Synthesis of Double-Modified Xyloside Analogs for Probing the

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Synthesis of Double-Modified Xyloside Analogs for Probing the #4GalT7 Active Site Daniel Willén, Dennis Bengtsson, Sebastian Clementson, Emil Tykesson, Sophie Manner, and Ulf Ellervik J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02809 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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The Journal of Organic Chemistry

Synthesis of Double-Modified Xyloside Analogs for Probing the β4GalT7 Active Site Daniel Willén, Dennis Bengtsson, Sebastian Clementson, Emil Tykesson, Sophie Manner, Ulf Ellervik*

Centre for Analysis and Synthesis, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden. * Corresponding author. E-mail address: [email protected].

Abstract Monosubstituted naphthoxylosides have been shown to function as substrates for, and inhibitors of, the enzyme β4GalT7, a key enzyme in the biosynthetic pathway leading to glycosaminoglycans and proteoglycans. In this article, we explore the synthesis of 16 xyloside analogs, modified at two different positions, as well as their function as inhibitors of and/or substrates for the enzyme. Seemingly simple compounds turned out to require complex synthetic pathways. A meta-analysis of the synthetic work shows that, regardless of the abundance of methods available for carbohydrate synthesis, even simple modifications can turn out to be problematic, and double modifications present additional challenges due to conformational, steric, and stereoelectronic effects.

Introduction Among the three most important classes of biopolymers, i.e. proteins, nucleic acids, and carbohydrates, the latter is the least explored and also the most complicated to sequence and synthesize. However, the last decades of research have emphasized the

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important roles of carbohydrates for cell-cell interactions as well as interactions with bacteria, fungi, and virus. Proteoglycans (PG) are large macromolecules that consist of a core protein decorated by large linear, negatively charged, carbohydrate chains called glycosaminoglycans (GAG). The PGs are found on the cell surface as well as in the extracellular matrix (ECM) where they have important roles in the regulation of growth factor signaling, inflammation, angiogenesis, and cell-cell interactions.1-3 The carbohydrate structures of the ECM and the cell surfaces are constantly changing, on a time scale of minutes, depending on the cells’ requirements of e.g. growth signals. β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the synthesis of two

different

classes

of

GAG

chains,

i.e.

heparan

sulfate

(HS)

and

chondroitin/dermatan sulfate (CS/DS). The first two steps in the synthesis of these GAG chains are xylosylation of a suitable serine residue of a proteoglycan core protein by xylosyltransferase (XT), followed by galactosylation by β4GalT7 (Figure 1a).4 By step-wise additions of another galactose unit and a glucuronic acid (by the enzymes β3GalT6 and GlcAT-I respectively), a tetrasaccharide linker is formed, which is a branching point for the biosynthesis of HS and CS/DS.

Figure 1. a) Biosynthesis of the linker tetrasaccharide of HS and CS/DS PGs. b) Galactosylation of XylNap (5) to form GalXylNap.

It is well known that simple xylosides, such as 2-naphthyl β-D-xylopyranoside (XylNap), can initiate the biosynthesis of soluble GAG-chains, i.e. structures not connected to a core protein (Figure 1b), and also inhibit the biosynthesis of the natural structures.5-7 We have recently developed assays for measurement of galactosylation


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by, as well as inhibition of, β4GalT7.8 Furthermore, from several series of xyloside analogs modified in the xylose moiety8,9 as well as by alterations in the aglycon,10 we have developed a pharmacophore model for β4GalT7 (Figure 2).

Figure 2. Pharmacophore model of β4GalT7

In our previous studies, we concluded that most alterations concerning the hydroxyl groups of the xylose moiety resulted in compounds without capability to function as substrates for β4GalT7. However, several analogs functioned as moderate inhibitors. Alterations of the aglycon did affect the priming ability, but did not increase inhibition.10 The xylose moiety is thus the prioritized target for design of efficient inhibitors. From our recent investigations,8,11 we conclude that position 3 is of high importance for the binding of the substrate to the active site, and modification of this position generally renders inactive compounds. However, exchange of the hydroxyl group at position 4 for fluoride as well as epimerization gave relatively strong (43 and 52% respectively) inhibitors of galactosylation by β4GalT7. Highest inhibitory effect, i.e. 64%, was achieved by deoxygenation of position 2. Here we explore a set of seemingly simple xylose derivatives with double modifications (Chart 1) to fine-tune the pharmacophore model for β4GalT7. Our hypothesis is that the combination of modifications in positions 2 and 4 may generate strong inhibitors. Despite the intrinsically low degree of complexity of these monosaccharides, as well as the profound knowledge on carbohydrate synthesis and readily available starting materials with defined stereochemistry, it turned out to be a complicated and time-consuming process. We thus want to discuss the chemistry


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associated with synthetic modifications of monosaccharides and point to the continuous need for further development of synthetic strategies.

