Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active

Article pubs.acs.org/jmc

Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active eIF4A3 Inhibitors Masahiro Ito,*,† Toshio Tanaka,† Douglas R. Cary,† Misa Iwatani-Yoshihara,† Yusuke Kamada,† Tomohiro Kawamoto,† Samuel Aparicio,‡ Atsushi Nakanishi,† and Yasuhiro Imaeda*,† †

Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan ‡ Department of Molecular Oncology, BC Cancer Agency, 675 W. 10th Avenue, Vancouver, BC V5Z 1L3, Canada S Supporting Information *

ABSTRACT: Eukaryotic initiation factor 4A3 (eIF4A3), a member of the DEAD-box RNA helicase family, is one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD). In order to identify molecular probes to investigate the functions and therapeutic relevance of eIF4A3, a search for selective eIF4A3 inhibitors was conducted. Through the chemical optimization of 1,4-diacylpiperazine derivatives identified via high-throughput screening (HTS), we discovered the first reported selective eIF4A3 inhibitor 53a exhibiting cellular NMD inhibitory activity. A surface plasmon resonance (SPR) biosensing assay ascertained the direct binding of 53a and its analog 52a to eIF4A3 and revealed that the binding occurs at a non-ATP binding site. Compounds 52a and 53a represent novel molecular probes for further study of eIF4A3, the EJC, and NMD.

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INTRODUCTION Eukaryotic initiation factor 4A3 (eIF4A3) is a member of the DEAD-box RNA helicase family.1−4 DEAD-box RNA helicases play essential roles in RNA metabolism5 (e.g., pre-mRNA splicing, RNA transport, translation, and RNA decay) and generally function as part of large multiprotein complexes. DEAD-box proteins possess ATPase activity that is stimulated by RNA, and the ATPase cycle (e.g., ATP binding, hydrolysis, and release of products) changes the affinity of DEAD-box proteins for RNA.4 These ATP-driven changes in RNA affinity can be used to unwind the short RNA duplexes, to promote rearrangements of complex and structured RNAs, to remodel RNA-protein complexes, or to form a long-lived, highly stable complex on RNA (RNA clamping). eIF4A3 is known to be a core component of the exon junction complex (EJC) and has been suggested as an ATP-dependent RNA clamp that can serve as a nucleation center to recruit other EJC components.1,6 The EJC is involved in splicing, transport, translation, and nonsensemediated RNA decay (NMD).7−14 Among these functions, the most widely studied is NMD, which is the translation-dependent surveillance mechanism that recognizes mRNAs containing © 2017 American Chemical Society

premature termination codons (PTC) to prevent the accumulation of truncated proteins.15 Since nonsense mutations often cause hereditary and sporadic diseases due to a deficiency of essential proteins, a combination of an NMD inhibitor and a PTC read-through therapy has the potential to treat a variety of genetic diseases via the restoration of full-length proteins.16,17 There also are several reports implying the possible application of NMD inhibitors to cancer therapy (e.g., induction of cancer immunity18 or sensitization of cancer cells to a chemotherapeutic agent).19 Although little is known about the detailed regulatory mechanisms of eIF4A3 involving the EJC, NMD, and other functions, it is known that small interfering RNA (siRNA)mediated knockdown of eIF4A3 leads to a defect in NMD.8−10 Currently, several NMD suppressors with a variety of mechanisms are known;20−25 however, there are no such compounds that act via the selective inhibition of eIF4A3. It is expected that selective eIF4A3 inhibitors with NMD suppression Received: January 5, 2017 Published: March 30, 2017 3335

DOI: 10.1021/acs.jmedchem.6b01904 J. Med. Chem. 2017, 60, 3335−3351

Journal of Medicinal Chemistry

Article

Figure 1. Chemical structures of eIF4A3 inhibitors.

Scheme 1. Synthesis of 7a−d and 8a,ba

Reagents and conditions: (a) Boc2O, Et3N, CH2Cl2, 0−15 °C, 66−83%; (b) Boc2O, THF, toluene, 0 °C to rt, 70%; (c) 4-chlorobenzoyl chloride or 4-bromobenzoyl chloride, Et3N or DIPEA, CH2Cl2, 0−15 °C, 78−95%; (d) 4 M HCl in EtOAc, 0 °C or rt, 66% to quant; (e) (i) aq NaHCO3, rt, 97%; (ii) chiral HPLC, 41−42%. a

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CHEMISTRY Unless otherwise specified, the compounds described herein are all in the racemic form. The synthesis of 1-acyl-2-phenylpiperazines 7a−d and 8a,b (enantiomers of 7b), which were employed as common intermediates in this work, is depicted in Scheme 1. The 2-phenylpiperazine compounds 4a and 4c,d were selectively protected31 at the 4-position by a Boc group and then treated with 4-chlorobenzoyl chloride or 4-bromobenzoyl chloride, followed by removal of the Boc group to give 1-acyl2-phenylpiperazines (7a−d). Chiral separation of the 4bromobenzoyl derivative 7b afforded 8a as the (S)-enantiomer and 8b as the (R)-enantiomer. The absolute stereochemistry of these compounds was determined by X-ray crystal structure analysis using the TsOH salt of 8a (see Supporting Information). Attachment of varied acyl moieties to 7a−d and 8a,b was conducted as shown in Scheme 2. Condensation of 1-acyl-2phenylpiperazines 7a−d and 8a,b with the corresponding acids or esters 9−21 and 24−28 under a variety of conditions afforded the 1,4-diacylpiperazine derivatives 3, 29−48, 52a,b, and 53a,b. Meanwhile, compounds 49−51 were synthesized using the following two-step procedures. Amidation of the intermediate 7a

activity will expedite our understanding of the NMD process and the function of the EJC in RNA metabolism. A marine natural product, hippuristanol (1),26−29 has been reported as a paneIF4A inhibitor (Figure 1), but NMD inhibitory activity of 1 has not been reported. We recently reported a novel eIF4A3 selective inhibitor (2) with an ATP-competitive mechanism.30 However, 2 was inactive at 30 μM in a cellular assay for the detection of NMD suppression because of low permeability caused by the indispensable carboxylic acid moiety. Since such ATP-competitive inhibitors showed a narrow structure−activity relationship (SAR) and did not appear amenable to further chemical optimization, our attention shifted to other chemical series. Through high-throughput screening (HTS), a class of 1,4diacylpiperazine derivatives (e.g., 3) was identified as a promising chemotype. In this report, we focus on the discovery of 1,4diacylpiperazine derivatives 52a and 53a, which bind to a nonATP binding site of eIF4A3 and demonstrate cellular NMD inhibitory activity. 3336



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Scheme 2. Synthesis of 3, 29−51, 52a,b, 53a,ba

a

Reagents and conditions: (a) 9−10, 12, 14, 15, 18, 20, or 21, HATU, DIPEA, DMF, rt, 24−61%; (b) 11, 13, 21, or 25−26, EDC·HCl, HOBt, DIPEA, DMF, rt, 34−79%; (c) (i) 16 or 19, LiOH·H2O, THF, MeOH, H2O, rt; (ii) HATU, DIPEA, DMF, rt, 17−71%; (d) (i) 17, TFA, CH2Cl2, rt; (ii) HATU, Et3N, DMF, rt, 53%; (e) (i) aq NaHCO3, rt; (ii) 24 or 27, (Me3Al)2·DABCO,32 THF, microwave, 130 °C, 47−78%; (f) (i) aq NaHCO3, rt; (ii) 28, Me3Al, THF, microwave, 110 °C, 55%; (g) 13 or 26, EDC·HCl, HOBt, DMF, rt, 50−81%; (h) (i) 22, EDC·HCl, HOBt, DMF, rt, 25%; (ii) 2-bromobenzonitrile, Pd(OAc)2, CuI, Cs2CO3, DMF, microwave, 150 °C, 16%; (i) (i) 23, HATU, DIPEA, DMF, 0 °C, 95%; (ii) 3cyanophenylboronic acid or 4-cyanophenylboronic acid, Na2CO3, Pd(PPh3)4, DME, H2O, reflux, 30−49%.

Scheme 3. Synthesis of 55−60a

Reagents and conditions: (a) 21, HATU, DIPEA, DMF, rt, 85%; (b) (i) BH3·Me2S, THF, 0 °C to rt, 31%; (ii) 4-chlorobenzoyl chloride, K2CO3, MeCN, rt, 56%; (c) 4-chlorobenzyl bromide, K2CO3, MeCN, 80 °C, 19%; (d) corresponding acyl chloride (3-chlorobenzoyl chloride for 57, 2chlorobenzoyl chloride for 58, and benzoyl chloride for 59), Et3N, CH2Cl2, 0 °C to rt, 41−46%; (e) 4-bromobenzoic acid, EDC, HOBt, DIPEA, THF, rt, 96%. a

with acids 22 and subsequent C−H activation reaction33 at the 2positon of oxazole (condition h) furnished 49, whereas

condensation of 7a with acid 23 followed by Suzuki coupling (condition i) afforded 50 and 51. 3337



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Scheme 4. Synthesis of 63, 65, and 68a

Reagents and conditions: (a) (i) 4-chlorobenzoyl chloride, Et3N, CH2Cl2, 0 °C to rt; (ii) 62, NaH, DMF, 0 °C, 12% for 2 steps; (b) (i) Boc2O, Et3N, THF, 0 °C to rt, 73%; (ii) 4-chlorobenzoyl chloride, DIPEA, THF, 0 °C, 99%; (iii) 4 M HCl in EtOAc, rt, 91%; (iv) 21, EDC·HCl, HOBt, DIPEA, DMF, 0 °C to rt, 24%; (c) (i) 1,3-propanediamine, MeONa, MeOH, rt to 80 °C, 23%; (ii) BH3·Me2S, THF, 0−80 °C; (iii) Boc2O, Et3N, CH2Cl2, 0 °C to rt, 72% for 2 steps; (d) (i) 4-chlorobenzoyl chloride, Et3N, CH2Cl2, rt, 93%; (ii) 4 M HCl in dioxane, rt; (iii) 21, HATU, Et3N, CH2Cl2, rt, 34% for 2 steps. a

Compounds 55−60 were synthesized as illustrated in Scheme 3. Selective amidation of piperazine 7a at the 4-position of the piperazine with carboxylic acid 21 yielded intermediate 54, followed by reduction of the amide carbonyl group and subsequent acylation at the 4-position of the piperazine to afford amide 55. Meanwhile, alkylation at the 4-position on the piperazine of 54 provided compound 56. Acylation of 54 with the corresponding acid chlorides or 4-bromobenzoic acid afforded compounds 57−60. The synthesis of compounds 63, 65, and 68 was conducted as depicted in Scheme 4. Acylation of commercially available piperazinone 61 with 4-chlorobenzoyl chloride at the 4-position of piperazinone followed by alkylation with 62 provided compound 63. Protection of the 4-position of 2-cyclohexylpiperazine 64 with a Boc group, acylation with 4-chlorobenzoyl chloride, removal of the Boc group, and condensation with 21 provided compound 65. Treatment of α-bromoester 6634 with 1,3-propanediamine provided the homopiperazinone, followed by the reduction of the amide carbonyl group and selective protection at the 4-position of the homopiperazine by a Boc group to afford intermediate 67. Subsequently, the acylation of 67 with 4-chlorobenzoyl chloride followed by the removal of the Boc group under acidic conditions and finally amide coupling with carboxylic acid 21 yielded compound 68.

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Figure 2. Design strategy around compound 3.

Biological evaluation was conducted using the ADP-Glo assay system (Promega) in which inhibition of the RNA-dependent ATPase activity of eIF4A3 was detected, and the results were expressed as IC50 values. At first, replacement of the indole ring of compound 3 with other fused heterocycles was examined (Table 1). As a result, pyrazolopyridine 30 showed similar potency as 3, whereas the structurally similar indazole derivative 29 showed significantly diminished activity. These results suggested that subtle structural differences between the indole and indazole rings have an effect on binding to eIF4A3. Next, we designed and synthesized a variety of biaryl amide derivatives inspired by compound 30. Phenylpyrazole 31 was equipotent to 30, while regioisomer 32 showed deteriorated activity. 2-Phenyloxazole-5yl derivative 34 turned out to be the most potent among the oxazole series 33−35. In addition to that, 5-methylpyrazole 36 exhibited the same level of inhibitory activity as 31. Considering these observations, the nitrogen atom at the 3-position of oxazole 34 or the 2-position of pyrazoles 31 and 36 was judged to be indispensable for inhibitory activity. This implies the existence of a hydrogen bonding interaction between the nitrogen atom in these compounds and the eIF4A3 protein.

