Discovery of Novel, Dual Mechanism ERK Inhibitors by Affinity

Oct 14, 2014 - Selection Screening of an Inactive Kinase. Yongqi Deng,*. ,†. Gerald W. Shipps, Jr.,. †. Alan Cooper,. ‡. Jessie M. English,. ‡...
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Discovery of Novel, Dual Mechanism ERK Inhibitors by Affinity Selection Screening of an Inactive Kinase Yongqi Deng,*,† Gerald W. Shipps, Jr.,† Alan Cooper,‡ Jessie M. English,‡ D. Allen Annis,† Donna Carr,‡ Yang Nan,† Tong Wang,† Hugh Y. Zhu,‡ Cheng-Chi Chuang,† Priya Dayananth,‡ Alan W. Hruza,‡ Li Xiao,‡ Weihong Jin,‡ Paul Kirschmeier,‡ William T. Windsor,‡ and Ahmed A. Samatar‡ †

Merck Research Laboratories, 33 Avenue Louis Pasteur, Boston, Massachusetts 02115, United States Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United States

‡

S Supporting Information *

ABSTRACT: An affinity-based mass spectrometry screening technology was used to identify novel binders to both nonphosphorylated and phosphorylated ERK2. Screening of inactive ERK2 identified a pyrrolidine analogue 1 that bound to both nonphosphorylated and phosphorylated ERK2 and inhibited ERK2 kinase activity. Chemical optimization identified compound 4 as a novel, potent, and highly selective ERK1,2 inhibitor which not only demonstrated inhibition of phosphorylation of ERK substrate p90RSK but also demonstrated inhibition of ERK1,2 phosphorylation on the activation loop. X-ray cocrystallography revealed that upon binding of compound 4 to ERK2, Tyr34 undergoes a rotation (flip) along with a shift in the poly-Gly rich loop to create a new binding pocket into which 4 can bind. This new binding mode represents a novel mechanism by which high affinity ATP-competitive compounds may achieve excellent kinase selectivity.

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INTRODUCTION The Ras/MAPK (RAF/MEK/ERK) pathway plays a central role in regulating mammalian cell growth, differentiation, and survival by relaying extracellular signals from ligand-bound cell surface tyrosine kinase receptors such as ErbB family, IGF-1R, PDGF, and VEGF receptor tyrosine kinase to the nucleus.1−5 Aberrant activation of this pathway through gain-of-function mutations has been associated with several human cancers. RAS genes are mutated in colorectal (50%), melanoma (20%), and pancreatic (90%) tumors, and mutations in BRAF have been identified in melanomas (60%), thyroid cancers (∼50%), and colorectal cancers.6−8 No oncogenic mutations have been found in MEK or ERK; however, gain-of-function mutations in RAS or RAF activate MEK and ERK which are downstream of RAS and RAF. Activation/phosphorylation of MEK and ERK promotes tumor growth through transcriptional activation and increased cell cycle progression and survival. Down-regulation of the MAPK pathway through inhibition of BRAF and MEK has been demonstrated to decrease cell growth and tumor formation.9,10 These observations have promoted the development of several small molecule inhibitors targeting BRAF and MEK, several of which are in clinical trials.11 Despite its important role in the MAPK pathway, there are only a few ERK inhibitors reported in the literature and no known ERK inhibitor has been advanced into late stage clinical trials.12−14 Our initial approach to the discovery of an ERK inhibitor was to screen an activated ERK2 (aERK2) protein using a traditional high-throughput enzymatic assay. While this approach yielded high potency inhibitors, the compounds © XXXX American Chemical Society

lacked a satisfactory degree of kinase selectivity and/or cellbased activity. Subsequent efforts to improve selectivity proved to be challenging, and alternative approaches were pursued. Recently, a new class of kinase inhibitors has been identified that bind to the inactive conformation of kinases (type II). The type II class of kinase inhibitors binds to the ATP binding cleft only when the conserved DFG amino acid sequence motif flips to the “out” conformation. The ability to achieve a DFG-out conformation frequently requires the activation loop to be unphosphorylated; thus, type II inhibitors are found by screening inactive kinases. Some of the recently developed type II kinase inhibitors include imatinib, nilotinib, lapatinib, and sorafenib.15−19 These inhibitors in general possess several advantages over active kinase inhibitors (type I) because they do not compete with high levels of intracellular ATP concentrations, have slower off-rates and the potential to have a different selectivity profile than ATP competitive compounds.20 For these reasons, we decided to develop assays to identify compounds that preferentially bound to the inactive state of ERK. Affinity selection−mass spectrometry (AS−MS) has emerged as an attractive technique for studying protein−ligand interactions and screening biomolecular receptors against pools of potential small molecule ligands.21 One example of this affinity-based approach is known as the automated ligand identification system (ALIS).22−24 This method allows interReceived: June 3, 2014

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3-carboxamide 20 in quantitative yield. The right-hand side ethyl acetate group was introduced by reacting 20 with ethyl glyoxylate under reductive amination conditions to give pyrrolidine-3-carboxamide acetate 21, which was hydrolyzed with lithium hydroxide in THF to give carboxylic acid 22. Subsquentely, 22 was converted to the final compounds 1−3 by reacting with the corresponding arylpiperidines. The pure enantiomers 4−11 were synthesized following the same route using the corresponding commercially available R and S Boc protected pyrrolidine-3-carboxylic acids. To make analogues with different substitutions on the left-hand side, a different synthetic route was developed. In Scheme 2, the Boc group of 23 was removed using TFA to give compound 24, which was alkylated with tert-butyl chloroacetate to give compound 25 in 80% yield. Treating tert-butyl ester 25 with TFA gave carboxylic acid 26, which was then reacted with the corresponding piperazine to give 27 under HATU coupling conditions. The methyl ester of 27 was hydrolyzed using LiOH in THF to provide 28, which was reacted with various substituted aminophenols to give compounds 12−16. Biochemical Activities and SAR. Two enzyme assays were developed to assess the activity of compounds 1−16 binding to the phosphorylated and unphosphorylated states of ERK2 (Table 1). A conventional active ERK2 assay (aERK2) was used to measure the activity of compounds binding to the phosphorylated state of ERK2. A coupled ERK2 assay (cERK2) was used to assess the activity of compounds binding to the unphosphorylated state. In this coupled assay, unphosphorylated ERK2 was preincubated with the test compounds for 30 min before it was activated by the addition of phosphorylated MEK1 and then was used to phosphorylate the ERK2 polypeptide substrate. Since the cERK2 assay comprises both the processes of activating ERK2 and substrate phosphorylation, the inhibition measured in this assay is a composite of inhibiting both inactive ERK2 and activated ERK2. Control experiments showed that the MEK inhibitor PD0325901,10 which does not bind to ERK, had no activity in the active ERK assay as expected. PD0325901, however, showed good activity in the coupled assay with an IC50 of 25 nM because it prevented the phosphorylation of ERK2 by MEK. The binding affinity of each of the compounds to both pERK2 and apo ERK2 was also determined by TdF. The IC50 values measured from both the aERK2 and cERK assays as well as the Kd values determined from the TdF assay are listed in Table 1. The IC50 values for the aERK2 and cERK2 assays ranged from 0.44 to 20 μM for active compounds, and the Kd values for pERK and apo ERK2 in the TdF assays ranged from 0.07 to 2.65 uM. The relative rank order was consistent for both assay types. A comparison of the IC50 values determined in the aERK2 and the cERK2 assays indicated compounds 1−15 have IC50 values approximately 2- to 4-fold lower in the cERK2 assay. This result suggests that the compounds bind preferentially to the inactive state of ERK2. TdF studies confirmed that the binding affinities of compounds bound to apo ERK2 were 2- to 3-fold higher compared to activated ERK2 (pERK2). The SAR of this series of compounds revealed that the addition of a chloro group at the 3-postion of the phenol of 1 improved both its binding affinities and biochemical activities (2). The stereocenter of the pyrrolidine core was found to be important, as the R isomer (4) had a higher affinity and potency than the S isomer 5. Relative activities to apo ERK2 and pERK2 were similar. Shifting the chloro group from the 3-

