Discovery of 1-Methyl-1H-imidazole Derivatives as Potent Jak2

Article pubs.acs.org/jmc

Discovery of 1‑Methyl‑1H‑imidazole Derivatives as Potent Jak2 Inhibitors Qibin Su,*,† Stephanos Ioannidis,† Claudio Chuaqui,† Lynsie Almeida,† Marat Alimzhanov,† Geraldine Bebernitz,† Kirsten Bell,† Michael Block,† Tina Howard,‡ Shan Huang,† Dennis Huszar,† Jon A. Read,‡ Caroline Rivard Costa,† Jie Shi,† Mei Su,† Minwei Ye,† and Michael Zinda† †

AstraZeneca, Oncology Innovative Medicines, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States Discovery Sciences Innovative Medicines, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom

‡

ABSTRACT: Structure based design, synthesis, and biological evaluation of a novel series of 1-methyl-1H-imidazole, as potent Jak2 inhibitors to modulate the Jak/STAT pathway, are described. Using the C-ring fragment from our first clinical candidate AZD1480 (24), optimization of the series led to the discovery of compound 19a, a potent, orally bioavailable Jak2 inhibitor. Compound 19a displayed a high level of cellular activity in hematopoietic cell lines harboring the V617F mutation and in murine BaF3 TEL-Jak2 cells. Compound 19a demonstrated significant tumor growth inhibition in a UKE-1 xenograft model within a well-tolerated dose range.

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2,4-diamine, which was progressed into clinical trials.17 We have also explored the role of Jak kinases in solid tumors where activation of the Jak/STAT pathway is not typically associated with activating mutations in Jak2.18 Herein we describe our structure based design efforts toward the discovery of novel potent ATP competitive Jak2 small molecule inhibitors and detail the structure−activity relationship within this series. Detailed results of preclinical biological and pharmacokinetic studies of the lead compounds are also discussed below.

INTRODUCTION Jak2, together with Jak1, Jak3, and Tyk2, belongs to the Jak (Janus-associated kinase) family of nonreceptor tyrosine kinases.1 They play important roles in cytokine and growth factor mediated signal transduction. Activated by cytokine or growth factor engagement, autophosphorylated Jaks stimulate the recruitment and phosphorylation of the signal transducers and activators of transcription (STATs).2 This in turn leads to the dimerization and translocation of phosphorylated STATs into the nucleus, hence increasing cellular proliferation and resistance to apoptosis.3 Discovery of a single activating somatic mutation (V617F) in the pseudokinase JH2 (Janus Homology 2) domain of Jak2 stimulated significant interest in developing selective Jak2 inhibitors for the treatment of myeloproliferative neoplasms (MPNs).4 Indeed, there is growing evidence to suggest that deregulation of constitutively activated Jak/STAT signaling is an important event in all patients with MPNs, comprising essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF).5 MF is associated with the lowest survival rate among MPNs and represents a significant unmet medical need.6 Thus, selective inhibitors capable of targeting the Jak/STAT pathway are highly desirable.7 Several Jak inhibitors have been evaluated in human clinical trials, notably (Figure 1) Ruxolitinib 1 (formerly INCB018424), which has received approval for clinical use in MF in the United States and Europe,8 SAR302503 2 (formerly TG101348),9 Lestaurtinib 3 (CEP701),10 CYT-387 4,11 Pacritinib 5,12 LY2784544 6,13 BMS-911543 7,14 and others.15 Our lab has pursued the discovery and development of Jak2 selective inhibitors as anticancer drugs,16 culminating in the discovery of drug candidate (S)-5-chloro-N2-(1-(5-fluoropyrimidin-2-yl)ethyl)-N4-(5-methyl-1H-pyrazol-3-yl)pyrimidine© 2013 American Chemical Society

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CHEMISTRY The nitro-imidazole 8 was regioselectively methylated using methyl iodide at elevated temperature to yield 9. Subsequent palladium mediated hydrogenation of 9 afforded amino imidazole 10 in excellent yields (Scheme 1).19 Coupling reaction of 1-methyl-1H-imidazol-4-amine 10 with the commercially or readily available dichloride intermediates 11a−g proceeded smoothly, and regioselectively, at 70 °C in the presence of DIPEA to afford intermediates 12a−g. However, the subsequent displacement of the second chloride by 14 required microwave irradiation at 150 °C in a basic alcoholic solution to yield intermediates 13a−g.20 Unfortunately, these forcing reaction conditions led to a decrease in the enantiomeric excess of the coupling products, and therefore the final compounds required chiral purification to afford the desired 13a−g (Scheme 2). The synthesis of pyrrolo-pyrimidine analogues started with the capping of the NH of commercially available 15a. Removal Received: October 3, 2013 Published: December 10, 2013 144

dx.doi.org/10.1021/jm401546n | J. Med. Chem. 2014, 57, 144−158

Journal of Medicinal Chemistry

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Figure 1. Selected Jak inhibitors in human clinic trials.

after final deprotection of the sulfonyl group under aqueous basic conditions (aq NaOH). Unfortunately, this reaction sequence resulted in the partial racemization of the final compounds, thus chiral purification by HPLC was required (see the Experimental Section for the chiral separation conditions). Pyrazolo-pyrimidine analogues 19d and 19e were synthesized using the sequence depicted in Scheme 3, starting from commercially available 15b and 17e, respectively. 23a and 23b were synthesized in a similar manner starting from the corresponding commercially available dichloride starting materials, 20a and 20b, in a moderate overall yield (Scheme 4).

Scheme 1. Preparation of 1-Methyl-1H-imidazol-4-amine Hydrochloride Salt 10a

Reagents and conditions: (a) MeI, K2CO3, 65 °C, CH3CN, 75%; (b) H2, Pd/C then HCl, 100%.

a

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of the active NH functionality proved necessary for the subsequent coupling reactions. Hence, 15a was treated with base (NaOH or NaH) and then quenched via the addition of either tosyl chloride or methyl iodide to afford 17a or 17b, respectively (Scheme 3). Copper(II) acetate promoted coupling reaction between cyclopropyl boronic acid, and 15a proceeded smoothly to yield 17c. With these intermediates in hand, sequential attachment of 1-methyl-1H-imidazol-4-amine 10 and (S)-1-(5-fluoropyrimidin-2-yl)ethanamine 1421 furnished the desired final compounds 19b−c. 19a was obtained

RESULTS AND DISCUSSION Our lab has a long-standing interest in the discovery of isosteric replacements of the pyrazol-3-yl amine, which acts as a hinge binder in a number of Jak2 inhibitors such as compound 25a.16b Previously, we have demonstrated that thiazol-2-yl amine may operate as an effective replacement for the amino pyrazole, leading to potent Jak2 inhibitors, e.g., 25b (Figure 2).16b

Scheme 2. Preparation of 13a−ga

a

Reagents and conditions: (a) 10, DIPEA, CH3CN, 70 °C; (b) 14, DIPEA, n-BuOH, 150 °C microwave then chiral separation. 145



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Scheme 3. Preparation of 19a−ea

a Reagents and conditions: (a) For 17a, 15a, NaOH, tetra-butylammonium hydrogen sulfate, TsCl; for 17b, 15a, NaH, MeI; for 17c, cyclopropyl boronic acid, Cu(OAc)2; for 17d, 15b, TsOH, DHP; (b) 10, DIPEA, CH3CN, 100 °C microwave; (c) for 19a, 14, DIPEA, n-BuOH, 150 °C microwave, then NaOH (aq), 55 °C, then chiral separation; for 19b−e, 14, DIPEA, n-BuOH, 150 °C microwave; then chiral separation.

Scheme 4. Preparation of 23a and 23ba

Reagents and conditions: (a) NaOH, tetra-butylammonium hydrogen sulfate, TsCl; (b) 10, DIPEA, CH3CN, 100 °C microwave; (c) 14, DIPEA, nBuOH, 150 °C microwave; then chiral separation; (d) NaOH (aq), 55 °C. a

Figure 2. Bioisosteric replacement of amino-pyrazole with aminothiazole led to potent Jak2 inhibitor 25b.

