Substituted amides of pyrazine-2-carboxylic acids: Synthesis and biological activity

Article abstract of DOI:10.3390/70300363

Condensation of 6-chloro-, 5-tert-butyl- or 6-chloro-5-tert-butylpyrazine- 2-carboxylic acid chloride with ring substituted anilines yielded a series of amides, which were tested for their in vitro antimycobacterial, antifungal and photosynthesis-inhibiting activities. The highest antituberculotic activity (72% inhibition) against Mycobacterium tuberculosis and the highest lipophilicity (log P = 6.85) were shown by the 3,5-bis-trifluoromethylphenyl amide of 5-tert-butyl-6-chloropyrazine-2-carboxylic acid (2o). The 3-methylphenyl amides of 6-chloro- and 5-tert-butyl-6-chloro-pyrazine-2-carboxylic acid (2d and 2f) exhibited only a poor in vitro (antifungal effect (MIC = 31.25-500 μmol·dm^-3) against all strains tested, although the latter was the most active antialgal compound (IC₅₀ = 0.063 mmol·dm^-3). The most active inhibitor of oxygen evolution rate in spinach chloroplasts was the (3,5-bis-trifluoromethylphenyl)amide of 6-chloropyrazine-2-carboxylic acid (2m, IC₅₀ = 0.026 mmol·dm^-3).

Full text of DOI:10.3390/70300363

Molecules 2002, 7, 363-373
molecules
ISSN 1420-3049
http://www.mdpi.org
Substituted Amides of Pyrazine-2-carboxylic acids: Synthesis
and Biological Activity.
Martin Dolezal 1*, Miroslav Miletin1, Jiri Kunes2, Katarina Kralova3
1
2
Department of Pharmaceutical Chemistry and Drug Control and Department of Inorganic and
Organic Chemistry, Faculty of Pharmacy, Charles University, 500 05 Hradec Králové, Czech
Republic, Tel.: +420 49 5067272; Fax +420 49 5512423.
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, 842 15 Bratislava, Slovak
Republic.
3
* Author to whom correspondence should be addressed; e-mail: dolezalm@faf.cuni.cz
Received: 24 October 2001; in revised form: 12 March 2002 / Accepted: 22 March 2002 / Published:
31 March 2002
Abstract: Condensation of 6-chloro-, 5-tert-butyl- or 6-chloro-5-tert-butylpyrazine-2-
carboxylic acid chloride with ring substituted anilines yielded a series of amides, which
were tested for their in vitro antimycobacterial, antifungal and photosynthesis-inhibiting
activities. The highest antituberculotic activity (72% inhibition) against Mycobacterium
tuberculosis and the highest lipophilicity (log P = 6.85) were shown by the 3,5-bis-
trifluoromethylphenyl amide of 5-tert-butyl-6-chloropyrazine-2-carboxylic acid (2o). The
3-methylphenyl amides of 6-chloro- and 5-tert-butyl-6-chloro-pyrazine-2-carboxylic acid
(2d and 2f) exhibited only a poor in vitro antifungal effect (MIC = 31.25-500 µmol·dm-3)
against all strains tested, although the latter was the most active antialgal compound (IC50
= 0.063 mmol·dm-3). The most active inhibitor of oxygen evolution rate in spinach
chloroplasts was the (3,5-bis-trifluoromethylphenyl)amide of 6-chloropyrazine-2-
carboxylic acid (2m, IC50 = 0.026 mmol·dm-3).
Keywords: Amides of pyrazinecarboxylic acid; antimycobacterial activity; antifungal
evaluation; photosynthesis inhibition.

