Synthesis and antibacterial activity of 1H-pyrazolo[3,4-b]pyrazine and -pyridine derivatives

Article abstract of DOI:10.1016/j.farmac.2005.05.002

The investigations of new pyrazine and pyridine derivatives showing an antibacterial activity have been made. Upon treatment of 3-chloro-2- cyanopyrazine [1] and 2-chloro-3-cyanopyridine with 1,1-dimethyl-hydrazine, 1-aminopiperidine and 1-amino-4-methylpiperazine, either the pyrazolo-pyrazine (1), and -pyridine (2) derivatives, or ammonium salts (3-8) were obtained, according to the reaction conditions. Compound 1 was obtained in the reaction of the initial nitrile with methylhydrazine as well. The reactions of 1 gave the following derivatives: acylation - (9), that with p-chlorobenzoic aldehyde-(10), and with phenyl-isothiocyanate - (11). 3-Chloro-2-cyanopyrazine treated with hydrazine hydrate gave amidrazone (12), which upon condensation with p-chlorobenzoic aldehyde produced (13). The compounds obtained were tested in vitro for their tuberculostatic activity. The minimal inhibitory concentration (MIC) values were within 22-100 μ g/cm³. Compounds 1, 5 and 6 were also tested in vitro for their activity towards 25 strains of anaerobic, and 25 strains of aerobic bacteria. They appeared to be of elevated activity towards the anaerobes and of low one towards the aerobes (Table 2).

Full text of DOI:10.1016/j.farmac.2005.05.002

Il Farmaco 60 (2005) 513–517
http://france.elsevier.com/direct/FARMAC/
Original article
Synthesis and antibacterial activity of 1H-pyrazolo[3,4-b]pyrazine
and -pyridine derivatives
Henryk Foks a,*, Danuta Pancechowska-Ksepko a, Anna KVdzia b, Zofia Zwolska c,
Mieczysław Janowiec c, Ewa Augustynowicz-Kopec´ c
a Department of Organic Chemistry, Medical University of Gdan´sk, 107, Al. Gen. J. Hallera, 80-416 Gdan´sk, Poland
b Department of Oral Microbiology, Medical University of Gdan´sk, 14, Smoluchowskiego, 80-214 Gdan´sk, Poland
c Department of Microbiology, Institute of Tuberculosis and Pulmonary Diseases, 26, Płocka, 01-138 Warszawa, Poland
Received 18 February 2005; received in revised form 7 April 2005; accepted 4 May 2005
Available online 13 June 2005
Abstract
The investigations of new pyrazine and pyridine derivatives showing an antibacterial activity have been made. Upon treatment of 3-chloro-
2-cyanopyrazine [1] and 2-chloro-3-cyanopyridine with 1,1-dimethyl-hydrazine, 1-aminopiperidine and 1-amino-4-methylpiperazine, either
the pyrazolo-pyrazine (1), and -pyridine (2) derivatives, or ammonium salts (3–8) were obtained, according to the reaction conditions. Com-
pound 1 was obtained in the reaction of the initial nitrile with methylhydrazine as well. The reactions of 1 gave the following derivatives:
acylation—(9), that with p-chlorobenzoic aldehyde—(10), and with phenyl-isothiocyanate—(11). 3-Chloro-2-cyanopyrazine treated with
hydrazine hydrate gave amidrazone (12), which upon condensation with p-chlorobenzoic aldehyde produced (13). The compounds obtained
were tested in vitro for their tuberculostatic activity. The minimal inhibitory concentration (MIC) values were within 22–100 µg/cm3. Com-
pounds 1, 5 and 6 were also tested in vitro for their activity towards 25 strains of anaerobic, and 25 strains of aerobic bacteria. They appeared
to be of elevated activity towards the anaerobes and of low one towards the aerobes (Table 2).
© 2005 Elsevier SAS. All rights reserved.
