Synthesis of 2-benzylthiopyridine-4-carbothioamide derivatives and their antimycobacterial, antifungal and photosynthesis-inhibiting activity

Article abstract of DOI:10.1016/S0223-5234(99)80093-2

A series of 2-benzylthiopyridine-4-carbonitriles 4 and a series of 2- benzylthiopyridine-4-carbothioamides 5 were prepared. Their chemical structures were proved by IR and ¹H NMR data and by elemental analysis. The MIC assessment was used for the estimation of their potential antimycobacterial and antifungal activity in vitro. The compounds were shown to be more active against mycobacterial strains than the tested fungi. Their antimycobacterial activity appears to be related mainly to the benzylthio moiety, while the antifungal activity appears to be related to the carbothioamide group. Substituents on the benzylthio moiety of derivatives 5 modified antifungal activity, whereas they did not play any significant role in antimycobacterial activity. The inhibiting effect of 4 and 5 on the photochemical activity of spinach chloroplasts was determined by IC₅₀ values. The compounds exhibited a moderate photosynthesis-inhibiting activity.

Full text of DOI:10.1016/S0223-5234(99)80093-2

Eur. J. Med. Chem. 34 (1999) 433−440
Elsevier, Paris
433
©
Short communication
Synthesis of 2-benzylthiopyridine-4-carbothioamide derivatives
and their antimycobacterial, antifungal and photosynthesis-inhibiting activity
a
a
a
b
V eˇ ra Klime sˇ ová *, Martin Svoboda , Karel Waisser , Jarmila Kaustová ,
c
d
Vladimír Buchta , Katarina Krá ul ová
a
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, 500 05 Hradec Králové, Czech Republic
b
National Reference Laboratory for Mycobacterium kansasii, Regional Institute of Hygiene, 728 92 Ostrava, Czech Republic
c
Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, 500 05 Hradec Králové, Czech Republic
d
Institute of Chemistry, Comenius University, 84215 Bratislava, Slovak Republic
(Received 26 February 1998; revised 28 October 1998; accepted 29 October 1998)
Abstract – A series of 2-benzylthiopyridine-4-carbonitriles 4 and a series of 2-benzylthiopyridine-4-carbothioamides 5 were prepared. Their
1
chemical structures were proved by IR and H NMR data and by elemental analysis. The MIC assessment was used for the estimation of their
potential antimycobacterial and antifungal activity in vitro. The compounds were shown to be more active against mycobacterial strains than
the tested fungi. Their antimycobacterial activity appears to be related mainly to the benzylthio moiety, while the antifungal activity appears
to be related to the carbothioamide group. Substituents on the benzylthio moiety of derivatives 5 modified antifungal activity, whereas they
did not play any significant role in antimycobacterial activity. The inhibiting effect of 4 and 5 on the photochemical activity of spinach
chloroplasts was determined by IC50 values. The compounds exhibited a moderate photosynthesis-inhibiting activity. © Elsevier, Paris
2
-benzylthiopyridine-4-carbonitriles / 2-benzylthiopyridine-4-carbothioamides / antifungal activity / antimycobacterial activity /
photosynthesis inhibition / structure-activity relationship
1
. Introduction
antifungal activity. This fact prompted us to systemati-
cally investigate both biological activities.
Increase in the occurrence of mycobacterial infections
In our previous work, a significant effect in compounds
bearing an alkylthio group (alkyl C -C ) on the pyridine
and the developing resistance to conventional antimyco-
bacterial agents [1] is a stimulating factor in the research
of new, more selective compounds. Of the mycobacterial
infections caused by atypical mycobacteria (non-
tuberculosis mycobacteria), the greatest problem has
become the Mycobacterium avium complex. Dissemi-
nated infection with the M. avium complex is the most
common systematic bacterial infection complicating
AIDS [2]. In the Czech Republic an increase in the
occurrence of M. kansasii has been observed in some
localities [3]. With regard to the above mentioned facts,
the compounds were tested against the strains Mycobac-
terium tuberculosis, M. avium, and M. kansasii.
