C-branched iminosugars: α-glucosidase inhibition by enantiomers of isoDMDP, isoDGDP, and isoDAB- l -isoDMDP compared to miglitol and miglustat

Article abstract of DOI:10.1021/jo4005487

The Ho crossed aldol condensation provides access to a series of carbon branched iminosugars as exemplified by the synthesis of enantiomeric pairs of isoDMDP, isoDGDP, and isoDAB, allowing comparison of their biological activities with three linear isomeric natural products DMDP, DGDP, and DAB and their enantiomers. l-IsoDMDP [(2S,3S,4R)-2,4-bis(hydroxymethyl)pyrrolidine-3,4-diol], prepared in 11 steps in an overall yield of 45% from d-lyxonolactone, is a potent specific competitive inhibitor of gut disaccharidases [K_i 0.081 μM for rat intestinal maltase] and is more effective in the suppression of hyperglycaemia in a maltose loading test than miglitol, a drug presently used in the treatment of late onset diabetes. The partial rescue of the defective F508del-CFTR function in CF-KM4 cells by l-isoDMDP is compared with miglustat and isoLAB in an approach to the treatment of cystic fibrosis.

Full text of DOI:10.1021/jo4005487

Subscriber access provided by University of Chicago Library
Featured Article
C-Branched Iminosugars: alpha-Glucosidase Inhibition
by Enantiomers of isoDMDP, isoDGDP and isoDAB
L-isoDMDP Compared to Miglitol and Miglustat
–
Sarah F. Jenkinson, Daniel Best, Alexander Waldo Saville, James Mui, R. Fernando Martinez, Shinpei
Nakagawa, Takahito Kunimatsu, Dominic S Alonzi, Terry D. Butters, Caroline Norez, Frédéric Becq,
Yves Bleriot, Francis X. Wilson, Alexander C. Weymouth-Wilson, Atsushi Kato, and George W. J. Fleet
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo4005487 • Publication Date (Web): 20 May 2013
Downloaded from http://pubs.acs.org on June 3, 2013
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
The Journal of Organic Chemistry is published by the American Chemical Society.
1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 72
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
C-Branched Iminosugars: α-Glucosidase Inhibition by Enantiomers of isoDMDP, isoDGDP
and isoDAB – L-isoDMDP Compared to Miglitol and Miglustat
†
a,b
†a
a
a
a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Sarah F. Jenkinson,
Daniel Best, A. Waldo Saville, James Mui, R. Fernando Martínez,
c
c
b
b
d
Shinpei Nakagawa, Takahito Kunimatsu, Dominic S. Alonzi, Terry D. Butters, Caroline Norez,
d
e
f
g
Frederic Becq, Yves Blériot, Francis X. Wilson, Alexander C. Weymouth-Wilson, Atsushi
c
a,b
Kato* and George W. J. Fleet*
a
Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road,
Oxford, OX1 3TA, UK
b
Oxford Glycobiology Institute, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK
c
Department of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
d
Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, CNRS, 40 avenue du Recteur
Pineau, 86022 Poitiers, France
e
Equipe “Synthèse Organique” IC2MP, UMR 7285, Equipe G2S, Université de Poitiers, 4 rue
Michel Brunet, 86022 Poitiers, France
f
Summit PLC, 91, Milton Park, Abingdon, Oxon., OX14 4RY, UK
g
Dextra Laboratories Ltd, The Science and Technology Centre, Whiteknights Road, Reading, RG6
6BZ, UK
george.fleet@chem.ox.ac.uk kato@med.u-toyama.ac.jp
H
N
H
N
H
N
H
N
HOH2C
HOH2C
CH2OH
HOH2C
CH OH
HOH C
2
2
HO
OH
HO
OH
HO
OH
HO
DAB
OH
isoDMDP
DMDP
isoDAB
NO inhibition
IC50 290 µM
IC50 24 µM
IC50 55 µM
H
N
H
N
H
N
H
N
HOH2C
HOH2C
CH2OH
HOH2C
CH OH
HOH C
2
2
HO
OH
HO
OH
HO
OH
HO
OH
L
-isoDMDP
L
-DMDP
isoLAB
NO inhibition
LAB
IC50 0.93 µM
IC50 0.19 µM
IC50 1.2 µM
Inhibition IC50 of rat intestinal maltase shown in purple D-sugars in blue L-sugars in red
†
These authors contributed equally
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract The Ho crossed aldol condensation provides access to a series of carbon branched
iminosugars as exemplified by the synthesis of enantiomeric pairs of isoDMDP, isoDGDP and
isoDAB, allowing comparison of their biological activities with three linear isomeric natural
products DMDP, DGDP and DAB and their enantiomers. L-isoDMDP [(2S,3S,4R)-2,4-
bis(hydroxymethyl)pyrrolidine-3,4-diol], prepared in 11 steps in an overall yield of 45% from D-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
i
lyxonolactone, is a potent specific competitive inhibitor of gut disaccharidases [K 0.081 µM for rat
intestinal maltase] and is more effective in the suppression of hyperglycaemia in a maltose loading
test than Miglitol, a drug presently used in the treatment of late onset diabetes. The partial rescue of
the defective F508del-CFTR function in CF-KM4 cells by L-isoDMDP is compared with Miglustat
and isoLAB in an approach to the treatment of cystic fibrosis.
Introduction
The two drugs derived from iminosugars presently on the market are N-alkyl derivatives of
1
deoxynojirimycin (DNJ) 1. Miglitol 2 is well established for the treatment of late on-set diabetes.
Miglustat (N-butyl DNJ, Zavesca) 3 is licensed for substrate reduction therapy (SRT) in Gaucher’s
2
3
disease with potential for the management of Niemann-Pick disease, and for the chemotherapeutic
4
5,6
treatment of cystic fibrosis (CF). The N-acetyl-hexosaminidase inhibitor Siastatin B 4, isolated
7
from Streptomyces verticillus var. quintum, is the sole example of a naturally occurring branched
8
C-iminosugar; all the iminosugars yet isolated from plants have a linear carbon chain. Usually,
introduction of a carbon branch into the pyrrolidine or piperidine ring of an iminosugar leads to a
ACS Paragon Plus Environment

Page 3 of 72
The Journal of Organic Chemistry
1
2
9
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
loss of glycosidase inhibition; however, a number of synthetic analogues with carbon branches
1
0
have significant bioactivity. DADMe-immucillinH 5 and its enantiomer are both nanomolar
11
inhibitors of purine nucleoside phosphorylase [Figure 1]. 4-C-MethylDAB 6 and its enantiomer
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
12
13
14
are specific micromolar inhibitors of α-glucosidases. Both isofagomine 7 and noeuromycin 8
were designed as β-glucosidase inhibitors; isofagomine 7 has been studied as a potential chemical
15
pharmacological chaperone for Gaucher’s disease and as an inhibitor of glycogen phosphorylase
1
6
for the treatment of diabetes.
OH
OH
O
N
OH
H
N
HO
OH
HO
OH
HO
COOH
HO
X
CH OH
2
NH
Me
CH
N
R
CH OH
AcHN
N
H
N
H
OH
2
N
H
2
HOH C
2
N
1
R = H
4
5
6
7 R = H
8 R = OH
HO
2 R = CH CH OH
2
2
3 R = (CH ) Me
2
3
Figure 1: Structures of iminosugars
This paper reports the syntheses of isoDMDP 9D, isoDGDP 10D, and isoDAB 11D, and their
enantiomers, 9L, 10L and 11L respectively, as branched analogues of the natural products DMDP
1
7
1
2 [the most widely naturally occurring iminosugar ], DGDP 13 [from the Thai traditional drug
1
8
"
Non tai yak" (Stemona tuberosa) ] and DAB 14 [isolated from Arachniodes standishii and
19
Angylocalyx boutiqueanus ] [Figure 2]. The branched iminosugars have been studied in
comparison to their linear carbon chain counterparts as inhibitors of a range of glycosidases; the
inhibition of a digestive α-glucosidase [rat intestinal maltase] is summarized in Figure 2. L-
isoDMDP 9L, one of the most potent and specific inhibitors of α-glucosidases, was compared to
Miglitol 2 in mice for the control of blood sugar levels and may have value in the study of late on-
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
set diabetes. Iminosugars have been identified as pharmacological chaperones which can stabilize
or correct the structure of proteins; the partial rescue by L-isoDMDP 9L of the defective F508del-
2
0
CFTR function in CF-KM4 cells is compared with Miglustat 3 and isoLAB 11L as potential
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
21
agents for the treatment of CF.
H
N
H
N
H
N
H
N
H
N
H
N
HOH
2
C
HOH
2
C
HOH
2
C
CH
2
OH HOH
2
C
2 2
CH OH HOH C
CH
OH
2
OH
CH
OH
2
OH HOH
2
C
HO
HO
HO
OH
HO
OH
HO
OH
HO
OH
9D isoDMDP
10D isoDGDP
11D isoDAB
IC50 24 µM
12D DMDP
IC50 290 µM
13D DGDP
IC50 138 µM
14D DAB
NO inhibition
NO inhibition
IC50 55 µM
H
N
H
N
H
N
H
N
H
N
H
N
HOH C
HOH C
HOH C
CH OH HOH C
CH OH HOH C
2 2
2
2
2
2
2
CH
OH
2
OH
CH
OH
2
OH HOH
2
C
HO
HO
HO
10L
OH
HO
12L
IC50 1.2 µM
OH
HO
OH
HO
14L LAB
IC50 0.93 µM
OH
9
L
L
-isoDMDP
L
-isoDGDP
11L isoLAB
L
-DMDP
13L
L
-DGDP
IC50 0.19 µM
IC50 175 µM
NO inhibition
IC50 48 µM
Inhibition IC50 of rat intestinal maltase shown in purple D-sugars in blue L-sugars in red
Figure 2: Synthetic targets and inhibition [IC50] of a digestive α-glucosidase [rat intestinal maltase] by pyrrolidines
Results and Discussion 1. Synthesis
Usually, treatment of an unprotected sugar with aqueous base gives complicated mixtures arising
from reverse aldol reactions, dehydration, epimerization, and Lobry de Bruyn rearrangements;
treatment of glucose with aqueous calcium hydroxide for 30 minutes produces more than fifty
22
23,24
identifiable compounds. However, the Ho crossed aldol
of aqueous formaldehyde in the
presence of a base with an aldose protected at C2 and C3 by an isopropylidene group reliably
introduces a branching hydroxymethyl group at C2 in good to excellent yield. For all the iso-
iminosugars 9, 10 and 11, the branching hydroxymethyl group is introduced by a Ho reaction.
ACS Paragon Plus Environment

