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

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).

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 2a–g 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.

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].

pK_a, 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 pK_a value is 6.9 (Figure 1), which indicates that it is more acidic than benzotriazole (pK_a = 8.38) [16], 1,2,3-triazole (pK_a = 9.26), 1,2,4-triazole (pK_a = 10.04) and significantly less acidic than tetrazole (pK_a = 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.

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].

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.

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.

Antibacterial activity

The results of in vitro antibacterial activity of compounds 1, 2a,b, 2d–i 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.

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.

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-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].