Synthesis and characterization of metabolites and potential impurities of balsalazide disodium, an anti-inflammatory drug

Article abstract of DOI:10.1080/00397910903221068

Balsalazide disodium (Colazide) is an oral prodrug of mesalamine (5-aminosalicylic acid) and possesses anti-inflammatory properties. During the process development for balsalazide disodium, we observed eight impurities, namely des-alanine balsalazide, balsalazide-alanine, balsalazide 3-isomer, decarboxy balsalazide, bis-azo salicylic acid, biphenyl-azo salicylic acid, bis-azo diacid, and bis-azo triacid. The present work describes the synthesis and characterization of these impurities. Copyrigh

Full text of DOI:10.1080/00397910903221068

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Synthesis and Characterization of
Metabolites and Potential Impurities
of Balsalazide Disodium, an Anti-
Inflammatory Drug
Md. Umar Khan a b , M. A. Baseer b , S. Ranjith Kumar a , M.
Saravanakumar a , A. G. Prasannanjali a , P. Badarinadh Gupta a
Vipin K. Kaushik a , Vijay K. Handa a & Aminul Islam a
,
a Chemical Research and Development Department, APL Research
Centre, Aurobindo Pharma Ltd., Hyderabad, India
b Organic Chemistry Research Laboratory, Yeshwant Mahavidyalaya,
Nanded, India
Version of record first published: 07 Jul 2010.
To cite this article: Md. Umar Khan , M. A. Baseer , S. Ranjith Kumar , M. Saravanakumar , A. G.
Prasannanjali , P. Badarinadh Gupta , Vipin K. Kaushik , Vijay K. Handa & Aminul Islam (2010):
Synthesis and Characterization of Metabolites and Potential Impurities of Balsalazide Disodium,
an Anti-Inflammatory Drug, Synthetic Communications: An International Journal for Rapid
Communication of Synthetic Organic Chemistry, 40:15, 2241-2253
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Synthetic Communications1, 40: 2241–2253, 2010
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397910903221068
SYNTHESIS AND CHARACTERIZATION
OF METABOLITES AND POTENTIAL IMPURITIES
OF BALSALAZIDE DISODIUM,
AN ANTI-INFLAMMATORY DRUG
Md. Umar Khan,1,2 M. A. Baseer,2 S. Ranjith Kumar,1
M. Saravanakumar,1 A. G. Prasannanjali,1
P. Badarinadh Gupta,1 Vipin K. Kaushik,1 Vijay K. Handa,1
and Aminul Islam1
1Chemical Research and Development Department, APL Research Centre,
Aurobindo Pharma Ltd., Hyderabad, India
2Organic Chemistry Research Laboratory, Yeshwant Mahavidyalaya,
Nanded, India
Balsalazide disodium (Colazide1) is an oral prodrug of mesalamine (5-aminosalicylic acid)
and possesses anti-inflammatory properties. During the process development for balsalazide
disodium, we observed eight impurities, namely des-b-alanine balsalazide, balsalazide
b-alanine, balsalazide 3-isomer, decarboxy balsalazide, bis-azo salicylic acid, biphenyl-azo
salicylic acid, bis-azo diacid, and bis-azo triacid. The present work describes the synthesis
and characterization of these impurities.
Keywords: 5-Aminosalicylic acid; balsalazide; diazo coupling; impurities; synthesis
INTRODUCTION
Mesalamine, also known as mesalazine or 5-aminosalicylic acid (5-ASA), is an
anti-inflammatory drug, used to treat inflammation of the digestive tract ulcerative
colitis and mild to moderate Crohn’s disease.[1–3] Balsalazide is the generic name of
5-[(1E)-[4-[[(2-carboxyethyl) amino]carbonyl]phenyl]azo] 2-hydroxybenzoic acid (1).
It is an oral prodrug of 5-ASA, having an inert carrier molecule, N-(4-
aminobenzoyl)b-alanine (4-ABBA).[4] After oral administration, balsalazide splits into
mesalamine and N-(4-aminobenzoyl)b-alanine via azo-reduction by the colonic micro-
flora.[5] Mesalamine acts directly on the colon to reduce the local inflammation of the
colonic mucosa, which is responsible for the symptoms of ulcerative colitis.[6–8] Balsa-
lazide appeared to be an effective treatment option when compared to salazopyrin.
The clinical use and dosage of salazopyrin are limited by adverse reactions,[9–11] several
of which are attributed to the absorption of the carrier metabolite sulfapyridine.[12]
Received May 14, 2009.
Address correspondence to Aminul Islam, Chemical Research and Development Department,
APL Research Centre, Aurobindo Pharma Ltd., Chemical R&D, 313, Bachupally, Quthubullapur (M),
R. R. District, Hyderabad 500 072, India. E-mail: aminulislam@aurobindo.com
2241

