Synthesis and biological evaluation of quinazoline-4-thiones

Article abstract of DOI:10.3390/81100756

Several 2,2-dimethyl-3-phenyl-1,2-dihydroquinazoline-4(3H)-thiones and 2-methyl-3-phenylquinazoline-4(3H)-thiones were synthesized and tested for their antimycobacterial, photosynthesis-inhibiting, and antialgal activity. Antimycobacterially active compounds were found among the 6-chloro substituted compounds. 6-Chloro-3-(4-isopropylphenyl)-2-methylquinazoline-4(3H)-thione exhibited higher activity than the isoniazid standard against Mycobacterium avium and M. kansasii. Most of the compounds possessed photosynthesis-inhibiting activity. 6-Chloro-2,2-dimethyl-3-phenyl-1,2-dihydro-quinazoline-4(3H)-thione and its 3′-chloro- and 3′,4′-dichloro analogs were most effective in the inhibition of oxygen evolution rate in spinach chloroplasts. Of compounds selected for toxicological screening, 6-chloro-3-(4-isopropylphenyl)- 2-methyl-quinazoline-4(3H)-thione was the only one active in the brine shrimp bioassay.

Full text of DOI:10.3390/81100756

Molecules 2003, 8, 756-769
molecules
ISSN 1420-3049
http://www.mdpi.org
Synthesis and Biological Evaluation of Quinazoline-4-thiones
Lenka Kubicová 1,*, Martin Šustr 1, Katarína Kráľová 2, Vladimír Chobot 1, Jitka Vytlačilová 1,
Luděk Jahodář 1, Pia Vuorela 3, Miloš Macháček 1, Jarmila Kaustová 4
1
Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1205, 500 05
Hradec Králové, Czech Republic. Tel. +420 49 5067339, Fax +420 49 5210002.
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-2, 842
2
15 Bratislava, Slovak Republic.
Department of Pharmacy, Division of Pharmacognosy, University of Helsinki, P.O. Box 56,
3
(Viikinkaari 5), Helsinki, Finland.
National Reference Laboratory for Mycobacterium Kansasii, Institute of Hygiene, 728 92 Ostrava,
4
Czech Republic.
*Author to whom correspondence should be addressed; e-mail: kubicova@faf.cuni.cz
Received: 25 August 2004; in revised form: 30 September 2003 / Accepted: 30 September 2003 /
Published: 15 November 2003
Abstract: Several 2,2-dimethyl-3-phenyl-1,2-dihydroquinazoline-4(3H)-thiones and 2-
methyl-3-phenylquinazoline-4(3H)-thiones were synthesized and tested for their
antimycobacterial, photosynthesis-inhibiting, and antialgal activity. Antimycobacterially
active compounds were found among the 6-chloro substituted compounds. 6-Chloro-3-(4-
isopropylphenyl)-2-methylquinazoline-4(3H)-thione exhibited higher activity than the
isoniazid standard against Mycobacterium avium and M. kansasii. Most of the compounds
possessed photosynthesis-inhibiting activity. 6-Chloro-2,2-dimethyl-3-phenyl-1,2-dihydro-
quinazoline-4(3H)-thione and its 3´-chloro- and 3´,4´-dichloro analogs were most effective in
the inhibition of oxygen evolution rate in spinach chloroplasts. Of compounds selected for
toxicological screening, 6-chloro-3-(4-isopropylphenyl)-2-methyl-quinazoline-4(3H)-thione
was the only one active in the brine shrimp bioassay.
Keywords: Quinazoline-4-thiones, mycobacteria, photosynthesis-inhibiting activity,
chloroplasts, alga, toxicological screening, Artemia salina.

