Photochemical and DNA degradation studies on tenoxicam, lornoxicam, and their photolysis products

Article abstract of DOI:10.1007/s00706-016-1765-z

Tenoxicam and lornoxicam are nonsteroidal anti-inflammatory drugs, were subjected to photoirradiation at 254?nm led to the photodegradation of the pharmaceutical agents. Both, the isolated photodegradation products and the pharmaceutical agents were exami

Full text of DOI:10.1007/s00706-016-1765-z

Monatsh Chem
DOI 10.1007/s00706-016-1765-z
ORIGINAL PAPER
Photochemical and DNA degradation studies on tenoxicam,
lornoxicam, and their photolysis products
1
1,2
1,2
•
Ibrahim Elghamry
1
•
Usama El-Ayaan
3
•
Magdy M. Youssef
4
Shar Al-Shihry
•
Matthias Letzel
•
Jochen Mattay
Received: 14 February 2016 / Accepted: 17 April 2016
Ó Springer-Verlag Wien 2016
Abstract Tenoxicam and lornoxicam are nonsteroidal
anti-inflammatory drugs, were subjected to photoirradia-
tion at 254 nm led to the photodegradation of the
pharmaceutical agents. Both, the isolated photodegradation
products and the pharmaceutical agents were examined
toward DNA binding and degradation. The photodegrada-
tion products degrade calf thymus in concentration
dependent manner.
Introduction
Oxicams are nonsteroidal anti-inflammatory drugs
(NSAIDs) which are used for the treatment of rheumatic
arthritis, osteoarthritis, and post-operative inflammations
[1, 2]. Several other functions of this group of drugs have
been reported which include UV-protection and sensitiza-
tion, chemoprevention and chemosuppression of cancer,
and finally they function as effective anti-oxidants [3–7]. In
the year 1980, piroxicam (PRX, 4-hydroxy-2-methyl-N-
Graphical abstract
(
pyridinyl-2-yl)-2H-1,2-benzothiazine-3-carboxamide 1,1-
2
1
dioxide; Scheme 1, R = Me, R = CONH-2-pyridinyl)
was launched into the European market for clinical use as a
lead compound of the oxicams [8]. Since then, new
members of these pharmaceutical agents have been syn-
thesized based on structure–activity relationships (SARs)
and the isosteric substitution concept, which starts by
substituting the lead compound with moieties of similar
stereoelectronic features which might improve the phar-
macological efficacy and other pharmacokinetic properties
Keywords Oxicams Á Photostability Á Photodegradation Á
Zwitterionic effect Á DNA degradation
[
9].
Shortly after the pharmaceutical agent became widely
available for the therapeutic use, it was associated with the
development of adverse light-induced draw-back which
was explained in terms of phototoxicity of either the pho-
toproducts or the metabolite end products of the drug
[10–14]. Therefore, studying the photochemical and pho-
tophysical properties and of course the photostability of the
oxicams were the main concern of many literature reports
[15–17]. Also, contradictory data concerning the photo-
stability of the oxicams can be found in the literature.
While piroxicam was proved to be stable to photolysis even
after prolonged exposure to light [13, 14, 18], tenoxicam
showed 50 % loss of the drug when exposed to sunlight for
3 h [19]. On the other hand, it was reported that direct
&
Ibrahim Elghamry
ielghamry@kfu.edu.sa
1
Department of Chemistry, College of Science, King Faisal
University, P. O. Box 400, Al Hufuf, Al Ahsa 31982,
Saudi Arabia
2
3
4
Department of Chemistry, Faculty of Science, Mansoura
University, Mansoura 35516, Egypt
Organisch-Chemisches Institut, Westf. Wilhelms-Universit a¨ t
M u¨ nster, Corrensstraße 40, 48149 M u¨ nster, Germany
Organische Chemie I, Fakult a¨ t f u¨ r Chemie, Universit a¨ t
Bielefeld, 33501 Bielefeld, Germany
123

I. Elghamry et al.
Scheme 1
Scheme 2
irradiation of piroxicam in dichloromethane afforded two
main photodegradation products namely, N-methylsaccha-
rin (2) (Scheme 1, pathway I) and N-(2-pyridinyl)oxamic
acid [20]. This was explained in terms of a plausible
mechanism via formation of dioxetane intermediate
2-pyridinylcarboxamide group in the piroxicam is replaced
by cyano or ester groups in the analogues. Furthermore, the
piroxicam (PRX) is benzothiazine oxicam, while tenoxi-
cam (TNX) and lornoxicam (LRX) are thienothiazine
oxicams (Scheme 2). All of these oxicams have the thi-
azine ring with the same substitution pattern as
2-pyridinylcarboxamide at position three in the thiazine
ring. This prompted us to study the photochemical behavior
of tenoxicam and lornoxicam under the same conditions
which could reveal some answers and explanation for what
seems a contradicted photochemical behavior of this group
of compounds.