Molecules 2002, 7
364
Introduction
Recent years have seen increased incidence of tuberculosis in both developing and industrialized
countries, the widespread emergence of drug-resistant strains and a deadly synergy with the human
immunodeficiency virus (HIV)[1,2]. Pyrazinamide (PZA) is a nicotinamide analogue that has been
used for almost 50 years as a first-line drug to treat tuberculosis [3]. PZA is bactericidal to
semidormant mycobacteria and reduces total treatment time [4]. Although the exact biochemical basis
of PZA activity in vivo is not known, under acidic conditions it is thought to be a prodrug of
pyrazinoic acid, a compound with antimycobacterial activity [5]. The finding that PZA-resistant strains
lose amidase (pyrazinamidase or nicotinamidase) activity and the hypothesis that amidase is required
to convert PZA to pyrazinoic acid intracellularly led to the recent synthesis and study of various
prodrugs of pyrazinoic acid [6]. Various compounds possessing –NHCO– grouping, e.g. substituted
amides, acyl and thioacyl anilides, benzanilides, phenyl carbamates, etc., were found to inhibit
photosynthetic electron transport [7—10]. Therefore, antifungal and photosynthesis-inhibiting
evaluations of newly prepared pyrazine-2-carboxylic acid derivatives were additional areas of interest
to us.
Amides of 2-alkylpyridine-4-carboxylic [11,12] and 2-alkylsulfanyl-4-pyridinecarboxylic [12,13]
acids inhibited oxygen evolution rate in Chlorella vulgaris and their inhibitory activity depended on
the lipophilicity of the compounds. Several esters of alkoxy substituted phenylcarbamic acids (APA)
showed antialgal activity against Chlorella vulgaris [14-16]. The inhibitory efficiency of APA
concerning chlorophyll production in Chlorella vulgaris depended on the lipophilicity of the alkoxy
substituent and also on its position on the aromatic ring [14-16]. The antialgal activity of APA
correlated with the antifungal activity of these compounds against Candida albicans [16]. We have
recently reported the synthesis of a series of substituted amides prepared from some pyrazine-2-
carboxylic acids and some aminophenols [17], halogenated or alkylated anilines [18].
The present study is concerned in the synthesis of another series of amides prepared from
substituted pyrazine-2-carboxylic acids and alkylated (2-, 3-methyl-, 2,6-dimethyl-), alkoxylated (2-
methoxy-) or halogenated (3-bromo-, 3,5-bis-trifluoromethyl-) anilines. The aim of this work is to
search for the structure-activity relationships and to determine the importance of increased lipophilicity
for antimycobacterial, antifungal and photosynthesis-inhibiting evaluation of newly prepared pyrazine-
2-carboxylic acid amides.
Results and Discussion
The synthesis of amides is shown in Scheme 1. Condensation of the chlorides of 6-chloropyrazine-
2-carboxylic (1a)[19], 5-tert-butyl-pyrazine-2-carboxylic (1b) [17] or 5-tert-butyl-6-chloropyrazine-2-
carboxylic (1c) [17] acids with ring substituted anilines yielded a series of 18 substituted amides 2a-r
of the aforementioned substituted pyrazine-2-carboxylic acids.

Molecules 2002, 7
365
Scheme 1. Preparation of substituted amides 2a-r of pyrazine-2-carboxylic acids
NH2
X
R
N
COOH
X
R
CONH
N
N
1. SOCl2
2.
Y
N
Y
1a-c
2a-r
Y = 2-CH3, 3-CH3, 2,6-CH3,
2-OCH3, 3-Br, 3,5-CF3
X = H, Cl
R = H, 1,1-tert-butyl
The melting points, yields, and elemental analyses of the compounds prepared 2a-r are given in
Table 3, and their spectral data in Tables 4 and 5. The structures were corroborated by 2D NMR
spectroscopy using gHSQC and gHMBC experiments. The biological activities of the prepared amides
2a-r with regards to in vitro antimycobacterial, antifungal and inhibition of oxygen evolution rate in
spinach chloroplasts were investigated. The highest antituberculotic activity (72% inhibition) against
Mycobacterium tuberculosis and also the highest lipophilicity (log P = 6,85) was shown by the 3,5-bis-
trifluoromethylphenyl amide of 5-tert-butyl-6-chloropyrazine-2-carboxylic acid (2o). Some other
amides (2d, 2f, 2k, 2l) with higher than 20% inhibition were investigated. Three of them contain a tert-
butyl moiety in position 5 of the pyrazine ring. The negative results of antimycobacterial screening
allow us to make no conclusions regarding potential structure-activity relationships. Results of their
antimycobacterial activity (MIC, % Inhibition) and calculated log P values of 2a-r are shown in Table
1.
