development of a supply route for an inos inhibitor and investigation of alternative synthetic...
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Development of a Supply Route for an iNOS Inhibitorand
Investigation of Alternative Synthetic Routes
Gerry Rassias
R&D/Chem Dev/Synthetic ChemistryStevenage, Herts, SG1 2NY, UK
Overview
Nitric Oxide and iNOS Inhibition
Development of the Supply RouteSynthesis
Key Step & Other Issues
Disconnections/Alternative RoutesThe Lactamide Approach
The Bromopropionic Acid Route
Summary
Nitric Oxide and iNOS Inhibition
eNOS: nanomolar NO at the endothelium
nNOS: nanomolar NO at the neurons
iNOS, the inducible isoform: micromolar NO mostly in the gut
NO metabolites cause cytotoxicity and tissue damage
Diseases: Septic shock, Neurodegenerative disorders, Inflammation,
Asthma, Rheumatoid arthritis, Multiple sclerosis
1998: Robert F Furchgott, Louis J Ignarro and Ferid Murad receive the
Nobel prize in Medicine for the signalling role of NO in biology
NO is synthesised by conversion of arginine to citrulline
Catalysed by specific enzymes, NO synthases which exist in 3 isoforms
Selective iNOS Inhibitor in Development
Small molecule, 4 functional groups, 2 chiral centres
Monophosphate-monohydrate salt identified as most suitable
Significant improvements introduced in original synthesis
5, 50 and 250kg made in 3 campaigns
Development of the Supply Route - Issues
S
O
tBuO
NHBoc
OH OH
SH SH
SH
O
tBuO
NHBoc
DCMNEt390%
1/2
2
NH2
OH
CbzHNOTs
i) K2CO3 CbzCl
ii) NEt3,NMe3.HCl TosCl
DCM
82%
pKa of NHP linked to SIDE-PRODUCTS
EXPENSIVE
Undesired Solvent
CbzHNS
NHBoc
O
OtBu
20% tBuOK in THFTBME, rt, 80%
Development of the Supply Route - Issues
COST due to 1 WT REQUIRED
TOXICEXPENSIVE
S Naphth
NH.HCl
NH
S
O
OtBu
NH.HCl NHBoc
EtOAc
NH
NH.H3PO4.H2O
S
NH3+
O
O
HCO2H.H2NS
O
OtBu
NHBoc
CbzHNS
O
OtBu
NHBocPd(OH)2, IMS
NH4HCO2
78%
i) K2CO3, tolii) H3PO4
70%
92%
Key Coupling Step - Issues
SH
NHBoc
O
OtBuCbzHN
S
NHBoc
O
OtBuCbzHNOTs
NHBoc
O
OtBu
S
NHBoc
O
tBuO
NHBoc
O
OtBu
CbzHNS
NHBoc
O
OtBu
CbzHNSH
Base-InducedProduct Degradation
Acrylate observed spectroscopically
Thiol and thioethers have been isolated
Higher T favours both SN2 and formation of impurities
Use kinetic base for thiol deprotonation
NHBoc
O
OtBuH2S
Base-InducedDegradation of Thiol
M+ -S
NHBoc
O
OtBu
Solubilityof Thiolate anion
Stability/Reactivityof Thiolate anion
At <18oC degradation faster than SN2
Li, Na thiolates insoluble in THF, Acetone, MeCN
K, Cs thiolate soluble even in TBME
Use K salt of kinetic base at >18oC
Exposure of tosylate to base results in aziridine
Authentic sample gives ca 1:1 ring opened products
New thiols also engage tosylate and aziridine
Unreacted tosylate difficult to remove. Use thiol in xs
Thiol pKa similar to NHCbz. Issue!
