Synthesis and Evaluation of Thiazolyl-1 H-benzo [ d ] imidazole Inhibitors of Mycobacterium tuberculosis Inosine Monophosphate Dehydrogenase

Using an orthologue-based design approach, we synthesized and assayed a series of thiazolyl-1H-benzo[d]imidazole derivatives as inhibitors of Mycobacterium tuberculosis inosine 5’-monophosphate dehydrogenase (MtIMPDH). From these experiments, a benzo[d] imidazole compound was described to inhibit the enzyme in the low micromolar range (Ki IMP = 0.55 ± 0.02 μM), which places this compound among the most potent in vitro MtIMPDH inhibitors developed to date. In addition, steady-state kinetic measurements and docking simulations were employed to determine its inhibition and interaction modes. The results described herein may be useful for the design and development of novel alternative therapeutics for tuberculosis that target MtIMPDH, a predicted to be essential (for optimal in vitro bacillus growth), druggable and assayable molecular target.


Introduction
"Tuberculosis (TB) remains a major global health problem".This affirmation opens the last TB report of the World Health Organization (WHO). 1 Declared since the 1990s to be a global public health emergency, human TB has stricken an estimated 9.0 million people, which resulted in 1.5 million deaths worldwide in 2013. 1 Moreover, it has been estimated that approximately one third of the world's population is currently infected with a latent or dormant form of Mycobacterium tuberculosis (Mtb), the main causative agent of TB in humans, thus increasing the number of people at risk for developing active TB due to infection reactivation. 2The emergence of multi and extensively drug-resistant strains of Mtb (MDR-TB and XDR-TB, respectively), 3 the variable efficacy of the current vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG), 4 and the increasing prevalence of TB-HIV co-infection 1 have highlighted the need for new control measures.Although innovative drugs such as bedaquiline 5,6 and repurposing old drug classes have been described as a promising strategy to address this problem, 7 the current therapeutic TB treatments are suboptimal and new drugs are still needed. 8ithin this context, the identification, characterization, and validation of new molecular targets for the proposition of new drug-like compounds has been a challenge in the search for anti-mycobacterial agents. 9Indeed, the rate of target innovation in the past few decade has remained low for all research drug areas, with an average of approximately 5 first-against-target drugs per year. 10The study of novel targets can conduce to new molecules with novel mechanisms of action that are active against resistant and latent forms of Mtb, both of which are crucial attrition points in TB treatment.
Supported by the essentiality of most of its biosynthetic steps for optimal in vitro Mtb growth 11 and by evidence of nucleotide synthesis maintenance in latent bacillus, 12 the gene products of the purine nucleotide biosynthesis pathway have emerged as potential targets for drug development against both active and latent TB. 13 Among the enzymes that compose this pathway, the inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205)catalyzes the penultimate and rate-limiting step in guanine nucleotide biosynthesis: the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) and the concomitant reduction of nicotinamide adenine dinucleotide (NAD + ) (Scheme 1).
Encoded by the guaB2 gene (Rv3411c), 14 Mycobacterium tuberculosis IMPDH (MtIMPDH) has been predicted to be essential for optimal in vitro growth of M. tuberculosis H37Rv. 11,15Additionally, prokaryotic and eukaryotic IMPDHs have distinct structural features and kinetic properties, which make the development of species-selective inhibitors feasible. 16The different inhibition patterns of MtIMPDH and human IMPDH type II (HsIMPDH-II) 17 have supported the proposition of this target for the development of alternative drugs with selective toxicity, a basic pharmacology principle.Moreover, both proteins share sequence similarity of approximately 38%, and the amino acids residues at the HsIMPDH-II nicotinamide sub site that account for sensitivity to potent drugs, such as mycophenolic acid, are not conserved in the MtIMPDH. 180][21] These findings have described active compounds in whole cell assays that generate lead molecules that are now candidates for further development. 14,19hiazolyl-1H-benzo[d]imidazole derivatives have been identified from high-throughput screens as selective inhibitors of Cryptosporidium parvum IMPDH (CpIMPDH) that target the diverged NAD + site of enzyme. 22iffractometry studies from the crystal structure of CpIMPDH (PDB code: 3KHJ) in complex with IMP and a benzo [d]imidazole-based compound (named C64) have highlighted amino acid interactions, which account for the selectivity of these compounds to CpIMPDH over human IMPDH. 23Amino acid sequence alignment of MtIMPDH and CpIMPDH showed that almost all of the identified residues that interact with C64 (except Ser354, which was replaced by Ala483 in MtIMPDH) are conserved between the sequences (Figure S1 of the Supplementary Information).Incidentally, MtIMPDH has been predicted to be sensitive to CpIMPDH inhibitors 16 because phylogenetic analysis points to lateral gene transfer from bacteria, thus making eukaryotic IMPDH closely related to their prokaryotic counterparts. 24herefore, there is an expected divergence between mycobacteria and host IMPDH enzymes that, taken together with the sequence conservation of binding sites, prompted us to investigate the interaction of benzo[d]imidazole derivatives with MtIMPDH.For this, we deemed it appropriate to synthesize a series of thiazolyl-1H-benzo[d]imidazoles and evaluate their in vitro potential as inhibitors of MtIMPDH in an orthologue-based design approach.