2242
Md. U. KHAN ET AL.
Balsalazide is a therapeutic agent for ulcerative colitis. It is used as disodium
dihydrate salt and has molecular formula C17H13N3O6Na2 ꢀ 2H2O and molecular
weight 437.32 amu. The commonly used dose of balsalazide is 6.75 g per day, which
is equivalent to 2.3 g of mesalamine.[13]
The presence of impurities in an active pharmaceutical ingredient (API) can
have a significant impact on the quality and safety of the drug products. Therefore,
it is necessary to study the impurity profile of the API to be used in the manufacturing
of a drug product. International Conference on Harmonization (ICH) guidelines
recommends identification and characterization of all impurities that are present in
an API at a level of ꢁ0.05% (based on daily dose).[14] In this context, we have under-
taken a comprehensive study to synthesize and characterize the impurities in balsala-
zide API. These impurities are 5-[[4-[carboxy]phenyl]azo]-2-hydroxybenzoic acid
(des-b-alanine balsalazide, 2); 5-[4-[2-(2-carboxyethyl carbamoyl)ethyl carbamoyl]
phenyl azo]-2-hydroxy-benzoic acid (balsalazide b-alanine, 3); 3-[[4-[[(2-
carboxyethyl)amino]carbonyl]phenyl]-azo]-2-hydroxybenzoic acid (balsalazide 3-
isomer, 4); 3-[4-(4-hydroxy phenylazo)benzoylamino]-propionic acid (decarboxy
balsalazide, 5); 2,4-bis-[[4-[(2-carboxyethyl)amino]carbonyl]-phenyl]azo salicylic acid
(bis-azo salicylic acid, 6); 5-[2-[40,5-bis[(2-carboxyethyl)carbamoyl]-biphenyl-2-
yl]diazenyl]-2-hydroxybenzoic acid (biphenyl-azo salicylic acid, 7); 3-[4-[5-[4-(2-car-
boxy ethyl carbamoyl)phenylazo]-2-hydroxy phenylazo]benzoyl amino]propionic
acid (bis-azo diacid, 8); and 2,4-bis[[4[[(2-carboxyethyl)amino]carbonyl]phenyl]azo]-
3-[4-[[(2-carboxyethyl)-amino]carbonyl]phenyl]phenol (bis-azo triacid, 9). In addition
to these, we have also synthesized and characterized two metabolites, namely 3-(4-
acetylamino benzoylamino)-propionic acid (N-acetyl-4-ABBA, 10) and 5-acetylamino
2-hydroxybenzoic acid (5-acetamino-SA, 11).
Balsalazide disodium was first disclosed in US Patent 4,412,992 (Nov. 1, 1983),
assigned to Biorex Laboratories Limited, England, claiming product ‘‘balsalazide’’
and process of its preparation. Optimization of this diazonium salt–based process
is reported in the literature,[15–19] but to best of our knowledge a detail impurity
profile study is not yet cited anywhere, except for four metabolites.[20]
RESULTS AND DISCUSSION
Balsalazide 1 has been synthesized as per Fig. 1.[21] b-Alanine was acylated with
4-nitrobenzoyl chloride 12 in aqueous alkaline solution to give N-(4-nitrobenzoyl)-
b-alanine 13.
Hydrogenolysis of 13 with Pd=C in methanol results in the compound N-(4-
aminobenzoyl)-b-alanine 14. Diazotization of N-(4-aminobenzoyl)-b-alanine 14 with
sodium nitrite in aqueous hydrochloric acid yields the diazonium salt 15, which was
coupled with salicylic acid 16 in alkaline medium to give the title product, balsalazide 1.
Des-b-alanine balsalazide 2, a carryover impurity, is a result of the presence of
4-nitrobenzoic acid 17 in intermediate 13 of the balsalazide process. This impurity
may also form as a result of the amide hydrolysis of balsalazide during the process.
Des-b-alanine balsalazide was prepared starting with 4-nitrobenzoic acid 17 accord-
ing to Fig. 2. Catalytic hydrogenation of 17 with Pd=C in methanol yielded 18, which
was diazotized with sodium nitrite in aqueous hydrochloride and coupled with
sodium salicylate to give the desired compound 2 in excellent yield. The electrospray

MESALAMINE, ALSO KNOWN AS MESALAZINE
2243
Figure 1. Synthesis of balsalazide 1. Reagents and conditions: (a) water, NaOH, b-alanine, 0–5 ꢂC, 30 min,
85–90%; (b) methanol, Pd=C, H2, rt, 2–3 h, 98%; (c) water, conc. HCl, 0–5 ꢂC, aq. NaNO2 solution, 0–5 ꢂC,
1 h, 100%; (d) water, NaOH=Na2CO3, salicylic acid, 0–5 ꢂC; and then addition of diazonium salt 15
solution, 1 h, 95%.
ionization (ESI) mass spectrum of 2 displayed peaks at m=z 285.2 [(M – H)þ] in
negative ion mode and as sodium ion adduct at m=z 307.0 [(M – H) þ Na]. In
positive ion mode, this compound appeared at m=z 287.2 [(M þ H)þ] in the mass
1
spectrum. In H NMR spectrum of compound 2, corresponding peaks of b-alanine
at d 2.61 ppm and d 3.56 ppm were absent. Similarly, in the 13C NMR spectrum,
peaks at d 34.6 ppm and d 36.6 ppm due to b-alanine moiety were also not observed.
The assigned structure for compound 2 is clearly confirmed the spectral data.
Like des-b-alanine balsalazide 2, balsalazide b-alanine 3 originates from N-
(4-nitrobenzoyl)di-b-alanine 20, which may present in intermediate 13 and carry
through the synthetic process to give this impurity in the final product.
Figure 2. Synthesis of compound 2. Reagents and conditions: (a) methanol, Pd=C, H2, rt, 2–3 h, 98%; (b)
water, conc. HCl, 0–5 ꢂC then aq. NaNO2 solution, 0–5 ꢂC, 1 h, ꢃ100%; (c) water, NaOH=Na2CO3,
salicylic acid, 0–5 ꢂC, then addition of diazonium salt 19 solution, 1 h, ꢃ100%.