1
16
ring was observed. Especially, an interesting level of
antimycobacterial and antifungal activities were demon-
strated in a group of 2-alkylthio, or 4-alkylthio pyridine-
carbothioamides [4–7]. The above activities showed good
correlation with photosynthesis-inhibiting activity of the
compounds in Chlorella vulgaris and spinach chloro-
plasts [8, 9]. Using electro-paramagnetic resonance
(
2
EPR)
spectroscopy
it
was
confirmed
that
-alkylthiopyridine-4-carbothioamides inhibit photosyn-
thetic electron transport in photosythesizing organisms
and they interact with the intermediates Z+ and D+, i.e.
with tyrosine radicals which are situated in D and D
1
2
During the studies on new potent antimycobacterial
agents we found that some compounds also showed
proteins on the donor site of photosystem 2 [8, 9].
Quantitative relationships between the structure and
antimycobacterial activity of 2-alkylthio derivatives
showed that the efficacy of a benzylthio derivative is
*
Correspondence and reprints

4
34
Figure 1. Synthetic pathways for obtaining the title compounds.
The structures of compounds 4a–p and 5a–p were
confirmed by elemental analyses and by IR and H-NMR
spectral data. The IR and NMR spectra are in agreement
1
with the proposed structures. All compounds 4 showed a
–1
strong band at 2 240 cm in their IR spectra, which is
characteristic of the C≡N group, and compounds 5
showed characteristic bands of amides (3 260–3 300 and
–1
1
1 640 cm ). The H NMR spectra of all compounds
show a signal at position 6 of the pyridine moiety, which
can be observed as doublets of doublets at 8.36–8.66 ppm
with a coupling constant J5,6 = 5.0–5.6 Hz, and the singlet
of the SCH moiety at 4.38–4.83 ppm. Methyl groups in
2
the 3 or 4 positions of the phenyl rings give a singlet at
Synthetic pathways to obtain the title compounds are
2
.27–2.46 ppm, the methoxy group in the 4 position of
illustrated in figure 1. Thiuronium salt 2 was used as the
key starting material and was prepared from commer-
cially available pyridine-4-carboxamide (Sigma-Aldrich)
in the first step according to Lieberman [11]. Halogen
derivative 1 was transformed into thiuronium salt 2 by a
reaction with thiourea. Crude product 2 was used for
further reactions. Condensation of 2 with the appropriate
benzylchlorides 3 in N, N-dimethylformamide produced
the corresponding benzyl derivatives 4, which were
further converted, by addition of hydrogen sulfide in
pyridine solution, into the related pyridine carbothioam-
ides 5 in good yields. Only compounds 5l and 5o, having
the nitro group in the benzyl moiety, were obtained by
reacting 6 with the nitrobenzyl chlorides. Compound 6
was accomplished by reacting thiuronium salt 2 with
hydrogen sulfide in pyridine solution.
1
the phenyl ring gives a singlet at 3.72–3.78 ppm. The H
NMR spectra of the pyridinecarothioamides 5 also con-
tain two broad singlets at 9.73–9.94 ppm and
1
0.17–10.22 ppm due to the amide groups. The other
protons of the pyridine and phenyl moiety form a
multiplet (overlapping signals) in the region 6.86–7.89
ppm. Chemical and physical data of compounds 4 and 5
are described in tables I and II.
3. Biological investigation
The antimycobacterial activity of the prepared com-
pounds was tested in vitro against Mycobacterium tuber-
culosis CNCTC My 331/88, Mycobacterium kansasii
CNCTC My 235/80, Mycobacterium kansasii 509/96

4
35
Table I. Chemical and physical data of the compounds 4a–p.
a
1
Compound
R
Formula
M.W.
