2.1. Assays

The details of the primary HTS and additional assays can be found in the “Assay Description” section in the PubChem BioAssay view under the AIDs as listed (Table 2). Additionally the details for the primary HTS are provided in the Appendix at the end of this probe report.

Table 2

Summary of assays, listed by title and AID, used for the development of ML336.

2.2. Probe Chemical Characterization

A. Probe chemical name, structure and physiochemical data

The IUPAC name of the probe ML336 is (E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide. The batch prepared and submitted to the MLSMR is archived as SID 144087340, corresponding to CID 71301451.

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

Proton and carbon NMR data for ML336/ SID 144087340/ CID 71301451: 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 for SID 152199106. The corresponding NMR data for SID 144087340 (a separate lot of ML336) was obtained and determined to match what is described for SID 152199106. The experimental proton and carbon spectra are included for reference (Appendix, Figures A6A and A6B, respectively).

Figure A6B

¹³CNMR spectrum for ML336, SID 152199106, CID 71301451.

Proton NMR Data for ML336/ SID 144087340/ CID 7130145:¹H NMR (500 MHz, CDCl₃) δ 10.99 (s, 1H), 9.15 (d, J = 2.8 Hz, 1H), 8.15 (dd, J = 8.8, 2.8 Hz, 1H), 7.65 – 7.60 (m, 2H), 7.38 – 7.33 (m, 2H), 7.12 (tt, J = 7.3, 1.2 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H), 3.47 (t, J = 5.7 Hz, 2H), 3.28 (s, 3H), 3.13 (s, 2H), 2.69 (t, J = 5.7 Hz, 2H), 2.26 (s, 3H).

Carbon NMR Data for ML336/ SID 144087340/ CID 71301451:¹³C NMR (126 MHz, CDCl₃) δ 162.63, 156.42, 153.97, 142.79, 138.19, 129.09, 127.64, 126.35, 126.16, 124.19, 123.75, 120.19, 55.12, 51.82, 49.66, 45.27, 36.88.

LCMS and HRMS Data for ML336, SID 144087340/ CID 71301451: 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 as follows:

For SID 152199106: LCMS retention time: 3.206 min. LCMS purity at 214 nm: 99.2%. HRMS m/z calculated for C₁₉H₂₂N₅O₃ [M⁺+H] 368.1723, found 368.1718. The experimental HRMS and LCMS spectra are included for reference (Appendix, Figure A6C and A6D, respectively).

Figure A6C

High resolution MS for ML336, SID 152199106, CID 71301451.

Figure A6D

LCMS purity data at 214 nM for ML336, SID 152199106, CID 71301451.

For SID 144087340: LCMS retention time: 3.213 min. LCMS purity at 214 nm: 96.5%. HRMS: m/z calculated for C₁₉H₂₂N₅O₃ [M⁺+H] 368.1723, found 368.1729. The experimental HRMS and LCMS spectra are included for reference (Appendix, Figure A6E and A6F, respectively).

Figure A6E

High resolution MS for ML336, SID 144087340, CID 71301451.

Figure A6F

LCMS purity data at 214 nM for ML336, SID 144087340, CID 71301451.

If available from a vendor, please provide details. This probe is not yet commercially available, as it was designed and prepared as an original analog for this project. A 20 mg sample of ML336 synthesized at the KUSCC has been deposited in the MLSMR; however, the compound is also available free of charge from the KUSCC.1

D. Stability

Chemical Stability: ML336 was evaluated for susceptibility to nucleophilic addition and formation of conjugates by treatment with dithiothreitol (DTT). Figure 1 represents the time course experiment with ML336 under various conditions. ML336 was dissolved at 10 μM in PBS at pH 7.4 (1% DMSO) and independently incubated at room temperature with no nucleophile present or 50 μM dithiothreitol (DTT). The test reactions were sampled every hour for eight hours and analyzed by LCMS. The analytical LCMS system utilized for the analysis was a Waters Acquity system with UV-detection and mass-detection (Waters LCT Premier). The analytical method conditions included a Waters Acquity HSS T3 C18 column (2.1 × 50mm, 1.8 μm) and elution with a linear gradient of 1% water to 100% CH₃CN at 0.6 mL/min flow rate. Peaks on the 214 nm chromatographs were integrated using the Waters OpenLynx software. Absolute areas under the curve were compared at each time point to determine relative percent parent remaining. The masses of potential adducts and dimers of ML336 were searched for in the final samples to determine if any detectable adduct formed or dimerization had occurred. In the case of ML336, no adducts were detected at any time point using LCMS detection. All samples were prepared in duplicate. Ethacrynic acid, a known Michael acceptor, was used as a positive control.3

Figure 1

Chemical stability of ML336 over 8 h in the presence of a 5-fold concentration of dithiothreitol.

