5248 J. Med. Chem. 2010, 53, 5248–5255 DOI: 10.1021/jm100198t
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Click Chemistry Approach to New N-Substituted Aminocyclitols as Potential Pharmacological Chaperones for Gaucher Disease† Lucı´ a Dı´ az,‡ Jordi Bujons, Josefina Casas,§ Amadeu Llebaria,§ and Antonio Delgado*,‡,§ ‡
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Facultat de Farm acia, Unitat de Quı´mica Farmac eutica (Unitat Associada al CSIC), Universitat de Barcelona, Avda. Joan XXIII, s/n, 08028 Barcelona, Spain, §Research Unit on Bioactive Molecules (RUBAM), Departament de Quı´mica Biom edica, Institut de Quı´mica Avanc-ada de Catalunya (IQAC-CSIC, Spanish National Research Council), Jordi Girona 18-26, 08034 Barcelona, Spain, and Department de Quı´mica Biol ogica i Modelitzaci o Molecular, Institut de Quı´mica Avanc-ada de Catalunya (IQAC-CSIC, Spanish National Research Council), Jordi Girona 18-26, 08034 Barcelona, Spain Received February 15, 2010
New N-alkylaminocyclitols bearing a 1,2,3-triazole system at different positions of the alkyl chain have been prepared as potential GCase pharmacological chaperones using click chemistry approaches. Among them, compounds 1d and 1e, with the shorter spacer (n = 1) between the alkyltriazolyl system and the aminocyclitol core, were the most active ones as GCase inhibitors, revealing a determinant effect of the location of the triazole ring on the activity. Furthermore, SAR data and computational docking models indicate a correlation between lipophilicity and enzyme inhibition and suggest “extended” and “bent” potential binding modes for the compounds. In the “bent” mode, the most active compounds could establish a hydrogen-bond interaction between the triazole moiety and enzyme residue Q284. Such an interaction would be precluded in compounds with a longer spacer between the triazole and the aminocyclitol core.
Introduction Gaucher disease is one of the most prevalent lysosomal storage disorders characterized by the accumulation of the sphingolipid glucosylceramide in the lysosomes. The disease is caused by the deficient activity elicited by several mutated forms of the enzyme glucocerebrosidase (GCase a), the β-glucosidase that hydrolyzes glucosylceramide into glucose and ceramide.1 Cellular levels of the mutated, misfolded enzyme are abnormally low because of its premature degradation by specific cytosolic endoproteases in the endoplasmic reticulum. Several therapeutic strategies for Gaucher disease have been developed over the past years.2 Among them, the use of pharmacological chaperones, competitive inhibitors of the target enzyme that assist the proper folding of the defective protein at subinhibitory concentrations,3-5 is an active field of research.6 In this context, several iminosugars and aminocyclitols have been reported in the literature (Figure 1). To date, a number of GCase structures in its native state7 with different degrees of glycosylation,8-10 under different pH conditions,11,12 or as a complex with different inhibitors13-16 have been reported. All these structures show a very similar 3D-arrangement for the protein with four loops (L1-L4) located at the entrance to the catalytic site that control open † Dedicated to Prof. Pelayo Camps on the occasion of his 65th anniversary. *To whom correspondence should be addressed. Phone: þ34934006108. Fax: þ34-932045904. E-mail:
[email protected]. a Abbreviations: A-C10, N-decylaminocyclitol; CC50, cytotoxic concentration for 50% of the cell population under study; clogP, calculated logarithm of the partition coeficient; GCase, β-glucocerebrosidase; IFG, isofagomine; NBDNJ, N-butyldeoxynojirimycin; NNDNJ, N-nonyldeoxynojirimycin ; NOV, N-octylvalienamine; SAR, structure-activity relationship.
pubs.acs.org/jmc
Published on Web 06/17/2010
Figure 1. Aminocyclitols reported in this study (1-3), together with representative iminosugars and aminocyclitols reported as GCase inhibitors.