2244
Md. U. KHAN ET AL.
Balsalazide b-alanine 3 was prepared by hydrogenating N-(4-nitrobenzoyl)di-
b-alanine 20 with Pd=C in methanol to give the corresponding amine 21, which on
diazotization followed by coupling with salicylic acid in an alkaline medium resulted
in the desired compound 3 (Fig. 3). The ESI mass spectrum of 3 displayed peaks at
m=z 427.2 [(M – H)–] and as sodium ion adduct at m=z 449.0 [(M – H)– þ Na] in –ve
ion mode; in þve ion mode, this impurity appeared at m=z 429.2 [(M þ H)þ]. The 1H
and 13C NMR spectra of compound 3 also support the assigned structure for 3 (di-b-
alanine analog of balsalazide).
Balsalazide 3-isomer 4 is a positional isomer of balsalazide 1 and can form as a
result of the coupling of diazonium salt 15 to the 3-position of salicylic acid 16
instead of the 5-position (Fig. 4).
N-(4-Aminobenzoyl)-b-alanine 14 as hydrochloride salt was diazotized with
aqueous sodium nitrite solution at 0–5 ꢂC and further treated with sodium salicylate
in water=isopropyl alcohol mixture at 0–5 ꢂC to yield compound 4 as a by-product
along with 1. The ESI mass spectrum of compound 4 is showing equal mass value
1
as that of balsalazide. The H NMR of 4 exhibits three doublet of doublets at d
6.61 ppm (dd, J ¼ 7.7 Hz, 1H), 7.57 ppm (dd, J ¼ 8.0 Hz and 1.7 Hz, 1H), and
7.87 ppm (dd, J ¼ 8.0 Hz and 1.7 Hz, 1H) corresponding to three hydrogens at the
4-, 5-, and 6-positions of the aromatic ring of salicylic acid. 13C NMR also confirmed
the assigned structure of compound 4.
Decarboxy balsalazide 5 was detected in 1 as a degradation product. Decar-
boxylation of balsalazide results in this impurity. We have prepared this impurity
from 4-hydroxybenzoic acid 23, wherein N-(4-aminobenzoyl) b-alanine 14 was
diazotized with aqueous sodium nitrite solution=hydrochloric acid at 0–5 ꢂC and
added to aqueous alkaline solution of 23 to obtain compound 5 (Fig. 5). The ESI
mass spectrum of compound 5 displayed peak at m=z 312.1 [(M – H)–] in –ve ion
mode, which is 44 mass units (amu) less than balsalazide (m=z 356.2). In þve ion
mode, its base peak appeared at m=z 314.0 [(MH)þ], and the peak corresponded
Figure 3. Synthesis of compound 3. Reagents and conditions: (a) methanol, Pd=C, H2, rt, 2–3 h, 98%; (b)
water, conc. HCl, 0–5 ꢂC aq. NaNO2 solution, 0–5 ꢂC, 1 h, 100%; (c) water, NaOH =Na2CO3, salicylic
acid, 0–5 ꢂC, then addition of diazonium salt 22 solution, 1 h, 82%.

MESALAMINE, ALSO KNOWN AS MESALAZINE
2245
Figure 4. Synthesis of compound 4. Reagents and condition: (a) water, conc. HCl, 0–5 ꢂC aq. NaNO2
solution, 0–5 ꢂC, 1 h, ꢃ100%; (b) water, NaOH=Na2CO3, salicylic acid, 0–5 ꢂC, then addition of diazonium
salt 15 solution, 1 h, ꢃ7%.
to the sodium ion adduct appeared at m=z 336.0 [(MH) þ Na]. The 1H NMR
spectrum of this compound shows signals at d 6.97 (d, J ¼ 8.8 Hz, 2H), 7.84 (d,
J ¼ 8.8 Hz, 2H), 7.88 (d, J ¼ 8.8 Hz, 2H), and 8.04 (d, J ¼ 8.8 Hz, 2H); these values
indicate that both aromatic rings are para-substituted. In addition, 13CNMR and
DEPT (distortionless enhancement by polarization transfer) spectra also support
the assigned structure for the compound, 116.1(CH), 121.9(CH), 125.2(CH), and
128.4(CH).
Bis-azo salicylic acid 6 forms as a result of the coupling of diazo intermediate
15 with balsalazide 1 on the ortho position with respect to the phenolic –OH group
during its preparation and carries through the process to give this impurity in balsa-
lazide. The synthetic sequence for the preparation of this impurity is depicted in
Fig. 6. Using half mole of salicylic acid in diazo coupling reaction yielded this
impurity, but because of the number of side reactions, formation of 6 was less.
The ESI mass spectrum of impurity 6 displayed a peak at m=z 577.2 [(MH)þ], 221
mass unit (amu) more than balsalazide (m=z 356.2), indicating attachment of diazo
intermediate 15 to balsalazide. The signals in 1H NMR spectrum of this impurity at d
7.90 (2d, J ¼ 8.8 Hz, 4H) and 8.01 (2d, J ¼ 8.8 Hz, 4H) represent two diazo inter-
mediate moieties, the remaining two aromatic peaks at d 8.15 (d, J ¼ 2.5 Hz, 1H)
and 8.51 (d, J ¼ 2.5 Hz, 1H), due to the benzene ring of salicylic acid. The smaller
coupling constant value (J ¼ 2.5 Hz) indicates the meta-coupling in the benzene ring,
which authenticates the assigned structure for compound 6.
Base-promoted free radical coupling (Gomberg–Bachmann reaction)[22–24]
between the aryl diazonium salt 15 and balsalazide leads to the formation of
biphenyl-azo salicylic acid 7. The synthetic sequence to prepare this impurity is
Figure 5. Synthesis of compound 5. Reagents and conditions: (a) water, conc. HCl, 0–5 ꢂC aq. NaNO2
solution, 0–5 ꢂC, 1 h, 100%; (b) water, NaOH=Na2CO3, salicylic acid, 0–5 ꢂC, then addition of solution
of diazonium salt 15, 1 h, ꢃ91%.