rogation of a ligand binding to protein surface without modification of its structure, does not require immobilization of the protein or the small molecule pool against which the target is screened, and consumes only a few milligrams of purified, soluble protein to screen millions of compounds. The ALIS process involves the incubation of samples containing thousands of small molecules in the presence of a target protein and identifies bound ligands by mass spectrometry following separation of unbound ligands using size exclusion chromatography (SEC). Since ALIS techniques directly measure noncovalent protein−ligand interactions, it is independent of the phosphorylation (activation) state of kinases to be studied and is ideal for screening inactive forms of kinases which may have a wide range of conformational states. Herein, we report a novel series of ERK inhibitors identified through screening of inactive ERK by ALIS technology and their unique binding and inhibition mechanism.

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RESULTS AND DISCUSSION Identification of Initial Hits through ALIS Screening. To identify selective small-molecule inhibitors of ERK2, an inhouse diverse chemical library of ∼5 million compounds was screened using ALIS. The high throughput screening campaign involved incubation of unphosphorylated ERK2 (apo ERK2) with ∼2000 compounds per well followed by SEC to separate the protein-bound small molecules from the unbounded molecules. Compounds that bound to ERK2 were transferred to the reversed phase column (RPC) where ligands were dissociated from the protein and trapped on the RPC stationary phase. The dissociated ligands were eluted into a high resolution time-of-flight mass spectrometer for molecular weight analysis. The structure of a bound ligand was identified based on its unique mass for a particular well from each library. One of the hits from this screen was compound 1 which has a pyrrolidine core (Figure 1). Compound 1 is ATP competitive

Figure 1. Initial ALIS screening hit 1.

as assessed by the decreased binding with the addition of ATP (as indicated by the reduction of returned mass counts); however, ample signal was still evident at 2.5 mM ATP, suggesting that the compounds had favorable affinity to one or more ERK conformations relative to ATP. The binding of 1 to unphosphorylated ERK2 was confirmed using temperaturedependent fluorescence (TdF), and the Kd was determined to be 1.1 μM.25,26 TdF studies determined that 1 also bound to phosphorylated ERK2 (pERK2) with a Kd of 1.9 μM. These studies indicate the ALIS screen has identified a compound that bound to both unphosphorylated and phosphorylated ERK2. Compound Synthesis. Two synthetic routes were developed to make analogues of 1 with variations on both right- and left-hand sides. Scheme 1 depicts the synthesis of 1 and its derivatives 2−11. In this route, reacting commercially available substituted 4-aminophenol 17 with Boc protected pyrrolidine-3-carboxylic acid 18 under standard amide formation conditions27 gave Boc protected pyrrolidine-3-carboxamide derivative 19, which was then deprotected using 50% TFA in dichloromethane to give the corresponding pyrrolidineB

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Scheme 1a

a

Reagents and conditions: (i) HATU, DIEA, DMF, rt, overnight; (ii) TFA, rt, 1 h; (iii) HOAc, ethyl glyoxylate, rt, 1 h; then sodium borohydride triacetoxy, rt, overnight; (iv) LiOH, THF, rt, overnight; (v) arylpiperazine, HATU, DIEA, DMF, rt, 2 h.

Scheme 2a

Reagents and conditions: (i) TMSCHN2, toluene/MeOH, 0−5 °C, 30 min; (ii) TFA, rt, 1 h; (iii) tert-butyl chloroacetate, DIEA, rt, 1 h; (iv) arylpiperidine, HATU, DIEA, DMF, rt, 2 h; (v) LiOH, THF, rt, 12 h; (vi) aminophenol, HATU, DIEA, DMF, rt, overnight.

a

two hydrogen bonds toward the hinge region with a hydrogen bond donor to the Met106 backbone amide NH and a hydrogen bond acceptor to the backbone carbonyl of Asp104. The 3-Cl phenyl substitution of 4 appears to interact favorably with Met106 with a distance of 3.43 Å to its carbonyl group. Substitutions of 3-Cl with Br or methyl, as seen in 14 and 16, led to comparable activities and are likely to provide similar favorable interactions. The hydrogen at the 5-position of the phenol is close to the Ile82 with a distance of 3.65 Å, which explains why replacing this hydrogen with a chlorine resulted in the loss of activity in 13. The CO of the left-hand side amide off the phenol group is involved in an extended hydrogen bond network through a water molecule with the gatekeeper Gln103 and the catalytic Lys52. The substitution at 2-position of the phenol in 12 most likely disrupts this hydrogen bond network by rotating the amide bond relative to the phenol and resulting in a compound that does not bind (Table 1). The pyrrolidine N

position to the 2-position reduced affinity and activity of 12, and an additional chlorine at the 5-position was also not tolerated (13). The chlorine of 4 can be replaced by a bromine (14), but a methyl (16) and a fluoride group (15) was found to be less optimal. On the right-hand side (RHS) of the molecule, replacing the 4-acetylphenyl group in 4 with 3-chlorophyl group resulted in 6 with similar affinity and potency. However, replacement of acetyl group in 4 with bromo, phenyl, 3thiophenyl, and 3-furanyl groups resulted in compounds with lower activity (7−10). Interestingly, a bicyclic group, benzothiazole, was found to restore the affinity and activity in compound 11. Binding Mode of 4 and Kinase Selectivity. The X-ray structure of the 4/ERK2 complex was determined at a resolution of 1.65 Å (Figure 2). As expected, 4, an ATPcompetitive inhibitor of ERK2, bound at the ATP binding pocket of ERK2, with the hydroxyl of the phenol group forming C

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Table 1. IC50 Values from the Coupled and Active ERK Assays and TdF Kd Values for Apo ERK2 and Active ERK for Compounds 1−16