[
20, 21]. Furthermore, in another literature report, when a
methanolic solution of piroxicam was subjected to pho-
tolysis, two more products beside N-methylsaccharin were
0
isolated and identified as N-methyl-N -(2-pyridinyl)ox-
alamide (3) and methyl (2-pyridinylcarbamoyl)formate (4)
(
Scheme 1, pathway I) [22].
In a previous study, we reported a different photo-
chemical behavior for this ring system. When 3-substituted
-hydroxy-1,2-benzothiazine-1,1-dioxides were subjected
to 254 nm aerobic/anaerobic irradiation in methanol for
h, a single photoisomerization product was obtained in
4
Results and discussion
7
moderate yield 75 % (Scheme 1) which was demonstrated
to be the 2-substituted 4-oxo-1,3-2H-benzothiazine-1,1-
dioxides 5 [23] (Scheme 1, pathway II). This was
explained in terms of two-step free radical mechanism via
homolytic cleavage at S–N bond in the thiazine ring.
The obvious question that arises, if they both have a
common benzothiazine ring system and photolyzed under
the same aerobic conditions with the same solvent and
wavelength, why do we have two different photochemical
behaviors? Such question has no straight forward answer.
But from the chemical structure point of view, the only
difference between the piroxicam and the analogue com-
pounds is the substitution pattern in the thiazine ring. The
When the methanolic solution of tenoxicam (TNX) and
lornoxicam (LRX) is irradiated at 254 nm for 7 h, five
main photodegradation products were identified namely N-
0
methyl-N -(pyridin-2-yl)oxalamide (3), methyl(pyridin-2-
0
ylcarbamoyl)formate (4), N-methyl-N -(pyridin-2-yl car-
bamoyl)methanethioamide (6), N-methyl-3-oxothieno[3,2-
d]-1,2-thiazole-1,1-dioxides 7a, 7b and methyl thiophene-
2-carboxylates 8a, 8b (Scheme 2).
The structures of the photodegradation products were
1
13
assigned by H NMR, C NMR, 2D-NMR, IR, and GC–
MS. Moreover, UV–Vis spectra for both tenoxicam (TNX),
lornoxicam (LRX), and the isolated photodegradation
products were measured (Fig. 1). A plausible mechanism
1
23

Photochemical and DNA degradation studies on tenoxicam, lornoxicam, and their photolysis…
yl)oxalamide (3) and methyl thiophene-2-carboxylates 8a,
b [24].
8
However, formation of photoproduct 6 as one of the aerobic
photolysis products of tenoxicam and lornoxicam (Scheme 2)
is quite abnormal and unexpected for such photodegradation,
and was not detected or reported as one of the photolysis, oxi-
dation or metabolites of such oxicams [25, 26]. The chemical
1
structure of compound 6 was assigned and confirmed by H
1
3
NMR, C NMR, 2D-NMR, IR, and GC–MS.
First, the other isomeric structures for compound 6 were
excluded by closer look into the MS fragmentation pattern
(
Fig. 2) which revealed fragment at m/z = 121 attributed
?
to [PyrNHCO ], as a base peak followed by a peak at m/
?
z = 166 attributed to [PyrNHCOCS ].
Second, the only difference between structure 3 and 6 is
that, one of the carbonyl oxygen atoms in 3 is replaced by
one sulfur atom. The mechanism of formation of this
product is not clear but its formation only on photolysis of
tenoxicam and lornoxicam reveals that, the thiophene ring
has a role in its formation as a source of sulfur. On the
other hand, the participation of the other sulfur atom in the
thiazine ring system in the formation of this product is
excluded based on compound 6 was not detected or iso-
lated on the photolysis of piroxicam [20–22].
