7th International Electronic Conference on Synthetic Organic Chemistry (ECSOC-7), http://www.mdpi.net/ecsoc-7, 1-30 November 2003

[C003]

QUINAZOLINE-4-THIONES: SYNTHESIS, BIOLOGICAL ACTIVITY, AND TOXICOLOGICAL SCREENING

Lenka Kubicová1 Martin Šustr1, Katarína Kráľová2, Vladimír Chobot1, Jitka Vytlačilová1, Vladimír Buchta1, Luis Silva1, Luděk Jahodář1, Pia Vuorela3, Miloš Macháček1, Jiří Kuneš1, Jarmila Kaustová4


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. E-mail: kubicova@faf.cuni.cz
2 Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-2, 842 15 Bratislava, Slovak Republic. E-mail: kralova@fns.uniba.sk
3 Department of Pharmacy, Division of Pharmacognosy, University of Helsinki, P. O. Box 56 (Viikinkaari 5), Helsinki, Finland. E-mail: pia.vuorela@helsinki.fi
4 National Reference Laboratory for Mycobacterium Kansasii, Institute of Hygiene, 728 92 Ostrava, Czech Republic. E-mail: Jarmila.Kaustova@zuova.cz

Abstract: 2,2-Dimethyl-3-phenyl-1,2-dihydroquinazoline-4(3H)-thiones (1a-k) and 2-methyl-3-phenylquinazoline-4(3H)-thiones (2a-g) were synthesized and tested for their antimycobacterial, antifungal, photosynthesis-inhibiting, and antialgal activity. Derivatives 1h, 2d, 2f, and 2g were moderately active against mycobacteria. Compounds 1 and 2 showed no antifungal activity with an exception of derivative 1h which was moderately active against Aspergillus fumigatus and Absidia corymbifera. Most of compounds 1 and 2 possessed photosynthesis-inhibiting activity. The unsubstituted, mono- and dichloro 6-chloro analogs 1g, 1h, and 1i were most effective in the inhibition of oxygen evolution rate in spinach chloroplasts. The antialgal activity of the compounds tested was relatively low. Of compounds 1h, 1i, 2b, and 2f, selected for toxicological screening, 2f was the only one active in the brine shrimp bioassay.

Keywords: Quinazoline-4-thiones, mycobacteria, fungi, photosynthesis-inhibiting activity, chloroplasts, alga, toxicological screening, Artemia salina.

INTRODUCTION

Infectious diseases rank among the most significant causes of morbidity and mortality in recent years. Tuberculosis 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]. Invasive fungal infections have increased in incidence during the last decade mainly in patients receiving chemotherapy, glucocorticoids, in HIV patients, and transplant recipients [4,5]. The search for novel drugs is consequently one of the primary tasks of present-day medicinal chemistry.

Quinazoline derivatives are pharmaceutically interesting compounds and many of them have been registered as drugs [6–8]. Quinazolin-4-ones can possess hypnotic, analgesic, antiallergic, anticonvulsant, antimalarial, and other effects [9]. In our previous study we found that some 3-phenylquinazolin-4(3H)-ones were active against atypical strains of mycobacteria [10]. The conversion of the oxo group into the thioxo function leads, in general, to an increase in antimycobacterial activity [11–14]. Although the antimicrobial activity of some substituted quinazoline-4-thiones is known [14,15], 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.

It was found previously that various compounds with carbamoyl or thiocarbamoyl group, e.g. acylanilides, thioacylanilides, and their cyclic analogs, inhibited the photosynthetic electron transport in autotrophic organisms [16–22].

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, antifungal, photosynthesis-inhibiting, and antialgal activity. The influence of structural modifications on biological activity is discussed. Based on the results of the biological tests, four compounds, 1h, 1i, 2b, and 2f, wereselected for toxicological screening bioassay and tested using the brine shrimp larvae (Artemia salina L.) as the sensitive organism [23].

RESULTS AND DISCUSSION

Chemistry

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 mixture was allowed to stand at room temperature for 24 h, then concentrated in vacuo, and the product 1 was 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 [24]. 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 Table 1 and 2.

Scheme 1

 

Compound

X

R

Compound

X

R

1a

H

H

1j

Cl

4-isoC3H7

1b

H

4-Cl

1k

Cl

4-C4H9

 

1c

H

3,4-Cl2

2a

Cl

H

1d

H

4-CH3

2b

Cl

3-Cl

1e

H

4-C2H5

2c

Cl

4-Cl

1f

H

4-isoC3H7

2d

Cl

4-Br

1g

Cl

H

2e

Cl

4-CH3

1h

Cl

3-Cl

2f

Cl

4-isoC3H7

1i

Cl

3,4-Cl2

2g

Cl

4-OCH3

 

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

Compd.

