(3R,4S,5R,6R,7S)-3,4,5,7-Tetrahydroxyconidine, an azetidine analogue of 6,7-diepicastanospermine and a conformationally constrained D- deoxyaltronojirimycin, from L-arabinose

Article abstract of DOI:10.1016/j.bmc.2013.03.004

The synthesis from l-arabinose of an azetidine analogue of 6,7-diepicastanospermine and its glycosidase inhibition profile are described.

Full text of DOI:10.1016/j.bmc.2013.03.004

Bioorganic & Medicinal Chemistry 21 (2013) 4813–4819
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
(3R,4S,5R,6R,7S)-3,4,5,7-Tetrahydroxyconidine, an azetidine analogue
of 6,7-diepicastanospermine and a conformationally constrained
D-deoxyaltronojirimycin, from L-arabinose
R. Fernando Martínez a, Noelia Araújo a, Sarah F. Jenkinson a, Shinpei Nakagawa b, Atsushi Kato b,
,
⇑
George W. J. Fleet a,c,
⇑
a Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK
b Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
c Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford OX1 3QU, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 13 March 2013
The synthesis from L-arabinose of an azetidine analogue of 6,7-diepicastanospermine and its glycosidase
inhibition profile are described.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
6,7-Diepicastanospermine
Glycosidase inhibition
Azetidine
L
-Arabinose
1. Introduction
as a conformationally restricted equivalent of 1-deoxyaltronojiri-
mycin (altro-DNJ) 7. altro-DNJ 7 has been isolated from Scilla sibiri-
ca,12 Angylcalyx pyrnaertii,13 and from a Thai medicinal plant
(Connarus ferrugineus).14 Azet-LAB 8, which has recently been
shown to be a good inhibitor of some non-mammalian glycosi-
dases,6 has the same aminotriol motif. Several stereoisomers of
castanospermine have been isolated from Castanospermum aust-
rale,15,16 including 6,7-diepicastanospermine.17 Many syntheses
of altro-DNJ 7 have been reported,18 but only one synthesis of
6,7-diepicastanospermine 1 has been described.19 Inhibition of
Iminosugars,1 where the oxygen of the sugar ring is replaced by
nitrogen, are carbohydrate mimics; their interactions with
enzymes and other receptors indicate their promise as chemother-
apeutic agents.2 Many hundreds of pyrrolidine, piperidine and aze-
pane monosaccharide analogues have been studied. In contrast,
there are few examples of carbohydrate-derived azetidines,3
although the properties of hydroxyazetidines are of current inter-
est.4 N-Alkyl hydroxyazetidines are potent inhibitors of purine
nucleoside phosphorylase with sub-nanomolar Ki.5 Azetidine imi-
nosugar analogues of pentoses have been found to be specific
inhibitors of non-mammalian glycosidases,6,7 and N-nonyl trihy-
droxy-azetidines are specific inhibitors of some ceramide-specific
glucosyl transferases and glucosidases.8 Bicyclic trihydroxy azeti-
dines with no hydroxyl group in the azetidine ring show no signif-
icant glycosidase inhibition;9 an azetidine analogue of swainsonine
a-glucosidases of N-linked glycoprotein processing by castano-
spermine has led to strategies for the chemotherapy of viral dis-
eases.20 Celgosivir, the 6-O-butyryl ester of castanospermine, is in
clinical trials as a chemotherapeutic agent for hepatitis C.21
Synthesis of many castanospermine analogues22 have allowed
the structural basis of the inhibition of human glycosidases by
castanospermines to be delineated.23 This paper provides the first
synthesis of a tetrahydroxyazetidine analogue 2 of indolizidine
iminosugars, together with other conidines, and reports on the
effects of 2 and related compounds on a panel of glycosidases.
showed no significant inhibition of a
-mannosidases.10
This paper reports the synthesis of tetrahydroxyconidine 2,11 an
azetidine analogue of 6,7-diepicastanospermine 1, via hydroxyl-
ation of the key intermediate 3 derived from
L-arabinose 4
(Fig. 1); it was not possible to achieve the alternative hydroxyl-
ation that would lead to the azetidine 6 as the corresponding ana-
logue of castanospermine 5. The azetidine 2 can also be considered
2. Results and discussion
2.1. Synthesis
For the synthesis of the conidine 2, the protected azetidine 9,6,24
derived from L-arabinose 4, underwent acid catalyzed hydrolysis
⇑
Corresponding authors.
E-mail addresses: kato@med.u-toyama.ac.jp (A. Kato), george.fleet@chem.ox.
c.uk (G.W.J. Fleet).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.004

4814
R. F. Martínez et al. / Bioorg. Med. Chem. 21 (2013) 4813–4819
OH
OH
7
MeO2C
OH
OHC
OH
HO
OH
HO
OH
OH
3
5
6
HO
HOH2C
BnN
N
N
1
7
OH
OH
OH
1
2
3
4
6,7 numbering for
1,..7 numbering
for conidine
castanospermine
OH
OH
OH
OH
OH
HO
HO
OH
HO
OH
CH2OH
OH
OH
N
N
N
H
HN
OH
OH
5
6
7
8
Figure 1. Synthetic strategy for azetidine analogue of castanospermine.
by aqueous trifluoroacetic acid to afford a 1:1 mixture of the lactols
10 in 77% yield (Scheme 1). Reduction of the lactol in situ, followed
by hydrogenolysis of the benzyl group afforded azet-LAB 8 as
previously described.6 Reaction of the purified lactols 10 with the
stabilized ylid Bu3P@CHCO2Me in 1,4-dioxane gave the trans-eno-
ate 3 in 52% yield. Transfer hydrogenation of 3 by ammonium
formate in the presence of palladium on carbon caused debenzyla-
tion and reduction of the C@C followed by cyclization to the lactam
11 (65%); the very low carbonyl stretch at 1607 cmꢀ1 is character-
istic of conidine lactams.25 All attempts to reduce the unprotected
lactam directly to the dihydroxy conidine 12 were unsuccessful, so
the hydroxyl groups in 11 were protected as their TBDMS ethers by
treatment with TBDMS triflate in DMF in the presence of 2,6-luti-
dine to yield the protected lactam 13 (73%). 13 was formed more
efficiently by prior silylation of azetidine enoate 3 to the corre-
sponding bis-TBDMS ether 14 (88%); subsequent transfer hydroge-
nation of 14 afforded the protected lactam 13 in 99% yield. Higher
yields of bicyclic lactam formation are generally formed when the
hydroxyl groups are protected (and in particular when such
protection precludes the formation of an alternative lactone).
