Scientific Rationale

The molecular chaperone Hsp90 has been previously linked to the evolution of antifungal drug resistance in various fungal strains (1). In these studies, the inhibition of Hsp90 can abrogate drug resistance in diverse fungi, including species of both Candida and Aspergillus. At concentrations that demonstrate no antifungal activity on their own, classical Hsp90 inhibitors dramatically reduce azole antibiotic resistance of clinical isolates and transform conventional fungistatic azole compounds into fungicidal treatments (1).

The resistance phenotype mediated by a wide variety of specific mutations is contingent on intact Hsp90-dependent fungal stress responses (1). Such responses allow the organism to tolerate the stresses imposed by drug exposure itself and/or accommodate mutations that mediate drug resistance. For example, genetic compromise of the key fungal stress signaling molecule calcineurin, itself an Hsp90 client protein, faithfully phenocopies the effects of Hsp90 inhibition (1). Modulators of the fungal stress response could greatly improve the ability to control otherwise lethal fungal infections in immunocompromised hosts, especially cancer patients undergoing high-dose chemotherapy and/or bone marrow transplant procedures.

Although Hsp90 or calcineurin inhibitors under current development appear to be well-tolerated in early phase cancer clinical trials, compromise of host chaperone protein function could have very deleterious effects in the context of active infection and the associated stresses of fever and cardiovascular instability. For example, host immune response is compromised by treatment with nonselective calcineurin inhibitors such as cyclosporin A and FK506. An obvious way to avoid this problem would be the identification of fungal-specific inhibitors that do not impair the host chaperone protein. To address these concerns, this project seeks to identify new fungal-selective chemosensitizers. In particular, the design of the screening cascade will also allow capture of compounds targeting still other, as yet unknown, components of fungal stress response pathways that enable the emergence and maintenance of resistance to current antifungal drugs. Facilitating this strategy, secondary binning assays were included in the screening path and were designed to characterize the phenotypic hits by their species specificity (fungal vs. human cells) and by apparent mode of action (Hsp90-related, calcineurin-related, or Unknown/Other mechanism).

Several compounds have been previously identified as chemosensitizers, increasing the susceptibility of various C.albicans strains to fluconazole treatment (2,3,4,5). Cernicka et al. previously reported that the compound 7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine (CTBT, Figure 1) was capable of chemosensitizing C.albicans strains to fluconazole (3). Against fluconazole-susceptible C.albicans strain 90028 and fluconazole-resistant C.albicans strain Gu5, CTBT was effective with an MIC value of 2.4 μM when combined with fluconazole. In the absence of fluconazole, CTBT demonstrated no activity against C.albicans strain 90028, but did inhibit growth of C.albicans strain Gu5 at concentrations greater than 2.4 μM. The anti-arrhythmic drug amiodarone was recently demonstrated to act synergistically with fluconazole in C.albicans with MIC values ranging between 1.6 μM to 18.8 μM (4). Plagiochin E, a natural product isolated from liverwort, increased yeast susceptibility to fluconazole at 2.4 μM (5). These agents were not considered for the current project because of their documented single-agent antifungal activity (amiodarone an MIC₅₀ of 3.1 μM [6]); plagiochin E an IC₅₀ value of 3.8 μM [5]).

Figure 1

Chemosensitizing Agents for Reversing Fluconazole Resistance.

The most potent compounds currently known are several HDAC inhibitors previously reported by Mai et al. (7). As depicted in Figure 2, compounds 4 and 5 are uracil-derived hydroxamic acids that exhibited MIC values ranging from 1.2 μM to 1.4 μM when combined with fluconazole. When tested independently, neither compound demonstrated activity against C. albicans at concentrations up to 368 μM. When compounds 4 and 5 were evaluated in a biochemical binding assay with murine HDAC1, their IC₅₀ values were measured at 37 nM and 51 nM, respectively (7). In addition, closely related analogs inhibit human HDAC1 and HDAC4 and were further shown to possess antiproliferative and cytotoxic effects against several human cell lines (8,9). These findings strongly suggest that compounds 4 and 5 would not be particularly selective for fungal protein targets, diminishing their potential as fungal-selective chemosensitizers. At the present time, compounds 4 and 5 have not been registered with MLSMR, are not sold commercially, and as such, are not available for evaluation in the current investigation. Ideally, screening the MLPCN compound collection would yield novel chemical hits that work by new modes of action that could be investigated by the Assay Provider, but one back-up option that is available is to identify fungal selective HDAC inhibitors.

Figure 2

Uracil-derived HDAC Inhibitors Capable of Reversing Antifungal Drug Resistance.