Yield
(%)
Mp
RF
H NMR (δ ppm)
b
c
(°C)
TLC
H-6
–CH2–
(s)
–CH3
(s)
(dd)
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
H
C H N S
75
69
22
69
61
54
60
60
64
89
45
85
80
75
95
72
69–71 [ ]
118.5–120.5
82.5–85
98.5–101
63–64.5
48–50
0.60
0.59
0.60
0.63
0.59
0.58
0.59
0.58
0.58
0.61
0.65
0.57
0.59
0.57
0.64
0.54
8.66
8.61
8.59
8.58
8.57
8.57
8.58
8.57
8.56
8.58
8.58
8.58
8.51
8.50
8.50
8.36
4.46
4.58
4.42
4.40
4.49
4.42
4.42
4.40
4.38
4.40
4.40
4.52
4.83
4.70
4.73
4.40
13 10 2
2
26.3
2-Cl
C H ClN S
13
9
2
2
60.7
3-Cl
C H ClN S
13
9
2
2
60.7
4-Cl
C H ClN S
13
9
2
2
60.7
2-F
C H FN S
13
9
2
2
44.3
3-F
C H FN S
13
9
2
2
44.3
g
h
i
4-F
C H FN S
65–67
13
9
2
2
44.3
3-Br
C H BrN S
84–87
13
9
2
3
05.2
4-Br
C H BrN S
108.5–111.5
34–38
13
9
2
3
05.2
j
3-CH3
4-CH3
4-NO2
2,4-NO2
2-F-6-Cl
C H N S
2.32
2.32
14
12 2
2
40.3
k
l
C H N S
95–97
14
12 2
2
40.3
C H N O S
114–116
137–139
86.5–88.5
133–136
85–86.5
13
9 3 2
2
71.3
m
n
o
p
C H N O S
13
8 4 4
3
16.3
C H ClFN S
13
8
2
2
78.7
2-F-6-NO2 C H FN O S
13
8
3 2
2
89.3
d
4-OCH3
C H N OS
3.78
14
12 2
2
56.3
a
b
c
d
Yields are of the crude products, ethylacetate/petrolether 2:3; 1H J(5,6) = 5.0–5.6 Hz (H-6, pyridine), OCH3.
(
clinical isolate) and Mycobacterium avium CNCTC My
30/88 using the micromethod for the determination of
IC50 values, i.e. molar concentrations of the studied
compounds causing a 50% decrease in the oxygen evalu-
ation rate in spinach chloroplasts.
3
the minimum inhibitory concentration. (CNCTC–Czech
National Collection of Type Cultures).
The antifungal activity in vitro was found against the
dermatophyte Trichophyton mentagrophytes 445, yeasts
Candida albicans ATCC 44859, Candida tropicalis 156,
Candida krusei E28, Candida glabrata 20/I, Trichos-
poron beigelii 1188, hyphomycete Aspergillus fumigatus
4. Results and discussion
The results of in vitro screening of 4 and 5 are
summarised in tables III and IV. In several cases the
minimum inhibitory concentration could not be deter-
mined due to limited solubility of the compounds in the
tested medium. Compounds 4a–p are inactive in the used
concentration range against all tested fungi strains except
Trichophyton mentagrophytes. To facilitate the biological
activity comparison, the relative activity (R.A.) of studied
231, and zygomycete Absidia corymbifera 272 using a
microdilution broth test. All strains, except C. albicans,
were clinical isolates, identified by conventional morpho-
logical and biochemical methods.
Photosynthesis-inhibiting activity was tested in a sus-
pension of spinach chloroplasts. It was expressed by the

4
36
Table II. Chemical and physical data of the compounds 5a–p.
a
1
Compound
R
Formula
Yield
%)
Mp (°C)
RF
H NMR (δ ppm)
b
c
(
TLC
-NH (bs)
H-6
–CH2–
(s)
–CH3
(s)
2
M.W.
