New isothiazole derivatives: Synthesis, reactivity, physicochemical properties and pharmacological activity

Article abstract of DOI:10.1002/ardp.200500040

The synthesis and biological investigation of the series of amide and ester derivatives 10-20 of 5-(4-chlorobenzoyl)amino-3-methyl-4-isothiazolecarboxylic acid 5 are presented. Because the amide series of 5-benzoylamino-3-methyl-4- isothiazolecarboxylic a

Full text of DOI:10.1002/ardp.200500040

Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
A. Regiec et al.
401
Full Paper
New Isothiazole Derivatives: Synthesis, Reactivity,
Physicochemical Properties and Pharmacological Activity
1
1
2
2
´
Andrzej Regiec , Zdzisław Machon , Ryszard Mie˛dzybrodzki , Stanisław Szymaniec
1 Department of Organic Chemistry, Faculty of Pharmacy, Medical Academy of Wroclaw, Wroclaw, Poland
2 Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
The synthesis and biological investigation of the series of amide and ester derivatives 10–20 of
5-(4-chlorobenzoyl)amino-3-methyl-4-isothiazolecarboxylic acid 5 are presented. Because the
amide series of 5-benzoylamino-3-methyl-4-isothiazolecarboxylic acid 2 has been studied extensi-
vely and from this series denotivir (vratizolin) 4 [1, 2] became the antiviral drug. The influence of
exchanging the N-benzoyl for a N-(4-chlorobenzoyl) group at position 5 of the isothiazole ring on
the pharmacological activity of 5-benzoylamino-3-methyl-4-isothiazolecarboxylic acid 2 deriv-
atives is dealt with here. The effect of structure modifications in the carboxylic group of the 5-(4-
chlorobenzoyl)amino-3-methyl-4-isothiazolecarboxylic acid 5 series of derivatives on their biolo-
gical activity is discussed. Some of the tested 5-(4-chlorobenzoyl)amino-3-methyl-4-isothiazolecar-
boxylamides revealed significant anti-inflammatory activity in carrageenan induced edema and
air-pouch inflammation tests. Physicochemical properties of 6-(4-chlorophenyl)-3-methylisothia-
zolo[5,4-d]-4H-1,3-oxazin-4-one 6 are described. Its use in the synthesis of isothiazole derivatives
and its reactivity are also presented.
Keywords: Anti-inflammatory activity / Cytotoxicity / Isothiazole / Isothiazolo[5,4-d]-4H-1,3-oxazine / Membrane stabili-
zation /
Received: October 12, 2005; accepted: February 28, 2006
DOI 10.1002/ardp.200500040
vratizolin) [2, 5] (see Fig. 1), which is the antiviral drug
with anti-inflammatory activity used mainly against
herpes virus infections [1, 8]. It is for external application
only because of its poor bioavailability after gastric
administration [10]. Denotivir also has immunotropic
activity [5].
The main aim of this study was to investigate if repla-
cing the benzoyl group at position 5 by a 4-chlorobenzoyl
group would influence the biological and pharmacologi-
cal activity of 5-benzoylamino-3-methyl-4-isothiazolecar-
boxylic acid 2 derivatives in order to find new biologi-
cally active isothiazole compounds with better pharma-
cological efficacy profiles. The influence of the structure
modifications in the carboxylic group of 5-(4-chloroben-
zoyl)amino-3-methyl-4-isothiazolecarboxylic acid 5 deriv-
atives on their biological activity was studied. The use of
6-(4-chlorophenyl)-3-methylisothiazolo[5,4-d]-4H-1,3-oxa-
zin-4-one 6 [3, 4] in the synthesis of isothiazole derivatives
10–26 and its reactivity with different nucleophilic
reagents are also presented.
Introduction
The different derivatives of 5-amino-3-methyl-4-isothiazo-
lecarboxylic acid 1 (Fig. 1) displayed remarkable anti-
viral, anti-inflammatory and, recently discovered, immu-
notropic action [1, 3–7]. So far, mainly the 5-N-benzoyl
series of this amino acid 1 has been extensively studied
[1, 5, 7, 8]. Of this series of compounds, 5-benzoylamino-
N-(4-ethoxyphenyl)-3-methyl-4-isothiazolecarboxamide
(ITF) 3 exhibited significant anti-inflammatory and broad
antiviral activity both in vitro and in animal models [9].
The same is true for 5-benzoylamine-N-(4-chlorophenyl)-3-
methyl-4-isothiazolecarboxamide 4, i.e. denotivir (ITCL,
Correspondence: Andrzej Regiec, Department of Organic Chemistry,
Faculty of Pharmacy, Medical Academy of Wroclaw, ul. Grodzka 9, 50-
137 Wroclaw, Poland.
E-mail: rykolf@mp.pl
Fax: +48 71 784-0341
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402
A. Regiec et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
Figure 2. Chemical structure of 7, 8, 9.
of intramolecular dehydration of the imide form 5a of 5-
(4-chlorobenzoyl)amino acid 5 is probably similar to that
described for acylantranilic acid cyclodehydration [14,
15] as well as those of proper furan, thiophene, pyrrole,
isoxazole, isoquinoline, and pyridine derivatives [16–21].
There are many papers presenting the synthesis and
reactivity of hetero-1,3-oxazinones [16–21], but not of iso-
thiazolo[5,4-d]-4H-1,3-oxazin-4-ones. The first synthesis of
this type of compound, i.e. 6-phenyl-3-methylisothia-
zolo[5,4-d]-4H-1,3-oxazin-4-one 7, containing the consid-
Figure 1. Derivatives of 5-amino-3-methyl-4-isothiazolecar-
boxylic acid 1.
´
ered ring system, was conducted by Machon and pre-
sented in 1969 [22], but at that time the interpretation of
its structure was incorrect; it was presented as 5-phenyl-
3-methylisothiazolo[5,4-b]azetin-4-one 8 (lactam) [22, 23]
(Fig. 2). In 1976, Rajappa and coworkers reported the
Results and discussion
Chemistry
Compounds 10–26 (Table 1) were prepared using the synthesis of 3-methyl-6-(1-morpholino)-isothiazolo[5,4-d]-
methods outlined in Schemes 1 and 2.
4H-1,3-oxazin-4-one 9, the structure of which was proven
The starting material used in the synthesis was the on the basis of IR spectrum [24]. However, this bicyclic
known amino acid 1 [11], which had given the earlier system has been investigated and documented very
described 5-(4-chlorobenzoyl)amino-3-methyl-4-isothiazo-
poorly so far. Therefore, its chemical properties are
lecarboxylic acid 5 after reaction with 4-chlorobenzoyl described here using 6-(4-chlorophenyl)-3-methylisothia-
chloride [12, 13]. Compound 5 was transformed into a zolo[5,4-d]-4H-1,3-oxazin-4-one 6 as example. Similarly to
semianhydride type compound, namely 6-(4-chlorophe- the earlier described pyrazolo[4,3-c][1,2,5]oxadiazinones
nyl)-3-methylisothiazolo[5,4-d]-4H-1,3-oxazin-4-one 6. The and 6-aminopyrazolo[3,4-d][1,3]thiazinones [25], com-
single strong band of stretching oscillations of the carbo- pound 6 has interesting biological properties as a human
nyl group at 1772 cm– 1 and the lack of other bands leukocyte elastase (HLE) inhibitor [3] (F. Russo, Z. Machon´,
between 1600–2800 cm– 1 and above 3050 cm– 1 in the IR A. Regiec, R. Jasztold-Howorko, S. Guccione, L. Monsu Sco-
spectrum (see section 4 Experimental) support a semian- laro, A. Edison, X. Huang, W. B. Knight – unpublished
hydride structure of compound 6. Oxazinone 6 was cre- data).
ated from 5 as a result of intramolecular dehydration of
The isothiazolooxazinone 6 was used to obtain the dia-
the imide form 5a under the influence of dehydrating mides 10–23 as a result of a proper amine nucleophilic
reagents such as SOCl2, dicyclohexylcarbodiimide (DCC), attack at the carbon atom of the carbonyl group in the
POCl3, and ethyl chloroformate in solvents such as ben- oxazinone ring (Scheme 2). The reactions were con-
zene, toluene, carbon tetrachloride, chloroform, methyl- ducted mainly in ethanol or pyridine solutions. Pyridine
ene chloride, and tetrahydrofuran (THF). The mechanism proved to be a much better solvent for this reaction, par-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
New Isothiazole Derivatives and Their Biological Activity
403
Table 1. Physical constants and analytical data of compounds 10–26.
