Synthesis , X-ray diffraction study and pharmacological evaluation of 3-amino-4-methylthiophene-2-acylcarbohydrazones

N-acylhydrazone is an interesting privileged structure that has been used in the molecular design of a myriad of bioactive compounds. In order to identify new antinociceptive drug candidates, we described herein the design, synthesis, X-ray diffraction study and the pharmacological evaluation of a series of 3-amino-4-methylthiophene-2-acylcarbohydrazone derivatives (8a-t). Compounds were prepared in good overall yields through divergent synthesis from a common key intermediate and were characterized by classical spectroscopy methods. X-ray diffraction study was employed for unequivocal determination of the imine double bond stereochemistry. 8a-t were evaluated in vivo through oral administration using the classical writhing test in mice. N-acylhydrazone derivatives 8j and 8l displayed relative potency similar to dipyrone, highlighting them as promising analgesic lead-candidates for further investigation.


INTRODUCTION
The original definition of a privileged scaffold dates back to the year 1988 and was first described by Evans and coworkers (Evans et al. 1988).This concept refers to a single molecular framework, correlated to a minimum structural subunit, which affects more than one bioreceptor or enzyme target.The adequate functionalization allows the modulation of the different biological activities and of the selectivity.Rational employment of molecular modification strategies, such as molecular hybridization, bioisosterism, molecular simplification, homologation and conformational

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SONJA HERRMANN et al. restriction are applied to adapt and adjust the selected scaffold to the desired pharmacological application.These structural modifications lead to different patterns of lipophilic/hydrophilic nature, H-bonding donor/acceptor properties, electron donating/withdrawing profile, acid/basic character and conformational behavior of the pharmacophoric groups.On the other hand, the previously described synthetic accessibility and the possibility of rational modifications offer access to a wide range of applications for these molecular frameworks, representing an opportunity for rapid, economic and optimized discovery of new drug candidates (Duarte et al. 2007, Evans et al. 1988, Jones 2017).A well-known example of a privileged structure is the imidazole scaffold.This heterocyclic ring is found in bioactive compounds affecting a wide range of targets, e.g.cimetidine, a H1-receptor antagonist; losartan, an AT1-receptor antagonist; clotrimazole, an antifungal agent; metronidazole, an antimicrobial drug; mercaptopurine, a purine nucleoside antimetabolite; and theophylline, a nonselective phosphodiesterase inhibitor and nonselective adenosine receptor antagonist (Duarte et al. 2007).
We described herein the synthesis, X-ray diffraction study and in vivo and in vitro pharmacological evaluation of new 3-amino-4methylthiophene-2-acylcarbohydrazones (8a-t), designed by the introduction of a methyl group at position 4 of the thiophene ring in order to explore the methylation effect (Barreiro et al. 2011) in the biological profile of this new series of NAH derivatives (Figure 2).Moreover, the design concept explored the possibility to employ the same starting material used in the synthesis of the local anesthetic articaine (i.e.methyl 3-amino-4methylthiophene-2-carboxylate, 9) (Li et al. 2013), already known as a safe structural framework, contributing to the synthetic accessibility and druglike properties of the NAHs (8a-t) described here.

CHEMISTRY
All commercially available reagents and solvents were used without further purification.Reactions were routinely monitored by thin-layer chromatography (TLC) in silica gel plates (KieselGel 60 F254 Merck).The mobile phase consisted of a mixture of 70% hexane/30% ethyl acetate or 95% dichloromethane/5% methanol.The developed plates were examined with UV lamps (in wavelength of 254 nm and 365 nm) or by employing the color reagents iodine, 2,4-dinitrophenylhydrazine (coloring ketones or aldehydes) or 4-dimethylaminobenzaldehyde (highlighting amines).The melting points (m.p.) of the final products were determined by differential scanning calorimetry (DSC) using a Shimadzu DSC-60, with a heating rate of 20 °C/min and a maximum temperature of 300 °C.The results were recorded as the onset temperatures (Tonset), which are defined as the point of intersection of the tangent of the peak with the extrapolated baseline.The apparatus DSC-60 was calibrated with indium (In, m.p. 157 °C).
Mass spectrometry (MS) was performed by positive ionization at Bruker AmaZon SL.EI-ion-trap MS (electrospray ionization ion trap mass spectrometry) and the data were analyzed by Compass 4.0 software.The relative purity of the final compounds (8a-t) was determined by high performance liquid chromatography (HPLC) using a Shimadzu apparatus-LC20AD, column Kromasil 100-5 C18 (4.6 mm x 250 mm) and detector SPD-M20A (Diode Array) in the substance-specific wavelength (240-370 nm), employing a constant flux of 1 mL/min with an injection volume of 20 µL.A mixture of 70% methanol/30% water (A) or 80% methanol/20% water (B) was used as solvent.Standard HPLC solvents were purchased from TEDIA ® .Data were acquired by software "LC solution" version 4.0.Infrared (IR) spectra were obtained using a Thermo Nicolet IS 10 FT-IR spectrometer equipped with smart iTR ATR accessory for direct measurements.
The 1 H and 13 C nuclear magnetic resonance spectra were determined in DMSO-d 6 solutions using a Bruker AC-200 spectrometer with 200
MHz for 1 H and 50 MHz for 13 C (LABRMN, UFRJ).The chemical shifts are given in parts per million (δ) from solvent residual peaks and the coupling constant values (J) are given in Hz.Signal multiplicities are represented by: s (singlet), d (doublet), t (triplet) and m (multiplet).The synthetic methodologies and the detailed structure analysis for the target compounds (8a-t) are described in the Experimental Section.

