Synthesis and biological evaluation of (E)-3-(nitrophenyl)-1-(pyrazin-2-yl) prop-2-en-1-ones

Article abstract of DOI:10.1135/cccc20060044

The title (E)-(3-nitrophenyl)-1-(pyrazin-2-yl)prop-2-en-1-ones were prepared by the Claisen-Schmidt condensation of acetylpyrazines and 2-nitro-, 3-nitro- and 4-nitrobenzaldehyde in pyridine using diethylamine as the catalyst. The compounds were bioassaye

Full text of DOI:10.1135/cccc20060044

44
Opletalová et al.:
SYNTHESIS AND BIOLOGICAL EVALUATION OF
(E)-3-(NITROPHENYL)-1-(PYRAZIN-2-YL)PROP-2-EN-1-ONES
c1
Veronika OPLETALOVÁa1,*, Milan POURb1, Jiří KUNEŠb2, Vladimír BUCHTA ,
c
d
a
Luís SILVA , Katarína KRÁꢀOVÁ , Marta CHLUPÁČOVÁa2, Dana MELTROVÁ ,
a
a
Milan PETERKA and Martina POSLEDNÍKOVÁ
a
Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy,
Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic;
e-mail: veronika.opletalova@faf.cuni.cz, marta.chlupacova@faf.cuni.cz
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; e-mail: milan.pour@faf.cuni.cz,
1
2
b
c
1
2
jiri.kunes@faf.cuni.cz
Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic;
e-mail: vladimir.buchta@faf.cuni.cz
1
d
Institute of Chemistry, Faculty of Natural Sciences, Comenius University,
Mlynská dolina, 842 15 Bratislava, Slovak Republic; e-mail: kralova@fns.uniba.sk
Received Jun e 28, 2005
Accepted Septem ber 3, 2005
Th e title (E)-(3-n itroph en yl)-1-(pyrazin -2-yl)prop-2-en -1-on es were prepared by th e Claisen –
Sch m idt con den sation of acetylpyrazin es an d 2-n itro-, 3-n itro- an d 4-n itroben zaldeh yde in
pyridin e usin g dieth ylam in e as th e catalyst. Th e com poun ds were bioassayed for in vitro
an tifun gal, an tim ycobacterial an d ph otosyn th esis-in h ibitin g activity. Th e h igh poten cy of
(E)-1-(5-tert-butylpyrazin -2-yl)-3-(4-n itroph en yl)prop-2-en -1-on e again st Mycobacterium tuber-
culosis (MIC 0.78 µg/m l) an d m oderate activities of several com poun ds again st Trichophyton
mentagrophytes an d Candida spp. do n ot support th e assum ption th at ph en olic groups are es-
sen tial for an tim ycobacterial an d an tifun gal activity of ch alcon es an d th eir an alogues. In
fact, th e n itro-substituted com poun ds were superior to th e previously described h ydroxyl-
ated con gen ers with an tim ycobacterial activity (MIC ≥ 12.5 µg/m l). Th e com poun ds also re-
duced ch loroph yll con ten t in green alga Chlorella vulgaris, an d som e of th em in h ibited
ph otosyn th etic electron tran sport in spin ach ch loroplasts as well. Th e ph otosyn th esis-
in h ibitin g activity of n itro derivatives was lower th an th at of th e correspon din g h ydroxyl-
ated an alogues.
Keyw ord s: Ch alcon es; Pyrazin es; Structure-activity relation sh ip; An tifun gal activity; An ti-
m ycobacterial activity; Ph otosyn th esis-in h ibitin g activity.
Ch alcon es can be regarded as open -ch ain flavon oids, in wh ich th e two aro-
m atic rin gs are lin ked by a th ree-carbon α,β-un saturated carbon yl system 1.
Sim ilar to oth er com poun ds with th e reactive α,β-un saturated carbon yl
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 1, pp. 44–58
© 2006 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20060044

Synthesis and Biological Evaluation of Chalcones
45
fun ction ality, th ey m ay serve as in term ediates in both classical2–5 an d com -
bin atorial syn th esis6. From th e biological poin t of view, ch alcon e (1,3-di-
ph en ylprop-2-en -1-on e) is a un ique tem plate; its derivatives h ave been th e
subject of in ten se research as poten tial th erapeutic agen ts of serious an d
often life-th reaten in g diseases. It is worth n otin g th at ch alcon es are struc-
turally related to curcum in (1) an d resveratrol (2) – h igh ly popular an d in -
ten sively studied n atural products7–10
.
OH
O
O
HO
H3CO
HO
OCH3
OH
1
2
OH
Xan th oh um ol (3), licoch alcon e A (4), isoliquiritigen in (5) an d butein (6)
belon g to th e m ost in ten sively studied ch alcon es. Th ese com poun ds as well
as th eir syn th etic an alogues an d h eterocyclic con gen ers exh ibited an tiviral,
an tibacterial, an tiprotozoal, an tioxidative, an ti-in flam m atory, estrogen ic,
ch em oprotective, an tican cer an d m an y oth er in terestin g biological activi-
ties11–21
.
OH
O
OH O
OH
HO
OCH3
HO
OH
3
5
O
OH
O
OH
OH
HO
H3CO
OH
HO
4
6
As both tuberculosis an d m ycoses represen t a sign ifican t m edical prob-
lem , search for n ew an tim ycobacterial an d an tifun gal com poun ds is on e of
th e m ost ch allen gin g tasks of curren t m edicin al ch em istry22–26. In spite of
th e progress in un derstan din g th e bioch em istry of both Mycobacterium an d
various path ogen ic fun gi as well as th e m ode of action of curren t th erapeu-
tical agen ts, it is extrem ely difficult to fin d n ovel com poun ds th at would be
superior to establish ed drugs in th eir activity, an d would sim ultan eously
sh ow low toxicity an d favourable ph arm acological properties. Th us, th e
structures of th e clin ically used drugs con tin ue to serve as suitable tem -
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 1, pp. 44–58

46
Opletalová et al.:
plates for th e developm en t of n ew drug can didates. Sin ce pyrazin am ide
(pyrazin e carboxam ide) is a first-lin e drug in tuberculosis th erapy24 an d
an tim ycobacterial properties of ch alcon es an d th eir pyridin e aza an alogues
h ave also been reported14, we h ave been in terested in isosteric replacem en t
of a ph en yl m oiety in ch alcon es with th e pyrazin e rin g. Our previous paper
sh owed th at (E)-3-ph en yl-1-(pyrazin -2-yl)prop-2-en -1-on es substituted with
3-CH3O, 4-OH; 2-OH; 4-OH; 4-(CH3)2N on th e ben zen e rin g possessed both
an tim ycobacterial an d an tifun gal properties27. It was also foun d th at th eir
m olecules ten d to be plan ar28, an d th e en on e-m oiety plays an im portan t
role in th eir an tifun gal effects, but does n ot seem to be n ecessary for th e
an tim ycobacterial activity of th ese substan ces29. Based on th e well kn own
fact th at various n itro-substituted com poun ds h ave been widely used as
an tiin fective an d an tiin vasive agen ts, we decided to prepare th ree n ew se-
ries of (E)-3-ph en yl-1-(pyrazin -2-yl)prop-2-en -1-on es bearin g a n itro group
in position 2 (7a–7e), 3 (8a–8e) an d 4 (9a–9e) of th e ben zen e rin g. Biologi-
cal assays sh owed th at an tim ycobacterial an d an tifun gal poten cies of th e
n ovel com poun ds were superior to th e previously described series27. Studies
con cern in g th e effects of pyrazin e derivatives, in cludin g (E)-3-ph en yl-
1-(pyrazin -2-yl)prop-2-en -1-on es, on ph otosyn th etic processes h ave lon g-
stan din g tradition am on g our research efforts, an d th e results presen ted in
th is paper en able us to com pare th e activities of th e n itro-substituted deriv-
atives with th ose of previously reported com poun ds27,30
.
