Prior Art

As discussed above, the only FDA approved small molecule therapeutic for the treatment of severe RSV infection is ribavirin, a nucleoside anti-metabolite prodrug with notable primary clinical toxicity (Figure 2). Ribavirin may also resemble GMP and decrease cellular GTP pools due to the inhibition of the enzyme inosine monophosphate dehydrogenase (IMPDH) [4]. Patients treated with ribavirin have suffered hemolytic anemia and worsening of cardiac disease that has led to myocardial infarctions. While the significant teratogenic and/or embryocidal effects that have been demonstrated in animals exposed to ribavirin may be less pertinent for the indication of RSV in young children [3], the drug’s long half-life is of concern. Ribavirin accumulates in erythrocytes and cannot be eliminated, thus requiring regeneration of the affected red blood cell population to remove the drug from circulation – a process estimated to take as long as 6 months [5]. These effects underscore the importance of finding safer and more effective treatments [6–10]. In our assay system, ribavirin had marginal potency, CPE EC₅₀ = 28.37 ± 3.75 μM and a CC₅₀ of 113.90 ± 38.52 μM, providing a SI of 3.6. In the plaque reduction assay, ribavirin demonstrated a log reduction of viral titer by 2.47, or ~ 300-fold, at 10 μM.

Figure 2

Ribavarin.

A search of the literature was performed prior to the start of the project to accurately gauge what properties the probe should have in order to out-perform existing lead compounds that have been reported in journals and in patents related to RSV. A SciFinder search and Prous Integrity search was performed using the terms: respiratory syncytial virus inhibtors, RSV, RSV inhibitors. Patents and journal articles were evaluated for relevant content. Additionally, a substructure SciFinder search was done once chemistry resources were assigned to this chemical class to evaluate the chemical space related to the probe. Many compounds have been reported in patent and journal forms; however, only those most relevant to and having bearing on the probe criteria are discussed. Several compounds have been the subject of development programs; however, all to date have failed to yield positive clinical data. Most of these fall under the category of entry inhibitors which are associated with the quick emergence of resistance. Some of these are discussed in more detail below. Wyeth has described a lead compound that is effective against RSV with an EC₅₀ of ~ 0.020 μM. The compound, RFI-641 (WAY-15641), is reportedly effective against both RSV A and RSV B (Figure 3). WAY-15641, characterized as an anionic sodium salt, has also been described in its protonated form (SID 28730297, CID 16130904) [11–13].

Figure 3

RFI-641 (WAY-15641) as a disodium salt.

Viropharma has described a bis-tetrazole, VP-14637, as an entry inhibitor for RSV with an EC₅₀ = 0.0014 μM (Figure 4A). The tautomeric, carbon analog of this structure has also been reported (SID 815578 CID 6479288). While VP-14637 is potent, both it and its analogs bear several reactive, electrophilic sites that may have toxicological implications. VP-14637 has been reportedly dropped from development [14–16].

Figure 4

A. VP-14637; B. Arrow Therapeutics/Novartis inhibitor; C. JNJ-2408068 (R-170591); D. BMS-433771.

Novartis and Arrow Therapeutics partnered to investigate a series of benzodiazepines as entry RSV inhibitors (Figure 4B). The potency of their lead compound, SID 8615639, in a whole cell RSV assay was reported with an EC₅₀ = 3.3 μM (17). Johnson and Johnson pursued benzimidazole-derived compounds as potential RSV inhibitors, describing JNJ-2408068 (previously known as R-170591, Figure 4C) as an entry inhibitor with an EC₅₀ = 0.16 μM and CC₅₀ > 100 μM [18–20]. Bristol-Myers-Squibb also reported entry inhibitors (Fig. 4D.) effective against RSV in the benzimidazole series with a potency of EC₅₀ = 0.010 μM; CC₅₀ = 218 μM [21–23].

Novartis recently identified a series of host-targeted, broad-spectrum antivirals, and potent, in vitro EC₅₀ values in the range of 0.002 to 0.3 μM were reported for RSV, influenza virus, hepatitis C virus (HCV), dengue virus, yellow fever virus, and HIV (Figure 5). The isoxazolylpyrazole and the proline series are identified. The observed efficacy was lost when exogenous UDP was added to the system. This suggests that dihydroorotate dehydrogenase (involved in the pyrimidine de novo biosynthesis pathway) is the target. In vivo studies showed that the compounds did not reduce mortality or viral load, and actually increased viral load in the late stage of infection [24].

