Synthesis and characterization of related substances of deferasirox, an iron (Fe3+) chelating agent

Article abstract of DOI:10.1080/00397911.2011.580068

Deferasirox is an orally active iron chelating agent, and during process development for deferasirox, we observed six related substances (impurities), namely deferasirox methyl ester, deferasirox salicylyl derivative, deferasirox ethyl ester, deferasirox methoxy carbonyl derivative, bis(salicyl)imide, and deferasirox-2-isomer. The present work describes the detection, origin, synthesis, and characterization of these related substances.

Full text of DOI:10.1080/00397911.2011.580068

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Synthesis and Characterization of
Related Substances of Deferasirox, an
Iron (Fe3+) Chelating Agent
Vascuri Janardhana Rao a b , Kagga Mukkanti b , N. A. Vekariya a , P.
Badrinadh Gupta a & Aminul Islam a
a Chemical Research and Development Department, Aurobindo
Pharma Ltd., Hyderabad, Andhra Pradesh, India
b Department of Chemistry, Jawaharlal Nehru Technological
University, Hyderabad, Andhra Pradesh, India
Accepted author version posted online: 12 Jan 2012.
To cite this article: Vascuri Janardhana Rao , Kagga Mukkanti , N. A. Vekariya , P. Badrinadh Gupta
& Aminul Islam (2012) Synthesis and Characterization of Related Substances of Deferasirox, an Iron
(Fe3+) Chelating Agent, Synthetic Communications: An International Journal for Rapid Communication
of Synthetic Organic Chemistry, 42:21, 3200-3210, DOI: 10.1080/00397911.2011.580068
To link to this article: http://dx.doi.org/10.1080/00397911.2011.580068
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Synthetic Communications1, 42: 3200–3210, 2012
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2011.580068
SYNTHESIS AND CHARACTERIZATION OF RELATED
SUBSTANCES OF DEFERASIROX, AN IRON (Fe3þ
CHELATING AGENT
)
Vascuri Janardhana Rao,1,2 Kagga Mukkanti,2
N. A. Vekariya,1 P. Badrinadh Gupta,1 and Aminul Islam1
1Chemical Research and Development Department, Aurobindo Pharma Ltd.,
Hyderabad, Andhra Pradesh, India
2Department of Chemistry, Jawaharlal Nehru Technological University,
Hyderabad, Andhra Pradesh, India
GRAPHICAL ABSTRACT
Abstract Deferasirox is an orally active iron chelating agent, and during process develop-
ment for deferasirox, we observed six related substances (impurities), namely deferasirox
methyl ester, deferasirox salicylyl derivative, deferasirox ethyl ester, deferasirox methoxy
carbonyl derivative, bis(salicyl)imide, and deferasirox-2-isomer. The present work
describes the detection, origin, synthesis, and characterization of these related substances.
Keywords Deferasirox; iron (Fe3þ) chelating agent; related substances and synthesis
Received December 17, 2010.
Address correspondence to Aminul Islam, Chemical Research and Development Department,
Aurobindo Pharma Ltd., 313, Bachupally, Hyderabad 500090, Andhra Pradesh, India. E-mail:
aminulislam@aurobindo.com
3200

DEFERASIROX RELATED SUBSTANCES
3201
INTRODUCTION
Iron (Fe3þ), an essential bioelement in the human body, becomes toxic when
present in excess. Iron (Fe3þ) overload disturbs the blood transfusion in the body.[1]
Deferasirox is an orally active iron (Fe3þ) chelating agent,[2] chemically known as
4-[3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzoic acid. It is useful in the
treatment of iron (Fe3þ) overload in warm-blooded animals.
Iron (Fe3þ) overloads as a result of blood transfusion such as b-thalassemia
and other chronic anemia (transfusional hemosiderosis). Deferasirox is a safe triden-
tate ligand that binds selectively with iron (Fe3þ) with high affinity in a 2:1 ratio[3]
from the soluble iron pool in the plasma, and it is marketed under the trade name
Exjade.