(dd)
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
H
C H N S
3 12 2 2
87
77
52
68
73
55
64
70
76
97
87
78
89
84
144–145 [ ]
204.5–207
139–141
0.38
0.41
0.42
0.43
0.43
0.43
0.43
0.41
0.38
0.39
0.40
0.56
0.38
0.35
10.21, 9.77
10.19, 9.80
10.20, 9.94
10.21, 9.76
10.19, 9.76
10.22, 9.77
10.21, 9.77
10.80, 9.73
10.21, 9.76
10.20, 9.74
10.18, 9.74
10.22, 9.77
10.21, 9.74
10.18, 9.73
8.52
4.46
4.52
4.47
4.46
4.49
4.49
4.46
4.46
4.45
4.42
4.42
4.61
4.47
4.74
1
260.4
2-Cl
3-Cl
4-Cl
2-F
C H ClN S
8.54
8.53
8.52
8.54
8.54
8.53
8.54
8.53
8.52
8.56
8.52
8.45
8.40
1
3
11
2
2
2
2
294.8
C H ClN S
1
3
11
2
294.8
C H ClN S
97.5–100
1
3
11
2
294.8
C H FN S
189.5–192
123.5–125.5
107.5–109.5
149–152
1
3
11
2
2
2
2
278.4
3-F
C H FN S
1
3
11
2
278.4
g
h
i
4-F
C H FN S
1
3
11
2
278.4
3-Br
4-Br
3-CH3
4-CH3
4-NO2
C H BrN S
1
3
11
2
2
2
338.0
C H BrN S
104–106.5
115–118
1
3
11
2
2
2
3
38.0
j
C H N S
2.27
2.46
14
14 2
2
74.4
k
l
C H N S
119.5–122.5
166–169
14
14 2
2
74.4
C H N O S
11 3 2 2
13
3
05.4
2-F-6-Cl C H ClFN S
2 2
n
o
219–222.5
152–155
1
3
10
312.8
2-F-6-
NO2
C H FN O S
1
3
10
3
2
2
3
23.4
d
5p
4-OCH3 C H N OS
90
157–159.5
0.41
10.17, 9.74
8.52
4.39
3.72
14
14 2
2
290.4
a
b
c
d
Yields are of the crude products, ethylacetate/petrolether 2:3; 1H, J(5,6) = 5.0–5.6 Hz (H-6, pyridine); OCH3.
compounds R.A. = MICcompound /MICketoconazole or R.A.
The studied compounds 4 and 5 proved to be more
active against mycobacterial species than fungi strains.
As far as the antimycobacterial activity is concerned,
several points are to be considered:
=
MIC compound/MICisoniazide is reported in table V. The
photosynthesis-inhibiting activity of 4 and 5, expressed
as IC50 values is presented in table VI.
–1
Table III. Antimycobacterial and antifungal activity of derivatives 4 as minimum inhibitory concentrations (MIC -µmol.L ).
a
b
Strains Compounds
KET
INH
4
a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
a
b
c
d
e
125
62
62
62
125
–
–
16
–
–
–
125
62
62
125
62
16
31
250
62
62
31
16
250
–
–
125
31
31
62
250
–
–
62
31
–
–
62
8
–
–
16
–
–
–
16
–
–
–
–
–
–
4
500
8
500
–
31
16
–
62
16
31
31
16
16
125
31
16
–
62
16
–
31
16
31
31
–
> 750 62.5
> 500 125
250
> 1000 > 1000 > 1000 > 500 > 500 250
0.98
Strains, a: Mycobacterium tuberculosis My 331/ 88; b: Mycobacterium kansasii My 235/ 80; c: Mycobacterium kansasii 509/96 (clinical
a
b
isolate); d: Mycobacterium avium My 330/88; e: Trichophyton mentagrophytes 445. Ketoconazole; Isoniazide.

4
37
–1
Table IV. Antimycobacterial and antifungal activity of derivatives 5 as minimum inhibitory concentrations (MIC -µmol.L ).
