Synthesis and structure analysis of cyclodehydration product of piroxicam: A metabolite detected in dogs and monkeys

Article abstract of DOI:10.1016/j.ejmech.2009.03.010

We report here a novel synthesis of 6-methyl-6H-7-oxopyrido[1,2-a]pyrimido[5,4-c]-1,2-benzothiazine-5,5-dioxide or cyclodehydration product of piroxicam, a metabolite detected in dogs and monkeys that was synthesized in 6% yield earlier. The reaction of b

Full text of DOI:10.1016/j.ejmech.2009.03.010

European Journal of Medicinal Chemistry 44 (2009) 3368–3371
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Laboratory note
Synthesis and structure analysis of cyclodehydration product of piroxicam:
A metabolite detected in dogs and monkeys
,
a,
Sarbani Pal a , P. Bindu b, P.K. Dubey b, Santu Chakraborty c, Alok Kumar Mukherjee c
*
a Department of Chemistry, MNR Degree and PG College, Opp. JNT University, Kukatpally, Hyderabad, Andhra Pradesh 500072, India
b JNT University, Kukatpally, Hyderabad 500072, India
c Department of Physics, Jadavpur University, Kolkata 700032, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We report here a novel synthesis of 6-methyl-6H-7-oxopyrido[1,2-a]pyrimido[5,4-c]-1,2-benzothiazine-
5,5-dioxide or cyclodehydration product of piroxicam, a metabolite detected in dogs and monkeys that
was synthesized in 6% yield earlier. The reaction of benzoyl chloride with piroxicam in the presence of
triethylamine afforded the piroxicam metabolite in good yield. A comparison of spectral data of the
synthesized compound with the reported values remained inconclusive. The structure of the compound
was confirmed unambiguously by single-crystal X-ray analysis.
Received 11 August 2008
Received in revised form
7 March 2009
Accepted 12 March 2009
Available online 24 March 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Piroxicam
Cyclodehydration
Single crystal
X-ray
1. Introduction
1 with acetic anhydride inpyridine under refluxing condition. The high
melting solid obtained after usual work-up followed by fractional
Piroxicam (1, Fig. 1) or 4-hydroxy-2-methyl-2H-1,2-benzothiazine-
1-(N-(2-pyridyl)carboxamide)-1,1-dioxide is well-known non-
crystallization from ethyl acetate was characterized as compound
3 based on NMR, IR, MS and HRMS along with the combustion
analytical data.
a
steroidal anti-inflammatory drug (NSAID) used in the treatment of
rheumatoid arthritis, osteoarthritis and other inflammatory disorders
[1]. It is a non-selective inhibitor of cyclooxygenase (COX) possessing
analgesic and antipyretic properties. Chemically, it belongs to the
‘‘oxicam’’ class of compounds that contain N-heterocyclic benzothia-
zine carboxamide moiety (Fig. 1). Pharmacokinetic and metabolism
studies on piroxicam showed that it is readily absorbed after oral or
rectal administration. Because of its long plasma half life of 35–60 h
piroxicam is usually given in doses of 20 mg daily [2]. It is extensively
metabolized by hepatic cytochrome P450 enzyme giving rise to several
metabolites including the hydroxylated derivative as a major one. It
was observed that hydroxylation predominantly took place at the C-5
position of the pyridyl ring followed by glucuronidation to generate
5-hydroxypiroxicam glucuronide. Among the several other metabo-
lites reported a cyclodehydration product of piroxicam i.e. 3 (6-methyl-
6H-7-oxopyrido[1,2-a]pyrimido[5,4-c]-1,2-benzothiazine-5,5-dioxide,
Fig. 1) was detected especially in dogs and monkeys [3]. The synthesis
of 3 has been reported earlier in 6% yield that involved the treatment of
2. Results and discussion
Due to our continuing interest in the chemical modifications of
existing cyclooxygenase inhibitors [4,5] we have recently reported
synthesis and pharmacological evaluation of a number of piroxicam
derivatives [6]. In this study when piroxicam was treated with
a number of aliphatic and aromatic acyl chlorides to afford the O-
acylated products formation of an unidentified side product was
observed. The formation of this unknown product (3) was found to
be significant particularly when piroxicam (1) was reacted with
benzoyl chloride (Scheme 1). Initially, the reaction was carried out
for 6 h using benzoyl chloride and triethylamine, 1.0 equivalent
each, when 1a was isolated as a major product [6]. However, by
increasing the reaction time from 6 h to 24 h and using 2.0 equiv-
alent of each of these reagents, we were able to generate the other
product 3 in significant quantity. After separating from the O-
benzoylated compound 1a the unexpected product 3 was charac-
terized by various spectral data. The 1H NMR data recorded for this
compound in various solvents are listed in Table 1 (Entries 1–3)
along with the reported values (Entry 4, Table 1). As evident from
* Corresponding author. Tel.: þ91 40 23041488; fax: þ91 08458 279305.
E-mail address: sarbani277@yahoo.com (S. Pal).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.03.010

