Glycosidase inhibition by novel guanidinium and urea iminosugar derivatives

Article abstract of DOI:10.1039/c2md20343j

Novel guanidinium and urea derivatives of 1-deoxynojirimycin were prepared using a concise synthetic protocol and tested against a panel of glycosidases for their inhibitory properties. Potent and selective inhibition was observed with both classes of compounds. Further investigation involving an expanded series of N^G-substituted guanidinium deoxynojirimycin analogues revealed distinct inhibitory profiles in the inhibition of the glycosidases tested. The Royal Society of Chemistry.

Full text of DOI:10.1039/c2md20343j

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Medicinal Chemistry Communications
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O
OH
R
N
N
H
HO
HO
NG-substituted
urea derivatives
OBn
NH
OH
BnO
BnO
Novel glycosidase
inhibitors
OBn
protected
deoxynojirimycin
NH
OH
R
N
N
H
HO
HO
NG-substituted
OH
guanidinium derivatives
Novel guanidinium and urea derivatives of 1-deoxynojirimycin (DNJ) were prepared using a concise
synthetic protocol. These DNJ derivatives exhibited potent and selective inhibition against a panel of
glycosidase enzymes.

Medicinal Chemistry Communications
Page 2 of 8
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Journal Name
Cite this: DOI: 10.1039/c0xx00000x
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ARTICLE TYPE
Glycosidase inhibition by novel guanidinium and urea iminosugar
derivatives
Raymond Kooij, Hilbert M. Branderhorst, Simon Bonte, Sara Wieclawska, Nathaniel I. Martin* and
Roland J. Pieters*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Novel guanidinium and urea derivatives of 1-deoxynojirimycin were prepared using a concise synthetic
protocol and tested against a panel of glycosidases for their inhibitory properties. Potent and selective
inhibition was observed with both classes of compounds. Further investigation involving an expanded
10 series of NG-substituted guanidinium deoxynojirimycin analogues revealed distinct inhibitory profiles in
the inhibition of the glycosidases tested.
Introduction
The potent and selective inhibition of glycosidases is an
15 important goal for the development of therapeutics.1 Glycosidases
cleave the glycosidic bonds in oligosaccharides and
compounds were also limited by the lack of charge
complementarity. Compounds with both an sp2 hybridized C-1
and an endocyclic nitrogen that is part of an amidine functional
glycoconjugates. Their blocking can aid in the treatment of 45 group as in 3, showed good activities, although poor selectivity.
diabetes,2 viral infections,3 lysosomal storage diseases,4 and
cancer.5 Iminosugars have been shown to be highly effective
20 inhibitors but their limited selectivity can lead to side effects
Based on comparisons with less basic derivatives it was
concluded that the flattened conformation was more important
than the charge.14 This notion was supported by weakly basic
inhibitors with a flattened ring such as 4.15
7
when applied therapeutically.6, It is clear that improving the
selectivity of iminosugars as glycosidase inhibitors is an
50
Nojirimycin derivatives in which only the endocyclic nitrogen
was (partially) sp2 hybridized have also been reported.16-18 In
these compounds the endocylic nitrogen is bridged to the C-6 as
in 5 by a guanidinium or a related linkage. Compounds of this
type have shown good inhibitory properties and tuneable
8-12
important goal.6,
The effectiveness of the iminosugar as a
glycosidase inhibitor in many cases depends on its ability to
25 mimic the relevant transition state in the cleavage process.8
Considering the vast rate enhancements of glycosidases, these
enzymes bind the transition state with very high affinity. 55 selectivities as a function of the hydroxyl configurations around
Consequently, a transition state mimic has the potential to be a
very strong inhibitor. The mimicry depends on the
30 complementarity with respect to charge and shape. These issues
are intimately linked to the hybridization state of the ‘anomeric
the ring. Our own recently developed synthetic methodology to
conveniently transform amines into guanidino groups,19-21
enables access to unconstrained derivatives of deoxynojirimycin.
Evaluation of such compounds would delineate the effects of
carbon’ and the endocyclic oxygen or in case of derivatives of 60 conformation and charge delocalisation due to the introduction of
deoxynojirimycin 1 (Figure 1) the nitrogen. These atoms should
have a considerable sp2 character. We here focused on the
35 hybridization aspect by straightforward derivatisation of the
nitrogen of deoxynojirimycin 1 to explore new structural motifs
for glycosidase inhibition.
the guanidinium or its neutral urea counterpart, on glycosidase
OH
OH
OH
NH
O
N
HO
HO
HO
HO
HO
HO
Previous investigations employing sp2 hybridized inhibitors
have yielded promising results. Inhibitors with an sp2 hybridized
40 C-1 such as D-gluconolactone 2,13 showed activity indicating the
NH2
O
OH
OH
OH
1
2
3
H
N
OH
N
importance of
a somewhat flattened ring, although these
N
N
N
HO
HO
HO
HO
N
*Department of Medicinal Chemistry & Chemical Biology, Utrecht
Institute for Pharmaceutical Sciences, University of Utrecht, David de
Wied Building, Office: 5.64, Universiteitsweg 99, 3584 CG Utrecht, The
Netherlands. E-mail: n.i.martin@uu.nl, r.j.pieters@uu.nl
† Electronic Supplementary Information (ESI) available: Experimental
protocols and product characterization. See DOI: 10.1039/b000000x/
OH
OH
N
OH
4
5
Figure 1. 1-Deoxynojirimycin (1) and a series of glycosidase inhibitors
with varying degrees of sp2 hybridization.
