New derivatives of 4,6-dimethylisoxazolo[3,4-b] pyridin-3(1H)-one: Synthesis, tautomerism, electronic structure and antibacterial activity

Article abstract of DOI:10.1515/hc-2014-0107

The electronic structure and prototropic tautomerism of 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) were studied theoretically with use of the B3LYP/6-31Gand ωB97X-D/6-31Gdensity functional methods and SM8 (H₂O, DMF) solvation models. Compound 1, which is a weak acid with a pK_a of 6.9, undergoes regioselective alkylation and sulfonylation under basic reaction conditions to give a series of N1-substituted products 2a-i. Later compounds were evaluated in vitro for antibacterial activity with the use of 68 strains of aerobic and anaerobic bacteria, including 12 reference strains.

Full text of DOI:10.1515/hc-2014-0107

Heterocycl. Commun. 2014; 20(4): 215–223
Jarosław Sączewski*, Anna Kędzia and Aleksandra Jalińska
New derivatives of 4,6-dimethylisoxazolo[3,4-b]
pyridin-3(1H)-one: synthesis, tautomerism,
electronic structure and antibacterial activity
Abstract: The electronic structure and prototropic tauto- the formation of a new class of photostable fluorescent
merism of 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) dyes with a [1, 2, 4]triazolo[3,4-b]pyridin-2-ium core struc-
were studied theoretically with use of the B3LYP/6-31G* ture [10–12].
and ωB97X-D/6-31G* density functional methods and
In the present work, we describe the N-alkylation and
SM8 (H2O, DMF) solvation models. Compound 1, which N-sulfonylation reactions of 1 and antibacterial properties
is a weak acid with a pKa of 6.9, undergoes regioselective of the newly obtained isoxazolo[3,4-b]pyridin-3(1H)-one
alkylation and sulfonylation under basic reaction condi- derivatives 2a–i. To understand the observed regioselec-
tions to give a series of N1-substituted products 2a–i. Later tivity of the above reactions, theoretical studies on tau-
compounds were evaluated in vitro for antibacterial activ- tomerism of 1 and electronic structure of the most stable
ity with the use of 68 strains of aerobic and anaerobic bac- tautomers 1H-oxo (1-A), 7H-oxo (1-B) have been carried
teria, including 12 reference strains.
out using the B3LYP/6-31G* and ωB97X-D/6-31G* density
functional methods.
Keywords: acidity; antibacterial activity; DFT quantum
chemical calculations; isoxazolo[3,4-b]pyridin-3(1H)-one;
N-alkylation; mesomerism.
Results and discussion
DOI 10.1515/hc-2014-0107
Received June 19, 2014; accepted June 22, 2014; previously pub-
lished online July 17, 2014
Synthesis
4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) was syn-
thesized according to a previously described procedure
[13] with minor modification. As shown in Scheme 1, the
condensation of N-hydroxy-3-(hydroxyamino)-3-imino-
propanamide [14] with acetylacetone in the presence of
Introduction
It is well known that substituted 2,1-isoxazol-3-ones possess piperidine resulted in a yellow-colored solution of salt A
interesting pharmacological properties such as antibacte- from which, upon neutralization with aqueous 10% HCl,
rial [1–4], antitubercular [1], fungicidal [5, 6], antileukemic the solid product 1 precipitated in good yield.
[4], antioxidant [3] and antiandrogenic [7] activities. They
also act as inhibitors of tumor necrosis factor-α (TNF-α) of compound 1 has revealed that acetylation and ben-
[8] and inhibitors of p38 MAP kinases [9]. zoylation take place exclusively at the N1 nitrogen atom
Previously, the exploration of chemical reactivity
Recently, we have used 4,6-dimethylisoxazolo[3,4-b] [13, 15]. We have now found evidence that alkylation
pyridin-3(1H)-one (1) as a substrate for the fluorogenic and sulfonylation of 1 also proceed regioselectively at
tandem Mannich-electrophilic amination reaction with the N1 nitrogen atom (Scheme 2). Due to poor solubil-
formaldehyde and secondary amines, which give rise to ity of 1 in aprotic organic solvents such as benzene or
chloroform, the alkylation reaction was carried out in
a mixture of chloroform and N,N-dimethylformamide
(DMF) in the presence of triethylamine (Scheme 2). Pure
products 2a–g were isolated by means of a preparative,
centrifugally accelerated, radial, thin-layer chromato-
graphy (chromatotron) in 62–84% yield. By contrast, the
methylsulfonyl derivative 2h was obtained by reacting
1 with methanesulfonyl chloride in aqueous 5% NaOH
*Corresponding author: Jarosław Sączewski, Department of Organic
Chemistry, Medical University of Gdańsk, Al. Gen. J. Hallera 107,
80-416, Gdańsk, Poland, e-mail: js@gumed.edu.pl
Anna Kędzia: Department of Oral Microbiology, Medical University
of Gdańsk, ul. Do Studzienki 38, 80-227 Gdańsk, Poland
Aleksandra Jalińska: Department of Chemical Technology of Drugs,
Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80-416,
Gdańsk, Poland
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216ꢀ
ꢀJ. Sączewski et al.: Derivatives of 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one
NMR measurements (lack of NOE between substituents at
the N1 position and protons of 6-CH3 group) and the previ-
ously reported single crystal X-ray diffraction analysis of
compound 2i [15].
Me
O
N
H
O
H2C
HN
NHOH
H2O
100oC, 15 min.
Me
Me
OH
NHOH
Me
O
Me
O
pK , prototropic tautomerism and electronic
straucture of 1
NHOH
O
- H2NOH
N
N
Me
N
N
NHOH
H
H
Potentiometric titration of the NH acid 1 with use of
0.1 N NaOH has shown that its aqueous pKa value is 6.9
(Figure 1), which indicates that it is more acidic than
benzotriazole (pKa ꢀ= ꢀ 8.38) [16], 1,2,3-triazole (pKa ꢀ= ꢀ 9.26),
1,2,4-triazole (pKa ꢀ= ꢀ 10.04) and significantly less acidic
than tetrazole (pKa ꢀ= ꢀ 4.89) [17]. Starting point of pH ꢀ= ꢀ 3
adjusted with 0.1 N HCl.
To obtain insight into the chemical properties of the
isoxazolo[3,4-b]pyridin-3-one ring system, we have the-
oretically analyzed all three possible tautomeric forms:
1H-oxo (1-A), 7H-oxo (1-B) and hydroxy (1-C) (Figure
2) with use of the B3LYP/6-31G* and ωB97X-D/6-31G*
density functional methods and SM8 (H2O, DMF) sol-
vation models [18–21]. It appears that in the gas phase
the most stable is the 1H-oxo tautomer 1-A, which also
has the smallest dipole moment. However, the DFT
calculations indicate that in a solution of high relative
permittivity (H2O and DMF) the 7H-oxo tautomer 1-B is
more stable than the 1H-oxo form 1-A by approximately
1.5 kcal mol-1. Therefore, according to the Boltzmann dis-
tribution, in solvents such as H2O or DMF an estimated
ratio of 1-A to 1-B may be 1:20, which is in agreement
A
Me
O
HCl
O
N
Me
N
-
H
Cl
N
H
H
1
Scheme 1ꢀSynthesis of 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-
one (1).
solution, followed by chromatographic purification on
silica gel. The acetyl derivative 2i, which was required
for biological tests, was synthesized according to the lit-
erature procedure [15].
