Development of Furanopyrimidine-Based Orally Active Third-Generation EGFR Inhibitors for the Treatment of Non-Small Cell Lung Cancer

Abstract

The development of orally bioavailable, furanopyrimidine-based double-mutant (L858R/T790M) EGFR inhibitors is described. First, selectivity for mutant EGFR was accomplished by replacing the (S)-2-phenylglycinol moiety of 12 with either an ethanol or an alkyl substituent. Then, the cellular potency and physicochemical properties were optimized through insights from molecular modeling studies by implanting various solubilizing groups in phenyl rings A and B. Optimized lead 52 shows 8-fold selective inhibition of H1975 (EGFR^L858R/T790M overexpressing) cancer cells over A431 (EGFR^WT overexpressing) cancer cells; western blot analysis further confirmed EGFR mutant-selective target modulation inside the cancer cells by 52. Notably, 52 displayed in vivo antitumor effects in two different mouse xenograft models (BaF3 transfected with mutant EGFR and H1975 tumors) with TGI = 74.9 and 97.5% after oral administration (F = 27%), respectively. With an extraordinary kinome selectivity (S(10) score of 0.017), 52 undergoes detailed preclinical development.

Introduction

Lung cancer is one of the leading causes of cancer deaths worldwide, accounting for a quarter of all cancer deaths.^1,2 Most lung cancer patients, around 80–85%, have the non-small cell lung cancer (NSCLC) disease, which is characterized by somatic epidermal growth factor receptor (EGFR) activating mutations in 10–15% of Caucasian and 30–50% of Asian patients.^3,4 Several drugs targeting mutated EGFR have been approved, such as the first-generation EGFR inhibitors gefitinib (1) and erlotinib (2), which were approved by the US FDA in 2003 and 2004 (Figure 1).⁵⁻¹⁰ Gefitinib and erlotinib effectively target the EGFR exon 19 deletion or exon 21 L858R single point mutation^11,12 and most patients respond well, but about half of them develop resistance due to a secondary T790M gatekeeper mutation.^13,14 Several second-generation EGFR inhibitors capable of overcoming resistance due to T790M mutations have also been approved,¹⁵ including afatinib (3),¹⁶ dacomitinib (4),¹⁷ and ceritinib (5). These second-generation inhibitors all bear a Michael acceptor warhead that forms an irreversible covalent bond with the cysteine residue (Cys797) in the active site of EGFR.¹⁸

Figure 1.

Known EGFR inhibitors approved or in advanced phase of drug development for NSCLC.

However, major drawbacks with the first- and second-generation drugs were their dose-limiting toxicities, a consequence of EGFR wild-type (EGFR^WT) enzyme inhibition. To address this problem, third-generation EGFR inhibitors capable of overcoming the T790M mutation and sparing the EGFR^WT enzyme were developed, including osimertinib (6, approved by US FDA),¹⁹⁻²¹ rociletinib (7, halted, Phase III),^22,23 olmutinib (8, approved in South Korea),²⁴ avitinib (9, Phase II),²⁵ nazartinib (10, Phase II),²⁶ etc.²⁷

Recently, we disclosed a second-generation furanopyrimidine EGFR inhibitor DBPR112 (11) able to overcome the T790M EGFR mutation.²⁸ Compound 11 bears a Michael acceptor group that makes a covalent bond with the Cys797 residue of EGFR,²⁸ has favorable in vitro and in vivo anticancer profiles, and is currently undergoing Phase Ib/II clinical trials to treat lung cancer with an exon 20 insertion. Herein, we describe our continuation of the design and development of third-generation mutant-selective EGFR inhibitor 52 with improved in vitro and in vivo profiles than those of 11; mainly, compound 52 is EGFR^WT sparing and T790M mutant-selective, which would result in lower toxicities during administration to patients. More importantly, inputs from molecular modeling and in vitro assay data had guided the development of mutant-selective EGFR inhibitors.

Results and Discussion

Preliminary SAR Exploration of 11/12 to Impart EGFR^L858R/T790M Selectivity and Cellular Potency

The newly synthesized compounds were initially investigated for their abilities to inhibit the EGFR wild-type and EGFR double-mutant (EGFR^L858R/T790M) enzymes at 10 and 1 μM concentrations. Compounds with potent inhibition (>90%) at 10 μM were further evaluated in a six-point dose–response assay to determine the IC₅₀ values. Furthermore, the compounds were investigated for their antiproliferative activity on two lung cancer cell lines: A431 cells overexpressing EGFR^WT and H1975 cells overexpressing double-mutant EGFR^L858R/T790M. DBPR112 (11) was found to inhibit the EGFR^WT better than the mutant EGFR kinase (EGFR^WT IC₅₀ = 15 nM vs EGFR^L858R/T790M IC₅₀ = 48 nM; Table 1). In the cell-based antiproliferative assay, it displayed moderate potency (A431 CC₅₀ = 1020 nM and H1975 CC₅₀ = 620 nM). Moreover, one of our initial lead 12 without the N,N-dimethylamino solubilizing functionality was also a nonselective (EGFR^WT IC₅₀ = 20 nM vs EGFR^L858R/T790M IC₅₀ = 27 nM) inhibitor.^28,29 The antiproliferative activity of 12 in both cell lines (CC₅₀ > 1 μM) was lower than that of 11. In addition, it was noted that clinical candidate 11 had a molecular weight (Ml. Wt.) of 533.6 and log D_7.4 of 4.0. Hence, lead optimization effort to develop mutant-selective EGFR inhibitors should also optimize the physicochemical properties to improve potent cellular activity and optimal pharmacokinetics profile. With these goals in mind, we choose 12 as the starting point for the lead optimization effort due to its lower Ml. Wt. (476.5). Moreover, the acrylamide functional group in 12 was also present in several other reported irreversible EGFR inhibitors, including 6–9.

Table 1. SAR Exploration of the Leads 11 and 12.

^aPhysicochemical properties were determined using Discovery Studio version 2021.

^bIC₅₀ values and inhibition ratio of EGFR^WT and EGFR^L858R/T790M were calculated using the in-house Kinase-Glo assay, and the IC₅₀ values represent the mean of at least two independent experiments and are within ±15%.

Having previously established the importance of an acrylamide moiety at the meta-position of the phenyl ring for potent EGFR^L858R/T790M inhibition,²⁸ we retained this functionality and first investigated modifications to the side chain attached to the quinazoline ring 4-position. Molecular docking of 6 and 11 with EGFR^WT showed that the phenyl ring in the side chain of 11 established more extensive σ–π hydrophobic interactions in the EGFR back pocket compared to the N-methylindole ring of 6. Hence, we attempted to attenuate this hydrophobic interaction with EGFR^WT to improve the selectivity for EGFR^L858R/T790M. Replacing the phenyl group in the side chain of 12 with the smaller isopropyl group to give 13 (EGFR^WT IC₅₀ = 44 nM and EGFR^L858R/T790M IC₅₀ = 56 nM) showed that the presence of an aromatic ring in the side chain is not essential for EGFR inhibition (Table 1). This was further confirmed by synthesizing compounds 14 and 15 bearing a methyl group and hydrogen atom instead of the phenyl group, respectively. Both 14 and 15 potently inhibited EGFR with EGFR^WT sparing activity, with 15 (EGFR^WT IC₅₀ = 393 nM vs EGFR^L858R/T790M IC₅₀ = 38 nM) showing over 10-fold selectivity toward mutant EGFR^L858R/T790M over the EGFR^WT kinase similar to 6. Collectively, these results validate the strategy of increasing the inhibition and selectivity for mutant EGFR vs EGFR^WT by decreasing the σ–π hydrophobic interactions in the back pocket of EGFR. Hence, these investigations spurred us to further explore the role of the N-amino ethanol side chain in the 4-position of the furanopyrimidine ring for EGFR selectivity. More importantly, the removal of the phenyl ring from the side chain also resulted in better physicochemical properties like lower Ml. Wt. (400.4) and log D_7.4 (3.5) for 15, as compared to 12.

As a next step, 15 was evaluated in an antiproliferative assay using A431 and H1975 lung cancer cell lines. Unexpectedly, 15 showed cellular potency with a CC₅₀ > 1 μM in both the cell lines, suggesting that both the enzymatic activity and the compound’s physicochemical properties need to be carefully considered for optimal cellular potency. Hence, to further decrease the Ml. Wt./log D_7.4, the phenyl group attached to the furan ring was removed to obtain 16 (Ml. Wt. = 324.3 and log D_7.4 = 1.7), which resulted in the complete loss of inhibition potency for the EGFR^WT and EGFR^L858R/T790M kinases (IC₅₀ > 1 μM). Subsequently, we focused on the modifications in the N-amino ethanol side chain attached to the 4-position quinazoline ring. Increasing the length of the side chain by one carbon (17; EGFR^WT IC₅₀ = 387 nM vs EGFR^L858R/T790M IC₅₀ = 54 nM) did not alter the inhibition as well as selectivity much for the EGFR enzyme. Next, to understand the importance of the hydroxy functionality, it was masked with methyl (18) or with a phenyl group (19), which resulted in lower levels of EGFR inhibition and the loss of activity was more prominent in the case of bulky phenyl ether 19. Also, attempts to replace the hydroxy group with an ester (20; EGFR^WT IC₅₀ = 376 nM vs EGFR^L858R/T790M IC₅₀ = 142 nM) and carboxylic acid (21; EGFR^WT IC₅₀ = 89 nM vs EGFR^L858R/T790M IC₅₀ = 61 nM), both resulted in lower levels of EGFR inhibition and a concomitant loss of selectivity toward the mutant EGFR^L858R/T790M enzyme. Third, replacement of the hydroxy group of 15 with an amino (22) or N,N-dimethylamino (23) group resulted in the loss of EGFR inhibition (IC₅₀ > 1 μM) for both the wild-type and mutant enzymes.

Unable to identify a suitable replacement for the hydroxy group, we next investigated if the entire ethanol side chain of 15 could be replaced. Simple N-alkyl substitutions such as methyl (24), N,N-dimethyl (25), isopropyl (26), cyclopropyl (27), cyclobutyl (28), cyclopentyl (29), and cyclohexyl (30) groups instead of the ethanol side chain were well-tolerated (EGFR^L858R/T790M IC₅₀ of 3–65 nM) and also showed varying levels of selectivity (3.1 to 60-fold) toward the mutant EGFR over the EGFR^WT (Table 1). It is particularly interesting that 29 with the cyclopentyl substitution at the amino group displayed around 61-fold EGFR^L858R/T790M (IC₅₀ of 41 nM) mutant-selective kinase inhibition. However, compounds 24–30 were poorly antiproliferative (CC₅₀ > 1 μM) in NSCLC cell lines, suggesting that their ability to enter the cells was poor due to unfavorable log D_7.4 of 4.0–5.9 for these compounds.

We next set out to improve the cellular potency of our compounds. Several studies, including ours, have shown that introducing suitable solubilizing functional groups at an appropriate place in the molecules could aid in increasing the cellular potency by altering the physicochemical property, particularly the log D_7.4 values.³⁰ For example, the introduction of N,N-dimethylamino solubilizing group in the acrylamide side chain of the initial lead compound 12 to develop 11 has resulted in a lowering of log D_7.4 value from 5.2 to 4.0. Even though both showed similar levels of EGFR kinase enzyme inhibitions, 11 showed better cellular antiproliferative activity in the H1975 cell line (CC₅₀ = 620 nM), but 12 was not active, suggesting that the presence of N,N-dimethylamino solubilizing group could potentially enhance cellular potency by improving the permeability of the compound into the cells.

Hence, we introduced the N,N-dimethylamino solubilizing group in the acrylamide side chain in 15 and 29 to prepare two analogues. Both 31 (with the N-ethylamino side chain; log D_7.4 = 2.4) and 32 (with the N-cyclopentylamino side chain; log D_7.4 = 4.4) showed lower levels of EGFR inhibition (both wild-type and mutant enzymes), compared to 15 and 29, respectively (Table 1). But both compounds retained selectivity (8 to 14-fold) toward the double-mutant EGFR compared to the EGFR^WT enzyme inhibition. Due to lower levels of EGFR enzyme inhibition, both 31 and 32 did not show antiproliferative activity in both the cell lines (CC₅₀ > 1 μM). Our preliminary SAR investigations of 11/12 suggest that smaller hydrophobic groups such as methyl, isopropyl or cyclopentyl group at the 4-position amino terminus of the furanopyrimidine ring could impart EGFR double-mutant selectivity. However, their enzymatic activity could not be translated to cellular potency due to the poor physicochemical property.

Molecular Docking and Design Strategy to Improve Cellular Potency

As the incorporation of a solubilizing functional group in compounds 31 and 32 lowered its EGFR kinase inhibition activity and failed to impart cellular potency, we used molecular docking studies to identify a site capable of bearing a solubilizing group without that group also affecting the EGFR kinase enzyme inhibitory activity. Osimertinib (6) was docked onto the crystal structure of DBPR112 (11) and their binding orientation in the EGFR protein was compared (Figure 2). It was found that the Michael acceptor group of 6 was placed near the Cys797 residue, similar to 11. While the solubilizing group N,N,N′-trimethylethyl amino side chain of 6 was placed next to phenyl ring A of 11, suggesting that this ring could be a suitable option for introducing the solubilizing group instead of phenyl ring B of 11. Moreover, phenyl ring A is directed toward the solvent-accessible area of the protein, and introducing a solubilizing group in this ring could be well-tolerated without altering the protein–ligand interactions. Additionally, the solubilizing group could also be introduced to the para-position of phenyl ring B, as this position is directed toward the solvent-accessible region of the protein. Hence, it was contemplated to move the solubilizing group to phenyl ring A (strategy I) and the para-position of phenyl ring B (strategy II) of furanopyrimidine, as outlined in Figure 2.

Figure 2.

Identification of the suitable substitution site for introducing the solubilizing group in the furanopyrimidine analogues. (A) Docked orientation of 6 superimposed onto the cocrystal binding orientation of 11 in the EGFR structure (PDB ID: 6JZ0). Solvent-exposed regions of 11 suitable for introducing solubilizing groups are shown with arrows. (B) Two strategies (I and II) for introducing solubilizing groups into the phenyl ring of furanopyrimidine analogues are shown.

Design Strategy I: SAR Exploration by Introducing Solubilizing Groups in Phenyl Ring A

Based on the results of the molecular docking studies, a selection of solubilizing groups was introduced at the para-position of phenyl ring A of 15 to give compounds 33–41, whose structure–activity relationships (SAR) were then studied (Table 2). Compound 33 bearing an N,N-dimethylamino group at this position (log D_7.4 = 3.6) was a potent and selective inhibitor of EGFR (EGFR^WT IC₅₀ = 69 nM vs EGFR^L858R/T790M IC₅₀ = 14 nM) with a mutant selectivity of 5. However, 33 did not exhibit sufficient antiproliferative activity in either cell lines (CC₅₀ > 1 μM). Next, morpholine and N-methyl piperazine groups were introduced at the para-position of phenyl ring A of 15 to give 34 (log D_7.4 = 3.3) and 35 (log D_7.4 = 3.4), respectively, both of which demonstrated potent EGFR enzyme inhibition activity in the low nanomolar range and mutant selectivities of 21-fold for 34 and 3.6-fold for 35. In the cell-based assay, 34 was poorly antiproliferative (CC₅₀ > 1 μM). However, 35 exhibited significantly enhanced antiproliferative activity in the H1975 cell line with an IC₅₀ of 126 nM and 21-fold selectivity toward the H1975 cell line as compared to A431 cell line, suggesting a strong EGFR^WT sparing cellular antiproliferative activity. With the encouraging results, we have also introduced the solubilizing functional group N,N,N′-trimethylethyl amino side chain, which is present in 6. Compound 36 (log D_7.4 = 2.6) with this solubilizing group at the para-position of phenyl ring A of 15 possessed potent and mutant-selective EGFR enzyme inhibition (EGFR^L858R/T790M IC₅₀ = 7 nM and 3.6-fold selectivity over EGFR^WT). However, 36 did not show potent antiproliferative activity in both A431 and H1975 cell lines (CC₅₀ > 1 μM). It should be noted that even though 36 had a lower log D_7.4 value, the number of rotatable bonds was more than 10 and could be a possible reason for poor cellular potency.

Table 2. SAR Exploration of 15 and 29 by the Introduction of Solubilizing Groups in Phenyl Ring A.

^aPhysicochemical properties were determined using Discovery Studio version 2021.

^bThe IC₅₀ values and inhibition ratio of EGFR^WT and EGFR^L858R/T790M were calculated using the in-house Kinase-Glo assay, and the IC₅₀ values represent the mean of at least two independent experiments and are within ±15%.

^cThe CC₅₀ values represent the mean of at least two independent experiments and are within ±15%.

Next, we introduced the solubilizing groups morpholine and N-methyl piperazine in the para-position of phenyl ring A of 29 to prepare 37 and 38, respectively (Table 2). Both compounds showed excellent EGFR inhibition and 5 to 8-fold selectivity for mutant EGFR over EGFR^WT. Once again, only the N-methyl piperazine analogue 38 showed good cellular potency with a CC₅₀ of 600 nM in the H1975 cell line. Interestingly, 38 had a CC₅₀ of 3961 nM in A431 cell line overexpressing the EGFR^WT, suggesting that its selectivity for mutant EGFR over EGFR^WT was about 8-fold. As N-methyl piperazine bearing compounds 35 and 38 displayed good cellular potency, the N-methyl piperazine solubilizing group was also introduced in 12, 24, and 30 to synthesize 39, 40, and 41, respectively. All three compounds exhibited potent and selective EGFR enzyme inhibition (EGFR^L858R/T790M IC₅₀ = 2–6 nM and 3.2 to 5.2-fold selectivity over EGFR^WT). More importantly, all three compounds exhibited antiproliferative activity in H1975 cell line with a CC₅₀ range of 502–739 nM and displayed 2 to 10-fold selectivity over the A431 cell line. In addition, we have also replaced phenyl ring A in 29 with a more hydrophilic 4-pyridine ring in 42, morpholine-substituted 3-pyridine ring in 43, and N-methyl piperazine-substituted 3-pyridine ring in 44. These replacements resulted in potent and selective EGFR^L858R/T790M inhibition (IC₅₀ of 13–32 nM and selectivity of 4.1 to 12-fold over the EGFR^WT), but did not significantly increase cellular potency (CC₅₀ > 1 μM). Overall, the order of cellular enhancement by the solubilizing group in the para-position of phenyl ring A is N-methyl piperazine > N,N,N′-trimethylethyl amino > morpholine.