RESULTS AND DISCUSSION

Our design strategy based on compound 3 is illustrated in Figure 2. First, we investigated the SARs of the following key substructures: the 1-acyl group (A), the 4-acyl group (B), the 3-phenyl moiety (C), and the 1,4-diacylpiperazine ring (D). Then, combinations of the most promising functional groups were examined to further enhance biological activity. 3338



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Table 1. SAR around the 1-Acyl Moiety

a

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

modifications around the 4-acyl moiety were conducted. Similar to the SAR of substituents at the 3-postion on the piperazine (37 and 38), the 3-chlorobenzoyl (57) and 2-chlorobenzoyl (58) derivatives exhibited reduced activity. The unsubstituted phenyl derivative 59 showed slightly decreased activity, while the 4bromo derivative 60 demonstrated slightly increased potency compared with the 4-chloro derivative 34. These observations imply the importance of a 4-chloro or 4-bromo group. These halogen atoms might fill in a small hydrophobic cavity of the eIF4A3 protein or might be involved in a halogen bonding.35 In consideration to the basic SAR information presented above, we conducted further modifications of the 1-acyl moiety on compound 30 (Table 4). To enhance potency through additional interactions with eIF4A3, the introduction of a bromine atom on the pyrazolopyridine ring of 30 was examined first. The 7-bromo derivative 39 was found to be equipotent to 30, while the 6-bromo (40) and 5-bromo (41) derivatives showed slightly increased potency. Contrary to the other isomers, only the 4-bromo derivative 42 exhibited decreased

Modifications to the diacylpiperazine scaffold of compound 34 are depicted in Table 2. All major modifications, including removal of a carbonyl group (55 and 56), relocation of a carbonyl group (63), and ring expansion to homopiperazine (68), resulted in substantially diminished inhibitory activity. These results indicate the importance of both carbonyl groups in the 1,4diacylpiperazine scaffold and the central ring size. We have two main hypotheses regarding the function of the 1,4-diacylpiperazine moiety for the expression of eIF4A3 inhibitory activity: either the carbonyl groups are necessary to form key hydrogen bonds with eIF4A3 or the 1,4-diacylpiperazine acts as a structural scaffold to direct the 1- and 4-aromatic rings along appropriate vectors toward hydrophobic pockets on the protein. Next, the SARs of substituents at the 3-position on the piperazine of compound 34 were investigated (Table 3). The 3chloro (37) and 2-chloro (38) derivatives showed decreased activity, and substitution with an aliphatic cyclohexyl moiety (65) was not tolerated. These results indicate that the 4-chlorophenyl group at the piperazine 3-position is essential for potency. Then, 3339



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through a mechanism of eIF4A3 inhibition. Of the most potent compounds from the reporter assay, compounds 43 and 48 were selected for chiral separation and further evaluation. The ATPase inhibitory activities of the (S)- and (R)enantiomers of compounds 43 and 48 are shown in Table 6. The (S)-isomers 52a and 53a turned out to be more potent (eutomers), and they showed >100-fold more potent inhibitory activity than the corresponding distomers 52b and 53b. The dramatic difference in activity between enantiomers was noteworthy, and this observation suggests that the stereocenter on the piperazine ring is clearly recognized by the eIF4A3 protein. In addition, 53a exhibited excellent selectivity over other helicases and showed significant NMD inhibition at 10 and 3 μM in the reporter assay, while its distomer 53b did not show any inhibitory activity at the same concentrations (Table 7, Figure 3). These results imply that the NMD inhibition of 53a is correlated with eIF4A3 ATPase inhibitory activity and supports the validity of 53a as a molecular probe for studying eIF4A3 function. Binding affinity and kinetics of compounds (52a,b and 53a) were analyzed using a surface plasmon resonance (SPR) biosensing assay (Table 8). The SPR experiments ascertained the direct binding of 53a and its analog 52a to eIF4A3. Furthermore, both 52a and 53a showed double-digit nanomolar binding affinity, and the KD values of these compounds appeared to correlate with ATPase inhibitory activity. It is noteworthy that the kinetic profiles of 52a and 53a are distinct from the previously reported ATP-competitive inhibitor 2, and they showed a slower koff and longer dissociation t1/2. The identification of two types of eIF4A3 inhibitors enabled us to study the binding modes of both types, using them in a complementary manner. As shown in Figure 4, the absence or presence of compound 52a at a saturated concentration did not affect the SPR binding signals of 2 against eIF4A3. In other words, the data suggest the formation of a ternary complex composed of 52a, 2, and eIF4A3 and that 52a binds to a nonATP binding site of eIF4A3. Next, the known pan-eIF4A inhibitor 1 was used for similar SPR experiments. The binding signals of 1 against eIF4A3 were detected in a dose dependent manner in the absence of 52a (Figure 5D−F). Contrary to that, in the presence of 52a, binding signals of 1 were clearly weakened (Figure 5A−C). Hence, the observations imply two possibilities: 52a might bind to the same binding site as 1 or bind to other nonATP binding site and induce a conformational change to prevent the binding of 1. These results regarding the binding affinity, kinetics, and mode analysis of 52a and 53a also demonstrate the validity of these eIF4A3 inhibitors as excellent molecular probes.

Table 2. SAR around the 1,4-Diacylpiperazine Moiety

a

compd

eIF4A3 ATPase IC50 (μM)a

55 56 63 68

>100 >100 >100 >100


Table 3. SAR around the 3-Position of the Piperazine and 4Acyl Moiety

compd

R1

R2


37 38 65 57 58 59 60

3-chlorophenyl 2-chlorophenyl c-hexyl 4-chlorophenyl 4-chlorophenyl 4-chlorophenyl 4-chlorophenyl

4-chlorophenyl 4-chlorophenyl 4-chlorophenyl 3-chlorophenyl 2-chlorophenyl phenyl 4-bromophenyl

>100 >100 >100 >100 >100 11 (7.1−16) 0.66 (0.38−1.1)

a


potency. These results indicate the existence of sufficient space to accept a bromo group around the 5- to 7-positions of the pyrazolopyridine ring. Replacement of the 4-chlorobenzoyl moiety of 40 with a 4-bromobenzoyl moiety (43) tended to improve potency slightly. Interestingly, further investigation of substituents at the 6-position revealed that the 6-methoxy derivative 44 showed a similar level of potency as the 6-bromo derivative 43, whereas the 6-cyano derivative 45 displayed less potent activity. Further modifications of the biaryl series 34 and 36 were also executed. Introduction of a cyano group into each position of the phenyl ring of 34 revealed that the 3-cyano derivative 50 is the most potent among them. Further optimization produced equipotent derivatives such as 4bromobenzoyl derivatives 46 and 47. Replacement of the oxazole ring in 46 with a 5-methylpyrazole also led to the identification of similarly potent compound 48. Compounds 43−44 and 46−48 were tested in a reporter gene assay to evaluate NMD suppression (Table 5).36 Intriguingly, all the compounds exhibited NMD inhibition at 3 μM. As far as we know, this is the first example of an NMD inhibitor operating

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CONCLUSION As a result of chemical modifications of various 1,4diacylpiperazine derivatives, we discovered the first selective eIF4A3 inhibitor 53a exhibiting cellular NMD inhibitory activity. Compound 53a displayed high selectivity in favor of eIF4A3 and not for the eIF4A1 and eIF4A2 protein subtypes of the eIF4A family or other helicases. The clear difference in eIF4A3 inhibitory activity between 53a and its distomer 53b revealed the importance of stereochemistry at the 3-position of the piperazine ring for eIF4A3 inhibition. The lack of significant NMD inhibition by 53b also supports the supposition that NMD inhibition of 53a is mediated by eIF4A3 inhibition (and demonstrates the utility of this compound as a good negative control). The SPR biosensing assay provided evidence of the direct binding of 52a and 53a with eIF4A3 and their distinct kinetic profiles from the ATP-competitive inhibitor 2. 3340



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Table 4. SAR of the Pyrazolo[1,5-a]pyridine, the 2-Aryl-1,3-oxazole, and 1-Aryl-5-methyl-1H-pyrazole Derivatives

a


cancer. Further biological investigations using our selective eIF4A3 inhibitors will be published soon.

Furthermore, the SPR experiments also revealed that 52a binds to a non-ATP binding site of eIF4A3. Among our eIF4A3 inhibitors, compounds 52a37 and 53a could be invaluable molecular probes to study the functions of eIF4A3 and the EJC, as well as for more detailed mechanistic studies of NMD. In addition, these compounds may ultimately help lead to the development of new treatments in the areas of genetic disease or

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EXPERIMENTAL SECTION

Proton nuclear magnetic resonance (1H NMR) spectra were recorded on Bruker AVANCE-300 (300 MHz) and Bruker AVANCE-400 (400 MHz) instruments 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 = 3341



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Table 5. NMD Reporter Assay Results for Selected Compounds compd

eIF4A3 ATPase IC50 (μM)

NMD reporter assay fold change of firefly to renilla luciferase activity against DMSO control, 10 μM, 6 h (SD)a

43 44 46 47 48

0.42 0.92 0.36 0.39 0.58

1.9 (0.20) 1.7 (0.06) 1.8 (0.03) 1.8 (0.06) 2.3 (0.12)

a

HEK293T cells transfected with the NMD reporter gene were treated with the indicated compounds at 10 μM for 6 h. An increase in the ratio of firefly to renilla luciferase activity, indicating NMD inhibition, was detected and normalized with DMSO treated samples. n = 2, SD values shown in parentheses.

Table 6. ATPase Inhibitory Activity of Enantiomerically Pure Compounds against eIF4A3

Figure 3. NMD inhibition by 53a and 53b in the reporter gene assay. HEK293T cells transfected with the NMD reporter gene were treated with the indicated compounds at 10 μM or 3 μM for 6 h. An increase in the ratio of firefly to renilla luciferase activity, indicating NMD inhibition, was detected and normalized with DMSO treated samples. The y-axis in the bar graph represents the fold increase in the ratio of firefly to renilla luciferase activity. n = 2, and error bars represent the SD values.

a

compd

stereochemistry


52a 52b 53a 53b

S R S R

0.20 (0.16−0.25) 60 (21−177) 0.26 (0.18−0.38) >100

Table 8. Binding Affinity and Kinetics of 52a,b and 53a compd

eIF4A3 ATPase IC50 (μM)

eIF4A3 SPR KD (μM)a

kon (M−1 s−1), koff (s−1)a

t1/2 (s)a

52a 52b 53a 2c 1c

0.20 60 0.26 0.97 40

0.057 16b 0.043 0.20 50

2.8 × 104, 1.6 × 10−3 1.5 × 103, 2.4 × 10−2b 1.6 × 104, 6.9 × 10−4 4.0 × 105, 7.8 × 10−2 1.1 × 103, 5.7 × 10−2

438.2 28.6b 1006.9 8.9 12.2


Table 7. Selectivity over Other Helicases ATPase IC50 (μM)

a

a

compd

eIF4A1a

eIF4A2b

DHX29b

BRR2b

53a

>100

>100

>100

>100

Sensorgrams of each compound are in Supporting Information Figure 1. bApproximate values due to compound precipitation. c Reference 30.

n = 2. bn = 4. min. Specific rotation values were carried out by Sumika Chemical Analysis Service and observed in MeOH (1 w/v %). The purities of all the compounds tested in biological systems were assessed as being >95% using elemental analysis or analytical HPLC. Purity data were collected by HPLC with NQAD (nanoquality analyte detector) or Corona CAD (charged aerosol detector). The column was an L-column 2 ODS (30 mm × 2.1 mm i.d., CERI) or a Capcell Pak C18AQ (50 mm × 3.0 mm i.d., Shiseido) with a temperature of 50 °C and a flow rate of 0.5 mL/min. Mobile phases A and B under a neutral conditions were a mixture of 50 mmol/L AcONH4, water, and MeCN (1/8/1, v/v/v) and a mixture of 50 mmol/L AcONH4 and MeCN (1/9, v/v), respectively. The ratio of mobile phase B was increased linearly from 5% to 95% over 3 min, 95% over the next 1 min. 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

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). Elemental analyses were carried out by the Elemental Microanalysis Team in the Medicinal Chemistry Research Laboratories, Sumika Chemical Analysis Service, or Toray Research Center and were within 0.4% of the theoretical values. 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 acidic conditions 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 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 3342



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Figure 4. SPR binding studies of 2 against eIF4A3 in the presence or absence of 52a. The binding signals of compound 2 against immobilized eIF4A3 were detected in the presence of 52a at a saturated concentration (A−C) or absence of 52a (D−F). Parts A and D represent the SPR response to varying concentrations of 2. y axis is SPR response (RU), and x axis is concentration of 2. Parts B and E represent the SPR sensorgrams. y axis is SPR response, and x axis is time. Parts C and F represent the schematics of the experiments.

