Synthesis, Characterization and Biological Evaluation of New 3,5-Disubstituted-Pyrazoline Derivatives as Potential Anti-Mycobacterium tuberculosis H37Ra Compounds

A total of fourteen pyrazoline derivatives were synthesized through cyclo-condensation reactions by chalcone derivatives with different types of semicarbazide. These compounds were characterized by IR, 1D-NMR (1H, 13C and Distortionless Enhancement by Polarization Transfer - DEPT-135) and 2D-NMR (COSY, HSQC and HMBC) as well as mass spectroscopy analysis (HRMS). The synthesized compounds were tested for their antituberculosis activity against Mycobacterium tuberculosis H37Ra in vitro. Based on this activity, compound 4a showed the most potent inhibitory activity, with a minimum inhibitory concentration (MIC) value of 17 μM. In addition, six other synthesized compounds, 5a and 5c–5g, exhibited moderate activity, with MIC ranges between 60 μM to 140 μM. Compound 4a showed good bactericidal activity with a minimum bactericidal concentration (MBC) value of 34 μM against Mycobacterium tuberculosis H37Ra. Molecular docking studies for compound 4a on alpha-sterol demethylase was done to understand and explore ligand–receptor interactions, and to hypothesize potential refinements for the compound.


Introduction
Pyrazoline is a five-membered heterocyclic ring that has an endocyclic double bond with two adjacent nitrogen atoms at position 1-and 2-. The first pyrazoline (4,5-dihydro-1H-pyrazole) was synthesized by Knorr in 1883 [1]. Pyrazolines are noted for the stability of their ring system and the reactivity of several sites that permit a series of substitution reactions to take place. Structural modifications of pyrazolines can be achieved by decorating the stable fragments with different functional groups and aromatic scaffolds to create benzylhydrazine moieties for the development of new potent compounds possessing biological activities. Previous studies have reported considerable biological activities when substitution occurs at position 1-, 3-and 5-of the pyrazoline (Figure 1), such as anticonvulsant, antimalarial [2], anti-cardiovascular [3], anticancer [4], anti-amoebic [5], antimicrobial [6,7] and anti-tumor activities [8]. Meanwhile, carbothioamide derivatives were found to have significant pharmacological activities, such as monoamine oxidase (MAO) inhibitors [9,10], antituberculosis [11][12][13] and anticonvulsant activities [14][15][16]. Tuberculosis (TB) is an infectious disease cau that affects the lungs [17]. According to the W Global Tuberculosis Report, about 1.5 million peo TB died in 2018 [18]. In addition, WHO has decl the most affected with TB cases (44%), followed Eastern Mediterranean (8%), Americas (3%) and Ministry (2019) has reported about 1500 to 2000 d of six deaths a day in 2018 [19]. The increase in medical treatment and low TB awareness. In add ration impairs patient adherence to the TB medic highly concerning multi-drug and extensively-d underscores the importance of developing a new In continuation of our efforts to develop an synthesis of new pyrazoline derivatives, along wi M. tuberculosis H37Ra. The attenuated Mycobacte due to biosafety constraints with using virulent M However, Heinrichs et al. showed that the H37R tibility to antibiotics as the H37Rv strain and clin tion of avirulent M. tuberculosis for this study. M tochrome P450 14 alpha-sterol demethylase (CYP hibitory property of potent compounds within th Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) that affects the lungs [17]. According to the World Health Organization (WHO) 2019 Global Tuberculosis Report, about 1.5 million people from a total of 10 million people with TB died in 2018 [18]. In addition, WHO has declared that the South-East Asian region is the most affected with TB cases (44%), followed by Africa (24%), Western Pacific (18%), Eastern Mediterranean (8%), Americas (3%) and Europe (3%). In Malaysia, the Health Ministry (2019) has reported about 1500 to 2000 deaths from TB per year, with an average of six deaths a day in 2018 [19]. The increase in cases is mainly due to delays in seeking medical treatment and low TB awareness. In addition, the current lengthy treatment duration impairs patient adherence to the TB medications, which in turn fuels the spread of highly concerning multi-drug and extensively-drug resistant M. tuberculosis. Hence, this underscores the importance of developing a new effective anti-TB regimen.