Chart 1. Overview of synthetic targets, i.e. xyloside analogs, modified in positions 2 and 4.

Results and discussion A. Synthesis Xylose is a pentopyranoside and thus lacking a primary hydroxyl group. The three remaining hydroxyl groups are secondary and equatorial, and consequently exhibit similar nucleophilicity. Despite the similarities between the three hydroxyl groups, there are minor differences in reactivity. In general, for α-xylosides, hydroxyl group O-2 is the more reactive, followed by O-4 and O-3. For β-xylosides, O-4 is the more reactive followed by O-3 and O-2.12 There are, however, contradicting reports regarding the reactivity order.9 Furthermore, xylose exhibit conformational flexibility, mainly between the 4C1-, the 1C4-, and the 2SO-conformations, which complicates structure determination but also opens up for new synthetic strategies.8 Although a number of methods have been developed for selective introduction of protective groups such as esters, ethers, and acetals,12 the selectivity is still highly dependent on the anomeric configuration, the aglycon, and the substitution pattern. Furthermore, aglycon and substituents may alter the conformation, which in turn has large impact on the reactivity, and enables creative protective group strategies.13 To summarize, despite the simplicity of xylose, it is difficult to generalize a synthetic protocol for the synthesis of xyloside analogs.


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In this project, we have focused on binary combinations of four different modifications of the xylose moiety, i.e. methylation, epimerization, deoxygenation, and fluorination. Methylation of any hydroxyl group does not significantly alter the stereoelectronics of the molecule or induce steric hindrance. Therefore, it can be performed at almost any time during the synthesis. On the other hand, deoxyfluorination consists of two distinctive steps, i.e. epimerization and fluorination and must be carefully planned into the synthetic scheme. The relative stereochemistry of the hydroxyl groups is of outermost importance and in general terms cis-diols allow for selective protective group manipulations, in contrast to trans-diols (Figure 3). Furthermore, a participating equatorial protective group in position 2 is crucial for stereoselective introduction of the aglycon. Thus, the aglycon should preferably be introduced early on in the synthesis. In addition, deoxygenation of position 2 generally renders acid labile compounds.9 Deoxygenation of position 2 alters the electronic properties of the molecule which leads to increased electron density at the anomeric position and therefore higher rates of hydrolysis.14 Unpublished data from our group indicate that 2-deoxy naphthyl xylosides are also base labile. Alkali sensitivity of phenyl glucosides have been known for a long time,15 and the hydrolysis is envisioned to proceed through an elimination of the neighbouring hydrogen, followed by rapid addition of solvent to the unsaturated sugar. To summarize, in order to facilitate the synthesis of binary modified xyloside analogs, the aglycon should be introduced early on, an axial hydroxyl group in position 4 is favorable, and deoxygenations should be performed late in the synthesis. methylation at any time cis-diol good selectivity

OH O HO

aglycon preferably introduced first

O

OH

deoxygenation acid lability trans-diol poor selectivity

HO HO

O

O

OH

epimerization necessary before fluorination

Figure 3. Synthetic considerations.


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Generally, starting materials for modified glycosides are provided by the chiral pool. Of the eight aldopentoses, four are disregarded due to unfavorable stereochemistry. The remaining pentoses are D-xylose, D-lyxose, L-arabinose, and L-ribose. Further on, the need for a participating group in position 2,10 makes D-lyxose and L-ribose less favorable as starting materials. This leaves D-xylose and L-arabinose as the starting materials of choice. Interestingly, D-xylosides can easily be converted to D-lyxosides as well as L-arabinosides by an oxidation-reduction pathway.16 Apart from the chiral pool approach, a de novo synthesis might be practical for some compounds, mainly dideoxy analogs, but less suitable for most other modifications due to the need for asymmetric introduction of stereocenters and/or enantiomeric purification.17 The discussion is divided into sections, first describing the initial protection scheme followed by specific transformations, i.e. methylations, formation of xanthate esters, fluorinations, epimerizations, and finally deoxygenations. An overview of the syntheses can be found as Scheme S1 (Supporting information).

