Synthesis and biological activity of (Z) and (E) isomers of 3-(3,4-diaryl-1,2,4-triazole-5-yl)prop-2-enoic acid

Article abstract of DOI:10.1007/s00706-008-0055-9

(Z)-3-(3,4-diaryl-1,2,4-triazole-5-yl)prop-2-enoic acid derivatives were obtained in the course of the reaction of N ³-substituted amidrazones with maleic anhydride, and isomerized into the (E) isomers by heating under reflux in acetic acid sol

Full text of DOI:10.1007/s00706-008-0055-9

Monatsh Chem (2009) 140:439–444
DOI 10.1007/s00706-008-0055-9
ORIGINAL PAPER
Synthesis and biological activity of (Z) and (E) isomers
of 3-(3,4-diaryl-1,2,4-triazole-5-yl)prop-2-enoic acid
_
´
Bozena Modzelewska-Banachiewicz Æ Barbara Michalec Æ Teresa Kaminska Æ
Liliana Mazur Æ Anna E. Kozioł Æ Jacek Banachiewicz Æ Marzena Ucherek Æ
´
Martyna Kandefer-Szerszen
Received: 30 July 2008 / Accepted: 23 August 2008 / Published online: 14 October 2008
ꢀ Springer-Verlag 2008
Abstract (Z)-3-(3,4-diaryl-1,2,4-triazole-5-yl)prop-2-enoic
acid derivatives were obtained in the course of the reaction of
N3-substituted amidrazones with maleic anhydride, and
isomerized into the (E) isomers by heating under reflux
in acetic acid solution. The molecular structure of the
Keywords (Z) and (E) isomers Á
3,4-Diaryl-1,2,4-triazoles Á Propenoic acid derivatives Á
Antiviral activity Á Immunomodulation
1
compounds obtained was confirmed by IR and H NMR
Introduction
spectroscopy, and by X-ray crystallography for (2E)-3-(4,
5-diphenyl-4H-1,2,4-triazol-3-yl)prop-2-enoic acid. The
antiviral and immunomodulating activity of several of the
compounds was examined.
1,2,4-Triazole derivatives are constituents of many phar-
maceuticals with diverse therapeutic action—antiviral,
antifungal, sedative, and anxiolytic [1]. Moreover, the
derivatives of carboxylic acids (e.g. acetic, propionic, and
benzoic) which contain aromatic systems in their structure
(phenyl, naphthyl, indolyl, 2-pyridyl) are used as non-ste-
roidal anti-inflammatory drugs (NSAIDs) and analgesic
drugs (e.g. ibuprofen, naproxen, piroxicam, diclofenac,
aspirin) [1]. Recently, the antiviral [2, 3] and anti-inflam-
matory [4–13] activity of 1,2,4-triazole derivatives has
been studied.
B. Modzelewska-Banachiewicz Á J. Banachiewicz Á
M. Ucherek
In the course of the reaction of N3-substituted amid-
razones 1–7 [14] with maleic anhydride, (Z)-8–14 of
3-(3,4-diaryl-1,2,4-triazole-5-yl)prop-2-enoic acid have
been obtained [15]. The presence of a double C=C bond
in compounds 8–14 enabled the formation of (Z) and (E)
isomers differing in their energetic stability, which could
affect their biological activity also [16, 17]. In this paper
we present a method of transformation of the (Z)-8–14
parent isomers into the (E)-15–21 diastereoisomers. The
reactions are presented in Scheme 1. Due to the presence
of the 1,2,4-triazole system, which has also been found in
the structure of numerous drugs with antiviral activity
(e.g. ribavirin), we decided to conduct antiviral and
virucidal tests. The prepared compounds were also bio-
assayed for their potential to stimulate or modulate
mitogen phytohaemaglutinin (PHA) with lipopolysaccha-
ride (LPS)-induced interferon gamma (IFN-c) and tumour
Department of Organic Chemistry,
Faculty of Pharmacy,
Nicolaus Copernicus University,
Jagiellonska 13, 85-067 Bydgoszcz, Poland
e-mail: maru@cm.umk.pl
B. Michalec (&)
Department of Molecular Biology,
Maria Curie-Sklodowska University,
Akademicka 19, 20-033 Lublin, Poland
e-mail: basiam@hektor.umcs.lublin.pl
´
´
T. Kaminska Á M. Kandefer-Szerszen
Department of Immunology and Virology,
Maria Curie-Sklodowska University,
Akademicka 19, 20-033 Lublin, Poland
L. Mazur Á A. E. Kozioł
Department of Crystallography,
Maria Curie-Sklodowska University,
M. Curie-Sklodowska Sq. 3, 20-031 Lublin, Poland
123

440
B. Modzelewska-Banachiewicz et al.
O
signal was moved downfield. Signals of methyl group
hydrogens of compounds 8 and 10 were observed as
singlets in the range 2.3–2.4 ppm.