Design Strategy II: SAR Exploration by Introducing Solubilizing Groups in Phenyl Ring B

Next, our attention turned to investigating the second strategy that is focused on introducing solubilizing functional groups in the para-position of phenyl ring B of 29. The introduction of the morpholine group resulted in EGFR^WT sparing potent enzyme inhibitor 45 with a selectivity ratio of 14 toward the double-mutant EGFR^L858R/T790M (IC₅₀ = 8 nM) (Table 3). At the same time, the introduction of N-methyl piperazine (46) resulted in only a selectivity of 1.8-fold for the double-mutant EGFR^L858R/T790M (IC₅₀ = 61 nM) over the EGFR^WT. Furthermore, two other solubilizing functional groups with alkylamino side chains were introduced to the para-position of phenyl ring B of 29 through an ether linkage to synthesize compounds 47 and 48. Even though both maintained good activity and selectivity for the mutant EGFR kinase (IC₅₀ of 26–43 nM), they were not active in the cellular antiproliferation assay (CC₅₀ > 1 μM). Also, the N,N,N′-trimethylethyl amino side chain, which is present in 6 was introduced in the para-position of phenyl ring B of 29. Compound 49 with this solubilizing group displayed potent double-mutant EGFR^L858R/T790M (IC₅₀ = 7 nM) activity along with 5-fold selectivity against the EGFR^WT enzyme. In the cellular antiproliferation assay, compound 49 selectively inhibited the proliferation of double-mutant overexpressing H1975 NSCLC cells with a CC₅₀ of 310 nM and 13-fold selectivity over A431 NSCLC cells overexpressing EGFR^WT kinase.

Table 3. SAR Exploration of 15 and 29 by the Introduction of Solubilizing Groups in Phenyl Ring B.

^aPhysicochemical properties were determined using Discovery Studio version 2021.

^bThe IC₅₀ values and inhibition ratio of EGFR^WT and EGFR^L858R/T790M were calculated using the in-house Kinase-Glo assay, and the IC₅₀ values represent the mean of at least two independent experiments and are within ±15%.

^cThe CC₅₀ values represent the mean of at least two independent experiments and are within ±15%.

With these encouraging results for 49, we synthesized compounds 50–53 bearing methyl (50), ethyl (51), isopropyl (52), and cyclopropyl (53) groups in place of the cyclopentyl ring (Table 3). Of these compounds, 52 and 53 showed potent enzymatic and cellular antiproliferative activities and EGFR^WT sparing activity. Of particular interest was compound 52, which displayed over 8-fold selectivity for H1975 over A431 cell lines (CC₅₀, A431 = 1373 nM and H1975 = 179 nM). Moreover, we also investigated the N-amino ethanol side chain in the furanopyrimidine ring 4-position and found that 54 showed about 2.7-fold selective EGFR^L858R/T790M enzyme inhibition over the EGFR^WT. However, 54 (analogue of 15 bearing the N,N,N′-trimethylethyl amino side chain in the para-position of phenyl ring B) showed poor cellular antiproliferative activity in both A431 and H1975 cell lines (CC₅₀ > 1 μM).

As a next step, we synthesized stereoisomers 55 and 56, hydroxylated versions of 49, to alter the physicochemical properties and improve cellular potency. Compounds 55 and 56 exhibited potent mutant EGFR kinase inhibition (IC₅₀ of 15 and 7 nM) and H1975 cellular proliferation inhibition (CC₅₀) of 119 and 298 nM, respectively. Moreover, both compounds were EGFR^WT sparing, with selectivities of 8.0 and 5.6 in the antiproliferation assay. Similarly, compounds 57 and 58, bearing a hydroxylated cyclobutyl ring, showed potent mutant EGFR kinase inhibition (IC₅₀ of 10 and 9 nM), with a selectivity of 7.6 and 8.7-fold over the EGFR^WT kinase enzyme, respectively. More importantly, 57 and 58 similar to 55 and 56 displayed potent (CC₅₀, 100 and 117 nM) and selective (15.5 and 13.5-fold over A431) antiproliferative activity in H1975 cell lines.

Several compounds bearing a solubilizing functional group in phenyl ring A (35, 38, 39, 40, and 41) and in phenyl ring B (49, 50, 51, 52, 53, 55, 56, 57, and 58) exhibited EGFR^WT sparing potent antiproliferative activity in H1975 cell lines. Analysis of the physicochemical properties of these compounds (Tables 2 and 3) showed that the log D_7.4 values were in the range of 3.4–5.8 for compounds in Table 2 (solubilizing group in phenyl ring A), while it was in the range of 2.7–4.6 for compounds in Table 3 (solubilizing group in phenyl ring B). It is interesting to note that the cellular antiproliferative activities of compounds bearing a solubilizing group on phenyl ring B were better than those bearing that group on phenyl ring A. Our SAR exploration clearly shows the benefit of introducing a solubilizing group in an appropriate position, N-methyl piperazine in phenyl ring A and N,N,N′-trimethylethyl amino in phenyl ring B, for modulating the enzymatic and sparing the EGFR^WT. The SAR findings to improve the EGFR mutant selectivity and cellular potency in furanopyrimidines are summarized in Figure 3.

Figure 3.

Suitable functional groups identified in this study to improve the EGFR mutant selectivity and cellular potency in furanopyrimidines.

In Vitro and In Vivo Pharmacokinetics Investigations

With several potent compounds in hand, we investigated a few of the most interesting compounds for their ability to resist drug metabolism using an in vitro microsomal stability assay. Compounds were individually incubated with rat liver microsomes for 1 h; then, the proportion of unmetabolized compound remaining was estimated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). It was found that 35, 49, 52, 55, 57, and 58 were 70.4, 33.1, 58.4, 26.1, 27.9, and 9%, unmetabolized, respectively (Tables 2 and 3). This assay supports the candidacy of 35, 49, 52, and 57 for in vivo pharmacokinetics (PK) evaluation as they were more than 25% unmetabolized after 1 h of incubation.

The in vivo PK profiles of four compounds, 35, 49, 52, and 57, as their free base and HCl salts were evaluated in rats (Table 4); the HCl salts were evaluated because they could improve the aqueous solubility of the compounds and, in turn, could improve the PK profile. Neither 35 nor 57 could be detected in plasma after PO administration, presumably due to their high plasma clearance (300 and 82.1 mL/min/kg). However, the HCl salts of 49 and 52 were orally bioavailable with an F% of 12.9 and 26.8, respectively. In particular, among the tested compounds, the HCl salt of 52 had the best T_1/2 of 5.1 and 6.9 h after IV and oral administration.

Table 4. In Vivo Pharmacokinetics Profiles of 35, 49, and 52 in Rats^a.

	IV (dose: 5 mg/kg)				PO (dose: 20 mg/kg)
comp.	T1/2 (h)	CL (mL/min/kg)	Vss (L/kg)	AUC(0-inf) (ng/mL·h)	Cmax (ng/mL)	Tmax (h)	T1/2 (h)	AUC(0-inf) (ng/mL·h)	F (%)
35	0.3	300	4.1	278	ND	ND	ND	ND	ND
49	2.9	60.6	4.7	1386	68.1	4.0	2.3	603	11
49S1	2.2	22	2.2	3912	253	1.7	2.3	2012	12.9
52S1	5.1	36.9	7.8	1967	255	0.3	6.9	2107	26.8
57S1	2.9	82.1	6.7	894	ND	ND	ND	ND	ND

^aS1 means the compound is a HCl salt; ND: not detected.

Investigation of Cellular Target Modulation by 49 and 52

The antiproliferative activities of 49 and 52 were investigated in BaF3 cell lines overexpressed with EGFR^L858R/T790M mutant kinase. Both compounds showed potent antiproliferative activity with CC₅₀ values of 26 and 20 nM, respectively, similar to that of 6, which had a CC₅₀ of 14 nM. The results suggested that both 49 and 52 could inhibit mutant EGFR at the cellular level to produce the antiproliferative activity.

Further, to confirm the ability of 49 and 52 to inhibit the mutant EGFR selectively inside the cells, A431 and H1975 NSCLC cells were treated with the compounds for 1 h and then the cell extracts were analyzed using western blot for downstream signaling molecule modulation. Treatment with 49, 52, and 6 resulted in lower levels of tyrosine¹⁰⁶⁸ phosphorylated EGFR [pEGFR(Tyr¹⁰⁶⁸)] in the H1975 cell line but not in the A431 cell line (Figure 4). An active EGFR kinase enzyme autophosphorylates the EGFR tyrosine¹⁰⁶⁸ residue. Lower levels of pEGFR(Tyr¹⁰⁶⁸) in H1975 indicate target (EGFR^L858R/T790M) inhibition in the cell lines. However, pEGFR(Tyr¹⁰⁶⁸) levels were not affected in A431 cell line, suggesting that the target (EGFR^WT) is not inhibited in this cell line. This further confirms the findings that 49 and 52 are mutant-selective EGFR inhibitors with EGFR^WT sparing activity. Concurrent testing of the initial lead molecule 11 showed that it is not a mutant-selective EGFR inhibitor with lower potency. Both 49 and 52 showed similar AKT and ERK1/2 inhibition profiles as that of 6.

Figure 4.

Differential inhibition of wild-type and T790M mutant EGFR in the cellular context by 49 and 52. Western blot analysis shows that pEGFR(Try¹⁰⁶⁸) levels were unaltered in A431 cells expressing EGFR^WT, but not in the case of H1975 cells expressing EGFR T790M mutant, suggesting mutant-selective inhibition of EGFR kinase activity by 49 and 52 similar to 6 inside the cells.

As additional in vitro data confirmed that 49 and 52 are potent and EGFR mutant-selective agents, they were subjected to in vivo evaluation to determine their ability to inhibit the growth of tumors bearing EGFR^L858R/T790M double mutation.

Evaluation of In Vivo Efficacy of Lead Compounds 49 and 52

For the in vivo efficacy evaluation, mice bearing tumors of BaF3 cells overexpressed with EGFR^L858R/T790M mutant kinase were used. Animals with the tumor were administered 100 mg/kg of 49 or 52 over two cycles (the first cycle on days 1–5 and the second cycle on days 8–12) of treatment. Tumor volume and body weight changes were measured till 18 days of the treatment initiation. It was found that 52 produced a sustained and significant tumor growth inhibition (TGI) during the treatment cycle (Figure 5A). However, 49 did not show significant TGI upon treatment. Neither treatment altered the body weight of the animals more than 5% (Figure S1). Based on these in vivo findings, 52 was chosen for further evaluation in the H1975 lung cancer xenograft mouse model.

Figure 5.

In vivo efficacy evaluation of orally administered 49 and 52 in mouse xenograft models. (A) Change in BaF3 EGFR^L858R/T790M tumor volume over the treatment period. Drug treatment was for two cycles of 5 days each, with 2 days gap. *P < 0.05, compared to vehicle group. (B) Change in H1975 tumor volume over the treatment period. Tumor growth inhibition (TGI) is shown as percentage, compared to the vehicle-treated group.

For preclinical evaluation in the lung cancer xenograft model, mice were injected with H1975 cells bearing the EGFR^L858R/T790M double mutation. After detectable tumor growth, animals were PO-administered with two doses of 52 (10 or 30 mg/kg, QD) daily for 35 days. Tumor volume and body weight were measured during the treatment. The group that received 10 mg/kg of 52 showed 74.9% TGI, while the group administered 30 mg/kg showed 97.5% TGI at the end of the treatment (Figure 5B). The results suggest that 52 can effectively suppress the growth of NSCLC with EGFR double mutation. Moreover, drug treatment did not result in overt body weight loss (<5% change in body weight; Figure S1) during drug treatment, suggesting overall good tolerance for the drug. The in vivo pharmacodynamics experiments demonstrate the utility of 52 as an agent to overcome EGFR double-mutant overexpressing non-small cell lung cancer.

Binding Mode Analysis of 52 by Molecular Docking

To understand the EGFR^WT sparing inhibition activity of 52, the molecular docking of 52 and 11 in EGFR^WT (PDB ID: 6JXT) and EGFR^T790M (PDB ID: 6JX0) structures was carried out. The wild-type and mutant EGFR proteins were in complex with 6, and the ligand binding site for docking was designated based on this ligand. Covalent docking of the ligand was carried out with a criterion to undergo nucleophilic addition of the Cys797 thiol group of EGFR protein to the Michael acceptor group present in the ligand. Both the ligands 52 and 11 bound at the active site, forming a H-bond between the furanopyrimidine N¹ and the hinge region Met793 residue. In addition, the protonated N,N-dimethylamino solubilizing group of 11 made a salt bridge with the Asp800 residue in both wild-type and mutant EGFR, and the protonated N,N,N′-trimethylethyl amino solubilizing group of 52 made salt bridge with the Asp800 residue in the wild-type EGFR and with the Asp855 residue in mutant EGFR (Figure S2). It is interesting to note that the N,N,N′-trimethylethyl amino solubilizing group of 52 bound to the mutant EGFR is projected in the solvent-exposed region of the protein as envisaged in strategy II (Figure 2).

Compound 11 docked to EGFR^WT with a docking score of −10.927 and to EGFR^T790M with a docking score of −7.667, suggesting a better binding to EGFR^WT compared to EGFR^T790M. This could be due to a steric clash between the phenyl ring substituent present in the furanopyrimidine 4-position of 11 and the Met790 residue of EGFR^T790M, a residue that is not present in wild-type EGFR (Figure 6A–C). In contrast, 52 was bound to EGFR^T790M with a better docking score (−8.118) than to EGFR^WT (−6.891), suggesting stronger binding to the mutant EGFR than EGFR^WT. Analysis of the binding orientation shows that 52 was flipped in the ATP binding site of EGFR^WT and did not form H-bond with the hinge residue Met793. Further analysis suggests that the pyrimidine ring atoms would make steric clash and/or unfavorable interaction with the polar Thr790 residue, resulting in flipped orientation of the ligand in EGFR^WT with concomitant lower binding. However, 52 was bound in the ATP binding site of EGFR^T790M with the essential hinge region interaction (Figure 6D–F). The results from covalent docking rationalize the observed EGFR^WT sparing activity of 52.

Figure 6.

Three-dimensional (3D) docked orientations of 11 (A–C) and 52 (D–F) in EGFR^WT (PDB ID: 6JXT) and EGFR^T790M (PDB ID: 6JX0) structures. Schrödinger Glide covalent docking module was used to do the docking in the EGFR active site with nucleophilic addition of the Cys797 SH group to the Michael acceptor group of the ligand. (A) 11 in EGFR^WT, (B) 11 in EGFR^T790M, (C) superimposition of panels (A) and (B) showing the docked orientation of 11 (in EGFR^WT) phenyl ring atoms making steric clash (black dotted lines) with Met790 residue of EGFR^T790M, (D) 52 in EGFR^WT, (E) 52 in EGFR^T790M, and (F) superimposition of panels (D) and (E) showing the docked orientation of 52 (in EGFR^T790M) pyrimidine ring atoms making steric clash (black dotted lines) with the Thr790 residue of EGFR^WT. H-bond is shown as yellow dotted lines.

Kinase Profiling of Lead 52

Further, to estimate the specificity and safety profile, a panel of 468 kinases (including 65 mutant kinases) were tested for binding of 52 at a screening concentration of 1 μM using KINOMEscan technology (Figure 7, Tables S1 and S2). The results revealed that 52 presented an extraordinary kinome selectivity with an S(10) score of 0.017 (7/403 nonmutant kinases). Apart from EGFR^WT (1.7%), 52 also showed high affinity toward six other kinases bearing a cysteine at the front region including BLK (0.9%), BTK (0%), ERBB2 (0%), ERBB4 (0%), JAK3 (0.4%), and TXK (9.9%).³¹ In addition to these targets, 52 only presented moderate affinity toward another cysteine-containing kinase TEK (14%). The strong affinity between 52 and EGFR^L858R/T790M (1.9%) is consistent with the results of enzymatic assays as well. Collectively, the kinase profiling outcome of 52 demonstrated a highly selective EGFR inhibitor, similar to the clinical trial counterpart 11.

Figure 7.

Kinase profiling of 52 using KINOMEscan technology at 1 μM.

Chemistry

Syntheses of compounds 13–15, 17–23, 31, and 45–58 are outlined in Scheme 1. Similar to our previous study, building block 59(32) was initially subjected to nucleophilic aromatic substitution reaction (S_NAr) with various amines to yield the desired compounds 60a–w. The amino group of 60h was protected with Boc anhydride to generate 60h′. Then, the bromo compounds 60a–w were coupled with 3-nitrophenylboronic acid derivatives or 3-nitro-4-substituted-phenylboronic acid derivatives via Suzuki coupling to give compounds 61a–i, 62j, 62r, 62s, 62u, and 63j–w. On the other hand, the 4-fluoro-substituted 62j, 62r, and 62s were reacted with different secondary amines or alcohols under basic conditions to prepare compounds 63j, 63r, and 63s. Reductions of 61a–i and 63j–w were carried out using palladium on charcoal in the presence of hydrogen gas, stannic chloride, or iron powder under acidic conditions to give the corresponding amino derivatives 64a–w. Finally, compounds 64a–w were reacted with acrylic acid or acryloyl chloride to introduce amide bonds in analogues 13–15, 17–23, 31, and 45–58.

Scheme 1. Synthetic Route for EGFR Inhibitors 13–15, 17–23, 31, and 45–58.