Reduction of the protected lactam 13 by borane in THF gave the
corresponding protected amine as the borane adduct (79%), treat-
ment of which with aqueous trifluoroacetic acid formed the
conidine diol 12 (94%).
The osmium(VIII)-catalyzed hydroxylation of the unprotected 3
and protected 14 enoates by a number of procedures was investi-
gated.26 All attempts to hydroxylate the unprotected diol 3 were
unsuccessful; the reactions were very slow and gave highly polar
compounds that were difficult to isolate and purify. Thus, no
reaction of 3 with the TMEDA/osmium tetroxide mixture was ob-
served.27a A wide variety of conditions for dihydroxylation of the
bis-TBDMS ether 14 gave only the diol 15; the optimum conditions
[K2OsO4ꢁ2H2O, K3Fe(CN)6, K2CO3, and (DHQ)2PHAL in tert-BuOH:-
H2O (3:1)] gave the diol 15 as a single stereoisomer in 77% yield.
There was no evidence for the formation of the distereoisomer
16 (which would have allowed the synthesis of the castanosper-
mine azetidine analogue 6) under any of the conditions, including
the use of other ligands, and different amounts and sources of
MeO2C
OH
O
OH
HO
OHC
BnN
OH
(iii)
O
OH
OH
(ii)
(i)
BnN
O
N
O
N
O
BnN
OH
OH
BnN
OH
OH
3
11
12
10
9
(v)
(iv)
(vii)
OH
OH
OTBDMS
OTBDMS
MeO2C
OTBDMS
OTBDMS
MeO2C
HO
OTBDMS
OTBDMS
(vi)
MeO2C
HO
OTBDMS
(viii)
O
N
BnN
BnN
15
BnN
OTBDMS
13
14
16
(ix)
OH
OR
OTBDMS
OTBDMS
OH
HO
OH
RO
O
OTBDMS
OTBDMS
TBDMSO
HO
OH
(xii)
(xi)
N
N
HO
H3B
N
OH
N
OTBDMS
OH
2
19
17
R = H
6
(x)
18
R = TBDMS
Scheme 1. (i) CF3COOH/H2O 1:1, RT, 24 h, 77% (ii) Bu3P@CHCO2Me, 1,4-dioxane, RT, 16 h, 52% (iii) HCO2ꢀNH4+, 10% Pd/C, MeOH, reflux, 1 h, 65% (iv) tert-BuMe2SiOSO2CF3,
2,6-lutidine, DMF, RT, 16 h, 73% (v) tert-BuMe2SiOSO2CF3, 2,6-lutidine, DMF, RT, 22 h, 88% (vi) HCO2ꢀNH4+, 10% Pd/C, MeOH, reflux, 45 min, 99% (vii) THF/BH3, THF, reflux, 3 h,
79%; then CF3COOH:H2O 9:1, RT, 3 d, 94% (viii) K2OsO4, K3Fe(CN)6, K2CO3, (DHQ)2PHAL, tert-BuOH:H2O 3:1, RT, 24 h, 77% (ix) HCO2ꢀNH4+, 10% Pd/C, MeOH, reflux, 1 h, 57% (x)
tert-BuMe2SiOSO2CF3, 2,6-lutidine, DMF, RT, 16 h, 88% (xi) THF:BH3, THF, reflux, 1 h, 84% (xii) H2, 10% Pd/C, CF3COOH/H2O 9:1, RT, 4 d, 80% from 18.

R. F. Martínez et al. / Bioorg. Med. Chem. 21 (2013) 4813–4819
4815
osmium. The dihydroxylation of 14 is an example of the Kishi mod-
OH
N
OH
OH
el27b in which the stereoselectivity of the Z-substituted alkene is
HO
OH
OH
HO
OH
HO
OH
controlled by 1,3-allylic strain.27c
N
N
H
CH2OH
Removal of the benzyl group in 15 by transfer hydrogenation
resulted in the formation of the hydroxylated lactam 17 (57%).
NOE analysis of 17 is consistent with its stereochemistry (Fig. 2).
There are significant NOEs from H3 to H4 as well as from H4 to
H5. However, there is no evidence of NOEs neither from H6 to
H4 nor from H3 to H5, which rules out the presence of the stereo-
isomer 20.
Attempts to reduce the diol 17 with a number of hydride reduc-
ing agents were unsuccessful; accordingly 17 was protected as the
tetrasilyl ether 18 by treatment with treatment with TBDMS
triflate in DMF with 2,6-lutidine (88%). Reduction of the lactam
18 did not give the corresponding amine directly; the initial reduc-
tion product was consistent with the structure of lactol/borane
complex 19 which could be isolated by flash chromatography as
an unstable non-polar intermediate. Evidence for the structure of
19 included the low resolution mass spectrum (ESI), the B–H
stretch at 2382 cmꢀ1 in the infra red, and the HSQC spectrum
which showed only one methylene group. Hydrogenation of 19
in aqueous trifluoroacetic acid in the presence of a 10% palladium
catalyst removed both the borane and silyl ether protecting groups,
and proceeded with intramolecular reductive amination to afford
the target tetrahydroxy conidine 2 in 80% yield from the protected
amide 18. Although conidine itself undergoes acid catalyzed poly-
merization,28 2 was stable to acid treatment.
