the m 1-9 series of compounds with immunotropic activity

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 65 No. 2 pp. 241ñ244, 2008
ISSN 0001-6837
Polish Pharmaceutical Society
A NEW CLASS OF ISOXAZOLE DERIVATIVES: THE M 1-9 SERIES
OF COMPOUNDS WITH IMMUNOTROPIC ACTIVITY
MARCIN M•CZY—SKI1*, MICHA£ ZIMECKI2 and STANIS£AW RYNG1
1
Wroc≥aw Medical University, Faculty of Pharmacy, Department of Organic Chemistry,
50-137 Wroc≥aw, 9 Grodzka Str., Poland
2
Laboratory of Immunobiology, Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences, 12 r. Weigla Str., 53-114 Wroc≥aw, Poland
Abstract: The isoxazole derivatives are interesting objects for synthesis in the search for various sorts of biological activity. Looking for more active immunomodulators we synthesized a series of 5-amino-3-methyl-4isoxazolecarboxylic acid semicarbazides and thiosemicarbazides in the reaction of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide with isocyanates and isothiocyanates. The biological effect of these compounds
on the proliferative response of human mononuclear peripheral blood cells to phytohemagglutinin A (PHA) was
described.
Keywords: immunotropic activity, PHA
consisting of RPMI 1640 medium, L-glutamine,
sodium pyruvate, 2-mercaptoethanol, antibiotics
and 10% fetal calf serum and distributed in 0.2 mL
aliquots (2 ◊ 105 cells/well) in flat-bottom 96-well
plates. PHA (Sigma) was used to stimulate cells at
concentration of 2.5 µg/mL. The compounds were
initially dissolved in DMSO and then in RPMI
medium and were applied at doses 1.0 and 10.0
µg/mL. After 3-day culture in a cell culture incubator the rate of cell proliferation was determined by a
colorimetric MTT method (15). The results are presented as mean optical density (OD) value at the
wavelength 550/630 nm from quadruplicate determinations, and standard error. The Student t-test was
applied for statistical analysis. The results were
regarded significant when p < 0.5.
Isoxazole derivatives exhibit a wide array of
biological activities and many of them have found
application in therapy. The immunosuppressive or
anti-inflammatory properties are most characteristic
among that group of compounds (1, 2). In the course
of our studies immunological activities of amide (38), ureilene (9), hydrazide (10,1 1), 3-methylisoxazole[5,4-d]1,2,3-triazin-4-one (12), 5-amino-3methylisoxazolo[5,4-d]4-pyrimidinone (13), and
thiosemicarbazide (14) isoxazole derivatives were
described. In a search to obtain new derivatives of
interesting, immunotropic activity, were synthesized
active semicarbazide- and thiosemicarbazide-substituted derivatives in position 4 of isoxazole ring. In
this report we present activities of nine derivatives
in the model of the proliferative response of human,
mononuclear blood cells to a T-cell mitogen, phytohemagglutinin A (PHA).
Chemistry
5-Amino-3-methyl-4-isoxazolecarboxylic acid
semicarbazides and thiosemicarbazides (M 1-9)
were synthesized. The reaction of 5-amino-3methyl-4-isoxazolecarboxylic acid hydrazide with
isocyanates and isothiocyanates were designed and
conducted. The isocyanates, in the reaction of nucleophilic addition with compounds containing the primary amino group, form urea derivatives and isothiocyanates the thiourea derivatives. Only the
hydrazide terminal group (-NH2) participates in this
reaction. The amino group in position 5 of isoxazole
EXPERIMENTAL
Immunology
Venous blood was taken from a single donor to
heparinized tubes, diluted twice with phosphate
buffered saline (PBS), applied on a ficoll-uropoline
gradient (density 1.077 g/L), centrifuged at 2000
rpm and cells from the interphase (lymphocytes and
monocytes) were washed with Hanksí medium. The
cells were then resuspended in a culture medium
* Corresponding author: e-mail: [email protected]
241
242
MARCIN M•CZY—SKI et al.
ring remains not reactive under the reaction conditions. The mechanism of the reaction consists in
nucleophilic attack of the nitrogen atom in the
hydrazide group (-NH2) on the carbon atom of isocyanate or isothiocyanate. The intermediate forms
appear which undergo amidoiminole tautomerization leading to formation of substituted 5-amino-3methyl-4-isoxazolecarboxylic acid semicarbazides
and thiosemicarbazides (M 1-9).
RESULTS
Table 1 presents the effects of the studied compounds on the PHA-induced proliferation of human
mononuclear blood cells. Cyclosporine A (CsA)
served as a reference, suppressive agent. The effects
of the compounds were compared with the appropriate concentrations of the solvent (DMSO). The
results indicate that among the studied compounds
only M-3 and M-4 exhibited statistically significant,
dose-dependent stimulatory effects in this experimental in vitro model.
DISCUSSION
The presented findings supported our earlier
observation that M-3 and M-4 derivatives were
stimulatory in the model of the proliferative
response of mouse splenocytes to concanavalin A.
Interestingly, M-3 and M-4 appeared to be strongly
suppressive in the model of the humoral immune
response in vitro of mouse splenocytes to sheep red
blood cells (to be published). That phenomenon
seems to be not unique among isoxazoles since
another derivative, synthesized in our laboratory,
RM-33, demonstrated strong inhibitory properties
with regard to both humoral and cellular immune
response in mice, adjuvant-induced inflammation
(16) and carrageenanñelicited foot pad edema in rats
(17), whereas it moderately stimulated mitogen-
Table 1. The effect of the compounds on the proliferative response of human mononuclear peripheral blood cells to PHA.
