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Design, Synthesis, and Evaluation of Stable and Taste-Free Erythromycin Proprodrugs Pranab K. Bhadra,† Gareth A. Morris,‡ and Jill Barber*,† School of Pharmacy and Pharmaceutical Sciences and Department of Chemistry, University of Manchester, Manchester, M13 9PL, U.K. Received October 21, 2004
Erythromycin A is normally formulated for children as its 2′-ethyl succinate, a taste-free prodrug. Unfortunately, the prodrug hydrolyzes at a measurable rate in the medicine bottle, leading to the vile-tasting erythromycin. We have prepared derivatives of erythromycin B as putative paediatric prodrugs, taking advantage of the much improved acid stability of erythromycin B relative to erythromycin A. Thus, erythromycin B enol ether ethyl succinate is very poorly soluble in water, and its hydrolysis is undetectable in conditions resembling the medicine bottle. In acid, however, it converts rapidly to erythromycin B 2′-ethyl succinate, and this is in turn hydrolyzed to erythromycin B in neutral and basic conditions. Derivatives of erythromycin B enol ether are therefore proposed as taste-free proprodrugs of erythromycin B. Erythromycin A (1) is one of the most successful drugs of all time and has been widely used in the treatment of bacterial infections for 50 years.1 However, there are problems associated with its use. These may be summarized as (i) its vile taste, (ii) its lack of activity against most Gram-negative organisms, (iii) its metabolism by liver enzymes, competing with other drugs such as theophylline and common hay fever remedies, leading to overdose of these drugs, and (iv) the gut motilide activity of both erythromycin A and its metabolites. These problems are all greatly exacerbated by the fact that erythromycin needs to be administered in large doses (typically 1-2 g per day for 7-14 days for adults). Large oral doses are required because of the chemical and metabolic instability of the drug. In particular, erythromycin is unstable below pH 6.9, equilibrating with erythromycin A enol ether (2) and degrading to erythromycin A anhydride (3), as shown in Scheme 1. Enteric-coated tablets and capsules of erythromycin are used clinically so that the drug is released in the intestine rather than in the stomach.4 Much of the use of erythromycin is, however, in pediatrics, requiring a liquid formulation. This has been achieved by esterification of the drug at the 2′-position to give compounds that act as prodrugs and are administered as oral suspensions. Esterification using a suitable acid chloride is highly selective and facile. Further, the resulting esters are generally taste-free and have enhanced acid stability relative to erythromycin itself.5 Erythromycin is normally administered to children as a suspension of erythromycin A 2′-ethyl succinate (4). Unfortunately, children are less enthusiastic about erythromycin 2′-ethyl succinate than chemists. The prodrug is activated by base-catalyzed hydrolysis in the blood stream, but the environment of the blood and the medicine bottle are not sufficiently different for good * To whom correspondence should be addressed. Phone: 44-161275-2369. Fax: 44-161-275-2396. E-mail:
[email protected]. † School of Pharmacy and Pharmaceutical Sciences. ‡ Department of Chemistry.
Scheme 1. Decomposition Pathway of Erythromycin A in Acidic Aqueous Solution2,3
selectivity to be obtained. From the moment the erythromycin 2′-ethyl succinate suspension is made, basecatalyzed hydrolysis proceeds at a measurable rate, and within 3 weeks there is considerable contamination by erythromycin free base. Most children find the bitter taste very nasty, and in some it causes vomiting.6 This leads to poor compliance with drug regimes, which in turn is associated with increased resistance to the drug. This paper describes the preparation and evaluation of some taste-free and stable prodrug and proprodrug alternatives to erythromycin A 2′-ethyl succinate that hydrolyze selectively in the body and not in the formulation.
10.1021/jm049155y CCC: $30.25 © 2005 American Chemical Society Published on Web 04/29/2005
Taste-Free Erythromycin Proprodrugs
Scheme 2. Degradation of Erythromycin B in Acidic Aqueous Solution3
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3879 Table 1. Solubility Data of the Enol Ether Ethyl Succinates in Comparison with the Parent Ethyl Succinates in Deuterated Phosphate Buffer (50 mM) solubility at 25 °C (mM)
Results Design of Proprodrugs of Erythromycins A and B. During investigations of the kinetics of the acidcatalyzed degradation of erythromycin B (Scheme 2), we demonstrated an equilibrium between erythromycin B (5) and erythromycin B enol ether (6) at apparent pH 2.5. (Erythromycin B degrades slowly in acid by loss of the cladinose sugar to give 7.3) Erythromycin B enol ether was found to have a solubility of 9.0 mM in aqueous solution at apparent pH 7.4 (25 °C), compared with 19.9 mM for erythromycin B itself. These observations suggested the possibility of preparing sparingly soluble enol ether esters of erythromycin B that would convert to the normal 14-membered ring esters on treatment with acid, for example, in the stomach. Such compounds might be expected to serve as taste-free proprodrugs of erythromycin B.