Molecules 2003, 8
757
Introduction
Tuberculosis continues to be a devastating disease worldwide and is believed to be present in about
one third of the world's population [1]. The increasing incidence of multi-drug-resistant tuberculosis is
emerging as a major infectious disease problem throughout the world [2]. Mycobacterial diseases
caused by the Mycobacterium avium - M. intracellulare complex show a rising occurrence among
children, the elderly, and HIV-infected patients, and they are frequently fatal [3]. The search for
potential antimycobacterial drugs is consequently one of the primary tasks of present-day medicinal
chemistry.
Various nitrogen containing heterocycles have been recently studied for their antibacterial or
antimycobacterial effects, e.g. 3,5-dinaphthyl-2-pyrazolines [4], 2-phenyl-5,5-dialkylimidazolinones
[5], 4-amino-5-aryl-1,2,4-triazoles [6], triazolo- or tetrazolopyrrolopyrimidines [7], 5-alkylsulfanyl-
tetrazoles [8-11], benzimidazoles [12], 1,3-benzoxazinediones [13-15], quinazolines [14, 16-19], and
quinoxalines [20, 21]. Reviews of antimycobacterially active derivatives containing one or more
nitrogen atom in the five- or six-membered ring have been published [16, 22-24].
The derivatives of quinazolin-4-one are potential drugs which can possess hypnotic, analgesic,
antiallergic, anticonvulsant, antimalarial, and other effects [25]. In our previous study we found that
some 3-phenylquinazolin-4(3H)-ones were active against atypical strains of mycobacteria [18]. The
conversion of the oxo group into the thioxo function leads, in general, to an increase in
antimycobacterial activity [14, 15, 26, 27]. Although the antimicrobial activity of some substituted
quinazoline-4-thiones is known [27, 28], the antimycobacterial activity of neither 2,2-dimethyl-3-
phenyl-1,2-dihydroquinazoline-4(3H)-thiones, nor 2-methyl-3-phenylquinazoline-4(3H)-thiones has
been studied yet.
In this paper, we describe the synthesis of two series of quinazoline-4-thiones, 2,2-dimethyl-3-
phenyl-1,2-dihydroquinazoline-4(3H)-thiones (1) and 2-methyl-3-phenylquinazoline-4(3H)-thiones
(2), and results of their testing for antimycobacterial activity against Mycobacterium tuberculosis, M.
avium, and M. kansasii.
Because of previous experience that various compounds with carbamoyl or thiocarbamoyl group,
e.g. acylanilides, thioacylanilides, and their cyclic analogs, can inhibit the photosynthetic electron
transport in autotrophic organisms [29-35], the photosynthesis-inhibiting and antialgal activity of the
compounds 1 and 2 was also determined.
Based on the results of the biological tests, four compounds, 1h, 1i, 2b, and 2f, were selected for a
toxicological screening bioassay and tested using brine shrimp larvae (Artemia salina L.) as the
sensitive organism [36].

Molecules 2003, 8
758
Results and Discussion
Chemistry
Routes for preparation of quinazoline-4-thione derivatives can involve cyclization of a convenient
precursor, thionation of the corresponding oxo analogs, or condensation reactions [25, 28, 37, 38].
Efficient methods for synthesis of quinazolin-4-ones are e.g. acylation of 2-aminobenzamides with an
appropriate acyl chloride followed by cyclization in basic medium [39], or one-pot synthesis under
solvent-free conditions [40]. Derivatives of 2,2-dimethyl-1,2-dihydroquinazoline-4(3H)-thione can be
prepared by condensation of 2-aminothiobenzamides with acetone under mild conditions [41]. Other
way to obtain quinazoline-4(3H)-thiones is the ring closure of 2-acylaminothiobenzamides in basic
medium [28,42].
2,2-Dimethyl-3-phenyl-1,2-dihydroquinazoline-4(3H)-thiones (1a-k) were synthesized by
condensation of the corresponding 2-amino-N-phenylthiobenzamides with acetone under the catalysis
by silica gel. The reaction mixtures were allowed to stand at room temperature for 24 h, then
concentrated in vacuo, and the products 1 were isolated by column chromatography on silica gel using
petroleum ether with acetone as the mobile phase. The starting 2-amino-N-phenylthiobenzamides were
prepared by a two-step process from 2-amino-N-phenylbenzamides. Treatment of 2-amino-N-
phenylbenzamide with phosphorus decasulfide in pyridine afforded the corresponding pyridinium salt.
Hydrolysis of the pyridinium salt in a toluene-water system gave 2-amino-N-phenylthiobenzamide
[42]. 2-Methyl-3-phenylquinazoline-4(3H)-thiones (2a-g) were prepared by thionation of the
corresponding 2-methyl-3-phenylquinazolin-4(3H)-ones with phosphorus decasulfide in pyridine. The
syntheses are outlined in Scheme 1. The characteristic data of compounds 1a-k and 2a-g are given in
Tables 1 and 2. Characteristic data of the intermediates were [43] or will be published elsewhere.
Scheme 1
S
O
R
R
X
X
NH
NH
NH2
NH
NH2
1. P4S10
Me2CO
2. HCl, H2O, toluene
N
X
R
S
1a-k
N
S
N
O
P4S10
N
N
X
X
R
R
2a-g

