Application of the Bischler-Napieralski-Pschorr radiosynthesis of (R)-(-)-[6a-14C]apomorphine, a non-selective D1/D 2 dopamine receptor agonist

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS

J Label Compd Radiopharm 2006; 49: 517–531.

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1070

Research Article

Application of the Bischler–Napieralski–Pschorr

radiosynthesis of (R)-(-)-[6a-¹⁴C]apomorphine,

a non-selective D₁/D₂dopamine receptor agonist

Sean L. Kitson* and Eric Knagg

GE Healthcare Limited, Amersham Place, Little Chalfont, Buckinghamshire

HP7 9NA, UK

Summary

A method has been developed for the carbon-14 radiosynthesis of non-narcotic

morphine derivative (R)-(-)-[6a-¹⁴C]apomorphine ð1Þ from the starting material

3,4-dimethoxy-2-nitrophenyl-N-phenethyl[carboxyl-¹⁴C]acetamide ð5Þ. The key to this

synthesis was the application of the Bischler–Napieralski cyclodehydration to 1-(3,4-

dimethoxy-2-nitrobenzyl)dihydro[1-¹⁴C]isoquinoline ð4Þ, followed by N-methylation

and reduction to 1-(3,4-dimethoxy-2-nitrobenzyl)-2-methyl-1,2,3,4-tetrahydro[1-¹⁴C]

isoquinoline ð3Þ. A ﬁnal Pschorr reductive ring closure followed by chiral separation

to give (R)-(-)-[6a-¹⁴C]apomorphine dimethyl ether ð2Þ and O-demethylation led to

(R)-(-)-[6a-¹⁴C]apomorphine ð1Þ with a speciﬁc activity of 55 mCi/mmol, radio-

chemical purity of >98% and chiral purity of >99%. Copyright # 2006 John Wiley

& Sons, Ltd.

Received 27 February 2006; Revised 13 March 2006; Accepted 13 March 2006

Key Words: apomorphine; cyclodehydration; Bischler–Napieralski and Pschorr

Introduction

(R)-(-)-[6a-¹⁴C]Apomorphine ð1Þ, (R)-(-)-(5,6,[6a-¹⁴C],7-tetrahydro-6-methyl-

4H-dibenzo-[de,g]-quinoline-10,11-diol)¹shown in Scheme 1 contains the

ortho-aryl phenol motif and belongs to a class of natural alkaloids called the

aporphines (e.g. apocodeine and bulbocapnine) which exhibit interesting

biological properties.²The structural elucidation of apomorphine was solved

*Correspondence to: S. L. Kitson, GE Healthcare Limited, The Maynard Centre, Forest Farm,

Whitchurch, Cardiﬀ CF14 7YT, UK. E-mail: sean.kitson@ge.com

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518

S. L. KITSON AND E. KNAGG

A

D

B

NH₂

bond

rotation

C

fix bond

HO

NH₂

HO

DOPAMINE

a rigid anologue of dopamine

6a

N

H

¹⁴C

CH₃

HO

(R)-(-)-[6a-¹⁴C]APOMORPHINE (1)

Scheme 1. Chemical structures of (R)-(-)-[6a-¹⁴C]apomorphine (1) and

dopamine

by Pschorr³in 1907 and its absolute conﬁguration was determined by Corrodi

et al. in 1955 and shown to have a R stereogenic centre.⁴The ﬁrst reported

total synthesis of ( ꢀ )apomorphine was carried out by Neumeyer et al.⁵in

1973 involving the alkylation of a Reissert isoquinoline with 3,4-dimethoxy-2-

nitrobenzylchloride ð8Þ.

This drug has been found to be a non-selective D₁/D₂dopamine receptor

agonist; showing more potent D₂-like eﬀects in the treatment of Parkinson’s

disease⁶resulting from dopamine imbalances in the brain. Dopamine can

adopt many conformations due to single bond rotation in the alkylamine side

chain. Apomorphine contains a rigid dopamine pharmacophore within its

framework to prevent rotation resulting in biological activity.^7,8Another

application of this drug is the treatment of erectile dysfunction.⁹Other co-

workers have demonstrated that the main metabolic in vivo pathways in rat

and human hepatocytes are sulphonation, glucuronidation and N-demethyla-

tion to give norapomorphine. Elimination of these conjugates is predomi-

nantly via the kidneys.¹⁰

Results and discussion

The retro-synthetic analysis for the incorporation of a single carbon-14 label at

the (R) stereogenic centre to give (R)-(-)-[6a-¹⁴C]apomorphine ð1Þ is shown in

Scheme 2. The disconnection of the ortho aryl carbon–carbon bond in (R)-(-)-

[6a-¹⁴C]apomorphine dimethyl ether ð2Þ gives the 1-(3,4-dimethoxy-

2-nitrobenzyl)-2-methyl-1,2,3,4-tetrahydro[1-¹⁴C]isoquinoline ð3Þ. A further

disconnection leads to 3,4-dimethoxy-2-nitrophenyl-N-phenethyl[carboxyl-

¹⁴C]acetamide ð5Þ. This can be synthesized from 3,4-dimethoxy-2-nitrophe-

Copyright # 2006 John Wiley & Sons, Ltd.