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 1

Page 3 of 8
Medicinal Chemistry Communications
Table 1. Glycosidase inhibition values obtained for deoxynojirimycin
derivatives.
inhibition potency and selectivity. Considering the cyclic
framework in which the nitrogen resides, the likely reluctance of
the nitrogen to adopt a full sp2 geometry would have an
unpredictable effect on its activity. Furthermore, in comparison
to 5, the free movement of the side chain could also lead to
additional preferences. Furthermore, as was shown for sialidase
inhibition, introducing guanidinium functions can enhance
potency.22
Enzyme
1
8
10
13
α-galactosidase
(green coffee beans)
β-galactosidase
(bovine liver)
23 ± 3
251 ± 38 >1000
18 ± 8
View Article Online
5
>1000
>1000
27 ± 2
15 ± 1
α-glucosidase
(baker’s yeast)
β-glucosidase
(almonds)
α-mannosidase
(Jack beans)
β-mannosidase
(Helix pomatia)
Naringinase
167 ± 27 134 ± 19 >1000
20 ± 10
41 ± 5
312 ± 62 59 ± 2
>1000
>1000
>1000
>1000
>1000
>1000
33 ± 4
>1000
>1000
6 ± 3
113 ± 11
408 ± 45
>1000
Results and discussion
10
Given the unique activities of the compounds described above,
we prepared a series of novel analogues of deoxynojirimycin 1
containing alkyl, urea and guanidino substituents at the endocylic
nitrogen (Scheme 1). To this end the protected deoxynojirimycin
(Penicilium decumbens)
30 IC50 values are reported in µM and are averages obtained from triplicate
analysis for each compound.
6
was alkylated by 1-bromohexane and deprotected by
15 hydrogenation to give 8. The synthesis of the urea counterpart 9
was accomplished by treatment of 623 with n-butyl isocyanate
followed by hydrogenolysis giving urea 10.
activities of the glycosidases of the appropriate p-nitrophenyl
35 glycoside substrate in the presence of a varying concentrations of
each iminosugar derivative.24 In this assay, the alkylated
compound 8 showed good inhibition of naringinase with an IC50
of 6 µM. This compound also showed inhibition of β-glucosidase
and α-galactosidase, with IC50 values of 59 and 251 µM
40 respectively. The neutral urea derivative 10 displayed selective
inhibition of β-galactosidase with an IC50 of 27 µM (Figure 2),
while none of the other glycosidases were inhibited by this
compound. Incorporation of the guanidinium group in 13 proved
to have a significant effect. With the exception of naringinase, 13
45 displayed more potent inhibition of the glycosidases tested
relative to the alkylated analogue 8. Of particular note is the
observation that both α- and β-galactosidase are effectively
inhibited by 13 with IC50 values in the low micromolar range.
While the potencies of the guanidinium derivative 13 were
50 promising, structural variation of the guanidinium substituent was
introduced to study the influence on selectivity. The series of
derivatives 14-19 (Figure 3), were synthesized using the same
method as for 13 and include alkyl, aryl hydrophilic groups along
The inhibitory activities of 8, 10 and 13 towards a broad panel
of 7 glycosidases were determined. The panel consisted of an α-
20 glucosidase, β-glucosidase, α-galactosidase, β-galactosidase, α-
mannosidase, β-mannosidase and a naringinase. The IC50 values
are summarized in Table 1. Deoxynojirimycin 1 was employed as
a reference compound. Inhibition assays were performed in
either phosphate or acetate buffer at the optimum pH for each
25 enzyme. Determination of IC50 values was carried out by
spectrophotometrically measuring the residual hydrolytic
OR
a, b
N
RO
RO
OR
7: R = Bn
8: R = H (HCl salt)
O
OR
OBn
NH
c, b
N
N
H
RO
RO
BnO
BnO
OR
OBn
6
9: R = Bn
10: R = H
R2
N
OR1
N
N
H
R1O
R1O
d, b
OR1
12: R1 = Bn, R2 = Cbz
13: R1,R2 = H (HCl salt)
S
e
Cbz
H2N
N
H
N
H
11
Scheme 1. Synthesis of N-alkylated, urea, guanidinium derivatives of
deoxynojirimycin 1. Reagents and conditions: (a) 6, 1-bromohexane,
K2CO3, DMF, 62%; (b) H2, Pd/C, EtOH, HCl (1M); (c) 6, n-butyl
isocyanate, dimethoxyethane, 70%; (d) 11, EDCI, NEt3, CH2Cl2, 64%;
(e) CbzNCS, CH2Cl2, 68%.
Figure 2. Representative data and fitted curves for the inhibition of β-
galactosidase (from bovine liver) by compounds 1, 8, 10, and 13 (for
complete inhibition data for all glycosidases tested see Supporting
Information section).
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Medicinal Chemistry Communications
Page 4 of 8
NH
NH
NH
NH
NH
NH
were able to inhibit β-galactosidase, while the non-guanidino
compounds 1 and 8 were not. Looking at the individual
compounds it is noticeable that a few compounds such as 14, 18
OH
N
N
H
HO
HO
OH
10 and 19 had a preference for one of the tested glycosidases,
14
15
16
17
18
19
View Article Online
indicating that the guanidino substituents can be used to direct the
selectivity.
OH
O
N
N
H
OH
HO
HO
Conclusions
OH
In summary, we have successfully devised a straightforward
15 route to a new class of iminosugar derivatives and investigated
their ability to inhibit a diverse set of glycosidases. First an
alkylated, a urea, and a guanidinium derivative were prepared and
their screening against a series of glycosidases showed striking
inhibition profiles. Interestingly, the neutral urea derivative 10
OH
OH
N
N
H
HO
HO
OH
20 proved to be
a very selective β-galactosidase inhibitor.
OH
Incorporation of the guanidine moiety in 13 led to enhanced
potency against several glycosidases. In order to introduce more
selectivity the arginine substituent was varied in a second series
of compounds. From this series it was shown that selective
25 compounds such as 14, 18 or 19 could be obtained. This further
indicates the promise of the guanidinium motif for the creation of
N
N
H
HO
HO
OH
OH
N
N
H
HO
HO
therapeutic glycosidase inhibitors.
incorporation seems to alter both the steric and electronic
properties of the iminosugar. Its charge is likely more
The guanidinium
OH
30 delocalized than in the case of the N-alkylated 8. Furthermore,
due to the degree of sp2 hybridization of the ring nitrogen, the
ring conformation is likely slightly altered. Taken together these
effects result in a potent inhibitory motif for a range of
OH
N
N
H
O
HO
HO
OH
glycosidases that can be introduced using
a concise and
Figure 3. NG-substituted guanidinium deoxynojirimycin analogues
prepared to examine the effect of various NG-substituents on glycoside
inhibtion activity (all HCl-salts).
