Synthesis and evaluation of antinociceptive and anti-inflammatory effects of nitro-porphyrins

Article abstract of DOI:10.1007/s00044-018-2191-z

This manuscript reports the anti-inflammatory and antinociceptive effects of 4 nitrophenyl-porphyrins: 5,10,15,20-tetra-(3-nitrophenyl)-porphyrin (TNPP), 5,10,15,20-tetra-(4-fluoro-3-nitrophenyl)-porphyrin (TpFNPP), 5,10,15,20-tetra-(4-chloro-3-nitrophenyl)-porphyrin (TpClNPP), and 5,10,15,20-tetra-(4-bromo-3-nitrophenyl)-porphyrin (TpBrNPP). The in vivo anti-inflammatory assays were tested on the acute and chronic TPA (12-O-tetradecanoylphorbol 13-acetate) induced ear edema. The in vitro anti-inflammatory assay was carried out using J774A.1 murine macrophages stimulated with LPS. All nitro-porphyrins decreased inflammation significantly in the acute model: 58.55% (TNPP), 67.49% (TpBrNPP), 67.49% (TpClNPP), and 71.32% (TpFNPP). TpFNPP (50 μM/ml) increased the production of the anti-inflammatory cytokine IL-10, and decreased the production of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in macrophages activated with LPS, with similar activity than indomethacin (50 μM/ml). All porphyrins showed antinociceptive activity and lacked visible toxicity in the acute toxicity test. These results open the possibility of further studies to determine mechanisms of action, and study the influence of the structure on the activity of such compounds.

Full text of DOI:10.1007/s00044-018-2191-z

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2191-z
ORIGINAL RESEARCH
Synthesis and evaluation of antinociceptive and anti-inflammatory
effects of nitro-porphyrins
1 ●
2 ●
1 ●
Juan Ramón Zapata-Morales Cuauhtémoc Pérez-González Angel Josabad Alonso-Castro
2 ●
2 ●
2
Miguel Martell-Mendoza Abigail Hernández-Munive Salud Pérez-Gutiérrez
Received: 21 February 2018 / Accepted: 6 May 2018
©
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
This manuscript reports the anti-inflammatory and antinociceptive effects of 4 nitrophenyl-porphyrins: 5,10,15,20-tetra-(3-
nitrophenyl)-porphyrin (TNPP), 5,10,15,20-tetra-(4-fluoro-3-nitrophenyl)-porphyrin (TpFNPP), 5,10,15,20-tetra-(4-chloro-
3
-nitrophenyl)-porphyrin (TpClNPP), and 5,10,15,20-tetra-(4-bromo-3-nitrophenyl)-porphyrin (TpBrNPP). The in vivo anti-
inflammatory assays were tested on the acute and chronic TPA (12-O-tetradecanoylphorbol 13-acetate) induced ear edema.
The in vitro anti-inflammatory assay was carried out using J774A.1 murine macrophages stimulated with LPS. All nitro-
porphyrins decreased inflammation significantly in the acute model: 58.55% (TNPP), 67.49% (TpBrNPP), 67.49%
(
TpClNPP), and 71.32% (TpFNPP). TpFNPP (50 µM/ml) increased the production of the anti-inflammatory cytokine IL-10,
and decreased the production of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 in macrophages activated with LPS,
with similar activity than indomethacin (50 µM/ml). All porphyrins showed antinociceptive activity and lacked visible
toxicity in the acute toxicity test. These results open the possibility of further studies to determine mechanisms of action, and
study the influence of the structure on the activity of such compounds.
●
●
●
●
Keywords Nitro-porphyrins Antinociception Inflammation TPA Synthesis
Introduction
osteoporosis, and bruising of the skin, with other symptoms
(Bjarnason 2012; Dugowson and Gnanashanmugam 2006).
Inflammation is a basic response to many injuries including
infections and tissue injury (Dung et al. 2009), and it is
characterized by redness, warmth, swelling, and pain.
The pharmacotherapy of inflammatory conditions is
based on the use of steroidal and nonsteroidal anti-
inflammatory drugs (NSAIDs), but these treatments have
severe side effects. NSAIDs may induce gastric and
intestinal ulcers, anemia, and platelet inhibition (Dung et al.
Another major health problem is pain, although this
symptom is associated with a host of ailments, pain per-
ception is subjective. Chronic pain affects about 100 million
adults per a year; this number of people is higher than those
affected by other conditions that receive much more atten-
tion such as heart disease, cancer, and diabetes (Gaskin and
Richard 2011; James 2013; Covarrubias-Gómez 2008).
Today the pain is treated depending on its severity with
some nonsteroidal analgesics. Nevertheless, when these
drugs are administered in chronic inflammatory diseases,
due to side effects of these drugs, search of new agents with
activity antinociceptive and anti-inflammatory continues
2
009; Wolfe et al. 1999), whereas steroidal anti-
inflammatory drugs prevent and suppress inflammation,
but long period of administration induces weight gain,
(Jensen et al. 2001).
As aforementioned, we are currently seeking new ther-
*
Cuauhtémoc Pérez-González
cperezg@correo.xoc.uam.mx
apeutic options, which are just as efficient as the one in use,
but with fewer side effects. Porphyrins are a good option for
the treatment of inflammation. Our working group has
found that some symmetrical porphyrins have anti-
inflammatory and antinociceptive activity (Alonso-Castro
et al. 2015). Among porphyrins, monohydroxy tetra-
phenylporphyrins and manganese porphyrins have
1
Departamento de Farmacia, División de Ciencias Naturales y
Exactas, Universidad de Guanajuato, Guanajuato C.P. 36050,
Mexico
2
Departamento de Sistemas Biológicos, Universidad Autónoma
Metropolitana unidad Xochimilco, Calzada del Hueso 1100,
Colonia Villa Quietud, Coyoacán D.F. 04960, Mexico

Medicinal Chemistry Research
displayed anti-inflammatory and antinociceptive effects
compared to reference drugs (Jelić et al. 2012; Celic et al.
