Structural insights into JAK2 inhibition by ruxolitinib, fedratinib, and derivatives thereof

Abstract

The discovery that aberrant activity of Janus kinase 2 (JAK2) is a driver of myeloproliferative neoplasms (MPNs) has led to significant efforts to develop small molecule inhibitors for this patient population. Ruxolitinib and fedratinib have been approved for use in MPN patients, while baricitinib, an achiral analogue of ruxolitinib, has been approved for rheumatoid arthritis. However, structural information on the interaction of these therapeutics with JAK2 remained unknown. Here, we introduce a new methodology for the large-scale production of JAK2 from mammalian cells, which enabled the first crystal structures of JAK2 bound to these drugs and derivatives thereof. Along with biochemical and cellular data, the results provide a comprehensive view of the shape complementarity required for chiral and achiral inhibitors to achieve highest activity, which may facilitate the development of more effective JAK2 inhibitors as therapeutics.

Graphical Abstract

INTRODUCTION

Protein kinases have become one of the most desired classes of drug targets given their crucial roles in the regulation of cellular proliferation, survival, signaling, metabolism, and homeostasis. As a mediator of cytokine receptor activation, Janus kinase 2 (JAK2) is a cytosolic tyrosine kinase that phosphorylates signal transducer and activator of transcription proteins (STATs) resulting in SH2-domain mediated dimerization and STAT activation. STATs govern many processes including cell proliferation, differentiation, and immunological responses vital for cell survival.¹ The JAK/STAT pathway has been listed as one of the twelve core cancer pathways demonstrating the importance of proper JAK2 regulation to maintain normal cell function.¹ JAK2 is a multi-domain protein (Supplementary Fig. S1) which undergoes trans-autophosphorylation on the activation loop of the kinase domain (KD) involving residues Tyr1007 and Tyr1008. The purpose of this phosphorylation is not fully known but is thought to aid in the recruitment and phosphorylation of STATs.² Type 1 inhibitors have been shown to bind to the ATP binding site of the active conformation of JAK2, but inhibition paradoxically leads to accumulation of phosphorylated residues on the activation loop with increasing inhibitor concentration.^{3, 4} This phenomenon has been attributed to the protection of activation loop phosphotyrosines from phosphatases.^{5, 6} Higher levels of phosphorylated Tyr1007/1008, and therefore higher levels of constitutively active JAK2, have also been shown to be caused by a single hyperactivating point mutation, V617F, found in the pseudokinase domain (PKD) of JAK2.⁷

Constitutively active JAK2 has been identified in several types of cancers including breast cancer, lymphomas, and myeloid malignancies.^{3, 8-10} The V617F mutation is the most commonly identified mutation found in myeloproliferative neoplasms (MPNs), in more than 95% of polycythemia vera patients, and in more than 50% of all thrombocythemia and primary myelofibrosis cases.^{8, 11} The FDA approved the pyrrolopyrimidine JAK2 inhibitor ruxolitinib in 2011 and the dianilinopyrimidine inhibitor fedratinib in 2019 for treating myelofibrosis.^12-14 Baricitinib, an achiral analogue of ruxolitinib was approved for rheumatoid arthritis in 2018.¹⁵ JAK2 inhibitors for MPN provide quality of life improvements but provide little efficacy at antagonizing the natural course of disease. To date, no crystal structure of ruxolitinib has been reported with any of the JAK family proteins, making ruxolitinib the oldest FDA-approved kinase inhibitor without a co-crystal structure with its target protein.^{16, 17} Likewise, structural information on the interaction of JAK2 with fedratinib and baricitinib remained unknown. Knowledge of these structures, however, is required for the development of more efficacious drugs to specifically target aberrant JAK2. Here we report the first crystal structures of ruxolitinib, fedratinib and baricitinib along with derivatives bound to the JAK2 kinase domain (KD). We introduce a new methodology to efficiently produce crystallization-grade JAK2 KD using mammalian cells, which enabled efficient structure-activity relationship (SAR) studies with inhibitors of different chemical scaffolds. A total of 14 high-resolution crystal structures were determined of JAK2 liganded with known and novel inhibitors. The data sets detail the requirements of small molecule inhibitors for shape complementarity with the ATP site of JAK2 and provide a structural basis for the stereoselective discrimination of enantiomers of ruxolitinib and derivatives thereof.

RESULTS

Ruxolitinib significantly increases the stability of JAK2 KD overexpressed in Expi293F cells

Previously, JAK2 KD has been predominantly purified from insect cells; but this process takes several weeks to generate and amplify the baculovirus to produce large enough quantities of purified protein. Upon transient expression of JAK2 KD in Expi293F cells, the presence of ruxolitinib during the transfection increased the levels of phosphorylated JAK2 (pY1007), as previously reported³, as well the overall amount of JAK2 KD in a concentration dependent manner (Fig. 1A). Protein levels of recombinant JAK2 KD from Expi293F cells were approximately 60-fold higher with 1 μM ruxolitinib than in untreated cells. Additionally, by including histone deacetylase (HDAC) inhibitors, valproic acid or butyric acid, during expression alongside ruxolitinib, JAK2 KD expression increased four-fold over that of ruxolitinib alone (Fig. 1B).

Figure 1: Ruxolitinib enhances the expression and purification of crystallization-grade JAK2 KD.

A) Expi293F cells were transiently transfected with wildtype JAK2 KD. Cells were treated with increasing concentrations of ruxolitinib during the 24-hour transfection. Cell lysates were subjected to immunoblotting to detect phosphorylated and unphosphorylated JAK2 KD along with the loading control GAPDH. Densitometry of the Western blot is depicted as a bar graph. B) Same as A except 2 mM and 4 mM butyric acid (BA) or valproic acid (VPA) were added to cells with 1 μM ruxolitinib. C) Expi293F cells were transiently transfected with JAK2 KD +/− 1 μM ruxolitinib and analyzed by cellular thermal shift assay (CETSA). Cells were incubated at the indicated temperatures for three minutes and lysates were probed for JAK2 KD and loading control actin. The graph shows the densitometry values relative to loading control as a function of temperature. Data were fit to a four parameter Hill equation, yielding EC₅₀ values of 45 and 47 °C for JAK2 KD expressed in the absence and presence of ruxolitinib, respectively. D) Cells were grown as in C) for isothermal dose response (ITDR) against ruxolitinib at 47°C for three minutes, and cell lysates were probed for JAK2 and GAPDH. The corresponding densitometry values were fit to a four parameter Hill equation yielding EC₅₀ = 3.8 μM. E) SDS-PAGE of a typical purification of JAK2 KD from a 1 L Expi293F cell culture grown in the presence of 1 μM ruxolitinib and 4 mM butyric acid for 24 hours. Lane 1 GenScript Broad Range Ladder, Lane 2 soluble lysate, Lane 3 GE HisTrap flow-through, Lane 4 GE HisTrap elution peak, Lane 5 GE S75 elution peak. F) Photograph of X-ray grade crystals grown from thus purified JAK2 KD.

Next, we investigated the thermostability of JAK2 KD upon transient expression in the presence of ruxolitinib using a cellular thermal shift assay (CETSA)¹⁸ and isothermal dose-response (ITDR). At 1 μM concentration, ruxolitinib elevated the cellular thermostability of JAK2 KD by 2 °C, from 45 – 47 °C (Fig. 1C). At 47 °C, the amount of expressed JAK2 KD increased in dose-response with ruxolitinib (EC₅₀ = 3.8 μM) (Fig. 1D). Expression in the absence of ruxolitinib yielded very little soluble protein and purity could not be achieved above 50% (data not shown). Adding ruxolitinib to cells during transfection resulted in significantly more protein, which could be purified to homogeneity using Ni-affinity and size exclusion chromatography with yields of 3 mg/L culture (Fig. 1E). Purified JAK2 KD yielded X-ray grade crystals within 24 hours (Fig. 1F).

Structure-activity relationship (SAR) studies of diverse small molecule JAK2 inhibitors

To assess the suitability of purified JAK2 KD for SAR studies for drug development campaigns, three series of JAK2 inhibitors (Fig. 2A-C) were subjected to binding studies by differential scanning fluorimetry (DSF). Series A consists of ruxolitinib enantiomers and FDA-approved derivatives baricitinib and tofacitinib, while series B consists of piperidine-aniline analogues of ruxolitinib. Series C comprises the FDA-approved JAK2 inhibitor fedratinib and other diaminopyrimidines, which were further developed as dual JAK2-BRD4 inhibitors from early lead compounds.¹⁹ The melting temperature of JAK2 in the absence of inhibitor was 50 °C, and thermal shifts in the presence of 100 μM inhibitor ranged from 3.3 to 5.5 °C for series A, 6.4 to 12 °C for series B and 2.2 to 5.2 °C for series C (Fig. 2D and Table 1).

Figure 2: Purified JAK2 KD from Expi293 cells is suitable for SAR studies by DSF.

Three series of JAK2 inhibitors were subjected to SAR studies: A) Ruxolitinib stereoisomers and FDA approved derivatives, B) Piperidine-phenylamine analogues of ruxolitinib, C) Fedratinib and derived dual JAK2-BRD4 inhibitors. D) DSF derivative plots of JAK2 KD in the absence (DMSO) and presence of 100 μM inhibitor. E) Viability of UKE-1 cells in response to increasing inhibitor concentration. F) DSF and biochemical enzyme inhibition data correlate significantly. G) Analysis of DSF and UKE-1 cell growth inhibition data shows high correlation for series A, but not for series B and C. Series B likely suffers from poor cell permeability. H) For series C, cell inhibition data correlate significantly with binding potential for BRD4-1, suggesting predominant activity through inhibition of BRD4.

