Discovery of Potent Irreversible Pan-Fibroblast Growth Factor

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Discovery of Potent Irreversible Pan-Fibroblast Growth Factor Receptor (FGFR) Inhibitors Yuming Wang, Lijun Li, Jun Fan, Yang Dai, Alan Jiang, Mei-Yu Geng, Jing Ai, and Wenhu Duan J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01843 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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Journal of Medicinal Chemistry

Discovery of Potent Irreversible Pan-Fibroblast Growth Factor Receptor (FGFR) Inhibitors Yuming Wang,†,§,# Lijun Li, ‡,§,# Jun Fan,†,§ Yang Dai,‡,§ Alan Jiang,‡,§Meiyu Geng,‡,§Jing Ai,*,‡,§ and Wenhu Duan*,†,§ †

Department of Medicinal Chemistry, Shanghai Institute of Materia Medica (SIMM), Chinese

Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, P. R. China ‡

Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute

of Materia Medica (SIMM), Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, P. R. China §

University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049, P. R. China

ABSTRACT Fibroblast growth factor receptors (FGFR1−4) are promising therapeutic targets in many cancers. With the resurgence of interest in irreversible inhibitors, efforts have been directed to the discovery of irreversible FGFR inhibitors. Currently, several selective irreversible inhibitors are being evaluated in clinical trials that could covalently target a conserved cysteine in the P-loop of FGFR. In this article, we used a structure-guided approach that is rationalized by a computer-aided simulation to discover the novel and irreversible pan-FGFR inhibitor, 9g, which provided superior FGFR in vitro activities and decent selectivity over VEGFR2 (vascular endothelia growth factor receptor 2). In in vivo studies, 9g displayed clear antitumor activities in NCI-H1581 and SNU-16 xenograft mice models. Additionally, the diluting method confirmed the irreversible binding of 9g to FGFR. INTRODUCTION

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Fibroblast growth factor receptors (FGFR1−4) are a family of transmembrane receptor tyrosine kinases (RTKs) that serve as high affinity receptors for fibroblast growth factors (FGFs).1−3 Ligand binding to the receptors induces FGFR dimerization, initiating the activations of a cascade of downstream signaling pathways, such as Ras-MAPK, PI3K-Akt, STATs and PLCγ.1−3 FGFR signaling has been demonstrated to mediate crucial physiological processes, such as embryogenesis, tissue repair, wound healing, and angiogenesis.4 Recently, accumulative evidence has indicated that aberrant signaling of the FGF/FGFR pathway has the potential to drive the pathogenesis of a broad range of human malignancies, including urothelial cancers, breast cancers, endometrial cancers, squamous lung cancers, ovarian cancers, and cholangiocarcinomas.5 Additionally, the dysregulation of FGFR has also been implicated in the poor prognosis, metastatic progression and resistance to both cytotoxic and targeted agents during clinical treatment.6−10 Therefore, the inhibition of FGF/FGFR signaling pathway presents a promising option for cancer therapeutics. Several multi-targeting tyrosine kinase inhibitors (TKIs) were originally adopted for the treatment of cancers that harbor aberrant FGFR, such as dovitinib (TKI-258)11, lucitanib (E-3810)12, nintedanib (BIBF-1120)13, and ponatinib (AP-24534)14; however, the efficacies of these are quite variable. Although some benefits have emerged in early stage clinical trials, it is uncertain whether these multitargeting TKIs can achieve efficacious concentrations that will inhibit FGFR in clinical trials due to the dose-limiting adverse effects (in particular, the hypertension that occurs as a result of VEGFR2 inhibition).4 Based on these considerations, there has been an increasing interest in developing selective FGFR TKIs. More recently, several selective FGFR TKIs that bear various scaffolds have been developed; some TKIs have been evaluated in clinical trials at different stages, such as 1 (AZD-454715, phase III), 2 2 ACS Paragon Plus Environment

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(BGJ-39816, phase II), 3 (JNJ-4275649317, phase II), 4 (Debio-134718, phase I), and 5 (LY-287445519, phase II) (Figure 1A). Most selective FGFR inhibitors are ATP-competitive inhibitors that bind to active forms of FGFRs. 20 Given the diverse binding modes with FGFR, the ATP-binding pocket is divided into five regions21 (Figure 1B and 1C): (i) hydrophobic region I, (ii) the adenine region, (iii) hydrophobic region II, (iv) the nucleotide domain, and (v) the phosphate region. Hydrophobic region I is a selective pocket that forms van der Waals interactions and crucial H bonds with hydrophobic groups (colored in blue, Figure 1A);16, 22−24 the adenine region is rendered as the major binding site in which heterocycle templates (colored in orange, Figure 1A) are anchored through several H bonds with a hinge;16−19, 22−24 hydrophobic region II is another hydrophobic area that plays an important role in binding small molecules by interacting with lipophilic moieties (colored in gray, Figure 1A);16, 22, 24−25

the nucleotide domain is rarely used in binding inhibitors, whereas compound 5 has been

reported to bond to this region via a H bond between the pyridine N atom (colored in yellow, Figure 1A) and Asn568 (FGFR1 residue numbering) by adapting a chair-like conformation;19, 26 and the phosphate region forms a H bond with the amino side chain (colored in green, Figure 1A) of 3, which may considerably contribute to the overall binding affinity and specificity.23 Most clinical FGFR inhibitors present strong bioactivities against FGFR1−3 due to the high structural similarity of the three kinase domains, while sparing FGFR4 as a result of its structural distinction. However, aberrations of the four FGFR paralogs have been identified in a variety of cancers. For instance, FGFR1 amplifications predominate in squamous cell lung, breast, ovarian, and urothelial cancers; FGFR2 alterations have been identified in 3.7% of gastric cancers and 7.5% of endometrial adenocarcinomas; mutations in FGFR3 are known in approximately 15% of urothelial tumors; and aberrant FGF19/FGFR4 are associated with hepatocellular carcinomas (HCCs).5, 27 More 3 ACS Paragon Plus Environment


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importantly, FGFR1−4 can easily compensate for each other when one of the single pathways is blocked. Taken together, it is reasonable to infer that a molecule targeting all FGFR paralogs would provide an opportunity to satisfy the broadest medical needs for cancer therapeutics. Since suitably designed irreversible inhibitors may have higher selectivity and better efficacy than reversible ones, the first irreversible inhibitor of FGFR1−4 (FIIN-1, Figure 2) originated via a structure-guided approach, which could covalently engage a conserved cysteine in all four FGFR paralogs.28 Based on the same mode, FIIN-2, -3, -4,29−30 and PRN137131 have also been reported as promising irreversible pan-FGFR inhibitors for the treatment of tumors with FGFR alterations (Figure 2). These successful covalent applications provided great inspiration for our pan-FGFR inhibitor design. First, the inhibitors covalently targeting the cysteine within FGFR1−4 could substantially and consistently suppress the activation of the four paralogs. Second, the irreversible binding mechanism is considered to be a practical solution for cases in which reversible inhibitors are less effective against drugresistant mutants as well as tyrosine kinases with a high affinity for ATP.29, 31 In addition, the great successes of the irreversible inhibitors, ibrutinib32 (BTK inhibitor) and osimertinib33 (EGFR inhibitor), also encouraged us to identify a new set of irreversible inhibitors that are attractive for anti-FGFR therapies in the future. In this paper, we describe our efforts to apply a structure-guided design to discover pyrrolopyrimidine-based, potent, and irreversible pan-FGFR inhibitors.


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Figure 1. Structural analysis of the FGFR and FGFR inhibitors. (A) Representative FGFR inhibitors. (B) X-ray crystal structure of FGFR1 in complex with AMP (PDB ID: 3GQI). (C) Five regions of the ATP binding site.

Figure 2. Chemical structures of reported irreversible FGFR inhibitors.

RESULTS AND DISCUSSION 5 ACS Paragon Plus Environment


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Design and SAR (Structure-Activity Relationship) Exploration. Commonly, covalent inhibitors have been developed by structure-guided attachment of an electrophilic moiety to a reversible inhibitor.34 To identify an appropriate chemical scaffold for our covalent design, a set of clinical molecules with various binding modes were inspected, which emphasized the adenine region and hydrophobic region I (Figure 1C) as the two pivotal binding sites that played important roles in binding FGFR inhibitors. It was reasonable to assume that chemical entities capable of occupying the two regions would provide acceptable binding affinity that is beneficial for covalent design.35 We initiated our study of the pyrrolopyrimidine fragment as the hinge-binding moiety due to its structural resemblance to purine (Figure 3). Analysis of the ATPbinding pocket suggested that introducing an appropriate aromatic moiety at the C-3 position of pyrrolopyrimidine would provide considerable interactions with hydrophobic region I; thus, we directed our attention to the 3,5-dimethoxyphenyl moiety because it was the optimal aromatic group that could interact productively with this hydrophobic pocket to provide selectivity and potency.16, 27−29, 36−40

Based on our assumption, two motifs (pyrrolopyrimidine and 3,5-dimethoxyphenyl) formed

our basic molecular scaffold (Figure 3), which was expected to fill the two pivotal regions and provide the reversible (noncovalent) binding affinity to FGFR. To determine how to best align the electrophile, the X-ray crystal structure of FGFR1 in complex with AMP (PDB ID: 3GQI) was analyzed; we determined that the ribose moiety (5-membered ring) of AMP was in close proximity to the P-loop, which was reported to be a flexible region where Cys488 (FGFR1 residue numbering) resided.31 Based on this observation, we envisioned that a pyrrolidine-based linker attached to the pyrrolopyrimidine core at the N-1 position may provide a suitable length for a Michael acceptor to


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engage the cysteine. Compound 6a, as the early product, was first prepared based on the design hypothesis (Figure 3).

Figure 3. Design of covalent FGFR inhibitors.

To identify the pan-FGFR inhibitors, an activity-based assay was carried out by evaluating the inhibitory activities of pyrrolopyrimidines on FGFR1 and FGFR4. Compounds with acceptable biochemical activities were further profiled in a FGFR1-amplified NCI-H1581 proliferation assay. Due to the fact that covalent inhibitors interact with their target proteins time-dependently, IC50 values for FGFRs were carried out based on the same incubation time (60 min). As shown in Table 1, compound 6a elicited promising potency in the biochemical assay of FGFR1 (IC50 = 30.9 nM). Moderate impacts on the FGFR4 kinase and NCI-H1581 cell line were also observed with IC50 values of 327.3 nM and 90.2 nM, respectively. Further analysis of hydrophobic region I suggested that an increased potency could be achieved by extending the aromatic moiety more deeply into this region. To test this hypothesis, a set of analogues with aromatic extensions were synthesized.

Table 1. SARs in the Modification of Hydrophobic Region I



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IC50 (nM)a

Compd

Ar

L

FGFR1 kinase

inhibition

FGFR4 kinase

inhibition

NCI-H1581 cell proliferation

inhibition

6a

30.9 ±9.6

327.3 ±53.2

90.2 ±0.1

6b

2.6 ±1.4

33.2 ±11.0

< 0.3

6c

0.8 ±0.0

19.4 ±2.3

< 0.3

6d

0.8 ±0.3

19.4 ±2.1

< 0.3

6e

2.9 ±1.9

38.8 ±3.7

86.1 ±64.4

6f

2.0 ±0.2

94.3 ±0.2

38.1 ±24.3

6g

2.1 ±1.5

90.6 ±24.4

< 0.3

6h

3.8 ±2.5

42.8 ±5.3

170.1 ±77.1

6i

4.4 ±1.5

145.0 ±1.5

15.9 ±4.4

6j

< 0.3

6.2 ±0.3

< 0.3

6k

389.3 ±299.6

779.0 ±106.6

-

6l

105.1 ±88.2

> 1000

-


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a


1

-

-

1.5 ±0.1

-

40.0 ±2.8

5

-

-

0.3 ±0.2

3.2 ±0.1

-

Values are the mean ±SD of two independent assays.

Table 2. Effects of Substitutions on Pyrrolo[2,3-d]pyrimidine

IC50 (nM)a

Compd

R1

R2

L

FGFR1 kinase

inhibition

a

FGFR4 kinase

inhibition


inhibition

6j

H

H

< 0.3

6.2 ±0.3

< 0.3

7a

Cl

H

9.0 ±0.6

64.3 ±31.9

207.4 ±5.6

7b

CH3

H

163.9 ±64.0

489.0 ±36.9

253.6 ±38.0

7c

NH2

H

40.3 ±28.6

155.0 ±54.9

25.7 ±4.2

7d

H

CH3

> 100

> 100

-

1

-

-

-

1.5 ±0.1

-

40.0 ±2.8

5

-

-

-

0.3 ±0.2

3.2 ±0.1

-




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Table 3. Activity Data for Varying the Electrophilic Motifs

IC50 (nM)a

Compd.

