New pyridine derivatives as potential antimicrobial agents

Article abstract of DOI:10.1016/S0014-827X(99)00078-6

A set of pyridine derivatives bearing a substituted alkylthio chain or a piperidyl ring in position 2 or 4 were synthesized, and their antimycobacterial and antifugal activities were evaluated. Chemical structures were confirmed by IR and NMR data, and by elemental analysis. Minimum inhibitory concentrations (MIC) were used for the evaluation of microbiological activity in vitro. The compounds were moderately active against both Mycobacterium tuberculosis and nontuberculous mycobacteria. The most active compound was 2-cyanomethylthiopyridine-4-carbonitrile (7) with MIC against Mycobacterium kansasii in the range of 8-4 μmol/l. The antifungal activities of the compounds were relatively low. Copyright (C) 1999 Elsevier Science S.A.

Full text of DOI:10.1016/S0014-827X(99)00078-6

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 666–672
New pyridine derivatives as potential antimicrobial agents
a,
Veˇra Klimesˇova´ *, Martin Svoboda a, Karel Waisser a, Milan Pour b,
c
Jarmila Kaustova´
a Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles Uni6ersity, 500 05 Hradec Kra´lo6e´, Czech Republic
b Laboratory of Structure and Interactions of Biologically Acti6e Molecules, Department of Inorganic and Organic Chemistry,
Faculty of Pharmacy, Charles Uni6ersity, Hradec Kra´lo6e´, Czech Republic
c National Reference Laboratory for Mycobacterium Kansasii, Regional Institute of Hygiene, Ostra6a, Czech Republic
Received 8 March 1999; accepted 15 June 1999
Abstract
A set of pyridine derivatives bearing a substituted alkylthio chain or a piperidyl ring in position 2 or 4 were synthesized, and
their antimycobacterial and antifugal activities were evaluated. Chemical structures were confirmed by IR and NMR data, and
by elemental analysis. Minimum inhibitory concentrations (MIC) were used for the evaluation of microbiological activity in vitro.
The compounds were moderately active against both Mycobacterium tuberculosis and nontuberculous mycobacteria. The most
active compound was 2-cyanomethylthiopyridine-4-carbonitrile (7) with MIC against Mycobacterium kansasii in the range of 8–4
mmol/l. The antifungal activities of the compounds were relatively low. © 1999 Elsevier Science S.A. All rights reserved.
Keywords: Pyridine; Alkylthiopyridine; Piperidylpyridine; Antimycobacterial activity; Antifungal activity
1. Introduction
For several years, research in our laboratories has
been directed towards the search for new compounds
with antimycobacterial and antifungal activity. As a
part of this programme, we have been investigating
pyridine derivatives. Recently, we reported the synthesis
and in vitro antimycobacterial and antifungal activities
Fig. 2.
of various alkylthiopyridine derivatives bearing an
alkylthio group in position 2 or 4 [1–4], as given by the
general formula in Fig. 1. Pyridinecarbothioamides in
particular showed significant activities. The most anti-
mycobacterially active compounds were found in the
series of 4-benzylthiopyridine-2-carbothioamides [3]
with minimum inhibitory concentrations (MICs) of 4
mmol/l. As regards the spectrum of the antimycobacte-
rial activity, they acted efficiently against both My-
cobacterium tuberculosis and atypical mycobacterial
strains. Significant antifungal activity against some
yeasts and dermatophytes was observed only in the case
of 2-alkylthiopyridine-4-carbothioamides [1], while the
other derivatives displayed a weak or no activity at all.
In this work we decided to study the modulation of
the alkylthio chain of pyridine carbothioamides and
pyridine carbonitriles by substitution with the following
groups: ꢀOH, ꢀCN, ꢀCSNH2, ꢀCOOR, ꢀCONHNH2.
Another modification of the molecule was the replace-
ment of the alkylthio chain both in pyridine carboni-
triles and pyridine carbothioamides with the piperidine
ring (Fig. 2).
Fig. 1.
* Corresponding author. Tel.: +420-49-5067111; fax: +420-49-
5210002.
E-mail address: klimeso@faf.cuni.cz (V. Klimesˇova´)
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00078-6

V. Klimesˇo6a´ et al. / Il Farmaco 54 (1999) 666–672
667
The target compounds were tested for their antimy-
cobacterial and antifungal activity in vitro. MIC ex-
pressed in mmol/l was used for the evaluation of
potential activity in vitro. Isoniazide and ketoconazole
were employed as reference substances for antimy-
cobacterial and antifungal activity, respectively.
