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Discovery of Allosteric Inhibitors Targeting the Spliceosomal RNA Helicase Brr2 Misa Iwatani-Yoshihara,*,†,# Masahiro Ito,*,‡,# Michael G. Klein,⊥ Takeshi Yamamoto,‡ Kazuko Yonemori,‡ Toshio Tanaka,‡ Masanori Miwa,† Daisuke Morishita,§ Satoshi Endo,‡ Richard Tjhen,⊥ Ling Qin,⊥ Atsushi Nakanishi,∥ Hironobu Maezaki,‡ and Tomohiro Kawamoto† †

Biomolecular Research Laboratories, ‡Medicinal Chemistry Research Laboratories, §Oncology Drug Discovery Unit, and ∥Shonan Incubation Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd., 26-1, Muraoka-Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan ⊥ Department of Structural Biology, Takeda California Inc., 10410 Science Center Drive, San Diego, California 92121, United States S Supporting Information *

ABSTRACT: Brr2 is an RNA helicase belonging to the Ski2-like subfamily and an essential component of spliceosome. Brr2 catalyzes an ATP-dependent unwinding of the U4/U6 RNA duplex, which is a critical step for spliceosomal activation. An HTS campaign using an RNA-dependent ATPase assay and initial SAR study identified two different Brr2 inhibitors, 3 and 12. Cocrystal structures revealed 3 binds to an unexpected allosteric site between the C-terminal and the N-terminal helicase cassettes, while 12 binds an RNA-binding site inside the N-terminal cassette. Selectivity profiling indicated the allosteric inhibitor 3 is more Brr2-selective than the RNA site binder 12. Chemical optimization of 3 using SBDD culminated in the discovery of the potent and selective Brr2 inhibitor 9 with helicase inhibitory activity. Our findings demonstrate an effective strategy to explore selective inhibitors for helicases, and 9 could be a promising starting point for exploring molecular probes to elucidate biological functions and the therapeutic relevance of Brr2.



INTRODUCTION In eukaryotic cells, protein-coding transcripts are produced as pre-mRNAs that contain introns. Splicing of pre-mRNAs is an essential step where introns are removed, and exons are joined together to form mature mRNA, which is a prerequisite step for protein translation. Splicing is catalyzed by a dynamic multimegadalton ribonucleoprotein (RNP) complex, called the spliceosome.1,2 The spliceosome consists of five small nuclear RNPs (snRNPs), U1, U2, U4, U5, and U6. Brr2 is an essential component of U5 and belongs to the Ski2-like RNA helicase family.3−5 Brr2 disrupts the base-pairing of the U4/U6 small molecular RNA duplex, which is the major driving force for catalytic activation of the spliceosome. Since Brr2 remains tightly associated with the spliceosome even after the splicing catalysis step, it is considered to play critical roles through the splicing event.6 Brr2 consists of two multidomain helicase cassettes. Both cassettes’ domains are composed of dual RecA-like domains, a © 2017 American Chemical Society

winged helix (WH) domain, and a Sec63 homology unit composed of a helical bundle (HB) domain, a helix−loop−helix (HLH) domain, and an immunoglobulin-like (IG) domain.7 Several reports have demonstrated that only the N-terminal cassette possesses ATPase activity and RNA unwinding activity.5,7 In contrast, the C-terminal cassette is reportedly inactive5,7−9 and functions as a platform mediating protein− protein interaction.10 Mutations in the BRR2 gene are implicated in autosomal-dominant retinitis pigmentosa, a group of progressive retinal degenerative disorders. The pathogenic mutations c.3260C>T (p.S1087L) and c.3269G>T (p.R1090L) impair the fidelity of gene expression accompanied by reduction of the ATPase and U4/U6 unwinding activity,11−13 indicating that the biological function of Brr2 is critically connected to the mechanism of disease. Received: March 24, 2017 Published: June 6, 2017 5759

DOI: 10.1021/acs.jmedchem.7b00461 J. Med. Chem. 2017, 60, 5759−5771

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Figure 1. Schematic flowchart summarizing the process of the discovery of Brr2 inhibitors.

In spite of its biological importance, molecular probes targeting Brr2 have not been reported to date. Selective small molecular Brr2 inhibitors will provide a new and attractive option for molecular biologists addressing splicing machinery. However, identification of specific inhibitors for helicases has been quite challenging because screening using helicase assays is generally low-throughput and yields many false positives such as nucleic acid binders.14 Here, we present the discovery of highly specific Brr2 inhibitors using RNA-dependent ATPase assay, X-ray cocrystal structure analysis, and structure based chemical optimization.

using SBDD led to the identification of the more potent compound 6. Further, structure based optimization of 6 culminated in the discovery of compound 9 with double digit nanomolar Brr2 ATPase inhibitory activity, excellent Brr2 selectivity, good aqueous solubility, and optimal Brr2 helicase inhibitory activity. Further details of each process are described in the following sections. HTS, Hit Validation, and Preliminary SAR. First screening of Brr2 inhibitors was conducted using full-length recombinant Brr2 protein and RNA-dependent ATPase activity assay, which was developed based on ADP-Glo assay. Then, a phosphate sensor assay, which detects inorganic phosphate, was used for the secondary screening. Compounds meeting target criteria of the first and secondary screening (see details in Table S1 in Supporting Information) were subjected to a dosedependent ATPase assay, and their IC 50 values were determined. After that, the identified single- or double-digit micromolar inhibitors were characterized using a substrate competition assay and a thermal shift assay to exclude nonspecific binders. Following this process, several chemical series, including compound 1 and compound 11, were identified (Table 1). Compounds 1 and 11 inhibited the ATPase activity of Brr2 with IC50 values of 36 μM and 4.2 μM, respectively. Additionally, 1 and 11 induced thermal shifts (ΔTm) of 2.0 and 2.3 °C, respectively. In order to elucidate the key functional groups for inhibitory activity of 1 and 11, preliminary SAR was investigated. As shown in Table 2, replacement of the trifluoroethyl group of 1 with a methyl group was not tolerated (2), whereas that with a larger benzyl



RESULTS AND DISCUSSION Outline of the Discovery Process of Brr2 Inhibitors. The discovery process of selective Brr2 inhibitors is summarized in Figure 1. At first, a high-throughput screening of in-house library yielded two structurally distinct chemical series, 4,6-dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione derivative 115 and 5-imino-7-oxo-5H-[1,3,4]thiadiazolo[3,2a]pyrimidin derivative 11. Next, the validity of these chemical series was further investigated using SAR study, X-ray cocrystal structure analysis, and selectivity assays. Compound 3, an analog of compound 1, was ascertained to bind an allosteric site of Brr2 by X-ray crystal structure analysis. Meanwhile, compound 12, an analog of compound 11, turned out to be a RNA site binder. Since Brr2 selectivity of the allosteric inhibitor 3 is superior to that of the RNA site inhibitor 12, we selected 3 as a lead compound for further chemical optimization. Then, structural simplification of compound 3 5760

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Table 1. In Vitro Profile of Compounds 1 and 11a

compd 1 11

NH of Ile1681 at a distance of 2.7 Å. (3) The benzyl moiety at the R1 in 3 occupies a large hydrophobic cavity formed by side chains of Ile1193, Phe1255, Phe1713, Phe1717, Leu1722, and Ile1681. (4) The distal 3-fluoro-pyridin-2-yl moiety occupies a small cavity formed by Thr1197, Pro1257, Asp1712, Phe1713, Lys1716, and Phe1717. The fact that compound 3 resides in the pocket formed between C-terminal helicase cassette and the N-terminal cassette suggests that compound 3 may affect Nand C-terminal cassette interactions7 of Brr2. However, apparent positional changes of the amino acid residues, essential for intercassette interactions,7 were not observed in the absence or presence of ligand 3 (Figure S1). Thus, the inhibitory mechanism of 3 remains unclear, and further investigation using other techniques (e.g., site mutation or NMR) is necessary to elucidate the exact inhibitory mechanism of compound 3. In contrast, the SAR in Table 2 can be clearly explained by the cocrystal structure. Specifically, the decreased activity of compound 2 (R1 = methyl), when compared to 1 and 3, is accounted for by insufficient bulkiness and hydrophobicity of the R1 group at the hydrophobic cavity. Meanwhile, a large unidentified blob of electron density was found between the urea carbonyl and residues Thr1666 and Asp1678. On the basis of their relative position to the cyclic urea of 3 and the SAR (3 vs 4), it is reasonable to assume that Thr1666 or Asp1678 could make a hydrogen bond to the NH group of 3 via water-mediated interactions, whereas the putative water molecule was undefined in the crystal, presumably due to limitations in the resolution of the diffraction data. Meanwhile, the methyl group (R3) of 3 directs toward the side chain of Asp1678 and has no positive interaction with Brr2 protein. The absence of interaction is consistent with the SAR between 3 and 5. The role of the dihydropyran ring of 3 was not clearly defined by the cocrystal structure. We speculated that the ring would not be important for binding and was just working as a linker between the phenyl ring and the 3-fluoropyridin-2-yl moiety. On the contrary, as shown in Figure 2B, compound 12 sits inside an RNA binding site, leading to reduction of RNAstimulated ATPase activity. The RNA binding site is formed by the dual RecA domains and the HB/HLH domains of the Nterminal helicase cassette and runs through the cassette as a large open-ended channel. Notably, a pyrimidine nitrogen atom in compound 12 forms a hydrogen bond with the backbone NH of residue Arg545, and an oxygen atom from the carboxylic acid moiety of compound 12 interacts with the side chain of Thr589. Especially, the latter observation is consistent with the carboxy group being essential for its activity. (Further details for the binding site of compound 12 are described in Supporting Information, Figure S2.) Thus, direct binding of 3 and 12 against Brr2 and their binding modes were ascertained. Then, 3 and 12 were subjected to selectivity profiling against other helicases. As a result, it turned out that allosteric inhibitor 3 is more selective than RNA-site binder 12 (Table 3).16 Hence, we selected compound 3 as a lead compound for further optimization. Structure-Based Drug Design of 4,6-Dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione Derivatives. With insights from crystallographic data of 3, a structurally simplified a ring-opened derivative 6 was designed and synthesized (Table 4). As we expected, compound 6 was equipotent to 5 and showed submicromolar Brr2 inhibitory activity. The cocrystal structure of 6 with Brr2 revealed that the dihydropyrido[4,3d]pyrimidine-2,7(1H,3H)-dione ring, the benzyl moiety on the

Brr2 ATPase IC50 (μM)b thermal shift ΔTm (°C), 100 μM/33 μM 36 (23−55) 4.2 (2.6−6.8)

2.0/1.2 2.3/1.3

a

The denaturation rates are plotted as indicated by green for DMSOtreated control, blue for 33.3 μM, and red for 100 μM of test compounds, respectively. bn = 2, 95% confidence intervals shown in parentheses.

