Glycosidase inhibitors of gem-diamine 1-N-iminosugars: Structures in media of enzyme assays

Article abstract of DOI:10.1016/S0968-0896(00)00330-8

The relationships between structures and inhibitory activities of glycosidase inhibitors of gem-diamine 1-N-iminosugars in media of enzyme assays have been investigated. It has been proved that gem-diamine 1-N-iminosugar smoothly undergoes a structural change to a hydrated ketone or its derivative via a hemiaminal in the media (pH 5.0-6.3), and that the products generated in the media as well as the parent gem-diamine 1-N-iminosugars potently inhibit glycosidases.

Full text of DOI:10.1016/S0968-0896(00)00330-8

Bioorganic & Medicinal Chemistry 9 (2001) 1091±1095
Glycosidase Inhibitors of gem-Diamine 1-N-iminosugars:
Structures in Media of Enzyme Assays
Ken-ichiro Kondo, Hayamitsu Adachi, Eiki Shitara, Fukiko Kojima and
Yoshio Nishimura*
Institute of Microbial Chemistry, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
Received 27 September 2000; accepted 21 November 2000
AbstractÐThe relationships between structures and inhibitory activities of glycosidase inhibitors of gem-diamine 1-N-iminosugars
in media of enzyme assays have been investigated. It has been proved that gem-diamine 1-N-iminosugar smoothly undergoes a
structural change to a hydrated ketone or its derivative via a hemiaminal in the media (pH 5.0±6.3), and that the products generated
in the media as well as the parent gem-diamine 1-N-iminosugars potently inhibit glycosidases. # 2001 Elsevier Science Ltd. All
rights reserved.
Introduction
examined the time-course alteration of structures and
1
activities in media by H NMR experiments and eva-
luation of the corresponding activities against enzymes.
Glycosidase inhibitors are tools for unraveling the
mechanism of action of glycosidases, and are of ther-
We chose the inhibitors of d-galacturonic acid-types
1
4b,5b
5h,6
apeutic and biotechnological relevance. To this end,
various types of natural and synthetic inhibitors have
(4±6)
and l-fucose-types (7±9)
as the typical
inhibitors of gem-diamine 1-N-imino-d- and l-sugars
(2) for reasons of the potent competitive activities and
di€erent stabilities in media. Figure 4 shows a time-
course alteration of the structure of the 2-tri¯uoro-
acetamide 4, a strong inhibitor of b-glucuronidase in
2
been developed in the last decade. Our interest directed
toward glycosidase inhibitor-based therapeutics mod-
3
eled on natural siastatin B (1) has led to a new class of
glycosidase inhibitor, gem-diamine 1-N-iminosugars
4
ꢀ
(
2), in which an anomeric carbon atom is replaced by a
acetate bu€er (pH 5.0) at 37 C used for the enzyme
nitrogen, and of which protonated form may mimic
hexopyranosyl cation (3) (Fig. 2). gem-Diamine 1-N-
iminosugars have proved to be potent and speci®c gly-
cosidase inhibitors.4 In the course of our study for the
mechanism of action of 2 against glycosidases, it has
become apparent that 2 changes the structures depen-
dently on pH value of solution. In this paper, we report
the results of the structural changes of 2 in the media of
enzyme assays and the inhibitory activities of the pro-
ducts generated in the media against enzymes.
assay. As shown in Figure 4, 4 has been proved to
change its structure time-dependently to the unknown
compounds (10 and 11) in the medium. Compound 4
has also shown the pH-dependent conversion of struc-
ture to 10 and 11, while 4 is quite stable for several
weeks in hydrochloric acid (pH<1). Next, we under-
took the isolation of compounds 10 and 11. After
adjustment of pH value of the aqueous solution of the
hydrochloride of 4 to 5±6 by the weak basic resin,
Dowex WGR, evaporation of the solvent gave the pure
,5
10. The large coupling constants (J2ax,3=9.2 and
J5,6=9.8 Hz) and the small coupling constants
1
(
clearly indicative of the same con®guration and C -
conformation as those of 4. The C NMR spectrum
of 10 shows the carbons at d 38.74 (C-6), 43.50 (C-5),
J3,4=J4,5=2.7 Hz) in H NMR spectrum of 10 are
4
Results and Discussion
1
4
b
13
In order to clarify the relationships between structures
and inhibitory activities in media of enzyme assays, we
68.99 (C-4), 71.50 (C-3), 173.41 (COOH) and a char-
acteristic carbon at low-®eld of d 79.14 (C-2) assigned to
7
a hemiaminal carbon instead of a carbon at d 60.71 (C-
2) assigned to a methanediamine carbon shown in that
*
3
Corresponding author. Tel.: +81-3-3441-4173, ext. 383; fax: +81-3-
441-7589; e-mail: mcrf@bikaken.or.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00330-8

1092
K. Kondo et al. / Bioorg. Med. Chem. 9 (2001) 1091±1095
of 4. This low-®eld chemical shift of C-2 is in accordance
8
with those of hemiaminal carbons of nojirimycin and
9
galactostatin. The spectrum also shows no carbons at
around d 115.85 and 160.52 corresponding to the tri-
¯
uoroacetamido groups. These results are indicative of
the hemiaminal as the structure of 10 shown in Scheme
. The mass spectra of 10 show m/e 178, 178 and 176 in
1
FAB (positive), ESI (positive) and ESI (negative)
modes, respectively, indicative of m/e 177 as a molecular
ion of 10 and clearly supportive of the hemiaminal of 10
Figure 1. Structure of siastatin B (1).
