Tuberculosis (TB) represents one of the top public health concerns worldwide. Over 2 billion people are infected with Mycobacterium tuberculosis (M. tb), the etiological agent of TB, resulting in 9.4 million new cases of active TB and 1.7 million deaths in 2009.¹ The 1970s multidrug regimen for treating TB, recommended today by the WHO, has not been sufficient to eliminate TB in part due to the appearance of HIV/AIDS, failure of treatment programs, and enhanced transmission in hospitals and prisons. The recommended combination therapy for TB is lengthy and cumbersome since it can require medication of up to four drugs daily for 6–12 months. Limited healthcare system resources often lead to treatment interruption or failure, exacerbating drug resistance problems in places least capable of combating this disease. Thus, the discovery of new types of anti-tubercular drugs acting on novel drug targets with no cross-resistance to any existing drugs is urgently needed. Screening of ~330,000 compounds from the NIH Small Molecule Repository for their ability to inhibit the growth of M. tb identified a number of novel scaffolds with anti-tubercular activity. In this report, we discuss the development of one of those scaffolds leading to the probe ML242 (CID 2792221).

Probe Structure & Characteristics

ML242

The M. tb inhibition data for two lots of the probe compound is summarized, i.e., compound lots SID 103147611 and SID 123101665. The M. tb inhibition IC₅₀ averages for the two compound lots are 306 nM (n = 10) and 21 nM (n = 3), respectively. The data for the probe compound from lot SID 103147611 varied over the five-month period for which replicate data was collected, from an initial low of <0.01 μM to a high of 0.37 μM. At this time, we may only speculate as to the cause for this potency variation, with the most likely potential explanations being: (1) the inherent variability in the assay, (2) compound instability, (3) diminishing compound solubility in the DMSO stock solution upon repeated freeze-thaw cycles,² or combinations of these factors. Regardless of the specific reason for the variability in the anti-tubercular activity assay outcome, the potencies for fresh samples of two lots of the probe compound are consistent (0.067 μM and 0.021 μM).

1. Recommendations for Scientific Use of the Probe

Tuberculosis is a bacterial disease that is present in one third of the world’s population, mostly in a latent form. Approximately 5–10% of these cases will convert to a highly contagious active form. The current treatment regimen consists of multiple pills per day of multiple medicines for a duration of at least six months and this treatment burden has led to compliance issues, which has contributed to the emergence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) strains. Due to the duration of treatment and prevalence of drug-resistant forms of TB, new treatments are urgently needed.

The probe candidate proposed in this report is designed to provide a novel approach to TB therapy. A phenotypic HTS screen was run, and our initial analysis excluded compounds of chemotypes similar to chemotypes for reported antitubercular drugs; thereby biasing our remaining compound hit set towards novel antitubercular compounds with novel mechanisms of action. Selection of a chemotype core structure, and subsequent SAR work led to our probe candidate, which has been shown to be extremely efficacious against M. tb (H37Rv) and has not had measurable toxicity to date.

Work is in progress to elucidate possible mechanisms of action for this compound. Development of an inhibitor with a validated mechanism of action on a novel M. tb target will allow us and other researchers to potentially develop the next generation of antitubercular drugs, a critical need as there has not been a TB drug with a novel mechanism of action since the 1960’s.

2. Materials and Methods

General synthesis and analysis experimental details: All reagents were used as received from commercial suppliers. The ¹H NMR spectra were recorded on a 400 MHz Bruker Avance spectrometer equipped with a broadband observe probe and the ¹³C NMR on a 500 MHz Bruker AVIII spectrometer equipped with a dual cryoprobe (at 125 MHz). Chemical shifts are reported in parts per million and were referenced to residual solvent signals or TMS. Flash column chromatography separations were performed using the Teledyne Isco CombiFlash R_f using RediSep R_f silica gel columns. TLC was performed on Analtech UNIPLATE silica gel GHLF or HLF plates. TLC plates were visualized under ultraviolet light. The analytical RPLC method used an Agilent 1200 RRLC system with UV detection (Agilent 1200 DAD SL) and mass detection (Agilent 6224 TOF). The analytical method conditions included a Waters Aquity BEH C18 column (2.1 × 50 mm, 1.7 μm) and elution with a linear gradient of 5% acetonitrile in pH 9.8 buffered aqueous ammonium formate to 100% acetonitrile at 0.4 mL/min flow rate. Compound purity was measured on the basis of peak integration (area under the curve) from UV-vis absorbance at 214 nm, and compound identity was determined on the basis of mass spectral and NMR analyses. IR spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer with a UATR polarization accessory; spectra were taken directly from solid state samples. Melting points were determined on a Stanford Research Systems OptiMelt Automated Melting Point System. All compounds used for biological studies have purity >90%.

