Derivatives of 3-cyano-6-phenyl-4-(3'-pyridyl)-pyridine-2(1H)-thione and their neurotropic activity

Article abstract of DOI:10.1016/S0223-5234(99)80081-6

3-Cyano-6-phenyl-4-(3'-pyridyl)pyridine-2(1H)-thione, the related 2,2'- bis-pyridyldisulfide, 2-alkylthiopyridines and 2-aminothieno[2,3-b]pyridines were synthesised and their neurotropic activities were examined. Bispyridyldisulfide exhibited low toxicity (LD₅₀ > 5 000 mg/kg, ICR mice, i.p.) and selective antiamesic activity at the doses of 0.05-0.5 mg/kg p.o. This effect was significantly higher than that induced by Piracetam at 50 mg/kg.

Full text of DOI:10.1016/S0223-5234(99)80081-6

Eur. J. Med. Chem. 34 (1999) 301−310
© Elsevier, Paris
301
Original article
Derivatives of 3-cyano-6-phenyl-4-(3′-pyridyl)-pyridine-2(1H)-thione
and their neurotropic activity
ˇ
Aivars Krauze*, Skaidrite Gqe¯rmane, Oja¯rs Eberlinsˇ, Igors Sturms, Vija Klusa¯, Gunárs Duburs
>
Latvian Institute of Organic Synthesis, 21 Aizkraukles, Riga, LV-1006, Latvia
(Received 8 July 1998; accepted 28 October 1998)
Abstract – 3-Cyano-6-phenyl-4-(3′-pyridyl)pyridine-2(1H)-thione, the related 2,2′-bis-pyridyldisulfide, 2-alkylthiopyridines and 2-amino-
thieno[2,3-b]pyridines were synthesised and their neurotropic activities were examined. Bispyridyldisulfide exhibited low toxicity
(LD50 > 5 000 mg/kg, ICR mice, i.p.) and selective antiamesic activity at the doses of 0.05–0.5 mg/kg p.o. This effect was significantly higher
than that induced by Piracetam at 50 mg/kg. © Elsevier, Paris
1,2-dihydro-2-thioxopyridine-3-carbonitrile / thieno[2,3-b]pyridine / bispyridyldisulfide / antiamnesic agent
1. Introduction
sponding 2-alkylthio derivatives [29, 30] and pyrido[3,2-
c]1,3-thiazin-4-ones [31] are central nervous system
depressants, and 1-piperazinopropylisothiazolo[5,4-b]
pyridines have antipsychotic activity [32].
Our concept was to introduce an additional pyridyl
moiety in pyridine-2(1H)-thiones and related compounds
3-Cyanopyridine-2(1H)-thione, 3-amino-2-carbofunc-
tionally substituted thieno[2,3-b]pyridine and related
compounds are found to be very reactive substances for
the synthesis of many different heterocyclic systems.
They have diverse biological activities.
(2-alkylthiopyridines,
2,2′-bispyridyldisulfides
and
3-Cyanopyridine-2(1H)-thione and their derivatives,
2-alkylthiopyridines, thienopyridines and isothiazolopy-
ridines exhibited antibacterial [1–5], pesticidal [6], anti-
fungal [3, 7] and acaricidal [3] properties. Pyridine-
2-aminothieno[2,3-b]-pyridines) and to study these com-
pounds as neurotropic agents. These compounds were
examined in routine neuropharmacological tests to char-
acterise their activity spectrum and to reveal the influence
of the structure on activity which may indicate further
steps in psychotropic drug design.
2(1H)-selenones
and
2-alkylthio(seleno)pyridines
revealed antiviral activity [8], and pyridothiophenes and
their derivatives (pyridothienopyrimidines, pyridothieno-
pyridazines and pyridothienotriazines) have diuretic [2],
analgesic [2, 9, 10], anti-inflammatory [2, 10] and anti-
allergenic (antianaphylactic) activities [11–20]. Thieno-
pyridines have also been examined as cholesterol biosyn-
thesis inhibitors [21]. Cardiovascular activity of
3-cyanopyridine-2(1H)-thiones was profoundly investi-
gated and vasodilating [22], antihypertensive [22, 23] and
cardiotonic [24–28] activities were revealed.
