Design, Synthesis, and StructureâActivity Relationship Study of 2-Oxo

Article Cite This: J. Med. Chem. 2018, 61, 2353−2371

pubs.acs.org/jmc

Design, Synthesis, and Structure−Activity Relationship Study of 2‑Oxo-3,4-dihydropyrimido[4,5‑d]pyrimidines as New Colony Stimulating Factor 1 Receptor (CSF1R) Kinase Inhibitors Qiuju Xun,†,‡,○ Zhang Zhang,§,○ Jinfeng Luo,† Linjiang Tong,# Minhao Huang,†,‡ Zhen Wang,† Jian Zou,§ Yingqiang Liu,‡,∇,# Yong Xu,† Hua Xie,# Zheng-Chao Tu,† Xiaoyun Lu,*,§ and Ke Ding*,†,§,∥,⊥ †

State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kaiyuan Avenue, Guangzhou 510530, China ‡ University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China § School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China ∥ Guangzhou City Key Laboratory of Precision Chemical Drug Development, Guangzhou 510632, China ⊥ International Cooperative Laboratory of Traditional Chinese Medicine Modernization and Innovative Drug Development, Ministry of Education (MOE) of People’s Republic of China, Guangzhou 510632, China # State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, No. 555 Zu-Chong-Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China ∇ School of Life Sciences, Shanghai University, No. 99 Shangda Road, Shanghai 200444, China S Supporting Information *

ABSTRACT: Colony stimulating factor 1 receptor kinase (CSF1R) is a well validated molecular target for anticancer drug discovery. Herein, we report the design, synthesis, and structure−activity relationship study of 2-oxo-3,4dihydropyrimido[4,5-d]pyrimidines as new orally bioavailable CSF1R inhibitors. One of the most promising compounds, 3bw, potently inhibits CSF1R kinase with an IC50 value of 3.0 nM, while it is less potent against structurally related epidermal growth factor receptor (EGFR) and other kinases. The kinase inhibition of 3bw was further validated by Western blotting analysis in RAW264.7 macrophages. The molecule also potently blocks macrophage infiltration, abrogates the protumorigenic influences of macrophages, and exhibits reasonable pharmacokinetic profile. Compound 3bw may serve as a new valuable lead compound for future anticancer drug discovery.

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INTRODUCTION

signaling is crucial for the differentiation and survival of macrophages8,9 and plays crucial roles in the tumor promoting and immune suppressive functions of tumor-associated macrophages (TAMs, also termed M2 macrophages).10,11 TAMs are highly plastic dominant immune cells expressing immunosuppressive molecules (e.g., interleukin 10 (IL-10), prostaglandin E2 (PGE2), and transforming growth factor β (TGFβ), etc.) to recruit T regulatory cells (TREG) via the C−C motif chemokine 22 (CCL22) in both early stage and metastatic stage of human cancers.12−14 TAMs also express the inhibitory ligands of programmed cell death protein 1 receptor (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) to suppress

Colony stimulating factor 1 receptor (CSF1R, also known as FMS)1 belongs to the type III growth factor receptor family that includes FMS-like tyrosine kinase 3 (FLT-3), stem cell factor receptor (KIT), and platelet-derived growth factor receptor (PDGFR) α and β, etc. CSF1R structurally distinguishes itself from the other family members by the presence of five immunoglobulin-like extracellular domains and a kinase insert dividing the intracellular kinase domain into two lobes.2 CSF1R is primarily expressed in macrophage lineage including monocytes,3 tissue macrophages, dendritic cells, osteoclasts and is involved in many fundamental biological processes, such as macrophage lineage’s tissue normal development,4 body’s immune defense, and embryonic development.5 Upon activation by the intracellular ligand CSF-1,6,7 CSF1R © 2018 American Chemical Society

Received: November 1, 2017 Published: March 2, 2018 2353

DOI: 10.1021/acs.jmedchem.7b01612 J. Med. Chem. 2018, 61, 2353−2371

Journal of Medicinal Chemistry

Article

Figure 1. Design of 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidines as CSF1R inhibitors by structural modification of an irreversible EGFR inhibitor.

Scheme 1. Synthesis of Compounds 3a

Reagents and conditions: (a) K2CO3 (2.0 equiv), N,N-dimethylformamide (DMF), 60 °C, 80−95%; (b) lithium aluminum hydride (LiAlH4) (2.0 equiv), tetrahydrofuran (THF), −40 to 0 °C, 45−58%; (c) MnO2 (5.0 equiv), dichloromethane (DCM), room temperature (rt), 80−95%; (d) R1NH2 (2.0 equiv), AcOH (1.0 equiv), NaBH4 (2.0 equiv), PhMe, 0 °C to rt, 60−80%; (e) triphosgene (0.34 equiv), triethylamine (Et3N) (3.0 equiv), DCM, 0 °C to rt, 80−95%; (f) 3-chloroperbenzoic acid (m-CPBA) (3.0 equiv), DCM, rt, 80−92%; (g) RNH2 (1.2 equiv), trifluoroacetic acid (F3CCOOH) (1.2 equiv), 2-butanol (2-BuOH), 110 °C; (h) F3CCOOH (10 equiv), DCM, rt, 45−60% (for two steps); (i) RCOOH (1.2 equiv), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) (1.2 equiv), ethyl diisopropylamine (DIPEA) (2.0 equiv), DCM, rt, 75−90%; (j) 1-iodopropane (1.5 equiv), DIPEA (1.5 equiv), DCM, 0 °C, 56%. a

the activation of T lymphocytes and natural killer cells (NK cells).15−17 Both CSF-1 expression and the intratumoral presence of CSF-1R+ macrophages have demonstrated close correlation with poor survival in various solid tumors and hematological malignancies.18 Moreover, genetic or pharmacological blockade of CSF-1 or its receptor has been proven to markedly decrease the infiltration of macrophages at the tumor site and to slow primary tumor growth, reduce metastatic potential, and improve long-term survival of prostate tumorbearing mice.19,20 CSF1R inhibition was also reported to alter macrophage polarization and blocked glioma progression.21 Thus, suppression of TAM survival/activation by inhibiting CSF-1/CSF1R signaling becomes a highly attractive strategy for cancer immunotherapy. In addition, studies also revealed the potential therapeutic applications of CSF1R inhibitors in several types of clinical diseases besides cancer, such as inflammatory disorders, autoimmunity, and bone diseases.22−24

A number of selective or nonselective small molecule CSF1R inhibitors have been reported to date,25 several of which have been advanced into different stages of clinical investigation. For instance, 5-((5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)N-((6-(trifluoromethyl)pyridin-3-yl)methyl)pyridin-2-amine (1, PLX3397 or pexidartinib),26 an oral tyrosine kinase inhibitor of CSF1R, c-KIT, and mutant Flt3, is undergoing phase 3 clinical studies in patients with diffuse type giant cell tumor (dt-GCT or PVNS). PLX7486 (structure undisclosed),27 ARRY-382 (structure undisclosed),28 and JNJ-40346527 (structure undisclosed)29 are also being investigated in solid tumors and classical Hodgkin’s lymphoma (cHL). However, there is no CSF1R inhibitor approved by U.S. Food and Drug Administration (FDA) to date. It is still highly desirable to identify new CSF1R inhibitors with distinct chemical scaffolds. Herein, we report the identification of 2-oxo-3,4dihydropyrimido[4,5-d]pyrimidines as new orally bioavailable 2354



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Figure 2. Binding mode analysis of compound 2 to EGFR kinase and CSF1R kinase. (A) Molecular docking of compound 2 (yellow) into EGFR kinase (PDB code 3VJO, orange). Hydrogen bonds and cation−π interaction are indicated by red and green dashed lines, respectively. (B) Molecular docking of compound 2 (yellow) docked into CSF1R kinase (PDB code 3DPK, blue). Hydrogen bonds and cation−π interaction are indicated by red and green dashed lines, respectively. (C) Superposition of EGFR (PDB code 3VJO, orange) and CSF1R kinase (PDB code 3DPK, blue). All the procedures were performed in Maestro (version 9.9, Schrödinger, LLC, New York, NY; 2014) implemented in the Schrödinger program.

A preliminary computational investigation suggested that compound 2 might bind to the active conformation of EGFR (PDB code 3VJO) with a typical type I binding mode, and the acrylamide warhead reacted with the Cys797 residue to form a critical covalent bond contributing greatly to the strong protein occupancy (Figure 2A). The anilinopyrimidine moiety of 2 forms bidentate hydrogen bonds with Met793 in the hinge region, and the 2-chlorophenyl is directed toward the back pocket (Glu762, Met766, Leu788 in the present structure) and forms a strong cation−π interaction with Lys745 of the protein. Study also suggested compound 2 could fit nicely into the ATP binding pocket of CSF1R with similar orientation and hydrogen bonding interactions (i.e., the bidentate hydrogenbonds with Cys666, a cation−π interaction with Lys616 etc.) to that in EGFR protein (Figure 2B and Figure 2C), which might be due to their high sequence identity (approximate 42%) (Supporting Information, Figure S2). However, there is no covalent bond formation between the molecule and CSF1R because of the absence of a cysteine residue in the corresponding position of CSF1R. On the basis of the structural observation, the acryl moiety in compound 2 was first saturated to yield compound 3a which exhibited significantly decreased inhibition against EGFR with an IC50 value of 15.7 nM. Interestingly, compound 3a displayed an obvious potency improvement against CSF1R with an IC50 value of 5.0 nM (Table 1). Further investigation revealed that the propionyl moiety in 3a could be replaced with a variety of substituted carbonyl groups (e.g., acetyl (3b), isobutyryl (3c), n-butyryl (3d), 3-methylbutyryl (3e)), without obviously affecting the CSF1R inhibitory potency. Compounds 3b, 3c, 3d, and 3e exhibited IC50 values which ranged from 5.5 to 10.3 nM against CSF1R kianse. However, when the propionyl moiety in 3a was replaced with a relatively large hydrophobic

and selective CSF1R inhibitors by structural modification of an irreversible EGFR inhibitor (Figure 1).

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CHEMISTRY Synthesis of the designed 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl analogues was outlined in Scheme 1. Commercially available ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate (4) was reacted with protected aliphatic amines (5) to produce adducts (6) in high yields. The reduction of (6) with lithium aluminum hydride (LiAlH4) gave alcohols (7) which were subsequently oxidized by MnO2 to yield aldehydes (8). Compounds 9 were obtained through reductive amination of aldehydes (8) with the corresponding amines. Intermediates (11) were prepared by direct cyclization using triphosgene and subsequent oxidation with 3-chloroperbenzoic acid (m-CPBA). A coupling of compounds (11) with different substituted amines generated intermediates (12) which were deprotected by trifluoroacetic acid to produce the key intermediates (13). With compounds (13) in hand, the designed molecules were readily prepared by coupling with various carboxylic acids or 1iodopropane.

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RESULTS AND DISCUSSION In order to identify a “hit” molecule as the starting point for the new CSF1R inhibitor discovery, a random screening was performed against our in-house kinase inhibitor library.30−37 A number of compounds displayed obvious inhibition against CSF1R kinase, among which compound 2, a previously designed irreversible EGFR inhibitor,37 exhibited the strongest suppression against CSF1R with an IC50 value of 30 nM under the conditions of a FRET-based Z′-Lyte assay.38 Not surprisingly, this molecule also potently suppressed the kinase activity of EGFR with an IC50 value of 0.5 nM. 2355



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resulting compound (3l) dramatically decreased the CSF1R inhibitory potency by a factor of 36-fold, indicating a potentially critical interaction between the corresponding carbonyl group with the protein. The investigation also suggested that Sconfiguration in 3a was important for the compound to display selective CSF1R inhibitory potency, and the R-isomer 3m displayed a 4-fold less potency against the target kinase. However, the enantiomers exhibited almost identical suppressive potencies against EGFR kinase. The computational study also suggested that CSF1R possessed a relatively larger 2-Cl-phenyl binding pocket than EGFR because of its outward shifting αC-helix domain (Figure 2C). Substitution optimization on the position might be a reasonable strategy for potency and selectivity improvement. We first investigated potential impact of the substituted position in the phenyl ring by moving the chloro substituent from 2-position to 3-position (3ae) or 4-position (3af), respectively. Both 3ae and 3af exhibited 2- to 3-fold less potency than the parent molecule 3a, suggesting the 2-position might be optimal for the compounds to suppress the kinase function of CSF1R. Similar phenomena were observed by using the substitution patterns of floro (3aa, 3ac, and 3ad) and methyl (3ab, 3ag, and 3ah) groups. However, the 3- or 4substitution induced greater potency decrease against EGFR than what it did against CSF1R. This differentiation caused compounds 3ae and 3af to exhibit improved target selectivity compared with 3a. Interestingly, the substituted position had no obvious impact on CSF1R kinase inhibition when a methoxyl substituent was utilized. Compounds 3ai, 3aj, and 3ak displayed almost equal potencies against CSF1R with IC50 values of 5.1, 4.5, and 5.8 nM, respectively. Similar to the above observation, compound 3aj demonstrated the most selective profile between CSF1R and EGFR with a factor of 92-fold. However, when the methoxyl group in 3aj was replaced with a metabolically more stable difloromethoxyl (3al) or trifloromethoxyl (3am) substituent, the resulting compounds exhibited 6to 33-fold less potency. These results suggested that the electron-donating effect of methoxyl might contribute greatly to the activity by potentially enhancing the cation−π interaction between the phenyl ring and Lys616 in CSF1R protein. The disubstituted derivatives (3an, 3ao, 3ap, and 3aq) were further designed and synthesized based on the SAR information obtained, all of which exhibited strong inhibition against CSF1R with IC50 values of 4.5, 2.0, 4.4, and 2.1 nM, respectively (Table 2). The disubstituted molecules also displayed 99- to 200-fold target selectivity over EGFR. To rationalize potency and selectivity improvement of the disubstituted derivatives, a computational study was conducted to investigate the potential binding mode of 3aq with the CSF1R protein (Figure 3). It was found that the disubstituted phenyl ring was orthogonal to the plane of the basic parent structure, within the favorable angle and distance range to form multihydrophobic interactions with the protein. In addition, the disubstituted phenyl ring also formed a strong cation−π interaction with Lys616 of the protein. The chloro atom was involved in favorable contacts with the gate keeper Thr663 and Val661, respectively, and the OMe moiety potentially formed an H-bond with the backbone NH of Asp796 located in the DFG motif with a measured distance of 2.8 Å, which further benefited the binding with CSF1R. Given their outstanding potency and target selectivity, inhibitors 3ao and 3aq were chosen for a preliminary pharmacokinetic (PK) investigation in Sprague-Dawley (SD)

Table 1. In Vitro CSF1R Kinase Inhibitory Activities of Inhibitors 3a−ma

a

CSF1R and EGFRWT kinase inhibition was determined by using a FRET-based Z′-Lyte assay according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA).38 The data are mean values from at least three independent experiments, and the variations are less than 20%.

2,2-dimethyl propionyl group (3f) or a cyclic alkycarbonyl group (e.g., cyclopropyl acyl (3g), cyclopentyl acyl (3h), or cyclohexyl acyl (3i)), the resulting molecules displayed 4- to 6fold potency loss. For instance, cyclohexyl acyl substituted analogue (3i) exhibited an IC50 value of 30.6 nM against CSF1R, which was approximately 6-fold less potent than 3a. The compounds with hydrophilic substituents (3j and 3k) also displayed less potency. It was noteworthy that when the propionyl moiety in 3a was replaced with a propyl group, the 2356



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Table 2. In Vitro CSF1R Kinase Inhibitory Activities of Inhibitors 3aa−aqa

Figure 3. Representation of the predicted binding mode of compound 3aq in the active site of CSF1R (PDB code 3DPK). Blue lines represent important amino acid residues of CSF1R. The ligand is shown in a yellow stick representation. Interactions of 3aq with D796 and the backbone of C666 are illustrated with red dash lines. Interaction of 3aq with K616 is illustrated with green dash lines. The docking model is visualized using PyMOL Molecular Graphics System, version 1.3 (Schrödinger, LLC).

and 5311 ((μg/L)·h) and oral bioavailability (F) values of 17.1% and 38.6%, respectively, after a 25 mg/kg oral administration. However, they demonstrated relatively low maximum plasma concentration (Cmax) of 396 and 988 μg/L, and short T1/2 values of 1.6 and 1.89 h, respectively. Further structural modification was then conducted with the aim to improve the PK profiles of 3ao and 3aq and maintain the CSF1R inhibitory potency by optimizing both the lipophilic and hydrophilic moieties at the left-hand phenyl substituents. It was found that a 3′-methyl group is optimal for the compounds to demonstrate strong CSF1R kinase inhibition. When the 3′methyl group in 3ao was removed (3ba) or changed to 2′position (3bb), the resulting compounds demonstrated 6.8- to 10-fold potency loss. The replacement of 3′-methyl group with the other substituents, e.g., 3′-methoxyl (3bd), 3′-F (3be), 3-Cl (3bf), 3′ CF3 (3bg), 3′-ethyl (3bh), or 3′-isopropyl (3bi), caused obvious potency loss ranged from 2.9- to 16-fold. Not surprisingly, a replacement of the solvent exposing Nmethylpiperazine moiety in compound 3ao with a less hydrophilic moiety, such as 1-piperdinyl (3bj), N-morpholino (3bk), or 1-(4-acetylpiperazinyl) (3bl) group, caused an approximately 13- to 20-fold potency loss. However, the 1(4-methylpiperazinyl) group (3ao) could be replaced by other solvent accessibly terminal hydrophilic groups, e.g., 4-methyl1,4-diazepan-1-yl (3bm), 4-(dimethylamino)piperidin-1-yl (3bn), 7-methyl-2,7-diazaspiro[4.4]nonan-2-yl (3bo), 9-methyl-3,9-diazaspiro[5.5]undecan-3-yl (3bp), 7-methyl-2, 7diazaspiro[3.5]nonan-2-yl (3bq), cis-5-methylhexahydropyrrolo[3, 4-c]pyrrol-2(1H)-yl (3br), 2-(dimethylamino)ethyl)(methyl)amino (3bs), without obviously affecting the CSF1R inhibitory potency and the target selectivity (Table 3). N-Demethylation is one of the common metabolic pathways for N-methylpiperazine containing drug, and introduction of steric hindrance is a feasible strategy to block the demethylation process.39 Compounds with 3,4-dimethylpiperazin-1-yl (3bt) or cis-3,4,5-trimethylpiperazin-1-yl (3bu) substituent were then designed and synthesized based on this consideration. However, both compounds 3bt and 3bu exhibited 3- to 4fold decreased potency compared with the parent molecule 3ao. Encouragingly, when the N-methyl group in 3ao was replaced with an N-oxetayl fragment, the resulting compound 3bv displayed comparable CSF1R inhibitory potency and target

a


rats (Table 4). Both of the compounds exhibited reasonable PK profiles with high plasma exposure AUC(0−∞) values of 1630 2357



