The synthesis and microbiological activity of 2-mercapto-4-methoxypyridine- 3-carbonitrile derivatives

Article abstract of DOI:10.3987/COM-08-11439

Synthesis of 2-(3-cyano-4-methoxypyridin-2-ylthio)acetic acid derivatives, starting either from 2-bromo-4-methoxypyridine-3-carbonitrile or 2-mercapto-4-methoxypyridine-3-carbonitrile is reported. The obtained products could be transformed by intramolecul

Full text of DOI:10.3987/COM-08-11439

HETEROCYCLES, Vol. 75, No. 11, 200
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HETEROCYCLES, Vol. 75, No. 11, 2008, pp. 2723 - 2734. © The Japan Institute of Heterocyclic Chemistry
Received, 15th May, 2008, Accepted, 3rd July, 2008, Published online, 7th July, 2008. COM-08-11439
THE SYNTHESIS AND MICROBIOLOGICAL ACTIVITY OF
2-MERCAPTO-4-METHOXYPYRIDINE-3-CARBONITRILE
DERIVATIVES
Agnieszka Miszke,*a Henryk Foks,a Kamil Brożewicz,a Anna Kędzia,b
Ewa Kwapisz,b and Zofia Zwolskac
a) Department of Organic Chemistry, Medical University of Gdańsk, Poland
E-mail address: miszke@amg.gda.pl
b) Department of Oral Microbiology, Medical University of Gdańsk, Poland
E-mail address: anak@amg.gda.pl
c) Department of Microbiology, Institute of Tuberculosis and Pulmonary Diseases,
Warsaw, Poland
Abstract – Synthesis of 2-(3-cyano-4-methoxypyridin-2-ylthio)acetic acid
derivatives, starting either from 2-bromo-4-methoxypyridine-3-carbonitrile or
2-mercapto-4-methoxypyridine-3-carbonitrile is reported. The obtained products
could be transformed by intramolecular Thorpe-Ziegler cyclization to related
thieno[2,3-b]pyridines.
Diazotization
of
3-amino-N-(4-chlorophenyl)-4-
methoxythieno[2,3-b]pyridine-2-carboxamide resulted in formation of a suitable
pyridothienotriazine derivative. Some of the prepared compounds demonstrated
noticeable bacteriostatic or tuberculostatic activity.
INTRODUCTION
Synthesis of substituted pyridine derivatives attracts much attention due to their interesting biological
activity, such as bacteriostatic,1 fungicidal2 or insecticidal.3 Derivatives of 4-substituted-2-thiopyridine
have been reported as antibacterial agents,4,5 what in connection with our previous results indicating their
tuberculostatic activity6 prompted us to synthesis of new compounds containing the 4-methoxy-
2-thiopyridine framework, and evaluation of their antimicrobial activity, especially the tuberculostatic
activity. Tuberculosis disease has staged a lethal comeback primarily because of resistance development
by the causative organism, Mycobacterium tuberculosis against all major anti-tuberculosis drugs, and due
to rising number of immuno-suppressed cases (through cancer chemotherapy, AIDS infection and
transplantation).7 For this reason new tuberculostatic drugs are urgently needed.

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HETEROCYCLES, Vol. 75, No. 11, 200
RESULTS AND DISCUSSION
The desired compounds were prepared starting either from 2-bromo-4-methoxypyridine-3-carbonitrile
(1),8 (Scheme 1), or from 2-mercapto-4-methoxypyridine-3-carbonitrile (9)9 (Scheme 2). The original
method of synthesis of the bromo derivative 1 was modified by replacement of gaseous HBr with the
easily accessible 33% HBr in acetic acid.
Reaction of 2-bromo-4-methoxypyridine-3-carbonitrile (1) with methyl 2-mercaptoacetate and 1 eq of
KOH at rt in DMF gave methyl 2-(3-cyano-4-methoxypyridin-2-ylthio)acetate (2). If an excess of KOH
was used, the reaction resulted in formation of thieno[2,3-b]derivative (3), apparently through the
Thorpe-Ziegler cyclization of the initially formed compound 2. The ester 3 was hydrolyzed to
3-amino-4-methoxythieno[2,3-b]pyridine-2-carboxylic acid (4) by reflux in NaOH solution followed by
acidification with acetic acid. The result stands in contradiction to Parnes report of unsuccessful attempt
of hydrolysis of related methyl 3-amino-4-(dimethylamino)thieno[2,3-b]pyridine-2-carboxylate.9
Analogous reactions of the 2-bromopyridine 1 with mercapto-N-arylacetamides and an excess of KOH
also gave products of substitution and subsequent Thorpe-Ziegler cyclization (5, 6). Heating of the
compound 5 in acetic anhydride lead to acylation of the amino group, accompanied, unexpectedly, by
decomposition of the amido group, to give 3-acetamido-4-methoxythieno[2,3-b]pyridine-2-carboxylic
acid (7). Diazotization of the amino derivative (5) with sodium nitrite in hydrochloric acid gave
3-(4-chlorophenyl)-9-methoxypyrido[3’,2’:4,5]thieno[3,2-d][1,2,3]triazin-4(3H)-one (8), apparently by
cyclization of the initially formed diazonium salt (Scheme 1).