and closed conformations (see below). Differences on the surface topology of GCase close to the entrance to the active site have been observed in structures of the enzyme complexed with different inhibitors. Thus, while in N-nonyldeoxynojirimycin (NNDNJ) and N-butyldeoxynojirimycin (NBDNJ)-GCase complexes the N-alkyl chains are stabilized by accommodation into the hydrophobic valley between L3 and L4, an additional hydrophobic groove between L1 and L3 is observed in the isofagomine (IFG)-GCase complex structure.14,15 Interestingly, these two valleys between L3 and L4 and between L1 and L3 have been modeled as possible binding locations for the two alkyl chains of the ceramide moiety in the natural substrate.15 Over the past years, we have been working actively on the development of new aminocyclitols with potential applicability r 2010 American Chemical Society
Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Table 1. Aminocyclitols Arising from Click Chemistry Reaction between ω-Alkynyl Alkylaminocyclitols and Azidesa alkyne azide (RN3) 5 5 5 5 5 5 5 5 7 7 7 9 9 9 a
23 25 26 27 29 30 31 32 25 26 28 24 25 27
R (in RN3) n-butyl n-octyl n-nonyl n-decyl n-dodecyl n-tetradecyl 6-(propoxy)hexyl 2-phenylethyl n-octyl n-nonyl n-undecyl n-hexyl n-octyl n-decyl
5249
Scheme 1a
click adduct aminocyclitol 10a 10b 10c 10d 10e 10f 10g 10h 11a 11b 11c 12a 12b 12c
1a 1b 1c 1d 1e 1f 1g 1h 2a 2b 2c 3a 3b 3c
See Scheme 3.
as chemical chaperones of the enzyme GCase.17,18 In our previous work, we found that aminocyclitol A-C10 (Figure 1), which stabilized GCase under thermal denaturation conditions19 and inhibited GCase in wild type fibroblasts, behaved as a pharmacological chaperone in Gaucher disease patient fibroblasts bearing the L444P/G202R and L444P;E326K/ G202R genotypes.20 However, exploration of the chemical diversity around the amino group has met with limited success so far.21 Thus, the most promising results arose from the regio- and stereoselective opening22,23 of conduritol B epoxide with a series of aliphatic amines of different chain lengths.19 In order to explore the role of the aminoalkyl side chain on the enzyme stabilization under thermal denaturation conditions,24 a fast and convenient methodology aimed at the parallel synthesis of small to medium sized libraries of N-substituted aminocyclitol derivatives would be desirable. In this context, click-chemistry approaches based on the Cu-catalyzed 1,3dipolar Huisgen cycloaddition of alkynes and azides are being widely used in drug design.25,26 However, the electronic properties of the resulting 1,2,3-triazole system make possible the operation of additional interactions of uncertain effects at the active site level. In order to unravel such effects, we have synthesized and tested a small collection of N-alkylaminocyclitols arising from the formal placement of the triazole ring along the N-alkyl side chain. From a structural standpoint, the aminocyclitols presented in this study can be grouped into three different subsets (1-3) as a function of the spacer length (n = 1-3) between the aminocyclitol core and the triazole system (Figure 1). Synthesis Aminocyclitols 1-3 were obtained by the Cu-catalyzed 1,3dipolar cycloaddition (Huisgen reation) of ω-alkynylaminocyclitols 5 (n = 1), 7 (n = 2), and 9 (n = 3) with a set of azides 23-32, followed by massive benzyl removal (see Scheme 3 and Table 1). Aminocyclitols 5, 7, and 9 were obtained, in turn, by the regio- and diastereoselective opening22 of epoxide 4 with the commercially available propargylamine or amines 20 and 22, followed by TMS removal (Schemes 1 and 2). The use of the TMS protecting group in amines 20 and 22 increased their boiling point, thus favoring their isolation, purification, and subsequent manipulation. Copper-catalyzed Huisgen cycloaddition reaction of scaffolds 5, 7, and 9 with the aliphatic azides shown in Table 1
a Reagents and conditions: (a) amine (propargylamine, 20, or 22), LiClO4, CH3CN; (b) KF, DMSO.
