Discovery of Highly Potent, Selective, and Efficacious Small Molecule

Article pubs.acs.org/jmc

Discovery of Highly Potent, Selective, and Efficacious Small Molecule Inhibitors of ERK1/2 Li Ren,*,† Jonas Grina,† David Moreno,† James F. Blake,† John J. Gaudino,† Rustam Garrey,† Andrew T. Metcalf,† Michael Burkard,† Matthew Martinson,† Kevin Rasor,† Huifen Chen,§ Brian Dean,§ Stephen E. Gould,§ Patricia Pacheco,§ Sheerin Shahidi-Latham,§ Jianping Yin,§ Kristina West,§ Weiru Wang,§ John G. Moffat,§ and Jacob B. Schwarz§ †

Array BioPharma Inc, 3200 Walnut Street, Boulder, Colorado 80301, United States Genentech Inc., 1 DNA Way, South San Francisco, California 94080-4990, United States

§

S Supporting Information *

ABSTRACT: Using structure-based design, a novel series of pyridone ERK1/2 inhibitors was developed. Optimization led to the identification of (S)-14k, a potent, selective, and orally bioavailable agent that inhibited tumor growth in mouse xenograft models. On the basis of its in vivo efficacy and preliminary safety profiles, (S)-14k was selected for further preclinical evaluation.

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INTRODUCTION The Ras/Raf/MEK/ERK (MAPK) signal transduction pathway is widely activated in a large subset of human cancers and thus has attracted significant interest as a therapeutic target for cancer.1 Constitutive activation of the MAPK pathway by overexpression of growth factors, as well as oncogenic Ras and Raf mutations, are frequently observed in tumor types such as colon, lung, pancreatic, ovary, and kidney.2 The downstream consequence of disregulation of this pathway is over and/or constitutive production of phosphorylated ERK1/2 (pERK). Activity of this pathway has been linked to disease progression and clinical outcome in several malignancies.2 Small molecule inhibitors of B-Raf and MEK have shown promising activities in the clinic for a variety of solid tumors. The approval of Zelboraf3 (vemurafenib), Telfinlar4 (Dabrafenib), and Mekinist5 (trametinib) as treatments for metastatic melanoma validate the approach of targeting the MAPK pathway as an effective way of treating cancer. In comparison to B-Raf and MEK inhibitors, the development of small molecule inhibitors of ERK1/2 has lagged behind with only few reports of preclinical development.6 ERK1 and 2, which show 89% sequence identity within the kinase domain, are pivotal in the pathway downstream of Ras, Raf, and MEK, acting as a central node where multiple signaling pathways converge to drive transcription. ERK1/2 are activated by MEK1/2 through phosphorylation of both a threonine and a tyrosine residue, namely Thr202 and Tyr204 of ERK1 and Thr173 and Tyr185 of ERK2. Once activated, ERK1/2 phosphorylates serine/threonine residues of more than 50 downstream substrates and activates both cytosolic and nuclear proteins that are responsible for cell growth, prolifer© XXXX American Chemical Society

ation, survival, angiogenesis, and differentiation, all hallmarks of the cancer phenotype.7 Thus, it may be beneficial to target the ERK proteins directly as a more effective way to block the MAPK signaling pathway. Furthermore, an ERK inhibitor may have utility in combination with other MAPK inhibitors. Recently, researchers at Genentech and Merck independently reported that dual inhibition of MEK and ERK by small molecule inhibitors acted to overcome acquired resistance to MEK inhibitors, providing further rationale for developing selective ERK1/2 inhibitors.8 Herein, we describe the medicinal chemistry efforts leading to the discovery of (S)-14k, a potent, selective, and efficacious inhibitor of ERK1/2.

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STRUCTURE-BASED DESIGN Previously, we disclosed a novel series of ATP competitive ERK1/2 inhibitors, exemplified by 1a and 1b (Figure 1) built upon a piperidinopyrimidine urea template.9 On the basis of excellent potency and good in vitro ADME properties (medium predicted CL in hepatocytes and moderate plasma protein binding),9 the pharmacokinetics of 1a was evaluated in mice, rats, beagle dogs, and cynomolgous monkeys. Unfortunately, compound 1a showed high clearance and low oral bioavailability in multiple species, a profile shared with most analogues from the series.9 To understand the underlying reason for the poor PK across the series, in vitro metabolic profilings were performed on representative analogues. Consistently, major metabolites were Received: December 11, 2014

A

DOI: 10.1021/jm501921k J. Med. Chem. XXXX, XXX, XXX−XXX

Article

Journal of Medicinal Chemistry

Figure 1. Stuctures of 1a and 1b from the piperidinopyrimidine urea series.