OH
2
7
1
OH
OH
OH
OH
OH
OH
N
O
N
N
HN
OH
OH
OH
8
11
Figure 3. Glycosidase inhibition discussion.
12
21
418 lM, respectively. 2 can be regarded as a conformationally
restricted bicyclic amine, which combines the stereochemical
motifs of both altro-DNJ 7 (piperidine-ring) and the azet-LAB 8
(azetidine-ring). The monocyclic azetidine, azet-LAB 8 is a good
inhibitor of A. niger
pus sp. amyloglucosidase, with IC50 39, 70 and 19
tively.6 In contrast, altro-DNJ 7 showed no inhibition against
these enzymes but does inhibit bovine kidney -fucosidase with
IC50 value of 194 M. Furthermore, this study revealed that the less
a
-glucosidase, rat intestinal lactase, and Rhizo-
l
M, respec-
a
-L
l
oxygenated indolizidine alkaloid conidine diol 12 was much better
and specific fungal glycosidase inhibitors than tetrahydroxyconi-
dine 2:
ases (IC50 930
and -rhamnosidase (P. decumbens) (IC50 117
the less oxygenated indolizidine alkaloid lentiginosine 21 also spe-
cifically inhibits fungal amyloglucosidase (A. niger) (IC50 32
The lactam 11 showed no inhibition of any glycosidase.
a
-glucosidase (A. niger) (IC50 307
l
l
M) and amyloglucosid-
M from Rhizopus sp.),
M); similarly,
l
M from A. niger, IC50 243
2.2. Biological activity
a-
L
l
Inhibition by the iminosugars 2, 7, 8, 11, and 12 of the following
l
M).31
glycosidases was studied (Fig. 3):29
a
-glucosidases (rice, yeast, rat
intestinal maltase, Aspergillus niger), b-glucosidases (almond, bo-
vine liver, A. niger), -galactosidase (coffee beans), b-galactosidases
(bovine liver, rat intestinal lactase), -mannosidase (Jack beans),
b-mannosidase (snail), -rhamnosidase (Penicillium decumbens),
-fucosidase (bovine kidney), b-glucuronidases (Escherichia coli,
a
a
3. Conclusion
a
-L
a-L
In summary, the synthesis of the first tetrahydroxyazetidine
analogue of castanospermine from arabinose demonstrates that
such structures are stable. Lentiginosine 21, although only a weak
glycosidase inhibitor, inhibits ATPase and chaperone activity of
bovine liver), trehalase (porcine kidney), and amyloglucosidases
(A. niger, Rhizopus sp.). The indolizidine alkaloid 6,7-diepicastano-
spermine 1 has been reported as moderate but specific inhibitor
Hsp90,32 where its enantiomer and
a further hydroxylated
of the fungal
84
M)30 in comparison with castanospermine 5 (IC50
This study (Table 1) revealed that the tetrahydroxyconidine 2
a-glucosidase, amyloglucosidase (A. niger) (IC50
derivative show a novel mechanism for apoptosis.33 It is likely that
azetidine analogues such as 2 and 12 may have interesting
biological properties other than glycosidase inhibition. A different
approach is necessary for the synthesis of the azetidine castano-
spermine analogue 6.
l
8 l
M).
showed no inhibition of amyloglucosidase from A. niger (31% inhi-
bition at 1 mM); 2 was a weak inhibitor of amyloglucosidase from
Rhizopus sp. and rat intestinal lactase, with IC50 values of 532 and
OH
OH
HO
O
OTBDMS
OTBDMS
HO
O
OTBDMS
OTBDMS
N
N
20
17
H7
H7
H6
[Si]O
H6
[Si]O
H4
OH
[Si]O
H8'
[Si]O
H8'
H8
H8
OH
H4
N
N
OH
H3
O
H5
O
H5
H3
OH
Figure 2. NOE enhancements on 20 and 17.

4816
R. F. Martínez et al. / Bioorg. Med. Chem. 21 (2013) 4813–4819
Table 1
Concentration of azetidine analogues of 6,7-diepicastanospermine and related compounds giving 50% inhibition of various glycosidases
IC50 M)
(l
OH
OH
OH
OH
OH
HO
OH
HO
OH
OH
5
4
3
2
4
N
N
O
OH
OH
6
N
HN
N
H
CH2OH
2
7
1
OH
OH
1
Enzyme
-Glucosidase
Rice
Yeast
Rat intestinal maltase
Aspergillus niger
b-Glucosidase
Almond
Bovine liver
Aspergillus niger
2
7
8
11
12
a
NI (13.8%)
NI (0%)
NI (30.3%)
NI (47.3%)
NI (7.2%)
NI (1.4%)
NI (24.2%)
NI (19.1%)
NI (12.1%)
NI (16.0%)
607
NI (2.7%)
NI (3.1%)
NI (25.2%)
NI (0%)
NIa (0%)b
NI (1.6%)
NI (14.3%)
39
307
NI (31.1%)
NI (18.9%)
NI (1.8%)
NI (18.8%)
NI (10.9%)
NI (1.8%)
347
997
NI (0.4%)
NI (0%)
NI (14.9%)
NI (1.2%)
NI (0%)
NI (9.7%)
NI (0%)
a-Galactosidase
Coffee beans
b-Galactosidase
Bovine liver
Rat intestinal lactase
b-Mannosidase
Snail
NI (3.5%)
NI (20.5%)
NI (7.6%)
NI (0%)
NI (8.4%)
NI (43.4%)
418
NI (19.9%)
NI (44.6%)
NI (25.2%)
70
NI (13.4%)
NI (0%)
NI (14.0%)
NI (32.2%)
NI (0%)
NI (0%)
NI (2.1%)
194
NI (0%)
634
NI (0%)
NI (0%)
117
a-
L
-Rhamnosidase
Penicillium decumbens
-Fucosidase
NI (2.0%)
NI (2.0%)
NI (11.1%)
NI (0%)
a-L
Bovine kidney
b-Glucuronidase
Escherichia coli
Bovine liver
NI (4.6%)
NI (0%)
NI (0%)
NI (7.8%)
NI (0%)
NI (7.4%)
NI (0%)
NI (7.4%)
NI (8.9%)
NI (4.2%)
NI (13.2%)
NI (0%)
a,a-Trehalase
Porcine kidney
Amyloglucosidase
Aspergillus niger
Rhizopus sp.
