Synthesis of the fluorinated D-glucuronic acid II

As 5-fluoro-D-glucuronic acid has to be linked with two N-acetylglucosamine molecules in position 1 and 4, the fluorinated compound should be protected in the positions 2 and 3, whereas the hydroxy groups in the positions 1 and 4 should be ether free for linkage or protected with a group that can be selectively cleaved before linkage (cf. structureII).

Two different strategies were envisaged for the synthesis of this building block (cf. Fig. 4).

Design and synthesis of a substrate analogue as a potential inhibitor of hylB4755

Fig. 4: Retrosynthetic routes for the synthesis of the fluorinated D-glucuronic acid

Starting from the commercially available methyl α-D-glucopyranoside according to route A, the double bond between the carbon atoms 5 and 6 will be intro-duced by an elimination reaction. By subsequent fluorination of this alkene, the fluorine atom would be introduced in position 5. Finally, the oxidation of the carbon atom in position 6 would lead to the desired fluorinated glucuronic acid, which can be linked with both other carbohydrate monomers IaandIb.

According to route B, the fluorine atom would be introduced by fluorination of an intermediate with C=C double bond in position 4. The pertinent alkene will be synthesised by an elimination reaction starting from the commercially avail-able methyl α-D-glucopyranoside, too. The carboxylic acid in position 6 should be obtained by oxidation reaction.

Design and synthesis of a substrate analogue as a potential inhibitor of hylB4755

61 Synthetic route A

The key reactions of route A are elimination, fluorination and oxidation. The elimination of HI on the protected 6-iodo-α-D-glucose derivative was the first method tried to introduce an alkene function. Therefore, the hydroxy group in position 6 of methyl α-D-glucopyranoside (8) was selectively substituted by io-dine and the remaining hydroxy groups were subsequently acetylated or ben-zoylated. The iodination of compound8 was successfully performed by treating the starting material with N-iodosuccinimide and triphenylphosphine in DMF (Garegg et al. 1978) or with iodine, triphenylphosphine and imidazole in aceto-nitrile (Garegg et al. 1982). The subsequent acetylation or benzoylation of the iodinated intermediate were carried out under standard conditions with acetic anhydride and sodium acetate or benzoyl chloride and pyridine, respectively to yield the products9aand 9b(cf. Scheme 4).

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Scheme 4:Synthesis of the protected methyl 6-deoxy-6-iodo-α-D-glucopyrano-side9a and9b; reaction conditions: for 9a i) NIS, Ph3P, DMF; ii) Ac2O, NaOAc;

for9bi) I2, Ph3P, imidazole; ii) BzCl, pyridine

Due to difficult separation of the product from starting materials and low yield, an alternative route was envisaged: first protection of the hydroxy groups in the positions 2, 3 and 4 and then substitution of position 6 with iodine. Several methods were tried for this alternative route (Garegg et al. 1978; Garegg et al.

1982; Mirza et al. 1985), however, neither the acetylated glucose derivative nor the benzoylated compound could be iodinated. Therefore, the route shown in Scheme 4 was applied despite the aforementioned disadvantages.

In the next step the elimination of HI was accomplished according to the method described by Mirza et al. (Mirza et al. 1985) using

1,8-diazabicyclo-R a Ac b Bz

Design and synthesis of a substrate analogue as a potential inhibitor of hylB4755

62

[5.4.0]undec-7ene (DBU) in THF. Compound 9a could be converted to com-pound 10 by elimination and deacetylation, whereas the reaction did not work with the compound 9b and could not be improved by variation of the reaction conditions (cf. Scheme 5).

Scheme 5: Synthesis of methyl 6-deoxy-α-D-xylo-hex-5-enopyranoside (10)

An alternative synthetic route for the synthesis of the alkene derivative started from the 6-tosylated glucose derivative 11, which was prepared by treating methyl α-D-glucopyranose (8) with tosyl chloride in pyridine (Cramer et al.

1959). Then, methyl 2,3,4-tri-O-benzoyl-6-O-tosyl-α-D-glucopyranoside (12) was prepared by a benzoylation of compound 11 under standard conditions with benzoyl chloride and pyridine (cf. Scheme 6).

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Scheme 6: Protection of methyl α-D-glucopyranoside (8) with the tosyl pro-tecting group in position 6 and the benzoyl group in the positions 2, 3 and 4;

reaction conditions: i) TsCl, pyridine, 0 °C→RT; ii) BzCl, pyridine, RT

Finally, compound 12 was treated with sodium iodide, tetrabutylammonium io-dide, molecular sieve and DBU in DMSO to give the unsaturated glucose de-rivative 13 (cf. Scheme 7) (Sato et al. 1988; Sato et al. 1991; Mereyala et al.