2. Chemistry
There are two satisfactory methods for the synthesis of
3-cyano-6-phenyl-4-(3′-pyridyl)pyridine-2(1H)-thione 1
(figure 1): A) the Michael reaction of 1-phenyl-3-(3′-
pyridyl)-2-propen-1-one and cyanothioacetamide with
the subsequent heterocyclisation, dehydration and dehy-
drogenation to yield 1 (80%), and B) the Knoevenagel
reaction of 1-phenyl-3-(3′-pyridyl)-propane-1,3-dione
and cyanothioacetamide with the subsequent heterocycli-
sation to yield 1 (72%). The shortcoming of the second
method is a rather complicated synthesis of the 1,3-
diketone [33].
There are only a few reports (preliminary data) of the
neurotropic activity of 3-cyano(carbamoyl, carboxy)
pyridine-2(1H)-thione related compounds: the corre-
* Correspondence and reprints

302
Figure 1. Synthesis of 3-cyano-6-phenyl-4-(3≠-pyridyl)pyridine-2(1H)-thione 1.

303
The treatment of pyridine-2(1H)-thione 1 with piperi-
dine yielded piperidinium thiolate 2 which was an inter-
mediate for further reactions. The oxidation of piperi-
dinium pyridine-2-thiolate 2 with iodine gave 2,2′-
bispyridyldisulfide 3 (70%) and small amount of
2-piperidylthiopyridine 4 (8%) as by-product. A slightly
higher yield of 3 (75%) was obtained by the oxidation of
thione 1 with iodine in ethanol solution in the presence of
sodium hydroxide.
assess their interaction with cholin-, dopamin- and sero-
toninergic processes. The compounds were administered
orally at 5 and 50 mg/kg (in separate experiments the
dose range was broadened). Table II shows that the
compounds 1 and 3 at the highest tested dose (50 mg/kg)
exerted a slight anticholinergic effect as they prolonged
time prior to arecoline-induced tremors. However, nico-
tine tremorogenic action was not antagonised by the
tested compounds (table II). When amphetamine, a
dopamine agonist, was used as a test-drug in stereotypy
tests, compounds 3 and 5a slightly reduced the duration
of stereotypic behaviour caused by amphetamine only at
5 mg/kg (figure 2); this effect was comparable to that of
Piracetam (100 mg/kg). No effect was observed at lower
and higher doses. These data indicate weak and dose-
independent antidopamine action for compounds 3 and
5a. An antagonistic influence on serotoninergic processes
(reduction of the number of head shakes induced by
5-hydroxytryptophane (5-HTP), a serotonin precursor),
was found only for compound 3 at the highest dose tested
(50 mg/kg); this effect was comparable to that caused by
Piracetam at 50 mg/kg (figure 3). Surprisingly, in the
memory test (passive avoidance response behaviour,
electroconvulsive shock-induced amnesia), only com-
pound 3 showed high antiamnesic activity in mice (fig-
ure 4). The effects were observed at 0.05 mg/kg and
maintained at the plateau level at 0.5–25 mg/kg doses,
and they were significantly higher than the effects caused
by Piracetam (a well-known nootropic drug) at 50 mg/kg.
Other tested compounds 1 and 5a were inactive. In the
open field test (table III) the compound 3 in doses which
exerted the high antiamnesic activity, did not change the
rats locomotion (with the exception of dose 5 mg/kg
which caused a slight reduction in vertical activity).
Compounds 1 and 5a also did not affect locomotor
responses at doses of 5 and 25 mg/kg (with the exception
of compound 1, which slightly increased vertical activity
at 25 mg/kg). It is worth noting that the antiamnesic
activity of compound 3 occurred at much smaller doses
(already at 0.05 mg/kg p.o.) than those which affected
muscle tone and body temperature (on average,
5–10 mg/kg i.p.). So, our preliminary pharmacological
data have revealed the selective antiamnesic activity of
compound 3; however, the question of which mecha-
nisms could be taken as crucial for this activity remains
open. A slight anticonvulsant activity (PTZ-test) which
can be related to GABAergic processes, and negligible
antidopamine (at 5 mg/kg, but not 50 mg/kg), antisero-
toninergic and anticholinergic (both 50 mg/kg) activities,
as well as Diazepine-like myorelaxing and hypothermic
effects, may indicate that these properties can appear at
2-Alkylthio-3-cyanopyridines 5 were obtained in high
yields by alkylation of piperidinium thiolate 2 with the
corresponding alkyl halides. The subsequent Thorpe cy-
clisation of 5c–f in the presence of sodium methylate
gave 3-aminothieno[2,3-b]pyridines 6c–f in high yields.
Compound 6e was also obtained by heating thione 1 with
iodoacetamide in the presence of an excess of sodium
methylate without isolation of 5e, but in this case the
yield was appreciably lower. The structures of synthe-
sised compounds were confirmed by elemental analysis
1
and IR and H NMR spectra.