Table 1. Antimycobacterial activity (MIC, % inhibition), IC50 values for inhibition of
oxygen evolution rate in spinach chloroplasts by compounds 2a-r and calculated log P
values of the compounds in comparison with standards rifampicine (RMP) and DCMU
(see Experimental).
Compd.
2a
MIC [µg ml-1] % Inhibition IC50[mmol dm-3]
log P
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
0
0
0
28
11
24
6
18
7
1.072
0.440
0.244
0.486
0.148
0.118
2.72 ± 0.41
3.28 ± 0.40
4.41 ± 0.42
2.72 ± 0.41
3.28 ± 0.40
4.41 ± 0.42
2.15 ± 0.42
2.72 ± 0.41
3.84 ± 0.43
3.46 ± 0.48
2b
2c
2d
2e
2f
a
-
2g
0.286
0.097
0.313
2h
2i
19
2j

Molecules 2002, 7
366
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
>6.25
0.125
-
39
20
12
10
72
0
2
13
0.081
0.107
0.026
0.114
0.241
0.649
0.229
0.242
-
4.03 ± 0.48
5.15 ± 0.50
5.16 ± 0.54
5.73 ± 0.53
6.85 ± 0.55
3.18 ± 0.41
3.75 ± 0.40
4.87 ± 0.42
− 0.37 ± 0.35
2.78 ± 0.38
2k
2l
2m
2n
2o
2p
2q
2r
100
RMP
DCMUc
-
0.0019
a not measured
The evaluation of in vitro antifungal activity of the synthetized compounds showed that only
compounds 2d and 2f, and partly compound 2l having a considerable antifungal effect on all the fungal
strains tested. The most susceptible was Trichophyton mentagrophytes strain (MIC = 62.5–1000
µmol·L-1), especially towards compounds 2f, 2h, 2i and 2l. Another susceptible strain was Absidia
fumigatus (MIC = 31.25–500 µmol·.L-1) towards compounds 2f and 2j.
The studied compounds inhibited photosynthetic electron transport in spinach chloroplasts, which
was reflected in the inhibition of oxygen evolution rate. The photosynthesis inhibitory activity of the
compounds has been expressed as IC50 values (see Table 1). The IC50 values varied in the range from
0.026 (2m) to 1.072 mmol·dm-3 (2a). In general, the photosynthesis-inhibiting activity of the studied
compounds depended on their lipophilicity showing a quasi-parabolic trend. However, the studied
compounds could be divided into two groups. The compounds with 2-CH3 substituents on the phenyl
ring (2a, 2b, 2c, 2p, 2q and 2r, squares in Figure 1) had lower biological activity than the other
investigated compounds with comparable log P values. Consequently, we assume that the methyl
substituent in ortho position of the benzene ring is disadvantageous from the viewpoint of interactions
with the photosynhetic apparatus. On the other hand, compound 2m exhibited higher inhibitory
activity than expected.
Figure 1. Quasi-parabolic dependence between photosynthesis inhibitory activity
and log P of studied amides 2a-r.
5.0
4.5
4.0
3.5
3.0
2.5
2.0
m
k
l
i
f
n
e
o
h
j
c
r
b
q
d
p
a
1
2
3
4
5
6
7
8
log P

Molecules 2002, 7
367
Additionally some inhibition of chlorophyll production in green algae Chlorella vulgaris was
studied at the compounds 2f, 2l, 2m, 2n, 2o and 2p. Results of their antialgal activity are given in
Table 2. The antialgal activity of these six studied compounds showed a quasi-parabolic dependence
upon log P with maximum activity for compounds having log P in the range from 3.18 to 5.16 (see
Figure 2). With the further increasing of the lipophilicity a dramatic decrease of antialgal activity was
observed.
