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. 2017 Dec 11;9(2):244–253. doi: 10.1039/c7md00571g

Allenamide as a bioisostere of acrylamide in the design and synthesis of targeted covalent inhibitors†

Deheng Chen a,b, Dexiang Guo a, Ziqin Yan a, Yujun Zhao a,✉
PMCID: PMC6083791  PMID: 30108918

graphic file with name c7md00571g-ga.jpgThe success of acrylamide-containing drugs in treating cancers has spurred a passion to search for acrylamide bioisosteres.

Abstract

The success of acrylamide-containing drugs in treating cancers has spurred a passion to search for acrylamide bioisosteres. In our endeavour, we have identified that an allenamide group can be a reactive bioisostere of the acrylamide group. In our development of allenamide-containing compounds, we found that the most potent compound, 14, inhibited the kinase activities of both T790M/L858R double mutant and wild type EGFR in a low nM range. 14 also inhibited the growth of NCI-H1975 lung cancer cells at IC50 = 33 nM, which is comparable to that of acrylamide-containing osimertinib. The western blot analysis showed that the phosphorylation of EGFR, AKT, and ERK1/2 was simultaneously inhibited in a dose-dependent manner when NCI-H1975 cells were treated with 14. By measuring the conjugate addition product formed by 14 and GSH, we obtained a reaction rate constant of 302.5 × 10–3 min–1, which is about 30-fold higher than that of osimertinib. Taken together, our data suggest that the allenamide-containing compounds inhibited EGFR kinases through covalent modifications. Our study indicates that the allenamide group could serve as an alternative electrophilic warhead in the design of targeted covalent inhibitors, and this bioisostere replacement may have broad applications in medicinal chemistry.

Introduction

Inactivation of target biomolecules by forming covalent bonds with selective small molecules has emerged as a promising approach for drug discovery since the 1990s, and this field has evolved dramatically in recent years.1–8 With the successful launch of 1 (afatinib), 2 (osimertinib), and 3 (ibrutinib) (Fig. 1) in the market, acrylamide has become one of the privileged scaffolds in the design of target covalent inhibitors (TCIs).2,4,9 The olefin moiety of acrylamides readily reacts with the thiol group of target proteins in vitro and in vivo, forming conjugate adducts and thereby, causing loss of function of the target proteins to achieve in vivo efficacy.10–12

Fig. 1. Representative approved and experimental drugs bearing acrylamide and α,β-unsaturated carbonyls.

Fig. 1

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The clinical success of 1–3 and other TCIs has spurred a passion to search for other α,β-unsaturated carbonyls as bioisosteres of the acrylamide moiety in drug discovery.2,3,5,9,13,14 For example, a but-2-ynamide moiety has been used as the warhead of 4 (acalabrutinib), which covalently bonds to the Cys-481 residue of BTK and was recently approved by the FDA for treating mantle cell lymphoma.11,15 Another earlier example, 5 (ethacrynic acid), has also been approved for the treatment of high blood pressure and swelling for more than 30 years, and it is currently being investigated in clinics for the treatment of bladder cancer.16 Mechanistically, 5 reacted with the thiol residues of the Na-K-2Cl cotransporter and resulted in its subsequent inactivation.17

In 6 (E6201),18 a vinyl ketone moiety functions as the warhead to carry out covalent addition to MEK,7 and 6 has been advanced into phase I/II clinical trials for the treatment of advanced hematologic malignancies with documented FLT3 mutation.19 More interestingly, a thiol group of Keap1 can react with the α-cyanoenone group of 7 (bardoxolone methyl) forming a covalent bond although such an addition reaction is reversible.20–23 7 was well tolerated in humans, and the drug is currently evaluated in multiple phase II/III clinical trials for the treatment of pulmonary hypertension.24 Taken together, the design and synthesis of novel bioisosteres to replace the acrylamide moiety are attractive and promising approaches in the development of novel TCIs in medicinal chemistry.25

Recently, Loh's group reported that allenamides selectively reacted with a cysteine residue in peptides forming conjugate adducts, which is useful for orthogonal labelling thiol-containing peptides and proteins.26 In view of the robust reactivity of allenamides towards thiol groups, we hypothesized that allenamide could be used as a bioisostere of the acrylamide moiety in small molecule drug design. In order to test this hypothesis, we replaced the acrylamide moiety of 2 with an allenamide group in an irreversible EGFR kinase inhibitor model system,10,27–39 which led to a series of novel TCIs.

In this study, we report our synthesis and optimization of allenamide-containing covalent inhibitors, which exhibit efficient inhibition of the enzymatic activity of T790M mutant EGFR kinase. The allenamide-containing TCIs also have potent cellular activity against the NCI-H1975 lung cancer cell line, which harbours T790M mutant EGFR kinase. We also found that the antitumor activity of the allenamide-containing compounds depends on the inhibition of EGFR phosphorylation and the subsequent inactivation of two downstream kinases, namely, PI3K-AKT and MAPK.40,41 Overall, our studies indicate that allenamide could serve as an alternative electrophilic warhead for the design of novel targeted covalent inhibitors and may have broader applications in medicinal chemistry.

Results and discussion

We started our investigations with the replacement of the acrylamide group of 2 (ref. 10) with an allenamide group to obtain compound 8 (Scheme 1). Based on 8, we also synthesized 9–19 in order to briefly examine the influence of other solubilizing groups on the enzymatic and cellular activities. The syntheses of 8–19 were straightforward and conducted by reacting the corresponding substituted aniline with but-3-ynoic acid and 2-Cl-1-methylpyridinium iodide in the presence of a base,26 followed by treatment with potassium carbonate. We also synthesized compounds 20–28 (Scheme 1) to explore whether different substitutes on the pyrimidine ring and indole ring significantly affect the in vitro activities of the allenamide-containing compounds. The syntheses of 20–28 were similar to that of 8 as shown in Scheme 1.