3. Pharmacological results and discussion
As it can be seen from table I, all tested compounds
showed low toxicity in mice (ED50 > 1 000 mg/kg i.p.). A
general pharmacological examination (i.p., in mice) of
the influence of all compounds on coordination and body
temperature revealed two groups of compounds. Group 1
[2-alkylthiopyridines 5 (with the exception of 5a) and
thienopyridines 6c, 6e and 6f] showed very weak (aver-
age ED50 doses > 100–500 mg/kg) depressing activity on
muscle tone and coordination (rota rod and traction tests)
and weak hypothermic effects (table I). At 50 mg/kg
these compounds possessed moderate anticonvulsant ac-
tivity, since pentylenetetrazol (PTZ) dose had to be
increased to cause the PTZ-induced clonic and tonic
seizures (table II). The anticonvulsant activity of these
compounds (with the exception of 6f) was slightly less
than Diazepam (5 mg/kg). Group 2 (pyridine-2(1H)-
thione 1, the corresponding 2,2 -bispyridyldisulfide 3 and
2-methylthiopyridine 5a) lacked anticonvulsant activity
in comparison with Group 1, with exception of 3, which
showed slight anticlonic action at 5 mg/kg (table II).
Moreover, they gave a sharp rise in hypothermic and
myorelaxing activities (in comparison to compounds of
group 1) at doses of about 3–10 mg/kg (table I). The
myorelaxing and hypothermic activities indicate that the
compounds 1, 3 and 5a may possess tranquillising
features, since their activities were comparable with
Diazepam (a well-known tranquilliser) effects (table I).
The data obtained in the studies allowed us to select the
compounds 1, 3 and 5a for more detailed examination to

304
Table I. Neurotropic activity and toxicity of 3-cyano-6-phenyl-4-(3′pyridyl)pyridine-2(1H)-thione derivatives administered i.p. in ICR mice
(n = 6 per group).
Activity
Toxicity
Compounds
(ED50, mg/kg)
Traction
(LD50, mg/kg)
Rota rod
Hypothermia
1
5.6*
(3.4-8.1)
9.2*
(3.3-17.5)
3.0
(1.6-4.9)
650*
6.5*
(4.4-8.9)
11.6*
(4.0-22.0)
3.0
(1.6-4.8)
224*
(144-285)
325*
(214-455)
258*
(168-357)
258*
(168-357)
282*
3.7
(1.1-7.7)
4.8*
(2.5-7.7)
4.5*
(2.6-6.4)
547*
(200-1027)
410*
(268-552)
224*
(144-298)
300*
(158-489)
325*
> 5000
> 5000
> 10000
> 2000
> 1000
> 1000
> 5000
> 5000
> 8000
> 5000
> 4000
3
5a
5b
5c
5d
5e
5f
6c
6e
6f
(438-886)
> 500*
274*
(99-524)
410*
(268-552)
564*
(342-814)
410*
(268-552)
346*
(120-662)
325*
(183-372)
325*
(219-455)
258*
(168-357)
129*
(172-502)
282*
(159-419)
282*
(159-419)
178*
(219-455)
(61-202)
(136-230)
Diazepam
2.6
3.3
1.6
-
(1.5-4.4)
(1.4-7.6)
(0.6-4.0)
* P < 0.05 vs. Diazepam.
higher doses than those required for antiamnesic action
and cannot be considered as essential for antiamnesic
action.
Taking into account the effectiveness and low toxicity
of the compound 3 (> 5 000 mg/kg, i.p.), it may be
considered as a leading compound with an eventual
nootropic repertoire in the pyridinethioether series tested,
and can be selected for the further studies to clarify its
mechanisms of action. One may suggest that the struc-
tural peculiarities (two bipyridyl moieties connected via a
Figure 2. Influence of the compounds 1, 3, 5a and Piracetam
(P) on amphetamine-induced stereotypy in Wistar rats (n = 6).
* P < 0.05 vs. Amphetamine control (C).
Figure 3. Influence of the compounds 1, 3, 5a and Piracetam
(P) on the 5-HTP-induced head shakes in BALB/c mice (n = 6).
* P < 0.05 vs. 5-HTP control (C).

305
Table II. Influence of the compoundsa and Diazepam (i.p.) on pentylenetetrazole (PTZ)-induced convulsions and nicotine (N)-, and arecoline
(A)-induced tremor in BALB/c mice (n = 6 per group).