A novel protein conformational change occurs at the RHS of the 4/ERK2 complex (Figure 3). The Gly-rich loop (green) undergoes a large conformation change (yellow) which flips the aromatic side chain of Tyr34 away from the RHS and toward the hinge binding site. This conformational change opens up a new side pocket (the Tyr34 side pocket) for the RHS of 4 to extend into. The pyrrolidine ring stacks directly under Tyr34, making a hydrophobic interaction with the aromatic side chain. Asp165 (a component of the DFG motif) serves as a floor beneath the compound at the methyl group connecting the RHS amide. The piperazine functions as a linker to turn the RHS into the side pocket, while the distal acetylphenyl interacts “face to face” with the Tyr62 side chain through an aromatic π−π interaction. Despite the large movement of the Gly-rich loop observed upon 4 binding to ERK2, no structural change occurs in the activation loop (not shown). In addition, there are no structural changes at the DFG motif where the Phe166 side chain of the DFG motif overlays well with the X-ray structures of unbound apo ERK2 and 4 bound to ERK2. In comparison to its apo form, Tyr62 shifts right to create extra space upon binding to 4 to accommodate the acetyl group. The flexibility of

Figure 2. X-ray crystal structure of ERK2 with bound inhibitor 4: inhibitor 4, carbon atoms, cyan; ERK2 carbon, gray; nitrogen, blue; oxygen, red; chloride, green; sulfur, yellow. The water molecule oxygen atom involved in the hydrogen bond network is in CPK representation. The hydrogen bonds are labeled with dashed lines.

in the center of 4 is in the neutral form and acts as a hydrogen bond acceptor to Lys52. The right-hand side amide CO of 4 also makes a hydrogen bond with Lys52. D

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binding to 4 might disrupt phosphorylation by MEK. To determine if 4 is capable of inhibiting ERK2 phosphorylation, a cell-based assay was used to measure the level of RSK and ERK1/2 phosphorylation in a melanoma cell line A2058 in the presence of the compound. In this assay, 4 inhibited the phosphorylation of RSK but also inhibited phosphorylation of ERK1/2 in a dose dependent manner with an EC50 of ∼800 nM (Figure 4). While phosphorylation of ERK can be affected

Figure 3. Superposition of the X-ray crystal structures of apo ERK2 and inhibitor 4 bound ERK2 complex: apo ERK2, carbon, green; inhibitor 4 bound ERK2 complex, carbon, gray; inhibitor 4, carbon, cyan. Tyr34 in ERK2/4 complex is highlighted with carbon atoms in yellow.

Figure 4. Compound 4 suppresses the phosphorylation ERK1,2. A2058 BRAF mutant cell lines were treated for 4 h at 37 °C with 0, 150, 312, 625, 1250, 2500, 5000, or 10000 nM compound 4. Cells were harvested after 4 h, and cell lysates containing equal amounts of protein were electrophoresed and immunoblotted for phosphoERK1,2 and total ERK1,2 using appropriate antibodies. Blots were developed using ECL reagent from Amersham Biosciences. Cells treated with MEK inhibitor PD0325901 were used as a positive control for the suppression of phosphorylated ERK1,2 proteins.

Tyr 62 is also evident in that substitutions to the RHS terminal groups are tolerated to some degree. For example, large terminal substitutions are tolerated off the RHS phenyl groups (7−10), while their activities are lower than compound 4. Although compound 6 has the terminal acetyl group eliminated, the 3-Cl substitution appears to provide compensating interactions with Tyr 62 that retains a similar activity as 4. Compound 4 displays good kinase selectivity when counterscreened against a panel of 30 kinases (Table 2).28,29

by upstream kinases and growth factors,30,31 A2058 is a BRAF mutant cell line, and the up-regulation of the MAP kinase pathway in this cell line is driven by the BRAF mutation. Thus, the inhibition of pERK in this cell line is not likely due to the inhibition of other upstream tyrosine kinase receptors or other growth factors upstream of BRAF. The kinase counterscreen assay results also suggest that 4 does not inhibit BRAF, and the TdF binding studies confirmed that 4 does not bind to MEK.32 These results, along with the observation that the compounds bind preferentially to the inactive state of ERK2 in the enzyme and TdF assays, suggest the inhibition of pERK1,2 in the cell based assay is likely to be due to the binding of the compounds to the inactive state of ERK1,2. Although the exact manner in which MEK binds and activates ERK is not known in detail, the interaction between MEK1,2 and ERK1,2 is very specific. MEK1,2 are the only known activators of ERK1,2, and ERK1,2 are the only known substrates of MEK1,2.33 MEK is one of a few kinases that require the full-length protein substrate in order to phosphorylate the activation loop. We postulated that the large conformational change in the glycine-loop of ERK2 may distort the surface and the docking site of ERK2 such that MEK would either not be able to bind to ERK2 or unable to complete the catalytic step of phosphotransfer to the activation loop residues on ERK2. The phosphorylation of ERK1,2 by MEK1,2 requires both the correct orientation of the activation loop residues and the native ERK1,2 tertiary fold for recognition and phosphorylation.34 Such stringent requirement for the precise conformation of ERK2 may be disrupted by the large conformational change at the G-loop of ERK2 upon the binding of compound 4, and as a result, the ERK2 phosphorylation is blocked. We have tested 4 in cell proliferation assays, and it shows inhibition of cell growth in several cell lines with BRAF mutation. The IC50 values of 4 were found to be 28.8, 11.5, and 10.8 μM for A2058, A375SM, and Colo-205, respectively.

Table 2. Kinase Selectivity of Compound 4 kinase

IC50 (μM)

kinase

IC50 (μM)

ABL AKT1 CDK2 B-RAF CHK1 CSNK1D TSSK2 EPHB4 FLT3 IGF1R IKKB IRAK4 JAK2 LCK

>30 >30 >50 >30 >30 >30 >30 >30 >30 >30 >30 >30 >30 >30

MET MST2 NEK2 PKCA PLK3 ROCK2 RSK2 EGFR p38β MEK1 VEGFR-1 CDK4 GSK3β CDK6

>30 >30 >30 >30 >30 >30 ∼27 >50 >50 >20 >50 >50 >50 >50

The favorable selectivity may be due in part to the unique binding mode of 4. Unlike other ERK2 inhibitors, which bind to the ATP pocket (between the hinge and Tyr34 12,13), 4 binds to ERK2 in an “induced fit” manner enabling the RHS of 4 to extend further to the right side of the ATP pocket and into a hydrophobic pocket due to the torsional rotation of the polyglycine loop and the side chain of Tyr34. As a result, 4 is able to occupy an expanded space between the hinge and Tyr62. The large conformational change of the Gly-rich loop caused by the flip of Tyr34 and the formation of a new binding pocket toward Tyr62 is unique and may contribute toward the good kinase selectivity of 4. Cellular Activities. While adopting a DFG-in mode, we speculated that the large conformational change of ERK2 upon E

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Moreover, 4 shows no activity (>100 μM) in a cell line (CHL1) with wild type BRAF.