NI (9.0%)
NI (2.1%)
NI (5.8%)
NI (5.2%)
NI (1.0%)
NI (31.3%)
532
NI (5.0%)
NI (7.0%)
105
19
NI (3.0%)
NI (3.8%)
930
243
a
NI–no inhibition (less than 50% inhibition at 1000 lM).
(–): inhibition% at 1000 lM.
b
except in the case of deuterium oxide, where acetonitrile was used
as the reference.
4. Experimental
4.1. Materials and methods
4.1.1. (2E)-Methyl-(4S,5S,6S)-N-benzyl-4,6-dihydroxy-5,7-
iminohept-2-enoate (3)
All commercial reagents were used as supplied. THF and DMF
were purchased dry from the Aldrich chemical company in sure-
seal bottles. Methanol and pyridine were purchased dry from the
Aldrich chemical company in sure-seal bottles over molecular
sieves. All other solvents were used as supplied (Analytical or HPLC
grade), without prior purification. Thin layer chromatography
(TLC) was performed on aluminum sheets coated with 60 F254
silica. Plates were visualized using a spray of 1% w/v potassium
permanganate in 1 M sodium hydroxide, or a 0.2% w/v cerium
(IV) sulfate and 5% ammonium molybdate solution in 2 M sulfuric
acid. Flash column chromatography was performed on Sorbsil C60
40/60 silica. Melting points were recorded on a Kofler hot block
and are uncorrected. Optical rotations were recorded on a Per-
kin–Elmer 341 polarimeter with a path length of 1 dm. Concentra-
tions are quoted in g 100 mlꢀ1. Infrared spectra were recorded on a
Perkin–Elmer 1750 IR Fourier Transform spectrophotometer. Only
the characteristic peaks are quoted. Low resolution mass spectra
(m/z) were recorded on a Waters LCT Premier spectrometer, and
high resolution mass spectra (HRMS m/z) on a Waters ZMD spec-
trometer. The technique used was electrospray ionization (ESI).
Nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker AVII500 (1H: 500 MHz and 13C: 125.7 MHz) and Bruker
DPX400 and AV400 spectrometers (1H: 400 MHz and 13C:
100.6 MHz) in the deuterated solvent stated. All chemical shifts
(d) are quoted in ppm and coupling constants (J) in Hz. Residual
signals from the solvents were used as an internal reference,
Compound 9 (970 mg, 3.71 mmol) was dissolved in a mixture of
trifluoroacetic acid (2.5 mL) and water (2.5 mL) and the resulting
solution was stirred at room temperature. After 24 h, TLC analysis
(2:1 cyclohexane/ethyl acetate) showed the complete consump-
tion of starting material and the formation of a major product (Rf
0.10). The solvents were removed in vacuo. Purification by flash
column chromatography (EtOAc/NEt3, 99:1) afforded the corre-
sponding lactol 10 (631 mg, 77%) as a yellow oil in a 1:1 mixture
of anomers.
Bu3PCH2CO2MeBr (1.12 g, 3.16 mmol) was dissolved in dichlo-
romethane (20 mL) and washed with sodium hydroxide (1 M aq,
20 mL) for 15 min. The aqueous layer was then washed with
dichloromethane (2 ꢂ 15 mL). The combined organics were dried
(MgSO4), filtered and concentrated in vacuo. The resulting ylid
was added to a solution of 10 (582 mg, 2.63 mmol) in 1,4-dioxane
(10 mL) and the reaction mixture stirred at room temperature for
16 h. After this time, TLC analysis (ethyl acetate) showed the com-
plete consumption of starting material (Rf 0.50) and the formation
of a major product (Rf 0.38). The reaction mixture was concen-
trated and the residue purified by flash column chromatography
on silica gel (1:1 cyclohexane/ethyl acetate) to afford 3 as a pale
yellow solid (380 mg, 52%); ½a D25
ꢃ
+23.4 (c 0.36 in CHCl3); mmax (film,
cmꢀ1): 3403 (s, br s, OH), 1721 (CO), 1658 (C@C); mp 108–110 °C;
0
dH (CDCl3, 400 MHz): 3.28–3.31 (1H, dd, H7, J7,6 6.4, J7,7 9.2), 3.40–
3.44 (2H, m, H70 and H5), 3.69 (2H, s, CH2Ph), 3.73 (3H, s, CH3),

R. F. Martínez et al. / Bioorg. Med. Chem. 21 (2013) 4813–4819
4817
4.08–4.10 (1H, dt, H4, J4,2 2.0, J4,5/J4,3 4.4), 4.47–4.51 (1H, dd, H6, J6,5
anhydrous methanol (10 mL) and the mixture was refluxed under
argon. After 45 min, TLC analysis (2:1 cyclohexane/ethyl acetate)
showed no-remaining starting material and formation of the prod-
uct (Rf 0.21). The mixture was filtered through Celite and washed
with methanol. The solvent was concentrated in vacuo and the res-
idue purified by flash column chromatography on silica gel (2:1
cyclohexane/ethyl acetate) to afford 13 as a colourless oil (98.4 mg,
0
1.6, J6,7/J6,7 6.4), 6.08–6.12 (1H, dd, H2, J2,4 2.0, J2,3 16), 6.91–6.96
(1H, dd, H3, J3,4 4.4, J3,2 16), 7.27–7.35 (5H, m, 5 ꢂ ArH); dC (CDCl3,
100 MHz): 51.7 (CH3), 61.2, 61.4 (CH2Ph and C7), 65.9 (C6), 70.3
(C4), 71.3 (C5), 121.2 (C2), 127.6 (p-ArCH), 128.6, 128.8 (2 ꢂ o-
ArCH and 2 ꢂ m-ArCH), 137.3 ppm (ipso-ArC), 146.4 (C3), 166.9
(C1); m/z (ESI+ve): 278 ([M+H]+, 69%), 300 ([M+Na]+, 64%), 577
([2M+Na]+, 100%); HRMS m/z (ESI+ve): found 278.1385 ([M+H]+);
99%); ½a 2D5
ꢃ
+32.7 (c 1.25 in CHCl3); m
max (film, cmꢀ1): 1667 (CO); dH
C15H20NO4 requires 278.1387.