Formula

M. w.

X

R

M.p. (°C)

Yield (%)

Elemental analysis

% Calc. / % Found

C

H

N

S

1a

C16H16N2S
268.4

H

H

212-214a
78

71.61
71.50

6.01
6.15

10.44
10.51

11.95
12.10

1b

C16H15ClN2S
6.15302.8

H

4-Cl

238-241
6.1582

63.46
63.54

4.99
5.10

9.25
9.15

10.59
10.70

1c

C16H14Cl2N2
337.3

H

3,4-Cl2

199-201
86

56.98
56.80

4.18
4.30

8.31
8.27

9.51
9.70

1d

C17H18N2S
282.4

H

4-CH3

229-231
80

72.30
72.27

6.42
6.46

9.92
9.84

11.35
11.30

1e
 

C18H20N2S
296.4

H

4-C2H5

187-188
76

72.93
72.79

6.80
6.85

9.45
9.50

10.82
10.85

1f

C19H22N2S
310.5

H

4-isoC3H7

199-200
85

73.51
73.61

7.14
7.05

9.02
9.10

10.33
10.39

1g

C16H15ClN2S
337.3

Cl

H

218-219
83

63.46
63.58

4.99
4.83

9.25
9.14

10.59
10.70

1h

C16H14Cl2N2S
337.3

Cl

3-Cl

157-158
77

56.98
56.82

4.18
4.25

8.31
8.45

9.51
9.53

1i

C16H13Cl3N2S
371.7

Cl

3,4-Cl2

189-190
81

51.70
51.72

3.53
3.57

7.54
7.44

8.63
8.71

1j

C19H21ClN2S
344.9

Cl

4-isoC3H7

205-207
79

66.17
66.35

6.14
6.01

8.12
8.10

9.30
9.47

1k

C20H23ClN2S
358.9

Cl

4-C4H9

183-184
82

66.93
66.90

6.46
6.41

7.80
7.87

8.93
8.79

2a

C15H11ClN2S
286.8

Cl

H

153-154
69

62.82
62.72

3.87
3.96

9.77
9.78

11.18
11.30

2b

C15H10Cl2N2S
321.2

Cl

3-Cl

172-173
74

56.09
56.06

3.14
3.35

8.72
8.54

9.98
9.95

2c

C15H10Cl2N2S
321.2

Cl

4-Cl

202-204
76

56.09
56.35

3.14
3.08

8.72
8.75

9.98
10.26

2d

C15H10BrClN2S
365.7

Cl

4-Br

212-214
73

49.27
49.39

2.76
2.74

7.66
7.59

8.77
8.90

2e

C16H13ClN2S
(300.8)

Cl

4-CH3

157-158
70

63.89
63.71

4.36
4.48

9.31
9.25

10.66
10.60

2f

C18H17ClN2S
328.9

Cl

4-isoC3H7

135-136
68

65.74
65.85

5.21
5.18

8.52
8.47

9.75
9.68

2g

C16H13ClN2OS
316.8

Cl

4-OCH3

145-146
59

60.66
60.70

4.14
4.12

8.84
8.91

10.12
10.07

a Reference [25] reports m. p. 212–214°C for compound 1a.

Table 2. 1H NMR and 13C NMR spectral data of compounds 1 and 2

Compd.

1H NMR (300 MHz, DMSO)

d (ppm), J (Hz)

13C NMR (75 MHz, DMSO)

d (ppm)

1a

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)

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

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

1c

8.14 (d, 1H, J=7.97, H5), 7.72 (d, 1H, J=8.51, H5´), 7.56 (d, 1H, J=2.20, H2´), 7.42-7.30 (m, 2H, NH, H7), 7.28-7.22 (m, 1H, H6´), 6.81-6.69 (m, 2H, H6, H8), 1.40 (s, 6H, CH3)

191.0, 142.8, 142.3, 134.3, 132.5, 131.7, 131.7, 131.2, 130.9, 130.2, 120.7, 117.9, 115.3, 73.0, 27.0

1d

8.19-8.12 (m, 1H, H5), 7.36-7.20 (m, 4H, NH, H7, H2´, H6´), 7.08-7.01 (m, 2H, H3´, H5´), 6.78-6.68 (m, 2H, H6, H8), 2.34 (s, 3H, CH3) 1.35 (s, 6H, CH3)