X-RAY DIFFRACTION STUDY
In order to obtain a good X-ray powder diffraction (XRPD) pattern, the sample preparation is an important step.The selected compound 8j was gently hand-grinded using a pestle and an agate mortar.The measurement was carried out at room temperature on a STADI-P powder diffractometer (Stoe ® , Darmstadt, Germany) using transmission geometry with CuK α1 (λ = 1.54056Å) radiation.
The wavelength was selected by a curved Ge (111) crystal, with a tube voltage of 40 kV and a current of 40 mA.The fine powdered sample was loaded into a 0.3 mm diameter special glass capillary nr.14 (Hilgenberg ® , Malsfeld, Germany), which was kept spinning during data collection.A Mythen 1K (Dectris ® , Baden, Switzerland) linear detector was used and the reflections were detected in the range from 3° to 61.785°, with step sizes of 0.015° and 600 s of integration time at each 1.05°.On the basis of previous procedures (Costa et al. 2015(Costa et al. , 2013)), the crystal structure of 8j was determined using the DASH software program (David et al. 2006).Topas-Academic v.5 (Coelho et al. 2011) was used to index the diffraction pattern as well as to refine the crystal structure.
The number of writhes, a response consisting of the contraction of the abdominal wall and pelvic rotation, followed by hind-limb extension, was counted during continuous observation for 20 min, beginning 5 min after the acetic acid injection.NAH derivatives 8a-t (100 μmol/kg, oral administration) were administered 40 min before the acetic acid injection.The control group received 10 mL/kg of the vehicle (distilled water with 20 μL of Tween 80) via the i.p. route.Antinociceptive activity was expressed as percentage of inhibition of the usual number of writhing observed in control animals (Collier et al. 1968).The Research Ethics Committee from the Federal University of Alagoas (UFAL) approved the animal experimental model presented in this study and the process protocol number is nº 55/2013.