PTZ (mean ± SEM, mg/kg) seizures
A (mean ± SEM), min
N (mean ± SEM, mg/kg)
Compounds
Dose (mg/kg)
Clonic
Tonic
Tremor
Tremor
Control (saline)
1
0
5
50
5
50
5
31.4 ± 0.6**
33.8 ± 3.0**
31.4 ± 0.6**
40.0 ± 3.8*
37.8 ± 0.4**
34.3 ± 4.1**
32.3 ± 2.3**
86.5 ± 6.2**
11.7 ± 1.1
17.5 ± 1.4
1.3 ± 0.1
1.4 ± 0.1
1.8 ± 0.3
1.5 ± 0.1
1.4 ± 0.2
1.3 ± 0.2
1.4 ± 0.1
108.9 ± 8.6**
77.3 ± 10.2**
107.3 ± 7.1**
102.1 ± 11.0**
98.5 ± 12.4**
113.9 ± 16.1**
19.2 ± 1.2***
11.3 ± 0.6**
19.5 ± 2.0***
7.6 ± 3.1**
10.7 ± 2.1
3
5a
50
5b
5c
5d
5e
5f
6c
6e
6f
50
50
50
50
50
50
50
50
39.3 ± 2.6*
45.2 ± 1.9*
43.6 ± 3.2*
39.3 ± 3.3*
50.0 ± 3.5*
43.3 ± 1.3*
42.0 ± 2.2*
46.2 ± 1.6*
133.3 ± 15.5**
155.0 ± 24.2***
166.7 ± 17.6***
106.8 ± 7.7***
106.8 ± 6.1***
102.6 ± 6.2**
177.8 ± 9.8***
207.6 ± 19.8*
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Diazepam
5
43.8 ± 2.6*
213 ± 10.4*
14.5 ± 1.3
1.2 ± 0.3
* P < 0.05 vs. control. ** P < 0.05 vs. Diazepam.
aIn PTZ-test, the compounds are administered i.p.; in (N)- and (A)-induced tremor they are administered p.o.
disulfide bridge) may play an important role in a series of
substituted pyridine-2(1H)-thione or its pyridyl-2-thiol
type derivatives for enhancing their neurotropic activi-
ties, and particularly for inducing the antiamnesic prop-
erties.
were within ± 0.4% of the theoretical values. IR spectra
were recorded on a Perkin-Elmer 580 B spectrometer (in
Nujol) and peak positions νmax are expressed as cm–1. 1H
NMR spectra were recorded on a Bruker WH-90 spec-
trometer and chemical shifts are reported as d values
(ppm) relative to tetramethylsilane.
4. Experimental protocols
4.1.1. 3-Cyano-6-phenyl-4-(3′-pyridyl)pyridine-2(1H)-
thione 1
4.1. Chemistry
Melting points were determined on a Boetius apparatus
and were uncorrected. Elemental analyses (C, H, N, S)
Method A: A mixture of 1-phenyl-3-(3′-pyridyl)-2-
propen-1-one [33] (4.19 g; 0.02 mol), cyanothioaceta-
mide (2.0 g; 0.02 mol) and piperidine (0.85 g; 0.01 mol)
in 25 mL of ethanol was refluxed for 30 min. Then 5 mL
of acetic acid were added and the reaction mixture was
stirred for 2 h at room temperature and cooled to 0 °C.
The precipitated dark yellow crystals were filtered and
washed with 20 mL of cold ethanol and 20 mL of water to
yield 1 (4.63 g, 80%); m.p. 232–234 °C (from ni-
1
tromethane); IR ν cm–1: 3 160 (NH), 2 220 (CN); H
NMR (DMSO-d6) δ: 7.26 (1H, s, 5-H); 7.4–8.8 (9H, m,
C6H5 and C5H4N); 14.31 (1H, br.s, NH). Anal.
C17H11N3S (C, H, N, S).
Method B: A mixture of 1-phenyl-3-(3′-pyridyl)
propane-1,3-dione [34] (2.3 g; 0.01 mol), cyanothioac-
etamide (1.0 g, 0.01 mol) and triethylamine (1.0 g;
0.01 mol) in 20 mL ethanol was refluxed for 1 h. After
Figure 4. Antiamnesic activity of the compounds 1, 3, 5a and
Piracetam (P) in ECS-amnesia tests in BALB/c mice (n = 6). *
P < 0.05 vs. control (C), saline; o P < 0.05 vs. Piracetam

306
Table III. Influence of the compounds 1, 3, 5a (p.o.) and Piracetam (p.o.), on locomotion behaviour in male Wistar rats in ″open field″ test
(n = 6 per group).
