Synthesis directed at the disruption of a protein-protein interaction in asthma

Alan C. Spivey

RSC/BMCS 2nd Symposium on Chemical Biology for Drug

Discovery AstraZeneca, Alderley Park

20th-21st March 2012

Format of presentation

• Asthma & allergic disorders – Background & biochemical cascade – The hIgE/FceRI protein protein interaction (PPI) – Therapeutic proof-of-concept - Xolair®

• Peptide-based antagonists – Synthesis & activity

• Development of the germyl-Stille Reaction

– A photolabile safety-catch system • The synthesis of aspercyclide A & its C19 methyl ether

– Racemic total synthesis, optical resolution – Asymmetric synthesis – Emerging SAR data

Asthma & allergic disorders – UK figures

• The prevalence of asthma is increasing worldwide:

– 10.8 million sufferers in the UK (~20%) – 12.5 GP consultations pa (183,000 bed days) – NHS annual asthma bill ~£890 million – Cost of lost work days estimated ~£1.2 billion

Nasser Allergy 2008, 1624 [DOI]

http://dx.doi.org/10.1039/b707517k

Asthma & allergic disorders - Medication

• The majority of current medications address ‘downstream’ symptoms, e.g. – Asthma: b2-adrenergic agonist bronohidilators e.g. albuterol

– Hay fever: H1 antagonist anti-histamines e.g. diphenhydramine

– Eczema: anti-inflammatories vs. leukotrienes e.g. zafirlukast

– Anaphylactic shock: injection of adrenaline (epinephrine)

Stinson C & EN 1997, Jan 6, 25 [DOI]

http://dx.doi.org/10.1039/b707517k

The allergic response – Overview of the cascade

Block PPI

Gould Ann.Rev. Immunol. 2003, 21, 579

Proof-of-principle – Antibody therapy

• XolairTM (Genentech, Novartis) was approved by the European Commission in Oct 2005 for the treatment of chronic asthma in all 25 EU member states

- Humanised monoclonal anti-IgE antibody (~149 kDa) - Prescribed for severe asthma/hey fever - Subcutaneous injections required every 2-4 weeks - Expensive (£10K/patient/year)

http://www.xolair.com/

http://www.xolair.com/

The IgE-FceRI protein-protein complex (1)

Gould & Sutton Nat. Rev. Immunol. 2008, 8, 205 [DOI]

• The ligand (IgE): – Y-shaped C2 symmetric dimer (L-H)2 – Heavy (e) chain is involved in FceRI

binding – Heavy chain comprises 1 variable (Ve)

and 4 constant (Ce1, Ce2, Ce3, & Ce4) domains

• The receptor (FceR1):

– membrane bound, comprising 4 subunits (abg2)

– extracellular a subunit is involved in IgE binding

– comprises two small (86 residue) globular domains a1 & a2

http://dx.doi.org/10.1038/nri2273

The IgE-FceRI protein-protein complex (2)

• Characteristics of PPI: – High affinity - Ka 10-10 M-1

– Biphasic interaction where initial ‘sensitisation’ is reversible

– Large surface area - 1830 Å2

– Only a small proportion of cross-linking is required to initiate exocytosis?

• Implications for antagonist design:

– require high binding affinity for competitive inhibition

– require good bio-availability to attain high intra-cellular concentration

PDB 1F6A Jardetzky 2000 [DOI]

Gould & Sutton Nat. Rev. Immunol. 2008, 8, 205 [DOI]

http://www.rcsb.org/pdb/home/home.dohttp://dx.doi.org/10.1038/nri2273

The IgE-FceRI protein-protein complex (3)

PDB 1F6A Jardetzky 2000 [DOI]

http://www.rcsb.org/pdb/home/home.do

Peptide-based antagonists of the IgE-FceRI PPI

PDB 1F6A Jardetzky 2000 [DOI]

http://www.rcsb.org/pdb/home/home.do

• Non-’epitope’ peptides:

• Epitope peptides:

Peptide antagonists of IgE-FceRI PPI

b-hairpin (binds to receptor)

IC50 = 1 μM

Starovasnik,, Biochemistry 2001, 40, 9828 [DOI]

Zeta-loop (binds to receptor)

IC50 = 36 nM

Starovasnik, Structure 2004, 12, 1289 [DOI]

