Fluorescent glycosidase inhibiting 1,5-dideoxy-1,5-iminoalditols

Article abstract of DOI:10.1016/j.bmcl.2006.01.095

1,5-Dideoxy-1,5-iminoalditols of various configurations as well as isofagomine were N-alkylated with non-polar straight chain spacer-arms by a set of simple standard procedures. The spacer-arms' terminal functional groups, primary amines, were employed to

Full text of DOI:10.1016/j.bmcl.2006.01.095

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2067–2070
Fluorescent glycosidase inhibiting 1,5-dideoxy-1,5-iminoalditols
a
a
b
c
a,
*
Peter Greimel, Herwig H a¨ usler, Inge Lundt, Karen Rupitz, Arnold E. St u¨ tz,
c
c
a
Chris A. Tarling, Stephen G. Withers and Tanja M. Wrodnigg
a
Glycogroup, Institut f u¨ r Organische Chemie, Technische Universit a¨ t Graz, Stremayrgasse 16, A-8010 Graz, Austria
b
Laboratory of Organic Chemistry, Department of Chemistry, Technical University of Denmark,
Building 201, DK-2800 Lyngby, Denmark
Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1
c
Received 6 December 2005; revised 17 January 2006; accepted 18 January 2006
Available online 14 February2006
Abstract—1,5-Dideoxy-1,5-iminoalditols of various configurations as well as isofagomine were N-alkylated with non-polar straight
chain spacer-arms by a set of simple standard procedures. The spacer-arms’ terminal functional groups, primary amines, were
employed to introduce fluorescent tags such as dansyl and dapoxyl moieties. Resulting derivatives in the D-xylo, D-gluco, D-galacto
as well as GlcNAc series showed distinctly improved glycosidase inhibitory activities compared to parent compounds and are
designed to be useful analytical tools.
Ó 2006 Elsevier Ltd. All rights reserved.
6
Iminosugars including iminoalditols and related bicyclic
alkaloids are well-known, (usually) competitive, glycosi-
inhibitors 1–7 according to Scheme 1. Starting from
the respective free inhibitor (Scheme 2), the ring nitro-
gen was alkylated with 5-bromohexanoic nitrile to give
1
dase inhibitors. Quite a few representatives of this class
of compounds have found important roles as diagnostic
compounds such as in the investigation of glycoprotein
R4
R
R 2
2
5
trimming glycosidases or as pharmaceutical substances
3
such as in the treatment of diabetes type II symptoms.
3
N
R
R
Other biological activities associated with their glycosi-
dase inhibitory properties are anti-viral, anti-cancer
and anti-metastatic, anti-infective as well as insect
HO
R1
R = H
4
anti-feedant and plant growth-regulating effects.
1
2
3
4
5
1
2
3
4
5
(Glu):
(Man):
(Gal):
R = OH, R = H, R = OH, R = H, R = CH
R = H, R = OH, R = OH, R = H, R = CH
2
OH
OH
OH
OH
1
2
3
4
5
2
Recently, we have found that some fluorescently labeled
derivatives of the glucosidase inhibitor 2,5-dideoxy-2,5-
imino-D-mannitol (or DMDP) are powerful inhibitors
exceeding the parent compound’s activity by two orders
1
2
3
4
5
R = OH, R = H, R = H, R = OH, R = CH
2
1
2
3
4
5
(GlcNAc): R = NHAc, R = H, R = OH, R = H, R = CH
2
1
2
3
4
5
(Xyl):
R = OH, R = H, R = OH, R = H, R = H
5
of magnitude. Such labeled inhibitors are deemed to be
OH
R
highly useful diagnostic tools for activity-based high-
throughput analyses as well as on-gel-staining. With
the aim to evaluate a more general means of tagging
of iminosugars than was operative in the mentioned
structurally quite restricted case, we resorted to the con-
nection of fluorophores to the iminosugars via non-po-
lar medium length spacer-arms. These moieties were
conveniently attached to the ring nitrogen of the
H C
N
3
HO
HO
OH
R
N
OH
OH
R = H
6
7
1a - 7a:
R = (CH
R = (CH
R = (CH
R = (CH
R = (CH
2
)
5
CN
NH
NHDansyl
NHDapoxyl
CONH(CH
1b – 7b:
1c – 7c:
1d, 2d:
1e:
2
)
)
6
2
2
6
2
)
)
6
Keywords: Glycosidase inhibitor; Iminoalditol; Fluorescent.
*
2
5
2 2
) NHDansyl
Corresponding author. Tel.: +43 316 873 8744; fax: +43 316 873
740; e-mail: stuetz@tugraz.at
8
Scheme 1. Inhibitors and intermediates.
