Synthesis and antimicrobial evaluation of purine substituted N-acyl-α-carboxamides via the Ugi four-component reaction

Synthesis

Compounds 2a–x were obtained by the reaction of 2-amino-6-chloropurine with various aldehydes, acids and tert-butyl isocyanide/cyclohexyl isocyanide at 35°C in good to excellent yield (72–90%), as shown in Scheme 1. The optimization of reaction conditions was done by varying reaction temperature, ratio of reactants and solvent. TLC monitoring of the progress of the reaction was done to observe complete consumption of 2-amino-6-chloropurine; the reaction was stopped thereafter to avoid decomposition of the product. The reaction was performed at 25°C and 35°C wherein the time for completion was 10–11 h and 6–8 h, respectively. The relative ratio of reactants was varied in the range of 1.0–1.5 to maximize the yield and purity of the desired compound. Best results were obtained using 1.1 equivalents of both amine and acid relative to 1.0 equivalent of aldehyde and isocyanide. For similar set of reaction conditions, the reaction was carried out in various solvents including dichloromethane, DMSO, methanol and DMF. Methanol as the reaction medium gave best results with easy workup, isolation, good yield and purity of the product.

Scheme 1

Synthesis of purine substituted N-acyl-α-carboxamides 2a–x via Ugi four-component reaction.

The structural confirmation was done by spectroscopic analysis. The characteristic spectral data of the representative compound 2a with a cyclohexyl substituent show peaks in the IR between 3292 and 3227 cm^-1 due to NH stretching vibration and between 1681 and 1688 cm^-1 due to C=O stretching. The ¹H NMR spectrum shows the most significant singlet peak at δ 3.36 corresponding to CH proton of the cyclohexyl substituent. For all compounds 2a–x, a peak at δ 6.22 ppm is observed due to Ar-CH moiety, the phenyl rings show a multiplet in the aromatic region of the spectra at δ 7.70–7.98, and a singlet for NH (D₂ O exchangeable) is observed at δ 8.99 and 9.20. The ¹³C NMR spectra show the presence of characteristically distinct resonances which appear at δ 51.7 and 58.7, corresponding to the carbon atom of the cyclohexyl substituent and Ar-C-, respectively. These results are further supported by the mass spectra and elemental analysis.

Antimicrobial activity

All compounds 2a–x were evaluated for their in vitro antimicrobial activity against Escherichia coli (clinical strain), Pseudomonas aeruginosa (PA01), Staphylococcus aureus (MRSA), and Staphylococcus epidermidis (clinical strain). The tube dilution method was followed as per the National Committee for Clinical Laboratory Standards guidelines [18, 19] and minimum inhibitory concentration (MIC) was calculated. All MIC experiments were performed in triplicates (n=3) for each test organism. The results are expressed as mean±standard deviation (n=3) for each sample and are presented in Table 1. Ampicillin trihydrate (CAS 7177-48-2) was used as a standard reference antibacterial drug. DMSO (dimethyl sulfoxide) was used as a solvent control with no inhibition activity.

A preliminary screening was done of the synthesized compounds 2a–x and parent pharmacophore 1 for determining the inhibition zone for antimicrobial activity against all Gram positive and Gram negative strains, as shown in Table 1. Encouraged by the results, a detailed study of the antimicrobial activity, as shown in Table 2, was carried out in which relative to the parent pharmacophore 1 all compounds 2a–x showed improved bioactivity against all Gram positive and Gram negative strains. Compounds 2h, 2l, 2t and 2x show good antimicrobial potency for Gram positive strain Pseudomonas aeruginosa as a close lead when compared with the standard drug ampicillin trihydrate (25 μg/mL), whereas 2b, 2f, 2n and 2r show moderate activity for the same bacteria. For the same category of Gram negative bacteria Escherichia coli 2h, 2l, 2t and 2x display good potency with respect to ampicillin trihydrate (25 μg/mL) and compounds 2h and 2l show moderate activity. For the Gram positive category of organisms, compounds 2t and 2x show marginal bactericidal activity for Staphylococcus aureus in comparison with the standard drug (125 μg/mL). Compounds 2q, 2w and 2x have good potency for Staphylococcus epidermidis, whereas for the same bacteria, the compounds 2k and 2t are moderately potent in comparison with ampicillin trihydrate (75 μg/mL).

