New amides of 5-acylamino-3-methyl-4-isothiazolecarboxylic acid and their immunotropic activity

Article abstract of DOI:10.1002/ardp.200400903

Several new amides 4 of 5-substituted 3-methyl-4-isothiazolecarboxylic acid were obtained. These compounds have acetylamino or benzoylamino groups in position 5 of the isothiazole ring. In position 4, the carboxylic group was transformed in the amides using amino-acid esters. Activities of the obtained derivatives were checked in the humoral immune response and delayed type hypersensitivity reaction to sheep red blood cells (SRBCs) in vivo.

Full text of DOI:10.1002/ardp.200400903

322
DOI 10.1002/ardp.200400903
Arch. Pharm. Chem. Life Sci. 2005, 338, 322−328
New Amides of 5-Acylamino-3-Methyl-4-Isothiazole-
carboxylic Acid and their Immunotropic Activity
Urszula Lipnickaa, Andrzej Regieca, Emil Sułkowskib, Michał Zimeckic
a Wrocław Medical University, Faculty of Pharmacy, Department of Organic Chemistry, Wrocław, Poland
b P. P. Farm. HASCO-LEK, Wrocław, Poland
c Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wrocław, Poland
Several new amides 4 of 5-substituted 3-methyl-4-isothiazolecarboxylic acid were obtained. These com-
pounds have acetylamino or benzoylamino groups in position 5 of the isothiazole ring. In position 4,
the carboxylic group was transformed in the amides using amino-acid esters. Activities of the obtained
derivatives were checked in the humoral immune response and delayed type hypersensitivity reaction
to sheep red blood cells (SRBCs) in vivo.
Keywords: 5-amino-3-methylisothiazolecarboxamides, synthesis, immunotropic activity
Received: May 17, 2004; Accepted: March 4, 2005 [FP903]
Introduction
Results and discussion
Chemistry
During the recent years, a variety of isothiazole derivatives
exhibiting pharmacological activity have been described.
Our research on the reactivity and biological activity of iso-
thiazole compounds showed that some of them had strong
antiviral, anti-inflammatory, and anticancer properties
[1Ϫ5]. The potent antiviral and anti-inflammatory activity
The starting material for the synthesis was the 6-phenyl-3-
methylisothiazolo[5,4-d]-4H-1,3-oxazin-4-one 3 obtained by
Regiec [10]. Isothiazolooxazinone 3, which has semianhy-
dride properties, was transformed into the proper amides 4
in reaction with amino-acid esters in protogenic (ethanol)
and aprotogenic solvents (pyridine or carbon tetrachloride
(CCl4)). Regiec [7, 10] found that the nucleophilic amines’
influence on isothiazolooxazinone ring disclosure depends
on the amine type. In an aprotogenic polar solvent, such as
anhydrous ethanol or 2-propanol, isothiazolo[5,4-d]-4H-1,3-
oxazine 3 reacts with primary aromatic amines, giving a
mixture of mainly diamides and isomeric amidines with less
efficiency. In a nonpolar solvent, such as carbon tetra-
chloride, the products were mainly amidines and traces of
amides, but the speed of the reaction was much slower than
in polar solvents. In a pyridine solution, only diamides were
obtained and the speed of the reaction was highest. The
application of secondary or aliphatic amines yielded only
diamides in all the solvents used.
of
Vratizolin
(5-benzoylamino-4-N(4-chlorophenyl)-3-
methyl-4-isthiazolecarboxamide) 1 (Figure 1) [1, 6], which
also has immunotropic activity, prompted Regiec and
´
Machon [7, 10, 11] to synthesize a series of diamides 2 con-
taining a 4-chlorobenzoyl moiety in position 5 of the iso-
thiazole ring. These compounds possess anti-inflammatory
activities comparable to ibuprofen, but stronger immuno-
suppressive properties than cyclosporine A [10, 11]. It was
also determined that replacement of the aromatic rings with
aliphatic substituents causes a decrease in or loss of this
type of pharmacological activity in these amides 2. It was
described that amino-acid esters were used in the amides’
synthesis [8, 9]. Cyclosporin A (CsA) is a cyclic oligopep-
tide, which contains eleven amino-acid moieties connected
by peptide linkage, and is broadly used in transplantation
and as a therapeutic agent against rheumatoid arthritis
[12Ϫ15]. These findings prompted us to obtain new isothi-
azole amides possessing the moieties of amino-acid ester in
position 4 of the isothiazole ring and to check the influence
of these changes on their immunosuppressive activity.