compd
apparent pH 6.0
4 (EAES) 9 (EAEEES) 10 (EBES) 8 (EBEEES)
1.9 0.2 5.3 0.3
apparent pH 7.0 0.8 0.1 1.2 Broad lines indicated aggregation. Solubility could not be measured.
The first lead compound selected for synthesis was erythromycin B enol ether 2′-ethyl succinate (8) because the 2′-ethyl succinate of erythromycin A is well-tolerated in the body. There was no direct evidence that the proprodrug approach would be successful in the erythromycin A series; indeed, we had previously observed slow conversion of erythromycin B enol ether to erythromycin B at neutral pH, under which conditions erythromycin A enol ether (2) is stable.7 Nevertheless, it was logical to prepare and test erythromycin A enol ether 2′-ethyl succinate (9) in parallel. Synthesis. Erythromycin B enol ether 2′-ethyl succinate (8) was prepared by the two methods shown in Scheme 3. Better yields (72% vs 44%) were obtained when erythromycin B enol ether (6, Scheme 3A), rather than erythromycin B 2′-ethyl succinate (10), was used as an intermediate. The same methodology was then also applied to erythromycin A, yielding erythromycin A enol ether 2′-ethyl succinate (9) in 75% yield from erythromycin A. Solubility Measurements. The solubilities of the two enol ether ethyl succinates at apparent pH 6.0 and apparent pH 7.0 (at 37 °C) were measured by NMR spectroscopy and are shown in Table 1. It can be seen that, as predicted, both enol ether derivatives are much less soluble than their parent ethyl succinates. Activation of Erythromycin B Enol Ether 2′Ethyl Succinate by Acid. The conversion of the enol ether ethyl succinates to the corresponding erythromycin 2′-ethyl succinates in acid is an essential part of the drug design strategy. It was tested by incubating 8 and 9 separately at apparent pH 2.0 and 37 °C in deuterated buffer and monitoring by 1H NMR spectroscopy at
Scheme 3. (A) Synthesis of Erythromycin Enol Ether 2′-Ethyl Succinates 8 and 9 via Erythromycin Enol Ethers and (B) Synthesis of Erythromycin B Enol Ether 2′-ethyl Succinate 8 via Erythromycin B 2′-Ethyl Succinate
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Bhadra et al.
Figure 1. Reactions of erythromycin B enol ether 2′-ethyl succinate, 8, in deuterated Britton-Robinson buffer at apparent pH 2.0 and 37 °C. 8 is activated within 10-15 min to give erythromycin 2′-ethyl succinate (10), which is then degraded to 5-Odesoaminylerythronolide B ethyl succinate (11). Signals A, B, and C represent H-13 in 8, 10, and 11, respectively, and D represents H3-19.
