synthetic strategies for 14 c labelling of drug molecules
DESCRIPTION
TRANSCRIPT
Objective
• This lecture will focus on a brief introduction to ‘what is’ carbon-14
• Then leading onto some synthetic strategies towards labelling potential drug molecules with carbon-14
Agenda• Introduction to 14C• 14C Synthetic Strategies:- [14C]XEN-D0401- [14C]Apomorphine- [14C]Combretastin-A1- [5-14C]Hex-5-ynoic acid- [14C]ZT-1• Conclusion
cpres/v1b/ca/1998-08/ 4
Introduction to 14C
Discovery of 14CMartin Kamen & Sam Ruben (27-FEB-1940)
T1/2 ~ 5730 Years
14C Starting Materials
Barium 14C carbonate staircaseOH
C14
NH
O
OHF3C
OH
Cl
NH
CH3
O
N
OH
ClMeO
CH3OH
OHOMe
MeO
OMe
Ba14CO3
14C6
14CO2
14CH3OH
14CH3I
H14CHO
Cu14CN
K14CN
14C6
AA
14C
14C6
H314CO
14CHH14C PEPTIDES
14C Radiotracer• In pharmaceutical research 14C is used as
a tracer to ensure that potential drugs are metabolized without forming harmful by-products – ADMET Studies
• The 14C label should ideally form part of the compounds molecular skeleton
14C Synthetic Strategies14C
labelling
Target: [14C]XEN-D0401
NH
O
F3C
Cl
OHOH
*
• XEN-D0401 is a novel and selective small molecule activator of the large-conductance calcium-activated potassium channel (known as the BK channel)• Currently in Phase 1 development for the treatment of overactive bladder (OAB) * = 14C Label
14C Starting Material
NH
O
F3C OHOH
Cl
Cl
O O
OMeO
*
*
[14C]XEN-D0401 [14C]-4
14CO2
Generation of ‘Dry’ 14CO2
XHIGH VACUUMPUMP
P2O5
c H2SO4Ba14CO3
14CO2 (wet)
Liquid Nitrogen
14CO2 (dry)
High Vacuum Manifold
14CO2
CarboxylationBr
OH
Cl
Br
Cl
O OLi
Cl
O O
Cl
O O
OOH*
(1) (2)
Ba14CO3
c H2SO414CO2
[14C]-3
1. NaH / DMF
2. MOM-Cl
n-BuLi/Hexane
THF-78 oC, 20 mins
-196 oC to rt
lithiated intermediate
>50 mCi/mmol
Radiochemical yield = 76%
250 mCi
O-Methylation
Cl
O O
OOH
Cl
O
OMeO
O**
[14C]-4
CH3I
[14C]-3
K2CO3 / Acetone
35oC
NHCOt-Bu
CF3
NHCOt-Bu
F3C
O
Cl
OMOM
NH2
CF3
O
Cl
Cl
OMOM
OMeO
CF3
LiN
LiO
*
*
(6)
[14C]-7
n-BuLi, Hex, THF
[14C]-4
(5)
Column chromatography
STEP 1
STEP 2
-78 oC
83%
71%
NH2
F3C
O
Cl
OMOM
NH
O
F3C
Cl
OHOMOM
NH
O
F3C
Cl
OHOH
O
O
**
*
[14C]-8
LiHMDS / THF
[14C]XEN-D0401
NaOH
EtOH
IPA, HCl
45 oCColumn Chromatography
STEP 3
STEP 4 [14C]-9
STEP 5* = Carbon-14 label
-20 oC to -10 oC
68%
63%
Overall Radiochemical Yield ~ 30%
S L Kitson, S J Jones et al. J Label Compd. Radiopharm 2010, 53, 140-146
Key Step: A one pot construction of the quinolin-2-one ring under strong base
N
F3C
O
OLi
Cl
LiOO O
Li
*
Cl
O
NLi
F3C
OLiO
H
O O*
OO
Cl
O O
NH
F3C
O
OHOH
H
Cl
OF3C
NH
H
O O
NH
F3C
O
OH
Cl
O O
*
*
Cl
O
NLi
F3C
OLiO
O O
-
*
condensation
cyclisation
LiOH
-H2O
LiHMDS (2eqv)
dianion
LiHMDS
H2O
dehydration
trianion
[14C]-8
[14C]-9
*
Aporphine skeleton‘naturally occurring’
Dicentra formosa'Bleeding heart'
C14N
R1
3
52
4
6
7
8
9
10
11
A B
C
D
6a
S L Kitson. J Label Compd Radiopharm; 2007, 50, 290-294
S L Kitson. J Label Compd Radiopharm; 2006, 49, 517-531
Structure & Function of (R)-(-)-Apomorphine• (R)-(-)-Apomorphine is a potent non-selective
D1/D2 agonist at the dopamine receptor.• (R)-(-)- Apomorphine (Apokyn™) is used to
control Parkinson’s disease (D2 receptor).• (R)-(-)- Apomorphine (Uprima®) is used to treat
erectile dysfunction (D1 receptor).