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Table 3. In Vitro CSF1R Kinase Inhibitory Activities of Inhibitors 3ba−bwa

a


Table 4. Pharmacokinetic Profile of Compounds 3ao, 3aq, and 3bw in Ratsa compd

route

AUC(0−∞) ((μg/L)·h)

Cmax (μg/L)

T1/2 (h)

3ao

po (25 mg/kg) iv (5 mg/kg) po (25 mg/kg) iv (5 mg/kg) po (25 mg/kg) iv (5 mg/kg)

1630 1902 5311 2768 6462 2911

396

1.6 2.3 1.89 3.48 1.08 1.08

3aq 3bw a

988 2515

CLz (L h−1 kg−1)

F (%) 17.1

2.64 38.6 1.84 44.4 1.73

SD rats (male, three animals per group) weighing 190−230 g were used for the study.

bioavailability, respectively, after a 25 mg/kg oral administration. Inhibitory activity of 3bw against the other members of the type III growth factor receptor family, i.e., FLT-3, KIT, and PDGFRα and PDGFRβ, was also evaluated utilizing our inhouse kinase assays. It was shown that compound 3bw suppressed the kinase functions of FLT-3, KIT, PDGFRα, and PDGFRβ with IC50 values of 37, 30, 74, 106.2 nM, respectively, which were approximately 10- to 35-fold less potent to its inhibition against CSF1R. The 2-oxo-3,4-

selectivity to the parent molecule. The 3-methyl-4-(4-(oxetan3-yl)piperazin-1-phenyl substituted analogue of 3aq was also designed and synthesized (3bw) to display high CSF1R inhibitory potency with an IC50 value of 3.0 nM and an approximate 120-fold target selectivity over EGFR. Further PK investigation suggested 3bw exhibited improved PK parameters in SD rats comparing with original lead compounds 3ao and 3aq. This compound demonstrated good plasma exposure with AUC(0−∞) value of 6462 (μg/L)·h, high maximum plasma concentration with Cmax value of 2515 μg/L, 44.4% oral 2358



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Figure 4. Compound 3bw dose dependently inhibited phosphorylation of CSF1R and its downstream signaling pathway in RAW264.7 cells. (A) Compound 3bw inhibited phosphorylation of CSF1R and its downstream signaling pathway in RAW264.7 cells as determined by Western blot analysis. (B) Immunoblot quantification of p-CSF1R and p-AKT. All bands were quantified and normalized by GAPDH. The data are expressed as the mean ± SEM from at least three independent experiments.

Figure 5. Inhibitory effect of compound 3bw on LNCaP-MDV-CM-mediated macrophage migration. (A) Illustration of the in vitro macrophages migration model. (B) Compound 3bw suppressed macrophage migration induced by the PCa cells CM. (C) Quantified results of B. The data are expressed as the mean ± SD (n = 3) and are representative of the results obtained from three independent experiments. Statistical significance relative to untreated is indicated: (∗) p < 0.05, (∗∗) p < 0.01.

drug discovery,44−46 strong inhibition against the “off targets” may cause some potential toxicity concerns for the further development of this compound. In view of its strong potency, reasonable target selectivity, and good PK properties, 3bw was selected as a representative molecule for further biological investigation. We first investigated the suppressing effect of 3bw against CSF1R signal pathway in RAW264.7 cells which harbor high level of the kinase (Figure 4). It was shown that 3bw dose-dependently inhibited the activation of CSF1R and the downstream target Akt. The compound demonstrated 50% inhibition against the CSF1R activation at an approximate concentration of 80 nM and almost totally abrogated the signal at 2.0 μM after a 6 h treatment. CSF-1-mediated macrophage infiltration contributes greatly to the clinical drug resistance against both targeted therapies and chemotherapy in prostate cancer (PCa) patients.19,20 CSF1R inhibitor 1 has been reported to overcome castrationresistance against an androgen receptor (AR) antagonist MDV3100 through blocking TAM influx.20 Therefore, the potential suppressing effect of compound 3bw against macrophages migration was investigated by using a well-established transwell assay (Figure 5A).47 The LNCaP prostate cancer cells

dihydropyrimido[4,5-d]pyrimidine scaffold in 3bw has been successfully applied to the development of reversible and irreversible fibroblast growth factor receptor (FGFR) inhibitors.40−42 Therefore, the potential “off-target” inhibition of 3bw against FGFRs was also determined. Not surprisingly, 3bw also potently inhibited FGFR1/2/3/4 with IC50 values of 10, 27, 40.2, and 166.9 nM, respectively. To further validate the target selectivity of 3bw, a kinase selectivity profiling study was conducted against a panel of 250 nonmutant kinases by utilizing the Nanosyn screening platform (Nanosyn, Santa Clara, CA).43 It was shown that 3bw showed 98.4% competition rate with CSF1R at 100 nM and displayed reasonable target selectivity with S(35) and S(10) scores of 0.14 and 0.06, respectively (Supporting Information Table S3). In addition to FGFRs, 3bw also exhibited obvious inhibition (inhibition rate >90% at 100 nM) against several cancer related kinases including Abelson kinase 1 (ABL1), BMX nonreceptor tyrosine kinase (BMX), ephrin type-A receptor 1 (EPH-A1), EPH-A8, kinase insert domain receptor (KDR), Lck/Yes tyrosine kinase A (LYNA), LYNB and proto-oncogene tyrosine-protein kinase receptor Ret (RET), etc. Notably, although some of these kinases (e.g., FGFR, ABL1, and KDR) have been well validated as molecular targets for anticancer 2359



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Figure 6. Compound 3bw reduced mRNA levels of ARG1, IL10, MMP9, and VEGFA in a dose-dependent manner in RAW264.7 cells. Each bar represents the mean ± SE of 3−5 independent experiments. Statistical significance relative to untreated is indicated: (∗) p < 0.05, (∗∗) p < 0.01.

Figure 7. Antiproliferation effects of 3bw in LNCaP cells. (A) 3bw inhibited the proliferation of LNCaP cells induced by RAW264.7. (B) Quantified results of (A). The data are expressed as the mean ± SD (n = 3) and are representative of the results obtained from three independent experiments. Statistical significance relative to untreated is indicated: (∗) p < 0.05, (∗∗) p < 0.01.

were treated with 10 μM anti-androgen agent MDV3100 (MDV), or vehicle control (NC), and incubated in 10% FBS 1640 medium for 48 h. The treated medium (CM) was collected and then placed into the lower chamber of transwell plates. The number of RAW264.7 cells that migrated to the other side of the filter membrane was quantified. It was shown that compound 3bw dose-dependently inhibited the migration of macrophage cells. Treatment of 3bw at 0.625, 1.25, 2.5, or 5.0 μM for 24 h inhibited macrophages invasion by ∼44.5%, ∼ 52.7%, ∼60.6%, or ∼90.7%, respectively (P < 0.05, compared to the PCa CM treatment; Figure 5B and Figure 5C). CSF1R kinase is one of the key players for the protumorigenic properties of TAMs.20 The potential immunotherapeutic effect of 3bw was then determined by measuring its capability to suppress the release of protumorigenic cytokines of macrophages. It was found that compound 3bw dosedependently suppressed the production of arginase 1 (ARG1),

interleukin 10 (IL10), matrix metallopeptidase 9 (MMP9), and vascular endothelial growth factor A (VEGFA), etc. in RAW264.7 macrophages as determined by quantitative realtime polymerase chain reaction (qRT-PCR) assays, suggesting its promising antitumor potential (Figure 6). The antitumor potential of 3bw was further examined by a binary prostate tumor cell−macrophage coculture system (Figure 7). It was shown that stimulation of the cocultured macrophages significantly enhanced the growth of LNCaP prostate cancer cells. After a 48 h treatment, 3bw demonstrated dose-dependent abrogation on the cancer cell growth enhancement of RAW264.7 macrophages and apparently inhibited the proliferation of LNCaP cells by ∼63.4%, ∼70.0%, ∼85.0%, or ∼94.6%, at concentrations of 0.016, 0.08, 0.40, and 2.0 μM, respectively (P < 0.05, compared to the LNCaP alone; Figure 7A and Figure 7B). These results collectively suggest the 2360



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(S)-1-(1-Acetylpyrrolidin-3-yl)-3-(2-chlorophenyl)-7-((3methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3b). White solid 1 (yield 82%). [α]20 D −21.818 (c 0.110, MeOH). H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.50 (t, J = 6.0 Hz, 1H), 7.37−7.32 (m, 5H), 7.09 (s, 0.6H), 7.02−7.01 (m, 1.4H), 5.59−5.56 (m, 1H), 4.65−4.60 (m, 1H), 4.51−4.46 (m, 1H), 4.20 (s, 0.2H), 4.08−4.03 (m, 0.8H), 3.98−3.91 (m, 1H), 3.79−3.72 (m, 1H), 3.52−3.40 (m, 1H), 2.93 (br s, 4H), 2.79−2.75 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.30 (s, 3H), 2.19−2.15 (m, 1H), 2.02 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 169.5, 169.2, 159.6, 159.5, 153.8, 153.7, 147.6, 139.1, 134.4, 134.3, 133.6, 132.8, 130.8, 129.6, 129.5, 130.8, 129.6, 129.5, 128.3, 123.2, 119.7, 118.6, 103.1, 55.8, 55.7, 52.7, 52.1, 52.0, 51.3, 48.4, 47.1, 46.3, 44.5, 33.3, 32.1, 31.6, 31.4, 30.4, 29.8, 29.5, 26.9, 22.8, 22.7, 22.6, 20.6, 18.1, 18.0. HRMS (ESI) for C30H35ClN8O2 [M + H]+, calcd: 575.2644. Found: 575.2648. HPLC analysis: MeOH−H2O (85:15), 8.57 min, 98.78% purity. (S)-3-(2-Chlorophenyl)-1-(1-isobutyrylpyrrolidin-3-yl)-7-((3methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3c). White solid 1 (yield 80%). [α]20 D −34.545 (c 0.110, MeOH). H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.52− 7.49 (m, 1H), 7.36−7.30 (m, 5H), 7.07 (s, 0.6H), 7.03−7.00 (m, 1H), 6.96 (s, 0.4H), 5.60−5.50 (m, 1H), 4.65−4.61 (m, 1H), 4.60−4.48 (m, 1H), 4.11−4.08 (m, 0.7H), 3,97−3.91 (m, 1.3H), 3.88−3.79 (m, 1H), 3.57−3.45 (m, 0.4H), 3.43−3.40 (m, 0.6H), 2.93−2.91 (m, 4H), 2.78−2.61 (m, 1H), 2.59−2.57 (m, 5H), 2.36 (s, 3H), 2.30 (s, 3H), 2.19−2.17 (m, 1H), 1.14−1.09 (m, 4H), 1.08−1.06 (m, 2H). 13C NMR (125 MHz, CDCl3) δ 176.0, 159.5, 153.8, 153.7, 147.5, 139.2, 134.3, 133.6, 130.8, 129.6, 129.5, 128.3, 123.0, 119.7, 118.5, 103.2, 55.8, 52.9, 52.1, 52.0, 51.5, 47.6, 47.1, 46.3, 44.8, 32.7, 31.9, 29.8, 26.6, 20.6, 19.3, 19.2, 19.0, 18.1, 18.0. HRMS (ESI) for C32H39ClN8O2 [M + H]+, calcd: 603.2957. Found: 603.2957. HPLC analysis: MeOH− H2O (85:15), 12.07 min, 97.79% purity. (S)-1-(1-Butyrylpyrrolidin-3-yl)-3-(2-chlorophenyl)-7-((3methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3d). White solid 1 (yield 81%). [α]20 D −16.049 (c 0.162, MeOH). H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.01 (s, 0.6H), 7.99 (s, 0.4H), 7.51− 7.49 (m, 1H), 7.35−7.34 (m, 5H), 7.21 (s, 0.6H), 7.11 (s, 0.4H), 7.01−6.99 (m, 1H), 5.57−5.56 (m, 1H), 4.63−4.60 (m, 1H), 4.50− 4.47 (m, 1H), 4.06−3.92 (m, 2H), 3.76−3.74 (m, 1H), 3.50−3.38 (m, 1H), 2.91 (br s, 4H), 2.79−2.76 (m, 1H), 2.57 (br s, 4H), 2.35 (s, 3H), 2.29 (s, 3H), 2.25−2.21 (m, 2H), 2.19−2.12 (m, 1H), 1.67−1.64 (m, 2H), 0.93−0.92 (m, 3H). 13C NMR (125 MHz, CDCl3) δ 172.1, 171.7, 159.6, 159.5, 153.8, 153.6, 152.7, 147.5, 139.1, 134.3, 133.5, 132.8, 130.7, 129.8, 129.6, 129.5, 128.3, 123.1, 119.6, 118.7, 118.5, 103.0, 55.8, 55.7, 52.8, 52.0, 51.2, 47.7, 47.0, 46.3, 44.5, 36.9, 36.2, 29.8, 26.6, 18.4, 18.3, 18.0, 14.2, 9.1. HRMS (ESI) for C32H39ClN8O2 [M + H]+, calcd: 603.2957. Found: 603.2919. HPLC analysis: MeOH−H2O (85:15), 7.33 min, 97.93% purity. (S)-3-(2-Chlorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1yl)phenyl)amino)-1-(1-(3-methylbutanoyl)pyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3e). White solid 1 (yield 80%). [α]20 D −33.333 (c 0.138, MeOH). H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.52− 7.48 (m, 1H), 7.35−7.32 (m, 5H), 7.11 (s, 0.6H), 7.02−6.99 (m, 1.4H), 5.57−5.54 (m, 1H), 4.64−4.61 (m, 1H), 4.50−4.45 (m, 1H), 4.09−3.91 (m, 2H), 3.77−3.76 (m, 1H), 3.53−3.40 (m, 1H), 2.92 (br s, 4H), 2.79−2.75 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.30 (s, 3H), 2.15−2.11 (m, 4H), 0.95 (d, J = 6.8 Hz, 6H). 13C NMR (125 MHz, CDCl3) δ 171.6, 171.3, 159.6, 159.5, 153.8, 153.7, 147.5, 139.2, 134.3, 133.6, 130.8, 129.6, 129.4, 128.3, 128.2, 123.1, 119.7, 118.5, 103.1, 55.8, 52.8, 52.1, 52.0, 51.2, 47.8, 47.1, 46.3, 46.0, 44.5, 43.9, 43.2, 29.8, 26.6, 25.6, 25.5, 22.9, 22.8, 18.1, 18.0. HRMS (ESI) for C33H41ClN8O2 [M + H]+, calcd: 617.3114. Found: 617.3111. HPLC analysis: MeOH−H2O (85:15), 7.64 min, 97.76% purity. (S)-3-(2-Chlorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1yl)phenyl)amino)-1-(1-pivaloylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3f). White solid 1 (yield 84%). [α]20 D −11.667 (c 0.240, MeOH). H NMR (400 MHz,

promising potential of compound 3bw to serve as a lead compound for drug discovery.

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CONCLUSIONS In summary, a series of 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidines were designed and synthesized as new CSF1R inhibitors. One of the most promising compounds, 3bw, strongly inhibited the CSF1R kinase activity with an IC50 value of 3.0 nM and dose-dependently suppressed the activation of CSF1R downstream signals in RAW264.7 macrophages. In addition, 3bw demonstrated reasonable pharmacokinetic properties in rats and exhibited promising antitumor effects in blocking macrophage infiltration and abrogating the protumorigenic influences of macrophages. Compound 3bw may serve as a new lead compound for antitumor drug discovery. Further structural optimization and in vivo antitumor efficacy of 3bw investigation are underway, and the results will be reported in due course.

