Patent Publication Number: US-10316019-B2

Title: Method for preparing the anhydrous crystalline form of isoniazid-derived hydrazone, thus produced crystalline polymorph of the anyhydrous form, use thereof for the treatment of alzheimer&#39;s disease, parkinsonism and other neurodegenerative disorders, and pharamceutical composition

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority on and the benefit of U.S. patent application Ser. No. 15/106,181 having a filing date of 17 Jun. 2016, which claims priority on and the benefit of and is the US National Phase under 35 USC 371 of International Application No. PCT/BR2014/000186 having an international filing date of 6 Jun. 2014, which claims priority on and the benefit of Brazilian Patent Application No. 10 2013 033006-0 having a filing date of 20 Dec. 2013. 
     BACKGROUND OF THE INVENTION 
     Field of the Invention 
     The present invention regards the preparation method of the anhydrous crystalline form of an isoniazid-derived hydrazone (namely, 8-hydroxyquinoline-2-carboxaldehyde isonicotinoyl hydrazone, or INHHQ), the polymorph produced and its use, in any pharmaceutical composition, for the treatment of Alzheimer&#39;s and Parkinson&#39;s diseases, as well as other neurodegenerative disorders. 
     Prior Art 
     With the general increase in life expectancy recorded in recent times, especially in developed countries, the prevalence of certain neurodegenerative diseases has been observed. Alzheimer&#39;s disease (AD), first characterized in 1906 by the german neuropathologist Alois Alzheimer, is currently the most common one. It is a primarily age-related disease and the most common cause of dementia in older people. Dementia is characterized by progressive loss of memory and cognitive functions, among other diagnostic criteria, being most of them present in the Diagnostic and Statistical Manual of Mental Disorders and described on the National Institute of Neurological and Communicative Disorders Association. 
     The disease has two general classifications: 1) late onset, which occurs with the highest incidence at about 60 years of age, and 2) early onset, occurring around 40 years. In the US and Great Britain, it represents about 50% of cases of dementia, being estimated that it is the fourth leading cause of deaths of elderly in these countries. Regarding the neuropathological aspect, patients show diffuse cortical atrophy, presence of senile plaques and neurofibrillary tangles, neurovascular degeneration and neuronal loss. 
     These senile plaques are characterized mainly by the presence of fibrillar deposits of β-amyloid peptide (Aβ), consisting of approx. 40 amino-acid residues. It has been observed high concentration of physiological metal ions such as Zn 2+  and Cu 2+ , in these plaques, which is considered indicative that the interaction of Aβ with these biometais is at the heart of events that lead to aggregation and toxicity of this peptide. The ferric ions, in turn, have also been related to the aggregation of neurofibrillary tangles, in addition to contributing to the oxidative processes that occur in the nerve cells of the body. Something similar occurs in Parkinson&#39;s disease with the proteina-synuclein. 
     The already approved drugs for the treatment of AD are intended to combat deficits associated with reduced cerebral function and fall into two classes: acetylcholinesterase inhibitors and inhibitors of NMDA (N-Methyl-D-Aspartate) receptors. Such drugs seem to act in enhancing the remain of the cognitive function, however, are not able to prevent the progression of the disease, being, therefore, important the development of new therapeutic agents that hold the advance of neurodegeneration as well as, as far as possible, promote their regression. In this sense, an interesting approach is to obtain compounds that inhibit, specifically, abnormal metal-protein interactions. This class of drugs is known as MPACs (metal-protein attenuating compounds) and relate to the allocation and distribution normalization of physiological metal ions. 
     A classic example is the clioquinol (CQ or PBT1), a substance belonging to the group of 8-hydroxyquinolines, which, however, was abandoned due to certain unwanted side effects, such as subacute myelo-optic neuropathy. 
     
       
         
         
             
             
         
       
     
     8-hydroxyquinoline-2-carboxaldehyde isonicotinoyl hydrazone (or INHHQ) was first described in a series of publications from the year 2009, in which its interactions with some rare earths ions, namely, dysprosium(III), europium(III), holmium(III), neodymium(III), and ytterbium(III) were examined. Erbium(III) and terbium(III) complexes were reported in 2010, while the samarium(III) compound was described in 2011. In those works, its rare earths complexes, and not INHHQ itself, were proposed as potential anticancer drugs, since they bind to DNA through an intercalation mechanism, besides they possess antioxidant properties, scavenging hydroxyl and superoxide radicals. Although the method of preparation described by the authors of those papers, Liu &amp; Yang, is similar to the one employed by us, some experimental details are different. Mainly, the use of acid catalysis (drops of concentrated hydrochloric acid) allow us to reduce the reflux period to just an hour. On the other hand, and more important yet, the use of methanol PA ACS (99.8%) in the recrystallization step, instead of the methanol:water 80:20 used by the Chinese authors, lead to the preparation of the anhydrous crystalline form of the compound, which is completely original. Although this is not commented in the original articles, data suggest that the Chinese authors worked with an hydrated form of INHHQ, different from the one obtained by us, which can be clearly seen in the major differences between the melting points of the compounds, as well as between the vibrational (FTIR and Raman) spectra of both samples. Besides, application of INHHQ in the treatment of neurodegenerative diseases is completely new. 
     The anhydrous crystalline form described herein is characterized by its melting point, elemental analysis, crystal structure (single-crystal diffraction), powder diffraction pattern, and vibrational spectra. 
     BRIEF SUMMARY OF THE INVENTION 
     In the search for new MPACs, more effective and safer for patients, a hydrazone derived from the mycobactericidal agent isoniazid was produced, characterized in its crystalline form and successfully tested in modulating in vitro the interaction between β-amyloid peptide (or α-synuclein) and the physiological metals copper and zinc, which can have great impact on the treatment of Alzheimer&#39;s and Parkinson&#39;s diseases, as well as in other related diseases. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The present invention will hereinafter be illustrated with reference to the attached figures, which represent: 
         FIG. 1 : ORTEP representation of the compound INHHQ, showing the identification scheme of the constituent atoms; 
         FIG. 2 : Perspective views of INHHQ showing: (a) the hydrogen-bonding network, (b) the π-π and the O1-H12 . . . π stacking interactions and (c) the 3D crystalline packing along the crystallographic axis a; 
         FIG. 3 : Simulated and experimental X-ray diffractograms for INHHQ; 
         FIG. 4 : Most stable calculated structure in the gas-phase (Conf. 5). Level of theory: B3LYP/6-311+G (d,p); 
         FIG. 5 : FTIR (above) and Raman (below) spectra of INHHQ; 
         FIG. 6 : 2D  1 H× 15 N HSQC contour plots showing that the addition of INHHQ (4 eq.) to a mixture containing the β-amyloid peptide and Zn(II) (1 eq.) leads to a partial intensity recovery for all Aβ signals, which can be considered as an indicative that the hydrazone INHHQ weakens Zn(II)-Aβ interactions; 
         FIG. 7 :  1 H NMR spectra showing that the addition of INHHQ (4 eq.) to a mixture containinga-synuclein and Cu(II)/Cu(I) (1 eq.) leads to the recovery of the original methionine signals&#39; position; it is worth noting that methionine residues are the interaction point of copper with α-synuclein, which constitutes an indicative that INHHQ weakens Cu(II) or Cu(I)-α-synuclein interactions. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The isonicotinoyl hydrazone of 8-hydroxyquinoline-2-carboxaldehyde (or INHHQ) was synthesized, completely characterized in its anhydrous crystalline form and successfully tested in modulating the in vitro interaction between β-amyloid peptide (or α-synuclein) and the physiological metals copper and zinc, which can have applications in the treatment of Alzheimer&#39;s and Parkinson&#39;s diseases, amongst other neurodegenerative diseases (such as Huntington&#39;s). 
     