Keywords: Cyclization; 1H-pyrazolo[3,4-b]pyrazines and -pyridines; Nitriles; Antibacterial activity
1. Introduction
to be an effective 1-xanthine oxidase inhibitor, and the
research on 4-hydroxypyra-zolo[3,4-b]pyridines proved their
high enzymatic activity [8,9].
The investigation of pyrazole derivatives condensed either
with the benzene ring (indazoles), or with various heterocy-
clic systems confirmed their comprehensive biological activ-
ity. The indazo-lylquinazoline and indazolylbenzotriazine
derivatives showed the antibacterial activity [2], just as the
pyrazolquinoline derivatives [3]. Hoffman and Rayner [4]
obtained the N-benzothienopyrazolo-amide derivatives, which
have been used for effective treatment of the rhinovirus dis-
eases. Japanese scientists obtained a series of aminoindazole
derivatives, which were tested for their anti-phlogistic activ-
ity in alimentary canal treatment [5]. The reports of Roch et
al. and Kuczyn´ski et al. [6,7] showed the anticoagulative,
hypotensive and antiarrythmic efficacy of pyrazolpyridines.
4-Hydroxypyrazolo[3,4-b]pyrimidine (allopurinol) appeared
2. Chemistry
While going on with the search for new pyrazine and pyri-
dine derivatives of antibacterial activity, 2-chloro-3-
cyanopyridine [1] was treated with methylhydrazine, and the
expected 1-methyl-1H-pyrazolo[3,4-b]pyrazinyl-3-amine (1)
was obtained. Surprisingly, the same product was formed in
the reaction with 1,1-dimethylhydrazine. The syntheses were
carried on in varied conditions. In ether, an hour’s heating
gave the ammonium salt (3), which was transformed, in boil-
ing ethanol, into compound 1. Prolonged to 24 h heating in
benzene allowed to obtain 1-methyl-1H-pyrazolo[3,4-
b]pyrazinyl-3-amine (1) at one go.
2-Chloronicotinonitrile was also treated with 1-methyl-
hydrazine, according to the method proposed by Kuczyn´ski
* Corresponding author.
E-mail address: hfoks@amg.gda.pl (H. Foks).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2005.05.002

514
H. Foks et al. / Il Farmaco 60 (2005) 513–517
Scheme 1
.
et al [7], which produced the aminopyrazolpyridine deriva-
tive (2). The reaction with 1,1-dimethylhydrazine, however,
gave only the corresponding ammonium salt (4).
The reactions of 3-chloro-2-cyanopyrazine and 2-chloro-
nicotinonitrile with cyclic hydrazine derivatives, 1-amino-
piperidine and 1-amino-4-methylpiperidine, were carried on
as well, and their only products were cyclic ammonium salts
(5–8).
3.1.1. 1-Methyl-1H-pyrazolo[3,4-b]pyrazin-3-ylamine (1)
A. To 3-chloro-2-cyanopyrazine (5 mmol) dissolved in ben-
zene (10 ml) methylhydrazine or 1,1-dimethylhydrazine
(10 mmol) was added and stirred for 24 h at ambient tempera-
ture, then yellow precipitate of product 1 was filtered off.
B. Compound 3 (2.5 mmol) dissolved in ethanol (5 ml)
was refluxed for 2 h; the solvent was evaporated then, and the
residual 1 purified by crystallization.
1-Methyl-1H-pyrazolo[3,4-b]pyrazino-3-yl-amine (1) was
exposed to acylation with acetic anhydride, which gave the
corresponding amide derivative (9), and to the action of
p-chlorobenzoic aldehyde, or phenyl isothiocyanate, which
gave, respectively, compound (10) or the thiourea derivative
(11).
3.1.2. 1-Methyl-1H-pyrazolo[3,4-b]pyridin-3-ylamine (2)
To 2-chloro-3-cyanopyridine (5 mmol) dissolved in dry
xylene (20 ml) calcined soda (0.8 g) and methylhydrazine
(10 mmol) were added and refluxed for 1 h. On cooling down,
precipitated (2) was filtered off and crystallized.
