Synthesis of lipophilic 1-deoxygalactonojirimycin derivatives as D-galactosidase inhibitors

Article abstract of DOI:10.3762/bjoc.6.21

N-Alkylation at the ring nitrogen of the D-galactosidase inhibitor 1-deoxygalactonojirimycin with a functionalised C₆ alkyl chain followed by modification with different aromatic substituents provided lipophilic 1-deoxygalactonojirimycin derivatives which exhibit inhibitory properties against β-glycosidases from E. coli and Agrobacterium sp. as well as green coffee bean α-galactosidase. In preliminary studies, these compounds also showed potential as chemical chaperones for GM1-gangliosidosis related β-galactosidase mutants.

Full text of DOI:10.3762/bjoc.6.21

Synthesis of lipophilic 1-deoxygalactonojirimycin
derivatives as D-galactosidase inhibitors
Georg Schitter1, Elisabeth Scheucher1, Andreas J. Steiner1,
Arnold E. Stütz1, Martin Thonhofer1, Chris A. Tarling2, Stephen G. Withers2,
Jacqueline Wicki2, Katrin Fantur3, Eduard Paschke3, Don J. Mahuran4,
Brigitte A. Rigat4, Michael Tropak4 and Tanja M. Wrodnigg*1
Full Research Paper
Open Access
Address:
Beilstein Journal of Organic Chemistry 2010, 6, No. 21.
1Glycogroup, Institute for Organic Chemistry, University of
Technology Graz, Stremayrgasse 16, A-8010 Graz, Austria,
2Chemistry Department, University of British Columbia, 2036 Main
Mall, Vancouver, BC, Canada V6T 1Z1, 3Department of Pediatrics,
Medical University of Graz, Auenbruggerplatz 30, A-8010 Graz,
Austria and 4Department of Laboratory Medicine and Pathobiology,
Sick Kids Hospital, 555 University Avenue, University of Toronto, Ont.,
Canada M5G 1X81
doi:10.3762/bjoc.6.21
Received: 03 December 2009
Accepted: 11 February 2010
Published: 01 March 2010
Guest Editor: T. K. Lindhorst
© 2010 Schitter et al; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Tanja M. Wrodnigg* - t.wrodnigg@tugraz.at
* Corresponding author
Keywords:
chemical chaperones; 1-deoxy-D-galactonojirimycin; iminosugars;
lipophilic galactosidase inhibitor; N-modified iminosugars
Abstract
N-Alkylation at the ring nitrogen of the D-galactosidase inhibitor 1-deoxygalactonojirimycin with a functionalised C6 alkyl chain
followed by modification with different aromatic substituents provided lipophilic 1-deoxygalactonojirimycin derivatives which ex-
hibit inhibitory properties against β-glycosidases from E. coli and Agrobacterium sp. as well as green coffee bean α-galactosidase.
In preliminary studies, these compounds also showed potential as chemical chaperones for GM1-gangliosidosis related
β-galactosidase mutants.
Introduction
Iminosugars such as compounds 1–4 (Figure 1) have been diabetes, anti-infective, as well as insect anti-feedant and plant
shown to be potent glycosidase inhibitors and useful tools for growth regulatory effects. Because of their diverse properties,
the study of glycoside-hydrolysing enzymes. These sugar iminosugars have enjoyed continuous interest since their
mimetics have been found to have anti-viral, anti-cancer, anti- discovery in the 1960s. Consequently, many different
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Beilstein Journal of Organic Chemistry 2010, 6, No. 21.
Figure 1: Typical representatives of iminosugars.
derivatives have been prepared for biological evaluations via a 1-Deoxygalactonojirimycin (4) was shown to be a candidate for
wide range of synthetic approaches and have been used for the treatment of Fabry disease, an X-linked inherited lysosomal
various medicinal and biomolecular applications [1-9].
storage disorder caused by the deficiency of α-galactosidase A
activity resulting in the accumulation of globotriaosylceramide,
Recently, iminosugars were found to have potential to serve as thereby affecting the lysosomes of vascular endothelial cells.
pharmacological chaperones for the treatment of lysosomal Iminosugar 4 can increase α-galactosidase A levels 1.5 to 28
storage diseases in chaperone mediated therapy (CMT) [10]. In fold in cultured Fabry patient cell lines (baseline α-Gal A levels
contrast to enzyme replacement therapy (ERT), where recom- range from 0–52%) after incubation for five days, as was
binant enzyme is given to the patient at regular intervals, the observed for 49 different missense mutant forms [18-21]. It can
iminosugars used for CMT (recently called pharmacological also reduce tissue globotriaosylceramide levels in a mouse
chaperone therapy, PCT) are able to cross the blood brain model [22].