Strains Compounds
KET a INH b
5
a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5n
5o
5p
a
b
c
d
e
f
g
h
i
16
16
31
62
31
16
16
–
16
31
31
31
31
16
16
16
16
31
31
62
31
31
62
31
31
62
62
31
31
31
16
31
31
16
16
31
31
31
62
31
62
31
31
31
16
62
31
31
–
–
62
–
250
31
31
31
62
31
31
31
62
–
–
–
–
4
500
8
500
–
–
–
–
–
–
60
500
31.25 15.62 > 500 31.25 31.25 15.62 15.62 31.25 31.25 500
62.5
31.25 0.98
125
250
120
120
120
120
120
> 1000 62.5
> 1000 125
> 1000 125
> 1000 125
> 1000 125
62.5
125
500
125
125
250
> 500 62.5
> 500 62.5
> 500 62.5
> 500 62.5
> 500 > 1000 125
> 500 > 1000 125
62.5
125
62.5
62.5
31.25 62.5
62.5
31.25 > 500 > 500 250
< 0.24
1.95
0.98
0.49
< 0.24
7.81
31.25
62.5
62.5
62.5
62.5
62.5
62.5
62.5
62.5
> 500 125
> 500 125
> 500 125
> 500 125
> 500 > 500 500
> 500 > 500 > 500 500
> 500 > 500 500 250
> 500 > 500 > 500 500
> 500 > 500 250
> 500 > 500 125
j
k
l
> 500 > 1000 62.5
250
> 500 62.5
31.25 15.62 62.5
125
250
62.5
125
62.5
–
–
> 1000 31.25 31.25 > 500 > 1000 62.5
Strains: a: Mycobacterium tuberculosis My 331/88; b: Mycobacterium kansasii My 235/80; c: Mycobacterium kansasii 509/96 (clinical
isolate); d: Mycobacterium avium My 330/88; e: Trichophyton mentagrophytes 445; f: Candida albicans 44859; g: Candida tropicalis 156; ha:
Candida krusei E28; i: Candida glabrata 20/I; j: Trichosporon beigelii 1188; k: Aspergillus fumigatus 231; l: Absidia corymbifera 272.
b
Ketoconazole; Isoniazide.
1
) All tested compounds are active against all myco-
electron-deficient carbon is the pharmacophore of anti-
mycobacterial activity. An introduction of other substitu-
ents into the benzylthio moiety does not markedly affect
the antimycobacterial activity. None of the substituents
increases the activity. The activity of prepared com-
pounds exhibited comparable efficacy against atypical
strains as against M. tuberculosis.
bacterial species. The values of MICs were within a range
–1
–1
of 8–125 µmol.L , mainly between 16–31 µmol.L .
) Derivatives 4 exhibit a higher activity against
2
atypical strains than against M. tuberculosis. The stron-
gest biological efficacy among all tested compounds was
observed in 2,4-dinitrobenzyl substituted derivatives
(
8
4m) against M. kansasii (clinical isolate), with MIC
By comparing MIC values of prepared compounds
with isoniazide as the standard, the new compounds
possess a much better activity against M. avium than
isoniazide. The compound 4m reached the activity of
isoniazide against the M. kansasii clinical isolate.
–1
µmol.L reaching the activity of isoniazide.
) Derivatives 5 show practically the same activity
3
against all tested mycobacteria.
On the basis of these considerations, it appears that
their antimycobacterial activity is related to the benzyl-
thio moiety. This is in agreement with our previous
hypothesis [12] that the alkylthio group bound to the
As far as antifungal activity is concerned, the following
conclusions can be drawn:
Table V. Comparison of activities of compounds 5 with Isoniazide or Ketoconazole.
Strains
5c
5d
5f
5g
5h
5i
5k
5p
R.A.
R.A.
R.A.
R.A.
R.A.
R.A.
R.A.
R.A.
a
b
c
d
e
g
h
k
l
4
0.06
4
0.06
32
64
128
8
8
0.03
2
0.03
16
64
510
32
1
8
0.06
8
0.06
32
32
64
–
8
0.12
8
0.06
32
64
64
16
2
8
0.06
2
0.06
16
32
64
–
8
0.03
2
0.06
16
32
64
8
16
0.06
4
0.06
32
64
128
32
2
8
0.06
4
0.12
32
128
510
16
1
–
1
0.5
2
R.A. = MICcompound/ MICIsoniazide or Ketoconazole. Strains, a: Mycobacterium tuberculosis My 331/ 88; b: Mycobacterium kansasii My 235/ 80;
c: Mycobacterium kansasii 509/96 (clinical isolate); d: Mycobacterium avium My 330/ 88; e: Trichophyton mentagrophytes 445; g: Candida
tropicalis 156; h: Candida krusei E28; k: Aspergillus fumigatus 231; l: Absidia corymbifera 272.