Table 1:

Binding and inhibitory potential of investigated JAK2 inhibitors

Series	Compound ID	Binding potential1	Biochemical inhibition2	UKE-1 cell growth inhibition3
		ΔTm (°C)	IC50 (nM)	IC50 (μm)
A	Ruxolitinib	5.5 ± 0.06	0.40 ± 0.005	0.1 ± 0.02
	(S)-Ruxolitinib	3.3 ± 0.04	5.0 ± 0.1	1.04 ± 0.09
	(rac)-Ruxolitinib	4.8 ± 0.05	0.92 ± 0.06	0.38 ± 0.08
	Baricitinib	5.2 ± 0.08	0.29 ± 0.13	0.19 ± 0.03
	Tofacitinib	3.5 ± 0.08	2.9 ± 0.39	0.91 ± 0.1
B	(R)-1	12 ± 0.02	0.15 ± 0.03	3.2 ± 0.76
	(S)-1	8.3 ± 0.08	0.099 ± 0.02	6.3 ± 5.4
	(rac)-1	11.8 ± 0.05	< 0.05	2.2 ± 1.9
	2	7 ± 0.03	0.13 ± 0.048	4.5 ± 5.9
	3	6.4 ± 0.04	0.26 ± 0.04	0.62 ± 0.04
C	Fedratinib	5.2 ± 0.07	0.75 ± 0.39	0.66 ± 0.06
	4	3.1 ± 0.09	6.5 ± 3.5	0.2 ± 0.02
	5	3.2 ± 0.04	8.2 ± 5.7	0.15 ± 0.01
	6	2.2 ± 0.06	11.4 ± 0.87	0.32 ± 0.02
	7	4.3 ± 0.05	3.6 ± 1.1	0.21 ± 0.02

¹Standard deviation (SD) from two DSF data sets, each performed in quadruplicate.

²SD from two data sets of radiometric assay by Reaction Biology.

³Standard error of the mean (SEM) for data from three experiments, each performed in hexaplicate.

To evaluate if the thermal shifts reflect inhibitory activity against JAK2 in cells, compounds were characterized for growth inhibitory activity of UKE-1 cells, which are driven by V617F mutant JAK2 (Fig. 2E).²⁰ The resulting IC₅₀ values ranged from 0.1 to 1.0 μM for series A, 0.6 to 6.3 μM for series B and 0.15 to 0.66 μM for series C. Additionally, compounds were characterized for inhibition of enzymatic JAK2 activity in a radiometric assay by Reaction Biology Corp., IC₅₀ values ranging from 0.29 to 5.0 nM for series A, 0.05 to 0.26 nM for series B, and 0.75 to 11.4 nM for series C (Table 1). Statistical significance of the data sets was evaluated by Pearson’s correlation analyses (Fig. 2F-H). Thermal shifts showed excellent correlation with enzymatic inhibition values across all compounds, except for (R)-1 and rac-1 of series B, the high inhibitory activity of which likely exceeded the sensitivity of the radiometric assay applied (IC₅₀ < 0.15 nM) (Fig. 2F). By contrast, only series A significantly correlated with cellular inhibition data (Fig. 2G). Series B probably suffers from poor cell permeability, while series C showed higher cell growth inhibitory activity than expected from JAK2 inhibition alone. The increased cellular activity of series C is likely caused by their dual activity against JAK2 and BRD4, as UKE-1 cells are highly sensitive to inhibition of BRD4.¹⁹ The thermal shifts exerted by these compounds towards BRD4 correlated significantly with inhibitory activity against UKE-1 cell growth (Fig. 2H).

Structural basis of JAK2 inhibition by ruxolitinib and baricitinib.

Structural information on the JAK2-ruxolitinib complex was previously limited to molecular dynamics simulations¹⁶ or by prediction based on the known structures of c-SRC with ruxolitinib²¹ and of JAK2 with tofacitinib.²² Crystals of JAK2 KD grown in the absence of added inhibitor (Fig. 1F) showed that ruxolitinib was still bound to protein despite ~100,000-fold dilution throughout the purification process, reflecting the high binding affinity of ruxolitinib for JAK2 KD. However, bound ruxolitinib could be readily displaced by other inhibitors through in-diffusion of crystals with 1 mM inhibitor for 72 hours prior to data collection. Ruxolitinib is housed deep inside the ATP site, anchored through H-bonding interactions between the pyrrolopyrimidine moiety and main chain atoms of Glu930 and Leu932 of the hinge region (Fig. 3A-D).

Figure 3: Structural basis of ruxolitinib interaction with JAK2.

Co-crystal structure of JAK2 KD liganded with ruxolitinib determined at 1.9 Å resolution (PDB 6VGL). A) Electrostatic surface potential of the JAK2-ruxolitinib complex. The inhibitor is shown as yellow spheres. B) Positioning of ruxolitinib in the ATP site. The hinge region is shown in orange, gatekeeper residue in red, DFG motif in green, P-loop in cyan, C-helix in magenta, and other residues in grey. C) Binding interactions of ruxolitinib in the ATP site. Potential H-bonding and hydrophobic VDW interactions are indicated as black and green dotted lines, respectively. D) 2Fo-Fc electron density map (1σ) of bound ruxolitinib at 1.9 Å resolution. E) Binding pose of ruxolitinib (beige) in c-SRC (PDB 4U5J) and upon superposition with ruxolitinib in JAK2 (yellow). F) 1.9 Å resolution co-crystal structure of JAK2 with baricitinib (magenta, PDB 6VN8) and superposition with ruxolitinib (yellow).

The inhibitor is held in place through several van-der-Waals (VDW) hydrophobic interactions with surrounding residues, including the P-loop (Leu855, Gly856) and the DFG motif (Asp994). The binding pose of ruxolitinib in JAK2 agrees with molecular dynamics predictions¹⁶, but significantly differs from that observed in the crystal structure of c-SRC (Fig. 3E).²¹ In SRC, ruxolitinib is rotated ~180° relative to the hinge region, likely caused by repulsion and/or steric hindrance with the gatekeeper residue Thr338. In JAK2, the hydrophobic and flexible gatekeeper residue Met929 accommodates ruxolitinib through multiple VDW interactions. Accordingly, ruxolitinib inhibitory activity is three orders of magnitudes higher for JAK2 over SRC.²¹ To confirm the pyrrolopyrimidine binding mode of ruxolitinib in JAK2, a co-crystal structure was determined with baricitinib showing almost identical positioning of the two inhibitors in the ATP site (Fig. 3F).

Stereoselective discrimination of ruxolitinib and aniline derivatives by JAK2

The high diffraction power of the JAK2 KD crystals prompted us to evaluate the structure-activity relationship of enantiomers of ruxolitinib and analogues thereof. The S-isomer of ruxolitinib is ~ 10-fold less active against JAK2 than the R-isomer (Table 1). The co-crystal structure revealed that (S)-ruxolitinib adopts a binding pose similar to (R)-ruxolitinib (Fig. 4A) and achieves shape complementarity with the ATP site through ~180° rotation about the chiral center (Fig. 4B). Using the racemic mixture of ruxolitinib, (rac)-ruxolitinib, the resulting co-crystal structure revealed exclusively the R-isomer bound (Fig. 4C). Likewise, the R- and S-enantiomers of aniline derivatives of ruxolitinib, (R)-1 and (S)-1, assumed the same orientation for the cyclopentyl and propionitrile moieties in the ATP site (Fig. 4D-F). Furthermore, the racemic mixture (rac-1) showed only the R-isomer bound (Fig. 4G). The data demonstrate that JAK2 discriminates between the R and S stereoisomers of ruxolitinib and derivatives by preferentially interacting with the R-isomer. Ruxolitinib derivatives devoid of a chiral center and the cyclopentyl group (2 and 3) exhibited the same preference of the respective propionitrile and butenyl moieties for positioning in the sub-pocket that accommodates the propionitrile of ruxolitinib (Fig. 4H, I). Notably, the aniline derivatives interact in the ATP site through an additional H-bond with the hinge region (Fig. 4D-I), which explains the substantial increase in JAK2 inhibitory activity of series B over ruxolitinib and series A (Table 1).

Figure 4: JAK2 discriminates between the R and S stereoisomers of ruxolitinib and derivatives thereof.

Distinct stereoisomers and the enantiomeric mixture of ruxolitinib and derivatives were subjected to crystallographic studies with JAK2 KD. A) Co-crystal structure of (S)-ruxolitinib (PDB 6VSN). B) Superposition of the R- and S-isomers of ruxolitinib (yellow and cyan, respectively) reveals that (S)-ruxolitinib adopts shape complementarity with the ATP site through ~180° rotation about the stereocenter. C) Co-crystal structure obtained with the racemic mixture of ruxolitinib, (rac)-ruxolitinib, PDB 6VNK), reveals only the R-isomer bound (green). The inset shows the superposition of (rac)-ruxolitinib with ruxolitinib. D) Co-crystal structure showing the H-bonding interactions of (R)-1 (yellow, PDB 6VNC) with the hinge region. E) Same as D) for the (S)-1 (cyan, PDB 6VNB). F) Superposition of (R)-1 and (S)-1 reveals the same adaptation as for ruxolitinib (B). G) Co-crystal structure obtained with (rac)-1 (green, PDB 6VS3) shows only the R-isomer bound. The inset is the superposition of (rac)-1 with (R)-1 (yellow). H) Co-crystal structure with derivative 2 devoid of a stereocenter (PDB 6VNJ) reveals the same binding pose and inhibitor conformation as (R)-1. I) Same as H) for derivative 3 (PDB 6VNM). The blue mesh is the 2Fo-Fc electron density of ligand contoured at 1σ. The electron density maps from refinement omitting the ligand are shown in Supplementary Fig. S2.

Structural basis of JAK2 inhibition by diaminopyrimidines

To further probe the ATP site, co-crystal structures were determined with dianilinopyrimidine-containing inhibitors of series C. All inhibitors interacted with the hinge region similarly to the aniline derivatives of ruxolitinib (Fig. 5).

Figure 5: Diaminopyrimidine inhibitors mimic the binding pose of ruxolitinib aniline derivatives.

A) Co-crystal structure of JAK2 with fedratinib (PDB 6VNE). The inset shows the superposition of fedratinib (yellow) with (R)-1 (magenta). B) Co-crystal structure with 4 (PDB 6VNG). The inset shows the superposition of 4 (yellow) with fedratinib (green). C) Co-crystal structure with 5 (PDB 6VNH). D) Co-crystal structure with 6 (PDB 6VNL). E) Co-crystal structure with 7 (PDB 6VNF).

