Novel Trifluoromethyl Pyrimidinone Compounds With Activity Against Mycobacterium tuberculosis

The identification and development of new anti-tubercular agents are a priority research area. We identified the trifluoromethyl pyrimidinone series of compounds in a whole-cell screen against Mycobacterium tuberculosis. Fifteen primary hits had minimum inhibitory concentrations (MICs) with good potency IC90 is the concentration at which M. tuberculosis growth is inhibited by 90% (IC90 < 5 μM). We conducted a structure–activity relationship investigation for this series. We designed and synthesized an additional 44 molecules and tested all analogs for activity against M. tuberculosis and cytotoxicity against the HepG2 cell line. Substitution at the 5-position of the pyrimidinone with a wide range of groups, including branched and straight chain alkyl and benzyl groups, resulted in active molecules. Trifluoromethyl was the preferred group at the 6-position, but phenyl and benzyl groups were tolerated. The 2-pyridyl group was required for activity; substitution on the 5-position of the pyridyl ring was tolerated but not on the 6-position. Active molecules from the series demonstrated low selectivity, with cytotoxicity against eukaryotic cells being an issue. However, there were active and non-cytotoxic molecules; the most promising molecule had an MIC (IC90) of 4.9 μM with no cytotoxicity (IC50 > 100 μM). The series was inactive against Gram-negative bacteria but showed good activity against Gram-positive bacteria and yeast. A representative molecule from this series showed rapid concentration-dependent bactericidal activity against replicating M. tuberculosis bacilli with ~4 log kill in <7 days. Overall the biological properties were promising, if cytotoxicity could be reduced. There is scope for further medicinal chemistry optimization to improve the properties without major change in structural features.


INTRODUCTION
Tuberculosis remains a major global health killer with >1.5 million deaths and 10 million new cases in 2018 (World Health Organization, 2020). The current drug treatment regimen involves multiple antibiotics over a lengthy period of at least 6 months. In addition, there are multidrug-resistant and extremely drug-resistant strains circulating, making the current drugs ineffective. Thus, there is an urgent need for new drugs active against the causative pathogen Mycobacterium tuberculosis (Gordon and Parish, 2018).
The identification and development of anti-tubercular agents has been an increasing priority in the research community; target-based and whole-cell screens have been developed, which enable the screening of large libraries to find novel scaffolds with desirable biological activities (Parish, 2020). Phenotypic screening in which compounds are tested directly against the virulent organism in order to find novel matter has been widely utilized in the last decade or so (Parish, 2020). From these screens, a large number of compounds series that inhibit the growth of M. tuberculosis have been identified and explored (Gold and Nathan, 2017;Grzelak et al., 2019;Parish, 2020). However, given the high attrition rate in drug discovery, the difficulty of killing M. tuberculosis (as opposed to inhibiting growth), and the fact that it exists in different physiological states during infection, there is still a need for additional chemical (Payne et al., 2007;Keiser and Purdy, 2017;Mandal et al., 2019).
We have previously developed high-throughput screening using fluorescent strains of M. tuberculosis, which enabled us to screen compound libraries (Ollinger et al., 2018). We identified several series with anti-tubercular activity; among these is the trifluoromethyl pyrimidinone series, which has potent activity in vitro. Pyrimidinones are an important class of heterocycles containing heteroatoms that are broadly useful in medicinal chemistry (Fruit and Besson, 2018). This scaffold contains a range of drug molecules in the disease areas of cancer (5-fluorouracil and tegafur) (Papanastasopoulos and Stebbing, 2014) and HIV such as raltegravir (MK0518) (Marinello et al., 2008). We report the synthesis and structure-activity relationship (SAR) of this novel heterocyclic scaffold.

