Synthesis of isotopically labelled [14C]ZT-1,

[d3]ZT-1 & (-)-[d3]huperzine A, a new generation of acetylcholinesterase inhibitors

Dr Sean KitsonШОН 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

cpres/v1b/ca/1998-08/ 3

A Brief Introduction to 14C

Discovery of 14CMartin Kamen & Sam Ruben (27-FEB-1940)

T1/2 ~ 5730 Years

Production of 14C

ReactorBombardment by Neutrons

Nitrogen 147 Protons7 Neutrons

N

PP

Radioactiveatom

Carbon 146 Protons8 Neutrons

Decay to stable daughter nuclide

Nitrogen 147 Protons7 Neutrons

N

ß-

P

ß-

P

14N7 + 1n0 → 14C6 + 1H1

14C Starting Materials

Ba(OH)2

Barium 14C carbonate staircaseOH

C14

NH

O

OHF3C

OH

Cl

OH

OHOMe

MeO

OMe

NCH3

OH

OH

C14

H

C14 OH

O

Ba14CO3

14C6

14CO2

14CH3OH

14CH3I

H14CHO

Cu14CN

K14CN

14C6

AA

14C

H314CO

14CHH14C

[14C]PEPTIDES

R T Brown et al. JLCR 2009, 52, 567-570

S L Kitson, S Jones et al. JLCR 2010, 53, 140-146

S L Kitson. JLCR 2007, 50, 290-294S L Kitson, E Knagg. JLCR 2006, 49, 517-531

[14C]Apomorphine [14C]Combretastatin A-1

[14C]XEN-D0401

[14C]Acetylenes

14C LabellingWhen 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 labelS L Kitson ‘Accelerated Radiochemistry’,PMPS Manufacturing 2010, 68-70

14C Drug Molecules14C 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 Alzheimer's Disease

4

NH

CH3

O

NH2

CH3

6

11

13

14

1

2

35

12

8

9

7

10

C M Yu et al. Canadian J Chem 1986, 64, 837-839D L Bai et al. Curr. Med. Chem; 2000, 7, 355-374

ZT-1: Pro-drug for (-)-Huperzine AA Novel Acetylcholinesterase Inhibitor

CHO

OH

Cl OMe

NH

CH3

O

NH2

CH3

NNO

Cl

H

MeOH O

H

CH3

CH3 in vivo progressive hydrolysis

active compound, (-)-huperzine A

+

Pro-drug ZT-1 metabolite, 5-chlorovanillin

exocyclic E-double bond

pyridin-2-one ring

hydrogen bond

R

bicyclo[3.3.1] double bond

L Leman, S L Kitson, R T Brown et al. JLCR 2011 (in press)

ZT-1 Implant

POLYMER DRUG

BLENDING

EXTRUSION

PLGA ZT-1

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

NH

CH3

O

NH2

D3C

NH

CH3

O

N

OH

ClMeO

CH3

NH

CH3

O

N

OH

ClMeO

D3C

*

(-)-[d3]Huperzine-A [14C]ZT-1 [d3]ZT-1

Synthesis of [14C]ZT-114C

labelling

Schiff base

OH

Cl

O

OMeH

NH

CH3

O

CH3 N

OH

ClMeO

*

NH

CH3

O

CH3

NH2

*

[14C]ZT-1

+

Schiff baseformation

(-)-Huperzine A 5-Chloro[U-14C]vanillin

Retro-synthetic pathwayOH

Cl

O

OMeH

OP

OMe

OP

I

OH

*

OP

OMeOHC

* **

*

[14C]-1

deprotection

halogenation

[14C]-7 A B

C

alkoxy de-halogenationortho-directed iodination

ortho-directed formylation

14C SynthesisOH

OH

OMe

Cl

O

H

OH

OMe

O

H

OMOM

OMe

OMOM

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, heat

[14C]-7

Step 7

Synthesis of

[d3]ZT-1 & (-)-[d3]huperzine

Retro-synthesis

NH

CH3

O

NH2

D3C

N

CH3

OMe

D3C CO2H

D3C PPh3

N

CH3

OMe

CO2MeO

Curtius rearrangement

Wittig

+

(-)-[d3]Huperzine A

(+)-8

isomerisation

N

CH3

CO2Me

OMe

O

N

CH3

CO2Me

OMe

CD3

N

CH3

CO2Me

OMe

D3C

N

CH3

CO2Me

OMe

CD3

PhS

.

Ph3P+CH2CD3.Br -

basePhSH, AIBN

(+)-8 [d3]-9

[d3]-10

Step 9 Step 10

Ph3P

D3CCH2Br

Step 8

Z > E

E > Z

via

radical

hydrogen abstraction from PhSHaddition of PhS. to cis-double bond180 degrees rotationejection of thiyl radical by beta-scission

Synthesis of (-)-[d3]huperzine A

C. Ferreri et al. Chem. Commun; 1999, 407-408

N

CH3

CO2Me

OMe

D3C

N

CH3

OMe

NHCO2MeD3C

NH

CH3

O

NH2

D3C

N

CH3

OMe

D3C CO2H

(-)-[d3]huperzine A

NaOH (PhO)2PON3 / Et3N

MeOH

TMS-I

[d3]-10[d3]-11

[d3]-12

Step 11

Step 12

Step 13

E > Z

MeOH

Synthesis of (-)-[d3]huperzine A

G A Olah, J. Org. Chem; 1979, 44, 1247-1251

S-I Yamada et al. J. Am. Chem. Soc; 1972, 94, 6203

NH

CH3

O

NH2

D3C

NH

CH3

O

D3C N

OH

ClMeO

Cl

OH

OMeH

O

NH

CH3

O

D3C NH

OH

ClMeO

NH

CH3

O

D3C NH

OH

ClMeO

[d3]-huperzine A [d3]ZT-1

Step 14

NaBH4

reduced-[d3]ZT-1 reduced-[d3]ZT-1.HCl

.HClMeOH HCl/Et2O

Step 15 Step 16

EtOH, heat

Synthesis of [d3]ZT-1&

reduced-[d3]ZT-1

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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The hexagonal shapes denote the famous Giant’s Causeway rock in Northern Ireland – these shapes also connect to the benzene ring used in science