NH2
N
O
+
NH
R2
R1
Isomerization to (E) diastereoisomers 15–21 occurred
when the (Z)-isomers were heated under reflux in the glacial
acetic acid. The spatial configuration at the double C=C
bond was confirmed, by spectroscopic means, to be (Z) for
compounds 8–14 and (E) for compounds 15–21. In the IR
spectra of both series of isomers, the characteristic
absorption band of a carbonyl group was present in the
1,698–1,710 cm-1 range. In the 1H NMR spectra, signals of
vinyl-type hydrogens were observed at 6.5–6.9 ppm. The
coupling constants for (Z)-8–14 and (E)-5–21 measured in
the spectra were in good accordance with the literature
J = 12–18 Hz for the (E) configuration [18]. The carbox-
ylic group hydrogens were observed as broad singlets in
the 12.9–13.8 ppm range. Only for compound 15, contain-
ing pyridinyl and methylphenyl substituents, the COOH
hydrogen signal was moved downfield to 15.0 ppm. Signals
of methyl group hydrogens were observed as singlets in the
range 2.3–2.4 ppm for compounds 15 and 17.
O
1-7
HO
O
O
N
N
N
N
CH3COOH
N
N
OH
R1
R1
R2
R2
8-14 (Z )
15-21 (E )
Cmpd.
1, 8, 15
R1
R2
2-pyridyl
4-pyridyl
4-pyridyl
2-pyridyl
2-pyridyl
phenyl
4-methylphenyl
phenyl
2, 9, 16
4-methylphenyl
phenyl
3, 10, 17
4, 11, 18
5, 12, 19
6, 13, 20
7, 14, 21
2-pyridyl
The molecular structure of the isomerization products
of (Z)-3-(3,4-diaryl-1,2,4-triazole-5-yl)prop-2-enoic acid
derivatives 8–14 into (E)-15–21 was confirmed by the
X-ray analysis of crystalline 20. The numbering scheme
and general view of two symmetrically independent mol-
ecules of 20 are shown in Fig. 1.
phenyl
2-pyridyl
4-nitrophenyl
Scheme 1
Equivalent bond distances and angles for both molecules
are equal within experimental error. The triazole N1=C5
and N2=C3 bonds have double-bond character, with
necrosis factor (TNF) production in human blood
leukocytes.
˚
respective bond distances of 1.312(8) A (molecule A),
˚
˚
1.311(9) A (molecule B) and 1.302(8) A (molecule A),
˚
1.316(9) A (molecule B), whereas the N4–C3 and N4–C3
Results and discussion
bonds have an intermediate character, indicating delocal-
ization of the electron cloud. The triazole and two phenyl
rings are each essentially planar but are not coplanar. The
symmetrically independent molecules are connected by
alternate O1a–H…N1 and O1–H…N1a short hydrogen
bonds to form extended ribbons along the crystallographic
a axis. These ribbons are joined by numerous C–H…O/N
and C–H…p interactions.
The synthesis of (Z)-3-(3,4-diaryl-1,2,4-triazole-5-yl)prop-
2-enoic acid derivatives was performed according to the
procedure described in a previous report [15]. Condensa-
tion of N3-substituted amidrazones 1–7 with maleic acid
anhydride was carried out at ambient temperature. Sub-
strates were dissolved in anhydrous diethyl ether prior to
mixing them together (Scheme 1).
