Synthesis of isotopically labelled [14C]ZT-1,
[d3]ZT-1 & (-)-[d3]huperzine A,
a new generation of acetylcholinesterase inhibitors
Dr Sean Kitson
ШОН КИТСОН
sean.kitson@almacgroup.com
Objective
• This lecture will focus on a brief introduction
to carbon-14
• Leading onto synthetic strategies towards
labelling (-)-huperzine A derivatives with
14C and 2H
A Brief Introduction to 14C
cpres/v1b/ca/1998-08/
3
Discovery of 14C
Martin Kamen & Sam Ruben (27-FEB-1940)
T1/2 ~ 5730 Years
Production of 14C
14N
1n →
+
7
0
14C
Reactor
1H
+
6
1
Radioactive
atom
Bombardment by Neutrons
Decay to stable
daughter nuclide
ß-
ß-
N
N
P
P
Nitrogen 14
7 Protons
7 Neutrons
Carbon 14
6 Protons
8 Neutrons
P
P
Nitrogen 14
7 Protons
7 Neutrons
14C
Starting Materials
Ba(OH)2
Barium 14C carbonate staircase
OH
OH
14
C
O
[14C]Acetylenes
14C
6
14C
14C
6
[14C]PEPTIDES
AA
H14C
[14C]Apomorphine
14CH
[14C]Combretastatin A-1
H14CHO
MeO
14CH I
3
14
C
HO
HO
N
H
CH3
14CH OH
3
H
N
O
OH
H314CO
OMe
Cu14CN
K14CN
14
F3C
C
S L Kitson. JLCR 2007, 50, 290-294
S L Kitson, E Knagg. JLCR 2006, 49, 517-531
Cl
OH
OH
OMe
R T Brown et al. JLCR 2009, 52, 567-570
14CO
2
OH
[14C]XEN-D0401
Ba14CO3
S L Kitson, S Jones et al. JLCR 2010, 53, 140-146
14C
Labelling
When 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
S L Kitson ‘Accelerated Radiochemistry’,PMPS Manufacturing 2010, 68-70
14C
Drug Molecules
14C
•
•
•
•
•
•
Labelled drugs are used in human mass
balance or AME studies to evaluate:
Mass balance and the routes of elimination
Identify circulatory and excretory metabolites
Determination of clearance mechanisms
To determine the exposure of parent compound
and its metabolites
Used to validate animal species used for
toxicological testing
To explore whether metabolites contribute to the
pharmacological / toxicological effects of the
drug
C Prakash et al. Biopharm. Drug Dispos; 2009, 30, 185-203
(-)-Huperzine & ZT-1
(-)-Huperzine A
• (-)-Huperzine A is a naturally occurring Lycopodium
alkaloid found in an extract from the club moss
Huperzia serrata
• (-)-Huperzine A is a potent, selective and reversible
inhibitor of acetyl cholinesterase, the enzyme that
breaks down or degrades acetylcholine
• (-)-Huperzine A is currently a prescription
medication in China for the treatment of
12
Alzheimer's Disease
CH3
H1
N
7
8
10
6
9
14
2
5
H3C
11
13
C M Yu et al. Canadian J Chem 1986, 64, 837-839
D L Bai et al. Curr. Med. Chem; 2000, 7, 355-374
O
NH2
4
3
ZT-1: Pro-drug for (-)-Huperzine A
A Novel Acetylcholinesterase Inhibitor
CH3
bicyclo[3.3.1] double bond
exocyclic E-double bond
H3 C
CH3
hydrogen bond
O
N R
MeO
H
O
NH2
active compound, (-)-huperzine A
in vivo
progressive hydrolysis
+
CH3
H
H
N
N
H
CHO
OH
O
pyridin-2-one ring
Cl
Pro-drug ZT-1
L Leman, S L Kitson, R T Brown et al. JLCR 2011 (in press)
Cl
OMe
metabolite, 5-chlorovanillin
ZT-1 Implant
PLGA
POLYMER
ZT-1
DRUG
BLENDING
EXTRUSION
ZT-1
IMPLANT
S. Capancioni et al. Preparation of a sustained-release implant of the acetylcholinesterase inhibitor ZT-1
by hot-melt extrusion (HME) and evaluation in rats Debiopharm (April 2006)
ZT-1 implant offers the following
advantages over oral ZT-1:
• Once-a-month dosing
• Implant-controlled progressive increase in
(-)-huperzine A plasma levels