UV absorbance at 230 nm. After a pause to allow the band to leave the first detector and enter a valving arrangement, the protein−ligand complex peak is automatically transferred to a reverse-phase chromatography column (Higgins Targa-C18, Higgins Analytical Inc., Mountain View, CA). Ligands are dissociated from the complex and trapped at the head of the RPC column, where they are desalted and eluted into the mass spectrometer using a gradient of 0−95% acetonitrile (0.1% formic acid) in water (0.1% formic acid) over 5 min using an Agilent capillary binary pump (G1376A) for eluent delivery at 20 μL/min. To promote dissociation of ligands from the complex, the RPC column is maintained at 60 °C using an Agilent G1316A column compartment. In this study, MS analysis was performed using a Waters LCT “classic” high resolution time-of-flight mass spectrometer (Manchester, U.K.) with positive-mode ionization occurring from a standard nebulized ESI source with the capillary at 3.5 kV, a desolvation temperature of 180 °C, a source temperature of 100 °C, and 30 V “cone” and 3 V extraction lens settings. AS−MS analyses were conducted by incubating 2500-member libraries at 2.5 μM cumulative compound concentration with 5 μM ERK2 protein in a final volume of 2 μL of pH 7.5 phosphate buffer containing 2.5% DMSO and 200 mM NaCl. As such, 2 pmol of each library component (at 1.0 μM/component) and 10 pmol of protein were used in a single analysis. The use of excess protein relative to each library member minimizes competition between multiple binders in a given library. Typical sample preparation protocol is as follows: to 1 μL of a DMSO solution of 100 mM 2500-member library was added 19 μL of prewarmed (37 °C) pH 7.5 50 mM phosphate buffer containing 200 mM NaCl. The resulting solution was mixed by repeated pipetting and centrifuged at 10000g for 10 min. A 1.0 μL aliquot of the supernatant was added to 1.0 μL of a 5 μM solution of purified ERK2 in pH 7.5 50 mM phosphate buffer containing 200 mM NaCl. Samples were incubated at room temperature for 30 min and then chilled at 4 °C pending AS−MS analysis. Discrete compound screening and competition experiments were prepared identically except that compound stock concentrations in DMSO were adjusted such that the final DMSO concentration in each protein-containing sample was 2.5%. tert-Butyl 3-(4-Hydroxyphenylcarbamoyl)pyrrolidine-1-carboxylate (19a). To a solution of 1-(tert-butoxycarbonyl)pyrrolidine-3-carboxylic acid (2.15 g, 10 mmol) and diisopropylethylamine (DIEA) (3.9 g, 30 mmol) in N,N-dimethylformamide (DMF) (20 mL) at 0 °C was added 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3tetramethyluronium (HATU) (4.56 g, 12 mmol). The reaction mixture was stirred for 10 min, and 4-aminophenol (1.09 g, 10 mmol) was added. The reaction mixture was allowed to warm to room temperature and stirred overnight. The crude product was poured onto ice and extracted with ethyl acetate (20 mL × 3). After drying over anhydrous sodium sulfate and removing the solvent under reduced pressure, the crude product was purified by flash chromatography on silica gel, eluting with dichloromethane and methanol (5%). The title compound 19a was isolated as a solid (2.8 g, 82%). 1H NMR (400 MHz, CDCl3) δ 7.802 (d, 2H), 6.75 (d, 2H), 3.70 (m, 1H), 3.55 (m, 1H), 3.30−3.40 (m, 2H), 2.48 (m, 1H), 1.80− 1.95 (m, 2H), 1.38 (s, 9H); LC−MS tR = 1.35 min (method A), 307.1 (M + H+). tert-Butyl 3-(3-Chloro-4-hydroxyphenylcarbamoyl)pyrrolidine-1-carboxylate (19b). 1H NMR (500 MHz, CDCl3) δ 7.88 (br s, 1H), 7.27 (m, 1H), 6.90 (d, 1H), 3.80 (m, 1H), 3.52 (m, 1H), 3.30−3.40 (m, 2H), 2.48 (m, 1H), 1.80−1.95 (m, 2H), 1.38 (s, 9H); LC−MS tR = 2.16 min (method A), 341.2 (M + H+). N-(4-Hydroxyphenyl)pyrrolidine-3-carboxamide Trifluoroacetate (20a). To a stirring solution of compound 19a (1.0 g, 3.26 mmol) in DCM (5 mL) was added TFA (3 mL) at room temperature. The reaction was monitored by LC−MS, and it was completed in about 1 h. After removal of TFA and solvent under reduced pressure, the crude product (0.95 g) was used directly in the next step. LC−MS tR = 0.25 min (method A), 207.2 (M + H+). N-(3-Chloro-4-hydroxyphenyl)pyrrolidine-3-carboxamide Trifluoroacetate (20b). LC−MS tR = 0.67 min (method A), 241.1 (M + H+).

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CONCLUSIONS Screening of inactive ERK2 utilizing an affinity-based screening technology has resulted in a series of novel, potent, and selective ERK2 inhibitors. It has been shown that compound 4 is a dual function inhibitor that is capable of inhibiting not only the kinase activity of ERK but also the phosphorylation of ERK by MEK in cells. The X-ray structure of compound 4 in complex with inactive ERK protein reveals that the inhibitory function of ERK phosphorylation is likely attained through the large shift of the glycine rich loop upon binding. This is quite different from the DFG-out type inhibitors, which inhibit the kinase activation through conformational changes at the activation loop. If this new active site conformational change is observed in other inhibitor−protein complexes, the polyglycine loop conformational change and torsional rotation of the Tyr32 side chain could be designated as binding mode type IV. We believe the methodology and observations made in this study can provide a new approach to the discovery and design of a new class of kinase inhibitors for additional targets of interest.

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EXPERIMENTAL SECTION

All reagents and anhydrous solvents were obtained from commercial sources and were used without further purification unless otherwise specified. Mass spectrometry samples were analyzed on an Agilent 1100 series LC−MS mass spectrometer with electrospray ionization. Samples were introduced into the mass spectrometer using flow injection or chromatography. The mobile phase for all mass analysis consisted of acetonitrile−water mixtures with either 0.1% formic acid or trifluoroacetic acid. FAB HRMS results were recorded on a Micromass QT of Ultima API US mass spectrometer. The following HPLC methods were used to obtain the reported retention times. (i) Method A: Varian XRS 5, 4.6 mm × 30 mm column; linear gradient from 0% to 90% CH3CN in H2O over 5 min (0.1% trifluoroacetic acid); flow rate 3.0 mL/min; 214 nm/254 nm. (ii) Method B: Varian XRS 5, 4.6 mm × 100 mm column; linear gradient from 10% to 90% CH3CN in H2O over 10 min (0.1% trifluoroacetic acid); flow rate 3.0 mL/min; detection diode array. The purity of all compounds was determined using method B, and all tested compounds were confirmed to have ≥95% purity. 1H NMR spectra (δ, ppm) were recorded using a Varian-400 (400 MHz) instrument. Column chromatography was performed using a Biotage instrument. Preparative reversed-phase chromatography was carried out using a Gilson 322 pump coupled to a UV−vis 156 Gilson detector with a Gilson 215 liquid handler, a Varian XRS 10, C18, 21.2 mm × 250 mm, a linear gradient from 10% to 90% acetonitrile in water over 10 min (0.1% trifluoroacetic acid); flow rate 20 mL/min. Affinity Selection−Mass Spectrometry Methods (AS−MS). The size exclusion chromatography (SEC) based AS−MS hardware configuration used in this study has been described previously. Briefly, this system uses continuous SEC to isolate protein−ligand complexes from unbound library members. Samples containing a target protein, protein−ligand complexes, and unbound compounds are injected onto an SEC column, where the complexes are separated from nonbinding component by a rapid SEC step. SEC is performed at 4 °C using phosphate-buffered or Tris-buffered saline, typically 50 mM pH 7.5 phosphate buffer containing 200 mM NaCl. SEC columns are prepared in-house by proprietary methods. Operationally comparable columns are available from Regis Technologies, Inc. (Morton Grove, IL).4 An Agilent (Palo Alto, CA) isocratic pump (G1310A) fitted with an Agilent online degasser (G1322A) is used for eluent delivery at 300 μL/min. The eluent from the SEC column is passed through a UV detector (Agilent G1314A using a G1313 microflow cell) where the band containing the protein−ligand complex is identified by its native F