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APPLICATION OF BISCHLER–NAPIERALSKI–PSCHORR RADIOSYNTHESIS

519

N

H

N

H

N

C

CH

C

CH

C

CH

HO

MeO

NO

OMe

(1)

(2)

(3)

NO

MeO

Cl

C

O

NO

N

H

C

MeO

N

MeO

C

O

(6)

+

MeO

NO

H N

(5)

OMe

(4)

(7)

Scheme 2. Retrosynthetic analysis of (R)-(-)-[6a-¹⁴C]apomorphine ð1Þ

nyl[carboxyl-¹⁴C]acetylchloride ð6Þ and the commercially available starting

material 2-phenethylamine ð7Þ. The application of the Bischler–Napieralski

cyclodehydration on ð5Þ will give 1-(3,4-dimethoxy-2-nitrobenzyl)dihydro

[1-¹⁴C]isoquinoline ð4Þ, followed by N-methylation and reduction to produce

ð3Þ. A ﬁnal Pschorr reductive ring closure followed by chiral separation will

give ð2Þ and O-demethylation to (R)-(-)-[6a-¹⁴C]apomorphine ð1Þ.

Synthesis of ¹⁴C-labelled compounds

The synthetic route to 3,4-dimethoxy-2-nitrophenyl-N-phenethyl[carboxyl-

¹⁴C]acetamide ð5Þ is shown in Scheme 3. The inactive starting material

3,4-dimethoxy-2-nitrobenzylchloride ð8Þ was prepared in ﬁve steps starting

from the commercially available vanillin acetate.¹¹Compound ð8Þ gave the

1

following H NMR (d₆-DMSO, 200 MHz) d ¼ 7:41 (d, 1H, J ¼ 8:7 Hz), 7.32

(d, 1H, J ¼ 8:7 Hz), 4.72 (s, 2H), 3.91 (s, 3H), 3.84 (s, 3H).

The incorporation of the carbon-14 radiolabel was facilitated via S_N2

displacement of the chloro in 3,4-dimethoxy-2-nitrobenzylchloride ð8Þ

using potassium[¹⁴C]cyanide (55 mCi/mmol) in aqueous DMF at 808C to

aﬀord 3,4-dimethoxy-2-nitrobenzyl[¹⁴C]cyanide ð9Þ in 90% yield from ð8Þ.

Acid hydrolysis of ð9Þ with 50% hydrochloric acid produced 86% yield of 3,4-

dimethoxy-2-nitrophenyl[carboxyl-¹⁴C]acetic acid ð10Þ.

Subsequent conversion to 3,4-dimethoxy-2-nitrophenyl[carboxyl-¹⁴C]

acetylchloride ð6Þ with oxalyl chloride followed by the reaction with

Copyright # 2006 John Wiley & Sons, Ltd.

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520

S. L. KITSON AND E. KNAGG

NO

K¹⁴CN/H₂O

DMF

MeO

OH

MeO

50% HCl

Cl

C

N

O

(8)

(10)

(9)

NO

NH

MeO

Cl

H

N

(COCl)₂/DMF

MeO

C

(7)

O

(6)

(5)

Scheme 3. Synthetic route to 3,4-dimethoxy-2-nitrophenyl-N-phenethyl

[carboxyl-¹⁴C]acetamide ð5Þ

2-phenethylamine ð7Þ produced the retro fragment 3,4-dimethoxy-2-nitrophe-

nyl-N-phenethyl[carboxyl-¹⁴C]acetamide ð5Þ in 86% yield.

Bischler–Napieralski cyclodehydration

A successful Bischler–Napieralski cyclodehydration was accomplished by

activating the amide function in 3,4-dimethoxy-2-nitrophenyl-N-phenethyl[-

carboxyl-¹⁴C]acetamide ð5Þ with P₂O₅/POCl₃in toluene (Scheme 4) to give

O-phosphorylation. There is evidence that this combination of P₂O₅/POCl₃

provides a strong dehydrating medium which leads to the imino phosphates

(X₁¼ OPO₂, X₂¼ OPOCl₂) and nitrilium phosphates (Y₁¼ PO^ꢁ₃,

12,13

ꢁ

Y₂¼ OPOCl₂) depending on which dehydration route is taken.

Fodor

and Nagubandi¹⁴have reported that a mixture of P₂O₅/POCl₃may give rise to

the imino phosphate ðX₁¼ OPO₂Þ which facilitates a better leaving group

than the dichlorophosphates ðX₂¼ OPOCl₂Þ. The nitrilium salt ðY₁¼ PO^ꢁ₃Þ

can be captured via an intramolecular cyclization by the phenethylamino

moiety to give the Bischler–Napieralski endocyclic product 1-(3,4-dimethoxy-

2-nitrobenzyl)dihydro[1-¹⁴C]isoquinoline ð4Þ.

1

This was conﬁrmed by H NMR (d₆-DMSO, 200 MHz) d ¼ 7:55 (d, 1H,

J ¼ 6:8 Hz), 7.39–7.10 (m, 5H), 4.05 (s, 2H), 3.87 (s, 3H), 3.85 (s, 3H), 3.48

(t, 2H, J ¼ 7:3 Hz), 2.59 (t, 2H, J ¼ 7:8 Hz) and positive ion electrospray

[LC–MS–ES] with a pseudo molecular ion [MH]⁺bundle at m=z ¼ 327=329

conﬁrming the structure as a single carbon-14 label. No triplet was observed

for the isoquinoline nitrogen in ð4Þ therefore indicating that the structure is the

imine tautomer.