35 straightforward synthetic approach. Future efforts will be aimed
at exploring the incorporation of the guanidine centre in other
iminosugar structures as a means of further enhancing both the
potency and selectivity of glycoside inhibition. In addition, future
investigations will explore the use of these guanidinium and urea
40 modified iminosugars for inhibiting enzymes of medicinal
interest. Targets may include trehalase enzymes for development
with the bulky adamantyl group. Evaluation of the series of
compounds as inhibitors of a panel of glycosidases revealed
interesting variations. For the α-galactosidase inhibition only the
saturated alkyl chain-containing 13 and 14 showed good
inhibition. The same was also true for α-glucosidase and to a
lesser extent for α-mannosidase. In contrast most compounds
of antifungal or antibiotic agents,25 α-glucosidases located in the
endoplasmic reticulum towards anti-cancer agents,17,
and
26
5
targets related to hepatitis,6 diabetes,6 and Gaucher disease.27
45
Table 2. Glycosidase inhibition values obtained for NG-substituted guanidinium deoxynojirimycin analogues.
Enzyme
1
13
14
15
16
17
18
19
α-galactosidase
(green coffee beans)
β-galactosidase
(bovine liver)
23 ± 3
18 ± 8
23 ± 8
>1000
540 ± 46 >1000
>1000
>1000
>1000
15 ± 1
2.6 ± 0.4 >1000
>1000
5.1 ± 0.3 6.2 ± 0.7 5.4 ± 0.4
α-glucosidase
(baker’s yeast)
β-glucosidase
(almonds)
α-mannosidase
(Jack beans)
167 ± 27 20 ± 10
312 ± 62 41 ± 5
31 ± 10
25 ± 2
251 ± 230 133 ± 34 >1000
>1000
457 ± 42 4.3 ± 0.2 67 ± 3
131 ± 23
489 ± 29
>1000
>1000
>1000
>1000
33 ± 4
113 ± 11 55 ± 14
>1000 67 ± 13
>1000
>1000
>1000
33 ± 6
Naringinase
(Penicilium decumbens)
172 ± 96 645 ±498 27 ± 3
22 ± 11
IC50 values are reported in µM and are averages obtained from triplicate analysis for each compound. For ease of
comparison, the IC50 values obtained for compounds 1 and 13 shown in Table 1 are again included here.
This journal is © The Royal Society of Chemistry [year]
Journal Name, [year], [vol], 00–00 | 3

Page 5 of 8
Medicinal Chemistry Communications
0.87 (t, CH3, J = 6.74 Hz, 3H), 1.11 – 1.40 (m, CH2, 8H), 2.19 –
2.31 (m, CH2NH, 2H), 2.51 – 2.68 (m, 2H), 3.09 (m, 1H), 3.43 –
3.70 (m, 5H), 4.41 – 4.98 (m, CH2Carom, 8H), 7.12 – 7.34 (m,
Experimental Section
60 CHarom, 20H). δC (75.5 MHz; CDCl3; Me4Si) 14.0 (CH3), 22.5,
General remarks
View Article Online
23.4, 27.1, 31.6 (CH2), 52.3, 54.4 (CH2NH), 72.6, 73.3, 75.1,
75.2 (CH2), 63.6, 78.5, 87.3 (C-2, C-3, C-4, C-5), 127.3, 127.4,
127.5, 127.7, 128.2 (CHarom), 137.7, 138.5, 139.0 (Cq). Rf = 0.38
Reagents, solvents and solutions. Unless stated otherwise,
chemicals were obtained from commercial sources and used
without further purification. All solvents were purchased from
Biosolve (Valkenswaard, The Netherlands) and were stored on
molecular sieves (4Å). 2,3,4,6-tetra-O-benzyl-D-glucopyranose
was obtained from Carbosynth Limited (MT06691). 2,3,4,6-tetra-
5
(EtOAc/hexane 1:5). HRMS Calcd for C40H49NO4 [M+H]+
,
65 608.3740, found 608.3729.
N-hexyl-1-deoxynojirimycin (8). A suspension of 7 (100 mg,
0.164 mmol) and Pd/C (10%) (100 mg) in EtOH (5 mL) was
adjusted to pH = 1 with aqueous HCl (1M). The reaction mixture
70 was stirred vigorously under a H2 (g) atmosphere for 18 h at rt.
The reaction mixture was next filtered over celite, washed with
10 O-benzyl-1-deoxynojirimycin 6,28 and Cbz-NCS19 were prepared
as previously described. The preparation of compounds 15, 16,
and 19 each required access to non-commercial amine building
blocks that were prepared according to established literature
procedures.29-31 28
EtOH and concentrated to dryness.
8 was obtained after
15
lyophilization as the HCl-salt (47 mg, quant.). δH (300 MHz;
D2O) 0.75 (br s, CH3, 3H), 1.20 (br m, CH2, 6H), 1.55 (br s, CH2,
75 2H), 2.73 – 2.84 (m, 2H), 2.95 (s, 1H), 3.06 (s, 1H), 3.32 (m, 2H),
3.46 (m, 1H), 3.51 (br s, 1H), 3.62 – 3.93 (m, 2H). δC (75.5 MHz;
D2O) 13.4 (CH3), 22.0, 22.6, 25.8, 30.7 (CH2), 52.9, 53.7, 55.0,
65.3, 67.0, 68.1, 76.7 (CH2NH), C-2, C-3, C-4, C-5, C-6). HRMS
Purification Techniques. All reactions and fractions from
column chromatography were monitored by thin layer
chromatography (TLC) using plates with a UV fluorescent
indicator (normal SiO2, Merck 60 F254). One or more of the
20 following methods were used for visualization: 10% H2SO4 in
MeOH or ninhydrine. Flash chromatography was performed
according to the method of Still et al. using Merck type 60, 230-
400 mesh silica gel. Removal of solvent was performed under
reduced pressure using a rotary evaporator.
for C12H25NO4 [M+H]+ 248.1862, found 248.1857.