Fig. 1 UV–Vis spectra of TNX, LRX, and photodegradation prod-
ucts 3, 4, 6 in methanol
explaining the formation of these products is depicted in
Scheme 3.
Photoexcited oxicam reacts with singlet oxygen at
C =C in the thiazine ring to form dioxetane intermediate
3
4
The formation of the other photodegradation products
9
1
. Subsequent dioxetane ring opening leads to intermediate
(
Scheme 3) is compatible with the previously reported
0. The later intermediate undergoes either nucleophilic
photolysis pattern of piroxicam in term of chemical struc-
ture similarity between the three pharmaceutical agents
attack by the solvent lone pair to form methyl (pyridin-2-
ylcarbamoyl)formate (4), and N-methyl-3-oxothieno[3,2-
d]-1,2-thiazole-1,1-dioxides 7a, 7b or extrusion of sulfur
[
20–22]. Piroxicam, tenoxicam, and lornoxicam have
benzothiazine ring with 2-pyridinyl carboxamide and
hydroxyl groups at position 3 and 4, respectively, show
0
dioxide and methylation to yield N-methyl-N -(pyridin-2-
Scheme 3
123

I. Elghamry et al.
Fig. 2 Mass spectrum of
compound 6
different tautomeric forms due to fast internal proton
transfers between the hydroxyl group and the 2-pyridinyl-
carboxamide group in the thiazine ring. This might cause
unusual sensitive behavior to any changes in, chemical
substitution, solvent and temperature [27, 28].
Pharmacology
The competency of the isolated photolysis products 3, 4,
and 6 to have aptitude to interact and cleave calf thymus
DNA was assessed and interrelated to lornoxicam (LRX)
and tenoxicam (TNX) as controls. Nonsignificant degra-
dation on the calf thymus DNA was recorded at 2 lM
concentration of both the photolysis products and the
controls as shown in Fig. 3a. At 4 lM concentration the
tenoxicam exhibited a considerable degradation effect on
the tested calf thymus DNA, Fig. 3b while, the photolysis
products 3, 4, 6 and the lornoxicam displayed a feeble
DNA degradation at the same concentration. The tenoxi-
cam completely degrade the calf thymus DNA at 6 lM
concentration, while, lornoxicam and the photolysis prod-
ucts 3, 4, 6 at 6 lM concentration displayed a considerable
degradation activities on the tested DNA (Fig. 3c).
Thus, piroxicam, tenoxicam, and lornoxicam show a zwit-
13
terionic nature in polar organic solvents as demonstrated by C
NMR spectroscopy and obtained results from molecular orbital
calculations [21, 29]. Therefore, by increasing the charge
density on C =C in the thiazine ring, the electrophilic attack of
3
4
singlet oxygen is enhanced leading to the formation of the
dioxetane intermediate 9 as a key step in the photodegradation
process (Scheme 1, pathway I). On the other hand, benzoth-
iazines with other substitution pattern at position 3 like CN or
CO Me reacted differently and gave the photoisomerization
2
product 5 via homolytic cleavage at S–N bond in the thiazine
ring (Scheme 1, pathway II). To summarize these results, in the
presence of oxygen, photodegradation (photo-oxidation)
mechanism is preferred over the photoisomerization mecha-
nism for oxicams with the 2-pyridinylcarboxamide group at
position three in the thiazine ring.
The tenoxicam has higher degradation activity on the
calf thymus DNA than the lornoxicam at 6 lM concen-
tration. The lower degradation effect of lornoxicam on the
DNA may be due to electronic effect of the chlorine atom
as an electron withdrawing moiety in the thiophene ring of
lornoxicam. It was reported that the presence of an electron
withdrawing groups in the chemical structure decrease the
DNA degradation activities [30].
To support these interpretations tenoxicam and
lornoxicam were subjected to anaerobic photolysis under
the same conditions from solvent and wavelength. There-
fore, the methanolic solutions of the pharmaceutical agents
were purged with argon for 1 h prior to photolysis at
wavelength 254 nm, and the photolysis process was mon-
itored by GC–MS. Neither the above photodegradation nor
the photoisomerization products were detected in the
photolysis solution during the course of photolysis.
