Prior Art

Cruzain inhibitors 2⁵,3⁶ and our recently reported ML probe (ML092), all utilize an electrophilic warhead moiety which is attacked by the active-site cysteine moiety to form a colavent interaction. Both the vinyl sulfone (compound 2) and the tetrafluorophenoxymethyl ketone (compound 3) act by irreversible inhibition, while the purine nitrile (ML092) can be classified as a reversible, covalent inhibitor of cruzain. In contrast, our other ML probe (ML091), was initially found to be a reversible, non-covalent inhibitor.⁷ All of the covalent inhibitors of cruzain (shown above) exhibit potent in vitro inhibition of cruzain (2: IC₅₀ = 1.5 nM, 3: Ki = 460 nM, and ML092: IC₅₀ = 0.2 nM, whereas the noncovalent inhibitor, ML091, has an IC₅₀ value of 1.2 μM. Despite the potent activity of compound 3, the T. Cruzi IC₅₀ was 5.1 μM and the compound had a host cell IC₅₀ value of >10 μM, indicating limited selectivity. Our previously reported probe compound, ML092, was found to have potent efficacy against T. Cruzi but was also fairly toxic to the host cell and non-covalent inhibitor, ML091, was found to have minimal effect on the T. Cruzi parasite. In contrast, the probe described in this report exhibits T. Cruzi activity comparable to irreversible covalent inhibitors (2 and 3), is non-toxic to the host cell, and displays in vivo activity in a Chagas disease mouse model at 10 mg/kg SID. While both 2 and 3 also show favorable activity in mouse models, the frequency of treatment and dosing levels are higher, at 100 mg/kg and 20 mg/kg BID respectively.

2.1. Assays

Kinetic Fluorogenic qHTS Assay for Inhibitors of Cruzain: Three μL of reagents were dispensed into 1536-well Greiner black solid-bottom assay plate. Compounds and controls (23 nL) were transferred via Kalypsys PinTool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, Palo Alto, CA). The plate was incubated for 15 min at room temperature, and then a 1 μL aliquot of 8 μM substrate solution was added to start the reaction. The plate was transferred to ViewLux high-throughput CCD imager (Perkin-Elmer, Waltham, MA), where kinetic measurements (4 reads, one read every 30 seconds) of the AMC fluorescence were acquired using standard 340 nm excitation and 450 nm emission filter sets. During dispense, reagent bottles were kept submerged into a 4 °C recirculating chiller bath and all liquid lines were covered with aluminum foil to minimize fluorophore degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys Inc, San Diego, CA) as described elsewhere. Plates containing DMSO only (instead of compound solutions) were included approximately every 50 plates throughout the screen to monitor any systematic trend in the assay signal associated with reagent dispenser variation, or any decrease in enzyme specific activity. Pubchem AID 1478.

Kinetic Fluorogenic qHTS Assay for Inhibitors of Cruzain (Detergent-Free Screen): Three μL of reagents were dispensed into 1536-well Greiner black solid-bottom assay plate. Compounds and controls (23 nL) were transferred via Kalypsys PinTool equipped with 1536-pin array (10 nL slotted pins, V&P Scientific, Palo Alto, CA). The plate was incubated for 15 min at room temperature, and then a 1 μL aliquot of 8 μM substrate solution was added to start the reaction. The plate was transferred to ViewLux high-throughput CCD imager (Perkin-Elmer, Waltham, MA), where kinetic measurements (4 reads, one read every 30 seconds) of the AMC fluorescence were acquired using standard 340 nm excitation and 450 nm emission filter sets. During dispense, reagent bottles were kept submerged into a 4 °C recirculating chiller bath, and all liquid lines were covered with aluminum foil to minimize fluorophore degradation. All screening operations were performed on a fully integrated robotic system (Kalypsys Inc, San Diego, CA) as described elsewhere. Plates containing DMSO only (instead of compound solutions) were included approximately every 50 plates throughout the screen to monitor any systematic trend in the assay signal associated with reagent dispenser variation or decrease in enzyme specific activity. Pubchem AID 1476.