N8
R
Formula
M.W.
Elemental analysis (calcd/found) (%)
(g/mol)
C
H
N
Cl
9.54
10
11
C18H14N3O2SCl
371.85
58.14
3.79
11.30
C18H13N3O2SCl2
406.30
53.21
52.31
3.23
3.48
10.34
10.13
17.45
12
13
C18H13N3O2SClF
389.84
55.46
55.24
3.36
3.17
10.78
10.52
9.10
9.14
C18H14N3O3SCl
387.85
55.74
54.17
3.64
3.48
10.83
10.18
14
15
16
C19H16N3O2SCl
385.88
59.14
56.96
4.18
4.20
10.89
9.05
9.19
10.00
C24H24N3O2SCl
453.998
63.50
64.08
5.33
5.42
9.26
9.51
7.01
16.87
10.08
C19H15N3O2SCl2
420.32
54.29
54.02
3.60
3.79
10.00
9.78
17
18
C16H18N3O2SCl
351.86
54.62
54.41
5.16
5.21
11.94
12.54
C15H16N3O2SCl
337.83
53.33
53.53
4.77
5.70
12.44
13.28
19
20
21
C18H20N3O2ClS
377.89
57.21
58.23
5.33
5.66
11.12
10.72
C19H22N3O2ClS
391.92
C18H15N4O2SCl
386.86
55.89
55.46
3.91
4.10
14.48
14.03
9.16
22
23
C18H15N4O2SCl
386.86
55.89
56.26
3.91
3.78
14.48
14.56
9.16
9.51
C17H13N4O2SCl
372.84
54.77
55.06
3.51
3.81
15.03
14.82
24
25
26
C18H13N2O3SCl2
407.28
53.08
52.83
2.97
3.12
6.88
6.69
17.41
10.92
10.47
C14H13N2O3SCl
324.79
51.77
52.30
4.03
4.55
8.63
7.94
C15H15N2O3SCl
338.82
53.18
53.15
4.46
4.53
8.27
8.32
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404
A. Regiec et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
Scheme 1. Synthesis of compounds 6, 10, 18, 21, 22 (oxazi-
none 6 and amides 10, 18, 21, 22).
ticularly for the less reactive amines such as 4-chloroani- looxazinone 6 reacted with alcohol, creating a proper
line, 4-bromoaniline, and the aminopyridines. Applica- ester depending on the alcohol used as a solvent. In an
tion of pyridine significantly shortened the reaction anhydrous ethanol solution, the reaction between oxazi-
time and increased the yield. The diamides 11, 21, and 22 none 6 and 5-methyl-2-aminopyridine, 6-methyl-2-amino-
were also obtained from 5-(4-chlorobenzoyl)amino-3- pyridine and 3-aminopyridine did not give the expected
methyl-4-isothiazolecarboxylic acid 5 using DCC in a N-pyridylamides 21, 22, 23, but the known ethyl ester of
dimethylformamide (DMF) or methylene chloride solu- 5-(4-chlorobenzoyl)amino-3-methyl-4-isothiazolecar-
tion at room temperature. N-3-pyridylamide 23 of 5-(4- boxylic acid 25, which had been obtained earlier by a
chlorobenzoyl)amino-3-methyl-4-isothiazolecarboxylic
completely different method, described in [13, 26]. It is
acid was obtained in the reaction of 6-(4-chlorophenyl)-3- known that the ethoxycarbonyl group of ester 25 is very
methylisothiazolo[5,4-d]-4H-1,3-oxazin-4-one 6 with 3- weakly reactive with nuclephilic agents, so for the ami-
aminopyridine in pyridine solution with a fair yield. The nolysis method, this derivative 25 is useless for obtaining
reaction (with 3-aminopyridine) had a much lower yield proper amides 10–23 [11, 13, 26]. In the presence of pyri-
when it was conducted in benzene solution (see dines, the yield of alcoholysis reactions of isothiazolooxa-
Scheme 2 and section 4). The reaction time was also in zinone 6 is quite good and depends on the alcohols used
benzene solvent. In an anhydrous alcoholic solution in and the degree of basicity of the ring nitrogen of the pyr-
the presence of pyridine or aminopyridines, isothiazo- idines present (see section 4). Only primary or secondary
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Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
New Isothiazole Derivatives and Their Biological Activity
405
Scheme 2. Reactions of oxazinone 6 with nucleophiles
(synthesis methods of amides 10–23 and esters 24–26).
alcohols reacted with oxazinone 6 under these condi-
tions. Reaction with isopropyl alcohol gave the proper
isopropyl ester 26 with a yield worse than in case of etha-
nol. Tertiary alcohols did not act on lactone 6 under these
conditions. After a few hours of heating oxazinone 6
with t-butyl alcohol in the presence of pyridine or amino-
pyridines (mentioned above), only substrate 6 was iso-
lated, with no t-butyl ester 27 created. This is probably
the result of steric hindrance of the reagents. The bulky t-
butyl chain is too large and is repulsed by the methyl
group at position 3 and the 4-chlorophenyl substituent
at position 6 of the isothiazoloxazine rings. Oxazinone 6
also easily reacts with phenols in pyridine solution, giv-
ing proper phenyl esters with good yield (in this study
presented for the reaction with 4-chlorophenol, leading
to ester 24). There is a convenient method of obtaining
phenyl 5-(4-chlorobenzoyl)amino-3-methyl-4-isothiazole-
carboxylates which are difficult to obtain in another way
(see section 4). This phenomenon, concerning the influ-
ence of pyridines on oxazinone 6 reactivity described
above, supports the semianhydric structure of the new
oxazine 6 because pyridine bases catalyze the reaction of
anhydrides with nucleophilic reagents such as alcohols
or amines, substantially increasing the reaction speed
and yield (Scheme 3). This is called nucleophilic catalysis
[27–31]. Compound 6 did not react with alcohols under
Scheme 3. Pyridine nucleophilic catalysis of oxazinone 6 with
nucleophiles.
normal acidic esterification conditions. Only the sub-
strate 6 was isolated under these reaction conditions.
Even long heating of oxazinone 6 with ethanol did not
cause any transformation of substrate 6 unless pyridines
were added to the reaction mixture, forming the ethyl
ester 25. Therefore, the isothiazolooxazinone 6 presented
here is, according to Errede's statement concerning the
case of benzo-1,3-oxazin-4-ones (commonly called acylan-
tranils) [15], an example of an interesting semiacid anhy-
,
dride, called here a semianhydride‘, that undergoes
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406
A. Regiec et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
many of the characteristic reactions of true acid anhy- Anti-inflammatory activity
drides, but at a much slower rate. More results of The influence of the proper molecular fragments at posi-
research on the reactivity of the new isothiazolo[5,4-d]- tion 4 on anti-inflammatory activity in the group of 5-(4-
4H-1,3-oxazin-4-ones will be presented in subsequent chlorobenzoylamino)-3-methyl-4-isothiazolecarboxylic
papers.
acid 5 derivatives was shown using compounds 10–15.