Scheme 1. Structure-Based Design: From Piperidinopyrimidine Ureas to Pyridones

First of all, modeling studies (Figure 2) suggested that the pyridone carbonyl mimics the urea oxygen and should make the same hydrogen bond interactions to both Lys54 and the bound water molecule that is complexed to Gln105, Glu71, and Asp167. Second, an overlap of the pyridone and the piperidinopyrimidine urea templates revealed that the left-hand-side pyrimidine and right-hand-side benzyl moieties occupy similar space and should maintain critical contacts with the hinge and the glycine-rich loop. It was reasoned that the pyridone series could have improved metabolic stability due to removal of the two metabolic soft spots present in the piperidinopyrimidine scaffold (C-5 and C-8). Another salient feature of the pyridone design is elimination of the urea linker. Although the urea oxygen is necessary for activity, the urea NH does not make any specific interaction with the protein and removal of the unnecessary hydrogen bond donor could positively impact permeability and absorption.

identified that resulted from oxidation of the benzylic C-5 and C8 positions on the core (Scheme 1). In addition to high clearance, 1a exhibited low permeability (Papp AB (10−6 cm/s) = 1.3) and efflux (efflux ratio BA/AB = 7.8), limiting its oral absorption. These inherent PK liabilities of the piperidinopyrimidine urea template prompted us to search for an alternative core. In designing a new template, it was highly desirable to maintain all the critical interactions between 1a and the ERK2 protein while removing the metabolic liability inherent in the piperidinopyrimidine core. As can be seen in the X-ray cocrystal structure of 1b bound to ERK2 (Figure 2A), the pyrimidine ring accepts a hydrogen bond from the backbone NH of Met108 while the amino group at the 2-position donates a H-bond to the carbonyl oxygen of the same residue. The oxygen of the tetrahydro-2H-pyran is hydrogen-bonded to the side chain of Lys114, although it appears that the bulk of the potency realized from this group is derived via hydrophobic interactions near the hinge region. Additionally, the urea oxygen accepts two hydrogen bonds: one from the side chain of Lys54 and another from a bound water molecule that is complexed to Gln105, Glu71, and Asp167 (Figure 2B). The latter H-bond is believed to be critical to ERK1/2 and a key feature of our strategy to achieve selectivity against other members of the closely related CMGC family of kinases. The (S)-CH2OH group is in the vicinity of Asp167 and Asn154 and can make a hydrogen bond interaction to either the side chain carboxylate of Asp167 or the amide carbonyl of Asn154. It is important to note that the stereochemistry of this chiral center is critical for potency, as observed by us9 and others.6 The 3-Cl-4-F phenyl moiety points toward the glycinerich loop, making largely hydrophobic contacts. The metabolically labile piperidine acts mainly as a spacer and a conformational control and could potentially be replaced. It was hypothesized that a 4-pyridone could serve as effective replacement for the piperidine urea linker, as shown in Scheme 1, based on the following considerations.

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CHEMISTRY

Pyrimidine pyridones 6 were prepared in a four-step procedure as described in Scheme 2.10 Suzuki coupling of commercially available 2,4-dichloropyrimidine and 2-fluoropyridin-4-ylboronic acid produced 3. Replacing the 2-Cl with tetrahydro-2H-pyran-4amine (THP amine) gave aminopyrimidine 4, which was converted to pyridone 5 under acidic conditions. Chemoselective N-alkylation with various benzyl bromides afforded 6a− 6f. Hydroxylated analogues such as 14 required a slightly different synthesis as illustrated in Scheme 3.10 Suzuki coupling of commercially available 4-bromo-2-(methylthio)pyrimidine and 2-fluoropyridin-4-ylboronic acid produced 7, which was converted to the key pyridone intermediate 8 via acidic hydrolysis. Chemoselective N-alkylation with mesylate 10 (prepared via aryl Grignard addition to commercially available TBS protected glycolaldehyde) gave racemic methyl sulfide 11, B


Article


Figure 2. (A) Pyridone 6a (orange) docked into the X-ray structure of 1b (green) with ERK2 (PDB code 4O6E, solved at 1.95 Å resolution). Hydrogenbonding interactions are illustrated with red dashed lines. (B) Same model as in (A), rotated to illustrate key water mediated H-bond interactions.