Table 2. SAR of Compounds 1−5

a

compd

R1

R2

R3

Brr2 ATPase IC50 (μM)a

1 (hit) 2 3 4 5

CH2CF3 Me Bn Bn Bn

H H H Me H

Me Me Me Me H

36 (23−55) >100 5.3 (2.9−9.4) >100 1.7 (0.96−3.2)

n = 2, 95% confidence intervals shown in parentheses.

group (3) showed increased activity. Methylation at the R2 position in compound 3 resulted in reduced activity (4), while demethylation at the R3 group (5) was tolerated for activity. Meanwhile, SAR study of 11 revealed that the carboxyl group was essential for activity (11 vs 13), and the distal propyl chain was replaceable with a cyclohexyl ring (12) (Table S2). Crystal Structural Analysis for Brr2 Inhibitors and Selectivity Study. The X-ray cocrystal structures of compound 3 and 12 in complex with Brr2 were determined as shown in Figure 2A. For crystallization trials, we used the truncated human Brr2 lacking the N-terminal extension and some C-terminal residues (395−2129 residues). Focused views of the binding modes of 3 and 12 are shown in Figure 2B. Notably, the Brr2 cocrystal structure revealed that compound 3 binds to a newly identified allosteric site located in the protein interface formed between the second RecA domain of the Cterminal helicase cassette and the Ig domain from the Nterminal helicase cassette (Figure 2C and Figure 2D). There are four key features of the cocrystal structure of 3 with Brr2: (1) Compound 3 binds in a U-shaped conformation and is surrounded by numerous hydrophobic residues including five aromatic side chains (i.e., Phe1254, Phe1255, Tyr1682, Phe1713, and Phe1717). (2) The carbonyl of the pyridone ring in 3 forms a hydrogen-bonding interaction with the main chain 5761

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Figure 2. (A) Overall cocrystal structure of Brr2 with compound 3 (PDB code 5URJ) and 12 (PDB code 5URM). (Upper) Ribbon plot of Brr2. A green ligand represents compound 3, a magenta ligand represents compound 12, and yellow ligands represent nucleotides from a published structure (PDB code 4F93). N-terminal cassette: RecA-1, orange; RecA-2, yellow; WH, gray; HB, blue; HLH, pink; IG, green. C-terminal cassette: RecA-1, pale red; RecA-2, pale yellow; WH, gray; HB, cyan; HLH, pale pink; IG, yellow green. (Lower) Schematic representation of Brr2 in domain borders. (B) Binding site of 3* (left) and 12 (right) in Brr2. (C) Binding mode of the left side of 3. (D) Binding mode of right side of 3. *The binding isomer is estimated to be 2R-form.

Table 3. Selectivity of 3 and 12 IC50 (μM)a

a

compd

Brr2 ATPase

eIF4A1 ATPase

eIF4A3 ATPase

DHX29 ATPase

3 12

5.3 (2.9−9.4) 5.3 (4.0−7.1)

>100 57 (48−69)

>100 32 (24−41)

NTb 58 (41−80)

n = 2 or 4, 95% confidence intervals shown in parentheses. bNT = not tested.

pyridone ring, and the phenyl group substituted on the pyrimidinone ring are well overlapped with those in the cocrystal structure of 3 (Figure 3A). In contrast, the distal phenyl ring of 6 oriented perpendicularly to the 6fluoropyridin-2-yl group of 3. Considering the conformationally

constrained structure of 3, it seemed that the phenyl group of 6 can reside in a more favorable position. To improve the activity and solubility, further modifications of 6 were examined. Compounds with a polar functional group were designed to make an additional hydrogen bonding with an 5762

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Table 4. SAR of Compounds 6−10

a

n = 2, 95% confidence intervals shown in parentheses. bParallel artificial membrane permeability assay. cDistribution coefficient.

Figure 3. X-ray cocrystal structure of inhibitors bound to Brr2. (A) Overlay of X-ray crystal structure of 3 in Brr2 (PDB code 5URJ, depicted in green) and 6 (PDB code 5URK, depicted in yellow). (B) Polar amino acid residues around benzyl group of 6.

amino acid residue and reduce the hydrophobicity. As suitable amino acids (Thr1197, Ser1196, and Lys1716) are located around the 3-position of the distal phenyl ring in compound 6 (Figure 3B), the phenyl group was replaced by a hydrogenbond-accepting heterocyclic group, such as pyridyl (7 and 8) and thiazolyl (9) groups as shown in Table 4. Compounds 8 and 9 bearing a nitrogen atom oriented toward the similar direction (meta-direction) showed enhanced activity, whereas 2-pyridyl derivative 7 did not show increased activity. As shown in Figure 4, the docking model of compound 9 suggested the formation of a hydrogen bond between the nitrogen atom of the thiazole ring and Thr1197,17 which should be geometrically inaccessible by 2-pyridiyl derivative 7. Compounds 8 and 9 not only exhibited the most potent activity among this chemical series but showed better physicochemical properties (log D value, solubility, and permeability). As can be expected by the cocrystal structure of compound 6, o-substituted derivative 10 showed deteriorated activity. On the basis of the structural similarity of 10 to the potent compounds 6, 8, and 9, we adopted 10 as a negative control compound. Characterization of Compound 9. In accordance with the cocrystal structural data of a near neighbor analogue,

Figure 4. Docking model of compound 9.

optimized compound 9 demonstrated noncompetitive Brr2 inhibition against either ATP or RNA (Figure S3). Selectivity study showed that compound 9 did not inhibit single-cassette RNA helicases such as DEAD box-family RNA helicase (eIF4A1 and eIF4A3) and DEAH box-family RNA helicase (DHX29), as shown in Table 5. In addition, compound 8 also did not affect other helicases, which indicates the high selectivity of this chemical series but still remains to be seen if they are Brr2-specific. We next investigated the effects of 9 on 5763

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could be an excellent starting point leading to molecular probes for Brr2. Furthermore, since there are several amino acids (e.g., Tyr 1682) not directly involved in the ligand−protein interactions, further enhancement of inhibitory activity could be possible by forming additional hydrogen bonding with these residues. Chemistry. The synthesis of compounds 2−5 is illustrated in Scheme 1. Reported intermediates 14a18 and 14b19 were subjected to a nucleophilic azidation reaction, followed by hydrolysis of the ester group to afford azido acid 15a,b. The carboxyl group in 15a,b was converted to Weinreb amide, and subsequent reduction using LiAlH4 provided amino aldehyde 16a,b. Reductive coupling reaction of 16a,b and 17a,b,15 followed by treatment with 1,1′-carbonyldiimidazole (CDI), furnished compounds 2, 3, and 5. Finally, methylation of the nitrogen atom of cyclic urea in 3 gave compound 4. The synthesis of compounds 6−10 is depicted in Scheme 2. Intermediate 16b was coupled with commercially available amine 18a and 18b, followed by treatment with CDI to afford compounds 6 and 19. Removal of p-methoxybenzyl group in 19 using TFA and subsequent alkylation gave compounds 7, 8, and 9. Reductive amination of 16b with 18c using tin chloride and polymethylhydrosiloxane (PMHS) followed by treatment with CDI gave compounds 10.

Table 5. Selectivity of 8 and 9 IC50 (μM)a compd

Brr2 ATPase

eIF4A1 ATPase

eIF4A3 ATPase

DHX29 ATPase

8 9

0.18 (0.15−0.22) 0.079 (0.069−0.091)

>100 >100

>100 >100

NTb >100

a

n = 2 or 4, 95% confidence intervals shown in parentheses. bNT = not tested.

Figure 5. RNA helicase activity of Brr2 monitored using fluorescentlabeled RNA. Single- or double-stranded RNAs were separated by electrophoresis and detected as fluorescent signals. Dose-dependent inhibition as indicated concentration of Brr2 inhibitor 9 is represented (lanes 4−8). Negative control 10 was also evaluated at 100 μM (lane 9). The enzymatic reaction without compounds was termed as 100% of control (lane 3), and that without enzyme was determined as 0% control (lane 10). Double- and single-stranded RNAs and total reactions without ATP are lanes 1, 2, and 11, respectively. The intensity of each band was measured, and inhibition rate was calculated. The IC50 value against helicase activity was estimated by plotting of each inhibition rate, which is described in the right graph.



