Scavenging activity of aminoantipyrines against hydroxyl radical

Article abstract of DOI:10.1016/j.ejmech.2010.01.071

The pyrazolone derivatives antipyrine and 4-(N,N-dimethyl)-aminoantipyrine (aminopyrine) have long been used as analgesic, antipyretic and anti-inflammatory drugs. However, in spite of its recognized therapeutic benefits, the use of pyrazolones has been associated with agranulocytosis. Though the oxidation of aminopyrine by neutrophil-generated hypochlorous acid (HOCl), leading to the formation of a cation radical, has been considered responsible for the potential bone marrow toxicity, the reaction mechanisms of pyrazolones against other reactive oxygen species (ROS) remains elusive. Thus, the reactions of 4-aminoantipyrine and methylated derivatives with hydroxyl radicals (HO^?) were studied as a model of their reactivity against ROS. The results show that 4-(N,N-dimethyl)-aminoantipyrine (aminopyrine) undergoes demethylation when reacting with HO^· radical, leading to 4-(N-methyl)-aminoantipyrine, which is further demethylated to 4-aminoantipyrine. In addition, it was also observed that another favorable reaction of 4-aminoantipyrines in these conditions is the hydroxylation on the aromatic ring, a reaction that is common to aminopyrine, 4-(N-methyl)-aminoantipyrine, and 4-aminoantipyrine. Whether these reaction mechanisms give rise to harmful reactive intermediates requires further chemico-biological evaluation.

Full text of DOI:10.1016/j.ejmech.2010.01.071

European Journal of Medicinal Chemistry 45 (2010) 2258–2264
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Scavenging activity of aminoantipyrines against hydroxyl radical
,
Pedro M.P. Santos a, Alexandra M.M. Antunes b, Joa˜o Noronha a, Eduarda Fernandes c, Abel J.S.C. Vieira a
*
a
´
ˆ
REQUIMTE, CQFB, Departamento de Quımica, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, 2829-516 Caparica, Portugal
b
c
´
´
´
Centro de Quımica Estrutural, Instituto Superior Tecnico, Universidade Tecnica de Lisboa, 1049-001 Lisboa, Portugal
REQUIMTE, Departamento de Qu´ımica-F´ısica, Faculdade de Farmacia, Universidade do Porto, Rua Anıbal Cunha, 164, 4099-030 Porto, Portugal
´
´
a r t i c l e i n f o
a b s t r a c t
Article history:
The pyrazolone derivatives antipyrine and 4-(N,N-dimethyl)-aminoantipyrine (aminopyrine) have long
been used as analgesic, antipyretic and anti-inflammatory drugs. However, in spite of its recognized
therapeutic benefits, the use of pyrazolones has been associated with agranulocytosis. Though the
oxidation of aminopyrine by neutrophil-generated hypochlorous acid (HOCl), leading to the formation of
a cation radical, has been considered responsible for the potential bone marrow toxicity, the reaction
mechanisms of pyrazolones against other reactive oxygen species (ROS) remains elusive. Thus, the
reactions of 4-aminoantipyrine and methylated derivatives with hydroxyl radicals (HOꢀ) were studied as
a model of their reactivity against ROS. The results show that 4-(N,N-dimethyl)-aminoantipyrine
(aminopyrine) undergoes demethylation when reacting with HO$ radical, leading to 4-(N-methyl)-ami-
noantipyrine, which is further demethylated to 4-aminoantipyrine. In addition, it was also observed that
another favorable reaction of 4-aminoantipyrines in these conditions is the hydroxylation on the
aromatic ring, a reaction that is common to aminopyrine, 4-(N-methyl)-aminoantipyrine, and 4-ami-
noantipyrine. Whether these reaction mechanisms give rise to harmful reactive intermediates requires
further chemico-biological evaluation.
Received 3 September 2009
Received in revised form
19 January 2010
Accepted 21 January 2010
Available online 2 February 2010
Keywords:
Antipyrines
Radical oxidation
Hydroxylation
Demethylation
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
In spite of its recognized therapeutic benefits, the use of pyr-
azolones has been associated with potential adverse effects char-
The pyrazolone derivatives antipyrine (1) and 4-(N,N-dimethyl)-
aminoantipyrine (aminopyrine; 4) (Fig. 1) have been used as
analgesic, antipyretic and anti-inflammatory drugs, since the end of
the 19th century [1]. These compounds are strong inhibitors of
cycloxygenase isoenzymes, platelet tromboxane synthesis, and
prostanoids synthesis [1]. The biological activity of these
compounds has also been attributed to its scavenging activity
against reactive oxygen and nitrogen species (ROS and RNS), as well
as to the inhibition of neutrophil’s oxidative burst. Indeed,
aminopyrine was demonstrated to be a highly efficient scavenger of
the ROS hydroxyl radical (HOꢀ) [1], hypochlorous acid (HOCl) [1–3],
peroxyl radical (ROOꢀ) [1], and singlet oxygen (1O2) [4] and of the
RNS nitric oxide (ꢀNO) and peroxynitrite (ONOOꢁ) [5], as well as to
effectively prevent the phorbol-12-myristate-13-acetate-induced
neutrophil oxidative burst [1]. Antipyrine was shown to be the
most effective pyrazolone in scavenging ROOꢀ, though it was inef-
ficient against the other ROS and RNS mentioned above, or to the
neutrophil’s oxidative burst [1,4,5].
acterized by leukopenia, most commonly of neutrophils, causing
neutropenia in the circulating blood (agranulocytosis) [6]. Note-
worthy, the oxidation of aminopyrine by neutrophil-generated
HOCl was previously reported, leading to the formation of a blue
cation radical, to which has been attributed the potentially fatal
bone marrow toxicity, leading to agranulocytosis [2,3]. From the
studies performed so far, it has been proposed that the oxidation of
aminopyrine to a radical cation, by HOCl, involves an N-chlorina-
tion followed by loss of a chlorine radical [3]. Another possible
mechanism is the formation of a dication by the loss of chloride ion
from N-chloroaminopyrine [6]. Considering the high reactivity of
aminopyrine with other ROS besides HOCl, as well as with RNS, it
may be considered feasible that potentially harmful reactive
intermediates may also rise from aminopyrine scavenging effects.
However, the reaction mechanisms of pyrazolones against the
other ROS and RNS remains elusive. In addition, the relative
potency of the different pyrazolones concerning their reaction with
ROS and RNS is also hitherto unknown.
The aim of the present study is to evaluate the postulate that the
much higher antioxidant activity of 4 as compared to 1 may be
attributed to the capacity of the former to undergo oxidation fol-
lowed by demethylation in the reaction with free radicals, giving
* Corresponding author. Tel.: þ351 21 2949682x10930; fax: þ351 21 2948550.
E-mail address: ajvieira@dq.fct.unl.pt (A.J.S.C. Vieira).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.071

