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BY-NC-ND 3.0 license Open Access Published by De Gruyter July 17, 2014

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

  • Jarosław Sączewski EMAIL logo , Anna Kędzia and Aleksandra Jalińska

Abstract

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/6-31G* and ωB97X-D/6-31G* density functional methods and SM8 (H2 O, DMF) solvation models. Compound 1, which is a weak acid with a pKa of 6.9, undergoes regioselective alkylation and sulfonylation under basic reaction conditions to give a series of N1-substituted products 2ai. Later compounds were evaluated in vitro for antibacterial activity with the use of 68 strains of aerobic and anaerobic bacteria, including 12 reference strains.

Introduction

It is well known that substituted 2,1-isoxazol-3-ones possess interesting pharmacological properties such as antibacterial [1–4], antitubercular [1], fungicidal [5, 6], antileukemic [4], antioxidant [3] and antiandrogenic [7] activities. They also act as inhibitors of tumor necrosis factor-α (TNF-α) [8] and inhibitors of p38 MAP kinases [9].

Recently, we have used 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) as a substrate for the fluorogenic tandem Mannich-electrophilic amination reaction with formaldehyde and secondary amines, which give rise to the formation of a new class of photostable fluorescent dyes with a [1, 2, 4]triazolo[3,4-b]pyridin-2-ium core structure [10–12].

In the present work, we describe the N-alkylation and N-sulfonylation reactions of 1 and antibacterial properties of the newly obtained isoxazolo[3,4-b]pyridin-3(1H)-one derivatives 2ai. To understand the observed regioselectivity of the above reactions, theoretical studies on tautomerism of 1 and electronic structure of the most stable tautomers 1H-oxo (1-A), 7H-oxo (1-B) have been carried out using the B3LYP/6-31G* and ωB97X-D/6-31G* density functional methods.

Results and discussion

Synthesis

4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) was synthesized according to a previously described procedure [13] with minor modification. As shown in Scheme 1, the condensation of N-hydroxy-3-(hydroxyamino)-3-iminopropanamide [14] with acetylacetone in the presence of piperidine resulted in a yellow-colored solution of salt A from which, upon neutralization with aqueous 10% HCl, the solid product 1 precipitated in good yield.

Scheme 1 Synthesis of 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1).
Scheme 1

Synthesis of 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1).

Previously, the exploration of chemical reactivity of compound 1 has revealed that acetylation and benzoylation take place exclusively at the N1 nitrogen atom [13, 15]. We have now found evidence that alkylation and sulfonylation of 1 also proceed regioselectively at the N1 nitrogen atom (Scheme 2). Due to poor solubility 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 2ag were isolated by means of a preparative, centrifugally accelerated, radial, thin-layer chromatography (chromatotron) in 62–84% yield. By contrast, the methylsulfonyl derivative 2h was obtained by reacting 1 with methanesulfonyl chloride in aqueous 5% NaOH solution, followed by chromatographic purification on silica gel. The acetyl derivative 2i, which was required for biological tests, was synthesized according to the literature procedure [15].

Scheme 2 Alkylation, acetylation and sulfonylation of 1.
Scheme 2

Alkylation, acetylation and sulfonylation of 1.

The regioselectivity of the above reactions, that is, formation of N1-substituted products, has been confirmed by NMR measurements (lack of NOE between substituents at the N1 position and protons of 6-CH3 group) and the previously reported single crystal X-ray diffraction analysis of compound 2i [15].

pKa, prototropic tautomerism and electronic structure of 1

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.

Figure 1 Potentiometric titration of compound 1 in water with use of 0.1 N NaOH.
Figure 1

Potentiometric titration of compound 1 in water with use of 0.1 N NaOH.

To obtain insight into the chemical properties of the isoxazolo[3,4-b]pyridin-3-one ring system, we have theoretically 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 (H2 O, DMF) solvation 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 (H2 O 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 distribution, in solvents such as H2 O or DMF an estimated ratio of 1-A to 1-B may be 1:20, which is in agreement with the following principles: (i) in polar solvents the dynamic equilibrium favors the more polar structure [22–25] and (ii) the most polar tautomer of a given compound is generally the one to be found in the solid state [23, 26]. According to our calculations, the hydroxy tautomer 1-C is generally the least stable which, in turn, is in accordance with the previous examinations of tautomeric equilibrium for simple 5-hydroxyisoxazoles [27–29].