Page 5 of 72
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
The acetonide 15D of D-lyxonolactone, readily available from alkaline oxygenation of D-
2
5
galactose, was the starting material for the synthesis of L-isoDMDP 9L [Scheme 1]. The primary
alcohol in 15D was protected under neutral conditions by treatment with diphenyldiazomethane in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
26
refluxing toluene as the benzhydryl ether 16D (92%); it was not possible to convert base-sensitive
1
5D to the corresponding benzyl ether, and silyl protection was inappropriate because of instability
on later treatment with aqueous base.
O
O
O
O
O
ROH C
O
BzhOH C
OH
BzhOH C
OH
CH OH
BzhOH C
O
ROH C
O
2
2
2
2
2
(ii)
(iii)
(iv)
(vi)
CH X
CH N
2 3
2
2
O
O
O
O
O
O
O
O
RO
OR
1
5D R = H
6D R = Ph CH
19D X = OH
20D X = N3
21D R = H
17D
18D
(
i)
(v)
(vii)
1
22D R = PhCH2
2
t
(viii)
Bzh = Ph CH [Si] = BuMe Si Ms = CH SO2
2
2
3
H
N
H
N
O
OMs
N3
OH
OR
HOH C
O
HOH C
HOH C
BnOH C
BnOH C
2
2
2
2
2
(
xi)
(x)
(i) - (xi)
CH OH
CH OH
CH O[Si]
CH N
2
2
2
2
3
O
O
HO
OH
HO
OH
BnO
OBn
BnO
OBn
23D R = H
4D R = [Si]
15L
9D isoDMDP
9L L-isoDMDP
25D
(
ix)
2
Scheme 1: Reagents and conditions: (i) Ph
2
CN
2
, PhMe, reflux, 2 h, 92%; (ii) DIBALH, CH
2
Cl
2
, -78 °C, 1 h, 99%; (iii) CH
Cl
2
O,
K
2
CO , MeOH, H O, reflux, 4 h, 74%; (iv) I
3
2
2
, K
2
CO , t-BuOH, reflux, 1 h, 85%; (v) (CF SO
3
3
2
)
2
O, pyridine, CH
2
2
, -30 °C, 1 h;
then NaN
3
, DMF, rt, 18 h, 100%; (vi) AcCl, MeOH, reflux, 18 h, 97%; (vii) BnBr, NaH, DMF, 0 °C, 4.5 h, 86%; (viii) NaBH
SiCl, imidazole, DMF, 0 °C to rt, 6 h, 94%; (x) MsCl, Et N, CH Cl , 0 °C to rt, 2 h, 99%; (xi) H
4
2
,
,
EtOH, rt, 3 h, 100%; (ix) t-BuMe
2
3
2
2
2 2 2
Pd (10% on C), NaOAc, H O, 1,4-dioxane, rt, 24 h; then H , Pd (10% on C), HCl, H O, 1,4-dioxane, rt, 48 h, 100%.
The lactol 17D, formed by reduction of 16D with DIBALH in dichloromethane in a 5:1 ratio of
anomers (99%), underwent the Ho reaction with aqueous formaldehyde in the presence of
potassium carbonate in aqueous methanol to afford the branched lyxose derivative 18D in an
anomeric 3:1 ratio in 74% yield. Oxidation of the lactol 18D by bromine was not compatible with a
benzhydryl protecting group; however, iodine in tert-butanol in the presence of potassium
2
7
carbonate formed the lactone 19D (85%). Esterification of the highly hindered neopentyl alcohol
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
in 19D with triflic anhydride in dichloromethane in the presence of pyridine gave the corresponding
triflate which, on treatment with sodium azide in DMF, afforded the azide 20D (100%). It was
necessary to exchange the isopropylidene protecting group since pyrrolidine ring closure was not
possible with the formation of a trans-fused acetonide. The protecting groups were removed from
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
0D by hydrogen chloride in methanol to give the deprotected azidolactone 21D (97%). The
unprotected azidolactone 21D, with no acidic proton at C2, was treated with benzyl bromide and
sodium hydride in DMF to afford the tribenzyl azide 22D (86%). Reduction of 22D by sodium
borohydride in ethanol formed the diol 23D (100%) which was then protected as the primary silyl
ether 24D (94%). The remaining free hydroxyl group in 24D was esterified with mesyl chloride in
dichloromethane in the presence of triethylamine to afford the mesylate 25D (99%). Hydrogenation
of 25D by palladium on carbon in aqueous dioxane in the presence of sodium acetate caused
reduction of the azide to the amine and subsequent cyclization; further hydrogenation after addition
of hydrochloric acid resulted in removal of both the silyl and benzyl protecting groups to give L-
isoDMDP 9L in quantitative yield. The overall yield for the synthesis of 9L from the protected
lyxonolactone 15D was 45%. The enantiomer isoDMDP 9D was prepared from L-lyxonolactone
28
1
5L, available on a kilogram scale from D-ribose, by an identical procedure (overall yield 30%).
ACS Paragon Plus Environment

