Studies on synthesis, chromatographic resolution, and antiinflammatory activities of some 2-thioxo-1,2,3,4-tetrahydropyrimidines and their condensed derivatives

Article abstract of DOI:10.1002/1521-4184(200012)333:12<415::AID-ARDP415>3.0.CO;2-E

In this study, the synthesis of some new 2-thioxo-1,2,3,4-tetrahydropyrimidines and their condensed derivatives, thiazolo[3,2-a]pyrimidines, are described. The structures of the compounds were confirmed by IR, ¹H-NMR, ¹³C-NMR, and mass spectroscopy. The direct high-performance liquid chromatographic separation of the compounds on derivatized cellulose chiral stationary phases such as cellulose tris(3,5-dimethylphenylcarbamate) (OD), cellulose tris(4-methylphenylcarbamate) (OG), and cellulose tris(4-methylbenzoate) (OJ) was studied. All of the compounds were screened for their antiinflammatory activity and also investigated histopathologically. Compounds 3 and 1a were found to be the most promising antiinflammatory agents in this group.

Full text of DOI:10.1002/1521-4184(200012)333:12<415::AID-ARDP415>3.0.CO;2-E

2-Thioxo-1,2,3,4-tetrahydropyrimidines
415
Studies on Synthesis, Chromatographic Resolution, and
Antiinflammatory Activities of Some 2-Thioxo-1,2,3,4-tetrahydro-
pyrimidines and Their Condensed Derivatives
a)
a)
a)
a)*
b)
c)
Birsen Tozkoparan , Mine Yarim , Selma Saraç , Mevlüt Ertan , Pelin Kelicen , Gülçin Altinok ,
b)
and Rümeysa Demirdamar
a) Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100-Ankara, Turkey
b) Hacettepe University, Faculty of Pharmacy, Department of Pharmacology, 06100-Ankara, Turkey
c) Hacettepe University, Faculty of Medicine, Department of Pathology, 06100-Ankara, Turkey
Key Words: Antiinflammatory agents; 2-thioxo-1,2,3,4-tetrahydropyrimidines; 5H-thiazolo[3,2-a]pyrimidines;
enantioseparation; morphological findings
Summary
Table 1. Melting points, yields and empirical formula of the compounds 1–4
and 1a–4b.
In this study, the synthesis of some new 2-thioxo-1,2,3,4-tetrahy-
dropyrimidines and their condensed derivatives, thiazolo[3,2-
a]pyrimidines, are desc1ribed. The structures of the compounds
were confirmed by IR, H-NMR, 13C-NMR, and mass spectros-
copy. The direct high-performance liquid chromatographic sepa-
ration of the compounds on derivatized cellulose chiral stationary
phases such as cellulose tris(3,5-dimethylphenylcarbamate) (OD),
cellulose tris(4-methylphenylcarbamate) (OG), and cellulose
tris(4-methylbenzoate) (OJ) was studied. All of the compounds
were screened for their antiinflammatory activity and also investi-
gated histopathologically. Compounds 3 and 1a were found to be
the most promising antiinflammatory agents in this group.
——————————————————————————————
Comp.
No
R
R′
Mp
(°C)
Yield Empirical
(%) formula
——————————————————————————————
1
2-CH3
–
–
–
–
192–193
178–179
235–236
198–199
130–131
186–187
133–134
172–173
187–188
217–218
97–98
32 C15H18N2O2S
27 C15H18N2O2S
47 C15H18N2O3S
38 C15H18N2O3S
37 C24H22N2O3S
34 C24H21ClN2O3S
40 C24H22N2O3S
41 C24H21ClN2O3S
45 C24H22N2O4S
57 C24H21ClN2O4S
25 C24H22N2O4S
57 C24H21ClN2O4S
2
3-CH3
3
2-OCH3
3-OCH3
2-CH3
4
1a
1b
2a
2b
3a
3b
4a
4b
-H
2-CH3
4-Cl
-H
3-CH3
3-CH3
4-Cl
-H
Introduction
2-OCH3
2-OCH3
3-OCH3
3-OCH3
2-Thioxo-1,2,3,4-tetrahydropyrimidine derivatives have
attracted recent interest due to their structural similarity to the
4-Cl
-H
[1]
dihydropyridine calcium channel blockers . These com-
4-Cl
146–147
pounds have been used as precursors for the synthesis of some
——————————————————————————————
condensed derivatives which are reported to possess antiin-
[2,3]
flammatory activity
. In our previous studies, the synthesis
of all the compounds using methylbenzoate ester or phenyl-
carbamate derivatives of cellulose.
