Discovery and Structural Optimization of N5-Substituted 6,7-Dioxo-6,7

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Discovery and Structural Optimization of N5-Substituted 6,7-dioxo-6,7dihydropteridines as Potent and Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors Against L858R/T790M Resistance Mutation Yongjia Hao, Xia Wang, Tao Zhang, Deheng Sun, Yi Tong, Yuqiong Xu, Haiyang Chen, Linjiang Tong, Lili Zhu, Zhenjiang Zhao, Zhuo Chen, Jian Ding, Hua Xie, Yufang Xu, and Honglin Li J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Jul 2016 Downloaded from http://pubs.acs.org on July 11, 2016

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Journal of Medicinal Chemistry

Discovery and Structural Optimization of N5-Substituted 6,7-dioxo-6,7dihydropteridines as Potent and Selective Epidermal Growth Factor Receptor (EGFR) Inhibitors Against L858R/T790M Resistance Mutation Yongjia Hao1,#, Xia Wang1,#, Tao Zhang2,#, Deheng Sun1, Yi Tong1, Yuqiong Xu1, Haiyang Chen1, Linjiang Tong2, Lili Zhu1, Zhenjiang Zhao1, Zhuo Chen1, Jian Ding2, Hua Xie2,*, Yufang Xu,1,* and Honglin Li1,* 1

Shanghai Key Laboratory of New Drug Design, Shanghai Key Laboratory of Chemical Biology,

State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science & Technology, Shanghai, 200237, China; 2Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.

*To

whom

correspondence

should

be

addressed.

Email:

[email protected], [email protected]

Please address correspondence and requests for reprints to: Prof. Honglin Li School of Pharmacy, East China University of Science and Technology 130 Meilong Road, Shanghai 200237 Phone/Fax: +86-21-64250213

ACS Paragon Plus Environment

[email protected],


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ABSTRACT EGFR-targeted inhibitors (gefitinib and erlotinib) provided an effective strategy for the treatment of non-small cell lung cancer. However, the EGFR T790M secondary mutation has become a leading cause of clinically acquired resistance to these agents. Herein, based on the previously reported irreversible EGFR inhibitor (compound 9), we present a structure-based design approach, which is rationalized via analyzing its binding model and comparing the differences of gatekeeper pocket between the T790M mutant and wild-type (WT) EGFR kinases. Guided by these results, a novel 6,7-dioxo-6,7-dihydropteridine scaffold was discovered and hydrophobic modifications at N5-postion were conducted to strengthen nonpolar contacts and improve mutant selectivity over EGFRWT. Finally, the most representative compound 17d was identified. This work demonstrates the power of structure-based strategy in discovering lead compounds, and provides molecular insights into the selectivity of EGFRL858R/T790M over EGFRWT, which may play an important role in designing new classes of mutant-selective EGFR inhibitors.


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INTRODUCTION Receptor tyrosine kinases (RTKs) play significant roles in cellular signaling pathways and regulate numerous cellular processes, such as cell proliferation, differentiation, metabolism and apoptosis.1-3 Moreover, RTKs as key regulators are involved in the development and progression of many types of human malignancies.4,5 As for the known RTKs, the epidermal growth factor receptor (EGFR) tyrosine kinase is one of the most important clinically validated targets for anticancer therapies, especially for non-small cell lung cancer (NSCLC) treatment.6,7 Up to now, EGFR-targeted tyrosine kinase inhibitors (TKIs), including first-generation EGFR inhibitors gefitinib (1) and erlotinib (2), have been approved by US Food and Drug Administration (FDA) for treatment of NSCLC patients with EGFR activating mutations (exon 19 deletion or exon 21 L858R substitution mutation) in 2003 and 2004, respectively (Figure 1).8,9 EGFR L858R point mutation (substitution of a leucine to an arginine at position 858) in exon 21 is one of the most common EGFR activating mutations, which appears adjacent to the highly conserved activation loop.10 These two small molecular EGFR inhibitors achieved excellent improvements in overall response rates (ORR) and median progression-free survival (PFS) for NSCLC patients harboring this activating mutation.11,12 Although the early results of first-generation EGFR inhibitors are impressive, unfortunately, most NSCLC patients with activating mutations initially responded to the TKIs and invariably developed acquired resistance to EGFR TKIs therapy within months. The most common mechanism of acquired resistance is the secondary T790M (substitution of threonine to methionine) gatekeeper point mutation in exon 20 that occurs in cis with an EGFR activating mutation (e.g., L858R), which accounts for approximately 60% of these acquired resistances.13 In EGFR T790M mutation kinase, the Met790 gatekeeper residue is a more bulky amino acid than Thr790. Because the volume of the side chain increases, the hydrophobic ATP-



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binding subpocket becomes smaller. Thus, the resulting steric impediment may reduce binding of gefitinib and erlotinib.14-16 To overcome the problem of acquired resistance to first-generation TKIs, several second- and third-generation irreversible EGFR TKIs have been developed. These irreversible EGFR TKIs were appended with a Michael receptor moiety which can form a covalent bond with the sulfhydryl group of Cys797 in the ATP binding cleft of EGFR, thereby increasing the potency of inhibition. For example, afatinib (3), one of the second-generation irreversible EGFR-TKIs, which received marketing authorization by FDA in 2013 and showed good potency in vivo in xenograft models driven by dual-mutant EGFR (EGFRL858R/T790M) (Figure 1).17,18 However, because of dose-limiting toxicities related to inhibition of wild-type (WT) EGFR, this agent displayed limited benefit clinically for NSCLC patients who have developed T790M acquired resistance.19,20 More recently, third-generation covalent EGFR inhibitors, such as compounds 4 (WZ4002),21 5 (CO-1686, Rociletinib),22 and 6 (AZD9291, Osimertinib),23,24 etc., have been developed as mutant-selective EGFR inhibitors that specifically target EGFRL858R/T790M mutation, while being less selective for WT EGFR (Figure 1). However, compound 4 did not progress into clinical development. Compound 5 is currently in phase I/II clinical trials in NSCLC patients with EGFR T790M-positive mutation. Recently, compound 6 has been granted accelerated approval by the FDA to treat advanced EGFR T790M mutation-positive NSCLC patients whose disease had progressed following prior treatment with other EGFR-blocking therapy.25 In addition, researchers in academia have also reported a series of EGFR inhibitors with varying degrees of selectivity for dual-mutant EGFR over WT EGFR26-30, such as compounds 7 and 8 developed by Ding’s group on the basis of compound 4, which showed potent enzymatic activities against EGFRL858R/T790M with low nanomolar IC50 or Kd values (7, IC50 = 0.9 nM; 8, Kd


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= 2.6 nM). Notably, their design strategy using conformational constraint to develop thirdgeneration EGFR inhibitors provide inspiration for researchers to design novel selective EGFR inhibitors to overcome EGFR gatekeeper mutations in NSCLC treatment. Herein, we describe the design and

optimization of 6,7-dioxo-6,7-dihydropteridine derivatives

as novel

EGFRL858R/T790M inhibitors with improved selectivity profiles over EGFRWT. Figure 1

