INTRODUCTION:

The drug discovery efforts for searching new antibacterial are mainly focusing on identifying molecules that block different targets and thus can inhibit the growth of bacteria. Analysis of microbial genomes has revealed an abundance of novel and potentially useful targets. But many bacterial enzymes have been well characterized and hold promise for the discovery of new antibacterial drugs. But on the other side the promiscuity become a big problem because in living cells, where a pool of proteins can easily bind to a great variety of ligands, including ions, small organic and inorganic molecules and macromolecules and the selectivity become more difficult to develop highly specific compounds against a particular target.

In the course of the research into potential antibacterial agents we have turned our attention to small heterocyclic ring systems which are richest sources for structural diversification, which in addition to often exhibiting biological activity and serves as rigid scaffold for further display of functionalities. Nitrogen-containing five membered rings are interesting synthetic targets as they are the basis of many natural and bioactive products ¹. Pyrrolidines are such an important class of bioactive molecules which often exhibiting wide range of biological activities such as anti-parkinsonian, anticonvulsant, antipsychotic², anti-inflammatory³, anti tumor⁴, antiviral⁵, antibacterial⁶, antimalarial⁷ and antifungal⁸ activities.

The increase in drug-resistant bacterial strain isolates during recent years presents a therapeutic challenge to physicians selecting anti-microbial agents. Thus, the development of new agents with potent antibacterial activities and fewer adverse effects is urgently desired. During this process we were seeking a rapid and efficient method for the synthesis of bicyclic pyrrolidines. Literature survey shows that many protocols have been developed for the synthesis of bicyclic pyrrolidines. Different protective reagents have been utilized such as fluorous alcohol protected amino acids ⁹, perfluoro alkyl sulfonyl protected hydroxy benzaldehydes ¹⁰have been employed in the synthesis of pyrrolidines. Recently it was reported in the literature that a polymer or resin supported scavengers were employed to remove un-reacted starting materials in the synthesis of bicyclic pyrrolidines ¹¹.

In the present communication, we wish to report a simple, one step microwave-mediated synthesis of some bicyclic pyrrolidines and their anti-bacterial activity.

EXPERIMENTAL:

Melting points were determined in open capillaries and are uncorrected. IR spectra (KBr) were recorded on a Perkin-Elmer 1800 (FTIR) spectrometer. ¹H NMR spectra (DMSO-d₆) were taken on a Bruker spectrometer (300 MHz) using TMS as internal standard and chemical shift are expressed in d ppm. The purity of the compounds determined by using Schimadzu RP HPLC system.

General procedure for preparation of compound 1a

A stoichiometric mixture of 0.01mol of maleimide, 0.012mol of benzaldehyde and 0.01mol of L-phenylalanine methyl ester were mixed in 5 ml of dimethylformamide and irradiated at 150W/160°C for 10 minutes in a monomode microwave system. The contents were cooled and poured onto crushed ice, filtered and washed with water. The resultant solid was crystallized

SCHEME 1

SCHEME 2

from ethanol to give compound 1a, m.p. 225-227°C, Yield 73%. IR: 1665 (C=O); 3350 (NH). ¹H NMR (DMSO-d₆): 7.65-8.18 (m, 10H), 3.11 (m, 2H), 3.85 (s, 3H), 3.27 (d, 1H), 3.55 (dd, 1H), 4.1 (m, 1H).

A similar method was adopted for the synthesis of compounds 1b-1i.

RESULTS AND DISCUSSION:

Azomethine ylide 1, 3-dipolar cycloaddition methodologies were used for the synthesis of these compounds without employing any protected reagents and polymer scavengers for isolation of the product. Bicyclic Pyrrolidines were synthesized using amino acid ester, aromatic aldehyde and maleimide in one pot fashion by using microwave irradiation under controlled temperature and power. The reaction mixture was irradiated at 150W/160°C for 10 minutes. The reaction proceeds via condensation of an amine and aldehyde to generate a reactive azomethine ylide, which then undergoes a [3+2] cycloaddition reaction with electron poor diene (maleimide) to generate the corresponding bicyclic pyrrolidines as a single diastereomer and the two ring-fused hydrogen atoms are cis to the R¹ and trans to the phenyl group. The typical crude purity of these compounds was between 70 to 85% in fairly good yields. The reaction sequence and suggested mechanism leading to bicyclic pyrrolidines has been out lined in the Scheme-1 and 2.

