Synthesis of some 2, 6-disubstituted 4-amidopyridines and -thioamidopyridines, and their antimycobacterial and photosynthesis-inhibiting activity

Article abstract of DOI:10.3390/50300208

A group of 26 new 2-halogeno-6-alkylsulfanyl- and 2,6-bis-alkylsulfanyl-4- amidopyridines and corresponding thioamidopyridines was synthesised. Some of the amidopyridines and all thioamidopyridines were tested for their antimycobacterial activity against atypical mycobacterial strains. Promising photosynthesis-inhibiting activity was also found for some of the amidopyridines.

Full text of DOI:10.3390/50300208

Molecules 2000, 5, 208-218
molecules
ISSN 1420-3049
http://www.mdpi.org
Synthesis of Some 2, 6-Disubstituted 4-Amidopyridines and
-Thioamidopyridines, and Their Antimycobacterial and
Photosynthesis-Inhibiting Activity
Miroslav Miletin*1, Jiri Hartl1, Martin Dolezal1, Z. Odlerova3, K. Kralova4 and Milos Machacek2
1Department of Medicinal Chemistry and Drug Control, Faculty of Pharmacy, Charles University,
Hradec Králové, Czech Republic
Tel.: +420 49 50 67 387, Fax: +420 49 551 24 23, E-mail: miletin@faf.cuni.cz
2Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Hradec
Králové, Czech Republic
3Institute of Preventive and Clinic Medicine, Bratislava, Slovak Republic
4Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic
*Author to whom correspondence should be addressed.
Received: 8 March 1999 / Revised and Accepted: 28 February 2000 / Published: 3 March 2000
Abstract: A group of 26 new 2-halogeno-6-alkylsulfanyl- and 2,6-bis-alkylsulfanyl-4-
amidopyridines and corresponding thioamidopyridines was synthesised. Some of the ami-
dopyridines and all thioamidopyridines were tested for their antimycobacterial activity
against atypical mycobacterial strains. Promising photosynthesis-inhibiting activity was also
found for some of the amidopyridines.
Keywords: Thioamidopyridines, antimycobacterial activity, photosynthesis-inhibiting ac-
tivity.
Introduction
Some events during the past decade have dramatically changed the nature and magnitude of the
problem of tuberculosis. The HIV epidemic and increasing resistance to antituberculous drugs dictate
the need of development of new antituberculotics [1-3].
© 2000 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.

209
Molecules 2000, 5
In our recent study [4], we modified the structure of therapeutically used antituberculous drugs ethi-
onamide and prothionamide. Some of the more lipophilic derivatives showed promising activity
against atypical mycobacterial strains. The present study extends the scope of lipophilic derivatives of
4-thioamidopyridines. Since it has been recently reported that 2-alkylsulfanyl-4-thioamidopyridines
showing antimycobacterial [5] and antifungal activity [6] inhibit photosynthetic processes in algae and
plant chloroplasts [7,8], the synthesised compounds were tested for their both antimycobacterial and
photosynthesis-inhibiting activity.
Results and Discussion
The synthesis of 2,6-disubstituted 4-amidopyridines and -thioamidopyridines is shown in Scheme 1.
2,6-Dichloro- or -dibromo-4-amidopyridine [9] was treated with an equimolar amount of the respective
thiolate to give 2-halogeno-6-alkylsulfanyl-4-amidopyridines (1-8). 2-Alkylsulfanyl-6-hexylsulfanyl-
4-amidopyridines (9-13) were prepared from 2-chloro-6-hexylsulfanyl-4-amidopyridine (3) in a similar
fashion. Thionation of 4-amidopyridines (1-13) with the Lawesson’s reagent afforded the 4-
thioamidopyridines (14-26). The melting points, yields, and elemental analyses for compounds 1-26
are given in Table 1, and IR and 1H NMR spectroscopic data in Table 2.