,
80
N-1-butylurea-2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin (9).
To a solution of 1-DNJ (6) (200 mg, 0.38 mmol, 1 eq.) in
dimethoxyethane (3 mL) was added n-butyl isocyanate (86 µL,
0.76 mmol, 2 eq.). The resulting reaction mixture was refluxed
25
Analytical HPLC runs were performed on a Shimadzu automated
HPLC system with a reversed phase column (Alltima, C8, 90Å, 5
µm, 250x4.6 mm) equipped with an evaporative light scattering
detector (PL-ELS 1000, Polymer Laboratories) and a UV/VIS
30 detector operating at 22 and 254 nm. Elution was effected using a
gradient of 5% MeCN and 0.1% TFA in H2O to 5% H2O and
0.1% TFA in MeCN.
o
85 overnight at 90 C. The reaction mixture was warmed up to rt,
concentrated in vacuo and the product was isolated via flash
chromatography (SiO2, 2:5 EtOAc/hexane) to yield 9 (168 mg,
70%) as a colorless oil. δH (300 MHz; CDCl3; Me4Si) 0.82 (t,
CH3, J = 7.01 Hz, 3H), 1.17 – 1.30 (m, CH2, 4H), 3.06 – 3.14 (m,
90 CH2NH, 2H), 3.29 (dd, JA = 3.71 Hz, JB = 10.18 Hz, 1H), 3.53 –
3.76 (m, 5H), 3.92 (dd, JA = 4.68 Hz, JB = 9.35 Hz, 1H), 3.99 –
4.05 (m, 1H), 4.44 (d, CH2Carom, J = 2.75 Hz, 2H), 4.47 (s,
Instrumentation for Compound Characterization. 1H NMR
35 spectra were recorded at 300 MHz with chemical shifts reported
in parts per million (ppm) downfield relative to tetramethylsilane
(Me4Si) or H2O (δ 4.8). 1H NMR data are reported in the
following order: number of protons, multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet and m, multiplet) and coupling
40 constant (J) in Hertz (Hz). When appropriate, the multiplicity is
preceded by br, indicating that the signal was broad. 13C NMR
spectra were recorded at 75.5 MHz with chemical shifts reported
relative to CDCl3 δ 77.0. 13C NMR spectra were recorded using
the attached proton test (APT) sequence. All literature
CH2Carom), 1H), 4.52 (d, J = 2.75 Hz, 1H), 4.56 (s, CH2Carom
,
1H), 4.62 (s, CH2Carom, 1H), 4.70 and 4.74 (2 x d, CH2Carom, 2 x
95 1H), 5.47 (t, J = 5.23 Hz, 1H), 7.20 – 7.36 (m, CHarom, 20H). δC
(75.5 MHz; CDCl3; Me4Si) 13.7 (CH3), 19.9, 31.8 (CH2), 40.5,
40.6 (CH2NH), 70.8, 71.0, 72.8, 73.3 (CH2), 56.9, 75.8, 78.8,
82.1 (C-2, C-3, C-4, C-5), 127.6, 127.7, 127.9, 128.2 (CHarom),
137.4, 137.8, 137.9, 138.0 (Cq), 159.2 (NHC(O)). Rf = 0.40
100 (EtOAc/hexane 2:1). HRMS Calcd for C39H46N2O5 [M+H]+
,
623.3485, found 623.3471.
1
45 compounds had H NMR, and mass spectra consistent with the
N-1-butylurea-1-deoxynojirimycin (10). A suspension of 9 (168
mg, 0.27 mmol) and Pd/C (10%) (168 mg) in EtOH (5 mL) was
105 adjusted to pH = 1 with aqueous HCl (2M). The reaction mixture
was stirred vigorously under a H2 (g) atmosphere for 18 h at rt.
The reaction mixture was next filtered over celite, washed with
EtOH and concentrated in vacuo. 10 was obtained after
lyophilization as the HCl-salt (81 mg, quant.). δH (300 MHz;
110 D2O) 0.89 (t, CH3, 3H), 1.35 (m, CH2, 2H), 1.48 (m, CH2, 2H),
3.14 – 3.21 (m, CH2NH, 2H), 3.41 (d, J = 12.1 Hz, 1H), 3.61 (br
s, 1H), 3.72 – 3.86 (m, 6H). δC (75.5 MHz; D2O) 13.3 (CH3),
19.6, 31.6 (CH2), 40.4, 44.2 (CH2NH), 60.0, 68.7, 71.1, 74.0 (C-
2, C-3, C-4, C-5, C-6), 160.5 (NHC(O)). HRMS Calcd for
assigned structures.
Experimental Procedures and Data for Compounds 7-19
N-hexyl-2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin (7). To a
50 solution of 1-DNJ (6) (500 mg, 0.95 mmol) in DMF (5 mL) was
added K2CO3 (400 mg, 2.86 mmol, 3 eq.) and 1-bromohexane
(200 µL, 1.43 mmol, 1.5 eq.). The resulting reaction mixture was
refluxed overnight at 85 oC. The reaction mixture was warmed up
to rt, filtered, concentrated in vacuo and the product was isolated
55 via flash chromatography (SiO2, 1:5 EtOAc/hexane) to yield 7
(363 mg, 62%) of a colorless oil. δH (300 MHz; CDCl3; Me4Si)
This journal is © The Royal Society of Chemistry [year]
[journal], [year], [vol], 00–00 | 4

Medicinal Chemistry Communications
Page 6 of 8
C11H22N2O5 [M+H]+, 263.1607, 263.1583.