2.1. Assays

Overview of assay strategy

The screening protocols described below are organized into three groups: First, there are the primary screening protocols in which growth inhibition of M. tb is monitored in complete 7H9 media with glycerol; second, are confirmatory assay protocols in which growth inhibition is assessed in the absence of glycerol to determine if any of the identified hits are glycerol dependent; third, are assay protocols designed to assess the cytotoxicity in mammalian cells of compounds identified as hits in the M. tb assays. This three-tiered screening strategy allows for compounds to be ranked to favor potent, non-toxic compounds, while excluding those compounds that are dependent on the presence of glycerol for growth inhibition. An overview of the assays can be found in Table 1.

Table 1

Listing of all Assays and AIDs for this project.

It has been shown previously that compounds demonstrating anti-mycobacterial efficacy can be metabolically dependent on the presence of glycerol in the assay media.³ Compounds that had been put forth from in vitro screening into in vivo models failed to show efficacy and it was determined through further studies that compounds identified in primary screening were dependent on the carbon source(s) present in the assay media. For this reason, compounds identified as active in our primary single dose and confirmatory dose response screens were screened in dose response in the identical assay with the assay media absent of glycerol.

The use of the Vero cell line as an initial cytotoxicity assessment in M. tb screening campaigns is primarily historical. This cell line has been used by many investigators in the field including screening funded by the Tuberculosis Antimicrobial Acquisition and Coordinating Facility program⁴ as well as previous MLSCN⁵ and MLPCN anti-mycobacterial screening campaigns (Summary AIDs: AID 1626, AID 488929, AID 434999). Compounds that are found to demonstrate limited cytotoxicity in this cell line while maintaining efficacy against M. tb are ranked higher through the calculation of selectivity index (SI). To collect more physiologically relevant cytotoxicity data, THP-1 and HepG2 cells were used also.

All anti-mycobacterial assays (both with and without glycerol) were performed using the M. tb strain H37Rv, all from the same frozen stock. All compound sets beyond the single dose primary screen were screened in cytotoxicity assays against Vero cells (Cercopithecus aethiops epithelial) from the same frozen stock of assay ready cells. All purified and synthesized compound sets were also run in cytotoxicity assays against THP-1 (Homo sapiens monocyte) and HepG2 (Homo sapiens hepatic epithelial) cell lines.

Detailed Protocol Descriptions

Primary and Confirmatory Screening Details

Table 2Assays Conducted Using Primary Assay Protocol

View in own window

Assay Name	AID	Assay Type
High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media	AID 449762	Primary/Secondary
High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds	AID 493198	Secondary
High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (2)	AID 504556	Secondary
High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (3)	AID 504646	Secondary
High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (4)	AID 504857	Secondary
High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (5)	AID 504897	Secondary
High Throughput Screening Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in 7H9 Media using Purified and Synthesized Compounds (6)	AID 504903	Secondary

Notes

• Positive control compound is Amikacin at 2.5 μg/mL
• All test compounds are screened at 25 μM concentration for single dose screens and in 10 point dose response with a high concentration of 100 μg/mL and serial dilutions of 1:2.
• Compounds and controls are added to the plates at 2X concentration and given to the BSL3 personnel in charge of the assay execution.
• Assay in 384-well plate format contains 25 μL of media and compounds, 25 μL of inoculum and 9 μL of Alamar Blue endpoint.

Assay Protocol

Assay Materials

1. 7H9 complete medium (1 L):
   • Middlebrook 7H9 powder (BD, catalog # 271310) - 4.7 g
   • Glycerol (Fisher, catalog # G33) - 2 mL
   • Tween-80 (Sigma, catalog # P1754) - 0.5 mL
   • Water up to 900 mL
   • Autoclave, cool to 50 °C and add
   • 10× ADS – 100 mL
2. 10× ADS:
   • BSA (Sigma, catalog # A7638) - 25 g
   • Glucose (Sigma, catalog # G7528) – 10 g
   • NaCl (Fisher, catalog # S271) – 4.25 g
   • Water up to 500 mL
   • Filter sterilize
3. Alamar Blue Endpoint:
   • Alamar Blue (Trek Diagnostics)
   • Tween 80 (7.8% final)

Assay Method

Day -5: M. tb culture

1. In the BSL3 laboratory, start a culture of M. tb H37Rv from frozen stock in 10 mL 7H9 medium w/o Tween-80 (1:20 dilution of stock culture).
2. Grow M. tb culture at 37 °C for five days in a shaking water bath, until OD₆₀₀ = 0.3–0.9.

