Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and

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Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor Jorge Garcia Fortanet, Christine Hiu-Tung Chen, Ying-Nan P Chen, Zhouliang Chen, Zhan Deng, Brant Firestone, Peter Fekkes, Michelle Fodor, Pascal D Fortin, Cary Fridrich, Denise Grunenfelder, Samuel Ho, Zhao B. Kang, Rajesh Karki, Mitsunori Kato, Nick Keen, Laura R. LaBonte, Jay Larrow, Francois Lenoir, Gang Liu, Shumei Liu, Franco Lombardo, Dyuti Majumdar, Matthew J Meyer, Mark Palermo, Lawrence B. Perez, Minying Pu, Timothy Ramsey, William R. Sellers, Michael David Shultz, Travis Stams, Christopher S. Towler, Ping Wang, Sarah L. Williams, Ji-Hu Zhang, and Matthew J. LaMarche J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00680 • Publication Date (Web): 27 Jun 2016 Downloaded from http://pubs.acs.org on July 1, 2016

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Journal of Medicinal Chemistry

Shultz, Michael; Novartis Institutes for Biomedical Research, Inc., Global Discovery Chemistry - Oncology Stams, Travis; Novartis Institutes for BioMedical Research, Inc., CPC Towler, Christopher; Novartis Institutes for BioMedical Research, Inc., CPP Wang, Ping; Novartis Institutes for BioMedical Research, Inc., Oncology Williams, Sarah; Novartis Institutes for BioMedical Research Inc, Zhang, Ji-Hu; Novartis Institutes for BioMedical Research, Inc., LDI LaMarche, Matthew; Novartis Institutes for Biomedical Resea, Oncology Medicinal Chemistry

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Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor Jorge Garcia Fortanet, Christine Hiu-Tung Chen, Ying-Nan P. Chen, Zhouliang Chen, Zhan Deng, Brant Firestone, Peter Fekkes, Michelle Fodor, Pascal D. Fortin, Cary Fridrich, Denise Grunenfelder, Samuel Ho, Zhao B. Kang, Rajesh Karki, Mitsunori Kato, Nick Keen, Laura R. LaBonte, Jay Larrow,

Francois Lenoir, Gang Liu, Shumei Liu, Franco Lombardo, Dyuti

Majumdar, Matthew J. Meyer, Mark Palermo, Lawrence Perez, Minying Pu, Timothy Ramsey, William R. Sellers, Michael D. Shultz, Travis Stams, Christopher Towler, Ping Wang, Sarah L. Williams, Ji-Hu Zhang, and Matthew J. LaMarche* ABSTRACT SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway.

SHP2 also

purportedly plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our labs, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the auto-inhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed


1

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to optimize for SHP2 inhibition and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor. Cl Cl

NH2 N

N

N NH2

1 SHP2 IC50 = 0.070 µM p-ERK IC50 = 0.250 µM

INTRODUCTION SHP2 phosphatase, encoded by the PTPN11 gene, is a nonreceptor PTP containing two Nterminal Src homology 2 (SH2) domains, a PTP domain, and a C-terminal tail. X-ray structures have demonstrated that SHP2 adopts an autoinhibited conformation in its basal state, whereby the N-terminal SH2 domain interacts with the PTP domain and blocks access to the catalytic site.1 Others have previously demonstrated that bis-phosphotyrosyl peptides (e.g., IRS-1) or proteins bind to the SH2 domains of SHP2 and activate the phosphatase,2 which imparts cancer dependence.3

In cells, SHP2 functions in the cytoplasm downstream of multiple receptor-

tyrosine kinases and is involved in numerous oncogenic cell signaling cascades (e.g., RAS-ERK, PI3K-AKT, JAK-STAT); however, its role in those pathways is not yet fully understood.4 More recently, SHP2 was reported to bind and dephosphorylate RAS and increase its association with effector protein RAF to activate downstream proliferative RAS/ERK/MAPK signaling.5 Furthermore, germline or somatic mutations in PTPN11 that cause hyperactivation of SHP2 have been identified in Noonan syndrome (50%),6 juvenile myelomonocytic leukemia (JMML, 35%),


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myelodysplastic syndrome (10%), B-cell acute lymphoblastic leukemia (7%), acute myeloid leukemia (AML, 4%),7 as well as in solid tumors including lung adenocarcinoma, colon cancer, neuroblastoma, melanoma, and hepatocellular carcinoma.8 SHP2 may also participate in the programed cell death pathway (PD-1/PD-L1) and contribute to immune evasion.9 The PD-1/SHP2/STAT1/T-bet signaling axis mediates the suppressive effects of PD-1 on Th1 immunity at tumor sites. Therefore, inhibition of PD-1 or SHP2 should be sufficient to restore robust Th1 immunity and T-cell activation, reversing immunosuppression in the tumor microenvironment.

Given recent clinical success of anti-PD-1 and PD-L1

therapeutics,10 investigating the inhibition of SHP2 for cancer immunotherapy is also of great interest. In view of the importance of SHP2 as a potential anticancer target, the discovery of SHP2 small molecule inhibitors has attracted wide interest in the scientific community.11 High protein sequence homology within the catalytic site among PTPs creates a distinct challenge in achieving selectivity among phosphatases.

In addition, the positive-charged environment of the PTP

catalytic pocket presents unique drug discovery challenges, as most catalytic site inhibitors require multiple ionizable functional groups in order to inhibit the enzyme. These functional groups, in turn, complicate drug discovery and development due to low cell permeability and bioavailability.12 Indeed many small molecule SHP2 inhibitors have been described in the literature (Figure 1), however these catalytic site inhibitors often lack robust selectivity among other PTPs (e.g., SHP1, PTP1B) and also suffer from poor cell permeability and oral bioavailability due to the presence of polar and ionic functional groups.13

Despite these

challenges, researchers have recently achieved moderately potent inhibitors (up to 0.200 µM) targeting the active site which have proven effective in intracranial xenograft models of glioma.14 Others have turned to allosteric modes of phosphatase inhibition and detection, but these efforts


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have yet to progress viable chemical matter to the clinic.15 Highly selective and potent SHP2 inhibitors with appropriate pharmacokinetic and pharmacological properties are needed as tools for further mechanistic studies and ultimately, for the development of agents to treat SHP2dependent malignancies. In this article we describe the identification and initial optimization of a new pyrazinyl class of SHP2 allosteric inhibitors, which targets the auto-inhibited conformation of SHP2.16 Crystal-guided optimization led to the discovery of 1,17 a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor. N

O ONa O S O H H N

N

N NH N

N H O N N NAT6-297775 IC 50 (SHP2): 2.5 M

N

HO2C

N

O N H IC 50 (SHP2): 0.8 M

O S O ONa

OH NSC-87877 IC 50 (SHP2): 0.3 M

O

I

N HN

Ph

N H

O

HO N N

HO O

N

N

O

H N O

HO

II-B08 IC 50 (SHP2): 5.5 M

CO2H

S

HO IC50 (SHP2): 0.2 M

Figure 1. Representative SHP2 inhibitors reported in the literature possessing ionizable functional groups RESULTS Our efforts towards the discovery of allosteric SHP2 inhibitors began with the identification of a phosphatase assay which exploited the auto-inhibitory mechanism of SHP2.16 We reasoned that screening compounds at 50% enzyme activation would allow for the protein to equilibrate and sample the continuum of conformations between the auto-inhibited (closed) and active (open) conformations (Figure 2A). Control of this equilibrium was achieved by titrating an activating bisphosphotyrosyl peptide in a well-precedented, fluorescence-based phosphatase assay measuring dephosphorylation of 6,8-difluoro-4-methylumbelliferyl phosphate (0.5 µM 2P-IRS-1,


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DIFMUP assay,18 Figure 2B). A focused library of the Novartis compound archive (100,000 compounds) was next evaluated in a high throughput screen.16 Figure 2. A. Equilibrium of SHP2 in closed (blocked active site) and open (free active site) states via a diphosphotyrosyl peptide.19 B. DIFMUP assay measuring phosphatase activity A.

B.

Compounds which inhibited phosphatase activity using a near full length construct (residues 1525) were also evaluated using a truncated construct of SHP2 (residues 237-529, PTP domain only). We utilized PTP domain activity in order to identify, and deprioritize, undesired catalytic


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site inhibitors. This process led to the identification of known ion channel inhibitor 2,20 which had an IC50 = 12 µM against the full length enzyme and an IC50 > 100 µM against the PTP domain (Figure 3A).