4-fluoro-3-nitrobenzaldehyde: Yellow solid; mp
1
44–47 °C; yield 63%; H NMR (600 MHz, CDCl ), 7.60 (d,
3
2
2
014; Li et al. 2014; Naveed-Umar et al. 2015; Stover et al.
016). Therefore, we have decided to test the anti-
J = 8.01, 1H, Ar–H), 8.26 (d, J = 8.01, 1H, Ar–H), 8.42 (t,
J = 8.01, 1H, Ar–H), 10.1 (s, 1H, –CHO).
inflammatory and antinociceptive capacity of some of the
synthetic porphyrins with a nitro benzene ring.
3-nitrobenzaldehyde: Yellow solid; mp 50–52 °C; yield
1
71%; H NMR (600 MHz, CDCl ), 7.80 (d, J = 8.3, 1H,
3
Ar–H), 8.26 (d, J = 8.3, 1H, Ar–H), 8.65 (t, J = 8.3,1H,
Ar–H), 10.20 (s, 1H, –CHO).
Materials and methods
Synthesis of nitro-porphyrins
Reagents
Freshly distilled pyrrole (0.08 M) was added dropwise into
a refluxing propionic acid (180 ml) solution of the corre-
sponding aldehyde (0.08 M). The reaction mixture was
refluxed for approximately 1.5 h. The solution quickly
changed color from yellow to black. After the reaction was
stopped, the solution was cooled at –4 ºC and left overnight
to allow the precipitation of the porphyrins. After filtration,
the solid was washed with methanol and dried under
vacuum. All porphyrins were preserved in amber flasks to
be protected from light. For all the experiments, PVP was
used as vehicle (Furniss et al. 1989; Adler et al. 1967; Adler
et al. 1968). Figure 2 shows the general synthesis reaction.
The analytical determination of purity of the porphyrins
was carried out using a Varian mod Pro Star 230 high
pressure liquid chromatography (HPLC) system. The HPLC
determination was made with an econosphere silica 250 X
4.6 mm 5 μm column, with an eluent system of chloroform-
ethyl acetate 70–30%, a flow rate of 0.6 ml/min and an
evaporative light scattering detector.
All chemicals used in the synthesis of porphyrins, poly-
vinylpyrrolidone (PVP), 12-O-tetradecanoylphorbol 13-
acetate (TPA), lipopolysaccharide (LPS) (Escherichia coli
0
111:B4), and indomethacin (IND) were purchased from
Sigma (St. Louis, MO, USA). Buprenorphine (BNP) from
Schering Plough Mexico (Distrito Federal, Mexico).
Naproxen sodium was obtained from Tripharma (Distrito
Federal, Mexico).
Porphyrin synthesis
Nitro-aldehydes preparation
The nitration of aldehydes was carried out in a three-necked
flask of 50 ml with magnetic stirrer. H SO 25 ml (0.47 M)
2
4
were cooled to 0 °C, 3.1 ml (0.070 M) of HNO were added,
3
and then the aldehyde (0.06 M) was slowly added. The
reaction was carried out at a temperature of 0–5 °C. Then,
the mixture was heated at 40 °C for 1 h. The reaction was
poured into ice water and filtered under a vacuum; the
nitrobenzaldehydes were purified by recrystallization. The
scheme of reaction is showed in Fig. 1 (Furniss et al. 1989).
The spectrums of H NMR and 13C NMR were obtained
1
in CDCl and TMS as reference.
3
5,10,15,20-tetra-(3-nitrophenyl)-porphyrin (TNPP):
−1
1
IR (ν, cm , solid): 3379 (NH), 1601 (NO ), 1540 (Ar) H
2
1
The spectrums of H NMR of nitrobenzaldehydes were
NMR (600 MHz, CDCl ): -2.84 (s, 2H, N–H), 8.80 (s, 4H,
3
obtained in CDCl and TMS as reference.
Ar–H), 8.08 (s, 8H, H pyrrole-Hβ), 8.71 (m, 12H, Ar–H).
3
1
3
4
-chloro-3-nitrobenzaldehyde: Yellow solid; mp
C NMR (600 MHz, CDCl ): 147.8, 146.6, 135.4, 135.0,
3
1
6
0–62 °C; yield: 70%; H NMR (600 MHz, CDCl ), 7.89
133.4, 129.5, 123.1, 120.1, 120.03. UV–Vis λmax: 423 nm;
yield 13.53%.
3
(
(
d, J = 8.16, 1H Ar–H), 8.25 (d, J = 8.16, 1H, Ar–H), 8.98
t, J = 8.16, 1H, Ar–H), 10.01 (s, 1H, –CHO).
5,10,15,20-tetra-(4-fluoro-3-nitrophenyl)-porphyrin
−
1
4
-bromo-3-nitrobenzaldehyde: Yellow solid; mp
(TpFNPP): IR (ν, cm , solid): 3309 (N–H), 1699 (NO ),
2
1
1
1
8
8
02–1004 °C; yield 55%; H NMR (600 MHz, CDCl ),
1540 (Ar), 1400 (C–F), 917, 840, 734 (Ar trisubstituted). H
3
.17 (d, J = 8.10, 1H, Ar–H), 8.21 (d, J = 8.10, 1H, Ar–H),
.90 (t, J = 8.10, 1H, Ar–H), 9.90 (s, 1H, –CHO).
NMR (600 MHz, CDCl ): -1.60 (s, 2H, N–H), 8.89 (s, 4H,
3
Ar–H), 8.67 (s, 8H, pyrrole-Hβ), 8.28, 8.29 (d, J = 8.20 Hz,
1
3
4
H, Ar–H), 8.20, 8.21 (d, J = 8.20 Hz, 4H, Ar–H). C NMR
(
600 MHz, CDCl ): 154.7, 146.6, 136.8, 135.4, 134.1, 129.0,
3
124.8, 119.03. UV–Vis λmax: 425 nm; yield 18.27%.