(CDCl3, 400 MHz): 0.03–0.06 (4 ꢂ 3H, m, 4 ꢂ CH3), 0.85–0.88
(2 ꢂ 9H, m, 2 ꢂ C(CH3)3), 1.85–2.03 (2H, m, H4), 2.27–2.35 (1H, m,
H3), 2.42–2.49 (1H, dt, H30, J3 ,4 5.2, J3 ,4 5.2, J3 ,3 17.6), 3.77–3.79
0
0
0
0
4.1.2. (5S,6S,7S)-5,7-Dihydroxy-1-azabicyclo[4.2.0]octane-2-one
(11)
(1H, d, H8, J8,8 10.5), 4.07–4.11 (1H, dd, H80, J8 ,7 4.6, J8 ,8 10.5),
4.21–4.26 (1H, m, H5), 4.34–4.37 (1H, t, H6, J6,7/J6,5 5.6), 4.52–4.54
(1H, m, H7); dC (CDCl3, 100 MHz): ꢀ4.7, ꢀ4.6, ꢀ4.3, ꢀ4.1 (4ꢂ CH3),
18.0, 18.1 (2 ꢂ C(CH3)3), 25.9 (2 ꢂ C(CH3)3), 29.4 (C3), 31.2 (C4),
59.8 (C8), 63.5 (C5), 68.2 (C7), 74.1 (C6), 171.9 (C2); m/z (ESI+ve):
386 ([M+H]+, 100%), 408 ([M+Na]+, 80%); HRMS m/z (ESI+ve): found
408.2352 ([M+Na]+); C19H39NNaO3Si2 requires 408.2361.
0
0
0
Compound 3 (100 mg, 0.36 mmol), 10% Pd on carbon (115 mg)
and ammonium formate (341 mg, 5.40 mmol) were suspended in
anhydrous methanol (10 mL) and the mixture was refluxed under
argon. After 1 h, TLC analysis (3:7 methanol/ethyl acetate) showed
no-remaining starting material (Rf 0.74) and formation of the prod-
uct (Rf 0.38). The mixture was filtered through Celite and washed
with methanol. The solvent was concentrated in vacuo and the res-
idue purified by flash column chromatography on silica gel (2:8
methanol/ethyl acetate) to afford 11 as a white solid (37.1 mg,
4.1.5. (5S,6S,7S)-5,7-Dihydroxy-1-azabicyclo[4.2.0]octane (12)
tert-Butyldimethylsilyl
trifluoromethanesulfonate
(44 lL,
65%); ½a 2D5
ꢃ
ꢀ42.0 (c 0.48 in methanol); mmax (film, cmꢀ1): 3363 (s,
0.19 mmol) and 2,6-lutidine (30 lL, 0.26 mmol) were added to a
br s, OH), 1607 (CO); mp 164–166 °C; dH (CD3OD, 400 MHz):
solution of 11 (10 mg, 0.06 mmol) in dimethylformamide (1 mL)
and the resulting solution was stirred at room temperature. After
16 h, TLC analysis (1:3 cyclohexane/ethyl acetate) showed the com-
plete consumption of starting material and the formation of a major
product (Rf 0.51). The mixture was diluted with ethyl acetate (5 mL),
washed with 1:1 water/brine (3 ꢂ 5 mL), and the organic layer dried
(MgSO4) and concentrated in vacuo. Purification by flash column
chromatography (1:1 cyclohexane/ethyl acetate) afforded 13
(18 mg, 73%) as a colourless oil. A solution of borane in tetrahydrofu-
ran (0.44 mL, 1 M) was added to a solution of the protected lactam
(17 mg, 0.044 mmol) in anhydrous tetrahydrofuran (1 mL) and the
resulting mixture stirred at reflux for 3 h under a nitrogen atmo-
sphere. The reaction mixture was cooled to room temperature, and
treated with methanol (2 mL). Removal of solvents gave a residue
which was purified by chromatography (7:1 cyclohexane/ethyl ace-
tate) to afford the protected amine as a colourless oil (13 mg, 79%).
The protected amine (11 mg, 0.03 mmol) was dissolved in a mixture
of trifluoroacetic acid (0.9 mL) and water (0.1 mL) and the resulting
solution was stirred at room temperature. After 3 days, the solvents
were removed in vacuo. The remaining residue was loaded onto a
Dowex H+ ion exchange resin. The column was flushed with water
and ethanol and then eluted with 2 M aqueous ammonia. The
ammoniacal fractionwas concentratedin vacuoto afford 12aswhite
1.86–1.93 (1H, m, H4), 2.03–2.10 (1H, m, H40), 2.33–2.42 (1H,
ddd, H3, J3,4 7.2, J3,4 10.8, J3,3 18), 2.45–2.52 (1H, ddd, H30, J3 ,4
0
0
0
0
0
0
1.6, J3 ,4 7.2, J3 ,3 18), 3.75–3.78 (1H, dt, H8, J8,8 10.8, J8,6/J8,7 1.6),
4.16–4.24 (2H, m, H5 and H80), 4.33–4.36 (1H, dd, H6, J6,7 5.2, J6,5
0
8.4), 4.55–4.58 (1H, td, H7, J7,8 2.0, J7,6/J7,8 5.2); dC (CD3OD,
100 MHz): 29.5, 29.6 (C3 and C4), 59.4 (C8), 62.2 (C5), 66.6 (C7),
73.2 (C6), 172.7 (CO); m/z (ESI+ve): 158 ([M+H]+, 16%), 180
([M+Na]+, 45%), 337 ([2 M+Na]+, 100%); HRMS m/z (ESI+ve): found
180.0631 ([M+Na]+); C17H11NNaO3 requires 180.0631.
