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Design, synthesis and biological evaluation of novel
hexosaminidase inhibitors
Design, synthesis and biological evaluation of novel
hexosaminidase inhibitors
Shane Rountree
(3rd Year DPhil)
Shane Rountree
(3rd Year DPhil)
Supervisors: Raymond Dwek, Terry Butters (Glycobiology)George Fleet (Organic Chemistry)
11th May 200511th May 2005
Why inhibition of hexosaminidases?Why inhibition of hexosaminidases?
• Family of glycosidases
• Have been studied less extensively
• Although involved in a number of key biological pathways
Inhibitors of both - and - types of hexosaminidase enzymes have potential medicinal applications
• Family of glycosidases
• Have been studied less extensively
• Although involved in a number of key biological pathways
Inhibitors of both - and - types of hexosaminidase enzymes have potential medicinal applications
-N-acetyl-galactosaminidase (anti-cancer?)-N-acetyl-galactosaminidase (anti-cancer?)
• Has been shown to accumulate in serum of cancer patients
• Results in deglycosylation of the vitamin D3-binding protein (DBP)
• DBP is a precursor for a potent macrophage activating factor (DBP-MAF)
• Activated macrophages have the potential to kill and eliminate cancerous cells
• Has been shown to accumulate in serum of cancer patients
• Results in deglycosylation of the vitamin D3-binding protein (DBP)
• DBP is a precursor for a potent macrophage activating factor (DBP-MAF)
• Activated macrophages have the potential to kill and eliminate cancerous cells
• Comparative Biochemistry and Physiology Part A, 2002, 132, 1–8• Comparative Biochemistry and Physiology Part A, 2002, 132, 1–8
Formation of DBP-MAF / DBP-MAF protein deglycosylation
Formation of DBP-MAF / DBP-MAF protein deglycosylation
-hexosaminidase (GSL Storage disorders)-hexosaminidase (GSL Storage disorders)
• Inhibitors are potential drug candidates for osteoarthritis via prevention of cartilage matrix glycosaminoglycan degradation (Chem.&Biol., 2001, 8, 701-711)
• May also have application for GSL lysosomal storage disorders:– Tay-Sachs (-hexosaminidase A)– Sandhoff (-hexosaminidase A and B)
Accumulation of GM2 ganglioside
• Rare autosomal recessive disorders that are often associated with profound mental deterioration in early infancy and in severe cases, death
• Inhibitors are potential drug candidates for osteoarthritis via prevention of cartilage matrix glycosaminoglycan degradation (Chem.&Biol., 2001, 8, 701-711)
• May also have application for GSL lysosomal storage disorders:– Tay-Sachs (-hexosaminidase A)– Sandhoff (-hexosaminidase A and B)
Accumulation of GM2 ganglioside
• Rare autosomal recessive disorders that are often associated with profound mental deterioration in early infancy and in severe cases, death
Chemical Chaperone ApproachChemical Chaperone Approach
• The deficiency is due to genetic defects that cause incorrect folding of lysosomal enzyme resulting in retention in ER and premature degradation
• This may cause the accumulation of GM2 gangliosides resulting in degeneration of the CNS
• Potential therapy is based on competitive inhibitors helping the mutant enzyme to fold properly to proceed to the lysosome
• Initial studies have shown adult Tay-Sachs fibroblasts grown in culture medium containing inhibitors have raised activity well above the critical level. (Tropak et al., J.Biol.Chem., 2004, 279, p13478-13487)