The presence of impurities in an active pharmaceutical ingredient (API) can
impact the quality and safety of the drug products. International Conference on
Harmonization (ICH) guidelines recommend identifying and characterizing all
impurities present in an API at a level of ꢀ 0.10%.[4] This limit is calculated based
on maximum daily dosage. However, in deferasirox, the limit is further tightened
to ꢀ0.05% because of the high maximum daily dosage (i.e., ꢁ2.4 g). These impurities
are required in pure form to check the analytical performance characteristics such as
specificity, linearity, range, accuracy, precision, limit of detection (LOD), limit of
quantification (LOQ), robustness, system suitability testing, and relative retention
factor.[5] In our current work, we identified six related substances in deferasirox
API. Their detection, origin, synthesis, and characterization are described in this
article.
Following are the six related substances identified in our deferasirox (1) synthetic
process: methyl-4-[3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzoate (defera-
sirox methyl ester) (2), 4-[3-(2-(2-hydroxybenzoyloxy)phenyl)-5-(2-hydroxyphenyl)-
1H-1,2,4-triazol-1-yl]benzoic acid (deferasirox salicylyl derivative) (3), ethyl
4-[3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzoate (deferasirox ethyl ester)
(4), 4-[5-(2-hydroxy-5-methoxycarbonylphenyl)-3-(2-hydroxyphenyl)-1H-1,2,4-triazol-
1-yl]benzoicacid (deferasirox methoxycarbonyl derivative) (5), 2-hydroxy-N-
(2-hydroxybenzoyl)benzamide
hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzoic acid (deferasirox-2-isomer) (7).
[bis(salicyl)-imide]
(6),
and
2-[3,5-bis(2-
RESULTS AND DISCUSSION
Deferasirox (1) was synthesized by the known literature synthetic procedure[6]
(Scheme 1). A key intermediate, 2-(2-hydroxyphenyl)-4H-1,3-benzoxazin-4-one (9),
was obtained by reacting salicylic acid (18) with salicylamide (8) in o-xylene and
thionyl chloride in presence of catalytic amount of pyridine[6,7] and aluminium chlor-
ide was used as catalytic amount for arresting self condensation reactions to yield 9
with the high reaction efficiency. Further, condensation of compound 9 with
4-Hydrazinobenzoic acid (10) in boiling ethanol results desired Deferasirox (1).[8]
Deferasirox methyl ester (2) formed during the preparation of deferasirox in
boiling methanol[9] with compounds 9 and 10 as hydrochloride salt to form 2 as
an impurity. Related substance 2 was prepared from deferasirox in boiling methanol
with few drops of concentrated sulfuric acid (Scheme 2). The mass spectrum showed

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V. J. RAO ET AL.
Scheme 1. Synthetic scheme of deferasirox 1. Reagents and conditions: (a) o-xylene, thionyl chloride, pyri-
dine, aluminium chloride, 120–125 ꢃC, 2 h, ethanol, 72–77%; (b) ethanol, 74–78 ꢃC, 70 min, 92–95%.
a molecular ion at m=z 388.3 [(M þ H)þ], and the infrared (IR) spectrum displayed
carbonyl stretching at 1716.5 cmꢂ1. In view of deferasirox, we observed an additional
singlet at d 3.87 ppm in 1H NMR and a 13C NMR characteristic signal at d 52.3 ppm
corresponding to the methyl group. This related substance was also spiked with
deferasirox sample containing deferasirox methyl ester and confirmed related sub-
stance. This impurity is formed up toꢁ0.30% [by high-performance liquid chroma-
tography (HPLC), by area normalization] in final reaction; however, during
isolation of crude product, ꢁ0.10% and ꢁ0.02% of this impurity was found in the
drug substance after final purification.
Deferasirox salicylyl derivative (3) is a carryover impurity, originating during
the preparation of 9, which on further condensation with 10 produces 3. This related
substance was prepared by compound 9 with salicylic acid 18 in the presence of
Scheme 2. Synthetic scheme of deferasirox methyl ester 2. Reagents and conditions: (a) methanol, concen-
trated H2SO4, reflux, 18 h, 87%.