The structures of compounds 6-26 have been proven by The activity of compounds 16 and 17 was investigated to
1
IR, H-NMR, MS spectroscopic and elemental analysis check the influence of blocking the free proton in the car-
(Experimental and Table 1).
boxamide fragment at position 4. The effect of the N-aro-
matic and N-heteroaromatic (heterocyclic) substituent of
the carboxamide moiety at position 4 of the isothiazole
Pharmacology
The biological activities of acid 5, oxazinone 6, and deriv- ring was shown with derivatives 14 and 21. The influence
atives 6–21 and 24–25 were compared in in vivo and in of the hydrogenation of the N-phenyl ring of the carboxa-
vitro experiments. Ibuprofen, a non-selective COX inhibi- mide fragment at position 4 on the activity was shown
tor widely used in clinical treatment, served as a refer- considering compounds 19 and 20 as example. The activ-
ence compound in tests in vivo because of its known anti- ity of the new ester derivatives of isothiazole was shown
inflammatory action, its reduction of swelling, and its for 4-chlorophenyl ester 24 and ethyl ester 25 and was
low toxicity similar to that of denotivir [1]. Anti-inflam- compared with the actions of their amide analogues.
matory activity was investigated in a carrageenan-
The acid 5, oxazinone 6, and derivatives 10–21 and
induced rat paw edema model and air-pouch inflamma- 24–25 were tested as potential anti-inflammatory agents
tion test. Erythrocyte membrane stabilization by com- by measuring their activity in the carrageenan-induced
pounds and its correlation with activity in the carragee- rat paw edema model. The results of the carrageenan test
nan test were investigated. Cytotoxicity of the new deriv- (Tables 2 and 3) showed a strong activity for some of the
atives was assessed in vitro on murine bone marrow cells.
tested diamides when areas under the curve of edema
Table 2. Inhibition of carrageenan-induced edema by compounds 5, 6, 10–17, 19–25 and ibuprofen after oral administration.
N8
Dosea) (mg/kg)
Inhibition of carrageenan-induced edema (%)b) after:
Activityc)
ꢀ
ꢁ
0–6 hd)
%
1 h
3 h
6 h
lmole=kg
5
6
10
11
26100
26100
26100
26100
2610
10.5 l 19.4
0.5 l 23.2
27.7 l 12.3
54.0 l 9.5
47.9 l 7.9
15.7 l 10.7
15.7 l 7.9
0.2 l 8.7
13.5 l 8.4
–4.9 l 15.1
7.7 l 15.5
44.9 l 19.8
7.8 l 14.3
10.4 l 10.1
5.3 l 12.4
22.8 l 12.0
3.1 l 14.1
26.6 l 17.7
8.8 l 14.7
11.5 l 18.6
23.0 l 15.1
22.2 l 13.2
7.8 l 15.3
35.1 l 11.0
37.1 l 11.4
6.7 l 6.8
6.7 l 11.2
8.2 l 11.1
14.5 l 13.7
–1.0 l 8.3
33.7 l 8.5
20.1 l 11.6
5.6 l 9.6
2.9 l 13.5
24.1 l 12.0
8.7 l 15.0
7.1 l 9.1
46.2 l 10.8
40.7 l 6.8
10.6 l 17.3
0.2 l 21.3
14.1 l 16.1
16.8 l 13.4
8.7 l 13.6
44.5 l 8.3*
40.7 l 11.1*
15.8 l 12.6
10.1 l 8.2
7.8 l 9.5
17.8 l 12.0
4.9 l 6.0
23.8 l 10.1*
25.2 l 9.9
5.3 l 7.8
4.7 l 10.9
21.9 l 11.0*
10.9 l 12.0
6.9 l 7.3
43.6 l 9.5*
32.9 l 6.9*
12.1 l 16.5
0.021
0.023
0.016
0.090
0.827
3.211
0.020
0.015
0.034
0.011
0.050
0.044
0.009
0.009
0.042
0.022
0.011
0.045
0.339
1.249
16.9 l 12.2
–7.6 l 16.2
53.6 l 10.8
41.1 l 18.7
28.9 l 11.7
13.2 l 10.9
14.0 l 9.6
30.8 l 13.0
24.0 l 8.5
17.5 l 14.9
18.5 l 15.3
2.4 l 12.3
3.1 l 14.4
32.7 l 11.8
3.6 l 23.8
9.8 l 9.2
261
12
13
14
15
16
17
19
20
21
24
25
26100
26100
26100
26100
26100
26100
26100
26100
26100
26100
26100
Ibuprofen 26100
53.8 l 4.9
43.8 l 10.8
14.7 l 15.9
2610
261
a)
Double dose given orally 9 and 2 h before carrageenan injection.
Mean value (n = 5) lSE.
Per cent paw thickness inhibition per dose expressed as micromole of compound per kilogram of body weight.
Calculated from area under curve, (*) Pa 0.05 (ANOVA) when mean increase of paw thickness in compound-treated group was
compared to the control.
b)
c)
d)
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Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
New Isothiazole Derivatives and Their Biological Activity
407
Table 3. Inhibition of carrageenan-induced edema by compound 10, 11, 13, 19, 24, denotivir, and ibuprofen after intra-peritoneal
administration.
N8
Dosea) (mg/kg)
Inhibition of carrageenan-induced edema (%)b) after:
Activityc)
ꢀ
ꢁ
0–6 hd)
%
1 h
3 h
6 h
lmole=kg
10
11
13
16
19
24
20
20
20
20
20
20
32.9 l 12.0
43.6 l 12.8
24.2 l 12.8
27.1 l 8.6
19.1 l 16.3
8.1 l 14.3
0.8 l 32.3
23.4 l 8.0
32.8 l 12.2
39.5 l 10.7
6.9 l 7.4
2.9 l 20.6
7.7 l 23.4
–3.3 l 6.5
26.2 l 19.7
17.7 l 14.6
16.9 l 14.1
38.7 l 11.6
1.8 l 8.6
19.4 l 15.3
8.8 l 16.6
15.3 l 12.9
23.5 l 21.6
44.1 l 12.6
28.5 l 11.5
40.1 l 9.8*
10.1 l 6.6
13.8 l 14.6
11.0 l 17.6
4.5 l 8.8
0.529
0.815
0.188
0.290
0.208
0.092
0.375
0.272
Denotivir 20
Ibuprofen 20
20.0 l 22.0
26.4 l 11.1
a)
Single dose given intra-peritoneally 2 h before carrageenan injection.
Mean value (n = 5) lSE.
Per cent paw thickness inhibition per dose expressed as micromole of compound per kilogram of body weight.
Calculated from area under curve, (*) Pa 0.05 (ANOVA) when mean increase of paw thickness in compound treated group was
compared to the control.
b)
c)
d)
inhibition during 0–6 h were compared. Derivatives 11, decreases the anti-inflammatory activity (compound 16).
16, and 21 showed statistically significant anti-edema- Also, replacing both amide protons in the carboxamide
tous activity at a dose of 26100 mg/kg of body weight fragment at position 4 with two aliphatic-ethyl substitu-
p.o. The 4-chloro-substituted 11 derivative proved to be ents gives derivative 17, an anti-carrageenan activity
the most potent, being even 2–3 times stronger than ibu- which is weaker than that of compound 11, but it does
profen (depending on the dose and route of administra- not vanish as in the N-benzoyl series [33]. Hydrogenation
tion). It should be taken into consideration that the mole- of the N-aromatic ring of the amide group or its exchange
cular masses of the tested compounds are about twice as for N-monoaliphatic (N-propyl) chain lowers the anti-car-
large as that of ibuprofen: results are expressed as per- rageenan activity or has it almost disappear (compounds
cent paw thickness inhibition per dose (represented here 18–20). Earlier research [33] concerning the structural-
as micromole of drug per kilogram of body weight) [32].
biological effect of the N-benzoyl series indicated the fol-
This indicates that optimal lipophilicity of the tested lowing relationships: The activity of the N-unsubstituted
molecules is required to show strong anti-inflammatory amide and N-alkyl amides diminished. Substitution of
activity in the carrageenan test. Increasing lipophilicity both amide hydrogen atoms by aliphatic radicals caused
above the optimum decreases this activity (compound complete disappearance of the compounds' activity.
