 475 (Mþ,
73%), 461 (100%). 1H NMR (600 MHz, DMSO-d6):
d
¼ 1.89, 2.22
(2s, 6H, 2CH3), 3.13, 3.72 (2t, 8H, 4CH2), 6.87e8.17 (m, 9H, Ar-H),
10.07 (s, 1H, NH), 11.88 (s, 1H, OH) ppm. 13C NMR (600 MHz,
DMSO-d6):
d
¼ 13.80, 21.60 (2CH3), 63.84 (C3, C5 of morpholine),
3.1.19. N-acetyl-N-(4-(4-morpholinophenyl) thiazol-2-yl)
acetamide 19
76.50 (C2, C6 of morpholine), 78.60 (CeC^N), 80.10 (thiazole-
C5), 111.80, 121.50, 122.80, 127.00, 129.40, 130.01, 142.00, 151.50 (2
phenyl-C), 114.50 (CeC^N), 148.00 (C]N), 170.01 (C]O), 172.40
(thiazole-C2), 176.50 (thiazole-C4)ppm. Elemental analysis for
A mixture of compound 17 (0.01 mol) and acetic acid anhydride
(30 ml) was refluxed for 24 h. The solid product which produced
after cooling was collected and recrystallized from ethanol as white
solid. Yield (50%); m.p. 272 ꢁC; IR (KBr, cmꢀ1): 2973, 2860 (aliph.
CH), 1961(C]O) and 1608 (C]N). 1HNMR (600 MHz, DMSO-d6):
C
25H25N5O3S. Calcd. C, 63.14; H, 5.30; N, 14.73; Found: C, 62.90;
H, 5.00; N, 14.50.
d
¼ 2.23, 2.40 (2s, 6H, 2CH3), 3. 19, 3.87 (2t, 8H, 4CH2), 6.92-7.78 (m,
3.1.14. Preparation of compounds (16a & 16b); general procedure
A mixture of compound 11 (0.01 mol), aryl isothiocyanate
(0.01 mol), sulfur metal (0.01 mol) and catalytic amount of trie-
thylamine were refluxed in ethanol (30 mL) for 6 h. The reaction
mixture was poured into ice/water and acidified with 0.1 N HCl at
pH 3e4 then the resulting precipitate was filtered off, dried, and
recrystallized from the proper solvent.
5H, Ar-H þ thiazole-H5) ppm. Elemental analysis for C17H19N3O3S.
Calcd. C, 59.11; H, 5.54; N, 12.17; Found: C, 59.00; H, 5.30; N, 11.90.
3.1.20. N-(1-(4-morpholinophenyl)ethylidene)-4-phenylthiazol-2-
amine 20
Equimolar amounts of compound 1 (0.01 mol), 4-phenylthiazol-
2-amine (0.01 mol) and a few drops of piperidine in ethanol (30 ml)
was refluxed for 5 h. The solid product which produced on heating
was collected and recrystallized from the acetic acid as white solid.
Yield (65%); m.p.304e6 ꢁC; IR (KBr, cmꢀ1): 2967, 2838 (aliph. CH),
1645 (C]N). MS: 363 (Mþ, 100%), 303 (100%). 1HNMR (600 MHz,
3.1.15. 4-Amino-N0-(1-(4-morpholinophenyl)ethylidene)-3-phenyl-
2-thioxo-2,3-dihydro-thiazole-5-carbohydrazide 16a
Yield (50%); brown solid (dioxane); m.p. 244e46 ꢁC; IR (KBr,
cmꢀ1): 3327, 3310, 3200 (NH/NH2), 1672 (C]O). MS: 453 (Mþ, 18%),
CDCl3):
d
¼ 2.53 (s, 2H, CH3), 3.29, 3.86 (2t, 8H, morphonyl-H), 6.71
246 (100%). 1H NMR (600 MHz, DMSO-d6):
d
¼ 2.11 (s, 3H, CH3),
(s,1H, thazole-H5), 6.85- 7.88 (m, 9H, Ar-H) ppm. Elemental analysis

M.H.M. Helal et al. / European Journal of Medicinal Chemistry 65 (2013) 517e526
525
for C21H21N3OS. Calcd. C, 69.39; H, 5.82; N,11.56; Found: C, 69.10; H,
5.50; N, 11.50.
zone of inhibition was measured after 24 h of incubation. The anti-
bacterial activity of a common standard antibiotic ampicillinwas also
recorded maintaining the same protocol as above and at the same
concentration and solvent. The antibacterial activity results of the
compounds were compared with the standard and % activity index
for the heterocyclic compounds was calculated by using the formula
as given below:
3.2. Molecular modeling
An attempt to gain a better insight on the molecular structure of
these synthesized thiosemicarbazone compounds, geometric
Zone of inhibition by test compound ðdiameterÞ
% Activity index ¼
ꢄ 100
Zone of inhibition by standard ðdiameterÞ
optimization and conformation analysis has performed using PM3
force field as implemented in HyperChem 7.5 [41].
