Research Article
Received 9 October 2014,
Revised 29 December 2014,
Accepted 5 January 2015
Published online 17 February 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3266
Synthesis of deuterium-labelled isotopomer
of deferasirox
*
Freddy H. Havaldar, Bhushan Vasant Dabholkar, Ganesh Baban Mule,
and Suhas Kulkarni
A d4-labeled isotopomer of deferasirox was synthesized as internal standard for use in a LC/mass spectroscopy (MS)/MS
method developed for the simultaneous quantitative determination of deferasirox in human serum. d4-deferasirox was
synthesized from d8-toluene.
Keywords: deferasirox; deuterium
acid-d4 (8) was condensed with 2-(2-hydroxyphenyl)-4H-
1,3-benzoxazin-4-one (3) under microwave irradiation to afford
deferasirox-d4 (9) having isotopic purity of 99%.
Introduction
Deferasirox, marketed as Exjade,1 is a rationally designed2 oral iron
chelator. Its main use is to reduce chronic iron overload in patients
who are receiving long-term blood transfusions for conditions
suchasbeta-thalassemiaandotherchronicanemias.2,3 Itisthefirst
oral medication approved in the USA for this purpose.4 Two
molecules of deferasirox are capable of binding to one atom of
iron that is subsequently eliminated by fecal excretion. Its low
molecular weight and high lipophilicity allow the drug to be taken
orally. There are many properties of deferasirox that make it an
attractive agent for use in other oxidative-stress-related diseases.
In a project aiming at the development of a bioanalytical
method based on high-performance liquid chromatography (LC)
with electrospray tandem mass spectrometry (MS/MS) for the
simultaneous quantitative determination of deferasirox in human
serum, deuterium-labeled deferasirox was required. While stable
isotope-labeled compounds exhibit the same stability, extraction,
and chromatographic behavior as their nonlabeled counterpart,
their different molecular weights make them distinguishable in
LC/MS/MS from the nonlabeled counterpart. Therefore, isotopically
labeled compounds are ideal internal standards in LC/MS/MS
assays used for the quantitation of drugs in biological matrices.
Experimental procedures
Materials and methods
The analytical HPLC verification was carried out on an Agilent HPLC
system (Make: 1290 infinity). UV detection was at 242 nm. The mobile
phase consisted of A (water with 0.1% trifluoroacetic acid (TFA)), B
(acetonitrile with 0.1% TFA), gradient: 0–5 min 0%, B, 5–30 min 0–50%
B, 30–30.1 min 50–100% B, and hold to 40 min. Precoated thin-layer
chromatography (TLC) sheets (silica gel 60F254) were obtained from
Merck. Toluene-d8 was purchased from Alfa Aesar Chemical. All other
reagents were purchased from Sigma-Aldrich.
Preparation of 2-(2-hydroxyphenyl)-4H-1,3-benzoxazin-4-
one (3)
Xylene (15 ml) and salicylic acid (1; 10 g, 0.072 mol) were added in a
round-bottom flask equipped with
a
mechanical stirrer and
thermocouple. Thionyl chloride (8.7 g, 0.073 mol) was added at 10–15°
C. After addition of thionyl chloride, the reaction mixture was stirred at
10–15°C for 30 min. The reaction mixture was heated at 35–40°C for
1 h. A solution of salicylamide (2; 10 g, 0.072 mol) in xylene (20 ml) was
added to the reaction mixture at 25–30°C. After addition, the reaction
mixture was gradually heated at 120°C and stirred for 2 h. After
completion of the reaction, excess of xylene was distilled out. Methanol
(20 ml) was added to the reaction mixture at 70–80°C and stirred for 1 h.
The reaction mixture was gradually cooled to 25–30°C. The solid was
collected by filtration and washed with methanol then dried under
vacuum at 55–60°C to give 2-(2-hydroxyphenyl)-4H-1,3-benzoxazin-4-
one (3) as a white powder (16.5 g, 95%). m.p. 239°C. ESI MS m/z [M + H]+
240.0.
Results and discussion
The benzoic acid site of deferasirox appears to be appropriate for
the labeling with deuterium because of the ready availability of
toluene-d8 (Schemes 1–3).
4-Nitrotoluene-d7 (5) was obtained in good yields by nitration
of toluene-d8 (4) in the presence of zeolite catalyst ZS-5 and
nitric acid. The oxidation of 4-nitrotoluene-d7 (5) was carried
out using potassium permanganate to afford 4-nitrobenzoic
acid-d4. 4-Nitrobenzoic acid-d4 (6) was reduced to
4-aminobenzoic acid-d4 (7) by in situ generated hydrogen gas
from ammonium formate in the presence of palladium catalyst. Nadkarni-Sacasa Research Laboratory, St. Xavier’s College, Mumbai, India
The reaction was carried out under microwave irradiation.
*Correspondence to: Bhushan Vasant Dabholkar, the Nadkarni-Sacasa Research
4-Aminobenzoic acid-d4 (7) was converted to its hydrazine via
Laboratory 5, St. Xavier’s College, Mahapalika Marg, Dhobi Talao, Mumbai,
Maharastra 400001, India.
to afford 4-hydrazinebenzoic acid-d4 (8). 4-Hydrazinebenzoic E-mail: bhushan_dabholkar@hotmail.com
diazotization and simultaneous reduction by stannous chloride
J. Label Compd. Radiopharm 2015, 58 163–165
Copyright © 2015 John Wiley & Sons, Ltd.