15). Decreasing the electron density of the carboxamide Introduction of a phenyl ring in the place of one of the
fragment at position 4 through the phenyl ring increases hydrogen atom of the amide group at position 4 mark-
this type of activity. The phenyl ring, due to its p-deloca- edly increased the activity. The presence of substituents
lized bonding molecular orbital, fulfills the role of a con- such as an electronegative atom (–Cl) or a fragment
ductive wire through which the electrons can freely flow. (–OEt) connected through the electronegative atom with
The electron properties of the substituents in the phenyl the phenyl ring ameliorated the activity even more.
ring are thus a factor that has a very significant influence Exchange of the N-phenyl group in the amide moiety at
on the activity of the tested derivatives. The carboxamide position 4 by a cyclohexyl or tetrahydrofuryl group mark-
moiety at position 4 of the isothiazole ring is also a neces- edly diminished the activity of this type of N-substituted
sary fragment for the compounds of this series to reveal amide [33].
anti-inflammatory activity. Replacement of this carboxa-
Proper volume of the fragment at position 4 of the iso-
mide group by an ester group (4-chlorophenyl ester 24 thiazole ring seems to be very important as well; a too
and ethyl one 25) or its removal (compounds 5, 6) signifi- voluminous substituent, as in the case of the 4-cyclohex-
cantly diminishes or abolishes this type of activity. The ylphenyl, is unfavorable for the considered activity (com-
amide proton in the carboxamide fragment must be at pound 15 has no anti-inflammatory activity). Lipophilic
position 4 of the isothiazole ring for this activity to be and electron-withdrawing substituents (like Cl) in the N-
strong. Exchange of this proton for a methyl group benzoyl fragment of the 5th isothiazole position magnify
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408
A. Regiec et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
basis of three different doses of the carrageenan-induced
edema inhibition results (ED50: 274 mg/kg). The summary
effective dose ED50 for ibuprofen is higher, 349 mg/kg.
The high anti-inflammatory activity of the compound
11 was confirmed in the air-pouch inflammation test
(Fig. 3). At dose of 36100 mg/kg body weight, the migra-
tion of inflammatory cells (mainly neutrophils) into the
site of inflammation is significantly inhibited, to a
degree comparable to that of ibuprofen used at the same
dose. The 4-chlorophenyl ester 24 insignificantly
decreased cytosis in the air-pouch fluid up to 74% of the
control. The other checked compounds, 10 and 17, were
not active in this test.
Membrane stabilization
Figure 3. Inhibition of carrageenan-induced air-pouch inflam-
mation by compounds 10, 11, 17, 24 and ibuprofen. (*) Statisti-
cally significant difference when mean cytosis in compound-trea-
ted group was compared to the control group (Pa 0.05; U
Mann—Whitney test).
Stabilization of the erythrocyte membrane is characteris-
tic of some nonsteroidal anti-inflammatory agents [34].
The N-aromatic amides of the 5-benzoyl derivative of
amino acid 1 predominately displayed this type of activ-
ity in previous tests [35]. Here, in the tested 5-(4-chloro-
benzoyl) derivatives, derivative 21 had the highest activ-
ity (EC50: 2.9 l 0.6 lM); i.e. the lowest EC50, the concentra-
tion at which relative hemolysis equals half of the con-
trol value (Table 4). Compound 14 was a little less active
(EC50: 10.0 l 3.2 lM). Compound 10, an unsubstituted
one, was characterized by a medium membrane-stabiliz-
ing effect (EC50: 24.0 l 10.9 lM). The other investigated
derivatives were much less active. There was no signifi-
cant correlation between anti-inflammatory activity
(% mmol/kg b.w.) of the tested compounds in the car-
rageenan-induced edema test after oral administration
and erythrocyte membrane stabilization (r = –0.085; P =
0.80).
the activity very much, by about a factor of two (compare
the activities of derivative 11 and denotivir 4 at a dose of
20 mg/kg i.p.; see Table 3). As mentioned in Introduction,
denotivir was not given per os because, according to ear-
lier tests, this compound was inactive after per os intro-
duction. It was probably not easily absorbed or it lost its
biological potency while in the stomach [10]. This sug-
gests that the 5-(4-chloro)benzoyl series of compounds of
this type should be better anti-inflammatory agents than
the non-substituted 5-benzoyl ones.
It was possible to calculate a value for the hypothetical
summary effective dose ED50 for compound 11 on the
Table 4. Erythrocyte membrane stabilization by compounds 10–17, 21, 24, and 25.
b)
N8
Relative hemolysisa) (%)
EC50
(lM)
0.001 lg/mL
0.01 lg/mL
0.1 lg/mL
1.0 lg/mL
10 lg/mL
10
11
12
13
14
15
16
17
21
24
25
1.00 l 0.06
0.97 l 0.04
0.95 l 0.03
1.04 l 0.03
0.93 l 0.15
1.05 l 0.01
1.03 l 0.02
1.12 l 0.04
1.01 l 0.03
1.04 l 0.03
1.09 l 0.03
1.00 l 0.04
0.90 l 0.06
0.94 l 0.05
0.95 l 0.03
0.89 l 0.11
1.05 l 0.02
0.98 l 0.02
1.07 l 0.05
0.95 l 0.02
1.00 l 0.03
1.03 l 0.01
0.81 l 0.11
0.87 l 0.03
0.93 l 0.07
1.00 l 0.06
0.91 l 0.14
1.02 l 0.03
0.97 l 0.01
1.04 l 0.04
0.81 l 0.13
0.98 l 0.02
0.97 l 0.02
0.78 l 0.08
0.81 l 0.05
0.87 l 0.06
0.86 l 0.05
0.80 l 0.16
0.97 l 0.01
0.95 l 0.02
1.10 l 0.07
0.57 l 0.07
0.82 l 0.04
0.90 l 0.05
0.47 l 0.17
0.57 l 0.05
0.81 l 0.05
0.86 l 0.08
0.29 l 0.09
0.77 l 0.03
0.72 l 0.03
0.91 l 0.02
0.13 l 0.05
0.70 l 0.10
0.65 l 0.09
24.0 l 10.9
76.3 l 41.6
A 1000
A 1000
10.0 l 3.2
70.3 l 60.9
84.7 l 28.5
A 1000
2.9 l 0.6
141 l 115
70.2 l 35.9
a)
Mean value; lSE.
b)
Concentration where the value of the relative hemolysis equals 0.5 € SE.
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Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
New Isothiazole Derivatives and Their Biological Activity
409
Table 5. Cytotoxic activity of compounds 10–17, 21, 24, and 25
tested with murine bone marrow cells.
better than cyclosporin A, and due to this activity profile,
compound 11 is subject of a patent notification [4] and is
now undergoing patent registration procedures.
a)
N8
Cytotoxicity (%)
0.1 lg/mL 1 lg/mL 10 lg/mL
CC50
(lM)
We would like to thank our technicians, Ms. Barbara Bubak,
MS, and Ms. Wieslawa Kilian for skilful assistance.
10
11
12
13
14
15
16
17
21
24
25
0.0
1.9
9.8
2.3
10.6
0.0
0.7
0.0
0.0
0.0
0.0
4.2
19.5
40.8
4.8
43.2
8.8
2.0
0.0
0.0
0.0
37.0
84.2
67.3
16.2
98.6
66.0
16.8
6.5
43.2 l 0.6
7.1 l 0.2
6.3 l 1.3
545 l 167
3.0 l 0.5
13.3 l 0.2
122 l 14
n.e.b)
Experimental
Chemistry
The melting points of all the new compounds were measured on
a Boetius hotstage (VEB Kombinat Negema Wꢀgetechnik Rapido,
Radebeul, Germany) or Bꢁchi apparatus (Bꢁchi Labortechnik,
Flawil, Switzerland) and were uncorrected. Elemental analysis:
Microlaboratory of the Department of Pharmacy, Medical Uni-
versity, Wroclaw. IR spectra: Pye-Unicam SP-1000 apparatus (Pye
49.1
1.2
15.5
n.e.b)
n.e.b)
19.6
n.e.b)
a)
50% cytotoxic concentration lSE.
Value very high or difficult to estimate.
b)
1
Unicam Ltd. Cambridge, England), nujol film. H-NMR spectra:
80 MHz Tesla BS 587 A (Tesla, Brno, Czech Republic) and 300.14
MHz Bruker apparatus (Bruker, Rheinstetten, Germany), TMS as
internal reference. MS: mass spectrometer AMD-604 (Perkin
Elmer). TLC made with “Silufol” and Polygram SIL G/U254 plates.