CONCLUSION By using RNA-dependent ATPase activity assays, crystal structure analysis, and SBDD, we successfully discovered a Brr2 specific inhibitor 9 with optimal helicase inhibitory activity. Our study demonstrated that the newly identified allosteric site in a protein−protein interface of Brr2 was a druggable pocket that led to highly selective and potent smallmolecule inhibitors of Brr2. Since compound 9 is endowed with not only potency and selectivity but also excellent physicochemical properties, it could be a promising starting point leading to molecular probes for elucidating biological function or therapeutic relevance of Brr2. Moreover, we recently reported another successful example using ATPase assay for a helicase inhibitor project;20 our studies demonstrate the

in vitro ATP-dependent RNA unwinding activity of Brr2 (Figure 5). Its activity was measured using fluorescent-labeled RNA with the single- or double-stranded form separated by electrophoresis. We set the assay conditions within the initial velocity phase, which allowed us to estimate the accurate IC50 values (Figure S4). Compound 9 inhibited helicase activity in dose-dependent manner with an IC50 value of 1.3 μM. On the contrary, its negative control, compound 10 did not inhibit the activity even at 100 μM, demonstrating a positive correlation between ATPase- and helicase-activity. Thus, compound 9 Scheme 1. Synthesis of 2−5a

Reagents and conditions: (a) (i) NaN3, DMF, 55 °C−60 °C; (ii) LiOH·H2O, EtOH (or EtOH and THF), H2O, rt, 71%−95% for 2 steps; (b) (i) MeNH(OMe)·HCl, T3P, DIPEA, DMF, 0 °C to rt; (ii) LiAlH4, THF, 0 °C to rt, 24% for 2 steps; (c) (i) MeNH(OMe)·HCl, EDC·HCl, HOBt, DIPEA, DMF, rt; (ii) LiAlH4, THF, 0 °C, 58% for 2 steps; (d) (i) 17a or 17b, Ti(i-PrO)4, THF, 50 °C, 16 h; (ii) NaBH(OAc)3, 50 °C; (iii) CDI, DBU, THF, rt, 6%−27% for 3 steps; (e) NaH, MeI, DMF, 0 °C to rt, 73%. a

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Scheme 2. Synthesis of 6−10a

Reagents and conditions: (a) (i) 18a or 18b, Ti(i-PrO)4, THF, 50 °C, 16 h; (ii) NaBH(OAc)3, 50 °C; (iii) CDI, DBU, THF, rt, 34%−67% for 3 steps; (b) TFA, anisole, rt, quant; (c) 21a or 21b, K2CO3, DMF, rt, 13%−36%; (d) 21c, K2CO3, KI, NMP, rt, 27%; (d) (i) 18c, PMHS, SnCl2· MeOH, rt, 31%; (ii) CDI, DBU, THF, rt, 81%. a

mmol/L AcONH4 and MeCN (9/1, v/v) and a mixture of 5 mmol/L AcONH4 and MeCN (1/9, v/v), respectively. The ratio of mobile phase B was increased linearly from 5% to 90% over 0.9 min, 90% over the next 1.1 min. The purities of all synthesized compounds tested in biological systems were assessed as being >95% using elemental analysis. Elemental analyses were carried out by Sumika Chemical Analysis Service or Toray Research Center and were within 0.4% of the theoretical values. Reaction progress was determined by thin layer chromatography (TLC) analysis on Merck Kieselgel 60 F254 plates or Fuji Silysia NH plates. Chromatographic purification was carried out on silica gel columns (Merck Kieselgel 60, 70−230 mesh or 230−400 mesh, Merck; Chromatorex NH-DM 1020, 100−200 mesh, Fuji Silysia Chemical; inject column and Universal column, YAMAZEN, http:// yamazenusa.com/products/columns/; or Purif-Pack Si or NH, Shoko Scientific, http://shoko-sc.co.jp/english2/). Preparative TLC was carried out on Merck Kieselgel 60 PLC plates. Preparative HPLC was acquired using a Gilson preparative HPLC system (conditions 1 and 2) or MassLynx UV prep system (condition 3) with UV detector (220 and 254 nm). Condition 1: Mobile phases A and B under a basic condition were 0.05% aqueous ammonia solution and MeCN, respectively. Condition 2: Mobile phases A and B under an acidic condition were 0.225% formic acid in water and MeCN, respectively. Condition 3: Mobile phases A and B under an acidic condition were 0.1% TFA in water and 0.1% TFA in MeCN, respectively. The ratio of mobile phase B was increased linearly between 7 and 12 min. The column used was a Gemini C18 (25 mm × 150 mm i.d., 10 μm or 150 mm × 25 mm i.d., 5 μm, Phenomenex) for condition 1, a Synergi C18

effectiveness and robustness of ATPase activity-based screening for exploring helicase inhibitors.



EXPERIMENTAL SECTION

Chemistry Procedure. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on Bruker AVANCE-300 (300 MHz), Bruker AVANCE-400 (400 MHz), and Bruker AVANCE-600 (600 MHz), and carbon nuclear magnetic resonance (13C NMR) spectra were recorded on Bruker AVANCE-300 (75 MHz), Bruker AVANCE400 (101 MHz), and Bruker AVANCE-600 (151 MHz) in CDCl3 or DMSO-d6 solution. Chemical shifts are given in parts per million (ppm) with tetramethylsilane as an internal standard. Abbreviations are used as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, dt = doublet of triplets, brs = broad singlet. Coupling constants (J values) are given in hertz (Hz). Low-resolution mass spectra (MS) were acquired using an Agilent LC/MS system (Agilent1200SL/Agilent6130MS, Agilent1200SL/ Agilent1956MS, or Agilent1200SL/Agilent6110MS) or Shimadzu UFLC/MS (Prominence UFLC high pressure gradient system/ LCMS-2020) operating in an electron spray ionization mode (ESI +). The column used was an L-column 2 ODS (3.0 mm × 50 mm i.d., 3 μm, CERI) with a temperature of 40 °C and a flow rate of 1.2 or 1.5 mL/min. Condition 1: mobile phases A and B under an acidic condition were 0.05% TFA in water and 0.05% TFA in MeCN, respectively. The ratio of mobile phase B was increased linearly from 5% to 90% over 0.9 min, 90% over the next 1.1 min. Condition 2: mobile phases A and B under a neutral condition were a mixture of 5 5765

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Journal of Medicinal Chemistry

Article

([30 mm × 150 mm i.d., 4 μm] or [25 × 150 mm i.d., 10 μm], Phenomenex) for condition 2, an L-column 2 ODS (20 mm × 150 mm i.d., 5 μm, CERI) for condition 3, and a flow rate of 25 mL/min (conditions 1 and 2) or 20 mL/min (condition 3). All commercially available solvents and reagents were used without further purification. Yields were not optimized. Compound 115 was used from the in-house library (>95% purity was confirmed by element analysis). Compounds 11−13 were purchased from ChemDiv. (±)-3-(2-(3-Fluoropyridin-2-yl)-6-methyl-3,4-dihydro-2Hchromen-7-yl)-6-methyl-4,6-dihydropyrido[4,3-d]pyrimidine2,7(1H,3H)-dione (2). To a mixture of 16a (120 mg, 0.789 mmol) and 17a15 (224 mg, 0.868 mmol) in THF (10 mL) was added Ti(iPrO)4 (0.47 mL, 1.6 mmol) at room temperature under N2. The reaction mixture was heated to 50 °C and stirred for 16 h. To the reaction mixture was added NaBH(OAc)3 (669 mg, 3.15 mmol), and the reaction mixture was stirred at 50 °C for 48 h. The reaction mixture was quenched with saturated aqueous NaHCO3, and the mixture was extracted with CH2Cl2/MeOH (10/1). The organic layer was separated, washed with brine, dried over anhydrous Na2SO4, and concentrated. The residue was purified by preparative TLC on silica gel (CH2Cl2/MeOH = 10/1) to afford (±)-4-amino-5-(((2-(3fluoropyridin-2-yl)-6-methyl-3,4-dihydro-2H-chromen-7-yl)amino)methyl)-1-methylpyridin-2(1H)-one (70 mg, 23%) as a yellow solid. MS m/z 395 (M + H)+. A mixture of (±)-4-amino-5-(((2-(3-fluoropyridin-2-yl)-6-methyl3,4-dihydro-2H-chromen-7-yl)amino)methyl)-1-methylpyridin-2(1H)one (70 mg, 0.18 mmol), CDI (95.0 mg, 0.586 mmol), and DBU (88 μL, 0.59 mmol) in THF (10 mL) was stirred at room temperature for 16 h under N2. Then the reaction mixture was allowed to warm to room temperature and stirred for 20 h. To the reaction mixture was added CDI (86.3 mg, 0.532 mmol) and the reaction mixture was stirred at room temperature for 20 h. The reaction mixture was diluted with water (20 mL), and the mixture was extracted with CH2Cl2/ MeOH (10/1). The organic layer was separated, washed with brine, dried over anhydrous Na2SO4, and concentrated. The residue was purified by preparative HPLC (condition 1, 0.05% aqueous ammonia solution/MeCN = 70/30 to 51/49) to afford 2 (44 mg, 59%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 2.04 (s, 3H), 2.13− 2.24 (m, 1H), 2.32−2.36 (m, 1H), 2.76−2.86 (m, 1H), 2.88−3.02 (m, 1H), 3.32 (s, 3H), 4.23 (dd, J = 13.6, 4.8 Hz, 1H), 4.60 (d, J = 13.6 Hz, 1H), 5.42 (d, J = 10.0 Hz, 1H), 5.68 (s, 1H), 6.76 (s, 1H), 7.02 (s, 1H), 7.48−7.62 (m, 2H), 7.76−7.84 (m, 1H), 8.48 (d, J = 4.4 Hz, 1H), 9.82 (brs, 1H). 13C NMR (101 MHz, DMSO-d6, T = 90 °C) δ 15.9, 23.2, 24.8, 35.4, 47.3, 73.0 (d, JC−F = 1.8 Hz, 1C), 95.7, 100.7, 114.4, 120.9, 123.5 (d, JC−F = 19.1 Hz, 1C), 124.9 (d, JC−F = 4.4 Hz, 1C) 126.5, 130.6, 134.8, 139.4, 144.7 (d, JC−F = 5.1 Hz, 1C), 146.2 (d, JC−F = 12.1 Hz, 1C), 147.8, 151.0, 152.6, 156.9 (d, JC−F = 258.6 Hz, 1C), 161.5. MS m/z 421 (M + H)+. Anal. Calcd for C23H21FN4O3· 0.5H2O: C, 64.33; H, 5.16; N, 13.05. Found: C, 64.49; H, 5.18; N, 13.05. (±)-6-Benzyl-3-(2-(3-fluoropyridin-2-yl)-6-methyl-3,4-dihydro-2H-chromen-7-yl)-4,6-dihydropyrido[4,3-d]pyrimidine2,7(1H,3H)-dione (3). To a mixture of 4-amino-1-benzyl-6-oxo-1,6dihydropyridine-3-carbaldehyde 16b (30 mg, 0.13 mmol) and 17a (34.0 mg, 0.13 mmol) in THF (3 mL) was added Ti(i-PrO)4 (59 μL, 0.20 mmol) at room temperature under N2. The reaction mixture was heated to 50 °C and stirred for 16 h. To the reaction mixture was added NaBH(OAc)3 (111 mg, 0.52 mmol) and the reaction mixture was stirred at 50 °C for 18 h. The reaction mixture was concentrated, and the residue was purified by preparative TLC (CH2Cl2/MeOH = 20/1) to afford (±)-4-amino-1-benzyl-5-(((2-(3-fluoropyridin-2-yl)-6methyl-3,4-dihydro-2H-chromen-7-yl)amino)methyl)pyridin-2(1H)one (10 mg, 16%) as a yellow solid. MS m/z 471 (M + H)+. A mixture of (±)-4-amino-1-benzyl-5-(((2-(3-fluoropyridin-2-yl)-6methyl-3,4-dihydro-2H-chromen-7-yl)amino)methyl)pyridin-2(1H)one (30 mg, 0.064 mmol), CDI (52 mg, 0.32 mmol), and DBU (48 μL, 0.32 mmol) in THF (3 mL) was stirred at room temperature for 18 h. The reaction mixture was concentrated. The residue was purified by preparative HPLC (condition 2, 0.225% aqueous formic acid