2246
Md. U. KHAN ET AL.
Figure 6. Synthesis of compounds 6 and 7. Reagents and conditions: (a) water, conc. HCl, 0–5 ꢂC, aq.
NaNO2 solution, 0–5 ꢂC, 1 h, ꢃ100%; (b) water, NaOH=Na2CO3, 0.5 mol eq. salicylic acid, 0–5 ꢂC, then
addition of solution of 15, 1 h, 3–7%.
Figure 7. Synthesis of compounds 8 and 9. Reagents and conditions: (a) water, conc. HCl, 0–5 ꢂC, aq.
NaOH solution, 0–5 ꢂC, 1 h, ꢃ100%; (b) water, NaOH=Na2CO3, 0.5 (for 8)=0.25 (for 9) mol
eq. 4-hydroxy benzoic acid 23, 0–5 ꢂC, solution of 15, 1 h, 8 (ꢃ32%) and 9 (ꢃ36%).

MESALAMINE, ALSO KNOWN AS MESALAZINE
2247
illustrated in Fig. 6. Using half mole of salicylic acid in the reaction yielded this
impurity. The formation of 7 was very less as there were various side reactions.
ESI mass spectrum of impurity 7 displayed peak at m=z 549.2 [(MH)þ], 28 mass unit
(amu) less than bis-azo salicylic acid 6, indicating diazo group detachment from
bis-azo salicylic acid 6.1H NMR data for compound 6 are 6.70 (d, J ¼ 8.8 Hz,
1H), 7.58 (d, J ¼ 8.5 Hz, 2H), 7.60 (dd, J ¼ 8.8 Hz and 2.5 Hz, 1H), 7.70 (d, J ¼
8.8 Hz, 1H), 7.93 (d, J ¼ 8.5 Hz, 2H), 7.97 (dd, J ¼ 8.8 Hz and 1.4 Hz, 1H), 8.05 (d,
J ¼ 1.4 Hz, 1H), 8.17 (d, J ¼ 2.5 Hz, 1H).
Bis-azo diacid 8 originates because of the decarboxylation of compound 6 and
contaminates the product. Bis-azo triacid 9 originates because of Gomberg–
Bachmann-type coupling of 15 and 8 (Fig. 7). The compound 8 was prepared by
reacting diazonium salt 15 with alkaline solution of para-hydroxybenzoic acid 23
(0.5 mol equivalent), whereas the compound 9 was obtained by reacting 15 with
alkaline solution of 0.25 mol equivalent of 23.
CONCLUSION
To have a thorough understanding of impurity formation pathways of the
anti-inflammatory drug balsalazide disodium, it is essential to have detailed infor-
mation about the various possible impurities, metabolites, and their synthetic routes.
In view of the regulatory importance of the impurities in the API, a detailed study on
various impurities in balsalazide was conducted. Different process-related impurities,
degradation products, and metabolites in balsalazide API were identified, synthe-
sized, and characterized by using various spectroscopic techniques like low-resolution
mass spectroscopy (LCMS), mass,1H NMR, 13C NMR, and infrared (IR).
EXPERIMENTAL
1H NMR, 13C NMR, and DEPT spectral data were performed on a Bruker-
Avance 300-MHz spectrometer in dimethylsulfoxide (DMSO-d6). The chemical shift
values were reported on the d scale in parts per million (ppm), downfield from tetra-
methylsilane (TMS) as an internal standard. IR spectra were recorded in the solid
state as KBr dispersion using a Perkin-Elmer Spectrum One Fourier transform
(FT)–IR spectrophotometer. Mass spectrum was recorded using a Perkin-Elmer
PE SCIEX-API 2000, equipped with ESI source used online with a HPLC system
after the ultraviolet (UV) detector. The solvents and reagents were used without
further purification.
5-[[4-Carboxy]phenyl]azo]-2-hydroxybenzoic Acid (Des-b-alanine
Balsalazide, 2)
Concentrated hydrochloric acid (30 mL, ꢃ36% w=w) was added to a suspension
of 4-aminobenzoic acid (30 g, 0.22 mol) in demineralized (DM) water (360 mL) at
20–25 ꢂC, and thereafter the resultant solution was cooled to 2 to ꢄ2 ꢂC. To this sol-
ution, aqueous solution of sodium nitrite (15.86 g, 0.23 mol) in DM water (60 mL) was
added at 2 to ꢄ2 ꢂC. The obtained diazotized solution was added to a precooled alka-
line solution of salicylic acid (36.26 g, 0.26 mol) in DM water (630 mL) containing