Figure 2. Quasi-parabolic dependence between antialgal activity and log P of
studied amides 2f, 2l, 2m, 2n, 2o and 2p.
f
4.2
l
4.0
p
m
3.8
n
3.6
o
3.4
3.2
2
3
4
5
6
7
8
log P
Table 2. IC50 values concerning inhibition of chlorophyll production in green algae Chlorella
vulgaris by the tested anilides 2f, 2l, 2m, 2n, 2o and 2p and calculated log P values of the
compounds in comparison with standard DCMU (see experimental).
Compd.
2f
IC50[mmol dm-3]
0.063
log P
4.41 ± 0.42
5.15 ± 0.50
5.16 ± 0.54
5.73 ± 0.53
6.85 ± 0.55
3.18 ± 0.41
2.78 ± 0.38
0.067
0.125
0.208
0.356
0.079
0.0073
2l
2m
2n
2o
2p
DCMU

Molecules 2002, 7
368
Acknowledgements.
This study was supported by the Ministry of Education of the Czech Republic (No. FRVS
1676/G4/2001, Research Plans No. 11160001), and by the Scientific Grant Agency of the Ministry of
Education of the Slovak Republic and the Slovak Academy of Sciences (Grant No. 1/7262/20).
Antimycobacterial data were provided by the Tuberculosis Antimicrobial Acquisition and
Coordinating Facility (TAACF) through a research and development contract with the U.S. National
Institute of Allergy and Infectious Diseases. The authors wish to thank Dr. V. Buchta, CSc.,
Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, Hradec
Králové, Czech Republic, for providing data of antifungal activities. We also thank Mrs. D. Karlíčková
and J. Žižková for their skillful technical assistance.
Experimental
General
Melting points were determined on a Kofler block, and are uncorrected. Elemental analyses were
obtained using an EA 1110 CHNS-O CE apparatus (Fisons Instruments S.p.A., Milan). The IR spectra
were recorded on a Nicolet Impact 400 spectrometer in KBr pellets. The 1H and 13C NMR spectra were
measured for CDCl3 solutions with a Varian Mercury - Vx BB 300 spectrometer operating at 300
MHz. Chemical shifts were recorded as δ values in parts per million (ppm), and were indirectly
referenced to tetramethylsilane via the solvent signal (7.26 for 1H and 77.0 for 13C). Multiplicities are
given together with the coupling constants (in Hz). Log P values were computed using a program
ACD/Log P ver. 1.0 (Advanced Chemistry Development Inc., Toronto).
Synthesis of amides 2a-r
A mixture of acid (i.e. 6-chloropyrazine-2-carboxylic [19], 5-tert-butylpyrazine-2-carboxylic [17]
or 5-tert-butyl-6-chloropyrazine-2-carboxylic [17] acid, 0.05 mol) and thionyl chloride (5.5 mL, 75
mmol) in dry benzene (20 mL) was refluxed for about 1 h. Excess thionyl chloride was removed by
repeated evaporation with dry benzene in vacuo. The crude acyl chloride dissolved in dry acetone (50
mL) was added dropwise to a stirred solution of the corresponding substituted aniline (50 mmol) in dry
pyridine (50 mL) kept at room temperature. After the addition was complete, stirring was continued
for another 30 min. The reaction mixture was then poured into cold water (200 mL) and the crude
amide was collected and recrystallized from aqueous ethanol.

Molecules 2002, 7
369
Table 3. Analytical data of the amides 2a-r.