Figure 2 Relative 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 (H2 O, DMF) solvation models [18, 19].
Figure 2

Relative enthalpies of formation Δ (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 (H2 O, DMF) solvation models [18, 19].

As shown in Figure 3, the alkylation reactions of both the heterocyclic amidine derivatives D, described as 1,3-dinucleophiles [30] or ‘protomeric ambident nucleophiles’ [31] and heterocyclic guanidines E, such as 2-aryliminoimidazolidines [32] or O-substituted imidazolidin-2-one oximes [33, 34], usually occur at a pyridine-type sp2 hybridized endo- or exo-cyclic nitrogen atom.

Figure 3 Alkylation sites at heterocyclic amidines D and guanidines E.
Figure 3

Alkylation sites at heterocyclic amidines D and guanidines E.

To identify reactive sites at the amidine moiety of compound 1, we have performed quantum-chemical calculations and molecular modeling, through which the electronic properties of the annular tautomers 1-A and 1-B as well as the anion F have been revealed. As shown in Figure 4, in all cases (1-A, 1-B and F) the magnitude of the calculated charges are higher at the N7 nitrogen atoms. By contrast, the HOMO orbital density [√(e/au3)] mapped on isodensity surface (0.002 e/au3), corresponding to the molecular size and shape, is greater at the vicinity of the isoxazolone N1 nitrogen atom than at the pyridine N7 atom. These results indicate that the orbital-controlled reactions of 1 take place exclusively because no N7-alkylation or acylation products resulting from electrostatically-controlled reaction were observed.

Figure 4 The 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.
Figure 4

The 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.

Antibacterial activity

The results of in vitro antibacterial activity of compounds 1, 2a,b, 2di against anaerobic bacterial strains and compounds 1, 2g, 2f, 2i against aerobic bacterial strains are presented in Tables 1 and 2, respectively. The tested compounds show moderate potencies against anaerobic bacteria as they inhibit growth of 15–47% strains at concentrations in the range ≤6.2–100 μg/mL. The most potent methyl propanoate 2g displays broad activity against 47% of bacterial strains, with minimal inhibitory concentration (MIC) values in the range 25–100 μg/mL. The unsubstituted 4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) inhibits growth of 15 strains (44%) at concentrations in the range 25–100 μg/mL, whereas the remaining seven congeners exhibit relatively weaker biological activities: the methyl derivative 2a is active against 13 bacterial strains (38%, MIC 50–100 μg/mL), 2b, 2f and 2h show potent activity against 12 strains (35%, MIC ≤6.2–100 μg/mL) and compounds 2i and 2e inhibit growth of 11 (32%, MIC ≤6.2–50 μg/mL) and seven strains (21%, 25–100 μg/mL), respectively. The benzyl (2d) derivative proved to be the least active, whereas the butyl congener (2c) does not exert any antibacterial effect, probably due to its poor solubility. All tested compounds are more active against Gram-positive cocci (12–32% susceptible strains) than Gram-positive rods (3–15% susceptible strains). The most susceptible genera among Gram-positive cocci and Gram-positive rods are Parvimonas micra and Bifidobacterium breve, respectively. Among anaerobes, the following strains exhibit high resistance towards all tested compounds (MIC ≥200 μg/mL): Parabacteroides distasonis (1), Prevotella bivia (1), Prevotella buccalis (1), Prevotella intermedia (2), Fusobacterium nucleatum (2), Fusobacterium necrophorum (2), Parabacteroides distasonis, ATCC 8503 and Fusobacterium nucleatum ATCC 25586.

Table 1

In vitro antibacterial activity of compounds 1, 2a,b, 2di against anaerobic bacterial strains.