Bischler–Napieralski products

During the reaction another product was isolated (Scheme 5) and character-

ized by MS and ¹H NMR. Positive ion [LC–MS–ES] gave a pseudo molecular

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APPLICATION OF BISCHLER–NAPIERALSKI–PSCHORR RADIOSYNTHESIS

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Y^-₁, Y^-₂

NO

H

N

P O₅/POCl₃

2

MeO

NH+

C

O

C

Toluene

reflux

X₁, X₂

(5)

-

X₁= OPO₂, Y₁= PO₃

X₂= OPOCl₂, Y₂= Cl^-

NO

-

Y₁, Y₂

MeO

N

-HOPO₂

-HCl

MeO

C

N

X₁, X₂

MeO

X₁= OPO₂, X₂= OPOCl₂

Y₁= PO₃-, Y₂= -OPOCl₂

X₁= imino phosphate

Y₁= nitrilium phosphate

X₂= imino dichlorophosphoric anhydride

Y₂= nitrilium dichlorophosphate

N

C

-HOPO₃

-HOPOCl₂

MeO

NO

OMe

(4)

Scheme 4. Mechanistic view of the Bischler–Napieralski cyclodehydration

1

ion [MH]⁺bundle at m=z ¼ 309=311 and H NMR (d₆-DMSO, 200 MHz)

d ¼ 7:74 (d, 1H, J ¼ 6:9 Hz), 7.66 (d, 1H, J ¼ 9:4 Hz), 7.57 (t, 1H, J ¼ 6:9 Hz),

7.50 (t, 1H, J ¼ 6:4 Hz), 7.42 (d, 1H, J ¼ 8:3 Hz), 7.23 (d, 1H, J ¼ 9:4 Hz),

4.04 (s, 3H), 3.94 (s, 3H), 3.94 (t, 2H, J ¼ 7:0 Hz), 2.81 (t, 2H, J ¼ 7:0 Hz).

The above data is consistent with structure ð12Þ having a substituted

anthranil ring from the cyclodehydration of the nitrophenyl moiety with the

reactive methylene group¹⁵in ð5Þ to give the intermediate 6,7-dimethoxy-

N-phenethylanthranil-3-[carboxy-¹⁴C]amide ð11Þ.

The amide moiety was further dehydrated to initiate the intramolecular

cyclization via the phenethylamino group to give 3-(6,7-dimethoxyanthranil)-

dihydro[1-¹⁴C]isoquinoline ð12Þ.

Hey and Palluel¹⁶have carried out the Bischler–Napieralski cyclodehydra-

tion on unlabelled ð5Þ and reported the by-product to be unlabelled ð11Þ.

Copyright # 2006 John Wiley & Sons, Ltd.

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S. L. KITSON AND E. KNAGG

NO

H

N

O

N

MeO

H

P O /POCl₃

C

O

2

5

N

MeO

C

O

Toluene

Reflux

(5)

(11)

O

N

P O₅/POCl₃

2

N

C

MeO

Toluene

Reflux

(12)

H

NO

N

NO

MeO

(13)

(14)

Scheme 5. Bischler–Napieralski dehydration products

This compound would have a molecular mass of 326 compared to ð12Þ which

has a molecular mass of 308 when unlabelled. The Hey and Palluel¹⁶

1

conclusion must be dismissed based on the MS and H NMR analysis.

Previous co-workers¹⁷had assigned the by-product to be the ketamine ð13Þ or

acetylene ð14Þ. No von Braun amide degradation products, i.e. 3,4-dimethoxy-

2-nitrobenzyl[¹⁴C]cyanide ð9Þ and styrene were observed under any of these

conditions.

When the cyclodehydration of ð5Þ was carried out with POCl₃in reﬂuxing

acetonitrile (Scheme 6) it gave only the non-basic dehydration product in the

exocyclic form, 1-(3,4-dimethoxy-2-nitrobenzal)-1,2,3,4-tetrahydro[1-¹⁴C]iso-

quinoline ð15Þ.¹⁸

This product was identiﬁed by ¹H-NMR (CDCl₃, 200 MHz) d ¼ 7:75

(d, 1H, J ¼ 9:4 Hz), 7.40–7.20 (m, 5H), 7.10 (d, 1H, J ¼ 9:4 Hz), 6.75

(bt, 1H, NH), 4.20 (s, 3H), 4.00 (s, 3H), 3.80 (q, 2H, J ¼ 7:3 Hz), 3.00

(t, 2H, J ¼ 7:3 Hz) and LRMS analysis by positive ion EI produced

the expected ion bundle [M⁺] at m=z ¼ 326=328 as expected for

a single carbon-14 label. This data is consistent with structure ð15Þ

having a ‘stilbene’ with extended conjugation between the two aromatic

rings. The enamine proton in the tetrahydroisoquinoline gave a triplet

and does not exchange with D₂O indicating ð15Þ was non-basic. This

product did not undergo N-methylation with methyl iodide under various

conditions.

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NO

H

N

CH O

NH

C

POCl₃/CH₃CN

C

O

(5)

MeO

NO

OMe

(15)

Scheme 6. The Bischler–Napieralski exocylic product ð15Þ

N-methylation and reduction

The Bischler–Napieralski product 1-(3,4-dimethoxy-2-nitrobenzyl)dihy-

dro[1-¹⁴C]-isoquinoline ð4Þ was then converted into the quaternary salt with

methyl iodide (Scheme 7). On isolation this gave 1-(3,4-dimethoxy-2-

nitrobenzyl)dihydro[1-¹⁴C]isoquinoline methiodide ð16Þ. MS by negative ion

fast atom bombardment (FAB) analysis produced the expected ion bundle

[M–I]^ꢁat m=z ¼ 127 for the iodide ion. Positive ion electrospray (ES)

produced the ion bundle [M⁺] at m=z ¼ 341=343 as expected for a single

carbon-14 label. The 3,4-dihydroisoquinolinium ring was reduced by sodium

borohydride in ethanol to give 1-(3,4-dimethoxy-2-nitrobenzyl)-2-methyl-

1,2,3,4-tetrahydro[1-¹⁴C]isoquinoline ð3Þ. The strategy employed for the

reduction of ð16Þ prevented the carbon–carbon bond cleavage between the

3,4-dihydroisoquinolinium ring and the 3,4-dimethoxy-2-nitrophenyl moiety.