2248
Md. U. KHAN ET AL.
sodium hydroxide (17.52 g) and sodium carbonate (46.42 g) at 2 to ꢄ2 ꢂC, maintain-
ing pH ꢁ 8. After completion of reaction, the reaction mass was heated to 60–65 ꢂC,
acidified, and stirred for 3 h, maintaining the same temperature. Thereafter, the
resulting slurry was cooled to 40 ꢂC, filtered, washed with DM water, and dried to give
compound 2 as brown crystalline solid (62.6 g, ꢃ100% yield). IR (KBr, cmꢄ1): 3425
=
=
(OꢄH), 3072 (Ar-CH), 1686 (C O), 1602 and 1587 (C C ring), 1457 (aliphatic
1
CH), 1208 (CꢄN), 1077 (CꢄO), 799 and 776 (Ar-H, out of plane bend). H NMR
(DMSO-d6, 300 MHz, d ppm): 6.96 (d, J ¼ 8.8 Hz, 1H), 7.90 (d, J ¼ 8.5 Hz, 2H),
7.98 (dd, J ¼ 8.8 Hz and 2.5 Hz, 1H), 8.11 (d, J ¼ 8.5 Hz, 2H), 8.34 (d, J ¼ 2.5 Hz,
1H), 13.20 (brs, 1H). 13C NMR and DEPT (DMSO-d6, 300 MHz, d ppm): 116.6
(C), 118.2(CH), 122.1 (CH), 126.5 (CH), 128.0 (CH), 130.6 (CH), 131.9 (C), 143.5
(C), 154.6 (C), 166.8 (C), 166.9 (C), 171.1 (C). MS m=z (ESI): 285.2 [(M – H)ꢄ],
307.0 [(M – H)–þNa].
5-[4-[2-(2-Carboxyethyl Carbamoyl)ethyl Carbamoyl]phenyl Azo]-2-
hydroxybenzoic Acid (Balsalazide b-alanine, 3)
Concentrated hydrochloric acid (6 mL, ꢃ36% w=w) was added to a suspension
of N-(4-aminobenzoyl)di-b-alanine (6 g, 0.02 mol) in DM water (72 mL) at 20–25 ꢂC,
and the resultant solution was cooled to 2 to ꢄ2 ꢂC. To this solution, an aqueous
solution of sodium nitrite (1.4 g, 0.02 mol) in DM water (6 mL) was added at 2 to
ꢄ2 ꢂC. Thereafter, the resulting diazotized solution was added to a precooled solution
of salicylic acid (3.2 g, 0.02 mol) in 120 mL DM water containing sodium hydroxide
(1.55 g) and sodium carbonate (4.15 g) at 2 to ꢄ2 ꢂC, maintaining pH ꢁ 8. After
completion of the reaction, the resultant mass was heated to 60–65 ꢂC and acidified.
The resulting suspension was cooled to 30–35 ꢂC, filtered, washed with DM water,
and dried to obtain the desired product 3 (7.7 g, ꢃ82% yield). IR (KBr, cmꢄ1):
=
=
3375 (O-H), 3019 (Ar-CH), 1675 (C O), 1600 and 1577 (C C ring), 1455 (aliphatic
1
CH), 1212(C-N), 1067 (C-O), 795 and 770 (Ar-H, out of plane bend). H NMR
(DMSO-d6, 300 MHz, d ppm): 2.40 (m, 4H, 2CH2), 3.26 (m, 2H, CH2), 3.47 (m,
2H, CH2), 6.77 (d, J ¼ 8.8 Hz, 1H), 7.83 (d, J ¼ 8.8 Hz, 1H), 7.85 (d, J ¼ 8.5 Hz,
2H), 7.97 (d, J ¼ 8.5 Hz, 2H), 8.02 (t, J ¼ 5.5 Hz, 1H), 8.28 (d, J ¼ 2.5 Hz, 1H), 8.62
(t, J ¼ 5.5 Hz, 1H), 12.21(brs, 1H). 13C NMR and DEPT (DMSO-d6, 300 MHz, d
ppm): 34.8 (CH2), 35.6 (CH2), 36.2 (CH2), 37.1 (CH2), 118.9 (CH), 119.8 (C), 122.5
(CH), 123.4 (C), 127.2 (CH), 127.8 (CH), 129.2 (CH), 135.9 (C), 134.1 (C), 154.7
(C), 166.5 (C), 170.8 (C), 171.3 (C), 173.7 (C). MS m=z (ESI): 427.2 [(M – H)ꢄ],
449.0 [(M – H)ꢄ þ Na].
3-[[4-[[(2-Carboxyethyl)amino]carbonyl]phenyl]azo]-2-
hydroxybenzoic Acid (Balsalazide 3-isomer, 4)
Concentrated hydrochloric acid (10 mL, ꢃ36% w=w) was added to a suspension
of N-(4-aminobenzoyl) b-alanine (10 g, 0.05 mol) in DM water (145 mL) at 20–25 ꢂC.
The obtained clear solution was cooled to 0 to ꢄ2 ꢂC, and a solution of sodium nitrite
(3.5 g, 0.05 mol) in DM water (20 mL) was added to it. Thus obtained, diazotized
solution was added to a solution of salicylic acid (8 g, 0.06 mol) in a water=isopropyl
alcohol mixture (1:1, 150 mL) containing sodium carbonate (18 g) at 2 to ꢄ2 ꢂC,