Formula
M. w.
% Calculated / % Found
M.p./°C
Yield/%
97-99
75
80-81
84
114-15
78
83-84
79
94-95
85
98-99
84
71-72
85
77-78
88
Compd. X
R
Y
C
H
N
F
Cl
Br
-
-
-
-
C12H10ClN3O
247.7
C16H19N3O
269,3
C16H18ClN3O
303.8
C12H10ClN3O
247.7
C16H19N3O
269,3
C16H18ClN3O
303.8
C12H10ClN3O2
263.7
C16H19N3O2
285.3
58.19 4.07 16.97
58.02 4.14 16.86
71.35 7.11 15.60
71.48 7.08 15.67
63.26 5.97 13.83
63.15 5.82 13.96
58.19 4.07 16.97
58.08 4.11 16.80
71.35 7.11 15.60
71.41 7.22 15.77
63.26 5.97 13.83
63.40 6.08 14.01
54.66 3.82 15.94
54.57 3.93 16.01
67.35 6.71 14.73
67.16 6.68 14.62
60.09 5.67 13.14
60.16 5.59 13.23
-
-
-
-
-
-
-
-
-
-
14.31
14.19
-
Cl
H
2-CH3
2a
2b
2c
2d
2e
2f
H (CH3)3C 2-CH3
Cl (CH3)3C 2-CH3
-
11.67
11.86
14.31
14.48
-
-
-
-
-
-
-
Cl
H
3-CH3
H (CH3)3C 3-CH3
Cl (CH3)3C 3-CH3
-
-
11.67
11.74
13.45
13.35
-
-
-
-
-
-
-
-
-
-
-
-
Cl
H
2-OCH3
2g
2h
2i
H (CH3)3C 2-OCH3
Cl (CH3)3C 2-OCH3
-
C16H18ClN3O2
319.8
-
-
11.09
11.07
-
-
118-19
82
C11H7BrClN3O 42.27 2.26 13.44
312.5 42.37 2.25 13.41
C15H16BrN3O 53.91 4.83 12.57
334.2 54.03 4.97 12.61
C15H15BrClN3O 48.87 4.10 11.40
368.7 48.79 4.22 11.28
-
-
-
-
-
-
11.34 25.56 99-100
11.48 25.60 83
23.91 113-14
23.77 75
Cl
H
3-Br
2j
-
-
H (CH3)3C 3-Br
Cl (CH3)3C 3-Br
2k
2l
9.62 21.67 104-105
9.77 21.78
62
132-133
88
135-137
89
C13H6ClF6N3O 42.24 1.64 11.37 30.84
9.59
9.46
-
-
-
-
Cl
H
3,5-CF3
2m
2n
2o
2p
2q
2r
369.7
C17H15F6N3O 52.18 3.86 10.74 29.13
391.3 52.02 3.84 10.72 29.17
C17H14ClF6N3O 47.96 3.31 9.87 26.77
425.8 48.01 3.41 9.63 26.56
C13H12ClN3O 59.66 4.62 16.06
42.21 1.66 11.33 30.77
H (CH3)3C 3,5-CF3
Cl (CH3)3C 3,5-CF3
-
-
8.33
8.51
13.55
13.67
-
-
-
-
-
-
98-99
88
121-122
75
84-85
78
-
-
-
-
-
Cl
H
2,6-CH3
361.7
C17H21N3O
283.4
59.70 4.70 16.09
72.06 7.47 14.83
72.09 7.45 14.84
H (CH3)3C 2,6-CH3
Cl (CH3)3C 2,6-CH3
-
-
C17H20ClN3O 64.25 6.34 13.22
317.8 64.19 6.40 13.18
11.16
11.17
-
-
145-146
68
-

Molecules 2002, 7
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Table 4. IR and 1H-NMR spectral data of the amides 2a-r.
IR (cm-1) 1H-NMR (δ, ppm; J in Hz)
Compd.
