Potent GH20 N-Acetyl-β-d-hexosaminidase Inhibitors: N-Substituted 3-acetamido-4-amino-5-hydroxymethyl-cyclopentanediols

From 1,2;3,4-di-O-isopropylidene-d-galactopyranose, a preliminary series of highly functionalized amino(hydroxymethyl)cyclopentanes was easily available. These amine-containing basic carbasugars featuring the d-galacto configuration are potent inhibitors of the GH20 β-d-hexosaminidases probed and may bear potential as regulators of N-acetyl-d-hexosaminidase activities in vivo.


Introduction
In humans, N-acetyl-D-hexosaminidases (HexA, HexB, and HexS) play essential roles in the lysosomal processing of degradation-bound glycolipids as well as glycans [1]. O-GlcNAcase [2] removes O-linked GlcNAc residues from serine or threonine in nucleocytoplasmic proteins. Another N-acetylhexosaminidase, HexD, was recently reported and has been mentioned in context with rheumatoid arthritis [3].
Potent and highly selective inhibitors are required for studying these enzymes and their physiological significance. In particular, potential therapeutic applications in context with lysosomal disorders [7], cancer [8], and Alzheimer's disease [9] require high degrees of selectivity for any of the enzymes mentioned above over the other respective N-acetylhexosaminidases.
Its structural identity can unambiguously be verified by X-ray structure determination ( Figure  2). The corresponding triflate 15 provided azidodeoxy derivative 16 by clean inversion of configuration. Intermediate 16, upon reduction with Zn under slightly acidic conditions and subsequent conventional N-acetylation, highly selectively furnished, via free amine 17, desired acetamido compound 18, which yielded crystals of sufficient quality for XRD ( Figure 2). Jäger and co-workers [21] have directed our attention toward cyclopentanoid basic sugar analogs as potentially useful inhibitors of lysosomal glycosidases. Relying on the pioneering synthetic work by Vasella and co-workers [22] based on guiding contributions by Padwa [23] as well as Oppolzer [24], we had thus investigated cyclopentane-based β-galactosidase inhibitors [25] and have recently extended our range of new compounds by addition of N-acetyl-β-D-galactosaminide and -β-D-glucosaminide analogs.
Its structural identity can unambiguously be verified by X-ray structure determination ( Figure 2). The corresponding triflate 15 provided azidodeoxy derivative 16 by clean inversion of configuration. Intermediate 16, upon reduction with Zn under slightly acidic conditions and subsequent conventional N-acetylation, highly selectively furnished, via free amine 17, desired acetamido compound 18, which yielded crystals of sufficient quality for XRD ( Figure 2).  From tricycle 18, by hydrogenolysis over Perlman's catalyst, cyclopentane 19 featuring a free amine was obtained in good yield. Subsequent acidic deprotection yielded free aminotriol 20.
Chemoselective N-alkylation of intermediate 19 provided the corresponding N-hexyl (21), Nmethoxycarbonylpentyl (22), as well as N-cyanopentyl (23) derivatives. By reduction of the nitrile function in 23, primary amine 24 became available, which was converted into fluorescent dansylaminohexyl derivative 25 with the aid of dansyl chloride.

Biological Evaluation
New compounds turned out particularly potent inhibitors of Streptomyces plicatus N-acetyl-βhexosaminidase (SpHex) with Ki-values in the sub-nanomolar range (Table 1). By introduction of a dansylamido moiety into the alkyl chain, this activity was further improved as shown by the 60 pM value determined for inhibitor 25 when compared to analogue 21. With the same enzyme, compound 1 exhibited Ki = 80 μM [26].
Inhibitors 21 and 25 exhibited excellent properties with this vital human enzyme, considerably exceeding the activities of the reference compounds probed in this study.
In conclusion, compounds of this new aminocyclopentane-derived family of Nacetylgalactosaminide mimetics represent a potentially interesting class of N-acetyl-Dhexosaminidase inhibitors and pharmacological chaperones for treatment of Tay Sachs disease, in particular, when also considering their simplicity of synthesis.   From tricycle 18, by hydrogenolysis over Perlman's catalyst, cyclopentane 19 featuring a free amine was obtained in good yield. Subsequent acidic deprotection yielded free aminotriol 20.
Chemoselective N-alkylation of intermediate 19 provided the corresponding N-hexyl (21), Nmethoxycarbonylpentyl (22), as well as N-cyanopentyl (23) derivatives. By reduction of the nitrile function in 23, primary amine 24 became available, which was converted into fluorescent dansylaminohexyl derivative 25 with the aid of dansyl chloride.