Day 0: Assay Setup

1. Test compounds are added to the assay plates before addition of inoculated media. Compounds are added in 50 μL Assay plates are then moved into the BSL-3 laboratory for bacteria addition.
2. M. tb culture is diluted to OD₆₀₀ = 0.001 in complete 7H9 media and 25 μL of inoculated media is added to each well of the assay plate using a Matrix WellMate.
3. Assay plates are incubated at 37 °C for 7 days.

Day 7: Endpoint Addition

1. Assay plates are removed from the incubator and 9 μL of Alamar Blue endpoint is added to each well.
2. Assay plates are returned to incubation for 18–24 hours.

Day 8: Endpoint Read

1. Plates are sealed with adhesive seals inside a biological safety cabinet.
2. Bacterial growth inhibition is assessed by reading fluorescence with an excitation at 530 nM and emission wavelength of 595 nm.

Counter Screen Assay Details

Table 3Assays Conducted Using Counter Screening Protocol

View in own window

Assay Name	AID	Assay Type
A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol	AID 449764	Target/Confirmatory Screen
A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds	AID 493181	Target/Confirmatory Screen
A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (2)	AID 504564	Target/Confirmatory Screen
A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (3)	AID 504645	Target/Confirmatory Screen
A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (4)	AID 504860	Target/Confirmatory Screen
A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (5)	AID 504898	Target/Confirmatory Screen
A High Throughput Confirmatory Assay used to Identify Novel Compounds that Inhibit Mycobacterium Tuberculosis in the absence of Glycerol using Purified and Synthesized Compounds (6)	AID 504901	Target/Confirmatory Screen

Cytotoxicity Secondary Assay Screening

Vero Cell Assay Protocol

Table 4Assays Conducted Using Vero Cell Cytotoxicity Screening Protocol

View in own window

Assay Name	AID	Assay Type
A Cell Based Secondary Assay To Explore Cytotoxicity of Compounds that Inhibit Mycobacterium Tuberculosis.	AID 435019	Anti-Target Screen Cytotoxicity
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis.	AID 504335	Anti-Target Screen Cytotoxicity
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (2)	AID 504562	Anti-Target Screen Cytotoxicity
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (3)	AID 504684	Anti-Target Screen Cytotoxicity
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (4)	AID 504854	Anti-Target Screen Cytotoxicity
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis (5)	AID 504909	Anti-Target Screen Cytotoxicity

Notes

• African green monkey kidney cells.
• Positive control compound is Hyamine at 100 μg/mL.
• All test compounds were screened in 10 point dose response with a high concentration of 40 μM and serial dilutions of 1:2.
• Compounds and controls are added to the plates at 5X concentration in 5 μL and then transferred to the screening center where diluted cells are added in 20 μL.
• Assay in 384-well plate format contains 5 μL media and compounds, 20 μL of cells and 25 μL of Cell-titer Glo endpoint reagent.

Assay Materials

1. ATCC complete growth medium (ATCC # 30-2003):
   • Minimum essential medium (Eagle)
   • 2 mM L-glutamine
   • Earle’s BSS adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM non-essential amino acids
   • 1.0 mM sodium pyruvate
   • Fetal bovine serum 10% (GIBCO # 16140-063)
   • Penicillin/Streptomycin (GIBCO # 15140-122)
   • 0.25% (w/v) Trypsin (GIBCO # 25200-056)
2. Vero Cells (ATCC# CCL-81)
3. Cell-Titer Glo (Promega)
4. Hyamine (Sigma)

Day 0: Assay Setup

1. Test compounds are added to the assay plates in 5 μL.
2. Frozen Vero cells are thawed and diluted in complete assay media. 20 μL of diluted cells are added to each well (2,500 cells/mL).
3. Assay plates are incubated at 37 °C with 5% CO₂ for 72 hours.