DEFERASIROX RELATED SUBSTANCES
3203
thionyl chloride and o-xylene in absence of aluminium chloride to give
2-(2-hydroxybenzoyloxy)phenyl-4H-1,3-benzoxazin-4-one (12). This compound 12
reacts with 4-hydrazinobenzoic acid 10, resulting in deferasirox salicylyl derivative
3. The mass spectra of 3 showed the molecular ion peak at m=z 492.1 [(M-H)ꢂ].
In comparison with Deferasirox 1H NMR, there were four additional aromatic pro-
tons, and in 13C NMR there were one additional carbonyl group at d 167.5 ppm and
four aromatic carbons with two quaternary carbons, indicating that an additional
1
salicylyl group might be added to compound 3 as shown in Scheme 3. H NMR,
13C NMR, and mass spectral (MS) data of prepared compound 3 were identical with
that of a sample isolated from deferasirox. The impurity 3 is formed ꢁ0.20%
(by HPLC, by area normalization) in the reaction but reduced to ꢁ0.12% during
crude product isolation, and finally ꢁ0.04% is observed in the API after the final
purification.
Deferasirox ethyl ester 4 is formed as an impurity in the synthesis of defera-
sirox. Ethyl 4-Hydrazinobenzoate (17),[10] which is a contaminant in 4-hydrazino-
benzoic acid 10, further reacts with compound 9 to produce deferasirox ethyl ester
4. Related substance 4 was prepared from ethyl 4-hydrazinobenzoate 17 with
compound 9 in boiling methanol (Scheme 4). The protonated molecular ion of
compound 4 appeared at m=z 402.1 [(MþH)þ]. In view of deferasirox, we observed
Scheme 3. Synthetic scheme of deferasirox salicylyl derivative 3. Reagents and conditions: (a) o-xylene,
thionyl chloride, DMF, 65–70 ꢃC, 8 h, ethanol; and (b) ethanol, 70–75 ꢃC, 70 min, ꢁ14.3%.

3204
V. J. RAO ET AL.
Scheme 4. Synthetic scheme of deferasirox ethyl ester 4. Reagents and conditions: (a) methanol, 65–70 ꢃC,
3 h, 76%.
1
an additional triplet at d 1.32 ppm (3H) and quartet at d 4.33 ppm (2H) in the H
NMR spectrum, and in 13C NMR characteristic signals at 14.1 ppm and 61.0 ppm
corresponding to additional ethyl groups. In 13C NMR, considerable change in
chemical shift of carbonyl group from 166.5 ppm to 164.8 ppm indicates ester forma-
tion. The IR spectrum displayed carbonyl stretching at 1715.9 cmꢂ1. This ethyl ester
analog 4 was spiked with deferasirox sample containing ethyl ester, and the related
substance was confirmed. Around ꢁ0.25% (by HPLC, by area normalization) of this
impurity is formed in the final reaction. In the crude product this impurity was
ꢁ0.12%, and final API after purification had ꢁ0.03% of this impurity.
Deferasirox methoxy carbonyl derivative 5 is observed as an impurity in defer-
asirox API. This related substance originated from salicylamide 8, and this salicyla-
mide was contaminated with 5-methoxy carbonyl salicylamide (15) because of its
route of synthesis, starting from salicylic acid. Generally, salicylic acid contaminates
4-hydroxyisopthalic acid 13 as an impurity in reaction sequences to produce 15 along
with 8. Compound 15 was prepared by a known literature method[11] and further
reaction with salicylic acid 18 with thionyl chloride in the presence of o-xylene, pyri-
dine, and aluminum chloride produces 2-(2-hydroxy-5-methoxycarbonylphenyl)-4H-
1,3-benzoxazin-4-one (16). Compound 16 was condensed with 10 to give deferasirox
methoxy carbonyl derivative 5 (Scheme 5). The mass spectra of resulted compound 5
1
showed the protonated molecular ion peak at m=z 431.9 [(M þ H)þ]. In H NMR
and 13C NMR, we observed the absence of one aromatic proton and an additional
singlet at d 3.86 ppm (3H) and corresponding carbon signal at d 52.0 ppm. In
addition to this, we observed carbonyl carbon signal at d 165.6 ppm and quaternary
carbon signal at d 114.0 ppm, indicating that the methoxy carbonyl group could be
attached to the para-position of the phenol group. The IR spectrum displayed car-
bonyl stretching at 1704.2 cmꢂ1. This deferasirox methoxy carbonyl derivative (5)
was spiked with deferasirox sample containing methoxy carbonyl derivative and con-
firmed related substance. Even though this impurity is formed ꢁ0.17% (by HPLC, by
area normalization) in the reaction, isolation procedure takes care of this impurity,
and finally ꢁ0.03% of this impurity was present in the drug substance.