(
MW=177). On the other hand, adjustment of pH of
the aqueous solution of the hydrochloride of 4 to pH 7±
by Dowex WGR a€orded the another product 11.
8
Compound 11 was also stable in an acidic aqueous
1
solution. H NMR spectrum of 11 in D O shows the
2
methylene protons of the two aminomethyl groups at C-
6
(2H, ABq, J=12.4 Hz, d=2.98 and 3.05) and at C-2
(1H, t, J2,2
J2,2
0
=J2,3=12.2 Hz, d 3.06, H-2; 1H, dd,
0
=4.4 Hz, d 3.21, H-2 ), a proton at
0
=12.2 and J
0
2 ,3
C-3 (1H, ddd, J3,2=12.2 Hz, J3,2
0
=4.4 Hz and J3,4
.2 Hz, d 2.88), a proton at C-4 (1H, d, J4,3=1.2 Hz, d
.98) and no proton at C-5, suggesting a boat-con-
=
1
3
1
3
formational lactal 11 shown in Scheme 1. The C NMR
spectrum of 11 in H O shows the carbons at d 40.88 (C-2),
2
4
4.76 (C-3), 71.28 (C-4), 177.57 (ÀC(=O)ÀOÀ), and
the two characteristic carbons at high-®eld of d 47.26 (C-
6) and at low-®eld of d 92.47 (C-5) di€erent from those of
1
0, indicative of a lactal. The mass spectra of 11 of APCI
Figure 2. gem-Diamine 1-N-iminosugars 2 and glycopyranosyl cation
(
(positive) mode and APCI (negative) mode show m/e 160
3), the presumed transition state of enzymatic glycosidic hydrolysis.
and 158, respectively, supportive of a formula of the cyclic
lactal of 11 (MW=159). Moreover, the methylene pro-
tons at C-2 of 11 were deuterated when the above adjust-
ment of pH of the hydrochloride of 4 to pH 7±8 was
carried out in D O. Evaporation of D O solution of 11
2
2
also deuterated the methylene protons at C-2.
These results con®rmed the cyclic lactal as the structure of
11, and also indicate that a sequence of dehydration of the
aminal 10, enolization to the enaminal 12 and protonation
on C-6 of 12 would lead to the lactal 11 as shown in
Scheme 1. This dehydration is commonly observed in
Figure 3. gem-Diamine 1-N-iminosugars of d-galacturonic acid-types
4±6) and l-fucose-types (7±9).
(
Scheme 1. Hemiaminal 10, lactal 11, hydrated carboxylic acid 12 and
the putative enolate 13 generated from gem-diamine 1-N-iminosugar
of d-galacturonic acid-type 4 in medium of acetate bu€er (pH 5.0).
Figure 4. The time-course of alteration of 4 in 0.1 M acetate bu€er
ꢀ
(
pH 5.0) at 37 C.

K. Kondo et al. / Bioorg. Med. Chem. 9 (2001) 1091±1095
1093
many hemiaminals, and the lactone 11 can be envisaged as
the reaction product of a cyclic Amadori rearrangement
bu€er, pH 5.0). Therefore, IC50 value (0.023 mg/mL) of
6 against b-glucuronidase should not be its own value,
but the value of the aminal 10. This result also supports
that the aminal 10 itself is a strong inhibitor of b-glu-
curonidase. Next, we turned our attention to the
another gem-diamine 1-N-iminosugars of l-fucose-types
(7±9), the strong inhibitors of l-fucosidase. Figure 7
shows the time-course conversion of the structure of the
2-tri¯uoroacetamide 7 in citrate-phosphate bu€er (pH 6.3)
1
0
of 10. Next, we attempted to clarify which species of 4,
0 and 11 greatly inhibit b-glucuronidase in the medium.