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Journal of Medicinal Chemistry

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Ethyl 2-(3-(4-Hydroxyphenylcarbamoyl)pyrrolidin-1-yl)acetate (21a). At room temperature, to a stirring solution of 20a prepared from previous step in the mixture of DCM and MeOH (1:1 ratio, 10 mL) were added ethyl glyoxylate (2.3 g, 50% in toluene, 11.7 mmol) and acetic acid (100 μL). After the reaction mixture was stirred at room temperature for 1 h, sodium triacetoxyborohydride (2.49 g, 11.7 mmol) was added in several portions and the reaction mixture was stirred at room temperature overnight. After removal of the solvent, ethyl acetate (50 mL) was added, followed by water (20 mL). The organic layer was collected, and the aqueous layer was extracted with ethyl acetate (20 mL). The two extractions were combined and were washed with saturated sodium bicarbonate solution (20 mL), water (30 mL × 3), and brine. After removal of the solvent under vacuum, the residue was purified by flash chromatography (10% methanol in DCM) to give the desired product (0.58 g) in 60% yield for the last two steps. 1H NMR (400 MHz, CDCl3) δ 7.75 (d, 2H), 6.75 (d, 2H), 4.18 (q, 2 H), 3.30 (s, 2H), 2.68 (m, 1H), 2.50 (m, 1H), 2.48 (m, 1H), 2.20−2.30 (m, 2H), 1.80−2.05 (m, 2H), 1.32 (t, 3H); LC−MS tR = 0.53 min (method A), 293.1 (M + H+); Ethyl 2-(3-(3-Chloro-4-hydroxyphenylcarbamoyl)pyrrolidin1-yl)acetate (21b). 1H NMR (500 MHz, CDCl3) δ 7.85 (br s, 1H), 7.36 (d, 1H), 6.82 (d, 1H), 4.15 (q, 2 H), 3.32 (s, 2H), 2.78 (m, 1H), 2.50 (m, 1H), 2.48 (m, 1H), 2.20−2.30 (m, 2H), 1.80−2.05 (m, 2H), 1.32 (t, 3H); LC−MS tR = 0.77 min (method A), 327.1 (M + H+); 2-(3-(4-Hydroxyphenylcarbamoyl)pyrrolidin-1-yl)acetic Acid (22a). To a solution of 21a (0.52 g, 1.77 mmol) in THF (5 mL) was added LiOH solution (1 M, 3.6 mL, 3.6 mmol). The reaction mixture was stirred at room temperature overnight. After acidification to pH ≈ 5 with 1 N HCl, THF was removed under vacuum and the mixture was extracted with dichloromethane (30 mL × 3). The combined extracts were washed with brine (20 mL) and dried over sodium sulfate. The solution was filtered and the solvent was removed under reduced pressure to afford the desired product as brown solid (0.34 g, 72% yield). LC−MS tR = 0.25 min (method B), 265.1 (M + H+). 2-(3-(3-Chloro-4-hydroxyphenylcarbamoyl)pyrrolidin-1-yl)acetic Acid (22b). LC−MS tR = 0.64 min (method B), 299.0 (M + H+); 1-(2-(4-(4-Acetylphenyl)piperazin-1-yl)-2-oxoethyl)-N-(4hydroxyphenyl)pyrrolidine-3-carboxamide Hydrochloride (1). To a solution of 22a (40 mg, 0.15 mmol) and DIEA (38 mg, 0.30 mmol) in DMF (1.5 mL) at 0 °C was added HATU (68 mg, 0.18 mmol). After the reaction mixture was stirred for 30 min at 0 °C, 1-(4(piperazin-1-yl)phenyl)ethanone (31 mg, 0.15 mmol) was added and the reaction mixture was allowed to stir at room temperature for 2 h. Water (10 mL) was added, and the mixture was extracted with ethyl acetate (5 mL × 3). The combined extracts were washed with water (10 mL) and brine (10 mL). The solvent was removed under reduced pressure, and the crude product was purified using preparative HPLC. The resulting product was then converted to hydrochloride salt to afford the title compound (25 mg) in 32% yield as white powder. 1H NMR (400 MHz, DMSO-d6) δ 11.0 (s, 1H), 7.82 (d, 2H), 7.40 (d, 2H) 7.02 (d, 2H), 6.64 (d, 2H), 4.52 (m, 2H), 3.84 (m, 1H), 3.62 (m, 4H), 3.50 (m, 4H), 3.45−3.02 (m, 4H), 2.45 (s, 3H), 2.12 (m, 1H), 2.02 (m, 1H); LC−MS purity, 97%, tR = 1.78 min (method B), 451.1 (M + 1); HRMS (ESI) calcd for C25H30N4O4 (M + H+) 451.2345, found 451.2349. 1-(2-(4-(4-Acetylphenyl)piperazin-1-yl)-2-oxoethyl)-N-(3chloro-4-hydroxyphenyl)pyrrolidine-3-carboxamide Hydrochloride (2). 1H NMR (400 MHz, DMSO-d6) δ 11.8 (s, 1H), 7.82 (d, 2H), 7.76 (m, 1H) 7.26 (m, 1H), 7.02 (d, 2H), 6.90 (d, 1H), 4.52 (m, 2H), 3.84 (m, 1H), 3.62 (m, 4H), 3.50 (m, 4H), 3.45−3.02 (m, 4H), 2.45 (s, 3H), 2.12 (m, 1H), 2.02 (m, 1H); LC−MS purity, 95%, tR = 2.67 min (method B), 485.2 (M + 1); HRMS (ESI) calcd for C25H30ClN4O4 (M + H+) 485.1956, found 485.1950. 1-(2-(4-(3-Chlorophenyl)piperazin-1-yl)-2-oxoethyl)-N-(4hydroxyphenyl)pyrrolidine-3-carboxamide Hydrochloride (3). 1 H NMR (400 MHz, DMSO-d6) δ 11.0 (s, 1H), 7.40 (d, 2H), 7.22 (m, 1H), 7.00 (s, 1H), 6.95 (d, 1H), 6.82 (d, 1H), 6.72 (d, 2H), 4.52 (m, 2H), 3.84 (m, 1H), 3.62 (m, 4H), 3.50 (m, 4H), 3.45−3.02 (m, 4H), 2.22 (m, 1H), 2.10 (m, 1H); LC−MS purity, 95%, tR = 2.28 min