Chemistry
In continuation of our efforts to develop antitubercular agents, here we report the synthesis of new pyrazoline derivatives, along with their potential anti-TB activity against M. tuberculosis H37Ra. The attenuated Mycobacterium tuberculosis H37Ra strain was used due to biosafety constraints with using virulent M. tuberculosis H37Rv and clinical strains. However, Heinrichs et al. showed that the H37Ra strain is able to equally predict susceptibility to antibiotics as the H37Rv strain and clinical isolates [20]. This justifies the selection of avirulent M. tuberculosis for this study. Molecular docking at the active site of cytochrome P450 14 alpha-sterol demethylase (CYP51) was performed to rationalize the inhibitory property of potent compounds within the active pocket.

Chemistry
The synthesis of the first series of 3,5-disubstituted-4,5-dihydro-N-phenyl-1H-pyrazole-1-carbothioamide (4a-g) was synthesized by the cyclo-condensation of chalcone derivatives (3a-g) with 4-phenyl-3-thiosemicarbazide in the presence of sodium hydroxide as a base (Scheme 1). Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) that affects the lungs [17]. According to the World Health Organization (WHO) 2019 Global Tuberculosis Report, about 1.5 million people from a total of 10 million people with TB died in 2018 [18]. In addition, WHO has declared that the South-East Asian region is the most affected with TB cases (44%), followed by Africa (24%), Western Pacific (18%), Eastern Mediterranean (8%), Americas (3%) and Europe (3%). In Malaysia, the Health Ministry (2019) has reported about 1500 to 2000 deaths from TB per year, with an average of six deaths a day in 2018 [19]. The increase in cases is mainly due to delays in seeking medical treatment and low TB awareness. In addition, the current lengthy treatment duration impairs patient adherence to the TB medications, which in turn fuels the spread of highly concerning multi-drug and extensively-drug resistant M. tuberculosis. Hence, this underscores the importance of developing a new effective anti-TB regimen.
In continuation of our efforts to develop antitubercular agents, here we report the synthesis of new pyrazoline derivatives, along with their potential anti-TB activity against M. tuberculosis H37Ra. The attenuated Mycobacterium tuberculosis H37Ra strain was used due to biosafety constraints with using virulent M. tuberculosis H37Rv and clinical strains. However, Heinrichs et al. showed that the H37Ra strain is able to equally predict susceptibility to antibiotics as the H37Rv strain and clinical isolates [20]. This justifies the selection of avirulent M. tuberculosis for this study. Molecular docking at the active site of cytochrome P450 14 alpha-sterol demethylase (CYP51) was performed to rationalize the inhibitory property of potent compounds within the active pocket.
Compound 4a is the representative compound from the first series. This new compound was synthesized by the cyclo-condensation of 3-(4-methylphenyl)-1-phenyl-2propen-1-one (3a) with 4-phenyl-3-thiosemicarbazide in the presence of sodium hydroxide as a base. Compound 4a formed as a cream-colored precipitate with 26% yield and a melting point of 133-136 • C. Compound 4a was characterized by various spectroscopic techniques including IR, 1D-NMR, 2D-NMR and HRMS. The important absorption bands in the IR spectrum of compound 4a were observed at 3291 cm −1 (N-H stretching), 1594 cm −1 (C=N stretching), 1520 cm −1 and 1449 cm −1 (C=C stretching of the aromatic), and 1399 cm −1 (C=S stretching). The absence of C=O and C=C bands, as well as the appearance of new C=N and C=S bands in the IR spectra of compound 4a suggests the complete formation of compound 4a via the cyclo-condensation reaction between chalcone 3a and 4-phenyl-3thiosemicarbazide (Scheme 1).