P.M.P. Santos et al. / European Journal of Medicinal Chemistry 45 (2010) 2258–2264
2259
Fig. 1. Structures of studied antipyrine derivatives.
rise to 4-(N-methyl)-aminoantipyrine (3), which can be further
demethylated to 4-aminoantipyrine (2) (Scheme 1).
detection. In some instances, reaction products isolation by semi-
preparative HPLC was used for further spectroscopic characteriza-
tion by NMR and MS.
Representative HPLC chromatograms, recorded with an isocratic
elution (methanol/ammonium acetate buffer 50 mM; 40:60), of the
reaction mixtures of 4, 3 and 2 are displayed in Fig. 2.
In the case of the oxidation of compound 4 (see Fig. 2a), it was
observed that changing the pH of the Fenton reaction mixture from
1 (data not shown) to 7 didn’t affect significantly the HPLC profile.
The only noticeable difference was that at pH 1 (HCl 0.1 N aqueous
medium) products 2 and 3 were formed after long reaction times
(about one day), while at pH 7 (phosphate buffered solution) these
products could be detected after 5 min of reaction.
With this objective, the reactions of 4-aminoantipyrine and
methylated derivatives with HOꢀ were studied as a model of their
antioxidant activity against reactive oxygen species (ROS). For this
purpose 4-(N-methyl)-aminoantipyrine (3) was synthesized to be
used as standard. Oxidation of aminoantipyrine derivatives by HOꢀ
was then carried out and the reaction products analyzed.
2. Results and discussion
2.1. Synthesis of 4-(N-methyl)-aminoantipyrine (3)
This result can be explained on the basis of the degree of
protonation of the molecule: at pH 1, the exocyclic amino group is
protonated (pKa ¼ 5.0) [8], which will difficult the oxidation on this
part of the molecule.
In order to prove that 3 stemmed from the reaction of 4 with
oxidizing radicals, compound 3 was synthesized to be used as
a standard. This compound was prepared by an adaptation of
a published methodology [7] involving initial formation of 4-
phthalimidoantipyrine, followed by semi-hydrolysis with sodium
hydroxide and subsequent methylation of the sodium salt of 4-(N-
phtalyl)-aminoantipyrine with dimethylsulfate. Acid hydrolysis of
4-(N-methyl-N-phtalyl)-aminoantipyrine gave 3 in 4% overall yield.
The product was fully characterized by mass spectrometry and 1H
and 13C NMR, being the assignment of all signals based on corre-
lations observed in HMQC and HMBC spectra (see Experimental
section).
Compounds 2 and 3 were identified as 4-aminoantipyrine and
4-(N-methyl)-aminoantipyrine, respectively, on the basis of indis-
tinguishable UV spectra, as well as identical retention times of the
corresponding commercially available and synthesized standards,
when recorded under analogous conditions. Due to low chro-
matographic resolution of peak of compound 2 and instability of
compound 3, it was not possible to isolate these compounds to
perform further analysis by 1H NMR and MS. However, it was
possible to confirm their structures by HPLC-MS analysis (Fig. 3).
As can be seen in Fig. 2a, apart the demethylation products three
other main products are formed on the reaction of 4 with HOꢀ (A, B
and C). The respective mass spectra (Fig. 3) show equal molecular
ions compatible with an additional OH group. Separation of this
reaction mixture by semi-preparative HPLC allowed isolation of
these products and their further analysis by spectroscopic methods.
Compounds B and C showed a molecular ion at m/z 247 in the TOF-
MS(EI) spectra and compound A showed a pseudo-molecular ion at
2.2. Oxidation of aminoantipyrine derivatives
by the hydroxyl radical
The reaction of 4-(N,N-dimethyl)-aminoantipyrine (4), 4-(N-
methyl)-aminoantipyrine (3) and 4-aminoantipyrine (2) with the
HOꢀ radical (generated by Fenton reaction) was performed in
aqueous solution, at different pH conditions. The reaction mixtures
were analyzed by HPLC-MS, with UV–vis (photodiode array)
Scheme 1. Oxidative demethylation of 4-(N,N-dimethyl)-aminoantipyrine.