Scheme 1. Syntheses of allenamide-containing compounds 8–28.

Scheme 1

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Next, we tested the enzymatic activity of 8–19 against wild type and T790M/L858R double mutant EGFR kinases, and the results are summarized in Table 1. 8 has an IC50 value of 1.4 nM, inhibiting the phosphorylation of T790M/L858R double mutant EGFR kinase, which is 2–3-fold more potent than 2. It also displays 20-fold selectivity against the wild type EGFR kinase (IC50 = 27.9 nM). In contrast, 2 shows 36-fold selectivity between the double mutant and wild type EGFR kinases in our assay. Although the selectivity profile of 8 slightly decreased compared to that of 2, it nevertheless efficiently inhibited the enzymatic activity of both the double mutant and wild type EGFR kinases in vitro. Since the aliphatic amine parts play a role in the activity of EGFR kinase inhibitors, we hypothesized that further optimization of this moiety might increase the in vitro potency and selectivity of 8. We thus synthesized a series of allenamide-containing analogues 9–19, and the results are summarized in Table 1.

Table 1. Optimization of allenamide-containing compounds.

Compound EGFR WT IC50 (nM) EGFR T790M/L858R IC50 (nM) Selectivity (in -fold)
2 142.1 ± 56.2 3.9 ± 1.0 36
8 27.9 ± 6.4 1.4 ± 0.3 20
9 25.4 ± 0.5 0.8 ± 0.1 32
10 42.0 ± 9.4 1.2 ± 0.3 35
11 29.1 ± 1.9 0.7 ± 0.1 42
12 13.9 ± 4.6 0.4 ± 0.4 34
13 30.4 ± 6.8 0.7 ± 0.1 43
14 6.3 ± 0.0 1.0 ± 0.1 6
15 6.3 ± 3.2 0.3 ± 0.1 21
16 13.5 ± 0.6 0.6 ± 0.4 22
17 8.6 ± 4.2 0.7 ± 0.1 12
18 5.8 ± 3.8 0.7 ± 0.1 8
19 18.9 ± 3.0 0.7 ± 0.0 27
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In general, the IC50 values of compounds 9–19 are all less than 2 nM for double mutant EGFR kinase with compound 15 as the most potent compound (IC50 = 0.3 nM). The activities of 9–19 against wild type EGFR kinase also improved, while the double mutant/wild type selectivity ratio ranges from 6-fold to 43-fold. In terms of the target selectivity between the double mutant and wild type kinases, compound 13 bearing an oxoethylmorpholine moiety is the most selective compound (43-fold).

To examine whether the selectivity of kinase activity translates into the difference in cellular activity, we further evaluated 8–19 against NCI-H1975 and A549 lung cancer cell lines,42 which harbour T790M/L858R double mutant and wild type EGFR, respectively (Table 2).

Table 2. Cellular activity of allenamide-containing compounds.

ID NCI-H1975 IC50 (μM) A549 IC50 (μM) Selectivity (in -fold)
2 0.013 ± 0.004 0.31 ± 0.23 23.8
8 0.29 ± 0.22 0.28 ± 0.1 1
9 0.23 ± 0.15 0.12 ± 0.12 0.52
10 0.36 ± 0.2 0.28 ± 0.16 0.77
11 1.42 ± 0.84 0.72 ± 0.61 0.51
12 0.31 ± 0.19 0.59 ± 0.42 1.90
13 1.93 ± 0.94 0.26 ± 0.042 0.13
14 0.033 ± 0.021 0.06 ± 0.036 2
15 0.34 ± 0.14 0.35 ± 0.2 1
16 0.49 ± 0.24 0.061 ± 0.001 0.12
17 0.45 ± 0.078 0.49 ± 0.41 1.1
18 0.15 ± 0.097 0.36 ± 0.24 2.4
19 5.28 ± 5.18 1.57 ± 1.59 0.30
20 0.78 ± 0.21 0.43 ± 0.38 0.55
21 0.4 ± 0.2 0.31 ± 0.24 0.78
22 0.15 ± 0.058 0.21 ± 0.19 1.4
23 0.19 ± 0.04 0.34 ± 0.26 1.79
24 0.16 ± 0.06 0.39 ± 0.38 2.43
25 0.35 ± 0.12 0.27 ± 0.19 0.77
26 0.22 ± 0.17 0.047 ± 0.043 0.21
27 0.24 ± 0.16 0.16 ± 0.09 0.67
28 0.29 ± 0.19 0.087 ± 0.056 0.3
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Compound 8 has an IC50 value of 0.29 μM in the NCI-H1975 cancer cell line, which is about 20-fold less potent than 2. The cellular potency of 8 in A549 is similar to that in the NCI-H1975 cancer cell line, whereas 2 shows about 24-fold selectivity between the two cell lines, but 8 has no cellular selectivity. In general, the differences in the cellular potency between double mutant and wild type EGFR-containing cells for 8–19 are less than threefold. Among these, 14 containing the N,N-dimethylpiperidin-4-amine moiety produces the most potent cellular activity in this series by inhibiting the growth of NCI-H1975 cancer cells at IC50 = 33 nM.

In the A549 lung cancer cell line, 14 inhibits cell growth at IC50 = 60 nM and shows 2-fold selectivity between the wild type and double mutant enzyme-containing cancer cell lines. Other compounds in this series all yield weaker cellular activities (>100 nM) against the NCI-H1975 cancer cell line. Taken together, the allenamide-containing analogues (8–19) have high kinase inhibition activities for both double mutant and wild type EGFR kinases. However, the selectivity varies between double mutant and wild type EGFR kinase-containing cancer cell lines. In this series, compound 14 gives the most potent cellular activities against the NCI-H1975 and A549 lung cancer cell lines but with decreased selectivity between the two cell lines when compared with 2.