The 1 H-NMR spectrum of compound 4a showed that two germinal protons, H-4α and H-4β, of the methylene group resonated as a doublet of doublet at δ H 3.26 and δ H 4.05, respectively. The appearance of these two signals could be attributed to the nonequivalent nature of the two germinal protons with a J coupling constant of J 4α4β = 18.0 Hz, J 4α5 = 3.5 Hz and J 4β5 = 11.5 Hz. Meanwhile, the vicinal proton (H-5) also appeared as a doublet of doublet at a slightly downfield region, δ H 6.14, due to vicinal coupling with the two neighboring germinal protons of the methylene group at position 4-of the pyrazoline ring. A singlet at δ H 9.93 in the downfield region indicated the presence of a thioamide proton. In compound 4a, some aromatic protons such as H-3 , H-4 and H-5 were observed at the same chemical shift, δ H 7.49, due to the overlapping of peaks in the similar environment. The 1 H-NMR spectrum also showed three signals for ring C. A triplet at δ H 7.33 (2H, J = 7.5 Hz) was attributed to H-10 and H-12. In addition, two multiplets at δ H 7.72 (2H) and δ H 7.16 were assigned to H-9 with H-13 and H-11. The 13 C-NMR spectrum of carbothioamide compound 4a showed seventeen carbon signals. One carbon signal each at δ C 42.3 and δ C 62.3 suggested the presence of pyrazoline ring carbons, which were assigned to 4-CH 2 and 5-CH, respectively. Also, a signal at δ C 155.3 was attributed to 3-C=N in the pyrazoline ring, which is a carbon attached to an electronegative nitrogen by a double bond and also to a benzene ring. Besides, a signal at δ C 174.2 was attributed to 6-C=S. The 2D-NMR correlation of 1 H-1 H COSY and 1 H-13 C HMBC spectra were used to assign the aromatic signals, especially in the case of the pyrazoline ring. The analysis of 1 H-1 H COSY showed the correlation between of 14-CH 3 -H-4 -H-3 and H-5 , 4α-CH with 4β-CH and 5-CH, H-8 with H-9-H-10-H-11-H-12 and H-2 with H-1 -H-6 -H-3 , H-4 with H-5 ( Figure 2). The 1 H- 13 C HMBC spectrum of 4a shows the cross-correlations of 7-NH with carbons C-9 and C-13, and H-10 with C-9, C-13, C-11, C-12 and C-8 ( Figure 2), and the cross-correlation of 14-CH 3 with C-3 , C-5 and C-4 . The HRMS spectrum of 4a revealed the molecular ion peak [M + Na] + at m/z 394.1156, which is consistent with the molecular formula C 23 H 21 N 3 S (calcd. 394.1354). According to the above spectra data analysis, compound 4a was identified as a new 2-pyrazoline compound and named 5-(4-methylphenyl)-N,3-diphenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide. Compound 4a is the representative compound from the first series. This new compound was synthesized by the cyclo-condensation of 3-(4-methylphenyl)-1-phenyl-2-propen-1-one (3a) with 4-phenyl-3-thiosemicarbazide in the presence of sodium hydroxide as a base. Compound 4a formed as a cream-colored precipitate with 26% yield and a melting point of 133-136 °C. Compound 4a was characterized by various spectroscopic techniques including IR, 1D-NMR, 2D-NMR and HRMS. The important absorption bands in the IR spectrum of compound 4a were observed at 3291 cm −1 (N-H stretching), 1594 cm −1 (C=N stretching), 1520 cm −1 and 1449 cm −1 (C=C stretching of the aromatic), and 1399 cm −1 (C=S stretching). The absence of C=O and C=C bands, as well as the appearance of new C=N and C=S bands in the IR spectra of compound 4a suggests the complete formation of compound 4a via the cyclo-condensation reaction between chalcone 3a and 4-phenyl-3thiosemicarbazide (Scheme 1).