Synthesis of suitably protected starting materials As mentioned earlier, there are only minor differences in reactivity between the three equatorial hydroxyl groups in xylopyranosides.12 For example, formation of the isopropylidene acetal by reaction with 2-methoxypropene and catalytic amounts of CSA gave a 10:3 distribution of the 2,3- and 3,4-protected compounds in 67% yield,16 while protection with 2,2,3,3-tetramethoxybutane/BF3⋅OEt2 gave the 2,3- and 3,4BDA-protected compounds in a 2:3 ratio in quantitative yield.9 Direct methylation of the unprotected xylopyranoside 5 using NaH and MeI gave a complex mixture of mono-, di-, and tri-substituted xylosides, with starting material still present. An acetylation-separation-deacetylation sequence was required to isolate the final 2,4-methoxy product 2b in 9% yield over three steps (Scheme 1a). However, the simplicity as well as the readily available starting material justified the use of this method.


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a) O

HO HO

i)

ONap

OH

9%

5

b)

ONap OMe

+ mix of methylated compounds

2b

OH O

HO HO

O

MeO HO

ONap

O O

ii) HO O 96%

6

O O

iii)

ONap

MeO 86% O

7

ONap

8

iv) 89% OH O

MeO HO

ONap

4b

Scheme 1. a) Protection of 2-naphthyl D-xyloside. Reagents and reaction conditions: i) NaH, MeI, DMF, 0 °C to r.t. b) Protection of 2-naphthyl D-lyxoside. Reagents and reaction conditions: ii) 2,2-dimethoxypropane, p-TsOH (cat.), DMF, rt; iii) NaH (60 % dispersion in mineral oil), MeI, DMF, 0 °C→rt; iv) 70% AcOH (aq.), 70 °C, 0.5 h. In contrast, the cis-diol in lyxose derivatives facilitates the subsequent protection scheme and the intermediate 7 was formed in 96% yield from 6 (Scheme 1b).9 Subsequent methylation to give 8, followed by deprotection using acetic acid, gave 4b. We then intended to use 4b as starting material for further transformations. However, the introduction of a benzoyl group on the equatorial hydroxyl 3-OH in 4b, using a borinic acid catalyst, surprisingly yielded the 2-O-benzoyl ester 9 as the major product (Scheme 2).18 Benzoylation using the standard method, i.e. BzCl/pyridine, gave a mixture of 9 and the disubstituted 10 in a 1:3 ratio according to 1H-NMR (Supporting information).


7


i) 50%

OBz O

MeO HO

Page 8 of 58

ONap

9

MeO HO

OH O

ONap

ii) (1:3)

OBz O

MeO HO

ONap

9

4b

OBz O

+ MeO BzO

ONap

10

OEt O

iii) 73% (two steps)

O

ONap O

OH O

MeO AcO

OMe

ONap

11

Scheme 2. Protection of 2-naphthyl 4-methoxy-L-lyxoside. Reagents and reaction conditions: i) BzCl, DIPEA, 2-aminoethyl diphenylborinate, CH3CN, rt; ii) BzCl, pyridine, CH2Cl2, 0 °C; iii) 1. triethylorthoacetate, p-TsOH (cat.), CH3CN; 2. 1M HCl

The reversed selectivity in the benzoyl protection indicates a conformational change during the reaction. Introduction of an orthoester to 4b, followed by mild acid hydrolysis formed 11 in 73%, accompanied by the regioisomer (21%). A crude NMR of the intermediate orthoester revealed a shift from the 4C1 to the 1C4 conformation, making 3-OH the axial position. Several attempts to introduce an orthoester to unprotected 6 failed. The arabinoside 12 can be synthesized from isopropylidene protected xyloside by Swern oxidation followed by in situ reduction using NaBH4 at 0 ºC.16 12 Can also be synthesized directly from readily available per-O-acetylated arabinose19 (supporting information). The cis-diol in arabinosides facilitates selective protection. Thus, isopropylidination, using 2,2-dimethoxypropane/p-TsOH yielded the 3,4protected compound 13 exclusively (Scheme 3), suitable for transformation of position 2-OH.20-22


8

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O

i) 95%

O ONap

O OH 13 OH

ii) 46% (1:1)

OH O

MeO ONap

O

O

+

OMe O ONap

O

O

OH

OMe 14

ONap

HO

OMe O

OH

15

OH

12

iii)

O ONap

BzO OH

64%

16 OH

iv)

ONap

60% BnO (1:1)

OH OH

v) 57% BnO

OBn O

+

O ONap

HO OH

17

18

O ONap OH 17

Scheme 3. Protection of 2-naphthyl L-arabinoside. Reagents and reaction conditions: i) 2,2-dimethoxypropane, p-TsOH (cat.), DMF, rt, 1 h; ii) 2,2,3,3-tetramethoxybutane, BF3⋅OEt2 (cat.), CH3CN; iii) 2-aminoethyl diphenylborinate, DIPEA, BzCl, CH3CN, rt, 29h; iv) 2-aminoethyl diphenylborinate, BnBr, Ag2O, CH3CN, 40 °C, 30 h; v) 2aminoethyl diphenylborinate, BnBr, KI, K2CO3, CH3CN, 60 °C, 22 h.