4
38
–
1
Table VI. Inhibition of oxygen evolution rate in spinach chloro-
and within a range of 108–294 µmol.L for 5. The most
active compounds 5h and 5i, i.e. 3-Br, 4-Br derivatives,
are in conformity with the most active antifungal agents
in the investigated sets.
–
1
plasts. (IC5 -µmol.L )
0
Compound
IC50
Compound
IC50
4
4
4
4
4
4
4
4
4
4
4
a
b
c
h
j
k
l
m
n
o
p
452
189
160
235
188
302
162
209
173
267
511
5a
5b
5c
5d
5e
5f
5g
5h
5i
274
167
143
146
197
152
235
108
115
182
251
220
118
112
294
The tested compounds show only a weak inhibiting
activity of photosynthetic electron transport in spinach
chloroplasts and they did not reach the activity of
2-alkylthiopyridine-4-carbo-
thioamides. The IC50 value of the most active octyl
derivative of 2-alkylthiopyridine-4-carbothioamides was
2 µmol.L [8]. Comparable IC50 values determined for
very effective herbicides are about 0.1 µmol.L for
Bromoxynil, 1 µmol.L for Atrazine and Dinoseb [13].
previously
studied
–1
7
–1
5j
5k
–1
5
5
5
5
l
n
o
p
5. Experimental protocols
5.1. Chemistry
The melting points were determined on a Kofler block
apparatus and are uncorrected. IR spectra were obtained
on a Nicolet Impact 400 spectrometer in KBr pellets.
H-NMR spectra were recorded on a spectrometer BS
87 Tesla (80 MHz) using tetramethylsilane as the
1
) Pyridinecarbonitriles 4 showed a weak activity only
against Trichophyton mentagrophytes while they were
practically inactive against other strains. The most active
compound was 4c.
1
5
2
) An introduction of a carbothioamide group on the
internal standard and CDCl for compounds 4 and DMSO
pyridine ring strongly extended the spectrum of anti-
fungal activity.
3
for compounds 5 as the solvent. Analyses indicated by
elemental symbols were within ± 0.4% of the theoretical
values and were performed on a CHNS-O instrument
FISONS EA 1110. Silufol aluminium oxide plates with a
fluorescent indicator were used for thin-layer chromatog-
raphy to check the purity of all compounds.
3
) The antifungal activity of pyridinecarbothioamides
5
depends on the substituents on the benzylthio moiety. A
bromine, fluorine, chlorine atom and a methyl group in
the meta or para position on the phenyl ring increases the
biological activity as compared to unsubstituted benzyl-
thioderivate 5a. On the other hand, 2-Cl, 2-F, 4-NO and
2
5
.1.1. General procedure for preparation of compounds
2
-F-6-Cl rapidly decreased the activity.
From the observations of biological activity, the
4a–p
structure-activity relationships can be derived: the thioa-
mide groups must be regarded as fundamental to anti-
fungal activity. This is in agreement with our previous
observations [4, 5]. The activity is modified by substi-
tuents on the benzylthio moiety and by their position on
the phenyl ring. An increase in the effect is achieved by
substituting a halogen atom in the para or meta position,
while substitution in the ortho position markedly de-
creases antifungal activity. A substituent in position 2
probably causes steric hindrance of free rotation of the
benzylthio moeity. By comparing the MIC values of
prepared compounds with Ketoconazole as the standard,
it can be seen that their activity against Absidia corymb-
ifera exceeds that of Ketoconazole.
To a stirred solution of 2 (7 mmol) in dry DMF (8 mL)
a solution of sodium (8 mmol) in dry methanol (2.5 mL)
was added at room temperature, followed by the appro-
priate benzyl derivative 3 (7 mmol). After stirring at room
temperature for 2–4 h, the solvent was evaporated in
vacuo and the residue was diluted with water (75 mL).
