Synthesis and biological evaluation of new 4,5-disubstituted-thiazolyl amides, derivatives of 4-hydroxy-piperidine or of 4-N-methyl piperazine

Article abstract of DOI:10.3390/80600472

4,5-disubstituted-thizolyl amides, derivatives of 4-hydroxy-piperidine and of 4-N-methyl piperazine, were synthesized and tested as anti-inflammatory agents. Log P values were theoretically calculated and experimentally determined. These compounds were tested for antioxidant activity, as hydroxyl radical scavengers and for their ability to interact with stable 1,1-diphenyl-2-picryl hydrazyl free radical (DPPH). The effect of the synthesized compounds on inflammation, using the carrageenin induced mice paw edema model was studied. Both anti-inflammatory and antioxidant activities depended on some structural characteristics of the synthesized compounds.

Full text of DOI:10.3390/80600472

Molecules 2003, 8, 472-479
molecules
ISSN 1420-3049
http://www.mdpi.org
Second Eurasian Meeting on Heterocyclic Chemistry
“Heterocycles in Organic and Combinatorial Chemistry“
Synthesis and Biological Evaluation of New 4,5-Disubstituted-Thiazolyl
Amides, Derivatives of 4-Hydroxy-Piperidine or of 4-N-Methyl
Piperazine
A. Geronikaki *, D. Hadjipavlou-Litina , C. Chatziopoulos and G. Soloupis
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotelian University of
Thessaloniki, Thessaloniki 54124, Greece; Tel. (+30)310997616, Fax (+30)310997612
*Author to whom correspondence should be addressed; e-mail: geronik@pharm.auth.gr
Received: 13 May 2003; in revised form: 15 May 2003 / Accepted: 16 May 2003 / Published: 30 June
2003
Abstract: 4,5-disubstituted-thizolyl amides, derivatives of 4-hydroxy-piperidine and of
4-N-methyl piperazine, were synthesized and tested as anti-inflammatory agents. Log P
values were theoretically calculated and experimentally determined. These compounds
were tested for antioxidant activity, as hydroxyl radical scavengers and for their ability to
interact with stable 1,1-diphenyl-2-picryl hydrazyl free radical (DPPH). The effect of the
synthesized compounds on inflammation, using the carrageenin induced mice paw edema
model was studied. Both anti-inflammatory and antioxidant activities depended on some
structural characteristics of the synthesized compounds.
Keywords: Thiazolyl amides, anti-inflammatories, antioxidants, lipoxygenase inhibitors.
Introduction
Various non-steroidal anti-inflammatory drugs (NSAIDs) are in widespread clinical use for the
treatment of inflammation diseases. However, despite their great number, their mechanism of action as

Molecules 2003, 8
473
well as their precise therapeutic activities are still under investigation. Furthermore, almost all of them
present a number of unwanted, often serious, side effects as consequence of interference with the
arachidonic cascade [1]. It is well known that free radicals play an important role in the inflammatory
process [2]. Superoxide anion radicals, hydrogen peroxide and hydroxyl radicals, produced by
activation of phagocytes, are considered to be involved in inflammation and tissue destruction. Free
radicals are also involved in the biosynthesis of prostaglandins, important mediators of inflammation
[2]. Compounds with antioxidant properties are generally expected to protect against inflammation.
Several substituted thiazolyl derivatives [3,4] are reported to possess anti-inflammatory and/or
antioxidant activities. This paper is an extension of previous work on the synthesis of thiazole
derivatives with structures justifying anti-inflammatory activity.
Results and Discussion
The structures of the synthesized compounds are given in Figure 1 and the general method used to
prepare the final compounds is shown in Figure 2.
Figure 1. Structures of synthesized compounds
R
N
NHCO(CH2)nN(R1)2
= H, CH3, C6H5, C6H4OCH3
S
R
N
N
CH3
N(R1)2=
N
OH
,
Figure 2. Synthetic procedure
R
R
N
N
abs. C2H5OH
Na2CO3
dry benzene
3h
+
ClCO(CH2)nCl
NH2
NHCO(CH2)nCl
S
S
R
N
12h
NHCO(CH2)nN(R1)2
S
Overall the reactions proceeded smoothly in good yields. The structure of the synthesized
compounds and their physicochemical properties are shown in Table 1. The amides were identified
both by elemental analyses as well as by their spectroscopic analyses.