2.1. Assays

A summary listing of completed assays and corresponding PubChem AID numbers is provided in Appendix A (Table A1). Refer to Appendix B for the detailed assay protocols.

2.2. Probe Chemical Characterization

After preparation as described in Section 2.3, the probe (ML212) was analyzed by UPLC, ¹H and ¹³C NMR spectroscopy, and high-resolution mass spectrometry. The data obtained from NMR and mass spectroscopy were consistent with the structure of the probe, and UPLC indicated an isolated purity of greater than 93%. The relevant data is provided in Appendix C.

The solubility of the probe (ML212) was experimentally determined to be 3.6 μM in 2% (v/v) DMSO/PBS solution. The probe is exceptionally stable in PBS solution (>99% remaining after a 48-hour incubation). The data from the PBS stability assay is provided in Figure 4. Plasma protein binding (PPB) was determined to be 95% bound in human plasma. The probe is unstable in human plasma with approximately 3% remaining after a 5-hour incubation period. Presumably, hydrolysis of the methyl ester is the primary contributor to instability. The solubility, PPB, and plasma stability results are summarized in Section 3.4 (entry 8, Table 5).

Figure 4

Stability of the Probe (ML212) in PBS at 23 °C.

The probe (ML212) and five additional analogs were submitted to the SMR collection MLS003271341 (probe), MLS003271339 (CID100493), MLS003271340 (CID49835836), MLS003271342 (CID49835857), MLS003271343 (CID3243873), and MLS003271344 (CID49835842).

2.3. Probe Preparation

The probe compound 11 (ML212) was prepared in a three-step sequence beginning with commercially available 3-iodo-1H-indazole (see Scheme 1). This material was first protected as its tert-butyl carbamate then coupled with 3-methoxyphenyl boronic acid. Conveniently, the thermal, alkaline conditions of the Suzuki coupling facilitated in situ decomposition of the Boc protecting group (11–13), and coupling product was isolated as only the free indazole. Subsequent alkylation with methyl bromoacetate completed the synthesis of the probe. Full experimental details are provided in this section.

Scheme 1

Synthesis of the Probe (ML212).

General details. All reagents and solvents were purchased from commercial vendors and used as received. NMR spectra were recorded on a Bruker 300 MHz spectrometer. Proton and carbon chemical shifts are reported in ppm (δ) relative to tetramethylsilane (¹H δ 0.00) or residual chloroform in CDCl₃ solvent (¹H δ 7.24, ¹³C δ 77.0). NMR data are reported as follows: chemical shifts, multiplicity (obs. = obscured, br = broad, s = singlet, d = doublet, t = triplet, m = multiplet); coupling constant(s) in Hz; integration.

Unless otherwise indicated, NMR data were collected at 25 °C. Flash chromatography was performed using 40–60 μm Silica Gel (60 Å mesh) on a Teledyne Isco Combiflash R_f system. Tandem Liquid Chromatography/Mass Spectrometry (LC/MS) was performed on a Waters 2795 separations module and 3100 mass detector. Analytical thin layer chromatography (TLC) was performed on EM Reagent 0.25 mm silica gel 60-F plates. Visualization was accomplished with UV light and aqueous potassium permanganate (KMnO₄) stain followed by heating. Microwave reactions were performed with a Biotage Initiator 2.5 Microwave Synthesizer. High-resolution mass spectra were obtained at the MIT Mass Spectrometry Facility with a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometer. Compound purity and identity were determined by UPLC-MS (Waters, Milford, MA). Purity was measured by UV absorbance at 210 nm. Identity was determined on an SQ mass spectrometer by positive electrospray ionization. Mobile Phase A consisted of either 0.1% ammonium hydroxide or 0.1% trifluoroacetic acid in water, while mobile Phase B consisted of the same additives in acetonitrile. The gradient ran from 5% to 95% mobile Phase B over 48 seconds at 0.45 ml/min. An Acquity BEH C18, 1.7 μm, 1.0 × 50 mm column was used with column temperature maintained at 65 °C. Compounds were dissolved in DMSO at a nominal concentration of 1 mg/ml, and 0.25 μl of this solution was injected.