employed to generate the fully protected NG-substituted guanidine
60 modified DNJ derivatives. In this procedure, the primary amine
of interest was first converted to the corresponding thiourea by
N-(benzyloxycarbonyl) N’-(butyl) thiourea (11). A solution of
n-butylamine (309 µL, 3.12 mmol) in CH2Cl2 (10 mL) was
treated with a solution of Cbz-NCS (5 mL, 0.5M in CH2Cl2). The
resulting reaction mixture was stirred for 30 min at rt. The
treatment with an equimolar quantity Cbz-NCS. Formation of the
View Article Online
5
thiourea is generally complete within minutes and can be
followed via TLC. A two-fold excess of the thiourea was then
reaction mixture was concentrated in vacuo and the product was 65 directly activated with EDCI/NEt3 and treated with DNJ building
isolated via flash chromatography (SiO2, EtOAc/hexane 1:6) to
yield 11 (456 mg, 68%) as a white powder. δH (300 MHz; CDCl3;
10 Me4Si) 0.93 (t, CH3, 3H), 1.26 – 1.41 (m, CH2, 2H), 1.44 – 1.66
(m, CH2, 2H), 3.62 (q, NHCH2, 2H), 5.13 (s, CH2Carom, 2H), 7.26
block 6 to yield protected NG-substituted guanidine modified DNJ
derivatives 14a-19a. Global deprotection of the intermediate
protected compounds was achieved via hydrogenation over 10%
Pd/C in slightly acidic media to yield final products 14-19. As an
– 7.40 (m, CHarom, 5H), 8.80 (br s, NH, 1H), 9.66 (br s, NH, 1H). 70 example, the preparation of compounds 14a and 14 are provided
δC (75.5 MHz; CDCl3; Me4Si) 13.5 (CH3), 19.8, 30.0 (CH2), 45.1
(NHCH2), 67.6 (CH2Carom), 127.5, 127.8, 128.1, 128.4, 128.7,
15 128.8 (CHarom), 134.4 (Cq), 152.5 (NHC(S)NH), 178.7 (NHC(O)).
Rf = 0.55 (EtOAc/hexane 1:5). HRMS Calcd for C13H18N2O2S
[M+H]+, 267.1167, found 267.1155.
below.
N-(benzyloxycarbonyl) N’-(octyl) 2,3,4,6-tetra-O-benzyl-
deoxynojirimycin (14a). A solution of Octylamine (198 mL, 1.2
75 mmol) in CH2Cl2 (10 mL) was treated with a equimolar quantity
of Cbz-NCS (delivered as 2.4 mL of a 0.5 M solution in CH2Cl2).
The resulting reaction mixture was stirred for 30 min at RT at
which point TLC indicated complete conversion to the thiourea.
To the crude thiourea was added the protected DNJ building
N-(benzyloxycarbonyl) N’-(butyl) 2,3,4,6-tetra-O-benzyl-
20 deoxynojirimycin (12). Thiourea 11 (305 mg, 1.14 mmol, 2 eq.)
in CH2Cl2 (10 mL) was treated with 1-DNJ (6) (300 mg, 0.57
mmol, 1 eq.), EDCI (220 mg, 1.14 mmol, 2 eq.) and Et3N (160 80 block 6 (314 mg, 0.6 mmol, 1 eq.), EDCI (230 mg, 1.2 mmol, 2
µL, 1.14 mmol, 2 eq.). The resulting reaction mixture was stirred
for 18 h at rt. The reaction mixture was concentrated in vacuo and
25 the product was isolated via flash chromatography (SiO2,
EtOAc/hexane 1:3  1:2  1:1  2:1) to yield 12 (270 mg,
eq.) and Et3N (167 µL, 1.2 mmol, 2 eq.). The resulting reaction
mixture was stirred for 18 h at rt. The reaction mixture was
concentrated in vacuo and the residue applied directly to a silica
column. The product was isolated by eluting with a gradient of
64%) as a colorless oil. δH (300 MHz; CDCl3; Me4Si) 0.77 (t, 85 EtOAc/hexane (1:2  1:1  2:1) to yield the fully protected 14a
CH3, J = 7.15 Hz, 3H), 1.08 – 1.32 (m, CH2, 4H), 2.93 – 3.04 and
3.06 – 3.14 (2 x m, CH2NH, 2 x 1H), 3.43 (dd, JA = 3.2 Hz, JB =
30 10.4 Hz, 1H), 3.59 – 3.79 (m, 6H), 3.98 – 4.03 (m, 1H), 4.40 (s,
CH2Carom, 2H, 4.47 and 4.51 (2 x d, CH2Carom, J = 6.85 Hz, 2 x
(255 mg, 52%) as a colorless oil. δH (300 MHz; CDCl3; Me4Si)
0.90 (t, J = 6.9 Hz, 3H), 1.06 – 1.37 (m, 12H), 2.93 – 3.19 (m,
2H), 3.45 (dd, JA = 3.2 Hz, JB = 13.5 Hz, 2H), 3.59 – 3.83 (m,
6H), 4.04 (q, J = 5.1 Hz, 1H), 4.43 (s, 2H), 4.50 (d, J = 6.3 Hz,
1H), 4.61 (s, CH2Carom, 2H), 4.64 and 4.68 (2 x d, CH2Carom, 2 x 90 1H), 4.54 (d, J = 6.2 Hz, 1H), 4.61 – 4.71 (m, 4H), 5.08 – 5.22
1H), 5.09 and 5.16 (2 x d, CH2Carom, J = 12.65 Hz, 2 x 1H), 7.20
– 7.42 (m, CHarom, 25H). δC (75.5 MHz; CDCl3; Me4Si) 13.5
35 (CH3), 19.7, 31.5 (CH2), 43.5, 44.3 (CH2NH), 66.4, 69.7, 71.1,
72.6, 72.9, 73.2 (CH2), 58.5, 75.2, 77.6, 80.4 (C-2, C-3, C-4, C-
(m, 2H), 7.22 – 7.46 (m, 25H). δC (75.5 MHz; CDCl3; Me4Si)
14.4, 22.9, 27.0, 29.4, 29.9, 32.0, 44.3, 44.7, 59.0, 66.8, 70.1,
71.6, 73.0, 73.4, 73.7, 75.7, 78.1, 81.0, 127.6, 128.2, 128.4,
128.6, 137.8, 138.1, 138.2, 138.3, 161.2, 163.3. HRMS Calcd for
5), 127.2, 127.6, 127.8, 128.0, 128.3 (CHarom), 137.4, 137.7, 95 C51H61N3O6 [M+H]+, 812.4633, found 812.4637.