Figure 5. SPR binding studies of 1 against eIF4A3 in the presence or absence of 52a. The binding signals of compound 1 against immobilized eIF4A3 were detected in the presence of 52a at a saturated concentration (A−C) or absence of 52a (D−F). Parts A and D represent the SPR response to varying concentrations of 1. y axis is SPR response (RU), and x axis is concentration of 1. Parts B and E represent the SPR sensorgrams. The y axis is the SPR response, and the x axis is time. Parts C and F represent the schematics of the experiments. system with UV detector (220 and 254 nm). The column used was a Symmetrix ODS-R (150 mm × 30 mm i.d., 5 μm, Boston Analytics), Synergi C18 (150 mm × 30 mm i.d., 4 μm, Phenomenex), Xbridge C18 (150 mm × 20 mm i.d., 5 μm, Waters), or Gemini C18 (150 mm × 25 mm i.d., 10 μm, Phenomenex), and a flow rate of 25 mL/min. Condition 1: Mobile phases A and B under acidic conditions were 0.225% formic acid in water and MeCN, respectively. Condition 2: Mobile phases A and B under acidic conditions were 1% TFA in water and MeCN, respectively. Condition 3: Mobile phases A and B under basic conditions were 0.05% aqueous ammonia solution and MeCN, respectively. The ratio of mobile phase B was increased linearly between 8 and 15 min. All commercially available solvents and reagents were used without further purification. Yields were not optimized.

Compound 1 (hippuristanol) was purchased from Centaurus Biopharma (fee-for-service).38 (4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(1Hindol-3-yl)methanone (3). HATU (153 mg, 0.40 mmol) was added to a solution of 7a (100 mg, 0.27 mmol), 9 (43 mg, 0.27 mmol), and DIPEA (70 mg, 0.54 mmol) in DMF (3 mL). After stirring at room temperature for 18 h, the reaction mixture was purified by preparative HPLC (condition 1, 0.225% aqueous formic acid solution/MeCN = 53/ 47 to 23/77) to give 3 (35 mg, 27%) as a white solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.39−3.43 (m, 2H), 3.72−3.74 (m, 1H), 3.77−3.78 (m, 1H), 3.85−3.87 (m, 1H), 4.66−4.70 (m, 1H), 5.49 (s, 1H), 7.05−7.08 (m, 1H), 7.12−7.15 (m, 1H), 7.34 (s, 4H), 7.41−7.44 (m, 1H), 7.56 (s, 5H), 7.58−7.59 (m, 1H), 11.29 (s, 1H). MS m/z 478 3343



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(M + H)+. Anal. Calcd for C26H21Cl2N3O2·1.25H2O: C, 62.34; H, 4.73; N, 8.39. Found: C, 62.27; H, 4.48; N, 8.38. tert-Butyl 3-(4-Chlorophenyl)piperazine-1-carboxylate (5a). To a solution of 4a (100 mg, 0.51 mmol) in CH2Cl2 (5 mL) were added Boc2O (111 mg, 0.51 mmol) and Et3N (51 mg, 0.51 mmol) at 0 °C. After being stirred at 15 °C for 2 h, the reaction mixture was diluted with water, extracted with CH2Cl2, and concentrated. The residue was purified by column chromatography on silica gel (CH2Cl2/MeOH = 50/1 to 30/1) to give 5a (100 mg, 66%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.45−1.48 (m, 9H), 2.68 (brs, 1H), 2.77−2.99 (m, 2H), 3.06 (d, J = 8.1 Hz, 1H), 3.68 (dd, J = 10.5, 3.0 Hz, 1H), 4.04 (brs, 2H), 7.27−7.38 (m, 4H). 1H was not observed. MS m/z 297 (M + H)+. tert-Butyl 3-(3-Chlorophenyl)piperazine-1-carboxylate (5c). To a solution of 4c (0.94 g, 4.78 mmol) in toluene (12 mL) and THF (2 mL) was added Boc2O (1.11 mL, 4.78 mmol) dropwise at 0 °C. After stirring at room temperature for 3 h, the reaction mixture was concentrated. The residue was purified by silica gel column chromatography (hexane/EtOAc = 50/1 to 7/3) to give 5c (0.99 g, 70%) as a yellow oil. 1H NMR (300 MHz, CDCl3) δ 1.47 (s, 9H), 2.71 (brs, 1H), 2.80−2.99 (m, 2H), 3.07 (d, J = 9.5 Hz, 1H), 3.69 (dd, J = 10.4, 2.9 Hz, 1H), 4.04 (brs, 2H), 7.19−7.33 (m, 3H), 7.44 (s, 1H). 1H was not observed. MS m/z 297 (M + H)+. tert-Butyl 3-(2-Chlorophenyl)piperazine-1-carboxylate (5d). Compound 5d was prepared from compound 4d in a similar manner to that described for the synthesis of compound 5a and obtained in 83% yield as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.48 (s, 9H), 2.64 (dd, J = 12.6, 10.1 Hz, 1H), 2.84−3.00 (m, 2H), 3.02−3.14 (m, 1H), 3.95−4.09 (m, 1H), 4.14 (dd, J = 10.1, 2.8 Hz, 1H), 4.21 (d, J = 12.1 Hz, 1H), 7.16−7.31 (m, 3H), 7.33−7.41 (m, 1H), 7.62 (d, J = 7.3 Hz, 1H). MS m/z 297 (M + H)+. tert-Butyl 4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazine-1-carboxylate (6a). To a solution of 5a (6.30 g, 21.0 mmol) in CH2Cl2 (50 mL) were added 4-chlorobenzoyl chloride (3.90 g, 22.0 mmol) and Et3N (4.20 g, 42.0 mmol) at 0 °C. After stirring at 15 °C for 2 h, the reaction mixture was diluted with water, extracted with CH2Cl2, and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2/MeOH = 50/1) to give 6a (8.65 g, 95%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.45 (brs, 9H), 3.03 (brs, 2H), 3.29 (brs, 1H), 3.78−4.20 (m, 2H), 4.64 (brs, 1H), 5.35−6.10 (m, 1H), 7.29−7.46 (m, 8H). MS m/z 457 (M + Na)+. tert-Butyl 4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazine-1-carboxylate (6b). Compound 6b was prepared from compounds 5a and 4-bromobenzoyl chloride in a similar manner to that described for the synthesis of compound 6a and obtained in 87% yield as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 1.33 (s, 9H), 2.77− 3.18 (m, 2H), 3.43−3.97 (m, 2H), 4.12−5.08 (m, 2H), 5.68 (brs, 1H), 7.08−7.53 (m, 6H), 7.67 (s, 2H). MS m/z 423 (M − (t-Bu) + H)+. tert-Butyl 4-(4-Chlorobenzoyl)-3-(3-chlorophenyl)piperazine-1-carboxylate (6c). Compound 6c was prepared from compound 5c in a similar manner to that described for the synthesis of compound 6a and obtained in 78% yield as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.49 (brs, 9H), 2.75−3.18 (m, 2H), 3.30 (d, J = 10.4 Hz, 1H), 3.79−4.14 (m, 1H), 4.67 (d, J = 14.0 Hz, 1H), 7.21−7.33 (m, 3H), 7.36−7.47 (m, 5H). 2H were not observed. MS m/z 379 (M − (t-Bu) + H)+. tert-Butyl 4-(4-Chlorobenzoyl)-3-(2-chlorophenyl)piperazine-1-carboxylate (6d). To a solution of 5d (1.00 g, 3.37 mmol) and DIPEA (871 mg, 6.74 mmol) in CH2Cl2 (30 mL) was added 4-chlorobenzoyl chloride (708 mg, 4.04 mmol) dropwise at 0 °C. The mixture was stirred at room temperature for 4 h. The reaction mixture was concentrated. The residue was purified by silica gel column chromatography (petroleum ether/EtOAc = 30/1 to 3/1) to give 6d (1.40 g, 95%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.17−1.50 (m, 9H), 3.17 (brs, 1H), 3.42−4.37 (m, 5H), 5.68 (s, 1H), 7.18−7.28 (m, 3H), 7.29−7.46 (m, 5H). MS m/z 379 (M − (t-Bu) + H)+. (4-Chlorophenyl)-(2-(4-chlorophenyl)piperazin-1-yl)methanone Hydrochloride (7a). A mixture of 6a (1.26 g, 2.89 mmol) and 4 M hydrogen chloride EtOAc solution (15 mL, 60.0 mmol) was stirred at 0 °C for 3 h. Then the mixture was concentrated to give 7a (1.06 g, 99%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 2.98−

3.28 (m, 3H), 3.52 (dd, J = 13.7, 4.6 Hz, 1H), 3.96 (d, J = 13.2 Hz, 2H), 5.60 (brs, 1H), 7.35−7.47 (m, 2H), 7.47−7.62 (m, 6H), 8.26−9.92 (m, 2H). MS m/z 335 (M + H)+. (4-Bromophenyl)-(2-(4-chlorophenyl)piperazin-1-yl)methanone Hydrochloride (7b). A mixture of 6b (7.00 g, 14.6 mmol) and 4 M hydrogen chloride EtOAc solution (20 mL, 80.0 mmol) was stirred at room temperature for 2 h. Then the mixture was concentrated to give 7b (4.00 g, 66%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 3.03−3.13 (m, 1H), 3.14−3.28 (m, 3H), 3.51−3.54 (m, 1H), 3.94−3.98 (m, 1H), 5.60 (s, 1H), 7.37−7.46 (m, 2H), 7.47− 7.55 (m, 4H), 7.69 (d, J = 8.0 Hz, 2H), 8.60 (s, 1H), 9.71 (s, 1H). MS m/ z 379 (M + H)+. (4-Chlorophenyl)-(2-(3-chlorophenyl)piperazin-1-yl)methanone Hydrochloride (7c). Compound 7c was prepared from compound 6c in a similar manner to that described for the synthesis of compound 7b and obtained quantitatively as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 3.01−3.36 (m, 3H), 3.53 (brs, 1H), 4.00 (d, J = 13.7 Hz, 2H), 5.62 (brs, 1H), 7.33 (d, J = 7.4 Hz, 1H), 7.39−7.52 (m, 3H), 7.57 (s, 4H), 8.60 (brs, 1H), 9.72 (d, J = 8.4 Hz, 1H). MS m/z 335 (M + H)+. (4-Chlorophenyl)-(2-(2-chlorophenyl)piperazin-1-yl)methanone Hydrochloride (7d). Compound 7d was prepared from compound 6d in a similar manner to that described for the synthesis of compound 7b and obtained in 84% yield as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 3.27 (d, J = 5.7 Hz, 2H), 3.38−3.50 (m, 1H), 3.51− 3.63 (m, 1H), 3.83−4.09 (m, 2H), 5.64 (dd, J = 8.5, 5.8 Hz, 1H), 7.31− 7.44 (m, 2H), 7.44−7.55 (m, 5H), 7.55−7.63 (m, 1H), 9.41 (brs, 2H). MS m/z 335 (M + H)+. (4-Bromophenyl)((2S)-2-(4-chlorophenyl)piperazin-1-yl)methanone (8a) and (4-Bromophenyl)((2R)-2-(4-chlorophenyl)piperazin-1-yl)methanone (8b). To a solution of 6b (3.40 g, 7.09 mmol) in THF (30 mL) was added 4 M hydrogen chloride EtOAc solution (53.1 mL, 213 mmol) at 0 °C. The resulting mixture was allowed to warm to room temperature and stirred at room temperature for 1 h. The reaction mixture was basified with saturated aqueous NaHCO3 solution at room temperature and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over MgSO4, and concentrated. The residue was purified by NH-silica gel column chromatography (hexane/EtOAc = 9/1 to 0/10) to give (4bromophenyl)-(2-(4-chlorophenyl)piperazin-1-yl)methanone (free form of 7b, 2.60 g, 97%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 2.69−3.01 (m, 2H), 3.04−3.30 (m, 2H), 3.62 (d, J = 13.0 Hz, 1H), 4.96−6.01 (m, 1H), 7.27−7.47 (m, 6H), 7.54 (d, J = 8.2 Hz, 2H). 2H were not observed. MS m/z 379 (M + H)+. The racemic mixture (2.60 g) was separated by HPLC (YMC K-prep system, CHIRALPAK AD (Daicel) 500 mm × 50 mm i.d., hexane/iPrOH/Et2NH = 400/600/1 (v/v/v), flow rate of 60 mL/min) to give 8a (1.13 g, 42%, 99.9% ee, retention time 14.2 min) as a colorless oil and 8b (1.10 g, 41%, 99.8% ee, retention time 24.0 min) as a colorless oil. 8a: 1H NMR (300 MHz, CDCl3) δ 2.70−2.99 (m, 2H), 3.01−3.28 (m, 2H), 3.62 (d, J = 12.6 Hz, 1H), 4.61−6.25 (m, 1H), 7.27−7.45 (m, 6H), 7.54 (d, J = 8.1 Hz, 2H). 2H were not observed. MS m/z 379 (M + H)+. 8b: 1 H NMR (300 MHz, CDCl3) δ 2.73−3.01 (m, 2H), 3.02−3.33 (m, 2H), 3.62 (d, J = 12.6 Hz, 1H), 5.01−6.06 (m, 1H), 7.27−7.46 (m, 6H), 7.54 (d, J = 8.1 Hz, 2H). 2H were not observed. MS m/z 379 (M + H)+. Analytical HPLC conditions for determination of the % ee values of 8a and 8b: Waters system, CHIRALPAK AD (Daicel) 250 mm × 4.6 mm i.d., hexane/i-PrOH/Et2NH = 400/600/1 (v/v/v), flow rate of 0.5 mL/min. Absolute stereochemistry was determined as a (2S)enantiomer by X-ray crystal structure analysis of TsOH salt of 8a (see Supporting Information). (4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(1Hindazol-3-yl)methanone (29). Compound 29 was prepared from compounds 7a and 10 in a similar manner to that described for the synthesis of compound 3 and obtained in 39% yield as a white solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.39−3.56 (m, 2H), 3.82− 4.02 (m, 2H), 4.37−4.52 (m, 1H), 4.99−5.10 (m, 1H), 5.54 (s, 1H,), 7.15−7.24 (m, 1H), 7.26−7.44 (m, 5H), 7.50 (s, 4H), 7.57−7.60 (m, 1H), 7.88 (d, J = 8.1 Hz, 1H), 13.12−13.45 (s, 1H). MS m/z 479 (M + 3344