The photolysis products 3, 4, 6 and the tested controls
have a powerful degradation effect on calf thymus DNA at
8 lM concentration (Fig. 3d). It is obvious from the results
that photolysis products 3, 4, 6 and tested controls bind and
interact with the DNA in a concentration dependent
1
23

Photochemical and DNA degradation studies on tenoxicam, lornoxicam, and their photolysis…
Experimental
The NMRs were recorded on a 500 MHz NMR spectrometer
Bruker, DRX500). EI GC/MS spectra were recorded using a
(
Shimadzu GC17A/GCMS-QP 5050A mass spectrometer
equipped with a standard EI source. EI mass spectra were
recorded using an Autospec X magnetic sector mass spec-
trometer with EBE geometry (Vacuum Generators,
Manchester, UK) equipped with a standard EI source. Sam-
ples were introduced by push rod in Aluminum crucibles.
Ions were accelerated by 8 kV. The spectra shown here were
recorded and processed with the opus software (V3.6
Micromass 1998) by the accumulation and averaging of
several single spectra. UV/Vis spectra were recorded on a
Perkin Elmer Lambda 40 UV/Vis spectrometer. Melting
points were measured on a Buchi B-540. Photodegradation
was performed with a RPR-100 Rayonet Photochemical
Chamber Reactor (Southern New England Ultraviolet Com-
pany, Branford, USA) at 254 nm using quartz tubes.
Tenoxicam and lornoxicam were purchased from Kemprotec
Limited United Kingdom and used directly without any fur-
ther purification. All the chromatographic separations
were performed on 20 9 20 cm or 5 9 20 cm Merck glass
plates covered with silica gel 60 F254 layer thicknesses
Fig. 3 A figure presenting the degradation effect of 2 lM (a), 4 lM
b), 6 lM (c), and 8 lM (d) of photolysis products 3, 4, and 6 on the
calf thymus DNA. Lane 1 DNA; lane 2 DNA DMSO; lane 3
lornoxicam (LRX); lane 4 tenoxicam (TNX); lanes 5, 6, and 7
photolysis products 3, 4, and 6, respectively
(
manner. Binding and interacting activities of the photolysis
products with calf thymus DNA have encouraged compe-
tency to performance as a nuclease like activities and
degrade calf thymus DNA. The existing study verifies that
both the photolysis products and tenoxicam have signifi-
cant degradation activities on calf thymus DNA without
any addition. The calf thymus DNA-binding assessments
are significant for the rational approach and get-together of
novel and additional-effective medicines directing the
heritable ingredients in eukaryotes. The DNA degradation
competence in the absence of any chemical is an appreci-
ated feature for TNX, 3, 4, and 6 which delivers a possible
demand of these compounds as chemotherapeutic appli-
cation in antitumor treatments.
0.25 mm.
General photolysis procedure
Tenoxicam (TNX) and lornoxicam (LRX) 100 mg were
3
dissolved in 100 mm methanol and irradiated at 254 nm
in a RPR-100 Rayonet Photochemical Chamber Reactor
for 7 h. After concentration, the residue was subjected to
chromatography on silica gel plates with 1 % methanol in
chloroform. The R values of the appropriate zones are
f
given below.
0
N-Methyl-N -(pyridin-2-yl)oxalamide (3)
It was obtained in 18–22 % conversion as colorless powder
after sublimation at 60–65 °C under 60 mbar vacuum, and
crystallization from diethyl ether and n-pentane (1:1).
Rf = 0.33; m.p.: 120–121 °C ([24, 25] 121 °C).
Conclusion
The photochemical behavior of oxicams and analogues is
very sensitive and depends on: (1) the type of the sub-
stituent on position three in the thiazine ring; (2) there is
a correlation between the 2-pyridinylcarboxamide group
at this position and the photo-oxidation mechanism. The
behavior of lornoxicam and tenoxicam is nearly com-
patible with that of piroxicam, except formation of
product 6 which is a novel photolysis product of oxi-
cams. The photolysis products 3, 4, 6, lornoxicam, and
tenoxicam bind and interact with the DNA in a con-
centration dependent manner and a powerful degradation
effect on calf thymus DNA at 8 lM concentration was
noticed.
Methyl (pyridin-2-ylcarbamoyl)formate (4)
It was obtained in 12–15 % conversion as colorless powder
after sublimation at 60–65 °C under 60 mbar vacuum, and
crystallization from diethyl ether and n-pentane (1:1).