The reaction of 3-chloro-2-cyanopyrazine with hydrazine
hydrate produced 3-hydrazinopyra-zino-2-amidrazone (12),
which, condensed then with p-chlorobenzoic aldehyde, gave
compound (13).
The reactions were shown on Scheme 1, and the charac-
teristics of the compounds newly obtained, was given in
Table 1.
3.1.3. 1,1-Dimethyl-1H-pyrazolo[3,4-b]pyrazin-3-ylamine
chloride (3)
To 3-chloro-2-cyanopyrazine (5 mmol) in diethyl ether
(10 ml) 1,1-dimethylhydrazine (5 mmol) was added and
refluxed for 1 h. Reaction mixture was cooled down then,
and the precipitated compound (3) filtered off.
3.1.4. 1,1-Dimethyl-1H-pyrazolo[3,4-b]pyridin-3-ylamine
chloride (4)
3. Experimental
To 2-chloro-3-cyanopyridine (5 mmol) dissolved in ben-
zene (10 ml) 1,1-dimethylhydrazine (10 mmol) was added,
stirred for 48 h at ambient temperature; the precipitated com-
pound (4) was filtered off then and recrystallized.
3.1. Chemistry
Melting points were determined with a Reichert apparatus
and are uncorrected. The IR spectra were taken with a Satel-
lite spectrophotometer. The 1H NMR spectra were taken with
a Varian Gemini 200 spectrometer. Reaction yields and the
physical constants of the compounds obtained are given in
Table 1. The results of elemental analyses for C and H for all
the compounds obtained are in good agreement with the data
calculated.
3.1.5. Spiro-piperidin-1,1′-1H-pyrazolo[3,4-b]pyrazin-3-
ylamine chloride (5) and spiro-4-methyl-piperazin-1,1-1H-
pyrazolo[3,4-b]pyrazin-3-ylamine chloride (6)
3.1.5.1. General method. To 3-chloro-2-cyanopyrazine
(5 mmol) dissolved in benzene (10 ml) either 1-amino-

H. Foks et al. / Il Farmaco 60 (2005) 513–517
515
Table 1
Characteristics of the newly synthesized compounds 1–13
Compound Formula molecular mass Yield M.p. (°C) solvent for
1H NMR solvent d (ppm)
(%)
67
crystallization
1
2
C6H7N5 149.16
C7H8N4 148.17
147–150 cyclohexane DMSO-d6: 3.81 (s, 3H, CH3); 5.9 (s, 2H, NH2); 8.37, 8.46 (2d, 2H, CH pyrazin)
100–104 cyclohexane CDCl3: 3.5 (s, 3H, CH3); 4.5 (s, 2H, NH); 6.7 (d, 1H, CH pyrydin); 7.7 (d, 1H,
CH pyrydin); 8.3 (m, 1H, CH pyrydin)
54
3
4
5
6
7
8
C7H10N5Cl 199.52
C8H11N4Cl 198.52
35
170 decompose
DMSO-d6: 3.65 (s, 6H, CH3); 8.41 (s, 2H, NH2); 8.97, 9.22 (2d, 2H, CH pyra-
zin)
155 decompose
DMSO-d6): 3.59 (s, 6H, CH3); 7.92, 8.91 (2 m, 3H, CH pyrydin); 8.5 (b.s, 2H,
NH2)
C
C
C
C
10H14N5Cl 239.55
10H15N6Cl 254.56
11H15N4Cl 238.56
11H16N5Cl 253.57
72
43
55
62
165–167 absolutes
EtOH/Et2O
DMSO-d6: 1.7–2.2 (m, 6H, CH2); 3.55, 4.05 (2 m, 4H, CH2); 8.5 (s, 2H, NH2);
9.0, 9.25 (2d, 2H, CH pyrazin)
164 decompose