However, the lack of a pyrrolo moiety allows only for the establishment of H-bonds with the main chain atoms of Leu932, but not with Glu930. This is reflected in a 10-fold reduced biochemical potency of inhibitors of series C against JAK2 (Table 1). Reversal of the sulfonamide moiety and addition of fluorine (4), which is beneficial for binding to the acetyl-lysine binding site of BRD4¹⁹, slightly tilts the aniline ring such that the tert-butyl moiety moves away from Val863 and positions close to the carboxyl group of Asp994 of the DFG motif (Fig. 5B). Although the reversed sulfonamide enables potential H-bonds with the side chain of Asn981, the unfavorably close distance between the hydrophobic tert-butyl and Asp994 appears to cause slightly reduced inhibitory activity for 4 and related compounds 5 and 6 (Fig. 5C, D). Constraining the sulfonamide moiety using an indoline moiety (7) was well tolerated (Fig. 5E) and showed highest inhibitory activity against JAK2 across series C (Table 1).

DISCUSSION AND CONCLUSIONS

JAK2 presents challenges for structural studies as it is not stable upon recombinant overexpression, and typical protein purifications require over 10 L insect cultures from bioreactors to obtain a few milligrams of crystallization-grade protein.^23-26 Recombinant overexpression of JAK2 KD using mammalian Expi293F cells requires only 24 hours growth post transfection and 1 liter of culture is sufficient to purify milligram quantities of crystallization-grade protein for biochemical and structural studies. This was achieved using ruxolitinib during expression, which significantly enhanced protein stability and purification yields and enabled the first crystal structures of JAK2 expressed in mammalian cells. The crystal structure data suggest that ruxolitinib improves the thermostability of JAK2 by decreasing flexible regions and causing a more rigid structure with well-defined activation and P-loops. Notably, ruxolitinib did not affect the proliferation of Expi293F or HEK293 cells even at 10 μM after 72 hours.¹⁹ This strategy provided faster, more efficient, and cost-effective production of recombinant JAK2, and may be applicable to other difficult to overexpress kinases provided that the inhibitor is specific, potent, and non-lethal over the timeframe of expression.

Robust crystallization conditions and ease of in-diffusion of inhibitors resulted in 14 novel co-crystal structures of JAK2 liganded with various inhibitors of three chemical scaffolds. Along with binding and inhibition data, this information provides a comprehensive view of the ATP site and the shape complementarity required for small molecules to achieve highest inhibitory activity. The (R) and (S) stereoisomers of ruxolitinib and aniline derivatives thereof were thoroughly characterized for binding potential and inhibitory activity against cancer cells driven by constitutively active V617F JAK2. While (S)-ruxolitinib is a formidable inhibitor of JAK2 (IC₅₀ = 5 nM), the (R)-isomer exerted > 10-fold higher activity with respect to binding and cell kill potential. Co-crystal structures obtained with the isolated isomers along with racemic mixtures demonstrate that JAK2 discriminates against the S-configuration. The (S)-isomer mimics the conformation of the preferred R-isomer through rotation about the chiral center, but the resulting binding pose is less compatible with the ATP site. Notably, the achiral analogue baricitinib adopts a conformation almost identical to that of ruxolitinib, which is reflected in the similar values obtained for binding, enzymatic and cellular activities of these FDA-approved inhibitors.

Piperidine aniline derivatives of both (R)- and (S)-ruxolitinib showed greatly enhanced binding and inhibitory potential towards JAK2; the picomolar activities could not be resolved by the steady-state assays employed here. This substantial increase in activity is attributable to an additional H-bond established with the hinge region. Although these compounds suffer from poor cell penetration as indicated by a significant loss of cell inhibitory activity, solubilizing groups other than piperidine may alleviate this drawback. Diaminopyrimidine inhibitors of JAK2, including fedratinib, mimic the binding pose of aniline derivatives of ruxolitinib. However, they lack the potential to establish an H-bond with Glu930 of the hinge region, which is reflected in reduced but still formidable inhibitory activities (IC₅₀ between 0.75 and 11.4 nM). These compounds also inhibit BET bromodomains to varying degrees and appear to exert strong cell growth inhibitory activity by predominantly targeting BRD4. Previously, structural modeling, binding and enzymatic assays using full-length JAK2 expressed in insect cells showed high affinity of fedratinib for the substrate-binding site.³ The co-crystal structure of JAK2 kinase domain with fedratinib (or any other compound studied herein) showed inhibitor bound exclusively to the ATP site. It is conceivable that fedratinib interaction with the substrate binding site occurs in the full-length enzyme, but not in the isolated kinase domain. Combined, our findings may serve the iterative drug design process in developing JAK2 inhibitors with increased efficacy to combat cancers caused by this enzyme.

EXPERIMENTAL SECTION

Compounds and reagents:

Reagents for biochemical and crystallographic experiments were purchased from Fisher Scientific and Hampton Research unless otherwise indicated. Ruxolitinib (phosphate) was from LC Laboratories (R-6688, >99%), Tofacitinib (citrate) from MedChemExpress (HY-40354A, 99.1%), Baricitinib (free base) from Combi-blocks (QJ-1094, 98%), Fedratinib from MedChemExpress (HY-10409, 99.9%). The following antibodies were used for immunoblotting: His-HRP (ProteinTech, HRP-66005, 1:5,000), pJAK2 (Y1007/1008) (Cell Signaling, 3771, 1:1,000), actin (Sigma, A5441, 1:1,000), GAPDH-HRP (ProteinTech, HRP-60004, 1:10,000), anti-mouse-HRP IgG (Jackson Immuno Research, 115-035-003, 1:2,000).

General Synthetic Methods:

All reagents were purchased from commercial suppliers and used without further purification. ¹H NMR spectra were recorded on Bruker 500 MHz spectrometer with CDCl₃, CD₃OD or DMSO-d₆ as the solvent. ¹³C NMR spectra were recorded at 125 MHz. All coupling constants are measured in Hertz (Hz) and the chemical shifts (δ_H and δ_C) are quoted in parts per million (ppm) relative to TMS (δ 0), which was used as the internal standard. High resolution mass spectroscopy was carried out on an Agilent 6210 LC/MS (ESI-TOF). HPLC-MS analysis was performed using Agilent 6120 single quadrupole 1220 LCMS equipped with Zorbax SB-C18 column (4.6 x 50 mm, 1.8 micron). The purity of final compounds that underwent biological assessment were >95% as measured by HPLC-MS. Thin layer chromatography was performed using silica gel 60 F254 plates (Fisher), with observation under UV when necessary. Anhydrous solvents (acetonitrile, dimethylformamide, ethanol, isopropanol, methanol and tetrahydrofuran) were used as purchased from Aldrich. Burdick and Jackson HPLC grade solvents (methanol, acetonitrile and water) were purchased from VWR for HPLC and high-resolution mass analysis. HPLC grade TFA was purchased from Fisher. Compound synthesis and characterization are detailed in the Supporting Information.

Compound synthesis and characterization

Pyrrolopyrimidine inhibitors of JAK2.

(S)-Ruxolitinib was prepared by a novel resolution of pyrazole-containing carboxylic acid 9 [from conjugate addition of 4-bromo-1H-pyrazole, and ester hydrolysis of 8 ²⁷] as shown in Supplementary Scheme S1. Reaction of 9 with the Evans oxazolidinone provided the diastereoisomeric oxazolidinones (4R,3R)-10 and (4R,3S)-10 which were easily separated to homogeneity by flash chromatography on silica gel. The faster-running fraction was identified as (4R,3S)-10 by correlation to the enantiomer of clinically used (R)-ruxolitinib using the synthetic route described in a reported patent²⁷. Treatment of (4R,3S)-10 with ammonium hydroxide²⁸ gave the primary amide 11, which upon dehydration with phosphorus pentoxide gave the nitrile 12. Formation of the boronate ester 13 followed by Suzuki reaction with 4-bromo-7H-pyrrolo[2,3-d]pyrimidine provided (S)-ruxolitinib. The enantiomeric excess of the (S)-ruxolitinib was determined to be >99.99% by chiral HPLC using (Supplementary Fig. S3). A sample of rac-ruxolitinib was prepared from rac-13 in the same way. The synthesis of the ruxolitinib analogs (R) and (S)-1 is shown in schemes S1 and S2. 2-Chloro-ruxolitinib (14) was prepared by Suzuki coupling of 13 with 2,4-dichloropyrrolopyrimidine (Supplementary Scheme S1). Substitution of the chlorine atom with the anilinopyrimidine subsituent required protection of the pyrrole NH group. Thereby, Buchwald-Hartwig amination of the boc-protected pyrrolopyrimidine 15 gave 16 bearing the anilinopiperidine at the 2-position of the pyrimidine ring. Finally, removal of the protecting groups gave the desired (S)-1 bearing the required piperidine group oriented towards the solvent accessible region. Again, chiral HPLC analysis indicated that the enantiomeric excess of (S)-1 was >99.99% (as determined by the e.e. of its bis-boc protected precursor (S)-16, Supplementary Fig. S4). The enantiomer (R)-1 was prepared in the same way using the diasteroisomer (4R,3R)-10 (Supplementary Scheme S2). In this case the enantiomeric excess of (R)-1 was 98.6% (as determined by the e.e. of its bis-boc protected precursor (R)-22. Ruxolitinib derivatives lacking the cyclopentyl group have been shown to retain potent JAK2 inhibitory activity.^{29, 30} Thus, the piperidine 2, incorporating the propionitrile substituted pyrazole, was prepared in a similar way by Suzuki reaction of addition of 2,4-dichloropyrrolopyrimidine and the boronic ester 27 [from acrylonitrile and 4-pyrazoleboronic pinacolate³¹] to give the nitrile 28 (Supplementary Scheme S3). A similar sequence of Buchwald-Hartwig amination of the boc-protected 2-chloropyrrolopyrimidine 29 followed by deprotection of 30 provided the piperidine 2. The piperidine 3, bearing a butenyl substituted pyrazole group was prepared by the same methods used to make the piperidine 2 (Supplementary Scheme S4).

Methyl 3-(4-bromo-1H-pyrazol-1-yl)-3-cyclopentylpropanoate (8):

This compound was prepared according to the reported method ²⁷. To a solution of 3-cyclopentyl methacrylate (4.56 g, 29.58 mol, 1.0 eq.), in anhydrous acetonitrile (100 mL) added 4-bromopyrazole (4.77 g, 32.49 mol, 1.1 eq), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (6.63 mL, 44.37 mol, 1.5 eq). The reaction mixture was stirred overnight at room temperature. The mixture was concentrated under reduced pressure and the residue was dissolved in ethyl acetate (500 mL), added 1N HCl to adjust pH value to 3 or 4, and then washed with water (100 mL) and brine (100 mL). The organic layer was dried (Na₂SO₄), filtered and concentrated under reduced pressure to obtain the title compound as a colorless oil (7.02 g, 80%). This compound was used in the next step without purification. HPLC 98% (t_R = 13.1 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 8.04 (s, 1H), 7.51 (s, 1H), 4.39 (m, 1H), 3.50 (s, 1H), 2.97 (m, 2H), 2.24 (m, 1H), 1.74 (m, 1H), 1.59 – 1.34 (m, 4H), 1.26 – 1.05 (m, 3H); HPLC–MS (ESI+): m/z 301.1 (M+H)⁺.