O
1
NO2
2"
O
1'
N
3
2'
1"
N
N
NO2
3"
4"
6'
2
3'
6"
R1
N
4'
5'
R1
5"
7a-7e
8a-8e
O
N
R1
N
NO2
9a-9e
RESULTS AND DISCUSSION
Th e title com poun ds were prepared by th e Claisen –Sch m idt con den sation
of acetylpyrazin es with n itroben zaldeh ydes usin g a m eth od, reported in our
previous paper27. Th e reaction is illustrated in Sch em e 1.
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Synthesis and Biological Evaluation of Chalcones
47
O
O
N
N
N
aldehyde
R2
R1
N
R1
pyridine; Et2NH
7-9
R1 = H, alkyl; R2 = 2-NO2, 3-NO2, 4-NO2
SCHEME 1
Structures of th e com poun ds were corroborated by NMR spectra; all NMR
assign m en ts are based on pulse field gradien t 2D NMR experim en ts
(gCOSY, gHSQC, an d gHMBC). Th e values of th e spin in teraction con stan t J
(15–16 Hz) were clearly in dicative of E-con figuration on th e double bon d.
Furth erm ore, th e carbon yl group can assum e s-cis or s-trans con form ation
with respect to th e vin yl double bon d31,32. Th e calculated stan dard h eats of
form ation for th e two con form ers of 3-(2-h ydroxyph en yl)-1-(pyrazin e-2-yl)-
prop-2-en -1-on e in dicated th at th e (E)-s-cis-con form er was m ore stable28
.
In frared spectra of th e propen on es 7a–7e, 8a–8e an d 9a–9e sh owed th at
(E)-s-cis-con form ers (νCO = 1676–1665 cm –1) with a m in or am oun t of
(E)-s-trans-con form ers (a sh oulder at 1654–1635 cm –1) were presen t in th e
solid state (KBr pellets).
In our previous paper, th e im portan ce of ph en olic h ydroxy groups for
an tim ycobacterial, an tifun gal an d ph otosyn th esis-in h ibitin g activity was
stressed27. Th e results of th is study sh ow th at th e substitution of th e basic
skeleton with a ph en olic h ydroxy group is n ot n ecessary for (E)-3-ph en yl-
1-(pyrazin -2-yl)prop-2-en -1-on es to sh ow an tim ycobacterial an d an tifun gal
activity. In a study perform ed by Lin an d co-workers33, th e h igh est an ti-
m ycobacterial activity (MIC = 6.80–31.5 µg/m l) was observed with
ch alcon es in wh ich th e aryl boun d to th e carbon yl group was h ydroxy-
ph en yl or h eteroaryl (pyridin -3-yl, furan -2-yl), an d th e oth er rin g was
lipoph ilic (ph en yl, 3-ch loroph en yl, 3-iodoph en yl or ph en an th ren -9-yl).
3-(Nitroph en yl)-1-(pyrazin -2-yl)prop-2-en -1-on es reported h ere also m eet
th ese structural criteria an d are even m ore poten t. Th eir MIC values
(0.78–6.25 µg/m l) are on e order of m agn itude lower. It is also im portan t to
stress th at both th e com poun ds prepared by Lin an d co-workers an d th e
propen on es described in th e presen t work were tested by th e Tuberculosis
An tim icrobial Acquisition Coordin atin g Facility (TAACF). Th erefore, th e
MICs were obtain ed un der th e sam e experim en tal con dition s an d are fully
com parable. Th us, substitution of th e ben zen e rin g with a n itro group
seem s to be favourable for th e an tim ycobacterial activity.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 1, pp. 44–58

48
Opletalová et al.:
In order to see wh eth er th e differen ce in lipoph ilicity could be respon si-
ble for th e h igh er an tim ycobacterial poten cy of 3-(n itroph en yl)-1-(pyrazin -
2-yl)prop-2-en -1-on es as com pared to 3-(h ydroxyph en yl)-1-(pyrazin -2-yl)-
prop-2-en -1-on es reported in our previous paper27, log P (th e logarith m of
th e partition coefficien t for n-octan ol/water) an d C log P (th e logarith m of
n-octan ol/water partition coefficien t based on establish ed ch em ical in terac-
tion s) of both series were calculated by m ean s of Ch em Draw Ultra, ver. 7.0
(Cam bridgeSoft, Cam bridge, MA, U.S.A.). It was foun d th at th e n itro deriva-
tives are on ly sligh tly m ore lipoph ilic (log P = 1.32–3.43; C log P =
1.71066–3.79666) th an th e h ydroxy derivatives (log P = 0.94–3.07; C log P =
1.30066–3.38667). In addition , for th e n on -alkylated 3-(4-n itroph en yl)-
1-(pyrazin -2-yl)prop-2-en -1-on e (9a) log P = 1.32 an d C log P = 1.71066, for
3-(4-n itroph en yl)-1-(5-tert-butylpyrazin -2-yl)prop-2-en -1-on e (9b) log P =
3.43 an d C log P = 3.53667, an d for 3-(4-n itroph en yl)-1-(5-propylpyrazin -
2-yl)prop-2-en -1-on e (9e) th e calculated lipoph ilicity param eters are 2.88
an d 3.26767, respectively. However, all th ese com poun ds exh ibited 100%
in h ibition of M. tuberculosis H37Rv in th e prim ary screen in g test (Table I). In
con trast, 2-n itro derivatives 7b–7e an d 3-n itro derivatives 8a–8e were less
effective th an th e correspon din g 4-n itro derivatives 9a–9e even th ough th e
calculated log P an d C log P values are th e sam e for th e th ree series. Hen ce,
lipoph ilicity probably plays a m in or role in th e an tim ycobacterial proper-
ties of ch alcon es an d th eir h eterocyclic an alogues, an d oth er structural fac-
tors m ust be taken in to accoun t. Th e n itro group probably gives rise to a
favourable electron distribution in th e con jugated system of ch alcon es due
to its stron g electron -with drawin g properties. Th is in fluen ce is especially
obvious with 4-n itro derivatives 9a–9e th at in h ibited th e growth of
M. tuberculosis alm ost com pletely (85–100%), wh ile th e correspon din g
4-dim eth ylam in o derivatives27 were practically in active (0–26%), an d
4-h ydroxy derivatives27 displayed in term ediate poten cy (35–72%). Regard-
in g substitution of th e pyrazin e rin g, both our previous results27 an d h igh
poten cy of com poun d 9b in dicate th at th e presen ce of a tert-butyl group is
favourable for an tim ycobacterial properties of th e studied com poun ds.