1
Based on results from spectral analysis (IR, H NMR)
and X-ray crystallography, it was revealed that the reaction
led directly to the formation of (Z)-3-(3,4-diaryl-1,2,4-tri-
azole-5-yl)prop-2-enoic acids (8–14). In the IR spectra of
cyclic compounds, characteristic absorption bands of the
carbonyl group were present in the range 1,700–
Antiviral activity
Anti-herpes simplex virus type 1 (HSV-1) activity of the
1,2,4-triazole derivatives was examined in a cytopathic
effect (CPE) reduction assay and as a result the virus titre
was calculated. All compounds examined inhibited viral
replication during 24 h, however, when CPE was estimated
after 48 h, only partial inhibition of viral replication was
observed. No significant differences in the antiviral activity
of (Z)-11 and (Z)-13 in comparison with (E)-18 and (E)-20
were detected. The compounds 18 and 20 most effectively
1
1,708 cm-1. In the H NMR spectra, signals of vinyl-type
hydrogens are observed at 6.2–6.5 ppm. The coupling
constants for (Z) diastereoisomers are 6–12 Hz. The car-
boxylic group hydrogens were observed as broad singlets
in the range 13.5–13.8 ppm. Only for compound 12, con-
taining two pyridyl rings, the carbonyl group hydrogen
123

Synthesis and biological activity of (Z)- and (E)-isomers of 3-(3,4-diaryl-1,2,4-triazole-5-yl)prop-2-enoic acid
441
Fig. 1 Molecular structure and
atom numbering scheme for two
symmetrically independent
molecules of 20. Displacement
ellipsoids are shown at the 50%
probability level, hydrogen
atoms are shown as spheres of
arbitrary size. Hydrogen
bonding is indicated by dashed
lines
reduced the viral CPE. Most potent antiviral activity was
exhibited by compound 20 with 50% of virus inhibition.
The compounds 11, 13, and 18 were less active with virus
inhibition above 30% (Table 1).
Table 1 The antiviral activity of compounds 11 and 13, and their (E)
isomers, 18 and 20, respectively (6 lg cm-3) in comparison with
ACV
HSV titre SD (log10 CCID50 cm-3
)
Incubation
24 h
48 h
Inhibition (%)
Virucidal activity
Virus control
2.7 0.4
4.0 0.5
0
ACV
11
0
0
0
0
0
The concentration of compounds used in this assay was
100 lg cm-3. After incubation of the viral suspension with
the compound for 1 h at 37ꢁC, the most potent virucidal
effect on HSV-1 was exerted by compound 14. Compounds
10 and 11 did not inactivate HSV-1 in direct contact
whereas the rest of the derivatives did reduce the titre of
HSV-1, but less effectively than compound 14 (Table 2).
2.7 0.4
2.5 0.4
2.5 0.3
2.0 0.5
33.2
37.5
37.5
50
13
18
20
Table 2 The virucidal activity of the compounds
Compound
Virus titre SD (log10 CCID50 cm-3
48 h 72 h 96 h
)
The immunomodulatory activity of the examined
derivatives
120 h
incubation incubation incubation incubation
The prepared compounds were bio-assayed for their
potency to stimulate or modulate mitogen (PHA with
LPS)-induced IFN-c and TNF production in human blood
leukocytes. The results indicated that compounds 8, 9, 11,
and 14 induced low but detectable amounts of TNF
(Table 3).
Control of virus 3.3 0.4 3.5 0.4 3.7 0.4 4.0 0.5
8
2.5 0.3 2.5 0.3 2.7 0.4 3.3 0.4
2.7 0.4 3.0 0.5 3.2 0.5 3.2 0.5
3.3 0.4 3.8 0.5 3.5 0.4 3.5 0.4
3.3 0.4 3.5 0.4 3.5 0.4 3.5 0.4
2.5 0.3 2.7 0.4 2.8 0.5 3.2 0.5
2.5 0.3 2.5 0.3 2.7 0.4 3.5 0.4
9
10
11
12
13
14
Moreover, compounds 9 and 12 slightly enhanced
mitogen-stimulated TNF production in human blood leu-
kocytes (Table 4).
0
0
2.0 0.5 3.0 0.5
None of the compounds induced IFN production (data
not shown) and compounds 8, 13, and 14 inhibited mito-
gen-stimulated level of IFN (Table 5).
On the other hand, overproduction of these cytokines may
lead to pathology [19] observed for example in autoim-
mune diseases (rheumatoid arthritis, lupus erythematosus,
insulin-dependent diabetes) or in sepsis, with fatal conse-
quences [20]. Thus, modulators of TNF and IFN-c
production have been sought for many years and it seems
that some of the compounds examined might be considered
as a candidates for such an agent. Further experiments in
this field are needed.