3-(4-Bromo-1H-pyrazol-1-yl)-3-cyclopentylpropanoic acid (9):

This compound was prepared according to the reported method ²⁷. To a solution of 8 (7.02 g, 23.40 mol, 1.0 eq.), in dioxane (140 mL), added sodium hydroxide (2.81 g, 70.20 mol, 3.0 eq) in water (70 mL). The reaction mixture was stirred overnight at room temperature. The mixture was concentrated under reduced pressure and added water (100 mL) followed by 1N HCl to adjust pH to 3 or 4. The aqueous layer extracted with ethyl acetate (3 × 150 mL), dried (Na₂SO₄) and filtered, and concentrated under reduced pressure to obtain title compound as a white solid (6.20 g, 95%). This compound was used in the next step without further purification. HPLC 99% (t_R = 12.3 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 12.21 (s, 1H), 8.02 (s, 1H), 7.51 (s, 1H), 4.37 (m, 1H), 2.86 (m, 2H), 2.23 (m, 1H), 1.75 (m, 1H), 1.60 – 1.35 (m, 4H), 1.26 – 1.06 (m, 3H); HPLC–MS (ESI+): m/z 287.2 (M+H)⁺.

(4R)-4-Benzyl-3-((3R)-3-(4-bromo-1H-pyrazol-1-yl)-3-cyclopentylpropanoyl)oxazolidin-2-one (4R,3R)-10:

To a solution of 9 (5.73 g, 20.0 mol, 1.0 eq.) in tetrahydrofuran (172 mL), added pivaloyl chloride (4.93 mL, 40.0 mol, 2.0 eq) and triethylamine (8.34 mL, 60.0 mol, 3.0 eq.) at 0 °C. The reaction mixture was stirred for 1 h at room temperature. To the reaction mixture, lithium chloride (1.70 g, 40.0 mol, 2.0 eq) and oxazolidinone (3.58 g, 20.0 mol, 1.0 eq) were added, and then the mixture was stirred overnight at room temperature. The reaction mixture was diluted with water (100 mL), evaporated under reduced pressure to remove tetrahydrofuran and extracted with ethyl acetate (3 × 150 mL). The organic layer evaporated under reduced pressure and the residue was purified by SiO₂ chromatography using ethyl acetate/hexane (20 %) as eluent to give the title compound as a white solid (3.9 g, 42%, TLC: R_f = 0.51 developed using ethyl acetate/hexane (2:3)). HPLC 97% (t_R = 13.40 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 8.10 (d, J = 0.7 Hz, 1H), 7.61 (s, 1H), 7.27 – 7.21 (m, 3H), 6.94 – 6.91 (m, 2H), 4.63 – 4.52 (m, 3H), 4.32 (t, J = 8.7 Hz, 1H), 4.17 (dd, J = 9.0, 3.1 Hz, 1H), 3.79 (dd, J = 17.6, 10.6 Hz, 1H), 3.20 (dd, J = 17.6, 2.9 Hz, 1H), 2.83 – 2.74 (m, 2H), 2.37 – 2.30 (m, 1H), 1.79 – 1.74 (m, 1H), 1.60 – 1.50 (m, 3H), 1.47 – 1.39 (m, 1H), 1.29 – 1.12 (m, 3H); HPLC–MS (ESI+): m/z 468.2 (M+Na)⁺.

(4R)-4-Benzyl-3-((3S)-3-(4-bromo-1H-pyrazol-1-yl)-3-cyclopentylpropanoyl)oxazolidin-2-one (4R,3S-10):

The (4R,3S)-10 was purified using the procedure described for (4R,3R)-10 to obtain the title product as a white solid (4.0 g, 43%, TLC: R_f = 0.62 developed using ethyl acetate/hexane (2:3). HPLC 98% (t_R = 13.60 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 8.10 (d, J = 0.8 Hz, 1H), 7.52 (d, J = 0.6 Hz, 1H), 7.34 – 7.30 (m, 2H), 7.28 – 7.24 (m, 1H), 7.22 – 7.19 (m, 2H), 4.61 – 4.49 (m, 2H), 4.28 (t, J = 8.5 Hz, 1H), 4.15 (dd, J = 8.8, 2.8 Hz, 1H), 3.53 (dd, J = 17.1, 10.4 Hz, 1H), 3.37 (dd, J = 17.1, 3.0 Hz, 1H), 2.97 (dd, J = 13.6, 3.2 Hz, 1H), 2.84 (dd, J = 13.5, 8.3 Hz, 1H), 2.34 – 3.31 (m, 1H), 1.82 – 1.73 (m, 1H), 1.64 – 1.50 (m, 3H), 1.47 – 1.41 (m, 1H), 1.36 – 1.12 (m, 1H); HPLC–MS (ESI+): m/z 446.2 (M+H)⁺.

(S)-3-(4-Bromo-1H-pyrazol-1-yl)-3-cyclopentylpropanamide (11):

The amide 11 was synthesized using the same procedure described for 17 from (4R,3S)-10 (7.6 mmol, 3.4 g) as a white solid (1.9 g, 87%). HPLC 97.2% (t_R = 11.4 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 7.92 (s, 1H), 7.51 (s, 1H), 7.30 (s, 1H), 6.76 (s, 1H), 4.41 (td, J = 9.7, 4.1 Hz, 1H), 2.76 (dd, J = 15.3, 10.0 Hz, 1H), 2.57 (dd, J = 15.3, 4.0 Hz, 1H), 2.24 (h, J = 8.6 Hz, 1H), 1.79 – 1.70 (m, 1H), 1.59 – 1.35 (m, 4H), 1.24 – 1.06 (m, 3H); HPLC–MS (ESI+): m/z 286.2 (M+H)⁺.

(S)-3-(4-Bromo-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile (12):

The nitrile 12 was synthesized using the same procedure described for 18, from amide 11 (6.6 mmol, 1.9 g) as a white solid (1.6 g, 94%). HPLC 97.2% (t_R = 12.5 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 8.13 (d, J = 0.7 Hz, 1H), 7.63 (s, 1H), 4.38 (td, J = 9.4, 4.7 Hz, 1H), 3.15 – 3.05 (m, 2H), 2.36 – 2.26 (m, 1H), 1.80 – 1.69 (m, 2H), 1.62 – 1.37 (m, 4H), 1.30 – 1.19 (m, 2H), 1.08 (dq, J = 12.7, 8.3 Hz, 1H); HPLC–MS (ESI+): m/z 268.0 (M+H)⁺.

(S)-3-Cyclopentyl-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)propanenitrile (13):

The boronic ester 13 was synthesized using the same procedure described for 13 from (S)-bromopyrazole 19 (1.1 mmol, 0.3 g), as a white solid (165 mg, 47%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.08 (s, 1H), 7.95 (s, 1H), 4.43 (td, J = 9.7, 4.3 Hz, 1H), 3.15 – 3.09 (m, 2H), 2.37 – 2.27 (m, 1H), 1.80 – 1.71 (m, 1H), 1.58 – 1.37 (m, 4H), 1.25 (s, 12H), 1.20 – 1.15 (m, 3H).

(S)-3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile [(S)-Ruxolitinib]:

(S)-Ruxolitinib was synthesized using the same procedure described for 14 from (S)-13 (0.15 mmol, 49 mg), as a white solid (35 mg, 74%). HPLC 99.17% (t_R = 12.57 min, CH₃OH 75% in 0.1% TFA water, 20 min); ¹H NMR (500 MHz, DMSO) δ 12.10 (s, 1H), 8.79 (s, 1H), 8.67 (s, 1H), 8.37 (s, 1H), 7.59 (dd, J = 3.5, 2.4 Hz, 1H), 6.98 (dd, J = 3.6, 1.7 Hz, 1H), 4.54 (td, J = 9.8, 4.0 Hz, 1H), 3.26 (dd, J = 17.2, 9.8 Hz, 2H), 2.42 (dd, J = 17.1, 8.3 Hz, 1H), 1.86 – 1.78 (m, 1H), 1.65 – 1.40 (m, 4H), 1.35 – 1.17 (m, 3H); HPLC–MS (ESI+): m/z 307.2 (M+H)⁺. HRMS (ESI+) m/z calculated for C₁₇H₁₉N₆ (M+H)⁺ 307.1666, found 307.1667.

(3RS)-3-(4-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile (rac-Ruxolitinib):

To a solution of 4-bromo-7H-pyrrolo[2,3-d]pyrimidine (198 mg, 1.0 mmol, 1.0 equiv.), 3-cyclopentyl-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)propanenitrile (378 mg, 1.2 mmol, 1.2 equiv.)[prepared from commercially available 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole and 3-cyclopentylprop-2-enenitrile according to a reported procedure ³²], and Na₂CO₃ (340 mg, 3.2 mmol, 3.2 equiv.) in dioxane (4 mL) and H₂O (1 mL) was added Pd(PPh₃)₄ (40 mg, 0.033 mmol, 0.033 equiv.). The pressure tube was purged with argon for 15 min, sealed and then placed in pre-heated oil bath 120 °C. The mixture was stirred at this temperature for 12 h and then cooled to room temperature and partitioned between saturated NH₄Cl (20 mL) and EtOAc (10 mL). The layers were separated, and the aqueous layer was extracted twice more with EtOAc (10 mL). The combined organic extracts were washed with brine, dried (MgSO₄) and concentrated under reduced pressure to give a yellow oil. Purification by flash chromatography (SiO₂, 75% EtOAc in hexane) provided the title compound rac-Ruxolitinib as a brown powder (0.224 g, 73%). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.11 (s, 1H), 8.80 (d, J = 0.7 Hz, 1H), 8.68 (s, 1H), 8.37 (s, 1H), 7.60 (dd, J = 3.6, 2.4 Hz, 1H), 6.99 (dd, J = 3.6, 1.8 Hz, 1H), 4.54 (td, J = 9.8, 4.1 Hz, 1H), 3.20 (m, 3H), 2.43 (m, 1H), 1.82 (m, 1H), 1.61 (m, 3H), 1.46 (m, 1H), 1.39 (m, 2H), 1.18 (m, 1H). HPLC-MS (ESI+): m/z 307.4 [100% (M+H)⁺], HRMS (ESI+) m/z calculated for C₁₇H₁₉N₆ (M+H)⁺ 307.1666, found 307.1662.

