Ring substituted 3-phenyl-1-(2-pyrazinyl)-2-propen-1-ones as potential photosynthesis-inhibiting, antifungal and antimycobacterial agents

Article abstract of DOI:10.1016/S0014-827X(01)01187-9

Four series of ring substituted (E)-3-phenyl-1-(2-pyrazinyl)-2-propen-1-ones were prepared by means of modified Claisen-Schmidt condensation of acetylpyrazines with aromatic aldehydes. The structures were confirmed by elemental analysis, IR, ¹H NMR and ¹³C NMR spectra. The compounds were tested for specific biological properties and some derivatives exhibited photosynthesis-inhibiting, antifungal and antimycobacterial properties. The most pronounced effects were observed with compounds substituted with phenolic groups. Ortho-hydroxyl substituted derivatives were more potent than the corresponding para-hydroxyl substituted analogues.

Full text of DOI:10.1016/S0014-827X(01)01187-9

Il Farmaco 57 (2002) 135–144
www.elsevier.com/locate/farmac
Ring substituted 3-phenyl-1-(2-pyrazinyl)-2-propen-1-ones as
potential photosynthesis-inhibiting, antifungal and
antimycobacterial agents
a,
Veronika Opletalova´ *, Jirˇ´ı Hartl a, Asmita Patel b, Karel Pala´t Jr. c,
Vladim´ır Buchta d
a Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy, Charles Uni6ersity in Prague,
500 05 Hradec Kra´lo6e´, Czech Republic
b School of Pharmacy and Biomedical Science, Uni6ersity of Portsmouth, Portsmouth PO1 2DT, UK
c Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles Uni6ersity in Prague, 500 05 Hradec Kra´lo6e´, Czech Republic
d Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles Uni6ersity in Prague, 500 05 Hradec Kra´lo6e´, Czech Republic
Received 15 February 2001; accepted 14 October 2001
Abstract
Four series of ring substituted (E)-3-phenyl-1-(2-pyrazinyl)-2-propen-1-ones were prepared by means of modified Claisen–
1
Schmidt condensation of acetylpyrazines with aromatic aldehydes. The structures were confirmed by elemental analysis, IR, H
NMR and 13C NMR spectra. The compounds were tested for specific biological properties and some derivatives exhibited
photosynthesis-inhibiting, antifungal and antimycobacterial properties. The most pronounced effects were observed with com-
pounds substituted with phenolic groups. Ortho-hydroxyl substituted derivatives were more potent than the corresponding
´
para-hydroxyl substituted analogues. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Ring substituted (E)-3-phenyl-1-(2-pyrazinyl)-2-propen-1-ones; Photosynthesis-inhibiting activity; Antifungal activity; Antimycobacte-
rial activity
1. Introduction
cinnamic acid [8] which have structural features in
common with the cinnamoylpyrazines in this study are
known to influence plant growth, antifungal properties
of chalcones and their heterocyclic analogues are well
documented in literature [2], and antimycobacterial
properties of chalcones and azachalcones have been
known since the 1950’s [9] and analogous derivatives
have recently been shown to be potential antitubercu-
lous agents [10–12]. The effect on plant growth was
monitored by quantifying the chlorophyll and oxygen
evolution rate (OER), as an indirect measure of the
effect on photosynthesis. To test the antifungal poten-
tial, eight pathogenic strains of fungi were included in
the programme of study. In recent years, tuberculosis
has again become a problem throughout the world due
to the increasing migration from developing countries
and the spread of the human immunodeficiency virus
infection. Furthermore, the built-up of resistance to the
antituberculous drugs by Mycobacterium tuberculosis
strains and non-tuberculous mycobacterial infections
Chalcones are natural products that may be used as
lead structures for the development of new drugs. Bio-
logical activities of chalcones and their heterocyclic
analogues have been reviewed recently [1–3]. Four
series of ring substituted (E)-3-phenyl-1-(2-pyrazinyl)-2-
propen-1-ones 1a–1e, 2a–2e, 3a–3e and 4a–4e were
prepared as a part of our continuing effort aimed at the
synthesis of biologically active pyrazine derivatives.
