Synthesis, Molecular Modeling and Biological Studies of Novel 1-Aryl-Thiazolo Benzimidazole Derivatives as Cytochrome (CYP51 MTb ) Type I Inhibitors & Anticancer Agents

Synthesis, Molecular Modeling and Biological Studies of Novel 1-Aryl-Thiazolo Benzimidazole Derivatives as Cytochrome (CYP51 MTb ) Type I Inhibitors & Anticancer Agents | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis, Molecular Modeling and Biological Studies of Novel 1-Aryl-Thiazolo Benzimidazole Derivatives as Cytochrome (CYP51 MTb ) Type I Inhibitors & Anticancer Agents Iten M. Fawzy, Hanan M. Refaat, Reem T. Attia, Heba A. Ibrahim, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1727366/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The prominent biological activity of polycyclic systems inspired the synthesis of novel 1-Aryl-thiazolo-benzimidazole Derivatives (2a-e), (3a-e) and (4a-e). In vitro antiproliferative assay was performed on colorectal adenocarcinoma (SW620) and the novel compounds showed superior anticancer activity with IC 50 in the range of 0.03–0.35 µM compared to reference compound Doxorubicin (DOX) with IC 50 (0.07 µM). The ability of the new molecules to interact with extracted cytochrome CYP51M Tb was also studied using absorption spectroscopy. Results showed mild type I spectral shift especially compound 4d displayed 3% shift in the spin state assay referring to the presence of electron withdrawing groups attached to benzene ring, thus that could be a promising nucleus for better cytochrome inhibition. The results were supported by molecular modeling studies to confirm general binding of the compounds to the hydrophobic region of the heme-protein. These outcomes encourage the new molecules to be candidates for new potential anticancer agents. Aryl-Thiazolo-Benzimidazoles Anticancer activity CYP51MTb Human Colon Cancer Molecular Modeling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Cytochrome-P 450 (CYPs) targeting is considered an effective anticancer chemotherapeutic approach [ 1 – 4 ], including different CYPs either as drug targets or drug-metabolizing enzymes. Literature survey presented specific types of CYPs that were targeted as anticancer agents; where aromatase (CYP19) is a key target for breast cancer treatment & also sterol 14α -demethylases (CYP51 Mtb ), is essential for membrane sterol biosynthesis either cholesterol in animals or ergosterol in fungi, thus is a more specific drug target [ 1 – 4 ]. CYP51 Mtb , the Mycobacterium tuberculosis CYP enzyme with a determined catalytic function, belongs to sterol 14α - demethylases which is responsible for demethylation of the 14α -methyl group from sterol molecules during the biosynthesis of sterol in most pathogenic fungi or protozoa [ 1 – 4 ]. Many drugs including azoles prevent fungal growth via inhibition of sterol 14α-demethylases and thus, inhibiting ergosterol biosynthesis [ 4 ]. From this point, azoles were further reported as potential chemotherapeutic agents and inhibitors with clear role in cancer management [ 5 ]. Based on literature, there are two types of binding for the CYP51 Mtb substrates which can be recorded via alteration of CYP51 Mtb optical spectra. If the ligand binding in the active site showed 390 nm peak and 420 nm trough difference in spectra then it is substrate binding (type I) while, inhibitor binding (type II) shows 416 nm trough and 436 nm peak in the difference spectra [ 2 ]. Substrates as Galeterone (Fig. 1 A ) , a benzimidazole derivative and a CYP 17 α hydrolase inhibitor - was investigated against human sterol 14α demethylase (CYP51A1), which catalyzes the main part of the sterol biosynthetic pathways. Differential absorption spectra showed type I spectral changes of CYP51A1 upon addition of Galeterone with sigmoidal dependence of ΔA (390–424) on ligand concentration [ 6 ]. On the other hand, Galeterone was confirmed to possess anticancer activity against various cancerous cell lines with IC 50 in range of 8–41 µM [ 7 ]. Also, prominent compound: (R)-N-(1-(3,4′-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VFV) showed in Fig. 1 B, which is a benzamide-imidazole derivative, showed prominent ability of interaction through binding and inhibition to CYP51 mtb together being as an efficient anti-proliferative agent [ 2 ]. The above findings supported the fact that the fused heterocycle containing nitrogen in the benzimidazole scaffold is the reason for their interaction with biomolecular targets via its nucleo-bioisostere activity [ 8 – 12 ] and hence allowing them to be multi-target inhibitors specifically against various critical enzymes involved in cell synthesis [ 13 ]. It was also concluded that not only the presence of benzimidazole nucleus is responsible for the anticancer activity, but also the proper substitution is very critical for the development of new cytotoxic candidates [ 11 ]. As a result, novel chemotherapeutic agents of various series of benzimidazoles have been reported and their proliferative assays confirmed their significant potent anticancer activity against various human cancer cell lines via different mechanisms [ 14 – 21 ]. This wide therapeutic potential exhibited by benzimidazole and its derivatives, opened a new approach to design and synthesize more potent derivatives with diverse pharmacological activities [ 8 ]. Guided by the above outlines, this research was focused on the design and synthesis of novel 1-Aryl-thiazolo-benzimidazole derivatives as new candidates with potential anticancer activity. NaHSO 4 -SiO 2 catalyst assisted synthesis method of benzimidazoles was adopted as reported [ 9 , 22 , 23 ], to fulfill the green chemistry requirements to protect both environment and human health. The new molecules were tested for their binding to extracted cytochrome CYP51 Mtb . Since in vitro CYP51 MTb absorbance assay was performed on the available non-human strain, it was essential to assure that the efficacy of the tested compounds will not deviate from those of human strain. Therefore, molecular modeling studies were performed on both human & non-human 14α-sterol demethylase enzymes (4UHL), (1EA1) respectively downloaded from PDB [ 24 , 25 ] and the type of binding was detected. The results coincide with literature survey which confirmed the insignificant difference between human and non-human strains thus would not affect the efficacy of the tested compounds [ 24 ]. Further, the active CYP51 inhibitors were subjected to in vitro anti-proliferative assay against colorectal adenocarcinoma (SW620) cell types. 2. Results And Discussion 2.1. Chemistry The study was designed to synthesize candidates based on the findings that 1,3-fused-5/6- benzimidazoles with thiourea derivatives exhibited remarkable anti-proliferative activity against tumor cells [ 9 , 10 ]. Introduction of strategic substitutions to the proposed moiety was adopted in the following Scheme 1 . The scheme of synthesis showed that 4-nitro-o-phenylenediamine was selected as a strategic starting chemical to react with 2-mercaptoacetic acid and diverse of aromatic aldehydes in one pot reaction using acetonitrile as solvent and NaHSO 4 -SiO 2 as a catalyst. The mixture was allowed to reflux at 75°C for 1 h followed by purification and recrystallization procedures as reported in literature [ 26 ]. The NaHSO 4 -SiO 2 catalyst was first prepared in situ applying the modified procedure of Nishiguchi’s method, where NaHSO 4 .H 2 O was allowed to stir in adequate amount of distilled water with SiO 2 of column chromatographic grade and gently heated till white solid was obtained then the catalyst was allowed to dry in oven for 48 h prior to use [ 26 ]. The obtained products (2a-e) with yield (89–95%) were further subjected to catalyzed reduction rendering the nitro into amino group using classical H 2 /Pd over carbon procedure as reported to yield intermediates (3a-e) [ 27 ]. Each obtained intermediate from the above (3a-e) was allowed to react with benzoyl chloride using TEA and methylene chloride as a solvent to obtain the targeted final compounds (4a-e) with yield (67–84%). The unreported above three series of compounds (2a-e), (3a-e) & (4a-e) were subjected to further structural elucidation analysis; Ft-IR, H 1 NMR, C 13 NMR & Mass analysis. 2.2. Biology 2.2.1. CYP51 MTb Spectrometric Absorbance Assay The ability of the synthesized molecules to interact with CYP51 MTb was studied using a dual-beam Shimadzu UV-2401PC spectrophotometer to record UV-visible absorption spectra. The binding affinity of ligand or inhibitor in the CYP active site is measured by the concentration of the spectral changes. The Concentration of CYP51 MTb used was 0.0015mM and measured at 418 nm with max absorbance = 0.2. The stock solutions of ligands were prepared 1-4mg in 1ml DMSO and volume used for each ligand = 0.1–0.2 µL [ 25 , 28 ]. The newly designed ligands induced type I spectral shift of the enzyme signifying a change from the low-spin state to the high-spin state of the heme iron as shown in supplementary 1 & 2 chromatograms. The minimum peak wavelength was 390 nm, while the maximum peak wavelength was 418 nm. The absorbance change (ΔA (A418 – A390)) and the shift percentage were calculated and summarized in Table 1 . Most of the ligands induced less than 5% shift with CYP51 MTb . The nitro derivatives (2a-e) displayed mild shift indicating agonist like binding of type I, while the amino derivatives of the novel nucleus (3a-e) showed no shift nor change in spectral troughs which indicated no binding. Meanwhile, the benzamide derivatives (4a-e) showed better shifts than (2a-e) with the same behavior of type I binding as shown in Fig. 2 . Table 1 Ligand binding parameters calculated from the spectral analysis of CYP51 mtb : Ligands ΔA * % Shift * 2a 0.01 1% 2b No change No shift 2c 0.01 1% 2d No change No shift 2e No change No shift 3a No change No shift 3b No change No shift 3c No change No shift 3d 0.01 1% 3e No change No shift 4a No change No shift 4b 0.02 2% 4c 0.01 1% 4d 0.03 3% 4e 0.04 4% * ΔA was calculated according to the following equation: ΔA=(A418 − A390) =(0.2−(obtained absorbance value), Shift percentage was calculated according to the following equation: %Shift = Calculated ΔA x 100 2.2.2. Anti-proliferative Assay The newly synthesized compounds of both active series in the CYP51 MTb assay (2a-e) and (4a-e) were screened for their anti-proliferative activities against colorectal adenocarcinoma (SW620) using sulfo-rhodamine-B (SRB) assay. The compounds were compared with doxorubicin ( DOX ), which is an anthracycline chemotherapeutic agent that has been used for the treatment of a variety of human cancers such as colorectal cancer [ 29 ]. From the observed results ( Table 2 & Fig. 3 ), DOX showed an IC 50 of 0.07 µM. It was also observed that most of the synthesized compounds showed high cytotoxic properties with IC 50 in the range of 0.03–0.35 µM. 4d which is the most potent compound, showed 57% significant decrease compared to DOX . By the same token, 4b showed 42.8% of significant decrease when compared to DOX treated group. Comparing the obtained results, it was concluded that the benzamide derivatives showed slightly more potent anti-cancer activity than the nitro derivatives. Also, the presence of electron donating groups on benzene ring attached to thiazole (tri-methoxy at ortho & para positions / hydroxy at para position) revealed the most effective anticancer activity as shown for compounds 4b & 4d. 2.3. Molecular Modeling Studies Molecular modeling studies were carried out to support the idea that benzamide compounds can interact effectively with CYP51 mtb leaning on the fact and as a reference ligand that VFV -a benzamide-imidazole derivative- revealed significant CYP51 Mtb inhibition together with prominent anti-proliferative activity in various cancer cell lines [ 2 ].Since in vitro CYP51 MTb absorbance assay was performed on the available non-human strain, the question that would evolve will be the difference between human and non-human strains, also if the results will be quite similar or would differ greatly. Literature survey showed difference in human strain than non-human one that could affect inhibition activity which is the middle portion of the human CYP51 I-helix (the core helix in the P450- fold) [ 30 , 31 ]. Where, this portion is distanced away from the heme iron by 1.5–2.0 Å in human rather than non-human and a phylum specific residue (small leucine residue) is subjected to CYP51 active site rather than a bulky phenylalanine residue, which renders the enzyme binding cavity with increased volume in human CYP51 rather than non-human, thus, bulky groups and enlarged volume of compound atomic structures will be essential. Also, the non-human CYP51 helix lacks loop like segments, where binding to its residues closes the channel and any non-polar moiety could perform inhibition via interrupting the threonine/serine succession. Meanwhile, in human CYP51 it lacks helical hydrogen bonds leaving loops in dynamic phases that could be inhibited via Van der Waals interactions with residues [ 24 ]. From this point, modeling studies were performed on both human and non-human cytochrome sterol 14-alpha demethylase to compare the binding interactions of the 15 novel compounds and reference VFV , also analyze their interactions difference on both enzyme strains. The crystal structure of human sterol 14-alpha demethylase (CYP51) in complex with VFV in P1 space group was downloaded from protein data bank pdb with code: (4UHL) [ 24 ]. While, the crystal structure of cytochrome P450–14 alpha-sterol demethylase (CYP51) from Mycobacterium tuberculosis was downloaded with pdb code: (1EA1) [ 25 ]. Enzymes were prepared for docking while; compounds together with VFV were simulated in dynamic phases. Docking protocol was applied using MOEv.2010 and interaction scores were obtained in Kcal/mol and displayed in Table 3 . Table 3 Docking score results of both enzymes (4UHL) & (1EA1) Ligands Score (Kcal/mol) against 4UHL Score (Kcal/mol) against 1EA1 VFV (Reference lead) -14.62 -14.35 2a 2b 2c 2d 2e -11.21 -12.11 -11.74 -13.79 -11.39 -10.77 -12.25 -11.87 -12.79 -11.73 3a 3b 3c 3d 3e -10.61 -12.09 -11.31 -13.98 -11.73 -10.52 -11.68 -10.39 -13.82 -12.27 4a 4b 4c 4d 4e -12.32 -13.30 -12.78 -15.62 -12.60 -12.44 -13.82 -12.88 -15.08 -13.68 From the scores above, it was obvious that interaction energies were comparable with that of VFV especially series 4(a-e) with even higher scores for compound 4d to both enzymes. Also, from the scores and interactions of 3D and 2D diagrams presented in Fig. (4–9) within channels, it was obvious that the volume of the occupying inhibitor is the contributing key factor in displaying inhibition to enzyme either human or non-human type. It was also reported in literature that (type I) substrate binding reflect low-to-high spin transition of the ferric P450 heme iron as due to displacement of the heme-coordinated water molecule towards the pouch while (type II) inhibitor binding reflect coordination of the basic heterocyclic nitrogen to the P450 heme iron towards the tail [ 2 ]. Figure 4 showed comparable occupation of helix-1 cavity of human enzyme by heme, VFV and compound 4d respectively where, 4d occupied the whole pouch of cavity together with the tail while VFV occupied the tail only and the heme-iron occupied the pouch. This stands up greatly for the fact that the benzamide group attached strategically to the novel thiazolo-imidazole derivatives occupied the large capacity of helix together with the trimethoxy group which were bulky enough to fill the space cavity. Also, this occupation displayed by 4d especially in the pouch clearly explains the reason that the novel synthesized compounds showed (type I) substrate binding shift in the absorbance assay rather than (type II) displayed mostly by azole inhibitors that occupy the tail. As for Figs. 5 & 6 , 2 D interactions were displayed to compare the key interaction residues with heme & VFV in the cavity to that with 4d . Heme showed metal contact of iron with Cys 449, arene-hydrogen Van der Waals interaction with Ala 311 and side chain acceptor for Lys 156 while, VFV displayed receptor contact and polar interactions with Gly 451, Phe 442 and Thr 315 as in reference literature for both heme & VFV [ 2 ], together with extra arene-hydrogen Van der Waals interaction with Gly 307 and His 236. On the other hand, 4d was able to occupy the whole cavity tightly with receptor contact, polar and basic interactions with Cys 449, Thr 315, Ala 311 & Lys 156. Also, it revealed tight binding via arene-hydrogen Van der Waals interactions with Gly 451 & Phe 442 from both opposite sides. Figure 7 showed the interaction of compound 3a with the enzyme (4UHL) which reflected the reason behind the low activity of this series of compounds, where it didn't occupy the whole cavity and there were hollow distances in between the opposite sides of the channel. Figure 8 presented VFV and compound 4d in the stacked helix of non-human (CYP51) (1EA1) , where we can find that 4d is mainly in the pouch region of the channel rather than the tail. Figure 9 revealed clearly the hydrophobic 2D interactions of VFV with Cys 394, Pro 386, Leu 324, Leu 321, Pro 320 and Ala 256while, compound 4d showed the same hydrophobic interactions with extra deeper hydrogen bonds in the channel with Thr 260 and Gln 72. 3. Conclusion The widely known anticancer activity of benzimidazoles encouraged the synthesis of new three series of thiazolo-benzimidazole derivatives (2a-e ), (3a-e) & (4a-e) with five different strategic groups (phenyl, hydroxyphenyl, chlorophenyl, trimethoxyphenyl & furo) attached to thiazole ring. Compounds (2a-e) are characterized by nitro group being attached to benzimidazole ring, (3a-e) characterized by amino while, (4a-e) characterized by benzamide group. The in vitro CYP51 mtb spectroscopic absorbance assay that was carried out showed that compounds exhibited mild shift indicating (type I) substrate binding with better binding exhibited by (4a-e) series than the other two series. Molecular modeling studies were then performed to explain more the binding mode of such compounds to (CYP51) using VFV as reference ligand. Two types of (CYP51) enzymes were downloaded from pdb (human (4UHL) & non-human (1EA1)). The (4a-e) series displayed also comparable interaction scores to reference compound better than the other two series, with highest score attributed to compound 4d . Helix-1 channel; the active site of both enzymes is constituted of two strategic regions; pouch which is occupied by heme while the tail region that mostly occupied by famous azole inhibitors of (type II) and VFV . (2a-e) & (3a-e) series were not fully occupying the channel, while series of (4a-e) which is characterized by large structural volumes occupied most of the channel as a tight belt specifically the pouch region and part of the tail, hence, provide its inhibitory activity to channel via (type I) substrate binding. The compound 4d synthesized with trimethoxyphenyl group attached to thiazole ring was recognized for its best score as well as binding mode. Further, in vitro SW620 colorectal adenocarcinoma cell lines assay was performed, and all 15 compounds revealed noticeable chemotherapeutic activities with IC 50 in the range of 0.35–0.03 µM compared to DOX with IC 50 of 0.07µM where, the highest activity was also accredited to (4a-e) series especially compounds 4d & 4b . 