This reductive cleavage occurs readily in Reissert 1,2-dihydroisoquinoline

derivatives.^19,20This subsequent reduction would have given 2-methylisoqui-

noline and 3,4-dimethoxy-2-nitrotoluene.

Reductive Pschorr cyclization^21,22

The reduction of the aryl nitro group in 1-(3,4-dimethoxy-2-nitrobenzyl)-2-

methyl-1,2,3,4-tetrahydro[1-¹⁴C]isoquinoline ð3Þ to the aryl amino group was

achieved using zinc dust/hydrochloric acid (Scheme 7). Diazotization of the

amino group to the corresponding arenediazonium salt followed by the

addition of copper facilitated an intramolecular Pschorr coupling (probably

via an aryl radical intermediate)^23,24to generate a new six-membered ring to

form the aporphine (R/S)-( ꢀ )-[6a-¹⁴C]apomorphine dimethyl ether ð17Þ. The

enantiomers of ð17Þ were separated by preparative chiral HPLC using a

Chiralcel-OD column to give (R)-(-)-[6a-¹⁴C]apomorphine dimethyl ether ð2Þ

and (S)-(+)-[6a-¹⁴C]apomorphine dimethyl ether ð18Þ.²⁵Assignment of the

enantiomers was established by reacting authentic (R)-(-)-apomorphine

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S. L. KITSON AND E. KNAGG

I

N

C

N

C

CH

C

CH

CH₃I/THF

Reflux

NaBH₄/EtOH

MeO

NO

MeO

NO

MeO

NO

OMe

(4)

(16)

(3)

N

H

1. 10% aq H₂SO₄/NaNO₂

2. Cu₂O

C

CH

N

H

C

CH

MeO

Chiral HPLC

Resolution

MeO

(17)

(18)

N

H

C

CH

C

CH

H

BBr₃/CH₂Cl₂

MeO

HO

(2)

(1)

Scheme 7. Radiosynthesis of (R)-(-)-[6a-¹⁴C]apomorphine ð1Þ

hydrochloride hemihydrate with diazomethane to give (R)-(-)-apomorphine

dimethyl ether.

O-demethylation

The O-demethylation of (R)-(-)-[6a-¹⁴C]apomorphine dimethyl ether ð2Þ was

accomplished with boron tribromide in dichloromethane (Scheme 7) to give

the non-selective D₁/D₂dopamine receptor agonist: (R)-(-)-[6a-¹⁴C]apomor-

phine ð1Þ as the hydrochloride hemihydrate [RCP ¼ 98:4%, optical

purity ¼ 99:6%, SA ¼ 55 mCi/mmol].

The structure of the ﬁnal product was conﬁrmed by comparison

(HPLC, NMR, MS) with authentic (R)-(-)-apomorphine hydrochloride

hemihydrate [41372-20-7] from Aldrich Chemical Company. The

other enantiomer (S)-(+)-apomorphine hydrochloride hemihydrate

[41035-30-7] is also available from Aldrich. (R)-(-)-apomorphine

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hydrochloride hemihydrate is commercialized as apomorphine SL (Ixense¹,

and Uprima¹).

Experimental procedures

Materials and methods

All radioactive experiments were carried out in the custom labelling special

synthesis laboratories in specially designed hoods dedicated to carbon-14 only.

Proton NMR spectra were recorded on a Bruker AC 200 or Bruker Avance

400 FT-NMR spectrometer. Chemical shifts were recorded in ppm (d) from an

internal tetramethylsilane standard in either d₆-DMSO or CDCl₃and coupling

constants (J) are reported in Hertz. All reagents were purchased from

commercial sources and were used without further puriﬁcation. 3,4-

dimethoxy-2-nitrobenzylchloride ð8Þ was prepared using reported methods.^5,11

Thin layer chromatography (TLC) was carried out using Merck Kieselgel

silica gel 60 F₂₅₄and Whatman LKC18F octadecyl-silica gel ODS glass plates.

Radio-TLC was carried out on a BIOSCAN System AR 2000. Compounds

were detected with UV light at 254 nm. Final products were dried in a vacuum

desiccator over phosphorus pentoxide. The radiochemical yield was deter-

mined by liquid scintillation counting using the WALLAC 1409 DSA.

Analytical HPLC was performed on the Hewlett Packard series 1100 using a

Berthold Scintillator Pump equipped with a Berthold LB507A monitor used

for the detection of radiochemical compounds. Radiochemical and chiral

purity by HPLC of the ﬁnal compound was performed on a Chiralcel OD-R

column (250 ꢂ 4.6 mm)²⁵with a ﬂow of 0.5 ml/min and 0.05 M sodium

perchlorate buﬀer pH 2.0:CH₃CN (65:35) as the mobile phase at UV 273 nm.

Low resolution mass spectrometry [LRMS] was performed on the following

instruments; Jeol JMS-DX300, Kratos MS25RF and Q-Tof2 spectrometers.