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EXPERIMENTAL SECTION

General Methods for Chemistry. All reagents and solvents used were purchased from commercial sources and were of analytical grade. Flash chromatography was performed using 300 mesh silica gel. All reactions were monitored by thin-layer chromatography (TLC), using silica gel plates with fluorescence F254 and ultraviolet (UV) light visualization. 1H NMR spectra were recorded on a Bruker AV-400 spectrometer at 400 MHz or a Bruker AV-500 spectrometer at 500 MHz. 13C NMR spectra were recorded on a Bruker AV-500 spectrometer at 125 MHz. Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per million (ppm) units relative to an internal standard (TMS). Low resolution ESI-MS data were recorded on an Agilent 1200 HPLCMSD mass spectrometer and high resolution ESI-MS data on an Applied Biosystems Q-STAR Elite ESI-LC−MS/MS mass spectrometer. Purity of all of the final compounds was determined by reversephase high performance liquid chromatography (HPLC) analysis to be >95%. HPLC instrument: Dionex Summit HPLC (column, Diamonsil C18, 5.0 μm, 4.6 mm × 250 mm (Dikma Technologies); detector, PDA-100 photodiode array; injector, ASI-100 autoinjector; pump, p680A). A flow rate of 1.0 mL/min was used with mobile phase of MeOH in H2O with 0.1% modifier (ammonia, v/v). (S)-3-(2-Chlorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3a). General Procedure for Syntheses of 3a−k, 3m, 3aa−aq, 3ba−bw. DIEA (98 μL, 0.56 mmol) and propanoic acid (25 μL, 0.337 mmol) were added to a solution of HATU (128 mg, 0.337 mmol) in anhydrous CH2Cl2 (10 mL) at rt. After stirring for 10 min, 13a (150 mg, 0.28 mmol) was added to the resulting mixture, and then the mixture was stirred at room temperature for 0.5 h. The resulting solution was poured into H2O and extracted with CH2Cl2. The organic layer was washed with 10% aqueous NaHCO3, brine and dried with Na2SO4. The resultant crude material was purified by column chromatography (SiO2, CH2Cl2/MeOH/NH4OH, 40:1:0.4) to give 3a (white solid, 132 mg, 1 yield 80%). [α]20 D −10.169 (c 0.118, MeOH). H NMR (400 MHz, CDCl3) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.50 (t, J = 6.8 Hz, 1H), 7.48−7.30 (m, 5H), 7.09 (s, 0.6H), 7.02−7.00 (m, 1.4H), 5.59−5.57 (m, 1H), 4.65−4.61 (m, 1H), 4.51−4.48 (m, 1H), 4.02−3.91(m, 2H), 3.75−3.73 (m, 1H), 3.49−3.42 (m, 1H), 2.93−2.91 (m, 4H), 2.78− 2.74 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.30−2.21 (m, 5H), 2.19− 2.16 (m, 1H), 1.14 (t, J = 7.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.6, 159.5, 153.8, 153.7, 152.6, 147.5, 147.4, 139.2, 134.4, 134.3, 133.6, 132.8, 130.8, 129.8, 129.6, 129.5, 128.3, 123.1, 119.7, 118.5, 103.1, 55.8, 52.8, 52.1, 51.2, 47.7, 47.1, 46.3, 45.8, 44.6, 29.0, 28.0, 27.4, 26.7, 18.1, 9.1. HRMS (ESI) for C31H37N8O2 [M + H]+, calcd: 589.2801. Found: 589.2805. HPLC analysis: MeOH−H2O (85:15), 9.29 min, 96.44% purity. 2361



Article

CDCl3, a mixture of rotamers) δ 8.01 (s, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.37−7.29 (m, 5H), 7.11 (s, 1H), 7.00 (d, J = 8.8 Hz, 1H), 5.53−5.49 (m, 1H), 4.62 (d, J = 13.6 Hz, 1H), 4.48 (d, J = 13.6 Hz, 1H), 4.12− 4.11 (m, 1H), 4.00−3.98 (m, 2H), 3.56−3.55 (m, 1H), 2.92 (t, J = 4.4 Hz, 4H), 2.71−2.70 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.30 (s, 3H), 2.19−2.14 (m, 1H), 1.23 (s, 9H). 13C NMR (125 MHz, CDCl3) δ176.7, 159.5, 157.6, 153.7, 152.7, 147.4, 139.2, 134.4, 133.5, 134.4, 133.5, 132.9, 132.8, 130.7, 129.7, 129.5, 128.3, 123.0, 119.7, 118.5, 103.1, 55.8, 52.0, 48.7, 47.1, 47.0, 46.3, 39.1, 32.0, 29.8, 29.7, 29.5, 27.7, 22.8, 18.1, 14.2. HRMS (ESI) for C33H41ClN8O2 [M + H]+, calcd: 617.3114. Found: 617.3116. HPLC analysis: MeOH−H2O (85:15), 14.12 min, 98.96% purity. (S)-3-(2-Chlorophenyl)-1-(1-(cyclopropanecarbonyl)pyrrolidin-3-yl)-7-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3g). White solid (yield 75%). [α]20 D −41.667 (c 0.072, MeOH). 1 H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.52−7.49 (m, 1H), 7.36−7.33 (m, 5H), 7.11 (s, 0.6H), 7.02−6.97 (m, 1.4H), 5.63 (t, J = 8.4 Hz, 1H), 4.64 (d, J = 13.6 Hz, 1H), 4.50 (d, J = 13.6 Hz, 1H), 4.25−4.22 (m, 0.6H), 4.00−3.96 (m, 2.4H), 3.72−3.70 (m, 0.4H), 3.48−3.40 (m, 0.6H), 2.93 (br s, 4H), 2.79−2.78 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.30 (s, 3H), 2.20− 2.18 (m, 1H), 1.66−1.65 (m, 0.4H), 1.58−1.57 (m, 0.6H), 1.00−0.99 (m, 2H), 0.88−0.86 (m, 1H), 0.75−0.71 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 172.5, 172.1, 159.6, 159.5, 157.4, 153.8, 152.8, 147.4, 139.2, 134.4, 133.6, 132.8, 130.8, 129.8, 129.6, 120.3, 123.2, 123.1, 123.0, 119.7, 118.7, 118.6, 118.4, 103.1, 55.8, 52.8, 52.1, 52.0, 47.1, 46.3, 45.0, 29.8, 26.8, 18.1, 18.0, 12.8, 12.1, 7.6, 7.5, 7.4. HRMS (ESI) for C32H37ClN8O2 [M + H]+, calcd: 601.2801. Found: 601.2798. HPLC analysis: MeOH−H2O (85:15), 10.11 min, 98.3% purity. (S)-3-(2-Chlorophenyl)-1-(1-(cyclopentanecarbonyl)pyrrolidin-3-yl)-7-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3h). White solid (yield 90%). [α]20 D −24.490 (c 0.098, MeOH). 1 H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.50−7.49 (m, 1H), 7.36−7.35 (m, 5H), 7.17 (s, 0.6H), 7.06−7.00 (m, 1.4H), 5.57−5.56 (m, 1H), 4.61−4.60 (m, 1H), 4.51− 4.47 (m, 1H), 4.23−4.22 (m, 0.1H), 4.09−4.08 (m, 0.6H), 3.93−3.82 (m, 2H), 3.65−3.57 (m, 0.1H), 3.44−3.43 (m, 0.3H), 3.11−3.10 (m, 0.7H), 3.15−3.12 (m, 0.2H), 2.94 (br s, 4H), 2.79−2.72 (m, 1H), 2.70 (br s, 4H), 2.40 (s, 3H), 2.29 (br s, 4H), 2.17−2.15 (m, 1H), 1.54− 1.52 (m, 3H), 1.42−1.40 (m, 1H), 1.28−1.25 (m, 4H). 13C NMR (125 MHz, CDCl3) δ 175.3, 175.0, 159.5, 159.4, 157.4, 153.8, 153.6, 152.7, 147.3, 147.2, 139.2, 134.6, 134.4, 133.6, 132.6, 130.8, 129.8, 129.6, 129.5, 128.3, 123.1, 119.7, 118.6, 103.1, 55.8, 55.7, 52.8, 52.0, 51.9, 51.2, 47.7, 47.1, 46.2, 46.1, 45.8, 44.8, 43.4, 42.6, 33.3, 32.0, 31.6, 31.4, 30.3, 30.1, 29.9, 29.8, 29.5, 26.6, 26.3, 25.2, 23.2, 22.8, 18.6, 18.2, 9.1. HRMS (ESI) for C30H36N8O3 [M + H]+, calcd: 629.3270. Found: 629.3088. HPLC analysis: MeOH−H2O (85:15), 16.39 min, 96.77% purity. (S)-3-(2-Chlorophenyl)-1-(1-(cyclohexanecarbonyl)pyrrolidin-3-yl)-7-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3i). White solid (yield 85%). [α]20 D −39.130 (c 0.092, MeOH). 1 H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.50 (t, J = 7.2 Hz, 1H), 7.35−7.32 (m, 5H), 7.14 (s, 0.7H), 7.02−6.99 (m, 1.3H), 5.56−5.55 (m, 1H), 4.64−4.61 (m, 1H), 4.51−4.47 (m, 1H), 4.20−4.21 (m, 0.1H), 4.11−4.07 (m, 0.9H), 3.91−3.81 (m, 1.6H), 3.64−3.61 (m, 0.6H), 3.56−3.40 (m, 0.4H), 3.09−3.08 (m, 0.4H), 2.93 (br s, 4H), 2.79−2.74 (m, 1H), 2.60 (br s, 4H), 2.37 (s, 3H), 2.29 (s, 3H), 2.17−2.16 (m, 2H), 1.49−1.42 (m, 2H), 1.39−1.28 (m, 2H), 1.25−1.21 (m, 6H). 13C NMR (125 MHz, CDCl3) δ 175.2, 174.8, 159.5, 159.4, 157.4, 153.8, 153.6, 152.7, 147.3, 147.2, 139.2, 134.6, 134.4, 133.6, 132.8, 130.8, 129.8, 129.6, 129.5, 128.3, 123.1, 119.7, 118.6, 103.1, 103.0, 55.8, 54.9, 52.9, 52.0, 51.9, 51.1, 47.5, 47.1, 46.2, 46.1, 45.7, 44.7, 43.1, 42.3, 32.0, 31.6, 31.4, 30.3, 29.8, 29.5, 29.1, 29.0, 28.8, 26.6, 26.0, 25.2, 23.2, 22.8, 18.6, 18.0, 9.1. HRMS (ESI) for C30H36N8O3 [M + H]+, calcd: 643.3270. Found: 643.3203. HPLC analysis: MeOH−H2O (85:15), 19.80 min, 98.22% purity.

(S)-3-(2-Chlorophenyl)-1-(1-(2-hydroxyacetyl)pyrrolidin-3yl)-7-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3j). White solid 1 (yield 82%). [α]20 D −21.311 (c 0.122, MeOH). H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.02 (s, 1H), 7.52−7.50 (m, 1H), 7.35−7.32 (m, 5H), 7.03−7.01 (m, 2H), 5.60 (q, J = 8.8 Hz, 1H), 4.64−4.60 (m, 1H), 4.50−4.47 (m, 1H), 4.09 (s, 1H), 4.03−3.94 (m, 2H), 3.64 (s, 1H), 3.56−3.51 (m, 2H), 3.38−3.36 (m, 1H), 2.93 (br s, 4H), 2.75−2.73 (m, 1H), 2.65 (br s, 4H), 2.41 (s, 3H), 2.29 (s, 3H), 2.25−2.22 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 170.3, 170.0, 159.6, 157.3, 153.9, 153.8, 152.5, 147.7, 139.0, 134.2, 133.5, 132.8, 130.8, 129.7, 129.6, 129.5, 128.3, 123.4, 119.6, 118.9, 102.9, 60.7, 60.4, 55.7, 52.4, 52.0, 51.9, 50.6, 47.1, 46.3, 44.9, 31.5, 31.4, 30.3, 29.8, 26.3, 20.6, 18.1, 18.0. HRMS (ESI) for C30H35ClN8O3 [M + H]+, calcd: 591.2593. Found: 591.2597. HPLC analysis: MeOH−H2O (85:15), 8.09 min, 98.57% purity. (S)-3-(2-Chlorophenyl)-1-(1-(2-(dimethylamino)acetyl)pyrrolidin-3-yl)-7-((3-methy l-4-(4-methylpiperazin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3k). White solid (yield 79%). [α]20 D −26.415 (c 0.106, MeOH). 1 H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.02 (s, 0.6H), 7.99 (s, 0.4H), 7.52−7.49 (m, 1H), 7.37−7.30 (m, 5H), 7.10 (s, 0.4H), 7.06 (s, 0.6H), 7.03−7.00 (m, 1H), 5.59−5.57 (m, 1H), 4.66−4.61 (m, 1H), 4.59−4.48 (m, 1H), 4.21−4.20 (m, 0.2H), 4.07−4.02 (m, 0.8H), 3.95−3.93 (m, 1.5H), 3. 89−3.85 (m, 0.5H), 3.61−3.59 (m, 0.4H), 3.49−3.46 (m, 0.6H), 3.11−3.06 (m, 1.4H), 3.05−3.01 (m, 0.6H), 2.98−2.93 (m, 4H), 2.76−2.1 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.31 (s, 6H), 2.30 (s, 3H), 2.19−2.15 (m, 1H). 13C NMR (125 MHz, CDCl3) δ 168.7, 168.3, 159.5, 159.4, 157.3, 153.7, 153.5, 152.5, 152.4, 147.3, 139.0, 134.4, 134.3, 133.3, 132.6, 132.5, 130.6, 130.5, 129.6, 129.5, 129.4, 129.3, 128.1, 123.3, 123.1, 119.4, 118.7, 118.4, 102.8, 62.4, 62.3, 62.1, 55.6, 55.5, 52.7, 51.9, 51.8, 50.8, 46.9, 46.2, 46.1, 45.8, 45.7, 45.6, 45.5, 44.7, 26.2, 26.1, 18.0, 17.9. HRMS (ESI) for C32H40ClN9O2 [M + H]+, calcd: 618.3066. Found: 618.3069. HPLC analysis: MeOH−H2O (85:15), 11.42 min, 96.13% purity. (S)-3-(2-Chlorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1yl)phenyl)amino)-1-(1-propylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3l). N,N-Diisopropylethylamine (49 μL, 0.28 mmol) and iodopropane (27 μL, 0.28 mmol) were added to a solution of 13a (100 mg, 0.19 mmol) in anhydrous CH2Cl2 (10 mL) with stirring at 0 °C. The reaction was allowed to warm to room temperature, and then, after stirring for 4 h, the reaction mixture was poured into a solution of 10% aqueous NaHCO3 and extracted with CH2Cl2. The organic layer was separated, washed with 10% aqueous NaHCO3 and brine, dried with Na2SO4, filtered, and concentrated in vacuo. The resultant crude material was purified by column chromatography (SiO2, CH2Cl2/MeOH/NH4OH, 40:1:0.4) to give 3n (60 mg, yield 56%). [α]20 D −27.027 (c 0.074, MeOH). 1H NMR (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.51−7.44 (m, 3H), 7.36−7.30 (m, 4H), 7.05 (d, J = 8.8 Hz, 1H), 5.69 (m, 1H), 4.66−4.4.42 (m, 2H), 3.52−3.43 (m, 1H), 3.16−3.15 (m, 3H), 2.94− 2.91 (m, 4H), 2.73−2.70 (m, 1H), 2.58 (br s, 4H), 2,43−2.41 (m, 1H), 2.35 (s, 3H), 2.31 (s, 3H), 1.67 (q, J = 8.0 Hz, 2H), 1.41−1.33 (m, 2H), 0.94 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 159.6, 157.6, 153.6, 152.6, 147.3, 139.2, 134.5, 133.6, 133.0, 132.8, 130.8, 129.6, 129.5, 128.4, 128.4, 123.1, 119.9, 118.6, 102.8, 57.9, 55.9, 55.1, 54.9, 53.9, 52.0, 51.3, 47.2, 46.3, 29.8, 28.5, 28.4, 20.9, 19.2, 18.1, 14.2, 11.9. HRMS (ESI) for C31H39ClN8O [M + H]+, calcd: 575.3008. Found: 575.3017. HPLC analysis: MeOH−H2O (85:15), 27.27 min, 97.44% purity. (R)-3-(2-Chlorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3m). White solid 1 (yield 87%). [α]20 D +14.736 (c 0.095, MeOH). H NMR (400 MHz, CDCl3, a mixture of rotamers) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.52− 7.49 (m, 1H), 7.37−7.34 (m, 5H), 7.04−7.00 (m, 1.6H), 6.94 (s, 0.4H), 5.60−5.53 (m, 1H), 4.65−4.61 (m, 1H), 4.51−4.45 (m, 1H), 4.08−3.88 (m, 2H), 3.78−3.70 (m, 1H), 3.52−3.41 (m, 1H), 2.94− 2.92 (m, 4H), 2.78−2.74 (m, 1H), 2.60 (br s, 4H), 2.37 (s, 3H), 2.30 (s, 3H), 2.27−2.21 (m, 2H), 2.19−2.14 (m, 1H), 1.12 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 162.8, 160.8, 159.6, 2362