Confirmatory Assay: Top inhibitors, including some potentially covalent modifiers such as triazine nitriles, were cherry-picked for follow-up confirmation using the primary screening protocol. Several commercially available compounds, including new benzimidazoles, were also purchased for follow-up based on the inhibitor clusters. A total of 599 compounds were tested in the confirmatory assay. All original qHTS actives confirmed while several of the newly purchased analogs were inactive. Pubchem AID 2158.

qHTS Counterscreen Assay for Papain Inhibition: Although high selectivity was not a stated prerequisite for probe nomination, we wished to evaluate the selectivity of our top small molecules against related proteases. Papain, another cysteine protease known to utilize the same Z-FR-AMC fluorogenic substrate, was selected as a convenient profiling target. The fluorogenic assay used for cruzain was used, with the exception of the reducing agent, which was 5 mM cystein instead of DTT. A total of 414 compounds were tested in this counter screen assay. The benzimidazole series was inactive against papain, though other series such as the triazine nitriles, inhibited papain. Pubchem AID 2161.

T. Cruzi Growth Inhibition Assay:⁸ NIH/3T3 cells and parasites were harvested, washed once and resuspended in DMEM supplemented with 2% FBS and Pen-Strep-Glut. DMEM did not contain phenol red to avoid interference with the assay absorbance readings at 590 nM. Different numbers of NIH/3T3 cells were seeded in 96-well plates. After 3 hours, compounds were added at the indicated concentrations and mixed by pipetting. BZN tablets (Rochagan, Roche) dissolved in DMSO and 4 μM Amphotericin B solution (Sigma-Aldrich) were used as positive controls. Different numbers of T. cruzi parasites were added in a final volume of 200 μl/well. After 4 days, 50 μL of PBS containing 0.5% of the detergent NP40 and 100 μM Chlorophenol Red-β-D-galactoside (CPRG) (Fluka) were added. Plates were incubated at 37 °C for 4 hours and absorbance was read at 590 nm using a Tecan Spectra Mini plate reader. To calculate the Z′ factor, we used the formula described by Zhang et al.:⁹ Z′ = 1−[(3σ_c++3σ_c−)/|μ_c+−μ_c−|] where σ_c+ = standard deviation (SD) of positive control, σ_c− = SD of negative control, μ_c+ = mean of positive control, μ_c− = mean of negative control. Subsequently, the best ratio was used for all growth inhibition assays (50,000 cells and parasites, multiplicity of infection (MOI) 1:1). To determine IC₅₀ values, β-gal activity (Abs₅₉₀) was plotted against compound concentration for each compound. The IC₅₀ was determined as the concentration at which the activity (absorbance) was half that in the absence of compound. Mean IC₅₀ values are the average of independent experiments performed in triplicate on three different days.

Cytotoxicity Assay: Cells (NIH/3T3) were washed, plated using 200 μL at a density of 50,000 cells/well in 96-well plates, and were allowed to adhere for 3 hours. Twenty-four hour assays were done in DMEM without phenol red supplemented with 10% FBS and Pen-Strep-Glut, while 4-day assays were done in the same medium containing 2% FBS. Drugs were added and mixed. After 1 or 4 days, 20 μL of Alamar Blue (Biosource, Invitrogen) was added. Plates were incubated for 4 – 6 hours (NIH/3T3) at 37 °C and fluorescence was read using a Labsystems Fluoroskan II plate reader (excitation: 544 nm, emission: 590 nm). To determine TC₅₀ values, fluorescence was plotted against inhibitor concentration. TC₅₀ was determined as the concentration at which cytotoxicity (fluorescence) was half that in the absence of inhibitor.

2.2. Probe Chemical Characterization

*Purity >95% as judged by LC/MS and ¹H NMR

N-(2-(1-(2-(p-tolyloxy)ethyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclohexanecarboxamide.¹H NMR (CDCl₃) δ 1.09–1.28 (m, 3 H), 1.29–1.51 (m, 2 H), 1.53–1.88 (m, 5 H), 1.96–2.11 (m, 2 H), 2.24 (s, 3 H), 3.17 (t, J = 6.0 Hz, 2 H), 3.86 (q, J = 6.0 Hz, 2 H), 4.22 (t, J = 5.0 Hz, 2 H), 4.49 (t, J = 4.6 Hz, 2 H), 6.66 (d, J = 8.6 Hz, 2 H), 6.94 (brs, 1 H), 7.01 (d, J = 8.6 Hz, 2 H), 7.20–7.32 (m, 1 H), 7.36–7.46 (m, 1 H) and 7.66–7.81 (m, 1 H). ¹³C (CDCl₃) δ 20.40, 20.44, 25.58, 25.70, 25.74, 27.31, 29.25, 29.53, 36.09, 43.23, 45.44, 66.01, 109.37, 114.10, 119.15, 119.18, 122.18, 122.46, 129.95, 130.73, 134.93, 142.39, 153.70, 155.76 and 176.31. HRMS (ESI) m/z (M+H)⁺ calcd. for C₂₅H₃₂N₃O₂N, 406.2489; found 406.2499.