solution/MeCN = 55/45 to 34/66) to afford 3 (12 mg, 38%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 2.03 (s, 3H), 2.11− 2.22 (m, 1H), 2.27−2.34 (m, 1H), 2.75−2.85 (m, 1H), 2.87−3.04 (m, 1H), 4.20−4.31 (m, 1H), 4.53−4.65 (m, 1H), 4.91−5.09 (m, 2H), 5.36−5.46 (m, 1H), 5.71 (s, 1H), 6.75 (s, 1H), 7.00 (s, 1H), 7.23− 7.29 (m, 3H), 7.30−7.36 (m, 2H), 7.48−7.56 (m, 1H), 7.65 (s, 1H), 7.74−7.85 (m, 1H), 8.47 (d, J = 4.4 Hz, 1H), 9.87 (s, 1H). 13C NMR (101 MHz, DMSO-d6, T = 90 °C) δ 15.9, 23.2, 24.7, 47.2, 50.0, 72.9 (d, JC−F = 1.8 Hz, 1C), 95.9, 101.3, 114.3, 120.8, 123.4 (d, JC−F = 19.4 Hz, 1C), 124.8 (d, JC−F = 4.4 Hz, 1C), 126.5, 126.8, 127.1(2C), 128.0 (2C), 130.5, 134.0, 137.3, 139.2, 144.6 (d, JC−F = 5.1 Hz, 1C), 146.1 (d, JC−F = 12.5 Hz, 1C), 147.8, 150.8, 152.5, 156.8 (d, JC−F = 258.6 Hz, 1C), 161.1. MS m/z 497 (M + H)+. Anal. Calcd for C29H25FN4O3· 2.5H2O: C, 64.32; H, 5.58; N, 10.35. Found: C, 64.34; H, 5.23; N, 10.36. (±)-6-Benzyl-3-(2-(3-fluoropyridin-2-yl)-6-methyl-3,4-dihydro-2H-chromen-7-yl)-1-methyl-4,6-dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione (4). To a solution of 3 (60 mg, 0.12 mmol) in DMF (2 mL) was added NaH (60%, 10 mg, 0.25 mmol) at 0 °C under N2. The reaction mixture was stirred at 0 °C for 0.5 h. To the reaction mixture was added iodomethane (34.3 mg, 0.24 mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 2 h. The reaction mixture was quenched with water at 0 °C, and the mixture was extracted with EtOAc. The organic layer was separated, washed with brine, dried over Na2SO4, and concentrated. The residue was purified by preparative HPLC (condition 2, 0.225% aqueous formic acid solution/MeCN = 55/45 to 25/75) to afford 4 (45 mg, 73%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 2.00 (s, 3H), 2.11−2.22 (m, 1H), 2.28−2.35 (m, 1H), 2.73−2.86 (m, 1H), 2.88−3.03 (m, 1H), 3.15 (s, 3H), 4.15−4.33 (m, 1H), 4.50−4.65 (m, 1H), 4.90−5.17 (m, 2H), 5.41 (d, J = 10.0 Hz, 1H), 5.86 (s, 1H), 6.74 (s, 1H), 7.00 (s, 1H), 7.23−7.38 (m, 5H), 7.49−7.56 (m, 1H), 7.65 (s, 1H), 7.76−7.84 (m, 1H), 8.46 (d, J = 4.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d6, T = 90 °C) δ 15.9, 23.1, 24.7, 29.6, 46.6, 50.0, 72.9 (d, JC−F = 2.2 Hz, 1C), 97.0, 102.5, 114.0, 120.8, 123.4 (d, JC−F = 19.1 Hz, 1C), 124.9 (d, JC−F = 4.0 Hz, 1C), 126.2, 126.9, 127.1 (2C), 128.0 (2C), 130.5, 133.0, 137.1, 139.8, 144.6 (d, JC−F = 5.1 Hz, 1C), 146.1 (d, JC−F = 12.1 Hz, 1C), 148.9, 151.4, 152.5, 156.8 (d, JC−F = 258.6 Hz, 1C), 161.2. MS m/z 511 (M + H)+. Anal. Calcd for C30H27FN4O3· 1.8H2O: C, 63.36; H, 5.68; N, 10.32. Found: C, 63.49; H, 5.55; N, 10.44. (±)-6-Benzyl-3-(2-(3-fluoropyridin-2-yl)-3,4-dihydro-2Hchromen-7-yl)-4,6-dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)dione (5). To a mixture of 16b (30 mg, 0.13 mmol) and 17b15 (32 mg, 0.13 mmol) in THF (3 mL) was added Ti(i-PrO)4 (59 μL, 0.20 mmol) at room temperature under N2. The reaction mixture was heated to 50 °C and stirred for 16 h. To the reaction mixture was added NaBH(OAc)3 (111 mg, 0.52 mmol), and the reaction mixture was stirred at 50 °C for 18 h. The reaction mixture was concentrated, and the residue was purified by preparative TLC (CH2Cl2/MeOH = 20/1) to afford (±)-4-amino-1-benzyl-5-(((2-(3-fluoropyridin-2-yl)3,4-dihydro-2H-chromen-7-yl)amino)methyl)pyridin-2(1H)-one (30 mg, 50%) as a yellow solid. MS m/z 457 (M + H)+. A mixture of (±)-4-amino-1-benzyl-5-(((2-(3-fluoropyridin-2-yl)3,4-dihydro-2H-chromen-7-yl)amino)methyl)pyridin-2(1H)-one (140 mg, 0.307 mmol), CDI (249 mg, 1.53 mmol), and DBU (0.23 mL, 1.54 mmol) in THF (5 mL) was stirred at room temperature for 18 h. The reaction mixture was concentrated, and the residue was purified by preparative HPLC (condition 2, 0.225% aqueous formic acid solution/MeCN = 65/35 to 35/65) to afford 5 (80 mg, 54%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 2.13−2.23 (m, 1H), 2.27−2.36 (m, 1H), 2.75−2.87 (m, 1H), 2.90−3.04 (m, 1H), 4.55 (s, 2H), 5.00 (s, 2H), 5.44 (dd, J = 10.0, 2.0 Hz, 1H), 5.71 (s, 1H), 6.74− 6.78 (m, 1H), 6.84 (dd, J = 8.4, 2.0 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.23−7.37 (m, 5H), 7.48−7.56 (m, 1H), 7.67 (s, 1H), 7.76−7.85 (m, 1H), 8.43−8.51 (m, 1H), 9.94 (s, 1H). 13C NMR (151 MHz, DMSOd6) δ 23.5, 24.8, 46.8, 50.3, 72.9 (d, JC−F = 2.2 Hz, 1C) 96.1, 101.8, 113.0, 117.3, 119.5, 124.0 (d, JC−F = 18.8 Hz, 1C) 125.5 (d, JC−F = 4.4 Hz, 1C) 127.3, 127.4 (2C), 128.4 (2 C), 129.2, 134.6, 137.6, 140.9, 145.2 (d, JC−F = 5.0 Hz, 1C) 146.1 (d, JC−F = 12.2 Hz, 1C), 147.8, 5766

