Synthesis , Characterization , Crystal Structure and Antimalarial Activity of ( 2 E )-2-( 1-{ 4-[ ( 7-chloroquinolin-4-yl ) amino ] phenyl } ethylidene ) hydrazine Carbothioamide

A simple synthesis and study by UV-vis, IR, NMR, ESI-CID-MS2 and X-ray diffraction of ((2E)2-(1-{4-[(7-chloroquinolin-4-yl)amino]phenyl}ethylidene)hydrazinecarbothioamide is reported. It was tested in vitro against chloroquine-resistant strain (W2) of Plasmodium falciparum, hemozoin (β-hematin) formation and cysteine protease falcipain-2. In general, it was found to possess a proved activity in its inhibitory power on the parasite but less active on the formation of hemozoin (β-hematin) and falcipain-2. Also, the X-ray analysis presented an unexpected electronic density that can be assigned like S(2). This electronic density can be attributed to autocondensation of thiosemicarbazide, generating H2S as a subproduct.


MATERIAL AND METHODS
Melting point was determined on a Stuart Scientific Digital Melting Point Apparatus SMP3.IR spectrum was determined as KBr pellet on a Shimadzu model 8400 spectrophotometer.The spectra in the UV-vis was recorded from solution of compound, at concentration 10 -5 M, in a Genesys 10S UV-vis scanning spectrophotometer (Thermo Electron Corporation) equipped with a high-intensity xenon lamp and quartz cell of 10 cm.The spectrum was taken in a 190-1100 nm scan range, at medium speed and a photometric absorbance range between 0.0-3.0Å.The 1 H NMR, 13 C NMR spectra were recorded using a Jeol Eclipse 270 (270 MHz/67.9MHz) spectrometer using DMSO-d6, and are reported in ppm downfield from the residual DMSO.Accurate mass measurement was performed on a Finnigan/Thermo TSQ Quantum triple Quadrupole Mass Spectrometer.Chemical reagents were obtained from Aldrich Chemical Co, USA.All solvents were distilled and dried in the usual manner.

Single Crystal X-ray Data Collection and Structure Determination
The single crystal X-ray diffraction data was carried out on a KAPPA APEX II DUO Diffractometer, with graphite monochromator and Mo-Kα radiation (λ=0.71069Å) operating at 50 kV and 30 mA.A total of 3387 frames were collected with ϕ and ω scans at every 0.30° for 10 s each.Data collections and unit cell refinement were carried out with SMART [7] and data reduction with SAINT [8].
The structure was solved and refined using the Bruker SHELXTL Software Package [9].The nonhydrogen atoms were refined anisotropically, while the hydrogen atoms bound to C atoms were placed geometrically and refined using a Riding model, with C-H = 0.93 Ǻ, Uiso(H) = 1.2 Ueq(C) for aryl H; C-H = 0.96 Ǻ, Uiso(H) = 1.2 Ueq(C) for methyl H; N-H = 0.86 Ǻ and Uiso(H) = 1.2Ueq(N).The final anisotropic full-matrix least-squares refinement on F2 with 263 variables converged at R1 = 4.60%, for the observed data and wR2 = 13.35% for all data.The goodness-of-fit was 1.05.The largest peak in the final difference electron density synthesis was 0.50 e-/Å 3 and the largest hole was -0.27 e-/Å 3 .On the basis of the final model, the calculated density was 1.436 g/cm 3 and F(000), 438 e-.The details of crystal data and refinement are given in Table 1.
Comprehensive crystallographic data (excluding structure factors) for the structural analysis of 3 have been deposited with the Cambridge Crystallographic Data Centre.Copies of the data (CIF file) can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, fax: +44-(0)1223-336033, or from https://summary.ccdc.cam.ac.uk/structure-summaryform, quoting deposition No. CCDC 1413401.