       
         
         
             
             
         
       
     
     Connected to the 8-hydroxyquinoline portion, characteristic of CQ, INHHQ also contains the mycobactericidal drug isoniazid (INH), resulting in a potentially interesting hydrazone capable of coordinating metal ions of biological importance through its various N/O-donor sites. Furthermore, linking two molecules that have, individually, specific activity, creating a single species, has been an attractive approach to rational drug development, since a combination of the two original activities can be expected of the hybrid molecule. Besides, isoniazid-derived hydrazones are well known as iron chelating agents. However, to the best of our knowledge, there are no studies in the literature involving the coordination of INHHQ to any transition metal. 
     In the context of the treatment of AD and Parkinson&#39;s disease, the coordination capacity of the ligand to essential transition metals (such as copper, zinc and iron) must be better understood. As a first approach to this problem, we present the results of a spectroscopic vibrational (FTIR/Raman) and a single-crystal X-ray diffraction structural study on the 8-hydroxyquinoline-2-carboxaldehyde isonicotinoyl hydrazone, obtained through the process herein described and claimed, including its crystal structure, vibrational spectra and their full assignment by computational methods based on the Density Functional Theory (DFT). 
     Preparation Methodology of the Anhydrous Crystalline Form of 8-hydroxyquinoline-2-carboxaldehyde isonicotinoyl hydrazone 
     To 25 mL of a 8-hydroxyquinoline-2-carboxaldehyde (1.04 g, 6.0 mmol) solution in ethanol, 25 mL of ethanolic solution of isoniazid (0.82 g, 6.0 mmol) were dropwise added. Two drops of concentrated hydrochloric acid were added as a catalyst for the reaction. After refluxing for 1 h, the mixture was cooled to room temperature and the yellow precipitate obtained was filtered and vacuum dried. Next, the product was recrystallized in hot methanol PA ACS (99.8%). A pale yellow crystalline solid was obtained after cooling and dried at room temperature. After few days, single-crystals of INHHQ were isolated from the mother liquor. Total yield of the process: 0.98 g (56%). 
     Characterization: m.p.: 246-249° C. 
     Elemental analysis: calculated for C 16 H 12 O 2 N 4 : C, 65.7%; H, 4.1%; N, 19.2%—found: C, 66.3%; H, 4.1%; N, 19.4%. 
     Main IR bands (KBr, cm −1 ): 3396 (v OH); 3183 (v NH); 1656 (v C═O+β NH); 1647 (v C═N azomethine group); 1556 (v C═N e v C═C from the quinolinic ring) and 1545 (v C═C e v C═N from the pyridinic ring). 
     X-Ray Diffraction Analysis 
     The X-ray diffraction (XRD) was performed using an appropriate single-crystal of INHHQ. The sample was measured on a diffractometer Enraf-Nonius Kappa-CCD with Mo K α  (A=0.71073 Å) radiation. The unit cell parameters were based on all reflections. Data were collected at room temperature (293 K) using the computer program Collect being the integration and scaling of reflections made with Denzo-Scalepach system of HKL programs. The crystal structure was solved by direct method with SHELXS-97 and the atoms, except hydrogens, were anisotropically refined by the least squares method on F2 using the SHELXL-97 program. All aromatic and hydroxyl group&#39;s hydrogen atoms were placed in the calculated positions (C—H: 0.98 Å, O—H: 0.82 Å). Shift factors were taken as U(H) isot =1.2/1.5 Uhost. The H atoms attached to the C7 carbon and N2 nitrogen were located in the Fourier difference map and refined freely. The computer programs ORTEP-3 and Mercury (version 2.3) were used to draw the structures. On the other hand, powder X-ray diffraction experiments were performed in a Bruker D8 Discover XRD equipment using copper radiation. Experimental conditions: tension of 40 kV and current of 40 mA. The Bragg-Brentano geometry was used. 
     Spectroscopic Analyses 
     IR spectra were obtained on a Perkin-Elmer 2000 FT-IR spectrometer, using KBr sampling. On the other hand, the Raman spectra of the solid sample were measured on a Perkin-Elmer 400 Station equipment, using the 785 nm line for excitation. 
     DFT Calculations 
     The first step was to conduct a search of the conformational space of 8-hydroxyquinoline-2-carboxaldehyde isonicotinoyl hydrazone using the semi-empirical PM3 method, as implemented in the SPARTAN&#39;02 software, with the following set of parameters: Max-Confs=100; window=10 kcal mol −1 ; model=PM3. A total of 12 different conformations were found. The lowest energy conformation was of 41.387 kcal mol −1 . From this distribution, 5 conformations were selected for analysis by DFT. These conformations will be called, from now on, from Conf. 1 to Conf. 5. 
     After the selection of these five conformations, a complete geometry optimization, without limitation, using the default convergence criteria and calculation of harmonic vibrational frequencies, was performed in gas phase, for each, using the Gaussian program package 03 Gauss. The three parameters of the exchange-correlation potential proposed by Becke with local and non-local correlations provided by Lee, Yang, and Parr (B3LYP functional) were selected with the basis set of triple zeta valence 6-311+G (d,p). Thermal contributions to the Gibbs free energy and other state functions were calculated at 298.15 K and 1 atm. The vibrational frequencies were scaled by a factor of 0.9381 for a better comparison with the experimental data. 
     Molecular Structure 
     Crystallographic Analysis 
     The main crystal parameters used, as well as data of information collected and structural refinement parameters are summarized in Table 1. INHHQ crystallizes in the orthorhombic system, space group Pbca. The asymmetric unit of INHHQ is shown in  FIG. 1 . The compound adopts an (E) configuration with respect to the C7=N3 bond of the hydrazonic group. The INHHQ molecule is almost planarin solid state (rms deviation=0.27010 for all atoms, except H) and shows an intramolecular H bond involving the phenolic hydroxyl and the quinolinic nitrogen group: the O1-H donor interacts with N4 [O1 . . . N4=2,689 Å] receptor. In this process, a pseudo five-membered ring is formed. The distances and bond angles (Table 2) are not significantly different from those observed in similar compounds. The crystal packing is maintained by intermolecular H bonds involving the carbonyl oxygen O2 (acceptor) of a molecule and the N2-H group of the following molecule [moderate N2 . . . O2 i =2,966 Å, symmetry codes: (i)−x+½, y−½, z], connecting the molecules of INHHQ in zigzag chains running parallel to the crystallographic b axis ( FIG. 2( a ) ). The molecules in each chain are interconnected by cross-stacking π-π interactions involving the quinoline rings. The calculated centroid-centroid distance is equal to 3.8303(9) Å. The adjacent chains are interconnected by O1-H12 . . . π interactions ( FIG. 2( b ) ), being the distance H12-centroid (N4-C8-C9-C10-C11-C12) of 3.5339(17) Å [symmetry code: −½+x, y, ½−z]. As a result of this last interaction, zigzag columns run parallel to the crystallographic axis a ( FIG. 2( c ) ). 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Selected crystallographic data for INHHQ 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Empirical formula 
                 C 16 H 12 O 2 N 4   
               