Step 1. Preparation of tert-butyl 3-iodo-1H-indazole-1-carboxylate (7): 3-Iodo-1H-indazole (5.00 g, 19.5 mmol) was placed in a round-bottom flask and dissolved in tetrahydrofuran (100 ml). 4-Dimethylaminopyridine (0.24 g, 1.9 mmol, 0.1 equiv) was then added, followed by di-tert-butyl dicarbonate (5.4 ml, 24 mmol, 1.2 equiv). Triethylamine (5.4 ml, 39 mmol, 2.0 equiv) was slowly added to the clear brown solution by syringe. The resulting solution was stirred at room temperature and monitored by TLC until complete. The reaction required approximately 2 hours. Once complete, the reaction was diluted with water (75 ml) and ethyl acetate (50 ml). After separating the layers, the aqueous phase was extracted with additional ethyl acetate (3 × 50 ml). The combined organic layers were washed with brine (100 ml), then shaken over magnesium sulfate, filtered, and concentrated under reduced pressure to give a dark red oil (8.40 g). The crude material was purified by column chromatography over silica gel (hexanes/ethyl acetate: 100/0 to 90/10) to give the title compound as an orange solid (6.20 g, 93%).

¹H NMR (300 MHz, CDCl₃): δ 8.12 (d, J = 8.4 Hz, 1H), 7.59 (t, J = 7.7 Hz, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 1.73 (s, 9H); ¹³C NMR (75 MHz, CDCl₃): δ 148.3, 139.6, 130.2, 129.9, 124.1, 121.9, 114.5, 102.8, 85.4, 28.1; ESI-MS (M-C₄H₉): m/z 288.

Step 2. Preparation of (3-(3-methoxyphenyl)-1H-indazole (9): tert-butyl 3-iodo-1H-indazole-1-carboxylate (100 mg, 0.29 mmol) was placed in a suitably sized microwave vial and dissolved in 1,4-dioxane (11.5 ml). 3-Methoxyphenyl boronic acid (88 mg, 0.58 mmol, 2.0 equiv) and tetrakis(triphenylphosphine) palladium (20 mg, 0.017 mmol, 0.06 equiv) were added, and the resulting turbid orange mixture was sparged thoroughly with nitrogen. An aqueous solution of sodium carbonate (2.0 M, 0.65 ml, 1.31 mmol, 4.5 equiv) was then added. The biphasic mixture was microwaved for 1 hour at a reaction temperature of 120 °C. The reaction was diluted with ethyl acetate (2 ml), and then filtered through a celite pad with additional ethyl acetate. The filtrate was concentrated under reduced pressure to give an oil. The crude material was purified by column chromatography over silica gel (hexanes/ethyl acetate: 100/0 to 30/70) to give the title compound as an orange oil (58.0 mg, 89%).

¹H NMR (300 MHz, CDCl₃): δ 12.71 (s, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.69 – 7.53 (m, 2H), 7.44 (t, J = 7.9 Hz, 1H), 7.31 – 7.22 (m, 1H), 7.17 (dd, J = 4.9, 13.0 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 7.03 – 6.98 (m, 1H), 3.79 (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ 160.1, 145.4, 141.7, 134.8, 130.0, 126.7, 121.3, 120.9, 120.8, 120.3, 114.2, 113.0, 110.5, 55.3; ESI-MS: m/z 224

Step 3. Preparation of methyl 2-(3-(3-methoxyphenyl)-1H-indazol-1-yl)acetate (11): 3-(3-methoxyphenyl)-1H-indazole (163 mg, 0.73 mmol) was dissolved in acetone (2.2 ml) and treated with methyl bromoacetate (0.21 ml, 2.2 mmol, 3.0 equiv). Finely powdered potassium carbonate (721 mg, 2.2 mmol, 3.0 equiv) was added in a single portion, and the resulting suspension was stirred overnight at 60 °C. Upon completion, the reaction was cooled to room temperature and filtered through celite with acetone. The clear orange filtrate was concentrated under reduced pressure to give the crude product as a dark orange oil. This material was purified by column chromatography over silica gel (hexanes/ethyl acetate: 100/0 to 70/30) to give the probe compound as an orange oil (186 mg, 86%).

¹H NMR (300 MHz, CDCl₃): δ 8.03 (d, J = 8.2 Hz, 1H), 7.53 (obs. t, J = 6.0 Hz, 1H), 7.50 (obs. s, 1H), 7.42 (obs. q, J = 7.7 Hz, 2H), 7.36 (obs. t, J = 8.1 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 6.96 (dd, J = 2.5, 8.2 Hz, 1H), 5.22 (s, 2H), 3.89 (s, 3H), 3.75 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 168.4, 160.0, 145.1, 141.7, 134.6, 129.8, 126.9, 122.2, 121.6, 121.5, 120.1, 114.2, 112.8, 109.0, 55.4, 52.6, 50.3. HRMS (ESI): calculated mass for C₁₇H₁₆N₂O₃ [M+H] 297.1234, found 297.1246.