4.1.3. (2E)-Methyl-(4S,5S,6S)-N-benzyl-4,6-bis(tert-
butyldimethylsilyloxy)-5,7-iminohept-2-enoate (14)
tert-Butyldimethylsilyl trifluoromethanesulfonate (0.21 mL,
0.91 mmol) and 2,6-lutidine (0.14 mL, 1.21 mmol) were added to
a solution of 3 (84 mg, 0.30 mmol) in dimethylformamide (6 mL)
and the resulting solution was stirred at room temperature. After
22 h, TLC analysis (2:1 cyclohexane/ethyl acetate) showed the
complete consumption of starting material and the formation of
a major product (Rf 0.85). The mixture was diluted with ethyl
acetate (10 mL), washed with 1:1 water/brine (3 ꢂ 10 mL) and
the organic layer dried (MgSO4) and concentrated in vacuo. Purifi-
cation by flash column chromatography (5:1 cyclohexane/ethyl
acetate) afforded 14 (135 mg, 88%) as a yellow oil; ½a D25
ꢃ
ꢀ1.6 (c
solid (4 mg, 94%); ½a D25
ꢃ
+52.5 (c 0.19 in methanol); m
max (film, cmꢀ1):
0.32 in CHCl3); mmax (film, cmꢀ1): 1727 (CO), 1472 (arom); dH
(CDCl3, 400 MHz): 0.03–0.08 (4 ꢂ 3H, m, 4 ꢂ CH3), 0.92–0.93
(2 ꢂ 9H, m, 2 ꢂ C(CH3)3), 2.92–2.96 (1H, m, H7, J7,5 2.4, J7,6 6.0,
3315 (s, br s, OH); mp >300 °C; dH (CD3OD, 500 MHz): 1.17–1.25 (1H,
0
0
dq, H4, J4,5 3.0, J4,3/J4,3 /J4,4 12.5), 1.53–1.57 (1H, m, H3), 1.69–1.78
(1H, m, H30), 1.96–2.01 (1H, m, H40), 2.51–2.54 (1H, m, H2), 3.26–
J7,7 8.8), 3.15–3.17 (1H, d, H70, J7 ,7 8.8), 3.23–3.30 (2H, m, CH2Ph
and H5), 3.74 (3H, s, OCH3), 3.82–3.85 (1H, d, CH2Ph, Jgem 13.2),
4.49–4.52 (1H, t, H6, J6,5 6.0, J6,7 6.0), 4.78–4.81 (1H, m, H4),
6.09–6.13 (1H, dd, H2, J2,4 1.6, J2,3 15.6), 7.23–7.30 (5H, m, 5 ꢂ ArH),
7.30–7.38 (1H, ddd, H3, J3,5 2.8, J3,4 4.8, J3,2 15.6); dC (CDCl3,
100 MHz): ꢀ4.5, ꢀ4.4, ꢀ4.3, ꢀ4.1 (4 ꢂ CH3), 18.2 (2 ꢂ C(CH3)3),
26.0 (2 ꢂ C(CH3)3), 51.5 (OCH3), 60.5 (C7), 62.1 (CH2Ph), 65.2
(C6), 70.4 (C4), 74.9 (C5), 120.1 (C2), 127.0 (p-ArCH), 128.2, 128.9
(2 ꢂ o-ArCH and 2 ꢂ m-ArCH), 138.3 ppm (ipso-ArC), 151.1 (C3),
167.2 (C1); m/z (ESI+ve): 506 ([M+H]+, 100%), 528 ([M+Na]+,
36%); HRMS m/z (ESI+ve): found 506.3124 ([M+H]+); C27H48NO4Si2
requires 506.3116.
3.29 (1H, m, H6), 3.33–3.35 (1H, t, H8, J8,7/J8,8 6.8), 3.37–3.40 (1H,
0
0
0
td, H80, J8 ,6 2.0, J8 ,7/J8 ,8 6.8), 4.21–4.26 (1H, m, H5), 4.51–4.55 (1H,
0
0
0
0
q, H7, J7,8/J7,8 /J7,6 6.8); dC (CD3OD, 125 MHz): 20.6 (C3), 31.2 (C4),
46.3 (C2), 58.9 (C8), 63.1 (C5), 63.3 (C7), 71.9 (C6); m/z (ESI+ve):
144 ([M+H]+, 100%); HRMS m/z (ESI+ve): found 144.1016
([M+H]+); C7H14NO2 requires 144.1019.