Th e im portan ce of electron -with drawin g groups in th e B-rin g of
ch alcon es for th eir an tifun gal activity h ave previously been reported by
López an d co-workers34,35. For (E)-3-ph en yl-1-(pyrazin -2-yl)prop-2-en -1-on es
prepared in our laboratory, th e depen den ce of an tifun gal poten cy on th e
substitution was less pron oun ced. Neverth eless, th e n itro derivatives were
m ore poten t th an th eir h ydroxylated an alogues in m ost cases. Wh ilst th e
ch alcon es studied by López et al. were effective on ly again st derm ato-
ph ytes34,35, som e (E)-3-(n itroph en yl)-1-(pyrazin -2-yl)prop-2-en -1-on es as
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 1, pp. 44–58

Synthesis and Biological Evaluation of Chalcones
49
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 1, pp. 44–58

50
Opletalová et al.:
well as th eir h ydroxylated con gen ers27,36, e.g. 10, 11 an d 12, were also ac-
tive again st Candida albicans, C. tropicalis, C. krusei an d C. glabrata (MIC =
7.81–125 µm ol/l). Furth erm ore, com poun d 11 in h ibited th e growth of
Trichosporon beigelii (MIC = 31.25 µm ol/l) an d Aspergillus fumigatus (MIC =
31.25–62.5 µm ol/l). Am on g th e fun gal strain s, Trichophyton mentagrophytes
was th e m ost susceptible to th e effects of (E)-3-ph en yl-1-(pyrazin -2-yl)-
prop-2-en -1-on es. Most studied com poun ds (except for 7b, 7e an d 8b) ex-
h ibited m oderate poten cy again st th is strain . In con trast to an tim yco-
bacterial activity, substitution of th e pyrazin e rin g with tert-butyl group
seem s to be un favorable for th e in h ibitory effect again st T. mentagrophytes
(see th e MICs of com poun ds 7b, 8b an d th e referen ce com poun d 13 in
Table I).
O
O
OH
N
N
OH
N
N
10
11
O
OH
O
N
N
N
N
OH
13
12
In th e oxygen evolution rate (OER) study with spin ach ch loroplasts, poor
water solubility of th e com poun ds was a serious problem (m ost com poun ds
precipitated after addin g th eir solution s in DMSO to th e suspen sion s of
ch loroplasts). Con sequen tly, on ly a few data could be obtain ed. Neverth e-
less, th e com parison of IC50 values (Table I) of com poun ds 7b, 7d an d 9d
with th ose reported for th e previously tested com parable derivatives 13, 10
27
an d 12 clearly sh ows th at n itro derivatives were approxim ately h alf as ac-
tive as th e correspon din g h ydroxylated com poun ds. Th e decreased aqueous
solubility lim itin g th e passage of th e com poun ds th rough th e h ydroph ilic
region s of th ylakoid m em bran es, an d h en ce a reduced access to th eir site of
action is probably respon sible for th e observed activity decrease. Th e stud-
ied 3-(2-n itroph en yl)-1-(pyrazin -2-yl)prop-2-en -1-on es 7a, 7c, 7d , 7e,
3-(3-n itroph en yl)-1-(pyrazin -2-yl)prop-2-en -1-on es 8a, 8c, 8e as well as th e
4-n itro com poun ds 9a an d 9e in h ibited ch loroph yll production in Chlorella
vulgaris (Table I). Due to low aqueous solubility of th e rem ain in g com -
poun ds, th e correspon din g IC50 values could n ot be determ in ed. In th e
studied con cen tration ran ge 0.83–100 µm ol/l, th ese com poun ds caused ap-
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 1, pp. 44–58

Synthesis and Biological Evaluation of Chalcones
51
proxim ately 7–15% reduction in ch loroph yll con ten t in th e suspen sion of
Chlorella vulgaris.
In spite of m uch research effort devoted to ch alcon es an d th eir h etero-
cyclic an alogues, little is kn own about th eir m ech an ism s of action at th e
m olecular level. In th e ph otosyn th etic apparatus of spin ach ch loroplasts,
th ese com poun ds probably in teract with th e tyrosin e radical in position
161 (TyrD), located in D2 protein at th e don or site of ph otosystem 2, wh ich
results in th e in h ibition of th e ph otosyn th etic electron tran sport37. Th e re-
duction of ch loroph yll con ten t in Chlorella vulgaris in th e presen ce of th e
studied com poun ds could h ave been caused by th e in h ibition of bioch em i-
cal processes associated with th e biosyn th esis of th is im portan t ph oto-
syn th etic pigm en t an d/or oth er con stituen ts of th e plan t cell, e.g. protein s
or lipids. Moreover, th e in h ibition of th e ph otosyn th etic electron tran sport
in Chlorella vulgaris by a series of related rin g-substituted (E)-3-ph en yl-
1-(pyrazin -2-yl)prop-2-en -1-on es was reported previously27. Regardin g an ti-
fun gal activities, it was suggested th at ch alcon e-like com poun ds m igh t act
on th e fun gal cell m em bran es38. In h ibition of β(1,3)-glucan an d ch itin
syn th ases by ch alcon e derivatives h as recen tly been reported34. It is well
kn own th at α,β-un saturated carbon yl com poun ds can react with n ucleo-
ph iles, e.g. th iol or am in o groups of en zym es, to give Mich ael-addition type
products39. Hen ce, en zym e in h ibition m igh t play a m ore gen eral role in th e
biological effects of ch alcon es an d th eir h eterocyclic an alogues. To th e best
of our kn owledge, n o study aim ed at determ in in g th e m olecular m ech a-
n ism s of an tim ycobacterial effects of ch alcon es an d th eir an alogues h as
been publish ed, even th ough th eir an tim ycobacterial properties h ave been
kn own sin ce th e 1950’s40. Sin ce th en on ly a few papers con cern in g th e ef-
fects of ch alcon e-like com poun ds on m ycobacteria33,41–44 h ave appeared,
an d th e relation sh ips between th eir structure an d an tim ycobacterial activ-
ity rem ain to be defin ed.