               
                 Molecular weight 
                 292.30 
               
               
                 Collecting temperature 
                 293(2) K. 
               
               
                 Wave length 
                 0.71073 Å 
               
               
                 Crystalline system 
                 Orthorhombic 
               
               
                 Space group 
                 Pbca 
               
               
                 Unit cell dimensions 
                 a = 17.0761(4) Å  
               
               
                   
                 b = 8.25480(10) Å 
               
               
                   
                 c = 19.3549(4) Å  
               
               
                 Cell volume 
                 2728.26(9) Å 3   
               
               
                 Z 
                 8 
               
               
                 ρ(calculated) 
                 1.423 g cm −3   
               
               
                 Absorption coefficient 
                 0.098 mm −1   
               
               
                 F(000) 
                 1216 
               
               
                 Crystal size utilized 
                 0.484 × 0.236 × 0.171 mm 3   
               
               
                 θ range used in collect 
                 2.94 a 27.48° 
               
               
                 Index ranges 
                 −22.19; −10.10; −25.23 
               
               
                 Collected reflections 
                 27142 
               
               
                 Unique 
                 3100 [R (int)  = 0.1167] 
               
               
                 Completeness to θ = 27, 48° 
                 99.0% 
               
               
                 Absorption correction 
                 None 
               
               
                 Refinement method 
                 “Full-matrix least-squares on F 2”   
               
               
                 Computational programs a   
                 COLLECT, HKL Denzo and Scalepack, 
               
               
                   
                 SHELXS−97, SHELXL−97 
               
               
                 Data/restraints/parameters 
                 3100/0/208 
               
               
                 Goodness-of-fit on F 2   
                 1.042 
               
               
                 Final Rindexes [I &gt; 2σ (I)]  
                 R 1  = 0.0517, wR 2  = 0.1188 
               
               
                 R index (all data) 
                 R 1  = 0.0968, wR 2  = 0.1427 
               
               
                 Largest diff. peak and hole 
                 0.198 and −0.234 e Å −3   
               
               
                   
               
               
                   a Used for data collection, data processing, structure solution, and structure refinement, respectively. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Main distances and bond angles, experimental and theoretical (Conf. 5), for 
               
               
                 INHHQ. The crystallographic identification scheme of the atoms was followed 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Experi- 
                   
                   
                 Experi- 
                   
               
               
                   
                 mental 
                 Calculated 
                   
                 mental 
                 Calculated 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Distances (Å) 
                   
                 Distances (Å) 
                   
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 O2—C6 
                 1.2268(18) 
                 1.211 
                 N4—C12 
                 1.362(2) 
                 1.355 
               
               
                 N2—C6 
                 1.345(2)  
                 1.388 
                 N3—C7 
                 1.270(2) 
                 1.280 
               
               
                 N2—N3 
                 1.3862(18) 
                 1.353 
                 O1—C13 
                 1.358(2) 
                 1.351 
               
               
                 N4—C8 
                 1.319(2)  
                 1.325 
                 N1—C1 
                 1.333(2) 
                 1.335 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Angles (°) 
                   
                 Angles (°) 
                   
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 C6—N2—N3 
                 118.44(13) 
                 120.9 
                 N2—C6—C5 
                 115.04(13) 
                 114.0 
               
               
                 C8—N4—C12 
                 117.83(13) 
                 118.6 
                 C1—N1—C2 
                 116.20(15) 
                 117.2 
               
               
                 C7—N3—N2 
                 115.54(13) 
                 117.4 
                 O1—C13—C12 
                 118.65(15) 
                 118.8 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 Dihedral angles (°) 
                   
                 Dihedral angles (°) 
                   
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 C8—C7—N3—N2 
                 −177.63 
                 179.7 
                 N3—N2—C6—C5 
                 −178.62 
                 −178.6 
               
               
                 C7—N3—N2—C6 
                 −163.79 
                 −175.4 
                 N2—C6—C5—C3 
                 144.93 
                 −151.6 
               
               
                   
               
            
           
         
       
     