Silica gel 60 F254 Aldrich (Sigma-Aldrich) for column chromato-
graphy purification of compounds, eluting agent was chloro-
form/acetone (9:1), detection of the compounds on the chroma-
tograms was done with UV light and/or by treatment with iodine
vapors.
Cytotoxic activity
The compounds 10–21, 24, and 25 were investigated in
the cytotoxic test in vitro. The amides 11, 12, 14, and 15
with the phenyl ring, p-substituted by lipophilic substitu-
ents and with an N–H bond in the carboxamide group at
position 4 of the isothiazole ring, had the highest cyto-
toxic activity against murine bone marrow cells, for
which the CC50 (50% cytotoxic concentration) values were
7.1 l 0.2, 6.3 l 1.3, 3.0 l 0.5, and 13.3 l 0.2 lM, respectively
(Table 5). The non-substituted derivative 10 showed med-
ium cytotoxic activity (CC50: 43.3 l 0.6 lM). Compound 21
was cytotoxic only at a concentration of 10 lg/mL. The
compounds 13, 16, and 25 had very weak cytotoxicity at
the tested concentrations of 0.1 to 10 lg/mL. The other
tested compounds 17 and 24 did not show significant
cytotoxic activity in the concentrations 0.1 to 10 lg/mL.
Synthesis of compounds
5-(4-Chlorobenzoyl)amino-3-methyl-4-isothiazole-
carboxylic acid 5 [12, 13]
Modification of the procedure described in [13]: 15.0 g
(0.095 mol) of aminoacid 1 was suspended in 60 mL of anhy-
drous acetone. The suspension was heated to the boiling point
and then 7.9 mL (0.114 mol) of pyridine was added. The mixture
was stirred and 15 mL (0.114 mol) of 4-chlorobenzoyl chloride in
20 mL of anhydrous acetone was added drop-wise for one hour.
The reflux was conducted for 10 h. After cooling of the precipi-
tate, it was collected by filtration, washed with acetone, put into
a beaker with 10% H2SO4, and then stirred for 1 h. The crude
product was collected by filtration and dried. 26.5 g (94%) of
crude product 5 was obtained. A sample for elemental analysis
was crystallized from ethanol, m.p. = 252–2538C (m.p. reported
in [13] = 250–2518C). IR (nujol): m [cm– 1] = 3192 (NH), 2668–2324
(COO–H), 1672 (C=O) (reported in [12] m [cm– 1] = 3400 (NH), 1720,
1670 (C=O)); 1H-NMR (Tesla, 80 MHz, DMSO-d6, TMS ) d [ppm] =
2.54 (s, 3H, CH3), 7.77 (d, J = 7.8 Hz, 2H, p-phenylen), 7.94 (d, J = 7.8
Hz, 2H, p-phenylen), 12.01 (s, 1H, NH).
Conclusion
In this tested series of new 5-(4-chlorobenzoyl)-deriv-
atives, the N-aromatic and N-heteroaromatic amides 11,
16, and 21, with electron-withdrawing, lipophilic, and
small substituents (such as chlorine) of the N-phenyl and
N-pyridyl groups in the carboxamide fragments at posi-
tion 4 of the isothiazole ring, displayed particularly sig-
nificant anti-inflammatory activity, stronger or compar-
able to that of ibuprofen. Free acid 5, methylisothia-
zolo[5,4-d]-4H-1,3-oxazin-4-one 5, and esters 24, 25 have
very weak or no anti-inflammatory activity. Among these
derivatives presented above, compound 11 exhibited
high anti-inflammatory potency, comparable to that of
ibuprofen. Our latest research showed that it also had
very interesting immunosuppressive properties, even
6-(4-Chlorophenyl)-3-methylisothiazolo[5,4-d]-4H-1,3-
oxazin-4-one 6
Procedure A: 20 g (0.067 mol) of 5-(4-chlorobenzoyl)amino-3-
methyl-4-isothiazolecarboxylic acid 5, 300 mL of anhydrous ben-
zene or toluene, and 66 mL (0.92 mol) of thionyl chloride were
refluxed for about 10 h. Then, the liquids were evaporated to
dryness in vacuo. The solid residue was dissolved in 200 mL of
dry benzene and the solution was cooled with ice to 0–58C and
saturated with gaseous ammonia. A precipitation was filtered
and the solution condensed to dryness. 16.83 g (90%) of crude
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410
A. Regiec et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
product 6 was obtained; Crystallization from ethanol or acet-
one.
washed with methanol, and dried. The crude product of 5.75 g
(99%) was crystallized from a mixture of chloroform-methanol
(1 : 2) to give 4.34 g (74%) of pure compound 11, m.p. = 190–
1928C. IR (nujol): = 3300 (m-NH), 1664 (m-CO-) cm– 1. 1H-NMR (DMSO-
d6, Tesla, 80 MHz) d: 2.52 (s, 3H, –CH3 at isothiazole position 3),
7.48–8.40 (2d, 8H, two phenyl rings H), 10.46 (s, 1H, –NH),
Procedure B: 0.50 g (1.685 mmol) of 5-(4-chlorobenzoyl)amino-
3-methyl-4-isothiazolecarboxylic acid 5, 20 mL of anhydrous
DMF, and 0.38 g (1.84 mmol) of DCC were stirred at room tem-
perature for 24 h. The precipitated DCU was filtered and then
the solution was evaporated to dryness. The crude residue
(0.58 g) was treated with CHCl3, filtered, and dried. 0.3 g of prod-
uct 6 was obtained (60%) and crystallized from ethanol.
1
11.925 (s, 1H, –NH) ppm. H-NMR (CDCl3, Bruker, 300 MHz) d:
2.86 (s, 3H, –CH3 at isothiazole position), 7.40 (d, J = 8.9 Hz, 2H,
2,6-H of p-phenylen at isothiazole position 4), 7.53 (d, J = 8.9 Hz,
2H, 3,5-H of p-chlorobenzoyl group at isothiazole position 5),
7.57 (d, J = 8.9 Hz, 2H, 3,5-H of p-phenylen at isothiazole position
4), 7.98 (d, J = 8.9 Hz, 2H, 2,6-H of p-chlorobenzoyl group at iso-
thiazole position 5), 7.84 (s, 1H, -NH, carboxamide group at iso-
thiazole position 4), 12.73 (s, 1H, –NH, amide group at isothia-
zole position 5, strong intramolecular hydrogen bond) ppm. MS
(electrospray ionization, ESI) m/e [Th] [%] = 410.1 (14.29)
[M+4+H]+ – isotope peak, 408.2 (73.8) [M+2+H]+ -isotope peak,
406.1 (100) [M+H]+ – Quasi-molecular ion and base peak.
Procedure C: 20.0 g (0.067 mol) of 5-(4-chlorobenzoyl)amino-3-
methyl-4-isothiazolecarboxylic acid 5, 120 mL of methylene
chloride and 10 mL of pyridine were mixed in a bulb with a mag-
netic stirrer. After the mixture was heated to boiling, ethyl
chloroformate (0.201 mol) in methylene chloride was added
drop-wise. Heating was conducted for 10 h and then the solution
was evaporated to dryness in vacuo. 500 mL of distilled water was
added to the dry residue and stirred for 1 h. The crude substance
was filtered off and the wet residue was taken up in 500 mL of a
5% aqueous solution of NaOH and stirred for 15 min, then the
whole was filtered off and dried. 18.25 g of crude product (which
can be used directly for the following synthesis) was obtained
(98%) and crystallized from ethanol, m.p. = 163–1658C. Anal
calcd. for C12H7ClN2O2S (278.72 g/mol) [%]: C, 51.71; H, 2.53; N,
10.05; Cl, 12.72. Found: C, 51.80; H, 2.17; N, 10.23; Cl: 2.39. IR
(nujol): m = 1776 (m-CO-, a, b, c, d-unsaturated lactone carbonyl),
1584 (mC=C, aromatic ring), 1552 (mC=N, conjugated imine bond),
5-(4-Chlorobenzoyl)amino-N-phenyl-3-methyl-4-
isothiazolecarboxamide 10 [12]
Method C: Yield = 92%. The synthesis was conducted according
to the method described in [12]. Yield = 86% m.p.= 221–2258C,
m.p. reported in [12] = 218–2208C. The spectral properties of 10
were not described before. IR (nujol): = 3325 (m-NH), 1678 (m-CO-
)
1
1512 (mC=C, aromatic ring), 1254 (mC-O-C, as) cm– 1. H-NMR (DMSO-
cm–1.