2. Chemistry
The synthetic pathways leading to the 2-alkylthio
derivatives are depicted in Scheme 1. The chloro deriva-
tive 1, that served as the starting material in the synthe-
ses, was prepared according to a literature procedure
[5]. Condensation of 1 with 3-mercaptopropan-1,2-diol
in N,N-dimethylformamide (in the presence of sodium)
afforded the hydroxyalkylthio derivative 2 which was
further converted into the corresponding pyridinecar-
bothioamide 3 by the addition of hydrogen sulfide. The
piperidyl derivative 4 was obtained by the treatment of
1 with piperidine, and then further converted into the
corresponding pyridinecarbothioamide 5 by addition of
hydrogen sulfide. The treatment of 1 with thiourea in
ethanol afforded the thiuronium salt 6 in 80% yield,
and the crude product was used in subsequent reac-
tions. The reactions with chloroacetonitrile and 3-
chloropropionitrile in NaOH solution gave the
cyanoalkylthio derivatives 7 and 8, respectively. The
reaction of 6 with 4-chlorobutyronitrile under the same
Scheme 2.
conditions did not lead to the cyanoalkylthio deriva-
tive, but furnished the amidinoalkylthio derivative 9.
The structural assignment for this last compound
is based on its 1H NMR and 13C NMR spectra.
The spectra show
a
disubstituted linear chain
ꢀCH2ꢀCH2ꢀCH2, two non-equivalent NH protons, a
nitrile group, and a thioguanidin carbon. Compounds 7
and 8 were treated with hydrogen sulfide in pyridine,
giving rise to the corresponding pyridinecarbothio-
amides 10 and 11. The cyano group in the
cyanomethylthio chain of 7 was transformed into the
thiocarbamoyl group (10), whereas the cyanoethylthio
chain of 8 remained intact (11).
Chloropyridine 12 prepared from 2-methylpyridine
by an established method [6,7] was employed as the
starting material for the synthesis of 4-alkylthio deriva-
tives (Scheme 2). The piperidyl derivatives 13 and 14
were obtained via the same synthetic pathways as the
2-piperidyl derivatives 4 and 5. The other 4-alkylthio
derivatives were prepared from the thiuronium salt 15
obtained upon treatment of 12 with thiourea in ethanol.
The reactions of 15 with chloroacetonitrile, 3-chloro-
propionitrile, and 4-chlorobutyronitrile in a NaOH so-
lution gave analogous compounds as in the case of
the 2-alkylthio derivatives described above. The
cyanoalkylthio derivatives 16 and 17 were prepared by
the reactions of 15 with chloroacetonitrile and 3-chloro-
propionitrile, respectively. They were subsequently sub-
jected to treatment with hydrogen sulfide to afford the
Scheme 1.

668
V. Klimesˇo6a´ et al. / Il Farmaco 54 (1999) 666–672
pyridinecarbothioamides 19 and 20. The reaction of 15
with 4-chlorobutyronitrile in NaOH solution led to the
amidinoalkylthio derivative 18. The condensation of 15
with ethyl chloroacetate in N,N-dimethylformamide in
the presence of sodium methoxide produced ethoxycar-
bonylmethylthio derivative 21, which was easily con-
verted into the related pyridinecarbothioamide 22 by
addition of hydrogen sulfide to the carbonitrile group
of the pyridine ring. The ester function on the alkyl
chain of 21 was converted into the hydrazide group by
the reaction with hydrazine hydrate (23).
8.52 (dd, J(6,5)=5.2 Hz, J(6,3)=1.1 Hz, 1H, H6),
7.50 (dd, J(3,5)=1.7 Hz, J(3,6)=1.1 Hz 1H, H3), 7.24
(dd, J(5.6)=5.2 Hz, J(5.3)=1.7 Hz 1H, H5), 4.10–
3.85 (m, 1H, H2-alkyl), 3.69 (dd overlapped,
J(3a,3b)=11.5 Hz, J(3a,2)=4.2 Hz, 1H, H3a-alkyl),
3.64 (dd overlapped, J(3b,3a)=11.5 Hz, J(3b,2)=4.9
Hz, 1H, H3b-alkyl), 3.41 (dd, J(1a,1b)=14.8 Hz,
J(1a,2)=5.1 Hz, 1H, H1a-alkyl), 3.33 (dd, J(1b,1a)=
14.8 Hz, J(1b,2)=6.3 Hz, 1H, H1b-alkyl), 2.87 (bs,
ꢀ1H, OH). Anal. Calc. C9H10N2O2S (C,H,N,S).
The structures of all compounds were confirmed by
elemental analyses, IR and H NMR spectra.
3.1.2. General procedure for the preparation of the
pyridinecarbothioamides (3, 5, 10, 11, 14, 19, 20, 22)
Dry triethylamine (0.7 ml) was added to the solution
of the appropriate pyridinecarbonitrile (2, 4, 7, 8,
13, 16, 17, 21) (1 mmol) in dry pyridine (7 ml), and dry
hydrogen sulfide was passed through the mixture at
40–50°C for 2–6 h. After cooling, the mixture was
diluted with water (75–100 ml), the precipitated solid
was filtered off, and crystallized from ethanol.