Figure 5

Novartis broad-spectrum antivirals.

Most recently, this team (SRI, KU SCC and the assay provider) developed an entry-inhibitor probe derived from a sulfonylpyrrolidine scaffold, ML232, which attenuated a CPE effect with an EC₅₀ = 2.25 μM with a selectivity index of 13.7, and showed ability to reduce viral plaques by a log reduction of 2 (Figure 6) [25a-b]. Comparatively, the probe described in this report, ML275, represents a significant improvement in potency, cytotoxicity, plaque reduction and novelty in mechanism of action.

Figure 6

Structure of ML232, an entry inhibitor for hRSV.

As discussed above, ribavirin has a narrow therapeutic index in humans, and while several potential therapeutics appear to be in development (in preclinical or clinical trials), none have been approved for use by regulatory agencies. Therefore, the identification of a novel chemotype with an improved profile of efficacy and safety as compared to ribavirin may provide a stage for further development and better treatment options.

Overall Assay Strategy

The inhibition of the CPE caused by RSV infection in HEp-2 cells was used as the primary assay to identify the antiviral effects of compounds screened. The phenotypic end-point assay measured the luminescence generated by cellular ATP as a marker of cell viability. A total of 313,816 compounds have been screened and Z values of the screen ranged between 0.6 and 0.9 with the median of 0.83. Seven thousand five hundred eighty-three (7583) compounds were evaluated as active using the criteria of the average of the negative control + 3 times SD (22.3%) of the entire screen. Two thousand four hundred sixty-five compounds (2465) were subjected to the dose response assays (using the primary assay methodology) to verify their activity and cytotoxicity. Confirmed, non-toxic compounds were further investigated and subjected to chemical optimization, followed by secondary assay evaluation. Secondary assays more closely characterized the ability of the compounds to reduce RSV replication and can be used to examine the mechanism of action of the compounds by determining their point of intervention in the viral life cycle. The combination of primary assay (to measure cytoprotection), counter assay (for general eukaryotic cell toxicity) and secondary assay (to measure reduction in viral replication rates and point of intervention) combine to allow a determination of probe efficacy, selectivity, and specificity.

A detailed description of all assays is located in the Appendix.

2.1. Assays

2.2. Probe Chemical Characterization

Probe Chemical Structure, Physical Parameters and Probe Properties

Figure 7Probe characteristics for ML275

Stability

Stability was measured under two distinct conditions with ML275 (SID 124756536, Figure 8). Stability, depicted as closed circles in the graph, was assessed at room temperature (23 °C) in PBS (no antioxidants or other protectants and DMSO concentration below 0.1%). Stability, illustrated with closed squares in the graph, was also assessed with 50% acetonitrile added as the solubility of the compound in PBS was 0.18 μg/mL. Stability data in each case is depicted as a graph showing the loss of compound with time over a 48 hr period with a minimum of 6 time points and providing the percent remaining compound at the end of the 48 hr [26, 28]. With no additives (closed circles), 27% of ML275 remains after 48 hours; however, this data is dependent on and misleading due to the solubility limitations in PBS buffer. With the addition of 50% acetonitrile to account for solubility (closed squares), 100% of ML275 remained after 48 hours.

Figure 8

Graph depicting stability of ML275 after 48 h under two separate conditions.

2.3. Probe Preparation

The probe and analogs were generally synthesized by the method shown (Figure 9A and 9B). Commercially available 2-aminobenzoic acid 1, coupled to the requisite benzylamine, afforded aminoamide 2 which was cyclized with CDI to provide quinazolinedione intermediate 3. Intermediate 3 was used to generate the probe and corresponding analogs in this report through two related methods, each offering selective, orthogonal, late stage diversification of key functionality. Most analogs were afforded by the same protocol (9A) leading to the probe which involved hydrolysis of the ester, subsequent coupling with the desired alkylamine (e.g.,5), and installation of the core N-alkyl appendage to generate final compounds (e.g.,6). For analogs bearing the oxetane moiety, it was advantageous to incorporate that structural entity late-stage (9B). The core N-alkyl appendage was introduced (e.g.,7) from intermediate 3, followed by routine coupling manipulation to deliver final compounds (e.g.,9). Specific experimental details for the probe are detailed in section 2.3, entitled, “Probe Preparation,” and experimental protocols for analogs are described in the Appendix.