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Fig. 2. HPLC-DAD chromatograms of the reaction mixtures of 4 (a), 3 (b) and 2 (c) with HOꢀ radical at pH 7, performed with a RP18e PurospherÒ column (250–4 mm, 5
mm, Merck)
with an isocratic elution (methanol/ammonium acetate buffer 50 mM; 40:60) and 1.00 mL minꢁ1 flow rate.
m/z 246 when analyzed by MS(ESI-). These data are completely
consistent with the formation of derivatives that resulted from the
insertion of a hydroxyl group in the molecule of 4.
HPLC with UV–vis (photodiode array, Fig. 2b) and mass detection
analysis (Fig. 3) when 3 was reacted with the radical HOꢀ is
a support of the successive demethylation process.
The position of the hydroxyl substitution was established based
on 1H NMR spectra, where the three compounds showed very
similar spectral profiles in the aliphatic region but very distinct
aromatic signals. The 1H NMR spectra of these three products (see
Experimental section)presentedthreesinglets(from3.5 to2.0 ppm)
compatible with the presence of twelve protons from four methyl
groups, indicating that no modification occurred in this part of the
molecule. Thus, structures of these three products were discrimi-
nated on basis of the pattern of 1H NMR signals from aromatic
protons. Product A presented in the 1H NMR spectrum two doublets
(at 6.93 and 6.65 ppm) each one integrating for two protons which is
evidence for para substitution on the aromatic moiety of 4. The 1H
NMR spectrum of product B presented one triplet at 7.32 ppm (40-H),
a doublet at 7.17 ppm (60-H) and a multiplet at 7.02–6.94 ppm
(integrating for two protons) that was simplified to one singlet and
a doublet when the triplet at 7.32 ppm was selectively irradiated.
Taking together, these data are evidence of a substitution in the ortho
position of the aromatic ring. Therefore, C, which presents only two
downfield 1H NMR signals at 7.29 ppm (triplet) and at 6.80–
6.78 ppm (a multiplet correspondent to three protons), results from
hydroxylation in the meta position. The formation of phenolic
derivatives was also observed when aqueous solutions of antipyrine
were submitted to 60Co irradiation [9].
The hydroxylation reaction is common to all compounds 2, 3 and
4. In the chromatograms of Fig. 2b and c some peaks could be
attributed to the corresponding hydroxylation products (assign-
ment not shown). In addition, poly-hydroxylation is also possible; in
the chromatograms 2 a, b and c peaks at retention times lower than
that of compound A correspond to products di- and tri-hydroxylated
and some of them are common to all reaction mixtures studied.
A possible mechanism for the formation of the demethylated
products is the one electron oxidation of the starting compounds
leading to the respective radical cations (Scheme 2). Deprotonation
of this species, with the formation of an aminomethyl radical, fol-
lowed by further oxidation gives the demethylated derivative with
loss of formaldehyde. The ESR spectrum of the radical cation of 4
(Fig. 4) has already been referred in the literature [10–12] and the
loss of a methyl group from a nitrogen atom, in the form of form-
aldehyde, is also a known process that occurs with this compound
under enzymatic oxidation in the presence of hydrogen peroxide
[11–13]. This suggests that HOꢀ radical is the primary oxidizing
species involved, which is confirmed by the results obtained by
gamma radiolysis. Irradiation of N2O saturated aqueous solutions of
4 in a 60Co source (data not shown) gave results very similar to
those obtained with the Fenton reaction described above.
These results suggest that the most favorable reaction between
4 and the HOꢀ radical is the hydroxylation on the aromatic ring.
There is also evidence that 4 undergoes demethylation leading to 3,
which is further demethylated to 2. The fact that 2 is detected by
2.3. Conclusions
From the obtained results, it may be concluded that one of the
possible pathways for the reaction between 4-(N,N-dimethyl)-