0
960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.095

2068
P. Greimel et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2067–2070
OH
OH
OH
H
N
N
a
b
HO
HO
HO
HO
(
CH ) CN
2 5
HO
HO
OH
N
N
c
HO
HO
(CH ) CH NH
HO
HO
(CH ) CH NHDansyl
2
5
2
2
2 5
2
HO
HO
Scheme 2. General reaction sequence example (D-gluco series). Reagents and conditions: (a) Br(CH
MeOH, 24 h; (c) dansylCl or dapoxylCl, NEt , DMF.
2
)
5
CN, Na
2
3 2
CO , DMF, 40 °C; (b) Raney-Ni, H ,
3
compounds 1a–7a followed by reduction of the nitriles
to the corresponding primary amines 1b–7b which, in
turn, were reacted with the dansyl or dapoxyl fluoro-
phors to yield compounds 1c–7c as well as 1d and 2d.
Introducing a variation of the spacer-arm, compounds
Contrasting these gratifying results, iminofucitol 6c
showed markedly decreased potency. The same was true
for tagged isofagomine 7c, which lost activity by three
orders of magnitude compared to parent compound 7,
in keeping with previous findings that N-alkylation of
this powerful b-glucosidase inhibitor results in signifi-
cant losses of inhibitory power.
8
1 were transformed into N-alkylated derivative 1e
Scheme 1).
(
Compounds investigated are reversible inhibitors of the
7
enzymes probed in this study. In the D-gluco (1c–e),
Acknowledgments
D-galacto (3c), as well as N-acetyl-D-glucosamine (4c)
series, practically independent of the fluorophores em-
ployed in this study, improvements over the parent com-
pounds were observed (Table 1). Comparing dansyl (1c
and 2c), and dapoxyl (1d and 2d) labeled compounds of
the same configuration, the respective dapoxyl deriva-
tive was found to be slightly less active than its corre-
sponding dansyl counterpart. In the D-manno series,
the N-substituent in 2c led to an acceptable four- to five-
fold reduction of inhibitory activity.
Financial support by the Austrian Fonds zur F o¨ rderung
der Wissenschaftlichen Forschung (FWF), Vienna,
(Project 15726-N03) as well as by the Protein Engineer-
ing Network of Centres of Excellence of Canada
(PENCE) is gratefully acknowledged.
References and notes
1
. For example: Iminosugars: Recent Insights into their
Bioactivity and Potential as Therapeutic Agents, Martin,
O. R., Compain, P. Eds.; Curr. Top. Med. Chem. 2003, 3,
pp 471–591; From lianas to glycobiology tools: twenty-five
years of 2,5-dideoxy-2,5-imino-D-mannitol Wrodnigg, T.
M. In Timely Research Perspectives in Carbohydrate
Chemistry; Schmid, W., St u¨ tz, A. E., Eds.; Springer:
Vienna, New York, 2002; pp 43–76; Heightman, T. D.;
Vasella, A. T. Angew. Chem., Int. Ed. Engl. 1999, 38, 750;
Iminosugars as Glycosidase Inhibitors; St u¨ tz, A. E., Ed.;
Wiley-VCH: Weinheim, 1999; Bols, M. Acc. Chem. Res.
i
Table 1. K values of new compounds in comparison with parent
inhibitors
Compound
i
K (lM)
a
1
12
0.32
a
1
1
1
2
2
2
3
3
4
4
5
5
6
6
7
7
c
d
e
a
1.0
0.14
a
b
18
1
1
998, 31, 1; Legler, G. Adv. Carbohydr. Chem. Biochem.
990, 48, 319.
b
c
78
b
d
65
2
. Spiro, R. G. In Carbohydrates in Chemistry and Biology,
Ernst, B., Hart, G. W., Sinay, P., Eds.; Part II: Biology of
Saccharides; Wiley-VCH: Weinheim, 2000; Vol. 3, pp 65–
c
c
12.5
0.95
c
c
c
c
c
d
80
1.8
79; Elbein, A. D.; Molyneux, R. J. In Iminosugars as
Glycosidase Inhibitors; St u¨ tz, A. E., Ed.; Wiley-VCH:
d
e
206
Weinheim, 1999; pp 216–251.
. Miglitol by BAYER.
e
7.6
0.01
Ò
3
f
4
. Simmonds, M. S. J.; Kite, G. C.; Porter, E. A. In
Iminosugars as Glycosidase Inhibitors; St u¨ tz, A. E., Ed.;
Wiley-VCH: Weinheim, 1999; pp 8–30.
f
0.5
0.007
a
a
9.0
5
. Hermetter, A.; Scholze, H.; St u¨ tz, A. E.; Withers, S. G.;
Wrodnigg, T. M. Bioorg. Med. Chem. Lett. 2001, 11, 1339.