In order to selectively protect arabinosides, several methods were tested. It is well known that BDA-acetals preferably are formed over trans-diols.9 To our surprise, BDA protection of 12 resulted in a 1:1 mix of the 2,3- and 3,4-protected compounds 14 and 15 in low yields (Supporting information).23 Instead, attempts were made to selectively protect 3-OH using a borinic acid catalyst.18,24 This gave the 3-Obenzoylated compound 16 in 63% yield. Benzylation using the borinic acid methodology with Ag2O formed a 1:1 mixture of the 3- and 4-benzylated sugars 17 and 18 in modest yields. However, a metal-free version, gave solely the 3-Obenzylated sugar 17. The 4-deoxy-L-threo-pentopyranoside 19 was synthesized from XylNap (5) as described before.9 Benzylation of 19 using the borinic acid methodology gave the two mono-benzylated compounds 20 and 21 in a 1:1 ratio with no di-substitution (estimated by 1H-NMR, Scheme 4, Supporting information). Attempts to introduce a


9


Page 10 of 58

MOM-ether gave low yields, in spite of elevated temperature and long reaction time. TBDMS protection using TBDMSCl with DMAP/Et3N and microwave heating gave 23, protected in position 3, as the major product (48%), with minor amounts of 22, disubstituted 24, and unreacted starting material (21%). Finally, methylation of 19 using NaH and MeI gave the 2-methoxy product 2a in 42% yield, accompanied by 17% of 26 and 6% of 25. i)

O HO

ONap

OBn

(1:1)

O +

BnO

20

O HO

ONap

OH 19

ii)

21

O

O

ONap + TBDMSO OTBDMS

HO

67% (15:48:4)

22

OH

ONap + OMe

HO

65% (42:6:17)

2a

ONap OTBDMS

24

O MeO

O

ONap + TBDMSO

23

O

iii)

ONap

OH

ONap

OH

O +

ONap OMe

MeO

25

26

Scheme 4. Protection of naphthyl 4-deoxy-L-threo-pentopyranoside. Reagents and reaction conditions: i) 2-aminoethyl diphenylborinate, BnBr, KI, K2CO3, CH3CN, N2, 60 °C, 12 h; ii) TBDMSCl, DMAP, Et3N, CH2Cl2, MW 100 °C, 50 min; iii) MeI, NaH (60% dispersion in mineral oil), DMF, 0 °C→rt, 4 days.

With a suitable set of protected sugars, we then initiated transformations into the target compounds.

Methylations As mentioned before, methylations can be performed at almost any time during the synthesis. The general method for methylation has been Williamson alkylations. However, multiple unprotected hydroxyl groups generally give selectivity problems, resulting in complex product mixtures. These compounds often co-elute during chromatographic separations. This was solved by an acetylation-purificationdeacetylation sequence. The isopropylidene protected arabinoside 13 was methylated to give 27 in 93% yield (Scheme 5). The isopropylidene acetal was deprotected by mild acid hydrolysis in 70% AcOH to give 2d. Mono-methylation of the D-threopentopyranoside 289 gave a complex mixture of 29, 1b, 30 as well as recovered ACS Paragon Plus Environment

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starting material (21%). Finally, the 2-deoxy-2-fluoro-D-xyloside 319 was methylated under phase-transfer catalysis (PTC) to give the two regioisomers 32 and 3b, as well as recovered starting material (56%). . O

O O

O

ONap

OH 13

O

88%

HO 2d

27

O

HO HO

ONap OMe

O

93%

OH

ii)

O

i)

ONap

i)

47% (10:16:21)

28

O

HO MeO

ONap

29

O

MeO HO

ONap

O F 31

iii)

ONap

36% (1:1)