The 1 H-NMR spectrum of compound 4a showed that two germinal protons, H-4α and H-4β, of the methylene group resonated as a doublet of doublet at δH 3.26 and δH 4.05, respectively. The appearance of these two signals could be attributed to the non-equivalent nature of the two germinal protons with a J coupling constant of J4α4β = 18.0 Hz, J4α5 = 3.5 Hz and J4β5 = 11.5 Hz. Meanwhile, the vicinal proton (H-5) also appeared as a doublet of doublet at a slightly downfield region, δH 6.14, due to vicinal coupling with the two neighboring germinal protons of the methylene group at position 4-of the pyrazoline ring. A singlet at δH 9.93 in the downfield region indicated the presence of a thioamide proton. In compound 4a, some aromatic protons such as H-3′', H-4′' and H-5′' were observed at the same chemical shift, δH 7.49, due to the overlapping of peaks in the similar environment. The 1 H-NMR spectrum also showed three signals for ring C. A triplet at δH 7.33 (2H, J = 7.5 Hz) was attributed to H-10 and H-12. In addition, two multiplets at δH 7.72 (2H) and δH 7.16 were assigned to H-9 with H-13 and H-11. The 13 C-NMR spectrum of carbothioamide compound 4a showed seventeen carbon signals. One carbon signal each at δC 42.3 and δC 62.3 suggested the presence of pyrazoline ring carbons, which were assigned to 4-CH2 and 5-CH, respectively. Also, a signal at δC 155.3 was attributed to 3-C=N in the pyrazoline ring, which is a carbon attached to an electronegative nitrogen by a double bond and also to a benzene ring. Besides, a signal at δC 174.2 was attributed to 6-C=S. The 2D-NMR correlation of 1 H-1 H COSY and 1 H-13 C HMBC spectra were used to assign the aromatic signals, especially in the case of the pyrazoline ring. The analysis of 1 13 C HMBC spectrum of 4a shows the cross-correlations of 7-NH with carbons C-9 and C-13, and H-10 with C-9, C-13, C-11, C-12 and C-8 ( Figure 2), and the crosscorrelation of 14-CH3 with C-3′, C-5′ and C-4′. The HRMS spectrum of 4a revealed the molecular ion peak [M+Na] + at m/z 394.1156, which is consistent with the molecular formula C23H21N3S (calcd. 394.1354). According to the above spectra data analysis, compound 4a was identified as a new 2-pyrazoline compound and named 5-(4-methylphenyl)-N,3diphenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide.  The second series of new (3,5-disubstituted-4,5-dihydro-1H-pyrazol-yl) (4-hydroxyphenyl)methanone (5a-g) was synthesized by the cyclo-condensation of chalcone derivatives (3a-g) with 4-hydroxybenzhydrazide in the presence of sodium hydroxide as a base.
Compound 5e was taken as the representative compound from the fourth series, which formed a pale-yellow powder with 17% yield and a melting point of 237-241 • C. Compound 5e was characterized by multiple spectroscopic techniques including IR, 1D-NMR, 2D-NMR, and HRMS. The important absorption bands in the IR spectrum of compound 5e were observed at 3137 cm −1 (O-H stretching), 3066 cm −1 (C sp 2 -H), 2926 cm −1 (C sp 3 -H), 1737 cm −1 (C=O stretching), 1618 cm −1 (C=N stretching), 1573 cm −1 and 1427 cm −1 (C=C stretching of the aromatic). Moreover, the hydroxy group of phenol in the para position is a good activating group [21]. The absence of C=C bands, as well as the appearance of new C=N and C-O bands in the IR spectrum of compound 5e suggests the complete formation of the compound 5e via a cyclo-condensation reaction between chalcone 3e and 4-hydroxybenzhydrazide (Scheme 1).