5
,10,15,20-tetra-(4-chloro-3-nitrophenyl)-porphyrin
−
1
(
8
TpClNPP): IR (ν, cm , solid): 3379 (NH), 1600 (NO ),
42 (C–Cl), 914, 830, 725 (Ar trisubstituted). H NMR
2
1
(
600 MHz, CDCl ): -2.93 (s, 2H, N–H), 8.69 (s, 4H, Ar–H),
3
8
.87 (s, 8H, pyrrole-Hβ), 8.35, 8.35 (d, J = 8 Hz, 4H,
1
3
Fig. 1 Nitration reaction
Ar–H), 8.01, 7.99 (d, J = 8 Hz, 4H, Ar–H). C NMR

Medicinal Chemistry Research
Fig. 2 Synthesis of porphyrins
(
600 MHz, CDCl ): 147.5, 146.6, 138.4, 135.4, 133.9,
dose (LD ) in mice was calculated by regression analysis
3
50
1
1
30.5, 129.6, 127.0, 121.4. UV–Vis λmax: 421 nm; yield
2.69%.
(percent death vs. log dose).
5
,10,15,20-tetra-(4-bromo-3-nitrophenyl)-porphyrin
Inflammation test
−
1
(
7
TpBrNPP): IR (ν, cm , solid): 3299 (N–H), 1690 (NO ),
42 (C–Br), 890, 842, 710 (Ar trisubstituted) H NMR (600
2
1
TPA-induced acute inflammation
MHz, CDCl ): -2.95 (s, 2H, N–H), 8.65 (s, 4H, Ar–H), 8.87
3
(
(
1
s, 8H, pyrrole-Hβ), 8.26, 8.27 (d, J = 8.29 Hz), 8.18, 8.18
The model of acute TPA-induced ear edema in mice was
carried out as described previously (De Young et al. 1989).
Groups of mice (n = 8) received topically vehicle, one of
the nitro-porphyrins (2.52 μM mg/ear) or 5.5 μM/ear IND.
Thirty minutes before the test; edema was induced on the
right ear (internal and external auricular pavilion) in mice
by topical application of 2.5 µg TPA dissolved in 20 µl of
acetone. The left ear (control) received the vehicle (acet-
one). After 6 h, the mice were sacrificed. A central section
6 mm in diameter was taken from the right (treated) and the
left (non-treated) ears. Sections were weighed to determine
the percentage of inhibition obtained using the expression
below:
1
3
d, J = 8.29). C NMR (600 MHz, CDCl ): 164.6, 150.3,
3
39.3, 137.4, 136.2, 132.4, 122.2, 120.1, 115.1. UV–Vis
λmax: 422 nm; 15.36%.
Animals
Male CD-1 mice (20–25 g), from the Universidad Autó-
noma Metropolitana-Xochimilco animal facility, were
housed under standard conditions (24 °C, under a light–dark
cycle of 12:12). The animals were supplied with food
(
LabDiet, St. Louis, MO, USA) and water ad libitum. The
experiments were carried out according to international and
local ethical standards (Pitts 2002; De Aluja 2002; Zim-
merman 1983). All animal procedures were approved by the
Research Bioethics Committee of the Universidad Autó-
noma Metropolitana-Xochimilco.
Δw control À Δw treated
% inhibition ¼
 100
Δw control
where Δw = wt - wnt; wt is the weight of the section of the
treated ear; wnt is the weight of the section of the non-
treated ear.
Acute toxicity
The acute toxicity was estimated according to the Lorke
Method (Lorke 1983). Nitro-porphyrins were orally admi-
nistered to groups of mice (n = 5) at single doses of
Mouse ear edema induced by multiple applications of TPA
The model of chronic TPA-induced ear edema in mice was
carried out as described previously (Stanley et al. 1991).
Groups of mice (n=8) were orally administered 30 min after
TPA application with vehicle, nitro-porphyrins each
(63.1–252.5 μM/kg p.o.), or 22.3 μM/kg IND. A solution of
TPA (2.5 mg/ear) was applied topically to both surfaces of
1
.2 mM, 3.1 mM, and 6.3 mM/kg. Observations were made
and recorded systematically during 120 h after treatment
administration. The visual observations included skin
changes, mobility, aggressively, sensitivity to sound and
pain, as well as respiratory movements. The median lethal

Medicinal Chemistry Research
each ear on alternate days for 10 days. At the end of the
experiment, the mice were sacrificed by cervical dislocation
and the percentage of edema inhibition was calculated as
described above.
Tail flick test
The tail flick test was carried out according to the method
described by D’Amour and Smith (D’Amour and Smith
1
941). A beam of light, emitted by an analgesiometer, was
focused on the dorsal surface of the tail. The latency time
in seconds) was recorded from the onset of the noxious
In vitro anti-inflammatory activity
Cell viability
(
stimulus to the withdrawal of the tail. Mice exhibiting basal
latency time between 2 and 5 s were chosen. A cutoff time
of 10 s was used as the maximum possible effect to prevent
tissue damage. The latency (in seconds) was determined
before, and 30 and 60 min after treatment administration in
six groups of mice (n = 8) that were administered with
nitro-porphyrins (63.1–252.5 μM/kg p.o.), 0.6 mM/kg NPX
p.o., 2.1 μM mg/kg BNP i.p., and saline solution (vehicle).
J774A.1 murine macrophages, obtained from ATCC
(
Manassas, VA, USA), were maintained in DMEM sup-
plemented by 7% fetal bovine serum and antibiotics
100 U/ml penicillin and 100 pg/ml streptomycin). Cell
cultures were grown at 37 °C, in a humidified atmosphere of
(
6
5
% CO .J774A.1 macrophages were seeded (1 × 10 cells/
2
well) in 96 well plates. After 24 h of incubation, macro-
phages were activated with 1 µg/ml LPS of E. coli and
treated with various concentrations of TpFNPP, dissolved in
Hot plate test
0
.1% DMSO, during 48 h following the method proposed
The hot plate test was carried out as previously described
(Woolfe and Mcdonald 1944) on a thermostatically con-
trolled heated metal plate (Analgesiometer, Ugo basile,
Italy). The temperature of the metal surface was maintained
at 55 ± 1 °C. The test was assessed on groups of eight mice.