1
3. Experimental
3.1. Chemistry
The melting points were determined on a Kofler
apparatus and are uncorrected. Analytical samples were
dried over P2O5 at 60°C and 30 Pa for 8–10 h. Elemen-
tal analyses were performed on CHNS–O CE instru-
ment (FISONS EA 1110). The results were within
90.4% of the calculated values. IR spectra were ob-
tained on a Nicolet Impact 400 spectrometer in KBr
pellets. The 1H NMR and 13C NMR spectra were
recorded for CDCl3 or d6-DMSO solutions at ambient
temperature on a Varian Mercury-VX BB 300 spec-
trometer operating at 300 MHz. Chemical shifts were
recorded as l values in parts per million (ppm), and
were indirectly referenced to tetramethylsilane via the
solvent signal (7.26 for 1H). Multiplicities are given
together with the coupling constant(s) (in Hz). The
signals were assigned to the corresponding protons only
if an unequivocal assignment could be made (1D de-
coupling experiments were done when necessary). The
reactions and purity of all the prepared compounds was
checked by TLC (Silufol UV254, Kavalier, Votice,
Czech Republic) in ethyl acetate–petroleum ether (2:3)
using UV detection.
3.1.2.1. 2-(2,3-Dihydroxypropylthio)pyridine-4-carbo-
thioamide (3). Yield 82%, m.p. 102–105°C; IR (KBr,
cm−1) 3166 and 1652 (NꢀH), 1432 (CꢂS), 3417 (OꢀH),
1
1037 and 1168 (CꢀO). H NMR300 (d6-DMSO, ppm) l
8.45 (bd, J(6,5)=5.2 Hz, 1H, H6), 7.52 (bs, 1H, H3),
7.38 (dd, J(5.6)=5.2 Hz, J(5.3)=1.7 Hz 1H, H5),
3.75–3.60 (m, 1H, H2-alkyl), 3.45–3.30 (m, 3H, H3a,
3b, 1a-alkyl), 3.11 (dd, J(1b,1a)=13.5 Hz, J(1b,2)=
7.2 Hz, 1H, H1b-alkyl). Anal. Calc. C9H12N2O2S2
(C,H,N,S).
3.1.2.2. 2-(1-Piperidyl)pyridine-4-carbothioamide (5).
Yield 86%, m.p. 161–163°C; IR (KBr, cm−1) 3274 and
1603 (NꢀH), 1458 (CꢂS), 2944 and 2852 (CH), 1241
1
(CꢀN). H NMR300 (CDCl3, ppm) l 8.19 (dd, J(6,5)=
5.2 Hz, J(6,3)=0.8 Hz, 1H, H6), 7.67 (bs, ꢀ1H,
CSNH2), 7.09 (dd, J(3,5)=1.7 Hz, J(3,6)=0.8 Hz, 1H,
H3), 6.72 (dd, J(5,6)=5.2 Hz, J(5,3)=1.7 Hz, 1H,
H5), 3.65–3.50 (m, 4H, H2,6-piperidine), 1.75–1.50 (m,
6H, H3,4,5-piperidine). Anal. Calc. C11H15N3S
(C,H,N,S).
3.1.1. 2-(2,3-Dihydroxypropylthio)pyridine-4-
carbonitrile (2)
3.1.2.3.
2-Thiocarbamoylmethylthiopyridine-4-carbo-
Sodium (0.4 g, 18 mmol) was added at room temper-
ature to a stirred solution of 3-mercaptopropan-1,2-diol
(1.5 ml, 18 mmol) in dry DMF (8 ml), followed by 1
(2.5 g, 18 mmol). After heating for 3 h at 50°C, DMF
was evaporated in vacuo and the residue was diluted
with water. The solution was acidified with 1 M H2SO4
to pH 6, and extracted with ether. The organic layer
was dried (Na2SO4) and the solvent was evaporated to
dryness under reduced pressure. The crude product was
recrystallized from ethanol (2.9 g, 76% yield), m.p.
70–73°C; IR (KBr, cm−1) 2238 (CꢁN), 3416 (O–H),
thioamide (10). Yield 90%, m.p. 135–138°C; IR (KBr,
cm−1) 3143 and 1615 (NꢀH), 1433 (CꢂS). H NMR300
1
(d6-DMSO, ppm) l 8.46 (dd, J(6,5)=5.2 Hz, J(6,3)=
0.7 Hz, 1H, H6), 7.58 (dd, J(3,5)=1.7 Hz, J(3,6)=0.7
Hz, 1H, H3), 7.41 (dd, J(5,6)=5.2 Hz, J(5,3)=1.7 Hz,
1H, H5), 4.28 (s, 2H, SCH2). Anal. Calc. C8H9N3S3
(C,H,N,S).