Biological Evaluation
New compounds turned out particularly potent inhibitors of Streptomyces plicatus N-acetyl-βhexosaminidase (SpHex) with Ki-values in the sub-nanomolar range ( Table 1). By introduction of a dansylamido moiety into the alkyl chain, this activity was further improved as shown by the 60 pM value determined for inhibitor 25 when compared to analogue 21. With the same enzyme, compound 1 exhibited Ki = 80 μM [26].
Inhibitors 21 and 25 exhibited excellent properties with this vital human enzyme, considerably exceeding the activities of the reference compounds probed in this study.
In conclusion, compounds of this new aminocyclopentane-derived family of Nacetylgalactosaminide mimetics represent a potentially interesting class of N-acetyl-Dhexosaminidase inhibitors and pharmacological chaperones for treatment of Tay Sachs disease, in particular, when also considering their simplicity of synthesis.

General Methods
Optical rotations were measured at 20 °C on a Perkin Elmer (Waltham, MA, USA) 341 polarimeter at a wave length of 589 nm and a path length of 10 cm.
NMR spectra were recorded on a Varian (Palo Alto, CA, USA) INOVA 500 operating at 499.82 MHz ( 1 H), and at 125.894 MHz ( 13 C), or on a Bruker (Billerica, MA, USA) Ultrashield spectrometer at 300.36 and 75.53 MHz, respectively. CDCl3 was employed for protected compounds and CD3OD as well as D2O for unprotected inhibitors. Chemical shifts are listed in delta employing residual, nondeuterated solvent as the internal standard. Signals were assigned unambiguously by COSY and HSQC analysis. The signals of the N-dansyl group are located in the expected regions and are not listed explicitly. For easier comparison with other N-acetylgalactosaminide analogues, interpretation of NMR-spectra was performed according to the carbohydrate-related numbering system depicted in Scheme 2. MALDI-TOF and EI-TOF mass spectrometry were performed on a Micromass (Waters Corporation, Milford, MA, USA) TofSpec 2E Time-of-Flight mass spectrometer. Analytical TLC was performed on pre-coated aluminum plates silica gel 60 F254 (E. Merck, Darmstadt, Germany 5554) and detected with UV light (254 nm). For staining, a solution of vanillin (9 g) in a mixture of H2O (950 mL)/EtOH (750 mL)/H2SO4 (120 mL) or ceric ammonium molybdate (100 g ammonium molybdate/8 g ceric sulfate in 1 l 10% H2SO4) were employed, followed by heating on a hotplate. For column chromatography, silica gel 60 (230-400 mesh, E. Merck 9385) or silica gel 60 (Acros Organics (Thermo Fisher Scientific Inc., Waltham, MA, USA), AC 24036) were used. CCDC contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk).

Biological Evaluation
New compounds turned out particularly potent inhibitors of Streptomyces plicatus N-acetyl-β-hexosaminidase (SpHex) with K i -values in the sub-nanomolar range (Table 1). By introduction of a dansylamido moiety into the alkyl chain, this activity was further improved as shown by the 60 pM value determined for inhibitor 25 when compared to analogue 21. With the same enzyme, compound 1 exhibited K i = 80 µM [26].

General Methods
Optical rotations were measured at 20 °C on a Perkin Elmer (Waltham, MA, USA) 341 polarimeter at a wave length of 589 nm and a path length of 10 cm.
NMR spectra were recorded on a Varian (Palo Alto, CA, USA) INOVA 500 operating at 499.82 MHz ( 1 H), and at 125.894 MHz ( 13 C), or on a Bruker (Billerica, MA, USA) Ultrashield spectrometer at 300.36 and 75.53 MHz, respectively. CDCl3 was employed for protected compounds and CD3OD as well as D2O for unprotected inhibitors. Chemical shifts are listed in delta employing residual, nondeuterated solvent as the internal standard. Signals were assigned unambiguously by COSY and HSQC analysis. The signals of the N-dansyl group are located in the expected regions and are not listed explicitly. For easier comparison with other N-acetylgalactosaminide analogues, interpretation of NMR-spectra was performed according to the carbohydrate-related numbering system depicted in Scheme 2. MALDI-TOF and EI-TOF mass spectrometry were performed on a Micromass (Waters Corporation, Milford, MA, USA) TofSpec 2E Time-of-Flight mass spectrometer. Analytical TLC was performed on pre-coated aluminum plates silica gel 60 F254 (E. Merck, Darmstadt, Germany 5554) and detected with UV light (254 nm). For staining, a solution of vanillin (9 g) in a mixture of H2O (950 mL)/EtOH (750 mL)/H2SO4 (120 mL) or ceric ammonium molybdate (100 g ammonium molybdate/8 g ceric sulfate in 1 l 10% H2SO4) were employed, followed by heating on a hotplate. For column chromatography, silica gel 60 (230-400 mesh, E. Merck 9385) or silica gel 60 (Acros Organics (Thermo Fisher Scientific Inc., Waltham, MA, USA), AC 24036) were used. CCDC contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12