Conformational analysis of the ortho-hydroxyl sub-
stituted derivatives 2a–2e based on spectral data and
molecular model calculations is available [4]. The
present study focuses on the synthetic and biological
results. Three types of bioassays were chosen based on
the following rationale: pyrazine derivatives [5–7] and
* Corresponding author.
E-mail address: opletalo@faf.cuni.cz (V. Opletalova´).
´
0014-827X/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0014-827X(01)01187-9

136
V. Opletalo6a´ et al. / Il Farmaco 57 (2002) 135–144
has made the classic armamentarium of antituberculous
drugs inefficacious and, thus, the research for new
drugs has become a public health priority [13,14]. In
1994, the National Institute of Allergy and Infectious
Diseases founded a drug acquisition and screening pro-
gramme, the TAACF, to facilitate the development,
testing, and commercialization of new antituberculous
agents. The compounds reported here were screened for
the antimycobacterial properties as part of the TAACF
project.
parameters were: Spectral width 3 (1H) and 18 kHz
(13C), pulse width 3 (1H) and 4.2 ms (13C), acquisition
time 5.46 or 0.90 s, number of scans 16 (1H) and 600
(13C). Chemical shifts are given in l, ppm and coupling
constants J in Hz. Initial geometries of the molecular
models were built by means of standard parameters and
then optimized by semi-empirical AM1 method using
Polak–Ribiere geometrical optimization, RMS gradient
−1
,
0.05 kcal A
mol−1. Molecular dynamics (with heat-
ing to 1200 K) in combination with manual rotation on
single bonds was used for finding the conformer with
the lowest energy. Other conformers were formed by
the manual rotation of the appropriate bonds in the
lowest energy model followed by the geometry opti-
2. Experimental
1
mization. Relative H NMR shifts were calculated by
Acetylpyrazines 5a–5e prepared as described pre-
viously [15] and commercially available aromatic alde-
hydes were used as starting materials. Silpearl (Kava-
lier, Votice) was used for flash column chromato-
graphy. Purity of the products was checked by TLC on
Silufol UV 254 plates (Kavalier, Votice). The following
solvent mixtures were used for TLC: C6H5CH3–C3H6O
50:50 (v/v), light petroleum–EtOAc 60:40 (v/v), and
light petroleum–EtOAc 80:20 (v/v). M.p. were deter-
mined with a Boe¨tius apparatus and are uncorrected.
Elemental analyses were performed with CHN Ana-
lyzer (Laboraton´ı prˇ´ıstroje, Prague). IR spectra were
recorded in KBr pellets with an IR spectrophotometer
Nicolet Impact 400. Characteristic wavenumbers are
given in cm−1. 1H and 13C NMR spectra were recorded
at room temperature (r.t.) in 5 mm tubes using a JOEL
GSX-270 (1H, 13C) FT spectrometer at 270.16 (1H) and
67.97 (13C) MHz, using the deuterium signal of the
solvent as the lock and TMS as internal standard. The
semi-empirical TNDO/2 method with special Slater ex-
ponents for shielding. HyperNMR software (version
2.0, Hypercube Inc.) was used for the calculation of
NMR spectra and the program HyperChem (release
5.11, HyperCube Inc.) for other quantum-chemical cal-
culations. Specord UV–Vis (Zeiss Jena, Germany) was
used in photosynthesis-inhibiting bioassays.
2.1. Chemistry
2.1.1. General procedure for the preparation of
compounds 1a–1e, 2a–2e, 3a–3e and 4a–4e
Acetylpyrazine (0.01 mol) and aromatic aldehyde
(0.01 mol) were dissolved in Py (4.4 ml). Diethylamine
(0.73 g, 0.01 mol) was added and the reaction mixture
was heated in the C3H8O3 bath at 80–120 °C for 2 h.
After cooling, the reaction mixture was poured into ice
water acidified to pH 3 with a few drops of AcOH, and

V. Opletalo6a´ et al. / Il Farmaco 57 (2002) 135–144
137
then refrigerated for 24 h. If the product separated as a
solid, it was filtered off and recrystallized several times
from absolute EtOH. If the product separated as an
oily mixture, it was extracted with Et2O and subjected
to the flash column chromatography on silica gel using
the mixture light petroleum–EtOAc 60:40 (v/v) as an
Table 1
Characteristic data and IR spectra of compounds 1, 2, 3 and 4
Comp.