The 1 H-NMR spectrum of compound 5e revealed the presence of one, mostly downfield, sharp singlet at δ H 10.11 for the hydroxy proton (13-OH). One doublet of doublet at δ H 7.40 (J = 2.5 and 8.5 Hz) was assigned to H-5 due to its meta coupling with H-3 and ortho coupling with H-6 in ring B. A doublet at δ H 7.22 (J = 8.0 Hz) was attributed to H-6 due to its ortho coupling to H-5 . In addition, a doublet at δ H 7.69 was attributed to H-3 due to its meta coupling to H-5. On the other hand, a doublet at δ H 7.91 (J = 8.0 Hz) integrating to two protons was assigned to H-9 and H-11, which occurred at the same position and can be considered chemically equivalent due to symmetry in the structure. The 13 C-NMR spectrum of compound 5e showed that signals corresponded to all twenty-two carbons in the compound. Signals at δ C 58.8, 40.1 and 155.3 were assigned to 5-CH, 4-CH 2 and 3-C=N of the pyrazoline ring, respectively, and the presence of the pyrazoline ring was further confirmed by 2D-NMR spectroscopy. The 1 H-1 H COSY spectrum of compound 5e showed a very clear correlation between 5-CH (δ C 5.95), 4α-CH (δ C 3.13) and 4β -CH (δ C 3.96). A strong correlation between H-8/H-12 (δ C 114.9) and H-9/H-11 (δ C 132.6) was also observed ( Figure 3). The 1 H- 13 C HMBC spectrum of 5e shows that 4α-CH (δ C 3.13) and 4β-CH (δ C 3.96) were correlated with 5-CH (δ C 58.8), C-1 (δ C 138.9) and 6-C=O (δ C 165.2), thus confirming the formation of a pyrazoline ring ( Figure 3). The cross-correlation of H-5 with C-3 and C-1 was found, while H-6 was found to correlate with 5-CH and C-2 . The HRMS of compound 5e showed a molecular ion peak [M + H] + at m/z 411.0694 (calcd. 411.0667) which corresponded to the molecular formula C 22 H 17 Cl 2 N 2 O 2 . In conclusion, based on the spectral data, it is proven that the compound 5e, N-(4-hydroxyphenyl)(5-(2,4-dichlorolphenyl)-3phenyl-4,5-dihydro-1H-pyrazol-1-yl)methanone, was successfully synthesized. The second series of new (3,5-disubstituted-4,5-dihydro-1H-pyrazol-yl) (4-hydroxyphenyl)methanone (5a-g) was synthesized by the cyclo-condensation of chalcone derivatives (3a-g) with 4-hydroxybenzhydrazide in the presence of sodium hydroxide as a base.
Compound 5e was taken as the representative compound from the fourth series, which formed a pale-yellow powder with 17% yield and a melting point of 237-241 °C. Compound 5e was characterized by multiple spectroscopic techniques including IR, 1D-NMR, 2D-NMR, and HRMS. The important absorption bands in the IR spectrum of compound 5e were observed at 3137 cm −1 (O-H stretching), 3066 cm −1 (Csp 2 -H), 2926 cm −1 (Csp 3 -H), 1737 cm −1 (C=O stretching), 1618 cm −1 (C=N stretching), 1573 cm −1 and 1427 cm −1 (C=C stretching of the aromatic). Moreover, the hydroxy group of phenol in the para position is a good activating group [21]. The absence of C=C bands, as well as the appearance of new C=N and C-O bands in the IR spectrum of compound 5e suggests the complete formation of the compound 5e via a cyclo-condensation reaction between chalcone 3e and 4-hydroxybenzhydrazide (Scheme 1).

Anti-Tuberculosis Activity
Two series of fourteen synthesized pyrazoline derivatives (4a-g and 5a-g) were screened for antituberculosis activity against M. tuberculosis H37Ra using the Tetrazolium bromide microplate assay (TEMA) method. Isoniazid was used as a positive control. Table  1
The in vitro antituberculosis evaluation revealed that the compounds which contained substituted para methyl (4a and 5a) in two series exhibited activity against M. tuberculosis. The result confirmed that methyl group substitution at the phenyl ring in pyrazoline analogues has a favorable effect, and a prominent improvement in inhibition was noticed [22]. However, it was found that compounds with a substituted para methoxy (4b and 5b) in both series showed no inhibition against M. tuberculosis H37Ra, even at the highest tested concentration of 200 µg/mL. This is not surprising, as previous studies have reported that (OCH 3 ) group substitution at the phenyl ring in pyrazoline analogues worsens the antituberculosis activity [23]. Moreover, it was discovered that all compounds (5a and 5c-g) from the second series (2-(4-hydroxyphenyl)-2-oxoethan-1-ide derivatives), except for compound 5b, exhibited moderate activity against M. tuberculosis H37Ra. These results confirmed that hydroxylphenyl substitution is necessary for antituberculosis activity [24].