2.4. Additional Analytical Analysis

Plasma Protein Binding. Plasma protein binding was determined by equilibrium dialysis using the Rapid Equilibrium Dialysis (RED) device (Pierce Biotechnology, Rockford, IL) for both human and mouse plasma. Each compound was prepared in duplicate at 5 μM in plasma (0.95% acetonitrile, 0.05% DMSO) and added to one side of the membrane (200 μl) with PBS pH 7.4 added to the other side (350 μL). Compounds were incubated at 37 °C for 5 hours in a 250-rpm orbital shaker. Following the incubation, the samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer.

Plasma Stability. Plasma stability was determined at 37 °C for 5 hours in both human and mouse plasma. Each compound was prepared in duplicate at 5 μM in plasma diluted 50/50 (v/v) with PBS pH 7.4 (0.95% acetonitrile, 0.05% DMSO). The compounds were incubated at 37 °C for 5 hours with a 250-rpm orbital shaker with time points taken at 0 hours and 5 hours. The samples were analyzed by UPLC-MS (Waters, Milford, MA) with compounds detected by SIR detection on a single quadrupole mass spectrometer.

Probe attributes

• Compounds that inhibit yeast growth in the presence, but not in the absence of 8 μg/ml fluconazole.
• Compounds that show at least 10-fold selectivity between the primary Candida test strain and mammalian cells.
• Compounds that show activity toward resistant clinical isolates at an IC₅₀ < 50 μM.
• IC₅₀ ≤1 μM in primary or resistant screen cell line.

3.1. Summary of Screening Results

Figure 6 displays the critical path for probe development.

Figure 6

Critical Path for Probe Development.

A high-throughput screen of 302,509 compounds (PubChem AID 1979) was performed in duplicate in the fluconazole-resistant C. albicans strain CaCi-2 in the presence of a sublethal concentration of fluconazole. Using a screening active cutoff of ≥75% inhibition at a screening concentration of 9.5 μM, 1893 hits were identified as Candida CaCi-2 growth inhibitors in the presence of fluconazole, and 1654 were available as cherry picks. These picked compounds were retested in dose against the C. albicans strain CaCi-2 to confirm their inhibitory activity and determine an IC₅₀ value. Of these, there were 622 compounds that met the criterion of inhibitory activity of less than or equal to 1 μM.

An orthogonal screen of these 1654 cherry picks against a more resistant C. albicans strain, CaCi-8 in the presence of a sub-lethal concentration of fluconazole (AID 2423), selected for compounds that were active with an IC₅₀ value less than 50 μM. There were 836 compounds that met this criterion, and 403 of these compounds were among the 622 actives from the CaCi-2 retest.

Murine 3T3 fibroblasts provided an assay for overt compound toxicity to mammalian cells. Of the 1654 cherry picks, 1012 compounds were inactive in this assay, indicating fungal selectivity, of which 350 also met the criteria in the prior CaCi-2 and CaCi-8 assays.

To eliminate cherry pick compounds that intrinsically inhibit Candida growth, an additional secondary screen of the 1654 cherry picks in the absence of fluconazole was included, with a 10 μM IC₅₀ cutoff. Of the 350 compounds of interest, 296 compounds met this criterion.

Figure 5Bar Chart of the Antifungal Screening Campaign

Two ancillary secondary assays were run to bin the remaining 296 compounds into three classes: Hsp90 inhibitors, calcineurin inhibitors, or other. Inhibition of either Hsp90 or calcineurin has been shown to restore fluconazole sensitivity in Saccharomyces cerevisiae as well as Candida albicans, but the specific mechanism(s) by which resistance is lost remains unknown. Identification of fungal-selective Hsp90 or calcineurin inhibitors would enable more detailed investigation into how Hsp90 engenders antifungal drug resistance in fungi.

The Hsp90 assay used a Saccharomyces cerevisiae strain engineered to express beta-galactosidase driven by glucocorticoid response element. The glucocorticoid hormone receptor depends heavily on Hsp90 for function. Of the 296 compounds of interest, 17 compounds were active as defined by a 10 μM upper threshold of inhibition.

The second binning assay for calcineurin inhibition was evaluated in a yeast carrying a construct encoding calcineurin-dependent response elements (CDRE) driving expression of beta-galactosidase. Reporter activity with or without the prior addition of test compounds was measured following challenge with the stressor calcium chloride (CaCl₂). Of the 296 compounds of interest, two compounds were active as defined by a 10 μM upper threshold of inhibition. The remaining 277 compounds functioned via alternative pathways and were categorized as “Other/Unknown mechanism.”

Thirty (30) compounds were chosen for initial dry powder confirmation studies from the 296 identified above by first clustering into small groups of related analogs, and then picking representative analogs from each of those families. After re-testing these dry powders in the test cascade, three compounds were chosen as potential probe candidates, and a first round of 31 analogs (plus three probe candidates) were obtained and assayed. Using the results from these assays as guidance, a second round of analogs (plus the probe candidate) was prepared for SAR analysis.