Table 3 summarizes the percent remaining of ML336 at the endpoints of each run in each experiment. The average amount of ML336 remaining after 8h when no nucleophile was added was 93.9%, and when 5X DTT was added, nearly 99% of parent ML336 remained after the same time period, indicating that ML336 was not generally susceptible to degradation or adduct formation in the presence of a thiol-based nucleophile.3

Table 3

Percent of ML336 remaining at the conclusion of the experiment (8h).

Aqueous Stability: Figure 2 represents the time course experiment with ML336 under various aqueous stability conditions. ML336 was dissolved at 10 μM in PBS at pH 7.4 (1% DMSO) or dissolved at 10 μM in 50% PBS/50% acetonitrile at pH 7.4 (1% DMSO). The samples were evaluated over 14 timepoints (0,1, 2, 3, 4, 5, 6, 7, 8, 16, 24, 32, 40, and 48 h) and analyzed by LCMS. The analytical LCMS system utilized for the analysis was a Waters Acquity system with UV-detection and mass-detection (Waters LCT Premier). The analytical method conditions included a Waters Acquity HSS T3 C18 column (2.1 × 50mm, 1.8 μm) and elution with a linear gradient of 1% water to 100% CH₃CN at 0.6 mL/min flow rate. Peaks on the 214 nm chromatographs were integrated using the Waters OpenLynx software. Absolute areas under the curve were compared at each time point to determine relative percent parent remaining.3

Figure 2

Aqueous stability of ML336 over 48 h in PBS or PBS/acetonitrile.

In PBS, ML336 showed and average 82.93% remaining after 48h; however, no other mass peaks were observed. In order to account for precipitation/solubility effects, ML336 was also evaluated in 50/50 PBS:acetonitrile and was shown to have an average of 95.89% remaining after 48 h (Table 4).3

Table 4

Percent of ML336 remaining at the conclusion of the experiment (48h).

2.3. Probe Preparation

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, bs = 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.

Probe ML336 was prepared by the route depicted in Figure 3. Anthranilic acid 1 was treated with 2-chloroacetyl chloride in the presence of triethylamine to afford 2-(chloromethyl)benzoxazinone 2. Dehydrative amidation was carried out by treating 2 with aniline and POCl₃.4 The resulting 2-(chloromethyl)phenylquinazolinone 3 was aminated with mono-BOC-protected tert-butyl methyl(2-(methylamino)ethyl)carbamate to provide 4 which was subsequently deprotected with TFA to afford ML336 in 11% overall yield over four steps.5 This process has been used to prepare ML336 on a 260 mg scale.

Figure 3

Synthetic scheme for preparation of ML336.

Detailed protocols used for the assembly of ML336 are as follows:

2-(chloromethyl)-6-nitro-4H-benzo[d][1,3]oxazin-4-one. Following a previously reported procedure,4 2-amino-5-nitro-benzoic acid (6.718 g, 36.9 mmol, 1 eq) was placed under nitrogen and dissolved in CH₂Cl₂ (100 mL). After addition of triethylamine (5.9 mL, 42.3 mmol, 1.15 eq), the mixture was lowered to 0 °C in an ice bath and a solution of chloroacetyl chloride (3.2 mL, 40.2 mmol, 1.1 eq) in CH₂Cl₂ (50 mL) was slowly added. The reaction mixture was stirred at 0 °C for 1 hour, then at rt for 1 additional hour. The solvent was removed in vacuo and water was added to the remaining solid. The solid was filtered and rinsed with water (3 × 20 mL), followed by 5% Et₂O/Hexanes (3 × 30 mL) to give 2-(chloromethyl)-6-nitro-4H-benzo[d][1,3]oxazin-4-one (8.87 g, 100%) along with residual triethylamine as a pale, yellow solid. The product was used in the following step without further purification. ¹H NMR (400 MHz, acetone-d₆) δ 8.96 – 8.91 (m, 2H), 8.48 (dd, J = 9.3, 2.8 Hz, 1H), 4.44 (s, 2H).