barrier. This gives the opportunity also to treat types of lyso-
somal storage diseases involving the central nervous system. Suzuki and co-workers found, that N-octyl-4-epi-β-valienamine
Furthermore, CMT is a cost-efficient alternative to ERT. In this (10) (Figure 3), a competitive inhibitor of lysosomal
context, N-alkylated derivatives of 1-deoxynojirimycin [11] β-galactosidase, when orally administered to GM1-ganglio-
such as 5 and 6 (Figure 2) as well as N-substituted D-glucono-δ- sidosis model mice, is able to enter the brain through the blood-
lactams 7 (Figure 2) [12] have been shown to be highly potent brain barrier and thereby enhancing β-galactosidase activity,
pharmacological chaperones for the potential treatment of reduce substrate storage, and clinically improve neurological
Gaucher [13] and Pompe [14] diseases by ‘rescuing’ the related deterioration [23].
mutant enzymes. Both Wong [15] and Overkleeft [16] have
shown that a rather large lipophilic substituent such as the
adamantyl group (8, Figure 2) attached via an alkyl chain with a
chain length from C3 up to C9 to the ring nitrogen of
1-deoxynojirimycin and isofagomine respectively, can increase
the interaction with the lysosomal glycosphingolipid gluco-
cerebrosidase. Interestingly, 5-N, 6-X-(N′-alkylimino-
methylene)nojirimycin derivatives where X is O, NH or S such
as in structure 9 (Figure 2) also have chaperone activity for
Figure 3: Structure of NOEV 10.
Gaucher related mutations [17].
Figure 2: N-Modified iminosugars 5–9 as potential pharmacological chaperones.
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Beilstein Journal of Organic Chemistry 2010, 6, No. 21.
Scheme 1: Three-step-synthesis of partially protected 1-deoxy-D-galactonojirimycin derivative 12 from 10 via L-arabino-hexos-5-ulose 11.
Our studies revealed that 1-deoxy-D-galactonojirimycin-lysine the 3,4-O-isopropylidene iminosugar 12. Starting from enol
hybrids, when carrying an aromatic substituent, such as a dansyl ether 10 [26,27], treatment with m-chloroperbenzoic acid gave
moiety, in its nature a lipophilic aromatic substituent, are potent the 5-O-chlorobenzoic ester via the corresponding 5,6-epoxide.
D-galactosidase inhibitors and also show activity with human This ester underwent hydrolysis under basic conditions to afford
lysosomal β-galactosidase, exhibiting improvements of the the L-arabino-hexos-5-ulose 11, which was immediately used
enzyme activity in mutant cell lines [24]. In the course of this for the next step after brief silica gel purification. The reductive
work, we became interested in the influence of other lipophilic amination and N-deprotection of 11 was carried out under an at-
aromatic substituents on the biological activity of such com- mosphere of H2 with benzylamine in methanol and Pd/C as
pounds. Different aromatic acid derivatives were prepared by catalyst to produce 3,4-O-isopropylidene-1-deoxy-D-galac-
coupling to the free amine at the terminus of the C6 alkyl chain tonojirimycin (12) in an overall yield of 75% (Scheme 1).
in compound 15, which is anchored to the ring nitrogen of
1-deoxygalactonojirimycin, to yield derivatives 16–19 and 22. Compound 12 underwent N-substitution upon treatment with
The spacer length of six carbon units has been proven suitable 1-O-tosyl-6-N-(tert-butoxycarbonyl)-6-aminohexanol (13) [28]
for enzyme recognition in previous studies [25] and was kept in DMF to give the 1-deoxygalactonojirimycin derivative 14 in
constant to compare the different aromatic substituents. Add- 64% yield. The isopropylidene and tert-butoxycarbonyl
itionally, Wong [15] as well as Suzuki [23] have shown from protecting groups were simultaneously removed under standard
computational studies, that in case of N-substitution on com- conditions to afford the desired free amine 15 [25], the key
pounds 8b and 10, the iminosugar and carbasugar units respect- building block for further modifications (Scheme 2).
ively, were found to interact with the active site of the corres-
ponding enzymes whereas the alkyl chains were located in the The chemoselective acylation of the free amine 15 was
distinctly hydrophobic entrance region to the active site. Thus, conducted with three different benzoic acid derivatives in order
for comparison, a lipophilic aliphatic tert-butyl group in com- to investigate the influence of the potential basicity of an add-
pound 20 was included in this study. In addition to the synthetic itional nitrogen at the aromatic substituent. For the synthesis of
approaches, the influence of the lipophilic substituents of the compound 16, 4-isopropylbenzoic acid was reacted with the
new N-modified 1-galactonojirimycin derivatives on their bio- primary amine under amide coupling conditions with O-(benzo-
logical interaction with respective glycoside hydrolases are triazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoro-borate
described.
(TBTU) as the coupling reagent in DMF and triethylamine.