p38 MAPK inhibition assay
The experimental settings and screening procedures of the employed nonradioactive immunosorbent p38α mitogen-activated protein kinase (p38α MAPK) activity assay have previously been described (Goettert et al. 2010).The ELISA assay was performed using 96-well plates (Nunc MaxiSorp®, Fisher Scientific), which were coated with 50 µL/well (10 µg/mL) in Tris-buffered saline (TBS) of the p38α substrate ATF-2 (ProQinase, Freiburg, Germany) and stored overnight at 4 °C.Subsequently, each plate was washed three times with bi-distilled water and remaining binding sites were blocked with blocking buffer (BB: 0.05 % Tween 20, 0.025 % bovine serum albumin (BSA) and 0.02 % NaN 3 in TBS) for 30 minutes at room temperature and washed again three times.A 10 mM stock solution of samples was prepared in dimethyl sulfoxide and further diluted in a kinase buffer (KB), which contained 12 ng / 50 µL activated p38α MAPK (Prof.Dr. J. Schultz, University of Tübingen, Germany), 50 mM Tris of pH 7.5, 10 mM MgCl 2 , 10 mM β-Glycerolphosphate, 100 µg/ mL BSA, 1 mM Dithiothreitol, 0.1 mM Na 3 VO 4 , and 100 µM ATP were used to dilute the samples and as control.50 µL of each dilution (0.01-10 µM) was pipetted into the corresponding wells and incubated 1 hour at 37 °C.After washing this dilution three times with bi-distilled water, blocking it for 15 minutes and washing it three more times, the 50 µL of diluted monoclonal anti-phospho-ATF-2 (Thyr69/71) peroxidase-conjugated antibody (1:5000) (Sigma, Germany) in blocking buffer adjusted to a pH of 6.5 was added into each well and incubated for 1 hour at 37 °C, followed by adding 50 µL of 3,3',5,5'-tetramethylbenzidine (TMB) (BD Bioscience, Europe) substrate into all wells.Then, the peroxide-labeled conjugates developed a definitive blue color, which was measured photometrically at 650 nm or was read at 450 nm with an ELISA reader (SOFTmax PRO software) after stopping with 25 µL of 2 N H 2 SO 4 .The inhibitor SB 203580 (Laboratory Prof. Dr. S.A. Laufer, University of Tübingen, Germany) was used as reference.
It's relevant to mention that NAHs may exist as E/Z geometrical isomers regarding the -C=Ndouble bond configuration, and may be observed as cis/trans amide (-CO-NH-) conformers (Abdel-Aziz et al. 2012, Khalil et al. 2013).To address this subject a careful analysis of the 1 H-NMR signals recorded for compounds 8a-t in DMSO-d 6 was conducted.Only one single signal related to the imine hydrogen (CH=N) was detected, with chemical displacement varying between 7.81 ppm and 8.21 ppm.These data indicate that the NAH derivatives 8a-t were synthesized in just one geometrical isomer.Based on chemical shifts of E/Z-isomer reported in literature (Palla et al. 1982), compounds 8a-t were assumed to have the E configuration for their imine double bond.To confirm this assumption, XRPD studies were performed using compound 8j as a model.As depicted in Figure 4, the crystal structure of 8j is arranged in a monoclinic space group (Cc), with unit cell dimensions a = 38.971(6)Å, b = 4.8314(6) Å, c = 18.843(2)Å, β = 109.964(4)° and V = 3334.6(8)Å 3 .The goodness of fit indicator and R-factors were, respectively: χ 2 = 3.153, R exp = 1.166%,R wp = 3.675% and R Bragg = 2.945%.The crystal structure of compound 8j is comprised by eight formula units per unit cell (Z = 8), accommodating two molecules in the asymmetric unit (Z' = 2).The relative configuration E about the imine double bond was observed, corroborating with the assignment based on NMR shifts.Regarding the conformation of the amide subunit (CONH), X-ray powder diffraction studies revealed a cis-conformation in the crystal structure of 8j.The hydrogen inter/intramolecular interactions contribute to the organization of the space arrangement in the unit cell.The intermolecular interactions can be observed between atoms Considering the previously described analgesic and anti-inflammatory properties of 3-aminothiophene-2-acylhydrazones 7a and 7b (Da Silva et al. 2014), we decided in a first approach to study the antinociceptive profiles of the methylated analogues 8a-t using the classical writhing test.
In this animal model, peripheral pain is induced in mice by a parenteral administration of acetic acid.Consequently, signals are sent to the central nervous system, causing release of several mediators, including prostaglandins, which contribute to increased sensitivity of nociceptors.
As summarized in Table I, the 3-amino-4methylthiophene-2-acylcarbohydrazones 8a-t were evaluated in a screening dose of 100 µmol/kg by oral administration, using dipyrone as standard.Among all evaluated compounds, ten derivatives (8b, 8c, 8d, 8f, 8h, 8i, 8m, 8p, 8s and 8t) were inactive in comparison to control, not being able to reduce the amount of constrictions.8e, 8o and 8r presented weak inhibitory activity, whereas 8a, 8g, 8k, 8n and 8q showed moderate antinociceptive effect at this dose.Explicitly, two derivatives (8j and 8l) demonstrated an expressive antinociceptive activity at the evaluated dose, with 70.0% and 70.2% of inhibition, comparable to the inhibition found for the standard dipyrone.
In comparison to the non-methylated original analgesic and anti-inflammatory prototypes 7a (Ar = phenyl; ID 50 = 3,5 µmol/kg in acetic acid-induced writhing model, p.o.) and 7b (Ar = 4-nitro-phenyl; ID 50 = 2,6 µmol/kg in acetic acid-induced writhing model, p.o.) (Da Silva et al. 2014), the methylation effect was deleterious for the antinociceptive profile of the methylated counterparts 8a (Ar = phenyl) and 8c (Ar = 4-nitro-phenyl).On the other hand, regarding the aromatic ring linked to the imine carbon, the introduction of more hydrophobic and bulky aromatic systems such as 4-biphenyl (8j) and 2-naphthyl (8l), not yet explored for the previously described 3-aminothiophene-2-acylhydrazones, proved to be useful for the optimization of the antinociceptive effect within the series.
Particularly, the p38 mitogen-activated protein kinase (p38 MAPK) plays a key role in inflammatory disorders, and it has been also implicated in the signal transduction cascade associated with chronic nociception and nociceptive sensitization (Anand et al. 2011, Lin et al. 2014, Zarubin and Han 2005).Our research group already described novel NAH derivatives designed as p38 MAPK inhibitors as orally active anti-inflammatory and antinociceptive drug candidates (Lacerda et al. 2012).Therefore, we also investigated the ability of compounds 8a-t to inhibit the enzymatic activity of p38α MAPK, using a nonradioactive immunosorbent assay and SB203590 as standard.As depicted in Table I, at the screening concentration of 10 µM, NAH derivatives 8a-t revealed weak p38α inhibition or were totally inactive.Although compounds 8a, 8d, 8m, 8q, 8s and 8t presented inhibitory percentages above 30%, none of the investigated NAH derivatives reached an inhibitory effect of 50%, indicating IC 50 values above the tested concentration of 10 µM.Moreover, no correlation was observed between the in vivo antinociceptive activities and the in vitro p38 MAPK inhibitory effect, indicating that this protein kinase is not the main biological target of the 3-amino-4-methylthiophene-2acylcarbohydrazone bioactive analogues, mainly 8j and 8l.