Th e results obtain ed in th is study clearly sh ow th at ch alcon es an d th eir
h eterocyclic an alogues bearin g electron -with drawin g groups in th e B-rin g
deserve furth er atten tion as poten tial an tituberculous an d an tim ycotic
agen ts. Th e best an tim ycobacterial activity was foun d with (E)-1-(5-tert-
butylpyrazin -2-yl)-3-(4-n itroph en yl)prop-2-en -1-on e (9b ), an d th e best
an tifun gal properties were exh ibited by (E)-3-(3-n itroph en yl)-1-(pyrazin -
2-yl)prop-2-en -1-on es (8a, 8d an d 8e). Com poun d 8a was effective n ot on ly
again st T. mentagrophytes, but also again st Candida spp. (MIC = 7.81–15.63
µm ol/l). Furth er testin g an d possible developm en t of th ese com poun ds will
depen d on th e results of cytotoxicity assays. In th e tests perform ed in
TAACF, evaluation of cytotoxicity h as been com plicated by th e poor solu-
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 1, pp. 44–58

52
Opletalová et al.:
bility in th e tissue culture m edium . Neverth eless, th e calculated lipo-
ph ilicity param eters in dicate th at th e com poun ds m eet th e criteria45 set by
th e Lipin ski’s rule of 5′, an d if n eed arises, th e solubility problem s could be
resolved usin g m odern approach es of ph arm aceutical tech n ology.
EXPERIMENTAL
Acetylpyrazin es prepared as described previously46 an d com m ercially available n itroben z-
aldeh ydes were used as th e startin g m aterials. Silpearl (Kavalier, Votice) was used for flash
colum n ch rom atograph y. Th e purity of th e products was ch ecked by TLC on Silufol UV 254
plates (Kavalier, Votice). Th e followin g solven t m ixtures were used for TLC: ligh t petroleum –
eth yl acetate 60:40 (v/v) an d ligh t petroleum –eth yl acetate 80:20 (v/v). An alytical sam ples
were dried over an h ydrous ph osph orus pen toxide un der reduced pressure at room tem pera-
ture. Meltin g poin ts were determ in ed on a Boëtius apparatus an d are un corrected. Elem en tal
an alyses were perform ed on an EA 1110 CHNS in strum en t (CE In strum en ts, Milan o, Italy).
In frared spectra were recorded in KBr pellets on a Nicolet Im pact 400 IR spectroph otom eter.
Ch aracteristic waven um bers are given in cm –1 1H an d 13C NMR spectra were recorded in
.
CDCl3 solution s at am bien t tem perature on a Varian Mercury-Vx BB 300 spectrom eter oper-
atin g at 300 MHz for 1H an d 75 MHz for 13C. Ch em ical sh ifts were recorded as δ values in
ppm , an d were in directly referen ced to tetram eth ylsilan e (TMS) via th e solven t sign al (7.26
for 1H, 77.0 for 13C in CDCl3). Couplin g con stan ts J are given in Hz.
Preparation of Com poun ds 7a–7e, 8a–8e an d 9a–9e. Gen eral Procedure
Acetylpyrazin e (0.01 m ol) an d a n itroben zaldeh yde (0.01 m ol) were dissolved in pyridin e
(4.4 m l). Dieth ylam in e (0.73 g, 0.01 m ol) was added, an d th e reaction m ixture was stirred at
80–120 °C for 2 h . After coolin g, th e m ixture was poured in to ice water (200 m l), acidified
to pH 3 with a few drops of acetic acid, an d th en refrigerated for 24 h . Th e separation of
crude products from water depen ded on th eir ch aracter. Solids were filtered off an d crystal-
lized wh ile oily m ixtures were extracted with dieth yl eth er an d subjected to flash ch rom a-
tograph y on silica gel. Ligh t petroleum –eth yl acetate 60:40 (v/v) was used as th e eluen t for
com poun ds 7a–7e, wh ilst 80:20 (v/v) ratio of th e two solven ts proved to be m ore suitable
for com poun ds 8a–8e an d 9a–9e. Th e fraction s con tain in g th e desired com poun ds were
com bin ed an d crystallized. Repeated crystallization from absolute eth an ol was sufficien t to
obtain an alytically pure crystals of com poun ds 7a–7e an d 8a–8e. Com poun ds 9a–9e h ad to
be crystallized from aceton e to rem ove th e startin g 4-n itroben zaldeh yde com pletely. Th e fi-
n al crystallization was th en perform ed from absolute eth an ol.
(E)-3-(2-Nitrophenyl)-1-(pyrazin-2-yl)prop-2-en-1-one (7a). Yield 22%, m .p. 144–145 °C (ref.31
196–198 °C). For C13H9N3O3 (255.2) calculated: 61.18% C, 3.55% H, 16.46% N; foun d:
60.93% C, 3.83% H, 16.50% N. IR: 1671 (C=O), 1608 (CH=CH). 1H NMR: 8.07 d, 1 H, J =
15.9 (H-2); 8.39 d, 1 H, J = 15.9 (H-3); 9.39 d, 1 H, J = 1.5 (H-3′); 8.80 d, 1 H, J = 2.5 (H-5′);
8.69 dd, 1 H, J = 1.5 an d 2.5 (H-6′,); 8.07 dd, 1 H, J = 1.4 an d 7.9 (H-3′′); 7.58 td, 1 H, J = 1.4
an d 7.9 (H-4′′); 7.70 td, 1 H, J = 1.4 an d 7.9 (H-5′′); 7.86 dd, 1 H, J = 1.4 an d 7.9 (H-6′′).
13C NMR: 188.7 (C-1); 124.7 (C-2); 140.6 (C-3); 147.8 (C-2′); 144.9 (C-3′); 147.6 (C-5′); 143.3
(C-6′); 130.9 (C-1′′); 148.8 (C-2′′); 124.9 (C-3"); 130.6 (C-4′′); 133.4 (C-5′′); 129.2 (C-6′′).