1
Firstly, the respective inhibitory-activities of 4, 10 and 11
were assayed by the ordinary method of incubation of a
substrate and a test sample with an enzyme in acetate
ꢀ
bu€er (pH 5.0) at 37 C for 60 min. The IC values of 4,
5
0
1
9
0 and 11 against b-glucuronidase were elucidated as
À8
À7
ꢀ
.2Â10 M (0.025 mg/mL), 1.6Â10 M (0.029 mg/mL)
at 37 C used for enzyme assay. As shown in Figure 7, it has
À6
and 3.1Â10 M (0.5 mg/mL), respectively. Secondly, we
attempted to evaluate the dynamic inhibitory-activity
in a change of 4 on standing in the medium shown in
Figure 4. The inhibitory activities of the mixtures over
become apparent that 7 rapidly converts the structure into
the unknown compounds (14 and 15) in the medium. We
speculated that the structural changes of 7 in citrate-
phosphate bu€er (pH 6.3) proceeded via the similar
manner to those of the above gem-diamine 1-N-imino-
sugars of d-galacturonic acid-types (4, 5, 6) in acetate
bu€er (pH 5.0) to give the corresponding hemiaminal 14
2
0, 40 and 60 min in Figure 4 were selected as the typical
ones for this purpose. In order to estimate these activities,
the evaluation was carried out by the similar method
mentioned above after pre-incubation of 4 in the medium
for 20, 40 and 60 min. The IC values after pre-incubation
5
0
for 20, 40 and 60 min were 0.022, 0.033 and 0.33 mg/mL,
respectively, appearing to be the activities correspond-
ing to the mixture of 4:10:11=ca. 50:50:0, 28:65:7 and
1
5:70:15, respectively as shown in Figure 4. These results
indicate that the 2-tri¯uoroacetamide of gem-diamine 1-
N-iminosugar 4 and the hemiaminal 10 are the equally
potent inhibitors against b-glucuronidase, and that both
4
and 10 contribute greatly to overall inhibitory activity
in a change of 4 with the passage of time in the medium.
Interestingly, the cyclic lactal 11 shows moderate inhi-
bition against b-glucuronidase. It is likely that 11 may
be slightly hydrolyzed in the medium and may be in
equilibrium with the carboxylic acid 13. Next, we
examined a time-course conversion of the 2-trichloro-
acetamide analogue 5 in a medium of b-glucuronidase
assay. Compound 5 itself has also proved to be very
stable in a solution of strong acid (pH<1). As shown in
Figure 5, 5 is converted into the aminal 10 and the lactal
Figure 5. The time-course of alteration of 5 in 0.1 M acetate bu€er
ꢀ
(pH 5.0) at 37 C.
1
The inhibitory activity (IC ) of 5 was evaluated as
1 in a similar fashion as the alteration of 4 in Figure 4.
5
0
0.033 mg/mL, and IC50 values of the mixtures over 10,
20, 35, 60 and 90 min in Figure 5 were also elucidated to
0.033, 0.032, 0.036, 0.038 and 0.050 mg/mL, respectively.
These results are indicative of a strong inhibitory-activity
of the 2-trichloroacetamide 5 as well as the 2-tri-
¯
uoroacetamide 4. Compound 5 has proved to be more
Figure 6. The time-course of alteration of 6 in 0.1 M acetate bu€er
ꢀ
stable than 4 in the medium of acetate bu€er (pH 5.0).
These results also indicate that both functional groups
of 2-tri¯uoroacetamido and 2-trichloroacetamido assist
the strong binding of gem-diamine 1-N-iminosugars of
d-galacturonic acid-types to the active pocket of b-glu-
curonidase. We further examined the time-course trans-
formation of the structure of the other gem-diamine 1-
N-iminosugar, 2-phthalimide 6 in the medium. Compound
(pH 5.0) at 37 C.
6, in contrast to 4 and 5 has been ascertained to be
unstable even in a solution of strong acid (pH<1). The
1
H NMR spectrum showed the disappearance of 6 and
ꢀ
the appearance of the aminal 10 for 3 h at 24 C in
hydrochloric acid (pHꢁ1). Interestingly, the hydrochloride
of 6 in methanol converted into 10 within 10 min at
ꢀ
2
4 C. Figure 6 shows the time-course conversion of 6
in the medium of enzyme assay (pH 5.0). As expected,
proved to be very unstable in the medium (acetate
Figure 7. The time-course of alteration of 7 in 0.025 M citrate-phos-
ꢀ
6
phate bu€er (pH 6.3) at 37 C.

