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

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Article

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 J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01612 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 3, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Design, Synthesis and Structure-Activity Relationship Study of 2-Oxo-3, 4dihydropyrimido[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, ‡,Ψ,║ Yong Xu, † Hua Xie, ║ Zheng-Chao Tu, † Xiaoyun Lu, §,* 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

▽

International Cooperative Laboratory of Traditional Chinese Medicine Modernization

and Innovative Drug Development , Ministry of Education (MOE) of People’s Republic of China ║

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Academy of Sciences, No. 555 Zu-Chong-Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China Ψ

School of Life Science, Shanghai University, #99 Shangda Road, Shanghai 200444,

China #

These authors contribute equally to this work.

KEYWORDS: CSF1R, inhibitor, structure-activity relationship (SAR), tumor-associated macrophages (TAMs) 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, 4-dihydropyrimido[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 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 pro-tumorigenic influences of macrophages, and exhibits reasonable pharmacokinetic profile. Compound 3bw may serve as a new valuable lead compound for future anticancer drug discovery. INTRODUCTION Colony stimulating factor-1 receptor (CSF1R, also known as FMS)1 belongs to the type III growth factor receptor family which 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 pres-


Page 2 of 74

Page 3 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ence 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 signaling is crucial for the differentiation and survival of macrophages,8,9 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 CC 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 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 have been proven to markedly decrease the infiltration of macrophages at the tumor site, and 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 immuno-



therapy. 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 CSF-1R, 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 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 US 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, 4-dihydropyrimido[4, 5-d] pyrimidines as new orally bioavailable and selective CSF1R inhibitors by structural modification of an irreversible EGFR inhibitor.

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


Page 4 of 74

Page 5 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 1-iodopropane. Scheme 1. Synthesis of Compounds 3 a O

O N S

N

S NH2 L Boc 5

Cl

4

N R2 H NH L Boc

N

d

N

S

9

N HN

N N

R3 R4

N L

O N

NH L Boc

Boc

S

c

OH N

N

e

N N

S

HN

N N

N L

R3

R2

N O N S O

f

N

R2 g

N O L Boc

11

R2 O

H

NH L Boc

8

10

h

N

S

NH L Boc

N O L Boc

N

CHO

N

7

6

R2 O

N

b

N HN

i or j R3

N N

N L

R2 O

R1

R4

R4 12

a

N

a

O

13

3a-3m;3aa-3aq;3ba-3bw

Reagents and conditions: (a) K2CO3 (2.0 equiv), N, N-dimethylformamide (DMF), 60



Page 6 of 74

o

C, 80-95%; (b) lithium aluminum hydride (LiAlH4) (2.0 equiv), tetrahydrofuran (THF), -

40 to 0 oC, 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 oC to rt, 60-80%; (e) triphosgene (0.34 equiv), triethylamine (Et3N) (3.0 equiv), DCM, 0 oC 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 o

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 oC, 56%. 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,27 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.


Page 7 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 ID: 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 ID: 3DPK, blue). Hydrogen bonds and cation-π interaction are given by red and green dashed lines, respectively. (C) Superposition of EGFR (PDB ID: 3VJO, orange) and CSF1R kinase (PDB ID: 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 І 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 hydrogen



bonds with Cys666, a cation−π interaction with Lys616 etc.) to that in EGFR protein (Figure 2B and 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. Based on 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), iso-butyryl (3c), n-butyryl (3d), 3-methyl-butyryl (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 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 4-6 fold 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 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 S-


Page 8 of 74

Page 9 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


configuration 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. Table 1. In Vitro CSF1R Kinase Inhibitory Activities of Inhibitors 3a-3m. a

N

N

N N

HN

N

O

Cl

HN

N N

N

Cl

N

N R1

N

O

R1

N N

N

II

I

Compds Formula

R1

CSF1R IC50 (nM)

EGFRWT IC50 (nM)

2

I

30

0.5

3a

I

5

15.7

3b

I

5.5

12.1

3c

I

6.7

8.5

3d

I

10.1

28.9

3e

I

10.3

22.2

3f

I

20.1

29.4

3g

I

20.7

28.7

3h

I

28.4

25.5

3i

I

30.6

24.9

3j

I

20.0

7

3k

I

22.7

26.6

3l

I

180.7

52.8



3m 1 a

II

Page 10 of 74

20.9

23.4

10.8

>10000

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 means from at least three independent experiments and the variations are less than 20%. The computational study also suggested that CSF1R possessed a relatively larger 2-Clphenyl binding pocket than EGFR because of its outwards 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(3ae), or 4- (3af) position, respectively. Both 3ae and 3af exhibited 2-3 folds less potencies 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 4- substitution 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


Page 11 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


with a metabolically more stable difloromethoxyl (3al) or trifloromethoxyl (3am) substituent, the resulting compounds exhibited 6-33 folds 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 di-substituted 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. The di-substituted molecules also displayed 99-200 folds target selectivity over EGFR. Table 2. In Vitro CSF1R Kinase Inhibitory Activities of Inhibitors 3aa-3aq. a

Compds

Formula

3aa

R2

CSF1R

EGFRWT

IC50 (nM)

IC50 (nM)

III

5.2

17.6

3ab

III

5.6

7.6

3ac

III

15.2

35.8

3ad

III

18.1

71.6

3ae

III

13.2

144

3af

III

14.8

383

3ag

III

16.4

365



a

Page 12 of 74

3ah

III

20.6

377

3ai

III

5.1

27.4

3aj

III

4.5

417

3ak

III

5.8

153

3al

III

28

257

3am

III

151

889

3an

III

4.5

421

3ao

III

2.0

362

3ap

III

4.4

413

3aq

III

2.1

373


assay according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA).38 The data are means from at least three independent experiments and the variations are less than 20%. To rationalize potency and selectivity improvement of the di-substituted derivatives, a computational study was conducted to investigate the potential binding mode of 3aq with the CSF1R protein (Figure 3). It was found the disubstituted phenyl ring was orthogonal to the plane of the basic parent structure, within the favorable angle and distance range to form multi-hydrophobic interactions with the protein. In addition, the disubstituted phenyl ring also formed a strong cation−π interaction with Lys616 of the protein. The chloroatom was involved in favorable contacts with the gate keeper Thr663 and Val661, respec-


Page 13 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tively, 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 2.8Å, which further benefited the binding with CSF1R.

