2,5-Bis-(2-hydroxybenzoylamino)pentanoic acid, a salicylic acid-metabolite isolated from chicken: Characterization and independent synthesis

Article abstract of DOI:10.1016/S0960-894X(02)01021-1

From excreta of chickens that had been treated with sodium salicylate, a new compound was detected and identified as a double conjugated ornithine metabolite. The structural assignment of this metabolite was further confirmed by an independent efficient 3-step synthesis from ornithine.

Full text of DOI:10.1016/S0960-894X(02)01021-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 335–337
2,5-Bis-(2-hydroxybenzoylamino)pentanoic Acid, a Salicylic
Acid-Metabolite Isolated from Chicken: Characterization and
Independent Synthesis
Ulrik Hillaert,a Kris Baert,b Jef Rozenski,c Siska Croubels,b Denis De Keukeleire,d
Patrick De Backerb and Serge Van Calenbergha,*
aGhent University, Faculty of Pharmaceutical Sciences, Laboratory for Medicinal Chemistry, Harelbekestraat 72, B-9000 Ghent,
Belgium
bGhent University, Faculty of Veterinary Medicine, Department of Pharmacology, Pharmacy and Toxicology, Salisburylaan 133,
B-9820 Merelbeke, Belgium
cKatholieke Universiteit Leuven, Rega Institute, Laboratory for Medicinal Chemistry, Minderbroedersstraat 10,
B-3000 Leuven, Belgium
dGhent University, Faculty of Pharmaceutical Sciences, Laboratory for Pharmacognosy and Phytochemistry, Harelbekestraat 72,
B-9000 Ghent, Belgium
Received 31 October 2002; accepted 22 November 2002
Abstract—From excreta of chickens that had been treated with sodium salicylate, a new compound was detected and identified as a
double conjugated ornithine metabolite. The structural assignment of this metabolite was further confirmed by an independent
efficient 3-step synthesis from ornithine.
# 2002 Elsevier Science Ltd. All rights reserved.
Knowledge of xenobiotic metabolism in birds is, in
contrast to mammals, very limited, especially with
regard to drugs containing a carboxylic acid group, such
as salicylates. In the body, a drug can be metabolized in
two phases. In the first phase (phase I), the reactions
occur which can be classified as oxydations, reductions
and hydrolyses. The products of the first phase may
then proceed to the second phase (phase II), which are
conjugation reactions. A wide range of carboxylic acids
compounds (drugs, herbicides, and pesticides) can con-
jugate to amino acids in living species. The fate of the
simplest aromatic carboxylic acid, benzoic acid, has
been studied extensively as a model compound for bio-
transformation reactions in animals, and it has been
demonstrated to undergo conjugation with sugars (glu-
curonic acid in vertebrates, glucose in insects) or amino
acids.1 The nature of the amino acid varies markedly
according to the animal species and the chemical struc-
ture of the carboxylic acid. In general, the amino acids
participating in conjugation reactions are glycine, glu-
tathione, taurine, and ornithine. Glycine and glu-
tathione are most utilized by a wide range of species,
including humans, for conjugation of carboxylic acids.
Taurine conjugation is relatively widespread in most
animal species, but is restricted to arylacetic and cholic
acid derivatives.
Ornithine conjugation occurs only in some birds and
reptiles, but it has not been reported in mammalian
species.2 Williams3 and Seymour4 reported that in birds
belonging to the order of Galliformes (chicken, turkey,
quail) and Anseriformes (duck, goose), the major meta-
bolite of benzoic acid is ornithurate (a,d-dibenzoyl-
ornithine), whereas in most mammals and in some bird
species belonging to the order of Columbiformes
(pigeon, woodpigeon, dove) conjugation with benzoyl-
glycine with the formation of hippurate is present.
Salicylic acid (SA), usually administered as its precursor
acetylsalicylic acid, is widely used for its analgesic,
antipyretic and antiphlogistic activities. Acetylsalicylic
acid is rapidly hydrolyzed to SA after absorption has
*Corresponding author. Tel.: +32-9-264-81-24; fax: +32-9-264-81-46;
e-mail: serge.vancalenbergh@rug.ac.be
0960-894X/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)01021-1