3.1.2.4. 2-(2-Cyanoethylthio)pyridine-4-carbothioamide
(11). Yield 83%, m.p. 110–113°C; IR (KBr, cm−1
)
1070 and 1120 (C–O). H NMR300 (CDCl3, ppm) l
3297 and 1637 (NꢀH), 2258 (CꢂN), 1400 (CꢂS). 1H
1

V. Klimesˇo6a´ et al. / Il Farmaco 54 (1999) 666–672
669
NMR300 (CDCl3, ppm) l 8.48 (dd, J(6,5)=5.2 Hz,
J(6,3)=1.0 Hz, 1H, H6), 7.68 (bs, ꢀ1H, CSNH2),
7.52 (dd, J(3,5)=1.7 Hz, J(3,6)=1.0 Hz, 1H, H3),
7.36 (dd, J(5,6)=5.2 Hz, J(5,3)=1.7 Hz, 1H, H5),
3.45 (t, J=7.1 Hz, 2H, SCH2), 2.86 (t, J=1.7 Hz, 2H,
CH2CN). Anal. Calc. C9H9N3S2 (C,H,N,S).
3.1.3.1. 2-(1-Piperidyl)pyridine-4-carbonitrile (4). Yield
74%, m.p. 53.5–55°C; IR (KBr, cm−1) 2234 (CꢁN),
1
2943 and 2853(CH), 1254 (CꢀN). H NMR300 (CDCl3,
ppm) l 8.24 (dd, J(6,5)=5.0 Hz, J(6,3)=1.0 Hz, 1H,
H6), 6.79 (bt, J(3,6)=J(3,5)=1.0 Hz, 1H, H3), 6.67
(dd, J(5,6)=5.0 Hz, J(5,3)=1.0 Hz, 1H, H5), 3.60–
3.50 (m, 4H, H2,6-piperidine), 1.75–1.55 (m, 6H,
H3,4,5-piperidine). Anal. Calc. C11H13N3 (C,H,N,S).
3.1.2.5. 4-(1-Piperidyl)pyridine-2-carbothioamide (14).
Yield 71%, m.p. 160–162°C; IR (KBr, cm−1) 3305 and
1596 (NꢀH), 1451 (CꢂS), 2940 and 2850 (CH), 1235
(CꢀN). 1H NMR300 (CDCl3, ppm) l 9.62 (bs, 1H,
CSNH2), 8.14 (d, J(3,5)=2.7 Hz, 1H, H3), 8.11 (d,
J(6,5)=6.0 Hz, 1H, H6), 7.33 (bs, ꢀ1H, CSNH2),
6.70 (dd, J(5,6)=6.0 Hz, J(5,3)=2.7 Hz, 1H, H5),
3.50–3.30 (m, 4H, H2,6-piperidine), 1.75–1.55 (m, 6H,
H3,4,5-piperidine). Anal. Calc. C11H15N3S (C,H,N,S).
3.1.3.2. 4-(1-Piperidyl)pyridine-2-carbonitrile (13). Yield
63%, m.p. 84–85.5°C; IR (KBr, cm−1) 2234 (CꢁN),
1
2918 and 2854 (CH), 1267 (C–N). H NMR300 (CDCl3,
ppm) l 8.22 (bd, J(6,5)=6.0 Hz, 1H, H6), 7.00 (bd,
J(3,5)=2.8 Hz, 1H, H3), 6.73(dd, J(5,6)=6.0 Hz,
J(5,3)=2.8 Hz, 1H, H5), 3.40–3.30 (m, 4H, H2,6-pipe-
ridine), 1.75–1.55 (m, 6H, H3,4,5-piperidine). Anal.
Calc. C11H13N3 (C,H,N,S).
3.1.4. General procedure for the preparation of the
thiuronium salts (6, 15)
Chloropyridinecarbonitrile (1, 12) (3.5 g, 25 mmol)
and thiourea (2.0 g, 26 mmol) were dissolved in ethanol
(20 ml), and the solution was heated at 100°C for 15
min. After cooling, the resultant salt was filtered off.
3.1.2.6.
4-Thiocarbamoylmethylthiopyridine-2-carbo-
thioamide (19). Yield 93%, m.p. 176–179°C; IR (KBr,
cm−1) 3174 and 1613 (NꢀH), 1432 (CꢂS). H NMR300
1
(d6-DMSO, ppm) l 8.40 (dd, J(6,5)=5.2 Hz, J(6,3)=
0.6 Hz, 1H, H6), 8.33 (bd, J(3,5)=1.9 Hz, 1H, H3),
7.49 (dd, J(5,6)=5.2 Hz, J(5,3)=1.9 Hz, 1H, H5),
4.24 (s, 2H, SCH2). Anal. Calc. C8H9N3S3 (C,H,N,S).
3.1.4.1. 4-Cyanopyridine-2-thiuronium salt (6). Yield
75%, m.p. 190–203°C.
3.1.2.7. 4-(2-Cyanoethylthio)pyridine-2-carbothioamide
(20). Yield 92%, m.p. 110–113°C; IR (KBr, cm−1
)
3.1.4.2. 2-Cyanopyridine-4-thiuronium salt (15). Yield
71%, m.p. 193–196°C.