[V6, A12] MCD

IC50 = 0.6 µM

Buku, Chem. Bio. Drug. Des. 2008, 72, 113 [DOI]

a2(C-C’) mimetic (binds to IgE)

IC50 = 30 μM

Sutton, Biochem. Soc. Trans. 1997, 25, 387 [DOI]

Ro 25-7162 a2(C-F) mimetic (binds to IgE)

IC50 = 40 μM

Danho, Proc. Am. Peptide Symp. 1997, June 14-19, 539 [DOI]

http://pubs.acs.org/doi/10.1021/bi0109360http://dx.doi.org/10.1016/j.str.2004.04.015http://www3.interscience.wiley.com/cgi-bin/fulltext/120750790/PDFSTARThttp://pubs.acs.org/doi/pdf/10.1021/ja963884o

The IgE-FceRI protein-protein complex - Hotspots

PDB 1F6A Jardetzky 2000 [DOI]

http://www.rcsb.org/pdb/home/home.do

The AB loop - Ce3/Ce4 Hinge region

• Site directed mutant hIgE (Phe349Ala) → just 10% mediator release (degranulation assay) – Presta J. Biol. Chem. 1994, 269, 26368 [DOI]

• Phe349 is situated in the middle of the ‘Omega’ loop at terminus of the Ce3 A-B b-strand:

PDB 1F6A Jardetzky 2000 [DOI]

http://www.jbc.org/content/269/42/26368.full.pdf+htmlhttp://www.rcsb.org/pdb/home/home.do

A disulfide-constrained A-B loop mimetic

• Simple disulfide constrained A-B loop epitope:

Helm Allergy 1997, 52, 1155 [DOI]

http://dx.doi.org/10.1021/jo026693e

Tolan amino acids as peptide loop constraints

• ...what if we introduced a more rigid constraint than a disulfide? – b-turn mimetics: e.g. ‘tolan’ amino acid: – Kemp Tetrahedron Lett. 1995, 36, 4175 [DOI]

• ...what about a suite of regioisomeric tolan amino acids & analogues?

With John McKendrick & Ratnasothy Srikaran J. Org. Chem. 2003, 68, 1843 [DOI]

http://dx.doi.org/10.1016/0040-4039(95)00775-8http://dx.doi.org/10.1021/jo026693e

A tolan-constrained A-B loop mimetic • Synthesis of 2,2’-tolan constrained peptide via ‘on-resin’ Sonogashira macrocyclisation:

– Only rink amide resin allows for cyclisation

With John McKendrick & Ratnasothy Srikaran J. Org. Chem. 2003, 68, 1843 [DOI]

http://dx.doi.org/10.1021/jo026693e

An array of tolan-constrained A-B loop mimetics

Synthesis of the tolan amino acids • NB. Installation of Ser353 allows for higher yielding subsequent macrocyclisations :

With Danny Offermann

Synthesis of hydrogenated 2,2’-tolans • The fully saturated 2,2’-bibenzyl amino acid:

• The partially saturated 2,2’-stilbene amino acid:

With Danny Offermann

Synthesis of the constrained A-B loop mimetics

With Danny Offermann

Constrained A-B loop mimetics – ELISA activity • ELISA data for the array of tolan & tolan-derived constrained A-B loop mimetics:

– linker & sequence dependent activity

with Danny Offermann & Jimmy Sejberg J. Org. Chem. 2012, 77, ASAP. IC50 = 660 ±70 mM

Ongoing work...

• Co-crystallisation with both proteins – with Brian Sutton & Mary Holdom, KCL

• Increase affinity by synthetic mutagenesis? – Use of germyl-Stille coupling...

with Bingli Mo & Jimmy Sejberg

The germyl-Stille reaction

O

GeR R

Cl

Where is germanium in the periodic table?

• Group 14: C, Si, Ge, Sn, Pb

Ge vs Si & Sn

• features: – Susceptibility to ipso-SEAr – intermediate between Si and Sn

– Better stability towards nucleophiles & bases cf. Si & Sn

• e.g. Vasella Helv. Chim. Acta, 1996, 79, 255

– Essentially non-toxic like Si cf. Sn! – Interesting and unique germylene etherate chemistry [Ge(II) cf. carbene) – Expensive: GeCl4 ~£300 /100 g...