General Chemistry Methods
Chemicals and solvents were purchased from commercial vendors. Analytical thin-layer chromatography (TLC) was performed with precoated TLC plates, air dried, and analyzed using a UV lamp (UV254/365 nm) and/or an aqueous solution of potassium permanganate for visualization. Flash chromatography was performed using a Combiflash Companion R f (Teledyne ISCO) and prepacked silica gel columns. Massdirected preparative high-performance liquid chromatography (HPLC) separations were performed using a Waters HPLC (2,545 binary gradient pumps, 515 HPLC makeup pump, 2,767 sample manager) connected to a Waters 2998 photodiode array and a Waters 3100 mass detector. Preparative HPLC separations were performed with a Gilson HPLC connected to a Gilson 155 UV/vis detector. HPLC chromatographic separations were conducted using Waters XBridge C18 columns 19 × 100 mm, 5 µm particle size, using 0.1% ammonia in water (solvent A) and acetonitrile (solvent B) as mobile phase. 1 H NMR and 13 C NMR spectra were recorded on a Bruker Advance II 500, 400, or 300 spectrometer operating at 500, 400, or 300 MHz (unless otherwise stated) using CDCl 3 or dimethyl sulfoxide (DMSO)-d 6 solutions. Chemical shifts (δ) are expressed in ppm recorded using the residual solvent as the internal reference in all cases. Signal splitting patterns described as singlet (s), doublet (d), triplet (t), multiplet (m), broad singlet (br, s), or a combination thereof. Coupling constants (J) quoted to the nearest 0.1 Hz. Low-resolution electrospray (ES) mass spectra were recorded on a Bruker Daltonics MicrOTOF mass spectrometer run in positive mode. High-resolution mass spectroscopy was performed using a Bruker Daltonics MicrOTOF mass spectrometer. Liquid chromatography-mass spectrometry (LC-MS) analysis and chromatographic separation were conducted with a Bruker Daltonics MicrOTOF mass spectrometer or an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole LC-MS where both instruments connected to an Agilent diode array detector. The columns used were Waters XBridge column (50 × 2.1 mm, 3.5 µm particle size), and the compounds were eluted with a gradient from 5 to 95% using acetonitrile in water with 0.1% ammonia. All final compounds had a purity of ≥95% as determined by the UV chromatogram (190-450 nm) obtained by LC-MS analysis. High-resolution ES measurements performed on a Bruker MicroT of mass spectrometer.

Minimum Inhibitory Concentration
The compound concentration required to inhibit growth of M. tuberculosis was determined by testing compounds as 2-fold serial dilutions, typically starting at 20 µM. Growth inhibition was measured after 5 days using a fluorescent strain of M. tuberculosis in 7H9-OADC-Tw, according to previously published methods (Ollinger et al., 2013(Ollinger et al., , 2018. Each 96-well plate contained positive and negative controls, as well as a standard curve for rifampicin.

Cytotoxicity
The compound concentration that resulted in 50% growth inhibition of HepG2 human liver cells (ATCC) was determined by testing compounds as 3-fold serial dilutions, typically starting at 100 µM. Cytotoxicity was measured after 72 h of compound exposure at 37 • C and 5% CO 2 using CellTiter-Glo (Promega). Each 384-well plate contained positive and negative controls, as well as a standard curve for staurosporine.

Spectrum
Minimum inhibitory concentration to inhibit growth of 99% (MIC 99 ) on solid medium was determined using the agar serial dilution method. Late log phase cultures were plated at ∼10 5 colony-forming units (CFU)/mL onto solid medium containing 2-fold serial dilutions of compounds. The MIC 99 is defined as the lowest concentration with ≤1% growth.

Kill Kinetics
M. tuberculosis was cultured to late log phase. Compound was added (final concentration 2% DMSO), and cultures were incubated standing at 37 • C. Viable bacteria were determined by serial dilutions and plating on Middlebrook 7H10 plus 10% vol/vol OADC. CFUs were counted after 3-4 weeks. The untreated control was 2% DMSO.