4. Experimental 4.1. Chemistry 4.1.1 General All melting points were determined on a Gallenkamp apparatus and are uncorrected. The progression of the reactions was monitored using TLC Merck Kieselgel 60 F254 aluminum packed plates. The IR spectra were measured on a Pye-UnicamSP300 instrument in potassium bromide discs. Bruker High performance Digital FT-NMR spectrometer Avance III 400 MHz was utilized for 1 H-NMR & 100 MHz for 13 C-NMR Faculty of Pharmacy Mansoura University. Mass spectra were run on a MAT Finnigan SSQ 7000 spectrometer, using the electron impact technique (EI). Initially, modification of Nishiguchi’s method procedure was applied to prepare NaHSO4-SiO2 Catalyst needed for further reactions. To a 100-mL beaker, 4.14 g (0.03 mol) of NaHSO 4 .H 2 O was dissolved in 20 mL of distilled water and 10 g of SiO 2 (column chromatographic grade, 60 Å, 200–400 mesh) was added. The solution was stirred for 15 min and was heated gently on a hot plate, with alternating shaker until a free-flowing white solid was produced. Further, the beaker was allowed to stay in oven to dry thoroughly at 120°C for at 48 h before use [ 27 ]. 4.1.2. General procedure for the Synthesis of novel 1-aryl-1H, 3H-thiazolo[3,4-a]-6-nitro-benzimidazole derivatives (2a-e) To a one pot reaction, (4 mmol) of 4-nitro-o-phenylenediamine was dissolved in MeCN (12 mL) and equivalent amount of 2-mercaptoacetic acid (4 mmol) together with (4 mmol) of aromatic aldehydes were added and allowed to stir in presence of (800 mg) of the previously prepared catalyst; NaHSO 4 -SiO 2 at 75°C for 1 h. The reaction mixture was then cooled at room temperature and washed several times with adequate amount of methylene chloride [ 27 ]. The catalyst was recovered and concentrated under reduced then purified using column chromatography over silica gel with eluent (hexane: dichloromethane (7:3) and finally recrystallized using ethanol. 1-(Phenyl)-1H, 3H-thiazolo[3,4-a]-6-nitro-benzimidazole (2a) 1 H-NMR (DMSO-d6): 6.55–6.57 (d, 2H, J = 8Hz , CH 2 -S), 7.45 (s, 1H, N-CH-S), 7.47–7.48 (d, 1H, Ar-H), 7.59–7.60 (t, 1H, Ar-H), 7.62 (s, 1H, Ar-H), 7.641–7.648 (t, 1H, Ar-H), 7.79–7.81 (d, 1H, Ar-H), 8.14–8.17 (dd, 1H, Ar-H), 8.23–8.25 (t, 1H, Ar-H), 8.514–8.519 (d, 1H, Ar-H) ppm; IR (KBr): 3090, 2988 cm − 1 ; MS:(M + 1): 279.06; yield 89%. 1-(4-Hydroxyphenyl)-1H,3H-thiazolo[3,4-a]-6-nitrobenzimidazole (2b) 1 H-NMR (DMSO-d6): 5.61 (s, 1H, OH (exchangeable peak)), 6.53–6.55 (d, 2H, J = 8Hz , CH 2 -S), 6.66–6.69 (d, 1H, Ar-H), 6.92 (s, 1H, N-CH-S), 7.41–7.45 (d, 1H, Ar-H), 7.70–7.73 (d, 1H, Ar-H), 8.05–8.09 (d, 1H, Ar-H), 8.10 (s, 1H, Ar-H), 8.11–8.15 (dd, 1H, Ar-H), 8.41–8.45 (d, 1H, Ar-H) ppm; IR (KBr): 3280 − 2850, 3030, 2920 cm − 1 ; MS:(M + 1): 313.05; yield 92%. 1-(4-Chlorophenyl)-1H, 3H-thiazolo[3,4-a]-6-nitro-benzimidazole (2c) 1 H-NMR (DMSO-d6): 6.52–6.54 (d, 2H, J = 8Hz , CH 2 -S), 7.40–7.41 (d, 1H, Ar-H), 7.419 (s, 1H, N-CH-S), 7.442–7.447 (d, 1H, Ar-H), 7.67–7.69 (d, 1H, Ar-H), 7.77–7.80 (d, 1H, Ar-H), 8.13–8.15 (dd, 1H, Ar-H), 8.22–8.24 (d, 1H, Ar-H), 8.49 (s, 1H, Ar-H) ppm; IR (KBr): 3060, 2962 cm − 1 ; MS: (M + 2): 333.02, (M + 1): 331.02; yield 93%. 1-(4-Chlorophenyl)-1H, 3H-thiazolo[3,4-a]-6-nitro-benzimidazole (2d) 1 H-NMR (DMSO-d6): 3.79 (s, 3H, OCH 3 ), 3.87 (s, 3H, OCH 3 ), 3.91 (s, 3H, OCH 3 ), 6.98 (s, 1H, CH 2 -S), 7.11 (s, 1H, CH 2 -S), 7.23 (s, 1H, N-CH-S), 7.54–7.56 (d, 1H, Ar-H), 7.58–7.59 (d, 1H, Ar-H), 8.13 (s, 1H, Ar-H), 8.15 (s, 1H, Ar-H), 8.18 (s, 1H, Ar-H), 8.57 (s, 1H, Ar-H) ppm; IR (KBr): 3075, 2925 cm − 1 ; MS: (M + 1): 387.09; yield 95%. 1-(2-Furo)-1H, 3H-thiazolo[3,4-a]-6-nitro-benzimidazole (2e) 1 H-NMR (DMSO-d6): 6.51–6.54 (dd, 2H, J = 8Hz , CH 2 -S), 7.38 (s, 1H, Ar-H), 7.39 (s, 1H, N-CH-S), 7.40–7.41 (d, 1H, Ar-H), 7.418 (s, 1H, Ar-H), 7.425–7.427 (d, 1H, Ar-H), 7.43 (s, 1H, Ar-H), 7.44 (s, 1H, Ar-H) ppm; IR (KBr): 3110, 3090, 2985 cm − 1 ; MS: (M + 1): 287.04; yield 89%. 4.1.3. General procedure for the synthesis of 1-aryl-1H, 3H-thiazolo[3,4-a]-6-amino-benzimidazole derivatives (3a-e) The solution of synthesized candidates (2a-e) (3 mmol) in ethanol was allowed to react with H 2 /Pd over C for 2 h with stirring at room temperature adopting a reported procedure of catalyzed reduction [28] followed by filtration and crystallization of the yielded compounds from ethanol and methylene chloride. The obtained products were directly headed to the following reaction to yield the desired final products (4a-e) . 4.1.4. General procedure for the synthesis of 1-aryl-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole derivatives (4a-e) The final targets were obtained via acylation reaction, where (3 mmol) of the previously prepared intermediates (3a-e) was allowed to stir in a rounded flask surrounded by ice with 15 mL methylene chloride. An equivalent amount of TEA (3 mmol) was slowly added to the stirring solution followed by dropwise addition of benzoyl chloride (5 mmol). The mixture was refluxed for further 1.5 h and the resultant was purified and washed with methylene chloride and ether then dried under vacuum. [ 28 ] 1-(Phenyl)-1H 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole (4a) 1 H-NMR (DMSO-d6): 7.32–7.34 (d, 2H, J = 8Hz , CH 2 -S), 7.45 (s, 1H, N-CH-S), 7.47–7.48 (d, 1H, Ar-H), 7.51–7.53 (t, 1H, Ar-H), 7.59–7.60 (t, 1H, Ar-H), 7.62 (s, 1H, Ar-H), 7.641–7.648 (t, 1H, Ar-H), 7.79–7.81 (d, 1H, Ar-H), 7.82–7.84 (t, 1H, Ar-H), (7.95–8.03 (d, 2H, Ar-H), 8.14–8.17 (dd, 1H, Ar-H), 8.23–8.25 (t, 1H, Ar-H), 8.34–8.36 ( t, 1H, Ar-H), 8.514–8.519 (d, 1H, Ar-H), 10.45 (s, 1H, NH(exchangeable peak)) ppm; 13 C-NMR (DMSO-d6): 39.3, 57.8, 128.9, 129.1, 129.6, 130.1, 130.2, 131.2, 133.4, 134.1, 167.7 ppm; IR (KBr): 3220, 3090, 2988 cm − 1 ; MS:(M + 1): 371.11; yield 72%. 1-(4-Hydroxyphenyl)-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole (4b) 1 H-NMR (DMSO-d6): 5.61 (s, 1H, OH (exchangeable peak)), 6.53–6.55 (d, 2H, J = 8hz , CH 2 -S), 6.66–6.69 (t, 1H, Ar-H), 6.84–6.87 (d, 1H, Ar-H), 6.92 (s, 1H, N-CH-), 6.94–6.97 (t, 1H, Ar-H), 7.41–7.42 (d, 1H, Ar-H), 7.45 (s, 1H, Ar-H), 7.64–7.73 (dd, 2H, J = 8Hz , Ar-H), 7.83–7.85 (d, 1H, Ar-H), 8.05–8.10 (t, 1H, Ar-H), 8.11–8.12 (d, 1H, Ar-H), 8.13–8.15 (d, 1H, Ar-H), 8.41–8.45 (d, 1H, Ar-H) ppm; 10.23 (s, 1H, NH(exchangeable peak)) ppm; 13 C-NMR (DMSO-d6): 39.2, 57.9, 128.9, 129.1, 129.6, 130.1, 130.2, 131.2, 133.4, 132.1, 134.1, 139.7, 156.2, 167.7 ppm; IR (KBr): 3288, 3280 − 2850, 3030, 2920 cm − 1 ; MS:(M + 1): 387.1; yield 67%. 1-(4-Chlorophenyl)-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole (4c) 1 H-NMR (DMSO-d6): 7.49–7.53 (t, 1H, Ar-H), 7.56–7.59 (t, 1H, Ar-H), 7.62–7.66 (t, 1H, Ar-H), 7.80–7.81 (d, 2H, J = 6Hz , CH 2 -S), 7.83 (s, 1H, N-CH-S), 7.94–7.97 (dd, J = 8Hz , 4H, Ar-H), 8.01–8.03 (dd, 2H, Ar-H), 8.27–8.29 (d, 2H, Ar-H), 10.60 (s, 1H, NH(exchangeable peak)) ppm; 13 C-NMR (DMSO-d6): 39.33, 57.69, 128.22, 128.95, 129.06, 129.74, 130.14, 131.19, 133.37, 167.80 ppm; IR (KBr): 3255, 3060, 2962 cm − 1 ; MS: (M + 2): 407.07, (M + 1): 405.07; yield 75%. 1-(4-Chlorophenyl)-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole (4d) 1 H-NMR (DMSO-d6): 3.02–3.03 (m, 3H, OCH 3 ), 3.05–3.06 (m, 3H, OCH 3 ), 3..07-3.08 (m, 3H, OCH 3 ), 7.49 (s, 1H, CH 2 -S), 7.50 (s, 1H, CH 2 -S), 7.51 (s, 2H, Ar-H), 7.53 (s, 1H, N-CH-S), 7.61–7.62 (t, 1H, Ar-H), 7.62–7.65 (t, 1H, Ar-H), 7.65–7.66 (t, 1H, Ar-H), 7.946–7.949 (d, 4H, Ar-H), 7.96 (s, 1H, Ar-H), 10.62 (s, 1H, NH (exchangeable peak)) ppm; 13 C-NMR (DMSO-d6): 8.853, 39.33, 45.67, 129.06, 129.73, 131.20, 133.36, 167.77 ppm;IR (KBr): 3290, 3075, 2925 cm − 1 ; MS: (M + 1): 461.14; yield 84% 1-(2-Furo)-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole (4e) 1 H-NMR (DMSO-d6): 5.17 (s, 1H, CH 2 -S), 5.32 (s, 1H, CH 2 -S), 7.04 (s, 1H, Ar-H), 7.16 (s, 1H, Ar-H), 7.29 (s, 1H, Ar-H), 7.59 (s, 1H, N-CH-S), 7.61 (t, 1H, Ar-H), 7.62 (d, 2H, Ar-H), 7.70 (t, 1H, Ar-H), 7.71 (t, 1H, Ar-H), 8.07 (s, 1H, Ar-H), 8.09 (d, 2H, Ar-H), 8.95 (s, 1H, NH (exchangeable peak)) ppm; 13 C-NMR (DMSO-d6): 39.7, 59.8, 128.9, 129.1, 129.6, 130.1,, 131.2, 133.4, 132.1, 139.7, 156.2, 167.7 ppm; IR (KBr): 3280, 3110, 3090, 2985 cm − 1 ; MS: (M + 1): 361.09; yield 80%. 4.2. Biological Assays 4.2.1. CYP51 MTb enzyme production and purification The pET26 plasmid containing CYP51 gene was transformed into BL21(DE3) competent E. coli and plated onto LBkan/LBkancm plates and left overnight. Colonies were harvested and cultured at 37°C in 8x600 mL of LB for approximately 16 hours at 120 rpm. The incubator temperature was lowered to 19°C, followed by the addition of IPTG (0.1 mM), Delta-ALA (10mg/flask) and further incubation at this temperature for 16 hours at 120 rpm. The cells were harvested by centrifugation (5,000 g , 15 min) and resuspended in 200 mL of 50 mM Tris, pH 7.4, containing 1 mM DTT. The resuspended cells were kept on ice and lysed by sonication (25 cycles at 20:40 seconds on:off, 70%, 19 mm probe, Sonics Vibra-Cell). The supernatant, containing the desired protein, was isolated from cell debris by centrifugation (40,000 g , 30 min). The protein was then loaded onto a DEAE Sepharose column, (XK50, 200 mm x 40 mm, GE Healthcare) and eluted using a linear salt gradient of 100 mM to 400 mM KClin50 mM Tris, pH 7.4. The fractions containing the desired protein (identified by red color of the P450 enzymes) were combined and the volume reduced using a Vivacell 100 (Sartorius Stedim, 10 kD membrane) aided by centrifugation (1,500 g ). The protein was desalted using a Sephadex G-25 medium grain column (250 mm x 40 mm) eluted with 50 mM Tris, pH 7.4. The desalted protein was then further purified by loading it onto a Source-Q ion-exchange column (XK26, 80 mm x 30 mm, GE Healthcare) and eluted with a gradient of 0 to 750 mM KCl in 50 mM Tris, pH 7.4. Fractions with the highest A 420 / A 280 ratio were retained. The protein was concentrated via ultrafiltration and an equal volume of 80% glycerol was added before filtration through a 0.22 µM syringe filter and storage at -20°C. The final A 420 / A 280 ratio was 1.1for CYP51 MTb [ 25 , 30 ]. 4.2.2. Spin state shift assays Glycerol in stored protein samples was removed via buffer exchange into Buffer T (50mm Tris pH 7.5 with 1 mM DTT, (dithiothreitol)) using a PD-10 desalting column (GE Healthcare). The P450 was diluted to approximately 1 µM using the same buffer and the spectrum was recorded on a UV/Vis spectrophotometer. Aliquots (1 to 2 µL) of various substrates/inhibitors (2a-e), (3a-e) and (4a-e) stock solutions in DMSO or EtOH (50 mM − 100 mM) were added. The absorbance between 600 nm and 250 nm recorded using a UV-Visible spectrophotometer until no further spectral change is observed. The high spin percentage (± 5%) was estimated by comparison to a set of spectra, generated by the sum of substrate-free (> 95% low spin) and substrate-bound (camphor, ≥ 95% high spin) CYP101A1 to the appropriate percentages. CYP51 MTb showed low spin state in the resting ferric form at 418 nm [ 25 , 30 ]. 4.2.3. In vitro cell lines assay The in vitro analysis was carried out by testing the effect of the compounds on SW620 colorectal adenocarcinoma cell lines, (cells were obtained from Nawah Scientific., Inc (Mokatam, Cairo, Egypt). Cells were maintained in DMEM media supplemented with 100mg/ml of streptomycin, 100 units/ml of penicillin and 10%of heat inactivated fetal bovine serum in humidified, 5% (v/v) CO2 atmosphere at 37°C. Cell viability was assessed by SRB assay. Seeding was done using aliquots of 100 µl cell suspension (5x10^3cells) which were added to 96-well plates. Then, cells were treated with the compounds at various concentrations (0.1,1,10,100,1000 ug/ml). After 72h of drug exposure, SRB assay cells were fixed by replacing media with 150 µL of 10% trichloroacetic acid (TCA) and incubated at 4°C for 1h. The TCA solution was removed, and the cells were washed 5 times with distilled water. Aliquots of 70 µL SRB solution (0.4%w/v) were added and incubated in a dark place at room temperature for 10 min. Plates were washed 3 times with 1% acetic acid and allowed to air-dry overnight. Then, 150 µL of TRIS (10mM) was added to dissolve protein-bound SRB stain for 5 min on a shaker at 1,600 rpm. The absorbance was then measured at 540 nm using a BMGLABTECH®-FLUO star Omega micro-plate reader (Ortenberg, Germany) [ 32 ]. 4.3. Computational Studies The molecular modeling studies were applied using MOEv.2010 -where the heme portion can be readable- following reported procedures to perform the addressed research. Results and 2D interactions were viewed using also MOEv.2010, while the 3D interactions were presented using Discovery Studio 4.1 client. 4.3.1. Enzymes Preparation Initially, the crystal structures of sterol cytochrome 14-alpha demethylase (CYP51) were downloaded from pdb under codes: (4UHL) for the human source one complexed with VFV [ 24 ], and (1EA1) for the one with the source from mycobacterium tuberculosis [ 25 ]. Add hydrogens was applied to the downloaded enzymes followed by automatic connect and type step which was applied to all atoms and bonds. Receptor was identified, selected, and potentially fixed. The unneeded sequential chains and ligands were removed meanwhile, the interaction site (pocket) was identified and selected together with the required complexed ligand and heme position in the channel [ 33 ]. 4.3.2. Dynamics Simulation The 15 designed and synthesized compounds' structures were built up using ChemDraw Ultra 8.0 and saved as readable (.mol) files. Connect and Type was then applied automatically to all the compounds together with the reference ligand VFV. Dynamic simulation was then performed using start point zero-till 250 checkpoints, velocity was applied, and time was set at (0.5s). Then, the forcefield applied was set as MMFF94x while, bonded, Van der Waals, electrostatics and restraints were all enabled, cut –offs were set at: on-8, off-10 and distance salvation was set at 1. Hydrogens and all partial charges were fixed, and the protocol used was NPA (Nose–Poincare–Andersen–Hamiltonian equations of motion). 510 different dynamic conformations were resulted for each compound that were further used in the docking study [ 33 ]. 4.3.3. Molecular Docking Docking study was finally computed for each enzyme separately with the dynamic molecules of each 15 compounds and the reference VFV , using placement: Alpha Triangle and rescoring 1: London dG with None refinement, None rescoring 2, and retaining scores resulted were 5010 for each compound due to the huge dynamic conformations possibilities. Scores were then sorted in descending and the best score pose was chosen to view its interaction against the required enzyme [ 33 ]. Declarations Acknowledgements The authors are grateful to Professor Nasser S.M. Ismail, for his great collaboration in this study also would like to express sincere thanks to Future University in Egypt for providing CADD Lab facility. Ethics approval and consent to participate Conflict of interest We declare that the authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper. Consent for publication The results/data/figures in this manuscript have not been published elsewhere, nor are they under consideration (from you or one of your Contributing Authors) by another publisher. Availability of data and materials All the material is owned by the authors and are available. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors' contributions Prof. H.R. put the research ideas and revised the whole content. I.M., H.R., R.T. and A.M. did the experiments and characterizations, analyzed the results, and organized the writing of the manuscript. Authors' information 1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Future University in Egypt, 11835, Cairo, Egypt Iten M. Fawzy 1 *, [email protected] , Hanan M. Refaat 1 , [email protected] , Asmaa A. Mandour 1 , [email protected] . 2 Department of Pharmacology and Toxicology and Biochemistry, Faculty of Pharmacy, Future University in Egypt, 11835, Cairo, Egypt Reem T. Attia 2 , [email protected] . 3 Department of Chemistry, School of Physical Sciences, University of Adelaide, 5000 SA, Australia. Heba A. Ibrahim 3 , [email protected] References J. Zhang, L. Li, Q. Lv, L. Yan, Y. Wang, Y. Jiang, Front. 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Supplementary Files HNMRSupportingMaterial.pdf Scheme1.png Scheme 1. Synthetic scheme of Novel 1-Aryl-Thiazolo Benzimidazole Derivatives TOC.png Novel compound 4d ( 1-(4-chlorophenyl)-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole) characterized by trimethoxy phenyl and benzamide groups attached strategically to thiazolo-benzimidazole ring represents a promising anticancer agent of (type I) substrate binding to CYP51 MTb. It displayed IC 50 of 0.03 µM on SW620 in vitro colorectal adenocarcinoma cell lines and 3% shift in the Spin state assay against CYP51 MTb. The bulky groups occupied the whole space of helix human cytochrome in silico and thus explaining their type I binding behavior. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Refaat","email":"","orcid":"","institution":"Future University in Egypt","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hanan","middleName":"M.","lastName":"Refaat","suffix":""},{"id":111901595,"identity":"9020f5f8-6fe4-4662-97f8-ff0b713db575","order_by":2,"name":"Reem T. Attia","email":"","orcid":"","institution":"Future University in Egypt","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Reem","middleName":"T.","lastName":"Attia","suffix":""},{"id":111901596,"identity":"1c2b4be1-58e9-406c-b822-3a387bb48e9f","order_by":3,"name":"Heba A. Ibrahim","email":"","orcid":"","institution":"University of Adelaide","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Heba","middleName":"A.","lastName":"Ibrahim","suffix":""},{"id":111901597,"identity":"26b440e5-4d92-4416-9c19-844729af1c0a","order_by":4,"name":"Asmaa A. Mandour","email":"","orcid":"","institution":"Future University in Egypt","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Asmaa","middleName":"A.","lastName":"Mandour","suffix":""}],"badges":[],"createdAt":"2022-06-05 11:59:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1727366/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1727366/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":22644398,"identity":"c86efedc-0acb-4733-a6c3-e6d36ef62912","added_by":"auto","created_at":"2022-06-14 16:11:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":92468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eChemical structures of\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e: \u003c/em\u003e\u003c/strong\u003e\u003cem\u003eGaleterone [6]\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e \u003c/em\u003eA)\u003c/strong\u003e\u003cem\u003e and R)-N-(1-(3,4′-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl) ethyl)- 4-(5-phenyl-1,3,4-oxadiazol-2-yl) benzamide (VFV)\u003c/em\u003e\u003csup\u003e\u003cem\u003e[2]\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e\u003cem\u003e \u003c/em\u003eB)\u003c/strong\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/c0cf20007409b4a24e35a6a2.png"},{"id":22645414,"identity":"94727ab4-508b-48d6-b8dc-02d2280a5d2b","added_by":"auto","created_at":"2022-06-14 16:16:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":97962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eChromatograms\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e \u003c/em\u003e\u003c/strong\u003e\u003cem\u003eof\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e (4a-e):\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e Absorbance Soret peaks and type of shifts for each compound.