336
U. Hillaert et al. / Bioorg. Med. Chem. Lett. 13 (2003) 335–337
been taken place. In humans, SA is metabolized to var-
ious metabolites. The main important metabolites are
salicyluric acid (SU), also called the glycine conjugate,
salicylic acid phenolic glucuronide (SAPG) and salicylic
acid acyl glucuronide (SAAG). SU is also further con-
jugated with glucuronic acid to form salicyluric acid
phenolic glucuronide (SUPG).5
The use of SA (a water soluble form of acetylsalicylic
acid) and non-steroidal anti-inflammatory drugs
(NSAID’s) in general in bird medicine is still con-
troversial. Economically important features such as
growth and egg production seem not to improve by the
use of NSAID’s. Therefore, up till today, the avail-
ability of commercial formulations for use in poultry
industry in most European countries is very limited.
Nevertheless, valid indications for the use of an NSAID
in birds may exist and treatment of certain pathological
processes with NSAID’s can be beneficial.6 The phar-
macokinetics of sodium salicylate in several bird species
have been described by Baert and De Backer.7 Hereby,
a marked species difference in pharmacokinetic para-
meters was found between chicken and pigeon. A half-
life of sodium salicylate in pigeons could be calculated
which was three times longer than in chickens. Accord-
ing to these authors this large difference in elimination
half-life would be mainly provided by a substantial dif-
ference in drug metabolism, as was already seen for
benzoic acid.
10
Scheme 1. Synthetic pathways of the SA-conjugate 1. Reagents and
conditions: (a) SOCl2, MeOH, reflux; (b) Et3N, CH2Cl2, o-anisoyl-
chloride, À15 ꢀC; (c) BBr3, CH2Cl2, rt.
acceleration time-of-flight (Q/oaTOF) tandem mass
spectrometer (qTof 2, Micromass, Manchester, UK)
equipped with a standard electrospray ionization (ESI)
interface operated in the positive ionisation mode.
Samples were infused in a propan-2-ol:water (1:1) mix-
ture at 3 mL/min. Fragment ion spectra were acquired
with a collision energy of 30 eV. Accurate mass mea-
surements were performed using the lock mass of the
protonated peptide YGGFL (m/z=556.2771), which
was added to the sample (10 pmol/mL). For accurate
mass measurements of the fragments, the precursor ion
peak was used as internal mass reference.
Results
From the aforementioned biological excreta, a com-
pound was detected with negative ionization tandem
mass spectrometry (LC-MS/MS) that had an m/z value
of 371.2.
Metabolite Isolation
From excreta samples of chickens that had been treated
with sodium salicylate, salicylic acid and its metabolites
were isolated by a liquid-liquid back-extraction with
diethyl ether as extraction solvent, followed by a solid-
phase extraction procedure using a SAX column (strong
anion exchange, Isolute, Sopachem, Brussels, Belgium).
Chromatographic separations were achieved on a
Nucleosil C18 column (100Â3.0 mm i.d., Chrompack,
Varian, Middelburg, The Netherlands), using a gradient
elution with 1% acetic acid in water and methanol. The
flow rate was 0.2 mL/min. A Quattro Ultima triple-
quadrupole instrument (Micromass), equipped with an
ESI Z sprayTM source, which was operated in the nega-
tive ion MS/MS mode, was used as detector.
HRMS in the positive mode (Fig. 1) suggested that this
metabolite has an elemental formula of C19H20N2O6
(calculated m/z 373.1399 for M+H), as this was the sole
feasible composition for the measured value of M+H
of 373.1407 (C0-30H0-40N0-5O0-10) with a tolerance of 5
ppm. The ready loss of a neutral 120 Da fragment from
the pseudo-molecular ion suggests cleavage of a salicyloyl
moiety. Based on these spectroscopic data, the substance
was proposed to be 2,5-bis-(2-hydroxybenzoylamino)-
pentanoic acid. All fragment ions (positive ion mode)
Synthesis
Since standard acylation methods using acetylsalicyloyl
chloride yielded complex reaction mixtures, we opted
for a synthetic sequence as depicted in Scheme 1.
Treatment of ornithine.HCl with thionylchloride in
methanol led to methyl 2,5-diaminopentanoate (2).8
Acylation of 2 with o-anisoylchloride furnished amide 3,
9
which was subsequently treated with BBr3 to give the
desired product 1 in an overall yield of 89%.
The structure of the desired product and the inter-
mediates was assigned by 1H NMR and 13C NMR.
Mass spectra were acquired on a quadrupole/orthogonal-
Figure 1. Mass spectrum (positive ion mode) of the salicyluric acid
metabolite 1.

