Synthesis and evaluation as glycosidase inhibitors of carbasugar-derived spirodiaziridines, spirodiazirines, and spiroaziridines

Article abstract of DOI:10.1002/hlca.200390178

The spirodiaziridines 6 and 9, potential inhibitors of α- and β-glucosidases, were prepared from the validoxylamine A-derived cyclohexanone 5. The trimethylsilyl protecting groups of 5 are crucial for the formation of 6 in good yields. Oxidation of 6 gave

Full text of DOI:10.1002/hlca.200390178

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Helvetica Chimica Acta Vol. 86 (2003)
Synthesis and Evaluation as Glycosidase Inhibitors of Carbasugar-Derived
Spirodiaziridines, Spirodiazirines, and Spiroaziridines
by Peter Kapferer, Ve¬ronique Birault 1), Jean-FranÁois Poisson2), and Andrea Vasella*
Laboratorium f¸r Organische Chemie, ETH-Hˆnggerberg, HCI, CH-8093 Z¸rich
Dedicated to Professor Wolfgang Steglich on the occasion of his 70th birthday
The spirodiaziridines 6 and 9, potential inhibitors of a- and b-glucosidases, were prepared from the
validoxylamine A-derived cyclohexanone 5. The trimethylsilyl protecting groups of 5 are crucial for the
formation of 6 in good yields. Oxidation of 6 gave 7. The diaziridine 6 (pKHA ˆ 2.6) and the diazirine 7 did not
inhibit the b-glucosidases from almonds, the b-glucosidase from Caldocellum saccharolyticum, and the a-
glucosidase from yeast. The N-benzyl diaziridine 9 is a very weak inhibitor of the a-glucosidase, but did not
inhibit the b-glucosidases. To see whether the weak inhibition is due to the low basicity of the diaziridines or to
geometric factors, we prepared the spiro-aziridines 21 and 25 and 1-epivalidamine (32). The known
cyclohexanone 10 was methylenated and epoxidised to 16 and 17. Azide opening of 16 and 17, mesylation,
LiAlH4 reduction, and deprotection gave the aziridines 21 and 25 respectively. 1-Epivalidamine (32) was
prepared from the known carba-glucose 29. The aziridine 25 (pKHA ˆ 6.8) is a weak irreversible inhibitor of the
b-glucosidase from Caldocellum saccharolyticum and a weak reversible inhibitor of the a-glucosidase from
yeast, but did not inhibit the b-glucosidases from almonds. The poorly stable aziridine 21 weakly inhibited the
three enzymes. Similarly, 1-epivalidamine (pKHA ˆ 8.4) proved only a weak inhibitor. The known cyclopentyl-
amine 34 (pKHA ˆ 7.9), however, is a micromolar inhibitor of these enzymes. The much stronger inhibition by 34
is related to the pseudoaxial orientation of its amino group.
Introduction. Alkoxydiaziridines of type 1 (Scheme 1) possess trans-oriented N-
atom lone-pairs located above and below the average plane of the pyranose ring and
oriented more or less parallel to the ring plane. These diaziridines may, thus, inhibit
both a- and b-glucosidases3). The inhibition of b-glycosidases by diaziridines of this
type may provide evidence for the correlation between the change of the pyranose-ring
conformation, orienting the scissile bond in a pseudoaxial orientation, and the
proton transfer to the glycosidic heteroatom characterising the reaction coordinate
linking the ground and transition states of the rate-determining step. The diaziridines 1
may be accessible by deprotection of the known benzyl ethers 2 [2]. We were, however,
concerned about the low basicity and the fragility of alkoxydiaziridines in aqueous
solution. Exploratory experiments indeed showed that the diaziridines 1 are not
sufficiently stable [3], so that we aimed at the synthesis of the carba analogues 6. To
differentiate between the effects of shape and charge on the inhibition, we also
required the related spiro-aziridines 21 and 254) (Scheme 2), and 1-epivalidamine
1
)
)
)
Postdoctoral fellow from January 1996 to October 1997.
Postdoctoral fellow from November 2000 to October 2001.
2
3
No diaziridines are known to inhibit glycosidases. Carbohydrate-derived diaziridines with the hydrazi
group spiro-linked to other centers have been synthesized by Kurz et al. as intermediates in the synthesis
of diazirines [1].
A range of carbohydrate-derived fused aziridines are known, and many of them inhibit glycosidases [4
12]. Carbohydrate derived spiro-aziridines have been prepared as intermediates in the syntheses of C(3)-
branched 3-amino sugars, as reviewed in [13].
4
)

Helvetica Chimica Acta Vol. 86 (2003)
2211
(325), Scheme 3) [14]; their basicities are expected to increase in the order 6 < 21, 25 <
32.
Results and Discussion. Synthesis of the Diaziridines and Diazirines. We planned
to synthesise the diaziridines 6 by treating the trimethylsilyl ether 5 of validone (4) [15]
with NH3 and hydroxylamine-O-sulfonic acid in MeOH [16 19], although Kurz et al.
[1] and Thieme [20] reported low yields for a related synthesis of a 4-hydrazipyranoside.
Oxidation of 6 should provide the diazirine 7.
Scheme 1
a) N-Bromosuccinimide (NBS), MeCN, H2O ([15]); 50%. b) Me3SiCl (TMSCl), 1,1,1,3,3,3-hexamethyldisil-
azane (HMDS), pyridine; 27% (chrom.). c) NH3, MeOH, then NH2OSO3H; 50%. d) I2, Et3N, MeOH; 89%. e)
Ac2O, pyridine; 84% from 6. f) BnNH2, MeOH, then NH2OSO3H; 27%. g) NH3, MeOH, then NH2OSO3OH;
35%. h) I2, Et3N, MeOH, CH2Cl2; 85%.
Validone (4) was prepared by N-bromosuccinimide (NBS) cleavage of validoxyl-
amine A6) (3) [15] and isolated in a yield of 50% (Scheme 1). Trimethylsilylation of 4
5
)
)
1-Epivalidamine (32) is a competitive inhibitor of pig kidney trehalase (Ki ˆ 1.2 mm) [14]. To the best of
our knowledge, the inhibition by this compound against the enzymes studied here has not been reported.
We thank Dr. A. G. O×Sullivan, Syngenta, Basel, for a generous gift of validoxylamine A.
6

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Helvetica Chimica Acta Vol. 86 (2003)
[21] gave crude 5 (94%) that was isolated by chromatography (27%)7). Treatment of a
solution of 5 in MeOH and NH3 with hydroxylamine-O-sulfonic acid gave crude 6
(69%), which oxidised iodide in acidic solution [22 24]. Flash chromatography gave a
solution of 6, pure by TLC, but complete evaporation of the solvent (AcOEt, i-PrOH,
H2O), even below 108, resulted in partial decomposition. Pure 6 was obtained in 50%
yield by ion-exchange column chromatography of a concentrated solution.
The diaziridine 6 was also obtained by treating validone (4) with NH3 and
hydroxylamine-O-sulfonic acid in MeOH. However in agreement with previously
reported results [1][20] the yield of 6 was lower (28% of crude 6), and the reaction
less clean, so that 6 could not be obtained pure. The Me3Si groups are cleaved off during
the diaziridine syntheses and appear to play an active role in the diaziridine formation.
This might be due to the in situ formation of 1,1,1,3,3,3-hexamethyldisilazane (HMDS),
or to sequestration of H2O formed in the condensation of NH3 with the ketone, rather
than to protection of the b-hydroxy ketone 4. To address this issue, we tested the
transformation of validone (4) in the presence of HMDS, and we also examined the
transformation of the benzylated validone 10 [15] into the benzyl-protected diaziridine
11 (Table 1). Treatment of 4 with HMDS and then with hydroxylamine-O-sulfonic acid
led to a lower yield of the diaziridine (20% of crude 6) than the reaction with NH3/
hydroxylamine-O-sulfonic acid (28%). Using HMDS and NH3 improved the yield
somewhat (38%), but it was still significantly lower than the one realised from 5 (69%).
Treating the benzylated validone 10 with NH3 and hydroxylamine-O-sulfonic acid
yielded 35% of 11, whereas the reaction with HMDS and NH3/hydroxylamine-O-
sulfonic acid provided 11 in slightly lower yield (29%). Treating 10 with excess Me3SiCl
and NH3/hydroxylamine-O-sulfonic acid resulted in a significantly higher yield (50%),
suggesting that the trimethylsilyl ether 5 acts as a H2O scavenger.
Table 1. Synthesis of 6 and 11 in the Presence of Silylating Agentsa)
Entry
Starting material
Reagents
Product (yield)
1
2
3
4
5
6
7
4
4
4
NH3, then NH2OSO3H
6 (28%)b)
6 (20%)b)
6 (38%)b)
6 (69%)b)
11 (35%)
11 (29%)
11 (52%)
HN(SiMe3)2 (4 equiv.), then NH2OSO3H
HN(SiMe3)2 (2 equiv.), NH3, then NH2OSO3H
NH3, then NH2OSO3H
NH3, then NH2OSO3H
HN(SiMe3)2 (2 equiv.), NH3, then NH2OSO3H
Me3SiCl (4 equiv.), NH3, then NH2OSO3H
5
10
10
10
a) Conditions: a solution of the starting material in MeOH was treated with HN(SiMe3)2 at r.t. (Entries 2, 3, and
6) or with Me3SiCl at À 208 (Entry 7), saturated with NH3 at À 208, treated dropwise with NH2OSO3H (1
equiv.), stirred at À 208 for 3 h, and allowed to warm to r.t. overnight. b) Crude after FC.
Oxidation of the diaziridine 6 with I2 in MeOH in the presence of Et3N gave a
mixture of the diazirine 7 and Et3N (2.5 :1), which could not be separated even by
repeated flash chromatography (FC); replacing Et3N with the more volatile Me3N led
7
)
An attempt to purify 5 by bulb-to-bulb distillation (1308, 0.5 bar) resulted in partial decomposition. Only
chromatographically purified samples of 5 gave good results in the subsequent step.

Helvetica Chimica Acta Vol. 86 (2003)
2213
to a mixture of 7 and several unidentified by-products. Acetylation gave the tetra-
acetate 8 (84% from 6). Pure diazirine 7 (89%) was obtained from 7 ¥ Et3N by weak
acid ion-exchange chromatography. Treatment of the silylated validone 5 with BnNH2
and hydroxylamine-O-sulfonic acid in MeOH gave, after FC, a ca. 1:4 mixture of the
N-benzyl diaziridine 9 and BnNH2, from which pure 9 (27%) was obtained by
chromatography on a weak acid ion exchanger. The N-benzyl diaziridine 9 results from
equatorial attack of hydroxylamine-O-sulfonic acid on the N-benzyl imine formed in
8
situ from 5 and BnNH2 ).
The diaziridines 6, 11, and 9 oxidise iodide to iodine in acidic medium [22 24], and 11 shows a
characteristic IR NÀH band at 3259 cmÀ1 (CHCl3) [27][28]. According to the 1H-NMR spectrum in
(D6)DMSO, 6 is a ca. 3 :2 mixture of two diastereoisomers. NH Groups of the major isomer appear as two d at
2.30 and 2.13 ppm (J ˆ 8.1 Hz) and NH of the minor isomer as two d at 2.24 and 2.07 ppm, respectively (J ˆ
8.1 Hz). Upon addition of CF3COOH, the two diastereoisomers equilibrate rapidly, and the spectrum of a single
compound is observed. C(3) of 6 appears as a s at 60.02 ppm (cf. [29]). The 1H-NMR spectrum (CDCl3) shows
that 11 is a ca. 4 :1 mixture of diastereoisomers. NH Groups exchanged with D2O; the signals of the major
diastereoisomer resonate as two d at 2.64 and 1.57 ppm, respectively (J ˆ 7.9 Hz), while only one d resonating at
1.44 ppm (J ˆ 8Hz) could be observed for the other isomer (the other signal is hidden between 2.26 2.06 ppm).
C(3) resonates as a s at 57.06 ppm. According to the 1H-NMR spectrum (D2O), the N-benzyl diaziridine 9 is a
single stereoisomer. Its configuration was determined by NOE difference spectra. Upon irradiation at 2.04 ppm
(HeqÀC(8)), a NOE of 4 and 6%, respectively, was observed for the benzylic H-atoms. Upon irradiation of the
benzylic H-atoms at 3.92 and 3.81 ppm, a NOE of 6% for HeqÀC(8) and one of 8% for HÀC(7) were observed.
Irradiation at 3.62 ppm (HÀC(4)) did not lead to a NOE of the benzylic H-atoms. C(3) appears as a s at
62.90 ppm and PhCH2 as a t at 57.27 ppm (cf. [30]). The UV spectrum of 7 (H2O) shows a typical maximum at
340 nm (e ˆ 68) [27][31]. A large chemical-shift difference is observed for the two HÀC(8) (Dd ꢀ 1.2 ppm).
The signal for the equatorial H-atom is shifted upfield, an observation characteristic for spiro-diazirines
[18][27][32]. The C(3) s (32.34 ppm) is shifted upfield by 27.7 ppm as compared to that of 6 (for similar upfield
shifts of 1-azipyranoses, see [2]). The UV spectrum of the diazirine 12 (obtained in 85% yield by oxidation (I2,
Et3N) of 11) in CH2Cl2 shows a maximum at 340 nm (e ˆ 92). The IR NˆN band (CHCl3) is observed at
1590 cmÀ1 [31][27]. The chemical-shift difference for the two HÀC(8) (Dd ˆ 1.38ppm) is again large. The C(3)
s of 12, (28.82 ppm) is shifted upfield by 28.2 ppm as compared to 11. The vicinal coupling constants for the ring
H of 6, 7, 8, 11, 12, and 9 evidence a 6C3 conformation.
Synthesis of the Spiro-aziridines. Wittig methylenation of the cyclohexanone 10 gave
the alkene 13 (77%), but, of our attempts to transform 13 into an aziridine9), only the
reaction with chloramine Tand phenyl(trimethyl)ammonium tribromide in MeCN [44]
gave the N-tosyl aziridine 14 (18%), along with 2% of the ring-opened tosyl amide 15
(Scheme 2). The low yield of the aziridination prompted us to investigate the synthesis
of the aziridines via the epoxides 16 and 17 (cf. [45][46]). These epoxides were
obtained in 25 and 61% yield, respectively, by treating the alkene 13 with m-
chloroperbenzoic acid (mCPBA)/NaHCO3. On a scale of 700 mg, their separation
required HPLC, and the yields were somewhat lower (19 and 48%, resp.). Treatment of
8
)
Equatorial attack of hydroxylamine-O-sulfonic acid on an intermediary imine has also been observed by
Shustov et al. in the synthesis of 5-methyl-3,3-pentamethylenediaziridine [25]. For a discussion of the
diastereoselective addition of NH3 or H2NMe to glyconolactone oxime mesylates in the formation of
alkoxydiaziridines, see [26].
The alkene 13 did not react with TsN3, toluene, 1108 [33][34]; TfN3, CH2Cl2 [35]; diphenylphosphoryl
azide, toluene, 708; NBS, NaN3, DME/H2O (or DMF) [36]; I-NCO, Et2O then KOH, MeOH [37 39]. I-
NCO, MeCN, then KOH, MeOH gave 16 (36%) and unidentified by-products. ICl/NaN3 ([40 43]) gave
34% of a mixture of IN3 addition products, which, on treatment with LiAlH4, gave 13 (43%) (cf. [40]; see
Exper. Part).
9
)