1
Cl
N
Cl
R2S
N
SR1
-
2
SR
Cl
N
-
+
1
+
R S Na
20
R S Na
50
DMF, °C
DMF, °C
CONH2
CONH2
CONH2
1-3
9-13
Lawesson's toluene,
Lawesson's toluene,
reagent
reflux
reagent
reflux
1
SR
R2S
N
1
SR
Cl
N
CSNH2
CSNH2
14-16
22-26
Br
N
Br
1
Br
N
SR
SR
Br
N
-
1
Lawesson's
reagent
toluene,
reflux
+
R S Na
20
DMF, °C
CONH2
CONH2
CSNH2
4-8
17-21
Scheme 1. Synthesis of 2,6-disubstituted 4-amidopyridines and thioamidopyridines.

210
Molecules 2000, 5
As the amidopyridines were originally considered to be intermediates, only selected compounds
were evaluated for their activity against Mycobacterium tuberculosis, M. kansasii, M. avium, and M.
fortuitum. The minimum inhibitory concentrations (MIC) for the tested compounds are listed in Table
3, along with isoniazid as a reference standard. The tested compounds in the amidopyridine series (1,
4, 7, 9, 12) were inactive with the exception of 9 and, in part, 7. Compound 9 showed greater activity
against atypical strains, especially M. kansasii (MIC = 60 mmol.dm-3), than isoniazid.
The thioamidopyridine series was more active in the antimycobacterial screening than the amidopy-
ridine one. 2-Halogeno-6-alkylsulfanyl-4-thioamidopyridines (14-21) exhibited increasing antimyco-
bacterial activity with prolonging the carbon chain in the alkylsulfanyl substituent up to seven carbons.
Overall, 2-chloro substituted thioamidopyridines were more active than their 2-bromo analogues.
Compounds 16 and 20 were the most promising, with MICs of 60 mmol.dm-3 against M. tuberculosis
and M. kansasii (as well as M. avium for 16).
Among 2-alkylsulfanyl-6-hexylsulfanyl-4-thioamidopyridines (22-26), compound 22 was found to
be the most active of the compounds studied. It showed, similar to compounds 23 and 24, moderate
activity against M. tuberculosis (MIC = 30 mmol.dm-3). Additionally, it exhibited greater activity
against M. kansasiii (MIC = 60 mmol.dm-3), M. avium (MIC = 60 mmol.dm-3 ), and M. fortuitum (MIC
= 250 mmol.dm-3) than isoniazid, as well as 16. Increasing the total number of carbon atoms in both
alkylsulfanyl side chains above ten caused a decrease in antimycobacterial activity, which is in agree-
ment with our previous findings [4].
To better understand the structure-activity relationships, log P values were calculated (Table 3). We
found that the lipophilicities of the most potent antimycobacterial compounds were different for all
four strains employed. In the 4-thioamidopyridine series (14-26), the highest activities against M. tu-
berculosis were observed for compounds 22-24 with log P values between 5.75 and 7.34. The antimy-
cobacterial activity of 4-thioamidopyridines against other three strains showed a sharp dependence on
lipophilicity. In the case of M. kansasii and M. avium, the most active 4-thioamidopyridines 16 and 22
showed log P values ranging from 4.86 to 5.75, while the compounds with the highest activity against
M. fortuitum, 23 and 24, exhibited log P values 6.81 and 7.34.
The tested compounds also inhibited photochemical activity of spinach chloroplasts. The IC50 val-
ues, i. e., concentrations of the compounds causing 50% decrease of oxygen evolution rate in spinach
chloroplasts with respect to the untreated control, are listed in Table 3. From the comparison of IC50
values of the 2-halogeno substituted 4-amido (1-7) and 4-thioamidopyridines (14-21) it can be con-
cluded that amidopyridines exhibit greater inhibitory activity than the corresponding thioamidopyri-
dines. For compounds 9-12, a pronounced decrease in photosynthesis-inhibiting activity with the in-
creasing lipophilicity of the compounds has been confirmed. This is in good agreement with the previ-
ously obtained results concerning photosynthesis-inhibiting activity of 2-alkylsulfanyl-4-
thioamidopyridines in spinach chloroplasts and Chlorella vulgaris [7,8]. In the 4-amidopyridine series,
the most active compounds 7, 9, 3, and 6 showed log P in the range of 3.12-5.0, whereas the inhibitory
activity of thioamidopyridines with log P > 3.27 showed a pronounced decrease.