137.9 (Cq), 160.8 (NHC(O)), 162.9 (NHC(N). Rf
= 0.59
(EtOAc/hexane 1:1). HRMS Calcd for C47H53N3O6 [M+H]+,
40 756.4013, found 756.3996.
N-1-octylguanidine-1-deoxynojirimycin (14). A suspension of
fully protected 14a (103 mg, 123 µmol) and Pd/C (10%) (100
mg) in EtOH (5 mL) was adjusted to pH = 1 with aqueous HCl
N-1-butylguanidine-1-deoxynojirimycin (13). A suspension of 100 (1M). The reaction mixture was stirred vigorously under a H2 (g)
12 (75 mg, 0.1 mmol) and Pd/C (10%) (75 mg) in EtOH (5 mL)
was adjusted to pH = 1 with aqueous HCl (1M). The reaction
45 mixture was stirred vigorously under a H2 (g) atmosphere for 18
h at rt. The reaction mixture was next filtered over celite, washed
atmosphere for 18 h at rt. The reaction mixture was next filtered
over celite, washed with EtOH and concentrated in vacuo. 14 was
obtained after lyophilization as the HCl-salt (44 mg, quant.). δH
(500 MHz; D2O) 0.74 – 0.86 (t, 3H), 1.16 – 1.34 (m, 12H), 1.50 –
with EtOH and concentrated in vacuo. 13 was obtained after 105 1.62 (m, 2H), 3.17 – 3.26 (m, 2H), 3.50 – 3.55 (m, 2H), 3.60 (d,
lyophilization as the HCl-salt (30 mg, quant.). δH (300 MHz;
D2O) 0.93 (t, CH3, J = 7.1 Hz, 3H), 1.35 – 1.43 (m, CH2, 2H),
50 1.60 – 1.64 (m, CH2, 2H), 3.28 – 3.33 (m, NCH2, 2H), 3.60 and
3.68 (2 x s, 2 x 2H), 3.85 – 3.99 (m, 4H). δC (75.5 MHz; D2O)
12.7 (CH3), 30.0, 42.0, 45.2 (CH2), 59.2 (CH2), 63.7, 68.0, 70.4, 110
72.9 (C-2, C-3, C-4, C-5). HRMS Calcd for C11H23N3O4 [M+H]+,
262.1767, found 262.1771.
2H), 3.76 – 3.82 (m, 2H), 3.82 – 3.90 (m, 2H). δHSQC (500 MHz;
D2O) 28.0, 40.3, 45.6, 36.6, 42.5, 42.8, 56.8, 59.8, 82.6, 87.5,
73.8, 78.3, 85.0. LRMS Calcd for C15H32N3O4 [M+H]+, 318.24,
found 318.50.
N-(benzyloxycarbonyl)
N’-(2-2-(benzyloxy)ethoxy)ethyl)
2,3,4,6-tetra-O-benzyl-deoxynojirimycin (15a). As described
above for compound 14a, working at a 0.6 mmol scale (relative
to protected DNJ building block 6), 15a (499 mg, 95%) was
55
General one-pot procedure for the preparation of NG-
substituted guanidine modified DNJ derivatives 14-19. In the 115 obtained as a colorless oil. δH (300 MHz; CDCl3; Me4Si) 3.20 –
preparation of compounds 14-19 a one-pot procedure was
3.47 (m, 5H), 3.49 (s, 4H), 3.58 – 3.68 (m, 2H), 3.69 – 3.82 (m,
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Medicinal Chemistry Communications
4H), 4.05 – 4.17 (m, 1H), 4.42 (s, 2H), 4.45 – 4.52 (m, 4H), 4.58
3.81 (m, 1H), 3.83 – 3.94 (m, 2H), 4.42 – 4.54 (m, 2H), 7.30 –
(s, 2H), 4.64 (d, J = 11.6 Hz, 1H), 5.14 (d, J = 7.2 Hz, 2H), 7.18 – 60 7.42 (m, 5H). δHSQC (500 MHz; D2O) 59.8, 60.2, 63.4, 73.7, 78.3,
7.47 (m, 30H). δC (75.5 MHz; CDCl3; Me4Si) 43.7, 44.5, 58.9,
66.9, 68.8, 69.8, 71.5, 72.9, 73.0, 73.3, 73.5, 75.5, 78.0, 80.9,
127.9, 128.0, 128.1, 128.2, 128.4, 128.6, 137.9, 138.1, 138.3,
160.7, 162.9. HRMS Calcd for C54H59N3O8 [M+H]+, 878.4375,
found 878.4413.
82.6, 84.7, 87.2, 141.6, 142.6, 143.5. LRMS Calcd for
C14H22N3O4 [M+H]+, 296.16, found 296.10.
View Article Online
5
N-(benzyloxycarbonyl) N’-(3-phenylpropyl) 2,3,4,6-tetra-O-
65 benzyl-deoxynojirimycin (18a). As described above for
compound 14a, working at a 0.6 mmol scale (relative to protected
DNJ building block 6), 18a (476 mg, 97%) was obtained as a
colorless oil. δH (300 MHz; CDCl3; Me4Si) 1.61 (m, 2H), 1.87
(m, 1H), 2.47 (dd, JA = 6.5 Hz, JB = 9.0 Hz, 2H), 2.98 – 3.27 (m,
N-1-(2-(2-hydroxyethoxy)ethyl)guanidine-1-deoxynojirimycin
10 (15). 15a (103 mg, 117 µmol) was deprotected as described
above for the preparation of 14 to yield 15 as the HCl-salt (39
mg, quant.). δH (500 MHz; D2O) 3.46 (t, 2H), 3.53 – 3.58 (m, 70 2H), 3.47 (dd, JA = 3.1 Hz, JB = 13.5 Hz, 1H), 3.63 – 3.86 (m,
2H), 3.58 – 3.64 (m, 4H), 3.64 – 3.73 (m, 4H), 3.78 – 3.85 (m,
2H), 3.85 – 3.92 (m, 2H). δHSQC (500 MHz; D2O) 57.1, 60.0, 73.8,
15 74.9, 78.6, 82.6, 83.8, 85.0, 86.4, 87.5. LRMS Calcd for
C11H24N3O6 [M+H]+, 294.17, found 294.35.