Day 3: Endpoint Read

1. Assay plates are taken out of incubation and equilibrated to room temperature for 10 minutes.
2. 25 μL of Cell-titer Glo is added to each well of the assay plates and the plates are incubated for 10 minutes at room temperature.
3. Cell viability is assessed by measuring luminescence on a PerkinElmer multimode Envision reader with an integration time of 0.1 second.

HepG2 Cell Assay Protocol

Table 5Assays Conducted Using HepG2 Cell Cytotoxicity Screening Protocol

View in own window

Assay Name	AID	Assay Type
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis in HepG2	AID 504642	Counter Screen Cytotoxicity
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis in HepG2 (2)	AID 504682	Counter Screen Cytotoxicity
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis in HepG2 (3)	AID 504853	Counter Screen Cytotoxicity
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis in HepG2 (4)	AID 504910	Counter Screen Cytotoxicity

Notes

• Human liver epithelials.
• Positive control compound is Hyamine at 100 μg/mL.
• All test compounds were screened in 10 point dose response with a high concentration of 20 μM and serial dilutions of 1:2.
• Compounds and controls are added to the plates at 5X concentration in 5 μL and then transferred to the screening center where diluted cells are added in 20 μL.
• Assay in 384-well plate format contains 5 μL media and compounds, 20 μL of cells and 25 μL of Cell-titer Glo endpoint reagent.
• HepG2 cells were subcultured every 7 days in complete E-MEM. Cells were passaged as needed, harvested from flasks using 0.25% trypsin-EDTA and maintained for no more than 20 passages.

Assay Materials

1. ATCC complete growth medium (ATCC # 30-2003):
   • Minimum essential medium (Eagle)
   • Fetal bovine serum 10% (GIBCO # 16140-063)
   • Penicillin/Streptomycin (GIBCO # 15140-122)
2. HepG2 Cells (ATCC# HB-8065)
3. Cell-Titer Glo (Promega)
4. Hyamine (Sigma)

Day 0: Assay Setup

1. Test compounds are added to the assay plates in 5 μL.
2. Cultured cells were harvested from 175 cm³ flasks and diluted in complete assay media. 20 μL of diluted cells are added to each well (3,000 cells/mL).
3. Assay plates are incubated at 37 °C with 5% CO₂ for 72 hours.

Day 3: Endpoint Read

1. Assay plates are taken out of incubation and equilibrated to room temperature for 10 minutes.
2. 25 μL of Cell-titer Glo is added to each well of the assay plates and the plates are incubated for 10 minutes at room temperature.
3. Cell viability is assessed by measuring luminescence on a PerkinElmer multimode Envision reader with an integration time of 0.1 second.

THP-1 Cell Assay Protocol

Table 6Assays Conducted Using THP-1 Cell Cytotoxicity Screening Protocol

View in own window

Assay Name	AID	Assay Type
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis in THP-1 Cells	AID 504640	Counter Screen Cytotoxicity
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis in THP-1 Cells (2)	AID 504683	Counter Screen Cytotoxicity
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis in THP-1 Cells (3)	AID 504852	Counter Screen Cytotoxicity
A Cell Based Secondary Assay To Explore Cytotoxicity of Purified and Synthesized Compounds that Inhibit Mycobacterium Tuberculosis in THP-1 Cells (4)	AID 504911	Counter Screen Cytotoxicity

Notes

• Human monocyte.
• Positive control compound is Hyamine at 100 μg/mL.
• All test compounds were screened in 10 point dose response with a high concentration of 40 μM and serial dilutions of 1:2.
• Compounds and controls are added to the plates at 5X concentration in 5 μL and then transferred to the screening center where diluted cells are added in 20 μL.
• Assay in 384-well plate format contains 5 μL media and compounds, 20 μL of cells and 25 μL of Cell-titer Glo endpoint reagent.
• THP-1 cells were subcultured every 7 days in complete RPMI. Cells were passaged as needed, harvested from flasks using 0.25% trypsin-EDTA and maintained for no more than 20 passages.

Assay Materials

1. ATCC Complete Assay Media (ATCC 30-2011):
   • RPMI-1640 (ATCC)
   • Fetal bovine serum 10% (GIBCO # 16140-063)
   • Penicillin/Streptomycin (GIBCO # 15140-122)
   • 0.05 mM β-mercaptoethanol (Sigma, M3148)
2. THP-1 Cells (ATCC# TIB-202)
3. Cell-Titer Glo (Promega)
4. Hyamine (Sigma)

Day 0: Assay Setup

1. Test compounds are added to the assay plates in 5 μL.
2. Cultured cells were harvested from 175 cm³ flasks and diluted in complete assay media. 20 μL of diluted cells are added to each well (3,000 cells/mL).
3. Assay plates are incubated at 37 °C with 5% CO₂ for 72 hours.