• Sub-inhibitory concentrations of competitive inhibitors required since inhibiton of lysosomal enzyme would pronounce the disease
• The deficiency is due to genetic defects that cause incorrect folding of lysosomal enzyme resulting in retention in ER and premature degradation
• This may cause the accumulation of GM2 gangliosides resulting in degeneration of the CNS
• Potential therapy is based on competitive inhibitors helping the mutant enzyme to fold properly to proceed to the lysosome
• Initial studies have shown adult Tay-Sachs fibroblasts grown in culture medium containing inhibitors have raised activity well above the critical level. (Tropak et al., J.Biol.Chem., 2004, 279, p13478-13487)
• Sub-inhibitory concentrations of competitive inhibitors required since inhibiton of lysosomal enzyme would pronounce the disease
• Sugar mimics in which the ring oxygen has been replaced by a nitrogen atom
• Many are naturally occurring and are known to be potent glycosidase inhibitors
• Sugar mimics in which the ring oxygen has been replaced by a nitrogen atom
• Many are naturally occurring and are known to be potent glycosidase inhibitors
HN
OHHO
OH
HOHN
OHHO
HO NHO
OHHO
deoxynojirimycin (DNJ)
piperidines
D-AB1
pyrrolidines
(-)-swainsonine
indolizidines
IminosugarsIminosugars
Pyrrolidines vs PiperidinesPyrrolidines vs Piperidines
• Pyrrolidines are the less studied
• But often better inhibitors than their corresponding piperdine analogues
• They are thought to be better TS mimics
• Pyrrolidines are the less studied
• But often better inhibitors than their corresponding piperdine analogues
• They are thought to be better TS mimics
O
HO
HOHO
O O
ORH
O
O
∂+
∂-
∂-
∂-
NH2HOHO
HO
HO
OH
O
O O
O∂-
∂-
Proposed oxonium ion TS Pyrrolidine mimic
General target structureGeneral target structure
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
ARABINO RIBO
XYLO LYXO
D D
DD
L
L L
L
First target compoundsFirst target compounds
Compare with these known iminosugars:Compare with these known iminosugars:
potent -D-glucosidase
some -D-glucosidase and -D-mannosidase activity
potent -D-glucosidase activity
HN
NHAcHO
HO
HN
NHAcHO
HO
DAB-NAc LAB-NAc
HN
OHHO
HO
HN
OHHO
HO
DAB1 LAB1
Naturally occurring:Angylocalyx boutiqueanus,Morus alba
Synthesis of LAB-NAcSynthesis of LAB-NAc
OOHO
OO
O
OHOPh
OO
N3
O
OPh
O
Ph
O
OTf
O
O
OH
N3
O
OPh
OMs
N3
O
OPh
H2/Pd-Black
PhCHO Tf2O
py
NaN3
LiBH4
DMF
OHOMs
THF
H2/Pd-C
HCl
OMs
N3
O
OPh
HN
BnNH2Bn
Ac2O
1,4-dioxane
Amberlyst-15
NaOMe
MeOH
N
N3HO
HO
Bn
N
NH2HO
HO
BnN
NHAcAcO
AcO
Bn
N
NHAcHO
HO
BnHN
NHAcHO
HO
MsCl
DCM, Et3N
THF
pyridine
1,4-dioxane/ H2O
1,4-dioxane/ H2O
90% 89% 92%
98%90%75%89%
99% 75% 96% 100%
OHHO
Crystal structure of NBn-LAB-NAcCrystal structure of NBn-LAB-NAc
C.C. Harding, J.S.S. Rountree, D.J. Watkin, T.D. Butters, M.R. Wormald,R.A. Dwek, G.W.J. Fleet, Acta Cryst., 2005, E61, in press C.C. Harding, J.S.S. Rountree, D.J. Watkin, T.D. Butters, M.R. Wormald,R.A. Dwek, G.W.J. Fleet, Acta Cryst., 2005, E61, in press
N
NHAcHO
HO
Bn
Synthesis of DAB-NAcSynthesis of DAB-NAc
OOHO
OO
O
OHOPh
OO
N3
O
OPh
O
Ph
O
OTf
O
O
OH
N3
O
OPh
OMs
N3
O
OPh
H2/Pd-Black
PhCHO Tf2O
py
NaN3
LiBH4
DMF
OHOMs
THF
H2/Pd-C
HCl
OMs
N3
O
OPh
HN
BnNH2Bn
Ac2O
1,4-dioxane