1094
K. Kondo et al. / Bioorg. Med. Chem. 9 (2001) 1091±1095
1
and hydrated ketone 15. The H NMR spectrum of
dilute solution (1 mg/mL) of 7 in D O also shows a
l-fucosidase inhibitors of gem-diamine 1-N-imino-
sugars, we attempted again to reveal which species sig-
ni®cantly inhibit the enzyme in a change with the passage of
time in the medium. It is already known that the 2-tri-
¯uoroacetamide 7, 2-trichloroacetamide 8 and 2-phtha-
2
mixture of 14 and 15 in a ratio of 3:1 after standing for
2
days at room temperature. Next, we analyzed the
1
13
structure of 14 utilizing this H NMR and its C NMR
spectra. The large coupling constants (J2,3=9.3 and
J5,6ax=12.7 Hz) and the small coupling constants
limide 9 inhibit very potently l-fucosidase as K and
i
À9
5h
IC values of 5Â10 M and 3 ng/mL, respectively,
50
1
ꢀ
(J3,4=J4,5=2.9 and J5,6eq=4.4 Hz) in the H NMR
spectrum indicate clearly the same con®guration and
C -conformation as those of 7. The C NMR
4
spectrum shows the carbons at d 13.67 (CH ), 32.45 (C-
), 43.37 (C-5), 71.61 (C-4), 72.15 (C-3) and a char-
acteristic carbon at low-®eld of d 78.85 (C-2) assigned to
an aminal carbon instead of a carbon at d 61.99
assigned to a methanediamine carbon shown in that of
assayed by the usual manner at 37 C. However, as the
structural change of 7 in the medium proceeds rapidly
as shown in Figure 7, so it has become apparent that the
usual method provides only poorly characterized species
which inhibit the enzyme. Therefore, we examined the
relationshipbetween the structure and the inhibitory
activity in the medium at low temperature, utilizing 7 as
a representative fucosidase-inhibitor. Figure 8 shows the
time-course conversion of 7 into 14 and 15 in citrate-
1
5h,6
13
3
6
7À9
7
. The spectrum also shows no carbons at around d
15.53 and 160.30 corresponding to the tri¯uoroacet-
ꢀ
1
amide group. These results indicate a cyclic aminal as
phosphate bu€er (pH 6.3) at 23 C, expressing the slow
structure-change. Firstly, the fucosidase inhibition assay
the structure of 14 shown in Scheme 2, and conse-
5
was carried out by incubation of a substrate and 7 with an
enzyme in citrate-phosphate bu€er (pH 6.3) at 23 C for
h
ꢀ
60 min. The IC50 value was elucidated as 5 ng/mL, indi-
quently, imply that our previous wrong conclusion of
generation of a cyclic methanediamine in the media of
enzyme assay should be revised. On the other hand, the
cative of that of 7 itself as shown in Figure 8. Secondly,
the inhibitory activity (IC ) of the mixture of 7, 14 and
1
H NMR spectrum of 7 in a D O solution of citrate-
2
50
phosphate bu€er (pH 6.3) shows only 15 on standing
for 30 min at room temperature as shown in Figure 7.
15 (about 1:1:1) was evaluated as 4 ng/mL by the similar
method after pre-incubation of 7 in the medium at 23 C
ꢀ
for 60 min as shown in Figure 8. Thirdly, the inhibitory
1
13
Therefore, we used this H NMR and its C NMR
1
spectra for analysis of the structure of 15. The H NMR
spectrum shows protons of C(2), C(4), C(5), C(6) and
activity (IC ) of the hydrated-ketone 15 was deter-
5
0
ꢀ
pre-incubation at 37 C for 40 min as shown in Fig. 7.
mined as 14 ng/mL by the assay at 23 C for 60 min after
ꢀ
CH at d 3.17 and 3.15 (total 1H, s each), 3.66 (1H, d,
3
J4,5=2.4 Hz), 2.36 (1H, m), 3.12 (1H, dd, J5,6eq=4.9
and J6ax,6eq=12.7 Hz) and 2.85 (1H, t, J5,6ax=J6ax,6eq=
On the whole, these results indicate that 7, 14 and 15
very strongly inhibit l-fucosidase, and that the hemi-
aminal 14 of these is proved to be the strongest inhibitor
against the enzyme. These results also suggest that a
hydroxyl group at C-2 play an important role for the
interaction of active-site residues. It is known that a
hydroxyl groupat C-2 stabilize the transition state of
enzyme-catalyzed glycoside hydrolysis by the interac-
1
shows no proton corresponding to C(3), indicative of a
2.7 Hz) and 1.03 (3H, d, J=6.8 Hz), respectively, and
1
C -conformational hydrated-ketone 15 bearing deuter-
4
ated methylene protons shown in Scheme 2. On the
1
3
other hand, the C NMR spectrum shows the carbons
at 14.10 (CH ), 30.80 (C-5), 43.96 (C-6), 72.57 (C-4),
3
À1 11
and the two characteristic carbons at low-®eld of d
tion with the catalytic nucleophile (30±40 kJ mol ).