(S)-3-(4-(2-Chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile (14):

The pyrrolopyrimidine 14 was synthesized using the same procedure described for 20 from (S)-boronic ester 13 (1.1 mmol, 0.3 g), as a white oil (151 mg, 41%). HPLC 78.0% (t_R = 11.99 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 12.32 (s, 1H), 8.84 (d, J = 0.7 Hz, 1H), 8.39 (s, 1H), 7.64 (dd, J = 3.6, 2.3 Hz, 1H), 7.05 (dd, J = 3.6, 1.8 Hz, 1H), 4.56 (td, J = 9.8, 4.1 Hz, 1H), 3.29 – 3.21 (m, 2H), 2.43 (q, J = 8.6 Hz, 1H), 1.82 (td, J = 11.9, 7.2 Hz, 1H), 1.66 – 1.41 (m, 4H), 1.33 – 1.23 (m, 3H); HPLC–MS (ESI+): m/z 341.2 (M+H)⁺.

tert-Butyl (S)-2-chloro-4-(1-(2-cyano-1-cyclopentylethyl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (15):

The boc-protected pyrrolopyrimidine 15 was synthesized by the same procedure described for 21 from (S)-pyrrolopyrimidine 14 (0.27 mmol, 92 mg) as a white solid (70 mg, 68%). HPLC 95.8% (t_R = 13.6 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 8.91 (d, J = 0.7 Hz, 1H), 8.44 (s, 1H), 7.92 (d, J = 4.1 Hz, 1H), 7.27 (d, J = 4.2 Hz, 1H), 4.56 (td, J = 9.7, 4.1 Hz, 1H), 3.29 – 3.21 (m, 2H), 2.43 (q, J = 8.6 Hz, 1H), 1.82 (td, J = 12.2, 7.4 Hz, 1H), 1.64 (s, 8H), 1.58 – 1.41 (m, 4H), 1.35 – 1.23 (m, 3H); HPLC–MS (ESI+): m/z 441.2 (M+H)⁺.

tert-Butyl (S)-2-((4-(1-(tert-butoxycarbonyl)piperidin-4-yl)phenyl)amino)-4-(1-(2-cyano-1-cyclopentylethyl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (16):

The bis-boc-protected pyrrolopyrimidine 16 was synthesized using the same procedure described for 22, from the (S)-pyrrolopyrimidine derivative 15 (0.09 mmol, 60 mg) as a yellow solid (47 mg, 76%). HPLC 97.0% (t_R = 15.04 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 10.85 (s, 1H), 9.43 (s, 2H), 8.75 (d, J = 0.8 Hz, 1H), 8.33 (s, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.56 (d, J = 4.1 Hz, 1H), 7.15 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 4.2 Hz, 1H), 4.56 (td, J = 9.6, 4.4 Hz, 2H), 3.26 – 3.21 (m, 2H), 2.65 – 2.62 (m, 1H), 2.45 – 2.40 (m, 1H), 1.85 – 1.80 (m, 1H), 1.76 (d, J = 13.1 Hz, 2zH), 1.66 – 1.52 (m, 15H), 1.43 (s, 9H), 1.36 – 1.26 (m, 5H); HPLC–MS (ESI+): m/z 681.4 (M+H)⁺.

(S)-3-Cyclopentyl-3-(4-(2-((4-(piperidin-4-yl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile [(S)-1]:

The anilinopiperidine (S)-1 was synthesized using the same procedure described for (R)-1 from (S)-pyrrolopyrimidine 16 (0.06 mmol, 42 mg) as a yellow solid (24 mg, 83 %). HPLC 97.6 % (t_R = 7.76 min, CH₃OH 50 % and water 50% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 11.49 (s, 1H), 9.04 (s, 1H), 8.69 (s, 1H), 8.30 (s, 1H), 7.82 – 7.78 (m, 2H), 7.22 (dd, J = 3.7, 2.1 Hz, 1H), 7.15 – 7.11 (m, 2H), 6.79 (dd, J = 3.6, 1.6 Hz, 1H), 4.57 (td, J = 9.7, 4.2 Hz, 1H), 3.26 – 3.20 (m, 2H), 3.08 (d, J = 11.9 Hz, 2H), 2.68 – 2.61 (m, 2H), 1.87 – 1.80 (m, 1H), 1.73 (d, J = 12.6 Hz, 2H), 1.65 – 1.42 (m, 7H), 1.38 – 1.20 (m, 6H); HPLC–MS (ESI+): m/z 481.5 (M+H)⁺. HRMS (ESI+) m/z calculated for C₂₈H₃₃N₈ (M+H)⁺ 481.2823, found 481.2830.

(R)-3-(4-Bromo-1H-pyrazol-1-yl)-3-cyclopentylpropanamide (17):

To a solution of (4R,3R)-10 (3.0 g, 6.7 mol, 1.0 eq.), in tetrahydrofuran (180 mL) was added ammonium hydroxide (30%, 120 mL). The reaction mixture was stirred for 2 days at room temperature. The mixture was diluted with methanol (250 mL), concentrated under reduced pressure and this process was repeated another 2 times. The residue obtained was purified by reverse phase chromatography over C-18 silica gel using methanol/dichloromethane (gradient elution 0 to 5 %) as eluent to give the title compound as a white solid (1.24 g, 64%). HPLC 95% (t_R = 11.44 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 7.92 (d, J = 0.7 Hz, 1H), 7.51 (d, J = 0.7 Hz, 1H), 7.29 (s, 1H), 6.75 (s, 1H), 4.41 (td, J = 9.7, 4.0 Hz, 1H), 2.79 – 2.57 (m, 1H), 2.27 – 2.20 (m, 1H), 1.78 – 1.72 (m, 1H), 1.60 – 1.36 (m, 4H), 1.24 – 1.04 (m, 3H); HPLC–MS (ESI+): m/z 286.1 (M+H)⁺.

(R)-3-(4-Bromo-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile (18):

This compound was prepared according to the reported method ²⁷. To a solution of 17 (1.24 g, 4.3 mol, 1.0 eq.), in tetrahydrofuran (50 mL), added phosphorus pentoxide (1.84 g, 13.0 mol, 3 eq.) under argon. The reaction mixture was stirred for 2 hours at 70 °C, diluted with ethyl acetate (200 mL), and quenched by adding saturated sodium bicarbonate (200 mL). The aqueous layer was back-extracted with ethyl acetate (2 × 100 mL). The organic phases were combined, washed with water, brine, dried over anhydrous Na₂SO₄ and filtered. The filtrate was concentrated under reduced pressure and purified by SiO₂ chromatography using methanol/dichloromethane (5 %) as eluent to provide the title compound as a white solid (1.13 g, 98%). HPLC 100% (t_R = 12.56 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 8.12 (d, J = 0.5 Hz, 1H), 7.63 (d, J = 0.5 Hz, 1H), 4.38 (td, J = 9.4, 4.7 Hz, 1H), 3.15 – 3.08 (m, 2H), 2.33 – 2.28 (m, 1H), 1.79 – 1.72 (m, 1H), 1.61 – 1.38 (m, 4H), 1.30 – 1.05 (m, 3H); HPLC–MS (ESI+): m/z 268.0 (M+H)⁺.

(R)-3-Cyclopentyl-3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)propanenitrile (19):

This compound was prepared according to the reported method ²⁷. To a 20 mL microwave vial, added 18 (0.30 g, 1.12 mmol, 1.0 eq.), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (0.30 g, 1.19 mmol, 1.1 eq.), potassium acetate (329 mg, 3.36 mmol, 3.0 eq.), and 1,4-dioxane (4.0 mL). The resulting reaction mixture was degassed by bubbling argon for 5 minutes before being treated with tetrakis(triphenylphosphine)palladium(0) (65 mg, 0.06 mmol, 0.05 eq.). The resulting reaction mixture was heated to 120 °C in a microwave reactor for 2 hours. The reaction mixture was filtered through a celite bed and the celite bed was washed with dichloromethane and the organic layer was diluted with water (10 mL). The aqueous layer was extracted with dichloromethane (2 × 10 mL). The combined organic layers were concentrated under reduced pressure, and the crude product was purified using SiO₂ chromatography (ethyl acetate 0-30% in hexanes) to yield the title compound as a yellow oil (153 mg, 43 %). ¹H NMR (500 MHz, DMSO-d₆) δ 8.09 (d, J = 0.7 Hz, 1H), 7.66 (d, J = 0.7 Hz, 1H), 4.43 (td, J = 9.6, 4.3 Hz, 1H), 3.19 – 3.05 (m, 2H), 2.33 – 2.31 (mz, 1H), 1.80 – 1.73 (m, 1H), 1.61 – 1.39 (m, 4H), 1.27 (s, 12H), 1.20 – 1.15 (m, 3H).