Compound 2 binding and thermal stabilization of the protein was

confirmed in biophysics experiments by differential scanning fluorometry (∆Tm = 3.02 °C) and furthermore, by X-ray crystallography (Figure 3B, PDB code 5EHP). Interestingly, 2 did not bind to the PTP catalytic site but to a tunnel-like region formed between the C-terminal SH2, Nterminal SH2, and PTP domains (Figure 3B). By binding to this allosteric site comprised of all three domains, 2 stabilizes the inactive conformation for SHP2 where the catalytic site is blocked and no longer accessible to substrate. This small molecule bound structure is reminiscent of the apo inactive structure (PDB code 2SHP),1 whereby the N-terminal SH2 domain binds the phosphatase domain and blocks the active site. Figure 3. A. pyrimidine HTS hit, 2. B. X-ray of 2 with SHP2, 1.85 Å, in the closed, inactive conformation; inset: allosteric binding pocket at the junction of three domains. C. Binding mode of 2, showing protein interactions (e.g., Glu250)


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A

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B

2 (SHP836) IC50 = 12 µM DSF ∆Tm = 3.0 °C C

The X-ray co-crystal for 2 with SHP2 (Figure 3 B, C) revealed several protein-ligand interactions. Aniline H-bonding occurs with backbone carbonyl of Glu250; the methyl groups make Van der Waal contacts with His114 and Glu249; the dichlorophenyl resides in a hydrophobic cleft (Leu254, Gln257, Pro491), and various interactions exist with waters (e.g., pyrimidine nitrogen, piperazine). Interestingly, Arg111 occupies two conformations in the unit cell: one resembling the apo structure and the other in a weak cationic-Pi stacking interaction with the dichlorophenyl (circled in orange, Figure 3C), perhaps indicating a suboptimal interaction (3.75 Å distance from R111 to centroid of the ring) and opportunity for optimization. With this information in-hand, we decided to initially maintain the Glu250 and water interactions


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of the central pyrimidine ring and independently explore the SAR of the dichlorophenyl and the piperazine regions. In our approach towards building SAR around the aminopyrimidine, we took advantage of the orthogonal reactivity of the two halogens in 5-bromo-2-chloropyrimidin-4-amine.

Thus,

introducing the hydrophobic phenyl regioselectively via Pd-catalyzed Suzuki-Miyaura was followed by introduction of the piperidine via SNAr in high overall yields (table 1, ca. 20-60%, 3 steps). The synthesis was flexible, as the synthetic steps were alternatively also carried out in reverse order. Pyrimidines and triazines (Tables 1, 2) were prepared in either order with similar, high yields. In the synthesis of the pyrazines however (Table 2, Scheme 1), Suzuki coupling followed by amine SnAr was preferred. Table 1. SAR of the amine and phenyl regions.


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Attempts to remove or reposition the chlorines in the phenyl ring resulted in a significant loss in phosphatase inhibition (e.g., 3, 4, 5). The difference in activity between 2 and 3 led us to conclude that the ortho-chlorine was required for activity. The activity difference between 2 and 5 was also striking, as Cl and Me functional groups are approximate isosteres.21 Upon further Xray analysis, however, the chlorines effectively fill a hydrophobic pocket formed by residues Arg111, T253, L254, Q257, and Q495. Our attention next turned to the piperazine motif which occupies a polar region of the allosteric binding pocket (e.g., Phe113, His114, Glu249, Glu250, Thr218, etc.) and is also solvent (water) exposed. We hypothesized that extending the terminal amine towards these polar residues would allow for new interactions and an increase in phosphatase inhibition. Morphing the piperazine ring to a 4-aminopiperidine motif increased the biochemical activity 10-fold (e.g., 6: IC50 = 1.3 µM).

Increasing the nitrogen substitution

reduced inhibition (e.g., 7: IC50 = 6.5 µM), however activity was further improved by stabilizing the pseudo-equatorial amine conformation by adding a geminal methyl in the piperidine ring (e.g. 8, IC50 = 0.26 µM). Importantly, compound 8 showed modulation of phospho-ERK (p-ERK) activity in the esophageal squamous cell carcinoma KYSE-520 (IC50 =1.98 µM). With preferred aromatic and amine fragments in-hand, we then turned our attention to the central pyrimidine ring. At the outset we preserved the aniline interaction with Glu250 and found that the 1,2,4-triazine retained biochemical inhibition (9: IC50 = 0.30 µM). Recognizing that the N-1 nitrogen (see numbering on 9) was tolerated vis à vis the triazine, and maintained a trajectory towards Arg111, we removed the adjacent nitrogen (N-2).

We hypothesized that this

modification would increase the basicity of the nitrogen and strengthen the interaction with Arg111. As expected, the measured pKa’s of the protonated triazine and pyrazine rings were 4.7 and 2.9, respectively. As a result, the pyrazine ring significantly increased both biochemical


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inhibition (1: IC50 = 0.07 µM) and p-ERK modulation in cells (IC50= 0.250 µM). Furthermore, upon extended incubation (5 days), 1 showed inhibition of cell proliferation (KYSE-520 model) with an IC50 of 1.4 µM. Removing the amine group (10) or increasing the steric hindrance around the amine (11) lessened inhibition, presumably due to disturbance of the Glu250 interaction (vide infra).

Table 2. SAR of the central ring.

Cl

het

Cl

N NH2

Compound

Core

SHP2 IC50 ( M) (p-ERK ( M))

NH2 9

N4 1N

2N

0.30 (0.62)

NH2 1

N N

0.07 (0.25)

N 10

5.7

N Me

NH N

11

22

N

The X-ray co-crystal for 1 with SHP2 (Figure 4, PDB code 5EHR) revealed a new interaction with the basic amine and the Phe113 backbone carbonyl.


In addition, the pyrazine core

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maintained the aniline-Glu250 H-bond, and a new H-bond to Arg111 was observed. This new H-bond interaction pre-organized Arg111 for cationic-Pi stacking with the dichlorophenyl motif. As previously observed, Arg111 occupied two conformations in the unit cell. The Pi stacking interaction with 1 appeared stronger than 2, as measured by the distance of Arg111 to the centroid of the ring (3.45 Å versus 3.75 Å, respectively).

We speculate that the H-bond

preorganization of Arg111 via the pyrazine accounts for this more optimal interaction. A

B

Figure 4. X-ray of 1 and SHP2, 1.70 Å. A. Arg111 cationic Pi stacking interaction. B. F113 interaction with 4-aminopiperidine motif of 1. The synthesis of 1 (scheme 1), started with the Suzuki-Miyaura coupling between (2,3dichlorophenyl)boronic acid (12) and 3-bromo-6-chloropyrazin-2-amino (13) affording intermediate 14 in good yield and regioselectivity. SNAr reaction on this biphenyl intermediate 14 with tert-butyl (4-methylpiperidin-4-yl)carbamate (15) provided the Boc-protected intermediate 16. Final deprotection using aq HCl in a mixture of THF/H2O gave high yields of 1 as free base after acid/base extraction (see supporting information). Scheme 1. Synthesis of 1.a


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a

Reagents and conditions: (a) PdCl2(dppf), K3PO4, MeCN:H2O, 120 °C, 4 h; 74% (b) K3PO4, NMP, 140 °C, 36 h,

55% (c) HCl, THF:H2O, 60 °C, 2 h, 95%.