Page 7 of 72
The Journal of Organic Chemistry
1
2
O
O
O
O
O
ROH C
O
BzhOH C
OH
BzhOH C
OH
BzhOH C
O
ROH C
O
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
2
2
2
2
(
ii)
(iii)
(iv)
(vi)
CH OH
CH X
CH N
2 3
OR
2
2
O
O
O
O
O
O
O
O
RO
2
2
6L R = H
7L R = Ph CH
30L X = OH
31L X = N3
32L R = H
28L
29L
(
i)
(v)
(vii)
33L R = PhCH2
2
t
(viii)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Bzh = Ph CH [Si] = BuMe Si Ms = CH SO
2
2
3
2
H
N
OMs
N3
OH
OR
HOH C
BnOH C
2
BnOH C
2
2
(xi)
(x)
CH OH
CH O[Si]
CH N
2
2
2
3
HO
OH
BnO
OBn
BnO
OBn
3
3
4L R = H
5L R = [Si]
1
0L L-isoDGDP
36L
(
ix)
Scheme 2: Reagents and conditions: (i) Ph
CO , MeOH, H
then NaN , DMF, rt, 18 h, 91%; (vi) AcCl, MeOH, reflux, 18 h, 66%; (vii) BnBr, NaH, DMF, 0 °C, 3.5 h, 65%; (viii) NaBH
rt, 3 h, 93%; (ix) t-BuMe SiOTf, 2,6-lutidine, DCM, -78 °C, 6 h, 93%; (x) MsCl, pyridine, CH Cl , 0 °C to rt, 15 h, 76%; (xi) H
10% on C), NaOAc, H O, 1,4-dioxane, rt, 5 h; then H , Pd (10% on C), HCl, H O, 1,4-dioxane, rt, 54 h, 100%.
2
CN
2
, PhMe, reflux, 1 h, 94%; (ii) DIBALH, CH
2
Cl
2
, -78 °C, 3 h, 98%; (iii) CH
2
O,
K
2
3
2
O, reflux, 2 h, 75%; (iv) I
2
, K
2
CO , t-BuOH, reflux, 1 h, 86%; (v) (CF SO
3
3
2
)
2
O, pyridine, CH
2
Cl
2
, -30 °C, 1 h;
, EtOH,
, Pd
3
4
2
2
2
2
(
2
2
2
29
The same strategy provided access to L-isoDGDP 10L from L-ribonolactone 26L, epimeric at C4
with D-lyxonolactone 15D [Scheme 2]. A sequence of protection 27L, reduction 28L, introduction
of the branching hydroxyl methyl group by the Ho crossed aldol reaction 29L, oxidation 30L and
introduction of the azide afforded the isopropylidene protected lactone 31L in an overall yield of
5
4%. Subsequent functional group manipulation analogous to the synthesis of 9L via 32L, 33L,
4L, 35L, and 36L, provided L-isoDGDP 10L in an overall yield of 15% in 11 steps from 26L.
3
A shorter synthesis, in which the pyrrolidine ring was formed by intramolecular reductive
amination of an azido ketone 40L, avoided the need for the switching of protecting groups but
required separation of isoDMDP 9D and isoDGDP 10D as the final step [Scheme 3]. Reduction of
3
1D, prepared from D-ribonolactone 26D as in Scheme 2, by sodium borohydride in ethanol:tert-
butanol afforded diol 37D (97%) in which the primary alcohol was selectively protected as the
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
TBDMS ether 38D (98%). Oxidation of 38D with Dess-Martin periodinane gave the corresponding
ketone which with TBAF formed the lactols 39L (92%). Reaction of 39L with p-toluenesulfonic
acid in aqueous dioxane removed both the benzhydryl and acetonide protecting groups to give the
unprotected azide 40L (63%). Hydrogenation of 40L in water in the presence of 10% palladium on
carbon caused reduction of the azide to the corresponding amine and intramolecular reductive
amination to produce a mixture of isoDMDP 9D and isoDGDP 10D in a ratio of 2:3 and a yield of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
3% [46% from 31D; 26% from 26D]. The crude reaction mixture of 9D and 10D was separated by
-
30
ion exchange chromatography using Dowex 1x2 (OH form).
H
N
H
N
O
OH
HO
(iii) R'OCH2
O
BzhOH C
O
BzhOH C
i)
OR
HOH C
HOH C
2
2
2
2
(
(v)
CH N
CH N
CH N
2 3
OR
CH2OH
OH
CH2OH
OH
2
3
2
3
O
O
O
O
RO
HO
HO
[
2 : 3]
3
3
7D R = H
8D R = TBS
39L R,R = Me2C
3
1D
9D isoDMDP
10D isoDGDP
(
ii)
(iv)
R' = Bzh
Bzh = Ph CH
2
40L R = R' = H
t
Scheme 3: Reagents and conditions: (i) NaBH
8%; (iii) Dess-Martin periodinane, CH Cl , rt, 2 h; then TBAF, THF, 0 °C to rt, 2 h, 92%; (iv) pTsOH, H
h, 63%; (v) H , Pd (10% on C), H O, rt, 18 h, 83%.
4
, EtOH: BuOH 13:2, rt, 1.5 h, 97%; (ii) t-BuMe
2
SiCl, imidazole, DMF, 0 °C, 5 h,
9
2
2
2
O, 1,4-dioxane, 85 °C, 18
2
2
The lack of diastereoselectivity in the hydrogenation of 40L was in contrast to the selectivity found
3
1
in the hydrogenations of the azido-fructose 41 to give only DMDP 12D, and of the azido-sorbose
3
2
4
3 to form solely DGDP 13D [Scheme 4]. In the case of intermediate imine 42, neither the Re nor
Si face of the C=N bond is more clearly sterically hindered. The exclusive formation of DMDP 12D,
as rationalized by Wong, is due to the accumulation of torsional strain associated with addition to
3
3
the Re face. In the case of imine 44, the cooperative combination of torsional strain and steric
hindrance gives DGDP 13D exclusively. In contrast, for branched imine 45 these steric and
ACS Paragon Plus Environment

Page 9 of 72
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
torsional factors oppose one another, resulting in a mixture of 9D and 10D with little
stereoselectivity.
Re
H
N
HOH C
CH OH
2
CH2OH
OH
2
O
HO
HO
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(i)
OH
N
HO
OH
N3
OH
N
OH
Si
DMDP 12D
exclusively
OH
41
42
Re face
HO
HO
H
H
Re face
hindered
N
CH2OH
CH OH
OH
HOH2C
2
O
(
(
i)
i)
Disfavored attack at
the Re face induces
torsional strain
OH
OH
43
HO
HO
OH
HO
OH
N3
N
OH
DGDP 13D
exclusively
44
H
N
H
N
OH
O
HOH2C
HOH2C
HO
HO
CH OH
OH
2
N H C
N
CH OH
2
CH OH
3
2
2
OH
HO
HO
HO
OH
OH
OH
Si face
hindered
4
0L
45
isoDMDP 9D
2:3
isoDGDP 10D
Scheme 4: Diastereoselectivity in the hydrogenation of azidoketoses. Reagents: (i) H
2
, Pd/C, H
2
O.
O
O
RO
H
N
O
O
OH
O
O
OH
O
O
RO
OH
HO
(
ii)
(iv)
(vi)
(viii)
R
CH2X
CH N
CH2N3
OR
CH2OH
OH
2
3
O
O
O
O
O
O
RO
HO
4
6D R = H
48D X = OH
49D X = N3
50D R,R = Me C
52D R,R = Me C
2
2
11L isoLAB
(
i)
(iii)
(v)
(vii)
47D R = CH OH
51D R,R = H
53D R,R = H
2
Scheme 5: Reagents and conditions: (i) CH
2
O, K
SO
, MeOH, 0 °C to rt, 2.5 h, 68% (v) H
O:1,4-dioxane, 4:1, 60 °C, 36 h, 91% (viii) Pd/C (10%), H
2
CO
3
, MeOH, H
2
O, reflux, 5 h, 74% (ii) Br
Cl , -30 °C; 2.5 h then NaN
O:AcOH, 1:1, rt, 16 h, 78% (vi) NaIO
, H O:AcOH, 9:1, rt, 24 h, 81%.
2
, BaCO
, DMF, rt, 3 h, 96% (iv) DIBALH,
, H O, rt, 18 h, 85%
3 2
, H O, 0 °C to rt, 32 h, 80%, or
t
I
2
, K
2
CO
3
, BuOH, 100 °C, 81% (iii) (CF
3
2
)
2
O, pyridine, CH
2
2
3
CH Cl
2
2
, -78 °C, 2 h; then NaBH
4
2
4
2
+
(
vii) Dowex (50W-X8 H form) H
2
2
2
3
4
IsoLAB 11L was prepared from diacetone mannose 46D in eight steps with an overall yield of
1
9% [Scheme 5]. The Ho aldol of 46D introduced the hydroxymethyl branch to form 47D (74%),
2
which on bromine or iodine oxidation under basic conditions gave the lactone 48D (80%, Br , 81%,
o
35
2
I ). In the early preparations of 48D the mp was 130-132 C. On later syntheses the mp was 107-
o
24
1
13
108 C as reported by Ho. Both the H and C NMR samples from all procedures were identical;
it is likely there are two different crystalline forms of 48D. Esterification of the neopentyl alcohol in
4
8D by triflic anhydride, followed by sodium azide in DMF, afforded the azide 49D (96%).
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Reduction of 49D by DIBALH in dichloromethane, followed by sodium borohydride in methanol
afforded the diol 50D (68%). The terminal acetonide in 50D underwent selective hydrolysis with
aqueous acetic acid to give the tetraol 51D (78%). Oxidative cleavage of the terminal diol of 51D
with sodium periodate gave the protected azido-apiose 52D (85%). Hydrolysis of the acetonide in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
2D with Dowex 50W-X8, H+ form, formed the lactol 53D (91%), hydrogenation of which in
aqueous acetic acid in the presence of palladium on carbon afforded isoLAB 11L (81%). The
preparation of 11L from D-mannose is experimentally easier than that previously reported from D-
2
1
tagatose.
H
N
O
O
O
O
HOH C
OH
HO
O
HO
2
(i)
(ii)
(iv)
(v)
CH OH
CH X
CH
OH
N
2
3
CH
OH
OH
2
2
2
O
O
O
O
O
O
HO
HO
5
6L X = OH
54D
55L
52L
11D isoDAB
(
iii)
57L X = N3
Scheme 6: Reagents and conditions: (i) CH
h, 84% (from 54D) (ii) Br , BaCO , H O, 0 °C to rt, 3 h, 90% (iii) (CF
4 h, 67% (iv) DIBALH, CH Cl , -78 °C, 1 h, 93% (v) Dowex (50W-X8 H form) H
10%), H , H O:AcOH, 9:1, 24 h, 71%.
2
O, K
2
CO
3
, MeOH, H
2
O, reflux, 6 h; then NaBH
4
, H
Cl
2
O:1,4-dioxane, 4:1, 75 °C, 18 h; then Pd/C
2
O, rt, 1.33 h; then NaIO
4
, H
2
O, rt, 1
2
3
2
3
SO O, pyridine, CH
2
)
2
2
2
, -30 °C, 1 h; then NaN
3
, DMF, rt,
+
2
2
2
(
2
2
For isoDAB 11D, the acetonide of D-ribose 54D was treated sequentially in water with
formaldehyde in the presence of potassium carbonate, sodium borohydride and sodium periodate to
provide the acetonide of L-apiose 55L on a multigram scale in an overall yield of 84% [Scheme 6].
Oxidation of 55L with bromine in the presence of barium carbonate gave 56L (90%) which was
converted to the triflate and treated with sodium azide in DMF to form the azidolactone 57L (67%).
Reduction of 57L with DIBALH in dichloromethane gave the lactol 52L (93%), which on acid
ACS Paragon Plus Environment