4.1.6. Methyl-(2S,3S,4R,5R,6S)-N-Benzyl-4,6-bis(tert-butyldi-
methylsilyloxy)-2,3-dihydroxy-5,7-iminoheptanoate (15)
Potassium ferricyanide (217 mg, 0.66 mmol), potassium carbon-
ate (91 mg, 0.66 mmol), methane sulphonamide (21 mg,
0.22 mmol), potassium osmate dihydrate (2.4 mg, 6.6 lmol) and
(DHQ)2PHAL (26 mg, 0.03 mmol) were dissolved in a mixture of
tert-butanol (1.7 mL) and water (1.7 mL). This mixture was added
to a solution of 14 (111 mg, 0.22 mmol) in tert-butanol (3.5 mL)
and the resulting solution was stirred at room temperature. After
24 h, TLC analysis (2:1 cyclohexane/ethyl acetate) showed the
4.1.4. (5S,6S,7S)-5,7-bis(tert-Butyldimethylsilyloxy)-1-
azabicyclo[4.2.0]octane-2-one (13)
Compound 14 (130 mg, 0.26 mmol), 10% Pd on carbon (82 mg)
and ammonium formate (243 mg, 3.85 mmol) were suspended in

4818
R. F. Martínez et al. / Bioorg. Med. Chem. 21 (2013) 4813–4819
formation of a major product (Rf 0.63). Solid Na2SO3 was added and
the mixture was stirred for a further 15 min. The reaction mixture
was concentrated to dryness and the residue was partitioned be-
tween ethyl acetate (10 mL) and water (10 mL). The organic layer
was dried (MgSO4) and concentrated in vacuo. Purification by flash
column chromatography (8:1 cyclohexane/ethyl acetate) afforded
ꢀ3.3 (8 ꢂ CH3), 18.9, 19.0, 19.1 (4 ꢂ C(CH3)3), 26.2, 26.3, 26.4,
26.5, 26.7 (4 ꢂ C(CH3)3), 60.9 (C8), 64.6 (C5), 70.5, 70.9 (C6 and
C7), 74.5 (C3), 78.9 (C4), 170.8 (C2); m/z (ESI+ve): 646 ([M+H]+,
76%), 668 ([M+Na]+, 100%); HRMS m/z (ESI+ve): found 668.3983
([M + Na]+); C31H67NNaO5Si4 requires 668.3989.
15 (91 mg, 77%) as a colourless oil; ½a D25
ꢃ
+28.0 (c 0.35 in CHCl3); mmax
4.1.9. (3R,4S,5R,6R,7S)-3,4,5,7-Tetrahydroxyconidine (2)
(film, cmꢀ1): 3563 (OH), 1748 (CO), 1496, 1472 (arom); dH (CDCl3,
400 MHz): 0.01–0.11 (4 ꢂ 3H, s, 4 ꢂ CH3), 0.91–0.92 (2 ꢂ 9H, s,
A solution of borane in tetrahydrofuran (1.42 mL, 1 M) was
added to a solution of 18 (46 mg, 0.071 mmol) in anhydrous tetra-
hydrofuran (4 mL) and the resulting mixture stirred at reflux for
1 h under a nitrogen atmosphere. The reaction mixture was cooled
to room temperature, and treated with methanol (2 mL). Removal
of solvents gave a residue which was purified by flash column
chromatography (50:1 cyclohexane/ethyl acetate) to afford 19 as
a colourless oil (39.4 mg, 84%). Selected data for 19: mmax (film,
cmꢀ1): 2382 (BH); dH (CDCl3, 400 MHz): 0.05–0.16 (8 ꢂ 3H, s,
8 ꢂ CH3), 0.87–0.96 (4 ꢂ 9H, s, 4 ꢂ C(CH3)3), 3.30–3.35 (2H, m, H8
and OH), 3.98–4.00 (3H, m, H3, H6 and H4), 4.05–4.09 (1H, t, H80,
J8´ ,8/J8´ ,7 8.0 Hz), 4.59-4.60 (1H, dd, H2, J2,3 2.0 Hz, J2,OH 4.4 Hz),
0
2 ꢂ C(CH3)3), 3.02–3.06 (1H, m, H7, J7,6 6.0, J7,7 9.2), 3.10–3.12
(1H, d, H70, J7 ,7 9.2), 3.45–3.48 (1H, d, CH2Ph, Jgem 12.8 Hz), 3.57–
0
3.60 (1H, m, H5), 3.81 (3H, s, OCH3), 3.94–3.97 (1H, d, H2, J2,3 9.2),
4.06–4.10 (1H, d, CH2Ph, Jgem 12.8), 4.32–4.34 (1H, dd, H3, J3,4 3.6,
J3,2 9.2), 4.35 (1H, m, H4), 4.48–4.51 (1H, t, H6, J6,5 6.0, J6,7 6.0),
7.25-7.34 (5H, m, 5 ꢂ ArH); dC (CDCl3, 100 MHz): ꢀ4.9, ꢀ4.8,
ꢀ4.3, ꢀ4.2 (4 ꢂ CH3), 18.1 (2 ꢂ C(CH3)3), 25.8, 25.9 (2 ꢂ C(CH3)3),
52.4 (OCH3), 60.4 (C7), 63.6 (CH2Ph), 64.9 (C6), 66.2 (C3), 70.7
(C4), 74.5 (C2), 76.7 (C5), 127.6 (p-ArCH), 128.6, 128.9 (2 ꢂ o-ArCH
and 2 ꢂ m-ArCH), 136.9 ppm (ipso-ArC), 174.7 (C1); m/z (ESI+ve):
540 ([M+H]+, 100%), 562 ([M+Na]+, 90%); HRMS m/z (ESI+ve): found
540.3177 ([M+H]+); C27H50NO6Si2 requires 540.3171.
0
4.82–4.84 (1H, m, H5), 5.08–5.13 (1H, q, H7, J7,8 /J7,6/J7,8 7.2 Hz);
dC (CDCl3, 100 MHz): ꢀ5.4, ꢀ5.0, ꢀ4.7, ꢀ4.6, ꢀ4.2, ꢀ4.1, ꢀ3.9,
ꢀ2.6 (8 ꢂ CH3), 17.9, 18.0, 18.1, 18.5 (4 ꢂ C(CH3)3), 25.7, 25.8,
25.9 (4 ꢂ C(CH3)3), 61.9 (C5), 62.9 (C7), 63.3 (C8), 72.3, 73.5 (C3
and C6), 75.4 (C4), 80.9 (C2); m/z (ESI+ve): 662 ([M+H]+, 100%),
684 ([M+Na]+, 80%), 1345 ([2 M+Na]+, 54%).
4.1.7. (3S,4S,5R,6R,7S)-5,7-bis(tert-Butyldimethylsilyloxy)-3,4-
dihydroxy-1-azabicyclo[4.2.0]octane-2-one (17)
Compound 15 (143 mg, 0.26 mmol), 10% Pd on carbon (85 mg)
and ammonium formate (251 mg, 3.97 mmol) were suspended in
anhydrous methanol (10 mL) and the mixture was refluxed under
argon. After 1 hour, TLC analysis (1:2 ethyl acetate/cyclohexane)
showed no-remaining starting material and formation of the prod-
uct (Rf 0.39). The mixture was filtered through Celite and washed
with methanol. The solvent was concentrated in vacuo and the res-
idue purified by flash column chromatography on silica gel (1:8
ethyl acetate/cyclohexane) to afford 17 as a white solid (63 mg,
10% Pd on carbon (16 mg) was added to a degassed solution of
19 in trifluoroacetic acid (1.8 mL) and water (0.2 mL) and the
resulting solution was stirred at room temperature under hydro-
gen. After 4 days, the reaction mixture was filtered through glass
microfibre (GF/C), the solvents were removed in vacuo, and the
remaining residue was loaded onto a Dowex H+ ion exchange resin.
The column was flushed with water, ethanol, more water, and then
eluted with 2 M aqueous ammonia. The ammoniacal fraction was
concentrated in vacuo to afford 2 as a brown oil (10.0 mg, 80% from
57%); ½a 2D5
ꢃ
ꢀ33.9 (c 0.96 in methanol); mmax (film, cmꢀ1): 3200–
3300 (OH), 1616 (CO); mp 184–186 °C; dH (CD3OD, 500 MHz):
0.11–0.16 (4 ꢂ 3H, s, 4 ꢂ CH3), 0.92–0.93 (2 ꢂ 9H, s, 2 ꢂ C(CH3)3),
19). ½a 2D5
ꢃ
ꢀ26.4 (c 0.15 in water);
m
max (film, cmꢀ1): 3283 (OH); dH
0
(D2O, 500 MHz): 2.58–2.61 (1H, d, H2, J2,2 15.0 Hz), 2.93–2.96 (1H,
3.76–3.78 (1H, td, H8, J8,6 1.5, J8,7 1.5, J8,8 10.5), 3.87–3.88 (1H, d,
d, H20, J2 ,2 15.0), 3.70–3.72 (1H, m, H6), 3.76 (1H, m, H8), 3.83–3.86
0
0
H3, J3,4 3.0), 4.01–4.02 (1H, t, H4, J4,3 3.0 Hz, J4,5 3.0 Hz), 4.22–
(1H, t, H80, J8 ,8/J8 ,7 7.0 Hz), 4.08 (2H, m, H3 and H4), 4.66–4.70 (2H,
m, H7 and H5); dC (D2O, 125 MHz): 45.0 (C2), 60.7 (C5), 61.8 (C8),
62.4 (C7), 65.9 (C6), 69.6, 70.7 (C3 and C4); m/z (ESI+ve): 176 ([M +
H]+, 37%); HRMS m/z (ESI+ve): found 176.0915 ([M + H]+);
C7H14NO4 requires 176.0917.
0
0
4.25 (1H, dd, H80, J8 ,7 5.0 Hz, J8 ,8 10.5 Hz), 4.27–4.29 (1H, dd, H5,
J5,4 3.0, J5,6 7.5 Hz), 4.63–4.66 (1H, m, H6), 4.67–4.70 (1H, dt, H7,
0
0
0
J7,8 1.5, J7,8 5.0, J7,6 5.0 Hz); dC (CD3OD, 125 MHz): ꢀ4.9, ꢀ4.8,
ꢀ4.4 (4 ꢂ CH3), 18.9, 19.0 (2 ꢂ C(CH3)3), 26.2, 26.3 (2 ꢂ C(CH3)3),
61.1 (C8), 64.6 (C5), 68.9 (C7), 70.1 (C6), 73.2 (C3), 78.2 (C4),
173.7 (C2); m/z (ESI+ve): 418 ([M+H]+, 19%), 440 ([M+Na]+, 37%),
857 ([2M+Na]+, 100%); HRMS m/z (ESI+ve): found 440.2257
([M+Na]+); C19H39NNaO5Si2 requires 440.2259.
Acknowledgments
This work was supported by Fundación Ramón Areces (R.F.M.),
Junta de Extremadura (N.A.), and Grant-in-Aid for Scientific Re-
search (C) (No: 23590127) (A.K.) from the Japanese Society for
the Promotion of Science (JSPS).
4.1.8. (3S,4R,5R,6R,7S)-3,4,5,7-Tetrakis(tert-
butyldimethylsilyloxy)-1-azabicyclo[4.2.0]octane-2-one (18)
tert-Butyldimethylsilyl trifluoromethanesulfonate (0.07 mL,
0.3 mmol) and 2,6-lutidine (0.05 mL, 0.4 mmol) were added to a
solution of 17 (42 mg, 0.1 mmol) in dimethylformamide (4 mL)
and the resulting solution was stirred at room temperature. After
16 h, TLC analysis (2:1 cyclohexane/ethyl acetate) showed the com-
plete consumption of starting material and the formation of a major
product (Rf 0.79). The mixture was diluted with ethyl acetate
(10 mL), washed with 1:1 water/brine (3 ꢂ 10 mL) and the organic
layer dried (MgSO4) and concentrated in vacuo. Purification by flash
column chromatography (95:5 cyclohexane/ethyl acetate) afforded
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.004.
References and notes
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ꢃ
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x 3H, s, 8 ꢂ CH3), 0.91–0.95 (4 ꢂ 9H, s, 4 ꢂ C(CH3)3), 3.72–3.74
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