CHEMISTRY As outlined in Scheme 1, compounds 17a-e were prepared starting from commercially available 1,3-benzendiamine (10), followed by treatment with di-tert-butyl dicarbonate to achieve Bocprotected amine (11). Reacting compound 11 with 2,4-dichloro-5-nitropyrimidine to yield tertbutyl (3-((2-chloro-5-nitropyrimidin-4-yl)amino)phenyl)carbamate (12) in a high yield (80%). Then, compound 12 was converted to the intermediate (13) by displacement of 2-chloro in the pyrimidine with appropriate arylamines in a good yield (69%), subsequent reduction of the nitro group and reaction with diethyl oxalate to obtain the ring-closure compound (15) in an acceptable 60% overall yield, which was deprotected with trifluoroacetic acid and reacted with acryloyl chloride to produce the compound 17a with moderate 41% overall yield of two-step. Meanwhile, compound 15 was alkylated according to the reported procedure with appropriate alkyl halides to afford the N5-alkylated derivatives (16b-e) in moderate 49-55% yields.31,32 Finally, compounds 16b-e were deprotected with trifluoroacetic acid in dichloromethane and reacted with acryloyl chloride to produce the final target compounds 17b−e with 31-46% overall



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yields. Compounds 17f-j were prepared by a method similar to that for compound 17b in similar overall yields. Scheme 1

RESULTS AND DISCUSSION Lead Compound and Design Strategy. In our previous studies, compound 9 (Figure 1) was identified by scaffold hopping as a potent irreversible EGFR kinase inhibitor, which exhibited competitive inhibitory enzymatic activities with single nanomolar IC50 values against both wildtype and L858R/T790M mutant EGFRs (3.8 nM vs. 1.1 nM).33 On the basis of molecular modeling studies, 9 irreversibly binds to the T790M mutant EGFR (PDB 3IKA) by targeting the Cys797 residue in the ATP binding site via the formation of a covalent bond, and the Nmethylpiperazine motif on the left-hand side is directed toward the solvent exposed region. The pteridin-7(8H)-one core forms an expected bidentate hydrogen-bonded interaction with the hinge residue Met793 while the double-ring system contacts the mutant gatekeeper Met790 residue. Besides, the EGFR L858R mutation adjacent to the activation loop is far from the ligand and would not affect the binding site (Figure 2A and 2B). The crystal structure of compound 4, which exhibited similar binding modes but mutant-selective activity, encouraged us to do the comparative crystal structure analysis for EGFRT790M mutant over EGFRWT. As shown in Figure 2B, overlay of the structure of WT EGFR (PDB 4G5J) into T790M mutant EGFR shows that selectivity of compound 4 for the gatekeeper EGFRT790M mutant was obtained through the 5position chlorine substitution that points directly toward the methionine residue at position 790 where it may achieve van der Waals contacts. However, in the EGFRWT, the bulkier methionine


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residue is replaced by the smaller and more polar threonine residue at position 790, and therefore, it reduced the hydrophobic interaction between the gatekeeper residue and the chlorine substituent, leading to lower affinity. Given these differences in the gatekeeper pocket between the T790M mutant and WT EGFR kinases, we conducted lead optimization studies with hydrophobic modifications at the 5-postion moiety to strengthen nonpolar contacts and improve selectivity over EGFRWT. Nevertheless, it is impossible to directly introduce substituents at the 5-position of compound 9. Therefore, a scaffold hopping of the pteridin-7(8H)-one was conducted. The pyrazinone part of the pteridin-7(8H)-one was replaced with piperazinediones in order to add hydrophobic substituents at the 5-position and thus occupy the lipophilic space within the gatekeeper pocket (Figure 2C). These efforts resulted in compound 17a. Moreover, the superposition of compound 4 and compound 9 suggests that little space remains in the binding site adjacent to the gatekeeper residue, and only small substitutions can be accommodated. We therefore designed a series of 6,7-dioxo-6,7-dihydropteridine derivatives with N5-substituents as a hydrophobic functional group to improve selectivity over EGFRWT, studied the interactions between EGFR kinases and the inhibitors by molecular modeling to provide us guidance for the structural optimization, and ultimately synthesized the inhibitors to verify our hypothesis. Figure 2

Binding Mode Analysis. The designed inhibitors (17a-e) were aligned to the crystal structure of compound 4 via in-house molecular 3D similarity calculation method SHAFTS34 and using MacroModel optimization to generate covalent protein-ligand complexes. As shown in Figure



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3A, the 6,7-dioxo-6,7-dihydropteridine ring extends into the hydrophobic pocket around the T790M gatekeeper residue, however, the hydrophobic contact between the N5-unsubstituted double-ring system and Met790 is not as tight as that of the chlorine substituent on the pyrimidine ring in compound 4, and would not significantly influence selectivity over EGFRWT. A N5-methyl moiety (17b) was then added as a hydrophobic substitution to target Met790 in EGFR on the basis of observing the overlay poses of 9 and compound 4 as well as the gatekeeper pocket differences. As expected, in the case of the T790M mutant EGFR, the 5-substituent is in van der Waals contacts (distance of 3.1 Å) with the methyl group of Met790, while for the WT EGFR the corresponding distance is much shorter (distance of 1.9 Å), leading to a steric clash between the 5-substituent and the hydroxyl side chain of Thr790 (Figure 3B) which would likely affect the potency and selectivity. Based on the above molecular simulations, we increased the bulkiness of the group at N-5 moiety in inhibitor 17a, such as 5-ethyl (17c), 5-isopropyl (17d) and 5-propyl (17e), to further explore how large a substituent would be tolerated in terms of potency in the mutant EGFR kinase and simultaneously benefit selectivity over EGFRWT. Modeling structure of compound 17d shows that the 5-isopropyl moiety extends to the methionine gatekeeper pocket and fits the pocket shape well, resulting in the formation of close hydrophobic interactions with the residues of Met790, Ala743, Leu844 and Met793 that contribute to the potency of 17d toward the L858R/T790M mutant (Figure 3C). However, in the model of EGFRWT/17d, different orientation of the 5-isopropyl group was adopted. The 5-isopropyl also participates in nonpolar interactions with the residues of Thr790, Thr854, Ala743 and Leu844 (Figure 3D), which may be responsible for the similarity in the EGFRWT potency of 17d compared to compound 17a. As for 17c and 17e, the molecular modeling analysis suggests that they are likely to form the same hydrophobic


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interactions with the gatekeeper Met790 residue (Figure S1A and S1C), but similar to that of 17d, the orientations of the 5-substituents would be influenced to match the gatekeeper Thr790 residue while binding to the active site of the WT EGFR (Figure S1B and S1D), thus possibly leading to a decrease of selectivity between EGFRL858R/T790M and EGFRWT. Figure 3

Enzyme and Cellular Activities. As a proof, these five compounds have been synthesized (as shown in Scheme 1) and their IC50 values are shown in Table 1. Compound 17a potently inhibited the enzymatic activity of EGFRWT and EGFRL858R/T790M mutant with IC50 values of 2.8 and 2.5 nM, respectively, which is similar to those of compound 9 and also showed no selectivity for L858R/T790M mutant EGFR over WT EGFR. The binding mode shown in Figure 3A also implied that the hydrophobic contact between the 6,7-dioxo-6,7-dihydropteridine core and the T790M gatekeeper residue is not as tight as that of the chlorine substituent on the pyrimidine ring in compound 4. The kinase inhibition of 17a was further validated by investigating its suppression on the growth of H1975 cells ectopically expressing EGFR L858R/T790M.22,24 However, it did not account for any significant inhibitory effect on H1975 cells at concentrations up to 10000 nM. The effects of 17a on A431 cells harboring WT EGFR kinase were also investigated. The results demonstrated that 17a poorly affected the activation of EGFR in the corresponding cells although it displayed strong inhibition against EGFR kinases under the in vitro screening assay, indicating the need for further investigation with respect to physicochemical properties, such as solubility and cell permeability. The solubility was measured at pH 7.4 and revealed an acceptable aqueous solubility of 839 µg/mL (Table 2).