Bis(salicyl)imide (6),[12,13] is the degradation product of compound 9 in acidic=
basic medium at room temperature. If any unreacted compound 9 is present in defer-
asirox API on storage, then it is converted into the compound 6. This related sub-
stance 6 was prepared by reaction of compound 9 with concentrated aqueous

DEFERASIROX RELATED SUBSTANCES
3205
Scheme 5. Synthetic scheme of deferasirox methoxy carbonyl derivative 5. Reagents and conditions: (a)
methanol, H2SO4; (b) AlCl3, (CH3)2S; (c) NH4OH; (d) CH3OH, H2SO4; (e) o-xylene, thionyl chloride,
pyridine, aluminium chloride, 110–115 ꢃC, 1 h, ethanol; and (f) ethanol, 74–78 ꢃC, 70 min, ꢁ52%.
hydrochloric acid as an acid source in absolute ethanol as a solvent (Scheme 6). The
mass spectrum of compound 6 showed molecular ion peak at m=z 255.9 [(M ꢂ H)ꢂ].
1
The H NMR spectrum showed brs at d 11.36 ppm (2H, 2 ꢄ OH) and brs at d
12.00 ppm (1H, NH). This was confirmed by D2O exchange. IR spectrum of related
substance 6 displayed carbonyl group stretching at 1701.0 cmꢂ1 and N-H and O-H
group stretching at 3247.7 cmꢂ1. This compound 6 was spiked with deferasirox sam-
ple containing compound 6 and confirmed the related substance. This impurity is
formed ꢁ0.15% (by HPLC, by area normalization) in the final reaction, with the iso-
lated crude having ꢁ0.09%. After purification the API contains ꢁ0.02% of this
impurity.
Deferasirox-2-isomer (7) is the positional isomer of deferasirox. The presence
of 2-hydrazinobenzoic acid (11) in the starting material, 4-hydrazinobenzoic acid
(10), leads to the formation of 7. Compound 7 was independently prepared by using
2-hydrazinobenzoic acid[14] 11 with compound 9 in boiling ethanol (Scheme 7). IR,
Scheme 6. Synthetic scheme of bis(salicyl)imide 6. Reagents and conditions: (a) ethanol, aqueous HCl,
74–78 ꢃC, 6 h, ꢁ71%.

3206
V. J. RAO ET AL.
Scheme 7. Synthetic scheme of deferasirox-2-isomer 7. Reagents and conditions: (a) ethanol, 74–78 ꢃC,
70 min, 77%.
1H NMR, and mass spectral data of prepared compound 7 are identical with those of
a sample isolated from deferasirox API. This impurity is formed in ꢁ0.28% (by
HPLC, by area normalization) in the final reaction, with the isolated crude having
ꢁ0.13%. After purification the API contains ꢁ0.05% of this impurity.
Deferasirox related substances (compounds 4, 5, and 7) are avoided by raw
material specifications=purifications. Any traces of these impurities are further
removed by purification. Related substances (compounds 2, 3 and 6) are the process-
related impurities. These impurities can be removed by purification of deferasirox.
We have done preliminary toxicology studies using Derek Nexus Report. As
per the report, compound 3 has alerts as hepatoxicity in mammals and compounds
2, 4, 5, 6, and have 7 have alerts as mammal skin sensitizers.
CONCLUSION
To have a thorough understanding of impurity formation for origin of iron-
chelating agent deferasirox, it is essential to have detailed information about various
potential impurities and their synthetic routes. In view of regulatory importance of the
impurities in the API, a detailed study on various impurities in deferasirox was con-
ducted. Different process-related impurities and degradation products in deferasirox
were identified. They were synthesized and characterized using various spectroscopic
techniques such as 1H NMR, 13C NMR, and IR. This characterization was supported
by liquid chromatography–mass spectrometry (LC-MS) and mass spectral data.
