A Cell Based HTS Approach for the Discovery of New Inhibitors of RSV

Respiratory Syncitial Virus (RSV) is a highly contagious member of the family Paramyxoviridae. It has been estimated that most children will be infected with RSV prior to their second birthday generating an estimated 75,000 – 125,000 hospitalizations in children. RSV is associated with substantial morbidity and mortality and is the most common cause of bronchiolitis and pneumonia among infants and children under one year of age. Nevertheless, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems. There are no vaccines commercially available. Existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis^® from MedImmune) that is limited to use in high risk pediatric patients. The economic impact of RSV infections due to hospitalizations and indirect medical costs is greater than $650 million annually; thus, finding inhibitors for RSV would be extensively valuable. The cell-based RSV inhibition screen produced novel compounds that may be developed further into potential prophylactic therapies. Of the 313,816 Molecular Libraries Small Molecule Repository (MLSMR) compounds screened, 51 compounds were selected, based on potency, selectivity and chemical tractability, for further evaluation in dose response and secondary assays. Collaboration between the assay provider, the screening center at Southern Research Institute and the University of Kansas Specialized Chemistry Center narrowed the SAR focus to three scaffolds. The probe, ML232, had an antiviral EC₅₀ value of 2.25 μM and a 13.7-fold Selectivity Index (SI) for antiviral activity over mammalian cell cytotoxicity.

Probe Structure & Characteristics

Figure 1Probe characterization

Table

1. Recommendations for Scientific Use of the Probe

Limitations in the current state of the art being addressed by the probe: There are currently two therapies approved for the treatment of RSV infection: ribavirin, a nucleoside analog used for therapeutic intervention that suffers from severe toxic liabilities that limits its use particularly in infants and children, and the prophylactic humanized monoclonal antibody (Synagis® from MedImmune) that is limited to use in high risk pediatric patients. Moreover, Synagis is expensive, used only for prophylaxis, and requires monthly injections. Several small molecules have been developed; 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. 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.

A useful probe from this project was defined as a small molecule with novel structure as compared to ribavirin and known compounds in development for RSV. Furthermore, the point of intervention in viral replication should be defined as an entry or post-entry inhibitor. Once the point of intervention for the probe has been determined as either entry inhibitors or post-entry inhibition, the required probe criteria were set as (for entry inhibitors) CPE EC₅₀ < 1 μM; (for post-entry inhibitors) CPE EC₅₀ < 5 μM; a selective index > 30× the EC₅₀ for the primary assay versus the observed cytotoxicity; titer reduction ≥ 1 log unit in virus reduction; and a differential over time in the time of addition assay. A probe that meets these expectations would represent an improvement over the use of ribavirin.

Use of the probe: The probe will be used as a novel tool for studying the replication cycle of RSV in human cells. Additional mechanisms of action studies that are outside the scope of the current project are in progress by the assay provider, Dr. Severson, and will define the specific host or viral component that is the target of the probe. This will provide preliminary data to support continued investigations and subsequent NIH/NIAID grant proposals to be submitted by Dr. Severson. Specifically, these studies will attempt to elucidate the antiviral mechanism and investigate the probe as a potential antiviral therapeutic using additional in vitro and in vivo models. The combination of mechanism of action studies and in vivo therapeutic data will allow chemistry optimization to enhance the pharmacological properties of the probe as the basis of identifying a novel treatment for RSV.

Use of the probe in the research community: The limitations associated with currently-approved RSV treatments, such as host toxicity and off-target effects, emphasize a special need for new probes that specifically target viral gene products and multiple stages of the viral propagation cycle. The probe will be used by the research community to isolate and study the functions of the viral targets in the context of host infection, and will provide new insights into the development of pathogen-specific antivirals. Alternatively, it is possible that the probe, whose mechanism has not yet been defined, modulates a host target that is essential for efficient viral replication. Similarly, this would allow the research community to study the host interactions with virus gene products and might form the basis for host-specific, broadly effective antiviral therapeutics.

