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