Rf = 0.63; m.p.: 102–103 °C ([24, 25] 102 °C).
0
N-Methyl-N -(pyridin-2-ylcarbamoyl)methanethioamide
(
6, C H N OS)
8 9 3
It was obtained in 12–14 % conversion as pale yellow
powder after sublimation at 60–65 °C under 60 mbar
vacuum, and crystallization from diethyl ether and n-
1
pentane (1:1). R = 0.78; m.p.: 133–134 °C; H NMR
f
3
(
CDCl ): d = 3.32 (d, J = 5 Hz, 3H, CH ), 7.14 (dd,
3
3
123

I. Elghamry et al.
3
3
3
3
J = 5 Hz, J = 5 Hz, 1H), 7.78 (dd, J = 8 Hz,
3
2. Kochevar IE, Morison WL, Lamm JL, McAuliffe DJ, Western A,
Hood AF (1986) Arch Dermatol 11:1283
3
J = 8 Hz, 1H), 8.22 (d, J = 8 Hz, 1H), 8.41 (d,
3
4
. Sporn MB, Suh N (2000) Carcinogenesis 21:525
. Ritland SR, Gendler SJ (1999) Carcinogenesis 20:51
J = 5 Hz, 1H), 9.56 (s, 1H, NHCH ), 10.59 (s, 1H, NH)
3
1
3
ppm; C NMR (CDCl ): d = 33.1 (NHCH ), 113.7 (CH),
1
5. Grossman ME, Longo WE, Panesar N, Kaminiski J (2000) Car-
cinogenesis 21:1403
3
3
20.8 (CH), 138.4 (CH), 148.5 (CH), 150.0 (CNHCO),
6
. Goldman AP, Williams CS, Sheng H, Lamps LW, Williams VP,
Pairet M, Morrow JD, DuBois RN (1998) Carcinogenesis
1
56.2 (CNHCO), 186.4 (CSNHCH ) ppm; IR (KBr):
3
-
V = 3265 (NH), 1683 (C=O), 1059 (C=S) cm ; MS: m/
1
1
9:2195
?
?
z (%) = 197 ([M ? 2] , 1), 196 ([M ? 1] , 2), 195
7. Vartiainen N, Huang CY, Salminen A, Goldsteins G, Chan PH,
Koistinaho J (2001) J Neurochem 62:480
8. Lombardino JG, Lowe JA (2004) Nat Rev Drug Discov 3:853
?
[M] , 14), 166 ([PyrNHCOCS] , 35), 121 ([PyrNHCO] ,
?
?
(
?
?
1
00), 94 ([PyrNH ] , 17), 78 ([Pyr] , 30), 74 ([CH
2 3-
9
. Roche VF (2009) Am J Pharm Educ 73:143
?
NHCS] , 23), 67 (14).
1
0. Serrano G, Aliaga JA, Gargallo E, Pelufo C (1984) J Am Acad
Dermatol 11:113
1. Fjellner B (1983) Acta Derm Venereol (Stockh) 63:557
2. Diffey BL, Diamond TJ, Fairgreaves H (1983) Br J Rheumatol
22:239
N-Methyl-3-oxothieno[2,3-d]-1,2-thiazole-1,1-dioxide
1
1
(7a)
It was obtained in 8–10 % conversion as white crystals from
1
3. Gon c¸ alo M, Figueiredo A, Tavares P, Fontes Ribeiro CA, Teix-
eira F, Polares Baptista A (1992) Contact Dermat 27:287
methanol/H O (1:1). M.p.: 141 °C ([32] 142–143 °C).
2
5
-Chloro-N-methyl-3-oxothieno[2,3-d]-1,2-thiazole-1,1-
14. Figueiredo A, Fontes Ribeiro CA, Goncalo S, Caldeira MM,
Polares-Baptista A, Teixeira FC (1987) Contact Dermat 17:73–75
dioxide (7b)
It was obtained in 7–10 % conversion as off white crystals
from ethanol. M.p.: 161 °C ([32, 33] 159 °C).