Figure 3. Representation of the predicted binding mode of compound 3aq in the active site of CSF1R (PDB ID: 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). Given their outstanding potency and target selectivity, inhibitors 3ao and 3aq were chosen for a preliminary pharmacokinetic (PK) investigation in Sprague-Dawley (SD) rats (Table 4). Both of the compounds exhibited reasonable PK profiles with high plasma exposure AUC(0-∞) values of 1630 and 5311(µg/L*h), oral bioavailability (BA) 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 hours, respectively. Table 3. In Vitro CSF1R Kinase Inhibitory Activities of Inhibitors 3ba-3bw. a



Compds Formula

R3

R4

Page 14 of 74

CSF1R IC50 (nM)

EGFRWT IC50 (nM)

3ba

IV

H

13.6

1100

3bb

IV

2’-CH3

20.2

2500

3bc

IV

2’-OCH

30.4

1600

3bd

IV

3’-OCH

5.8

600

3be

IV

3’-F

17.1

1300

3bf

IV

3’-Cl

14.8

1200

3bg

IV

3’-CF

31.9

3400

3bh

IV

3’-ethyl

10

518

3bi

IV

3’-isopropyl

25.8

365

3bj

IV

3’-CH3

41.2

467

3bk

IV

3’- CH3

25.2

626

3bl

IV

3’-CH3

25.9

148

3bm

IV

3’-CH3

4.9

209

3

3

3


Page 15 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a

4.7

286

6.8

295.7

3’-CH3

6.2

224.5

IV

3’-CH3

6.5

218

3br

IV

3’-CH3

7.0

216

3bs

IV

3’-CH3

10.3

1300

3bt

IV

3’-CH3

7.2

715

3bu

IV

3’-CH3

7.8

888.2

3bv

IV

3’-CH3

3.4

302

3bw

V

3.0

360

3bn

IV

3’-CH3

3bo

IV

3’-CH3

3bp

IV

3bq


assay according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA).38 The data are means from at least three independent experiments and the variations are less than 20%. 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 6.8-10 folds potency loss. The re-



Page 16 of 74

placement 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 folds. Not surprisingly, a replacement of the solvent exposing N-methyl piperazine 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-(4methylpiperazinyl) group (3ao) could be replaced by other solvent accessibly terminal hydrophilic groups, e.g. 4-methyl-1, 4-diazepan-1-yl (3bm), 4-(dimethylamino)piperidin1-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, 7-diazaspiro[3.5]nonan-2-yl (3bq), cis-5methylhexahydropyrrolo[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. N-demethylation is one of the common metabolic pathways for N-methyl piperazine containing drug and introduction of steric hindrance is a feasible strategy to block the de-methylation 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 3-4 folds decreased potency compared with the parent molecule 3ao. Encouragingly, when the Nmethyl group in 3ao was replaced with an N-oxetayl fragment, the resulting compound 3bv displayed comparable CSF1R inhibitory potency and target selectivity to the parent molecule. The 3-methyl-4-(4-(oxetan-3-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.


Page 17 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 bioavailability, respectively, after a 25 mg/kg oral administration. Table 4. Pharmacokinetic Profile of Compounds 3ao, 3aq and 3bw in Rats. a Cpds 3ao

3aq

3bw a

Route

AUC(0-∞) (µg/L*h)

Cmax (µg/L)

T1/2 (h)

CLz (L/h/kg)

po (25 mg/Kg)

1630

396

1.6

--

iv (5 mg/Kg)

1902

--

2.3

2.64

po (25 mg/Kg)

5311

988

1.89

--

iv (5 mg/Kg)

2768

--

3.48

1.84

po (25 mg/Kg)

6462

2515

1.08

--

iv (5 mg/Kg)

2911

--

1.08

1.73

F (%) 17.1 38.6 44.4

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

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 in-house 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 10-35 folds less potent to its inhibition against CSF1R. The 2-oxo-3, 4-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 non-mutant 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 non-receptor 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 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-hour treatment.


Page 18 of 74

Page 19 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 pAKT. All bands were quantified and normalized by GAPDH. The data are expressed as the mean ± SEM from at least three independent experiments. 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 castration-resistance 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 were treated with 10 µM of the anti-Androgen agent MDV3100 (MDV), or vehicle control (NC), and incubated in 10% FBS 1640 medium for 48 hrs. The treated medium (CM) was collected, and then placed into the lower chamber of transwell plates. The numbers of RAW264.7 cells that migrated to the other side of the filter membrane were 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 hrs 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 5C).

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. CSF1R kinase is one of the key players for the pro-tumorigenic properties of TAMs.20 The potential immunotherapeutic effect of 3bw was then determined by measuring its capability to suppress the release of pro-tumorigenic cytokines of macrophages. It was found that compound 3bw dose-dependently suppressed the production of Arginase 1 (ARG1), Interleukin 10 (IL10), Matrix Metallopeptidase 9 (MMP9) and Vascular Endo-


Page 20 of 74

Page 21 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


thelial Growth Factor A (VEGFA) etc. in RAW264.7 macrophages as determined by Quantitative Real-time-polymerase chain reaction (qRT-PCR) assays, suggesting its promising antitumor potential (Figure 6).