Article

127.3, 127.2, 123.3, 123.1, 119.7, 118.7, 118.6, 116.3, 116.2, 116.1, 116.0, 103.1, 55.8, 53.1, 52.1, 52.0, 51.5, 47.6, 47.1, 47.0, 46.3, 45.7, 44.6, 28.7, 28.1, 27.4, 26.6, 18.1, 18.0, 14.2, 9.2. HRMS (ESI) for C31H37FN8O2 [M + H]+, calcd: 573.3096. Found: 573.3078. HPLC analysis: MeOH−H2O (85:15), 8.84 min, 97.92% purity. (S)-3-(3-Chlorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ae). White solid 1 (yield 89%). [α]20 D −14.063 (c 0.128, MeOH). H NMR (400 MHz, CDCl3) δ 8.03 (s, 0.6H), 8.01 (s, 0.4H), 7.34−7.32 (m, 4H), 7.25− 7.23 (m, 2H), 7.14 (s, 0.5H), 7.01−6.99 (m, 1.5H), 5.55−5.47 (m, 1H), 4.65−4.56 (m, 2H), 4.07−4.01 (m, 0.6H), 3.95−3.92 (m, 1.4H), 3.75−3.70 (m, 1H), 3.51−3.39 (m, 1H), 2.92 (br s, 4H), 2.84−2.73 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.29 (s, 3H), 2.24 (q, J = 7.2 Hz, 2H), 2.18−2.15 (m, 1H), 1.16 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.6, 159.5, 157.5, 157.2, 153.8, 153.3, 153.2, 152.9, 147.6, 147.5, 143.1, 143.0, 134.8, 134.7, 134.3, 134.2, 133.6, 130.2, 130.1, 127.0, 126.9, 125.6, 123.6, 123.5, 123.2, 123.1, 119.7, 118.7, 118.5, 103.1, 103.0, 55.8, 53.1, 52.1, 52.0, 51.5, 47.6, 47.1, 47.0, 46.3, 45.7, 44.6, 28.7, 28.1, 27.4, 26.6, 18.1, 18.0, 9.2. HRMS (ESI) for C31H37ClN8O2 [M + H]+, calcd: 589.2801. Found: 589.2802. HPLC analysis: MeOH−H2O (85:15), 9.53 min, 97.38% purity. (S)-3-(4-Chlorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3af). White solid 1 (yield 82%). [α]20 D −14.583 (c 0.096, MeOH). H NMR (400 MHz, CDCl3) δ 8.04 (s, 0.6H), 8.02 (s, 0.4H), 7.39−7.33 (m, 5H), 7.03− 7.01 (m, 1.6H), 6.93 (s, 0.4H), 7.06−7.00 (m, 1.3H), 5.52 (q, J = 8.0 Hz, 1H), 4.65−4.62 (m, 2H), 4.04−3.94 (m, 1H), 3.77−3.67 (m, 1H), 3.52−3.48 (m, 1H), 2.97 (br s, 4H), 2.77−2.74 (m, 1H), 2.67 (br s, 4H), 2.42 (s, 3H), 2.30 (s, 3H), 2.27−2.23 (m, 3H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.5, 159.6, 159.5, 157.7, 157.6, 157.3, 153.6, 153.3, 153.0, 147.6, 147.5, 140.5, 134.3, 134.2, 133.6, 132.4, 132.3, 129.4, 129.3, 126.7, 126.6, 123.3, 123.1, 119.6, 118.7, 118.6, 103.0, 55.8, 53.1, 52.1, 52.0, 51.5, 47.6, 47.1, 47.0, 46.3, 45.7, 44.6, 28.7, 28.1, 27.4, 26.6, 18.1, 18.0, 9.2. HRMS (ESI) for C31H37ClN8O2 [M + H]+, calcd: 589.2801. Found: 589.2804. HPLC analysis: MeOH−H2O (85:15), 13.49 min, 99.12% purity. (S)-7-((3-Methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3-(m-tolyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ag). White solid (yield 88%). [α]20 D −16.000 (c 0.100, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.01 (s, 0.6H), 7.99 (s, 0.4H), 7.35−7.27 (m, 3H), 7.15−7.09 (m, 4H), 7.00 (d, J = 9.2 Hz, 1H), 5.54−5.49 (m, 1H), 4.61−4.59 (m, 2H), 4.09− 4.04 (m, 0.6H), 3.96−3.93 (m, 1.4H), 3.74−3.70 (m, 1H), 3.44−3.41 (m, 1H), 2.93−2.92 (m, 4H), 2.91−2.75 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 6H), 2.32 (s, 3H), 2.30 (s, 3H), 2.27 (q, J = 5.2 Hz, 2H), 2.23−2.21 (m, 1H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.5, 157.8, 157.4, 153.6, 153.4, 153.1, 147.5, 142.0, 139.4, 134.4, 133.5, 129.1, 127.8, 127.1, 126.2, 126.1, 123.2, 123.0, 119.7, 118.7, 118.5, 103.4, 55.8, 53.0, 52.1, 52.0, 47.3, 46.3, 44.6, 29.8, 28.7, 28.1, 27.4, 26.6, 21.5, 18.1, 9.2. HRMS (ESI) for C32H40N8O2 [M + H]+, calcd: 569.3347. Found: 569.3352. HPLC analysis: MeOH−H2O (85:15), 11.43 min, 96.13% purity. (S)-7-((3-Methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3-(p-tolyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ah). White solid (yield 78%). [α]20 D −19.672 (c 0.122, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.00 (s, 0.6H), 7.98 (s, 0.4H), 7.34−7.31 (m, 2H), 7.22−7.16 (m, 4.7H), 7.12 (s, 0.3H), 7.00−6.99 (m, 1H), 5.54−5.50 (m, 1H), 4.61−4.58 (m, 2H), 4.08−4.04 (m, 0.6H), 3.95−3.91 (m, 1.4H), 3.73−3.69 (m, 1H), 3.46−3.40 (m, 1H), 2.93−2.91 (m, 4H), 2.80−2.74 (m, 1H), 2.57 (br s, 4H), 2.36 (s, 6H), 2.29 (s, 3H), 2.24 (q, J = 7.6 Hz, 2H), 2.21−2.18 (m, 1H), 1.13 (t, J = 7.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.7, 172.4, 159.6, 159.5, 157.7, 157.4, 153.6, 153.4, 153.1, 147.4, 147.4, 140.5, 139.6, 139.5, 136.9, 136.7, 134.5, 134.4, 133.6, 129.9, 129.8, 125.3, 125.2, 123.2, 123.1, 119.6, 118.7, 118.6, 103.4, 103.3, 55.8, 52.9, 52.1, 52.0, 51.5, 47.6, 47.3, 47.2, 46.3, 45.7, 44.6, 32.1, 29.8, 29.5, 28.7, 28.1, 27.4, 26.6, 22.8, 21.1, 18.1, 18.0, 9.2. HRMS (ESI) for

159.5, 157.3, 153.9, 153.7, 152.7, 147.6, 147.5, 135.5, 134.3, 133.6, 130.9, 130.7, 130.6, 123.3, 123.2, 123.1, 119.7, 118.8, 118.7, 118.6, 118.2, 118.0, 115.6, 115.4, 102.9, 55.8, 55.7, 528, 52.1, 52.0, 51.2, 47.6, 47.2, 46.3, 44.6, 28.0, 27.4, 26.7, 18.1, 18.0, 9.1. HRMS (ESI) for C31H37ClN8O2 [M + H]+, calcd: 589.2801. Found: 589.2804. HPLC analysis: MeOH−H2O (85:15), 11.93 min, 98.56% purity. (S)-3-(2-Fluorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3aa). White solid 1 (yield 84%). [α]20 D −12.766 (c 0.094, MeOH). H NMR (400 MHz, CDCl3) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.36−7.32 (m, 4H), 7.23− 7.17 (m, 2H), 7.03−7.00 (m, 1.7H), 6.92 (s, 0.3H), 5.59−5.50 (m, 1H), 4.59−4.57 (m, 2H), 4.07−3.92 (m, 2H), 3.75−3.71 (m, 1H), 3.50−3.41 (m, 1H), 2.95−2.93 (m, 4H), 2.81−2.74 (m, 1H), 2.61 (br s, 4H), 2.38 (s, 3H), 2.30 (s, 3H), 2.23 (q, J = 7.6 Hz, 2H), 2.19−2.16 (m, 1H), 1.14 (t, J = 7.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.6, 159.5, 159.1, 157.8, 157.4, 157.1, 153.8, 153.6, 152.9, 152.6, 147.5, 147.4, 134.4, 134.3, 133.5, 129.5, 129.4, 129.3, 128.8, 128.6, 124.9, 123.3, 123.1, 119.7, 118.7, 118.5, 117.0, 116.9, 103.2, 103.1, 55.8, 55.7, 53.0, 51.9, 51.4, 47.6, 47.2, 47.1, 46.3, 45.7, 44.6, 28.7, 28.0, 27.4, 26.6, 18.1, 18.0, 9.2. HRMS (ESI) for C31H37N8O2 [M + H]+, calcd: 573.3096. Found: 573.3113. HPLC analysis: MeOH−H2O (85:15), 8.61 min, 98.59% purity. (S)-7-((3-Methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3-(o-tolyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ab). White solid (yield 85%). [α]20 D −19.149 (c 0.094, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.01 (s, 0.6H), 7.99 (s, 0.4H), 7.35−7.32 (m, 2H), 7.31−7.27 (m, 4H), 7.02− 7.01 (m, 1.6H), 6.92 (s, 0.4H), 5.57−5.54 (m, 1H), 4.66−4.63 (m, 1H), 4.43−4.39 (m, 1H), 4.08−3.96 (m, 1.7H), 3.76−3.71 (m, 1.3H), 3.50−3.39 (m, 2H), 2.95 (br s, 4H), 2.78−2.74 (m, 1H), 2.62 (br s, 4H), 2.39 (s, 3H), 2.30 (s, 3H), 2.28−2.23 (m, 5H), 2.15−2.13 (m, 1H), 1.12 (t, J = 8.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.6, 159.5, 157.9, 157.6, 153.6, 153.5, 152.6, 147.5, 147.4, 140.8, 140.7, 135.9, 135.7, 135.6, 134.4, 134.3, 133.6, 131.4, 128.3, 128.2, 127.5, 127.4, 127.0, 126.9, 126.8, 126.7, 123.2, 123.1, 119.7, 118.6, 118.5, 103.2, 55.8, 52.8, 52.7, 52.1, 52.0, 51.1, 47.6, 47.4, 46.3, 45.7, 44.6, 44.5, 29.0, 28.8, 28.1, 28.0, 27.4, 26.7, 26.6, 18.1, 18.0, 17.9, 17.8, 9.1. HRMS (ESI) for C32H40N8O2 [M + H]+, calcd: 569.3347. Found: 569.3352. HPLC analysis: MeOH−H2O (85:15), 10.88 min, 97.36% purity. (S)-3-(3-Fluorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ac). White solid 1 (yield 81%). [α]20 D −11.475 (c 0.122, MeOH). H NMR (500 MHz, CDCl3) δ 8.03 (s, 0.6H), 8.02 (s, 0.4H), 7.38−7.35 (m, 3H), 7.32 (s, 0.6H), 7.14−7.06 (m, 2H), 7.01−6.97 (m, 2.4H), 5.51 (q, J = 8.5 Hz, 1H), 4.65−4.58 (m, 2H), 4.07−4.03 (m, 0.6H), 3.96−3.94 (m, 1.4H), 3.73 (t, J = 9.0 Hz, 1H), 3.49−3.40 (m, 1H), 2.92 (br s, 4H), 2.83− 2.74 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.30 (s, 3H), 2.25−2.24 (m, 2H), 2.23−2.21 (m, 1H), 1.14 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.5, 163.9, 161.9, 159.7, 159.6, 157.6, 157.2, 153.8, 153.6, 153.2, 152.9, 147.6, 147.5, 143.5, 143.4, 134.3, 134.2, 133.6, 130.4, 130.3, 130.2, 123.3, 123.2, 123.1, 120.8, 119.7, 118.7, 118.6, 113.9, 113.7, 113.5, 112.9, 112.8, 103.1, 55.8, 53.1, 52.1, 52.0, 51.5, 47.6, 47.1, 47.0, 46.3, 45.7, 44.6, 28.7, 28.0, 27.4, 26.6, 18.1, 18.0, 9.2. HRMS (ESI) for C31H37FN8O2 [M + H]+, calcd: 573.3096. Found: 573.3098. HPLC analysis: MeOH−H2O (85:15), 10.82 min, 98.45% purity. (S)-3-(4-Fluorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ad). White solid 1 (yield 87%). [α]20 D −14.286 (c 0.070, MeOH). H NMR (400 MHz, CDCl3) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.35−7.27 (m, 4H), 7.13− 7.07 (m, 2.6H), 7.01−6.99 (m, 1.4H), 5.56−5.50 (m, 1H), 4.60−4.58 (m, 2H), 4.06−4.02 (m, 0.6H), 3.95−3.92 (m, 1.4H), 3.74−3.69 (m, 1H), 3.49−3.41 (m, 1H), 2.93−2.91 (m, 4H), 2.80−2.75 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.29 (s, 3H), 2.23 (q, J = 7.2 Hz, 2H), 1.94−1.92 (m, 1H), 1.14 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 162.2, 160.2, 159.6, 159.5, 157.6, 157.3, 153.7, 153.6, 153.5, 153.1, 147.6, 147.5, 138.1, 134.4, 134.3, 127.5, 127.4, 2363



Article

C32H40N8O2 [M + H]+, calcd: 569.3347. Found: 569.3352. HPLC analysis: MeOH−H2O (85:15), 11.66 min, 98.72% purity. (S)-3-(2-Methoxyphenyl)-7-((3-methyl-4-(4-methylpiperazin1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ai). White solid 1 (yield 81%). [α]20 D −10.000 (c 0.140, MeOH). H NMR (400 MHz, CDCl3) δ 7.98 (s, 0.6H), 7.96 (s, 0.4H), 7.35−7.29 (m, 3H), 7.25− 7.22 (m, 1H), 7.01−6.98 (m, 3.7H), 6.91 (s, 0.3H), 5.59−5.53 (m, 1H), 4.62−4.60 (m, 2H), 4.08−4.03 (m, 0.5H), 3.96−3.92 (m, 1.5H), 3.85−3.84 (m, 3H), 3.74−3.69 (m, 1H), 3.49−3.44 (m, 1H), 2.93− 2.91 (m, 4H), 2.80−2.77 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.30 (s, 3H), 2.26 (q, J = 8.0 Hz, 2H), 2.23−2.21 (m, 1H), 1.13 (t, J = 8.0 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.4, 155.3, 153.5, 153.3, 153.2, 152.9, 147.4, 147.3, 134.6, 134.5, 133.6, 130.3, 129.4, 129.3, 128.9, 123.1, 123.0, 121.3, 119.7, 118.6, 118.5, 112.4, 103.8, 55.9, 55.8, 52.8, 52.1, 52.0, 51.2, 47.6, 47.4, 46.3, 45.8, 44.6, 28.8, 28.1, 28.0, 27.4, 26.7, 26.6, 18.1, 18.0, 9.2. HRMS (ESI) for C32H40N8O3 [M + H]+, calcd: 585.3296. Found: 585.3293. HPLC analysis: MeOH−H2O (85:15), 8.54 min, 99.99% purity. (S)-3-(3-Methoxyphenyl)-7-((3-methyl-4-(4-methylpiperazin1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3aj). White solid 1 (yield 84%). [α]20 D −13.000 (c 0.200, MeOH). H NMR (400 MHz, CDCl3) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.35−7.28 (m, 3H), 7.05− 6.99 (m, 1.7H), 6.95 (s, 0.3H), 6.89−6.83 (m, 3H), 5.56−5.47 (m, 1H), 4.65−4.56 (m, 2H), 4.08−4.04 (m, 0.7H), 3.96−3.94 (m, 1.3H), 3.82 (s, 3H), 3.75−3.70 (m, 1H), 3.49−3.39 (m, 1H), 2.93 (t, J = 4.4 Hz, 4H), 2.80−2.75 (m, 1H), 2.60 (br s, 4H), 2.36 (s, 3H), 2.30 (s, 3H), 2.25 (q, J = 7.2 Hz, 2H), 2.22−2.21 (m, 1H), 1.14 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 160.4, 160.3, 159.6, 159.5, 157.8, 157.4, 153.6, 153.5, 153.3, 153.1, 147.5, 147.4, 143.2, 134.4, 134.3, 133.6, 130.0, 129.9, 123.2, 123.1, 119.7, 118.7, 118.5, 117.7, 112.6, 112.5, 111.7, 111.6, 103.4, 55.8, 55.6, 53.0, 52.1, 52.0, 51.5, 47.6, 47.3, 46.3, 45.7, 44.6, 28.7, 28.1, 27.4, 26.6, 18.1, 18.0, 9.2. HRMS (ESI) for C32H40N8O3 [M + H]+, calcd: 585.3296. Found: 585.3298. HPLC analysis: MeOH−H2O (85:15), 9.34 min, 99.42% purity. (S)-3-(4-Methoxyphenyl)-7-((3-methyl-4-(4-methylpiperazin1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ak). White solid 1 (yield 85%). [α]20 D −11.494 (c 0.174, MeOH). H NMR (400 MHz, CDCl3) δ 8.01 (s, 0.6H), 7.99 (s, 0.4H), 7.34−7.32 (m, 2H), 7.24− 7.21 (m, 2H), 7.02−6.99 (m, 2H), 6.95−6.92 (m, 2H), 5.57−5.49 (m, 1H), 4.59−4.57 (m, 2H), 4.06−4.04 (m, 0.6H), 3.95−3.93 (m, 1.4H), 3.81 (s, 3H), 3.73−3.71 (m, 0.6H), 3.46−3.43 (m, 1.6H), 2.93−2.91 (m, 4H), 2.82−2.77 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.30 (s, 3H), 2.24 (q, J = 7.6 Hz, 2H), 2.16−2.15 (m, 1H), 1.14 (t, J = 7.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.6, 159.5, 158.4, 158.3, 157.8, 157.4, 153.6, 153.4, 153.3, 147.5, 147.4, 134.9, 134.5, 134.4, 133.6, 126.9, 126.8, 123.2, 123.1, 119.6, 118.7, 118.5, 114.7, 103.4, 55.8, 55.7, 52.9, 52.1, 52.0, 51.3, 47.6, 47.2, 46.3, 45.8, 44.6, 28.7, 28.1, 27.4, 26.6, 18.1, 18.0, 9.2. HRMS (ESI) for C32H40N8O3 [M + H]+, calcd: 585.3296. Found: 585.3300. HPLC analysis: MeOH− H2O (85:15), 10.48 min, 97.65% purity. (S)-3-(3-(Difluoromethoxy)phenyl)-7-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3al). White 1 solid (yield 89%). [α]20 D −18.750 (c 0.096, MeOH). H NMR (400 MHz, CDCl3) δ 8.04 (s, 0.6H), 8.03 (s, 0.4H), 7.43−7.32 (m, 3H), 7.20−7.19 (m, 1H), 7.18−7.17 (m, 0.8H), 7.12−6.93 (m, 2.2H), 6.92 (s, 0.3H), 6.72 (s, 0.2H), 6.53 (s, 0.4H), 6.35 (s, 0.1H), 5.53−5.49 (m, 1H), 4.67−4.62 (m, 2H), 4.07−3.93 (m, 2H), 3.76−3.74 (m, 1H), 3.50−3.45 (m, 1H), 2.93−2.91 (m, 4H), 2.80−2.76 (m, 1H), 2.59 (br s, 4H), 2.36 (s, 3H), 2.31 (s, 3H), 2.30−2.28 (m, 2H), 2.26−2.22 (m, 1H), 1.15 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.7, 159.6, 159.6, 157.6, 157.2, 153.8, 153.6, 153.3, 153.0, 151.6, 147.6, 147.5, 143.4, 134.4, 134.3, 133.6, 130.3, 130.2, 123.3, 123.2, 122.2, 122.1, 119.7, 118.7, 118.6, 117.7, 117.6, 116.9, 116.8, 115.9, 113.8, 103.1, 55.8, 53.2, 52.1, 52.0, 51.6, 47.6, 47.1, 47.0, 46.3, 45.7, 44.6, 29.8, 28.7, 28.1, 27.4, 26.6, 18.1, 18.0, 9.2. HRMS (ESI) for