The regioselectivity of the above reactions, that is, for-
mation of N1-substituted products, has been confirmed by
Me
O
O
N
Me
N
O
Me
2i
Ac2O
pyridine
Me
Me
O
O
10
9
RX
O
O
CHCl3, DMF
Et3N, rt, 24 h
N
H
N
Me
N
Me
N
R
F
1
2a-g
8
7
MeSO2Cl
PhSO2Cl
pKa
H2O, NaOH
6
Me
Me
O
O
5
O
O
1
N
N
Me
N
Me
N
4
3
2
SO2
SO2
Me
Ph
2h
2f
: R = CH2CH2OH
: R = CH2CH2COOMe
: R =SO2Me
2a
2b
2c
2d
2e
: R = Me
: R = Et
: R = nBu
: R = Bn
2g
2h
0
0.200 0.400 0.600 0.800 1.000 1.200 1.400
V/mL
2i
: R = Ac
: R = CH2C6H3(3,5-diOMe)
Figure 1ꢀPotentiometric titration of compound 1 in water with use of
0.1 N NaOH.
Scheme 2ꢀAlkylation, acetylation and sulfonylation of 1.
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J. Sączewski et al.: Derivatives of 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-oneꢀ
ꢀ217
Me
N
Me
Me
N
O
O
OH
O
O
O
N
Me
N
N
Me
N
H
Me
H
1-A
1H-oxo
1-B
1-C
7H-oxo
hydroxy
o
o
o (µ)
H
(µ)
(µ)
∆
∆
∆
H
vacuum
H
B3LYP/6-31G*
0 (4.27)
0 (4.34)
5.8 (8.94)
6.2 (9.06)
18.0 (4.63)
18.1 (4.79)
ω
B97X-D/6-31G*
H2O
B3LYP/6-31G*
1.8 (5.88)
1.8 (5.89)
0 (13.80)
0 (13.94)
16.6 (6.90)
17.2 (7.01)
ω
B97X-D/6-31G*
DMF
B3LYP/6-31G*
1.6 (5.23)
1.4 (5.28)
0 (12.72)
0 (12.86)
18.4 (6.56)
18.8 (6.67)
ω
B97X-D/6-31G*
Figure 2ꢀRelative enthalpies of formation ΔH° (kcal mol-1, 298.15 K) and dipole moments μ (debye) of tautomeric forms 1-A, 1-B and 1-C
calculated using B3LYP and ωB97X-D density functional methods and SM8 (H2O, DMF) solvation models [18, 19].
H
R
N
As shown in Figure 3, the alkylation reactions of
both the heterocyclic amidine derivatives D, described as
1,3-dinucleophiles [30] or ‘protomeric ambident nucleo-
philes’ [31] and heterocyclic guanidines E, such as
2-aryliminoimidazolidines [32] or O-substituted imidazoli-
din-2-one oximes [33, 34], usually occur at a pyridine-type
sp2 hybridized endo- or exo-cyclic nitrogen atom.
N
NH2
R = aryl, O-alkyl
N
N
H
D
E
Figure 3ꢀAlkylation sites at heterocyclic amidines D and guani-
dines E.
To identify reactive sites at the amidine moiety of
with the following principles: (i) in polar solvents the compound 1, we have performed quantum-chemical
dynamic equilibrium favors the more polar structure calculations and molecular modeling, through which
[22–25] and (ii) the most polar tautomer of a given com- the electronic properties of the annular tautomers 1-A
pound is generally the one to be found in the solid state and 1-B as well as the anion F have been revealed. As
[23, 26]. According to our calculations, the hydroxy tau- shown in Figure 4, in all cases (1-A, 1-B and F) the mag-
tomer 1-C is generally the least stable which, in turn, nitude of the calculated charges are higher at the N7
is in accordance with the previous examinations of nitrogen atoms. By contrast, the HOMO orbital density
tautomeric equilibrium for simple 5-hydroxyisoxazoles [√(e/au3)] mapped on isodensity surface (0.002 e/au3),
[27–29].
corresponding to the molecular size and shape, is
0.021
0.018
0.016
0.013
0.010
0.008
0.005
0.003
0
1-A
1-B
F
ω
B97X-D/6-31G*
1H-oxo
7H-oxo
anion
N7
-0.500
N1
N7
-0.570
N1
N7
-0.538
N1
-0.281
natural
-0.314
-0.222
electrostatic
-0.627 -0.385
-0.544 -0.377
-0.624 -0.410
-0.769 -0.297
-0.725 -0.501
-0.581 -0.359
Mulliken
Figure 4ꢀThe HOMO absolute values [√e/au3] mapped on the isodensity surface (0.002 e/au3) and N1/N7 atomic charges for the tautomers
1-A (left) and 1-B (middle) as well as the anion F (right) calculated with the ωB97X-D/6-31G* method.
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218ꢀ
Table 1ꢀIn vitro antibacterial activity of compounds 1, 2a,b, 2d–i against anaerobic bacterial strains.