M.p. (°C) Yield (%)
Formula M.w.
w (cm−1
)
CꢀO s-cis
CꢀO s-trans
EꢁC(O)CHꢀCH
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
182–184 a
23
C
256.3
14H22N2O3
1664
1637
990
151–152
33
C18H20N2O3
321.4
1667
1664
1664
1664
1665
1652
1652
1661
1651
1674
1663
1665
1663
1664
1658
1651
1656
1652
1652
N
985
987
987
1009
992
993
997
987
998
986
987
989
1002
1010
994
996
1003
990
992
143–145
25
C18H20N2O3
321.4
N
148–150
8
C
321.4
18H20N2O3
1637
1636
1636
1630
N
182–185.5
8
C
298.3
17H18N2O3
172.5–174.5 b
35
C13H10N2O2
226.2
182–185.5
35
C17H18N2O2
282.3
152–156
28
C17H18N2O2
282.3
132–134
36
C
282.3
17H18N2O2
1637
N
148–150.5
26
C16H16N2O2
268.3
188–190 c
44
C
226.2
13H10N2O2
1636
1637
N
182–185.5
46
C17H18N2O2
282.3
151–153.5
35
C17H18N2O2
282.3
126–127
36
C17H18N2O2
282.3
N
135–137
37
C
268.3
16H16N2O2
N
162–165
20
C15H15N3O
253.3
1635
N
108–118
10
C19H23N3O
309.4
78–81
7
C19H23N3O
309.4
1636
1629
N
90–92
10
C19H23N3O
309.4
128.5–130.5
44
C18H21N3O
295.4
N, not observed.
a Ref. [23] 183–185 °C.
b Ref. [23] 173–175 °C.
c Ref. [23] 191–194 °C.

138
V. Opletalo6a´ et al. / Il Farmaco 57 (2002) 135–144

V. Opletalo6a´ et al. / Il Farmaco 57 (2002) 135–144
139
Table 3
13C NMR spectra of compounds 1, 2, 3, and 4
Comp.
Chemical shifts (l (ppm))
Substituents
C-1
C-2
C-3
C-2%
C-3%
C-5%
C-6%
C-1¦
C-2¦
C-3¦
C-4¦
C-5¦
C-6¦
la a
188.2
188.7
188.2
188.1
116.9
119.5
116.6
114.5
146.4
141.6
146.0
146.3
150.2
148.7
148.6
149.3
144.5
144.4
144.3
144.7
147.3
147.2
147.3
146.9
143.4
143.4
143.4
143.2
126.6
121.7
125.9
122.5
110.8
148.7
130.7
131.1
147.9 c 148.7 c 115.7
124.4
129.1
130.7
131.1
56.0 (OCH3)
2a b
3a b
4a b
116.5
116.2
111.7
132.2
160.8
152.3
119.5
116.2
111.7
40.1 (N(CH3)2)
a Chemical shifts for alkyl groups were: 29.7 ((CH3)3C), 37.1 ((CH3)3C); 22.4 ((CH3)2CHCH2), 29.1 ((CH3)2CHCH2), 44.7 ((CH3)2CHCH2); 13.8
(CH3CH2CH2CH2), 22.4 (CH3CH2CH2CH2), 31.5 (CH3CH2CH2CH2), 35.5 (CH3CH2CH2CH2); 13.7 (CH3CH2CH2), 22.4 (CH3CH2CH2), 37.5
(CH3CH2CH2). The remaining values were close to that of 1a (94.2 ppm) with exception of C-5% that were shifted slightly downfield, i.e. 167.5
for 1b, 160.3 for 1c, 161.2 for 1d and 160.7 for 1d.
b Chemical shifts for alkylated compounds showed a pattern similar to that of alkylated derivatives of series 1.
c Assignments are reversible.
Table 4
Photosynthesis-inhibiting properties of compounds 2b–2e and 3b–3e
Comp.