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H)+. Anal. Calcd for C25H20Cl2N4O2·H2O: C, 60.37; H, 4.46; N, 11.26. Found: C, 60.11; H, 4.32; N, 11.14. (4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(pyrazolo[1,5-a]pyridin-3-yl)methanone (30). A mixture of 7a (100 mg, 0.30 mmol), 11 (53 mg, 0.33 mmol), EDC·HCl (69 mg, 0.36 mmol), HOBt (55 mg, 0.36 mmol), and DIPEA (0.16 mL, 0.89 mmol) in DMF (3 mL) was stirred at room temperature overnight. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over MgSO4, and concentrated. The residue was purified by NH-silica gel column chromatography (hexane/EtOAc = 95/5 to 20/80) to give 30 (112 mg, 78%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 3.23− 3.46 (m, 2H), 3.52−4.51 (m, 3H), 4.98 (d, J = 14.1 Hz, 1H), 5.36−6.06 (m, 1H), 6.89−6.98 (m, 1H), 7.27−7.47 (m, 9H), 7.86 (d, J = 9.0 Hz, 1H), 7.92 (s, 1H), 8.49 (d, J = 6.9 Hz, 1H). MS m/z 479 (M + H)+. Anal. Calcd for C25H20Cl2N4O2: C, 62.64; H, 4.21; N, 11.69. Found: C, 62.34; H, 4.20; N, 11.48. (4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(1phenyl-1H-pyrazol-4-yl)methanone (31). Compound 31 was prepared from compounds 7a and 18 in a similar manner to that described for the synthesis of compound 3 and obtained in 50% yield as a white solid. 1H NMR (400 MHz, DMSO-d6, T = 80 °C) δ 3.42−3.51 (m, 2H), 3.73−3.79 (m, 1H), 3.79−3.94 (m, 1H), 4.05−4.12 (m, 1H), 4.60−4.66 (m, 1H), 5.50 (brs, 1H), 7.35−7.40 (m, 5H), 7.50−7.55 (m, 6H), 7.81−7.88 (m, 3H), 8.59 (s, 1H). MS m/z 505 (M + H)+. Anal. Calcd for C27H22Cl2N4O2·0.3H2O: C, 63.49; H, 4.46; N, 10.97. Found: C, 63.38; H, 4.43; N, 10.95. (4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(1phenyl-1H-pyrazol-3-yl)methanone (32). A solution of 19 (45 mg, 0.21 mmol) and lithium hydroxide hydrate (26 mg, 0.62 mmol) in THF (1 mL), MeOH (1 mL), and water (1 mL) was stirred at 40 °C for 1 h. The reaction mixture was concentrated, acidified to pH 3 with 1 M hydrochloric acid, and extracted with EtOAc. The separated organic layer was dried over Na2SO4 and concentrated. The residue was dissolved in DMF (2 mL), then 7a (77 mg, 0.21 mmol), DIPEA (81 mg, 0.62 mmol), and HATU (119 mg, 0.31 mmol) were added. After being stirred at room temperature for 1.5 h, the reaction mixture was purified by preparative HPLC (condition 1, 0.225% aqueous formic acid solution/MeCN = 37/63 to 16/84) to give 32 (75 mg, 71%) as a white solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.32−3.53 (m, 2H), 3.77−4.04 (m, 2H), 4.34−4.53 (m, 1H), 4.94−5.06 (m, 1H), 5.54 (s, 1H), 6.75 (d, J = 2.1 Hz, 1H), 7.37 (s, 5H), 7.50 (s, 6H), 7.75−7.85 (m, 2H), 8.42 (s, 1H). MS m/z 505 (M + H)+. Anal. Calcd for C27H22Cl2N4O2·0.5H2O: C, 63.04; H, 4.51; N, 10.89. Found: C, 63.00; H, 4.42; N, 10.74. (4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(2phenyl-1,3-oxazol-4-yl)methanone (33). Compound 33 was prepared from compounds 7a and 20 in a similar manner to that described for the synthesis of compound 3 and obtained in 24% yield as a white solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.27−3.41 (m, 2H), 3.70−3.81 (m, 1H), 3.84−3.96 (m, 1H), 4.22−4.37 (m, 1H), 4.84−4.97 (m, 1H), 5.41−5.51 (m, 1H), 7.25−7.35 (m, 4H), 7.41 (s, 4H), 7.47 (m, 3H), 7.86−7.95 (m, 2H), 8.38 (s, 1H). MS m/z 506 (M + H)+. Anal. Calcd for C27H21Cl2N3O3·0.2H2O: C, 66.52; H, 4.86; N, 8.31. Found: C, 66.47; H, 4.90; N, 8.40. (4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(2phenyl-1,3-oxazol-5-yl)methanone (34). Compound 34 was prepared from compounds 7a and 21 in a similar manner to that described for the synthesis of compound 30 and obtained in 79% yield as a white solid. 1H NMR (300 MHz, CDCl3) δ 3.21−3.84 (m, 4H), 4.35− 4.49 (m, 1H), 4.93−5.22 (m, 1H), 5.50−6.08 (m, 1H), 7.29−7.56 (m, 11H), 7.69 (s, 1H), 8.00 (dd, J = 7.8, 1.6 Hz, 2H). MS m/z 506 (M + H)+. Anal. Calcd for C27H21Cl2N3O3·0.1H2O: C, 63.81; H, 4.20; N, 8.27. Found: C, 63.71; H, 4.09; N, 8.23. (4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(5phenyl-1,3-oxazol-2-yl)methanone (35). Compound 7a (170 mg, 0.46 mmol) was basified with saturated aqueous NaHCO3 solution and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over MgSO4, and concentrated. The residue was azeotroped with toluene and dissolved in THF (3 mL). To the solution

were added 24 (99 mg, 0.46 mmol) and (Me3Al)2·DABCO32 (94 mg, 0.37 mmol) at room temperature. The resulting mixture was heated at 130 °C for 1 h under microwave irradiation. The reaction mixture was diluted with THF and added Na2SO4 decahydrate (147 mg, 0.46 mmol) at room temperature. After stirring at room temperature overnight, the insoluble material was removed by filtration and washed with THF. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (hexane/EtOAc = 95/5 to 30/70) to give 35 (180 mg, 78%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 3.10− 3.52 (m, 3H), 4.01 (d, J = 10.9 Hz, 1H), 4.50 (d, J = 10.9 Hz, 1H), 5.29 (t, J = 15.0 Hz, 1H), 5.77 (d, J = 15.0 Hz, 1H), 7.29−7.54 (m, 12H), 7.74 (d, J = 6.8 Hz, 2H). MS m/z 506 (M + H)+. Anal. Calcd for C27H21Cl2N3O3: C, 64.04; H, 4.18; N, 8.30. Found: C, 64.08; H, 4.26; N, 8.22. (4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(5methyl-1-phenyl-1H-pyrazol-4-yl)methanone (36). Compound 36 was prepared from compounds 7a and 25 in a similar manner to that described for the synthesis of compound 30 and obtained in 34% yield as a white solid. 1H NMR (400 MHz, CDCl3) δ 2.31 (s, 3H), 3.28−4.22 (m, 5H), 5.09−5.89 (m, 2H), 7.33−7.50 (m, 14H). MS m/z 519 (M + H)+. Anal. Calcd for C28H24Cl2N4O2: C, 64.74; H, 4.66; N, 10.79. Found: C, 64.52; H, 4.75; N, 10.83. (4-(4-Chlorobenzoyl)-3-(3-chlorophenyl)piperazin-1-yl)(2phenyl-1,3-oxazol-5-yl)methanone (37). Compound 37 was prepared from compounds 7c and 21 in a similar manner to that described for the synthesis of compound 30 and obtained in 51% yield as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 3.37−3.88 (m, 3H), 4.17 (brs, 1H), 4.76 (d, J = 13.2 Hz, 1H), 5.72 (brs, 1H), 7.34 (brs, 4H), 7.57 (d, J = 6.1 Hz, 7H), 7.82 (brs, 1H), 8.02 (dd, J = 7.5, 2.1 Hz, 2H). 1H was not observed. MS m/z 506 (M + H)+. Anal. Calcd for C27H21Cl2N3O3·0.2H2O: C, 63.59; H, 4.23; N, 8.24. Found: C, 63.46; H, 4.11; N, 8.17. (4-(4-Chlorobenzoyl)-3-(2-chlorophenyl)piperazin-1-yl)(2phenyl-1,3-oxazol-5-yl)methanone (38). Compound 38 was prepared from compound 7d and 21 in a similar manner to that described for the synthesis of compound 3 and obtained in 31% yield as a white solid. 1H NMR (400 MHz, CDCl3) δ 3.43−4.88 (m, 6H), 5.31− 6.30 (m, 1H), 7.18−7.41 (m, 7H), 7.42−7.57 (m, 5H), 8.03 (brs, 2H). MS m/z 506 (M + H)+. Anal. Calcd for C27H21Cl2N3O3·0.2H2O: C, 63.59; H, 4.23; N, 8.24. Found: C, 63.46; H, 4.11; N, 8.17. (7-Bromopyrazolo[1,5-a]pyridin-3-yl)(4-(4-chlorobenzoyl)-3(4-chlorophenyl)piperazin-1-yl)methanone (39). Compound 39 was prepared from compounds 7a and 12 in a similar manner to that described for the synthesis of compound 3 and obtained in 43% yield as a white solid. 1H NMR (400 MHz, DMSO-d6, T = 80 °C) δ 3.40−3.51 (m, 2H), 3.80−4.11 (m, 3H), 4.60−4.66 (m, 1H), 5.49 (brs, 1H), 7.28− 7.33 (m, 5H), 7.43−7.52 (m, 5H), 7.80 (d, J = 8.8 Hz, 1H), 8.25 (s, 1H). MS m/z 557 (M + H)+. Anal. Calcd for C25H19BrCl2N4O2·0.4H2O: C, 53.10; H, 3.53; N, 9.91. Found: C, 53.16; H, 3.47; N, 9.96. (6-Bromopyrazolo[1,5-a]pyridin-3-yl)(4-(4-chlorobenzoyl)-3(4-chlorophenyl)piperazin-1-yl)methanone (40). Compound 40 was prepared from compounds 7a and 13 in a similar manner to that described for the synthesis of compound 30 and obtained in 68% yield as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 3.37−3.46 (m, 2H), 3.76 (brs, 2H), 4.04−4.22 (m, 1H), 4.68 (d, J = 14.3 Hz, 1H), 5.63 (brs, 1H), 7.32 (brs, 4H), 7.42−7.62 (m, 5H), 7.71 (brs, 1H), 8.21 (s, 1H), 9.19 (s, 1H). MS m/z 557 (M + H)+. Anal. Calcd for C25H19BrCl2N4O2· 0.75C4H8O2: C, 53.87; H, 4.04; N, 8.97. Found: C, 54.03; H, 4.11; N, 9.05. Purity 98.6% (HPLC). (5-Bromopyrazolo[1,5-a]pyridin-3-yl)(4-(4-chlorobenzoyl)-3(4-chlorophenyl)piperazin-1-yl)methanone (41). Compound 41 was prepared from compounds 7a and 14 in a similar manner to that described for the synthesis of compound 3 and obtained in 55% yield as a white solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.40−3.54 (m, 2H), 3.84 (dd, J = 15.0, 6.0 Hz, 1H), 3.92−4.10 (m, 2H), 4.60 (dd, J = 15.0, 3.0 Hz, 1H), 5.47 (brs, 1H), 7.20 (dd, J = 9.0, 3.0 Hz, 1H), 7.32 (s, 4H), 7.48 (s, 4H), 7.95 (d, J = 3.0 Hz, 1H), 8.15 (s, 1H), 8.64 (d, J = 6.0 Hz, 1H). MS m/z 557 (M + H)+. Anal. Calcd for C25H19BrCl2N4O2· 0.7H2O: C, 52.60; H, 3.60; N, 9.81. Found: C, 52.52; H, 3.44; N, 9.96. 3345