Number of locomotor responses (mean ± SEM)
Compounds
Dose mg/kg
Horizontal activity
Vertical activity
Exploratory activity
Control (saline)
1
0
5
25
29.3 ± 3.7
32.5 ± 6.0
36.0 ± 4.8
6.8 ± 1.0
5.7 ± 0.5**
13.5 ± 2.7*
1.7 ± 0.6
1.3 ± 1.8
1.2 ± 0.8
3
0.005
0.05
0.5
36.2 ± 3.2
29.0 ± 4.2
34.2 ± 4.0
35.0 ± 8.1
5.6 ± 1.1
3.2 ± 1.4
3.5 ± 2.0
3.6 ± 0.4***
2.8 ± 0.8
2.6 ± 0.5
3.5 ± 1.0
3.6 ± 0.7
5
5a
5
25
34.3 ± 7.2
33.3 ± 4.5
6.0 ± 2.3
4.5 ± 1.8**
1.1 ± 1.3
1.2 ± 1.4
Piracetam
5
25
27.4 ± 5.4
26.3 ± 6.2
9.5 ± 1.8
10.5 ± 1.9
2.3 ± 1.8
1.7 ± 2.1
* P < 0.05 vs. control. ** P < 0.05 vs. Piracetam (corresponding dose).
that, 2 mL of acetic acid were added and then the reaction
mixture was treated as in method A to yield 1 (2.08 g,
72%).
(3.75 g, 0.01 mol) in 50 mL of ethanol and stirred for 1 h.
The reaction mixture was cooled to 0 °C, the precipitate
was filtered and washed with 10 mL of ethanol and
50 mL of water. The crude product (3 and 4) was
recrystallised from the chloroform-ethanol (2:1) mixture.
After separation of 3 (1.73 g; 60%), the filtrate was
evaporated to approx. 1/5 of its initial volume. The
precipitate was filtered and washed with 10 mL of hot
ethanol. After separation of the additional 3 (0.29 g,
10%), the second filtrate was concentrated to approx. 1/3
of its initial volume and compound 4 (0.31 g; 8%) was
filtered. M.p. 195–197 °C (from ethanol); IR ν cm–1:
4.1.2. Piperidinium 3-cyano-6-phenyl-4-(3′-pyridyl)
pyridine-2-thiolate 2
Similar to 1 (method A), after refluxing the reaction
mixture was stirred at room temperature. For 10 d the
precipitated crystals were filtered, combined and washed
with cold ethanol and dry ether to yield 2 (68%); m.p.
240–241 °C (from ethanol); IR ν cm–1: 2 530, 2 448 (+
NH2); 2 216 (CN). 1H NMR (DMSO-d6) δ: 1.60 [6H, m,
(CH2)3]; 3.02 [4H, m, N(CH2)2]; 7.08 (1H, s, 5-H);
7.4–8.8 (9H, m, C6H5 and C5H4N). Anal. C22H22N4S (C,
H, N, S).
1
2 216 (CN); H NMR (CDCl3) δ: 1.62 [6H, m, (CH2)3];
3.64 [4H, m, N(CH2)2]; 7.50 (1H, s, 5-H); 7.8–8.9 (9H,
m, C6H5 and C5H4N). Anal. C22H20N4S (C, H, N, S).
4.1.3. 2,2′-Bis[3-cyano-6-phenyl-4-(3′-pyridyl)] disulfide
3
4.1.5. General method for the synthesis of 2-alkylthio-3-
cyano-6-phenyl-4-(3′-pyridyl)pyridines 5
A solution of 0.5 N ethanolic iodine (4 mL; 0.01 mol)
was added under vigorous stirring to a suspension of
thione 1 (2.9 g; 0.01 mol) and sodium hydroxide (0.48 g;
0.012 mol) in 70 mL of ethanol and stirred for 1 h. The
reaction mixture was cooled to 0 °C, the precipitate was
filtered and washed with 20 mL of ethanol and 50 mL of
water. After drying in the oven, the crude product was
recrystallised from chloroform to yield 3 (2.16 g, 75%);
m.p. 239-241 °C; IR ν cm–1: 2 220 (CN); 1H NMR
(DMSO-d6) δ: 8.24 (1H, s, 5-H); 7.2–9.0 (9H, m, C6H5
and C5H4N). Anal. C34H20N6S2 (C, H, N, S).
A mixture of thiolate 2 (3.75 g; 0.01 mol) and the
corresponding
R-substituted
alkyl
halide
(0.011–0.04 mol) in 30–40 mL of ethanol was refluxed
for 5–10 min and filtered. The reaction mixture was
cooled to 0 °C, the precipitated crystals were filtered,
washed with ethanol (5–10 mL) and water (20–30 mL)
and recrystallised from ethanol.