and antiinflammatory activities of some 2-benzylidene-2,3-
dihydro-5H-thiazolo[3,2-a]pyrimidine-3-one derivatives
[4,5]
have been described
. The promising pharmacological
properties of some derivatives led us to further investigate the
effects of the substituents on the thiazolo[3,2-a]pyrimidine
ring on the activity of this group compounds. In this study we
describe the synthesis and biological activities of new thia-
zolo[3,2-a]pyrimidines (Table 1).
Results and Discussion
Synthesis of the precursor 2-thioxo-1,2,3,4-tetrahy-
dropyrimidine derivatives (1–4) were achieved by direct
Biginelli condensation using methyl 3-oxopentanoate instead
Since all compounds (1–4, 1a–4b) have a center of chirality of ethyl acetoacetate as β-ketoester component of classical
[19]
and stereoisomers often show different pharmacological ac- Biginelli reaction . The reaction of 1–4 with chloroacetic
[6,7]
tivities
, resolution of the racemic mixtures seemed advis- acid and aromatic aldehydes in the presence of sodium ace-
able to investigate the biological properties of each tate, acetic acid, and acetic anhydride in one step led to
enantiomer. Enantioselective high-performance liquid chro- 2-benzylidene-7-ethyl-5-(substituted phenyl)-3-oxo-2,3-di-
matography (HPLC) and related methods are well established hydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid
[8–11]
[4]
techniques for the analysis of enantiomers
ester and carbamate derivatives coated onto large pore silica
gel have proved to be extremely useful stationary phases for bands at 3333–3192 cm (N–H), 1734–1662 cm (C=O),
. Cellulose methyl esters (1a–4b) (Scheme 1).
In the IR spectra, compounds 1–4 showed strong absorption
–1
–1
[12–
–1
1
chiral resolution essentially used in normal-phase mode
and 1188–1177 cm (C=S), respectively. In the H-NMR
. We also reporthere the direct analytical enantioseparation spectra of the compounds the H-4 proton of the ring was seen
18]
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
0365-6233/00/1212/0415 $17.50 +.50/0

416
Ertan and co-workers
as a doublet approximately 5.15–5.50 ppm. The signals of the
other protons were appeared at the expected chemical shifts
and integral values (Table 2).
As has already been explained before
[4,5]
, there are two
possibilities for the ring closure. The site of cyclization can
1
be determined by comparison of the H-NMR spectral data
for both compounds 1–4 and 1a–4b. When ring closure
occurred at nitrogen atom N-3, the C-5 proton of the con-
densed ring could be expected to shift to downfield compared
with H-4 proton of the starting compounds due to the strong
[20]
electronegative effect of the carbonyl group . Indeed, the
signal for the C-4 proton collapsed from a doublet in 1–4
(δ = 5.15–5.50 ppm; J = 3.41–3.88 Hz) to a singlet in 1a–4b
13
(δ = 6.18–6.35 ppm). C-NMR data of compounds 2, 3, 1a,
and 4b are shown in Table 3.
Scheme 1. Synthesis of the compounds 1–4 and 1a–4b.
Table 2. IR and 1H-NMR spectral data of the compounds synthesized.