Bacteria (number of strains)
ꢀ
ꢀ
Minimum inhibitory concentration (MIC, μg mL-1)
Metronidazoleꢀ
1ꢀ
2aꢀ
2bꢀ
2d
Gram-positive cocci:
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃꢀ≤ ꢀꢃ0.4ꢃ
ꢃꢀ≤ ꢀꢃ0.4ꢃ
1.6ꢃ
ꢃ
200(2), 50ꢃ
100ꢃ
25ꢃ
ꢃ
200, 100, 50ꢃ
100ꢃ
ꢃ
ꢀFinegoldia magna (3)
100, 12.5, ꢀ< ꢀ6.2ꢃ
50, ꢀ< ꢀ6.2ꢃ
100ꢃ
ꢃꢀ≥ ꢀꢃ200
200, 100
100
ꢀParvimonas micra (2)
ꢀPeptostreptococcus anaerobius (1)
Gram-positive rods:
50ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢀBifidobacterium breve (2)
ꢀPropionibacterium acnes (1)
ꢀPropionibacterium granulosum (2)
Gram-negative rods:
50, 25ꢃ
ꢀ> ꢀ100ꢃ
ꢃꢀ≥ ꢀꢃ100ꢃ
ꢃ
ꢀ< ꢀ0.4ꢃ
ꢀ< ꢀ0.4ꢃ
1.6ꢃ
ꢀ< ꢀ0.4ꢃ
ꢀ< ꢀ0.4ꢃ
ꢀ< ꢀ0.4ꢃ
ꢀ< ꢀ0.4ꢃ
ꢀ< ꢀ0.4ꢃ
ꢃ
ꢀ< ꢀ0.4ꢃ
ꢀ< ꢀ0.4ꢃ
ꢀ< ꢀ0.4ꢃ
50ꢃ
100ꢃ
50ꢃ
100ꢃ
ꢃ
100ꢃ
50ꢃ
50ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
50ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
100ꢃ
100, 50
ꢃꢀ≥ ꢀꢃ200
ꢃꢀ≥ ꢀꢃ200
200, 100ꢃ
ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢃ
ꢀBacteroides fragilis (1)
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
200ꢃ
100ꢃ
100ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
100ꢃ
ꢀ> ꢀ200
ꢀ> ꢀ200
ꢀ> ꢀ200
100
ꢀBacteroides uniformis (1)
ꢀBacteroides ureolyticus (1)
ꢀBacteroides vulgatus (1)
ꢀPrevotella levii (1)
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
50ꢃ
200ꢃ
ꢀ> ꢀ200
ꢀ> ꢀ200
200
ꢀPrevotella loescheii (1)
ꢀPorphyromonas asaccharolytica (1)
ꢀPorphyromonas gingivalis (1)
Reference strains:
ꢀBacteroides fragilis ATCC 25285
ꢀFinegoldia magna ATCC 29328
ꢀPeptostreptococcus anaerobius ATCC 27331 ꢃ
ꢀBifidobacterium breve ATCC 15700
ꢀParabacteroides distasonis ATCC 8503
ꢀFusobacterium nucleatum ATCC 25586
ꢀ> ꢀ200ꢃ
100ꢃ
200ꢃ
50ꢃ
50ꢃ
100ꢃ
100ꢃ
200
ꢃ
ꢃ
ꢃ
200ꢃ
200ꢃ
100ꢃ
200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
100ꢃ
ꢀ> ꢀ200ꢃ
50ꢃ
ꢀ> ꢀ200
200
200
200
ꢀ> ꢀ200
ꢀ> ꢀ200
100ꢃ
200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
100ꢃ
ꢃ
ꢃ
ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ< ꢀ0.4ꢃ
ꢀ< ꢀ0.4ꢃ
ꢀ
2eꢀ
2fꢀ
2gꢀ
2hꢀ
2i
Gram-positive cocci:
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
100, 25ꢃ
100ꢃ
ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
50, 25ꢃ
100ꢃ
ꢃ
100ꢃ
100ꢃ
100ꢃ
ꢃ
ꢀ> ꢀ200ꢃ
100ꢃ
ꢀ> ꢀ200ꢃ
200ꢃ
100ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢀ> ꢀ200ꢃ
ꢃ
ꢀ> ꢀ200ꢃ
100ꢃ
100ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
ꢃ
ꢃ
200, 100(2)ꢃ
25, 12.5ꢃ
100ꢃ
ꢀFinegoldia magna (3)
ꢃꢀ≥ ꢀꢃ200, 100, 25ꢃ
50, 12.5, ꢀ< ꢀ6.2
25, 12.5
25
ꢀParvimonas micra (2)
Peptostreptococcus anaerobius (1)
Gram-positive rods:
50, 25ꢃ
50ꢃ
ꢃ
ꢃ
ꢃ
ꢀBifidobacterium breve (2)
ꢀPropionibacterium acnes (1)
ꢀPropionibacterium granulosum (2)
Gram-negative rods:
ꢀBacteroides fragilis (1)
ꢀBacteroides uniformis (1)
ꢀBacteroides ureolyticus (1)
ꢀBacteroides vulgatus (1)
ꢀPrevotella levii (1)
ꢀPrevotella loescheii (1)
ꢀPorphyromonas asaccharolytica (1)
ꢀPorphyromonas gingivalis (1)
Reference strains:
ꢀBacteroides fragilis ATCC 25285
ꢀFinegoldia magna ATCC 29328
ꢀPeptostreptococcus anaerobius ATCC 27331 ꢃ
ꢀBifidobacterium breve ATCC 15700
ꢀParabacteroides distasonis ATCC 8503
ꢀFusobacterium nucleatum ATCC 25586
200, 100ꢃ
100ꢃ
200, 100ꢃ
ꢃ
100, 50ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢃ
100, 50ꢃ
100ꢃ
12.5, ꢀ< ꢀ6.2
100
100ꢃ
ꢃꢀ≥ ꢀꢃ200
ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
100ꢃ
200ꢃ
ꢀ> ꢀ200ꢃ
200ꢃ
ꢀ> ꢀ200ꢃ
ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
200ꢃ
50ꢃ
25ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
100ꢃ
ꢀ> ꢀ200
ꢀ> ꢀ200
ꢀ> ꢀ200
100
ꢀ> ꢀ200
200
ꢃꢀ≥ ꢀꢃ200ꢃ
50ꢃ
50ꢃ
ꢀ> ꢀ200ꢃ
50ꢃ
ꢀ> ꢀ200ꢃ
ꢃ
100ꢃ
100ꢃ
100ꢃ
100ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
200ꢃ
200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
ꢃ
ꢀ> ꢀ200ꢃ
200ꢃ
100ꢃ
100ꢃ
200
ꢀ> ꢀ200
200
200
100
200
ꢀ> ꢀ200
ꢀ> ꢀ200
ꢃ
ꢃ
ꢃ
200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
greater at the vicinity of the isoxazolone N1 nitrogen exclusively because no N7-alkylation or acylation prod-
atom than at the pyridine N7 atom. These results indi- ucts resulting from electrostatically-controlled reaction
cate that the orbital-controlled reactions of 1 take place were observed.
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ꢀ219
Table 2ꢀIn vitro antibacterial activity of compounds 1, 2g, 2f, 2i against aerobic bacterial strains.