General Methods
Optical rotations were measured at 20 °C on a Perkin Elmer (Waltham, MA, USA) 341 polarimeter at a wave length of 589 nm and a path length of 10 cm.
NMR spectra were recorded on a Varian (Palo Alto, CA, USA) INOVA 500 operating at 499.82 MHz ( 1 H), and at 125.894 MHz ( 13 C), or on a Bruker (Billerica, MA, USA) Ultrashield spectrometer at 300.36 and 75.53 MHz, respectively. CDCl3 was employed for protected compounds and CD3OD as well as D2O for unprotected inhibitors. Chemical shifts are listed in delta employing residual, nondeuterated solvent as the internal standard. Signals were assigned unambiguously by COSY and HSQC analysis. The signals of the N-dansyl group are located in the expected regions and are not listed explicitly. For easier comparison with other N-acetylgalactosaminide analogues, interpretation of NMR-spectra was performed according to the carbohydrate-related numbering system depicted in Scheme 2. MALDI-TOF and EI-TOF mass spectrometry were performed on a Micromass (Waters Corporation, Milford, MA, USA) TofSpec 2E Time-of-Flight mass spectrometer. Analytical TLC was performed on pre-coated aluminum plates silica gel 60 F254 (E. Merck, Darmstadt, Germany 5554) and detected with UV light (254 nm). For staining, a solution of vanillin (9 g) in a mixture of H2O (950 mL)/EtOH (750 mL)/H2SO4 (120 mL) or ceric ammonium molybdate (100 g ammonium molybdate/8 g ceric sulfate in 1 l 10% H2SO4) were employed, followed by heating on a hotplate. For column chromatography, silica gel 60 (230-400 mesh, E. Merck 9385) or silica gel 60 (Acros Organics (Thermo Fisher Scientific Inc., Waltham, MA, USA), AC 24036) were used. CCDC contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12

General Methods
Optical rotations were measured at 20 °C on a Perkin Elmer (Waltham, MA, USA) 341 polarimeter at a wave length of 589 nm and a path length of 10 cm.
NMR spectra were recorded on a Varian (Palo Alto, CA, USA) INOVA 500 operating at 499.82 MHz ( 1 H), and at 125.894 MHz ( 13 C), or on a Bruker (Billerica, MA, USA) Ultrashield spectrometer at 300.36 and 75.53 MHz, respectively. CDCl3 was employed for protected compounds and CD3OD as well as D2O for unprotected inhibitors. Chemical shifts are listed in delta employing residual, nondeuterated solvent as the internal standard. Signals were assigned unambiguously by COSY and HSQC analysis. The signals of the N-dansyl group are located in the expected regions and are not listed explicitly. For easier comparison with other N-acetylgalactosaminide analogues, interpretation of NMR-spectra was performed according to the carbohydrate-related numbering system depicted in Scheme 2. MALDI-TOF and EI-TOF mass spectrometry were performed on a Micromass (Waters Corporation, Milford, MA, USA) TofSpec 2E Time-of-Flight mass spectrometer. Analytical TLC was performed on pre-coated aluminum plates silica gel 60 F254 (E. Merck, Darmstadt, Germany 5554) and detected with UV light (254 nm). For staining, a solution of vanillin (9 g) in a mixture of H2O (950 mL)/EtOH (750 mL)/H2SO4 (120 mL) or ceric ammonium molybdate (100 g ammonium molybdate/8 g ceric sulfate in 1 l 10% H2SO4) were employed, followed by heating on a hotplate. For column chromatography, silica gel 60 (230-400 mesh, E. Merck 9385) or silica gel 60 (Acros Organics (Thermo Fisher Scientific Inc., Waltham, MA, USA), AC 24036) were used. CCDC contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12  The latter two compounds were also screened with Tay-Sachs disease-related human lysosomal N-acetyl-β-hexosaminidase A. For comparison, pyrimethamine [27], which under the same screening conditions exhibited IC 50 = 62 µM, as well as 2-acetamido-1,2-dideoxynojirimycin,1 (IC 50 31 µM) [28] were included in these studies.
Inhibitors 21 and 25 exhibited excellent properties with this vital human enzyme, considerably exceeding the activities of the reference compounds probed in this study.
In conclusion, compounds of this new aminocyclopentane-derived family of N-acetylgalactosaminide mimetics represent a potentially interesting class of N-acetyl-D-hexosaminidase inhibitors and pharmacological chaperones for treatment of Tay Sachs disease, in particular, when also considering their simplicity of synthesis.