Probe in vitro ADME properties

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Compound	aq. Kinetic sol. (PBS @ pH 7.4)	Caco-2 (Papp 10−6 m/s @ pH 7.4)	efflux ratio (B→A)/(A→B)	mouse liver microsome stability (T1/2)	PBS-pH 7.4 Stability:% remaining after 48 hrs	Mouse plasma stability:% remaining after 48 hrs
8	5.0	4.1	1.1	<10 min.	100%	95

Figure 1Buffer Stability (48 hours at 25 °C) of ML217

Percent remaining after 48h = 100%

2.3. Probe Preparation

Preparation of tert-butyl 2-(1H-benzo[d]imidazol-2-yl)ethylcarbamate (Step 1). BOC-anhydride (0.99 mL, 4.27 mmol), 1 N NaOH (15 mL, 14.95 mmol) was added to a suspension of commercially available 2-(1H-benzo[d]imidazol-2-yl)ethanamine dihydrochloride (Sigma Alrich) (1.00 g, 4.27 mmol) in CH₂Cl₂. The biphasic reaction was stirred at room temperature for 5 hrs, then the solids were removed by fitration, washed with CH₂Cl₂, concentration under reduced pressure then under high vacuum to give a greater than 98% pure product in a 70% yield. LC-MS (min) = 2.69 min; ¹H NMR (DMSO-d₆) δ 1.35 (s, 9 H), 2.91 (t, J = 7.3 Hz, 2 H), 3.36 (m, 2 H), 6.95 (brs, 1 h), 7.08 (dd, J = 6.0 Hz and 3.2 Hz, 2 H) and 7.44 (dd, J = 6.0 Hz and 3.2 Hz, 2 H).

Preparation of tert-butyl-2-(1-(2-(p-tolyloxy)ethyl)-1H-benzo[d]imidazol-2-yl)ethylcarbamate (step 2). General procedure for alkylation: A suspension of tert-butyl 2-(1H-benzo[d]imidazol-2-yl)ethylcarbamate (0.52 mg, 1.99 mmol), 1-(2-bromoethoxy)-4-methylbenzene (0.43 g, 1.99 mmol), and cesium carbonate (1.95 g, 5.97 mmol) in acetonitrile/DMF (10 ml/5 ml) was heated to 8 5°C in for 4 hrs. The reaction mixture was cooled to room temperature, filtered and the solids were washed with acetone, and concentrated leaving only DMF remaining. The resulting residue was diluted with ethyl acetate and washed three times with water, dried with Na₂SO₄, filtered, and concentrated to yield clear glass-like oil in 89% yield. LC-MS (min) 3.14; ¹H NMR (CDCl₃) δ 1.14 (s, 9 H), 2.23 (s, 3H), 3.15 (t, J = 6.0 Hz, 2 H), 3.75 (d, J = 6.0 Hz, 2 H), 4.21 (t, J = 5.4 Hz, 2 H), 4.49 (t, J = 5.3 Hz, 2 H), 6.67 (d, J = 6.0 Hz, 2 H), 7.00 (d, J = 6.0 Hz, 2 H), 7.18–7.27 (m, 2H), 7.43 (d, J = 4.3 Hz, 1H) and 7.70 (d, J = 4.2 Hz, 1 H).

Preparation of 2-(1-(2-(p-tolyloxy)ethyl)-1H-benzo[d]imidazol-2-yl)ethanamine dihydrochloride (step 3). Tert-butyl-2-(1-(2-(p-tolyloxy)ethyl)-1H-benzo[d]imidazol-2-yl)ethylcarbamate was treated with 4M HCl/Dioxane and stirred for 2 hrs. The reaction mixture was then filtered and the solids were washed with CH₂Cl₂. The resulting colorless solid was dried under high vacuum and carried used in the next reaction without further purification. LC-MS (min) = 2.73.