EXPERIMENTAL
Solvents and reagents were obtained from commercial sources and used without
purification. The IR spectra (v max cmꢂ1) were recorded in solid-state KBr dispersion
using a Perkin-Elmer Fourier transform (FT)–IR spectrometer. The 1H NMR and 13
C
NMR spectra were recorded on a Bruker-Avance 300-MHz spectrometer. The chemi-
cal shifts were reported in d ppm relative to tetramethylsilane (TMS). The mass spectra
were recorded on a API 2000 Perkin-Elmer PE-SCIEX mass spectrometer. Melting
points were determined on a Polman melting-point apparatus (model no. MP-96). Dif-
ferential scanning calorimetry (DSC) was performed using model DSC-821e (Mettler
Toledo) with a heating rate of 10 ꢃC per minute under a nitrogen atmosphere. DSC

DEFERASIROX RELATED SUBSTANCES
3207
temperature scale was calibrated by the indium standard. The compounds 2, 3, and 5
are novel and 4, 6, and 7 are known in the literature.[8,12,13,15] We synthesized all these
compounds as described in this experimental section.
Preparation of Methyl 4-[3,5-Bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-
yl]benzoate (2)
Deferasirox 1 (4.00 g, 10.72 mmol) was added to methanol (25 mL) followed by
addition of concentrated sulfuric acid (five drops) at 25–30 ꢃC and refluxed for 18 h.
The reaction was cooled to 15–20 ꢃC, and the resulting product was filtered and
washed with chilled methanol (2 ꢄ 5 mL, 2–8 ꢃC) to give compound 2 (3.60 g,
86.80%); mp: 172–175 ꢃC; IR (KBr pellet), cmꢂ1: 3255.8 (O-H, str.), 3064.7 (aromatic
C-H, str.), 1716.5 (C¼O, str.), 1622.9, 1607.6, 1587.7, 1515.4 (C¼N & C¼C, str.),
1290.6 (C-N, str.), 768.7 and 754.1 (aryl CH out-of-plane bend); 1H NMR
(300 MHz, DMSO-d6): d 3.87 (s, 3H), 6.86 (d, 1H, J ¼ 8.23 Hz), 7.00 (m, 2H), 7.02
(m, 1H), 7.38 (m, 2H), 7.57 (m, 1H), 7.58 (d, 2H, J ¼ 8.50 Hz), 8.01 (d, 2H,
J ¼ 8.50 Hz), 8.03 (m, 1H), 10.04 and 10.78 (2 brs,2xOH); 13C NMR (75 MHz,
DMSO-d6): d 52.3, 113.6, 114.4, 116.1, 117.0, 119.5, 119.7, 123.4, 126.8, 129.3,
130.2, 131.0, 131.5, 132.6, 141.5, 152.1, 155.1, 156.3, 159.9, 165.4; MS m=z: 388.2
[(M þ H)þ]. Anal. calcd. for C22H17N3O4 (387.3): C, 68.21; H, 4.41; N, 10.84. Found:
C, 68.11; H, 4.37; N, 10.79. HPLC retention time ꢁ42.3 min (RRT ꢁ1.49).
Preparation of 4-[2-(2-Hydroxybenzoyloxy)phenyl]-5-
(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzoic Acid (3)
Salicylic acid 18 (2.50 g, 18.11 mmol) was suspended in o-xylene (20 mL) con-
taining
a few drops of N,N-dimethylformamide. Thionyl chloride (5 mL,
68.48 mmol) was slowly added over 10 min at 28–35 ꢃC and stirred for 1 h.
