Design and Synthesis of Imidazole and Triazole Pyrazoles as Mycobacterium Tuberculosis CYP121A1 Inhibitors

Abstract The emergence of untreatable drug‐resistant strains of Mycobacterium tuberculosis is a major public health problem worldwide, and the identification of new efficient treatments is urgently needed. Mycobacterium tuberculosis cytochrome P450 CYP121A1 is a promising drug target for the treatment of tuberculosis owing to its essential role in mycobacterial growth. Using a rational approach, which includes molecular modelling studies, three series of azole pyrazole derivatives were designed through two synthetic pathways. The synthesized compounds were biologically evaluated for their inhibitory activity towards M. tuberculosis and their protein binding affinity (K D). Series 3 biarylpyrazole imidazole derivatives were the most effective with the isobutyl (10 f) and tert‐butyl (10 g) compounds displaying optimal activity (MIC 1.562 μg/mL, K D 0.22 μM (10 f) and 4.81 μM (10 g)). The spectroscopic data showed that all the synthesised compounds produced a type II red shift of the heme Soret band indicating either direct binding to heme iron or (where less extensive Soret shifts are observed) putative indirect binding via an interstitial water molecule. Evaluation of biological and physicochemical properties identified the following as requirements for activity: LogP >4, H‐bond acceptors/H‐bond donors 4/0, number of rotatable bonds 5–6, molecular volume >340 Å3, topological polar surface area <40 Å2.


Introduction
Tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent, ranking above HIV/AIDS. [1] In 2016, 6.3 million new cases of TB were reported; of these there were an estimated 1.3 million deaths among HIV-negative people and an estimated 374,000 deaths among HIV-positive people. [1] Drug-resistant TB is a continuing challenge, with an estimated 4.1 % of new cases and 19 % of previously treated cases presenting with multidrug resistant (MDR)/rifampicin resistant (RR) TB in 2016. [1] China, India and the Russian Federation have the largest number of MDR/RR-TB cases accounting for 47 % of the global total. [1] Drugs currently used to treat TB target cell wall synthesis (e. g. isoniazid, ethambutol, cycloserine), protein synthesis (e. g. capreomycin, kanamycin), RNA synthesis (e. g. rifampicin), DNA gyrase (e. g. fluoroquinolones) and ATP synthase (e. g. bedaquiline and delamanid) in Mycobacterium tuberculosis (Mtb), the causative agent of TB. [2] The first line TB drug regimen includes a combination of isoniazid, ethambutol, rifampicin and pyrazinamide over a 6-9 month period and, whilst effective, this therapy is associated with drug intolerance and toxicity. [3] Treatment of MDR-TB and extensively drug resistant (XDR) TB is more complex and also associated with increased side effects. [2,4] The emergence of MDR-TB, XDR-TB and totally drug resistant (TDR) Mtb strains has led to intensified research to identify new anti-TB drugs over the past decade.
Mtb encodes twenty cytochrome P450 enzymes (CYPs or P450s), three of these (CYP121A1, CYP125 A1 and CYP128 A1) were shown to be essential for mycobacterial growth or survival in the host. [5] CYP121A1 is of considerable interest from both a biochemical and drug design perspective. CYP121A1 catalyses a
Reduction of the aldehydes (7) with NaBH 4 resulted in high yields of the corresponding alcohols (8). Thionyl chloride was the chlorinating reagent used to convert the alcohols to the  Binding with the heme is through an interstitial water molecule with Ser237, Gln385, Arg386 and Thr77 as key binding amino acids. chlorides (9), different equivalents of thionyl chloride were used to optimize the reaction conditions (1, 4 and 10 equivalents), and the maximum yield was obtained when 10 equivalents of the reagent were used. Reaction of the chlorides (9) with either the potassium salt of imidazole or triazole, prepared in situ by treatment of imidazole or triazole with potassium carbonate in acetonitrile at 45°C for 1 h, [12,13] and overnight reflux at 70°C gave the required final imidazole (10 a and 10 b) and triazole (11 a and 11 b) pyridyl-pyrazole derivatives (Scheme 1) in yields of 67, 54, 48 and 55 % respectively.
In series 2, one of the phenyl rings was replaced with a methyl group to determine whether the second phenyl ring was significant for enzyme binding and inhibitory activity.
The Series 3 biarylpyrazole imidazole (10 c-j) compounds were obtained via the five-step synthetic route described in Scheme 1 and were prepared to explore the effect of different aryl and biaryl groups on binding and their 'fit' within the active site, and also to evaluate the correlation between MIC and lipophilicity previously observed. Of note, although the yields obtained for the imines (6 c-j) varied between 84-99 %, they were generally found to be highly sensitive to light and moisture, especially those with low melting points, so were used immediately in the next step of the reaction scheme. Also, in the chlorination step of the indole derivative 8 j a C-2 chlorination of the indole ring occurred in addition to the conversion of the alcohol to chloride.