Molecules 2003, 8
759
Compound
X
H
H
H
H
H
H
Cl
Cl
Cl
R
H
4-Cl
Compound
X
R
4-isoC3H7
4-C4H9
H
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
2a
2b
2c
2d
2e
2f
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3,4-Cl2
4-CH3
4-C2H5
4-isoC3H7
H
3-Cl
4-Cl
4-Br
4-CH3
4-isoC3H7
4-OCH3
3-Cl
3,4-Cl2
2g
Biological activity
Antimycobacterial activity
Antimycobacterial activity of the compounds was tested in vitro against Mycobacterium
tuberculosis, M. avium, and M. kansasii, obtained from the Czech National Collection of Type
Cultures (CNCTC), and a clinical isolate of M. kansasii, using the micromethod for the determination
of the minimum inhibitory concentration (MIC). The MIC values of the compounds are given in Table
3. Antimycobacterially active compounds were found only among the 6-chloro derivatives (X = Cl).
Derivatives 1h, 2d, 2f, and 2g demonstrated moderate activity against mycobacteria. The activity of
derivative 2f (X = Cl, R = 3-isopropyl), the most active compound, against M. avium and M. kansasii
is worth mentioning. In some cases, the antimycobacterial activity observed after 14 days weared off
after 21 days of incubation (1c, 1j, 1k). The other compounds showed no activity in the range of
concentrations tested (data not given).
Photosynthesis-inhibiting activity in spinach chloroplasts
Most of the tested compounds inhibited the photosynthetic electron transport in spinach
chloroplasts. The photosynthesis-inhibiting activity of the compounds was investigated as inhibition of
oxygen evolution rate (OER) in spinach chloroplasts. IC50 values are given in Table 4. The 6-chloro
analog 1g was the most effective inhibitor of OER. Its IC50 value was comparable to that of the
standard diuron (DCMU). 6-Unsubstituted compound 1a was 60-fold less potent than 1g. Substitution
on the phenyl ring was unfavourable. Whereas mono- and dichloro derivatives 1h and 1i were
approximately twice less potent than compound 1g, alkyl derivatives 1j and 1k were more than 100-
fold less potent. The relatively low photosyntesis-inhibiting activity of compounds 2 is probably a
consequence of their low aqueous solubility, and hence their restricted passage through the hydrophilic
regions of thylakoid membranes. A comparison of compounds 2a and 2b with their analogs 1g and 1h

Molecules 2003, 8
760
indicates 75- to 100-fold decrease in activity. Photosynthesis-inhibiting activity of compounds 1d, 1e,
and 1f could not be determined due to their incomplete solubility.
Reduction of chlorophyll content in the green algae Chlorella vulgaris Beij.
Some of the compounds under study reduced the chlorophyll content in Chlorella vulgaris Beij.
IC50 values could be determined only for compounds 1a (IC50 = 49.61 µmol dm-3), 1b (IC50 = 40.91
µmol dm-3), 1c (IC50 = 34.88 µmol dm-3), 1d (IC50 = 155.09 µmol dm-3), and 1g (IC50 = 74.4 µmol dm-
3). IC50 value for the standard, a selective herbicide 1,1-dimethyl-3-(3,4-dichlorophenyl)urea (Diuron),
was 7.3 µmol dm-3. The other compounds were inactive (less than 5% reduction) or weakly active
(27% (1h), 22% (1k), and 15% (2b) reduction of chlorophyll content) in the concentration range from
0.83 to 99.0 umol dm-3. This could be due to their too low aqueous solubility.
Toxicological screening bioassay
Four compounds, 1h, 1i, 2b, and 2f, were selected according to their biological activity in
antifungal [19], antimycobacterial, photosynthesis-inhibiting, and antialgal tests for toxicological
screening bioassay using brine shrimp larvae (Artemia salina L.) as the sensitive organism. Only
compound 2f was found toxic. Its value of EC50 was 155.20 µmol dm-3 (EC50 of MnCl2 was 41.44
mmol dm-3). Other compounds tested demonstrated no significant toxicity in the range of used
concentrations.
Conclusions
A series of novel 2,2-dimethyl-3-phenyl-1,2-dihydroquinazoline-4(3H)-thiones 1 and 2-methyl-3-
phenylquinazoline-4(3H)-thiones 2 was synthesized and tested for their antimycobacterial,
photosynthesis-inhibiting, and antialgal activity. Compound 2f (X = Cl, R = 3-isopropyl) exhibited
better activity than isoniazid against Mycobacterium avium and M. kansasii. Unfortunately, it was
found toxic in the brine shrimp bioassay (EC50 = 155.20 µmol dm-3).
Acknowledgements
This work was supported by the Grant Agency of the Czech Republic (grant No. 203/02/P013), the
Grant Agency of Charles University in Prague (grant No. 158/2003/B-BIO/Faf), the Slovak Scientific
Grant Agency (grant VEGA No. 1/0089/03), and the Ministry of Education of the Czech Republic
(research projects No. MSM 111600001 and No. MSM 111600003). The authors wish to thank Mrs.
D. Karlíčková from the Department of Pharmaceutical Chemistry and Drug Control, Faculty of
Pharmacy, Charles University, for elemental analyses. Thanks are due to Ms. K. Kissová from the
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic,
for her technical assistance in antialgal testing.