Reagents and conditions: (a) appropriate amines, Et₃N, ethanol, reflux, 53–97%; (b) appropriate amines, ⁿBuOH, reflux, 62–75%; (c) appropriate amines, DIPEA, ethanol, reflux, 92–98%; (d) appropriate boronic acids or boronic esters, Pd(dppf)Cl₂, Na₂CO₃, 1,4-dioxane, H₂O, 80 °C, reflux, 2–16 h, 47–93%; (e) (3-nitrophenyl)boronic acid, Pd(dppf)Cl₂, Na₂CO₃, DMF, H₂O, 150 °C, 1 h, microwave, 60%; (f) appropriate amines, Et₃N, 1,4-dioxane, 80 °C, reflux, 1–12 h, 60%–quant.; (g) appropriate alcohols, NaH, THF, rt, 16 h, 46–60%; (h) H₂, Pd/C, methanol or ethanol, rt, 30–99%; (i) from 60d, (3-aminophenyl)boronic acid, Pd(dppf)Cl₂, Na₂CO₃, 1,4-dioxane, H₂O, 100 °C, 16 h, 89%; (j) from 60e, (3-aminophenyl)boronic acid, Pd(dppf)Cl₂, Na₂CO₃, 1,4-dioxane, H₂O, 150 °C, 1 h, microwave, 43%; (k) SnCl₂, ethanol, reflux, 1–4 h, 87–94%; (l) iron powder, ethanol, CH₂Cl₂, H₂O, sat. NH₄Cl_(aq), 80 °C, 0.5–16 h, 52–94%; (m) SnCl₂·2H₂O, CH₂Cl₂, methanol, reflux, 1.5 h, 31–47%; (n) acrylic acid, EDCI, CH₂Cl₂, rt, 4–16 h, 14–78%; (o) acryloyl chloride, DIPEA or Et₃N, CH₂Cl₂, rt, 1–2 h, 42–70%; (p) LiOH_(aq), THF, rt, 3 h, 96%; (q) TFA, CH₂Cl₂, rt, 1 h, 96%; (r) (i) 4-bromocrotonoic acid, EDCI, CH₂Cl₂, rt, 16 h; (ii) N,N-dimethylamine, THF, rt, 4 h, 72%.

For the synthesis of final compounds 16 and 33–35 (Scheme 2), previously reported building block 65(28) was first substituted with the OTBS-protected ethanolamine to give 66a, which was then brominated at 6-position with N-bromosuccinimide in acetonitrile to yield 66b. Then, Suzuki coupling reaction of 66b was carried out under microwave irradiation with phenylboronic acids containing different substituents to afford 66c–e. Reduction of 66a and 66c–e was accomplished by either stannic chloride, iron powder, or palladium on charcoal in the presence of hydrogen gas to achieve 67a and 67c–e. The acrylation of 67a with acryloyl chloride in tetrahydrofuran gave the desired compound 16. On the other hand, the anilines 67c–e were treated with acryloyl chloride in dichloromethane followed by desilylation with trifluoroacetic acid to obtain the target compounds 33–35.

Scheme 2. Synthetic Route of EGFR Inhibitors 16 and 33–35.

Reagents and conditions: (a) 2-[(tert-butyldimethylsilyl)oxy]ethan-1-amine, DIPEA, ethanol, reflux, 64%; (b) NBS, ACN, 100 °C, microwave, time, 99%; (c) 4-substituted-phenylboronic acid, Pd(dppf)Cl₂·CH₂Cl₂, Na₂CO₃, 100 °C, 60–80%; (d) SnCl₂, HCl, CH₂Cl₂, reflux, 96%; (e) iron powder, ethanol, CH₂Cl₂, sat. NH₄Cl_(aq), 80 °C, 94%; (f) H₂, Pd/C, ethanol, rt, 60%; (g) acryloyl chloride, DIPEA, THF, rt, 38%; (h) acryloyl chloride, Et₃N, CH₂Cl₂, rt, then TFA, CH₂Cl₂, 34–66%.

The final compounds 24–30, 32, and 36–44 were obtained from the previously reported building block 68(28) and the synthetic route is illustrated in Scheme 3. To generate various intermediates 71 efficiently from 68, two different routes were employed. For compounds 24–29 and 41, building block 68 was initially coupled with phenylboronic acid or 4-(4-methylpiperazin-1-yl)phenylboronic acid via Suzuki cross-coupling to achieve compounds 69a and 69b. Then, 69a was treated with different amines through nucleophilic aromatic substitution to give 71a–f. The 4-chloride of furanopyrimidine 69b was coupled with methylamine to afford 71m. On the other hand, building block 68 was also reacted with various amines under basic conditions to yield amines 70a–d in which 70b was subsequently transformed into OTBS-protected 70b′. Then, 70a–d was treated with numerous amines to give 71g–o. The nitro functional group of intermediates 71a–p was then reduced to amino groups with stannic chloride or iron powder to afford anilines 72a–p. Finally, amide bond formation was achieved by treatment of 72a–p with acrylic acid to obtain the desired EGFR inhibitors 24–30, 32, and 36–44.

Scheme 3. Synthetic Route of EGFR Inhibitors 24–30, 32, and 36–44.

Reagents and conditions: (a) appropriate boronic acids, Pd(dppf)Cl₂, Na₂CO₃, 1,4-dioxane, H₂O, 75–80 °C, 3–12 h, 53–55%; (b) appropriate amines, Et₃N, ethanol or IPA, reflux, 1.5–16 h, 84–95%; (c) TBSCl, Et₃N, DMF, CH₂Cl₂, rt, 16 h, 79%; (d) appropriate amines, Et₃N or DIPEA, ethanol, reflux, 4–16 h, 51–95%; (e) appropriate boronic acids or boronic esters, Pd(dppf)Cl₂, Na₂CO₃, 1,4-dioxane, H₂O, 80 °C, reflux, 1–16 h, 65–96%; (f) H₂, Pd/C, methanol, rt, 1–16 h, 83–98%; (g) SnCl₂·2H₂O, CH₂Cl₂, methanol or ethanol, 70 °C, reflux, 2–4 h, 64–99%; (h) iron powder, ethanol, CH₂Cl₂, H₂O, sat. NH₄Cl_(aq), 80 °C, 2 h, 38–85%; (i) acrylic acid, EDCI, CH₂Cl₂, rt, 1–16 h, 14–86%; (j) (i) 4-bromocrotonoic acid, EDCI, CH₂Cl₂, rt, 16 h; (ii) N,N-dimethylamine, THF, rt, 5 h, 35%.

Conclusions

The propensity of NSCLC to resist EGFR inhibitors must be addressed if the burden of the disease is to be mitigated. The acquired T790M mutation results in resistance to first-generation EGFR inhibitors, making these drugs ineffective. Second-generation inhibitors targeting Cys797 such as compound 11 can overcome resistance due to the T790M mutation, but their poor selectivity for mutant EGFR kinase over wild-type EGFR kinase may cause clinical toxicity. Here, through structure and property-guided lead optimization efforts, we first attenuated hydrophobic interaction in the back pocket by replacing (S)-2-phenylglycinol to N-amino ethanol or cyclopentylamino substituent at the 4-position of the furanopyrimidine ring to achieve mutant selectivity. Furthermore, based on molecular modeling studies, two different strategies were applied to enhance cellular potency by implanting solubilizing groups, such as N-methyl piperazine in phenyl ring A and N,N,N′-trimethylethyl amino group in phenyl ring B to obtain various EGFR mutant-selective inhibitors (35, 38, 39, 40 and 41, 49, 50, 51, 52, 53, 55, 56, 57, and 58) that selectively inhibited the proliferation of H1975 over A431 NSCLC cells. After DMPK assessment by in vitro microsomal stability and in vivo pharmacokinetics studies, 52 is selected as an orally active agent that effectively inhibits tumor formation in animal models with T790M-mutated EGFR. Further preclinical evaluation of 52 is underway to develop it as a backup compound for clinical trial agent 11 (DBPR112).

Experimental Section

General Methods for Chemistry

All commercial chemicals and solvents are of reagent grade and used without further purification unless otherwise stated. All reactions were carried out under dry nitrogen or argon atmosphere and were monitored for completion by TLC using Merck 60 F254 silica gel glass-backed plates or aluminum plates; zones were detected visually under UV irradiation (254 nm) or by spraying with potassium permanganate reagent (Aldrich) followed by heating at 80 °C. Flash column chromatography was carried out using silica gel (Silicycle SiliaFlash P60, R12030B, 230–400 mesh or Merck Grade 9385, 230–400 mesh). ¹H and ¹³C NMR spectra were recorded with Varian Mercury-300 or Varian Mercury-400 spectrometers or Bruker 400 or 600 MHz AVANCE III spectrometers. Data analysis was done using Mnova software (Mestrelab Research). Chemical shift (δ) was reported in ppm and referenced to solvent residual signals as follows: DMSO-d₆ at 2.50 ppm, CDCl₃ at 7.26 ppm, and CD₃OD at 3.31 ppm for ¹H NMR; DMSO-d₆ at 39.5 ppm and CDCl₃ at 77.0 ppm for ¹³C NMR. Splitting patterns are indicated as follows: s = singlet; d = doublet; t = triplet; q = quartet; quin = quintet; dd = doublet of doublets; dt = doublet of triplets; dq = doublet of quartet; dquin = doublet of quintet; dset = doublet of septet; td = triplet of doublets; tt = triplet of triplets; qd = quartet of doublets, ddd = doublet of doublets of doublets; br = broad; m = multiplet. Coupling constants (J) were given in hertz (Hz). Low-resolution mass spectra (LRMS) data were measured with an Agilent MSD-1100 ESI-MS/MS system or Agilent Infinity II 1290 LC/MS (ESI) systems. High-resolution mass spectra (HRMS) data were measured with a Varian 901-MS FT-ICR HPLC/MS–MS system. Purity of the final compounds was determined using a high-performance liquid chromatography (HPLC) system (Hitachi 2000 series) equipped with a C18 column (Agilent ZORBAX Eclipse XDB-C18 5 μm. 4.6 mm × 150 mm) and operating at 25 °C. For method A, elution was carried out using acetonitrile as mobile phase A and water containing 0.1% formic acid + 10 mmol NH₄OAc as mobile phase B. Elution conditions: at 0 min, phase A 10% + phase B 90%; at 45 min, phase A 90% + phase B 10%; at 50 min, phase A 10% + phase B 90%; at 60 min, phase A 10% + phase B 90%. For method B, elution was carried out using acetonitrile as mobile phase A and water containing 0.1% formic acid + 2 mmol NH₄OAc as mobile phase B. Elution conditions: at 0 min, phase A 10% + phase B 90%; at 25 min, phase A 90% + phase B 10%; at 30 min, phase A 90% + phase B 10%; at 30.5 min, phase A 10% + phase B 90%; at 37 min, phase A 10% + phase B 90%. The flowrate of the mobile phase was 0.5 mL/min, and the injection volume of the sample was 10 or 20 μL. Peaks were detected at 254 nm. The purity of all tested compounds was determined and confirmed to be greater than 95% by HPLC analysis except for compounds 15 (84.5%), 19 (91.5%), 23 (92.7%), 25 (93.1%), 27 (93.8%), 32 (91.6%), 36 (89.6%), 39 (83.7%), 40 (94.2%), 49 (91.9%), 50 (92.2%), and 54 (93.4%). IUPAC nomenclature of the compounds was obtained with Mnova software (Mestrelab Research).

N-[3-(4-{[(2S)-1-Hydroxy-3-methylbutan-2-yl]amino}-6-phenylfuro[2,3-d]pyrimidin-5-yl)phenyl]prop-2-enamide (13)

To a solution of 64a (200 mg, 0.51 mmol, 1.0 equiv) in dichloromethane (5.0 mL) were added acrylic acid (40 μL, 0.58 mmol, 1.1 equiv) and EDCI (109 mg, 0.57 mmol, 1.1 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 4 h, to the reaction mixture was added H₂O (10 mL), extracted with dichloromethane (10 mL × 3), and washed with brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (2–5% methanol in dichloromethane) to yield the title compound 13 (33 mg, 0.07 mmol, 14%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.36 (s, 1H), 8.32 (s, 1H), 7.90 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.52–7.47 (m, 2H), 7.39–7.34 (m, 2H), 7.32 (ddd, J = 8.4, 7.2, 1.8 Hz, 1H), 7.27 (d, J = 7.2 Hz, 1H), 6.43 (dd, J = 17.1, 10.2 Hz, 1H), 6.26 (dd, J = 17.1, 1.8 Hz, 1H), 5.76 (dd, J = 10.2, 1.8 Hz, 1H), 4.93 (d, J = 9.6 Hz, 1H), 4.64 (t, J = 5.4 Hz, 1H), 4.05 (td, J = 9.6, 3.6 Hz, 1H), 3.42–3.37 (m, 1H), 3.30–3.24 (m, 1H), 1.86–1.74 (m, 1H), 0.77 (d, J = 6.6 Hz, 3H), 0.57 (d, J = 6.6 Hz, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.4, 163.4, 157.5, 154.1, 145.5, 140.3, 132.1, 131.6, 130.4, 129.0, 128.9, 128.7, 127.4, 125.8, 124.3, 119.8, 119.6, 115.1, 102.3, 60.4, 56.1, 27.7, 19.4, 16.8. LRMS (ESI) m/z: 443.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₆H₂₇N₄O₃, 443.2083 [M + H]⁺; found, 443.2085. HPLC purity (method A): 100% (t_R = 32.42 min).

N-[3-(4-{[(2S)-1-Hydroxypropan-2-yl]amino}-6-phenylfuro[2,3-d]pyrimidin-5-yl)phenyl]prop-2-enamide (14)

To a solution of 64b (20 mg, 0.06 mmol, 1.0 equiv) in dichloromethane (0.5 mL) were added acrylic acid (4.2 μL, 0.06 mmol, 1.1 equiv) and EDCI (12 mg, 0.06 mmol, 1.1 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo and purified by thin-layer chromatography (2–15% methanol in dichloromethane) to yield the title compound 14 (3 mg, 0.01 mmol, 14%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.33 (s, 1H), 8.35 (s, 1H), 7.90–7.78 (m, 2H), 7.59–7.44 (m, 3H), 7.43–7.29 (m, 3H), 7.24 (d, J = 7.6 Hz, 1H), 6.42 (dd, J = 17.0, 10.0 Hz, 1H), 6.25 (dd, J = 17.0, 2.2 Hz, 1H), 5.77 (dd, J = 10.0, 2.2 Hz, 1H), 5.18 (d, J = 7.6 Hz, 1H) 4.70 (t, J = 5.2 Hz, 1H), 4.23–4.08 (m, 1H), 3.32–3.27 (m, 2H), 1.02 (d, J = 6.8 Hz, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.4, 156.7, 154.1, 145.5, 140.1, 131.9, 131.6, 130.3, 129.0, 128.8, 128.7, 127.3, 126.0, 124.3, 119.9, 119.6, 115.1, 102.1, 63.5, 47.6, 17.0. LRMS (ESI) m/z: 415.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₄H₂₂N₄NaO₃, 437.1590 [M + Na]⁺; found, 437.1587. HPLC purity (method A): 98.1% (t_R = 28.29 min).

N-(3-{4-[(2-Hydroxyethyl)amino]-6-phenylfuro[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (15)

To a solution of 64c (100 mg, 0.29 mmol, 1.0 equiv) in dichloromethane (5.0 mL) were added acrylic acid (30 μL, 0.44 mmol, 1.5 equiv) and EDCI (83 mg, 0.43 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo and purified by thin-layer chromatography (0–10% methanol in dichloromethane) to yield the title compound 15 (75 mg, 0.19 mmol, 65%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.32 (s, 1H), 8.34 (s, 1H), 7.86 (ddd, J = 8.4, 2.4, 1.5 Hz, 1H), 7.80 (dd, J = 2.4, 1.5 Hz, 1H), 7.53 (t, J = 8.4 Hz, 1H), 7.48–7.44 (m, 2H), 7.38–7.30 (m, 3H), 7.24–7.21 (m, 1H), 6.43 (dd, J = 16.8, 10.2 Hz, 1H), 6.26 (dd, J = 16.8, 1.8 Hz, 1H), 5.77 (dd, J = 10.2, 1.8 Hz, 1H), 5.50 (t, J = 5.4 Hz, 1H), 4.64 (t, J = 4.8 Hz, 1H), 3.49–3.41 (m, 4H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.4, 157.2, 154.0, 145.6, 140.1, 131.8, 131.7, 130.4, 129.0, 128.8, 128.7, 127.3, 126.0, 124.4, 120.0, 119.7, 115.2, 102.1, 59.1, 42.8. LRMS (ESI) m/z: 401.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₃H₂₁N₄O₃, 401.1614 [M + H]⁺; found, 401.1610. HPLC purity (method A): 84.5% (t_R = 26.43 min).

N-(3-{4-[(2-Hydroxyethyl)amino]furo[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (16)

To a solution of 67a (59 mg, 0.22 mmol, 1.0 equiv) in THF (10.0 mL) at 0 °C were added triethylamine (36 μL, 0.26 mmol, 1.2 equiv) and a solution of acryloyl chloride (19 μL, 0.24 mmol, 1.1 equiv) in THF (5.0 mL); then, the reaction mixture was stirred at room temperature. After stirring for 3 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (5% methanol in dichloromethane) to yield the title compound 16 (27 mg, 0.08 mmol, 38%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.33 (s, 1H), 8.34 (s, 1H), 8.00 (s, 1H), 7.91 (dd, J = 2.4, 1.8 Hz, 1H), 7.66 (ddd, J = 8.4, 2.4, 1.2 Hz, 1H), 7.48 (dd, J = 8.4, 7.2 Hz, 1H), 7.26 (ddd, J = 7.2, 1.8, 1.2 Hz, 1H), 6.46 (dd, J = 17.1, 10.2 Hz, 1H), 6.29 (dd, J = 17.1, 1.5 Hz, 1H), 6.05 (t, J = 5.4 Hz, 1H), 5.79 (dd, J = 10.2, 1.5 Hz, 1H), 4.70 (td, J = 5.4, 1.2 Hz, 1H), 3.60–3.50 (m, 4H). ¹³C NMR (150 MHz, DMSO-d₆) δ 166.4, 163.5, 157,5, 153.8, 139.7, 138.4, 131.7, 131.2, 129.9, 127.3, 123.3, 120.5, 119.2, 119.1, 99.0, 59.2, 43.0. LRMS (ESI) m/z: 325.1 [M + H]⁺. HRMS (ESI) m/z: calcd for C₁₇H₁₇N₄O₃, 325.1301 [M + H]⁺; found, 325.1303. HPLC purity (method A): 96.0% (t_R = 18.46 min).