2-(2-Hydroxyphenyl)-4H-1,3-benzoxazin-4-one 9 (4.30 g, 17.99 mmol) was slowly
added to the salicylyl chloride at 28–32 ꢃC, and the reaction was maintained at
65–70 ꢃC for 8 h. The reaction mass was concentrated under reduced pressure to
obtain a residue. Absolute alcohol (60 mL) was added, stirred for 10 min, filtered,
and washed with absolute alcohol (2 ꢄ 5 mL). The pH of the filtrate was adjusted
to 4.8 with triethylamine and concentrated under reduced pressure to an oily mass
of 2-(2-hydroxybenzyloxy)phenyl)-4H-1,3-benzoxozin-4-one 12. Absolute alcohol
(50 mL) was added to compound 12 followed by addition of 4-hydrazino-benzoic
acid 10 (1.00 g, 6.58 mmol) at 28–30 ꢃC, and the reaction was maintained at
70–75 ꢃC for 70 min. The reaction mass was concentrated to a residue and crystal-
lized from hexanes and ethyl acetate mixture. Compound 3 was obtained as a pale
yellow gummy solid, having purity ꢁ50%, which was chromatographed on silica
gel (100–200 mesh) using a mixture of ethyl acetate and hexanes (1:1 ratio) as the
eluent to yield 3 in pure form (1.25 g, 14.28%); mp: 102–104 ꢃC; 1H NMR
(300 MHz, DMSO-d6): d 6.80 (d, 1H, J ¼ 7.68 Hz), 6.88 (dd, 1H, J ¼ 7.68 Hz), 7.04
(m, 2H), 7.10 (d, 2H, J ¼ 8.50 Hz), 7.34 (m, 1H), 7.35 (d, 1H, J ¼ 7.68 Hz), 7.50
(m, 2H), 7.61 and 7.66 (2dd, 4H, J ¼ 7.68 Hz, 1.65 Hz), 7.68 (d, 2H, J ¼ 8.78 Hz),
8.14 (dd, 1H, J ¼ 7.96 Hz, 1.65 Hz), 8.32 (dd, 1H, J ¼ 7.68 Hz, 1.65 Hz); 13C NMR
(75 MHz, DMSO-d6): d 113.5, 115.3, 115.9, 117.5, 119.5, 119.6, 121.9, 123.1,

3208
V. J. RAO ET AL.
124.3, 127.0, 129.4, 129.9, 131.0, 131.5, 132.3, 136.0, 141.1, 147.6, 152.6, 154.9, 157.9,
160.1, 166.5, 167.5; MS m=z: 492.1 [(M-H)ꢂ]. Anal. calcd. for C28H19N3O6 (493.4):
C, 68.15; H, 3.87; N, 8.51. Found: C, 68.05; H, 3.81; N, 8.47. HPLC retention time
ꢁ39.2 min (RRT ꢁ1.38).
Preparation of Ethyl 4-[3,5-Bis (2-hydroxyphenyl)-1H-1,2,4-triazol-1-
yl]benzoate (4)
2-(2-Hydroxyphenyl)-4H-1,3-benzoxazin-4-one 9 (4.00 g, 16.73 mmol) and
ethyl 4-hydrazinobenzoate 17 (3.07 g, 17.05 mmol) were added to methanol
(20 mL). The reaction was maintained at 60–65 ꢃC for 3 h. The solvent was removed
under reduced pressure, and 50% v=v aqueous methanol (20 mL) was added. The
product was cooled filtered off, and washed with precooled methanol (1 ꢄ 5 mL,
2–8 ꢃC). Deferasirox ethyl ester 4 was obtained as white crystals (5.10 g, 76.03%);
mp: 147–149 ꢃC (lit.[18] mp 148–149 ꢃC); IR (KBr pellet), cmꢂ1: 3275.1 (O-H, Str.),
3059.6 (aromatic C-H, str.), 2987.8, 2904.2 (aliphatic C-H, str.), 1715.9 (C¼O,
str.), 1622.5, 1607.8, 1586.9, 1515.1 (C¼N and C¼C, str.), 1254.2 (C-N, str),
1
772.6, 752.2 (aromatic CH out-of-plane bend); H NMR (300 MHz, DMSO-d6): d
1.32 (t, 3H, CH3 of ethyl ester, J ¼ 7.12), 4.33 (q, 2H, CH2 of ethyl ester,
J ¼ 7.12), 6.86 (d, 1H, J ¼ 8.20 Hz), 7.02 (m, 3H), 7.39 (m, 2H), 7.56 (d, 1H,
J ¼ 8.78 Hz), 7.59 (d, 2H, J ¼ 8.50 Hz), 8.01 (d, 2H, J ¼ 8.50 Hz), 8.05 (d, 1H,
J ¼ 7.68 Hz), 10.03 and 10.79 (2brs, 2 ꢄ OH); 13C NMR (75 MHz, DMSO-d6): d
14.1, 61.0, 113.6, 114.4, 116.1, 117.0, 119.5, 119.7, 123.4, 126.8, 129.6, 130.1,
131.0, 131.5, 132.6, 141.4, 152.1, 155.1, 156.3, 159.9, 164.8; MS m=z: 402.1
[(M þ H)þ]; Anal. calcd. for C23H19N3O4 (401.4); C, 68.82; H, 4.76; N, 10.46. Found:
C, 68.76; H, 4.71; N, 10.41. HPLC retention time ꢁ47.6 min (RRT ꢁ1.68).