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Synthesis and Biological Evaluation of Chalcones
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(E)-1-(5-tert-Butylpyrazin-2-yl)-3-(2-nitrophenyl)prop-2-en-1-one (7b). Yield 20%, m .p.131–134 °C.
For C17H17N3O3 (311.3) calculated: 65.58% C, 5.50% H, 13.50% N; foun d: 65.16% C, 5.36% H,
13.32% N. IR: 1672 (C=O), 1607 (CH=CH). 1H NMR: 8.04 d, 1 H, J = 15.9 (H-2); 8.33 d, 1 H,
J = 15.9 (H-3); 9.26 d, 1 H, J = 1.4 (H-3′); 8.71 d, 1 H, J = 1.4 (H-6′); 8.03 dd, 1 H, J = 1.3 an d
7.9 (H-3′′); 7.56 td, 1 H, J = 1.3 an d 7.9 (H-4′′); 7.67 td, 1 H, J = 1.3 an d 7.9 (H-5′′); 7.84 dd,
1 H, J = 1.3 an d 7.9 (H-6′′); 1.43, 9 H (tert-butyl). 13C NMR: 188.1 (C-1); 125.1 (C-2); 140.1
(C-3); 145.4 (C-2′); 143.6 (C-3′); 168.2 (C-5′); 140.2 (C-6′); 131.3 (C-1′′); 149.0 (C-2′′); 125.4
(C-3′′); 130.7 (C-4′′); 133.6 (C-5′′); 129.5 (C-6′′); 30.0, 37.5 (tert-butyl).
(E)-1-(5-Isobutylpyrazin-2-yl)-3-(2-nitrophenyl)prop-2-en-1-one (7c). Yield 16%, m .p. 103–104 °C.
For C17H17N3O3 (311.3) calculated: 65.58% C, 5.50% H, 13.50% N; foun d: 65.66% C, 5.64% H,
13.75% N. IR: 1673 (C=O), 1608 (CH=CH). 1H NMR: 8.06 d, 1 H, J = 15.9 (H-2); 8.36 d, 1 H,
J = 15.9 (H-3); 9.30 d, 1 H, J = 1.5 (H-3′); 8.48 d, 1 H, J = 1.5 (H-6′); 8.05 dd, 1 H, J = 1.4 an d
8.0 (H-3′′); 7.57 td, 1 H, J = 1.4 an d 8.0 (H-4′′); 7.68 td, 1 H, J = 1.4 an d 8.0 (H-5′′); 7.85 dd,
1 H, J = 1.4 an d 8.0 (H-6′′); 0.97 d, 6 H, J = 6.6, 2.18 m , 1 H, 2.79 d, 2 H, J = 7.1 (isobutyl).
13C NMR: 187.9 (C-1); 125.0 (C-2); 140.1 (C-3); 145.5 (C-2′); 144.2 (C-3′); 160.8 (C-5′); 143.2
(C-6′); 131.0 (C-1′′); 148.8 (C-2′′); 124.9 (C-3′′); 130.5 (C-4′′); 133.4 (C-5′′); 129.3 (C-6′′);
22.4, 29.2, 44.8 (isobutyl).
(E)-1-(5-Butylpyrazin-2-yl)-3-(2-nitrophenyl)prop-2-en-1-one (7d ). Yield 20%, m .p. 75–76 °C.
For C17H17N3O3 (311.3) calculated: 65.58% C, 5.50% H, 13.50% N; foun d: 65.66% C, 5.60% H,
13.66% N. IR: 1668 (C=O), 1611 (CH=CH). 1H NMR: 8.05 d, 1 H, J = 15.8 (H-2); 8.36 d, 1 H,
J = 15.8 (H-3); 9.29 d, 1 H, J = 1.5 (H-3′); 8.52 d, 1 H, J = 1.5 (H-6′); 8.06 dd, 1 H, J = 1.4 an d
8.0 (H-3′′); 7.57 td, 1 H, J = 1.4 an d 8.0 (H-4′′); 7.69 td, 1 H, J = 1.4 an d 8.0 (H-5′′); 7.85 dd,
1 H, J = 1.4 an d 8.0 (H-6′′); 0.96, 3 H, J = 7.3, 1.42 m , 2 H, 1.78 m , 2 H, 2.93 t, 2 H, J = 8.0
(butyl). 13C NMR: 187.8 (C-1); 125.1 (C-2); 140.0 (C-3); 145.5 (C-2′); 144.1 (C-3′); 161.6
(C-5′); 142.7 (C-6′); 131.0 (C-1′′); 148.7 (C-2′′); 124.9 (C-3′′); 130.4 (C-4′′); 133.3 (C-5′′);
129.2 (C-6′′); 13.9, 22.5, 31.4, 35.6 (butyl).
(E)-3-(2-Nitrophenyl)-1-(5-propylpyrazin-2-yl)prop-2-en-1-one (7e). Yield 21%, m .p. 129–131 °C.
For C16H15N3O3 (297.3) calculated: 64.64% C, 5.09% H, 14.13% N; foun d: 64.38% C, 5.17% H,
14.26% N. IR: 1671 (C=O), 1606 (CH=CH). 1H NMR: 8.05 d, 1 H, J = 15.9 (H-2); 8.34 d, 1 H,
J = 15.9 (H-3); 9.27 d, 1 H, J = 1.5 (H-3′); 8.51 d, 1 H, J = 1.5 (H-6′); 8.04 dd, 1 H, J = 1.3 an d
7.8 (H-3′′); 7.56 td, 1 H, J = 1.3 an d 7.8 (H-4′′); 7.68 td, 1 H, J = 1.3 an d 7.8 (H-5′′); 7.84 td,
1 H, J = 1.3 an d 7.8 (H-6′′); 1.00 t, 3 H, J = 7.3, 1.82 m , 2 H, 2.89 t, 2 H, J = 8.0 (propyl).
13C NMR: 187.8 (C-1); 125.0 (C-2); 140.0 (C-3); 145.5 (C-2′); 144.1 (C-3′); 161.4 (C-5′); 142.8
(C-6′); 131.0 (C-1′′); 148.7 (C-2′′); 124.8 (C-3′′); 130.4 (C-4′′); 133.3 (C-5′′); 129.2 (C-6′′);
13.8, 22.6, 37.8 (propyl).