8H), 4.03 – 4.12 (m, 1H), 4.44 (s, 2H), 4.53 (dd, JA = 5.5 Hz, JB =
11.5 Hz, 2H), 4.61 – 4.73 (m, 4H), 5.08 – 5.25 (m, 2H), 6.95 –
7.68 (m, 30H). δC (75.5 MHz; CDCl3; Me4Si) 25.9, 31.7, 33.3,
43.8, 44.7, 59.0, 66.9, 68.2, 70.1, 71.6, 73.0, 73.3, 73.7, 75.6,
75 78.0, 80.6, 126.1, 127.7, 128.0, 128.1, 128.2, 128.5, 128.6, 128.7,
137.8, 138.1, 138.2, 138.3, 141.5, 161.3, 163.3. HRMS Calcd for
C52H55N3O6 [M+H]+, 818.4164, found 818.4155.
N-(benzyloxycarbonyl) N’-(2-(benzyloxy)ethyl) 2,3,4,6-tetra-
O-benzyl-deoxynojirimycin (16a). As described above for
20 compound 14a, working at a 0.6 mmol scale (relative to protected
DNJ building block 6), 16a (403 mg, 80%) was obtained as an
N-1-(3-phenylpropyl)guanidine-1-deoxynojirimycin (18). The
yellowish oil. δH (300 MHz; CDCl3; Me4Si) 3.21 – 3.34 (m, 1H), 80 protected intermediated 18a (100 mg, 122 µmol) was deprotected
3.36 – 3.49 (m, 4H), 3.58 – 3.82 (m, 6H), 4.04 – 4.19 (m, 1H),
4.39 (d, J = 4.2 Hz, 2H), 4.48 (dd, JA = 3.6 Hz, JB = 11.6, 2H),
25 4.60 (d, J = 4.5 Hz, 2H), 4.62 – 4.69 (m, 2H), 5.10 – 5.24 (m,
2H), 7.14 – 7.51 (m, 30H). δC (75.5 MHz; CDCl3; Me4Si) 43.6,
as described above for the preparation of 14. Compound 18 was
thus obtained as the HCl-salt (42 mg, quant.). δH (300 MHz; D2O)
1.89 – 2.05 (m, 2H), 2.73 (t, 2H), 3.24 – 3.39 (m, 2H), 3.52 (d,
2H), 3.60 – 3.69 (m, 2H), 3.75 – 3.98 (m, 4H), 7.25 – 7.43 (m,
44.6, 58.8, 66.9, 69.6, 69.8, 70.4, 71.4, 72.9, 73.3, 73.4, 73.5, 85 5H). δC (75.5 MHz; D2O) 29.4, 32.0, 41.6, 45.1, 48.8, 59.2, 63.7,
75.4, 78.0, 80.9, 128.0, 128.1, 128.2, 128.4, 128.5, 128.6, 138.0,
138.1, 138.3, 138.4, 160.7, 162.9. HRMS Calcd for C52H55N3O7
30 [M+H]+, 834.4113, found 834.4135.
68.0, 70.3, 72.8, 126.2, 128.4, 128.7, 141.4, 158.0, 163.2. LRMS
Calcd for C16H26N3O4 [M+H]+, 324.19, found 324.10.
N-(benzyloxycarbonyl) N’-(3-adamantylpropyl) 2,3,4,6-tetra-
N-1-(2-hydroxyethyl)guanidine-1-deoxynojirimycin (16). The 90 O-benzyl-deoxynojirimycin (19a). As described above for
protected intermediated 16a (102 mg, 123 µmol) was deprotected
as described above for the preparation of 14. Compound 16 was
35 thus obtained as the HCl-salt (35 mg, quant.). δH (500 MHz; D2O)
3.36 – 3.42 (m, 2H), 3.50 – 3.58 (m, 2H), 3.58 – 3.65 (m, 2H),
compound 14a, working at a 0.45 mmol scale (relative to
protected DNJ building block 6), 19a (379 mg, 94%) was
obtained as a colorless oil. δH (300 MHz; CDCl3; Me4Si) 2.87 (s,
2H), 3.11 – 3.23 (m, 2H), 3.24 – 3.33 (m, 2H), 3.44 (dd, JA = 3.1
3.65 – 3.75 (m, 2H), 3.77 – 3.93 (m, 4H). δHSQC (500 MHz; D2O) 95 Hz, JB = 13.3 Hz, 1H), 3.59 – 3.81 (m, 6H), 4.01 – 4.11 (m, 1H),
59.2, 59.9, 73.8, 74.7, 78.5, 82.6, 84.9, 87.4. LRMS Calcd for
C9H20N3O5 [M+H]+, 250.14, found 250.35.
4.41 (s, 2H), 4.51 (dd, JA = 4.3 Hz, JB = 11.6 Hz, 2H), 4.59 – 4.71
(m, 4H), 5.07 – 5.20 (m, 2H), 7.19 – 7.45 (m, 25H). δC (75.5
MHz; CDCl3; Me4Si) 28.4, 29.9, 34.4, 37.4, 39.9, 41.9, 66.9,
69.6, 71.5, 73.1, 73.3, 73.6, 75.5, 78.1, 81.2, 82.5, 128.0, 128.1,
40
N-(benzyloxycarbonyl) N’-(benzyl) 2,3,4,6-tetra-O-benzyl-
deoxynojirimycin (17a). As described above for compound 14a, 100 128.2, 128.4, 128.5, 128.6, 137.9, 138.2, 138.3, 161.0, 163.0.
working at a 0.6 mmol scale (relative to protected DNJ building
block 6), 17a (433 mg, 91%) was obtained as a colorless oil. δH
HRMS Calcd for C57H67N3O7 [M+H]+, 906.5052, found
906.5039.
45 (300 MHz; CDCl3; Me4Si) 3.44 – 3.53 (m, 1H), 3.61 – 3.70 (m,
2H), 3.74 (m, 2H), 3.78 (d, J = 4.9 Hz, 2H), 4.04 – 4.12 (m, 1H),
N-1-(3-adamantylpropyl)guanidine-1-deoxynojirimycin (19).