We hypothesized that other heterocycles, with a similar array of hydrogen bond interactions in a cis-donor/acceptor/donor pattern, could provide analogous binding in the hinge region of the ATP binding site. We therefore designed the 1-methyl-1Himidazol-4-amine analogue 26 (Figure 3), reasoning that the required hydrogen bond interactions could be achieved with the C1-NH, 2-N, and C3-H of the amino imidazole. Moreover, the imidazole C5-H is available to form an intramolecular hydrogen bond with the nitrogen of the pyrimidine (B ring), thereby favoring a coplanar conformation with reduced intramolecular ligand strain while making the requisite interactions with the hinge. We envisioned that a small hydrophobic substituent (N4-Me) could engage the methionine (M929) gatekeeper in the Jak2 kinase and provide the critical hydrophobic interaction. To test the hypothesis, compound 26 was synthesized and evaluated in the Jak2

Figure 3. Bioisosteric replacement of the A ring. aMeasured at high ATP concentration (5 mM); for experimental details, see ref 17. bAll values are an average of at least three independent dose−response curves. cBiochemical and cellular activity of 24 was reported in ref 17.

biochemical and cellular assays. Compound 26 was shown to be less potent against Jak2 (IC50 = 0.12 μM), compared to 24 146



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(IC50 = 0.058 μM), but maintained activity in our cellular assay (TEL-Jak2 GI 50 = 0.27 μM, Figure 3), albeit at a correspondingly reduced level, suggesting that amino imidazole could be an effective replacement for amino pyrazole. Our hypothesis that amino imidazole could be a viable replacement of amino pyrazole was strengthened when we obtained cocrystal structures of 2716d and 2822 (Figure 4)

bound into the ATP binding site of Jak2. The binding mode observed for 28 was consistent with that observed with 24 (Figure 3)17 and its close pyrazole analogues, 27.16d The imidazole ring interacts with the hinge via an acceptor−donor hydrogen-bonding motif to the backbone of leucine (L932) and glutamate (E930), while the donor N−H hydrogen bond to the hinge in 27 is replaced by a C−H in 28. The imidazole ring in 28 shows a small shift away from the hinge, which results in a movement of ∼0.6 Å of the N-1 methyl group toward the M929 gatekeeper residue. The tolerance of a polar C−H interacting with the backbone carbonyl at this position of the hinge is not uncommon.23 For example, it is observed in the well characterized bis-aminopyrimidine24 and 4-anilinoquinazoline class of kinase inhibitors.25 With these encouraging results in hand, we decided to hold the 1-methyl-1H-imidazol-4-amine constant as the hingebinding motif and explore the SAR of the other region (B ring, Figure 3) of the molecule with the hope of further improving Jak2 potency. We hypothesized that fused bicyclic B rings would fill the hydrophobic pocket around the hinge area, thus leading to a significant boost of Jak2 potency. To prioritize the syntheses of bicyclic B-rings, docking studies of the newly designed compounds into the structure of 24 bound to Jak2 (PDB code 2XA4) were carried out using Glide,26 followed by post processing of docked poses using a MM-GBSA workflow27 that approximates ΔG of binding and ligand strain using a continuum solvent approximation for desolvation.28 Top scoring poses were then inspected visually to ensure that binding modes were consistent with the known X-ray structures of this class of Jak2 inhibitors.16d,17 From these docking studies for [6−6]-bicyclic B-rings, several interaction preferences were observed that helped prioritize the design and synthesis of analogues. In particular, hydrogen bonding complementarity between the B ring and the hinge backbone carbonyl of leucine (L932) was key to achieving a high ΔG of binding, reduced ligand strain, and a consistent binding mode with observed Xray structures (Figure 5a). B rings with a hydrogen bond donor (N−H or polar C−H) at the C5 position were predicted to be superior to sp2 N or O-containing rings. The predicted SAR trends are shown in Figure 5b.

Figure 4. X-ray crystal structure overlay of Jak2 in complex with pyrazole hinge binding compound 27 (yellow carbon atoms, PDB code 3zmm)16d and Jak2 in complex with imidazole hinge binding compound 28 (blue carbon atoms, PDB code 4c62). Protein backbone for the complex of Jak2 with compound 28 is shown as a cartoon. Selected residues are shown as sticks. Hydrogen bonds are shown as dotted lines in the same color as the carbon atoms.

Figure 5. (a) Predicted binding mode of [6−6]-bicyclic B-rings (the structure of 24 bound to Jak2 was used for all docking studies (PDB code 2XA4)).17 (b) Predicted SAR from docking studies where the surface coloring scheme denotes hydrogen bonding preferences (blue, acceptor; red, donor). 147



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Table 1. Biochemical and Cellular Activity of [6,6]-Fused B-Ring Analogues

a

Measured at high ATP concentration (5 mM); for experimental details, see ref 17. bAll values are an average of at least three independent dose− response curves. cDenotes not tested. dCompound is a mixture of enantiomers; enantiomeric excess (ee) was not determined.

Table 2. Biochemical and Cellular Data of [5,6]-Fused B-Ring Analogues

a

Measured at high ATP concentration (5 mM); for experimental details, see ref 17. bAll values are an average of at least three independent dose− response curves. cCompound is a mixture of enantiomers; enantiomeric excess (ee) was not determined.

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To optimize Jak2 potency of 26 based on the predicted binding mode, analogues with a [6,6]-fused pyrimidine B ring were synthesized and evaluated (Table 1). We were pleased to find that analogue 13a, which contained a quinazoline B ring, was potent (IC50 = 0.003 μM, Table 1) in both the Jak2 biochemical assay at high ATP concentration and in the cellular assay (TEL-Jak2 GI50 = 0.034 μM), which was comparable to 24. With this encouraging result, we elected to explore the SAR around this specific scaffold. Compound 13c with a 7-OMe substituent exhibited potent Jak2 enzyme activity (0.024 μM), while substitution on the 6-position was detrimental to Jak2 potency as 13b was less potent in Jak2 enzyme assay (IC50 = 0.67 μM) and inactive in the cellular assay (3 μM), implying a steric clash between the 6-OMe group in the Jak2 protein. Compounds 13d and 13h displayed potent Jak2 activity (Jak2 IC50 = 0.033 μM and IC50 = 0.012 μM, respectively), while incorporation of a sp2 nitrogen at the C5 position (13g) greatly decreased Jak2 potency (IC50 = 0.80 μM), suggesting that there may be electronic repulsion between this sp2 nitrogen and the backbone carbonyl of leucine (L932). This is consistent with our predicted binding mode depicted in Figure 5. Diminishing Jak2 activity (IC50 = 0.49 μM) of 13e confirmed the limited space available for substitution and hydrogen bonding preferences shown in Figure 5b. Being cognizant of the analogues with [6,6]-fused B rings, which were potent against Jak2, we set out to explore the SAR of [5,6]-fused B ring analogues in an attempt to diversify the SAR while maintaining Jak2 activity. We elected to synthesize the analogues 19a−e and 23a−b (Table 2), which contain different fused 5-membered ring heterocycles. As shown in Table 2, the fused B-ring analogue 19a retained the Jak2 enzymatic potency (IC50 < 0.003 μM). 19a exhibited excellent cellular activity in the BaF3 TEL-Jak2 cell line (GI50 = 0.012 μM). Unsurprisingly, the alkylation of the 7-position of pyrrolopyrimidine (19b,c) was tolerated and maintained Jak2 potency because this position is solvent accessible and points toward the solvent channel. Pyrazolo-analogues 19d and 19e were synthesized to lower the hydrophobicity and, to our delight, 19e demonstrated a combination of favorable enzymatic (IC50 = 0.015 μM) and cellular activity (GI50 = 0.026 μM). Interestingly, analogue 23a with a hydrogen bond donor toward the hinge region exhibited excellent Jak2 enzyme potency (IC50 < 0.003 μM), implying a productive interaction between the NH and protein, thus further supporting our binding mode hypothesis. To avoid the immunosuppression associated with Jak3 deficiency, we have been focusing on the development of Jak2 inhibitors with high level of selectivity against Jak3.17 Gratifyingly, a significant level of selectivity against Jak3 was observed in this series, however to a smaller extent against Jak1 (Tables 1 and 2). Interestingly, sulfur containing bicycle 23b was predicted by MM-GBSA to be preferred and was subsequently shown to be a potent Jak2 inhibitor (IC50 = 0.004 μM and GI50 = 0.037 μM). Finally, an X-ray crystal structure of compound 23b bound into Jak2 was solved and was shown to be in good agreement with the predicted binding mode of this series (Figure 6). In both the predicted binding modes (Figure 6a) and the solved X-ray cocrystal structure (Figure 6b) of [5,6]-bicyclic B-rings, the key hydrogen bonding interactions to the hinge are consistent with the pyrimidine B ring analogues shown in Figure 4. The fused B-rings make an additional interaction with the backbone carbonyl of leucine (L932) where a hydrogen bond donor or polar C−H is preferred. In the case of 23b, we postulate that

Figure 6. (a) Predicted binding mode of [5,6]-bicyclic B-rings (The structure of 24 bound to Jak2 was used for all docking studies (PDB code 2XA4)).17 (b) X-ray crystal structure of compound 23b bound to JAK2 (PDB code 4c61). Carbon atoms of the complex are colored in green. 2FoFc electron density contoured at 1.0σ and shown as a wire mesh. Hydrogen bonds are shown as yellow dotted lines.

the sulfur interacts favorably with the carbonyl oxygen via a back-bonding mechanism.29 PK Profiling of Leads. Having achieved highly potent Jak2 inhibitors (such as the analogues in Table 3), the Table 3. Rat PKa Properties of Selected Potent Leads compd

F%

CLobs (mL/min/kg)

Vdss (L/kg)

t1/2 (h)

13c 13d 19a 19e 23b

NAb NAb 64 90 NAb

110 100 36 25 44

6 2.5 2.2 1.8 1.2

0.98 0.92 1.6 1.3 0.64

a

Han Wistar rat male; 10 mg/kg po (0.1% HPMC); 3 mg/kg iv (40% DMA/40%PEG/20% saline). bDenotes not tested.

pharmacokinetic (PK) properties of these leads were evaluated and shown to display different metabolic stabilities in vivo depending on the nature of the B-ring. While the compounds with fused [6,6] B-rings (13c and 13d) were highly cleared in vivo in the rat, analogues with [5,6]-fused B-rings, e.g., 19a and 19e, showed low in vivo clearance with a moderate half-life and excellent oral bioavailability (Table 3). Because of their favorable rat PK properties and excellent cellular potencies, compounds 19a and 19e were progressed into further in vivo pharmacokinetic studies in dog. We were pleased to find that both compounds displayed excellent metabolic stability with prolonged half-life, as well as excellent oral bioavailability in dogs (Table 4). Table 4. Dog PK Propertiesa of 19a and 19e compd

route

19a

iv po iv po

19e

F%

AUC (μM·h)

64

45b

139

49b

CLobs (mL/min/kg)

Vdss (L/kg)

t1/2 (h)

2.07

1.9

4.56

2.1

12.4 7.6 6.2 7.7

a

Beagle dog male; 10 mg/kg po (0.1% HPMC, pH 2); 2.5 mg/kg iv (20% TEG/D5W). bAUC0−12h.