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Furthermore, we built simple prediction models of cell permeability described by QPPCaco2 values calculated using Qikprop in Maestro software package. As shown in Table 2, 17a exhibited poor cell permeability with the QPPCaco2 value of 22.4, which was outside the recommended ranges (< 25 poor), thus providing a reasonable explanation for the compound’s loss of cellular efficacy as compared to the inhibitory activity with respect to isolated kinases. Table 1

Introduction of a methyl group at the 5-position led to a compound (17b) which was roughly equipotent to compound 17a in the kinase inhibitory activities against EGFRL858R/T790M (IC50 = 4.7 nM) and also approximately 101-fold selective over the EGFRWT (IC50 = 474 nM). These results were consistent with the modeling structure of EGFRT790M in complex with compound 17b (Figure 3B), showing that the 5-methyl moiety was directed toward the gatekeeper region of the ATP-binding site, and was detrimental to the kinase inhibition of EGFRWT due to the steric clashes, thus leading to a 170-fold potency loss compared with the parent compound 17a (474 nM vs. 2.8 nM). Furthermore, 17b displayed a decreased solubility of 392 µg/mL and an increased QPPCaco2 value of 47.4, with the suppressive potency against H1975 cell lines increasing to 1772 nM, but still suffering a significantly drop in potency from enzyme to cell, which might be due to the poor octanol/water partition coefficient log P (Table 2). As expected, the introduction of bulkier substitutions at the 5-position resulted in further enhancement of inhibitory activities against EGFRL858R/T790M as observed for the 5-isopropyl (17d) and 5-propyl (17e) derivatives. Both of these compounds strongly inhibited EGFRL858R/T790M with IC50 values below 1 nM. However, their EGFRWT inhibitory potency (2.0


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and 2.2 nM, respectively) were also improved relative to compound 17b, resulting in a slight decrease of selectivity between EGFRL858R/T790M and EGFRWT. As shown in the predicted binding mode (Figure 3C and 3D), the 5-isopropyl group adopted different orientations in binding with the T790M mutant and WT EGFR kinases, and could form nonpolar interactions with both Thr790 and Met790, which may account for the increased EGFR potency and decreased selectivity. Additionally, 5-ethyl derivative (17c) displayed an IC50 value of 1.2 nM on EGFRL858R/T790M, but its IC50 value on EGFRWT (IC50 = 81 nM) was 6 times greater than that of 17b, further highlighting that larger groups than methyl at the 5-position might strengthen the hydrophobic interactions with the threonine gatekeeper, and thus showing decreased selectivity over WT EGFR. The effects of all three compounds were further investigated on the activation of H1975 and A431 cells. It was shown that compounds 17c and 17e exhibited moderate potency in suppressing the growth of H1975 cells with IC50 values of 126 and 119 nM, respectively, whereas their corresponding values for A431 cells were about 18-70-fold greater. Besides, the most enzymatically potent compound 17d also exhibited potent growth-inhibitory activities in the H1975 cells with an IC50 value of 18 nM, which was comparable to that of compound 5. More significantly, the corresponding antiproliferative activity on A431 cells was about 7773 nM and was less potent by over 432 fold than that against H1975 cells, suggesting that 17d may serve as an excellent selective inhibitor for EGFR mutations. The pKa, especially the aqueous solubility of compounds 17c, 17d, and 17e were much lower than that of compound 17a, along with their higher QPPCaco2 values, leading to improved cellular antiproliferative activities. Besides, in agreement with the findings that the mutant EGFRs exhibit substantially increased auto-phosphorylating activity relative to WT EGFR35, 17d suffered a 3887-fold drop in potency of WT EGFR from enzyme to cell, yet a relatively less drop for L858R/T790M mutant EGFR



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(60-fold), indicating the enhanced catalytic activity and increased sensitivity of EGFR mutants compared to EGFRWT. On the basis of compound 17a, we introduced alkyl groups, such as methyl and ethyl at the N5 moiety to improve selectivity over EGFRWT kinase, the resulting compounds 17b and 17c indeed showed 101- and 67-fold selectivity between EGFRL858R/T790M and EGFRWT. These results confirmed our initial hypothesis that hydrophobic optimization at 5-position to strengthen nonpolar contacts is extremely important to inhibitors of L858R/T790M EGFR mutants with selectivity over WT EGFR. Furthermore, substituting groups at the 5-position are actually sizeconstrained, namely introduction of bulkier substitutions, such as isopropyl (17d) and propyl (17e) at the 5-position resulted slight decrease of selectivity over EGFRWT kinase, suggesting that bulkier substituents may improve the inhibitory activities against EGFRWT through the hydrophobic interactions within the gatekeeper pocket, and therefore resulting in a decrease of selectivity between EGFRL858R/T790M and EGFRWT.

Modifications to the Left-Hand Side Chain. Considering compound 17d containing a 5isopropyl moiety showed remarkable enzymatic and cellular inhibitory activities against L858R/T790M mutant EGFR, we selected it as a new lead compound for further structural optimization. Previous investigations have demonstrated that the 2-position side chain bearing hydrophobic interactions with the backbone of the hinge region has a significant impact on the inhibitory activity.27,33,36 We synthesized a set of compounds with different left-hand side chains while maintaining the 5-isopropyl substitution on the dihydropteridine ring. The resulting IC50 values are listed in Table 1. Replacements of N-methylpiperazine moiety with N,N,N’-


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trimethylethylenediamine (compound 17f), N,N-dimethylethanolamine (compound 17g) and 1methyl-4-(piperidin-4-yl)piperazine (compound 17h), produced compounds that showed improved pKa and aqueous solubility relative to 17d (Table 2), while these compounds demonstrated diminished potency in the enzyme and proliferation assays compared to 17d. Next, we kept N-methylpiperazine motif constant, removed the methoxyl at 2-position of benzene and introduced methyl or methoxy groups at 3-position and produced compounds 17i and 17j. Their pKa and aqueous solubility were also greater than 17d, which was in accordance with the previous study.28 However, these two compounds also exhibited a slight drop in enzymatic and cellular assays in comparison to 17d (Table 1). These molecules displayed modest efficiency but less selectivity in inhibiting the growth of H1975 cancer cells than 17d, indicating that the initial left-hand side chain was most favourable for mutant EGFR potency and selectivity. Table 2

The Reactivity to Glutathione of 17d. In order to preliminarily explore its reactivity to other thiols sources, such as glutathione, 17d was selected as representative compound to perform the experiment. According to the reported literature, the experiment was performed with compound 17d (1 mM), glutathione (10 mM) in PBS (0.1 M, pH 7.4) at 37 oC in the presence of dimethylacetamide (10%).37 However, we did not detect the glutathione adduct using highresolution mass spectra (HRMS) even though the reaction has been mixed for 24 h. Furthermore, considering the intracellular concentrations of glutathione are quite low, the reactivity of 17d to glutathione might be lower.