Preparation ofN-(2-(1-(2-(p-tolyloxy)ethyl)-1H-benzo[d]imidazol-2-yl)ethyl) cyclohexanecarboxamide [ML217)] (Step 4). Diisopropylethylamine (71.0 μL, 0.41 mmol) was added to a solution containing 2-(1-(2-(p-tolyloxy)ethyl)-1H-benzo[d]imidazol-2-yl)ethanamine dihydrochloride (0.050 g, 0.14 mmol) in CH₂Cl₂ (1.5 mL). The resulting suspension was stirred until clear, then cyclohexanecarbonyl chloride (74 μL, 0.14 mmol) was added. The reaction mixture for 30 min. at room temperature was then diluted with CH₂Cl₂ and washed with 1N NaOH, brine and dried over Na₂SO₄. The organic layer was filtered then concentrated under reduced pressure and purified by reversed-phase HPLC (see methods section for details).

General Synthesis for Various Phenoxy-Derivatives (Mitsunobu Reaction). A solution containing DIAD (0.13 mL, 0.67 mmol), and Ph₃P (0.18g, 0.67 mmol) in toluene/THF was stirred at room temperature for 15 min. The requisite phenol (0.62 mmol) was added and the reaction mixture was stirred at room temperature for 15 min before cooling in an ice bath. N-(2-(1-(2-hydroxyethyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclohexanecarboxamide (0.14 g, 0.44 mmol) was then added to the cooled solution and allowed to stir at this temperature for 30 min, at which time was warmed to room temperature and stirred for an additional 3 hrs. The resulting orange solution was poured into ethyl acetate and washed with water, 1 N NaOH, and brine, then dried over Na₂SO₄, filtered and concentrated under reduced pressure to yield an orange oil, which was purified by reversed-phase HPLC.

3.1. Summary of Screening Results

To measure the enzymatic activity of cruzain, we utilized its model fluorogenic substrate Z-Phe-Arg-AMC, which is converted to a highly fluorescent 7-amino-4-methylcoumarin reporter upon cruzain-catalyzed hydrolysis. Assay optimization was performed directly in 1,536-well format at a final reaction volume of 4 μL. For the detergent-present screen, Triton X-100 was used at 0.01%, and the cruzain concentration selected for screening was 1.5 nM. During the optimization of the detergent-free assay, low stability and high variability in the specific activity of cruzain was noted, likely due to protein denaturation promoted by the large surface-to-volume ratio in the 1,536-well polystyrene assay plates. In order to stabilize the enzyme’s performance, its final concentration was raised to 3 nM and a trace of Triton X-100 (final concentration of 0.00005%) was included in the detergent-free assay; we note that a similar adjustment step was needed for the AmpC beta-lactamase aggregation screen described earlier. At the conditions selected – 1.5 nM or 3 nM final cruzain concentration and 2 μM final substrate concentration (the latter was chosen to match previously reported conditions and being close to the K_m value for this substrate) – the signal evolution was robust, and low substrate conversion was monitored over the course of 1 minute, making the assay highly sensitive to cruzain inhibitors. All assay components were tested and found stable for at least 24 hours when formulated as stock solutions at their working concentrations, in both the detergent-present and the detergent-free buffers (data not shown). Such demonstrated stability permitted the implementation of an unattended overnight screening operation.

The screens of the 197,864-compound collection were completed within a period of two separate workweeks. During the first week, approximately 60% of the 1,107 1,536-well plates library were screened in the detergent-free assay first; this was immediately followed by a screen of the same set of library plates in the detergent-present assay. The remaining 40% of the library was screened in the same manner during the second week. The screening of each compound against the two assays in close succession minimized the possibility for sample-age related differences in results. Overall, two sets of 1,107 1,536-well assay plates were run under the detergent-free and detergent-present conditions, respectively, leading to the generation of 197,864 concentration responses consisting of at least seven points per compound per assay, and corresponding to a total of approximately 1.5 million samples tested per detergent condition.

The Z′ screening factors associated with each plate and each screening condition remained high and stable throughout the two screens (Figure 2): the average Z′ for the detergent-free screen was 0.78, while the corresponding average for the detergent-present screen was 0.93. Of the two screens comprising a total of 2,214 plates, only a group of six plates failed and was re-screened immediately using the same batches of enzyme and substrate. As a further quality control measure, we included a concentration response of the known vinyl sulfone cruzain inhibitor K1177721 (2), added as a 16-point dilution series in duplicate between 5.7 μM and 0.175 nM into the second column of every assay plate. The shape and quality of the concentration response remained consistent throughout both screens (Figure 2b, green data points) with the associated minimum significant ratios¹⁰ of 2.5 and 1.4 for the detergent-free and detergent-present conditions, respectively, further indicating stable runs.