Anti-bacterial activity

Synthesized compounds were evaluated for their in-vitro antibacterial activity against Gram-positive organisms, Staphylococcus aureus (ATCC 6538), Bacillus subtilis (ATCC 6633) and Gram-negative organisms, Escherichia coli (ATCC 8739) and Pseudomonas aeruginosa (ATCC 25619) by the conventional agar dilution procedures¹² and compared with that of ciprofloxacin. Drugs were dissolved in dimethylsulfoxide (DMSO; 1 ml) and the solution was diluted with water (9 ml). Further progressive double dilutions with melted Muller–Hinton agar were performed to obtain the required concentrations.

The agar dilution method was performed using Muller-Hinton agar (Hi-Media) medium. Suspension of each microorganism was prepared and applied to plates with serially diluted compounds (DMF, solvent control) to be tested and incubated (approx. 20 h) at 37 °C. The minimum inhibitory concentration (MIC) was considered to be the lowest concentration that was completely inhibited growth on agar plates. To ensure that the solvent had no effect on bacterial growth, a control test was performed with test medium supplemented with DMSO at the same dilutions as used in the experiment. The MIC of these compounds is listed in table 2. The tested compounds showed antibacterial activity with a range of MICs between 12.5 to 50 mg/mL (Table 2). The results

showed that the synthesized compounds possessed moderate antibacterial activity.

Spectral data of the compounds:

1b: m.p. 183-185°C, Yield 70%. IR: 1655 (C=O); 3350 (NH). ¹H NMR (DMSO-d₆): 7.27-8.04 (m, 10H), 3.14 (m,

Table-1

Compound	R¹	R²	R³	R⁴	Mol. Form	Mol. Wt
1a	C₆H₅CH₂	H	H	H	C₂₁H₂₁N₂O₄	364.39
1b	C₆H₅CH₂	H	H	C₂H₅	C₂₃H₂₄N₂O₄	392.45
1c	C₆H₅CH₂	H	H	C₆H₅	C₂₇H₂₄N₂O₄	440.49
1d	C₆H₅CH₂	H	H	C₆H₅CH₂	C₂₈H₂₆N₂O₄	454.49
1e	C₆H₅CH₂	H	H	C₆H₅CH₂CH₂	C₂₉H₂₈N₂O₄	468.54
1f	C₆H₅CH₂	OCH₃	H	H	C₂₂H₂₂N₂O₅	394.15
1g	C₆H₅CH₂	OCH₃	H	C₂H₅	C₂₄H₂₆N₂O₅	422.47
1h	C₆H₅CH₂	OCH₃	OCH₃	H	C₂₃H₂₄N₂O₆	424.45
1i	C₆H₅CH₂	OCH₃	OCH₃	C₂H₅	C₂₅H₂₈N₂O₆	452.5

All compounds gave correct elemental data

Table 2: Antimicrobial activity of the compounds MIC’s in μg/mL^*

Microorganisms

Compounds (MIC Values)

Ciprofloxacin

S. aureus

(ATCC 6538)

3.9

B. subtilis

(ATCC 6633)

12.5

3.9

E. coli

(ATCC 8739)

3.9

P. aeruginosa (ATCC 25619)

12.5

3.9

^*MIC- Minimum inhibitory concentration

2H), 3.72 (s, 3H), 3.24 (d, 1H), 3.58 (dd, 1H), 4.21 (m, 1H), 3.44 (m, 2H), 1.40 (t, 3H).

1c: m.p.229-231°C, Yield 72%. IR: 1660 (C=O); 3340 (NH). ¹H NMR (DMSO-d₆): 7.57-7.80 (m, 15H), 3.18 (m, 2H), 3.73 (s, 3H), 3.26 (d, 1H), 3.53 (d, 1H), 4.18 (m, 1H).

1d: m.p.166-168°C, Yield 85%. IR: 1645 (C=O); 3350 (NH). ¹H NMR (DMSO-d₆): 7.12-7.84 (m, 15H), 3.12 (m, 2H), 4.16 (m, 2H), 3.68 (s, 3H), 3.19 (d, 1H), 3.41 (dd, 1H), 4.01 (m, 1H).