regular intervals. A typical time course for the treatment of 8 with acid is shown in Figure 1 and demonstrates that conversion of 8 to erythromycin B 2′-ethyl succinate (10) is very rapid and essentially complete within 10-15 min. Unfortunately, a second reaction, resulting in the degradation of 10, can also be detected. In this experiment, 10 showed a half-life of 66 min. This degradation is likely to reduce the bioavailability of the drug significantly (a typical residence time in the stomach is 30-120 min). Since the erythromycins are nontoxic, this is acceptable, though not ideal. The degradation product was identified as 5-O-desosaminyl erythronolide B 2′-ethyl succinate (11) by diffusion-ordered spectroscopy (DOSY) NMR8-10 and electrospray ionization mass spectrometry. In the DOSY NMR spectrum (Figure 2), NMR signals are separated according to chemical shift in one dimension and according to their self-diffusion coefficients in the other. It shows that the signals for the cladinose ring exhibit much faster diffusion than those due to the macrolide and the desosamine sugar, demonstrating that cladinose had been hydrolyzed from the macrolide. Electrospray ionization mass spectrometry showed signals at m/z 831, 851 (70%), and 693 (100%) corresponding to 8, 10, and 11, respectively (each containing two or three deuterated hydroxyl groups because exchange of OD to OH is incomplete in the mass spectrometer), in agreement with the results obtained from the DOSY experiment. The effect of acid on erythromycin B enol ether 2′-ethyl succinate (8) is therefore analogous to the effect of acid
on erythromycin B enol ether (6), as is illustrated in Scheme 2. Treatment of Erythromycin A Enol Ether 2′Ethyl Succinate with Acid. The NMR time course at apparent pH 2 was repeated using erythromycin A enol ether 2′-ethyl succinate (9). Both the initial conversion and the subsequent loss of cladinose were slower. As can be seen in Figure 3, 9 had a half-life of 41 min under these conditions. Loss of cladinose was not seen at apparent pH 2 but was evident at apparent pH 1; the half-time for loss of cladinose at this pH was 35 min. Unfortunately, however, Figure 3 shows that the initial hydrolysis product was not erythromycin A 2′ethyl succinate (4). The electrospray ionization mass spectrum gave an M + H+ peak at m/z 848, suggesting that 8 is converted to anhydroerythromycin A 2′-ethyl succinate (12) under acidic conditions (see Scheme 1). (12 had incorporated on average four deuteriums from the deuterated solvent, and these were not washed out in the mass spectrometer.) When 4 was incubated at 37 °C and pH 2, the 1H NMR spectrum showed that it too was converted to 12. Thus, although esterification at the 2′ position is reported to slow anhydride formation, it remains fast compared with the conversion of 8 to 4. This result means that the proprodrug approach is unlikely to be successful for the delivery of erythromycin A. Degradation of Erythromycin B Enol Ether 2′Ethyl Succinate in Acidic H2O. It can be inferred from the preceding data that under acidic conditions the cladinose sugar is lost from erythromycin B 2′-ethyl
Taste-Free Erythromycin Proprodrugs
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3881
Figure 2. DOSY NMR spectrum at 500 MHz of erythromycin B enol ether 2′-ethyl succinate at 25 °C after 2 h at 37 °C. Signals due to the cladinose ring clearly show faster diffusion than those due to the macrolide and desosamine rings.
Figure 3. Reactions of erythromycin A enol ether 2′-ethyl succinate, 9, in deuterated Britton-Robinson buffer at apparent pH 2.0 and 37 °C. Signals A and B represent H3-15 in 9 and anhydroerythromycin A 2′-ethyl succinate (12), respectively. Signal C is due to H3-19 of 9 and is highly characteristic of erythromycin enol ethers.
succinate (10) rather than from the enol ether ethyl succinate (8). Further, loss of cladinose from erythromycin B esters in acid is much slower than anhydride formation by erythromycin A esters. This was now confirmed by 1H NMR spectroscopy in 95% protiated buffer (to avoid solvent and any other isotope effects). Compounds 8 and 10 were incubated separately in Britton Robinson buffer at pH 2 containing 5% D2O. The WATERGATE pulse sequence was used to obtain time courses, which were confirmed by electrospray ioniza-
tion mass spectrometry. The half-life of 10 under these conditions was 71 ( 5 min; that of 10 generated in situ from 8 was 73 ( 5 min. Thus, compounds 8 and 10 lose cladinose at essentially the same rate, supporting the proposal that 8 is rapidly converted to 10 under acidic conditions and that this conversion is followed by slow loss of the cladinose sugar to give 11. An apparent pH 2 in D2O-based buffer corresponds to an actual pD of about 2.4, so a shorter half-life in H2O at pH 2 was expected, whereas similar half-lives (71 ( 5 vs 66 ( 5
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Table 2. Half-Lives for the Acid-Catalyzed Activation of Erythromycin B Enol Ether 2′-Ethyl Succinate (8) to Erythromycin B 2′-Ethyl Succinate (10) and Its Subsequent Degradation to 5-O-Desosaminylerythronolide B 2′-Ethyl Succinate (11) and the Degradation of Erythromycin A Enol Ether 2′-Ethyl Succinate (9) to Anhydroerythromycin A 2′-Ethyl Succinate (12) under the Same Conditions half-life (min) reaction
pH 1.0
pH 2.0
pH 3.0
pH 4.0
pH 5.0
8 f 10 10 f 11 9 f 12
a