Inhibition of chlorophyll production in
Inhibition of oxygen evolution rate IC50 (mmol l−1
)
C. 6ulgaris at 0.1 mmol l−1 (%)
Spinach chloroplasts
C. 6ulgaris
2b
2c
2d
2e
3b
3c
3d
3e
27
92
89
68
39
35
10
12
100 a
N
0.167
0.144
0.184
0.187
0.315
0.235
0.306
0.399
0.072 b
0.105 d
0.478 d
0.078
0.063
0.147
0.100
0.279
0.232
0.265
0.514
0.048 c
0.099 d
0.329 d
KlimCS-8
Dol-2
Dol-5
N
a Ref. [34], KlimCS-8=2-octylthio-4-pyridinecarbothioamide.
b Ref. [29].
c Ref. [35].
d Ref. [36], Dol-2=5-tert-butyl-6-chloro-N-(3-chloro-5-hydroxyphenyl)-2-pyrazinecarboxamide, Dol-5=5-tert-butyl-N-(3-hydroxyphenyl)-2-
pyrazinecarboxamide.
N, not determined.
eluent. After separation the product was crystallized
from absolute EtOH. The microanalyses (C, H, N)
were within 90.4% for all new compounds. IR spectra
of the products are given in Table 1 and the NMR data
in Tables 2 and 3.
described in Ref. [16]. The samples contained 0.1 mmol
l−1 of the studied compounds and the resulting solvent
concentration in the samples as well as in the control
was adjusted to 1%. Seven days after addition of the
test compounds, chlorophyll content in the algal sus-
pensions was determined spectrophotometrically after
extraction into N,N dimethylformamide [17].
2.2. Biological e6aluation
The OER in spinach chloroplasts (Cchloroplasts=30
mg l−1) was determined spectrophotometrically using
2,6-dichlorophenol–indophenol as an electron acceptor
according to the method reported previously [18].
OER in the algal suspension of C. 6ulgaris
(Cchlorophyll=20 mg l−1) was measured at 24 °C by
Clark type electrode (SOPS 31 atp., Chemoprojekt,
Prague) in a chamber constructed according to a
reported method [19]. The suspensions were
accommodated in the dark (4 h) prior to the OER
2.2.1. E6aluation of photosynthesis-inhibiting acti6ity
The compounds were dissolved in dimethyl sulfoxide
(DMSO) rather than water due to their restricted solu-
bility in the latter solvent. DMSO concentration in the
samples was found not to affect the photochemical
activity of spinach chloroplasts and algal suspensions.
The algae, Chlorella 6ulgaris, were statically culti-
vated (photoperiod 16 h light/8 h dark; illumination: 60
mEs−1 m−2 PAR) at r.t. according to the method

140
V. Opletalo6a´ et al. / Il Farmaco 57 (2002) 135–144
measurements. The samples were then illuminated with
a 250 W halogen lamp from 0.3 m distance through a
water filter (400 mEs−1 m−2 PAR) at r.t. [20]. The
results are summarized in Table 4.
SDA and fungal inocula were prepared by suspending
yeasts or conidia or sporangiospores in sterile 0.85%
saline. The cell density was adjusted by means of a
Bu¨rker’s chamber to yield a stock suspension of 1.0–
5.0×105 cfu ml−1. The final inoculum was made by
1:20 dilution of the stock suspension with the test
medium.
Antifungal activity of the compounds 1a–1e, 2a–2e,
3a–3e and 4a–4e, dissolved in DMSO, was determined
in tissue culture medium RPMI 1640 (Sevac) buffered
to pH 7.0 with 0.165 M 3-(N-morpholino)propane-
sulfonic acid (Sigma). Controls without the test com-
pounds were also included. The minimum inhibitory
concentrations (MIC), defined as the 80% inhibition
of fungal growth compared to control wells, were
2.2.2. E6aluation of in 6itro antifungal acti6ity
Antifungal susceptibility of all the compounds was
assessed by microdilution broth method against Can-
dida albicans ATCC 44859, Candida tropicalis 156,
Candida krusei E 28, Candida glabrata 20/I, Tri-
chosporon beigelii 1188, Trichophyton mentagrophytes
445, Aspergillus fumigates 231 and Absidia corymbifera
272. All strains were subcultured on Sabouraud dex-
trose agar (SDA) and maintained on the same medium
at 4 °C. Prior to testing, each strain was passaged onto
Table 5
Inhibitory activity of compounds 1, 2, 3 and 4 against T. mentagrophytes 445 and M. tuberculosis H37Rv
Comp.
