Perspectiva política

In an effort to synthesise a quinolone derivative with enhanced activity, the well-known quinolone, levofloxacin, was chosen as the quinolone derivative for modification with the hydroxamic acid. The synthesis of a C-3 hydroxamic acid derivative of levofloxacin, 3.25, was first attempted using the coupling reagents HOBt and TBTU, in the presence of Et3N, with DMF as the reaction solvent (Scheme 3.22).

Scheme 3.22: Synthesis of levofloxacin C-3 hydroxamic acid 3.25. Reaction carried out under nitrogen, (i) HOBt, TBTU, Et3N, DMF, 10 min (ii) NH2OH.HCl, Et3N,

DMF, 24 h, rt.

Coupling reagents are most commonly used in the synthesis of peptides.¹⁴⁰ TBTU is a (1H)-benzotriazole-based coupling reagent that is believed to exist as both the uronium and aminium salt in solution (Figure 3.25).^140-141 These uronium/aminium salts work by activating the carboxylic acid moiety which in turn facilitates the nucleophilic attack of an amine, resulting in formation of an amide bond.¹⁴¹

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Figure 3.25: TBTU (a) uronium salt and (b) aminium salt.^140-141

As shown in Scheme 3.23, the deprotonated carboxylic acid nucleophile attacks the electrophilic centre of TBTU. The resulting intermediate reacts with HOBt, generating the activated ester. Attack at the carbonyl carbon of the activated ester, by the amine, results in the formation of the amide bond. When carrying out the

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After 24 hours the solvent was removed under reduced pressure and the resulting residue analysed by ¹H NMR spectroscopy. In the ¹H NMR spectrum, only the starting material appeared to be present with no shifts in the ¹H signals being observed. Thus, an alternative coupling method was attempted.

COMU, shown in Figure 3.26, is a relatively new coupling reagent.¹⁴² The presence of the oxyma leaving group in place of a benzotriazole along with the replacement of a dimethylamino group by a morpholino moiety enhances stability, solubility and reactivity of the reagent.^142-143 As mentioned earlier, the benzotriazole-type coupling reagents, such as TBTU, are believed to co-exist as the uronium and aminium salts.

However, COMU exists solely as the more reactive uronium structure.^142,144 Additionally, the by-products formed by COMU are water soluble, allowing their removal by simple extraction.^143,145

Figure 3.26: COMU coupling reagent with the oxyma moiety shown in red.

The synthesis of 3.25 was attempted using COMU coupling as described in Scheme 3.24. After 24 hours, the solvent was removed under reduced pressure and the resulting residue was analysed by ¹H NMR spectroscopy. As with the previous HOBt/TBTU coupling reaction, in the ¹H NMR spectrum, no shifts were observed for the levofloxacin ¹H signals, suggesting that only starting material was returned.

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Scheme 3.24: Synthesis of 3.25 under COMU coupling conditions.¹⁴³ The reaction was carried out under nitrogen, (i) DIEA, 0 ^oC, 10 min, (ii) NH₂OH.HCl, DIEA, 10

min, (iii) COMU, 0 ^oC, 1 h followed by 24 h, rt.

After the unsuccessful attempts to synthesise 3.25, using two different coupling reagents, a trial coupling reaction was carried out in an effort to determine if a simple amine could be coupled to the C-3 carboxylic acid of levofloxacin. As shown in Scheme 3.25, a reaction of levofloxacin with n-butylamine using standard coupling conditions of HOBt/TBTU in the presence of Et₃N was carried out.

Scheme 3.25: Synthesis of 3.26. (i) HOBt, TBTU, ⁿBuNH2, DMF, 24 h, rt.

In the ¹H NMR spectrum, the appearance of a triplet at 0.92 ppm for the protons of the n-butylamine –CH₃ group was observed, together with mulitplets at ca. 1.58 and 1.40 ppm for the protons of the n-butyl –CH₂ groups, indicating formation of 3.26. A triplet at 9.95 ppm was also observed for the NH proton confirming generation of the amide bond. A small shift in the ¹H signal of H-5 was also observed. An upfield shift of the ¹³C signal of the C-3 carbonyl carbon, from 177.0 ppm to 175.5 ppm, was observed in the ¹³C NMR spectrum, further confirming formation of the amide derivative.