211
Molecules 2000, 5
Using EPR spectroscopy it was found that in the suspension of spinach chloroplasts the studied 4-
thioamidopyridines interact with D+ intermediate, i.e., with the radical of tyrosine 161 (TyrD) which is
located in D2 protein on the donor side of photosystem 2 [10], and due to this interaction the photo-
synthetic electron transport from the oxygen evolving complex to the core of photosystem 2 is im-
paired. The same site of action in the photosynthetic apparatus of spinach chloroplasts has also been
confirmed for the structurally similar 2-alkylsulfanyl-4-thioamidopyridines [7].
Table 1. Analytical data of the prepared compounds.
R2
N
SR1
X
NH2
Compd.
Formula
M. w.
R1,
R2
X
O
O
O
O
O
O
O
O
O
O
M. p. °C
Yield %
Calculated / Found
% H % N % S % Cl(Br)
% C
1
2
C8H9ClN2OS
(216.7)
C2H5,
Cl
162-163 44.34 4.19 12.93 14.80
75 44.21 4.12 13.11 14.69
112-113 46.85 4.81 12.14 13.90
76 46.65 4.73 12.35 13.62
129-131 52.84 6.28 10.27 11.75
72 52.76 6.21 10.39 11.64
178-180 34.02 2.86 11.34 12.97
70 33.96 2.78 11.46 12.89
160-162 36.80 3.47 10.73 12.28
73 36.66 3.35 10.85 12.18
113-115 41.53 4.53
71 41.31 4.41
120-122 47.13 5.78
68 47.01 5.71
123-125 48.70 6.13
16.36
16.51
15.37
15.50
13.00
13.14
32.34
32.48
30.60
30.76
27.63
27.81
24.12
24.26
23.14
23.26
-
C9H11ClN2OS
(230.7)
C3H7,
Cl
3
C12H17ClN2OS
(272.8)
C6H13,
Cl
4
C7H7BrN2OS
(247.1)
CH3,
Br
5
C8H9BrN2OS
(261.1)
C2H5,
Br
6
C10H13BrN2OS
(289.2)
C4H9,
Br
9.69 11.09
9.90 10.81
7
C13H19BrN2OS
(331.3)
C7H15,
Br
8.46
8.57
8.11
8.31
9.68
9.58
9.28
9.11
8
C14H21BrN2OS
(345.3)
C8H17,
Br
65
83-84
75
48.43 6.01
56.34 7.43
56.29 7.41
58.86 8.03
58.93 8.09
9
C14H22N2OS2
(298.5)
C6H13,
SC2H5
C6H13,
SC4H9
9.39 21.48
9.45 21.43
8.58 19.64
8.51 19.71
10
C16H26N2OS2
(326.5)
86-88
72
-

212
Molecules 2000, 5
Continuation of the Table 1.
Compd.