Dose
µg/mL
No mitogen
X
OD 550 nm
± SE
Student
t-test
0.096
0.004
PHA control
5
0.259
0.009
DMSO solvent
control
1
10
0.265
0.266
0.012
0.010
NS
NS
M1
1
10
0.261
0.257
0.003
0.009
NS
NS
M2
1
10
0.260
0.257
0.016
0.006
NS
NS
M3
1
10
0.293
0.289
0.006
0.005
< 0.05
< 0.001
M4
1
10
0.342
0.309
0.008
0.012
< 0.001
< 0.001
M5
1
10
0.258
0.254
0.006
0.009
NS
NS
M6
1
10
0.255
0.259
0.011
0.005
NS
NS
M7
1
10
0.255
0.260
0.007
0.007
NS
NS
M8
1
10
0.240
0.250
0.013
0.008
NS
< 0.05
M9
1
10
0.248
0.256
0.008
0.008
NS
NS
CsA
1
10
0.155
0.008
0.003
0.003
< 0.001
< 0.001
The compounds were initially dissolved in DMSO, then in RPMI1640 culture medium
The results are expressed as the mean values from 4 determinations (wells) ± standard error
NS-not significant
A new class of isoxazole derivatives: the M 1-9 series of compounds with immunotropic activity
Table 2. Structure of the obtained compounds M 1-9.
Compound
R
M1
243
ity of M-3 and M-4 the thiosemicarbazide group and
the aliphatic substituent may be responsible. The
compounds with semicarbazide group and phenyl
substituent did not exhibit any immunological activity. This investigation indicates that these two derivatives may be further investigated in animal models
relevant to clinical situations.
Acknowledgment
These studies were supported by the Wroc≥aw
Medical University, grant 1312.
REFERENCES
M2
M3
M4
M5
M6
M7
M8
M9
induced cell proliferation. T-cell proliferation is also
moderately affected in the case of another strong
immunosuppressor ñ leflunomide. In this respect,
the immunosuppressive mechanism of action of
some isoxazoles differs from that of CsA (18, 19). In
the proliferative test (Table 1) only 4-aliphatic
thiosemicarbazide 5-amino-3-methylisoxazole-4carboxylic
acid
derivatives
demonstrated
immunotropic activity. The structure-activity analysis suggests that for the distinct immunotropic activ-
1. Fischereder M., Kretzler M.: J. Nephrol. 17,
1366 (2004).
2. Ibrahim A., Park S., Feldman J., Karim A.,
Kharasch E.D.: Anesthesiology 96, 88 (2002).
3. Ryng S., MachoÒ Z., Wieczorek Z., Zimecki
M., G≥owiak T.: Arch. Pharm. Pharm. Med.
Chem. 330, 319 (1997).
4. Ryng S., Zimecki M., Fedorowicz A., Koll A.:
Pol. J. Pharmacol. 51, 257 (1999).
5. Ryng S., Zimecki M., Sonnenberg Z., Mokrosz
M. J.: Arch. Pharm. Pharm. Med. Chem. 332,
158 (1999).
6. Ryng S., Zimecki M., Fedorowicz A., Koll A.:
Quant. Struct. Act. Relat. 18, 236 (1999).
7. Ryng S., Sonnenberg Z., Zimecki M.: Arch.
Immunol. Ther. Exp. 48, 127 (2000).
8. Fedorowicz A., Jezierska A., Ryng S.: Internet
J. Chem. 4, 7 (2001).
9. Ryng S., MachoÒ Z., Wieczorek Z., Zimecki
M.: Pharmazie 54, 359 (1999).
10. Ryng S., Zimecki M., Fedorowicz A., Jezierska
A.: Arch. Pharm. Pharm. Med. Chem. 334, 71
(2001).
11. Jezierska A., Panek J., Ryng S., Fedorowicz A.,
Koll A.: Pol. J. Chem. 76, 1255 (2002).
12. MπczyÒski M., Jezierska A., Zimecki M., Ryng
S.: Acta Pol. Pharm. Drug Res. 60, 147 (2003).
13. MπczyÒski M., Zimecki M., Drozd-Szczygie≥
E., Ryng S., Cell. Mol. Biol. Lett. 10, 613
2005).
14. MπczyÒski M., Zimecki M., Ryng S.: Acta Pol.
Pharm. 61, 82 (2004).
15. Hansen M.B., Nielsen S.E., Berg K.: J.
Immunol. Methods 119, 203 (1989).
16. Ryng S, Zimecki M, MπczyÒski M, Chodaczek
G, KociÍba M.: Pharmacol. Rep. 57, 195
(2006).
17. Zimecki M., Ryng S., MπczyÒski M.,
Chodaczek G., KociÍba M., Kuryszko J., Kaleta
K.: Pharmacol. Rep. 58, 236 (2006).
244
MARCIN M•CZY—SKI et al.
18. Ruckemann K., Fairbanks C.D., Carrey E.A.,
Hawrylowocz
C.M.,
Richards
D.F.,
Kirschbaum B, Simmonds H.A.: J. Biol. Chem.
273, 21682 (1998).
19. Lang R., Wagner H., Heeg K., Transplantation
59, 382 (1995).
Received: 24.10.2007