     Moreover, an analysis of the INHHQ diffraction peaks was performed using Bragg-Brentano geometry (Table 3). The ultimate goal is to characterize, unambiguously, the obtained polymorph. The crystal structure-based simulated diffractogram in the range of 5&lt;2θ&lt;70°, was also calculated.  FIG. 3  displays the superposition of the experimental (red) and simulated (blue) diffractograms. 
                     TABLE 3                  Information on the diffraction peaks of INHHQ        for 2θ ranging from 10.5 to 70.5°                                                         Interplanar       Structure                                 Plane (h, k, l)   Multiplicity   distance (Å)   2 theta (°)   factor                                         h   k   l   m   d       F 2                                                   1   0   0   2   8.34877   10.58783   1128.338       0   1   0   2   7.07920   12.49361   331.562       0   1   1   4   5.94931   14.87873   4309.398       1   0   −2   2   5.93863   14.90564   3564.964       1   −1   −1   4   5.50433   16.08920   1058.563       0   0   2   2   5.48852   16.13588   185.920       1   1   0   4   5.39942   16.40394   3734.208       1   −1   −2   4   4.54974   19.49495   3562.456       1   1   1   4   4.37713   20.27167   81.922       2   0   −2   2   4.37659   20.27421   2544.993       0   1   2   4   4.33757   20.45853   131.263       2   0   0   2   4.17439   21.26742   743.827       2   −1   −1   4   3.87923   22.90666   5227.304       1   0   2   2   3.87110   22.95541   2615.373       2   −1   −2   4   3.72262   23.88427   1558.836       2   1   0   4   3.59579   24.73979   177.345       0   2   0   2   3.53960   25.13890   1609.181       1   −1   −3   4   3.53090   25.20189   4821.653       1   1   2   4   3.39646   26.21681   4682.200       0   2   1   4   3.36879   26.43604   783.016       1   −2   −1   4   3.28146   27.15290   1838.082       1   2   0   4   3.25881   27.34522   1594.341       2   −1   −3   4   3.25355   27.39035   1187.558       0   1   3   4   3.25049   27.41659   1027.571       3   0   −2   2   3.08971   28.87348   81.686       2   1   1   4   3.08859   28.88417   2178.500       1   −2   −2   4   3.04049   29.35123   126085.008       1   0   −4   2   3.03231   29.43221   46210.547       1   2   1   4   2.98732   29.88574   3959.848       0   2   2   4   2.97465   30.01599   23814.010       2   0   −4   2   2.96931   30.07124   20257.656       3   −1   −2   4   2.83175   31.56923   1316.031       2   −2   −1   4   2.81367   31.77739   2147.033       3   −1   −1   4   2.78865   32.07025   962.328       1   −1   −4   4   2.78737   32.08534   5017.017       2   0   2   2   2.78466   32.11737   5954.872       3   0   0   2   2.78292   32.13796   198029.766       2   −2   −2   4   2.75217   32.50702   166846.703       0   0   4   2   2.74426   32.60332   78569.977       2   −1   −4   4   2.73820   32.67747   7683.784       2   2   0   4   2.69971   33.15667   5524.309       3   −1   −3   4   2.69759   33.18355   5725.885       1   1   3   4   2.69411   33.22766   3144.983       1   −2   −3   4   2.67192   33.51166   5080.103       1   2   2   4   2.61222   34.30092   184684.922       3   0   −4   2   2.61018   34.32857   75081.414       2   1   2   4   2.59139   34.58541   10269.332       3   1   0   4   2.58999   34.60470   3123.402       0   1   4   4   2.55873   35.04100   3632.206       2   −2   −3   4   2.54551   35.22898   128.840       0   2   3   4   2.54404   35.24991   2518.650       2   2   1   4   2.46414   36.43237   9364.604       3   −1   −4   4   2.44902   36.66531   6171.995       0   3   0   2   2.35973   38.10499   2742.504       3   −2   −2   4   2.32767   38.65065   49774.492       1   0   4   2   2.32176   38.75283   27728.582       4   0   −2   2   2.31874   38.80530   8126.180       3   1   1   4   2.31772   38.82317   7422.130       0   3   1   4   2.30703   39.01032   1802.321       3   −2   −1   4   2.30355   39.07167   4033.272       1   −2   −4   4   2.30283   39.08438   4588.360       2   −1   −5   4   2.30097   39.11728   4454.373       1   −3   −1   4   2.27839   39.52090   11520.123       2   −2   −4   4   2.27487   39.58469   7746.600       1   −1   −5   4   2.27151   39.64563   598.446       1   3   0   4   2.27077   39.65907   2802.268       3   −2   −3   4   2.25142   40.01449   3656.189       1   2   3   4   2.24939   40.05204   2882.448       1   1   4   4   2.20614   40.87197   4367.143       4   −1   −2   4   2.20355   40.92216   1606.162       1   −3   −2   4   2.19295   41.12888   5896.017       2   2   2   4   2.18856   41.21509   90978.344       4   0   −4   2   2.18829   41.22042   43250.957       3   2   0   4   2.18772   41.23171   13279.063       4   −1   −3   4   2.18705   41.24487   37423.781       2   1   3   4   2.18427   41.29979   20309.209       1   3   1   4   2.17275   41.52890   27249.758       0   2   4   4   2.16879   41.60831   20608.090       3   −1   −5   4   2.16859   41.61228   15481.041       0   3   2   4   2.16786   41.62688   3233.373       4   −1   −1   4   2.13480   42.30230   2642.791       3   0   2   2   2.13258   42.34844   20.261       2   −3   −1   4   2.10312   42.97079   2666.499       3   −2   −4   4   2.10076   43.02150   1828.689       0   1   5   4   2.09689   43.10495   17605.400       4   −1   −4   4   2.09069   43.23919   2405.904       4   0   0   2   2.08719   43.31522   1974.789       2   −3   −2   4   2.07706   43.53728   2901.627       2   3   0   4   2.05423   44.04625   3037.751       3   1   2   4   2.04194   44.32549   1874.683       1   −3   −3   4   2.04191   44.32613   1474.088       2   0   −6   2   2.03690   44.44102   2853.471       3   2   1   4   2.01612   44.92393   1408.763       1   3   2   4   2.01489   44.95273   793.879       2   −2   −5   4   2.00506   45.18528   8021.569       4   1   0   4   2.00199   45.25840   3930.259       1   −2   −5   4   1.98548   45.65592   1014.055       