3.3.2. Determination of minimum inhibitory concentration (MIC)
value
The antibacterial screening concentrations of the compounds to
be used were estimated from the minimum inhibitory concentra-
tion (MIC) value. The MIC was determined using the disc diffusion
technique.
3.3. Biological activity
3.3.1. Antibacterial activity
Antimicrobialactivityof the tested samples was determined using
3.3.3. Anti-inflammatory (in vivo)
a modified KirbyeBauer disc diffusion method [42]. Briefly, 100
the test bacteria/fungi were grown in 10 ml of fresh media until they
reached a count of approximately 108 cells/ml [43]. A 100 l of mi-
ml of
All the synthesized compounds were screened for the in vivo
anti-inflammatory activity by carrageenan induced rat paw edema
method.
m
crobial suspension was spread onto agar plates corresponding to the
broth in which they were maintained. Isolated colonies of each or-
ganism that might be playing a pathogenic role should be selected
from primary agar plates and tested for susceptibility by disc diffu-
sion method of the National Committee for Clinical Laboratory
Standards (NCCLS) [44]. Among the available media available, NCCLS
recommends MuellereHinton agar due to: it results in good batch-
to-batch reproducibility. Plates inoculated with Gram (þ) bacteria
as S. aureus and B. cereus; Gram (ꢀ) bacteria as P. mirabilis and S.
marcesens, they were incubated at35e37 ꢁC for 24e48 h and thenthe
diameters of inhibition zones were measured in millimeters [42].
Standard discs of ampicillin (antibacterial agent served as positive
controls for antimicrobial activity but filter discs impregnated with
_ Method: Inhibition of carrageenan induced inflammation in
rat paw.
_ Animals used: Albino Wister rats.
_ Number of animals used: 6.
_ Dose of test compounds: 50 mg/kg.
_ Dose of standard drug: 10 mg/kg (indomethacin).
_ Route of administration: oral (1% w/v Tween 80 suspension).
_ Carrageenan suspension: sub planter (0.1 ml of 1% w/v sus-
pension in 0.9% saline solution).
The method developed by Winter et al. [47] was employed.
Albino Wistar rats of either sex (130e150 g) were divided into
various groups (6 rats per group). Animals were deprived of food
for 12 h prior to experiment and only water was given ad libitum.
First group was used as a control group (treated with 1 ml of 20% v/
v DMSO solution), the second group (treated with 1 ml of 20% v/v
DMSO solution of indomethacin (10 mg/kg) orally) and the rest of
groups (treated with DMSO solution of test compounds at a dose of
50 mg/kg orally). One hour after the administration of the com-
pounds, carrageenan suspension (0.1 ml of 1% w/v suspension in
0.9% saline solution) was injected into the plantar region of left
hind paw of animals. Immediately, the paw volume was measured
using plethysmometer (UGO Basile 21025 Comerio, Italy, initial paw
volume, Vc). Thereafter, the paw volume was measured after 1e4 h
after carrageenan administration. The difference between initial
and subsequent readings gave the change in edema volume for the
corresponding time. Edema volume of control (Vc) and volume of
treated (Vt) were used to calculate percentage (%) inhibition and (%)
edema volume by using following formula.
10 ml of solvent were used as a negative control. The agar used is
MuellereHinton agar that is rigorously tested for composition and
pH. Further the depth of the agar in the plate is a factor to be
considered in the disc diffusion method. This method is well docu-
mented and standard zones of inhibition have been determined for
susceptible and resistant values. Blank paper disks (Schleicher and
Schuell, Spain) with a diameter of 8.0 mm were impregnated with
10 ml of tested concentration of the stock solutions. When a filter
paper disc impregnated with a tested chemical is placed on agar, the
chemical will diffuse from the disc into the agar. This diffusion will
place the chemical in the agar only around the disc. The solubility of
the chemical and its molecular size will determine the size of the area
of chemical infiltration around the disc. If an organism is placed on
the agar, it will not grow in the area around the disc if it is susceptible
to the chemical. This area of no growth around the disc is known as a
“Zone of inhibition” or “Clear Zone”. For the disc diffusion, the zone
diameters were measured with slipping calipers of the (NCCLS) [44],
results are presented in Table 3. Agar-based methods such as E-test
and disk diffusion can be good alternatives because they are simpler
and faster than the broth-based methods [45,46]. The diameter of the
% Inhibition ¼ ½1 ꢀ ðVt=VcÞꢃ ꢄ 100
% Edema volume ¼ 100 ꢄ ðEdema volume after drug treatment=Initial volumeÞ:

526
M.H.M. Helal et al. / European Journal of Medicinal Chemistry 65 (2013) 517e526
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3.3.3.1. Statistical analysis. Values were expressed as means ꢂ S.E.
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ANOVA followed by least significant difference (LSD) and Turkey
multiple comparisons test.
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