3243 and 1597 (NꢀH), 2248 (CꢁN), 1429 (CꢂS). 1H
NMR300 (CDCl3, ppm) l 9.64 (bs, ꢀ1H, CSNH2), 8.55
(dd, J(3,5)=1.9 Hz, J(3,6)=0.8 Hz, 1H, H3), 8.36
(dd, J(6,5)=5.2 Hz, J(6,3)=0.8 Hz 1H, H6), 7.68 (bs,
ꢀ1H, CSNH2), 7.30 (dd, J(5,6)=5.2 Hz, J(5,3)=1.9
Hz, 1H, H5), 3.36 (t, J=7.1 Hz, 2H, SCH2), 2.80 (t,
J=7.1 Hz, 2H, CH2CN). Anal. Calc. C9H9N3S2
(C,H,N,S).
3.1.5. General procedure for the preparation of the
cyanoalkylthio pyridinecarbonitriles (7, 8, 16, 17)
Sodium hydroxide (0.75 g, 18 mmol) followed by
chloroacetonitrile (0.5 ml, 9 mmol) or 3-chloropropio-
nitrile (0.7 ml, 9 mmol) were added to a suspension of
the appropriate thiuronium salt (1, 15) (2 g, 9 mmol) in
hot water (50 ml). The solution was heated for 5 min (7
and 16) or 0.5 h (8 and 17), the resultant solid was
collected, and recrystallized from ethanol.
3.1.2.8. Ethyl-(2-thiocarbamoylpyridine-4-ylthio)acetate
(22). Hydrogen sulfide was passed through at room
temperature. 83% yield, m.p. 123–124°C; IR (KBr,
cm−1) 3282 and 1571 (NꢀH), 1428 (CꢂS), 1721 (CꢂO).
1H NMR300 (d6-DMSO, ppm) l 8.39 (dd, J(6,5)=5.2
Hz, J(6,3)=0.6 Hz, 1H, H6), 8.30 (dd, J(3,5)=1.9 Hz,
J(3,6)=0.6 Hz 1H, H3), 7.45 (dd, J(5.6)=5.2 Hz,
J(5.3)=1.9 Hz 1H, H5), 4.12 (q overlapped, J1=7.1
Hz, J2=14.0 Hz, 2H, OCH2), 4.11 (s overlapped, 2H,
SCH2), 1.17 (t, J=7.1 Hz, 3H, CH3). Anal. Calc.
C10H12N2O2S2 (C,H,N,S).
3.1.5.1. 2-Cyanomethylthiopyridine-4-carbonitrile (7).
Yield 80%, m.p. 94–96°C; IR (KBr, cm−1) 2241 and
2248(CꢁN). 1H NMR300 (CDCl3, ppm) l 8.68 (dd,
J(6,5)=5.2 Hz, J(6,3)=1.1 Hz, 1H, H6), 7.47 (t,
J(3,6)=J(3,5)=1.1 Hz, 1H, H3), 7.31 (dd, J(5,6)=5.2
Hz, J(5,3)=1.1 Hz, 1H, H5), 4.02 (s, 2H, SCH2). Anal.
Calc. C8H5N3S (C,H,N,S).
3.1.5.2. 2-(2-Cyanoethylthio)pyridine-4-carbonitrile (8).
Yield 51%, m.p. 98–101°C; IR (KBr, cm−1) 2239 and
2253(CꢁN). 1H NMR300 (CDCl3, ppm) l 8.58 (dd,
J(6,5)=5.0 Hz, J(6,3)=1.2 Hz, 1H, H6), 7.42 (t,
J(3,6)=J(3,5)=1.2 Hz, 1H, H3), 7.23 (dd, J(5,6)=5.0
Hz, J(5,3)=1.2 Hz, 1H, H5), 3.45 (t, J=7.0 Hz, 2H,
SCH2), 2.85 (t, J=7.0 Hz, 2H, CH2CN). Anal. Calc.
C9H7N3S (C,H,N,S).
3.1.3. General procedure for the preparation of the
piperidyl deri6ati6es of the pyridinecarbonitriles (4, 13)
A mixture of chloropyridinecarbonitrile (1, 12) (0.7 g,
5 mmol) and piperidine (1.25 ml, 15 mmol) was heated
under reflux for 1 h. The resultant solid was then
collected and recrystallized from ethanol.

670
V. Klimesˇo6a´ et al. / Il Farmaco 54 (1999) 666–672
3.1.5.3. 4-Cyanomethylthiopyridine-2-carbonitrile (16).
stirred solution of 15 (2.5 g, 12 mmol) in dry DMF (8
ml). After stirring at room temperature for 2 h, the
solvent was evaporated in vacuo, and the residue was
diluted with water (50 ml). The precipitated solid was
filtered off, washed with cold water, and crystallized
from ethanol (1.1 g, 42% yield), m.p. 45–47°C; IR
(KBr, cm−1) 2240 (CꢁN), 1727 (CꢂO). 1H NMR300
(CDCl3, ppm) l 8.49 (dd, J(6,5)=5.4 Hz, J(6,3)=0.8
Hz, 1H, H6), 7.55 (dd, J(3,5)=1.9 Hz, J(3,6)=0.8 Hz,
1H, H3), 7.37 (dd, J(5,6)=5.4 Hz, J(5,3)=1.9 Hz, 1H,
H5), 4.24 (q, J1=7.1 Hz, J2=14.3 Hz, 2H, OCH2),
3.76 (s, 2H, SCH2), 1.28 (t, J=7.1 Hz, 3H, CH3). Anal.
Calc. C10H10N2O2S (C,H,N,S).
Yield 86%, m.p. 130–132°C; IR (KBr, cm−1) 2245
1
(CꢁN). H NMR300 (CDCl3, ppm) l 8.64 (dd, J(6,5)=
5.2 Hz, J(6,3)=0.7 Hz, 1H, H6), 7.60 (dd, J(3,5)=1.9
Hz, J(3,6)=0.7 Hz, 1H, H3), 7.43 (dd, J(5,6)=5.2 Hz,
J(5,3)=1.9 Hz, 1H, H5), 3.81 (s, 2H, SCH2). Anal.
Calc. C8H5N3S (C,H,N,S).
3.1.5.4. 4-(2-Cyanoethylthio)pyridine-2-carbonitrile (17).
Yield 62%, m.p. 95–98°C; IR (KBr, cm−1) 2233 and
2243(CꢁN). 1H NMR300 (CDCl3, ppm) l 8.54 (dd,
J(6,5)=5.4 Hz, J(6,3)=0.6 Hz, 1H, H6), 7.51 (t,
J(3,5)=1.9 Hz, J(3,6)=0.6 Hz, 1H, H3), 7.33 (dd,
J(5,6)=5.4 Hz, J(5,3)=1.9 Hz, 1H, H5), 3.33 (t, J
=7.1 Hz, 2H, SCH2), 2.79 (t, J=7.1 Hz, 2H, CH2CN).
Anal. Calc. C9H7N3S (C,H,N,S).
3.1.8. (2-Cyanopyridine-4-ylthio)acethydrazide (23)
Some 30% hydrazine hydrate (7.5 ml) was added to a
solution of 21 (1 g, 4.5 mmol) in dry ethanol, and the
reaction mixture was maintained at room temperature
for 3 h. The solution was evaporated to dryness, and
the resultant solid was recrystallized from ethanol (0.65
g, 68% yield), m.p. 154–157°C; IR (KBr, cm−1) 2240
3.1.6. General procedure for the preparation of the
butanamidine deri6ati6es (9, 18)
Sodium hydroxide (0.5 g, 12 mmol) followed by
4-chlorobutyronitrile (0.5 ml, 6 mmol) were added to a
suspension of the appropriate thiuronium salt (1, 15)
(1.3 g, 6 mmol) in hot water (70 ml), and the solution
was heated at reflux for 3 h. After cooling, the precipi-
tate was collected, and recrystallized from ethanol.
1
(CꢁN), 1651 (CꢂO). H NMR300 (d6-DMSO, ppm) l
8.49 (dd, J(6,5)=5.5 Hz, J(6,3)=0.6 Hz, 1H, H6),
7.99 (dd, J(3,5)=1.9 Hz, J(3,6)=0.6 Hz, 1H, H3),
7.61 (dd, J(5,6)=5.5 Hz, J(5,3)=1.9 Hz, 1H, H5),
3.81 (s, 2H, SCH2). Anal. Calc. C8H9N4OS (C,H,N,S).
3.1.6.1. 4-(4-cyanopyridine-2-ylthio)butanamidine (9).
Yield 23%, m.p. 103–106°C; IR (KBr, cm−1) 2250
3.2. Microbiology
1
(CꢁN), 3378 and 1657 (NꢀH), 1697 (CꢂN). H NMR300
(d6-DMSO, ppm) l 8.55 (dd, J(6,5)=5.2 Hz, J(6,3)=
0.5 Hz, 1H, H6), 8.20 (bs, ꢀ1H, NH), 7.71 (bs, ꢀ1H,
NH), 7.65 (dd, J(3,5)=1.5 Hz, J(3,6)=0.5 Hz, 1H,
H3), 7.48 (dd, J(5,6)=5.2 Hz, J(5,3)=1.5 Hz, 1H,
H5), 3.24 (t, J =7.1 Hz, 2H, SCH2), 2.62 (t, J=7.1
Hz, 2H, CH2), 1.96 (p, J=7.1 Hz, 2H, CH2CN). 13C
NMR (d6-DMSO, ppm) l 165.97, 158.67, 150.22,
142.06, 120.22, 119.74, 117.83, 28.28, 25.10, 15.66.
Anal. Calc. C10H12N4S (C,H,N,S).
3.2.1. Antimycobacterial acti6ity
For the evaluation of the antimycobacterial activity
of the substances in vitro, the following strains were
used: M. tuberculosis CNCTC My 331/88, M. kansasii
CNCTC My 235/80, Mycobacterium a6ium CNCTC
My 330/88, obtained from the Czech National Collec-
tion of Type Cultures (CNCTC), National Institute of
Public Health, Prague, and a clinical isolate of M.
kansasii 6 509/96. The antimycobacterial activities of
the compounds against these strains were determined in
3.1.6.2. 4-(2-cyanopyridine-4-ylthio)butanamidine (18).
Yield 30%, m.p. 99–102°C; IR (KBr, cm−1) 2245
&
the Sula’s semisynthetic medium (SEVAC, Prague). The
1
(CꢁN), 3328 (NꢀH), 1694 (CꢂN). H NMR300 (CDCl3,
compounds were added to the medium in dimethylsul-
foxide solutions. The following concentrations were
used: 1000, 500, 250, 125, 62, 31, 16, 8, and 4 mmol/l.
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 of
mycobacteria occurred.
ppm) l 8.37 (dd, J(6,5)=5.3 Hz, J(6,3)=0.6 Hz, 1H,
H6), 8.03 (dd, J(3,5)=2.1 Hz, J(3,6)=0.6 Hz, 1H,
H3), 7.85 (bs, ꢀ1H, NH), 7.27 (dd, J(5,6)=5.3 Hz,
J(5,3)=2.1 Hz, 1H, H5), 6.14 (bs, ꢀ1H, NH), 3.20 (t,
J=7.1 Hz, 2H, SCH2), 2.57 (t, J=7.1 Hz, 2H, CH2),
2.10 (p, J=7.1 Hz, 2H, CH2CN). 13C NMR (CDCl3,
ppm) l 166.40, 149.71, 149.47, 147.93, 123.35, 118.94,
118.49, 29.08, 24.20, 16.09. Anal. Calc. C10H12N4S
(C,H,N,S).
3.2.2. Antifungal acti6ity
In vitro antifungal activities against Trichophyton
mentagrophytes 445, Candida albicans ATCC 44859,
Candida tropicalis 156, Candida krusei E28, Candida
glabrata 20/I, Trichosporon beigelii 1188, Aspergillus
fumigatus 231, and Absidia corymbifera 272 were deter-
mined using the microdilution broth test. All strains,
3.1.7. Ethyl-(2-cyanopyridine-4-ylthio)acetate (21)
A solution of sodium (0.3 g, 15 mmol) in dry
methanol (5 ml) followed by ethyl chloroacetate (1.2
ml, 12 mmol) were added at room temperature to a

V. Klimesˇo6a´ et al. / Il Farmaco 54 (1999) 666–672
671
except of C. albicans, were clinical isolates, identified by
conventional morphological and biochemical methods.
All substances were dissolved in dimethylsulfoxide. A
two-fold dilution range of the solutions was used with
the first concentration being 1 mmol/l provided that a
given compound was soluble in dimethylsulfoxide and
stable in the culture tissue medium RPMI 1640 (Sevac,
Prague). The test medium was buffered to pH 7.0 with
0.165 M morpholine-4-propanesulfonic acid. Drug-free
controls were included. The yeast inocula were pre-
pared from 24–72 h colonies grown on Sabouraud agar
at 37°C. Conidia from 5- to 10-day colonies were used
to obtain suspension in filamentous fungi. The cell
density in sterile 0.85% saline was adjusted by means of
the Bu¨rker’s chamber. Antifungal activity of the com-
pounds in vitro was expressed as MIC which was
determined after 24 and 48 h of static incubation at
35°C. In the case of Trichophyton mentagrophytes, the
MICs were recorded after 72 and 120 h incubation.
efficient against M. kansasii (MIC in the range of 8–4
mmol/l). By comparing of MIC values, 7 turned out to
be six to seven times more potent than the standard. It
appears that the antimycobacterial activities of these
compounds are related to the presence of the 2-
cyanomethylthio moiety. This is in agreement with our
previous hypothesis [8] that the alkylthio group bound
to an electron-deficient carbon is the pharmacophore of
antimycobacterial activity. Prolonging of the alkyl
chain (8) caused a substantial decrease of activity. The
4-cyanoalkylthio isomers (16, 17) did not turn out to be
particularly active either. Moderately active compounds
(10, 11, 19, 20) with MIC values in the range of 31–250
mmol/l were obtained upon replacing the nitrile group
on the pyridine ring with the thiocarbamoyl group, but
all of them turned out to be more potent against
nontuberculous mycobacteria than isoniazide. The
other variations of the alkylthio chain as well as its
replacement with the piperidine ring led to weakly
active or inactive compounds.
As far as the antifungal activity is concerned, some of
the compounds were found to be slightly active against
the tested strains. Only T. mentagrophytes, C. albicans,
A. fumigatus and A. corymbifera are generally suscepti-
ble to the new pyridine derivatives, which exhibited
activity within the range of 125–1000 mmol/l. The rest
of the fungi strains were not susceptible up to the
concentration of 1000 mmol/l against all the com-
pounds. Most of the moderately active compounds
possess the thioamide group which we regard as a
4. Results and discussion
The antimycobacterial activities of the compounds
compared to isoniazide (INH) are shown in Table 1.