190.5, 142.7, 140.2, 137.2, 133.9, 132.7, 129.8, 128.9, 121.1, 117.7, 115.2, 72.7, 27.1, 20.9

1e

8.16 (d, 1H, J=7.96, H5), 7.36-7.24 (m, 4H, NH, H7, H2´, H6´), 7.11-7.04 (m, 2H, H3´, H5´), 6.74 (t, overlapped, 1H, J=7.83, H6), 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)

190.5, 143.3, 142.7, 140.3, 133.9, 132.7, 129.0, 128.6, 121.1, 117.7, 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´), 7.11-7.04 (m, 2H, H3´, H5´), 6.78-6.68 (m, 2H, H6, H8), 3.01-2.84 (m, 1H, CH), 1.34 (s, 6H, CH3), 1.23 (d, 6H, J=6.87, CH3)

190.5, 147.8, 142.7, 140.4, 133.9, 132.7, 128.9, 127.1, 121.1, 117.7, 115.1, 72.7, 33.2, 27.1, 24.0

1g

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 (m AA´, BB´, 2H, H3´, H5´), 6.81 (d, 1H, J=8.79, H8), 1.37 (s, 6H, CH3)

189.0, 142.4, 141.5, 133.6, 131.3, 129.4, 129.1, 128.2, 121.9, 121.4, 117.4, 73.0, 27.0

1h

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)

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

1i

8.09 (d, 1H, J=2.47, H5), 7.73 (d, 1H, J=8.79, H5´), 7.63 (bs, 1H, NH), 7.59 (d, 1H, J=2.20, H2´), 7.38 (dd, 1H, J=8.79, J=2.47, H7), 7.26 (dd, 1H, J=8.51, J=2.47, H6´), 6.82 (d, 1H, J=8.51, H8), 1.40 (s, 6H, CH3)

189.6, 142.1, 141.5, 134.0, 131.8, 131.6, 131.3, 131.1 130.1, 121.5, 121.4, 117.5, 73.3, 27.0

1j

8.13 (d, 1H, J=2.75, H5), 7.52 (bs, 1H, NH), 7.36 (dd, 1H, J=8.51, J=2.47, H7),7.34-7.29 (m AA´, BB´, 2H, H2´, H6´), 7.13-7.05 (m AA´, BB´, 2H, H3´, H5´), 6.80 (d, 1H, J=8.79, H8), 3.02-2.85 (m, 1H, CH), 1.35 (s, 6H, CH3), 1.23 (d, 6H, J=6.87, CH3)

189.0, 148.1, 141.5, 140.1, 133.5, 131.3, 128.8, 127.2, 121.9, 121.3, 117.3, 73.0, 33.2, 27.1, 24.0

1k

8.12 (d, 1H, J=2.47, H5), 7.52 (bs, 1H, NH), 7.36 (dd, 1H, J=8.79, J=2.47, H7), 7.29-7.23 (m AA´, BB´, 2H, H2´, H6´), 7.10-7.04 (m 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)

189.0, 142.2, 141.5, 140.1, 133.5, 131.3, 129.2, 128.8, 121.9, 121.3, 117.4, 73.0, 34.6, 33.1, 27.0, 22.0, 14.0

2a

8.49 (d, 1H, J=2.48, H5), 7.90 (dd, 1H, J=8.79, J=2.47, H7), 7.73 (d, 1H, J=8.79, H8), 7.63-7.48 (m, 3H, H3´, H4´, H5´), 7.44-7.37 (m, 2H, H2´, H6´), 2.17 (s, 3H, CH3)

188.1, 154.5, 142.4, 141.5, 135.2, 132.4, 130.2, 130.0, 129.3, 129.1, 127.9, 25.3

2b

8.49 (d, 1H, J=2.47, H5), 7.95-7.89 (m, 1H, H7), 7.44 (d, 1H, J=8.52, H8), 7.66-7.57 (m, 3H, H2´, H5´, H6´), 7.46-7.40 (m, 1H, H4´), 2.19 (s, 3H, CH3)

188.1, 154.2, 143.5, 141.5, 135.3, 134.2, 132.5, 131.8, 130.1, 129.5, 129.2, 129.1, 128.3, 127.1, 25.3

2c

8.48 (d, 1H, J=2.47, H5), 7.91 (dd, 1H, J=8.79, J=2.47, H7), 7.73 (d, 1H, J=8.79, H8), 7.70-7.62 (m AA´, BB´, 2H, H2´, H6´), 7.52-7.44 (m AA´, BB´, 2H, H3´, H5´), 2.18 (s, 3H, CH3)