Molecules 2003, 8
761
Experimental
General
The melting points were determined on a Kofler block and are uncorrected. The samples for
elemental analyses and biological tests were dried over P4O10 at 61 °C and 66 Pa for 24 h. Elemental
analyses were performed on a C,H,N,S analyzer (FISONS AE 1110, Milano, Italy). The purity of the
compounds was checked by TLC using petroleum ether-ethyl acetate (9:1) and petroleum ether-
acetone (7:3) as the mobile phases. Column chromatography was performed on Silica gel Merck 60
13
with petroleum ether-acetone (9:1) or toluene. 1H- and C-NMR spectra were recorded for DMSO-d6
solutions at ambient temperature on a Varian Mercury-Vx BB 300 spectrometer (operating at 300 and
75 MHz, respectively). Chemical shifts were recorded as δ values in parts per million (ppm), and were
1
indirectly referenced to tetramethylsilane via the solvent signal (2.49 for H and 39.7 for 13C).
Multiplicities are given together with coupling constants (J, in Hz).
2-Amino-N-phenylthiobenzamides
A 100-mL flask was charged with the appropriate 2-amino-N-phenylbenzamide (0.05 mol),
tetraphosphorus decasulfide (0.05 mol), and pyridine (35 mL). Reaction mixture was refluxed for 4-6 h
and after cooling poured into ice water (250 mL). The obtained precipitate was placed in a 500-mL
flask, toluene (150 mL), water (150 mL), and conc. hydrochloric acid (5 mL) were added and the
mixture was refluxed for 8–18 h. After cooling to room temperature, the toluene layer was separated
and the solvent evaporated in vacuo. The residue was chromatographed on silica gel (toluene) and the
product recrystallized from aqueous ethanol (yield 25–45 %).
2,2-Dimethyl-3-phenyl-1,2-dihydroquinazoline-4(3H)-thiones 1a-k
2-Amino-N-phenylthiobenzamide (0.01 mol) was dissolved in acetone (50 mL) at room temperature
and silica gel (4 g) was added to the solution under stirring. The reaction mixture was stirred for 24 h
at room temperature and then concentrated in vacuo. The residue was chromatographed on silica gel
using petroleum ether-acetone (9:1) as the mobile phase. The product was recrystallized from ethanol.
1
The yields, melting points, H- and 13C-NMR spectral data as well as elemental analyses are
summarized in Tables 1 and 2.
2-Methyl-3-phenylquinazoline-4(3H)-thiones 2a-g
6-Chloro-2-methyl-3-phenylquinazolin-4(3H)-one (0.01 mol) was dissolved in pyridine (10 ml) and
tetraphosphorus decasulfide (0.01 mol) was added. The reaction mixture was refluxed under stirring
for 4 h. After cooling, the mixture was poured into ice water, the crude product was filtered off,

Molecules 2003, 8
762
washed with water, and dried. 6-Chloro-2-methyl-3-phenylquinazoline-4(3H)-thione was isolated by
column chromatography on silica gel using petroleum ether-acetone (9:1) as the mobile phase and
1
recrystallized from ethanol. The yields, melting points, H- and 13C-NMR spectral data as well as
elemental analyses are summarized in Tables 1 and 2.
Table 1. Analytical data of 2,2-dimethyl-3-phenyl-1,2-dihydroquinazoline-4(3H)-thiones (1)
and 2-methyl-3-phenylquinazoline-4(3H)-thiones (2).
Elemental analysis
% Calc. / % Found
Formula
M. w.
M.p. (°C)
Yield (%)
Compd.
X
R
C
H
N
S
1a
1b
1c
1d
1e
1f
C16H16N2S
268.4
C16H15ClN2S
302.8
C16H14Cl2N2
337.3
C17H18N2S
282.4
C18H20N2S
296.4
C19H22N2S
310.5
C16H15ClN2S
337.3
C16H14Cl2N2S
337.3
C16H13Cl3N2S
371.7
C19H21ClN2S
344.9
C20H23ClN2S
358.9
C15H11ClN2S
286.8
C15H10Cl2N2S
321.2
C15H10Cl2N2S
321.2
H
H
H
4-Cl
212-214a
78
238-241
82
199-201
86
229-231
80
187-188
76
199-200
85
218-219
83
157-158
77
189-190
81
205-207
79
183-184
82
153-154
69
172-173
74
202-204
76
71.61 6.01 10.44 11.95
71.50 6.15 10.51 12.10
63.46 4.99
63.54 5.10
56.98 4.18
56.80 4.30
72.30 6.42
72.27 6.46
72.93 6.80
72.79 6.85
73.51 7.14
73.61 7.05
63.46 4.99
63.58 4.83
56.98 4.18
56.82 4.25
51.70 3.53
51.72 3.57
66.17 6.14
66.35 6.01
66.93 6.46
66.90 6.41
62.82 3.87
62.72 3.96
56.09 3.14
56.06 3.35
56.09 3.14
56.35 3.08
9.25 10.59
9.15 10.70
8.31
8.27
H
3,4-Cl2
4-CH3
4-C2H5
4-isoC3H7
H
9.51
9.70
H
9.92 11.35
9.84 11.30
9.45 10.82
9.50 10.85
9.02 10.33
9.10 10.39
9.25 10.59
9.14 10.70
8.31
8.45
7.54
7.44
8.12
8.10
7.80
7.87
H
H
1g
1h
1i
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3-Cl
9.51
9.53
8.63
8.71
9.30
9.47
8.93
8.79
3,4-Cl2
4-isoC3H7
4-C4H9
H
1j
1k
2a
2b
2c
9.77 11.18
9.78 11.30
8.72
8.54
8.72
3-Cl
9.98
9.95
9.98
4-Cl
8.75 10.26