3.2. Dose Response Curves for Probe

Figure 7Dose-dependent Activity of the Probe (ML212) Against Various Cell Lines

C.albicans CaCi-2 in the presence of fluconazole (IC₅₀ = 440 nM, AID 493080) (A); C.albicans CaCi-8 in the presence of fluconazole (IC₅₀ = 1210 nM, AID 493149) (B); Murine 3T3 fibroblasts in the absence of fluconazole (inactive, AID 493147) (C); C.albicans CaCi-2 in the absence of fluconazole (inactive, AID 493070) (D); S. cerevisiae with Hsp90 construct (IC₅₀ = 4.18 μM, AID 493134) (E).

3.3. Scaffold/Moiety Chemical Liabilities

A search of PubChem for the probe (ML212) indicated that the probe has not been previously evaluated in any other assay. A structure-based search in SciFinder and Reaxys did not identify any publications or patents in which the probe appeared. The only potential chemical liability associated with the probe is possible hydrolysis of the methyl ester.

3.4. SAR Tables

In order to investigate the activity of this structural class, a collection of 58 structurally related analogs were synthesized and evaluated for their ability to reverse fluconazole resistance in the C.albicans test strains. Figure 8 depicts the diversification points selected for modification. Three diversity points (highlighted in purple, blue, and green) were explored, and the number of analogs screened for each site is specified. Several analogs did not fit into these three clusters and are classified as “miscellaneous” analogs.

Figure 8

Summary of analogs prepared to investigate the SAR profile of the HTS hit. Key SAR findings for each site of diversification are provided in italics.

The biological assay data and physical properties of these analogs are presented in Tables 1–6. Characterization data (¹H NMR spectra and UPLC chromatograms) of these analogs are provided in Appendix C.

Table 1

Evaluation of 12 Synthetic Ester and Amide Analogs.

Table 2

Synthetic Replacements of the Methyl Acetate Side Chain (10 analogs).

Table 3

Biological and Physical Properties of Substituted Indazoles (10 analogs).

Table 4

Alkyl Replacements of the 3-phenyl Ring (3 analogs).

Table 5

Effect of Substitution on the 3-phenyl Ring System (12 analogs).

Table 6

Miscellaneous Analogs of the 3-phenyl-1H-indazole Scaffold (11 analogs).

The initial hit from the HTS campaign described in Section 3.1 was methyl 3-phenyl-1H-indazole acetate (entry 1, Table 1). The side chain methyl ester of the hit was predicted to be susceptible to hydrolysis, and a series of ester and amide analogs were prepared accordingly to evaluate the necessity of this latent acid (Table 1). From this screen, it was determined that replacement of the methyl ester with metabolically more stable derivatives was detrimental to cellular activity. The inactivity of these compounds suggested that perhaps the free acid was the active species. Unfortunately, when this predicted metabolite was prepared and tested, it also proved ineffective at countering fluconazole resistance in C.albicans (entry 12, Table 1).

Following the investigation of alternative esters and amides, efforts were undertaken to substantially modify the entire side chain. To this end, 10 analogs were prepared and tested (Table 2). The absence of the methyl acetate was not tolerated (entry 1, Table 2), and excision of any oxygen atoms from the ester system (entries 2–4, Table 2) was also not tolerated. Extension of the side chain length was similarly ineffective (entries 5 and 6, Table 2). Several compounds incorporating substitutions adjacent to the ester were also prepared (entries 7–9, Table 2). However, only the mono-methyl derivative (entry 7, Table 2) retained any activity in the cellular assay; but, this analog was less potent than the original hit. While the γ-lactone (entry 9, Table 2) was clean by NMR spectroscopy, UPLC indicated hydrolysis to the seco-acid readily occurs; this phenomenon may have contributed to the inactivity of this compound.

Finally, an oxazole conjugate was investigated as a possible ester surrogate. Although this derivative displayed very weak activity against CaCi-8, there was no measurable efficacy towards the less resistant CaCi-2 (entry 10, Table 2). Based on the results summarized in Table 1 and Table 2, it appears that the methyl acetate system is critical to activity and further perturbation of this functionality is discouraged.