d6, Tesla, 80 MHz): 2.68 (s, 3H), 7.96 (d, J = 8.0 Hz, 2H, 3,5-H p-phe-
nylene), 8.33 (d, J = 8.0 Hz, 2H, 2,6 –H p-phenylene) ppm. 1H-NMR
(CDCl3, Bruker, 300 MHz) : 2.93 (s, 3H, –Me), 8.01 (d, J = 8.8 Hz, 2H,
3,5 -H of p-phenylen), 8.24 (d, J = 8.8 Hz, 2H, 2,6-H p-phenylen)
ppm. MS (electrospray ionization, ESI) m/e [Th] [%] = 279.2 (100)
[M+H]+ – Quasi-molecular ion and base peak.
5-(4-Chlorobenzoyl)amino-N-(4-fluorophenyl)-3-methyl-
4-isothiazolecarboxamide 12
Method A: The crude solid was washed with methanol to give 12;
yield: 58%, m.p. = 1938C. IR (nujol): = 3324 (m-NH), 1664 (m-CO-) cm– 1
.
General methods for the synthesis of
the amides 10–23
5-(4-Chlorobenzoyl)amino-N-(4-hydroxyphenyl)-3-
5-(4-Chlorobenzoyl)amino-N-(4-chlorophenyl)-3-methyl-
methyl-4-isothiazolecarboxamide 13
Method A: Yield: 69%, m. p. = 297–2998C. IR (nujol): = 3460 (m-OH),
4-isothiazolecarboxamide 11
Procedure A: 1.0 g (3.59 mmol) of 6-(4-chlorophenyl)-3-methyli-
3272 (m-NH), 1664 (m-CO-), 1634 (m-CO-) cm– 1
.
sothiazolo[5,4-d]-4H-1,3-oxazin-4-one
6
was refluxed with
872.4 mg (7.18 mmol) of 4-chloroaniline in 36 mL of anhydrous
ethanol for 10 h. Then the mixture was condensed to half the
volume and set to precipitate. The crude solid was collected and
washed with ethanol to give 957 mg of 11 (66%); it was crystal-
lized from a mixture of chloroform-methanol (1:1). Yield after
purification: 44%.
5-(4-Chlorobenzoyl)amino-N-(4-methylphenyl)-3-methyl-
4-isothiazolecarboxamide 14
Method A: The crude solid 14 (84%) was crystallized from a mix-
ture of ethanol-chloroform (1:2), m.p. = 200–2048C; IR (nujol): =
1
3272 (m-NH), 1664, 1632 (m-CO-) cm-1. H-NMR (DMSO-d6, Tesla, 80
Procedure B: 2.0 g (6.74 mmol) of 5-(4-chlorobenzoyl)amino-3-
methyl-4-isothiazolecarboxylic acid 5 was dissolved in DMF at
70–808C. Then 946 mg (7.41 mmol) of 4-chloroaniline was
added and a solution of DCC (7.41 mmol) in DMF was added
drop-wise. The reaction was carried out at 708C for 3 h and then
left for 24 h at room temperature. The precipitated DCU was fil-
tered out and the filtrate was condensed to dryness in vacuo.
2.98 g of crude product 11 was obtained which was crystallized
from a mixture of chloroform-methanol (1:1) to give 1.84 g of
pure compound 11 (62%).
Procedure C: 4.0 g (14.33 mmol) of 6-(4-chlorophenyl)-3-methy-
lisothiazolo[5,4-d]-4H-1,3-oxazin-4-one 6 was refluxed with 3.66 g
(28.66 mmol) of 4-chloroaniline in 18 mL of anhydrous pyridine
for 10 h. The mixture was evaporated to dryness and the residue
was treated with a small amount of methanol, filtered off,
MHz) d: 2.30 (s, 3H, –CH3), 2.54 (s, 3H, –CH3 at isothiazole position
3), 7.13–8.25 (4d, 8H, two phenyl rings H), 10.35 (s, 1H, –NH),
11.94 (s, 1H, –NH) ppm.
5-(4-Chlorobenzoyl)amino-N-(4-cyclohexylphenyl)-3-
methyl-4-isothiazolecarboxamide 15
Method A: The crude solid was washed with methanol to give 15;
yield: 80%. The product 15 was purified by chromatography
with silica gel (eluent: chloroform), m.p. = 230–2318C. IR (nujol):
1
= 3324 (m-NH), 1664 (m-CO-), 1632 (m-CO-) cm– 1; H-NMR (CDCl3, Tesla,
80 MHz) d: 1.12–2.38 (m, 11H, cyclohexyl), 2.82 (s, 3H, –CH3),
7.21–8.27 (4d, 8H, two phenyl rings H), 10.31 (s, 1H, –NH),
12.79(s, 1H, –NH, strong intramolecular hydrogen bond) ppm.
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Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
New Isothiazole Derivatives and Their Biological Activity
411
5-(4-Chlorobenzoyl)amino-N-(4-chlorophenyl)-N-methyl-
5-(4-Chlorobenzoyl)amino-N-(5-methyl-2-pyridyl)-3-
3-methyl-4-isothiazolecarboxamide 16
methyl-4-isothiazolocarboxamide 21
Method B: A crude product was crystallized from chloroform to
give the pure 21 (Yield: 20%).
Method C: Crystallization was from a mixture of chloroform-
methanol (1:1), yield: 66%, m.p. = 223–2258C. 1H-NMR (CDCl3,
Bruker, 300 MHz) d: 1.92 (s, 3H, –CH3 ), 3.51 (s, 3H, N-CH3), 6.94 (d,
J = 8.5 Hz, 2H, 2,6-H of p-phenylen at isothiazole position 4), 7.21
(d, J = 8.5 Hz, 2H, 3,5-H of p-phenylen of benzoyl group at isothia-
zole position 4), 7.51 (d, J = 8.5 Hz, 2H, 3,5-H of p-phenylen at iso-
thiazole position 5), 7.95 (d, J = 8.5 Hz, 2H, 2,6-H of p-phenylen at
isothiazole position 5), 11.08 (s, 1H, –NH, amide group at isothia-
zole position 5, strong intramolecular hydrogen bond) ppm.
Method C: A precipitated product was filtered and washed
with methanol and ether to give compound 21 with 41% yield,
m.p. = 196–1988C. IR (nujol): = 3289 (m-NH), 1694, 1660 (m-CO-) cm– 1
.
1H-NMR (CDCl3, Tesla, 80 MHz) d: 2.24 (s, 3H, –CH3), 2.73 (s, 3H, –
CH3 at isothiazole position 3), 6.60–6.70 (2 6 m, 2H), 7.48 (d, J1 =
8.3 Hz, 2H, 3,5-H phenyl), 7.54 (s, 1H), 7.97 (d, J1 = 8.3 Hz, 2H, 2,6-
H phenyl), 8.45 (s, 1H, NH), 12.75 (s, 1H, NH, strong intramolecu-
lar hydrogen bond) ppm. MS (electrospray ionization, ESI) m/e
[Th] [%] = 389.2 (7) [M+2]+-isotope peak, 387.2 (21) [M]+ - molecular
ion, 108.9 (100) [M-279+H]+ - base peak.