——————————————————————————————————————————————————
Cmpd
No
IR (KBr)
(cm–1
1H-NMR
ppm
)
——————————————————————————————————————————————————
1
3274 (N–H)
1733 (C=O)
1188 (C=S)
(DMSO-d6) 1.15 (3H, t, CH3-), 2.45 (3H, s, Ar-CH3), 2.74 (2H, q, -CH2-),
3.45 (3H, s, CH3O-), 5.40 (1H, d, J: 3.71 Hz, H-4), 7.20 (4H, m, aromatic
protons), 9.45 (1H, s, N3-H), 10.25 (1H, s, N1-H)
2
3333 (N–H)
1734 (C=O)
1188 (C=S)
(DMSO-d6) 1.10 (3H, t, CH3-), 2.50 (3H, s, Ar-CH3), 2.70 (2H, q, -CH2-),
3.55 (3H, s, CH3O-), 5.15 (1H, d, J: 3.56 Hz, H-4), 6.90–7.30 (4H, m,
aromatic protons), 9.60 (1H, s, N3-H), 10.30 (1H, s, N1-H)
3
3192 (N–H)
1713 (C=O)
1177 (C=S)
(DMSO-d6) 1.10 (3H, t, CH3-), 2.70 (2H, q, -CH2-), 3.50 (3H, s, CH3O-), 3.75
(3H, s, Ar-OCH3), 5.50 (1H, d, J: 3.41 Hz, H-4), 6.80–7.30 (4H, m,
aromatic protons), 9.10 (1H, s, N3-H) ,10.20 (1H, s, N1-H)
4
3328 (N–H)
1662 (C=O)
1186 (C=S)
(DMSO-d6) 1.10 (3H, t, CH3-), 2.70 (2H, q, -CH2-), 3.55 (3H, s, CH3O-),
3.70 (3H, s, Ar-OCH3), 5.15 (1H, d, J: 3.88 Hz, H-4), 6.70–7.30 (4H, m,
aromatic protons), 9.60 (1H, s, N3-H), 10.30 (1H, s, N1-H)
1a
1b
2a
2b
3a
3b
4a
4b
1715 (C=O)
1615 (C=N)
(CDCl3) 1.26 (3H, t, CH3-), 2.70 (3H, s, -C6H4CH3), 2.75–3.00 (2H, m, -CH2-),
3.65 (3H, s, CH3O-), 6.35 (1H, s, H-5), 7.13–7.47 (m, 9H, aromatic protons),
7.70 (1H, s, =C-H)
1712 (C=O)
1602 (C=N)
(CDCl3) 1.25 (3H, t, CH3-), 2.70 (3H, s, -C6H4CH3), 2.80 (1H, q, -CH2-),
2.95 (1H, q, -CH2-), 3.65 (3H, s, CH3O-), 6.35 (1H, s, H-5), 7.10–7.50 (m, 8H,
aromatic protons), 7.65 (1H, s, =C-H)
1714 (C=O)
1615 (C=N)
(CDCl3) 1.25 (3H, t, CH3-), 2.30 (3H, s, -C6H4CH3), 2.90 (2H, q, -CH2-),
3.70 (3H, s, CH3O-), 6.20 (1H, s, H-5), 7.00–7.50 (m, 9H, aromatic protons),
7.75 (1H, s, =C-H)
1716 (C=O)
1608 (C=N)
(CDCl3) 1.28 (3H, t, CH3-), 2.35 (3H, s, -C6H4CH3), 2.90 (2H, q, -CH2-),
3.70 (3H, s, CH3O-), 6.20 (1H, s, H-5), 7.10–7.50 (m, 8H, aromatic protons),
7.70 (1H, s, =C-H)
1707 (C=O)
1605 (C=N)
(CDCl3) 1.25 (3H, t, CH3-), 2.65 (1H, q, -CH2-), 2.95 (1H, q, -CH2-),
3.65 (3H, s, CH3O-), 3.80 (3H, s, -C6H4OCH3), 6.30 (1H, s, H-5), 6.80–7.50
(m, 9H, aromatic protons), 7.70 (1H, s, =C-H)
1708 (C=O)
1621 (C=N)
(CDCl3) 1.25 (3H, t, CH3-), 2.65 (1H, q, -CH2-), 2.95 (1H, q, -CH2-),
3.65 (3H, s, CH3O-), 3.80 (3H, s, -C6H4OCH3), 6.30 (1H, s, H-5), 6.80–7.45
