Overall Assay Strategy

Both the quinazoline and pyrimidine scaffolds were originally identified from a cell-based assay aimed at identifying modulators of Lamin A splicing (AID 1487). Based upon these agents’ structures, we considered that kinase targets may be the mechanistic basis for the activity within this assay. From the literature it was determined that the cdc2-like kinases were likely targets and we followed up this phenotypic assay with an in vitro assay to determine these agents’ activity versus Clk4. In addition to this assay, we utilized commercially available assays from Reaction Biology (www.reactionbiology.com) and DiscoveRx (www.kinomescan.com/) to assess the selectivity of these agents.

Clk 4 qHTS Assays

The application of bioluminescence to ATPase assays has relied on a substrate depletion format. In these assays, the ATP dependence of firefly luciferase is used to measure the remaining ATP concentration, where the luminescence signal is inversely proportional to kinase activity.^12–16 To provide a signal-to-background of approximately 2-fold, the substrate must be depleted by at least 50%. Operating enzyme assays under these high conversion conditions is not at all optimal for classical enzymological studies. However, this is acceptable for HTS, as shifts in potency are typically less than 2-fold with a percent conversion < 80%. Given that HTS assays typically show variability in potency determinations between ~2–3-fold, shifts due to high conversions in the range of 50–80% will not be easily discernible from the assay noise even if the assay is performed at lower conversions. Therefore, ATP depletion has become a popular choice to perform generic HTS assays for ATPases, particularly protein kinases.

We used two bioluminescent assays for Clk4 qHTS (Figure 1). Measurement of ATP depletion was assessed using the Kinase-Glo™ assay system (AID 1770), where a firefly luciferase detection reagent containing D-luciferin and buffer components are added to detect the remaining ATP following the Clk4 kinase assay (Figure 1a). The second system, ADP-Glo^® measures kinase activity by quantifying the amount of ADP formed after the kinase reaction (AID 1771). Bioluminescent detection of ADP levels is achieved through the addition of two different detection reagents (Figure 1b). First, a reagent that stops the protein kinase reaction and depletes the remaining ATP is added. Then, a second reagent is added to stop ATP degradation. In addition, the second reagent also contains an enzyme such as pyruvate kinase that efficiently converts the ADP to ATP, and the same firefly luciferase/D-luciferin components present in Kinase-Glo, which generate a luminescent signal proportional to the ADP concentration produced. Therefore, the two assay formats show opposite luminescent signal changes in response to protein kinase inhibitors.

Figure 1

Bioluminescent assays used for Clk4 qHTS. a. Bioluminescent measurement of ATP depletion using Kinase-Glo. b. Bioluminescent measurement of ADP formation using ADP-Glo.

All compounds were screened using a qHTS approach,¹⁵ in which compounds were assayed using at least seven concentrations to generate concentration-response curves for each compound tested. The methodology for creating a concentration-titration series between successive copies of library plates for the purpose of large-scale titration-based screening has been described. Briefly, qHTS uses an inter-plate dilution method where the first plate contains the highest concentration of a set of compounds in DMSO, while subsequent plates contain the same compounds in the same well locations, but at successively lower concentrations. Using the protocol outlined above, we calculated a plate throughput of 18 plates/hr, or approximately 7 samples/sec, on the Kalypsys robotic system, which means that a 7 point CRC was obtained every second on the robotic system.

Table 1Assay protocol: The optimized 1536-well protocol

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Kinase-Glo				ADP-Glo
Step	Parameter	Value	Description	Parameter	Value	Description
1	Reagent	2 μL	ATP/peptide	Reagent	2 μL	ATP/peptide
2	Library	23 nL	0.5 nM- 46 μM	Library	23 nL	0.6 nM- 55.2 μM
3	Controls	23 nL	TG003	Controls	23 nL	TG003
4	Reagent	1 μL	Clk4	Reagent	0.5 μL	Clk4
5	Time	4.5 hrs	r.t. incubation	Time	1 hr	r.t. incubation
6	Reagent	3 μL	Kinase-Glo	Reagent	2.5 μL	Deplete ATP
7	Read	2 sec	ViewLux	Time	45 min	r.t. incubation
8				Reagent	5 μL	ADP→ATP/Luc
9				Time	30 min	r.t. incubation
10				Read	2 sec	ViewLux
Step	Notes			Notes
1	100 μM RS peptide, 1 μM ATP (final) concentration in buffer; FRD dispense			100 μM RS peptide, 1 μM ATP (final) concentration in buffer; FRD dispense
2	Pin-tool transfer compound library for a (final) range of 46 μM to 0.5 nM			Pin-tool transfer compound library for a (final) range of 55.2 μM to 0.6 nM
3
4	Clk4 at 25 nM final, FRD dispense			Clk4 at 25 nM final, FRD dispense

The assay showed excellent performance (the signal-to-background ratio was 3.2 +/− 0.07, the average Z′ screening factor associated with each plate was 0.86 +/− 0.02 and the CV was 7.2 +/− 1.9, indicating a robust performance of the screen).