(R)-3-(4-(2-Chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile (20):

To a 50 mL round bottom flask added 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (87.0 mg, 0.46 mmol, 0.95 eq.), 19 (153.0 mg, 0.49 mmol, 1 eq.), sodium carbonate (191 mg, 1.38 mmol, 3 eq.), water (0.5 mL), 1,4-dioxane (2.0 mL), and the resulting reaction mixture was degassed by bubbling argon for 5 minutes before adding Pd(PPh₃)₄ (17 mg, 0.01 mmol, 0.03 eq.). The resulting reaction mixture was heated to 100 °C under argon overnight. The reaction mixture was gradually cooled down to ambient temperature and filtered through a celite bed. The celite bed was washed with dichloromethane (10 mL) and the organic layer was diluted with water (10 mL). The aqueous layer was extracted with dichloromethane (2 × 10 mL). The combined organic layers were concentrated under reduced pressure to remove solvents, and the crude product was purified by SiO₂ chromatography (ethyl acetate 0-60% in hexanes) to yield the title compound as a yellow solid (94 mg, 68%). HPLC 74.7% (t_R = 11.93 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (600 MHz, DMSO-d₆) δ 12.32 (s, 1H), 8.84 (d, J = 0.8 Hz, 1H), 8.39 (s, 1H), 7.64 (d, J = 3.6, 1H), 7.05 (d, J = 3.6, 1H), 4.56 (td, J = 9.8, 4.0 Hz, 1H), 3.27 – 3.18 (m, 2H), 2.45 – 2.38 (m, 1H), 1.86 – 1.78 (m, 1H), 1.64 – 1.44 (m, 4H), 1.35 – 1.24 (m, 3H); HPLC–MS (ESI+): m/z 341.2 (M+H)⁺.

tert-Butyl (R)-2-chloro-4-(1-(2-cyano-1-cyclopentylethyl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (21):

To a solution of pyrrolopyrimidine 20 (94.0 mg, 0.28 mmol, 1.0 eq.), in dichloromethane (2.0 mL), added N,N-diisopropylethylamine (0.058 mL, 0.33 mmol, 1.2 eq) and then added di-tert-butyl dicarbonate (90.3 mg, 0.41 mmol, 1.5 eq) and 4-dimethylaminopyridine (7.0 mg, 0.06 mmol, 0.2 eq). The reaction mixture was stirred for 2 hours at room temperature. The reaction mixture was diluted with water (10 mL) and extracted with dichloromethane (3 × 10 mL). The organic phase was evaporated under reduced pressure and the residue was purified by SiO₂ chromatography using ethyl acetate/hexane (50 %) as eluent to give the title compound as a white solid (98 mg, 80%). HPLC 95.2% (t_R = 13.63 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (600 MHz, DMSO-d₆) δ 8.90 (d, J = 0.8 Hz, 1H), 8.43 (s, 1H), 7.91 (d, J = 4.1 Hz, 1H), 7.26 (d, J = 4.2 Hz, 1H), 4.55 (td, J = 9.8, 4.0 Hz, 1H), 3.24 – 3.16 (m, 2H), 1.85 – 1.78 (m, 1H), 1.58 – 1.40 (m, 4H), 1.35 – 1.22 (m, 12H); HPLC–MS (ESI+): m/z 441.2 (M+H)⁺.

tert-Butyl (R)-2-((4-(1-(tert-butoxycarbonyl)piperidin-4-yl)phenyl)amino)-4-(1-(2-cyano-1-cyclopentylethyl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (22):

A mixture of the boc-pyrrolopyrimidine 21 (47.0 mg, 0.11 mmol, 1.0 eq.), tert-butyl 4-(4-aminophenyl)piperidine-1-carboxylate (30.0 mg, 0.11 mmol, 1.0 eq.) and potassium carbonate (30.0 mg, 2 mmol, 2.0 eq.) in anhydrous tert-butyl alcohol (2 mL) was degassed by bubbling argon. To this mixture, added 2-dicyclohexylphosphino-2’,4’,6’-triisopropylbiphenyl (10.0 mg, 0.021 mmol, 0.2 eq.) and tris(dibenzylideneacetone)dipalladium (10.0 mg, 0.01 mmol, 0.1 eq.). The reaction was refluxed overnight, then allowed to cool to room temperature, water was added (10 mL) and the mixture was extracted with dichloromethane (3 × 10 mL). The combined organic extracts were washed with brine, dried (Na₂SO₄), filtered and the solvent was removed under reduced pressure. The residue was purified by SiO₂ chromatography using ethyl acetate/hexane (50 %) as eluent to give the title compound as a yellow solid (54 mg, 89 %). HPLC 97.1% (t_R = 15.13 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 9.43 (s, 1H), 8.75 (d, J = 0.7 Hz, 1H), 8.33 (s, 1H), 7.93 – 7.90 (m, 2H), 7.56 (d, J = 4.2 Hz, 1H), 7.15 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 4.2 Hz, 1H), 4.56 (td, J = 9.6, 4.3 Hz, 1H), 3.27 – 3.20 (m, 2H), 2.46 – 2.43 (m, 1H), 1.87 – 1.79 (m, 1H), 1.76 (d, J = 13.0 Hz, 2H), 1.66 (s, 9H), 1.57 – 1.45 (m, 6H), 1.43 (s, 9H), 1.37 – 1.16 (m, 8H); HPLC–MS (ESI+): m/z 681.5 (M+H)⁺.

(R)-3-Cyclopentyl-3-(4-(2-((4-(piperidin-4-yl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile [(R)-1)]:

The bis-boc-protected derivative 16 (54.0 mg, 0.08 mmol, 1.0 eq.) was suspended in dichloromethane (2 mL) and trifluoroacetic acid (0.12 mL, 1.59 mmol, 20.0 eq.) was added. The mixture was stirred at room temperature for 2 hours and concentrated under reduced pressure. The residue was dissolved in chloroform and washed with 10% potassium carbonate (aq.). The aqueous layer was extracted with chloroform (3 × 10 mL) and combined organic phase was dried (Na₂SO₄) and filtered. The filtrate was concentrated under reduced pressure and the product was dried under vacuum to afford the title compound as a white solid (47 mg, 95%). HPLC 98.7% (t_R = 11.00 min, CH₃OH in 0.1% TFA water 5%~95% in 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 11.49 (s, 1H), 9.01 (s, 1H), 8.68 (s, 1H), 8.30 (s, 1H), 7.80 – 7.76 (m, 2H), 7.21 (d, J = 3.6 Hz, 1H), 7.14 – 7.10 (m, 2H), 6.78 (d, J = 3.6 Hz, 1H), 4.56 (td, J = 9.6, 4.1 Hz, 1H), 3.22 – 3.17 (m, 2H), 3.02 (d, J = 11.9 Hz, 1H), 2.44 – 2.42 (m, 1H), 1.81 – 1.85 (m, 1H), 1.68 (d, J = 13.0 Hz, 2H), 1.61 – 1.44 (m, 6H), 1.36 – 1.22 (m, 8H); HPLC–MS (ESI+): m/z 681.5 (M+H)⁺. HRMS (ESI+) m/z calculated for C₂₈H₃₃N₈ (M+H)⁺ 481.2823, found 481.2830.

3-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)propanenitrile (27)³³:

Using the reported method ³¹, a 100 mL round bottom flask was charged with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (5.0 g, 25.8 mmol, 1.0 equiv.), acrylonitrile (1.04 g, 52.0 mmol, 2.0 equiv.), DBU (1.97 g, 12.9 mmol, 0.5 equiv.), and acetonitrile (50 mL). The mixture was stirred at room temperature overnight. After cooling, the mixture was diluted with ethyl acetate, washed with water twice and brine once, and dried over Na₂SO₄. After filtration the filtrate was concentrated under reduced pressure to yield the nitrile 27 as yellow oil (3.9 g, 62%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.04 (d, J = 0.6 Hz, 1H), 7.65 (d, J = 0.7 Hz, 1H), 4.41 (t, J = 6.4 Hz, 2H), 3.07 (t, J = 6.4 Hz, 2H), 1.26 (s, 9H).

3-(4-(2-Chloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile (28):

To a 50 mL round bottom flask was added 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (3.0 g, 12.0 mmol, 1.2 equiv.), 3-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-yl)propanenitrile 27 (0.51 g, 10.0 mmol, 1.0 equiv.), sodium carbonate (3.4 g, 31.6 mmol, 3.16 equiv.), water (H₂O, 13 mL) and 1,4-dioxane (53 mL), the resulting reaction mixture was degassed by bubbling argon for 5 min before being treated with Pd(PPh₃)₄ (384 mg, 0.333 mmol). The resulting reaction mixture was heated to 100 °C under argon overnight. The reaction mixture was gradually cooled to ambient temperature before being filtered through a Celite bed. The Celite bed was washed with dichloromethane before the filtrate and washes were combined. The two layers were separated, and the aqueous layer was extracted with dichloromethane. The combined organic layers were concentrated under reduced pressure to remove solvents, and the residue was purified by column chromatography (EtOAc 0 to 50% in hexanes) to yield the title compound 28 as a brown solid (1.8 g, 69%). ¹H NMR (500 MHz, DMSO-d₆) δ: 12.33 (s, 1H), 8.81 (d, J = 0.7 Hz, 1H), 8.37 (d, J = 0.7 Hz, 1H), 7.64 (dd, J = 3.6, 2.3 Hz, 1H), 7.03 (dd, J = 3.6, 1.7 Hz, 1H), 4.53 (t, J = 6.4 Hz, 2H), 3.19 (t, J = 6.4 Hz, 2H). HPLC-MS (ESI+): m/z 273.4 (M+1)⁺, 567.3 (2M+Na)⁺.

tert-Butyl-2-chloro-4-(1-(2-cyanoethyl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (29):

To a solution of the chloropyrrolopyrimidine 28 (850 mg, 3.12 mmol, 1.2 equiv.) in DCM (15 mL) was added (Boc)₂O (1.0 g, 4.68 mmol, 1.5 equiv.), DIPEA (0.49 g, 3.75 mmol, 1.2 equiv.) and DMAP (76 mg, 0.624 mmol, 0.2 equiv.). The reaction mixture was stirred at room temperature for 12 h, quenched with water and extracted with EtOAc (2 × 20 mL). The organic layer was dried (Na₂SO₄) and concentrated in vacuo to yield the title compound 29 as a brown solid (1.12 g, 96%). ¹H NMR (500 MHz, DMSO-d₆) δ: 8.87 (d, J = 0.9 Hz, 1H), 8.40 (d, J = 0.7 Hz, 1H), 7.91 (d, J = 4.1 Hz, 1H), 7.25 (d, J = 4.2 Hz, 1H), 4.53 (t, J = 6.4 Hz, 2H), 3.19 (t, J = 6.4 Hz, 2H), 1.64 (s, 9H). HPLC-MS (ESI+): m/z 373.1 [60%, (M+H)⁺], 769.2 [100% (2M+Na)⁺].

tert-Butyl-2-((4-(1-(tert-butoxycarbonyl)piperidin-4-yl)phenyl)amino)-4-(1-(2-cyanoethyl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (30):

A mixture of tert-butyl-2-chloro-4-(1-(2-cyanoethyl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine-7-carboxylate (29) (0.37 g, 1.0 mmol, 1.2 equiv.), tert-butyl-4-(4-aminophenyl)piperidine-1-carboxylate (0.33 g, 1.2 mmol, 1.2 equiv.) and K₂CO₃ (0.21 g, 1.5 mmol) in anhydrous ^tBuOH (10 mL) was degassed under argon. To this mixture was added XPhos (48 mg, 0.1 mmol,) and Pd₂(dba)₃ (46 mg, 0.05) and the reaction was heated under for 6 h, then allowed to cool. To this mixture was added water (40 mL) and the mixture was extracted with CH₂Cl₂. The combined organic extracts were washed with brine, dried, filtered and the solvent removed under reduced pressure. The residue was purified by flash chromatography (50% EtOAc/hexanes) to yield the title compound 30 as a brown solid (0.40 g, 79%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.43 (s, 1H), 8.72 (d, J = 1.6 Hz, 1H), 8.31 (d, J = 1.5 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 4.2 Hz, 1H), 7.14 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 4.2 Hz, 1H), 4.53 (m, 2H), 4.02 (m, 2H), 3.16 (m, 2H), 2.80 (m, 2H), 2.6 (m, 1H), 1.75 (m, 2H), 1.65 (s, 9H), 1.47 (m, 2H), 1.42 (s, 9H). HPLC–MS (ESI+): m/z 613.1 [100%, (M+H)⁺].