1e: m.p.204-206°C, yield 77%. IR: 1635 (C=O); 3345 (NH). ¹H NMR (DMSO-d₆): 7.15-8.1 (m, 15H), 3.16 (m, 2H), 2.51 (t, 2H), 4.08 (t, 2H). 3.58 (s, 3H), 3.27 (d, 1H), 3.48 (dd, 1H), 4.23 (m, 1H).

1f: m.p.148-150°C, yield 73%. IR: 1650 (C=O); 3340 (NH). ¹H NMR (DMSO-d₆): 7.08 (m, 5H), 3.18 (m, 2H), 7.15 (t, 2H), 7.35 (d, 2H), 33.94 (9s, 3H), 3.53 (s, 3H), 3.18 (d, 1H), 3.34 (d, 1H), 4.2 (m, 1H).

1g: m.p.218-220°C, yield 71%. IR: 1635 (C=O); 3355 (NH). ¹H NMR (DMSO-d₆): 8.1 (m, 5H), 3.21 (m, 2H), 7.64 (t, 2H), 7.82 (d, 2H), 3.93 (s, 3H), 3.61 (s, 3H), 3.21 (d, 1H), 3.32 (dd, 1H), 4.23 (m, 1H), 3.48 (m, 2H), 1.43 (t, 3H).

1h: m.p.192-194°C, yield 73%. IR: 1640 (C=O); 3350 (NH). ¹H NMR (DMSO-d₆): 7.81 (m, 5H), 3.24 (m, 2H), 7.52 (m, 3H), 3.68 (s, 6H), 3.84 (s, 3H), 3.18 (d, 1H), 3.41 (d, 1H), 4.28 (m, 1H).

1i: m.p.225-227°C, yield 79%. IR: 1640 (C=O); 3360 (NH). ¹H NMR (DMSO-d₆): 7.80 (m, 5H), 3.22 (m, 2H), 7.39 (m, 3H), 3.72 (s, 6H), 3.8 (s, 3H), 3.18 (d, 1H), 3.36 (d, 1H), 4.32 (m, 1H), 3.51 (m, 2H), 1.52 (t, 3H).

REFERENCES:

1. S. E. Denmark, A.Thorarensen, D. S. Middleton, J. Am. Chem. Soc. 118 (1996), 8266.

2. D.T. Witiak, B. R. Vishnuvajjala, W. L. Cook, J. A. Minatelli, T. K. Gupta and M. C. Gerald, J. Med. Chem, 20(6) (1977), 801-805.

3. T. D. Penning, N. S. Chandrakumar, B. N. Desai, S. W. Djuric, A. F. Gasiecki, and C. Liang, Bioorg. Med. Chem. Lett, 12(23) (2002), 3383-3386.

4. L. Li, H. Wang, S. Kuo, T. Wu, A. Mauger and C. M. Lin, J. Med. Chem, 37(20) (1994), 3400-3407.

5. George Burton, W. Thomas, J. Thomas, Terry Kiesow, Robert T. Sarisky, J. L. Goerke, et al., Bioorg. Med. Chem. Lett, 17(7) (2007), 1930-1933.

6. H. Egawa, T. Miyamoto, A. Minamida, Y. Nishimura, H. Okada, and H. Uno, et al. J. Med. Chem, 27(12) (1984), 1543-1548.

7. S. Batra, P. Srivastava, K. Roy, V. C. Pandey, and A. P. Bhaduri, J. Med. Chem, 43(18) (2000), 3428-3433.

8. Y.Rajendra Prasad, M.Srinivas and S.Chandrashekaran, Ind.J.Het.Chem, 18(2009), 255-258.

9. W. Zhang, Curr. Opin. Drug Disc. Dev, 7 (2004), 784.

10. W. Zhang, C.H. Tung Chen, Tetrahedron. Lett, 46 (2005), 1807-1810.

11. 11. N.S. Wilson, C.R. Sarko, G.P. Roth, Tetrahedron. Lett, 42 (2001), 8939-8941.

12. 12. E.J. Baron, S.M. Finegold, in: Bailey and Scott’s Diagnostic Microbiology,8th ed, Mosby, St. Louis, MO, 1990 184–188.

Received on 04.09.2009 Modified on 05.09.2009

Asian J. Research Chem. 2(3): July-Sept., 2009, page 357-359