Scheme 2. Preparation of Compounds 6a−6fa

Reaction conditions: (a) 5% Pd(dppf)Cl2(DCM), Na2CO3, 1,4-dioxane/H2O (4:1), 80 °C, 3 h, 96%; (b) tetrahydro-2H-pyran-4-amine, N-ethyl-Nisopropylpropan-2-amine, 2-butanol, 100 °C, 16 h, 49%; (c) 2 N HCl, 100 °C, 16 h, 94%; (d) benzyl bromide, K2CO3, DMF, 25 °C, 65%. a

pyridone N-alkylation between 18 and 8 would occur via a SN2 mechanism with complete inversion at the newly created chiral center (19) and thus would require a precursor such as 16. The absolute (R)-stereochemistry of the chiral center in diol 16 could be set via the well-known Sharpless dihydroxylation by using AD-

which was oxidized to sulfone 12. Installation of the THP amine followed by TBS deprotection afforded 14a−14o. The synthetic route shown in Scheme 3 to produce racemic compounds was modified to obtain the desired (S)-enantiomers with high ee (Scheme 4).10 It was hypothesized that the key C


Article

Journal of Medicinal Chemistry Scheme 3. Preparation of Compounds 14a−14oa

Reaction conditions: (a) 5% Pd(dppf)Cl2(DCM), Na2CO3, 1,4-dioxane/H2O (4:1), 80 °C, 2 h, 90%; (b) 2 N HCl, 100 °C, 2 h, 50%; (c) THF, 0 °C, 1 h, 93%; (d) MsCl, NEt3, DCM, 0 °C, 1 h, 96%; (e) KHMDS, THF, 65 °C, 30 h, 65%; (f) mCPBA, DCM, 0 °C, 2 h, 89%; (g) tetrahydro-2Hpyran-4-amine, 2-butanol, 120 °C, 4 h, 90%; (h) TBAF, THF, 25 °C, 16 h, 90%.

a

Scheme 4. Preparation of Compounds (S)-14k and 22a−22ia

a Reaction conditions: (a) NaH, methyltriphenylphosphonium bromide, THF, 25 °C, 16 h, 47%; (b) AD-mix-β, t-BuOH/H2O (1:1), 25 °C, 16 h, 64%; (c) TBSCl, imidazole, DCM, 0 °C, 1 h, 58%; (d) MsCl, NEt3, DCM, 0 °C, 1 h, 96%; (e) KHMDS, THF, 65 °C, 30 h, 65%; (f) mCPBA, DCM, 0 °C, 2 h, 89%; (g) R4NH2, 120 °C, 4 h, 90%; (h) TBAF, THF, 25 °C, 16 h, 90%.

mix-β.11 Indeed, treatment of styrene 15 with AD-mix-β afforded 16 with 95% ee (determined from 17 by chiral chromatography). TBS protection of the primary alcohol (17) followed by mesylation of the benzylic hydroxyl group gave 18. With KHMDS as a base, displacement of mesylate by intermediate 8

occurred predominantly on nitrogen to afford 19. Although the enantiomeric excess of 19 was not measured, it was inferred to be >95% because no ee loss was observed during the transformation from 17 (95% ee) to (S)-14k (96% ee, determined by chiral chromatography). The absolute stereochemistry of (S)-14k was D