Results and Discussion
The synthesis of thiazolyl-1H-benzo[d]imidazoles was carried out in two reaction steps in accordance to previously described protocol (Scheme 2). 25 It is noteworthy that proposed structural modifications were aimed primarily at the creation of an electron density gradient, variation of the molecular volume, and to explore the importance of the aromaticity and planarity of the substituents in a preliminary SAR (structure activity relationship) study.
First, the classical substitution reaction of primary and secondary amines 1 at the carbonyl group of bromoacetyl chloride 2 in the presence of catalytic The effect of each compound on catalytic activity of recombinant MtIMPDH was evaluated by monitoring the production of NADH (nicotinamide adenine dinucleotide).First, all of the synthesized compounds were assayed at a final-fixed concentration of 10 μM, and when the inhibition was greater than 50% of the initial enzymatic activity, the IC 50 values (concentration of inhibitor that reduces enzyme velocity by half) were determined (Table 1).
The results shown in the Table 1 demonstrate that MtIMPDH inhibition is highly sensitive to minor modifications of the thiazolyl-1H-benzo[d]imidazoles substituent groups.Additionally, the electron withdrawing groups generally showed better inhibitory activity on recombinant MtIMPDH.Compared with the non-substituted 5a, the replacement of hydrogen by halogens at the 4-position of the benzene ring furnished molecules with improved inhibitory activity.This strategy has been extensively explored in medicinal chemistry programs aiming for more lipophilic molecules and the addition of a new site for hydrogen and/or halogen bonding.It is noteworthy that as the atomic volume of halogens increased, the inhibitory action of the compounds became Observed at a final concentration of 10 μmol L -1 ; b IC 50 ≥ 20 μmol L -1 for HsIMPDH-II. 23ore potent (Table 1).The compound 4-fluoro substituted 5b inhibited the enzyme activity with an IC 50 > 10 μmol L -1 (25% at 10 μmol L -1 ), whereas both 4-chloro (5c) and 4-bromo (5e) substituted compounds conduced to IC 50 values of 4.5 ± 0.4 μmol L -1 and 3.0 ± 0.3 μmol L -1 , respectively.Interestingly, the iodine substituted compound 5f showed less inhibitory activity on MtIMPDH than did its isostere molecules 5c and 5e; its IC 50 was determined for HsIMPDH-II. 23ccordingly, K i (inhibition constant) measurements were carried out using classic Michaelis-Menten experiments.The K i values were determined for benzo[d]imidazole 5e, the lead of the series of synthesized analogs.Lineweaver-Burk plots suggested the mode of inhibition based on the effects on V max (maximal velocity) and K m (Michaelis constant) values for each inhibition type, and data fitting to appropriate equations led to values for the inhibition constants (K is and/or K ii ; K ii is the overall inhibition constant for the enzyme-substrateinhibitor complex, and K is is the overall inhibition constant for the enzyme-inhibitor complex).Particular signatures and mechanism of inhibition for MtIMPDH inhibitors were investigated and provided insights into the interaction mode for the enzyme. 14,17,19 F benzo[d]imidazole 5e, the types of inhibition were characterized as uncompetitive toward IMP and noncompetitive to NAD + (Figure 1).Compound 5e showed K is values of 0.55 ± 0.02 μmol L -1 for IMP and values of K is = 2 ± 1 μmol L -1 and K ii = 0.7 ± 0.1 μmol L -1 for NAD + .It is important to note that the inhibition profile exhibited by 5e cannot be overcome by improving the substrate concentrations, a detrimental condition for the success of inhibitors aiming for in vivo activity.
A steady-state ordered BiBi kinetic mechanism has been suggested for MtIMPDH, in which IMP binds first and is followed by the binding of NAD + and NADH dissociates first and is followed by XMP release. 26After the NAD + binding event, a rapid electron transference forming NADH and the covalent intermediate E-XMP* for IMPDHs has been reported. 27imilar to the analogous compounds already reported to be CpIMPDH inhibitors, the inhibition pattern showed by 5e indicates that this benzo[d]imidazole binds to both E•IMP and E-XMP*, with a slight preference for the E-XMP* state (K is > K ii ). 22It is interesting to note that the experimental findings concerning the benzo[d]imidazole potencies suggest a similar interaction mode of compounds with MtIMPDH to that shown by C64 with CpIMPDH. 23inally, 5e inhibits the MtIMPDH with 50-fold less potency than CpIMPDH (IC 50 = 0.06 ± 0.03 μmol L -1 ). 25 This difference may be related to the replacement of Ser354 residue in the eukaryotic protein by Ala483 in the MtIMPDH.This modification alters the physicochemical properties of the binding site because alanine is a non-polar residue replacing a polar one.Thus, the hydrogen-bonding network formed among Glu329, Ser354, Thr221 and amide group 23 would not likely occur in MtIMPDH.
In order to explore the binding phenomenon, a three dimensional protein model was constructed and used for docking simulations.The necessary coordinates for the protein structure was prepared using popular method of homology modeling, details of which has been provided in the Supplementary Information.Interestingly, compound 5e (most active out of 12) was found to be binding to the protein with best ranking and docking score of −6.69 kcal moL -1 .The compound was found to be involved in hydrogen bonding with residues Ser83, Ser339, Ile340 and Tyr421 and also in cation-π interaction between Arg443 and phenyl ring of compound (Figure 2).Moreover, the 4-bromo phenyl group of compound was found to be stabilized by strong hydrophobic interactions with residues Met85, Cys341, Met424 and Arg443.These strong interactions of 5e at the active site can be reasoned for its activity towards MtIMPDH.While analyzing the docking pattern of inactive compounds such as 5a and 5d (Figure S7 of the Supplementary Information), the compounds were found to be lacking some of the polar contacts with Ile340 and Tyr421 and also Ser339.The compounds were also found to be orienting slightly in different manner, compared to 5e, which moved the thiazole ring away from Tyr341 making it unavailable for hydrogen bonding.