Figure 2

Cruzain dual-qHTS Screening Performance (Z′ trend (A) and intraplate control titrations (B)). Detergent-sensitive Z′ trend and control titration is shown on left and detergent-insensitive on the right.

The cumulative effect of all library compounds on the cruzain activity at each screening condition is shown in Figure 3. On the plots, concentration responses were color-coded and positionally sorted based on activity with inactive samples (flat concentration responses) represented by the black dots, activators in red, and inhibitors in blue; green points in the very front of the 3D plots represented the 1,107 duplicate responses of the intra-plate control titration of K11777 (2). Similar to our AmpC beta-lactamase profiling, the outcomes from the detergent-free versus detergent-present screens were strikingly different: the detergent-free screen yield over 15 times more hits than its detergent-present counterpart. Also, as noted previously, we observed a large number (~12,000) of apparent activators in the detergent-free screen. Of these samples, almost all turned completely inactive upon inclusion of detergent. Over 85% of the activators were associated with partial or single-point top concentration responses, indicating that the condition-dependent activation was being observed only at the highest compound concentrations where complicating phenomena such as transient precipitation, light scatter, and compound aggregate-assisted enzyme stabilization have been known to lead to false positive effects. We did not consider those compounds further.

Figure 3

Full qHTS activity data for detergent-sensitive (left) and detergent-insensitive (right) screens.

The two primary screens led to a large number of potential actives that needed substantial filtering. A large percentage of the actives (12,746) were detergent-sensitive ‘activators’ as mentioned above. These were identified and filtered out using our curve classification method.¹¹ The screen with detergent helped eliminate another 10,399 detergent-sensitive weak curves. An additional 3,844 detergent-sensitive inhibitors gave significant response but were inactive in the presence of detergent. These were considered likely aggregators and were filtered out. Next, the kinetic reads captured in the primary screen provided background auto-fluorescence data, which were used to reject 507 false positive concentration response curves. Substructure patterns of 243 reactive and problematic functional groups were used to filter out another 428 compounds. Finally, 550 weak inhibitors were filtered out due to low potency. The remaining 493 compounds were clustered, and among the chemotypes identified was a benzimidazole-based series.

3.2. Dose Response Curves for Probe

T. Cruzi growth inhibition activity vs. cytotoxicity is shown in Figure 4.

Figure 4

T. Cruzi growth inhibition assay for compound 15. Solid line represents parasite killing; IC₅₀ values were determined as the concentration at which the activity (Abs₅₉₀) of β-gal was half that in the absence of compound. Dotted line represents (more...)

3.3. Scaffold/Moiety Chemical Liabilities

While many of the series found in the qHTS contained reactive functional groups, the series chosen for probe development did not contain any serious liabilities.

3.4. SAR Tables

Table 1Cruzain inhibition of representative analogs

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Entry	CID	SID	NCGC IDs	IC50 (μM)
4	17756962	29216479	NCGC00164730	>57
5	6625411	10422459	NCGC00241489	6.4
6	49853116	10422460	NCGC00241503	>57
7	49853253	10422449	NCGC00238716	>57
8	49853280	10422451	NCGC00238727	16
9	49853229	10422449	NCGC00238715	>57
10	49853176	10422459	NCGC00241495	8.1

Note: all compounds in table were synthesized at NCGC. IC₅₀ values represent the average of three separate experiments.