Previous results showed that the indole, pyrimidine, and benzene moieties in 2 are important for the target selectivity;10 we thus further optimized these three moieties in our allenamide-containing compounds to increase their cellular potency and selectivity. The results are summarized in Table 3.

Table 3. Optimization of indole and pyrimidine cores of allenamide-containing compounds.

Compound EGFR WT IC50 (nM) EGFR T790M/L858R IC50 (nM) Selectivity (in -fold)
20 29.9 ± 6.1 2.7 ± 0.3 11
21 22.8 ± 9.8 1.5 ± 0.5 15
22 14.7 ± 3.9 0.8 ± 0.2 18
23 6.3 ± 1.8 0.7 ± 0.2 9
24 5.2 ± 3.1 1.0 ± 0.6 5
25 3.2 ± 1.1 0.5 ± 0.1 6
26 10.5 ± 1.0 1.0 ± 0.1 10
27 30.6 ± 3.5 2.6 ± 0.4 12
28 22.4 ± 0.1 1.3 ± 0.1 17
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We found that R1 can tolerate a hydrogen, a methyl, or an ethoxyl group other than the methoxyl group because 20–22 have similar kinase inhibition potency compared to 8. The R2 position tolerates OMe and Me groups as the kinase inhibition IC50 values of 23 and 24 are 1 nM for double mutant EGFR. The removal of the methyl group from the indole N–H led to 25, which has an IC50 value of 0.5 nM against double mutant EGFR kinase and is threefold more potent than 8. The F-substituted indole-containing compound 26 has an IC50 value of 1.0 nM against double mutant EGFR kinase, which is about 2–3-fold more potent than its chlorine substituted analogue 27 (IC50 = 2.6 nM). Pyrazolo[1,5-a]pyridine-containing 28 has similar enzymatic activity against double mutant EGFR kinase to 8. For compounds 20–28, the selectivity between the double mutant and wild type kinases is in the range of 5–18-fold, which is less than that of 2. In our evaluation of the cellular activities against NCI-H1975 and A549 cancer cell lines, we found no significant improvement in these compounds when compared with 14 (Table 2). Although the growth inhibition IC50 values of 20–28 are in the range of 0.15–0.78 μM against NCI-H1975 cancer cell lines, compounds 20, 21, and 25–28 were more potent against the A549 cancer cell line than the NCI-H1975 cancer cell line. The selectivity for the EGFR wild type cancer cell line of 20, 21, and 25–28 is surprising. Overall, the optimization of the indole, pyrimidine, and benzene moieties of 2 did not yield a more potent compound than 14 in the inhibition of cell growth of the NCI-H1975 cell line.

In order to find out whether the cellular activity of 14 is a result of the inhibition of the EGFR signal pathway in intact cells, we performed the western blot analysis of the NCI-H1975 cancer cell line treated with 14 using compound 2 as the positive control. As shown in Fig. 2, the NCI-H1975 cells were first treated with 2 and 14 at increasing doses for 16 h, followed by EGF stimulation for another 15 min. The cells were then harvested, and the proteins were blotted for analysis. We observed that 14 dose-dependently inhibited the phosphorylation of EGFR, AKT, and ERK1/2, indicating that this compound inhibited cell growth through the inhibition of EGFR phosphorylation, and caused subsequent inactivation of PI3K–AKT and RAF–MEK–ERK pathways.

Fig. 2. Western blot analysis of EGFR and downstream proteins in the NCI-H1975 cancer cell line.

Fig. 2

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The western blot also shows that 2 had the same effect on the phosphorylation of EGFR, AKT, and ERK1/2 and is 10-fold more potent than 14, which is consistent of the cellular activity difference between 2 and 14 observed in the NCI-H1975 cell line data. In conclusion, the action of 14 on cell growth inhibition of NCI-H1975 cells is mediated by the inhibition of EGFR phosphorylation and the subsequent inactivation of two downstream signal pathways, namely, PI3K–AKT and RAF–MEK–ERK.10,27–36

In order to understand the biochemical reactivity profile of allenamide as a covalent warhead, we measured the pseudo-first-order reaction9 rate constant of GSH addition to 14 or acrylamide containing marketed drugs 1–3 (Table 4). The rate constant, kpseudo 1st, observed for GSH addition to 14 is 302.5 × 10–3 min–1 under our experimental conditions, which is about 7 to 28-fold higher than that of 1–3. These data indicate that 14 is expected to react with the Cys-797 residue of T790M-containing EGFR mutants at a faster rate than 1–3.

Table 4. The pseudo-first-order reaction rate of GSH addition to 14 or acrylamide containing marketed drugs 1–3.

Inline graphic
Compound k pseudo 1st (min–1 × 10–3)
1 43.6
2 10.6
3 11.4
14 302.5
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However, the higher reactivity of the allenamide moiety also led to low oral exposure and poor pharmacokinetic properties of 8 in mice (ESI†). In order to understand the reasons for their poor pharmacokinetic properties, we measured the stability of compounds 8 and 14 in fetal bovine serum (FBS) with 2 as the positive control. Compounds 2, 8, and 14 reacted with FBS at pseudo-first-order reaction rate constants of 0.35 × 10–3 min–1, 4.40 × 10–3 min–1, and 5.46 × 10–3 min–1, respectively. The data show that 8 decomposed 12.6-fold faster than 2 in FBS and 14 decomposed even faster (15.6-fold), which is in line with the quick clearance of 8 in mice. Therefore, we conclude that the poor pharmacokinetic properties of allenamide-containing compounds are partially because of their low stability observed in plasma and likely in vivo as well.