Probe Chemical Structure, Physical parameters, and Properties

Structure Verification and Purity: ¹H NMR, ¹³C NMR, RP HPLC/UV/HRMS Data

Proton and carbon NMR data for ML315: Detailed analytical methods and associated instrumentation are described in section 2.3, entitled “Probe Preparation”, under general experimental and analytical details. The numerical experimental proton and carbon NMR data are presented.

Proton NMR Data for ML315:¹H NMR (500 MHz, DMSO-d₆): δ 8.41 (s, 1H), 7.96 (s, 1H), 7.41 (t, J = 2.0 Hz, 1H), 7.35-7.32 (m, 3H), 7.05 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 1.5 Hz, 1H), 6.90 (dd, J = 8.0 Hz, 1.5 Hz, 1H), 6.09 (s, 2H), 4.54 (d, J = 6.5 Hz, 2H).

Carbon NMR Data for ML315:¹³C NMR (125 MHz, DMSO-d₆): δ 158.9, 156.9, 153.4, 147.8, 147.3, 144.7, 133.8 (2), 127.6, 126.2, 125.9 (2), 122.6, 119.1, 109.4, 109.0, 101.3, 42.8.

RP HPLC/UV/HRMS Data for ML315: Detailed analytical methods and associated instrumentation are described in section 2.3, entitled “Probe Preparation”, under general experimental and analytical details. Purity assessment by RP HPLC/UV/HRMS at 214 nm for ML315 revealed purity of 97.6% (retention time = 3.37 minutes). HRMS (m/z): calcd for C₁₈H₁₄Cl₂N₃O₂ (MH⁺) 374.0465; found 374.0486.

Solubility

Aqueous solubility for the probe was assessed in phosphate-buffered saline (PBS) at room temperature. PBS by definition is 137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic at a pH of 7.4 (ref to SBMRI). Under these conditions, the solubility of ML315 was determined to be 1.15 μM. Solubility was also assessed in the assay media of the primary assay (25 mM Tris pH7.5, 10 mM MgCl₂, 0.5 mM EGTA, 2.5 mM DTT, 0.01% Triton X-100). Under these conditions, the solubility of probe ML315 was determined to be 1.71 μM. Though the solubility of ML315 is limited, the compound concentration was 17-fold greater in PBS than the measured IC₅₀ as determined in the AMBIT panel, and in the assay media, the compound concentration was 25-fold greater than the measured IC₅₀, allaying concerns about solubility-limited potency and SAR.

Stability

Aqueous stability for the probe was assessed using two solvent systems (100% aqueous PBS, and 50:50 aqueous PBS:acetonitrile). The probe’s stability was measured in aqueous PBS (no antioxidants or other protectants, DMSO concentration below 1%, room temperature) and the results are reported as circles in the graph in Figure 2. The probe’s stability was also measured in 50:50 aqueous PBS and acetonitrile and the results are reported as triangles in the graph in Figure 2. Stability data in each case is depicted as the loss of compound with time over 48 hours with a minimum of six time points and providing the percent compound remaining after 48 hours. The apparent compound instability in aqueous PBS is much more likely attributed to compound insolubility in that medium, because 100% of the compound was detected after 48 hours in PBS with 50% acetonitrile (50% ACN).

Figure 2

Stability studies for ML315 in aqueous PBS (circles) and 50:50 aqueous PBS:acetonitrile (triangles).

Synthesis Route

The probe compound ML315 was synthesized according to Scheme 1. Bromination of 4-aminopyrimidine afforded 5-bromo-4-amino pyrimidine, which was subjected to Suzuki cross-coupling conditions with 3,4-(methylenedioxy)phenylboronic acid in a microwave reactor to produce S-1. Reductive amination of S-1 with 3,5-dichlorobenzaldehyde delivered ML315, which was purified by preparative reverse-phase HPLC.

Scheme 1

Synthetic route to ML315.

Benzodioxole and benzodioxane analogs were synthesized via the sequence shown in Scheme 2. Suzuki cross-coupling of 2-amino-4-chloropyrimidine with either 3,4-(methylenedioxy)phenylboronic acid or 1,4-benzodioxane-6-boronic acid in a microwave reactor afforded S-2 or S-3, respectively. Subjecting the cross-coupling products to reductive amination conditions with the appropriate aldehyde in each case produced the desired analogs, which were purified by preparative reverse-phase HPLC.