Relevant biology to which the probe can be applied: All stages of the RSV life cycle (attachment, entry, genomic replication, assembly, and budding) are valid targets for the actions of the probe. In addition, cellular functions that are preempted by the virus and participate in viral replication, trafficking, and release are also possible targets. This includes, but is not limited to, kinase-regulated host cell signaling, metabolic regulation, cell cycle regulation, microtubule and actin cytoskeleton modeling, and innate immunity.

We seek to identify small molecule compounds that inhibit the virus-induced cytopathic effect (CPE) by reducing respiratory syncytial virus replication. To do so, a well-characterized in vitro human cell/virus infection model has been combined with a simple, phenotypic, cytoprotection high-throughput screening (HTS) assay.

RSV was discovered ~40 years ago, and was initially isolated from chimpanzees during an epizootic upper respiratory tract disease outbreak. RSV, which belongs to the family Paramyxoviridae, was subsequently found to be the most important cause of infectious pulmonary disease in human infants, and is a major causative agent of respiratory tract infections among children worldwide. Infants, immune-compromised children, or those with underlying respiratory disorders are at a particularly high risk of developing severe and lethal RSV respiratory tract infections that can be complicated by the resultant viral pneumonia and respiratory distress. Elderly and immune-compromised individuals are also susceptible to severe respiratory infections, thereby highlighting the importance of medical intervention in the form of early diagnosis and implementation of supportive or antiviral therapies [1].

The RSV non-segmented RNA genome encodes eleven proteins. These are NS1 and NS2 (which inhibit type I interferon activity); N, which is the genome-coating nucleocapsid protein; M, which encodes the matrix protein; and SH, G and F (fusion protein) which are incorporated into the viral coat. The G protein is a post-translationally glycosylated surface protein and also recognizes cell surface components during viral attachment. The F protein mediates entry of the virus into the cell cytoplasm and also promotes the formation of syncytia. Typically, antibodies directed at the F protein are neutralizing because this protein is conserved in both subtypes of RSV, while the G protein varies in sequence considerably between the two subtypes. The M2 gene encodes the elongation factor M2-1 and transcription factor M2-2. The L gene encodes the viral RNA polymerase, and the phosphoprotein P, which is a cofactor for L function [2].

Existing therapies for acute RSV infections in infants are ribavirin and the prophylactic humanized monoclonal antibody (Synagis^® from MedImmune) that is limited to use in high risk pediatric patients. Although the side effects of ribavirin use are an increased risk for hemolytic anemia and teratogenicity, it remains an approved antiviral therapy. Multiple potential antivirals (discussed in prior art) are under clinical investigation, but none of these have been governmentally approved as therapies or are available to the research community as tools to study the life cycle of the virus or host/virus interactions. Therefore, there are significant research opportunities for the discovery of additional probes and potential therapeutics for RSV [2, 3].

The probe described in this report is a small molecule that demonstrates useful single-digit micromolar potency against RSV, with a reasonable margin of selectivity against mammalian cell cytotoxicity, potentially providing a basis for evaluation in small animal toxicity and efficacy studies.

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 1). 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 [4]. These effects underscore the importance of finding safer and more effective treatments [5–9]. In our assay system, ribavirin had marginal potency, EC₅₀ = 28.38 ± 3.75 μM and a CC₅₀ of 113.90 ± 38.52 μM, providing a SI of 3.6.

Figure 1

Ribavarin.

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) (Figure 2), 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) [10–12].

Figure 2

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

Figure 3

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

Viropharma has described a bis-tetrazole, VP-14637, as an entry inhibitor for RSV with an EC₅₀ = 0.0014 μM (Figure 3A). 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 [13–15].

Novartis and Arrow Therapeutics partnered to investigate a series of benzodiazepines as entry RSV inhibitors (Figure 3B). The potency of their lead compound, SID 8615639, in a whole cell RSV assay was reported with an EC₅₀ = 3.3 μM (16). Johnson and Johnson pursued benzimidazole-derived compounds as potential RSV inhibitors, describing JNJ-2408068 (previously known as R-170591, Figure 3C) as an entry inhibitor with an EC₅₀ = 0.16 μM and CC₅₀ > 100 μM [17–19]. Bristol-Myers-Squibb also reported entry inhibitors (Figure 3D.) effective against RSV in the benzimidazole series with a potency of EC₅₀ = 0.010 μM; CC₅₀ = 218 μM [20–22].