2   −3   −3   4   1.98380   45.69690   16302.775       0   3   3   4   1.98310   45.71378   11086.845       3   0   −6   2   1.97954   45.80066   5989.497       1   0   −6   2   1.97817   45.83434   9677.354       2   −1   −6   4   1.95748   46.34679   2033.378       2   3   1   4   1.94455   46.67316   3814.112       4   −1   −5   4   1.94222   46.73259   2465.540       1   2   4   4   1.94138   46.75385   1044.347       4   −2   −2   4   1.93962   46.79894   71647.414       2   0   4   2   1.93555   46.90307   22593.117       4   −2   −3   4   1.92834   47.08917   2359.305       2   2   3   4   1.92643   47.13862   3496.081       3   −2   −5   4   1.91564   47.42022   3565.626       3   −1   −6   4   1.90641   47.66401   3852.061       1   −1   −6   4   1.90518   47.69668   126.165       4   −2   −1   4   1.89223   48.04363   2471.013       3   −3   −2   4   1.87535   48.50388   728.923       2   1   4   4   1.86703   48.73407   2582.125       0   2   5   4   1.86568   48.77150   687.112       3   −3   −1   4   1.86266   48.85561   371.683       1   −3   −4   4   1.86228   48.86626   3642.781       4   −2   −4   4   1.86131   48.89351   961.915       1   1   5   4   1.85776   48.99309   2897.553       2   −3   −4   4   1.84740   49.28595   2366.935       5   0   −2   2   1.83827   49.54725   191.546       4   1   1   4   1.83748   49.56990   893.848       3   −3   −3   4   1.83478   49.64793   34517.039       1   3   3   4   1.83368   49.67962   3586.474       4   0   −6   2   1.83168   49.73747   1341.276       0   0   6   2   1.82951   49.80075   5427.721       5   0   −4   2   1.82704   49.87241   2611.138       3   2   2   4   1.82666   49.88361   15035.101       2   3   2   4   1.80028   50.66584   852.734       3   3   0   4   1.79981   50.68000   7635.262       4   2   0   4   1.79790   50.73774   31.252       5   −1   −3   4   1.79778   50.74113   3492.466       3   1   3   4   1.79572   50.80350   1004.864       0   3   4   4   1.78922   51.00132   203.157       5   −1   −2   4   1.77926   51.30747   668.832       4   −1   −6   4   1.77329   51.49295   1637.459       0   1   6   4   1.77131   51.55465   738.791       0   4   0   2   1.76980   51.60187   118322.898       5   −1   −4   4   1.76908   51.62454   2002.556       2   −2   −6   4   1.76545   51.73845   267467.688       4   −2   −5   4   1.75423   52.09423   1767.949       3   −3   −4   4   1.75045   52.21513   447.095       0   4   1   4   1.74724   52.31836   2620.549       1   −4   −1   4   1.73470   52.72556   2457.296       1   4   0   4   1.73133   52.83614   1390.317       3   −2   −6   4   1.72771   52.95534   624.514       1   −2   −6   4   1.72680   52.98560   2870.138       5   −1   −1   4   1.71763   53.29042   3992.833       4   0   2   2   1.71625   53.33669   171.427       3   3   1   4   1.70057   53.86813   4463.794       5   −1   −5   4   1.69944   53.90672   1663.483       2   2   4   4   1.69823   53.94829   3506.858       1   −4   −2   4   1.69608   54.02211   2307.989       2   −3   −5   4   1.69392   54.09682   2349.578       1   2   5   4   1.69125   54.18921   1409.942       2   −1   −7   4   1.69108   54.19489   787.935       1   4   1   4   1.68669   54.34788   882.257       0   4   2   4   1.68440   54.42786   182.510       1   −3   −5   4   1.68206   54.50961   4158.873       3   −1   −7   4   1.68013   54.57753   2978.968       4   2   1   4   1.67591   54.72649   1026.918       5   0   0   2   1.66975   54.94516   2190.528       4   1   2   4   1.66794   55.01012   1131.238       1   3   4   4   1.65500   55.47716   297.480       4   −3   −2   4   1.65390   55.51703   92.102       2   −4   −1   4   1.65348   55.53225   2628.066       4   −3   −3   4   1.64689   55.77391   1703.457       2   3   3   4   1.64570   55.81771   14933.787       5   −2   −3   4   1.64562   55.82063   904.931       5   0   −6   2   1.64485   55.84915   11045.036       3   2   3   4   1.64404   55.87900   537.543       1   0   6   2   1.64248   55.93667   4104.017       2   −4   −2   4   1.64073   56.00177   6555.738       3   −3   −5   4   1.63896   56.06742   3117.231       1   −1   −7   4   1.63562   56.19224   7479.478       5   −2   −2   4   1.63138   56.35117   139974.406       2   4   0   4   1.62941   56.42557   2336.839       3   0   4   2   1.62858   56.45687   79060.617       4   −2   −6   4   1.62677   56.52512   231.031       0   2   6   4   1.62525   56.58301   200.360       5   1   0   4   1.62516   56.58630   1372.377       4   −3   −1   4   1.62423   56.62165   941.537       5   −2   −4   4   1.62352   56.64860   977.446       1   −4   −3   4   1.62324   56.65939   3828.351       2   1   5   4   1.62060   56.76011   661.635       1   4   2   4   1.60956   57.18482   2848.325       0   3   5   4   1.60734   57.27112   4687.052       4   −1   −7   4   1.60639   57.30817   785.019       4   −3   −4   4   1.60455   57.38024   979.594       5   −1   −6   4   1.60217   57.47322   229.132       1   1   6   4   1.59998   57.55912   756.968       2   −4   −3   4   1.59358   57.81228   6432.516       0   4   3   4   1.59322   57.82655   993.033       3   1   4   4   1.58712   58.06987   1480.846       5   −2   −1   4   1.58349   58.21592   1232.501       3   3   2   4   1.58219   58.26825   878.980       2   4   1   4   1.57302   58.64091   2737.882       5   −2   −5   4   1.56920   58.79766   1757.640       4   3   0   4   1.56339   59.03785   1224.589       2   −2   −7   4   1.56261   59.07000   858.527       3   −2   −7   4   1.55396   59.43196   388.934       6   0   −4   2   1.54485   59.81773   40226.578       4   2   2   4   1.54429   59.84161   132527.313       2   −3   −6   4   1.54191   59.94356   919.952       3   −4   −2   4   1.53571   60.21080   3337.484       4   −3   −5   4   1.53442   60.26657   2142.301       0   1   7   4   1.53103   60.41363   4066.389       3   −4   −1   4   1.52872   60.51473   530.741       1   −4   −4   4   1.52851   60.52394   