C32H38F2N8O3 [M + H]+, calcd: 621.3108. Found: 621.3105. HPLC analysis: MeOH−H2O (85:15), 10.34 min, 99.71% purity. (S)-7-((3-Methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3-(3-(trifluoromethoxy)phenyl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H) -one (3am). White 1 solid (yield 86%). [α]20 D -7.895 (c 0.152, MeOH). H NMR (400 MHz, CDCl3) δ 8.05 (s, 0.6H), 8.04 (s, 0.4H), 7.46−7.41 (m, 1H), 7.37− 7.34 (m, 2H), 7.33−7.29 (m, 1H), 7.21−7.18 (m, 1H), 7.15−7.13 (m, 1H), 7.08 (s, 0.6H), 7.02−6.98 (m, 1.4H), 5.53−5.49 (m, 1H), 4.68− 4.63 (m, 2H), 4.05−4.04 (m, 0.6H), 3.96−3.93 (m, 1.4H), 3.76−3.74 (m, 1H), 3.49−3.47 (m, 1H), 2.98−2.96 (m, 4H), 2.76−2.74 (m, 1H), 2.69 (br s, 4H), 2.45 (s, 3H), 2.32 (s, 3H), 2.24 (q, J = 7.2 Hz, 2H), 2.21−2.20 (m, 1H), 1.12 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.6, 172.3, 159.6, 159.5, 157.5, 157.1, 153.8, 153.6, 153.2, 152.9, 149.5, 147.2, 143.3, 143.2, 134.6, 134.4, 133.5, 130.2, 130.1, 123.5, 123.4, 123.2, 121.5, 119.6, 119.5, 118.9, 118.7, 118.6, 118.0, 117.4, 103.1, 103.0, 55.7, 53.1, 51.6, 51.5, 47.5, 47.1, 46.9, 46.0, 45.7, 44.5, 28.6, 28.0, 27.3, 26.5, 18.0, 17.9, 9.1. HRMS (ESI) for C32H37F3N8O3 [M + H]+, calcd: 639.3014. Found: 639.3020. HPLC analysis: MeOH−H2O (85:15), 11.14 min, 96.01% purity. (S)-3-(2-Fluoro-3-methoxyphenyl)-7-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3an). White 1 solid (yield 87%). [α]20 D −17.308 (c 0.104, MeOH). H NMR (400 MHz, CDCl3) δ 8.00 (s, 0.6H), 7.98 (s, 0.4H), 7.35−7.33 (m, 2H), 7.22 (s, 0.6H), 7.11−7.09 (m, 1.4H), 7.00−6.89 (m, 3H), 5.58−5.51 (m, 1H), 4.57−4.52 (m, 2H), 4.06−4.02 (m, 0.7H), 3.99−3.90 (m, 4.3H), 3.74−3.70 (m, 1H), 3.51−3.39 (m, 1H), 2.92 (br s, 4H), 2.78− 2.73 (m, 1H), 2.58 (br s, 4H), 2.36 (s, 3H), 2.28−2.24 (m, 5H), 2.22− 2.20 (m, 1H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.6, 159.5, 157.8, 157.4, 153.7, 153.6, 152.8, 152.5, 149.5, 149.0, 148.9, 147.5, 147.4, 134.4, 134.3, 133.5, 130.1, 124.2, 124.1, 123.2, 123.1, 119.9, 119.7, 118.7, 118.5, 112.9, 103.2, 103.1, 56.7, 55.8, 52.9, 52.1, 52.0, 51.3, 47.6, 47.2, 46.3, 45.7, 44.6, 29.8, 28.8, 27.4, 26.6, 18.1, 18.0, 9.1. HRMS (ESI) for C32H39FN8O3 [M + H]+, calcd: 603.3202. Found: 603.3197. HPLC analysis: MeOH−H2O (85:15), 8.95 min, 98.91% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ao). White 1 solid (yield 88%). [α]20 D −22.222 (c 0.100, MeOH). H NMR (400 MHz, CDCl3) δ 8.00 (s, 0.6H), 7.98 (s, 0.4H), 7.35−7.31 (m, 2H), 7.27 (s, 0.7H), 7.16 (s, 0.3H), 7.08−7.06 (m, 1H), 7.00−6.98 (m, 1H), 6.84−6.80 (m, 2H), 5.58−5.49 (m, 1H), 4.60−4.55 (m, 2H), 4.06− 4.02 (m, 0.6H), 3.96−3.92 (m, 1.4H), 3.78−3.70 (m, 4H), 3.49−3.38 (m, 1H), 2.93−2.91 (m, 4H), 2.82−2.73 (m, 1H), 2.59 (br s, 4H), 2.36 (s, 3H), 2.28−2.25 (m, 5H), 2.24−2.22 (m, 1H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.6, 159.5, 157.7, 157.4, 153.7, 153.6, 153.4, 152.8, 152.6, 151.5, 147.5, 147.4, 134.4, 134.3, 133.5, 129.7, 129.5, 123.2, 123.1, 119.6, 118.7, 118.5, 117.2, 117.0, 114.6, 114.5, 114.4, 114.3, 113.7, 113.6, 103.2, 103.1, 56.0, 55.8, 53.0, 52.1, 52.0, 51.3, 47.6, 47.2, 47.1, 46.3, 45.7, 44.6, 29.8, 28.7, 28.0, 27.4, 26.6, 18.1, 18.0, 9.1. HRMS (ESI) for C32H39FN8O3 [M + H]+, calcd: 603.3202. Found: 603.3195. HPLC analysis: MeOH−H2O (85:15), 10.23 min, 99.24% purity. (S)-3-(2-Chloro-3-methoxyphenyl)-7-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ap). White 1 solid (yield 89%). [α]20 D −22.973 (c 0.148, MeOH). H NMR (400 MHz, CDCl3) δ 8.03 (s, 0.6H), 8.01 (s, 0.4H), 7.39−7.29 (m, 3.3H), 7.03 (s, 0.7H), 7.00−6.97 (m, 3H), 5.60−5.52 (m, 1H), 4.66−4.63 (m, 1H), 4.51−4.48 (m, 1H), 3.98−3.94 (m, 5H), 3.77−3.75 (m, 1H), 3.47−3.38 (m, 1H), 2.96−2.94 (m, 4H), 2.78−2.76 (m, 1H), 2.62 (br s, 4H), 2.39 (s, 3H), 2.32−2.25 (m, 5H), 2.24−2.20 (m, 1H), 1.12 (t, J = 7.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.5, 159.4, 157.8, 156.6, 153.8, 153.7, 147.5, 140.5, 134.4, 133.6, 128.1, 123.3, 123.2, 121.0, 119.7, 118.6, 111.9, 103.1, 56.8, 55.8, 52.8, 52.1, 52.0, 51.2, 51.0, 47.6, 47.0, 46.3, 44.6, 29.1, 28.1, 27.4, 26.7, 18.1, 9.2. HRMS (ESI) for C32H39ClN8O3 [M + H]+, calcd: 619.2906. Found: 619.2885. HPLC analysis: MeOH−H2O (85:15), 10.65 min, 97.52% purity. 2364



Article

(S)-3-(2-Chloro-5-methoxyphenyl)-7-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3aq). White 1 solid (yield 83%). [α]20 D −41.071 (c 0.112, MeOH). H NMR (400 MHz, CDCl3) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.40−7.32 (m, 3H), 7.03−6.99 (m, 1.6H), 6.89 (s, 0.4H), 6.88−6.85 (m, 2H), 5.58−5.49 (m, 1H), 4.64−4.60 (m, 1H), 4.49−4.46 (m, 1H), 4.03−3.99 (m, 2H), 3.80 (s, 1H), 3.75−3.73 (m, 1H), 3.49−3.42 (m, 1H), 2.94−2.92 (m, 4H), 2.78−2.73 (m, 1H), 2.59 (br s, 4H), 2.37 (s, 3H), 2.30 (s, 3H), 2.26 (q, J = 7.6 Hz, 2H), 2.22−2.21 (m, 1H), 1.12 (t, J = 7.6 Hz, 3H). 13 C NMR (125 MHz, CDCl3) δ 172.6, 172.4, 159.6, 159.4, 157.7, 157.4, 153.7, 153.6, 152.5, 152.3, 152.1, 147.4, 147.2, 139.6, 134.4, 134.3, 133.4, 130.9, 124.1, 123.8, 123.2, 123.1, 119.5, 118.6, 118.5, 115.6, 115.3, 114.9, 114.8, 102.9, 55.8, 55.7, 52.7, 52.0, 51.9, 51.0, 47.5, 47.0, 46.2, 45.7, 44.5, 28.9, 28.7, 28.0, 27.3, 26.6, 18.1, 18.0, 9.1. HRMS (ESI) for C32H39ClN8O3 [M + H]+, calcd: 619.2906. Found: 619.2902. HPLC analysis: MeOH−H2O (85:15), 12.56 min, 97.72% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((4-(4-methylpiperazin1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ba). White solid 1 (yield 82%). [α]20 D +8.333 (c 0.072, MeOH). H NMR (400 MHz, CDCl3) δ 7.99 (s, 0.6H), 7.97 (s, 0.4H), 7.39−7.37 (m, 2H), 7.15 (s, 0.6H), 7.11−7.03 (m, 1.4H), 6.90−6.88 (m, 2H), 6.87−6.83 (m, 2H), 5.47 (q, J = 8.5 Hz, 1H), 4.60−4.51 (m, 2H), 4.09−4.05 (m, 0.6H), 3.92−3.91 (m, 1.4H), 3.78 (s, 3H), 3.71−3.66 (m, 1H), 3.54−3.52 (m, 1H), 3.42−3.37 (m, 1H), 3.19 (br s, 4H), 2.79−2.77 (m, 1H), 2.60 (br s, 4H), 2.36 (s, 3H), 2.24 (q, J = 7.2 Hz, 2H), 1.13 (t, J = 7.2 Hz, 3H). 13 C NMR (125 MHz, CDCl3) δ 172.6, 172.4, 159.8, 159.7, 157.6, 157.2, 156.1, 153.7, 153.5, 153.4, 152.9, 152.6, 151.4, 147.8, 147.7, 134.5, 134.4, 129.6, 129.5, 122.3, 121.8, 117.1, 116.9, 116.7, 116.5, 114.5, 114.4, 114.3, 113.7, 113.6, 102.8, 65.8, 55.9, 55.2, 55.1, 52.8, 51.2, 49.6, 49.4, 47.1, 47.0, 46.1, 46.0, 45.6, 44.4, 42.0, 30.2, 29.7, 29.2, 28.4, 27.9, 27.3, 26.2, 23.4, 23.1, 14.1, 11.1, 9.1. HRMS (ESI) for C31H37FN8O3 [M + H]+, calcd: 589.3045. Found: 589.3040. HPLC analysis: MeOH−H2O (85:15), 7.12 min, 97.35% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((2-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3bb). White 1 solid (yield 80%). [α]20 D +8.108 (c 0.074, MeOH). H NMR (400 MHz, CDCl3) δ 7.97 (s, 0.6H), 7.96 (s, 0.4H), 7.44−7.39 (m, 1H), 7.09−7.08 (m, 1H), 6.83−6.78 (m, 4H), 6.61 (s, 0.6H), 6.53 (s, 0.4H), 5.46−5.39 (m, 1H), 4.59−4.49 (m, 2H), 4.01−3.97 (m, 0.5H), 3.87− 3.85 (m, 1H), 3.79 (s, 3H), 3.63−3.61 (m, 1H), 3.54 (s, 0.5H), 3.39− 3.36 (m, 1H), 3.20 (br s, 4H), 2.72−2.70 (m, 1H), 2.60 (br s, 4H), 2.36 (s, 3H), 2.27 (s, 3H), 2.26−2.20 (m, 3H), 1.12 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.5, 172.4, 160.8, 160.7, 157.7, 157.5, 156.2, 153.9, 153.8, 153.0, 153.0, 152.8, 152.5, 151.5, 149.2, 133.6, 133.0, 129.7, 129.2, 128.9, 126.4, 125.9, 118.7, 117.2, 117.0, 114.5, 114.4, 114.3, 114.2, 113.7, 113.6, 102.7, 56.0, 55.3, 55.2, 52.7, 51.1, 49.4, 49.2, 47.2, 47.0, 46.7, 46.2, 46.1, 45.4, 44.3, 28.2, 28.0, 27.2, 26.1, 18.7, 9.2, 9.1. HRMS (ESI) for C32H39FN8O3 [M + H]+, calcd: 603.3202. Found: 603.3202. HPLC analysis: MeOH−H2O (85:15), 7.36 min, 96.26% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((2-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3bc). White solid (yield 83%). [α]20 D +9.836 (c 0.122, MeOH). 8.01−7.99 (m, 2H), 7.41−7.40 (m, 0.6H), 7.30−7.29 (m, 0.4H), 7.08−7.05 (m, 1H), 6.83−6.80 (m, 2H), 6.54−6.49 (m, 2H), 5.58−5.53 (m, 1H), 4.59− 4.51 (m, 2H), 4.16−4.12 (m, 1H), 4.00−3.96 (m, 4H), 3.88−3.68 (m, 4H), 3.48−3.44 (m, 1H), 3.18 (br s, 4H), 2.91 (s, 0.5H), 2.89 (s, 0.5H), 2.59 (br s, 4H), 2.35 (s, 3H), 2.27 (q, J = 7.5 Hz, 2H), 2.14− 2.11 (m, 1H), 1.12 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.7, 172.4, 160.0, 159.4, 157.6, 157.4, 156.2, 153.8, 153.6, 153.5, 153.0, 152.7, 151.5, 150.0, 147.9, 147.8, 129.7, 129.6, 123.7, 121.5, 120.9, 120.4, 117.2, 117.0, 114.6, 114.5, 114.4, 114.3, 113.7, 113.6, 108.1, 107.9, 102.9, 102.8, 100.6, 100.3, 56.0, 55.8, 55.3, 55.2, 52.9, 51.2, 50.1, 49.9, 47.3, 47.2, 46.8, 46.2, 45.8, 44.3, 32.0, 30.3, 29.7, 29.0, 28.4, 28.1, 27.7, 15.2, 9.1. HRMS (ESI) for C32H39FN8O4 [M + H]+,

calcd: 619.3151. Found: 619.3144. HPLC analysis: MeOH−H2O (85:15), 8.58 min, 97.14% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((3-methoxy-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2 (1H)-one (3bd). White 1 solid (yield 87%). [α]20 D −15.190 (c 0.158, MeOH). H NMR (400 MHz, CDCl3) δ 8.03 (s, 0.6H), 8.01 (s, 0.4H), 7.18−7.15 (m, 0.6H), 7.12−7.06 (m, 3H), 6.98 (s, 0.4H), 6.91−6.90 (m, 1H), 6.85−6.83 (m, 2H), 5.70−5.60 (m, 1H), 4.61−4.52 (m, 2H), 4.04−3.92(m, 2H), 3.87 (s, 3H), 3.78 (s, 3H), 3.76−3.71 (m, 1H), 3.50−3.38 (m, 2H), 3.11 (br s, 4H), 2.80−2.75 (m, 1H), 2.69 (br s, 4H), 2.40 (s, 3H), 2.28 (t, J = 7.6 Hz, 2H), 1.13 (t, J = 7.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.5, 159.6, 159.4, 157.7, 157.3, 156.2, 153.7, 153.6, 153.5, 152.9, 152.7, 152.5, 151.6, 137.4, 137.3, 134.6, 129.7, 129.5, 118.5, 117.2, 117.0, 114.6, 114.5, 114.4, 113.8, 113.7, 112.9, 112.5, 105.7, 104.8, 103.3, 56.0, 55.7, 55.6, 55.5, 52.9, 51.2, 50.9, 50.8, 47.9, 47.2, 47.1, 46.2, 45.7, 44.7, 32.0, 31.6, 30.3, 29.8, 29.5, 28.7, 28.1, 27.4, 26.7, 15.1, 9.0. HRMS (ESI) for C32H39FN8O4 [M + H]+, calcd: 619.3151. Found: 619.3095. HPLC analysis: MeOH−H2O (85:15), 7.64 min, 99.09% purity. (S)-7-((3-Fluoro-4-(4-methylpiperazin-1-yl)phenyl)amino)-3(2-fluoro-5-methoxyphenyl)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3be). White 1 solid (yield 86%). [α]20 D −10.938 (c 0.128, MeOH). H NMR (400 MHz, CDCl3) δ 8.04 (s, 0.6H), 8.02 (s, 0.4H), 7.55−7.54 (m, 0.5H), 7.51−7.50 (m, 0.5H), 7.19−7.07 (m, 3H), 6.93−6.89 (m, 1H), 6.85− 6.83 (m, 2H), 5.60−5.45 (m, 1H), 4.62−4.54 (m, 2H), 4.05−3.91 (m, 2H), 3.79−3.73 (m, 4H), 3.53−3.37 (m, 1H), 3.11 (br s, 4H), 2.84− 2.73 (m, 1H), 2.63 (br s, 4H), 2.37 (s, 3H), 2.27 (t, J = 7.6 Hz, 3H), 2.22−2.17 (m, 1H), 1.13 (t, J = 7.6 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 173.0, 172.6, 159.2, 159.1, 157.8, 157.4, 153.6, 153.5, 153.4, 152.8, 152.6, 151.5, 135.7, 135.6, 129.6, 129.5, 119.4, 119.3, 119.2, 119.1, 117.2, 117.0, 115.7, 114.6, 114.5, 114.4, 114.3, 113.7, 113.6, 108.9, 108.8, 108.7, 108.6, 103.7, 103.6, 56.0, 55.3, 53.1, 51.6, 50.9, 47.9, 47.2, 47.0, 46.2, 45.7, 44.8, 32.0, 31.6, 30.3, 29.8, 28.5, 28.1, 27.4, 26.7, 18.2, 18.1, 15.1, 9.1. HRMS (ESI) for C31H36F2N8O3 [M + H]+, calcd: 607.2951. Found: 607.2947. HPLC analysis: MeOH−H2O (85:15), 8.72 min, 98.92% purity. (S)-7-((3-Chloro-4-(4-methylpiperazin-1-yl)phenyl)amino)-3(2-fluoro-5-methoxyphenyl)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3bf). White 1 solid (yield 80%). [α]20 D −25.758 (c 0.132, MeOH). H NMR (500 MHz, CDCl3) δ 8.05 (s, 0.6H), 8.03 (s, 0.4H), 7.84−7.83 (m, 1H), 7.38−7.37 (m, 0.7H), 7.28−7.27 (m, 1.3H), 7.13−7.09 (m, 1H), 7.04−7.02 (m, 1H), 6.86−6.80 (m, 2H), 5.59−5.50 (m, 1H), 4.60− 4.57 (m, 2H), 4.06−3.95 (m, 2H), 3.81−3.80 (m, 4H), 3.54−3.43 (m, 1H), 3.07 (br s, 4H), 2.81−2.79 (m, 1H), 2.64 (br s, 4H), 2.38 (s, 3H), 2.32−2.22 (m, 3H), 1.13 (t, J = 5.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.9, 172.5, 159.2, 159.1, 157.9, 157.5, 156.2, 156.1, 153.7, 153.6, 153.5, 152.8, 152.4, 151.5, 144.8, 144.7, 135.2, 135.0, 129.6, 129.5, 129.1, 122.3, 122.2, 120.7, 120.6, 119.2, 119.1, 117.2, 117.0, 114.6, 114.5, 114.4, 114.3, 113.8, 113.7, 103.8, 103.7, 56.0, 55.4, 53.1, 51.6, 51.5, 51.4, 47.8, 47.2, 47.0, 46.2, 45.7, 44.6, 32.0, 31.6, 30.3, 29.8, 29.5, 28.7, 28.1, 27.4, 26.7, 18.8, 18.2, 18.1, 9.1. HRMS (ESI) for C31H36FN8O3 [M + H]+, calcd: 623.2656. Found: 623.2653. HPLC analysis: MeOH−H2O (85:15), 10.95 min, 97.73% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((4-(4-methylpiperazin1-yl)-3-(trifluoromethyl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3bg). White solid (yield 88%). [α]20 D −20.588 (c 0.068, MeOH). 1 H NMR (500 MHz, CDCl3) δ 8.05 (s, 0.6H), 8.03 (s, 0.4H), 8.00 (s, 1H), 7.64−7.63 (m, 0.4H), 7.58−7.56 (m, 0.4H), 7.54−7.53 (m, 0.6H), 7.52−7.49 (m, 1.6H), 7.10−7.08 (m, 1H), 6.84−6.83 (m, 2H), 5.60−5.50 (m, 1H), 4.60−4.58 (m, 2H), 4.23−4.22 (m, 0.2H), 4.00− 3.98 (m, 1.2H), 3.94−3.92 (m, 0.6H), 3.78−3.77 (m, 4H), 3.51−3.41 (m, 1H), 2.93 (br s, 4H), 2.80−2.70 (m, 0.5H), 2.68−2.66 (m, 0.5H), 2.57 (br s, 4H), 2.36 (s, 3H), 2.30−2.24 (m, 3H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 173.0, 172.6, 159.1, 159.0, 157.7, 157.5, 153.7, 156.2, 153.7, 153.6, 153.5, 152.7, 152.3, 151.6, 147.4, 147.3, 136.3, 129.5, 127.9, 125.0, 124.9, 124.1, 123.6, 123.5, 118.3, 117.2, 117.0, 114.7, 114.6, 114.5, 114.4, 113.8, 113.7, 103.2, 104.1, 2365