N-(3-{4-[(3-Hydroxypropyl)amino]-6-phenylfuro[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (17)

To a solution of 64d (31 mg, 0.09 mmol, 1.0 equiv) in dichloromethane (10.0 mL) at 0 °C were added DIPEA (36 μL, 0.17 mmol, 2.0 equiv) and a solution of acryloyl chloride (8 μL, 0.10 mmol, 1.2 equiv) in dichloromethane (1.0 mL); then, the reaction mixture was stirred at room temperature. After stirring for 2 h, the reaction mixture was concentrated in vacuo and purified by thin-layer chromatography (5% methanol in dichloromethane) to yield the title compound 17 (25 mg, 0.06 mmol, 70%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.33 (s, 1H), 8.34 (s, 1H), 7.84 (dd, J = 8.4, 1.8 Hz, 1H), 7.79 (dd, J = 1.8, 1.8 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.45 (d, J = 7.8 Hz, 2H), 7.38–7.29 (m, 3H), 7.21 (d, J = 7.8 Hz, 1H), 6.43 (dd, J = 17.4, 10.2 Hz, 1H), 6.26 (dd, J = 17.4, 2.1 Hz, 1H), 5.77 (dd, J = 10.2, 2.1 Hz, 1H), 5.46 (t, J = 6.0 Hz, 1H), 4.39 (t, J = 5.7, 1H), 3.46 (td, J = 6.6, 5.7 Hz, 2H), 3.37–3.33 (m, 2H), 1.56 (quin, J = 6.6 Hz, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.4, 157.1, 154.0, 145.5, 140.0, 131.8, 131.6, 130.3, 129.0, 128.8, 128.7, 127.3, 126.0, 124.5, 120.1, 119.7, 115.2, 102.1, 58.6, 38.2, 31.6. LRMS (ESI) m/z: 415.1 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₄H₂₃N₄O₃, 415.1770 [M + H]⁺; found, 415.1769. HPLC purity (method A): 99.6% (t_R = 26.85 min).

N-(3-{4-[(2-Methoxyethyl)amino]-6-phenylfuro[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (18)

To a solution of 64e (150 mg, 0.42 mmol, 1.0 equiv) in dichloromethane (5.0 mL) at 0 °C were added triethylamine (70 μL, 0.50 mmol, 1.2 equiv) and a solution of acryloyl chloride (40 μL, 0.49 mmol, 1.2 equiv) in dichloromethane (2.0 mL); then, the reaction mixture was stirred at room temperature. After stirring for 1 h, the reaction mixture was concentrated in vacuo and purified by CombiFlash automated flash chromatography (2% methanol in dichloromethane) to yield the title compound 18 (148 mg, 0.36 mmol, 86%) as a light-yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.36 (s, 1H), 8.35 (s, 1H), 7.87 (ddd, J = 8.4, 2.4, 1.2 Hz, 1H), 7.83 (dd, J = 2.4, 2.4 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.39–7.30 (m, 3H), 7.20 (ddd, J = 7.8, 2.4, 1.2 Hz, 1H), 6.44 (dd, J = 16.8, 10.2 Hz, 1H), 6.26 (dd, J = 16.8, 1.8 Hz, 1H), 5.78 (dd, J = 10.2, 1.8 Hz, 1H), 5.35 (t, J = 5.4 Hz, 1H), 3.52 (td, J = 5.4, 5.4 Hz, 2H), 3.10 (s, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.4, 157.0, 154.1, 145.6, 140.2, 131.9, 131.6, 130.4, 129.0, 128.9, 128.8, 127.4, 126.0, 124.4, 119.8, 119.6, 115.1, 102.3, 69.8, 57.9, 39.9. LRMS (ESI) m/z: 415.1 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₄H₂₂N₄NaO₃, 437.1590 [M + Na]⁺; found, 437.1593. HPLC purity (method A): 100% (t_R = 31.83 min).

N-(3-{4-[(2-Phenoxyethyl)amino]-6-phenylfuro[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (19)

To a solution of 64f (34 mg, 0.08 mmol, 1.0 equiv) in dichloromethane (15.0 mL) at 0 °C were added triethylamine (17 μL, 0.12 mmol, 2.2 equiv) and a solution of acryloyl chloride (8 μL, 0.10 mmol, 1.2 equiv) in dichloromethane (5.0 mL); then, the reaction mixture was stirred at room temperature. After stirring for 1 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (100% dichloromethane) to yield the title compound 19 (16 mg, 0.03 mmol, 42%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.31 (s, 1H), 8.39 (s, 1H), 7.85–7.81 (m, 2H), 7.51–7.44 (m, 3H), 7.39–7.30 (m, 3H), 7.23 (dd, J = 8.7, 7.5 Hz, 2H), 7.19 (ddd, J = 7.2, 1.8, 1.2 Hz, 1H), 6.91 (td, J = 7.5, 1.2 Hz, 1H), 6.80 (dd, J = 8.7, 1.2 Hz, 1H), 6.39 (dd, J = 16.8, 10.2 Hz, 1H), 6.25 (dd, J = 16.8, 1.8 Hz, 1H), 5.76 (dd, J = 10.2, 1.8 Hz, 1H), 5.53 (t, J = 6.0 Hz, 1H), 4.03 (t, J = 6.0 Hz, 2H), 3.79 (td, J = 6.0, 5.4 Hz, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.4, 158.0, 157.0, 154.0, 145.8, 140.1, 131.8, 131.6, 130.3, 129.4, 128.9, 128.83, 128.77, 127,2, 126.0, 124.4, 120.7, 119.9, 119.7, 115.1, 114.4, 102.4, 65.6, 39.5. LRMS (ESI) m/z: 477.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₉H₂₅N₄O₃, 477.1927 [M + H]⁺; found, 477.1923. HPLC purity (method A): 91.5% (t_R = 40.64 min).

Ethyl 3-({5-[3-(Acryloylamino)phenyl]-6-phenylfuro[2,3-d]pyrimidin-4-yl}amino)propanoate (20)

To a solution of 64g (400 mg, 0.99 mmol, 1.0 equiv) in dichloromethane (10.0 mL) at 0 °C were added triethylamine (170 μL, 1.22 mmol, 1.2 equiv) and a solution of acryloyl chloride (99 μL, 1.23 mmol, 1.2 equiv) in dichloromethane (5.0 mL); then, the reaction mixture was stirred at room temperature. After stirring for 1 h, the reaction mixture was concentrated in vacuo and purified by CombiFlash automated flash chromatography (2% methanol in dichloromethane) to yield the title compound 20 (261 mg, 0.57 mmol, 58%) as a brown solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.33 (s, 1H), 8.36 (s, 1H), 7.83 (ddd, J = 8.4, 2.4, 1.5 Hz, 1H), 7.80 (dd, J = 1.8, 1.5 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.47–7.43 (m, 2H), 7.38–7.30 (m, 3H) 7.18–7.15 (m, 1H), 6.43 (dd, J = 17.1, 10.2 Hz, 1H), 6.35 (dd, J = 17.1, 1.8 Hz, 1H), 5.77 (dd, J = 10.2, 1.8 Hz, 1H), 5.55 (t, J = 6.0 Hz, 1H), 3.96 (q, J = 7.2 Hz, 2H), 3.63 (td, J = 6.0, 6.0 Hz, 2H), 2.52 (t, J = 6.6 Hz, 2H), 1.09 (t, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 171.5, 164.6, 163.4, 156.9, 154.0, 145.7, 140.1, 131.7, 131.6, 130.3, 129.0, 128.84, 128.79, 127.3, 126.1, 124.5, 120.0, 119.7, 115.1, 102.3, 60.0, 36.2, 33.3, 14.0. LRMS (ESI) m/z: 457.1 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₆H₂₅N₄O₄, 457.1876 [M + H]⁺; found, 457.1799. HPLC purity (method A): 98.6% (t_R = 35.47 min).

3-({5-[3-(Acryloylamino)phenyl]-6-phenylfuro[2,3-d]pyrimidin-4-yl}amino)propanoic Acid (21)

To a solution of 20 (164 mg, 0.36 mmol, 1.0 equiv) in THF (10.0 mL) at 0 °C was added 0.5 M LiOH_(aq) (2 mL, 1.02 mmol, 2.8 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 3 h, the reaction mixture was concentrated in vacuo and purified by CombiFlash automated flash chromatography (5% methanol in dichloromethane) to yield the title compound 21 (142 mg, 0.33 mmol, 92%) as a light-yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.40–10.31 (m, 1H), 8.36 (s, 1H), 7.87 (ddd, J = 8.4, 2.4, 1.2 Hz, 1H), 7.76–7.71 (m, 1H), 7.51 (t, J = 8.4 Hz, 1H), 7.47–7.43 (m, 2H), 7.39–7.30 (m, 3H) 7.19–7.15 (m, 1H), 6.43 (dd, J = 16.8, 10.2 Hz, 1H), 6.26 (dd, J = 16.8, 2.1 Hz, 1H), 5.76 (dd, J = 10.2, 2.1 Hz, 1H), 5.69–5.63 (m, 1H), 3.60 (td, J = 6.6, 6.0 Hz, 2H), 2.47–2.41 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 173.3, 164.6, 163.4, 156.9, 154.0, 145.7, 140.0, 131.7, 131.6, 130.3, 129.0, 128.8, 128.7, 127.2, 126.1, 124.4, 120.1, 119.7, 115.2, 102.2, 36.4, 33.4. LRMS (ESI) m/z: 429.1 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₄H₂₁N₄O₄, 429.1563 [M + H]⁺; found, 429.1560. HPLC purity (method A): 98.8% (t_R = 26.74 min).

N-(3-{4-[(2-Aminoethyl)amino]-6-phenylfuro[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (22)

To a solution of 22′ (215 mg, 0.43 mmol, 1.0 equiv) in dichloromethane (5.0 mL) was added trifluoroacetic acid (1.0 mL); then, the reaction mixture was stirred at room temperature. After stirring for 1 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (3–20% methanol in dichloromethane) to yield the title compound 22 (165 mg, 0.41 mmol, 96%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.48 (s, 1H), 8.40 (s, 1H), 7.90 (s, 1H), 7.81–7.68 (m, 3H), 7.50 (td, J = 7.8, 1.8 Hz, 1H), 7.44 (d, J = 7.8 Hz, 2H), 7.40–7.31 (m, 3H), 7.19 (dd, J = 7.8, 1.2 Hz, 1H), 6.47 (dd, J = 17.1, 10.2 Hz, 1H), 6.29 (dd, J = 17.1, 2.1 Hz, 1H), 5.88 (t, J = 6.0 Hz, 1H), 5.79 (dd, J = 10.2, 2.1 Hz, 1H), 3.66 (td, J = 6.3, 6.0 Hz, 2H), 3.02 (td, J = 6.3, 1.8 Hz, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.7, 163.6, 157.3, 153.7, 146.1, 139.8, 131.6, 131.5, 130.2, 128.92, 128.90, 128.8, 127.5, 126.4, 124.9, 120.6, 119.9, 115.3, 102.5, 38.6, 38.2. LRMS (ESI) m/z: 400.1 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₃H₂₂N₅O₂, 400.1774 [M + H]⁺; found, 400.1779. HPLC purity (method A): 96.9% (t_R = 19.93 min).

N-(3-(4-{[2-(Dimethylamino)ethyl]amino}-6-phenylfuro[2,3-d]pyrimidin-5-yl)phenyl)prop-2-enamide (23)

To a solution of 64i (324 mg, 0.87 mmol, 1.0 equiv) in dichloromethane (5.0 mL) were added triethylamine (160 μL, 1.15 mmol, 1.3 equiv) and acryloyl chloride (85 μL, 1.05 mmol, 1.2 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 4 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (1–5% methanol in dichloromethane) to yield the title compound 23 (185 mg, 0.43 mmol, 50%) as a pale orange solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.62 (s, 1H), 8.39 (s, 1H), 7.93 (dd, J = 2.4, 1.8 Hz, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.49 (t, J = 8.4 Hz, 1H), 7.45 (d, J = 7.2 Hz, 2H), 7.39–7.31 (m, 3H), 7.18 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 6.53 (dd, J = 17.4, 10.2 Hz, 1H), 6.27 (dd, J = 17.4, 2.1 Hz, 1H), 5.89 (t, J = 6.0 Hz, 1H), 5.77 (dd, J = 10.2, 2.1 Hz, 1H), 3.73 (s, 2H), 3.11 (s, 2H), 2.64 (s, 6H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.7, 163.5, 157.1, 153.8, 146.0, 140.1, 131.7, 131.4, 130.2, 128.9, 128.82, 128.81, 127.2, 126.2, 124.8, 120.2, 119.7, 115.3, 102.5, 55.8, 42.8, 40.0, 36.2. LRMS (ESI) m/z: 428.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₅H₂₆N₅O₂, 428.2087 [M + H]⁺; found, 428.2089. HPLC purity (method A): 92.7% (t_R = 21.41 min).

N-{3-[4-(Methylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (24)

To a solution of 72a (40 mg, 0.13 mmol, 1.0 equiv) in dichloromethane (3.0 mL) were added DIPEA (4 μL, 0.02 mmol, 18 mol %), acrylic acid (11 μL, 0.16 mmol, 1.3 equiv), and EDCI (29 mg, 0.15 mmol, 1.2 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture was added ethyl acetate (5 mL) and washed with NaHCO_3(aq) (10 mL) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo and purified by flash column chromatography (33–66% ethyl acetate in hexane) to yield the title compound 24 (32 mg, 0.09 mmol, 68%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.34 (s, 1H), 8.36 (s, 1H), 7.85 (ddd, J = 8.4, 2.1, 1.2 Hz, 1H), 7.75 (dd, J = 2.1, 1.8 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 7.2 Hz, 2H), 7.38–7.29 (m, 3H), 7.17 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 6.44 (dd, J = 17.1, 10.2 Hz, 1H), 6.27 (dd, J = 17.1, 2.4 Hz, 1H), 5.77 (dd, J = 10.2, 2.4 Hz, 1H), 5.54 (q, J = 4.8 Hz, 1H), 2.89 (d, J = 4.8 Hz, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.4, 157.6, 154.0, 145.5, 140.0, 131.8, 131.6, 130.3, 129.0, 128.8, 128.7, 127.3, 126.1, 124.7, 120.2, 119.8, 115.3, 102.1, 28.0. LRMS (ESI) m/z: 371.1 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₂H₁₇N₄O₂, 369.1352 [M – H]⁺; found, 369.1351. HPLC purity (method B): 97.0% (t_R = 21.52 min).

N-{3-[4-(Dimethylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (25)

To a solution of 72b (40 mg, 0.12 mmol, 1.0 equiv) in dichloromethane (3.0 mL) were added DIPEA (4 μL, 0.02 mmol, 18 mol %), acrylic acid (11 μL, 0.16 mmol, 1.3 equiv), and EDCI (28 mg, 0.15 mmol, 1.2 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture was added ethyl acetate (5 mL) and washed with NaHCO_3(aq) (10 mL) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (17–66% ethyl acetate in hexane) to yield the title compound 25 (27 mg, 0.07 mmol, 58%) as a light-yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.29 (s, 1H), 8.36 (s, 1H), 7.85 (ddd, J = 8.4, 2.1, 1.2 Hz, 1H), 7.69 (dd, J = 2.1, 1.8 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.41 (d, J = 7.2 Hz, 2H), 7.37–7.29 (m, 3H), 7.14 (ddd, J = 7.2, 1.8, 1.2 Hz, 1H), 6.42 (dd, J = 16.8, 10.2 Hz, 1H), 6.24 (dd, J = 16.8, 1.8 Hz, 1H), 5.76 (dd, J = 10.2, 1.8 Hz, 1H), 2.71 (s, 6H). ¹³C NMR (150 MHz, DMSO-d₆) δ 166.3, 163.3, 159.8, 152.3, 145.6, 139.7, 134.2, 131.7, 129.8, 129.3, 128.7, 128.6, 127.2, 126.5, 125.4, 120.7, 119.2, 116.1, 103.5, 40.2. LRMS (ESI) m/z: 385.1 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₃H₂₁N₄O₂, 385.1665 [M + H]⁺; found, 385.1677. HPLC purity (method B): 93.1% (t_R = 23.09 min).

N-{3-[6-Phenyl-4-(propan-2-ylamino)furo[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (26)

To a solution of 72c (30 mg, 0.09 mmol, 1.0 equiv) in dichloromethane (3.0 mL) were added DIPEA (3 μL, 0.02 mmol, 20 mol %), acrylic acid (8 μL, 0.12 mmol, 1.4 equiv), and EDCI (20 mg, 0.10 mmol, 1.2 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture was added ethyl acetate (5 mL) and washed with NaHCO_3(aq) (10 mL) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (17–50% ethyl acetate in hexane) to yield the title compound 26 (27 mg, 0.07 mmol, 58%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.38 (s, 1H), 8.36 (s, 1H), 7.93 (dd, J = 2.1, 1.8 Hz, 1H), 7.79 (ddd, J = 7.8, 2.1, 1.2 Hz, 1H), 7.56 (t, J = 8.4 Hz, 1H), 7.50 (d, J = 7.8 Hz, 2H), 7.40–7.31 (m, 3H), 7.24 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 6.43 (dd, J = 16.8, 10.2 Hz, 1H), 6.27 (dd, J = 16.8, 2.1 Hz, 1H), 5.78 (dd, J = 10.2, 2.1 Hz, 1H), 4.85 (d, J = 7.2 Hz, 1H), 4.18 (dset, J = 7.2, 6.3 Hz, 1H), 1.03 (d, J = 6.3 Hz, 6H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.5, 156.4, 154.1, 145.5, 140.1, 132.0, 131.6, 130.3, 128.94, 128.86, 128.8, 127.4, 126.0, 124.5, 119.9, 119.7, 115.0, 102.0, 42.3, 22.0. LRMS (ESI) m/z: 399.1 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₄H₂₃N₄O₂, 399.1821 [M + H]⁺; found, 399.1814. HPLC purity (method B): 99.1% (t_R = 25.66 min).