(m, 8H, aromatic protons), 7.65 (1H, s, =C-H)
1706 (C=O)
1603 (C=N)
(CDCl3) 1.28 (3H, t, CH3-), 2.92 (1H, q, -CH2-), 2.95 (1H, q, -CH2-),
3.67 (3H, s, CH3O-), 3.78 (3H, s, -C6H4OCH3), 6.19 (1H, s, H-5), 6.80–7.48
(m, 9H, aromatic protons), 7.75 (1H, s, =C-H)
1713 (C=O)
1604 (C=N)
(CDCl3) 1.26 (3H, t, CH3-), 2.91 (1H, q, -CH2-), 2.95 (1H, q, -CH2-),
3.67 (3H, s, CH3O-), 3.77 (3H, s, -C6H4OCH3), 6.18 (1H, s, H-5), 6.80–7.46
(m, 8H, aromatic protons), 7.68 (1H, s, =C-H)
———————————————————————————————————————————————————
Arch. Pharm. Pharm. Med. Chem. 333, 415–420 (2000)

2-Thioxo-1,2,3,4-tetrahydropyrimidines
417
Table 3. 13C-NMR spectral data of the compounds 2, 3, 1a, and 4b.
————————————————————————————————————————————————————————
Comp.
C2
C3
C4
C5
C6
C7
C8
C8a
C9
C10
C11
C12
C13
C14
————————————————————————————————————————————————————————
2
174.51
174.58
–
–
50.98 99.59 123.30 23.38 12.88
50.80 98.24 120.10 23.25 12.93
–
–
53.73 165.21 21.05
55.37 165.19 49.44
–
–
–
–
–
–
3
1a
4b
120.41 165.59
120.39 165.05
–
–
51.48 109.45 156.89
51.60 108.47 156.06
–
–
165.41 28.90 12.43 52.24 165.96 19.22 139.48
159.95 28.71 12.45 55.45 165.79 55.30 141.32
————————————————————————————————————————————————————————
Table 4. Chromatographic data obtained by direct enantioseparation of compounds 1–4 and 1a–4b.
—————————————————————————————————————————————————————
Comp.
R/R′
CSP
k′1
k′2
α
Rs
N1
N2
—————————————————————————————————————————————————————
1
2
2-CH3/–
3-CH3/–
OJa
1.87
0.49
1.63
0.71
2.25
0.75
3.28
3.27
0.58
3.72
0.84
3.85
0.88
6.72
–
1.75
1.18
2.28
1.18
1.71
1.17
2.05
–
0.87
0.73
1.60
0.8
81
2500
124
2916
124
1936
125
–
89
1965
127
1495
100
1547
119
–
ODa
OJa
3
2-OCH3/–
3-OCH3/–
2-CH3/-H
ODa
OJa
ODa
OJa
ODa
OGa
OGb
ODa
OGa
ODa
OGb
OGa
ODa
ODa
1.04
0.73
1.56
–
4
c
1a
–
c
–
–
–
–
–
–
1b
2a
2-CH3/4-Cl
3-CH3/-H
1.96
2.70
–
1.38
–
0.58
–
132
–
107
–
c
–
c
–
–
–
–
–
–
2b
3a
3b
4a
4b
3-CH3/4-Cl
2-OCH3/-H
2-OCH3/4-Cl
3-OCH3/-H
3-OCH3/4-Cl
0.61
3.24
1.92
0.70
1.96
0.92
3.96
2.82
0.79
1.24
1.52
1.22
1.47
1.13
0.63
3.20
0.36
0.82
0.63
1.89
6561
84
4182
70
144
2862
3844
154
3136
3181
—————————————————————————————————————————————————————•
a Eluent, hexane:ethanol (85:15, v/v). b Eluent, hexane:ethanol (95:5, v/v). c No separation.
+.