2-(chloromethyl)-6-nitro-3-phenylquinazolin-4(3H)-one. To a microwave vial was added 2-(chloromethyl)-6-nitro-4H-benzo[d][1,3]oxazin-4-one (1.47 g, 6.12 mmol, 1 eq) under Ar, and the solid was dissolved in acetonitrile (13 mL). Phosphorus oxychloride (1.15 mL, 12.34 mmol, 2 eq) was added, followed by the addition of solution of aniline (0.73 mL, 8.00 mmol, 1.3 eq) in acetonitrile (4 mL). The mixture was heated in a MW reactor at 150 °C for 15 min. The reaction mixture was transferred to a larger flask and slowly quenched with saturated aq. NaHCO₃ (20 mL). The precipitate was filtered and rinsed with water (3 × 20 mL) to give 2-(chloromethyl)-6-nitro-3-phenylquinazolin-4(3H)-one (1.24 g, 64%) as a burnt-orange solid. ¹H NMR (400 MHz, CDCl₃) δ 9.13 (d, J = 2.6 Hz, 1H), 8.59 (dd, J = 8.9, 2.6 Hz, 1H), 7.91 (d, J = 9.0 Hz, 1H), 7.65 – 7.58 (m, 3H), 7.39 – 7.35 (m, 2H), 4.28 (s, 2H).

tert-butyl methyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate. Following a previously reported procedure,5 2-(chloromethyl)-6-nitro-3-phenylquinazolin-4(3H)-one (1.40 g, 4.43 mmol, 1 eq) was dissolved in acetonitrile (18 mL). Potassium carbonate (1.226 g, 8.87 mmol, 2 eq), tert-butyl methyl(2-(methylamino)ethyl)carbamate (ChemBridge, 1.18 g, 6.25 mmol, 1.4 eq), and potassium iodide (0.280 g, 1.687 mmol, 0.4 eq) were added sequentially, and the mixture was heated in a MW reactor at 80 °C for 5 min. The crude reaction mixture was adsorbed onto Celite®, and the product was purified (2×) by flash chromatography (CombiFlash, 40 g silica, 0-10% MeOH/CH₂Cl₂, followed by CombiFlash, 80 g silica, 0-80% EtOAc/Hexanes) to give tert-butyl methyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate (0.72 g, 35%) as a pale orange solid. ¹H NMR (400 MHz, CDCl₃) δ 9.13 (d, J = 2.6 Hz, 1H), 8.56 (dd, J = 9.0, 2.7 Hz, 1H), 7.89 (d, J = 9.0 Hz, 1H), 7.60 - 7.52 (m, 3H), 7.32 – 7.27 (m, 2H), 3.36 (s, 2H), 3.22 - 3.06 (m, 2H), 2.75 (br s, 3H), 2.48 (br s, 2H), 2.20 (s, 3H), 1.39 (br s, 9H).

PROBE ML336: (E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide. To a stirred solution of tert-butyl methyl(2-(methyl((6-nitro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)methyl)amino)ethyl)carbamate (678 mg, 1.45 mmol) in CH₂Cl₂ (20 mL), TFA (9.5 mL, 124 mmol) was slowly added, and the resulting mixture was stirred at rt for 45 min. Water (40 mL) and CH₂Cl₂ (40 mL) were then added, and the mixture was adjusted to pH 10 using saturated aq. Na₂CO₃ (40 mL). The organic phase was separated and the aqueous layer was extracted with CH₂Cl₂ (2 × 40 mL). The combined organic phase was concentrated and purified by flash chromatography (CombiFlash, 40 g silica, 0-5% MeOH/ CH₂Cl₂) to give (E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide (265 mg, 49%) as a pale yellow solid. ¹H NMR (500 MHz, CDCl₃) δ 10.99 (s, 1H), 9.15 (d, J = 2.8 Hz, 1H), 8.15 (dd, J = 8.8, 2.8 Hz, 1H), 7.65 – 7.60 (m, 2H), 7.38 – 7.33 (m, 2H), 7.12 (tt, J = 7.3, 1.2 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H), 3.47 (t, J = 5.7 Hz, 2H), 3.28 (s, 3H), 3.13 (s, 2H), 2.69 (t, J = 5.7 Hz, 2H), 2.26 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 162.63, 156.42, 153.97, 142.79, 138.19, 129.09, 127.64, 126.35, 126.16, 124.19, 123.75, 120.19, 55.12, 51.82, 49.66, 45.27, 36.88. LCMS retention time: 3.206 min, purity at 214 nm = 99.2%. HRMS m/z calculated for C₁₉H₂₂N₅O₃ [M⁺+H] 368.1723, found 368.1718. Pale yellow needles, mp 168-173 °C (recrystallized from CH₂Cl₂).