Molecules 2003, 8
763
Elemental analysis
% Calc. / % Found
Formula
M. w.
M.p. (°C)
Yield (%)
Compd.
X
R
C
H
N
S
2d
2e
2f
C15H10BrClN2S
365.7
C16H13ClN2S
(300.8)
C18H17ClN2S
328.9
C16H13ClN2OS
316.8
Cl
Cl
Cl
Cl
4-Br
4-CH3
212-214
73
157-158
70
135-136
68
145-146
59
49.27 2.76
49.39 2.74
63.89 4.36
63.71 4.48
65.74 5.21
65.85 5.18
60.66 4.14
60.70 4.12
7.66
7.59
9.31 10.66
9.25 10.60
8.52
8.47
8.84 10.12
8.91 10.07
8.77
8.90
4-isoC3H7
4-OCH3
9.75
9.68
2g
aReference [41] reports m.p. 212–214°C for compound 1a.
Table 2. 1H-NMR and 13C-NMR spectral data
Compd.
1a
1H-NMR δ (ppm), J (Hz)
8.16 (dd, 1H, J=7.96, J=1.37, H5), 7.50-7.41 (m, 2H, H3´, H5´),
7.41-7.28 (m, 3H, NH, H7, H4´), 7.21-7.14 (m, 2H, H2´, H6´),
6.79-6.69 (m, 2H, H6, H8), 1.37 (s, 6H, CH3)
13C-NMR δ (ppm)
190.4, 142.7, 142.6, 133.9, 132.6,
129.3, 129.2, 128.0, 121.0, 117.8,
115.2, 72.7, 27.1
1b
1c
8.15 (dd, 1H, J=7.97, J=1.37, H5), 7.54-7.47 (m AA´, BB´, 2H,
H2´, H6´), 7.38-7.29 (m, 2H, NH, H7), 7.26-7.19 (m AA´, BB´,
2H, H3´, H5´), 6.80-6.68 (m, 2H, H6, H8), 1.37 (s, 6H, CH3)
190.8, 142.7, 141.4, 134.1, 132.6,
132.5, 131.3, 129.4, 120.9, 117.8,
115.3, 72.8, 27.0
8.14 (d, 1H, J=7.97, H5), 7.72 (d, 1H, J=8.51, H5´), 7.56 (d, 1H,
191.0, 142.8, 142.3, 134.3, 132.5,
J=2.20, H2´), 7.42-7.30 (m, 2H, NH, H7), 7.28-7.22 (m, 1H, H6´), 131.7, 131.7, 131.2, 130.9, 130.2,
6.81-6.69 (m, 2H, H6, H8), 1.40 (s, 6H, CH3) 120.7, 117.9, 115.3, 73.0, 27.0
1d
1e
8.19-8.12 (m, 1H, H5), 7.36-7.20 (m, 4H, NH, H7, H2´, H6´), 7.08- 190.5, 142.7, 140.2, 137.2, 133.9,
7.01 (m, 2H, H3´, H5´), 6.78-6.68 (m, 2H, H6, H8), 2.34 (s, 3H,
CH3) 1.35 (s, 6H, CH3)
132.7, 129.8, 128.9, 121.1, 117.7,
115.2, 72.7, 27.1, 20.9
8.16 (d, 1H, J=7.96, H5), 7.36-7.24 (m, 4H, NH, H7, H2´, H6´),
190.5, 143.3, 142.7, 140.3, 133.9,
7.11-7.04 (m, 2H, H3´, H5´), 6.74 (t, overlapped, 1H, J=7.83, H6), 132.7, 129.0, 128.6, 121.1, 117.7,
6.71 (d, overlapped, 1H, J=7.83, H8), 2.64 (q, 2H, J=7.55, CH2),
1.35 (s, 6H, CH3), 1.21 (t, 3H, J=7.55, CH3)
115.2, 72.7, 27.9, 27.1, 15.5
1f
8.19-8.13 (m, 1H, H5), 7.36-7.25 (m, 4H, NH, H7, H2´, H6´), 7.11- 190.5, 147.8, 142.7, 140.4, 133.9,
7.04 (m, 2H, H3´, H5´), 6.78-6.68 (m, 2H, H6, H8), 3.01-2.84 (m, 132.7, 128.9, 127.1, 121.1, 117.7,
1H, CH), 1.34 (s, 6H, CH3), 1.23 (d, 6H, J=6.87, CH3)
115.1, 72.7, 33.2, 27.1, 24.0