6. Typical procedures: a 3% solution of the respective imino-
a
b
c
d
e
f
b-Glucosidase Agrobacterium sp.
a-Mannosidase jack beans.
b-Galactosidase Agrobacterium sp.
b-D-Hexosaminidase Streptomyces plicatur.
b-Xylosidase Thermoanaerobact. Sacch.
a-L-Fucosidase human liver.
alditol in dry DMF was stirred with x-bromohexanoic
nitrile (2 equiv) in the presence of Na
40 °C for 72 h. Removal of the solvent and chromato-
graphic purification (CHCl /MeOH, 1:1), gave products
2 3
CO (1.3 equiv) at
3

P. Greimel et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2067–2070
2069
0
0
0
0
1
1a–7a in yields ranging between 45% and 90%. Standard
hydrogenation of a 5% solution of the respective hexanoic
nitrile under an atmosphere of H (50 bar) in MeOH in the
2
25.6, 24.9, 22.3, 16.0 (C-2 , C-3 , C-4 , C-5 ). H NMR: d
3.78 (m, 1H, H-2), 3.73–3.55 (m, 2H, H-6a, H-6b), 3.54 (dd,
1H, J3,4 = J4,5 9 Hz, H-4), 3.24 (dd, 1H, J2,3 2.6 Hz, H-3),
3.10 (dd, 1H, J1a,1e 12.8 Hz, J1e,2 4 Hz, H-1e), 3.10–2.80 (m,
presence of excess Raney-Ni for 24 h, followed by chro-
matography (CHCl /MeOH/concd NH OH, 50:50:1) gave
0 0 0
3H, H-1 , H-5 ), 2.68 (m, 1H, H-5 ), 2.20 (m, 2H, H-1a, H-
3
4
0
0
0
primary amines 1b–7b in yields between 20% and 60%.
Treatment of primary amines 1b–7b, with 3% solution in
dry DMF with the respective fluorophor (dansyl or
5), 1.60–1.10 (m, 6H, H-2 , H-3 , H-4 ). MS (API-ES): Calcd
for [C H N O ]: m/z 258.3201. Found: m/z 257.20 [MꢀH].
1
2
22
2
4
1
3
Compound 2b: C NMR: d 75.3, 68.4, 68.3, 66.0, 57.8, 55.2
0 0
(C-1, C-2, C-3, C-4, C-5, C-6), 52.3 (C-1 ), 39.8 (C-6 ), 28.2,
dapoxylchloride, 1.2 equiv) in the presence of Et
2 equiv) gave the corresponding fluorescently tagged
inhibitors 1c–7c, 1d, and 2d in yields ranging between
0% and 60%. Chromatography: Silica gel 60 (Merck),
CHCl /MeOH/NH OH (300:100:1 or 500:100:1). Electro-
3
N
0
0
0
0
(
26.6, 25.9, 23.9 (C-2 , C-3 , C-4 , C-5 ). MS (API-ES): Calcd
for [C12
H
26
2
N O
4
]: m/z 262.3520. Found: m/z 261.30 [MꢀH].
1
3
3
Compound 2c: C NMR: d 74.7, 67.9, 67.8, 66.0, 57.2, 55.0
0
3
4
(C-1, C-2, C-3, C-4, C-5, C-6), 52.7 (C-1 ), 44.7 (Me
2
N-aryl),
0
0
0
0
0
1
42.5 (C-6 ), 29.2, 26.5, 26.0, 23.6 (C-2 , C-3 , C-4 , C-5 ). H
spray mass spectra were recorded with a HP 1100 series
MSD, Hewlett Packard. Samples were dissolved in aceto-
nitrile/MeOH mixtures. The scan mode for negative ions
NMR: d 3.91 (m, 2H, H-2, H-6a), 3.84 (dd, 1H, J5,6b 2 Hz,
6a,6b 11.7 Hz, H-6b), 3.73 (dd, 1H, J3,4 8.8 Hz, J4,5 9.3 Hz,
J
(mass range 100–1000 D) was employed varying the
fragmentation voltage from 30 to 130 V. NMR spectra
H-4), 3.37 (dd, 1H, J2,3 2.4 Hz, H-3), 3.04 (br s, 1H, H-1e),
0
2.90 (s, 6H, Me N-aryl), 2.86 (t, 2H, H-1 ), 2.76 (m, 1H, H-
2
1
were recorded at 200 as well as 500 MHz ( H), and at 50
unless stated
0
0
6 a), 2.44 (m, 2H, H-1a, H-6 b), 2.32 (br s, 1H, H-5), 1.40–
0
1
3
and 125 MHz ( C) from samples in MeOH-d
0
0
0
4
1.10 (m, 8H, H-2 , H-3 , H-4 , H-5 ). MS (API-ES): Calcd
for [C H N SO ]: m/z 495.6430. Found: m/z 494.60
otherwise. Chemical shifts are listed in d employing
residual, not deuterated, solvent as the internal standard.