ONap

30

O

MeO HO

O

MeO MeO

1b

HO HO

ONap OMe

ONap

F 3b

O

HO MeO

ONap

F 32

Scheme 5. Methylations. Reagents and reaction conditions: i) a) NaH (60% dispersion in mineral oil, DMF, 0 °C, 0.5 h b) MeI; ii) 70% acetic acid in H2O, 60 °C; iii) TBAB, MeI, CH2Cl2, 0.5 M NaOH. Formation of xanthate esters The introduction of a methyl xanthate ester on substrates with only one unprotected hydroxyl is generally straightforward, and gives the desired product in high yields (33, 35) (Scheme 6). The isopropylidene acetals were then deprotected by mild acid hydrolysis in 70% AcOH to give 34 and 36. Formation of the xanthate ester in the 3-OTBDMS protected 23 gave both the expected compound 37 as well as 38 as an inseparable mixture (2:1) according to 1HNMR (Supporting information). We speculate in that the unprotected deprotonated hydroxyl causes migration of the TBDMS group, followed by xanthate ester formation. Addition of carbon disulfide prior to deprotonation gave the desired product 37. Removal of the TBDMS group in 37 using alkaline conditions with TBAF in THF resulted in xanthate ester migration and a complex mixture of byproducts. Acidic deprotection with AcOH was not successful either. 1% HCl in EtOH gave the desired product 3917 in 85% yield.


11


O

O O

O

i)

ONap

OH

87%

O

13

HO

ONap OCS 2Me

85%

HO

33

O O O

MeS 2CO O 85%

ONap

ONap

O

ONap

36

ONap OCS 2Me

O +

ONap OTBDMS

MeS 2CO 38

37

ONap

OH O

MeS 2CO HO 95%

O TBDMSO

ONap

OCS 2Me

ii)

35 i) (2:1)

OH O 34

O O

i)

7

TBDMSO

ii)

O

Page 12 of 58

iv, v)

OH

O

23 iii) 83%

ONap OCS 2Me

TBDMSO 37

O vi)

ONap OCS 2Me

HO

85%

39

Scheme 6. Formation of xanthate esters. Reagents and reaction conditions: i) 1. NaH (60% dispersion in mineral oil), THF, 0 °C→rt, 0.5 h; 2. CS2; 3. MeI; ii) 70% acetic acid in H2O, 60 °C; iii) 1.CS2, THF, 0 °C, 2. NaH (60% dispersion in mineral oil), 0 °C →rt, 0.5 h; 3. MeI; iv) 1M NaOH, MeOH, CH2Cl2, rt, 1 h; v) HCl (conc., 22 eq.), Pd/C (10 mol%), DMF, H2 (1 atm); vi) TBAF, THF, 0 °C. The introduction of bulky substituents in position 2 of arabinosides often results in conformational changes from the 4C1 conformation to the 1C4 conformation.25-27 Thus, protection of 2-OH as a xanthate (34) resulted in a ring-flip (Scheme 6). Subsequent benzoylation using the borinic acid methodology, gave a 1:5 mixture of the 3- and 4O-benzoylated compounds 40 and 41 (Scheme 7), according to 1H-NMR (Supporting information). However, benzoylation using BzCl in pyridine at -42 °C,26 gave 3-OBz 40 as the major product accompanied by recovered starting material (24%). However, migration of the benzoyl group to 4-OH occurred during purification by chromatography. Introduction of an orthoester followed by mild acidic conditions gave the 4-O-acetylated compound 42 (Supporting information).28,29


12

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i)

OBz O

HO

(1:5) 40

HO

OH O 34

ONap

OCS 2Me

ii)

HO

40

EtO iii)

BzO

OCS 2Me

OBz O

59% (5:1)

ONap

41

ONap BzO

OCS 2Me

O

O

ONap OCS 2Me

AcO

OCS 2Me ONap

OH O 41

O

ONap

OH O

OCS 2Me

OH O

ONap

OCS 2Me 42

Scheme 7. Formation of 2-xanthate protected L-arabinosides. Reagents and reaction conditions: i) BzCl, DIPEA, 2-aminoethyl diphenylborinate, CH3CN, rt; ii) BzCl, pyridine, -35 °C, 4 h; iii) triethyl orthoacetate, p-TsOH (cat.), CH3CN, rt, 1 h.

Most attempts to introduce a single xanthate ester on a starting material with more than one free hydroxyl group gave either no or minor amounts of the target compound, accompanied by varying amounts of cyclic thionocarbonates, tri-, di-, and mono-xanthate products depending on the reaction conditions. Attempts were also made to deliberately introduce a cyclic thiocarbonate, intended for radical deoxygenation. The same reagents as for methyl xanthate ester introduction were used and a large excess of reagents together with unprotected 12 gave the thiocarbonate (cis only) in approximately 10% yield, in addition to all possible mono-, di-, and tri-xanthate ester products except for the 2-OH xanthate ester (Supporting information).