L

Warhead

FGFR1 kinase

inhibition

a

FGFR4 kinase

inhibition


inhibition

6j

< 0.3

6.2 ±0.3

< 0.3

8a

0.8 ±0.1

55.4 ±4.4

49.7 ±12.9

8b

< 0.3

1.7 ±0.1

> 1000

8c

0.3 ±0.1

3.8 ±0.4

> 1000

8d

0.8 ±0.1

35.6 ±5.3

< 0.3

8e

0.9 ±0.5

78.9 ±6.2

< 0.3

1

-

-

1.5 ±0.1

-

40.0 ±2.8

5

-

-

0.3 ±0.2

3.2 ±0.1

-


Table 4. SARs for Optimization of the Linker Region


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IC50 (nM)a

Compd

L

FGFR1 kinase

inhibition

a

FGFR4 kinase

inhibition


inhibition

6j

< 0.3

6.2 ±0.3

< 0.3

9a

0.6 ±0.4

31.2 ±1.0

< 0.3

9b

2.6 ±1.3

179.5 ±6.4

227.2 ±25.2

9c

1.4 ±0.2

145.7 ±9.5

67.9 ±2.3

9d

1.7 ±0.3

85.2 ±16.0

25.6 ±1.5

9e

7.2 ±1.6

312.9 ±50.5

254.7 ±62.1

9f

2.5 ±1.3

25.9 ±8.8

< 0.3

9g

< 0.3

5.3 ±1.2

< 0.3

9h

0.6 ±0.1

82.7 ±28.1

21.7 ±10.5

9i

0.6 ±0.3

5.4 ±0.3

< 0.3

1

-

1.5 ±0.1

-

40.0 ±2.8

5

-

0.3 ±0.2

3.2 ±0.1

-


As shown in Table 1, incorporating the 5,7-dimethoxybenzo[b]thiophene group at the C-3 position of pyrrolopyrimidine (6b) noticeably improved the FGFR1/4 inhibitory activities compared with 6a with IC50 values of 2.6 nM and 33.2 nM, respectively; a significant increase in the cellular potency was also observed with 6b when it was tested against the NCI-H1581 cell line (IC50 < 0.3 nM). The 11 ACS Paragon Plus Environment


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sharp increase in inhibiting FGFR strongly supported our aforementioned hypothesis; thus, we then surveyed the influences of substituents on the benzo[b]thiophene ring. We found that removal of the 5-methoxy at benzo[b]thiophene (6b) to give compound 6c further increased the biochemical potencies against FGFRs; the corresponding monomethoxy analogue, 6d, with a lower molecular weight, was equipotent to 6c in FGFR suppression, which indicated the similar impacts of pyrrolidine and azetidine on FGFR potencies. A comparison of 6d−f revealed that introducing a methoxy at different positions in benzo[b]thiophene was tolerated for the biochemical activities towards FGFR1, whereas the phenomenon was variable with FGFR4. Reduced potencies on FGFR4 were observed when the methoxy group shifted from the 7-position (6d) to other positions (6e−f). To further understand the substitution pattern, compound 6g with the 7-methyl at the benzo[b]thiophene and 6h with no substituents were subsequently assessed. Both compounds displayed modest inhibitory activities against FGFR4, and a substantial drop in cellular potency with 6h was observed. The obvious preference for 7-methoxy implied that a H bond may be involved within hydrophobic region I, which was supported by molecular modeling (Figure 4A). However, the substantial losses in cellular potencies of 6e−f indicated that an improper introduction of the methoxy may cause unfavorable interactions with hydrophobic region I and weaken the potency against FGFR. Intrigued by the 7-methoxy substitution pattern, we conducted further investigation on dimethoxy-substituted 6i; the resulting compound displayed no highlights but moderate inactivation of FGFR4 with an IC50 of 145.0 nM, which was 4-fold less potent than 6b. Given the similar impact of pyrrolidine and azetidine on FGFR inhibition, the observed increase in 6b was directly linked to the effect of the methoxy substituents. According to the molecular modeling (Figure 4B), we rationalized that the improved potency for FGFR4 was presumably provoked by the interaction between the 5-methoxy 12 ACS Paragon Plus Environment

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at the benzo[b]thiophene (6b) and Met524 (FGFR4 residue numbering) within hydrophobic region I. Considering that 6c was almost 2-fold better than 6b against FGFR4, we proposed that further improving the potency could be feasible by switching the 5-methoxy (6b) to a smaller methyl; this improvement may not only retain the interaction with Met524 but also cripple the possible steric interference between the spatially demanding methionine and the 5-methoxy. Interestingly, the replacement of 5-methoxy with 5-methyl to give 6j considerably improved the FGFR1/4 inhibitory activities with IC50 values of less than 0.3 nM and 6.2 nM, respectively, thus underscoring the importance of the interaction between the 5-methyl and methionine. A similar methyl-methionine interaction was also observed in compound 4, which not only contributed to the potency to FGFR but also improved the specificity over VEGFR2.18,

24

Attempts to alter benzo[b]thiophene with

benzofuran caused considerable losses of FGFR inhibitory activities (6k−l). Therefore, the 5-methyl7-methoxybenzo[b]thiophene segment of 6j represented the optimal aromatic motif and formed the basis for further structural optimization.

Figure 4. Modeled interactions of benzo[b]thiophene motifs with hydrophobic region I in FGFR4 (PDB ID: 4R6V): (A) 6c with hydrophobic region I; (B) 6b with hydrophobic region I.

Having produced an optimized aromatic group, we turned our attention to the 4-amino pyrrolo[2,3d]pyrimidine core. As summarized in Table 2, any addition to the pyrrolopyrimidine ring (7a−c) 13 ACS Paragon Plus Environment


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suffered losses in biochemical and cellular assays, which indicated that even minor modifications in pyrrolopyrimidine would impair H bonding with the hinge and result in attenuated potencies. Compared to 6j, the methylated analogue, 7d, completely lost potencies for FGFR despite losing one hydrogen, which emphasized the critical nature of the amino group. Then, the SARs with respect to the electrophiles were investigated (Table 3). For propylamide 8a, notable losses in activities were observed compared with the irreversible counterpart, 6j, indicating the significance of covalent binding for FGFR inhibition. However, the reasonable biochemical/cellular activities with 8a reflected a considerable reversible binding affinity to FGFR, which strongly confirmed our design. Vinylsulfonamide 8b and propiolamide 8c exhibited inspiring inhibitions on FGFR kinases; however, their biochemical impacts translated into poor cellular potencies in the NCI-H1581 cell line. Attachments of water-solubilizing groups at the terminal position of the electrophiles (8d−e) provided good biochemical and cellular activities that were relevant to FGFR1, whereas FGFR4 was less addressed. To further systematically optimize the electrophilic system, compounds containing various linkers were tested. As shown in Table 4, increasing the size of the linker (9a−c) led to moderate potencies against FGFR4, and the cellular impacts of 9b−c were also attenuated. Compounds with a flexible linker (9d−e) were unfavorable for the suppression of FGFR; in contrast, the more constrained analogue, 9f, demonstrated improved potencies in both biochemical and cellular evaluations. This phenomenon was observed with other irreversible inhibitors and was attributed to the conformational flexibility, which may impede the formation of the covalent bond between the protein and the acrylamide moieties.41−42 In accordance with 6c and 6d, the pyrrolidine- and azetidine-based linkers (6j and 9i) were well tolerated according to the excellent activities in both biochemical and cellular 14 ACS Paragon Plus Environment

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evaluations. Our investigation also revealed that the S configuration (9g) was more favorable than the R configuration (9h) due to the higher potencies observed with 9g. With superior biochemical and cellular potencies, 9g and 9i were chosen for a pharmacokinetic study. Pharmacokinetic Study for Compound Selection. The preliminary pharmacokinetic evaluations were conducted in Sprague-Dawley (SD) rats. Compounds 9g and 9i were orally administered by gavage at a 10 mg/kg dose to male SD rats, and the pharmacokinetic parameters are summarized in Table 5. Both compounds displayed moderate maximum concentrations and oral exposures; however, 9g was more favorable. Therefore, compound 9g was selected for further evaluation.

Table 5. Pharmacokinetic Data of 9g and 9i in Ratsa

a

Compound

Dose (mg/kg)

Tmax (h)

Cmax (ng/mL)

AUC (ng·h/mL)

T1/2 (h)

9g

10

2.75

77.7

277

1.73

9i

10

1.67

12.0

46.4

2.11

Tmax, time of maximum concentration; Cmax, maximum concentration; T1/2, elimination half-life;

AUC, area under the plasma concentration time curve. Experiments were carried out in male Sprague Dawley rats (n = 3). Data are the mean values.

Selectivity Profile of 9g. Compound 9g was tested against a panel of tyrosine kinases to determine its kinase selectivity. As summarized in Table 6, 9g effectively inhibited the activities of FGFR1−4 with IC50 values of less than 0.3, 0.8, 9.4, and 5.3 nM, respectively, indicating that 9g is a potent pan-FGFR inhibitor. 15 ACS Paragon Plus Environment


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Compared with 1 (FGFR4 IC50 = 165 nM, literature reported data15), 9g was significantly more potent in inhibiting FGFR4, suggesting its potential use for the treatment of FGFR4-driven cancer types, such as FGF19/FGFR4-driven hepatocellular carcinoma. Moreover, 9g displayed a more than 350fold selectivity over recombinant VEGFR2 relative to FGFR1; and no obvious inhibitory effects were observed with the other closely related tyrosine kinases (Table 6). These data indicated that 9g was a selective pan-FGFR kinase inhibitor.

Table 6.

a

Kinase Selectivity Profile of 9g Tyrosine kinase

IC50 (nM)a

Tyrosine kinase

IC50 (nM)a

FGFR1

< 0.3

c-Src

> 1000

FGFR2

0.8 ±0.1

ErbB2

> 1000

FGFR3

9.4 ±1.6

c-Met

> 1000

FGFR4

5.3 ±1.2

ABL

> 1000

VEGFR2

113.4 ±0.6

EGFR

> 1000

PDGFR-α

> 1000

EPH-A2

> 1000

PDGFR-β

> 1000

ErbB4

> 1000

ALK

> 1000

IGF1R

> 1000

Flt-1

> 1000

BTK

> 1000

Values are the mean ±SD of two or more independent assays.

In vitro Activity of Compound 9g.


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To assess the cellular potency of 9g, we profiled its efficacies on FGFR-mediated cancer cell lines. As shown in Table 7, 9g significantly inhibited the proliferation of FGFR-driven cancer cell lines with IC50 values of less than 1 nM against NCI-H1581, SNU-16, OPM-2, and RT-112, respectively. Moreover, the potent impact of 9g on FGFR4 translated into pronounced antiproliferative effects on the Huh-7 and BaF3/TEL-FGFR4 cell lines, affording IC50s of less than 1.5 nM and less than 4.6 nM, respectively; this outcome further confirmed the potential for treatment of cancers that harbor FGF19/FGFR4 alterations. Moreover, 9g demonstrated substantially weaker activity against the BaF3/TEL-KDR cell line (293.5 nM). Collectively, these results highlighted the antiproliferative activities of 9g on FGFR-altered cancer cell lines and the selectivity over VEGFR2 on the cellular level.

Table 7. Antiproliferative Activity of 9g on FGFR-Addicted Cell Lines IC50 (nM)a Cell lines

FGFR alteration 9g

1

2

NCI-H1581

FGFR1 amplification

< 0.5

37.9 ±2.8

-

SNU-16

FGFR2 amplification

< 0.1

1.5 ±0.2

-

OPM-2

FGFR3 amplification

< 0.5

83.6 ±5.2

-

RT-112

FGFR3 amplification

< 0.5

36.4 ±3.2

< 0.5

Huh-7

FGF19/FGFR4

< 1.5

284.3 ±22.6

57.2 ±4.8



a

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BaF3/TEL-FGFR4

TEL-FGFR4

< 4.6

339.2 ±30.0

132.6 ±9.8

BaF3/TEL-KDR

TEL-KDR

293.5 ±32.9

543.5 ±140.2

3343.8 ±33.0

Values are the mean ±SD of two or more independent assays.

To further elucidate the impact of 9g on FGFR signaling, we analyzed its effects on the phosphorylation of FGFR and the major downstream signaling molecules, PLCγ and Erk. Three representative human cancer cell lines with different mechanisms of FGFR activation were used, including FGFR1-translocated myeloid leukemia cancer cells (KG-1), FGFR2-amplified gastric cancer cells (SNU-16), and FGFR3-mutated bladder cancer cells (UMUC-14). As shown in Figure 5, 9g significantly inhibited the phosphorylation of FGFR1 in the KG-1 cell line. In accordance with the suppressed FGFR1 phosphorylation, the phosphorylation of PLCγ and Erk (two key downstream effectors of FGFR signaling) were also substantially inhibited. Similar results were obtained in 9gtreated SNU-16 and UMUC-14 cells. These results suggested that 9g effectively blocked FGFR signaling, regardless of the mechanistic complexity of the FGFR activation.


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Figure 5. Compound 9g effectively inhibits the phosphorylation of FGFR and the downstream effectors PLCγ and Erk in KG-1 (A), SNU-16 (B), and UMUC-14 (C). Cells treated with 9g for 2 h at the indicated concentrations were lysed and subjected to Western blot analysis.

Irreversible assay of 9g. To ascertain if compound 9g inhibited FGFR via irreversible binding, we used a diluting method43 to assess this intrinsic property of 9g. In this assay, FGFR1 kinase was pre-incubated with excess 9g (or 1) at a final concentration of 100-fold IC50 at room temperature for 30 min. Then, the enzyme/inhibitor mixture was diluted to 100-fold with a reaction buffer containing ATP and peptide, and the FGFR1 kinase activity was continuously measured. The enzyme activity was determined by the percentage of peptide converted (phosphorylated) from substrate to product. After incubation with FGFR1, the recovery curve of reversible inhibitor 1 (Figure 6) coincided with the non-pre-incubation control, whereas the recovery curve of 9g (Figure 6) considerably decreased compared with the nonpre-incubation control and was almost comparable with the control group. This assay suggested that 9g could irreversibly bind to FGFR1 and suppress kinase activity.

Figure 6. Compound 9g irreversibly binds to FGFR1. Enzyme activity of FGFR1 was assayed by a Caliper EZ Reader under three different conditions: without the enzyme (background), without the



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compound (non-pre-incubation control) and pre-incubated with the compound. “Conversion” here represents the enzyme activity and means “the percent of conversion of the substrate peptide”.

In vivo Antitumor Efficacy of Compound 9g. We assessed the in vivo efficacies of 9g on tumors with FGFR alterations. Mice bearing xenograft tumors derived from NCI-H1581 (FGFR1-amplified) and SNU-16 (FGFR2-amplified) cells were treated orally with 9g once daily for 14 consecutive days in the NCI-H1581 model and for 21 consecutive days in the SNU-16 model. As illustrated in Figure 7, 9g dose-dependently suppressed the tumor progression in both xenograft models with a maximum tumor growth inhibition rate (TGI) of 98.9% at a dose of 100 mg/kg in the NCI-H1581 model and 85.8% at a dose of 50 mg/kg in the SNU-16 model. No severe weight loss was observed during the treatment. These results demonstrated that inhibitor 9g was effective against tumors with FGFR genetic alterations, exhibiting potential for further study.


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Figure 7. 9g significantly inhibited FGFR-mediated tumor growth in vivo. (A) Tumor growth was inhibited upon 9g treatment in NCI-H1581 xenografts. The RTVs are expressed as the mean ±SEM. Significant difference from the vehicle group was determined using a t-test, * P < 0.05, ** P < 0.01, *** P < 0.001. (B) Tumor weights are shown for day 14 after the mice were administered 9g. (C) Body weight measurements during treatment. (D) Tumor growth was inhibited upon 9g treatment in the SNU-16 xenografts. The RTVs are expressed as the mean ±SEM. Significant difference from the vehicle group was determined using a t-test, * P < 0.05, ** P < 0.01, *** P < 0.001. Tumor weights (E) are shown for day 21 after the mice were administered 9g. (F) Body weight measurements during treatment.