Due to the interesting biochemical and cellular activity of 1, extensive phosphatase selectivity profiling was performed over a panel of 21 human phosphatases (see supporting information). And due to recent reports of aminopyrazines as kinase inhibitors22 we also evaluated 1 against a panel of 66 serine-threonine and tyrosine kinases. In both phosphatase and kinase panels, no biochemical inhibitory activity was evident, suggesting that the aminopyrazine series was quite selective for SHP2. Moreover, 1 showed high solubility (> 0.5 mM in pH 6.8 buffer) and high permeability with no apparent efflux in Caco-2 cells (Papp A-B: 13.2 x 10-6 cm/sec).23 Pharmacokinetic studies in mice for 1 showed acceptable oral exposure (5 mg/kg PO: 565 µM/h) and bioavailability (46 % F). We next evaluated 1 in the EGFR amplified human esophageal squamous cell carcinoma KYSE520 xenograft model subcutaneously implanted into immunocompromised mice (Figure 5A,B). After a single doses of 30 and 100 mg/kg (red and blue lines, respectively), dose-dependent exposure and modulation of the pharmacodynamic marker p-ERK was observed in the xenografts (Figure 5A). The anti-tumor activity of 1 was then evaluated in a multi-dose efficacy


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study with mice bearing KYSE-520 xenografts (Figure 5b). A daily oral dose of 10 or 30 mg/kg yielded 19% and 61% tumor growth inhibition, respectively, relative to vehicle control treated mice. Tumor stasis was achieved at 100 mg/kg, which compared equally with Erlotinib administered 80 mg/kg daily (see supporting information). A

B

Figure 5. A. Dose-dependent exposure and p-ERK modulation of 1 in KYSE-520 tumor bearing nude mouse. B. Dose-dependent efficacy of 1 in KYSE-520 tumor bearing nude mouse. DISCUSSION AND CONCLUSIONS Small molecule modulation of SHP2 is of considerable therapeutic interest given its importance in known oncogenic pathways and emerging role in immuno-oncology. A high throughput screen was designed and conducted on full length SHP2 in order to identify allosteric inhibitors. Identification and deprioritization of undesired orthosteric inhibitors was possible via evaluation of hits against the PTP domain of SHP2. This methodology allowed for the identification of aminopyrimidine 2. X-ray analysis of 2 with SHP2 revealed a new allosteric binding mode with ligand interactions to all three domains of SHP2. This new, allosteric mode of binding stabilizes SHP2 in the auto-inhibited and inactive conformation. The aminopyrimidine template was optimized and morphed via structure based drug design, and several new protein-ligand


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interactions were identified. These studies culminated in the identification of pyrazine 1: a potent, selective, highly soluble, orally bioavailable, and efficacious SHP2 inhibitor exhibiting dose-dependent pathway inhibition and antitumor activity in xenograft models. The studies described herein illustrate that appropriate pharmacokinetic and pharmaceutical properties can be achieved by inhibitors of the allosteric site of SHP2. The methodology described to identify allosteric inhibitors also avoids the drug discovery challenges related to the polarity and homology of the orthosteric binding pocket of SHP2. Significantly, this new molecular tool 1 should enable further interrogation of the multifaceted roles of SHP2 in general signal transduction and related molecular pathologies. EXPERIMENTAL SECTION Compound synthesis and characterization.

Compound purity was assessed by HPLC to

confirm >95% purity. All solvents employed were commercially available anhydrous grade, and reagents were used as received unless otherwise noted. A Biotage Initiator™ Sixty system was used for microwave heating.

Flash column chromatography was performed on either an

Analogix Intelliflash 280 using Si 50 columns (32-63 µm, 230-400 mesh, 60Å) or on a Biotage SP1 system (32-63 µm particle size, KP-Sil, 60 Å pore size). Preparative high pressure liquid chromatography (HPLC) was performed using a Waters 2525 pump with 2487 dual wavelength detector and 2767 sample manager. Columns were Waters C18 OBD 5µm, either 50x100 mm Xbridge or 30x100 mm Sunfire. NMR spectra were recorded on a Bruker AV400 (Avance 400 MHz) or AV600 (Avance 600 MHz) instruments. Analytical LC-MS was conducted using an Agilent 1100 series with UV detection at 214 nm and 254 nm, and an electrospray mode (ESI) coupled with a Waters ZQ single quad mass detector. One of two methods was used: Method A) 5-95% acetonitrile/H2O with 5 mM ammonium formate with a 2 min run, 3 µL injection through


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an inertisil C8 3 cm x 5 mm x 3µm; Method B) 20-95% acetonitrile/H2O with 10 mM ammonium formate with a 2 min run, 3 µL injection through an inertisil C8 3 cm x 5 mm x 3µm. Purity of all tested compounds was determined by LC/ESI-MS Data recorded using an Agilent 6220 mass spectrometer with electrospray ionization source and Agilent 1200 liquid chromatography. The mass accuracy of the system has been found to be < 5 ppm. HPLC separation was performed at 75 mL/min flow rate with the indicated gradient within 3.5 min with an initial hold of 10 seconds. 10 mM ammonia hydroxide or 0.1 M TFA was used as the modifier additive in the aqueous phase. General method A for compounds 3-5 and 8. 2-(4-amino-4-methylpiperidin-1-yl)-5-(2,3dichlorophenyl)pyrimidin-4-amine

(8).

A

solution

of

tert-butyl-4-methylpiperidine-4-

ylcarbamate (3.21 g, 14.99 mmol), 5-bromo-2-chloropyrimidin-4-amine (2.5 g, 11.99 mmol), and N-methylmorpholine (1.58 mL, 14.39 mmol) in NMP (15 mL) was stirred in a microwave reactor at 130 °C for 2 h. After cooling to RT, the resulting mixture was poured into H2O (600 mL), the suspension was stirred at RT for 5 min and then filtered to afford tert-butyl (1-(4-amino-5bromopyrimidin-2-yl)-4-methylpiperidin-4-yl)carbamate (7.5 g, 9.71 mmol). A

suspension

of

tert-butyl

(1-(4-amino-5-bromopyrimidin-2-yl)-4-methylpiperidin-4-

yl)carbamate (966 mg, 2.50 mmol), (2,3-dichlophenyl)boronic acid (596 mg, 3.13 mmol), potassium phosphate (1.59 g, 7.50 mmol), and PdCl2(dppf)·DCM adduct (204 mg, 0.25 mmol) in MeCN:H2O (9:1, 10 mL, degassed) was stirred in a microwave reactor for 3 h at 110 °C. After cooling to RT, the reaction was filtered through a pad of Celite followed by EtOAc (50 mL) wash. The combined filtrates were concentrated and the resulting residue was purified by silica chromatography (0 to 5% gradient of MeOH/DCM) to give tert-butyl (1-(4-amino-5-(2,3dichlorophenyl)pyrimidin-2-yl)-4-methylpiperidin-4-yl)carbamate (1.07 g, 2.37 mmol). MS m/z 452.4 (M+H)+. To a solution of tert-butyl (1-(4-amino-5-(2,3-dichlorophenyl)pyrimidin-2-yl)-4-


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methylpiperidin-4-yl)carbamate (2.14 g, 4.73 mmol) in DCM (50 mL), was added TFA (31.7 mL). After stirring at 0 oC until no starting material remained (30 min, monitored by LC/MS), the volatiles were removed under reduced pressure and the resulting residue was purified by HPLC (gradient elution 10-30% acetonitrile in water, 0.1% TFA modifier) to give 2-(4-amino-4methylpiperidin-1-yl)-5-(2,3-dichlorophenyl)pyrimidin-4-amine (8, 657 mg, 1.69 mmol; HCl salt). 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.49-7.63 (m, 2 H) 7.34 (t, J=7.71 Hz, 1 H) 7.26 (d, J=7.33 Hz, 1 H) 4.18 (d, J=12.13 Hz, 2 H) 3.38-3.61 (m, 2 H) 1.88 (br. s., 4 H) 1.45 (s, 3 H). HRMS calcd for C16H20Cl2N5 (M+H)+ 352.1096, found 352.1086. 5-(3-chlorophenyl)-2-(cis-3,5-dimethylpiperazin-1-yl)pyrimidin-4-amine (3). Prepared following the general method A (no Boc-deprotection necessary). 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.71 (s, 1 H) 7.36-7.47 (m, 2 H) 7.26-7.36 (m, 2 H) 4.59 (dd, J=13.05, 2.01 Hz, 2 H) 2.81 (td, J=6.78, 3.26 Hz, 2 H) 2.43 (dd, J=13.05, 11.04 Hz, 2 H) 1.14 (d, J=6.53 Hz, 6 H). HRMS calcd for C16H21ClN5 (M+H)+ 318.1485, found 318.1283. 5-(3,4-dichlorophenyl)-2-(cis-3,5-dimethylpiperazin-1-yl)pyrimidin-4-amine

(4).

Prepared

following the general method A (no Boc-deprotection necessary). 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.72 (s, 1 H) 7.46-7.61 (m, 2 H) 7.30 (dd, J=8.28, 2.01 Hz, 1 H) 4.59 (dd, J=13.05, 2.26 Hz, 2 H) 2.80 (ddd, J=10.29, 6.65, 3.14 Hz, 2 H) 2.42 (dd, J=13.05, 10.79 Hz, 2 H) 1.13 (d, J=6.27 Hz, 6 H). HRMS calcd for C16H20Cl2N5 (M+H)+ 352.1097, found 352.1096. 5-(3,4-dimethylphenyl)-2-(cis-3,5-dimethylpiperazin-1-yl)pyrimidin-4-amine

(5).