2a
1692 (C=O) 2.40s (CH3), 7.10-7.17m (H5´), 7.22-7.33m (H3´- H4´), 8.11-8.15m (H6´), 8.81s (H5),
3377 (NH) 9.40s (H3), 9.42bs (NH)
1685 (C=O) 1.45s [C(CH3)3], 2.40s (CH3), 7.10td (J=7.70, H5´), 7.20-7.33m (H3´- H4´), 8.26d
3358 (NH) J=7.70, H6´), 8.65dd (J=1.37, H5), 9.41dd (J=1.37, H3), 9.71bs (NH)
1695 (C=O) 1.56s [C(CH3)3], 2.40s (CH3), 7.12td (J=7.41, 1.37, H5´), 7.21-7.32m (H3´-H4´), 8.18-
3360 (NH) 8.13m (H6´), 9.28s (H3), 9.42s (NH)
1692 (C=O) 2.39s (CH3), 6.98-7.03m (H6´), 7.28t (J=7.96, H5´), 7.52-7.61m (H2´-H4´), 8.80s
3369 (NH) (H5), 9.35bs (NH), 9.39bs (H3)
1684 (C=O) 1.45s[C(CH3)3], 2.38s(CH3), 6.98d (J=7.69, H6´), 7.27t (J=7.69, H5´), 7.50-7.56m
3356 (NH) (H4´), 7.61-7.65m (H2´), 8.62d (J=1.51, H6), 9.40d (J=1.51,H3), 9.61s (NH)
1694 (C=O) 1.55s [C(CH3)3], 2.39s (CH3), 6.97-7.02m (H6´), 7.28t (J=7.69, H5´), 7.51-7.57m
3374 (NH) (H4´), 7.59-7.63m (H2´), 9.27s (H3), 9.32bs (NH)
1690 (C=O) 3.97s (OCH3), 6.94dd (J=7.96, 1.64, H3´), 7.03td (J=7.69, 1.51, H5´), 7.13td (J=7.69,
3377 (NH) 1.51, H4´), 8.52dd (J=7.96, 1.64, H6´), 8.78s (H5), 9.38s (H3), 10.04s (NH)
1691 (C=O) 1.45s [C(CH3)3], 3.96s (OCH3), 6.94dd (J=7.96, 1.64, H3´), 7.02td (J=7.69, 1.53, H5´),
2b
2c
2d
2e
2f
2g
2h
3356 (NH)
7.11td (J=7.69, 1.53, H4´), 8.59dd (J=7.96, 1.64, H6´), 8.68d (J=1.37, H6), 9.39d
(J=1.37, H3), 10.27bs (NH)
1695 (C=O) 1.55s [C(CH3)3], 3.97s (OCH3), 6.94dd (J=7.97, 1.51, H3´), 7.02td (J=7.97, 1.51, H5´),
3369 (NH) 7.12td (J=7.97, 1.51, H4´), 8.53dd (J=7.97, 1.51, H6´), 9.26s (H3), 10.01bs (NH)
1701 (C=O) 7.35-7.22m (H5´, H6´), 7.67ddd (J=7.96, 1.92, 1.37, H4´), 8.01t (J=1.92, H2´), 8.82s
3369 (NH) (H5), 9.38bs (H3), 9.38bs (NH)
1692 (C=O) 1.45s [C(CH3)3], 7.21-7.32m (H5´,H6´), 7.66dt (J=7.65, 1.92, H4´), 8.03t (J=1.92,
3352 H2´), 8.62d (J=1.65, H6), 9.38d (J=1.65, H3), 9.66bs (NH)
1697 (C=O) 1.55s [C(CH3)3], 7.22-7.34m (H5´, H6´), 7.66dt (J=7.69, 1.92, H4´), 8.02t (J=1.92,
3360 (NH) H2´), 9.26s (H3), 9.36bs (NH)
2i
2j
2k
2l
1681 (C=O) 7.70bs (H4´), 8.87bs (H5, H2´, H6´) 9.41s (H3), 9.66bs (NH)
3368 (NH)
1699 (C=O) 1.46s[C(CH3)3], 7.66bs (H4´), 8.28bs (H2´, H6´), 8.64d (J=1.51, H6), 9.41d
2m
2n
2o
2p
2q
2r
3346 (NH)
(J=1.51, H3), 9.94bs (NH)
1686 (C=O) 1.56s[C(CH3)3], 7.68bs (H4´), 8.29bs (H2´, H6´), 9.29s (H3), 9.63bs (NH)
3370 (NH)
1691 (C=O) 2.28s (CH3), 7.10-7.21m (H3´, H4´, H5´), 8.83s (H5), 8.94bs (NH), 9.39s (H3)
3356 (NH)
1667 (C=O) 1.46s [C(CH3)3], 2.29s (CH3), 7.09-7.19m (H3´, H4´, H5´), 8.65d (J=1.37, H6), 9.16bs
3370 (NH)
(NH), 9.40d (J=1.37, H3)
1710 (C=O) 1.57s [C(CH3)3], 2.28s (CH3), 7.07-7.20m (H3´, H4´, H5´), 8.91bs (NH), 9.27s (H3)
3291 (NH)

Molecules 2002, 7
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Table 5. 13C NMR spectral data of the amides 2a-r.