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Molecular Docking of Compound 9g to FGFR4 To investigate the binding modes of our inhibitors, representative compound 9g was docked into the ATP-binding pocket of FGFR4 (PDB ID: 4R6V). As depicted in Figure 8, the 5-methyl-7methoxybenzo[b]thiophene motif was anchored to hydrophobic region I via van der Waals interactions by adopting the perpendicular orientation with respect to the plane of the pyrrolopyrimidine core; a H bond was present between the oxygen atom of 7-methoxy and the backbone of Asp630. The 4-aminopyrrolopyrimidine core interacted effectively with Glu551 and Ala553 through two H bonds at the adenine region. Pyrrolidine-linked electrophilic acrylamide was poised in a suitable position to engage Cys477 (FGFR4 residue numbering) in a covalent bond. The simulated results generated a reasonable binding mode of 9g, further strengthening our concept of this rational design.

Figure 8. Proposed binding mode of 9g with FGFR4 (PDB ID: 4R6V). (A) The docked protein structure of FGFR4 is shown on the surface. (B) The atoms of 9g are colored as follows: carbon, green; oxygen, red; nitrogen, blue; sulfur, dark yellow. The protein is shown in the cartoon, and key residues are colored as follows: carbon, yellow; oxygen, red; nitrogen, blue, sulfur, dark yellow. H bonds are indicated by red dotted lines.


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Chemistry. The pyrrolopyrimidine compounds were prepared according to Schemes 1−3. Commercially available 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (10) or 14 were modified with N-Bocsubstituted aliphatic amines via Mitsunobu or SN2 reactions, which were transformed to the 4-amino5-iodopyrrolo[2,3-d]pyrimidines (12 and 16) by treatment with aqueous ammonia (Schemes 1−2). Intermediates 13, 17, and 18 were prepared from iodopyrrolo[2,3-d]pyrimidines (12 or 16) and boronic acids (or boronate esters) using conventional Suzuki coupling conditions. Then, intermediates 13, 17, and 18 were deprotected using HCl/dioxane and subsequently coupled with various electrophiles to provide the desired products. CONCLUSION We described the design and SAR exploration of pyrrolopyrimidine-based irreversible pan-FGFR inhibitors. Structural optimizations that were rationalized by computer-aided simulations led to the identification of 9g, which exhibited excellent potencies against FGFR1−4 and acceptable selectivity over VEGFR2. On the cellular level, 9g displayed superior antiproliferative effects on various cell lines associated with FGFR dysregulation, which was supported by the Western blot study. Despite the moderate pharmacokinetic profile of 9g, we proposed that only a sufficient concentration pulse was required for irreversible inhibitors to bind to the target kinases and to exert an antitumor effect in vivo. The pharmacodynamics evaluations of 9g confirmed our assumption via dose-dependent TGIs in NCI-H1581 and SNU-16 xenograft models. Moreover, the irreversible assay demonstrated that 9g covalently bonded to the FGFR kinase. Although our newly-designed irreversible pan-FGFR inhibitor exhibited good selectivity over VEGFR2, it seems difficult to avoid the on-target side effect (i.e. hyperphosphatemia) which is 23 ACS Paragon Plus Environment


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commonly seen in patients treated with pan-FGFR inhibitors. Luckily, the preliminary data from clinic suggests that the issue can be managed by diet modification and drug intervention,44 and we believe that this new pharmacophore and binding mode-based inhibitor, such as 9g, would be a potential FGFR inhibitor for further drug development. Scheme 1. Synthesis of Target Compounds 6a−la

a

Reagents and conditions: (a) HO-linker-Boc, PPh3, DIAD, THF, 0 oC to rt or tert-butyl 2-

bromoethylcarbamate, cesium carbonate, 80 oC; (b) ammonia (aq), 110 oC; (c) boronic acids or boronate esters, Pd(PPh3)4, Na2CO3, dioxane/H2O, 80 oC; (d) (i) HCl/dioxane, rt; (ii) acryloyl chloride, TEA, DCM, rt.


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Scheme 2. Synthesis of Target Compounds 7a−da

a

Reagents and conditions: (a) HO-linker-Boc, PPh3, DIAD, THF, 0 oC to rt; (b) ammonia or

methylamine (aq), 110 oC; (c) (7-methoxy-5-methylbenzo[b]thiophen-2-yl) boronic acid, Pd(PPh3)4, Na2CO3, dioxane/H2O, 80 oC; (d) (i) HCl/dioxane, rt; (ii) acryloyl chloride, TEA, DCM, rt.

Scheme 3. Synthesis of Target Compounds 8a−e, 9a−ia



a

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Reagents and conditions: (a) (7-methoxy-5-methylbenzo[b]thiophen-2-yl) boronic acid, Pd(PPh3)4,

Na2CO3, dioxane/H2O, 80 oC; (b) (i) HCl/dioxane, rt; (ii) acyl chloride, TEA, DCM, rt or corresponding carboxylic acid, HATU, DMF, DIPEA, rt.

EXPERIMENTAL SECTION Chemistry All starting materials and reagents were obtained from commercial suppliers or prepared according to known procedures. All starting materials and solvents were used without further purification unless otherwise noted. Chemical reactions were monitored by thin-layer chromatography (TLC), using silica gel plates with fluorescence F254 and visualized under UV light. Flash chromatography was conducted using silica gel (300−400 mesh). 1H NMR and 13C NMR were generated in CDCl3, DMSOd6, or CD3OD on Varian Mercury 300, 400, or 500 NMR spectrometers. ESI (Low resolution mass spectra) were recorded on a Thermo Fisher LCQ-DECA spectrometer; ESI (High resolution mass spectra) were determined on an Agilent G6520 Q-TOF spectrometer. All tested compounds were purified to ≥ 95% purity as determined by the Agilent infinity 1260 HPLC system coupled with a diode array detector (DAD) and the ZORBAX Eclipse Plus column (C18, 4.6×150 mm, 5 μm). General Procedure A: Synthesis of compounds 11a−g, 11i−j, 15a−c. To a stirred solution of 4chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (10) or 14a−c (0.5 mmol, 1.0 eq), requisite N-Bochydroxylsubstituted amine (0.75 mmol, 1.5 eq), and triphenylphosphine (1.0 mmol, 2.0 eq) in anhydrous tetrahydrofuran (5 mL) was slowly added diisopropyl azodicarboxylate (1.0 mmol, 2.0 eq) at 0 oC under argon atmosphere. The resulting mixture was subsequently warmed up to room temperature and stirred for additional 2 h (24 h for 11d). The reaction mixture was concentrated and 26 ACS Paragon Plus Environment

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the resulting residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 4:1) to afford compounds 11a−g, 11i−j, 15a−c. General Procedure B: Synthesis of compounds 12a−j, 16a−d. A mixture of 11 or 15 (2.5 mmol, 1.0 eq), and aqueous ammonia (3 mL) (aqueous methylamine for 16d) in dioxane (4 mL) was heated in a seal tube at 110 oC for 16 h (48 h for 16c). After cooling, the mixture was distributed in water and ethyl acetate. The organic layer was separated and washed with water and brine, dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 99:1 to 98:2) to afford compounds 12a−j, 16a−d. General Procedure C: Synthesis of compounds 13a−l, 17a−d, 18a−i. A mixture of appropriate boronic acid or boronate ester (3.4 mmol, 1.3 eq), corresponding 12 or 16 (2.3 mmol, 1.0 eq), Na2CO3 (4.7 mmol, 2.0 eq), and tetrakis(triphenylphosphine)palladium (0.12 mmol, 0.05 eq) in dioxane (20 mL)/water (5 mL) was heated to 90 oC under argon for 6 h. After cooling, the mixture was distributed in water and ethyl acetate. The organic layer was separated and washed with water and brine, dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 98:2) to afford compounds 13a−l, 17a−d, 18a−i. General Procedure D: Synthesis of target compounds 6a−l, 7a−d, 8a, 9a−i. To a reaction flask containing 13, 17, or 18 (0.21 mmol, 1.0 eq) was added 4 N HCl/dioxane (4 mL). The reaction mixture was stirred at room temperature for 3 h, then dioxane was removed to dryness. The deprotected amine was taken up in dry dichloromethane (5 mL) and cooled to 0 oC. The solution was treated with triethylamine (0.63 mmol, 3.0 eq) and stirred for 15 min at 0 oC. Acryloyl chloride or propionyl chloride (0.23 mmol, 1.1 eq) was then added slowly, and the mixture was stirred at 0 oC for 30 min. After completion, the mixture was distributed in water and dichloromethane. The organic layer was 27 ACS Paragon Plus Environment


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separated and washed with water and brine, dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 98:2) to afford target compounds 6a−l, 7a−d, 8a, 9a−i. General Procedure E: Synthesis of target compounds 8c−e. To a reaction flask containing 13j or 18i (0.21 mmol, 1.0 eq) was added 4 N HCl/dioxane (4 mL). The reaction mixture was stirred at room temperature for 3 h, then dioxane was removed to dryness. The deprotected amine was taken up in dimethylforamide (4 mL) and cooled to 0 oC. Then corresponding carboxylic acid (1.2 mmol, 1.2 eq) and 2-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (1.5 mmol, 1.5 eq) were added, and the solution was stirred for 15 min at 0 oC. After that N,N-diisopropyl ethylamin (4.0 mmol, 4.0 eq) was added dropwise to the mixture at 0 oC, and the resulting mixture was stirred at room temperature overnight. After completion, the mixture was distributed in water and ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated.

The

resulting

residue

was

purified

by

silica

gel

chromatography

(dichloromethane/methanol = 98:2 + 1% of aqueous ammonia) to afford target compounds 8c−e. 1-(3-(4-Amino-5-(3,5-dimethoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidin-1yl)prop-2-en-1-one (6a). The title compound was prepared from 13a following general procedure D. Yield: 47%. 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.58 (s, 2H), 6.52−6.39 (m, 3H), 5.78−5.70 (m, 1H), 5.57−5.45 (m, 1H), 5.36 (s, 2H), 4.25−4.00 (m, 2H), 3.83 (s, 6H), 3.84−3.75 (m, 2H), 2.60−2.35 (m, 2H).

13

C NMR (126 MHz, DMSO-d6) δ 163.52, 163.44,

160.76, 157.20, 151.65, 151.62, 150.38, 136.45, 136.42, 129.47, 129.10, 127.02, 126.97, 120.87, 120.78, 115.99, 115.90, 106.39, 106.35, 100.06, 99.99, 98.83, 55.23, 52.89, 51.62, 50.36, 49.99, 44.58, 44.05, 31.06, 29.28. HRMS m/z C21H24N5O3: calcd 394.1874, found 394.1877 [M + H]+. 28 ACS Paragon Plus Environment

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1-(3-(4-Amino-5-(5,7-dimethoxybenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7yl)pyrrolidin-1-yl)prop-2-en-1-one (6b). The title compound was prepared from 13b following general procedure D. Yield: 41%. 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 7.21 (s, 1H), 7.13 (s, 0.5H), 7.09 (s, 0.5H), 6.87 (s, 1H), 6.50−6.46 (m, 2H), 6.42 (d, J = 5.6 Hz, 1H), 5.80−5.70 (m, 1H), 5.60 (s, 2H), 5.55−5.45 (m, 1H), 4.20−4.00 (m, 2H), 3.97 (s, 3H), 3.88 (s, 3H), 3.88−3.75 (m, 2H), 2.60−2.40 (m, 2H). HRMS m/z C23H24N5O3S: calcd 450.1594, found 450.1600 [M + H]+. 1-(3-(4-Amino-5-(7-methoxybenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7yl)pyrrolidin-1-yl)prop-2-en-1-one (6c). The title compound was prepared from 13c following general procedure D. Yield: 62%. 1H NMR (300 MHz, CDCl3) δ 8.34 (s, 1H), 7.45−7.31 (m, 2H), 7.29 (d, J = 1.7 Hz, 1H), 7.14 (s, 0.5H), 7.11 (s, 0.5H), 6.81 (d, J = 7.8 Hz, 1H), 6.51−6.45 (m, 1H), 6.45−6.39 (m, 1H), 5.81−5.68 (m, 1H), 5.58 (s, 2H), 5.55−5.45 (m, 1H), 4.25−4.04 (m, 1H), 4.02 (s, 3H), 3.95−3.75 (m, 3H), 2.62−2.38 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 164.93, 164.83, 157.27, 157.22, 154.35, 152.58, 152.52, 151.08, 151.01, 142.08, 136.73, 136.58, 128.80, 128.70, 128.57, 128.18, 127.91, 126.37, 126.33, 123.23, 123.19, 121.13, 121.06, 116.28, 109.99, 109.81, 104.57, 104.52, 101.64, 101.58, 55.87, 53.74, 52.24, 51.57, 50.44, 45.00, 44.47, 32.72, 30.06. HRMS m/z C22H22N5O2S: calcd 420.1489, found 420.1495 [M + H]+. 1-(3-(4-Amino-5-(7-methoxybenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7yl)azetidin-1-yl)prop-2-en-1-one (6d). The title compound was prepared from 13d following general procedure D. Yield: 45%. 1H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H), 7.44−7.30 (m, 4H), 6.81 (d, J = 7.5 Hz, 1H), 6.42 (dd, J = 16.9, 1.6 Hz, 1H), 6.24 (dd, J = 17.0, 10.2 Hz, 1H), 5.80−5.57 (m, 4H), 4.84−4.45 (m, 4H), 4.02 (s, 3H).

13

C NMR (126 MHz, CDCl3) δ 166.04, 157.32, 154.37,

152.83, 151.45, 142.06, 136.43, 128.62, 128.45, 126.39, 125.75, 123.33, 120.88, 116.31, 110.69, 29 ACS Paragon Plus Environment


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104.61, 101.62, 57.81, 55.88, 55.26, 43.34. HRMS m/z C21H20N5O2S: calcd 406.1332, found 406.1342 [M + H]+. 1-(3-(4-Amino-5-(6-methoxybenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7yl)azetidin-1-yl)prop-2-en-1-one (6e). The title compound was prepared from 13e following general procedure D. Yield: 48%. 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.34 (s, 2H), 7.24 (s, 1H), 7.07−7.00 (m, 1H), 6.42 (d, J = 17.0 Hz, 1H), 6.25 (dd, J = 16.9, 10.3 Hz, 1H), 5.80−5.65 (m, 2H), 5.52 (s, 2H), 4.85−4.75 (m, 1H), 4.72−4.62 (m, 1H), 4.60−4.45 (m, 2H), 3.90 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 166.05, 157.81, 157.34, 152.88, 151.40, 141.47, 134.38, 133.16, 128.48, 125.75, 124.31, 122.63, 120.62, 115.05, 110.71, 104.92, 101.67, 57.86, 55.82, 55.29, 43.23. HRMS m/z C21H20N5O2S: calcd 406.1332, found 406.1343 [M + H]+. 1-(3-(4-Amino-5-(5-methoxybenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7yl)azetidin-1-yl)prop-2-en-1-one (6f). The title compound was prepared from 13f following general procedure D. Yield: 57%. 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.37 (s, 1H), 7.26 (s, 2H), 7.02 (dd, J = 8.8, 2.4 Hz, 1H), 6.43 (d, J = 15.2 Hz, 1H), 6.25 (dd, J = 17.0, 10.2 Hz, 1H), 5.80−5.60 (m, 4H), 4.85−4.45 (m, 4H), 3.89 (s, 3H). HRMS m/z C21H20N5O2S: calcd 406.1332, found 406.1342 [M + H]+. 1-(3-(4-Amino-5-(7-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7yl)pyrrolidin-1-yl)prop-2-en-1-one (6g). The title compound was prepared from 13g following general procedure D. Yield: 49%. 1H NMR (300 MHz, CDCl3) δ 8.34 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.37−7.29 (m, 2H), 7.20−7.08 (m, 2H), 6.51−6.39 (m, 2H), 5.80−5.68 (m, 1H), 5.60 (s, 2H), 5.54−5.46 (m, 1H), 4.25−4.05 (m, 1H), 3.80−3.75 (m, 3H), 2.58 (s, 3H), 2.54−2.35 (m, 2H).