(E)-3-(3-Nitrophenyl)-1-(pyrazin-2-yl)prop-2-en-1-one (8a). Yield 42%, m .p. 165–168 °C (ref.31
166–168 °C). For C13H9N3O3 (255.2) calculated: 61.18% C, 3.55% H, 16.46% N; foun d:
61.13% C, 3.65% H, 16.34% N. IR: 1676 (C=O), 1609 (CH=CH). 1H NMR: 7.97 d, 1 H, J =
16.1 (H-2); 8.30 d, 1 H, J = 16.1 (H-3); 9.38 d, 1 H, J = 1.5 (H-3′); 8.81 d, 1 H, J = 2.5 (H-5′);
8.72 dd, 1 H, J = 1.5 an d 2.5 (H-6′); 8.57 t, 1 H, J = 1.9 (H-2′′); 8.02–7.96 m , 1 H (H-4′′);
7.62, 1 H, J = 8.0 (H-5′′); 8.29–8.24 m , 1 H (H-6′′). 13C NMR: 188.1 (C-1); 125.0 (C-2); 142.3
(C-3); 147.8 (C-2′); 144.9 (C-3′); 147.8 (C-5′); 143.4 (C-6′); 136.1 (C-1′′); 122.7 (C-2′′); 148.7
(C-3′′); 122.8 (C-4′′); 134.6 (C-5′′); 130.0 (C-6′′).
(E)-1-(5-tert-Butylpyrazin-2-yl)-3-(3-nitrophenyl)prop-2-en-1-one (8b ). Yield 40%, m .p.
160–162 °C. For C17H17N3O3 (311.3) calculated: 65.58% C, 5.50% H, 13.50% N; foun d:
65.21% C, 5.56% H, 13.70% N. IR: 1676 (C=O), 1610 (CH=CH). 1H NMR: 7.95 d, 1 H, J =
15.9 (H-2); 8.30 d, 1 H, J = 15.9 (H-3); 9.29 d, 1 H, J = 1.7 (H-3′); 8.76 d, 1 H, J = 1.7 (H-6′);
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8.57 t, 1 H, J = 1.8 (H-2′′); 8.02–7.96 m , 1 H (H-4′′); 7.62 t, 1 H, J = 8.0 (H-5′′); 8.29–8.23 m ,
1 H (H-6′′); 1.46 s, 9 H (tert-butyl). 13C NMR: 188.2 (C-1); 124.8 (C-2); 140.0 (C-3); 145.2
(C-2′); 143.4 (C-3′); 168.2 (C-5′); 141.8 (C-6′); 136.6 (C-1′′); 122.8 (C-2′′); 148.7 (C-3′′); 123.2
(C-4′′); 134.5 (C-5′′); 130.0 (C-6′′); 29.7, 37.2 (tert-butyl).
(E)-1-(5-Isobutylpyrazin-2-yl)-3-(3-nitrophenyl)prop-2-en-1-one (8c). Yield 40%, m .p. 103–104 °C.
For C17H17N3O3 (311.3) calculated: 65.58% C, 5.50% H, 13.50% N; foun d: 65.23% C, 5.56% H,
13.65% N. IR: 1676 (C=O), 1610 (CH=CH). 1H NMR: 7.95 d, 1 H, J = 15.9 (H-2); 8.30 d, 1 H,
J = 15.9 (H-3); 9.30 d, 1 H, J = 1.5 (H-3′); 8.52 d, 1 H, J = 1.5 (H-6′); 8.58 t, 1 H, J = 2.2
(H-2′′); 8.02–7.96 m , 1 H (H-4′′); 7.62 t, 1 H, J = 8.0 (H-5′′); 8.29–8.23 m , 1 H (H-6′′); 0.98 d,
6 H, J = 6.9, 2.27–2.11 m , 1 H, 2.80 d, 2 H, J = 7.1 (isobutyl). 13C NMR: 188.2 (C-1); 124.9
(C-2); 141.9 (C-3); 145.6 (C-2′); 144.1 (C-3′); 161.0 (C-5′); 143.4 (C-6′); 136.6 (C-1′′); 122.8
(C-2′′); 148.7 (C-3′′); 123.1 (C-4′′); 134.6 (C-5′′); 130.0 (C-6′′); 22.3, 29.2, 44.8 (isobutyl).
(E)-1-(5-Butylpyrazin-2-yl)-3-(3-nitrophenyl)prop-2-en-1-one (8d ). Yield 30%, m .p. 102-104 °C.
For C17H17N3O3 (311.3) calculated: 65.58% C, 5.50% H, 13.50% N; foun d: 65.15% C, 5.56% H,
13.76% N. IR: 1672 (C=O), 1609 (CH=CH). 1H NMR: 7.95 d, 1 H, J = 16.1 (H-2); 8.30 d, 1 H,
J = 16.1 (H-3); 9.28 d, 1 H, J = 1.4 (H-3′); 8.55 d, 1 H, J = 1.4 (H-6′); 8.57 t, 1 H, J = 2.0
(H-2′′); 8.01–7.96 m , 1 H (H-4′′); 7.62 t, 1 H, J = 8.0 (H-5′′); 8.26 ddd overlapped, 1 H, J =
8.24 an d 2.0 an d 1.1 (H-6′′); 0.96 t, 3 H, J = 7.6, 1.50–1.35 m , 2 H, 1.84–1.72 m , 2 H, 2.93 t,
2 H, J = 7.6 (butyl). 13C NMR: 188.2 (C-1); 124.8 (C-2); 141.9 (C-3); 145.6 (C-2′); 144.1
(C-3′); 161.9 (C-5′); 142.9 (C-6′); 136.6 (C-1′′); 122.8 (C-2′′); 148.7 (C-3′′); 123.1 (C-4′′); 134.6
(C-5′′); 130.0 (C-6′′); 13.8, 22.4, 31.4, 35.6 (butyl).
(E) 3-(3-Nitrophenyl)-1-(5-propylpyrazin-2-yl)prop-2-en-1-one (8e). Yield 18%, m .p. 91–92 °C.
For C16H15N3O3 (297.3) calculated: 64.64% C, 5.09% H, 14.13% N; foun d: 64.70% C, 5.20% H,
14.13% N. IR: 1669 (C=O), 1609 (CH=CH). 1H NMR: 7.90 d overlapped, 1 H, J = 15.8 (H-2);
8.30 d overlapped, 1 H, J = 15.8 (H-3); 9.28 d, 1 H, J = 1.6 (H-3′); 8.55 d, 1 H, J = 1.6 (H-6′);
8.57 t, 1 H, J = 2.1 (H-2′′); 8.29–8.22 m , 1 H (H-4′′); 7.62 t, 1 H, J = 8.0 (H-5′′); 7.95–8.00 m ,
1 H (H-6′′); 1.01 t, 3 H, J = 7.3, 1.90–1.75 m , 2 H, 2.91 t, 2 H, J = 7.6 (propyl). 13C NMR:
188.2 (C-1); 124.8 (C-2); 141.9 (C-3); 145.6 (C-2′); 144.1 (C-3′); 161.6 (C-5′); 142.9 (C-6′);
136.6 (C-1′′); 122.8 (C-2′′); 148.7 (C-3′′); 123.1 (C-4′′); 134.6 (C-5′′); 130.0 (C-6′′); 13.8, 22.6,
37.7 (propyl).