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Helvetica Chimica Acta Vol. 86 (2003)
Scheme 2
a) Ph3P ˆ CH2, THF; 77%. b) Chloramine T, PhMe3NBr3, MeCN; 18% of 14, 2% of 15. c) 3-Chloroperbenzoic
acid (mCPBA), NaHCO3, CH2Cl2; 25% of 16, 61% of 17. d) NaN3, DMF; 91% of 18; 8 8 % of22. e) MsCl,
DMAP, 2,5-lutidine; 95% of 19, 4% of 26; 99% of 23. f) LiAlH4, THF; 83% of 20; 74% of 24. g) Na, NH3,
THF; 45% of 25; (yield of 21: see text).
16 and 17 with NaN3 in DMF gave the azido alcohols 18 (91%) and 22 (88%).
Mesylation (MsCl, 4-(dimethylamino)pyridine (DMAP), 2,5-lutidine) [47] of 18
yielded 95% of the methanesulfonate 19 besides 4% of the elimination product 26,
while mesylation of 22 gave the methanesulfonate 23 (99%) exclusively. The azido
methanesulfonates 19 and 23 were transformed into aziridines 20 (83%) and 24 (74%),
respectively, by treatment with LiAlH4 in THF (cf. [48]). Debenzylation of the
aziridine 24 under Birch conditions gave the aziridine 25, which was was isolated in a
yield of 45% by chromatography first on an ion exchanger and then on a CN phase.
Birch debenzylation of the aziridine 20, followed by chromatography, led to the crude
aziridine 21, which decomposed on the CN phase column. Even just evaporating a

Helvetica Chimica Acta Vol. 86 (2003)
2215
methanolic solution of some batches of 21 led to decomposition10). Crude 21
(containing small amounts of an unidentified by-product and some salts) was obtained
by chromatography of the reaction product over a Sephadex G 10 column, and used for
the enzyme assay. Hydrogenation of the benzylated aziridine 20 over Pd/C in MeOH/
HCl gave a mixture of the ring-opened 27 and 28 as their hydrochlorides (1.6 :1).
Elution of crude 21 from a Sephadex C 25 column with aqueous HCl gave 27 ¥ HCl11).
1
The H-NMR spectrum of the N-tosyl aziridine 14 shows two br. s at 2.63 and 2.59 ppm (CH2(2)). Their
small coupling constants are typical for aziridines [53]. C(3) appears as a s at 51.73 ppm and C(2) as a t at
28.77 ppm. The configuration of 14 was determined by NOE difference spectra. Upon irradiation at 2.63 ppm
(HÀC(2)), a NOE of 2% was observed for HÀC(5), and irradiation at 2.59 ppm (H'ÀC(2)) led to a NOE of
1% for HÀC(7) and for the CH2(8) multiplet. Irradiation at 3.78ppm (H ÀC(4)) did not lead to an observable
NOE for CH2(2). Irradiation at 3.39 ppm (HÀC(5)) caused an NOE of 1.5% for HÀC(2). The vicinal coupling
constants evidence a 6C3 conformation. The mass spectrum of the ring-opened tosyl derivative 15 shows two
‡
[M ‡ Na] peaks at m/z 808 and 806 of about equal intensity. CH2ÀC(1) resonate as two dd at 3.18ppm ( J ˆ
13.4, 6.5 Hz) and 3.04 ppm (J ˆ 13.4, 8.1 Hz), and NH as a dd at 3.62 ppm (J ˆ 7.8, 6.2 Hz), evidencing that the
NHTs moiety is bound to CH2ÀC(1) and not to C(1). HÀC(2) appears as a d at 3.13 ppm (J ˆ 9.0 Hz),
indicating that C(1) is fully substituted. The configuration of C(1) was deduced from the large downfield shifts
for HÀC(5) (2.29 2.15 ppm) and HÀC(3) (4.03 ppm), which indicate that BrÀC(1) is axial.
The configuration of the epoxides was determined by NOE difference spectra. Irradiation of HÀC(2) of 16
at 3.18ppm caused a NOE of 1% for H ÀC(5). Upon irradiation at 2.56 ppm (H'ÀC(2)), a NOE of 7% was
observed for HeqÀC(8). For 17, irradiation at 2.98ppm (H ÀC(2)) led to a NOE of 1% for HÀC(4). Irradiation
at 2.59 ppm (H'ÀC(2)) led to a NOE of 4% for HeqÀC(8). The pseudo-axial OÀC(3) of 17 leads to a downfield
shift for HÀC(5) (Dd ˆ 0.29 ppm) and HÀC(7) (Dd ꢀ 0.2 ppm), as compared to 16. This shift confirms the
assignment of the configuration of 16 and 17. The azido alcohols 18 and 22 show strong IR bands for the N3 and
OH groups. CH2ÀN3 resonate as two d at 3.74 and 3.34 ppm (J ˆ 12.8Hz) for 18, at 3.29 and 3.22 ppm (J ˆ
11.8Hz) for 22, at 4.10 and 3.45 ppm (J ˆ 14.0 Hz) for the methanesulfonate 19, and at 4.17 and 3.90 ppm (J ˆ
12.1 Hz) for the methanesulfonate 23. HÀC(3) and HÀC(5) of 22 and 23 (axial OÀC(1)) are shifted downfield
as compared to 18 (Dd ꢀ 0.3 ppm for HÀC(3) and Dd ꢀ 0.4 ppm for HÀC(5)) and 19 (Dd ˆ 0.37 ppm for
HÀC(3) and Dd ꢀ 0.4 ppm for HÀC(5)). HÀC(2) (Dd ꢀ 0.8ppm) and H axÀC(6) (Dd ˆ 0.94 ppm) of 19 are
shifted downfield as compared to 18. A downfield shift of ca. 20 ppm is observed for the C(1) s of 19 (95.31 ppm)
and 23 (94.00 ppm) as compared to 18 and 22, confirming that the O-atom is bound to C(1). The coupling
constants for the ring H-atoms evidence a 4C1 conformation for 18, 22, 19, and 23. The geminal coupling
constants for the axial CH2ÀN3 substituent of 18 and 19 are larger than those of the equatorial CH2ÀN3 of 22
and 23 (DJ ˆ 1.0 to 1.9 Hz). The tertiary alcohol 22 and the methanesulfonate 23 can adopt a conformation in
which the RO group is gauche to both vicinal CH2 H-atoms, an orientation of an electron-withdrawing
substituent that leads to a decreased (absolute) geminal coupling constant [54][55]. The structure of the
elimination product 26 is evidenced by a strong IR band at 2109 cmÀ1 for the N3 group, by a br. t at 6.36 ppm (J ˆ
‡
1.2 Hz) for CHÀN3, and by the mass spectrum (m/z 570, corresponding to [M ‡ Na À N2] . The 13C-NMR
spectrum shows alkene signals at 126.55 and 120.32 ppm. The coupling constants for the ring H-atoms evidence a
flattened 3C6 conformation. The (E)-configuration of the olefinic CˆC bond was not proven. CH2(2) of the
aziridines 20 and 24 appear as two br. s at 1.87 and 1.40 ppm for 20, and at 1.98and 1.29 ppm for 24. NH of 20 and
24 appear as a br. signal at 1.17 0.87 and 1.18 0.85 ppm, respectively. C(3) resonates as a s at 37.42 ppm for 20
and at 37.86 ppm for 24, and C(2) as a t at 27.17 ppm for 20 and at 26.97 ppm for 24. The coupling constants for
6
the ring H-atoms of 20 evidence a C3 conformation. Signal overlap for the ring H-atoms of 24 prevented a
conformational analysis. CH2(2) of the aziridine 25 resonate as two br. s at 2.02 and 1.34 ppm, respectively. C(3)
appears as a s at 39.90 ppm and C(2) as a t at 27.36 ppm. The coupling constants for the ring H-atoms evidence a
6C3 conformation. The pKHA value of 25 is 6.8, and equilibration of the N-configurational isomers should be fast
at pH 7. CH2(2) of the crude aziridine 21 resonate as two br. s at 1.96 and 1.53 ppm, respectively. The coupling
10
)
)
For other examples of related, unstable aziridines see Martin and Saavedra [9], and Paulsen et al. [11].
Hassner et al. reported that some aziridines decompose upon concentration of their solution in organic
solvents, unless scrupulously dried [40].
11
For similar acid-catalysed regioselective ring openings of aziridines, cf. [49 52].

2216
Helvetica Chimica Acta Vol. 86 (2003)
‡
constants evidence a 6C3 conformation. In the HR-MS, the [M ‡ 1] peak appears at 190.1076 (calc. 190.1079).
The structure of the ring-opened 27 ¥ HCl and 28 ¥ HCl was assigned on the basis of MS and 1H-NMR data. The
‡
‡
ESI -MS spectrum of the mixture of 27 ¥ HCl and 28 ¥ HCl shows peaks at 224 ([M ‡ MeOH ‡ 1] ) and 192
‡
([M ‡ 1] ) for 28, and pairs of peaks with an intensity ratio of 1:2 (typical for chloro compounds) at 250 and 248
‡
‡
‡
‡
([M ‡ Na] ), and 228and 226 ([ M ‡ 1] ), and peaks at 222 ([M À Cl ‡ MeOH] ) and 190 ([M À Cl] , i.e.,
[M(21) ‡ 1] ) for 27. In the 1H-NMR spectrum of the mixture 27 ¥ HCl/28 ¥ HCl, Me of 28 ¥ HCl resonates as a s
‡
at 1.43 ppm. CH2ÀC(1) of 27 ¥ HCl resonates as two d (J ˆ 12.3 Hz) at 4.11 and 3.85 ppm. The large geminal
coupling constant excludes the structure of an aziridinium ion. The large downfield shift indicates that Cl and
‡
not NH3 is bound to CH2ÀC(1).
1-Epivalidamine. Asano et al. prepared 1-epivalidamine (32) by reductive amina-
tion of validone (4), thus obtaining 32 (22%) and 33 (36%) after separation by ion-
exchange chromatography [14] (Scheme 3). To avoid this separation, we prepared 32
from the known axial alcohol 29 [56] that was transformed into the methanesulfonate
30 (93%). Substitution of the methanesulfonate with NaN3 in DMF gave the azide 31
(94%) that was hydrogenated over Pd/C in MeOH/HCl to yield 1-epivalidamine (32)
in an almost quantitative yield.
Scheme 3
a) MsCl, pyridine; 93%. b) NaN3, DMF; 94%. c) H2 (6 bar), Pd/C, MeOH, HCl; 100%.
Enzyme-Inhibition Studies. The diaziridines 6 and 9, and the aziridines 21 and 25 are
weak inhibitors of the b-glucosidase from Caldocellum saccharolyticum, the b-
glucosidases from almonds, and the a-glucosidase from brewer×s yeast (Table 2). At
pH 6.8, 6 did not inhibit the b-glucosidases, while the a-glucosidase was inhibited only
very weakly at a concentration of 8.8 mm. At pH 4.2, 6 inhibited the b-glucosidase from
Caldocellum saccharolyticum very weakly and proved inactive against the sweet
almond b-glucosidases. At pH 6.8, the N-benzyl diaziridine 9 was a weak inhibitor of
the a-glucosidase (IC50 ˆ 1.7 mm); as expected, the hydrophobic aglycon mimic leads to
a (slightly) stronger inhibition [57]. The diazirine 7 did not significantly inhibit any of
the enzymes tested. Also, the aziridine 25 did not inhibit the almond b-glucosidases
(pH 6.8). It proved a weak irreversible inhibitor of the b-glucosidase from Caldocellum
saccharolyticum, and a weak reversible inhibitor of the a-glucosidase from brewer×s
yeast; the inhibition of the a-glucosidase was abolished at pH 5.0. The crude aziridine
21 appears to be a weak reversible inhibitor of the b-glucosidases, and a weak