Scheme 2

Synthetic route to oxygenated analogues.

Indazole analogs were synthesized according to the sequence shown in Scheme 3. 2,4-Dichloropyrimidine was reacted with 3,4-dichlorobenzylamine via S_NAr reaction in a microwave reactor at 110 °C, producing S-4 and S-5 in a ratio of 72:28. The isomeric mixture was readily separated using silica gel column chromatography. The individual isomers S-4 and S-5 were then used in microwave-assisted Suzuki cross-coupling reactions with the appropriate indazolylboronic acids to afford the desired indazole analogs, which were purified by preparative reverse-phase HPLC. These compounds were also synthesized via Suzuki cross-coupling/reductive amination sequences starting from either 2-amino-4-chloropyrimidine or 4-amino-2-chloropyrimidine to confirm the identities of the desired analogs.

Scheme 3

Synthetic route to indazole analogues.

Submission of Probe and Five Supporting Analogues to the MLSMR

Samples of the probe and five analogs were prepared, analytically characterized, and shipped to the MLSMR. The structures for the five supporting analogues are shown in Table 2. The screening results from NCGC in-house bioluminescent luciferase-based assays are shown in parentheses, while the remaining data (IC₅₀ values, nM) was generated using a commercial vendor for AMBIT subtype selectivity profiling against the Clk and Dyrk kinases.

Table 2

Supporting analogs with IC₅₀ values (nM) against Clk and Dyrk kinases.

General experimental and analytical details

All reagents were used as received from commercial suppliers. Microwave reactions were carried out using a Biotage Initiator. The ¹H and ¹³C spectra were recorded on Bruker Avance 400 MHz or 500 MHz spectrometers. Chemical shifts are reported in parts per million and referenced to the center line of residual solvent signals: CDCl₃ (7.26 ppm for ¹H and 77.23 ppm for ¹³C), d₃-MeCN (1.94 ppm for ¹H and 1.39 ppm for ¹³C), and d₆-DMSO (2.50 ppm for ¹H and 39.51 ppm for ¹³C). Flash column chromatography separations were performed using Sorbent Technologies standard grade silica gel (40–63 μm particle size, 230 × 400 mesh) with compressed nitrogen as a source of positive pressure or the Teledyne Isco CombiFlash R_F using RediSep R_F silica gel columns. TLC was performed on Analtech UNIPLATE silica gel GHLF plates (gypsum inorganic hard layer with fluorescence). TLC plates were developed using UV light or aqueous KMnO₄. HPLC/MS analysis was carried out with gradient elution (5% CH₃CN to 100% CH₃CN) on an Agilent 1200 RRLC with a photodiode array UV detector and an Agilent 6224 TOF mass spectrometer (also used to produce high resolution mass spectra). Purification was carried out by mass-directed fractionation with gradient elution (a narrow CH₃CN gradient was chosen based on the retention time of the target from LCMS analysis of the crude sample) on an Agilent 1200 instrument with photodiode array detector, an Agilent 6120 quadrupole mass spectrometer, and a HTPAL LEAP autosampler. Fractions were triggered using an MS and UV threshold determined by HPLC/MS analysis of the crude sample. The conditions for HPLC analysis included the following: Waters BEH C-18, 1.7 μm, 2.1 × 50mm column; 0.6 ml/min flow rate; and pH 9.8 NH₄OH aqueous mobile phase. The conditions for purification included: Waters XBridge C18 5μm, 19 × 150mm column; 20 ml/min flowrate pH 9.8 NH₄OH aqueous mobile phase.

The probe was prepared using the following protocols

5-Bromopyrimidin-4-amine. This compound was synthesized according to the published procedure.¹⁷ 4-Aminopyrimidine (1.00 g, 10.5 mmol, 1.0 equiv) and CaCO₃ (263 mg, 2.63 mmol, 0.25 equiv) were combined in H₂O (20 mL) and Br₂ (1.06 mL, 20.5 mmol, 2.0 equiv) was added dropwise. The reaction mixture was stirred at 60 °C for 45 minutes, cooled to room temperature, and then poured into CH₂Cl₂ (20 mL). The layers were separated and the aqueous layer was extracted with CH₂Cl₂ (2 × 20 mL). The combined organic layers were set aside. The aqueous layer was treated with 20% aqueous K₂CO₃ until pH 9–10 was achieved, and the product precipitated. The precipitate was removed via filtration and dried, affording the product (1.08 g, 6.22 mmol, 59%) as a light tan solid. ¹H NMR (500 MHz, DMSO-d₆): δ 8.32 (s, 2H), 7.87–6.62 (v br s, 2H). ¹³C NMR (125 MHz, DMSO-d₆): δ 160.1, 156.8_, 156.0, 102.5.