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Kinase Selectivity Profile of 17d. As compound 17d demonstrated promising enzymatic potencies, we next evaluated its kinase selectivity profile against a diverse panel of 468 kinases at a concentration of 1000 nM by using the profiling platform of DiscoveRx KINOMEscan (Figure 4).38 The results demonstrated that 17d only showed similar strong binding interaction (percent of control value 95%). HPLC instrument: Hewlett-Packard 1100 HPLC equipped with a photodiode array detector using a Zorbax RP-18, 5 µm, 4.6 mm×250 mm column (reverse phase). The mobile phase A was acetonitrile and mobile phase B was 10 mM NH4OAc in water (pH 6.0). A gradient of 10−100% A over 20 min was run at a flow rate of 1.0 mL/min. tert-Butyl (3-Aminophenyl)carbamate (11). To a mixture of 1,3-phenylenediamine (10.800 g, 100 mmol), Et3N (10.100 g, 100 mmol) in methanol (150 mL) was added (Boc)2O (21.800 g, 100 mmol) at 0 oC. The reaction solution was stirred for 24 h at room temperature. Then, the solution was concentrated with a rotary evaporator. The crude product was purified by silica gel chromatography (petroleum ether/ethylacetate = 4:1, v/v) to obtain the title product as white powder (13.310 g, 64%). 1H NMR (400 MHz, CDCl3) δ 7.03 (t, J = 8.0 Hz, 1H), 6.96 (s, 1H), 6.55 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.43 (s, 1H), 6.36 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H), 3.54 (s, 2H), 1.51 (s, 9H). LC-MS: m/z: 209.1 (M+H)+. tert-Butyl

(3-((2-Chloro-5-nitropyrimidin-4-yl)amino)phenyl)carbamate

(12).

To

a

mixture of compound 11 (1.940 g, 10 mmol), Na2CO3 (1.590 g, 15 mmol) in DMF (20 mL) was added dropwise to a solution of tert-butyl(3-aminophenyl) carbamate (2.080 g, 10 mmol) in DMF (20 mL) at -70 oC. After stirring for 1 h, the reaction mixture was added to ice water (300 mL). The precipitate was filtered and dried in a vacuum oven to give the title product as a yellow solid (2.918 g, 80%), which was used without further purification. 1H NMR (400 MHz, CDCl3): δ 10.19 (s, 1H), 9.20 (s, 1H), 7.84 (s, 1H), 7.36 (d, J = 5.2 Hz, 2H), 7.21-7.18 (m, 1H), 6.63 (s, 1H), 1.56 (s, 9H). LC-MS: m/z: 366.1 (M+H) +.



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tert-Butyl (3-((2-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-nitropyrimidin4-yl)amino)phenyl)carbamate (13). To a mixture of compound 12 (2.555 g, 7 mmol), DIPEA (1.806 g, 14 mmol) in THF (30 mL) was added 2-methoxy-4-(4-methylpiperazin-1-yl)aniline (1.547 g, 7 mmol), and the reaction solution was stirred at room temperature overnight. The resulting precipitate was filtered and washed with dichloromethane to give the title product as a yellow solid (2.655 g, 69%), which was used without further purification. 1H NMR (400 MHz, DMSO-d6): δ 10.24 (s, 1H), 9.42 (s, 1H), 9.19 (s, 1H), 9.03 (s, 1H), 7.54 (s, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.22 (t, J = 8.8 Hz, 2H), 7.09 (t, J = 8.0 Hz, 1H), 6.62-6.60 (m, 1H), 6.34 (d, J = 7.6 Hz, 1H), 3.76 (s, 3H), 3.15 (t, J = 4.4 Hz, 4H), 2.47 (t, J = 4.4 Hz, 4H), 2.24 (s, 3H), 1.47 (s, 9H). LC-MS: m/z: 551.4 (M+H) +. tert-Butyl (3-((5-Amino-2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimid in-4-yl)amino)phenyl)carbamate (14). A solution of compound 13 (2.600 g, 4.7 mmol) in methanol (80 mL) was treated with 10% Pd/C (10 wt %) and hydrogenated for 10 h. The catalyst was filtered off through Celite, and the filtrate was concentrated under reduced pressure. The resulting crude product was recrystallized from ethanol to give the title compound 14 (2.110 g, 86%). 1H NMR (400 MHz, DMSO-d6): δ 9.32 (s, 1H), 8.17 (s, 1H), 7.99 (d, J = 8.8 Hz, 1H), 7.96 (s, 1H), 7.59 (s, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.16 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.98 (s, 1H), 6.60 (d, J = 2.4 Hz, 1H), 6.38 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 4.39 (s, 2H), 3.82 (s, 3H), 3.07 (t, J = 4.4 Hz, 4H), 2.48 (t, J = 4.4 Hz, 4H), 2.25 (s, 3H), 1.48 (s, 9H). LC-MS: m/z: 521.3 (M+H) +. tert-Butyl (3-(2-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-6,7-dioxo-6,7-dihy dropteridin-8(5H)-yl)phenyl)carbamate (15). To a mixture of compound 14 (2.100 g, 4 mmol), Et3N (0.808 g, 8 mmol) in ethanol (25 mL) was added diethyl oxalate (1.168 g, 8 mmol).


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The reaction mixture was stirred at reflux for 30 h. After being cooled to room temperature, the resulting precipitate was filtered and washed with ethanol to give the title product as a yellow solid (1.606 g, 70%). 1H NMR (400 MHz, DMSO-d6): δ 9.64 (s, 1H), 8.14 (s, 1H), 7.59 (s, 1H), 7.57 (s, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 6.55 (d, J = 2.4 Hz, 1H), 6.06 (d, J = 8.0 Hz, 1H), 3.77 (s, 3H), 3.03 (t, J = 4.4 Hz, 4H), 2.44 (t, J = 4.4 Hz, 4H), 2.22 (s, 3H), 1.45 (s, 9H). HRMS(ESI) (m/z): (M+H)+ calcd for C29H35N8O5 575.2730, found, 575.2725. N-(3-(2-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-6,7-dioxo-6,7-dihydropte ridin-8(5H)-yl)phenyl)acrylamide (17a). To a solution of compound 15 (0.574 g, 1 mmol) in CH2Cl2 (5 mL) was added trifluoroacetic acid (1 mL). The mixture was stirred for 5 h at room temperature. The reaction solution was neutralized with saturated aqueous NaHCO3, and extracted with CH2Cl2 (3×15 mL). The organic layer was washed with saturated aqueous NaCl, dried over anhydrous Na2SO4 and concentrated in vacuo to obtain a yellow solid (0.355 g, 75%), which was used in the next reaction without further purification. To a solution of 8-(3-aminophenyl)-2-((2-methoxy-4-(4-methylpiperazin-1-yl) phenyl)amino)5,8-dihydropteridine-6,7-dione (0.340 g, 0.7 mmol) in NMP (1.5 mL) was added acryloyl chloride (74 µL, 0.9 mmol) dissolved in CH3CN (1.0 mL) at 0 oC. The reaction solution was stirred overnight at room temperature. The mixture was poured into saturated aqueous NaHCO3, and extracted with CH2Cl2 (3×20 mL). The organic layer was washed with saturated aqueous NaCl, dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography (dichloromethane/methanol = 15:1, v/v) to give the product 17a as a yellow solid (0.200 g, 54%). mp >300 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.69 (s, 1H), 8.22 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.64 (s, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.28 (d, J =



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8.8 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.59-6.52 (m, 2H), 6.26 (dd, J = 16.8 Hz, J = 1.2 Hz, 1H), 6.07 (d, J = 8.4 Hz, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.2 Hz, 1H), 3.78 (s, 3H), 3.31-3.29 (m, 4H), 3.20-3.18 (m, 4H), 2.74 (s, 3H).