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 mean ± SE of 3–5 independent experiments. Statistical significance relative to untreated is indicated: *p < 0.05, **p < 0.01. The antitumor potential of 3bw was further examined by a binary prostate tumor cellmacrophage coculture system (Figure 7). It was shown that stimulation of the co-cultured macrophages significantly enhanced the growth of LNCaP prostate cancer cells. After a 48-hour 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, 0.40and 2.0 µM, respectively (P < 0.05, compared to the LNCaP alone; Fig-



ure 7A and 7B). These results collectively suggest the promising potential of compound 3bw to serve as a lead compound for drug discovery.

Figure 7. Anti-proliferation 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. 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 dosedependently suppressed the activation of CSF1R downstream signals in RAW264.7 macrophages. In addition, 3bw demonstrated reasonable pharmacokinetic properties in rats and exhibited promising anti-tumor effects in blocking macrophage infiltration and abrogating the pro-tumorigenic influences of macrophages. Compound 3bw may serve as a new lead compound for anti-tumor drug discovery. Further structural optimization and in


Page 22 of 74

Page 23 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


vivo antitumor efficacy of 3bw investigation are underway, and the results will be reported in due course. 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 500MHz.

13

C 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 were recorded on an Agilent 1200 HPLC-MSD mass spectrometer and high resolution ESI-MS on an Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer. Purity of all of the final compounds was determined by reverse-phase high performance liquid chromatography (HPLC) analysis to be >95%. HPLC instrument: Dionex Summit HPLC (Column: Diamonsil C18, 5.0µm, 4.6×250 mm (Dikma Technologies); detector: PDA-100 photodiode array; injector: ASI-100 autoinjector; pump: p-680A). 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-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one

(3a).

General procedure for syntheses of 3a−3k, 3m, 3aa-3aq, 3ba-3bw. DIEA (98 µL, 0.56 mmol) and propanoic acid (25 µL, 0.337 mmol) were added to a solution of HATU (128



Page 24 of 74

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 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, yield 80%).

[α]D20-

10.169 (c 0.118, MeOH). 1H 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).

13

C 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. (S)-1-(1-acetylpyrrolidin-3-yl)-3-(2-chlorophenyl)-7-((3-methyl-4-(4-methylpipera zin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3b). White solid (yield 82%).

[α]D20-21.818 (c 0.110, MeOH). 1H 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),


Page 25 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.19-2.15 (m, 1H), 2.02 (s, 3H).

13

C 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-((3-methyl-4-(4-methyl piperazin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3c). White solid (yield 80%).

[α]D20-34.545 (c 0.110, MeOH). 1H 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), 2.592.57 (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-((3-methyl-4-(4-methylpipe razin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one White solid (yield 81%).

(3d).


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,



Page 26 of 74

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).

13

C 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-1-yl)phenyl)amino)-1(1-(3-methylbutanoyl)pyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3e). White solid (yield 80%).

[α]D20-33.333 (c 0.138, MeOH). 1H 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), 7.357.32 (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).

13

C 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-1-yl)phenyl)amino)-1-


Page 27 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(1-pivaloylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one White solid (yield 84%).

(3f).


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).

13

C 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-methyl4-(4-methylpiperazin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1 H)-one (3g). White solid (yield 75%).

[α]D20-41.667 (c 0.072, MeOH). 1H 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.



Page 28 of 74

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-methyl4-(4-methylpiperazin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1 H)-one (3h). White solid (yield 90%).

[α]D20-24.490 (c 0.098, MeOH). 1H 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).

13

C 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]D20-39.130 (c 0.092, MeOH). 1H 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),


Page 29 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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).

13

C 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: MeOHH2O (85:15), 19.80 min, 98.22% purity. (S)-3-(2-chlorophenyl)-1-(1-(2-hydroxyacetyl)pyrrolidin-3-yl)-7-((3-methyl-4-(4methylpiperazin-1-yl)phenyl)amino)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3j). White solid (yield 82%).

[α]D20-21.311 (c 0.122, MeOH). 1H 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), 7.037.01 (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%).

Page 30 of 74

[α]D20-26.415 (c 0.106, MeOH). 1H 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), 3.953.93 (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-1-yl)phenyl)amino)-1(1-propylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3l). N, NDiisopropylethylamine (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 hrs, 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

CH2Cl2/MeOH/NH4OH, 40:1:0.4) to give 3n (60 mg, yield 56%).


(SiO2,

[α]D20-27.027 (c 0.074,

Page 31 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3m). White solid (yield 87%).

[α]D20+14.736 (c 0.095, MeOH). 1H 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), 2.192.14 (m, 1H), 1.12 (t, J = 7.2 Hz, 3H).

13

C NMR (125 MHz, CDCl3) δ 172.8, 172.4,

162.8, 160.8, 159.6, 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-1-yl)phenyl)amino)-1-



(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3aa). White solid (yield 84%).

[α]D20-12.766 (c 0.094, MeOH). 1H 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). 13

C 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-propionylpyrrolidi n-3-yl)-3-(o-tolyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ab). White solid (yield 85%).

[α]D20 -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).

13

C 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,


Page 32 of 74

Page 33 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ac). White solid (yield 81%).


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), 7.016.97 (m, 2.4H), 5.51 (q, J = 8.5 Hz, 1H), 4.65-4.58 (m, 2H), 4.07-4.03 (m, 0.6H), 3.963.94 (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).

13

C 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-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ad). White solid (yield 87%).


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).

13

C 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, 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-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ae). White solid (yield 89%).


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), 7.016.99 (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).

13

C 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-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3af). White solid (yield 82%).



Page 34 of 74

Page 35 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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).

13

C 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-propionylpyrrolidi n-3-yl)-3-(m-tolyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one solid (yield 88%).

(3ag).

White

[α]D20-16.000 (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), 3.743.70 (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).

13

C 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.