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/8d1066b7fec735b14f0cfe8c.png"},{"id":22646189,"identity":"cb5962de-db09-456e-b1a2-567aff2c5b45","added_by":"auto","created_at":"2022-06-14 16:21:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":29855,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxicity results of the newly synthesized compounds on SW620 cells, Data represent mean ± SD; n = 3 (Each experiment was done in triplicate; in each run 6 wells were used for each concentration). Statistical analysis was done using prism version 8.4.3 and the means were compared using t-test at P ˂0.05, compared to \u003cstrong\u003eDOX\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/96c158e411dc45ef2d5b1f2d.png"},{"id":22645420,"identity":"7ec5b1c0-1e32-4171-8b37-129f902502d1","added_by":"auto","created_at":"2022-06-14 16:16:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":84085,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cspan class=\"ql-cursor\"\u003e\u003c/span\u003e3D structure of \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eHeme-iron\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e embedded in helix-I channel of human (CYP51) \u003c/em\u003e\u003cstrong\u003e\u003cem\u003e(4UHL) \u003c/em\u003eA)\u003c/strong\u003e\u003cem\u003e, 3D structure of \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eVFV\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e embedded in helix-I channel of human (CYP51) \u003c/em\u003e\u003cstrong\u003e\u003cem\u003e(4UHL) \u003c/em\u003eB)\u003c/strong\u003e\u003cem\u003e, 3D structure of compound \u003c/em\u003e\u003cstrong\u003e\u003cem\u003e4d\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e embedded in helix-I channel of human (CYP51) \u003c/em\u003e\u003cstrong\u003e\u003cem\u003e(4UHL) \u003c/em\u003eC)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/f223c910bdb0dc2d49db0008.png"},{"id":22645417,"identity":"2800e131-53f5-4279-87ed-e5ac817c323e","added_by":"auto","created_at":"2022-06-14 16:16:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":151856,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e2D interaction diagram between \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eHeme-iron\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e, \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eVFV\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026amp; channel residues of helix-1 human sterol (CYP51) \u003c/em\u003e\u003cstrong\u003e\u003cem\u003e(4UHL)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e. Color Code; Purple dots: Metal contact, Green arrow: side chain acceptor, Green dotted with H: Hydrogen-arene Van der Waals \u0026amp; Dark blue circle: basic interaction.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/b5f90a839fcc2bc46051b22d.png"},{"id":22645419,"identity":"295efe6b-9224-47e6-9ca4-d61cbce2d3f1","added_by":"auto","created_at":"2022-06-14 16:16:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":88610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e2D interaction diagram between \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eNovel compound 4d\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026amp; channel residues of helix-1 human sterol (CYP51) \u003c/em\u003e\u003cstrong\u003e\u003cem\u003e(4UHL)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e. Color Code; Green dotted with H: Hydrogen-arene Van der Waals \u0026amp; Dark blue circle: basic interaction.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/9c368845d94900976bf51be3.png"},{"id":22644410,"identity":"94af870d-8804-49cd-962a-baef3b29b3ce","added_by":"auto","created_at":"2022-06-14 16:11:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":52563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e2D interaction diagram between \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ecompound (3a) \u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026amp; channel residues of non-human cytochrome (CYP51)\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e (1EA1)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e. Color Code; Green arrow: side chain acceptor, Green dotted with H: Hydrogen-arene Van der Waals \u0026amp; Dark blue circle: basic interaction.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/7e6257a38f7f1286b9320118.png"},{"id":22645446,"identity":"7c0c166e-f0e9-4fc2-9383-72971cf26576","added_by":"auto","created_at":"2022-06-14 16:16:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":95892,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eA)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e 3D structure of \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eVFV\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e embedded in channel of non-human cytochrome (CYP51)\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e (1EA1)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eB)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e3D structure of \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ecompound (4d)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e embedded in channel of non-human cytochrome (CYP51)\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e (1EA1)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/3ab3de43fff6244bfc47a31f.png"},{"id":22644443,"identity":"03576d9a-d06b-4983-a6f1-6afc8ed37009","added_by":"auto","created_at":"2022-06-14 16:11:00","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":72643,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eA\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e) 2D interaction diagram between \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eVFV\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026amp; channel residues of non-human cytochrome (CYP51)\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e (1EA1)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e. \u003c/em\u003e\u003cstrong\u003e\u003cem\u003eB)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e 2D interaction diagram between \u003c/em\u003e\u003cstrong\u003e\u003cem\u003ecompound (4d) \u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026amp; channel residues of non-human cytochrome (CYP51)\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e (1EA1)\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e. Color Code; Blue arrows: H-bond-interactions.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/797c2a0fd751d5ecd9770a86.png"},{"id":23115983,"identity":"89b6205a-530a-4a9f-9ba2-82bb4a1ff076","added_by":"auto","created_at":"2022-06-27 09:44:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1510225,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/2c484675-8af5-44d5-aceb-5c5f2a6c2119.pdf"},{"id":22644401,"identity":"431dcbbe-d4be-44a7-ab17-6225b78b5782","added_by":"auto","created_at":"2022-06-14 16:11:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":605361,"visible":true,"origin":"","legend":"","description":"","filename":"HNMRSupportingMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/31d4501dee07f301d1e28dcf.pdf"},{"id":22645416,"identity":"177e88fa-a9bd-4892-b236-adca9ca48e5d","added_by":"auto","created_at":"2022-06-14 16:16:00","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":81481,"visible":true,"origin":"","legend":"\u003cp\u003e\t\u003cstrong\u003eScheme 1.\u003cem\u003e \u003c/em\u003e\u003c/strong\u003e\u003cem\u003eSynthetic scheme of Novel 1-Aryl-Thiazolo Benzimidazole Derivatives\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/b917816902bbf742dfc29b9f.png"},{"id":22644440,"identity":"0aa90a3e-1b5e-4e5d-8caf-0e30c28f38cc","added_by":"auto","created_at":"2022-06-14 16:11:00","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":274814,"visible":true,"origin":"","legend":"\u003cp\u003eNovel compound \u003cstrong\u003e4d (\u003c/strong\u003e\u003cem\u003e1-(4-chlorophenyl)-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole)\u003c/em\u003e characterized by trimethoxy phenyl and benzamide groups attached strategically to thiazolo-benzimidazole ring represents a promising anticancer agent of (type I) substrate binding to CYP51\u003csub\u003eMTb.\u003c/sub\u003e It displayed IC\u003csub\u003e50\u003c/sub\u003e of 0.03 µM on \u003cstrong\u003eSW620\u003c/strong\u003e\u003cem\u003ein vitro\u003c/em\u003ecolorectal adenocarcinoma cell lines and 3% shift in the Spin state assay against CYP51\u003csub\u003eMTb.\u003c/sub\u003eThe bulky groups occupied the whole space of helix human cytochrome \u003cem\u003ein silico\u003c/em\u003e and thus explaining their type I binding behavior.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"TOC.png","url":"https://assets-eu.researchsquare.com/files/rs-1727366/v1/fc0d3f9d399ec8cd1db37936.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis, Molecular Modeling and Biological Studies of Novel 1-Aryl-Thiazolo Benzimidazole Derivatives as Cytochrome (CYP51 MTb ) Type I Inhibitors \u0026 Anticancer Agents","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCytochrome-P 450 (CYPs) targeting is considered an effective anticancer chemotherapeutic approach [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], including different CYPs either as drug targets or drug-metabolizing enzymes. Literature survey presented specific types of CYPs that were targeted as anticancer agents; where aromatase (CYP19) is a key target for breast cancer treatment \u0026amp; also sterol 14α -demethylases (CYP51\u003csub\u003eMtb\u003c/sub\u003e), is essential for membrane sterol biosynthesis either cholesterol in animals or ergosterol in fungi, thus is a more specific drug target [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. CYP51\u003csub\u003eMtb\u003c/sub\u003e, the Mycobacterium tuberculosis CYP enzyme with a determined catalytic function, belongs to sterol 14α - demethylases which is responsible for demethylation of the 14α -methyl group from sterol molecules during the biosynthesis of sterol in most pathogenic fungi or protozoa [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Many drugs including azoles prevent fungal growth via inhibition of sterol 14α-demethylases and thus, inhibiting ergosterol biosynthesis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. From this point, azoles were further reported as potential chemotherapeutic agents and inhibitors with clear role in cancer management [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBased on literature, there are two types of binding for the CYP51\u003csub\u003eMtb\u003c/sub\u003e substrates which can be recorded via alteration of CYP51\u003csub\u003eMtb\u003c/sub\u003e optical spectra. If the ligand binding in the active site showed 390 nm peak and 420 nm trough difference in spectra then it is substrate binding (type I) while, inhibitor binding (type II) shows 416 nm trough and 436 nm peak in the difference spectra [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSubstrates as Galeterone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e, a benzimidazole derivative and a CYP 17 α hydrolase inhibitor - was investigated against human sterol 14α demethylase (CYP51A1), which catalyzes the main part of the sterol biosynthetic pathways. Differential absorption spectra showed type I spectral changes of CYP51A1 upon addition of Galeterone with sigmoidal dependence of ΔA (390\u0026ndash;424) on ligand concentration [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. On the other hand, Galeterone was confirmed to possess anticancer activity against various cancerous cell lines with IC\u003csub\u003e50\u003c/sub\u003e in range of 8\u0026ndash;41 \u0026micro;M [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlso, prominent compound: (R)-N-(1-(3,4\u0026prime;-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide \u003cb\u003e(VFV)\u003c/b\u003e showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, which is a benzamide-imidazole derivative, showed prominent ability of interaction through binding and inhibition to CYP51\u003csub\u003emtb\u003c/sub\u003etogether being as an efficient anti-proliferative agent [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe above findings supported the fact that the fused heterocycle containing nitrogen in the benzimidazole scaffold is the reason for their interaction with biomolecular targets via its nucleo-bioisostere activity [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and hence allowing them to be multi-target inhibitors specifically against various critical enzymes involved in cell synthesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It was also concluded that not only the presence of benzimidazole nucleus is responsible for the anticancer activity, but also the proper substitution is very critical for the development of new cytotoxic candidates [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs a result, novel chemotherapeutic agents of various series of benzimidazoles have been reported and their proliferative assays confirmed their significant potent anticancer activity against various human cancer cell lines via different mechanisms [\u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19 CR20\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This wide therapeutic potential exhibited by benzimidazole and its derivatives, opened a new approach to design and synthesize more potent derivatives with diverse pharmacological activities [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGuided by the above outlines, this research was focused on the design and synthesis of novel 1-Aryl-thiazolo-benzimidazole derivatives as new candidates with potential anticancer activity. NaHSO\u003csub\u003e4\u003c/sub\u003e-SiO\u003csub\u003e2\u003c/sub\u003e catalyst assisted synthesis method of benzimidazoles was adopted as reported [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], to fulfill the green chemistry requirements to protect both environment and human health.\u003c/p\u003e \u003cp\u003eThe new molecules were tested for their binding to extracted cytochrome CYP51\u003csub\u003eMtb\u003c/sub\u003e. Since \u003cem\u003ein vitro\u003c/em\u003e CYP51\u003csub\u003eMTb\u003c/sub\u003e absorbance assay was performed on the available non-human strain, it was essential to assure that the efficacy of the tested compounds will not deviate from those of human strain. Therefore, molecular modeling studies were performed on both human \u0026amp; non-human 14α-sterol demethylase enzymes \u003cb\u003e(4UHL), (1EA1)\u003c/b\u003e respectively downloaded from PDB [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and the type of binding was detected. The results coincide with literature survey which confirmed the insignificant difference between human and non-human strains thus would not affect the efficacy of the tested compounds [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurther, the active CYP51 inhibitors were subjected to \u003cem\u003ein vitro\u003c/em\u003e anti-proliferative assay against colorectal adenocarcinoma \u003cb\u003e(SW620)\u003c/b\u003e cell types.\u003c/p\u003e"},{"header":"2. Results And Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. Chemistry\u003c/h2\u003e\n \u003cp\u003eThe study was designed to synthesize candidates based on the findings that 1,3-fused-5/6- benzimidazoles with thiourea derivatives exhibited remarkable anti-proliferative activity against tumor cells [\u003cspan class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e]. Introduction of strategic substitutions to the proposed moiety was adopted in the following Scheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eThe scheme of synthesis showed that 4-nitro-o-phenylenediamine was selected as a strategic starting chemical to react with 2-mercaptoacetic acid and diverse of aromatic aldehydes in one pot reaction using acetonitrile as solvent and NaHSO\u003csub\u003e4\u003c/sub\u003e-SiO\u003csub\u003e2\u003c/sub\u003e as a catalyst. The mixture was allowed to reflux at 75\u0026deg;C for 1 h followed by purification and recrystallization procedures as reported in literature [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe NaHSO\u003csub\u003e4\u003c/sub\u003e-SiO\u003csub\u003e2\u003c/sub\u003e catalyst was first prepared \u003cem\u003ein situ\u003c/em\u003e applying the modified procedure of Nishiguchi\u0026rsquo;s method, where NaHSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO was allowed to stir in adequate amount of distilled water with SiO\u003csub\u003e2\u003c/sub\u003e of column chromatographic grade and gently heated till white solid was obtained then the catalyst was allowed to dry in oven for 48 h prior to use [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eThe obtained products \u003cstrong\u003e(2a-e)\u003c/strong\u003e with yield (89\u0026ndash;95%) were further subjected to catalyzed reduction rendering the nitro into amino group using classical H\u003csub\u003e2\u003c/sub\u003e/Pd over carbon procedure as reported to yield intermediates \u003cstrong\u003e(3a-e)\u003c/strong\u003e [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eEach obtained intermediate from the above \u003cstrong\u003e(3a-e)\u003c/strong\u003e was allowed to react with benzoyl chloride using TEA and methylene chloride as a solvent to obtain the targeted final compounds \u003cstrong\u003e(4a-e)\u003c/strong\u003e with yield (67\u0026ndash;84%).\u003c/p\u003e\n \u003cp\u003eThe unreported above three series of compounds \u003cstrong\u003e(2a-e), (3a-e) \u0026amp; (4a-e)\u003c/strong\u003e were subjected to further structural elucidation analysis; Ft-IR, H\u003csup\u003e1\u003c/sup\u003eNMR, C\u003csup\u003e13\u003c/sup\u003eNMR \u0026amp; Mass analysis.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. Biology\u003c/h2\u003e\n \u003cdiv class=\"Section3\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.2.1. CYP51\u003csub\u003eMTb\u003c/sub\u003eSpectrometric Absorbance Assay\u003c/h2\u003e\n \u003cp\u003eThe ability of the synthesized molecules to interact with CYP51\u003csub\u003eMTb\u003c/sub\u003e was studied using a dual-beam Shimadzu UV-2401PC spectrophotometer to record UV-visible absorption spectra. The binding affinity of ligand or inhibitor in the CYP active site is measured by the concentration of the spectral changes. The Concentration of CYP51\u003csub\u003eMTb\u003c/sub\u003e used was 0.0015mM and measured at 418 nm with max absorbance\u0026thinsp;=\u0026thinsp;0.2. The stock solutions of ligands were prepared 1-4mg in 1ml DMSO and volume used for each ligand\u0026thinsp;=\u0026thinsp;0.1\u0026ndash;0.2 \u0026micro;L [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e]. The newly designed ligands induced type I spectral shift of the enzyme signifying a change from the low-spin state to the high-spin state of the heme iron as shown in \u003cstrong\u003esupplementary 1 \u0026amp; 2\u003c/strong\u003e chromatograms. The minimum peak wavelength was 390 nm, while the maximum peak wavelength was 418 nm. The absorbance change (\u0026Delta;A (A418 \u0026ndash; A390)) and the shift percentage were calculated and summarized in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Most of the ligands induced less than 5% shift with CYP51\u003csub\u003eMTb\u003c/sub\u003e.