Inhibition of T. mentagrophytes 445 MIC
Inhibition of M. tuberculosis H37Rv
at 12.5 mg ml−1 (%)
(mmol l−1
)
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
125
\125
250
\100
\125
15.62
\250
31.25
3.91
N
N
N
N
35
77
53
45
20
55
94
20
33
50
42
72
63
51
35
26
7
N
31.25
\250
N
15.62
7.81
125
\250
\500
62.5
62.5
\62.5
\250
\250
\250
\250
N
7.81
62.5
\250
\500
31.25
31.25
\62.5
\250
250
\250
\250
N
4b
4c
4d
4e
10
0
8
pyrazineamide
100
N, not determined
Table 6
Antifungal activity of compound 2a
Comp.
MIC (mmol l−1
)
CA
CT
CK
CG
TB
TM
AF
AC
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
72 h
120 h
24 h
48 h
24 h
48 h
2a
62.5
62.5
125
250
125
250
125
250
125
250
15.62
31.25
125
250
125
250
Ketoconazole
50.06
50.06
3.91
7.81
1.95
1.95
0.49
1.95
50.06
50.06
0.24
1.95
7.81
7.81
15.63
15.63
CA, Candida albicans ATCC 44859; CT, Candida tropicalis 156; CK, Candida krusei E 28; CG, Candida glabrata 20/I; TB, Trichosporon beigelii
1188; TM, Trichophyton mentagrophytes 445; AF, Aspergillus fumigates 231; AC, Absidia corymhifera 272.

V. Opletalo6a´ et al. / Il Farmaco 57 (2002) 135–144
141
determined after 24 and 48 h of static incubation at
35 °C. In the case of T. mentagrophytes the MICs were
recorded after 72 and 120 h. Ketoconazole (batch no. E
3401, Janssen Pharmaceutica) was used as a reference
antifungal drug. The results for this part of the study
are given in Tables 5 and 6.
taken to indicate the presence of a small amount of the
unfavoured s-trans conformer [4].
The chemical shift assignments (1H and 13C NMR) of
the four series of compounds were made using DEPT
and 2D-Hetero (1H, 13C) COSY techniques. The magni-
tude of the olefinic vicinal coupling constant between
H-2 and H-3 (J=15–16 Hz) in the 1H NMR spectra of
all the compounds in the series 1, 2 and 3 is indicative
of E-configuration. In contrast, the corresponding pro-
tons of compounds in the 4 series with the para-
dimethylamino substituent show a singlet. However, the
13C NMR shifts for C-2 and C-3 are highly comparable
to the values obtained for compounds in the series 1, 2
and 3. In order to investigate the effect of solute–sol-
2.2.3. E6aluation of antimycobacterial acti6ity
Primary screening of all the compounds was con-
ducted at 12.5 mg ml−1 against M. tuberculosis H37Rv
(ATCC 27294) in BACTEC 12B medium using the
BACTEC 460-radiometric system (Table 5). Compound
2b demonstrating 94% inhibition in the primary screen
was re-tested at lower concentrations against M. tuber-
culosis H37Rv to determine the actual MIC in a broth
microdilution assay, the Microplate Alamar Blue Assay
(MABA). The MIC is defined as the lowest concentra-
tion effecting a reduction in fluorescence of 90% rela-
tive to controls. This compound was also tested against
M. a6ium using the same technique and procedure.
1
vent interactions, the H NMR spectra of 4a and 4d
were obtained in CDC13 after a 50-fold dilution. Both
the resulting spectra then gave the expected AB quar-
tets (JH2,H3=15.8 Hz). Attempts were also made to
obtain spectra of 4a, 4b and 4d in C6D6. Sample
solubility was limited in each case, but the spectra of
the solutions also showed clear AB quartets (4a: l 8.37
and 8.40; 4b: l 8.46 and 8.52; 4d: l 8.43 and 8.53;
J=15.8 Hz).