Figure 9

Synthetic route for probe and analog generation. Reagents: (a) DIPEA, HATU, methyl 4-(aminomethyl) benzoate hydrochloride, DMF, rt, 2h; (b) DIPEA, 1,1-carbonyldiimidazole, DCM, reflux, 16 h; (c) LiOH.H₂O, THF, 40 °C, 1h; (d) DIPEA, HATU, 3-methoxypropylamine, (more...)

General experimental and analytical details:¹H and ¹³C NMR spectra were recorded on a Bruker AM 400 spectrometer (operating at 400 and 101 MHz respectively) or a Bruker AVIII spectrometer (operating at 500 and 126 MHz respectively) in CDCl₃ with 0.03% TMS as an internal standard or DMSO-d₆. The chemical shifts (δ) reported are given in parts per million (ppm) and the coupling constants (J) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, br. s = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet and m = multiplet. The LCMS analysis was performed on an Agilent 1200 RRL chromatograph with photodiode array UV detection and an Agilent 6224 TOF mass spectrometer. The chromatographic method utilized the following parameters: a Waters Acquity BEH C-18 2.1 × 50mm, 1.7 um column; UV detection wavelength = 214 nm; flow rate = 0.4ml/min; gradient = 5 - 100% acetonitrile over 3 minutes with a hold of 0.8 minutes at 100% acetonitrile; the aqueous mobile phase contained 0.15% ammonium hydroxide (v/v). The mass spectrometer utilized the following parameters: an Agilent multimode source which simultaneously acquires ESI+/APCI+; a reference mass solution consisting of purine and hexakis(1H, 1H, 3H-tetrafluoropropoxy) phosphazine; and a make-up solvent of 90:10:0.1 MeOH:Water:Formic Acid which was introduced to the LC flow prior to the source to assist ionization. Melting points were determined on a Stanford Research Systems OptiMelt apparatus.

The probe was prepared using the following protocols:

Methyl 4-((2-aminobenzamido)methyl)benzoate: To a solution of 2-aminobenzoic acid (1.50 g, 10.94 mmol) in N,N-dimethylformamide (12 mL) was added methyl 4-(aminomethyl)benzoate hydrochloride (2.21 g, 10.94 mmol), HATU (4.57 g, 12.03 mmol) and N,N-diisopropylethylamine (5.42 mL, 32.80 mmol). The reaction mixture was stirred for 16 h at room temperature, then diluted with dichloromethane (50 mL) and washed sequentially with 1M HCl (40 mL), sat. aqueous NaHCO3 (40 mL) and water (2 × 200 mL). The separated organic extract was dried (MgSO4), filtered, and concentrated under reduced pressure to afford a crude product which was purified by silica gel flash column chromatography (0 – 60% v/v EtOAc/Hexane), yielding the product as a white solid (1.88 g, 6.61 mmol, 61% yield). ¹H NMR (400 MHz; CDCl₃): δ (ppm) 8.01 (d, J = 8.4 Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H), 7.35 (dd, J = 7.9 and 1.4 Hz, 1H), 7.25–7.19 (m, 1H), 6.70 (dd, J = 8.3 and 0.9 Hz, 1H), 6.67–6.61 (m, 1H), 6.43 (broad s, 1H), 5.56 (broad s, 2H), 4.66 (d, J = 5.9 Hz, 2H), 3.91 (s, 3H).

Methyl 4-((2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)benzoate: After stirring a solution of methyl 4-((2-aminobenzamido)methyl)benzoate (3.05 g, 10.73 mmol) and N,N-diisopropylethylamine (8.87 ml, 53.60 mmol) in CH₂Cl₂ (125 mL) for 10 minutes at room temperature under nitrogen, 1,1′-carbonyldiimidizaole (5.22 g, 32.20 mmol) was added, and the reaction mixture was heated 16 h at reflux. The formed precipitate was filtered, dried under vacuum and the desired product was furnished as a white solid without further purification (3.16 g, 10.18 mmol, 95% yield). ¹H NMR (400 MHz; DMSO): δ (ppm) 11.58 (s, 1H), 7.94 (dd, J = 8.3 and 1.4 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.72–7.64 (m, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.26–7.18 (m, 2H), 5.15 (s, 2H), 3.83 (s, 3H).