Molecules 2003, 8
764
Compd.
1H-NMR δ (ppm), J (Hz)
13C-NMR δ (ppm)
1g
1h
1i
8.11 (d, 1H, J=2.48, H5), 7.58 (bs, 1H, NH), 7.54-7.48 (m AA´,
BB´, 2H, H2´, H6´), 7.37 (dd, 1H, J=8.79, J=2.47, H7), 7.27-7.20 129.4, 129.1, 128.2, 121.9, 121.4,
189.0, 142.4, 141.5, 133.6, 131.3,
(m AA´, BB´, 2H, H3´, H5´), 6.81 (d, 1H, J=8.79, H8), 1.37 (s,
6H, CH3)
8.11 (d, 1H, J=2.48, H5), 7.60 (bs, 1H, NH), 7.51-7.46 (m, 2H,
H2´, H6´), 7.38 (dd, 1H, J=8.79, J=2.47, H7), 7.34-7.31 (m, 1H,
H5´), 7.23-7.18 (m, 1H, H4´), 6.81 (d, 1H, J=8.79, H8), 1.37 (s,
6H, CH3)
117.4, 73.0, 27.0
189.3, 143.5, 141.5, 133.8, 133.5,
131.2, 131.0, 129.3, 128.4, 128.3,
121.6, 121.4, 117.5, 73.1, 27.0
8.09 (d, 1H, J=2.47, H5), 7.73 (d, 1H, J=8.79, H5´), 7.63 (bs, 1H, 189.6, 142.1, 141.5, 134.0, 131.8,
NH), 7.59 (d, 1H, J=2.20, H2´), 7.38 (dd, 1H, J=8.79, J=2.47, 131.6, 131.3, 131.1 130.1, 121.5,
H7), 7.26 (dd, 1H, J=8.51, J=2.47, H6´), 6.82 (d, 1H, J=8.51, H8), 121.4, 117.5, 73.3, 27.0
1.40 (s, 6H, CH3)
1j
8.13 (d, 1H, J=2.75, H5), 7.52 (bs, 1H, NH), 7.36 (dd, 1H, J=8.51, 189.0, 148.1, 141.5, 140.1, 133.5,
J=2.47, H7),7.34-7.29 (m AA´, BB´, 2H, H2´, H6´), 7.13-7.05 (m 131.3, 128.8, 127.2, 121.9, 121.3,
AA´, BB´, 2H, H3´, H5´), 6.80 (d, 1H, J=8.79, H8), 3.02-2.85 (m, 117.3, 73.0, 33.2, 27.1, 24.0
1H, CH), 1.35 (s, 6H, CH3), 1.23 (d, 6H, J=6.87, CH3)
1k
8.12 (d, 1H, J=2.47, H5), 7.52 (bs, 1H, NH), 7.36 (dd, 1H, J=8.79, 189.0, 142.2, 141.5, 140.1, 133.5,
J=2.47, H7), 7.29-7.23 (m AA´, BB´, 2H, H2´, H6´), 7.10-7.04 (m 131.3, 129.2, 128.8, 121.9, 121.3,
AA´, BB´, 2H, H3´, H5´), 6.80 (d, 1H, J=8.79, H8), 2.61 (t, 2H,
J=7.41, CH2), 1.64-1.51 (m, 2H, CH2), 1.40-1.24 (m, 2H, CH2),
1.35 (s, overlapped, 6H, CH3), 0.90 (t, 3H, J=7.42, CH3)
117.4, 73.0, 34.6, 33.1, 27.0,
22.0, 14.0
2a
2b
2c
2d
2e
8.49 (d, 1H, J=2.48, H5), 7.90 (dd, 1H, J=8.79, J=2.47, H7), 7.73 188.1, 154.5, 142.4, 141.5, 135.2,
(d, 1H, J=8.79, H8), 7.63-7.48 (m, 3H, H3´, H4´, H5´), 7.44-7.37 132.4, 130.2, 130.0, 129.3, 129.1,
(m, 2H, H2´, H6´), 2.17 (s, 3H, CH3)
127.9, 25.3
8.49 (d, 1H, J=2.47, H5), 7.95-7.89 (m, 1H, H7), 7.44 (d, 1H,
188.1, 154.2, 143.5, 141.5, 135.3,
J=8.52, H8), 7.66-7.57 (m, 3H, H2´, H5´, H6´), 7.46-7.40 (m, 1H, 134.2, 132.5, 131.8, 130.1, 129.5,
H4´), 2.19 (s, 3H, CH3) 129.2, 129.1, 128.3, 127.1, 25.3
8.48 (d, 1H, J=2.47, H5), 7.91 (dd, 1H, J=8.79, J=2.47, H7), 7.73 188.2, 154.3, 141.5, 141.2, 135.3,
(d, 1H, J=8.79, H8), 7.70-7.62 (m AA´, BB´, 2H, H2´, H6´), 7.52- 133.9, 132.4, 130.3, 130.1, 129.8,
7.44 (m AA´, BB´, 2H, H3´, H5´), 2.18 (s, 3H, CH3)
129.2, 129.1, 25.4
8.49 (d, 1H, J=2.47, H5), 7.92 (dd, 1H, J=8.79, J=2.47, H7), 7.83- 188.1, 154.3, 141.7, 141.5, 135.3,
7.77 (m AA´, BB´, 2H, H2´, H6´), 7.74 (d, 1H, J=8.79, H8), 7.45- 133.3, 132.4, 130.4, 130.1, 129.2,
7.38 (m AA´, BB´, 2H, H3´, H5´), 2.18 (s, 3H, CH3)
129.1, 122.5, 25.4
8.50 (d, 1H, J=2.47, H5), 7.90 (dd, 1H, J=8.65, J=2.47, H7), 7.72 188.2, 154.7, 141.5, 139.9, 138.7,
(d, 1H, J=8.65, H8), 7.41-7.34 (m AA´, BB´, 2H, H2´, H6´), 7.30- 135.1, 132.3, 130.7, 130.1, 129.3,
7.23 (m AA´, BB´, 2H, H3´, H5´), 2.39 (s, 3H, CH3), 2.17 (s, 3H, 129.2, 127.6, 25.3, 21.0
CH3)