Kinase Selectivity of Compound 19a. Compound 19a was evaluated against a panel of 82 kinases using Upstate Biotechnologies Kinase Profiler Service (Millipore Corporation, Charlottesville, VA) at three concentrations (0.01, 0.10, and 1.0 μM) at or near the Km for ATP using a radiometric filterbinding assay. The set of kinases tested were selected based on 149



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inhibition of phosphorylation of STAT5 in both cell lines upon treatment with 19a (Figures 8 and 9). 19a Demonstrated a Dose Dependent Pharmacodynamic (PD) Effect in a Mouse TEL-Jak2 Model. To evaluate further the inhibitory effect of 19a in vivo, we measured pSTAT5 levels in the spleen of mice implanted with BaF3 TELJAK2 cells as an in vivo biomarker of Jak2 kinase inhibition as described previously.17 A single dose of 19a was given to tumor-bearing animals orally at 3, 5, and 10 mg/kg, and mice were sacrificed at different time points postdose. A single 10 mg/kg dose of 19a achieved almost complete inhibition of pSTAT5 levels at 6 h and about 50% inhibition at 12 h post dose. With a 3 mg/kg dose of 19a about 80% pSTAT5 inhibition was seen at 6 h post dose, while only 30% inhibition was detected at 8 h (Figure 10). This demonstrated a clear dose/time-dependent modulation of pSTAT5 levels in spleen lysates of tumor-bearing mice was achieved by compound 19a. 19a Showed Tumor Growth Inhibition in a UKE-1 Xenograft Model. Having established PD activity of 19a in vivo, we proceeded with efficacy testing in the UKE-1 xenograft mouse model. In a multidose tolerability study in naı̈ve NCr nude female mice,34 19a was tolerated at doses up to 10 mg/kg given orally twice daily (20 mg/kg/day) for 10 days.35 UKE-1 xenograft tumors were established in SCID female mice36 by subcutaneous implantation of UKE-1 cells mixed with matrigel. On day 15, animals were randomized into groups at an average tumor volume of ∼150 mm3. Mice were treated twice daily by oral gavage with either 3 or 10 mg/kg of 19a and vehicle for 14 days. We observed a significant dose-dependent tumor growth inhibition with 19a-treated animals (Figure 11). At the end of the dosing phase, a maximum tumor growth inhibition of 77% was achieved with a 10 mg/kg dose level twice daily. No significant body weight loss was observed in any of the study groups (Figure 11).

kinase binding site similarity and similarity in the gatekeeper residue, thus representing the diversity of the kinome. The selectivity profile of 19a (Jak2 IC50 < 3 nM) in this representative kinase panel is shown in Table 5.30 More than Table 5. Kinase Profiling of Lead Compound 19a compd 19a target

IC50 (μM)a (enzyme fold selectivity)b

JAK2(h) CK2α2(h) KDR(h) Abl(h) Fgr(h) JAK3(h) TrkA(h) Flt4(h) Flt3(h) Ret(h) ALK(h) FGFR1(h) Aurora-A(h) LIMK1(h) SIK(h) PKCζ(h)

10 min with a gradient mixture of H2O−acetonitrile with formic or trifluoroacetic acid at wavelengths of 220, 254, and 280 nm. All compounds analyzed were >95% pure. Reverse-phase chromatography was performed with Gilson systems using a YMC-AQC18 151



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Figure 11. 19a showed dose-dependent tumor growth inhibition in a UKE-1 xenograft model. 1-Methyl-1H-imidazol-4-amine Hydrochloride Salt (10). 1Methyl-4-nitro-1H-imidazole (9, 27 g) was dissolved in EtOH (800 mL), and Pd(OH)2 (2.5 g) was added. The mixture was subjected to an atmosphere of hydrogen for 3 h at room temperature. The mixture was filtered and the organic layer was concentrated to give 1-methyl1H-imidazol-4-amine. This amine was dissolved in EtOH (800 mL) and stirred at room temperature. A saturated solution of EtOH with HCl (g) (750 mL) was added. The mixture was stirred for 30 min, and the solution was concentrated under reduced pressure to 100 mL. The white solid formed. The solid was collected via filtration and washed with ether to give 1-methyl-1H-imidazol-4-amine hydrochloride salt (10, 28.4 g, 100%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.44 (1H, d), 6.59 (1H, d), 3.70 (3H, s). m/z: 98 [M + H]+. 2,4-Dichloro-6-methoxyquinazoline (11b). A solution of commercially available 6-methoxyquinazoline-2,4-diol (1.91 g, 9.94 mmol) and N,N-dimethylaniline (1.26 mL, 9.94 mmol) in POCl3 (13.90 mL, 149.09 mmol) was heated at reflux for 4 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure to give 2,4-dichloro-6-methoxyquinazoline (11b), which was used directly in the next step without any further purification. m/z: 230 [M + H]+. 2-Chloro-6-methoxy-N-(1-methyl-1H-imidazol-4-yl)-quinazolin-4-amine (12b). A solution of 1-methyl-1H-imidazol-4-amine hydrochloride salt (10, 1.9 g, 14.3 mmol) was added to a solution of 2,4-dichloro-6-methoxyquinazoline (11b, 2.3 g, 9.94 mmol) (12.2 mL) and DIPEA (8.7 mL, 49.70 mmol) in MeCN. The resulting mixture was stirred at 70 °C overnight. The reaction mixture was diluted with water and extracted with DCM/MeOH (3 × 100 mL, 10%). The organic layer was concentrated to a volume of 20 mL. White solid formed. 2-Chloro-6-methoxy-N-(1-methyl-1H-imidazol-4-yl)-quinazolin-4-amine 12b (710 mg, 26%) was collected after filtration as white fluffy solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 10.89 (1H, s), 8.16 (1H, d), 7.64 (1H, m), 7.55 (1H, s), 7.47 (1H, m), 3.93 (3H, s), 3.73 (3H, s). m/z: 291 [M + H]+. The following compounds were synthesized in a similar fashion to 12b. 2-Chloro-N-(1-methyl-1H-imidazol-4-yl)quinazolin-4-amine (12a). 1-Methyl-1H-imidazol-4-amine hydrochloride salt (10, 429 mg, 3.38 mmol) and 2,4-dichloroquinazoline (11a, 560 mg, 2.81 mmol) were reacted using the same procedure as the one described for the synthesis of 12b, providing 12a (530 mg, 72%), m/z 260 [M + H]+. 2-Chloro-7-methoxy-N-(1-methyl-1H-imidazol-4-yl)quinazolin-4-amine (12c). 1-Methyl-1H-imidazol-4-amine hydrochloride salt (10, 1.3 g, 10.6 mmol) and 2,4-dichloro-7-methox-