Conclusions
In summary, we have reported the efficient preparation of benzo [d]imidazole derivatives and their use as inhibitors on catalytic activity of MtIMPDH.The proposition of these compounds as possible MtIMPDH inhibitors was based on their previously described action on CpIMPDH conducing to selective inhibitors at a low micromolar range.To the best of our knowledge, the titled compounds are among the most potent MtIMPDH inhibitors described to date.Therefore, the data herein presented may provide key insights into the proposition of new compounds that can be used in the further development of novel TB drugs that target MtIMPDH.

Experimental
All common reactants and solvents were used as obtained from commercial suppliers without further purification.All reactions involving reactants, reagents or intermediates sensitive to air or moisture were performed under an inert atmosphere of argon.Melting points were determined using a Microquímica MQAPF-302 apparatus. 1H NMR spectra were acquired on an Anasazi EFT-60 spectrometer ( 1 H at 60.13 MHz) and at 30 °C. 13 C NMR spectra were acquired on a Fourier 300 spectrometer of University of Caxias do Sul (UCS) in Brazil ( 13 C at 75.48 MHz) and at 25 °C.DMSO-d 6 was used as the solvent, and TMS was used as an internal standard in 5 mm samples tubes.Chemical shifts are expressed in ppm, and J values are given in Hz.High-resolution mass spectra (HRMS) were obtained for all compounds on an LTQ Orbitrap Discovery mass spectrometer from Thermo Fisher Scientific.This hybrid system combines the LTQ XL linear ion trap mass spectrometer with an Orbitrap mass analyzer.The experiments were performed using direct infusion of the sample in a solution of acetonitrile (50%), methanol (50%), and formic acid (0.1%), with a flow of 5 μL min -1 in positive-ion mode using electrospray ionization.Elemental composition calculations were performed using a specific tool included in the Qual Browser module of the Xcalibur (Thermo Fisher Scientific, release 2.0.7)software.Fourier transform infrared (FTIR) spectra were recorded using a universal attenuated total reflectance (UATR) attachment on a PerkinElmer Spectrum 100 spectrometer in the wavenumber range of 650-4000 cm -1 with a resolution of 4 cm -1 .Purity of compounds was determined by HPLC using an Äkta HPLC system of GE Healthcare ® Life Sciences.HPLC analysis conditions: RP column 5 μm Nucleodur C-18 (250 × 4.6 mm); flow rate 1.5 mL min -1 ; UV detection at 254 nm; 100% water (0.1% acetic acid) for 7 minutes followed by linear gradient from 100% water (0.1% acetic acid) to 90% acetonitrile/methanol (1:1, v/v) in 16 min; the last partition was maintained by 15 min and subsequently returned to 100% water (0.1% acetic acid) in 5 min remaining for additional 6 min.All evaluated compounds were > 95% pure.