Article

151.5, 147.2, 147.0, 134.7, 134.6, 133.5, 129.9, 129.5, 123.2, 123.1, 119.5, 118.6, 118.5, 117.1, 117.0, 114.6, 114.5, 114.4, 114.3, 113.7, 113.6, 103.2, 103.1, 67.5, 56.0, 52.9, 52.6, 52.5, 51.3, 47.4, 47.2, 45.7, 44.5, 28.7, 28.0, 27.4, 26.5, 18.1, 18.0, 9.1. HRMS (ESI) for C31H36FN7O4 [M + H]+, calcd: 590.2886. Found: 590.2884. HPLC analysis: MeOH−H2O (85:15), 9.38 min, 96.49% purity. (S)-7-((4-(4-Acetylpiperazin-1-yl)-3-methylphenyl)amino)-3(2-fluoro-5-methoxyphenyl)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3bl). White 1 solid (yield 89%). [α]20 D -6.316 (c 0.190, MeOH). H NMR (500 MHz, CDCl3) δ 8.01 (s, 0.6H), 8.00 (s, 0.4H), 7.37−7.29 (m, 2H), 7.28 (s, 0.5H), 7.22 (s, 0.5H), 7.09−7.07 (m, 1H), 6.97−6.95 (m, 1H), 6.84−6.80 (m, 2H), 5.58−5.55 (m, 1H), 4.57−4.54 (m, 2H), 4.06− 4.04 (m, 0.6H), 3.93−3.92 (m, 1.4H), 3.78−3.71 (m, 6H), 3.60−3.58 (m, 2H), 3.56−3.54 (m, 1H), 3.13−3.11 (m, 2H), 2.78−2.59 (m, 3H), 2.32−2.30 (m, 3H), 2.28−2.21 (m, 3H), 2.21 (s, 3H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 169.3, 159.6, 159.5, 157.7, 157.3, 156.1, 153.7, 153.5, 153.4, 152.8, 152.5, 151.4, 148.0, 147.9, 134.1, 134.0, 133.5, 133.4, 129.6, 129.5, 123.2, 123.0, 119.4, 118.6, 118.3, 117.1, 116.9, 115.7, 114.5, 114.4, 114.3, 114.2, 113.7, 113.6, 102.9, 62.3, 62.2, 56.0, 52.9, 52.1, 51.3, 47.3, 47.1, 45.6, 44.5, 41.8, 29.2, 28.6, 27.9, 27.3, 26.4, 21.4, 18.0, 9.1. HRMS (ESI) for C33H39FN8O4 [M + H]+, calcd: 631.3151. Found: 631.3134. HPLC analysis: MeOH−H2O (85:15), 16.96 min, 98.90% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((3-methyl-4-(4-methyl1,4-diazepan-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2 (1H)-one (3bm). 1 White solid (yield 84%). [α]20 D −23.684 (c 0.076, MeOH). H NMR (500 MHz, CDCl3) δ 8.01 (s, 0.6H), 7.99 (s, 0.4H), 7.32−7.29 (m, 2H), 7.19−7.18 (m, 0.6H), 7.09−7.07 (m, 1.4H), 7.03−7.01 (m, 1H), 6.84−6.82 (m, 2H), 5.58−5.54 (m, 1H), 4.57−4.54 (m, 2H), 4.05− 3.96 (m, 0.6H), 3.94−3.92 (m, 1.4H), 3.80 (s, 3H), 3.78−3.72 (m, 1H), 3.69−3.54 (m, 1H), 3.19−3.17 (m, 2H), 3.15−3.12 (m, 2H), 2.79−2.72 (m, 5H), 2.41 (s, 3H), 2.30 (s, 3H), 2.29−2.21 (m, 3H), 1.99−1.93 (m, 2H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.4, 159.6, 159.5, 157.7, 157.4, 153.6, 153.4, 152.8, 149.9, 149.8, 133.9, 133.8, 133.5, 129.6, 129.5, 123.1, 123.0, 121.4, 121.1, 118.7, 118.5, 117.1, 117.0, 114.4, 114.3, 113.7, 113.6, 103.1, 103.0, 57.0, 56.0, 55.2, 55.0, 54.5, 54.4, 52.9, 51.3, 47.4, 47.2, 47.1,47.0, 45.7, 44.5, 29.8, 28.7, 28.4, 28.3, 28.0, 27.4, 26.5, 19.0, 18.9, 9.1. HRMS (ESI) for C33H41FN8O3 [M + H]+, calcd: 617.3358. Found: 617.3343. HPLC analysis: MeOH−H2O (85:15), 25.42 min, 99.41% purity. (S)-7-((4-(4-(Dimethylamino)piperidin-1-yl)-3methylphenyl)amino)-3-(2-fluoro-5-methoxyphenyl)-1-(1propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3bn). White solid (yield 77%). [α]20 D −13.636 (c 0.176, MeOH). 1H NMR (500 MHz, CDCl3) δ 8.00 (s, 0.6H), 7.98 (s, 0.4H), 7.37−7.36 (m, 0.6H), 7.35−7.30 (m, 1.4H), 7.28−7.27 (m, 0.8H), 7.09−7.07 (m, 1.2H), 6.97−6.95 (m, 1H), 6.86−6.82 (m, 2H), 5.56−5.51 (m, 1H), 4.56−4.54 (m, 2H), 4.06−4.04 (m, 0.6H), 3.93− 3.91 (m, 1.4H), 3.78−3.71 (m, 4H), 3.46−3.44 (m, 1H), 3.13−3.11 (m, 2H), 2.81−2.77 (m, 1H), 2.64−2.59 (m, 2H), 2.32 (s, 6H), 2.30− 2.24 (m, 2H), 2.23−2.21 (m, 1H), 1.92−1.89 (m, 2H), 1.67−1.65 (m, 2H), 1.31−1.13 (m, 3H). 13C NMR (125 MHz, CDCl3) δ 172.4, 159.5, 159.4, 157.7, 157.5, 156.1, 153.5, 153.4, 153.3, 152.8, 152.5, 151.5, 149.0, 148.9, 133.8, 133.7, 133.5, 133.4, 129.6, 129.4, 123.3, 123.0, 119.4, 118.7, 118.4, 117.1, 116.9, 115.7, 114.6, 114.5, 114.4, 114.3, 113.7, 113.6, 102.8, 56.0, 53,7, 53.6, 52.9, 51.3, 47.3, 47.1, 45.7, 44.5, 38.7, 29.7, 28.6, 28.0, 27.3, 26.7, 26.4, 24.5, 24.4, 18.0, 17.9, 9.3, 9.1. HRMS (ESI) for C34H43FN8O3 [M + H]+, calcd: 631.3515. Found: 631.3478. HPLC analysis: MeOH−H2O (85:15), 19.95 min, 96.89% purity. 3-(2-Fluoro-5-methoxyphenyl)-7-((3-methyl-4-(7-methyl2,7-diazaspiro[4.4]nonan-2-yl)phenyl)amino)-1-((S)-1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin2(1H)-one (3bo). White solid (yield 76%). [α]20 D -9.615 (c 0.104, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.00 (s, 0.6H), 7.98 (s, 0.4H), 7.23−7.20 (m, 2H), 7.10−6.93 (m, 1H), 6.85−6.80 (m, 4H), 5.58−5.51 (m, 1H), 4.57−4.55 (m, 2H), 4.06−3.91 (m, 2H), 3.79− 3.71 (m, 4H), 3.49−3.42 (m, 1H), 3.27−3.10 (m, 4H), 2.65−2.63 (m, 1H), 2.53−2.51 (m, 4H), 2.36 (s, 3H), 2.30 (s, 3H), 2. 28−2.23 (m,

80.2, 65.8, 56.0, 55.6, 53.6, 53.1, 51.6, 48.2, 47.2, 46.3, 45.7, 44.8, 33.3, 32.1, 31.8, 31.6, 31.4, 30.4, 29.8, 29.5, 28.7, 28.1, 27.4, 26.9, 26.8, 26.6, 18.2, 18.1, 9.1. HRMS (ESI) for C32H36F4N8O3 [M + H]+, calcd: 657.2919. Found: 657.2919. HPLC analysis: MeOH−H2O (85:15), 12.57 min, 98.79% purity. (S)-7-((3-Ethyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-3(2-fluoro-5-methoxyphenyl)-1-(1-propionylpyrrolidin-3-yl)3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3bh). White 1 solid (yield 89%). [α]20 D −14.063 (c 0.128, MeOH). H NMR (400 MHz, CDCl3) δ 8.03 (m, 0.6H), 8.01 (s, 0.4H), 7.41−7.32 (m, 2H), 7.10−7.07 (m, 2.6H), 6.96 (m, 0.4H), 6.85−6.84 (m, 2H), 5.61−5.55 (m, 1H), 4.58−4.56 (m, 2H), 4.08−3.92 (m, 2H), 3.79−3.65 (m, 4H), 3.50−3.41 (m, 1H), 2.92 (br s, 4H), 2.78−2.75 (m, 1H), 2.70 (q, J = 6.4 Hz, 2H), 2.60 (br s, 4H), 2.37 (s, 3H), 2.28 (q, J = 7.2 Hz, 2H), 2.24−2.22 (m, 1H), 1.24 (t, J = 6.4 Hz, 3H), 1.12 (t, J = 7.2 Hz, 3H). 13 C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.5, 159.4, 157.4, 156.2, 153.7, 153.6, 152.8, 152.5, 151.5, 147.0, 146.9, 140.3, 135.2, 135.1, 129.6, 129.5, 121.0, 120.9, 118.5, 118.4, 117.2, 117.0, 114.4, 114.3, 113.7, 113.6, 103.1, 56.0, 55.9, 55.8, 52.9, 52.8, 52.7, 51.3, 47.6, 47.2, 46.2, 46.1, 45.7, 44.6, 28.7, 28.0, 27.4, 26.6, 23.7, 19.1, 15.0, 13.8, 9.1. HRMS (ESI) for C33H41FN8O3 [M + H]+, calcd: 617.3358. Found: 617.3347. HPLC analysis: MeOH−H2O (85:15), 13.64 min, 96.90% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((3-isopropyl-4-(4methylpiperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3bi). 1 White solid (yield 83%). [α]20 D −21.429 (c 0.140, MeOH). H NMR (400 MHz, CDCl3) δ 8.02 (m, 0.6H), 8.00 (s, 0.4H), 7.44−7.28 (m, 3H), 7.11−7.06 (m, 2H), 6.85−6.81 (m, 2H), 5.63−5.59 (m, 1H), 4.60−4.57 (m, 2H), 4.07−3.98 (m, 1H), 3.95−3.86 (m, 2H), 3.78− 3.72 (m, 4H), 3.49−3.40 (m, 2H), 2.97 (br s, 4H), 2.86−2.74 (m, 4H), 2.37 (s, 3H), 2.33−2.23 (m, 2H), 2.22−2.21 (m, 1H), 1.21 (d, J = 2.4 Hz, 6H), 1.12 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.7, 159.3, 157.6, 153.7, 156.1, 153.6, 153.2, 152.7, 152.4, 151.3, 145.5, 145.4, 145.1, 136.3, 136.2, 129.5, 129.4, 121.3, 121.2, 118.3, 118.1, 118.0, 117.0, 114.7, 114.4, 113.5, 103.1, 103.0, 55.9, 55.4, 52.5, 52.1, 51.1, 47.4, 47.3, 47.0, 45.7, 45.2, 45.1, 44.5, 28.6, 27.9, 27.2, 26.9, 26.4, 24.0, 23.9, 9.1, 9.0. HRMS (ESI) for C34H43FN8O3 [M + H]+, calcd: 631.3515. Found: 631.3481. HPLC analysis: MeOH−H2O (85:15), 14.76 min, 98.01% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((3-methyl-4-(piperidin1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3bj). White solid 1 (yield 85%). [α]20 D -6.154 (c 0.260, MeOH). H NMR (500 MHz, CDCl3) δ 8.00 (s, 0.6H), 7.97 (s, 0.4H), 7.36−7.28 (m, 3H), 7.09 (s, 0.2H), 7.07−7.06 (m, 0.8H), 6.98−6.96 (m, 1H), 6.83−6.82 (m, 2H), 5.57−5.49 (m, 1H), 4.56−4.55 (m, 2H), 4.06−4.04 (m, 0.5H), 3.94− 3.91 (m, 1.5H), 3.78 (s, 3H), 3.77−3.72 (m, 1H), 3.50−3.38 (m, 1H), 2.81−2.77 (m, 5H), 2.37−2.30 (m, 0.3H), 2.29 (s, 3H), 2.24 (q, J = 7.0 Hz, 2H), 2.19−2.15 (m, 0.7H), 1.71−1.69 (m, 4H), 1.56−1.55 (m, 2H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.8, 172.4, 159.5, 159.4, 157.7, 157.5, 156.1, 153.5, 153.4, 153.2, 152.8, 152.5, 151.5, 149.0, 148.9, 133.8, 133.7, 133.5, 133.4, 129.6, 129.4, 129.3, 123.3, 123.0, 119.4, 118.7, 118.4, 117.4, 116.9, 115.6, 114.5, 114.4, 114.3, 113.7, 113.6, 56.0, 53.7, 53.6, 52.9, 51.3, 47.3, 47.1, 45.6, 44.7, 38.7, 29.7, 28.6, 27.9, 27.3, 26.7, 26. 4, 24.5, 24.4, 18.0, 17.9, 9.3, 9.0. HRMS (ESI) for C32H38FN7O3 [M + H]+, calcd: 588.3093. Found: 588.3037. HPLC analysis: MeOH−H2O (85:15), 11.44 min, 96.03% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((3-me thyl-4morpholinophenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3bk). White solid 1 (yield 86%). [α]20 D −17.143 (c 0.140, MeOH). H NMR (500 MHz, CDCl3) δ 8.01 (s, 0.6H), 7.99 (s, 0.4H), 7.38−7.31 (m, 2.7H), 7.09− 7.07 (m, 0.3H), 7.06−7.05 (m, 1H), 7.00−6.97 (m, 1H), 6.84−6.81 (m, 2H), 5.57−5.51 (m, 1H), 4.57−4.55 (m, 2H), 4.07−4.03 (m, 0.6H), 3.96−3.92 (m, 1.4H), 3.85−3.83 (m, 4H), 3.78−3.77 (m, 3H), 3.74−3.72 (m, 1H), 3.43−3.41 (m, 1H), 2.89−2.87 (m, 4H), 2.79− 2.77 (m, 1H), 2.31 (s, 3H), 2.28 (q, J = 12.0 Hz, 2H), 2.23−2.21 (m, 1H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.4, 159.5, 159.4, 157.7, 157.4, 156.1, 153.6, 153.5, 153.4, 152.8, 152.5, 2366