5-(4-Chlorobenzoyl)amino-N,N-diethyl-3-methyl-4-
isothiazolecarboxamide 17
Method A: Yield of crude compound 17 was 86%. Crystallization
was from tetrachloromethane (CCl4), m.p. = 179–1818C. IR
5-(4-Chlorobenzoyl)amino-N-(6-methyl-2-pyridyl)-3-
methyl-4-isothiazolocarboxamide 22
1
(nujol): = 3204 (m-NH), 1666 (m-CO-) cm– 1. H-NMR (CDCl3, Tesla, 80
Method B: A crude residue (yield: 34%) was crystallized from a
mixture methanol-chloroform (1:1) to give the pure compound
22 with a yield of 14%, m.p. = 219–2228C. IR (nujol): = 3300 (m-NH),
MHz) d: 1.20 (t, 6H, 2 6 CH3), 2.04 (s, 3H, –CH3 at isothiazole posi-
tion 3), 3.46 (q, 4H, 2 6 –CH2-), 7.45 (d, J1 = 8.8 Hz, 2H, 3,5-H phe-
nyl), 7.86 (d, J1 = 8.8 Hz, 2H, 2,6-H phenyl), 8.84 (s, 1H, –NH) ppm.
1
1686, 1654 (m-CO-) cm–1. H-NMR (CDCl3, Tesla, 80 MHz) d: 2.75 (s,
3H, –CH3 at isothiazole position 3), 2.92 (s, 3H, –CH3), 6.50–8.25
(m, 7H, aromatic range), 8.41 (s H, NH), 12.63 (s, H, NH, strong
intramolecular hydrogen bond) ppm.
5-(4-chlorobenzoyl)amino-3-methyl-N-propyl-4-
isothiazolocarboxamide 18
Method A: The crude solid (92%) was crystallized from methanol
to give the pure compound 18 with 62% yield. Method B: 47%
yield after crystallization from methanol, m.p. = 144–1478C. IR
5-(4-Chlorobenzoyl)amino-N-(3-pyridyl)-3-methyl-4-
isothiazolecarboxamide 23
(nujol): (cm– 1) = 3312 (m-NH), 1676 (m-CO-) cm– 1 1H-NMR (CDCl3,
.
1.0 g (3.59 mmol) of 6-(4-chlorophenyl)-3-methylisothiazolo[5,4-
d]-4H-1,3-oxazin-4-one 6 was refluxed with 675 mg (7.18 mmol)
of 3-aminopyridine in 4.5 mL of anhydrous benzene for 20 h.
The precipitated crude product 23 was filtered and washed with
methanol and then cleaned by chromatography with silica gel.
Chloroform was used as the eluent. 391 mg of pure compound
was obtained (30%).
Tesla, 80 MHz) d: 1.03 (t, J = 7.3 Hz, 3H, CH3), 1.67 (m, 2H, –CH2–),
2.70 (s, 3H, –CH3 at isothiazole position 3), 3.48 (t, J1 = 5.9 Hz, 2H,
–CH2–), 6.20 (s, 1H, –NH), 7.50 (d, J1 = 8.8 Hz, 2H, 3,5-H phenyl),
7.98 (d, J1 = 8.8 Hz, 2H, 2,6-H phenyl) ppm.
5-(4-chlorobenzoyl)amino-3-methyl-N-cyclohexyl-4-
isothiazolocarboxamide 19 [12]
Method A: The product was purified by column chromatography
with silica gel (eluent: chloroform). Yield: 56%, m.p. = 238–
2398C; m.p. = 235–2378C (reported in [12]). IR (nujol): (cm– 1) =
3292 (m-NH), 1648, 1626 (m-CO-) (Spectral properties of 19 were not
described before).
Compound 23 was also obtained in accordance with the
method C described for compound 11 using 3-aminopyridine
instead of 4-chloroaniline. The precipitated product was filtered
and washed with methanol and ether. Yield: 49%, m.p. = 202–
2048C. IR (nujol): = 3392–3112 (m-NH), 1656 (m-CO-), 1630 (m-C=N-),
1
1594, 1534 (m-C=C-, aromatic) cm– 1. H-NMR (DMSO-d6, Tesla, 80
MHz) d: 2.55 (s, 3H, –CH3 at isothiazole position 3), 7.23–8.35 (m,
8H, aromatic range), 8.94 (s, 1H, –NH), 11.33 (s, 1H, –NH, strong
intramolecular hydrogen bond) ppm.
5-(4-chlorobenzoyl)amino-3-methyl-N-(4-
methyl)cyclohexyl-4-isothiazolocarboxamide 20
(as equimolar mixture of isomer cis and trans)
4-Chlorophenyl-5-(4-chlorobenzoyl)amino-3-methyl-4-
isothiazolecarboxylate 24
Synthesis was conducted according to method A described for
compound 11 using the equimolar mixture of cis,trans-(4-
methyl)cyclohexylamine instead of 4-chloroaniline. The crude
product (80%) was purified by column chromatography with
silica gel (eluent: chloroform), m.p. = 166–1778C (dec.). 1H-NMR
(CDCl3, Bruker, 300 MHz) d: 0.77–2.38 (m; 12H, protons at posi-
tion 2,3,4,5,6 and methyl group of 4-methylcyclohexyl ring of
both cis and trans isomers), 3.86, 4.22 (m, 2 6 0.5H, N-CHa, proton
at position 1 of cyclohexyl ring of both cis and trans isomers),
2.60, 2.66 (26s; 261.5 H, –CH3 at isothiazole position 3 of both
cis and trans isomers), 7.42 (d, 2H, 3,5-H of 4-chlorobenzoyl group
at isothiazole position 5), 7.90 (d, 2H, 2,6-H of 4-chlorobenzoyl
group at isothiazole position 5), 6.10, 6.35 (26s,1H, –NH, carbox-
amide group at isothiazole position 4 of both isomers), 11.15 (s,
1H, –NH, amide group at isothiazole position 5, strong intramo-
lecular hydrogen bond) ppm.
2.0 g (7.18 mmol) of 6-(4-chlorophenyl)-3-methylisothiazolo[5,4-
d]-4H-1,3-oxazin-4-ones
6
was refluxed with 920 mg
(10.77 mmol) of 4-chlorophenole in 20 mL of anhydrous pyri-
dine for 10 h. Then the mixture was condensed to dryness in
vacuo. The crude solid was stirred with a 5% aqueous solution of
NaOH for 1 h, then the product has been filtered off, washed
with a small amount of methanol, and dried, giving 1.82 g (62%)
of compound 24. Crystallization from pyridine, m.p. = 1808C.
1H-NMR (CDCl3, Bruker, 300 MHz) d: 2.76 (s, 3H, –CH3), 7.18 (d, J
= 8.5 Hz, 2H, 2,6-H of p-phenylen isothiazole position 4), 7.44 (d, J
= 8.5 Hz, 2H, 3,5-H p-phenylen of 4-chlorobenzoyl group at iso-
thiazole position 5), 7.48 (d, J = 8.5 Hz, 2H, 3,5-H of p-phenylen at
isothiazole position 4), 7.91 (d, J = 8.5 Hz, 2H, 2,6-H of p-phenylen
at isothiazole position 5), 11.82 (s, 1H, –NH, amide group at iso-
thiazole position 5, strong intramolecular hydrogen bond) ppm.
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412
A. Regiec et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
pounds 9 h and 2 h before carrageenan injection or one dose i.p.
2 h before. The control group (five rats) received a suspension of
methylcellulose. Paw thickness was measured using calipers
(Schnelltaster, Kroepelin) before (initial paw thickness) and 1, 3,
and 6 h after carrageenan injection. Intensity of edema for each
animal was expressed as the mean increase in the left and right
paw thickness at each time-point compared with the initial paw
thickness.
Mean results of the relative increase in paw thickness for each
group (compared with the initial paw thickness) at each time-
point were compared with the results of the control group. Anti-
inflammatory/anti-edematous effect of the investigated com-
pounds was expressed as the percentage of inhibition of carra-
geenan-induced edema (H) calculated by the formula: H = (DC–
DT)/DC6100%, where DC is the relative increase in paw thick-
ness in the control group and DT the relative increase in paw
thickness in the compound-treated group.