Molecules 2003, 8
474
Table 1. Characterization data of the synthesized amides
R1
N
Z
N
NHCO(CH2)n
R2
S
N
1
R1
R2
Z
n
1
1
1
1
1
1
2
2
2
2
2
2
1
1
1
2
m.p. oC
85-86
MS
241
254
316
512
346
326
255
269
331
360
526
343
240
316
326
382.5
Mol. Structure b
C12H14N3O2S
C11H16N3O2S
C16H18N3O2S
C30H46N3O2S
C17H20N3O4S
C14H20N3O4S
C11H16N3O2S
C12H19N3O2S
C17H21N3O2S
C18H22N3O2S
C31H48N3O2S
C15H23N3O4S
C13H16N3OS
C17H20N3OS
C15H22N3O3S
C15H22N3O3S
H
CH3
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
CH3
CH3
CH3
CH3
2
H
89-92
3
Ph
H
89.5-92
79-80
4
Ph
CH3(CH2)13
5
Ph-OCH3
CH2COOEt
H
H
115-117
79-82c
6
H
7
H
semisolid
148-150
227-8
8
CH3
H
9
Ph
H
10
11
12
13a
14a
15a
16a
Ph-OCH3
Ph
H
193-5
CH3(CH2)13
semisolid
116-8
CH2COOEt
H
H
H
H
H
H
225-228
semisolid
semisolid
248-50
Ph
CH2COOEt
Ph-OCH3
Notes: a Piperazinyl – ring; b Elemental analyses for molecular formula; c m.p for hydrochloride salt
Physicochemical parameters, e.g. log P have been calculated theoretically, as an expression of lipo-
philicity (Table 2). The in vivo antiinflammatory activity of the synthesized compound was determined
using carrageenin induced mice paw edema (26.1-64.3 %) (see Table 2). The compounds have also
been screened for their in vitro reducing activity towards the free stable radical DPPH (15.6-26.6 %)
and for their hydroxyl free radical scavenging activity (44.6-100 %) (Table 3). For the 4-OH-
piperidinyl amides the inhibition on the soybean LOX has been performed (77.5-100 %). The
preliminary results reveal that the tested compounds exhibit in generally good biological activity. The
results are discussed in terms of structural characteristics. The presence of a R1 = phenyl group seems
to be crucial for high activity in vivo. The length of the chain also plays an important role. The
derivatives with n = 1 are stronger inhibitors. The nature of the alicyclic amine is an important feature,
since the 4-N-methyl piperazinyl derivatives possess higher in vivo results.

Molecules 2003, 8
Table 2. Lipophilicity values a) experimentally performed RM values; b) theoretically
475
calculated lipophilicity values using: i) Suzuki-Kudo’s method ii) the clog P program
from Biobyte. In vivo carrageenin rat paw edema % inhibition after 3.5 h (CPE %).
N
1
RM
logPsk Clog P
CPE%
52.3
59
-0.494± 0.03
-4.287
-1.878
-0.434
6.322
-1.062
-2.473
-1.751
-0.365
1.081
0.453
6.5
-0.34
0.159
1.758
6.322
1.777
-0.133
-0.143
0.212
-1.0955
1.974
8.684
0.014
1.102
3.2
2
nt
3
-0.578± 0.031
62.6
44.2
45
4
0.911± 0.037
5
-0.490± 0.031
6
nt
33.4
26.1
27.5
47.5
57
7
-0.504± 0.021
8
-0.534± 0.037
9
-0.589± 0.039
10
11
12
13
14
15
16
-0.504± 0.024
0.894± 0.033
32.2
59.6
61
nt
nt
nt
nt
nt
-0.958
-3.747
-1.969
-2.161
0.97
64.3
47.9
38.5
1.309
3.219
Table 3. Biological testing results. In vitro effects of the examined and reference drugs
(in % at 0.1 mmol/L and 0.2 mmol/L) on reducing ability (DPPH), on hydroxyl radical
scavenging activity (. OH) and on soybean lipoxygenase activity (LOX).
DPPH
DPPH
DPPH
N
(0.1 mmol/L)
(0.1 mmol/L)
(0.2 mol/L)
. OH
LOX
20min
25.4
13.5
nt
1hr
24.6
17.3
nt
1 hr
23.5
18.1
nt
1
2
3
4
5
6
7
8
9
97.2
100
75.3
78.4
44.6
no
no
100
100
100
100
100
100
nt
15.6
no
29.8
27.6
nt
17.5
no
nt
nt
nt
nt
nt
no
nt
nt
nt
nt
23.7
25.1
36
no
100