Conclusions

In summary, we have demonstrated that allenamide-containing compounds, such as 14, inhibited T790M/L858R double mutant and wild type EGFR kinases in a low nM range. Compound 14 inhibits the cell growth of the NCI-H1975 lung cancer cell line with IC50 = 33 nM, which is twofold more selective over wild type EGFR containing A549 lung cancer cells. Upon treatment of 14 in NCI-H1975 cells, the phosphorylation of EGFR, AKT, and ERK1/2 was simultaneously inhibited as is evident in the western blot analysis. The reaction of 14 and GSH readily forms a conjugate adduct with a reaction rate constant of 302.5 × 10–3 min–1, which is about 7 to 28-fold higher than the marketed acrylamide-containing covalent drugs afatinib, osimertinib, and ibrutinib. Taken together, our data indicated that allenamide-containing compounds inhibit EGFR kinases through covalent modifications. These results suggest that the allenamide moiety could serve as an alternative electrophilic warhead in the design of novel targeted covalent inhibitors and may have broader applications in medicinal chemistry.

Experimental section

General

All reactions were conducted in a round-bottom flask equipped with a magnet stirring bar. Commercially available reagents and anhydrous solvents were used without further purification. The crude reaction products were purified by column chromatography packed with silica gel. Further purifications were performed by preparative HPLC (SunFire Prep C18 OBRTM 5 μm). The mobile phases were a gradient flow of solvent A (water, 0.1% of TFA) and solvent B (CH3CN, 0.1% of TFA) at a flow rate of 10 mL min–1. Proton nuclear magnetic resonance (1H NMR) was performed using a Bruker Avance 400 NMR spectrometer. Carbon nuclear magnetic resonance (13C NMR) was performed using a Bruker Avance 500 NMR spectrometer. Low resolution ESI mass spectrum analysis was performed using an Advion expression CMS-L (TLC) mass spectrometer. All final compounds were purified to ≥95% purity as determined by analytical HPLC analysis, except for 9 (94%), 27 (94%) and 28 (94%).

Typical procedure for the synthesis of allenamide-containing compounds

The synthesis of 8 was used as an example. But-3-ynoic acid (21 mg, 0.25 mmol, 1.1 equiv.) was dissolved in dichloromethane (10 mL), and 2-Cl-1-methylpyridinium iodide (74 mg, 0.29 mmol, 1.3 equiv.) was added. The reaction solution was stirred at ambient temperature for 1 hour. A mixture of N1-(2-(dimethylamino)ethyl)-5-methoxy-N1-methyl-N4-(4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)benzene-1,2,4-triamine (100 mg, 0.23 mmol, 1.0 equiv.) and DIEA (0.1 mL) in dichloromethane was added dropwise to the aforementioned reaction solution. The reaction mixture was stirred for another 30 minutes and then concentrated. The resulting syrup was dissolved in acetonitrile (10 mL), and anhydrous potassium carbonate (47 mg, 0.34 mmol, 1.5 mmol) was added. The mixture was heated at 85 °C for 3 hours, then concentrated and purified by HPLC to yield 8 as a salt of trifluoroacetate (51 mg, 35%).

N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)buta-2,3-dienamide (8)

1H NMR (MeOD-d4, 400 MHz): 8.50 (s, 1H), 8.37–8.11 (m, 2H), 7.92 (d, J = 6.81 Hz, 1H), 7.50–7.44 (m, 1H), 7.37–7.30 (m, 1H), 7.29–7.20 (m, 2H), 7.08 (s, 1H), 6.16 (t, J = 6.48 Hz, 1H), 5.42 (d, J = 6.48 Hz, 2H), 3.96 (s, 3H), 3.88 (s, 3H), 3.52 (t, J = 5.95 Hz, 2H), 3.37 (t, J = 5.95 Hz, 2H), 2.93 (s, 6H), 2.80 (s, 3H). ESI-MS calculated for C29H33N7O2 [M + H]+ = 512.3. Obtained: 512.2.

N-(2-((2-(Dimethylamino)ethyl)(ethyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)buta-2,3-dienamide (9)

Yield 51%. 1H NMR (MeOD-d4, 400 MHz): 8.50–8.42 (m, 1H), 8.35–8.08 (m, 2H), 7.88 (d, J = 6.59 Hz, 1H), 7.43 (d, J = 8.01 Hz, 1H), 7.27–7.33 (m, 1H), 7.25–7.17 (m, 2H), 7.07 (m, 1H), 6.11 (t, J = 6.59 Hz, 1H), 5.42 (d, J = 6.59 Hz, 2H), 3.94 (s, 3H), 3.84 (s, 3H), 3.55 (t, J = 6.09 Hz, 2H), 3.35–3.28 (m, 2H), 3.20–3.10 (m, 2H), 2.89 (s, 6H), 1.15–1.06 (m, 3H). ESI-MS calculated for C30H35N7O2 [M + H]+ = 526.3. Obtained: 526.1.

N-(4-Methoxy-2-(methyl(2-morpholinoethyl)amino)-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)buta-2,3-dienamide (10)

Yield 6%. 1H NMR (MeOD-d4, 400 MHz): 8.51 (m, 1H), 8.33–8.20 (m,1H), 8.08–7.91 (m, 2H), 7.55–7.49 (m, 1H), 7.39–7.31 (m, 2H), 7.28–7.21 (m, 1H), 7.07 (m, 1H), 6.11 (t, J = 6.60 Hz, 1H), 5.44 (d, J = 6.60 Hz, 2H), 4.15–4.02 (m, 4H), 3.95 (s, 3H), 3.94 (s, 3H), 3.63–3.56 (m, 2H), 3.40–3.33 (m, 2H), 3.48–3.08 (m, 4H), 2.80 (s, 3H). ESI-MS calculated for C31H35N7O3 [M + H]+ = 554.3. Obtained: 554.5.