Prepared

following the general method A (no Boc-deprotection necessary). 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.60 (s, 1 H) 7.08-7.24 (m, 2 H) 6.96 (d, J=7.28 Hz, 1 H) 4.79 (d, J=14.05 Hz, 2 H) 3.18 (br. s., 2 H) 2.60-2.79 (m, 2 H) 2.33 (s, 3 H) 2.08 (s, 3 H) 1.30 (d, J=6.27 Hz, 6 H). HRMS calcd for C16H21ClN5 (M+H)+ 312.2188, found 312.2189.


16


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General

method

B

for

compounds

6-7.

Page 18 of 35

2-(4-aminopiperidin-1-yl)-5-(2,3-

dichlorophenyl)pyrimidin-4-amine (6). A mixture of Xphos 2nd generation precatalyst (72.3mg, 0.098 mmol), 2,3-dichlorophenylboronic acid (411 mg, 2.153 mmol), potasium phosphate (0.5 M in H2O, 11.75 mL, 5.87 mmol), and 2-chloro-5-iodopyrimidin-4-amine (500 mg, 1.957 mmol) in THF (5 mL) was stirred for 16 h at 40 °C. After cooling to RT, the organic phase was separated and the aqueous phase was washed with EtOAc (3 x 5 mL), the combined organic phases were dried over MgSO4, filtered and the volatiles were removed under reduced pressure. The resulting residue was purified by silica chromatography (10 to 50% gradient of EtOAc in heptane) to give 2-chloro-5-(2,3-dichlorophenyl)pyrimidin-4-amine (398 mg, 1.087 mmol). A mixture of 2-chloro-5-(2,3-dichlorophenyl)pyrimidin-4-amine (41 mg, 0.188 mmol) and tert-butyl piperidin-4-ylcarbamate (38 mg, 0.188 mmol) in H2O (2 mL) was stirred in a microwave reactor for 90 min at 125 °C followed by 30 min at 200 °C (Boc deprotection occurs under the reaction conditions). After cooling to RT, the volatiles were removed under reduced pressure and the resulting residue was purified by HPLC (gradient elution 25-50% acetonitrile in water, 5 mM NH4OH modifier) to give 5-(2,3-dichlorophenyl)-2-(4-(dimethylamino)piperidin-1yl)pyrimidin-4-amine (11.4 mg, 0.031 mmol). 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.64-7.70 (m, 1 H), 7.58 (dd, J=8.03, 1.51 Hz, 1 H), 7.38 (t, J=7.78 Hz, 1 H), 7.27 (dd, J=7.78, 1.51 Hz, 1 H), 4.90 (d, J=14.05 Hz, 2 H), 3.20 (s, 1 H), 2.81 - 2.97 (m, 2 H), 2.73 (s, 6 H), 2.042.13 (m, 2 H), 1.60 (dd, J=12.30, 4.27 Hz, 2 H). HRMS calcd for C17H22Cl2N5 (M+H)+ 366.1252, found 366.1249 2-(4-aminopiperidin-1-yl)-5-(2,3-dichlorophenyl)pyrimidin-4-amine (7). Prepared following the general method B. 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.60-7.69 (m, 1 H) 7.57 (dd, J = 8.03, 1.76 Hz, 1 H) 7.32-7.43 (m, 1 H) 7.24-7.32 (m, 1 H) 4.70 (d, J = 13.30 Hz, 2 H) 2.86-3.08


17

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(m, 3 H) 1.84-1.97 (m, 2 H) 1.38 (qd, J = 12.00, 4.14 Hz, 2 H). HRMS calcd for C15H18Cl2N5 (M+H)+ 338.0939, found 338.0931 3-(4-amino-4-methylpiperidin-1-yl)-6-(2,3-dichlorophenyl)-1,2,4-triazin-5-amine (9). A solution of 6-azauracil (1.0 g, 8.84 mmol), POCl3 (10 mL, 107 mmol), and N,N-dimethylaniline (2 mL, 1.784 mmol) was stirred in a microwave reactor for 25 min at 90 oC. After cooling to RT, the reaction was poured into a beaker containing heptane (200 mL) stirred for 5 min at RT and the phases were separated. This procedure was repeated twice (200 mL of heptane each). The heptane phases were filtered through a pad of Celite and MgSO4, the volatiles were removed under reduced pressure and the resulting residue was treated with NH3 (7 N in MeOH, 5 mL in 10 mL of MeOH) precooled at 0 oC. The mixture was stirred for 5 min at RT, then, the volatiles were removed to give 3-chloro-1,2,4-triazin-5-amine (200 mg, 17.3% yield). This compound was used in next step without further purification. A solution of 3-chloro-1,2,4-triazin-5-amine (165 mg, 1.264 mmol), tert-butyl (4methylpiperidin-4-yl)carbamate (271 mg, 1.264 mmol), and N-methylmorpholine (208 µL, 1.896 mmol) in NMP (5 mL) was stirred in a microwave reactor for 3 h at 130 °C. After cooling to RT, the resulting residue was purified by HPLC (gradient elution 15-40% acetonitrile in water, 5 mM NH4OH modifier) to give tert-butyl (1-(5-amino-1,2,4-triazin-3-yl)-4-methylpiperidin-4yl)carbamate (71.0 mg, 0.23 mmol). MS m/z 308.4 (M+H)+. A solution of tert-butyl (1-(5-amino-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate (71 mg, 0.230 mmol) and NBS (41 mg, 0.230 mmol) in CHCl3 (2 mL) was stirred 16 h at RT. The volatiles were removed under reduced pressure and the resulting residue was purified by silica chromatography (0 to 5% gradient of MeOH (containing 1% NH3)/DCM) to give tert-butyl (1-(5amino-6-bromo-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate (87 mg, 0.23 mmol). MS m/z 387.3 (M+H)+.


18


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Page 20 of 35

A suspension of (1-(5-amino-6-bromo-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate (250 mg, 0.646 mmol), 2,3-dichlorophenylboronic acid (154 mg, 0.807 mmol), potassium phosphate (411 mg, 1.937 mmol), and XPhos 2nd generation precatalyst (25.4 mg, 0.032 mmol) in MeCN:H2O (9:1, 5 mL, degassed) was stirred in a microwave reactor for 2 h at 120 °C. After cooling to RT, the reaction was filtered through a pad of Celite followed by EtOAc (20 mL) wash. The combined filtrates were concentrated and the resulting residue was purified by silica chromatography (0 to 5% gradient of MeOH(containing 1% NH3)/DCM) to give tert-butyl (1-(5amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-3-yl)-4-methylpiperidin-4-yl)carbamate (78% purity). This mixture was further purified by HPLC (gradient elution 35-60% acetonitrile in water, 5 mM NH4OH modifier) to give tert-butyl (1-(5-amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-3-yl)-4methylpiperidin-4-yl)carbamate (155 mg, 0.646 mmol). MS m/z 453.0 (M+H)+. To

a

solution

of

tert-butyl

(1-(5-amino-6-(2,3-dichlorophenyl)-1,2,4-triazin-3-yl)-4-

methylpiperidin-4-yl)carbamate (155 mg, 0.342 mmol) in dioxane (2 mL), was added HCl (4 M in dioxane, 4 mL). After stirring at RT until no starting material remained (monitored by LCMS), the volatiles were removed under reduced pressure and the resulting residue was purified by silica chromatography (0 to 5% gradient of MeOH(containing 1% NH3)/DCM) to give 3-(4amino-4-methylpiperidin-1-yl)-6-(2,3-dichlorophenyl)-1,2,4-triazin-5-amine (130 mg, 0.342 mmol, HCl salt). 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.80 (dd, J=7.78, 2.01 Hz, 1 H), 7.42-7.58 (m, 2 H), 4.29 (m., 2 H), 3.64 (ddd, J=14.24, 9.47, 4.64 Hz, 2 H), 1.92-2.06 (m, 4 H), 1.57 (s, 3 H). HRMS calcd for C15H19Cl2N6 (M+H)+ 353.1048, found 353.1042. 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (1). A mixture of 3bromo-6-chloropyrazine-2-amine (1.5 g, 7.2 mmol), (2,3-dichlorophenyl)boronic acid (1.37 g, 7.2 mmol), PdCl2(dppf).DCM adduct (294 mg, 0.36 mmol), and potassium phosphate (4.58 g, 21.59 mmol) in MeCN:H2O (9:1, 15 mL) was stirred for 4 h at 120 °C. After cooling to RT, the