General procedure for the preparation of 2-bromo-N-phenylacetamides 3a-l
To a solution containing aniline 1 (0.382 g, 4.1 mmol) and a catalytic amount of dimethylaminopyridine (DMAP) (0.150 g, 30 mmol%) in dry dichloromethane (20 mL) at 0 °C was added bromoacetyl chloride 2 (0.425 mL, 5.1 mmol) dropwise.The resulting solution was stirred at 0 °C for 30 min, and the temperature was then increased to 25 °C.After stirring for an additional 3 h, the reaction mixture was diluted with diethyl ether (50 mL).All of the stirring time was accomplished under argon atmosphere.The organic layers were washed sequentially with a solution of HCl (1 mol L -1 , 2 × 50 mL), water (1 × 100 mL), saturated aqueous NaHCO 3 (3 × 50 mL), and brine (5% m/v, 1 × 50 mL).Finally, the organic solution was dried over anhydrous MgSO 4 and evaporated under vacuum, and the residue was purified by flash chromatography on silica gel eluting with chloroform:methanol (40:1).

Enzymatic activity assays
The recombinant MtIMPDH was expressed and purified as previously described. 26The effect of each compound on steady-state velocities was evaluated using a UV-2550 UV-Visible spectrophotometer (Shimadzu) by monitoring NAD + reduction to NADH at 340 nm (ε β−NADH : 6.22 M -1 cm -1 ) and corrected for non-catalyzed chemical reactions in the absence of MtIMPDH.Assays were performed at 37 °C, in 50 mmol L -1 Tris pH 8.5, 1 mmol L -1 DTT, and 200 mmol L -1 KCl.Enzyme-catalyzed reaction started with the addition of MtIMPDH at 145 nM to the assay mixture (500 μL of final volume) and data were collected for 5 min.The inhibitors were dissolved in dimethyl sulfoxide (DMSO) and added to the reaction mixture at a final concentration of 10 μmol L -1 .Enzyme velocity was used to determine the % inhibition and, as a control, the maximal rate of the enzymatic reaction (100% of MtIMPDH activity) was determined in the absence of inhibitor and in the presence of fixed non-saturating concentrations of IMP (100 μmol L -1 , K 0.5 ca.120 μmol L -1 ) and NAD + (1 mmol L -1 , K m ca.887 μmol L -1 ), in the presence of 1% DMSO. 26Compounds able to reduce the enzyme activity by more than 50% were selected for further IC 50 determination.

IC 50 determinations
The IC 50 values were determined by adding different concentrations of the compounds (dissolved in DMSO) to the reaction mixture above described and the enzyme velocity was used to determine the % inhibition.The IC 50 values were estimated using equation 1, where [I] is the inhibitor concentration, v i is the initial velocity in the presence of the inhibitor, v o is the initial velocity in the absence of inhibitor, and the IC 50 value is defined as the concentration of the inhibitor that reduces the enzyme velocity by half.

Time-dependent inhibition
To evaluate whether or not enzyme inhibition may be time dependent, recombinant MtIMPDH was preincubated at room temperature with 10 μmol L -1 inhibitor (final concentration), which was then added at different times (up to 30 min) to the reaction mixture, as previously described.This analysis was performed to determine if inhibition follows a rapid equilibrium mode (classical competitive, uncompetitive and noncompetitive inhibition) or if there is a slow step in the equilibrium process.