13C NMR (75 MHz, CDCl3) δ, ppm, J in Hz
Compd.
159.3, 147.5, 147.4, 144.2, 142.1, 134.9, 130.6, 128.6, 127.0, 125.5, 121.9, 17.6
167.7, 160.9, 142.9, 141.7, 139.1, 135.5, 130.4, 127.9, 126.9, 124.8, 121.4, 37.0, 29.7, 17.6
164.5, 159.7, 145.7, 141.3, 140.2, 135.1, 130.5, 128.5, 126.9, 125.3, 121.7, 39.0, 28.2, 17.6
159.2, 147.4, 147.4, 144.0, 142.2, 139.2, 136.7, 129.0, 126.0, 120.5, 117.1, 21.5
167.6, 161.0, 142.9, 141.5, 139.1, 138.9, 137.3, 128.9, 125.4, 120.3, 116.8, 37.0, 29.7, 21.5
164.4, 159.7, 145.7, 141.2, 140.2, 139.1, 137.0, 128.9, 125.7, 120.5, 117.0, 39.0, 28.3, 21.5
159.2, 148.7, 147.5, 147.3, 144.5, 142.1, 126.6, 124.8, 121.1, 120.0, 110.1, 55.9
167.4, 161.0, 148.6, 142.9, 141.9, 139.2, 127.2, 124.2, 121.1, 119.7, 110.1, 55.8, 37.0, 29.7
164.2, 159.7, 148.7, 145.8, 141.6, 140.1, 126.9, 124.6, 121.0, 119.9, 110.1, 55.9, 38.9, 28.3
159.4, 147.8, 147.5, 143.5, 142.2, 138.1, 130.5, 128.1, 122.9, 122.8, 118.4, 29.7
168.1, 161.1, 143.0, 141.0, 139.0, 138.7, 130.4, 127.5, 122.8, 122.6, 118.1, 37.1, 29.7
164.9, 159.9, 145.8, 140.7, 140.3, 138.3, 130.4, 127.9, 122.8, 118.4, 39.0, 28.2
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
159.9, 148.4, 147.7, 142.9, 142.4, 138.3, 132.7 (q, J=33.5 Hz), 123.0 (q, J=272.8 Hz), 119.7, 118.5 (q,
J=3.7 Hz)
2m
168.7, 161.6, 143.2, 140.3, 139.1, 138.9, 132.6 (q, J=33.7 Hz), 123.1 (q, J=272.9 Hz), 119.4 (d, J=2.9
Hz), 117.8 (q, J=3.8 Hz), 37.2, 29.7
165.6, 160.4, 146.0, 140.4, 140.0, 138.5, 132.6 (q, J=33.2 Hz), 123.0 (q, J=272.9 Hz), 119.6 (q, J=3.2
Hz), 118.1 (q, J=4.0 Hz), 39.2, 28.2
2n
2o
159.8, 147.5, 143.9, 142.3, 135.3, 132.7, 128.3,, 127.7, 30.9, 18.5
167.6, 161.4, 143.0, 141.4, 139.1, 135.3, 133.3, 128.2, 127.4, 37.0, 29.7, 18.5
164.5, 160.2, 145.9, 141.0, 140.3, 135.4, 132.9, 128.3, 127.6, 39.0, 28.3, 18.5
2p
2q
2r
Antimycobacterial Assay
Antimycobacterial evaluation was carried out in Tuberculosis Antimicrobial Acquisition
and Coordinating Facility (TAACF), Southern Research Institute, Birmingham, Alabama, USA, which
is a part of National Institutes of Health (NIH). Primary screening of all compounds were conducted at
6.5 or 12.5 µg·ml-1 against Mycobacterium tuberculosis H37Rv in BACTEC 12B medium using
the BACTEC 460 radiometric system [20]. The MIC was defined as the lowest concentration effecting
a reduction in fluorescence of 99% relative to controls. For the results see Table 1.