Molecules 2003, 8
765
Compd.
1H-NMR δ (ppm), J (Hz)
13C-NMR δ (ppm)
2f
8.51 (d, 1H, J=2.48, H5), 7.90 (dd, 1H, J=8.79, J=2.47, H7), 7.73 188.1, 154.8, 149.3, 141.5, 140.1,
(d, 1H, J=8.79, H8), 7.48-7.41 (m AA´, BB´, 2H, H2´, H6´), 7.33- 135.1, 132.3, 130.0, 129.3, 129.2,
7.27 (m AA´, BB´, 2H, H3´, H5´), 3.06-2.90 (m, 1H, CH), 2.16 (s, 128.0, 127.6, 33.3, 25.3, 24.0
3H, CH3), 1.26 (d, 6H, J=7.14, CH3)
2g
8.50 (d, 1H, J=2.47, H5), 7.90 (dd, 1H, J=8.79, J=2.47, H7), 7.72 188.5, 159.4, 155.1, 141.5, 135.1,
(d, 1H, J=8.79, H8), 7.35-7.28 (m, AA´, BB´, 2H, H2´, H6´), 7.14- 135.1, 132.3, 130.0, 129.3, 129.3,
7.07 (m AA´, BB´, 2H, H3´, H5´), 3.83 (s, 3H, OCH3), 2.19 (s,
3H, CH3)
129.0, 115.2, 55.6, 25.4
Biological assays
Antimycobacterial activity
For the in vitro evaluation of antimycobacterial activity of the substances, the following strains
were used: Mycobacterium tuberculosis CNCTC My 331/88, M. kansasii CNCTC My 235/80, and M.
avium CNCTC My 330/88, obtained from the Czech National Collection of Type Cultures (CNCTC),
National Institute of Public Health, Prague, and a clinic isolate of M. kansasii 6509/96.
Antimycobacterial activity of the compounds against these strains was determined in Šula
semisynthetic medium (SEVAPHARMA, Prague). Each strain was simultaneously inoculated into a
Petri dish containing Löwenstein-Jensen medium for the control of sterility of the inoculum and its
growth. The compounds were added to the medium in dimethyl sulfoxide (DMSO) solutions. The
following concentrations were used: 250, 125, 62.5, 31, 16, 8, 4, 2, 1, and 0.5 µmol dm-3. The
o
minimum inhibitory concentrations (MICs) were determined after incubation at 37 C for 14 and 21
days. MIC was the lowest concentration of a substance at which the inhibition of the growth occurred.
The compound is considered active, when its MIC is lower than 1000 µmol dm-3. Isoniazid was used
as the standard.
Table 3. Antimycobacterial activity of compounds 1 and 2 expressed as MIC (µmol dm-3)
MIC (µmol dm-3)
M. tbc.
My 331/88
14d/21d
62.5/>62.5
62.5/62.5
>31/>125
>62.5/>125
M. avium
My 330/88
14d/21d
62.5/>62.5
125/>250
>62.5/>250
>31/>125
M. kansasii
My 235/80
14d/21d
>62.5/>62.5
62.5/125
31/>62.5
31/>62.5
M. kansasii
6 509/96
14d/21d
>250/>250
>62.5/>62.5
>62.5/>125
62.5/>62.5
Compound
X
R
1c
1h
1j
H
3,4-Cl2
3-Cl
4-isoC3H7
4-C4H9
Cl
Cl
Cl
1k