188.2, 154.3, 141.5, 141.2, 135.3, 133.9, 132.4, 130.3, 130.1, 129.8, 129.2, 129.1, 25.4

2d

8.49 (d, 1H, J=2.47, H5), 7.92 (dd, 1H, J=8.79, J=2.47, H7), 7.83-7.77 (m AA´, BB´, 2H, H2´, H6´), 7.74 (d, 1H, J=8.79, H8), 7.45-7.38 (m AA´, BB´, 2H, H3´, H5´), 2.18 (s, 3H, CH3)

188.1, 154.3, 141.7, 141.5, 135.3, 133.3, 132.4, 130.4, 130.1, 129.2, 129.1, 122.5, 25.4

2e

8.50 (d, 1H, J=2.47, H5), 7.90 (dd, 1H, J=8.65, J=2.47, H7), 7.72 (d, 1H, J=8.65, H8), 7.41-7.34 (m AA´, BB´, 2H, H2´, H6´), 7.30-7.23 (m AA´, BB´, 2H, H3´, H5´), 2.39 (s, 3H, CH3), 2.17 (s, 3H, CH3)

188.2, 154.7, 141.5, 139.9, 138.7, 135.1, 132.3, 130.7, 130.1, 129.3, 129.2, 127.6, 25.3, 21.0

2f

8.51 (d, 1H, J=2.48, H5), 7.90 (dd, 1H, J=8.79, J=2.47, H7), 7.73 (d, 1H, J=8.79, H8), 7.48-7.41 (m AA´, BB´, 2H, H2´, H6´), 7.33-7.27 (m AA´, BB´, 2H, H3´, H5´), 3.06-2.90 (m, 1H, CH), 2.16 (s, 3H, CH3), 1.26 (d, 6H, J=7.14, CH3)

188.1, 154.8, 149.3, 141.5, 140.1, 135.1, 132.3, 130.0, 129.3, 129.2, 128.0, 127.6, 33.3, 25.3, 24.0

2g

8.50 (d, 1H, J=2.47, H5), 7.90 (dd, 1H, J=8.79, J=2.47, H7), 7.72 (d, 1H, J=8.79, H8), 7.35-7.28 (m AA´, BB´, 2H, H2´, H6´), 7.14-7.07 (m AA´, BB´, 2H, H3´, H5´), 3.83 (s, 3H, OCH3), 2.19 (s, 3H, CH3)

188.5, 159.4, 155.1, 141.5, 135.1, 135.1, 132.3, 130.0, 129.3, 129.3, 129.0, 115.2, 55.6, 25.4



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).

Table 3.Antimycobacterial activity of compounds 1 and 2 expressed as MIC ( umol dm-3)

Compound

X

R

MIC (umol dm-3)

M. tbc.

My 331/88

14d/21d

M. avium

My 330/88

14d/21d

M. kansasii

My 235/80

14d/21d

M. kansasii

6 509/96

14d/21d

1c

H

3,4-Cl2

62.5/>62.5

62.5/>62.5

>62.5/>62.5

>250/>250

1h

Cl

3-Cl

62.5/62.5

125/>250

62.5/125

>62.5/>62.5

1j

Cl

4-isoC3H7

>31/>125

>62.5/>250

31/>62.5

>62.5/>125

1k

Cl

4-C4H9

>62.5/>125

>31/>125

31/>62.5

62.5/>62.5

2d

Cl

4-Br

31/31

>31/>31

>31/>31

62.5/62.5

2f

Cl

4-isoC3H7

31/31

31/31

31/31

62.5/62.5

2g

Cl

4-OCH3

31/>62.5

62.5/125

31/62.5

31/62.5

Isoniazid

-

-

0.5/1

>250/>250

>250/>250

4/4

Antifungal activity

Antifungal activity was tested in vitro against Candida albicans ATCC 44859, C. tropicalis 156, C. krusei E28, C. glabrata 20/I, Trichosporon beigelii 1188, Trichophyton mentagrophytes 445, Aspergillus fumigatus 231, and Absidia corymbifera 272 using broth microdilution method. 2,2-Dimethyl-3-phenyl-1,2-dihydroquinazoline-4(3H)-thiones 1 and 2-methyl-3-phenylquinazoline-4(3H)-thiones 2 were inactive, with an exception of derivative 1h (X = Cl, R = 3-Cl) which showed a moderate activity against Aspergillus fumigatus (MIC = 62.5 umol dm-3) and Absidia corymbifera (MIC = 62.5 umol dm-3) after both 24 h and 48 h of incubation.