In vitro antifungal susceptibility testing
Broth microdilution test [21,22] was used for the assessment of in vitro antifungal activity of
ketoconazole (standard) and the synthetized compounds against Candida albicans ATCC 44859,
Candida tropicalis 156, Candida krusei E28, Candida glabrata 20/I, Trichosporon beigelii 1188,
Trichophyton mentagrophytes 445, Aspergillus fumigatus 231, and Absidia corymbifera 272. The

Molecules 2002, 7
372
procedure was performed with twofold compound dilutions in RPMI 1640 buffered to pH 7.0
with 0.165 mol morpholinopropanesulfonic acid. The final concentrations of the compounds ranged
from 1000 to 0.975 µmol/L. Drug free controls were included. The MICs were determined after 24 and
48 h of static incubation at 35oC. In the case of Trichophyton mentagrophytes the MICs were
determined after 72 and 120 h of incubation.
Study of inhibition of oxygen evolution rate in spinach chloroplasts
The oxygen evolution rate in spinach chloroplasts was investigated spectrophotometrically
(Specord UV VIS, Zeiss, Jena) in the presence of an electron acceptor 2,6-dichlorophenol-indophenol,
by method described in Ref. [23]. The compounds were dissolved in dimethyl sulfoxide (DMSO)
because of their low water solubility. The DMSO volume fractions used (up to 5 vol. %) did not affect
the oxygen evolution. The inhibitory efficiency of the studied compounds has been expressed by IC50
values, i.e. by molar concentration of the compounds causing 50 % decrease in the oxygen evolution
relative to the untreated control. IC50 value for the standard, a selective herbicide 3-(3,4-
dichlorophenyl)-1,1-dimethylurea, DCMU (DIURON) was measured about 1.9 µmol dm-3.
For the results see Table 1.
Study of inhibition of chlorophyll production in green algae Chlorella vulgaris
The algae Chlorella vulgaris were cultivated statically at room temperature according to Sidóová
et al. [24] (photoperiod 16 h light/8 h dark; illumination 4000 lx; pH = 7.2). The effect of compounds
2f, 2l, 2m, 2n, 2o and 2p on algal chlorophyll (Chl) content was determined after 4-day cultivation in
the presence of the tested compounds, expressing the response as percentage of the corresponding
values obtained for control. The Chl content in the algal suspension was determined
spectrophotometrically (Specord UV VIS, Zeiss Jena, Germany) after extraction into N,N-
dimethylformamide according to Inskeep and Bloom [25]. The Chl content in the suspensions at the
beginning of cultivation was 0.5 mg dm-3. Because of their low water solubility, the tested compounds
were dissolved in DMSO. DMSO concentration in the algal suspensions did not exceed 0.25 v/v %
and the control samples contained the same DMSO amount as the suspensions treated with the tested
compounds. IC50 value for the standard, a selective herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea,
DCMU (DIURON) was measured about 7.3 µmol dm-3. For the results see Table 2.
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Sample availability: Samples of the compounds mentioned in this paper are available from MDPI.
© 2002 by MDPI (http://www.mdpi.org). Reproduction is permitted for non commercial purposes.