Chemistry
We developed and conducted a whole-cell screen that identified the trifluoromethyl pyrimidinone series as having good growth inhibitory activity against virulent M. tuberculosis. We identified 15 compounds with activity in the primary screen. We determined the MIC for each. The primary hits are shown in Figure 1 with MICs. Among the initial set of 15 analogs, several compounds displayed good activity with an IC 90 of <5.0 (Figure 1). In general, the series had low molecular weight (<500), relatively low hydrophobicity (clogP < 5) and complied with Lipinski's Rule of 5, making this an attractive series for assessment. The ease of synthesis was another attractive feature.
Based on the attractive properties of this series, we expanded our studies to investigate the SAR of the series. Our objective was to explore the limits of the chemical space making structurally diverse molecules to determine if the series had dynamic SAR (>50-fold changes in MIC) and/or if we could improve the anti-tubercular potency (to <1 µM) and reduce cytotoxicity (selectivity index >10). We synthesized a series of substituted pyrimidinone to establish SAR. A small number of publications describe the synthesis of substituted trifluoromethyl pyrimidinones (Fruit and Besson, 2018). We used a procedure similar to those of Tice and Bryman to obtain substituted trifluoromethyl pyrimidinones (Tice and Bryman, 2001); we introduced trifluoromethyl substituents by the condensation of a variety of amidines with substituted ethyl 4,4,4-trifluoro-3oxobutanoate.
We used amidine as the starting material for the synthesis of trifluoromethyl pyrimidinones. Treatment of the substituted aryl or pyridine nitrile, such a picolinonitrile, with lithium bis(trimethylsilyl)amide in tetrahydrofuran at −30 • C and warming the reaction mixture to an ambient temperature afforded the amidine in moderate to high yield. Reaction of the amidine with ethyl 4,4,4-trifluoro-3-oxobutanoate in the presence of a base in ethanol under refluxing conditions furnished the trifluoromethyl pyrimidinone compound 16 with a high yield (Scheme 1). Following this synthetic pathway, all compounds were prepared in two or three steps from commercially available and inexpensive starting materials.

Biological Testing
We tested all compounds for activity against M. tuberculosis in aerobic culture, as well as for cytotoxicity using the HepG2 cell line.
We first looked at substitutions on the 2-position of the pyrimidinone. The initial SAR is summarized in Table 1. In this set, the most attractive molecule was compound 16, which had good activity (IC 90 = 4.9) and a lack of cytotoxicity (IC 50 >100), as well as good physicochemical properties with clogP of 0.63 and topological polar surface area of 53.8. It was interesting that this compound had good activity, even though it had a low clogP, as the activity of molecules against M. tuberculosis is often limited by their ability to penetrate the waxy cell wall (Machado et al., 2018). Compounds 7 and 8 with substitution of the 2pyridine rings showed slightly improved potency (IC 90 = 3.3 and 1.5 µM, respectively), as compared to compound 16. Inclusion of a polar hydroxyl group at the 3-position of the pyridine ring was tolerated (compound 17, IC 90 = 3.3 µM). However, all of these substitutions increased cytotoxicity with IC 50 < 20 µM against HepG2 cells. Addition of a benzyloxy at the 4-position of pyridyl ring retained potency (compound 6, IC 90 = 1.3 µM) but did not improve cytotoxicity (IC 50 = 2.5 µM). Incorporating a nitrile on the benzyloxy group (compound 9) or the reverse benzyloxy group (15) similarly retained activity (IC 90 = 3.7 and 5.3 µM, respectively), but with no improvement on cytotoxicity (IC 50 = 6.1 and 7.4 µM, respectively). Replacing the oxygen atom linker on the pyridine ring with a carbon chain such as 2-(4-(4-hydroxyphenethyl)pyridin-2-yl in compound 4 decreased potency (IC 90 = 7.8 µM). However, replacing the 2-pyridyl ring with a 2-pyrimidine ring in 18 abolished activity and cytotoxicity.
In order to rationalize SAR and obtain additional information on cytotoxicity liability, we designed a small set of library analogs around compound 16. Analogs with phenyl rings were prepared using cyclocondensation conditions using a variety of amidines as described in Scheme 1. Interestingly, moving the nitrogen around the pyridyl ring and substitution around the pyridyl ring led to loss of anti-tubercular activity (IC 90 >20 µM) as shown in 19-23. We replaced the pyridyl ring with a substituted phenyl ring in 24-31. As we had seen a trend toward increased potency with a 2-pyridine ring and any substitution at the C-4 position, we used this strategy to increase potency. However, we did not see improved potency with either unsubstituted or substituted phenyl groups (24-31), where all compounds were inactive (IC 90 >20 µM).
We next explored substitution on the pyrimidinone ring at C-5 using a methyl moiety as R1 (32-46, Table 2) and varying substitution at C-2 as R2 (47-50, Table 3). Several compounds were synthesized following the same cyclocondensation conditions with a variety of amidines and substituted ethyl 4,4,4-trifluoro-3-oxobutanoate as depicted in Scheme 1 to obtain 32-46. Compound 32 without substitution at C-3 on the pyrimidinone core had no antibacterial activity. Similarly, loss of potency was observed for compounds 34-46 ( Table 2) with trifluoromethyl and methyl groups at C-5 and C-6, respectively, and different substitutions at C-3. The methyl group did not appear to affect potency, as the matched pair of compounds with 3 and 4 had similar potency and cytotoxicity (within 2-fold). Similar to the SAR observed for the 2-pyridine with hydrogen as R1, we found a trend toward increased potency with 2-pyridine analogs; compounds 10 and 34 had similar potency (IC 90 = 2.9 and 2.5 µM, respectively) as 16 and 17. However, both 10 and 33 were cytotoxic with IC 50 of 5.4 and 3.2 µM, respectively. As with compound 18, compound 43 containing a pyrimidine group showed no cytotoxicity, but no antibacterial activity either.
The SAR clearly shows a preference for a 2-pyridine substitution on the pyrimidinone core at C-2 position and the tolerance for a methyl group at the C-5 position of the pyrimidinone moiety. We used this information to design compounds to investigate the role of trifluoromethyl group at the C-6 position. We prepared several compounds (47-50) containing a 2-pyridine at C-2 and various group at C-6 position of the pyrimidinone ring (Table 3). Compounds were synthesized using the same scheme as for compound 16. Compound 47 containing benzyl group at C-6 position was slightly less active, then the matched pair compound with the trifluoromethyl group (10), with an IC 90 = 5.7 µM compared to IC 90 = 2.9 µM. Similarly, shortening the linker of the benzyl group to phenyl afforded 48, which was 3-fold less active (IC 90 = 9.4 µM). Neither modification improved cytotoxicity (IC 50 of 9.8 and 9.7 µM, respectively). Another set of matched pairs demonstrated the importance of the trifluoromethyl at C-5 vs. an electron-rich methyl group at the same position (compounds 49 and 51): 2 | Activity of compounds with substitutions at the 2-and 5-positions of the pyrimidinone.