DOI: 10.1021/acs.jmedchem.7b00461 J. Med. Chem. 2017, 60, 5759−5771

Journal of Medicinal Chemistry

Article

6-Benzyl-3-(3-(1,3-thiazol-5-ylmethoxy)phenyl)-4,6dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione (9). To a solution of 20 (82 mg, 0.24 mmol) and 21c (60 mg, 0.35 mmol) in NMP (3 mL) were added K2CO3 (147 mg, 1.06 mmol) and KI (5.9 mg, 0.036 mmol) at room temperature. The mixture was stirred at room temperature overnight, diluted with EtOAc and 5% aqueous citric acid, and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (EtOAc/MeOH = 100/0 to 88/12) and subsequently by preparative HPLC (condition 3, 0.1% TFA in water/0.1% TFA in MeCN = 80/20 to 0/100) to give 9 (28 mg, 27%) as a white solid. 1H NMR δ (300 MHz, DMSO-d6) 4.59 (s, 2H), 5.01 (s, 2H), 5.39 (s, 2H), 5.75 (s, 1H), 6.88−6.98 (m, 2H), 7.04 (s, 1H), 7.23−7.38 (m, 6H), 7.68 (s, 1H), 8.01 (s, 1H), 9.12 (s, 1H), 10.00 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 46.8, 50.3, 61.7, 96.2, 101.8, 112.1, 112.3, 118.1, 127.3, 127.5 (2C), 128.4 (2C), 129.2, 133.8, 134.7, 137.6, 142.9, 143.2, 147.7, 151.8, 155.2, 157.6, 161.3. MS m/z 445 (M + H)+. Anal. Calcd for C24H20N4O3S·0.5H2O: C, 63.56; H, 4.67; N, 12.35. Found: C, 63.84; H, 4.89; N, 12.52. 6-Benzyl-3-(2-(benzyloxy)phenyl)-4,6-dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione (10). To a solution of 4-amino-1benzyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde 16b (100 mg, 0.438 mmol), 2-(benzyloxy)aniline (0.085 mL, 0.481 mmol), and SnCl2· 2H2O (20 mg, 0.089 mmol) in MeOH (10 mL) was added polymethylhydrosiloxane (81 μL) at room temperature. The mixture was stirred at room temperature for 3 d, diluted with EtOAc, passed through a short column (Na2SO4 and silica gel, EtOAc), and concentrated. The residue was purified by silica gel column chromatography (EtOAc/MeOH = 100/0 to 88/12) to give 4amino-1-benzyl-5-(((2-(benzyloxy)phenyl)amino)methyl)pyridin2(1H)-one (55 mg, 31%) as a colorless oil. 1H NMR (300 MHz, DMSO-d6) δ 4.03 (d, J = 6.7 Hz, 2H), 4.89 (s, 2H), 5.10 (s, 2H), 5.32 (s, 1H), 5.38 (t, J = 5.8 Hz, 1H), 6.11 (s, 2H), 6.45−6.54 (m, 1H),6.60 (s, 1H), 6.65−6.73 (m, 1H), 6.86 (d, J = 8.1 Hz, 1H), 7.13−7.20 (m, 2H), 7.22−7.40 (m, 6H), 7.47 (d, J = 9.9 Hz, 3H). MS m/z 412 (M + H)+. To 4-amino-1-benzyl-5-(((2-(benzyloxy)phenyl)amino)methyl)pyridin-2(1H)-one (52 mg, 0.13 mmol) and CDI (102 mg, 0.63 mmol) in THF (10 mL) was added DBU (95 μL, 0.63 mmol) at room temperature. The mixture was stirred at room temperature for 4 h, diluted with EtOAc, acidified with 5% citric acid, and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4, and concentrated. The residual solid was suspended in EtOAc, collected by filtration, washed with EtOAc, and dried to give 10 (45 mg, 81%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 4.45 (2H, s), 5.00 (2H, s), 5.14 (2H, s), 5.73 (1H, s), 6.98 (1H, t, J = 7.5 Hz), 7.16 (1H, d, J = 8.3 Hz), 7.21−7.41 (12H, m),7.64 (1H, s), 9.91 (1H, s). 13C NMR (151 MHz, DMSO-d6) δ ppm 47.2, 50.3, 69.4, 96.2, 101.7, 113.7, 120.8, 126.9 (2C)127.3, 127.4 (2C), 127.6, 128.2 (2C), 128.4 (2C), 128.5, 129.0, 130.7, 134.6, 136.9, 137.6, 148.2, 151.7, 153.9, 161.3. MS m/z 438 (M + H)+. Anal. Calcd for C27H23N3O3·0.2H2O: C, 73.52; H, 5.35; N, 9.53. Found: C, 73.64; H, 5.28; N, 9.53. 4-Azido-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylic Acid (15a). A mixture of 14a18 (3.25 g, 15.1 mmol) and sodium azide (1.96 g, 30.1 mmol) in DMF (32 mL) was heated to 55 °C and stirred for 5 d. The reaction mixture was diluted with saturated aqueous NaHCO3 and saturated aqueous Na2CO3, and the mixture was extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over anhydrous Na2SO4, and concentrated to afford ethyl 4-azido-1-methyl-6-oxo-1,6-dihydropyridine-3-carboxylate (6.60 g) as a crude product. The crude (6.60 g) and lithium hydroxide monohydrate (2.18 g, 52.0 mmol) in EtOH (30 mL) and water (30 mL) was stirred at room temperature for 4 h. The reaction mixture was acidified with conc hydrochloric acid to pH 2. The resultant precipitate solid was collected by filtration to afford 15a (2.08 g, 71% for 2 steps) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 3.46 (s, 3H), 6.16 (s, 1H), 8.45 (s, 1H). 1H was not observed. MS m/z 195 (M + H)+.

151.8, 154.1, 157.1 (d, JC−F = 258.2 Hz, 1C), 161.34. MS m/z 483 (M + H)+. Anal. Calcd for C28H23FN4O3·2.6H2O: C, 63.53; H, 5.37; N, 10.58. Found: C, 63.85; H, 5.05; N, 10.64. 6-Benzyl-3-(3-(benzyloxy)phenyl)-4,6-dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione (6). To a mixture of 18a (30.0 mg, 0.151 mmol) and 16b (34.4 mg, 0.151 mmol) in THF (3 mL) was added Ti(i-PrO)4 (68 μL, 0.23 mmol) at room temperature under N2. The reaction mixture was heated to 50 °C and stirred for 16 h. To the reaction mixture was added NaBH(OAc)3 (128 mg, 0.603 mmol), and the reaction mixture was stirred at 50 °C for 18 h. The reaction mixture was concentrated. The residue was purified by preparative TLC (CH2Cl2/MeOH = 20/1) to afford 4-amino-1-benzyl-5-(((3(benzyloxy)phenyl)amino)methyl)pyridin-2(1H)-one (40.0 mg, 65%) as a yellow solid. MS m/z 421 (M + H)+. A mixture of 4-amino-1-benzyl-5-(((3-(benzyloxy)phenyl)amino)methyl)pyridin-2(1H)-one (160 mg, 0.39 mmol), CDI (315 mg, 1.94 mmol), and DBU (0.290 mL, 1.94 mmol) in THF (10 mL) was stirred at room temperature for 18 h. The reaction mixture was concentrated, and the residue was purified by preparative HPLC (condition 2, 0.225% aqueous formic acid solution/MeCN = 50/50 to 29/71) to afford 6 (90 mg, 53%) as a white solid. 1H NMR (400 MHz, DMSOd6) δ 4.59 (s, 2H), 5.01 (s, 2H), 5.09 (s, 2H), 5.75 (s, 1H), 6.87−6.96 (m, 2H), 7.00−7.05 (m, 1H), 7.23−7.30 (m, 4H), 7.31−7.36 (m, 3H), 7.37−7.42 (m, 2H), 7.43−7.48 (m, 2H), 7.68 (s, 1H), 10.00 (s, 1H). 13 C NMR (151 MHz, DMSO-d6) δ 46.8, 50.3, 69.3, 96.2, 101.8, 111.9, 112.1, 117.6, 127.3, 127.5 (2C), 127.6 (2C), 127.8, 128.3 (2C), 128.4 (2C), 129.2, 134.6, 136.8, 137.6, 143.2, 147.7, 151.8, 158.4, 161.4. MS m/z 438 (M + H)+. Anal. Calcd for C27H23N3O3·1.9H2O: C, 68.75; H,5.73; N, 8.91. Found: C, 68.84; H, 5.60; N, 9.09. 6-Benzyl-3-(3-(pyridin-2-ylmethoxy)phenyl)-4,6dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione (7). A mixture of 20 (200 mg, 0.576 mmol), 2-(bromomethyl)pyridine hydrobromide (21a, 160 mg, 0.638 mmol), and K2CO3 (318 mg, 2.30 mmol) in DMF (5 mL) was stirred at room temperature for 15 h. The reaction mixture was diluted with water and extracted with CH2Cl2/MeOH (10/1). The organic layer was separated, washed with brine, dried over anhydrous Na2SO4, and concentrated. The residue was purified by preparative TLC (CH2Cl2/MeOH = 12/1) and subsequently by preparative HPLC (condition 1, 0.05% aqueous ammonia solution/MeCN = 75/25 to 45/55) to afford 7 (91 mg, 36%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 4.59 (s, 2H), 5.01 (s, 2H), 5.17 (s, 2H), 5.75 (s, 1H), 6.90 (dd, J = 8.0, 2.0 Hz, 1H), 6.94 (dd, J = 8.0, 1.2 Hz, 1H), 7.05−7.06 (m, 1H), 7.25−7.36 (m, 7H), 7.52 (d, J = 7.6 Hz, 1H), 7.68 (s, 1H), 7.82−7.86 (m, 1H), 8.55− 8.60 (m, 1H), 10.00 (brs, 1H). 13C NMR (75 MHz, DMSO-d6) δ 47.3, 50.8, 70.9, 96.8, 102.3, 112.4, 112.7, 118.3, 122.2, 123.5, 127.9, 128.0 (2C) 129.0 (2C) 129.8, 135.2, 137.5, 138.2, 143.7, 148.3, 149.6, 152.4, 157.0, 158.8, 161.9. MS m/z 439 (M + H)+. Anal. Calcd for C26H22N4O3·1.0H2O: C, 68.41; H, 5.30; N, 12.27. Found: C, 68.50; H, 5.41; N, 12.31. 6-Benzyl-3-(3-(pyridin-3-ylmethoxy)phenyl)-4,6dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione (8). A mixture of 20 (200 mg, 0.576 mmol), 21b (160 mg, 0.638 mmol), and K2CO3 (318 mg, 2.30 mmol) in DMF (2 mL) was stirred at room temperature for 14 h under N2. The reaction mixture was poured into water and extracted with CH2Cl2/MeOH (10/1). The organic layer was separated, washed with brine, dried over Na 2SO4 , and concentrated. The residue was purified by preparative TLC (CH2Cl2/MeOH = 20/1) and subsequently by preparative HPLC (condition 1, 0.05% aqueous ammonia solution/MeCN = 68/32 to 40/60) to afford 8 (33 mg, 13%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 4.59 (s, 2H), 5.01 (s, 2H), 5.14 (s, 2H), 5.75 (s, 1H), 6.87−6.98 (m, 2H), 7.01−7.08 (m, 1H), 7.25−7.35 (m, 6H), 7.38− 7.48 (m, 1H), 7.68 (s, 1H), 7.82−7.91 (m, 1H), 8.50−8.59 (m, 1H), 8.64−8.71 (m, 1H), 9.99 (brs, 1H). 13C NMR (75 MHz, DMSO-d6) δ ppm 47.4, 50.8, 67.5, 96.8, 102.3, 112.5, 112.7, 118.4, 124.1, 127.9, 128.0 (2C), 129.0 (2C), 129.8, 133.0, 135.2, 136.2, 138.2, 143.8, 148.3, 149.6, 149.7, 152.4, 158.7, 161.9. MS m/z 439 (M + H)+. Anal. Calcd for C26H22N4O3·1.5H2O: C, 67.08; H, 5.41; N, 12.04. Found: C, 67.22; H, 5.30; N, 12.08. 5767