Ethyl-5-(4-chlorobenzoyl)amino-3-methyl-4-
isothiazolecarboxylate 25 [13, 26]
Procedure A: 1.0 g (3.59 mmol) of 6-(4-chlorophenyl)-3-methyli-
sothiazolo[5,4-d]-4H-1,3-oxazin-4-one 6 was refluxed with 465 mg
(4.30 mmol) of 2-amino-5-methylpyridine in 36 mL of anhydrous
ethanol for 5 h. Then the precipitated product was filtered.
1.04 g of compound 25 was obtained. Yield: 75%. Its physico-
chemical parameters are the same as the compound described
in [13, 26] obtained by a different method of synthesis.
Procedure B: As above, using 2-amino-6-methylpyridine
instead of 2-amino-5-methylpyridine. Yield of the reaction 79%.
Procedure C: As above, using 3-aminopyridine instead of 2-
amino-5-methylpyridine. Yield of the reaction 60%, m.p. = 147–
1488C (m.p. reported in [26] = 149–1518C, in [13] = 148–1498C).
IR (nujol): = 3260 (m-NH), 1678, 1664 (m-CO-), 1260, 1224 (mC-O ester)
cm– 1 (reported in [26] = 1695 (m-CO), 1250 (mC-O ester) cm– 1). 1H-NMR
(CDCl3, Tesla, 80 MHz) d: 1.46 (t, J = 7.1 Hz, 3H, CH3), 2.64 (s, 3H,
–CH3 at isothiazole position 3), 4.45 (q, J = 7.2 Hz, 2H, –CH2–), 7.52
(d, J1 = 8.8 Hz, 2H, 3,5-H phenyl), 7.96 (d, J1 = 8.8 Hz, 2H, 2,6-H phe-
nyl), 12.05 (s, 1H, –NH, strong intramolecular hydrogen bond)
ppm (shifts reported in [26] are in t, here converted to d [ppm]:
1.4 (t, J = 7.5, 3H, CH3), 2.6 (s, 3H, –CH3 at isothiazole position 3),
4.4 (q, J = 7.1 Hz, 2H, –CH2–).
Air-pouch inflammation test
Air-pouches were induced by hypodermic injection of sterile air
(5 mL, needle 23G611/4, 0.44 lm-Millipore filter) to female
CB6F1 mice under halothane anesthesia in the dorsal region
[38]. Five days later the pouches were supplemented with 3 mL
of air. Inflammation was induced on the sixth day by injection
of a 1% carrageenan solution in saline (0.5 mL) to each pouch.
The tested compounds were administered p.o. in a dose of
100 mg per body weight 24, 12, and 1 h before carrageenan
injection. The control group of mice was treated with 3% Tween
80 solution. Four hours after carrageenan injection the mice
were sacrificed and the air-pouches were washed with 2 mL of
saline. The mean cytosis of the aspirated fluids was counted (8–
10 animals in a group). Compound activity was expressed as the
percentage of the relative mean cytosis of the compound-treated
group to the control group.
Isopropyl-5-(4-chlorobenzoyl)amino-3-methyl-4-
isothiazolecarboxylate 26
500 mg (1.79 mmol) of 6-(4-chlorophenyl)-3-methylisothia-
zolo[5,4-d]-4H-1,3-oxazin-4-one
6 was refluxed with 388 mg
(3.59 mmol) of 2-amino-5-methylpyridine in 36 mL of anhydrous
isopropyl alcohol for 10 h. Then the mixture was condensed to
half volume. The precipitated product was filtered. 400 mg
(66%) of compound 19 was obtained, m.p.= 108–109.58C; IR
(nujol): = 3260 (m-NH), 1670, 1660 (m-CO-), 1262, 1218 (C–O ester)
cm–1. 1H-NMR (CO(CD3)2, Tesla, 80 MHz) d: 1.46 (d, J = 6.4 Hz, 6 H, 2
6 CH3), 2.58 (s, 3H, –CH3 at isothiazole position 3), 5.34 (sept, J =
6.4 Hz, 1H, –CH–), 7.71 (d, J1 = 8.8 Hz, 2H, 3,5-H phenyl), 8.045 (d,
J1 = 8.8 Hz, 2H, 2,6-H phenyl) ppm.
Membrane stabilization
Stabilization of erythrocyte membranes was determined by
hemoglobin release according to Seeman and Weinstein [39].
Rabbit erythrocytes were used. The compounds were dissolved
in DMSO and further diluted with hypotonic phosphate buffer
to a concentration ranging from 0.001 to 10 lg/mL. Inhibition of
hemoglobin release of more than 20% compared with controls
(control hemolysis was set at around 50%) was considered as a
positive effect. All experiments were done in duplicate or tripli-
cate.
Pharmacological tests
Biological activity was tested in mice and rats and in vitro with
rabbit and murine cells. The substances were given to the ani-
mals p.o. and i.p. in a suspension of 0.5% methylcellulose in H2O
in a volume of 0.5 mL per 100 g of body weight (rats) or in a 3%
solution of Tween 80 (Serva) in a volume of 0.1 mL per 10 g of
body weight (mice). Ibuprofen (POLFA Pabianice) was used as a
reference compound. The animals come from the Inbred Ani-
mals Breeding Center at the Institute of Immunology and
Experimental Therapy, Polish Academy of Sciences (Wroclaw,
Poland). They were housed under standard conditions with food
and water ad libitum. For in vitro experiments the compounds
were dissolved in DMSO (5 mg/5610– 3 mL) and then diluted in
tissue culture medium or buffers.
Cytotoxic activity
Cytotoxic activity was tested on bone marrow cells from 6- to 8-
week-old female CBA mice. Briefly, bone marrow cells obtained
from the femur were washed three times in culture medium
(RPMI supplemented with 10% fetal bovine serum, 2 lM L-gluta-
mine, 100 U/mL penicillin, and 100 lg/mL streptomycin). Next,
the cells in culture medium were incubated (5% CO2, 378C) in 96-
well culture plates (26105 cells/well) for 20 h with the com-
pounds in concentrations of 0.1, 1 and 10 lg/mL. Mean cell viabi-
lity of a total of 100 or 200 cells counted from two wells for each
compound dilution was assessed with an acridine orange/ethi-
dium bromide mixture solution with a fluorescence microscope
(viable cells appear green, dead cells orange) [40]. The cytotoxic
activity of the compounds was determined as the percentage
decrease in cell viability compared with the viability of control
cells incubated with DMSO solution.
Carrageenan-induced edema test
Carrageenan-induced edema tests were conducted according to
the Winter, Risley, and Nuss method [36, 37], with slight modifi-
cation. Swelling of Wistar rats' paws was induced by subcuta-
neous injection of 50 lL of a 1% carrageenan solution in saline
(378C) into the right and left hind footpads of female Wistar rats
(body weight 150–300 g). Groups of five rats were given two
doses (1, 10 or 100 mg per body weight, p.o.) of the tested com-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 401–413
New Isothiazole Derivatives and Their Biological Activity
413
[16] H. M. F. Madkour, M. A. I. Salem, T. M. Abdelrahman, M.
Data analysis
E. Arab, Heterocycles 1994, 38, 57–69.
IC50 and EC50 concentrations were calculated using a model of a
sigmoid curve with Hill slope (Microsoft Excel 97 and LSW Data
Analysis Toolbox ver. 1.1.1). Standard error (SE) of the inhibition
of carrageenan-induced edema (H) was calculated according to
the formula for SE of the ratio of the means: SEH = (DT/
DC)6[(SEDT/DT)2 + (SEDC/DC)2]1/26100%, where DC is the relative
increase in paw thickness in the control group and DT the rela-
tive increase in paw thickness in the compound-treated group
[21]. Differences between group means were determined at a sig-
nificance level of P a 0.05 using two-way ANOVA and post hoc
comparison (Fisher's LSD test) or the U Mann-Whitney test. Nor-
mality and homogeneity of the variance assumptions were
determined using the Shapiro-Wilk's W and Brown-Forsythe's
tests, respectively. Correlation between the membrane stabiliz-
ing effect (logEC50) and activity of compounds in the carragee-
nan test (logActivity) was determined using linear regression
and Pearson coefficient (r). Statistical analysis was done using
Statistica (release 4.0) for Windows.
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www.archpharm.com