Studies on pyrazine derivatives, XLII: Synthesis and tuberculostatic activity of 6-(1,4-dioxa-8-azaspiro-[4,5]-decano)- and 6-(1- ethoxycarbonylpiperazino)-pyrazinocarboxylippc acid derivatives

Article abstract of DOI:10.1080/104265090902741

The 2-cyano-6-chloropyrazine's chlorine atom reactivity was substituted with amines 1,4-dioxo-8-azaspiro-[4,5]-decane and 1-ethoxycarbonylpiperazine. The substrates thus obtained were used in the syntheses of the new derivatives, which were tested for the

Full text of DOI:10.1080/104265090902741

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Studies on Pyrazine
Derivatives, XLII: Synthesis
and Tuberculostatic Activity
of 6-(1,4-Dioxa-8-azaspiro-
[4, 5]-decano)- and 6-(1-
Ethoxycarbonylpiperazino)-
pyrazinocarboxylic Acid
Derivatives
Barbara Milczarska a , Henryk Foks a & Zofia Zwolska
b
a Department of Organic Chemistry , Medical
University of Gdańsk , Gdańsk, Poland
b Department of Microbiology , Institute of
Tuberculosis and Pulmonary Diseases , Warsaw,
Poland
Published online: 19 Aug 2006.
To cite this article: Barbara Milczarska , Henryk Foks & Zofia Zwolska (2005) Studies
on Pyrazine Derivatives, XLII: Synthesis and Tuberculostatic Activity of 6-(1,4-Dioxa-8-
azaspiro- [4, 5]-decano)- and 6-(1-Ethoxycarbonylpiperazino)- pyrazinocarboxylic
Acid Derivatives, Phosphorus, Sulfur, and Silicon and the Related Elements, 180:9,
1977-1992, DOI: 10.1080/104265090902741
To link to this article: http://dx.doi.org/10.1080/104265090902741
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Phosphorus, Sulfur, and Silicon, 180:1977–1992, 2005
Copyright © Taylor & Francis Inc.
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/104265090902741
Studies on Pyrazine Derivatives, XLII: Synthesis and
Tuberculostatic Activity of 6-(1,4-Dioxa-8-azaspiro-
[4,5]-decano)- and 6-(1-Ethoxycarbonylpiperazino)-
pyrazinocarboxylic Acid Derivatives
Barbara Milczarska
Henryk Foks
Department of Organic Chemistry, Medical University of Gdan´sk,
Gdan´sk, Poland
Zofia Zwolska
Department of Microbiology, Institute of Tuberculosis and Pulmonary
Diseases, Warsaw, Poland
The 2-cyano-6-chloropyrazine’s chlorine atom reactivity was substituted with
amines 1,4-dioxo-8-azaspiro-[4,5]-decane and 1-ethoxycarbonylpiperazine. The
substrates thus obtained were used in the syntheses of the new derivatives, which
were tested for their tuberculostatic activity. Minimum inhibitory concentration
(MIC) value of the most active ones (5b, 12a, 14b, 16b, 21b) was 25 µg/mL.
Keywords
2-Cyano-6-chloropyrazine; 6-aminosubstituted-2-cyanopyrazine; N1-
substituted thioamidopyrazincarboxyamidrazones; thioamides
INTRODUCTION
The tuberculostatic activity of pyrazine and its derivatives has been,
among other properties, a subject of intensive studies. Most of the ac-
tive compounds made the pyrazino-carboxylic acid derivatives.1−5 An
introduction of the 1,3,4-oxathiazolino-2-one system into the pyrazine
molecule increased the tuberculostatic activity 16 times in comparison
with that of pyrazinamide.6
The earlier reports of Foks and colleagues7,8 on some new 2-cyano-
6-substituted pyrazine derivatives showed the considerable tuberculo-
static activity of these compounds as well.
Received September 7, 2004; accepted September 16, 2004.
Address correspondence to Henryk Foks, Medical University of Gdan´sk, Depart-
ment of Organic Chemistry, Al Gen J Hallera 107, Gdan´sk, PL 80-416, Poland. E-mail:
hfoks@farmacja.amg.gda.pl
1977