Alternatively to the approach presented on Scheme 1, the 2-thiopyridine derivatives were prepared
starting from a suitable 2-mercaptopyridine (Scheme 2).
Reaction of 2-mercapto-4-methoxypyridine-3-carbonitrile (9) with chloroacetamides or methyl
3-bromopropanoate at rt in DMF containing equimolar amount of KOH gave S-substituted thiopyridines
(10-12) with 20-30% yield. Similarly as for the reactions starting from 2-bromo derivative 1, reaction of
the 2-mercaptopyridine 9 with suitable halo compounds in the presence of excess of KOH gave directly
products of substitution and subsequent Thorpe-Ziegler cyclization (13 – 19) (Scheme 2).
The structures of all new compounds were established based on elemental analysis and spectroscopic data.
Some of the synthesized compounds were evaluated for their antimicrobiological activities against
aerobic bacteria, anaerobic bacteria and Mycobacterium tuberculosis strains.
MICROBIOLOGICAL ACTIVITY
The investigations included 27 strains of anaerobic bacteria and 26 strains of aerobic bacteria isolated
from the oral cavity, respiratory system and abdominal cavity as well as 9 standard strains. The anaerobes
belonged to the following genera: Finegoldia (2 strains), Micromonas (3 strains), Propionibacterium

HETEROCYCLES, Vol. 75, No. 11, 200
2725
OMe
N
OMe
OMe
CN
NH2
CN
2 eq KOH
1 eq KOH
CO2Me
HSCH2CO2Me
HSCH2CO2Me
S
S
CO2Me
N
N
Br
1
2
3
HSCH2CONHC6H4Cl
HSCH2CONHC6H3(CH3)2
1. NaOH/MeOH
2. H
CH3
CH3
OMe
OMe
N
OMe
NH2
NH2
NH2
HN
HN
Cl
CO2H
S
S
S
O
O
N
N
4
5
6
Ac2O
NaNO2/HCl
OMe
O
Me
OMe
NH
N
N
Cl
N
O
CO2H
S
N
S
N
7
8
Scheme 1
OMe
N
NH2
R2
O
ClCH2COR2
OMe
CN
S
S
ClCH2COR1
13-17
R1
N
OMe
OMe
CN
10-11
O
NH2
1eq KOH
BrCH2COPh
2eq KOH
S
O
N
N
SH
OMe
18
CN
S
9
Br(CH2)2CO2Me
CO2Me
OMe
NH2
N
O
ClCH(COMe)2
12
S
Me
N
19
No
R1
10
11
N
N
N
N
N
No
R2
13
14
NH2
15
H2C
16
17
O
N
N
Me
N
N
F
OEt
Scheme 2
(4 strains), Prevotella (6 strains), Porphyromonas (2 strains), Bacteroides (4 strains) and standard strains:
Bacteroides fragilis ATCC 25285, Fusobacterium nucleatum ATCC 25586, Peptostreptococcus
anaerobius ATCC 27337 and Propionibacterium acnes ATCC 11827. There were also the following
aerobes: Staphylococcus (4 strains), Enterococcus (3 strains), Corynebacterium (3 strains), Acinetobacter
(4 strains), Escherichia (4 strains), Klebsiella (1 strain), Pseudomonas (7 strains) and 5 standard strains:
Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Klebsiella pneumonia ATCC

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HETEROCYCLES, Vol. 75, No. 11, 200
13883, Acinetobacter baumannii ATCC 19606 and Escherichia coli ATCC 25922. The susceptibility of
the anaerobic bacteria was determined by means of the plate dilution technique in Brucella agar
supplemented with 5% lamb blood.10-12 For aerobic bacteria experiments agar dilution technique with
Miller-Hinton agar was used. The derivatives were dissolved in 1 mL of DMSO immediately before the
experiment. Sterile, distilled water was used for further dilutions. The following concentrations were
used: 200, 100, 50, 25, 12.5 and 6.2 µg/mL. The inoculums containing 106 CFU/spot was applied to the
agar plates with Steers replicator. For aerobes the inoculated agar plates with derivatives were incubated
for 24 h at 37 ºC. For anaerobes agar plates were incubated in anaerobic jars for 48 h at 37 ˚C in 10% CO2,
10% H2 and 80% N2 with palladium catalyst and indicator for anaerobiosis. The minimal inhibitory
concentration (MIC) was defined as the lowest concentration of derivative that inhibited growth of
bacteria. Metronidazole (for anaerobes) and Amikacin (for aerobes) were used as the reference
substances.