1993; Sato et al. 1993).

Design and synthesis of a substrate analogue as a potential inhibitor of hylB4755

63

O

OBz

OBz OCH₃

O

OBz

OBz OCH₃

OTs

BzO BzO

1) NaI / Bu₄NI DMSO / MS (4 A) 2) DBU

12 13

Scheme 7: Synthesis of methyl 2,3,4-tri-O-benzoyl-6-deoxy-α-D-xylo-hex-5-enopyranoside (13)

Following this synthetic route, the elimination product13 could be obtained in a three step reaction instead of a four step reaction compared to the first stra-tegy. Furthermore, the handling and the purification of the synthesised product were much more convenient.

The crucial step of the synthetic route Ais the introduction of the fluorine atom in position 5 of the D-xylo-hex-5-eno-pyranoside 13. In the literature, several methods are published for the introduction of a fluorine atom by an addition reaction. From the list of commercially available reagents 1-fluoropyridinium tetrafluoroborate, 1-fluoro-2,4,6-trimethylpyridinium triflate, N-fluorobenzene-sulfonimide, selectfluor^TMand silver fluoride were the most common fluorinating agents (Maguire et al. 1993; Burkart et al. 1997; Albert et al. 1998; Vincent et al. 1999). With exception of silver fluoride, all other fluorination agents intro-duce the fluorine atom in position 6 instead of position 5, i.e. not adjacent to the oxygen atom. Maguire et al. (Maguire et al. 1993) reported the iodofluorination of an adenosine derivative with silver fluoride and iodine in acetonitrile at - 40 °C. All attempts to transfer this method to the preparation of the 5-fluo-rinated hexose derivative were unsuccessful. Variation of the reaction tem-perature from – 20 °C over – 10 °C to 0 °C or room temtem-perature did not lead to the fluorinated compound.

Design and synthesis of a substrate analogue as a potential inhibitor of hylB4755

64 Synthetic route B

In parallel to route A, the synthesis of the fluorinated D-glucuronic acid II via route B was explored. The hydroxy groups in position 4 and 6 of methyl α-D-glucopyranoside (8) were protected with benzaldehyde dimethyl acetal and camphorsulphonic acid in DMF according to a method of Mallet et al.(Mallet et al. 1993) with some modifications. The subsequent protection of the remaining hydroxy groups was carried out with benzyl bromide and sodium hydroxide to give methyl 2,3-di-O-benzyl-4,6-O-benzyliden-α-D-glucopyranoside (14) (cf.

Scheme 8).

Scheme 8: Formation of the 4,6-benzylidene acetal and subsequent benzyla-tion of posibenzyla-tions 2 and 3; reacbenzyla-tion condibenzyla-tions: i) PhHC(OCH3)2, DMF, camphor-sulfonic acid; ii) BnBr, DMF, NaH

The reductive opening of benzylidene acetals using lithium aluminium hydride-aluminium chloride or sodium cyanoborohydrate has been reported (Bhattacharjee et al. 1969; Liptak et al. 1975; Garegg et al. 1981; Gelas 1981;

Johansson et al. 1984). For 4,6-benzylidene acetals with bulky substituents such as benzyl groups at position 3, the regioselectivity of the dioxane ring opening is dependent on the reducing agents. By the reaction with LiAlH4/AlCl3

the benzyl group is directed to position 4 and position 6 remains free (Liptak et al. 1975), whereas with NaCNBH3/HCl and NaCNBH3/TFA the inverse result is achieved (Garegg et al. 1981; Garegg et al. 1982; Johansson et al. 1984).

To synthesise the sugar derivative 15 with the benzyl group in position 6, the NaCNBH3method was chosen. According toJohansson et al.(Johansson et al.

1984) the regioselective reductive ring-opening of the benzylidene acetals

Design and synthesis of a substrate analogue as a potential inhibitor of hylB4755

65

should be achieved with NaCNBH3/TFA. Unfortunately, this reaction failed.

However, a modified procedure (Garegg et al. 1981; Garegg et al. 1982) work-ing at room temperature instead of 0 °C, changwork-ing the 3 Å molecular sieve from powdered to spherical shape and by extending the reaction time from 10 min to 16 h, gave compound15(cf. Scheme 9).

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Scheme 9: Regioselective deprotection at position 4 by cleavage of the ben-zylidene acetal NaCNBH3under acidic conditions

With respect to the introduction of a C=C double bond, the hydroxy group in position 4 was mesylated to obtain an efficient leaving group. Methyl 2,3,6-tri-O-benzyl-α-D-glucopyranoside (15) was treated with mesyl chloride and triethyla-mine in anhydrous dichloromethane according to a method described byBernet et al.(Bernet et al. 1979) with some modifications (cf. Scheme 10).

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