MESALAMINE, ALSO KNOWN AS MESALAZINE
2249
maintaining pH ꢁ 8. After completion of reaction, the reaction mass was acidified,
filtered, washed, and dried. Thereafter, the desired impurity balsalazide 3-isomer 4
was purified by flash chromatography (ethyl acetate=methanol as eluant) to get
brown solid. (1.2 g, 7% yield). IR (KBr, cmꢄ1): 3426 and 3185 (OH and NH), 1717
=
=
=
and 1711 (C O), 1631(C O amide), 1572 and 1545 (C C aromatic), 1444 and
1399 (aliphatic C-H), 1229 (C-N), 1071 (C-O), 771 and 736 (Ar-H out of plane bend).
1H NMR (DMSO-d6, 300 MHz, d ppm): 2.52 (m, 2H, CH2), 3.47 (m, 2H, CH2), 6.61
(dd, J ¼ 7.7 Hz, 1H), 7.57 (dd, J ¼ 8.0 Hz and 1.7 Hz, 1H), 7.86 (d, J ¼ 8.2 Hz, 2H),
7.87 (dd, J ¼ 8.0 Hz and 1.7 Hz, 1H), 8.00 (d, J ¼ 8.5 Hz, 2H), 8.70 (t, J ¼ 5.5 Hz,
1H), 18.43 (brs, 1H). 13C NMR and DEPT (DMSO-d6, 300 MHz, d ppm): 35.0
(CH2), 36.7 (CH2), 115.1 (CH), 119.3 (CH), 122.8 (CH), 123.5 (C), 129.1 (CH),
135.0 (CH), 136.1 (C), 142.1 (C), 155.0 (C), 165.5 (C), 166.4 (C), 171.4 (C), 174.1
(C). MS m=z (ESI): 356.2 [(M – H)ꢄ], 378.0 [(M – H)ꢄ þ Na].
3-[4-(4-Hydroxy Phenyl Azo)benzoyl Amino]propionic Acid
(Decarboxy Balsalazide, 5)
Concentrated hydrochloric acid (20 mL, ꢃ36% w=w) was added to a suspension
of N-(4-aminobenzoyl)-b-alanine (20 g, 0.1 mol) in DM water (300 mL) at 20–25 ꢂC,
and the resulting clear solution was cooled to 2 to ꢄ2 ꢂC. To the obtained solution,
an aqueous solution of sodium nitrite (6.88 g, 0.1 mole) in DM water (20 mL) was
added at 2 to ꢄ2 ꢂC. Thus obtained, diazotized solution was added slowly to a pre-
cooled solution of 4-hydroxybenzoic acid (13.53 g, 0.1 mole) in DM water (200 mL)
containing (8.15 g) of sodium hydroxide and (15.9 g) of sodium carbonate at 2 to
ꢄ2 ꢂC, maintaining pH ꢁ 8. After completion of the reaction, the reaction mass
was acidified, filtered, washed, and dried to obtain the desired impurity 5 (27.38 g,
91% yield). IR (KBr, cmꢄ1): 3336 (OH and NH), 3048 (Ar-H), 2969 (aliphatic
=
=
C-H), 1707 and 1606 (C O), 1589 and 1543 (C C aromatic), 1464 (aliphatic C-H),
1228 (C-N) 1087 (C-O), 774 and 724 (Ar-H out of plane bend). 1H NMR (DMSO-d6,
300 MHz, d ppm): 2.54 (m, 2H, CH2), 3.50 (m, 2H, CH2), 6.97 (d, J ¼ 8.8 Hz, 2H),
7.84 (d, J ¼ 8.8 Hz, 2H), 7.88 (d, J ¼ 8.8 Hz, 2H), 8.04 (d, J ¼ 8.8 Hz, 2H), 8.72 (t,
J ¼ 5.5 Hz, 1H), 10.47 (brs, 1H), 12.30 (brs, 1H). 13C NMR and DEPT (DMSO-d6,
300 MHz, d ppm): 33.8 (CH2), 35.7 (CH2), 116.1 (CH), 121.9 (CH), 125.2 (CH),
128.4 (CH), 135.6 (C), 145.3 (C), 153.6 (C), 161.5 (C), 165.6 (C), 172.9 (C). MS
m=z (ESI): 314.0 [(MH)þ], 336.0 [Mþ þ Na].
2,4-bis-[[4-[(2-Carboxyethyl)amino]carbonyl]phenyl]azo Salicylic
Acid (bis-Azo Salicylic Acid, 6)
Concentrated hydrochloric acid (20 mL, ꢃ36% w=w) was added to a suspension
of N-(4-aminobenzoyl)-b-alanine (20 g, 0.1 mol) in DM water (250 mL) at 20–25 ꢂC.
The resulting clear solution was cooled to 2 to ꢄ2 ꢂC, and sodium nitrite (6.7 g,
0.1 mol) in DM water (30 mL) was added to it. The diazotized solution was added
to a precooled solution of salicylic acid (6.6 g, 0.05 mol) in DM water (300 mL) con-
taining sodium hydroxide (7.68 g) and sodium carbonate (15.26 g) at 2 to ꢄ2 ꢂC,
maintaining pH above 8. After addition, the reaction mass was acidified, filtered,
washed with DM water, and dried. Thereafter, the desired impurity was purified by