(S)-N-(4-Methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-((tetrahydrofuran-3-yl)oxy)phenyl)buta-2,3-dienamide (11)

Yield 4%. 1H NMR (MeOD-d4, 400 MHz): 8.54 (s, 1H), 8.48–8.22 (m, 2H), 7.95–7.85 (m, 1H), 7.51 (d, J = 8.24 Hz, 1H), 7.39–7.19 (m, 3H), 6.87 (s, 1H), 5.97 (t, J = 6.56 Hz, 1H), 5.41 (d, J = 6.56 Hz, 2H), 5.27–5.22 (m, 1H), 4.14–4.02 (m, 2H), 4.02–3.91 (m, 2H), 3.93 (s, 3H), 3.90 (s, 3H), 2.41–2.30 (m, 1H), 2.28–2.18 (m, 1H). ESI-MS calculated for C28H27N5O4 [M + H]+ = 498.2. Obtained: 498.4.

N-(4-Methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl) amino)-2-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)buta-2,3-dienamide (12)

Yield 0.7%. 1H NMR (MeOD-d4, 400 MHz): 8.56 (s, 1H), 8.47–8.32 (m, 1H), 8.28–8.05 (m, 1H), 7.99–7.89 (m, 1H), 7.54 (d, J = 8.46 Hz, 1H), 7.39–7.32 (m, 2H), 7.30–7.20 (m, 1H), 6.94 (s, 1H), 6.01 (t, J = 6.68 Hz, 1H), 5.41 (d, J = 6.68 Hz, 2H), 4.40 (t, J = 5.14 Hz, 2H), 3.97 (s, 3H), 3.92 (s, 3H), 3.41–3.28 (m, 4H), 3.14 (t, J = 5.14 Hz, 2H), 3.16–2.98 (m, 4H), 2.90 (s, 3H). ESI-MS calculated for C31H35N7O3 [M + H]+ = 554.3. Obtained: 554.3.

N-(4-Methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-(2-morpholinoethoxy)phenyl)buta-2,3-dienamide (13)

Yield 8%. 1H NMR (MeOD-d4, 400 MHz): 8.51 (s, 1H), 8.41–8.22 (m, 1H), 8.05–7.90 (m, 2H), 7.54–7.49 (m, 1H), 7.38–7.31 (m, 2H), 7.29–7.21 (m, 1H), 6.99 (s, 1H), 6.06 (t, J = 6.65 Hz, 1H), 5.41 (d, J = 6.65 Hz, 2H), 4.64 (t, J = 4.78 Hz, 2H), 4.17–3.96 (m, 4H), 3.95 (s, 3H), 3.94 (s, 3H), 3.71 (t, J = 4.92 Hz, 2H), 3.80–3.53 (m, 4H). ESI-MS calculated for C30H32N6O4 [M + H]+ = 541.3. Obtained: 541.4.

N-(2-(4-(Dimethylamino)piperidin-1-yl)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)buta-2,3-dienamide (14)

Yield 30%. 1H NMR (MeOD-d4, 400 MHz): 8.88–8.73 (m, 1H), 8.55 (s, 1H), 8.09–7.96 (m, 1H), 7.85 (d, J = 6.31 Hz, 1H), 7.42–7.34 (m, 1H), 7.30–7.23 (m, 1H), 7.22–7.14 (m, 1H), 7.08 (d, J = 6.31 Hz, 1H), 6.92 (s, 1H), 5.96 (t, J = 6.51 Hz, 1H), 5.50 (d, J = 6.51 Hz, 2H), 3.85 (s, 3H), 3.79 (s, 3H), 3.42–3.33 (m, 1H), 3.22–3.10 (m, 2H), 2.94 (s, 6H), 2.92–2.82 (m, 2H), 2.30–2.16 (m, 2H), 2.07–1.89 (m, 2H). 13C NMR (MeOD-d4, 125 MHz): 214.2, 167.6, 164.4, 154.7, 149.1, 147.0, 140.9, 140.0, 139.5, 127.1, 127.0, 124.3, 123.6, 122.7, 116.7, 113.7, 111.7, 107.4, 105.3, 92.0, 81.3, 64.7, 56.7, 52.1, 40.6, 33.9, 28.4. ESI-MS calculated for C31H35N7O2 [M + H]+ = 538.3. Obtained: 538.1.

N-(4-Methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl) amino)-2-(4-methylpiperazin-1-yl)phenyl)buta-2,3-dienamide (15)

Yield 2%. 1H NMR (MeOD-d4, 400 MHz): 8.80–8.59 (m, 1H), 8.52 (s, 1H), 8.03–7.81 (m, 1H), 7.72 (d, J = 6.90 Hz, 1H), 7.35–7.28 (m, 1H), 7.27–7.20 (m, 1H), 7.20–7.11 (m, 1H), 7.04–6.99 (m, 1H), 6.93 (s, 1H), 6.07 (t, J = 6.47 Hz, 1H), 5.45 (d, J = 6.47 Hz, 2H), 3.84 (s, 3H), 3.70 (s, 3H), 3.75–3.60 (m, 2H), 3.34–3.32 (m, 2H), 3.27–3.18 (m, 4H), 3.02 (s, 3H). ESI-MS calculated for C29H31N7O2 [M + H]+ = 510.3. Obtained: 510.2.

N-(4-Methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-morpholinophenyl)buta-2,3-dienamide (16)

Yield 5%. 1H NMR (MeOD-d4, 400 MHz): 8.74–8.44 (m, 2H), 8.40–8.22 (m, 1H), 8.00–7.92 (m, 1H), 7.53 (d, J = 8.46 Hz, 1H), 7.40–7.32 (m, 2H), 7.28–7.21 (m, 1H), 7.07 (s, 1H), 5.92 (t, J = 6.60 Hz, 1H), 5.52 (d, J = 6.60 Hz, 2H), 3.96 (s, 3H), 3.94–3.88 (m, 7H), 3.04–2.96 (m, 4H). ESI-MS calculated for C28H28N6O3 [M + H]+ = 497.2. Obtained: 497.3.