11
Formula
R1,
R2
X
O
O
O
S
S
S
S
S
S
S
S
S
S
S
S
S
M. p. °C
Yield %
Calculated / Found
M. w.
C17H28N2OS2
(340.6)
% C
% H
% N
% S % Cl(Br)
C6H13,
SC5H11
C6H13,
SC7H15
C6H13,
SC8H17
C2H5,
Cl
109-111 59.96 8.29
70 60.05 8.34
106-108 61.91 8.75
8.23 18.83
8.17 18.92
7.60 17.40
7.55 17.43
7.32 16.76
7.38 16.67
-
-
-
12
C19H32N2OS2
(368.6)
67
96-98
64
61.93 8.72
62.78 8.96
62.67 8.90
13
C20H34N2OS2
(382.6)
14
C8H9ClN2S2
(232.8)
80-81
89
41.28 3.90 12.04 27.55
41.16 3.81 12.17 27.41
43.81 4.49 11.35 25.98
43.73 4.45 11.43 25.87
15.23
15.38
14.37
14.49
12.27
12.42
30.36
30.48
28.83
28.98
26.18
26.39
23.01
23.27
22.11
22.35
-
15
C9H11ClN2S2
(246.8)
C3H7,
Cl
oil
87
16
C12H17ClN2S2
(288.9)
C6H13,
Cl
45-47
91
49.90 5.93
49.69 5.78
9.70 22.20
9.81 22.01
17
C7H7BrN2S2
(263.2)
CH3,
Br
127-129 31.95 2.68 10.64 24.36
85 31.82 2.61 10.51 24.22
107-108 34.66 3.27 10.11 23.13
18
C8H9BrN2S2
(277.2)
C2H5,
Br
92
53-55
90
34.58 3.22 10.03 22.96
19
C10H13BrN2S2
(305.3)
C4H9,
Br
39.35 4.29
39.15 4.20
44.96 5.51
44.78 5.47
46.53 5.86
46.31 5.77
53.46 7.05
53.41 7.02
56.10 7.65
56.25 7.71
57.26 7.91
57.02 7.78
59.33 8.39
59.47 8.42
60.25 8.60
60.03 8.47
9.18 21.01
9.07 20.89
8.07 18.46
8.15 18.27
7.75 17.74
7.92 17.59
8.91 30.58
9.03 30.41
8.18 28.08
8.03 28.23
7.86 26.97
8.05 26.72
7.28 25.00
7.15 24.78
7.03 24.12
7.25 23.91
20
C13H19BrN2S2
(347.3)
C7H15,
Br
43-45
89
21
C14H21BrN2S2
(361.4)
C8H17,
Br
44-46
91
22
C14H22N2S3
(314.5)
C6H13,
SC2H5
C6H13,
SC4H9
C6H13,
SC5H11
C6H13,
SC7H15
C6H13,
SC8H17
oil
90
23
C16H26N2S3
(342.6)
58-60
91
-
-
-
-
24
C17H28N2S3
(356.6)
62-63
89
25
C19H32N2S3
(384.7)
71-73
89
26
C20H34N2S3
(398.7)
62-64
87

213
Molecules 2000, 5
Table 2. IR and 1H NMR spectroscopic data of the prepared compounds.
Compd.
IR, (cm-1)
3019, 2972
(CH aliph.)
1690 (C=O)
3013, 2968
(CH aliph.)
1689 (C=O)
1H NMR, d (ppm)
7.38 d, J = 1, 1 H, arom.; 7.27 d, J = 1, 1 H, arom.; 6.09 bs, 1 H,
1
NH; 5.90 bs, 1 H, NH; 3.18 q, J = 7, 2 H, SCH2; 1.37 t, J = 7, 3 H, CH3
2
3
7.38 d, J = 1, 1 H, arom.; 7.27 d, J = 1, 1 H, arom.; 6.53 bs, 1 H, NH;
6.42 bs, 1 H, NH; 3.14 t, J = 7, 2 H, SCH2; 1.73 sext, J = 7, 2 H, CH2;
1.02 t, J = 7, 3 H, CH3
3014, 2959, 2931 7.38 d, J = 1, 1 H, arom.; 7.27 d, J = 1, 1 H, arom.; 6.19 bs, 2 H, NH2;
(CH aliph.)
1689 (C=O)
3019, 2970
(CH aliph.)
1696 (C=O)
3.15 t, J = 7, 2 H, SCH2; 1.69-1.26 m, 4 H, (CH2)2; 0.87 t, J = 7, 3 H,
CH3
7.44-7.46 m, 2 H, arom.; 6.15 bs, 1 H, NH; 5.75 bs, 1 H, NH; 2.60 s,
3 H, CH3
4
5
3018, 2969, 2936 7.40-7.42 m, 2 H, arom.; 6.06 bs, 1 H, NH; 5.88 bs, 1 H, NH; 3.18 q,
(CH aliph.)
1695 (C=O)
J = 7, 2 H, SCH2; 1.37 t, J = 7, 3 H, CH3
6
3014, 2962, 2933 7.40-7.42 m, 2 H, arom.; 6.24 bs, 2 H, NH2; 3.14 t, J = 7, 2 H, SCH2;
(CH aliph.)
1694 (C=O)
1.73 m, 2 H, CH2; 1.44 m, 2 H, CH2; 0.92 dist. t, J = 5, 3 H, CH3
7
3014, 2958, 2930 7.40-7.42 m, 2 H, arom.; 6.19 bs, 2 H, NH2; 3.14 t, J = 7, 2 H, SCH2;
(CH aliph.)