Day 3: Endpoint Read

1. Assay plates are taken out of incubation and equilibrated to room temperature for 10 minutes.
2. 25 μL of Cell-titer Glo is added to each well of the assay plates and the plates are incubated for 10 minutes at room temperature.
3. Cell viability is assessed by measuring luminescence on a PerkinElmer multimode Envision reader with an integration time of 0.1 second.

2.2. Probe Chemical Characterization

Probe Structure and Properties

Figure 1Synthetic Route

¹H NMR (DMSO-d₆; 400 MHz) δ 12.67 (s, 1H), 8.15 (br d, J = 7.8 Hz, 1H), 8.04 (dd, J = 1.2, 7.9 Hz, 1H), 7.78 (m, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.42 (m, 1H), 3.92 (s, 2H), 3.54 (m, 1H), 1.78 – 1.61 (m, 4H), 1.53 (m, 1H), 1.31 – 1.06 (m, 5H).

¹³C NMR (DMSO-d₆; 125 MHz) δ 165.9, 161.1, 155.5, 148.3, 134.7, 126.1, 125.9, 125.7, 120.0, 48.0, 34.2, 32.3, 25.2, 24.4.

LC-MS (ESI⁺): Purity at 214 nm is 100%. HRMS: 318.1271 (calcd for C₁₆H₂₀N₃O₂S⁺); 318.1268 (found/[M+H]⁺).

Elemental analysis (%): calcd for C₁₆H₁₉N₃O₂S: C 60.54, H 6.03, N 13.24, S 10.10; found: C 60.27, H 5.93, N 13.16, S 10.10.

Solubility

Aqueous solubility was measured in phosphate buffered saline (PBS) at room temperature (23 °C). PBS by definition is 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pH of 7.4. The solubility of probe SID 13147611 (CID 2792221) was determined to be 5.5 μg/mL at pH 7.4.

Stability

Aqueous stability was measured at room temperature (23 °C) in PBS (no antioxidants or other protectants and DMSO concentration below 0.1%). Stability data are depicted as a graph showing the loss of compound versus time over a 48 hour period, with a minimum of 6 time points and the percent compound remaining at the end of 48 hours (Figure 3). Since the starting compound concentration for this procedure used the kinetic solubility as determined in Figure 2, it is not uncommon to observe apparent compound instability, which in reality, may be more appropriately assigned to compound insolubility. For this reason, the compound stability measurement was repeated with the addition of acetonitrile (50% v/v final). The stability data for these two experiments are depicted as a graph showing the loss of compound versus time over a 48 hour period with a minimum of 6 time points and the percent compound remaining at the end of 48 hours.

Figure 3

Stability for compound SID 103147611 (CID 2792221) in 100% aqueous and 50% acetonitrile/aqueous.

Figure 2

Structure Verification.

The apparent stability of probe compound SID 103147611 (CID 2792221), determined as the percent of compound remaining after 48 hours, was 66% in 100% aqueous solution versus 100% in 50% acetonitrile/aqueous solution. We propose that the difference observed for these measurements is due to gradual compound precipitation in 100% aqueous solution.

Submission of Probe and Five Related Analogs to the MLSMR

The structures for the supporting analogs of ML242 are shown in Figure 4. The associated compound identifier, source, and amount information for the probe and supporting analogs is in provided in Table 7.

Figure 4

Probe analogs for which samples were submitted to the MLSMR.

Table 7

Probe and analog submissions to MLSMR (BioFocus DPI) for inhibitors of M. tb in 7H9 media.