9
2.30 (C-3) assigned to a hydrated-ketone and at high-
®
eld of d 46.1±46.9 (m) (C-2) assigned to a deuterated
In summary, glycosidase inhibitors of gem-diamine 1-N-
iminosugars change the structures pH-dependently to
the hydrated-ketones or their derivatives via the aminals
in the media of enzyme assay. The products generated in
the media as well as the parent gem-diamine 1-N-imino-
sugars are shown to have the potent inhibition against gly-
cosidases. That these gem-diamine 1-N-iminosugars are
potent inhibitors of glycosidases further supports the
hypothesis of our design of the new type inhibitors.
methylene-carbon, clearly supportive of a hydrated-
ketone 15. This result is accordance with the structural
alteration of 4 in the media mentioned above. In the
Scheme 2. Hemiaminal 14, and keto-hydrate 15 generated from gem-
diamine 1-N-iminosugar of l-fucose type 8 in medium of citrate-
phosphate bu€er (pH 6.3).
Figure 8. The time-course of alteration of 7 in 0.025 M citrate-phos-
ꢀ
phate bu€er (pH 6.3) at 23 C.

K. Kondo et al. / Bioorg. Med. Chem. 9 (2001) 1091±1095
1095
Experimental
was measured at 405 nm. The IC50 value was elucidated
by the same way as that of d-glucuronidase inhibition
assay.
General methods
1H NMR and 13C NMR spectra were recorded with
JEOL GX-400 and a-500 spectrometers. Chemical shifts
are expressed in d values (ppm) with CD HOD (3.30)
2
References
for CD OD and with HDO (4.65) for D O as an internal
3
2
standard. The mass spectra were taken by JEOL SX102
for FAB and by Hitachi M-1200H for APCI (atmo-
spheric pressure chemical ionization).
1
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4
b,5b,5h,6
viously developed by us.
glucuronidase (bovine kidney) and a-l-fucosidase
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2
9, 340. (d) Heightman, T. D.; Vasella, A. T. Angew. Chem.,
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(
mono-b-glucuronic acid and p-nitrophenyl a-l-fuco-
pyranoside were purchased from Sigma Chemical Co.
3
4
General procedures for D-glucuronidase inhibition assay
ꢀ
7
at 37 C
5
b-d-Glucuronidase inhibition was assayed using phenol-
À4
Azetaka, M.; Fukuyasu, H.; Iizuka, Y.; Shibahara, S. J. Anti-
biot 1994, 47, 840. (b) Satoh, T.; Nishimura, Y.; Kondo, S.;
Takeuchi, T.; Azetaka, M.; Fukuyasu, H.; Iizuka, Y.; Ohuchi,
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phthalein mono-b-glucuronic acid (3.3Â10 M) as a
substrate at pH 5.0 (0.1M acetate bu€er). The reaction
mixture contained 0.075 mL of bu€er, 0.01 mL of sub-
strate solution and 0.05 mL of water or aqueous solution
containing the test compound. The mixture was incu-
ꢀ
bated at 37 C, and 0.015 mL of enzyme was added.
After 60 min of reaction, 0.15 mL of 0.6M glycine-
sodium hydroxide bu€er (pH 10.5) was added and the
absorbance of the liberated phenolphthalein was mea-
sured at 570 nm. The percentage inhibition was calculated
by the formula (AÀB)/AÂ100, where A is the phenol-
phtalein liberated by the enzyme without an inhibitor
and B is that with an inhibitor. The IC50 value is the
concentration of inhibitor at 50% of enzyme activity.
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23 C
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at pH 6.3 (0.025 M citrate-phosphate bu€er). The reac-
tion mixture contained 0.05 mL of bu€er, 0.03 mL of
substrate solution and 0.095 mL of water or aqueous
solution containing the test compound. The mixture
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1
1. (a) White, A.; Tull, D.; Johns, K.; Withers, S. G.; Rose,
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ꢀ
was incubated at 23 C for 3 min, and 0.025 mL of
enzyme was added. After 60 min of reaction, 0.1 mL of
0.6 M glycine-sodium hydroxide bu€er (pH 10.5) was
added and the absorbance of the liberated p-nitrophenol