Page 11 of 72
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
hydrolysis and hydrogenation in the presence of palladium on carbon formed isoDAB 11D in 71%
yield [34% from ribose 54D].
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Results and Discussion 2. Biological assays
Glycosidase inhibition of the iso-D-iminosugars [9D, 10D, and 11D] was compared with that of
their naturally occurring unbranched analogues [DMDP 12D, DGDP 13D, and DAB 14D] [Table 1];
3
6
a similar study on the corresponding L-enantiomers is shown [Table 2]. The natural products
DMDP 12D, DGDP 13D, and DAB 14D are modest - and not very specific - competitive inhibitors
of many α-glucosidases; DMDP 12D and DAB 14D show rare potent inhibition of yeast α-
glucosidase. DMDP 12D is a much more potent inhibitor of β-glucosidase and β-galactosidase.
isoDMDP 9D showed weak but specific inhibition against β-mannosidase, whereas isoDGDP 10D
did not inhibit any glycosidase.
Unnaturally-configured enantiomers of iminosugars frequently inhibit the same enzymes as their
37
natural product analogues. L-DMDP 12L, L-DGDP 13L, and LAB 14L - are much more potent
36c
and specific inhibitors of α-glucosidases than their natural D-enantiomers.
For the iso-
iminosugars, both enantiomers of isoDGDP 10D and 10L showed only weak inhibition toward rat
intestinal lactase. isoDAB 11D is a better and more specific α-glucosidase inhibitor than DAB 14D,
but its enantiomer isoLAB 11L did not inhibit any glycosidase unlike the potent inhibition shown
by LAB 14L. In startling contrast, L-isoDMDP 9L is the most potent inhibitor of α-glucosidases by
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a parent iminosugar yet reported, and is completely specific; L-isoDMDP 9L showed strong
inhibition of rat intestinal maltase with IC50 value of 0.19 µM. This value is significantly better than
clinically available Miglitol 2. The kinetic analysis showed that L-isoDMDP 9L is a competitive
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
i
inhibitor of rat intestinal maltase with a K of 0.081 µM. Of the 12 iminosugars only DAB 14D
(
IC50 = 0.15 µM) and DMDP 12D (IC50 = 0.71 µM) show potent inhibition of yeast α-glucosidase;
as is true for most iminosugars, none of the branched iso-iminosugars have any significant
inhibition of yeast α-glucosidase.
Table 1. Concentration of D-iminosugars giving 50% inhibition of various glycosidases
IC50 (µM)
H
N
H
N
H
N
H
N
H
N
H
N
HOH2C
HOH2C
CH2OH HOH2C
HOH2C
CH2OH
HOH2C
CH2OH
OH
CH2OH
OH
HOH2C
HO
HO
HO
OH
HO
HO
OH
OH
HO
OH
enzyme
isoDMDP (9D)
DMDP (12D)
isoDGDP (10D)
DGDP (13D)
isoDAB (11D)
DAB (14D)
!-glucosidase
a
yeast
NI
0.71
214
290
91
NI
NI
NI
NI
639
167
131
138
320
143
NI
41
24
20
15
0.15
250
55
5.8
16
rice
NI
NI
NI
NI
rat intestinal maltase
rat intestinal isomaltase
rat intestinal sucrase
40
"
-glucosidase
almond
bovine liver
NI
NI
10
9.7
NI
NI
256
523
NI
NI
250
638
!-galactosidase
coffee beans
NI
NI
NI
NI
NI
NI
"
-galactosidase
bovine liver
rat intestinal lactase
NI
NI
3.3
7.9
NI
791
361
41
NI
NI
NI
323
!-mannosidase
"-mannosidase
jack beans
NI
206
NI
NI
721
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
320
NI
snail
!
!
!
-L-fucosidase
,!-trehalase
bovine kidney
porcine kidney
Penicillium decumbens
NI
NI
NI
200
379
NI
4.8
NI
-L-rhamnosidase
NI
NI
a
NI : No inhibition (less than 50% inhibition at 1000 µM).
ACS Paragon Plus Environment

Page 13 of 72
The Journal of Organic Chemistry
1
Table 2. Concentration of
L-iminosugars giving 50% inhibition of various glycosidases
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
IC50 (µM)
H
H
H
H
H
H
N
N
N
N
N
N
HOH2C
HOH2C
CH2OH HOH2C
HOH2C
CH2OH
2
HOH C
CH2OH
OH
CH2OH
OH
2
HOH C
HO
HO
OH
HO
HO
OH
HO
OH
HO
OH
enzyme
L-isoDMDP (9L)
L-DMDP (12L)
L-isoDGDP (10L)
L-DGDP (13L)
isoLAB (11L)
LAB (14L)
!
-glucosidase
a
yeast
rice
NI
NI (34.9%)
NI
NI
175
961
127
NI
60
48
9.9
11
NI
NI
NI
NI
NI
70
3.2
0.93
0.36
1.0
2.0
0.19 (Ki = 0.081)
8.8
5.8
1.2
0.1
1.4
rat intestinal maltase
rat intestinal isomaltase
rat intestinal sucrase
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.38
"
-glucosidase
almond
bovine liver
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
!-galactosidase
"-galactosidase
coffee beans
NI
NI
NI
216
NI
NI
bovine liver
rat intestinal lactase
NI
NI
NI
NI
NI
743
NI
652
NI
NI
NI
415
!-mannosidase
jack beans
NI
NI
NI
NI
NI
NI
NI
NI
48
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
"
-mannosidase
snail
!-L-fucosidase
!,!-trehalase
bovine kidney
porcine kidney
Penicillium decumbens
NI
131
NI
!-L-rhamnosidase
a
NI : No inhibition (less than 50% inhibition at 1000 µM).
To confirm that L-isoDMDP 9L had significant effects on blood glucose levels in vivo, a maltose
loading test was conducted, using Miglitol 2 as a positive control [Figure 3]. The animal
experimental protocols in this study were approved by the Animal Experiments Committee of the
University of Toyama. Male ddY mice (29-33 g) after an overnight fast were used for acute
TM
disaccharide loading tests. The blood glucose levels were measured by the StatStrip Xpress kit
(
Nova Biochemical Co. Ltd. Massachusetts, USA). A control group was loaded with saline only.
Administration of maltose (2.5 g/kg body weight p.o.) to fasted mice resulted in a rapid increase in
blood glucose concentrations from 95 ± 12 to a maximum of 253 ± 24 mg/dL after 15 min.
Thereafter blood glucose levels recovered to the pretreatment level at 120 min. A significant
suppressive effect of the blood glucose level was achieved with 1.0 mg/kg body weight of L-
isoDMDP 9L after 15 and 30 min. 1.0 mg/kg body weight Miglitol 2 also significantly decreased
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the blood glucose concentrations at 15 min after maltose-loading. However, the suppression effects
were clearly weaker than L-isoDMDP 9L. These results suggested that L-isoDMDP 9L is worthy of
study as a potential therapeutic agent for the treatment of diabetes.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
None of isoDMDP 9D, L-isoDMDP 9L, isoDAB 11D, or isoLAB 11L showed any significant
effect on endoplasmic reticulum (ER)-resident α-glucosidase I and II activity in cells at 0.5 mM
3
8
using a free oligosaccharide assay. isoDMDP 9D had no effect at the highest concentration tested
100 µM) where L-isoLDMDP 9L at the same concentration showed very modest inhibition (20 to
00-fold less effective than Miglustat 3). At 1 mM, isoDAB 11D showed weak inhibition of α-
(
1
glucosidase II. These data suggest that the lack of significant inhibition of glucosidase processing
enzymes is due to the inability to administer sufficient concentrations of these weak inhibitors to
cells to observe any effects. DMDP 12D and L-DMDP 12L are not inhibitors of α-glucosidase I at
1
mM using in vitro assays. DAB 14D and LAB 14L are presumed inhibitors of processing
3
9
glucosidases; their N-butyl analogues are also weak inhibitors (IC50, 319 µM and 769 µM,
4
0
respectively) of α-glucosidase I and are consequently ineffective at inhibiting glucosidase activity
in cellular assays at concentrations of 1 mM or less.
ACS Paragon Plus Environment