N-(4-Methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-(4-morpholinopiperidin-1-yl)phenyl)buta-2,3-dienamide (17)

Yield 15%. 1H NMR (MeOD-d4, 400 MHz): 8.82–8.33 (m, 2H), 8.10–7.84 (m, 1H), 7.74 (d, J = 6.96 Hz, 1H), 7.37–7.30 (m, 1H), 7.30–7.22 (m, 1H), 7.21–7.11 (m, 1H), 7.08–7.00 (m, 1H), 6.93 (s, 1H), 5.95 (t, J = 6.42 Hz, 1H), 5.50 (d, J = 6.42 Hz, 2H), 4.19–4.07 (m, 2H), 3.92–3.83 (m, 2H), 3.85 (s, 3H), 3.75 (s, 3H), 3.65–3.53 (m, 2H), 3.45–3.37 (m, 1H), 3.33–3.12 (m, 4H), 2.94–2.82 (m, 2H), 2.37–2.24 (m, 2H), 2.11–1.94 (m, 2H). ESI-MS calculated for C33H37N7O3 [M + H]+ = 580.3. Obtained: 580.2.

N-(4-Methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)buta-2,3-dienamide (18)

Yield 62%. 1H NMR (MeOD-d4, 400 MHz): 8.73–8.50 (m, 2H), 8.25–8.06 (m, 1H), 7.88 (d, J = 7.03 Hz, 1H), 7.46 (d, J = 8.27 Hz, 1H), 7.35–7.29 (m, 1H), 7.26–7.17 (m, 2H), 7.00 (s, 1H), 5.96 (t, J = 6.57 Hz, 1H), 5.52 (d, J = 6.57 Hz, 2H), 3.88 (s, 6H), 3.67–3.44 (m, 8H), 3.28–3.16 (m, 3H), 2.96 (s, 3H), 2.95–2.85 (m, 2H), 2.31–2.19 (m, 2H), 2.07–1.89 (m, 2H). ESI-MS calculated for C34H40N8O2 [M + H]+ = 593.3. Obtained: 593.2.

N-(2-(3-(Dimethylamino)pyrrolidin-1-yl)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)buta-2,3-dienamide (19)

Yield 29%. 1H NMR (MeOD-d4, 400 MHz): 8.53 (s, 1H), 8.47–8.31 (m, 1H), 8.00–7.74 (m, 2H), 7.53 (d, J = 8.25 Hz, 1H), 7.39–7.25 (m, 3H), 6.80 (s, 1H), 6.00 (t, J = 6.50 Hz, 1H), 5.40 (d, J = 6.62 Hz, 2H), 4.08–3.99 (m, 1H), 3.96 (s, 3H), 3.92 (s, 3H), 3.65–3.58 (m, 2H), 3.57–3.50 (m, 1H), 3.41–3.33 (m, 1H), 3.00 (s, 6H), 2.60–2.48 (m, 1H), 2.33–2.21 (m, 1H). ESI-MS calculated for C30H33N7O2 [M + H]+ = 524.3. Obtained: 524.1.

N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methyl-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)buta-2,3-dienamide (20)

Yield 16%. 1H NMR (MeOD-d4, 400 MHz): 8.45 (s, 1H), 8.22–8.01 (m, 1H), 7.99–7.91 (m, 1H), 7.87 (s, 1H), 7.51 (d, J = 8.38 Hz, 1H), 7.38–7.28 (m, 3H), 7.20–7.10 (m, 1H), 6.07 (t, J = 6.54 Hz, 1H), 5.40 (d, J = 6.54 Hz, 2H), 3.93 (s, 3H), 3.50 (t, J = 5.52 Hz, 2H), 3.35 (t, J = 5.56 Hz, 2H), 2.90 (s, 6H), 2.79 (s, 3H), 2.35 (s, 3H). ESI-MS calculated for C29H33N7O [M + H]+ = 496.3. Obtained: 496.1.

N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)buta-2,3-dienamide (21)

Yield 39%. 1H NMR (MeOD-d4, 400 MHz): 8.45 (s, 1H), 8.37–8.31 (m, 1H), 8.17–8.10 (m, 1H), 7.82 (d, J = 6.91 Hz, 1H), 7.46–7.42 (m, 1H), 7.41–7.31 (m, 1H), 7.33–7.25 (m, 2H), 7.21–7.15 (m, 2H), 6.16 (t, J = 6.44 Hz, 1H), 5.42 (d, J = 6.44 Hz, 2H), 3.83 (s, 3H), 3.46–3.42 (m, 2H), 3.36–3.32 (m, 2H), 2.91 (s, 6H), 2.74 (s, 3H). ESI-MS calculated for C28H31N7O [M + H]+ = 482.3. Obtained: 482.3.

N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-ethoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2yl)amino)phenyl)buta-2,3-dienamide (22)

Yield 34%. 1H NMR (MeOD-d4, 400 MHz): 8.55 (s, 1H), 8.42–8.25 (m, 1H), 8.23–8.01 (m, 2H), 7.55 (d, J = 8.02 Hz, 1H), 7.40 (d, J = 6.97 Hz, 1H), 7.38–7.32 (m, 1H), 7.30–7.23 (m, 1H), 7.05 (s, 1H), 6.06 (t, J = 6.40 Hz, 1H), 5.42 (d, J = 6.40 Hz, 2H), 4.21 (q, J = 7.09 Hz, 2H), 3.97 (s, 3H), 3.58–3.48 (m, 2H), 3.38–3.32 (m, 2H), 2.91 (s, 6H), 2.81 (s, 3H), 1.40 (t, J = 7.09 Hz, 3H). ESI-MS calculated for C30H35N7O2 [M + H]+ = 526.3. Obtained: 526.1.