(method B), 443.2 (M + 1); HRMS (ESI) calcd for C23H28ClN4O3 (M + H+) 443.1850, found 443.1844. (R)-1-(2-(4-(4-Acetylphenyl)piperazin-1-yl)-2-oxoethyl)-N-(3chloro-4-hydroxyphenyl)pyrrolidine-3-carboxamide Hydrochloride (4). 1H NMR (400 MHz, DMSO-d6) δ 11.8 (s, 1H), 7.82 (d, 2H), 7.76 (m, 1H) 7.26 (m, 1H), 7.02 (d, 2H), 6.90 (d, 1H), 4.52 (m, 2H), 3.84 (m, 1H), 3.62 (m, 4H), 3.50 (m, 4H), 3.45−3.02 (m, 4H), 2.45 (s, 3H), 2.12 (m, 1H), 2.02 (m, 1H); LC−MS purity, 98%, tR = 2.67 min (method B), 485.2 (M + 1); HRMS (ESI) calcd for C25H30ClN4O4 (M + H+) 485.1956, found 485.1950. (S)-1-(2-(4-(4-Acetylphenyl)piperazin-1-yl)-2-oxoethyl)-N-(3chloro-4-hydroxyphenyl)pyrrolidine-3-carboxamide Hydrochloride (5). 1H NMR (400 MHz, DMSO-d6) δ 11.8 (s, 1H), 7.82 (d, 2H), 7.76 (m, 1H) 7.26 (m, 1H), 7.02 (d, 2H), 6.90 (d, 1H), 4.52 (m, 2H), 3.84 (m, 1H), 3.62 (m, 4H), 3.50 (m, 4H), 3.45−3.02 (m, 4H), 2.45 (s, 3H), 2.12 (m, 1H), 2.02 (m, 1H); LC−MS purity, 95%, tR = 2.67 min (method B), 485.2 (M + 1); HRMS (ESI) calcd for C25H30ClN4O4 (M + H+) 485.1956, found 485.1950. N-(3-Chloro-4-hydroxyphenyl)-1-(2-(4-(3-chlorophenyl)piperazin-1-yl)-2-oxoethyl)pyrrolidine-3-carboxamide (6). HRMS (ESI) calcd for C23H27Cl2N4O3 (M + H+) 477.1460 (R)-1-(2-(4-(4-Bromophenyl)piperazin-1-yl)-2-oxoethyl)-N(3-chloro-4-hydroxyphenyl)pyrrolidine-3-carboxamide Hydrochloride (7). 1H NMR (400 MHz, DMSO-d6) δ 11.00 (s, 1H), 7.75 (s, 1H), 7.40 (d, 2H) 7.30 (m, 1H), 6.95 (d, 2H), 6.82 (d, 1H), 4.50 (m, 2H), 3.82 (m, 1H), 3.62 (m, 4H), 3.50 (m, 4H), 3.45−3.02 (m, 4H), 2.22 (m, 1H), 2.10 (m, 1H); LC−MS purity, 95%, tR = 2.50 min (method B), 521.1 (M + 1); HRMS (ESI) calcd for C23H27BrClN4O3 (M + H+) 521.0950, 523.0935, found 521.0947, 523.0925 (R)-1-(2-(4-(Biphenyl-4-yl)piperazin-1-yl)-2-oxoethyl)-N-(3chloro-4-hydroxyphenyl)pyrrolidine-3-carboxamide (8). 1H NMR (400 MHz, DMSO-d6) δ 11.80 (s, 1H), 7.72 (s, 1H), 7.60 (d, 2H) 7.54 (d, 2H), 7.41 (m, 2H), 7.30 (m, 1H), 7.28 (m, 1H), 7.08 (d, 2H), 6.82 (d, 1H), 4.50 (m, 2H), 3.90 (m, 1H), 3.62 (m, 4H), 3.50 (m, 2H), 3.45−3.02 (m, 6H), 2.22 (m, 1H), 2.10 (m, 1H); LC−MS purity, 95%, tR = 2.69 min (method B), 519.1 (M + 1); HRMS (ESI) calcd for C29H32ClN4O3 (M + H+) 519.2157, found 519.2156. (R)-N-(3-Chloro-4-hydroxyphenyl)-1-(2-oxo-2-(4-(4-(thiophen-3-yl)phenyl)piperazin-1-yl)ethyl)pyrrolidine-3-carboxamide (9). 1H NMR (400 MHz, DMSO-d6) δ 11.80 (s, 1H), 7.72 (s, 1H), 7.68 (s, 1H), 7.60 (d, 2H), 7.58 (m, 1H), 7.46 (d, 1H), 7.26 (m, 1H), 7.02 (d, 2H), 6.92 (d, 1H), 4.50 (m, 2H), 3.84 (m, 1H), 3.62 (m, 4H), 3.50 (m, 2H), 3.45−3.02 (m, 6H), 2.22 (m, 1H), 2.10 (m, 1H); LC−MS purity, 98%, tR = 2.60 min (method B), 525.2 (M + 1); HRMS (ESI) calcd for C27H30ClN4O3S (M + H+) 525.1722, found 525.1721. (R)-N-(3-Chloro-4-hydroxyphenyl)-1-(2-(4-(4-(furan-3-yl)phenyl)piperazin-1-yl)-2-oxoethyl)pyrrolidine-3-carboxamide (10). 1H NMR (400 MHz, DMSO-d6) δ12.00 (s, 1H), 8.04 (s, 1H), 7.72 (d, 1H), 7.68 (s, 1H), 7.48 (d, 2H), 7.26 (m, 1H), 7.02 (d, 2H), 6.92 (d, 1H), 6.88 (s, 1H), 4.50 (m, 2H), 3.84 (m, 1H), 3.66 (m, 2H), 3.50 (m, 2H), 3.40 (m, 2H), 3.45−3.02 (m, 6H), 2.22 (m, 1H), 2.10 (m, 1H); LC−MS purity, 96%, tR = 2.42 min (method B), 509.1 (M + 1); HRMS (ESI) calcd for C27H30ClN4O4 (M + H+) 509.1950, found 509.1950. (R)-1-(2-(4-(Benzo[d]thiazol-2-yl)piperazin-1-yl)-2-oxoethyl)N-(3-chloro-4-hydroxyphenyl)pyrrolidine-3-carboxamide (11). 1 H NMR (400 MHz, DMSO-d6) δ 12.40 (s, 1H), 7.82 (d, 1H), 7.76 (s, 1H), 7.54 (d, 1H), 7.36 (m, 2H), 7.14 (m, 1H), 6.92 (d, 1H), 4.52 (m, 2H), 3.84 (m, 1H), 3.66 (m, 2H), 3.50 (m, 2H), 3.40 (m, 2H), 3.45−3.02 (m, 6H), 2.22 (m, 1H), 2.10 (m, 1H); LC−MS purity, 95%, tR = 2.10 min (method B), 501.1 (M + 1); HRMS (ESI) calcd for C24H27ClN5O3S (M + H+) 500.1518, found 500.1518. (R)-Methyl Pyrrolidine-3-carboxylate (24). (R)-1-N-Boc-β-proline (1 g, 4.65 mmol) was dissolved in the mixture of toluene (8 mL) and MeOH (2 mL). The mixture was cooled to 0−5 °C, and TMSCHN2 (2 M in hexanes) (∼2.5 mL) was added slowly until yellow color was sustained. After stirring for additional half hour, the reaction was quenched with drops of acetic acid and concentrated under reduced pressure. The residue was taken into dichloromethane (10 mL), and 4 N HCl in dioxane (4 mL) was added. The resulting G

dx.doi.org/10.1021/jm500847m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

