Fase: Evaluación del riesgo

There have been numerous reports of the synthesis of p-glucuronide conjugates (for a review see Stachulski and Jenkins, 1998). This is because many drugs and natural products are glucuronidated prior to excretion in the bile or urine. In addition, a number of these glucuronides, notably morphine glucuronides (Berrang et al., 1997), have been shown to have pharmacological effects which are at least as important as their parent compounds. Of particular interest are a number of reports on the synthesis of glucoside or glucuronide conjugates of vitamin E type compounds (Yoshioka et al., 1991; Lahmann and Thiem, 1997; Uhrig et al., 2000). These reports suggest that, owing to their ease of oxidation, particular care must be taken in the glucosylation or glucuronidation of vitamin E type compounds.

There are a number of possible intermediates that can be used to glucuronidate a substrate of interest. The preparation of common glucuronic acid donors is shown in scheme 5.22, starting with the commercially available D-glucurono-6,3-lactone (47).

Treatment of the glucuronolactone (47) with sodium methoxide produces the glucuronic acid methyl ester, which can be acetylated with acetic anhydride (Bollenback et al., 1955; Leu et al., 1999) to give a mixture of a /p anomers (48). These epimers can be easily separated if required by kinetic recrystallisation. In some studies, the p-acetylated anomer (48) has been coupled directly to an aglycone moiety (Lahmann and Thiem, 1997; Stachulski and Jenkins, 1998).

From the tetra-acetate sugar (48) a number of more reactive intermediates can be made. The hydroxy sugar (49) can be prepared from the a/p protected sugar (48) using

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tributyltin methoxide (Nudelman et al., 1987) and can either be coupled directly to an hydroxy aglycone using Mitsunobu conditions (Laurin et al., 1999) or can in turn be converted to the trichloroacetimidate intermediate (50) (Jacquinet, 1990; Brown et al., 1997). Glucuronidation with this intermediate (50) is particularly attractive because it has very high p-stereoselectivity and it requires relatively mild catalysis. It has been used in the synthesis of aryl glucuronides (Brown et al., 1997; Ferguson et al., 2000). The common catalysts used for trichloroacetimidate glucuronidation are BFj-Et2 0 or more occasionally trifluoromethanesulphonate (TMSOTf) and the reaction is carried out at reduced temperature (Brown et al., 1997; Ferguson et al., 2000).

The other possible intermediate shown in the scheme is the bromo sugar (51). This sugar derivative (51) is probably still the most popular glucuronidation intermediate and has been used in the synthesis of both alkyl and aryl glucuronides using the Koenigs- Knorr conditions (Bowering and Timell, 1960; Berrang et al., 1997). The P-bromo sugar (51) can be directly synthesised from the tetra-acetate sugar (48) using reagents such as titanium bromide or hydrogen bromide. The common catalysts used to couple the bromo sugar (51) with an hydroxy aglycone are Ag(I) salts such as AgzO or Ag2C0 3. Both the bromo (51) and trichloroacetimidate intermediates (50) are unstable and care must be taken to avoid exposure to air, heat and water. Storage under desiccation at -20°C is advised.

Once the appropriate intermediate has been coupled to the aglycone, the protected glucuronide can be saponified with sodium or potassium hydroxide in aqueous methanol. The resulting glucuronide can often be re-crystallised from ethanol (Brown et al., 1997).

HO 2)Ac20.pyr (47): D-glucurono-6,3-lactone (48) ( 5 1 ) (glucorone) COgMe BUgSnOMe 58% * ^ ° À æ ^ O H D g u ACO^J^-^ 59% ° > = N H CI3C (49) (50)

Scheme 5J22. Synthesis of glucuronic acid donors

5.4.1.1. Synthesis of glucuronide donors

The glucuronide donors were synthesised as described above in the yields given in scheme 5.22. Briefly, the tetra-acetate sugar (48) was made from glucuronolactone (47) using sodium methoxide and acetic anhydride followed by two recrystallisations in ethanol in order to isolate the two anomers (48). The a-amomer (48) was then used in the synthesis of the p-bromo sugar (51) using titanium bromide. The residual mixture of anomers (48) from the second recrystallisation was used to synthesise the hydroxyl sugar (49) by treatment with tin methoxide. Finally the p-imidate intermediate (50) was synthesised from the hydroxyl sugar using trichloroacetonitrile and 1,8-Diazabicyclo- undec-7-ene (DBU) (Jacquinet, 1990). No major problems were encountered, except in the synthesis of the bromo sugar (51). Attempts at purification were made but the bromo

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sugar (51) could not be characterised, possibly because it decomposed during the purification procedure. The compound produced was therefore coupled directly to the aglycone without prior purification.

The usefulness and efficiency of the various glucuronide donors were then assessed on a model compound in order to optimise the procedure prior to glucuronidation of the a - tocopherol metabolites.

5 4.1.2. G lucuronidation of trim ethylhydroquinone (TMHQ) as a mode! compound TrimethyIhydroquinone (TMHQ) (5) was chosen as an appropriate model compound because of its similar structure to both a-tocopheronolactone (38) and a-CEHC (9). Owing to the poor yields obtained using the tetra acetate sugar (48), as reported in earlier studies (Yoshioka et al., 1991), initial attempts were made using the hydroxyl sugar (49). Coupling of the hydroxyl sugar (49) with TMHQ (5) using Mitsunobu conditions (TributyIphosphine (BugP)/ 1, T -(azodicarbonyl)dipiperidine (ADDP) in THF, at room temperature (rt) overnight) was tried. However TLC analysis indicated that little or no product was formed and TMHQ was present solely in the oxidised trimethyl benzoquinone form (52) (TMBQ).

Glucuronidation using the bromo sugar (51) was then attempted. The bromo sugar (51) was synthesised and then coupled directly to TMHQ (5), without purification. The Koenigs-Knorr method was used and both silver oxide and silver carbonate were tried as catalysts (Berrang et al., 1997; Stachulski and Jenkins, 1998). The TLC data obtained was very similar to that for the Mitsunobu coupling above, with very little or no product spots appearing and the major spot corresponding to trimethyl benzoquinone

(52) (Scheme 5.23). The failure of both these methods, using the hydroxyl (49) and bromo sugar (51), was probably due to the facile oxidation of TMHQ (5) leading to formation of the unconjugatable benzoquinone (52) (Scheme 5.23).

Lahmaim and Thiem (1997) suggested that one of the main problems in the synthesis of a-tocopheryl oligosaccharides is the easy oxidation of a-tocopherol to the corresponding open chain quinone and other oxidation products. They, therefore, suggested that methods such as the Koenigs-Knorr glycosylation employing silver salts were not suitable for tocopherol type compounds. In contrast, they successfully used a combination of trichloroacetamide (50) and boron trifluoride diethyl etherate since this did not oxidise tocopherol (Lahmann and Thiem, 1997). Consequently, further attempts at glucuronidation of the model compound were performed employing the imidate (50).

(5)

AcO

COgMe

AcO

(53) (52) TMBQ

substrate glycosyl donor conditions products

(5) (49) ADDP/BU3P