Mode of inhibition and determination of the overall inhibition constant for 5e
The determination of K i values and the mode of inhibition were performed for 5e inhibitor towards both MtIMPDH substrates.Lineweaver-Burk (double-reciprocal) plots were employed to determine the mode of inhibition (competitive, noncompetitive or uncompetitive) and data fitting to appropriate equations gave values for the inhibition constants (K is and/or K ii ).In short, the inhibition mode was proposed based on the effects on V max and K m values for each inhibition type, resulting in plots with distinct straight line patterns toward either IMP or NAD + as follows: lines intercept on y axis for competitive inhibition (does not affect the apparent V max and increases apparent K m ), lines intercept on left of y axis for noncompetitive inhibition (decreases apparent V max , and does not affect apparent K m values if K is = K ii , increases the apparent K m values if K is < K ii , or decreases the K m values if K is > K ii ), and parallel lines for uncompetitive inhibition (decreases both apparent V max and K m values). 28he inhibition studies were carried out at varying concentrations of one substrate, a fixed non-saturating concentration of the other substrate, and in the absence of inhibitor and in the presence of three different concentrations of each inhibitor 5e (0.5, 1 and 1.5 μmol L -1 ).For IMP substrate analysis, the experiment was carried out with IMP concentrations ranging from 40 μmol L -1 to 1000 μmol L -1 and NAD + at 1 mmol L -1 ; for NAD + substrate analysis, the NAD + concentrations ranged from 0.2 mmol L -1 to 4 mmol L -1 , with IMP fixed at 100 μmol L -1 .The enzyme concentration was constant at 550 nM throughout the assays.Noncompetitive and uncompetitive inhibition data were fitted to equations 2 and 3, respectively: In these equations, [I] is the inhibitor concentration, [S] is the substrate concentration, K m is the Michaelis constant, V max is the maximal velocity, and K ii is the overall inhibition constant for the enzyme-substrate-inhibitor complex, and K is is the overall inhibition constant for the enzymeinhibitor complex. 28

Computational studies
Homology modeling is used to predict the three-dimensional structure of a protein sequence.
at 9.0 ± 3.0 μmol L -1 .The lower activity may be related to a possible steric hindrance at the 4-position of the benzene ring of thiazolyl-1H-benzo[d]imidazoles because a molecular volume-improving limit may have been achieved with the bromo-substituted 5e.It is important to note that the replacement of the chloro group attached at the 4-to 3-position of the benzene ring led to compound 5d, which lacked inhibition capacity on MtIMPDH in our experimental conditions.This result emphasizes the fine tuning of the halogen's position relative to the MtIMPDH binding site.The 4-nitro and 4-cyanobenzo[d]imidazole compounds (5g, 5h) both showed similar inhibition activity, with an IC 50 of 8.0 ± 0.3 μmol L -1 and ≥ 10 μmol L -1 , respectively.Focusing on the compounds substituted with electron donating groups, the results for benzo[d] imidazoles 5i-k showed that only the compound carrying the 3-propoxy group (5j) exhibited a significant inhibition of MtIMPDH activity.The IC 50 of 5j was 5.1 ± 0.5 μmol L -1 , similar to lead compound 5e, suggesting the existence of a hydrophobic portion accessed by the mobile 3-propoxy group.Finally, the benzo[d]imidazole derivative from piperidine (5l) did not show action on catalytic MtIMPDH activity in our assay conditions.Therefore, the initial screening of 12 molecules showed that five compounds (5c, 5e-g, and 5j) displayed reasonably good inhibition (IC 50 < 10 μmol L -1 ) on MtIMPDH activity.These five benzo[d]imidazoles showed no time-dependent inhibition up to 30 min of preincubation with MtIMPDH suggesting rapid equilibrium processes (data not shown).It is noteworthy that compounds 5c and 5e showed selective MtIMPDH inhibition because both have been described to show an IC 50 ≥ 20 μmol L-1

Figure 1 .
Figure 1.Double-reciprocal plots of inhibition assay for 5e.Patterns of parallel lines indicate uncompetitive inhibition towards IMP (a), whereas intersecting lines on the left of the y-axis are indicative of noncompetitive inhibition towards NAD + (b).Substrate IMP was varied from 40 to 1000 μmol L -1 in presence of NAD + 1 mmol L -1 (a); substrate NAD + was varied from 0.2 to 4 mmol L -1 in the presence of IMP 100 μmol L -1 (b).Enzyme concentration was 550 nmol L -1 throughout the assays.

Figure 2 .
Figure 2. Interaction profile of compound 5e (green sticks) at MtIMPDH active site.Pink: residues involved in hydrogen bonding; yellow: residues involved in hydrophobic interactions; blue dashed lines indicate hydrogen bonds.