(E)-3-(4-Nitrophenyl)-1-(pyrazin-2-yl)prop-2-en-1-one (9a). Yield 43%, m .p. 188–192 °C (ref.31
189–192 °C). For C13H9N3O3 (255.2) calculated: 61.18% C, 3.55% H, 16.46% N; foun d:
61.47% C, 3.67% H, 16.22% N. IR: 1672 (C=O), 1612 (CH=CH). 1H NMR: 7.96 d, 1 H, J =
16.2 (H-2); 8.30 d, 1 H, J = 16.2 (H-3); 9.39 d, 1 H, J = 1.5 (H-3′); 8.82 d, 1 H, J = 2.6 (H-5′);
8.72 dd, 1 H, J = 1.5 an d 2.6 (H-6′); 7.86 d, 2 H, J = 8.6 (H-2′′ an d H-6′′); 8.28, 2 H, J = 8.6
(H-3′′ an d H-5′′). 13C NMR: 188.1 (C-1); 123.8 (C-2); 142.2 (C-3); 147.9 (C-2′); 144.7 (C-3′);
147.8 (C-5′); 143.4 (C-6′); 140.8 (C-1′′); 129.4 (C-2′′ an d C-6′′); 124.2 (C-3′′ an d C-5′′); 148.7
(C-4′′).
(E)-1-(5-tert-Butylpyrazin-2-yl)-3-(4-nitrophenyl)prop-2-en-1-one (9b ). Yield 43%, m .p.
142–144 °C. For C17H17N3O3 (311.3) calculated: 65.58% C, 5.50% H, 13.50% N; foun d:
65.74% C, 5.69% H, 13.75% N. IR: 1672 (C=O), 1613 (CH=CH). 1H NMR: 7.92 d, 1 H, J =
16.0 (H-2); 8.28 d, 1 H, J = 16.0 (H-3); 9.27 d, 1 H, J = 1.5 (H-3′); 8.73 d, 1 H, J = 1.5 (H-6′);
7.84 d, 2 H, J = 8.8 (H-2′′ an d H-6′′); 8.26 d, 2 H, J = 8.8 (H-3′′ an d H-5′′); 1.44 s, 9 H (tert-
butyl). 13C NMR: 188.1 (C-1); 124.1 (C-2); 141.6 (C-3); 145.1 (C-2′); 143.4 (C-3′); 168.2
(C-5′); 139.9 (C-6′); 140.9 (C-1′′); 129.2 (C-2′′ an d C-6′′); 124.1 (C-3′′ an d C-5′′); 148.6 (C-4′′);
29.7, 37.2 (tert-butyl).
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Synthesis and Biological Evaluation of Chalcones
55
(E)-1-(5-Isobutylpyrazin-2-yl)-3-(4-nitrophenyl)prop-2-en-1-one (9c). Yield 45%, m .p. 141–143 °C.
For C17H17N3O3 (311.3) calculated: 65.58% C, 5.50% H, 13.50% N; foun d: 65.23% C, 5.67% H,
13.38% N. IR: 1672 (C=O), 1610 (CH=CH). 1H NMR: 7.94 d, 1 H, J = 16.0 (H-2); 8.30 d, 1 H,
J = 16.0 (H-3); 9.29 d, 1 H, J = 1.5 (H-3′); 8.51 d, 1 H, J = 1.5 (H-6′); 7.85 d, 2 H, J = 8.8 (H-2′′
an d H-6′′); 8.27 d, 2 H, J = 8.8 (H-3′′ an d H-5′′); 0.97 d, 6 H, J = 6.6, 2.18 m , 1 H, 2.80 d,
2 H, J = 7.3 (isobutyl). 13C NMR: 188.2 (C-1); 124.2 (C-2); 141.7 (C-3); 145.5 (C-2′); 144.2
(C-3′); 161.1 (C-5′); 143.4 (C-6′); 140.9 (C-1′′); 129.3 (C-2′′ an d C-6′′); 124.2 (C-3′′ an d C-5′′);
148.7 (C-4′′); 22.4, 29.2, 44.8 (isobutyl).
(E)-1-(5-Butylpyrazin-2-yl)-3-(4-nitrophenyl)prop-2-en-1-one (9d). Yield 40%, m .p. 117–119 °C.
For C17H17N3O3 (311.3) calculated: 65.58% C, 5.50% H, 13.50% N; foun d: 65.47% C, 5.56% H,
13.71% N. IR: 1665 (C=O), 1609 (CH=CH). 1H NMR: 7.93 d, 1 H, J = 16.0 (H-2); 8.29 d, 1 H,
J = 16.0 (H-3); 9.28 d, 1 H, J = 1.4 (H-3′); 8.54 d, 1 H, J = 1.4 (H-6′); 7.85 d, 2 H, J = 8.8 (H-2′′
an d H-6′′); 8.27 d, 2 H, J = 8.8 (H-3′′ an d H-5′′); 0.96 t, 3 H, J = 7.4, 1.42 m , 2 H, 1.78 m ,
2 H, 2.93 t, 2 H, J = 7.7 (butyl). 13C NMR: 188.1 (C-1); 124.2 (C-2); 141.7 (C-3); 145.5 (C-2′);
144.2 (C-3′); 161.9 (C-5′); 142.9 (C-6′); 140.9 (C-1′′); 129.3 (C-2′′ an d C-6′′); 124.2 (C-3′′ an d
C-5′′); 148.6 (C-4′′); 13.8, 22.4, 31.4, 35.6 (butyl).
(E)-3-(4-Nitrophenyl)-1-(5-propylpyrazin-2-yl)prop-2-en-1-one (9e). Yield 40%, m .p. 124–127 °C.