In the EI-MS spectra, molecular ion peaks (M ) were
The chromatographic results of the chiral separations of
detected and confirmed the molecular weight of the examined compounds 1–4 and 1a–4b are summarized in Table 4. The
compounds (2, 3, 4, 1a, 2a, 4a, and 4b). Cleavage of the C–C enantiomers of compounds 2, 3, and 4 were well separated on
bond adjacent to the N-3 nitrogen atom is the general frag- the OJ column with hexane:ethanol (85:15 v/v) as the mobile
mentation pattern leading to the base peak. Molecular ion phase. The introduction of a substituent in the 3-position of
peaks were the base peaks for compounds 3 and 4. The C-4 phenyl ring led to a strongly improved separation on the
prominent peaks are noted at m/z 134 (R′ = -H) and m/z 168 OJ column. Compound 4 was baseline separated on the OJ
+.
(R′ = 4-Cl) resulting from R′C H CH=C=S ion in the spec- column, giving separation (α) and resolution factor (R ) val-
6
4
s
tra of the condensed derivatives.
Analytical HPLC methods using derivatized cellulose chi-
ues of 2.05 and 1.56, respectively (Figure 1a).
In the case of condensed tetrahydropyrimidines 1a–4b,
ral stationary phases were developed for the separation of the compounds 1a and 2a could not be separated on cellulose
enantiomers of compounds 1–4 and 1a–4b. Resolution was carbamate derivatives chiral stationary phases used in this
achieved using normal-phase methodology with mobile study. Partial or baseline resolutions were observed for com-
phases consisting of hexane:ethanol in various ratios and a pounds 1b, 2b–4b using carbamoylated cellulose derivatives,
silica-based chiral stationary phase, such as cellulose tris(3,5- OD and OG. Among these compounds, compounds 2b and
dimethylphenylcarbamate (OD), cellulose tris(4-methyl- 4b were baseline separated on the OD column with resolution
phenylcarbamate) (OG), or cellulose tris(4-methylbenzoate) factor values of 3.20 and 1.89, respectively. As seen in Table
(OJ).
4, chiral recognition of OD coated on silica gel depended on
Arch. Pharm. Pharm. Med. Chem. 333, 415–420 (2000)

418
Ertan and co-workers
Table 5. Antiinflammatory activity of 1–4 and 1a–4b.
——————————————————————————————
Compound
No
R
R′
Antiinflam- ED50
matory
activity%a
(n = 6)
——————————————————————————————
1
2-CH3
3-CH3
2-OCH3
3-OCH3
2-CH3
2-CH3
3-CH3
3-CH3
2-OCH3
2-OCH3
3-OCH3
3-OCH3
–
–
–
–
–
–
–
2
–
–
3
36
–
34.36
–
4
1a
-H
33
26
6
50.00
50.00
80.61
–
1b
4-Cl
-H
2a
2b
4-Cl
-H
–
3a
–
–
3b
4-Cl
-H
15
29
–
50.00
59.72
–
4a
4b
4-Cl
–
Figure 1. Chromatographic resolution of a) compound 4 on cellulose tris(4-
methylphenyl benzoate) (OJ) and b) compound 2b on cellulose tris(3,5-di-
methylphenylcarbamate) (OD). Column, 250 × 4.6 mm I.D.; eluent,
hexane:ethanol (85:15, 0.5 ml/min); detection wavelength, 320 nm.
Indomethacinb
32.00
1.98
——————————————————————————————
a100 mg/kg dose p.o. b10 mg/kg dose p.o.
the position of substituents on the C-5 phenyl ring and C-2
Acute pyelonephritis should be considered in the differen-
benzylidene structure of the condensed tetrahydropyrimidine tial diagnosis of the TIN due to the drug effect but none of
our cases showed interstitial suppurative inflammation with
microabscesses, tubular necrosis, or deformation of the un-
derlying calyx and pelvis. Therefore, the presented mor-
phologic findings were accepted as the reflection of the
effects of the drugs.