CYP121A1 Ligand Binding Affinity
The CYP121A1 binding affinity (K D ) of the various compounds was determined by UV-vis optical titration. For CYP121A1, the typical low-spin ligand free spectrum shows five characteristic peaks; a protein peak from the aromatic amino acid residues at 280 nm, a Soret peak at 416.5 nm, the β band at 538 nm, the α band at 565 nm, and small charge transfer band at~650 nm owing to a residual amount of high-spin heme iron. A heme iron low-spin (LS) to high-spin (HS) type I shift occurs as cYY binds to CYP121A1, with the Soret band shifting from 416.5 to 393 nm (the cYY K D is 5.82 � 0.16 μM [13] ). Other features of note in the spectral titration are apparent isosbestic points at approximately 420 nm and 492 nm, small red shifts in the alpha and beta band maxima at~572 and 547 nm, and loss of the small high-spin signal at~650 nm. Coordination binding of azoles to the CYP121A1 heme iron results in type II (red) spectral shifts of the Soret peak to wavelengths typically between~418-424 nm (according to the specific azole and whether it binds directly to the heme iron or via a retained water ligand to the heme iron), accompanied by spectral changes in both the α and β bands (e.g. 10f, Figure 4). [19] In Series 1, replacing the phenyl ring of the lead imidazole compound (1) or triazole compound (2) with a pyridyl ring resulted in decreased binding affinity compared with the lead phenyl imidazole (1) and triazole (2)    . Compound 10 f binding to CYP121A1 using UV-Vis optical binding titration The left-hand panel shows spectra for compound 10 f titrated with CYP121A1 (ligand-free spectrum is a thick black line, spectra following progressive additions of compound 10 f are thin solid lines, and the final near-saturated protein spectrum is a thick red line). The inset shows overlapped difference spectra generated by subtracting the starting spectrum from each consecutive ligand-bound spectrum. The right-hand panel shows a plot of compound 10 f induced-absorbance change calculated as the difference between the peak and trough in the difference spectra in the left-hand panel, using the same wavelength pair (429 and 392 nm, respectively). Data was fitted using the Hill equation giving a K D value of 0.22 � 0.01 μM for 10 f.  (Table 1). Series 3 biaryl derivatives with branched butyl substitutions (10 f and 10 g), the biphenyl substitution (10 h) and the indole substitution (10 j) gave the highest affinity for CYP121A1, having K D values < 5 μM (K D = 0.22 � 0.01 μM, 4.81 � 0.31 μM, 2.91 � 0.14 μM and 3.31 � 0.05 μM, respectively) ( Table 1). The aryl derivatives with shorter alkyl substitutions, 4-ethyl (10 c), 4propyl (10 d), 4-isopropyl (10 e) and benzo [d] [1,3] dioxole (10 i) had moderate binding affinity (K D = 18.3 � 3.4 μM, 53.7 � 5.9 μM, 22.5 � 3.5 μM, and 10.1 � 0.3 μM, respectively). The values were compared with the K D values of fluconazole (8.6 � 0.2 μM) and clotrimazole (0.07 � 0.01 μM). [19] Compounds 10 e, 10 f, 10 h, 10 i, 10 j and 13 a induce the most extensive Soret absorbance shifts, with the Soret maxima shifting by 6.5 nm (in the case of 10 e) and by 7 nm (in the case of 10 f, 10 h, 10 j and 13 a). The shifts observed are comparable with those for azole antifungal drugs (clotrimazole, econazole, fluconazole, ketoconazole and miconazole), which bind tightly to Mtb CYP121A1, inducing a Soret peak shift to between 421-423.5 nm. [20] Less extensive Soret red shifts were observed for imidazole pyrazole regioisomers (23 and 25), the triazole pyrazole regioisomers (24 and 26), the 4-ethylbenzene (10 c), 4propylbenzene (10 d) and 4-tert-butylbenzene (10 g) and the Series 1 3-pyridyl and 4-pyridyl (10 a and 10 b) imidazole derivatives on binding to the CYP121A1 heme iron.

MIC Determination Against Mycobacterium Tuberculosis
The derivatives were screened against M. tuberculosis H37Rv by the REMA (Resazurin Microtiter Assay) method. [21] In Series 1 only the imidazole pyridyl derivatives displayed antimicrobial activity (10 a and 10 b, MIC = 12.5 μg/mL), retaining the inhibitory activity observed for the phenyl imidazole lead compound (1).