Molecules 2003, 8
476
Table 3. Cont.
DPPH
DPPH
DPPH
N*
(0.1 mmol/L)
20min
26.7
(0.1 mmol/L)
(0.2 mol/L)
. OH
LOX
1hr
32
17.7
14
–
1 hr
35.3
16.1
20.6
–
10
11
95.9
40
87
–
100
100
77.5
–
17.8
12
13.3
ASA
NDGA
80.5
94
–
–
–
91
Conclusions
We have presented a facile route for the formation of new 4,5-disubstituted thiazolyl amides,
possessing anti-inflammatory activity. These compounds also present mild antioxidant activity. They
highly compete with DMSO for hydroxyl radical and strongly inhibit LOX. A phenyl group in R1, the
length of the chain and a value of n = 1 are all significant structural features for the activity observed.
Acknowledgements
This work was supported by INTAS (grant No. INTAS 00-0711). Thanks are also due to Drs. C.
Hansch and A. Leo for providing the Mac Clog P software, to the Theagenium Anticancer Hospital for
providing mice and to D. Rigas, Lab. of Organic Chemistry Dept., for providing accurate mass spectra.
Experimental
General
Melting points were obtained with a MELTEMP II capillary apparatus (LAB. Devices Holliston,
MA, USA) and are reported without correction. Infrared spectra (Nujol mulls) were recorded on a
Perkin Elmer 597 spectrophotometer. UV-Vis were determined on a Perkin Elmer 554 UV-Vis spec-
trophotometer. Proton NMR spectra were obtained on a Brucker AW 80 apparatus at 80 MHz and are
reported in ppm downfield from tetramethylsilane (TMS). Analyses indicated by the symbols of ele-
ments were within 0.4 % of theoretical values. Mass spectra (MS) were determined on a VG-250 in-
strument (VG Labs. Tritech, England) with the ionization energy maintained at 70 eV. The reactions
were monitored by TLC on silica gel 60 F254 (Merck). All reagents were obtained from commercial
sources. 2-Aminothiazole, 2-amino-4-methylthiazole, 2-amino-4-phenylthiazole and 2-amino-4-(4-
methoxyphenyl)thiazole were synthesized as described previously [5].

Molecules 2003, 8
477
General synthetic procedure for the synthesis of the 2-chloroacetamido- and 3-chloropropionylamido
thiazoles [5].
To a solution of 2-aminothiazole or suitable 4-substituted aminothiazole (0.02 mol) in dry benzene
a cooled solution of chloroacetyl or 3-chloropropionyl chloride (0.033 mol) in dry benzene (7.5 mL)
was added dropwise. The reaction mixture was refluxed in a water bath at 80 oC for 3 h. Benzene and
excess 3-chloropropionyl chloride were removed by distillation. The residue was washed with aqueous
sodium bicarbonate (5 % w/v) followed by cold water. The crude product was dried and crystallized
from ethanol.
General synthetic procedure for the synthesis of the 2-(N-substituted aminoacetamido)/3-(N-
substituted aminopropioamido) thiazoles [5].
A mixture of 2-chloroacetamido or 3-chloropropionamido thiazole (0.006mol), amine (4-OH-
piperidine or N-methyl-piperazine, 0.7 mol), absolute ethanol (15 mL) and anhydrous sodium carbona-
te (1.48 g) was heated under reflux in a water bath for 12 h. The excess of amine and ethanol was re-
moved by distillation and the residue was treated with 5 % sodium bicarbonate solution to remove acid
impurities, filtered, washed with water and dried. It was crystallized from ethanol (95 %) to give white
crystals.
Preparation of the hydrochlorides of 2-(N-substituted- aminoacetamido) / 3-(N-substituted-amino-
propionamido) thiazoles.
A solution of the base in anhydrous ethanol was saturated with dry hydrogen chloride gas. The salt
was filtered off, washed with dry ether and recrystallized from absolute ethanol to give white crystals.
The structure of all compounds was identified both by elemental analyses as well as by spectroscopic
analysis (IR, 1H-NMR, MS).
Spectral Data: 1H-NMR (CDCl3, DMSO) for representative compounds ( δ):
Compound 1: 1.94-2.16 (d, 4H, piperidyl C3-H,), 2.157 (s, 2H, COCH2), 3.2 (s, 1H, piperidyl C4-H),
3.2-3.38 (d, 4H, piperidyl NCH2), 6.4 (d, 1H, thiazolyl C5-H), 6.86 (d, 1H, thiazolyl C4-H).
Compound 9: 1.71-1.89 (d, 4H, piperidyl C3-H), 2.49-2.53 (m, 8H, NCH2, COCH2CH2 ), 7.42-7.45
(m, 3H, phenyl C3,4,5-H), 7.46-7.48 (m, 2H, phenyl C-2.6-H), 9.05 (b, 1H, NH). Compound 16: 2.78
(s, 3H, NCH3), 2.78-3.53 (m, 8H, piperidyl), 3.35 (s, 2H, COCH2), 3.71 (s, 3H, OCH3), 6.96-6.99 (d,
2H, phenyl C2,6-H), 7.51 (s, 1H, thiazolyl C5),7.8-7.89 (d, 2H, phenyl C3,5-H); IR cm-1: 3190-3230,
1680, 1620