Page 36 of 74

(S)-7-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1-(1-propionylpyrrolid in-3-yl)-3-(p-tolyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one solid (yield 78%).

(3ah).

White

[α]D20-19.672 (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).

13

C 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 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-methylpiperazin-1-yl)phenyl)amino)1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ai). White solid (yield 81%).

[α]D20 -10.000 (c 0.140, MeOH). 1H 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).

13

C 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,


Page 37 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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-methylpiperazin-1-yl)phenyl)amino)1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3aj). White solid (yield 84%).

[α]D20 -13.000 (c 0.200, MeOH). 1H 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).

13


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-methylpiperazin-1-yl)phenyl)amino)1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ak). White solid (yield 85%).

[α]D20 -11.494 (c 0.174, MeOH). 1H 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



Page 38 of 74

(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).

13


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 solid (yield 89%).

[α]D20 -18.750 (c 0.096, MeOH). 1H 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), 3.503.45 (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-propionylpyrrolid in-3-yl)-3-(3-(trifluoromethoxy)phenyl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)


Page 39 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-one (3am). White solid (yield 86%).

[α]D20-7.895 (c 0.152, MeOH). 1H 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), 5.535.49 (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). 13

C 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 solid (yield 87%).

[α]D20-17.308 (c 0.104, MeOH). 1H 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.



Page 40 of 74

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 solid (yield 88%).

[α]D20 -22.222 (c 0.100, MeOH). 1H 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 solid (yield 89%).

[α]D20 -22.973 (c 0.148, MeOH). 1H 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), 3.773.75 (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).


13

C NMR

Page 41 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(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: MeOHH2O (85:15), 10.65 min, 97.52% purity. (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 solid (yield 83%).

[α]D20-41.071 (c 0.112, MeOH). 1H 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), 4.033.99 (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). 13C 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-methylpiperazin-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3ba). White solid (yield 82%).

[α]D20+8.333 (c 0.072, MeOH). 1H 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), 4.094.05 (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). 13C 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 solid (yield 80%).

[α]D20+8.108 (c 0.074, MeOH). 1H 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


Page 42 of 74

Page 43 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


min, 96.26% purity. (S)-3-(2-fluoro-5-methoxyphenyl)-7-((2-methoxy-4-(4-methylpiperazin-1-yl)phen yl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1 H)-one (3bc). White solid (yield 83%).

[α]D20+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), 6.546.49 (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)phen yl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2 (1H)-one (3bd). White solid (yield 87%).

[α]D20-15.190 (c 0.158, MeOH). 1H 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).

13

C 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-methoxyp henyl)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3be). White solid (yield 86%).

[α]D20-10.938 (c 0.128, MeOH). 1H 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), 7.197.07 (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: MeOHH2O (85:15), 8.72 min, 98.92% purity. (S)-7-((3-chloro-4-(4-methylpiperazin-1-yl)phenyl)amino)-3-(2-fluoro-5-methoxyp henyl)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3bf). White solid (yield 80%).

[α]D20-25.758 (c 0.132, MeOH). 1H NMR (500 MHz,


Page 44 of 74

Page 45 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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), 7.287.27 (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).

13

C 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-methylpiperazin-1-yl)-3-(trifluorometh yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimi din-2(1H)-one (3bg). White solid (yield 88%).

[α]D20-20.588 (c 0.068, MeOH). 1H 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), 6.846.83 (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), 2.802.70 (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).

13

C 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, 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-methoxyph enyl)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3bh). White solid (yield 89%).

[α]D20-14.063 (c 0.128, MeOH). 1H 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). 13C 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-(4-methylpiperazin-1-yl)phen yl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1 H)-one (3bi). White solid (yield 83%).

[α]D20-21.429 (c 0.140, MeOH). 1H 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


Page 46 of 74

Page 47 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(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-(piperidin-1-yl)phenyl)amino)-1(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3bj). White solid (yield 85%).


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), 2.812.77 (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).

13

C 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-methyl-4-morpholinophenyl)amino)-1-(1propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one


(3bk).


White solid (yield 86%).


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), 4.074.03 (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, 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), 9.38min, 96.49% purity. (S)-7-((4-(4-acetylpiperazin-1-yl)-3-methylphenyl)amino)-3-(2-fluoro-5-methoxyp henyl)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)one (3bl). White solid (yield 89%).

[α]D20-6.316 (c 0.190, MeOH). 1H 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).

13

C 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,


Page 48 of 74

Page 49 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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-methyl-1,4-diazepan-1-yl)phe nyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2 (1H)-one (3bm). White solid (yield 84%).

[α]D20-23.684 (c 0.076, MeOH). 1H 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), 3.693.54 (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).

13

C 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)-3-methylphenyl)amino)-3-(2-fluoro-5methoxyphenyl)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidi n-2(1H)-one (3bn). White solid (yield 77%).

[α]D20-13.636 (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).

13

C 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-methyl-2,7-diazaspiro[4.4]nonan -2-yl)phenyl)amino)-1-((S)-1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]p yrimidin-2(1H)-one (3bo). White solid (yield 76%).

[α]D20-9.615 (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, 3H), 2.00-1.90 (m, 4H), 1.13 (t, J = 7.2Hz, 3H).

13

C 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-methyl-3,9-diazaspiro[5.5]


Page 50 of 74

Page 51 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


undecan-3-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido [4,5-d]pyrimidin-2(1H)-one (3bp). White solid (yield 78%).

[α]D20 -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 = 7.2Hz, 3H).

13

C 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-methyl-2,7-diazaspiro[3.5]non an-2-yl)phenyl)amino)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]py rimidin-2(1H)-one (3bq). White solid (yield 89%).