Molecular Modelling
The Molecular Operating Environment (MOE) program [22] was used to perform molecular docking and was found to closely replicate the position and binding interactions of cYY and fluconazole, as observed in the crystal structures PDB 3G5H and PDB 2IJ7, respectively. The 4-and 3-pyridyl derivatives (10/11 a and 10/11 b) both interacted with the heme iron through an interstitial water molecule bonded with Ser237, and the 4pyridyl (10 a) also formed a direct hydrogen bond with Arg386 (e. g. 10 a, Figure 6). Both pyridyl imidazoles (10) and triazoles (11) formed additional binding interactions through the pyridine nitrogen, for the 3-pyridyl via a water molecule with Met62 and for the 4-pyridyl via a water molecule with Met62, Met86 and Val83 ( Figure 6).
The imidazole derivatives (13) with the 5-atom linker between the imidazole and the pyrazole ring were found to form more interactions with the CYP121A1 active site compared with the 4-pyridyl derivatives (14) with the 3-atom linker between the 4-pyridyl and pyrazole ring. However, the  extended pyridine compounds (13 and 14) were found to form numerous docking conformations owing to the increased flexibility of the extended linker between the heme-bonding group, imidazole for 13 and 4-pyridyl for 14. Series 2 imidazole (23 and 25) and triazole (24 and 26) derivatives, with the smaller methyl replacing a phenyl ring, were found to interact with the heme via an interstitial water molecule bound to Ser237, and for imidazole 23 a direct hydrogen bond interaction with Arg386 was formed. Additional hydrophobic interactions were observed with hydrophobic amino acid residues including Thr77, Val78, Val82, Val83 and Met86.
The ethyl (10 c) and propyl (10 d) imidazoles were found to bind in the same manner through interactions with the heme iron via an interstitial water molecule linked to Ser237 and with the phenyl ring interacting with Thr77 via a water molecule. The isopropyl (10 e) and isobutyl (10 f) imidazoles interacted with the heme in the same manner as described for 10 c and 10 d, but also formed an additional interaction between the pyrazole ring either with Thr77 via a water molecule in the case of 10 e, or with Gln385 and Arg386 via a water molecule for 10 f. The tert-butyl imidazole (10 g) and the biphenyl imidazole (10 h) both formed a direct hydrogen bond with Arg386 and interacted with the heme through an interstitial water molecule bonded with Ser237. The phenyl ring of the biphenyl imidazole (10 h) also formed an interaction with Thr77 via a water molecule. The positioning of the additional binding interactions of the branched alkyl and biphenyl imidazoles (10 e-10 h) resulted in a better filling of the binding pocket (Figure 7), especially in the case of 10 f and 10 g, and therefore these compounds would be predicted to be more efficient at blocking binding of the cYY natural substrate.
The heterobiaryl benzo[d] [1,3]dioxole (10 i) and 2-chloro-1Hindole (10 j) derivatives showed binding of the imidazole with the heme via an interstitial water molecule through Ser237, and additional H-bond interactions via Thr77, Arg386 and Gln385. The more extended pyrazole derivatives (10 f, 10 g, 10 h, 10 i and 10 j) either formed additional binding interactions and/or more completely blocked the active site by occupying a similar binding site as the natural substrate cYY and fluconazole, resulting in improved binding affinity.

X-Ray Crystallographic Studies
Two compounds were successful co-crystallized with CYP121A1, 10 j and 14 a (PDB 6GEO and 6GEQ respectively). Both crystals contained two molecules of sulfate resulting from the crystallography process, one of which is positioned above the heme ( Figure 8).
The indole derivative 10 j forms an arene-H interaction between the imidazole ring and Gln385, and between the indole benzene ring and Asn85. The indole NH and Cl act as Hdonors with two water molecules and the imidazole N acts as a H-acceptor with a water molecule. The pyrrole aryl ring is positioned to form a π-π interaction with Phe168 (   The extended 4-pyridyl derivative 14 a makes a H-bond interaction between the carbonyl oxygen and Gln385, and interacts indirectly with Thr77. Both 4-pyridyl groups form indirect H-bond interactions: the amide pyridyl with Thr65 and Arg72, and the pyrrole pyridyl with Val83 and Asn65. The benzene ring forms an arene-H interaction with Trp182 (Figure 8B).
Alignment of the crystal structure of 10 j co-crystallized with CYP121A1 (PDB 6GEO) with the crystal structures of CYP121A1 co-crystallized with fluconazole (PDB 2IJ7) and cYY (PDB 3G5H) showed a comparable position of all three structures in the enzyme ( Figure 9). All three compounds are positioned to form a π-π interaction with Phe168 and interact either directly or indirectly with amino acids Met62, Val83, Asn85. In particular, Hbonding interactions with Gly385 (10 j and fluconazole) or Arg386 (cYY) would appear to effectively block access to the active site.