3. Results and discussion
1
The relative H NMR chemical shift positions of H-2
3.1. Chemistry
and H-3 are reversed for compounds of series 2 on the
basis of 2D-Hetero COSY spectra (compared to that of
other three series). NOE experiments and molecular
model calculations [4] indicated that there is a lack of
free rotation of the phenyl ring in the compounds of
All the 3-phenyl-1-(2-pyrazinyl)-2-propen-1-ones re-
ported in the present paper are novel with exception of
1a, 2a and 3a that have been reported previously [21–
24]. Compounds 1a, 2a, 3a and 4a were isolated directly
as solids since acetylpyrazine 5a is freely soluble in
water and does not contaminate the required products
after pouring the reaction mixture into acidified water.
In the case of derivatives with an alkylated pyrazine
ring (1b–1e, 2b–2e, 3b–3e and 4b–4e), an oily mixture
of the product and unreacted acetylpyrazine resulted.
Each compound was then obtained by means of flash
column chromatographic separation using silica gel.
The products recovered are crystalline, coloured solids;
compounds 1a–1e, 2a–2e and 3a–3e are yellow or
orange, compounds 4a–4e are red.
1
this series. Experimental H NMR data of compounds
2a–2e were, therefore, compared with those calculated
for various conformers of compound 2a (Fig. 1). The
calculated relative 1H NMR absorptions are l 7.040
(H-2) and 8.665 (H-3) for conformer I, in which H-3
and OH-2¦ are in close proximity. In contrast, the
1
calculated relative H NMR signals for H-2 and H-3
are reversed (l 8.025 and 7.198, respectively) for con-
former II, in which H-2 is in close proximity to the
OH-2¦ group. Since the presence of a mixture of (E)-s-
cis- and (E)-s-trans-conformers was expected in a chlo-
roform solution on the basis of literature data [26], the
1H NMR shifts were also calculated for the two (E)-s-
trans-conformers III and IV. For the results see Fig. 1.
Structural confirmation of the products was based on
elemental analysis, and IR, H and 13C NMR spectro-
1
1
scopic analysis. IR spectra of all compounds conformed
to literature data for analogous structures [23–26].
Carbonyl group can present an s-cis or s-trans confor-
mation with respect to the vinyl double bond. A con-
formational equilibrium between the two conformers is
dependent on their structures and the properties of
environment, e.g. solvent and temperature. A strong
absorption band in the region of 1651–1674 cm−1 was
found in the IR spectra of all the present 3-phenyl-1-(2-
pyrazinyl)-2-propen-1-ones, and it was concluded that
the more stable s-cis-conformer prevailed in the solid
state (KBr pellets). A shoulder at 1636 cm−1 was
observed in the spectra of several compounds. This was
The observed relative H NMR chemical shifts posi-
tions of H-2 and H-3 for compounds of series 2 (proton
H-3 further downfield than proton H-2) conform to the
calculated values for conformer I. According to the
results of molecular model calculations reported previ-
ously conformer I is the most stable one [4]. Similar
conformation has been reported for structurally related
ortho-hydroxyl
substituted
bis(benzylidene)cyclo-
alkanones [27]. The phenolic protons are probably in-
volved in weak hydrogen bonds (most likely with the
solvent or between two or more solute molecules), since
their chemical shifts are observed between 9 and 10
ppm.

142
V. Opletalo6a´ et al. / Il Farmaco 57 (2002) 135–144
Fig. 1. The relative 1H NMR shift position (in l, ppm) for conformers of compound 2a calculated using TNDO/2 method.
port IC50 values for some known herbicides can be
found in Ref. [32], e.g. for various urea derivatives IC50
are in the range 39.81–0.05 mmol l−1 and for atrazine
between 0.79 and 0.25 mmol l−1. According to Carpen-
tier et al. [33], IC50 of atrazine is about 1 mmol l−1. In
both types of tests, inhibitory activity of ortho-hydroxyl
substituted derivatives 2b–2e was greater than that of
para-hydroxyl substituted ones 3b–3e. The lowest IC50
values were found with compounds having a branched
alkyl group on the pyrazine ring (2b, 2c).
All the compounds under study were also subjected
to an assessment of in vitro antifungal susceptibility.
