Synthesis and antimicrobial activity of new 4-(benzylsulfanyl)pyridine derivatives

Article abstract of DOI:10.1135/cccc19990417

A series of 4-(benzylsulfanyl)pyridine-2-carbonitriles 2 and 4-(benzylsulfanyl)pyridine-2-carbothioamides 3 was synthesized and their antimycobacterial and antifungal activities were tested. The prepared compounds exhibit significant activity both against M. tuberculosis and nontuberculous mycobacteria. The most active compounds show the MICs of 4 μmol/1. The antifungal activities of 2 are very weak, compounds 3 are inactive against all the tested fungi.

Full text of DOI:10.1135/cccc19990417

4-(Benzylsulfanyl)pyridine Derivatives
417
SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF NEW
4-(BENZYLSULFANYL)PYRIDINE DERIVATIVES
a4
Věra KLIMEŠOVÁa1,*, Martin SVOBODAa2, Karel WAISSERa3, Milan POUR
b
and Jarmila KAUSTOVÁ
a
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
500 05 Hradec Králové, Czech Republic; e-mail: klimeso@faf.cuni.cz, svobodam@faf.cuni.cz,
1
2
3
4
waisser@faf.cuni.cz, pour@faf.cuni.cz
b
National Reference Laboratory for Mycobacterium Kansasii, Regional Institute of Hygiene,
728 92 Ostrava, Czech Republic; e-mail: khsova@ha-vel.cz
Received July 16, 1998
Accepted Septem ber 22, 1998
Dedicated to the memory of Dr Miroslav Protiva, one of the greatest Czech medicinal chemists.
A series of 4-(ben zylsulfan yl)pyridin e-2-carbon itriles 2 an d 4-(ben zylsulfan yl)pyridin e-2-
carboth ioam ides 3 was syn th esized an d th eir an tim ycobacterial an d an tifun gal activities
were tested. Th e prepared com poun ds exh ibit sign ifican t activity both again st M. tuberculosis
an d n on tuberculous m ycobacteria. Th e m ost active com poun ds sh ow th e MICs of 4 µm ol/l.
Th e an tifun gal activities of 2 are very weak, com poun ds 3 are in active again st all th e tested
fun gi.
Key w ord s: Pyridin ecarbon itriles; Pyridin ecarboth ioam ides; Tuberculostatics; An tim yco-
bacterial activity; An tifun gal activity; QSAR.
Th e curren t search for n ew an tim ycobacterial agen ts is very urgen t as tuber-
culosis h as becom e th e m ajor em ergin g opportun istic in fection . Th e devel-
opin g resistan ce to con ven tion al an tim ycobacterial agen ts is a stim ulatin g
factor in th e research of n ew, m ore selective com poun ds1. In addition , also
th e in fection s caused by atypical m ycobacteria (n on tuberculosis m ycobac-
teria) sh ow a risin g frequen cy, th e greatest problem beein g th e Mycobacte-
rium avium com plex. Dissem in ated in fection with th e M. avium com plex is
th e m ost com m on system atic bacterial in fection com plicatin g AIDS (ref.2).
In th e Czech Republic an in crease in th e occurren ce of M. kansasii h as been
observed in som e areas3.
Durin g our studies on n ew poten t an tim ycobacterial agen ts, we foun d
th at som e com poun ds sh owed also an tifun gal activity. Th is fact prom pted
us to in vestigate system atically both of th ese biological activities.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

418
Klimešová, Svoboda, Waisser, Pour, Kaustová:
In our previous work con cern in g th e pyridin e derivatives, we h ave re-
ported syn th esis an d an tim icrobial activity of com poun ds of gen eral struc-
ture 1.