Helvetica Chimica Acta Vol. 86 (2003)
2217
irreversible inhibitor of the a-glucosidase12). Irreversible inhibition by the aziridines
most likely results from an acyloxylating aziridine ring opening. Also 1-epivalidamine
(32) proved only a millimolar inhibitor of all three enzymes, approximately as potent as
its epimer 33 [58].
Table 2. Inhibition of the b-Glucosidases from Sweet Almonds, the b-Glucosidase from Caldocellum
saccharolyticum and the a-Glucosidase from Brewer×s Yeast ([IC50] in mm at pH 6.8) by the Diaziridines 6
and 9, the Diazirine 7, the Aziridines 21 and 25, Epivalidamine (32), Validamine (33) [58 ],and the
Cyclopentylamine 34 [61]
Inhibitor (pKHA
)
b-Glucosidase
b-Glucosidase
a-Glucosidase
(sweet almonds)
(C. saccharolyticum)
(brewer×s yeast)
6 (2.6)
9
no inh. at 8.8 mm
no inh. at 3.7 mm
no inh. at 5.8m m
ca. 30% inh.
no inh. at 1.3 mm
1.7
no inh. at 2.2 mma)
no inh. at 8.8 mm
ca. 1.7
no inh. at 11.6 mm
ca. 50% inh.d)
ca. 1f)
b
)
)
b
7
21c)
ca. 10% inh.
3e) (irreversible)
1.0
25 (6.8)
32 (8.4)
33g)
0.7
1.5
0.58
34h) (7.9)
0.0034e)
0.00018e)
0.013e)
a) At pH 4.2, ca. 20% inhibition at 6.6 mm. b) Not determined. c) Ca. 100 mm solution. d) After 20 min,
f
irreversible. e) Ki Value. ) At pH 5.0: no inhibition at 1.3 mm. g) Data from [58]. h) Inhibition data from [61a].
The very low inhibition by the diaziridines, aziridines, and epivalidamine suggests
that structural factors rather than basicity13) are at the root of the weak interaction of
the glycosidases tested. This is highlighted by the striking differences in the inhibitory
action of the cyclohexane derivative 32 ((IC50 ca. 1 mm; pKHA ˆ 8.4) and the
cyclopentane derivative 34 (Scheme 3) [60] (Ki between 0.2 and 13 mm [61]; pKHA
ˆ
7.9 ). The small difference in the pKHA of the two compounds cannot account for this
difference in the affinity to the enzymes. Both inhibitors will be mostly in the
protonated form at pH 6.8. To further study the inhibition of the b-glucosidases from
almonds by 34, we examined the pH dependence of the inhibition between pH 5.0 and
7.8( Figure). Between pH 6.2 and 7.8, the inhibition was nearly constant, but it was
significantly reduced at pH 5.0. This behaviour evidences that the protonated form of
34 binds to the enzyme; it is similar to the one of b-glucosyl pyridinium ion [62].
The stronger inhibition by the cyclopentane derivative may be traced back to the
ease with which it adapts a conformation with a pseudoaxial amino group, whereas the
cyclohexane derivatives are fixed in a chair conformation. On the basis of stereo-
electronic effects, it was proposed that b-glucosidases cleave their substrate from a
conformation where a lone pair of the ring O-atom is antiperiplanar to the scissile bond
[63]. Crystal-structure analyses have shown such substrate distortion in enzymeÀsub-
strate complexes for hen eggwhite lysozyme [64], the endoglucanase I from Fusarium
oxysporum (family 7) [65], the chitobiase from Serratia marcescens (family 20) [66],
12
)
)
The concentration of 21 in the enzyme tests was assumed 100 mm. This is an upper limit, as this
concentration implies a quantitative yield of the Birch reduction of 20.
Basicity does have some effect, and the lower basicity of 6 as compared to the one of 25 may contribute to
the particularly low inhibition by 6. Legler has reported that only glycosylamines with a pKHA higher than
5 are strong glycosidase inhibitors [59].
13

2218
Helvetica Chimica Acta Vol. 86 (2003)
Figure. pH Dependence of the inhibition of almond b-glucosidases by 34.
and the b-glycosidase Cel5A from Bacillus agaradhaerens (family 5) [67]. The
transition state for the glycoside cleavage by these enzymes lies on the reaction
coordinate between the distorted substrate and an oxycarbenium cation (with
F(C(5)ÀOÀC(1)ÀC(2) ꢀ 08), with the exact location of the transition state probably
depending on the glycosidase family. Substrate distortion has not been demonstrated
for family 1 glycosidases, but the aminocyclopentitol 34 is suggested to be a stronger
inhibitor of the b-glucosidases on account of the pseudoaxial NH2 group. The
cyclopentylamine thereby mimics the distorted substrate and is, thus, closer to the
transition state.
We thank the Swiss National Science Foundation and F. Hoffmann-La Roche AG, Basel, for generous
support, Dr. B. Bernet for a sample of 34 and for double-checking the manuscript, and Dr. N. Panday for double-
checking the inhibition data for 6.
Experimental Part
General. Solvents were freshly distilled from CaH2 (CH2Cl2, MeOH, DMF), Na/benzophenone (THF,
toluene), Na (BnNH2), or BaO (2,6-lutidine). Reactions were carried out under Ar, unless stated otherwise.
Anal. TLC: Merck precoated silica-gel 60 F-254 plates; detection by treatment with a soln. of 5%
(NH4)6Mo7O26 ¥ 4 H2O, 0.1% Ce(SO4)2 ¥ H2O in 10% H2SO4 soln. Flash chromatography (FC): silica gel 60
(40 63 mm). M.p.: uncorrected. Optical rotations: 1-dm cell, 589 nm. FT-IR Spectra: absorption in cmÀ1. NMR
Spectra: chemical shifts in ppm relative to TMS; coupling constants in Hz. FAB-MS in 3-nitrobenzyl alcohol
(NOBA) matrix. HR-MALDI-MS: in 2,5-dihydroxybenzoic acid (DHB) matrix. The pKHA values of 25, 32, and
34 were determined by potentiometric titration in H2O. The pKHA value of 6 was estimated as the pH, at which
c(6) ˆ c(6 ¥ HCl) as described in [62]. The b-glucosidases from almonds (Fluka), the b-glucosidase from

Helvetica Chimica Acta Vol. 86 (2003)
2219
Caldocellum saccharolyticum (Sigma), and the a-glucosidase from brewer×s yeast (Sigma) were used without
further purification.
(2R,3S,4R,5R)-2,3,4-Trihydroxy-5-(hydroxymethyl)cyclohexanone (4).
A suspension of 3 (2.0 g,
5.96 mmol) in MeCN (30 ml) and H2O (6 ml) was treated with NBS (1.6 g, 8.98 mmol), stirred for 15 h, and
evaporated. FC (90 g of silica gel; AcOEt/i-PrOH/H2O 8:2 :1) gave 4 (520 mg, 50%) as a brown amorphous
solid. A pure sample (14 mg) was obtained by reversed-phase C8 HPLC (H2O) and lyophilisation. Rf (PrOH/
AcOH/H2O 4 :1:1) 0.53. 1H-NMR (200 MHz, D2O): 4.25 (d, J ˆ 10, HÀC(2)); 3.84 3.60 (m, HÀC(4),
CHÀC(5), CH'ÀC(5)); 3.50 3.30 (m, HÀC(3)); 2.63 2.41 (m, 2 HÀC(6)); 2.00 1.44 (m, HÀC(5)).
13C-NMR (50 MHz, D2O): 212.9 (s, C(1)); 81.1, 80.8, 74.4 (3d, C(2), C(3), C(4)); 64.3 (t, CH2ÀC(5)); 43.6
‡
‡
‡
(d, C(5)); 41.7 (t, C(6)). CI-MS (NH3): 194 (77, [M ‡ NH4] ), 176 (7, M ), 141 (26, [M ‡ 1 À 2 H2O] ), 123 (100,
‡
[M ‡ 1 À 3 H2O] ), 110 (75), 98(16), 86 (17), 73 (22), 55 (19).
(2R,3S,4R,5R)-2,3,4-Tris(trimethylsilyloxy)-5-[(trimethylsilyloxy)methyl]cyclohexanone (5). At 258, a soln.
of 4 (630 mg, 3.59 mmol) was treated with HMDS (10 ml, 48.0 mmol) and Me3SiCl (5 ml, 39.5 mmol), stirred
for 6.5 h, evaporated, and dried in h.v. The residue was digested with CH2Cl2 (3 Â 50 ml) and filtered. The org.
phase was washed with ice-cold H2O (75 ml), dried (MgSO4), and evaporated. FC (30 g of silica gel; hexane/
AcOEt/Et3N 400 :10 :0.4) of the residue (1.28g, brown oil) gave 5 (448mg, 27%). Colourless oil. Rf (hexane/
AcOEt 9 :1) 0.63. 1H-NMR (300 MHz, CDCl3): 4.03 (dd, J ˆ 9.5, 1.1, HÀC(2)); 3.76 (t, J ˆ 9.8, HÀC(4)); 3.76
(dd, J ˆ 10.0, 3.1, CHÀC(5)); 3.47 (dd, J ˆ 10.0, 2.5, CHÀC(5)); 3.46 (t, J ˆ 9.0, HÀC(3)); 2.40 (td, J ˆ 14.0, 1.1,
HaxÀC(6)); 2.30 (dd, J ˆ 14.0, 4.7, HeqÀC(6)); 1.70 1.56 (m, HÀC(5)); 0.19, 0.15, 0.14, 0.09 (4s, 4 Me3Si).
13C-NMR (75 MHz, CDCl3): 207.24 (s, CˆO); 80.20, 79.76, 73.50 (3d, C(2), C(3), C(4)); 61.76 (t, CH2ÀC(5));
‡
‡
‡
41.57 (t, C(6)); 39.45 (d, C(5)). FAB-MS (NOBA): 465 (14, [M ‡ 1] ), 464 (31, M ), 447 (42, [M À OH] ), 359
‡
(68, [M À TMSÀO2] ), 331 (32), 305 (13).
(4S,5S,6R,7R)-7-(Hydroxymethyl)-1,2-diazaspiro[2.5]octane-4,5,6-triol (6). At À 208, a soln. of 5 (260 mg,
0.56 mmol) in MeOH (15 ml) was saturated with NH3, treated dropwise with a soln. of hydroxylamine-O-
sulfonic acid (63 mg, 0.56 mmol) in MeOH (5 ml), stirred for 3 h, allowed to warm to r.t., and stirred overnight.
Filtration, evaporation, and FC (60 g of silica gel; AcOEt/i-PrOH/H2O 4 :2 :1) gave a colourless soln. of 6. After
removal of most of the AcOEt in vacuo below 308, this soln. was applied to an ion-exchange column (5 ml of
‡
Dowex 50-WX-8, H form, washed with MeOH (50 ml) and H2O to pH 7). After washing with H2O (200 ml),
elution with 2% aq. NH3, and lyophilisation gave colourless, amorphous 6 (53 mg, 50%). Rf (AcOEt/i-PrOH/
H2O 4 :2 :1) 0.13. Rf (acetone/H2O 2 :1) 0.72. Rf (i-PrOH/H2O 4 :1) 0.43. M.p. 68 76 8. [a]2D5 ˆ 22.2 (c ˆ 1.50,
H2O). pKHA ˆ 2.6. 1H-NMR (300 MHz, (D6)DMSO; 3 :2 mixture of diastereoisomers): 4.87 4.68 (m, 1.4 OH);
4.61 4.52 (m, 1.2 OH); 4.41 4.27 (m, 1.4 OH); 3.61 2.85 (m, HÀC(4), HÀC(5), HÀC(6), CH2ÀC(7)); 2.30
(br. d, J ˆ 8.1, 0.6 H), 2.24 (br. d, J ˆ 8.1, 0.4 H), 2.13 (br. d, J ˆ 8.1, 0.6 H), 2.07 (br. d, J ˆ 8.1, 0.4 H) (2 NH);
1.80 (t, J ˆ 13.4, 0.4 HaxÀC(8)); 1.60 (t, J ˆ 13.0, 0.6 HaxÀC(8)); 1.57 1.37 (m, HÀC(7)); 1.27 1.15
(m, HeqÀC(8)). 1H-NMR (300 MHz, (D6)DMSO/CF3COOH): 3.70 (d, J ˆ 9.0, HÀC(4)); 3.56 (dd, J ˆ 10.6,
3.1, CHÀC(7)); 3.38( dd, J ˆ 10.6, 6.2, CH'ÀC(7)); 3.16 (t, J ˆ 9.0, HÀC(6)); 3.06 (t, J ˆ 9.0, HÀC(5)); 1.95
1
(br. t, J ˆ 14.9, HaxÀC(8)); 1.52 1.47 (m, HÀC(7), HeqÀC(8)). H-NMR (500 MHz, CD3OD; 4 :3 mixture of
diastereoisomers): 3.74 3.63 (m, 2.57 H); 3.48( d, J ˆ 9.1, 0.43 HÀC(4)); 3.38 3.34 ( m, 1.43 H); 3.19 (t, J ˆ 9.3,
0.57 H); 2.04 (t, J ˆ 13.6, 0.43 HÀC(8)); 1.89 (t, J ˆ 13.3, 0.57 HÀC(8)); 1.80 1.71 (m, 0.57 HÀC(7)); 1.71
1.63 (m, 0.43 HÀC(7)); 1.37 (td, J ˆ 14.5, 3.9, H'ÀC(8)). 13C-NMR (75 MHz, D2O; ca. 1:1 mixture of
diastereoisomers): 80.37, 79.87 (2d); 75.50, 75.21 (2d); 72.19 (d); 64.70 (t, CH2ÀC(7)); 60.02 (s, C(3)); 43.78,
‡
‡
43.50 (2d, C(7)); 34.91 (t, C(8)). ESI-MS: 445 (4, [2M ‡ 1 ‡ 2 MeOH] ), 419 (8, [2M ‡ K] ), 403 (100, [2M ‡
‡
‡
‡
‡
Na] ), 381 (12, [2M ‡ 1] ), 360 (6), 338(4), 245 (16, [ M ‡ Na ‡ MeOH] ), 229 (10, [M ‡ K] ), 213 (26, [M ‡
‡
‡
‡
‡
Na] ), 191 (23, [M ‡ 1] ). HR-FAB-MS (NOBA): 213.0858 (C7H14N2NaO4 , [M ‡ Na] ; calc. 213.0851),
‡
‡
191.1026 (C7H15N2O4 , [M ‡ H] ; calc. 191.1027).
(4S,5S,6R,7R)-7-(Hydroxymethyl)-1,2-diazaspiro[2.5]oct-1-ene-4,5,6-triol (7). A soln. of I2 (ca. 30 mg) in
MeOH (1 ml) was added dropwise at 258 to a soln. of 6 (55 mg, 0.29 mmol) in MeOH (8ml) and Et 3N (0.1 ml,
0.72 mmol) until the brown colour persisted. Evaporation and FC (20 g of silica gel; CH2Cl2/MeOH 9 :1) gave a
mixture of 7 and Et3N (2.5:1, 60 mg). This mixture was applied to an ion-exchange column (7 ml of Dowex-
‡
CCR-2, H form, washed with MeOH (100 ml) and H2O to pH 7). Elution with H2O and lyophilisation gave 7
(48.8 mg, 89%). Colourless syrup. Rf (AcOEt/i-PrOH/H2O 4 :2 :1) 0.75. [a]2D5 ˆ 37.2 (c ˆ 1.03, H2O). UV (H2O):
340 (68). 1H-NMR (300 MHz, D2O): 3.86 (d, J ˆ 9.3, HÀC(4)); 3.77 (dd, J ˆ 11.2, 3.1, CHÀC(7)); 3.69 3.66
(m, CH'ÀC(7)); 3.54 (t, J ˆ 9.3, HÀC(5)); 3.48( t, J ˆ 9.3, HÀC(6)); 2.00 1.85 (m, HÀC(7), HaxÀC(8)); 0.72
(dd, J ˆ 13.1, 2.8, HeqÀC(8)). 13C-NMR (75 MHz, D2O): 80.60, 75.38, 71.93 (3d, C(4), C(5), C(6)); 64.68
‡
(t, CH2ÀC(7)); 43.46 (d, C(7)); 32.34 (s, C(3)); 31.76 (t, C(8)). HR-ESI-MS (MeOH): 211.069 (C7H12NaN2O4 ,
‡
[M ‡ Na] ; calc. 211.0695).