• Sustained plasma levels
• A prolonged release of the (-)-huperzine A
over several weeks
S. Capancioni et al. Preparation of a sustained-release implant of the acetylcholinesterase inhibitor ZT-1
by hot-melt extrusion (HME) and evaluation in rats Debiopharm (April 2006)
[14C] & [d3] - Targets
CH3
H
N
CH3
H
N
D3C
CH3
NH2
O
H3C
H
N
O
D3C
N
HO
N
HO
*
MeO
(-)-[d3]Huperzine-A
[14C]ZT-1
Cl
MeO
Cl
[d3]ZT-1
O
Synthesis of [14C]ZT-1
14C
labelling
Schiff base
CH3
H
N
H3C
O
CH3
Schiff base
formation
H
N
N
O
O
+
HO
H3C
NH2
*
MeO
OH
OMe
H
*
Cl
Cl
[14C]ZT-1
(-)-Huperzine A
5-Chloro[U-14C]vanillin
Retro-synthetic pathway
O
OH
OMe
H
OP
deprotection
OMe
OHC
*
ortho-directed
formylation
OP
OMe
*
*
A
B
halogenation
Cl
[14C]-7
OP
alkoxy de-halogenation
I
*
C
ortho-directed
iodination
OH
*
[14C]-1
14C
Synthesis
NaH/DMF
*
I
n-BuLi
*
MOM-Cl
[14C]-1
OMOM
OMOM
OH
[14C]-2
Step 1
*
I2
Step 2
[14C]-3
OMOM
CuBr/DMF
OMe
OMOM
O
n-BuLi
OMe
H
*
NaOMe/MeOH
[14C]-4
Step 3
DMF
*
Step 4
[14C]-5
O
O
OH
OH
HCl/MeOH
OMe
H
*
ICl / CH2Cl2
OMe
H
*
NaHCO3
Cl
Step 5
[14C]-6
Step 6
[14C]-7
* = U-14C
14
[ C]ZT-1
O
OH
OMe
H
*
CH3
H
N
H3C
CH3
NH2
H
N
[14C]-7
O
Cl
H3C
N
EtOH, heat
HO
Step 7
*
MeO
(-)-Huperzine A
Cl
[14C]ZT-1
O
Synthesis
of
[d3]ZT-1 & (-)-[d3]huperzine
Retro-synthesis
CH3
CH3
H
N
D3C
O
Curtius
rearrangement
N
D3C
NH2
CO2H
(-)-[d3]Huperzine A
CH3
N
Wittig
OMe
+
isomerisation
O
CO2 Me
(+)-8
D3C
PPh3
OMe
Synthesis of (-)-[d3]huperzine A
D3CCH2Br
Step 8
CH3
Ph3P
CH3
N
O
OMe Ph P+CH CD .Br 3
2
3
base
CO2 Me
Step 9
(+)-8
N
PhSH, AIBN
OMe
Step 10
CO2 Me
CD3
[d3]-9
Z>E
CH3
CH3
N
OMe
N
via
D3C
CO2 Me
[d3]-10
E>Z
hydrogen abstraction from PhSH
addition of PhS. to cis-double bond
180 degrees rotation
ejection of thiyl radical by beta-scission
.
PhS
OMe
CO2 Me
CD3
radical
C. Ferreri et al. Chem. Commun; 1999, 407-408
Synthesis of (-)-[d3]huperzine A
CH3
CH3
N
D3C
OMe
N
NaOH
Step 11 D C
3
CO2 Me
[d3]-10
CO2 H
Step 12
S-I Yamada et al. J. Am. Chem. Soc; 1972, 94, 6203
CH3
CH3
H
N
N
D3C
OMe
TMS-I
MeOH
NHCO2 Me
[d3]-12
(PhO)2PON3 / Et3N
MeOH
[d3]-11
E>Z
OMe
D3C
O
NH2
Step 13
G A Olah, J. Org. Chem; 1979, 44, 1247-1251
(-)-[d3]huperzine A
O
OH
CH3
OMe
H
H
N
CH3
H
N
O
D3C
N
EtOH, heat
D3C
NH2
Step 14
HO
MeO
[d3]-huperzine A
[d3]ZT-1
CH3
H
N
D3C
H
N
O
D3C
HN
HN
.HCl
MeOH
Synthesis of [d3]ZT-1
&
reduced-[d3]ZT-1
Cl
CH3
NaBH4
O
Cl
HCl/Et2O
HO
HO
Step 16
Step 15
MeO
reduced-[d3]ZT-1
Cl
MeO
Cl
reduced-[d3]ZT-1.HCl
O
Conclusion
• [14C]ZT-1 was isolated with a radiochemical purity of
>98%area and a gravimetric specific activity of 129 μCi/mg
in a seven step synthesis starting from [U-14C]phenol in
7% yield
• Subsequently, the deuterium labelled target
(-)-[d3]huperzine A was achieved in 6 steps with an
overall yield of 15% and gave an isotopic distribution of
d2 (1.65% huperzine A) and d3 (97.93% huperzine A) with
a chemical purity of 98.5%
Conclusion
• Condensation of the substrate (-)-[d3]huperzine A with
5-chlorovanillin gave the Schiff’s base [d3]ZT-1 in a
chemical yield of 80%
• Reduction of the Schiff’s base gave reduced-[d3]ZT-1
which was converted into the hydrochloride salt with an
isotopic distribution of d2 (1.60%) and d3 (98.02%)
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connect to the benzene ring used in science