(DMSO-d6: 2.41 (s, 3H, CH3); 2.89 (s, 2H, CH2); 3.12 (s, 2H, CH2); 3.61 (s,
2H, CH2); 4.14 (s, 2H, CH2); 8.6 (s, 2H, NH2); 9.02, 9.26 (2d, 2H, CH pyrazin)
DMSO-d6: 1.5–2.25 (m, 6H, CH2); 2.95 (m, 2H, CH2); 4.05 (m, 2H, CH2); 7.96
(m, 1H, CH pyrydin); 8.25–8.75 (m, 2H, NH2); 8.9 (m, 2H, CH) pyrydin)
DMSO-d6: 2.4 (s, 3H, CH3); 2.89 (m, 2H, CH2); 3.14 (m, 2H, CH2); 3.46 (m,
2H, CH2); 4.16 (m, 2H, CH2); 7.99 (m, 1H, CH pyrydin); 8.35–8.7 (m, 2H,
NH2); 8.77 (m, 1H, CH pyrydin); 8.91 (m, 1H, CH pyrydin)
DMSO-d6: 2.1 (s, 3H, CH3); 4.02 (s, 3H, CH3); 8.61 (s, 2H, CH pyrazin)
CDCl3: 3.3 (s, 1H, CH); 3.9 (s, 3H, CH3); 7.1–7.4 (m, 2H, Ph); 7.7–7.9 (m, 2H,
Ph); 8.3–8.5 (m, 2H pyrazin)
164–170 absolutes
EtOH/Et2O
183–187 absolutes
EtOH/Et2O
9
10
C8H9N5O 191.19
43
66
167–169 benzene
182–183 MeOH
C
13H10N5Cl 271.76
11
12
13
C
13H12N6S 284.27
48
56
70
168–170 EtOH
169–175 EtOH
205–210 acetone
CDCl3: 4.0 (m, 3H, CH3); 7.6 (m, 5H, Ph); 8.4 (s, 2H pyrazin); 11.2 (m, 2H,
NH)
C5H9N7 167.18
19H15N7Cl2 412.25
DMSO-d6: 4.3–4.7 (b.s, 2H, NH); 5.4–5.68 (b.s, 2H, NH); 5.83 (s, 2H, NH);
7.71, 8.01 (2d, 2H, CH pyrazin); 9.83 (s, 1H, NH)
C
CDCl3: 1.26 (s, 2H, CH); 7.44, 7.8 (2 m, 8H, Ph); 8.62 (s, 2H, CH pyrazin)
piperidine or 1-amino-4-methylpiperazine (5 mmol) was
added and stirred for 24 h at ambient temperature. The fil-
tered off precipitates of compounds 5 or 6, respectively, were
recrystallized.
3.1.9. 1-(1-Methyl-1H-pyrazolo[3,4-b]pyrazin-3-yl)-3-phe-
nyl-thiourea (11)
To compound 1 (5 mmol) dissolved in anhydrous pyridine
(5 ml) phenyl isothiocyanate (5 mmol) was added and refluxed
for 2 h. The product 11 was precipitated with petroleum ether
and crystallized.
3.1.6. Spiro-piperidin-1,1’-1H-pyrazolo[3,4-b]pyridin-3-
ylamine chloride (7) and spiro-4-methyl-piperazin-1,1’-
1H-pyrazolo[3,4-b]pyridin-3ylamine chloride (8)
3.1.10. 3-Hydrazinepyrazine-2-amidrazone (12)
To 3-chloro-2-cyanopyrazine (5 mmol) dissolved in etha-
nol (10 ml) and cooled with ice, hydrazine hydrate (15 mmol)
was added drop wise. After 1 h the precipitated compound 12
was filtered off.
3.1.6.1. General method. To 2-chloro-3-cyanopyridine
(5 mmol) dissolved in benzene (10 ml) either 1-amino-
piperidine or 1-amino-4-methylpiperazine (5 mmol) was
added and stirred for 20 h at ambient temperature. The pre-
cipitated compounds 7 or 8, respectively, were filtered off
then.
3.1.11. 2-N (4-Chlorobenzylideneamidrazone)-3-(4-
chlorobenzylidenehydrazin)-pyrazine (13)
To compound 12 (2.5 mmol) dissolved in ethanol,
p-chlorobenzoic aldehyde (5 mmol) was added and refluxed
for 1 h. On cooling down, the precipitated compound 13 was
filtered off.