1690 (C=O)
1.70-1.25 m, 10 H, (CH2)5; 0.87 dist. t, J = 5, 3 H, CH3
8
3013, 2957, 2929 7.39-7.41 m, 2 H, arom.; 6.14 bs, 2 H, NH2; 3.14 t, J = 7, 2 H, SCH2;
(CH aliph.)
1689 (C=O)
1.70-1.26 m, 12 H, (CH2)6; 0.87 dist. t, J = 5, 3 H, CH3
9
3013, 2960, 2931 7.14 s, 2 H, arom.; 6.32 bs, 2 H, NH2; 3.21 q overlapping with 3.17 t,
(CH aliph.)
1686 (C=O)
4 H both, 2 ´ SCH2; 1.1 - 1.9 m, 8 H, 4 ´ CH2; 1.38 t, J = 7, 3 H,
SCH2CH3; 0.90 dist. t, J = 5, 3 H, CH3
10
11
12
13
3010, 2961, 2932 7.14 s, 2 H, arom.; 6.03 bs, 2 H, NH2; 3.20 t, J = 7, 4 H, 2 ´ SCH2; 1.1 -
(CH aliph.)
1686 (C=O)
1.9 m, 12 H, 6 ´ CH2; 0.95 t, J = 6, 3 H, S(CH2)3CH3; 0.90 dist. t,
J = 5, 3 H, CH3
3010, 2960, 2931 7.14 s, 2 H, arom.; 6.14 bs, 2 H, NH2; 3.19 t, J = 7, 4 H, 2 ´ SCH2; 1.1 -
(CH aliph.)
1686 (C=O)
1.9 m, 14 H, 7 ´ CH2; 0.90 dist. t, J = 5, 6 H, 2 ´ CH3
3009, 2959, 2930 7.14 s, 2 H, arom.; 6.02 bs, 2 H, NH2; 3.19 t, J = 7, 4 H, 2 ´ SCH2; 1.1 -
(CH aliph.)
1686 (C=O)
1.9 m, 18 H, 9 ´ CH2; 0.90 dist. t, J = 5, 6 H, 2 ´ CH3
3010, 2959, 2929 7.14 s, 2 H, arom.; 6.20 bs, 2 H, NH2; 3.19 t, J = 7, 4 H, 2 ´ SCH2; 1.1 -
(CH aliph.)
1686 (C=O)
1.9 m, 20 H, 10 ´ CH2; 0.90 dist. t, J = 5, 6 H, 2 ´ CH3

214
Molecules 2000, 5
Continuation of the Table 2.
Compd.
IR, (cm-1)
1H NMR, d (ppm)
14
2991, 2931, 2874 7.64 bs, 1 H, NH; 7.37 d, J = 1, 1 H, arom.; 7.29 d, J = 1, 1 H, arom.;
(CH aliph.)
1603 (C=O)
7.3 bs, 1 H, NH; 3.18 q, J = 7, 2 H, SCH2; 1.38 t, J = 7, 3 H, CH3
15
16
17
18
19
20
21
22
23
24
25
26
2996, 2968, 2934 7.7 bs, 1 H, NH; 7.38 d, J = 1, 1 H, arom.; 7.29 d, J = 1, 1 H, arom.; 7.2
(CH aliph.)
1603 (C=O)
bs, 1 H, NH; 3.17 t, J = 7, 2 H, SCH2; 1.74 sext, J = 7, 2 H, CH2;
1.05 t, J = 7, 3 H, CH3
2996, 2959, 2931 7.82 bs, 1 H, NH; 7.36 d, J = 1, 1 H, arom.; 7.28 d, J = 1, 1 H, arom.;
(CH aliph.)
1603 (C=O)
7.3 bs, 1 H, NH; 3.17 t, J = 7, 2 H, SCH2; 1.69-1.26 m, 8 H, (CH2)4;
0.90 dist. t , J = 5, 3 H, CH3
3001, 2932 (CH 7.76 bs, 1 H, NH; 7.40 d, J = 1, 1 H, arom.; 7.29 d, J = 1, 1 H, arom.;
aliph.)