Fluorous-tagged arylgermanes - Synthesis

OOGeCl2·

1)BrMg

OMe

2) 4M HCl in dioxane

(>2eq)

RMgX (Xs.)

R = 2-NapMe [86%]

IC8F17

mW 300 WH2O, c.HCl

20 min

C8F17GeCl3

C8F17Ge

OMe

ClCl

[78%]

[84%]

£

C8F17Ge

OMe

RR1) 4M HCl in dioxane2) Ar-MgBr

R = 2-NapMe, Ar = p-Tol [71%]R = 2-NapMe, Ar = Ph [83%]R = 2-NapMe, Ar = p-ClC6H4 [80%]R = 2-NapMe, Ar = p-CF3C6H4 [84%]R = 2-NapMe, Ar = o-MeOC6H4 [87%]

C8F17Ge

RR

Ar

features • can ‘tune’ steric and electronic

properties of R groups for specific applications

• fluorous-tag allows rapid purification by FC on fluorous SiO2

with David Whitehead & Joseph Hannah

Zhang Chem. Rev. 2004, 104, 2531 [DOI]

http://dx.doi.org/10.1021/cr030600r

Pd(0) catalysed cross-coupling

• Sn couples readily as SnR3: Stille coupling – review: Stille Angew. Chem. Int. Ed. 1986, 98, 508 [DOI]

• Si requires an eletronegative ligand and nucleophilic activation: Hiyama-Denmark coupling – review: Denmark Aldrichimica Acta 2003, 36, 75 [DOI]

• what is required for Ge coupling? – review: with Chris Gripton & Joseph Hannah Curr. Org. Synth. 2004, 1, 111 [DOI]

http://dx.doi.org/10.1002/anie.198605081http://www.sigmaaldrich.com/aldrich/acta/al_acta_36_3.pdfhttp://www.ch.ic.ac.uk/spivey/publicationpdfs/currorgsynth20041211.pdf

Requirement for di-halogermanes

• Ge requires 2 × electronegative ligands and nucleophile activation – initial results: (cf. conditions of Hiyama Tetrahedron Lett. 1997, 38, 439 [DOI]

– optimised conditions:

GeR R

MePd(OAc)2 (5 mol%)

PPh3 (10 mol%)NaOH (6 eq)

THF, 70 °C, 24 h

O

Br CF3

CF3(1 eq) Me

CF3

CF3

R2 = Me2 [0%]R2 = Me,Cl [0%]R2 = Cl2 [36%]

OEt

with Chris Gripton

# R Ar Yield/% 1 Me 4-AcC6H4 60 2 Me 3,5-(CF3)2C6H3 63 3 Me 1-Nap 79 4 OMe Ph 36 5 OMe 3-CF3C6H4 51 6 OMe 3,5-(CF3)2C6H3 71 7 OMe 1-Nap 56 8 OMe 3-Py 44 9 OMe 4-NO2C6H4 47

GeCl Cl

R PdCl2(MeCN)2 (10 mol%)dppp (20 mol%)

KF (6 eq)DMF, 120 °C, 8 h

O R

Ar-Br (1eq)ArOEt

http://dx.doi.org/10.1016/S0040-4039(96)02320-9

A safety-catch germyl-Stille reaction?

• Target:

• Safety-catch R groups must:

• confer stability to a wide range of reaction conditions prior to activation (i.e. retain unique advantages of Ge vs. Si or Sn vis-à-vis stability to strong bases and nucleophiles)

• be selectively activated with in presence of wide range of FGs – ‘magic bullet’ conditions

Photo-oxidative ‘activation’ of benzylic ‘R’ groups

• 2-naphthylmethyl groups can be selectively 'activated' by photo-oxidation with Cu(II)

• mechanism: – cf. Otsuji Chem. Lett. 1988, 229 [DOI]

with Chris Gripton, Joseph Hannah & Chih-Chung Tseng Appl. Organomet. Chem. 2007, 21, 572 [DOI]

GePh Ph

OMeO

OEt

Ge2-Nap 2-Nap

OMeO

OEt

http://dx.doi.org/10.1246/cl.1988.229http://dx.doi.org/10.1002/aoc.1270

The photolysis apparatus • ...a tower PC case:

...a Cathodeon HPW 125W lamp

Convenient monitoring – 19F NMR

• sequential photooxidation of both groups monitored by 19F NMR:

with Chih-Chung Tseng

C8F17Ge

Cu(BF4)2 (4 eq)MeCN:MeOH (3:1)

h, pyrex filter10 min

OMe

2-Nap 2-Nap

OMe ~1 h

OMe

C8F17GeF F

OMe

C8F17Ge

F2-Nap

OMe

Scope of germyl-Stille aryl-aryl cross-coupling

with Chih-Chung Tseng, Joseph Hannah & Chirs Gripton Chem. Comm. 2007, 2926 [DOI]

Ar

Ge2-Nap2-Nap

R

GeF F

R

R

Ar-Br (2 eq)

C8F17

C8F17

(2 eq)

Cu(BF4)2 (4 eq)MeCN:MeOH (3:1)

h, pyrex filter~2 h

PdCl2(MeCN)2 (10 mol%)P(2-Tol)3 (15 mol%)

TBAF·3H2O (2.7 eq), CuI (1 eq)DMF, 120 °C, 16 h

OMe

# R Ar Yield/% 1 4-OMe 3,5-(CF3)2C6H3 96 2 4-OMe 4-ClC6H4 85 3 4-OMe 4-BnOC6H4 65 4 4-OMe 1-Nap 75 5 4-Me 3,5-(CF3)2C6H3 84 6 4-Me 4-ClC6H4 69 7 4-Me 4-BnOC6H4 48 8 4-Me 1-Nap 71 9 H 3,5-(CF3)2C6H3 74 10 H 4-ClC6H4 63 11 H 4-BnOC6H4 40 12 H 1-Nap 60 13 4-Cl 3,5-(CF3)2C6H3 71 14 4-Cl 4-BnOC6H4 42 15 4-Cl 1-Nap 75 16 2-OMe 3,5-(CF3)2C6H3 65 17 2-OMe 4-ClC6H4 49 18 2-OMe 4-BnOC6H4 11 19 2-OMe 1-Nap 27 20 4-CF3 3,5-(CF3)2C6H3 26 21 4-CF3 4-ClC6H4 11

http://dx.doi.org/10.1039/b707517k

Total synthesis of aspercyclide A – a natural product antagonist of the

IgE-FceRI PPI

Aspergillus sp.

Small molecule antagonists of IgE-FceRI PPI

• Fluorescein dyes MW ~700–1100 Da

– e.g. Na2-Rose Bengal – Cheng (Heska Corp.) US patent 5,965,605 12th Oct 1999 – IC50 = 0.5 mM (MW = 1017 Da)

• (+) Aspercyclide A MW 410 Da – Soil fungus metabolite ex. Aspergillus sp. – Singh (Merck) Tetrahedron Lett. 2004, 45, 7605 [DOI] – IC50 of 200 mM (MW = 410 Da)

X-ray

MM2

http://dx.doi.org/10.1016/j.tetlet.2004.08.116

(±)-Aspercyclide A – Syntetic strategy

• Features & strategy – 11 membered ring – labile paraquinol – trans-alkene – anti-1,2-diol – hindered ester – di-ortho-substituted biaryl ether

• Aryl-alkene Germyl-Stille key step?

Anti-1,2-diol formation & ring B esterification

• Boeckman modified Takai-Utimoto Condensation – Boeckman J. Org. Chem. 1998, 63, 3524 [DOI]

• Hindered ester formation with di-ortho-substituted benzoate

X-ray with James Carr

via

http://dx.doi.org/10.1021/jo980160h

Ring A synthesis and vinyl germane formation

• (E)-Vinyl germane synthesis – cf. (E)-selective hydroboration: Srebnik Tetrahedron Lett. 1996, 37, 3283 [DOI]

• Ring A synthesis

– Porco Jr Org. Lett. 2001, 3, 1649 [DOI]

X-ray

with James Carr

http://dx.doi.org/10.1016/0040-4039(96)00576-Xhttp://dx.doi.org/10.1021/ol0159367

Biaryl ether formation & germyl-Stille macrocyclisation

• Biaryl ether formation – vinyl germane substrate – cf. Kulkarni Tetrahedron 1988, 44, 5145 [DOI]

• germyl-Stille macrocyclisation

with James Carr & Jimmy Sejberg Org. Biomol.Chem. 2011, 6814

http://dx.doi.org/10.1016/S0040-4020(01)86020-8

Biaryl ether formation & Heck macrocyclisation

• Biaryl ether formation – terminal alkene substrate – Kulkarni Tetrahedron 1988, 44, 5145 [DOI]

• Heck macrocyclisation

– cf. Nolan J. Organomet. Chem. 2003, 687, 269 [DOI]

Entry Additive Ratio A:B:C

Conversion

ArBr - 0 : 1 : 4.2 100%

ArBr AgI (1 eq.) 10 : 5 : 1 30%

ArI AgI (1 eq.) 2.5 : 10 : 1 75% (52% isolated)

Aromatic Finklestein:

Buchwald J. Am. Chem. Soc. 2002, 124, 14884 [DOI] with James Carr

http://dx.doi.org/10.1016/S0040-4020(01)86020-8http://dx.doi.org/10.1016/S0022-328X(03)00375-9http://dx.doi.org/10.1021/ja028865v

Deprotection & oxidation • Acetal hydrolysis & benzylic oxidation...

• Need a new C19 OH protecting group…

X-ray

with James Carr

Use of PMB protection – synthesis of (±)-aspercyclide

with James Carr & Daniel Offermann Chem. Commun., 2010, 46, 1777 [DOI]

http://dx.doi.org/10.1039/c003190a

Aspercyclide A & its C19 Me ether – ELISA activity

• Racemic ‘Nat Prod’ vs. C19 Me ether – ELISA – (±)-C19 Me ether is equipotent wrt parent! – IC50 ~50 mM

• Enantiomers of C19 Me ether – Separation by CSP-HPLC (Chiralpak IA) – ELISA – (+)-C19 Me ether has natural configuration – 10 fold more IC50 ~50 mM

Entry Compound (% ee)

IC50 (µM)

1 (+)-asp (98.4) 40 ± 1

2 (–)-31 (98.8) 483 ± 105

3 (±)-31 56 ± 2

with Jimmy Sejberg, Helena Dennison & Mary Holdom (KCL)

CD curve for (+)-aspercyclide

Enantioselective synthesis of ()-aspercyclide A

with Jimmy Sejberg

cf. Krische J. Org. Chem. 2011, 76, 2350 [DOI]

http://dx.doi.org/10.1021/jo200068q

SAR studies - Ongoing work...

• Synthesis of analogues → SAR – with Helena Dennison & Jimmy Sejberg

• Co-crystallisation with protein, SPR , NMR... – with Lucy Smith, IC – with Brian Sutton & Andrew Beavil, KCL

SAR studies – aspercyclide C19 OMe analogues... • A-ring analogues → SAR

• B-ring analogues → SAR

with Jimmy Sejberg, Helena Dennison & Lucy Smith

SAR studies – aspercyclide C19 OMe analogues... • Macrocycle ‘strap’ analogues → SAR

• Dibenzofuran A-ring & ‘strap’ analogues → SAR

with Jimmy Sejberg, Helena Dennison & Lucy Smith

SAR studies – aspercyclide C19 OMe analogues... • Dibenzofuran B-ring & ‘strap’ analogues → SAR

with Jimmy Sejberg, Helena Dennison & Lucy Smith

Acknowledgements • peptides: John McKendrick & Ratnasothy Srikaran (Birgit Helm @MBB, Sheffield), Daniel Offermann, Bingli Mo,

Jimmy Sejberg & Lucy Smith (Robin Leatherbarrow @IC; Andrew Beavil @KCL; Brian Sutton @KCL; Mary Holdom @KCL)

• Germyl-Stille: Chris Gripton, (Jan Scinski @GSK), Joseph Hannah & Chih-chung (Jimy) Tseng • aspercyclide A: James Carr (Steve Lindell @)Bayer CropScience), Daniel Offermann , Jimmy Sejberg & Helena

Dennison (Keith Spencer & Kevin Foote@Arrow Therapeutics/AstraZeneca; Fabienne Saab @KCL) • X-ray: Chris Frampton @ Pharmorphix & Andrew J.P. White @IC

• EPSRC, Pfizer, Roche, Bayer CropScience, AstraZeneca, Novartis, Wellcome Trust, MRC, EU FP7 Marie Curie

http://portal.bayercropscience/http://www.epsrc.ac.uk/