3.1.7. N-(1-Methyl-1H-pyrazolo[3,4-b]pyrazin-3-yl)-aceta-
mide (9)
Compound 1 (5 mmol) dissolved in acetic anhydride (5 ml)
was refluxed for 1 h. On cooling down the mixture was poured
on ice, neutralized with saturated Na2CO3 solution and
extracted thrice with chloroform. The combined extracts were
dried with anhydrous MgSO4 and the solvent evaporated. Pre-
cipitated compound 9 was crystallized.
3.2. Pharmacology
The compounds newly obtained (1–13) were tested for their
tuberculostatic activity towards the standard Myc. tbc H37Rv
strain, as well as two wild strains isolated from tuberculotic
patients: one resistant to p-aminosalicylic acid (PAS), isoni-
cotinic acid hydrazide (INH), ethambutol (EMB) and rifampy-
cine (RFP), the other, fully susceptible to the drugs adminis-
tered.
3.1.8. (4-Chloro-benzylidene)-(1-methyl-pyrazolo[3,4-
b]pyrazin-3-yl)-amine (10)
To compound 1 (5 mmol) dissolved in 10 ml ethanol,
5 mmol p-chlorobenzaldehyde was added and refluxed for
1 h. On cooling down, the precipitated compound 10 was fil-
tered off.
Tuberculostatic activity was tested in vitro with classical
test tube method onYouman’s liquid medium containing 10%

516
H. Foks et al. / Il Farmaco 60 (2005) 513–517
Table 2
The MIC for the compounds 1, 5, 6
Anaerobic bacteria
Number of
strains
MIC (µg/ml)
Metronidazol
Compound 1
Compound 5
Compound 6
Gram-positive: Peptococcus
niger
1
1
1
1
1
Peptostreptococcus magnus
Peptostreptococcus micros
Actinomyces israelii
2
2
1
1
2
1
2
2
1
2
1
2
3
2
2
2
1
1
2
2
1
1
1
2
2
1
1
1
1
1
1
1
1
1
1
1
1
2
Actinomyces naeslundii
Propionibacterium granulosum
Gram-negative: Prevotella bivia
Prevotella buccalis
2
1
1
1
1
1
1
1
1
2
1
1
1
1
1
2
1
1
1
2
2
2
2
Prevotella intermedia
1
1
Prevotella loescheii
1
1
Porphyromonas asaccharolytica
Fusobacterium nucleatum
Fusobacterium necrophorum
Bacteroides forsythus
2
1
2
3
2
2
1
2
3
2
2
1
1
1
1
2
1
1
Bacteroides ureolyticus
of bovine serum [10]. The obtained values of minimum growth
inhibiting concentration (MIC): 25–100 µg/ml allowed to con-
clude, that the group of compounds tested is of rather weak
tuberculostatic activity.
Compounds 1, 5 and 6 were also tested in vitro towards
25 strains of anaerobic and 25 of aerobic bacteria. The results
were given in Table 2.
susceptibility of the same bacteria to metronidazol. Despite
of having been in therapeutic use for many years, metronida-
zol maintains high activity towards the anaerobes, esp. The
Gram-negative ones, incl. Bacterioides genus [13–20]. It
seems to be important, as the rod-bacteria strains from Bac-
terioides gen. are frequently involved in the infections, and
appear to be resistant towards commonly used antibiotics.
The tests showed, that 22 from among 25 strains used, i.e.
88% were susceptible to metronidazol. This is consistent to
the other reports [13–15,17–20]. The highest refractoriness
exhibited the Gram-positive rod-bacteria strains from Propi-
onibacterium gen. (MIC > 200 µg/ml). This is confirmed by
other reports [15,16,19].