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(4-Bromopyrazolo[1,5-a]pyridin-3-yl)(4-(4-chlorobenzoyl)-3(4-chlorophenyl)piperazin-1-yl)methanone (42). Compound 42 was prepared from compounds 7a and 15 in a similar manner to that described for the synthesis of compound 3 and obtained in 61% yield as a white solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.21−3.34 (m, 2H), 3.58−3.64 (m, 1H), 3.86−3.89 (m, 2H), 4.60 (brs, 1H), 5.49 (brs, 1H), 6.89 (t, J = 6.0 Hz, 1H), 7.35−7.40 (m, 4H), 7.48 (s, 4H), 7.58 (d, J = 9.0 Hz, 1H), 7.91 (s, 1H), 8.71 (d, J = 6.0 Hz, 1H). MS m/z 557 (M + H)+. Anal. Calcd for C25H19BrCl2N4O2·H2O: C, 52.11; H, 3.67; N, 9.72. Found: C, 52.24; H, 3.51; N, 9.86. (4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(6bromopyrazolo[1,5-a]pyridin-3-yl)methanone (43). Compound 43 was prepared from compounds 7b and 13 in a similar manner to that described for the synthesis of compound 30 and obtained in 76% yield as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 3.43 (brs, 2H), 3.75 (brs, 2H), 4.03 (q, J = 7.2 Hz, 1H), 4.68 (d, J = 12.0 Hz, 1H), 5.67 (brs, 1H), 7.20−7.50 (m, 6H), 7.55 (dd, J = 9.4, 1.3 Hz, 1H), 7.68 (d, J = 7.6 Hz, 3H), 8.21 (s, 1H), 9.19 (s, 1H). MS m/z 601 (M + H)+. Anal. Calcd for C25H19Br2ClN4O2·0.2C6H14: C, 50.76; H, 3.54; N, 9.04. Found: C, 50.78; H, 3.71; N, 8.90. Purity 97.7% (HPLC). (4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(6methoxypyrazolo[1,5-a]pyridin-3-yl)methanone (44). To a solution of 16 (80 mg, 0.39 mmol) in MeOH (1 mL), THF (1 mL), and water (1 mL) was added lithium hydroxide hydrate (98 mg, 2.33 mmol) at room temperature. After stirring at 50 °C for 2 h, the reaction mixture was concentrated to give lithium 6-methoxypyrazolo[1,5a]pyridine-3-carboxylate (70 mg, 91%) as a white solid. A solution of lithium 6-methoxypyrazolo[1,5-a]pyridine-3-carboxylate (70 mg, 0.35 mmol), 7b (148 mg, 0.35 mmol), HATU (148 mg, 0.39 mmol), and DIPEA (183 mg, 1.41 mmol) in DMF (2 mL) was stirred at room temperature for 2 h. The reaction mixture was purified by preparative HPLC (condition 1, 0.225% aqueous formic acid solution/MeCN = 53/ 47 to 23/77) to give 44 (33 mg, 17%) as a white solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.42−3.48 (m, 2H), 3.78−3.94 (m, 5H), 4.07−4.11 (m, 1H), 4.57−4.63 (m, 1H), 5.46 (s, 1H), 7.21−7.31 (m, 1H), 7.38 (s, 4H), 7.41 (d, J = 8.4 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H), 7.71 (d, J = 9.6 Hz, 1H), 8.00 (s, 1H), 8.37 (d, J = 1.5 Hz, 1H). MS m/z 553 (M + H)+. Anal. Calcd for C26H22BrClN4O3·1.5H2O: C, 53.76; H, 4.34; N, 9.65. Found: C, 53.48; H, 3.98; N, 9.51. 3-((4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)carbonyl)pyrazolo[1,5-a]pyridine-6-carbonitrile (45). To a solution of 17 (80 mg, 0.33 mmol) in CH2Cl2 (3 mL) was added TFA (3 mL). The resulting mixture was stirred at room temperature for 1 h and then concentrated. To a solution of the residue, 7b (94 mg, 0.23 mmol), and Et3N (83 mg, 0.82 mmol) in CH2Cl2 (10 mL) was added HATU (86 mg, 0.23 mmol). The resulting mixture was stirred at room temperature for 5 h and concentrated. The residue was purified by preparative HPLC (condition 1, 0.225% aqueous formic acid solution/ MeCN = 40/60 to 10/90) to give 45 (60 mg, 53%) as a white solid. 1H NMR (400 MHz, DMSO-d6, T = 80 °C) δ 3.38−3.55 (m, 2H), 3.83 (dd, J = 14.0, 4.4 Hz, 1H), 3.94 (d, J = 10.0 Hz, 1H), 4.08 (d, J = 11.6 Hz, 1H), 4.62 (d, J = 14.0 Hz, 1H), 5.48 (s, 1H), 7.31 (s, 4H), 7.42 (d, J = 8.4 Hz, 2H), 7.58 (d, J = 9.2 Hz, 1H), 7.65 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 9.2 Hz, 1H), 8.36 (s, 1H), 9.54 (s, 1H,). MS m/z 548 (M + H)+. Anal. Calcd for C26H19BrClN5O2·0.3H2O: C, 56.35; H, 3.56; N, 12.64. Found: C, 56.44; H, 3.52; N, 12.45. 3-(5-((4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazin-1yl)carbonyl)-1,3-oxazol-2-yl)benzonitrile (46). Compound 46 was prepared from compounds 7b and 27 in a similar manner to that described for the synthesis of compound 35 and obtained in 47% yield as a white solid. 1H NMR (300 MHz, CDCl3) δ 3.00−3.80 (m, 3H), 3.97− 4.56 (m, 2H), 4.85−5.25 (m, 1H), 5.50−6.05 (m, 1H), 7.27−7.42 (m, 6H), 7.55−7.69 (m, 4H), 7.74−7.83 (m, 1H), 8.20−8.32 (m, 2H). MS m/z 575 (M + H)+. Anal. Calcd for C28H20BrClN4O3: C, 58.40; H, 3.50; N, 9.73. Found: C, 58.41; H, 3.73; N, 9.69. 2-(5-((4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazin-1yl)carbonyl)-1,3-oxazol-2-yl)isonicotinonitrile (47). To a solution of 6b (3.40 g, 7.09 mmol) in THF (30 mL) was added 4 M hydrogen chloride EtOAc solution (53.1 mL, 213 mmol) at 0 °C. The mixture was allowed to warm to room temperature and stirred at room temperature

for 1 h. The mixture was basified with saturated aqueous NaHCO3 solution at room temperature and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over MgSO4, and concentrated. The residue was purified by NH-silica gel column chromatography (hexane/EtOAc = 9/1 to 0/100) to give (4bromophenyl)-(2-(4-chlorophenyl)piperazin-1-yl)methanone (free form of 7b, 2.60 g, 97%) as a colorless oil. A mixture of (4bromophenyl)-(2-(4-chlorophenyl)piperazin-1-yl)methanone (312 mg, 0.82 mmol), 28 (200 mg, 0.82 mmol), and 1 M Me3Al in CH2Cl2 (1.64 mL, 1.64 mmol) in THF (10 mL) was stirred at 110 °C under microwave irradiation for 1 h. The reaction mixture was diluted with water and extracted with EtOAc. The separated organic layer was washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by preparative HPLC (condition 1, 0.225% aqueous formic acid solution/MeCN = 52/48 to 22/78) to give 47 (260 mg, 55%) as a white solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.44−3.62 (m, 2H), 3.86−4.03 (m, 2H), 4.08−4.16 (m, 1H), 4.66 (dd, J = 14.8, 3.9 Hz, 1H), 5.53 (brs, 1H), 7.33−7.45 (m, 6H), 7.61−7.67 (m, 2H), 7.82 (s, 1H), 7.97 (dd, J = 4.8, 1.5 Hz, 1H), 8.30 (s, 1H), 9.03 (dd, J = 4.8, 0.9 Hz, 1H). MS m/z 576 (M + H)+. Anal. Calcd for C27H19BrClN5O3·0.7H2O: C, 55.02; H, 3.49; N, 11.88. Found: C, 54.94; H, 3.45; N, 12.04. 3-(4-((4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazin-1yl)carbonyl)-5-methyl-1H-pyrazol-1-yl)benzonitrile (48). Compound 48 was prepared from compounds 7b and 26 in a similar manner to that described for the synthesis of compound 30 and obtained in 64% yield as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 2.20 (brs, 3H), 2.95−3.41 (m, 2H), 3.82−4.13 (m, 2H), 4.90 (brs, 1H), 5.73 (brs, 1H), 7.19−7.55 (m, 6H), 7.62−7.82 (m, 4H), 7.87−7.99 (m, 2H), 8.07 (s, 1H). 1H was not observed. MS m/z 588 (M + H)+. Anal. Calcd for C29H23BrClN5O2·0.1H2O: C, 58.97; H, 3.96; N, 11.86. Found: C, 59.16; H, 4.33; H, N, 11.74. 2-(5-((4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1yl)carbonyl)-1,3-oxazol-2-yl)benzonitrile (49). A mixture of 7a (70 mg, 0.19 mmol), 22 (29.8 mg, 0.26 mmol), EDC·HCl (50.5 mg, 0.26 mmol), and HOBt (35.6 mg, 0.26 mmol) in DMF (3 mL) was stirred at room temperature overnight. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by NH-silica gel column chromatography (hexane/ EtOAc = 98/2 to 50/50) to give (4-(4-chlorobenzoyl)-3-(4chlorophenyl)piperazin-1-yl)(1,3-oxazol-5-yl)methanone (20 mg, 25%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 3.54 (brs, 2H), 4.02 (brs, 1H), 4.70 (brs, 1H), 5.62 (brs, 1H), 7.40 (brs, 4H), 7.54 (brs, 4H), 7.67 (brs, 1H), 8.54 (s, 1H). 2H were not observed. MS m/z 430 (M + H)+. A mixture of (4-(4-chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1yl)(1,3-oxazol-5-yl)methanone (100 mg, 0.232 mmol), 2-boromobenzonitrile (64 mg, 0.348 mmol), Pd(OAc)2 (3 mg, 12 μmol), Cs2CO3 (151 mg, 0.465 mmol), and CuI (44 mg, 0.232 mmol) in DMF (2 mL) was bubbled with N2 for 2 min, then heated at 150 °C for 5 min under microwave irradiation. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by preparative HPLC (condition 2, 1% TFA in water/MeCN = 90/10 to 20/80) to give 49 (20 mg, 16%) as a white solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.44−3.64 (m, 2H), 3.85−4.00 (m, 2H), 4.20−4.25 (d, J = 16.8 Hz, 1H), 4.68−4.74 (dd, J = 18.8, 4.0 Hz, 1H), 5.53 (brs, 1H), 7.38 (s, 4H), 7.50 (s, 4H), 7.73−7.79 (m, 1H), 7.86− 7.91 (m, 2H), 7.99−8.01 (m, 1H), 8.17−8.19 (m, 1H). MS m/z 531 (M + H)+. Anal. Calcd for C28H20Cl2N4O3·0.3H2O: C, 62.65; H, 3.87; N, 10.44. Found: C, 62.57; H, 3.90; N, 10.46. 3-(5-((4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1yl)carbonyl)-1,3-oxazol-2-yl)benzonitrile (50). To a solution of 23 (354 mg, 1.48 mmol) in DMF (10 mL) were added HATU (844 mg, 2.22 mmol) and DIPEA (0.773 mL, 4.44 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 5 min, and then 7a (550 mg, 1.48 mmol) was added to the mixture at 0 °C. After stirring at 0 °C for 30 min, the reaction mixture was poured into water at room temperature and extracted with EtOAc. The organic layer was washed with water and 3346