Sample introduction was by DCI, Direct Insertion or De-absorption Probes,

LC or infusion with the instruments set to detect positive or negative ions to

obtain electron impact (EI), electrospray (ES), chemical ionization (CI) and

fast atom bombardment (FAB) spectra.

Carbon-14 radiosynthesis

Synthesis of 3,4-dimethoxy-2-nitrobenzyl[¹⁴C]cyanide (9). To a solution of

3,4-dimethoxy-2-nitrobenzylchloride ð8Þ (8.1 g, 26 mmol) in DMF (273 ml) was

added a solution of potassium[¹⁴C]cyanide (1450 mCi, 55 mCi/mmol, 26 mmol)

in water (27 ml). The reaction was heated to 808C and held for 90 min with

stirring. HPLC (Genesis AQ column, CH₃CN/H₂O) analysis of the reaction

mixture gave 87% product conversion. The reaction mixture was quenched

with water (273 ml) and the product extracted into ether (10 ꢂ 130 ml). The

combined organic extracts were washed with 1 N sodium hydroxide (150 ml),

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S. L. KITSON AND E. KNAGG

separated, dried [MgSO₄], ﬁltered and evaporated in vacuo to give a pale

orange oil. This was puriﬁed by column chromatography on ﬂash silica eluting

with a mixture of ether/pentane. The appropriate fractions were combined and

concentrated in vacuo to aﬀord 3,4-dimethoxy-2-nitrobenzyl[¹⁴C]cyanide ð9Þ

(1310 mCi, 55 mCi/mmol, 90%) as a white solid. Analytical HPLC (Genesis

AQ column, CH₃CN/H₂O) analysis gave a single radioactive peak at

RTꢃ21 min and no starting material was observed. TLC (silica gel, ether/

hexane, 50:50) R_f¼ 0:38 gave a single spot.

Synthesis of 3,4-dimethoxy-2-nitrophenyl[carboxyl-¹⁴C]acetic acid (10). 3,4-

Dimethoxy-2-nitrobenzyl[¹⁴C]cyanide ð9Þ (1310 mCi, 23.8 mmol, 55 mCi/

mmol) was added to 50% hydrochloric acid (200 ml) and the heterogeneous

reaction mixture was heated to 1268C for 5 hours. Analytical HPLC (Genesis

AQ column, CH₃CN/H₂O) of the reaction mixture gave 98% product

conversion. The reaction mixture was cooled to ambient temperature and

quenched with ice water (200 ml) and the product was extracted into ether

(5 ꢂ 100 ml). The ether extracts were then combined and extracted with

saturated sodium bicarbonate (5 ꢂ 100 ml). The basic extract was acidiﬁed

with concentrated hydrochloric acid and the product extracted back into ether

(3 ꢂ 250 ml). The ether extracts were combined, dried over [Na₂SO₄], ﬁltered

and concentrated in vacuo to give the crude product. This was puriﬁed by

column chromatography on silica eluting with a mixture of ether/hexane/

acetic acid. The appropriate fractions were combined and concentrated in

vacuo to give 3,4-dimethoxy-2-nitrophenyl[carboxyl-¹⁴C]acetic acid ð10Þ

(1130 mCi, 55 mCi/mmol, 20.5 mmol, 86%) as a yellowish solid. TLC

RCP ¼ 99:5%, R_f¼ 0:73 [silica gel, ether/acetic acid (100:1)].

Synthesis of 3,4-dimethoxy-2-nitrophenyl-N-phenethyl[carboxyl-¹⁴C]acetamide

(5). 3,4-Dimethoxy-2-nitrophenyl[carboxyl-¹⁴C]acetic acid (10) (20.5 mmol,

1130 mCi, 55 mCi/mmol) was dissolved in anhydrous dichloromethane

(200 ml). Excess oxalyl chloride (15 g, 118 mmol) was added to the stirred

yellow solution, followed by one drop of DMF. The reaction mixture was

stirred at ambient temperature for one hour. After complete consumption of

the starting material by TLC analysis, the excess oxalyl chloride was removed

in vacuo to give the yellow oil 3,4-dimethoxy-2-nitrophenyl[carboxyl-¹⁴C]

acetylchloride ð6Þ. The oil was taken up into anhydrous dichloromethane

(200 ml) and the solution was cooled down in an ice water bath. 2-

Phenethylamine ð7Þ (12.2 g, 101 mmol) was added dropwise to the cold

solution. After complete addition the reaction mixture was allowed to warm to

ambient temperature and left stirring overnight. The mixture was quenched

with 2 N hydrochloric acid (200 ml) and dichloromethane (200 ml). The

organic layer was separated and washed with 2 N hydrochloric acid

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(3 ꢂ 500 ml) followed by 2 N sodium hydroxide (500 ml) and brine (500 ml).

The organic layer was dried over [Na₂SO₄], ﬁltered and concentrated

in vacuo to give the crude product as brown oil (1052 mCi). The product

was puriﬁed by ﬂash column chromatography on silica eluting with a mixture

of hexane/ether. The product fractions were combined and concentrated in

vacuo to aﬀord an oﬀ-white solid 3,4-dimethoxy-2-nitrophenyl-N-phenethyl[-

carboxyl-¹⁴C]acetamide ð5Þ (6.2 g, 968 mCi, 55 mCi/mmol, 86%): TLC

RCP ¼ 99:5%, R_f¼ 0:74, [silica gel, ethyl acetate/acetic acid (100:1)]; TLC

RCP ¼ 99:1%, R_f¼ 0:74, [ODS, CH₃CN/H₂O (8:2)]; LRMS positive ion EI

[M⁺]:344/346; speciﬁc activity 55 mCi/mmol; ¹H-NMR (d₆-DMSO, 400 MHz)

d ¼ 8:11 (bt, 1H, NH), 7.33–7.17 (m, 6H), 7.12–7.08 (d, 1H, J ¼ 8:3 Hz), 3.88

(s, 3H), 3.70 (s, 3H), 3.38 (s, 2H), 3.23 (t, 2H, J ¼ 6:4 Hz), 2.69 (t, 2H,

J ¼ 7:8 Hz).