Must pre-form thiolate anion
Aziridine formationand ring opening
CbzHNS
NHBoc
O
OtBu
CbzHNS
NHBoc
O
OtBu
CbzN
Dimerisation of thiols
Elemental sulphurThiolate anions dimerise in the presense of oxygen
Both disulfides hard to remove
2 more disulfides from isomeric thiols also observed
Parent thiolate in xs. pH must be lowered at quench
Must de-gas solventsS
O
tBuO
NHBoc
SNHCbz
2
2
Development of the Supply Route - Issues
SH
NHBoc
O
OtBuCbzHN
S
NHBoc
O
OtBuCbzHNOTs
20% tBuOK in THFTBME
80%
1st time in plant: 100 kg of intemediate in 2nd Campaign
>½ Tonne of Intermediate in 3rd Campaign
But is this the best route?Sensitive Reactions,Toxic/Expensive Reagents
CbzHNSH
CbzN
CbzHNS
2
CbzHNS
NHCbz
CbzHNS
NHCbzCbzHNS
NHCbz
NHBoc
O
OtBu S
NHBoc
O
tBuO2
NHBoc
O
OtBu
CbzHNS
NHBoc
O
OtBuCbzHN
S
NHBoc
O
OtBu S
NHBoc
O
tBuO
NHBoc
O
OtBu
CbzHNS
NHBoc
O
OtBu
CbzHNS
NHBoc
O
OtBuCbzHN
S
NHBoc
O
OtBu
CbzHNSHCbzHN
SHNHBoc
O
OtBu
SH
NHBoc
O
OtBuCbzHN
S
NHBoc
O
OtBuCbzHNOTs
New Routes- The Lactamide Approach
NH2
S
O
OtBu
NHBoc
NH2
S
O
OtBu
NHBocO
SH
O
OtBu
NHBoc
NH2
OH
O
The Lactamide Approach-Issues
SH
O
OtBu
NHBoc
NH2
OTs
O
S
O
OtBu
NHBoc
NH2
OtBuOKTHF, rt
95%
S
O
OtBu
NHBoc
NCS OH
NHBoc
NH2
O
NH
S
OOtBu
O
Amide does not reduce successfully to the 1o amine.(8 borane complexes, 8 hydride reagents, 33 Lewis acids)
Dehydrates to Nitrile successfully but… does not reduce under borane or hydrogenation conditions
Approach abandoned
New Routes - The 2-Bromo-propionic acid Approach
NH
S
O
OtBu
NH.HCl NHBoc
NH2
NH.HCl
LGS
O
OtBu
NHBoc
OHS
O
OtBu
NHBocO
SH
O
OtBu
NHBoc
OHBr
O
NH2
S
O
OtBu
NHBoc
S+
O
OtBu
NHBoc
LG-
Features of the expected Thiiranium species
S+
O
MeO OMeS
+
OMe
OR
MsO-
MsO- ClS
+
Cl
S+
OtBut
O
NHBoc
S+
OtBut
O
NHBoc H
BocHN CO2tBu
S+
H H
anti isomer more favoured than syn E2 elimination requiresdisfavoured conformer
Stable enough but …. Regioselectivity?
Limited examples of unsymmetrical thiiranium salts
Synthesis of the Alcohol Intermediate
SH
O
OtBu
NHBoc
OHBr
O
S
O
OtBu
NHBoc
OH
Oi) HMDS, THFii) tBuOK, THF
90%
S
O
OtBu
NHBoc
OH
NaBH4, BF3.OEt2THF, rt
90%
Clean, robust and high yielding.
No other isomers by NMR. No loss of ee
Acid: crystallineAlcohol: Low melting point solid.
Solution after work up works equally well in next stage as columned material
Demonstrated on 100g scaleIdentified potential process improvements
Alcohol intermediate is easily made
Product Distribution from Alcohol Activation
S
O
OtBu
NHBoc
MsOS
O
OtBu
NHBoc
MsO
S
O
OtBu
NHBoc
ClS
O
OtBu
NHBoc
Cl
S+
OtBut
O
NHBoc
X-
S
O
OtBu
NHBoc
OH
MsClNEt3DCM, rt
t=30minExclusive product
t=2hMajor component
t=5hMajor Component
t=24hExclusive product
IntimateION PAIR
Reactivity of the chloride and mesylate
S
O
OtBu
NHBoc
XS
O
OtBu
NHBoc
XS
O
OtBu
NHBoc
NH2
S
O
OtBu
NHBoc
NH2
MeCN7M NH3
MeOH
X=Cl
80% 20% 40% trace
0% 100% 0% 0%
X=Ms
80% 20% 20% trace
20% 80% 20% trace
2o Species (X=Ms, Cl) unreactive (towards NH3/MeOH)(although thermodynamically favoured)
Leaving group ability of X determines reversibility from 2o Species to the 1o Species
As MeOH/MeCN increases, rate increases-Not Classic Sn2
Mechanistic Hypothesis
S
O
OtBu
NHBoc
NH2
S
O
OtBu
NHBoc
X
S
O
OtBu
NHBoc
XS
O
OtBu
NHBoc
NH2
S+
OtBut
O
NHBoc
XNH3
Nu-=NH3
NH3
X- Nu-
Thermodynamic
Kinetic MAJOR PRODUCT
TRACE
via Intimate ion pair
Dominant Pathway
Minor Pathway
Key equilibriumto control
Testing The Hypothesis with the Bromide
S+
OtBut
O
NHBocBr-
S
O
OtBu
NHBoc
OH
S
O
OtBu
NHBoc
BrS
O
OtBu
NHBoc
Br
MsCl, LiBrMeCNNEt3
S
O
OtBu
NHBoc
NH2
NH3/MeOH
<5% >95%
70% TRACE
S
O
OtBu
NHBoc
NH2
NH3/MeOH
A New Procedure for Amidine Synthesis
S