S. Pal et al. / European Journal of Medicinal Chemistry 44 (2009) 3368–3371
3369
Table 3
Selected bond distances (Å) and bond angles (ꢀ) for C15H11N3O3S$2H2O (3).
N
O
OH
S
N
N
O
OH
S
S1–O1
S1–O2
S1–N1
S1–C14
N1–C1
N1–C15
N2–C3
N2–C2
N3–C3
N3–C7
N3–C8
O3–C2
1.423(2)
1.432(2)
1.637(2)
1.749(2)
1.407(2)
1.471(3)
1.328(2)
1.357(2)
1.384(2)
1.391(2)
1.409(2)
1.237(2)
O1–S1–O2
O1–S1–N1
O2–S1–N1
N1–S1–C14
C1–N1–S1
C15–N1–S1
C1–N1–C15
C3–N2–C2
C3–N3–C7
C3–N3–C8
C7–N3–C8
C9–C14–S1
119.1(1)
108.2(1)
107.6(1)
100.7(1)
114.2(1)
120.6(1)
121.0(2)
120.5(1)
119.9(1)
118.5(1)
121.4(1)
116.3(1)
S
N
H
O
N
N
H
N
N
N
S
O
O
O
O
O
O
3
2
1
Fig. 1. Piroxicam (1), meloxicam (2) and a metabolite of piroxicam (3).
O
OCOPh
N
N
PhCOCl
N
N
H
O
1
+
N
Et3N, CHCl3
N
S
S
O
O
O
O
3
1a
Scheme 1. Benzoylation of piroxicam.
Table 1
Comparison of spectral data of compound 3.
Entry Spectra Solvent values (ppm)
d
1.
1H NMR
(400 MHz) DMSO-d6
CF3CO2H– 9.29 (d, J ¼ 6.9 Hz, 1H), 8.43 (t, J ¼ 7.3 Hz, 1H), 8.15
(d, J ¼ 7.8 Hz, 2H), 8.06–7.9 (m, 2H), 7.80 (d,
J ¼ 8.7 Hz, 1H), 7.70 (t, J ¼ 7.4 and 1.0 Hz,1H), 3.40 (s,
3H)
2.
3.
1H NMR
CDCl3
8.30 (d, J ¼ 7.3 Hz, 1H), 8.10–7.60 (m, 4H), 7.40 (d,
J ¼ 7.8 Hz, 1H), 6.80 (t, J ¼ 6.9 Hz, 1H), 3.50 (s, 3H)
9.10 (d, J ¼ 6.9 Hz, 1H), 8.30 (d, J ¼ 7.9 Hz, 1H), 8.14
(t, J ¼ 8.4 Hz, 2H), 8.10–7.90 (m, 2H), 7.90 (d,
J ¼ 8.7 Hz, 1H), 7.50 (t, J ¼ 6.4 Hz, 1H), 3.20 (s, 3H)
9.3–7.6 (m, 8H), 3.47 (s, 3H) [doublets observed at
9.24 (d, J ¼ 3.5 Hz) and 8.50 (d, J ¼ 3.5 Hz)]
(200 MHz)
1H NMR
DMSO-d6
(300 MHz)
4.
1H NMRa
(60 MHz)
CF3COOD
Fig. 2. ORTEP view of molecule in 3 with atom-labeling scheme. Displacement ellip-
soids are drawn at the 50% probability level. Solvent water molecules are omitted for
the sake of clarity.
Data collected from literature [as reported in ref [3]: 1H NMR (60 MHz)
3H, CH3), a multiplet at 2.4–0.7 (8H, aromatic protons) in which a pair of doublets at
0.76 and 1.50 (J ¼ 3.5 Hz) could be distinguished] after converting the ‘‘ ’’ values into
‘‘ ’’ values.
s 6.53 (s,
a
s
Table 1 that comparison of the present data with the reported one
remained inconclusive mainly due to the difference in MHz of the
NMR instrument used and solvent for the preparation of NMR
sample.
d
Analysis of other data for compound 3 [13C NMR (DMSO-d6)
157.3, 148.3, 141.9, 135.4, 134.2, 133.4, 132.8, 128.2, 127.8, 123.8,
123.9, 123.3, 118.6, 117.2, 34.6 (NCH3); MS: 314; IR: 1701 (C]O)] did
not provide enough information or evidence to confirm its struc-
ture. The structure of 3 was finally established from single-crystal
X-ray analysis. Single crystals of 3 suitable for crystallographic
study were obtained by slow crystallization from methanol. Rele-
vant crystal data and structure refinement parameters are
summarized in Table 2. Selected geometrical parameters of the
molecule are listed in Table 3 [7].
Table 2
Crystal data and structure refinement parameters for C15H11N3O3S$2H2O (3).
Empirical formula
Formula weight
C15H11N3O3S$2H2O
349.36
Temperature
298(2) K
Wavelength
0.71073 Å
Crystal system, space group
Unit cell dimensions
Monoclinic, P21/c
a ¼ 8.3027(8) Å
b ¼ 27.641(3) Å
c ¼ 7.2861(7) Å
b
¼ 113.04(1)ꢀ
Volume
1538.7(3) Å3
Z, Calculated density
Absorption coefficient
F(000)
4, 1.508 mg/m3
0.243 mmꢁ1
Table 4
728
Selected hydrogen bonds for C15H15N3O5S (3).
Crystal size
0.22 ꢂ 0.28 ꢂ 0.38 mm
D–H/A
D–H (Å)
H/A (Å)
D/A (Å)
D–H/A (ꢀ)
Theta range for data collection
Reflections collected/unique
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2
R indices (all data)
Largest diff. peak and hole
2.67–25.97ꢀ
15,721/3006 [R(int) ¼ 0.0259]
Full-matrix least-squares on F2
3006/5/233
O5–H5A/O2
O5–H5B/O4
C6–H6/O3i
0.97
0.97
0.93
0.97
0.97
0.96
2.15
2.07
2.41
1.89
1.95
2.49
3.029(3)
2.811(4)
3.131(2)
2.855(3)
2.920(2)
3.365(3)
149
131
134
174
176
152
O4–H4A/O3i
O4–H4B/N2ii
C15–H15B/O1iii
1.017
s
(I)]
R1 ¼0.0449, wR2 ¼ 0.1205
R1 ¼0.0485, wR2 ¼ 0.1237
0.321 and ꢁ0.388 e Aꢁ3
Symmetry codes: (i) x þ 1, þy, þz þ 1; (ii) ꢁx þ 1, ꢁy, ꢁz þ 1, (iii) x, ꢁyþ1/2, zꢁ1/2
.