Page 15 of 72
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3: Effect of L-isoDMDP 9L and Miglitol 2 on Blood Glucose Levels. Blood glucose concentrations of male ddY mice after
an oral load with maltose, 2.5 g/kg body weight, with 1.0 mg/kg body weight L-isoDMDP 9L (green circle). 1.0 mg/kg body weight
miglitol 2 (red circle) was used as positive control. A control group was loaded with saline (blue circle). Each value represents the
mean ± SEM (n = 5).
4
1
Pharmacological chaperones (PC) are small molecules that target protein misfolding. In particular,
4
2
iminosugars have shown promise in the treatment of lysosomal storage diseases (LSD). Fabry’s
4
3
disease is due to a deficiency of the α-galactosidase, α-GAL A. Deoxygalactonojirimycin (DGJ),
a competitive inhibitor of α-GAL A, is the C4 epimer of DNJ 1 and is in phase 3 clinical trials for
4
4
the treatment of Fabry’s disease. The enantiomer of DGJ, a non-competitive inhibitor of α-GAL
A, showed additive benefits with DGJ for stabilization of α-GAL A; thus it is not necessary in
4
5
iminosugar PC therapy for the agent to bind to the active site of the enzyme. This implies that
there are other sites distinct from the active site where small molecules, such as iminosugars, can
bind and increase thermal stabilization of the protein. The protein responsible for cystic fibrosis
transmembrane conductance regulator (CFTR) function in CF is glycosylated, even though it does
not involve any sugar metabolism; CFTR is an ABC transporter-class protein and ion channel that
transports chloride ions across the apical membrane of epithelial cells. Mutations of the CFTR gene
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
affect folding and/or functioning of the chloride channels in these cell membranes, causing CF. The
most common CF mutation F508del causes misfolding of the protein and intracellular retention by
the endoplasmic reticulum quality control and premature degradation; iminosugars may help in the
correction of structure of the misfolded CFTR protein.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Inhibition of glycosyl transferase by Miglustat 3 is the basis for its use in substrate reduction
4
6
47
therapy (SRT) for the treatment of Gaucher’s disease. Both 3 (an α-glucosidase inhibitor) and
2
1
isoLAB 11L have been found to show significant rescue of the defective F508del-CFTR function
as assessed by single-cell fluorescence imaging and sensitivity to the CFTR selective inhibitor
4
8
CFTR -172, L-isoDMDP 9L was compared with Miglustat 3 and isoLAB 11L for their corrector
inh
4
9
50
effect on CFTR function in CF-KM4 cells using single-cell fluorescence imaging [Figure 4].
The correcting effect of L-isoDMDP 9 on F508del-CFTR function in CF-KM4 cells was tested by
similar experiments; CFTR activity was first stimulated by a cocktail of Forskolin (Fsk) + Genistein
(
Gst) and subsequently inhibited by the selective inhibitor CFTRinh-172. As expected, the panel on
the left [Figure 4] shows an absence of Fsk+Gst induced response on untreated cells (blue circles).
In contrast, after 4 h of treatment with 3 (orange circles) or 9L (green circles), a significant increase
of the fluorescence after addition of the cocktail was observed, demonstrating the restoration of
CFTR activity to the plasma membrane in CF-KM4 cells. The histograms in Figure 4 report the
variation of fluorescence notes F(x) induced by CFTR stimulation in CF-KM4 after treatment by
1
1L, 3 or 9L and compare the relative effectiveness of their correction. Although L-isoDMDP 9L
ACS Paragon Plus Environment

Page 17 of 72
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
shows significant rescue of F508del-CFTR activity, it has less effect than either isoLAB 11L [the
most potent corrector] or Miglustat 3. The majority of iminosugars which are glucosidase inhibitors,
for example, Miglitol 2 shows no rescue of F508del-CFTR activity. The mechanism by which
iminosugars show such effects are not clear but may involve binding to the glycosylated site of the
protein; although α-glucosidase inhibitors such as 3 and 9L are effective, it is noteworthy that
isoLAB 11L is the most effective iminosugar corrector so far described, even though it is not an α-
glucosidase inhibitor.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4: Effect of L-isoDMDP 9L, isoLAB 11L and Miglustat 3 on the activity of F508del-CFTR assessed by single-cell
fluorescence imaging using the potential-sensitive fluorescent probe bis-(1,3-diethylthiobarbituric acid) trimethine oxonol, and its
sensitivity to the CFTR selective inhibitor CFTRinh-172 using the human airway epithelial CF-KM4 cell line.
(A) Cells were incubated with the given iminosugar for 4h at 100 µM and the activity of CFTR channels were assessed by
stimulation with 10 µM forskolin (Fsk) and 30 µM genistein (Gst), followed by inhibition with 10 µM CFTRinh-172. In cells with
rescued CFTR function, stimulation results in chloride ion transport and a change in cell potential that translates to a change in
fluorescence in the presence of the potential-sensitive fluorescent probe. Subsequent treatment with CFTR-selective inhibitor
CFTRinh-172 results in a return to the base potential, suggesting that active CFTR is responsible for the change in potential following
stimulation. Untreated cells with inactive CFTR do not respond to inhibition or stimulation. Each value represents the mean ± SEM
(n=48). See reference 21 for analogous assessment of 11L.
(B) Histograms summarizing the results collected from separate experiments; comparison of 11L, 3 and 9L.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Conclusion
The value of the Ho crossed aldol condensation for the controlled introduction and functional group
manipulation of incipient diastereomeric hydroxymethyl groups for the reliable synthesis of C-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
branched iminosugars is firmly established. The S 2 displacement of triflate derived from a
neopentyl alcohol by azide consistently proceeds in high yield.
The inhibition profile of α-glucosidases by branched iminosugar pyrrolidines is complex. L-
isoDMDP 9L is the most potent parent iminosugar of digestive enzymes yet described, whereas
there is no inhibition by isoDMDP 9D; this parallels - though exceeds - the comparative behavior of
L-DMDP 12L and DMDP 12D. In contrast isoDAB 11D is a good inhibitor whereas isoLAB 11L
shows no inhibition of the digestive enzymes; this is counter to the potent glucosidase inhibition by
LAB 14L and the relatively weak inhibition of glucosidases – other than yeast – shown by DAB
14D. DMDP 12D and DAB 14D are the only iminosugars to show any significant inhibition of
yeast α-glucosidase. None of the iso-iminosugars show any inhibition of ER glucosidases,
indicating that in the treatment of CF and diabetes unwanted side effects would be avoided.
Miglitol 2 is a well established drug for the treatment of late-onset diabetes. The specificity and
potency of L-isoDMDP 9L, together with the demonstration of superior control of blood glucose
levels, indicate there may be a second generation of iminosugars for the treatment of the disease.
Although it is not clear by what mechanism iminosugars are effective in the partial rescue of the
defective F508del-CFTR function in CF-KM4 cells, it may well be that binding to other
glycosylated sites of the protein not associated with enzyme activity may provide effective PC
ACS Paragon Plus Environment

Page 19 of 72
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
control. Further studies of iminosugars that have no glycosidase activity, and therefore potentially
fewer side effects, may have a significant role in the treatment of diseases involving mis-folded
proteins. Such bioactivity by small 5 or 6 carbon pyrrolidines indicate iminosugars will have a
major role in disease therapy that is not associated with glycosidase inhibition.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Experimental Section
General experimental
All commercial reagents were used as supplied. Thin layer chromatography (TLC) was performed
on aluminium sheets coated with 60 F254 silica. Plates were visualized using a spray of 0.2% w/v
cerium (IV) sulfate and 5% ammonium molybdate solution in 2 M aqueous sulfuric acid. Flash
chromatography was performed on Sorbsil C60 40/60 silica. Melting points were recorded on a
3
2
-1
Kofler hot block and are uncorrected. Optical rotations are quoted in 10 .deg.cm .g at
-1
1
13
concentrations (c) in g.100 mL . H and C NMR spectra were assigned by utilizing 2D COSY
and HSQC spectra. All chemical shifts (δ) are quoted in ppm and coupling constants (J) in Hz.
51
2
Residual signals from the solvents were used as an internal reference. For solutions in D O
A
acetonitrile was used as an internal reference. For anomeric mixtures, denotes the major anomer,
B
and the minor. HRMS measurements were made using a microTOF mass analyzer.
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A. Synthesis of L-iso-DMDP 9L and isoDMDP 9D
-O-Benzhydryl-2,3-O-isopropylidene-D-lyxono-1,4-lactone 16D
5
Diphenyldiazomethane (2.80 g, 14.4 mmol) was added to a refluxing solution of lactone 15D (1.81
g, 9.63 mmol) in toluene (180 mL). The reaction mixture was stirred at reflux for 1 h after which no
purple color remained and TLC analysis (1:1 EtOAc/cyclohexane) revealed the presence of a small
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
f
amount of unreacted starting material (R 0.10) and one major product (0.70). A further portion of
diphenyldiazomethane (0.93 g, 4.8 mmol) was added and after a further hour the reaction mixture
was concentrated in vacuo and the residue purified by flash column chromatography (1:50 → 1:10
→
1:8 EtOAc/cyclohexane) to afford the title lactone 16D (3.12 g, 92%) as a colorless oil.
+
25
HRMS (ESI+ve): found 377.1363 [M+Na] ; C21
H
22NaO
5
requires 377.1359; [α]
D
+35.7 (c 1.00,
CHCl
3
); vmax (thin film): 1789 (s, C=O); δ
H
(400 MHz, CDCl
3
): 1.38 (3H, s, CH
3
), 1.42 (3H, s,
3
CH ), 3.82 (1H, dd, H5a, Jgem 10.7, J5a,4 6.9), 3.88 (1H, dd, H5b, Jgem 10.9, J5b,4 5.1), 4.72 (1H, ddd,
2
H4, J4,5a 7.1, J4,5b 4.7, J4,3 3.0), 4.80-4.84 (2H, m, H2, H3), 5.48 (1H, s, CHPh ), 7.25-7.39 (10H, m,
C 3 3 3
ArH); δ (100.6 MHz, CDCl ): 25.9 (CH ), 26.7 (CH ), 66.8 (C5), 75.9 (C2), 76.0 (C3), 78.2 (C4),
8
1
1
2 3 2
4.4 (CHPh ), 114.2 (C(CH ) ), 126.9, 127.2, 127.7, 127.7, 128.4 (ArCH), 141.4, 141.5 (ArC),
+
37
-
35
-
73.6 (C1); m/z (ESI +ve): 731 ([2M+Na] , 100%), (ESI-ve): 391 ([M+ Cl] , 35%), 389 ([M+ Cl] ,
-
00%), 371 ([M+OH] , 70%).
25
[
Enantiomer 16L: [α]
D
3
-37.9 (c 1.80, CHCl ).]
ACS Paragon Plus Environment