• Features:• Contains an ortho-phenol motif.• Tetracyclic carbon skeleton.• Tetrahydroisoquinoline nucleus.• ‘Planar’ structure.• Contains an embedded dopamine
pharmacophore.
R
S
Retrosynthesis of Apomorphine
CH3
NO2OMe
MeO
N NO2
MeO
MeO NH
O
NCH3
MeO
MeO
*
*
*
A B
C
D
A B
DD A
N-[carboxyl-14C]AcetamideTetrahydro[14C]isoquinoline
(R/S)-[6a-14C]Apomorphine dimethyl ether
=14C
RING BFORMATION
RING CFORMATION
*
Pschorrcoupling
Bischler-Napieralskicyclodehydration
HO
HO
FGI
Synthesis of N-[carboxyl-14C]acetamide
ClMeO
NO2
MeO
MeONO2
MeO
CNMeO
NO2
OH
OMeO
MeONO2
Cl
OMeO
MeONO2
O
NH
MeO
NH2
*
*
*
*(COCl)2
DMF
55mCi/mmolK14CN
AD
D
A
N-[carboxyl-14C]Acetamide
acidhydrolysis
90% 86%
86%
Radiochemical yield = 67%
(2 steps)
D
Bischler-Napieralski Cyclodehydration
MeO
MeO
NO2
O
NH
N+
NO2MeO
H
OMe
*
*
Cl N
NO2MeOOMe
*
N+
MeO
MeO
NO2
MeO
MeO
NO2
OP
ND *
*
Toluene
-OP
D A
nitrilium salt
imine
N-[carboxyl-14C]Acetamide
A
AD
basic work up
Dihydro[14C]isoquinoline
A B
D
Radiochemical yield = 43%
P2O5/POCl3
A B
D
RING BFORMATION
N-Methylation Reduction StepN
MeO
OMe
NO2
N+
MeO
OMe
NO2
CH3
MeO
OMe
NO2
CH3
N* * *
Bischler-Napieralski
I-
NaBH4/EtOHCH3I/THF
Dihydro[14C]isoquinoline [14C]Methiodide Tetrahydro[14C]isoquinoline
Radiochemical yield = 77%
Endocyclic Product
Reduction-Pschorr Coupling
MeO
OMe
NO2
CH3
N
NCH3
MeO
MeO
H
CH3
N
N+
NMeO
OMe
MeO
OMe
CH3
N
NH2
*
*
*
*+ Cu2O
PSCHORR COUPLING
Zn/HCl 10% H2SO4
NaNO2
Tetrahydro[14C]isoquinoline
(R/S)-[6a-14C]Apomorphine dimethyl ether
A B
D
A B
C
D
HSO4-
aryldiazonium salt
RING C FORMATION
DD
Chiral Separation
NCH3
MeO
MeO
H*
NCH3
MeO
MeO
H*
(R/S)-(+/-)-[6a-14C]Apomorphine dimethyl ether
NCH3
MeO
MeO
H*
(S)-(+)-[6a-14C]Apomorphine dimethyl ether(R)-(-)-[6a-14C]Apomorphine dimethyl ether
HPLC Conditions: Chiralcel OD column, eluting with hexane/propan-2-ol/dimethylamine [950:50:5]
S R
Radiochemical yield = 11% fromTetrahydro[14C]isoquinoline
Radiochemical yield = 16% fromTetrahydro[14C]isoquinoline
(R)-(-)-[6a-14C]Apomorphine
• PLRP-S column (CH3CN:HCl aq)• Specific Activity: 55mCi/mmol• Radiochemical purity => 98%• Radiochiral purity => 99%
NCH3
MeO
MeO
HN
CH3OH
OH
H* *BBr3
(R)-(-)-[6a-14C]Apomorphine(R)-(-)-[6a-14C]Apomorphine dimethyl ether
CH2Cl2
Combretastatin A-1
OMe
MeO
OH
OH
OMe
MeO
• Combretastatin A-1 is a natural productfirst isolated from the African bush willow tree in the 1980s
• Chemotherapeutic properties by acting on thetumour vasculature, leading to blood flow shut down and ultimately tumour death