13

C NMR (100 MHz, DMSO-d6) δ 163.36, 156.77, 154.48,

153.10, 148.88, 146.75, 145.19, 143.85, 139.99, 135.80, 131.78, 129.41, 127.08, 123.52, 121.72, 119.47, 119.36, 113.10, 106.82, 100.31, 55.74, 52.37, 46.39, 42.33. HPLC purity: 97.63%, retention time = 9.50 min. HRMS(ESI) (m/z): (M+H)+ calcd for C27H29N8O4 529.2312, found, 529.2312. tert-Butyl (3-(5-Ethyl-2-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-6,7-dioxo6,7-dihydropteridin-8(5H)-yl)phenyl)carbamate (16c). To a mixture of compound 15 (0.861 g, 1.5 mmol), Cs2CO3 (0.587 g, 1.8 mmol) in DMF (8 mL) was added iodoethane (155 µL, 1.9 mmol). The mixture was stirred at room temperature overnight. The solution was added ice water and extracted with CH2Cl2 (3×30 mL). The organic layer was washed with saturated aqueous NaCl, dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography (dichloromethane/methanol = 15:1, v/v) to give the title product 16c as a yellow solid (0.477 g, 53%). 1H NMR (500 MHz, DMSO-d6) δ 9.68 (s, 1H), 8.54 (s, 1H), 7.69 (s, 1H), 7.65 (s, 1H), 7.60 (d, J = 6.4 Hz, 1H), 7.49 (t, J = 6.4 Hz, 1H), 7.30 (d, J = 6.8 Hz, 1H), 7.00 (d, J = 6.4 Hz, 1H), 6.61 (s, 1H), 6.12 (d, J = 6.0 Hz, 1H), 4.18 (q, J = 5.2 Hz, 2H), 3.82 (s, 3H), 3.19 (t, J = 4.4 Hz, 4H), 2.88 (t, J = 4.4 Hz, 4H), 2.54 (s, 3H), 1.49 (s, 9H), 1.30 (t, J = 5.6 Hz, 3H). HRMS(ESI) (m/z): (M+H)+ calcd for C31H39N8O5 603.3043, found, 603.3047. The obtained 2D HMBC spectrum was provided in the Supporting Information. N-(3-(2-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-ethyl-6,7-dioxo-6,7-dihy dropteridin-8(5H)-yl)phenyl)acrylamide (17c). To a solution of compound 16c (0.421 g, 0.7 mmol) in CH2Cl2 (5 mL) was added trifluoroacetic acid (1 mL). The mixture was stirred for 5 h


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at room temperature. The reaction solution was neutralized with saturated aqueous NaHCO3, and extracted with CH2Cl2 (3×15 mL). The organic layer was washed with saturated aqueous NaCl, dried over anhydrous Na2SO4 and concentrated in vacuo to obtain a yellow solid (0.299 g, 85%), which was used in the next reaction without further purification. To

a

solution

of

8-(3-aminophenyl)-5-ethyl-2-((2-methoxy-4-(4-methylpiperazin

-1-

yl)phenyl)amino)-5,8-dihydropteridine-6,7-dione (0.299 g, 0.6 mmol) in NMP (1.5 mL) was added acryloyl chloride (65 µL, 0.8 mmol) dissolved in CH3CN (1.0 mL) at 0 oC. The reaction solution was stirred overnight at room temperature. The mixture was poured into saturated aqueous NaHCO3, and extracted with CH2Cl2 (3×20 mL). The organic layer was washed with saturated aqueous NaCl, dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel chromatography (dichloromethane/methanol = 15:1, v/v) to give the product 17c as a yellow solid (0.178 g, 53%). mp >300 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.57 (s, 1H), 7.92 (s, 1H), 7.89 (s, 1H), 7.70 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 6.45 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H ), 6.26 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 6.07-6.05 (m, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 4.42 (q, J = 6.8 Hz, 2H), 3.77 (s, 3H), 3.02 (t, J = 4.4 Hz, 4H), 2.45 (t, J = 4.4 Hz, 4H), 2.23 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H).

13

C NMR (100 MHz, DMSO-d6) δ 163.26, 155.10,

152.45, 151.89, 149.55, 139.88, 135.39, 131.63, 129.52, 127.20, 123.59, 120.27, 119.41, 117.32, 106.24, 99.71, 63.02, 55.62, 54.53, 48.56, 45.64, 14.06. HPLC purity: 98.90%, retention time = 10.74 min. HRMS(ESI) (m/z): (M+H)+ calcd for C29H33N8O4 557.2625, found, 557.2615. The following compounds 17b, 17d-j were prepared by a method similar to that for compound 17c.



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N-(3-(2-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-methyl-6,7-dioxo-6,7dihydropteridin-8(5H)-yl)phenyl)acrylamide (17b). Yellow solid (yield 43%). mp >300 oC. 1

H NMR (400 MHz, DMSO-d6) δ 10.46 (s, 1H), 8.48 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.69 (s,

1H), 7.66 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.22 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.53 (d, J = 1.6 Hz, 1H), 6.46 (dd, J = 16.8 Hz, J = 10.0 Hz, 1H ), 6.26 (dd, J = 17.2 Hz, J = 1.6 Hz, 1H ), 6.02 (d, J = 8.4 Hz, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.2 Hz, 1H ), 3.77 (s, 3H), 3.553.53 (m, 4H), 3.06-3.02 (m, 4H), 2.57 (s, 3H), 2.32 (s, 3H).

13

C NMR (100 MHz, DMSO-d6) δ

163.24, 155.48, 154.76, 153.13, 146.80, 143.85, 139.96, 135.82, 131.67, 129.56, 127.14, 123.50, 120.84, 119.36, 119.26, 114.92, 106.37, 99.84, 55.63, 54.34, 48.43, 45.31, 29.03. HPLC purity: 95.77%, retention time = 10.81 min. HRMS(ESI) (m/z): (M+H)+ calcd for C28H31N8O4 543.2468, found, 543.2470. N-(3-(2-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-isopropyl-6,7-dioxo-6,7dihydropteridin-8(5H)-yl)phenyl)acrylamide (17d). Yellow solid (yield 49%). mp >300 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.63 (s, 1H), 8.57 (s, 1H), 7.96 (s, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 6.60 (d, J = 2.0 Hz, 1H), 6.54 (dd, J = 16.8 Hz, J = 10.0 Hz, 1H), 6.27 (dd, J = 16.8 Hz, J = 1.6 Hz, 1H), 6.12-6.10 (m, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 5.38-5.30 (m, 1H), 3.79 (s, 3H), 3.313.29 (m, 4H), 3.26-3.24 (m, 4H), 2.79 (s, 3H), 1.40 (d, J = 6.4 Hz, 6H).

13

C NMR (100 MHz,

DMSO-d6) δ 163.35, 155.00, 152.01, 151.87, 149.40, 139.96, 135.40, 131.74, 129.45, 127.11, 123.56, 120.95, 119.45, 117.43, 106.61, 100.13, 69.98, 55.71, 52.99, 46.98, 43.30, 21.49. HPLC purity: 97.32%, retention time = 11.84 min. HRMS(ESI) (m/z): (M+H)+ calcd for C30H35N8O4 571.2781, found, 571.2780.