CSNH
2
R
S
R = alkyl C - C
1 16
cycloalkyl C
N
6
1
Especially 2-(alkylsulfan yl)pyridin e-4-carboth ioam ides an d 4-(alkyl-
sulfan yl)pyridin e-2-carboth ioam ides sh owed an in terestin g level of activity
in vitro4. Regardin g th e spectrum of th e an tim ycobacterial activity, th ey
exh ibited efficacy both again st M. tuberculosis an d again st atypical m yco-
bacterial strain s. Moreover, in vivo results in dicated th at th e activity of
2-(propylsulfan yl)pyridin e-4-carboth ioam ide was com parable with th e ac-
tivity of ison iazide. A study of quan titative relation sh ips between th e struc-
ture an d activity of 2-alkylsulfan yl derivatives brough t us to an idea to
m odify th e alkylsulfan yl group an d a series of ben zylsulfan yl derivatives
bearin g various substituen ts on th e ph en yl rin g was prepared.
In th e presen t paper, we report th e results of an tim ycobacterial an d
an tifun gal activities of n ew 4-(ben zylsulfan yl)pyridin e-2-carbon itriles 2 an d
4-(ben zylsulfan yl)pyridin e-2-carboth ioam ides 3.
Nitrile 4 as th e key startin g com poun d was prepared from 2-m eth yl-
pyridin e by a m eth od described in th e literature5,6. Treatm en t of 4 with
th iourea in eth an ol afforded th iuron ium salt 5 in 71% yield. Crude product
5 was used in furth er reaction s. Con den sation of 5 with th e appropriate
ben zyl ch lorides 6 in N,N-dim eth ylform am ide in th e presen ce of sodium
m eth oxide produced correspon din g ben zyl derivatives 2, wh ich were fur-
th er con verted by addition of h ydrogen sulfide in pyridin e in to th e related
pyridin ecarboth ioam ides 3 in good yields. On ly com poun ds 3s, 3t, an d 3u ,
h avin g at least on e n itro group in th e ben zyl rin g, were obtain ed by react-
in g 7 with th e correspon din g n itroben zyl ch lorides. Com poun d 7 was ob-
tain ed by a treatm en t of th iuron ium salt 5 with h ydrogen sulfide in
pyridin e (Sch em e 1).
Structures of com poun ds 2 an d 3 were con firm ed by elem en tal an alyses,
1
IR an d H NMR spectral data. All com poun ds 2 sh owed in th eir IR spectra a
stron g ban d at 2 240 cm –1, wh ich is ch aracteristic of th e C≡N group, an d
com poun ds 3 sh owed ch aracteristic ban ds of am ides at 3 260–3 300 an d
1 640 cm –1 (Tables I an d II). In th e 1H NMR spectra th e sign als of th e
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

4-(Benzylsulfanyl)pyridine Derivatives
419
pyridin e m oiety appeared separate from th ose of th e substituted ph en yl
rin g. In both th e n itrile an d th e carboth ioam ide sets, th e pyridin e proton s
were observed as doublets of doublets coupled with on e an oth er; th e larger
couplin g con stan t between H-5 an d H-6 was in th e ran ge of 5.2–5.5 Hz
wh ile both th ese h ydrogen s h ad sm aller couplin gs with H-3. Hydrogen
atom H-6 was sh ifted m ost down field in th e n itrile series due to th e pres-
en ce of th e pyridin e n itrogen . On th e oth er h an d, in th e carboth ioam ide
series, H-3 lies even m ore down filed sin ce it is desh ielded by th e th io-
carbam oyl group. Ch em ical sh ifts an d m ultiplicities of proton s in th e
ph en yl m oiety depen d on th e substitution (Tables III an d IV).