[α]D20-4.819 (c 0.166, MeOH). 1H

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 = 7.5Hz, 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]+,



Page 52 of 74

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)-5-methylhexahydro pyrrolo[3,4-c]pyrrol-2(1H)-yl)phenyl)amino)-1-((S)-1-propionylpyrrolidin-3-yl)-3,4dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (3br). White solid (yield 79%).

[α]D20 -

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 = 7.5Hz, 3H).

13

C 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)-3-methylphenyl)amino)-3-(2fluoro-5-methoxyphenyl)-1-(1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5d]pyrimidin-2(1H)-one (3bs). White solid (yield 88%). 1

[α]D20-18.519 (c 0.108, MeOH).

H 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), 2.262.25 (m, 8H), 1.13 (t, J = 7.2 Hz, 3H).

13

C NMR (125 MHz, CDCl3) δ 172.4, 159.5,


Page 53 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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-5methoxyphenyl)-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), 4.054.01 (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).

13

C 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)-3,4,5-trimethylpiperazin-1yl)phenyl)amino)-1-((S)-1-propionylpyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]



pyrimidin-2(1H)-one (3bu). White solid (yield 88%).

[α]D20-26.684 (c 0.076, MeOH). 1H

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), 2.772.73 (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]pyrimidin2(1H)-one (3bv). White solid (yield 84%).

[α]D20-14.063 (c 0.128, MeOH). 1H 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,


Page 54 of 74

Page 55 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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]pyrimidin2(1H)-one (3bw). White solid (yield 88%).

[α]D20-46.000 (c 0.100, MeOH). 1H 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), 3.713.69 (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 (m, 1H), 1.13 (t, J = 7.2 Hz, 3H).

13

C 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-car boxylate (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, icewater (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)pyrrole dine-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 hr 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-car boxylate (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


Page 56 of 74

Page 57 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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), 3.733.72 (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-4yl)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 hrs. 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-oxo-3,4-dihydropyrimido [4, 5-d]pyrimidin-1(2H)-yl)pyrrolidine-1-carboxylate (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 hrs. 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)-2-oxo-3,4-dihydropyrimid o[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 hrs. 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), 2.252.24 (m, 1H), 1.45 (s, 9H). MS (ESI) m/z 507.1 [M + H]+. (S)-3-(2-chlorophenyl)-7-((3-methyl-4-(4-methylpiperazin-1-yl)phenyl)amino)-1(pyrrolidin-3-yl)-3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one (13a). 2-Methyl-4-


Page 58 of 74

Page 59 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(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-butylalcohol (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 hrs. 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, and 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), 2.942.92 (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), phosphorAKT (SC16646R) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).



MDV3100 were purchased from ACROS organic company, 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 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 ℃ 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 concentration was: EGFR (PV3872, Invitrogen), 0.287µg/µL, CSF1R (PV3249, Invitrogen) 0.055 µg/µL. First, the compounds were diluted three-fold from 10-10 M to 1x10-4 M in DMSO and a 400 µM compound solution was prepared (4 µL compound dissolved in 96 µL 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 phospho-peptide 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 were 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 one hour at room temperature. Then 5 µL development solution was added to each well and the plate was incubated for 1h at room temperature; the nonphosphopep-


Page 60 of 74

Page 61 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tides were cleaved at this time. In the end, 5 µL stop reagent was loaded to stop the reaction. For the control setting, 5 µL phospho-peptide solution instead of kinase-peptide mixture was used as 100% phosphorylation control. 2.5 µL 1.33×Kinase Buffer instead of ATP solution was used as 100% inhibition control, and 2.5 µL 4% DMSO instead of compound solution was used as the 0% inhibitor control. The plate was measured on an EnVision Multilabel Reader (Perkin-Elmer). Curve fitting and data presentations were performed using Graph Pad Prism, version 5.0. Every experiment was repeated at least 3 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 com-



Page 62 of 74

pounds 3bw for 6 hrs and then stimulated with or without CSF-1 (50 ng/ml) for 20 mins. 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). 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 hrs. The migrating cells were counted as described above. The experiments shown are representative of three independent experiments.


62

Page 63 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 hrs. 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). Realtime PCR was performed in triplicate using gene-specific primers on a Stratagene BioRad 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 Supplementary 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 cocultured for 48 hrs. 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 com-


63


Page 64 of 74

mittee. The 4- to 6-week-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 in -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. ASSOCIATED CONTENT Supporting Information 1

H NMR, and 13C NMR for final compounds, biological data, assay details, and molecu-

lar formula strings (CSV). This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION


64

Page 65 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Corresponding Author *Tel: +86-20-85223764. Fax: +86-20-85224766. Email: [email protected] (K.D.); [email protected] (X.L.) Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors appreciate the financial support from National Natural Science Foundation of China (21572230, 81425021 and 81673285), Guangdong Province (2014TQ01R341, 2015A030306042, 2015A030312014, 2016A050502041 and Nanyue-Baijie Award), Guangzhou City (201508030036) and Jinan University. ABBREVIATIONS CSF1R, colony stimulating factor-1 receptor; TAMs, tumor-associated macrophages; TME, tumor microenvironment; PGE2, prostaglandin E2; TGFβ, transforming growth factor β; TREG, T regulatory cells; 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, FMS-like tyrosine kinase-3; KIT, stem cell factor receptor; PDGFR, plateletderived 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,