MIC values fall within the range of 1000–4 mmol/l. As
compared to isoniazide, the compounds did not match
the activity of INH against M. tuberculosis, but some of
them displayed remarkable activity against nontubercu-
lous mycobacteria. The most active derivative was 2-
cyanomethylthiopyridine-4-carbonitrile (7) being highly
Table 1
Antimycobacterial in vitro activity expressed as MIC (mmol/l)
Comp.
M. tuberculosis 331/81
14 d 21 d
1000
M. kansasii 235/80
M. kansasii 509/96
M. a6ium 330/88
14 d
21 d
14 d
21 d
14 d
21 d
250
2
3
4
5
7
100
500
1000
500
500
500
4
1000
500
500
500
4
250
250
250
250
8
500
500
250
500
8
1000
–
500
500
62
250
500
500
31
250
500
500
500
500
31
125
8
9
250
\1000
125
31
1000
500
500
125
500
62
250
\1000
125
31
1000
500
500
250
1000
62
250
500
500
250
62
500
125
500
500
500
31
500
500
250
125
1000
250
500
500
1000
31
125
1000
500
500
500
500
500
62
125
500
250
500
250
500
31
125
500
250
500
500
500
500
125
125
500
500
500
250
500
62
125
500
500
500
500
500
\1000
62
125
1000
125
500
250
1000
62
250
1000
250
250
500
1000
\1000
125
125
1000
250
1000
500
\1000
62
250
1000
500
10
11
13
14
16
17
18
19
20
21
22
23
INH
250
500
500
500
4
62
1000
1000
1000
4
500
125
500
500
500
500

672
V. Klimesˇo6a´ et al. / Il Farmaco 54 (1999) 666–672
Table 2
Antifungal in vitro activity expressed as MIC (mmol/l)
Comp.
Trichophyton mentagrophytes 445 Candida albicans 44859
Aspergillus fumigatus 231
Absidia corymbifera 272
72 h
120 h
24 h
48 h
24 h
48 h
24 h
48 h
10
14
250
125
250
0.98
250
125
250
1.95
1000
500
500
1000
\1000
1000
\1000
250
\1000
1.95
\1000
250
\1000
3.91
\1000
500
\1000
7.81
\1000
500
\1000
7.81
19
Ket a
B0.24
B0.24
a Ketoconazole.
fundamental condition for the antifungal activity in
compounds studied by us [1,4]. The most susceptible
strain was T. mentagrophytes, and the most antifungally
activecompoundturnedouttobe4-(1-piperidyl)pyridine-
2-carbothioamide (14). The results of antifungal ac-
tivity of selected compounds are summarized in
Table 2.
References
[1] V. Klimesˇova´, M. Otcˇena´sˇek, K. Waisser, Potential antifungal
agents. Synthesis and activity of 2-alkylthiopyridine-4-carbo-
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[2] V. Klimesˇova´, M. Svoboda, K. Waisser, M. Macha´cˇek, V.
&
Buchta, Z. Odlerova´, Research on antifungal and antimycobac-
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438–442.
[3] V. Klimesˇova´, M. Svoboda, K. Waisser, M. Pour, J. Kaustova´,
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[4] V. Klimesˇova´, M. Svoboda, K. Waisser, J. Kaustova´, V.
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bothioamide derivatives and their antimycobacterial, antifungal
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(1999) 433–440.
[5] D. Liberman, N. Rist, F. Grumbach, S. Cals, M. Moyeux, A.
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Acknowledgements
This work was supported by Grant 203/99/0030 of the
Grant Agency of the Czech Republic, and the Grant
Agency of Charles University (No. 21/1999) NMR
spectra were recorded and interpreted in the Laboratory
of Structure and Interactions of Biologically Active
Molecules, Faculty of Pharmacy, Charles University,
Hradec Kra´love´, which is supported by the Ministry of
Education of the Czech Republic project No.: VS97124.
The authors would also like to thank Mrs D. Karl´ıcˇkova´
(Department of Pharmaceutical Chemistry and Drug
Control, Faculty of Pharmacy, Charles University) for
&
[7] M. Celadn´ık, Z. Kosˇt’a´lova´, S. J´ısˇka, K. Waisser, K. Kubala,
K. Pala´t, Antituberkulotika. XVII. Funkcˇn´ı deriva´ty kyseliny
&
performing elemental analyses, and Mrs J Zizˇkova´ (De-
4-halogenpikolinove´
181–185.
a jejich N-oxidu`, Cesk Farm 25 (1976)
partment of Inorganic and Organic Chemistry, Faculty
of Pharmacy, Charles University) for recording the IR
spectra. We are indebted to Dr V. Buchta (Department
of Biological and Medical Sciences, Faculty of Pharmacy,
Charles University) for providing the antifungal data.
&
[8] K. Waisser, V. Klimesˇova´, Z. Odlerova´, The alkylthio group
bound to the electron-deficient atom of carbon as the pharma-
cophore of antituberculotic activity, Folia Pharm. Univ. Carol.
18 (1995) 31–34.
.
.