\u003c/p\u003e\n \u003cp\u003eThe nitro derivatives \u003cstrong\u003e(2a-e)\u003c/strong\u003e displayed mild shift indicating agonist like binding of type I, while the amino derivatives of the novel nucleus \u003cstrong\u003e(3a-e)\u003c/strong\u003e showed no shift nor change in spectral troughs which indicated no binding. Meanwhile, the benzamide derivatives \u003cstrong\u003e(4a-e)\u003c/strong\u003e showed better shifts than \u003cstrong\u003e(2a-e)\u003c/strong\u003e with the same behavior of type I binding as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eLigand binding parameters calculated from the spectral analysis of CYP51\u003csub\u003emtb\u003c/sub\u003e:\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLigands\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026Delta;A\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e% Shift\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo shift\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo shift\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo shift\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo shift\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo shift\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo shift\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo shift\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo shift\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003csup\u003e\u0026nbsp;\u003cspan class=\"BoldItalic\" name=\"Emphasis\" type=\"BoldItalic\"\u003e*\u003c/span\u003e \u003cem\u003e\u0026Delta;A was calculated according to the following equation: \u0026Delta;A=(A418 \u0026minus; A390) =(0.2\u0026minus;(obtained absorbance value), Shift percentage was calculated according to the following equation: %Shift = Calculated \u0026Delta;A x 100\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.2.2. Anti-proliferative Assay\u003c/h2\u003e\n \u003cp\u003eThe newly synthesized compounds of both active series in the CYP51\u003csub\u003eMTb\u003c/sub\u003e assay \u003cstrong\u003e(2a-e) and (4a-e)\u003c/strong\u003e were screened for their anti-proliferative activities against colorectal adenocarcinoma \u003cstrong\u003e(SW620)\u003c/strong\u003e using sulfo-rhodamine-B (SRB) assay. The compounds were compared with doxorubicin (\u003cstrong\u003eDOX\u003c/strong\u003e), which is an anthracycline chemotherapeutic agent that has been used for the treatment of a variety of human cancers such as colorectal cancer [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. From the observed results \u003cstrong\u003e(\u003c/strong\u003eTable \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cstrong\u003e\u0026amp;\u003c/strong\u003e Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cstrong\u003e), DOX\u003c/strong\u003e showed an IC\u003csub\u003e50\u003c/sub\u003e of 0.07 \u0026micro;M. It was also observed that most of the synthesized compounds showed high cytotoxic properties with IC\u003csub\u003e50\u003c/sub\u003ein the range of 0.03\u0026ndash;0.35 \u0026micro;M.\u003cstrong\u003e4d\u003c/strong\u003e which is the most potent compound, showed 57% significant decrease compared to \u003cstrong\u003eDOX\u003c/strong\u003e. By the same token, \u003cstrong\u003e4b\u003c/strong\u003e showed 42.8% of significant decrease when compared to \u003cstrong\u003eDOX\u003c/strong\u003e treated group. Comparing the obtained results, it was concluded that the benzamide derivatives showed slightly more potent anti-cancer activity than the nitro derivatives. Also, the presence of electron donating groups on benzene ring attached to thiazole (tri-methoxy at ortho \u0026amp; para positions / hydroxy at para position) revealed the most effective anticancer activity as shown for compounds \u003cstrong\u003e4b\u003c/strong\u003e \u0026amp; \u003cstrong\u003e4d.\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cimg 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wIAQQiYBSpqEEEKIneieJiF26O7uRldXF/85ICAA06dPd2NEhBB3oJImBjtl37JlC9avX4/jx4+Pah7/+Mc/0NzcbPf4vr6+o1oOcY/KykosXLgQ27dvx/bt2xEQEEC9OxEyCVHSBFBeXo6VK1fi4MGDKCsrc2has9mM//mf/8HHH38MlUoFhUIBk8k04nQXL15EbW0t+vr6Rhs2GWcKhQKVlZWorKzE9u3b8d5777k7JELIOKOkCeDuu+8eMqynp2fEFrE1NTUICwtDfHw89u3bh9DQUDz55JNYsWIFVCrVsAnx6NGjkMvlEIlEKC0tdco6ENf76quv0NraitbWVlRXVyMsLMzdIRFCxhklTQBxcXE4ceIEtmzZgk2bNgEY7Cd26dKl2Llz55CWsSaTCStWrMDu3btRXV2N8vJytLW1Yf78+VCr1Xj66afxwAMPICwsDPv37+en4+ZZXl6O6upq5OfnT8gu9OLj4+Hj48P/i4+Px61bt9wd1pjp9Xqkp6dj4cKFSExMxMaNG90dEiFkvDEyxIULF5hIJGI7duxg2dnZLCQkhFVVVbGBgQGWm5vLQkJCWHV1tc1pe3t7+Wnee+89lpuby0wmE0tNTWWhoaGsvr5+nNdm/CkUClZcXMwYY6y/v5/NnTuXHTt2zM1RjU1xcTFTKBSMscH1mzt3Luvv7+c/A2DvvvuuO0MkhIwDKmnaEBISgqamJnz55ZfQarVQqVR47bXXcP/99wMA2trahu39h3v7SXV1NSoqKnDw4EEsW7YM8+fPR1tbG6RS6XiuCnGBvXv34u9//zvKy8vR2tqKr776CvX19aisrHR3aIQQV3N31vZ0ly9fZnFxcezOO+9k+/fvd3j6Rx55hP35z392QWSeiyt5cf+2b9/u7pDG7NixYxbr8e6777J169bxnzMyMry+NE0IGdmkKmlyj5O0traiu7vbrmlEIhGqqqrw8MMP48EHH3R4mT/60Y8wZcoUh6fzdsXFxbh06RIAYNasWfzw1tZW+Pj4uCusUVu1ahVeeukl/vMzzzyDgwcPAgC2bNmC6OhorFq1yl3hEULGyaRKmmVlZWhtbYVWq7V4UN0eP/nJT+x6lIR8Jzg4GMXFxfjVr36F7u5utLa2ora21t1hOVVDQwN27dqFJ598EvHx8e4OhxDiYpMqaQJAc3MzDAaDu8OYNNasWYO5c+fizTffRHh4uEVpbSKQSqVgjIExRvc0CZkEJl03evSYgOsJk8eUKVPQ0dHhxmicr729HaGhoe4OgxDiBpMqaXInc0qcZCzCwsLAGHN3GIQQN5hU1bPHjx/H+vXrsWXLFocfth/tOzO//fZbfPvttw5PN5FRwiGEeKtJlTTfeOMNHDx4ENHR0airq3No2lu3buGLL75weJnXr1/Hs88+C61W6/C0hBBCPMukSpr33nvvqKYrKirC+++/j4qKCpvd6tnS3t6Oxx57DF9//TVeffVVHDt2DJGRkQ69CWWiam9vt7slstlsRkNDA4qKipCSkoK5c+dCp9O5OEJCCLFtUiXN5ORk7NixAzqdDjExMbh48SKKioqGTYLNzc0ICwvDlStXkJ2djcrKSty8eRNhYWE4evSozWl6enqQkpKClJQUqNVqfPbZZ/jrX/+Knp4epKWlQaVS8a8gm2gNZOzl5+cHhUIxpEP7vr4+NDQ0IC8vD0lJSXjooYdwzz33YOvWrbhy5QqWLFmCQ4cOITMzExcvXnRT9ISQSc29fSu418DAAEtPT+f7luV0d3ez5ORkJpFI2B/+8AcmkUhYamoqGxgYYIx910uQRCJhTU1N/Lxyc3OZSCRipaWlQ5ZVX1/PQkNDWXZ2Nlu+fDmrr69n69atY5cuXRqflfUwTU1NLDw8nGVlZTG5XM5EIhHz8/NjUqmU5ebmMo1Gwy5cuGAxTW9vLxsYGGCXL19mEomEdXd3uyX2SX7YEDKp0dHPvkuCUqmUbd68mYWEhLCioiI+cXKJ0VpTUxOTSCRMIpGwWbNmsdzcXD6xDqe0tJStWrWKMcZYS0sL37H5ZBQUFMR+8YtfsOrqanb58uURx6+vr2dyuZwxxlhbWxuTSCQjbm9n6u3tZYmJiezHP/4xy87OHrflEkI8ByVNgT/84Q9sypQpbO/evSwkJMRmiVFoYGCAlZSUsDlz5jCtVmv3ctatW8fq6+uZQqGYtCXNy5cvM5FIxP9tr5KSEpaamsoYY6y6uppPoq5WXV3NQkJCmEajYYwxlp+f79bSLiHEPShpCnAn8ra2Ntbb22v3+FKp1KFXfvX397OWlhbW1dU1lnC9Wn5+PsvOzmYDAwPM39/fru3Nyc7OZvn5+YwxyyTqCgMDAyw1NZXJ5fIhCbKpqem2r4kjhEw8k6ohkL1CQ0Nd+nLoKVOmIDw8HNOnT3fZMjzdkSNH8NRTT6GhocHh7Z2fn49z585Bq9UiNTUVfn5+2Llzp8MxtLa24r333uM/Hz9+HK2trfzn5uZmREZGYv78+aiuroa/v7/F9BKJBE1NTdi9ezdUKtWQBmWOvBiAEOIdKGmScXfx4kWYzWaEhoaivLwca9ascXgepaWlKC8vR0NDg0USdURzczN+9atf8YmyrKyMfySopqYGKpUKGo0Gqampw87Dz88P1dXVmD17NlasWME/ShMdHY309HQEBARM2lbShExEk6obPVcRiUT0BhQHaLVaPPXUUzCbzaipqUFpaanD8/D19YVGo8GKFStQWlqK0tJSJCUlwc/Pb9gXhA8nPT19yLOfUqnUofmkp6dDIpGgoaEBjzzyCPR6Pfr7+7Fnzx5cvHgRwcHBDsVECPFM45Y0uW7rHH23ZHd3t8PVmKNdFhkf5eXlqK+vx9GjRyGXy+Hr6zuq+fj5+UGj0SAlJYVPnAqFAv7+/nZ3qB4VFQW9Xs+/a5UzmpgkEgkkEgm//6Wnp+P111+f1NXwhEw0Tq2e5d4nuHfvXot7Q8ePH0d6ejqeffZZu+/x3Lp1C/Hx8aisrHToPYXcstLT09HQ0ODYCvybyWQa8uA9cY7m5mb4+/tDJBKNumpWSCQSobCwECkpKQCAgoICPPbYY/Dx8bntv7CwMACDvUQVFxcjIyNjzOvGmTJlClpaWnDp0iUEBAQMSciEEO/l9Hua8fHx2L17NwCgo6MD3d3dKCsrw8GDB1FZWWn3VfelS5ewbNkybNy4EQsXLrT7vhC3rKKiIotGHvb4/PPP8fXXX+Ovf/0rIiMjsX///tuOLxKJcODAAeh0Onz88ccOLWuy4hoA9fT04OLFi5BKpWOeZ2hoKAoLC+Hn54fW1lasXbuWf8flcP/a2tr46bnEXVVVNeZYgMHakalTp0Kn02H79u0oKytzynwJIe7n9KRZWVmJzZs3Axhs8PHmm29CLBajo6MD7733Hp/8tFrtbftwDQgIQFtbG27duoWWlhYEBATYtXxuWS0tLQgMDAQw2Kjjdssym83YuXMn1q9fD6lUih07diA1NRXnzp1DWFgYampqbE5XU1ODrKwsSKVS9PT0YOnSpdS37Ai0Wi0SExPHXDVrjauOHU3pdcqUKdi1a5dT4gCArq4uPPjgg2hoaIDRaMS0adOcNm9CiJs58/mVY8eOMcYY/wwi1+NNf38/Ky4uZu+++y5jbLA3l9DQ0GGfh6yqqmJxcXGsvr6eFRcXs5aWFsYYY7m5uSw/P99mLzB/+tOf2LRp09jx48f5ZfX39/M9x3z88cfDLiskJIRlZ2fz33Pd6IWGhrK3336byeVyJpfL+YfwL1++zA8TdvVWX1/PJBIJS0xMZNevX2fbt29n27dvZ/39/WPcshNDfX09k0qljDHm8LOt9hB2mGCPlpYWfp9ljFnsa2NVXFzMFAoFy8jIoN+fkAnEpZ0bvPvuuzZPjFVVVaytrY2JRCJmMpn4xHPhwgUmlUpZXFyczV5ient7WXZ2tsUD5QMDA2zJkiXMx8eHRUREDJmutLSUffzxx0wikbDGxkb++wsXLjC5XD7sshgbTO5cPG+99Rarra0dsnxbNBoNW758Obt06RJraWlh27dvt2+DTXDJycmstLTU4eRmL67DBEIIcRW39gjU3d3NqqurmUajYatXr2YhISF2lT64kt5DDz3E7rrrLiaTyZjJZBpxGo1Gw6qrq/ll2duTS1VVFZs9ezb74Q9/OGxJ15pCoWCMDfb+s27dOruWM9EFBQWxxsZGxphjXefZi6u9GK3e3l6nlH67u7tZSUnJmOdDCPE8Powx5u4qYpPJhIiICHz55ZcOTbdkyRKsWLEC2dnZLl9WQ0MDsrKy7G7w09DQwDdEeuaZZ5zS4MXbmUwm/vEQkUjk1HlfvHgRCoUCFy5cGPU8enp6EBYWNuZefHbu3ImbN28iPz9/TPMhhHgej+nc4K677nJ4msDAwCFdm7lqWQBw55132j2uVCqlRGlF+HhIVVWVU7sq5DpMGAt/f3+EhISgoaFhTL/dsWPHUFhYOKZYCCGeibrRcxHuJdMdHR30nJ5AaGgocnNzkZSUdNsWzY46cuQIEhMTxzwfmUyG2traUU9vMpnQ09MDiUQy5lgIIZ7HY0qaE821a9f4rtmcXRXp7bhHdFJSUqDRaMY8v/b2dvj6+iIkJGTM80pMTMTSpUtHXbVaVlY25g4bCCGei0qaLjR//nzMnz/f3WF4pMTERMyfPx8vvvii3dOYzWY0NDRg586dSEpK4t9swnWY4AwikQi+vr64ePHiqKZ3VomXEOKZKGnayd/fHz09PXaPP3PmTERERCAiIgIzZ850YWTeKzs7G2azGUVFRUO+6+vrQ0NDA/Ly8qBQKPDQQw/hnnvuwdatW3Hz5k08+eSTSE5OBvBdhwljxf2+Tz31lMNvTAGcW+IlhHgmqp61k6+vr0P34FatWsX/TW+4GF5hYSEUCgVEIhHi4uLQ3t4OhUKBvr4+hIaGYsmSJVizZg1CQkIsklFDQwO0Wi1OnDiBf/7zn2OuAt+5cyeOHTuG+vp6yOVyrFmzxuHGQH/4wx+cVuIlhHgmlyRNrqP1vXv3QiKRIDw8/Lbj+/r6oq+vDyaTye6TX19fH86dO4cFCxY4HN/AwIDD01y/fp1/ewVxLu4VX/7+/pBIJKivr+f3g76+PrS3t+Po0aM4d+4c2tvb+T5rQ0ND8fTTT+Phhx/G0qVLodFoHG5NffHiRaSkpEAqlaK+vh5msxlbt27FwMAAtm7d6tC8urq6kJub69A0hBAv44qHPxUKBVMoFGzu3LnDdks2MDDAqqurWVVVFcvPz2cVFRVDurMbTklJCQsJCWG5ubkOx9bb28t+/vOfM4lEYteD7AMDAyw3N5eFhIQwjUbj8PKIfXp7e5lEImHl5eUsNzeXxcXFMZFIxPz8/JhUKmXZ2dlMo9FYdFsoVF9fz0JDQx3qnKC0tJSFhoaypqYmxhhj1dXVDnV6QQiZfFyWNBn7ri/P/v5+i35Yc3Nz+X5aL1y4wPewMzAwwPLz81lISIjNHlW4E2Nqairr7u4eU4xNTU237bKPse9Oorm5uXb1AkTG5ty5c2zBggUsNzeXVVVVOdxrUHd3N5NKpXZdTCUnJ7Pk5GQ2MDDAent7WXJyMpPL5WPerwghE5tLkqZ1x+3r1q2z6IeV67B9uBJld3c3S01N5UsOly9fZnFxcUwqlY6pmzRbhuuwPS4uzqKTduI9cnNzmVQqZd3d3ezYsWMW3Rhu376dHTt2jP+tm5qamEQiYaWlpe4KlxDiRcal71lb/bDak4za2tpYREQEmz59OquqqnJZfMIS7hNPPMFEIpFLl0dcr76+nqWmprLi4mIGgL+QUygUrLi4mB+Hq+0ghBB7jEvfs2Pph7Wvrw99fX3j0kHAlStXsHDhQpw4cQIREREuXx5xvb179+KFF14AAPT39+PZZ5/lX25OCCGOGpdHTsbSD2t7ezva29uRnp7u5KiGys7ORnl5OZ5//nm0tbW5fHlkfCgUCnz22WcoLy93dyiEEC/n8Z0bmEwmly/DbDYjKacIZawAACAASURBVCkJzz//PB5//HFIJBLs37/f5csl42fXrl144YUX8NVXX7k7FEKIF/P4pOnn5+fUt2FYM5vNKCsrg0qlQl9fHwAgNzcXBw4ccKgHIOLZVq1aBYVCAb1e7+5QCCFezKOTptlsxosvvgilUjnqvkABoLu7G7du3cKtW7cs3pXY0NCA5uZm9PT0YM2aNZDL5QAGu8x7/vnnHX64nXi2l19+2d0hEEK8nMcmzY6ODrzyyiu4cOECXnnlFXzve9/jX7G1Y8cOh+ZVWVmJS5cu4dKlS6isrOTncf36dfz3f/83goKC0NbWBl9fX36a1NRUNDY28m8qId4pPj6e79g9PDwcLS0tiI+Pd3NUhBBv5bF9z/b39+PYsWOIjo5GS0sLZDIZysrKcO3aNbS0tDg8P64hEdexd0tLC1566SUAgyfWZ555Zsg0fn5+WLlyJR544AGb3bN9++23+MEPfoAPP/zQ4XjI+Jg+fTqmT5/Ofx6pS0dCCLkdj02aALB582aLFziLxWJIJBKcOnUK3d3dqKysxE9/+lO7WuZyb9Jobm4GACxbtgzvvfcerl69arPkcfToUdx3330IDQ1FbGwsoqKibM5327ZtqKmp4at2CSGETFwemzSnTp2KmTNn4uc//zkqKiowdepUREdHIzw8HAsXLsT06dPtfuVWfHw8pk6dCgAICAgAAGzcuBGtra2IiIiw+RaSrVu34je/+Q327duHnJycYeft7++PpKQkSKVSi+pdQgghE8+4dG7gCq2trUhPT0dRUZHTq9yKiopw5coVNDQ0oLS0FKGhobcdX6VSYfbs2ePyLCkhhBD38dqk6SpmsxlhYWFITU1Fe3s7SktL7ZrmoYceQlNTk8OvpiKEEOI9PLb17Hi7desWduzYgTfffBN/+ctf0NzcjPz8fLum9fX1RVxcHNRqNd+yt7W1FXv37gUAaq1JCCETBCXNf0tPT0dCQgJkMhmOHDni0AuNW1tbce+996KwsBC9vb38s6C7d++mhEkIIRMIJc1/u3HjBoKDg/Hggw/CaDQ6NC3XyMja5s2b+edCCSGEeD+PbT073jZt2oT169cDgM1nNm+Ha327Y8cOiEQi/rlAiUQCAEhOTnZipIQQQtyFGgIRQgghdqLqWUIIIcROlDQJIYQQO1HSJIQQQuxESZMQQgixEyVNQgghxE6UNAkhhBA7UdIkhBBC7ERJk9yWXC6Hj48P/6+goMDdIXmNgoICi21H71wlxPtR0iQjkslkYIxBrVYjKyvL3eF4HcYYdDodamtrodfr3R0OIWQMKGkSQgghdqK+Z8mIamtr4ePjA2Cw1MQ5cOAAEhIS8KMf/chdoXkFbtvpdDpERUXBaDSioqICDz/8MB5//HE3R0cIcQSVNMmIuOpZAPw9zQMHDuCNN97AhQsX3BmaV2CMQSaTYdu2bQCArq4uPPzww1i5cqWbIyOEOIqSJrGb8J7m888/j6CgIDdH5D1ycnL4e5pRUVF44IEHIBaL3R0WIcRBlDSJ3RISEgAAWq3WzZF4n6ioKIjFYhw+fBhff/01srKy8Kc//cndYRFCHESvBiOj8vLLL6OkpARisRiffPKJu8PxKomJiWhpaUFQUBDUajXmz5/v7pAIIXaipEkIIYTYiapnCSGEEDtR0iSEEELsREmTEEIIsRMlTUIIIcROlDQJIYQQO1HSJIQQQuxESZMQQgixEyVNQgghxE6UNAkhhBA7UdIkhBBC7ERJkxBCCLETJU1CCCHETt8fzUTcm+gJIYSQiU74XpNRJU16Mcr4+nvvLXeHQAghBFQ9SwghhNhtQiRNHx+fIf8IIe7x9p4iBE+biuXh8yyGLw+fZ3O4I05UVtg1j7wMFU5UVox6Oa7CbRtb67AuQYF1CQoAwDWTiR8veNrUIfPhtoPw+5Gm8XTctuG2gTXh+l0zmYYMu920zjQhkqbBYAAAqNVqvuqYEich4487mXXc6MdTa9byietEZQWili5Dx41+RC1dNmxCa21uGva7ayYTCre/ho4b/VC9/Cre3lM07DwOHzrohLVxjcKDpei40Y8PWz/lh52orMBMkYj/PFMkQseNfnTc6MfqtevR2tw0ZD5b8l7nx7GeZkve6x550TCc1uYmNDd+hI4b/ZAsXmIz9pRfrMKps+dx6ux55GWoAACfnm3h1/nU2fOQLF7i8lgnRNLs6uoCACxatAgAoNFoAABardZtMREyWT23KR0A0Nz4EVbGJ/DDuYQKAP4BDzg830/PtuCpNWsBACvjE3Ck/JDN8f5c8T5Wr13v8PzHi2p9ypBSUeH217Duhc0W461LUCB42lQ8MGs2wiWRQ+bzRt4rw5a6mxs/wrwFC50fvIucPfMx4lc/DQCQr4pD5eE/Wnx/zWTC7DlizBSJ+IuLayaTxTp+erYFCyIec3msEyJpnj59GgAQFRVl83uj0chX28rlcgDfVekajUYUFBTwfxNCRm+mSMRXmQlxyTN42lQ8uuhnNpPASLquX0fAjBn856uXLw8Z5+09RUOSjyd5blO6RemwtbkJeRkqqPceGDLuOxVV/HjWpeqV8Qn8fKxL7usSFIhf/bRFydXTfX71isWF1JXLBovvv+zpHrI+1sM+Of23Ue1XjpoQSbOurg5KpZL/fPXqVQDAjBkzYDQaIRaLodFooNPpUFtbC+C7FsBpaWn858DAwHGOnJCJh6smnCkSWZzsJYuXYPXa9Sjc/tqQabj7nUmPx/IlMUfvy3ElWW9KFvf5T4e+/hSSHo/FsgWPQFf3IV/1yBFeKAyHSzjrEhRIy8i0KOET55oQSbO2thaLFy/mP2dlZUEsFiMqKgppaWmQyWRITEzE6dOnIRaL+fGUSiU6OzuRmZnpjrAJmdDWvbAZn1+9AmDwnl3AjBnI21WIp9asHVJy+rD1U3Tc6Ifmg5P8PT+ulMUJmDEDXdevAxhMkLPmzLH4vub4Ub7K8vChg1CtT/G4+3pclWvwtKmQLF6CmSIRv+6nzp5HdMxy5O0qtGjoU7j9Nb7Km7uQyMtQWVxYhEsi0drcBF3dh0h6PBbB06YOSb6e7IFZs9HT9TmAwRLk7Dlii+/v859uUb1/5bIB9/lP5z+fqKzAfyb817jE6vVJU6/XAwAiIiIAAEFBQQCAkydPAgA6OzsRExMDvV6PrKwsbNu2zWJ6g8EAvV7PlzgJIc7x6dkWPLroZwAGq1Y/Of03AIPJj0umjpi3YCF/H1N4f5MjrPpcvXY9Cg+WelyJi6ty7bjRzydCzkyRCO9UVAGwrH4VNhjiLiTydhXy3+ftKgQwmDi5YcLh3mBBxGP8fUzh/U3OTJEIVy4bcM1ksri/yRmvqllglJ0beIq0tDSUlJQAAF+CFIvFFp0vbNiwAVlZWQAAnU6HqKgovmWtwWCA0WhEdHQ0dDodfHx8oFQqsW/fvnFeE0ImhhOVFVCtTwEArF67nj9xP7cpnS8BAhhSilwePs/iHiU3D+F4M0UiPLVmLYKnTcWsOXP4ZMKVqLwpSRBL4ZJIzBSJEDxtKqJjlvMXD+sSFJAsXoLnNqVDvfcAli14BABw6ux5t8Xqw6h7H49HPQIRQoj7zL1nCv+311fPEkIIIeOFkiYhhBBiJ0qahBBCiJ0oaRJCCCF2oqRJCCGE2ImSJiGEEGInSpqEEEKInShpEkIIIXaizg0IIYQQO1FJk5Bx0NPTg7vvvpt/JZ0j/9RqtbvDJ4T824RPmsJ3adJLqW3j3idaUFDAD9Pr9fx244YLtyVtT8eoVCocOHAAjDGH/g0MDGD//v3o6elx9yoMKygoyOJdtUIFBQUW+xXHel+yNa01uVw+ZD8dbr7cixuEcYw0rTsJjzfhcaXVavnh3MsphOP6+Ph49csmhOsyHG7/Em4X7vcUbhcA43NuYhOcTCZjjDGmVquZWCx2czSey2AwMABMp9NZDNNoNIwxxnQ6HQPADAYD/z0A/nsyvPr6eiaVShljjGk0GiaVSu36l56ezk+TmJjozlUYllqt5vcJsVhssT9w+5RarR4ynfV41uMolcohy+HGkclkFvshh9tHhfswNy9PP/a59eXWgbHB7cLFLfxbuO10Op1XH4Pcuup0uiG/OWOD24VbP+FvKJyOO8cLp3fl7+3VbzmxR01NDQBg1qxZbo7E86nVakRHR1u8JYazbds2qNVqixd1azQa5OTkIDExcTzD9CpmsxkqlQoajQYAIJVK4e/vb9e0fn5+AIDExEQcOHAADQ0NkEqlLot1NITvot22bRv/AngA2LVr17BVy9yr/ADgzJkzWLRo0W2XU1dXx799KDk5GRUVFUPegxsdHQ2NRoOoqCh+mFarRUlJic192pNw6xYVFQWZTAaj0Yiuri7ExsYCAAIDAxEUFAS9Xm+x7U6fPo2EBM96/Zm99Ho9lEolgMH1Tk5OHjLOyZMn+W2zYcMGaLVa/nxjNBoBgD8nZWRk8PPdsGGDy+Ke8NWznMbGRosNyVUJ3a5aYDLS6XQ2q8o6OzuHXHhERETwr1cjtu3cuRNPPvkkQkJCAAD+/v6QSqV2/QsNDeXnU1hYCJXK818qzO0jWq0Wq1evHnY84cVXY2OjRaKzpbOz02Iak+CFxNzyxGIxcnJyLKrnysrKIJPJRqwC9DTcunKvPrT1HTC4HYSfvcn169chErwTE8CI5xLuokyj0UAsFmPbtm18Ug0MDERaWhqio6NHvAgbi0mRNI1GI0pKSvgrU7lcDqVSieTkZP5KhwyKiopCTEyMw/d+PvjgA6SlpeHcuXMuisz7mEwmHDlyBNnZ2WOeV2hoKKRSKYqKipwQmWs0NjbypQB7EqE14X2qkpISh+5PXb16FbGxsTh58iR0Oh1ycnIADCbb5ORkMMagVCo99p4mR6/X8yWuqKgoKJVKfjvU1tYiICCAH9doNA5JOpPFjBkzoFarUVtba3FPc9++fTAYDIiOjnbdwl1W8etBhPXbwntzarXaq+8HOJP1fSWZTMY0Gg2/fWQy2ZD7ThqNxuKey//8z//YvC8xWcnlclZdXe20+fX29rKQkBDW3d3ttHk6i/DepkajYQAs/tm6r8mNa30PkrGh9zSF9zGF9zeFyxcO405tYrHYIi5P3z+Hi0+j0fD37jjCbe6NrO9j2kpHwnO38P4mty2E93qFlEqly7bNhE+a3EGj0+mYTqfjGwRxjRS8eadzJluNMSBo6GOrkQWsGgIZDAZ24sSJ8QnYw1VVVbG4uDinz7ekpISlpqY6fb5jIUxYIyUza8MlCUcbAlk3mhE2ABTG5skXycIEL9xmwsZBQp5+AWAPjKIhEHfutp6HkPUFhjNN2KTJ7WjcP24jclfBYrGY/87Wle5kwu2E1iUC65Z51tvU+gTkySek8TQwMMBEIpHLSoShoaGsra3NJfN2lFKptNgnbpfsbF2oCsdXq9VDSqjC/Yw7Zq1LlNzxK5xeiJvOk5OM9TrrdDp+e9kqSdm6yPVGwloJjlqttkh6ts43wv3Oev9wdWGIegQiTvHyyy/j7rvvBoAhrRonG5VKhdmzZyM9Pd0l829vb0dKSgra2tpcMn9CyPAoaRLiRO3t7VAqlaivr4evr6/LlpOSkoIlS5bYbKZPCHGdSdF6lhBH9fX14Z577nG4y7uIiAhs3LgRvr6+UCqVMJvNTo2L6x0oPz8fW7Zsceq8CSEjo6RJiA1+fn7o7e11uNu7kydP4p133gEAzJ49G1u3bnVaTO3t7SgvL4efnx8aGhqwePFip82bEGIfqp4lxMkUCgXWrFkDuVyOsLAwVFdXO+V5uqVLlyI3NxcSiQQPPfQQmpqa7O5diBDiHJQ0CXGynp4eREZG4sKFC6ipqUF5eTmqqqrGNM+ysjJ89NFHKC0tdXlDI0LI8ChpEuICO3fuxM2bN5Gfn8+XPOPi4kY1r76+PkRGRqK+vh49PT3UcpYQN6KkSYgLmM1mREZG8h21JyUloampaVQtapVKJebPn4/U1FRERkaisLAQEonE2SETQuxADYEIcQFfX1/k5+dDpVIhJCQEcrl8VP3Gtre3o7m5Gampqdi/fz9CQ0MpYRLiRlTSJMSFhI2CRtN4JywsDKWlpfD398eKFStQXV1NjX8IcSNKmoS4kMlkwooVK9DW1oajR4/i2LFjfJXtSPbv349z586hpKSEOjMgxENQ9SwhLiQSifDUU09h586dSExMhNlsRl9fn13THjlyBPn5+WhubsbFixcpYRLiAaikSYiLmc3mUT+vaTabsXTpUpSUlFi8lJoQ4h7fd3cAhEx0vr6+eO211xAdHY2goCCHpv3yyy8RGxtLCZMQD0ElTULGyYkTJ3DXXXc5NE1PTw8SExNdFBEhxFGUNAkhhBA7UfWsF/h77y13h0AIIZPW3Hum8H9T0iRklNSv5uCd4t0OTxfxsyi89+dqF0RECHE1SpqEjMJXX36BmuNVON/9Fe64w7Gu8dJ+lYQPT/wFy1c+4aLoCCGuMuGf0zQajfwLgrVaLQCgoKAAPj4+SEtLc3N0nuHtPUUInjYVb+/5rpu31uYmBE+bajH8msnEDwueNhUnKivcFbLb5f42HS9vVwMYTIK/WvX4iP/W/zIen1+9ioxXt+L3Bfn417+c+4Jqd8nLUA3ZH7h96nb7yroEBYKnTUVehsqu5XDjC/dTW4KnTeX/PlFZYRHHSNO6k61txcVvvY2Wh8/z+PVxFLfPrEtQ2PxeeP65ZjJZfMedr4ab1pkmfEkzLS0NjDEUFBQgJycHiYmJyMzMRFZWFlavXu3u8DzCc5vSIV8Vh2ULHsGCiMcQLolEuCQSp86ex6dnW7AyPgGtzU1IejwWp86ex8x/P2vInZxWxie4M/xx13jqr/jW/C++pLhh82/wL7N9CfDH99+HO+7wxWNR0dAcegfJyo2uDHVc5O0qHDIsYMYMdNzoBzB4Mpy3YKHF9ycqK5CWkYl3KqqwLkGBayYTv18Bg4lYON+39xRBsngJP758VZzF+MLprHFxnKisgH/AA6NbSRfLy1DxcS4Pn8cfU9z/n5z+Gz/u23uKUPqn45gpEmF5+Dw8t8n7XxHX2tyE5saP0HGjH2/vKcKJyooh55WUX6zCqbPnAQxur3cqBl+3d6KyAqr1KRbnJlea8EmzpqYGADBr1ix+mF6vBwBERUW5JSZPtSXvdSQ9HssfvEL7dhVgS97rFjtl4cFSFG5/bVIlzX/9y4zfvZaHwoOl/LDQhY86PJ/fvpoH+WPhWPmLBNx73/3ODNEjCJPk51evDDmZzVuw0GLYSCe75saP+CQav/pp1Bw/OiRZtDY34dFFP8PhQwdtxvHJ6b/ZTPCeYN0LmwEMrsNTa9bedlzuguGayYSopcvGIzyXO3vmY8SvfhrA4PrlZagszivXTCbMniO22E+40qZqfQo0H5wcl4QJTILqWU5jYyM2bNgAADh9+jRkMhlfbWs0Gt0cnefQfHDSZhXHlcsGBMyYYTFs3oKFuHr58pCqkomsrGQfFi9bjsC5wWOazx13+OLXmS/hd6/lOikyz8KdwK6ZTHhg1myb33NVj5LFS0ac35XLBouT4udXrwwZ588V7w+5gBuvE+lYzRSJkJehQtLjsVgQ8diI4769pwjLFjyCRxf9bJwidK3Pr16xqAW4ctlg8f2XPd1Dfssve7pRc/woomOWI+nxWARPm4rW5iaXxzopkqbRaERJSQkyMzMBAHV1dejs7ARjDGKxGBUVk/fenLVwSSQki5c4fK/ko7/WIi9DhYufnXdRZO731ZdfQFv2Dl7IetEp84tf/TQunD+PC+c/dcr8PNGnZ1uGTQIr4xPQcaMfR8oPobW5yeI+6OFDBx26d/72niK+tGYLVwr1ZHm7CnHq7HkkPR474rjPbUpHx41+qNanTKqLVmtcLUbHjX4UHizFvl0FLl/mpEiasbGxEPbhUFtbi5MnT8JoNMJgMGDRokVujM7zPLcpHc2NH+HTsy38sNlzxOi6ft1ivE/PtmDWnDmYKRIhcO6DSH/pFWjLDo13uOOGa/zjaGvZ29lZXILsF5QOT/fPb77Bsfe1+OPBt/BZexs//KsvevDHg2/xnz9rb8Op6g+cEutofHL6bwiXRN52HNXLr6Kn63M+EXTc6Mfqtev5v1fGJ2D2HDGfHLquXx9Sej1SfgjLFjzC32cXNgYCbJdCPdFMkQir1663OxFuyXsdX/Z0uzgq13tg1mz0dH0OYLAEOXuO2OL7+/ynW2yTK5cNuM9/OgDwF0PzFiwcUkJ1hQmfNIOCgnDy5Eno9Xr+n1gsRmBgINLS0iAWi+nepg3vVFRBtT6F/5yWkYk38l6xqP5QrU+B6uVXAQwe7P3/+AeWyuTjHut4sG784ywPPTIPgXODHWqJ/NUXPQid6Y8zeh0A4DfPpWD/m7sAAF90d2Nr5m/5cc+1fIJKzXtOjdnZPjn9tyENhaxJFi9BzfGjAAbvb8pXxVl8/2Hrp3ySBWDzvry3sG4UdTvNjR+NeFHiDRZEPIbKw38EYHl/kzNTJMKVywZcM5ks7m/+Z8J/8dN92dM9Lvd4J2xDIL1ej+joaACAWCyGTCZDTU0NCgoKYDAY4OPjA7FYjM7OTjdH6n7XTCYsW/AI/5lrYKH54CR/9RcuiYTmg5MWVUeFB0strt65lrYTja3GP8700o6d+K/YGCxf+YRdpdhDe4vxy2eexfY9ewEAP1sag9hHw/CLp59xSXwjyctQ4fChgzh86CD8Ax7gT+InKivwnwn/xY/H7Wenzp7HO8W7+QY7hQdL+ft0b+S9wo8v/P65TelYHj4Pb+S9YtEgLXjaVGg+OHnbxOENVbPLw+fh6uXLAMC3EAW+axkKDJbGntuUjnUJCujqPgQweIxOBOGSSMwUiRA8bSqiY5bzLWPXJSggWbwEz21Kh3rvAf48xW0j4XSz5szBh62uv9VBfc96AW/oRu/NbVsx9Yd3A8CEaAIvdKDoTdz6x0389tWtHrGMjc+uxqLFUjy9fgM/LHjaVFTWNQIA4mMWI7fgdwCA040NAIC9fzjsgqgJmRyoGz3idL/J8fxWoJ9fvYqloT91eLof/OBOnDFewYXzn+Jjvc6pz1Y2nvorfni3H5KVaZDMnYOP9Trc4Tt8aXPZipVOWzYhxHGUNMmk8cCsWaO617Xj5WxoDr2DpLXrkP2CEouX/3zMj5wAg9W+ub9Nx/sn62Ds6IB/wAMjljRnzJqNL7q78f99eo4fZjIM3mK4f/p0fNE92ChEWArlSpuEkLGjpEnICJ7brML6X8Zj5S8S8OvMF7HrtVzse1cz5vn+7rU8rHk+Dffedz/W/zIeb759CA89Mm/E6dZufAE/e2guAOAn8+Zja+Zv8ZucXNx7vz+fNAkhrjHhW88SMlb33nc/FImr8fbuQr717Icn/jKmeV44/ynaPzmDpLXroCl9B6ELH7UrYQLAvff7o/1aDyKiBhu6VdY1IvU3GQAGS5vc/UwAmL/wUcQnuaeBECETETUE8gLe0BBoovvXv8z41X8+jq2/G+z0Ife36Xj3zx+M+pnNX616HC9kvojA4GCs/2U8Dv6/ygnZnR4hE4GwIRCVNAmxwx13+OKFrBfxu9fy8NAj8xD6aAQ0h94Z1bwqD/8RD8yahceiolGszkdi8lpKmIR4CUqahNhp8bKf4z9878CHJ/6CFzJfhLbsEL768guH5vGPmzfx1u438dtXtw5W0bZ8gqSUdS6KmBDibJQ0CXEA9y7MO3zvQGLyWry927G3ZhQXfFeyzH5BiZ3FJS6KlBDiCpQ0CXFA4Nxgi3dhfqzX2d3huvA5T0cb/xBCPAM1BPIC1BDIs/zrX2bIHwvnn6888zcdfp310ojT5f42HYqkp/HArFnU+IcQLyJsCERJ0wtQ0vQ8lYf/iDOnddhZvN/haXN/m46Qhx+he5mEeAlKml6GkqZnWjr/p/jRvffiB3feafc0t/r78b//+7/4i77ZhZERQpyJ+p4lxAn2Hz6Cvt5eh6b55zffIPgnP3FRRIQQV6OkScgoPfjTh90dAiFknFHrWUIIIcROdE+TEEIIsROVNAkZo5ycHPj4+Dj8LyQkxN2hE0IcNCmSZlBQEHx8fJCWlubuUDxSQUEBfHx8UFBQwA/T6/X8yZ0bbjQaLU76Wq3WXSF7jJ6eHvzpT39Cb28vGGMO/VuxYgWKiorcvQpO4+Pjw//N7VO321ds7WO3I5fL7RpXGAf32XqYJ7K1rbRarc34uXOa0Wgc7zBdhttn5HK5ze+5bWHrPG40GhEUFOTqEAexCU6pVDKDwcB0Oh0DwAwGg7tD8kgGg4EBYDqdzmKYRqNhjDGb2w8A//1klZyczEpLS/m/pVLpiP/kcjm7fPky6+7uZqGhoay7u9vNazF2SqWSCU8nwv1CrVbbPO5ut+8olUqLz2q1mqnVasYYYzKZbNjj2DoOjqef6oTrKxaLhwzXaDT8+gvHmyjnM51Ox2QyGWNs8Lcebt/QaDRD9g3GBvcJ4XZzpQnfenbfvn3uDsFrqNVqREdHg9m4zb1t2zao1WoEBgbywzQaDXJycpCYmDieYXqM5uZmXLx4EaWlpQCAzZs3o6+vz65p/f394evrizVr1kCtVqOw0LE+bD2JXq/H4sWLUVLyXT+6ERER/N8mk8livwEGSwZJSUlISkqyub9Zq6ur44/l5ORkVFRUIDMzc8Q4vEVGxuD7UPV6PTZs2ABgcBtx69zY2MiPMxGdPn0aycnJAICEhASkpaXZfV7RarVITk5GTk6OK0PkTYrqWQA4c+YMZDIZf/AKq48mUhXHWOl0OpvVI52dnZg1a5bFsIiICBgMhkm5/cxmM1QqlcUJOjQ0FFKp1K5/vr6D7+FMT09HQ0MD2tvb3bUqY3b48OEhJzjuODMajRCJRDanY4xBo9HYVXXa2dlpkXhNJpNdcXiLwMBApKWlITo6GosWLeKHAYPVtidP25qAGwAAIABJREFUnhxy4TGRmEwmzJgxg//c2dlp97SNjY0WF2muNmmSZk5ODn8lkpaWhrfeeou/wu3q6nJnaB4lKioKMTExdt1jEvrggw+QlpaGc+fOuSgyz7J//35IJBKEhoaOeV6FhYVQqVROiGr8FRQU3LYEdObMGT4JCHEJIDExEWq1Glqt1uJCtqSkxKF75yPF4Q327dsHg8GA6Ohoi+GMMWzYsIHaZNiQlpY27rWJkyJppqWloaysDFFRUTAajSgpKUFZWRmAwR0yKirKzRF6lszMTNTV1eHMmTP8sKCgIFy9etVivDNnzkAsFiMwMBAhISF4/fXXceDAgfEOd9z19PRg9+7dyM/Pd8r8pFIp/Pz8cPToUafMbzy99dZbEIvFfGnRutTY2Ng44vHFJdXMzEy+kZRSqeT/TkxMRFBQEF+jcfXq1SGl15Hi8BaBgYFQKpVDam8yMzMndI2OSCTC9evXAQwWYuxt1MNdXInFYhgMhnFpDDThk2ZaWhoWL16MqKgoFBQU8KXKgIAAGI3GYVtqTXY1NTVISkriP+fk5CArKwt6vZ4flpSUhG3btgEYPNhv3ryJJ554YtxjHW8qlQr5+fl8FaszlJSUQKVSwWw2Ozxta2sr9u7di+PHj+PWrVsWw1yts7OTT24A7Lo/ac2eatWYmBhUVFQAGLy/mZCQ4PQ4PIXRaBxSFavX6xETE+OmiFxv0aJFfEFGeH9zJNxvbjAYIBaLHarWHbVxaW7kJgAs/nHEYjEDMG6trTwd13IWgEULPZ1OZ9GKjWtBy/2zbuE2GVrSVldXM7lc7pJ5Z2dns/z8fIem2b59O5s7dy4rLi5mCoWCRUVFMcYYKy4uHvcWo9bL02g0Q1pj498tsNVq9ZD9SDjM1n7GHbfCfRRWLb5txcHNx5OPd27duO3DmOXxxrUstTX+RMG1fBauq0wm439vjUZj8zzF2OC+NV6/L/UIRJzi5Zdfxt133w0AQ1o1ThRmsxmRkZHQaDQu6ZjAbDbjoYceQlNTE/z9/Uccv7u7GwEBAejq6sL06dNx69YtlJeXIz4+HpWVlXjhhRe8usRFiCeipEmInXbu3ImbN2867V6mLVqtFseOHYNGoxlx3NbWVixcuNBmYty7dy8lTUJcYMI/p0mINZPJhDlz5jg83Z133omvv/4aFy9exNGjR5Gdne20mNrb22EymZCYmAiVSoXQ0FDcc889w47v5+eHzZs3O235hBD7UNIkk45IJBpVCUylUmH//v1IT0/HkSNHIJfLnfLICQCkpKSgtLQUPT09+P73v4+CggL8x3/8x22nefDBBwEMVtNOnz4dwGAJMz4+3ikxEUKGoqRJiJ2ysrKwYsUKJCYmoqSkBEqlEk1NTWOeb1FREaRSKUJDQ6FQKPD73/8esbGxdk2bkZGBJUuWYPPmzTh16hS++uorbNy4ccwxEUJso3uahDigqKgIV65cQWFhIZKSkvDkk0+OqReanp4eLF26FE1NTWhubsbu3btRXV3t0DxaW1vR3NyMmTNnIiYmBlOmTOGHUQIlxLkoaRLioLCwMJSWlsLf3x+RkZG4cOHCqJ/ZTElJwZIlS5CYmOjSlrmEEOeY8J0bEOJsXLd3/v7+WLNmDXbu3Dmq+TQ0NMBkMiE5ORlFRUWQy+WUMAnxcFTSJGQUFAoF1qxZA7lc7tCzlRyz2YylS5eipKTEKSVWQsj4oJImIaPAdXsHDJY8lUqlQ9MLO3xXKpUoLCykhEmIF6CkScgoCKtm4+Li0NPTg+bmZrumFXb4XlNTA7PZjLi4OBdHTAhxBqqeJWSUhN3e9fT0oLy83K6XSefl5SE0NBRyuZwa/xDiZShpEjIGR48eRXl5Oaqqqhyedjy65SOEOBclTULGKCwsDN/73vfwwx/+0O5pzGYzPv/8c3R0dNC9TEK8CCVNQsboypUruHTp0ojd3gl9++238Pf3x7x581wYGSHE2ShpEkIIIXaivme9wN97b7k7BEIImbTm3jOF/5seOSGEEELsREmTkHHyz2++gfrVHARPm4rgaVOhfjUH//zmG5vf7X9zFz9d8LSp+Ky9DQDwWXsbgqdNdUv8hJBJkDSNRiN8fHzg4+MDrVY77HdGo9FNEbrf23uKEDxtKt7eU8QPa21u4k/g3PBrJhM/LHjaVJyorHBXyF7p9+p8tLecQfu1HrRf68FVkxG/V+fz3/2jrxft13rwtwt/x5vbtuJU9Qdujtg+wn1iefhgw6YTlRX8sNbm4V+fds1k4qcZyboExZD91Hpe3DLzMlT8cG7/HikWd7N1XAm3IzdceGxar6s3436ndQkKm99z28J6fbn9wt79aKwmfNJMS0sDYwxqtRo5OTkW3wUGBkKtVkMmkyEwMNBNEbrfc5vScersebyR9wp/UgmXROLU2fMoPFiK5zalo7W5CcsWPIJTZ8+j40Y/Om70Q7U+hRKnA94p3o2Xd6hx51134c677oLyN1vwTvFu/ruktetx51134d77/dFxox/LVjzu5ohHdqKygt8fOm70I2rpMgDAJ6f/ho4b/Sg8WIqsjc8PO/1wJ3zr4W/vKYJk8RJ03OhHc+NHuGYyDZnm07MtfBx5u77rZCJgxgx+eLgkcjSr6XJ5GSo+xsLtr/HDC7e/hlNnzw8ei/8e3tP1OT+u5oOTeHTRz9wVttO0NjehufEjdNzoh2TxEpvnlZXxCSg8WGoxTLhfPLVm7bAXVM404ZNmTU0NAGDWrFk2v6+rq0NMTMx4huSxtuS9jqTHbb/8eN+uAmzJex0zRSJ+WOHBUosDnLhOfMxiBE+biviYxe4OxcK8BQv5v1ubm/gTOJe0VsYnDDvticoKxK9+2q7lNDd+BPmqwa4G41c/jZrjR4eMo1qfguBpUy0S6onKCqjWpwxbevEU617YDGBwGz61Zq3Fd1/2dAMAZs8RA7Dc5mfPfGzx2VudPfMxvy/IV8Wh8vAf7Zru86tXsCDiMX66I+WHXBYjZ8InTU5jYyM2bNjAfw4KCoKPjw9qa2uxaNEiN0bmWTQfnLR5grly2YCAGTMshs1bsBBXL1+2edVPnKuyrhEdN/pRWdfIDzMZOrHx2dU49r72NlO6lvAi6s8V79tMktzJ3tonp/9m9wn/ymWDxbI+v3rF4vtrJhNf8lq24BF+n5y3YCE6bvRjpkjk0YlzpkiEvAwVkh6P5ZMAAKj3HkDS47FI+cUqvFNRxY/L+fzqFYvP3urzq1fgH/AA//nKZYPd0/654n1XhDSsSZE0jUYjSkpKkJmZCQCQy+WIjY2FTqcDAERFRbkzPI8SLomEZPESh6s5PvprLfIyVLj42XkXReb91r2wGdtfysI/v/kG//zmG2x/KYsvYfzymWehOXQQ//zmG3z1RQ9iHw0d8Z7m27sLsWixFFtSnxuP8EflRGUF0jIyhwzPy1BZVKEClvceDx866NC9cy5xhEsiUXiwlC+JcsO5ZXnyBV7erkKcOnveorbnPv/p2JL3Oq5evjxkO1wzmfDArNnjHaZHydtVyO8ryxY8MuwFmjNNiqQZGxsLrg8H/f/P3p3HNXXn++N/5f7uLVwVoX6r4oaRIIN3qgPFIlpARGVxh6EdotMKxS1IKyjK5ghYBUK1YouiVoUuGtoyYK0L4gKFVFBBGZ3fxUESUlQW7SgIKszc+z3fP5icJqwJZCHh/Xw8eEDOyfnwPifJeeeznM8Ri3HhwgVERETg6tWr8PLy0nN0g8/aD8NQWvQjbt8sY5dNnsJD3YMHSs+7fbMMVlOmYBKXC+upv0FYzJ+Qlan95hFD9UFkNOxnOsF+kmXHz0wnfBAZDQCITUrBSItXYT/JEm9Nmwo//h/77NMMi/0TRowciYVLluoi/F4pNs0qunH1p277ERVPdLU1NVjgOANrPwxj++pWvr+G/Xuxnz8mT+GxCa/uwYNek0VPtVdnt7n93DvdmcTlYuX7a9h9jY8IZ49L+JogpefmnT7FNlkbuglWk9FQ9xBAR3O0OslP/j5x9VigcnP/QBh90rSxsUF+fj7EYjHEYjGuXr0KHq/jBYmMjKT+zB4cy85V+pCGRGxTGigEdPQhhcfuANDxYW959gzzvLx1HquhGDZ8OCJ37mI/5JE7d2HY8OHdrtuwOYLdrupJC163dwAAvG7vgKonLQCA4wfSwLP9DZ788gtkkmrd75CC7vrWbEeZsTW8zi0X8v28fPMOrKZMwaXy272W7+w2l609KvZvdqenZGIoTZn3ZTI2zuIrl9gEajVlilJN2VD2RxVvOM1i+zEV+zdVFR8Rjklcbq996JpitElTLBaDw+FAIpGAx+Nh165dcHFxgZWVFSQSCTw9O5pAIiMjkZKSoudo9eu+TIb5b0zHx/F/Ujq5ic7ls387Os+G6Fw++Is82WazfUczlN6kt2+WYe5Cqrn35Jq4GJfOntFYeeYWFti8NgjlpSUYPmJE3xtokeIJXH7pB/DrZRTey1awy3tqIu2teXbth2H45ovjsB1lBme3uez/kl9GonhpBvBrs6z8cgTbUWZsU/hgtMBxxq+XkCg0W2+N/wjz35gO21Fm+MPq95WOsTE1zTo6z8YkLhe2o8xQWvQje14J9vdlz0nyQV0njx9ll8nfMxOsJndp7tcWmnvWABjCNHqf7EqA2UhzAB3Nu0RZe3sbvGc54tv8Kxg9ZqzGyv1rxS1YT7Vla6yEEM1TnEaPkqYBMISkSXr3mTARAPBBZIyeIyGEqEsxadKE7YRo2eNHjcgRnUDetXJ9h0IIGSCj7dMkZLCI2xKG2N1CmJjQzaYJMXSUNAnRomviYrQ0N2PB4iX6DoUQogHUPEuIFiXGRiE5LV3fYRBCNISSJiEqeG/5YpQWF/X9xE7emOWMadN1c/cFQoj2UdIkpA+Vd26juamJnVRAVY8fNWLN2354/KhRo5eZEEL0hy45MQB0yYl+LZ/7FpLT0jHBajK+OHRApW3MRpojULARmekHUPfgPmJ2J2s5SkKItiheckIDgQjphSjjGOxnvolp02dgpLk5O4FDX0aaWwAA+O8Ho+LGdVTe6X2aOEKIYaCapgGgmqZ+PGtuxjueHvjqh3MDal69dPYMcrJO4OBXIg1GRwjRFappEqKCtJQkBAS+P+D+yAWLl+Afbe0ounxRQ5ERQvSFkiYh3ai8cxvXxMUIFGzUSHlbdsRj7854jZRFCNEfSpqEdCMxNkqjg3emTZ+BWS6uyExXbSBRZ3+tuIW/VtzqcZ3ircFePH+u9NzHjQ16v3UYIcaCLjkhpJNLZ8/AzNwcs1xcNVru2k3heHfpIvit/CNGmqs2oEgmqca6AH/8n9FjAAB/f/wIR7KyweXZKK37++NH+D+jx+DYd7mQ3quCn4cbe4lM/g+ncbWoEAe+PKnR/SFkKKKaJiEK2tvbsDs2Egl7U/t+sppGjxmLgMD3kZaSpPI2HyfswHyfJRCdy4foXD78+H/Exwk72HWr14dAdC4f+TcqYG0zFXdu3dR43ISQX1HSJERBmjAJAYHBWpuMgP9+MK6JiyG9V6XS8y+e+QGL/X7PPnbxmI+LZ35g1/1u5pvsut2fHlCqHcubdB/U1mooekIINc8So1R55zYSY6PU2qa9rQ337lai9F4NromLMdLcXKNT4BVdvohZLq74YFs0gv194bdyVa/P9+P/cUD/L/2TjwEA9yr/G1On/Re7nG5cTUj/UdIkRmna9BkI3Rat9nbxEeGQVlVhotVkBL/ti++LftLILb0eP2pE3JYw5F0rx+NHjXj1tddU3vZ5a2u3f3d+LJNUY/iIX68nk/dhnjh6BFeLCtm/vzh8EACQf6NC/R0hZIijpEmMVn8G8kTvTsLenfE4+l0O3BYshOj4MY1cdiK/p2Z7Wzu+OHRQ5QkT4lL24t1li/DV6XMAgHeXLUJcyl4AwObtcUhN/AhhMX9Ca0sLBKv+gJwrfU8q/9G+z/DuskUD2yFChihKmoQocJu/EFmZx3Hp7Bls2REP71mOWPx7/wH1cSreUzMxNkqtCRNWrVkHywkT8eWRjtuLpZ/4BvN9OhLehs0RmDrtv/DlkXRYWLyKr06fw+v2DpBJqrFwyVK2DMsJEzHd/g0AwFvzPCCpqsLCJUvx4vlzaqIlRE00jZ4BoGn0dEt6rwrha4Lwbf5l5Jw8gbt/vdPv0bTt7W14d+kidvu4LWH46odzGmny7Q/+Ik8s8fPHF4cP4pPPM/C6vYNe4iDEkNA0eoT0wnqqLWa5uEJ0/Bj4QcGoKLvR7wnXRcePwf5NJ0ybPgN7d8YjNDJabwkTAJI+O4irRYVYvT6EEiYh/UA1TQNANU3dk98L8+h3Oai8cxtfHkrH0e9y1C7j3aWL8G3+FVwXF9Ok7YQYKKppEtKH0WPGwjdgJT7fvw9u8xfiFVMTXDp7Rq0y0oRJWL0hBCamJtizMw6xu4VaipYQoiuUNAnpgeK9MCN2JOCzlCS0t7eptG3lnduoKLsBflAwjqR+gsV+v8cEKystR0wI0TZKmoT0wMTEFOs2bcZnKUlsP2fOyRMqbRu3JQwJe1Px+FEjckQnsC5ss5ajJYToAvVpGgDq09SvNW/74b0NAsxycUV7W7tKk60/rK3FBCsrhLzLh1/AKixYvEQHkRJCtEGxT5Ou0ySkD1t2xCN8TZDal51c+OEUe30mIcQ4UNIkpA/Tps/AtOkz8Gnybvzbv6neo3HvbiUCAt/XYmSEEF2j5lkDQM2zhulZczPe8fRQeco8QsjgRJecEKIDI83NERD4Pj7fv0/foRBCNISSJiFaFCjYiGvi4n7PKEQIGVwoaRKiZVt2xGPvznh9h0EI0QBKmoRoWX9nFCKEDD6UNAnRAXVnFCKEDE40etYA0OhZ45C0PQYcDgf3f66BhcWr4L+/hr3TyIvnz5ErOoGrRYWw4lrjD6sDweXZ4PL5c7hX+d/YsDkCAHDokz2YOu2/2HtqEkK0j0bPEqIHD2pl+Ev5DQg2b4WHz2L4ebjhrxW3AABbBWvx37f/AsHmrTC3sIDnmw548fw5Gh4+wJ2Km2wZdypuouHhA33tAiFDHk1uQIgOyCTVuHjmB1Tcb8Cw4cPxur0DNm+Pg+j4UazdFN5lnYvHfH2HTAjpBiVNQnSgtaUFADBs+HB2mdnIkWhqetrtOsUbRF888wM2vreS/XuOm7suQiaEdIOSJiGDnKPzbAg2bwUAPPnlF3a5TFKNnwquwHPpMowea6mv8AgZUqhPkxAdsJ5qCwC4fP4cgI6BP2dysjHHzZ1dJ+/ffPH8OTa+txKPGxsAAKNeew2v2zvgdXsHjHrtNbbMdQH++O/bf0Hq7o90uSuEDGmUNAnRgWHDhyP9xDcQrPoDNr63Eivc38Ko116DL38Vu87Pww0b31sJ+0mWsOJa91l73Lg1Cv8143eYxJ2io70ghNAlJwaALjkxHi+eP4f0XhVGmJmBy7Ppdt3YcePYhPm4sQHPW1vZ58ok1Rg+YgSGjzDDxbM/YITZSBz9LBWic/k63xdChgrFS04oaRoASpqkO7ajzBAcugnH0vaj6kmLvsMhxGjRdZqEGIH8G7cw0coKP1Xe03cohAwZVNM0AFTTJIQQ/aGaJiGEENIPlDQJIYQQFVHSJIQQQlRESZMQQghRESVNQgghREWUNAkhhBAVUdIkhBBCVETXaRJCCCEqopomIYQQoiJKmoToUEhICGbMmIGGhgZ9h0II6YchkTRtbGzA4XAQEhKi71AGpZSUFHA4HKSkpLDLxGIxOByO0nKpVMou43A4yMrK0lfIBqehoQHz5s2DlZUVUlJS4OPjg8LCQn2HpTHy94u3t7fS8qysLHA4HNjYKN/RRf6ZVOU9pPi+k0ql3T5H/h7mcDgQi8VK/9sQPveKnzfFYyLfB8Xlis81lP1Thfw17PweklM8Foq6O1dpFWPkBAIBI5FImOLiYgYAI5FI9B3SoCSRSBgATHFxsdIykUjEMAzT7fEDwK4nPSsoKGDs7e2ZgoICdll9fT3j7u7OREVF6TEyzSouLma8vLzYxxKJhOHxeAzDMIxIJGKEQiHDMB2fSfn7Rr5esYzO7ykej8dIJBJGIpEola+op/ehSCRiBAJB/3ZIh+Qxyj9ncor7rngs5bo7XoZI8b0jFAq73Sf5MVJ8L8kf65LR1zQPHjwIa2trjB8/Xt+hDHpCoRCurq7drtu1axeEQiGsra3ZZSKRCNu3b9dVeAYpOTkZCQkJOH/+PNzd3SGTyeDj4wMAKCgogLm5OWbPng2ZTKbnSDXv+vXrWLduHQAgICAAR44cAQDk5+cjICAAALBu3bpea5tSqRQ2NjawtrZm33uda5tZWVng8/k91lAMwcGDBwEALi4u8PLyUtrHuro6AGBr605OTuy6q1evKj02VFevXkVgYCAAwN/fH5mZmUrrpVIpe4yKiorg7+/PLufz+V1qn9pk9ElT7vr16/Dy8mI/eIpVfXlzDgGKi4u7PflUV1fDyspKaZmTkxMkEkmPTWZDwenTp+Hn5wc/Pz+cPn2aXd7U1AQfHx80NzejoKAAlpYdN5XmcrnYtGkTfHx8kJeXh6ioKOzbtw++vr44deqUvnZDK2pra5XeMxKJpMfn9aSurk7pi5p8mSInJycwDANra2uDTpyK5PucmZkJV1dXeHp6Ii8vT2kdAMhksi7HxxDJZDJMnDiRfVxdXa20Xr6PHA4H+fn5SvvMMAxEIpHOEueQSZrbt29na0UpKSkoKipCcXExAFAtVIGLiws8PDzU7hs4d+4cQkJC8Je//EVLkQ0+Bw4cwPLly/Hhhx/iww8/REREBA4cOIC2tjbMmzcPmzZtQlJSUpftvL29cf78eezfvx/R0dGwt7dHQUEBvvjiCwgEArS1telhb/RL3sfp6urK1hxUPQnKT6Dymoghf4kTi8VsjQvoODcJhUJIJJIuNXKpVAoul6vrEPWKYRisW7eO7ceVv/YBAQEQCoU6GWcxJJJmSEgIMjMz4eLiAgCIjIxEeno6XF1d2W+o5Ffbtm3DlStXcP36dXaZjY1NlxrB9evXwePxYG1tDTs7O3z00Uc4fPiwrsPVm9DQUBQUFMDd3R3u7u44cuQIQkNDYWpqipKSkl5rPZaWljh//jzMzc3h4+ODhoYG5Obm4ne/+x1mz56Nu3fv6nBPtMPKyop9z0ilUvB4vC7PkclksLKyQnV1NRiGQXFxMUQiERiGAcMwGD9+vFISrK6u7vVLroeHh+Z3RIdOnjzJNl0DHeeubdu2gWEY8Pl8pedmZ2ezzZSGjsvl4sGDBwA6WhI6DxxTtG3btm6/GM2ZM0dr8Sky+qQZEhICNzc3uLi4ICUlhW2KlUgkkEgkRtOco2l5eXlKH9Lt27cjMjJSqSmbz+dj165dADq+8TU3N2PJkiU6j1WfzMzMuv3b1NRUpe2joqKQlJSEoKAgZGZmYsOGDRCJRODz+Th+/LjG49UlJycnth9TsX/T09OTrREo9m92x9raGtXV1ZBKpUr9mz0x5OZKGxsbREREAADb0nPhwgU2QfB4PKVkYcj72tmcOXPYfkzF/s3uiMXibr8cdf7CoTU6HXakYwCUfjov7zxyb6iSj5wFoDQqrfPIPPnIPvlP51FrxjCKTx0AmLKyMvZxWVkZ09+P1NOnT5nAwEAmMDCQefnyJfPy5UvG3Nycefr0qabC1SrF94biaFWhUNjtZ63ze4jH43X5vMqPpWLZ8tHbIpGILdPLy6vLevlzuntfD0ad91s+il1+/Drvg0QiGfT7pC6BQMAAUBoh7eXlxQiFQqX3gOJ6xeOjq/MPTaNHNCI2Nhbm5uYAOppPhgI/Pz/weDx8/PHHAICtW7dCIpEgJyen32VmZmbC2dkZbW1tCAoKwq1btzQVLiFEAyhpEtJPVVVVWLJkCcaOHQsAaGxsxJkzZ2BrazvgsuPj45V+E0IGB0qahAxQeXk5AMDR0VFjZTo4OCAjIwP29vYaK5MQMnCUNAnRoLa2NpUHAfVEJpNh3rx5qKmp0VBUhBBNMfrRs4ToSlNTExwcHAZcTl5eHo3qJmSQoqRJiIZYWFjAwsICpaWlAyrn+++/x/LlyzUUFSFEkyhpEqJBy5cvx/fff9/v7ZuamlBRUQF3d3cNRkUI0RRKmoRoUEBAwICm8srLy4O7u/uA+0UJIdpBSZMQDeJyubC0tOx3E+2FCxfg5eWl4agIIZpCo2cJ0bDk5GQ0NzcjKSkJfD4fDQ0NSusrKirQ1NSktCw3NxcrVqzAq6++ipqaGlhYWOgyZEKIiihpEqJhipeM3L17t0vStLe37zYpFhYWIiEhAQUFBboKlRCipn/XdwCEGBsul4tXXnkFQUFBmDx5cpf1hYWF3W534cIFLFu2TNvhEUIGgPo0CdGwpqYmPH/+XO3BPHPmzIFAINBSVIQQTaDmWUI0LCgoCHPnzu319kaEEMNENU1CNOjUqVNoamqihEmIkaKaJiEa0tDQAB8fH5w/fx6Wlpb6DocQogVU0yREQ4KCgpCUlEQJkxAjRkmTEA04dOgQuFwuTbROiJGj5llCBuju3bvg8/koKSmh6e8IMXJU0yRkgIKCgpCenk4Jk5AhgJImIQMQHx8PLy8vODs76zsUQogO0IxAhPRTaWkpfvzxR5w/f17foRBCdIT6NAnph7a2NsyePRsikQh2dnb6DocQoiOUNAnpp4qKCtjb2+s7DEKIDlHSJIQQQlREfZoG4N7TVn2HQAghQ9bUV0ewf9PoWUIIIURFlDRJv7x4/hzCHdthO8oMtqPMINyxHS+eP9d3WIPW40eNeGPyePZ4qfNzZP8+fYdPCPmXIZU0bWxsIBaL9R2GUfhMmISKsuuouN+AivsNqJVJ8ZkwSd9hDVqJMVH4aN+nqHrSotbPnfrHEGUcxeNHjfreBSXB/r6wHWWGBY4zlJbHR4TDdpQZ4iPCu91ugeMMpS8Eis7mZPegbDaOAAAgAElEQVS6rSL59uWlJT0+p3N5n3+aym53Xybr83/omjy2sznZ7DL5ce4ct/w4Dsb96C/56xPs79vt+vsyWY+vX3lpSa/batKQSZryOUFdXFz0HIlxOJa2H7GJQgwbPhzDhg+HYPNWHEvbr++wBqVr4mL88qgRi/38cTYnG+8uW6TST2JsFExMTBGxIwGJMVH63g3W2ZxshERsQ9WTFkyewsPnn6ayywGg6kkLAHRJaOWlJcj482n2C8HK99ew6z7/NBXha4JQ9aQF8Xt+rVnfl8nY8uWC/X0hOpePqictiNy4vtsYO5cnP8lWPWnB1viPVErMuhQfEc4el327dwLo2Hf5cZYfr0lcLgDgUvltWE2Zos+QNaq8tASlRT+i6kkLnN3mKn1xkAv6/TJcvnkHl2/eUXr9zuZkg7/IE5dv3sGx7FytxzokBgKFhIQAANatW6fnSMhQ097ehsTYKOw7mgEAcHJxxWtjxqq07UhzcwDAYj9/ZGUexzVxMWa5uGotVlUt9vNn/w6J2IYfsr8FANy4+hOW+r8DAFjq/w5+yP4Wjs6z2eeOsRzHnvTvy2SYYDUZQMcJ8+P4P+HyzTsq/f+fayRsuS7z5qO8tETp/3RX3iQuF2s/DAMArP0wDN98cVzt/dam4NBNADpi/8Pq99nlivtlzG5evwa/lasAAN7LViA+IlzpfXZfJsPkKTz2/SNfBgDha4IgOpevtE6bjD5phoSEwM3NDVKpFHPmzGGXczgceHl54cKFC6Crboi2HEn9BAsWLYb1VFsAwOgxYzFaxaSpKGZ3MqJCBfj+x580HeKAyZPffZkMYyzHscs7N6EpntTyTp+C97IVAIAfsr+Fq8cCzH9jOgDg8s07PZ4A5SdPRQ11D5Ueq1Je5zL0bRKXi/iIcJw8fhSic/nsMrmzOdnsFxJj9LD2Z7zhNIt9/HONRGn9o4b6Lq/ho4Z63Lx+Da4eC8Bf5AkAEJ3L1/oXDaNunk1JSYGbmxucnJxQXV3NNs16e3tDIBAgMDAQAoFAz1EapuDQTdgdE4kXz5/jxfPn2B0TyX5bJh0e1tbibM6fsS5s84DLmjZ9Bma5uCIz/YAGItOcH7K/ZWtw6nhY+7NSrdPZbS7bdHosbT/blzf/jen4OP5PbF9W/cMHfZbdXXmKPv80VakJeLCI37MPl2/eYROAohtXfxoytU51yN9HVU9asO9oBg7uSdH6/zTapCmVShEZGQk+nw8ejweJRAKxWAyxWIwLFy4gIiICtbW1cHNz03eoBumDyGjYz3SC/STLjp+ZTvggMlrfYQ0qcVvCEJOYDBMTzdz9JHRbNLIyjw+aQUGdaz+TuFw8aqgH0FH76622KK+dyslrGW84zcJ9mQzHsnNR9aQFl2/ewdb4j9h+Pae3XJRqIfdlMliOn9Dlf3QuT/H58lgHo0lcLla+v8aoBvioYoLVZLbF4FFDfZeWgDGW45SOyc81ErZV4805bwEAZrwxs0sNVRuMNmlaW1uDYRgwDAMvLy8UFxfDxcUFV69eBY/X8YJERkbCyclJz5EapmHDhyNy5y72ZBa5cxeGDR+u77AGjUtnz+AVUxO4zV+osTJHmptj9YYQpA2CUcpnc7LZ2s/ZnGzcl8nw5py3uu3f7Oz2zTKlpji/lavY7RrqHsLZbW6v/3vyFB47yEixf7Ov8u7LZAj6/TKs/TAM92WybgebDAb3ZTKlpF5eWsImBmP1htMs5Jw8AUC5f1NuEpeLn2skuC+TKfVvLvV/h93uUUM9XObN13qsRps05aRSqVLTrJWVFSQSCTw9O5pAeDweXYYyAO3tbUq/Scex2B0biYS9qX0/WU38oGBUlN1A5Z3bGi9bVfKRqSePH4XtKDPknDyBSVwuFvv5s5cFAL8OYlngOEMpQXVualTcLufkCaz9MKzH5tn7Mhni9+wDf5Fnx/XBBw4D+PWSg57KKy8twfw3pqO2poYtV3Ggib4pXorTuen4h+xvu8S6wHEGamtq2H5bQ+foPBuTuFzYjjJDadGP7P4G+/uyo6eFBw5j/hvTMf+N6ewxUtwucuN6nTS709yzBmCwTqP3+FEj1rzth+9//Alr3vbDlh3xmDZ9Rt8bGrnE2CiMnzgJgYKNWim/8s7tQTsoiBBjRNPoEY34R1s7njU3AeioXT1rbtZzRPpXeec2Km5cB//9YK39j2nTZ2Da9OlssxQhRHeM/pITQvrjWXMzPOx/q/YXgX//j/9A0mcHYWJiqvGBQAAgyjiGBYuXYMuOBCx5a1aXvh9CiHZRTZOQbow0N0dZzQO1p73L+PP3yP76SwDA+ElWGh20U3nnNnJFJzDS3BzXxcVGPziEkMGIkiYhGjTLxRVm5ua4dPYMAgUhuHj2DB7W1mqk7MTYKGzZkQAA2LMzDnEff6KRcgkhqqOkSYiGJexNxe7YSABAxI4E9u+ByDl5AhOsrDDLxRV7d8Zj9fqQfs0sRAgZGEqahGjY6DFjERAYjDRhEhYsXgKg47rN/nrW3Iwj+z/Blh0JqLxzG9fExVobmUsI6R0lTUK0IFAQgqLLlyC9V4WIHQn4LCWp39ey7t0Zh9UbOmqWcVvCtHL9JyFENZQ0h4Ciyxfh9rod4iPCNX5ZyPPWVng4vI72NprcQJGJiSm27IhHYkwUrKfawm3+AmSmH1S7nMo7t1FRdgP8oGCIMo5h2vQZsJ/5phYiJoSogpKmEZPeq8Kat/3w5aF0ZOaeBpdng+Vz52hk0u9r4mJsWPUHTLWbhv3Hv0CNpBptL19oIGrj4TZ/IV4xNcGls2cQGhmNrMxjas8bGxUqQHJaOh4/akRW5nGE0vy+hOgVJU0j9Ky5GXt3xiF8TRDe2yDA0e9yYGZujkDBRnz/41XUPbgP71mOKLp8Ue2yHz9qRPiaIKSlJCHl4GGcOJOH6Q6O+PbCZRz9bL/GRooai9jdQuzZGQcAat9MWpRxDPYz38S06TM6mmhp8A8hekdJ08iIMo7hHU8PjBhpjm/zL7MThl86ewbL576Fyju3EbM7GQe/FuHLQ+lY87YfpPeq+iy3vb0NnwkT8Y6nBxYsWoyvTp9TmjJvis1UJKcdQlToBkqcCiZYWWGx3+9xJPUTLPbzV2vmpHO5f8aWHQmoKLsBaVUVTWRAyCBAc88aAFXmnr0mLkZibBTsZ76J0MjobmsklXduIzE2Cmbm5ojdLcQEKysUXb6IxJgoLFy8BGs3bcZIc/Mu253NycaenXHw46/CurDNvc5w87C2FnFbwnDg65ManQnHkLW3t2G521s49l0uJlhZqb3tu0sXIWFvKs3rS4ieKM49S9PoGbjHjxoRtyUMLc3NSE5L7/XEOm36DHx1+hwunT2D4Ld92USZd60ch1M/wTueHli9IQT8oI55UyvKbiAxJhITrCbj2/wrPTYNXj5/DmPHjcPr9g6YYGWFeV7eeNbUMSdt/g+n4ctfhWHDh+OvFbfQWF+P+T6LNH8gBjETE1NsitkO/iJPTLa2Vmvbvz9+DBeP+ZQwCRkkqKZpAHqqae7YvAnFVy4hdreQvR5QVe3tbchMP4hc0Qk2UT5+1Ig0YRLKr5VirOU4tDxrRkyisM/RmhvfW4k5bu5YtWYdAHTckulKEQDAz8MNcSl7sWrNOpw4egRXiwpx4MuTasVqLArzL+A/hw1Ta5tfHjUOqltYETIUUU3TCNRU30Pe6VMo/v//1q9mUBMTU6wP2wy/lauQJkzC8rlvIWZ3MhL2pmLvR/EouJCHM+JSjcSasG0LPJcu00hZhszd00vfIRBCBoiSpoF65RUTDB8xAiYmpghfE4QPIqNhPdVW7XJGjxmLhL2pqLxzG3t3xsPa1hZTeFPx2L5BrXKuFhX2uG7hkqU4fiANE9XszyOEkMGGkqYR+OVRIx43NvYracpNmz4DR7/L0WBUvxJs3go/DzcEh25il53NyUZW5vE+tw3dFo1ZLq5aiYsQQtRFSZNohGKfZsK2LUrrXrd3wNt/fA/H0vZj4ZKlAAAnF1e8psI1hzQAhhAymFDSJDoRFvsnfPev+0wCHc3CdKE+IcTQUNIkA+bH/yPGjhvHPo5L2cs+jkvZCwAYPdYS6Se+0Ut8hBCiKZQ0yYB1vu5S3kzb+e+hdn0mIcT40DR6hBBCiIooaRJCCCEqoqRJCCGEqIiSJiGEEKIiSpqEJco4pvJtqwghZCiipGnA/u///l8AQHLaoQHNmlN0+SK8Zzmi7n4te2uwf/7jnxqJkRBCjAklTQP12tgx4HA4iNsShldMTfpVhvReFda87YcvD6Xj4NcibNmRgKLLF3FgTzJGj7XUcMSEEGL46DpNA2ViYorC2/8NUcYxvLt0EXz5q7A+bLNK2z5rbkZaShKKLl1ETGIy3OYvZBMoAHz+zZ8HNI8tIYQYK6ppGjh+UDC+zb+C1mfN8J7liKLLF3t9fmb6ASyfOwfjJ05C3rVy2M90QmJsFEL+yMd7GwQ4+l0OJUxCCOkBJU0jMNLcHFt2JODg1yJ8eSgda972g/ReldJzromLO/otH9zH9z9eRaBgY5cE6jZ/oZ72gBBCDAOHYRhG30GQ3t172qrW84suX0RiTBTcFixE6LZojDQ3x6WzZ2Btawvrqba4Ji5G3JYwpfWEEEK6N/XVEezflDQNgLpJUy4z/QC+OHwQ6zZtBj8oGA9ra7E7NhItzc1I2JtKzbCEEKICSpoGpr9JE+gY9LN3ZxyKLl3EKyYm2Bq3EwsWL9FgdIQQYtwoaRqYgSRNufLSErzu4AATE1MNREQIIUMHJU0Do4mkSQghpH8oaRJCCCH9QJecENJPW7duBYfDUfvH3d1d36ETQvqJkibpt9bWVqXEsXXrVrS2Do2m5IaGBmRnZ+Ply5dgGEatn1dffRWnTp3S9y4QQvphSCVNGxsbiMVipKSkgMPhICQkRN8hGbSEhASUlpaipaUFLS0tkEgkSEhI0HdYOiEQCLBv3z4AgK+vL+bNm9fnj4+PD2QyGZKSkpCQkIC2tjY974X2hISEgMPhICsrS2m5/LPH4XAgFot7LUMqlbLPlUql3T6nu/KysrIM9vOdlZWlFHdPx0C+j90dY0Mlfy29vb27Xd/b+0EqlcLGxkYXYQ6duWflL4SLiwtcXFwQGRmJlStX6jkqw7Znzx6UlZVhxIiOTvLY2FjMnDkTH3/8sZ4j0668vDy0tbVhxYoVAIDIyEiVE6ClpSVMTU3h7u6OQ4cOISwsTJuh6s3Bgwe7XW5lZYXuhlGIxWI8ePAAAQEB7DJPT09IJBIAHUk4Ly9PpfLkZRQVFfU7fn3h8/kQCATs496OgTENRxGLxbhy5QoYhkFKSgqysrKU3gtA78dCl1+QhkTSlB/QdevWAQD7jdTFxUVvMRHD1NbWhujoaIhEInaZs7Oz2uUkJSVh2rRpCAgIgKXl0LijTFZWFvh8PjIzM7tNgIrkNQdra2ulZYqP1SnPEISEhEAkErHJvqdjUFdXBz6fj+3bt6O6ulpf4WrU1atXERgYCADw9/dHSEiIUtLs7f2QlZWFwMBAbN++XSexGn3zbEhICNzc3AAAc+bMAdDxAnl5efXZ9ENIZ6mpqfD29oadnd2AyjE1NUVcXByio6M1FNng5+TkBIZhYG1t3WMTnFxdXZ3SCVK+rL/lDXZisZg9T8n1dAzGjx8PhmGwbt06nTVJaptMJsPEiRPZx52/DPT2figqKoKTk5P2g/wXo06aKSkpcHNzg5OTE6qrq9ma5ZUrV1BdXQ2GYcDj8ZCdna3nSA1TREQEwsLC0NraitbWVoSFhSEiIkLfYWlNQ0MDDh8+jLi4OI2UFxgYiIqKClRUVGikvMFOftKTN93Kaw8cDgeurq7g8/nsF9n+lmeoTp482aU5sify/d62bRs8PT377Bs2ZiEhIT12BWiL0SZNqVSKyMhI8Pl88Hg8SCQS9s114cIF5OfnQyqVQiKRsDVQop64uDg4OzvDzMwMZmZmcHZ21lhCGYzkg39MTTU3q1JGRgaCgoLU3q61tRVff/01Dhw4gPLycnZ5fX09Dhw4wD4uLy/H6dOnNRKrJnl4eAAA++W1uLgYIpGIHWE8fvx4pSRYXV2N8ePH91meIRKLxUhPTweHwwGfz0d6ejpCQkJUOgada6eGisvl4sGDBwA6apCda9A9HQv5cZOf43VR8zbapGltbc1+AL28vFBcXAwXFxeIxWLweDxYW1sjJCQEPB6P+jb7acSIEfj444/Z4/zxxx+zg4KMTefBP5pib28POzs7tUZA1tfXw8zMDIWFhQA6Bo8kJiYC6DjhhIaGss8tLS1FZmamRmPWBJlM1qW5TZG1tTWqq6shlUq77c9St7zBzMXFhf0MiUQiCAQCHDx4UKVjUFRUZBTnrzlz5rDvU8X+TbmejoX8uEkkEvB4PN308TJGTiKRMDwej30sFAoZAAwApeWk/16+fKn029i8fPmSsbe3ZyorK7VSfn19PcPlclU+fhEREUxwcDD7+G9/+xsDgKmrq2PKysoYxY91Wloa4+vrq/GY+yIQCNjPWXFxMcMwDOPl5cUuk0gkDMMwDI/HY5cp/jAMwxQXF3d5vkgkYj+33ZUnf458uVAo1OVuD5hIJGIEAgH7uLtj0N2xNQby/fLy8mKXeXl5sa9hd8dCrvN5XptoGj0yIA0NDfDx8cGtW7fg4+ODpKQk2Nvb6zssjUpOTkZzczOSkpIGxf/w8/PD/PnzsXHjRnYZh8NBWVkZAGDmzJlIS0sDAFy+fBkAkJOTo4WoCRl6hsQlJ0R72tra0NTU1OXvwUgmk2HKlClqbzds2DD8/e9/R0VFBQoLCzV6bWVeXh4sLCwQFhaGMWPGoLCwsNc+0+XLl2vsfxNC1EdJkwwZXC63XxeEh4eH49ChQ9iwYQOCgoI0cskJ0PElQyAQoKSkBHfv3sXEiRP7rGlyuVw8fPgQt27dYpdVVVUB6BgsIR+Gr1gLldc2CSEDR0mTkD5ERkbCx8cHAQEB7LWVubm5Ay43OjoamzZtgqWlJXx8fHDy5EmVmrY3b97MjqJ0cHBAaGgodu/ejXHjxnW5lpEQollGO3qWEE2xtLTE6tWrIRQK2dGzA51wvaKiAqWlpdiwYQMOHToEZ2dnlfuCx40bh5aWFvZuKWVlZYiJiQHQUduU92cCHbMVdR6JSAjpPxoIRAZEJpNh3rx5qKmpwbx58xAXF2eUt75qa2vDvHnzkJ6eDqDjms2CgoJ+X7MpP1Z2dnbw8fHB+fPnh8x0eoQYMqppEqICxWnv7O3t4ezsjEOHDvWrrMzMTHC5XLi7uyMhIQHr16+nhEmIgaCkSYiKvL29YWpqilOnTiEuLg6HDx9GQ0ODWmU0NTVBKBQiKSlJqYmWEGIYKGkOEXl5eZg0aRJCQkI0fllIS0sLrK2tjfr+kHLye2Gamppi/fr1EAqFam2vWLMMCgpCRkaGliIlhGgDJU0jd/fuXfj4+GD//v24ePEibG1t4eDggNTU1AGXXVhYiOXLl+O3v/0tvvnmG1RVVeHFixcaiHrwsrOzU7oXZmFhocoTrite56nu4B9CyCChk3mHiM49ffqUiYqKYuzt7Znz588zDNMxXZt8XVhYGGNnZ8euU0d9fT0TEBDAuLu7M7du3WKX3717l3F3d2dqamo0sxOD1MuXLxkul8vU19czBQUFTFxcnErbbdiwgSkpKWHq6+sZe3t79vUghBgOSppGKD09nbGzs2OSkpKU5jNNT09n7O3tmYKCAoZhGKayspLx9vZmvL29VZpX9eXLl0xcXBzD5XIZkUjU7XNqamqGROLMyMhgAgMD+7Xthg0bmPT0dA1HRAjRBbrkxIgUFhYiPDycvUVXdyMyKyoqEB4eDgsLC+zbtw9cLhd5eXkIDw/HihUrEBkZCQsLiy7bZWVlITo6GqtXr0ZUVFSvl1rIZDIIBALk5uZq9DZagw2Xy8XYsWMxbNgwlbd59uwZ/vnPf+L27dtajIwQoi00I5ARaGhogEAgQFNTEzIyMnrtJ7O3t0dBQQFOnToFHx8fNlFWVlYiOTkZs2fPxqZNm9gRnaWlpQgPDweXy0VJSUmPl0acPn0aEyZMgKOjI7hcLpYsWYKnT58C6JgsfPXq1RgxYgTKy8vx8OFDLFu2TPMHQsdOnz6NJ0+eqLVNa2srpk+frqWICCFap++qLhmY9evXM1wul8nNzVV725cvXzJJSUmMnZ0d21xYX1/PbNiwgZk+fTrj5eXFODs7MyUlJX2W5evry6SlpbGPATBlZWXsrark6/R1qypCCNEEGj1rwP72t78hOzsblZWV/bo5sqmpKaKiolBQUIC//OUvcHBwwN27d5Geno4lS5agrq4OJSUlcHZ2HnCsoaGhqK+vH3A5hBCiT9Q8a8BMTExgZmYGU1NT8Pl8dlo2dVlaWiI9PR0VFRWIjo6GnZ0dbG1t4ejoqFY5vd1Nw9fXF5988gm4XK7a8RFCyGBBSdNINDQ0oKGhYUC3rLK3t8f58+c1GNWvYmNjMXPmTERERLDLsrKycPjw4T63Ndb5bAkhhoeSJtGY+fPns/dxDA0NVVrn6OiI4OBg7NmzB76+vgAAd3d3leZcpQkACCGDBSVNojMfffQRjh07xj62tLSkicoJIQaFkibRiMDAQEyYMIF9nJaWxt4oWX5/x3HjxuH777/XS3yEEKIJlDSJRnS+7lLeTNv5b2O4PpMQMnTRJSeEEEKIiihpEkIIISqipEkIIYSoiJImIYQQoiJKmkTJoUOH0NTUpO8wCCFkUKKkaeD+93//FwCQkZExoFlz8vLyMG3aNPz888/srcH+8Y9/aCRGQggxFpQ0DZilpSU4HA4EAkG/71t59+5d+Pj4YP/+/cjNzUVSUhLy8vLw0UcfYdy4cRqOmBBCDBslTQNmamqKn3/+Gb/73e8wb948JCcnq7xtU1MTwsPD4evri02bNrFzzsoT6NmzZ7Fnzx5thU4IIQaJkqYR2LBhA0pKStDc3Ixp06YhLy+v1+enpqbCwcEBkydPRmVlJZydnbsk0IFM/E4IIcaKkqaRsLCwQFJSEnJzc7F//374+Pjg7t27Ss8pLCxk+y1v3bqFsLCwLgnU29tbT3tACCGDH02jZ2Ts7Oxw/vx55OXlwdfXF97e3oiLi4OFhQWampqQm5sLOzs7FBYWQiAQwNvbG7du3WIH/xBCCOkZh2EYRt9BEO1JTU3F/v37ERkZiQ0bNkAmkyE8PBxNTU1IT0+nZlhCCFEDJc0hoKmpCdHR0Th//jxMTU2RnJyMFStW6DssQggxOJQ0hxCxWIyZM2f2+/IUQggZ6ihpEkIIISqi0bOEEEKIiihpEkIIISqipEkIIYSoiJImIYQQoiJKmoQQQoiKKGkSQgghKqKkSQghhKiI5p41APeetuo7BEIIGbKmvjqC/ZuSJiEDtG/3TqTv/Vjt7abYTMWF6ze1EBEhRFuoeZb024vnzyHcsR22o8xgO8oMwh3b8eL5c32HpVOPHzXiwunvUVbzAFVPWtT6mbvQE5npB/S9C4QQNQyZpMnhcCCVSvUdhlH5TJiEirLrqLjfgIr7DaiVSfGZMEnfYenU3p1xWLdpM0aamyMqdAPeXbaoz581b/vhYW0t1m4KR27WSTx+1Kjv3dCIsznZsB1lhviIcKXl8RHh3S6Xf9kqLy3ps+zy0hL2+d25L5N1W94CxxmwHWWGYH/ffuyRbsnjP5uTzS6TH1PF5YrHorvjaqg+/zS119dK/j7q7j0gPya6eJ2HRPOsjY0NvLy8YG1tre9QjMqxtP3IuVKEYcOHAwAEm7fCz8MNkTt36Tky3agouwFpVRWS0w4BAFavD8Gz5maVtn1t7BiYmJjCN2AlPt+/DzG7k7UZqk4s9vMHANy4+hO7TH6ir3rSgviIcJSXlsDReTaC/X0hOpcPR+fZWOA4A5fKbyttYzl+AhydZ7PL+Is8UfWkBeWlJYiPCEf8nn1K//tY2n5UPWnBfZkM8RHhOJadi88/TUXGn09jEpeLBY4zcDYnm41xsImPCEfVkxYAHYleHue+3Ttx+eYdAEDQ75dhsZ8/Guoess8tLy1BQ91D/QStQeWlJSgt+hFVT1rw+aepXV6r8tISvDnnLcTv2dfx+iu8B87mZCN8TRAu37yDSVyu1mM1+qTp7e0NGxsbBAYG6jsUYkTa29uQGBOJhL2p7LJp02eoXU6gYCOWz30LlXdu92v7we7G1Z+w1P8dAMBS/3fwQ/a3cHSejZ9rJGxSdJk3n02m3SkvLcHK99cAABydZyNy4/ouzwkO3QQAuH2zDH4rVwEA1n4Yxq4Pj92BugcPNLdjGiaPv7y0BH9Y/b7SukcN9RhjOQ6Tp/AAADPemMmuu3n9GryXGf5t/m5ev8a+bt7LViA+IlwpaSq+N4JDN7G16/syGcLXBEF0Ll8nCRMw8uZZb29vbN++HdXV1XBycmKX29jYgMPhwNvbW4/REUMmOn4M9m86aSTRxexORmJslAaiGnzuy2QYYzlO6fF9mYxNAHK91ZYa6h5igtXkLuUqmsTlItjfF+FrgpSSiqLxEyeqG77OTOJyER8RDv4iT7zhNItdLjxwGPxFngj6/TIcy85lnyv3sPZnnSULbXpY+zMsx09gH/9cI+n1+fJ9zjt9Cq4eC8Bf5KlyU/9AGW3SDAkJwfbt2wF0JEl50yyHw8G6desgEolQXV2tzxANXnDoJuyOicSL58/x4vlz7I6JZL8xG7PHjxrxxeGD2LIjXiPlzXJxhZm5OS6dPaOR8gyVvL8qfE0QexJc4Kj6l5Jj2bkQnctH0O+XdVl34+pPg7ZpVi5+zz5cvnkH/EWe7LIxluOwNf4j1NbUKPV1Ah1fHDp/mRgKjqXtZ5tm5V8aqp60YN/RDBzck6L1/2+USVMsFiM9PR2urq5wdXXFhQsXIJVKkZKSAh6Ph23btiEgIICS5gB9EJFHPQIAACAASURBVBkN+5lOsJ9k2fEz0wkfREbrOyytS4yJQsSOBJiYaO5m3gl7U7E7NhLt7W1qb/vXils4cfQILp8/x45eli/Tt0lcLh411APoqDFO4nIxictVqkncl8lgOX4CO6p439EMiM7lo+pJCy6V34bl+Al4WPsz+/zampoea1eOzrO71GI//zTVYL7MTeJysfL9NWxNOj4iHGs/DEPVkxaErwlSem7e6VNG0TQLABOsJrOtDY8a6ru8hnLyvk1F8scz3pjZZw1VE4wyabq4uIBhGDAMAx6PB4lEAmtra8hkMtjY2AAAUlJSkJWVpedIDduw4cMRuXMXe7KL3LmLHRRkrIouX0RLc7PGay2jx4zFEj9/ZKYfVGu7Q5/swea1HSfTHNHXCH67Y/TgX8puIGHbFo3G2B9vznkLP2R/C0C5f3PyFB7blKbYv9kdR+fZOHn8KADl/k1VfP5pR5/zJC6X/Xuwuy+TsV8Kiq9cYhOo1ZQpSs3SxtI0CwBvOM1CzskTAJT7NxWVl5bg4J4ULPbzR3lpCcpLS7DU/x12u0cN9XCZN1/rsRr1QCCxWKzUNLty5Uq4urqCw+FAKBQiICBAzxEah/b2NpiYmLK/jVV7exv27ozHvqMZWik/NDIa3rMc4bdyFUaPGdvn8x83NuCTXQn4qfIeRo+1hC9/FXJFJ/C4sUEr8fVFPooR6Kg5rP0wDIv9/JFz8gRsR5lh5ftr2OQYv2cf5r8xHQAgOpcPAN1eSmA1ZQould/GvqMZ7Hr5yFH5YJD4PfuUtlVcL0+2H8f/Sa1kq2sLHGegtqYGANjRsgCwNf4j9jhtjf+ITZLG1jTr6Dwbk7hc2I4yg6vHArb/NtjfF85uczF+4kT2vWU7yox9XwBgt1Ncpk0chmEYrf8XMiCDeRq9x48aseZtP3z/409Y87YftuyIN8pRoABwOPUTtD5rxpYdCVr7H2dzsnHp3FmVEvNfK27Bz8ONTRKKThw9goRtW7pdRwhRD02jRzTmH23teNbcBKCjJqbqdYr69LC2FvPsf6v2dv/5n8NwXfozpPeqcPHsGawP26yxmCrv3MbD2los9vNHYkwUlrnNgbmFRY/PNzExRWDIRo39f0KIaihpkiFngpVVv2pgibFREB0/hkDBRpzL/TPc5i/QWK06KlSA5LR0PH7UiP/v3/8d2xI+wn/8xyu9bmM9dSqAjmba0WMtAXTUMD2Xdh09SgjRDEqahKho7aZwrHnbD4t/74+EvamI2xKGb/OvDLjczPQDmOXiimnTZyDkXT52CPeoPKAhOHQTVi3xxur1IbhaVIgnv/yCVWvWDTgmQkj3jHL0LCHaMHrMWHbaO/uZb2KC1eQu186p6/GjRmRlHkfotmgUXb6If7S1Y8HiJSpvH7lzFz75vKP/04//Rxz7rmMAxe9mvom4lL0Dio0Q0hXVNAlRg+K0dzGJyXjH0wMLFi/p96hh+YTvJqYm/R6Z+7q9A163d+hzGSFk4KimSYia5NPejR4zFn78VTiS+km/yrkmLsbD2lr4rVyFzPSDcJu/ANZTbTUcLSFEkyhpEqImxWnv1oVtRo7ohNq39+q45jMOMbuT/9VEewyhQ2A2JUIMHSXNIaLo8kW4vW6H+IhwjV8W8ry1FR4Or6O9Tf0p4AyVfNo7AIjdLUTclrA+tlCmOOF73JYwxO4WGvXEEIQYC0qaRk56rwpr3vbDl4fSkZl7GlyeDZbPnYPM9AMDLvuauBgbVv0BU+2mYf/xL1AjqUbbyxcaiHrwU2yaXbB4CX5pbERF2Q2VtlWc8L0/g38IIfpDMwIZgP7MCPSsuRmf7/8ERZcvYcuOeLjNX4jHjxoxesxYPGtuRlpKEoouXURMYjLc5i9Uq+zHjxqRGBOFXx41ImZ3MnutYk31PezYvAnJaYcwwcpK7ZgNTXt7G7xnOeLb/Cv4pbERuVknVbqZ9GfCREx7fQZcFyzAO57zse9oBvVlEjKIKc4IREnTAKibNEUZx/DFoYPw5a9CoCCEbfYTZRxDVuZxxOxOxiwXV0jvVSExpuM+jjGJyX2euNvb23Ak9RPkiE4gYkdCt5OWP6ytRVTohiGTOC+dPYOcrBM4+JVI7W11MS0fIWTgKGkaGFWT5jVxMRJjo2A/802ERkZ3O+l35Z3bSIyNgpm5OWJ3CzHBygpFly8iMSYKCxcvwdpNmzHS3LzLdmdzsrFnZxz8+KuwLmxzr/1vD2trEbclDAe+Pjkk+umWz30L//Zv/4YRZl0nHO9Je1sbGuvrkV92a0gcI0IMGc09a2QeP2pE3JYwtDQ3Izktvdep3aZNn4GvTp/DpbNnEPy2L5so866V43DqJ3jH0wOrN4SAHxQMAKgou4HEmEhMsJqMb/Ov9Hj3jcvnz2HsuHF43d4BE6ysMM/LG8+aOuakzf/hNHz5qzBs+HD8teIWGuvrMd9nkeYPhJ4c/FqEmup7fU57p+if//wHRo8ZSwmTEANDNU0D0FtNc8fmTSi+cgmxu4VqDyZpb29DZvpB5IpOsIny8aNGpAmTUH6tFGMtx6HlWTNiEoWwn/lmr2VtfG8l5ri5s1O42Y4yQ86VIgCAn4cb4lL2YtWadThx9AiuFhXiwJcn1YqVEEL0RbGmSaNnDVhN9T3knT6FvGvl/Rp9aWJiivVhm/HVD+dw9693sHzuW5BWVSFhbyrmeXmjsaEe3+Zf6TNhqiJh2xa93eeREEI0hZpnDdgrr5hg+IgRMDExRfiaIHwQGd2vUZijx4xFwt5UVN65jb0742Fta4spvKl4bK9ekrtaVNjjuoVLluL4gTRMHAKDgwghxouSppH45VEjHjc2DujShWnTZ+DodzkajOpXgs1b4efhhuDQTeyysznZyMo83ue2oduiMcvFVStxEUKIOihpEo1R7NNM2LZFad3r9g54+4/v4VjafixcshQA4OTiitd6GFikSFP3rCSEkIGipEl0Jiz2T/ju6y/Zx6PHjO1xNC4hhAxGlDSJRvjx/4ix48axj+NS9rKP5fd1HD3WEuknvtFLfIQQogmUNIlGdL7uUt5M2/lvY7o+kxAy9NAlJ4QQQoiKKGkSQgghKqKkSQghhKiIkiYhhBCiIkqaRIko4xieNTfrOwxCCBmUKGkauP/55/8AAJLTDg1o1pyiyxfhPcsRdfdrMdLcHP/zP/9E28s2TYVJCCFGgZKmATMzN8d/DhuG8DVBeMXUpF9lSO9VYc3bfvjyUDoOfi3Clh0JuHT2DNJSkmE5foKGIyaEEMNG12kasJHm5si/cQtnc7LxjqeHSjeIlnvW3Iy0lCQUXbqImMRkuM1fiMo7t/HuskUwMzfHn6/8SLP1EEJIJ1TTNAKL/fyRd60cALDc7S1cOnum1+dnph/A8rlzMH7iJORdK8e06TMQtyUMUaEChG6LxsGvRJQwCSGkG5Q0jYSJiSk+iIzBse9ykZN1Au8uW4TKO7eVnnNNXNzRb/ngPr7/8SoCBRuRmX4A7y5dBLvXp+P7H3+iu4kQQkgvOAzDMPoOgvTu3tNWtbe5Ji5GYmwUZrm4InRbNEaam+PS2TOwtrWF9VRbFF2+iMSYKLgtWMiuJ4QQ0tXUV0ewf1OfppGa5eKK73/8iW2KXb0+BIGCjezAHwA4+LVoQPffJISQoYZqmgagPzVNRfJBPxfP/IARI0dia/xOuM1fqKHoCCHEuCnWNClpGoCBJk258tISvO7goNLoWkIIIR0oaRoYTSVNQggh6lNMmjR6lhBCCFERJU1CCCFERZQ0CSGEEBVR0iSEEEJUREmTEEIIURElTUIIIURFlDQJIYQQFdF1moQQQoiKqKZJCCGEqIiSJumX1tZWbN26FRwOBxwOB1u3bkVrK81c1JvejlnndYmJiex2HA4H5eUd90stLy8Hh8PRS/yEkCGSNMViMWxsbJSWhYSEgMPhICQkRE9RGbaEhASUlpaipaUFLS0tkEgkSEhI0HdYg1pvxywhIQFPnz5FS0sL6urqEBsbi9OnT+s54v5JSUkBh8NBSkoKu0wqlbJfCBSXd7dd588q0PEZ9vb27vN/i8VicDgclZ47GGVlZXV7TrKxsQGHw4FUKu32sTGQv/49vXaK7yHF/fb29tbtuZwxchKJhAHACIXCLusAMMXFxXqIyvABYMrKytjHZWVlzBB4Ow1Ib8es87qetjOU4ywUCpU+cwKBgGGYXz+PEolE6fkSiYR9vlAoZEQikdJ6Ho/HeHl59biNouLi4i7PNRQA2GOluKy7/eTxeF2Oo6FSfM26e/0Z5tf9lUgk7HNFIhF7Dvfy8tLJ8TD6mqanpyd4PB78/f2VlovFYgCAi4uLPsIiRC0zZ84Eh8PBzJkz9R1Kvxw8eBAAYG1tDYFAgLq6ui7P2bZtGwDgypUrCAgIYJenpKRg165duglUj0JCQiASiZSWeXt7QyAQsMfGWF29ehWBgYEAAH9/f2RmZiqtl0qlsLGxgbW1NaytrdllTk5OSudw+TptMuqkyeFwkJ+fD+DXg5mVlQUOhwNXV1d4eXnpMzxCVFZWVgaGYVBWVsYuq6qqgp+fH77++ms9RtY/48ePV3psbW3NNr8pkjfDTZw4UWex6YNYLIabm5vSMqlUigsXLrDHxZi7kmQymdJrXF1drbS+rq6uS0KUL5Of0z08PHQSq9EmTRsbGzAMg+zsbKxbtw5AxxuTz+dDIpHAy8tLZwfZGEVERCAsLAytra1obW1FWFgYIiIi9B3WoNbbMQsODkZ6ejpaW1tRX18PW1vbPvs0U1JSMH/+fLz77ru6CF8jpFIpuFxutzUCa2trMAwDa2trtt9zz549XWpZ8j4sHo+HyMjIbvu5DM3JkyeVatdAR1Lw8vLC9u3bwTAM0tPTDXoftSUgIAAMw+DIkSNsC6JWab0BWA+EQiEDgP3h8XgMw3S0ecv7C0D9mQPS0tLCREREsMc4IiKCaWlp0XdYg1pvx6zzut27d7PboYc+zbq6Ouarr75ifH19db8zfejcpynXub+uOxKJhBEIBGz/p+KPYl+lsfRpFhcXd9lPgUDQZT+8vLyUzlnG1Kep2I/Z3eun2I/JMN3vu0gk6rYvVNOMMmnKSSQSNmEyTMfJRyQSMQKBwCAGUxDSm4iICKasrIxxcXFh/va3v+k7HCXdJU3Fk353yU6uu5NfTydSY0iaiuTnJznF5KB4Luu8ztD1dyCQIvkXLW0z6szR+YPL4/HYmqf8Gx0ZmJcvXyr9Jj0rKChgcnNzNVbe7t27malTpzIAmLq6Oo2VO1CKLT3yk5/iZ67zcnmSVKxldaZ4UvXy8upSM8O/RuQq1tpUqdUONp2TpuL+dK5lGts5TF6Z6Vy7lp/DFY+FPDnKt1F8T2kbTaNH+q2hoQE+Pj64desWfHx8kJSUBHt7e32HNSi1tbVh2rRpKCkpgaWlpcbKLS8vx29+8xuMGDFCY2USQnr27/oOgBiutrY2NDU1dfmbdJWcnIzVq1drNGECgKOjo0bLI4T0jpImIVrW0NCAL774ApWVlfoOhRAyQEZ7yQkhg4VAIMC+fftgamqq71AIIQNESZMQLSosLERTUxNWrFih71AIIRpAzbOEaFF4eDgyMjL0HQYhREOopkmICjw8PNiZZ9T5GT58OI0oJsSIUNIkpA8VFRV4+vQpmI7rmlX+qa+vx/Pnz9HQ0KDvXSCEaAg1zw4BeXl5WLt2LZYuXYrExERYWFhorOyWlhZYW1tj7NixGitzsAkKCkJGRgaampqQmpqq0jYWFhYICwvD6tWrIRQKsW/fPi1HSQjRBappGrG7d+/Cx8cH+/fvx8WLF2FrawsHBweVT/y9KSwsxPLly/Hb3/4W33zzDaqqqvDixQsNRD24HDp0CM7OzrC3t4eFhYXKXzjkz9uwYQNKS0tRUVGhzTAJIbqik3mHiE49ffqUiYqKYuzt7Znz588zDMMw9fX17LqwsDDGzs6OXaeO+vp6JiAggHF3d2du3brFLr979y7j7u7O1NTUaGYnBoGnT58ydnZ27LHrr9zcXGbFihUaiooQok9U0zQyhw4dwuzZs2Fubo6SkhJ4e3sDAE6dOgUHBwdUVFRg3759yM3Nxf79++Hj44O7d+/2WW5bWxvi4+Mxe/ZsLF++HAUFBUoDXH7zm98gIyMDQUFBkMlkWts/XUpISPh/7d19WJRV3gfwL7s9MU864bKhqKGTyjjuo7sgZNiCgagMSilzqYFmYFKAWoCgvKnD6AKSL4ANDaaErhVsmYDJiwJiwIZuuJDagmMKYsAIrYGDCu229/MHO7dMgsIwzDDD73NdXc79dvzdN8Zvzjn3OQf+/v6DnsVn2bJl6OzsREFBgZYiI4Tojb6zNtGOkpISxsbGhgkICOizZlRVVcU4Ozszy5YtY2uE+fn5jEAgYCIiIpgff/yx1+syMjIYHo/HiMXix07MXldXxwiFQoOfwL2qqoqxsbEZtuURQvSDkqaBa25uZpYtW/ZQc+mjZGVlPZQo4+PjGYFAwMhkMva8iooKxsHBgfHy8npkE2VOTg673iPDMIxUKmWampqYpqYmRiqVsmtGVlZWMjk5OZrcps45OzszJSUlWi0zODiYSUxM1OjayspKtWf8y2M9lwZTKpVq5zY1NQ27pcMIMVSUNA2Yv78/w+PxNFpu6v79+w8lyubmZiYgIICZNWsW4+bmxjg4ODAVFRWPLcvT05ORSqXsNv67aLJqwWTVMalUOiwXTP6loeqDbG5uZgQCQZ81+t5cuXKFsba2ZhwdHRlHR0fG2tqaTYA9j6n+VCXMno1IhvLcCTEE1KdpoK5cuYJjx46hpqZGoynaOBwOIiIiUFJSgm+++Qa2traora2FTCaDh4cHmpqaUFFRAQcHh0HHunHjRjQ3Nw+6HF3o7OxESEgIZDKZ1su2tLSEv78/JBJJv6+JiIjA0qVLUVZWhrKyMvj6+iIiIoI9FhQUhLKyMsjlckyfPh2VlZVaj5sQ8gCN0zRQpqam4HK54HA48Pb2hlgshkAgGHA5lpaWkMlkqK6uRmRkJAQCAfh8/oCXnCouLu7zmKenJ/bt2wcejzfg+HRNWy//9CUgIABz585FbW1tv35eWVlZaonQzc0N0dHR7DHVZwA4dOgQgO41Nnv+aSwvZhEyHFDSNAIKhQIKhUKjpKliY2OD/Px8LUb1QHR0NOzt7REWFsbuy8zMxIEDBx57rVgshrOz84D/zurqaoSEhAzoms7OTnz77bdoaWnB2bNnMWbMGK1OgVdQUABnZ2eIxWIIhUL4+vo+8vzHHX+c2NhYAMDly5cxc+ZMdj8tXE2I5ihpEq1wdXXFhg0bAHQ3x/ZkZ2eHdevWYc+ePfD09AQAODs796s2p2nSsrGxgVgsHvB169evR21tLXg8Htzd3VFVVaWVJb0UCgUCAwNRU1MDhUIBCwuLfl+rVCp7/fzLbblcDi6Xy24fP34cAJCSksK2BKSkpCA5OZk9nxAyMJQ0iU7s3LkTaWlp7LalpeWQNYGqaFJD3bdvHyIjI5Gfnw+hUIjU1FQEBwcPOhbVmpqdnZ1ITk5GSUlJv+5fKpXCxcUFJSUlAAAXFxdIpVIA3TXJbdu2YefOnbhz5w6WLl3arz7NDz74AC4uLoO7IUJGKEqaZNB8fX0xceJEdlsqlWLChAnsZwAYP348cnJy9BLfQAiFQhw4cADZ2dmIj4/HjBkz4OXlNagE33NNzZCQkAH1mW7YsAFWVlbYv38/ACAnJwevvPIKACAqKgozZ87E/v37YW5ujpKSEtjZ2UEul7M1egCwsrKCvb09AGDhwoWora2Fp6cnOjo6qImWkIHS9+u7RDN1dXUMj8djGEb7YwrT09MZX19frZVnaGpqahgbGxvm/v37jEwmYwICAjQu6/79+4yDgwNTVVXFVFVVMQ4ODnqd+MHR0ZGRSqWMtbV1n+M+CSF9oyEnhPyCQCCAs7MzUlNTBz3hes8J3yMjIyEWi7XSR6qptLQ0FBcXIygoaMBvSBNCqHmWkF6Fh4fD3d0dXl5eiI+PZ/s5B0KhUODAgQOoqKhAdnY2OBwOOxewvvD5fPYFIULIwFFNk5BeWFpasmthCoVCcDgcZGdnD6gMiUSCoKAgcDgcREZG0pqahBgBSpqE9KFn02x8fDwkEgk6Ozv7dW11dTXOnTuHgIAA7Nq1C6+++qpBTO5ACHk0SpqElZqaira2Nn2HMWxwOByEh4dDIpGw/ZyHDx/u17WBgYGQyWRQKBQ4cuQIO/UdIcSwUdI0YD///DMAID09XaMxiSoFBQWYMWMGbty4gTFjxgAAfvrpJ63EaOh6roUZHx8PLy+vfl2XkZEBBwcHdnymPl/+IYRoD70IZKAsLS1hYmKCwMBAjWa+AYDa2lp2qrmsrCwIBAIUFBRg586dauP8Rrr4+Hh4e3sPeBL3zz//nB2fSQgxDpQ0DRSHw8GNGzeQmpoKFxcX+Pj49LsJsK2tDRKJBAUFBUhMTIRQKERtbS3c3d0BALm5uYOax9bY2NjYsNPy/epX/W+c+fbbb+Hv7z+EkRFCdM2EYRhG30GQwWlra0NCQgKys7PZJNiXpKQkJCcnIygoCMHBwb0mUKIdbW1tmDt3br+nzCOEDH/Up2kExowZg/j4eGRlZSE5ORnu7u6ora1VO+fs2bNsv2VVVRWCg4ORlJQEW1tbTJ48GTU1NZQwtWzMmDHw9/dHQkKCvkMhhGgJ1TSNUEFBAUJCQiAUCiEWizFmzBhkZ2dDIBBAIBDg7NmzCAwMVDtOho6trS3S09O1uswYIUQ/KGkaMVVTbHh4OAICAlBfX4+QkBC0tbVBJpNRv6WOFBQUIDk5ecjWKyWE6A41zxqx4OBgVFVV4ZtvvgGPx4NQKISPjw9KSkooYeqQpjMKEUKGH6ppjhDl5eWwt7en8YJ6UltbC29vb1RUVNDPgBADRkmTEB0JDQ2FiYkJrl+/DnNzcwQGBrIrjXR0dODIkSMoLi7G1KlT8eabb4LP5+PEiRO4fPkyoqKiAABxcXGYOXMmu6YmIUS3aJwmITpSV1eH1tZWJCUlobGxEfb29qisrISdnR1ef/11mJubIzo6GqdOncL06dOhVCpx8+ZNVFZWsmVUVlbCzMxMj3dByMhGSZMQHZDL5cjKyoJSqcTo0aNhZ2eH2NhYyGQybNmy5aFjbm5u+g6ZENILSpqE6IBSqQQAjB49mt1nZmaG27dv93qs5wLRWVlZEIlE7GdXV1ddhEwI6QUlTUKGOUdHR0RHRwMAWltb2f1yuRyFhYUQiUQYP368vsIjZEShISeE6MD06dMBACdOnADQ/eJPZmYmXF1d2WMXLlxgj4lEIjQ3NwMALCwsYGdnBzs7O1hYWLBlenh4oKqqCtu2bdPlrRAyolHSJEQHRo8ejZycHCxduhQikQizZ8+GhYUFfHx82GP29vYQiUTgcrmYOnXqY2uP27dvh62tLaZMmaKjuyCE0JATQnSoo6MDV65cAZfLBZ/P7/XYhAkT2ITZ3NwMpVLJniuXy8HlcsHlcpGdnY2nn34au3fvRllZmc7vhZCRiJImIQbKxMQEYWFh2LNnD+h/Y0J0g5pnCTFQV65cAY/HQ1NTk75DIWTEoJomIYQQ0k9U0ySEEEL6icZpGoCrP3boOwRCCBmxrH/zYOIRqmkSokOfHT2i7xAIIYNASZNo5N7du0jYvhV8cy745lwkbN+Ke3fv6jusYaurqxPi0GD85Ug6Vi6aj8aGBn2HRAjRwIhJmqolmYh2vJcQj+rKv6H6pgLVNxVoqL+O9xLi9R3WsHT9qhxrXl4MwcxZOFZ0FlFxCVi/xhu5x4/pO7QhcXB/EvjmXBzcn6S2PyYsBHxzLmLCQtT2q754XThX8diyVWX09ewunKsA35yLdcs9Nb8BPVE9h97ubYHd78E35+JmfX2v28ZA9e+mr5+d6mfPN+eq7V+33LPX/UNlRCTNadOmwc3NjWZO0aI0aTKi4xLw1KhReGrUKARu2ow0abK+wxp2co8fQ8SGAEj2JsF77TqcLy/DjFmzcPREHorychGxMQBdXZ36DlOr3nwnGJtjdqrtUyUC+e3uyelVCXLdck9k5J2G/LYS4Rv81a45uD/poaQQsycRq97w6/PvtnOYi4y804O+B12LCQuB/LYS8ttKJMbuUDvGN+fiVZ83IL+thBWPBwAounARk557Th+hDokL5ypwrvRLyG8r4TDvpYe+OFw4V4HnX/wj5LeVWPWGH/vFS/XvSH5bic0xOx/6ojYUjD5pCoVCTJs2Db6+vvoOhRih4vw8fHzoAxTn56ntVzXHFuXl4tBnWZgx6/cAgMaGBqxc5IofWm4h8VA65rzohJWLXFFz6aI+wteZr7/6K15evhIA8PLylfji2KcAgBt112DnMBcA4Oji2q/apjFatzEIQHcSeNXnjQf7l3ti1Rt+ePOdYH2FphN//9t5iFatBgAIX1mG4598rHbczmEuloiWA+h+Vj2/TN2ou8Z+nvDss0Meq1EnTaFQiK1bt+K7777DnDlz1PabmJhg2rRpeoyOGDrvxYtw6L3ub7bHMz6C9+JFALoTo6o5NvFQOp7usWi0aNVqJB5KR4jfWmSkp7Hb4tBgHJal6OU+dOFmfT3GWo5X275ZX4/Jz01VO0/R1Kjr0IYFKx4PMWEh8F68CLPnvACg+xmVnSnCzfr6Xpu1jUljww1YTpjIbvdMhL1R1bjtHObC0cWVbZpVJdahZLRJc/369di6dSuA7uZZVdPstGnTsHXrVgQGBlLSHIR1G4MQGxWOe3fv4t7du4iNCme/LY8Exfl5+GdrC9I+y8Jqv7ewW3YQ/2xtQXF+HmouXWSbY3szxZqPT08Xo/byJaxf441nxo7D0S/y0PT9TfitEOFOe7uO72b4+GdrK9s/tTtmG1xnzzLYPsqBitmTiOK/X2K/fLUomuE0fwHWh22B/LYSn3x4YCjBvgAACrJJREFUyKj6MDWVJk1GzJ5EdnvipMnYHLMTu2N0s9qPUSbN8vJyyGQyODk5wcnJCadOncL169eRmZmJa9euwcnJCQBQUFCg50gN19vhkbCxnwMbK8vu/+zn4O3wSH2HpTOKxu9hPeN3eGrUKADAU6NGwXrG76Bo/B4LlniwzbF9MTXlQLI3CSKv1Vi5aD5qLl1CVOwuvB4QiJWL5uN8uXFNwG7F46FF0b3UmaKpEVY8Hqx4PLUaxc36evzfH2zYvr3NMTtR/PdLkN9WIu1Ylr5C1ykrHg+r3vBTS46q5mun+QvYZ2hsJk6azLYytCiaH2qBUFH1bfbcBrr70RMPpeukNm6USdPR0REMw4BhGEydOhXXrl3DlClTUFpaCjc3NzAMAx6Ph8zMTH2HarCeGjUK4Tv+xP6CC9/xJzaBkP5bsMQDR7/IQ1xUON5LiMM814U4+kUedsdsw65t0foOT2uef/GPbD9mz/7Nyc9NZX/x9ezfHMlu1tfDiseDncNc3Ki7xiZQY34+s+e8wPZj9uzf7OnCuQq8v+ddLBEtx4VzFbhwrgKKpkacK/0SAGA5YaJOauJGmTRVysvL1Zpm582bh1OnTsHExAQA4OXlpc/wjILqzU9jewP0cSwnPourNf9gx6beu3sXV2v+AcuJA38RwWLsOHx6+gwAIMRvLSzGjsPceS9h1GjD/BJycH8Sdsdsw+6YbexbkEtEy9m+OeBB7SlmTyK8Fy/qHuubcoA9p6/m2ZiwEHzy4SGE+K1lk21MWIja25Teixeh7EyRQfUBqoaQ8M25ak2PCSkH2GeQkHJA7fyGujq4zp6lj3C1zs5hLqx4PPDNuThX+uWDl36We+Lg/iTkHj/G/lz55lyEb/BnXw66UXcNfHMuvBcvUnt2Q4UmbDcAw3UavdaWW/BbIULOl3+F3woRQrfHPLZZ0pio+p48RMtx8r/JYbDDHbq6OmFqyoHwBTu8/1EGpljzH38RIWRI9ZxGj+aeJRr7qbMLd9rbAHT/sh9pL7Bk5J1GcX4eFI3fw+/tYLi6Lx50maamHFy/KgcASpiEDEOUNAkZBG0kyl8qzD2JhUs8tF4uIWTwjLpPkxBdKso9qZU5ZcvPFMFx/gItREQI0TZKmoRoybWrcmQeThtUGXfa21Fz6SJecHTSUlSEEG2ipEmIlnS/EPT5oMooyj2JBdQ0S8iwRUmTEC2ZOGkSLMaORXXl1xqXUZR/EgvcKWkSMlxR0iREi1wXe6A476RG13Z1deJv5WVwWkD9mYQMV/T27AhQWlyIrUFvY77QHZu2xahNID5Ydzs6MN92Jp6xGKu1Mg2Zh2g5XntlMUK3S9j+yZ6U7e2ouay+b6LVZIhWrcb58jLY2M+BqSlHlyETQgaAkqYRu35VjrioCADA4awTKC0qxNKXXoSP/3r4Bm4YVNnny8vwp8gtsBbMQMTOWLyx3BOd9+9pI2yD1rOJtquzE9J31Rfm5pqZYcZM9VlcVGNdi/Ny4bp4ic5iJYQMHM0IZAAGOiPQnfZ2HEzeh9LiIoRuj8E814VobbkFi7HjcKe9HdJ341FaVIiouF2Y57pwQGW3ttxCXFQEfmi5hajYXewMQHXfXcX2TUHYJU3FxEmTBlSmsTmQtA8dd9oRul0yoOv+KJiG7NK/wmLsuCGKjBCiiZ4zAlHSNAADSZoZ6Wk4kvo+PL1XwzdwPdvUl5GehszDHyIqdhdecHRSq4VGxe167OwzXV2d+CBpH45nfIyw7ZJe161rbGhAxMaAEZ84z5V+ibXLl+Hnf/97QNdNmy5AXoXmLxERQoYGJU0D05+keb68DHHREbCxfx4bwyN7ra3UXLqIuOgIcM3MEB2bgImTJqG0uBBxURFYuMQDbwZt6rW/M/f4MezZIYbIezXeCt70yD63xoYGiEODkfLRJyO2b27NK4uxcUskjbUkxEj0TJq/jomJidFjLKQfbnf+1Oex1pZb2BzwJsrPFEGyNwkr1vhg1KjRvZ5rMW4cRN6rYWrKQcTGANxqbsIS0XKs2/gOLlVVYWd4GH79xBOYZTsbAFBd+TXe8V2DH1pasP/wUbi4ueOJJx7uBi/Oz8O9ux0YazkeT5uZQXmnHc9Onox7dzuQnfkJpk0X4H+efBKXq6twqaoKU6yttfNghqEDSftg/tvfYsUaH32HQgjRkt/+75PsZ3oRyIBt3xSEsjNFiI5NGNCA+AVLPOC0YAEOy97HykXz4ROwHv7BmyBatRrShHh4ODpgnOV4KO+0IyouATb2zz+yvOMZH+HFec6YaWMLAJBsCcUf/nuNZEsoAGC131v4pvJrfFV6dkjmax0Oai5dRHHeSRz9Ik/foRBChgiN0zRQdd9dRcGJbBScv6DRDDKmphz4B2/C0S/yUHv5Epa+9Edcl8sh2ZsEFzchbima8enpM49NmP0h2RKK1luKQZcznHV1dUIcGgzJ3qQR2yxNyEhANU0D9eSTphg1ejRMTTkI8VuLt8MjNVpKymLsOEj2JqHm0kXs3RGDKXw+nptqjVabgSW5r0rP9nlsocfL+DBFimeN+OUgaUI8XBd7jKj1RAkZiShpGoEfWm6h9datQa2/OGPW73Hos+NajOqBwE2bIZo/D+s2BrH7co8fQ+bhDx97rSG8UFNd+TXOl5fh09Nn9B0KIWSIUdIkWvHiPGes9nsLwIN+TJWZNrZY8drrSJMmY6HHywCAOY5OeKYf4xGHe82tq6sTERsCkPZZlr5DIYToACVNohPB0dvw2Ud/Zrctxo4zikH8cVER8AlYP6LHpRIyklDSJIMm8n4N48aPZ7fF7+5lt8Xv7gUAWIyzhOzjv+glvqFSWlyIxoYGSPYm6TsUQoiOUNIkg/bLISSqZtpffjamoSatLbewd0fMkPUDE0KGJxpyQogGinJPInR7jFE0MRNC+o9qmoRowHvtOn2HQAjRA6ppEkIIIf1ESZMQQgjpJ0qahJWRnoY77e36DoMQQoYtSpoG7D8//wcAsEuaOqhZc0qLCyF8wQ5NNxvYpcH+9dO/tBIjIYQYE0qaBuqZcWNhYmICcWgwnuSYalTG9aty+K0Q4c+pMrz/UQZCt0tQWlyIlD27YDHOUssRE0KI4aO3Zw2UqSkHZy/+AxnpaVjz8mJ4eq+Gf/Cmfl17p70d0nfjUVpUiKi4XZjnupBNoABw8C+fD2oeW0IIMVZU0zRw3mvX4dPTZ9Bxpx3CF+xQWlz4yPMPy1Kw9KUXMeFZKxScvwAb+zmIi47A+te88XpAIA59dpwSJiGE9IGSphF42swModsleP+jDPw5VQa/FSJcvypXO+d8eVl3v+X3N5Hz5VfwDdzwUAKd57pQT3dACCGGwYRhGEbfQZBHu/pjx4DOLy0uRFxUBOYtWIiNWyLxtJkZinJPYgqfjynWfJwvL4M4NFjtOCGEkN5Z/2Y0+5mSpgEYaNJUOSxLwZED7+OtoE3wXrsOjQ0NiI0Oh7K9HZK9SdQMSwgh/UBJ08BomjSB7pd+9u4Qo7SoEE+ammKzeAcWLPHQYnSEEGLcKGkamMEkTZUL5yow09YWpqYcLURECCEjByVNA6ONpEkIIUQzPZOmRuM0TUxMtBYMIYQQMpz1rFtqlDSpckoIIWQkonGahBBCSD9R0iSEEEL6iZImIYQQ0k//D3yPWCRrBHhyAAAAAElFTkSuQmCC\"\u003e\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.3. Molecular Modeling Studies\u003c/h2\u003e\n \u003cp\u003eMolecular modeling studies were carried out to support the idea that benzamide compounds can interact effectively with CYP51\u003csub\u003emtb\u003c/sub\u003e leaning on the fact and as a reference ligand that \u003cstrong\u003eVFV\u003c/strong\u003e -a benzamide-imidazole derivative- revealed significant CYP51\u003csub\u003eMtb\u003c/sub\u003e inhibition together with prominent anti-proliferative activity in various cancer cell lines [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e].Since \u003cem\u003ein vitro\u003c/em\u003eCYP51\u003csub\u003eMTb\u003c/sub\u003e absorbance assay was performed on the available non-human strain, the question that would evolve will be the difference between human and non-human strains, also if the results will be quite