N-{3-[4-(Cyclopropylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (27)

To a solution of 72d (30 mg, 0.09 mmol, 1.0 equiv) in dichloromethane (3.0 mL) were added DIPEA (3 μL, 0.02 mmol, 20 mol %mol %), acrylic acid (8 μL, 0.12 mmol, 1.3 equiv), and EDCI (20 mg, 0.10 mmol, 1.2 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture was added ethyl acetate (5 mL) and washed with NaHCO_3(aq) (10 mL) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (33–66% ethyl acetate in hexane) to yield the title compound 27 (27 mg, 0.07 mmol, 78%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.35 (s, 1H), 8.42 (s, 1H), 7.84 (dd, J = 2.4, 1.8 Hz, 1H), 7.79 (ddd, J = 8.4, 2.4, 1.2 Hz, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.48 (d, J = 10.2 Hz, 2H), 7.39–7.31 (m, 3H), 7.20 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 6.43 (dd, J = 17.1, 10.2 Hz, 1H), 6.27 (dd, J = 17.1, 2.1 Hz, 1H), 5.78 (dd, J = 10.2, 2.1 Hz, 1H), 5.26 (d, J = 3.3 Hz, 1H), 2.79 (dset, J = 3.6, 3.3 Hz, 1H), 0.73–0.67 (m, 2H), 0.34–0.29 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.4, 163.5, 158.1, 154.0, 145.8, 140.0, 131.9, 131.6, 130.3, 128.9, 128.84, 128.83, 127.4, 126.1, 124.6, 120.0, 119.7, 115.0, 102.4, 23.9, 7.0. LRMS (ESI) m/z: 397.1 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₄H₂₁N₄O₂, 397.1665 [M + H]⁺; found, 397.1663. HPLC purity (method B): 93.8% (t_R = 22.85 min).

N-{3-[4-(Cyclobutylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (28)

To a solution of 72e (38 mg, 0.11 mmol, 1.0 equiv) in dichloromethane (3.0 mL) were added DIPEA (4 μL, 0.02 mmol, 22 mol %), acrylic acid (10 μL, 0.15 mmol, 1.4 equiv), and EDCI (27 mg, 0.14 mmol, 1.3 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture was added ethyl acetate (5 mL) and washed with NaHCO_3(aq) (10 mL) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (17–33% ethyl acetate in hexane) to yield the title compound 28 (31 mg, 0.08 mmol, 71%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.40 (s, 1H), 8.34 (s, 1H), 7.93 (dd, J = 2.1, 1.8 Hz, 1H), 7.80 (ddd, J = 8.4, 2.1, 1.2 Hz, 1H), 7.57 (t, J = 8.4 Hz, 1H), 7.50 (d, J = 7.2 Hz, 2H), 7.40–7.32 (m, 3H), 7.25 (ddd, J = 7.2, 1.8, 1.2 Hz, 1H), 6.44 (dd, J = 17.1, 10.2 Hz, 1H), 6.27 (dd, J = 17.1, 1.8 Hz, 1H), 5.78 (dd, J = 10.2, 1.8 Hz, 1H), 5.26 (d, J = 7.5 Hz, 1H), 4.50 (dquin, J = 7.8, 7.5 Hz, 1H), 2.27–2.20 (m, 2H), 1.70–1.57 (m, 4H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.6, 163.5, 156.1, 154.0, 145.7, 140.0, 132.0, 131.6, 130.3, 128.9, 128.85, 128.81, 127.4, 126.1, 124.6, 120.1, 119.8, 115.0, 102.0, 45.5, 30.5, 14.6. LRMS (ESI) m/z: 411.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₅H₂₃N₄O₂, 411.1821 [M + H]⁺; found, 411.1813. HPLC purity (method B): 99.2% (t_R = 26.92 min).

N-{3-[4-(Cyclopentylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (29)

To a solution of 72f (37 mg, 0.10 mmol, 1.0 equiv) in dichloromethane (3.0 mL) were added DIPEA (3 μL, 0.02 mmol, 17 mol %), acrylic acid (9 μL, 0.14 mmol, 1.4 equiv), and EDCI (23 mg, 0.12 mmol, 1.2 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture was added ethyl acetate (5 mL) and washed with NaHCO_3(aq) (10 mL) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and washed with cold methanol (5 mL) to yield the title compound 29 (20 mg, 0.05 mmol, 47%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.39 (s, 1H), 8.36 (s, 1H), 7.93 (dd, J = 2.1, 1.8 Hz, 1H), 7.80 (ddd, J = 8.4, 2.1, 1.2 Hz, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.52 (d, J = 7.8 Hz, 2H), 7.40–7.32 (m, 3H), 7.25 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 6.43 (dd, J = 16.8, 10.2 Hz, 1H), 6.26 (dd, J = 16.8, 1.8 Hz, 1H), 5.78 (dd, J = 10.2, 1.8 Hz, 1H), 4.89 (d, J = 7.2 Hz, 1H), 4.39–4.30 (m, 1H), 1.87–1.77 (m, 2H), 1.54–1.44 (m, 2H), 1.43–1.33 (m, 2H), 1.26–1.17 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.4, 163.5, 156.6, 154.2, 145.4, 140.2, 132.2, 131.5, 130.4, 128.94, 128.89, 128.79, 127.5, 125.9, 124.5, 119.8, 119.6, 115.0, 102.3, 52.0, 32.5, 22.7. LRMS (ESI) m/z: 425.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₆H₂₅N₄O₂, 425.1978 [M + H]⁺; found, 425.1920. HPLC purity (method B): 97.8% (t_R = 27.60 min).

N-(3-(4-(Cyclohexylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl)phenyl)prop-2-enamide (30)

To a solution of 72g (66 mg, 0.17 mmol, 1.0 equiv) in dichloromethane (0.9 mL) were added DIPEA (6 μL, 0.03 mmol, 20 mol %), acrylic acid (14 μL, 0.20 mmol, 1.2 equiv), and EDCI (49 mg, 0.26 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (22% ethyl acetate in hexane) to yield the title compound 30 (43 mg, 0.10 mmol, 57%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.39 (s, 1H), 8.34 (s, 1H), 7.95 (dd, J = 2.1, 1.8 Hz, 1H), 7.80 (ddd, J = 8.4, 2.1, 1.2 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.51 (d, J = 7.2 Hz, 2H), 7.40–7.31 (m, 3H), 7.26 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 6.44 (dd, J = 16.8, 10.2 Hz, 1H), 6.26 (dd, J = 16.8, 1.8 Hz, 1H), 5.78 (dd, J = 10.2, 1.8 Hz, 1H), 4.93 (d, J = 7.8 Hz, 1H), 4.04–3.95 (m, 1H), 1.78–1.69 (m, 2H), 1.45–1.20 (m, 4H), 1.19–1.03 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.5, 156.3, 154.2, 145.5, 140.2, 132.1, 131.5, 130.4, 128.94, 128.87, 128.77, 127.4, 125.9, 124.5, 119.74, 119.66, 115.0, 102.1, 47.9, 31.3, 25.0, 23.1. LRMS (ESI) m/z: 439.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₇H₂₆N₄NaO₂, 461.1953 [M + Na]⁺; found, 461.1951. HPLC purity (method B): 99.5% (t_R = 28.82 min).

(2E)-4-(Dimethylamino)-N-(3-{4-[(2-hydroxyethyl)amino]-6-phenylfuro[2,3-d]pyrimidin-5-yl}phenyl)but-2-enamide (31)

To a solution of 64c (100 mg, 0.29 mmol, 1.0 equiv) in dichloromethane (5.0 mL) were added 4-bromocrotonoic acid (71 mg, 0.43 mmol, 1.5 equiv) and EDCI (83 mg, 0.43 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo, dissolved in THF (5.0 mL), and added 2 M N,N-dimethylamine (250 μL, 0.50 mmol, 1.7 equiv) in THF. Then, the reaction mixture was stirred at room temperature for 4 h, concentrated in vacuo, and purified by thin-layer chromatography (0–15% methanol in dichloromethane) to yield the title compound 31 (95 mg, 0.21 mmol, 72%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.42 (s, 1H), 8.34 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.82 (s, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.46 (d, J = 6.9 Hz, 2H), 7.36 (dd, J = 7.8, 6.9 Hz, 2H), 7.34–7.30 (m, 1H), 7.19 (d, J = 7.8 Hz, 1H), 6.73 (dt, J = 15.3, 5.7 Hz, 1H), 6.30 (d, J = 15.3 Hz, 1H), 5.50 (t, J = 5.4 Hz, 1H), 4.69 (s, 1H), 3.49–3.39 (m, 4H), 3.04 (d, J = 5.7 Hz, 2H), 2.16 (s, 6 H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.5, 157.2, 154.0, 145.5, 141.8, 131.7, 130.3, 129.0, 128.8, 128.7, 126.0, 125.7, 124.1, 119.9, 119.6, 115.3, 102.1, 59.7, 59.1, 45.1, 42.8. LRMS (ESI) m/z: 458.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₆H₂₇N₅NaO₃, 480.2012 [M + Na]⁺; found, 480.2008. HPLC purity (method A): 97.4% (t_R = 19.17 min).

(2E)-N-{3-[4-(Cyclopentylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]phenyl}-4-(dimethylamino)but-2-enamide (32)

To a solution of 72f (50 mg, 0.13 mmol, 1.0 equiv) in dichloromethane (3.0 mL) were added 4-bromocrotonoic acid (29 mg, 0.18 mmol, 1.3 equiv) and EDCI (83 mg, 0.43 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture was added ethyl acetate (5 mL) and washed with brine (10 mL). The combined organic layers were dried over MgSO₄ and concentrated in vacuo. The residue was dissolved in THF (5.0 mL), cooled to 0 °C, and added 2 M N,N-dimethylamine (202 μL, 0.41 mmol, 3.0 equiv) in THF. The reaction mixture was stirred at room temperature for 5 h, concentrated in vacuo, and purified by flash column chromatography (3–9% methanol in dichloromethane) to yield the title compound 32 (23 mg, 0.05 mmol, 35%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 8.35 (s, 1H), 7.86–7.81 (m, 3H), 7.59 (t, J = 8.4 Hz, 1H), 7.52 (d, J = 7.2 Hz, 2H), 7.40–7.29 (m, 5H), 6.38 (d, J = 8.4 Hz, 1H), 5.70 (t, J = 6.0 Hz, 1H), 4.84 (d, J = 7.2 Hz, 1H), 4.40–4.26 (m, 1H), 3.32 (s, 6H), 1.86–1.78 (m, 2H), 1.52–1.36 (m, 4H), 1.26–1.17 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 173.6, 164.3, 156.7, 154.1, 145.4, 139.3, 131.9, 130.0, 129.0, 128.8, 128.7, 125.9, 125.8, 125.7, 122.3, 122.1, 114.9, 102.4, 52.0, 32.4, 32.3, 29.8, 28.0, 22.8. LRMS (ESI) m/z: 482.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₉H₃₂N₅O₂, 482.2556 [M + H]⁺; found, 482.2557. HPLC purity (method B): 91.6% (t_R = 16.99 min).

N-(3-{6-[4-(Dimethylamino)phenyl]-4-[(2-hydroxyethyl)amino]furo[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (33)

To a solution of 67c (155 mg, 0.31 mmol, 1.0 equiv) in dichloromethane (15.0 mL) were added triethylamine (60 μL, 0.43 mmol, 1.4 equiv) and a solution of acryloyl chloride (30 μL, 0.37 mmol, 1.2 equiv) in dichloromethane (15.0 mL) dropwise at 0 °C, then the reaction mixture was stirred at room temperature. After stirring for 6 h, to the reaction mixture was added H₂O (10 mL) and extracted with dichloromethane (10 mL × 3). The organic layers were combined, washed with brine (10 mL), dried over MgSO₄, concentrated in vacuo, and purified by thin-layer chromatography (2% methanol in dichloromethane) to yield the TBS-protected compound 33′. To the TBS-protected compound 33′ in dichloromethane (8.0 mL) was added trifluoroacetic acid (0.4 mL). The reaction mixture was stirred at room temperature for 6 h and then concentrated in vacuo. The obtained residue was neutralized with 10% NH₄OH in methanol, then concentrated in vacuo again and purified by flash column chromatography (3% methanol in dichloromethane) to yield the title compound 33 (90 mg, 0.20 mmol, 66%) as a light-yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.30 (s, 1H), 8.28 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.76 (dd, J = 1.8, 1.8 Hz, 1H), 7.51 (dd, J = 8.4, 7.8 Hz, 1H), 7.29 (d, J = 9.0 Hz, 2H), 7.20 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 6.65 (d, J = 9.0 Hz, 2H), 6.43 (dd, J = 17.1, 10.2 Hz, 1H), 6.26 (dd, J = 17.1, 2.1 Hz, 1H), 5.77 (dd, J = 10.2, 2.1 Hz, 1H), 5.39 (t, J = 5.4 Hz, 1H), 4.62 (t, J = 4.8 Hz, 1H), 3.50–3.40 (m, 4H), 2.90 (s, 6H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.1, 163.4, 156.7, 152.9, 150.2, 147.0, 139.9, 132.5, 131.7, 130.2, 127.2, 127.1, 124.6, 120.3, 119.3, 116.1, 111.8, 102.4, 59.2, 42.8, 39.9. LRMS (ESI) m/z: 324.1 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₅H₂₅N₅NaO₃, 466.4968 [M + Na]⁺; found, 466.1994. HPLC purity (method A): 97.5% (t_R = 27.85 min).

N-(3-{4-[(2-Hydroxyethyl)amino]-6-[4-(morpholin-4-yl)phenyl]furo[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (34)

To a solution of 67d (94 mg, 0.17 mmol, 1.0 equiv) in dichloromethane (8.0 mL) were added triethylamine (34 μL, 0.25 mmol, 1.4 equiv) and a solution of acryloyl chloride (17 μL, 0.21 mmol, 1.2 equiv) in dichloromethane (8.0 mL) dropwise at 0 °C, then the reaction mixture was stirred at room temperature. After stirring for 6 h, to the reaction mixture was added H₂O (10 mL) and extracted with dichloromethane (10 mL × 3). The organic layers were combined, washed with brine (10 mL), dried over MgSO₄, and concentrated in vacuo to yield the TBS-protected compound 34′. To the TBS-protected compound 34′ in dichloromethane (4.0 mL) was added trifluoroacetic acid (0.2 mL); then, the reaction mixture was stirred at room temperature. After stirring for 12 h, the reaction mixture was concentrated in vacuo, neutralized with 10% NH₄OH in methanol, then concentrated in vacuo again and purified by flash column chromatography (3% methanol in dichloromethane) to yield the title compound 34 (40 mg, 0.08 mmol, 48%) as a light-yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.32 (s, 1H), 8.29 (s, 1H), 7.86 (ddd, J = 8.1, 2.4, 1.2 Hz, 1H), 7.77 (dd, J = 2.4, 1.8 Hz, 1H), 7.51 (dd, J = 8.1, 7.8 Hz, 1H), 7.32 (d, J = 9.0 Hz, 2H), 7.20 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 6.90 (d, J = 9.0 Hz, 2H), 6.44 (dd, J = 16.8, 10.2 Hz, 1H), 6.26 (dd, J = 16.8, 2.1 Hz, 1H), 5.77 (dd, J = 10.2, 2.1 Hz, 1H), 5.42 (t, J = 5.4 Hz, 1H), 4.63 (t, J = 4.8 Hz, 1H), 3.74–3.66 (m, 4H), 3.49–3.40 (m, 4H), 3.18–3.10 (m, 4H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.2, 163.4, 156.8, 153.2, 150.8, 146.4, 140.0, 132.3, 131.7, 130.2, 127.2, 127.0, 124.5, 120.2, 119.4, 119.0, 114.3, 112.4, 102.3, 65.9, 59.1, 47.3, 42.8. LRMS (ESI) m/z: 486.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₇H₂₇N₅NaO₄, 508.1961 [M + Na]⁺; found, 508.1961. HPLC purity (method A): 97.6% (t_R = 24.57 min).

N-(3-{4-[(2-Hydroxyethyl)amino)-6-[4-(4-methylpiperazin-1-yl)phenyl]furo[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (35)

To a solution of 67e (140 mg, 0.25 mmol, 1.0 equiv) in dichloromethane (7.5 mL) were added triethylamine (40 μL, 0.29 mmol, 1.1 equiv) and a solution of acryloyl chloride (20 μL, 0.25 mmol, 1.0 equiv) in dichloromethane (7.5 mL) dropwise at 0 °C, then the reaction mixture was stirred at room temperature. After stirring for 3 h, to the reaction mixture was added H₂O (10 mL) and extracted with dichloromethane (10 mL × 3). The organic layers were combined, washed with brine (10 mL), dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (2.5% methanol in dichloromethane with 0.3% NH₄OH) to yield the TBS-protected compound 35′. To the TBS-protected compound 35′ in dichloromethane (4.0 mL) was added trifluoroacetic acid (0.4 mL); then, the reaction mixture was stirred at room temperature. After stirring for 8 h, the reaction mixture was concentrated in vacuo, neutralized with 10% NH₄OH in methanol, then concentrated in vacuo again and purified by flash column chromatography (5% methanol in dichloromethane with 0.5% NH₄OH) to yield the title compound 35 (43 mg, 0.09 mmol, 34%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.30 (s, 1H), 8.29 (s, 1H), 7.86 (dd, J = 8.4, 2.4 Hz, 1H), 7.76 (dd, J = 2.4, 1.8 Hz, 1H), 7.51 (dd, J = 8.4, 7.5 Hz, 1H), 7.30 (d, J = 9.0 Hz, 2H), 7.20 (ddd, J = 7.5, 1.8, 1.2 Hz, 1H), 6.88 (d, J = 9.0 Hz, 2H), 6.43 (dd, J = 17.1, 10.2 Hz, 1H), 6.26 (dd, J = 17.1, 1.8 Hz, 1H), 5.77 (dd, J = 10.2, 1.8 Hz, 1H), 5.41 (t, J = 5.4 Hz, 1H), 4.63 (s, 1H), 3.50–3.40 (m, 4H), 3.17 (dd, J = 4.8, 4.8 Hz, 4H), 2.39 (dd, J = 4.8, 4.8 Hz, 4H), 2.19 (s, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.2, 163.4, 156.8, 153.2, 150.7, 146.5, 140.0, 132.3, 131.7, 130.2, 127.2, 127.0, 124.5, 120.2, 119.4, 118.5, 114.5, 112.2, 102.3, 59.1, 54.3, 47.0, 45.7, 42.8. LRMS (ESI) m/z: 499.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₈H₃₁N₆O₃, 499.2458 [M + H]⁺; found, 499.2460. HPLC purity (method A): 99.2% (t_R = 15.46 min).