2220
Helvetica Chimica Acta Vol. 86 (2003)
(4S,5S,6R,7R)-4,5,6-Tris(acetoxy)-7-[(acetoxy)methyl]-1,2-diazaspiro[2.5]oct-1-ene (8). At 08, a soln. of a
mixture of 7 and Et3N (84 mg, prepared as described above from 6 (79 mg, 0.42 mmol)) in pyridine (10 ml) was
treated with Ac2O (1 ml), allowed to warm to r.t. overnight, and poured into ice-water (50 ml). The aq. phase
was extracted with CH2Cl2 (4 Â 50 ml). Drying (Na2SO4), evaporation, and FC (30 g of silica gel; hexane/
AcOEt 3:1) gave 8 (124 mg, 84%). Colourless oil. Rf (hexane/AcOEt 3 :1) 0.15. [a]2D5 ˆ 47.4 (c ˆ 1.54, CHCl3).
UV (CH2Cl2): 229 (130), 334 (83). FT-IR (1.5%, CHCl3): 3038w, 2954w, 1751s, 1594w, 1443w, 1377m, 1248s,
1131w, 1064m, 1033m, 978w, 932w, 8 96w, 8 46w. 1H-NMR (300 MHz, CDCl3): 5.33 (t, J ˆ 9.7, HÀC(5)); 5.15
(d, J ˆ 9.7, HÀC(4)); 5.13 (dd, J ˆ 10.6, 9.3, HÀC(6)); 4.07 (dd, J ˆ 11.5, 5.6, CHÀC(7)); 3.90 (dd, J ˆ 11.5, 3.1,
CH'ÀC(7)); 2.40 2.29 (m, HÀC(7)); 2.05, 2.03, 1.98, 1.87 (4s, 4 AcO); 2.02 (dd, J ˆ 15.1, 9.0, HaxÀC(8)); 0.79
(dd, J ˆ 15.1, 4.4, HeqÀC(8)). 13C-NMR (75 MHz, CDCl3): 170.86, 170.11, 170.00, 169.03 (4s, 4 C ˆ O); 73.69,
71.07, 68.26 (3d, C(4), C(5), C(6)); 62.66 (t, CH2ÀC(7)); 37.28( d, C(7)); 29.67 (t, C(8)); 26.75 (s, C(3)); 20.68,
‡
‡
20.58, 20.55, 20.11 (4q, 4 Me). FAB-MS (NOBA): 379 (11, [M ‡ Na] ), 357 (24, [M ‡ 1] ), 297 (9, [M À
‡
‡
AcO] ), 269 (100, [M À AcO À N2] ), 227 (21), 208(11), 167 (93). Anal. calc. for C 15H20N2O8 (356.33): C 50.56,
H 5.66, N 7.86; found: C 50.80, H 5.58, N 7.64.
(1R,2R,3S,4S,5S,6R,7R)-1-Benzyl-7-(hydroxymethyl)-1,2-diazaspiro[2.5]octane-4,5,6-triol (9). A soln. of 5
(466 mg, 1.0 mmol) in MeOH (30 ml) was treated at 258 with BnNH2 (4.5 ml, 41.2 mmol), stirred for 1 h, cooled
to 08, treated dropwise with a soln. of hydroxylamine-O-sulfonic acid (116 mg, 1.00 mmol) in MeOH (5 ml),
allowed to warm to r.t. and stirred overnight. Filtration, evaporation, and repeated FC (50 g of silica gel; AcOEt/
MeOH 1:0 ! 4 :1) gave a mixture of 9 and BnNH2 (1:4, 487 mg), which was dissolved in H2O, applied on an
‡
ion-exchange column (15 g of Dowex-CCR-2, H form, washed with MeOH (100 ml) and H2O to pH 7), and
eluted with H2O. Lyophilisation gave colourless, amorphous 9 (76 mg, 27%). Rf (AcOEt/i-PrOH/H2O 4 :2 :1)
0.51. M.p. 52 588. [a]2D5 ˆ 59.7 (c ˆ 1.53, H2O). 1H-NMR (300 MHz, D2O): 7.49 7.38( m, 5 arom. H); 3.92
(d, J ˆ 14.0, irrad. at 2.04 ! NOE of 4%, PhCH); 3.81 (d, J ˆ 14.0, irrad. at 2.04 ! NOE of 6%, PhCH); 3.75
(dd, J ˆ 11.5, 3.4, CHÀC(7)); 3.64, (dd, J ˆ 11.5, 5.9, CH'ÀC(7)); 3.62 (d, J ˆ 9.3, HÀC(4)); 3.43 (t, J ˆ 9.0,
irrad. at 3.62 ! NOE of 9%, HÀC(6)); 3.36 (t, J ˆ 9.0, HÀC(5)); 2.04 (dd, J ˆ 14.6, 4.1, irrad. at 3.92 and 3.81 !
NOE of 6%, HeqÀC(8)); 1.93 (br. t, J ˆ 14.0, irrad. at 3.62 ! NOE of 4%, HaxÀC(8)); 1.53À1.40 (m, irrad. at
3.81 and 3.75 ! NOE of 8%, HÀC(7)). 13C-NMR (75 MHz, CD3OD): 140.07 (s); 129.83 (d, 2 C); 129.65
(d, 2 C); 128.52 (d); 79.66, 74.45, 73.10 (3d, C(4), C(5), C(6)); 63.96 (t, CH2ÀC(7)); 62.87 (s, C(3)); 57.19
‡
‡
(t, PhCH2); 43.02 (d, C(7)); 27.44 (t, C(8)). HR-MALDI-MS (DHB): 303.1310 (C14H20NaN2O4 , [M ‡ Na] ;
‡
‡
calc. 303.1316), 281.1492 (C14H21N2O4 , [M ‡ H] ; calc. 281.1496). Anal. calc. for C14H20N2O4 ¥ 0.25H2O: C 59.04,
H 7.26, N 9.84; found: C 58.79, H 7.31, N 10.00.
(4S,5S,6R,7R)-4,5,6-Tris(benzyloxy)-7-[(benzyloxy)methyl]-1,2-diazaspiro[2.5]octane (11). At À 208, a
suspension of 10 [15] (115 mg, 0.21 mmol) in MeOH (5 ml) was saturated with NH3, treated dropwise with a
soln. of hydroxylamine-O-sulfonic acid (24.3 mg, 0.21 mmol) in MeOH (2.5 ml), stirred for 3 h, allowed to warm
to r.t., and stirred overnight. Filtration, evaporation, and FC (17 g of silica gel; hexane/AcOEt 3 :1) gave 11
(41 mg, 35%). Colourless, amorphous. Rf (hexane/AcOEt 1 :1) 0.49. M.p. 76 788. [a]2D5 ˆ 29.4 (c ˆ 0.98, CHCl3).
FT-IR (1%, CHCl3): 3259w, 3089w, 3066w, 3008s, 2908m, 2862m, 1951w, 1876w, 1811w, 1730w, 1603w, 1497m,
1454s, 1404w, 1358s, 1274w, 1175m, 1149s, 1097s, 1028s, 1016w, 913w, 8 72w, 8 55w. 1H-NMR (200 MHz, CDCl3, 4 :
1 mixture of diastereoisomers) 7.35 7.22 (m, 20 arom. H); 5.00 (d, J ˆ 10.0, PhCH); 4.91 (d, J ˆ 10.8, PhCH);
4.89 (d, J ˆ 11.6, PhCH); 4.82 (d, J ˆ 10.8, PhCH); 4.70 (d, J ˆ 10.4, PhCH); 4.59 (d, J ˆ 11.2, PhCH); 4.47
(s, PhCH2); 3.90 (d, J ˆ 9.6, HÀC(4)); 3.77 (dd, J ˆ 9.1, 3.7, CHÀC(7)); 3.69 (t, J ˆ 10.0, HÀC(6)); 3.56 (t, J ˆ
9.6, HÀC(5)); 3.48( dd, J ˆ 9.1, 2.5, CH'ÀC(7)); 2.64 (br. d, J ˆ 7.9, 0.8H, exchange with D 2O, NH); 2.26 2.06
(0.2 H, exchange with D2O, NH); 2.26 (br. t, J ˆ 12.9, HaxÀC(8)); 2.19 2.06 (m, HÀC(7)); 1.57 (d, J ˆ 7.9,
0.8H, exchange with D 2O, NH); 1.44 (d, J ˆ 8, 0.2 H, exchange with D2O, NH); 1.35 (dd, J ˆ 12.9, 2.9,
HeqÀC(8)). 13C-NMR (75 MHz, CDCl3, one set of signals): 139.00, 138.88, 138.62, 138.50 (4s); 128.62 127.79
(several d); 88.20, 80.33, 79.49 (3d, C(4), C(5), C(6)); 76.38, 76.01, 75.51, 73.21 (4t, 4 PhCH2); 69.42
‡
(t, CH2ÀC(7)); 57.06 (s, C(3)); 39.96 (d, C(7)); 34.80 (t, C(8)). FAB-MS (NOBA): 551 (100, [M ‡ 1] ), 534 (14,
‡
‡
‡
[M À NH2] ), 531 (10), 461 (18), 459 (22, [M À Bn] ), 443 (96, [M À BnO] ), 428(27). Anal. calc. for
C35H38N2O4 (550.70): C 76.34, H 6.95, N 5.09; found: C 76.04, H 6.66, N 4.98.
(4S,5S,6R,7R)-4,5,6-Tris(benzyloxy)-7-[(benzyloxy)methyl]-1,2-diazaspiro[2.5]oct-1-ene (12). At 08, a
soln. of 11 (61 mg, 0.11 mmol) in MeOH (10 ml) and CH2Cl2 (10 ml) was treated with Et3N (0.1 ml, 0.72 mmol)
and, dropwise, with a soln. of I2 (ca. 30 mg) in MeOH (1 ml), until the brown colour persisted. Evaporation and
FC (10 g of silica gel; hexane/AcOEt 10 :1) gave 12 (52 mg, 85%). Colourless, amorphous. Rf (hexane/AcOEt
3 :1) 0.73. M.p. 59 618. [a]2D5 ˆ 49.7 (c ˆ 1.03, CHCl3). UV (CH2Cl2): 232 (846), 252 (846), 258 (906), 340 (92).
FT-IR (1%, CHCl3): 3089w, 3066w, 3007m, 2907m, 2864m, 2791w, 1950w, 1869w, 1810w, 1750w, 1590m , 1558w,
1497m, 1454s, 1400w, 1357m, 1329w, 1309w, 1150m, 1131s, 1096s, 1043s, 1028s, 1005m, 912w, 8 68w. 1H-NMR