Article

3H), 2.00−1.90 (m, 4H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.3, 159.8, 157.4, 156.1, 153.8, 153.6, 152.9, 145.7, 131.6, 129.3, 124.7, 124.5, 119.2, 118.9, 117.1, 117.0, 115.9, 115.8, 114.4, 113.7, 113.6, 102.7, 68.1, 68.0, 63.9, 63.8, 56.3, 56.0, 52.9, 51.3, 50.7, 50.6, 48.6, 47.5, 47.2, 47.1, 45.7, 44.4, 38.9, 37.8, 37.7, 37.6, 28.7, 28.0, 27.4, 26.4, 20.6, 20.5, 9.1. HRMS (ESI) for C35H43FN8O3 [M + H]+, calcd: 643.3515. Found: 643.3512. HPLC analysis: MeOH−H2O (85:15), 19.29 min, 97.11% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((3-methyl-4-(9-methyl3,9-diazaspiro[5.5]undecan-3-yl)phenyl)amino)-1-(1propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3bp). White solid (yield 78%). [α]20 D −15.789 (c 0.076, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.01 (s, 0.6H), 7.99 (s, 0.4H), 7.38−7.30 (m, 2H), 7.10−6.95 (m, 3H), 6.85−6.82 (m, 2H), 5.56−5.52 (m, 1H), 4.58−4.55 (m, 2H), 4.06−3.92 (m, 2H), 3.79−3.72 (m, 4H), 3.47−3.45 (m, 1H), 2.82−2.80 (m, 5H), 2.39− 2.32(m, 4H), 2.29−2.21 (m, 9H), 1.62−1.61 (m, 8H), 1.12 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.3, 159.6, 159.5, 157.7, 157.4, 156.1, 153.7, 153.6, 153.6, 152.8, 152.5, 148.4, 148.3, 134.0, 133.9, 133.4, 133.3, 123.3, 123.0, 119.3, 118.6, 118.3, 117.1, 117.0, 114.4, 114.3, 113.7, 113.6, 103.0, 102.9, 56.0, 52.9, 51.4, 51.3, 48.2, 48.1, 47.4, 47.2, 47.1, 46.4, 45.6, 44.5, 36.1, 28.7, 28.6, 28.0, 27.3, 26.5, 18.0, 17.9, 9.1. HRMS (ESI) for C37H47FN8O3 [M + H]+, calcd: 671.3828. Found: 671.3833. HPLC analysis: MeOH−H2O (85:15), 34.80 min, 99.86% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((3-methyl-4-(7-methyl2,7-diazaspiro[3.5]nonan-2-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3bq). White solid (yield 89%). [α]20 D -4.819 (c 0.166, MeOH). 1 H NMR (500 MHz, CDCl3) δ 8.01 (s, 0.6H), 7.99 (s, 0.4H), 7.36− 7.30 (m, 2H), 7.12−7.10 (m, 1H), 7.08−6.91 (m, 2H), 6.83−6.83 (m, 2H), 5.58−5.51 (m, 1H), 4.58−4.55 (m, 2H), 4.07−3.92 (m, 2H), 3.79−3.71 (m, 4H), 3.49−3.42 (m, 1H), 3.09−3.08 (m, 4H), 2.79− 2.75 (m, 5H), 2.36 (s, 3H), 2. 92−2.16 (m, 6H), 1.89−1.86 (m, 4H), 1.12 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.3, 159.6, 159.5, 157.7, 157.4, 156.1, 153.7, 153.6, 153.5, 152.8, 152.6, 151.5, 148.2, 134.1, 133.6, 129.6, 123.2, 122.9, 119.5, 118.6, 118.3, 117.2, 117.0, 114.4, 113.7, 113.6, 103.0, 66.6, 56.0, 52.9, 51.3, 49.8, 47.5, 47.2, 46.4, 45.7, 44.5, 37.0, 33.9, 28.7, 28.0, 27.4, 26.5, 18.0, 9.1. HRMS (ESI) for C35H43FN8O3 [M + H]+, calcd: 643.3515. Found: 643.3516. HPLC analysis: MeOH−H2O (85:15), 27.79 min, 96.23% purity. 3-(2-Fluoro-5-methoxyphenyl)-7-((3-methyl-4-((3aR,6aS)-5methylhexahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)phenyl)amino)1-((S)-1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3br). White solid (yield 79%). [α]20 D −10.000 (c 0.140, MeOH). 1H NMR (500 MHz, CDCl3) δ 8.01 (s, 0.6H), 7.99 (s, 0.4H), 7.32−7.25 (m, 2H), 7.09−6.93 (m, 3H), 6.83−6.82 (m, 2H), 5.57−5.51 (m, 1H), 4.57−4.55 (m, 2H), 4.07−3.92 (m, 2H), 3.79−3.71 (m, 4H), 3.47−3.41 (m, 1H), 2.98−2.77 (m, 9H), 2.35 (s, 3H), 2.31 (s, 3H), 2.28−2.15 (m, 5H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.3, 159.6, 159.5, 157.7, 157.4, 156.1, 156.0, 153.7, 153.6, 152.8, 152.5, 151.5, 144.2, 133.6, 133.5, 132.3, 129.6, 129.5, 123.6, 123.4, 118.7, 118.5, 117.1, 117.0, 114.4, 114.3, 113.7, 113.6, 102.9, 62.5, 62.4, 56.5, 56.4, 56.3, 56.0, 52.9, 51.3, 47.3, 47.2, 45.7, 44.4, 42.5, 41.8, 28.7, 28.0, 27.4, 26.4, 19.3, 19.2, 9.1. HRMS (ESI) for C34H41FN8O3 [M + H]+, calcd: 629.3358. Found: 629.3356. HPLC analysis: MeOH−H2O (85:15), 20.56 min, 99.45% purity. (S)-7-((4-((2-(Dimethylamino)ethyl)(methyl)amino)-3methylphenyl)amino)-3-(2-fluoro-5-methoxyphenyl)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin2(1H)-one (3bs). White solid (yield 88%). [α]20 D −18.519 (c 0.108, MeOH). 1H NMR (400 MHz, CDCl3) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.39−7.37 (m, 0.7H), 7.33−7.32 (m, 1.3H), 7.10−7.06 (m, 2.6H), 7.04−7.03 (m, 0.4H), 6.85−6.83 (m, 2H), 5.57−5.53 (m, 1H), 4.57−4.56 (m, 2H), 4.05−3.93 (m, 2H), 3.79−3.72 (m, 4H), 3.55− 3.45 (m, 2H), 3.03−2.99 (m, 2H), 2.79−2.77 (m, 1H), 2.68 (s, 3H), 2.48−2.44 (m, 2H), 2.30 (s, 3H), 2.26−2.25 (m, 8H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.4, 159.5, 159.4, 157.3, 156.1, 153.6, 153.5, 152.8, 147.8, 134.3, 134.1, 129.6, 129.5, 122.9, 122.8, 120.5, 118.4, 118.2, 117.1, 116.9, 114.4, 114.3, 113.7, 113.6, 103.0, 65.1, 57.5, 57.4, 55.9, 54.6, 54.6, 52.8, 51.3, 47.4, 47.2, 47.1,

45.8, 45.7, 45.6, 44.5, 42.9, 42.7, 42.1, 30.2, 29.7, 28.2, 28.7, 28.0, 27.3, 26.4, 23.4, 23.1, 18.4, 18.3, 14.1, 11.1, 9.1. HRMS (ESI) for C32H41FN8O3 [M + H]+, calcd: 605.3358. Found: 605.3356. HPLC analysis: MeOH−H2O (85:15), 14.95 min, 99.79% purity. 7-((4-((S)-3,4-Dimethylpiperazin-1-yl)-3-methylphenyl)amino)-3-(2-fluoro-5-methoxyphenyl)-1-((S)-1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3bt). White solid (yield 86%). 1H NMR (400 MHz, CDCl3) δ 8.00 (s, 0.6H), 7.98 (s, 0.4H), 7.33−7.31 (m, 2.5H), 7.18−7.17 (m, 0.5H), 7.10−7.08 (m, 1H), 7.06−6.98 (m, 1H), 6.83−6.82 (m, 2H), 5.55−5.52 (m, 1H), 4.56−4.54 (m, 2H), 4.05−4.01 (m, 0.6H), 3.95− 3.91 (m, 1.4H), 3.77−3.69 (m, 4H), 3.45−3.40 (m, 1H), 2.98−2.95 (m, 4H), 2.91−2.86 (m, 1H), 2.78−2.72 (m, 3H), 2.56 (s, 3H), 2.50 (s, 3H), 2.47−2.20 (m, 3H), 1.12 (m, 6H). 13C NMR (125 MHz, CDCl3) δ 172.7, 172.4, 159.5, 159.4, 157.6, 157.3, 156.0, 153.6, 153.5, 153.4, 152.7, 152.5, 147.1, 134.4, 134.3, 133.3, 129.5, 129.4, 129.3, 123.2, 123.1, 123.0, 119.4, 118.6, 118.5, 118.4, 117.0, 116.9, 115.7, 114.3, 114.2, 113.6, 113.5, 102.9, 59.2, 59.1, 58.2, 55.9, 52.8, 52.2, 52.1, 51.2, 47.4, 47.1, 45.6, 44.4, 42.6, 42.5, 28.6, 27.9, 27.3, 26.4, 18.0, 17.9, 9.0. HRMS (ESI) for C33H41FN8O3 [M + H]+, calcd: 617.3358. Found: 617.3344. HPLC analysis: MeOH−H2O (85:15), 6.9 min, 99.24% purity. HRMS (ESI) for C33H41FN8O3 [M + H]+, calcd: 617.3358. Found: 617.3344. HPLC analysis: MeOH−H2O (85:15), 6.9 min, 99.24% purity. 3-(2-Fluoro-5-methoxyphenyl)-7-((3-methyl-4-((3R,5S)-3,4,5trimethylpiperazin-1-yl)phenyl)amino)-1-((S)-1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one 1 (3bu). White solid (yield 88%). [α]20 D −26.684 (c 0.076, MeOH). H NMR (500 MHz, CDCl3) δ 8.01 (s, 0.6H), 7.99 (s, 0.4H), 7.32−7.27 (m, 2H), 7.18−7.17 (m, 0.6H), 7.11−7.10 (m, 0.4H), 7.08−7.05 (m, 1H), 6.96−6.94 (m, 1H), 6.84−6.83 (m, 2H), 5.57−5.52 (m, 1H), 4.59−4.55 (m, 2H), 4.05−4.02 (m, 0.7H), 3.95−3.91 (m, 1.3H), 3.88 (s, 3H), 3.78−3.72 (m, 1H), 3.50−3.40 (m, 1H), 2.95−2.94 (m, 2H), 2.79−2.78 (m, 2H), 2.77−2.73 (m, 4H), 2.46 (s, 3H), 2.30 (s, 3H), 2.28−2.13 (m, 2H), 1.28−1.25 (m, 6H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.6, 159.5, 157.7, 157.4, 156.1, 153.7, 153.6, 153.4, 152.8, 152.6, 151.5, 146.5, 146.4, 134.8, 134.6, 133.5, 133.4, 129.6, 129.5, 123.1, 123.0, 119.5, 118.7, 118.5, 117.1, 117.0, 114.4, 114.3, 113.7, 113.6, 103. 1, 56.0, 52.9, 51.3, 47.5, 47.3, 47.2, 45.7, 44.6, 29.8, 28.7, 28.0, 27.4, 26.5, 18.1, 18.0, 17.5, 17.4, 9.2, 9.1. HRMS (ESI) for C34H43FN8O3 [M + H]+, calcd: 631.3515. Found: 631.3513. HPLC analysis: MeOH−H2O (85:15), 15.06 min, 98.49% purity. (S)-3-(2-Fluoro-5-methoxyphenyl)-7-((3-methyl-4-(4-(oxetan-3-yl)piperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one 1 (3bv). White solid (yield 84%). [α]20 D −14.063 (c 0.128, MeOH). H NMR (400 MHz, CDCl3) δ 8.02 (s, 0.6H), 8.00 (s, 0.4H), 7.37−7.34 (m, 2H), 7.11−7.10 (m, 1.7H), 7.08−7.00 (m, 1.3H), 6.85−6.82 (m, 2H), 5.56−5.52 (m, 1H), 4.71−4.70 (m, 4H), 4.68−4.55 (m, 2H), 4.05−4.00 (m, 0.6H), 3.94−3.92 (m, 1.4H), 3.79−3.72 (m, 4H), 3.60−3.58 (m, 1H), 3.57−3.56 (m, 1H), 2.96−2.94 (m, 4H), 2.79− 2.78 (m, 1H), 2.50 (br s, 4H), 2.28−2.25 (m, 5H), 2.29−2.21 (m, 1H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.3, 159.5, 159.4, 157.7, 157.3, 156.1, 156.0, 153.7, 153.5, 153.4, 152.8, 152.5, 151.4, 147.2, 147.0, 134.5, 133.4, 129.6, 129.5, 123.1, 123.0, 119.6, 118.6, 118.5, 117.1, 116.9, 114.3, 114.2, 113.7, 113.6, 103.0, 59.3, 55.9, 52.9, 51.6, 51.5, 51.3, 50.2, 47.4, 47.1, 45.6, 44.4, 28.6, 28.0, 27.3, 26.4, 18.0, 17.9, 9.1. HRMS (ESI) for C34H41FN8O4 [M + H]+, calcd: 645.3308. Found: 645.3316. HPLC analysis: MeOH−H2O (85:15), 7.99 min, 96.27% purity. (S)-3-(2-Chloro-5-methoxyphenyl)-7-((3-methyl-4-(4-(oxetan-3-yl)piperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one 1 (3bw). White solid (yield 88%). [α]20 D −46.000 (c 0.100, MeOH). H NMR (400 MHz, CDCl3) δ 8.01 (s, 0.6H), 8.00 (s, 0.4H), 7.38−7.32 (m, 3H), 7.20−7.19 (m, 0.6H), 7.03−7.02 (m, 0.4H), 7.00−6.98 (m, 1H), 6.89−6.83 (m, 2H), 5.58−5.56 (m, 1H), 4.71−4.60 (m, 5H), 4.49−4.45 (m, 1H), 4.05−3.90 (m, 2H), 3.80 (s, 3H), 3.79−3.74 (m, 1H), 3.71−3.69 (m, 1H), 3.60−3.40 (m, 1H), 2.96−2.94 (m, 4H), 2.79−2.78 (m, 1H), 2.49 (br s, 4H), 2.28−2.25 (m, 5H), 2.29−2.21 2367



Article

(m, 1H), 1.13 (t, J = 7.2 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 172.6, 172.3, 159.5, 159.4, 159.3, 153.7, 153.6, 147.2, 139.6, 134.6, 134.5, 133.5, 130.9, 123.9, 123.1, 119.7, 118.5, 115.3, 115.2, 115.0, 114.9, 103.0, 59.4, 55.8, 52.8, 51.7, 51.6, 51.1, 50.2, 47.5, 47.0, 45.8, 44.5, 29.8, 28.0, 27.4, 26.6, 18.0, 17.9, 9.1. HRMS (ESI) for C34H41ClN8O4 [M + H]+, calcd: 661.3012. Found: 661.2989. HPLC analysis: MeOH−H2O (85:15), 8.44 min, 99.74% purity. (S)-tert-Butyl-3-((5-formyl-2-(methylthio)pyrimidin-4-yl)amino)pyrrolidine-1-carboxylate (6a). Compound 5a (22.0 g, 118.2 mmol) and potassium carbonate (29.64 g, 214.8 mmol) were added to a solution of 4a (24.98 g, 107.4 mmol) in DMF (50 mL). The resulting solution was stirred at 60 °C overnight. After cooling to room temperature, ice−water (500 mL) was added to the reaction mixture. The precipitate was filtered, and the filter cake was rinsed with additional cold water and then dried in a vacuum oven to give 6a (white solid, 30.4 g, yield 74%), which was used without further purification. 1H NMR (400 MHz, CDCl3) δ 8.63 (s, 1H), 8.34 (s, 1H), 4.72 (s, J = 2.4 Hz, 1H), 4.31 (q, J = 7.2 Hz, 2H), 3.77−3.73 (m, 1H), 3.49−3.48 (m, 2H), 3.36−3.34 (m, 0.5H), 3.23−3.22 (m, 0.5H), 2.52 (s, 3H), 2.30−2.21 (m, 1H), 1.94−1.93 (m, 1H), 1.45 (s, 9H), 1.36 (t, J = 7.2 Hz, 3H). MS (ESI) m/z 383.2 [M + H]+. (S)-tert-Butyl-3-((5-(hydroxymethyl)-2-(methylthio)pyrimidin-4-yl)amino)pyrrolidine-1-carboxylate (7a). A suspension of LiAlH4 (6.04 g, 158.3 mmol) in anhydrous THF (50 mL) was added dropwise to a solution of 6a (30.4 g, 79.15 mmol) in anhydrous THF (200 mL) under stirring for 0.5 h at −40 °C. The reaction mixture was stirred until the temperature was slowly warmed to 0 °C, and then 6 mL of water was added to decompose unreacted LiAlH4. Then, a further 18 mL of water was added to the reaction mixture followed by 15% aqueous NaOH (6 mL). The resulting mixture was stirred for 10 min at room temperature and filtered through a pad of Celite. The Celite was washed with THF, and the washes were combined and then concentrated in vacuo. The concentrated mixture was poured into water and extracted with CH2Cl2. The organic layer was separated, washed with brine, dried with Na2SO4. The crude material was purified by column chromatography (SiO2, CH2Cl2/ MeOH stepwise elution, 40:1 to 20:1) to give 6a (white solid, 13 g, yield 48%). 1H NMR (400 MHz, CDCl3) δ 7.74 (s, 1H), 6,02 (s, 1H), 4.68 (d, J = 5.6 Hz, 1H), 4.52 (s, 2H), 3.72−3.71 (m, 1H), 3.46−3.45 (m, 2H), 3.27−3.21 (m, 1H), 2.50 (s, 3H), 2.25−2.23 (m, 1H), 1.92− 1.91 (m, 1H), 1.46 (s, 9H). MS (ESI) m/z 341.2 [M + H]+. (S)-tert-Butyl-3-((5-formyl-2-(methylthio)pyrimidin-4-yl)amino)pyrrolidine-1-carboxylate (8a). MnO2 (16.6 g, 191.10 mmol) was added to a solution of 7a (13 g, 38.2 mmol) in CH2Cl2 (100 mL). The mixture was stirred at room temperature overnight. The mixture was filtered and the filtrate was concentrated in vacuo and further purified by flash chromatography on silica gel (SiO2, petroleum ether/EtOAc stepwise elution, 3:1 to 1:1) to give 8a (white solid, 11.2 g, yield 87%). 1H NMR (400 MHz, CDCl3) δ 9.78 (s, 1H), 8.64 (s, 1H), 8.30 (s, 1H), 4.72 (s, 1H), 3.73−3.72 (m, 1H), 3.47−3.46 (m, 2H), 3.73−3.72 (m, 0.5H), 3.34−3.21 (m, 0.5H), 2.52 (s, 3H), 2.26− 2.23 (m, 1H), 1.97−1.96 (m, 1H), 1.44 (s, 9H). MS (ESI) m/z 339.2 [M + H]+. (S)-tert-Butyl-3-((5-(((2-chlorophenyl)amino)methyl)-2(methylthio)pyrimidin-4-yl)amino)pyrrolidine-1-carboxylate (9a). 2-Chloroaniline (1.51 g, 11.84 mmol) and AcOH (0.1 mL) were added at room temperature to a solution of 8a (2.0 g, 5.92 mmol) in toluene (50 mL). After the amine was converted completely to the imine as determined by TLC, NaBH4 (1.119 g, 29.6 mmol) was added to the reaction mixture at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 4 h. The resulting mixture was concentrated in vacuo, partitioned between CH2Cl2 and saturated NaHCO3. The organic layer was separated, and washed with brine, dried with Na2SO4. The resulting crude material was subjected to flash column chromatography (SiO2, petroleum ether/EtOAc, 3:1) to give 9a (white solid, 1.86 g, yield 70%). 1H NMR (400 MHz, CDCl3) δ 7.96 (s, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.21−7.17 (m, 1H), 6.81−6.77 (m, 2H), 5.98 (s, 1H), 4.71−4.64 (m, 1H), 4.20−4.10 (m, 3H), 3.72− 3.70 (m, 1H), 3.43−3.42 (m, 1H), 3.31−3.30 (m, 0.5H), 3.15−3.14