Bacteria (number of strains)Minimum inhibitory concentration (MIC, μg mL-1)
Metronidazole12a2b2d
Gram-positive cocci:
 Finegoldia magna (3)≤0.4200(2), 50200, 100, 50100, 12.5, <6.2≥200
 Parvimonas micra (2)≤0.410010050, <6.2200, 100
 Peptostreptococcus anaerobius (1)1.62550100100
Gram-positive rods:
 Bifidobacterium breve (2)50, 25100≥200≥200100, 50
 Propionibacterium acnes (1)>1005050100≥200
 Propionibacterium granulosum (2)≥100100200, 100≥200≥200
Gram-negative rods:
 Bacteroides fragilis (1)<0.4100>200>200>200
 Bacteroides uniformis (1)<0.450>200>200>200
 Bacteroides ureolyticus (1)1.650200>200>200
 Bacteroides vulgatus (1)<0.4>200100100100
 Prevotella levii (1)<0.4>200100200>200
 Prevotella loescheii (1)<0.4>200>200200>200
 Porphyromonas asaccharolytica (1)<0.45010050200
 Porphyromonas gingivalis (1)<0.450100100200
Reference strains:
 Bacteroides fragilis ATCC 25285<0.4200>200>200>200
 Finegoldia magna ATCC 29328<0.420010050200
 Peptostreptococcus anaerobius ATCC 27331<0.4100100100200
 Bifidobacterium breve ATCC 1570050200200≥200200
 Parabacteroides distasonis ATCC 8503<0.4>200>200>200>200
 Fusobacterium nucleatum ATCC 25586<0.4>200>200>200>200
2e2f2g2h2i
Gram-positive cocci:
 Finegoldia magna (3)≥200≥200≥200, 100, 25200, 100(2)50, 12.5, <6.2
 Parvimonas micra (2)100, 2550, 2550, 2525, 12.525, 12.5
Peptostreptococcus anaerobius (1)1001005010025
Gram-positive rods:
 Bifidobacterium breve (2)200, 100100100, 50100, 5012.5, <6.2
 Propionibacterium acnes (1)100100≥200100100
 Propionibacterium granulosum (2)200, 100100≥200100≥200
Gram-negative rods:
 Bacteroides fragilis (1)>200>20050>200>200
 Bacteroides uniformis (1)>20010025>200>200
 Bacteroides ureolyticus (1)>200>200≥200>200>200
 Bacteroides vulgatus (1)10020050100100
 Prevotella levii (1)20010050200>200
 Prevotella loescheii (1)>200≥200>200200200
 Porphyromonas asaccharolytica (1)200≥20050>200200
 Porphyromonas gingivalis (1)>200>200>200>200>200
Reference strains:
 Bacteroides fragilis ATCC 25285>200>200100>200200
 Finegoldia magna ATCC 29328>200100100200200
 Peptostreptococcus anaerobius ATCC 27331200100100100100
 Bifidobacterium breve ATCC 15700200≥200100100200
 Parabacteroides distasonis ATCC 8503>200>200>200>200>200
 Fusobacterium nucleatum ATCC 25586>200>200>200>200>200
Table 2

In vitro antibacterial activity of compounds 1, 2g, 2f, 2i against aerobic bacterial strains.

Bacteria (number of strains)Minimum inhibitory concentration (MIC, μg mL-1)
Amikacin12g2f2i
Gram-positive cocci:
 Staphylococcus aureus (3)≤6.2≥200100100≥200
 Staphylococcus aureus (MRSA) (3)≥200≥200100≥200(2), 100≥200
 Staphylococcus epidermidis (2)≤6.2>200, 50100, 50100, 50>200, 25
 Streptococcus pyogenes (1)50100100100>200
 Streptococcus anginosus (2)100, 50100, 5050
 Enterococcus faecalis (2)50, 2550100100200, 50
Gram-positive rods:
 Corynebacterium ulcerans (2)50, 25100100100≥200, 100
 Corynebacterium xerosis (1)501002550100
Gram-negative rods:
 Escherichia coli (2)12.5, ≤6.2100≥200≥200≥200
 Acinetobacter baumannii (1)<6.2>200>200100>200
 Citrobacter freundii (2)≤6.2200>200, 100200, 100≥200
 Klebsiella pneumoniae (2)≤6.2≥200100≥200, 100≥200
 Serratia marcescens (1)<6.250100100>200
 Pseudomonas aeruginosa (2)12.5≥200>200, 100>200, 100≥200
 Pseudomonas stutzeri (2)≤6.2≥200100100≥200
Reference strains:
 Staphylococcus aureus ATCC 25923<6.2>200>200>200>200
 Enterococcus faecalis ATCC 2921225100>200>200>200
 Corynebacterium xerosis ATCC 37325200100100100
 Klebsiella pneumoniae ATCC 13883<6.2>200100>200>200
 Acinetobacter baumannii ATCC 19606<6.2>200>200≥200>200
 Escherichia coli ATCC 25922<6.2200>200>200>200