The signals of aromatic groups were found in the expected
regions and are not listed explicitly.
2
4
37
3
6
[MꢀH].
Compound 2d: d 75.4, 68.5, 68.4, 65.8, 58.0, 55.3 (C-1, C-2,
0
0
2
C-3, C-4, C-5, C-6), 52.5 (C-1 ), 42.8 (C-6 ), 39.2 (Me N-
1
3
0
0 0 0 0
1
aryl), 29.4, 26.9, 26.3, 24.0 (C-2 , C-3 , C-4 , C-5 ). H
Compound 1a: C NMR: d 120.0 (C-6 ), 78.9, 70.3, 69.0,
0
6
6.3, 57.6, 56.0 (C-1, C-2, C-3, C-4, C-5, C-6), 52.3 (C-1 ),
NMR: d 3.87 (dd, 1H, J5,6a 2.4 Hz, J6a,6b 11.7 Hz, H-6a),
3.83 (dd, 1H, J5,6b 2.4 Hz, H-6b), 3.79 (m, 1H, H-2), 3.64
(dd, 1H, J3,4 9.3 Hz, J4,5 8.8 Hz, H-4), 3.28 (dd, 1H, J2,3
0
0
0
0
1
6.3, 25.1, 23.2, 16.1 (C-2 , C-3 , C-4 , C-5 ). H NMR
2
(
D
2
O): d 3.74 (m, 2H, H-6a, H-6b), 3.44 (ddd, 1H, J1a,2
1
9
J
0.1 Hz, J1e,2 4.8 Hz, J2,3 9.7 Hz, H-2), 3.27 (dd, 1H, J3,4
.2 Hz, H-3), 3.14 (dd, 1H, J4,5 8.8 Hz, H-4), 2.96 (dd, 1H,
3.3 Hz, H-3), 3.01 (s, 6H, Me N-aryl), 2.92 (m, 3H, H-1e, H-
2
0
0
0
1 ), 2.71 (m, 1H, H-6 a), 2.51 (m, 1H, H-6 b), 2.40 (dd, 1H,
J1a,2 1.5 Hz, J1a,1e 12.2 Hz, H-1a), 2.07 (ddd, 1H, H-5), 1.50–
0
1a,1e 11.9 Hz, H-1e), 2.80–2.50 (m, 2H, H-1 ), 2.40–2.22
0
0
0
0
0
0
0
(
m, 4H, H-1a, H-5, H-5 ), 1.62–1.20 (m, 6H, H-2 , H-3 , H-
0
1.16 (m, 8H, H-2 , H-3 , H-4 , H-5 ). MS (API-ES): Calcd
for [C H N O S]: m/z 588.7288. Found: m/z 587.70
4
). MS (API-ES): Calcd for [C H N O ]: m/z 258.3201.
4
1
2
22
2
29 40
4
7
Found: m/z 257.30 [MꢀH].
[MꢀH].
1
3
13
Compound 3a: C NMR: d 120.1 (C-6 ), 75.6, 71.7, 67.6,
63.5, 61.1, 56.5, 52.5 (C-1, C-2, C-3, C-4, C-5, C-6, C-1 ),
0
Compound 1c: C NMR: d 78.6, 69.5, 68.5, 66.1, 58.3, 56.7
0
0
(
aryl), 42.5 (C-6 ), 29.2, 26.2, 26.0, 23.5 (C-2 , C-3 , C-4 , C-
C-1, C-2, C-3, C-4, C-5, C-6), 49.1 (C-1 ), 44.6 (Me N-
0
2
0
0
0
0
0
0
0
26.4, 25.2, 23.1, 16.2 (C-2 , C-3 , C-4 , C-5 ). MS (API-ES):
Calcd for [C12H N O ]: m/z 258.3201. Found: m/z 257.30
22 2 4
0
1
5
(
). H NMR: d 3.91 (br d, 1H, J6a,6b 12.0 Hz, H-6a), 3.83
dd, 1H, J5,6b 2.4 Hz, H-6b), 3.53 (m, 1H, H-2), 3.42 (dd,
[MꢀH].
1
3
1
3
H, J3,4 = J4,5 9 Hz, H-4), 3.20 (dd, 1H, J 9 Hz, H-3),
2,3
Compound 3b: C NMR: d 76.1, 71.1, 67.8, 64.2, 61.3, 56.9,
0
0 0
52.7 (C-1, C-2, C-3, C-4, C-5, C-6, C-1 ), 40.2 (C-6 ), 29.5,
.08 (m, 1H, H-1e), 2.90 (m, 7H, H-1 a, Me
0
2
N-aryl), 2.66
0
0
0
0
1
(
m, 1H, H-1 b), 2.38 (m, 2H, H-1a, H-5), 1.42–1.05 (m, 8H,
0
27.0, 26.3, 23.9 (C-2 , C-3 , C-4 , C-5 ). H NMR: d 3.98 (dd,
1H, J3,4 3 Hz, J4,5 1.5 Hz, H-4), 3.84–3.77 (m, 3H, H-2, H-
6a, H-6b), 3.22 (dd, 1H, J2,3 9.5 Hz, H-3), 2.99 (dd, 1H,
0
0
0
H-2 , H-3 , H-4 , H-5 ). MS (API-ES): Calcd for
C H N SO ]: m/z 495.6430. Found: m/z 494.60 [MꢀH].