Molecular Docking
The free binding energy and interaction modes between compound 4a and residues in the active site of cytochrome P450 14 alpha-sterol demethylase (CYP51) were identified by molecular docking studies using AutoDock Vina version 1.14. The crystal structure of CYP51 complexed with fluconazole (Protein Data Bank ID -PDB ID 1EA1) was used as a reference structure for the docking process [25]. The structure of isoniazid (PDB ID: 2VCF) was used as an experimental control to investigate molecular docking with CYP51. The docking results indicated binding energy between CYP51 and the control inhibitor (fluconazole) or the experimental control (isoniazid) or compound 4a, ranging between −6.2 to −7.1, −6.0 to −5.0 and −6.3 to −6.7 respectively ( Table 2). The active site(s) of CYP51 responsible for the binding of compound 4a and the controls (isoniazid and fluconazole) is shown in Figure 4.  The interaction modes of compound 4a with CYP51 are shown in Figure 5a-d. These structures detail the interactions in the active sites that are listed in Table 3. The interaction modes of the most potent pose in CYP51 (Figure 5a) showed that all phenyl rings at position 1 and 5, including the thioamide group, interacted with ILE27, TRP267, HIS318 and ARG354 via hydrophobic interactions. The second most potent pose (Figure 5b) demonstrated that the pyrazoline ring and thioamide group interacted with ARG274, GLU424 and TYR426, respectively, via non-conventional hydrogen bonding. Five hydrophobic interactions were formed between the thioamide group with ARG274, and at the phenyl rings at position 1 and 3 with TRP267, GLU271, ARG274, and ARG427. The interaction modes of compound 4a with CYP51 are shown in Figure 5a-d. These structures detail the interactions in the active sites that are listed in Table 3. The interaction modes of the most potent pose in CYP51 (Figure 5a) showed that all phenyl rings at position 1 and 5, including the thioamide group, interacted with ILE27, TRP267, HIS318 and ARG354 via hydrophobic interactions. The second most potent pose (Figure 5b) demonstrated that the pyrazoline ring and thioamide group interacted with ARG274, GLU424 and TYR426, respectively, via non-conventional hydrogen bonding. Five hydrophobic interactions were formed between the thioamide group with ARG274, and at the phenyl rings at position 1 and 3 with TRP267, GLU271, ARG274, and ARG427.    The third pose indicated the compound also docked inside active site 2 (Figure 5c) with residue thioamide and pyrazoline ring interacted with LEU317 and ARG354, respectively. Meanwhile, three hydrophobic interactions were occurred between phenyl rings with ILE27, ARG274, and ARG247. The remaining hydrophobic interactions were established between the thioamide group and HIS318, ARG354, and HIS430. In contrast, poses 4 and 5 showed that compound 4a preferred to bind at active site 3 of CYP51, with established interactions between residues THR80 and ARG96 with the phenyl group, and GLU94 with the thioamide group (Figure 5d-e). Only one non-conventional hydrogen bond was formed between the thioamide group and GLY84 in pose 4 (Figure 5d).

Chemistry
All chemicals and reagents were obtained from Acros Organic (Geel, Belgium), QRec (Penang, Malaysia, Asia), Sigma-Aldrich Chemical Co. or Merck (Darmstadt, Germany). Thin-layer chromatography (TLC) was performed on alumina plates pre-coated with silica gel (Merck 60 F254). The progress of reactions was determined by the appearance of product and disappearance of reactant spots under UV radiation (λ max = 254 nm, Muttenz, Switzerland), respectively. Melting points were determined on open capillary tubes and are uncorrected. All spectral data were obtained on the following instruments: infrared (IR) spectra were recorded on a Perkin-Elmer System FTIR-ATR spectrometer; 1D-and 2D-NMR spectra were recorded on a 500 FT-NMR Bruker Advance spectrometer (Bruker Bioscience, Billerica, MA, USA) in CD 3 COCD 3 , DMSO-d 6 , CDCl 3 and tetramethylsilane (TMS) as internal standards. Chemical shifts are reported in part per million (δ-scale) and the coupling constants, J, are reported in Hertz (Hz). High resolution HRMS mass spectra were obtained from a Waters Xevo QTOF MS system.