similar or would differ greatly. Literature survey showed difference in human strain than non-human one that could affect inhibition activity which is the middle portion of the human CYP51 I-helix (the core helix in the P450- fold) [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. Where, this portion is distanced away from the heme iron by 1.5\u0026ndash;2.0 \u0026Aring; in human rather than non-human and a phylum specific residue (small leucine residue) is subjected to CYP51 active site rather than a bulky phenylalanine residue, which renders the enzyme binding cavity with increased volume in human CYP51 rather than non-human, thus, bulky groups and enlarged volume of compound atomic structures will be essential. Also, the non-human CYP51 helix lacks loop like segments, where binding to its residues closes the channel and any non-polar moiety could perform inhibition via interrupting the threonine/serine succession. Meanwhile, in human CYP51 it lacks helical hydrogen bonds leaving loops in dynamic phases that could be inhibited via Van der Waals interactions with residues [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\n \u003cp\u003eFrom this point, modeling studies were performed on both human and non-human cytochrome sterol 14-alpha demethylase to compare the binding interactions of the 15 novel compounds and reference \u003cstrong\u003eVFV\u003c/strong\u003e, also analyze their interactions difference on both enzyme strains. The crystal structure of human sterol 14-alpha demethylase (CYP51) in complex with \u003cstrong\u003eVFV\u003c/strong\u003e in P1 space group was downloaded from protein data bank pdb with code: \u003cstrong\u003e(4UHL)\u003c/strong\u003e [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e]. While, the crystal structure of cytochrome P450\u0026ndash;14 alpha-sterol demethylase (CYP51) from Mycobacterium tuberculosis was downloaded with pdb code: \u003cstrong\u003e(1EA1)\u003c/strong\u003e [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e]. Enzymes were prepared for docking while; compounds together with \u003cstrong\u003eVFV\u003c/strong\u003e were simulated in dynamic phases. Docking protocol was applied using \u003cstrong\u003eMOEv.2010\u003c/strong\u003e and interaction scores were obtained in Kcal/mol and displayed in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eDocking score results of both enzymes \u003cstrong\u003e(4UHL) \u0026amp; (1EA1)\u003c/strong\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLigands\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eScore (Kcal/mol) against 4UHL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eScore (Kcal/mol) against 1EA1\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eVFV (Reference lead)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldUnderline\" name=\"Emphasis\" type=\"BoldUnderline\"\u003e-14.62\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cspan class=\"BoldUnderline\" name=\"Emphasis\" type=\"BoldUnderline\"\u003e-14.35\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e2a\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2b\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2c\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2d\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e2e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-11.21\u003c/p\u003e\n \u003cp\u003e-12.11\u003c/p\u003e\n \u003cp\u003e-11.74\u003c/p\u003e\n \u003cp\u003e-13.79\u003c/p\u003e\n \u003cp\u003e-11.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-10.77\u003c/p\u003e\n \u003cp\u003e-12.25\u003c/p\u003e\n \u003cp\u003e-11.87\u003c/p\u003e\n \u003cp\u003e-12.79\u003c/p\u003e\n \u003cp\u003e-11.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e3a\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3b\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3c\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3d\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-10.61\u003c/p\u003e\n \u003cp\u003e-12.09\u003c/p\u003e\n \u003cp\u003e-11.31\u003c/p\u003e\n \u003cp\u003e-13.98\u003c/p\u003e\n \u003cp\u003e-11.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-10.52\u003c/p\u003e\n \u003cp\u003e-11.68\u003c/p\u003e\n \u003cp\u003e-10.39\u003c/p\u003e\n \u003cp\u003e-13.82\u003c/p\u003e\n \u003cp\u003e-12.27\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e4a\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e4b\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e4c\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e4d\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e4e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-12.32\u003c/p\u003e\n \u003cp\u003e-13.30\u003c/p\u003e\n \u003cp\u003e-12.78\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"BoldUnderline\" name=\"Emphasis\" type=\"BoldUnderline\"\u003e-15.62\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e-12.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-12.44\u003c/p\u003e\n \u003cp\u003e-13.82\u003c/p\u003e\n \u003cp\u003e-12.88\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"BoldUnderline\" name=\"Emphasis\" type=\"BoldUnderline\"\u003e-15.08\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003e-13.68\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003eFrom the scores above, it was obvious that interaction energies were comparable with that of \u003cstrong\u003eVFV\u003c/strong\u003e especially series \u003cstrong\u003e4(a-e)\u003c/strong\u003e with even higher scores for compound \u003cstrong\u003e4d\u003c/strong\u003e to both enzymes. Also, from the scores and interactions of 3D and 2D diagrams presented in \u003cstrong\u003eFig.\u0026nbsp;(4\u0026ndash;9)\u003c/strong\u003e within channels, it was obvious that the volume of the occupying inhibitor is the contributing key factor in displaying inhibition to enzyme either human or non-human type. It was also reported in literature that (type I) substrate binding reflect low-to-high spin transition of the ferric P450 heme iron as due to displacement of the heme-coordinated water molecule towards the pouch while (type II) inhibitor binding reflect coordination of the basic heterocyclic nitrogen to the P450 heme iron towards the tail [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e]. Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e showed comparable occupation of helix-1 cavity of human enzyme by heme, \u003cstrong\u003eVFV\u003c/strong\u003e and compound \u003cstrong\u003e4d\u003c/strong\u003e respectively where, \u003cstrong\u003e4d\u003c/strong\u003e occupied the whole pouch of cavity together with the tail while \u003cstrong\u003eVFV\u003c/strong\u003e occupied the tail only and the heme-iron occupied the pouch. This stands up greatly for the fact that the benzamide group attached strategically to the novel thiazolo-imidazole derivatives occupied the large capacity of helix together with the trimethoxy group which were bulky enough to fill the space cavity. Also, this occupation displayed by\u0026nbsp;\u003cstrong\u003e4d\u003c/strong\u003e especially in the pouch clearly explains the reason that the novel synthesized compounds showed (type I) substrate binding shift in the absorbance assay rather than (type II) displayed mostly by azole inhibitors that occupy the tail.\u003c/p\u003e\n \u003cp\u003eAs for Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e \u0026amp; \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD interactions were displayed to compare the key interaction residues with heme \u0026amp;\u003cstrong\u003eVFV\u003c/strong\u003e in the cavity to that with \u003cstrong\u003e4d\u003c/strong\u003e. Heme showed metal contact of iron with Cys 449, arene-hydrogen Van der Waals interaction with Ala 311 and side chain acceptor for Lys 156 while, \u003cstrong\u003eVFV\u003c/strong\u003e displayed receptor contact and polar interactions with Gly 451, Phe 442 and Thr 315 as in reference literature for both heme \u0026amp;\u003cstrong\u003eVFV\u003c/strong\u003e [\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e], together with extra arene-hydrogen Van der Waals interaction with Gly 307 and His 236. On the other hand, \u003cstrong\u003e4d\u003c/strong\u003e was able to occupy the whole cavity tightly with receptor contact, polar and basic interactions with Cys 449, Thr 315, Ala 311 \u0026amp; Lys 156. Also, it revealed tight binding via arene-hydrogen Van der Waals interactions with Gly 451 \u0026amp; Phe 442 from both opposite sides. Figure \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e showed the interaction of compound \u003cstrong\u003e3a\u003c/strong\u003e with the enzyme \u003cstrong\u003e(4UHL)\u003c/strong\u003e which reflected the reason behind the low activity of this series of compounds, where it didn\u0026apos;t occupy the whole cavity and there were hollow distances in between the opposite sides of the channel.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e presented \u003cstrong\u003eVFV\u003c/strong\u003e and compound \u003cstrong\u003e4d\u003c/strong\u003e in the stacked helix of non-human (CYP51) \u003cstrong\u003e(1EA1)\u003c/strong\u003e, where we can find that \u003cstrong\u003e4d\u003c/strong\u003e is mainly in the pouch region of the channel rather than the tail. Figure \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e revealed clearly the hydrophobic 2D interactions of \u003cstrong\u003eVFV\u003c/strong\u003e with Cys 394, Pro 386, Leu 324, Leu 321, Pro 320 and Ala 256while, compound \u003cstrong\u003e4d\u003c/strong\u003e showed the same hydrophobic interactions with extra deeper hydrogen bonds in the channel with Thr 260 and Gln 72.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eThe widely known anticancer activity of benzimidazoles encouraged the synthesis of new three series of thiazolo-benzimidazole derivatives \u003cb\u003e(2a-e\u003c/b\u003e), \u003cb\u003e(3a-e) \u0026amp; (4a-e)\u003c/b\u003e with five different strategic groups (phenyl, hydroxyphenyl, chlorophenyl, trimethoxyphenyl \u0026amp; furo) attached to thiazole ring. Compounds \u003cb\u003e(2a-e)\u003c/b\u003e are characterized by nitro group being attached to benzimidazole ring, \u003cb\u003e(3a-e)\u003c/b\u003e characterized by amino while, \u003cb\u003e(4a-e)\u003c/b\u003e characterized by benzamide group. The \u003cem\u003ein vitro\u003c/em\u003e CYP51\u003csub\u003emtb\u003c/sub\u003e spectroscopic absorbance assay that was carried out showed that compounds exhibited mild shift indicating (type I) substrate binding with better binding exhibited by \u003cb\u003e(4a-e)\u003c/b\u003e series than the other two series. Molecular modeling studies were then performed to explain more the binding mode of such compounds to (CYP51) using \u003cb\u003eVFV\u003c/b\u003e as reference ligand. Two types of (CYP51) enzymes were downloaded from pdb (human \u003cb\u003e(4UHL)\u003c/b\u003e \u0026amp; non-human \u003cb\u003e(1EA1)).\u003c/b\u003e The \u003cb\u003e(4a-e)\u003c/b\u003e series displayed also comparable interaction scores to reference compound better than the other two series, with highest score attributed to compound \u003cb\u003e4d\u003c/b\u003e. Helix-1 channel; the active site of both enzymes is constituted of two strategic regions; pouch which is occupied by heme while the tail region that mostly occupied by famous azole inhibitors of (type II) and \u003cb\u003eVFV\u003c/b\u003e. \u003cb\u003e(2a-e) \u0026amp; (3a-e)\u003c/b\u003e series were not fully occupying the channel, while series of \u003cb\u003e(4a-e)\u003c/b\u003e which is characterized by large structural volumes occupied most of the channel as a tight belt specifically the pouch region and part of the tail, hence, provide its inhibitory activity to channel via (type I) substrate binding. The compound \u003cb\u003e4d\u003c/b\u003e synthesized with trimethoxyphenyl group attached to thiazole ring was recognized for its best score as well as binding mode. Further, \u003cem\u003ein vitro\u003c/em\u003e \u003cb\u003eSW620\u003c/b\u003e colorectal adenocarcinoma cell lines assay was performed, and all 15 compounds revealed noticeable chemotherapeutic activities with IC\u003csub\u003e50\u003c/sub\u003e in the range of 0.35\u0026ndash;0.03 \u0026micro;M compared to \u003cb\u003eDOX\u003c/b\u003e with IC\u003csub\u003e50\u003c/sub\u003e of 0.07\u0026micro;M where, the highest activity was also accredited to \u003cb\u003e(4a-e)\u003c/b\u003e series especially compounds \u003cb\u003e4d\u003c/b\u003e\u0026amp;\u003cb\u003e4b\u003c/b\u003e.\u003c/p\u003e"},{"header":"4. Experimental","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Chemistry\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e4.1.1 General\u003c/h2\u003e \u003cp\u003eAll melting points were determined on a Gallenkamp apparatus and are uncorrected. The progression of the reactions was monitored using TLC Merck Kieselgel 60 F254 aluminum packed plates. The IR spectra were measured on a Pye-UnicamSP300 instrument in potassium bromide discs. Bruker High performance Digital FT-NMR spectrometer Avance III 400 MHz was utilized for \u003csup\u003e1\u003c/sup\u003eH-NMR \u0026amp; 100 MHz for \u003csup\u003e13\u003c/sup\u003eC-NMR Faculty of Pharmacy Mansoura University. Mass spectra were run on a MAT Finnigan SSQ 7000 spectrometer, using the electron impact technique (EI).\u003c/p\u003e \u003cp\u003eInitially, modification of Nishiguchi\u0026rsquo;s method procedure was applied to prepare NaHSO4-SiO2 Catalyst needed for further reactions. To a 100-mL beaker, 4.14 g (0.03 mol) of NaHSO\u003csub\u003e4\u003c/sub\u003e.H\u003csub\u003e2\u003c/sub\u003eO was dissolved in 20 mL of distilled water and 10 g of SiO\u003csub\u003e2\u003c/sub\u003e (column chromatographic grade, 60 \u0026Aring;, 200\u0026ndash;400 mesh) was added. The solution was stirred for 15 min and was heated gently on a hot plate, with alternating shaker until a free-flowing white solid was produced. Further, the beaker was allowed to stay in oven to dry thoroughly at 120\u0026deg;C for at 48 h before use [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e4.1.2. General procedure for the Synthesis of novel 1-aryl-1H, 3H-thiazolo[3,4-a]-6-nitro-benzimidazole derivatives (2a-e)\u003c/h2\u003e \u003cp\u003eTo a one pot reaction, (4 mmol) of 4-nitro-o-phenylenediamine was dissolved in MeCN (12 mL) and equivalent amount of 2-mercaptoacetic acid (4 mmol) together with (4 mmol) of aromatic aldehydes were added and allowed to stir in presence of (800 mg) of the previously prepared catalyst; NaHSO\u003csub\u003e4\u003c/sub\u003e-SiO\u003csub\u003e2\u003c/sub\u003e at 75\u0026deg;C for 1 h. The reaction mixture was then cooled at room temperature and washed several times with adequate amount of methylene chloride [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The catalyst was recovered and concentrated under reduced then purified using column chromatography over silica gel with eluent (hexane: dichloromethane (7:3) and finally recrystallized using ethanol.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1-(Phenyl)-1H, 3H-thiazolo[3,4-a]-6-nitro-benzimidazole (2a)\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6): 6.55\u0026ndash;6.57 (d, 2H, \u003cem\u003eJ\u0026thinsp;=\u0026thinsp;8Hz\u003c/em\u003e, CH\u003csub\u003e2\u003c/sub\u003e-S), 7.45 (s, 1H, N-CH-S), 7.47\u0026ndash;7.48 (d, 1H, Ar-H), 7.59\u0026ndash;7.60 (t, 1H, Ar-H), 7.62 (s, 1H, Ar-H), 7.641\u0026ndash;7.648 (t, 1H, Ar-H), 7.79\u0026ndash;7.81 (d, 1H, Ar-H), 8.14\u0026ndash;8.17 (dd, 1H, Ar-H), 8.23\u0026ndash;8.25 (t, 1H, Ar-H), 8.514\u0026ndash;8.519 (d, 1H, Ar-H) ppm; IR (KBr): 3090, 2988 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; MS:(M\u003csup\u003e+\u003c/sup\u003e1): 279.06; yield 89%.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1-(4-Hydroxyphenyl)-1H,3H-thiazolo[3,4-a]-6-nitrobenzimidazole (2b)\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6): 5.61 (s, 1H, OH (exchangeable peak)), 6.53\u0026ndash;6.55 (d, 2H, \u003cem\u003eJ\u0026thinsp;=\u0026thinsp;8Hz\u003c/em\u003e, CH\u003csub\u003e2\u003c/sub\u003e-S), 6.66\u0026ndash;6.69 (d, 1H, Ar-H), 6.92 (s, 1H, N-CH-S), 7.41\u0026ndash;7.45 (d, 1H, Ar-H), 7.70\u0026ndash;7.73 (d, 1H, Ar-H), 8.05\u0026ndash;8.09 (d, 1H, Ar-H), 8.10 (s, 1H, Ar-H), 8.11\u0026ndash;8.15 (dd, 1H, Ar-H), 8.41\u0026ndash;8.45 (d, 1H, Ar-H) ppm; IR (KBr): 3280\u0026thinsp;\u0026minus;\u0026thinsp;2850, 3030, 2920 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; MS:(M\u003csup\u003e+\u003c/sup\u003e1): 313.05; yield 92%.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1-(4-Chlorophenyl)-1H, 3H-thiazolo[3,4-a]-6-nitro-benzimidazole (2c)\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6): 6.52\u0026ndash;6.54 (d, 2H, \u003cem\u003eJ\u0026thinsp;=\u0026thinsp;8Hz\u003c/em\u003e, CH\u003csub\u003e2\u003c/sub\u003e-S), 7.40\u0026ndash;7.41 (d, 1H, Ar-H), 7.419 (s, 1H, N-CH-S), 7.442\u0026ndash;7.447 (d, 1H, Ar-H), 7.67\u0026ndash;7.69 (d, 1H, Ar-H), 7.77\u0026ndash;7.80 (d, 1H, Ar-H), 8.13\u0026ndash;8.15 (dd, 1H, Ar-H), 8.22\u0026ndash;8.24 (d, 1H, Ar-H), 8.49 (s, 1H, Ar-H) ppm; IR (KBr): 3060, 2962 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; MS: (M\u003csup\u003e+\u003c/sup\u003e2): 333.02, (M\u003csup\u003e+\u003c/sup\u003e1): 331.02; yield 93%.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1-(4-Chlorophenyl)-1H, 3H-thiazolo[3,4-a]-6-nitro-benzimidazole (2d)\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6): 3.79 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.87 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.91 (s, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 6.98 (s, 1H, CH\u003csub\u003e2\u003c/sub\u003e-S), 7.11 (s, 1H, CH\u003csub\u003e2\u003c/sub\u003e-S), 7.23 (s, 1H, N-CH-S), 7.54\u0026ndash;7.56 (d, 1H, Ar-H), 7.58\u0026ndash;7.59 (d, 1H, Ar-H), 8.13 (s, 1H, Ar-H), 8.15 (s, 1H, Ar-H), 8.18 (s, 1H, Ar-H), 8.57 (s, 1H, Ar-H) ppm; IR (KBr): 3075, 2925 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; MS: (M\u003csup\u003e+\u003c/sup\u003e1): 387.09; yield 95%.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1-(2-Furo)-1H, 3H-thiazolo[3,4-a]-6-nitro-benzimidazole (2e)\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6): 6.51\u0026ndash;6.54 (dd, 2H, \u003cem\u003eJ\u0026thinsp;=\u0026thinsp;8Hz\u003c/em\u003e, CH\u003csub\u003e2\u003c/sub\u003e-S), 7.38 (s, 1H, Ar-H), 7.39 (s, 1H, N-CH-S), 7.40\u0026ndash;7.41 (d, 1H, Ar-H), 7.418 (s, 1H, Ar-H), 7.425\u0026ndash;7.427 (d, 1H, Ar-H), 7.43 (s, 1H, Ar-H), 7.44 (s, 1H, Ar-H) ppm; IR (KBr): 3110, 3090, 2985 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; MS: (M\u003csup\u003e+\u003c/sup\u003e1): 287.04; yield 89%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e4.1.3. General procedure for the synthesis of 1-aryl-1H, 3H-thiazolo[3,4-a]-6-amino-benzimidazole derivatives (3a-e)\u003c/h2\u003e \u003cp\u003eThe solution of synthesized candidates \u003cb\u003e(2a-e)\u003c/b\u003e (3 mmol) in ethanol was allowed to react with H\u003csub\u003e2\u003c/sub\u003e/Pd over C for 2 h with stirring at room temperature adopting a reported procedure of catalyzed reduction \u003csup\u003e[28]\u003c/sup\u003e followed by filtration and crystallization of the yielded compounds from ethanol and methylene chloride. The obtained products were directly headed to the following reaction to yield the desired final products \u003cb\u003e(4a-e)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e4.1.4. General procedure for the synthesis of 1-aryl-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole derivatives (4a-e)\u003c/h2\u003e \u003cp\u003eThe final targets were obtained via acylation reaction, where (3 mmol) of the previously prepared intermediates \u003cb\u003e(3a-e)\u003c/b\u003e was allowed to stir in a rounded flask surrounded by ice with 15 mL methylene chloride. An equivalent amount of TEA (3 mmol) was slowly added to the stirring solution followed by dropwise addition of benzoyl chloride (5 mmol). The mixture was refluxed for further 1.5 h and the resultant was purified and washed with methylene chloride and ether then dried under vacuum. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1-(Phenyl)-1H 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole (4a)\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6): 7.32\u0026ndash;7.34 (d, 2H, \u003cem\u003eJ\u0026thinsp;=\u0026thinsp;8Hz\u003c/em\u003e, CH\u003csub\u003e2\u003c/sub\u003e-S), 7.45 (s, 1H, N-CH-S), 7.47\u0026ndash;7.48 (d, 1H, Ar-H), 7.51\u0026ndash;7.53 (t, 1H, Ar-H), 7.59\u0026ndash;7.60 (t, 1H, Ar-H), 7.62 (s, 1H, Ar-H), 7.641\u0026ndash;7.648 (t, 1H, Ar-H), 7.79\u0026ndash;7.81 (d, 1H, Ar-H), 7.82\u0026ndash;7.84 (t, 1H, Ar-H), (7.95\u0026ndash;8.03 (d, 2H, Ar-H), 8.14\u0026ndash;8.17 (dd, 1H, Ar-H), 8.23\u0026ndash;8.25 (t, 1H, Ar-H), 8.34\u0026ndash;8.36 ( t, 1H, Ar-H), 8.514\u0026ndash;8.519 (d, 1H, Ar-H), 10.45 (s, 1H, NH(exchangeable peak)) ppm; \u003csup\u003e13\u003c/sup\u003eC-NMR (DMSO-d6): 39.3, 57.8, 128.9, 129.1, 129.6, 130.1, 130.2, 131.2, 133.4, 134.1, 167.7 ppm; IR (KBr): 3220, 3090, 2988 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; MS:(M\u003csup\u003e+\u003c/sup\u003e1): 371.11; yield 72%.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1-(4-Hydroxyphenyl)-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole (4b)\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6): 5.61 (s, 1H, OH (exchangeable peak)), 6.53\u0026ndash;6.55 (d, 2H, \u003cem\u003eJ\u0026thinsp;=\u0026thinsp;8hz\u003c/em\u003e, CH\u003csub\u003e2\u003c/sub\u003e-S), 6.66\u0026ndash;6.69 (t, 1H, Ar-H), 6.84\u0026ndash;6.87 (d, 1H, Ar-H), 6.92 (s, 1H, N-CH-), 6.94\u0026ndash;6.97 (t, 1H, Ar-H), 7.41\u0026ndash;7.42 (d, 1H, Ar-H), 7.45 (s, 1H, Ar-H), 7.64\u0026ndash;7.73 (dd, 2H, \u003cem\u003eJ\u0026thinsp;=\u0026thinsp;8Hz\u003c/em\u003e, Ar-H), 7.83\u0026ndash;7.85 (d, 1H, Ar-H), 8.05\u0026ndash;8.10 (t, 1H, Ar-H), 8.11\u0026ndash;8.12 (d, 1H, Ar-H), 8.13\u0026ndash;8.15 (d, 1H, Ar-H), 8.41\u0026ndash;8.45 (d, 1H, Ar-H) ppm; 10.23 (s, 1H, NH(exchangeable peak)) ppm; \u003csup\u003e13\u003c/sup\u003eC-NMR (DMSO-d6): 39.2, 57.9, 128.9, 129.1, 129.6, 130.1, 130.2, 131.2, 133.4, 132.1, 134.1, 139.7, 156.2, 167.7 ppm; IR (KBr): 3288, 3280\u0026thinsp;\u0026minus;\u0026thinsp;2850, 3030, 2920 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; MS:(M\u003csup\u003e+\u003c/sup\u003e1): 387.1; yield 67%.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1-(4-Chlorophenyl)-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole (4c)\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6): 7.49\u0026ndash;7.53 (t, 1H, Ar-H), 7.56\u0026ndash;7.59 (t, 1H, Ar-H), 7.62\u0026ndash;7.66 (t, 1H, Ar-H), 7.80\u0026ndash;7.81 (d, 2H, \u003cem\u003eJ\u0026thinsp;=\u0026thinsp;6Hz\u003c/em\u003e, CH\u003csub\u003e2\u003c/sub\u003e-S), 7.83 (s, 1H, N-CH-S), 7.94\u0026ndash;7.97 (dd, \u003cem\u003eJ\u0026thinsp;=\u0026thinsp;8Hz\u003c/em\u003e, 4H, Ar-H), 8.01\u0026ndash;8.03 (dd, 2H, Ar-H), 8.27\u0026ndash;8.29 (d, 2H, Ar-H), 10.60 (s, 1H, NH(exchangeable peak)) ppm; \u003csup\u003e13\u003c/sup\u003eC-NMR (DMSO-d6): 39.33, 57.69, 128.22, 128.95, 129.06, 129.74, 130.14, 131.19, 133.37, 167.80 ppm; IR (KBr): 3255, 3060, 2962 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; MS: (M\u003csup\u003e+\u003c/sup\u003e2): 407.07, (M\u003csup\u003e+\u003c/sup\u003e1): 405.07; yield 75%.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1-(4-Chlorophenyl)-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole (4d)\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6): 3.02\u0026ndash;3.03 (m, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3.05\u0026ndash;3.06 (m, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 3..07-3.08 (m, 3H, OCH\u003csub\u003e3\u003c/sub\u003e), 7.49 (s, 1H, CH\u003csub\u003e2\u003c/sub\u003e-S), 7.50 (s, 1H, CH\u003csub\u003e2\u003c/sub\u003e-S), 7.51 (s, 2H, Ar-H), 7.53 (s, 1H, N-CH-S), 7.61\u0026ndash;7.62 (t, 1H, Ar-H), 7.62\u0026ndash;7.65 (t, 1H, Ar-H), 7.65\u0026ndash;7.66 (t, 1H, Ar-H), 7.946\u0026ndash;7.949 (d, 4H, Ar-H), 7.96 (s, 1H, Ar-H), 10.62 (s, 1H, NH (exchangeable peak)) ppm; \u003csup\u003e13\u003c/sup\u003eC-NMR (DMSO-d6): 8.853, 39.33, 45.67, 129.06, 129.73, 131.20, 133.36, 167.77 ppm;IR (KBr): 3290, 3075, 2925 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; MS: (M\u003csup\u003e+\u003c/sup\u003e1): 461.14; yield 84%\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003e1-(2-Furo)-1H, 3H-thiazolo[3,4-a]-6-benzamide-benzimidazole (4e)\u003c/span\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH-NMR (DMSO-d6): 5.17 (s, 1H, CH\u003csub\u003e2\u003c/sub\u003e-S), 5.32 (s, 1H, CH\u003csub\u003e2\u003c/sub\u003e-S), 7.04 (s, 1H, Ar-H), 7.16 (s, 1H, Ar-H), 7.29 (s, 1H, Ar-H), 7.59 (s, 1H, N-CH-S), 7.61 (t, 1H, Ar-H), 7.62 (d, 2H, Ar-H), 7.70 (t, 1H, Ar-H), 7.71 (t, 1H, Ar-H), 8.07 (s, 1H, Ar-H), 8.09 (d, 2H, Ar-H), 8.95 (s, 1H, NH (exchangeable peak)) ppm; \u003csup\u003e13\u003c/sup\u003eC-NMR (DMSO-d6): 39.7, 59.8, 128.9, 129.1, 129.6, 130.1,, 131.2, 133.4, 132.1, 139.7, 156.2, 167.7 ppm; IR (KBr): 3280, 3110, 3090, 2985 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; MS: (M\u003csup\u003e+\u003c/sup\u003e1): 361.09; yield 80%.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Biological Assays\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e4.2.1. CYP51\u003csub\u003eMTb\u003c/sub\u003e enzyme production and purification\u003c/h2\u003e \u003cp\u003eThe pET26 plasmid containing CYP51 gene was transformed into BL21(DE3) competent \u003cem\u003eE. coli\u003c/em\u003e and plated onto LBkan/LBkancm plates and left overnight. Colonies were harvested and cultured at 37\u0026deg;C in 8x600 mL of LB for approximately 16 hours at 120 rpm. The incubator temperature was lowered to 19\u0026deg;C, followed by the addition of IPTG (0.1 mM), Delta-ALA (10mg/flask) and further incubation at this temperature for 16 hours at 120 rpm. The cells were harvested by centrifugation (5,000 \u003cem\u003eg\u003c/em\u003e, 15 min) and resuspended in 200 mL of 50 mM Tris, pH 7.4, containing 1 mM DTT. The resuspended cells were kept on ice and lysed by sonication (25 cycles at 20:40 seconds on:off, 70%, 19 mm probe, Sonics Vibra-Cell). The supernatant, containing the desired protein, was isolated from cell debris by centrifugation (40,000 \u003cem\u003eg\u003c/em\u003e, 30 min). The protein was then loaded onto a DEAE Sepharose column, (XK50, 200 mm x 40 mm, GE Healthcare) and eluted using a linear salt gradient of 100 mM to 400 mM KClin50 mM Tris, pH 7.4. The fractions containing the desired protein (identified by red color of the P450 enzymes) were combined and the volume reduced using a Vivacell 100 (Sartorius Stedim, 10 kD membrane) aided by centrifugation (1,500 \u003cem\u003eg\u003c/em\u003e). The protein was desalted using a Sephadex G-25 medium grain column (250 mm x 40 mm) eluted with 50 mM Tris, pH 7.4. The desalted protein was then further purified by loading it onto a Source-Q ion-exchange column (XK26, 80 mm x 30 mm, GE Healthcare) and eluted with a gradient of 0 to 750 mM KCl in 50 mM Tris, pH 7.4. Fractions with the highest \u003cem\u003eA\u003c/em\u003e\u003csub\u003e420\u003c/sub\u003e/\u003cem\u003eA\u003c/em\u003e\u003csub\u003e280\u003c/sub\u003e ratio were retained. The protein was concentrated via ultrafiltration and an equal volume of 80% glycerol was added before filtration through a 0.22 \u0026micro;M syringe filter and storage at -20\u0026deg;C. The final \u003cem\u003eA\u003c/em\u003e\u003csub\u003e420\u003c/sub\u003e/\u003cem\u003eA\u003c/em\u003e\u003csub\u003e280\u003c/sub\u003e ratio was 1.1for CYP51\u003csub\u003eMTb\u003c/sub\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e4.2.2. Spin state shift assays\u003c/h2\u003e \u003cp\u003eGlycerol in stored protein samples was removed via buffer exchange into Buffer T (50mm Tris pH 7.5 with 1 mM DTT, (dithiothreitol)) using a PD-10 desalting column (GE Healthcare). The P450 was diluted to approximately 1 \u0026micro;M using the same buffer and the spectrum was recorded on a UV/Vis spectrophotometer. Aliquots (1 to 2 \u0026micro;L) of various substrates/inhibitors \u003cb\u003e(2a-e), (3a-e)\u003c/b\u003e and \u003cb\u003e(4a-e)\u003c/b\u003e stock solutions in DMSO or EtOH (50 mM \u0026minus;\u0026thinsp;100 mM) were added. The absorbance between 600 nm and 250 nm recorded using a UV-Visible spectrophotometer until no further spectral change is observed. The high spin percentage (\u0026plusmn;\u0026thinsp;5%) was estimated by comparison to a set of spectra, generated by the sum of substrate-free (\u0026gt;\u0026thinsp;95% low spin) and substrate-bound (camphor, \u0026ge;\u0026thinsp;95% high spin) CYP101A1 to the appropriate percentages. CYP51\u003csub\u003eMTb\u003c/sub\u003e showed low spin state in the resting ferric form at 418 nm [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e4.2.3. \u003cem\u003eIn vitro\u003c/em\u003e cell lines assay\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e analysis was carried out by testing the effect of the compounds on \u003cb\u003eSW620\u003c/b\u003e colorectal adenocarcinoma cell lines, (cells were obtained from Nawah Scientific., Inc (Mokatam, Cairo, Egypt). Cells were maintained in DMEM media supplemented with 100mg/ml of streptomycin, 100 units/ml of penicillin and 10%of heat inactivated fetal bovine serum in humidified, 5% (v/v) CO2 atmosphere at 37\u0026deg;C. Cell viability was assessed by SRB assay. Seeding was done using aliquots of 100 \u0026micro;l cell suspension (5x10^3cells) which were added to 96-well plates. Then, cells were treated with the compounds at various concentrations (0.1,1,10,100,1000 ug/ml). After 72h of drug exposure, SRB assay cells were fixed by replacing media with 150 \u0026micro;L of 10% trichloroacetic acid (TCA) and incubated at 4\u0026deg;C for 1h. The TCA solution was removed, and the cells were washed 5 times with distilled water. Aliquots of 70 \u0026micro;L SRB solution (0.4%w/v) were added and incubated in a dark place at room temperature for 10 min. Plates were washed 3 times with 1% acetic acid and allowed to air-dry overnight. Then, 150 \u0026micro;L of TRIS (10mM) was added to dissolve protein-bound SRB stain for 5 min on a shaker at 1,600 rpm. The absorbance was then measured at 540 nm using a BMGLABTECH\u0026reg;-FLUO star Omega micro-plate reader (Ortenberg, Germany) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Computational Studies\u003c/h2\u003e \u003cp\u003eThe molecular modeling studies were applied using MOEv.2010 -where the heme portion can be readable- following reported procedures to perform the addressed research. Results and 2D interactions were viewed using also MOEv.2010, while the 3D interactions were presented using Discovery Studio 4.1 client.\u003c/p\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e4.3.1. Enzymes Preparation\u003c/h2\u003e \u003cp\u003eInitially, the crystal structures of sterol cytochrome 14-alpha demethylase (CYP51) were downloaded from pdb under codes: \u003cb\u003e(4UHL)\u003c/b\u003e for the human source one complexed with \u003cb\u003eVFV\u003c/b\u003e [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and \u003cb\u003e(1EA1)\u003c/b\u003e for the one with the source from mycobacterium tuberculosis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Add hydrogens was applied to the downloaded enzymes followed by automatic connect and type step which was applied to all atoms and bonds. Receptor was identified, selected, and potentially fixed. The unneeded sequential chains and ligands were removed meanwhile, the interaction site (pocket) was identified and selected together with the required complexed ligand and heme position in the channel [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e4.3.2. Dynamics Simulation\u003c/h2\u003e \u003cp\u003eThe 15 designed and synthesized compounds' structures were built up using ChemDraw Ultra 8.0 and saved as readable (.mol) files. Connect and Type was then applied automatically to all the compounds together with the reference ligand \u003cb\u003eVFV.\u003c/b\u003e Dynamic simulation was then performed using start point zero-till 250 checkpoints, velocity was applied, and time was set at (0.5s). Then, the forcefield applied was set as MMFF94x while, bonded, Van der Waals, electrostatics and restraints were all enabled, cut \u0026ndash;offs were set at: on-8, off-10 and distance salvation was set at 1. Hydrogens and all partial charges were fixed, and the protocol used was NPA (Nose\u0026ndash;Poincare\u0026ndash;Andersen\u0026ndash;Hamiltonian equations of motion). 510 different dynamic conformations were resulted for each compound that were further used in the docking study [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e4.3.3. Molecular Docking\u003c/h2\u003e \u003cp\u003eDocking study was finally computed for each enzyme separately with the dynamic molecules of each 15 compounds and the reference \u003cb\u003eVFV\u003c/b\u003e, using placement: Alpha Triangle and rescoring 1: London dG with None refinement, None rescoring 2, and retaining scores resulted were 5010 for each compound due to the huge dynamic conformations possibilities. Scores were then sorted in descending and the best score pose was chosen to view its interaction against the required enzyme [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful to Professor Nasser S.M. Ismail, for his great collaboration in this study also would like to express sincere thanks to Future University in Egypt for providing CADD Lab facility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003ch3\u003eConflict of interest\u003c/h3\u003e\n\u003cp\u003eWe declare that the authors have no competing interests as defined by Springer, or other interests that might be perceived to influence the results and/or discussion reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results/data/figures in this manuscript have not been published elsewhere, nor are they under consideration (from you or one of your Contributing Authors) by another publisher.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the material is owned by the authors and are available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProf. H.R. put the research ideas and revised the whole content. I.M., H.R., R.T. and A.M. did the experiments and characterizations, analyzed the results, and organized the writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003e1\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Future University in Egypt, 11835, Cairo, Egypt\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIten M. Fawzy \u003csup\u003e1\u003c/sup\u003e*, [email protected], Hanan M. Refaat\u003csup\u003e1\u003c/sup\u003e, [email protected], Asmaa A. Mandour \u003csup\u003e1\u003c/sup\u003e,\u0026nbsp;[email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003e2\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003eDepartment of Pharmacology and Toxicology and Biochemistry, Faculty of Pharmacy, Future University in Egypt, 11835, Cairo, Egypt\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eReem T. Attia\u003csup\u003e2\u003c/sup\u003e, [email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003e3\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003eDepartment of Chemistry, School of Physical Sciences, University of Adelaide, 5000 SA, Australia.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeba A. Ibrahim\u003csup\u003e3\u003c/sup\u003e, [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJ. Zhang, L. Li, Q. Lv, L. Yan, Y. Wang, Y. Jiang, Front. Microbiol.\u003cem\u003e \u003c/em\u003e10\u003cstrong\u003e (2019\u003c/strong\u003e) 691. 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DOI:https://doi.org/10.1074/jbc.M110.133215 \u003c/li\u003e\n\u003cli\u003eJ. Rivero, O.P. Luzardo, L.A. Henr\u0026iacute;quez-Hern\u0026aacute;ndez, R.P. Mach\u0026iacute;n, J. Pestano, M. Zumbado, L.D. Boada, M. Camacho, P.F. Valer\u0026oacute;n, Sci. Total Environ. 537\u003cstrong\u003e (2015\u003c/strong\u003e) 197-202. https://doi.org/10.1016/j.scitotenv.2015.08.016 \u003c/li\u003e\n\u003cli\u003eS.A. Fahmy, I.M. Fawzy, B.M. Saleh, M.Y. Issa, U. Bakowsky, H.M. Azzazy, J. Nanomater.\u003cem\u003e \u003c/em\u003e(4) (\u003cstrong\u003e2021\u003c/strong\u003e) 965. https://doi.org/10.3390/nano11040965\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Aryl-Thiazolo-Benzimidazoles, Anticancer activity, CYP51MTb, Human Colon Cancer, Molecular Modeling","lastPublishedDoi":"10.21203/rs.3.rs-1727366/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1727366/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe prominent biological activity of polycyclic systems inspired the synthesis of novel 1-Aryl-thiazolo-benzimidazole Derivatives \u003cb\u003e(2a-e), (3a-e)\u003c/b\u003e and \u003cb\u003e(4a-e).\u003c/b\u003e \u003cem\u003eIn vitro\u003c/em\u003e antiproliferative assay was performed on colorectal adenocarcinoma \u003cb\u003e(SW620)\u003c/b\u003e and the novel compounds showed superior anticancer activity with IC\u003csub\u003e50\u003c/sub\u003e in the range of 0.03\u0026ndash;0.35 \u0026micro;M compared to reference compound Doxorubicin (DOX) with IC\u003csub\u003e50\u003c/sub\u003e (0.07 \u0026micro;M). The ability of the new molecules to interact with extracted cytochrome \u003cb\u003eCYP51M\u003c/b\u003e\u003csub\u003e\u003cb\u003eTb\u003c/b\u003e\u003c/sub\u003e was also studied using absorption spectroscopy. Results showed mild type I spectral shift especially compound \u003cb\u003e4d\u003c/b\u003e displayed 3% shift in the spin state assay referring to the presence of electron withdrawing groups attached to benzene ring, thus that could be a promising nucleus for better cytochrome inhibition. The results were supported by molecular modeling studies to confirm general binding of the compounds to the hydrophobic region of the heme-protein. These outcomes encourage the new molecules to be candidates for new potential anticancer agents.\u003c/p\u003e","manuscriptTitle":"Synthesis, Molecular Modeling and Biological Studies of Novel 1-Aryl-Thiazolo Benzimidazole Derivatives as Cytochrome (CYP51 MTb ) Type I Inhibitors \u0026amp; Anticancer Agents","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-06-14 16:10:58","doi":"10.21203/rs.3.rs-1727366/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0579969f-2751-4433-965e-fd636f37cf10","owner":[],"postedDate":"June 14th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-06-27T09:44:25+00:00","versionOfRecord":[],"versionCreatedAt":"2022-06-14 16:10:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1727366","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1727366","identity":"rs-1727366","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}