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. 6-Chloro analog 1g was the most effective inhibitor of OER. It was 60-fold more potent than 6-unsubstituted compound 1a. 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 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.

Table 4. Inhibition of oxygen evolution rate in spinach chloroplasts by compounds 1 and 2 expressed as IC50 (umol dm-3)

Compd.

X

R

IC50
(umol dm-3)

1a

H

H

93.4

1b

H

4-Cl

72.2

1c

H

3,4-Cl2

29.8

1d

H

4-CH3

-a)

1e

H

4-C2H5

-a)

1f

H

4-isoC3H7

-a)

1g

Cl

H

1.5

1h

Cl

3-Cl

3.5

1i

Cl

3,4-Cl2

3.0

1j

Cl

4-isoC3H7

251

1k

Cl

4-C4H9

280

2a

Cl

H

140

2b

Cl

3-Cl

267

2c

Cl

4-Cl

146

2d

Cl

4-Br

295

2e

Cl

4-CH3

260

2f

Cl

4-isoC3H7

351

2g

Cl

4-OCH3

268

aThe value could not be determined.



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 =49.6 umol dm-3), 1b (IC50 = 40.9 umol dm-3), 1c (IC50 = 34.9 umol dm-3), 1d (IC50 = 155.1 umol dm-3), and 1g (IC50 = 74.4 umol 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 antimycobacterial, antifungal, photosynthesis-inhibiting, and antialgal tests for toxicological screening bioassay using the brine shrimp larvae (Artemia salina L.) as the sensitive organism. Only compound 2f was found toxic. Its value of EC50 was 155.20 umol dm-3 (EC50 of MnCl2 was 41.44 mmol dm-3). Other compounds tested demonstrated no significant toxicity in the range of used concentrations.

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 with petroleum ether-acetone (9:1) or toluene. 1H and 13C NMR spectra were recorded for DMSO-d6 solutions at ambient temperature on a Varian Mercury-Vx BB 300 spectrometer (operating at 300 MHz). Chemical shifts were recorded as d values in parts per million (ppm), and were indirectly referenced to tetramethylsilane via the solvent signal (2.49 for 1H and 39.7 for 13C). Multiplicities are given together with coupling constants (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). To the obtained precipitate 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 evaporatedin 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 the 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.

2-Methyl-3-phenylquinazoline-4(3 H)-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, 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 recrystallized from ethanol.

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 umol dm-3. The minimum inhibitory concentrations (MICs) were determined after incubation at 37 oC 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 umol dm-3. Isoniazid was used as the standard.

Antifungal activity

Antifungal activity was tested in vitro against Candida albicans ATCC 44859, C. tropicalis 156, C. krusei E28, C. glabrata 20/I, Trichosporon beigelii 1188, Trichophyton mentagrophytes 445, Aspergillus fumigatus 231, and Absidia corymbifera 272 using broth microdilution method. Prior to testing, each strain was passaged onto SDA. Fungal inocula were prepared by suspending yeast or conidia in sterile water to obtain a final inoculum of 5.0 ± 0.2 . 103 cfu cm-3. Antifungal activity of the compounds in vitro was determined in tissue culture medium RPMI 1640 (SEVAPHARMA) buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (Sigma). Each substance was dissolved in DMSO. The concentrations tested were 1000, 500, 250, 125, 62.5, 31.25, 15.6, 8, 4, and 2 umol dm-3 provided a given compound was soluble in DMSO and did not precipitate in RPMI. Drug-free controls were included. The minimum inhibitory concentrations (MICs) were determined after 24 and 48 h of static incubation at 35 oC. In the case of T. mentagrophytes, MICs were recorded after 72 and 120 h of incubation [26].

Photosynthesis-inhibiting activity in spinach chloroplasts

Spinach chloroplasts were prepared by the procedure described by Walker [27]. 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. [28]. 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.

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. [29] (photoperiod 16 h light/8 h dark; irradiation: 90 ummol 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 [30]. 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 umol dm-3).

Artemia screening bioassay

Artemia salina L. eggs were obtained from JBL NovoTermia (Germany). The method of Eppley [31] was applied for A. salina larvae hatching. The test was arranged according to Kiviranta et al. [32]. 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 uL 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.

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, No. MSM 111600002, and No. MSM 111600003). The authors wish to thank Ms. K. Kissová from the Institute of Chemistry, Faculty of Natural Sciences, ComeniusUniversity, Bratislava, SlovakRepublic, for her technical assistance in antialgal testing.

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