yquinazoline (11c, 1.62 g, 7.1 mmol) were reacted using the same procedure as the one described for the synthesis of 12b, providing 12c (0.71 g, 35%), m/z 290 [M + H]+. 2-Chloro-N-(1-methyl-1H-imidazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine (12d). 1-Methyl-1H-imidazol-4-amine hydrochloride salt (10, 167 mg, 1.72 mmol) and 2,4-dichloropyrido[3,4d]pyrimidine (11d, 500 mg, 2.50 mmol) were reacted using the same procedure as the one described for the synthesis of 12b, providing 12d (421 mg, 64%). 1H NMR (300 MHz, DMSO-d6) δ ppm 11.33 (s, 1 H), 9.16 (d, 1 H), 9.01 (s, 1 H), 7.54−7.72(m, 3H), 3.75 (s, 3 H). m/z 261 [M + H]+. 2-Chloro-6-fluoro-N-(1-methyl-1H-imidazol-4-yl)pyrido[2,3d]pyrimidin-4-amine (12e). 1-Methyl-1H-imidazol-4-amine hydrochloride salt (10, 0.770 g, 6.05 mmol) and 2,4-dichloro-6-fluoropyrido[2,3-d]pyrimidine (11e, 1.1 g, 5.1 mmol) were reacted using the same procedure as the one described for the synthesis of 12b, providing 12e (1.01 g, 72%), m/z 279 [M + H]+. 1H NMR (300 MHz, DMSO-d6) δ ppm 9.06−9.39 (m, 2 H), 7.49−7.81 (m, 2 H), 3.82 (s, 3 H). 2,7-Dichloro-N-(1-methyl-1H-imidazol-4-yl)pyrido[2,3-d]pyrimidin-4-amine (12f). 1-Methyl-1H-imidazol-4-amine hydrochloride salt (10, 261 mg, 2.06 mmol) and 2,4,7-trichloropyrido[2,3d]pyrimidine (402 mg, 1.71 mmol) were reacted using the same procedure as the one described for the synthesis of 12b, providing 12f (365 mg, 72.1%), m/z 295 [M + H]+. 2-Chloro-N-(1-methyl-1H-imidazol-4-yl)pteridin-4-amine (12g). 1-Methyl-1H-imidazol-4-amine hydrochloride salt (10, 400 mg, 3.15 mmol) and 2,4-dichloropteridine (11g, 633 mg, 3.15 mmol) were reacted using the same procedure as the one described for the synthesis of 12b, providing 12g (135 mg, 16%), m/z 262 [M + H]+. N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imidazol-4-yl)quinazoline-2,4-diamine, Trifluoroacetic Acid Salt (13a). 2-Chloro-N-(1-methyl-1H-imidazol-4-yl)quinazolin-4-amine (12a, 460 mg, 1.77 mmol), DIPEA (5 mmol), and (1S)-1-(5fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 378 mg, 2.13 mmol)21 in n-BuOH (4 mL) were heated at 150 °C under microwave irradiation for 6 h. The mixture was cooled at room temperature, and the volatiles were evaporated in vacuo. Residue was purified by reversed phase HPLC (Gilson chromatography, MeCN/0.1%TFA in H2O 5% → 50%) to give N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1methyl-1H-imidazol-4-yl)quinazoline-2,4-diamine, trifluoroacetic acid salt (13a, 527 mg, 60%) as a mixture of enantiomers. The racemic mixture were separated using chiral SFC using the following analytical instrument and conditions. Chiral SFC column, Chiralpak AD (21 mm × 250 mm, 5 μm); solvent condition, mobile 152



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the synthesis of 12a, providing 13b (390 mg, 63%) after purification by reversed phase HPLC (Gilson chromatography, MeCN/0.1%TFA in H2O 5% → 55%). 1H NMR (300 MHz, MeOD) δ ppm 8.77 (s, 2 H), 7.98 (d, 1 H), 7.87 (s, 1 H), 7.52 (d, 2 H), 5.37−5.59 (m, 1 H), 3.97 (s, 3 H), 3.93 (s, 3 H), 1.74 (d, 3 H). m/z: 395 [M + H]+. N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-7-methoxy-N4-(1-methyl-1H-imidazol-4-yl)quinazoline-2,4-diamine, Trifluoroacetic Acid Salt (13c). 2-Chloro-7-methoxy-N-(1-methyl-1H-imidazol-4yl)quinazolin-4-amine (12c, 383 mg, 1.32 mmol) and (1S)-1-(5fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 468 mg, 2.64 mmol) were reacted using the same procedure as the one described for the synthesis of 13a, providing 13c (160 mg, 24%) as a mixture of enantiomers after purification utilizing reversed phase HPLC: Column, Waters XBridge C18 100 mm × 19 mm, particle size 5 um; mobile phase, 0.1% NH4OH in water/MeCN; gradient, 10−60% acetonitrile in 10 min. The racemic mixture was separated using chiral SFC. Chiral SFC column, Chiralpak AD (21 mm × 250 mm, 5 μm); solvent condition, mobile phase A, carbon dioxide 65%; mobile phase B, 1:1 methanol:ethanol; additive, 0.4% diethylamine 35%; flow rate (mL/ min), 60; detection (nm), 254; temperature (°C), 40; outlet pressure (bar), 100. Enantiomeric excess (ee) of 13c (retention time 4.46 min) was >98% determined by the following analytical instrument and conditions. Chiral SFC column: Chiralpak AD (column dimensions: 4.6 mm × 250 mm, 5 μm); mobile phase, 60:44:0.4% carbon dioxide:methanol:dimethylethylamine; flow rate (mL/min), 2.5; detection, 254 nm. 1H NMR (300 MHz, MeOD) δ ppm 8.71 (s, 2 H), 7.85−8.00 (m, 1 H), 7.59 (br s, 1 H), 7.44 (d, 1H), 6.80 (dq, 2 H), 5.48 (q, 1 H), 3.89 (s, 3 H), 3.82 (s, 3 H), 1.64 (d, 3H). m/z: 394 [M + H]+. N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imidazol-4-yl)pyrido[3,4-d]pyrimidine-2,4-diamine, Trifluoroacetic Acid Salt (13d). 2-Chloro-N-(1-methyl-1H-imidazol-4-yl)pyrido[3,4-d]pyrimidin-4-amine (12d, 404 mg, 1.55 mmol) and (1S)-1-(5Fluoropyrimidin-2-yl)ethanamine hydrochloride (14) were reacted using the same procedure as the one described for the synthesis of 13a, providing 13d (209 mg, 28%) as a mixture of enantiomers, after purification by reversed phase HPLC (Gilson chromatography, MeCN/0.1%TFA in water 5% → 40%). The racemic mixture were separated using Chiral HPLC. Chiral SFC column: Chiralpak AD (20 mm × 250 mm, 10 μm); solvent condition, 1:1 methanol:ethanol; additive, 0.1% diethylamine 35%; flow rate (mL/min), 20; detection 220 (nm). Enantiomeric excess (ee) of 13d (retention time 7.48 min) was >98% determined by the following chiral HPLC instrument and conditions: Chiral HPLC column, Chiralpak AD (4.6 mm × 250 mm, 10 μm); mobile phase, 1:1:1 methanol:ethanol:0.1% dimethylethylamine; flow rate (mL/min), 1; detection, 254 nm. 1H NMR (300 MHz, MeOD) δ ppm 8.57 (s, 1 H), 8.27 (d, 1 H), 8.08 (d, 1 H), 7.83 (d, 1 H), 7.43−7.63 (m, 2 H), 7.27 (br s, 1 H), 5.61 (q, 1 H), 3.60 (br s, 3 H), 1.51 (d, J = 6.97 Hz, 3H). m/z: 367.0 [M + H]+. 6-Fluoro-N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl1H-imidazol-4-yl)pyrido[2,3-d]pyrimidine-2,4-diamine, Trifluoroacetic Acid Salt (13e). 2-Chloro-6-fluoro-N-(1-methyl-1Himidazol-4-yl)pyrido[2,3-d]pyrimidin-4-amine (12e, 90 mg, 0.32 mmol) and (1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14) were reacted using a procedure similar to the one described for the synthesis of 13a, providing 13e (139 mg, 86%) as a mixture of enantiomers, after purification by reversed phase HPLC (Gilson chromatography, MeCN/0.1%TFA in water 5%→30%). The racemic mixture were separated using chiral HPLC. Chiral HPLC: Chiralpak IC (20 mm × 250 mm, 5 μm); mobile phase A, hexane 70%, mobile phase B, 1:1 methanol:ethanol; additive, 0.1% diethylamine; flow rate (mL/min), 20; detection (nm), 220. Enantiomeric excess (ee) of 13e (retention time 16.02 min) was >98% determined by the following chiral HPLC instrument and conditions. Chiral HPLC: Chiralpak IC (4.6 mm × 250 mm, 5 μm); mobile phase, 1:1 methanol:ethanol 0.1% dimethylethylamine; flow rate (mL/min), 1; detection, 254 nm. 1H NMR (300 MHz, DMSO-d6) δ ppm 10.26 (br s, 1 H), 8.79 (s, 2 H), 8.70 (d, 1 H), 8.55 (br s, 1 H), 7.59 (d, 1 H), 7.41 (s, 1 H), 5.17−5.50 (m, 1 H), 3.65 (s, 3H), 1.50 (d, 3 H). m/z: 394 [M + H]+.

Table 7. Data Processing and Refinement Statistics for X-ray Crystallographic Data Collected for Structures of Jak2 in Complex with Compounds 23b and 28a 23b

28

PDB code space group cell constants: a, b, c (Å), b (deg) resolution range (Å)

4c61 C2 43.9, 127.1, 135.2, 97.3

completeness overall (%) total no. of observations (unique reflns) multiplicity Rmerge overallb I/σIc Roverall value (%)d Rfree value (%)e non-hydrogen protein atoms non-hydrogen ligand atoms solvent molecules rms deviations from ideal values bond lengths (Å) bond angles (deg) average B values (Å2) protein main chain atoms protein all atoms ligand solvent Φ, Ψ angle distribution for residuesf in most favored regions (%) in additional allowed regions (%) in generously regions (%) in disallowed regions (%)

96.2(96.4) 109283(25971)

4c62 C2 44.5, 126.9, 135.9, 97.5 21.2−2.75(2.90− 2.75) 99.4(98.7) 315953(19353)