[
2
4
37
3
6
1
3
0
Compound 1d: C NMR: d 79.4, 70.9, 69.6, 66.2, 58.3, 56.2
J1a,1e 11.0 Hz, J1e,2 4.9 Hz, H-1e), 2.81 (t, 2H, H-1 ), 2.79 (m,
0
0
0
0
(
C-1, C-2, C-3, C-4, C-5, C-6), 52.5 (C-1 ), 42.8 (C-6 ), 39.2
0
1H, H-6 a), 2.50 (m, 1H, H-6 b), 2.38 (ddd, 1H, H-5), 2.11
0
0
0
0
0
(
Me N-aryl), 29.5, 26.9, 26.3, 24.0 (C-2 , C-3 , C-4 , C-5 );
2
(dd, 1H, J
0
10.5 Hz, H-1a), 1.65–1.26 (m, 8H, H-2 , H-3 ,
H-4 , H-5 ). MS (API-ES): Calcd for [C H N O ]: m/z
1a,2
0
1
H NMR: d 3.85 (dd, 1H, J5,6a 2 Hz, J6a,6b 12.7 Hz, H-6a),
12
26
2
4
3
9
3
.81 (dd, 1H, J5,6b 3 Hz, H-6b), 3.45 (ddd, 1H, J1a,2 = J2,3
262.3520. Found: m/z 261.35 [MꢀH].
1
3
.8 Hz, J1e,2 4.9 Hz, H-2), 3.33 (dd, 1H, J3,4 8.8 Hz, H-4),
10.8 Hz, H-1e), 2.90
Compound 3c: C NMR: d 74.6, 70.3, 66.4, 64.7, 60.2, 54.9,
0
.20 (dd, 1H, H-3), 2.96 (dd, 1H, J
0
52.9 (C-1, C-2, C-3, C-4, C-5, C-6, C-1 ), 44.6 (Me N-aryl),
1
a,1e
2
0
0
0
0
0
0
0
1
(
(
t, 2H, H-1 ), 2.76 (m, 1H, H-6 a), 2.50 (m, 1H, H-6 b), 2.14
0
42.4 (C-6 ), 29.2, 26.3, 25.9, 23.1 (C-2 , C-3 , C-4 , C-5 ). H
NMR: d 3.97 (dd, 1H, H-4), 3.78 (ddd, 1H, H-2), 3.74 (m,
2H, H-6a, H-6b), 3.20 (dd, 1H, J2,3 9.3 Hz, J3,4 3.4 Hz, H-3),
dd, 1H, H-1a), 2.08 (m, 1H, H-5), 1.50–1.17 (m, 8H, H-2 ,
0
0
0
40 4 7
H-3 , H-4 , H-5 ). MS (API-ES): Calcd for [C29H N O S]:
m/z 588.7288. Found: m/z 587.70 [MꢀH].
2.91 (dd, 1H, J1a,1e 11.3 Hz, J
0
4.9 Hz, H-1e), 2.90 (s, 6H,
0
1e,2
1
3
Compound 1e: C NMR: d 175.3 (C@O), 79.2 (C-3), 70.6
Me N-aryl), 2.85 (t, 2H, H-1 ), 2.58 (m, 1H, H-6 a), 2.37 (m,
0
1H, H-6 b), 2.52 (m, 1H, H-5), 2.06 (dd, 1H, J1a,2 10.3 Hz,
2
(
1
2
C-4), 69.3 (C-2), 66.2 (C-5), 57.9 (C-6), 56.3 (C-1), 52.4 (C-
0
0
0
0
0
0
0
0
), 44.7 (Me N-aryl), 42.1 (C-5 ), 39.1 (C-7 ), 35.6 (C-8 ),
2
H-1a), 1.35–0.97 (m, 8H, H-2 , H-3 , H-4 , H-5 ). MS (API-
ES): Calcd for [C H N SO ]: m/z 495.6430. Found: m/z
0
0
0
1
6.8, 25.4, 23.7 (C-2 , C-3 , C-4 ). H NMR: d 3.86 (m, 2H,
2
4
37
3
6
H-6a, H-6b), 3.49 (ddd, 1H, H-2), 3.37 (dd, 1H, J3,4 10.3 Hz,
0
494.40 [MꢀH].