7.26 bs, 1 H, NH; 2.59 s, 3 H, CH3
1603 (C=O)
2992, 2932 (CH 7.79 bs, 1 H, NH; 7.39 d, J = 1, 1 H, arom.; 7.29 d, J = 1, 1 H, arom.;
aliph.)
7.26 bs, 1 H, NH; 3.19 q, J = 7, 2 H, SCH2; 1.38 t, J = 7, 3 H, CH3
1603 (C=O)
2999, 2962, 2933 7.88 bs, 1 H, NH; 7.37 d, J = 1, 1 H, arom.; 7.29 d, J = 1, 1 H, arom.;
(CH aliph.)
1603 (C=O)
7.3 bs, 1 H, NH; 3.18 t, J = 7, 2 H, SCH2; 1.25-1.86, 4 H, (CH2)2; 0.95 t,
J = 6, 3 H, CH3
2997, 2958, 2929 7.85 bs, 1 H, NH; 7.38 d, J = 1, 1 H, arom.; 7.29 d, J = 1, 1 H, arom.;
(CH aliph.)
1603 (C=O)
7.3 bs, 1 H, NH; 3.17 t, J = 7, 2 H, SCH2; 1.25-1.86, 10 H, (CH2)5; 0.89
dist. t, J = 5, 3 H, CH3
2998, 2957, 2928 7.77 bs, 1 H, NH; 7.37 d, J = 1, 1 H, arom.; 7.28 d, J = 1, 1 H, arom.;
(CH aliph.)
1603 (C=O)
7.26 bs, 1 H, NH; 3.16 t, J = 7, 2 H, SCH2; 1.2-1.8 m, 12 H, (CH2)6; 0.88
dist. t, J = 5, 3 H, CH3
3004, 2960, 2930 7.13 s, 2H, arom.; 6.1 bs, 1 H, NH; 5.8 bs, 1 H, NH; 3.20 q overlapping
(CH aliph.)
1601 (C=O)
with 3.19 t, 4 H both, 2 ´ SCH2; 1.2 -1.8 m, 8 H, 4 ´ CH2; 1.34 t, J = 7,
3 H, SCH2CH3; 0.90 dist. t, J = 5, 3 H, CH3
2996, 2961, 2931 7.76 bs, 1 H, NH; 7.13 s, 2H, arom.; 7.2 bs, 1 H, NH; 3.19 t, J = 7, 4
(CH aliph.)
1601 (C=O)
H, 2 ´ SCH2; 1.1-1.9 m, 12 H, 6 ´ CH2; 0.95 t, J = 6, 3 H, S(CH2)3CH3;
0.90 dist. t, J = 5, 3 H, CH3
2995, 2960, 2931 7.76 bs, 1 H, NH; 7.13 s, 2H, arom.; 7.2 bs, 1 H, NH; 3.19 t, J = 7, 4
(CH aliph.)
1601 (C=O)
H, 2 ´ SCH2; 1.1-1.9 m, 14 H, 7 ´ CH2; 0.90 dist. t, J = 5, 6 H, 2 ´ CH3
2996, 2959, 2930 7.61 bs, 1 H, NH; 7.13 s, 2H, arom.; 7.2 bs, 1 H, NH; 3.19 t, J = 7, 4
(CH aliph.)
1601 (C=O)
H, 2 ´ SCH2; 1.1-1.9 m, 18 H, 9 ´ CH2; 0.90 dist. t, J = 5, 6 H, 2 ´ CH3
2995, 2958, 2929 7.66 bs, 1 H, NH; 7.13 s, 2H, arom.; 7.2 bs, 1 H, NH; 3.18 t, J = 7, 4
(CH aliph.)
1601 (C=O)
H, 2 ´ SCH2; 1.1-1.9 m, 20 H, 10 ´ CH2; 0.90 dist. t, J = 5, 6 H,
2 ´ CH3

215
Molecules 2000, 5
Table 3. MIC of the tested compounds against used mycobacterial strains, IC50 values concerning inhi-
bition of oxygen evolution rate in spinach chloroplasts by the tested compounds and calculated logP
values of the prepared compounds.