Conclusions
For optimal binding interactions, inhibitors that effectively fill the CYP121A1 binding site and exhibit strong binding affinity are those that should most effectively inhibit CYP121A1 substrate (cYY) binding. This is observed for the branched alkyl derivatives 10 f and 10 g, and for the biphenyl 10 h. Compound flexibility also has a notable effect, with the extended pyridine derivatives 13 and 14 displaying very weak binding affinity. Lipophilicity is a major contributor to antimycobacterial activity, as shown by Hansch analysis ( Figure 5). The challenge in designing a compound with both good binding affinity and antimycobacterial activity is to combine the most favourable properties for each component. The physicochemical properties of the prepared compounds and reference compounds (cYY, fluconazole and clotrimazole) were calculated, the cLogP was determined using Crippen's fragmentation [23] and the number of H-bond acceptors (nON), H-bond donors (nOHNH) rotatable bonds (nrot), along with the molecular volume (MV) and topological polar surface area (TPSA) were calculated using Molinspiration software. [24] Those compounds with cLogP > 4 and TPSA < 40 Å 2 had optimal antimycobacterial activity (shaded green, Table 2), while the compounds with 5-6 rotatable bonds, 4-5 H-bond acceptors, no H-bond donors and molecular volume > 340 Å 3 had optimal binding affinity (shaded red, Table 2). Therefore, the optimal combined physicochemical properties for antimycobacterial activity and binding affinity (shaded green and red, Table 2) identified Series 3 isobutyl (10 f) and tert-butyl derivatives (10 g) as fulfilling these requirements. These and other high affinity compounds generated in this study will undergo further testing for their ability to kill pathogenic strains of M. tuberculosis.

Experimental Section Chemistry
All reagents and solvents were of general purpose or analytical grade and purchased from Sigma-Aldrich Ltd, Fisher Scientific, Fluka and Acros. 1 H and 13 C NMR spectra were recorded with a Bruker Avance DPX500 spectrometer operating at 500 and 125 MHz, with Me 4 Si as internal standard. Elemental analysis was performed by MEDAC Ltd (Chobham, Surrey, UK). High resolution mass spectra (HRMS) were determined at the EPSRC National Mass Spectrometry Facility at Swansea University and Medac Ltd (Chobham, Surrey, UK), using ESI (Electrospray Ionisation) in positive and negative modes, and a TOF (Time-of-Flight) analyser. Flash column chromatography was performed with silica gel 60 (230-400 mesh) (Merck) and TLC was carried out on precoated silica plates (kiesel gel 60 F 254 , BDH). Compounds were visualised by illumination under UV light (254 nm) or by the use of potassium permanganate stain followed by heating. Melting points were determined on an  electrothermal instrument and are uncorrected. All solvents were dried prior to use and stored over 4 Å molecular sieves, under nitrogen. All the compounds were � 95 % pure.

General Method for the Preparation of Hydrazine Derivatives (6)
To a solution of the acetyl reagent (4)

General Method for the Preparation of Alcohol Derivatives (8)
To an ice-cooled solution of carbaldehyde (7) (1 mmol) in EtOH (10 mL) was added NaBH 4 (1 mmol) in portions, then the reaction was then stirred at room temperature for 1 h. The solvent was evaporated and H 2 O (20 mL) was added slowly and the reaction stirred for 30 min. The reaction mixture was extracted with EtOAc (2 × 25 mL), then the combined organic layers washed with H 2 O (3 × 25 mL), dried (MgSO 4 ) and evaporated under reduced pressure to give the crude alcohol (17), which was was further purified by gradient column chromatography.

General Method for the Preparation of Alcohol Derivatives (19) and (20)
To an ice-cooled solution of ethyl carboxylate (17 or 18) [18] (5 mmol) in dry THF (15 mL) was added LiAlH 4 (1 M in THF, 7.5 mmol) dropwise over 25 min. The reaction was then stirred at room temperature overnight then cooled in an ice-bath and carefully quenched with H 2 O until cessation of effervescence. The reaction mixture was extracted with EtOAc (2 × 50 mL), then the combined organic layers washed with H 2 O (3 × 50 mL), dried (MgSO 4 ) and evaporated under reduced pressure to give the crude alcohol (5) or (6) which was was further purified by recrystallisation from CH 2 Cl 2hexane 1 : 1 v/v.

General Method for the Preparation of Chlorides (9), (21) and (22)
To an ice-cooled solution of alcohol (8 or 19 or 20) (1 mmol) in dry CH 2 Cl 2 (5 mL) was added thionyl chloride (10 mmol) dropwise over 25 min. The reaction was stirred at room temperature overnight and then cooled in an ice-bath and carefully quenched with saturated aqueous NaHCO 3 in portions until slightly basic (pH 8.0).
The organic layer was separated, washed with brine (3 × 10 mL), H 2 O (2 × 10 mL), dried (MgSO 4 ) and evaporated under reduced pressure to give the crude chloride, which was was purified by petroleum ether -EtOAc gradient column chromatography.