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These results prove that a simple amine can be coupled to the carboxylic acid of levofloxacin. However, 3.26 was obtained in a low yield of 42% suggesting that this reaction, although successful, is not very efficient in the synthesis of a simple levofloxacin carboxamide. Therefore, an alternative synthetic approach was sought.

Reddy et al.¹⁴⁶ have demonstrated the synthesis of a variety of aliphatic and aromatic hydroxamic acid derivatives, in yields of 80-95%, using ethylchloroformate and NH2OH.HCl (Scheme 3.26). The reaction of the carboxylic acid with ethylchloroformate in the presence of base should produce the ester derivative bearing a better leaving group in comparison to the carboxylic acid –OH (Scheme 3.26). This, in turn, should facilitate attack by the hydroxylamine, resulting in formation of the hydroxamic acid derivative.

Scheme 3.26: Synthesis of 3.25.

Hydroxylamine hydrochloride was dissolved in MeOH and added to a stirred solution of KOH in MeOH at 0 ^oC. After stirring for 15 minutes the solution was filtered. Levofloxacin was dissolved in DCM followed by the addition of EtCO2Cl and NMM (as base) at 0 ^oC. The solution was stirred for 10 minutes followed by filtration. The resulting filtrate was added to the freshly prepared hydroxylamine followed by stirring at room temperature for 30 minutes after which the solvent was removed under reduced pressure. The resulting ¹H NMR spectrum of the crude

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product appeared to be a mixture of reactants, with no shifts observed in the ¹H signals of levofloxacin.

In a final attempt to synthesise the C-3 hydroxamic acid derivative of levofloxacin, a reaction of the C-3 ethyl ester of levofloxacin with hydroxylamine hydrochloride was carried out (Scheme 3.27).¹⁴⁷ Levofloxacin ethyl ester, 3.24, was synthesised as described earlier in section 3.2.13. A methanol solution of 3.24 was added slowly to a solution of hydroxylamine and allowed to stir at room temperature for 24 hours.

After 24 hours, the reaction mixture was acidified (pH 6) using concentrated HCl.

The solvent was concentrated under reduced pressure and the resulting precipitate collected by filtration.

Scheme 3.27: Synthesis of 3.25.

In the ¹H NMR spectrum of the resulting white solid, the disappearance of the ¹H signals for the ethyl group protons indicated formation of the hydroxamic acid. A downfield shift of the ¹³C signals for C-2 (145.1 ppm to 146.4 ppm) and the C-3 carbonyl carbon (172.8 ppm to 176.4 ppm) was observed in the ¹³C NMR spectrum.

The strong absorption bands of the carboxylic acid and ketone (C=O) at 1719 cm^-1 and 1617 cm^-1, respectively, had shifted to 1722 cm^-1 and 1623 cm^-1, further indicating formation of 3.25 (Scheme 3.27). LC/TOF-MS also confirmed formation

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of the C-3 hydroxamic acid derivative of levofloxacin returning a (M+H⁺) of 390.1843.

3.2.15 In vitro antibacterial activity

Each of the compounds, the quinolones and their precursors (Appendix B, Table B1-B3), described in section 3.2, were evaluated for their antibacterial activity against E.

coli, P. aeruginosa and S. aureus using the susceptibility assay described in section 1.2.5.

In general, the quinolone precursors, the phenylamino malonates and acrylates, were inactive against all three bacteria. The 1-H-quinolones also exhibited little or no activity against all three bacteria. Alkylation at the N-1 position of the quinolones did not improve activity, in comparison to the 1-H-quinolones, with the N-ethyl quinolone derivatives also exhibiting little or no activity against each of the three bacteria tested.