2250
Md. U. KHAN ET AL.
flash chromatography. IR (KBr, cmꢄ1): 3440 and 3178 (OH and NH), 2968 and 2975
=
=
(aliphatic C-H), 1713 and 1631 (C O), 1548 and 1493 (C C aromatic), 1451
(aliphatic C-H), 1242 (C-N), 1074 (C-O), 772 and 739 (Ar-H out of plane bend).
1H NMR (DMSO-d6, 300 MHz, d ppm): 2.47 (m, 4H, 2CH2), 3.48 (m, 4H, 2CH2),
7.90 (2d, J ¼ 8.8 Hz, 4H), 8.01 (2d, J ¼ 8.8 Hz, 4H), 8.15 (d, J ¼ 2.5 Hz, 1H), 8.51
(d, J ¼ 2.5 Hz, 1H), 8.70 and 8.74 (2 t, J ¼ 5.2 Hz, 2H), 18.89 (brs, 1H). 13C NMR
and DEPT (DMSO-d6, 300 MHz, d ppm): 34.2 (CH2), 35.9 (CH2), 113.7 (CH),
121.2 (C), 121.7 (CH), 122.2 (CH), 128.3 (CH), 128.8 (CH), 135.2 (C), 135.6 (C),
139.5 (C), 142.4 (C), 153.7 (C), 154.0 (C), 165.5 (C), 169.5 (C), 171.2 (C), 173.2 (C).
MS m=z (ESI): 577.2 [(MH)þ], 599.0 [Mþ þ Na].
5-[2-[40,5-bis[(2-Carboxyethyl)carbamoyl]biphenyl-2-yl]diazenyl]-2-
hydroxybenzoic Acid (Biphenyl-Azo Salicylic Acid, 7)
This related substance was prepared in laboratory as described in previous
example. IR (KBr, cmꢄ1): 3446 and 3180 (OH and NH), 2960 and 2971 (aliphatic
=
=
C-H), 1720 and 1635 (C O), 1552 and 1490 (C C aromatic), 1443 (aliphatic C-H),
1248 (C-N), 1076 (C-O), 775 and 731 (Ar-H out of plane bend). 1H NMR (DMSO-d6,
300 MHz, d ppm): 2.54 (m, 4H, 2CH2), 3.49 (m, 4H, 2CH2), 6.70 (d, J ¼ 8.8 Hz, 1H),
7.58 (d, J ¼ 8.5 Hz, 2H), 7.60 (dd, J ¼ 8.8 Hz and 2.5 Hz, 1H), 7.70 (d, J ¼ 8.8 Hz, 1H),
7.93 (d, J ¼ 8.5 Hz, 2H), 7.97 (dd, J ¼ 8.8 Hz and 1.4 Hz, 1H), 8.05 (d, J ¼ 1.4 Hz, 1H),
8.17 (d, J ¼ 2.5 Hz, 1H), 8.64 (t, J ¼ 5.2 Hz, 1H), 8.77 (t, J ¼ 5.2 Hz, 1H), 18.34 (brs,
1H). 13C NMR and DEPT (DMSO-d6, 300 MHz, d ppm): 34.6 (CH2), 36.5 (CH2),
116.9 (CH), 119.2 (CH), 119.9 (C), 126.2 (CH), 127.5 (CH), 128.6 (CH), 129.4
(CH), 130.4 (CH), 131.3 (CH), 134.0 (C), 135.6 (C), 138.7 (C), 141.9 (C), 143.5 (C),
151.8 (C), 166.4 (C), 166.8 (C), 171.1 (C), 173.8 (C). MS m=z (ESI): 549.2 [(MH)þ],
571.0 [Mþ þ Na].
3-[4-[5-[4-(2-Carboxy Ethyl Carbamoyl)phenylazo]-2-hydroxy
Phenylazo]benzoyl Amino] Propionic Acid (Bis-Azo Diacid, 8)
Concentrated hydrochloric acid (5 mL, ꢃ36% w=w) was added to a suspension
of N-(4-aminobenzoyl)-b-alanine (5 g, 0.024 mol) in DM water (75 mL) at 20–25 ꢂC,
and the resulting solution was cooled to 2 to ꢄ2 ꢂC. A solution of sodium nitrite
(1.74 g, 0.025 mol) in DM water (10 mL) was added at 2 to ꢄ2 ꢂC. The resulting
diazotized solution was added to a precooled solution of 4-hydroxybenzoic acid
(1.65 g, 0.012 mole) in DM water (100 mL) containing sodium carbonate (9.1 g) at 2
to ꢄ2 ꢂC, maintaining pH above 8. After completion of the reaction, the reaction mass
was acidified, filtered, washed with DM water, and dried. Thereafter, the obtained
product was suspended in aqueous acetic acid (400 mL) and refluxed for 20 min, then
cooled to 25–30 ꢂC, filtered, and dried to obtain impurity 8 (4.28 g, 32% yield). IR
(KBr, cmꢄ1): 3298 (NH), 3057 (Ar-H), 2921 (aliphatic C-H), 1630 and 1604 (C O),
=
=
1577 and 1538 (C C aromatic), 1430 (C-H), 1214 (C-N), 1085 (C-O), 799 and 772
1
(Ar-H out of plane bend). H NMR (DMSO-d6, 300 MHz, d ppm): 2.57 (m, 4H,
2CH2), 3.52 (m, 4H, 2CH2), 7.32 (d, J ¼ 8.8 Hz, 1H), 7.94 (d, J ¼ 8.2 Hz, 1H),
7.95–8.15 (m, 8H), 8.24 (s, 1H), 8.72 and 8.75 (2t, J ¼ 5.2 Hz, 2H), 11.54 (brs, 1H),
12.27 (brs, 2H). 13C NMR and DEPT (DMSO-d6, 300 MHz, d ppm): 33.7 (CH2),