The production of inflammatory mediators by mono-
cytes and macrophages is a crucial step in the early
response to infections and tissue damage. TNF and IFN-c
are proinflammatory cytokines produced mainly by acti-
vated monocytes, macrophages, and Th1 lymphocytes and
play a key role in immunological response, which causes
rapid neutralization of factors dangerous to the organism.
123

442
B. Modzelewska-Banachiewicz et al.
Elemental analysis (C, H, N) was conducted in the
Department of Organic Chemistry, Medical University of
Lublin (Poland). Their results were in good agreement with
calculated values. All reactions were monitored by thin-
layer chromatography (TLC). Chromatography was per-
formed on 10 cm 9 10 cm TLC plates precoated with
silica gel RP-18 F254 and silica gel Si 60 F254 (Merck,
Darmstadt, Germany). The mobile phase for the normal-
phase system was acetone–toluene–acetic acid 8:2:1; for
the reversed-phase system the mixture methanol–water–
formic acid 5:5:0.5) was applied. Compounds were dis-
solved in methanol (5 mg cm-3) and 10-lL samples of
these solutions were spotted on the plates. After develop-
ment in horizontal Teflon DS chambers (Chromdes, Lublin,
Poland) and drying, the spots were visualized under UV
light at k = 254 nm. Chemicals were purchased from
Merck or Lancaster and used without further purification.
Table 3 Examined compounds as inducers of TNF production
Compound
TNF (pg cm-3
)
SD
Control (without induction)
0
8
84.6 107.7
30.9 27.8
\5
9
10
11
12
13
14
30.4 52.5
\5
\5
53.3 76.5
Table 4 Examined compounds as modulators of TNF production
Compound
TNF (pg cm-3
)
SD
Control (%)
Control (mitogen induced)
638 295
648 283
752 211
649 352
611 258
757 327
650 243
685 423
100
102
118
102
96
8
9
Synthesis of (E)-3-(3,4-diaryl-1,2,4-triazole-5-yl)prop-
2-enoic acids derivatives 15–21: general procedure
10
11
12
13
14
119
102
107
A mixture of 0.01 mol (Z)-3-(3,4-diaryl-1,2,4-triazole-5-
yl)prop-2-enoic acid 8–14 and 5 cm3 glacial acetic acid
was heated under reflux for 2.5 h (8–11, 13, 14) or 0.5 h
(12). Acetic acid was removed under reduced pressure. The
residue was washed with cold methanol and purified by
crystallization from methanol.
Compounds 8–14, 18, 20 (10 lg cm-3
(5 lg cm-3) ? PHA (10 lg cm-3
) with inducers: LPS
)
(2E)-3-[4-(4-methylphenyl)-5-pyridin-2-yl-4H-1,2,
4-triazol-3-yl]prop-2-enoic acid (15, C17H14N4O2)
Table 5 Examined compounds as modulators of IFN-c production
Compound
IFN-c (pg cm-3
)
SD Control (%)
ꢀ
Yield 2.45 g (80%); mp 266–268ꢁC; IR (KBr): m = 3,450
(OH), 2,971, 1,450 (CHal.), 1,700 (C=O), 1,191 (C–O)
Control (mitogen induced) 1,614 426
100
78
82
95
89
90
73
76
1
cm-1; H NMR (DMSO-d6): d = 2.5 (s, CH3), 6.6, 6.7
(d, J = 15.9 Hz, 2HC2=), 7.0–7.5 (m, 8Ar-H), 15.00
(s, COOH).
8
1,259 597
1,329 376
1,529 648
1,442 714
1,450 766
1,186 243
1,226 202
9
10
11
12
13
14
(2E)-3-(4-phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-
yl)prop-2-enoic acid (16, C16H12N4O2)
ꢀ
Yield 2.05 g (70%); mp 240–242ꢁC; IR (KBr): m = 3,433
(OH), 1,705 (C=O), 1,199 (C–O) cm-1; 1H NMR (DMSO-
d6): d = 6.7, 6.8 (d, J = 15.9, 2HC2=); 7.2–8.5 (m, 9Ar-H);
13.8 (s, COOH).
Control of inducers: LPS (5 lg cm-3) ? PHA (10 lg cm-3); com-
with inducers: LPS
pounds 8–14, 18, 20 (10 lg cm-3
)
(5 lg cm-3) ? PHA (10 lg cm-3
)
(2E)-3-[4-(4-methylphenyl)-5-pyridin-4-yl-4H-1,2,4-
triazol-3-yl]prop-2-enoic acid (17, C17H14N4O2)
Experimental
ꢀ
Yield 2.08 g (68%); mp 243–245ꢁC; IR (KBr): m = 3,430
(OH), 2,970, 1,450 (CHal.), 1,705 (C=O), 1,190 (C–O)
Chemistry
1
cm-1; H NMR (DMSO-d6): d = 2.5 (s, CH3), 6.7, 6.9
(d, J = 15.9 Hz, 2HC2=), 7.1–8.2 (m, 8Ar-H), 12.9
(s, COOH).
Melting points were measured on a Boetius apparatus.
1H NMR spectra were recorded on a Tesla BS 567A
(300 MHz) apparatus in DMSO-d6 with TMS as an
external standard. Chemical shifts are given in ppm
(d-scale), coupling constants in Hz. IR spectra (v, cm-1
were recorded in KBr using a Specord IR-75 spectrometer.
(2E)-3-(4-phenyl-5-pyridin-2-yl-4H-1,2,4-triazol-3-
yl)prop-2-enoic acid (18, C16H12N4O2)
)
Yield 2.45 g (72%); m.p. 268–270ꢁC; IR (KBr):
-1
ꢀ
m = 3,468 (OH), 1,698 (C=O), 1,190 (C–O) cm .
123

Synthesis and biological activity of (Z)- and (E)-isomers of 3-(3,4-diaryl-1,2,4-triazole-5-yl)prop-2-enoic acid
443
1H NMR (DMSO-d6): d = 6.6, 6.9 (d, J = 15.8 Hz,
2HC2=), 7.3–8.3 (m, 9Ar-H), 12.9 (s, COOH).
d = 6.5, 6.9 (d, J = 15.6, 2H (C2=), 7.3–8.4 (m, 8Ar-H),
13.1 (s, COOH).
(2E)-3-(4,5-dipyridin-2-yl-4H-1,2,4-triazol-3-yl)
prop-2-enoic acid (19, C15H11N5O2)
Pharmacology
ꢀ
Yield 1.9 g (65%); mp 168–170;8C; IR (KBr): m = 3,420
Compounds
(OH), 1,708 (C=O), 1,180 (C–O) cm-1; 1H NMR (DMSO-
d6): d = 6.4, 6.6 (d, J = 15.8 Hz, 2HC2=), 6.9–7.9
(m, 8Ar-H), 13.2 (s, COOH).
Compounds 8–21 were dissolved in DMSO at a concen-
tration of 1,000 lg cm-3 as a stock solution. Just before
experiments compounds were diluted with 2% minimal
essential medium (MEM).
(2E)-3-(4,5-diphenyl-4H-1,2,4-triazol-3-yl)
prop-2-enoic acid (20, C17H13N3O2)
ꢀ
Yield 2.1 g (72%), mp 262–264ꢁC. IR (KBr): m = 3,434
The cells and viruses
(OH); 1,702 (C=O); 1,191 (C–O) cm-1; 1H NMR (DMSO-
d6): d = 6.6, 6.9 (d, J = 15.9 2H C2=), 7.3–7.6 (m, 10Ar-H),
12.90 (s, COOH).
HSF were obtained by a routine method of trypsynization
of an adult human skin fragment and cultured in MEM
(Sigma Chemical, St Louis, MO, USA) supplemented with
10% FCS (Life Technologies, Karlsruhe, Germany),
100 U cm-3 penicillin, and 100 lg cm-3 streptomycin.
HeLa-human cells of cervical carcinoma were cultured in
MEM supplemented with 5% FCS (Gibco, BRL),
100 U cm-3 penicillin, and 100 lg cm-3 streptomycin.
HSV-1 McIntyre strain was obtained from the National
Institute of Health (Warsaw, Poland). Virus stock was
Crystal data for 20: monoclinic space group P21/n, unit-
˚
cell parameters a = 10.498(2) A, b = 18.977(4) A,
˚
3
˚
˚
c = 14.997(3) A, b = 100.17(2)ꢁ, V = 2,940.8(10) A ;
Z = 8, dcalc = 1.316 g cm-3, l = 0.725 mm-1
.
Diffraction data for 20 were measured at 293(2) K on a
KM4 diffractometer using variable scan speed (x - 2h
scan mode) and graphite-monochromatized CuKa radiation
˚
single crystal of dimensions
0.68 9 0.35 9 0.06 mm3 was used. Reflections were col-
lected up to hmax = 80.21ꢁ; 6,480 reflections were
measured. Crystal structure was solved by direct methods
using the SHELXS97 program [21] and refined by the full-
matrix least squares on F2 using the SHELXL97 program
[22].
prepared from infected HeLa cells. The titre of the virus in
HeLa cells was 104.71
(k = 1.54,178 A).
A
0.3
CCID50 cm-3
.
The blood donors
The heparinized blood samples were obtained from vol-
unteers aged 21–23 (two men and eight women). None of
the volunteers reported any history of acute or chronic
medical problems.
Non-hydrogen atoms of the phenyl substituents were
refined with isotropic displacement parameters. The
remaining non-hydrogen atoms were refined anisotropi-
cally. Hydrogen atoms of the carboxyl groups were located
in a difference map and their positional parameters were
not refined. All the other hydrogen atoms were positioned
geometrically and were given isotropic factors of 1.2 Ueq of
the bonded C/O atoms; the ‘‘riding’’ model was used in the
refinement. Final discrepancy factors are R1 = 0.1068,
wR2 = 0.2049, and S = 0.93, for 1,299 reflections with
I [ 2r(I).
Crystallographic data for the structure reported in this
paper have been deposited with the Cambridge Crystallo-
graphic Data Centre, CCDC No. 271179. Copies of the
data may be obtained on application to The Director,
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax:
?44-1223-336-033; e-mail: deposit@ccdc.cam.ac.uk or
www: http://www.ccdc.cam.ac.uk).
Cytopathic effect reduction assay
The compounds were examined for their antiviral activity in
the CPE reduction assay. Human skin fibroblasts (HSF) were
seeded into 96-well plates at a density of 1 9 105 cells cm-3
in MEM supplemented with 10% foetal calf serum (FCS)and
grown at 37ꢁC for 24 h. The cells were covered with HSV-1
suspension at a multiplicity of infection (MOI) of 0.01 and
with non-toxic concentrations of triazole derivatives
(6 lg cm-3) diluted in MEM supplemented with 2% FCS.
As a control ACV (6 lg cm-3) was used. The cells were then
incubated for 24 h and 48 h at 37ꢁC in 5% CO2 atmosphere.
After incubation, the virus CPE was read under the light
microscope and virus titre—log10 Cell Culture Infecting
Dose (CCID50) cm-3—was calculated.
The virucidal activity of the compounds
The virucidal activity of the compounds was estimated by
incubation of undiluted stock virus samples with equal
volumes of the compounds used at a concentration of
100 lg cm-3. After 1 h of incubation at 37ꢁC, the titre of
the virus (log10 CCID50 cm-3) was estimated in HSF cells.
(2E)-3-[4-(4-nitrophenyl)-5-pyridin-2-yl-4H-1,2,4-triazol-
3-yl]prop-2-enoic acid (21, C16H11N5O4)
Yield 2.36 g (70%); mp 266–268ꢁC; IR (KBr):ꢀm = 3,419
(OH), 1,710(C=O), 1,182(C–O)cm-1; 1H NMR(DMSO-d6):
123

444
B. Modzelewska-Banachiewicz et al.
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The influence of the compounds on cytokine production
The ability of compounds to induce IFN-c and TNF pro-
duction was also tested. Heparinized blood obtained from
healthy donors was diluted in MEM (supplemented with
antibiotics) to obtain 1 9 106 leukocytes cm-3
. The
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examined derivatives were added to diluted blood at
10 lg cm-3 concentration, samples of blood from donors
were incubated for 24 h at 37ꢁC in 5% CO2 atmosphere.
After centrifugation, the harvested supernatants were ana-
lysed for cytokines by enzyme-linked immunosorbent
assay (ELISA) techniques with commercially available kits
and according to the manufacturer’s instructions. The IFN-
c kit was obtained from Endogen (Woburn, MA, USA) and
the TNF kit was purchased from Bender MedSystem
(Vienna, Austria). The lower limits of detection for the
individual assays were IFN-c 2 pg cm-3 and TNF
5 pg cm-3. To estimate the ability of the compounds to
modulate IFN-c and TNF production, a mixture of mito-
gens (LPS at a concentration of 5 lg cm-3 and PHA at a
concentration of 10 lg cm-3) was used. The mixture of
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