4.2(4.3) 0.07(0.30) 13.4(2.5) 18.1 21.7 4334

4(3.7) 0.10(0.50) 11.6(2.1) 19.7 24.6 4356

54 199

58 117

0.01 1.12

0.01 1.7

54.4

50.5

56.9 39.4 51.5

51.7 34.6 36

92.4

89.9

7.2

9.7

0.4

0.4

0

0

134.1−2.45(2.58−2.45)

a

Numbers in parentheses represent data collection statistics for the highest resolution shell unless marked otherwise. bRmerge = ∑hkl [(∑i | Ii − ⟨I⟩|)/∑i Ii]. cI/σI average is the mean I/σ for the unique reflections in the output file. dRvalue = ∑hkl||Fobs| − |Fcalc||/∑hkl|Fobs. e Rfree is the cross-validation R factor computed for the test set of 5% of unique reflections. fRamachandran statistics as defined by PROCHECK. phase A, carbon dioxide 75%; mobile phase B, 1:1 methanol:ethanol; additive, 0.4% diethylamine 25%; flow rate (mL/min), 60; detection (nm), 254; temperature (°C), 40; outlet pressure (bar), 100. Enantiomeric excess (ee) of 13a (retention time 3.86 min) was >98% determined by the following analytical instrument and conditions. Chiral SFC column: Chiralpak AD (4.6 mm × 100 mm, 5 μm); mobile phase A, carbon dioxide 80%; mobile phase B, 1:1 methanol:ethanol; additive, 0.4% diethylamine 20%; flow rate (mL/ min), 5; detection (nm), 220; temperature (°C), 35; outlet pressure (bar), 120. 1H NMR (300 MHz, MeOD) δ ppm 8.66 (s, 2 H), 8.21 (d, 2 H), 8.00 (br s, 1 H), 7.79 (td, 1 H), 7.47 (m, 2 H), 5.38 (q, 1 H), 3.84 (s, 3 H), 1.61 (d, 3 H). m/z: 383 [M + H]+. N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-6-methoxy-N4-(1-methyl-1H-imidazol-4-yl)quinazoline-2,4-diamine, Trifluoroacetic Acid Salt (13b). 2-Chloro-6-methoxy-N-(1-methyl-1H-imidazol-4yl)quinazolin-4-amine (12b, 350 mg, 1.21 mmol) and (1S)-1-(5fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 428 mg, 2.42 mmol) were reacted using the same procedure as the one described for 153



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7-Chloro-N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl1H-imidazol-4-yl)pyrido[2,3-d]pyrimidine-2,4-diamine, Trifluoroacetic Acid Salt (13f). 2,7-Dichloro-N-(1-methyl-1H-imidazol-4-yl)pyrido[2,3-d]pyrimidin-4-amine (12f, 385 mg, 1.30 mmol) and (1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 323 mg, 1.30 mmol) were reacted using the same procedure as the one described for the synthesis of 13a, providing 13f (181 mg, 27%) as a mixture of enantiomers, after purification by reversed phase HPLC (Gilson chromatography, MeCN/0.1%TFA in water 5% → 35%).1H NMR (300 MHz, MeOD) δ ppm 8.67 (s, 2 H), 8.57 (d, 1 H), 7.90 (s, 1 H), 7.45−7.59 (m, 1 H), 7.39 (d, 1 H), 5.40 (q, 1 H), 3.82 (s, 3 H), 1.62 (d, 3 H). m/z: 400 [M + H]+. N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imidazol-4-yl)pteridine-2,4-diamine (13g). 2-Chloro-N-(1-methyl-1Himidazol-4-yl)pteridin-4-amine (12g, 135 mg, 0.52 mmol) and (1S)-1(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 183 mg, 1.03 mmol) were suspended in butan-1-ol (2 mL), and DIPEA (0.360 mL, 2.06 mmol) was added. The reaction was irradiated in a microwave at 150 °C for 1 h. The reaction mixture was concentrated in vacuo, leaving an amber oil (423 mg). This material was purified by ISCO (2−10% MeOH/DCM). Concentration of the fractions in vacuo provided 13g as a mixture of enantiomers as a yellow solid (72 mg, 29%). The racemic mixture were separated using chiral SFC. Chiral SFC: Chiralpak AD (21 mm × 250 mm, 5 μm); mobile phase, 20% methanol with 0.4% dimethylethylamine; flow rate (mL/min), 40; detection (nm), 254; outlet pressure (bar), 100. Enantiomeric excess (ee) of 13g (retention time 15.52 min) was >98% determined by the following chiral SFC instrument and conditions. Chiral SFC: Chiralpak AD (4.6 mm × 250 mm, 5 μm); mobile phase: 20% methanol with 0.4% dimethylethylamine; flow rate (mL/min), 2.5; detection, 254 nm; pressure (bar), 120. 1H NMR (300 MHz, MeOD) δ ppm 8.49−8.88 (m, 3 H), 8.37 (d, 1 H), 7.88 (br s, 0.5 H), 7.56 (br s, 0.5 H), 7.46 (d, 1 H), 5.37−5.70 (m, 1 H), 3.81 (d, 3 H), 1.67 (d, 3 H). m/z: 367 [M + H]+. 2,4-Dichloro-7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3d]pyrimidine (17a). 2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (15a, 1.00 g, 5.32 mmol), 4-methylbenzene-1-sulfonyl chloride (1.115 g, 5.85 mmol), and tetra-butylammonium hydrogen sulfate (0.090 g, 0.27 mmol) were dissolved in DCM (20 mL) at room temperature, and NaOH (50% aq, 1 mL) was added. The reaction mixture stirred at room temperature for 30 min. After completion of the reaction as indicated by TLC, the reaction mixture was diluted with H2O and DCM. The organic layer was evaporated in vacuo to obtain a lightyellow solid, which was purified by column chromatography (100% DCM) to provide 17a (1.76 g, 97%) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 8.14 (d, 2 H), 7.78 (d, 1 H), 7.39 (d, 2 H), 6.70 (d, 1 H), 2.45 (s, 3 H). m/z: 342 [M + H]+. 2,4-Dichloro-7-methyl-7H-pyrrolo[2,3-d]pyrimidine (17b). 2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (15a, 2.37 g, 12.61 mmol) was dissolved in acetonitrile (8.32 mL), and sodium hydride (529 mg, 13.24 mmol) was added portionwise. The reaction mixture was stirred at room temperature for 0.5 h until gas evolution ceased. Methyl iodide (0.87 mL, 13.87 mmol) was added, and the resulting mixture was stirred for 0.5 h. The reaction mixture was then poured into water and extracted with DCM/MeOH. Concentration of the organic layers under reduced pressure provided a residue, which was purified utilizing ISCO (0% → 100% DCM/EtOAc) to afford 17b (2.1g, 84%). 1H NMR (300 MHz, DMSO-d6) δ ppm 7.75 (s, 1 H), 6.71 (s, 1 H), 3.82 (s, 3 H). m/z: 204 [M + H]+. 2,4-Dichloro-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidine (17c). 2,4-Dichloro-7H-pyrrolo[2,3-d]pyrimidine (15a, 1 g, 5.32 mmol), copper(II) acetate (1.45 g, 7.98 mmol), pyridine (2.15 mL, 26.59 mmol), and cyclopropylboronic acid (1.1 g, 13.30 mmol) were heated at 90 °C under dry air for 36 h. The reaction mixture was concentrated under reduced pressure, and the residue was partitioned between EtOAc and water. The organic layer was collected, dried, and concentrated under reduced pressure to provide a crude mixture, which was purified utilizing ISCO (0% → 30% hexanes/EtOAc) to afford 17c (270 mg, 22%). 1H NMR (300 MHz, MeOD) δ ppm 7.53