3H), 7.89 (s,1H), 7.83 (d, 1H, J = 9.2 Hz), 7.30 (m, 1H), 7.0 (d, 1H, J = 8.8 Hz), 6.93 (m, 1H), 4.50 (m, 2H), 3.20−3.90 (m, 13H), 2.46(s, 3H), 2.0−2.4(m,2H); LC−MS purity, 97%, tR = 2.07 min (method B), 528.9 (M + 1); HRMS (ESI) calcd for C25H30BrN4O4 (M + H+) 529.1450 and 567.1197, found 529.1450 and 567.1197. (R)-1-(2-(4-(4-Acetylphenyl)piperazin-1-yl)-2-oxoethyl)-N-(3fluoro-4-hydroxyphenyl)pyrrolidine-3-carboxamide Hydrochloride (15). 1H NMR (DMSO-d6, 400 MHz) δ 10.2−10.5 (m, 2H), 7.83 (d, 1H, J = 9.2 Hz), 7.57 (dd, J = 14 Hz, J = 2.4 Hz), 6.89− 7.16 (m, 3H), 4.50(m, 2H), 3.20−3.90 (m, 13H), 2.46 (s, 3H), 2.0− 2.4 (m, 2H); LC−MS purity, 98%, tR = 1.90 min (method B), 468.9 (M + 1); HRMS (ESI) calcd for C25H31ClFN4O4 (M + H+) 505.2018, found 505.2016. (R)-1-(2-(4-(4-Acetylphenyl)piperazin-1-yl)-2-oxoethyl)-N-(4hydroxy-3-methylphenyl)pyrrolidine-3-carboxamide Hydrochloride (16). 1H NMR (DMSO-d6, 400 MHz) δ 10.1−10.4 (m, 2H), 7.83 (d, 2H, J = 9.2 Hz), 7.30 (d, 2H, J = 2.0 Hz), 7.22 (m, 1H), 7.01 (d, 1H, J = 8.8 Hz), 6.71 (m, 1H), 4.47−4.55 (m, 2H), 3.16−3.99 (m, 13H), 2.46 (s,3H), 2.1−2.44(m, 2H), 2.08 (d, 3H, J = 4 Hz); LC− MS purity, 98%, tR = 1.94 min (method B), 464.9 (M + 1); HRMS (ESI) calcd for C26H33N4O4 (M + H+) 465.2496, found 465.2493. Temperature Dependent Fluorescence (TdF) Assay. The TdF assay was mainly conducted in the 96-well based CHROMO-4 real time fluorescence plate reader (Bio-Rad). The Sypro Orange (SigmaAldrich), an environmentally sensitive fluorescence dye, was used to monitor the protein folding−unfolding transition. Protein−ligand binding was gauged by the change (or shift) in the unfolding transition temperature (ΔTm) acquired at protein alone with respect to protein in the presence of ligand of interest. Compound of interest was first prepared in DMSO stock (typical concentration, 10 mM). Sample of 20 μL was then added into the 96-well PCR plate, where it consisted of 3 μM ERK protein and 15, 50, or 100 μM compound (depending on compound’s solubility) in buffer (25 mM HEPES, 200 mM NaCl, pH 7.5, and 1 mM DTT) incorporated with Sypro Orange dye (5× final concentration). Final percentage of DMSO resided in the sample was 2%. The sample plate was heated from 30 to 90 °C with thermal ramping rate of 1 °C/min. The fluorescence signals were acquired with excitation and emission wavelengths centered at 490 and 560 nm, respectively. The instrument thermal stability was ±0.2 °C. The melting temperatures (Tm) for inactive ERK2 protein and phosphorylated ERK2 under aforementioned conditions occurred at 56.3 ± 0.2 and 58.6 ± 0.2 °C respectively. Coupled ERK2 Assay. Activity of compounds against inactive ERK2 was tested in a coupled MEK1/ERK2 IMAP assay as follows: Compounds were diluted to 25× final test concentration in 100% DMSO. Then 14 μL of kinase buffer (10 mM Tris-HCl, pH 7.2, 10 mM MgCl2, 0.01% Tween-20, 1 mM DTT) containing 0.4 ng of unphosphorylated mouse ERK2 protein was added to each well of a black 384-well assay plate. An amount of 1 μL of 25× compound was added to each well and incubated at room temperature for 30 min to allow an opportunity for the compound to bind to the inactive enzyme. DMSO concentration during initial incubation is 6.7%. ERK2 activity was determined to be insensitive to DMSO concentrations up to 20%. ERK2 was then activated and its kinase activity measured by the addition of 10 μL of kinase buffer with the following components (final concentration per reaction): 2 ng of active (phosphorylated) human MEK1 protein and 4 μM (total) ERK2 IMAP substrate peptides (3.9 μM unlabeled IPTTPITTTYFFFK-CONH2 and 100 nM IPTTPITTTYFFFK (5-carboxyfluorescein)-CONH2) and 30 μM ATP. DMSO concentration during ERK activation was 4%. After 1 h, reactions were terminated by addition of 60 μL of IMAP detection beads in binding buffer (Molecular Devices). Binding was allowed to equilibrate for 30 min before reading the plate on an LJL Analyst fluorescence polarization plate reader. Compound inhibition was calculated relative to DMSO and fully inhibited standards. Active compounds were reconfirmed in two independent assays (N = 2). Active ERK2 Assay. Activated ERK2 activity was also determined in the IMAP assay format using the procedure outlined above. An amount of 1 μL of 25× compound was added to 14 μL of kinase buffer containing 0.25 ng of fully phosphorylated active mouse ERK2 protein.