ring. This may indicate that compounds 2b and 4b having
3-substituted phenyl ring at the C-5 position and 4-chloro
substituent on the benzylidene structure can adopt a suitable
conformation for interaction with the chiral selector. The
separation of compound 2b on OD column using hex-
ane:ethanol (85:15) as the mobile phase is shown in Fig-
ure 1b. The peaks observed at 10.36 and 12.37 minutes may
belong to the enantiomeric pairs of other geometric isomer
Gastritis is characterized by the inflammation of the gastric
mucosa and we also evaluated the severity of the gastritis for
each compound. In none of our cases erosion (disruption of
the integrity and sloughing of the superficial mucosa) was
observed, but the cases showing severe gastritis are shown in
Table 6. In such cases hemorrhaging into the mucosa was
also noted. The reflux esophagitis was seen in both control
group and the other groups and is not accepted as an effect of
the drugs, but only a sign of reflux of the contents.
As a result of our morphological findings, compounds 3 and
1a were found to be reliable and less harmful to the kidney
parenchyme and the mucosa of the stomach (Table 6). The
other compounds such as 1b, 2a, 3b, and 4a displayed mod-
erate tubulointertitial nephritis and gastritis in some cases but
none of them were as severe as seen in the case of in-
domethacin.
As expected, naproxen (30 mg/kg) and phenylbutazone
(100 mg/kg) did not show any inflammatory reaction.
In conclusion, 3 and 1a were the most promising com-
pounds as they produced 33% and 36% antiinflammatory
activity at a dosage of 100 mg/kg, respectively, when com-
pared the others. They also proved to be more reliable than
indomethacin as they did not cause any reaction in the kidney
and stomach.
[4]
due to the double bond in the benzylidene structure .
All compounds were screened for their antiinflammatory
activity by carrageenan hind paw edema test using in-
domethacin as reference compound. Among the compounds
tested in this study, compounds 3, 1a, 1b, 3b, and 4a showed
consistent antiinflammatory activity. As seen in Table 5
compound 3 (R: 2-OCH ), one of the starting compounds,
3
was found as the most potent antiinflammatory compound,
showing 36% inhibition. Test results revealed that, cycliza-
tion of the tetrahydropyrimidines to their corresponding sub-
stituted thiazolo[3,2-a]pyrimidines resulted in an increase in
antiinflammatory activity of only three compounds (1a, 1b,
and 4a).
[4]
Taking our previous and present findings into considera-
tion, it is worth noting that enhancing the length of the alkyl
chain at position 7 generally causes a decrease in activity
(except 1a, 3b, and 4a).
On microscopic examination, the morphologic findings in
the kidney, such as pronounced edema and interstitial
mononuclear cell infiltration, were characteristic for tubu-
lointerstitial nephritis (TIN). The presence of the scattered
eosinophil leucocytes also provided evidence of the drug
effect. Focal areas displaying variable but generally mild
degree of tubular regeneration were also present. The
glomeruli were all normal and granulomatous reaction was
not observed in the interstitium. The compounds associated
with moderate to severe interstitial nephritis are shown in
Table 6.
Acknowledgements
This research was supported by the Scientific and Technical Council of
Turkey (TÜBITAK) (Project No: 198S082; SBAG-AYD-217).
Arch. Pharm. Pharm. Med. Chem. 333, 415–420 (2000)

2-Thioxo-1,2,3,4-tetrahydropyrimidines
419
Table 6. Morphological findings seen in kidney, esophagus, and stomach.
——————————————————————————————————————————————————————————
Kidney
Esophagitis
Stomach
—————————————————————————————
————————————
Congestion – Edema
—————————
Inflammation
———————————————————
Erosion
Gastritis
+
++
+++
MNL
++
PMNL
++ +++
+
++ +++
+
+++
+
——————————————————————————————————————————————————————————
3
1a
1b
2a
3b
4a
✔
✔
–
+
+
–
+
+
–
–
–
–
–
–
–
–
✔
✔
✔
✔
✔
✔
✔
✔
✔
✔
✔
✔
✔
✔
Naproxen
(30 mg/kg)
Phenylbutazone
(100 mg/kg)
Indomethacin
(10 mg/kg)
Control
✔
✔
–
+
+
–
–
–
✔
✔
✔
✔
——————————————————————————————————————————————————————————
(+): mild, (++): moderate, (+++): severe activity, (MNL): mononuclear inflammatory cell, (PNML): polymorphonuclear leucocytes.