Preparation of 4-[5-(2-Hydroxy-5-methoxycarbonylphenyl)-
3-(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl]benzoic Acid (5)
5-Methoxycarbonyl salicylamide 15 (3.00 g, 15.38 mmol) and salicylic acid 18
(2.30 g 16.66 mmol) were added to o-xylene (25 mL) at 25–30 ꢃC. A catalytic amount
of pyridine (0.5 mL) and aluminium chloride (200 mg) was added to the suspension.
Thionyl chloride (3.30 g, 27.78 mmol) was slowly added at 110–120 ꢃC over 35 min
and maintained at 110–120 ꢃC for 50 min. The reaction was cooled to 25–30 ꢃC,
and ethanol (absolute alcohol, 15 mL) was added and stirred for 1 h. The solid
was filtered off and washed with precooled ethanol (absolute alcohol, 2 ꢄ 5 mL,
2–8 ꢃC) to obtain 2-(2-hydroxy-5-methoxy carbonyl phenyl)-4H-1,3-benzoxazin-4-
one 16. Compound 16 (wet) and 4-hydrazionobenzoic acid 10 (1.46 g, 9.60 mmol)
were added to the boiling absolute ethanol (30 mL) and maintained at boiling tem-
perature for 70 min. The resulting precipitation was filtered and washed with pre-
cooled ethanol (absolute alcohol, 2 ꢄ 5 mL, 2–8 ꢃC) to produce compound 5,
which further recrystallized in methanol to give the pure form of 5 as white crystal-
line powder (3.40 g, 51.28%); mp: 250–255 ꢃC (dec); IR (KBr pellet), cmꢂ1: 3403.7
(O–H, str.), 1704.3 (C¼O, str.), 1601.7, 1562.0 (C¼C and C¼N, str.), 1228.8
1
(C-N, str.), 769.2, 704.5 (aromatic CH out-of-plane bend); H NMR (300 MHz,
DMSO-d6): d 3.86 (s, 3H, COOCH3), 6.90 (dd, 1H, J ¼ 7.68 Hz, 1.65 Hz), 6.92(dd,

DEFERASIROX RELATED SUBSTANCES
3209
1H, J ¼ 7.68 Hz, 1.65 Hz), 7.15 (d, 1H, J ¼ 8.78 Hz), 7.34 (d, 2H, J ¼ 8.23 Hz), 7.37
(dd, 1H, 7.40 Hz, 1.50 Hz), 7.46 (dd, 1H, J ¼ 7.40 Hz, 1.50 Hz), 7.85 (d, 2H,
J ¼ 8.23 Hz), 7.95 (dd, 1H, J ¼ 8.50 Hz), 8.67 (d, 2 H, J ¼ 2.20 Hz), 10.57 and
11.62 (2 brs, 2 ꢄ OH); 13C NMR (75 MHz, DMSO-d6): d 52.0, 116.2, 117.6, 119.2,
119.5, 121.2, 123.4, 128.6, 130.3, 130.7, 131.1. 132.4, 132.6, 141.1, 152.3, 155.2,