N-{3-[6-(4-{[2-(Dimethylamino)ethyl](methyl)amino}phenyl)-4-[(2-hydroxyethyl)amino]furo[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (36)

To a solution of 72h (125 mg, 0.28 mmol, 1.0 equiv) in dichloromethane (3.0 mL) were added acrylic acid (21 μL, 0.31 mmol, 1.1 equiv) and EDCI (80 mg, 0.42 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was quenched with NaHCO_3(aq) (10 mL) and extracted with ethyl acetate (10 mL × 3). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by thin-layer chromatography (5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 36 (40 mg, 0.08 mmol, 29%) as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.33 (s, 1H), 7.80 (s, 1H), 7.73 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.44 (dd, J = 8.0, 8.0 Hz, 1H), 7.38 (d, J = 8.8 Hz, 2H), 7.28–7.24 (m, 1H), 6.55 (d, J = 9.2 Hz, 2H), 6.47 (dd, J = 16.8, 1.4 Hz, 1H), 6.28 (dd, J = 16.8, 10.4 Hz, 1H), 5.82 (dd, J = 10.4, 1.4 Hz, 1H), 5.46 (t, J = 5.0 Hz, 1H), 3.76 (dd, J = 5.8, 5.0 Hz, 2H), 3.63 (s, 1H), 3.43 (dd, J = 7.6, 7.4 Hz, 2H), 2.95 (s, 3H), 2.44 (dd, J = 7.6, 7.4 Hz, 2H), 2.27 (s, 6H), 0.88 (dd, J = 6.8, 5.8 Hz, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.1, 163.4, 156.7, 152.9, 149.0, 147.0, 140.0, 132.6, 131.7, 130.2, 127.3, 127.2, 124.6, 120.3, 119.3, 115.7, 111.4, 111.2, 102.5, 59.2, 55.6, 49.5, 45.5, 42.8, 30.9. LRMS (ESI) m/z: 501.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₈H₃₂N₆NaO₃, 523.2434 [M + Na]⁺; found, 523.2434. HPLC purity (method B): 89.6% (t_R = 12.57 min).

N-{3-[4-(Cyclopentylamino)-6-[4-(morpholin-4-yl)phenyl]furo[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (37)

To a solution of 72i (84 mg, 0.18 mmol, 1.0 equiv) in dichloromethane (3.0 mL) were added DIPEA (6 μL, 0.03 mmol, 19 mol %), acrylic acid (16 μL, 0.24 mmol, 1.3 equiv), and EDCI (42 mg, 0.22 mmol, 1.2 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture was added ethyl acetate (5 mL) and washed with NaHCO_3(aq) (10 mL) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (25–66% ethyl acetate in hexane) to yield the title compound 37 (45 mg, 0.09 mmol, 48%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.37 (s, 1H), 8.30 (s, 1H), 7.89 (dd, J = 2.1, 1.8 Hz, 1H), 7.79 (dd, J = 7.8, 2.1 Hz, 1H), 7.55 (dd, J = 7.8, 7.8 Hz, 1H), 7.37 (d, J = 9.0 Hz, 2H), 7.24–7.21 (m, 1H), 6.92 (d, J = 9.0 Hz, 2H), 6.43 (dd, J = 16.8, 10.2 Hz, 1H), 6.27 (dd, J = 16.8, 2.1 Hz, 1H), 5.78 (dd, J = 10.2, 2.1 Hz, 1H), 4.83 (d, J = 7.2 Hz, 1H), 4.33 (dquin, J = 7.2, 4.8 Hz, 1H), 3.70 (dd, J = 12.0, 4.2 Hz, 4H), 3.15 (dd, J = 12.0, 3.6 Hz, 4H), 1.86–1.77 (m, 2H), 1.53–1.33 (m, 4H), 1.26–1.15 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.1, 163.4, 156.2, 153.4, 150.9, 146.2, 140.1, 132.6, 131.6, 130.3, 127.4, 126.9, 124.6, 119.9, 119.4, 119.0, 114.4, 112.2, 102.4, 65.9, 51.9, 47.3, 32.5, 22.7. LRMS (ESI) m/z: 510.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₀H₃₁N₅NaO₃, 532.2325 [M + Na]+; found, 532.2318. HPLC purity (method B): 99.7% (t_R = 26.25 min).

N-{3-[4-(Cyclopentylamino)-6-[4-(4-methylpiperazin-1-yl)phenyl]furo[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (38)

To a solution of 72j (40 mg, 0.09 mmol, 1.0 equiv) in dichloromethane (5 mL) at 0 °C were added acrylic acid (9 μL, 0.13 mmol, 1.5 equiv) and EDCI (25 mg, 0.42 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was quenched with NaHCO_3(aq) (10 mL) and extracted with dichloromethane (10 mL × 3). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 38 (35 mg, 0.07 mmol, 78%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.37 (s, 1H), 8.30 (s, 1H), 7.89 (dd, J = 2.1, 1.8 Hz, 1H), 7.79 (dd, J = 8.1, 2.1, 1.2 Hz, 1H), 7.54 (dd, J = 8.1, 7.5 Hz, 1H), 7.35 (d, J = 9.0 Hz, 2H), 7.22 (ddd, J = 7.5, 1.8, 1.2 Hz 1H), 6.90 (d, J = 9.0 Hz, 2H), 6.44 (dd, J = 16.8, 10.2 Hz, 1H), 6.27 (dd, J = 16.8, 2.1 Hz, 1H), 5.78 (dd, J = 10.2, 2.1 Hz, 1H), 4.82 (d, J = 6.9 Hz, 1H), 4.33 (dquin, J = 6.9, 5.4 Hz, 1H), 3.18 (dd, J = 5.4, 5.1 Hz, 4H), 2.40 (dd, J = 5.1, 4.8 Hz, 4H), 2.19 (s, 3H), 1.86–1.77 (m, 2H), 1.52–1.32 (m, 4H), 1.25–1.16 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.1, 163.4, 156.2, 153.3, 150.8, 146.3, 140.1, 132.7, 131.6, 130.2, 127.4, 126.9, 124.6, 119.9, 119.4, 118.5, 114.6, 112.0, 102.4, 54.3, 51.9, 47.0, 45.7, 32.5, 22.7. LRMS (ESI) m/z: 523.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₁H₃₅N₆O₂, 523.2822 [M + H]+; found, 523.2836. HPLC purity (method B): 96.9% (t_R = 16.13 min).

N-(3-{4-[((1S)-2-Hydroxy-1-phenylethyl)amino]-6-[4-(4-methylpiperazin-1-yl)phenyl]furo[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (39)

To a solution of 72k (100 mg, 0.19 mmol, 1.0 equiv) in dichloromethane (2 mL) at 0 °C were added acrylic acid (16 μL, 0.23 mmol, 1.2 equiv) and EDCI (51 mg, 0.27 mmol, 1.4 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was quenched with NaHCO_3(aq) (10 mL) and extracted with dichloromethane (10 mL × 3). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by thin-plate chromatography (5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 39 (35 mg, 0.06 mmol, 32%) as a light-yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.36 (s, 1H), 8.23 (s, 1H), 7.94 (s, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.56 (dd, J = 8.0, 7.6 Hz, 1H), 7.40–7.10 (m, 8H), 6.92 (d, J = 8.8 Hz, 2H), 6.45 (dd, J = 17.0, 10.2 Hz, 1H), 6.28 (dd, J = 17.0, 2.0 Hz, 1H), 5.78 (dd, J = 10.2, 2.0 Hz, 1H), 5.66 (d, J = 7.6 Hz, 1H), 5.24–5.16 (m, 1H), 4.82 (t, J = 5.2 Hz, 1H), 3.62–3.54 (m, 1H), 3.47–3.38 (m, 1H), 3.32–3.14 (m, 4H), 2.44–2.36 (m, 4H), 2.20 (s, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.2, 163.4, 156.2, 153.2, 150.8, 146.6, 140.8, 140.2, 132.5, 131.7, 130.4, 128.1, 127.3, 126.9, 126.8, 126.5, 124.5, 119.9, 119.3, 118.5, 114.6, 112.1, 102.8, 64.4, 55.3, 54.3, 47.0, 45.7. LRMS (ESI) m/z: 575.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₄H₃₄N₆NaO₃, 597.2590 [M + Na]⁺; found, 597.2593. HPLC purity (method B): 83.7% (t_R = 15.16 min).

N-{3-[4-(Cyclohexylamino)-6-[4-(4-methylpiperazin-1-yl)phenyl]furo[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (40)

To a solution of 72l (130 mg, 0.27 mmol, 1.0 equiv) in dichloromethane (5 mL) at 0 °C were added acrylic acid (28 μL, 0.41 mmol, 1.5 equiv) and EDCI (77 mg, 0.40 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was quenched with NaHCO_3(aq) (10 mL) and extracted with dichloromethane (10 mL × 3). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 40 (67 mg, 0.12 mmol, 46%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.37 (s, 1H), 8.28 (s, 1H), 7.91 (dd, J = 2.4, 1.8 Hz, 1H), 7.80 (dd, J = 8.1, 1.8 Hz, 1H), 7.55 (dd, J = 8.1, 7.8 Hz, 1H), 7.34 (d, J = 9.0 Hz, 2H), 7.22 (d, J = 7.8 Hz 1H), 6.90 (d, J = 9.0 Hz, 2H), 6.44 (dd, J = 16.8, 10.2 Hz, 1H), 6.27 (dd, J = 16.8, 2.1 Hz, 1H), 5.77 (dd, J = 10.2, 2.1 Hz, 1H), 4.86 (d, J = 7.8 Hz, 1H), 4.02–3.92 (m, 1H), 3.17 (dd, J = 5.4, 4.8 Hz, 4H), 2.40 (dd, J = 5.4, 4.8 Hz, 4H), 2.19 (s, 3H), 1.77–1.69 (m, 2H), 1.45–1.22 (m, 5H), 1.17–1.02 (m, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.1, 163.4, 156.0, 153.4, 150.8, 146.4, 140.1, 132.7, 131.6, 130.3, 127.4, 126.9, 124.6, 119.9, 119.4, 118.5, 114.5, 112.0, 102.3, 54.3, 47.8, 47.0, 45.7, 31.4, 25.0, 23.1. LRMS (ESI) m/z: 537.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₂H₃₇N₆O₂, 537.2978 [M + H]⁺; found, 537.2941. HPLC purity (method B): 94.2% (t_R = 17.49 min).

N-{3-[4-(Methylamino)-6-[4-(4-methylpiperazin-1-yl)phenyl]furo[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (41)

To a solution of 72m (58 mg, 0.14 mmol, 1.0 equiv) in dichloromethane (1 mL) were added acrylic acid (12 μL, 0.18 mmol, 1.3 equiv) and EDCI (40 mg, 0.21 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 1 h, the reaction mixture was quenched with NaHCO_3(aq) (10 mL) and extracted with ethyl acetate (10 mL × 3). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (5% methanol in dichloromethane) to yield the title compound 41 (27 mg, 0.06 mmol, 41%) as colorless oil. ¹H NMR (600 MHz, DMSO-d₆) δ 10.34 (s, 1H), 8.31 (s, 1H), 7.84 (ddd, J = 8.1, 1.8, 1.2 Hz, 1H), 7.72 (dd, J = 1.8, 1.8 Hz, 1H), 7.50 (dd, J = 8.1, 7.8 Hz, 1H), 7.27 (d, J = 9.0 Hz, 2H), 7.15 (d, J = 7.8, 1.8, 1.2 Hz 1H), 6.88 (d, J = 9.0 Hz, 2H), 6.44 (dd, J = 16.8, 10.2 Hz, 1H), 6.27 (dd, J = 16.8, 1.8 Hz, 1H), 5.77 (dd, J = 10.2, 1.8 Hz, 1H), 5.42 (q, J = 4.8 Hz, 1H), 3.17 (dd, J = 5.4, 4.8 Hz, 4H), 2.87 (d, J = 4.8 Hz, 3H), 2.39 (dd, J = 5.4, 4.8 Hz, 4H), 2.19 (s, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.1, 163.4, 157.3, 153.2, 150.7, 146.4, 139.9, 132.3, 131.7, 130.2, 127.2, 127.0, 124.8, 120.4, 119.6, 118.5, 114.4, 112.4, 102.3, 54.3, 46.9, 45.7, 27.9. LRMS (ESI) m/z: 469.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₇H₂₈N₆NaO₂, 491.2171 [M + Na]⁺; found, 491.2173. HPLC purity (method B): 99.4% (t_R = 13.31 min).

N-{3-[4-(Cyclopentylamino)-6-(pyridin-4-yl)furo[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (42)

To a solution of 72n (71 mg, 0.19 mmol, 1.0 equiv) in dichloromethane (5 mL) were added acrylic acid (19 μL, 0.28 mmol, 1.4 equiv) and EDCI (52 mg, 0.27 mmol, 1.4 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was quenched with NaHCO_3(aq) (10 mL) and extracted with dichloromethane (10 mL × 3). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 42 (70 mg, 0.16 mmol, 86%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.43 (s, 1H), 8.55 (d, J = 6.0 Hz, 2H), 8.41 (s, 1H), 7.97 (dd, J = 2.4, 1.8 Hz, 1H), 7.82 (ddd, J = 8.1, 2.4, 1.2 Hz, 1H), 7.61 (dd, J = 8.1, 7.8 Hz, 1H), 7.41 (d, J = 6.0 Hz, 2H), 7.30 (ddd, J = 7.8, 1.8, 1.2 Hz 1H), 6.44 (dd, J = 16.8, 10.2 Hz, 1H), 6.27 (dd, J = 16.8, 1.8 Hz, 1H), 5.79 (dd, J = 10.2, 1.8 Hz, 1H), 4.97 (d, J = 6.9 Hz, 1H), 4.35 (dquin, J = 6.9, 4.8 Hz, 1H), 1.88–1.77 (m, 2H), 1.53–1.33 (m, 4H), 1.27–1.17 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.7, 163.5, 156.9, 155.2, 150.3, 142.6, 140.3, 135.9, 131.5, 131.3, 130.6, 127.5, 124.2, 120.1, 119.5, 119.2, 119.0, 102.2, 52.1, 32.4, 22.7. LRMS (ESI) m/z: 426.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₅H₂₃N₅NaO₂, 448.1749 [M + Na]⁺; found, 448.1756. HPLC purity (method B): 96.9% (t_R = 23.20 min).

N-{3-[4-(Cyclopentylamino)-6-[6-(morpholin-4-yl)pyridin-3-yl]furo[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (43)

To a solution of 72o (60 mg, 0.13 mmol, 1.0 equiv) in dichloromethane (0.7 mL) were added acrylic acid (12 μL, 0.18 mmol, 1.3 equiv) and EDCI (42 mg, 0.22 mmol, 1.7 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 2 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (5% methanol in dichloromethane) to yield the title compound 43 (17 mg, 0.03 mmol, 25%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.38 (s, 1H), 8.32 (s, 1H), 8.24 (dd, J = 2.1, 0.9 Hz, 1H), 7.92 (dd, J = 2.1, 1.8 Hz, 1H), 7.70 (ddd, J = 8.1, 2.1, 1.2 Hz, 1H), 7.60 (dd, J = 9.3, 2.4 Hz, 1H), 7.55 (dd, J = 8.1, 7.8 Hz, 1H), 7.23 (ddd, J = 7.8, 1.8, 1.2 Hz, 1H), 6.85 (dd, J = 9.3, 0.9 Hz, 1H), 6.43 (dd, J = 16.8, 10.2 Hz, 1H), 6.27 (dd, J = 16.8, 1.8 Hz, 1H), 5.78 (dd, J = 10.2, 1.8 Hz, 1H), 4.90 (d, J = 6.9 Hz, 1H), 4.34 (dquin, J = 6.9, 4.8 Hz, 1H), 3.65 (dd, J = 6.0, 5.1 Hz, 4H), 3.48 (dd, J = 6.0, 5.1 Hz, 4H), 1.87–1.77 (m, 2H), 1.54–1.36 (m, 4H), 1.26–1.18 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.3, 163.4, 158.2, 156.3, 153.6, 145.5, 144.6, 140.1, 134.9, 132.1, 131.5, 130.3, 127.4, 124.5, 119.9, 119.5, 114.5, 112.8, 106.6, 102.1, 65.8, 52.0, 44.6, 32.5, 22.7. LRMS (ESI) m/z: 511.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₉H₃₀N₆NaO₃, 533.2277 [M + Na]⁺; found, 533.2278. HPLC purity (method B): 99.8% (t_R = 24.56 min).

N-{3-[4-(Cyclopentylamino)-6-[6-(4-methylpiperazin-1-yl)pyridin-3-yl]furo[2,3-d]pyrimidin-5-yl]phenyl}prop-2-enamide (44)

To a solution of 72p (75 mg, 0.16 mmol, 1.0 equiv) in dichloromethane (0.8 mL) were added acrylic acid (13 μL, 0.19 mmol, 1.2 equiv) and EDCI (46 mg, 0.24 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 2 h, the reaction mixture was concentrated in vacuo, dissolved in ethyl acetate, and washed with NaHCO_3(aq) (10 mL) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (5% methanol in dichloromethane) to yield the title compound 44 (12 mg, 0.02 mmol, 14%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.38 (s, 1H), 8.31 (s, 1H), 8.22 (d, J = 2.4 Hz, 1H), 7.91 (dd, J = 1.8, 1.8 Hz, 1H), 7.77 (dd, J = 8.4, 1.8 Hz, 1H), 7.57–7.53 (m, 2H), 7.24–7.21 (m, 1H), 6.84 (d, J = 9.0 Hz, 1H), 6.43 (dd, J = 17.1, 10.2 Hz, 1H), 6.27 (dd, J = 17.1, 1.8 Hz, 1H), 5.78 (dd, J = 10.2, 1.8 Hz, 1H), 4.89 (d, J = 7.2 Hz, 1H), 4.34 (dquin, J = 7.2, 4.8 Hz, 1H), 3.51 (dd, J = 5.1, 4.8 Hz, 4H), 2.35 (dd, J = 5.1, 4.8 Hz, 4H), 2.19 (s, 3H), 1.87–1.77 (m, 2H), 1.53–1.35 (m, 4H), 1.26–1.17 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.3, 163.5, 158.0, 156.2, 153.5, 145.5, 144.7, 140.1, 134.8, 132.2, 131.6, 130.3, 127.4, 124.5, 119.9, 119.5, 114.0, 112.6, 106.6, 102.2, 54.2, 52.0, 45.7, 44.1, 32.5, 22.7. LRMS (ESI) m/z: 524.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₀H₃₃N₇NaO₂, 546.2593 [M + Na]⁺; found, 546.2593. HPLC purity (method B): 95.7% (t_R = 15.56 min).