MESALAMINE, ALSO KNOWN AS MESALAZINE
2251
35.7 (CH2), 114.1 (CH), 119.3 (CH), 122.2 (CH), 122.8 (CH), 128.0 (CH), 128.4 (CH),
128.5 (CH), 136.1 (C), 136.5 (C), 139.0 (C), 145.1 (C), 153.2 (C), 153.3 (C), 159.0 (C),
165.5 (C), 165.5 (C), 172.9 (C). MS m=z (ESI): 533.3 [(MH)þ], 555.1 [Mþ þ Na].
2,4-bis[[4[[(2-Carboxyethyl)amino]carbonyl]phenyl]azo]-3-[4-[[(2-
carboxyethyl)amino]carbonyl]-phenyl]phenol (Bis-Azo Triacid, 9)
N-(4-Aminobenzoyl)-b-alanine (10 g, 0.05 mol) was suspended in DM water
(120 mL) at 20–25 ꢂC. Concentrated hydrochloric acid (10 mL, ꢃ36% w=w) was
added to the obtained suspension and cooled to 2 to ꢄ2 ꢂC. A solution of sodium
nitrite (3.5 g, 0.05 mol) in DM water (20 mL) was added at 2 to ꢄ2 ꢂC. The resulting
diazotized solution was added to a solution of 4-hydroxybenzoic acid (1.65 g,
0.01 mol) in DM water (100 mL) containing sodium carbonate (18 g) at 2 to
ꢄ2 ꢂC, maintaining pH above 8. After completion of reaction, the temperature of
the reaction mass was raised to 35–40 ꢂC and acidified. Thereafter, the precipitated
product was filtered, washed, and dried. The obtained product was suspended in
ethyl methyl ketone (100 mL) having 10% v=v water and refluxed for 20 min. There-
after, the suspension was cooled to 20–25 ꢂC, filtered, and dried to obtain product 9
(3.15 g, 36% yield). IR (KBr, cmꢄ1): 3261 (OH and NH), 1714 and 1639 (C O), 1548
=
=
and 1494 (C C aromatic), 1228 (aliphatic C-N), 1070 (C-O), 789 and 760 (Ar-H out
1
of plane bend). H NMR (DMSO-d6, 300 MHz, d ppm): 2.41 (m, 4H, 2CH2), 2.47
(m, 2H, CH2), 3.43 (m, 4H, 2CH2), 3.49 (m, 2H, CH2), 6.98 (d, J ¼ 9.3 Hz, 1H),
7.43 (d, J ¼ 8.2 Hz, 2H), 7.51 (2d, J ¼ 8.8 Hz and 2.5 Hz, 4H), 7.87 (d, J ¼ 8.0 Hz,
6H), 7.93 (d, J ¼ 9.3 Hz, 1H), 8.55, 8.64 and 8.72 (3 t, J ¼ 5.2 Hz, 3H). 13C NMR
and DEPT (DMSO-d6, 300 MHz, d ppm): 37.3 (CH2), 37.4 (CH2), 38.4 (CH2),
121.6 (CH), 123.8 (CH), 124.2 (CH), 124.3 (CH), 127.4 (CH), 130.4 (CH), 130.6
(CH), 134.6 (CH), 135.1 (C), 136.5 (C), 137.7 (C), 138.4 (C), 139.8 (C), 144.3 (C),
146.5 (C), 154.0 (C), 155.7 (C), 158.2 (C), 167.6 (C), 167.8 (C), 168.6 (C), 176.5
(C), 176.8 (C). MS m=z (ESI): 724.1 [(MH)þ], 746.2 [Mþ þ Na].
3-(4-Acetylamino Benzoylamino)-propionic Acid (N-Acetyl-4-ABBA, 10)
N-(4-Aminobenzoyl)-b-alanine (30 g, 0.15 mol) was added to an acetic acid=
water mixture (2:1, 90 mL) at 25–30 ꢂC. Acetic anhydride (30 g, 0.29 mol) was added
to the obtained suspension, maintaining temperature at 30–45 ꢂC. The reaction mass
was stirred at 35–40 ꢂC for 1 h and diluted with acetone (90 mL). The precipitated
product was filtered, washed with DM water followed by acetone, and dried to
obtain 10 (32.46 g, 90% yield). IR (KBr, cmꢄ1): 3326 and 3196 (OH and NH),
=
3064 (Ar-H), 2907 (aliphatic C-H), 1706, 1693 and 1626 (C O), 1590 and 1537
=
(C C aromatic), 1429 (aliphatic C-H), 1262 (C-N), 1083 (C-O), 813 and 763
1
(Ar-H out of plane bend). H NMR (DMSO-d6, 300 MHz, d ppm): 2.06 (s, 3H,
CH3), 2.48 (t, 2H, CH2), 3.44 (m, 2H, CH2), 7.62 (d, J ¼ 8.5 Hz, 2H), 7.77 (d,
J ¼ 8.5 Hz, 2H), 8.38 (t, J ¼ 5.2 Hz, 1H), 10.13 (s, 1H).
5-Acetamido-2-hydroxybenzoic Acid (5-Acetamido-SA, 11)
5-Aminosalicylic acid (50 g, 0.33 mol) was added to an acetic acid=acetic anhy-
dride mixture (1:1, 100 mL), stirred at reflux for 30 min, and cooled to 20–25 ꢂC. The

2252
Md. U. KHAN ET AL.
precipitated product was filtered, washed with DM water, suspended in 10% w=w
aqueous sodium hydroxide solution, and stirred for 1 h at 25–30 ꢂC. The pH was
adjusted to 2 to precipitate the product. The product 11 was filtered, washed with
DM water, and dried (59.7 g, 94% yield). IR (KBr, cmꢄ1): 3357 and 3106 (OH
=
=
and NH), 2874 (aliphatic C-H), 1685 and 1603 (C O), 1539 and 1516 (C C aro-
matic), 1420 (aliphatic C-H), 1238 (C-N), 807 and 792 (Ar-H out of plane bend).
1H NMR (DMSO-d6, 300 MHz, d ppm): 2.14 (s, 3H, CH3), 7.07 (dd, J ¼ 8.78 Hz
and 1.92 Hz, 1H), 7.32 (d, J ¼ 1.92 Hz, 1H), 7.74 (d, J ¼ 8.78 Hz, 1H), 9.60 (s, 1H),
11.32 (brs, 1H).
ACKNOWLEDGMENTS
The authors are grateful to colleagues in the Analytical Research Department
of APL Research Centre, a division of Aurobindo Pharma Limited, Hyderabad, for
their valuable contribution to this work. The authors also thank the management for
permission to publish this work.
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