DOI: 10.1021/acs.jmedchem.7b00461 J. Med. Chem. 2017, 60, 5759−5771

Journal of Medicinal Chemistry

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4-Azido-1-benzyl-6-oxo-1,6-dihydropyridine-3-carboxylic Acid (15b). To a solution of 14b19 (10.5 g, 36.0 mmol) in DMF (100 mL) was added sodium azide (4.75 g, 73.1 mmol) at room temperature. The mixture was stirred at 60 °C for 2 d, cooled to room temperature, diluted with water, and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4, and concentrated to give ethyl 4-azido-1-benzyl-6-oxo-1,6dihydropyridine-3-carboxylate (10.7 g) as a crude product. To a solution of the crude (10.7 g) in THF (50 mL) and EtOH (50 mL) was added 1 M NaOH (43 mL, 43.0 mmol) at room temperature. The mixture was stirred at room temperature for 1 h, and THF was removed by evaporation. The residual solution was cooled to 0 °C and acidified with 1 M HCl to pH 2. The resultant precipitated solid was collected by filtration, washed with water, and dried to give 15b (9.20 g, 95% for 2 steps) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 5.18 (s, 2H), 6.24 (s, 1H), 7.25−7.41 (m, 5H), 8.58 (s, 1H), 12.92 (brs, 1H). MS m/z 243 (M − N2 + H)+. 4-Amino-1-methyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde (16a). To a mixture of 15a (700 mg, 3.61 mmol), N,Odimethylhydroxylamine hydrochloride (700 mg, 7.21 mmol) and DIPEA (3.77 mL, 21.6 mmol) in DMF (5 mL) was added T3P (50% in ethyl acetate, 4.28 mL, 7.21 mmol) dropwise at 0 °C. The reaction mixture was warmed to room temperature and stirred for 16 h. The reaction mixture was diluted with water, and the mixture was extracted with CH2Cl2/MeOH (10/1). The organic layer was separated, washed with brine, dried with anhydrous Na2SO4, filtered, and concentrated to afford 4-azido-N-methoxy-N,1-dimethyl-6-oxo-1,6-dihydropyridine-3carboxamide (800 mg) as a crude product. To a solution of the crude (800 mg) in THF (20 mL) was added LiAlH4 (384 mg, 10.1 mmol) portionwise at 0 °C under N2. The reaction mixture was warmed to room temperature and stirred for 2 h. The reaction mixture was poured into saturated aqueous NH4Cl at 0 °C, and the mixture was extracted with CH2Cl2/MeOH (10/1). The organic layer was separated, washed with brine (30 mL), dried over anhydrous Na2SO4, and concentrated. The residue was purified by preparative TLC on silica gel (CH2Cl2/MeOH = 10/1) to afford 16a (130 mg, 24% for 2 steps) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 3.38 (s, 3H), 5.29 (s, 1H), 7.05 (brs, 2H), 8.35 (s, 1H), 9.44 (s, 1H). MS m/z 153 (M + H)+. 4-Amino-1-benzyl-6-oxo-1,6-dihydropyridine-3-carbaldehyde (16b). To a solution of 15b (9.19 g, 34.0 mmol), N,Odimethylhydroxylamine hydrochloride (4.98 g, 51.0 mmol), HOBt H2O (7.81 g, 51.0 mmol), and EDC HCl (9.78 g, 51.0 mmol) in DMF (120 mL) was added DIPEA (26.7 mL, 153 mmol) at room temperature. The mixture was stirred at room temperature for 3 days. The reaction mixture was concentrated to one-fourth volume, diluted with water, and extracted with EtOAc. The organic layer was separated, washed with 5% aqueous citric acid, water, 5% aqueous NaHCO3, water, and brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (hexane/ EtOAc = 50/50 to 0/100) to give 4-azido-1-benzyl-N-methoxy-Nmethyl-6-oxo-1,6-dihydropyridine-3-carboxamide (9.00 g, 84%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 3.19 (s, 3H), 3.52 (s, 3H), 5.09 (s, 2H), 6.29 (s, 1H), 7.23−7.41 (m, 5H), 8.10 (s, 1H). MS m/z 314 (M + H)+. To a suspension of LiAlH4 (807 mg, 21.3 mmol) in THF (30 mL) was added dropwise a solution of 4-azido-1-benzyl-N-methoxy-Nmethyl-6-oxo-1,6-dihydropyridine-3-carboxamide (3.33 g, 10.6 mmol) in THF (30 mL) at 0 °C. The mixture was stirred at 0 °C for 0.5 h and quenched with EtOH in THF at 0 °C. To the mixture was added EtOAc and saturated aqueous potassium sodium (+)-tartarate at 0 °C. The mixture was filtered through a pad of Celite, and the filtrate was extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4, passed through a short column (silica gel, EtOAc), and concentrated to give 16b (1.64 g, 68%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 5.06 (s, 2H), 5.32 (s, 1H), 7.10 (s, 2H), 7.24−7.38 (m, 5H), 8.49 (1H, s), 9.47 (1H, s). MS m/z 229 (M + H)+. 6-Benzyl-3-(3-((4-methoxybenzyl)oxy)phenyl)-4,6dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione (19). To a