The investigation of susceptibility of aerobic and anaerobic bacteria to the synthesized
2-mercaptopyridine derivatives are summarized in Table 1.
Of the tested 2-mercaptopyridine derivatives (16 samples) only compound 4 did not exhibit any activity
against anaerobic bacteria. The anaerobes were most susceptible (at concentrations in the range from ≤ 6.2
to 25 µg/mL) to the compounds 12 (37% of strains were susceptible), 2 (30% were susceptible) and 14
(22% of susceptible strains). Moreover, from 4 to 15 % of the screened anaerobic strains were susceptible
at concentration in the range from ≤ 6.2 to 25 µg/mL to derivatives 3, 5, 10, 11, 13, 15, 17 and 18.
Derivatives 2-3, 5-8, 10-15 and 17-19 inhibited growth of 8 to 35% of aerobic bacteria strains at
concentration in the range from ≤ 6.2 to 100 µg/mL. All derivatives, which were active towards anaerobic
bacteria, were more effective against Gram-positive strains.
Aerobic bacteria were less susceptible to the tested compounds than anaerobes. The aerobes were the
most susceptible (at concentrations in the range from ≤ 6.2 to 25 µg/mL) to derivatives 2 and 19 (8% of
susceptible strains) and compounds 3 and 12 (4% of susceptible strains). Derivatives 2, 3, 5, 12-14 and 19
inhibited growth of 8 to 35% of aerobic bacteria at concentration in the range from ≤ 6.2 to 100 µg/mL.
The standard strains of aerobic types of bacteria exhibited rather high resistance towards tested compound
(MIC ≥ 200 µg/mL), only for the aerobic Enterococcus faecalis ATCC 29212, compounds 2, 12 and 13
(MIC 100 µg/mL) and 3 and 5 (MIC 50 µg/mL) were active.
In the case of aerobic Staphylococcus aureus ATCC 25923 compounds (3, 5) (MIC 100 µg/mL) were
active. Derivative (2) induced the growth inhibition of Klebsiella pneumonia ATCC 13883 at
concentration of 50 µg/mL. In the case of anaerobic Bacteroides fragilis ATCC 25285, compounds (5,
11) (MIC 100 µg/mL) were active. Derivative (3, 5, 8, 10, 11, 12, 13) induced the growth inhibition of
Fusobacterium nucleatum ATCC 25586 at concentration of 100 µg/mL and compound (2) at

HETEROCYCLES, Vol. 75, No. 11, 200
2727
concentration of 50 µg/mL. Compound (2) inhibited the growth of Peptostreptococus anaerobius ATCC
27337 at ≤ 6.2 µg/mL, compounds (10, 14) at 12.5 µg/mL, derivatives (3, 5, 11, 13, 18) at 50 µg/mL and
(6, 7, 8, 12, 15) at 100 µg/mL. In the case of aerobic Propionibacterium acnes ATCC 11827 compounds
(2) (MIC ≤ 6.2 µg/mL), (14) (MIC 12.5 µg/mL), (12, 18) (MIC 25 µg/mL), (3, 5, 10, 11, 13, 19) (MIC 50
µg/mL) and (6, 7, 8, 15) (MIC 100 µg/mL) were active.
Selected compounds were tested for their tuberculostatic activity towards the standard Mycobacterium
tuberculosis H37Rv strain and two wild strains isolated from the tuberculotic patients: Myc. Species 210
and Myc. Species 192. The tuberculostatic activity was determined in vitro by classical test tube method
with Youman’s liquid medium containing 10% of bovine serum. Antituberculosis drugs: isonicotinic acid
hydrazide (MIC 0.5 μg/mL), viomycin (MIC 6.2 μg/mL), cycloserine (MIC 5 μg/mL) and pyrazinamid
(MIC 25 μg/mL) (towards Mycobacterium tuberculosis H37Rv) were used as references. The results are
given in Table 2. Of the tested group, only compounds 3, 13 and 17 exhibited activity at MIC 12.5 μg/mL.
In summary, the tested derivatives exhibited diversified activity against anaerobic bacteria. The anaerobes
were the most susceptible at concentrations in ranges from 6.2 to 100 µg/mL to derivatives 8 (44%), 2
(41%) and 12 (41%). The lowest susceptible in the same range of concentration to derivative 3, 13, 14, 15
(26%). The highest activity against the investigated strains of aerobic bacteria showed derivatives 2 and 3
in range from ≤ 6.2 to 100 µg/mL (35% of strains were susceptible).