For C16H15N3O3 (297.3) calculated: 64.64% C, 5.09% H, 14.13% N; foun d: 64.66% C, 5.12% H,
14.09% N. IR: 1672 (C=O), 1612 (CH=CH). 1H NMR: 7.94 d, 1 H, J = 16.0 (H-2); 8.29 d, 1 H,
J = 16.0 (H-3); 9.29 d, 1 H, J = 1.4 (H-3′); 8.54 d, 1 H, J = 1.4 (H-6′); 7.85 d, 2 H, J = 8.8 (H-2′′
an d H-6′′); 8.27d, 2 H, J = 8.8 (H-3′′ an d H-5′′); 1.01 t, 3 H, J = 7.4, 1.83 m , 2 H, 2.91 t, 2 H,
J = 7.7 (propyl). 13C NMR: 188.1 (C-1); 124.2 (C-2); 141.7 (C-3); 145.6 (C-2′); 144.3 (C-3′);
161.7 (C-5′); 142.9 (C-6′); 140.9 (C-1′′); 129.3 (C-2′′ an d C-6′′); 124.2 (C-3′′ an d C-5′′); 148.6
(C-4′′); 14.0, 22.8, 38.0 (propyl).
Evaluation of in vitro An tifun gal Activity
An tifun gal activity of all com poun ds again st Candida albicans ATCC 44859, Candida
tropicalis 156, Candida krusei E 28, Candida glabrata 20/I, Trichosporon beigelii 1188, Aspergillus
fumigatus 231, Absidia corymbifera 272 an d Trichophyton mentagrophytes 445 was evaluated by
th e m icrodilution broth m eth od. All strain s were subcultured on Sabouraud dextrose agar
(SDA, Difco) an d m ain tain ed on th e sam e m edium at 4 °C. Prior to testin g, each strain was
passaged on to SDA an d fun gal in ocula were prepared by suspen din g yeasts or con idia or
sporan giospores in sterile 0.85% salin e. Th e cell den sity was adjusted, usin g th e Bürker’s
ch am ber, to yield a stock suspen sion of (1.0 ± 0.2) × 105 CFU/m l. Th e fin al in oculum was
m ade by 1:20 dilution of th e stock suspen sion with th e test m edium .
Th e com poun ds were dissolved in dim eth yl sulfoxide (DMSO) an d an tifun gal activity was
determ in ed in th e tissue culture m edium RPMI 1640 (Sevaph arm a, Prague, Czech Republic)
buffered to pH 7.0 with 0.165 M 3-m orph olin opropan e-1-sulfon ic acid (Sigm a). Con trols
were in cluded as well. Th e fin al con cen tration of DMSO in th e test m edium did n ot exceed
1% (v/v) of th e total solution com position . Th e m in im um in h ibitory con cen tration (MIC),
defin ed as 80% in h ibition of fun gal growth com pared to con trol, were determ in ed after 24
an d 48 h of static in cubation at 35 °C. In th e case of T. mentagrophytes th e MICs were re-
corded after 72 an d 120 h . Ketocon azole (batch No. E 3401, Jan ssen Ph arm aceutica) was
used as a referen ce an tifun gal drug. Th e results are given in Table I.
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Evaluation of An tim ycobacterial Activity
Prim ary screen in g of all com poun ds was con ducted at 6.25 µg/m l again st Mycobacterium
tuberculosis H37Rv (ATCC 27294) in th e Bactec 12B m edium usin g a broth m icrodilution as-
say (Microplate Alam ar Blue Assay, MABA)47. Com poun ds exh ibitin g fluorescen ce were
tested in th e Bactec 460 radiom etric system 47. Th e com poun ds displayin g at least 90% in h i-
bition in th e prim ary screen were reevaluated at lower con cen tration s again st M. tuberculosis
H37Rv to determ in e th e m in im um in h ibitory con cen tration (MIC) usin g MABA. Th e MIC is
defin ed as th e lowest con cen tration effectin g th e 90% reduction in fluorescen ce relative to
con trol. For th e results, see Table I.
Evaluation of Cytotoxicity
Th e com poun ds th at exh ibited prom isin g an tim ycobacterial activity (7a, 9a an d 9b) were
tested for cytotoxicity (IC50) in VERO cells at con cen tration s less th an or equal to 10 tim es
th e MIC for M. tuberculosis H37Rv. After 72-h exposure, viability was assessed on th e basis of
cellular con version of 1-(4,5-dim eth ylth iazol-2-yl)-2,5-diph en yltetrazolium (MTT) in to a
form azan product usin g th e Prom ega CellTiter 96 Non -radioactive Cell Proliferation Assay.
For com poun d 7a, IC50 was 0.86 µg/m l an d IC50 values for th e rem ain in g two derivatives
could n ot be determ in ed due to solubility problem s.
Evaluation of Ph otosyn th esis-In h ibitin g Activity in Ch loroplasts
Th e oxygen evolution rate (OER) in spin ach ch loroplasts (ch loroph yll con ten t 30 m g/l) was
determ in ed spectroph otom etrically (Specord UV VIS, Zeiss Jen a, Germ an y) in th e presen ce
of 2,6-dich loroph en olin doph en ol as an electron acceptor usin g th e m eth od described48. Due
to low water solubility th e studied com poun ds were dissolved in DMSO so th at th e applied
DMSO con cen tration (up to 5%, v/v) did n ot affect oxygen evolution . Th e in h ibitory activ-
ity was expressed by IC50 values, i.e. con cen tration s causin g 50% OER reduction relative to
un treated con trols. Th e results are given in Table I.
Evaluation of In h ibition of Ch loroph yll Con ten t in Chlorella vulgaris
Th e algae Chlorella vulgaris were statically cultivated (7 days, ph otoperiod 16 h ligh t/8 h dark;
irradian ce 80 µm ol/m 2/s2; pH 7.2) at room tem perature accordin g to ref.49 Ch loroph yll con -
ten t in th e algal suspen sion was determ in ed spectroph otom etrically after extraction with
eth an ol accordin g to Wellburn 50. At th e begin n in g of th e cultivation , th e ch loroph yll con -
ten t in th e suspen sion s was 0.1 m g/l. Th e tested com poun ds were dissolved in DMSO, th e
con cen tration of wh ich in th e algal suspen sion s did n ot exceed 0.5% (v/v). Th e con trol sam -
ples con tain ed th e sam e DMSO am oun t as th e suspen sion s treated with th e tested com -
poun ds. For th e results, see Table I.
Chemical part of this study was supported by the Ministry of Education, Youth and Sports of the
Czech Republic (Research project MSM 0021620822), antifungal tests were performed with the
support of Ministry of Health of the Czech Republic (IGA 1A/8238-3), and photosynthetic assays were
supported by the Slovak Grant Agency (VEGA 1/0089/03). Antimycobacterial data were provided by
the Tuberculosis Antimicrobial Acquisition Coordinating Facility (TAACF) through a research and
development contract with the U.S. National Institute of Allergy and Infectious Diseases.
Collect. Czech. Chem. Commun. 2006, Vol. 71, No. 1, pp. 44–58

Synthesis and Biological Evaluation of Chalcones
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