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14.3 min) as a white solid. 1H NMR (400 MHz, DMSO-d6, T = 90 °C) δ 2.21 (s, 3H), 3.24−3.40 (m, 2H), 3.68 (dd, J = 14.1, 3.8 Hz, 1H), 3.90 (d, J = 9.3 Hz, 1H), 3.98 (d, J = 10.3 Hz, 1H), 4.64 (d, J = 13.4 Hz, 1H), 5.48 (brs, 1H), 7.28−7.33 (m, 2H), 7.35−7.44 (m, 4H), 7.61−7.65 (m, 3H), 7.73 (t, J = 8.0 Hz, 1H), 7.83−7.90 (m, 2H), 7.97 (t, J = 1.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6, T = 90 °C) δ 11.0, 41.5, 44.5, 46.0, 54.1, 112.6, 116.0, 117.7, 123.2, 128.1, 128.48 (2C), 128.49 (2C), 128.9 (2C), 129.6, 130.7, 131.6 (2C), 131.8, 132.1, 134.9, 137.5, 139.2, 139.6, 140.5, 164.0, 169.4. MS m/z 588 (M + H)+. Anal. Calcd for C29H23BrClN5O2· 0.25C6H14O·0.1H2O: C, 59.45; H, 4.37; N, 11.36. Found: C, 59.37; H, 4.34; N, 11.25. Purity 98.6% (HPLC). [α]D25 = +11.2°. 3-(4-(((3R)-4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)carbonyl)-5-methyl-1H-pyrazol-1-yl)benzonitrile (53b). Compound 53b was prepared from compounds 26 and 8b in a similar manner to that described for the synthesis of compound 52a and obtained in 74% yield (>99.0% ee, retention time 17.7 min) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 2.20 (brs, 3H), 3.38−3.71 (m, 2H), 3.94 (brs, 2H), 4.94 (brs, 1H), 5.78 (brs, 1H), 7.19−7.54 (m, 6H), 7.62−7.83 (m, 4H), 7.93 (dd, J = 14.2, 7.8 Hz, 2H), 8.08 (s, 1H). 1H was not observed. MS m/z 588 (M + H)+. Anal. Calcd for C29H23BrClN5O2·0.25C6H14O·0.1H2O: C, 59.45; H, 4.37; N, 11.36. Found: C, 59.43; H, 4.28; N, 11.34. Purity 98.1% (HPLC). [α]D25 = −11.3°. Analytical HPLC conditions for determination of the % ee values of 53a and 53b: Waters system, CHIRALPAK AS-3 (Daicel) 250 mm × 4.6 mm i.d., EtOH/hexane = 60/40, flow rate of 0.5 mL/min. (3-(4-Chlorophenyl)piperazin-1-yl)(2-phenyl-1,3-oxazol-5yl)methanone (54). To a solution of 4a (1.00 g, 5.08 mmol) in DMF (10 mL) were added HATU (2.81 g, 7.39 mmol), DIPEA (1.79 g, 13.9 mmol), and 21 (874 mg, 4.62 mmol). The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was diluted with EtOAc, washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2/MeOH = 10/1) to give 54 (1.45 g, 85%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 1.77−2.17 (m, 1H), 2.62−3.54 (m, 4H), 3.85 (dd, J = 10.6, 2.9 Hz, 1H), 4.56 (brs, 2H), 7.29−7.59 (m, 8H), 7.68 (s, 1H), 8.06 (d, J = 7.2 Hz, 1H). MS m/z 368 (M + H)+. (4-Chlorophenyl)(2-(4-chlorophenyl)-4-((2-phenyl-1,3-oxazol-5-yl)methyl)piperazin-1-yl)methanone (55). To a solution of 54 (200 mg, 0.54 mmol) in THF (10 mL) was added borane dimethylsulfide complex (10 M in THF, 54 μL, 0.54 mmol) in one portion at 0 °C under N2. After being stirred at 0 °C for 5 min and at room temperature for 16 h, the reaction mixture was cooled to 0 °C and quenched with MeOH (15 mL). The resulting mixture was stirred at 0 °C for 0.5 h and then acidified with 4 M hydrochloric acid to pH 3−4. After stirring at 80 °C for 0.5 h, the reaction mixture was neutralized with saturated aqueous Na2CO3 solution and extracted with CH2Cl2. The separated organic phase was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by preparative TLC (CH2Cl2/ MeOH = 10/1) to give 5-((3-(4-chlorophenyl)piperazin-1-yl)methyl)2-phenyloxazole (60 mg, 31%) as a yellow gum. 1H NMR (400 MHz, CDCl3) δ 2.23 (t, J = 10.8 Hz, 1H), 2.40 (t, J = 9.8 Hz, 1H), 2.94 (d, J = 10.8 Hz, 2H), 3.04−3.14 (m, 2H), 3.72 (s, 2H), 3.93 (d, J = 9.6 Hz, 1H), 7.04 (s, 1H), 7.27−7.30 (m, 2H), 7.35−7.36 (m, 2H), 7.45 (brs, 3H), 8.04 (d, J = 4.0 Hz, 2H). 1H was not observed. MS m/z 354 (M + H)+. To a mixture of 5-((3-(4-chlorophenyl)piperazin-1-yl)methyl)-2phenyloxazole (45 mg, 0.13 mmol) and 4-chlorobenzoyl chloride (33 mg, 0.19 mmol) in MeCN (2 mL) was added K2CO3 (53 mg, 0.38 mmol) in one portion at room temperature. The resulting mixture was stirred at room temperature for 16 h. The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by preparative HPLC (condition 3, 0.05% aqueous ammonia solution/ MeCN = 43/57 to 13/87) to give 55 (35 mg, 56%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 2.30−2.38 (m, 1H), 2.62 (dd, J = 12.3, 4.2 Hz, 1H), 2.89−2.93 (m, 1H), 3.08−3.17 (m, 1H), 3.45 (d, J = 12.3 Hz, 1H), 3.70−3.84 (m, 3H), 5.46 (s, 1H), 7.14 (s, 1H), 7.32−7.34 (m, 2H), 7.40−7.53 (m, 9H), 7.94−7.97 (m, 2H). MS m/z 492 (M + H)+. Anal. Calcd for C27H23Cl2N3O2·0.25H2O: C, 65.26; H, 4.77; N, 8.46. Found: C, 65.30; H, 4.82; N, 8.54.

brine, dried over MgSO4, and concentrated. The residue was purified by silica gel column chromatography (hexane/EtOAc = 95/5 to 30/70) to give (4-(4-chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(2-iodo1,3-oxazol-5-yl)methanone (785 mg, 95%) as a pale yellow solid. 1H NMR (300 MHz, CDCl3) δ 2.90−3.86 (m, 3H), 4.26 (brs, 1H), 5.00 (brs, 1H), 5.39−6.03 (m, 1H), 7.27−7.45 (m, 8H), 7.51 (s, 1H). 1H was not observed. MS m/z 556 (M + H)+. To a solution of (4-(4-chlorobenzoyl)-3-(4-chlorophenyl)piperazin1-yl)(2-iodo-1,3-oxazol-5-yl)methanone (140 mg, 0.252 mmol) and 3cyanophenylboronic acid (74 mg, 0.503 mmol) in DME (3 mL) and H2O (1 mL) were added Pd(PPh3)4 (29 mg, 25 μmol) and Na2CO3 (53 mg, 0.503 mmol) under N2. The resulting mixture was refluxed for 12 h. The reaction mixture was concentrated, and the residue was purified by preparative TLC (EtOAc/petroleum ether =1/1) to give 50 (40 mg, 30%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 3.45−3.60 (m, 2H), 3.87−4.02 (m, 2H), 4.16−4.20 (m, 1H), 4.68−4.73 (dd, J = 14.0, 3.2 Hz, 1H), 5.53 (brs, 1H), 7.39 (s, 4H), 7.52 (s, 4H), 7.78−7.80 (m, 2H), 8.01 (dt, J = 8.0, 1.2 Hz, 1H), 8.30 (dt, J = 8.0, 1.2 Hz, 1H), 8.34 (t, J = 1.2 Hz, 1H). MS m/z 531 (M + H)+. Anal. Calcd for C28H20Cl2N4O3· 0.4H2O: C, 62.44; H, 3.89; N, 10.40. Found: C, 62.40; H, 3.92; N, 10.50. 4-(5-((4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1yl)carbonyl)-1,3-oxazol-2-yl)benzonitrile (51). Compound 51 was prepared from (4-(4-chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1yl)(2-iodo-1,3-oxazol-5-yl)methanone in a similar manner to that described for the synthesis of compound 50 and obtained in 49% yield as a white solid. 1H NMR (300 MHz, CDCl3) δ 3.11−3.81 (m, 3H), 3.96− 4.50 (m, 2H), 4.77−5.18 (m, 1H), 5.39−6.02 (m, 1H), 7.28−7.46 (m, 8H), 7.67 (s, 1H), 7.77 (d, J = 8.2 Hz, 2H), 8.11 (d, J = 8.2 Hz, 2H). MS m/z 531 (M + H)+. Anal. Calcd for C28H20Cl2N4O3·0.5H2O: C, 62.23; H, 3.92; N, 10.37. Found: C, 62.15; H, 3.94; N, 10.42. ((3S)-4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazin-1yl)(6-bromopyrazolo[1,5-a]pyridin-3-yl)methanone (52a). A solution of 8a (200 mg, 0.53 mmol), 13 (165 mg, 0.68 mmol), EDC· HCl (131 mg, 0.68 mmol), and HOBt (93 mg, 0.68 mmol) in DMF (5 mL) was stirred at room temperature overnight. The reaction mixture was poured into water and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by NH-silica gel column chromatography (hexane/EtOAc = 8/2 to 2/8) to give 52a (232 mg, 73%, >99.9% ee, retention time 28.6 min) as colorless needles. 1H NMR (400 MHz, DMSO-d6, T = 90 °C) δ 3.35−3.51 (m, 2H), 3.80 (dd, J = 14.1, 4.5 Hz, 1H), 3.91 (d, J = 11.0 Hz, 1H), 4.03−4.12 (m, 1H), 4.60 (dd, J = 14.1, 2.6 Hz, 1H), 5.45 (brs, 1H), 7.26−7.33 (m, 4H), 7.37− 7.42 (m, 2H), 7.49 (dd, J = 9.3, 1.7 Hz, 1H), 7.60−7.65 (m, 2H), 7.71 (d, J = 10.0 Hz, 1H), 8.12 (s, 1H), 9.01−9.03 (m, 1H). 13C NMR (101 MHz, DMSO-d6, T = 90 °C) δ 41.7, 44.7, 46.2, 54.4, 106.3, 107.4, 119.2, 123.1, 128.4 (2C), 128.4 (2C), 128.9 (2C), 129.26, 129.32, 131.5 (2C), 132.0, 135.0, 137.8, 139.0, 141.7, 163.2, 169.4. MS m/z 601 (M + H)+. Anal. Calcd for C25H19Br2ClN4O2·0.25C6H14O: C, 50.66; H, 3.61; N, 8.92. Found: C, 50.54; H, 3.52; N, 8.95. Purity 100% (HPLC). [α]D25 = +4.7°. ((3R)-4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazin-1yl)(6-bromopyrazolo[1,5-a]pyridin-3-yl)methanone (52b). Compound 52b was prepared from compounds 13 and 8b in a similar manner to that described for the synthesis of compound 52a and obtained in 50% yield (>99.9% ee, retention time 33.9 min) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 3.36−3.55 (m, 2H), 3.63−3.90 (m, 2H), 4.07 (brs, 1H), 4.68 (d, J = 13.5 Hz, 1H), 5.66 (brs, 1H), 7.33 (brs, 4H), 7.45 (brs, 2H), 7.55 (d, J = 9.4 Hz, 1H), 7.68 (d, J = 7.3 Hz, 3H), 8.21 (s, 1H), 9.20 (s, 1H). MS m/z 601 (M + H)+. Anal. Calcd for C25H19Br2ClN4O2·0.25C6H14O: C, 50.66; H, 3.61; N, 8.92. Found: C, 50.57; H, 3.66; N, 8.70. Purity 100% (HPLC). [α]D25 = −4.9°. Analytical HPLC condition for determination of the % ee values of 52a and 52b: Waters system, CHIRALPAK IC-3 (Daicel) 250 mm × 4.6 mm i.d., EtOH 100%, flow rate of 0.5 mL/min. 3-(4-(((3S)-4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)carbonyl)-5-methyl-1H-pyrazol-1-yl)benzonitrile (53a). Compound 53a was prepared from compounds 26 and 8a in a similar manner to that described for the synthesis of compound 52a and obtained in 81% yield (>99.0% ee, retention time 3347