A glance into the structure-activity relationship (SAR) of the antimicrobial screening results suggests that the compounds with R¹ as p-methoxy and p-methyl groups, R³ as the tert-butyl and cyclohexyl groups and R² as the alkyl group at the para-position tends to attenuate antimicrobial potency in Gram negative bacteria Pseudomonas aeruginosa and Escherichia coli. Against Staphylococcus aureus, compounds containing R¹ as p-methoxy and p-methyl along with R² as an alkyl chain and R³ as the tert-butyl group display good antimicrobial activity, whereas for the same organism a moderate activity has been displayed where R³ is the cyclohexyl group. For Staphylococcus epidermidis, compounds containing R³ as the tert-butyl group and R¹ as p-chloro and p-methyl groups, and R² as an alkyl chain, displayed good antimicrobial activity. For the same organism, moderate activity is displayed in compounds where R³ is cyclohexyl, R² is an alkyl group and R¹ is the p-methoxy substituent.

General procedure for N-acyl-α-carboxamides

To a dry magnetically stirred 10-mL round-bottom flask in oil bath was added 2-amino-6-chloropurine (1, 1.1 mmol), freshly distilled methanol (5.0 mL) and a substituted aromatic benzaldehyde (1.0 mmol), and the mixture was stirred for 2 h at 35°C. To this reaction mixture was further added a substituted carboxylic acid (1.1 mmol) and an alkyl isocyanide (1.0 mmol), and the stirring was continued for an additional 6–8 h at 35°C. At the completion of the reaction, as monitored by TLC, water (5 mL) was added, and the mixture was further stirred for 10 min, resulting in separation of the solid product. The aqueous layer was decanted and the product was dissolved in ethyl acetate (15 mL). The organic solution was washed with water (2×5 mL), dried over sodium sulfate and concentrated. The residue of the crude product was crystallized from hexanes/ethyl acetate (8:2).

Preliminary determination of the antimicrobial activity

For primary screening, each compound and the standard drug were diluted with DMSO to obtain a solution of 500 μg/mL concentration. Individual wells were made in a sterile agar plate and inoculated with compound solution of a precise concentration of 500 μg/mL/well. The antibacterial ampicillin trihydrate 200 μg/mL/well was also placed on the agar culture plate that had been inoculated earlier with the microorganism. The plates were incubated at 37°C and the diameter of the microbial growth inhibition was measured after 24 h, as shown in Table 1.

Determination of the minimal inhibitory concentration (MIC)

All compounds 2a–x were evaluated for their in vitro antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis. Compound dilutions (ranging from 25 μg/mL to 800 μg/mL) were prepared in nutrition media (Luria broth) using the serial double dilution protocol. For secondary screening serial dilutions were prepared. The compounds found active at the primary screening stage were freshly diluted to obtain 500, 250, 200, 150, 100, 75 and 50 μg/mL concentrations for secondary screening of the compounds. To check the antimicrobial activity of these compounds, mid-log phase culture was used for inoculation. A 1% culture was added to the media containing different concentrations of the compounds under observation. After inoculation, incubation was done at 36°C, for 24 h. Growth inhibition was visually checked by observing the tubes for presence or absence of turbidity due to microbial growth. A set of tubes containing only nutrition medium and solvent control were maintained under identical conditions to substantiate that the solvent has no influence on the strain growth. Further confirmation of the results was done by streaking on Luria agar plates. Lowest concentration of the compound showing complete inhibition (no growth) as detected by the naked eye was taken as MIC.