2.3. Probe Preparation

N-Cyclohexyl-2-(4-oxo-3,4-dihydroquinazolin-2-ylthio)acetamide: Neat chloroacetyl chloride (180 μL; 2.26 mmol) was added to a solution of cyclohexylamine (209 mg; 2.11 mmol) and DIPEA (0.80 mL; 4.84 mmol) in DMF (2 mL) cooled in an ice-water bath. Solid 2-thioxo-2,3-dihydroquinazolin-4(1H)-one (413 mg; 2.32 mmol) was added over 1 min. A precipitate formed over time. After 99 h, the material was suspended in DMF and the solid collected by filtration. The filter cake resulted in pure product as a grey solid after drying in vacuo (106 mg; 16%). Water was added to the filtrate and the precipitate was collected on a Büchner funnel, washed with water, then dried in vacuo to afford N-cyclohexyl-2-(4-oxo-3,4-dihydroquinazolin-2-ylthio)acetamide as an off-white solid (326 mg; 49%; SID 103147611). Total yield: 432 mg (65%). A subsequent lot (SID 123101665) was similarly prepared, except that 0.2 M citric acid was added to the crude reaction in DMF and the precipitate collected by filtration and dried in vacuo. This crude material was purified by MPLC (silica gel using a 0–10% i-PrOH in DCM gradient). The column fractions were pooled and concentrated, suspended in methanol and collected by filtration, dried, then recrystallized from 3:1 95% ethanol:water yielding pure material after drying in vacuo at 40 °C. Melting point (mp 232.9–237.6 °C), elemental analysis and an infrared spectrum were taken on this later lot. See Section 2.2.3 for analytical characterization data.

3. Results

3.1. Dose Response Curves for Probe

The concentration-dependence curve for inhibition of M. tb growth by the probe ML242 (CID 2792221) is displayed in Figure 5. The average IC₅₀ value for the initial compound lot (SID 103147611) in 7H9 media is 306 nM; the IC₉₀ value is 476 nM. The IC₅₀ and IC₉₀ values for the second compound lot (SID 123101665) are 21 nM and 80 nM, respectively.

Figure 5

Inhibition of Mycobacterium tuberculosis growth by ML242. Colored symbols and lines are for SID 103147611, the initial synthesis of the probe. Black and white symbols and lines are for SID 123101665, the second synthesis of the probe.

The probe has little to no cytotoxicity in mammalian cells (Figure 6).

Figure 6

Effect of ML242 (CID 2792221) on three mammalian cell lines.

3.3. Profiling Assays

In the absence of a known biomolecular target, the “selectivity” for the probe compound ML242 was evaluated using the LeadProfilingScreen (Ricerca Biosciences). This screen employs a suite of assays across 68 biomolecular targets. In summary, at a concentration of 10 μM, the probe compound did not inhibit any of the 68 biomolecular targets at greater than 29%, and the majority of targets were not inhibited at greater than 10%.

In Vitro Pharmacology Profiles (see Table 8) of probe ML242 (CID 2792221):

Table 8

Summary of in vitro ADME/T Properties for probe ML242.

ML242 was soluble at >10 μM across all pHs tested, which is >100 times the IC₅₀ measured against M. tb in 7H9 media.

The Parallel Artificial Membrane Permeability Assay (PAMPA) assay is used as an in vitro model of passive, transcellular permeability. An artificial membrane immobilized on a filter was placed between a donor and acceptor compartment. At the start of the test, compound was introduced in the donor compartment. Following the permeation period, the concentration of drug in the donor and acceptor compartments were measured using UV spectroscopy. In this assay, probe ML242 (CID 2792221) had good permeability.

Plasma protein binding is a measure of a drug’s efficiency to bind to the proteins within blood plasma. The less bound a drug is, the more efficiently it can traverse cell membranes or diffuse. Compounds that exhibit high plasma protein binding are confined to the vascular space, thereby having a relatively low volume of distribution. In contrast, compounds that remain largely unbound in plasma are generally available for distribution to other organs and tissues. ML242 (CID 2792221) was highly bound to human plasma (>99%) and much less highly bound to mouse plasma (85%).

Plasma stability is a measure of the stability of small molecules and peptides in plasma and is an important parameter, which can influence the in vivo efficacy of a test compound. Drug candidates are exposed in plasma to enzymatic processes (proteinases, esterases, etc.), and they can undergo intramolecular rearrangement or bind irreversibly (covalently) to proteins. ML242 (CID 2792221) showed excellent stability in mouse plasma (100%) and marginal stability in human plasma (65%).

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. This assay addresses the pharmacologic question of how long the parent compound will remain circulating in plasma within the body. ML242 (CID 2792221) exhibited acceptable stability upon incubation with human microsomes, and was much more rapidly metabolized by mouse microsomes.

ML242 (CID 2792221) showed no toxicity toward human hepatocyctes.

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

The efficacy and cytotoxicity for drugs currently used for the treatment of TB in our assays are shown in Table 9. The probe, ML242, compares favorably to all of these compounds in terms of in vitro efficacy and cytotoxicity towards the three mammalian cell lines studied.