4.1.6.
3-Cyano-2-methylthio-6-phenyl-4-(3′-pyridyl)
pyridine 5a
From thiolate 2 and methyl iodide (1:4). M.p.
1
202–204 °C; yield: 94%; IR ν cm–1: 2 220 (CN); H
4.1.4. 3-Cyano-6-phenyl-2-piperidylthio-4-(3′-pyridyl)
pyridine 4
A solution of 0.5 N ethanolic iodine (4 mL; 0.01 mol)
was added under vigorous stirring to a solution of salt 2
NMR (CDCl3) d: 2.80 (3H, s, SCH3); 7.57 (1H, s, 5-H);
7.5–8.9 (9H, m, C6H5 and C5H4N). Anal. C18H13N3S (C,
H, N, S).

307
4.1.7.
pyridine 5b
3-Cyano-2-ethylthio-6-phenyl-4-(3′-pyridyl)
4.1.13. 3-Amino-2-ethoxycarbonyl-6-phenyl-4-(3′-pyri-
dyl)-thieno[2,3-b]-pyridine 6c
M.p. 235–236 °C; yield 93%; IR ν cm–1: 3 494, 3 344
From thiolate 2 and ethyl bromide (1:4). M.p.
1
137–139 °C; yield: 82%; IR ν cm–1: 2 216 (CN); H
1
(NH2); 1 680 (CO); H NMR (CDCl3) δ: 1.38 (3H, t,
CH2CH3); 4.36 (2H, q, CH2CH3); 5.52 (2H, s, 3-NH2),
7.50 (1H, s, 5-H); 7.4–8.8 (9H, m, C6H5 and C5H4N).
Anal. C21H17N3O2S (C, H, N, S).
NMR (CDCl3) δ: 1.48 (3H, t, CH2CH3); 3.43 (2H, q,
CH2CH3); 7.48 (1H, s, 5-H); 7.4–8.8 (9H, m, C6H5 and
C5H4N). Anal. C19H15N3S (C, H, N, S).
4.1.14. 3-Amino-2-cyano-6-phenyl-4-(3′-pyridyl)thieno
[2,3-b]pyridine 6d
4.1.8. 3-Cyano-2-ethoxycarbonylmethylthio-6-phenyl-4-
(3′-pyridyl)pyridine 5c
M.p. 238–240 °C; yield 90%; IR ν cm–1: 3 284 (NH2);
2 192 (CN); 1H NMR (DMSO-d6) δ: 5.68 (2H, s,
3-NH2); 7.96 (1H, s, 5-H); 7.5–8.8 (9H, m, C6H5 and
C5H4N). Anal. C19H12N4S (C, H, N, S).
From thiolate 2 and ethyl bromoacetate (1:1.2). M.p.
164–165 °C; yield 55%; IR ν cm–1: 1 736 (CO);
1
2 218(CN); H NMR (CDCl3) δ: 1.25 (3H, t, CH2CH3);
4.21 (2H, q, CH2CH3); 4.13 (2H, s, SCH2); 7.57 (1H, s,
5-H); 7.4–8.9 (9H, m, C6H5 and C5H4N). Anal.
C21H17N3O2S (C, H, N, S).
4.1.15.
3-Amino-2-carbamoyl-6-phenyl-4-(3′-pyridyl)
thieno[2,3-b]pyridine 6e
Method A (general method): M.p. 245–246 °C; yield
91%; IR ν cm–1: 3 444, 3 324, 3 152 (NH2); 1 660 (CO);
1H NMR (DMSO-d6) δ: 5.90 (2H, s, 3-NH2); 7.30 (2H, s,
CONH2); 7.85 (1H, s, 5-H); 7.5–8.8 (9H, m, C6H5 and
C5H4N). Anal. C19H14N4OS (C, H, N, S).
Method B: A 20 mL solution of 1 N sodium methylate
in methanol was added to a mixture of thione 1 (2.90 g;
0.01 mol) and iodoacetamide (2.04 g, 0.011 mol) in
20 mL of methanol, then the reaction mixture was re-
fluxed for 30 min and cooled to 0 °C. The precipitated
crystals were filtered and recrystallised from chloroform
ethanol (1:1) to yield 6e (2.40 g, 69%); m.p. 245–246 °C.
4.1.9. 3-Cyano-2-cyanomethylthio-6-phenyl-4-(3′-pyri-
dyl)pyridine 5d
From thiolate 2 and chloroacetonitrile (1:1.2). M.p.