Article


analogues such as 6d and 6e remained similarly active, extending the pyridone side chain from benzyl to phenylethyl (6f) resulted in substantial lost in cellular potency. Because preliminary SAR on the phenyl ring tracked well with the piperidinopyrimidine ureas,9 we decided to focus on the hydroxymethyl substituent at the benzylic position as we9 and others6 have shown that this group is near optimal in terms of both potency and kinase selectivity (Table 2). Although the cellular IC50 of racemic 14a remained unchanged in comparison to 6a at first glance, it was important to realize that potency should improve by 2-fold if only the active (S)-enantiomer was obtained. To expedite the SAR exploration of other regions of the molecule, we elected to proceed with testing racemic material due to ease of synthesis. Once an optimal racemic compound was identified, the more active (S)-enantiomer was prepared and profiled. Using this strategy, SAR of the terminal phenyl group was further expanded (Table 2). Again, exploring chlorine substitution on the aryl ring quickly revealed that 3-Cl (14d vs 14b and 14c) was favored in both biochemical and cellular assays. Further SAR exploration at this position showed 3-F (14e), 3-Me (14f), and 3-CF3 (14g) were less potent in the cellular assay and polar substituents were not tolerated (14h and 14i), likely due to unfavorable interaction with the largely lipophilic P-loop. On the basis of these results, we prepared dihalo analogues 14j−14o. Most of these compounds retained cellular activity in comparison to 14d, with the exception of 14m, which is 2× less potent in the cellular assay. Because of the combination of good cellular potency and CDK2 selectivity, the active (S)-enantiomer of 14d, 14e, 14k, and 14l were obtained by asymmetric synthesis as described in Scheme 4. As expected, all of the (S)-enantiomers were potent and selective (>70× over CDK2). These compounds showed reasonable in vitro ADME properties, such as medium metabolic stability as predicted by mouse liver microsomes and medium to high permeability as measured in the MDCK cell line, and were advanced to mouse (CD-1 strain) pharmacokinetic studies (Table 3). Among these compounds, the one bearing the 3-F-4Cl substitution pattern ((S)-14k) showed the best overall mouse PK profile with low clearance and high bioavailability and was chosen as the preferred piece on the right-hand-side of the molecule. Having chosen the 3-F-4-Cl-Ph as the preferred right-handside substituent, central pyridone ring SAR was investigated next (Table 4). Again, we elected to proceed with testing racemic material due to ease of synthesis. Only small substitutions, especially at the 3- and 5-position of the pyridone were considered, as conformational analysis suggested that the pyrimidine and the pyridone core would prefer to be coplanar when bound. 3-F substitution (23a) retained potency, albeit with a loss of selectivity over CDK2. Substitutions at other positions were not well tolerated, and 5-F (23b), 5-Me (23c), and 6-Me (23d) were substantially less active. Changing the pyridone template to either the pyrimidinone (23e) or the pyridazinone (23f) resulted in a drop in potency. The decrease in activity of 23e and 23f was likely due to the increased polarity (decreased lipophilicity) of the linker. SAR of the pyrimidine ring was examined next (Table 5). Other hinge binding templates such as pyridine (24a) as well as an isomeric pyrimidine (24b) were both less effective. Substitution at the 5-position of the pyrimidine ring with fluorine (24e) or chlorine (24d) largely retained cellular potency, albeit with a loss of selectivity over CDK2. The loss

confirmed by X-ray crystallography (Figure 3), indicating that the N-alkylation to produce 19 occurred with clean inversion.

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RESULTS AND DISCUSSION Compounds were screened for potency against ERK2 enzymatic activity, Cdk2/Cyclin A binding, and inhibition of ERKdependent p90RSK Serine 380 phosphorylation in HepG2 cells. To enable determination of subnanomolar enzyme potencies, ERK2 kinase activity was assayed in the presence of 2 mM ATP (80× the ATP Km(apparent) of 25 μM) and Ki values derived by the Cheng−Prusoff formula.12 Given that the residues in the ATP site are conserved between ERK1 and ERK2, compounds were not routinely tested against ERK1, however, follow-up testing of several compounds confirmed that the ERK1 activity (Ki or IC50 values) were consistently within 3-fold of ERK2.13 Cyclin-dependent kinase 2 (CDK2) was employed as a representative kinase selectivity assay due to its relatively similar sequence homology with ERK2 in the ATP binding pocket and previous experience that this kinase was a frequent and biologically significant off-target.9 Pyridone 6a, with an unsubstituted benzyl group, showed promising activity with an enzyme Ki = 0.6 nM and cellular IC50 = 120 nM. This encouraging result confirmed the validity of our docking experiments and gave us further confidence in using this model to drive SAR. We first proceeded to introduce small, mostly lipophilic substituents onto the phenyl ring to optimize interaction with the glycine-rich loop (Table 1). In general, halogens at the 3- and/or 4-position afforded a modest increase in potency and selectivity over CDK2. 3-Chlorophenyl (6b) was favored over 4-chlorophenyl (6c), affording a 4-fold improvement in cellular activity over 6a. While 3,4-disubstituted Table 1. SAR of the Right-Hand-Side Phenyl Ring