Bacteria (number of strains)
ꢀ
ꢀ
Minimum inhibitory concentration (MIC, μg mL-1)
Amikacinꢀ
1ꢀ
2gꢀ
2fꢀ
2i
Gram-positive cocci:
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃ
ꢃꢀ≤ ꢀꢃ6.2ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢃꢀ≤ ꢀꢃ6.2ꢃ
50ꢃ
100, 50ꢃ
50, 25ꢃ
ꢃ
ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢀ> ꢀ200, 50ꢃ
100ꢃ
100, 50ꢃ
50ꢃ
ꢃ
100ꢃ
ꢃ
ꢀStaphylococcus aureus (3)
ꢀStaphylococcus aureus (MRSA) (3)
ꢀStaphylococcus epidermidis (2)
ꢀStreptococcus pyogenes (1)
ꢀStreptococcus anginosus (2)
ꢀEnterococcus faecalis (2)
Gram-positive rods:
100ꢃ
ꢃꢀ≥ ꢀꢃ200
ꢃꢀ≥ ꢀꢃ200
ꢀ> ꢀ200, 25
ꢀ> ꢀ200
50
200, 50
100ꢃ
ꢃꢀ≥ ꢀꢃ200(2), 100ꢃ
100, 50ꢃ
100ꢃ
100, 50ꢃ
100ꢃ
–ꢃ
100ꢃ
–ꢃ
100ꢃ
ꢃ
ꢃ
ꢃ
ꢀCorynebacterium ulcerans (2)
ꢀCorynebacterium xerosis (1)
Gram-negative rods:
ꢀEscherichia coli (2)
ꢀAcinetobacter baumannii (1)
ꢀCitrobacter freundii (2)
ꢀKlebsiella pneumoniae (2)
ꢀSerratia marcescens (1)
ꢀPseudomonas aeruginosa (2)
ꢀPseudomonas stutzeri (2)
Reference strains:
ꢀStaphylococcus aureus ATCC 25923
ꢀEnterococcus faecalis ATCC 29212
ꢀCorynebacterium xerosis ATCC 373
ꢀKlebsiella pneumoniae ATCC 13883
50, 25ꢃ
50ꢃ
100ꢃ
100ꢃ
100ꢃ
ꢃꢀ≥ ꢀꢃ200, 100
100ꢃ
25ꢃ
ꢃ
50ꢃ
ꢃ
100
ꢃ
ꢃ
12.5, ꢃꢀ≤ ꢀꢃ6.2ꢃ
ꢀ< ꢀ6.2ꢃ
ꢃꢀ≤ ꢀꢃ6.2ꢃ
ꢃꢀ≤ ꢀꢃ6.2ꢃ
ꢀ< ꢀ6.2ꢃ
12.5ꢃ
ꢃꢀ≤ ꢀꢃ6.2ꢃ
ꢃ
100ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200, 100ꢃ
100ꢃ
100ꢃ
ꢀ> ꢀ200, 100ꢃ
100ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
100ꢃ
200, 100ꢃ
ꢃꢀ≥ ꢀꢃ200, 100ꢃ
100ꢃ
ꢀ> ꢀ200, 100ꢃ
100ꢃ
ꢃꢀ≥ ꢀꢃ200
ꢀ> ꢀ200
ꢃꢀ≥ ꢀꢃ200
ꢃꢀ≥ ꢀꢃ200
ꢀ> ꢀ200
ꢃꢀ≥ ꢀꢃ200
ꢃꢀ≥ ꢀꢃ200
ꢀ> ꢀ200ꢃ
200ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
50ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢃꢀ≥ ꢀꢃ200ꢃ
ꢃ
ꢀ> ꢀ200ꢃ
100ꢃ
200ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
200ꢃ
ꢃ
ꢃ
ꢀ< ꢀ6.2ꢃ
25ꢃ
25ꢃ
ꢀ< ꢀ6.2ꢃ
ꢀ< ꢀ6.2ꢃ
ꢀ< ꢀ6.2ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
100ꢃ
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
100ꢃ
ꢀ> ꢀ200
ꢀ> ꢀ200
100
ꢀ> ꢀ200
ꢀ> ꢀ200
ꢀ> ꢀ200
100ꢃ
ꢀ> ꢀ200ꢃ
ꢀ≥ ꢀꢃ200ꢃ
ꢀ> ꢀ200ꢃ
ꢀAcinetobacter baumannii ATCC 19606ꢃ
ꢀEscherichia coli ATCC 25922
ꢀ> ꢀ200ꢃ
ꢀ> ꢀ200ꢃ
ꢃ
Antibacterial activity
exert any antibacterial effect, probably due to its poor
solubility. All tested compounds are more active against
The results of in vitro antibacterial activity of compounds Gram-positive cocci (12–32% susceptible strains) than
1, 2a,b, 2d–i against anaerobic bacterial strains and Gram-positive rods (3–15% susceptible strains). The most
compounds 1, 2g, 2f, 2i against aerobic bacterial strains susceptible genera among Gram-positive cocci and Gram-
are presented in Tables 1 and 2, respectively. The tested positive rods are Parvimonas micra and Bifidobacterium
compounds show moderate potencies against anaerobic breve, respectively. Among anaerobes, the following
bacteria as they inhibit growth of 15–47% strains at con- strains exhibit high resistance towards all tested com-
centrations in the range ꢀꢀ≤ ꢀꢀ6.2–100 μg/mL. The most potent pounds (MIC ꢀꢀ≥ ꢀꢀ200 μg/mL): Parabacteroides distasonis
methyl propanoate 2g displays broad activity against 47% (1), Prevotella bivia (1), Prevotella buccalis (1), Prevotella
of bacterial strains, with minimal inhibitory concentration intermedia (2), Fusobacterium nucleatum (2), Fusobacte-
(MIC) values in the range 25–100 μg/mL. The unsubsti- rium necrophorum (2), Parabacteroides distasonis, ATCC
tuted 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) 8503 and Fusobacterium nucleatum ATCC 25586.
inhibits growth of 15 strains (44%) at concentrations in the
Aerobic bacterial strains exhibit susceptibility to only
range 25–100 μg/mL, whereas the remaining seven con- four tested compounds at the concentrations in the range
geners exhibit relatively weaker biological activities: the of 25–100 μg/mL. The most potent derivative 2g exhib-
methyl derivative 2a is active against 13 bacterial strains its activity against 22 (65%) bacterial strains, with MIC
(38%, MIC 50–100 μg/mL), 2b, 2f and 2h show potent values in the range of 50–100 μg/mL. The unsubstituted
activity against 12 strains (35%, MIC ꢀꢀ≤ ꢀꢀ6.2–100 μg/mL) and hydroxyethyl congeners 1 and 2f inhibit growth of 13
and compounds 2i and 2e inhibit growth of 11 (32%, (38%) and 20 strains (59%), respectively, at concentra-
MICꢀꢀ≤ ꢀꢀ6.2–50μg/mL)andsevenstrains(21%,25–100μg/mL), tions in the range of 25–100 μg/mL.
respectively. The benzyl (2d) derivative proved to be the
Gram-positive aerobic cocci and rods proved to be
least active, whereas the butyl congener (2c) does not more susceptible than Gram-negative rods to the four tested
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220ꢀ
pressure and the residue was treated with water (5 mL) and dichlo-
romethane (10 mL). The organic layer was dried over anhydrous
sodium sulfate and concentrated. The crude compound was purified
on a chromatotron eluting with the solvent indicated below.
compounds. Among Gram-positive bacteria the Staphylococ-
cus epidermidis, Enterococcus faecalis and Corynebacterium
xerosis are the most susceptible genera (MIC in the range
25–100 μg/mL). By contrast, Staphylococcus aureus and
methicillin resistant Staphylococcus aureus (MRSA) are sus- 1 , 4 ,6 -Tr i m e t hyl i s ox a z o l o [ 3, 4- b ] p y r i d i n -3 ( 1 H) - o n e
(2a)ꢁ4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) (0.3 g,
ceptible to compounds 2g and 2f. Generally, the Gram-neg-
1.83 mmol), methyl iodide (0.34 mL, 5.49 mmol) and triethylamine
ative rods are most resistant to the genera tested as growths
(0.51 mL, 3.66 mmol) were added to the mixture of chloroform (3 mL)
of only a few strains of Citrobacter freundii, Klebsiella pneu-
and DMF (1 mL); eluent: petroleum ether-dichloromethane, 1:1, v/v;
moniae, Serratia marcescens and Pseudomonas stutzeri are
inhibited at concentrations of 50–100 μg/mL. Among all the
tested compounds, only 1, 2f, 2g and 2i are active against
both aerobic and anaerobic bacterial strains. The most active
compound in this category is the methyl propanoate 2g,
which inhibits 56% of the bacterial strains tested.