222–224 °C; yield 95%; IR ν cm–1: 2 214 (3-CN); 2 248
1
(CH2CN); H NMR (DMSO-d6) δ: 4.55 (2H, s, SCH2);
8.14 (1H, s, 5-H); 7.5–9.0 (9H, m, C6H5 and C5H4N).
Anal. C19H12N4S (C, H, N, S).
4.1.10. 2-Carbamoylmethylthio-3-cyano-6-phenyl-4-(3′-
pyridyl)pyridine 5e
From thiolate 2 and iodoacetamide (1:1.1). M.p.
245–247 °C; yield 96%; IR ν cm–1: 1 654, 1 697 (CO);
4.1.16. 3-Amino-2-benzoyl-6-phenyl-4-(3′-pyridyl)thieno
[2,3-b]pyridine 6f
General method: M.p. 190–191 °C; yield: 91%; IR ν
cm–1: 3 484, 3 300 (NH2); 1 600 (CO); 1H NMR (CDCl3)
δ: 6.65 (2H, s, 3-NH2); 7.54 (1H, s, 5-H); 7.4–8.8 (14H,
m, C6H5 and C5H4N). Anal. C15H12N3OS (C, H, N, S).
1
2 216 (CN); 3 180 (NH2); H NMR (DMSO-d6) δ: 4.08
(2H, s, SCH2); 7.28 and 7.50 (2H, s and s, CONH2); 8.00
(1H, s, 5-H); 7.5–9.0 (9H, m, C6H5 and C5H4N). Anal.
C19H14N4OS (C, H, N, S).
4.1.11. 2-Benzoylmethylthio-3-cyano-6-phenyl-4-(3′-pyri-
dyl)pyridine 5f
4.2. Biological evaluation
From thiolate 2 and 2-bromoacetophenone (1:1.1).
The experiments were performed in winter/spring sea-
son on BALB/c and ICR mice of both sexes weighing
18–24 g, and on Wistar rats weighing 200 ± 20 g. Ani-
mals used were from the breeding colony of the GRIN-
DEX, Riga, Latvia. The animals were kept in plastic
cages which were equipped with drinking bowls and
bunkers-feeder. The animals had free access to water and
food (Altromin Standard Diets, Germany). Ambient tem-
perature in the animal colony and laboratory during
experiments was maintained at 21 ± 2 °C, air humidity
50–70%, lighting from 7 a.m. to 7 p.m. The compounds
1, 3, 5a-5f, 6c, 6e, 6f were prepared as aqueous suspen-
sions (by using 1–2 drops of 0.6% Tween-80) and
injected intraperitoneally (i.p.) 1 h prior to testing in ICR
M.p. 201–204 °C; yield 96%; IR ν cm–1: 1 692 (CO);
1
2 222 (CN); H NMR (CDCl3) δ: 4.81 (2H, s, SCH2);
7.47 (1H, s, 5-H); 7.2–8.8 (14H, m, 2C6H5 and C5H4N).
Anal. C15H12N3OS (C, H, N, S).
4.1.12. General method for the synthesis of 3-amino-6-
phenyl-4-(3′-pyridyl)thieno-[2,3-b]pyridines 6
A 5 mL solution of 1 N sodium methylate in methanol
was added to a mixture of 0.05 mol of the corresponding
2-alkylthio-3-cyanopyridine 5 c–f in 30 mL of ethanol.
The reaction mixture was refluxed for 5 min and cooled
to 0 °C. The precipitated crystals were filtered and
recrystallised from ethanol-chloroform (1:1) mixture.

308
mice for general examination of the pharmacological
profile of these compounds. In more detailed studies, the
compounds 1, 3, and 5a were administered orally (p.o.) as
aqueous suspensions in BALB/c mice or Wistar rats 1 h
prior to testing performance. Control animals received a
corresponding volume of solvent. Piracetam (p.o.) and
Diazepam (i.p.) were used as reference drugs, and they
were administered according to the same schedule as the
tested compounds. For general pharmacological exami-
nation of the compounds, the tests according to the
Pharmacological Screening Program PANLABS, Inc,
1973, were used [35]. Brief descriptions of the proce-
dures are given below. Some specific tests (e.g. antiam-
nesic) were performed according to methods described
elsewhere and are explained in detail.
50 mg/kg. Diazepam (5 mg/kg, i.p.) was used as a refer-
ence drug. The PTZ dose (1% solution, i.v.) required for
the inducing clonic and tonic seizures in the compound-
treated and non-treated mice (PTZ control) was mea-
sured. The compounds were administered i.p. 1 h prior to
the PTZ infusion.