Page 21 of 72
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5
-O-Benzhydryl-2,3-O-isopropylidene-D-lyxofuranose 17D
DIBALH (1.5 M in toluene, 0.87 mL, 1.3 mmol) was added to a solution of lactone 16D (417 mg,
.18 mmol) in DCM (6 mL) at -78 °C. The reaction was stirred at -78 °C for 1 h after which time
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
f
TLC (1:3 EtOAc/cyclohexane) showed the complete consumption of the starting material (R 0.24)
f
and the formation of one major product (R 0.29). The reaction was quenched with methanol (0.5
2 2
mL) diluted with CH Cl (5 mL) and stirred with sodium potassium tartrate (sat. aq., 15 mL) at rt
2 2
until two layers formed. The aqueous layer was extracted with CH Cl (3 x 20 mL) and the
4
combined organics were dried (MgSO ), filtered and concentrated in vacuo to give the lactol 17D
as a colorless, viscous oil (416 mg, 99%) in a 5:1 ratio of anomers, which was used without further
purification.
+
25
HRMS (ESI+ve): found 379.1510 [M+Na] ; C21
H24NaO
5
requires: 379.1516; [α]
D
-4.6 (c 1.34,
A
CHCl
3
); vmax (thin film): 3383 (s, br, OH); δ
H
(400 MHz, CDCl
3
): 1.30 (3H, s, CH
3
), 1.37 (3H, s,
B
A
B
A
A
CH
3
), 1.40 (3H, s, CH
3
), 1.48 (3H, s, CH
3
), 2.76 (1H, d, OH1 , JOH,1 2.3), 3.73 (1H, dd, H5a ,
B
B
B
A
Jgem 10.4, J5a,4 7.3), 3.71-3.76 (1H, m, H5a ), 3.80-3.83 (2H, m, H5b , H4 ), 3.82 (1H, dd, H5b ,
B
A
Jgem 10.4, J5b,4 4.1), 3.89 (1H, d, OH1 , JOH,1 12.1), 4.46 (1H, dt, H4 , J4,5 7.5, J4,3 4.0), 4.50 (1H,
B
A
B
dd, H2 , J2,3 6.1, J2,1 3.5), 4.60 (1H, d, H2 , J2,3 5.8), 4.71 (1H, dd, H3 , J3,2 6.1, J3,4 2.8), 4.76 (1H,
A
B
A
dd, H3 , J3,2 5.8, J3,4 3.8), 5.02 (1H, dd, H1 , J1,OH 12.1, J1,2 3.5), 5.41 (1H, d, H1 , J1,OH 2.0), 5.46
A+B
A+B
A
B
(
2H, s, CHPh
2
), 7.16-7.41 (20H, m, ArH ); δ
C
(100.6 MHz, CDCl
3
3 3
): 24.8 (CH ), 25.0 (CH ),
B
A
B
A
B
B
A
B
2
8
5.8 (CH
3
), 26.0 (CH
3
), 66.7 (C5 ), 67.4 (C5 ), 75.0 (C4 ), 78.5 (C2 ), 79.4 (C4 ), 79.7 (C3 ),
B
A+B
A
B
A
A
0.1 (C3 ), 84.2 (x2) (CHPh
2
3 2
), 85.4 (C2 ), 96.8 (C1 ), 101.3 (C1 ), 112.5 (C(CH ) ), 113.1
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
B
A+B
(
3 2
C(CH ) ), 127.1, 127.2, 127.4 (x2), 128.2, 128.3 (x2), 128.6, 129.0 (ArCH ), 137.9, 141.9,
A+B
+
+
1
42.0 (ArC ); m/z (ESI+ve): 735 ([2M+Na] , 100%), 379 ([M+Na] , 72%).
o
25
[
Enantiomer 17L: m.p. 68-71 C; [α]
D
3
+3.1 (c 1.56, CHCl ).]
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
-O-Benzhydryl-2-C-hydroxymethyl-2,3-O-isopropylidene-D-lyxofuranose 18D
Potassium carbonate (496 mg, 3.59 mmol) was added to a solution of lactol 17D (810 mg, 2.28
mmol) in methanol (8 mL). Aqueous formaldehyde (39.5%, 5.1 mL, 68 mmol) was added slowly
and the reaction was heated to reflux. After 4 h TLC (1:1 EtOAc/cyclohexane) showed the
f f
formation of one major product (R 0.60) and only a trace of starting material (R 0.87) remaining.
The reaction mixture was concentrated in vacuo and the residue was partitioned between ethyl
acetate (10 mL) and sodium bicarbonate (sat. aq., 10 mL). The aqueous layer was extracted with
ethyl acetate (2 x 10 mL) and the combined organics were washed with brine (20 mL), dried
(
(
MgSO
4
), filtered and concentrated in vacuo. The crude was purified by column chromatography
1:4 → 1:2 EtOAc/cyclohexane) to give the lactol 18D as a colorless oil (650 mg, 74%) in a 3:1
ratio of anomers.
+
25
A
HRMS (ESI+ve): found 409.1621 [M+Na] ; C22
H
26NaO
6
requires: 409.1622; [α]
D
-6.0 (c 0.77,
CHCl
3
); vmax (thin film): 3424 (s, br, OH); δ
H
(400 MHz, CDCl
3
): 1.36 (3H, s, CH
3
), 1.39 (3H, s,
A
B
B
B
CH
3
), 1.45 (3H, s, CH
3
), 1.50 (3H, s, CH
3
), 1.97 (1H, dd, OH2' , J 6.6, J 5.3), 2.61 (1H, dd,
A
A
A+B
A+B
A+B
OH2' , J 7.8, J 6.3), 3.71 (1H, d, OH1 , JOH,1 2.5), 3.69-3.82 (7H, m, H5a , H5b , H2'a
,
B
B
B
H2'b ), 3.84 (1H, d, OH1 , JOH,1 11.9), 3.84-3.88 (1H, ddd, H4 , J 6.6, J 5.3, J4,3 3.0), 3.92 (1H, dd,
ACS Paragon Plus Environment