Helvetica Chimica Acta Vol. 86 (2003)
2221
(300 MHz, CDCl3): 7.37 7.19 (m, 20 arom. H); 4.93 (d, J ˆ 10.9, PhCH); 4.88 (d, J ˆ 10.9, PhCH); 4.82 (d, J ˆ
10.9, PhCH); 4.57 (d, J ˆ 10.9, PhCH); 4.42 (s, PhCH2); 4.26 (d, J ˆ 11.2, PhCH); 4.21 (d, J ˆ 11.2, PhCH); 3.82
(t, J ˆ 9.2, HÀC(5)); 3.70 (d, J ˆ 9.2, HÀC(4)); 3.65 3.57 (hidden by other signals, HÀC(6)); 3.63 (dd, J ˆ 9.0,
4.4, CHÀC(7)); 3.42 (dd, J ˆ 9.0, 3.4, CH'ÀC(7)); 2.13 2.05 (m, HÀC(7)); 2.05 (br. t, J ˆ 12.8, HaxÀC(8); 0.67
(br. d, J ˆ 10.6, HeqÀC(8)). 13C-NMR (75 MHz, CDCl3): 138.94, 138.79, 138.49, 137.39 (4s); 128.67 127.75
(several d); 86.39, 80.55, 78.10 (3d, C(4), C(5), C(6)); 75.86, 75.57, 73.23, 73.19 (4t, 4 PhCH2); 69.46
‡
(t, CH2ÀC(7)); 39.66 (d, C(7)); 30.21 (t, C(8)); 28.82 (s, C(3)). FAB-MS (NOBA): 549 (60, [M ‡ 1] ), 457 (15,
‡
‡
‡
[M À Bn] ), 441 (18, [M À BnO] ), 413 (22, [M À BnO À N2] ), 307 (33). Anal. calc. for C35H36N2O4 (548.68):
C 76.62, H 6.61, N 5.11; found: C 76.75, H 6.73, N 4.88.
(1S,2S,3R,4R)-1,2,3-Tris(benzyloxy)-4-[(benzyloxy)methyl]-6-methylidenecyclohexane (13). A soln. of 10
(1.17 g, 2.18mmol) in THF (20 ml) was treated dropwise at À 208 with a soln. of Ph3PˆCH2 (prepared at 08
from Ph3PMeBr (1.17 g, 3.27 mmol) and BuLi (2.05 ml, 1.6m in hexane) in THF (15 ml)), allowed to warm
slowly for 3 h, treated with acetone (5 ml), and poured into sat. aq. NH4Cl soln. (150 ml). The aq. phase was
extracted with CH2Cl2 (3 Â 100 ml), and the extract was dried (Na2SO4), and evaporated. FC (150 g of silica gel;
cyclohexane/AcOEt 20 :1) gave 13 (0.90 g, 71%). Colourless oil. Rf (cyclohexane/AcOEt 3 :1) 0.75. [a]2D5 ˆ 56.7
(c ˆ 1.53, CHCl3). FT-IR (1.5%, CHCl3): 3089w, 3066m, 3008m, 2908m, 2862m, 2789w, 1951w, 1876w, 1811w,
1657w, 1605w, 1496m, 1454s, 1400w, 1356m, 1309w, 1149m, 1100s, 1028s, 912m, 8 65w. 1H-NMR (300 MHz,
CDCl3): 7.45 7.24 (20 arom. H); 5.22 (br. d, J ˆ 0.9, CHˆC(6)); 5.05 (d, J ˆ 10.9, PhCH); 4.99 (d, J ˆ 1.6,
CH' ˆ C(6)); 4.96 (d, J ˆ 10.9, PhCH); 4.86 (d, J ˆ 10.9, PhCH); 4.81 (d, J ˆ 11.5, PhCH); 4.73 (d, J ˆ 11.2,
PhCH); 4.58( d, J ˆ 10.9, PhCH); 4.50 (s, PhCH2); 3.96 (br. d, J ˆ 9.3, HÀC(1)); 3.66 (dd, J ˆ 9.0, 1.9,
CHÀC(4)); 3.64 (dd, J ˆ 9.0, 7.2, HÀC(3)); 3.56 (dd, J ˆ 9.0, 2.8, CH'ÀC(4)); 3.53 (t, J ˆ 9.0, HÀC(2)); 2.48
(dd, J ˆ 13.4, 4.4, HeqÀC(5)); 2.16 (br. t, J ˆ 13.4, HaxÀC(5)); 1.84 1.75 (m, HÀC(4)). 13C-NMR (75 MHz,
CDCl3): 144.38( s, C(6)); 139.34 (s); 139.09 (s); 138.83 (s, 2 C); 128.65 127.72 (several d); 108.28
(t, CH2ˆC(6)); 88.37, 83.82, 81.23 (3d, C(1), C(2), C(3)); 75.81, 75.44, 73.68, 73.26 (4t, 4 PhCH2); 70.31 (t);
‡
43.36 (d, C(4)); 33.95 (t, C(5)). FAB-MS (NOBA): 535 (3, [M ‡ 1] ), 335 (9), 280 (15), 279 (17), 263 (13), 262
(10), 221 (13), 207 (22). Anal. calc. for C36H38O4 (534.69): C 80.87, H 7.16; found: C 80.60, H 7.17.
(3S,4S,5S,6R,7R)-4,5,6-Tris(benzyloxy)-7-[(benzyloxy)methyl]-1-[(4-methylphenyl)sulfonyl]-1-azaspiro-
[2.5]octane (14) and 4-Methyl-N-{[(1S,2R,3S,4R,5R)-2,3,4-tris(benzyloxy)-5-[(benzyloxy)methyl]-1-bromocy-
clohexyl]methyl}benzenesulfonamide (15). A soln. of 13 (500 mg, 0.94 mmol) in MeCN (50 ml) was treated with
chloramine T (2.13 g, 9.35 mmol) and PhMe3N ¥ Br3 (123 mg, 0.33 mmol). The mixture was stirred for 4.5 h,
diluted with AcOEt (250 ml), washed with H2O (150 ml) and brine (150 ml), dried (Na2SO4), and evaporated.
FC (80 g of silica gel; cyclohexane/AcOEt 9 :1) of the residue (1.2 g) gave a mixture of 14 and 15 (283 mg),
which was separated by prep. HPLC (silica gel; toluene/AcOEt 40 :1) to yield 14 (120 mg, 18%) and 15 (15 mg,
2%).
Data of 14: Colourless oil. Rf (toluene/AcOEt 10:1) 0.47. Prep. HPLC (toluene/AcOEt 40 :1): tR 22.8min.
[a]2D5 ˆ À9.2 (c ˆ 1.56, CHCl3). FT-IR (1.5%, CHCl3): 3064w, 3008m, 2866m, 1952w, 1810w, 1599w, 1496w,
1454m, 1357m, 1321s, 1158s, 1099s, 995s, 910w, 8 59m. 1H-NMR (300 MHz, CDCl3): 7.84 (d, J ˆ 8.4, 2 arom. H);
7.40 7.08(22 arom. H); 4.87 ( d, J ˆ 10.6, PhCH); 4.84 (d, J ˆ 10.0, PhCH); 4.74 (d, J ˆ 10.6, PhCH); 4.73 (d, J ˆ
10.3, PhCH); 4.59 (d, J ˆ 10.3, PhCH); 4.53 (d, J ˆ 10.9, PhCH); 4.51 (d, J ˆ 11.8, PhCH); 4.44 (d, J ˆ 12.1,
PhCH); 3.78( d, J ˆ 9.3, HÀC(4)); 3.70 (dd, J ˆ 9.0, 4.4, CHÀC(7)); 3.63 (dd, J ˆ 9.3, 10.6, irrad. at 3.78 ! NOE
of 3%, HÀC(6)); 3.51 (dd, J ˆ 9.0, 2.5, CH'ÀC(7)); 3.39 (t, J ˆ 9.2, irrad. at 2.63 ! NOE of 2%, HÀC(5)); 2.63
(br. s, irrad. at 3.39 ! NOE of 1.5%, HÀC(2)); 2.59 (br. s, irrad. at 2.63 ! NOE of 19%, H'ÀC(2)); 2.45
(s, Me); 2.42 2.35 (m, irrad. at 3.78 ! NOE of 1%, irrad. at 2.59 ! NOE of 1%, 2 HÀC(8)); 1.88 1.78
(m, irrad. at 3.39 ! NOE of 9%, irrad. at 2.59 ! NOE of 1%, HÀC(7)). 13C-NMR (75 MHz, CDCl3): 144.25
(s); 138.83, 138.55, 138.16 (3s, 1s hidden by noise or other signals); 129.82 127.58 (several d); 86.49, 80.96, 80.18
(3d, C(4), C(5), C(6)); 76.11, 75.93, 75.54, 73.35 (4t, 4 PhCH2); 69.50 (t, CH2ÀC(7)); 51.73 (s, C(3)); 41.58
‡
(d, C(7)); 35.82 (t, C(8)); 28.77 (t, C(2)); 21.65 (q, Me). MALDI-MS (DHB): 742 (67, [M ‡ K] ), 726 (100,
‡
[M ‡ Na] ). Anal. calc. for C43H45NO6S (703.90): C 73.37, H 6.44, N 1.99; found: C 73.36, H 6.44, N 1.96.
Data of 15: Colourless oil. Rf (toluene/AcOEt 10:1) 0.45. Prep. HPLC (toluene/AcOEt 40 :1): tR 27.2 min.
1H-NMR (300 MHz, CDCl3): 7.56 (d, J ˆ 8.4, 2 arom. H); 7.40 7.18 (22 arom. H); 4.94 (d, J ˆ 10.9, PhCH); 4.92
(d, J ˆ 11.8, PhCH); 4.89 (d, J ˆ 11.2, PhCH); 4.87 (d, J ˆ 10.9, PhCH); 4.73 (d, J ˆ 12.1, PhCH); 4.55 (d, J ˆ
10.9, PhCH); 4.44 (s, PhCH2); 4.03 (t, J ˆ 9.2, HÀC(3)); 3.70 (dd, J ˆ 9.0, 4.1, CHÀC(5)); 3.62 (dd, J ˆ 7.8, 6.2,
irrad.. at 3.04 ! d, NH); 3.57 (dd, J ˆ 10.9, 9.3, HÀC(4)); 3.40 (dd, J ˆ 9.0, 2.2, CH'ÀC(5)); 3.18( dd, J ˆ 13.4,
5.6, CHÀC(1)); 3.13 (d, J ˆ 9.0, irrad. at 4.03 ! s, HÀC(2)); 3.04 (dd, J ˆ 13.4, 8.1, CH'ÀC(1)); 2.34 (s, Me);
‡
2.29 2.15 (m, HÀC(5)); 2.09 1.91 (m, 2 HÀC(6)). FAB-MS (NOBA): 808 (9, [M(81Br) ‡ Na] ), 806 (8,