Compounds 2a, 2h, 2d and 2i were also evaluated for
their cytotoxic activity [35, 36] against five human cancer cell
lines: uterine cervical adenocarcinoma SISO, lung cancer
LCLC and A-427, pancreas adenocarcinoma DAN-G and
neural cell line RT-4. However, the tested compounds did not
exhibit cytotoxic activity at concentrations of up to 20 μm.
yield 0.16 g (49%); mp 72–75°C; IR: 2921, 1749, 1618, 1598, 1437, 11371,
1284, 1180, 1110, 1035, 1009, 869, 800 cm-1; 1H NMR (CDCl3): δ 2.57 (s,
3H, CH3), 2.60 (s, 3H, CH3), 3.47 (s, 3H, CH3), 6.81 (s, 1H, CH); 13C NMR
(CDCl3): δ 17.5, 25.4, 40.1, 102.4, 121.2, 151.1, 166.6, 166.8, 168.6; MS
(ESI): m/z 179 [M+1]+. Anal. Calcd for C9H10N2O2: C, 60.66; H, 5.66; N,
15.72. Found: C, 60.51; H, 5.78; N, 15.56.
1-Ethyl-4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one
(2b)ꢁ4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) (0.3 g, 1.83
mmol), ethyl iodide (0.29 mL, 3.66 mmol) and triethylamine (0.51 mL,
3.66 mmol) were added to the mixture of chloroform (3 mL) and DMF
(1 mL); eluent: petroleum ether-dichloromethane, 1:1, v/v; yield 0.267
g (76%); mp 55–58°C; IR: 2982, 1758, 1613, 1371, 1278, 1172, 1082, 1018,
1
931, 845, 806, 737, 702, 613, 548, 501 cm-1; H NMR (CDCl3): δ 1.26 (t,
J ꢀ= ꢀ 7.0 Hz, 3H, CH3), 2.56 (s, 3H, CH3), 2.59 (s, 3H, CH3), 3.87 (q, J ꢀ= ꢀ 7.0
Hz, 2H, CH2), 6.79 (s, 1H, CH); 13C NMR (CDCl3): δ 11.7, 17.5, 25.5, 48.3,
102.8, 121.0, 150.9, 166.8 (two signals), 167.6; MS (ESI): m/z 193 [M+1]+.
Anal. Calcd for C10H12N2O2: C, 62.49; H, 6.29; N, 14.57. Found: C, 62.57;
H, 6.33; N, 14.34.
Experimental
All chemicals were purchased from commercial sources and used
without further purification. Silica gel chromatography was per-
formed with use of silica gel 60 PF254 containing gypsum for prepara-
1-Butyl-4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one
(2c)ꢁ4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) (0.3 g, 1.83
mmol), butyl iodide (0.41 mL, 3.66 mmol) and triethylamine (0.51 mL,
3.66 mmol) were added to the mixture of chloroform (3 mL) and DMF
(1 mL); eluent: petroleum ether-dichloromethane, 1:1, v/v; yield 0.25
g (62%); mp 22–25°C; IR: 2961, 2934, 2873, 1762, 1613, 1593, 1443, 1376,
1
tive layer chromatography and chromatotron. H and 13C NMR data
were obtained using 200 MHz or 50 MHz spectrometers, respectively.
1H NMR data were internally referenced to TMS (0.0 ppm) or DMSO-d6
(2.5 ppm); 13C NMR spectra were referenced to CDCl3 (77.23 ppm) or
DMSO-d6 (39.50 ppm). The IR spectra were recorded using KBr pellets.
1
1283, 1173, 1024, 802, 702, 612 cm-1; H NMR (CDCl3): δ 0.95 (t, J ꢀ= ꢀ 7.0
Hz, 3H, CH3), 1.37–1.48 (m, 2H, CH2), 1.66–1.77 (m, 2H, CH2), 2.56 (s, 3H,
CH3), 2.60 (s, 3H, CH3), 3.79 (q, J ꢀ= ꢀ 7.0 Hz, 2H, CH2), 6.78 (s, 1H, CH);
13C NMR (CDCl3): δ 14.2, 17.5, 20.4, 25.5, 29.0, 53.1, 102.3, 120.9, 151.0,
166.8 (two signals), 167.6; MS (ESI): m/z 221 [M+1]+. Anal. Calcd for
C12H16N2O2: C, 65.43; H, 7.32; N, 12.72. Found: C, 65.64; H, 7.19; N, 12.48.
4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-
one (1)
1-Benzyl-4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one
(2d)ꢁ4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) (0.2 g,
1.22 mmol), benzyl bromide (0.16 mL, 1.34 mmol) and triethylamine
(0.17 mL, 1.34 mmol) were added to the mixture of chloroform (3 mL)
and DMF (1 mL); eluent: dichloromethane; yield 0.26 g (84%); mp
100–103°C; IR: 3030, 2950, 1761, 1612, 1592, 1434, 1376, 1174, 1031, 798,
760, 738, 703, 637 cm-1; 1H NMR (CDCl3): δ 2.56 (s, 3H, CH3), 2.61 (s, 3H,
CH3), 4.99 (s, 2H, CH2), 6.80 (s, 1H, CH), 7.27–7.39 (m, 5H, CH); 13C NMR
(CDCl3): δ 17.5, 25.5, 57.0, 103.0, 121.2, 128.7, 129.0, 129.7, 134.2, 151.0,
166.5, 166.8, 167.4; MS (ESI): m/z 255 [M+1]+. Anal. Calcd for C15H14N2O2:
C, 70.85; H, 5.55; N, 11.02. Found: C, 70.73; H, 5.76; N, 10.81.
N-Hydroxy-3-(hydroxyamino)-3-iminopropanamide [14] (1.3 g, 9.8
mmol), acetylacetone (1 mL, 9.8 mmol) and piperidine (1 mL, 9.8
mmol) were heated under reflux in water (30 mL) for 15 min. Afer
cooling to room temperature the reaction mixture was acidified
with 5% HCl and the resultant yellow precipitate was filtered off
and washed with water (2 ꢀ× ꢀ 3 mL); yield 1.2 g (75%); mp 204–207°C
(lit. mp 204°C [13]); IR: 3372, 2725, 1694, 1670, 1640, 1619, 1531, 1350,
1
1289, 1051, 1030, 980, 938, 823, 784 cm-1; H NMR (DMSO-d6): δ 2.38
(s, 3H, CH3), 2.45 (s, 3H, CH3), 6.38 (s, 1H, CH), 13.00 (bs, 1H, NH); 13
C
NMR (DMSO-d6): δ 16.7, 21.3, 100.8, 112.7, 155.9, 158.1, 159.9, 169.0; MS
(ESI): m/z 163 [M-1]-.