CONCLUSION
We identified the trifluoropyrimidinone pharmacophore as active against M. tuberculosis in aerobic culture with rapid bactericidal activity and have determined SAR. This series has not been previously tested against M. tuberculosis. The series shows a good dynamic range of activity, with the most active being 5 (4fluorobenzyl), 6 (2-(4-(benzyloxy)pyridin-2-yl), and 8 (methyl 3-((2-(6-oxo-4-(trifluoromethyl)-1,6-dihydropyrimidin-2yl)pyridin-4-yl) methoxy) benzoate) with IC 90 s of 1.6, 1.3, and 1.5 µM, respectively. The molecules have good calculated properties with low molecular weight, clogP < 5, and druglikeness features of pyrimidinone. Cytotoxicity was an issue with the series, but there is significant scope for further FIGURE 2 | Kill kinetics for a representative compound. M. tuberculosis was cultured aerobically and exposed to compound 55. Viable bacteria were determined by serial dilution and plating onto agar plates for 3 to 4 weeks. Independent experiments are shown. The control was 2% DMSO. The upper and lower limits of detection are indicated.
medicinal chemistry optimization to explore SAR and improve in cytotoxicity without any major change in structural features.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
EH, JE, JO, MM, TM, PH, and TP contributed to conception and design of the study. CS, OA, JG, LF, DD, MM, AK, and YO designed and conducted the experimental work. TP wrote the first draft of the manuscript. All authors contributed to data analysis, manuscript revision, read, and approved the submitted version.

FUNDING
This work was funded in part by Eli Lilly and Company in support of the mission of the Lilly TB Drug Discovery Initiative and by funding from the Bill and Melinda Gates Foundation, under grant OPP1024038.