The highest, from three tested, activity towards the anaer-
obes showed derivative 6. Low concentration ranging from
12.5 to 25 µg/ml inhibited growth of four (16%) strains, and
the higher ones –50 and 100 µg/ml inhibited growth of four
(16%) and three (12%) strains. Gram-positive rod-bacteria
from Propionibacterium gen., whose was inhibited by met-
ronidazol in concentrations higher than 200 µg/ml, appeared
to be susceptible to the concentrations 50–200 µg/ml. Within
the limits 12.5–200 µg/ml derivative 6 showed the activity
towards 16 (16%) from among all the anaerobes tested. To
inhibiting growth of the remaining nine (36%) strains the con-
centrations > 200 µg/ml were necessary. The growth inhibi-
tion of two from the standard strains, i.e. Propionibacterium
acnesATCC 11827, and Peptostreptococcus anaerobiusATCC
27337 was induced by concentration of 200 µg/ml.
3.2.1. Aerobic bacteria
The investigations included 25 strains of aerobes isolated
from the oral cavity, respiratory tract and abdominal cavity,
as well as six standard strains. There were following aerobes:
Staphylococcus aureus (four strains), Corynbacterium spp.
(2), Klebsiella pneumoniae (3), Acinetobacter baumanii (2),
Escherichia coli (6), Pseudomonas aeruginosa (6),
Pseudomonas stutzeri (2) and six standard strains: Staphylo-
coccus aureus ATCC 25923, Entorococcus faecalis ATCC
29212, K. pneumoniae ATCC 13883, A. baumanii ATCC
19606, E. coli ATCC 25922 and P. aeruginosa ATCC 27853.
The susceptibility of the aerobic bacteria to the derivatives
was determined using agar dilution technique with Mueller–
Hinton agar [11,12]. It uses sterile distilled water for ultimate
dilutions. The inoculum containing 106 CFU per spot was
applied to the agar plates with Steers replicator. The inocu-
lated agar plates and “blank” agar plates were incubated for
24 h at 37 °C. The minimal incubatory concentration MIC
was defined as the lowest concentration of the derivative that
inhibited growth of aerobic bacteria.
The derivatives 1 and 5 were of similar activity towards
the anaerobes. The activity of 1 appeared to be slightly higher.
The lowest concentrations ≤ 6.2–12.5 µg/ml inhibited growth
of three (12%) strains, and 50 µg/ml of one (4%) strain. From
among the remaining 21 strains, 10 (40%) were inhibited by
concentration 200 µg/ml and the last 11 (44%)
strains—concentrations > 200 µg/ml.
4. Results and discussion
The investigations of anaerobic bacteria susceptibility to
the compounds 1, 5 and 6 were summarized in Table 2. The
results were compared with that obtained while testing the

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low concentrations 12.5–25 µg/ml. The remaining 22 (88%)
strains required for growth inhibition the use of concentra-
tions ≥ 200 µg/ml. MIC value for seven (25%) strains was
200 µg/ml.
All the derivatives evaluated were more active towards the
tested Gram-positive microbes. The strains of Gram-positive
rod-bacteria from Propionobacterium granulosum genus were
more sensitive to the derivatives tested than to metronidazol.
The growth of above-mentioned bacteria was inhibited by
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25–200 µg/ml, while by 1 within 12.5–200 µg/ml. The nec-
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susceptible to metronidazol in concentrations ≤ 6.2 µg/ml,
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[13,17,18]. However, the lower in comparison with ours
—susceptibility results were obtained by Goldstein et al. [15],
Hoellman et al. [16] and Wexler et al. [20] (MICs for 90%
strains were 0.25–2.0 µg/ml). From among the derivatives
tested the highest activity towards the strains from Peptostrep-
tococcus gen. was shown by compound 6, which inhibited
growth of three (60%) strains in concentrations from 12.5 to
25 µg/ml.
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growth inhibiting metronidazol concentration for this strain
was 6.2 µg/ml, while that of derivatives 1 and 5 –12.5 µg/ml:
• the three derivatives evaluated 1, 5, and 6 showed the dif-
ferentiated activity towards anaerobic bacteria;
• the highest activity towards Gram-negative microbes was
shown by derivative 6;
• all the derivatives evaluated showed higher activity towards
the Gram-positive anaerobes;
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