1-(3,5-Dimethoxybenzyl)-4,6-dimethylisoxazolo[3,4-b]pyridin-
3(1H)-one (2e)ꢁ4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1)
(0.2 g, 1.22 mmol), 3,5-dimethoxybenzyl bromide (0.28 mL, 1.34 mmol)
and triethylamine (0.17 mL, 1.34 mmol) were added to DMF (3 mL);
eluent: dichloromethane; yield 0.32 g (83%); mp 108–113°C; IR: 2947,
2843, 1761, 1601, 1474, 1452, 1438, 1393, 1375, 1354, 1296, 1276, 1206,
General procedure for compounds 2a–g
4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1), alkyl halide and
triethylamine were allowed to react at room temperature as indi-
cated below. Afer 12 h the mixture was concentrated under reduced
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ꢀ221
1170, 1161, 1068, 1054, 1032, 983, 962, 912, 837, 806, 743, 720 cm-1;
Antibacterial activity tests
1H NMR (CDCl3): δ 2.58 (s, 3H, CH3), 2.60 (s, 3H, CH3), 3.75 (s, 6H, CH3),
4.92 (s, 2H, CH2), 6.38 (s, 1H, CH), 6.52 (s, 2H, CH), 6.80 (s, 1H, CH);
13C NMR (CDCl3): δ 17.3, 25.2, 55.6, 56.8, 100.6, 102.8, 107.1, 121.0, 136.2,
150.9, 161.0, 166.2, 166.4, 167.0; MS (ESI): m/z 315 [M+1]+. Anal. Calcd
for C17H18N2O4: C, 64.96; H, 5.77; N, 8.91. Found: C, 64.75; H, 6.16; N,
8.62.
The investigations included 28 strains of anaerobic bacteria and 28
strains of aerobic bacteria isolated from the oral cavity, respiratory
system and intestinal tract as well as 12 reference strains. The anaer-
obes belonged to the following genera: Finegoldia magna (3), Parvi-
monas micra (2), Peptostreptococcus anaerobius (1), Bifidobacterium
breve (2), Propionibacterium acnes (1), Propionibacterium granulosum
(2), Bacteroides fragilis (1), Bacteroides uniformis (1), Bacteroides ure-
olyticus (1), Bacteroides vulgatus (1), Parabacteroides distasonis (1),
Prevotella bivia (1), Prevotella buccalis (1), Prevotella intermedia (2),
Prevotella levii (1), Prevotella loescheii (1), Porphyromonas asaccha-
rolytica (1), Porphyromonas gingivalis (1), Fusobacterium nucleatum
(2), Fusobacterium necrophorum (2) and following reference strains:
Bacteroides fragilis ATCC 25285, Parabacteroides distasonis ATCC
8503, Fusobacterium nucleatum ATCC 25586, Finegoldia magna ATCC
29328, Peptostreptococcus anaerobius ATCC 27331, Bifidobacterium
breve ATCC 15700. There were also the following aerobes used: Staph-
ylococcus aureus (3), Staphylococcus aureus methicillin resistant
(MRSA) (3), Staphylococcus epidermidis (2), Streptococcus pyogenes
(1), Streptococcus anginosus (2), Enterococcus faecalis (2), Corynebac-
terium ulcerans (2), Corynebacterium xerosis (1), Escherichia coli (2),
Acinetobacter baumannii (1), Citrobacter freundii (2), Klebsiella pneu-
moniae (2), Serratia marcescens (1), Pseudomonas aeruginosa (2),
Pseudomonas stutzeri (2) and six reference strains: Staphylococcus
aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Corynebac-
terium xerosis ATCC 373, Klebsiella pneumoniae ATCC 13883, Acine-
tobacter baumannii ATCC 19606, 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% defibrinated sheep’s blood [37, 38]. For aerobic bacteria experi-
ments, the agar dilution technique with Mueller-Hinton agar was
used [39, 40]. The derivatives were dissolved in 1 mL of DMSO imme-
diately before the experiment. Sterile distilled water was used for
further dilutions. The following concentrations of derivatives were
used: 200, 100, 50, 25, 12.5 and 6.2 μg/mL. The inoculum containing
105 CFU/spot was applied to the appropriate agar plates with Steers
replicator. For aerobes, the inoculated agar plates and agar plates
without derivatives were incubated for 24 h at 37°C. 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 MIC was defined as the lowest concentration of the
derivative that inhibited growth of the tested bacteria [39–41].
1-(2-Hydroxyethyl)-4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-
one (2f)ꢁ4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) (0.1 g,
0.61 mmol), 2-bromoethanol (0.38 mL, 3.04 mmol) and triethylamine
(0.17 mL, 1.22 mmol) were added to DMF (3 mL); eluent: ethyl acetate-
dichloromethane, 5:95, v/v; yield 0.08 g (63%); mp 84–85°C; IR: 3489,
3044, 2923, 1743, 1613, 1590, 1445, 1407, 1373, 1359, 1277, 1180, 1073,
1028, 881, 801, 703, 614 cm-1; 1H NMR (CDCl3): δ 2.56 (s, 3H, CH3), 2.60
(s, 3H, CH3), 3.87–3.92 (m, 2H, CH2), 4.01–4.06 (m, 2H, CH2), 6.82 (s, 1H,
CH); 13C NMR (CDCl3): δ 17.6, 25.4, 56.4, 59.9, 102.4, 121.3, 151.7, 166.1,
166.6, 167.4; MS (ESI): m/z 209 [M+1]+. Anal. Calcd for C10H12N2O3: C,
57.68; H, 5.81; N, 13.45. Found: C, 57.32; H, 6.08; N, 13.23.
Methyl 3-(4,6-dimethyl-3-oxoisoxazolo[3,4-b]pyridin-1(3H)-yl)
propanoate (2g)ꢁ4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1)
(0.1 g, 0.61 mmol), methyl 3-bromopropionate (0.20 mL, 1.83 mmol)
and triethylamine (0.17 mL, 1.22 mmol) were added to DMF (3 mL);
eluent: ethyl acetate-dichloromethane, 1:99, v/v; yield 0.12 g (79%);
mp 33–34°C; IR: 3010, 2959, 2926, 1760, 1727, 1619, 1591, 1445, 1372,
1
1269, 1248, 1198, 1173, 1032, 1017, 803 cm-1; H NMR (CDCl3): δ 2.56 (s,
3H, CH3), 2.59 (s, 3H, CH3), 2.77 (t, 2H, CH2, J ꢀ= ꢀ 7.0 Hz), 3.70 (s, 3H,
CH3), 4.10 (t, 2H, CH2, J ꢀ= ꢀ 7.0 Hz), 6.82 (s, 1H, CH); 13C NMR (CDCl3):
δ 17.5, 25.5, 31.8, 49.1, 52.4, 102.7, 121.5, 151.1, 166.4, 167.0, 167.6, 171.7;
MS (ESI): m/z 251 [M+1]+. Anal. Calcd for C12H14N2O4: C, 57.59; H, 5.64;
N, 11.19. Found: C, 57.30; H, 5.96; N, 11.01.