65


Page 66 of 74

clearance rate; FGFR, fibroblast growth factor receptor; ABL1; abelson kinase 1; BMX, BMX non-receptor tyrosine kinase; EPH-A1, Ephrin type-A receptor 1; KDR, kinase insert domain receptor; LYNA, Lck/Yes tyrosine kinase A; RET, Proto-oncogene tyrosineprotein 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-timepolymerase chain reaction; TLC, thin-layer chromatography; UV, ultraviolet; ppm, parts per million; SRB, sulforhodamine B. REFERENCES (1) Sherr, C. J.; Roussel, M. F.; Rettenmier, C. W. Colony-stimulating factor-1 receptor (c-fms). J. Cell. Biochem. 1988, 38, 179-187. (2) Hubbard, S. R.; Till, J. H. Protein tyrosine kinase structure and function. Annu. Rev. Biochem 2000, 69, 373-398. (3) Woolford J, R. V.; Rohrschneider L. Characterization of the human c-fms gene product and its expression in cells of the monocyte-macrophage lineage. Mol. Cell. Biochem. 1985, 5, 3458–3466. . (4) Yu, W.; Chen, J.; Xiong, Y.; Pixley, F. J.; Yeung, Y. G.; Stanley, E. R. Macrophage proliferation is regulated through CSF-1 receptor tyrosines 544, 559, and 807. J. Biol. Chem. 2012, 287, 13694–13704. (5) Sweet, M. J.; Hume, D. A. CSF-1 as a regulator of macrophage activation and immune responses. Arch. Immunol. Ther. Exp. 2003, 51, 169-177.


66

Page 67 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(6) Sherr, C. J. R.; C. W.; Roussel, M. F. Macrophage colony-stimulating factor, CSF-1, and its proto-oncogene-encoded receptor. Cold Spring Harbor Symp. Quant. Biol. 1988, 53, 521-530. (7) Chitu, V.; Stanley, E. R. Colony-stimulating factor-1 in immunity and inflammation. Curr. Opin. Immunol. 2006, 18, 39-48. (8) Fiona, J. P. Macrophage migration and its regulation by CSF-1. Int. J. Cell Biol. 2012, 2012, 501962. (9) Stanley, E. R.; Chitu, V. CSF-1 receptor signaling in myeloid cells. Cold Spring Harb. Perspect. Biol. 2014, 6, a021857. (10) Noy, R.; Pollard, J. W. Tumor-associated macrophages: from mechanisms to therapy. Immunity 2014, 41, 49-61. (11) Ostuni, R.; Kratochvill, F.; Murray, P. J.; Natoli, G. Macrophages and cancer: from mechanisms to therapeutic implications. Trends Immunol. 2015, 36, 229-239. (12) Ding, L.; Linsley, P. S.; Huang, L. Y.; Germain, R. N.; Shevach, E. M. IL-10 inhibits macrophage costimulatory activity by selectively inhibiting the up-regulation of B7 expression. J. Immunol. 1993, 151, 1224-1234. (13) Oh, S. A.; Li, M. O. TGF-β: guardian of T cell function. J. Immunol. 2013, 191, 3973-3979. (14) Curiel, T. J.; Coukos, G.; Zou, L.; Alvarez, X.; Cheng, P.; Mottram, P.; EvdemonHogan, M.; Conejo-Garcia, J. R.; Zhang, L.; Burow, M.; Zhu, Y.; Wei, S.; Kryczek, I.; Daniel, B.; Gordon, A.; Myers, L.; Lackner, A.; Disis, M. L.; Knutson, K. L.; Chen, L.; Zou, W. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat. Med. 2004, 10, 942-949.


67


Page 68 of 74

(15) Butte, M. J.; Keir, M. E.; Phamduy, T. B.; Sharpe, A. H.; Freeman, G. J. Programmed death-1 ligand 1 interacts specifically with the B7-1 costimulatory molecule to inhibit T cell responses. Immunity 2007, 27, 111-122. (16) Daurkin, I.; Eruslanov, E.; Stoffs, T.; Perrin, G. Q.; Algood, C.; Gilbert, S. M.; Rosser, C. J.; Su, L.-M.; Vieweg, J.; Kusmartsev, S. Tumor-associated macrophages mediate immunosuppression in the renal cancer microenvironment by activating the 15lipoxygenase-2 pathway. Cancer Res. 2011, 71, 6400-6409. (17) Greenwald, R. J.; Latchman, Y. E.; Sharpe, A. H. Negative co-receptors on lymphocytes. Curr. Opin. Immunol. 2002, 14, 391-396. (18) Zhang, Q. W.; Liu, L.; Gong, C. Y.; Shi, H. S.; Zeng, Y. H.; Wang, X. Z.; Zhao, Y. W.; Wei, Y. Q. Prognostic significance of tumor-associated macrophages in solid tumor: a meta-analysis of the literature. PloS one 2012, 7, e50946. (19) Xu, J.; Escamilla, J.; Mok, S.; David, J.; Priceman, S.; West, B.; Bollag, G.; McBride, W.; Wu, L. CSF1R signaling blockade stanches tumor-infiltrating myeloid cells and improves the efficacy of radiotherapy in prostate cancer. Cancer Res. 2013, 73, 2782-2794. (20) Escamilla, J.; Schokrpur, S.; Liu, C.; Priceman, S. J.; Moughon, D.; Jiang, Z.; Pouliot, F.; Magyar, C.; Sung, J. L.; Xu, J.; Deng, G.; West, B. L.; Bollag, G.; Fradet, Y.; Lacombe, L.; Jung, M. E.; Huang, J.; Wu, L. CSF1 receptor targeting in prostate cancer reverses macrophage-mediated resistance to androgen blockade therapy. Cancer Res. 2015, 75, 950-962. (21) Pyonteck, S. M.; Akkari, L.; Schuhmacher, A. J.; Bowman, R. L.; Sevenich, L.; Quail, D. F.; Olson, O. C.; Quick, M. L.; Huse, J. T.; Teijeiro, V.; Setty, M.; Leslie, C. S.;