19

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reaction mixture was filtered through a pad of Celite followed by EtOAc wash. The solvent was removed under reduced pressure and the resulting residue was purified by silica chromatography (5 to 30% gradient of EtOAc in heptane) to give 6-chloro-3-(2,3-dichlorophenyl)pyrazin-2-amine (1.46 g, 5.32 mmol) as yellow solid. MS m/z 453.0 (M+H)+. A mixture of 6-chloro-3-(2,3-dichlorophenyl)pyrazin-2-amine (125 mg, 0.455 mmol), tert-butyl (4-methylpiperidin-4-yl)carbamate (195 mg, 0.911 mmol), and potassium phosphate (97 mg, 0.455 mmol) in NMP (1 mL) was stirred for 36 h at 140 °C. After cooling to RT, the mixture was poured into a separation funnel containing aq. sat NH4Cl and it was extracted with EtOAc (3 x 5 mL). The combined organic phases were dried over MgSO4, filtered and the solvents were removed under reduced pressure. The resulting residue was by silica chromatography (5 to 30% gradient of EtOAc in heptane) to give tert-butyl (1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2yl)-4-methylpiperidin-4-yl)carbamate (113 mg, 0.250 mmol) as yellow solid. MS m/z 452.4 (M+H)+. A solution of tert-butyl (1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4yl)carbamate (113 mg, 0.250 mmol) in THF:H2O (4:1, 2.5 mL) was treated with HCl (4 M in dioxane, 230 µL, 0.928 mmol). The resulting mixture was stirred for 2 h at at 140 °C. After cooling to RT, the volatiles were removed under reduced pressure, and the resulting residue was diluted with EtOAc (10 mL), H2O (10 mL). The phases were separated and the aqueous was futher extracted with EtOAc (2 x 5 mL). The combined organic phases were discarded, the aqueous phase was basified to pH 9 with NaOH 2 M, and extracted with EtOAc (3 x 10 mL). The combined organic phases were dried over MgSO4, filtered and the solvent was removed under

reduced

pressure

to

afford

6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-

dichlorophenyl)pyrazin-2-amine (84 mg, 0.238 mmol) as a yellow solid. 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.61 (dd, J=7.91, 1.63 Hz, 1 H) 7.47 (s, 1 H) 7.40 (t, J=7.78 Hz, 1 H)


20


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Page 22 of 35

7.34 (dd, J=7.65, 1.63 Hz, 1 H) 3.78 (ddd, J=13.43, 7.15, 4.27 Hz, 2 H) 3.50-3.64 (m, 2 H) 1.551.75 (m, 4 H) 1.25 (s, 3 H) HRMS calcd for C16H20Cl2N (M+H)+ 352.1096, found 352.1099. 1-(5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-amine (10). A mixture of 2,5dichloropyrazine (466 mg, 3.13 mmol), (2,3-dichlorophenyl)boronic acid (477 mg, 2.5 mmol), PdCl2(dppf).DCM adduct (102 mg, 0.125 mmol), and potassium phosphate (1.6 g, 7.5 mmol) in MeCN:H2O (9:1; 10 mL) was stirred in a microwave reactor for 1 h at 120 °C. After cooling to RT, the volatiles were removed under reduced pressure and the resulting residue was purified by silica chromatography (5 to 30% gradient of EtOAc in heptane) to give 2-chloro-5-(2,3dichlorophenyl)pyrazine (435 mg, 1.676 mmol). MS m/z 261.0 (M+H)+. A mixture of 2-chloro-5-(2,3-dichlorophenyl)pyrazine (100 mg, 0.385 mmol), tert-butyl (4methylpiperidin-4-yl)carbamate (165 mg, 0.771 mmol), and N-methylmorpholine (127 µL, 1.156 mmol) was stirred in a microwave reactor for 2 h at 120 °C and 30 min at 220 °C (Boc deprotection under the reaction conditions). After cooling to RT, the volatiles were removed under reduced pressure and the resulting residue was purified by HPLC (gradient elution 25-50% acetonitrile in water, 5 mM NH4OH modifier) to give 1-(5-(2,3-dichlorophenyl)pyrazin-2-yl)-4methylpiperidin-4-amine (17.2 mg, 0.05 mmol). 1H NMR (400 MHz, METHANOL-d4) δ ppm 7.61-7.71 (m, 2 H) 7.51 (dd, J = 7.65, 1.63 Hz, 1 H) 7.44 (t, J = 7.78 Hz, 1 H) 7.37 (d, J = 9.79 Hz, 1 H) 3.82-3.96 (m, 2 H) 3.65-3.80 (m, 2 H) 1.58-1.77 (m, 4 H) 1.27 (s, 3 H). HRMS calcd for C16H19Cl2N4 (M+H)+ 337.0987, found 337.0983. 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)-N-methylpyrazin-2-amine (11). To a 0 °C solution of 2-chloro-5-(2,3-dichlorophenyl)pyrazine (80 mg, 0.291 mmol) in DMF (1 mL) was added NaH (60% in mineral oil, 13 mg, 0.321 mmol). After stirring at RT for 15 min, methyliodide was added at 0 °C and the mixture was stirred at RT for 1 h. Water (1 mL) was added to quench the reaction and the resulting mixture was purified by HPLC (gradient elution


21

Page 23 of 35

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35-60 % acetonitrile in water, 5 mM NH4OH modifier) to give 6-chloro-3-(2,3-dichlorophenyl)N-methylpyrazin-2-amine (31 mg, 0.107 mmol). MS m/z 289.9 (M+H)+. A mixture of 6-chloro3-(2,3-dichlorophenyl)-N-methylpyrazin-2-amine

(31

mg,

0.107

mmol),

tert-butyl

(4-

methylpiperidin-4-yl)carbamate (46 mg, 0.215 mmol), and N-methylmorpholine (24 µL, 0.215 mmol) was stirred in a microwave reactor for 1 h at 250 °C (Boc deprotection under the reaction conditions). After cooling to RT, the volatiles were removed under reduced pressure and the resulting residue was purified by HPLC (gradient elution 25-50% acetonitrile in water, 5 mM NH4OH

modifier)

to

give

6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)-N-

methylpyrazin-2-amine (16.0 mg, 0.041 mmol). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.64 (dd, J=8.03, 1.51 Hz, 1 H) 7.34-7.47 (m, 2 H) 7.29 (dd, J=7.65, 1.63 Hz, 1 H) 5.72-5.85 (m, 1 H) 3.60-3.75 (m, 2 H) 3.49 (ddd, J=12.92, 8.78, 3.89 Hz, 2 H) 2.72 (d, J=4.52 Hz, 3 H) 1.59 (br. s., 2 H) 1.34-1.52 (m, 4 H) 1.09 (s, 3 H). HRMS calcd for C17H22Cl2N5 (M+H)+ 366.1252, found 366.1237. Protein Expression and Purification. The gene encoding human SHP2 from residues Met1Leu525 was inserted into a pET30 vector. A coding sequence for a 6X histidine tag followed by a TEV protease consensus sequence was added 5’ to the SHP2 gene sequence. The construct was transformed into BL21 Star™ (DE3) cells and grown at 37 °C in Terrific Broth containing 100 µg/mL kanamycin. At an OD600 of 4.0, SHP2 expression was induced using 1 mM IPTG. Cells were harvested following overnight growth at 18 °C. Cell pellets were resuspended in lysis buffer containing 50 mM Tris-HCl pH 8.5, 25 mM imidazole, 500 mM NaCl, 2.5 mM MgCl2, 1 mM TCEP, 1 µg/mL DNase1, and complete EDTAfree protease inhibitor and lysed using a microfluidizer, followed by ultracentrifugation. The supernatant was loaded onto a HisTrap HP chelating column in 50 mM Tris-HCl, 25 mM