Table 3 presents several derivatives of the initial hit wherein the indazole core was modified to incorporate additional functional groups. This series of analogs focused predominantly on the 5- and 6-positions of the aromatic system. With regards to the 5-position (i.e. R₁), only the methyl or fluoro derivatives displayed low micromolar potency against CaCi-2 (entries 1 and 3, Table 3). Conversely, no activity was recorded for the methoxy or chloro counterparts (entries 2 and 4, Table 3). R₂, or the 6-position, appeared more amenable to manipulation as four of five conjugates demonstrated mild inhibitory effects towards CaCi-2 (entries 5–9, Table 3); only the 6-trifluoromethyl variant was inactive (entry 9, Table 3). It is notable that all of the 5- and 6-substituted variants were slightly active against the more resistant CaCi-8 strain (IC₅₀ values 5.3 μM to 32.1 μM). SAR of the indazole core suggests the 6-position is an attractive site for future exploration, in particular for further optimization of aqueous solubility.

The last point of diversity explored was the 3-phenyl ring of the parent indazole scaffold. Fifteen analogs were synthesized to probe the SAR of this region, and the results are summarized in Table 4 and Table 5. Table 4 presents several nonaromatic analogs that were prepared to replace the benzene ring. Removal of the phenyl ring adversely affected potency; both the unsubstituted 1H-indazole and 3-methyl compounds were inactive in the primary assay (entries 1 and 3, Table 4). The 3-iodo derivative was likewise an ineffective chemosensitizing agent (entry 2, Table 4). Incidentally, this series of analogs boasted high PBS solubility, but sacrificed all cellular potency.

To complement the previous nonaromatic series, a collection of monosubstituted phenyl conjugates was prepared (Table 5). Substitution at the para-position was generally not productive (entries 1–6, Table 5); only the methoxy and fluoro compounds displayed measurable activity in the primary screen (entries 2 and 3, Table 5). This trend stands in stark contrast to modification of the meta site. All three indazoles bearing a meta-substituted phenyl ring were very potent chemosensitizers (entries 7–9, Table 5). Both the 3-(3-tolyl) and 3-(3-anisolyl)-1H- indazoles demonstrated nanomolar potency against CaCi-2 (IC₅₀= 650 nM and 440 nM, respectively) and low micromolar activity against CaCi-8 (IC₅₀ = 1.3 μM and 1.2 μM, respectively). The 3-fluoro counterpart was marginally less effective (IC₅₀ = 1.4 μM against CaCi-2 and 2.1 μM against CaCi-8). The meta-position was clearly the pivotal site for potency; the two ortho-substituted analogs were considerably less potent than the meta-series although there were small gains in solubility (entries 10–11, Table 5).

SAR evaluation of the original scaffold was concluded with a series of compounds encompassing larger structural perturbations (Table 6). For several analogs, the aromatic and acetate side chains were relocated to alternative sites around the indazole system (entries 1–6, Table 6). Not surprisingly, these drastic alterations failed to yield any beneficial increase to cellular potency. Entries 7 and 8 (Table 6) re-affirmed SAR trends previously observed – neither the 5- chloro indazole nor acetamide analogs were efficacious chemosensitizers (cf. Table 3 and Table 1, respectively), and their corresponding hybrids were just as impotent. Various fragmented indazole systems did not exhibit any bioactivity when evaluated (entries 9–11, Table 6).

It can be concluded from these SAR studies that the methyl acetate side chain is invaluable to cellular bioactivity. Similarly, replacement of the 3-phenyl ring system with nonaromatic functionalities strongly attenuated potency. However, it was identified that meta-substituted phenyl rings were equipotent to or more potent than the original HTS hit. Modification of the para- or ortho-positions was generally counterproductive. The indazole core itself could tolerate functionalization at the 5-position to a limited degree while the adjacent 6-position could accommodate substituents with only a small loss in activity. Most of the compounds evaluated exhibited poor solubility in PBS (<1 μM); the few compounds that were soluble were often completely inactive.

As a result of these synthetic efforts, it was possible to increase the potency of the original hit (CID 3243873) from 1.8 μM to 0.44 μM and led to the identification of 3-(3-methoxyphenyl)-1H-indazole as a new probe compound (ML212).

3.5. Cellular Activity

All assays were performed with whole cells. A murine 3T3 fibroblast mammalian cell toxicity assay was included as a secondary screen. Experimental details are provided above in Section 2.1.2. The probe (ML212) clearly met the established probe criteria specified for this project (Table 7).

Table 7

Comparison of Probe to Project Criteria.

3.6. Profiling Assays

The probe (ML212) was evaluated for inhibitory activity against calcinuerin and Hsp90. Both proteins have been linked to the acquisition and maintenance of antifungal drug resistance in C. albicans. Assay results indicated the probe was a mild inhibitor of the Hsp90 pathway (IC₅₀ = 4.18 μM). Additional details are provided below in Section 4.2.