solution was stirred for 1 h and concentrated to provide 0.8 g of crude 24. (R)-Methyl 1-(2-tert-Butoxy-2-oxoethyl)pyrrolidine-3-carboxylate (25). To a reaction mixture containing 24 (750 mg, 4.53 mmol), DIEA (2.4 mL, 13.8 mmol), and Cs2CO3 (1.5 g, 4.6 mmol) in DMF (10 mL) was added tert-butyl bromoacetate (748 μL, 5.06 mmol) slowly, and the reaction mixture was stirred at room temperature for 1 h. The mixture was diluted with EtOAc (70 mL), washed with water (3 × 15 mL), brine, and dried over MgSO4. The solvent was evaporated under reduced pressure, and the residue was purified on silica gel. Eluting with hexane/EtOAc (0−50%) provided the desired product (830 mg, 75%) as colorless oil. 1H NMR (CDCl3, 400 MHz) δ 3.69 (s, 3H), 3.16(m, 2H), 3.11 (m, 2H), 2.93 (m,1H), 2.74 (m,1H), 2.58 (m,1H), 2.11 (m, 2H), 1.48 (s, 9H). (R)-2-(3-(Methoxycarbonyl)pyrrolidin-1-yl)acetic Acid (26). (R)-Methyl 1-(2-tert-butoxy-2-oxoethyl)pyrrolidine-3-carboxylate (830 mg, 3.41 mmol) was dissolved in dichloromethane (5 mL), and TFA (5 mL) was added. The solution was stirred for 2 h and concentrated to a residue which was exchanged with hydrochloric acid by dissolving in acetonitrile (5 mL) and adding 4 N HCl in dioxane (3 mL) to provide the crude title compound as HCl salt (1.49 g) after concentration. (R)-Methyl 1-(2-(4-(4-Acetylphenyl)piperazin-1-yl)-2oxoethyl)pyrrolidine-3-carboxylate (27). To a solution of (R)-2(3-(methoxycarbonyl)pyrrolidin-1-yl)acetic acid (3.2 mmol), 1-(4(piperazin-1-yl)phenyl)ethanone (650 mg, 3.2 mmol), and HATU (1.2 g, 3.2 mmol) in DMF (4 mL) was added DIEA (1.7 mL, 9.8 mmol). The reaction mixture was stirred for 10 min. 1-(4-(Piperazin1-yl)phenyl)ethanone (673 mg, 3.3 mmol) was added, and the reaction mixture was allowed to stir at room temperature for 2 h. Water (30 mL) was added, and the mixture was extracted with EtOAc (3 × 30 mL). The organic extracts were combined and washed with water (3 × 20 mL), brine (20 mL) and dried over MgSO4. Solvent was evaporated under reduced pressure and the residue was purified on silica gel. Elution with MeOH/EtOAc (0−20%) provided the title compound (728 mg, 59%) as colorless oil. 1H NMR (CDCl3, 400 MHz) δ 7.89 (d, 2H), 6.87 (d, 2H), 3.80 (m,3H), 3.66 (s,3H), 3.66 (m,4H), 3.36 (m,6H), 3.06 (m, 2H), 2.90 (m, 2H), 2.71 (m,2H), 2.53 (s, 3H), 2.13 (2H). (R)-1-(2-(4-(4-Acetylphenyl)piperazin-1-yl)-2-oxoethyl)pyrrolidine-3-carboxylic Acid (28). To a solution of (R)-methyl 1(2-(4-(4-acetylphenyl)piperazin-1-yl)-2-oxoethyl)pyrrolidine-3-carboxylate (728 mg, 1.95 mmol) in THF/water (3:1, 4 mL) was added LiOH monohydrate (98 mg, 2.33 mmol), and the resulting mixture was stirred at room temperature for 12 h. The reaction mixture was acidified to pH ≈ 3 by adding slowly 4 N HCl and was lyophilized to provide the crude product which was used next step without further purification. (R)-1-(2-(4-(4-Acetylphenyl)piperazin-1-yl)-2-oxoethyl)-N-(2chloro-4-hydroxyphenyl)pyrrolidine-3-carboxamide Hydrochloride (12). To a solution of (R)-1-(2-(4-(4-acetylphenyl)piperazin-1-yl)-2-oxoethyl)pyrrolidine-3-carboxylic acid (72 mg, 0.2 mmol) in DMF (2 mL) were added HATU (76 mg, 0.2 mmol) and DIEA (104 uL, 0.6 mmol), followed by 4-amino-3-chlorophenol (36 mg, 0.25 mmol). The mixture was allowed to stir at room temperature overnight, and the reaction mixture was directly purified by HPLC to provide the title product. 1H NMR (DMSO-d6, 400 MHz) δ 10.6 (m, 1H), 7.83 (d, 1H), 7.23 (dd, 1H), 6.96−7.02 (m, 4H), 4.53 (d, 2H), 3.25−3.98 (m, 13H), 2.46 (s, 3H), 2.0−2.34 (m, 2H); LC−MS purity, 98%, tR = 2.16 min (method B), 485.0 (M + 1). HRMS (ESI) calcd for C25H30ClN4O4 (M + H+) 485.1956, found 485.1950. (R)-1-(2-(4-(4-Acetylphenyl)piperazin-1-yl)-2-oxoethyl)-N(3,5-dichloro-4-hydroxyphenyl)pyrrolidine-3-carboxamide (13). 1H NMR (DMSO-d6, 400 MHz) δ 10.2−10.9 (m, 2H), 7.83 (d, 2H), 7.72 (d, 2H), 7.00 (d, 2 H), 6.75 (s, 1H), 4.53 (m, 2H), 3.25− 4.00 (m, 13H), 2.46 (s, 3H), 2.0−2.6 (m, 2H); LC−MS purity, 95%, tR = 2.18 min (method B), 519.0 (M + 1); HRMS (ESI) calcd for C25H29Cl2N4O4 (M + H+) 519.1566, found 519.1568. (R)-1-(2-(4-(4-Acetylphenyl)piperazin-1-yl)-2-oxoethyl)-N-(3bromo-4-hydroxyphenyl)pyrrolidine-3-carboxamide Hydrochloride (14). 1H NMR (DMSO-d6, 400 MHz) δ 10.2−10.4 (m, H

dx.doi.org/10.1021/jm500847m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Following a 30 min incubation, the reactions were initiated by addition of 10 μL of kinase buffer containing 1 μM ERK2 IMAP substrate peptide (0.9 μM unlabeled IPTTPITTTYFFFK-CONH2 and 100 nM IPTTPITTTYFFFK (5-carboxyfluorescein)-CONH2) and 30 μM ATP. Reactions proceeded for 30 min before termination by addition of 60 μL of IMAP detection beads in binding buffer. Plates were read as above after a 30 min binding equilibration. Active compounds were repeated in two independent assays (N = 2). Proliferation Assay. Melanoma (A2058 and A375) and colon cancer (Colo-205) cell lines were grown and maintained in RPMI1640 medium containing 100 U/mL penicillin−streptomycin and 10% fetal bovine serum. Cells were seeded on the day of treatment in 40 μL of growth medium in white/opaque bottom 384-well collagen-coated Biocoat plates at 2000 cells/well (A2058, Colo-205) or 1500 cells/well (A375SM) and allowed to attach to the plates for 2 h before treatment. Compounds were diluted in 100% DMSO in a Labcyte Echo qualified plate and delivered using an Echo 555 so that the final concentration of DMSO in the assay was 1.0% or 0.1%. Plates were placed in 37 °C, 5% CO2 for 3 days before they were developed by adding 40 μL of CellTiter-Glo reagent (Promega). Plates were shaken briefly after reagent addition and read on an EnVision 2104 plate reader (PerkinElmer). Data were analyzed in GraphPad Prism.

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ASSOCIATED CONTENT

S Supporting Information *

Crystallization data table and additional kinase selectivity data for 4. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 617-992-3182. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Stuart Black, Kimberly Gray, Tom Hesson, and Catherine Smith for providing ERK and MEK proteins for this study.

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ABBREVIATIONS USED ERK, extracellular regulated kinase; ATP, adenosine 5′triphosphate; Ras, renin−angiotensin system; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; Boc, tert-butoxycarbonyl; HATU, (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate); SAR, structure− activity relationship

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