13

C

NMR (126 MHz, CDCl3) δ 164.95, 164.85, 157.27, 157.23, 152.63, 152.56, 151.10, 151.03, 140.30, 30 ACS Paragon Plus Environment

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140.17, 135.80, 135.65, 131.98, 128.81, 128.73, 128.18, 127.93, 125.45, 125.42, 125.01, 124.95, 123.62, 123.60, 121.26, 121.12, 121.02, 110.13, 109.93, 101.67, 101.58, 53.72, 52.25, 51.65, 50.43, 45.01, 44.51, 32.78, 30.11, 20.53. HRMS m/z C22H22N5OS: calcd 404.1540, found 404.1541 [M + H]+. 1-(3-(4-Amino-5-(benzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidin-1yl)prop-2-en-1-one (6h). The title compound was prepared from 13h following general procedure D. Yield: 53%. 1H NMR (300 MHz, DMSO-d6) δ 8.19 (s, 1H), 7.98 (d, J = 7.3 Hz, 1H), 7.92 (s, 1H), 7.87 (d, J = 7.0 Hz, 1H), 7.46 (s, 1H), 7.45−7.32 (m, 2H), 6.56 (s, 2H), 6.38 (dd, J = 17.0, 10.2 Hz, 1H), 6.17 (dd, J = 18.0, 3.2 Hz, 1H), 5.71 (dd, J = 9.8, 3.0 Hz, 1H), 5.67−5.57 (m, 1H), 4.79−4.66 (m, 2H), 4.50−4.35 (m, 2H). HRMS m/z C20H18N5OS: calcd 376.1227, found 376.1232 [M + H]+. 1-(3-(4-Amino-5-(4,7-dimethoxybenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7yl)azetidin-1-yl)prop-2-en-1-one (6i). The title compound was prepared from 13i following general procedure D. Yield: 63%. 1H NMR (300 MHz, DMSO-d6) δ 8.19 (s, 1H), 7.95 (s, 1H), 7.40 (s, 1H), 6.86 (s, 2H), 6.54 (s, 2H), 6.45−6.30 (m, 1H), 6.15 (d, J = 17.1 Hz, 1H), 5.71 (d, J = 10.2 Hz, 1H), 5.68−5.55 (m, 1H), 4.78−4.65 (m, 2H), 4.48−4.35 (m, 2H), 3.91 (s, 3H), 3.88 (s, 3H). HRMS m/z C22H22N5O3S: calcd 436.1438, found 436.1446 [M + H]+. 1-(3-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)pyrrolidin-1-yl)prop-2-en-1-one (6j). The title compound was prepared from 13j following general procedure D. Yield: 49%. 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 7.21 (s, 2H), 7.11 (d, J = 10.6 Hz, 1H), 6.63 (s, 1H), 6.50−6.39 (m, 2H), 5.79−5.68 (m, 1H), 5.60−5.42 (m, 3H), 4.24−4.02 (m, 1H), 3.99 (s, 3H), 3.93−3.70 (m, 3H), 2.62−2.50 (s, 1H), 2.48 (s, 3H), 2.45−2.33 (m, 1H).

13

C

NMR (126 MHz, CDCl3) δ 164.92, 164.82, 157.25, 157.19, 153.99, 152.46, 152.37, 151.00, 150.92, 31 ACS Paragon Plus Environment


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142.12, 136.61, 136.59, 136.54, 136.48, 128.77, 128.67, 128.18, 127.90, 125.82, 122.99, 122.96, 121.03, 120.95, 116.19, 110.14, 109.98, 106.47, 106.42, 101.62, 101.57, 55.79, 53.71, 52.21, 51.54, 50.42, 44.99, 44.46, 32.69, 30.03, 22.05. HRMS m/z C23H24N5O2S: calcd 434.1645, found 434.1647 [M + H]+. 1-(3-(4-Amino-5-(benzofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidin-1-yl)prop-2en-1-one (6k). The title compound was prepared from 13k following general procedure D. Yield: 57%. 1H NMR (300 MHz, CDCl3) δ 8.34 (s, 1H), 7.61−7.55 (m, 1H), 7.51 (d, J = 7.1 Hz, 1H), 7.35 (d, J = 9.1 Hz, 1H), 7.31−7.25 (m, 2H), 6.85 (d, J = 5.5 Hz, 1H), 6.53−6.46 (m, 1H), 6.46−6.41 (m, 1H), 6.22 (s, 2H), 5.81−5.71 (m, 1H), 5.60−5.46 (m, 1H), 4.25−3.76 (m, 4H), 2.64−2.37 (m, 2H). HRMS m/z C21H20N5O2: calcd 374.1612, found 374.1622 [M + H]+. 1-(3-(4-Amino-5-(7-methoxy-5-methylbenzofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7yl)azetidin-1-yl)prop-2-en-1-one (6l). The title compound was prepared from 13l following general procedure D. Yield: 50%. 1H NMR (300 MHz, CD3OD) δ 8.14 (s, 1H), 8.00 (s, 1H), 6.94 (s, 2H), 6.71 (s, 1H), 6.48−6.25 (m, 2H), 5.79 (d, J = 10.0 Hz, 1H), 5.68−5.58 (m, 1H), 4.90−4.78 (m, 2H), 4.66−4.48 (m, 2H), 3.98 (s, 3H), 2.42 (s, 3H). HRMS m/z C22H22N5O3: calcd 404.1717, found 404.1718 [M + H]+. 1-(3-(4-Amino-2-chloro-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3d]pyrimidin-7-yl)azetidin-1-yl)prop-2-en-1-one (7a). The title compound was prepared from 17a following general procedure D. Yield: 49%. 1H NMR (300 MHz, CDCl3) δ 7.36 (s, 1H), 7.23 (s, 2H), 6.66 (s, 1H), 6.42 (d, J = 16.9 Hz, 1H), 6.30−6.19 (m, 1H), 5.80−5.65 (m, 4H), 4.85−4.60 (m, 2H), 4.50−4.35 (m, 2H), 4.00 (s, 3H), 2.49 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 166.07, 158.08, 154.38, 154.00, 152.22, 141.98, 136.77, 135.55, 128.65, 125.87, 125.62, 123.32, 120.55, 116.26, 111.43, 32 ACS Paragon Plus Environment

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106.65, 99.97, 57.88, 55.81, 55.31, 43.05, 22.07. HRMS m/z C22H21ClN5O2S: calcd 454.1099, found 454.1094 [M + H]+. 1-(3-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-2-methyl-7H-pyrrolo[2,3d]pyrimidin-7-yl)pyrrolidin-1-yl)prop-2-en-1-one (7b). The title compound was prepared from 17b following general procedure D. Yield: 54%. 1H NMR (300 MHz, CDCl3) δ 7.21 (s, 1H), 7.19 (s, 1H), 7.05 (d, J = 9.7 Hz, 1H), 6.63 (s, 1H), 6.50−6.40 (m, 2H), 5.78−5.68 (m, 1H), 5.61−5.41 (m, 3H), 4.21−4.07 (m, 1H), 3.98 (s, 3H), 3.96−3.70 (m, 3H), 2.55 (s, 3H), 2.48 (s, 3H), 2.47−2.31 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 164.90, 164.81, 161.69, 161.51, 156.91, 156.83, 153.98, 152.03, 151.95, 142.17, 136.96, 136.83, 136.53, 136.48, 128.70, 128.61, 128.23, 127.94, 125.72, 122.69, 122.67, 120.39, 120.25, 116.17, 110.16, 109.97, 106.40, 106.36, 99.27, 99.21, 55.79, 53.25, 51.84, 51.62, 50.58, 45.08, 44.53, 32.67, 30.17, 25.71, 25.66, 22.05. HRMS m/z C24H26N5O2S: calcd 448.1802, found 448.1809 [M + H]+. 1-(3-(2,4-Diamino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3d]pyrimidin-7-yl)pyrrolidin-1-yl)prop-2-en-1-one (7c). The title compound was prepared from 17c following general procedure D. Yield: 47%. 1H NMR (300 MHz, CDCl3) δ 7.19 (s, 1H), 7.18 (s, 1H), 6.82 (d, J = 9.5 Hz, 1H), 6.62 (s, 1H), 6.50−6.36 (m, 2H), 5.78−5.66 (m, 1H), 5.39 (s, 2H), 5.35−5.20 (m, 1H), 4.71 (s, 2H), 4.17−4.02 (m, 1H), 3.99 (s, 3H), 3.94−3.65 (m, 3H), 2.47 (s, 3H), 2.45−2.30 (m, 2H).

13

C NMR (126 MHz, CDCl3) δ 164.88, 164.82, 159.50, 159.45, 157.89, 157.84, 153.99,

153.57, 153.51, 142.23, 137.57, 137.43, 136.42, 136.37, 128.60, 128.50, 128.31, 128.00, 125.54, 122.17, 122.13, 118.24, 118.16, 116.10, 110.54, 110.35, 106.29, 106.24, 95.86, 95.81, 55.78, 53.23, 51.74, 51.38, 50.37, 45.05, 44.51, 32.48, 29.95, 22.05. HRMS m/z C23H25N6O2S: calcd 449.1754, found 449.1754 [M + H]+. 33 ACS Paragon Plus Environment


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1-(3-(5-(7-Methoxy-5-methylbenzo[b]thiophen-2-yl)-4-(methylamino)-7H-pyrrolo[2,3d]pyrimidin-7-yl)pyrrolidin-1-yl)prop-2-en-1-one (7d). The title compound was prepared from 17d following general procedure D. Yield: 44%. 1H NMR (300 MHz, DMSO-d6) δ 8.28 (s, 1H), 7.58 (s, 0.5H), 7.52 (s, 0.5H), 7.29 (s, 1H), 7.27 (s, 1H), 6.79 (s, 1H), 6.71−6.53 (m, 1H), 6.22−6.08 (m, 2H), 5.78−5.62 (m, 1H), 5.45−5.25 (m, 1H), 4.21−3.99 (m, 1H), 3.94 (s, 3H), 3.80−3.65 (m, 2H), 3.57−3.45 (m, 1H), 2.95 (d, J = 3.7 Hz, 3H), 2.43 (s, 3H), 2.42−2.32 (m, 2H). 13C NMR (126 MHz, CDCl3) δ 164.89, 164.80, 157.47, 157.44, 154.00, 152.76, 152.71, 150.06, 149.99, 142.12, 137.11, 136.96, 136.60, 136.55, 128.73, 128.63, 128.20, 127.93, 125.90, 122.95, 122.91, 120.28, 120.21, 116.18, 109.64, 109.47, 106.42, 106.37, 101.79, 101.75, 55.80, 53.61, 52.09, 51.61, 50.46, 44.99, 44.47, 32.77, 30.05, 28.01, 22.07. HRMS m/z C24H26N5O2S: calcd 448.1802, found 448.1798 [M + H]+. 1-(3-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)pyrrolidin-1-yl)propan-1-one (8a). The title compound was prepared from 13j following general procedure D. Yield: 45%. 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 7.23−7.19 (m, 2H), 7.12 (s, 0.5H), 7.07 (s, 0.5H), 6.63 (s, 1H), 5.68 (s, 2H), 5.52−5.39 (m, 1H), 4.10−4.02 (m, 1H), 4.00 (s, 3H), 3.93−3.80 (m, 1H), 3.74−3.62 (m, 2H), 2.48 (s, 3H), 2.44−2.22 (m, 4H), 1.25−1.13 (m, 3H). C NMR (126 MHz, CDCl3) δ 172.95, 172.78, 157.26, 157.20, 153.99, 152.47, 152.38, 150.99,

13

150.91, 142.14, 142.12, 136.69, 136.60, 136.55, 125.82, 122.97, 122.93, 121.08, 120.98, 116.19, 110.11, 109.90, 106.47, 106.42, 101.62, 101.55, 55.80, 53.72, 52.34, 51.59, 50.30, 44.88, 44.26, 32.74, 30.19, 28.10, 27.76, 22.06, 9.05. HRMS m/z C23H26N5O2S: calcd 436.1802, found 436.1797 [M + H]+.


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Synthesis of 5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7-(1-(vinylsulfonyl)azetidin-3-yl)7H-pyrrolo[2,3-d]pyrimidin-4-amine (8b). To a reaction flask containing 18i (100 mg, 0.21 mmol, 1.0 eq) was added 4 N HCl/dioxane (4 mL). The reaction mixture was stirred at room temperature for 3 h, then dioxane was removed to dryness. The deprotected amine was taken up in dry dichloromethane (5 mL). The resulting solution was treated with triethylamine (0.63 mmol, 3.0 eq) and stirred for 15 min at 0 oC. Then 2-chloroethanesulfochloride (0.23 mmol, 1.1 eq) was added dropwise to the mixture at 0 oC and the mixture was stirred at room temperature overnight. After completion, the mixture was distributed in water and dichloromethane. The organic layer was separated and washed with water and brine, dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel chromatography (dichloromethane/methanol = 98:2) to afford target compound 8b. Yield: 64%. 1H NMR (300 MHz, CDCl3) δ 8.30 (s, 1H), 7.39 (s, 1H), 7.24 (s, 2H), 6.73 (dd, J = 16.6, 9.9 Hz, 1H), 6.65 (s, 1H), 6.43 (d, J = 16.6 Hz, 1H), 6.24 (d, J = 9.9 Hz, 1H), 5.65−5.50 (m, 3H), 4.46−4.33 (m, 4H), 4.01 (s, 3H), 2.49 (s, 3H).