3.1. Dose Response Curves for Probe

Figure 3Each data point represents the mean of % cell viability from triplicate runs

Dose response curve and IC₅₀ were generated using the Four Parameter Logistic Model or Sigmoidal Dose-Response model. A. Fourteen point CPE assay dose response curve for ML336 using TC-83 VEEV strain. B. CPE assay dose response curves for ML336 using wild type Trinidad donkey VEEV strain (triangles), V3526 VEEV strain (circles), and Chikungunya virus (squares)

3.2. Cellular Activity

All of the assays utilized in this project were cell-based (see Table 2, section 2.1). The probe and many analogs show potent inhibition of a VEEV-induced cytopathic effect and dramatic attenuation of viral turnover at sub- to low micromolar concentrations in cellular titer reduction assays. Cytotoxic effects were not observed for the series. As the mechanism of action appears to target the viral nsP2, ML336 and its analogs must penetrate host cells to engage cytoplasmic targets involved in viral replication.

3.3. Profiling Assays

Profiling against host targets. Probe ML336 was submitted to Eurofins PanLabs (formerly Ricerca Biosciences) to evaluate it in radioligand binding assays against a panel of 67 GPCRs, ion channels and transporters at a single concentration of 10 μM, each in duplicate. ML336 inhibited the human norepinephrine transporter with 91% inhibition at 10 μM, and an IC₅₀ determination has been ordered and will be reported in due course. With the exception of this one target in the panel, inhibition was not observed above 36%. Norepinephrine inhibition (or reuptake inhibitors) is associated with treatment of depression, attention deficit hyperactivity disorder (ADHD), as well as other brain-related disorders. Given that ML336 is blood-brain barrier penetrant and has potential in the development as a PET imaging agent, the activity against the norepinephrine transporter (and notable lack of inhibition on dopamine and seratonin transporters) may indicate other therapeutic significance. For the full report, see Appendix.

NCI-60 panel. Probe ML336 was submitted to the NCI-60 panel for anti-cancer profiling. The NCI looks for one of two scenarios when evaluating a test compound at a single dose: (A) at least 8 cell lines with a growth percent of 40 or less (at least 60% inhibition), or (b) very specific and maybe strong inhibition of only a few cell lines. At a single dose of ML336 at 10 μM concentration, neither of these criteria was met to warrant confirmation in the standard, follow-up dose response assay. For the full report, see Appendix.

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

Prior Art Overview: A number of compounds were identified from patent and literature sources with reported VEEV inhibitory activity (Figure 4, Table 5); however, closer inspection of the data and/or testing in the CPE assay and considering their dependency on host mediated mechanisms of action revealed these compounds were severely limited and not particularly relevant given the remarkable results seen with ML336.

Figure 4

Structures for which VEEV activity is reported.

Table 5

Data and liabilities of compounds with reported VEEV activity.