Table 9

Data for marketed and select literature antitubercular drugs in assays used in this report.

Close examination of the literature has revealed a small number of instances where the thioacetamide substructure has been used in the context of antitubercular studies. Murav’eva et al. reported data for six compounds, the most active of which was VI.⁶ Murav’eva et al. concluded that “None of the prepared compounds had significant tuberculostatic activity against strain H-37 in vitro.”⁷ The authors did not specify which isomer “butyl” or “C₄H₉” refers to. In our hands, compound VI (Table 9, Entry 10), where “C₄H₉” = n-butyl, had modest activity (IC₅₀ = 1.1 μM).

More recently, and subsequent to our HTS screen and initiation of work on the thioacetamide chemotype, Vasan et al. reported on an HTS screen run against purified salicylate synthase from M. tb.⁸ In this work, there was a single instance of the thioacetamide chemotype in a data table and it was not otherwise noted or followed up on. It is reasonable to think that salicylate synthase (MbtI) could be a biological target of our proposed probe; however, the exact compound reported in the literature as a 2–3 micromolar inhibitor of salicylate synthase from M. tb was screened as part of the NIH MLSMR compound collection during our primary HTS, and the compound showed 6.9% inhibition at 25 μM concentration. In our dose response assay, this compound did not show measurable activity (Table 9, Entry 11). Given the much greater potency of the probe compound (ML242; 306 nM) compared to the relatively low potency reported for CID 3237435 in the biochemical salicylate synthase assay and our phenotypic screen, it would be surprising if the present probe confirms as functioning via salicylate synthase inhibition.

5. References

1.

Organization WH. Global tuberculosis control 2010. World Health Organization; 2010.

2.

Di L, Kerns EH. Biological assay challenges from compound solubility: strategies for bioassay optimization. Drug discovery today. 2006;11(9–10):446–51. [PubMed: 16635808]

3.

Pethe K, Sequeira PC, Agarwalla S, Rhee K, Kuhen K, Phong WY, Patel V, Beer D, Walker JR, Duraiswamy J, Jiricek J, Keller TH, Chatterjee A, Tan MP, Ujjini M, Rao SP, Camacho L, Bifani P, Mak PA, Ma I, Barnes SW, Chen Z, Plouffe D, Thayalan P, Ng SH, Au M, Lee BH, Tan BH, Ravindran S, Nanjundappa M, Lin X, Goh A, Lakshminarayana SB, Shoen C, Cynamon M, Kreiswirth B, Dartois V, Peters EC, Glynne R, Brenner S, Dick T. A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy. Nature communications. 2010;1:57. [PMC free article: PMC3220188] [PubMed: 20975714]

4.

Ananthan S, Faaleolea ER, Goldman RC, Hobrath JV, Kwong CD, Laughon BE, Maddry JA, Mehta A, Rasmussen L, Reynolds RC, Secrist JA 3rd, Shindo N, Showe DN, Sosa MI, Suling WJ, White EL. High-throughput screening for inhibitors of Mycobacterium tuberculosis H37Rv. Tuberculosis (Edinb). 2009;89(5):334–53. [PMC free article: PMC3255569] [PubMed: 19758845]

5.

Maddry JA, Ananthan S, Goldman RC, Hobrath JV, Kwong CD, Maddox C, Rasmussen L, Reynolds RC, Secrist JA 3rd, Sosa MI, White EL, Zhang W. Antituberculosis activity of the molecular libraries screening center network library. Tuberculosis (Edinb). 2009;89(5):354–63. [PMC free article: PMC2792876] [PubMed: 19783214]

6.

Murav’eva KM, Arkhangel’skaya NV, Shchukina MN, Zykova TN, Pershin GN. The synthesis and tuberculostatic activity of derivatives of 4-oxoquinazolyl-2-mercaptoacetic acid. Khimiko-Farmatsevticheskii Zhurnal. 1967;1:12–4.

7.

Chemical Abstracts Service Accession Number. 69:19117.

8.

Vasan M, Neres J, Williams J, Wilson DJ, Teitelbaum AM, Remmel RP, Aldrich CC. Inhibitors of the salicylate synthase (MbtI) from Mycobacterium tuberculosis discovered by high-throughput screening. Chem Med Chem. 2010;5(12):2079–2087. [PMC free article: PMC3021963] [PubMed: 21053346]