Aerobic bacterial strains exhibit susceptibility to only four tested compounds at the concentrations in the range of 25–100 μg/mL. The most potent derivative 2g exhibits activity against 22 (65%) bacterial strains, with MIC values in the range of 50–100 μg/mL. The unsubstituted and hydroxyethyl congeners 1 and 2f inhibit growth of 13 (38%) and 20 strains (59%), respectively, at concentrations in the range of 25–100 μg/mL.

Gram-positive aerobic cocci and rods proved to be more susceptible than Gram-negative rods to the four tested compounds. Among Gram-positive bacteria the Staphylococcus 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 susceptible to compounds 2g and 2f. Generally, the Gram-negative rods are most resistant to the genera tested as growths of only a few strains of Citrobacter freundii, Klebsiella pneumoniae, 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.

Experimental

All chemicals were purchased from commercial sources and used without further purification. Silica gel chromatography was performed with use of silica gel 60 PF254 containing gypsum for preparative layer chromatography and chromatotron. 1H 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.

4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1)

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. After 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, 1289, 1051, 1030, 980, 938, 823, 784 cm-1; 1H NMR (DMSO-d6): δ 2.38 (s, 3H, CH3), 2.45 (s, 3H, CH3), 6.38 (s, 1H, CH), 13.00 (bs, 1H, NH); 13C 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]-.

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 indicated below. After 12 h the mixture was concentrated under reduced pressure and the residue was treated with water (5 mL) and dichloromethane (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.

1,4,6-Trimethylisoxazolo[3,4-b]pyridin-3(1H)-one (2a)

4,6-Dimethylisoxazolo[3,4-b]pyridin-3(1H)-one (1) (0.3 g, 1.83 mmol), methyl iodide (0.34 mL, 5.49 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 0.16 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 C9 H10 N2 O2: 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, 931, 845, 806, 737, 702, 613, 548, 501 cm-1; 1H 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 C10 H12 N2 O2: C, 62.49; H, 6.29; N, 14.57. Found: C, 62.57; H, 6.33; N, 14.34.

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, 1283, 1173, 1024, 802, 702, 612 cm-1; 1H 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 C12 H16 N2 O2: C, 65.43; H, 7.32; N, 12.72. Found: C, 65.64; H, 7.19; N, 12.48.

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 C15 H14 N2 O2: C, 70.85; H, 5.55; N, 11.02. Found: C, 70.73; H, 5.76; N, 10.81.

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, 1170, 1161, 1068, 1054, 1032, 983, 962, 912, 837, 806, 743, 720 cm-1; 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 C17 H18 N2 O4: C, 64.96; H, 5.77; N, 8.91. Found: C, 64.75; H, 6.16; N, 8.62.

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 C10 H12 N2 O3: 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, 1269, 1248, 1198, 1173, 1032, 1017, 803 cm-1; 1H 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 C12 H14 N2 O4: 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 C9 H10 N2 O4 S: C, 44.62; H, 4.16; N, 11.56. Found: C, 44.37; H, 4.35; N, 11.47.

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 filtered 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, 604 cm-1; 1H 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]+.