General Procedure for the Preparation of Chalcone Derivatives (3a-g) by the Claisen-Schmidt Condensation Reaction
Chalcone derivatives (3a-g) were synthesized following the method described in the literature. A mixture of acetophenone (1) (0.01 mol) and substituted benzaldehyde (2a-g) (0.015 mol) in methanol (10 mL) was refluxed in the presence of few drops of piperidine for 72 h [26,27]. The solution was kept in an ice bath until a solid was obtained, and the chalcone compounds were filtered, washed with cold water, dried and recrystallized from ethanol. The results were compared with the literature [28][29][30][31].   The carbothioamide compounds (4a-g) were synthesized according to a previously reported method with slight modifications. The cyclo-condensation of chalcone derivatives (3a-g) (1 mmol) with 4-phenyl-3-thiosemicarbazide (1.5 mmol) was carried out in ethanol (6 mL) in the presence of NaOH (3 mmol) for 3 to 8 h [32]. The reaction progress was observed on a TLC plate. After the reaction was over, the reaction mixture was left at room temperature overnight. A solid was formed after crushed ice was added. The solid was filtered, washed with cold water, dried and recrystallized from ethanol. Please refer the Supplementary Data for full spectra.  3.1.4. General Procedure for the Preparation of (3,5-disubstituted-4,5-dihydro-1Hpyrazol-yl) (4-hydroxyphenyl)methanone (5a-g) The new methanone compounds (5a-g) were synthesized by the cyclo-condensation of chalcone (3a-g) (1 mmol) with 4-hydroxybenzhydrazide (1.5 mmol) in ethanol (6 mL) in the presence of NaOH (3 mmol) for 3 to 8 h. The reaction progress was observed on a TLC plate. After the reaction was over, the reaction mixture was left at room temperature overnight. The reaction mixture was neutralized by adding 1% v/v of HCl and monitored by pH paper until the precipitate formed. A solid was formed after crushed ice was added. The solid was filtered, washed with cold water and dried. The products were purified by CC using silica gel with the eluent n-hexane:ethyl acetate to give the compounds 5a-g. Please refer the Supplementary Data for full spectra. The tetrazolium microplate assay (TEMA) method was performed to evaluate the anti-mycobacterial activity of the derivatives as described by Caviedes et al. with minor modifications [34]. The assay was performed in 96-well plates in duplicate and at least three times independently. The derivatives were dissolved in DMSO and serially diluted to the desired concentration in complete Middlebrook 7H9 media enriched with albumin dextrose catalase supplement to reduce the DMSO concentration below 1%. DMSO at this concentration did not inhibit the growth of M. tuberculosis H37Ra (unreported data). Briefly, 200 µL of sterile distilled water was added to the outer wells of the microplate and 100 µL of Middlebrook 7H9-ADC media into the wells in columns C to G, rows 2 to 11. Then, 100 µL of working solution containing the compounds was added into the wells in columns B and C, rows 2 to 11, in duplicate. A two-fold serial dilution of the compounds was made by transferring 100 µL from the wells in column C to column D, the content was mixed well, and the dilutions were continued until column G, where 100 µL of the excess medium from the wells in column G was discarded. Log phase M. tuberculosis H37Ra at a density of (~1.5 × 107 CFU/mL) was added and incubated at 37 • C with 5% CO 2 for 5 days. On day 5, 50 µL of tetrazolium reagent mixture was added to all wells, and the plates were re-incubated for 24 h. The results were read visually the following day. The MIC is defined as the lowest drug concentration that prevented the color change from yellow to purple. A small volume of the culture from the 96-well plate was transferred into Middlebrook 7H10 agar media and the plates were incubated at 37 • C with 5% CO 2 for 28 days. The MBC is defined as the lowest concentration of compound that did not show any bacterial colony growth.

Molecular Docking
The three-dimensional coordinates of the reference structure, cytochrome P45014 alpha-sterol demethylase (CYP51) complexed with fluconazole (PDB ID: 1EA1), and the structure of the experimental control, isoniazid (PDB ID: 2VCF), were fetched by ID from the Protein Data Bank (PDB) database in UCSF Chimera version 1.14 [35]. Before docking, the crystal PDBs were processed using the Dock Prep tool, starting with the removal of water molecules and unrelated hetam (i.e., refer to any ions molecules and any atoms that not belong to the protein) followed by separation of the receptor and inhibitor from the complexes into individual structures and finally the minimization of individual