P.M.P. Santos et al. / European Journal of Medicinal Chemistry 45 (2010) 2258–2264
2261
Fig. 3. Mass spectra (ESIþ) of demethylation products (2, 3) and hydroxylation products (A, B, C) from reaction of 4 with hydroxyl radical at pH 7 (HPLC profile shown in Fig. 2a).
aminoantipyrine (4) and the HOꢀ radical is the hydroxylation on the
aromatic ring. This reaction, common to compounds 2, 3 and 4, has
been observed for aromatic iso- and heterocyclic systems, namely
from biologically relevant compounds [14–16].
information about the putative reactivity of the studied pyr-
azolones and HOꢀ. Thus, in the event of oxidative stress-related
inflammatory processes, where HOꢀ may be involved, the therapy
with pyrazolone NSAIDs implies the hydroxylation of the aromatic
ring, as well as the demethylation reactions. These byproducts
should be taken into account, either for their putative toxicity or
the contribution to healing effects. The demethylation reactions can
probably play a major biological role, as compared to the hydroxy-
lation reaction, since it may configure an antioxidizing cascade in
which compounds 4, 3 and 2 all behave as antioxidants.
There is also evidence that compound 4 undergoes a new reaction
consisting in
a demethylation leading to 4-(N-methyl)-amino-
antipyrine (3), which is furtherdemethylated to 4-aminoantipyrine (2).
The pathophysiological importance of the observed reaction
products and possible reactive intermediates is hitherto unre-
ported, though these results add a significant amount of new

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P.M.P. Santos et al. / European Journal of Medicinal Chemistry 45 (2010) 2258–2264
Scheme 2. Reaction mechanism of successive demethylation of 4 and formation of hydroxylated derivatives.

P.M.P. Santos et al. / European Journal of Medicinal Chemistry 45 (2010) 2258–2264
2263
Fig. 4. ESR spectrum of 4-(N,N-dimethyl)-aminoantipyrine radical cation obtained by Fenton reaction (4 30 mM, H2O2 30 mM, FeSO4 0.5 mM, pH 1).
3. Experimental section
chromatograph was equipped with a silica capillary column
(30 m ꢃ 0.32 mm i.d.; df: 0.25 m) covered with 5% phenyl-
m
3.1. Separation and spectroscopic techniques
polisiloxane/95% dimethylpolisiloxane (DB-5ms, Agilent-J&W
Scientific). GC temperatures: 200 ꢂC isothermal 3 min, 3 ꢂC/min–
250 ꢂC. The Helium flow was 1.5 mL/min and the head pressure was
85 kPa. The ion source and the transference line were kept at 150
and 220 ꢂC respectively. The MS spectra were obtained by Elec-
tronic Impact (EI) at 70 eV by using MassLabÔ software (Micro-
mass). The spectral mass/charge range used was 50–350 Da.
1H NMR and 13C NMR spectra were recorded on a Bruker ARX
400, operating at 400 and 100.62 MHz, respectively. Chemical shifts
(d) are reported in ppm downfield from tetramethylsilane, and
coupling constants (J) are reported in Hz.
Infrared (IR) spectra were recorded on a Perkin Elmer 683 FT-IR
spectrometer; group frequencies are reported in cmꢁ1
.
Reverse phase chromatography RP18 (LiChrospherÒ, column
250–4 mm, 5 m, Merck) of reaction mixtures was performed with
3.2. Preparation of 4-(N-methyl)-aminoantipyrine
m
DAD (Diode Array Detector) detection from 200 to 600 nm with
a 1.0 mL/min flow, using a well degassed mobile phase with
methanol (20–80%)/ammonium acetate buffer 50 mM (80–20%)
with a gradient elution. HPLC-DAD-ESI-MS were carried out in
a HPLC system (Thermo Surveyor) and ESI-ion trap MS system with
a LCQ ion trap mass spectrometer (Thermofinnigan) equipped with
electrospray source and run by Xcalibur (Thermofinnigan) version
1.3 software. The spectral mass/charge range used was 50–1000 Da.
The sample analyzed by mass spectroscopy was dissolved in
methanol and delivered by an infusion pump (Harvard Apparatus)
at 0.6 mL/h to a quadrupole VG Platform (Micromass, UK Ltd)
spectrometer equipped with an electrospray ionization source
operating in negative mode. Capillary temperature was kept
between 100 ꢂC and 120 ꢂC, using a cone voltage of 140 V and
capillary voltage of 3.5 kV. Nitrogen was used as drying and
nebulizing gas at 300 L/h and 10 L/h, respectively. Spectral mass/
charge range used was 150–300 Da.
4-(N-Methyl)-aminoantipyrine (4% overall yield) was prepared
by adaptation of a published technique [7] as a yellow oil. IR
nmax(NaCl)/cmꢁ1 1651 (C]O). NMR dH(CDCl3) 7.38–7.32 (4 H, m,
Ph), 7.15 (1 H, t, J 6.9, 40-H), 2.77 (3 H, s, 1-Me), 2.73 (3 H, s, N4-Me),
2.16 (3 H, s, 5-Me); dC(CDCl3) 162.7 (C3), 140.2 (C5), 135.7 (C10),
129.5 (C30 þ C50), 124.4 (C40), 123.4 (C20 þ C60), 38.1 (1-Me), 35.1
(N4-Me), 11.3 (5-Me). GC–MS m/z (EI) 217 (Mþ, 7%), 202 (0.5,
M ꢁ CH3), 98 (4, M ꢁ C6H5NCO), 83 (28, M ꢁ C6H5NCO–CH3), 77 (7,
C6H5), 56 (100, CH3CNCH3).
3.3. Oxidation of 4-(N,N-dimethyl)-aminoantipyrine by HOꢀ
(Fenton reaction)
Reaction mixtures at pH 7 were prepared by using the antipy-
rine derivative 10 mM, H2O2 10 mM, FeEDTA 2 mM and ascorbic
acid 1 mM buffered with Na2HPO4 and KH2PO4. At pH 1, solutions
were prepared in an HCl 1 M medium with FeSO4 1 mM as catalyst.
Samples were collected at different reaction times.
Samples analyzed by mass spectroscopy (electron impact) were
delivered by direct probe to a GCT-MS(TOF) (Micromass, UK Ltd)
spectrometer.
For detection of MSTFA derivatized antipyrines by GC/MS the
3.4. Gamma radiolysis
derivatization was carried out in vials with 50 mL of N-methyl-N-
trimethylsilyltrifluoroacetamide (MSTFA) for 30 min at 60 ꢂC. The
separation and detection of the trimethylsilyl derivative of the
antipyrine were achieved using a GC/MS system: GC 6000 (Carlo
Erba Vega Series 2) and MS, QMD1000 Carlo Erba. The gas
Solutions were prepared in MilliQ water saturated with N2O
typically containing the antipyrine derivative 10 mM at pH 7 or pH
1 and were irradiated in a 60Co gamma source with doses ranging
from 0.9 to 2.7 kGy.

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3.5. ESR spectroscopy
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Prof. Joa˜o Paulo Telo (IST) for ESR measurements.
ˆ
Funding from Fundaça˜o para a Ciencia e Tecnologia through
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nucleosides and nucleotides. Characterization of two types of isomeric OH
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2867/200 (PMPS) is also gratefully acknowledged.
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Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2010.01.071.
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