3-(4-(2-((4-(Piperidin-4-yl)phenyl)amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)propanenitrile (2):

To a solution of the bis-boc-protected pyrrolopyrimidine 30 (0.306 g, 0.5 mmol) in DCM (3 mL) was added TFA (3 mL) under argon atmosphere and the reaction mixture was stirred at room temperature for 4 h. After this time there was no starting material present (as measured by HPLC), and the reaction was quenched with sat. NaHCO₃ solution and extracted with DCM (2 × 10 mL). The combined organic layers were dried over (Na₂SO₄) and evaporated to dryness in vacuo to yield the title compound 2 as a brown solid (0.121 g, 81%). ¹H NMR (500 MHz, DMSO-d₆) δ: 11.49 (s, 1H), 9.06 (s, 1H), 8.67 (s, 1H), 8.29 (s, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.22 (d, J = 3.6 Hz, 1H), 7.13 (d, J = 8.3 Hz, 2H), 6.77 (d, J = 3.6 Hz, 1H), 4.53 (t, J = 6.4 Hz, 2H), 3.13 (m, 4H), 2.69 (m, 2H), 2.60 (m, 1H), 1.77 (dd, J = 13.6, 3.4 Hz, 2H), 1.58 (m, 2H). HPLC-MS (ESI+): m/z 207.2 [100% (1/2M+H)²⁺], 413.3 [40% (M+H)⁺]. m/z calculated for C₂₃H₂₅N₈ (M+H)⁺ 413.2197, found 413.2191.

1-(But-3-en-1-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (31):

A 100 mL round bottom flask was charged with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.5 g, 2.58 mmol, 1.0 eq.), 4-bromobutene (0.48 g, 3.6 mmol, 1.4 eq.), cesium carbonate (1.67 g, 5.16 mmol, 2 eq.), and acetonitrile (10 mL). The reaction mixture was stirred at 90 °C overnight. After cooling, the mixture was quenched with water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The organic layers were combined, washed with brine (50 mL), dried (Na₂SO₄) and filtered. The solvent was evaporated under reduced pressure to yield the title product as yellow oil. ¹H NMR (500 MHz, chloroform-d) δ 7.78 (s, 1H), 7.67 (s, 1H), 5.78 (m, 1H), 5.09 (m, 2H), 4.20 (t, J = 7.5 Hz, 2H), 2.63 (m, 2H), 1.31 (s, 12H).

4-(1-(But-3-en-1-yl)-1H-pyrazol-4-yl)-2-chloro-7H-pyrrolo[2,3-d]pyrimidine (32):

A 50 mL round bottom flask was charged with 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (0.3 g, 1.6 mmol, 1.0 eq.), 1-(but-3-en-1-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole 31 (0.51 g, 2.08 mmol, 1.5 eq.), sodium carbonate (0.33 g, 3.2 mmol, 2.0 eq.), water (H₂O, 5 mL) and 1,4-dioxane (15 mL).The reaction mixture was degassed by bubbling argon for 5 min before treating with Pd(PPh₃)₄ (199 mg, 0.172 mmol, 0.107 eq.). The mixture was heated to 100 °C under argon overnight. The reaction mixture was gradually cooled down to ambient temperature, quenched with water (20 mL) and the mixture was extracted with ethyl acetate (3 × 30 mL). The organic layers were combined, washed with brine (50 mL), dried (Na₂SO₄) and filtered. The solvent was evaporated under reduced pressure and the residue was triturated with Et₂O and hexanes to give 32 as a yellow solid (0.32 g, 82%). ¹H NMR (500 MHz, DMSO-d₆) δ 12.28 (s, 1H), 8.69 (s, 1H), 8.28 (s, 1H), 7.60 (dd, J = 3.0, 1.5 Hz, 1H), 7.02 (d, J = 3.0 Hz, 1H), 5.84 (m, 1H), 5.08 (m, 2H), 4.31 (t, J = 7.5 Hz, 2H), 2.64 (m, 2H). HPLC-MS (ESI+): m/z 274.2 (M+1)⁺, 569.2 (2M+Na)⁺.

4-(1-(But-3-en-1-yl)-1H-pyrazol-4-yl)-2-chloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine (33):

To a solution of 4-(1-(but-3-en-1-yl)-1H-pyrazol-4-yl)-2-chloro-7H-pyrrolo[2,3-d]pyrimidine 32 (1.0 g, 5.32 mmol. 1.0 eq.), p-toluenesulfonyl chloride (1.1 g, 5.85 mmol, 1.1 eq.) and tetra-butylammonium hydrogen sulfate (0.090 g, 0.27 mmol, 0.05 eq.) in dichloromethane (20 mL) was added NaOH (50% aq, 0.2 mL). The reaction mixture was stirred at room temperature for 30 minutes. After completion of the reaction, (as indicated by TLC) the reaction mixture was diluted with H₂O (20 mL). The organic layer was separated, washed with brine (50 mL), dried (Na₂SO₄) and filtered. The organic layer was evaporated under reduced pressure to obtain a light yellow solid, which was purified by SiO₂ chromatography using hexanes/ethyl acetate (5:1) as eluent to give the title product (1.3 g, 85%). ¹H NMR (500 MHz, DMSO-d₆) δ 8.75 (s, 1H), 8.29 (s, 1H), 8.04 (d, J = 4.5 Hz, 1H), 8.03 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 4.5 Hz, 1H), 5.81 (m, 1H), 5.05 (m, 2H), 4.29 (t, J = 7.5 Hz, 2H), 2.61 (m, 2H). 2.37 (s, 3H). HPLC-MS (ESI+): m/z 428.1 (M+1)⁺, 877.1 (2M+Na)⁺.

tert-Butyl 4-(4-((4-(1-(But-3-en-1-yl)-1H-pyrazol-4-yl)-7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)phenyl)piperidine-1-carboxylate (34):

A mixture of 4-(1-(but-3-en-1-yl)-1H-pyrazol-4-yl)-2-chloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine 33 (300 mg, 0.7 mmol, 1.0 eq.), tert-butyl 4-(4-aminophenyl)piperidine-1-carboxylate (200 mg, 0.74mmol, 1.06 eq.) and K₂CO₃ (200 mg, 1.4 mmol, 2.0 eq.) in anhydrous ^tBuOH (15 mL) was degassed by bubbling argon for 5 min. XPhos (33 mg, 0.07 mmol, 0.1 eq.) and Pd₂(dba)₃ (32 mg, 0.035, 0.05 eq.) were added and the mixture was refluxed overnight. The mixture was allowed to cool to room temperature and quenched with water (40 mL). The mixture was extracted with CH₂Cl₂ (3 × 30 mL). The combined organic extracts were washed with brine, dried (Na₂SO₄), filtered and the solvent was removed under reduced pressure. The residue was purified by SiO₂ chromatography (0 to 60% EtOAc in Hexanes) to afford the title compound as yellow solid (330 mg, 85%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.52 (s, 1H), 8.60 (s, 1H), 8.19 (s, 1H), 8.02 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 4.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 5.79 (m, 1H), 5.07 (m, 2H), 4.30 (t, J = 7.5 Hz, 2H), 4.09 (br s, 2H), 2.82 (br s, 2H), 2.69 – 2.58 (m, 4H), 2.32 (s, 3H), 1.80 (m, 2H), 1.53 (m, 2H), 1.48 (s, 9H). HPLC-MS (ESI+): m/z 668.3 (M+1)⁺.

tert-Butyl 4-(4-((4-(1-(but-3-en-1-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)phenyl)piperidine-1-carboxylate (35):

To a solution of tert-butyl 4-(4-((4-(1-(but-3-en-1-yl)-1H-pyrazol-4-yl)-7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)phenyl)piperidine-1-carboxylate 34 (165 mg, 0.29 mmol, 1.0 eq.) in MeOH (5 mL) and H₂O (3 mL) was added K₂CO₃ (165 mg, 1.20 mmol, 4.0 eq.) and the mixture was refluxed for 3 h. The mixture was diluted with H₂O (30 mL) and extracted with EtOAc (3 × 30 mL). The combined organic extracts were washed with brine, dried (Na₂SO₄), filtered and the solvent was removed under reduced pressure to give a yellow residue. The residue was purified by SiO₂ flash chromatography (0 to 70% EtOAc in hexanes) to give the title compound (105 mg, 87%) as a white solid. ¹H NMR (500 MHz, DMSO-d₆) δ 11.47 (s, 1H), 9.03 (s, 1H), 8.55 (s, 1H), 8.20 (s, 1H), 7.81 (d, J = 9.0 Hz, 2H), 7.19 (m, 1H), 7.14 (d, J = 9.0 Hz, 2H), 6.76 (m, 1H), 5.86 (m, 1H), 5.10 (m, 2H), 4.32 (t, J = 7.0 Hz, 2H), 4.10 (br s, 2H), 2.79 (br s, 2H), 2.65 – 2.58 (m, 3H), 1.76 (m, 2H), 1.49 (m, 2H), 1.42 (s, 9H). HPLC-MS (ESI+): m/z 514.4 (M+1)⁺.