afforded the expected fully benzylated triazolylalkylaminocyclitols 10-12 (Scheme 3). Massive O-benzyl deprotection was carried out either by hydrogenolysis or by reaction with BCl3, following reported protocols.17,27 Results and Discussion A pharmacological chaperone binds to the active site of the target enzyme at the neutral pH of the endoplasmic reticulum (ER) to assist in protein folding and also to enhance the enzyme transport to lysosomes. However, it partly dissociates at the low pH of the lysosomal environment while still stabilizing the enzyme at that acidic pH. Consequently, compounds were evaluated as inhibitors of recombinant GCase (imiglucerase (Cerezyme)) at both neutral and acidic pH. Compounds 1, bearing the shorter spacer (n = 1) (see Tables 1 and 2) gave IC50 values lower than those of compounds 2 and 3, with a longer spacer (n = 2 and 3, respectively), resulting from the formal placement of the triazole ring along the aliphatic chain (compare the couples 1b/3a and 1c/2a and the triplets 1d/2b/ 3b and 1e/2c/3c). Inhibition constants (Ki) for the most active compounds in each series showed, in all cases, a competitive inhibition pattern, as illustrated in Figure 2 for 1d (Table 2).28 It is worth mentioning that the low Ki values found for compounds 1d-1f were in the 60-90 nM range. Interestingly, none of the above aminocyclitols showed significant activity on glucosyl ceramide synthase (GCS) from cell homogenates at 250 μM, thus indicating a selectivity toward the hydrolytic enzyme.29 Moreover, the above aminocyclitols were inactive at 0.1 mM against a panel of commercially available R- and β-glycosidases,30 reinforcing the selectivity against GCase. Regarding neutral pH, IC50 values for compounds 1 were similar to or slightly lower than those at acidic pH, with the exception of 1e and 1h (Table 2). Interestingly, compounds 2 and 3 showed lower IC50 values at neutral pH, an indication of the ability of the inhibitors to interact with the enzyme at the cellular pH of both ER and lysosomes. Enzyme stabilization under thermal denaturation conditions, expressed as the stabilization ratio (see Table 2), was used as an indication of the potential of the target compounds to behave as pharmacological chaperones.24 An IC50 threshold of 15 μM at pH 7.4 was set as selection criteria for the thermal stabilization assay. Recovery of GCase activity was measured at 48 °C in the presence and in the absence of increasing concentrations (from 0.50 to 150 μM or from 50 to 150 μM, as appropriate) of selected aminocyclitols at different incubation times (Figure 3). For comparative purposes, NNDNJ and our previously reported aminocyclitol A-C1019 (Figure 1) were also assayed under the same experimental conditions.
5250 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 14
Dı´az et al.
Scheme 2. Synthesis of Aminesa
a
Reagents and conditions: (a) TMSCl, BuLi, THF, -78 °C; (b) MsCl, Et3N, THF, 0 °C; (c) NaN3, DMF; (d) LiAlH4, THF.
Scheme 3a
a
Reagents and conditions: (a) R-N3 (23-32; see Table 1), Cu2SO4, sodium ascorbate, H2O/THF (1:1); (b) BCl3, CH2Cl2 (-78 °C); (c) H2, Pd/C.
Table 2. Inhibitory Activity of Aminocyclitols 1a-h, 2a-c, and 3a-c against Imiglucerasea IC50 (μM) compd pH 7.4 pH 5.2 Ki (μM)b 1a 1b 1c 1d 1e 1f 1g 1h 2a 2b 2c 3a 3b 3c NNDNJ A-C10
nd 1.1 0.20 0.09 0.06 0.10 6.2 25.0 55.9 11.0 4.9 44.6 18.2 1.2 0.30
178 1.0 0.20 0.10 0.03 0.09 7.4 10.8 167 44.7 25.4 257 35.4 3.5 1.3, 0.66 j 1.8
nd 1.8 0.33 0.09 0.06 0.08 2.4 19.1 nd nd 20.2 nd 19.9 7.6 0.30 j 0.30
stabilization % inhibition GCase ratioc,d in human wt fibroblastse nd 1.0 13.0 21.0 28.7 13.2 1.1 nd nd 7.6 11.6 nd nd 1.4 9.2 4.9