3370
S. Pal et al. / European Journal of Medicinal Chemistry 44 (2009) 3368–3371
Fig. 3. Formation of polymeric chain in C15H15N3O5S (3).
Et3N.HCl
HN
N
S
HN
HO
N
S
PhCOCl
OCOPh
OCOPh
3
1
Et3N, CHCl3
O
N
N
O
O
O
O
1a
E
Scheme 2. Probable mechanism for the formation of 3.
The molecular view of compound 3 is shown in Fig. 2. The
compound 3 consists of 2-methyl-2H-benzo[e][1,2]thiazine-1,1-
dioxide moiety and a pyrido[1,2-a]pyrimidin-2-one system fused
across C1–C8. The ring-puckering parameters [9] of the thiazine
preparation of 1a (Scheme 1) hence intermediacy of 1a during the
formation of 3 cannot be ruled out completely. Accordingly,
formation of 3 was mediated by the reactivity of the pyridinium
nitrogen towards the benzoyloxy bearing carbon of the benzo-
thiazine ring and a possible mechanistic path leading to compound
3 via 1a is shown in Scheme 2. Thus pyridine undergoes a Michael
type of addition through its nitrogen with the olefinic moiety of the
benzothiazine ring to form a six-membered ring intermediate E
which on elimination of benzoyloxy group provided the compound
3. Similarly, in a biological system such as dog or monkey, pirox-
icam is expected to undergo metabolism at the enolic hydroxyl
group providing the corresponding O-sulphate or O-glucuronide
derivative (Fig. 4) via a physiological process common to many
well-known phenolic drugs [14–16]. This in turn can facilitate
attack of the pyridinium nitrogen on the carbon bearing the
sulfonated/glucuronated enolic hydroxyl group to provide the
metabolite 3.
ring (S1/N1/C1/C8–C9/C14), Q ¼ 0.585(2) Å,
q
¼ 110.0(2)ꢀ and
4
¼ 197.3(2)ꢀ, indicate a twist-boat conformation [10]. The dihedral
angle between the planar benzene (C9–C14) and pyridine (N3,
C3–C7) rings in 3 is 44.5(1)ꢀ. The S–O and S–N bond distances in 3
[Table 3] are similar to that reported for analogous structures
[11–13]. The crystal packing in 3 is stabilized by intermolecular
O–H/O, O–H/N and C–H/O hydrogen bonds (Table 4). Pairs of
intermolecular O–H/O and O–H/N connect molecules of 3
forming R44(12) ring, in which the lattice water molecule (O4) acts as
a double donor. Further linking of dimeric rings via C6–H6/O3
hydrogen bonds generates a one-dimensional polymeric chain
(Fig. 3) along the [101] direction. Additional reinforcement between
the molecules forming R44(12) rings and C(8) chains is provided by
O–H/O and O–H/N hydrogen bonds in which the water molecule
(O5) acts as a double donor.
Mechanistically, it was not clearly understood how the
compound 3 formed during the reaction of piroxicam 1 with
benzoyl chloride. Since the compound 3 was detected during the
3. Conclusion
In summary, the present work reports a novel synthetic root for
preparing the cyclodehydration product of piroxicam, a metabolite
detected in dogs and monkeys. The procedure involves the reaction
of benzoyl chloride with piroxicam in the presence of triethylamine
affording the piroxicam metabolite in good yield. As the spectro-
scopic characterization of the metabolite was ambiguous, the
crystal structure of the compound has been established by single-
crystal X-ray analysis. The cyclodehydration product of piroxicam
consists of a 2-methyl-2H-benzo[e][1,2]thiazine-1,1-dioxide moiety
and a pyrido[1,2-a]pyrimidin-2-one system fused across the C–C
bond. In the solid state, the intermolecular hydrogen bonds connect
the molecules to form R44(12) rings, in which one lattice water
molecule acts as a double donor. A probable mechanism for
formation of the compound under the present reaction condition as
well as the physiological condition has been proposed. The present
HN
N
Z
O
O
N
S
O
O
HO
OH
OH
CO2H
Z = -SO3H or
O
Fig. 4. Sulphate and glucuronide derivative of piroxicam.