4-(1-(But-3-en-1-yl)-1H-pyrazol-4-yl)-N-(4-(piperidin-4-yl)phenyl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (3):

tert-Butyl-4-(4-((4-(1-(but-3-en-1-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)phenyl)piperidine-1-carboxylate 35 (0.22 g, 0.43 mmol, 1.0 eq.) was suspended in CH₂Cl₂ (2 mL) and TFA (0.65 mL, 8.5 mmol, 20 eq.) was added. The mixture was stirred at r.t. for 2 h and concentrated under reduced pressure. The residue was dissolved in CHCl₃ (20 mL) and washed with 10% K₂CO₃ (aq). The aqueous layer was extracted with CHCl₃ (3 × 20 mL). The organic layers were combined, dried (Na₂SO₄) and filtered. The filtrate was concentered under reduced pressure and the residue was dried under vacuum to afford the titled compound as an off-white solid (0.13 g, 75%). HPLC 99.8% (t_R = 8.90 min, CH₃OH 45% in H₂O (0.1% TFA), 1 mL/min, 20 min); ¹H NMR (500 MHz, DMSO-d₆) δ 11.47 (s, 1H), 9.00 (s, 1H), 8.55 (s, 1H), 8.20 (s, 1H), 7.79 (d, J = 7.5 Hz, 2H), 7.19 (d, J = 4 Hz, 1H), 7.12 (d, J = 7.5 Hz, 2H), 6.76 (d, J = 4 Hz, 1H), 5.86 (m, 1H), 5.10 (m, 2H), 4.32 (t, J = 7.0 Hz, 2H), 3.02 (br d, J = 12.0 Hz, 2H), 2.65 (q, J = 7 Hz, 2H), 2.59 (t, J = 12.5 Hz, 2H), 1.68 (d, J = 12.0 Hz, 2H), 1.53 (m, 2H); ¹³C NMR (125 MHz, DMSO-d₆) δ 156.4, 154.2, 151.3, 140.0, 139.3, 139.1, 135.3, 131.3, 126.9, 123.6, 121.2, 118.7, 117.7, 107.6, 100.5, 51.3, 47.2, 42.4, 35.0, 34.3; HPLC-MS (ESI+): m/z 414.3 (M+1)⁺; HRMS (ESI+) m/z calculated for C₂₄H₂₈N₇ (M+H)⁺ 414.2401, found 414.2402.

Dianilinopyrimidine inhibitors of JAK2

N-(2-Fluoro-5-((2-((3-fluoro-4-(1-methylpiperidin-4-yl)phenyl)amino)-5-methylpyrimidin-4-yl)amino)phenyl)-2-methylpropane-2-sulfonamide (4).

This was prepared according the method reported.³⁴

N-(5-((2-((3,5-Difluoro-4-(1-methylpiperidin-4-yl)phenyl)amino)-5-methylpyrimidin-4-yl)amino)-2-fluorophenyl)-2-methylpropane-2-sulfonamide (5).

This was prepared according the method reported.³⁴

4-((4-((4-Chloro-3-((1,1-dimethylethyl)sulfonamido)phenyl)amino)-5-methylpyrimidin-2-yl)amino)-2-fluoro-N-(1-methylpiperidin-4-yl)benzamide (6).

This was prepared according the method reported.³⁵

N⁴-(1-(tert-Butylsulfonyl)indolin-6-yl)-N²-(3-fluoro-4-(1-methylpiperidin-4-yl)phenyl)-5-methylpyrimidine-2,4-diamine (7).

This was prepared according the method reported.³⁴

Cloning and expression of JAK2 kinase domain:

The DNA of human JAK2 KD (JH1), encoding amino acids 840-1132, was synthesized and cloned into pcDNA 3.3 (GeneArt). The construct was expressed using a CMV promoter and contained a His₈ N-terminal affinity tag followed by a TEV cleavage site. Expi293F cells (Invitrogen) were grown, maintained, and treated in shaking culture at 37°C with 8% CO₂ in Expi293 medium (Invitrogen). Recombinant JAK2 was expressed in Expi293F cells using Transporter 5 transfection reagent as described by the manufacturer (PolySciences). One hour after adding the plasmid:transfection complex, varying concentrations of JAK2 inhibitor or transcription enhancer were added as indicated in Fig. 1. The transfection proceeded for 24 hours before harvest. Cells used for the preparation of crystallization-grade JAK2 were incubated with 1 μM Ruxolitinib and 4mM butyric acid for 23 hours.

Purification of JAK2 kinase domain:

Purification of JAK2 KD was performed at 4°C. Expi293F cells were centrifuged at 1,000xg for 30 minutes at 4°C. Cells were resuspended in lysis buffer (50mM HEPES pH 7.5, 250mM NaCl, 10% glycerol, 5mM β-mercaptoethanol, 5mM MgCl₂, 0.1mM ATP, 10mM imidazole, 1mM PMSF, 0.5% Triton X100). The cells were sonicated at 40% power for 2 minutes on ice and then centrifuged at 40,000xg for 1 hour at 4°C to clarify the lysate. The soluble lysate was loaded onto two 5mL HisTrap FF columns in tandem (GE Healthcare). The columns were washed with 10mM imidazole, 40mM imidazole, 100mM imidazole, and 200mM imidazole before applying a gradient up to 600mM imidazole. The protein was concentrated to 10mL using a 10,000 MWCO filter and loaded onto an S75 gel filtration column (GE Healthcare) that was pre-equilibrated with 40mM bicine pH 8.6, 100mM NaCl, 10% glycerol. The single JAK2 peak, as determined by SDS-PAGE and western blot analysis (>99% purity), was collected, concentrated to 8.1mg/mL using a 3,000 MWCO spin concentrator, flash-frozen in liquid N₂ and stored at −80°C.

Cellular Thermal Shift Assay:

Cellular thermal shift assay (CETSA) was used to compare melting curves from ligand-based thermal stabilization of JAK2 KD. Expi293F cells were transfected with His-JAK2 KD from pcDNA 3.3 as described above. Ruxolitinib (20 μM), or the equivalent amount of DMSO, was added to the cells and incubated for one hour at 37°C. After one hour, the cells were centrifuged at 300xg for three minutes at room temperature. The cells were washed in 15mL PBS and centrifuged again at 300xg for three minutes. The cells were resuspended in 1mL PBS and approximately 3x10⁶ cells were added to PCR tubes that were incubated at 40, 43, 46, 49, 52, 55, 58, 61, 64, or 67°C for three minutes. The tubes were then flash-frozen in liquid N₂ and thawed twice at 25°C. The cells were vortexed at 20,000xg for 20 minutes at 4°C. The soluble lysate was transferred to a new tube, 5x SDS-PAGE loading buffer was added, and 13μL (equivalent of 3.3x10⁵ cells) was added to an SDS-PAGE gel. The gel was transferred using the eBlot transfer system (GenScript), blocked with 5% BSA in TBS-T, and blotted for anti-His-HRP, anti-actin or anti-GAPDH-HRP for 2 hours at room temperature. The anti-actin blot was washed then incubated with anti-mouse-HRP conjugated secondary antibody at 1:2,000 for one hour at room temperature. The blots were incubated with SignalFire ECL (Cell Signaling, 6883) and imaged on a GE Healthcare AmerSham Imager 600.

Isothermal Dose-Response:

Isothermal dose-response (ITDR) measures protein stabilization as a function of increasing inhibitor concentration. After performing the CETSA assay described above, the data were graphed and a temperature at the IC₅₀ value (47°C) was used for the isothermal dose-response experiment. Expi293F cells were transfected with His-JAK2 KD. The cells were centrifuged at 300xg for three minutes at room temperature. Cells were resuspended at a density of 4x10⁷ cells/mL. Serial dilutions were performed yielding three-fold dilutions ranging from 10nM to 20μM of inhibitor with constant amounts of DMSO. Cells (approximately 1.2x10⁶ cells) were added to the compounds and were incubated for 30 minutes at 37°C with shaking every 10 minutes. The tubes were heated at 47°C for three minutes. The cells were then vitrified and thawed twice at 25°C before being vortexed at 20,000xg for 20 minutes at 4°C. The soluble supernatant was transferred to a new tube, mixed with 5x SDS-loading dye, and resolved on SDS-PAGE. The western blot transfer and incubation is the same as described above. Data were normalized to 0% and 100%.

Cell Viability Assays:

UKE-1 cells were cultured in RPMI-1640 with 10% FBS at 37°C with 5% CO₂. Suspension cells were seeded at 20,000 cells per well. Cells were incubated with increasing concentrations of compound ranging from 1 nM to 10 μM (as denoted in the figure legends) in the presence of vehicle (0.2% DMSO) with six replicates per concentration for 72 hours at 37°C. After drug treatment, 15 μL of CellTiter Blue (Promega) was added to each well, mixed for one minute using an orbital shaker, and incubated at 37°C for three hours. Fluorescence was measured using a Wallac EnVision 2130 plate reader (PerkinElmer). Excitation and emission filters of 570 nm and 615 nm were used, respectively. Cell viability data were analyzed using GraphPad Prism6.

Protein Crystallography:

Purified His-JAK2 KD at 8.1mg/mL was incubated with 1mM Ruxolitinib and then added to 0.2M NaCl, 0.1M Bis-Tris pH 5.5, and 25% PEG 3350 in a 1:1 v/v ratio on a hanging drop coverslip with the crystallization solution at 293K. Rod-like crystals formed within ten hours and grew to a maximum size over five days. Ligand exchange was performed by moving JAK2-ruxolitinib crystals into the crystallization solution containing 1mM inhibitor of interest for 3 days. The crystals were preserved in a cryoprotectant containing the crystallization solution and 30% glycerol. All data was collected on the 23-ID beamline, GM/CA at the Advanced Photon Source (Argonne, IL). Data were processed and scaled with XDS. Molecular replacement (using PDB 2XA4 as the search model) and refinements were carried out using PHENIX, and model building was performed using Coot. Figures were prepared using PyMOL (Schrödinger, LLC). All structures were validated and deposited in the PDB with accession codes listed in Supplementary Tables S1-S2.

ABBREVIATIONS

JAK2

Janus kinase 2

MPN

myeloproliferative neoplasms

KD

kinase domain

SAR

structure-activity relationship

STATs

signal transducer and activator of transcription proteins