NH
2
Cl
NH
2
Cl
S
S
R
Cl
6
R
SC(NH )
2 2
ethanol
DMF, CH ONa
3
N
C
N
C
N
C
N
N
N
4
5
2
Py H S
2
Py H S
2
S
SH
R
6
DMF, CH ONa
3
N
C
NH
S
N
C
S
2
NH
2
3
7
R
R
2, 3, 6
2, 3, 6
H
4-CH
3
a
b
c
d
e
f
k
l
2-Cl
3-Cl
4-Cl
2-F
3-F
4-F
3-Br
4-Br
3-NO
4-NO
2
2
m
n
o
p
r
4-OCH
3,4-Cl
3
2
3,4-F
2
2-Cl-6-F
g
h
i
2-F-6-NO
s
t
2
2,4-(NO )
2 2
3-CH
3,5-(NO )
2 2
j
u
3
SCHEME 1
An tim ycobacterial activity of th e prepared com poun ds was tested in vitro
again st Mycobacterium tuberculosis CNCTC 331/88, Mycobacterium kansasii
CNCTC 235/80, Mycobacterium kansasii 509/96 (clin ical isolate), an d Myco-
bacterium avium CNCTC 33/88 usin g th e m icrom eth od for th e determ in a-
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

420
Klimešová, Svoboda, Waisser, Pour, Kaustová:
tion of th e m in im um in h ibitory con cen tration (MIC). In vitro an tigun gal
activity again st Trichophyton mentagrophytes 445, Candida albicans ATCC
44859, Candida tropicalis 156, Candida krusei E28, Candida glabrata 20/I,
Trichosporon beigelii 1188, Aspergillus fumigatus 231, an d Absidia corymbifera
272 was determ in ed usin g th e m icrodilution broth test. All strain s, except
of C. albicans, were clin ical isolates, iden tified by con ven tion al m orph ologi-
cal an d bioch em ical m eth ods.
EXPERIMENTAL
Th e m eltin g poin ts were determ in ed on a Kofler apparatus an d are un corrected. An alytical
sam ples were dried over P4O10 at 60 °C an d 30 Pa for 8–10 h . Elem en tal an alyses were per-
form ed on CHNS-O CE in strum en t (FISONS EA 1110). IR spectra were obtain ed on a Nicolet
Im pact 400 spectrom eter in KBr pellets. Th e NMR spectra were recorded in CDCl3 solution s
(with th e exception of 4-sulfan ylpyridin e-2-carboth ioam ide wh ich was m easured in CD3OD)
at am bien t tem perature on a Varian Mercury-VX BB 300 spectrom eter operatin g at 300 MHz.
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). Couplin g con stan ts (J) are given in Hz. Th e sign als were assign ed
to th e correspon din g proton s on ly if an un equivocal assign m en t could be m ade (1D decoup-
lin g experim en ts were don e wh en n ecessary). Th e reaction s an d purity of all th e prepared
com poun ds was ch ecked by TLC (Silufol UV254, Kavalier, Votice, Czech Republic) in eth yl
acetate–ligh t petroleum (2 : 3) usin g UV detection .
2-Cyan opyridin e-4-th iuron ium Ch loride (5)
4-Ch loropyridin e-2-carbon itrile (4; 3.5 g, 25 m m ol) an d th iourea (2.0 g, 26 m m ol) were dis-
solved in eth an ol (20 m l) an d th e solution was stirred at 100 °C for 15 m in . After coolin g,
th e resultin g salt was filtered off, affordin g 5 as a grey-green product; yield 3.9 g (71%), m .p.
193–196 °C.
Preparation of 4-(Ben zylsulfan yl)pyridin e-2-carbon itriles 2.
Gen eral Procedure
To a stirred solution of 5 (1.5 g, 7 m m ol) in dry DMF (8 m l) a solution of sodium (0.2 g,
8 m m ol) in dry m eth an ol (2.5 m l) was added at room tem perature, followed by th e appro-
priate ben zyl ch loride 6 (7 m m ol). After stirrin g at room tem perature for 2–4 h , th e solven t
was evaporated in vacuo an d th e residue was diluted with water (75 m l). Th e precipitated
solid was filtered off, wash ed with cold water an d crystallized from eth an ol or eth an ol–water
(Table I).
4-Sulfan ylpyridin e-2-carboth ioam ide (7)
Dry h ydrogen sulfide was passed th rough a solution of 5 (2.2 g, 10 m m ol) in a m ixture of
dry pyridin e (18 m l) an d trieth ylam in e (1.8 m l) at 70 °C for 3 h . After coolin g, th e m ixture
was diluted with water (75 m l), th e solution was acidified with 1 M H2SO4 to pH 6, th e
form ed precipitate was filtered off an d crystallized from water to give 7 as yellow crystals;
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

4-(Benzylsulfanyl)pyridine Derivatives
421
TABLE I
Yields an d ph ysicoch em ical properties of com poun ds 2a–2u
Calculated/Foun d
Com -
poun d
Form ula
M.w.