Likewise, nicotinic acid under the same conditions gave com-
pound 17 in 20% yield. Reaction of 15 with
Results and Discussion
The key intermediate for the synthesis of N-modified lipophilic 4-(dimethylamino)benzoyl chloride in DMF and triethylamine
1-deoxygalactonojirimycin derivatives 16–20 as well as 22 was afforded derivative 18 in 22% yield (Scheme 3). These unusu-
Scheme 2: Synthesis of N-(6-aminohexyl)-1-deoxygalactonojirimycin (15) from 12 via 14.
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Beilstein Journal of Organic Chemistry 2010, 6, No. 21.
Scheme 3: Synthesis of lipophilic 1-deoxy-D-galactonojirimycin derivatives 16–18 by chemoselective acylation of 15.
ally low yields for the standard coupling reactions were due to because of its potential to serve as diagnostic tool. Conven-
the formation of very polar side products as well as material tional BOC-protection of amine 15 gave derivative 20 in 81%
losses during column chromatography.
yield (Scheme 4).
For increased lipophilicity as well as for analytical purposes, Aromatic derivative 22 was synthesised from 4 [29-42]
compound 15 was also coupled to 1-pyrenebutyric acid with (conveniently obtained by deprotection of compound 12 under
TBTU in DMF in the presence of triethylamine to give com- acidic conditions), in 77% yield by ring nitrogen alkylation with
pound 19 in 56% yield. The pyrenyl substituent was chosen tosylate 21 (Scheme 5) [43].
Scheme 4: Synthesis of compounds 19 as well as 20 from primary amine 15.
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Beilstein Journal of Organic Chemistry 2010, 6, No. 21.
Scheme 5: Synthesis of compound 22.
Inhibition constants of the compounds synthesised are presented 10.9 µM (Ki = 2.0 µM) and 3.26 µM (Ki = 0.72 µM), respect-
in Table 1. The 1-deoxygalactonojirimycin analogues were ively, with human lysosomal β-galactosidase.
tested as inhibitors of Agrobacterium sp. β-glucosidase/
galactosidase and E. coli β-galactosidase as well as green coffee In preliminary studies compounds 17 as well as 22 served as
bean α-galactosidase (Table 1). All new compounds inhibited chemical chaperone and increased the enzyme activity of a
the Agrobacterium sp. enzyme better than the parent iminosugar β-galactosidase mutant feline fibroblast cell line up to 5.5 fold
4. The pyrenyl substituted compound 19 with an extended when applied at a concentration of 100 µM. Compound 18 was
aromatic system turned out to be the most active inhibitor with a significantly better chemical chaperone for this mutant
a Ki value of 60 nM against Agrobacterium sp. β-glucosidase/ increasing the relative enzyme activity 4.8 fold at a concentra-
galactosidase and 0.25 µM against E. coli β-galactosidase. tion of 2 µM.
However, the toxicity of this compound clearly requires further
evaluation. In general, N-substitution does not dramatically Conclusion
affect the inhibitory properties of the derivatives against We have synthesised new 1-deoxygalactonojirimycin
β-galactosidase from E. coli, with Ki values distributed in the derivatives 16–20, as well as 22, which feature a C6 chain
range of the parent iminosugar 4. No particular trend could be anchored to the ring nitrogen. Different lipophilic aromatic and
observed in a comparison of compounds 16–19 as regards the aliphatic substituents at the N-alkyl chain were introduced
presence or absence of the additional nitrogen at the aromatic resulting in an interesting Ki-value profile against
substituent. Iminosugars 16–20, as well as 22, were less active β-galactosidases from Abg and E. coli, respectively, as well as
than the parent compound with α-galactosidase from green with α-galactosidase from green coffee beans. The Ki values
coffee beans. However, the Ki values are still in the low µM with human lysosomal β-galactosidase and preliminary data for
range and thus, suitable for use as chemical chaperones. Grati- chaperone activity in a cat fibroblast model of the new com-
fyingly, compounds 20 and 22 exhibited IC50 values of pounds suggest that such iminosugar derivatives have interest-
ing potential in the chaperone-mediated therapy of lysosomal
storage diseases, such as, for example, GM1 gangliosidosis as
Table 1: Inhibitory activities of compounds 16–20 and 22 with β-glyc-
well as Morquio B disease, possibly also Fabry’s disease.
Further bio-medicinal evaluation and toxicity studies are
currently in progress.
osidases from Agrobacterium sp. (β-glu/gal Abg), E. coli (β-gal E. coli)
as well as with the α-galactosidase from green coffee beans (α-gal
GCB).
Compound
Ki [µM]
Ki [µM]
Ki [µM]
β-glu/gal Abg β-gal E. coli
α-gal GCB
Supporting Information
4
100
13
13
0.013a
2.6
Supporting Information File 1
16
17
18
19
20
22
1.3
0.83
1.1
0.25
2.4
0.4
Full experimental details and characterisation data
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-6-21-S1.pdf]
1.5
3.8
0.06
3.0
6.0
2.2
0.49
7.0
4.3
2.2
Acknowledgements
G. S. thanks the Institute of Organic Chemistry for financial
aSee reference [7].
support (January–June 2009).
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