4.1. Comparison to existing art and how the new probe is an improvement

Investigation into relevant prior art entailed searching the following databases: SciFinder, Reaxys, PubChem, PubMed, US Patent and Trademark Office (USPTO), PatFT, AppFT, and World Intellectual Property Organization (WIPO). The search terms applied and hit statistics are provided in Table 8. The searches were performed on and are current as of February 7, 2011.

Table 8

Search Strings and Databases Employed in the Prior Art Search.

Several compounds have been previously identified as chemosensitizers, increasing the susceptibility of various C.albicans strains to fluconazole treatment (2–7). Of these, the most potent belonged to a series of HDAC inhibitors reported by Mai et al. (7). Depicted in Figure 9, these compounds are uracil-derived hydroxamic acids and exhibited MIC values ranging from 1.2 μM to 2.8 μM when combined with fluconazole. When tested in the absence of fluconazole, neither compound demonstrated activity against C.albicans at concentrations up to 368 μM. However, the potent activity of these compounds against murine HDAC1 (IC₅₀ <51 nM), combined with the antiproliferative activity of similar analogs in human cells (8,9), indicates they would not possess any significant species selectivity. The absence of selectivity disqualified compounds 4 and 5 as probe candidates. In addition, neither compound could be readily obtained for direct comparison to ML212.

Figure 9

Chemosensitizers for Reversing Fluconazole Resistance (MIC=1.2–2.8 μM).

The probe (ML212) has a potency of 440 nM against the C.albicans strain CaCi-2, and does not exhibit any effect in the absence of fluconazole or against mammalian fibroblasts.

4.2. Mechanism of Action Studies

Previous studies have demonstrated that functional Hsp90 and calcineurin are critical to drug resistance. In order to determine if ML212 affects the Hsp90 pathway or calcineurin itself, secondary assays were established.

Potential inhibition of the Hsp90-based chaperone machinery was evaluated using yeast reporter assays involving the glucocorticoid hormone receptor and the tyrosine kinase, v-Src. Both of these well established client proteins depend heavily on Hsp90 for their function. Refer to Section 2.1.4 for method details.

Potential inhibition of calcineurin function was evaluated in yeast carrying a construct encoding calcineurin-dependent response elements (CDRE) driving expression of a reporter enzyme. Reporter activity with or without the prior addition of test compounds was measured following challenge with the stressor CaCl₂.

The probe (ML212) exhibited Hsp90 pathway inhibition with IC₅₀ = 4.18 μM in the S. cerevisiae model. The inactivity of the compound in the mammalian fibroblast assay (IC₅₀ > 26 μM) suggests that ML212 may be a fungal-selective inhibitor of the Hsp90 pathway. Further investigations will evaluate this possibility and determine if this is the mode of action in C.albicans.

4.3. Planned Future Studies

The goal of probe optimization is to generate potent, fungal-specific agents for future mechanistic studies and target identification projects. The Lindquist lab is currently performing C.albicans resistance studies with ML212, and preliminary results suggest that generation of a ML212-resistant strain is possible. Successful induction of resistance would enable further investigation of ML212’s cellular target through the use of powerful genetic approaches that are available, including genome-wide, over expression, and deletion libraries, both in parallel arrayed format and pooled bar-coded format. To complement genetic approaches, affinity precipitation and proteomic approaches based on Stable Isotope Labeling with Amino acids in Cell culture (SILAC) technology, which are already being employed in other projects, may be used. The drug metabolism/pharmacokinetic (DMPK) properties of the probe can be further optimized, and the probe (ML212) could be studied in well-established mouse models of invasive, drug-resistant Candida fungemia, which are available and have been employed in the Whitehead Institute in conjunction with the laboratory of Gerald Fink. There is currently an IACUC-approved protocol in place to enable such experimentation in future work and to evaluate the therapeutic potential of probes generated by this screen.

Primary CaCi-2 (AID No. 1979) and CaCi-2 Dose-Response Retest (AID Nos. 2467, 488836)

Orthogonal Resistant Strain Dose Response (AID Nos. 2423, 488807, 493064, 493069, 493082, 493149)

Counterscreen Mammalian Cell Toxicity Dose Response (AID Nos. 2327, 488809, 493099, 493147)

Secondary Single Agent (No Fluconazole) Activity Assay Protocol (AID 488802)

Hsp90 Binning Protocol

Spectroscopic Data for SAR Analogs

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 3243873 (Table 3, entry 1)

UPLC-MS of Analog CID 3243873

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 13330959 (Table 3, entry 2)

UPLC-MS of Analog CID 13330959

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835820 (Table 3, entry 3)

UPLC-MS of Analog CID 49835820

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835839 (Table 3, entry 4)