mixture of 18b (510 mg, 2.22 mmol) and 16b (460 mg, 2.01 mmol) in THF (10 mL) was added Ti(i-PrO)4 (0.89 mL, 3.0 mmol) at room temperature under N2. The reaction mixture was heated to 50 °C and stirred for 16 h. To the reaction mixture was added NaBH(OAc)3 (1.70 g, 8.02 mmol), and the reaction mixture was stirred at 50 °C for 18 h. The reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (CH2Cl2/MeOH = 100/1 to 20/1) to afford 4-amino-1-benzyl-5(((3-((4-methoxybenzyl)oxy)phenyl)amino)methyl)pyridin-2(1H)one (700 mg, 79%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 3.75 (s, 3H), 3.89 (d, J = 5.6 Hz, 1H), 4.06−4.08 (m, 1H), 4.86− 4.92 (m, 4H), 5.35−5.43 (m, 1H), 5.88−5.93 (m, 2H), 6.17−6.33 (m, 3H), 6.90−6.96 (m, 3H), 7.04−7.08 (m, 1H), 7.16−7.28 (m, 5H), 7.30−7.34 (m, 2H), 7.38−7.46 (m, 1H). MS m/z 442 (M + H)+. To a 4-amino-1-benzyl-5-(((3-((4-methoxybenzyl)oxy)phenyl)amino)methyl)pyridin-2(1H)-one (4.64 g, 10.5 mmol) and CDI (8.52 g, 52.6 mmol) in THF (150 mL) was added DBU (7.92 mL, 52.6 mmol) at room temperature. The mixture was stirred at room temperature overnight. The reaction mixture was diluted with EtOAc, acidified with 5% citric acid and extracted with EtOAc. The organic layer was separated, washed with water, brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (EtOAc/MeOH = 100/0 to 88/12) to give 19 (4.20 g, 85%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 3.75 (s, 3H), 4.59 (s, 2H), 5.00 (brs, 2H), 5.01 (brs, 2H), 5.74 (s, 1H), 6.85−6.98 (m, 4H), 7.00 (s, 1H), 7.24−7.40 (m, 8H),7.68 (s, 1H), 9.97 (s, 1H). MS m/z 468 (M + H)+. 6-Benzyl-3-(3-hydroxyphenyl)-4,6-dihydropyrido[4,3-d]pyrimidine-2,7(1H,3H)-dione (20). To a mixture of 19 (300 mg, 0.642 mmol) and anisole (697 μL, 6.42 mmol) was added TFA (3.0 mL, 39 mmol) at room temperature. The mixture was stirred room temperature for 1 h, diluted with EtOAc and basified with 5% aqueous NaHCO3, and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4, and concentrated. The residual solid was suspended in hexane/EtOAc (4/1), collected by filtration, washed with hexane/EtOAc (4/1), and dried to give 20 (223 mg, quant) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 4.55 (s, 2H), 5.00 (s, 2H), 5.73 (s, 1H), 6.63 (d, J = 8.0 Hz, 1H), 6.70−6.77 (m, 2H), 7.15 (t, J = 8.3 Hz, 1H), 7.22−7.40 (m, 5H), 7.68 (s, 1H), 9.51 (s, 1H), 9.93 (s, 1H). MS m/z 348 (M + H)+. Evaluation of Solubility. Small volumes of compound solution dissolved in DMSO were added to the aqueous buffer solution (pH 6.8). After incubation, precipitates were separated by filtration. The solubility was determined by UV absorbance of each filtrate. Parallel Artificial Membrane Permeability Assay (PAMPA). The donor wells were filled with 200 μL of PRISMA HT buffer (pH 7.4, pION Inc.) containing 10 μmol L−1 test compound. The filter on the bottom of each acceptor well was coated with 4 μL of a GIT-0 lipid solution (pION Inc.) and filled with 200 μL of acceptor sink buffer (pION Inc.). The acceptor filter plate was put on the donor plate and incubated for 3 h at room temperature. After the incubation, the amount of test compound in both the donor and acceptor wells was measured by LC/MS/MS. Evaluation of log D. The log D7.4, which is a partition coefficient between 1-octanol and aqueous buffer pH 7.4, of the compounds was measured using the chromatographic procedure whose condition was developed based on a published method.21,22 Preparation of Enzymes for Biochemical Assays. For ATPase assay, the human recombinant full-length Brr2 and full-length DHX29 were expressed in Sf-9 insect cells as fusion proteins with His and HisFLAG tag at the N-terminus, respectively, using the BaculoDirect Cterm baculovirus expression system (Thermo Fisher Scientific Inc., Waltham, MA, USA). His-Brr2 and His-FLAG-DHX29 were purified by Ni-NTA superflow affinity column and Superdex 200 gel-filtration column. The human recombinant proteins, full-length eIF4A3, MLN51 (residues 137−283), full-length eIF4A1, full-length eIF4B, and eIF4G (residues 712−1451) were expressed in Escherichia coli BL21(DE3) as fusion proteins with 6× His- SUMO or His tag followed by a tobacco etch virus (TEV) protease cleavage site at the N terminus and purification by Ni-NTA superflow affinity column 5768

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(QIAGEN, Venlo, The Netherlands) and Superdex 200 gel-filtration column (GE Healthcare, Chicago, IL, USA). The His-SUMO or His tags were cleaved with SUMO protease or TEV protease. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific Inc.) with bovine serum albumin as a standard. RNA-Dependent ATPase Assay. The RNA-dependent ATPase assay was performed using the ADP-Glo assay system (Promega Corp., Madison, WI, USA) for primary screening and a phosphate sensor (Thermo Fisher Scientific Inc.) for secondary screening. Singlestranded RNA poly(U) was purchased from MP Biomedicals, LLC (Solon, OH, USA). The assay buffer comprised 20 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2, 100 mM KCl, 1 mM dithiothreitol (DTT), and 0.01% (v/v) Tween 20. As for ADP-Glo assay, after the addition of 20 μM ATP and 2.5 μg mL−1 poly(U) and test compounds, the ATPase reactions were started by the addition of 6.25 nM Brr2. They were incubated at room temperature for 30 min, and then the enzymatic reactions were terminated by ADP-Glo reagent. Following addition of ADP-Glo detection reagent, luminescent signals were measured using an EnVision 2102 multilabel plate reader (PerkinElmer, Waltham, MA, USA). For an assay using a phosphate sensor, 2 nM Brr2 was mixed with test compounds and substrates, and enzymatic reaction was terminated by addition of EDTA after 120 min. Following addition of 1 μM phosphate sensor, fluorescent signals (excitation 430 nm/ emission 450 nm) were detected using EnVision 2102 multilabel plate reader (PerkinElmer). We defined the luminescent or fluorescent signals of the reaction without enzyme as 100% inhibitory activity and those of the complete reaction mixture as 0% inhibitory activity. Curve fittings and calculations of IC50 values were performed using the program XLfit version 5 (ID Business Solutions Ltd., Guildford, Surrey, U.K.). For evaluating selectivity, we also conducted ATPase assay for eIF4A1, eIF4A3, and DHX29. To enhance ATPase activity for eIF4A, the equivalent molar concentrations of MLN51 for 150 nM eIF4A3 or eIF4B and eIF4G for 100 nM eIF4A1 or eIF4A2 were added. Regarding the ATPase assays for DHX29, the optimal concentrations were 6.3 nM. Concentrations of ATP or RNA were set at the Km value of each substrate for each enzyme as follows: 35 μM ATP and 1.5 μg mL−1 poly(U) for eIF4A1 and eIF4A3; 30 μM ATP and 1.8 μg mL−1 poly(U) for DHX29. Detection of luminescent signals or estimation of IC50 values was performed as described above. Thermal Shift Assay. A mixture of 500 nM Brr2 and 500-diluted SYPRO Orange (Thermo Fisher Scientific Inc.) in assay buffer containing 20 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2, 100 mM KCl, and 1 mM DTT was mixed with test compounds in 384-well plates. Fluorescent signals were measured by ABI 7900HT FAST real-time PCR system (Thermo Fisher Scientific Inc.). The temperature was held for 1 min per degree from 40 to 70 °C. The maximum fluorescent signal within detected ones at various temperatures is defined as 100% denaturation rate, and the minimum signal is defined as 0% denaturation rate. Tm values were calculated using the program Graphfit Prism version 5.03 (GraphPad Software, Inc., San Diego, CA, USA). Substrate-Competition Assay. ATP or RNA competition assays for Brr2 were performed using the ADP-Glo assay system. The enzymatic reactions were performed under identical conditions to those used for RNA-dependent ATPase assay except for substrate concentrations. ATP or RNA concentrations were set at the Km values (25 μM or 2.5 μg mL−1, respectively), and 500, 200, or 2.5 μM ATP and 50 μg mL−1 or 0.25 μg mL−1 poly(U) were used to examine ATP or RNA competitive inhibition. The enzymatic reactions were performed at room temperature for 30 min except for 45 min at 25 μM ATP/0.25 μg mL−1 poly(U). Curve fittings and calculations of IC50 values were performed, as previously described in the section of RNA-dependent ATPase assay. RNA Unwinding Assay. RNA unwinding assays were performed in assay buffer containing 40 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 30 mM NaCl, 1 mM DTT, and 0.01% (v/v) Tween 20. Duplex RNAs were generated with fluorescence-labeled 14-mer (5′-[FITC]UUCCCCUGCAUAAC-3′) and 36-mer (5′-GUUAUGCAGGGGAACCAACGCAUAUCAGUGAGGAUU-3′) oligo RNAs purchased