N-{5-[4-(Cyclopentylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]-2-(morpholin-4-yl)phenyl}prop-2-enamide (45)

To a solution of 64j (47 mg, 0.10 mmol, 1.0 equiv) in dichloromethane (3.0 mL) were added DIPEA (4 μL, 0.02 mmol, 22 mol %), acrylic acid (9 μL, 0.14 mmol, 1.3 equiv), and EDCI (24 mg, 0.13 mmol, 1.2 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture was added ethyl acetate (5 mL) and washed with NaHCO_3(aq) (10 mL) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (33–50% ethyl acetate in hexane) to yield the title compound 45 (32 mg, 0.06 mmol, 61%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 9.35 (s, 1H), 8.34 (s, 1H), 8.31 (d, J = 1.8 Hz, 1H), 8.17 (s, 1H), 7.56–7.53 (m, 3H), 7.40–7.31 (m, 4H), 7.26 (dd, J = 8.4, 1.8 Hz, 1H), 6.71 (dd, J = 16.8, 10.2 Hz, 1H), 6.24 (dd, J = 16.8, 1.8 Hz, 1H), 5.78 (dd, J = 10.2, 1.8 Hz, 1H), 4.92 (d, J = 6.9 Hz, 1H), 4.34 (dquin, J = 6.9, 4.8 Hz, 1H), 3.85 (dd, J = 4.8, 4.2 Hz, 4H), 2.91 (dd, J = 4.8, 4.2 Hz, 4H), 1.84–1.76 (m, 2H), 1.52–1.43 (m, 2H), 1.42–1.32 (m, 2H), 1.31–1.23 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.3, 163.4, 156.6, 154.1, 145.3, 132.7, 131.8, 129.1, 128.9, 128.7, 127.3, 126.5, 126.1, 125.8, 123.1, 121.0, 114.9, 102.5, 79.2, 66.0, 54.9, 52.0, 51.5, 32.3, 22.7. LRMS (ESI) m/z: 510.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₀H₃₂N₅O₃, 510.2505 [M + H]⁺; found, 510.2457. HPLC purity (method B): 98.6% (t_R = 25.93 min).

N-{5-[4-(Cyclopentylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]-2-(4-methylpiperazin-1-yl)phenyl}prop-2-enamide (46)

To a solution of 64k (51 mg, 0.10 mmol, 1.0 equiv) in dichloromethane (5 mL) were added acrylic acid (8 μL, 0.11 mmol, 1.1 equiv) and EDCI (31 mg, 0.16 mmol, 1.6 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo and purified by thin-plate chromatography (5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 46 (18 mg, 0.03 mmol, 34%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 9.24 (s, 1H), 8.34 (s, 1H), 8.12 (s, 1H), 7.55 (d, J = 7.2 Hz, 2H), 7.40–7.30 (m, 4H), 7.24 (dd, J = 8.4, 2.4 Hz, 1H), 6.67 (dd, J = 16.8, 10.2 Hz, 1H), 6.23 (dd, J = 16.8, 1.8 Hz, 1H), 5.77 (dd, J = 10.2, 1.8 Hz, 1H), 4.91 (d, J = 6.9 Hz, 1H), 4.34 (dquin, J = 6.9, 2.4 Hz, 1H), 2.92 (s, 4H), 2.58 (s, 4H), 2.27 (s, 3H), 1.84–1.75 (m, 2H), 1.52–1.22 (m, 6H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.3, 163.3, 156.6, 154.1, 145.3, 144.2, 132.5, 131.8, 129.1, 128.8, 128.7, 127.2, 126.2, 126.0, 125.8, 123.1, 120.9, 114.9, 102.6, 54.5, 52.0, 50.9, 45.7, 32.4, 22.7. LRMS (ESI) m/z: 523.4 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₁H₃₅N₆O₂, 523.2822 [M + H]⁺; found, 523.2829. HPLC purity (method B): 98.6% (t_R = 16.83 min).

N-{5-[4-(Cyclopentylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]-2-[2-(dimethylamino)ethoxy]phenyl}prop-2-enamide (47)

To a solution of 64l (16 mg, 0.03 mmol, 1.0 equiv) in dichloromethane (5 mL) were added acrylic acid (4 μL, 0.06 mmol, 1.7 equiv) and EDCI (10 mg, 0.05 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture were added water (15 mL), dichloromethane (5 mL), and sat. NaHCO_3(aq) (2 mL) and extracted with dichloromethane (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 47 (14 mg, 0.03 mmol, 78%) as a yellow oil. ¹H NMR (600 MHz, DMSO-d₆) δ 9.76 (s, 1H), 8.34 (s, 1H), 8.31 (d, J = 2.4 Hz, 1H), 7.54 (d, J = 7.5 Hz, 2H), 7.37 (dd, J = 8.4, 7.5 Hz, 2H), 7.35–7.30 (m, 2H), 7.21 (dd, J = 8.4, 2.4 Hz, 1H), 6.60 (dd, J = 16.8, 10.2 Hz, 1H), 6.23 (dd, J = 16.8, 1.8 Hz, 1H), 5.77 (dd, J = 10.2, 1.8 Hz, 1H), 4.99 (d, J = 7.2 Hz, 1H), 4.35 (dquin, J = 7.2, 5.4 Hz, 1H), 4.25 (t, J = 6.0 Hz, 2H), 2.69 (t, J = 6.0 Hz, 2H), 2.27 (s, 3H), 1.87–1.77 (m, 2H), 1.52–1.36 (m, 4H), 1.30–1.22 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.3, 163.4, 156.7, 154.1, 148.8, 145.4, 131.7, 129.5, 129.1, 128.8, 128.6, 127.2, 125.83, 125.76, 123.9, 122.1, 115.5, 114.9, 102.4, 67.9, 57.2, 52.0, 45.2, 32.4, 22.8. LRMS (ESI) m/z: 512.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₀H₃₄N₅O₃, 512.2662 [M + H]⁺; found, 512.2658. HPLC purity (method B): 99.0% (t_R = 16.14 min).

N-{5-[4-(Cyclopentylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl)-2-[(1-methylpiperidin-4-yl)oxy]phenyl]prop-2-enamide (48)

To a solution of 64m (16 mg, 0.03 mmol, 1.0 equiv) in dichloromethane (5 mL) were added acrylic acid (3 μL, 0.04 mmol, 1.3 equiv) and EDCI (10 mg, 0.05 mmol, 1.6 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture were added water (15 mL), dichloromethane (5 mL), and sat. NaHCO_3(aq) (2 mL) and extracted with dichloromethane (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 48 (9 mg, 0.02 mmol, 51%) as a yellow oil. ¹H NMR (600 MHz, DMSO-d₆) δ 9.36 (s, 1H), 8.34 (s, 1H), 8.21 (s, 1H), 7.55 (d, J = 7.2 Hz, 2H), 7.37 (dd, J = 7.8, 7.2 Hz, 2H), 7.35–7.30 (m, 2H), 7.20 (dd, J = 8.4, 2.4 Hz, 1H), 6.72 (dd, J = 17.4, 10.2 Hz, 1H), 6.22 (dd, J = 17.4, 2.1 Hz, 1H), 5.76 (dd, J = 10.2, 2.1 Hz, 1H), 4.95 (d, J = 7.2 Hz, 1H), 4.53 (dquin, J = 8.4, 4.8 Hz, 1H), 4.38–4.33 (m, 1H), 2.66 (s, 1H), 2.18 (s, 3H), 2.00–1.92 (m, 2H), 1.86–1.75 (m, 4H), 1.53–1.32 (m, 6H), 1.30–1.20 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.3, 163.5, 156.7, 154.1, 147.8, 145.3, 131.8, 129.2, 129.1, 128.8, 128.6, 127.1, 125.8, 125.7, 123.2, 123.0, 115.5, 114.9, 102.6, 54.9, 52.4, 51.9, 45.8, 32.4, 30.3, 22.8. LRMS (ESI) m/z: 538.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₂H₃₅N₅NaO₃, 560.2638 [M + Na]⁺; found, 560.2641. HPLC purity (method B): 96.1% (t_R = 17.10 min).

N-{5-[4-(Cyclopentylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]-2-[(2-(dimethylamino)ethyl](methyl)amino]phenyl}prop-2-enamide (49)

To a solution of 64n (28 mg, 0.06 mmol, 1.0 equiv) in dichloromethane (3.0 mL) were added DIPEA (2 μL, 0.01 mmol, 19 mol %), acrylic acid (5 μL, 0.08 mmol, 1.3 equiv), and EDCI (14 mg, 0.07 mmol, 1.2 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, to the reaction mixture was added ethyl acetate (5 mL) and washed with NaHCO_3(aq) (10 mL) and brine (10 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (2–3% methanol in dichloromethane) to yield the title compound 49 (13 mg, 0.02 mmol, 42%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.22 (s, 1H), 8.43 (s, 1H), 8.34 (s, 1H), 7.56 (d, J = 7.2 Hz, 2H), 7.47 (d, J = 8.4 Hz, 1H), 7.40–7.31 (m, 3H), 7.22 (dd, J = 8.4, 2.4 Hz, 1H), 6.52 (s, 1H), 6.25 (dd, J = 16.8, 1.8 Hz, 1H), 5.80 (dd, J = 10.2, 1.8 Hz, 1H), 4.95 (d, J = 6.6 Hz, 1H), 4.35 (dquin, J = 6.6, 4.2 Hz, 1H), 2.97 (s, 2H), 2.76 (s, 1H), 2.36–2.20 (m, 6H), 1.86–1.77 (m, 2H), 1.54–1.32 (m, 4H), 1.31–1.18 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.3, 163.4, 156.6, 154.1, 145.3, 143.6, 131.6, 129.1, 128.8, 128.7, 127.3, 126.9, 125.8, 125.3, 122.8, 115.0, 102.4, 54.9, 51.9, 41.4, 32.5, 22.8. LRMS (ESI) m/z: 525.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₁H₃₇N₆O₂, 525.2978 [M + H]⁺; found, 525.2954. HPLC purity (method B): 96.1% (t_R = 17.10 min).

N-(2-{[2-(Dimethylamino)ethyl](methyl)amino}-5-[4-(methylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]phenyl)prop-2-enamide (50)

To a solution of 64o (26 mg, 0.06 mmol, 1.0 equiv) in dichloromethane (5 mL) were added acrylic acid (6 μL, 0.09 mmol, 1.4 equiv) and EDCI (17 mg, 0.09 mmol, 1.4 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 4 h, to the reaction mixture were added water (3 mL), dichloromethane (10 mL), and sat. NaHCO_3(aq) (1 mL) and extracted with dichloromethane (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 50 (12 mg, 0.03 mmol, 41%) as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.41 (s, 1H), 8.54 (s, 1H), 8.43 (s, 1H), 7.58–7.53 (m, 3H), 7.30–7.20 (m, 4H), 7.10 (dd, J = 9.8, 2.4 Hz, 1H), 6.45 (d, J = 16.8 Hz, 1H), 5.75 (dd, J = 9.8 Hz, 1H), 5.41 (s, 1H), 3.05 (d, J = 4.8 Hz, 3H), 2.95 (s, 2H), 2.80 (s, 3H), 2.50–2.26 (m, 8H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.6, 157.7, 153.9, 145.4, 133.7, 131.6, 129.2, 128.7, 128.6, 128.4, 128.2, 127.3, 126.2, 125.6, 122.5, 122.4, 115.4, 102.2, 56.7, 55.4, 45.5, 28.1, 24.9. LRMS (ESI) m/z: 471.2 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₇H₃₁N₆O₂, 471.2509 [M + H]⁺; found, 471.2539. HPLC purity (method B): 92.2% (t_R = 14.44 min).

N-(2-{[2-(Dimethylamino)ethyl](methyl)amino}-5-[4-(ethylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]phenyl)prop-2-enamide (51)

To a solution of 64p (18 mg, 0.04 mmol, 1.0 equiv) in dichloromethane (5 mL) were added acrylic acid (4 μL, 0.06 mmol, 1.4 equiv) and EDCI (12 mg, 0.06 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 4 h, to the reaction mixture were added water (3 mL), dichloromethane (10 mL), and sat. NaHCO_3(aq) (1 mL) and extracted with dichloromethane (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 51 (10 mg, 0.02 mmol, 49%) as a yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 10.33 (s, 1H), 8.69 (s, 1H), 8.40 (s, 1H), 7.58 (d, J = 8.0 Hz, 2H), 7.32–7.23 (m, 5H), 7.11 (dd, J = 8.4, 2.4 Hz, 1H), 6.44 (dd, J = 17.2, 2.0 Hz, 1H), 6.35 (s, 1H), 5.74 (dd, J = 10.0, 2.4 Hz, 1H), 5.07 (s, 1H), 3.49 (dq, J = 7.2, 5.2 Hz, 2H), 2.94 (s, 2H), 2.80 (s, 3H), 2.50–2.24 (m, 8H), 1.09 (t, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.5, 157.0, 154.0, 145.4, 144.0, 133.9, 131.6, 129.2, 128.8, 128.7, 127.3, 126.1, 125.6, 122.5, 115.2, 102.1, 56.6, 55.2, 45.4, 41.0, 35.4, 14.2. LRMS (ESI) m/z: 485.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₈H₃₃N₆O₂, 485.2665 [M + H]⁺; found, 485.2665. HPLC purity (method B): 98.0% (t_R = 15.32 min).

N-(2-{[2-(Dimethylamino)ethyl](methyl)amino}-5-[6-phenyl-4-(propan-2-ylamino)furo[2,3-d]pyrimidin-5-yl]phenyl)prop-2-enamide (52)

To a solution of 64q (19 mg, 0.04 mmol, 1.0 equiv) in dichloromethane (5.0 mL) were added acrylic acid (4 μL, 0.06 mmol, 1.4 equiv) and EDCI (12 mg, 0.06 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 4 h, to the reaction mixture were added water (3 mL), dichloromethane (10 mL), and sat. NaHCO_3(aq) (1 mL) and extracted with dichloromethane (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 52 (10 mg, 0.02 mmol, 47%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.27 (s, 1H), 8.41 (d, J = 2.1 Hz, 1H), 8.34 (s, 1H), 7.55 (d, J = 6.6 Hz, 2H), 7.45 (d, J = 8.4 Hz, 1H), 7.41–7.31 (m, 3H), 7.19 (dd, J = 8.4, 2.1 Hz, 1H), 6.47 (dd, J = 17.4, 10.2 Hz, 1H), 6.25 (dd, J = 17.4, 2.1 Hz, 1H), 5.80 (dd, J = 10.2, 2.1 Hz, 1H), 4.93 (d, J = 7.2 Hz, 1H), 4.17 (dset, J = 7.2, 6.6, 6.3 Hz, 1H), 2.93 (t, J = 6.0 Hz, 2H), 2.78 (s, 3H), 2.38 (t, J = 6.0 Hz, 2H), 2.21 (s, 6H), 1.03 (d, J = 6.3 Hz, 6H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.4, 163.4, 156.5, 154.1, 145.3, 143.7, 134.4, 131.6, 129.1, 128.8, 128.7, 127.3, 126.6, 126.0, 125.4, 122.7, 121.5, 115.1, 102.2, 56.7, 55.5, 45.4, 42.3, 41.3, 22.0. LRMS (ESI) m/z: 499.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₉H₃₅N₆O₂, 499.2822 [M + H]⁺; found, 499.2824. HPLC purity (method B): 99.0% (t_R = 16.31 min).

N-{5-[4-(Cyclopropylamino)-6-phenylfuro[2,3-d]pyrimidin-5-yl]-2-{[2-(dimethylamino)ethyl](methyl)amino}phenyl}prop-2-enamide (53)

To a solution of 64r (29 mg, 0.07 mmol, 1.0 equiv) in dichloromethane (5.0 mL) were added acrylic acid (7 μL, 0.10 mmol, 1.6 equiv) and EDCI (19 mg, 0.10 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 2.5 h, to the reaction mixture were added water (3 mL), dichloromethane (10 mL), and sat. NaHCO_3(aq) (1 mL) and extracted with dichloromethane (10 mL × 3). The combined organic layers were washed with brine (10 mL), dried over MgSO₄, concentrated in vacuo, and purified by flash column chromatography (3% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 53 (17 mg, 0.03 mmol, 52%) as a yellow oil. ¹H NMR (600 MHz, DMSO-d₆) δ 10.25 (s, 1H), 8.40 (s, 1H), 8.32 (d, J = 2.4 Hz, 1H), 7.53 (d, J = 6.6 Hz, 2H), 7.41 (d, J = 8.4 Hz, 1H), 7.38–7.31 (m, 3H), 7.15 (dd, J = 8.4, 2.1 Hz, 1H), 6.47 (dd, J = 16.8, 10.2 Hz, 1H), 6.25 (dd, J = 16.8, 1.8 Hz, 1H), 5.80 (dd, J = 10.2, 1.8 Hz, 1H), 5.32 (d, J = 3.6 Hz, 1H), 2.93 (t, J = 6.0 Hz, 2H), 2.86–2.80 (m, 1H), 2.77 (s, 3H), 2.40 (t, J = 6.0 Hz, 2H), 2.22 (s, 6H), 0.72–0.66 (m, 2H), 0.35–0.32 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.4, 163.4, 158.1, 154.0, 145.6, 143.8, 134.1, 131.6, 129.1, 128.8, 128.7, 127.3, 126.4, 126.1, 125.4, 122.6, 121.7, 115.1, 102.6, 56.7, 55.4, 45.5, 41.1, 23.9, 6.8. LRMS (ESI) m/z: 497.4 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₉H₃₃N₆O₂, 497.2665 [M + H]⁺; found, 497.2659. HPLC purity (method B): 96.4% (t_R = 14.46 min).