Synthesis of Methyl 6-Ethyl-4-(substituted phenyl)-2-thioxo-1,2,3,4-tetrahy-
dropyrimidine-5-carboxylates (1–4)
Experimental Part
A mixture of methyl 3-oxopentanoate (0.0375 mol), thiourea (0.025 mol),
and substituted benzaldehyde (0.025 mol) in absol. ethanol (20 ml) was
treated with 8 drops of 37% HCl as catalyst and then refluxed for an
appropriate period. The crude product which precipitated on cooling was
filtered and washed with 50% ethanol. Then, it was recrystallized several
times from ethanol.
Chemistry
Melting points were estimated with a Thomas Hoover capillary melting
point apparatus and are uncorrected. IR spectra (KBr) were recorded on a
Perkin Elmer 1720X FT-IR spectrometer. H-NMR spectra were obtained
with a Bruker 200 MHz FT NMR instrument using CDCl3/DMSO-d6 and
tetramethylsilane as internal standard. Elemental analyses of the compounds
were performed on a Leco CHNS 932 analyzer at the ScientificandTechnical
Research Council of Turkey, Instrumental Analysis Laboratory at Ankara.
Elemental analyses for C, H, and N were within ±0.4% of theoretical values.
1
Synthesis of Methyl 2-Benzylidene-7-ethyl-3-oxo-5-phenyl-2,3-dihydro-5H-
thiazolo[3,2-a] pyrimidine-6-carboxylates (1a–4b)[4,5]
Equimolar amounts of 1–4, chloroacetic acid, and benzaldehyde or 4-chlo-
robenzaldehyde were refluxed in presence of anhydrous sodium acetate (1.5
mmol), acetic anhydride (3 ml), and of acetic acid (2.5 ml) for 3 h. The
mixture was poured into ice-water and the resulting precipitate was dissolved
in dichloromethane. The organic layer was washed using 6% NaHCO3 and
brine, and was then evaporated under reduced pressure. The crude product
was recrystallized from appropriate solvents.
HPLC Experiment
The HPLC analysis was performed at room temperature with a Spec-
traSYSTEM P1500 Pump, a Rheodyne injector with a 20 µl loop, a Spec-
traSYSTEM UV1000 variable wavelength UV detector set at 320 nm, a
Hewlett Packard HP 670C integrator, and a IBM PC computer using PC1000
system software. Chromatographic resolution of the compounds was per-
formed on cellulose-based chiral stationary phases such as OD cellulose
tris(3,5-dimethylphenylcarbamate) coated on silica gel, OG cellulose tris(4-
dimethylphenyl carbamate) coated on silica gel and OJ cellulose trisbenzoate
coated on silica gel at a flow rate of 0.5 ml/min. These column materials were
synthesized in our laboratory as described previously[21,22]. The separation
was achieved with n-hexane:ethanol (85:15 v/v) and (95:5 v/v) as the mobile
phases. Toluene was used as a non-retained compound to estimate the dead
time (t0) of the chiral columns.
Melting points, yields, and formula of the compounds are shown in
Table 1.
Pharmacology
Local breed albino mice of either sex (Refik Saydam Hifzissihha Institute,
Animal Care Unit, Ankara, Turkey) weighing approximately 20–25 g were
used. Randomly chosen six mice were housed in two days for acclimatization
to room conditions and were maintained on standard diet and water ad
libitum. The food was withdrawn on the day before the experiment, but free
access to water was allowed.