13

C NMR (126 MHz,

CDCl3) δ 157.34, 154.01, 152.60, 151.32, 142.08, 136.67, 136.20, 131.59, 130.80, 125.88, 123.19, 121.31, 116.24, 110.86, 106.54, 101.70, 57.38, 55.82, 42.84, 22.08. HRMS m/z C21H22N5O3S2: calcd 456.1159, found 456.1158 [M + H]+. 1-(3-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)pyrrolidin-1-yl)prop-2-yn-1-one (8c). The title compound was prepared from 13j following general procedure E. Yield: 34%. 1H NMR (300 MHz, CDCl3) δ 8.31 (d, J = 3.0 Hz, 1H), 7.22 (s, 2H), 7.10 (d, J = 9.7 Hz, 1H), 6.64 (s, 1H), 5.75 (s, 2H), 5.53−5.42 (m, 1H), 4.32−4.23 (m, 0.5H), 4.12−4.02 (m, 1H), 3.99 (s, 3H), 3.96−3.84 (m, 2H), 3.75−3.66 (m, 0.5H), 3.12 (s, 0.5H), 3.07 (s, 0.5H), 2.57−2.50 (m, 1H), 2.48 (s, 3H), 2.45−2.37 (m, 1H). 13C NMR (126 MHz, CDCl3) δ 157.22, 35 ACS Paragon Plus Environment


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157.19, 154.01, 152.44, 152.38, 151.89, 151.69, 151.02, 150.94, 142.12, 136.64, 136.61, 136.48, 136.40, 125.85, 123.05, 120.93, 120.90, 116.23, 110.32, 110.23, 106.51, 106.48, 101.65, 101.62, 78.47, 78.31, 76.43, 76.34, 55.82, 52.96, 52.71, 52.66, 43.93, 46.48, 43.99, 31.87, 30.55, 22.08. HRMS m/z C23H22N5O2S: calcd 432.1489, found 432.1496 [M + H]+. (E)-1-(3-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3d]pyrimidin-7-yl)pyrrolidin-1-yl)-4-(dimethylamino)but-2-en-1-one (8d). The title compound was prepared from 13j following general procedure E. Yield: 39%. 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 7.22 (s, 2H), 7.11 (d, J = 11.4 Hz,1H), 7.03–6.86 (m, 1H), 6.78 (d, J = 15.9 Hz, 0.5H), 6.61 (d, J = 17.7 Hz, 1.5H), 5.57 (s, 2H), 5.53–5.39 (m, 1H), 4.33–3.81 (m, 4H), 3.99 (s, 3H), 3.52 (s, 2H), 2.63 (s, 3H), 2.57 (s, 2H), 2.53–2.44 (m, 6H). HRMS m/z C26H31N6O2S: calcd 491.2224, found 491.2212 [M + H]+. 1-(3-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)pyrrolidin-1-yl)-2-((dimethylamino)methyl)prop-2-en-1-one (8e). The title compound was prepared from 13j following general procedure E. Yield: 41%. 1H NMR (300 MHz, CD3OD) δ 8.18 (s, 1H), 7.45 (d, J = 2.6 Hz, 1H), 7.25 (s, 1H), 7.22 (s, 1H), 6.73 (s, 1H), 5.92 (d, J = 10.7 Hz, 1H), 5.84 (d, J = 11.0 Hz, 1H), 5.46−5.32 (m, 1H), 4.25−3.58 (m, 9H), 2.70 (s, 3H), 2.62 (s, 3H), 2.58−2.47 (m, 2H), 2.46 (s, 3H). HRMS m/z C26H31N6O2S: calcd 491.2224, found 491.2210 [M + H]+. 1-(4-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)piperidin-1-yl)prop-2-en-1-one (9a). The title compound was prepared from 18a following general procedure D. Yield: 53%. 1H NMR (300 MHz, CDCl3) δ 8.30 (s, 1H), 7.20 (s, 1H), 7.19 (s, 1H), 7.13 (s, 1H), 6.68−6.56 (m, 2H), 6.31 (dd, J = 16.7, 1.7 Hz, 1H), 5.80−5.68 (m, 3H), 5.04−4.84 (m, 2H), 4.18 (d, J = 11.4 Hz, 1H), 3.98 (s, 3H), 3.40−3.32 (m, 1H), 2.95−2.75 (m, 1H), 2.47 (s, 3H), 36 ACS Paragon Plus Environment

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2.25−1.80 (m, 4H). 13C NMR (126 MHz, CDCl3) δ 165.67, 157.25, 153.95, 152.10, 150.32, 142.14, 136.93, 136.48, 128.35, 127.58, 125.72, 122.67, 121.01, 116.12, 109.57, 106.33, 101.38, 55.76, 51.66, 45.29, 41.66, 33.28, 31.95, 22.03. HRMS m/z C24H26N5O2S: calcd 448.1802, found 448.1805 [M + H]+. 1-(3-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)piperidin-1-yl)prop-2-en-1-one (9b). The title compound was prepared from 18b following general procedure D. Yield: 43%. 1H NMR (300 MHz, CDCl3) δ 8.30 (s, 1H), 7.21 (s, 1H), 7.20 (s, 1H), 7.16 (s, 1H), 6.66−6.57 (m, 2H), 6.31 (dd, J = 16.9, 1.8 Hz, 1H), 5.82−5.66 (m, 3H), 4.90−4.55 (m, 2H), 4.40−4.25 (m, 0.5H), 3.99 (s, 3H), 3.45−3.10 (m, 2H), 2.90−2.70 (m, 0.5H), 2.48 (s, 3H) 2.35−1.65 (m, 4H). HRMS m/z C24H26N5O2S: calcd 448.1802, found 448.1804 [M + H]+. N-(4-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)cyclohexyl)acrylamide (9c). The title compound was prepared from 18c following general procedure D. Yield: 44%. 1H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H), 7.23−7.18 (m, 3H), 6.64 (s, 1H), 6.35 (d, J = 16.6 Hz, 1H), 6.27−6.13 (m, 2H), 5.69 (d, J = 10.1 Hz, 1H), 5.55 (s, 2H), 4.70−4.55 (m, 1H), 4.40−4.30 (m, 1H), 3.99 (s, 3H), 2.48 (s, 3H), 2.15−2.20 (m, 6H), 1.90−1.80 (m, 2H). HRMS m/z C25H28N5O2S: calcd 462.1958, found 462.1964 [M + H]+. 1-(3-((4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)methyl)pyrrolidin-1-yl)prop-2-en-1-one (9d). The title compound was prepared from 18d following general procedure D. Yield: 48%. 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 7.22 (s, 2H), 7.11 (d, J = 7.2 Hz, 1H), 6.64 (s, 1H), 6.45−6.30 (m, 2H), 5.72−5.62 (m, 1H), 5.55 (s, 2H), 4.38−4.15 (m, 2H), 4.00 (s, 3H), 3.83−3.35 (m, 4H), 3.00−2.80 (m, 1H), 2.49 (s, 3H), 2.17−1.96 (m, 1H), 1.88−1.81 (m, 1H). 13C NMR (126 MHz, CDCl3) δ 164.75, 157.23, 157.18, 154.01, 152.62, 152.51, 37 ACS Paragon Plus Environment


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151.04, 151.00, 142.20, 136.83, 136.79, 136.60, 136.51, 128.54, 128.39, 127.99, 127.95, 125.82, 125.77, 124.33, 124.31, 122.81, 122.76, 116.18, 109.51, 109.45, 106.43, 106.39, 101.34, 101.32, 55.81, 50.00, 49.30, 46.91, 46.85, 45.84, 45.22, 40.46, 38.43, 29.75, 28.13, 22.07. HRMS m/z C24H26N5O2S: calcd 448.1802, found 448.1803 [M + H]+. N-(2-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)ethyl)acrylamide (9e). The title compound was prepared from 18e following general procedure D. Yield: 50%. 1H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H), 7.21 (s, 1H), 7.20 (s, 1H), 7.11 (s, 1H), 7.07 (s, 1H), 6.64 (s, 1H), 6.25 (d, J = 17.3 Hz, 1H), 6.14−6.01 (m, 1H), 5.67−5.56 (m, 3H), 4.47−4.36 (m, 2H), 4.00 (s, 3H), 3.80−3.70 (m, 2H), 2.48 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 166.15, 157.31, 154.01, 152.40, 151.01, 142.17, 136.70, 136.56, 130.95, 126.56, 125.82, 125.17, 122.81, 116.19, 109.22, 106.41, 101.60, 55.80, 44.89, 40.99, 22.06. HRMS m/z C21H22N5O2S: calcd 408.1489, found 408.1491 [M + H]+. 1-(2-((4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)methyl)pyrrolidin-1-yl)prop-2-en-1-one (9f). The title compound was prepared from 18f following general procedure D. Yield: 55%. 1H NMR (300 MHz, CDCl3) δ 8.37−8.30 (m, 1H), 7.23−7.16 (m, 3H), 6.63 (s, 1H), 6.52−6.42 (m, 2H), 5.78−5.72 (m, 1H), 5.62−5.45 (m, 3H), 4.53 (s, 3H), 3.99 (s, 3H), 3.55−3.43 (m, 2H), 2.48 (s, 3H), 2.00−1.55 (m, 3H). HRMS m/z C24H26N5O2S: calcd 448.1802, found 448.1803 [M + H]+. (S)-1-(3-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3d]pyrimidin-7-yl)pyrrolidin-1-yl)prop-2-en-1-one (9g). The title compound was prepared from 18g following general procedure D. Yield: 46%. 1H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H), 7.20 (s, 2H), 7.11 (d, J = 10.8 Hz, 1H), 6.62 (s, 1H), 6.49−6.38 (m, 2H), 5.78−5.64 (m, 3H), 5.55−5.40 (m, 38 ACS Paragon Plus Environment

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1H), 4.22−3.68 (m, 4H), 3.99 (s, 3H), 2.62−2.32 (m, 2H), 2.48 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 164.88, 164.78, 157.32, 157.28, 153.96, 152.59, 152.54, 151.02, 150.94, 142.11, 136.67, 136.55, 136.52, 136.50, 128.72, 128.61, 128.17, 127.90, 125.78, 122.95, 122.91, 120.97, 120.90, 116.17, 110.06, 109.89, 106.43, 106.38, 101.61, 101.56, 55.77, 53.67, 52.17, 51.50, 50.39, 44.97, 44.44, 32.65, 29.99, 22.03. HRMS m/z C23H24N5O2S: calcd 434.1645, found 434.1647 [M + H]+. (R)-1-(3-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3d]pyrimidin-7-yl)pyrrolidin-1-yl)prop-2-en-1-one (9h). The title compound was prepared from 18h following general procedure D. Yield: 40%. 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 7.21 (s, 2H), 7.11 (d, J = 10.6 Hz, 1H), 6.63 (s, 1H), 6.50−6.39 (m, 2H), 5.79−5.68 (m, 1H), 5.60−5.42 (m, 3H), 4.24−4.02 (m, 1H), 3.99 (s, 3H), 3.93−3.70 (m, 3H), 2.62−2.50 (m, 1H), 2.48 (s, 3H), 2.45−2.33 (m, 1H). HRMS m/z C23H24N5O2S: calcd 434.1645, found 434.1647 [M + H]+. 1-(3-(4-Amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)azetidin-1-yl)prop-2-en-1-one (9i). The title compound was prepared from 18i following general procedure D. Yield: 53%. 1H NMR (300 MHz, CDCl3) δ 8.30 (s, 1H), 7.35 (s, 1H), 7.25−7.21 (m, 2H), 6.64 (s, 1H), 6.47−6.37 (m, 1H), 6.24 (dd, J = 17.0, 10.1 Hz, 1H), 5.79−5.61 (m, 4H), 4.83−4.73 (m, 1H), 4.72−4.62 (m, 1H), 4.60−4.45 (m, 2H), 3.99 (s, 3H), 2.48 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 166.04, 157.33, 154.02, 152.83, 151.42, 142.10, 136.63, 136.36, 128.45, 125.89, 125.76, 123.11, 120.77, 116.23, 110.83, 106.52, 101.64, 57.82, 55.81, 55.26, 43.31, 22.07. HRMS m/z C22H22N5O2S: calcd 420.1489, found 420.1493 [M + H]+. tert-Butyl

3-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate

(11a). The title compound was prepared from 10 following general procedure A. Yield: 78%. 1H



Page 40 of 78

NMR (300 MHz, CDCl3) δ 8.62 (s, 1H), 7.39 (s, 1H), 5.47 (s, 1H), 3.95−3.85 (m, 1H), 3.78−3.52 (m, 3H), 2.52−2.38 (m, 1H), 2.30−2.18 (m, 1H), 1.49 (s, 9H). MS (ESI) m/z 449.1 [M + H]+. tert-Butyl 3-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidine-1-carboxylate (11b). The title compound was prepared from 10 following general procedure A. Yield: 75%. 1H NMR (300 MHz, CDCl3) δ 8.61 (s, 1H), 7.69 (s, 1H), 5.63−5.53 (m, 1H), 4.50 (t, J = 8.9 Hz, 2H), 4.25−4.18 (m, 2H), 1.48 (s, 9H). MS (ESI) m/z 435.1 [M + H]+. tert-Butyl

4-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)piperidine-1-carboxylate

(11c). The title compound was prepared from 10 following general procedure A. Yield: 51%. 1H NMR (300 MHz, CDCl3) δ 8.60 (s, 1H), 7.43 (s, 1H), 4.95−4.82 (m, 1H), 4.40−4.20 (m, 2H), 3.00−2.85 (m, 2H), 2.10−2.00 (m, 2H), 1.97−1.80 (m, 2H), 1.49 (s, 9H). MS (ESI) m/z 463.1 [M + H]+. tert-Butyl

3-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)piperidine-1-carboxylate

(11d). The title compound was prepared from 10 following general procedure A. Yield: 19%. 1H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H), 7.47 (s, 1H), 4.82−4.72 (m, 1H), 4.25−4.12 (m, 1H), 4.00−3.85 (m, 1H), 3.38−3.24 (m, 1H), 3.09−2.97 (m, 1H), 2.20−2.01 (m, 2H), 1.86−1.65 (m, 2H), 1.46 (s, 9H). MS (ESI) m/z 463.1 [M + H]+. tert-Butyl (4-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)carbamate (11e). The title compound was prepared from 10 following general procedure A. Yield: 73%. 1H NMR (300 MHz, CDCl3) δ 8.59 (s, 1H), 7.50 (s, 1H), 4.78 (s, 1H), 4.75−4.65 (m, 1H), 3.91 (s, 1H), 2.08−1.88 (m, 6H), 1.87−1.74 (m, 2H), 1.47 (s, 9H). MS (ESI) m/z 477.1 [M + H]+. tert-Butyl