13
Compound 4a: C NMR: d 172.5 (CO), 120.0 (C-6 ), 76.3,
71.2, 66.2, 58.0, 54.1, 52.0 (C-1, C-2, C-3, C-4, C-5, C-6),
0
H-4), 3.15 (dd, 1H, J2,3 8.8 Hz, H-3), 3.17 (t, 2H, H-6 ), 3.02
10.9 Hz, J1e,2 4.9 Hz, H-1e), 2.92 (t, 2H, H-
(
7
2
(
4
dd, 1H, J1
a,1e
0
0
0
0
0
0
0
), 2.88 (s, 6 H, Me N-aryl), 2.85–2.79 (m, 1H, H-1 ), 2.63–
0
.58 (m, 1H, H-1 ), 2.23 (dd, 1H, J1a,2 11.2 Hz, H-1a), 2.19
2
50.4 (C-1 ), 26.4, 25.2, 23.6, 21.6 (C-2 , C-3 , C-4 , C-5 ),
25 3 4
16.1 (COMe). MS (API-ES): Calcd for [C14H N O ]: m/z
0
0
m, 1H, H-5), 2.01 (t, 2H, H-5 ), 1.56–1.45 (m, 4H, H-3 , H-
0
299.3731. Found: m/z 298.20 [MꢀH].
0
13
Compound 4b: C NMR: d 172.4 (CO), 76.2, 71.6, 66.4,
58.6, 54.5, 52.2 (C-1, C-2, C-3, C-4, C-5, C-6), 50.8 (C-1 ),
), 1.30–1.20 (m, 2H, H-2 ). MS (API-ES): Calcd for
SO
0
0
[
C
26
H
40
N
4
7
]: m/z 552.6953. Found: m/z 552.60 [MꢀH].
1
3
0
0
0
0
0
Compound 2a: C NMR: d 119.9 (C-6 ), 73.0, 66.4, 66.3,
40.8 (C-6 ), 31.1, 27.1, 26.5, 24.4 (C-2 , C-3 , C-4 , C-5 ),
2.5 Hz, J6a,6b
,6a
0
1
6
6.2, 55.5, 54.5 (C-1, C-2, C-3, C-4, C-5, C-6), 52.8 (C-1 ),
21.6 (COMe). H NMR: d 3.90 (dd, 1H, J
5

2070
P. Greimel et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2067–2070
0
0
1
1
1.7 Hz, H-6a), 3.83 (dd, 1H, J5,6b 3.2 Hz, H-6b), 3.81 (m,
H, H-2), 3.40 (dd, 1H, J3,4 9.3 Hz, J4,5 9.0 Hz, H-4), 3.21
H-4 , H-5 ), 1.12 (d, 3H, H-6). MS (API-ES): Calcd for
SO
]: m/z 479.6436. Found: m/z 478.4 [MꢀH].