Chromatographic Characteristics
Antiinflammatory Activity: Carrageenan Induced Edema
The separation factor (α), which represents a measure of relative peak
separation is expressed as follows: α = k2′/k1′ where k1′ and k2′ are capacity
factor for the first and second eluting enantiomers, respectively. The capacity
factor (k′) is calculated as follows:
k1′ = (t1 – t0)/t0 and k2′ = (t2 – t0)/t0 where t0, t1, and t2 refer to the retention
time in seconds for the non-retained compound, the first and second eluting
enantiomers, respectively.
The stereochemical resolution factor (Rs) of the first and second eluting
enantiomer peaks is calculated by the ratio of the distance between the
retention times (t1 and t2) and arithmetic mean of the two peak widths
Rs = 2(t2 – t1)/(w1 + w2).
The method of Winter et al.[23] was employed with some modifications.
All test samples were administered to animals in a 100 mg/kg dosage as a
suspension in 0.5% carboxymethyl cellulose by using a gastric lavage
apparatus. 1 h after the oral administration of test sample, each mouse was
injected with 2% carrageenan solution (0.01 ml) into subplantar tissue of the
right hind paw. The volume of the paw was measured immediately and 2 h
after carrageenan injection with Peacock thickness gauge. The control group
animals received appropriate volumes of the dosing vehicle only. The per-
centage inhibition effects of drugs was calculated according to the following
equation:
Arch. Pharm. Pharm. Med. Chem. 333, 415–420 (2000)

420
Ertan and co-workers
[6] D. E. Drayer, Clin. Pharmacol. Ther. 1986, 40, 125–133.
Antiinflammatory Activity (%) = (n – n’)/n × 100
[7] D. F. Smith, Pharmacol. Toxicol. 1989, 65, 321–331.
n = the difference in thickness between first and second measurement of
paw in the control group; n’ = the difference in thickness between first and
second measurement of paw in the group which had been administered the
test sample.
[8] G. Blaschke, Angew. Chem. Int. Ed. Engl. 1980, 19, 13–24.
[9] E. Francotte, A. Junker-Buchheit, J. Chromatogr. 1992, 576, 1–45.
Indomethacin (10 mg/kg) was used as reference compound.
The experiment was repeated in two different dose levels (25 and
50 mg/kg) for the compounds which have significant statistical differences
between the treatments and the control group. ED50 values were calculated
by using Lichtfield-Wilcoxon method. Two way Analysis of Variance
(ANOVA) was employed as the statistical method. All statistical analysis
were performed by using a PC program ”Pharmacological Calculations
System Version 4.1”.
[10] M. Schulte, J. N. Kinkel, R.-M. Nicoud, F. Charton, Chem. Ing. Tech.
1996, 68, 670–683.
[11] M. Schulte, K.-H. Derwenskus, R. Ditz, Nachr. Chem. Tech. Lab. 1997,
45, 487–492.
[12] A. Ichida, T. Shibata, I. Okamoto, Y. Yuki, H. Namikoshi, Y. Toga,
Chromatographia 1984, 19, 280–284.
[13] Y. Okamoto, M. Kawashima, K. Hatada, J. Chromatogr. 1986, 363,
173–186.
Histopathological Examination
[14] Y. Okamoto, R. Aburatani, K. Hatada, J. Chromatogr. 1987, 389,
After the paw edema experiments, mice were sacrificed after 4 h and the
stomachs and kidneys were removed and placed in formalin solution for
histopathological examination. The sections taken from these specimens
were stained with haemotoxilen eosine and examined under a light micro-
scope.
95–102.
[15] Y. Okamoto, R. Aburatani, K. Hatano, K. Hatada, J. Liq. Chromatogr.
1988, 11, 2147–2163.
[16] K. Ikeda, T. Hamasaki, H. Kohno, T. Ogawa, T. Matsumoto, J. Sakai,
Chem. Lett. 1989, 1089–1090.
[17] T. Shibata, K. Mori, Y. Okamoto in Chiral Separation by HPLC (Ed.:
A. M. Krstulovic), Ellis Horwood Ltd., Chichester 1989, chapter 13.
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Received: July 10, 2000 [FP505]
Arch. Pharm. Pharm. Med. Chem. 333, 415–420 (2000)