2222
Helvetica Chimica Acta Vol. 86 (2003)
‡
‡
‡
‡
[M(79Br) ‡ Na] ), 786 (59, [M(81Br) ‡ H] ), 784 (100, [M(81Br) À H] , [M(79Br) ‡ H] ), 782 (51, [M(79Br) À
‡
H] ), 695 (14), 694 (33), 693 (12), 692 (29), 614 (11), 613 (23), 461 (15), 460 (54).
Iodoazidation and Reduction of 13. A soln. of NaN3 (61 mg, 94 mmol) in MeCN (1 ml) at 08 was treated with
ICl (7.3 mg, 45 mmol), stirred for 10 min, treated with 13 (20 mg, 37 mmol), allowed to warm to r.t., stirred for
20 h, and poured into H2O (20 ml). The aq. phase was extracted with AcOEt (2 Â 20 ml), and the combined org.
phases were washed with 5% Na2S2O3 (20 ml) and H2O (2 Â 20 ml), and dried (Na2SO4). Evaporation and FC
(20 g of silica gel; cyclohexane/AcOEt 20 :1) gave a mixture (1H-NMR) of iodo azides (9 mg, 34%). Colourless
oil. Rf (toluene/AcOEt 10 :1) 0.69. FT-IR (1%, CHCl3): 2106s (N3). HR-MS (MALDI): 726.1802
‡
‡
(C36H38IN3NaO4 [M ‡ Na] ; calc. 726.1805).
A soln. of this mixture (9 mg, 13 mmol) in THF (2 ml) was treated at 08 with LiAlH4 (2.5 g, 64 mmol), stirred
for 70 min, and treated with MeOH (1 ml), and 1m NaOH (0.5 ml). Filtration through Celite, evaporation, and
FC (20 g of silica gel; cyclohexane/AcOEt 20 :1) gave 13 (3 mg, 43%).
(3S,4R,5S,6R,7R)- and (3R,4R,5S,6R,7R)-4,5,6-Tris(benzyloxy)-7-[(benzyloxy)methyl]-1-oxaspiro-
[2.5]octane (16 and 17, resp.). A soln. of 13 (20 mg, 37.4 mmol) in CH2Cl2 (1 ml) was treated with NaHCO3
(7 mg, 84.2 mmol) and mCPBA (23 mg, 93.5 mmol), and stirred for 220 min. The mixture was diluted with
CH2Cl2 (25 ml), washed with sat. aq. NaHCO3 soln. (3 Â 20 ml) and brine (20 ml), dried (Na2SO4), and
evaporated. FC (20 g of silica gel; cyclohexane/AcOEt 9 :1) gave 16 (5.2 mg, 25%) and 17 (61%).
Data of 16: Colourless oil. Rf (cyclohexane/AcOEt 3 :1) 0.66. [a]2D5 ˆ 36.0 (c ˆ 1.32, CHCl3). FT-IR (1.3%,
CHCl3): 3065w, 3008m, 2922m, 2863m, 1953w, 1877w, 1811w, 1726w, 1603w, 1496w, 1454m, 1358m, 1099s, 8 41w.
1H-NMR (300 MHz, CDCl3): 7.37 7.19 (20 arom. H); 4.91 (d, J ˆ 10.9, 2 PhCH); 4.78( d, J ˆ 10.9, PhCH); 4.77
(d, J ˆ 10.9, PhCH); 4.62 (d, J ˆ 10.9, PhCH); 4.54 (d, J ˆ 10.9, PhCH); 4.45 (s, PhCH2); 3.73 (br. d, J ˆ 9.3,
HÀC(4)); 3.61 3.54 (m, HÀC(5), HÀC(6), CHÀC(7), irrad. at 3.18 ! NOE of 1% for t at 3.54 of HÀC(5));
3.50 (dd, J ˆ 8.7, 2.8, CH'ÀC(7)); 3.18( dd, J ˆ 5.3, 1.3, HÀC(2)); 2.56 (d, J ˆ 5.3, H'ÀC(2)); 2.09 (td, J ˆ 13.1,
2.0, HaxÀC(8)); 1.93À1.81 (m, HÀC(7)); 1.46 (dd, J ˆ 13.4, 3.4, irrad. at 2.56 ! NOE of 7%, HeqÀC(8)).
13C-NMR (75 MHz, CDCl3): 138.81 (br. s); 128.62 127.79 (several d); 86.83, 80.75, 80.26 (3d, C(4), C(5),
C(6)); 75.93, 75.56, 75.51, 73.23 (4t, 4 PhCH2), 69.89 (t, CH2ÀC(7)); 59.77 (s, C(3)); 49.61 (t, C(2)); 40.06
‡
(d, C(7)); 32.64 (t, C(8)). FAB-MS (NOBA): 1213 (21), 663 (35), 647 (19), 572 (16, [M ‡ Na À 1] ), 550 (44,
‡
‡
‡
M ), 549 (20, [M À 1] ), 548(84, [ M À 2] ), 480 (20), 459 (13), 458(30), 444 (15), 442 (36), 391 (12), 338(14),
306 (19), 288 (15), 271 (13), 260 (11), 259 (40), 245 (16), 242 (12), 219 (47), 217 (12), 203 (13), 197 (12), 181
(100).
Data of 17: Colourless, amorphous. Rf (cyclohexane/AcOEt 3 :1) 0.55. [a]2D5 ˆ 16.5 (c ˆ 1.47, CHCl3). FT-IR
(1.5%, CHCl3): identical to that of 16. 1H-NMR (300 MHz, CDCl3): 7.36 7.22 (20 arom. H); 4.94 (d, J ˆ 10.9,
PhCH); 4.92 (d, J ˆ 10.9, PhCH); 4.88 (d, J ˆ 10.9, PhCH); 4.86 (d, J ˆ 11.5, PhCH); 4.61 (d, J ˆ 11.5, PhCH);
4.58( d, J ˆ 10.9, PhCH); 4.43 (s, PhCH2); 3.83 (t, J ˆ 9.3, HÀC(5)); 3.75 (dd, J ˆ 9.0, 3.4, CHÀC(7)); 3.69
(d, J ˆ 9.6, irrad. at 2.98 ! NOE of 1%, HÀC(4)); 3.66 (br. t, J ˆ 9.6, HÀC(6)); 3.41 (dd, J ˆ 9.0, 1.9,
CH'ÀC(7)); 2.98( d, J ˆ 5.0, HÀC(2)); 2.59 (d, J ˆ 5.0, H'ÀC(2)); 2.09 2.00 (m, HÀC(7), HaxÀC(8)); 1.36
(br. d, J ˆ 10.0, irrad. at 2.59 ! NOE of 4%, HeqÀC(8)). 13C-NMR (75 MHz, CDCl3): 138.94, 138.70, 138.07
(3s, 1s hidden by other signal or noise); 128.68 127.79 (several d); 86.75, 80.81, 78.55 (3d, C(4), C(5), C(6));
75.93, 75.47, 75.38, 73.18 (4t, 4 PhCH2); 69.54 (t, CH2ÀC(7)); 58.76 (s, C(3)); 49.70 (t, C(2)); 39.80 (d, C(7));
‡
‡
32.17 (t, C(8)). FAB-MS (NOBA): 662 (16), 646 (12), 572 (22, [M ‡ Na À 1] ), 550 (32, M ), 549 (26, [M À
‡
‡
1] ), 548(77, [ M À 2] ), 459 (10), 458(24), 442 (28), 335 (9), 288(8), 271 (17), 260 (8), 259 (32), 245 (14), 241
(8), 219 (32), 217 (8), 203 (9), 197 (11), 181 (100). Anal. calc. for C 36H38O5 (550.69): C 78.52, H 6.95; found: C
78.67, H 6.99.
(1S,2R,3S,4R,5R)-1-(Azidomethyl)-2,3,4-tris(benzyloxy)-5-[(benzyloxy)methyl]cyclohexan-1-ol (18). A
soln. of 16 (174 mg, 0.32 mmol) in DMF (25 ml) was treated with NaN3 (255 mg, 3.93 mmol), stirred at 1008
for 20 h, cooled, diluted with AcOEt (200 ml), washed with H2O (100 ml) and brine (100 ml), dried (Na2SO4),
and evaporated. FC (40 g of silica gel; cyclohexane/AcOEt 9:1) gave colourless, amorphous 18 (170 mg, 91%).
Rf (cyclohexane/AcOEt 3 :1) 0.52. M.p. 79 808. [a]2D5 ˆ 16.2 (c ˆ 1.50, CHCl3). FT-IR (1.5%, CHCl3): 3555w,
3066w, 3008m, 2867m, 2107s , 1954w, 1812w, 1589w, 1496w, 1453m, 1359m, 1282m, 1065s, 912w, 8 71w. 1H-NMR
(300 MHz, CDCl3): 7.38 7.19 (20 arom. H); 4.91 ( d, J ˆ 11.5, PhCH); 4.87 (d, J ˆ 10.9, PhCH); 4.85 (s, PhCH2);
4.79 (d, J ˆ 10.6, PhCH); 4.54 (d, J ˆ 10.6, PhCH); 4.46 (s, PhCH2); 3.74 (br. d, J ˆ 12.8, 1 H, CH2N3); 3.66
(dd, J ˆ 9.0, 4.1, CHÀC(5)); 3.62 3.49 (m, HÀC(2), HÀC(3), HÀC(4)); 3.45 (dd, J ˆ 9.0, 2.5, CH'ÀC(5));
3.34 (d, J ˆ 12.8, 1 H, CH2N3); 2.13 (s, OH); 2.06 (dd, J ˆ 12.8, 2.5, HeqÀC(6)); 1.75 1.62 (m, HÀC(5)); 1.58
(br. t, J ˆ 13.1, HaxÀC(6)). 13C-NMR (75 MHz, CDCl3): 138.76, 138.60 (2 br. s); 128.88 127.86 (several d);
86.81, 85.41, 81.06 (3d, C(2), C(3), C(4)); 76.06, 75.78(2 t, 2 PhCH2); 75.68( s, C(1)); 75.54, 73.26 (2t, 2 PhCH2);
69.62 (t, CH2ÀC(5)); 54.49 (t, CH2N3); 38.64 (d, C(5)); 33.80 (t, C(6)). MALDI-MS (DHB): 616 (68,

Helvetica Chimica Acta Vol. 86 (2003)
2223
‡
‡
‡
[M ‡ Na] ), 590 (34), 588 (45, [M ‡ Na À N2] ), 573 (19), 566 (20, [M ‡ 1 À N2] ), 559 (100), 480 (20, [M ‡
‡
‡
Na À N2 À BnOH] ), 460 (19), 458(60, [ M ‡ 1 À N2 À BnOH] ). Anal. calc. for C36H39N3O5 (593.72): C 72.83,
H 6.62, N 7.08; found: C 72.65, H 6.62, N 6.91.
(1R,2R,3S,4R,5R)-1-(Azidomethyl)-2,3,4-tris(benzyloxy)-5-[(benzyloxy)methyl]cyclohexan-1-ol (22). As
described above for 18, 22 (186 mg, 88%) was obtained from 17 (195 mg, 0.35 mmol) and NaN3 (280 mg,
4.31 mmol). Colourless oil. Rf (cyclohexane/AcOEt 3 :1) 0.45. [a]2D5 ˆ 9.1 (c ˆ 1.50, CHCl3). FT-IR (1.5%,
CHCl3): apart from a band at 3559m identical to the spectrum of 18. 1H-NMR (300 MHz, CDCl3): 7.61 7.20 (20
arom. H); 4.98( d, J ˆ 10.9, PhCH); 4.95 (d, J ˆ 11.2, PhCH); 4.85 (d, J ˆ 11.1, 2 PhCH); 4.62 (d, J ˆ 11.2,
PhCH); 4.57 (d, J ˆ 10.9, PhCH); 4.48( d, J ˆ 12.1, PhCH); 4.44 (d, J ˆ 12.1, PhCH); 3.87 (t, J ˆ 9.3, HÀC(3));
3.76 (dd, J ˆ 9.0, 4.1, CHÀC(5)); 3.54 (dd, J ˆ 10.9, 9.3, HÀC(4)); 3.46 (d, J ˆ 9.3, HÀC(2)); 3.43 (dd, J ˆ 9.0,
2.5, CH'ÀC(5)); 3.29 (d, J ˆ 11.8, 1 H, CH2N3); 3.22 (d, J ˆ 11.8, 1 H, CH2N3); 2.41 (d, J ˆ 2.2, OH); 2.21 2.09
(m, HÀC(5)); 1.80 (dd, J ˆ 14.3, 4.1, HeqÀC(6)); 1.67 (td, J ˆ 14.3, 2.2, HaxÀC(6)). 13C-NMR (75 MHz, CDCl3):
139.02, 138.73, 138.13 (3s, 1s hidden by noise or other signals); 128.76 127.81 (several d); 85.67, 81.54, 81.01
(3d, C(2), C(3), C(4)); 75.78(2 t), 75.31 (t, 3 PhCH2); 74.67 (s, C(1)); 73.27 (t, PhCH2); 69.71 (t, CH2ÀC(5));
‡
57.89 (t, CH2N3); 37.39 (d, C(5)); 33.02 (t, C(6)). MALDI-MS (DHB): 616 (47, [M ‡ Na] ), 590 (37), 588 (72,
‡
‡
‡
[M ‡ Na À N2] ), 573 (24), 566 (12, [M ‡ 1 À N2] ), 559 (100), 480 (18, [M ‡ Na À N2 À BnOH] ), 460 (8), 458
‡
(14, [M ‡ 1 À N2 À BnOH] ). Anal. calc. for C36H39N3O5 (593.72): C 72.83, H 6.62, N 7.08; found: C 72.81, H
6.66, N 6.99.
(1S,2R,3S,4R,5R)-1-(Azidomethyl)-2,3,4-tris(benzyloxy)-5-[(benzyloxy)methyl]cyclohexane-1-methane-
sulfonate (19) and (1S,2S,3R,4R)-6-(Azidomethylidene)-1,2,3-tris(benzyloxy)-4-[(benzyloxy)methyl]cyclohex-
ane (26). A soln. of 18 (165 mg, 0.28mmol) in 2,6-lutidine (2.2 ml) was treated at 0 8 with MsCl (0.1 ml,
1.25 mmol) and DMAP (2 mg), stirred for 7.5 h, diluted with Et2O (120 ml), washed with cold brine (2 Â 60 ml),
dried (Na2SO4), and evaporated. FC (25 g of silica gel; cyclohexane/AcOEt 9 :1) gave 26 (6 mg, 4%) and 19
(177 mg, 95%).
Data of 19: Colourless oil. Rf (toluene/AcOEt 10 :1) 0.49. 1H-NMR (300 MHz, CDCl3): 7.41 7.17 (20
arom. H); 4.98( d, J ˆ 10.9, PhCH); 4.92 (d, J ˆ 10.9, PhCH); 4.88 (d, J ˆ 10.3, PhCH); 4.87 (d, J ˆ 10.9, PhCH);
4.84 (d, J ˆ 11.2, PhCH); 4.55 (d, J ˆ 10.9, PhCH); 4.54 (d, J ˆ 12.5, PhCH); 4.49 (d, J ˆ 12.1, PhCH); 4.38
(d, J ˆ 9.3, HÀC(2)); 4.10 (br. d, J ˆ 13.4, 1 H, CH2N3); 3.67 (dd, J ˆ 9.0, 4.7, CHÀC(5)); 3.66 (t, J ˆ 9.2,
HÀC(4)); 3.53 (t, J ˆ 9.3, HÀC(3)); 3.51 (dd, J ˆ 9.0, 2.5, CH'ÀC(5)); 3.45 (d, J ˆ 14.0, 1 H, CH2N3); 3.02
(s, MsO); 2.66 (dd, J ˆ 13.4, 3.7, HeqÀC(6)); 2.52 (br. t, J ˆ 13.1, HaxÀC(6)); 1.79 1.66 (m, HÀC(5)). 13C-NMR
(75 MHz. CDCl3): 138.62, 138.47 (2 br. s); 128.70 127.79 (several d); 95.31 (s, C(1)); 85.91, 84.70, 80.28
(3d, C(2), C(3), C(4)); 76.22, 75.91, 75.59, 73.26 (4t, 4 PhCH2); 69.18( t, CH2ÀC(5)); 52.94 (t, CH2N3); 41.14
‡
(q, MsO); 38.64 (d, C(5)); 30.94 (t, C(6)). MALDI-MS (DHB): 570 (100, [M ‡ Na À HOMs À N2] ).
Data of 26: Colourless oil. Rf (toluene/AcOEt 10 :1) 0.67. FT-IR (1.5%, CHCl3): 3089w, 3067w, 3008m,
2914w, 2862m, 2109s, 1950w, 1732w, 1668w, 1604w, 1496w, 1454m, 1358m, 1327w, 1275w, 1151m, 1130m, 1095m,
1028m, 909w, 8 43w. 1H-NMR (300 MHz, CDCl3): 7.36 7.18(20 arom. H); 6.36 (br. t, J ˆ 1.2, CHN3); 4.93
(d, J ˆ 10.9, PhCH); 4.87 (d, J ˆ 10.9, PhCH); 4.81 (d, J ˆ 10.9, PhCH); 4.75 (d, J ˆ 11.5, PhCH); 4.69 (d, J ˆ
11.5, PhCH); 4.53 (d, J ˆ 10.9, PhCH); 4.46 (s, PhCH2); 3.92 (br. d, J ˆ 7.5, HÀC(1)); 3.65 (dd, J ˆ 9.0, 4.7,
CHÀC(4)); 3.58( t, J ˆ 9.5, HÀC(3)); 3.52 3.47 (dd, J ˆ 9.9, 2.5, partly hidden by other signals, CH'ÀC(4));
3.45 (t, J ꢀ 8.7, HÀC(2)); 2.73 (dd, J ˆ 13.7, 3.7, HeqÀC(5)); 1.80 (br. t, J ˆ 13.4, HaxÀC(5)); 1.72 1.60
(m, HÀC(4)). 13C-NMR (75 MHz, CDCl3): 139.07; 138.73; 138.39; 128.73 127.81; 126.55; 120.32; 88.59;
82.58; 80.80; 75.67; 75.36; 74.02; 73.24; 69.93; 42.24; 30.39. MALDI-MS (DHB): 588 (5), 586 (1, [M ‡ K À
‡
‡
‡
N2] ), 570 (100, [M ‡ Na À N2] ), 566 (4), 548(17, [ M ‡ 1 À N2] ).
(1R,2R,3S,4R,5R)-1-(Azidomethyl)-2,3,4-tris(benzyloxy)-5-[(benzyloxy)methyl]cyclohexane-1-methane-
sulfonate (23). As described above, treatment of 22 (103 mg, 0.17 mmol) with MsCl (0.06 ml, 0.77 mmol) and
DMAP (1 mg) in 2,6-lutidine (3 ml) at 08 gave 23 (116 mg, 99%). Colourless oil. Rf (toluene/AcOEt 10 :1) 0.38.
1H-NMR (300 MHz, CDCl3): 7.42 7.19 (20 arom. H); 4.96 (d, J ˆ 11.2, PhCH); 4.91 (d, J ˆ 10.9, PhCH); 4.89
(d, J ˆ 10.6, PhCH); 4.85 (d, J ˆ 10.9, PhCH); 4.69 (d, J ˆ 11.2, PhCH); 4.56 (d, J ˆ 10.9, PhCH); 4.48( d, J ˆ
11.8, PhCH); 4.43 (d, J ˆ 12.1, PhCH); 4.17 (d, J ˆ 11.8, 1 H, CH2N3); 3.90 (t, J ˆ 9.3, HÀC(3)); 3.90 (d, J ˆ 12.1,
1 H, CH2N3); 3.79 (dd, J ˆ 9.3, 3.7, CHÀC(5)); 3.62 (dd, J ˆ 10.9, 9.3, HÀC(4)); 3.54 (d, J ˆ 9.7, HÀC(2)); 3.43
(dd, J ˆ 9.3, 2.5, CH'ÀC(5)); 3.07 (s, MsO); 2.40 (dd, J ˆ 14.9, 3.4, HeqÀC(6)); 2.25 2.13 (m, HÀC(5)); 1.89
(dd, J ˆ 14.6, 13.4, HaxÀC(6)). 13C-NMR (75 MHz, CDCl3): 138.83, 138.76, 138.55, 138.29 (4s); 128.70 127.87
(several d); 94.00 (s, C(1)); 84.66, 81.06, 80.54 (3d, C(2), C(3), C(4)); 76.04, 75.77, 75.62, 73.34 (4t, 4 PhCH2);
69.00 (t, CH2ÀC(5)); 53.46 (t, CH2N3); 40.71 (q, MsO); 37.83 (d, C(5)); 32.67 (t, C(6)). MALDI-MS (DHB):
‡
570 (100, [M ‡ Na À HOMs À N2] ).