1
5. Bartsch H, Eiper A, Habiger K, Kopelent-Frank H (1999) J
Chromatogr A 846:207
16. Becker RS, Chakravorti S, Minjoong Y (1990) Photochem Pho-
tobiol 51:151
Methyl thiophene-2-carboxylate (8a)
It was obtained in 8–10 % conversion as viscous oil. B.p.:
1
1
1
7. Andrade SM, Costa SM, Pansu R (2000) Photochem Photobiol
71:405
8. Mihalic M, Hofman H, Kajfez F, Kuftinec J, Blazevic N, Zinic M
8
0–82 °C/10 mbar ([34] 120–121 °C/13 mbar, [35]
0–44 °C/0.7 mbar).
(
1982) Acta Pharma Jugosol 32:13
4
9. Heizmann P, K o¨ rner J, Zinapold K (1986) J Chromatogr Biomed
Appl 374:95
Methyl 5-chlorothiophene-2-carboxylate (8b)
It was obtained in 9–10 % conversion as viscous oil. B.p.:
2
2
0. Miranda MA, Gunther F (1991) J Photochem Photobiol B 8:199
1. Lemp E, Zanocco AL (2001) J Photochem Photobiol B 65:165
9
9
5–96 °C/9 mbar ([34] 87–88 °C/7 mbar, [36] 95–97 °C/
mbar).
22. Glass BD, Brown ME, Daya S, Worthington MS, Drummond P,
Antunes E, Lebete M, Anoopkumar-Dukie S, Mahrajmm D
(
2001) Int J Photoenergy 3:205
23. Elghamry I, Doepp D, Henkel G (2007) J Heterocycl Chem
4:849
DNA degradation and agarose gels electrophoresis
The photolysis products 3, 4, and 6 in DMSO were added
independently to 1 lg of calf thymus DNA (life technolo-
gies) and incubated at 37 °C for 60 min. The lornoxicam
4
2
2
2
2
4. Elghamry I, Letzel M, Mattay J (2012) Trends Photochem Pho-
tobiol 14:21
5. Ichihara S, Tomisawa H, Fukazawa H, Tateishi M, Joly R, Heintz
R (1989) Drug Metab Dispos 17:463
6. Nascimento AL, Escobar JA, Cilento G (1993) Photochem Pho-
tobiol 57:362
7. Tasi R-S, Carrupt PA, Etayar N, Giroud Y, Andrade P, Testa B,
Br e´ e F, Tillement JP (1993) Helv Chim Acta 76:842
(
LRX) and the tenoxicam (TNX) were used as parental and
controls for this assay. The DNA was examined by
utilizing agarose gels electrophoresis [31]. The elec-
trophoresis performance was achieved through 1 % (w/v)
agarose gels in Tris–acetate-EDTA buffer (0.04 M Tris–
HCl, 5 mM sodium acetate, and 1 mM EDTA (pH 8). The
28. Bordner J, Hammen PD, Whipple EB (1989) J Am Chem Soc
11:6572
1
3
29. Tak a´ cs-Nov a´ k K, K o¨ k o¨ si J, Pod a´ nyi B, Nosz a´ l B, Tsai R-S, Lisa
G, Carrupt P-A, Testa B (1995) Helv Chim Acta 78:553
ethidium bromide (0.5 lg/cm ) was added to agarose gels
for staining. The DNA in agarose gels was visualized with
3
0. Ismail MA, Arafa RK, Youssef MM, El-Sayed WM (2014) Drug
Des. Dev Ther 8:1659
1. Youssef MM, Al-Omair MA, Ismail MA (2012) Med Chem Res
UV transilluminator and photographed with digital camera.
3
2
1:4074
Acknowledgments The authors are grateful to the Deanship of Sci-
entific Research at King Faisal University for the financial support
3
3
2. Unterhalt B, Moghaddam S (1994) Pharmazie 49:317
3. Modhave D, Handa T, Shah R, Singh S (2011) J Pharm Biomed
Anal 56:538
(
Project Number 160025).
3
4. Khusnutdinov R, Schandneva N, Bayguzina A, Matkova Y,
Smirnov A, Burangulova R, Dzhemilev U (2004) Arkivoc (xi):53
5. Penga M, Wang PC (1984) Synth Commun 14:77
6. Hurd CD, Kreuz K (1952) J Am Chem Soc 74:2965
References
3
3
1
. Brogden RN, Heel RC, Speight TM, Avery GS (1984) Drugs
8:292
2
1
23