Neither the C-3 carboxylic acid derivative 3.13 nor the C-3 tetrazole derivative 3.20 exhibited an MIC50 against any of the bacteria, with the greatest bacteriostatic activity (although minimum) achieved only at the highest concentration of 100

g/mL (Table 3.5). However, on comparing 3.13 to 3.20, similar activity was observed for both compounds against E. coli, P. aeruginosa and S. aureus (Table 3.5). This result suggests that although the replacement of the carboxylic acid with the tetrazole bioisostere did not improve activity, it also does not appear to have decreased the activity of the quinolone. This result contradicts the earlier studies by Gilis et al.¹⁴⁸, wherein the presence of a tetrazole at C-3 of nalidixic acid diminished the antibacterial activity exhibited by nalidixic acid.

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Table 3.5: Percentage growths of 3.13 versus 3.20 as a function of concentration of each compound. greatest activity against P. aeruginosa (Graph 3.2), producing an MIC50 in the range of 12.50-18.75 g/mL. A similar trend in activity was also observed with the addition of the piperazine to 3.10 to give 3.16. The addition of the N-methylpiperazine at C-7 of 3.14 also improved the antibacterial activity against both P. aerugionsa and S. aureus but resulted in a decrease in activity against E. coli (Graph 3.1, 3.2, and 3.3). For the nitrile derivative, 3.18, the presence of the C-7 N-methylpiperazine moiety resulted in similar activity to that of 3.16. Although the addition of the piperazine (and N-methylpiperazine) was advantageous for activity against all three bacteria, the nitrile derivatives, 3.16 and 3.18, were less active in comparison to the carboxylic acid derivatives, 3.17 and 3.19.

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Graph 3.1: Activity profile for the C-7 quinolone derivatives versus E. coli.

Graph 3.2: Activity profile for the C-7 quinolone derivatives versus P. aerugionsa.

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Graph 3.3: Activity profile for the C-7 quinolone derivatives versus S. aureus.

The bacteriostatic activity of levofloxacin versus the C-3 hydroxamic acid derivative of 3.25, are summarised in Table 3.6. The results are expressed as the MIC50, the minimum inhibitory concentration that is required to inhibit 50% of bacterial growth.

As can be seen in Table 3.6, 3.25 exhibited similar activity against both the Gram-negative bacteria, E. coli and P. aeurginosa, as well as the Gram-positive bacterium, S. aureus. In comparison to levofloxacin, 3.25 demonstrated similar activity against E. coli, S. aureus and P. aeruginosa (Table 3.6). Furthermore, the bacteriostatic activity of 3.25 increased with increasing concentration, resulting in an MIC₉₀ in the range of 1.17-1.56 g/mL against P. aeruginosa, which is an MIC₉₀ value similar to that exhibited by levofloxacin.

Table 3.6: Antibacterial activity as MIC₅₀ range, values are mean of three experiments.

Compound E. coli P. aeruginosa S. aureus

M g/ml M g/ml M g/ml

Levofloxacin 0.54-1.08 0.2-0.39 1.62-2.16 0.59-0.78 3.24-4.33 1.17-1.56 3.25 1.56-2.08 0.59-0.78 1.04-1.56 0.39-0.59 1.56-2.08 0.59-0.78

152 3.2.16 Conclusion

In this study, the synthesis of a (1H)-tetrazole and hydroxamic acid quinolone derivatives was undertaken. For comparison, the carboxylic acid analogues were also synthesised. The structure of each synthesised compound was elucidated by means of LC/TOF-MS, ¹H and ¹³C NMR and IR spectroscopies.

The synthesis of the quinolone compounds involved formation of the phenylamino acrylates or malonates, which were then cyclised, followed by alkylation at N-1. It was found that cyclisation of the phenylamino acrylates and malonates bearing a fluorine substituent resulted in the generation of two regioisomers, the 5-fluoro and 7-fluoro quinolones. Additionally, with the carboxylate derivatives, both N-ethyl and O-ethyl derivatives were generated during alkylation, the structures of which were confirmed by NOEdiff NMR experiments. The carboxylate derivatives were hydrolysed and the fluoro quinolones were then used in the synthesis of the 7-piperazine derivatives. 1-Ethyl-3-(1H-tetrazol-5-yl)quinolin-4(1H)-one (3.20) was successfully synthesised with a yield of 83%. However, the synthesis of a 7-piperazine derivative bearing a tetrazole moiety proved difficult and could not be generated using the same method.

The X-ray crystal structure was obtained for 3.20, and revealed that the quinolone