2-((4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)pyrrolidine-1-

carboxylate (11f). The title compound was prepared from 10 following general procedure A. Yield: 40 ACS Paragon Plus Environment

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45%. 1H NMR (300 MHz, CDCl3) δ 8.59 (s, 1H), 7.41 (s, 0.5H), 7.30 (s, 0.5H), 5.09−4.95 (m, 1H), 4.53−4.28 (m, 2H), 4.22−4.10 (m, 1H), 3.48−3.15 (m, 2H), 1.90−1.70 (m, 3H), 1.50−1.36 (m, 9H). MS (ESI) m/z 463.1 [M + H]+. tert-Butyl

3-((4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)pyrrolidine-1-

carboxylate (11g). The title compound was prepared from 10 following general procedure A. Yield: 51%. 1H NMR (300 MHz, CDCl3) δ 8.61 (s, 1H), 7.37 (s, 1H), 4.28−4.20 (m, 2H), 3.49−3.30 (m, 3H), 3.16−3.05 (m, 1H), 2.80−2.70 (m, 1H), 1.98−1.85 (m, 1H), 1.70−1.60 (m, 1H), 1.45 (s, 9H). MS (ESI) m/z 463.1 [M + H]+. Synthesis of tert-butyl (2-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)carbamate (11h). In a reaction flask, 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine 10 (100 mg, 0.4 mmol, 1.0 eq), tert-butyl 2-bromoethylcarbamate (108 mg, 0.5 mmol, 1.2 eq), and cesium carbonate (200 mg, 0.6 mmol, 1.5 eq) were suspended in dimethylforamide (2 mL), and the mixture was stirred at 80 oC under argon for 6 h. After cooling, the mixture was distributed in water and ethyl acetate. The organic layer was separated and washed with water and brine, dried over anhydrous sodium sulfate and concentrated. The resulting residue was purified by silica gel chromatography (petroleum ether/ethyl acetate = 4:1) to afford compound 11h. Yield: 63%. 1H NMR (300 MHz, CDCl3) δ 8.60 (s, 1H), 7.38 (s, 1H), 4.75 (s, 1H), 4.40 (t, J = 6.0 Hz, 2H), 3.55−3.45 (m, 2H), 1.40 (s, 9H). MS (ESI) m/z 423.1 [M + H]+. tert-Butyl (S)-3-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (11i). The title compound was prepared from 10 following general procedure A. Yield: 75%. 1H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H), 7.39 (s, 1H), 5.47 (s, 1H), 3.95−3.85 (m, 1H), 3.78−3.52 (m, 3H), 2.52−2.38 (m, 1H), 2.30−2.18 (m, 1H), 1.49 (s, 9H). MS (ESI) m/z 449.1 [M + H]+. 41 ACS Paragon Plus Environment


tert-Butyl

Page 42 of 78

(R)-3-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-

carboxylate (11j). The title compound was prepared from 10 following general procedure A. Yield: 76%. 1H NMR (300 MHz, CDCl3) δ 8.62 (s, 1H), 7.39 (s, 1H), 5.47 (s, 1H), 3.95−3.85 (m, 1H), 3.78−3.52 (m, 3H), 2.52−2.38 (m, 1H), 2.30−2.18 (m, 1H), 1.49 (s, 9H). MS (ESI) m/z 449.1 [M + H]+. tert-Butyl

3-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate

(12a). The title compound was prepared from 11a following general procedure B. Yield: 95%. 1H NMR (300 MHz, CDCl3) δ 8.26 (s, 1H), 7.06 (s, 1H), 5.72 (s, 2H), 5.39 (s, 1H), 3.90−3.80 (m, 1H), 3.70−3.46 (m, 3H), 2.46−2.35 (m, 1H), 2.26−2.14 (m, 1H), 1.48 (s, 9H). MS (ESI) m/z 430.2 [M + H]+. tert-Butyl 3-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidine-1-carboxylate (12b). The title compound was prepared from 11b following general procedure B. Yield: 93%. 1H NMR (300 MHz, CDCl3) δ 8.22 (s, 1H), 7.35 (s, 1H), 5.99 (s, 2H), 5.56−5.46 (m, 1H), 4.45 (t, J = 8.6 Hz, 2H), 4.20−4.12 (m, 2H), 1.47 (s, 9H). MS (ESI) m/z 416.1 [M + H]+. tert-Butyl

4-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)piperidine-1-carboxylate

(12c). The title compound was prepared from 11c following general procedure B. Yield: 91%. 1H NMR (300 MHz, CDCl3) δ 8.25 (s, 1H), 7.08 (s, 1H), 5.79 (s, 2H), 4.85−4.73 (m, 1H), 4.40−4.20 (m, 2H), 2.98−2.82 (m, 2H), 2.06−1.96 (m, 2H), 1.91−1.77 (m, 2H), 1.47 (s, 9H). MS (ESI) m/z 444.2 [M + H]+. tert-Butyl

3-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)piperidine-1-carboxylate

(12d). The title compound was prepared from 11d following general procedure B. Yield: 92%. 1H NMR (300 MHz, CDCl3) δ 8.25 (s, 1H), 7.12 (s, 1H), 5.85 (s, 2H), 4.74−4.62 (m, 1H), 4.25−4.06 (m, 42 ACS Paragon Plus Environment

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1H), 3.95 (d, J = 13.0 Hz, 1H), 3.30−3.15 (m, 1H), 3.05−2.90 (m, 1H), 2.18−2.08 (m, 1H), 2.05−1.90 (m, 1H), 1.88−1.73(m, 1H), 1.72−1.60 (m, 1H), 1.45 (s, 9H). MS (ESI) m/z 444.2 [M + H]+. tert-Butyl (4-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)cyclohexyl)carbamate (12e). The title compound was prepared from 11e following general procedure B. Yield: 89%. 1H NMR (300 MHz, DMSO-d6) δ 8.07 (s, 1H), 7.69 (s, 1H), 7.07 (d, J = 8.1 Hz, 1H), 6.57 (s, 2H), 4.63−4.50 (m, 1H), 3.78 (s, 1H), 2.10−1.95 (m, 2H), 1.73−1.59 (m, 6H), 1.42 (s, 9H). MS (ESI) m/z 458.2 [M + H]+. tert-Butyl

2-((4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)pyrrolidine-1-

carboxylate (12f). The title compound was prepared from 11f following general procedure B. Yield: 90%. 1H NMR (300 MHz, CDCl3) δ 8.21 (s, 1H), 7.06 (s, 0.5H), 6.95 (s, 0.5H), 5.95 (s, 2H), 5.00−4.87 (m, 1H), 4.41−4.07 (m, 3H), 3.41−3.15 (m, 2H), 1.90−1.70 (m, 3H), 1.50−1.35 (m, 9H). MS (ESI) m/z 444.2 [M + H]+. tert-Butyl

3-((4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl)pyrrolidine-1-

carboxylate (12g). The title compound was prepared from 11g following general procedure B. Yield: 90%. 1H NMR (300 MHz, CDCl3) δ 8.23 (s, 1H), 7.01 (s, 1H), 5.87 (s, 2H), 4.28−4.04 (m, 2H), 3.55−3.40 (m, 2H), 3.35−3.23 (m, 1H), 3.17−3.03 (m, 1H), 2.80−2.65 (m, 1H), 1.95−1.85 (m, 1H), 1.69−1.55 (m, 1H), 1.44 (s, 9H). MS (ESI) m/z 444.2 [M + H]+. tert-Butyl (2-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl)carbamate (12h). The title compound was prepared from 11h following general procedure B. Yield: 90%. 1H NMR (300 MHz, CDCl3) δ 8.24 (s, 1H), 7.04 (s, 1H), 5.66 (s, 2H), 5.03−4.94 (m, 1H), 4.29 (t, J = 5.3 Hz, 2H), 3.49 (dd, J = 11.7, 5.9 Hz, 2H), 1.41 (s, 9H). MS (ESI) m/z 404.2 [M + H]+.



Page 44 of 78

tert-Butyl (S)-3-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (12i). The title compound was prepared from 11i following general procedure B. Yield: 93%. 1H NMR (300 MHz, CDCl3) δ 8.26 (s, 1H), 7.06 (s, 1H), 5.72 (s, 2H), 5.39 (s, 1H), 3.90−3.80 (m, 1H), 3.70−3.46 (m, 3H), 2.46−2.35 (m, 1H), 2.26−2.14 (m, 1H), 1.48 (s, 9H). MS (ESI) m/z 430.2 [M + H]+. tert-Butyl

(R)-3-(4-amino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-

carboxylate (12j). The title compound was prepared from 11j following general procedure B. Yield: 95%. 1H NMR (300 MHz, CDCl3) δ 8.26 (s, 1H), 7.06 (s, 1H), 5.72 (s, 2H), 5.39 (s, 1H), 3.90−3.80 (m, 1H), 3.70−3.46 (m, 3H), 2.46−2.35 (m, 1H), 2.26−2.14 (m, 1H), 1.48 (s, 9H). MS (ESI) m/z 430.2 [M + H]+. tert-Butyl

3-(4-amino-5-(3,5-dimethoxyphenyl)-7H-pyrrolo[2,3-d]pyrimidin-7-

yl)pyrrolidine-1-carboxylate (13a). The title compound was prepared from 12a following general procedure C. Yield: 77%. 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 7.00 (s, 1H), 6.60 (s, 2H), 6.47 (s, 1H), 5.52−5.40 (m, 1H), 5.28 (s, 2H), 3.95−3.88 (m, 1H), 3.83 (s, 6H), 3.65−3.50 (m, 3H), 2.50−2.38 (m, 1H), 2.35−2.23 (m, 1H), 1.47 (s, 9H). MS (ESI) m/z 440.3 [M + H]+. tert-Butyl 3-(4-amino-5-(5,7-dimethoxybenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)pyrrolidine-1-carboxylate (13b). The title compound was prepared from 12a and 23b following general procedure C. Yield: 76%. 1H NMR (300 MHz, CDCl3) δ 8.37 (s, 1H), 7.30−7.27 (m, 1H), 7.18 (s, 1H), 6.91 (s, 1H), 6.52 (s, 1H), 5.70 (s, 2H), 5.55−5.45 (m, 1H), 4.02 (s, 3H), 3.98−3.89 (m, 1H), 3.93 (s, 3H), 3.80−3.50 (m, 3H), 2.55−2.40 (m, 1H), 2.40−2.25 (m, 1H), 1.52 (s, 9H). MS (ESI) m/z 496.3 [M + H]+.


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tert-Butyl

3-(4-amino-5-(7-methoxybenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-

yl)pyrrolidine-1-carboxylate (13c). The title compound was prepared from 12a and 23c following general procedure C. Yield: 72%. 1H NMR (300 MHz, CDCl3) δ 8.34 (s, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.30 (s, 1H), 7.16 (s, 1H), 6.81 (d, J = 7.7 Hz, 1H), 5.59 (s, 2H), 5.50−5.42 (m, 1H), 4.02 (s, 3H), 3.98−3.88 (m, 1H), 3.76−3.52 (m, 3H), 2.51−2.37 (m, 1H), 2.36−2.24 (m, 1H), 1.48 (s, 9H). MS (ESI) m/z 466.3 [M + H]+. tert-Butyl


yl)azetidine-1-carboxylate (13d). The title compound was prepared from 12b and 23c following general procedure C. Yield: 75%. 1H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H), 7.44 (s, 1H), 7.42−7.31 (m, 3H), 6.81 (d, J = 7.6 Hz, 1H), 5.59 (s, 3H), 4.51 (t, J = 8.9 Hz, 2H), 4.30−4.21 (m, 2H), 4.02 (s, 3H), 1.49 (s, 9H). MS (ESI) m/z 452.2 [M + H]+. tert-Butyl


yl)azetidine-1-carboxylate (13e). The title compound was prepared from 12b and 23d following general procedure C. Yield: 69%. 1H NMR (300 MHz, CDCl3) δ 8.30 (s, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.41 (s, 1H), 7.33 (s, 1H), 7.25 (s, 1H), 7.04 (d, J = 8.7 Hz, 1H), 5.73 (s, 2H), 5.65−5.58 (m, 1H), 4.51 (t, J = 8.9 Hz, 2H), 4.32−4.20 (m, 2H), 3.90 (s, 3H), 1.49 (s, 9H). MS (ESI) m/z 452.2 [M + H]+. tert-Butyl


yl)azetidine-1-carboxylate (13f). The title compound was prepared from 12b and 23e following general procedure C. Yield: 72%. 1H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.42 (s, 1H), 7.26 (s, 2H), 7.02 (d, J = 8.8 Hz, 1H), 5.65 (s, 2H), 5.61−5.54 (m, 1H), 4.51 (t, J = 8.8 Hz, 2H), 4.30−4.20 (m, 2H), 3.89 (s, 3H), 1.48 (s, 9H). MS (ESI) m/z 452.3 [M + H]+. 45 ACS Paragon Plus Environment


tert-Butyl

Page 46 of 78

3-(4-amino-5-(7-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-

yl)pyrrolidine-1-carboxylate (13g). The title compound was prepared from 12a and 23f following general procedure C. Yield: 78%. 1H NMR (300 MHz, CDCl3) δ 8.34 (s, 1H), 7.65 (d, J = 7.3 Hz, 1H), 7.40−7.29 (m, 2H), 7.20−7.14 (m, 2H), 5.67 (s, 2H), 5.48 (s, 1H), 4.00−3.87 (m, 1H), 3.80−3.53 (m, 3H), 2.58 (s, 3H), 2.50−2.40 (m, 1H), 2.35−2.25 (m, 1H), 1.49 (s, 9H). MS (ESI) m/z 450.3 [M + H]+. tert-Butyl 3-(4-amino-5-(benzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidine1-carboxylate (13h). The title compound was prepared from 12b and 2-benzothienylboronic acid following general procedure C. Yield: 73%. 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 7.88−7.78 (m, 2H), 7.45−7.32 (m, 4H), 5.66−5.58 (m, 1H), 5.56 (s, 2H), 4.51 (t, J = 8.8 Hz, 2H), 4.29−4.22 (m, 2H), 1.49 (s, 9H). MS (ESI) m/z 422.2 [M + H]+. tert-Butyl 3-(4-amino-5-(4,7-dimethoxybenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)azetidine-1-carboxylate (13i). The title compound was prepared from 12b and 23a following general procedure C. Yield: 71%. 1H NMR (300 MHz, CDCl3) δ 8.30 (s, 1H), 7.47 (s, 1H), 7.42 (s, 1H), 6.71 (s, 2H), 5.65 (s, 2H), 5.62−5.55 (m, 1H), 4.50 (t, J = 8.8 Hz, 2H), 4.29−4.15 (m, 2H), 3.97 (s, 3H), 3.93 (s, 3H), 1.49 (s, 9H). MS (ESI) m/z 482.2 [M + H]+. tert-Butyl