Compound 7a: C NMR: d 119.9 (C-6 ), 71.8, 69.3, (C-3,
[C24
H
37
N
3
5
1
3
00
(
1
dd, 1H, J2,3 10.0 Hz, H-3), 3.03 (dd, 1H, J
0
4.6 Hz, J
0
1e,2
1a,1e
0 00
C-4) 59.7, 56.8, 55.5, 53.6, 41.9 (C-2, C-6, C-5, C-5 , C-1 ),
25.6, 24.8, 23.7, 16.0 (C-2 , C-3 , C-4 , C-5 ). H NMR: d
3.83 (dd, 1H, J5
1H, H-3), 3.70 (dd, 1H, J
1.2 Hz, H-1e), 2.83 (m, 1H, H-1 a), 2.72 (t, 2H, H-6 ), 2.54
0
00 00 00 00
1
(m, 1H, H-1 b), 2.13 (m, 1H, H-5), 2.09 (dd, 1H, J1a,2 11 Hz,
0
H-1a), 1.97 (s, 3H, COMe), 1.57–1.26 (m, 8H, H-2 , H-3 , H-
0
0
0
11:2 Hz, H-5 a), 3.76 (ddd,
5 a;5 b
0
6:6 Hz, J
0
;5 a
0
0
0
0
3:4 Hz, H-5 b), 3.57–3.45 (m,
5;5 b
4
3
, H-5 ). MS (API-ES): Calcd for [C H N O ]: m/z
4
1
4
29
3
03.4049. Found: m/z 302.3 [MꢀH].
2H, H-2e, H-6e), 3.41 (dd, 1H, J3,4 9.5 Hz, J4,5 9.2 Hz, H-4),
00
1
3
Compound 4c: C NMR: d 172.4 (CO), 76.5, 71.5, 66.2,
3.11 (m, 2H, H-1 ), 2.87 (dd, 1H, J5,6a 11.7 Hz, J6a,6e
0
5
4
3
8.4, 54.4, 52.2 (C-1, C-2, C-3, C-4, C-5, C-6), 50.7 (C-1 ),
0
12.7 Hz, H-6a), 2.75 (dd, 1H, J
00
= J2a,3 11.5 Hz, H-2a),
a,2e
2
0
4.7 (Me N-aryl), 42.6 (C-6 ), 29.3, 26.7, 26.1, 24.1 (C-2 , C-
2
2.52 (t, 2H, H-5 ), 1.99 (m, 1H, H-5), 1.86–1.45 (m, 6H, H-
00
0
0
0
1
00
00
, C-4 , C-5 ), 21.6 (COMe). H NMR: d 3.86–3.78 (m, 3H,
22 2 3
2 , H-3 , H-4 ). MS (API-ES): Calcd for [C12H N O ]: m/z
H-2, H-6a, H-6b), 3.39 (dd, 1H, J3,4 = J4,5 9.3 Hz, H-4), 3.21
(
242.3207. Found: m/z 241.2 [8MꢀH].
1
3
dd, 1H, J2,3 9.8 Hz, H-3), 2.97 (dd, 1H, J1e,2 4.4 Hz, J
Compound 7b: C NMR: d 74.9, 71.9 (C-3, C-4), 61.5, 58.6,
1a,1e
0
0 00 00
58.2, 55.3, 43.8 (C-2, C-5, C-5 , C-6, C-1 ), 41.0 (C-6 ), 27.3,
1
2
2
1
1.2 Hz, H-1e), 2.89 (s, 6H, Me
0
2
N-aryl), 2.84 (t, 2H, H-1 ),
0
00 00 00 00
1
.68 (m, 1H, H-6 a), 2.43 (m, 1H, H-6 b), 2.12 (m, 1H, H-5),
.07 (dd, 1H, J1a,2 11.2 Hz, H-1a), 1.98 (s, 3H, COMe),
26.6, 26.5, 26.4 (C-2 , C-3 , C-4 , C-5 ). H NMR: d 3.83
0
0
11:2 Hz, H-5 b), 3.53 (dd, 1H,
5 a;5 b
(dd, 1H, J5
J5
0
3:9 Hz, J
0
;5 b
0
0
0
0
0
0
7 Hz, H-5 a), 3.52 (m, 1H, H-3), 3.09 (dd, 1H, J
;5 a
3,4
.34–0.65 (m, 8H, H-2 , H-3 , H-4 , H-5 ). MS (API-ES):
S]: m/z 536.696. Found: m/z 535.5
MꢀH].For compounds 5a and b, see H a¨ usler, H.; Rupitz,
K.; St u¨ tz, A. E.; Withers, S. G. Chem. Mon. 2002, 133, 555;
Calcd for [C26
[
H
40
N
4
O
6
9.2 Hz, J4,5 9.8 Hz, H-4), 3.04 (m, 2H, H-2a, H-6e), 2.68 (br
00 00
s, 2H, H-1 ), 2.39 (m, 2H, H-2a, H-6a), 1.84 (m, 2H, H-6 ),
0
0
00
00
1.75 (m, 1H, H-5), 1.58–1.30 (m, 8H, H-2 , H-3 , H-4 , H-
00
1
3
compound 5c: C NMR: d 79.2 (C-3), 70.2 (C-2, C-4), 58.3
5 ). MS (API-ES): Calcd for [C H N O ]: m/z 246.3526.
1
2
26
2
3
0
0
0
13
Found: m/z 245.3 [MꢀH].Compound 7c: C NMR: d 74.9,
(
2
C-1, C-5), 57.6 (C-1 ), 44.7 (Me
0
2
0
N-aryl), 42.5 (C-6 ), 29.2,
0
1
0 00
6.7, 26.3, 26.1 (C-2 , C-3 , C-4 , C-5 ). H NMR: d 3.23
71.9 (C-3, C-4), 61.5, 58.8, 58.0, 55.4 (C-2, C-5 , C-6, C-1 ),
00
44.7 (Me N-aryl), 43.8, 42.5 (C-5, C-6 ), 29.2, 26.8,
2
(
ddd, 2H, J2,3 = J3,4 8.8 Hz, H-2, H-4), 2.84 (dd, 1H, H-3),
.68 (m, 2H, J1e,2 = J4,5e 4.7 Hz, H-1e, H-5e), 2.64 (s, 6H,
Me N-aryl), 2.60 (dd, 2H, J1a,2 = J4,5a 10.3 Hz, J1a,1e = J5a,5e
1.0 Hz, H-1a, H-5a), 2.01 (t, 2H, H-1 ), 1.70–0.77 (m, 8H,
00 00 00 00
1
2
26.7, 24.1 (C-2 , C-3 , C-4 , C-5 ). H NMR: d 3.82 (dd,
0
0
10:7 Hz, H-5 b), 3.52 (dd, 1H,
5 a;5 b
1H, J5
0
3:7 Hz, J
0
2
;5 b
0
0
0
7:0 Hz, H-5 a), 3.49 (m, 1H, H-3), 3.08 (dd, 1H, J3,4
;5 a
1
J5
0
0
0
0
H-2 , H-3 , H-4 , H-5 ). MS (API-ES): Calcd for
]: m/z 242.3207. Found: m/z 241.3 [MꢀH].