Synthesis of 1-(3,4-dimethoxy-2-nitrobenzyl)-dihydro[1-¹⁴C]isoquinoline (4). A

solution of 3,4-dimethoxy-2-nitrophenyl-N-phenethyl[carboxyl-¹⁴C]acetamide ð

5Þ (906 mCi, 55mCi/mmol, 16.5 mmol) in toluene (200ml) was added dropwise

over 20 min to a suspension of phosphorous pentoxide (49 g) and phosphorous

oxychloride (12 ml) in toluene (120ml). On complete addition, the reaction was

reﬂuxed for a further 30min and then allowed to cool to ambient temperature.

The active orange-coloured toluene solution was decanted oﬀ and the residual

solid was washed with toluene (3ꢂ 100 ml). The residual solid was then

decomposed with ice and the product was extracted into chloroform (7 ꢂ 50ml).

The combined chloroform extracts were shaken with 2 N sodium hydroxide,

dried over [Na₂SO₄], ﬁltered and concentrated in vacuo to give a dark oil. The

crude product was puriﬁed by ﬂash chromatography on silica eluting with a

mixture of ether/hexane to give the Bischler–Napieralski product 1-(3,4-

Dimethoxy-2-nitrobenzyl)-dihydro[1-¹⁴C]isoquinoline ð4Þ (396 mCi, 43%) as

an oil. TLC R_f¼ 0:46 (silica gel, ethyl acetate), ¹H-NMR (d₆DMSO, 400MHz)

d ¼ 7:55 (d, 1H, J ¼ 6:8 Hz), 7.39–7.10 (m, 5H), 4.05 (s, 2H), 3.87 (s, 3H), 3.85

(s, 3H), 3.48 (t, 2H, J ¼ 7:3 Hz), 2.59 (t, 2H, J ¼ 7:8 Hz); Positive ion LC–MS–

ES: [MH]⁺327/329; speciﬁc activity 55 mCi/mmol.

Synthesis of 1-(3,4-dimethoxy-2-nitrobenzyl)dihydro[1-¹⁴C]isoquinoline meth-

iodide (16). 1-(3,4-Dimethoxy-2-nitrobenzyl)dihydro[1-¹⁴C]isoquinoline ð4Þ

(396 mCi, 55 mCi/mmol) was taken up in anhydrous tetrahydrofuran

(100 ml) and methyl iodide (5 ml, 80.3 mmol) was added. The reaction mixture

was heated under reﬂux for 12 hours. TLC analysis (ODS, CH₃CN/H₂O 8:2)

indicated complete consumption of the starting material. The cooled reaction

mixture was evaporated in vacuo to give a yellow solid 1-(3,4-dimethoxy-2-

nitrobenzyl)dihydro-[1-¹⁴C]isoquinoline methiodide ð16Þ. LRMS positive ion

ES: [MH]⁺341/343 and negative ion FAB: 127 for iodide counter ion.

J Label Compd Radiopharm. 2006; 49:517–531

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528

S. L. KITSON AND E. KNAGG

Synthesis of 1-(3,4-dimethoxy-2-nitrobenzyl)-2-methyl-1,2,3,4-tetrahydro[1-¹⁴C]

isoquinoline (3). 1-(3,4-Dimethoxy-2-nitrobenzyl)dihydro-[1-¹⁴C]isoquinoline

methiodide ð16Þ was taken up in ethanol (125 ml) and treated with sodium

borohydride (1.33 g, 35.2 mmol). The reaction was stirred at ambient

temperature for 1.5 hours. The excess sodium borohydride was destroyed by

the addition of 2 N hydrochloric acid and the reaction was basiﬁed with 2 N

sodium hydroxide. The basic solution was extracted with ether and the

combined ether extracts were dried over [MgSO₄], ﬁltered and concentrated in

vacuo to give the crude product. This was puriﬁed by reverse phase HPLC

using a Phenomenex Prodigy column eluting the product oﬀ with a mixture of

acetonitrile/water to aﬀord a pale yellow solid 1-(3,4-dimethoxy-2-nitroben-

zyl)-2-methyl-1,2,3,4-tetrahydro[1-¹⁴C]isoquinoline ð3Þ (306 mCi, 55 mCi/

mmol). RCP TLC 98.2% at R_f¼ 0:31 (silica gel, ethyl acetate) and 97.5%

at R_f¼ 0:50 [ODS, acetonitrile:water (8:2)]; ¹H-NMR (CDCl₃, 200 MHz) d ¼

7:10 (m, 3H), 6.86 (d, 1H, J ¼ 8:7 Hz), 6.85 (s, 1H), 6.79 (d, 1H, J ¼ 8:7), 3.93

(s, 3H), 3.88 (s, 3H), 3.77 (t, 1H), 3.30–2.50 (m, 6H), 3.02 (s, 3H).