4.2.5. Nicotine tremor test
The i.v. titration was carried out with nicotine base
(0.01% solution) and the doses required for inducing
tremor were registered in treated and non-treated (nico-
tine control) mice. The compounds were administered
p.o. 1 h prior to nicotine infusion.
4.2.6. Arecoline-tremor test
Arecoline-tremor tests were performed by subcutane-
ous (s.c.) administration of arecoline, 25 mg/kg. The
compounds were administered p.o. 1 h prior to arecoline
injection. The time (min) before the arecoline-induced
tremors began in the compound-treated and non-treated
(arecoline control) mice was registered. The data ob-
tained in the tests (4–6) were expressed as the mean
values ± SEM and were statistically analysed by Stu-
dent’s t-test at P < 0.05.
4.2.1. Coordination and muscle tone
Coordination and muscle tone were evaluated in ICR
mice by using routine rota rod and traction tests. The
compounds were administered i.p. 1 h prior to testing.
Animals were placed on rota rod (Ugo Basile apparatus,
Model Nr. 7600, Italy) and the number of animals which
had fallen from the rotating cylinder (diameter 3 cm,
rotating speed 8 rpm) during 2 min was registered. In the
traction test, animals (compounds administered i.p.1 h
prior to testing) were forced to hang by their forelegs on
a metal wire (diameter 2 mm). The number of animals
unable to hang for 30 s was registered.
4.2.7. Amphetamine stereotypy
Amphetamine stereotypy in Wistar rats was induced by
administration of D-amphetamine in a dose of 10 mg/kg,
s.c., 30 min following compound p.o. administration.
Compounds 1, 3 and 5a were administered at 0.5, 5 and
50 mg/kg p.o. (with the exception of 1 which was
administered at 5 and 50 mg/kg). The duration (min) of
stereotypic behaviour was registered 1 h after the com-
pound administration and compared to the data obtained
in amphetamine control rats (without compound-
treatment). Piracetam (100 mg/kg) served as reference
drug. The mean values in doses ± SEM were calculated
and statistical significance was evaluated vs. amphet-
amine control at P < 0.05.
4.2.2. Body temperature
Body temperature was measured by electrothermom-
eter in ICR mice rectum before the experiment and 1 h
after i.p. administration of each compound. The number
of animals with a drop in temperature of 3 °C or more
was considered as a hypothermic effect. Statistical evalu-
ation of the results obtained in coordination and tempera-
ture tests was carried out by calculation of mean effective
doses (ED50) and confidence interval according to Litch-
field and Wilcoxon.
4.2.3. Acute toxicity
4.2.8. 5-HTP-induced head shake test
Acute toxicity in ICR mice was determined at doses of
5-HTP-induced head shake tests were performed in
BALB/c mice by i.p. administration of 5-HTP at a dose of
300 mg/kg. Compounds 1, 3 and 5a were administered at
5 and 50 mg/kg, p.o. 1 h prior to 5-HTP administration.
The number of head shake incidences was registered for
1 min at 10, 20, 30 and 40 min after 5-HTP injection.
Total number of head shakes (4 min) was calculated. A
statistical analysis of the treated and non-treated (5-HTP
control) animal groups was the same as in amphetamine
test.
1 000–10 000 mg/kg i.p. The animals were observed for 3
d.
4.2.4. Anticonvulsant activity (pentylenetetrazol (PTZ)-
titration test)
Anticonvulsant activity (pentylenetetrazol (PTZ)-
titration test) was evaluated in BALB/c mice. The com-
pounds 1, 3, and 5a were administered in doses of 5 and
50 mg/kg, compounds 5b–5f and 6c, 6e, 6f in doses of

309
4.2.9. Antiamnesic activity
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passive avoidance response (PAR) tests according to De
Wied [36]. Amnesia was induced by electroconvulsive
shock (ECS). The following method was used. PAR
training was started by placing the mice individually into
the lighted side of the two-compartment (light-dark) PAR
apparatus (Model Nr. 7530, Ugo Basile, Italy). Following
10 min accommodation in the light compartment, the
door was opened to provide access to the dark compart-
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closed completely. Mice not entering the dark compart-
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5 s an inescapable foot-shock (1.0 mA, 5s) was applied
immediately through the steel grid floor of the dark
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corneal electrodes and returned to its home cage. The
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mean values ± SEM were statistically evaluated by Stu-
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saline and Piracetam controls).
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