Target: [methyl-14C]Combretastatin A-1 diphosphate
• Treatment of solid tumours• The pro-drug undergoes in vivo de-phosphorylation
to generate the active agent combretastatin A-1
MeO
OMe OMe
O
O
P
P
O
O
OH
O
OOH
14CH3O
.2K+
O
O
TBSO
MeO
OMe OMe
O
OMeO
OMe OMe
OH
OHMeO
OMe OMe
OP(O)(OBn)2
OP(O)(OBn)2MeO
OMe OMe
MeO
OMe OMe
O
O
P
P
O
O
OH
O
O
OH
14CH3I TBAF CH3CN
75%
[methyl-14C]-Combretastatin A1 diphosphate
14CH3O
TiCl4 / DCM
27%14CH3O
(BnO)2P(O)H
CCl4 / CH3CN
DIPEA / DMAP
72%
14CH3O
1. TMSBr / MeCN2. KOMe, MeOH3. HCl/H2O/EtOH
57%14CH3O
.2K+
14C Synthetic routeR T Brown et al. J Label Compd. Radiopharm 2009, 52, 567-570
Target: [5-14C]Hex-5-ynoic acid
C14 OH
O
[14C]Acetylenes
[14C]Acetylenes: Ohira Bestmann Reagent
RC14
H
O
CH3
N
N
O
P
O
MeO
MeO
C14 HR
CH3
N
N
P
O
MeO
MeO O OMe
N
N
P
O
MeO
MeO
+
-
Ohira-Bestmann Reagent
MeOH+
-
K2CO3
rt, 4-16 h +
-
-
BrOH
OPO
N+
N
OMeOMe
BrOTHP
OTHPC14
O
H
OTHPC14
O
OTHPNC*
OHC14
O
K2CO3, MeOH
p-TsOHEtOH
K14CN, EtOH
DiBAL
CrO3, H2SO4,
H2O, acetone
67 %
71 %
80 %
68 % 74 %
14C Acetylene Synthesis
Overall Radiochemical Yield 29 % from [14C]KCN
[14C]ZT-1: a new generation of acetylcholinesterase (AChE) inhibitors
L Leman, S L Kitson et al. J. Label Compd. Radiopharm 2011, 720-730
CHO
OH
Cl OMe
NH
CH3
O
NH2
CH3
NNO
Cl
H
MeOH O
H
CH3
CH3 in vivo progressive hydrolysis
active metabolite, (-)-huperzine A
+
Pro-drug (-)ZT-1 inactive metabolite, 5-chloro-o-vanillin
exocyclic E-double bond
pyridin-2-one ring
hydrogen bond
R
bicyclo[3.3.1] double bond
14C SynthesisOH
OH
OMe
Cl
O
HOH
OMe
O
H
OMOM
OMeOMOM
OMe
O
H
OMOM OMOM
I
* * *
* *
**
[14C]-1
[14C]-7[14C]-6
[14C]-2 [14C]-3
[14C]-4 [14C]-5
NaH/DMF
MOM-Cl
n-BuLi
I2
CuBr/DMF
NaOMe/MeOH
n-BuLi
DMF
HCl/MeOH ICl / CH2Cl2
NaHCO3
Step 1 Step 2
Step 5
Step 3 Step 4
Step 6
* = U-14C
[14C]-ZT-1
NH
CH3
O
NH2
CH3
OH
Cl
O
OMeH
NH
CH3
O
CH3 N
Cl
OH
MeO
*
*
(-)-Huperzine A [14C]-ZT-1
EtOH
[14C]-7
Step 7
ConclusionWhen designing a 14C labelled synthesis it is important to consider the following:
• Identify simple starting materials from the barium 14C carbonate ‘staircase’ which are commercially available or alternatively easily made
• Plan, develop and execute the synthetic methodology to the final drug substance. This approach can often restrict the position of the label in the drug and will cause a change in the drug purity profile from the original laboratory synthesis route
• Locate a biologically stable position for the 14C label
14C Radiochemistry Laboratory