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Helvetica Chimica Acta Vol. 86 (2003)
(3R,4S,5S,6R,7R)-4,5,6-Tris(benzyloxy)-7-[(benzyloxy)methyl]-1-azaspiro[2.5]octane (20). A soln. of 19
(165 mg, 0.25 mmol) in THF (15 ml) was treated at 08 with LiAlH4 (49 mg, 1.2 mmol), stirred for 3.5 h, treated
with MeOH (14 ml) and 1m NaOH (3 ml), filtered through Celite, and evaporated. FC (20 g of silica gel;
cyclohexane/AcOEt 1:1) gave 20 (112 mg, 83%). Colourless oil. Rf (cyclohexane/AcOEt 1:1) 0.14. [a]2D5 ˆ 32.1
(c ˆ 1.50, CHCl3). FT-IR (1.5%, CHCl3): 3296w , 3066m, 3007s, 2911m, 2863m, 1952w, 1877w, 1811w, 1725w,
1585w, 1497m, 1453m, 1359m, 1152s, 1095s, 909m, 8 69w. 1H-NMR (300 MHz, CDCl3): 7.38 7.24 (20 arom. H);
4.94 (d, J ˆ 10.9, PhCH); 4.92 (br. d, J ˆ 12.1, 2 PhCH); 4.86 (d, J ˆ 10.9, PhCH); 4.62 (d, J ˆ 10.9, PhCH); 4.57
(d, J ˆ 11.2, PhCH); 4.48( d, J ˆ 12.1, PhCH); 4.44 (d, J ˆ 12.1, PhCH); 3.79 (dd, J ˆ 9.0, 3.7, CHÀC(7)); 3.78
(d, J ˆ 9.0, HÀC(4)); 3.68( t, J ˆ 9.5, HÀC(6)); 3.60 (t, J ˆ 9.2, HÀC(5)); 3.45 (dd, J ˆ 9.0, 2.2, CH'ÀC(7));
2.15 2.06 (m, HÀC(7)); 2.01 (t, J ˆ 12.9, HaxÀC(8)); 1.87 (br. s, HÀC(2)); 1.40 (br. s, H'ÀC(2)); 1.29 1.21
(m, HeqÀC(8)); 1.17 0.87 (br. s, NH). 13C-NMR (75 MHz, CDCl3): 139.05, 138.86, 138.21 (3s, 1s hidden by
noise or other signals); 128.72 127.75 (several d); 87.79, 81.25, 79.20 (3d, C(4), C(5), C(6)); 75.96, 75.88, 75.44,
73.19 (4t, 4 PhCH2); 69.84 (t, CH2ÀC(7)); 40.29 (d, C(7)); 37.42 (s, C(3)); 33.62 (t, C(8)); 27.17 (t, C(2)).
‡
‡
‡
MALDI-MS (DHB): 588 (2, [M ‡ K] ), 572 (100, [M ‡ Na] ), 550 (4, [M ‡ 1] ). Anal. calc. for C36H39NO4
(549.71): C 78.66, H 7.15, N 2.55; found: C 78.57, H 7.12, N 2.48.
(3S,4S,5S,6R,7R)-4,5,6-Tris(benzyloxy)-7-[(benzyloxy)methyl]-1-azaspiro[2.5]octane (24). As described
above, a soln. of 23 (116 mg, 0.17 mmol) in THF (11 ml), was treated with LiAlH4 (35 mg, 0.86 mmol) at 08 for
9 h to yield 24 (70 mg, 74%). Colourless oil. Rf (cyclohexane/AcOEt 1 :1) 0.22. [a]2D5 ˆ 8.8 (c ˆ 1.42, CHCl3). FT-
1
IR (1.4%, CHCl3): identical to that of 20. H-NMR (300 MHz, CDCl3): 7.38 7.23 (20 arom. H); 4.93 ( d, J ˆ
10.9, PhCH); 4.92 (d, J ˆ 11.8, PhCH); 4.91 (d, J ˆ 10.9, PhCH); 4.86 (d, J ˆ 10.9, PhCH); 4.59 (d, J ˆ 10.9,
PhCH); 4.58( d, J ˆ 11.5, PhCH); 4.46 (s, PhCH2); 3.65 3.57 (m, HÀC(4), HÀC(5), HÀC(6), CHÀC(7)); 3.49
(dd, J ˆ 9.0, 2.8, CH'ÀC(7)); 2.00 (br. t, J ꢀ 12.5, HaxÀC(8)); 1.98 (br. s, HÀC(2)); 1.93 1.81 (m, HÀC(7));
1.31 1.25 (m, HeqÀC(8)); 1.29 (br. s, H'ÀC(2)); 1.18 0.85 (br. s, NH). 13C-NMR (75 MHz, CDCl3): 138.89,
138.71, 138.45 (3s, 1s hidden); 128.85 127.78 (several d); 88.61, 81.54, 79.60 (3d, C(4), C(5), C(6)); 75.75, 75.64,
75.51, 73.21 (4t, 4 PhCH2); 70.02 (t, CH2ÀC(7)); 40.92 (d, C(7)); 37.86 (s, C(3)); 34.32 (t, C(8)); 26.97 (t, C(2)).
MALDI-MS (DHB): identical to that of 20. Anal. calc. for C36H39NO4 (549.71): C 78.66, H 7.15, N 2.55; found:
C 78.86, H 7.32, N 2.60.
(3R,4S,5S,6R,7R)-7-(Hydroxymethyl)-1-azaspiro[2.5]octane-4,5,6-triol (21).
A soln. of 20 (10 mg,
18mmol) in NH 3 (5 ml) and THF (2 ml) at À 788 was treated with Na in small pieces (ca. 20 mg), until the
blue colour persisted for 1 h, treated with NH4Cl (ca. 40 mg), until the blue colour disappeared, allowed to warm
to r.t. overnight, and evaporated. The residue was dried in h.v., suspended in abs. MeOH, and filtered. After
evaporation, the suspension of the residue in abs. EtOH was filtered. Evaporation and chromatography on
Sephadex G-10 (20 Â 1 cm, eluant H2O), and lyophilisation gave a mixture of 21, an unidentified by-product,
and inorganic material (13 mg, colourless powder). Rf (PrOH/AcOH/H2O 4 :1 :1) 0.31. 1H-NMR (300 MHz,
D2O): 3.80 (d, J ˆ 9.3, HÀC(4)); 3.78( dd, J ˆ 11.5, 3.1, CHÀC(7)); 3.70 (dd, J ˆ 11.2, 5.0, CH'ÀC(7)); 3.45
(t, J ˆ 9.5, HÀC(6)); 3.36 (t, J ˆ 9.2, HÀC(5)); 1.96 (br. s, HÀC(2)); 1.94 (s, impurity); 1.87 1.78 (m, HÀC(7),
HaxÀC(8)); 1.53 (br. s, H'ÀC(2)); 1.35 (d, J ˆ 6.9, impurity); 1.26 (br. d, J ˆ 10.6, HeqÀC(8)). HR-MALDI-MS
‡
‡
(DHB): 190.1076 (C8H16NO4 , [M ‡ H] ; calc. 190.1079).
(1S,2S,3S,4R,5R)-1-Amino-1-(chloromethyl)-5-(hydroxymethyl)cyclohexane-2,3,4-triol Hydrochloride
(27 ¥ HCl) and (1S,2S,3S,4R,5R)-1-Amino-5-(hydroxymethyl)-1-methylcyclohexane-2,3,4-triol Hydrochloride
(28 ¥ HCl). A soln. of 20 (12 mg, 21.8mmol) in MeOH (2 ml) was acidified (pH 4) by dropwise addition of a
soln. of conc. HCl (1 drop) in MeOH (1 ml) (20 drops), treated with 10% Pd on C (10 mg), and stirred under a
H2 atmosphere for 110 min. Filtration through a pad of Celite (washed with 20 ml of 0.1n HCl in MeOH and with
50 ml of MeOH) with MeOH, and evaporation gave a mixture of 27 ¥ HCl and 28 ¥ HCl (7.6 mg, 3 :2). Rf (PrOH/
AcOH/H2O 4 :1:1) 0.35. 1H-NMR (300 MHz, D2O; 27 ¥ HCl/28 ¥ HCl 3 :2): 4.06, 3.79 (2d, J ˆ 11.8, 1.2 H); 3.83
3.33 (m, 5 H); 2.32 (br. d, J ˆ 12.1, 0.6 H, HÀC(6)); 2.04 (dd, J ˆ 14.8, 3.3, 0.4 H, HÀC(6)); 1.81 1.57 (m, 2 H,
‡
‡
‡
HÀC(5), H'ÀC(6)); 1.43 (s, 1.2 H, Me). ESI -MS: 27: 250 (6, [M(37Cl) ‡ Na] ), 248(11, [ M(35Cl) ‡ Na] ), 228
‡
‡
‡
‡
(14, [M(37Cl) ‡ 1] ), 226 (34, [M(35Cl) ‡ 1] ), 222 (34, [M À Cl ‡ MeOH] ), 190 (100, [M À Cl] ); 28: 224 (8,
‡
‡
[M ‡ MeOH ‡ 1] ), 192 (94, [M ‡ 1] ).
Compound 27 ¥ HCl. At À 608, Na (70 mg, 2 mmol) was dissolved in NH3 (3 ml) and THF (5 ml), stirred for
15 min, treated dropwise with a soln. of 20 (31 mg, 0.05 mmol) in THF (1 ml), and stirred for 30 min. at À 508.
The blue mixture was treated with NH4Cl in small portions, until it became colourless, and NH3 was allowed to
evaporate. The remaining soln. was diluted with MeOH, filtered through Celite, and evaporated. The residue
was purified by chromatography on Sephadex C-25 (elution with HCl 0.01m 1m). Lyophilisation of the eluate
1
gave a mixture of 27 ¥ HCl and NH4Cl (64 mg). H-NMR (300 MHz, D2O): 4.11 (d, J ˆ 12.3, CHÀC(1)); 3.85