3-(4-amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-

d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (13j). The title compound was prepared from 12a and 23h following general procedure C. Yield: 73%. 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 7.22 (s, 1H), 7.21 (s, 1H), 7.14 (s, 1H), 6.64 (s, 1H), 5.63 (s, 2H), 5.50−5.40 (m, 1H), 4.00 (s, 3H), 3.97−3.88 (m, 1H), 3.75−3.52 (m, 3H), 2.49 (s, 3H), 2.47−2.39 (m, 1H), 2.35−2.25 (m, 1H), 1.48 (s, 9H). MS (ESI) m/z 480.3 [M + H]+. 46 ACS Paragon Plus Environment

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tert-Butyl

3-(4-amino-5-(benzofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-

carboxylate (13k). The title compound was prepared from 12a and benzofuran-2-boronic acid following general procedure C. Yield: 70%. 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 7.60−7.55 (m, 1H), 7.54−7.47 (m, 1H), 7.37 (s, 1H), 7.30−7.25 (m, 2H), 6.84 (s, 1H), 6.25 (s, 2H), 5.47 (s, 1H), 3.99−3.87 (m, 1H), 3.80−3.50 (m, 3H), 2.53−2.40 (m, 1H), 2.36−2.20 (m, 1H), 1.49 (s, 9H). MS (ESI) m/z 420.3 [M + H]+. tert-Butyl 3-(4-amino-5-(7-methoxy-5-methylbenzofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin7-yl)azetidine-1-carboxylate (13l). The title compound was prepared from 12b and 23g following general procedure C. Yield: 75%. 1H NMR (300 MHz, CDCl3) δ 8.28 (s, 1H), 7.66 (s, 1H), 6.96 (s, 1H), 6.80 (s, 1H), 6.62 (s, 1H), 6.52 (s, 2H), 5.62 (s, 1H), 4.51 (t, J = 9.0 Hz, 2H), 4.24 (s, 2H), 3.99 (s, 3H), 2.45 (s, 3H), 1.50 (s, 9H). MS (ESI) m/z 450.3 [M + H]+. tert-Butyl

3-(2,4-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidine-1-carboxylate

(15a). The title compound was prepared from 14a following general procedure A. Yield: 39%. 1H NMR (300 MHz, DMSO-d6) δ 8.37 (s, 1H), 5.535.43 (m, 1H), 4.35−4.20 (m, 4H), 1.42 (s, 9H). MS (ESI) m/z 468.5 [M + H]+. tert-Butyl

3-(4-chloro-5-iodo-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-

carboxylate (15b). The title compound was prepared from 14b following general procedure A. Yield: 56%. 1H NMR (300 MHz, CDCl3) δ 7.27 (s, 1H), 5.52−5.36 (m, 1H), 3.95−3.83 (m, 1H), 3.72−3.48 (m, 3H), 2.73 (s, 3H), 2.47−2.36 (m, 1H), 2.28−2.14 (m, 1H), 1.49 (s, 9H). MS (ESI) m/z 463.1 [M + H]+. tert-Butyl 3-(4-chloro-5-iodo-2-pivalamido-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1carboxylate (15c). The title compound was prepared from 14c following general procedure A. Yield: 47 ACS Paragon Plus Environment


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55%. 1H NMR (300 MHz, CDCl3) δ 8.15 (s, 1H), 5.52 (s, 1H), 3.88 (dd, J = 11.8, 6.8 Hz, 1H), 3.70−3.45 (m, 3H), 2.50−2.40 (m, 1H), 2.23−2.12 (m, 1H), 1.48 (s, 9H), 1.34 (s, 9H). tert-Butyl

3-(4-amino-2-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)azetidine-1-

carboxylate (16a). The title compound was prepared from 15a following general procedure B. Yield: 93%. 1H NMR (300 MHz, CDCl3) δ 7.37 (s, 1H), 5.91 (s, 2H), 5.55−5.45 (m, 1H), 4.45 (t, J = 8.9 Hz, 2H), 4.13−4.06 (m, 2H), 1.48 (s, 9H). MS (ESI) m/z 450.1 [M + H]+. tert-Butyl

3-(4-amino-5-iodo-2-methyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-

carboxylate (16b). The title compound was prepared from 15b following general procedure B. Yield: 92%. 1H NMR (300 MHz, CDCl3) δ 6.98 (s, 1H), 5.64 (s, 2H), 5.45−5.32 (m, 1H), 3.91−3.82 (m, 1H), 3.65−3.45 (m, 3H), 2.53 (s, 3H), 2.45−2.30 (m, 1H), 2.22−2.10 (m, 1H), 1.48 (s, 9H). MS (ESI) m/z 444.2 [M + H]+. tert-Butyl 3-(2,4-diamino-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (16c). The title compound was prepared from 15c following general procedure B. Yield: 58%. 1H NMR (300 MHz, CDCl3) δ 6.76 (s, 1H), 5.51 (s, 2H), 5.18 (s, 1H), 4.67 (s, 2H), 3.85−3.70 (m, 1H), 3.68−3.40 (m, 3H), 2.40−2.24 (m, 1H), 2.20−2.08 (m, 1H), 1.45 (s, 9H). MS (ESI) m/z 445.3 [M + H]+. tert-Butyl

3-(5-iodo-4-(methylamino)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-

carboxylate (16d). The title compound was prepared from 11a following general procedure B. Yield: 88%. 1H NMR (300 MHz, CDCl3) δ 8.35 (s, 1H), 6.98 (s, 1H), 6.08 (s, 1H), 5.38 (s, 1H), 3.90−3.80 (m, 1H), 3.70−3.48 (m, 3H), 3.16 (d, J = 4.9 Hz, 3H), 2.45−2.30 (m, 1H), 2.26−2.10 (m, 1H), 1.47 (s, 9H). MS (ESI) m/z 444.1 [M + H]+.


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tert-Butyl

3-(4-amino-2-chloro-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-

pyrrolo[2,3-d]pyrimidin-7-yl)azetidine-1-carboxylate (17a). The title compound was prepared from 16a and 23h following general procedure C. Yield: 70%. 1H NMR (300 MHz, CDCl3) δ 7.42 (s, 1H), 7.23 (s, 2H), 6.65 (s, 1H), 5.69 (s, 2H), 5.63−5.53 (m, 1H), 4.55−4.43 (m, 2H), 4.22−4.15 (m, 2H), 4.00 (s, 3H), 2.49 (s, 3H), 1.48 (s, 9H). MS (ESI) m/z 500.3 [M + H]+. tert-Butyl

3-(4-amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-2-methyl-7H-

pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (17b). The title compound was prepared from 16b and 23h following general procedure C. Yield: 76%. 1H NMR (300 MHz, CDCl3) δ 7.21 (s, 1H), 7.20 (s, 1H), 7.08 (s, 1H), 6.63 (s, 1H), 5.74 (s, 2H), 5.48 (s, 1H), 3.99 (s, 3H), 3.95−3.88 (m, 1H), 3.73−3.50 (m, 3H), 2.59 (s, 3H), 2.48 (s, 3H), 2.47−2.37 (m, 1H), 2.30−2.20 (m, 1H), 1.48 (s, 9H). MS (ESI) m/z 494.3 [M + H]+. tert-Butyl

3-(2,4-diamino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-

d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (17c). The title compound was prepared from 16c and 23h following general procedure C. Yield: 72%. 1H NMR (300 MHz, CDCl3) δ 7.19 (s, 1H), 7.18 (s, 1H), 6.86 (s, 1H), 6.61 (s, 1H), 5.38 (s, 2H), 5.30−5.20 (m, 1H), 4.71 (s, 2H), 3.99 (s, 3H), 3.93−3.80 (m, 1H), 3.73−3.60 (m, 1H), 3.60−3.47 (m, 2H), 2.47 (s, 3H), 2.43−2.32 (m, 1H), 2.28−2.18 (m, 1H), 1.47 (s, 9H). MS (ESI) m/z 495.3 [M + H]+. tert-Butyl

3-(5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-4-(methylamino)-7H-

pyrrolo[2,3-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (17d). The title compound was prepared from 16d and 23h following general procedure C. Yield: 75%. 1H NMR (300 MHz, CDCl3) δ 8.42 (s, 1H), 7.24 (s, 1H), 7.17 (s, 1H), 7.07 (s, 1H), 6.65 (s, 1H), 5.58 (s, 1H), 5.51−5.37 (m, 1H), 4.01 (s,



Page 50 of 78

3H), 3.96−3.87 (m, 1H), 3.70−3.52 (m, 3H), 3.07 (d, J = 4.6 Hz, 3H), 2.50 (s, 3H), 2.48−2.38 (m, 1H), 2.34−2.24 (m, 1H), 1.47 (s, 9H). MS (ESI) m/z 494.3 [M + H]+. tert-Butyl


d]pyrimidin-7-yl)piperidine-1-carboxylate (18a). The title compound was prepared from 12c and 23h following general procedure C. Yield: 77%. 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 7.22 (s, 1H), 7.21 (s, 1H), 7.17 (s, 1H), 6.63 (s, 1H), 5.59 (s, 2H), 4.93−4.81 (m, 1H), 4.40−4.20 (m, 2H), 4.00 (s, 3H), 3.02−2.88 (m, 2H), 2.49 (s, 3H), 2.14−2.05 (m, 2H), 1.96−1.84 (m, 2H), 1.49 (s, 9H). MS (ESI) m/z 494.3 [M + H]+. tert-Butyl


d]pyrimidin-7-yl)piperidine-1-carboxylate (18b). The title compound was prepared from 12d and 23h following general procedure C. Yield: 80%. 1H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H), 7.23−7.18 (m, 3H), 6.63 (s, 1H), 5.73 (s, 2H), 4.80−4.70 (m, 1H), 4.35−4.20 (m, 1H), 4.10−4.00 (m, 1H), 3.99 (s, 3H), 3.29−3.16 (m, 1H), 3.00−2.85 (m, 1H), 2.48 (s, 3H), 2.25−2.15 (m, 1H), 2.10−1.95 (m, 1H), 1.90−1.80 (m, 1H), 1.75−1.65 (m, 1H), 1.45 (s, 9H). MS (ESI) m/z 494.5 [M + H]+. tert-Butyl

(4-(4-amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-

d]pyrimidin-7-yl)cyclohexyl)carbamate (18c). The title compound was prepared from 12e and 23h following general procedure C. Yield: 74%. 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 7.23 (s, 1H), 7.22−7.19 (m, 2H), 6.63 (s, 1H), 5.68 (s, 2H), 4.88 (s, 1H), 4.70 (s, 1H), 3.99 (s, 3H), 3.92 (s, 1H), 2.48 (s, 3H), 2.05−1.96 (m, 4H), 1.95−1.75 (m, 4H), 1.47 (s, 9H). MS (ESI) m/z 508.3 [M + H]+. tert-Butyl

3-((4-amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-

d]pyrimidin-7-yl)methyl)pyrrolidine-1-carboxylate (18d). The title compound was prepared from 12g and 23h following general procedure C. Yield: 76%. 1H NMR (300 MHz, CDCl3) δ 8.31 (s, 1H), 50 ACS Paragon Plus Environment

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7.22 (s, 2H), 7.10 (s, 1H), 6.63 (s, 1H), 5.70 (s, 2H), 4.30–4.15 (m, 2H), 4.00 (s, 3H), 3.60−3.42 (m, 2H), 3.40−3.25 (m, 1H), 3.23−3.10 (m, 1H), 2.86−2.74 (m, 1H), 2.49 (s, 3H), 2.02−1.90 (m, 1H), 1.74−1.62 (m, 1H), 1.45 (s, 9H). MS (ESI) m/z 494.3 [M + H]+. tert-Butyl

(2-(4-amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-

d]pyrimidin-7-yl)ethyl)carbamate (18e). The title compound was prepared from 12h and 23h following general procedure C. Yield: 78%. 1H NMR (300 MHz, CDCl3) δ 8.29 (s, 1H), 7.19 (s, 2H), 7.12 (s, 1H), 6.63 (s, 1H), 5.70 (s, 2H), 5.14 (t, J = 5.3 Hz, 1H), 4.36 (t, J = 5.2 Hz, 2H), 3.99 (s, 3H), 3.61−3.52 (m, 2H), 2.48 (s, 3H), 1.41 (s, 9H). MS (ESI) m/z 454.3 [M + H]+. tert-Butyl

2-((4-amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-

d]pyrimidin-7-yl)methyl)pyrrolidine-1-carboxylate (18f). The title compound was prepared from 12f and 23h following general procedure C. Yield: 71%. 1H NMR (300 MHz, CDCl3) δ 8.32 (s, 1H), 7.22 (s, 1H), 7.20 (s, 1H), 7.16 (s, 0.5H), 7.07 (s, 0.5H), 6.63 (s, 1H), 5.64 (s, 2H), 4.50−4.15 (m, 3H), 4.00 (s, 3H), 3.50−3.21 (m, 2H), 2.49 (s, 3H), 2.30−1.78 (m, 4H), 1.52−1.38 (m, 9H). MS (ESI) m/z 494.4 [M + H]+. tert-Butyl

(S)-3-(4-amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-

d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (18g). The title compound was prepared from 12i and 23h following general procedure C. Yield: 73%. 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 7.22 (s, 1H), 7.21 (s, 1H), 7.15 (s, 1H), 6.64 (s, 1H), 5.64 (s, 2H), 5.50−5.40 (m, 1H), 4.00 (s, 3H), 3.97−3.88 (m, 1H), 3.75−3.52 (m, 3H), 2.49 (s, 3H), 2.47−2.39 (m, 1H), 2.35−2.25 (m, 1H), 1.48 (s, 9H). MS (ESI) m/z 480.3 [M + H]+. tert-Butyl

(R)-3-(4-amino-5-(7-methoxy-5-methylbenzo[b]thiophen-2-yl)-7H-pyrrolo[2,3-

d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (18h). The title compound was prepared from 12j and 51 ACS Paragon Plus Environment