(d, J = 3.77 Hz, 1 H), 6.62 (d, J = 3.77 Hz, 1 H), 3.49−3.67 (m, 1 H), 1.01−1.31 (m, 4 H). m/z: 230 [M + H]+. 2,6-Dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4d]pyrimidine (17d). To a solution of 4,6-dichloro-1H-pyrazolo[3,4d]pyrimidine (15b, 2 g, 10.58 mmol) and p-Ts-OH (0.201 g, 1.06 mmol) in DCM (30 mL) and THF (30.0 mL) was added 3,4-dihydro2H-pyran (1.335 g, 15.87 mmol). The resulting solution was stirred overnight at ambient temperature, whereupon the volatiles were removed under reduced pressure. The residue was dissolved in DCM, and the organic layer was washed with saturated aqueous sodium carbonate solution, water, brine, and dried MgSO4. Evaporation of the volatiles under reduced pressure gave 17d (2.80g, 100%). m/z: 273 [M + H]+. 2-Chloro-N-(1-methyl-1H-imidazol-4-yl)-7-[(4methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (18a). 1-Methyl-4-nitro-1H-imidazole hydrochloride salt (10, 50 mg, 0.39 mmol) was added to a solution of 4-dichloro-7-[(4methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidine (17a, 108 mg, 0.31 mmol) and TEA (0.11 mL, 0.79 mmol) in CH3CN. The reaction mixture was stirred at 100 °C in a microwave reactor for 2 h. After completion of the reaction as indicated by TLC, the reaction mixture was evaporated in vacuo to obtain a light-yellow solid which was purified by column chromatography (3% MeOH, 0.3% NH4OH in DCM) to provide 18a (90 mg, 72%) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 8.92 (s, 1 H), 8.03 (d, 2 H), 7.41 (s, 1 H), 7.39 (d, 1 H), 7.25 (d, 2H), 6.48 (s, 1 H), 3.67 (s, 3 H), 2.33 (s, 3 H). m/z: 403 [M + H]+. The following compounds were synthesized in a similar fashion to 18a. 2-Chloro-7-methyl-N-(1-methyl-1H-imidazol-4-yl)-7Hpyrrolo[2,3-d]pyrimidin-4-amine, Trifluoroacetic Acid Salt (18b). 1-Methyl-4-nitro-1H-imidazole hydrochloride salt (10, 1.38 g) was added to a solution of 2,4-dichloro-7-methyl-7H-pyrrolo[2,3d]pyrimidine (17b, 500 mg) and DIPEA (0.92 mL, 5.32 mmol). The resulting mixture was stirred at 90 °C for 15 h. The reaction mixture was diluted with water and extracted with DCM/MeOH (10%). Evaporation of the volatiles under reduced pressure gave a residue which was purified by reverse phase HPLC (Gilson chromatography, MeCN/0.1%TFA in water 5%→45%) to provide 18b (300 mg, 21%). m/z: 265 [M + H]+. 2-Chloro-7-cyclopropyl-N-(1-methyl-1H-imidazol-4-yl)-7Hpyrrolo[2,3-d]pyrimidin-4-amine, Trifluoroacetic Acid Salt (18c). 2,4-Dichloro-7-cyclopropyl-7H-pyrrolo[2,3-d]pyrimidine (17c, 270 mg, 1.18 mmol) and 1-methyl-1H-imidazol-4-amine hydrochloride (10, 604 mg, 3.55 mmol) were reacted using a procedure similar to the one described for the synthesis of 18a, providing 18c (200 mg, 42%) after purification by reverse phase HPLC (Gilson chromatography, MeCN/0.1%TFA in water 0%→50%).m/z: 291 [M + H]+ . 6-Chloro-N-(1-methyl-1H-imidazol-4-yl)-1-(tetrahydro-2Hpyran-2-yl)-1H pyrazolo[3,4-d]pyrimidin-4-amine (18d). To a solution of 4,6-dichloro-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-d]pyrimidine (17d, 3.17 g, 11.60 mmol) in ethanol (60 mL) was added TEA (4.04 mL, 29.00 mmol) followed by 1-methyl-1Himidazol-4-amine hydrochloride (10, 1.549 g, 11.60 mmol). The resulting mixture was heated at 60 °C for 2 h. Evaporation of the volatiles under reduced pressure gave a residue which was purified utilizing ISCO (EtOAc/hexanes 0→80%) to give 18d (1.56 g, 41%). m/z: 334 [M + H]+. 6-Chloro-1-methyl-N-(1-methyl-1H-imidazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-4-amine (18e). Commercially available 4,6-dichloro-1-methyl-1H-pyrazolo[3,4-d]pyrimidine (17e, 4.49 g, 22.12 mmol) and 1-methyl-1H-imidazol-4-amine hydrochloride (10, 2.96 g, 22.12 mmol) were suspended in ethanol (104 mL), and TEA (6.17 mL, 44.25 mmol) was added. The reaction mixture was heated at 70 °C overnight. The reaction mixture was cooled to 0 °C. Solid formed and was collected via filtration to provide 18e as a purple−gray solid (2.94 g, 50%). 1H NMR (300 MHz, MeOD) δ ppm 8.69 (s, 2 H) 7.85 (br s, 1 H) 7.53 (br s, 1 H) 7.42 (s, 1 H) 5.42 (q, 1 H) 3.65−3.89 (m, 6 H) 1.61 (d, 3 H). m/z: 264 [M + H]+. N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imidazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (19a). In a 154



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microwave tube, 2-chloro-N-(1-methyl-1H-imidazol-4-yl)-7-[(4methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (18a, 90 mg), (1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 881 mg, 4.96 mmol), and DIPEA (1.084 mL, 6.21 mmol) were dissolved in n-BuOH (5 mL). The reaction mixture was heated in a microwave reactor at 150 °C for 3 h. After completion of the reaction as indicated by LCMS, the reaction mixture was evaporated in vacuo to obtain a brown residue. This residue was purified by column chromatography (4% MeOH, 0.4% NH4OH in DCM) to provide the N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imidazol-4-yl)7-[(4 methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (350 mg, 56%) as a mixture of enantiomers as a yellow solid. m/z: 508 [M + H]+. 1H NMR (400 MHz, DMSO-d6) δ ppm 9.35 (s, 1H), 8.79 (s, 2H), 7.91−7.97 (m, 2H), 7.46 (s, 1H), 7.33 (t, 3H), 7.08 (d, 1H), 6.90 (d, 1H), 6.67 (d, 1H), 5.28−5.35 (m, 1H), 3.67 (s, 3H), 2.36 (s, 3H), 1.56 (d, 3H). A solution of N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl1H-imidazol-4-yl)-7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (7.61 g, 15 mmol) and NaOH (12g, 300.00 mmol) in water (10 mL), methanol (10 mL), and 1,4-dioxane (52 mL) was heated at 55 °C overnight. The reaction mixture was acidified with HCl to pH = 3 and washed with DCM. The aqueous layer was neutralized with NaHCO3 to pH = 8 and extracted with DCM/MeOH (10%). The organic layer was concentrated under reduced pressure to give a residue. This residue was purified utilizing ISCO (0%→80% DCM/acetone/2% NH4OH) to provide 19a (3.91 g, 74%) as a mixture of enantiomers. The racemic mixture was separated using chiral HPLC. Chiral HPLC: Chiralpak AD (50 mm × 250 mm, 5 μm); mobile phase, 1:1:0.1% methanol:ethanol:diethylamine; flow rate (mL/min), 120; detection (nm), 220. Enantiomeric excess (ee) of 19a (retention time 3.21 min) was >98% determined by the following chiral HPLC instrument and conditions. Chiral HPLC: Chiralpak AD (4.6 mm × 250 mm, 5 μm); mobile phase, 60:40:0.4% carbon dioxide:methanol; flow rate (mL/min), 5; detection, 220 nm. 1H NMR (300 MHz, MeOD) δ ppm 8.68 (s, 2 H), 7.47 (d, 1 H), 7.39 (d, 1 H), 6.74 (d, 1 H), 6.37 (d, 1 H), 5.39 (q, 1 H), 3.80 (s, 3H), 1.59 (d, 4 H). m/z: 354 [M + H]+. N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-7-methyl-N4-(1-methyl1H-imidazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine, Trifluoroacetic Acid Salt (19b). (1S)-1-(5-Fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 303 mg, 1.71 mmol) and 2-chloro-7methyl-N-(1-methyl-1H-imidazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4amine, trifluoroacetic acid salt (18b, 300 mg, 1.14 mmol), were reacted using a procedure similar to the one described for the synthesis of 19a, providing 19b (211 mg, 38%) as a mixture of enantiomers, after purification by reverse phase HPLC (Gilson chromatography, MeCN/ 0.1%TFA in water 5%→45%). The racemic mixture were separated using chiral SFC. Chiral SFC: Chiralpak AD (21 × 250 mm, 5 μm); mobile phase, 65:35:0.4% carbon dioxide:methanol; flow rate (mL/ min), 60; detection (nm), 254. Enantiomeric excess (ee) of 19b (retention time 4.33 min) was >98% determined by the following chiral HPLC instrument and conditions. Chiral HPLC: Chiralpak AD (4.6 × 250 mm, 5 μm); mobile phase, 60:40:0.4% carbon dioxide:methanol; flow rate (mL/min), 2.5; detection, 254 nm. 1H NMR (300 MHz, MeOD) δ ppm 8.58 (s, 2 H), 7.36 (d, 1 H), 7.28 (d, 1 H), 6.58 (d, 1 H), 6.26 (d, 1 H), 5.30 (q, 1 H), 3.68 (s, 3 H), 3.47 (s, 3 H), 1.50 (d, 3 H). m/z: 368 [M + H]+. 7-Cyclopropyl-N2-[1-(5-fluoropyrimidin-2-yl)ethyl]-N4-(1methyl-1H-imidazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine, Trifluoroacetic Acid Salt (19c). (1S)-1-(5-Fluoropyrimidin2-yl)ethanamine hydrochloride (14, 252 mg, 1.42 mmol) and 2chloro-7-cyclopropyl-N-(1-methyl-1H-imidazol-4-yl)-7H-pyrrolo[2,3d]pyrimidin-4-amine, trifluoroacetic acid salt (18c, 205 mg, 0.71 mmol), were reacted using a procedure similar to the one described for the synthesis of 19a, providing 19c (40 mg, 14%) as a mixture of enantiomers after purification by reverse phase HPLC (Gilson chromatography, MeCN/0.1%TFA in water 5%→45%). The racemic mixture were separated using chiral SFC. Chiral SFC: Chiralpak AD (21 × 250 mm, 5 μm); mobile phase, 75:25:0.4% carbon dioxide:methanol; flow rate (mL/min), 60; detection (nm), 254.