EXPERIMENTAL
Melting points were obtained with a Boёtius apparatus and are uncorrected. Elemental analyses for C, H,
N and S were performed on Carlo-Erba 1108 instrument. The IR spectra were taken with a Satellite
spectrophotometer and the 1H NMR spectra were taken with a Varian Gem 200 MHz apparatus.
2-Bromo-4-methoxypyridine-3-carbonitrile (1)8
To 1,1-dicyano-4-(N,N-dimethyloamino)-2-methoxy-1,3-butadiene (0.885 g,
5
mmol), 33%
HBr/CH3CO2H (5 mL) was added and the mixture was left at rt for 48 h. Then ice (10 g) and concentrated
ammonium hydroxide (10 mL) were added. The precipitated solid was filtered off and crystallized from
MeOH to give 2-bromo-4-methoxypyridine-3-carbonitrile (1) (0.73 g, 69%) as a white solid.
Methyl 2-(3-cyano-4-methoxypyridin-2-ylthio)acetate (2)
Method A:
2-Bromo-4-methoxypyridine-3-carbonitrile (1) (0.639 g, 3 mmol) was dissolved in MeOH (20 mL), KOH
(0.168 g, 3 mmol) in water (3 mL) followed by methyl 2-mercaptoacetate (0.318 g, 3 mmol) were added,
and the mixture was refluxed for 2 h. Next, ice (20 g) was added, the precipitated solid was filtered off

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HETEROCYCLES, Vol. 75, No. 11, 200
2729
Table 2. Tuberculostatic Activity [μg/mL]
Compound
no.
Myc.tbc
H37Rv
Myc.spec.
192
Myc.spec.
210
2
25
12.5
25
25
25
50
100
25
25
25
25
25
50
25
25
25
50
50
25
50
50
50
100
50
50
50
25
50
50
25
25
50
-
25
25
25
25
25
25
100
25
25
25
12.5
25
25
12.5
25
25
-
3
4
5
6
7
8
10
11
12
13
14
15
17
18
19
Isonicotinic acid hydrazide 0.5
Cycloserine
Viomycin
Pyrazinamid
5
6.2
25
-
-
-
-
-
-
and crystallized from MeOH/H2O (1:3) to give methyl 2-(3-cyano-4-methoxypyridin-2-ylthio)acetate (2)
1
as a white solid (0.142 g, 20%), mp 129-131 ˚C. H NMR (DMSO-d6) δ 3.75 (3H, s), 4.00 (3H, s), 4.11
(2H, s), 6.68 (1H, d, J = 5.8 Hz), 8.43 (1H, d, J = 5.8 Hz); IR (KBr) νmax 2225, 1735, 1559, 1475, 1303,
1163, 1042, 1025, 825 cm-1. Anal. Calcd for C10H10N2O3S: C, 50.41; H, 4.23; N, 11.76; S, 13.46. Found:
C, 50.29; H, 4.22; N, 11.73; S, 13.42.
Method B
2-Bromo-4-methoxypyridine-3-carbonitrile (1) (0.639 g, 3 mmol) was dissolved in DMF (20 mL), KOH
(0.168 g, 3 mmol) in water (3 mL) followed by methyl 2-mercaptoacetate (0.318 g, 3 mmol) were added,
and the mixture was stirred at rt for 15 min. Next, ice (20 g) was added, the precipitated solid was filtered
off and crystallized from MeOH/H2O (1:3) to give methyl 2-(3-cyano-4-methoxypyridin-2-ylthio)acetate
(2) as a white solid (0.385 g, 54%).
General procedure for synthesis of thieno[2,3-b]pyridine derivatives 3, 5, and 6
2-Bromo-4-methoxypyridine-3-carbonitrile (1) (0.213 g, 1 mmol) was dissolved in DMF (15 mL), and
KOH (0.056 g, 1 mmol) in water (3 mL) was added. Then methyl 2-mercaptoacetate (0.106 g, 1 mmol)
(for 3), N-(4-chlorophenyl)-2-mercaptoacetamide (0.201 g, 1 mmol) (for 5) or N-(3,4-dimethylphenyl)-
2-mercaptoacetamide (0.195 g, 1 mmol) (for 6) was added and the mixture was stirred at rt for 15 min.
Next, KOH (0,056 g, 1 mmol) in water (3 mL) was added and the mixture was stirred for additional 45
min. Then ice (10 g) was added and the precipitated solid was filtered off and crystallized from
MeOH/H2O (2:3).

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HETEROCYCLES, Vol. 75, No. 11, 200
Methyl 3-amino-4-methoxythieno[2,3-b]pyridine-2-carboxylate (3)
Reaction with methyl 2-mercaptoacetate. Product 3 was isolated as a white solid (yield 46%), mp 162-164
˚C. 1H NMR (DMSO-d6) δ 3.76 (3H, s), 4.01 (3H, s), 6.95 (2H, s), 7.01 (1H, d, J = 5.6 Hz), 8.48 (1H, d,
J = 5.6 Hz); IR (KBr) νmax 3501, 3355, 1678, 1603, 1512, 1436, 1287, 1127, 1037, 842, 763, 573 cm-1.