Biochemical Methods
Streptomyces plicatus N-acetyl-β-hexosaminidase, SpHex, was expressed and purified in E. coli as described previously [27]. Kinetic studies were performed at 25 • C in the assay buffer (sodium phosphate (50 mM), sodium citrate (50 mM), NaCl (100 mM), BSA (2 mg/mL), pH = 6.0). The enzyme was incubated with different concentrations of the inhibitors for 2-5 min prior to the start of the reaction by addition of the substrate (4-nitrophenyl N-acetyl-β-D-glucosaminide) and the initial rates were measured by monitoring the increase in absorbance at 405 nm for three to five minutes using a microplate reader (Synergy H1, BioTek, VT, USA). K i determinations were performed using two or three different substrate concentrations. For each one of these substrate concentrations a range of five to eight different inhibitor concentrations bracketing the ultimately determined K i value were Human skin fibroblasts (wild type) were grown in minimal essential medium (MEM) with Earle's Salts (Sigma Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum, 400 µM L-glutamine, and 50 µg/mL gentamycin at 37 • C and 5% CO 2 . All cells used in this study were between the third and nineteenth passages.
All inhibitors were dissolved in DMSO in a concentration of 10 mM and diluted in 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl, 0.01% NaN 3 , and 0.01% Triton for the IC 50 -measurements.
Modified β-hexosaminidase A assays were used to estimate the half maximal inhibitory concentration (IC 50 ) of the particular inhibitor. For IC 50 determination, 0.001 to 100 µM of inhibitor was added to the assay mixture.
Activity was measured in normal fibroblasts. Data analysis was performed with Microcal™ Origin ® v6.0 (Origin Lab, Northampton, MA, USA) using a non-linear curve fitting module based on sigmoid curve fitting. 22 mmol) in CH 2 Cl 2 was added and the reaction was stirred for 15 min when Et 3 N (2.89 mL, 20.9 mmol) was added. The reaction mixture was allowed to reach ambient temperature and methanol (50 mL) and NaBH 4 (0.39 g, 10.4 mmol) were added. When completed conversion of cyclopentanone 13 was observed (tlc, 30 min), solvents were removed under reduced pressure and crude alcohol 14 was dissolved in CH 2 Cl 2 . The organic layer was extracted with saturated aqueous NaHCO 3 , dried (Na 2 SO 4 ), filtered and concentrated under reduced pressure. Purification on silica gel (cyclohexane/ethyl acetate 3:1 v/v) provided compound 14 as a pale yellow syrup (848 mg, 2.91 mmol, 55.8% from epimer 12).
(b) Via Dess-Martin oxidation: A 10% solution (w/v) of alcohol 12 (1.05 g, 3.60 mmol) in CH 2 Cl 2 was stirred with Dess-Martin periodinane (1.68 g, 3.96 mmol) at ambient temperature for 10 min. After completed conversion, the reaction mixture was washed with saturated aqueous NaHCO 3 , dried (Na 2 SO 4 ), and filtered. Removal of solvents under reduced pressure gave the crude product 13.
To a solution of crude ketone 13 in methanol (20 mL), NaBH 4 (0.273 g, 7.21 mmol) was added. When completed conversion was detected (tlc, 30 min), solvents were removed under reduced pressure, and the crude product was dissolved in CH 2 Cl 2 . The organic layer was extracted with saturated aqueous NaHCO 3 , dried (Na 2 SO 4 ), filtered, and concentrated under reduced pressure. Purification on silica gel (cyclohexane/ethyl acetate 3:1 v/v) provided compound 14 as a pale yellow syrup (0.769 g, 2.64 mmol, 73.2% over two steps).
After extended storage, a compound sample provided a few minute crystals, one of which could be exploited for X-ray structure determination (CCDC 1826202).
[α] A solution of alcohol 14 (848 mg, 2.91 mmol) in CH 2 Cl 2 (20 mL) was cooled to 0 • C. Pyridine (0.940 mL, 11.6 mmol) and trifluoromethanesulfonyl anhydride (0.637 mL, 3.78 mmol) were added. When completed conversion of the starting material was observed (10 min), the reaction mixture was washed consecutively with HCl (6%) and saturated aqueous NaHCO 3 . After drying with Na 2 SO 4 , the suspension was filtered, and the solvent was removed at room temperature under reduced pressure. Resulting crude triflate 15 was dissolved in DMF (20 mL), NaN 3 (1.14 g, 17.5 mmol) was added and the mixture was stirred at ambient temperature for 60 min. The reaction mixture was then concentrated under reduced pressure, the residue was dissolved with CH 2 Cl 2 , and the solution was washed with brine. The organic layer was dried (Na 2 SO 4 ), filtered, and concentrated under reduced pressure. Purification of the remaining residue on silica gel (cyclohexane/ethyl acetate 10: