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]
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]
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/
The IgE-FceRI protein-protein complex (1)
•
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
Gould & Sutton Nat. Rev. Immunol. 2008, 8, 205 [DOI]
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]
The IgE-FceRI protein-protein complex (3)
PDB 1F6A Jardetzky 2000 [DOI]
Peptide-based antagonists of the
IgE-FceRI PPI
PDB 1F6A Jardetzky 2000 [DOI]
Peptide antagonists of IgE-FceRI PPI
•
•
Non-’epitope’ peptides:
Zeta-loop (binds to receptor)
b-hairpin (binds to receptor)
[V6, A12] MCD
IC50 = 36 nM
IC50 = 1 μM
IC50 = 0.6 µM
Starovasnik, Structure 2004, 12,
1289 [DOI]
Starovasnik,, Biochemistry 2001,
40, 9828 [DOI]
Buku, Chem. Bio. Drug. Des. 2008,
72, 113 [DOI]
Epitope peptides:
a2(C-C’) mimetic (binds to IgE)
Ro 25-7162 a2(C-F) mimetic (binds to IgE)
IC50 = 30 μM
IC50 = 40 μM
Sutton, Biochem. Soc. Trans.
1997, 25, 387 [DOI]
Danho, Proc. Am. Peptide Symp. 1997, June 1419, 539 [DOI]
The IgE-FceRI protein-protein complex - Hotspots
PDB 1F6A Jardetzky 2000 [DOI]
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]
A disulfide-constrained A-B loop mimetic
•
Simple disulfide constrained A-B loop epitope:
Helm Allergy 1997, 52, 1155 [DOI]
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]
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]
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
IC50 = 660 ±70 mM
with Danny Offermann & Jimmy Sejberg J. Org. Chem. 2012, 77, ASAP.
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
R R
Ge
Cl
O
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
C8F17
GeCl2·O
O
I
£
GeCl3
C8F17
mW 300 W
H2O, c.HCl
20 min
[78%]
OMe
1)
(>2eq)
BrMg
2) 4M HCl in dioxane
C8F17
R R
Ge
Ar
1) 4M HCl in dioxane
2) Ar-MgBr
C8F17
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%]
RMgX (Xs.)
•
Cl Cl
Ge
C8F17
R = 2-NapMe [86%]
•
Zhang Chem. Rev. 2004, 104, 2531 [DOI]
R R
Ge
OMe
[84%]
OMe
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
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]




Requirement for di-halogermanes
•
Ge requires 2 × electronegative ligands and nucleophile activation
–
initial results: (cf. conditions of Hiyama Tetrahedron Lett. 1997, 38, 439 [DOI]
Br
OEt
R R
Ge
CF3
O
–
OEt
O
CF3
Me
Me
(1 eq)
CF3
Pd(OAc)2 (5 mol%)
PPh3 (10 mol%)
NaOH (6 eq)
THF, 70 °C, 24 h
R2 = Me2 [0%]
R2 = Me,Cl [0%]
R2 = Cl2 [36%]
CF3
optimised conditions:
Cl Cl
Ge
Ar-Br (1eq)
R
PdCl2(MeCN)2 (10 mol%)
dppp (20 mol%)
KF (6 eq)
DMF, 120 °C, 8 h
Ar
R
#
1
R
Me
2
3
4
5
6
7
8
9
Me
Me
OMe
OMe
OMe
OMe
OMe
OMe
Ar
4-AcC6H4
3,5-(CF3)2C6H3
1-Nap
Ph
3-CF3C6H4
3,5-(CF3)2C6H3
1-Nap
3-Py
4-NO2C6H4
Yield/%
60
63
79
36
51
71
56
44
47
with Chris Gripton
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]
OEt
2-Nap
Ge
2-Nap
O
OMe
OEt
O
Ph
Ge
Ph
OMe
with Chris Gripton, Joseph Hannah & Chih-Chung Tseng Appl. Organomet. Chem. 2007, 21, 572 [DOI]
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:
OMe
2-Nap
C8F17
Ge
2-Nap
OMe Cu(BF4)2 (4 eq)
MeCN:MeOH (3:1)
h, pyrex filter
10 min
OMe
2-Nap
C8F17
F
Ge
OMe
~1 h
C8F17
FF
Ge
OMe
with Chih-Chung Tseng
Scope of germyl-Stille aryl-aryl cross-coupling
2-Nap
C8F17
2-Nap
Ge
R
Cu(BF4)2 (4 eq)
MeCN:MeOH (3:1)
h, pyrex filter
~2 h
C8F17
OMe
(2 eq)
F F
Ge
R
PdCl2(MeCN)2 (10 mol%)
P(2-Tol)3 (15 mol%)
TBAF·3H2O (2.7 eq), CuI (1 eq)
DMF, 120 °C, 16 h
Ar-Br (2 eq)
Ar
R
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
R
4-OMe
4-OMe
4-OMe
4-OMe
4-Me
4-Me
4-Me
4-Me
H
H
H
H
4-Cl
4-Cl
4-Cl
2-OMe
2-OMe
2-OMe
2-OMe
4-CF3
4-CF3
Ar
3,5-(CF3)2C6H3
4-ClC6H4
4-BnOC6H4
1-Nap
3,5-(CF3)2C6H3
4-ClC6H4
4-BnOC6H4
1-Nap
3,5-(CF3)2C6H3
4-ClC6H4
4-BnOC6H4
1-Nap
3,5-(CF3)2C6H3
4-BnOC6H4
1-Nap
3,5-(CF3)2C6H3
4-ClC6H4
4-BnOC6H4
1-Nap
3,5-(CF3)2C6H3
4-ClC6H4
Yield/%
96
85
65
75
84
69
48
71
74
63
40
60
71
42
75
65
49
11
27
26
11
with Chih-Chung Tseng, Joseph Hannah & Chirs Gripton Chem. Comm. 2007, 2926 [DOI]
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
(±)-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]
via
•
Hindered ester formation with di-ortho-substituted benzoate
X-ray
with James Carr
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
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
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
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]
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
IC50 (µM)
1
Compound
(% ee)
(+)-asp (98.4)
2
(–)-31 (98.8)
483 ± 105
3
(±)-31
56 ± 2
40 ± 1
CD curve for
(+)-aspercyclide
with Jimmy Sejberg, Helena Dennison & Mary Holdom (KCL)
Enantioselective synthesis of ()-aspercyclide A
cf. Krische J. Org. Chem. 2011, 76, 2350 [DOI]
with Jimmy Sejberg
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
•
•
•