Anal. Calcd for C10H10N2O3S: C, 50.41; H, 4.23; N, 11.76; S, 13.46. Found: C, 50.29; H, 4.22, N, 11.74;
S, 13.42.
3-Amino-N-(4-chlorophenyl)-4-methoxythieno[2,3-b]pyridine-2-carboxamide (5)
Reaction with N-(4-chlorophenyl)-2-mercaptoacetamide. Product 5 was isolated as a yellow solid (yield
1
58%), mp 285-287 ˚C. H NMR (DMSO-d6) δ 4.01 (3H, s), 6.99 (1H, d, J = 5.6 Hz), 7.17 (2H, s),
7.33-7.37 (2H, m), 7.69-7.74 (2H, m), 8.48 (1H, d, J = 5.6 Hz), 9.47 (1H, s); IR (KBr) νmax 3471, 3331,
1637, 1581, 1489, 1314, 1241, 1043, 841, 803 cm-1. Anal. Calcd for C15H12ClN3O2S: C, 53.97; H, 3.62; N,
12.59; S; 9.61. Found: C, 53.86; H, 3.60; N, 12.56; S, 9.59.
3-Amino-4-methoxy-N-(3,4-dimethylphenyl)thieno[2,3-b]pyridine-2-carboxamide (6)
Reaction with N-(3,4-dimethylphenyl)-2-mercaptoacetamide. Product 6 was isolated as a yellow solid
(yield 50%), mp 225-227 ˚C. 1H NMR (DMSO-d6) δ 2.17 (3H, s), 2.20 (3H, s), 4.02 (3H, s), 7.02 (1H, d,
J = 5.8 Hz), 7.06 (1H, d, J = 7.9 Hz), 7.11 (2H, s), 7.36 (1H, d, J = 7.9 Hz), 7.46 (1H, s), 8.48 (1H, d, J =
5.8 Hz), 9.18 (1H, s); IR (KBr) νmax 3462, 3325, 1636, 1588, 1500, 1313, 1258, 1046, 802, 756 cm-1. Anal.
Calcd for C17H17N3O2S: C, 62.36; H, 5.23; N, 12.83; S, 9.79. Found: C, 62.23, H, 5.21, N, 12.80; S, 9.77.
3-Amino-4-methoxythieno[2,3-b]pyridine-2-carboxylic acid (4)
Methyl 3-amino-4-methoxythieno[2,3-b]pyridine-2-carboxylate (3) (0.238 g, 1 mmol) was dissolved in
MeOH (15 mL) and then NaOH (0.200 g, 5 mmol) was added. The mixture was refluxed for 1 h. Then
the mixture was evaporated and ice (10 g) was added to the residue. Then glacial acetic acid was added,
the precipitate was filtered off and crystallized from MeOH/H2O (1:1) to give 3-amino-4-
1
methoxythieno[2,3-b]pyridine-2-carboxylic acid (4) (0.103 g, 46%) as a white solid, mp 230-232 ˚C. H
NMR (DMSO-d6) δ 4.15 (3H, s), 7.11 (1H, d, J = 5.3 Hz), 7.16 (2H, s), 8.46 (1H, d, J = 5.3 Hz), 9.46
(1H, s); IR (KBr) νmax 3392, 1571, 1538, 1360, 1290, 1195, 1054, 799 cm-1. Anal. Calcd for C9H8N2O3S:
C, 48.21; H, 3.60; N, 12.49; S, 14.30. Found: C, 48.09; H, 3.59; N, 12.47; S, 14.26.
3-Acetamido-4-methoxythieno[2,3-b]pyridine-2-carboxylic acid (7)
3-Amino-N-(4-chlorophenyl)-4-methoxythieno[2,3-b]pyridine-2-carboxamide (5) (0.333 g, 1 mmol) was
refluxed in acetic anhydride (15 mL) for 6 h. The mixture was evaporated, ice (10 g) was added to the
residue and the precipitated solid was filtered off and crystallized from MeOH/H2O (1:1) to give
3-acetamido-4-methoxythieno[2,3-b]pyridine-2-carboxylic acid (7) as a white solid (0.095 g, 36%), mp
1
240-242 ˚C. H NMR (DMSO-d6) δ 2.02 (3H, s), 3.92 (3H, s), 7.0 (1H, d, J = 5.5 Hz), 8.51 (1H, d, J =
5.5 Hz), 9.7 (1H, s); 13.20 (1H, bs); IR (KBr) νmax 3359, 1698, 1518, 1476, 1282, 1211, 1056, 828, 752,

HETEROCYCLES, Vol. 75, No. 11, 200
2731
567 cm-1. Anal. Calcd for C11H10N2O4S: C, 49.62; H, 3.79; N, 10.52; S, 12.04. Found: C, 49.52; H, 3.77;
N, 10.49; S, 12.01.