Fluorinations We have previously performed deoxy-fluorinations on each one of the three hydroxyl groups in xylosides, using a sequence of hydroxyl group inversion followed by fluorination by diethylaminosulfur trifluoride (DAST).9 The obvious starting material for the synthesis of compound 3d is a suitably protected riboside. The 3,4-isopropylidene protected riboside 43 (Scheme 13) was found to reside in the 1C4 conformation and fluorination, using DAST, gave exclusive rearrangement to the anomeric fluoride 44 (Scheme 8) instead of 45. This kind of rearrangement has been observed earlier.30,31 TLC analysis indicated that the reaction intermediate formed at 0 °C, but rearranged at higher temperatures. No reaction was


13


Page 14 of 58

observed using the alternative reagent XtalFluor-M™ (activated by Et3N·3HF, Et3N·2HF or DBU).32,33 The intermediate formed readily with PyFluor™ but the reaction did not proceed into product.

O

O O

ONap O F

i)

O

O O

S

N F

ONap

O O

ii)

OH

O

29%

43

F ONap

44

iii)

O

O O

ONap

O

O

S O O

ONap

O O

N

45

F

v)

iv)

O

O O

ONap OTf

vii)

O O

vi) (80%)

ONap

O 46

H

Scheme 8. Attempts to synthesize 3d. Riboside pathway. Reagents and reaction conditions: a) i) DAST, CH2Cl2, 20 ºC, iii) PyFluor™, toluene, rt; iv) Tf2O, pyridine, -42 °C→rt; v) Et3N⋅3HF, THF, rt →40 °C; vi) DBU, THF, reflux (DBU); vii) SelectFluor™, CH3CN, rt. All attempts of fluorinations via the triflate were equally unsuccessful (Scheme 8). The relatively mild nucleophilic fluorination reagent triethylamine trihydrofluoride (TREAT) gave no reaction and the more basic tetrabutylammonium fluoride (TBAF) gave 46 in trace amounts. Instead, 46 could be formed in good yield using a nonnucleophilic base (DBU). Electrophilic fluorine (SelectFluor™) gave only trace amounts of the wanted compound.


14

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O

O O

O

i)

ONap

OH O

O

96%

OH

ii)

ONap

O

HO

OTs

13

47

OH

F

ONap

OTs 48

OH O

OH O

iv)

ONap

iii) 86% (two steps)

O

ONap

60% 50

49

Scheme 9. Attempts to synthesize 3d. Arabinoside pathway. Reagents and reaction conditions: i) pTsCl, ZnBr2, pyridine, -40 °C→rt; ii) 70% AcOH (aq.), 12 h then iii) 0.05 M NaOMe, MeOH, rt; iv) TBAF, p-TsOH (cat.), toluene, 80 °C.

The next approach was epoxidation of the arabinoside 13 (Scheme 9). Tosylation of the 2-OH in 13, with addition of ZnBr2,34 gave 47 in 96% yield. Deprotection of the isopropylidene acetal gave 48 which was ring closed to form the 2,3-anhydro-Lriboside 49. There are several nucleophilic fluoride reagents that have been used to open epoxides, such as TREAT,35 TBAF,36 and potassium hydrogen difluoride (KHF2).37,38 The epoxide 49 was quite inert and neither fluorination, nor naphthol cleavage occured using treatment with TBAF, Et3N•3HF or Et3N•2HF. However, addition of pTsOH to TBAF in toluene at 80 °C, opened the epoxide to give the 3-fluorinated Lxyloside 50 (Supporting information). KHF2 at 130 °C in ethylene glycol, gave a similar outcome, but with decreased reaction rate. Finally, the 2-deoxy-2-fluoro xyloside 319 was used as starting material (Scheme 10). Position 4 was selectively tosylated to give 51 and subsequently inverted into the 2-deoxy-2-fluoro-L-arabinoside 52, which was immediately deprotected, to avoid acetate migration, to give 3d in 27% over-all yield. OAc O

HO HO

F 31

i)

O

TsO ONap HO 24%

F

ONap

ii)

O HO

ONap

F

51

52 iii) 27% (two steps)