Synthesis of (R/S)-( ꢀ )-[6a-¹⁴C]apomorphine dimethyl ether (17). A solu-

tion of 1-(3,4-dimethoxy-2-nitrobenzyl)-2-methyl-1,2,3,4-tetrahydro[1-¹⁴C]iso-

quinoline ð3Þ (306 mCi, 55 mCi/mmol) in ethanol (140 ml) and concentrated

hydrochloric acid (63 ml) was cautiously treated with zinc dust (7.9 g,

120 mmol) and the reaction heated under reﬂux for 1 hour. On complete

consumption of the starting material (by TLC analysis on silica eluting with

ethyl acetate), the reaction was cooled and basiﬁed with 20% aqueous sodium

hydroxide and then extracted with dichloromethane (500 ml). The organics

were washed with water, saturated sodium bicarbonate solution and brine then

dried over [MgSO₄], ﬁltered and evaporated in vacuo to give a dark residue.

The residue was dissolved in 10% aqueous sulphuric acid (47.5 ml) and cooled

to 08C. A solution of sodium nitrite (638 mg, 9.38 mmol) in water (24 ml) was

then added dropwise. The reaction was stirred for 30 min at ꢁ38C to give the

diazonium salt suspension. Urea was added to destroy the excess nitrous acid.

Copper(I)oxide powder (638 mg, 9.38 mmol) was added to the diazonium salt

suspension and the reaction was left to warm to ambient temperature. The

reaction was basiﬁed with 2 N sodium hydroxide and the product extracted

into ethyl acetate (2 ꢂ 200 ml) and dichloromethane (1 ꢂ 200 ml). The

combined organic extracts were dried over [MgSO₄], ﬁltered and concentrated

in vacuo to give the crude product. This was puriﬁed by preparative HPLC on

a Dynamax silica column eluting with 2% methanol in dichloromethane to

aﬀord a pale yellow oil (R/S)-( ꢀ )-[6a-¹⁴C]apomorphine dimethyl ether ð17Þ.

¹H-NMR (CDCl₃, 200 MHz) d ¼ 8:24 (d, 1H, J ¼ 7:9 Hz), 7.28 (t, 1H,

J ¼ 7:8 Hz), 7.08 (d, 1H, J ¼ 7:5 Hz), 7.02 (d, 1H, J ¼ 8:2 Hz), 6.85 (d, 1H,

J ¼ 8:2 Hz), 4.99 (s, 3H), 3.90 (s, 3H), 3.70 (s, 3H), 3.35–2.40 (bm, 7H).

J Label Compd Radiopharm. 2006; 49:517–531

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APPLICATION OF BISCHLER–NAPIERALSKI–PSCHORR RADIOSYNTHESIS

529

Chiral resolution of (R/S)-( ꢀ )-[6a-¹⁴C]apomorphine dimethyl ether

(17). (R/S)-( ꢀ )-[6a-¹⁴C]Apomorphine dimethyl ether ð17Þ was separated

by normal phase preparative HPLC using a cellulose-based chiral column

(Chiralcel OD) eluting with hexane/propan-2-ol/dimethylamine (950:50:5).

This gave (R)-(-)-[6a-¹⁴C]apomorphine dimethyl ether ð2Þ (49 mCi, 55 mCi/

mmol) which co-eluted with authentic (6aR)-(-)-apomorphine dimethyl ether

and the other unwanted enantiomer (S)-(+)-[6a-¹⁴C]apomorphine dimethyl

ether ð18Þ (34 mCi, 55 mCi/mmol).

Synthesis of (R)-(-)-[6a-¹⁴C]apomorphine (1). To a solution of (R)-(-)-

[6a-¹⁴C]apomorphine dimethyl ether ð2Þ (42 mCi, 55 mCi/mmol) in anhydrous

dichloromethane (42 ml), boron tribromide (1.0 ml, 1.0 mmol, 1.0 M solution

in dichloromethane) was added. The reaction was stirred at ambient

temperature for 90 min. Methanol (3 ꢂ 50 ml) was added and the solvent

removed by evaporation in vacuo. The crude product was puriﬁed on a reverse

phase PLRP-S column eluting the product with a mixture of acetonitrile/

aqueous hydrochloric acid. The solvent was evaporated in vacuo to give the

ﬁnal product (R)-(-)-[6a-¹⁴C]apomorphine ð1Þ (32 mCi, 76% yield) as a white

crystalline hydrochloride salt. Radiochemical purity: 98.4%, Chiral purity:

99.6% [Chiralcel OD-R, 0.05 M sodium perchlorate buﬀer pH 2.0/acetonitrile

(65:35)]; LRMS positive ion EI: [M⁺] 36/38 as expected for the hydrochloride

counter ion and by DCI-MNH⁺₄gave a pseudo molecular ion [MH]⁺bundle

at m=z ¼ 268=270 conﬁrming the structure as a single carbon-14 label. Speciﬁc

activity 55 mCi/mmol; ¹H-NMR (D₂O, 400 MHz) d ¼ 8:10 (d, 1H,

J ¼ 7:7 Hz), 7.32 (t, 1H, J ¼ 7:8 Hz), 7.07 (d, 1H, J ¼ 7:6 Hz), 6.61 (m, 2H),

3.75 (m, 1H), 3.57 (bm, 1H), 3.16–3.09 (m, 2H), 2.98 (s, 3H), 2.88–2.81 (m,

2H), 2.49 (m, 1H).