Molecules 2003, 8
478
Biological assays
1. Physicochemical Studies [4]
Lipophilicity is an important factor affecting the distribution and fate of drug molecules. Increased
lipophilicity is correlated with increased biological activity and more rapid metabolism. Theoretical
calculations of lipophilicity as clog P and Suzuki-Kudo’s method [10] were performed (Table 2). The
program CLOG P [11], has been designed to calculate the lipophilicity of a molecule using the
additivity method. Reversed phase TLC (RPTLC) was performed on silica gel plates impregnated
with 55 (v/v) liquid paraffin in light petroleum ether. Mobile phase: methanol/water mixture (70/30,
v/v) containing 2 % aqueous ammonia (27 %). The plates were developed in closed chromatography
o
tanks saturated with the mobile phase at 24 C. Spots were detected under UV light or by iodine
vapours. RM values were determined from the corresponding Rf values (from ten individual
measurements) using the equation RM = log [( 1/Rf ) -1 ]. For results see Table 2.
2. Inhibition of carrageenin induced paw oedema [4]
All tested compounds were suspended in water with few drops of Tween-80 and ground in a mortar
before use. Groups of 5-6 AKR mice weighing 20-30 g were used. A single dose of 0.2 mmol/Kg or
indomethacin (0.11 mmol/Kg) was administered intraperitoneally i.p., simultaneously to the adminis-
tration of the phlogistic agent : carrageenin (2%, 0.05 ml was injected intradermally i.d. into the right
foot pad, the left serving as control). Results are given in Table 2.
3. Competition of the tested compounds with DMSO for hydroxyl radicals [6]
The hydroxyl radicals generated by the Fe3+/ascorbic acid system were detected by the determi-
nation of formaldehyde produced from the oxidation of DMSO. The reaction mixture contained EDTA
(0.1mM), Fe3+(167 µM, as a 1:2 mixture with EDTA) and DMSO (33mM) in phosphate buffer (50
mM, pH 7.4) and the tested compounds (final concentration 1mM - final volume of the samples 1 mL).
Ascorbic acid (150 µL, 10 mM in phosphate buffer) was added at the end in order to initiate the
reaction. The mixture was incubated at 37 oC for 30 min. The reaction was stopped by the addition of
trichloroacetic acid (250 µL, 17.5 % w/v) and the formaldehyde formed was detected
spectrophotometrically at 412 nm by the method of Nash [7].
Interaction of the synthesized compounds with DPPH [8]
To a solution of DPPH (0.1 mM) in absolute ethanol, an equal volume of the compounds dissolved
in ethanol was added (0.1 mM). A control solution containing ethanol was also used. After 20 and 60
mins at room temperature, absorbance was recorded at 517 nm. For the results see Table 3.

Molecules 2003, 8
479
4. Soybean lipoxygenase inhibition [9]
The conversion of sodium linoleate to 13-hydroxy-peroxylinoleic acid at 234 nm, was recorded and
compared with appropriate standard, see Table 3.
Each in vitro experiment was performed at least in triplicate and the standard deviation in absor-
bance was less than ±10 %. Acetyl salicylic acid (ASA) and/or nor-dihydroguaeretic acid (NDGA)
were used as standards.
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