OH O HO

ONap

F 3d


15


Page 16 of 58

Scheme 10. Synthesis of 3d. 2-deoxy-2-fluoroxyloside pathway. Reagents and reaction conditions: i) TBAHSO4, p-TsCl, CH2Cl2 /5%NaOH; ii) AcOH-DBU (12:6 eq), toluene, 90 °C then 0.05 M NaOMe, MeOH, rt. Regarding the remaining 2-F compounds, fluorination using DAST on the 3-acetyl-4methoxy-lyxoside 11 gave only 15% of the desired product 53 whereas the analogous xanthate ester 36 gave 54 in 53% yield over two steps (Scheme 11). Interestingly, fluorination of the isopropylidene protected 5516 gave no reaction, only rearrangement into the anomeric fluoride 56, according to 1H-NMR (Supporting information), whereas the analogous BDA protected compound 57 gave 40% of the fluorinated compound 58.9 The fluorination reactions gave incomplete conversion and starting materials were usually recovered. OH O

MeO AcO

ONap

i)

11

53

OH O

MeS 2CO HO

ii)

ONap

36

OH O

O O

iii)

54

O

O

OMe 57

ONap O F

56

OH O

ONap

F

O O

ONap

ONap

F O

MeS 2CO AcO

53% (two steps)

55

OMe

O

MeO AcO

15%

OMe i)

ONap

40%

O

O O

OMe 58

ONap

F

Scheme 11. Fluorination of position 2. Reagents and reaction conditions: i) DAST, CH2Cl2, 40 °C),9 ii) triethyl orthoacetate, p-TsOH (cat.), CH3CN, rt, 1.5 h, then DAST, CH2Cl2, 40 °C). iii) DAST, CH2Cl2, sealed tube, 50 °C. Due to the axial configuration of 4-OH in L-arabinosides, they could serve as starting materials for analogs with fluorine in position 4. Fluorination of the monoprotected arabinosides 16 or 17 gave the corresponding fluoro analogs 59 and 60 in modest yields, leaving 2-OH open for further transformations (Scheme 12). The rather low yields are justified by the synthetic simplicity. In the synthesis towards the 4-deoxy-4-fluoro analog 1c, the xanthate group was used as a protective group39 that facilitated fluorination, similar to the


16

Page 17 of 58 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


fluorination of 36. Thus, fluorination of 40 gave the corresponding fluoro analog 61 in 22% yield together with recovered 40 (47%). The 2-O-methylated arabinoside 2d was fluorinated without additional protecting groups to give the final product 2c in 10% yield. Fluorination of the Lerythropentopyranoside 1d (Scheme 15) with DAST led to fluorination solely in position 3, to give 62 in 31% yield. This suggests that the conformation of the starting material, under these reaction conditions, is skewed in favor of the axial 3-OH, leading to fluorination in position 3 due to better orbital positioning. OH O BzO

i)

ONap

OH

22%

O

F BzO

ONap

OH

16

59

OH O BnO

ONap

i) 28%

OH

O

F BnO

17

HO

ONap

OH 60

OBz O

ONap

OBz O

i)

ONap

47% 40

OCS 2Me

F

OCS 2Me

61

OH O HO 2d

ONap OMe

i) 10%

O

F HO

ONap OMe

2c

OH

OH O

HO

ONap

1d

O

i) 31%

F

ONap

62

Scheme 12. Fluorination of position 4. Reagents and reaction conditions: i) DAST, CH2Cl, 40 °C. Epimerizations Previous work involving inversions of different positions of naphthoxylosides have involved the Latrell-Dax reaction with nitrite on positions 3 and 4, triflate displacement with CsOAc on position 2,9 as well as Swern oxidation-reduction sequences.16 Several methods were attempted to invert the hydroxyl group in position 2 of the L-arabinoside 13 (Scheme 13). Using the same methodology as for inversion of position 2 in xylose, i.e. displacement of the triflate with CsOAc, gave only 11% of the inverted product 63. Attempts using Mitsunobu conditions were unsuccessful. The Latrell-Dax reaction gave a modest increase of the yield of 43 (21%). These low