Yield
%
M.p.
°C
IR
cm –1
R
% C % H % N % S
2a
2ba
2cb
2d c
2e
H
2-Cl
C13H10N2S
226.3
68
83
61
76
46
30
65
82
80
65
63
83
85
83
81
90.5–92.5 69.01 4.46 12.39 14.17 2 234
69.03 4.80 12.53 14.08
C13H9ClN2S
260.7
100–101 60.00 3.49 10.77 12.30 2 235
60.13 3.43 10.82 12.42
3-Cl
C13H9ClN2S
260.7
76.5–78
60.00 3.49 10.77 12.30 2 237
60.25 3.40 10.73 12.14
4-Cl
C13H9ClN2S
260.7
102.5–104 60.00 3.49 10.77 12.30 2 239
59.90 3.64 10.70 12.34
2-F
C13H9FN2S
244.3
103–105 63.92 3.71 11.47 13.12 2 236
64.27 3.65 11.59 13.26
2f
3-F
C13H9FN2S
244.3
67–69
58–60
70–73
63.92 3.71 11.47 13.12 2 240
63.76 3.78 11.57 13.19
2g
4-F
C13H9FN2S
244.3
63.92 3.71 11.47 13.12 2 235
63.81 3.66 11.70 12.87
2h d
2ie
2j
3-Br
C13H9BrN2S
305.2
51.16 2.97
51.47 2.95
9.18 10.50 2 235
9.16 10.41
4-Br
C13H9BrN2S
305.2
112–113 51.16 2.97
51.24 3.02
9.18 10.50 2 236
8.80 10.11
3-CH3
4-CH3
3-NO2
4-NO2
4-OCH3
3,4-Cl2
C14H12N2S
240.3
52–54
69.97 5.03 11.66 13.34 2 237
69.96 4.88 11.92 13.35
2k
C14H12N2S
240.3
78–81.5
69.97 5.03 11.66 13.34 2 238
69.80 4.99 11.57 13.28
2l
C13H9N3O2S
271.3
142–144 57.56 3.34 15.49 11.82 2 240
57.73 3.54 15.50 11.66
2m
2n
C13H9N3O2S
271.3
140.5–143 57.56 3.34 15.49 11.82 2 240
57.72 3.23 15.78 11.49
C14H12N2OS
256.3
107–109.5 65.60 4.72 10.93 12.51 2 234
65.44 4.65 11.29 12.51
2o
C13H8Cl2N2S
295.2
85–87
52.90 2.73
52.69 3.02
9.49 10.86 2 240
9.29 10.68
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

422
Klimešová, Svoboda, Waisser, Pour, Kaustová:
TABLE I
(Continued)
Calculated/Foun d
Com -
poun d
Form ula
M.w.
Yield
%
IR
M.p.
°C
R
cm –1
% C % H % N % S
2p
2rf
2s
2t
3,4-F2
C13H8F2N2S
262.3
84
87
60
46
82
73–75
59.53 3.07 10.68 12.22 2 243
59.71 3.31 10.56 11.91
2-Cl-6-F
C13H8ClFN2S
278.7
121.5–125 56.02 2.89 10.05 11.50 2 234
55.92 2.80 10.00 11.39
2-F-6-NO2 C13H8FN3O2S
289.3
115.5–117.5 53.98 2.79 14.53 11.08 2 235
53.91 2.76 14.23 11.07
2,4-(NO2)2 C13H8N4O4S
316.3
118.5–120 49.37 2.55 17.71 10.14 2 238
49.55 2.54 17.50 10.11
2u
3,5-(NO2)2 C13H8N4O4S
316.3
148–151
49.37 2.55 17.71 10.14 2 245
49.32 2.80 17.72 9.79
a
b
c
% Cl calculated: 13.45, foun d: 13.18; % Cl calculated: 13.45, foun d: 13.33; % Cl calcu-
d
e
lated: 13.45, foun d: 13.58; % Br calculated: 26.18, foun d: 25.97; % Br calculated: 26.18,
foun d: 26.24; % Cl calculated: 12.72, foun d: 12.52.
f
yield 1.4 g (89%), m .p. 212–215 °C. For C6H6N2S2 (170.3) calculated: 42.33% C, 3.55% H,
16.45% N, 37.66% S; foun d: 42.24% C, 3.52% H, 16.38% N, 37.65% S. 1H NMR (CD3OD):
7.83 d, 1 H, J(3,5) = 1.9 (H-3); 7.46 dd, 1 H, J(5,6) = 6.3, J(5,3) = 1.9 (H-5); 7.68 d, 1 H,
J(6,5) = 6.3 (H-6).