The reported compounds are either weakly inhibitory against VEEV and/or do not act selectively on viral components. The interaction of these compounds via host mechanisms can lead to toxicity and was an undesirable characteristic for a new probe. Ribavirin (5) and carbodine (6) are nucleoside anti-metabolite prodrugs with notable clinical toxicities and are not specific in their mechanism of action to viral targets. For example, ribavirin has a broad in vitro inhibitory activity against RNA viruses. The activities include 1) depleting the cellular GTP pool (IMPDH inhibitor), 2) increasing mutations in the viral genome, and 3) inhibiting the GTP capping enzyme. Direct, antiviral activity for ribavirin6 against Sindbis virus (another alphavirus) has been disclosed, but inhibitory activity for VEEV has not yet been reported. Our internal assessment of ribavirin against VEEV in a CPE assay showed an IC₅₀ of 126 μM. Carbodine (6) has been shown to have in vitro activity, but has associated toxicity and is only moderately efficacious in vivo.7 Urea VX-497 (7) is a potent, reversible uncompetitive IMP dehydrogenase (IMPDH) inhibitor with modest VEEV activity.8 IMPDH catalyzes an essential step in the de novo biosynthesis of guanine nucleotides, and as such, VX-497 is known to target cellular processes.

The quinazolinone structure (8) had a reported VEEV CPE IC₅₀ of 16.7 μM, and importantly, it is the most closely related structurally to selected chemotypes for our investigation.9 The KU SCC synthesized the compound for assessment in our internal CPE assay; however, we found the IC₅₀ for this compound to be > 25 μM. Completed SAR studies for our quinazolinone series leading to ML336 do not support that quinazolinone (8) should be active for VEEV.

Thienylpyrrole (9) is reported to inhibit WEEV viral replication with an IC₅₀ = 9.3 μM.10 VEEV inhibition is referred to (< 100 μM) for the compound class but is not explicitly reported for any one compound. To dispel ambiguity, the KU SCC synthesized this lead compound and assessed it in our internal CPE assay, corroborating the finding with a VEEV IC₅₀ > 25 μM.

Antioxidant 80 (10) is a broad spectrum antiviral agent11 due to its antioxidant activity, and it has notable CYP450 (2C9, 2C19) inhibition to the tune of about 3 μM.12 This compound was purchased and purified prior to submitting to the CPE assay, which resulted in a VEEV IC₅₀ of > 50 μM. Didemnin (11) was reported in Pubchem13 as an active compound for a VEEV screen; however, it is a large cyclic peptide and does not represent prior art against which ML336, a small molecule, should be compared. Lastly, BIOder (12) is a recently disclosed compound14 (April 2012) that was reported during the course of our own project investigation and should not be considered prior art according to NIH; however, it is worth noting that BIOder inhibited VEEV via the host protein GSK-3β and was only partially efficacious in protecting mice from succumbing to VEEV infection. Moreover, BIOder has significant structural liabilities such as the presence of a diazodiimine and the electrophilic character of the dimeric core that are a cause for concern.

In summary, many of the compounds with disclosed anti-VEEV activity are weakly potent against VEEV, and when four selected compounds were assessed in an internal TC-83 CPE assay, we found them to have CPE IC₅₀ values > 25 μM. Of the small molecule prior art compounds disclosed, quinazolinone compound 8 (CID 13182904) and thienylpyrrole 9 (CID 3240671) do not have described mechanisms of action and they have TC-83 CPE IC₅₀ values > 25 μM. The depsipeptide didemnin 11 (CID 44287859) also has an unknown mechanism of action, but is not a small molecule against which ML336 can be compared. Moreover, didemnin is exceedingly cytotoxic which led to it being dropped from clinical evaluation.15 The remaining compounds (carbodine 6, VX-497 7, and recently disclosed BIOder 12) have mechanisms of action that involve host cellular targets. ML336 is a first-in-class probe that represents a significant improvement over the prior art landscape in terms of CPE nanomolar potency (∼ 16-fold more potent than BIOder), lack of notable cytotoxicity (> 50 μM in Vero 76 cells), ability to reduce VEEV viral titer by more than 7.2 log at 1 μM, and according to mechanistic data, ML336 appears to inhibit VEEV replication by means of viral nsP2 interference and not host-mediated targets.

Quality control ¹H-NMR and ¹³C-NMR spectra for ML336

Figure A6A¹H NMR spectrum for ML336, SID 152199106, CID 71301451

Quality control LCMS Purity and Mass ID data: HRMS and HPLC for ML336

Eurofins Panlabs Target Profiling Report

Summary of Significant Results

Biochemical assay results are presented as the percent inhibition of specific binding or activity throughout the report. All other results are expressed in terms of that assay's quantitation method.