compd no. 6a 6b 6c 6d 6e 6f

R1 −CH2Ph −CH2-3Cl-Ph −CH2-4Cl-Ph −CH2-3Cl-4-FPh −CH2-3F-4-ClPh −CH2 CH2-3F-4-ClPh

ERK2 enzyme Ki (nM)a,b

CDK2 enzyme Kd (nM)b,c

phosphop90RSK IC50 (nM)b,d

CDK2 Kd/ERK2 Ki

0.6 ± 0.3 0.4 ± 0.1

21 ± 11 20 ± 13

120 ± 26 30 ± 12

35 67

0.8 ± 0.1

49 ± 22

85 ± 6

61

0.4 ± 0.1

37 ± 12

29 ± 6

93

0.3 ± 0.1

21 ± 9

34 ± 10

70

1.5 ± 0.3

103 ± 32

615 ± 9

69

a

Ki values were determined by carrying out ERK2 enzymatic assays in the presence of ATP at 80× Km(app) and applying Cheng−Prusoff correction to convert IC50 to Ki. bAll assay results represent the mean of at least three independent determinations. cKd values were determined by competitive displacement of an ATP-analogue probe. d Cellular assay in PMA-stimulated HepG2 cells. See Supporting Information for details. E


Article

Journal of Medicinal Chemistry Table 2. Expanded SAR of the Right-Hand-Side Phenyl Ring

compd no.

R3



phospho-p90RSK IC50 (nM)b,d

CDK2 Kd/ERK2 Ki

14a 14b 14c 14d 14e 14f 14g 14h 14i 14j 14k 14l 14m 14n 14o

H 2-Cl 4-Cl 3-Cl 3-F 3-Me 3-CF3 3-OMe 3-CN 3,4-diF 3-F-4-Cl 3-Cl-4-F 3,5-diF 3,5-diCl 3-F-5-Cl

0.7 ± 0.1 1.1 ± 0.2 1.5 ± 0.1 0.4 ± 0.1 0.1 ± 0.03 1.3 ± 0.1 1.3 ± 0.1 1.6 ± 0.2 2.3 ± 0.3 0.1 ± 0.01 0.6 ± 0.1 0.5 ± 0.1 0.4 ± 0.1 0.3 ± 0.02 0.5 ± 0.04

36 ± 19 108 ± 15 69 ± 15 43 ± 15 19 ± 4 155 ± 129 177 ± 116 179 ± 124 219 ± 127 16 ± 8 57 ± 29 70 ± 28 22 ± 3 40 ± 16 37 ± 13

148 ± 18 249 ± 43 98 ± 43 39 ± 16 79 ± 23 100 ± 31 82 ± 31 227 ± 62 484 ± 149 58 ± 31 49 ± 15 37 ± 21 83 ± 34 27 ± 9 41 ± 19

51 98 46 108 190 119 136 112 95 160 95 140 55 133 74

a

Ki values were determined by carrying out ERK2 enzymatic assays in the presence of ATP at 80× Km(app) and applying Cheng−Prusoff correction to convert IC50 to Ki. bAll assay results represent the mean of at least three independent determinations. cKd values were determined by competitive displacement of an ATP-analogue probe. dCellular assay in PMA-stimulated HepG2 cells. See Supporting Information for details.

Table 3. In Vitro ADME Properties and Mouse PK Parameters of (S)-14d, (S)-14e, (S)-14k and (S)-14l compd no.