Amberlyst-15
NaOMe
MeOH
N
N3HO
HO
Bn
N
NH2HO
HO
BnN
NHAcAcO
AcO
Bn
N
NHAcHO
HO
BnHN
NHAcHO
HO
MsCl
DCM, Et3N
THF
pyridine
1,4-dioxane/ H2O
1,4-dioxane/ H2O
95% 100% 78%
83%90%79%94%
99% 90% 89% 100%
O O
Biological evaluationBiological evaluation
• No significant inhibition for -N-acetyl-galactosaminidase or any of the other common - and -glycosidases
• No significant inhibition for -N-acetyl-galactosaminidase or any of the other common - and -glycosidases
-Hexosaminidase IC50 Ki IC50 Ki IC50 Ki IC50 Ki
bovine kidney 0.36 0.277 0.64 0.095 41.2 16.9 326 104
jack bean 7.72 3.11 3.52 4.5 446 ND - -
human placenta 2.8 ND 13 ND 320 ND - -
N
NHAcHO
HO
BnHN
NHAcHOHO
N
NHAcHO
HO
Bn HN
NHAcHO
HO
Showing IC50 and Ki values for the new inhibitors in M
[“ND” = not determined, “-” = no inhibition]
NBn-LAB-NAc LAB-NAc NBn-DAB-NAc DAB-NAc
Overlay of DAB-NAc and LAB-NAc with -GlcNAcOverlay of DAB-NAc and LAB-NAc with -GlcNAc
DAB-NAc LAB-NAc
GlcNAc GlcNAc
Produced using InsightII and Discover software (Accelrys Inc., San Diego, USA)Produced using InsightII and Discover software (Accelrys Inc., San Diego, USA)
Non-competitive inhibition of LAB-NAcNon-competitive inhibition of LAB-NAc
Control
2.5 M
5 M
0
0.25
0.5
0.75
1
1.25
-0.5 0 0.5 1 1.5 2
1/[ ] ( )S mM
1/ (v
_ / / )mol mg min
Control
2.5 M
5 M
0
0.25
0.5
0.75
1
1.25
-0.5 0 0.5 1 1.5 2
1/[ ] ( )S mM
1/ (v
_ / / )mol mg min
NBn-LAB-NAc vs -Hex (jack bean)
L-iminosugars as enzyme inhibitorsL-iminosugars as enzyme inhibitors
• Both LAB1 and L-DMDP are more potent inhibitors of plant and mammalian -D-glucosidases than their enantiomers, DAB1 and DMDP (Chem.Comm., 2004, 1936; Tetrahedron Asymm., 2005, 16, 223)
• The D-enantiomers are competitive and the L- are non-competitive - this has also been shown for DNJ and DGJ (J. Med. Chem., 2005, 48, 2036)
• It is thought that the L-enantiomers bind somewhere other than the active site (regulatory site), thereby changing its shape so that substates cannot fit
• Both LAB1 and L-DMDP are more potent inhibitors of plant and mammalian -D-glucosidases than their enantiomers, DAB1 and DMDP (Chem.Comm., 2004, 1936; Tetrahedron Asymm., 2005, 16, 223)
• The D-enantiomers are competitive and the L- are non-competitive - this has also been shown for DNJ and DGJ (J. Med. Chem., 2005, 48, 2036)
• It is thought that the L-enantiomers bind somewhere other than the active site (regulatory site), thereby changing its shape so that substates cannot fit
HN
OHHO
HN
OHHO
HO
DMDP L-DMDP
OHOHHO
HN
OHHO
HO
HN
OHHO
HO
DAB1 LAB1
Part II Project - Barry GibneyPart II Project - Barry Gibney
• Following the discovery that LAB-NAc was a -hexosaminidase inhibitor, it was decided to introduce to a cell (mouse macrophage)
• Preliminary work has shown that 0.5 mM concentration of inhibitor exhibited better inhibition of the lysosomal enzyme than -GlcNAc
• This caused a deficiency of the lysosomal enzyme and induced a Sandhoff phenotype
• Following the discovery that LAB-NAc was a -hexosaminidase inhibitor, it was decided to introduce to a cell (mouse macrophage)
• Preliminary work has shown that 0.5 mM concentration of inhibitor exhibited better inhibition of the lysosomal enzyme than -GlcNAc
• This caused a deficiency of the lysosomal enzyme and induced a Sandhoff phenotype
0
100
200
300
400
500
600
700
800
900