The above results [7, 10] are supported in this investigation,
whereas treatment of isothiazolooxazinone 3 with esters of
aliphatic aminoacid, should yield diamides 4 as the sole
products, regardless of the solvents used (Scheme 1),
though, in the case of anestesine, the products were depen-
dent on the applied solvents. Compounds 4e and 4i were
also obtained in another way: 5-amino-3-methyl-4-isothi-
azolecarboxylic acid 5 was treated with thionyl chloride
(SOCl2) to give a proper acid chloride 6 that was trans-
formed into amide 7 in reaction with anesthesin. Next, di-
amides 4e and 4i were obtained as a result of the reaction
of amide 7 with benzoyl chloride and acetyl chloride, respec-
tively (Scheme 2).
Correspondence: Urszula Lipnicka, Wrocław Medical University,
Faculty of Pharmacy, Department of Organic Chemistry, Grodzka
9, 50-137 Wrocław, Poland. Phone: ϩ48 71 784-0340, Fax: ϩ48 71
784-0341, e-mail: urszula.lipnicka@mars.bf.uni.wroc.pl
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 322−328
New Isothiazole Derivatives with Immunotropic Activity
323
Figure 1.
Scheme 1. Preparation of compounds 4aϪd and 4fϪn.
Pharmacology
compound 4i, which was supported by earlier studies [7,
10]. The weak activity of compound 4f could result from
instability of the cyclic lactone group that under disclosure
conditions produces a compound with free hydroxylic and
carboxylic fragments that also have hydrophilic properties.
Biological tests showed that product 4b, which contains an
alanine moiety in position 4, had the highest immunosup-
pressive activity in this series of compounds. It exhibited
a 1.5-times stronger activity than Vratizolin [6], which is
comparable to that of CsA in the humoral and cellular im-
mune responses (Tables 1 and 2). These data indicate that
introducing a lipophilic amino-acid chain substituent of the
proper size and shape in position 4 of the carboxamide frag-
ment of the isothiazole ring conditions the immunosup-
pressive activity of the isothiazole derivatives. However, re-
placement of the benzoyl group in position 5 with an acetyl
and the carboethoxyphenyl group in position 4 with an ala-
nine moiety eliminates the immunosuppressive activity of
At this stage of the investigation it is difficult to predict
properties of compound 4b which could have a potential
therapeutic value. It is most likely that the exceptionally
strong immunosuppressive action of this compound on the
generation of both humoral and cellular immune responses
could also be associated with its anti-inflammatory activity,
as in the case of Vratizolin [6]. Our unpublished data on an
isoxazole derivative RM33 revealed that its strong immuno-
suppressive activity on the generation of an immune re-
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Lipnicka et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 322−328
Scheme 2. Preparation of compounds 4i and 4e.
Table 1. Number of the plaque-forming cells (PFC) in the
spleens of CBA mice, immunized with SRBCs and treated i.p.
with the study preparation 2 h before antigen administration†.
Table 2. DTH reaction (footpad test) in CBA mice sensitized
with SRBCs and treated i.p. with the preparation 2 h before
administration of sensitizing dose of the antigen†.
Preparation
PFC/106
SE
P
Preparation
Dose
[μg/mL]
Units
SE
P
Student test
[10؊2 cm]
Student test
Control
4a
4640
1440
640
3540
3210
2770
3940
3290
3830
620
266
Ϫ
Control
4a
100
”
”
”
”
”
”
”
”
13.1
7.5
5.5
7.2
7.6
8.1
8.2
10.7
11.5
4.4
0.16
0.13
0.20
0.22
0.17
0.47
0.22
0.47
0.35
0.21
Ϫ
86.71
105.07
96.02
136.18
76.32
131.22
70.88
90.24
43.81
< 0.001
< 0.001
< 0.01
< 0.001
< 0.001
< 0.05
< 0.01
< 0.05
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
< 0.001
4b
4b
4c
4c
4d
4d
4e
4e
4f
4f
4h
4h
4i
4I
CsA
CsA
”
†
†
The results are expressed as a mean SE of five mice. 2.5 mg of
the preparation was dissolved in 0.1 mL DMSO and then diluted
in 0.9% NaCl to proper concentration.
The results are expressed as the mean SE of 10 determinations.
2.5 mg of the preparation were dissolved in 0.1 mL DMSOand
then diluted in 0.9% NaCl to the proper concentration.
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 322−328
New Isothiazole Derivatives with Immunotropic Activity
325
using toluene/methanol (9:1) as eluting agent. Detection of com-
pounds on the chromatograms was made with UV light and/or by
treatment with iodine vapor.
sponse was also associated with its ability to suppress the
inflammatory reaction elicited by Freund’s complete adju-
vant and carrageenan-induced inflammation. It is highly
conceivable that compound 4b may exhibit similar proper-
ties.
3,6-dimethylisothiazolo[5,4-d]-4H-1,3-oxazin-4-one 3b and 6-phenyl-
3-methylisothiazolo[5,4-d]-4H-1,3-oxazin-4-one 3a
were obtained according to the method described for 6-(4-chlororo-
phenyl)-3-methylisothiazolo[5,4-d]-4H-1,3-oxazin-4-one analogue
[7, 10, 11]. 3a: mp. 128Ϫ129°C, yield 71%, 1H-NMR δ ppm 2.73
(-CH3 3H, s), 7.5Ϫ8.10 (Ar-5H, m). 3b: mp. 80Ϫ82°C, yield 31%,
1H-NMR δ ppm 2.37 (-CH3, 3H, s), 2.76 (-CH3, 3H, s).
Effects of the compounds on the generation
of the humoral immune response to SRBCs
in vivo
Table 1 shows the results of the effects of a single intraperi-
toneal (i.p.) administration Ϫ 2 h before immunization of
mice Ϫ of 100 μg of the compounds studied on the magni-
tude of the humoral immune response. The data are ex-
pressed as the mean number of antibody-forming cells in
General methods of 5-acylamino-3-methyl-4-isothiazolecarboxamides
synthesis
For results see also Tables 3 and 4.
the spleen
the standard error, calculated per 106 viable
Method A
splenocytes. CsA served as a reference drug. The results
show that, generally, all compounds exhibit suppressive ac-
tivities to various degrees. Compound 4b requires particular
attention since its suppressive activity is comparable with
that of CsA. Compound 4a was also strongly inhibitory in
this model.
0.01 mol of 3a or 3b and methyl amino-acid ester (0.01 mol) dis-
solved in 10 mL of anhydrous pyridine and the solution was heated
for 3 h. Then, the reaction mixture was left for 12 h at room tem-
perature. From the mixture a crude product was precipitated with
cold water. The solid was filtered, then dried and crystallized from
the proper solvent.
Method B
To 40 mL of anhydrous ethanol, 0.01 mol of compound 3a or 3b,
amino-acid ester hydrochloride (0.01 mol) and 0,05 mol of anhy-
drous potassium were added. The whole mixture was heated in re-
flux for 2Ϫ4 h in anhydrous conditions. Then, the solvent was eva-
porated in vacuum to 1/3 of volume and the cool solution was
poured into cold water. The precipitated solid was washed with
water and after drying, crystallized.
Effects of the compounds on the generation
of delayed type hypersensitivity to SRBCs
in vivo
Table 2 shows the effect of i.p. treatment of mice before
sensitization with SRBCs on the expression of delayed type
hypersensitivity (DTH). The data are presented as mean val-
ues of foot-pad swelling (in DTH units) SE. The result
indicates that compound 4b is the most potent inhibitor in
this experimental model, with an activity close to that of
CsA. Strong suppressive activity also characterizes com-
pounds 4a, 4c, and 4d.
5-Benzoylamino-N-Methoxycarbonylmethyl-3-Methyl-4-Isothiazole-
carboxamide 4a
Method A: mp. 177°C, yield 37.7%; method B: mp. 176Ϫ177°C,
yield 39.5%.
5-Benzoylamino-N-(α-Methoxycarbonyl)Ethyl-3-Methyl-4-Isothi-
azolecarboxamide 4b
Acknowledgments
Method A: mp. 168Ϫ170°C, yield 33.5%; method B: mp.
168Ϫ169°C, yield 32%.
The presented study was supported by the Wrocław Univer-
sity of Medicine (internal project number 979).
5-Benzoylamino-N-(α-Methoxycarbonyl-γ-Methyl)Buthyl-3-Methyl-
4-Isothiazolecarboxamide 4c
Experimental
Chemistry
Method A: mp. 136Ϫ137°C, yield 26.8%; method B: mp.
135Ϫ136°C, yield 47.7%.
General
5-Benzoylamino-N-(α-Methoxycarbonyl-β-Phenyl)Ethyl-3-Methyl-
4-Isothiazolecarboxamide 4d
The melting points of all new compounds were measured on a
Boethius hot-stage (VEB Kombinat Negema Wägetechnik Rapido,
Radebeul, Germany) and were uncorrected. Elemental analysis: car-
ried out in the Microlaboratory of the Pharmacy Department,
Wrocław Medical University, showed that all the analytical results
were according to the calculated ones within 0.5%. The following
equipment was employed: IR spectra: Specord M80 apparatus, KBr.
1H-NMR spectra: 80 MHz Tesla BS 587 A apparatus (Tesla, Brno,
Czech Republik), TMS as internal reference. TLC was made with
Polygram SIL G/UV254 plates (Mocherey-Nagel, Düren, Germany),
Method A: mp. 162Ϫ164°C, yield 35.8%; method B: mp.
162Ϫ163°C, yield 61.3%.
5-Benzoylamino-N-(2-Oxotetrahydrofuranyl-3)-3-Methyl-4-Isothi-
azolecarboxamide 4f
Method A: mp. 200Ϫ201°C, yield 15.1%; method B: mp.
201Ϫ203°C, yield 37.8%.
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Lipnicka et al.
Arch. Pharm. Chem. Life Sci. 2005, 338, 322−328
Table 3. Physical and analytical data for compounds 4aϪi.
Compound
Formula,
molecular weight
Mp. [°C]
Method A/B/C
Yield [%]
Method A/B/C
Elemental analysis
Calculated/Found
H [%]
C [%]
N [%]
4a
4b
4c
4d
4e
4f
C15H15N3O4S
333.21
A)177
B)176-177
(methanol)
37.7
39.5
54.07
54.38
4.50
4.58
12.60
12.73
C16H17N3O4S
347.22
A) 168Ϫ170
B) 168Ϫ169
(methanol)
33.5
32.0
55.34
55.30
4.90
4.96
12.10
12.00
C19H23N3O4S
389.25
A) 136Ϫ137
B) 135Ϫ136
(methanol:ethanol 3:1)
26.8
47.7
58.62
58.56
5.91
5.90
10.79
10.93
C22H21N3O4S
423.28
A) 162Ϫ164
B) 162Ϫ163
(methanol:ethanol 3:1)
35.8
61.3
62.42
62.36
4.96
5.03
9.92
10.00
C21H19N3O4S
409.27
A) 173Ϫ174
B) 172Ϫ174
(methanol)
41.5
36.8
61.62
60.83
4.64
4.91
10.26
10.27
C16H15N3O4S
345.22
A) 200Ϫ201
B) 201Ϫ203
15.1
37.8
55.66
55.64
4.35
4.09
12.17
11.68
(methanol:chloroform 4:1)
4g
4h
C10H13N3O4S
271.16
A) 233Ϫ235
(chloroform:methanol 5:1)
22.0
44.29
44.48
4.79
4.88
15.49
15.29
C17H19N3O4S
361.23
A) 168Ϫ169
B) 168Ϫ169.5
23.7
25.3
56.52
56.25
5.26
5.20
11.63
11.12
(chloroform:methanol 5:1)
4i
C16H17N3O4S
347.22
C) 260Ϫ261
(chloroform:methanol 5:1)
37.6
55.34
55.35
4.90
4.96
12.10
12.16
Table 4. Spectroscopic data for compounds 4aϪi.