1978
B. Milczarska et al.
RESULTS AND DISCUSSION
In continuation of our studies on the reactivity and applications of
pyrazine derivatives, the new 2,6-disubstituted pyrazines have been
synthesized (Scheme).1
SCHEME 1
The reaction of 2-cyano-6-chloropyrazine with amines 1,4-dioxa-
8-azaspiro-[4,5]-decane and 1-ethoxycarbonylpiperazine produced the
initial pyrazinonitriles 1a and 1b, which were converted into
thioamides (2), amidoximes (3), imidoesters (4), and amidrazones (5).
The amidoximes obtained thus were allowed to react with aromatic
isothiocyanates (p-chloro-, p-bromo-, and p-methyloisothiocyanate) the
derivatives (13–15). The reaction course and time were controlled by
means of thin layer chromatography.
In the reactions of imidoesters (4) with the substituted thiosemi-
carbazides A–E the N1-substituted thioamidopyrazincarboxyamidra-
zones (7–12) were formed (see Table I). The attempts at obtaining
the same compounds by the earlier preparation of 3-carbonimido-
yldithiocarbazoic acid S-methyl esters, followed by their reactions with
the corresponding secondary amines, failed.
The condensation of amidrazones (5a, b) with aromatic aldehy-
des p-bromo-; o-, m-, and p-chloro-; p-nitro-; o-methoxy-; and 2,5-
dimethoxybenzoic) produced the corresponding Schiff bases (16–21)
(see Table II). The addition of piperidine increased the reaction
yields.

1979

1980

1981

1982

1983

1984

1985

1986

1987

1988

Studies on Pyrazine Derivatives
1989
The structure of the newly obtained compounds was established by
the analyses of IR and 1H NMR spectra. The spectral data are given in
the Tables I and II.
MICROBIOLOGY
The newly obtained derivatives were tested for their tuberculostatic ac-
tivity towards the standard Mycobacterium tuberculosis H37Rv strain,
as well as two strains isolated from tuberculotic patients (Table III).
One strain, Myc. species 210, was resistant to P-Aminosalicylic Acid
(PAS), Isonicotinic Acid Hydrazide (INH), Ethambutol (EMB) and Ri-
fampycine (RFP); the other strain, Myc. species 192, was fully suscep-
tible to the drugs administered.
Tuberculostatic activity was tested in vitro with the classical test
tube method on Youman’s liquid medium containing 10% of a bovine
serum.9 The Minimum Growth Inhibiting Concentration (MIC) values
obtained showed that some compounds were noteworthy. MIC values
of the compounds 5b, 12a, 14b, 16b, and 21b were within the limits of
25–50 µg/mL. The MIC values obtained for pyrazinamide were within
30–60 µg/mL using the same method.
TABLE III Tuberculostatic Activity [µg/mL]
Myc. spec. Myc. spec. Comp-
192 210 ound Myc. tbc.
H37Rv susceptible resistant
Myc. spec. Myc. spec.
192 210
H37Rv susceptible resistant
Compound Myc. tbc.
no.
no
1a
1b
2a
2b
3a
3b
5a
5b
6a
6b
7a
7b
8a
8b
9b
10a
10b
11a
11b
50
50
100
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
100
100
50
50
100
50
100
50
50
50
50
50
100
50
50
50
50
50
50
50
50
50
50
50
50
50
25
50
50
50
50
50
50
50
100
50
50
50
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
18a
18b
19a
19b
20a
20b
21a
21b
25
50
50
50
100
50
100
50
50
50
100
50
50
50
50
50
100
50
100
25
50
50
50
50
100
50
100
50
50
50
50
25
50
100
50
100
25
50
50
50
25
100
50
50
50
50
50
50
25
50
50
50
50
50