Mice were treated with nitro-porphyrins (63.1–252.5 μM/kg),
0.6 mM/kg NPX, 2.1 μM/kg BNP i.p., or saline solution
(vehicle). The latency (in seconds) to a discomfort reaction
(licking paws or jumping) was determined before, and 30 and
60 min after treatment administration. The cutoff time was
30 s to prevent any thermal injury to mice.
by Mueller, (Mueller et al. 2010), with some modifications.
Cytokines production
Tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10,
and IL-1β serum levels were measured using a commer-
cially available ELISA kit according to the manufacturer’s
instructions (Peprotech, London, UK). The OD was mea-
sured in a microplate reader at 405 nm with a wavelength
correction set at 650 nm.
J774A.1 macrophages were grown at a density of 1 ×
6
1
0 . Then, the cells were treated separately with TpFNPP at
Statistical analysis
a concentration of 50 µM, whereas another group was
treated with IND at a concentration of 50 µg/ml. The cells
were incubated for 2 h. After treatment with LPS (1 µg/ml)
and incubated for 24 h, the cell-free supernatants were
collected and stored at 20 °C until they were analyzed by
immunoassay for the quantification of cytokines.
All experimental values are expressed as the mean ± stan-
dard error of mean (SEM). Statistical significance was
analyzed by comparison between treated groups and control
groups were carried out by ANOVA and post hoc Tukey
test. The level of P < 0.05 was used to determine statistical
significance. All calculations were done using the NCSS8
program (Hintze 2012).
Antinociceptive assays
Formalin test
Results and discussions
The antinociceptive effect of porphyrins was evaluated by
the formalin test (Dubuisson and Dennis 1977). The nitro-
porphyrins (63.1–252.5 μM/kg p.o.), 0.6 mM/kg naproxen
In a previous work, we synthesized and evaluated the
pharmacological activity of some symmetrical porphy-
rins: 5,10,15,20-tetra-(3-nitrophenyl)-porphyrin, 5,10,15,20-
tetra-(4-fluorophenyl)-porphyrin, 5,10,15,20-tetra-(4-chlor-
ophenyl)-porphyrin, and 5,10,15,20-tetra-(4-bromophenyl)-
porphyrin. The results showed that those porphyrins
exerted good antinociceptive and anti-inflammatory activity
(Alonso-Castro et al. 2015). In recent years, several com-
pounds containing the nitro group in their structure have
been synthesized and their anticonvulsant, antidepressant,
(
NPX) as positive control, and the vehicle (saline solution)
as negative control were administrated one prior to formalin
injection. Mice (n = 8, per group) were injected with 30 µl
of 2% formalin into the subplantar space of the right hind
paw, placed into an acrylic box, and the time of pain
reaction (time for licking the paw) was recorded in two
periods: 0–15 min (first phase: neurogenic pain) and
1
5–45 min (second phase: inflammatory pain).

Medicinal Chemistry Research
Table 1 Effects of the topical
application of indomethacin,
Treatment
Dose (mg/ear) Edema formation (mg) Inhibition (%)
5
,10,15,20-tetra-(3-nitrophenyl)-
Vehicle
0
2
2
2
15.38 ± 0.97
3.14 ± 0–61*
6.38 ± 0.68*
5.00 ± 0.66*
-
porphyrin, 5,10,15,20-tetra-(4-
bromo- 3-nitrophenyl)-
porphyrin, 5,10,15,20-tetra-(4-
chloro-3-nitrophenyl)-
Indomethacin
79.56 ± 3.99
58.55 ± 4.41
67.49 ± 4.32
5,10,15,20-tetra(3-nitrophenyl)-porphyrin
5
,10,15,20-tetra(4-bromo-3-nitrophenyl)-
porphyrin
,10,15,20-tetra(4-cloro-3-nitrophenyl)-
porphyrin, and 5,10,15,20-tetra-
(
4-fluoro-3-nitrophenyl)-
5
2
2
5.00 ± 0.96*
4.41 ± 0.44*
67.49 ± 6.23
71.31 ± 2.89
porphyrin on inflammation in
acute TPA-induced ear edema in
mice
porphyrin
5,10,15,20-tetra(4-fluoro-3-nitrophenyl)-
porphyrin
Results are mean ± S.E.M, *P < 0.05. According ANOVA followed by Tukey test
Table 2 Effects of the orally administrated indomethacin, 5,10,15,20-
tetra-(3-nitrophenyl)-porphyrin, 5,10,15,20-tetra-(4-bromo- 3-nitro-
phenyl)-porphyrin, 5,10,15,20-tetra-(4-chloro-3-nitrophenyl)-porphyrin,
and 5,10,15,20-tetra-(4-fluoro-3-nitrophenyl)-porphyrin on inflamma-
tion in chronic TPA-induced ear edema in mice
analgesic, anti-inflammatory, antiplatelet, and antiviral
activities, have been evaluated (Chiroli et al. 2003; Wu et al.
2
004). In models of nociception such as the acetic acid
assay, these molecules have presented antinociceptive
effects. Presence of nitro group in the structure enhanced
antinociceptive activity. In this work, four new porphyrins
were synthetized with nitro-aldehydes in their structure
Treatment
Dose
mg/Kg
Edema
formation
(mg)
Inhibition (%)
(
p.o.)
(
NO ), which could possibly contribute to the modulation of
Vehicle
0
21.63 ± 2.09
9.70 ± 1.18*
-
2
the release of nitric oxide (Chiroli et al. 2003; Duarte et al.
007; Since et al. 2013).
Indomethacin
8
55.14 ± 5.47
2
5
,10,15,20-tetra(3-
50
13.98 ± 1.80*
20.44 ± 1.98*
19.15 ± 1.23*
35.38 ± 8.31
14.58 ± 4.62
13.15 ± 4.95
nitrophenyl)-porphyrin 100
00
2
Biological evaluation
5
,10,15,20-tetra(4-
50
100
200
13.74 ± 1.36*
13.75 ± 1.04*
15.74 ± 1.42*
36.46 ± 6.30
36.40 ± 4.80
27.21 ± 6.57
bromo-3-nitrophenyl)-
porphyrin
Acute toxicity of nitro-porphyrins in mice
5
,10,15,20-tetra(4-
50
100
200
20.29 ± 1.17*
15.66 ± 1.68*
19.15 ± 1.25*
10.36 ± 3.20
31.68 ± 7.50
14.99 ± 4.86
After 1 h of administration, toxicological symptoms such as
drowsiness and sedation were observed in mice treated with
cloro-3-nitrophenyl)-
porphyrin
3
.1 or 6.3 mM/kg p.o. of TNPP, TpBrNPP, TpClNPP, and
5,10,15,20-tetra(4-
fluoro-3-nitrophenyl)-
porphyrin
50
100
200
19.73 ± 0–94* 10.58 ± 3.17
6.56 ± 0.51*
6.55 ± 0.42*
69.66 ± 2.33
69.71 ± 1.97
TpFNPP, each. The drowsiness continued for 2 h. No visible
damage to organs was shown in any mice treated with these
porphyrins. TpClNPP had a LD = 4.7 mmol/kg, and for the
Results are mean ± S.E.M, *P < 0.05. According ANOVA followed by
Tukey test
50
others four porphyrins showed a LD > 6.3 mmol/kg.
50
Anti-inflammatory activity of nitro-porphyrins in acute and
chronic TPA-induced ear edema
mice. Nitro-porphyrins significantly decreased the edema
and ear weight, which suggests that nitro-porphyrins might
be a good alternative in the treatment of inflammation.
Nevertheless, additional experiments, such as carrageenan-
kaolin induced arthritis, should be performed to analyze the
anti-inflammatory effects of nitro-porphyrins in long-term
assays. Compared with the other porphyrins, TpFNPP
showed a better anti-inflammatory activity in the TPA-
chronic assay. Further anti-inflammatory experiments were
carried out with TpFNPP.
The four nitro-porphyrins showed anti-inflammatory activ-
ity similar compared to IND (79.56%) in the following
order 58.55% (TNPP), 67.49% (TpBrNPP), 67.49%
(
TpClNPP), and 71.32% (TpFNPP) (Table 1).
In the chronic assay, only TpFNPP at 126.2 mM/kg
69.66%) and 252.5 mM/kg (69.77 %) showed anti-
(
inflammatory effects (P ≤ 0.05) with similar activity com-
pared to 22.3 μM/kg IND (55.14%). The other nitro-
porphyrins showed lower anti-inflammatory activity
TpFNPP exerts anti-inflammatory effects in vitro
(
Table 2). Repeated applications of TPA on the mouse ear
produces edema with hyperplasia and the infiltration of
polymorphonuclear leukocytes (Stanley et al. 1991). The
nitro-porphyrins showed anti-inflammatory activity in the
acute and chronic TPA-induced inflammation models in
TpFNPP (50 µM) decreased significantly (P ≤ 0.05), com-
pared to the control group, the concentration of TNF-α, IL-
6, and IL-1β by 56%, 50%, and 60% respectively. These
effects were similar compared with those found with the

Medicinal Chemistry Research
(A)
(B) 500
5
4
3
2
1
00
00
00
00
00
0
4
00
3
00
**
*
*
*
*
200
100
0
**
**
*
*
*
*
**
LPS
IND (μg/ml)
TpFNPP (μg/ml)
-
-
-
+
-
-
+
+
+
LPS
-
-
-
+
-
-
+
+
-
+
-
17
-
-
IND (μg/ml)
17
-
25
50
TpFNPP (μg/ml)
-
25
50
Treatments
Treatments
(C)
(D) 250
400
300
200
100
0
2
00
*
*
**
**
1
50
**
**
**
100
**
5
0
0
LPS
IND (μg/ml)
TpFNPP (μg/ml)
-
-
-
+
-
-
+
+
-
+
LPS
-
-
-
+
-
-
+
+
+
17
-
IND (μg/ml)
17
-
-
-
25
50
TpFNPP (μg/ml)
25
50
-
Treatments
Treatments
Fig. 3 TpFNPP induces anti-inflammatory effects in vitro. The pro-
duction of TNF-α (a), IL-1β (b), IL-6 (c), and IL-10 (d) were measured
as described in Materials and methods. Data are representative of three
independent experiments in quadruplicate. Results represent the mean
± standard deviation. *Denotes P ≤ 0.05, compared to LPS treatment
positive control IND (50 µM), which decreased significantly
in vitro results obtained with TpFNPP corroborate the
(
6
P ≤ 0.05) the concentration of TNF-α, IL-6, and IL-1β by
2%, 55%, and 61% respectively, (P ≤ 0.05) (Fig. 3a, b, c).
in vivo findings.
In the case of IL-10, TpFNPP (50 µM) and IND (50 µM)
Antinociceptive activity of nitro-porphyrins
significantly increased the concentration of this cytokine by
4
2% and 36%, respectively (Fig. 3d).
LPS, a lipopolysaccharide of the outer membrane of
In the formalin test, the results demonstrated that all four
nitro-porphyrins reduced the nociception during the phase
2. TpBrNPP and TpFNPP had the highest antinociceptive
effects compared to the other two nitro-porphyrins. At the
three doses, TpBrNPP and TpFNPP exerted antinociceptive
effects in the phase 2 with similar activity than NPX
(0.6 mM/kg) (Fig. 3). In the formalin test, nociception
occurs in two phases. The neurogenic pain or first phase
(0–15 min) with the involvement of mediators including
substance P, bradykinin, histamine and serotonin (Dubuis-
son and Dennis 1977; Munron 2007). The second type of
Gram-negative bacteria, stimulates the production of pro-
inflammatory cytokines (IL-1β, TNF-α, and IL-6) in mac-
rophages (Poon et al. 2013). IL-10 is a well-known anti-
inflammatory cytokine (de Waal Malefyt et al. (1991)). The
results indicated that TpFNPP (50 µM) increase the pro-
duction of the anti-inflammatory cytokine IL-10, and
decreased the production of the pro-inflammatory cytokines
IL-1β, TNF-α, and IL-6 in macrophages activated with LPS,
with similar activity than indomethacin (50 µM). The

Medicinal Chemistry Research
Fig. 4 Effect of nitro-porphyrins (50, 100, and 200 mg/kg p.o.) and
naproxen (150 mg/kg p.o.) on the licking time of formalin test-induced
pain, in mice, in the first phase (0–15 min) and the second phase
(15–45 min). Vertical bars represent mean ± S.E.M. of reaction time
pain, in seconds. *P < 0.05 vs. vehicle (n = 8). According ANOVA
followed by Tukey test
pain or the second phase (15–45 min), is associated with the
production and release of various pro-inflammatory med-
iators such as histamine, bradykinin, serotonin, pros-
taglandins, tachykinins, and glutamate by cells activated
after formalin administration (Fujimaki et al. 1992). Drugs
that act primarily on the central nervous system inhibit both
phases equally, while peripherally acting drugs inhibit the
second phase, such as NPX (Shibata et al. 1989). The nitro-
porphyrins showed antinociceptive effect in the phase 2 of
the formalin test, which involves the possibility of the
inhibition of inflammatory mediators.
induced hyperalgesia is specific for centrally mediated
nociception (Pinardi et al. 2002).
In the results of the nociception test, in the formalin test
the nitro-porphyrins was better for those previously sym-
metrical porphyrins (TNPP, TpClNPP, and TpFNPP),
which is indicative that the four nitro-porphyrins have
peripheral antinociceptive effects, whereas in the hot plate
and tail flick, the previous symmetrical porphyrins have a
better effect that the nitro-porphyrins, the results show
central antinociceptive effects (Alonso-Castro et al. 2015).
Further studies are necessary to elucidate the molecular
mechanisms by which nitro-porphyrins exert their anti-
nociceptive effects (Fig. 6).
In the tail flick test, at 60 and 120 min TNPP, TpClNPP,
and TpFNPP display significant antinociception activity,
similar to that produced by NPX (0.6 mM/kg p.o.) and
antinociceptive activity lower than that produced by BNP
In conclusion, the results suggest that nitro-porphyrins
might be an alternative for the treatment of inflammatory
diseases because of its low toxicity and good anti-
nociceptive and anti-inflammatory effects.
(
2.1 μM mg/kg).
In the hot plate test, at 60 and 120 min of treatment, the
positive control BNP (2.1 μM mg/kg) manifest the highest
antinociceptive effects compared to the vehicle group (Fig.
Porphyrin synthesis
4
). NPX only showed antinociceptive activity at 60 min,
and TpClNPP presented activity antinociceptive at doses of
The synthesis of the porphyrins is outlined in Scheme 1 and
2, and their structures are shown in Fig. 1. In all the cases
the purity of porphyrins was higher than 98%. Porphyrins
before pharmacological studies were washed with methanol
to ensure high purity.
2
52.2 μM/kg at 60 and 120 min (Fig. 5).
The hot plate test and tail flick are models commonly
used to evaluate the centrally acting antinociceptive
including sedatives and narcotic agents. Thermal stimulus-

Medicinal Chemistry Research
Fig. 5 Effect of nitro-porphyrins (50, 100, and 200 mg/kg p.o.)
in the latency time response in mice in the tail flick test. Naproxen
control (n = 10). Vertical bars represent mean ± S.E.M. of reaction
time pain, in seconds. *P < 0.05. According ANOVA followed by
Tukey test
(
150 mg/kg p.o.) and buprenorphine (1 mg/kg) were used as positive
In the synthesis of 5,10,15,20-tetra-(3-nitrophenyl)-por-
ring, 129.0 ppm and 124.8 ppm for the aromatic ring,
120.0 ppm for the β carbons of pyrrole, and 116.3 ppm for
the four carbons of the union between the α carbons of the
pyrroles. In the synthesis of (TpClNPP), the yield obtained
phyrin (TNPP), the yield was 13.53%; the NMR spectrum
1
contained the following signals: H NMR (CDCl3,
6
00 MHz), 8.803 (s), which corresponds to hydrogen 2 of
the benzene ring, 8.080 (s) corresponding to pyrrole β
1
was 12.69%, the NMR spectrum show the next signals, H
hydrogens, 8.71 (m), -2.84 (s), this signal due for the
NMR (CDCl , 600 MHz): 8.69 (s), which corresponds to
3
13
hydrogens of the nitrogen of pyrrole. The C NMR
CDCl , 600 MHz) data comprised the following signals:
hydrogen 2 of the benzene ring, 8.87 (s) corresponding to
pyrrole β hydrogens, 8.35, 8.35 (d, J = 8 Hz), 8.01, 7.99 (d,
J = 8 Hz), -2.938 (s), signal for the hydrogens of the pyrrole
(
3
1
1
47.8 ppm for the α carbons that support the nitro group,
46.6 ppm for the α carbon of pyrrole and 135.4 ppm for the
1
3
nitrogen. C NMR (CDCl , 600 MHz) 147.5 ppm for the
3
β carbons of pyrrole; 135.0, 129.5, 123.1, and 120.1 ppm
signals that correspond to a benzene ring; and 119.03 ppm
for the four carbons of the union between the α carbons of
the pyrroles. For the synthesis of (TpFNPP), the yield was
carbon attached to nitro group, 138.4 ppm corresponding to
carbon bonded with the chlorine, 146.6 ppm corresponding
for α carbons of pyrrole, 135.4, 130.5, 129.6 ppm signals
that corresponding to benzene, 127.0 ppm for β carbons of
pyrrole, 121.4 ppm for the four carbons of the union
between the α carbons of pyrroles. Synthesis of (TpBrNPP),
1
8.27%, and the NMR spectrum contained the following
1
signals: H NMR (CDCl , 600 MHz) 8.89 (s), which cor-
3
1
responds to hydrogen 2 of the benzene ring, 8.67 (s) cor-
the yield obtained was 15.36%, H NMR (CDCl3,
responding to pyrrole β hydrogens, 8.28, 8.29 (d, J =
600 MHz): 8.65 (s), which corresponds to hydrogen 2 of the
benzene ring, 8.87 (s) corresponding to pyrrole β hydro-
gens, 8.26, 8.27 (d, J = 8.29 Hz), 8.18, 8.18 (d, J = 8.29),
-2.95 (s), signal due for the hydrogens bonded to nitrogen of
8
.20 Hz), 8.20, 8.21 (d, J = 8.20 Hz), -1.60 (s), signal due
1
3
for the hydrogens attached at the nitrogen of pyrrole;
C
NMR (CDCl , 600 MHz) 136.8 ppm for the carbon attached
3
1
3
at NO group, 154.7 ppm corresponding to the carbon bond
pyrrole, C NMR (CDCl , 600 MHz) 164.3 ppm for the
2
3
with the fluorine, 146.6 ppm for the α carbons of pyrrole,
carbon bonded to nitro group, 139.3 ppm for the carbon
attached to bromine, 150.3 ppm for α carbons of pyrrole,
1
35.4 ppm for the aromatic ring, 134.1 ppm for the aromatic

Medicinal Chemistry Research
Fig. 6 Effect of nitro-porphyrins (50, 100, and 200 mg/kg p.o.) in the
latency time in mice in the hot plate test. Naproxen (150 mg/kg p.o.)
and buprenorphine (1 mg/kg) were used as positive control (n = 10).
Vertical bars represent mean ± S.E.M. of reaction time pain, in sec-
onds. *P < 0.05. According ANOVA followed by Tukey test
References
1
36.2 ppm for the benzene ring, 132.4 ppm for the benzene
ring, 137.4 ppm for β carbons of pyrrole, 122.2 ppm for the
benzene ring, 115.1 ppm for the four carbons of the union
between the α carbons of pyrroles. All porphyrins exhibited
a band in UV–Vis at 423 nm TNPP, 425 nm TpFNPP,
Adler AD, Longo FR, Finarelli JD, Goldmacher J, Assour J, Korsakoff
LJ (1967) A simplified synthesis for meso-tetraphnylporphine.
Org Chem 32:476
Adler AD, Sklar L, Longo FR, Finarelli JD, Finarelli MGJ (1968) A
mechanistic study of the synthesis of meso-tetraphenylporphin. J
HeterocyclChem 5:669
Alonso-Castro AJ, Zapata-Morales JR, Hernández-Munive A,
Campos-Xolalpa N, Pérez-Gutiérrez S, Pérez-González C (2015)
Synthesis, antinociceptive and anti-inflammatory effects of por-
phyrins. Bioorg Med Chem 23(10):2529–2537
Bjarnason I (2012) Gastrointestinal safety of NSAIDs and over the
counter analgesics. Int J ClinPract 67(178):37–42
Celic T, Španjol J, Bobinac M, Tovmasyan A, Vukelic I, Reboucas JS,
Batinic-Haberle I, Bobinac D (2014) Mn porphyrin-based SOD
mimic, MnTnHex-2-PyP(5+), and non-SOD mimic, MnTBAP(3-),
suppressed rat spinal cord ischemia/reperfusion injury via NF-κB
pathways. Free Radic Res 48(12):1426–1442
4
21 nm TpClNPP and 422 nm TpBrNPP, which corre-
sponds to the Soret band, a particular signal of porphyrins
Shkirman et al. 1999).
(
Acknowledgements JRZM was supported with a postdoctoral fel-
lowship from CONACYT (Number 205614, from October 2014 to
January 2015). AJAC was supported with a postdoctoral fellowship
from CONACYT (Number 174493, from February 2014 to January
2
015). We wish to thank the Directorate for Research Support and
Postgraduate Programs at the University of Guanajuato for their sup-
port in the editing of the English-language version of this article.
Chiroli V, Benedini F, Ongini E, Del Soldato P (2003) Nitric oxide-
donating non-steroidal anti-inflammatory drugs: the case of
nitroderivatives of aspirin. Eur J Med Chem 38(4):441–446
Covarrubias-Gómez A (2008) Papel del anestesiólogo en el manejo del
dolor crónico. Rev Mex Anest 31:S235–S237. (Suppl. 1)
D’Amour FE, Smith DL (1941) A method for determining loss of pain
sensation. J Pharmacol Exp Ther 72:74–79
De Aluja AS (2002) Animales de laboratorio y la Norma Oficial
Mexicana (NOM-062-ZOO-1999). Gac Med Mex 138
(3):295–298
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval All applicable international, national, and/or institu-
tional guidelines for the care and use of animals were followed. All
procedures performed in studies involving animals were in accordance
with the ethical standards of the institution or practice at which the
studies were conducted.

Medicinal Chemistry Research
de Waal Malefyt R, Abrams J, Bennett B, Figdor C, de Vries JE
1991) Interleukin 10(IL-10) inhibits cytokine synthesis by
Lorke D (1983) A new approach to partial acute toxicity testing. Arch
Toxicol 54(4):275–287
(
human monocytes: an autoregulatory role of IL-10 produced by
monocytes. J Exp Med 174:1209–1220
Mueller M, Hobiger S, Jungbauer A (2010) Anti-inflammatory activity
of extracts from fruits, herbs and species. Food Chem
122:987–996
Munron G (2007) Dopamine D1 and D2 receptor agonism enhances
antinociception in mediated by the serotonin and noradrenaline
reuptake inhibitor duloxetine the rat formalin test. Eur J Ethol
575:66–74
Naveed-Umar M, Ur-Rashid H, Gul-Sayed M, Karim N, Ghaffar R,
Utrera-Martines MA, Shoaib M (2015) Synthesis and evaluation
of analgesic, anti-inflammatory and anti-bacterial activity of
synthetic porphyrin derivatives. Rev Roum Chim 60(9):861–866
Pinardi G, Sierralta F, Miranda HF (2002) Adrenergic mechanisms in
antinociceptive effects of non steroidal anti-inflammatory drugs
in acute thermal nociception in mice. Inflamm Res 51(5):219–222
Pitts M (2002) Institutional Animal care and use committee guide-
book, 2nd edn. Office of Laboratory Animal Welfare National
Istitutes of Health, Maryland
De Young LM, Kheifets JB, Ballaron SJ, Young JM (1989) Edema
and cell infiltration in the phorbol ester-treated mouse ear are
temporally separate and can be differentially modulated by
pharmacologic agents. Agents Actions 26(3-4):335–341
Duarte CD, Tributino JL, Lacerda DI, Martins MVM, Alexandre-
Moreira MS, Dutra F, Bechara EJ, De-Paula FS, Goulart MO,
Ferreira J, Calixto JB, Nunes MP, Bertho AL, Miranda AL,
Barreiro EJ, Fraga CA (2007) Synthesis, pharmacological eva-
luation and electrochemical studies of novel 6-nitro-3,4-methy-
lenedioxyphenyl-N-acylhydrazone derivatives: Discovery of
LASSBio-881, a new ligand of cannabinoid receptors. Bioorg
Med Chem 15(6):2421–2433
Dubuisson D, Dennis SG (1977) The formalin test: A quantitative
study of the analgesic effects of morphine, meperidine, and brain
stem stimulation in rats and cats. Pain 4(2):161–174
Dugowson CE, Gnanashanmugam
P
(2006) Nonsteroidal anti-
Poon DC, Ho YS, Chiu K, Chang RC (2013) Cytokines: how
important are they in mediating sickness. Neurosci Biobehav Rev
37:1–10
Shibata M, Ohkubo T, Takahashi H, Inoki R (1989) Modified formalin
test: Characteristic biphasic pain response. Pain 38:347–352
Shkirman SF, Solovev KN, Kachura TF, Arabei SA, Skakovskii ED
(1999) Interpretation of the soret band of porphyrins based on the
polarization spectrum of N-methyltetraphenylporphin fluores-
cence. J Appl Spectrosc 66:68–75
inflammatory drugs. Phys Med Rehabil Clin N AM 17:347–354
Dung NT, Bajpai VK, Yoon JI, Kang SC (2009) Anti-inflammatory
effects of essential oil isolated from the buds of Cleistocalyx
operculatus (Roxb.) Merr and Perry. Food Chem Toxicol
4
7:449–453
Fujimaki H, Kawagoe A, Bissonnette E, Befus D (1992) Mast cell
response to formaldehyde. 1. Modul Mediat Release Int Arch
Allergy Immunol 98(4):324–331
Furniss BS, Hannaford AJ, Smith PWG, Tatchell AR (1989) Textbook
of practical organic chemistry Vogel’s, 5a Ed. Longman Scien-
tific & Technical, New York
Gaskin DJ, Richard P (2011) The economic costs of pain in the United
States. In: Institute of Medicine (US) Committee on Advancing
Pain Research, Care, and Education. Relieving Pain in America: A
blueprint for transforming prevention, care, education, and research.
National Academies Press (US), Washington (DC), 113–158
Hintze J (2012) NCSS 8. NCSS, LLC. Kaysville, Utah, USA. www.
ncss.com
Since M, Freret T, Nee G, Terme T, Vanelle P, Boulouard M (2013)
New orally effective 3-(2-nitro)phenylpropanamide analgesic
derivatives: synthesis and antinociceptive evaluation. Eur J Med
Chem 69:728–734
Stanley PL, Steiner S, Havens M, Tramposch KM (1991) Mouse skin
inflammation induced by multiple topical applications of 12-O-
tetradecanoylphorbol-13-acetate. Skin Pharmacol 4(4):262–271
Stover K, Fukuyama T, Young AT, Daniele MA, Oberley R, Crapo
JD, Bäumer W (2016) Topically applied manganese-porphyrins
BMX-001 and BMX-010 display a significant anti-inflammatory
response in a mouse model of allergic dermatitis. Arch Dermatol
Res 308(10):711–721
James S (2013) Human pain and genetics: some basics. Br J Pain 7
(
4):171–178
Jelić D, Tatić I, Trzun M, Hrvačić B, Brajša K, Verbanac D,
Tomašković M, Čulić O, Antolović R, Glojnarić I, Weygand-
Đurašević I, Vladimir-Knežević S, Mildner B (2012) Porphyr
Wolfe MM, Lichtenstein DR, Singh G (1999) Gastrointestinal toxicity
of nonsteroidal anti-inflammatory drugs. N Engl J Med 340
(24):1888–1899
Woolfe G, Mcdonald AD (1944) The evaluation of analgesic action of
pethidine hydrochloride (Demerol). J Pharmacol Exp Ther
80:300–307
Wu WP, Hao JX, Ongini E, Impagnatiello F, Presotto C, Wiesenfeld-
Hallin Z, Xu XJ (2004) A nitric oxide (NO)-releasing derivative
of gabapentin, NCX 8001, alleviates neuropathic pain-like
behavior after spinal cord and peripheral nerve injury. Br J
Pharmacol 141(1):65–74
New Endog anti-Inflamm Agents Ur
J
Pharmacol 691
(
1-3):251–260
Jensen TS, Gottrup H, Kasch H, Nikolajsen L, Terkelsen AJ, Witting
N (2001) Has basic research contributed to chronic pain treat-
ment? Acta Anaesthesiol Scand 45:1128–1135
Li BQ, Dong X, Li N, Gao JY, Yuan Q, Fang SH, Gong XC, Wang SJ,
Wang FS (2014) In vitro enzyme-mimic activity and in vivo
therapeutic potential of HSJ-0017, a novel Mn porphyrin-based
antioxidant enzyme mimic. Exp Biol Med (Maywood) 239
Zimmerman M (1983) Ethical guidelines for investigations on
experimental pain in conscious animals. Pain 16:109
(
10):1366–1379