4-((2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)benzoic acid: To a solution of methyl 4-((2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)benzoate (1.00 g, 3.23 mmol) in THF (20 mL) was added 1 M lithium hydroxide in water (19.35 mL, 19.35 mmol). The reaction mixture was stirred at 40 °C for 1 hr, at which point TLC confirmed reaction completion. Then 1 M HCl was cautiously added until the reaction mixture reached pH 2, at which point the product precipitated out of solution. The precipitate was collected by filtration, washed with water (2 × 25 mL), dried under high vacuum to afford the desired product as a white solid (0.84 g, 2.84 mmol, 88% yield). ¹H NMR (400 MHz; DMSO): δ (ppm) 12.90 (broad s, 1H), 11.58 (s, 1H), 7.95 (d, J = 7.3 Hz, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.74–7.65 (m, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.28–7.18 (m, 2H), 5.15 (s, 2H).

4-((2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-methoxypropyl)benzamide: To a solution of 4-((2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)benzoic acid (1.00 g, 3.38 mmol) in N,N-dimethylformamide (15 mL) was added 3-methoxypropylamine (0.35 mL, 3.38 mmol), HATU (1.41 g, 3.71 mmol) and N,N-diisopropylethylamine (1.67 mL, 10.13 mmol). The reaction mixture was stirred for 16 h at room temperature, then diluted with CH₂Cl₂ (90 ml) and washed sequentially with 1 M HCl (60 mL), sat. aqueous NaHCO3 (60 mL) and water (2 × 180 mL). The organic extract was separated, dried (MgSO4), filtered, and concentrated under reduced pressure to afford a crude product which was purified by silica gel flash column chromatography (0 – 5% v/v MeOH/CH₂Cl₂) yielding the desired product as a white solid (0.91 g, 2.48 mmol, 73% yield). ¹H NMR (400 MHz; DMSO): δ (ppm) 11.56 (s, 1H), 8.40 (t, J = 5.6 Hz, 1H), 7.95 (d, J = 7.3 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.71–7.63 (m, 1H), 7.36 (d, J = 8.4 Hz, 2H), 7.26–7.17 (m, 2H), 5.13 (s, 2H), 3.35 (t, J = 6.3 Hz, 2H), 3.31–3.24 (m, 2H), 3.22 (s, 3H), 1.78–1.67 (m, 2H).

PROBE ML275: 4-((1-(4-isopropylbenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-methoxypropyl)benzamide (SID 124756536, CID 53301904). To a solution of 4-((2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-methoxypropyl)benzamide (0.050 g, 0.14 mmol) in N,N-dimethylformamide (2 mL) was added 4-isopropylbenzyl bromide (0.028 mL, 0.16 mmol) and potassium carbonate (0.056 g, 0.41 mmol). The resulting reaction mixture was stirred at 40 °C for 16 h. The formed residue was dissolved in CH₂Cl₂ (6 mL) and sequentially washed with 1 M HCl (4 mL), water (3 × 20 mL) and brine (8 mL). The organic layer was separated, dried (MgSO4), filtered, and concentrated under reduced pressure to give a crude product which was purified by silica gel flash column chromatography (0 – 5% v/v MeOH/DCM) yielding 4-((1-(4-isopropylbenzyl)-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)methyl)-N-(3-methoxypropyl)benzamide as a white solid (0.030 g, 0.060 mmol, 44% yield). ¹H NMR (500 MHz; CDCl₃): δ (ppm) 8.24 (dd, J = 7.9 and 1.6 Hz, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.61–7.53 (m, 3H), 7.22 (apparent t, J = 7.5 Hz, 1H), 7.20–7.13 (m, 5H), 6.89 (t, J = 4.5 Hz, 1H), 5.36 (s, 2H), 5.33 (broad s, 2H), 3.60–3.52 (m, 4H), 3.37 (s, 3H), 2.92–2.81 (m, 1H), 1.87 (quintet, J = 5.9 Hz, 2H), 1.21 (d, J = 6.9 Hz, 6H). ¹³C NMR (126 MHz; CDCl₃): δ (ppm) 166.98, 161.89, 151.45, 148.51, 140.38, 140.13, 135.36, 134.12, 132.86, 129.22, 129.07, 127.15, 127.14, 126.53, 123.26, 115.71, 114.65, 72.55, 59.06, 47.31, 44.90, 39.27, 33.84, 28.91, 24.01. LCMS retention time: 3.422 min. LCMS purity at 214 nm: 98.8%. HRMS: m/z calcd for C₃₀H₃₃N₃O₄ (M + H⁺) 500.2544, found 500.2540. Melting point: 171–173 °C.