Preparation of 4-(Ben zylsulfan yl)pyridin e-2-carboth ioam ides 3a–3r.
Gen eral Procedure
A 4-(ben zylsulfan yl)pyridin e-2-carbon itrile 2 (1 m m ol) was dissolved in dry pyridin e (6 m l),
th en dry trieth ylam in e (0.6 m l) was added an d dry h ydrogen sulfide was passed th rough th e
m ixture at 50 °C for 3–5 h . After coolin g, th e m ixture was diluted with water (75 m l), th e
precipitated solid was filtered off an d crystallized from eth an ol (Table II).
Preparation of 4-(Ben zylsulfan yl)pyridin e-2-carboth ioam ides 3s–3u .
Gen eral Procedure
To a stirred solution of 7 (1.5 g, 9 m m ol) in dry DMF (8 m l) a solution of sodium (0.2 g,
9 m m ol) in dry m eth an ol (3 m l) was added, followed by th e correspon din g ben zyl ch loride 6
(9 m m ol). After stirrin g at room tem perature for 1–2.5 h , th e solven t was evaporated in
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

4-(Benzylsulfanyl)pyridine Derivatives
423
TABLE II
Yields an d ph ysicoch em ical properties of com poun ds 3a–3u
Calculated/Foun d
Com -
poun d
Form ula
M.w.
Yield
%
M.p.
°C
IR
R
cm –1
% C % H % N % S
3a
3ba
3cb
3d c
3e
H
2-Cl
C13H12N2S2
260.4
87
90
92
95
85
80
87
92
90
125–127
59.97 4.65 10.76 24.63
60.05 4.71 10.71 24.72
3 232
1 598
C13H11ClN2S2
294.8
169.5–172 52.96 3.76 9.50 21.75
52.73 3.74 9.64 21.72
3 231
1 597
3-Cl
C13H11ClN2S2
294.8
143–145.5 52.96 3.76 9.50 21.75
52.91 3.75 9.69 21.73
3 284
1 616
4-Cl
C13H11ClN2S2
294.8
163–166
52.96 3.76 9.50 21.75
52.95 3.73 9.69 21.81
3 247
1 602
2-F
C13H11FN2S2
278.4
152–153.5 56.09 3.98 10.06 23.03
56.14 4.19 10.02 23.56
3 242
1 595
3f
3-F
C13H11FN2S2
278.4
121–123
56.09 3.98 10.06 23.03
55.91 3.90 9.95 23.18
3 316
1 615
3g
4-F
C13H11FN2S2
278.4
154–155.5 56.09 3.98 10.06 23.03
55.87 3.97 9.89 23.50
3 297
1 600
3h d
3ie
3j
3-Br
C13H11BrN2S2
338.0
152–155.5 46.16 3.28 8.29 18.92
46.01 3.21 8.26 19.00
3 259
1 617
4-Br
C13H11BrN2S2
338.0
157–161
46.16 3.28 8.29 18.92
45.94 3.55 8.56 18.95
3 299
1 637
3-CH3
4-CH3
3-NO2
4-NO2
4-OCH3
3,4-Cl2
C14H14N2S2
274.4
92 132.5–134.5 61.28 5.14 10.21 23.37
61.57 5.42 10.07 23.42
3 299
1 640
3k
C14H14N2S2
274.4
95
77
68
92
89
137–138
61.28 5.14 10.21 23.37
61.14 5.10 9.97 23.46
3 309
1 624
3l
C13H11N3O2S2
305.4
167–169
51.13 3.63 13.76 21.00
51.44 3.50 14.05 21.17
3 286
1 637
3m
3n
C13H11N3O2S2
305.4
139.5–142 51.13 3.63 13.76 21.00
50.83 3.55 13.65 21.27
3 321
1 619
C14H14N2OS2
290.4
131–133
57.90 4.86 9.65 22.08
57.95 4.73 9.73 22.23
3 324
1 610
3o
C13H10Cl2N2S2
328.0
135–137
47.57 3.07 8.54 19.50
47.54 2.71 8.34 19.41
3 258
1 609
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

424
Klimešová, Svoboda, Waisser, Pour, Kaustová:
TABLE II
(Continued)
Calculated/Foun d
Com -
poun d
Form ula
M.w.