N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((5-methoxy-4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)buta-2,3-dienamide (23)

Yield 5%. 1H NMR (MeOD-d4, 400 MHz): 8.63 (s, 1H), 8.45 (d, J = 7.96 Hz, 1H), 7.98 (s, 1H), 7.68 (s, 1H), 7.53–7.46 (m, 1H), 7.34 (t, J = 7.76 Hz, 1H), 7.21 (t, J = 7.41 Hz, 1H), 7.08 (s, 1H), 6.07 (t, J = 6.52 Hz, 1H), 5.40 (d, J = 6.52 Hz, 2H), 3.98 (s, 3H), 3.93 (s, 3H), 3.89 (s, 3H), 3.55 (t, J = 6.04 Hz, 2H), 3.37 (t, J = 6.04 Hz, 2H), 2.93 (s, 6H), 2.82 (s, 3H). ESI-MS calculated for C30H35N7O3 [M + H]+ = 542.3. Obtained: 542.5.

N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((5-methyl-4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)buta-2,3-dienamide (24)

Yield 40%. 1H NMR (MeOD-d4, 400 MHz): 8.42–8.32 (m, 1H), 8.21 (s, 1H), 7.98 (s, 1H), 7.83 (s, 1H), 7.50–7.44 (m, 1H), 7.36–7.29 (m, 1H), 7.20–7.14 (m, 1H), 7.06 (s, 1H), 6.05 (t, J = 6.59 Hz, 1H), 5.38 (d, J = 6.59 Hz, 2H), 3.90 (s, 3H), 3.89 (s, 3H), 3.56–3.48 (m, 2H), 3.38–3.32 (m, 2H), 2.90 (s, 6H), 2.79 (s, 3H), 2.38 (s, 3H). ESI-MS calculated for C30H35N7O2 [M + H]+ = 526.3. Obtained: 526.3.

N-(5-((4-(1H-Indol-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)buta-2,3-dienamide (25)

Yield 5%. 1H NMR (MeOD-d4, 400 MHz): 8.49 (s, 1H), 8.36–8.24 (m, 1H), 8.05–7.93 (m, 2H), 7.49 (d, J = 7.68 Hz, 1H), 7.41 (d, J = 6.82 Hz, 1H), 7.30–7.23 (m, 1H), 7.23–7.15 (m, 1H), 7.06 (s, 1H), 6.05 (t, J = 6.49 Hz, 1H), 5.40 (d, J = 6.49 Hz, 2H), 3.94 (s, 3H), 3.54 (t, J = 5.62 Hz, 2H), 3.36–3.32 (m, 2H), 2.90 (s, 6H), 2.80 (s, 3H). ESI-MS calculated for C28H31N7O2 [M + H]+ = 498.3. Obtained: 498.3.

N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-5-((4-(6-fluoro-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-4-methoxyphenyl)buta-2,3-dienamide (26)

Yield 5%. 1H NMR (MeOD-d4, 400 MHz): 8.49 (s, 1H), 8.30–8.06 (m, 2H), 8.03–7.94 (m, 1H), 7.33–7.28 (m, 1H), 7.28–7.22 (m, 1H), 7.09–7.04 (m, 1H), 7.04–6.95 (m, 1H), 6.09 (t, J = 6.54 Hz, 1H), 5.41 (d, J = 6.54 Hz, 2H), 3.95 (s, 3H), 3.87 (s, 3H), 3.53 (t, J = 5.78 Hz, 2H), 3.35 (t, J = 5.78 Hz, 2H), 2.92 (s, 6H), 2.79 (s, 3H). ESI-MS calculated for C29H32FN7O2 [M + H]+ = 530.3. Obtained: 530.4.

N-(5-((4-(6-Chloro-1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)-2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxyphenyl)buta-2,3-dienamide (27)

Yield 14%. 1H NMR (MeOD-d4, 400 MHz): 8.51 (m, 1H), 8.31–8.14 (m, 1H), 8.14–7.97 (m, 2H), 7.55 (m, 1H), 7.33 (dd, J = 6.93, 2.46 Hz, 1H), 7.23–7.17 (m, 1H), 7.07 (d, J = 8.19 Hz, 1H), 6.07 (t, J = 6.55 Hz, 1H), 5.43 (d, J = 6.55 Hz, 2H), 3.95 (s, 3H), 3.90 (s, 3H), 3.56–3.51 (m, 2H), 3.38–3.33 (m, 2H), 2.92 (s, 6H), 2.80 (s, 3H). ESI-MS calculated for C29H3235ClN7O2 [M + H]+ = 546.2. Obtained: 546.2.

N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(pyrazolo[1,5-a]pyridin-3-yl)pyrimidin-2yl)amino)phenyl)buta-2,3-dienamide (28)

Yield 6%. 1H NMR (MeOD-d4, 400 MHz): 8.82 (s, 1H), 8.78–8.75 (m, 1H), 8.55–8.41 (m, 1H), 8.09 (d, J = 6.57 Hz, 1H), 7.88 (m, 1H), 7.66–7.58 (m, 1H), 7.45 (d, J = 6.85 Hz, 1H), 7.27–7.20 (m, 1H), 7.07 (s, 1H), 6.06 (t, J = 6.53 Hz, 1H), 5.41 (d, J = 6.53 Hz, 2H), 3.94 (s, 3H), 3.55–3.52 (m, 2H), 3.37–3.33 (m, 2H), 2.91 (s, 6H), 2.79 (s, 3H). ESI-MS calculated for C27H30N8O2 [M + H]+ = 499.3. Obtained: 499.2.

Materials and methods

EGFR kinase activity assay

The kinase activity was evaluated with enzyme-linked immunosorbent assay (ELISA). Wild type EGFR and T790M/L858R double mutant EGFR were purchased from Eurofins.