Enantiomeric excess (ee) of 19c (retention time 3.44 min) was >98% determined by the following chiral SFC instrument and conditions. Chiral SFC: Chiralpak AD (4.6 × 100 mm, 5 μm); mobile phase, 60:40:0.4% carbon dioxide:methanol; flow rate (mL/min), 5; detection, 254 nm. 1H NMR (300 MHz, MeOD) δ ppm 8.70 (s, 2 H), 7.47 (d, 1 H), 7.39 (d, 1 H), 6.70 (d, 1 H), 6.33 (d, 1 H), 5.27− 5.52 (m, 1 H), 3.80 (s, 3 H), 3.17−3.29 (m, 1 H), 1.62 (d, 3 H), 0.74− 1.07 (m, 4 H). m/z: 394 [M + H]+. N6-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imidazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (19d). 6Chloro-N-(1-methyl-1H-imidazol-4-yl)-1-(tetrahydro-2H-pyran-2-yl)1H-pyrazolo[3,4-d]pyrimidin-4-amine (18d, 158 mg, 0.47 mmol) and (1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 84 mg, 0.47 mmol) were dissolved in butan-1-ol (2.5 mL), followed by the addition of triethylamine (0.165 mL, 1.18 mmol). The reaction mixture was heated under microwave irradiation at 160 °C for 6 h. LCMS analysis indicated that the protecting group was cleaved under the employed conditions. The volatiles were evaporated under reduced pressure, and the residue left was purified to give 19d (14.2 mg, 8%) as a mixture of enantiomers. 1H NMR (300 MHz, MeOD) δ ppm 8.70 (s, 2 H), 7.87 (br s, 1 H), 7.55 (br s, 1 H), 7.56 (br s, 1H), 5.40 (q, 1 H), 3.81 (s, 3 H), 1.62 (d, 3 H). m/z: 355 [M + H]+. N6-[1-(5-Fluoropyrimidin-2-yl)ethyl]-1-methyl-N4-(1-methyl1H-imidazol-4-yl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (19e). 6-Chloro-1-methyl-N-(1-methyl-1H-imidazol-4-yl)-1Hpyrazolo[3,4-d]pyrimidin-4-amine (18e, 2 g, 7.58 mmol) and (1S)-1(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 1.482 g, 8.34 mmol) were suspended in butan-1-ol (21.05 mL), and TEA (4.23 mL, 30.34 mmol) was added. The reaction mixture was subjected to microwave irradiation at 150 °C for 3 h. The reaction mixture was filtered, and the filtrate was concentrated in vacuo, leaving a brown semisolid (3.504 g). This material was purified by ISCO (5% MeOH/ DCM, isocratic) to provide 19e as a mixture of enantiomers in the form of a yellow solid (1.58 g, 57%). The racemic mixture were separated using chiral HPLC. Chiral HPLC: Chiralpak AD (50 mm × 500 mm, 20 μm); mobile phase, 100% methanol; flow rate (mL/min), 120; detection (nm), 220. Enantiomeric excess (ee) of 19e (retention time 2.30 min) was >98% determined by the following chiral SFC instrument and conditions. Chiral SFC: Chiralpak AD (4.6 mm × 100 mm, 5 μm); mobile phase, 40% methanol with 0.1% dimethylethylamine; flow rate (mL/min), 5; detection, 220 nm. 1H NMR (300 MHz, MeOD) δ ppm 8.69 (s, 2 H), 7.85 (br s, 1 H), 7.53 (br s, 1 H), 7.42 (s, 1 H), 5.42 (q, 1 H), 3.65−3.89 (m, 6 H), 1.61 (d, 3 H). m/z: 369 [M + H]+. 2,4-Dichloro-5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[3,2d]pyrimidine (21). Commercially available 2,4-dichloro-5H-pyrrolo[3,2-d]pyrimidine (20a, 500 mg, 2.66 mmol) and 4-methylbenzene-1sulfonyl chloride (558 mg, 2.93 mmol) were reacted using a procedure similar to the one described for the synthesis of 17a, providing 21 (900 mg, 100%). 1H NMR (400 MHz, CDCl3) δ ppm 8.34 (d, 1 H), 7.75 (d, 2 H), 7.34 (d, 2 H), 6.87 (d, 1H), 2.44 (s, 3 H). m/z: 342 [M + H]+. 2-Chloro-7-methyl-N-(1-methyl-1H-imidazol-4-yl)thieno[3,2d]pyrimidin-4-amine (22a). 1-Methyl-1H-imidazol-4-amine hydrochloride salt (10, 194 mg, 2 mmol) and 2,4-dichloro-7-methylthieno[3,2-d]pyrimidine (20a, 438 mg, 2 mmol) were reacted using a procedure similar to the one described for the synthesis of 18a, providing 22a (294 mg, 52%). 1H NMR (300 MHz, MeOD) δ ppm 7.85 (s, 1H), 7.53 (s, 1H), 7.40 (s, 1 H), 3.71 (s, 3 H), 2.30 (s, 3H). m/z: 280 [M + H]+. 2-Chloro-N-(1-methyl-1H-imidazol-4-yl)-5-[(4methylphenyl)sulfonyl]-5H-pyrrolo[3,2-d]pyrimidin-4-amine (22b). 2,4-Dichloro-5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[3,2-d]pyrimidine (21, 240 mg, 0.70 mmol) and 1-methyl-1H-imidazol-4amine (10, 1.5 equiv) were reacted using a procedure analogous to that described for the synthesis of 18a, providing the title product 22b (90 mg, 32%). 1H NMR (400 MHz, CDCl3) δ ppm 9.92 (s, 1 H), 7.71 (d, 1 H), 7.61 (d, 2 H), 7.44 (br.s., 1 H), 7.24 (s, 1H), 7.16 (d, 2 H), 6.62 (d, 1 H), 3.68 (s, 3 H), 2.28 (s, 3 H). m/z: 403 [M + H]+. N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-7-methyl-N4-(1-methyl1H-imidazol-4-yl)thieno[3,2-d]pyrimidine-2,4-diamine, Tri155



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fluoroacetic Acid Salt (23a). 2-Chloro-7-methyl-N-(1-methyl-1Himidazol-4-yl)thieno[3,2-d]pyrimidin-4-amine (22a, 276 mg, 0.99 mmol) and (1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 175 mg, 0.99 mmol) were reacted using a procedure similar to the one described for the synthesis of 19a, providing 23a as a mixture of enantiomers, in the form of a yellow solid (126 mg, 25%). 1H NMR (300 MHz, MeOD) δ ppm 8.79 (s, 2 H), 7.97 (bs, 1H), 7.86 (s, 1H), 7.52 (s, 1H), 5.46 (q, 1 H), 3.91 (s, 3 H), 2.40 (s, 3H), 1.70 (d, 3 H). m/z: 385 [M + H]+. N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imidazol-4-yl)-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (23b). 2Chloro-N-(1-methyl-1H-imidazol-4-yl)-5-[(4-methylphenyl)sulfonyl]5H-pyrrolo[3,2-d]pyrimidin-4-amine (22b, 65 mg, 0.16 mmol) and (1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 114 mg, 0.64 mmol) were reacted using a procedure similar to the one described for the synthesis of 19a, providing N2-[1-(5-fluoropyrimidin2-yl)ethyl]-N4 -(1-methyl-1H-imidazol-4-yl)-5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (25 mg) as a mixture of enantiomers. 1H NMR (400 MHz, MeOD) δ ppm 8.60 (s, 1 H), 8.54 (s, 2 H), 7.62 (d, 1 H), 7.54 (d, 2 H), 7.34 (s, 1 H), 7.14 (d, 2H), 6.33 (d, 1 H), 5.21 (q, 1 H), 3.69 (s, 3 H), 2.18 (s, 3 H), 1.46 (d, 3 H). m/z: 508 [M + H]+. N2-[1-(5-Fluoropyrimidin-2-yl)ethyl]-N4-(1-methyl-1H-imidazol-4yl)-5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (25 mg, 0.05 mmol) was reacted using a procedure similar to the one described for the synthesis of 19a, providing 23b (13 mg, 23% for 2 steps) as a mixture of enantiomers. 1H NMR (400 MHz, MeOD) δ ppm 8.59 (s, 2 H), 7.39 (s, 1 H), 7.31 (s, 1 H), 7.21 (d, 1 H), 6.09 (d, 1 H), 5.29 (q, 1H), 3.70 (s, 3 H), 1.52 (d, 3 H). m/z: 354 [M + H]+. 5-Chloro-N2-(1-(5-fluoropyrimidin-2-yl)ethyl)-N4-(1-methyl1H-imidazol-4-yl)pyrimidine-2,4-diamine (26). A mixture of 1methyl-1H-imidazol-4-amine hydrochloride salt (10, 500 mg) was added to a solution of 2,4,5-trichloro-pyrimidine (0.36 mL) in MeCN (12.2 mL) and TEA (1.10 mL, 7.87 mmol) and the resulting mixture stirred at room temperature overnight. 2,5-Dichloro-N-(1-methyl-1Himidazol-4-yl)pyrimidin-4-amine (710 mg, 39%) was isolated as a white fluffy solid after filtration. m/z: 236 [M + H]+. In a microwave vessel, a suspension of 2,5-dichloro-N-(1-methyl-1H-imidazol-4-yl)pyrimidin-4-amine (100 mg, 0.41 mmol) and (1S)-1-(5-fluoropyrimidin-2-yl)ethanamine hydrochloride (14, 91 mg, 0.51 mmol) in butan-1-ol was treated with DIPEA (0.29 mL, 1.6 mmol). The mixture was subjected to microwave irradiation at 150 °C for 5 h. The mixture was concentrated and purified using an ISCO system (2−10% MeOH/DCM) to provide the desired compound 26 (100 mg, 70%). 1 H NMR (300 MHz, MeOD) δ ppm 8.70 (s, 2 H), 7.83 (s, 1 H), 7.40 (s, 1 H), 5.25 (q, 1H), 3.78 (s, 3 H), 1.58 (d, 3 H). m/z: 349 [M + H]+. Computational Chemistry. The structure of 24 bound to Jak2 was used for all docking studies (ref 17, PDB code 2XA4). The structure was prepared for docking using the Schrödinger protein preparation utilities prep and impref, applying the appropriate sidechain protonation states, refine, and structure minimization. Docking grids were generated and defined based on the centroid of 24 in the ATP binding site incorporating hydrogen-bonding constraints to the hinge. Ligands were prepared using proprietary in-house software. Ligand receptor docking was carried out using XP-Glide26 and constrained to satisfy all three of the hydrogen bonds to hinge illustrated in Figure 4. The top five poses generated by Glide were retained and used to compute estimates of the ΔG of binding and ligand strain energies via the MM-GBSA.27 Protein Expression, Purification, Crystallization, and Structure Determination. Protein expression and purification, crystallization, and cryoprotection procedures have been described previously.17 Pictures of the crystal structures of Jak2 kinase with compounds have been generated by PYMOL.37 Diffraction data for complexes of JAK2 with compounds 23b and 28 were collected ”inhouse” using a Rigaku MM007 X-ray generator equipped with a MAR345 image plate X-ray detector, using Cu Kα radiation at a wavelength of 1.5418 Å focused using Rigaku VariMax HF mirrors.