Antibacterial activity tests

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 anaerobes belonged to the following genera: Finegoldia magna (3), Parvimonas micra (2), Peptostreptococcus anaerobius (1), Bifidobacterium breve (2), Propionibacterium acnes (1), Propionibacterium granulosum (2), Bacteroides fragilis (1), Bacteroides uniformis (1), Bacteroides ureolyticus (1), Bacteroides vulgatus (1), Parabacteroides distasonis (1), Prevotella bivia (1), Prevotella buccalis (1), Prevotella intermedia (2), Prevotella levii (1), Prevotella loescheii (1), Porphyromonas asaccharolytica (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: Staphylococcus aureus (3), Staphylococcus aureus methicillin resistant (MRSA) (3), Staphylococcus epidermidis (2), Streptococcus pyogenes (1), Streptococcus anginosus (2), Enterococcus faecalis (2), Corynebacterium ulcerans (2), Corynebacterium xerosis (1), Escherichia coli (2), Acinetobacter baumannii (1), Citrobacter freundii (2), Klebsiella pneumoniae (2), Serratia marcescens (1), Pseudomonas aeruginosa (2), Pseudomonas stutzeri (2) and six reference strains: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Corynebacterium xerosis ATCC 373, Klebsiella pneumoniae ATCC 13883, Acinetobacter 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 experiments, the agar dilution technique with Mueller-Hinton agar was used [39, 40]. The derivatives were dissolved in 1 mL of DMSO immediately 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].


Corresponding author: Jarosław Sączewski, Department of Organic Chemistry, Medical University of Gdańsk, Al. Gen. J. Hallera 107, 80-416, Gdańsk, Poland, email:

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.

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.Search in Google Scholar

[2] Yu, M.; Wang, J.; Tang, K.; Shi, X.; Wang, S.; Zhu, W.-M.; Zhang, X.-H. Purification and characterization of antibacterial compounds of Pseudoalteromonasflavipulchra JG1. Microbiology (Reading, UK)2012, 158, 835–842.Search in Google Scholar

[3] Padmavathi, V.; Subbaiah, D. R. C. V.; Mahesh, K.; Lakshmi, T. R. Synthesis and bioassay of amino-pyrazolone, amino-isoxazolone and amino-pyrimidinone derivatives. Chem. Pharm. Bull.2007, 55, 1704–1709.Search in Google Scholar

[4] Wierenga, W.; Evans, B. R.; Zurenko, G. E. Benzisoxazolones: antimicrobial and antileukemic activity. J. Med. Chem.1984, 27, 1212–1215.Search in Google Scholar

[5] Braunholtz, J. T.; Freeman, P. F. H. Fungicidal isoxazolones. GB1074803, 1967.Search in Google Scholar

[6] Das, N. P.; Mishra, P. K.; Sahu, S. Fungicidal activity of some substituted 5-isoxazolones. Acta Cienc. Indica Chem.2011, 37, 239–243.Search in Google Scholar

[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.Search in Google Scholar

[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.Search in Google Scholar

[9] Laufer, S. A.; Margutti, S. Isoxazolone based inhibitors of p38 MAP kinases. J. Med. Chem.2008, 51, 2580–2584.Search in Google Scholar

[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.Search in Google Scholar

[11] Saczewski, J.; Korcz, M. Synthesis and reactivity of heterocyclic hydroxylamine-O-sulfonates. Heterocycl. Commun.2014, 20, 133–148.Search in Google Scholar

[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.Search in Google Scholar

[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. Perkin Trans. I1975, 693–694.10.1039/p19750000693Search in Google Scholar

[14] Bauer, L.; Nambury, N. N. V. Synthesis of aminoisoxazolones from α-cyano esters and hydroxylamine. J. Org. Chem.1961, 26, 4917–4922.Search in Google Scholar

[15] Hamid, M. A.; Hempel, A. 1-Acetyl-4,6-dimethylisoxazolo[3,4-b]pyridin-3(1H)-one. Acta Crystallogr. Sect. B1979, B35, 470–471.10.1107/S0567740879003848Search in Google Scholar

[16] Alkorta, I.; Sánchez-Sanz, G.; Trujillo, C.; Elguero, J.; Claramunt, 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. Arkivoc2012, ii, 85–106.10.3998/ark.5550190.0013.209Search in Google Scholar

[17] Kolodobskii, G. I.; Oastrovskii, V. A. Acid-base properties of five-membered nitrogen-containing heterocycles. Chem. Heterocycl. Compounds1988, 24, 469–480.Search in Google Scholar

[18] Spartan ’08 for Windows, Irvine, CA: Wavefunction, Inc.; www.wavefun.com.Search in Google Scholar

[19] Marenich, A. V.; Olson, R. M.; Kelly, C. P.; Cramer, C. J.; Truhlar, D. G. Self-consistent reaction field model for aqueous and nonaqueous solutions based on accurate polarized partial charges. J. Chem. Theory Comput.2007, 3, 2011–2033.Search in Google Scholar