5-(Benzo[d][1,3]dioxol-5-yl)pyrimidin-4-amine. 5-Bromopyrimidin-4-amine (400 mg, 2.30 mmol, 1.0 equiv) was dissolved in dioxane (7 mL) in a 20-mL microwave vial and to it was added 3,4-(methylenedioxy)-phenylboronic acid (763 mg, 4.60 mmol, 2.0 equiv), aqueous Na₂CO₃ (2.0 M, 3.45 mL, 6.90 mmol, 3.0 equiv), and Pd(PPh₃)₄ (266 mg, 0.23 mmol, 0.10 equiv). The sealed vial was placed in a microwave reactor and heated at 110 °C for 30 minutes. The crude reaction mixture was partitioned between CH₂Cl₂ (20 mL)/H₂O (20 mL) and the layers were separated. The aqueous layer was extracted with CH₂Cl₂ (2 × 20 mL), and the combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude reaction mixture was purified via silica gel column chromatography using MeOH/CH₂Cl₂ (1:19), affording the product (220 mg, 1.02 mmol, 44%) as a light tan solid. ¹H NMR (500 MHz, DMSO-d₆): δ 8.33 (s, 1H), 7.95 (s, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.97 (d, J = 1.5 Hz, 1H), 6.87 (dd, J = 8.0 Hz, 1.5 Hz, 1H), 6.60 (br s, 2H), 6.06 (s, 2H) ¹³C NMR (125 MHz, DMSO-d₆): δ 160.8, 157.0, 154.0, 147.7, 147.0, 128.3, 122.2, 117.7, 109.0, 108.9, 101.2.

5-(Benzo[d][1,3]dioxol-5-yl)-N-(3,5-dichlorobenzyl)pyrimidin-4-amine. 5-(Benzo[d][1,3]dioxol-5-yl)pyrimidin-4-amine (150 mg, 0.70 mmol, 1.0 equiv) was suspended in MeCN (15 mL), and then 3,5-dichlorobenzaldehyde (610 mg, 3.49 mmol, 5.0 equiv), ClTi(Oi-Pr)₃ (95%, 1.05 mL, 4.20 mmol, 6.0 equiv) and AcOH (5 drops) were added. The reaction mixture was stirred for 5 minutes at room temperature and then Na(OAc)₃BH (95%, 779 mg, 3.49 mmol, 5.0 equiv) was added. The mixture was stirred for two hours and then aqueous NH₄OH (15%, 15 mL) was added. The mixture was stirred for a further one hour and then solids were filtered off and washed with CH₂Cl₂ (20 mL) and H₂O (20 mL). The layers of the biphasic mixture were separated and the aqueous layer was extracted with CH₂Cl₂ (2 × 20 mL). The combined organic layers were dried over Na₂SO₄ and then concentrated under reduced pressure. The crude reaction mixture was submitted for preparative reverse-phase HPLC purification, affording the product as a white solid (86.1 mg, 0.23 mmol, 33%). ¹H NMR (500 MHz, DMSO-d₆): δ 8.41 (s, 1H), 7.96 (s, 1H), 7.41 (t, J = 2.0 Hz, 1H), 7.35-7.32 (m, 3H), 7.05 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 1.5 Hz, 1H), 6.90 (dd, J = 8.0 Hz, 1.5 Hz, 1H), 6.09 (s, 2H), 4.54 (d, J = 6.5 Hz, 2H). ¹³C NMR (125 MHz, DMSO-d₆): δ 158.9, 156.9, 153.4, 147.8, 147.3, 144.7, 133.8 (2), 127.6, 126.2, 125.9 (2), 122.6, 119.1, 109.4, 109.0, 101.3, 42.8. HRMS (ESI/APCI) Calcd for C₁₈H₁₄Cl₂N₃O₂ ([M+H]⁺): 374.0465. Found: 374.0486.

In vitro pharmacokinetics profiling

The in vitro pharmacokinetic (PK) properties of the probe (ML315) were profiled using a standard panel of assays (Table 3). The results from this profile suggested that ML315 has modest aqueous and assay media solubility, good permeability across an artificial membrane (PAMPA), good plasma stability but low microsomal stability.

Table 3

Summary of in vitro ADME properties of ML315.

Broad-spectrum target profiling: ML315 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 were noted as ≥ 50% inhibition or stimulation for biochemical assays (see Table 4).

Table 4

Off-target pharmacology data for ML315 highlighting targets with ≥ 50% inhibition or stimulation.