N-(2-{[2-(Dimethylamino)ethyl](methyl)amino}-5-{4-[(2-hydroxyethyl)amino]-6-phenylfuro[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (54)

To a solution of 64s (56 mg, 0.13 mmol, 1.0 equiv) in dichloromethane (5.0 mL) were added acrylic acid (9 μL, 0.13 mmol, 1.1 equiv) and EDCI (36 mg, 0.19 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (2–5% methanol in dichloromethane with 0.1% NH₄OH) to yield the title compound 54 (36 mg, 0.07 mmol, 57%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.32 (s, 1H), 8.36 (d, J = 2.1 Hz, 1H), 8.33 (s, 1H), 7.52 (d, J = 7.2 Hz, 2H), 7.41 (d, J = 8.1 Hz, 1H), 7.38–7.30 (m, 3H), 7.19 (dd, J = 8.1, 2.1 Hz, 1H), 6.44 (dd, J = 17.1, 10.2 Hz, 1H), 6.24 (dd, J = 17.1, 2.1 Hz, 1H), 5.79 (dd, J = 10.2, 2.1 Hz, 1H), 5.50 (t, J = 5.4 Hz, 1H), 3.50–3.38 (m, 4H), 2.91 (t, J = 6.0 Hz, 2H), 2.76 (s, 3H), 2.40 (t, J = 6.0 Hz, 2H), 2.23 (s, 6H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.4, 157.2, 154.0, 145.3, 143.7, 134.4, 131.7, 129.2, 128.8, 128.6, 127.2, 126.5, 126.0, 125.1, 122.7, 121.6, 115.3, 102.3, 59.1, 56.7, 55.7, 45.5, 42.8, 41.1. LRMS (ESI) m/z: 501.4 [M + H]⁺. HRMS (ESI) m/z: calcd for C₂₈H₃₃N₆O₃, 501.2614 [M + H]⁺; found, 501.2593. HPLC purity (method B): 93.4% (t_R = 13.60 min).

N-(2-{[2-(Dimethylamino)ethyl](methyl)amino}-5-{4-[((1S,2R)-2-hydroxycyclopentyl)amino]-6-phenylfuro[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (55)

To a solution of 64t (88 mg, 0.18 mmol, 1.0 equiv) in dichloromethane (0.9 mL) were added acrylic acid (15 μL, 0.22 mmol, 1.2 equiv) and EDCI (52 mg, 0.27 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (3% methanol in dichloromethane) to yield the title compound 55 (55 mg, 0.10 mmol, 56%) as a yellow solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.32 (s, 1H), 8.45 (s, 1H), 8.31 (s, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 1H), 7.38–7.29 (m, 3H), 7.20 (dd, J = 8.4, 2.4 Hz, 1H), 6.41 (dd, J = 16.8, 10.2 Hz, 1H), 6.22 (dd, J = 16.8, 2.1 Hz, 1H), 5.77 (dd, J = 10.2, 2.1 Hz, 1H), 5.63 (d, J = 7,2 Hz, 1H), 4.63 (s, 1H), 4.15 (t, J = 7.2 Hz, 1H), 3.94–3.88 (m, 1H), 2.95–2.84 (m, 2H), 2.76 (s, 3H), 2.39 (t, J = 5.4 Hz, 2H), 2.33 (s, 6H), 2.00–1.85 (m, 1H), 1.76–1.68 (m 1H), 1.64–1.54 (m, 1H), 1.46–1.29 (m, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.4, 163.3, 156.8, 154.1, 145.1, 143.3, 134.9, 131.8, 129.2, 128.8, 128.6, 127.1, 126.8, 125.8, 124.8, 122.9, 115.5, 102.5, 70.3, 56.7, 55.9, 54.4, 45.4, 41.3, 32.3, 29.4, 20.0. LRMS (ESI) m/z: 541.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₁H₃₇N₆O₃, 541.2927 [M + H]⁺; found, 541.2916. HPLC purity (method B): 98.2% (t_R = 15.07 min).

N-(2-{[2-(Dimethylamino)ethyl](methyl)amino}-5-{4-[((1R,2R)-2-hydroxycyclopentyl)amino]-6-phenylfuro[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (56)

To a solution of 64u (54 mg, 0.11 mmol, 1.0 equiv) in dichloromethane (1.5 mL) were added acrylic acid (9 μL, 0.13 mmol, 1.2 equiv) and EDCI (32 mg, 0.17 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (5% methanol in dichloromethane) to yield the title compound 56 (38 mg, 0.07 mmol, 63%) as colorless oil. ¹H NMR (600 MHz, DMSO-d₆) δ 10.30 (s, 1H), 8.45 (d, J = 2.4 Hz, 1H), 8.35 (s, 1H), 7.55 (d, J = 6.6 Hz, 2H), 7.45 (d, J = 8.1 Hz, 1H), 7.38–7.30 (m, 3H), 7.20 (dd, J = 8.1, 2.4 Hz, 1H), 6.46 (dd, J = 16.8, 10.2 Hz, 1H), 6.24 (dd, J = 16.8, 1.8 Hz, 1H), 5.80 (dd, J = 10.2, 1.8 Hz, 1H), 4.90–4.80 (m, 2H), 4.16–4.10 (m, 1H), 3.74–3.67 (m, 1H), 2.90 (t, J = 6.0 Hz, 2H), 2.76 (s, 3H), 2.40 (t, J = 6.0 Hz, 2H), 2.22 (s, 6H), 2.03–1.95 (m, 1H), 1.65–1.55 (m 1H), 1.54–1.46 (m, 1H), 1.42–1.34 (m, 1H), 1.32–1.16 (m, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.3, 163.4, 156.7, 154.1, 145.4, 143.6, 134.6, 131.6, 129.1, 128.8, 128.7, 127.4, 126.8, 125.8, 125.2, 122.8, 121.0, 115.0, 102.5, 75.8, 58.7, 56.7, 55.6, 45.5, 41.2, 31.6, 29.4, 20.2. LRMS (ESI) m/z: 541.5 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₁H₃₇N₆O₃, 541.2927 [M + H]⁺; found, 541.2907. HPLC purity (method B): 95.2% (t_R = 14.85 min).

N-(2-{[2-(Dimethylamino)ethyl](methyl)amino}-5-{4-[(3-hydroxycyclobutyl)amino]-6-phenylfuro[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (57)

To a solution of 64v (75 mg, 0.16 mmol, 1.0 equiv) in dichloromethane (0.8 mL) were added acrylic acid (13 μL, 0.19 mmol, 1.2 equiv) and EDCI (46 mg, 0.24 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (3% methanol in dichloromethane) to yield the title compound 57 (35 mg, 0.07 mmol, 42%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.34 (s, 1H), 8.40 (d, J = 2.4 Hz, 1H), 8.33 (s, 1H), 7.54 (d, J = 7.2 Hz, 2H), 7.46 (d, J = 8.1 Hz, 1H), 7.39–7.31 (m, 3H), 7.21 (dd, J = 8.1, 2.4 Hz, 1H), 6.46 (dd, J = 17.4, 10.2 Hz, 1H), 6.27 (dd, J = 17.4, 1.8 Hz, 1H), 5.80 (dd, J = 10.2, 1.8 Hz, 1H), 5.21 (d, J = 7,2 Hz, 1H), 5.00 (s, 1H), 4.03–3.95 (m, 1H), 3.81 (ddd, J = 7.2, 7.2, 7.2 Hz, 1H), 2.94 (t, J = 6.0 Hz, 2H), 2.79 (s, 3H), 2.60–2.53 (m, 2H), 2.41 (t, J = 6.0 Hz, 2H), 2.22 (s, 6H), 1.51–1.44 (m 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 163.5, 156.3, 154.0, 145.5, 143.9, 134.3, 131.6, 129.1, 128.8, 128.7, 127.4, 126.5, 126.0, 125.4, 122.6, 121.7, 115.1, 102.3, 59.1, 56.8, 55.6, 45.5, 41.4, 41.2, 37.3. LRMS (ESI) m/z: 527.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₀H₃₄N₆NaO₃, 549.2590 [M + Na]⁺; found, 549.2589. HPLC purity (method B): 98.1% (t_R = 14.06 min).

N-(2-{[2-(Dimethylamino)ethyl](methyl)amino}-5-{4-[(3-hydroxycyclobutyl)amino]-6-phenylfuro[2,3-d]pyrimidin-5-yl}phenyl)prop-2-enamide (58)

To a solution of 64w (66 mg, 0.16 mmol, 1.0 equiv) in dichloromethane (0.7 mL) were added acrylic acid (11 μL, 0.16 mmol, 1.1 equiv) and EDCI (40 mg, 0.21 mmol, 1.5 equiv); then, the reaction mixture was stirred at room temperature. After stirring for 16 h, the reaction mixture was concentrated in vacuo and purified by flash column chromatography (5% methanol in dichloromethane) to yield the title compound 58 (16 mg, 0.03 mmol, 22%) as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.32 (s, 1H), 8.42 (d, J = 2.4 Hz, 1H), 8.34 (s, 1H), 7.56 (d, J = 6.6 Hz, 2H), 7.45 (d, J = 8.1 Hz, 1H), 7.40–7.31 (m, 3H), 7.21 (dd, J = 8.1, 2.4 Hz, 1H), 6.47 (dd, J = 17.4, 10.5 Hz, 1H), 6.26 (dd, J = 17.4, 2.4 Hz, 1H), 5.80 (dd, J = 10.5, 2.4 Hz, 1H), 5.30 (d, J = 6.6 Hz, 1H), 5.02 (s, 1H), 4.50–4.42 (m, 1H), 4.12–4.05 (m, 1H), 2.93 (t, J = 6.0 Hz, 2H), 2.78 (s, 3H), 2.40 (t, J = 6.0 Hz, 2H), 2.22 (s, 6H), 2.17–2.11 (m, 2H), 1.95–1.87 (m 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 164.4, 163.4, 156.5, 154.0, 145.4, 143.8, 134.3, 131.6, 129.1, 128.8, 128.7, 127.3, 126.6, 126.0, 125.4, 122.7, 121.5, 115.1, 102.6, 63.0, 56.7, 55.6, 45.5, 45.4, 42.1, 41.2. LRMS (ESI) m/z: 527.3 [M + H]⁺. HRMS (ESI) m/z: calcd for C₃₀H₃₅N₆O₃, 527.2771 [M + H]⁺; found, 527.2781. HPLC purity (method B): 98.2% (t_R = 14.10 min).

Molecular Docking Studies

For carrying out the noncovalent docking of ligand 6 in EGFR^WT (PDB ID: 6JZ0), Schrodinger Glide XP³³ was used. Ligands were prepared at pH 7 ± 2 using Ligprep module before docking in the active site defined by the cocrystal ligand 11 present in the EGFR protein. For carrying out the covalent docking of 11 and 52 to EGFR^WT (PDB ID: 6JXT) and EGFR^T790M (PDB ID: 6JX0), covalent docking module of Schrodinger was used. Ligands were prepared at pH 7±2 using Ligprep module before docking in the active site defined by the cocrystal ligand 6 present in the EGFR protein. Nucleophilic addition reaction and Cys797 residue were chosen to perform the covalent docking. Top scoring poses were retrieved and analyzed.

EGFR^WT and EGFR^L858R/T790M Enzyme Inhibition Assays

Kinase-Glo Plus Luminescent Kinase Assay (Promega) was used to test the inhibition potential of the newly synthesized compounds for GST-EGFR (G696-G1022) wild-type and L858R/T790M double-mutant recombinant proteins. Detailed methodology for carrying out the enzyme inhibition assay could be found in our previous publication.²⁸

Cell Proliferation Assay and Western Blot Analysis

A431 (wild-type EGFR overexpressing) and H1975 (double-mutant overexpressing) cells were exposed to test compounds for 96 h and the cell proliferation was determined by the MTS method. Western blot analysis was performed by exposing the cells (A431 and H1975) to the test compound for 1 h. Drug-treated cells were lysed and subjected to western blot analysis. Refer to our previous publication for detailed methods of cell proliferation and western blot analysis.^28,34

In Vitro Microsomal Stability Assay

In vitro drug metabolism (hCyt450) assay was performed as reported earlier by us.³⁵

In Vivo Pharmacokinetics Study

Male Sprague Dawley rats (300–400 g) were obtained from BioLASCO Taiwan Co., Ltd. (Ilan, Taiwan) and used for pharmacokinetics studies as reported earlier.³⁵ The animal studies were conducted according to NHRI institutional animal care and committee-approved procedures.

In Vivo BaF3 EGFR^L858R/T790M Xenograft Study

Adult male nude mice (Nu-Fox1nu) purchased from BioLasco, Taiwan Co., Ltd. were used for this study. Animals had access to food and water ad libitum. Institutional Animal Care and Use Committees (IACUC) of the National Health Research Institutes approved the study using animals. EGFR^L858R/T790M expressing human BaF3 cells were cultured in RPMI-1640 supplemented with 10% (v/v) fetal bovine serum (FBS) at 37 °C in a humidified atmosphere consisting of 5% CO₂. Each 9-week-old nude mouse was inoculated subcutaneously with 1 × 10⁶ BaF3 EGFR^L858R/T790M cells in 50% Matrigel (Becton Bickinson) in 0.1 mL injection volume via a 24-gauge needle. Tumor volume was measured with the help of an electronic caliper and calculated using the formula, length × width² × 0.5. When the size of a growing tumor reached ∼280 mm³, the tumor-bearing mice were administered compound 49 or 52 (dissolved in 20% 2-hydroxypropyl-β-cyclodextrin in water) by oral gavage 5 days a week for 2 consecutive weeks at 100 mg/kg doses. 6 (AZD9291) was administered orally at 10 mg/kg doses in the same regimen for comparison. The control group received vehicle alone (20% 2-hydroxypropyl-β-cyclodextrin in water) by oral gavage.

In Vivo H1975 Xenograft Study

To assess the antitumor activity of 52, five-week-old male nude mice (National Laboratory Animal Center, NLAC, Taipei, Taiwan) were subcutaneously injected with 1 × 10⁷ H1975 cells. When the tumor sizes reached 100 mm³, mice were divided into four groups randomly (control, 6 (AZD9291) 10 mg/kg, 52 10 and 30 mg/kg groups, with 9–10 mice in each group). Mice in control groups were dosed with the vehicle (20% 2-hydroxypropyl-β-cyclodextrin, HP-β-CD) by oral administration (po) once daily (qd); 6 (AZD9291) group was dosed with 10 mg/kg of 6 (AZD9291) po, qd; 52 groups were dosed with 10 or 30 mg/kg of 52 po, qd. The tumor size and body weight were measured twice per week during the experiment, and the tumor volume (mm³) was calculated as mentioned above. Tumor growth inhibition (% TGI) = [1 – (T_t – T₀)/(C_t – C₀) × 100], where C₀ and C_t are mean tumor volumes of the control group by first data point and day t, respectively, while T₀ and T_t are mean tumor volumes of the treatment group by first data point and day t, respectively. This study was approved by Animal Use and Management Committee of Taipei Medical University (IACUC number LAC-2017-0444).

Glossary

Abbreviations Used

3D

three-dimensional

ACN

acetonitrile

ATP

adenosine triphosphate

AUC

area under the curve

BLK

B lymphocyte kinase

Boc

di-tert-butyl dicarbonate

BTK

Bruton’s tyrosine kinase

ⁿBuOH

n-butanol

CC₅₀

half-maximal cytotoxic concentration

DIPEA

N,N-diisopropylethylamine

DMF

N,N-dimethylformamide

DMPK

drug metabolism and pharmacokinetics

DMSO

dimethyl sulfoxide

EDCI

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EGFR

epidermal growth factor receptor

equiv

equivalence

ERBB2

receptor tyrosine-protein kinase erbB-2

ERBB4

receptor tyrosine-protein kinase erbB-4

ESI

electrospray ionization

Et₃N

triethylamine

F

oral bioavailability

FBS

fetal bovine serum

FDA

food and drug administration

GI

gastrointestinal

HCl

hydrochloride salt

HPLC

high-performance liquid chromatography

HRMS

high-resolution mass spectrometry

IC₅₀

half-maximal inhibitory concentration

IPA

isopropyl alcohol

IV

intravenous administration

JAK3

Janus kinase 3

LC-MS

liquid chromatograph/mass spectrometer

LRMS

low-resolution mass spectrometry

MHz

megahertz

Ml. Wt.

molecular weight

NBS

N-bromo succinimide

ND

not detected/determined

NHRI

National Health Research Institutes

NMR

nuclear magnetic resonance

NSCLC

non-small cell lung cancer

OTBS

tert-butyldimethylsilyl ether

PDB

protein data bank

PK

pharmacokinetics

PO

oral administration

QD

once daily

RBN

number of rotatable bonds

rt

room temperature

SAR

structure–activity relationship

S_NAr

aromatic substitution reaction

TBSCl

tert-butyldimethylsilyl chloride

TFA

trifluoroacetic acid

TGI

tumor growth inhibition

THF

tetrahydrofuran

TLC

thin-layer chromatography

vs

versus

WT

wild-type

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01434.

• ¹H NMR, ¹³C NMR, and HPLC purity spectra of final compounds 13–58, synthesis procedure and compound characterization data for intermediate compounds 60–64, 66, 67, and 69–72, rat body weight change during drug treatment, and kinase profiling data of 52 (PDF)

• SMILES string for compounds 13–58 (CSV)

• 6JX0_11 (Docking pose of 11 in EGFR T790M) (PDB)

• 6JX0_52 (Docking pose of 52 in EGFR T790M) (PDB)

• 6JXT_11 (Docking pose of 11 in EGFR WT) (PDB)

• 6JXT_52 (Docking pose of 52 in EGFR WT) (PDB)

• 6JZ0_6 (Docking pose of 6 in EGFR WT) (PDB)

Author Contributions

^∇ M.-C.L. and M.S.C. contributed equally to this work.

The authors declare no competing financial interest.