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N-(3-(2-((2-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-6,7-dioxo-5-propyl-6,7dihydropteridin-8(5H)-yl)phenyl)acrylamide (17e). Yellow solid (yield 47%). mp 298.1-299.6 o

C. 1H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 8.58 (s, 1H), 7.97 (s, 1H), 7.91 (d, J = 8.0

Hz, 1H), 7.77 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H), 6.59-6.52 (m, 2H), 6.26 (dd, J = 16.8 Hz, J = 1.2 Hz, 1H), 6.11-6.09 (m, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 4.32 (t, J = 6.8 Hz, 2H), 3.78 (s, 3H), 3.31-3.29 (m, 4H), 3.16-3.14 (m, 4H), 2.72 (s, 3H), 1.85-1.77 (m, 2H), 1.01 (t, J = 7.2 Hz, 3H). HPLC purity: 97.20%, retention time = 12.17 min. HRMS(ESI) (m/z): (M+H)+ calcd for C30H35N8O4 571.2781, found, 571.2780. N-(3-(2-((4-((2-(Dimethylamino)ethyl)(methyl)amino)-2-methoxyphenyl)amino)-5-isopro pyl-6,7-dioxo-6,7-dihydropteridin-8(5H)-yl)phenyl)acrylamide (17f). Red-orange solid (yield 44%). mp >300 oC. 1H NMR (400 MHz, CDCl3) δ 8.58 (s, 1H), 8.42 (s, 1H), 7.65-7.63 (m, 2H), 7.40 (t, J = 8.0 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.32 (dd, J = 16.8 Hz, J = 1.2 Hz, 1H), 6.22 (d, J = 2.4 Hz, 1H), 6.11 (dd, J = 16.8 Hz, J = 10.0 Hz, 1H), 5.90-5.85 (m, 1H), 5.62 (dd, J = 10.0 Hz, J = 1.2 Hz, 1H), 5.51-5.45 (m, 1H), 3.79 (s, 3H), 3.53 (t, J = 7.2 Hz, 2H), 2.87 (s, 3H), 2.42 (t, J = 7.6 Hz, 2H), 2.29 (s, 6H), 1.50 (d, J = 6.0 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 163.56, 156.66, 156.01, 153.36, 151.60, 149.14, 145.62, 140.35, 134.86, 131.69, 129.93, 127.03, 123.24, 120.37, 119.22, 118.61, 117.41, 104.11, 96.16, 72.22, 55.95, 55.53, 51.53, 49.93, 38.97, 21.69. HPLC purity: 95.07%, retention time = 10.56 min. HRMS(ESI) (m/z): (M+H)+ calcd for C30H37N8O4 573.2938, found, 573.2939. N-(3-(2-((4-(2-(Dimethylamino)ethoxy)-2-methoxyphenyl)amino)-5-isopropyl-6,7-dioxo6,7-dihydropteridin-8(5H)-yl)phenyl)acrylamide (17g). Yellow solid (yield 46%). mp 207.7209.1 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H), 8.57 (s, 1H), 8.01 (s, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 7.2 Hz, 1H), 7.11 (d, J = 8.0 Hz,



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1H), 6.60 (d, J = 2.4 Hz, 1H), 6.54 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H), 6.26 (dd, J = 17.2 Hz, J = 2.0 Hz, 1H), 6.16-6.14 (m, 1H), 5.76 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 5.37-5.31 (m, 1H), 4.21 (t, J = 4.8 Hz, 2H), 3.78 (s, 3H), 3.26 (t, J = 4.8 Hz, 2H), 2.69 (s, 6H), 1.40 (d, J = 6.0 Hz, 6H). 13

C NMR (100 MHz, DMSO-d6) δ 163.37, 155.02, 152.00, 151.92, 149.39, 140.01, 135.39,

131.78, 129.40, 127.02, 123.55, 121.97, 119.46, 117.53, 104.48, 99.21, 70.00, 63.39, 55.82, 43.37, 21.48. HPLC purity: 95.28%, retention time = 11.59 min. HRMS(ESI) (m/z): (M+H)+ calcd for C29H34N7O5 560.2621, found, 560.2625. N-(3-(5-Isopropyl-2-((2-methoxy-4-(4-(4-methylpiperazin-1-yl)piperidin-1-yl)phenyl)ami no)-6,7-dioxo-6,7-dihydropteridin-8(5H)-yl)phenyl)acrylamide (17h). Yellow solid (yield 49%). mp >300 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 8.56 (s, 1H), 7.94 (d, J = 8.0 Hz, 1H), 7.91 (s, 1H), 7.72 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 6.55-6.48 (m, 2H), 6.27 (dd, J = 17.2 Hz, J = 2.0 Hz, 1H), 6.08-6.06 (m, 1H), 5.78 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 5.36-5.30 (m, 1H), 3.77 (s, 3H), 3.62-3.60 (m, 2H), 3.01-2.92 (m, 6H), 2.69 (s, 3H), 2.56 (t, J = 11.6 Hz, 4H), 2.42-2.39 (m, 2H), 1.82-1.81 (m, 2H), 1.54-1.49 (m, 1H), 1.39 (d, J = 6.4 Hz, 6H).

13

C NMR (100 MHz, DMSO-d6) δ 163.31, 155.01, 152.02,

151.82, 149.40, 139.98, 135.40, 131.76, 129.46, 127.07, 123.55, 120.27, 119.39, 119.35, 117.35, 106.84, 100.27, 69.95, 55.63, 48.52, 21.49. HPLC purity: 99.03%, retention time = 12.34 min. HRMS(ESI) (m/z): (M+H)+ calcd for C35H44N9O4 654.3516, found, 654.3512. N-(3-(2-((3-Methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-isopropyl-6,7-dioxo-6,7dihydropteridin-8(5H)-yl)phenyl)acrylamide (17i). Yellow solid (yield 54%). mp >300 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.54 (s, 1H), 9.61 (s, 1H), 8.61 (s, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.76 (s, 1H), 7.55 (t, J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.13 (d, J = 8.0 Hz, 1H), 6.69 (d, J = 8.8 Hz, 1H), 6.50 (dd, J = 17.2 Hz, J = 10.4 Hz, 1H), 6.26 (dd, J = 17.2 Hz, J = 2.0 Hz, 1H), 5.76 (dd, J


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= 10.0 Hz, J = 1.6 Hz, 1H), 5.37-5.31 (m, 1H), 2.94-2.91 (m, 4H), 2.85-2.82 (m, 4H), 2.58 (s, 3H), 1.99 (s, 3H), 1.40 (d, J = 6.0 Hz, 6H).

13

C NMR (100 MHz, DMSO-d6) δ 163.29, 156.39,

155.06, 152.12, 151.81, 149.31, 144.40, 140.07, 135.68, 135.60, 131.91, 131.71, 129.56, 127.14, 123.54, 119.51, 119.28, 118.60, 117.30, 116.66, 69.94, 53.87, 49.66, 43.61, 21.50, 17.42. HPLC purity: 99.38%, retention time = 11.50 min. HRMS(ESI) (m/z): (M+H)+ calcd for C30H35N8O3 555.2832, found, 555.2833. N-(3-(2-((3-Methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-5-isopropyl-6,7-dioxo-6,7dihydropteridin-8(5H)-yl)phenyl)acrylamide (17j). Yellow solid (yield 43%). mp >300 oC. 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.57 (s, 1H), 8.62 (s, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.75 (s, 1H), 7.54 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 7.6 Hz, 1H), 7.04-6.99 (m, 2H), 6.55-6.49 (m, 2H), 6.27 (dd, J = 17.2 Hz, J = 2.0 Hz, 1H), 5.77 (dd, J = 10.0 Hz, J = 1.6 Hz, 1H), 5.37-5.31 (m, 1H), 3.57 (s, 3H), 3.19-3.07 (m, 8H), 2.74 (s, 3H), 1.40 (d, J = 6.0 Hz, 6H). 13C NMR (100 MHz, DMSO-d6) δ 163.35, 156.39, 155.00, 152.10, 151.85, 151.66, 149.33, 140.07, 135.99, 135.49, 133.96, 131.75, 129.50, 127.15, 123.59, 119.49, 119.43, 117.69, 117.43, 110.44, 69.97, 55.24, 52.85, 47.49, 42.47, 21.49. HPLC purity: 98.62%, retention time = 10.41 min. HRMS(ESI) (m/z): (M+H)+ calcd for C30H35N8O4 571.2781, found, 571.2782. 3. In Vitro Enzymatic Activity Assay. Kinases domain of wild-type EGFR (EGFRWT), dualmutant EGFR (EGFRL858R/T790M) were expressed using the Bac-to-Bac™ baculovirusexpression system (Invitrogen, Carlsbad, CA, USA) and purified in Ni-NTA columns (QIAGEN Inc., Valencia, CA, USA). The kinase activity was evaluated with enzyme-linked immunosorbent assay (ELISA). The detailed characterization are provided in the Supporting Information.