U. Hillaert et al. / Bioorg. Med. Chem. Lett. 13 (2003) 335–337
Table 1. Assignment of all fragment ions (positive ion mode) in the mass spectrum of 1
337
Ion (ppm)
Relative
abundance (%)
m/z
Measured
m/z
Calculated
Elemental
composition
Error
M+H–H2O
M+H–2H2O
1.9
7.0
100
31.3
22.5
2.0
355.1288
337.1180
253.1178
235.1068
190.0858
133.0985
121.0310
115.0897
355.1294
337.1189
253.1188
235.1083
190.0868
133.0977
121.0289
115.0872
C19H19N2O5
C19H17N2O4
C12H17N2O4
C12H15N2O3
C11H12NO2
C5H13N2O2
C7H5O2
À1.7
À2.9
À3.9
À6.4
À5.2
6.0
M+H–salicyloyl+H
M+H–salicyloyl–H2O+H
M+H–HCOOH–salicylamide
M+H–2 salicyloyl+2H
Salicyloyl
6.5
2.8
17
23
M+H–2 salicyloyl+2H
C5H11N2O
present in the mass spectrum can be assigned (Table 1).
The structure may arise from double conjugation of
salicylic acid to ornithine.
M. R.; Shah, P.; Upton, R. J.; Walls, S. B. J. Org. Chem. 1999,
14, 5166.
9. Obrecht, D.; Lehmann, C.; Ruffieux, R.; Schonholzer, P.;
Muller, K. Helv. Chim. Acta 1995, 78, 1567.
10. All new compounds were characterized by spectroscopic
means. 3: white solid; TLC Rf=0.37 (ethyl acetate/hex-
ane=50:50); H NMR (300 MHz, DMSO-d6) d 1.60 (2H, m,
Structure 1 was confirmed by comparison of relevant
spectroscopic data with those of the synthetic com-
pound. The synthesized ornithine conjugate will further
serve as a valuable tool (analytical standard) to investi-
gate the excretion of salicylic acid and its metabolites.
1
CH2–CH), 1.87 (2H, m, CH2–CH2–CH2), 3.29 (2H, dt, app
dd, J=6.45 and 12.61, NH-CH2), 3.67 (3H, s, COCH3), 3.84
(3H, s, OCH3), 3.90 (3H, s, OCH3), 4.53 (1H, ddd, J=5.28,
7.92 and 12.61, CH-NH), 6.96–7.18 (4H, m, arom.), 7.40–7.52
(2H, m, arom.), 7.67 (1H, dd, J=2.05 and 7.91, arom.), 7.76
(1H, dd, J=1.76 and 7.62), 8.16 (1H, t, J=5.57, NH-CH2),
8.47 (1H, d, J=7.33, CH–NH–CO); 13C NMR (75 MHz,
DMSO-d6) d 25.67, 28.73, 38.68, 52.10, 52.38, 55.88, 56.20,
112.05, 112.39, 120.52, 120.75, 122.30, 123.87, 130.26, 130.66,
131.98, 132.78, 156.93, 157.33, 164.94, 165.25, 172.56; Exact
mass (ESI-MS) for C22H27N2O6 [M+H]+: found 415.1861
calculated 415.1868. 1: white solid; TLC Rf=0.24 (ethyl ace-
tate/hexane/formic acid=80:20:1); 1H NMR (300 MHz,
DMSO-d6) d 1.63 (2H, m, CH2–CH), 1.88 (2H, m, CH2–CH2–
CH2), 3.34 (2H, dt, app dd, J=6.81 and 12.81, NH–CH2),
4.49 (1H, ddd, J=5.18, 8.45 and 12.54, CH-NH), 6.82-6.95
(4H, m, arom.), 7.33–7.44 (2H, m, arom.), 7.82 (1H, dd,
J=1.64 and 8.45, arom.), 7.95 (1H, dd, J=1.64 and 8.45,
arom.), 8.80 (1H, t, J=5.72, NH-CH2), 8.93 (1H, d, J=7.63,
References and Notes
1. Burke, A. B.; Millburn, P.; Huckle, K. R.; Hutson, D. H.
Drug Metab. Dispos. 1987, 15, 581.
2. Igarashi, K.; Suzuki, R.; Kasuya, F.; Fukui, M. Chem.
Pharm. Bull. 1992, 40, 2196.
3. Williams, R. T. Federation Proceedings 1967, 26, 1029.
4. Seymour, M. A.; Millburn, P.; Tait, G. H. Biochem. Soc.
Trans. 1988, 16, 1021.
5. Vree, T. B.; van Ewijk-Beneken Kolmer, E. W. J.; Verwey-
van Wissen, C. P. W. G. M.; Hekster, Y. A. J. Chromatogr. B
1994, 652, 161.
6. Baert, K.; De Backer, P. Vlaams Diergeneeskundig Tijds-
chrift 2002, 71, 117.
7. Baert, K.; De Backer, P. Comp. Biochem. Physiol. C 2002,
accepted for publication.
8. Macdonald, S. J. F.; Clarke, G. D. E.; Dowle, M. D.;
Harrison, L. A.; Hodgson, S. T.; Inglis, G. G. A.; Johnson,
CH–NH–CO), 12.14 (1H, br s, OH), 12.65 (1H, br s, OH); 13
C
NMR (75 MHz, DMSO-d6) d 25.83, 28.46, 38.69, 52.28,
115.25, 115.72, 117.43, 117.56, 118.62, 118.93, 127.69, 128.78,
133.81, 133.95, 159.50, 160.41, 168.48, 169.29, 173.34; Exact
mass (ESI-MS) for C19H21N2O6 [M+H]+: found 373.1407
calculated 373.1399.