Compd.
MIC
IC50
(mmol.dm-3)
spinach
chloroplasts
101.5
58.4
10.2
76.7
34.2
10.6
5.9
(mmol.dm-3)
calculated
logP
tuberculosis
M. kansasii M. avium M. fortuitum
H37Rv
500
-
PKG8
1000
-
80/72
1000
-
1021
1000
-
1
2
1.96 ± 0.38
2.49 ± 0.38
4.08 ± 0.38
1.52 ± 0.42
2.06 ± 0.42
3.12 ± 0.42
4.71 ± 0.42
5.24 ± 0.42
5.00 ± 0.41
6.06 ± 0.41
6.59 ± 0.41
7.66 ± 0.41
8.19 ± 0.41
2.73 ± 0.41
3.27 ± 0.41
4.86 ± 0.41
2.34 ± 0.48
2.87 ± 0.48
3.93 ± 0.48
5.52 ± 0.48
6.06 ± 0.48
5.75 ± 0.47
6.81 ± 0.47
7.34 ± 0.47
8.41 ± 0.47
8.94 ± 0.47
-0.89 ± 0.24
3
-
-
-
-
4
>1000
-
>1000
-
>1000
-
>1000
-
5
6
-
-
-
-
7
250
-
250
-
>1000
-
>1000
-
8
-
9
60
-
60
250
-
250
-
9.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
isoniazid
-
203.5
249.3
543.6
258.8
104.8
9.3
-
-
-
-
1000
-
>1000
-
>1000
-
>1000
-
125
125
60
500
250
125
60
125
30
30
30
60
125
7
250
125
60
250
250
60
500
500
250
1000
500
500
500
500
250
125
125
500
500
1000
29.8
187.7
19.6
20.9
61.0
105.1
-
500
250
250
60
500
500
250
125
250
60
125
60
125
250
250
250
250
125
125
250
250
1000
99.7
157.5
-
-
-

216
Molecules 2000, 5
Experimental
General
Melting points were determined on a Kofler block, and are uncorrected. IR spectra were recorded
on a Nicolet Impact 400 spectrometer in chloroform. 1H NMR spectra were determined for solutions in
CDCl3 with TMS as the internal standard with a BS 587 ( Tesla, Brno ) 80 MHz apparatus. Column
chromatography was performed on silica gel (Silpearl, Kavalier Votice). Elemental analyses were per-
formed on a EA 1110 CHNS-O CE INSTRUMENTS elemental analyser.
Lipophilicity of the compounds was computed using a program ACD/LogP version 1.0 (Advanced
Chemistry Development Inc., Toronto).
Synthesis of 2-halogeno-6-alkysulfanyl-4-amidopyridines 1-8
2,6-Dichloro- or 2,6-dibromo-4-amidopyridine [9] (10 mmol) and the appropriate thiol (10 mmol)
were dissolved in anhydrous N,N-dimethylformamide (10 mL). To the stirred solution sodium
methoxide (10 mmol) in methanol (5 ml) was added dropwise. (Preparing 2-bromo-6-methylsulfanyl-
4-amidopyridine, sodium methanethiolate (10 mmol) was added in several portions to the stirred solu-
tion of 2,6-dibromo-4-amidopyridine (10 mmol) in anhydrous N,N-dimethylformamide.) The reaction
mixture was stirred at room temperature until TLC indicated a complete reaction. TLC was performed
using petroleum ether : ethyl acetate (2:1) as the mobile phase. The mixture was poured into cold wa-
ter. The crude product was filtered off, purified by column chromatography (petroleum ether : ethyl
acetate, 2:1), and recrystallised from aqueous ethanol. The yields and melting points are given in Table
1, and the IR and NMR spectroscopic data in Table 2.