(m, 0.5H), 2.52 (s, 3H), 2.23−2.15 (m, 1H), 1.84−1.83 (m, 1H), 1.43 (s, 9H). MS (ESI) m/z 450.2 [M + H]+. (S)-tert-Butyl-3-(3-(2-chlorophenyl)-7-(methylthio)-2-oxo3,4-dihydropyrimido[4,5-d]pyrimidin-1(2H)-yl)pyrrolidine-1carboxylate (10a). DIEA (2.2 mL, 12.42 mmol) and triphosgene (0.49 g, 1.66 mmol) were added to a solution of 9a (1.86 g, 4.14 mmol) in CH2Cl2 at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 0.5 h. The mixture was poured into a solution of 10% aqueous NaHCO3 and extracted with CH2Cl2. The organic layer was washed with 10% aqueous NaHCO3, brine, and dried with Na2SO4. Then the organic phase was concentrated in vacuo to give 10a (yellow oil, 1.90 g, yield 97%), which was used without further purification. 1H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.36−7.31 (m, 3H), 5.59−5.54 (m, 1H), 4.70− 4.66 (m, 1H), 4.56−4.51 (m, 1H), 3.88−3.83 (m, 1H), 3.72−3.67 (m, 2H), 3.44−3.39 (m, 1H), 2.77−2.71 (m, 1H), 2.54 (s, 3H), 2.15−2.14 (m, 1H), 1.45 (s, 9H). MS (ESI) m/z 476.1 [M + H]+. (S)-tert-Butyl-3-(3-(2-chlorophenyl)-7-(methylsulfonyl)-2oxo-3,4-dihydropyrim ido[4,5-d]pyrimidin-1(2H)-yl)pyrrolidine-1-carboxylate (11a). m-CPBA (1.74 g, 8.66 mmol) was added to a solution of 10a (1.9 g, 4.33 mmol) in anhydrous CH2Cl2 (50 mL) at 0 °C under argon. The reaction mixture was allowed to warm to room temperature and stirred for 4 h. The solution was diluted with CH2Cl2 and then treated with 50% Na2S2O3/NaHCO3 solution. The organic layer was separated, washed with brine, dried with Na2SO4, and concentrated in vacuo. The resulting crude material was subjected to flash column chromatography (SiO2, CH2Cl2/MeOH stepwise elution, 40:1 to 20:1) to give 11a (white solid, 1.71g, yield 78%). 1H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H), 7.53 (d, J = 6.0 Hz, 1H), 7.39−7.38 (m, 3H), 5.64−5.59 (m, 1H), 4.87−4.82 (m, 1H), 4.72−4.67 (m, 1H), 3.80−3.79 (m, 3H), 3.78−3.74 (m, 1H), 3.33 (s, 3H), 2.68−2.67 (m, 1H), 2.25−2.24 (m, 1H), 1.45 (s, 9H). MS (ESI) m/z 507.1 [M + H]+. (S)-3-(2-Chlorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1yl)phenyl)amino)-1-(pyrrolidin-3-yl)-3,4-dihydropyrimido[4,5d]pyrimidin-2(1H)-one (13a). 2-Methyl-4-(4-methylpiperazin-1-yl)aniline (172 mg, 0.84 mmol) was added to a solution of 11a (3 27 mg, 0.64 mmol) in 2-butyl alcohol (5 mL), followed by trifluoroacetic acid (62 μL, 0.84 mmol). The reaction mixture was stirred for 18 h at 110 °C in a sealed tube. The reaction mixture was cooled to room temperature and concentrated in vacuo to give crude product 12a which was used in the next step without further characterization. Then trifluoroacetic acid (2 mL) was added to a solution of 12a in CH2Cl2 (5 mL), and the resulting mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with CH2Cl2, and basified with saturated aqueous NaHCO3 to pH = 9. The organic layer was separated and washed with 10% aqueous NaHCO3, brine, dried over Na2SO4, and then concentrated in vacuo. The resultant crude material was purified by column chromatography (SiO2, CH2Cl2/MeOH/ NH4OH, 40:1:0.4) to give 13a (white solid, 150 mg, yield 44% for twosteps). 1H NMR (400 MHz, CDCl3) δ 7.98 (s, 1H), 7.51 (d, J = 7.2 Hz, 1H), 7.42 (s, 1H), 7.37−7.31 (m, 4H), 7.10 (s, 1H), 7.04−7.02 (m, 1H), 5.49−5.47 (m, 1H), 4.73−4.66 (m, 1H), 4.50−4.45 (m, 1H), 3.30−3.27 (m, 2H), 3.09−3.04 (m, 1H), 2.94−2.92 (m, 4H), 2.74− 2.69 (m, 1H), 2.67−2.65 (m, 4H), 2.36 (s, 3H), 2.32 (s, 3H), 1.89− 1.88 (m, 2H). MS (ESI) m/z 533.3 [M + H]+. Reagents and Antibodies. The compound was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at a concentration of 10 mmol/L and stored at −20 °C. Primary antibodies against CSF1R (3152), phosphor-CSF1R (Tyr723, 3151), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 2118), and anti-rabbit or anti-mouse IgG horseradish peroxidase (HRP)-linked secondary antibodies were purchased from Cell Signaling Technology (CST, Boston, MA, USA). Primary antibodies against akt (AKT, SC8312), phosphor-AKT (SC16646R) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MDV3100 was purchased from ACROS Organic Company, and CSF1R protein, EGFR protein, and the Z′-Lyte kinase assay kit were purchased from Invitrogen. Cell Lines. The murine macrophage RAW264.7 (RAW) cells (ATCC) were cultured in DMEM, while LNCaP (ATCC) cells were 2368



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In Vitro Migration Assay. Cell migration assays were evaluated in transwell chambers (BD Falcon). First, (2−4) × 105 RAW264.7 macrophage cells (200 μL) were added in the hanging cell culture insert (BD Falcon: 353097) without FBS. A total of 800 μL of the conditioned media from LNCaP cells treated with 10 μM MDV3100 or DMSO vehicle was added in the lower chamber. After incubation for 24 h at 37 °C, migrated cells were immediately fixed in 100% methanol and stained with 0.25% crystal violet, and the cells that had not migrated from the top surface of the filters were removed with cotton. Migrated cells were quantitated by counting cells in six randomly selected fields on each filter under a microscope at 200× magnification and graphed as the mean of three independent experiments. To block CSF1 signaling, four doses of 3bw of 0.625, 1.25, 2.5, and 5.0 μM were added to the top chamber, and macrophage cells in the hanging insert were incubated for 24 h. The migrating cells were counted as described above. The experiments shown are representative of three independent experiments. Real-Time RT-PCR. RAW264.7 macrophage cells were treated with 3bw of 0.08, 0.4, and 2.0 μM or DMSO vehicle for 48 h. Total RNA was extracted using TRIzol (Takara, Japan) according to TRIzol protocol. One microgram of total RNA was used for complementary DNA synthesis using a cDNA reverse transcription kit (Takara, Japan). Real-time PCR was performed in triplicate using gene-specific primers on a Stratagene Bio-Rad CFX 3.1 real-time PCR systems (Bio-Rad Laboratories) machine. The mRNA expression levels were normalized to GAPDH. All analysis was performed using Microsoft Excel 2010 and GraphPad Prism 5 software. The experiment was performed three times and in triplicate. The gene-specific primers are listed in Supporting Information Table 1. In Vitro Coculture Assay. Cell coculture assays were evaluated in transwell chambers (BD Falcon). First, (2−4) × 105 LNCaP cells were seeded in the lower chamber (BD Falcon: 353502). After incubation for 2 h at 37 °C, (1−2) × 106 RAW264.7 cells were seeded in the 0.4 μm hanging cell culture insert (BD Falcon: 353090). After coculture for 48 h at 37 °C, the top chamber was removed, and the images of the bottom LNCaP cells were captured by bright-field microscopy (CKX41; Olympus), and three images fields were randomly chosen. Also, the LNCaP cells was digested and counted by cell counting kit 8 (CCK8, CK04, Dojindo Laboratories, Japan). To block CSF1 signaling, four doses of 3bw of 0.016, 0.08, 0.4, and 2.0 μM were added to the top chamber, and cells were cocultured for 48 h. The LNCaP cells were counted as described above. All analysis was performed using Microsoft Excel 2010 and GraphPad Prism 5 software. The experiment was performed three times and in triplicate. Determination of Pharmacokinetic Parameters in Rats. All animal studies were performed according to the protocols and guidelines of the institutional care and use committee. The 4- to 6week-old male Sprague-Dawley rats 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 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. Rats were housed at an ambient temperature of 21−23 °C with 50−60% relative humidity and 12 h/12 h light/dark cycle. Rats were fed with pelleted rat chow and reverse osmosis (RO) water at libitum. After adaptation for 1 week the rats (180−220 g) were dosed with the test compounds intravenously (iv) at 5 mg/kg and by oral gavage (po) at 25 mg/kg. The compound was dissolved in a vehicle of 2% DMSO, 4% EtOH, 4% castor oil, and 90% H2O. Blood samples (0.3 mL) were then obtained via orbital sinus puncture at 2 min, 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 21 h, 24 h, 30 h, 36 h, 48 h, and 72 h time points and collected into heparinized tubes. Blood samples were centrifuged for cell removal, and the plasma supernatant was then transferred to a clean vial and subsequently stored at −20 °C prior to analysis. Test compound concentrations were determined by LC/MS using propranolol as an internal standard. Pharmacokinetic parameters were calculated using DAS 2.0 software.

cultured in RPMI 1640 medium. Both media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (P/S). The cells were maintained at 37 °C in 5% CO2 incubator. In Vitro Kinase Enzyme Assay. CSF1R, EGFR, and the Z′-Lyte kinase assay kit were purchased from Invitrogen. The experiments were performed according to the instructions of the manufacturer. The concentrations of kinases were determined by optimization experiments and the respective concentrations were the following: EGFR (PV3872, Invitrogen), 0.287 μg/μL; CSF1R (PV3249, Invitrogen) 0.055 μg/μL. First, the compounds were diluted 3-fold from 10−10 M to 1 × 10−4 M in DMSO and a 400 μM compound solution was prepared (4 μL of compound dissolved in 96 μL of water). Second, a 100 μM ATP solution in 1.33× kinase buffer was prepared. Third, a kinase/peptide mixture containing 2× kinase and 4 μM Tyr4 peptide (Invitrogen, PV3193) was prepared right before use. Kinase/peptide mixture was prepared by diluting Z′-LYTE Tyr4 peptide (Invitrogen, PV3193) and kinase in 1× kinase buffer, and 0.2 μM Tyr4 phosphopeptide solution was made by adding Z′-LYTE Tyr4 phospho-peptide to 1× kinase buffer. The final 10 μL reaction consists of 0.002 ng of kinase, 2 μM Tyr4 peptide in 1× kinase buffer. For each assay, 10 μL kinase reactions was made at first (including 2.5 μL compound solution, 5 μL kinase/peptide mixture, and 2.5 μL ATP solution). Mixed the plate thoroughly and incubated for 1 h at room temperature. Then 5 μL development solution was added to each well and the plate was incubated for 1 h at room temperature; the nonphospho-peptides were cleaved at this time. In the end, 5 μL of stop reagent was loaded to stop the reaction. For the control setting, 5 μL of phospho-peptide solution instead of kinase−peptide mixture was used as 100% phosphorylation control. 2.5 μL of 1.33× kinase buffer instead of ATP solution was used as 100% inhibition control, and 2.5 μL of 4% DMSO instead of compound solution was used as the 0% inhibitor control. The plate was measured on an EnVision multilabel reader (PerkinElmer). Curve fitting and data presentations were performed using GraphPad Prism, version 5.0. Every experiment was repeated at least three times. Computational Study. All procedures were performed in Maestro 9.9 (Schrodinger LLC). The crystal structures of CSF1R and EGFR protein were taken from the PDB (3DPK and 3VJO). The protein was processed using the “Protein Preparation Wizard” workflow in Maestro 9.9 (Schrodinger LLC) to add bond orders and to add hydrogens. All heteroatom residues and crystal water molecules beyond 5 Å from heteroatom group were removed. Inhibitors were built in the LigPrep module using the OPLS-2005 force field. Glide module was used as the docking program. The grid-enclosing box was placed on the centroid of the binding ligand in the optimized crystal structure as described above, and a scaling factor of 1.0 was set to van der Waals (vdW) radius of those receptor atoms with partial atomic charges of less than 0.25. Standard precision (SP) approach of Glide was adopted to dock 2 into CSF1R and EGFR with the default parameters, and the top-ranking pose was selected for energy minimization using Prime MM-GBSA, under the solvation model of VSGB. Western Blot Assay. RAW264.7 macrophage cells were seeded into six-well plates and incubated overnight and then treated with or without different concentrations of compounds 3bw for 6 h and then stimulated with or without CSF-1 (50 ng/mL) for 20 min. Cell samples were then lysed in 1× SDS lysis buffer (CST recommended). After being sonicated and boiled, the supernatant of cell lysate was used for Western blot analysis. Cell lysates were loaded to 8−12% SDS−PAGE and separated by electrophoresis. Separated proteins were then electrically transferred to polyvinylidene difluoride membranes (Millipore), which were blocked with 5% bovine serum albumin/TBST for 1 h and then hybridized with specific primary antibodies. The bands were visualized using a SuperSignal West Dura Extended Duration Substrate kit (Thermo Scientific, USA) after hybridization with a HRP-conjugated secondary antibody and then quantified by ImageJ software (1.51j8, National Institutes of Health, USA). 2369



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REFERENCES

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01612. 1 H NMR and 13C NMR for final compounds, biological data, and assay details (PDF) Compound characterization checklist (XLS) Molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*X.L.: e-mail, [email protected]. *K.D.: phone, +86-20-85223764; fax, +86-20-85224766; e-mail, [email protected]. ORCID

Yong Xu: 0000-0003-3601-0246 Ke Ding: 0000-0001-9016-812X Author Contributions ○

Q.X. and Z.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors appreciate the financial support from National Natural Science Foundation of China (Grants 21572230, 81425021, and 81673285), Guangdong Province (Grants 2014TQ01R341, 2015A030306042, 2015A030312014, 2016A050502041 and Nanyue-Baijie Award), Guangzhou City (Grant 201508030036), and Jinan University.

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ABBREVIATIONS USED CSF1R, colony stimulating factor 1 receptor; TAM, tumorassociated macrophage; TME, tumor microenvironment; PGE2, prostaglandin E2; TGFβ, transforming growth factor β; TREG, T regulatory cell; CCL22, C−C motif chemokine 22; PD-1, death protein 1; CTLA-4, cytotoxic T-lymphocyte antigen 4; HLA-G, human leukocyte antigen G; FLT-3, FMSlike tyrosine kinase-3; KIT, stem cell factor receptor; PDGFR, platelet-derived growth factor receptor; EGFR, epidermal growth factor receptor; SAR, structure−activity relationship; PCa, prostate cancer; IC50, half maximal (50%) inhibitory concentration (IC) of a substance; C, cysteine; V, valine; T, threonine; M, methionine; D, aspartic acid; F, phenylalanine; R, arginine; E, glutamic acid; K, lysine; PK, pharmacokinetics; F, oral bioavailability; DMF, N,N-dimethylformamide; DIPEA, ethyldiisopropylamine; THF, tetrahydrofuran; TFA, trifluoroacetic acid; DCM, dichloromethane; PDB, Protein Data Bank; Cmax, maximum serum concentration; AUC, area under curve; CLz, clearance rate; FGFR, fibroblast growth factor receptor; ABL1, Abelson kinase 1; BMX, BMX nonreceptor tyrosine kinase; EPH-A1, ephrin type-A receptor 1; KDR, kinase insert domain receptor; LYNA, Lck/Yes tyrosine kinase A; RET, proto-oncogene tyrosine-protein kinase receptor Ret; iv, intravenous administration; po, oral gavage; Akt, protein kinase B; ARG1, arginase 1; IL10, interleukin 10; MMP9, matrix metallopeptidase 9; VEGFA, vascular endothelial growth factor A; qRT-PCR, quantitative real-time polymerase chain reaction; TLC, thin-layer chromatography; UV, ultraviolet; ppm, parts per million; SRB, sulforhodamine B 2370



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