CDK2 Kd/ERK2 Ki

in vitro predicted CL (mL/min/kg)e

permeability Papp ABf

in vivo CL (mL/min/kg) IVg

AUC (μM·h)h

F (%)

(S)-14d (S)-14e (S)-14k (S)-14l

0.2 ± 0.03 0.1 ± 0.03 0.3 ± 0.02 0.2 ± 0.03

16 ± 2 23 ± 19 21 ± 8 31 ± 6

27 ± 7 42 ± 24 17 ± 4 38 ± 8

80 230 70 165

71 59 60 66

8.5 5.5 11.3 4.6

114 39 33 63

0.59 1.72 3.05 2.18

38 36 66 76

a Ki values were determined by carrying out ERK2 enzymatic assays in the presence of ATP at 80× Km(app) and applying Cheng−Prusoff correction to convert IC50 to Ki. bAll assay results represent the mean of at least three independent determinations. cKd values were determined by competitive displacement of an ATP-analogue probe. dCellular assay in PMA-stimulated HepG2 cells. See Supporting Information for details. eMouse hepatic CL predicted from liver microsomes. fPermeability measurement determined in MDCK cell line, in units of 10−6 cm/s. gIV clearance measured at 1 mg/kg for CD-1 mouse, dosed as a solution in 40% PEG400/60% H2O. hCD-1 mouse PO PK at 5 mg/kg, dosed as a solution in 40% PEG400/60% of 10% HP-b-CD in H2O.

Table 4. SAR of the Central Pyridone Ring

compd no.

X

Y

Z




CDK2 Kd/ERK2 Ki

14k 23a 23b 23c 23d 23e 23f

C−H C−F C−H C−H C−H N C−H

C−H C−H C−F C-Me C−H C−H C−H

C−H C−H C−H C−H C-Me C−H N

0.6 ± 0.1 0.3 ± 0.03 41 ± 7 3.3 ± 0.8 6.8 ± 2.2 2.4 ± 0.2 1.7 ± 0.4

57 ± 29 10 ± 4 723 ± 179 643 ± 229 758 ± 262 202 ± 38 92 ± 42

49 ± 15 82 ± 35 >5000 687 ± 122 1619 ± 213 697 ± 156 230 ± 58

95 33 18 195 111 84 54

a Ki values were determined by carrying out ERK2 enzymatic assays in the presence of ATP at 80× Km(app) and applying Cheng−Prusoff correction to convert IC50 to Ki. bAll assay results represent the mean of at least three independent determinations. cKd values were determined by competitive displacement of an ATP-analogue probe. dCellular assay in PMA-stimulated HepG2 cells. See Supporting Information for details.

F


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Journal of Medicinal Chemistry Table 5. SAR of the Pyrimidine Ring

compd no.