22


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Page 24 of 35

imidazole, 500 mM NaCl, 1 mM TCEP and protein was eluted with the addition of 250 mM imidazole. The N-terminal histidine tag was removed with an overnight incubation using TEV protease at 4 °C. The protein was subsequently diluted to 50 mM NaCl with 20 mM Tris-HCl pH 8.5, 1 mM TCEP then applied to a HiTrap Q FastFlow column equilibrated with 20 mM Tris pH 8.5, 50 mM NaCl, 1 mM TCEP. The protein was eluted with a 10 column volume gradient from 50-500 mM NaCl. Fractions containing SHP2 were pooled and concentrated then loaded onto a HiLoad Superdex200 PG 16/100 column, exchanging the protein into the crystallization buffer, 20 mM Tris-HCl pH 8.5, 150 mM NaCl and 3 mM TCEP. The protein was concentrated to 15 mg/mL for use in crystallization. Crystallization, DSF, and high throughput screening assays used the 1-525 construct of SHP2, while biochemical assays used the 2-593 construct. Biochemical assay. SHP2 is allosterically activated through binding of bis-tyrosylphorphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 PTP active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format. More specifically, the phosphatase reactions were performed at room temperature in 384-well black polystyrene plate, flat bottom, low flange, non-binding surface (Corning, Cat# 3575) using a final reaction volume of 25 µL and the following assay buffer conditions : 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, 5 mM DTT. The inhibition of SHP2 from the tested compounds (concentrations varying from 0.003 – 100 µM) was monitored using an assay in which 0.5 nM of SHP2 was incubated with of 0.5 µM of peptide

IRS1_pY1172(dPEG8)pY1222(sequence:H2N-

LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide). After 30-60 minutes incubation at 25 oC,


23

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the surrogate substrate DiFMUP (Invitrogen, cat# D6567, 200 µM) was added to the reaction and incubated at 25 oC for 30 minutes (200 µM for 2-593, 100 µM for 1-525 construct). The reaction was then quenched by the addition of 5 µL of a 160 µM solution of bpV(Phen) (Enzo Life Sciences cat# ALX-270-204). The fluorescence signal was monitored using a microplate reader (Envision, Perki-Elmer) using excitation and emission wavelengths of 340 nm and 450 nm, respectively. The inhibitor dose response curves were analyzed using normalized IC50 regression curve fitting with control based normalization. Cellular assay. p-ERK cellular assay using the AlphaScreen® SureFire™ Phospho-ERK 1/2 Kit (PerkinElmer): KYSE-520 cells (30,000 cells/well) were grown in 96-well plate culture overnight and treated with SHP2 inhibitors at concentrations of 20, 6.6, 2.2, 0.74, 0.24, 0.08, 0.027 µM for 2 h at 37 °C. Incubations were terminated by addition of 30 µL of lysis buffer (PerkinElmer) supplied with the SureFire phospho-extracellular signal-regulated kinase (p-ERK) assay kit (PerkinElmer). Samples were processed according to the manufacturer's directions. The fluorescence signal from p-ERK was measured in duplicate using a 2101 multilabel reader (Perkin Elmer Envision). The percentage of inhibition was normalized by the total ERK signal and compared with the DMSO vehicle control. Cell proliferation assay. Cells (1500-cells/well) were plated onto 96-well plates in 100 µL medium (RPMI-1640 containing 10% FBS, Lonza). Compounds with various concentrations (1.25, 2.5, 5, 10, 20 µM) were added 24 h after cell plating. At day 5, 50 µL Celltiter-Glo reagent (Promega) was added, and the luminescent signal was determined according to the supplier’s instruction (Promega). Selectivity assays. Activity of 1 vs other phosphatases and kinases is found in the supporting information.


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Page 26 of 35

Differential Scanning Fluorimetry. Differential Scanning Fluorimetry (DSF) was used as a method to identify compounds that stabilize SHP2 from thermal denaturation. The following assay conditions were used: 100 µg/mL SHP2, 5× SYPRO Orange dye (5000× concentrate in DMSO; Life Technologies), 100 mM Bis-Tris (pH 6.5), 100 mM NaCl, 0.25 mM TCEP, and 5 % DMSO. The final compound concentration evaluated was 100 µM. To carry out the experiment, 9.5 µL DSF assay solution was dispensed into an assay plate (LightCycler; 480 Multiwell Plate 384 White) containing 500 nL of compound dissolved in DMSO then mixed. The final assay volume was 10 µL per well in a 384-well format. Plates were then sealed after reagent addition, centrifuged at 1000 rpm for 1 minute, and read on a Bio-Rad C1000 Thermal Cycler with a CFX384 Real Time System using an excitation of 465 nm and an emission at 580 nm. The temperature was ramped from 25 °C to 75 °C and measurements were taken at 0.5 °C increments. The melting temperature (Tm) of the raw fluorescence data was identified as the midpoint of the transitions via a semi-parametric fit. The ∆Tm was determined by comparing the individual Tm values for each compound with the mean Tm of the apo SHP2 protein controls (32 per plate) containing DMSO only. Crystallization and Structure Determination. Sitting drop vapor diffusion method was used for crystallization, with the crystallization well containing 17% PEG 3350 and 200 mM ammonium phosphate and a drop with a 1:1 volume of SHP2 protein and crystallization solution. Crystals were formed within five days, and subsequently soaked in the crystallization solution with 2.5 mM of 2. This was followed by cryoprotection using the crystallization solution with the addition of 20% glycerol and 1 mM compound 2, followed by flash freezing directly into liquid nitrogen.


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Diffraction data for the SHP2/compound 1 complex is reported elswhere16 and SHP2/compound 2 complex were collected on a Dectris Pilatus 6M Detector at beamline 17ID (IMCA-CAT) at the Advanced Photon Source at Argonne National Laboratories. The data were measured from a single crystal maintained at 100 K at a wavelength of 1 Å, and the reflections were indexed, integrated, and scaled using XDS.24 The spacegroup of the complex was P21 with 2 molecules in the asymmetric unit. The structure was determined with Fourier methods, using the SHP2 apo structure1 (PDB accession 2SHP) with all waters removed.

Structure determination was

achieved through iterative rounds of positional and simulated annealing refinement using BUSTER,25 with model building using COOT.26 Individual B-factors were refined using an overall anisotropic B-factor refinement along with bulk solvent correction.

The solvent,

phosphate ions, and inhibitor were built into the density in later rounds of the refinement. Data collection and refinement statistics are shown in Table 1 found in the supporting information. Pharmacokinetics. All animal related procedures were conducted under a Novartis IACUC approved protocol in compliance with Animal Welfare Act regulations and the Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice were obtained from Harlan Labs. Following IV administration (via tail vein) at 1 mg/kg, approximately 50 µL of whole blood was collected via tail transection, at 0.083, 0.5, 1, 2, 4, and 7 h post-dose and transferred to an Eppendorf microcentrifuge tube containing EDTA.

Oral administration (at 5 mg/kg) and

collection procedures were similar to IV, except with whole blood collection at 0.25, 0.5, 1, 2, 4 and 7 h. The blood was centrifuged at 5000 rpm and plasma was transferred to a Matrix 96 well plate, capped and stored frozen (-20 °C) for parent compound analysis.

Samples were

precipitated and diluted with acetonitrile containing internal standard and prepared for


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Page 28 of 35

LC/MS/MS. An aliquot (20 µL) of each sample was injected into an API4000 LC/MS/MS system for analysis, and transitions of 352.05 amu (Q1) and 267.10 amu (Q3) were monitored. All pharmacokinetic (PK) parameters were derived from concentration-time data by noncompartmental analyses. All PK parameters were calculated with the computer program WinNonlin (Version 6.4) purchased from Certara Company (St. Louis, MO). For the intravenous dose, the concentration of unchanged compound at time 0 was calculated based on a log-linear regression of first two data points to back-extrapolate C(0). The area under the concentration-time curve (AUClast) was calculated using the linear trapezoidal rule. The bioavailability was estimated as following equation: %F =

AUC inf, p.o. AUC inf,i.v.

⋅

Dosei.v . Dose p.o..

Results are expressed as mean. No further statistical analysis was performed.

Tumor Xenograft Experiments. All animal studies were carried out according to the Novartis Guide for the Care and Use of Laboratory Animals.