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.

2. Materials and Methods

2.2. Probe Chemical Characterization

A. Probe Chemical Structure, Physical Parameters and Probe Properties

Figure 4Probe characteristics for ML232

B. Structure Verification and Purity: ¹H NMR, ¹³C NMR, LCMS, HRMS and Chiral HPLC Data

Proton and carbon NMR data for ML232 SID 10416954/CID 49842897: Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The numerical experimental proton and carbon data are represented below. The experimental proton and carbon spectra are included for reference (Appendix, Figures A1A and A1B, respectively).

Proton Data for ML232 SID 10416954/CID 49842897: ¹H NMR (500 MHz; CDCl₃): δ (ppm) 9.54 (s, 1H), 8.88 (dd, J = 4.3 and 1.8 Hz, 1H), 8.62 (dd, J = 7.4 and 1.4 Hz, 1H), 8.28 (dd, J = 8.4 and 1.7 Hz, 1H), 8.11 (dd, J = 8.2 and 1.3 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.63 (s, 1H), 7.53 (dd, J = 8.3 and 4.3 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 5.41 (dd, J = 7.9 and 2.0 Hz, 1H), 3.44-3.32 (m, 2H), 2.52-2.40 (m, 1H), 2.33 (s, 6H), 1.96-1.82 (m, 2H), 1.82-1.72 (m, 1H).

Carbon Data for ML232 SID 10416954/CID 49842897: ¹³C NMR (126 MHz; CDCl₃): δ (ppm) 170.66, 151.58, 143.91, 137.12, 136.37, 135.88, 135.29, 134.92, 134.62, 130.45, 129.29, 127.65, 126.34, 125.84, 124.23, 122.51, 63.20, 49.29, 30.19, 24.98, 21.24, 17.65.

LCMS and HRMS Data for ML232 SID 10416954/CID 49842897: Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. The numerical experimental LCMS and HRMS data are represented below. LCMS retention time: 3.068 min. LCMS purity at 214 nm: 100%. HRMS: m/z calcd for C₂₂H₂₃N₃O₃S (M + H⁺) 410.1533, found 410.1532. The experimental LCMS and HRMS spectra are included for reference (Appendix, Figure A1C and A1D, respectively). The LCMS and HRMS data for the other lot of ML232 (SID 123058985) was also determined: LCMS retention time: 3.119 min. LCMS purity at 214 nm: 95.5%. HRMS: m/z calcd for C₂₂H₂₃N₃O₃S (M + H⁺) 410.1533, found 410.1535. Spectra for SID 123058985 was analogous to that obtained for SID 10416954 (not shown).

Chiral HPLC data for ML232 SID 10416954/CID 49842897: Detailed analytical methods and instrumentation are described in section 2.3, entitled “Probe Preparation” under general experimental and analytical details. HPLC analysis of enantiomeric excess was experimentally determined to be ≥ 99% ee. The chiral HPLC traces of racemic and probe compound are included for reference (Appendix, Figure A1E). The HPLC analysis of enantiomeric excess was experimentally determined to be ≥ 99% ee for the second lot of ML232 (SID 123058985). Spectra for SID 123058985 was analogous to that obtained for SID 10416954 (not shown).

Solubility

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 [23, 24]. Probe ML232 (SID 123058985) was found to have an acceptable solubility measurement of 92.7 μg/mL under these conditions.

Stability

Stability was measured under two distinct conditions with ML232 (SID 123058985, Figure 5). Stability, depicted as closed circles in the graph, was assessed at room temperature (23 ºC) in PBS (no antioxidants or other protectants and DMSO concentration below 0.1%). Stability, illustrated with closed triangles in the graph, was also assessed with 50% acetonitrile added in case the solubility of the compound was an issue (note that the solubility data was not available prior to this experiment and has since shown that the probe is highly soluble). 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 end of the 48 hr [23, 25]. With no additives (closed circles), 80.6% of ML232 remains after 48 hours. With the addition of 50% acetonitrile (closed triangles), 95.4% of ML232 remains after 48 hours.