4.20 – 4.34 (m, 2H), 4.37 (d, J = 2.0 Hz, 2H), 4.48 (dd, JA = 2.3 105 The protected intermediated 19a (99 mg, 109 µmol) was
Hz, JB = 11.6 Hz, 2H), 4.55 – 4.69 (m, 4H), 5.09 – 5.25 (m, 2H),
7.06 – 7.56 (m, 30H). δC (75.5 MHz; CDCl3; Me4Si) 44.8, 48.1,
50 59.0, 67.0, 69.8, 71.6, 72.9, 73.3, 73.5, 75.4, 80.4, 128.0, 128.6,
137.8, 138.0, 138.2, 161.1, 163.0. HRMS Calcd for C50H51N3O6
[M+H]+, 790.3851, found 790.3839.
deprotected as described above for the preparation of 14.
Compound 19 was thus obtained as the HCl-salt (49 mg, quant.).
δH (500 MHz; D2O) 1.21 (t, 1H), 1.46 (s, 6H), 1.54 – 1.70 (m,
6H), 1.77 – 1.86 (m, 2H), 1.88 (s, 3H), 2.98 – 3.02 (s, 2H), 3.25 –
110 3.41 (m, 2H), 3.44 – 3.53 (m, 2H), 3.52 – 3.57 (m, 2H), 3.62 (s,
2H), 3.75 – 3.84 (m, 2H), 3.84 – 3.92 (m, 2H). δHSQC (500 MHz;
D2O) 42.6, 42.7, 51.3, 53.7, 53.8, 59.8, 61.2, 63.4, 73.8, 78.5,
82.6, 82.2, 87.7, 84.8, 96.4. LRMS Calcd for C21H38N3O5
[M+H]+, 412.28, found 412.20.
N-1-benzylguanidine-1-deoxynojirimycin (17). The protected
55 intermediated 17a (99 mg, 125 µmol) was deprotected as
described above for the preparation of 14. Compound 17 was thus
obtained as the HCl-salt (41 mg, quant.). δH (500 MHz; D2O)
3.29 (s, 1H), 3.51 – 3.60 (m, 2H), 3.60 – 3.67 (m, 2H), 3.74 –
115
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Medicinal Chemistry Communications
Page 8 of 8
7. E. Borges de Melo, A. da Silveira Gomes and I. Carvalho,
General Procedure for Enzyme Inhibition Assays
Tetrahedron, 2006, 62, 10277-10302.
8. a) T. M. Gloster and G. J. Davies, Org. Biomol. Chem., 2010, 8, 305-
320; b) Gloster, T. M.; Vocaldo, D. J.; Nature Chem. Biol. 2012, 8,
Glycosidases used in the inhibition studies were purchased
from Sigma; α-galactosidase (from green coffee beans; G8507),
β-galactosidase (from bovine liver; G1875), α-glucosidase (from
bakers yeast; G5003), β-glucosidase (from almonds; G4511), α-
mannosidase (from Jack beans; M7257), β-mannosidase (from
Helix pomatia; M9400) and Naringinase (from Penicillium
decumbens; N1385). The corresponding p-nitrophenyl glycosides
substrates were purchased from Carbosynth Limited.
Inhibition assays were performed in either phosphate or acetate
buffer at the optimum pH for each enzyme. Determination of the
IC50 values of the iminosugars were carried out by
spectrophotometrically measuring the residual hydrolytic
activities of the glycosidases on the corresponding p-nitrophenyl
60
683.
View Article Online
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5
65 10. J. C. Lee, S. Francis, D. Dutta, V. Gupta, Y. Yang, J.-Y. Zhu, J. S.
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70 12. H. S. Overkleeft, G. H. Renkema, J. Neele, P. Vianello, I. O. Hung,
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M. Aerts, J. Biol. Chem., 1998, 273, 26522-26527.
10
13. J. Conchie, A. J. Hay, I. Strachan and G. A. Levvy, Biochem. J.,
1967, 102, 929-941.
15 glycosides substrate in the presence of a concentration range of
iminosugar derivatives. The incubation mixture consisted of 15
µL of inhibitor solution in water (0.1 U/mL) and 15 µL of
enzyme solution. The concentrations of the enzyme were adjusted
so that the reading for the final absorbance was in the range of 0.5
20 – 1.5 units.
Inhibitor and enzyme solutions were mixed in a disposable 96-
well microtiter plate and then incubated at room temperature for 5
minutes. Next the reactions were initiated by addition of 75 µL of
a solution of the corresponding p-nitrophenyl glycosides substrate
25 solution in the appropriate buffer at the optimum pH for the
enzyme. After the reaction mixture was incubated at 37 oC for 30
min, the reaction was quenched with 0.5M Na2CO3 (240 µL) and
the absorbance of 4-nitrophenol released from the substrate was
read immediately at 405 nm using a BioTek µQuant Microplate
30 Spectrophotometer.
75 14. G. Papandreou, M. K. Tong and B. Ganem, J. Am. Chem. Soc., 1993,
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85
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Fernandez, Chem. Commun., 2002, 848-849.
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Med. Chem., 2008, 51, 924-931.
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IC50 values were determined as a concentration of the
iminosugars that inhibits 50% of the enzyme activity under the
assay conditions. IC50 values were determined graphically with
GraphPad Prism (version 5.0) by making a plot of percentage
35 inhibition versus the log of inhibitor concentration, using at least
8 different inhibitor concentrations (triplicate).
100 25. D. Bini, F. Cardona, M. Forcella, C. Parmeggiani, P. Parenti, F.
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Acknowledgements
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Ther. Pat., 2011, 21, 885-903.
We would like to thank Cees Versluis and Mirjam Damen of
Biomolecular Mass Spectrometry and Proteomics (Utrecht
40 University) for measuring HRMS. We gratefully acknowledge
the Dutch Technology Foundation STW, applied science division
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of NWO and the Technology Program of the Ministry of 110
Economic Affairs, for the financial support.
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