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(4-(4-Chlorobenzyl)-3-(4-chlorophenyl)piperazin-1-yl)(2phenyl-1,3-oxazol-5-yl)methanone (56). To a mixture of 54 (200 mg, 0.54 mmol) and 4-chlorobenzyl bromide (112 mg, 0.54 mmol) in MeCN (3 mL) was added K2CO3 (225 mg, 1.63 mmol) in one portion at room temperature. The resulting mixture was stirred at 80 °C for 16 h. The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by preparative TLC (petroleum ether/EtOAc = 5/ 1) and preparative HPLC (condition 3, 0.05% aqueous ammonia solution/MeCN = 32/68 to 2/98) to give 56 (52 mg, 19%) as a pale yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 2.25−2.34 (m, 1H), 2.91−2.97 (m, 1H), 3.08 (d, J = 14.1 Hz, 1H), 3.18 (t, J = 11.7 Hz, 1H), 3.31 (t, J = 11.3 Hz, 1H), 3.52 (dd, J = 10.5, 3.0 Hz, 1H), 3.63 (d, J = 13.8 Hz, 1H), 4.32 (t, J = 15.9 Hz, 2H), 7.31 (q, J = 4.6 Hz, 4H), 7.43−7.46 (m, 2H), 7.56−7.58 (m, 5H), 7.76 (s, 1H), 7.98−8.02 (m, 2H). MS m/z 492 (M + H)+. Anal. Calcd for C27H23Cl2N3O2: C, 65.86; H, 4.71; N, 8.53. Found: C, 65.69; H, 4.78; N, 8.60. (4-(3-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(2phenyl-1,3-oxazol-5-yl)methanone (57). To a solution of 54 (100 mg, 0.27 mmol) and Et3N (55 mg, 0.54 mmol) in CH2Cl2 (5 mL) was added 3-chlorobenzoyl chloride (95 mg, 0.54 mmol) dropwise at 0 °C. The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was quenched with water and extracted with EtOAc. The separated organic phase was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by preparative HPLC (condition 1, 0.225% aqueous formic acid solution/MeCN = 45/55 to 15/85) to give 57 (63 mg, 46%) as a white solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.50 (m, 2H), 3.88−3.98 (m, 2H), 4.02 (d, J = 12.3 Hz, 1H), 4.64 (dd, J = 12.3, 4.2 Hz, 1H), 5.52 (brs, 1H), 7.39 (m, 11H), 7.72 (s, 1H), 7.99 (dd, J = 7.8, 2.7 Hz, 2H). MS m/z 506 (M + H)+. Anal. Calcd for C27H21Cl2N3O3·0.3H2O: C, 63.36; H, 4.25; N, 8.21. Found: C, 63.37; H, 4.23; N, 8.35. (4-(2-Chlorobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(2phenyl-1,3-oxazol-5-yl)methanone (58). Compound 58 was prepared from compounds 54 and 2-chlorobenzoyl chloride in a similar manner to that described for the synthesis of compound 57 and obtained in 41% yield as a pale yellow solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.43 (m, 3H), 3.84 (d, J = 14.4 Hz, 1H), 4.15 (d, J = 14.1 Hz, 1H), 4.67 (d, J = 14.1 Hz, 1H), 5.84 (brs, 1H), 7.39 (m, 11H), 7.73 (s, 1H), 7.98 (dd, J = 7.8, 2.7 Hz, 2H). MS m/z 506 (M + H)+. Anal. Calcd for C27H21Cl2N3O3·0.3H2O: C, 63.36; H, 4.25; N, 8.21. Found: C, 63.16; H, 4.05; N, 8.08. (4-Benzoyl-3-(4-chlorophenyl)piperazin-1-yl)(2-phenyl-1,3oxazol-5-yl)methanone (59). Compound 59 was prepared from compounds 54 and benzoyl chloride in a similar manner to that described for the synthesis of compound 57 and obtained in 45% yield as a white solid. 1H NMR (300 MHz, DMSO-d6, T = 80 °C) δ 3.38−3.63 (m, 2H), 3.87 (dd, J = 14.1, 4.5 Hz, 1H), 4.02 (d, J = 12.3 Hz, 1H), 4.17 (d, J = 12.3 Hz, 1H), 4.69 (dd, J = 14.1, 4.5 Hz, 1H), 5.56 (brs, 1H), 7.38 (s, 4H), 7.46 (s, 5H), 7.51−7.63 (m, 3H), 7.71 (s, 1H), 8.00 (dd, J = 7.2, 3.0 Hz, 2H). MS m/z 472 (M + H)+. Anal. Calcd for C27H22ClN3O3· 0.3H2O: C, 67.94; H, 4.77; N, 8.80. Found: C, 67.99; H, 4.73; N, 8.60. (4-(4-Bromobenzoyl)-3-(4-chlorophenyl)piperazin-1-yl)(2phenyl-1,3-oxazol-5-yl)methanone (60). A solution of 4-bromobenzoic acid (48 mg, 0.24 mmol), 54 (80 mg, 0.22 mmol), DIPEA (0.11 mL, 0.65 mmol), HOBt (50 mg, 0.33 mmol), and EDC (0.06 mL, 0.33 mmol) in THF (3 mL) was stirred at room temperature overnight. The reaction mixture was poured into water at room temperature and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over MgSO4, and concentrated. The residue was purified by NH-silica gel column chromatography (hexane/EtOAc = 20/1 to 3/7) to give 60 (115 mg, 96%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 3.11−3.77 (m, 3H), 4.42 (d, J = 10.8 Hz, 1H), 4.85− 5.23 (m, 1H), 5.25−6.15 (m, 1H), 7.28−7.41 (m, 6H), 7.43−7.55 (m, 3H), 7.59 (d, J = 8.3 Hz, 2H), 8.00 (dd, J = 7.8, 1.6 Hz, 1H), 8.00 (dd, J = 7.8, 1.6 Hz, 2H). 1H was not observed. MS m/z 550 (M + H)+. Anal. Calcd for C27H21BrClN3O3: C, 58.87; H, 3.84; N, 7.63. Found: C, 58.73; H, 3.99; N, 7.73. 4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)-1-((2-phenyl-1,3-oxazol-5-yl)methyl)piperazin-2-one (63). To a solution of 61 (2.00 g, 6.65 mmol) in CH2Cl2 (20 mL) was added a solution of 4-

chlorobenzoyl chloride (1.28 g, 7.31 mmol) in CH2Cl2 (20 mL) at 0 °C. The mixture was stirred at room temperature for 16 h. To the reaction mixture was added saturated aqueous NaHCO3. The mixture was concentrated and extracted with EtOAc. The separated organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by preparative TLC (CH2Cl2/MeOH = 20/1) to give 4-(4-chlorobenzoyl)-3-(4-chlorophenyl)piperazin-2-one (2.00 g) as a crude product. To a solution of the crude (240 mg) in DMF (5 mL) at 0 °C was added NaH (60% in oil, 27 mg, 0.68 mmol). The mixture was stirred at 0 °C for 0.5 h. Then to the mixture was added a solution of 62 (130 mg, 0.67 mmol) in DMF (5 mL) at 0 °C. After stirring at 0 °C for 0.5 h, the reaction mixture was poured into water and extracted with EtOAc. The separated organic layer was dried over Na2SO4 and concentrated. The residue was purified by preparative HPLC (condition 3, 0.05% aqueous ammonia solution/MeCN = 53/47 to 23/77) to give 63 (49 mg, 12% for 2 steps) as a white solid. 1H NMR (400 MHz, CDCl3) δ 3.31−3.54 (m, 2H), 3.56−3.78 (m, 1H), 4.57 (d, J = 15.6 Hz, 1H), 4.84−5.20 (m, 1H), 5.51 (brs, 1H), 6.46 (brs, 1H), 7.16 (s, 1H), 7.30−7.55 (m, 11H), 8.01 (brs, 2H). MS m/z 506 (M + H)+. Anal. Calcd for C27H21Cl2N3O3·0.25H2O: C, 63.48; H, 4.24; N, 8.22. Found: C, 63.58; H, 4.22; N, 8.26. (4-(4-Chlorobenzoyl)-3-cyclohexylpiperazin-1-yl)(2-phenyl1,3-oxazol-5-yl)methanone (65). To a solution of 2-cyclohexylpiperazine 64 (250 mg, 1.49 mmol) in THF (10 mL) were added Et3N (0.62 mL, 4.46 mmol) and Boc2O (0.34 mL, 1.48 mmol) at 0 °C. After stirring at 0 °C to room temperature overnight, the reaction mixture was poured into water at room temperature and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by NH-silica gel column chromatography (hexane/EtOAc = 95/5 to 20/80) to give tertbutyl 3-cyclohexylpiperazine-1-carboxylate (290 mg, 73%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 0.88−1.11 (m, 2H), 1.11−1.35 (m, 4H), 1.46 (s, 9H), 1.62−1.88 (m, 5H), 2.27−2.38 (m, 1H), 2.41− 2.62 (m, 1H), 2.62−2.89 (m, 2H), 2.97 (d, J = 11.4 Hz, 1H), 3.80−4.30 (m, 2H). 1H was not observed. MS m/z 269 (M + H)+. To a solution of tert-butyl 3-cyclohexylpiperazine-1-carboxylate (100 mg, 0.37 mmol) in THF (3 mL) were added DIPEA (0.20 mL, 1.14 mmol) and 4-chlorobenzoyl chloride (0.071 mL, 0.559 mmol) at 0 °C. After stirring at 0 °C for 1 h, the reaction mixture was poured into water at room temperature and extracted with EtOAc. The organic layer was separated, washed with water and brine, dried over Na2SO4, and concentrated. The residue was purified by NH-silica gel column chromatography (hexane/EtOAc = 95/5 to 20/80) to give tert-butyl 4(4-chlorobenzoyl)-3-cyclohexylpiperazine-1-carboxylate (150 mg, 99%) as a white solid. 1H NMR (300 MHz, CDCl3) δ 0.94−1.33 (m, 5H), 1.47 (s, 9H), 1.60−2.10 (m, 6H), 2.56−2.98 (m, 2H), 3.05−3.56 (m, 2H), 3.98−4.55 (m, 3H), 7.30 (d, J = 8.1 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H). MS m/z 351 (M − (t-Bu) + H)+. To a solution of tert-butyl 4-(4-chlorobenzoyl)-3-cyclohexylpiperazine-1-carboxylate (260 mg, 0.64 mmol) in EtOAc (10 mL) was added 4 M hydrogen chloride EtOAc solution (3.2 mL, 12.8 mmol) at room temperature. After stirring at room temperature for 5 h, the reaction mixture was concentrated to give (4-chlorophenyl)-(2cyclohexylpiperazin-1-yl)methanone hydrochloride (200 mg, 91%) as a colorless oil. 1H NMR (300 MHz, DMSO-d6) δ 0.75−1.37 (m, 5H), 1.45−1.86 (m, 5H), 2.03−2.20 (m, 1H), 2.85−3.29 (m, 4H), 3.37−3.66 (m, 2H), 4.27−4.63 (m, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 8.1 Hz, 2H), 9.29 (brs, 2H). MS m/z 307 (M + H)+. To a solution of (4-chlorophenyl)-(2-cyclohexylpiperazin-1-yl)methanone hydrochloride (60 mg, 0.17 mmol), 21 (40 mg, 0.21 mmol), HOBt (28 mg, 0.21 mmol), and DIPEA (113 mg, 0.87 mmol) in DMF (3 mL) was added EDC·HCl (40 mg, 0.21 mmol) at 0 °C. After stirring at 0 °C to room temperature for 12 h, the reaction mixture was poured into water at room temperature and extracted with EtOAc. The combined organic layer was concentrated, and the residue was purified by preparative HPLC (condition 1, 0.225% aqueous formic acid solution/MeCN = 40/60 to 10/90) to give 65 (20 mg, 24%) as a light gray powder. 1H NMR (400 MHz, CDCl3) δ 0.52−0.99 (m, 1H), 1.26− 1.36 (m, 4H), 1.61−1.78 (m, 6H), 3.04 (s, 1H), 3.31−3.54 (m, 2H), 3.66 (d, J = 10.8 Hz, 1H), 4.48−4.66 (m, 3H), 7.35−7.37 (m, 2H), 3348