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4. Cell Proliferation Inhibition Assay. H1975 and A431 cells were obtained from the American Type Culture Collection. Cell lines were cultured according to the suppliers' instructions. Cells were seeded in 96-well plates (3,000 cells per well) and grown overnight. The cells were treated with various concentrations of the compounds for 72 h, and the cells were then fixed with 10% pre-cooled trichloroacetic acid (TCA) for 2 h at 4 oC and stained for 15 min at room temperature with 100 µL of 4 mg/mL sulforhodamine B (SRB, Sigma) solution in 1% acetic acid. After washing the plates for three times, the SRB solution was dissolved in 150 µL of 10 mmol/L Tris base for 5 min and measured at 515 nm using a multiwell spectrophotometer (VERSAmax, Molecular Devices). The inhibition rate for cell proliferation was calculated as [1– (A515 treated/A515 control)]×100%. The IC50 value was obtained using the Logit method. 5. Kinase Selectivity Profile. The kinase selectivity profile was performed by using the DiscoveRx KINOMEscan platform (http://www.kinomescan.com/).38 The compound was screened at a concentration of 1000 nM against a panel of 468 kinases. The results were defined as a percentage of signal between the negative (DMSO, 100% control) and the positive (control compound, 0% control) control, where the “% control” was calculated as follows: (%) = [(test compound signal - positive control signal)/(negative control signal - positive control signal) ×100]. 6. Western Blot Analysis. Cells were collected and suspended in lysis buffer (100 mmol/L Tris-HCl, pH6.8, 200 mmol/L DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol). Equivalent amounts of proteins were loaded and separated by 8% SDS-PAGE followed by transfer to nitrocellulose membranes. Western blot analysis was subsequently performed using standard procedures. Antibodies used for immune detection of proteins were p-EGFR (Y1068;


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#3777S), EGFR (#4267S), p-ERK (T202/Y204; #4370L), ERK (#4695S), and Tubulin (#2128L; Cell Signaling). 7. Mouse Liver Microsomal Stability Assay. This assay was performed by Shanghai Medicilon, Inc. Briefly, 1 µM compound was incubated with mouse liver microsomes (0.5 mg/mL) and NADPH (1 mg/mL) at 37 oC. Then samples were collected at time points 0 min, 10 min, 15 min, 30 min, 45 min, and 60 min and were terminated by the addition of cold methanol. Samples were separated by centrifugation (15000 r/min for 5 min) and analyzed by using LCMS/MS (Waters UPLC system; Applied Biosystems mass spectrometer). 8. In Vivo Pharmacokinetics Study in Mice. The pharmacokinetic parameters of the compound in mice were conducted by Shanghai Medicilon, Inc. A 0.2 mg/mL dosing solution was prepared by dissolving 0.6 mg of the compound in mixed solvent (0.15 mL DMSO+1.200 mL PEG400+1.650 mL Saline) for intravenous administration, and the 1 mg/mL dosing solution was prepared by dissolving 6.0 mg of the compound in 0.5% CMC-Na aqueous solution for oral administration. Male ICR mice were separately administered to a group of three mice per time point for intravenous (1 mg/kg) or oral administration (10 mg/kg). At time points 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h after dosing, a blood sample was collected from each mouse and separated by centrifugation (8000 r/min for 6 min). Then the samples were analyzed by LC−MS/MS (SIMADZU LC system; Applied Biosystems mass spectrometer), and the acquired data were analyzed by using the WinNonlin (v5.2). 9. In Vivo Efficacy for Mouse Tumor Xenografts. 4-6 weeks-old BALB/c mice were purchased from the Shanghai Laboratory Animal Research Center (Shanghai, China). All the procedures related to animal handling, care and treatment in this article were performed in



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compliance with Agreement of the ethics committee on laboratory animal care and the Guidelines for the Care and Use of Laboratory Animals in Shanghai, China. H1975 and A431 cell lines were cultured in RPMI-1640 (GE Healthcare) supplemented with 10% v/v fetal bovine serum (Gibico). Approximately 2×106 cells were implanted subcutaneously into the right flank in a total volume of 0.1 mL/mouse. Mice were randomized into vehicle and treated groups with a group mean tumor size of ∼0.2−0.4 cm3. For efficacy studies, mice were dosed once daily by oral gavage with either vehicle or inhibitors using the indicated doses for maximum 14 days. The average tumor volume was measured with vernier calipers every 2 or 3 days, which then was calculated with the formula V = (L×W2)/2, and L stands for length and W is width. 10. Immunohistochemical (IHC) Assays. After the treatment, H1975 xenograft mouse models were harvested and fixed in formalin. Tumor samples were embedded in paraffin and prepared in sections (4 µm). The immunohistochemical analysis was conducted by Wuhan goodbio technology Ltd, and the assay was operated according to the manufacturer’s instructions. Before incubated with the primary antibodies (Cell Signaling Technology) at room temperature, sections were deparaffinized and rehydrated, and then developed in the solution of 3,3diaminobenzidine (DAB) for 10 minutes for independent analysis. The quantification of p-EGFR and Ki-67 were performed by using the Image Pro. 11. Measurements of Thermodynamic Solubility and pKa. Determination of the thermodynamic solubility was performed according to the published literature.42 The measurements of pKa were performed using the Sirius T3 instrument (Sirius Analytical Ltd.). The study was conducted according to the supplier's instructions, and under nitrogen to eliminate the interruption of dissolved CO2. Methanol (80%, dissolve 2.795 g KCl in 50 mL of deionised water and make up to 250 mL with analytical grade methanol) was chosen as the cosolvent


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(gradient varied from 60 to 40%). The pH-metric titration was performed from low to high (pH 2−12), and the temperature throughout the experiment was controlled at about 25 oC.

ASSOCIATED CONTENT Supporting Information Superposition simulations, molecular docking studies, MacroModel optimization, full experimental details, the predicted binding mode analysis, in vitro enzymatic activity assay. This material is available free of charge via the Internet at http://pubs.acs.org. · Molecular formula strings and the associated biological data (CSV).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (H.X.). Phone: +86-21-50805897. *E-mail: [email protected] (Y.X.). Phone: +86-21-64251399. *E-mail: [email protected] (H.L.). Phone/Fax: +86-21-64250213.

Author Contributions #These authors contributed equally. Notes The authors declare no competing financial interest.



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ACKNOWLEDGMENTS The research is supported in part by the Fundamental Research Funds for the Central Universities, the National Natural Science Foundation of China (Grants 21302054, 81222046 and 81230076) (Z.C., H.L.), the Shanghai Committee of Science and Technology (Grant 14431902100, and 13ZR1453100) (Y.X., Z.C.), the National Key Research and Development Program (Grant 2016YFA0502304), the National S&T Major Project of China (Grant 2013ZX09507004), and the Twelfth Five-Year National Science & Technology Support Program (Grant 2012BAI29B06) (H.L.). H.L. is also sponsored by Specialized Research Fund for the Doctoral Program of Higher Education (Grant 20130074110004), the Innovation Program of Shanghai Municipal Education Commission (grant 13SG32) and Fok Ying Tung Education Foundation (Grant 141035).