1990
B. Milczarska et al.
EXPERIMENTAL
All melting points were obtained with a Boe¨tius apparatus and are
uncorrected. The elemental analysis results for C and H of all the com-
pounds obtained were in good agreement with the data calculated.
The IR spectra were taken with a Satellite spectrophotometer, and
the 1H NMR spectra were taken with a Varian Unity 500 MHz appara-
tus. The reaction yields and physical constants of the new compounds
are given in the Tables I and II.
Syntheses of Substituted 2-Cyanopyrazine Derivatives (1a, 1b)
To a solution of 2-cyano-6-chloropyrazine (0.1 mol) in benzene (150 cm3),
triethylamine (0.15 mol) and corresponding amine (0.1 mol), i.e.,
1,4-dioxa-8-azaspiro-[4,5]-decane or 1-ethoxycarbonylpiperazine, was
added. The mixture was refluxed for 2 h. On cooling down, water
(60 cm3) was added and the mixture was extracted with benzene. The
benzene extracts were dried over anhydrous MgSO4. The solution ob-
tained was thickened by evaporation under vacuum and allowed to
stand for crystallization. The precipitate was filtered off and recrys-
tallized.
Syntheses of Thioamides (2a, 2b)
The corresponding nitrile (2 mmol) was dissolved in ethanol (8 cm3) and
ammonium polysulfide was added until turbidity appeared. After 12 h,
the precipitate was filtered off and crystallized.
Syntheses of Amidoximes (3a, 3b)
To a solution of hydroxylamine hydrochloride (17.0 mmol) in absolute
methanol (10 cm3) the solution of KOH (22 mmol) in absolute methanol
(10 cm3) was added. The precipitated KCl was filtered off and the filtrate
was treated with the corresponding nitrile 1a or 1b (2.5 mmol). The
whole was refluxed for 2 h. The solvent was then evaporated under
reduced pressure and the residue treated with acetic acid (0.8 cm3) in
water (10 cm3). The precipitate was filtered off and recrystallized.
Syntheses of Imidoesters (4a, 4b)
To the solution of nitrile 1a or 1b (4 mmol) in benzene (60 cm3) the solu-
tion of NaOH (10 g) in water (10 cm3) was added, followed by methanol
(10 cm3) and a small amount of triethylbenzylamine (TEBA). The whole

Studies on Pyrazine Derivatives
1991
was stirred for 2 h at ambient temperature. The water (70 cm3) was
then added and the aqueous layer after separation was extracted with
benzene. The combined benzene extracts were dried over anhydrous
MgSO4. The benzene solution was thickened under reduced pressure
and the oily residue was treated with petroleum ether. The precipitates
were filtered off and recrystallized.
Syntheses of Amidrazones (5a, 5b)
To the solution of nitrile 1a or 1b (2.5 mmol) in methanol (8 cm3),
80%—hydrazine (1.3 cm3) was added. The solution was heated to
50◦C for 1 h. The solvent and an excess of hydrazine were evaporated
under reduced pressure. The residue was cooled and treated with a
small amount of water. The precipitate obtained was filtered off and
crystallized.
The Reactions of Amidoximes with Aryloisothiocyanates
(13a, b–15a, b)
Amidoxime 3a or 3b (0.5 mmol) and corresponding isothiocyanate
(0.5 mmol) were suspended in DMF (5 cm3) and stirred at ambient
temperature for 3–5 days. The mixture was ice-cooled and treated with
water. The precipitate was filtered off and crystallized.
Condensation of Amidrazones with Aromatic Aldehydes
(16a, b–21a, b)
To the solution of the corresponding amidrazone (0.64 mmol) in absolute
methanol (8 cm3) aromatic aldehyde (0.64 mmol) and a few drops of
piperidine were added. The whole was refluxed for 4 h. On cooling down,
the precipitates obtained were filtered off and crystallized.
Syntheses of 3-Carbonimidoyldithiocarbazonic Acid S-Methyl
Esters (6a, 6b)
Imidoester 4a or 4b (1 mmol) in absolute methanol (8 cm3) was treated
with methyl dithiocarbazones (1 mmol) dissolved in methanol (3 cm3).
The whole was stirred at ambient temperature for 24 h. The precipitate
was filtered off and crystallized.
Syntheses of Thiosemicarbazides A–E
A suspension of methyl dithiocarbazone (2 mmol) in water (5 cm3)
was treated with the corresponding amine (6 mmol), i.e., pyrrolidine

1992
B. Milczarska et al.
(A), morpholine (B), phenylpiperazine (C), 1,4-dioxa-8-azaspiro-[4,5]-
decane (D), or 1-benzylpiperazine (E), dissolved in water (3 cm3). The
whole was heated with a water bath for 6 h. On cooling down, the pre-
cipitate was filtered off and washed with water.
Thiosemicarbazide: A M.p. 165–9◦C (45% yield); B M.p. 186–6◦C
(23%); C M.p. 170–3◦C (42%); D M.p. 130–6◦C (15%); E M.p. 161–5◦C
(34%).
Syntheses of N1-Substituted
Thioamidopyrazincarboxyamidrazones (7a, b–12a, b)
A solution of imidoester 4a or 4b (1 mmol) and the corresponding
thiosemicarbazide A–E (1 mmol) in methanol (20 mcm3) was stirred at
ambient temperature for 4 days. The solvent excess was distilled off. On
cooling down, the oily residue was treated with benzene and petroleum
ether. The precipitate obtained was filtered off and crystallized.
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