159.0, 160.2, 165.6, 166.4; MS m=z: 431.9 [(M þ H)þ]. Anal. calcd. for
C23H17N3O6 (431.3): C, 64.04; H, 3.97; N, 9.74. Found: C, 63.97; H, 3.92; N,
9.69. HPLC retention time ꢁ30.70 min (RRT ꢁ1.08).
Preparation of 2-Hydroxy-N-(2-hydroxybenzoyl)benzamide (6)
2-(2-Hydroxyphenyl)-4H-1,3-benzoxazin-4-one 9 (5.00 g, 21.00 mmol) was
added to ethanol (absolute alcohol, 50 mL). Concentrated aqueous hydrochloric acid
(2 mL) was added at 25–30 ꢃC and refluxed for 6 h. The reaction mixture was cooled
to 25–30 ꢃC. The resulting solid was filtered and washed with precooled ethanol
(absolute alcohol, 2 ꢄ 10 mL, 2–8 ꢃC) to give the product 6 as off-white crystꢂal1s
(3.80 g, 70.69%); mp 204–205.5 ꢃC (lit.[15] mp 203–204 ꢃC); IR (KBr pellet), cm
:
3247.7 (N-H, O-H, str.), 1701.0 (C¼O, str.), 1608.0, 1592.8 (aromatic C¼C, str.),
1249.1 (C-N, str.), 754.7, 745.7 (aromatic CH out-of-plane bend); 1H NMR
(300 MHz, DMSO-d6): d 6.96–7.04 (m, 2 ꢄ 2 H, O- and P-position of phenol),
7.47 (dd, 2 ꢄ 1H, J ¼ 7.96 Hz, 1.90 Hz, m-phenol), 7.84 (d, 2 ꢄ 1H, J ¼ 7.96 Hz),
11.36 (brs, 2 ꢄ OH), 12.00 (brs, 1H, NH); 13C NMR (75 MHz, DMSO-d6): d
117.1, 119.0, 119.7, 130.6, 134.3, 156.9, 164.5; MS m=z: 255.9 [(M ꢂ H)ꢂ]. Anal.
calcd. for C14H11NO4 (257.2): C, 65.37; H, 4.30; N, 5.44. Found: C, 65.29; H,
4.23; N, 5.41. HPLC retention time ꢁ8.1 min (RRT ꢁ0.29).
Preparation of 2-[3,5-Bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-
yl]benzoic Acid (7)
2-(2-Hydroxyphenyl)-4H-1,3-benzoxazin-4-one 9 (5.00 g, 20.92 mmol) and
2-hydrazinobenzoic acid 11 (3.24 g, 21.31 mmol) were added to ethanol (absolute
alcohol, 40 mL) at 25–30 ꢃC and refluxed for 1 h. The resulting precipitate was fil-
tered and washed with precooled ethanol (absolute alcohol, 2 ꢄ 5 mL, 2–8 ꢃC) to give
deferasirox-2-isomer (7) as a white crystalline powder (6.00 g, 76.92%). DSC thermo-
gram showed a sharp endothermal peak at 225.7 ꢃC; IR (KBr pellet), cmꢂ1: 3226.2
(O-H, str.), 3070.2 (aromtic C-H, str.), 1691.3 (C¼O, str.), 1622.2, 1603.0, 1585.8,
1514.7 (C¼N and C¼C, str.), 1248.9 (C-N, str.), 756.2, 727.1 (aromatic CH, out-of-
plane bend); 1H NMR (300 MHz, DMSO-d6): d 6.82 (dd, 1H), 6.83 (d, 1H,
J ¼ 7.68 Hz), 6.98 (m, 1H), 7.02 (d, 1H, J ¼ 7.68 Hz), 7.21 (dd, 1H, J ¼ 7.68 Hz,
1.37 Hz), 7.28 (dd, 1H, J ¼ 7.40, 1.65 Hz), 7.36 (dd, 1H, J ¼ 7.90 Hz, 1.65 Hz), 7.63
(dd, 2 H, J ¼ 7.90 Hz, 1.65 Hz), 7.73 (dd, 1H, J ¼ 7.68 Hz, 1.65 Hz), 7.87 (dd, 1H,
J ¼ 7.68 Hz, 1.90 Hz), 7.99 (dd, 1H, J ¼ 7.96 Hz, 1.65 Hz), 10.21 and 10.93 (2brs, 2
x OH), 13.14 (brs, 1H, COOH); 13C NMR (75 MHz, DMSO-d6): d 113.5, 113.9,
116.2, 117.0, 119.0, 119.6, 126.6, 128.3, 128.7, 130.0, 130.6, 130.7, 131.2, 132.2,
132.7, 136.6, 152.9, 155.7, 156.3, 159.2, 165.8; MS m=z: 374.2 [(M þ H)þ]. Anal.
calcd. for C21H15N3O4 (373.3): C, 67.56; H, 4.05; N, 11.25. Found: C, 67.50; H,
4.01; N, 11.25. HPLC retention time ꢁ26.5 min (RRT ꢁ0.94).

3210
V. J. RAO ET AL.
ACKNOWLEDGMENTS
The authors gratefully acknowledges the management of Aurobindo Pharma
Limited for allowing us to carry out this research work. The authors are also thank-
ful to the colleagues of the Chemical Research Department and Analytical Research
Department for their cooperation.
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