4,6-Dimethyl-1-(methylsulfonyl)isoxazolo[3,4-b]pyridin-3(1H)-
one (2h)ꢁ4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) (0.2 g,
1.22 mmol), NaOH (0.6 g, 15 mmol) and methanesulfonyl chloride
(0.45 mL, 5.8 mmol) were added to water (12 mL), and the resulting
mixture was stirred at room temperature for 0.5 h and then extracted
with dichloromethane (2 ꢀ× ꢀ 10 mL). The combined organic layers were
dried over anhydrous sodium sulfate and concentrated to give crude
compound 2i, which was further purified by use of chromatotron;
eluent: dichloromethane; yield 0.17 g (58%); mp 130–136°C; IR: 3020,
3010, 2927, 1783, 1762, 1618, 1585, 1387, 1364, 1334, 1321, 1284, 1243,
1181, 1172, 1033, 966, 815, 797, 759, 737, 697, 660, 607, 552, 514 cm-1;
1H NMR (CDCl3): δ 2.67 (s, 3H, CH3), 2.69 (s, 3H, CH3), 3.36 (s, 3H, CH3),
7.13 (s, 1H, CH); 13C NMR (CDCl3): δ 17.3, 25.5, 39.2, 105.1, 124.8, 151.7,
162.2, 164.4, 167.9; MS (ESI): m/z 243 [M+1]+. Anal. Calcd for C9H10N2O4S:
C, 44.62; H, 4.16; N, 11.56. Found: C, 44.37; H, 4.35; N, 11.47.
Acknowledgments: This work was supported by Polish
Ministry of Science and Higher Education and National
Science Centre, research grant IP2012 055472. We thank
Professor Patrick J. Bednarski (University of Greifswald,
Germany) for cytotoxicity assessments.
1-Acetyl-4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (2i)ꢁTo
the solution of 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1)
(0.2 g, 1.22 mmol) in pyridine (0.6 mL) acetic anhydride (0.4 mL,
4.24 mmol) was added and the resulting orange mixture was stirred
at room temperature to discolor. The precipitated white solid was fil-
tered and dried under reduced pressure; yield 0.17 g (68%); mp 188–
194°C (lit. mp 195°C [13, 15]); IR: 3048, 2953, 1785, 1709, 1615, 1591,
1438, 1395, 1376, 1298, 1264, 1176, 1164, 1070, 1034, 887, 852, 791, 618,
References
[1] Chande, M. S.; Verma, R. S.; Barve, P. A.; Khanwelkar, R. R.;
Vaidya, R. B.; Ajaikumar, K. B. Facile synthesis of active
antitubercular, cytotoxic and antibacterial agents: a Michael
addition approach. Eur. J. Med. Chem. 2005, 40, 1143–1148.
1
604 cm-1; H NMR (CDCl3): δ 2.62 (s, 3H, CH3), 2.63 (s, 3H, CH3), 2.69
(s, 3H, CH3), 6.98 (s, 1H, CH); 13C NMR (CDCl3): δ 17.7, 24.1, 25.8, 102.2,
122.8, 151.7, 157.2, 162.0, 163.2, 167.8; MS (ESI): m/z 207 [M+1]+.
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ꢀJ. Sączewski et al.: Derivatives of 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one
222ꢀ
[2] Yu, M.; Wang, J.; Tang, K.; Shi, X.; Wang, S.; Zhu, W.-M.; Zhang,
X.-H. Purification and characterization of antibacterial com-
pounds of Pseudoalteromonas flavipulchra JG1. Microbiology
(Reading, UK) 2012, 158, 835–842.
[3] Padmavathi, V.; Subbaiah, D. R. C. V.; Mahesh, K.; Lakshmi, T.
R. Synthesis and bioassay of amino-pyrazolone, amino-isox-
azolone and amino-pyrimidinone derivatives. Chem. Pharm.
Bull. 2007, 55, 1704–1709.
[4] Wierenga, W.; Evans, B. R.; Zurenko, G. E. Benzisoxazolones:
antimicrobial and antileukemic activity. J. Med. Chem. 1984,
27, 1212–1215.
[5] Braunholtz, J. T.; Freeman, P. F. H. Fungicidal isoxazolones.
GB1074803, 1967.
nonaqueous solutions based on accurate polarized partial
charges. J. Chem. Theory Comput. 2007, 3, 2011–2033.
[20] Gupta, R.; Chaudhary, R. P. Synthesis, antimicrobial and DFT
studies of novel fused thiazolopyrimidine derivatives. Hetero-
cycl. Commun. 2013, 19, 207–214.
[21] Guemues, S.; Tuerker, L. Substituent effect on the aromaticity
of 1,3-azole systems. Heterocycl. Commun. 2012, 18, 11–16.
[22] Antonov, L. Tautomerism: Methods and Theories; Wiley-VCH
Verlag GmbH & Co. KGaA: Hoboken, NJ, 2014.
[23] Katritzky, A.; Karelson, H.; Harris, P. Prototropic tautomerism of
heteroaromatic-compounds. Heterocycles 1991, 32, 329–369.
[24] Kang, Y. K.; Sook Park, H. Ab initio conformational study of
N-acetyl-L-proline-N′,N′-dimethylamide: a model for polypro-
line. Biophys. Chem. 2005, 113, 93–101.
[25] Shabanian, M.; Hajibeygi, M.; Moghanian, H.; Mohamadi, A.
Theoretical investigation on tautomerism and NBO analysis of
3-hydroxy-1,2,5-thiadiazole derivatives: solvent and substitu-
ent effects. Heterocycl. Commun. 2012, 18, 161–164.
[26] Hempel, A.; Hamid, M. A. 4,6-Dimethylisoxazolo[3,4-b]pyridin-
3(7H)-one monohydrate. Acta Crystallogr. Sect. B 1979, B35,
471–473.
[27] Katritzky, A. R.; Ramsden, Ch.; Joule, J.; Zhdankin, V. Handbook
of Heterocyclic Chemistry; Elsevier: Philadelphia, PA, 2010.
[28] Karelson, M. M.; Katritzky, A. R.; Szafran, M.; Zerner, M. C. A
theoretical treatment of solvent effects on the tautomeric equi-
libria of five-membered rings with two heteroatoms. J. Chem.
Soc. Perkin Trans. 2 1990, 1, 195–201.