Data were processed using MOSFLM, SCALA, and reduced using CCP4 software.38 The structures were solved by molecular replacement using coordinates of the JAK2 kinase domain (PDB entry 2XA4) as a trial model using CCP4 software. Protein and inhibitor were modeled into the electron density using COOT.39 The models were refined using Refmac40 and BUSTER.41 Atomic coordinates and structure factors for the Human JAK2 complex42,43 with compound 23b and 28 have been deposited in the Protein Data Bank (4c61 and 4c62, respectively), together with structure factors and detailed experimental conditions (Table 7).

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AUTHOR INFORMATION

Corresponding Author

*Phone: +1 781 839 4683. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Nancy DeGrace and Kanayochukwu Azogu for chiral separations work. Special thanks to Melissa Vasbinder, Andrew Mortlock, James Dowling, and Neil Grimster for valuable discussions during the preparation of the manuscript.

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ABBREVIATIONS USED DIAD, diisopropylazodicarboxylate; DHP, dihydropyran; DIPEA, diisopropylethyl amine; D5W, 5% dextrose (w/v) in water; ET, essential thrombocythemia; HPMC, hydroxypropylmethylcellulose; Jak2, Janus kinase 2; Jak1, Janus kinase 1; Jak3, Janus kinase 3; MF, idiopathic myelofibrosis; MM-GBSA, molecular mechanics-generalized Born solvation approximation; mpk, mg/kg; MPNs, myeloproliferative neoplasms; PV, polycythemia vera; STAT, signal transducers and activators of transcription; p-Ts-OH, p-toluenesulfonic acid; pSTAT, phosphorylated signal transducers and activators of transcription; SCID, severe combined immunodeficiency; tSTAT, total signal transducers and activators of transcription; TGI, tumor growth inhibition; Tyk2, tyrosine kinase 2

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REFERENCES

(1) Quintas-Cardama, A.; Vertovsek, S. New JAK2 inhibitors for myeloproliferative neoplasms. Expert Opin. Invest. Drugs 2011, 20, 961−972. (2) Levine, R. L.; Wadleigh, M.; Cools, J.; Ebert, B. L.; Wernig, G.; Huntly, B. J. P.; Boggon, T. J.; Wlodarska, I.; Clark, J. J.; Moore, S.; Adelsperger, J.; Koo, S.; Lee, J. C.; Gabriel, S.; Mercher, T.; D’Andrea, A.; Frohling, S.; Deohner, K.; Marynen, P.; Vandenberghe, P.; Mesa, R. A.; Tefferi, A.; Griffin, J. D.; Eck, M. J.; Sellers, W. R.; Meyerson, M.; Golub, T. R.; Lee, S. J.; Gilliland, D. G. Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. Cancer Cell 2005, 7, 387− 397. (3) Anand, S.; Huntly, B. Disordered signaling in myeloproliferative neoplasms. Hematol. Oncol. Clin. North Am. 2012, 26, 1017−1035. (4) Baxter, E. J.; Scott, L. M.; Campbell, P.; East, C.; Fourouclas, N.; Swanton, S.; Vassiliou, G. S.; Bench, A. J.; Boyd, E. M.; Curtin, N.; Scott, M. A.; Erber, W. N.; Green, A. R. Cancer Genome Project. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet 2005, 365, 1054−1061. (5) James, C.; Ugo, V.; Le Couedic, J. P.; Staerk, J.; Delhommeau, F.; Lacout, C.; Garc-on, L.; Raslova, H.; Berger, R.; Bennacaur-Griscelli, A.; Villeval, J. L.; Constantinescu, S. N.; Casadevall, N.; Vainchenker, W. A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera. Nature 2005, 434, 1144−1148. (6) Passamonti, F.; Cervantes, F.; Vannucchi, A. M.; Morra, E.; Rumi, E.; Pereira, A.; Guglielmelli, P.; Pungolino, E.; Caramella, M.; Maffioli, M.; Pascutto, C.; Lazzarino, M.; Cazzola, M.; Tefferi, A. A dynamic

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(41) Bricogne, G.; Blanc, E.; Brandl, M.; Flensburg, C.; Keller, P.; Paciorek, W.; Roversi, P.; Sharff, A.; Smart, O. S.; Vonrhein, C.; Womack, T. O. BUSTER, version 2.11.5; Global Phasing Ltd: Cambridge, United Kingdom, 2011. (42) Crystallographic statistics for JAK2 bound to 23b are as follows. Numbers in parenthesis characterize the higher resolution shell. Space group C2, unit cell a, b, and c dimensions 43.9, 127.1, 135.2, 97.3 Å, β = 97.3°, resolution 134−2.45 Å (2.58−2.45 Å), 25971 unique reflections with an overall redundancy of 4.2 (4.3), 96.2% (96.4%) completeness with Rmerge of 7% (30%) and mean I/σ(I) of 13.4 (2.5). The final model containing 4334 proteins, 1199 solvents, and 54 compound atoms has an R-factor of 18.1% (Rfree using 5% of the data 21.7%). Mean temperature factors for the protein and the ligand are 43.0 and 50.1 Å2, respectively. (43) Crystallographic statistics for the JAK2 bound to 28 are as follows. Numbers in parenthesis characterize the higher resolution shell. Space group C2, unit cell a, b, and c dimensions 44.5, 126.9, 135.9 Å, β = 97.5°, resolution 21−2.75 Å (2.9−2.75 Å), 19353 unique reflections with an overall redundancy of 4.0(3.7), 99.4% (98.7%) completeness with Rmerge of 10% (50%) and mean I/σ(I) of 11.6 (2.1). The final model containing 4356 proteins, 117 solvents, and 58 compound atoms has an R-factor of 19.7% (Rfree using 5% of the data 24.6%). Mean temperature factors for the protein and the ligand are 50.5 and 51.7 Å2, respectively.

(31) For assay conditions and experimental details: Dowling, J. E.; Alimzhanov, M.; Bao, L.; Block, M. H.; Chuaqui, C.; Cooke, E. L.; Denz, C. R; Hird, A.; Huang, S.; Larsen, N. A.; Peng, B.; Pontz, T. W.; Rivard-Costa, C.; Saeh, J. C; Thakur, K.; Ye, Q.; Zhang, T.; Lyne, P. D. Structure and property based design of pyrazolo[1,5-a]pyrimidine inhibitors of CK2 kinase with activity in vivo. ACS Med. Chem. Lett. 2013, 4, 800−805. (32) Cells were treated with decreasing concentration of 19a for 1 and 24 h, harvested into SDS lysis buffer, and run on SDS PAGE. Phosphorylated and total STAT5 proteins were assayed by Western blot and quantitated using a Licor Odyssey imager. For a detailed experimental, see ref 18a. (33) Quentmeier, H.; MacLeod, R. A.; Zaborski, M.; Drexler, H. G. JAK2 V617F tyrosine kinase mutation in cell lines derived from myeloproliferative disorders. Leukemia 2006, 20, 471−476. (34) Giovanella, B. C.; Stehlin, J. S. Heterotransplantation of human malignant tumors in “nude” thymusless mice. I. Breeding and maintenance of “nude” mice. J Natl Cancer Inst. 1973, 51, 615−619. The nude mice were obtained from Taconic: http://www.taconic. com/wmspage.cfm?parm1=873. (35) No significant body weight loss was observed when 19a was dosed up to 10 mg/kg given orally twice daily (20 mg/kg/day) for 10 days.

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