3-(4-Chlorophenyl)-9-methoxypyrido[3’,2’:4,5]thieno[3,2-d][1,2,3]triazin-4(3H)-one (8)
3-Amino-N-(4-chlorophenyl)-4-methoxythieno[2,3-b]pyridine-2-carboxamide (5) (0.333 g, 1 mmol) was
dissolved in hydrochloric acid (6 mL) and cooled down to 0 ºC. Then aqueous solution of NaNO2 (0.138
g, 2 mmol) was added, and the mixture was stirred at 0 ºC for 10 min. The precipitated solid was filtered
off and crystallized from acetic acid to give 3-(4-chlorophenyl)-9-methoxypyrido[3’,2’:4,5]thieno-
1
[3,2-d][1,2,3]triazin-4(3H)-one (8) as a white solid (46%), mp 243-245 ˚C; H NMR (DMSO-d6) δ 4.16
(3H, s), 7.63 (1H, d, J = 5.7 Hz), 7.71-7.83 (4H, m), 8.77 (1H, d, J = 5.7 Hz); IR (KBr) νmax 1677, 1562,
1488, 1290, 1037, 944, 828, 520 cm-1. Anal. Calcd for C15H9ClN4O2S: C, 52.25; H, 2.63; N, 16.25; S,
9.30. Found: C, 52.11; H, 2.62; N, 16.21; S, 9.18.
2-Mercapto-4-methoxypyridine-3-carbonitrile (9)9
Thiourea (0.760 g, 10 mmol) was added to a stirred solution of 2-bromo-4-methoxypyridine-
3-carbonitrile (1) (1.065 g, 5 mmol) in MeOH (25 mL), the mixture was refluxed for 3 h, and next cooled
to rt. The precipitated solid was filtered off, dissolved in 10% aqueous NaOH (20 mL) and the solution
was refluxed for 15 min. The mixture was cooled down, acidified with glacial acetic acid, the precipitate
was filtered off, and crystallized from MeOH to give 2-mercapto-4-methoxypyridine-3-carbonitrile (9)
(0.639 g, 77%) as a white solid.
General procedure for the synthesis of 10 and 11
2-Mercapto-4- methoxypyridine-3-carbonitrile (9) (0.332 g, 2 mmol) was dissolved in DMF (15 mL) and
KOH (0.224 g, 4 mmol) was added to the solution. Then 2-chloro-1-(4-phenylpiperazin-1-yl)ethanone
hydrochloride (0.55 g, 2 mmol) (for 10) or 2-chloro-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone
hydrochloride (0.55 g, 2 mmol) (for 11) was added, and the mixture was stirred at rt for 30 min. The
formed precipitate was filtered off and crystallized from MeOH.
2-(2-Oxo-2-(4-phenylpiperazin-1-yl)ethylthio)-4-methoxypyridine-3-carbonitrile (10)
Reaction with 2-chloro-1-(4-phenylpiperazin-1-yl)ethanone hydrochloride. Product 10 was isolated as a
1
white solid (26%), mp 188-190 ˚C. H NMR (DMSO-d6) δ 3.21-3.24 (4H, m), 3.84-3.92 (4H, m), 3.97
(3H, s), 4.23 (2H, s), 6.64 (1H, d, J = 5.9 Hz), 6.94-7.35 (5H, m), 8.37 (1H, d, J = 5.9 Hz); IR (KBr) νmax
2828, 2218, 1629, 1559, 1472, 1309, 1233, 1153, 1043, 812, 761, 691 cm-1. Anal. Calcd for
C19H20N4O2S: C, 61.94; H, 5.47; N, 15.21; S, 8.70. Found: C, 61.82; H, 5.45; N, 15.17; S, 8.68.
2-(2-Oxo-2-(4-(pyridin-2-yl)piperazin-1-yl)ethylthio)-4-methoxypyridine-3-carbonitrile (11)
Reaction with 2-chloro-1-(4-(pyridin-2-yl)piperazin-1-yl)ethanone hydrochloride. Product 11 was
isolated as a white solid (21%), mp 203-205 ˚C. 1H NMR (DMSO-d6) δ 3.45-3.65 (8H, m), 3.98 (3H, s),
4.36 (2H, s), 6.63-6.69 (1H, dd, J1 = 4.8 Hz, J2 = 6.6 Hz), 6.82 (1H, d, J = 8.5 Hz), 7.05 (1H, d, J = 6.1

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Hz), 7.51-7.59 (1H, m), 8.10-8.14 (1H, dd, J1 = 1.6 Hz, J2 = 4.8 Hz), 8.49 (1H, d, J = 6.1 Hz); IR (KBr)
νmax 2846, 2217, 1628, 1560, 1474, 1306, 1236, 1158, 1046, 979, 810, 781, 735 cm-1. Anal. Calcd for
C18H19N5O2S: C, 58.52; H, 5.18, N, 18.96; S, 8.68. Found: C, 58.39; H, 5.16; N, 18.92; S, 8.66.