Page 23 of 72
The Journal of Organic Chemistry
1
2
A
A
B
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H2'b , Jgem 11.9, J2'b,OH 6.1), 4.48 (1H, a-quin, H4 , J 3.7), 4.60 (1H, d, H3 , J3,4 2.8), 4.61 (1H, d,
A
B
A
A+B
H3 , J3,4 3.0), 4.87 (1H, d, H1 , J1,OH 11.9), 5.41 (1H, d, H1 , J1,OH 2.3), 5.44 (2H, s, CHPh
2
),
A+B
B
B
A
7
.22-7.39 (20H, m, ArH ); δ
C
(100.6 MHz, CDCl
3
): 26.9 (CH
3
), 27.1 (CH
3
), 27.3 (CH
3
), 27.5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A
B
A
B
A
B
A
B
(
(
(
CH
3
), 62.7 (C2' ), 63.5 (C2' ), 66.6 (C5 ), 67.2 (C5 ), 75.0 (C4 ), 80.0 (C4 ), 82.1 (C3 ), 83.2
A
B
A
B
A
B
A
C3 ), 84.2 (CHPh
2
), 84.3 (CHPh
2
), 89.5 (C2 ), 93.6 (C2 ), 97.4 (C1 ), 103.7 (C1 ), 113.7
A
B
A+B
C(CH
3
)
2
), 113.9 (C(CH
3
)
2
), 127.0 (x2), 127.1, 127.5 (x2), 127.6, 128.3, 128.4 (x2) (ArCH ),
A+B
+
+
1
41.7, 141.8 (ArC ); m/z (ESI+ve): 795 ([2M+Na] , 100%), 409 ([M+Na] , 18%).
25
[
Enantiomer 18L: [α]
D
3
+8.3 (c 0.54, CHCl ).]
5-O-Benzhydryl-2-C-hydroxymethyl-2,3-O-isopropylidene-D-lyxono-1,4-lactone 19D
Potassium carbonate (354 mg, 2.56 mmol) and iodine (651 mg, 2.56 mmol) were added to a hot
solution of lactol 18D (495 mg, 1.29 mmol) in tert-butanol (10 mL). The reaction was stirred at
reflux for 1 h after which time TLC (1:1 EtOAc/cyclohexane) showed the complete conversion of
f f
the starting material (R 0.60) to one major product (R 0.75). Sodium thiosulfate (sat. aq., ~5 mL)
and ethyl acetate (10 mL) were added and the mixture stirred at rt until colorless. The aqueous layer
4
was extracted with ethyl acetate (3 x 10 mL) and the combined organics were dried (MgSO ),
filtered and concentrated in vacuo. The crude lactone was purified by column chromatography (1:8
→
1:2 EtOAc/cyclohexane) to give the lactone 19D as a white crystalline solid (420 mg, 85%).
+
HRMS (ESI+ve): found 407.1464 [M+Na] ; C22
H24NaO
6
requires: 407.1465; m.p. 126-129 °C;
25
[
α]
D
+26.7 (c 1.07, CHCl
3
H
); vmax (thin film): 3475 (m, br, OH), 1786 (s, C=O); δ (400 MHz,
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3 3 3
CDCl ): 1.39 (3H, s, CH ), 1.41 (3H, s, CH ), 2.48 (1H, dd, OH, JOH,2'b 7.3, JOH,2'a 4.3), 3.82 (1H,
dd, H5a, Jgem 10.6, J5a,4 6.8), 3.86 (1H, dd, H5b, Jgem 10.7, J5b,4 4.7), 3.90 (1H, dd, H2'a, Jgem 11.4,
J2'a,OH 4.1), 3.97 (1H, dd, H2'b, Jgem 11.4, J2'b,OH 7.6), 4.73-4.78 (1H, m, H4), 4.78 (1H, d, H3, J3,4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2 C 3 3
.5), 5.47 (1H, s, CHPh ), 7.24-7.40 (10H, m, ArH); δ (100.6 MHz, CDCl ): 26.4 (CH ), 26.9
(
CH
3
), 61.2 (C2'), 66.6 (C5), 78.5 (C4), 78.7 (C3), 84.3 (CHPh
2
), 86.0 (C2), 113.7 (C(CH
3
)
2
), 127.0,
+
1
1
27.2, 127.7 (x2), 128.4 (ArCH), 141.4, 141.5 (ArC), 175.5 (C=O); m/z (ESI+ve): 791 ([2M+Na] ,
+
00%), 407 ([M+Na] , 78%).
2
5
[
Enantiomer 19L: m.p. 132-134 °C; [α]
D
3
-28.3 (c 0.98, CHCl ).]
2-C-Azidomethyl-5-O-benzhydryl-2,3-O-isopropylidene-D-lyxono-1,4-lactone 20D
Trifluoromethanesulfonic anhydride (0.28 mL, 1.6 mmol) was added dropwise to a solution of
2 2
lactone 19D (395 mg, 1.03 mmol) and pyridine (0.25 mL, 3.1 mmol) in CH Cl (10 mL) at -30 °C.
The reaction was stirred between -30 and -10 °C for 1 h after which time TLC (2:3
f
EtOAc/cyclohexane) showed complete conversion of the starting material (R 0.35) to one major
f 2 2
product (R 0.72). The reaction mixture was diluted with CH Cl (10 mL) and washed with HCl (2
2 2
M, 10 mL). The aqueous layer was extracted with CH Cl (2 x 5 mL) and the combined organics
4
were washed with brine (20 mL), dried (MgSO ), filtered and concentrated in vacuo. The crude
triflate (531 mg, 1.0 mmol) was dissolved in DMF (11 mL) and sodium azide (94 mg, 1.4 mmol)
was added. The reaction was stirred at rt for 18 h after which TLC (2:3 EtOAc/cyclohexane)
f f
showed the complete conversion of the triflate (R 0.72) to one major product (R 0.78). The
ACS Paragon Plus Environment

Page 25 of 72
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
reaction mixture was diluted with ethyl acetate (20 mL) and washed with 1:1 brine/water (2 x 15
4
mL). The organic layer was dried (MgSO ), filtered and concentrated in vacuo. The crude azide was
purified by column chromatography (1:20 → 1:8 EtOAc/cyclohexane) to give the azide 20D as a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
colorless oil (421 mg, quant).
+
25
D
HRMS (ESI+ve): found 432.1531 [M+Na ]; C22
H23NaN
3
O
5
requires: 432.1530; [α]
-2.6 (c 0.93,
3 3 H 3 3
CHCl ); vmax (thin film): 2110 (s, N ), 1788 (s, C=O); δ (400 MHz, CDCl ): 1.41 (3H, s, CH ),
1
3
.45 (3H, s, CH ), 3.55 (1H, d, H2'a, Jgem 12.9), 3.81 (1H, d, H2'b, Jgem 12.9), 3.82 (1H, dd, H5a,
Jgem 10.7, J5a,4 7.0), 3.86 (1H, dd, H5b, Jgem 10.7, J5b,4 5.1), 4.67-4.72 (1H, ddd, H4, J4,5a 6.8, J4,5b
5
2 C
.1, J4,3 3.4), 4.73 (1H, d, H3, J3,4 3.5), 5.47 (1H, s, CHPh ), 7.27-7.39 (10H, m, ArH); δ (100.6
3 3 3 2
MHz, CDCl ): 26.1 (CH ), 26.9 (CH ), 50.4 (C2'), 66.5 (C5), 78.2 (C4), 78.4 (C3), 84.4 (CHPh ),
3 2
84.9 (C2), 114.3 (C(CH ) ), 126.9, 127.1, 127.7 (x2), 128.5 (ArCH), 141.3, 141.4 (ArC), 174.0
+
+
(C=O); m/z (ESI+ve): 841 ([2M+Na] , 100%), 432 ([M+Na] , 16%).
25
[
Enantiomer 20L: [α]
D
3
+3.5 (c 1.04, CHCl ).]
2
-C-Azidomethyl-D-lyxono-1,4-lactone 21D
A solution of acetyl chloride (0.8 mL) in methanol (16 mL) was added to azide 20D (864 mg, 2.11
mmol) and the solution was stirred at reflux for 18 h. After this time, TLC (1:1 EtOAc/cyclohexane)
indicated the complete consumption of the starting material and the formation of one major product
f 2 2
(R 0.11). The reaction was concentrated in vacuo and coevaporated with CH Cl . The solid was
triturated at rt with ether (x3) to give the lactone 21D as a white crystalline solid (417 mg, 97%).
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 26 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
HRMS (ESI+ve): found 226.0433 [M+Na] ; C
6
H
9
NaN
3
O
5
requires: 226.0434; m.p. 124-128 °C;
25
[
(
α]
D
3 H
+42.3 (c 0.78, MeOH); vmax (thin film): 3406 (s, br, OH), 2114 (s, N ), 1774 (s, C=O); δ
400 MHz, MeOD): 3.48 (1H, d, H2'a, Jgem 13.1), 3.56 (1H, d, H2'b, Jgem 13.0), 3.88 (2H, a-d, H5, J
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
.1), 4.34 (1H, d, H3, J3,4 4.7), 4.58 (1H, a-q, H4, J 5.0); δ
C
(100.6 MHz, MeOD): 54.2 (C2'), 61.2
-
(
(
[
C5), 71.4 (C3), 78.0 (C2), 82.7 (C4), 177.3 (C=O); m/z (ESI-ve): 405 ([2M-H] , 65%), 240
37
-
35
-
-
[M+ Cl] , 50%), 238 ([M+ Cl] , 97%), 202 ([M-H] , 100%).
2
5
Enantiomer 21L: m.p. 130-132 °C; [α]
D
-43.9 (c 0.77, MeOH).]
2
-C-Azidomethyl-2,3,5-tri-O-benzyl-D-lyxono-1,4-lactone 22D
Benzyl bromide (1.1 mL, 9.3 mmol) and sodium hydride (60% in mineral oil, 150 mg, 3.75 mmol)
were added to a solution of azide 21D (190 mg, 0.94 mmol) in DMF (4 mL) at -5 °C. The reaction
was stirred at 0 °C for 3.5 h after which time TLC (1:3 EtOAc/cyclohexane) showed almost
f f
complete conversion of the starting material (R 0.07) to one major product (R 0.50). A further
portion of NaH (38 mg, 0.95 mmol) was added and the reaction stirred for a further 1 h. The
reaction was quenched with acetic acid, diluted with ethyl acetate (10 mL) and washed with 1:1
4
brine/water (3 x 10 mL). The organic layer was dried (MgSO ), filtered and concentrated in vacuo.
The crude residue was purified by column chromatography (1:99 → 1:9 EtOAc/cyclohexane) to
give the tribenzyl lactone 22D as a colorless oil (379 mg, 86%).
+
25
HRMS (ESI+ve): found 496.1843 [M+Na ]; C27
H
27NaN
3
O
5
requires: 496.1843; [α]
D
-11.7 (c
1
3 3 H 3
.61, CHCl ); vmax (thin film): 2106 (s, N ), 1779 (s, C=O); δ (400 MHz, CDCl ): 3.42 (1H, d,
ACS Paragon Plus Environment