Compound
H1-NMR (δ [ppm])
2.77 (-CH3, 3H, s); 3.84 (-CH3, 3H, s); 4.25Ϫ4.31 (-CH2-NH-, 2H, d); 7.58Ϫ8.05 (phenyl, 5H Ar, m)
4a
4b
1.51Ϫ1.63 (-CH-CH3, 3H, d); 2.81 (-CH3, 3H, s); 3.86 (-CH3, 3H, s); 4.63Ϫ4.89 (-CH-CH3, 1H, q); 6.92Ϫ8.07
(phenyl, 5H Ar, m)
4c
4d
4e
0.99Ϫ1.05 (CH3-CH-CH3, 6H, d); 1.78Ϫ1.81 (-CH-CH2-CH-, 2H, m); 2.79 (-CH3, 3H, s); 3.81 (-CH3, 3H, s);
4.70Ϫ5.03 (CH3-CH- CH3, 1H m); 6.62Ϫ6.82 (-CH-CH2-, 1H, t); 7.60Ϫ8.00 (phenyl, 5H Ar, m)
2.49 (-CH3, 3H, s); 3.28Ϫ3.34 (-CH2-CH-, 2H, d); 3.83 (-CH3, 3H, s); 5.00Ϫ5.20 (-CH-CH2-, 1H, t); 7.18Ϫ8.07 (2
ϫ phenyl, 2 ϫ 5H Ar, m); 12.83 (-NH-, 1H, s)
1.31Ϫ1.49 (CH3-CH2-, 3H, t); 2.79 (-CH3, 3H, s); .24Ϫ4.50 (-CH2-CH3, 2H, q); 7.27Ϫ8.11 (2 ϫ phenyl, 4H Ar I
5H Ar, m)
4f
4g
4h
2.75 (-CH3, 3H, s); 4.22Ϫ5.12 (lactone, 5H, m); .50Ϫ8.12 (phenyl, 5H Ar, m)
2.46 (-CH3, 3H, s); 3.30 (-CH3, 3H, s); 3.40 (-CH3, 3H, s); 3.75Ϫ3.95 (-NH-CH2-, 2H, d); 8.5 (-NH-, 1H, s)
2.55 (-CH3, 3H, s); 2.92 (-CH3, 3H, s); .22Ϫ3.35 (-CH2-CH-, 2H, d); 3.86 (-CH3, 3H, s); 4.99Ϫ5.18 (-CH-CH2-, 1H,
t); 6.56Ϫ7.26 (phenyl, 5H, Ar, m); 12.5 (-NH-, 1H, s)
4i
1.31Ϫ1.53 (CH3-CH2-, 3H, t); 3.34 (-CH3, 3H, s); 2.69 (-CH3, 3H, s); 4.25Ϫ4.53 (-CH2-CH3, 2H, q); 7.60Ϫ8.16
(phenyl, 4H Ar, m); 10.0 (-NH-, 1H, s)
© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2005, 338, 322−328
New Isothiazole Derivatives with Immunotropic Activity
327
5-Acetylamino-N-Methoxycarbonylmethyl-3-Methyl-4-Isothi-
azolecarboxamide 4g
SRBCs. CsA was used at the same dosage. Control mice were un-
treated or given solvent 2 h before immunization. Four days later,
the number of plaque-forming cells (PFCs) in the spleens was deter-
mined by the method described by Michell and Dutton [16]. The
Method A: mp. 233Ϫ235°C (crystallized from chloroform : meth-
anol (5:1)), yield 22%.
results are presented as mean values of PFCs per 106 cells
standard error (SE).
the
5-Acetylamino-N-(α-Methoxycarbonyl-β-Phenyl)Ethyl-3-Methyl-4-
Isothiazolecarboxamide 4h
Delayed type hypersensitivity (DTH) reaction
Method A: mp. 168Ϫ169°C, yield 23.7%; method B: mp.