O
OtBu
NHBoc
NH2
S
O
OtBu
NHBoc
NH
NH.HCl
NH2
NH.HCl
S
O
tBuO
NHBoc
NS
O
OtBu
NHBoc
NH
.HCl
+
7M NH3In MeOH
slow7M NH3In MeOH
XSamine
Acetamidine hydrochloride:A new amidination reagent
“Green” + Inexpensive
In methanolic solution of NH3An equilibrating mixture of
Amidine, amidine dimer and the parent amine is established
Difficult to get selectivity for middle component
Currently investigating Dynamic Crystallisation
Authentic sample prepared
The Emergence of a Promising Synthesis
O
OtBu
NHBoc
OHS S
O
OtBu
NHBoc
NH
NH HBF4i) MsCl, LiBr, NEt3MeCN, rt
ii) 7M NH3 in MeOH
NH2
NH.HBr
iii) NH4BF4
O
OtBu
NHBoc
MsOS
S+
OtBut
O
NHBoc
O
OtBu
NHBoc
MsOS
O
OtBu
NHBoc
BrS
O
OtBu
NHBoc
BrS S
O
OtBu
NHBoc
NH2
S
O
OtBu
NHBoc
NH
NH.HBr
42%
Requires 6 forward reactionsoperating at av.>85% eachInvolves 7 intermediatesand 3 sets of equilibria
The Emergence of a Promising Synthesis
O
OtBu
NHBoc
NH
S
NH.HBF4
O
O
NH3+
NH
S
NH.H3PO4.H2OH3PO4
Water, Acetone
78%
O
OtBu
NHBoc
BrS
O
OtBu
NHBoc
OHS
O
OtBu
NHBoc
OHS
O
O
OtBu
NHBoc
SHOH
O
Br
NH2
NH.HBr
MsCl, LiBrNEt3, MeCN
85%
20% tBuOK in THF
90%
NaBH4
BF3.OEt2THF, 90%
7M NH3
in MeOHNH4BF4, 50%
Summary
Original route delivered 300kg of drug substance
Despite intense development work, the inherent cost, safety and process issues
render it unsuitable as manufacturing route
(S)-2-Bromopropionic acid route has been demonstrated on 100g scale
Substantially “greener” and operates at 50% of cost.
Dithiothreitol, Pd(OH)2, CbzCl, Naphthyl methyl thioacetamidine
eliminated from synthesis
Development work on new route led to the discovery of a new amidination procedure and
the counteranion-controlled reactivity of thiiranium intermediates
“The First and the Best Syntheses count”EJ Corey
SYNTHETIC CHEMISTRY
Gerry Rassias, Steve Hermitage, Mahesh Sanganee, Pete Kincey
Neil Smith, Graham Slater, Gregory Stepney
Rachel Tughan, Gary Borett, Ian Andrews
Chemical Development, Stevenage
R&D
Attempts to further Improve the Supply Route
NH2
S
O
OtBu
NHBoc
CO2Me
CO2Me
XNH2NH2
S
NHBoc
O
tBuO
SH
NHBoc
O
tBuO
2
PPh3 Acetone/water
88%
1/2
PhthNOTs
PhthNS
O
OtBu
NHBoc
X
Dithiothreitol/DCM in disulfide “cracking” replaced withgreener and cheaper TPP/acetone
Phthalimide would address aziridine issue but deprotectionnot possible without severe degradation and epimerisation
Mechanistic Hypothesis
S
O
OtBu
NHBoc
NH2
S
O
OtBu
NHBoc
X
S
O
OtBu
NHBoc
XS
O
OtBu
NHBoc
NH2
S+
OtBut
O
NHBoc
S+
OtBut
O
NHBoc
S+
OtBut
O
NHBoc
X-Nu-
XNH3
Nu-=NH3
NH3
X- Nu-
Nu-=NH3
Thermodynamic
Kinetic MAJOR PRODUCT
TRACE
Intimateionpair
Individually solvated ions
Intimateionpair
Dominant Pathway
Minor Pathway
Issue of Chemoselective Reduction(we did try)
X in X-BH3
NH3 tButNH2
R2NH
R3N
Py
Ph3P
R2S
THF
Hydrides
LiBH4
NaBH4
KBH4
Super Hydride
Selectride
AlH3.HNEt2
DIBAL
Red-Al
Lewis acids
H+ MgBr2 BF3.Et2O SiMe3Cl ZnCl2LiCl CaCl2 BF3.MeOH SiMe2Cl2 CuCl2 I2 AlCl3 BF3.AcOH SiCl4 BiCl3
NH2
S
NHBoc
O
OtBut
O
NH2
S
NHBoc
O
OtBut
CeF4 TiF3 VOCl3 ScTfO3
CeCl4 TiCl3 ZrOCl2 YbCl3 CeBr4 TiOiPr4 ZrCl4 ZrH4
CeTfO4 TiCl2Cp2 ZrCl2Cp2 TiH2
Amberlyst 15 Resin (NH4) 2Ce(NO3) 6
Desired chemoselectivity Undesired chemoselectivityNo reaction/complex mixtures
CORK PILOT PLANTGrainne Geraghty
Steve BallColin Chapman
Christina McSweeney
STEV. PILOT PLANTBrian Broadbelt
Julian NashDavid Box
ANALYSTSGuy WellsAsha Patel
Simone BrownsellNisha Mistry
PROCESS SAFETYSteve Gooding
PHYS PROPERTIESSarah Thomson
PARTICLE SCIENCESPeter Aspin
SYNTH CHEMISTRYGerry Rassias
Mahesh SanganeePete Kincey
Graham SlaterNeil Smith
Rachel TughanGregory Stepney
Gary BorettIan Andrews
PROJECT LEADERSteve Hermitage