Female nude mice were inoculated

subcutaneously (3 x 106 cells) in a suspension containing 50% phenol red-free matrigel (BD Biosciences) in Hank’s balanced salt solution with parental KYSE-520 cells. For PK/PD studies, mice were administered a single dose of vehicle control or 1 by oral gavage once tumors reached roughly 500 mm3. Mice were subsequently euthanized at predetermined time points following a single dose of compound at which point plasma and xenograft fragments were harvested for determination of 1 concentrations and p-ERK modulation, respectively. For efficacy studies, mice were calipered twice weekly by calipering in two dimensions. Once tumors reached roughly 200 mm3, mice were randomly assigned to treatment groups. For the efficacy study, mice were assigned to receive either vehicle, 1 (10, 30, or 100 mg/kg qd), or erlotinib (80 mg/kg qd) by oral gavage. Tumor volume and mouse body weight was assessed twice weekly. To


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assess MAPK pathway modulation in xenograft protein lysates, total and phospho-ERK1/2 was assessed using a commercially available kit (Meso Scale Discovery catalog number K15107D). The assay was conducted as recommended by Meso Scale Discovery with the exception that protein lysate was incubated overnight.

ANCILLARY INFORMATION Supporting information is available which includes selectivity assays, Xray data table, and additional pharmacology results. PDB ID codes. 5EHP for SHP2 in compex with compound 2. 5EHR for SHP2 in complex with compound 1. Authors will release the atomic coordinates and experimental data upon article publication. Corresponding author information. email: [email protected]; telephone: 617871-7729.

Novartis Institutes for Biomedical Research, Inc. 250 Massachusetts Avenue,

Cambridge, MA 02139. Acknowledgements: Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Hauptman-Woodward Medical Research Institute. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. The authors thank the entire SHP2 team. Abbreviations used. PTP, protein tyrosine phosphatase; RAS, rat sarcoma protein; AKT, protein kinase B; JAK, Janus kinase; STAT, Signal Transducer and Activator of Transcription proteins.


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REFERENCES.

1. a) Hof, P.; Pluskey, S.; Dhe-Paganon, S.; Eck, M. J.; Shoelson, S. E. Crystal structure of the tyrosine phosphatase SHP-2. Cell, 1998, 92, 441-450. b) Barford, D.: Neel B. G. Revealing mechanisms for SH2 domain mediated regulation of the protein tyrosine phosphatase SHP-2. Structure, 1998, 6, 249-254. 2. Plusky, S.; Wandless, T. J.; Walsh, C. T.; Shoelson, S. E.; Potent Stimulation of SH-PTP2 Phosphatase Activity by Simultaneous Occupancy of Both SH2 Domains. J. Biol. Chem., 1995, 270, 2897-2900. 3. (a) Prahallad, A.; Heynen, G. J.; Germano, G.; Willems, S.M.; Evers, B.; Vecchione, L.; Gambino, V.; Lieftink, C.; Beijersbergen, R.L.; Di Nicolantonio, F.; Bardelli, A.; Bernards, R. PTPN11 Is a Central Node in Intrinsic and Aquired Resistance to Targeted Cancer Drugs. Cell Reports, 2015, 12, 1-8. (b) Wang, H.; Chiang, W.; Huang, H.; Shen, Y.; Chiang, H.; Src Homology 2 domain-containing tyrosine phosphatase 2 promotes oral cancer invasion and metastasis. BMC Cancer, 2014, 14, 442. 4. a) Neel, B. G.; Gu, H.; Pao, L. The ‘Shp’ing News: SH2 Domain-Containing Tyrosine Phosphatases in Cell Signaling. Trends Biochem. Sci., 2003, 28, 284-293. b) Chan, R. J.; Feng, G. S. PTPN11 is the First Identified Proto-Oncogene That Encodes a Tyrosine Phosphatase. Blood 2007, 109, 862-867. c) Tiganis, T.; Bennett, A. M. Protein Tyrosine Phosphatase Function: the Substrated Perspective. Biochem. J. 2007, 402, 1-15. 5. Bunda, S.; Burrell, K.; Heir, P.; Zeng, L.; Alamsahebpour, A.; Kano, Y.; Raught, B.; Zhang, Z-Y.; Zadeh, G.; Ohh, M.; Inhibition of SHP2-Mediated Dephosphorylation of Ras Suppresses Oncogenesis. Nat. Commun., 2015, 6, 8859.


29

Page 31 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6. Tartaglia, M.; Mehler, E. L.; Godberg, R.; Zampino, G.; Brunner H. G.; Kremer, H.; Van Der Burgt, I.; Crosby, A. H.; Ion, A.; Jeffery, S.; Kalidas, K.; Patton, J. A.; Kucherlapati, R. S.; Gelb, B. D. Mutations in PTPN11, Encoding the Protein Tyrosine Phosphatase SHP-2, Cause Noonan syndrome. Nat. Genet. 2001, 29, 465-468. 7. Tartaglia, M.; Niemeyer, C. M.; Fragale, A.; Song, X.; Buechner, J.; Jung, A.; Hählen, K.; Hasle, H.; Licht, J. D.; Gelb, B. D. Somatic Mutations in PTPN11 in Juvenile Myelomonocytic Leukemia, Myelodysplactic Syndromes and Acute Myeloid Leukemia. Nat. Genet. 2003, 34, 148-150. 8. a) Chan, G.; Kalaitzidis, D.; Neel, B. G. The Tyrosine Phosphoatase Shp2 (PTPN11) in Cancer. Cancer Metastasis Rev. 2008, 27, 179-192. b) Miyamoto, D.; Miyamoto, M.; Takahashi, A.; Yomogita, Y.; Hagashi, H.; Kondo, S.; Hatakeyama, M. Isolation of a Distinct Class of Gainof-Function SHP-2 mutants with Oncogenic RAS-like Transforming Activity From Solid Tumors. Oncogene, 2008, 27, 3508-3515. 9. Li, J.; Jie, H.-B.; Lei, Y.; Gildener-Leapman, N.; Trivedi, S. PD-1/SHP-2 Inhibits Tc1/Th1 Phenotypic Responses and the Activation of T Cells in the Tumor Microenvironment. Cancer Res. 2015, 75, 508-518. 10. (a) Topalian, S.L.; Hodi, F.S; Brahmer, J.R.; Gettinger, S.N.; Smith, D.C.; McDermott, D.F.; Powderly, J.D.; Carvajal, R.D.; Sosman, J.A.; Atkins, M.B.; Leming, P.D.; Spigel, D.R.; Antonia, S.J.; Horn, L.; Drake, C.G.; Pardoll, D.M.; Chen, L.; Sharfman, W.H.; Anders, R.A.; Taube, J.M.; McMiller, T.L.; Xu,H.; Korman, A.J.; Jure-Kunkel, M.; Agrawal, S.; McDonald, D.; Kollia, G.D.; Gupta, A.; Wigginton, J.M.; Sznol, M. Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer. New Engl. J. Med., 2012, 366, 2443–2454. (b) Ohigashi, Y.; Sho, M.; Yamada, Y.; Tsurui, Y.; Hamada, K.; Ikeda, N.; Mizuno, T.; Yoriki, R.; Kashizuka, H.;