1000
1 2 3
Amount as a % of control
GA2GM2GM1a
Glycolipid storage in inhibitor-treated cellsGlycolipid storage in inhibitor-treated cells
NBn-LAB-NAc
100 M 100 M(no inhibitor)
Control DNJ-NAc
Second target compoundsSecond target compounds
Compare with the known iminosugars:Compare with the known iminosugars:
potent -D-glucosidase (yeast)and -D-galactosidase (lactase)
also potent against some nucleoside hydrolases, eukaryotic DNA polymerases and modest inhibition of HIV replication
modest inhibition of HIV replication
HN
NHAcHO
HO
HN
NHAcHO
HO
DRB-NAc LRB-NAc
HN
OHHO
HO
HN
OHHO
HO
DRB1 LRB1
Naturally occurring:Morus alba
OOHO
OO
O
OHOPh
OO
N3
O
OPh
O
Ph
O
OTf
O
O
OH
N3
O
OPh
OMs
N3
O
OPh
H2/Pd-Black
PhCHO Tf2O
py
NaN3
L-Selectride
DMF
OHOMs
THF
HCl
OMs
N3
O
OPh
HN
BnNH2Bn
Ac2O
Amberlyst-15
NaOMe
MeOH
N
N3HO
HO
Bn
N
NH2HO
HO
Bn
N
NHAcAcO
AcO
Bn
N
NHAcHO
HO
BnHN
NHAcHO
HO
MsCl
DCM, Et3N
pyridine
OHHO
PPh3, H2O
90% 86% (2 steps)
57%91%80%89%
THF
89% 85% 91% 88%
1,4-dioxane/ H2O
1,4-dioxane/ H2O
Synthesis of LRB-NAcSynthesis of LRB-NAc
Crystal structure of NBn-LRB-NAcCrystal structure of NBn-LRB-NAc
C.C. Harding, D.J. Watkin, J.S.S. Rountree, T.D. Butters, M.R. Wormald,R.A. Dwek, G.W.J. Fleet, Acta Cryst., 2005, E61, o930-o932 C.C. Harding, D.J. Watkin, J.S.S. Rountree, T.D. Butters, M.R. Wormald,R.A. Dwek, G.W.J. Fleet, Acta Cryst., 2005, E61, o930-o932
N
NHAcHO
HO
Bn
Biological evaluation of DRB-NAc and LRB-NAcBiological evaluation of DRB-NAc and LRB-NAc
• NBn-DRB-NAc and NBn-LRB-NAc were screened against the following enzymes and showed no significant activity:
yeast -glucosidasealmond -glucosidasecoffee bean -galactosidasejack bean -galactosidasejack bean -mannosidasesnail -mannosidasebovine -fucosidaseAspergillus niger -xylosidase
jack bean -hexosaminidaseCharonia lampas -N-acetyl-galactosaminidase
• NBn-DRB-NAc and NBn-LRB-NAc were screened against the following enzymes and showed no significant activity:
yeast -glucosidasealmond -glucosidasecoffee bean -galactosidasejack bean -galactosidasejack bean -mannosidasesnail -mannosidasebovine -fucosidaseAspergillus niger -xylosidase
jack bean -hexosaminidaseCharonia lampas -N-acetyl-galactosaminidase
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
HN
NHAcHO
HO
ARABINO RIBO
XYLO LYXO
D D
DD
L
L L
L
Target structuresTarget structures
Compare with these known iminosugars:Compare with these known iminosugars:
potent -D-galactosidase
some -D-glucosidase, -D-galactosidase and
-D-mannosidase activity
HN
NHAcHO
HO
HN
NHAcHO
HO
D-lyxo L-lyxo
HN
NHAcHO
HO
HN
NHAcHO
HO
D-xylo L-xylo
HN
OHHO
HO
HN
OHHO
HO
D-lyxo L-lyxo
HN
OHHO
HO
HN
OHHO
HO
D-xylo L-xylo
Naturally occurring:Angylocalyx pynaertii
Third and fourth target compoundsThird and fourth target compounds
Current synthesis problems in the L-lyxo routeCurrent synthesis problems in the L-lyxo route
O
OH
OO
OPh
O
N3
OO
OPh
O
HOO
N3HO
(i) Tf2O, py
(ii) NaN3, DMF
TFA/H2O
2:1
60% (2 steps) 87%
O
TBDMSOO
N3TBDMSO
73%
imidazole, DMF
TBDMSCl
O
TBDMSOO
N3TBDMSO
H-mixture of two inseparable
diols
silyl migration
epimerisation
OH
TBDMSOOH
N3TBDMSO
OTBDMS
HOOH
N3TBDMSO
+
OH
TBDMSOOH
N3TBDMSO
OH
TBDMSOOH
N3TBDMSO
+
OR
O
ROO
RO N3
HB
O
ROO
N3RO
BH
O
ROO
N3RO
O
ROO
N3RO
+
enolisation lactonisation
Epimerisation mechanism:
However, not all bad… (?!)However, not all bad… (?!)