Page 27 of 72
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H2'a, Jgem 12.9), 3.70 (1H, d, H2'b, Jgem 12.9), 3.82 (1H, dd, H5a, Jgem 10.9, J5a,4 6.8), 3.86 (1H, dd,
a
H5b, Jgem 10.9, J5b,4 4.8), 4.34 (1H, d, H3, J3,4 5.6), 4.54 (1H, d, CH
2
Ph , Jgem 11.9), 4.59 (1H, d,
a
b
CH
2
Ph , Jgem 11.9), 4.60 (1H, d, CH
2
Ph , Jgem 11.6), 4.65 (1H, a-dt, H4, J4,5a 6.7, J4,3 = J4,5b 5.3),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
c
4
.78 (1H, d, CH
2
Ph , Jgem 11.6), 4.92 (2H, a-s, CH
2
Ph ), 7.20-7.24 (2H, m, ArH), 7.28-7.40 (13H,
c
a
m, ArH); δ
C
(100.6 MHz, CDCl
3
2 2
): 53.0 (C2'), 68.1 (C5), 69.7 (CH Ph ), 73.7 (CH Ph ), 74.3
b
(
(
(
[
CH
2
Ph ), 77.9 (C3), 79.6 (C4), 81.4 (C2), 127.5, 127.7, 127.9 (x2), 128.1, 128.2, 128.3, 128.5 (x2)
+
ArCH), 136.8, 137.5, 137.6 (ArC), 171.8 (C=O); m/z (ESI+ve): 969 ([2M+Na] , 60%), 496
+
[M+Na] , 100%).
25
Enantiomer 22L: [α]
D
3
+10.6 (c 1.73, CHCl ).]
2-C-Azidomethyl-2,3,5-tri-O-benzyl-D-lyxitol 23D
Sodium borohydride (37 mg, 0.96 mmol) was added to a solution of lactone 22D (181 mg, 0.383
mmol) in ethanol (3 mL). The reaction was stirred at rt for 2 h after which a further portion of
sodium borohydride (22 mg, 0.58 mmol) was added. After a further hour, TLC analysis (1:3
f
EtOAc/cyclohexane) showed complete consumption of the starting material (R 0.64). The reaction
was quenched with ammonium chloride (sat. aq.), diluted with ethyl acetate (10 mL) and washed
with brine (10 mL). The aqueous layers were extracted with ethyl acetate (2 x 5 mL) and the
4
combined organics were dried (MgSO ), filtered and concentrated in vacuo. The crude was purified
by column chromatography (1:9 → 7:13 EtOAc in cyclohexane) to afford the diol 23D as a
colorless oil (184 mg, quant.) (R
f
0.17, 1:3 EtOAc/cyclohexane).
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 28 of 72
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
25
HRMS (ESI+ve): found 500.2156 [M+Na] ; C27
H
31NaN
3
O
5
requires: 500.2156; [α]
D
-15.2 (c 1.1,
CHCl
3
); νmax (thin film): 3418 (m, br, OH), 2103 (s, N
3
); δ
H
(400 MHz, CDCl
3
): 2.82 (1H, d, OH4,
J
OH,4 8.1), 2.99 (1H, a-br-t, OH1, J 5.3), 3.44 (1H, dd, H5a, Jgem 9.4, J5a,4 6.6), 3.54 (1H, dd, H5b,
gem 9.4, J5b,4 5.8), 3.72 (1H, d, H2'a, Jgem 13.9), 3.79 (1H, d, H2'b, Jgem 13.7), 3.82 (1H, dd, H1a,
gem 12.4, J1a,OH 7.3), 3.99 (1H, s, H3), 4.02 (1H, dd, H1b, Jgem 12.4, J1b,OH 3.3), 4.05-4.09 (1H, m,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
J
J
a
a
c
H4), 4.50 (1H, d, CH
2
Ph , Jgem 11.9), 4.55 (1H, d, CH
2
Ph , Jgem 11.9), 4.58 (1H, d, CH
2
Ph , Jgem
b
b
c
1
1
1.1), 4.69 (1H, d, CH
2
Ph , Jgem 11.1), 4.72 (1H, d, CH
2
Ph , Jgem 10.9), 4.78 (1H, d, CH
2
Ph , Jgem
b
0.9), 7.21-7.39 (15H, m, ArH); δ
C
(100.6 MHz, CDCl
3
): 51.7 (C2'), 61.5 (C1), 65.3 (CH
2
Ph ), 68.1
a
c
(
C4), 71.9 (C5), 73.4 (CH
2
Ph ), 75.9 (CH
2
Ph ), 78.6 (C3), 81.7 (C2), 127.5, 127.6, 127.9, 128.0,
+
128.1, 128.2, 128.4, 128.5 (x2) (ArCH), 137.7 (x2), 138.3 (ArC); m/z (ESI+ve): 977 ([2M+Na] ,
+
+
100%), 500 ([M+Na] , 100%), 495 ([M+NH
4
] , 32%).
25
[Enantiomer 23L: [α]
D
3
+13.6 (c 0.97, CHCl ).]
2
-C-Azidomethyl-2,3,5-tri-O-benzyl-1-O-tert-butyldimethylsilyl-D-lyxitol 24D
Imidazole (168 mg, 2.47 mmol) and TBSCl (248 mg, 1.65 mmol) were added to a solution of diol
3D (392 mg, 0.822 mmol) in DMF (8 mL) with 3Å powdered molecular sieves. The reaction was
2
stirred at rt for 4 h after which a further portion of imidazole (84 mg, 1.2 mmol) and TBSCl (124
mg, 0.823 mmol) were added. After a further 2 h TLC (1:2 EtOAc/cyclohexane) showed almost
f f
complete conversion of the starting material (R 0.23) to one major product (R 0.77). The reaction
was diluted with ethyl acetate (20 mL), washed with 1:1 brine/water (3 x 20 mL) and the organic
ACS Paragon Plus Environment

Page 29 of 72
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
layer was then concentrated in vacuo and the residue purified by column chromatography (1:99 →
3
:17 EtOAc/cyclohexane) to give the silyl ether 24D as a colorless oil (455 mg, 94%).
+
25
HRMS (ESI+ve): found 614.3023 [M+Na] ; C33
H
45NaN
3
O
5
Si requires: 614.3021; [α]
D
+3.4 (c
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3 3 H 3
.0, CHCl ); vmax (thin film): 3533 (w, br, OH), 2103 (s, N ); δ (400 MHz, CDCl ): 0.09 (3H, s,
3 3 3 3
CH Si), 0.11 (3H, s, CH Si), 0.94 (9H, s, (CH ) CSi), 3.02 (1H, d, OH4, J 6.6), 3.48 (1H, dd, H5a,
Jgem 9.4, J5a,4 7.1), 3.53 (1H, dd, H5b, Jgem 9.4, J5b,4 5.8), 3.57 (1H, d, H2'a, Jgem 13.1), 3.84 (1H, d,
H2'b, Jgem 13.1), 3.91 (1H, d, H1a, Jgem 11.1), 4.00 (1H, d, H1b, Jgem 11.1), 4.12 (1H, d, H3, J3,4 1.3),
a
a
4
.21 (1H, ddt, H4, J4,5a 6.9, J4,5b 5.7, J4,3 1.3), 4.49 (1H, d, CH
2
Ph , Jgem 11.9), 4.56 (1H, d, CH
2
Ph ,
b
c
b
J
gem 11.9), 4.63 (1H, d, CH
2
Ph , Jgem 11.1), 4.71 (1H, d, CH
2
Ph , Jgem 11.1), 4.72 (1H, d, CH
2
Ph ,
c
J
gem 11.1), 4.82 (1H, d, CH
2
Ph , Jgem 11.1), 7.25-7.37 (15H, m, ArH); δ
C
3
(100.6 MHz, CDCl ): -5.7,
c
-5.6 (CH
3
Si), 18.1 ((CH
3
)
3
CSi), 25.9 ((CH
3
)
3
CSi), 52.1 (C2'), 63.0 (C1), 66.4 (CH
2
Ph ), 67.8 (C4),
a
b
71.9 (C5), 73.3 (CH
2
Ph ), 75.3 (CH
2
Ph ), 76.9 (C3), 82.0 (C2), 127.4, 127.7 (x2), 127.8, 128.3,
+
+
1
4
4
28.4 (x2) (ArCH), 138.0 (x2), 138.6 (ArC); m/z (ESI+ve): 614 ([M+Na] , 100%), 609 ([M+NH ] ,
+
8%), 592 ([M+H] , 12%).
25
[
Enantiomer 24L: [α]
D
3
-2.9 (c 1.11, CHCl ).]
ACS Paragon Plus Environment