17


Page 18 of 58

yields can be attributed to the instability of the intermediate triflate. Interestingly, the Latrell-Dax reaction has been used to make simultaneous inversion of multiple hydroxyl groups.40 Therefore, this method was attempted on 3-O-benzoylated Larabinoside 21. In our hands, the reaction only gave single inversion, i.e. the xylose configuration (64) according to 1H-NMR (Supporting information). Oxidation of 13 to the ketone using Dess-Martin periodinane, followed by NaBH4 reduction gave 43 in 60% yield over two steps. Oxidation using TPAP with NMO in acetonitrile resulted in naphthol cleavage. A modified Albright-Goldman oxidation41 under microwave irradiation, followed by reduction by NaBH4 gave 43 in 75% yield over two steps. The isopropylidene acetal was cleaved off by mild acid hydrolysis in 70% AcOH 4d. Attempts to use the Albright-Goldman procedure for inversion of 2-OH of 4fluorinated xyloside 59 gave an unexpected outcome. After reduction, the product obtained was not the expected 4-fluoro-3-O-benzoylated lyxoside, but rather the 4deoxygenated compound 65. The Albright-Goldman oxidation is accomplished under alkaline conditions and we speculate in that the reaction proceed through an E1cB mechanism with abstraction of the acidic α-proton and subsequent elimination of the fluorine to form the α,β-unsaturated compound. 1H-NMR of the crude reaction mixture revealed a vinylic proton that couples to the H-5eq and H-5ax. The acetate ester in compound 65 was cleaved by alkaline hydrolysis using NaOMe/MeOH to give 4a. O

O i)

O O

ii)

ONap

OTf

O

O

11% iii)

63

ONap

OH

O

iv)

13

or v)

O O

vi)

ONap

60% (route iv) 75% (route v)

O

O O

O OH i)

ONap

O BzO

OH

OH

59%

OH OH O HO

ONap

4d

iii)

ONap

OTf

O

HO BzO

ONap

OH 64

O OH 59

vii)

43

21

F BzO

ONap

OTf O

BzO

ONap

21%

O O

OAc O

v)

O

ONap BzO

O

ONap

OH O

vi) 45%

BzO

90% 65

OH O

viii)

ONap

HO

ONap

4a

Scheme 13. Epimerizations. Reagents and reaction conditions: i) Tf2O, pyridine; ii) CsOAc, DMF, 50 °C; iii) TBANO2, DMF, 50 °C; iv) Dess-Martin periodinane,


18

Page 19 of 58 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NaHCO3, CH2Cl2; v) DMSO, Ac2O, MW 80 °C, 5 min.; vi) NaBH4, MeOH, 0 °C; vii) 70% acetic acid in H2O, 60 °C; viii) 1M NaOH, MeOH, CH2Cl2, rt, 1 h.

A mild nucleophilic displacement of the 3-O-benzylated 60 (Scheme 14) using triflate formation followed by addition of AcOH:DBU in heated toluene,42 gave 67% of fluorinated 66. The acetate ester was cleaved by alkaline hydrolysis using NaOMe/MeOH to give 67, and subsequent debenzylation using a procedure with DMF/HCl to avoid saturation of the naphthyl moiety43 gave 4c. Fluorination of 67 gave the difluoro compound 68 in 61% yield. The benzyl group in 68 was then hydrogenolyzed in DMF/HCl to give 3c. The relatively high yield in this fluorination procedure, in comparison to the fluorinations in Scheme 14, indicate that an electron-withdrawing group in position 4 facilitates fluorination of position 2.

O

F BnO

i)

O

F BnO

ONap

OH

ii)

ONap

OTf

OAc O

F BnO

67%

iii, iv)

ONap

78%

F HO

OH O

ONap

4c

60

66 iii) 88%

O

F HO

ONap

F

iv) 79%

O

F BnO

3c

F

ONap

v) 61%

OH O

F BnO

ONap

67

68

Scheme 14. Synthesis of naphthyl 2,4-difluoroxyloside. Reagents and reaction conditions: i) Tf2O, pyridine; ii) AcOH-DBU (12:6 eqs), toluene, 85 °C; iii) 1M NaOMe, MeOH, rt; iv) HCl (conc., 22 eq.), Pd/C (10 mol%), DMF, H2 (1 atm). v) DAST, CH2Cl2, 40 °C).9

Deoxygenations The most common procedure for deoxygenation of a secondary alcohol is the BartonMcCombie method.44,39 Deoxygenation of methyl xanthates is normally performed using a hydride source in combination with a radical initiator. For environmental reasons, hypophosphorous acid with AIBN and Et3N was used in these deoxygenations, rather than the more common organic tin hydrides.9 The success of the radical deoxygenation with these compounds appears to be highly dependent on which position to be deoxygenated, the substituents present at


19


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other positions, the concentration and rate of addition of AIBN solution, and the pH of the reaction mixture. The use of too weak AIBN solution (

Synthesis of Double-Modified Xyloside Analogs for Probing the

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