Some compounds with phenolic groups inhibited
growth of T. mentagrophytes 445. MIC are listed in
Table 5. As with the photosynthesis-inhibiting activity
the most pronounced antifungal effects were observed
with ortho-hydroxyl substituted derivatives. This is in
accordance with literature data pointing out the impor-
tance of hydroxyl groups for antibacterial [3,37] and
antifungal [2,38,39] effects of chalcones. In contrast to
the observations made with the photosynthesis-inhibi-
ting bioassay, the hydroxyl derivatives with non-
branched alkyl substituents on the pyrazine ring (2d,
2e) were most potent. Activity against Candida species
(MIC=62.5 mmol l−1) was exhibited by compounds 2d
and 3d. Compound 2a showed weak to moderate acti-
vity against all testing strains (Table 6).
3.2. Biological e6aluation
With regard to the influence of pyrazine derivatives
on plant growth, one study [28] has shown with the aid
of EPR spectroscopy that the site of action of 3-phenyl-
1-(2-pyrazinyl)-2-propen-1-ones is in the photosynthetic
apparatus of spinach chloroplasts. The intensity of the
EPR signal II, mainly that of its constituent signal
IIslow, was decreased. This implied that these com-
pounds interact predominantly with D+ intermediate,
i.e. with the tyrosine radical in position 161 (TyrD)
which is located in D2 protein on the donor side of
photosystem (PS) 2. Due to this interaction the photo-
synthetic electron transport from the oxygen evolving
complex to the primary donor of PS 2 is impaired and
consequently the electron transport between PS 1 and
PS 2 is inhibited as well. A similar site of action (TyrD)
has been confirmed for other inhibitors with a hetero-
cyclic skeleton, for example 2-alkylthio-4-pyridinecar-
bothioamides [29] and anilides of 2-pyrazinecarboxylic
acid [30], whereas the anilides of 2-alkyl-4-pyridinecar-
boxylic acids are claimed to interact also with the Z+
intermediate [31]. The effects of the diazachalcones in
the present study on statically cultured C. 6ulgaris were
found to lower the chlorophyll content (10–92%) by
compounds 2b–2e and 3b–3e, compared to untreated
controls (Table 4). The compounds of the series 2 and
3 also reduced the rate of oxygen evolution in spinach
chloroplasts and C. 6ulgaris. IC50 values of these com-
pounds, i.e. concentrations causing 50% inhibition of
OER, were compared with IC50 values of several com-
pounds known to exhibit the same mechanism of action
and are given in Table 4. Photosynthetic electron trans-
In the antimycobacterial bioassay, para-dimethy-
lamino substituted derivatives (with exception of com-
pound 4a) were less active than compounds with the
phenolic group (series 1, 2 and 3). The highest activity
was displayed by compounds 1b, 2b and 3b. Thus, the
presence of a tert-butyl group on the pyrazine nucleus

V. Opletalo6a´ et al. / Il Farmaco 57 (2002) 135–144
143
their derivatives and analogues), Farm. Obzor LXI (1992) 341–
350.
seems favourable for antimycobacterial activity (Table
5). MIC of the most active derivative 2b was 12.5 mg
ml−1, while rifampicin in the same test was 0.06 mg
ml−1. Compound 2b was also tested against Mycobac-
terium a6ium, a naturally drug-resistant opportunistic
pathogen, and it inhibited the growth of this strain by
32%, MIC\12.5 mg ml−1. Clarithromycin (MIC=4
mg ml−1) was used as the positive control against M.
a6ium. It is important to note that the most active
compound 2b also belongs to the ortho-hydroxyl substi-
tuted series. Further studies are underway aimed at the
detailed elucidation of structure–activity relationships
in the class of chalcones and their analogues to discover
a suitable drug candidate.
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Acknowledgements
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[16] L’. Mitterhauszerova´, K. Kra´l’ova´, F. Sersˇenˇ, V. Blana´rikova´, J.
This study was supported by the Internal Grant
Agency of Charles University (Grant no. 26/1998-BCH
and the Research Project MSM 111600001. Antimy-
cobacterial data were provided by the Tuberculosis
Antimicrobial Acquisition Coordinating Facility
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