68

Page 69 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Oei, Y.; Pedraza, A.; Zhang, J.; Brennan, C. W.; Sutton, J. C.; Holland, E. C.; Daniel, D.; Joyce, J. A. CSF-1R inhibition alters macrophage polarization and blocks glioma progression. Nat. Med. 2013, 19, 1264-1272. (22) Genovese, M. C.; Hsia, E.; Belkowski, S. M.; Chien, C.; Masterson, T.; Thurmond, R. L.; Manthey, C. L.; Yan, X. D.; Ge, T.; Franks, C.; Greenspan, A. Results from a phase IIA parallel group study of JNJ-40346527, an oral CSF-1R inhibitor, in patients with active rheumatoid arthritis despite disease-modifying antirheumatic drug therapy. J. Rheumatol. 2015, 42, 1752-1760. (23) Uemura, Y.; Ohno, H.; Ohzeki, Y.; Takanashi, H.; Murooka, H.; Kubo, K.; Serizawa, I. The selective M-CSF receptor tyrosine kinase inhibitor Ki20227 suppresses experimental autoimmune encephalomyelitis. J. Neuroimmunol. 2008, 195, 73-80. (24) Ohno, H.; Kubo, K.; Murooka, H.; Kobayashi, Y.; Nishitoba, T.; Shibuya, M.; Yoneda, T.; Isoe, T. A c-fms tyrosine kinase inhibitor, Ki20227, suppresses osteoclast differentiation and osteolytic bone destruction in a bone metastasis model. Mol. Cancer Ther. 2006, 5, 2634-2643. (25) Cannarile, M. A.; Weisser, M.; Jacob, W.; Jegg, A. M.; Ries, C. H.; Ruttinger, D. Colony-stimulating factor 1 receptor (CSF1R) inhibitors in cancer therapy. J. Immunother. 2017, 5, 53-66. (26) Tap, W. D.; Anthony, S. P.; Chmielowski, B.; Staddon, A. P.; Cohn, A. L.; Shapiro, G.; Puzanov, I.; Kwak, E. L.; Wagner, A. J.; Peterfy, C.; Hsu, H. H.; Gee, C.; Lin, P. S.; Tong, S.; Wainberg, Z. A. A pilot study of PLX3397, a selective colony-stimulating factor 1 receptor (CSF1R) kinase inhibitor, in pigmented villonodular synovitis (PVNS). J. Clin. Oncol. 2014, 32, 10503.


69


Page 70 of 74

(27) Mok, S.; Duffy, C.; Du, R.; Allison, J. P. Abstract PR01: blocking colony stimulating factor 1 receptor (CSF-1R) and tropomyosin receptor kinase (Trk) improves antitumor efficacy of immunotherapy. Cancer Immunol. Res. 2017, 5, PR01. (28) Bendell, J. C.; Tolcher, A. W.; Jones, S. F.; Beeram, M.; Infante, J. R.; Larsen, P.; Rasor, K.; Garrus, J. E.; Li, J.; Cable, P. L.; Eberhardt, C.; Schreiber, J.; Rush, S.; Wood, K. W.; Barrett, E.; Patnaik, A. Abstract A252: a phase 1 study of ARRY-382, an oral inhibitor of colony-stimulating factor-1 receptor (CSF1R), in patients with advanced or metastatic cancers. Mol. Cancer Ther. 2013, 12, A252. (29) Von Tresckow, B.; Morschhauser, F.; Ribrag, V.; Topp, M. S.; Chien, C.; Seetharam, S.; Aquino, R.; Kotoulek, S.; de Boer, C. J.; Engert, A. An open-label, multicenter, phase I/II study of JNJ-40346527, a CSF-1R inhibitor, in patients with relapsed or refractory hodgkin lymphoma. Clin. Cancer Res. 2015, 21, 1843-1850. (30) Xu, S.; Xu, T.; Zhang, L.; Zhang, Z.; Luo, J.; Liu, Y.; Lu, X.; Tu, Z.; Ren, X.; Ding, K. Design, synthesis, and biological evaluation of 2-oxo-3,4-dihydropyrimido[4,5d]pyrimidinyl derivatives as new irreversible epidermal growth factor receptor inhibitors with improved pharmacokinetic properties. J. Med. Chem. 2013, 56, 8803-8813. (31) Chang, S.; Zhang, L.; Xu, S.; Luo, J.; Lu, X.; Zhang, Z.; Xu, T.; Liu, Y.; Tu, Z.; Xu, Y.; Ren, X.; Geng, M.; Ding, J.; Pei, D.; Ding, K. Design, synthesis, and biological evaluation of novel conformationally constrained inhibitors targeting epidermal growth factor receptor threonine790 → methionine790 mutant. J. Med. Chem. 2012, 55, 2711−2723. (32) Xu, T.; Zhang, L.; Xu, S.; Yang, C.-Y.; Luo, J.; Ding, F.; Lu, X.; Liu, Y.; Tu, Z.; Li, S.; Pei, D.; Cai, Q.; Li, H.; Ren, X.; Wang, S.; Ding, K. Pyrimido[4,5-d]pyrimidin-