mixture of two inseparable
diols
MsCl, Et3N
DCM
OMs
TBDMSOOMs
N3TBDMSO
OTBDMS
MsOOMs
N3TBDMSO
OMs
TBDMSOOMs
N3TBDMSO
+ OR
N
N3TBDMSO
TBDMSO
BnN
N3TBDMSO
TBDMSON
N3TBDMSO
TBDMSO
Bn Bn
HN
NHAcHO
HO
NH
NHAcHO
HO
HN
NHAcHO
HO
L-lyxo L-xylo
EPIMERISATION MIGRATION
Future WorkFuture Work
• Continue new work to make both enantiomers of the pyrrolidine iminosugars with lyxo- and xylo- stereochemistry
• Evaluate these compounds for biological activity
• Look at different N-alkylated analogues of some inhibitors including photoaffinity labelling
• Continue new work to make both enantiomers of the pyrrolidine iminosugars with lyxo- and xylo- stereochemistry
• Evaluate these compounds for biological activity
• Look at different N-alkylated analogues of some inhibitors including photoaffinity labelling
ConclusionsConclusions
• Completed the syntheses of DAB-NAc, LAB-NAc, DRB-NAc and LRB-NAc in good overall yields
• LAB-NAc has been shown to be a very potent and selective -hexosaminidase inhibitor
• NBn-LAB-NAc is currently being used in the Butters group as a -hexosaminidase inhibitor for biological studies
• Hopefully some of the other targets that we are making will inhibit -N-acetyl-galactosaminidase - our main target enzyme!
• Completed the syntheses of DAB-NAc, LAB-NAc, DRB-NAc and LRB-NAc in good overall yields
• LAB-NAc has been shown to be a very potent and selective -hexosaminidase inhibitor
• NBn-LAB-NAc is currently being used in the Butters group as a -hexosaminidase inhibitor for biological studies
• Hopefully some of the other targets that we are making will inhibit -N-acetyl-galactosaminidase - our main target enzyme!
AcknowledgementsAcknowledgements
Supervisors:
Raymond DwekTerry ButtersGeorge Fleet
Supervisors:
Raymond DwekTerry ButtersGeorge Fleet
NMR/ Molecular Modeling:
Mark Wormald (OGI)Mukram Mackeen (OGI)Barbara Odell (CRL)
NMR/ Molecular Modeling:
Mark Wormald (OGI)Mukram Mackeen (OGI)Barbara Odell (CRL)
Enzyme screening:
Robert Nash (MNLpharma)Naoki Asano (Hokuriku University)
Enzyme screening:
Robert Nash (MNLpharma)Naoki Asano (Hokuriku University)
Co-workers:
Fleet Group1st Floor Glycobiology
Co-workers:
Fleet Group1st Floor Glycobiology
Funding: Oxford Glycobiology Institute
X-ray Crystallography:
Chris HardingDave Watkin
X-ray Crystallography:
Chris HardingDave Watkin
Cell Line Work:
Barry Gibney
Cell Line Work:
Barry Gibney