Methyl 3-(3-cyano-4-methoxypyridin-2-ylthio)propanoate (12)
2-Mercapto-4- methoxypyridine-3-carbonitrile (9) (0.332 g, 2 mmol) was dissolved in DMF (20 mL) and
KOH (0.112 g, 2 mmol) was added to the solution. Then methyl 3-bromopropanoate (0.334 g, 2 mmol)
was added, and the mixture was stirred at rt for 45 min. Then ice (10 g) was added to the mixture and the
precipitated solid was filtered off and crystallized from MeOH/H2O (2:3) to give methyl
3-(3-cyano-4-methoxypyridin-2-ylthio)propanoate (12) as a white solid (0.151 g, 30%), mp 102-104 ˚C.
1H NMR (DMSO-d6) δ 2.72 (2H, t, J = 6.9 Hz), 3.42 (2H, t, J = 6.9 Hz), 3.61 (3H, s), 3.97 (3H, s), 7.06
(1H, d, J = 5.8 Hz), 8.53 (1H, d, J = 5.8 Hz); IR (KBr) νmax 2949, 2221, 1741, 1562, 1468, 1362, 1304,
1202, 1040, 814 cm-1. Anal. Calcd for C11H12N2O3S: C, 52.37; H, 4.79; N, 11.10; S, 12.71. Found: C,
52.21; H, 4.57; N, 11.00; S, 12.68.
General procedure for the synthesis of compounds 13, 15-19
2-Mercapto-4-methoxypyridine-3-carbonitrile (9) (0.332 g, 2 mmol) was dissolved in DMF (15 mL) and
KOH [(0.224 g, 4 mmol) or (0.336 g, 6 mmol for compound 16 and 17)] was added. Then ethyl
2-chloroacetate (2 mmol) (for 13), (adamantan-1-yl)methyl chloroacetate (for 15), 2-chloro-1-
(4-methylpiperazin-1-yl)ethanone hydrochloride (for 16), 1-(2-chloroacetyl)-4-(4-fluorophenyl)piperazine
hydrochloride (for 17), 2-bromo-1-phenylethanone (for 18) and 3-chloropentane-2,4-dione (for 19) was
added, and the mixture was stirred at rt for 30 min. The precipitated solid was filtered and recrystallized
from MeOH/H2O (1:1).
Ethyl 3-amino-4-methoxythieno[2,3-b]pyridine-2-carboxylate (13)
1
Reaction with ethyl 2-chloroacetate. Product 13 was isolated as a white solid (33%), mp 123-124 ˚C. H
NMR (DMSO-d6) δ 1.26 (3H, t, J = 7.3 Hz), 4.02 (3H, s), 4.22 (2H, q, J = 7.3 Hz), 6.94 (2H, s), 7.01 (1H,
d, J = 5.3 Hz), 8.49 (1H, d, J = 5.3 Hz); IR (KBr) νmax 3502, 3368, 1671, 1566, 1509, 1366, 1290, 1131,
1043, 972, 764, 571 cm-1. Anal. Calcd for C11H12N2O3S: C, 52.37; H, 4.79; N, 11.10; S, 12.71. Found: C,
52.29; H, 4.77; N, 11.08; S, 12.68.
(Adamantan-1-yl)methyl 4-methoxythieno[2,3-b]pyridine-2-carboxylate (15)
Reaction with (adamantan-1-yl)methyl chloroacetate. Product 15 was isolated as a white solid (51%), mp
225-226 ˚C. 1H NMR (DMSO-d6) δ 1.52-1.91 (15H, m), 3.80 (2H, s), 4.02 (3H, s), 6.91 (2H, s), 7.04 (1H,
d, J = 5.7 Hz), 8.48 (1H, d, J = 5.7 Hz); IR (KBr) νmax 3438, 3322, 2904, 1675, 1600, 1510, 1294, 1051,
811, 762 cm-1. Anal. Calcd for C20H24N2O3S: C, 64.49; H, 6.49; N, 7.52; S, 8.61. Found: C, 64.32; H,
6.47; N, 7.50; S, 8.59.