30


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 35

Yane, K.; Tsushima, F.; Otsuki, N.; Yagita, H.; Azuma, M.; Nakajima, Y.; Clinical Significance of Programmed Death-1 Ligand-1 and Programmed Death-1 Ligand-2 Expression in Human Esophageal Cancer. Clin. Cancer Res., 2005, 11, 2947–2953. 11. Butterworth, S.; Overduin, M.; Barr, A. J.; Targeting Protein Tyrosine Phosphatase SHP2 for Therapeutic Intervention. Future Med. Chem., 2014 , 6, 1423-1437. 12. Scott, L. M.; Lawrence, H. R.; Sebti, S. M.; Lawrence, N. J.; Wu, J. Targeting Protein Tyrosine Phosphatases for Anticancer Drug Discovery. Curr. Pharm. Des. 2010, 16, 1843-1862. 13. He, R.; Zeng, L.-F.; He, Y.; Zhang, S.; Zhang, Z.-Y. Small Molecule Tools for Functional Interrogation of Protein Tyrosine Phosphatases. FEBS J. 2013, 280, 731-750. 14. (a) Zhang, X.; He, Y.; Liu, S.; Yu, Z.; Jiang, Z.X.; Yang, Z.; Dong, Y.; Nabinger, S.C.; Wu, L.; Gunawan, A.M.; Wang, L.; Chan, R.J.; Zhang, Z.Y. Salicylic Acid Based Small Molecule Inhibitor for the Oncogenic Src Homology-2 Domain Containing Protein Tyrsosine Phosphatase2 (SHP2). J. Med. Chem, 2010, 53, 2482-2493. (b) Zeng, L.F.; Zhang, R.Y.; Yu, Z.H.; Li, S.; Wu, L.; Gunawan, A.M.; Lane, B.S.; Mali, R.S.; Li, X.; Chan, R.J.; Kapur, R.; Wells, C.D.; Zhang, Z.Y. Therapeutic Potential of Targeting the Oncogenic SHP2 Phosphatase. J. Med. Chem. 2014, 57, 6594-6609. (c) Bunda, S.; Burrell, K.; Heir, P.; Zeng, L.; Alamsahebpour, A.; Kano, Y.; Raught, B.; Zhang, Z.Y.; Zadeh, G.; Ohh, M. Nature Comm. 2015, 6, 8859. 15. (a) Gilmartin, A.G.; Faitg, T.H.; Richter, M.; Groy, A.; Seefeld, M.A.; Darcy, M.G.; Peng, X.; Federowicz, K.; Yang, J.; Zhang, S.Y.; Minthorn, E.; Jaworski, J.P.; Schaber, M.; Martens, S.; McNulty, D.E.; Sinnamon, R. H.; Zhang, H.; Kirkpatrick, R.B.; Nevins, N.; Cui, G.; Pietrak, B.; Diaz, E.; Jones, A.; Brandt, M.; Schwartz, B.; Heerding, D.A.; Kumar, R. Allosteric WIP1 Phosphatase Inhibition Through Flap-Subdomain Interaction. Nat. Chem. Biol. 2014, 10, 181187. (b) Chio, C. M.;Yu, X.; Bishop, A. C. Rational Design of Allosteric-Inhibition Sites in


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Classical Protein Tyrosine Phosphatases. Bioorg. Med. Chem. 2015, 23, 2828-2838. (c) Schneider, R.; Beumer, C.; Simard, J. R.; Grutter, C.; Rauh, D. Selective Detection of Allosteric Phosphatase Inhibitors J. Am. Chem. Soc. 2013, 135, 6838-6841. 16. Chen, Y. P.; LaMarche, M.J.; Fekkes, P.; Garcia-Fortanet, J.; Acker, M.; Chan, H.; Chen, Z.; Deng, Z.; Fei, F.; Firestone, B.; Fodor, M.; Gao, H.; Ho, S.; Hsiao, K.; Kang, Z.; Keen, .N; Labonte, l.; Liu, S.; Meyer, M.; Pu, M.; Price, E.; Ramsey, T.; Slisz, J.; Wang, P.; Yang, G.; Zhang, J.; Zhu, P.; Sellers, W.R.; Stams, T.; Fortin, P.D. Discovery of an Allosteric SHP2 Inhibitor for Cancer Therapy. Nature, in press. 17. Chen, Christine Hiu-Tung; Chen, Zhuoliang; Fortanet, Jorge Garcia; Grunenfelder, Denise; Karki, Rajesh; Kato, Mitsunori; Lamarche, Matthew J.; Perez, Lawrence Blas; Stams, Travis Matthew;

Williams,

Sarah.

Preparation

of

1-pyridazin-/triazin-3-yl-piper(-azine)/idine

/pyrrolidine derivatives for inhibiting the activity of SHP2. WO2015107493. 18. Gee, K. R.; Sun, W. C.; Bhalgat, M. K.; Upson, R. H.; Klaubert, D. H.; Latham, K. A.; Haugland, R. P. Fluorogenic Substrates Based on Fluorinated Umbelliferones for Continuous Assays of Phosphatases and Beta-Galactosidases. Anal. Biochem. 1999, 273, 41. 19. SHP2 activation has been extensively reviewed: (a) Barford, D.; Neel, B. G.; Revealing Mechanisms for SH2 Domain Mediated Regulation of the Protein Tyrosine Phosphatase SHP-2. Structure, 1998, 6, 249-254. (b) Chan, G.; Kalaitzidis, Neel, B. G. The Tyrosine Phosphatase SHP2 (PTPN11) in Cancer. Cancer Metastasis Rev. 2008, 27, 179-182. (c) Poole, A.W.; Jones, M.L. A SHPing Tale: Perspectives on the Regulation of SHP-1 and SHP-2 Tyrosine Phosphatases by the C-terminal Tail. Cell. Signal., 2005, 17, 1323-1332. (d) Butterworth, S.; Overduin, M.; Barr, A. J. Targeting Protein Tyrosine Phosphatase SHP2 for Therapeutic Intervention. Future Med. Chem. 2014, 6, 1423-1437.


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Page 34 of 35

20. (a) Clare, J.J.; Tate, S.N.; Nobbs, M.; Romanos, M.A. Voltage-Gated Sodium Channels as Therapeutic Targets. Drug Discov. Today 2000, 5, 506-520. (b) Large, C.H.; Kalinichev, M.; Lucas, A.; Carignani, C.; Bradford, A.; Garbati, N.; Sartori, I.; Austin, N.E.; Ruffo, A.; Jones, D.N.C.; Giuseppe A., Kevin D.R.. The Relationship Between Sodium Channel Inhibition and Anticonvulsant Activity in a Model of Generalised Seizure in the Rat. Epilepsy Res.2009, 85, 96106. 21. Patani, G.A.; Lavoie, E.J.; Bioisosterism: a Rationale Approach in Drug Design. 1996, Chem. Rev., 96, 3147-3176. 22. For example: (a.) Burger, M.T.; Han, W.; Lan, J.;Nishiguchi, G.; Bellamacina, C.; Lindval, M.; Atallah, G.; Ding, Y.; Mathur, M.; McBride, C.; Beans, E.L.; Muller, K.; Tamez, V.; Zhang, Y.; Huh, K.; Feucht, P.; Zavorotinskaya, T.; Dai, Y.; Holash, J.; Castillo, J.; Langowski, J.; Wang, Y.; Chen, M.Y.; Garcia, P.D.; Structure Guided Optimization, in Vitro Activity, and in Vivo Activity of Pan-PIM Kinase Inhibitors. ACS Med. Chem. Lett. 2013, 4, 1193-1197. (b.) Eriksson A.L; Höglund, M.; Lindhagen, E.; Aleskog, A.; Hassan, S.B.; Ekholm, C.; Fhölenhag, K.; Jensen, A.J.; Löthgren, A.; Scobie, M.; Larsson, R.; Parrowm V. Identification of AKN-032, a Novel 2-Aminopyrazine Tyrosine Kinase Inhibitor, with Significant Preclinical Activity in Acute Myeloid Leukemia. Biochem Pharmacol., 2010, 80, 1507-1516. (c.) Lin, S.; Malkani, S.; Lombardo, M.; Yang, Y.; Mills, S.G.; Chapman, K.; Thompson, J.E.; Zhang, W.X.; Wang, R.; Cubbon, R.M.; O’Neill, E.A.; Hale, J.J.; Design, Synthesis, and Biological Evaluation of Aminopyrazine Derivatives as Inhibitors of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK-2). Bioorg. Med. Chem. Lett. 2015, 25, 5402–5408. 23. (a) Artursson P.; Tavelin, S.; Caco-2 and Emerging Alternatives for Prediction of Intestinal Drug Transport: A General Overview. In: Methods and Principles in Medicinal Chemistry. 2003,


33

Page 35 of 35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


18, Drug Bioavailability. 72-82. (b) Ungell A.; Karlsson, J.; Cell Cultures in Drug Discovery: an Industrial Perspective. Methods and Principles in Medicinal Chemistry, 2003, 18, Drug Bioavailability: Estimation of Solubility, Permeability, Absorption, and Bioavailability, 90-131. 24. Kabsch, W. XDS. Acta Cryst. 2010, D66, 125-132. 25. Bricogne, G.; Blanc, E.; Brandl, M.; Flensburg, C.; Keller, P.; Paciorek, W.; Roversi, P.; Smart, O.S.; Vonrhein, C.; Womack, T.O. BUSTER, version 2.8.0. Cambridge, United Kingdom: Global Phasing Ltd. 2009. 26. Emsley, P.; Lohkamp, B.; Scott, W.G.; Cowtan, K. Features and Devlopment of Coot. Acta Cryst. 2010, D66, 486-501.


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TABLE OF CONTENTS GRAPHIC.

SHP2 Phosphatase

Allosteric pocket

Orally efficacious phosphatase inhibitor


35

Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and

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