Helvetica Chimica Acta Vol. 86 (2003)
2225
(d, J ˆ 12.3, CH'ÀC(1)); 3.81 (dd, J ˆ 11.4, 3.3, CHÀC(5)); 3.75 (dd, J ˆ 11.1, 5.4, CH'ÀC(5)); 3.71 3.61
(m, 2 H); 3.44 (ddd, J ˆ 9.6, 7.8, 1.5, 1 H); 2.37 (dd, J ˆ 14.1, 2.4, HÀC(6)); 1.87 1.65 (m, HÀC(5), H'ÀC(6)).
(3S,4S,5S,6R,7R)-7-(Hydroxymethyl)-1-azaspiro[2.5]octane-4,5,6-triol (25).
A soln. of 24 (33 mg,
60 mmol) in NH3 (10 ml) and THF (2.5 ml) was treated at À 788 with Na in small pieces (ca. 30 mg), until
the blue colour persisted for 1 h, treated with NH4Cl (ca. 50 mg), until the blue colour disappeared, and allowed
to warm to r.t. overnight. The solvent was evaporated, the residue dried in h.v., suspended in abs. MeOH, and
filtered. After evaporation, the suspension of the resulting residue in abs. EtOH was filtered, and the filtrate
evaporated. The residue (138mg) was adsorbed on neutral Dowex 50-WX-8 (3 ml, washed with H2O). After
washing with H2O (50 ml), elution with 2% aq. NH3 gave crude 25 (6 mg). Chromatography (25 g of Nucleoprep
20 CN, MeOH) gave colourless, amorphous 25 (5.2 mg, 45%). Rf (PrOH/AcOH/H2O 4 :1 :1) 0.26. [a]2D5 ˆ 6.0
(c ˆ 0.31, H2O). pKHA ˆ 6.78. 1H-NMR (300 MHz, CD3OD): 3.75 (dd, J ˆ 10.9, 3.7, CHÀC(7)); 3.58( dd, J ˆ
10.9, 5.9, CH'ÀC(7)); 3.48( d, J ˆ 9.0, HÀC(4)); 3.30 (t, J ˆ 9.2, HÀC(6)); 3.22 (t, J ˆ 9.0, HÀC(5)); 2.02
(br. s, HÀC(2)); 1.73 (br. t, J ˆ 12.5, HaxÀC(8)); 1.68À1.57 (m, HÀC(7)); 1.34 (br. s, H'ÀC(2)); 1.29 (dd, J ˆ
12.5, 2.8, HeqÀC(8)). 13C-NMR (75 MHz, CD3OD): 80.44, 75.30, 72.11 (3d, C(4), C(5), C(6)); 64.25
(t, CH2ÀC(7)); 43.77 (d, C(7)); 39.90 (s, C(3)); 34.27 (t, C(8)); 27.36 (t, C(2)). HR-MALDI-MS (DHB): 401
‡
‡
‡
‡
(69, [2 M ‡ Na] ), 379 (71, [2 M ‡ 1] ), 212.0891 (19) (C8H15NNaO4 , [M ‡ Na] ; calc. 212.0899), 207.1337 (23)
‡
‡
‡
‡
(C8H19N2O4 , [M ‡ NH4] ; calc. 207.1345), 190.1074 (100) (C8H16NO4 , [M ‡ H] ); calc. 190.1079).
(1S,2S,3S,4R,5R)-2,3,4-Tris(benzyloxy)-5-[(benzyloxy)methyl]cyclohexane-1-methanesulfonate (30). A
soln. of 29 [56] (200 mg, 0.37 mmol) in pyridine (6 ml) at 08 was treated with MsCl (0.12 ml, 1.49 mmol),
stirred for 45 min at 08 and for 90 min at r.t., diluted with AcOEt (100 ml), washed with H2O (100 ml) and brine
(100 ml), dried (Na2SO4), and evaporated. FC (20 g of silica gel; cyclohexane/AcOEt 9 :1) gave colourless,
amorphous 30 (212 mg, 93%). Rf (cyclohexane/AcOEt 3 :1) 0.40. M.p. 137 1388. 1H-NMR (300 MHz, CDCl3):
7.38 7.21 (20 arom. H); 5.22 5.18( m, HÀC(1)); 4.94 (d, J ˆ 10.9, PhCH); 4.90 (d, J ˆ 9.7, PhCH); 4.84 (d, J ˆ
10.6, PhCH); 4.78( d, J ˆ 11.2, PhCH); 4.69 (d, J ˆ 10.9, PhCH); 4.55 (d, J ˆ 10.9, PhCH); 4.43 (s, PhCH2); 3.84
(t, J ˆ 9.3, HÀC(3)); 3.77 (dd, J ˆ 9.0, 3.4, CHÀC(5)); 3.59 (dd, J ˆ 10.9, 9.3, HÀC(4)); 3.49 (dd, J ˆ 9.7, 2.8,
HÀC(2)); 3.40 (dd, J ˆ 9.3, 2.2, CH'ÀC(5)); 3.01 (s, MsO); 2.18 2.05 ( m, HÀC(5), HeqÀC(6)); 1.77 (br. t, J ˆ
13.7, HaxÀC(6)). 13C-NMR (75 MHz, CDCl3): 138.9, 138.55, 137.78 (3s, 1s hidden by other signals); 128.76
127.84 (several d); 83.39, 81.20, 80.34, 79.07 (4d, C(1), C(2), C(3), C(4)); 75.89, 75.54, 73.40, 73.21 (4t, 4 PhCH2);
‡
69.08( t, CH2ÀC(5)); 39.03 (d, C(5)); 37.42 (q, MsO); 30.32 (t, C(6)). FAB-MS (NOBA): 639 (25, [M ‡ Na] ),
‡
615 (100, [M À 1] ), 525 (48), 435 (6), 391 (7). Anal. calc. for C36H40O7S (616.77): C 70.11, H 6.54; found: C
69.88, H 6.47.
(1R,2S,3S,4R,5R)-1-Azido-2,3,4-tris(benzyloxy)-5-[(benzyloxy)methyl]cyclohexane (31). A soln. of 30
(210 mg, 0.34 mmol) in DMF (15 ml) was treated with NaN3 (221 mg, 3.4 mmol), stirred at 1208 for 3 h, cooled
to r.t., diluted with AcOEt (60 ml), washed with H2O (50 ml) and brine (50 ml), dried (Na2SO4), and
evaporated. FC (30 g of silica gel; cyclohexane/AcOEt 20 :1) gave colourless, amorphous 31 (181 mg, 94%). Rf
(cyclohexane/AcOEt 9 :1) 0.36. M.p. 69 708. [a]2D5 ˆ 49.5 (c ˆ 1.49, CHCl3). FT-IR (1.5%, CHCl3): 3066w,
3006w, 2865w, 2103s, 1497w, 1454m, 1359m, 1260w, 1089m, 1028m, 910w. 1H-NMR (300 MHz, CDCl3): 7.38 7.18
(20 arom. H); 4.93 4.83 (m, 5 PhCH); 4.53 (d, J ˆ 10.9, PhCH); 4.46 (s, PhCH2); 3.60 (dd, J ˆ 9.0, 5.0,
CHÀC(5)); 3.59 3.35 (m, 5 H); 2.04 (dt, J ˆ 13.4, 3.7, HeqÀC(6)); 1.82 1.69 (m, HÀC(5)); 1.48( q, J ˆ 13.0,
HaxÀC(6)).13C-NMR (75 MHz, CDCl3): 138.84, 138.63, 138.50, 138.24 (4s); 128.67 127.86 (several d); 86.91,
85.05, 80.62 (3d, C(2), C(3), C(4)); 75.91 (t, 2 PhCH2); 75.52, 73.34 (2t, 2 PhCH2); 69.71 (t, CH2ÀC(5)); 63.21
‡
(d, C(1)); 40.01 (d, C(5)); 30.68( t, C(6)). FAB-MS (NOBA): 614 (29), 565 (38, [M ‡ 2] ), 554 (13), 540 (11),
537 (20), 510 (18), 496 (12), 461 (100), 452 (13), 444 (20), 429 (19), 422 (17), 408 (14), 392 (10), 339 (29), 329
(12). Anal. calc. for C35H37N3O4 (563.70): C 74.58, H 6.62, N 7.45; found: C 74.61, H 6.71, N 7.23.
(1R,2S,3S,4R,6R)-4-Amino-6-(hydroxymethyl)cyclohexane-1,2,3-triol (32). A soln. of 31 (50 mg, 88 mmol)
in MeOH (12 ml) was treated with conc. aq. HCl (2 drops) and Pd on C (10%, 40 mg), stirred in a H2
atmosphere (6 bar) for 15 h, filtered through Celite, and evaporated. The residue was adsorbed on neutral
Dowex 50 WX8 (3 ml, washed with H2O). After washing with H2O (70 ml), elution with 2% aq. NH3 gave 32
(16 mg, 100%). Rf (PrOH/AcOH/H2O 4 :1:1) 0.31. [a]2D5 ˆ 17 (c ˆ 0.42, H2O; [14]: 17.2 (c ˆ 0.57, H2O)).
1H-NMR (300 MHz, D2O): 3.77 (dd, J ˆ 11.2, 3.4, CHÀC(6)); 3.62 (dd, J ˆ 11.2, 6.2, CH'ÀC(6)); 3.31 3.26
(m, 2 H); 3.14 3.08( m, 1 H); 2.74 (ddd, J ˆ 13.1, 9.65, 4.0, HÀC(4)); 1.92 (dt, J ˆ 13.1, 4.0, HeqÀC(5)); 1.75
1.60 (m, HÀC(6)); 1.15 (br. q, J ˆ 12.6, HaxÀC(5)). 13C-NMR (75 MHz, D2O): 80.26, 80.10, 75.68 (3d, C(1),
C(2), C(3)); 65.09 (t, CH2ÀC(6)); 54.81 (d, C(4)); 44.05 (d, C(6)); 33.77 (t, C(5)).
Inhibition Studies. The IC50 values were determined based on a range of inhibitor concentrations (typically
4
8concentrations) that bracket the IC50 value.

2226
Helvetica Chimica Acta Vol. 86 (2003)
a) Inhibition of the b-Glucosidase from Almonds in the pH Range of 6.2 7.8. IC50 values were determined
at 378 in 0.08m KH2PO4/K2HPO4 or NaH2PO4/Na2HPO4 buffer, with 4-nitrophenyl b-d-glucopyranoside as the
substrate ([S] ꢀ KM). The enzymatic reaction was started after incubation of the enzyme for 10 60 min in the
presence of the inhibitor by the addition of substrate. The increase of absorption per min at 400 nm was taken as
the relative rate for the hydrolysis of the substrate. The increase was linear during all measurements (1 min).
IC50 Values were determined by plotting the relative rate of substrate hydrolysis vs. the inhibitor concentration.
Determination of the inhibitor concentration corresponding to half the relative rate measured in the absence of
the inhibitor gave the appropriate IC50 value. To check for slow inhibition, the dependence of the inhibition on
the incubation time was determined.
b) Inhibition of the b-Glucosidase from Almonds at pH 4.2. See a. IC50 Values were determined at 378 in
0.08m citric acid/Na2HPO4 buffer (pH 4.2). The enzymatic reaction was started after incubation of the enzyme
for 15 min in the presence of the inhibitor by the addition of substrate. The mixture was incubated at 378 for
5 min, and the reaction was quenched by the addition of 0.2m borate buffer (pH 9.0). The absorption at 400 nm
was measured immediately and taken as the relative rate for the hydrolysis of the substrate.
c) Inhibition of the b-Glucosidase from Caldocellum saccharolyticum. As described in a or b, respectively.
All measurements were performed at 558.
d) Inhibition of the a-Glucosidase from Brewer×s Yeast at pH 6.8. As described in a. All measurements were
performed at 378, with 4-nitrophenyl a-d-glucopyranoside as substrate.
e) Inhibition of the a-Glucosidase from Brewer×s Yeast and the b-Glucosidase from Almonds at pH 5.0. As
described in a, b, and d, with 0.08m AcOH/AcONa buffer (pH 5.0).
f) Determination of Ki and ki Values for the Irreversible Inhibition of the b-Glucosidase from Caldocellum
saccharolyticum by 25. Cf. [6][68][69]. The enzyme was incubated with various concentrations of inhibitor
(5.0 0.5 mm) at 558 in 0.08m KH2PO4/K2HPO4 buffer (pH 6.8). Aliquots (50 ml) were taken at appropriate time
intervals, diluted into 900 ml of buffer, and the residual enzyme activity was measured by the increase of the
absorbance at 400 nm, which was observed after the addition of substrate. For each inhibitor concentration, ln of
the residual activity was plotted vs. time, fitted to a straight line, and the first-order rate constant for the
inactivation determined from the slope of the line. Replotting the reciprocal of these rate constants vs. the
reciprocal of the inhibitor concentrations gave a straight line, from which Ki and ki values were determined.
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Received March 5, 2003