Yield
%
IR
M.p.
°C
R
cm –1
% C % H % N % S
3p
3rf
3s
3t
3,4-F2
C13H10F2N2S2
296.0
90
88
67
80
34
131–134 52.69 3.40 9.45 21.61 3 258
52.82 3.17 9.25 21.68 1 610
2-Cl-6-F
C13H10ClFN2S2
312.8
146–148.5 49.92 3.22 8.96 20.50 3 237
49.79 3.11 8.92 20.68 1 605
2-F-6-NO2
2,4-(NO2)2
3,5-(NO2)2
C13H10FN3O2S2
323.4
147–149.5 48.29 3.12 12.99 19.83 3 274
48.18 3.13 12.76 19.78 1 621
C13H10N4O4S2
350.4
147.5–150 44.57 2.88 15.99 18.30 3 312
44.34 2.78 15.90 18.36 1 604
3u
C13H10N4O4S2
350.4
160–163 44.57 2.88 16.00 18.27 3 255
44.20 3.11 15.80 18.05 1 594
a
b
c
% Cl calculated: 12.03, foun d: 11.90; % Cl calculated: 12.03, foun d: 11.78; % Cl calcu-
d
e
lated: 12.03, foun d: 12.16; % Br calculated: 23.35, foun d: 23.50; % Br calculated: 23.35,
foun d: 23.75; % Cl calculated: 11.33, foun d: 11.35.
f
vacuo an d th e residue was diluted with water (100 m l). Th e precipitated dolid was filtered
off an d crystallized from eth an ol (Table II).
An tim ycobacterial Activity
For th e evaluation of an tim ycobacterial activity of th e substan ces in vitro, th e followin g
strain s were used: Mycobacterium tuberculosis CNCTC My 331/88, Mycobacterium kansasii
CNCTC My 235/80, Mycobacterium avium CNCTC My 330/88, obtain ed from th e Czech Na-
tion al Collection of Type Cultures (CNCTC), Nation al In stitute of Public Health , Prague, an d
a clin ical isolate Mycobacterium kansasii 6 509/96. An tim ycobacterial activity of th e com -
poun ds again st th ese strain s was determ in ed in Šula’s sem isyn th etic m edium (SEVAC,
Prague). Th e com poun ds were added to th e m edium as dim eth yl sulfoxide solution s. Th e
followin g con cen tration s were used: 1 000, 500, 250, 125, 62, 31, 16, 8, an d 4 µm ol/l. Th e
m in im um in h ibitory con cen tration s were determ in ed after in cubation at 37 °C for 14 an d
21 days. MIC was th e lowest con cen tration of a substan ce, at wh ich th e in h ibition of th e
growth of m ycobacteria occurred.
Collect. Czech. Chem. Commun. (Vol. 64) (1999)

4-(Benzylsulfanyl)pyridine Derivatives
425
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An tifun gal Activity
Th e m icrodilution broth test was used for th e assessm en t of th e an tifun gal effect. All th e
tested substan ces were dissolved in dim eth yl sulfoxide. A two-fold dilution ran ge of th e
com poun ds was used so th at th e first con cen tration was 1 m m ol/l provided a given com -
poun d was soluble in dim eth yl sulfoxide an d stable in th e culture tissue m edium RPMI 1640
(Sevac, Prague). Th e test m edium was buffered to pH 7.0 with 0.165
M m orph olin e–
4-propan esulfon ic acid. Drug-free con trols were in cluded. Th e yeast in ocula were prepared
from 24–72 h colon ies grown on Sabouraud agar at 37 °C. Con idia from 5–10 day colon ies
were used to obtain suspen sion in filam en tous fun gi. Th e cell den sity in sterile 0.85% salin e
was adjusted by m ean s of a Bürker’s ch am ber. An tifun gal activity of th e com poun ds in vitro
was expressed as th e m in im um in h ibitory con cen tration (MIC) wh ich was read after 24 an d
48 h of static in cubation at 35 °C. In th e case of Trichophyton mentagrophytes, th e MICs were
recorded after 72 an d 120 h in cubation .