Molecules 2003, 8
766
MIC (µmol dm-3)
M. tbc.
My 331/88
14d/21d
31/31
31/31
31/>62.5
0.5/1
M. avium
M. kansasii
My 235/80
14d/21d
>31/>31
31/31
M. kansasii
Compound
X
R
My 330/88
14d/21d
>31/>31
31/31
62.5/125
>250/>250
6 509/96
14d/21d
62.5/62.5
62.5/62.5
31/62.5
4/4
2d
2f
2g
Cl
Cl
Cl
-
4-Br
4-isoC3H7
4-OCH3
-
31/62.5
>250/>250
Isoniazid
Photosynthesis-inhibiting activity in spinach chloroplasts
Spinach chloroplasts were prepared by the procedure described by Walker [45]. The effect of the
compounds studied on oxygen evolution rate (OER) in spinach chloroplasts was investigated
spectrophotometrically in the presence of the electron acceptor 2,6-dichlorophenol-indophenol
(DCPIP) according to Kráľova et al. [46]. The rate of photosynthetic electron transport was monitored
as a photoreduction of DCPIP. The chlorophyll (Chl) content was 30 mg dm-3. Samples were irradiated
from the distance of 1 dm with a halogen lamp (250 W) through a 4-cm water filter to prevent
overheating of the samples. The activity of compounds 1 and 2 was expressed as IC50 values, i. e.
molar concentration causing a 50% decrease of OER with respect to the untreated control. For low
solubility of the studied compounds in water, these were dissolved in DMSO. The applied solvent
content (up to 4 v/v %) did not affect the photochemical activity in spinach chloroplasts. Diuron was
used as the standard.
Table 4. Inhibition of oxygen evolution rate in spinach chloroplasts by compounds
1 and 2 expressed as IC50 (µmol dm-3)
IC50
Compd.
X
R
(µmol dm-3)
93.4
1a
1b
1c
1d
1e
1f
1g
1h
1i
H
H
H
H
H
H
4-Cl
3,4-Cl2
4-CH3
4-C2H5
4-isoC3H7
H
72.2
29.8
a
-
-
-
a
a
H
Cl
Cl
Cl
Cl
Cl
1.5
3.5
3.0
251
280
3-Cl
3,4-Cl2
4-isoC3H7
4-C4H9
1j
1k

Molecules 2003, 8
767
IC50
(µmol dm-3)
140
Compd.
X
R
2a
2b
2c
2d
2e
2f
2g
Cl
Cl
Cl
Cl
Cl
Cl
Cl
-
H
3-Cl
4-Cl
267
146
295
260
351
268
4-Br
4-CH3
4-isoC3H7
4-OCH3
-
Diuron
1.9
aThe value could not be determined.
Reduction of chlorophyll content in the green algae Chlorella vulgaris Beij.
The green algae Chlorella vulgaris Beij. were cultivated statically at room temperature according
to Kráľová et al. [47] (photoperiod 16 h light/8 h dark; irradiation: 90 µmol m-2 s-1 PAR; pH 7.2). The
effect of the compounds on algal chlorophyll (Chl) content was determined after 7-day cultivation in
the presence of the compounds tested. The Chl content in the algal suspension was determined
spectrophotometrically after extraction into methanol according to Wellburn [48]. The Chl content in
the suspensions at the beginning of the cultivation was 0.1 mg dm-3. Because of their low water
solubility, the tested compounds were dissolved in dimethyl sulfoxide (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. The antialgal activity of compounds was
expressed as IC50 (the concentration of the inhibitor causing a 50% decrease in content of chlorophyll
as compared with the control sample) or by the percentage of reduction in the investigated
concentration range (0.89 – 99.0 µmol dm-3). Diuron was used as the standard.
Artemia screeing bioassay
Artemia salina L. eggs were obtained from JBL NovoTermia (Germany). The method of Eppley
[49] was applied for A. salina larvae hatching. The test was arranged according to Kiviranta et al. [50].
24-h old larvae were pipetted into 96-well plates (15-20 larvae per a well). The microcrystalline
suspensions of tested substances were prepared by sonication for 1 h in an ultrasonic bath. The solvent
was 1% DMSO in artificial seawater (pH 8.0 ± 0.1). The substances were tested in 11 concentrations
with 8 repetitions. The final volume was always 150 µL per well. Every experiment was repeated twice
at least. The negative control was 1% DMSO solution. The sensitivity of the organism was specified
by a solution of MnCl2. The mortality was determined after 24 h.

Molecules 2003, 8
768
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Sample Availability: Available from the authors.
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