[29] Yi, P. G.; Liang, Y. H.; Tang, Z. Q. Theoretical study of intermo-
lecular proton transfer reaction in isolated 5-hydroxyisoxazole-
water complexes. Chem. Phys. 2006, 322, 387–391.
[30] Beak, P.; Lee, J.; McKinnie, B. G. Methylation of protomeric
ambident nucleophiles with methyl fluorosulfonate: a regio-
specific reaction. J. Org. Chem. 1978, 43, 1367–1372.
[31] Abarghaz, M.; Kerbal, A.; Bourguignon, J.-J. Regioselective
alkylation of the exocyclic nitrogen of heterocyclic amidines
via the Mitsunobu reaction. Tetrahedron Lett. 1995, 36,
6463–6466.
[6] Das, N. P.; Mishra, P. K.; Sahu, S. Fungicidal activity of some
substituted 5-isoxazolones. Acta Cienc. Indica Chem. 2011, 37,
239–243.
[7] Ishioka, T.; Tanatani, A.; Nagasawa, K.; Hashimoto, Y. Anti-
androgens with full antagonistic activity toward human
prostate tumor LNCaP cells with mutated androgen receptor.
Bioorg. Med. Chem. Lett. 2003, 13, 2655–2658.
[8] Laughlin, S. K.; Clark, M. P.; Djung, J. F.; Golebiowski, A.;
Brugel, T. A.; Sabat, M.; Bookland, R. G.; Laufersweiler, M. J.;
VanRens, J. C.; Townes, J. A.; et al. The development of new
isoxazolone based inhibitors of tumor necrosis factor-alpha
(TNF-α) production. Bioorg. Med. Chem. Lett. 2005, 15,
2399–2403.
[9] Laufer, S. A.; Margutti, S. Isoxazolone based inhibitors of p38
MAP kinases. J. Med. Chem. 2008, 51, 2580–2584.
[10] Saczewski, J.; Hinc, K.; Obuchowski, M.; Gdaniec, M. The
tandem Mannich-electrophilic amination reaction: a versatile
platform for fluorescent probing and labeling. Chem. Eur. J.
2013, 19, 11531–11535.
[11] Saczewski, J.; Korcz, M. Synthesis and reactivity of heterocyclic
hydroxylamine-O-sulfonates. Heterocycl. Commun. 2014, 20,
133–148.
[12] Sączewski, J.; Gdaniec, M. Synthesis and molecular structure
of (Z)-1H-purin-6-ylideneaminooxysulfonic acid: a possible
secondary metabolite of adenine. Heterocycl. Commun. 2012,
18, 109–112.
[32] Novak, L.; Hornyanszky, G.; Kiraly, I.; Rohaly, J.; Kolonits, P.;
Szantay, C. Preparation of new imidacloprid analogues. Het-
erocycles 2001, 55, 45–48.
[13] Khan, M. A.; Rafala, F. K. Synthesis of isoxazolo[3,4-b]pyridin-
3(1H)-one and isoxazolo[5,4-b]-pyridin-3(2H)-one. J. Chem. Soc. [33] Saczewski, J.; Gdaniec, M. Regioselective reaction of 2-hydrox-
Perkin Trans. I 1975, 693–694.
yliminoimidazolidine-O-sulfonate with benzyl bromides. Pol. J.
Chem. 2008, 82, 2107–2113.
[34] Saczewski, J.; Hudson, A.; Laird, S.; Rybczyńska, A.;
Boblewski, K.; Lehmann, A.; Ma, D.; Maze, M.; Watts, H.;
Gdaniec, M. N-(Imidazolidin-2-ylidene)-1-arylmethanamine
oxides: synthesis, structure and pharmacological evaluation.
Arch. Pharm. 2012, 345, 33–42.
[14] Bauer, L.; Nambury, N. N. V. Synthesis of aminoisoxazolones
from α-cyano esters and hydroxylamine. J. Org. Chem. 1961,
26, 4917–4922.
[15] Hamid, M. A.; Hempel, A. 1-Acetyl-4,6-dimethylisoxazolo[3,4-
b]pyridin-3(1H)-one. Acta Crystallogr. Sect. B 1979, B35,
470–471.
[16] Alkorta, I.; Sánchez-Sanz, G.; Trujillo, C.; Elguero, J.; Clara-
munt, R. M. A theoretical study of the parent NH-benzazoles
(benzimidazoles, indazoles and benzotriazoles): geometries,
energies, acidity and basicity, NMR properties and molecular
electrostatic potentials. Arkivoc 2012, ii, 85–106.
[17] Kolodobskii, G. I.; Oastrovskii, V. A. Acid-base properties of
five-membered nitrogen-containing heterocycles. Chem. Het-
erocycl. Compounds 1988, 24, 469–480.
[35] Bracht, K.; Boubakari, Grünert, R.; Bednarski, P. J. Correlations
between the activities of 19 anti-tumor agents and the intracel-
lular glutathione concentrations in a panel of 14 human cancer
cell lines: comparisons with the National Cancer Institute data.
Anticancer Drugs 2006, 17, 41–51.
[36] Ivanova, Y. B.; Momekov, G. T.; Petrov, O. I. New heterocyclic
chalcones. Part 6. Synthesis and cytotoxic activities of 5- or
6-(3-aryl-2-propenoyl)-2(3H)-benzoxazolones. Heterocycl. Com-
mun. 2013, 19, 23–28.
[18] Spartan ’08 for Windows, Irvine, CA: Wavefunction, Inc.; www.
wavefun.com.
[37] Balows, A.; Hausler, H. J.; Herrmann, K. L.; Isenberg, H. D.;
Shadomy, H. J. Manual of Clinical Microbiology; 5th Edition.
American Society for Microbiology: Washington, DC, 1991.
[19] Marenich, A. V.; Olson, R. M.; Kelly, C. P.; Cramer, C. J.; Truhlar,
D. G. Self-consistent reaction field model for aqueous and
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[38] Baron, E. J.; Finegold, S. M. Bailey and Scott’s Diagnostic
Microbiology; 8th Edition. C.V. Mosby Co.: St. Louis, MO, 1990.
[39] Forbes, B. A.; Sahn, D. F.; Weissfeld, A. S. Bailey and Scott’s
Diagnostics Microbiology; 12th Edition. Mosby Elsevier, St.
Louis, MO, 2007.
[40] Clinical and Laboratory Standards Institute/NCCLS. Methods
for Dilution Antimicrobial Susceptibility Testing for Bacteria
that Grow Aerobically; Approved standards 7th Edition. CLSI
document M7-A7. Clinical and Laboratory Standards Institute:
Wayne, PA, 2006.
[41] Clinical and Laboratory Standards Institute/NCCLS. Methods
for Antimicrobial Susceptibility Testing of Anaerobic Bacteria;
Approved standards 7th Edition. CLSI document M11-A7. Clinical
and Laboratory Standards Institute: Wayne, PA, 2007.
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