Calculation
All calculation s were carried out usin g th e Multireg H program (Klem era) for Microsoft Ex-
cel. Th e values of th e substituen t con stan ts (σ an d π) were taken from th e literature7.
RESULTS AND DISCUSSION
Th e results of an tim ycobacterial activity are collected in Tables V an d VI. In
several cases, th e m in im um in h ibitory con cen tration could n ot be deter-
m in ed due to th e lim ited solubility of th e com poun ds in th e tested m e-
dium .
Th e prepared com poun ds, in particular pyridin ecarboth ioam ides 3, ex-
h ibit sign ifican t in vitro activity again st all tested m ycobacterial strain s. Th e
values of MICs are with in th e ran ge of 4–125 µm ol/l for 2 an d 4–62 µm ol/l
for 3. By com parin g th eir MIC values with ison iazide (INH), th e studied
com poun ds reach th e activity of INH again st M. tuberculosis (MIC 4 µm ol/l)
an d th eir activities again st n on tuberculosis m ycobacteria exceed th at of
INH. Wh ereas n on tuberculosis m ycobacteria are m oderately susceptible to-
wards INH, th e n ewly prepared com poun ds display practically th e sam e ac-
tivity again st all th e tested strain s. Pyridin ecarbon itriles 2 exh ibit a better
efficacy again st both strain s of M. kansasii th an agaisn t M. tuberculosis an d
M. avium. Th e h igh est activity was observed with 3,5-din itroben zyl substi-
tuted derivative 2u (MIC 4–8 µm ol/l), wh ich is com parable with th e activity
of th e correspon din g pyridin ecarboth ioam ide 3u . Th e lowest MICs were ob-
served for pyridin ecarboth ioam ides 3 towards all tested strain s. Com -
poun ds 3u , 3m , 3d , an d 3h are th e m ost active com poun ds of th e series.
Th e results of an tifun gal activity, sum m arized in Table VII, in dicate th at
on ly five of th e eigh t fun gi strain s are sligh tly susceptible to som e of th e
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Klimešová, Svoboda, Waisser, Pour, Kaustová:
tested pyridin ecarbon itriles 2. Th e rem ain in g com poun ds 2 an d 3 were es-
sen tially in active again st all groups of th e fun gi tested.
For quan titative evaluation of th e structure–an tim ycobacterial activity re-
lation sh ips, a th ree-param eter regression equation was em ployed. Sim ilarly
to th e previous work8, th e Ham m ett con stan t σ of th e substituen ts on th e
ph en yl rin g served as on e param eter, th e secon d param eter bein g th e
TABLE V
In vitro an tim ycobacterial activity of com poun ds 2a–2u expressed as MIC (µm ol/l)
M. tuberculosis
M. kansasii 235/80
M. kansasii 509/96
M. avium 330/88
331/81
Com -
poun d
14 d
21 d
14 d
21 d
14 d
21 d
14 d
21 d
2a
2b
2c
2d
2e
2f
125
–
125
–
31
16
8
62
31
16
31
31
62
31
16
–
62
–
62
–
125
62
31
16
125
62
31
16
–
125
–
62
31
125
62
62
62
31
62
62
–
62
31
125
125
125
62
31
125
62
–
31
16
62
62
31
16
16
31
31
–
31
16
62
62
62
31
16
62
62
–
62
31
125
125
62
31
–
16
16
31
16
16
–
2g
2h
2i
2j
16
16
–
16
62
–
31
31
250
–
31
62
–
2k
2l
2m
2n
2o
2p
2r
–
–
31
31
8
–
31
–
62
–
–
–
–
–
–
–
62
125
–
–
16
62
–
16
16
–
16
62
–
31
16
–
–
250
–
31
16
62
16
8
31
–
2s
–
–
62
31
8
62
16
4
125
16
8
125
31
31
500
125
31
62
500
2t
62
16
4
62
31
4
2u
INH
500
500
500
500
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4-(Benzylsulfanyl)pyridine Derivatives
431
substituen t h ydroph obic con stan ts π. Th e th ird param eter was th e in dicator
variable I, wh ich acquired on ly two values, 1 in th e case of pyridin e-2-carbo-
n itriles 2, an d 0 in th e case of pyridin e-2-carboth ioam ides 3 (see Eqs (1)
an d (2)). Equation s (1) an d (2) sh ow th e correlation for th e m easured values
of an tim ycobacterial activity again st Mycobacterium tuberculosis after 14 an d
21 days of in cubation , respectively.