The precipitated solid was filtered off, washed with cold
water and crystallized from ethanol (table I).
5.1.2. 2-Mercaptopyridine-4-carbothioamide 6
Dry hydrogen sulfide was passed into a solution of 2
(10 mmol) in a mixture of dry pyridine (18 mL) and
triethylamine (1.8 mL) at 70 °C for 3 h. After cooling, the
mixture was diluted with water (75 mL), the solution was
acidified with 1 M H SO to pH 6, the formed precipitate
was filtered off and crystallized from water to give 6
(1.05 g, 88%) as yellow crystals m.p. 232–235 °C
(232–235 °C [14]).
As far as the photosynthesis-inhibiting activity is
concerned, it can be seen that pyridincarbothioamides 5
were more active than pyridincarbonitriles 4. The IC
2
4
50
–1
values vary within a range of 160–511 µmol.L for 4,

4
39
5
5
.1.3. General procedure for preparation of compounds
a–k, n and p
Trichophyton mentagrophytes the MICs were recorded
after 72 h incubation.
An appropriate 2-benzylthiopyridine-4-carbonitrile 4
1 mmol) was dissolved in dry pyridine (6 mL), then dry
triethylamine (0.6 mL) was added and dry hydrogen
sulphide was passed into the mixture at 50 °C for 3–5 h.
After cooling, the mixture was diluted with water
75 mL) The precipitated solid was filtered off and
crystallized from ethanol (table II).
5
.2.3. Photosynthesis-inhibiting activity
The oxygen evolution rate in spinach chloroplasts was
(
investigated spectrophotometrically (Specord UV-VIS,
Zeiss Jena, Germany) in the presence of the electron
acceptor 2,6-dichlorophenolindophenol according to the
method described [16]. Due to low water solubility the
studied compounds were dissolved in dimethyl sulfoxide
(
(
DMSO) so that the applied DMSO concentration (up to
% v/v) did not affect oxygen evolution. The inhibitory
.1.4. General procedure for preparation of compounds
5
efficiency of the studied compounds was expressed by
IC50 values.
To a stirred solution of 6 (9 mmol) in dry DMF (8 mL)
was added, followed by a nitrobenzyl derivative 6
(
9 mmol). After stirring at room temperature for 3–5 h,
Acknowledgements
the solvent was evaporated in vacuo and the residue was
diluted with water (100 mL). The precipitated solid was
filtered off and crystallized from ethanol (table II).
This work was supported by Grant 203/96/0262 of the
Grant Agency of the Czech Republic and by the Grant
No.1/4013/97 from the Scientific Grant Agency of the
Ministry of Education, Slovak Republic. We are indebted
to Mrs A. Kozmova from the Institute of Chemistry,
Comenius University, Bratislava, Slovak Republic, for
obtaining the NMR spectra. We also thank Mrs D.
Karlí cˇ ková (Department of Pharmaceutical Chemistry
and Drug Control, Faculty of Pharmacy, Charles Univer-
sity) for performing elemental analyses and Mrs J Z{ i ˇz k-
ová (Department of Inorganic and Organic Chemistry,
Faculty of Pharmacy, Charles University) for recording
the IR spectra.
5
5
.2. Microbiological methods
.2.1. Antimycobacterial activity
Antimycobacterial activity of the compounds was de-
ˇ
termined in Sula semisynthetic medium (SEVAC, Pra-
gue). The compounds were added to the medium as
dimethyl sulfoxide solutions. The following concentra-
tions were used: 1 000, 500, 250, 125, 62, 31, 16, 8, and
–1
4
µmol.L . The minimum inhibitory concentrations were
determined after incubation at 37 °C for 14 d. MIC was
the lowest concentration of a substance at which the
inhibition of the growth of mycobacteria occurred.
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5.2.2. Antifungal activity
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1
mmol.L , provided the given compound was soluble in
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4
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[
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[15] National Committee for Clinical Laboratory Standards: Reference
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1
8 (1995) 31–34.
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ˇ
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ˇ
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2
3