Conclusion

A wide array of experimental conditions (i.e. temperature and duration of

heating) and condensing agents have been used to facilitate the Bischler–

Napieralski cyclodehydration on 3,4-dimethoxy-2-nitrophenyl-N-phenethyl

[carboxyl-¹⁴C]acetamide ð5Þ. The dehydrating agent phosphorous oxychloride,

either alone or in an inert solvent (i.e. toluene), was found to be a mild reagent

and gave exclusively 1-(3,4-dimethoxy-2-nitrobenzal)-1,2,3,4-tetrahydro

[1-¹⁴C]isoquinoline ð15Þ. In our case we found utilizing a mixture of

phosphorous pentoxide and phosphorous oxychloride in reﬂuxing toluene

gave better results in generating 1-(3,4-dimethoxy-2-nitrobenzyl)dihydro

[1-¹⁴C]isoquinoline ð4Þ than would be when used separately. This cyclode-

hydration step is important in the synthesis of the aporphine alkaloids. When

the substituted aromatic ring to which ring closure is to occur is not strongly

activated as in ð5Þ neutral compounds have been isolated. These neutral

J Label Compd Radiopharm. 2006; 49:517–531

DOI: 10.1002/jlcr

530

S. L. KITSON AND E. KNAGG

products have been assigned various structures containing an acetylene ð14Þ,

ketamine ð13Þ and anthranil ð11Þ moieties. Leading from the work of

11

Neumeyer,⁵Hey and Lob

we have modiﬁed the synthesis to enable the

incorporation of a single carbon-14 radiolabel in (R)-(-)-[6a-¹⁴C]apomorphine

ð1Þ and in the course of this work we have identiﬁed the neutral compound to

be 3-(6,7-dimethoxyanthranil)-dihydro[1-¹⁴C]isoquinoline ð12Þ. This synthetic

methodology can be applied to the synthesis of other carbon-14 aporphines

which would be useful in ADME and pharmacokinetic studies on neuro-

degenerative diseases.

Acknowledgements

The authors would like to thank Dr David Hendry, Gary Shemilt, Dr Derek

Wallis and Alan Simmonds for their technical assistance in this project and

Mark Newby for chiral HPLC work. ¹H NMR interpretation was done by Dr

Steven Reynolds and Lindsey Jones. Final product analysis was carried out by

our Quality Control Department.

References

1. Roth HJ, Eger K, Troschute R. Pharmaceutical Chemistry, vol. 12. Ellis

Horwood: Chichester; UK, 1991; 633–635.

2. Rios JL, Manez S, Giner RM, Recio MC. The Alkaloids, vol. 53. Academic Press:

New York, 2000.

3. Pschorr R. Berichte 1907; 40: 1984–1995.

4. Corrodi H, Hardegger E. Helv Chim Acta 1955; 38: 2038–2043.

5. Neumeyer JL, Neustadt BR, Oh KH, Weinhardt KK, Boyce CB. J Med Chem

1973; 16: 1223–1228.

6. Thomas F, Muir A, Stirton J, Macphee G, Hudson S. Pharm J 2001; 267:

600–612.

7. Borrelli B. Neurosci Biobehav Rev 2000; 24: 125–132.

8. Lal S. Prog Neuropsychopharmacol Biol Psychiatry 1988; 12: 117–164.

9. Segraves RT. J Urol 1991; 145: 1174–1175.

10. LeWitt PA. Neurology 2004; 62: S8–S11.

11. Hey DH, Lobo LC. J Chem Soc 1954; 2246–2256.

12. EI-Sayraﬁ S, Rayyan S. Molecules 2001; 6: 279–286.

13. Fodor G, Gal G, Phillips BA. Angew Chem Int Ed Engl 1972; 11: 919–920.

14. Fodor G, Nagubandi S. Tetrahedron 1980; 36: 1279–1300.

15. Wunsch KH, Boulton AJ. Adv Heterocyclic Chem 1967; 8: 277–379.

16. Hey DH, Palluel AL. J Chem Soc 1956; 4123–4129.

17. Callow RK, Gulland JM, Haworth RD. J Chem Soc 1929; 1444–1456.

18. Weisbach JA, Burns C, Macko E, Douglas B. J Med Chem 1963; 6: 91–97.

19. Neumeyer JL, McCarthy M, Weinhardt KK, Levins PL. J Org Chem 1968;

2890–2894.

J Label Compd Radiopharm. 2006; 49:517–531

DOI: 10.1002/jlcr

APPLICATION OF BISCHLER–NAPIERALSKI–PSCHORR RADIOSYNTHESIS

531

20. Neumeyer JL, Oh KH, Weinhardt KK, Neustadt BR. J Org Chem 1969;

3786–3788.

21. Preston HL. Chem Rev 1956; 56: 27–48.

22. Duclos RL, Tung JS, Rapoport H. J Org Chem 1984; 49: 5243–5246.

23. Gadallah FF, Cantu AA, Elofson RM. J Org Chem 1973; 38: 2386–2393.

24. Kobori N, Kobayashi M, Minato H. Bull Chem Soc Jpn 1970; 43: 223–225.

25. Ameyibor E, Stewart JT. J Chromatogr B 1996; 686: 297–300.

J Label Compd Radiopharm. 2006; 49:517–531

DOI: 10.1002/jlcr

Article Doi

Application of the Bischler-Napieralski-Pschorr radiosynthesis of (R)-(-)-[6a-14C]apomorphine, a non-selective D1/D 2 dopamine receptor agonist

DOI: 10.1002/jlcr.1070

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Article abstract of DOI:10.1002/jlcr.1070

Full text of DOI:10.1002/jlcr.1070

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