Table 2Cruzain inhibition of representative analogs

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Entry	CID	SID	NCGC IDs	R1	R2	Y	IC50 (μM)
11	49853104	104224595	NCGC00241494	cyclohexane	4-Me	CH2CH2	5.7
12	49852854	104224505	NCGC00238722	cyclopentane	4-Me	CH2O	2.6
13	49852929	104224586	NCGC00241485	cycloheptane	4-Me	CH2O	2.6
14	16975202	104224511	NCGC00238728	cyclopropane	4-Me	CH2O	>57
15	3159995	104219745	NCGC00114713	cyclohexane	4-Me	CH2O	1.8
16	16012645	104219743	NCGC00114709	cyclohexane	2,5-dimethyl	CH2O	1.3
17	49853384	104224488	NCGC00238705	cyclohexane	4-CF3	CH2O	9.1
18	3159992	85736068	NCGC00168420	cyclohexane	H	CH2O	1.8
19	49853102	104224736	NCGC00241883	cyclohexane	3-NH2	CH2O	3.2
20	49853027	104224733	NCGC00241880	cyclohexane	2-NH2	CH2O	4.6
21	49853316	104224724	NCGC00241871	cyclohexane	3-Br	CH2O	3.6
22	16975770	104224489	NCGC00238706	phenyl	4-Me	CH2O	12.9
23	49852783	104224598	NCGC00241497	2-fluorophenyl	4-Me	CH2O	6.5
24	49852821	104224599	NCGC00241498	3-fluorophenyl	4-Me	CH2O	6.5
25	49853364	104224597	NCGC00241496	2,4-difluorophenyl	4-Me	CH2O	5.1
26	49853319	104224663	NCGC00241724	3,5-difluorophenyl	4-Me	CH2O	14.5
27	49852983	104224648	NCGC00241709	2-bromophenyl	4-Me	CH2O	12.9
28	16977961	104224650	NCGC00241711	3-chlorophenyl	4-Me	CH2O	9.1
29	16977185	104224654	NCGC00241715	4-chlorophenyl	4-Me	CH2O	7.2
30	16977497	104224655	NCGC00241716	4-bromophenyl	4-Me	CH2O	>57
31	16975932	104224652	NCGC00241713	2-methylphenyl	4-Me	CH2O	20
32	16976571	104224653	NCGC00241714	4-methylphenyl	4-Me	CH2O	>57
33	49852888	104224671	NCGC00241732	cyclohexane	2,5-difluoro	CH2O	2.0
34	49853353	104224669	NCGC00241730	cyclohexane	2-fluoro	CH2O	2.9
35	49853159	104224670	NCGC00241731	cyclohexane	7-quinoline	CH2O	2.6
36	49852796	104224668	NCGC00241729	3-aminophenyl	4-Me	CH2O	36
37	49853250	104224662	NCGC00241723	4-aminophenyl	4-Me	CH2O	18
38	49853108	104224641	NCGC00241703	2-CF3-phenyl	4-Me	CH2O	16
39	3164541	104224513	NCGC00238730	2-Furan	4-Me	CH2O	32
40	16978123	104224647	NCGC00241708	3-OMe-phenyl	4-Me	CH2O	14.5
41	49852803	104224490	NCGC00238707	3-thiophene	4-Me	CH2O	16
42	3159991	29216484	NCGC00164735	cyclohexane	4-Me	CH2O	1.8
43	16451029	104219749	NCGC00164736	cyclohexane	4-Me	CH2CH2O	3.2
44	5769809	104224665	NCGC00241726	cyclohexane	4-Me	CHCH	1.8

Note: all compounds in table were synthesized at NCGC. IC₅₀ values represent the average of three separate experiments.

3.5. Cellular Activity

Figure 5

A. Whole-body imaging of mice infected with bioluminescent T. Cruzi parasite, both the control (no treatment) and those treated with benznidazole at 30 mg/kg. B. Ratio of parasite burden after 12 days of treatment for analogs 15 and 16. Treatment starts 7 days post infection. Compounds were dosed at 10 mpk SID.

3.6. Profiling Assays

See in vitro ADME profiling properties in Section 2.2.

T. Cruzi Activity

Despite the moderate in vitro potency of these compounds against cruzain, we were quite encouraged by experiments performed in the lab of Professor Ana Rodriquez, where they investigated activity against the T. Cruzi parasite and activity against the host cells (cytotoxicity) as shown in Figure 4. At the time of these studies, only a few reasonably potent benzimidazole analogs had been synthesized (e.g. compounds 15 and 16), so these were tested along with our previously reported purine and triazine nitrile covalent inhibitors. Despite being much less potent in the in vitro assay, these non-covalent inhibitors displayed encouraging IC₅₀ values against the T. Cruzi parasite and were not very toxic against the host cell as shown in Figure 4 (at levels over 10-fold the EC₅₀ value against the parasite). Having an acceptable EC₅₀/TC₅₀ ratio prompted us to investigate these compounds in the T. Cruzi in vivo mouse model. This in vivo model utilizes bioluminescent T. Cruzi, in which the firefly luciferase enzyme is stably expressed within the parasite.¹² This method allows for rapid analysis of potential drugs for the treatment of Chagas disease (Figure 5A). Our compounds were dosed at 10 mg/kg, once/day (SID) and monitored compared to a control. Treatment began 7 days post infection and the parasite burden was analyzed at day 12 (data not shown) and day 19 (Figure 5B). We found a significant reduction of parasite burden at day 19, compared to the control and after 5 days treatment. These results are significant as other reported in vivo T. Cruzi parasite killing assays were conducted with higher doses (20–100 mg/kg) and more frequent treatment regiments (BID). These results suggest that over a longer treatment time, and using comparable dosing, these compounds would have an even more pronounced effect.