9.0 Hz, J4,5 10.0 Hz, H-4), 2.97 (m, 2H, H-2e, H-6e), 2.90 (s,
0
0
[C
12
H
22
N
2
O
3
6H, Me
6a), 1.97 (m, 2H, H-6 ), 1.73 (m, 1H, H-5), 1.36–1.02 (m,
2
N-aryl), 2.86 (t, 2H, H-1 ), 2.23 (m, 2H, H-2a, H-
00
1
3
0
Compound 6a: C NMR: d 120.0 (C-6 ), 73.9, 71.8, 65.7,
0
00
00
00
00
6
2
J
3
1
3.0 60.5 (C-1, C-2, C-3, C-4, C-5), 52.5 (C-1 ), 25.7, 24.9 (C-
0
8H, H-2 , H-3 , H-4 , H-5 ). MS (API-ES): Calcd for
0
0
0
1
, C-3 , C-4 , C-5 ), 16.1 (C-6). H NMR: d 4.01 (ddd, 1H,
[C H N SO ]: m/z 479.6436. Found: m/z 478.5 [MꢀH].
2
4
37
3
5
1a,2 = J2,3 10 Hz, J1e,2 4.6 Hz, H-2), 3.90 (br s, 1H, H-4),
.53 (m, 1H, H-3), 3.40–3.32 (m, 2H, H-1e, H-5), 3.14 (m,
H, H-6 a), 3.05 (m, 1H, H-6 b), 2.83 (dd, 1H, H-1a), 2.51
7. Assays were conducted as previously published: b-glucosi-
dase: Namchuk, M. N.; Withers, S. G. Biochemistry 1995,
34, 16194; a-mannosidase: Withers, S. G.; Rupitz, K.;
Street, I. P. J. Biol. Chem. 1988, 263, 7929; b-N-acetylhex-
osaminidase: Mark, B. L.; Vocadlo, D. J.; Zhao, D.;
Knapp, S.; Withers, S. G.; James, M. N. G. J. Biol. Chem.
2001, 276, 42131; a-L-fucosidase: Sulzenbacher, G.; Bignon,
C.; Nishimura, T.; Tarling, C. A.; Withers, S. G.; Henrissat,
B.; Bourne, Y. J. Biol. Chem. 2004, 279, 13119; b-
xylosidase: H a¨ usler, H.; Rupitz, K.; St u¨ tz, A. E.; Withers,
S. G. Chem. Mon. 2002, 133, 555.
0
0
0
0
0
0
0
(
1
m, 2H, H-1 ), 1.80–1.46 (m, 8H, H-2 , H-3 , H-4 , H-5 ),
.40 (d, 3H, J5,6 6.6 Hz, H-6). MS (API-ES): Calcd for
]: m/z 242.3207. Found: m/z 241.20 [MꢀH].
12 22 2 3
[C H N O
1
3
Compound 6c: C NMR: d 76.4, 73.9, 68.1, 58.3, 56.9, 52.6
0 0
C-1, C-2, C-3, C-4, C-5, C-1 ), 42.5 (C-6 ), 29.6, 26.9, 26.2,
(
2
0 0 0 0
1
3.3 (C-2 , C-3 , C-4 , C-5 ), 15.6 (C-6). H NMR: d 3.75
(
ddd, 1H, J1a,2 = J2,3 10.3 Hz, J1e,2 4.9 Hz, H-2), 3.60 (dd,
1
H, J3,4 2.9 Hz, J4,5 1.4 Hz, H-4), 3.20 (dd, 1H, H-3), 2.89
0
(
2
m, 1H, H-1e), 2.88 (s, 6H, Me
0
2
N-aryl), 2.85 (t, 2H, H-1 ),
0
8. Lundt, I.; Steiner, A. J.; St u¨ tz, A. E.; Tarling, C. A.; Ully,
S.; Withers, S. G.; Wrodnigg T. M. Bioorg. Med. Chem.
2006, 14, in press.
.49 (m, 1H, H-6 a), 2.35–2.24 (m, 2H, H-5, H-6 b), 1.99
0
0
(
dd, 1H, J1a,1e 10.7 Hz, H-1a), 1.34–0.94 (m, 8H, H-2 , H-3 ,