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23h following general procedure C. Yield: 70%. 1H NMR (300 MHz, CDCl3) δ 8.33 (s, 1H), 7.22 (s, 1H), 7.21 (s, 1H), 7.15 (s, 1H), 6.64 (s, 1H), 5.64 (s, 2H), 5.50−5.40 (m, 1H), 4.00 (s, 3H), 3.97−3.88 (m, 1H), 3.75−3.52 (m, 3H), 2.49 (s, 3H), 2.47−2.39 (m, 1H), 2.35−2.25 (m, 1H), 1.48 (s, 9H). MS (ESI) m/z 480.3 [M + H]+. tert-Butyl


d]pyrimidin-7-yl)azetidine-1-carboxylate (18i). The title compound was prepared from 12b and 23h following general procedure C. Yield: 74%. 1H NMR (300 MHz, CDCl3) δ 8.30 (s, 1H), 7.42 (s, 1H), 7.24 (s, 1H), 7.23 (s, 1H), 6.64 (s, 1H), 5.68−5.57 (m, 3H), 4.50 (t, J = 8.8 Hz, 2H), 4.29−4.22 (m, 2H), 4.00 (s, 3H), 2.49 (s, 3H), 1.48 (s, 9H). MS (ESI) m/z 466.3 [M + H]+. ELISA kinase assay. The effects of indicated compounds on the activities of various tyrosine kinases were determined using enzyme-linked immunosorbent assays (ELISAs) with purified recombinant proteins. Briefly, 20 μg/mL poly (Glu, Tyr) 4:1 (Sigma, St Louis, MO, USA) was precoated in 96-well plates as a substrate. A 50-μL aliquot of 10 μM ATP solution diluted in kinase reaction buffer (50 mM HEPES [pH 7.4], 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, and 1 mM DTT) was added to each well; 1 μL of various concentrations of indicated compounds diluted in 1% DMSO (v/v) (Sigma, St Louis, MO, USA) were then added to each reaction well. 1% DMSO (v/v) was used as the negative control. The kinase reaction was initiated by the addition of purified tyrosine kinase proteins diluted in 49 μL of kinase reaction buffer. After incubation for 60 min at 37 o

C, the plate was washed three times with phosphate-buffered saline (PBS) containing 0.1% Tween

20 (T-PBS). Anti-phosphotyrosine (PY99) antibody (100 μL; 1:500, diluted in 5 mg/mL BSA T-PBS) was then added. After a 30-min incubation at 37 oC, the plate was washed three times, and 100 μL horseradish peroxidase-conjugated goat anti-mouse IgG (1:1000, diluted in 5 mg/mL BSA T-PBS) 52 ACS Paragon Plus Environment

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was added. The plate was then incubated at 37 oC for 30 min and washed 3 times. A 100-μL aliquot of a solution containing 0.03% H2O2 and 2 mg/mL o-phenylenediamine in 0.1 M citrate buffer (pH 5.5) was added. The reaction was terminated by the addition of 50 μL of 2 M H2SO4 as the color changed, and the plate was analyzed using a multi-well spectrophotometer (SpectraMAX190, from Molecular Devices, Palo Alto, CA, USA) at 490 nm. The inhibition rate (%) was calculated using the following equation: [1-(A490/A490 control)] ×100%. The IC50 values were calculated from the inhibition curves in two separate experiments. Cell proliferation assay. Cells were seeded in 96-well cell culture plates. On the day when seeding, the cells were exposed to various concentrations of compounds and further cultured for 72 h at 37 oC. Cell proliferation was then determined using Cell Counts Kit-8 (CCK8) or the thiazolyl blue tetrazolium bromide (MTT, from Sigma-Aldrich, St. Louis, MO, USA) assay. The IC50 values were calculated by concentration-response curve fitting using the four-parameter method. Pharmacokinetic Parameters Obtained in Rats. Compound 9g or 9i was administrated to male Sprague-Dawley (SD) rats (n = 3) orally by gavage as a solution in 5% DMSO/95% (0.5% CMC-Na) at a 10 mg/kg dose. Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8 and 24 h following oral dosing. The blood samples were placed on wet ice, and serum was collected after centrifugation. Serum samples were frozen and stored at -20 oC. The serum samples were analyzed utilizing HPLCcoupled tandem mass spectrometry (LC-MS/MS). All animal experiments were performed according to the institutional ethical guidelines on animal care and approved by the Institute Animal Care and Use Committee at Shanghai Institute of Materia Medica (approval number: 2015-08-ZDF-39). Cell culture. Human lung cancer cell line NCI-H1581, human acute myelogenous leukemia cell line KG-1, human gastric cancer cell line SNU-16 were purchased from American Type Culture 53 ACS Paragon Plus Environment


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Collection (ATCC, Manassas, VA, USA). Human bladder cancer cell line RT-112, Human multiple myeloma cell line OPM-2 and Mouse Pre-B cell line BaF3 were purchased from DSMZ-German collection of microorganisms and cell cultures. Human bladder cancer cell line UMUC-14 was purchased from European Collection of Cell Cultures (ECACC). All the cell lines were routinely maintained in media according to the suppliers’ recommendations and authenticated via short tandem repeats analysis by Genesky Biopharma Technology (last tested in 2016). Western blot analysis. KG-1, SNU-16 and UMUC-14 cells were treated with the indicated dose of 9g for 2 h, and then lysed in 1×sodium dodecyl sulfate (SDS) sample buffer. The cell lysates were subsequently resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with the appropriate primary antibodies [phospho-FGFR, FGFR1, FGFR2, FGFR3, phospho-ERK, ERK, PLCγ (all from Cell Signaling Technology, Beverly, MA, USA), phospho-PLCγ, GAPDH (KangChen Biotech, Shanghai, China)] and then with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG. The immunoreactive proteins were detected using an enhanced chemilluminescense detection reagent (Thermo Fisher Scientific, Rockford, IL, USA). Irreversible assay. Rapid dilution experiment was used to demonstrate reversible binding of 9g to FGFR1. FGFR1 kinase (2 nM) was preincubated with excessive 9g at a final concentration of 100fold IC50 under room temperature for 30 min, then the mixed solution was diluted with reaction solution containing substrate peptide (5-FAM-KKSRGDYMTMQIG-CONH2) and ATP (262 μM) to a hundred times. Then the enzyme activity of this mixture was assayed by the EZ Reader (Caliper Life Sciences, MA). Wells were repeatedly read for 1 h.


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In vivo antitumor activity assay. Female nude mice (4–6 weeks old) were housed and maintained under specific pathogen-free conditions. Animal procedures were approved by the Institutional Animal Care and Use Committee at Shanghai Institute of Materia Medica (approval number: 201504-DJ-17 for NCI-H1581 model, and 2016-04-DJ-21 for SNU-16 model). The tumor cells at a density of 5 × 106 in 200 μL were injected subcutaneously (s.c.) into the right flank of nude mice and then allowed to grow to 700–800 mm3, which was defined as a well-developed tumor. Subsequently, the well-developed tumors were cut into 1-mm3 fragments and transplanted s.c. into the right flank of nude mice using a trocar. When the tumor volume reached 100–150 mm3, the mice were randomly assigned into a vehicle control group (n = 12) and treatment groups (n = 6 per group). The control groups were given vehicle alone, and the treatment groups received 9g at the indicated doses via oral administration once daily for 2−3 weeks. AZD4547 was a positive drug. The sizes of the tumors were measured twice per week using a microcaliper. Tumor volume (TV) = (length × width 2)/2, and the individual relative tumor volume (RTV) was calculated as follows: RTV = Vt / V0, where Vt is the volume on a particular day and V0 is the volume at the beginning of the treatment. The RTV was shown on indicated days as the median RTV ±SE indicated for groups of mice. Percent (%) inhibition (TGI) values were measured on the final day of study for the drug-treated mice compared with vehicle-treated mice and were calculated as 100 × {1 - [(VTreated Final day - VTreated Day 0)/(VControl Final day - VControl Day 0)]. Significant differences between the treated versus the control groups (P ≤ 0.05) were determined using Student’s t test. Modeling Method. The docking studies were carried out on AutoDock 4.2 using the X-ray crystal structure of FGFR4 in complex with FIIN-3 (PDB ID: 4R6V). The enzyme preparation and the hydrogen atoms adding were performed in the prepared process. After moving the native ligand and 55 ACS Paragon Plus Environment


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the irrelevant water, the FGFR4 enzyme was defined as a receptor and the enclosing box centered in native ligand was set on the basis of the binding site of FIIN-3. In the docking protocol, the Lamarchian genetic algorithm (GA) and the default parameters of GA were applied. The designed compounds were docked into the protein, then the docked complexes were clustered and sorted according to the binding energy, the binding models with the lowest binding energy were used to further reveal the binding mechanism. Figure 4 and 8 were generated with PyMOL version 1.3.

ASSOCIATED CONTENT Supporting Information Synthesis of requisite boronic acids 23a−h; Molecular formula strings. Accession Codes PDB ID: 4R6V. Authors will release the atomic coordinates and experimental data upon article publication. AUTHOR INFORMATION Corresponding Authors *(J.A.) E-mail: [email protected]. Phone: +86-21-50806600-2413. *(W.D.) E-mail: [email protected]. Phone: +86-21-50806032. Author Contributions #

These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT 56 ACS Paragon Plus Environment

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This work was supported by the funds from the National Natural Science Foundation (Nos. 81573271 and 81773762), Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA12020303), and the Shanghai Science and Technology Commission (No.15431901300). ABBREVIATIONS USED FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; KDR, kinase insert domain receptor, also known as VEGFR2; RTK, receptor tyrosine kinase; TKI, tyrosine kinase inhibitor; VEGFR, vascular endothelial growth factor receptor; PLCγ, phospholipase Cγ; STAT, signal transduction and activation of transcription; BTK, bruton’s tyrosine kinase; PDGFR, platelet-derived growth factor receptor; ABL, abelson tyrosine kinase; ErK, extracellular regulated protein kinases; TGI, tumor growth inhibition; RTV, relative tumour volume; IGF1R, insulin-like growth factor 1 receptor;

DIAD,

diisopropyl

azodicarboxylate;

TEA,

triethylamine;

DIPEA,

N,N-

diisopropylethylamine; HATU, 3-tetramethyluronium hexafluorophosphate REFERENCES 1.

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Table of Contents Graphic


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Figure 1. Structural analysis of the FGFR and FGFR inhibitors. (A) Representative FGFR inhibitors. (B) X-ray crystal structure of FGFR1 in complex with AMP (PDB ID: 3GQI). (C) Five regions of the ATP binding site. 97x65mm (600 x 600 DPI)

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Figure 2. Chemical structures of reported irreversible FGFR inhibitors. 71x36mm (600 x 600 DPI)


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Figure 3. Design of covalent FGFR inhibitors. 42x14mm (600 x 600 DPI)



Figure 4. Modeled interactions of benzo[b]thiophene motifs with hydrophobic region I in FGFR4 (PDB ID: 4R6V): (A) 6c with hydrophobic region I; (B) 6b with hydrophobic region I. 42x14mm (300 x 300 DPI)


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Figure 5. Compound 9g effectively inhibits the phosphorylation of FGFR and the downstream effectors PLCγ and Erk in KG-1 (A), SNU-16 (B), and UMUC-14 (C). Cells treated with 9g for 2 h at the indicated concentrations were lysed and subjected to Western blot analysis. 192x439mm (300 x 300 DPI)



Figure 6. Compound 9g irreversibly binds to FGFR1. Enzyme activity of FGFR1 was assayed by a Caliper EZ Reader under three different conditions: without the enzyme (background), without the compound (non-preincubation control) and pre-incubated with the compound. “Conversion” here represents the enzyme activity and means “the percent of conversion of the substrate peptide”. 64x49mm (300 x 300 DPI)


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Figure 7. 9g significantly inhibited FGFR-mediated tumor growth in vivo. (A) Tumor growth was inhibited upon 9g treatment in NCI-H1581 xenografts. The RTVs are expressed as the mean ± SEM. Significant difference from the vehicle group was determined using a t-test, * P < 0.05, ** P < 0.01, *** P < 0.001. (B) Tumor weights are shown for day 14 after the mice were administered 9g. (C) Body weight measurements during treatment. (D) Tumor growth was inhibited upon 9g treatment in the SNU-16 xenografts. The RTVs are expressed as the mean ± SEM. Significant difference from the vehicle group was determined using a t-test, * P < 0.05, ** P < 0.01, *** P < 0.001. Tumor weights (E) are shown for day 21 after the mice were administered 9g. (F) Body weight measurements during treatment. 152x158mm (300 x 300 DPI)



Figure 8. Proposed binding mode of 9g with FGFR4 (PDB ID: 4R6V). (A) The docked protein structure of FGFR4 is shown on the surface. (B) The atoms of 9g are colored as follows: carbon, green; oxygen, red; nitrogen, blue; sulfur, dark yellow. The protein is shown in the cartoon, and key residues are colored as follows: carbon, yellow; oxygen, red; nitrogen, blue, sulfur, dark yellow. H bonds are indicated by red dotted lines. 64x26mm (300 x 300 DPI)


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Scheme 1. Synthesis of Target Compounds 6a−la aReagents and conditions: (a) HO-linker-Boc, PPh3, DIAD, THF, 0 °C to rt or tert-butyl 2-bromoethylcarbamate, cesium carbonate, 80 °C; (b) ammonia (aq), 110 °C; (c) boronic acids or boronate esters, Pd(PPh3)4, Na2CO3, dioxane/H2O, 80 °C; (d) (i) HCl/dioxane, rt; (ii) acryloyl chloride, TEA, DCM, rt. 107x70mm (600 x 600 DPI)



Scheme 2. Synthesis of Target Compounds 7a−da aReagents and conditions: (a) HO-linker-Boc, PPh3, DIAD, THF, 0 °C to rt; (b) ammonia or methylamine (aq), 110 °C; (c) (7-methoxy-5methylbenzo[b]thiophen-2-yl) boronic acid, Pd(PPh3)4, Na2CO3, dioxane/H2O, 80 °C; (d) (i) HCl/dioxane, rt; (ii) acryloyl chloride, TEA, DCM, rt. 114x85mm (600 x 600 DPI)


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Scheme 3.Synthesis of Target Compounds 8a−e, 9a−ia aReagents and conditions: (a) (7-methoxy-5methylbenzo[b]thiophen-2-yl) boronic acid, Pd(PPh3)4, Na2CO3, dioxane/H2O, 80 °C; (b) (i) HCl/dioxane, rt; (ii) acyl chloride, TEA, DCM, rt or corresponding carboxylic acid, HATU, DMF, DIPEA, rt. 93x52mm (600 x 600 DPI)



Table of Contents Graphic 44x24mm (600 x 600 DPI)


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Discovery of Potent Irreversible Pan-Fibroblast Growth Factor

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