from Integrated DNA Technologies, Inc. (Coralville, IA, USA). The RNAs in assay buffer (30 nM) were annealed by heating at 95 °C for 5 min and cooling to 4 °C at 0.1 °C/s. Helicase activity was initiated with 30 nM duplex RNAs, 200 nM Brr2, and 20 μM ATP at 37 °C for 10 min. Unlabeled 14-mer oligo RNA (1.5 μM; 5′-UUCCCCUGCAUAAC-3′) was included to capture any free 36-mer oligo RNAs. Reactions were terminated with 40 mM EDTA, 3.2% (w/v) SDS, and 40% (v/v) glycerol. The reaction solutions were electrophoresed with 15% polyacrylamide gel (ATTO Corp.) at 10.5 mV for 45 min under native running buffer (25 mM Tris and 192 mM glycine). The fluorescence-labeled RNAs were visualized using a Typhoon 9400 (GE Healthcare). The intensity of each band was measured using ImageQuant TL (GE Healthcare). The intensity of single-stranded RNAs as products after enzymatic reaction was normalized with the total of the intensity of single- and doublestranded RNAs, and background was subtracted. We defined the fluorescent signals of the reaction without enzyme as 100% inhibitory activity and those of the complete reaction mixture as 0% inhibitory activity. Curve fittings and calculations of IC50 values were performed using the program Graphfit Prism version 5.03 (GraphPad Software, Inc.). Crystallography. The generation of recombinant human Brr2 protein is detailed in the Supporting Information. Prior to crystallization, the purified Brr2 was diluted to 10 mg mL−1 using delivery buffer (20 mM Tris, pH 7.6, 200 mM NaCl, 1 mM TCEP), and complexes were generated by addition of stock DMSO solutions of ligands to the protein solution at a final concentration of 1 mM (the final DMSO concentrations were 10% for Brr2-compound 12 and 1% for Brr2-compound 3 and Brr2-compound 6). After addition of compounds, the mixture was placed on ice for 30 min to promote complex formation. Crystallization of Brr2 was performed using the hanging drop vapor diffusion method using a published protocol.7 Briefly, the crystallization was performed with a reservoir solution composed of 0.1 M sodium citrate (pH 5.0−5.3) and 1.2−1.6 M sodium malonate (pH 7.0) (Hampton Research, Aliso Viejo, CA, USA). Crystal trials were set up and stored at room temperature, with crystals appearing overnight and reaching maximum size after 1−2 weeks. Crystals were harvested directly from the drops (with the high concentration of sodium malonate functioning as a cryoprotectant) and flash-cooled into ALS-style pucks submerged in liquid nitrogen. Diffraction data were collected at Advanced Light Source beamline 5.0.2 (compound 3) and 5.0.3 (compound 6) (Lawrence Berkeley’s National Laboratory, Berkeley, CA) and at the Diamond Light Source beamline I04 (Didcot, U.K.) (compound 12). Diffraction data were processed with XDS23 and Aimless.24 The structures were solved by molecular replacement with Phaser25 using the coordinates of human Brr2 (PDB code 4F91) as a search model. The graphics program COOT26 was used for model building and refinement was performed with REFMAC527 and PHENIX.28 Phaser and REFMAC5 are distributed as part of CCP4.29 Structure validation was performed using Molprobity.30 X-ray diffraction data collection and refinement statistics are reported in Table S3, and omit density and ligplots corresponding to the ligands are shown in Figures S5−S7. The coordinates of the three cocomplex structures were deposited in the PDB under the accession codes 5URJ, 5URK, and 5URM corresponding to complexes with compounds 3, 6, and 12, respectively.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00461. Scheme to explore Brr2 inhibitors, SAR of compounds 11−13, protein expression and purification for crystallization, comparison of the crystal structure of Brr2 with or without compound 3, crystal structure of Brr2 in complex with compound 12, substrate-competitive assay 5769

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(4) Raghunathan, P. L.; Guthrie, C. RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2. Curr. Biol. 1998, 8, 847−855. (5) Kim, D. H.; Rossi, J. J. The first ATPase domain of the yeast 246kDa protein is required for in vivo unwinding of the U4/U6 duplex. RNA 1999, 5, 959−971. (6) Small, E. C.; Leggett, S. R.; Winans, A. A.; Staley, J. P. The EF-Glike GTPase Snu114p regulates spliceosome dynamics mediated by Brr2p, a DExD/H box ATPase. Mol. Cell 2006, 23, 389−399. (7) Santos, K. F.; Jovin, S. M.; Weber, G.; Pena, V.; Luhrmann, R.; Wahl, M. C. Structural basis for functional cooperation between tandem helicase cassettes in Brr2-mediated remodeling of the spliceosome. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 17418−17423. (8) Pena, V.; Jovin, S. M.; Fabrizio, P.; Orlowski, J.; Bujnicki, J. M.; Luhrmann, R.; Wahl, M. C. Common design principles in the spliceosomal RNA helicase Brr2 and in the Hel308 DNA helicase. Mol. Cell 2009, 35, 454−466. (9) Zhang, L.; Xu, T.; Maeder, C.; Bud, L. O.; Shanks, J.; Nix, J.; Guthrie, C.; Pleiss, J. A.; Zhao, R. Structural evidence for consecutive Hel308-like modules in the spliceosomal ATPase Brr2. Nat. Struct. Mol. Biol. 2009, 16, 731−739. (10) van Nues, R. W.; Beggs, J. D. Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae. Genetics 2001, 157, 1451−1467. (11) Cvačková, Z.; Matějů, D.; Staněk, D. Retinitis pigmentosa mutations of SNRNP200 enhance cryptic splice-site recognition. Hum. Mutat. 2014, 35, 308−317. (12) Zhao, C.; Bellur, D. L.; Lu, S.; Zhao, F.; Grassi, M. A.; Bowne, S. J.; Sullivan, L. S.; Daiger, S. P.; Chen, L. J.; Pang, C. P.; Zhao, K.; Staley, J. P.; Larsson, C. Autosomal-dominant retinitis pigmentosa caused by a mutation in SNRNP200, a gene required for unwinding of U4/U6 snRNAs. Am. J. Hum. Genet. 2009, 85, 617−627. (13) Ledoux, S.; Guthrie, C. Retinitis Pigmentosa mutations in Bad Response to Refrigeration 2 (Brr2) impair ATPase and helicase activity. J. Biol. Chem. 2016, 291, 11954−11965. (14) Shadrick, W. R.; Ndjomou, J.; Kolli, R.; Mukherjee, S.; Hanson, A. M.; Frick, D. N. Discovering new medicines targeting helicases: challenges and recent progress. J. Biomol. Screening 2013, 18, 761−781. (15) Ogino, M.; Ikeda, Z.; Fujimoto, J.; Ohba, Y.; Ishii, N.; Fujimoto, T.; Oda, T.; Taya, N.; Yamashita, T.; Matsunaga, N. Heterocyclic compound. PCT Int. Appl. WO2014030743 A1, February 27, 2014. (16) Since helicase RNA binding sites are less similar to each other, one might expect RNA site binder would have higher selectivity. However, it was not correct at least in our case. The reason for the lower selectivity of 12 is still unclear, and further investigation such as cocrystal structure analysis with other helicases is necessary. (17) We could not rule out the possibility of the formation of hydrogen bonding interaction with Lys2500 in place of Thr1197 so far. (18) Wallace, E. M.; Lyssikatos, J.; Blake, J. F.; Seo, J.; Yang, H. W.; Yeh, T. C.; Perrier, M.; Jarski, H.; Marsh, V.; Poch, G.; Livingston, M. G.; Otten, J.; Hingorani, G.; Woessner, R.; Lee, P.; Winkler, J.; Koch, K. Potent and selective mitogen-activated protein kinase kinase (MEK) 1,2 inhibitors. 1. 4-(4-bromo-2-fluorophenylamino)-1- methylpyridin-2(1H)-ones. J. Med. Chem. 2006, 49, 441−444. (19) Kai, H.; Endoh, T.; Jikihara, S.; Asahi, K.; Horiguchi, T. Novel heterocyclic derivatives and pharmaceutical composition containing same. PCT Int. Appl. WO2012020742 A1, February 16, 2012. (20) Ito, M.; Iwatani, M.; Kamada, Y.; Sogabe, S.; Nakao, S.; Tanaka, T.; Kawamoto, T.; Aparicio, S.; Nakanishi, A.; Imaeda, Y. Discovery of selective ATP-competitive eIF4A3 inhibitors. Bioorg. Med. Chem. 2017, 25, 2200−2209. (21) Nakashima, S.; Yamamoto, K.; Arai, Y.; Ikeda, Y. Impact of physicochemical profiling for rational approach on drug discovery. Chem. Pharm. Bull. 2013, 61, 1228−1238. (22) Masumoto, K.; Takeyasu, A.; Oizumi, K.; Kobayashi, T. Studies of novel 1,4-dihydropyridine Ca antagonist CS-905. I. Measurement of partition coefficient (log P) by high performance liquid chromatography (HPLC). Yakugaku Zasshi 1995, 115, 213−220.

for compound 9, time course of RNA helicase activity of Brr2, X-ray data collection and refinement statistics, omit electron density of Brr2 inhibitors and the ligand interactions, and helicase inhibitory activity of compound 12 (PDF) Molecular formula strings for all the compounds reported in this study (CSV) Coordinate file of docking model for compound 9 bound to Brr2 (PDB) Accession Codes

PDB codes are the following: 5URJ (compound 3); 5URK (compound 6); 5URM (compound 12). Authors will release the atomatic coordinates and experimental data upon article publication.



AUTHOR INFORMATION

Corresponding Authors

*M.I.-Y.: phone, +81 466 32 2809; e-mail, misa.iwatani@ takeda.com. *M.I.: phone, +81 466 32 1196; e-mail, masahiro.ito@takeda. com. ORCID

Misa Iwatani-Yoshihara: 0000-0001-8083-3560 Masahiro Ito: 0000-0001-5965-535X Author Contributions #

M.I.-Y. and M.I. contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Hiromichi Kimura for starting this project, Shoichi Okubo for preparing plasmids, Takashi Ito for preparing proteins, Tsutomu Henta and Takashi Santou for conducting the high-throughput screening, and Motomi Oonishi for compound evaluation. We thank Yusuke Ohba, Jun Fujimoto, Masaki Ogino, and Shuji Kitamura for providing us with synthetic intermediates and for advice on synthetic protocols. We also thank Yasumi Kumagai and Motoo Iida for their assistance with NMR spectroscopic experiments, Haruyuki Nishida for supervising of structure determination, Kouichi Iwanaga and Izumi Nomura for conducting high-throughput synthesis, and Michiko Tawada, Ryosuke Tokunoh, Keiji Kamiyama, Akinori Toita, and Nobuo Cho for their cooperation in helping us execute this work.



ABBREVIATIONS USED Brr2, bad response to refrigeration 2; SAR, structure−activity relationship; HTS, high throughput screening; SBDD, structure based drug design; DHX29, DEAH box protein 29; eIF4A1, eukaryotic initiation factor 4A-I; eIF4A3, eukaryotic initiation factor 4A-III; SUMO, small ubiquitin-like modifier; TEV, tobacco etch virus



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