TABLE VI
In vitro an tim ycobacterial activity of com poun ds 3a–3u expressed as MIC (µm ol/l)
M. tuberculosis
M. kansasii 235/80
M. kansasii 509/96
M. avium 330/88
331/81
Com -
poun d
14 d
21 d
14 d
21 d
14 d
21 d
14 d
21 d
3a
3c
3d
3e
3f
16
8
31
8
8
4
31
8
16
8
31
8
16
8
31
31
8
8
8
8
8
8
8
8
4
16
16
8
16
8
31
16
16
8
62
16
8
–
16
16
16
8
–
8
31
16
8
62
31
–
3g
3h
3i
8
16
8
8
8
8
31
8
31
31
31
4
31
8
–
16
8
16
8
8
–
3j
8
8
31
31
16
8
3k
3l
16
4
16
4
31
16
–
16
16
4
16
16
4
16
8
3m
3n
3o
3p
3r
4
4
8
4
–
–
16
8
31
8
16
8
31
8
16
4
–
8
16
8
16
31
–
8
16
16
–
31
–
16
16
16
4
31
–
8
8
31
31
8
–
3t
8
–
–
16
8
31
31
500
3u
INH
4
4
8
4
4
4
500
500
500
500
500
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4-(Benzylsulfanyl)pyridine Derivatives
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log MICM.t.14d = –0.308(±0.094)σ + 0.012(±0.006)π + 0.801(±0.080)I
+ 0.998(±0.062)
R = 0.922 s = 0.20 F = 45.28 n = 28
(1)
log MICM.t.21d = –0.323(±0.121)σ + 0.017(±0.007)π + 0.734(±0.104)I
+ 1.160(±0.079)
R = 0.878 s = 0.26 F = 25.74 n = 27
(2)
From th e calculated equation s it can be con cluded th at th e activity of th e
com poun ds un der study in creases with in creasin g electron -acceptor proper-
ties an d decreasin g lipoph ilicity of th e substituen ts on th e ben zyl group. In -
fluen ce of th e electron ic param eters is predom in an t. Th ioam ides 3 are
usually m ore active th an th e correspon din g n itriles 2. Sim ilar equation s
were calculated for oth er strain s of Mycobacterium but th e statistical sign ifi-
can ce was worse (but still on th e level of sign ifican ce 0.05).
This work was supported by the Grant Agency of the Czech Republic (grant No. 203/96/0262) and
by the Higher Education Development Fund (grant No. 1371). 1H NMR spectra were recorded in the
NMR Centre, Faculty of Pharmacy, Charles University, supported by the Ministry of Education of the
Czech Republic (projekt No. VS97124). We are indebted to Ms D. Karlíčková (Department of Pharma-
ceutical Chemistry and Drug Control, Faculty of Pharmacy, Charles University) for performing ele-
mental analyses and Ms J. Žižková (Department of Inorganic and Organic Chemistry, Faculty of
Pharmacy, Charles University) for recording the IR spectra. The authors’ thanks are due to Dr V.
Buchta (Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University) for
providing the antifungal data.
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