UPLC-MS of Analog CID 49835839

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835870 (Table 3, entry 5)

UPLC-MS of Analog CID 49835870

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835857 (Table 3, entry 6)

UPLC-MS of Analog CID 49835857

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 13330955 (Table 3, entry 7)

UPLC-MS of Analog CID 13330955

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835882 (Table 3, entry 8)

UPLC-MS of Analog CID 49835882

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 20877577 (Table 3, entry 9)

UPLC-MS of Analog CID 20877577

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 20877568 (Table 3, entry 10)

UPLC-MS of Analog CID 20877568

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 20877557 (Table 3, entry 11)

UPLC-MS of Analog CID 20877557

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 5309553 (Table 3, entry 12)

UPLC-MS of Analog CID 5309553

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 300385 (Table 4, entry 1)

UPLC-MS of Analog CID 300385

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835816 (Table 4, entry 2)

UPLC-MS of Analog CID 49835816

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835825 (Table 4, entry 3)

UPLC-MS of Analog CID 49835825

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835848 (Table 4, entry 4)

UPLC-MS of Analog CID 49835848

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835835 (Table 4, entry 5)

UPLC-MS of Analog CID 49835835

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835881 (Table 4, entry 6)

UPLC-MS of Analog CID 49835881

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835813 (Table 4, entry 7)

UPLC-MS of Analog CID 49835813

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835873 (Table 4, entry 8)

UPLC-MS of Analog CID 49835873

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835834 (Table 4, entry 9)

UPLC-MS of Analog CID 49835834

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835836 (Table 4, entry 10)

UPLC-MS of Analog CID 49835836

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835874 (Table 5, entry 1)

UPLC-MS of Analog CID 49835874

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835837 (Table 5, entry 2)

UPLC-MS of Analog CID 49835837

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835889 (Table 5, entry 3)

UPLC-MS of Analog CID 49835889

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 9551137 (Table 5, entry 4)

UPLC-MS of Analog CID 9551137

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835872 (Table 5, entry 5)

UPLC-MS of Analog CID 49835872

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835812 (Table 5, entry 6)

UPLC-MS of Analog CID 49835812

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835815 (Table 5, entry 7)

UPLC-MS of Analog CID 49835815

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835818 (Table 5, entry 8)

UPLC-MS of Analog CID 49835818

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835849 (Table 5, entry 9)

UPLC-MS of Analog CID 49835849

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835842 (Table 5, entry 10)

UPLC-MS of Analog CID 49835842

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835852 (Table 6, entry 1)

UPLC-MS of Analog CID 49835852

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835832 (Table 6, entry 2)

UPLC-MS of Analog CID 49835832

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 46856254 (Table 6, entry 3)

UPLC-MS of Analog CID 46856254

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 46856255 (Table 7, entry 1)

UPLC-MS of Analog CID 46856255

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835864 (Table 7, entry 2)

UPLC-MS of Analog CID 49835864

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835876 (Table 7, entry 3)

UPLC-MS of Analog CID 49835876

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 46856253 (Table 7, entry 4)

UPLC-MS of Analog CID 46856253

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835854 (Table 7, entry 5)

UPLC-MS of Analog CID 49835854

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835863 (Table 7, entry 6)

UPLC-MS of Analog CID 49835863

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835823 (Table 7, entry 7)

UPLC-MS of Analog CID 49835823

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835858 (Table 7, entry 9)

UPLC-MS of Analog CID 49835858

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835865 (Table 7, entry 10)

UPLC-MS of Analog CID 49835865

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835850 (Table 7, entry 11)

UPLC-MS of Analog CID 49835850

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 49835868 (Table 7, entry 12)

UPLC-MS of Analog CID 49835868

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 12312246 (Table 8, entry 1)

UPLC-MS of Analog CID 12312246

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 100493 (Table 8, entry 2)

UPLC-MS of Analog CID 100493

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 46856252 (Table 8, entry 3)

UPLC-MS of Analog CID 46856252

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 14420692 (Table 8, entry 4)

UPLC-MS of Analog CID 14420692

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 46378661 (Table 8, entry 5)

UPLC-MS of Analog CID 46378661

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 4131200 (Table 8, entry 6)

UPLC-MS of Analog CID 4131200

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 20877381 (Table 8, entry 7)

UPLC-MS of Analog CID 20877381

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 20877322 (Table 8, entry 8)

UPLC-MS of Analog CID 20877322

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 46856251 (Table 8, entry 9)

LC-MS of Analog CID 46856251

¹H NMR Spectra (300 MHz, CDCl₃) of Analog CID 9855970 (Table 8, entry 10)

UPLC-MS of Analog CID 9855970