23281.965       3   0   −8   2   1.52498   60.67867   13248.033       2   −4   −4   4   1.52025   60.88755   1880.706       1   −2   −7   4   1.51854   60.96338   1753.461       6   0   −2   2   1.51698   61.03271   2012.546       3   −3   −6   4   1.51658   61.05053   1466.016       5   1   1   4   1.51638   61.05931   161.170       2   0   −8   2   1.51616   61.06934   3710.299       1   −3   −6   4   1.51596   61.07813   2665.123       3   −4   −3   4   1.51319   61.20197   419.151       1   4   3   4   1.51257   61.22953   10115.472       6   −1   −3   4   1.51024   61.33418   363.616       5   2   0   4   1.51016   61.33806   617.463       6   −1   −4   4   1.50933   61.37515   989.558       4   1   3   4   1.50872   61.40297   297.840       2   2   5   4   1.50649   61.50344   588.937       2   3   4   4   1.49652   61.95818   590.113       4   −2   −7   4   1.49506   62.02536   423.413       2   4   2   4   1.49366   62.09007   106.506       3   4   0   4   1.49339   62.10246   123565.250       1   3   5   4   1.49174   62.17898   478.546       5   −2   −6   4   1.49166   62.18267   105324.078       5   −1   −7   4   1.49157   62.18692   1581.715       3   −1   −8   4   1.49078   62.22328   974.226       1   2   6   4   1.48989   62.26457   134633.219       0   4   4   4   1.48733   62.38401   83526.117       4   0   −8   2   1.48466   62.50882   50996.125       6   −1   −2   4   1.48331   62.57221   539.855       2   −1   −8   4   1.48254   62.60831   1032.783       4   3   1   4   1.48118   62.67218   6016.078       6   −1   −5   4   1.48072   62.69367   7655.220       3   2   4   4   1.47949   62.75186   5174.257       3   −4   −4   4   1.46483   63.45251   92852.609       1   0   −8   2   1.46062   63.65703   34382.762       5   −3   −3   4   1.46015   63.67986   19231.883       3   3   3   4   1.45904   63.73375   2083.547       6   0   −6   2   1.45886   63.74255   104.403       2   0   6   2   1.45666   63.85023   5589.541       4   −1   −8   4   1.45305   64.02798   501.111       5   −3   −2   4   1.45017   64.17003   1181.973       4   −3   −6   4   1.44693   64.33096   2199.269       0   3   6   4   1.44586   64.38455   3392.815       5   −3   −4   4   1.44464   64.44527   603.990       3   4   1   4   1.43522   64.91979   856.294       0   2   7   4   1.43374   64.99512   3941.112       6   −1   −1   4   1.43271   65.04772   9958.013       5   0   2   2   1.43178   65.09530   464.878       2   −4   −5   4   1.43122   65.12385   1343.613       1   −1   −8   4   1.43049   65.16140   2849.458       6   −1   −6   4   1.42884   65.24586   1632.203       2   1   6   4   1.42677   65.35220   113.830       1   −4   −5   4   1.42405   65.49281   2715.529       5   2   1   4   1.42169   65.61510   2686.276       6   −2   −3   4   1.41663   65.87928   845.347       5   −3   −1   4   1.41622   65.90063   1809.560       6   −2   −4   4   1.41588   65.91867   654.705       0   5   0   2   1.41584   65.92052   1197.549       4   2   3   4   1.41537   65.94542   244.526       3   1   5   4   1.41356   66.04055   4426.744       1   4   4   4   1.40751   66.36081   21200.830       4   −4   −2   4   1.40684   66.39668   396.245       5   −3   −5   4   1.40597   66.44291   29.975       5   0   −8   2   1.40574   66.45520   3142.987       0   5   1   4   1.40421   66.53712   7893.484       5   1   2   4   1.40336   66.58237   970.894       1   1   7   4   1.40288   66.60856   170.244       4   −4   −3   4   1.40251   66.62790   1310.150       2   4   3   4   1.40178   66.66737   2531.638       2   −3   −7   4   1.40123   66.69715   8505.064       5   −2   −7   4   1.40118   66.69978   401.337       3   −2   −8   4   1.40053   66.73479   5147.162       1   −5   −1   4   1.39767   66.88894   1543.957       3   −4   −5   4   1.39761   66.89248   1963.978       1   5   0   4   1.39591   66.98464   1123.055       3   −3   −7   4   1.39498   67.03543   1584.166       6   −2   −2   4   1.39432   67.07096   6423.831       2   −2   −8   4   1.39368   67.10576   2221.480       4   0   4   2   1.39233   67.17963   9756.798       6   −2   −5   4   1.39218   67.18803   123.361       6   0   0   2   1.39146   67.22715   61458.719       4   −4   −1   4   1.38844   67.39312   1208.544       4   3   2   4   1.38797   67.41877   146.200       5   −1   −8   4   1.37882   67.92709   1203.279       0   4   5   4   1.37785   67.98154   710.651       1   −5   −2   4   1.37724   68.01559   3062.095       4   −4   −4   4   1.37608   68.08057   27359.787       1   5   1   4   1.37219   68.30019   694.557       0   0   8   2   1.37213   68.30382   15200.482       0   5   2   4   1.37096   68.37016   828.054       1   −3   −7   4   1.36918   68.47118   3214.390       4   −2   −8   4   1.36910   68.47589   45.553       4   1   4   4   1.36616   68.64389   519.685       6   1   0   4   1.36534   68.69093   1607.419       5   3   0   4   1.36303   68.82355   2112.606       3   4   2   4   1.36190   68.88850   1753.492       6   −1   −7   4   1.36065   68.96085   2649.621       2   3   5   4   1.36033   68.97921   2510.422       2   −5   −1   4   1.35414   69.33989   3657.629       6   −2   −1   4   1.35205   69.46230   243.801       4   −3   −7   4   1.35190   69.47100   6915.945       1   −2   −8   4   1.35018   69.57237   1099.760       4   4   0   4   1.34986   69.59140   705.399       5   −3   −6   4   1.34938   69.61938   756.755       6   −2   −6   4   1.34879   69.65417   2227.248       1   3   6   4   1.34807   69.69667   318.911       2   −5   −2   4   1.34710   69.75419   61.678       0   1   8   4   1.34706   69.75684   1471.690       2   2   6   4   1.34705   69.75717   576.139       2   5   0   4   1.34082   70.12915   2349.962       3   3   4   4   1.34035   70.15689   35.647       1   −5   −3   4   1.33737   70.33644   2229.274       2   −4   −6   4   1.33596   70.42175   3337.051       3   2   5   4   1.33592   70.42439   156.499                    
Gas-Phase DFT Calculations
 