Solubility improvement

Our current probe molecule shows activity against cruzain, T. Cruzi parasite killing, is non-toxic to the host cell (NIH/3T3 cells), and shows significant reduction of parasite burden in Chagas disease mouse models. However, this compound has only limited aqueous solubility (5 μM), and thus could have poor absorption and oral bioavailability. As such, we are encouraged by the improved solubility of compounds 19 and 20, which have much improved solubility in PBS buffer (pH 7.4) of >150 μM while maintaining comparable potency of the probe compound (15). These compounds have not yet been tested in the T. Cruzi assay or the Chagas disease mouse model, but the preliminary results suggest that the solubility liability has already been addressed.

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

While there are numerous small molecules reported to have efficacy against the T. Cruzi parasite, treatment is still limited to nitroaromatic compounds, benznidazole and nifurtimox. Both of these compounds are not only toxic, but are also ineffective against the frequently occurring chronic stage of Chagas disease. The only known cruzain inhibitors that have progressed into animal models for Chagas infection include the vinyl sulfone 2 (and related analogs) and a tetrafluorophenoxymethyl ketone containing inhibitor 3, both of which are irreversible covalent modifiers of cruzain. Compound 2 has shown to be effective in curing T. Cruzi infection in both cell and animal models, but is plagued by low oral bioavailabilty and a short half-life. Both of these compounds have thus far proven save in animal models, but the potential for toxicity associated with irreversible modification exists. As such, this report represents to the best of our knowledge the first example of a reversible, non-covalent inhibitor of cruzain showing substantial efficacy in a Chagas disease mouse model. Considering our compounds were dosed at 10 mg/kg SID compared to 20 mg/kg BID (both via IP) for compound 2, the potential exists for even greater reduction in parasite burden for our compounds if we use a comparable dosing regimen. Additionally, these compounds had over 10-fold selectivity.

4.2. Mechanism of Action Studies

See earlier discussion section on co-crystallization studies of compound related to the probe molecule.

4.3. Planned Future Studies

Currently, Ana Rodriguez is testing approximately 80 new benzimidazole analogs in the high-throughput T. Cruzi assay and host cell toxicity studies, most of which are highlighted in this report. The top candidates which emerged as having optimal IC₅₀/TC₅₀ ratios and ADME properties will be progressed to her in vivo model for Chagas disease (as described above). We will also investigate whether these compounds are trypanostatic (inhibit replication within the host cell) or trypanocidal (induce lysis of intracellular amastigotes). Trypanostatic compounds may be more effective at treating Chagas during the acute phase of the disease, whereas typanocidal compounds may be required for activity against chronic forms of the disease. To investigate whether the parasites have been completed eliminated, the mice will be immuno-suppressed, and we will wait to see if the parasites come back. One of the final studies would be to see if these compounds act against the Chronic stage of the disease. This is important because the majority of cases are found in this stage; however, this requires long protocols (3 month wait after infection) followed by treatment. As such, this study will be limited only to compound(s), which have been analyzed in the other studies. Importantly, none of the compounds used today are effective towards this stage of the disease.

Concurrently, we will be investigating the in vitro ADME properties of several of the top actives to determine which compounds are best suited for in vivo PK. We have already discovered compounds that improved solubility compared to our probe molecule, while maintaining potency as shown above. Given the apparent metabolic liability of this probe compound, we plan to investigate METID studies to help establish the metabolic soft-spots. Preliminary analysis seems to indicate that the cyclohexane is the primary spot for metabolism. A compound with improved ADME properties would presumably translate to improved in vivo efficacy. We plan to submit an extended characterization proposal to provide funds for the studies mentioned above.