A

B




CDK2 Kd/ERK2 Ki

14k 24a 24b 24c 24d 24e

N C−H C−H N N N

C−H C−H N C-Me C−Cl C−F

0.6 ± 0.1 1.8 ± 0.3 14 ± 1.0 2.2 ± 0.2 0.2 ± 0.02 0.1 ± 0.01

57 ± 29 567 ± 274 763 ± 209 136 ± 63 4.2 ± 1.4 1.8 ± 0.8

49 ± 15 385 ± 119 4428 ± 990 801 ± 116 116 ± 60 30 ± 15

95 315 55 62 21 18


3B) forms a H-bond to the pyridone carbonyl (2.85 Å), which also interacts with the side chain of Lys54 (3.01 Å). The methyl hydroxyl substituent interacts with the carboxylate side chain of Asp167 (2.70 Å). The 3-F-4-Cl phenyl group occupies a small hydrophobic pocket under the P-loop that is formed when Tyr36 is displaced toward the C-helix. In Vitro ADME and Pharmacokinetics. The in vitro ADME properties of (S)-14k were determined (Table 8). Liver hepatocytes predicted low to medium stability across species, similar to what was observed with our pervious lead 1a. Permeability measurement in the MDCK cell line showed high permeability without efflux, a significant improvement over 1a, suggesting better absorption potential. Other properties such as solubility (45 μg/mL @ pH 7.4) and human plasma protein binding (95.8%) were deemed sufficient although less desirable than 1a. Compound (S)-14k displayed no inhibition of cytochrome P450 enzymes (IC50 > 10 μM for CYP3A4, CYP1A2, CYP2C9, CYP2C19, CYP2D6) and was negative in the time-dependent CYP inhibition (TDI) assay for major CYP isoforms (CYP3A4, CYP1A2, CYP2C9, CYP2C19, and CYP2D6). In addition, minimal hERG channel inhibition was observed with an IC50 = 14.5 μM. On the basis of the combination of good potency, selectivity, and in vitro ADME profile, the PK of (S)-14k was evaluated in nude mice (Table 9). The clearance was modest (CL = 40 mL/ min/kg) at 1 mg/kg IV, and oral bioavailability was excellent at 5 mg/kg (F = 139%). More extensive PK evaluations were performed in rats, dogs, and cynomolgous monkeys. The results of these experiments were summarized in Table 9. The overall PK profiles of (S)-14k looked quite promising across species. For example, the IV clearances were modest in all three species, an improvement over the previous lead 1a (near hepatic blood flow CL in rat, dog, and cyno).9 These observations confirmed our hypothesis that by removing metabolic soft spots in the previous template, the pyridones should have improved metabolic stability over the piperidinopyrimidine ureas. Furthermore, oral bioavailability of (S)-14k in rat (F = 44%) and dog (F = 46%) at 5 mg/kg were superior to 1a (11% F in rat and 26% in dog),9 partly due to improved absorption. In vitro ADME profiling revealed that while 1a was an efflux substrate (efflux ratio BA/AB = 7.8) with low intrinsic permeability (Papp AB (10−6 cm/s) = 1.3), (S)-14k was a highly permeable compound without efflux. This was attributed to replacing the urea linker with a pyridone template, eliminating an

of activity with a larger methyl group (24c) was attributed to an unfavorable change in the dihedral angle between the pyrimidine and pyridone rings, resulting in increased strain energy upon binding. Final areas of SAR investigation focused on exploring other amino groups at the 2-position of the pyrimidine. This area is solvent exposed and can potentially be used to design inhibitors with improved physiochemical properties (Table 6). To maintain potency, substituents capable of interacting with the side chain of Lys114 (as the oxygen of the THP moiety) were evaluated. In this exercise, we proceeded with enantiomerically enriched material because large quantities of intermediate 20 had become available at this stage of the project. A variety of structural changes were explored, such as varying the ring size (22a, 22b, 22c), ring substitution (22e), THP oxygen atom replacement (22d, 22f) as well as ring opening (22g, 22h, 22i), without improving upon the potency of (S)-14k. Although the activity of oxetane 22c looked promising initially, it was chemically unstable toward acid (pH = 1) and was not advanced. To confirm our earlier hypothesis that the (S)-enantiomer was critical for potency, the opposite epitope (R)-14k was obtained (by chiral separation) and profiled (Table 7). As anticipated, (R)14k was 25-fold less active in the cellular assay in comparison to (S)-14k. Importantly, the more active enantiomer also maintained CDK2 selectivity. When compared to our previous lead 1a, (S)-14k has equivalent cellular potency with improved CDK2 selectivity. With an optimal combination of activity, selectivity, and mouse pharmacokinetics, (S)-14k emerged as a promising lead. The kinase selectivity profile of compound (S)14k was determined by screening against a panel of 170 kinases. No kinase showed >70% inhibition other than ERK1 and ERK2 at a test concentration of 100 nM (Supporting Information). Furthermore, (S)-14k showed no significant inhibition when screened against a panel of 40 receptors at a concentration of 10 μM except for mild adenosine (64% inhibition) A1 agonism (Supporting Information). X-ray Crystal Structure. An X-ray structure of (S)-14k bound to ERK2 at 2.58 Å resolution was obtained (Figure 3). The crystal structure confirms earlier modeling conclusions that the 2-aminopyrimidine interacts via a pair of H-bonds with the hinge region of ERK2 at Met108. The 4-THP interacts with Lys114 via the ether oxygen, although it appears that the bulk of the potency is derived from hydrophobic interactions in this region. As we hypothesized above, a key water molecule (Figure G


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Journal of Medicinal Chemistry Table 6. SAR of the Amino-pyrimidine


evaluated in a proof-of-concept tumor growth inhibition (TGI) study. (S)-14k was administered twice daily (BID) orally in nude mice bearing HCT116 colorectal cancer xenografts for 21 days (Figure 6). At the 75 mg/kg dose, significant tumor growth inhibition (70%) was obtained and the treatment was well tolerated. Body weight losses were within an acceptable range (

Discovery of Highly Potent, Selective, and Efficacious Small Molecule

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