CONCLUSIONS
In an attempt to identify new antinociceptive drug candidates, twenty 3-amino-4-methylthiophene-2-acylcarbohydrazones (8a-t) were synthesized in good overall yields and, based on X-ray powder diffraction studies realized with compound 8j, the relative configuration of their imine double bond was elucidated.Moreover, these studies revealed a cis-conformation for the amide subunit in the crystal structure of 8j.The compounds described herein were overall less potent than the nonmethylated original series, with exception of the new NAH derivatives 8j and 8l, which displayed relative potency similar to dipyrone, a traditional analgesic used worldwide for the treatment of acute and chronic pain.These results suggest that the introduction of more hydrophobic and bulky aromatic systems linked to the imine carbon, such as 4-biphenyl (8j) and 2-naphthyl (8l), represents a favorable structural modification for optimization of the antinociceptive effect within the described series.

Synthesis of the key intermediate (3-amino-4methylthiophene-2-carbohydrazide, 10)
In a 100 mL flask equipped with a magnetic stirrer and a reflux condenser, methyl 3-amino-4- methylthiophene-2-carboxylate (9) (1.02 g; 5.96 mmol) was dissolved in absolute ethanol (10.0 mL) and heated at 80 °C.Afterwards, an 80% solution of hydrazine monohydrate (9.36 g; 3.07 mL; 292 mmol) was slowly added to the reaction medium, which was constantly stirred at 80°C.The reaction mixture was maintained under reflux for 80 hours, at which time TLC (70% hexane/30% ethyl acetate; 4-dimethylaminobenzaldehyde) indicated the end of the reaction.After cooling, reaction media was concentrated under reduced pressure.By adding crushed ice to the flask, the desired carbohydrazide (10) precipitated as yellow crystalline solid and was collected by vacuum filtration.The key

General procedure for synthesis of target compounds 8a-t
In a 50 mL flask equipped with a magnetic stirrer, 3-amino-4-methylthiophene-2-carbohydrazide (10) (0.15-0.30g; 0.87-1.75mmol) was dissolved in absolute ethanol (12.0 mL).Next, the corresponding aldehyde (0.12-0.29 g; 0.97-1.85mmol) and three drops of hydrochloric acid 37 % were added and the reaction mixture was stirred at room temperature for 0.5-3.5 hours, until TLC examination indicated the end of reaction.Isolation was performed by product precipitation after adding crushed ice to the flask.Products 8a-t were collected by vacuum filtration and purified properly by recrystallization from absolute ethanol if necessary.

Figure 4 -
Figure 4 -(Top) Unit cell representation of compound 8j recognized by X-ray powder diffraction.(Bottom) The hydrogen inter/intramolecular interactions between the atoms from different equivalent symmetry operations are displayed in cyan lines.

TABLE I Effect of the target compounds (8a-t) and dipyrone (100 µmol/kg; p.o.) on the 0.6% acetic acid-induced abdominal constriction model in mice (period of 25 min), and, in the last column, percentage of inhibition of p38α MAPK enzymatic activity for the compounds (8a-t) and positive standard SB-203580 (10µM) in the nonradioactive immunosorbent assay. Compound Abdominal constrictions P38αMAPK Inhibition [%] (Mean ± S.E.M.) Percentage of Inhibition [%]
Data are expressed as mean ± S.E.M. Statistical differences between the treated and the control group were evaluated by ANOVA and Dunnett tests; the asterisks denote the levels of significance in comparison with control groups.*p<0.05;**p < 0.01; ***p< 0.001.intermediate 10 was collected with a yield of 74.1 %.The 1 H NMR and13C NMR data for compound 10 are consistent with previous reports (Al-Nuri and Husein 2011).