S. Pal et al. / European Journal of Medicinal Chemistry 44 (2009) 3368–3371
3371
study would undoubtedly help in better understanding of the
chemistry of piroxicam and its metabolite.
solvent water molecules were located from the difference Fourier
map. Thermal parameters of all non-hydrogen atoms were refined
anisotropically and hydrogen atoms were treated as riding (except
the H-atoms of the water molecules) with fixed isotropic thermal
parameters. After convergence of least-squares refinement based
on 3006 unique reflections final R-values R1 ¼0.045 [for 2736
4. Experimental
4.1. General methods
reflections with I > 2
s
(I)] and Rw ¼ 0.121 were obtained.
Reactions were monitored by thin layer chromatography (TLC)
on silica gel plates (60 F254), visualizing with ultraviolet light or
iodine spray. 1H NMR spectra were determined in a solvent as
specified on 200 and 400 MHz spectrometers. Proton chemical
Acknowledgements
The authors thank Mr. M.N. Raju, the chairman of M.N.R.
Educational Trust for his constant encouragement.
shifts (
d
) are relative to tetramethylsilane (TMS,
d
¼ 0.00) as internal
standard and expressed in ppm. Spin multiplicities are given as s
(singlet), d (doublet) and m (multiplet) as well as b (broad).
Coupling constants (J) are given in hertz. A 13C NMR spectrum was
recorded in DMSO-d6 at 50 MHz. Infrared spectra were recorded on
an FT-IR spectrometer. Melting points were determined by using
melting point apparatus and are uncorrected. MS spectra were
obtained on a JMS-D 300 spectrometer.
Appendix. Supplementary material
Supplementary data associated with this article can be found in
the online version, at doi: 10.1016/j.ejmech.2009.03.010.
References
4.2. Preparation of 6-methyl-6H-7-oxopyrido[1,2-a]pyrimido[5,4-
c]-1,2-benzothiazine-5,5-dioxide (3)
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(40 mL) was added benzoyl chloride (2.0 g, 0.014 mol) followed by
triethylamine (2.0 mL, 0.014 mol) dropwise at room temperature.
The mixture was then stirred at 30 ꢀC for 24 h, poured into ice and
extracted with chloroform (2ꢂ 25 mL). The organic layers were
collected, washed with 5% NaOH (20 mL) solution followed by 5%
HCl (20 mL) and then with water (2ꢂ 30 mL), dried over anhydrous
Na2SO4, filtered and concentrated. The residue was then treated
with methanol and the insoluble solid was isolated by filtration to
afford the title compound 3 (1.0 g, 50% yield) as a light yellow
powder, mp 282 ꢀC (lit. [3] 286–287 ꢀC).
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The compound 3 crystallizes in the monoclinic space group P21/
c with Z ¼ 4. The intensity data were recorded using a Bruker
SMART CCD area-detector diffractometer with graphite mono-
chromated MoKa radiation (
l
¼ 0.71073 Å). The crystal structure
was solved by direct methods, SHELXS97 [8] and refined on F2 using
SHELXL97 [8]. The hydrogen atoms associated with the carbon
atoms were positioned geometrically and hydrogen atoms of the