Synthesis of radio- and stable-labeled Fasidotril

Full text of DOI:10.1002/jlcr.901

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 1029–1033.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.901
Research Article
Synthesis of radio- and stable-labelled Fasidotrily
Tapan Ray, Grazyna Ciszewska*, Alban J. Allentoff, Amy Wu and Lawrence
Jones
Department of Preclinical Safety, Isotope Laboratory, Novartis Pharmaceuticals
Corporation, One Health Plaza, East Hanover, NJ 07936, USA
Key Words: vasopeptidase inhibitor; tritium labelled; deuterium labelled; 14C labelled
Introduction
Fasidotril (Figure 1) is a vasopeptidase inhibitor currently under develop-
ment for treatment of hypertension. Radiolabelled Fasidotril was re-
quired for ADME and whole-body autoradiography studies. Stable-labelled
Fasidotril (with an appropriately high number of labelled mass units) was also
needed as an LC/MS internal standard for use in bioanalytical assays.
Details of the synthesis of [14C]Fasidotril (with carbon-14 labelling in the
benzylic position of the piperonyl portion of the molecule), and stable-labelled
Fasidotril (with deuteriums situated in the benzyl ester moiety) are described
below.
Attempted bromination for tritium–halogen interchange
Initially, we envisioned preparing tritium-labelled Fasidotril via a tritium–
halogen interchange of a brominated analog of the drug substance. It was
thought that the methylene dioxophenyl ring of the drug substance would offer
suitable aromatic positions for facile bromination. Unfortunately, attempted
bromination of Fasidotril gave none of the desired bromo-analog. In-
stead, the thiol acetyl group was unstable under all bromination condi-
tions tried, affording mixtures of deactylated products and disulfur-containing
dimers.
*Correspondence to: G. Ciszewska, Department of Preclinical Safety, Isotope Laboratory, Novartis
Pharmaceuticals Corporation, One Health Plaza, East Hanover, NJ 07936, USA. E-mail: grazyna.ciszewska
@pharma.novartis.com
yThis paper was presented at the 8th International Symposium, Boston, USA, 1–5 June 2003 but was
inadvertently left out of the proceedings.
Copyright # 2004 John Wiley & Sons, Ltd.

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Figure 1.
Synthesis of [14C]Fasidotril
Since tritium–halogen interchange of the drug substance was not a viable
option, we developed a synthesis of carbon-14 labelled Fasidotril which was
accomplished in 11 steps.
Synthesis of the key precursor [14C]piperonylacrylic acid (10, Schemes 1 and
2). [14C]Piperonylacrylic acid (10) was a key intermediate in the radio-
synthesis. It was synthesized following the route displayed in Schemes 1 and 2.
Initially, benzylbromide 5 (Scheme 1) was prepared starting with 5-bromo-1,3-
benzodioxazole (1) which reacted with magnesium in THF to form the
Grignard reagent 2. Reagent 2, without isolation, was treated with 14CO2
generated from Ba14CO3 to furnish acid 3. Reduction of the carboxylic group
of 3 afforded piperonyl-a-14C-alcohol (4). Substitution of the hydroxyl group
with bromine was achieved by treatment of 4 with a 48% aqueous solution of
HBr yielding 5.
Scheme 1. Synthesis of [14C] benzyl bromide (5)
Carbanion 7 (Scheme 2), which was generated from malonic acid (6),
reacted with 5 to furnish 8. Hydrolysis of the ester groups was achieved by
treatment of 8 with a 10% aqueous solution of KOH forming diacid 9. Finally,
[14C]Piperonylacrylic acid (10) was obtained by the reaction of 9 with
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J Label Compd Radiopharm 2004; 47: 1029–1033

SYNTHESIS OF
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Scheme 2. Synthesis of piperonyl acrylic acid (10)
formaldehyde in the presence of triethylamine. Overall, 60.5 mCi of 10 was
obtained starting from 500 mCi of [14C] barium carbonate.
Completion of the synthesis of [14C] Fasidotril (15, Scheme 3). The synthesis
of labelled Fasidotril was completed in four steps as shown in Scheme 3.
[14C]Piperonylacrylic acid (10) was converted to acid chloride (11) by
treatment with thionyl chloride in DMF. The resulting [14C]Piperonylacryloyl
chloride reacted with L-alanine phenylmethyl ester (12) in the presence of
triethylamine in toluene, affording ester 13. Treatment of 13 with thiolacetic
Scheme 3. Completion of the synthesis of [14C]Fasidotril
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 1029–1033

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acid (14) afforded crude 15 (via a stereoselective Michael addition), which was
purified by crystallization from isopropyl alcohol yielding 40 mCi of
[14C]Fasidotril.
Analysis of the diastereomeric purity of [14C] Fasidotril by HPLC. In order to
establish the enantiomeric excess associated with the second chiral center
formed from the reaction of thiolacetic acid with 13, a reverse phase chiral
HPLC method was developed. This method utilized an acetonitrile/water
mobile phase with a Chiralcel AS-RH column to maximise the separation of
the diastereomers. Using this method, the diastereomeric purity of the desired
(S,S) labelled drug substance was established as >97%.
Synthesis of [2H7]Fasidotril
Attempted synthesis via direct hydrolysis and reesterification. Initially, it was
envisioned that [2H7] Fasidotril could be directly synthesized from unlabelled
Fasidotril by first hydrolyzing the ester groups, followed by reesterification
2
with labelled benzyl alcohol affording the H7 benzyl group and replacing the
acetyl group. This approach was unsuccessful since the resulting acid after
base hydrolysis (containing the free thiol) was extremely unstable and
dimerized during work-up.
Preparation of [2H7]Fasidotril (Scheme 4). The synthesis of [2H7]Fasidotril
was achieved (starting from intermediate 16) in three steps. The benzyl group
of 16 was removed by stirring a solution of 16 in methanol with 10% aqueous
NaOH at room temperature for three hours. After purification, the resulting
acid 17 was tested for the absence of any benzyl alcohol. Acid 17 was then
converted to the [2H7]benzyl ester by stirring with [2H7]benzyl alcohol in the
2
Scheme 4. Synthesis of H7-Fasidotril
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SYNTHESIS OF
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presence of 1,3-dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine in
methylene chloride at room temperature for 24 h. Finally, the labelled ester
was converted to [2H7]Fasidotril (18) via reaction with thiolacetic acid at 90 8C
for 5 h.
Conclusions
1. The vasopeptidase inhibitor Fasidotril with carbon-14 labelling was
efficiently synthesized in an 11-step synthesis starting with [14C]barium
carbonate.
2. Deuterium-labelled Fasidotril suitable for use as an MS internal standard
was prepared via a three-step synthesis starting from a readily available
intermediate.
3. The intrinsic instability of the thiol acetyl group can offer considerable
synthetic challenge in the synthesis of isotopically labelled compounds
containing this group. Thiol acetyl groups are often unstable to hydrolysis
and/or halogenation and the resulting free thiol groups readily dimerize.
Therefore, these common reactions may need to be circumvented when
designing a successful syntheticroute for labelled thiol acetyl-containing
drug substances.
Copyright # 2004 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2004; 47: 1029–1033