     The INHHQ structure was optimized in gas phase, using the DFT methodology, level of theory B3LYP/6-311+G (d,p). 
     As described above, a total of 5 conformations with the smallest PM3 energy differences were selected to perform a DFT optimization and frequency calculations. Cis-trans isomerism was contemplated in this treatment. Energy values found for each of these structures indicated that there are, in fact, only 3 different conformations. Conf.5 is the one with the lowest free energy (ΔG) when compared to the other conformations. However, the energy differences between the structures are not appreciable, especially between the Conf. ½ with respect to the Conf. 5 (only 0.06 kcal mmol −1 ), indicating that, indeed, all five conformations are possible, particularly Conf. 5. This last one was chosen for further deeper studies. 
     It is shown (Table 2) that there is excellent agreement between the structural parameters found in the calculations (Conf. 5) and the refined X-ray structure and that, in turn, there is no significant difference between  FIGS. 1 and 4 . Crystallography shows that the phenolic hydrogen points in the direction of the quinolinic nitrogen, since a connection involving these H atoms is formed, producing a 2.691 Å donor-acceptor distance, in perfect agreement with the X-ray data (O1 . . . N4=2.689 Å). 
     Vibrational Analyses 
     The experimental FTIR and Raman spectra of INHHQ, in the solid state, are shown in  FIG. 5 . The observed and calculated frequencies, as well as an attempt to assign the main bands, are given in Table 4. 
                     TABLE 4                  Assignment of the FTIR and Raman spectra of INHHQ (scale factor: 0.9381)                                     Theoretical (cm −1 )                   B3LYP/6-311+G(d,p)                                     Experimental (cm −1 )   Unscaled   Scaled               FTIRRaman   (cm −1 )   (cm −1 )   IR Intensity   Vibrational Assignment                                             3396 m   3400 br   3662   3435   104.3584   v OH       3208 sh   3205 w               —       3183 w   —   3502   3285   5.5099   v NH       3148 sh   —   3211/3205   3012/3007   1.8906/3.2991   v CH(Quin + Py)ip       3073 sh   3081 w   3198   3000   9.2441   v CH(Quin)ip       3059 sh   3055 w   3185/3178   2988/2981   18.7464/9.9528   v CH(Quin + Py)op       3042 w   —               —       3028 sh   3030 w               —       3016 w   —               —       —   2996 w               —       2959 sh   —               —       2923 w   —               —       2852 w   —   3048   2859   40.4835   vCH(Azomethine)       2835 sh   —               —       1656 s   1660 sh   1767   1658   350.6350   vC = O + βNH       1647 vs   1646 w   1673   1569   20.7477   v C = N(Azomethine) + v                           C = C(Quin) + βC − OH       1604 w   1603 vs   1659   1556   2.3397   v C = N(Azomethine) + v                           C = C(Quin) + βC − OH       1595 w   1595 s   1634   1533   5.5690   v C = C(Quin)       1556 m   1555 w   1597   1498   18.4149   v C = N(Quin) + vC = C(Quin)       1545 m   —   1630   1529   14.2312   v C = C(Py) + vC = N(Py)       1507 m   1507 w   1555   1459   371.3416   βNH + βCH(Quin)       1490 w   1488 vw               —       1465 m   1468 w   1541   1446   168.8474   Ring stretch(Quin) +                           βC − OH + β NH       1437 m   1435 s   1519   1425   6.8860   βCH(Py)       1407 w   —               —       1394 sh   1396 m               —       1371 w   1371 m   1495   1402   176.2051   β C − OH + β NH + β                           CH(Quin)       1330 m   1329 m               —       1299 s   1304 m   1360   1276   34.4059   β CH(Quin +                           Azomethine) + v                           C = N(Quin) + v C − OH       1280 m   1279 sh   1347   1264   31.7632   β NH + β                           CH(Azomethine + Py) + β C − OH       1270 sh   —               —       1252 m   1252 s   1309   1228   15.0576   β CH(Quin +                           Azomethyne) + β C − OH       1232 m   —   1284   1205   117.6910   v C − OH + β CH(Quin +                           Azomethine)       1217 w   1220 w   1278   1199   32.0853   v C = N(Py) + vC = C(Py) + β NH       1204 w   1204 vw   1267   1189   56.8637   β CH(Azomethine +                           Quin) + β C − OH       1170 w   1172 sh   1253   1175   303.5474   β CH(Py) + βNH       1156 m   1156 m   1172   1099   380.9402   v N − N + βCH(Py + Quin)       1136 w   1133 w   1112   1043   10.8679   βCH(Py)       1122 sh   —               —       1105 w   1105 vw               —       1090 w   1093 vw   1109   1040   19.7634   βCH(Py + Quin)       1072 w   1076 w   1092   1024   9.3109   βCH(Py)       —   1063 vw   1079   1012   11.9293   βCH(Quin) + δ NNC       1044 vw   1044 vw   1068   1002   1.6514   βCH(Quin)       1007 vw   —               —       992 vw   992 w   1010   947   1.7715   Ring breath(Py)       981 vw   —   1008   946   1.5921   yCH(Py)       950 vw   952 w   959   900   14.8080   yCH(Azomethine)       931 w   932 w   914   857   2.9777   Ring-deformation(Quin + Py)       895 w   897 vw               —       881 vw   —               —       867 w   869 w   898   842   0.0665   yCH(Quin)       856 m   855 sh   880   826   11.8041   Ring-deformation(Quin + Py)       837 m   835 vw   892   837   2.8606   yCH(Py)       —   812 vw   804   754   2.0048   y C = C − C(Quin) + y                           C = N − C(Quin)       792 vw   780 w   789   740   5.7350   β C = C − C(Quin + Azomethine)       766 s   768 vw   767   720   9.2858   yCH(Py)       720 m   720 m   734   689   18.7702   Ring-deformation(Quin)       696 w   696 vw   763   716   38.2602   y CH(Quin)       677 s   —   719   674   19.3631   β C = N − C(Py) + β C = C − (Quin)       670 sh   668 w   698   655   2.0785   y C = C − C(Quin) + β                           C = N − C(Py)       656 w   657 w   693   650   58.0279   Ring-deformation(Py)       644 sh   —   681   639   1.5003   Ring-deformation(Py)       616 vw   —   627   588   2.3638   Ring-deformation(Quin)       587 m   —   604   567   90.9456   γ OH       575 w   —               —       547 sh   553 w   588   552   10.5255   β C = C − C(Quin)       532 sh   533 vw   559   524   1.6705   β C = C − C(Quin) + y NH       522 w   523 vw   551   517   14.1393   yCH(Quin) + β C = C − C(Quin) +                            β C − OH       482 w   485 w   539/534   506/501   16.0281/30.1104   γ NH + y C = C − C(Quin)                    
Quin: quinoline ring; Py: pyridine ring; vs: very strong; s: strong; m: medium; w: weak; vw: very weak; br: broad; sh: shoulder; ip: in-phase; op: out-of-phase; v: stretching; p: in-plane bending; y: out-of-plane bending.
 