168Ϫ169.5°C, yield 25.3%, crystallized from chloroform : meth-
anol (5:1).
The influence of the preparation on the inductive or effector phase
of DTH was measured according to Lagrange et al. [17]. Mice were
sensitized intravenously (i.v.) with 105 SRBCs in 0.9% NaCl. The
animals were treated 2 h before sensitization with a 100 μg dose of
each preparation. After four days, the reaction was elicited by the
intradermal administration of 108 SRBCs into the left hind footpad.
Unsensitized mice, administered with 108 SRBCs into the left hind
footpad, were used as a negative control (background). The DTH
reaction was measured after 24 h as the increase in footpad thick-
ness after the challenging dose of the antigen. The data are ex-
pressed in units (1 unit ϭ 10Ϫ2 cm of the increase in footpad thick-
ness). The background, nonspecific response, was subtracted.
Method C (Scheme 2)
5-Amino-N-(4-Carboethoxy)Phenyl-3-Methyl-4-Isothiazole-
carboxamide 7
1.6 g of amino acid 5 was treated with 16 mL of thionyl chloride
(SOCl2) and heated under reflux for 4 h. Then, thionyl chloride was
evaporated, and benzene was added to the residue and evaporated
(2 ϫ 15 mL C6H6) as well. Crude acid chloride was dissolved in 10
mL of benzene, and ethyl 4-aminobenzoate was added to the solu-
tion. The entire mixture was refluxed for 1 h and left at 4°C for 12
h. The precipitated compound was filtered off and the filtrate was
evaporated to dryness. The collected residues were washed with
water and crystallized after drying from chloroform : methanol
(5:1). Mp. 215Ϫ17°C, yield 48%.
Proliferation assay
A single spleen cell suspension was prepared by pressing the spleens
through a plastic screen into 0.83% NH4Cl to lyse the erythrocytes.
After 5 min, the cells were washed 3 x with Hank’s medium and
resuspended in a culture medium consisting of RPMI 1640, 10%
fetal calf serum, 2mM l-glutamine, sodium pyruvate, 2-mercapto-
ethanol, and antibiotics to a final density 2 · 106/mL. 0.1 mL ali-
quots (2 · 105 cells) were distributed in 96-well flat-bottom culture
plates. Concanavalin A (ConA) was used at a concentration of 2.5
μg/mL. The preparations were added to the cell cultures at doses
0.1, 1.0, and 5.0 μg/mL at the beginning of a four-day incubation
in a cell culture incubator. Cell proliferation was determined using
the MTT (methyl tetrazolium bromide, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) colorimetric method [18].
5-Benzoylamino-N-(4-Carboethoxy)Phenyl-3-Methyl-4-Isothiazole-
carboxamide 4e
2.5 mmol of compound 7 were suspended in 10 mL of anhydrous
acetone, and 0.5 mL of anhydrous pyridine was added. While stir-
ring, 5 mmol of benzoyl chloride in 5 mL acetone were added drop-
wise to the mixture. The entire mixture was heated for 2 h; then, all
solvents were evaporated. The solid residue was dissolved in 5 mL
of methanol and this solution was poured into 120 mL of water.
Precipitated sediment was filtered, dried, and crystallized from
methanol giving 36.8% yield, mp. 172-174°C.
MTT colorimetric assay for cell growth and kill
5-Acetylamino-N-(4-Carboethoxy)Phenyl-3-Methyl-4-Isothiazole-
carboxamide 4i
The assay was performed according to Hansen et al. [18]. Briefly,
25 μL of the MTT (5 mg/mL) stock solution was added per well at
the end of cell incubation and the plates were incubated for another
3 h in a cell culture incubator. Then, 100 μL of the extraction buffer
(20% SDS with 50% DMF, pH 4.7) was added. After additional
overnight incubation, the optical density was measured at 550 nm
(Dynatech).
The method of obtaining is similar to the procedure described for
compound 4e, using acetyl instead of benzoyl chloride. Crystalliza-
tion from chloroform : methanol (5:1) gave a white product, mp.
260Ϫ261°C, yield 37.6%.
Pharmacology
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´
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