Introduction

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. 2009; Wolfe et al. 1999), whereas steroidal anti-inflammatory drugs prevent and suppress inflammation, but long period of administration induces weight gain, osteoporosis, and bruising of the skin, with other symptoms (Bjarnason 2012; Dugowson and Gnanashanmugam 2006).

Another major health problem is pain, although this symptom is associated with a host of ailments, pain perception 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 attention 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 (Jensen et al. 2001).

As aforementioned, we are currently seeking new therapeutic 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 tetraphenylporphyrins and manganese porphyrins have displayed anti-inflammatory and antinociceptive effects compared to reference drugs (Jelić et al. 2012; Celic et al. 2014; Li et al. 2014; Naveed-Umar et al. 2015; Stover et al. 2016). Therefore, we have decided to test the anti-inflammatory and antinociceptive capacity of some of the synthetic porphyrins with a nitro benzene ring.

Materials and methods

Reagents

All chemicals used in the synthesis of porphyrins, polyvinylpyrrolidone (PVP), 12-O-tetradecanoylphorbol 13-acetate (TPA), lipopolysaccharide (LPS) (Escherichia coli 0111: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. H2SO4 25 ml (0.47 M) were cooled to 0 °C, 3.1 ml (0.070 M) of HNO3 were added, 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).

Fig. 1
figure 1

Nitration reaction

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The spectrums of 1H NMR of nitrobenzaldehydes were obtained in CDCl3 and TMS as reference.

4-chloro-3-nitrobenzaldehyde: Yellow solid; mp 60–62 °C; yield: 70%; 1H NMR (600 MHz, CDCl3), 7.89 (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).

4-bromo-3-nitrobenzaldehyde: Yellow solid; mp 102–1004 °C; yield 55%; 1H NMR (600 MHz, CDCl3), 8.17 (d, J = 8.10, 1H, Ar–H), 8.21 (d, J  = 8.10, 1H, Ar–H), 8.90 (t, J = 8.10, 1H, Ar–H), 9.90 (s, 1H, –CHO).

4-fluoro-3-nitrobenzaldehyde: Yellow solid; mp 44–47 °C; yield 63%; 1H NMR (600 MHz, CDCl3), 7.60 (d, 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).

3-nitrobenzaldehyde: Yellow solid; mp 50–52 °C; yield 71%; 1H NMR (600 MHz, CDCl3), 7.80 (d, J = 8.3, 1H, 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).

Synthesis of nitro-porphyrins

Freshly distilled pyrrole (0.08 M) was added dropwise into a refluxing propionic acid (180 ml) solution of the corresponding 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.

Fig. 2
figure 2

Synthesis of porphyrins

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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.

The spectrums of 1H NMR and 13C NMR were obtained in CDCl3 and TMS as reference.

5,10,15,20-tetra-(3-nitrophenyl)-porphyrin (TNPP): IR (ν, cm−1, solid): 3379 (NH), 1601 (NO2), 1540 (Ar) 1H NMR (600 MHz, CDCl3): -2.84 (s, 2H, N–H), 8.80 (s, 4H, Ar–H), 8.08 (s, 8H, H pyrrole-Hβ), 8.71 (m, 12H, Ar–H). 13C NMR (600 MHz, CDCl3): 147.8, 146.6, 135.4, 135.0, 133.4, 129.5, 123.1, 120.1, 120.03. UV–Vis λmax: 423 nm; yield 13.53%.

5,10,15,20-tetra-(4-fluoro-3-nitrophenyl)-porphyrin (TpFNPP): IR (ν, cm−1, solid): 3309 (N–H), 1699 (NO2), 1540 (Ar), 1400 (C–F), 917, 840, 734 (Ar trisubstituted). 1H NMR (600 MHz, CDCl3): -1.60 (s, 2H, N–H), 8.89 (s, 4H, Ar–H), 8.67 (s, 8H, pyrrole-Hβ), 8.28, 8.29 (d, J = 8.20 Hz, 4H, Ar–H), 8.20, 8.21 (d, J = 8.20 Hz, 4H, Ar–H). 13C NMR (600 MHz, CDCl3): 154.7, 146.6, 136.8, 135.4, 134.1, 129.0, 124.8, 119.03. UV–Vis λmax: 425 nm; yield 18.27%.

5,10,15,20-tetra-(4-chloro-3-nitrophenyl)-porphyrin (TpClNPP): IR (ν, cm−1, solid): 3379 (NH), 1600 (NO2), 842 (C–Cl), 914, 830, 725 (Ar trisubstituted). 1H NMR (600 MHz, CDCl3): -2.93 (s, 2H, N–H), 8.69 (s, 4H, Ar–H), 8.87 (s, 8H, pyrrole-Hβ), 8.35, 8.35 (d, J = 8 Hz, 4H, Ar–H), 8.01, 7.99 (d, J = 8 Hz, 4H, Ar–H). 13C NMR (600 MHz, CDCl3): 147.5, 146.6, 138.4, 135.4, 133.9, 130.5, 129.6, 127.0, 121.4. UV–Vis λmax: 421 nm; yield 12.69%.

5,10,15,20-tetra-(4-bromo-3-nitrophenyl)-porphyrin (TpBrNPP): IR (ν, cm−1, solid): 3299 (N–H), 1690 (NO2), 742 (C–Br), 890, 842, 710 (Ar trisubstituted) 1H NMR (600 MHz, CDCl3): -2.95 (s, 2H, N–H), 8.65 (s, 4H, Ar–H), 8.87 (s, 8H, pyrrole-Hβ), 8.26, 8.27 (d, J = 8.29 Hz), 8.18, 8.18 (d, J = 8.29). 13C NMR (600 MHz, CDCl3): 164.6, 150.3, 139.3, 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 Autónoma 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; Zimmerman 1983). All animal procedures were approved by the Research Bioethics Committee of the Universidad Autónoma Metropolitana-Xochimilco.

Acute toxicity

The acute toxicity was estimated according to the Lorke Method (Lorke 1983). Nitro-porphyrins were orally administered to groups of mice (n = 5) at single doses 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 dose (LD50) in mice was calculated by regression analysis (percent death vs. log dose).

Inflammation test

TPA-induced acute inflammation

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 (acetone). 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:

$${\mathrm{\% }}\,{\mathrm{inhibition}} = \frac{{{{\Delta w}}\,{\mathrm{control}} - {{\Delta w}}\,{\mathrm{treated}}}}{{{{\Delta w}}\,{\mathrm{control}}}} \times 100$$

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.

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 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.

In vitro anti-inflammatory activity

Cell viability

J774A.1 murine macrophages, obtained from ATCC (Manassas, VA, USA), were maintained in DMEM supplemented 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 5% CO2.J774A.1 macrophages were seeded (1 × 106 cells/well) in 96 well plates. After 24 h of incubation, macrophages were activated with 1 µg/ml LPS of E. coli and treated with various concentrations of TpFNPP, dissolved in 0.1% DMSO, during 48 h following the method proposed 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 commercially available ELISA kit according to the manufacturer’s instructions (Peprotech, London, UK). The OD was measured 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 × 106. Then, the cells were treated separately with TpFNPP at 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.

Antinociceptive assays

Formalin test

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 (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 15–45 min (second phase: inflammatory pain).

Tail flick test

The tail flick test was carried out according to the method described by D’Amour and Smith (D’Amour and Smith 1941). 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 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).

Hot plate test

The hot plate test was carried out as previously described (Woolfe and Mcdonald 1944) on a thermostatically controlled 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.

Statistical analysis

All experimental values are expressed as the mean ± standard 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).

Results and discussions

In a previous work, we synthesized and evaluated the pharmacological activity of some symmetrical porphyrins: 5,10,15,20-tetra-(3-nitrophenyl)-porphyrin, 5,10,15,20-tetra-(4-fluorophenyl)-porphyrin, 5,10,15,20-tetra-(4-chlorophenyl)-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 compounds containing the nitro group in their structure have been synthesized and their anticonvulsant, antidepressant, analgesic, anti-inflammatory, antiplatelet, and antiviral activities, have been evaluated (Chiroli et al. 2003; Wu et al. 2004). 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 (NO2), which could possibly contribute to the modulation of the release of nitric oxide (Chiroli et al. 2003; Duarte et al. 2007; Since et al. 2013).

Biological evaluation

Acute toxicity of nitro-porphyrins in mice

After 1 h of administration, toxicological symptoms such as drowsiness and sedation were observed in mice treated with 3.1 or 6.3 mM/kg p.o. of TNPP, TpBrNPP, TpClNPP, and 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 LD50 = 4.7 mmol/kg, and for the others four porphyrins showed a LD50 > 6.3 mmol/kg.

Anti-inflammatory activity of nitro-porphyrins in acute and chronic TPA-induced ear edema

The four nitro-porphyrins showed anti-inflammatory activity 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).

Table 1 Effects of the topical application of indomethacin, 5,10,15,20-tetra-(3-nitrophenyl)-porphyrin, 5,10,15,20-tetra-(4-bromo- 3-nitrophenyl)-porphyrin, 5,10,15,20-tetra-(4-chloro-3-nitrophenyl)-porphyrin, and 5,10,15,20-tetra-(4-fluoro-3-nitrophenyl)-porphyrin on inflammation in acute TPA-induced ear edema in mice
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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 compared to 22.3 μM/kg IND (55.14%). The other nitro-porphyrins showed lower anti-inflammatory activity (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 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.

Table 2 Effects of the orally administrated indomethacin, 5,10,15,20-tetra-(3-nitrophenyl)-porphyrin, 5,10,15,20-tetra-(4-bromo- 3-nitrophenyl)-porphyrin, 5,10,15,20-tetra-(4-chloro-3-nitrophenyl)-porphyrin, and 5,10,15,20-tetra-(4-fluoro-3-nitrophenyl)-porphyrin on inflammation in chronic TPA-induced ear edema in mice
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TpFNPP exerts anti-inflammatory effects in vitro

TpFNPP (50 µM) decreased significantly (P ≤ 0.05), compared 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 positive control IND (50 µM), which decreased significantly (P ≤ 0.05) the concentration of TNF-α, IL-6, and IL-1β by 62%, 55%, and 61% respectively, (P ≤ 0.05) (Fig. 3a, b, c). In the case of IL-10, TpFNPP (50 µM) and IND (50 µM) significantly increased the concentration of this cytokine by 42% and 36%, respectively (Fig. 3d).

Fig. 3
figure 3

TpFNPP induces anti-inflammatory effects in vitro. The production 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

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LPS, a lipopolysaccharide of the outer membrane of Gram-negative bacteria, stimulates the production of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) in macrophages (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 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). The in vitro results obtained with TpFNPP corroborate the in vivo findings.

Antinociceptive activity of nitro-porphyrins

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 (Dubuisson and Dennis 1977; Munron 2007). The second type of pain or the second phase (15–45 min), is associated with the production and release of various pro-inflammatory mediators such as histamine, bradykinin, serotonin, prostaglandins, 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.

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 (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. 4). NPX only showed antinociceptive activity at 60 min, and TpClNPP presented activity antinociceptive at doses of 252.2 μM/kg at 60 and 120 min (Fig. 5).

Fig. 4
figure 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

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Fig. 5
figure 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 (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 seconds. *P < 0.05. According ANOVA followed by Tukey test

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The hot plate test and tail flick are models commonly used to evaluate the centrally acting antinociceptive including sedatives and narcotic agents. Thermal stimulus-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 symmetrical 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 antinociceptive effects (Fig. 6).

Fig. 6
figure 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 seconds. *P < 0.05. According ANOVA followed by Tukey test

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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 antinociceptive and anti-inflammatory effects.

Porphyrin synthesis

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.

In the synthesis of 5,10,15,20-tetra-(3-nitrophenyl)-porphyrin (TNPP), the yield was 13.53%; the NMR spectrum contained the following signals: 1H NMR (CDCl3, 600 MHz), 8.803 (s), which corresponds to hydrogen 2 of the benzene ring, 8.080 (s) corresponding to pyrrole β hydrogens, 8.71 (m), -2.84 (s), this signal due for the hydrogens of the nitrogen of pyrrole. The 13C NMR (CDCl3, 600 MHz) data comprised the following signals: 147.8 ppm for the α carbons that support the nitro group, 146.6 ppm for the α carbon of pyrrole and 135.4 ppm for the 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 18.27%, and the NMR spectrum contained the following signals: 1H NMR (CDCl3, 600 MHz) 8.89 (s), which corresponds to hydrogen 2 of the benzene ring, 8.67 (s) corresponding to pyrrole β hydrogens, 8.28, 8.29 (d, J = 8.20 Hz), 8.20, 8.21 (d, J = 8.20 Hz), -1.60 (s), signal due for the hydrogens attached at the nitrogen of pyrrole; 13C NMR (CDCl3, 600 MHz) 136.8 ppm for the carbon attached at NO2 group, 154.7 ppm corresponding to the carbon bond with the fluorine, 146.6 ppm for the α carbons of pyrrole, 135.4 ppm for the aromatic ring, 134.1 ppm for the aromatic 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 was 12.69%, the NMR spectrum show the next signals, 1H NMR (CDCl3, 600 MHz): 8.69 (s), which corresponds to 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 nitrogen. 13C NMR (CDCl3, 600 MHz) 147.5 ppm for the 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), the yield obtained was 15.36%, 1H NMR (CDCl3, 600 MHz): 8.65 (s), which corresponds to hydrogen 2 of the benzene ring, 8.87 (s) corresponding to pyrrole β hydrogens, 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 pyrrole, 13C NMR (CDCl3, 600 MHz) 164.3 ppm for the carbon bonded to nitro group, 139.3 ppm for the carbon attached to bromine, 150.3 ppm for α carbons of pyrrole, 136.2 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, 421 nm TpClNPP and 422 nm TpBrNPP, which corresponds to the Soret band, a particular signal of porphyrins (Shkirman et al. 1999).