[20] Gupta, R.; Chaudhary, R. P. Synthesis, antimicrobial and DFT studies of novel fused thiazolopyrimidine derivatives. Heterocycl. Commun.2013, 19, 207–214.Search in Google Scholar

[21] Guemues, S.; Tuerker, L. Substituent effect on the aromaticity of 1,3-azole systems. Heterocycl. Commun.2012, 18, 11–16.Search in Google Scholar

[22] Antonov, L. Tautomerism: Methods and Theories; Wiley-VCH Verlag GmbH & Co. KGaA: Hoboken, NJ, 2014.Search in Google Scholar

[23] Katritzky, A.; Karelson, H.; Harris, P. Prototropic tautomerism of heteroaromatic-compounds. Heterocycles1991, 32, 329–369.Search in Google Scholar

[24] Kang, Y. K.; Sook Park, H. Ab initio conformational study of N-acetyl-L-proline-N′,N′-dimethylamide: a model for polyproline. Biophys. Chem.2005, 113, 93–101.Search in Google Scholar

[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 substituent effects. Heterocycl. Commun.2012, 18, 161–164.Search in Google Scholar

[26] Hempel, A.; Hamid, M. A. 4,6-Dimethylisoxazolo[3,4-b]pyridin-3(7H)-one monohydrate. Acta Crystallogr. Sect. B1979, B35, 471–473.10.1107/S056774087900385XSearch in Google Scholar

[27] Katritzky, A. R.; Ramsden, Ch.; Joule, J.; Zhdankin, V. Handbook of Heterocyclic Chemistry; Elsevier: Philadelphia, PA, 2010.Search in Google Scholar

[28] Karelson, M. M.; Katritzky, A. R.; Szafran, M.; Zerner, M. C. A theoretical treatment of solvent effects on the tautomeric equilibria of five-membered rings with two heteroatoms. J. Chem. Soc. Perkin Trans. 21990, 1, 195–201.Search in Google Scholar

[29] Yi, P. G.; Liang, Y. H.; Tang, Z. Q. Theoretical study of intermolecular proton transfer reaction in isolated 5-hydroxyisoxazole-water complexes. Chem. Phys.2006, 322, 387–391.Search in Google Scholar

[30] Beak, P.; Lee, J.; McKinnie, B. G. Methylation of protomeric ambident nucleophiles with methyl fluorosulfonate: a regiospecific reaction. J. Org. Chem.1978, 43, 1367–1372.Search in Google Scholar

[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.Search in Google Scholar

[32] Novak, L.; Hornyanszky, G.; Kiraly, I.; Rohaly, J.; Kolonits, P.; Szantay, C. Preparation of new imidacloprid analogues. Heterocycles2001, 55, 45–48.Search in Google Scholar

[33] Saczewski, J.; Gdaniec, M. Regioselective reaction of 2-hydroxyliminoimidazolidine-O-sulfonate with benzyl bromides. Pol. J. Chem.2008, 82, 2107–2113.Search in Google Scholar

[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.Search in Google Scholar

[35] Bracht, K.; Boubakari, Grünert, R.; Bednarski, P. J. Correlations between the activities of 19 anti-tumor agents and the intracellular glutathione concentrations in a panel of 14 human cancer cell lines: comparisons with the National Cancer Institute data. Anticancer Drugs2006, 17, 41–51.Search in Google Scholar

[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. Commun.2013, 19, 23–28.Search in Google Scholar

[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.Search in Google Scholar

[38] Baron, E. J.; Finegold, S. M. Bailey and Scott’s Diagnostic Microbiology; 8th Edition. C.V. Mosby Co.: St. Louis, MO, 1990.Search in Google Scholar

[39] Forbes, B. A.; Sahn, D. F.; Weissfeld, A. S. Bailey and Scott’s Diagnostics Microbiology; 12th Edition. Mosby Elsevier, St. Louis, MO, 2007.Search in Google Scholar

[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.Search in Google Scholar

[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.Search in Google Scholar

Received: 2014-6-19
Accepted: 2014-6-22
Published Online: 2014-7-17
Published in Print: 2014-8-1

©2014 by De Gruyter

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