Figure 5

Graph depicting stability of ML232 after 48 h under two separate conditions.

2.3. Probe Preparation

The probe was synthesized by the method shown (Figure 6). Commercially available 8-quinolinesulfonyl chloride 1 was treated with L-proline under basic Schotten-Baumann conditions to afford acid 2. Coupling of acid 2 with substituted anilines furnished the desired sulfonamides 3.

Figure 6

Synthetic route for probe and analog generation.

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 (d) 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:

(S)-1-(quinolin-8-ylsulfonyl)pyrrolidine-2-carboxylic acid: L-proline (0.5 g, 4.34 mmol, 1 eq) was dissolved in a mixture of 10% K₂CO₃ (10 mL) and THF (10 mL). To this reaction mixture was added 8-quinolinesulfonyl chloride (1.98 g, 8.68 mmol, 2 eq), and the resulting mixture was stirred at 50 °C for 5 h, followed by acidification with 3 N HCl to pH 2, and extraction with EtOAc (3 × 30 mL). Drying (MgSO₄) of the separated organic extracts and removal of solvent under reduced pressure afforded the product as a white solid (0.8 g, 2.61 mmol, 60% yield). ¹H NMR (400 MHz; DMSO-d₆): δ (ppm) 9.09 (dd, J = 4.2 and 1.8 Hz, 1H), 8.56 (dd, J = 8.4 and 1.7 Hz, 1H), 8.41 (dd, J = 7.4 and 1.4 Hz, 1H), 8.31 (dd, J = 8.2 and 1.3 Hz, 1H), 7.76 (apparent t, J = 7.8 Hz 1H), 7.71 (dd, J = 8.3 and 4.2 Hz, 1H), 5.19 (t, J = 6.0 Hz, 1H), 3.45-3.35 (m, overlapping with water peak in DMSO, 1H), 3.13-3.03 (m, 1H), 1.96-1.86 (m, 2H), 1.86-1.72 (m, 1H), 1.60-1.48 (m, 1H).

PROBE ML232: (S)-N-(2,5-dimethylphenyl)-1-(quinolin-8-ylsulfonyl)pyrrolidine-2-carboxamide (SID 10416954/CID 49842897). To a solution of (S)-1-(quinolin-8-ylsulfonyl)pyrrolidine-2-carboxylic acid (0.06 g, 0.20 mmol, 1 eq) in DMF (0.75 mL) was added 2,5-dimethylaniline (0.024 mL, 0.2 mmol, 1 eq), HATU (0.082 g, 0.22 mmol, 1.1 eq), and DIPEA (0.097 mL, 0.59 mmol, 3 eq). The reaction mixture was stirred for 2 h at room temperature, diluted with CH₂Cl₂ (5 mL) and washed sequentially with aqueous 10% HCl (2 × 5 mL), saturated aqueous NaHCO₃ (2 × 5 mL), and water (2 × 5 mL). The separated organic extracts were dried over MgSO₄, and evaporated to give the crude product which was purified by silica gel flash column chromatography (2% MeOH in CH₂Cl₂) to give the desired (S)-N-(2,5-dimethylphenyl)-1-(quinolin-8-ylsulfonyl)pyrrolidine-2-carboxamide as a colorless oil (0.05 g, 0.122 mmol, 62% yield). ¹H NMR (500 MHz; CDCl₃): δ (ppm) 9.54 (s, 1H), 8.88 (dd, J = 4.3 and 1.8 Hz, 1H), 8.62 (dd, J = 7.4 and 1.4 Hz, 1H), 8.28 (dd, J = 8.4 and 1.7 Hz, 1H), 8.11 (dd, J = 8.2 and 1.3 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.63 (s, 1H), 7.53 (dd, J = 8.3 and 4.3 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 5.41 (dd, J = 7.9 and 2.0 Hz, 1H), 3.44-3.32 (m, 2H), 2.52-2.40 (m, 1H), 2.33 (s, 6H), 1.96-1.82 (m, 2H), 1.82-1.72 (m, 1H). ¹³C NMR (126 MHz; CDCl₃): δ (ppm) 170.66, 151.58, 143.91, 137.12, 136.37, 135.88, 135.29, 134.92, 134.62, 130.45, 129.29, 127.65, 126.34, 125.84, 124.23, 122.51, 63.20, 49.29, 30.19, 24.98, 21.24, 17.65. LCMS retention time: 3.068 min. LCMS purity at 214 nm: 100%. HRMS: m/z calcd for C₂₂H₂₃N₃O₃S (M + H⁺) 410.1533, found 410.1532. Enantiomeric excess was determined by HPLC analysis using the following conditions: Chiralcel OD-H, 150×4.6mm column; solvents: hexane/propan-2-ol in a ratio of 90:10 (0.5 mL/min); wavelength = 254 nm, temperature = rt; Racemic material was found with retention times as follows: S-isomer: 43.97 min., R-isomer: 52.08 min.; Enantiomerically pure probe ML232 prepared from L-proline: [α]_D²⁵ -31.5 (c 0.0039 CHCl₃), > 99% ee, (S-isomer, 43.80 min).