3.1. Dose Response Curves for Probe

The primary assay methodology (Summary AID: 2440; Assigned AID: 504526, 504820, 504823) was used to measure both probe efficacy and cytotoxicity. The probe ML275 potency in the RSV CPE assay was determined: EC₅₀ = 0.81 ± 0.75 μM, and the CC₅₀ > 200 μM. The calculated selectivity was determined as (CC₅₀/EC₅₀) > 247. The ML275 dose response profiles for efficacy and cytotoxicity curves are graphed in Figure 10.

Figure 10

Dose response efficacy (green circles) and cytotoxicity (red squares) for ML275.

3.2. Cellular Activity

A cell-based assay was employed to measure the cytotoxicity of the probes in cells using a luminescent cell viability assay readout. This assay is detailed in Summary AID: AID 2440 and additionally assigned AIDs: AID 2410, AID 488976, AID 492968, AID 493015, AID 493090, AID 504509, AID 504674, AID 504818, AID 504826, and AID 504825. This assay directly measures phenotypic cytotoxicity, but indirectly measures the solubility of the probe and its ability to pass through the cellular membrane. Insoluble or impermeable probes will not result in cell death.

3.3. Profiling Assays

Antiviral selectivity and tested cell types: The ML275 probe class was tested in two different human cell types. The primary assay was performed in HEp2 cells and the secondary assays were performed in HEp2 and A549 cells. The probe class was effective against RSV in both cell lines. The probe itself was also tested for activity against vaccinia, influenza A, Venezuelan equine encephalitis, and dengue viruses. The probe showed no efficacy in inhibiting these viruses, thus showing selectivity for RSV.

Broad spectrum target profiling: ML275 was submitted for assessing off-target pharmacology using a Ricerca LeadProfiling® screen made up of 67 assays. The probe was assayed in duplicate at a concentration of 10 μM for all targets, and the following responses (Table 1) were noted as ≥ 50% inhibition or stimulation for biochemical assays:

Table 1

Targets against which ML275 demonstrated > 50% inhibition.

Determination of actual IC₅₀ information for any of these will be handled as part of a MLPCN extended characterization proposal. Inhibition for the human adenosine A₃ receptor and human platelet activating factor was noted at 44% and 43%, respectively; however, all other inhibition levels were reported below 34 percent. Full results, including target profile list, percent inhibition for each assay, methods and experimental details are provided in the Appendix [29].

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

Currently, two FDA approved drugs are available for RSV infections. The first is Virozole, the brand name for ribavirin, and is delivered in aerosolized form directly to the lungs [3,4]. Ribavirin causes hemolytic anemia by the accumulation of ribavirin triphosphate and subsequent depletion of intracellular ATP in red blood cells. It has a narrow therapeutic index in humans. The second therapeutic (Synagis, MedImmune) is a humanized monoclonal antibody used prophylactically to protect at risk infants through their first two RSV seasons. The prophylactic opportunity is limited due to high treatment cost, and the therapeutic efficacy of this application has not been defined. While several potential therapeutics appear to be in development (in preclinical or clinical trials), none have been approved for use by regulatory agencies.

The discovery of novel, small molecular antiviral therapeutics would potentially fill this therapeutic gap. Understanding the probe’s mechanism of action may deconvolute the nuances of virus-host interaction, thus aiding in finding scaffold modifications that effectively target these interactions and increase efficacy and therapeutic potential. A side-by-side comparison of ML275 and ribavirin is provided in Table 2.

Table 2

Comparison of ribavirin to ML275.

Compared to ribavirin, probe ML275 has an improved CPE EC₅₀ of 0.81 μM and selective index of >247 (ribavirin: 28.37 μM and 3.6, respectively). ML275 was shown to reduce viral replication in a cell-based assay by 6.7 log, intervenes at a different stage in the viral life cycle than ribavirin, and has strong potential for therapeutic use instead of as a prophylactic agent. Therefore, probe ML275 represents an improvement both in its therapeutic index and potential.

Mechanism-of-action studies have classified this probe as a post-entry inhibitor of late stage infection processes. Ribavirin is a post-entry inhibitor of viral replication, but its toxicological profile (small therapeutic index) prohibits its use except for the most severe cases of lower respiratory infections of RSV in infants. Most of the other known compounds that have been described in the literature are entry inhibitors, prone to induce rapid emergence of viral resistance and none have progressed to reach FDA approval. Therefore, a probe which inhibits post-entry processes and has a low cytotoxicity (large therapeutic index) will address an unmet need.

Ricerca LeadProfiling® Report for ML275