Synthesis of 2-alkylsulfanyl-6-hexylsulfanyl-4-amidopyridines 9-13
To a stirred solution of 2-chloro-6-hexylsulfanyl-4-amidopyridine (3) (10 mmol) and the appropri-
ate thiol (10 mmol) in anhydrous N,N-dimethylformamide (10 mL) sodium methoxide (10 mmol) in
methanol (5 ml) was added dropwise. The reaction mixture was heated to about 50°C, stirred and
maintained at this temperature until TLC indicated a complete reaction. TLC was performed using pe-
troleum ether : ethyl acetate (2:1) as the mobile phase. The mixture was poured into cold water. The
crude product was filtered off, purified by column chromatography (petroleum ether : ethyl acetate,
2:1) and recrystallised from aqueous ethanol. The yields and melting points are given in Table 1, and
the IR and NMR spectroscopic data in Table 2.
Synthesis of 2,6-disubstituted 4-thioamidopyridines 14-26
To a stirred solution of 2,6-disubstituted 4-amidopyridine (10 mmol) in anhydrous toluene (10 ml)
Lawesson’s reagent (5 mmol) was added and the reaction mixture was heated at reflux until TLC indi-

217
Molecules 2000, 5
cated a complete reaction. TLC was performed using petroleum ether : ethyl acetate (4:1) as the mobile
phase. The mixture was then evaporated under reduced pressure, the crude product was purified by
column chromatography (petroleum ether : ethyl acetate, 4:1), and recrystallised from aqueous ethanol.
The melting points and yields are given in Table 1, and the IR and NMR spectroscopic data in Table 2.
Biological assays
Antimycobacterial evaluation was carried out on a semisynthetic liquid protein-containing Šula me-
dium (IMUNA, Šarišské Michal’any) buffered to pH 7.2. The following mycobacterial strains were
used: Mycobacterium tuberculosis H37Rv, M. kansasii PKG8, M. avium 80/72 and M. fortuitum 1021.
The concentrations of the compounds in the medium were 1000, 500, 250, 125, 60 and 30 mmol.dm-3.
The MIC values were determined after 14 days of incubation at 37°C.
The oxygen evolution rate (OER) in spinach chloroplasts was determined spectrophotometrically
(Specord UV VIS Zeiss Jena, Germany) by the Hill reaction. The measurements were carried out in
phosphate buffer (20 mmol, pH = 7.2) containing sucrose (0.4 mol.dm-3), MgCl2 (5 mmol.dm-3) and
NaCl (15 mmol.dm-3) using 2,6-dichlorophenol-indophenol as electron acceptor. Chlorophyll content
in the samples was 30 mg.dm-3 and the samples were irradiated (~100 W.m-2) from 10cm distance with
a halogen lamp (250 W) using a water filter to prevent warming of the samples (suspension tempera-
o
ture 22 C). The compounds were dissolved in dimethyl sulfoxide (DMSO) because of their limited
water solubility. The applied DMSO concentration (up to 5%) did not affect OER.
Acknowledgements: This study was supported by the Grants No. 204/1996 and 26/1998 from the Grant
Agency of Charles University and by the Grant No. 1/4013/97 from the Scientific Grant Agency of
Ministry of Education of Slovak Republic.
References and Notes
1. Raviglione, M. C.; Snider, D. E.; Kochi, A. Global epidemiology of tuberculosis: morbidity and
mortality of a worldwide epidemic. J. Am. Med. Ass. 1995, 273, 220-226.
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3. Buschauer, A. Pharmakotherapie der Tuberkulose: Wirkmechanismen und Resistenzen. Pharm. Z.
1997, 142, 11-25.
4. Miletin, M.; Hartl, J.; Odlerova, Z.; Machacek, M. Synthesis of some 2,6-bis(alkylthio)-4-pyri-
dinecarboxamides and carbothioamides and their antimycobacterial and antialgal activity. Phar-
mazie 1997, 52, 558-559.
5. Waisser, K.; Klimesova, V.; Odlerova Z. Relationships between the chemical structure of sub-
stances and their antimycobacterial activity to atypical strains. VII. 2-Alkythio-4-pyridinecarbo-
thioamides. Folia Pharm. Univ. Carol. 1996, 20, 59-62.

218
Molecules 2000, 5
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2-alkylthiopyridine-4-carbothioamides. Eur. J. Med. Chem. 1996, 31, 389-395.
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8. Kralova, K.; Sersen, F.; Klimesova, V.; Waisser, K. Relationships between photosynthesis-
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Samples Availability: Available from the authors.
© 2000 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes.