3-Aminothiophene-2-Acylhydrazones: Non-Toxic, Analgesic and Anti-Inflammatory Lead-Candidates

Different chemotypes are described as anti-inflammatory. Among them the N-acylhydrazones (NAH) are highlighted by their privileged structure nature, being present in several anti-inflammatory drug-candidates. In this paper a series of functionalized 3-aminothiophene-2-acylhydrazone derivatives 5a–i were designed, synthesized and bioassayed. These new derivatives showed great anti-inflammatory and analgesic potency and efficacy. Compounds 5a and 5d stand out in this respect, and were also active in CFA-induced arthritis in rats. After daily treatment for seven days with 5a and 5d (50 µmol/Kg), by oral administration, these compounds were not renal or hepatotoxic nor immunosuppressive. Compounds 5a and 5d also displayed good drug-scores and low risk toxicity calculated in silico using the program OSIRIS Property Explorer.


Introduction
Inflammation is an adaptive response that is triggered by noxious stimuli and conditions, such as infection and tissue injury, and can be classified into acute and chronic [1,2]. The role of inflammation in the etiology of several diseases is well-documented [3,4]. Many pathways are involved in acute and chronic inflammatory processes, among them the cyclooxygenase (COX) and p38 mitogen-activated protein kinase (p38 MAPK). These enzymes have been studied as important targets for the design of novel anti-inflammatory drugs [5][6][7][8]. COX and its isoenzymes (COX-1; COX-2 and COX-3) are the molecular targets of non-steroidal anti-inflammatory drugs (e.g., indomethacin, diclofenac and celecoxib). COX-3 is believed to be responsible for the antipyretic and analgesic effects of dipyrone and paracetamol [9]. Distinct chemotypes are described as anti-inflammatory, and among them the N-acylhydrazones (NAH) are especially noteworthy [10]. These privileged structures [11], exemplified by compounds 1-4 ( Figure 1), are present in several anti-inflammatory drug-candidates acting by different mechanism of action [12][13][14][15][16][17][18]. In a continuous effort to identify new anti-inflammatory drug-candidates a series of functionalized 3-aminothiophene-2-acylhydrazone derivatives 5a-i were designed by molecular modifications of the prototype 1. These modifications aimed to preserve the N-acylhydrazone moiety (CONHN=CH) and were based on application of ring isosterism between the pyridine and thiophene nucleus, and on molecular simplification with consequent elimination of the methyl group of the original prototype 1 (Figure 2). The aromatic pattern of compound 5a was later modified following classical isosteric replacement of monovalent groups and equivalent rings exchange [19] (Figure 2).

Chemistry
The functionalized 3-aminothiophene-2-acylhydrazone derivatives 5a-i were synthesized as depicted in Scheme 1, following previously described methodology [20][21][22][23]. Compounds were characterized by IR, 1 H-and 13 C-NMR and mass spectroscopy. The purity was determined by HPLC. The stereochemistry of the imine double bond (N=CH) was assigned as the E-isomer. This isomer was assigned based on the chemical shift of the imine-hydrogen singlet signal visualized in the 1 H-NMR spectra of NAH derivatives 5a-i and considering similar data previously reported in the literature [21][22][23]. Scheme 1. Synthesis of 3-aminothiophene-2-acylhydrazones derivatives 5a-i. Reagents

a) b)
As showed in Figure 3A,B, with exception of compounds 5g and 5d, the 3-aminothiophene-2acylhydrazones 5a-i were predicted to have similar druglikeness as dipyrone and dexamethasone, and inferior to that of indomethacin. Examining the drug-score parameter, OSIRIS predicted all compound 5a-i as being better than dipyrone. Likewise, derivatives 5a, 5e, 5h and 5i were predicted to have similar drug-score as dexamethasone, while compounds 5c, 5f and 5g were similar to indomethacin. Despite the close structural similarity between compounds 5a-i, the in silico study predicted different toxic profiles (Table 1). Compounds 5a, 5c, 5d, 5g and 5i were predicted with low risk of toxicity; whereas derivatives 5b, 5e, 5f and 5h showed moderate to high theoretical toxicity risk, for at least one of the parameters analyzed by the Osiris program.
The anti-inflammatory activity of compounds 5a-i was evaluated using a carrageenan-induced peritonitis test [28]. Animals were treated with 5a-i by oral administration in doses of 100, 30, 10, 3 and 1 µmol/Kg. The anti-inflammatory potency of compounds 5a-i is listed in Table 3. All compounds, with exception of 5e and 5i promoted a significant reduction of cell recruitment, being derivatives 5a (ID 50 = 7.2 ± 1.8 µmol/Kg) and 5d (ID 50 = 5.2 ± 2.0 µmol/Kg) the most promising, showing anti-inflammatory potency and efficacy similar to the standard indomethacin (Table 3).
One the anti-inflammatory profile of 3-aminothiophene-2-acylhydrazones 5a-i was confirmed in a murine acute inflammatory model, compounds 5a and 5d were selected in order to investigate their activity in a chronic model of inflammation. Therefore, these compounds (50 µmol/Kg, pathway orally) were evaluated in an arthritis model induced by complete Freund's adjuvant (CFA) in rats [29], and the results were compared to those obtained for the standard dexamethasone (5 µmol/Kg, pathway orally, Figure 5). As demonstrated in Figure 5, the daily treatment for seven days with compounds 5a and 5d was able to reduce paw edema on the 17th to 21st days of experiment, although, this reduction was less prominent than that caused by the standard dexamethasone. Statistical differences between the treated and the control groups were evaluated by ANOVA and Dunnett tests and the asterisks denote the levels of significance in comparison with control groups. *** p < 0.001; ** p < 0.01 and * p < 0.05. After the 21st day of the arthritis experiment, animals were anesthetized and blood was collected to investigate whether treatment with 5a and 5d had generated any liver or kidney toxicity. To assess liver and renal functions the AST/ALT and urea/creatinine levels were determined on serum of animals treated with 5a and 5d ( Figures 6 and 7). No alterations were observed in the levels of AST/ALT ( Figure 6) and/or urea/creatinine ( Figure 7). These data indicated that, in these conditions, compounds 5a and 5d were neither hepatotoxic nor nephrotoxic. After the daily treatment for seven days with 5a and 5d (50 µmol/Kg, pathway orally) the stomach of the animals was macroscopically analyzed [30] and no signal of redness or bleeding was observed (data not shown).

Figure 5.
Effect of compounds 5a (50 µmol/Kg, p.o.) and 5d (50 µmol/Kg, p.o.) and dexamethasone (5 µmol/Kg; p.o.) on the CFA-induced arthritis in rats. Each point represents the mean ± S.E.M. of six animals. Statistical differences between the treated and the control groups were evaluated by ANOVA and Dunnett tests and the asterisks denote the levels of significance in comparison with control groups. *** p < 0.001; ** p < 0.01 and * p < 0.05. Figure 6. Effect of compounds 5a (50 µmol/Kg, p.o) and 5d (50 µmol/Kg, p.o) and dexamethasone (5 µmol/Kg) on serum ALT and AST levels. Statistical differences between the treated and the control groups were evaluated by ANOVA and Dunnett tests and the asterisks denote the levels of significance in comparison with control groups. *** p < 0.001 and ** p < 0.01. In order to discount an eventual immunosuppressive profile and considering that several indicators of immunosuppression can be observed in standard nonclinical toxicology studies, such as alterations in organ weight and hypocellularity of immune system tissues [31][32][33], spleens of animals treated daily for seven days with compounds 5a, 5d and dexamethasone were also analyzed. No change in spleen weight was observed after treatment with 5d (50 µmol/Kg, p.o.), whereas a slight alteration was detected with 5a (50 µmol/Kg, p.o.). These results indicate that 5d doesn't promote immunosuppression, while the standard drug dexamethasone does (5 µmol/Kg, p.o.), as indicated by its ability to change the spleen's weight ( Figure 8).

Figure 8.
Effect of compounds 5a (50 µmol/Kg, p.o.) and 5d (50 µmol/Kg, p.o.) and dexamethasone (5 µmol/Kg, p.o.) on weight of spleen. Statistical differences between the treated and the control groups were evaluated by ANOVA and Dunnett tests and the asterisks denote the levels of significance in comparison with control groups. *** p < 0.001 and * p < 0.05.

General Procedure for the Preparation of 3-Aminothiophene-2-Carbohydrazide [23]
To a solution of methyl 3-aminothiophene-2-carboxylate (1 mmol) in ethanol (5 mL) was added hydrazine monohydrate (35 mmol). The reaction mixture was maintained under reflux for 24 h, at which time TLC (hexane-ethyl acetate (1:1)) indicated the end of the reaction. Then the reaction mixture was poured on ice and the resulting white precipitate was filtered out affording the title compound. This compound was prepared by Reinecke and coworkers using a similar procedure [22].

General Procedure for the Preparation of 3-Aminothiophene-2-Carbohydrazide Derivatives 5a-i
Under anhydrous conditions 3-aminothiophene-2-carbohydrazide (1.0 mmol), ethanol (5 mL), the corresponding benzaldehyde derivative (1.0 mmol) and a catalytic amount of hydrochloric acid were mixed together and stirred for 1 h at reflux and then the solvent was evaporated under reduced pressure. Next the reaction mixture was added to ice and the precipitate obtained was purified by column chromatography on silica gel with a CH 2 Cl 2 /MeOH 2% mobile phase.

In Silico Toxicological Evaluation and Drug-Like Profile
The in silico toxicity and drug-like profile of 3-aminothiophene-2-acylhydrazone derivatives 5a-i were calculated using the Program OSIRIS Property Explorer [24]. Data were generated on-line in the Osiris Program, accessed by the link (http://www.organic-chemistry.org/prog/peo/) [25] and represented by toxicity risks (mutagenic, irritant, tumorigenic and reproductive effects), druglikeness and drug-score. Druglikeness was calculated based on equation summing up score values of the fragments present in the molecule under investigation. The fragments were identified from a list of 5300 distinct substructure fragments with associated druglikeness scores [25]. Drug-score was calculated combining the druglikeness, cLogP, logS, molecular weight and toxicity risks data [25].

Animals
Experiments were conducted using Swiss mice obtained from the BIOCEN-UFAL breeding unit, weighing 20-30 g each, males, adult, with 6-8 weeks of age, distributed in groups up to 6-8 animals for treatment. Wistar rats (130-170 g), males, were used in the experiment of induction of arthritis. The animals were maintained with free access to food and water and kept at 25-28 °C under a controlled 12 h light/dark cycle. All animals were manipulated according to the norms established by the Ethics Commission-UFAL for handling animals (Protocol number: 14/2013).

Acetic Acid-Induced Abdominal Constriction Test
The peripheral analgesic activity was evaluated in male mice using the acetic acid-induced writhing test [23]. The acetic acid-induced abdominal constriction test was carried out as described previously by Coolier, 1968 with minor modifications in groups of six animals. In order to induce pain in mouse peritoneal cavity, 0.6% of acetic acid (10 mL/Kg) was injected intraperitoneally, 40 min after the oral administration of 3-amino-thiophene-2-acylhydrazone derivatives 5a-i (dose = 100, 30, 10, 3 and 1 µmol/Kg) and the standard dipyrone (dose = 100, 30, 10, 3 and 1 µmol/Kg). The ID 50 was calculated by nonlinear regression. The abdominal constriction resulting from the injection of acetic acid consists of a contraction of the abdominal region together with a stretching of the hind limbs or all limbs. The number of abdominal constrictions was counted cumulatively over a period of 20 min, commencing 5 min after acetic acid administration. The animals were then placed immediately to individual in a transparent plastic box. Anti-nociception response was indicated by the reduction in the mean of 3-amino-thiophene-2-acylhydrazone derivatives 5a-i number of abdominal constrictions in the test groups compared to the control group. Dipyrone was used as reference drugs while control group received vehicle (Arabic gum) that was used to dissolve compounds.

Formalin Test
The method used for this test was similar to that described by Hunskaar and Hole [24] with minor modifications. Adult Swiss mice were divided in groups of six mice each and pretreated with 3-amino-thiophene-2-acylhydrazone derivatives 5a-i (dose = 30 µmol/Kg, p.o.) or indomethacin (dose = 10 µmol/Kg, p.o.). Forty minutes after this treatment they were administered with 20 µL of a 2.5% solution of formalin, subcutaneously under the plantar surface of the left hind-paw. Using a chronometer, the total time spent in licking and biting the injected paw is recorded, quantifying thus the nociceptive behavior. Anti-nociceptive effect was determined in two phases, the early phase from 0 to 5 min and the late phase 15 to 30 min with a 10 min lag period in between both phases.

Carrageenan-Induced Peritonitis
Peritoneal inflammation in male mice was produced according to the method described by Ferrándiz and Alcaraz, 1991 [25]. Carrageenan was freshly prepared (10 mg/mL) in sterile 0.9% w/v saline, and 250 μL were injected i.p., After 4 h, the animals were killed by cervical dislocation. The peritoneal cavity was washed with 3.0 mL cold PBS, and after a gentle manual massage, the exudate was retrieved and its volume was measured. The number of recruit leukocytes to the peritoneum was counted in a Newbauer chamber and results were expressed as cells × 10 6 /mL. The 3-amino-thiophene-2-acylhydrazone derivatives 5a-i were tested orally in doses of 100, 30, 10, 3 and 1 µmol/Kg. The carrageenan group (Arabic gum, p.o.) and the reference drug (indomethacin, 100, 30, 10, 3 and 1 µmol/Kg, p.o.) were administered 30 min before the carrageenan injection. These five doses were perfomed in order to calculate the ID 50 . The ID 50 was calculated by nonlinear regression. In the negative control group, animals have just received the same dose of a vehicle (arabic gum, p.o.) 30 min before the saline injection by intraperitoneal route.

Arthritis Model Induced by Complete Freund's Adjuvant (CFA) in Rats
Before the onset of arthritis, Wistar rats were randomly divided separately into five groups: the normal control group, the AA model group, the positive control group (i.e., dexamethasone; 5 µmol/Kg, pathway orally, daily), 5a (50 µmol/Kg, pathway orally, daily), 5d (50 µmol/Kg, pathway orally, daily), and Complete Freund's adjuvant (CFA) (0.2 mL/paw) was injected into the right paw of the rats. The CFA solution was prepared as a 1 mg/mL suspension of heat-killed Mycobacterium butyricum in 0.85 mL of paraffin oil and 0.15 mL of mannide monooleate (Sigma-Aldrich) [26]. At 14 days after the CFA injection the animals received during seven days treatment with derivatives 5a, 5d and dexamethasone. The paw volumes were measured on the 1st, 14th, 15th, 16th, 17th, 18th, 19th, 20th and 21st days using a digital caliper (Mitutoyo). Mean paw volumes were obtained daily for treated groups and compared with the paw volumes of the positive control group. At the 21st day the animals were anesthetized with a solution ketamine/diazepam (75 mg/Kg and 5 mg/Kg, i.p.), blood was collected by cardiac puncture for biochemical measurements using an Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Urea and Creatinin, methods kit (Doles-Brazil). After collection deeper anesthesia was applied to euthanize the animals. The arthritic paw was collected for histological analysis and the spleen was collected for weighing.

Biochemical Measurements
The blood was collected by cardiac puncture using 50 µL of EDTA as an anticoagulant. The blood was centrifuged after collection was centrifuged at 1500 rpm for 5 min. After centrifugation the serum was aliquoted and used for carrying out the biochemical levels of urea, creatinine, AST and ALT. The level of AST/ALT was performed as follows. Two hundred µL of working reagent was placed in the wells of a 96 well plate, 20 µL of serum was added and homogenized performed as reading spectrophotometer wavelength at 340 nm. This was done with the initial reading and repeated three readings at intervals of 1, 2, and 3 min to calculate the Delta Abs. The formula used to calculate the dose of ALT/AST is below: ALT UI/L = ΔA/minute × 1746 AST UI/L = ΔA/minute × 1746 The urea was carried expressed as follows: 100 µL of reagent 1 was placed in the cavity of a 96 well plate, and 5 µL of urease and 1 µL of serum sample or standard were added. The plate was incubated in a spectrophotometer for 5 min at 37 °C and then added to 100 µL of reagent 2. After that the reading plate with a wavelength of 600 nm was performed. The quantity of urea was calculated following the formula below: Factor (F) = 80 Absorbance of the standard Urea (mg/dL) = Absorbance test × F The serum creatinine test was performed according to the following protocol: 200 µL of working reagent was aliquoted into a 96-well plate, and 20 µL of serum were added. The plate was incubated for 30 s in the spectrophotometer. After incubation took place after the initial reading and the final reading of 60 s. The formula used to calculate the amount of creatinine in the samples is as follows: Factor (F) = 5 (Absorbance initial − Absorbance final) standard Creatinine (mg/dL) = (Abs ini − Abs fin) sample × F 3.5.7. Macroscopic Analyses of the Stomach After euthanasia, the stomachs were removed for macroscopic examination of the occurrence of lesions with daily treatment for 7 days with dexamethasone (5 µmol/Kg, pathaway orally), 5d (50 µmol/Kg, pathway orally), and 5a (50 µmol/Kg, pathway orally), The mucosal damage was examined by means of a magnifying glass. For each stomach, the mucosal damage was assessed according to the following scoring system [28]. 0.5 Redness 1.0 Spot ulcers 1.5 Hemorrhagic streaks 2.0 Ulcers < 3, but ≤ 5 3.0 Ulcers > 5

Spleen Weight
In order to evaluate a possible immunosuppression promoted by the daily treatment for 7 days with dexamethasone (5 µmol/Kg, orally), 5d (50 µmol/Kg, orally), and 5a (50 µmol/Kg, orally), after euthanasia we proceeded with the removal of the spleen. The spleen was washed with PBS and weighed on an analytical balance. Treated groups were compared to the arthritic group [31,32].

Statistical Analysis
Data obtained from animal experiments are represented by mean ± standard error of the mean (mean ± S.E.M.). Statistical differences between the treated and the control groups were evaluated by test t of Student or ANOVA in the tutorial Prisma ® . Values were considered significant if * p < 0.05, ** p < 0.01 and *** p< 0.001.

Conclusions
In summary a series of functionalized 3-aminothiophene-2-acylhydrazone derivatives 5a-i were designed and synthesized. These new N-acylhydrazone derivatives showed potent analgesic and anti-inflammatory activities, with 5a and 5d standing out in this respect. These compounds were active in acute and chronic inflammation models. After animals' daily treatment for seven days with 5a and 5d, with a dose of 50 µmol/Kg by oral administration, they were unable did not present renal or hepatic toxicity. Moreover, 5d didn't demonstrate an immunosuppressive profile. Taken together, these data suggest the 3-aminothiophene-2-acylhydrazones 5a and 5d are new non-toxic, analgesic and anti-inflammatory lead-candidates. The mechanism of action of these two bioactive compounds is now being investigated.