ABBREVIATIONS USED EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; WT, wild-type; DM, dual-mutant; TKIs, tyrosine kinase inhibitors; (Boc)2O, Di-tert-butyl dicarbonate; DIPEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; NMP, N-methyl-2-pyrrolidone; PO, per os; IV, intravenous; IOD, integrated optical density


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FIGURE CAPTIONS Figure 1. Representative examples of first-, second- and third-generation EGFR inhibitors.

Figure

2.

Design

concept

of

6,7-dioxo-6,7-dihydropteridine

scaffold.

(A)

Surface

representations that show compound 9 (yellow) and compound 4 (pink) binding to T790M mutant EGFR (PDB 3IKA), of which compound 9 was aligned to the crystal structure of compound 4 by SHAFTS. (B) Aligned pose of compound 9 (yellow) bound to the catalytic domain of EGFRT790M (shown in dark green), overlaid with the structure of EGFRWT (shown in blue, PDB 4G5J). (C). Discovery of selective and covalent 6,7-dioxo-6,7-dihydropteridinesbased inhibitors of EGFR containing the T790M resistance mutation.

Figure 3. Modeling studies. Panels A and B show the aligned poses of compounds 17a and 17b bound to the ATP binding pocket of T790M mutant EGFR, respectively, with their carbons colored in purple. The crystal structure of compound 4 bound to EGFRT790M (shown in drak blue, PDB 3IKA) was overliad with a reported EGFRWT structure (shown in cyan, PDB 4G5J). Panels C and D show the binding model of compound 17d (yellow) bound to EGFRT790M and EGFRWT, respectively.

Figure 4. Kinase selectivity profile of compound 17d against 468 kinases with DiscoveRx KINOMEscan profiling platform. Measurements were performed at a concentration of 1000 nM, and the affinity was defined as a percent of the DMSO control (% control), where the lower of the percent represent stronger hits.


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Figure 5. Effects of 17d on EGFR signaling in H1975 cells (A) and A431 cells (B). Cells were treated with the indicated concentrations of 17d or compound 6 for 2 h and stimulated by EGF for 15 min. Cell lysates were harvested for Western blot analysis for EGFR phosphorylation and downstream signaling.

Figure 6. Plasma concentration vs time curves after IV (1 mg/kg) or PO (10 mg/kg) administration of 17d to mice. Data are represented as mean ± SD (n = 3).

Figure 7. Preliminary in vivo antitumor efficacy of 17d against H1975 and A431 NSCLC xenograft mouse models at a concentration of 50 mg/kg/day. (A) H1975 xenograft mouse model (n = 3) tumor volumes, (B) A431 xenograft mouse model (n = 6) tumor volumes were recorded every 2-3 days. All values represent mean ± SEM. The tumor growth inhibition (TGI, %) was measured at the final day of the treatment for the drug-treated group versus the vehicle control, where *P < 0.05 (Student’s t test) compared to the vehicle group. (C) 17d suppressed cell proliferation (Ki-67) and p-EGFR expression in H1975 xenograft model. Tumor tissue sections were stained by hematoxylin, eosin (H&E) (blue) and anti-phospho EGFR antibody (light brow). Tumor growth was evaluated by Ki-67 staining (dark brown). (D) In 17d treated tumor, p-EGFR and Ki-67 labeling was significantly less than that of untreated control tumor (*P < 0.05 vs. DMSO control).



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SCHEMES Scheme 1. Synthetic Route of the 6,7-dioxo-6,7-dihydropteridine Derivativesa

a

Reagents and conditions: (a) (Boc)2O, Et3N, CH3OH, r.t., 24 h, 64%; (b) 2,4-dichloro-5-

nitropyrimidine, Na2CO3, DMF, -70 oC, 1 h, 80%; (c) arylamine, DIPEA, THF, r.t., overnight, 69%; (d) H2, Pd/C, MeOH, r.t., 10 h, 86%; (e) diethyl oxalate, triethylamine, EtOH, reflux, 30 h, 70%; (f) haloalkane, Cs2CO3, DMF, r.t., overnight, 49-55%; (g) trifluoroacetic acid, CH2Cl2, r.t., 5 h, 71-85%; (h) acrylyl chloride, NMP/CH3CN, 0 oC to r.t., overnight, 43-54%.


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FIGURES Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5



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Figure 6


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Figure 7



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TABLES Table 1. In Vitro EGFR Inhibition and Antiproliferation Activities of Compounds 17a-ja

Enzyme inhibitory activity Compd.

R

R1

Enzyme selectivity

(IC50, nM)

R2

L858R/ WT

Cellular antiproliferative activity

Cellular selectivity

(IC50, nM)

WT:DMb

A431

H1975

A431: H1975

T790M

17a

H

2-OCH3

2.8±0.7

2.5±0.8

1.1

>10000

>10000

NDc

17b

-CH3

2-OCH3

474±49

4.7±1.2

101

>10000

1772±189

ND

17c

2-OCH3

81±3

1.2±0.3

67

8815±1444

126±50

70

17d

2-OCH3

2.0±0.6

0.3±0.1

6.7

7773±2260

18±19

432


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17e

2-OCH3

2.2±0.8

0.6±0.2

3.7

2143±587

119±97

18

17f

2-OCH3

223±13

17±4.0

1.4

1529±1160

440±408

3.5

17g

2-OCH3

68±17

143±31

ND

1500±1266

415±579

3.6

17h

2-OCH3

28±8

7.8±3.3

3.6

2260±1157

1188±1405

1.9

17i

3-CH3

1.4±0.7

1.5±0.4

ND

309±199

151±230

2.0

17j

3-OCH3

8.6±5.2

1.3±0.3

6.6

669±583

213±301

3.1

20±2.9

14

641±282

272±127

5

d

(K i: 303) a

(K i: 22)

d

(GI50: 547)

31±34 d

(GI50: 32)

21 d

Kinase activity assays were examined by using the ELISA-based EGFR-TK assay. The

antiproliferation activity of the compounds were employed by using the sulforhodamine B (SRB) colourimetric assay. Data are averages of at least two independent determinations and reported as the means ± SDs (standard deviations). bDual-mutant (EGFRL858R/T790M). cNot determined. d

Reported data.22



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Table 2. Physicochemical Parameters of the 6,7-dioxo-6,7-dihydropteridine Derivatives.

Compd.

Solubility (µg/mL)

pKaa

b

QPlogPo/wc

QPPCaco2d

@PBS (pH 7.4)

a

17a

7.8

839

1.3

22.4

17b

7.3

392

1.8

47.4

17c

7.4

< 10

2.1

55.4

17d

7.7

41

2.3

58.1

17e

7.5

< 10

2.4

55.5

17f

8.9

1621

2.5

76.5

17g

8.8

437

2.6

72.8

17h

8.8

109

2.7

17.5

17i

8.1

190

2.7

68.1

17j

8.4

298

2.5

68.1

The measured pKa of compounds 17a-j were conducted using Sirius T3 instrument. bAqueous

solubility of these derivatives was examined by using UV-visible spectrophotometer in PBS buffer (0.1 M, pH 7.4).

c,d

The QPlogPo/w and QPPCaco2 of compounds 17a-j were predicted

using QikProp in Maestro software package.


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Table 3. Mouse Pharmacokinetics Parameters for Compound 17d

Dose (route)

T1/2 (h)

Tmax (h)

Cmax (ng/mL)

AUC(0-t) (ng·h/mL)

AUC(0-∞) (ng·h/mL)

Vz (mL/kg)

CL (mL/h/kg)

F (%)

1 mg/kg (IV)

0.8

0.9

258

140

143

8014

7013

-

10 mg/kg (PO)

1.1

2.0

136

673

680

-

-

48



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Table of Contents Graphic


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Discovery and Structural Optimization of N5-Substituted 6,7-Dioxo-6,7

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