• For primary assays, only the lowest concentration with a significant response judged by the assays' criteria, is shown in this summary.
• Where applicable, either the secondary assay results with the lowest dose/concentration meeting the significance criteria or, if inactive, the highest dose/concentration that did not meet the significance criteria is shown.
• Unless otherwise requested, primary screening in duplicate with quantitative data (e.g., IC₅₀ ± SEM, K_i ± SEM and n_H) are shown where applicable for individual requested assays. In screening packages, primary screening in duplicate with semi-quantitative data (e.g., estimated IC₅₀, K_i and n_H) are shown where applicable (concentration range of 4 log units); available secondary functional assays are carried out (30 mM) and MEC or MIC determined only if active in primary assays >50% at 1 log unit below initial test concentration. Significant responses (≥ 50% inhibition or stimulation for Biochemical assays) were noted in the primary assays listed in Table 6:

Table 6

Results of Eurofins Profiling with ML336, Compound: CID60156253.

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Catalog #	Assay Name	Species	Concentration	% Inhibition
204410	Norepinephrine Transporter (NET)	human	10 μM	91

Table 7Results of Eurofins Profiling with ML336, Compound: CID60156253 continued

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Cat #	Assay name	Batch	Species	Rep.	Conc.	% inhibition
241000	Imidazoline I2, Central	326329	rat	2	10 μM	14
243520	Interleukin IL-1	326355	hum	2	10 μM	2
250460	Leukotriene, Cysteinyl CysLT1	326336	hum	2	10 μM	11
251600	Melatonin MT1	326356	hum	2	10 μM	18
252610	Muscarinic M1	326408	hum	2	10 μM	1
252710	Muscarinic M2	326409	hum	2	10 μM	2
252810	Muscarinic M3	326348	hum	2	10 μM	3
257010	Neuropeptide Y Y1	326330	hum	2	10 μM	26
257110	Neuropeptide Y Y2	326331	hum	2	10 μM	-6
258590	Nicotinic Acetylcholine	326298	hum	2	10 μM	11
258700	Nicotinic Acetylcholine α1, Bungarotoxin	326300	hum	2	10 μM	1
260130	Opiate δ1 (OP1, DOP)	326357	hum	2	10 μM	-2
260210	Opiate κ(OP2, KOP)	326371	hum	2	10 μM	10
260410	Opiate μ(OP3, MOP)	326372	hum	2	10 μM	25
264500	Phorbol Ester	326373	mouse	2	10 μM	0
265010	Platelet Activating Factor (PAF)	326383	hum	2	10 μM	7
265600	Potassium Channel [KATP]	326384	ham	2	10 μM	5
265900	Potassium Channel hERG	326385	hum	2	10 μM	30
268420	Prostanoid EP4	326343	hum	2	10 μM	5
268700	Purinergic P2X	326287	rabbit	2	10 μM	14
268810	Purinergic P2Y	326288	rat	2	10 μM	6
270000	Rolipram	326374	rat	2	10 μM	35
271110	Serotonin (5-Hydroxytryptamine) 5-HT1A	326289	hum	2	10 μM	2
271700	Serotonin (5-Hydroxytryptamine) 5-HT2B	326386	hum	2	10 μM	10
271910	Serotonin (5-Hydroxytryptamine) 5-HT3	326387	hum	2	10 μM	2
278110	Sigma σ1	326284	hum	2	10 μM	7
279510	Sodium Channel, Site 2	326318	rat	2	10 μM	9
255520	Tachykinin NK1	326382	hum	2	10 μM	8
285900	Thyroid Hormone	326388	rat	2	10 μM	2
220320	Transporter, Dopamine (DAT)	326376	hum	2	10 μM	11
226400	Transporter, GABA	326281	rat	2	10 μM	0
204410	Transporter, Norepinephrine (NET)	326375	hum	2	10 μM	91
274030	Transporter, Serotonin (5-Hydroxytryptamine)	326377	hum	2	10 μM	-6

Note: Items meeting criteria for significance (≥50% stimulation or inhibition) are highlighted.

*

Batch: Represents compounds tested concurrently in the same assay(s).

ham=Hamster; hum=Human;

NCI-60 Panel Profile

Table 8Single dose profile of ML336 at 10 micromolar concentration