(3-Amino-4-methoxythieno[2,3-b]pyridin-2-yl)(4-methylpiperazin-1-yl)methanone (16)

HETEROCYCLES, Vol. 75, No. 11, 200
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Reaction with 2-chloro-1-(4-methylpiperazin-1-yl)ethanone hydrochloride. Product 16 was isolated as a
1
white solid (47%), mp 174-176 ˚C. H NMR (DMSO-d6) δ 2.18 (3H, s), 2.31 (4H, s), 3.60 (4H, s), 4.01
(3H, s), 6.24 (1H, s); 6.32 (1H, s), 6.93 (1H, d, J = 5.3 Hz), 8.38 (1H, d, J = 5.3 Hz); IR (KBr) νmax 3481,
3346, 1617, 1591, 1499, 1424, 1295, 1141, 1050, 1002, 807, 752 cm-1. Anal. Calcd for C14H18N4O2S: C,
54.88; H, 5.92; N, 18.29; S, 10.47. Found: C, 54.74; H, 5.91; N, 18.25; S, 10.44.
(3-Amino-4-methoxythieno[2,3-b]pyridin-2-yl)(4-(4-fluorophenyl)piperazin-1-yl)methanone (17)
Reaction with 1-(2-chloroacetyl)-4-(4-fluorophenyl)piperazine hydrochloride. Product 17 was isolated as
1
a yellow solid (50%), mp 152-153 ˚C. H NMR (DMSO-d6) δ 3.11 (4H, m), 3.72 (4H, m), 4.00 (3H, s),
6.38 (2H, s), 6.97-7.06 (5H, m), 8.43 (1H, d, J = 5.8 Hz); IR (KBr) νmax 3494, 3328, 1586, 1511, 1432,
1291, 1159, 1059, 1010, 823, 523 cm-1. Anal. Calcd for C19H19FN4O2S: C, 59.05; H, 4.96; N, 14.50; S,
8.30. Found: C, 58.90; H, 4.94; N, 14.48; S, 8.28.
(3-Amino-4-methoxythieno[2,3-b]pyridin-2-yl)(phenyl)methanone (18)
Reaction with 2-bromo-1-phenylethanone. Product 18 was isolated as a yellow solid (52%), mp 145-147
˚C. 1H NMR (DMSO-d6) δ 4.06 (3H, s), 7.04 (1H, d, J = 5.3 Hz), 7.51-7.59 (3H, m), 7.75 (2H, d, J = 7.3
Hz), 8.12 (2H, bs), 8.52 (1H, d, J = 5.3 Hz); IR (KBr) νmax 3490, 3447, 3308, 1602, 1488, 1452, 1323,
1039, 809, 725, 693 cm-1. Anal. Calcd for C15H12N2O2S: C, 63.36; H, 4.25; N, 9.85; S, 11.28. Found: C,
63.24; H, 4.33; N, 9.71; S, 11.11.
1-(3-Amino-4-methoxythieno[2,3-b]pyridin-2-yl)ethanone (19)
Reaction with 3-chloropentane-2,4-dione. Product 19 was isolated as a yellow solid (67%), mp 138-141
1
˚C. H NMR (DMSO-d6) δ 2.30 (3H, s), 4.02 (3H, s), 7.02 (1H, d, J = 5.5 Hz), 7.49-7.81 (2H, bs), 8.53
(1H, d, J = 5.5 Hz); IR (KBr) νmax 3326, 1606, 1579, 1496, 1460, 1367, 1295, 1050, 803, 720, 576 cm-1.
Anal. Calcd for C10H10N2O2S: C, 54.04; H, 4.53; N, 12.60; S, 14.43. Found: C, 53. 96; H, 4.51; N, 12.58;
S, 14.39. 3-Amino-4-methoxythieno[2,3-b]pyridine-2-carboxamide (14)
2-Mercapto-4-methoxypyridine-3-carbonitrile (9) was dissolved in MeOH (20 mL) and MeONa (92 mg,
4 mmol Na/MeOH 5 mL) was added. Then 2-chloroacetamide (0.35 mL, 2 mmol) was added and the
mixture was refluxed for 3 h. The mixture was cooled down, the precipitated solid was filtered off and
crystallized from MeOH/H2O (1:1) to give 3-amino-4-methoxythieno[2,3-b]pyridine-2-carboxamide (14)
1
as a white solid (14%), mp 238-240 ˚C. H NMR (DMSO-d6) δ 4.99 (3H, s), 6.94 (d, 1H, J = 5.7 Hz),
6.97 (2H, s) 7.05 (2H, s), 8.43 (1H, d, J = 5.7 Hz); IR (KBr) νmax 3488, 3325, 1667, 1583, 1503, 1289,
1045, 743, 478 cm-1. Anal. Calcd for C9H9N3O2S: C, 48.42; H, 4.06; N, 18.82; S, 14.36. Found: C, 48.32;
H, 4.4; N, 18.79; S, 14.32.
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