70

Page 71 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4(1H)-one derivatives as selective inhibitors of EGFR threonine790 to methionine790 (T790M) mutants. Angew. Chem. Int. Ed. 2013, 52, 8387−8390. (33) Wang, Z.; Bian, H.; Bartual, S. G.; Du, W.; Luo, J.; Zhao, H.; Zhang, S.; Mo, C.; Zhou,Y.; Tu, Z.; Ren, X.; Lu, X.; Brekken, R. A.; Yao, L.; Bullock, A. N.; Su, J.; Ding, K. Structure-based design of tetrahydroisoquinoline-7-carboxamides as selective discoidin domain receptor 1 (DDR1) inhibitors. J. Med. Chem. 2016, 59, 5911-5916. (34) Wang, Z.; Zhang, Y.; Bartual, S. G.; Luo, J.; Xu, T.; Du, W.; Xun, Q.; Tu, Z.; Brekken, R. A.; Ren, X.; Bullock, A. N.; Liang, G.; Lu, X.; Ding, K. Tetrahydroisoquinoline-7-carboxamide derivatives as new selective discoidin domain receptor 1 (DDR1) inhibitors. ACS Med. Chem. Lett. 2017, 8, 327–332. (35) Tan, L.; Zhang, Z.; Gao, D.; Luo, J.; Tu, Z.; Li, Z.; Peng, L.; Ren, X.; Ding, K. 4Oxo-1,4-dihydroquinoline-3-carboxamide derivatives as new Axl kinase inhibitors. J. Med. Chem. 2016, 59, 6807–6825. (36) Chang, Y.; Lu, X.; Shibu, M. A.; Dai, Y.; Luo, J.; Zhang, Y.; Li, Y.; Zhao, P.; Zhang, Z.; Xu, Y.; Tu, Z.; Zhang, Q.; Yun, C.; Huang, C.; Ding, K. Structure based design of N(3-((1H-Pyrazolo[3,4-b]pyridin-5-yl)ethynyl)benzenesulfonamides as selective leucinezipper and sterile-α motif kinase (ZAK) inhibitors. J. Med. Chem. 2017, 60, 5927–5932. (37) Mo, C.; Zhang, Z.; Guise, C. P.; Li, X.; Luo, J.; Tu, Z.; Xu, Y.; Patterson, A. V.; Smaill, J. B.; Ren, X.; Lu, X.; Ding, K. 2-Aminopyrimidine derivatives as new selective fibroblast growth factor receptor 4 (FGFR4) inhibitors. ACS Med. Chem. Lett. 2017, 8, 543–548.


71


Page 72 of 74

(38) Rodems, S. M. H., B. D.; Lin, C.; Zhao, J.; Shah, S.; Heidary, D.; Makings, L.; Stack, J. H.; Pollok, B. A. A FRET-based assay platform for ultra-high density drug screening of protein kinases and phosphatases. Assay Drug Dev. Technol. 2002, 1, 9−19. (39) Cruciani, G.; Carosati, E.; De Boeck, B.; Ethirajulu, K.; Mackie, C.; Howe, T.; Vianello, R. MetaSite: understanding metabolism in human cytochromes from the perspective of the chemist. J. Med. Chem. 2005, 48, 6970-6979. (40) Zhou, W.; Hur, W.; McDermott, U.; Dutt, A.; Xian, W.; Ficarro, S. B.; Zhang, J.; Sharma, S. V.; Brugge, J.; Meyerson, M.; Settleman, J.; Gray, N. S. A structure-guided approach to creating covalent FGFR inhibitors. Chem. Biol. 2010, 17, 285-295. (41) Tan, L.; Wang, J.; Tanizaki, J.; Huang, Z.; Aref, A. R.; Rusan, M.; Zhu, S. J.; Zhang, Y.; Ercan, D.; Liao, R. G.; Capelletti, M.; Zhou, W.; Hur, W.; Kim, N.; Sim, T.; Gaudet, S.; Barbie, D. A.; Yeh, J. R.; Yun, C. H.; Hammerman, P. S.; Mohammadi, M.; Janne, P. A.; Gray, N. S. Development of covalent inhibitors that can overcome resistance to firstgeneration FGFR kinase inhibitors. Proc. Natl. Acad. Sci. 2014, 111, 4869−4877. (42) Li, X.; Guise, C. P.; Taghipouran, R.; Yosaatmadja, Y.; Ashoorzadeh, A.; Paik, W.K.; Squire, C. J.; Jiang, S.; Luo, J.; Xu, Y.; Tu, Z.-C.; Lu, X.; Ren, X.; Patterson, A. V.; Smaill, J. B.; Ding, K. 2-Oxo-3, 4-dihydropyrimido[4, 5-d]pyrimidinyl derivatives as new irreversible pan fibroblast growth factor receptor (FGFR) inhibitors. Eur. J. Med. Chem. 2017, 135, 531-543. (43) Elkins, J. M.; Fedele, V.; Szklarz, M.; Abdul Azeez, K. R.; Salah, E.; Mikolajczyk, J.; Romanov, S.; Sepetov, N.; Huang, X. P.; Roth, B. L.; Al Haj Zen, A.; Fourches, D.; Muratov, E.; Tropsha, A.; Morris, J.; Teicher, B. A.; Kunkel, M.; Polley, E.; Lackey, K. E.; Atkinson, F. L.; Overington, J. P.; Bamborough, P.; Muller, S.; Price, D. J.; Willson,


72

Page 73 of 74 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


T. M.; Drewry, D. H.; Knapp, S.; Zuercher, W. J. Comprehensive characterization of the published kinase inhibitor set. Nat. Biotechnol. 2016, 34, 95-103. (44) Dienstmann, R.; Rodon, J.; Prat, A.; Perez-Garcia, J.; Adamo, B.; Felip, E.; Cortes, J.; Iafrate, A. J.; Nuciforo, P.; Tabernero, J. Genomic aberrations in the FGFR pathway: opportunities for targeted therapies in solid tumors. Ann. Oncol. 2014, 25, 552-563. (45) Greuber, E. K.; Smith-Pearson, P.; Wang, J.; Pendergast, A. M. Role of ABL family kinases in cancer: from leukemia to solid tumors. Nat. Rev. Cancer 2013, 13, 559-571. (46) Luo, W.; Wang, X.; Zheng, L.; Zhan, Y.; Zhang, D.; Zhang, J.; Zhang, Y. Brucine suppresses colon cancer cells growth via mediating KDR signalling pathway. J. Cell. Mol. Med. 2013, 17, 1316-1324. (47) Marshall, J. Transwell invasion assays. Methods Mol. Biol. 2011, 769 (1940-6029 (Electronic)), 97−110.


73


Page 74 of 74

Table of Contents graphic


74

Design, Synthesis, and StructureâActivity Relationship Study of 2-Oxo

Recommend Documents

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