The reaction substrate poly(Glu, Tyr) 4 : 1 was diluted with PBS (10 mM phosphate buffer, 150 mM NaCl, pH 7.2–7.4) to reach the final concentration 20 μg mL–1 in the reaction plate. The plate was subsequently incubated at 37 °C for 12–16 hours before use. To each well of the plate, a kinase reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, and 1 mM DTT), serial diluted inhibitors, and ATP were added to achieve a final ATP concentration of 5 μM. The kinases were added and the plates were incubated at 37 °C for 1 h, and the wells were washed three times with PBS containing 0.1% Tween 20 (T-PBS). An anti-phosphotyrosine (PY99) antibody was added and the plate was incubated at 37 °C for 30 min. The plate was then washed three times before horseradish peroxidase-conjugated goat anti-mouse IgG was added, followed by incubation at 37 °C for 30 min. The plate was again washed, and 2 mg mL–1o-phenylenediamine was added, followed by incubation at 25 °C for 1–10 min. The reaction was quenched immediately by adding 2 M H2SO4. Subsequently, the plate was read using a SPECTRA MAX 190 at 492 nm. The IC50 values were calculated from the inhibitory curves.

Cell growth inhibition assay

The cell lines were purchased from Stem Cell Bank, Chinese Academy of Science. The cell lines were validated by short tandem repeat assay.

In a 96-well plate, the compounds were serially diluted in corresponding media (NCI-H1975 and A549 were maintained in DMED). The cells were seeded at a density of 3000–5000 cells per well. The plate was incubated at 37 °C in an atmosphere of 5% CO2 for 4 days. Cell viability was determined using a Cell Counting Kit-8 (Dojinda) according to the manufacturer's protocols. The absorbance (optical density, OD) was read at a wavelength of 450 nm using a multimode microplate reader (TECAN SPARK 10M). The untreated cells served as an indicator of 100% cell viability. The IC50 was calculated by nonlinear regression analysis using GraphPad Prism 6 software.

Western blotting assay

NCI-H1975 cells were seeded in 6-well plates and cultured 12 hours before use (37 °C, 5% CO2). The compounds were diluted to 10, 100, and 1000 nM and transferred into the assay plates accordingly. The cells were incubated for 16 hours followed by stimulation with EGF in fresh medium (10 ng ml–1) for 15 min before lysis. The medium was removed and 100 μL of ice-cold lysis buffer was added to treat the cells for 30 min (4 °C). After cell lysis, lysates were centrifuged at 13 000 × g for 30 min at 4 °C. The supernatant was collected for subsequent analysis. Proteins were normalized to 20 μg per lane and separated using 10% SDS-PAGE gels. The membrane was blocked in TBST buffer (20 mM Tris-HCl, pH 7.6, 0.1% Tween 20, 150 mM NaCl) containing 5% non-fat milk for 1 hour. Subsequently, primary antibodies (EGFR, pEGFR, AKT, pAKT, ERK, pERK and GAPDH) were added, and the membranes were shaken at 4 °C for 12 h. The membranes were then washed thoroughly with TBST and incubated for 1 h with secondary antibodies. Finally, the membranes were washed in TBST (5 × 5 min), and immunoreactive proteins were visualized using an enhanced chemiluminescence detection reagent (Thermo Scientific).

The EGFR antibody was purchased from Santa Cruz. Phospho-EGF Receptor (Tyr1068), AKT, Phospho-Akt (Ser473), p44/42 MAPK (Erk1/2) (137F5), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), and GAPDH antibodies were purchased from Cell Signaling Technology.

Method to determine pseudo-first-order rate constants

The analysis method was adopted from reported procedures9 with modifications. All reactions were carried out in 1.5 mL Eppendorf tubes. The reaction temperature was 37 °C in all cases.

Twenty five μL of electrophile in dimethylacetamide (DMA) was transferred to an Eppendorf tube. Twenty five μL of 2-amino-5-methoxybenzoic acid (20.0 mM in DMA, used as the internal standard for LC analysis) was transferred to the Eppendorf tube. A fresh-prepared solution (450 μL) of glutathione (11.1 mM) in potassium phosphate buffer (100 mM, pH 7.4) was transferred into the aforementioned Eppendorf tube, and thus the reaction was initiated. After the above operations were performed, each reaction tube contained 1 mM electrophile, a 1 mM internal standard, and 10 mM GSH in 100 mM potassium phosphate buffer and DMA (90 : 10), in a total volume of 500 μL.

The amounts of components at each time point were measured by taking out 20 μL of the reaction mixture and analysing this by liquid chromatography using a Sunfire C18 column (4.6 × 150 mm, 5 μm). The LC mobile phase consisted of solvent A (0.1% TFA in water) and solvent B (0.1% TFA in MeCN). LC was initiated with 10% solvent B, which increases to 100% in 30 min (3% min–1). The pseudo-first-order rate constants were determined by plotting the natural log of the electrophile/internal standard ratio as a function of time. The negative slope of the straight line is the pseudo-first-order rate constant.

Conflicts of interest

The authors declare no competing interests.

Supplementary Material

Click here for additional data file. (356.1KB, pdf)

Acknowledgments

This work was supported by the “Personalized medicines-molecular signature-based drug discovery and development”, a strategic priority research program of the Chinese Academy of Sciences (grant no. XDA12020322), and the National Natural Science Foundation of China (grant 81673295). We also thank the Shanghai Institute of Materia Medica startup-grant and the Recruitment Program of Global Youth Experts (The 1000 Youth Talents Program) for financial support. We thank Profs. Jian Ding and Hua Xie (Shanghai Institute of Materia Medica) for testing the enzymatic activity of our compounds. We also thank Chao-Yie Yang (University of Michigan, Ann Arbor) for proofreading.

Footnotes

†Electronic supplementary information (ESI) available: Synthetic methods and spectral characterization data of the final compounds, 1H and 13C NMR spectra of 14, HPLC purity report, procedures for enzymatic assay, western blot, and GSH addition assay, procedures and data for the pharmacokinetic study. See DOI: 10.1039/c7md00571g

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