Synthesis
of 4-[(7-chloroquinolin-4yl)amino]acetophenone (1) was carried out by refluxing of 4,7-dichloroquinoline with 4aminoacetophenone in ethanol [6].Thiosemicarbazone (3) was obtained from a solution of equimolar amounts of acetophenone 1 and thiosemicarbazide (2) in methanol, with constant stirring for 7 hours at 65 °C and pH 4.0.TLC (EtAc:Hx 7:3) showed only a compound produced (Scheme 1).Spectroscopic data ( 1 H and 13 C NMR) show that compound 3 was a product of a reaction of nucleophilic addition, because of the following spectral evidence: three 1 H resonances are observed due to chemical function NH 2 and NH (N-NH-CS) at 8.02, 8.33 and 10.30 ppm; respectively.Also, as can be seen, a remarkable upfield shifting effect on the chemical shift value of protons and carbon in the methyl group, with a 1 H resonance at 2.62 ppm in 1 and 2.34 ppm in 3, and a 13  This upfield shifting suggests a protective effect (shielding) on the carbon and hydrogen atoms due to the steric effect exerted by the -NHCSNH 2 group.The chemical shift value found for the carbon atom of the methyl group falls within the range reported in the literature for thiosemicarbazones derived from acetophenones, ranging between 10 to 15 ppm when methyl group is located in syn position relative to NHCSNHR group [10][11][12].NMR data suggest that compound 3 has E geometry.CID-MS 2 mass spectrum shows that a proton transfers from the quinolinic-N atom to N-4 atom of the thiosemicarbazone occur before fragmentation of quasi-molecular ion (ion precursor).Subsequently, relevant fragmentations were essentially based on concomitant loss of small molecules and radicals (such as ammonia, thiocyanate radical, methyl radical and hydrogen cyanide) in the sequence: 370 → 353 → 295 → 280 → 254 (Figure 2).These peaks agree with molecular mass and structure of thiosemicarbazone 3.    The X-ray analysis confirmed the molecular structure of compound 3.A previous search in the Cambridge Structural Database (CSD) [13] has not produced any results.Tables 2 and 3 contain relevant bond lengths and angles for compound 3, as well as hydrogen bond geometries as calculated with PLATON [14].Figure 3 show the molecular structure of compound 3 with the atom numbering scheme.Graphics were obtained using Diamond 3.0 [15].Like shown in Figure 3, compound 3 not present geometry planar, and the dihedral angle between the A/C ring and B ring is 26.56º.This compound exhibit one molecule of water in its crystal packing.Furthermore, also present an electronic density that can be assigned like S(2).This electronic density can be attributed to autocondensation of thiosemicarbazide, generating H 2 S as a subproduct.
Compound 3 was investigated to determine its in vitro antimalarial activity against chloroquineresistant strain (W2) of Plasmodium falciparum, hemozoin (β-hematin) formation and cysteine protease falcipain-2; using methodology previously reported [4,16,17].The evaluated compound shows antiplasmodial activity at low 50% inhibitory concentration (IC 50 ), with value of 0.45 µM ± 0.0699.This confirms that our medicinal chemistry strategy of molecular hybridization based in leader compounds is effective against this parasitic disease.
Compound 3 also showed a 58.89 ± 0.281% inhibition of the formation of hemozoin (% IFβH), a low value when compared to the control of chloroquine (91.44 ± 0.02 %IFβH).The inhibitory capacity of 3 on the cysteine protease recombinant falcipain-2 was not significant (IC 50 > 50 µM).In general, thiosemicarbazone 3 proved to be very active in its inhibitory power on the parasite but less active on the formation of hemozoin (β-hematin) and falcipain-2, which allows us to suggest that the biochemical mechanism that justifies the antiplasmodial activity of this compound must be other than to interactions with the ferriprotoporphyrin IX (heme) and the cysteine protease.

CONCLUSION
The synthesis and obtaining of ((2E)-2-(1-{4-[(7-chloroquinolin-4-yl)amino]phenyl}ethylidene) hydrazinecarbothioamide was confirmed by their physical constants, UV-vis, IR, ESI-CID-MS 2 , NMR spectral data and single crystal X-ray diffraction data.In general, it was found to possess a proved activity in its inhibitory power on the parasite but less active on the formation of hemozoin (β-hematin) and falcipain-2.Also, the X-ray analysis presented an unexpected electronic density that can be assigned like S(2).This electronic density can be attributed to autocondensation of thiosemicarbazide, generating H 2 S as a subproduct.

Figure 3 .
Figure 3. Molecular structure of compound 3 showing the atomic numbering.The displacement ellipsoids are drawn at 50% probability.

Figure 4 .
Figure 4. Part of the crystal structure of compound 3 showing the formation of a hydrogen-bonded R 2 2 (8) dimer.

Table 1 .
C resonance at 27.21 ppm in 1 and 13.30 ppm in compound 3. Crystal data, intensity data collection parameters and final refinement results for compound 3.