Carbonylic C═O Stretching
 
     The v C═O absorption is usually one of the most representative in an infrared spectrum and is also likely its most intense spectral feature. It appears in a wavenumber region relatively free of other vibrations (1800-1600 cm −1 ). On the other hand, this mode gives only weak or very weak absorptions in Raman spectroscopy. In our study, as expected, v C═O vibration originates one of the strongest bands of the infrared spectrum, at 1656 cm −1 , which is in excellent agreement with the calculated value of 1658 cm −1  (DFT calculations show a coupling between v C═O and β NH vibrations). 
     This mode was assigned at 1663 cm −1  by Liu and Yang. 
     Azomethine C═N Stretching 
     The C═N stretchings of azomethine groups show absorptions close to that of carbonyl stretching. This fact can difficult an accurate assignment. For example, the C═N stretching bands of alkylated Schiff bases are usually found in the range 1674-1649 cm −1 , inside the common region of v C═O absorption. If conjugations of the C═N moiety with phenyl groups are present, the stretching frequency shifts to 1650-1600 cm −1 . In this work, two frequencies involving azomethine C═N vibrations were calculated (1569/1556 cm −1 ), both of them coupled to v C═C of the quinoline ring and, to a lesser extent, to the phenol v C—OH. These values are in good agreement with the experimental frequencies observed in the infrared, at 1647 (vs) and 1604 (w) cm −1 , and Raman spectra, at 1646 (w) and 1603 (vs) cm −1 , respectively. 
     Liu and Yang, though, attributed this mode to a single band at 1613 cm −1  in the IR spectrum, which was not observed in our study. 
     OH and NH Stretching Vibrations 
     OH and NH groups are very characteristic and their stretching vibrations are observed, in many cases, around 3500-3300 cm −1 . This absorption, however, is highly influenced by chemical environment, mainly when OH or NH groups are involved in hydrogen bonding. This can occur within the same molecule (intramolecular H bonding) or with adjacent molecules (intermolecular H bonding). The presence of intramolecular H bonding causes a thinning of the band and makes its position unaffected by concentration changes. In the IR spectrum of INHHQ, we observed a sharp band of medium intensity located at 3396 cm −1 , assigned to v OH. A similar absorption, at 3418 cm −1 , was reported by Krishnakumar and Ramasamy in the infrared spectrum of 8-hydroxyquinoline (8-HQ). On the other hand, intermolecular hydrogen bonding usually leads to a broadening of the band, as can be seen in the case of the v NH absorption of INHHQ, which was attributed to the weak IR band at 3183 cm −1 . In a previous study on the isonicotinoyl hydrazone of 2-hydroxy-3-methoxybenzaldehyde, published by us, v NH vibration was observed as a weak band at 3157 cm −1 . Here, we found serious discrepancies concerning the assignments made by Liu and Yang, since these authors attributed an absorption of higher frequency (reported by them at 3576 cm −1 ) to the NH stretching mode, whereas the lower frequency band at 3193 cm −1  was credited to the OH stretching movement. 
     Phenol C—OH Vibrations 
     In this work, the C—OH stretching mode was assigned to the medium intensity infrared band at 1232 cm −1 . This vibration is Raman inactive and had its frequency calculated at 1205 cm −1 . A coupled mode involving this movement was also predicted at 1276 cm −1  [experimental: 1299 (infrared) cm −1  and 1304 (Raman) cm −1 ].Another important vibration concerning the phenol group is the in-plane bending, which typically appears in the region 1440-1260 cm −1 , attributed to the weak infrared band (medium in the Raman spectrum) at 1371 cm −1 . Coupled modes are observed in FTIR at 1465, 1280, 1252 and 1202 cm −1 . Theoretical and experimental frequencies show good agreement (Table 4). 
     Biological Activity Studies 
     This hydrazonic compound has the ability, as proved by 1D and 2D NMR experiments, of compete with the β-amyloid peptide (or α-synuclein), key-targets, respectively, in Alzheimer&#39;s and Parkinson&#39;s diseases, by physiological ions such as Zn 2+  Cu 2+  and Cu + , which can cause their precipitation or oligomerization and contribute to worsen the oxidative stress condition observed in the brains of patients suffering from these neurodegenerative disorders. INHHQ does not interact directly with the β-amyloid peptide and α-synuclein, nonetheless inhibits the interactions between these targets and metals through a mechanism that probably involves metal ion sequestering. 
     In silico pharmacological analyses (mandatory studies in the development of new therapeutic agents, whose aim is to predict pharmacokinetic properties of novel molecules with a potential pharmacological action) show that the Lipinski&#39;s parameters presented by INHHQ (Table 5), calculated via 1 D-QSAR method, are in agreement to ideal values, suggesting that this compound constitutes an excellent candidate to new drug, with good oral absorption and cellular permeability. The model also indicates that INHHQ possesses structural features which can allow it crossing the blood-brain barrier (BBB), resulting in therapeutic actions inside the patients&#39; brains. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Lipinski parameters calculated for  
               
               
                 INHHQ, along with reference values 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Ref. Oral 
                 Ref. BBB 
               
               
                   
                 Parameters 
                 INHHQ 
                 Biodisponibility 
                 Crossing 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 HBD 
                 2 
                 ≤5 
                 ≤3 
               
               
                   
                 HBA 
                 6 
                 ≤10 
                 ≤7 
               
               
                   
                 MW 
                 292 
                 ≤500 
                 ≤400 
               
               
                   
                 log P 
                 2.34 
                 −1 a 5 
                 −1 a 5 
               
               
                   
                 log D (pH = 7.4) 
                 2.27 
                 −1 a 5 
                 −1 a 5 
               
               
                   
                 log S 
                 −3.36 
                 −4 a 2 
                 −4 a 2 
               
               
                   
                 PSA 
                 66.077 Å 
                 ≤140 Å 
                 ≤90 Å 
               
               
                   
                 Rotatable bonds 
                 4 
                 ≤10 
                 ≤10 
               
               
                   
               
            
           
         
       
     
     Furthermore, both the INHHQ molecule itself and its potential metabolites were shown, in theoretical comparison with the toxic fragments of over 3000 comercially available drugs, completely non-toxic. 
     In fact, the intraperitoneal injection of up to 300 mg kg −1  of the compound, using 10% DMSO/saline solution as vehicle of injection, in Wistar male rats (acute toxicity test) showed that INHHQ was apparently not toxic to the animals throughout the 72 h of the experiment: no animals died and there were no behavioral changes noted in the injected rats. Also, after the animals&#39; sacrifice, there were no macroscopic abnormalities observed during its organs dissection.