3. Results

3.1. Dose Response Curves for Probe

The primary assay methodology (Summary AID: AID 2440; Assigned AID: AID 504526, AID 504820, AID 504823) was used to measure both probe efficacy and cytotoxicity. The probe ML232 potency in the RSV CPE assay was derived as an average of two separate lots: EC₅₀ = 2.25 μM, and the CC₅₀ = 30.91 μM. The calculated selectivity was determined as (CC₅₀/EC₅₀) = 13.7. The dose response profiles for efficacy and cytotoxicity curves are graphed for both lots of ML232 (Figure 7).

Figure 7

Dose response efficacy and cytotoxicity profile for probe ML232 (SID 104169543 is depicted by neon green triangles and red circles, respectively) and the second lot of ML232 (SID 123058985 is depicted by dark green triangles and purple circles, respectively). (more...)

The data collected for the individual lots of probe ML232 are represented below, along with the averaged calculations. Good agreement was found between the two lots of probe ML232.

Table

4. Discussion

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 de-convolute 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 ML232 and ribavirin is provided in Table 1.

Table 1

Comparison of ribavirin to ML232.

Compared to ribavirin, probe ML232 has an improved CPE EC₅₀ of 2.25 μM and selective index of 13.7 (ribavirin: 28.37 μM and 3.6 μM, respectively). ML232 was shown to reduce viral replication in a cell-based assay by 100-fold, 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.

Mechanism-of-action studies have classified this probe as an inhibitor of viral entry or early infection. Ribavirin is a post-entry inhibitor of viral replication, but its toxicological profile 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 resistance and none have progressed to reach FDA approval. Therefore, a probe of either category will be useful in identifying effective agents with the potential of addressing an unmet need. In particular, the potential of an entry or early-infection inhibitor to be used in combination with ribavirin is an attractive scenario.

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6. APPENDIX: NMR, LCMS, HRMS and HPLC Data for Probe ML232 SID 10416954 CID 49842897

Figure A1A. Proton data for ML232 SID 104169543 (CID 49842897)

Figure A1B. Carbon data for ML232 SID 104169543 (CID 49842897)

Figure A1C. LCMS purity data at 214 nm for ML232 SID 104169543 (CID 49842897): LCMS retention time: 3.068 min; purity at 214 nm = 100%

Figure A1D. HRMS data for ML232 SID 104169543 (CID 49842897): HRMS m/z calculated for C₂₂H₂₃N₃O₃S [M⁺ + H]: 410.1533, found 410.1532

Figure A1E. Analytical chiral HPLC trace of racemic compound compared to enantiomerically pure ML232 SID 104169543 (CID 49842897)