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7.43−7.45 (m, 2H), 7.51−7.54 (m, 3H), 7.75 (s, 1H), 8.07 (d, J = 5.6 Hz, 2H). MS m/z 478 (M + H)+. Anal. Calcd for C27H28ClN3O3· 0.75H2O: C, 65.98; H, 6.05; N, 8.55. Found: C, 65.84; H, 5.81; N, 8.41. tert-Butyl 3-(4-Chlorophenyl)-1,4-diazepane-1-carboxylate (67). To a mixture of propane-1,3-diamine (2.25 g, 30.4 mmol) in MeOH (50 mL) was added 66 (4.00 g, 15.2 mmol) portionwise at 0 °C. The mixture was stirred at room temperature for 1 h. Then to the mixture was added NaOMe (2.05 g, 38.0 mmol). After stirring at 80 °C for 16 h, the reaction mixture was concentrated. The residue was purified by silica gel column chromatography (CH2Cl2/MeOH = 10/1) to give 3-(4-chlorophenyl)-1,4-diazepan-2-one (800 mg, 23%) as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ 1.72−1.84 (m, 3H), 3.02−3.09 (m, 1H), 3.33−3.47 (m, 3H), 4.57 (s, 1H), 6.08 (s, 1H), 7.31−7.37 (m, 4H). MS m/z 225 (M + H)+. To a mixture of 3-(4-chlorophenyl)-1,4-diazepan-2-one (800 mg, 3.56 mmol) in THF (30 mL) at 0 °C was added borane dimethylsulfide complex (10 M, 5.00 mL, 50.0 mmol) dropwise. The mixture was stirred at 80 °C for 16 h. To the mixture was added MeOH (20 mL) dropwise, and the mixture was stirred at room temperature for 1 h. Then the mixture was poured into 4 M hydrochloric acid (30 mL). After stirring at 80 °C for 1 h, the mixture was concentrated. To a solution of the residue (752 mg) and Et3N (1.08 g, 10.7 mmol) in CH2Cl2 (20 mL) was added Boc2O (545 mg, 2.5 mmol) dropwise at 0 °C. After stirring at room temperature for 16 h, the reaction mixture was concentrated. The residue was purified by silica gel column chromatography on silica gel (petroleum ether/ethyl acetate = 10/1) to give 67 (800 mg, 72% for 2 steps) as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ 1.51 (s, 9H), 1.77−2.16 (m, 3H), 2.67−2.98 (m, 2H), 3.13−3.33 (m, 2H), 3.80−3.92 (m, 2H), 7.29−7.40 (m, 4H). 1H was not observed. MS m/z 311 (M + H)+. (4-(4-Chlorobenzoyl)-3-(4-chlorophenyl)-1,4-diazepan-1yl)(2-phenyl-1,3-oxazol-5-yl)methanone (68). To a mixture of 67 (351 mg, 1.13 mmol) and Et3N (342 mg, 3.38 mmol) in CH2Cl2 (20 mL) was added 4-chlorobenzoyl chloride (218 mg, 1.24 mmol) in CH2Cl2 (2 mL) dropwise at room temperature. After stirring at room temperature for 3 h, the reaction mixture was concentrated, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate = 1/1) to give tert-butyl 4-(4-chlorobenzoyl)-3-(4chlorophenyl)-1,4-diazepane-1-carboxylate (472 mg, 93%) as a light yellow solid. 1H NMR (300 MHz, CDCl3) δ 1.29 (s, 3H), 1.48 (s, 6H), 1.60−1.69 (m, 1H), 2.07−2.11 (m, 1H), 2.66−3.07 (m, 3H), 4.04−4.10 (m, 1H), 4.26−4.31 (m, 1H), 4.59−4.65 (m, 1H), 4.90−5.23 (m, 1H), 6.82−7.07 (m, 4H), 7.08−7.18 (m, 2H) 7.24−7.33 (m, 2H). MS m/z 449 (M + H)+. To a mixture of tert-butyl 4-(4-chlorobenzoyl)-3-(4-chlorophenyl)1,4-diazepane-1-carboxylate (520 mg, 1.16 mmol) in 1,4-dioxane (10 mL) was added 4 M hydrogen chloride in dioxane (10.0 mL, 40.0 mmol). After stirring at room temperature for 5 h, the reaction mixture was concentrated to give (4-chlorophenyl)-[2-(4-chlorophenyl)-1,4diazepan-1-yl]methanone hydrochloride (320 mg) as a crude product. To a mixture of the crude (77 mg), 21 (38 mg, 0.20 mmol), and Et3N (61 mg, 0.60 mmol) in CH2Cl2 (20 mL) was added HATU (84 mg, 0.22 mmol) at room temperature. After stirring at room temperature for 16 h, the reaction mixture was concentrated and purified by preparative HPLC (condition 1, 0.225% aqueous formic acid solution/MeCN = 58/ 42 to 12/88) to give 68 (36 mg, 34% for 2 steps) as a white solid. 1H NMR (400 MHz, DMSO-d6, T = 80 °C) δ 1.63−2.25 (m, 2H), 3.17− 3.93 (m, 3H), 4.03−4.90 (m, 3H), 4.91−6.12 (m, 1H), 6.71−7.72 (m, 11H), 7.76−8.42 (m, 3H). MS m/z 520 (M + H)+. Anal. Calcd for C28H23Cl2N3O3·0.75H2O: C, 62.99; H, 4.63; N, 7.87. Found: C, 62.98; H, 4.52; N, 7.93. Preparation of Enzymes. The human recombinant proteins, fulllength eIF4A3, MLN51 (residues 137−283), full-length eIF4A1, fulllength eIF4B, and eIF4G (residues 712−1451) were expressed in Escherichia coli BL21(DE3) as fusion proteins with 6 × histidine (His)small ubiquitin-like modifier (SUMO) or His tags followed by a tobacco etch virus (TEV) protease cleavage site at the N-terminus and purification by Ni-NTA superflow affinity column (QIAGEN) and Superdex 200 gel-filtration column (GE Healthcare). The His-SUMO or His tags were cleaved with SUMO protease or TEV protease. Human

recombinant full-length DHX29 and full-length BRR2 were expressed in Sf-9 insect cells as fusion proteins with FLAG-His or His tags at the N terminus, respectively, using the BaculoDirect C-term Baculovirus expression system (Thermo Fisher Scientific). FLAG-His-DHX29 and His-BRR2 were purified by Ni-NTA superflow affinity column and Superdex 200 gel-filtration column. Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific) with bovine serum albumin as a standard. Recombinant human HiseIF4A2 protein was purchased from GeneCopoeia. RNA-Dependent ATPase Assay. The RNA-dependent ATPase assay was performed using the ADP-Glo assay system (Promega). Single-stranded RNA poly(U) was purchased from MP Biomedicals. The assay buffer comprised 20 mM Tris-HCl (pH7.5), 2.5 mM MgCl2, 100 mM KCl, 1 mM dithiothreitol (DTT), and 0.01% (v/v) Tween20. To enhance ATPase activity for eIF4A, the equivalent molar concentration of MLN51 for 150 nM eIF4A3 or eIF4B and eIF4G for 100 nM eIF4A1 or eIF4A2 were added. Regarding the ATPase assays for DHX29 or BRR2, the optimal concentrations were 6.4 nM and 6.25 nM, respectively. 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 poly(U) for eIF4A1 and eIF4A3; 20 μM ATP and 3.0 μg/mL poly(U) for eIF4A2; 30 μM ATP and 1.8 μg/mL poly(U) for DHX29; and 20 μM ATP and 2.5 μg/mL poly(U) for BRR2. After the addition of the substrates and test compounds, the ATPase reactions were started by the addition of the enzymes. They were incubated at room temperature for 30 min for eIF4A3, DHX29, and BRR2 or 40 min for eIF4A1 and eIF4A2. The enzymatic reactions were terminated by ADP-Glo reagent, and then ADP-Glo detection reagent was added to detect ADP. Luminescent signals were measured using an EnVision 2102 multilabel plate reader (PerkinElmer). We defined the luminescent 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) with the maximum and minimum of the curve constrained to 100 and 0, respectively. Reporter Plasmids. The reporter gene plasmid was constructed as described in the literature.36 Briefly, the pGL4.12 SV40-Luc2CP-BGG vector was amplified by polymerase chain reaction (PCR) from pGL4.13 (Promega), and the β-globin genomic sequence from Exon II to the poly(A) signal was amplified by PCR from a human genomic library (Clontech Laboratories). Both PCR products were inserted into pGL4.12 (Promega). A pRL-TK vector as an internal control was purchased from Promega. NMD Reporter Assay. HEK293T cells were transfected with the pGL4.12 SV40-Luc2CP-BGG vector and a pRL-TK vector as an internal control using Lipofectamine LTX (Thermo Fisher Scientific). The compounds were treated for 6 h after transfection of the two vectors for 24 h. Relative firefly to renilla luciferase activity was determined using the dual luciferase kit (Promega). Luminescent signals were detected by EnVision 2102 multilabel plate reader (PerkinElmer). Surface Plasmon Resonance (SPR) Biosensing Assay. SPR biosensing experiments were performed on BiacoreS200 and BiacoreT100 instruments equipped with CM7 sensor chips (GE Healthcare). Immobilization of eIF4A3 onto the CM7 sensor surface was performed following the standard amine coupling procedure according to the manufacturer’s instructions. HBS-P+ (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Surfactant P20) supplemented with 1 mM DTT was used as the running buffer for immobilization. Typical immobilization levels ranged from 5000 to 10 000 RU. All binding experiments were performed in 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.2 mM tris(2carboxyethyl)phosphine (TCEP), 0.02% Surfactant P20 (v/v), and 1− 2% DMSO (v/v) at 25 °C. Compound solutions at several concentrations (6−12 points) were injected for 60−120 s at a flow rate of 50 μL/min, and the dissociation was followed for up to 120− 1200 s depending on the compound’s off-rate. Competition experiments were performed in the presence of blocking compound 52a at a saturated concentration, diluted in the running buffer.39 Data processing and analyzing were performed using BiacoreS200 and BiacoreT100 evaluation software (GE Healthcare). Sensorgrams were doublereferenced prior to global fitting the concentration series to a 1:1 3349



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binding model. Dissociative half-lives t1/2 were calculated from the dissociation rate constants koff (t1/2 = ln 2/koff).

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bis(trimethylaluminum) 1,4-diazabicyclo[2.2.2]octane adduct; MeCN, acetonitrile; MeOH, methanol; MTBE, methyl tert-butyl ether; NaOMe, sodium methoxide; Pd(OAc)2, palladium(II) acetate; Pd(PPh3)4, tetrakis(triphenylphosphine)palladium(0); i-PrOH, 2-propanol; SAR, structure−activity relationships; SPR, surface plasmon resonance; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TsOH, p-toluenesulfonic acid

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01904. Molecular formula strings and some data (CSV) SPR sensorgrams; X-ray crystal structure analysis of 8a; synthetic procedure for intermediates 17, 23, 26−28, and 62 (PDF)

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REFERENCES

(1) Linder, P.; Jankowsky, E. From unwinding to clamping  the DEAD box RNA helicase family. Nat. Rev. Mol. Cell Biol. 2011, 12, 505− 516. (2) Tanner, N. K.; Linder, P. DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol. Cell 2001, 8, 251−262. (3) Jankowsky, E. RNA Helicases at work: binding and rearranging. Trends Biochem. Sci. 2011, 36, 19−29. (4) Jarmoskaite, I.; Russell, R. RNA helicase proteins as chaperones and remodelers. Annu. Rev. Biochem. 2014, 83, 697−725. (5) Rocak, S.; Linder, P. DEAD-box proteins: the driving forces behind RNA metabolism. Nat. Rev. Mol. Cell Biol. 2004, 5, 232−241. (6) Saulière, J.; Murigneux, V.; Wang, Z.; Marquenet, E.; Barbosa, I.; Le Tonquèze, O.; Audic, Y.; Paillard, L.; Crollius, H. R.; Le Hir, H. CLIPseq of eIF4AIII reveals transcriptome-wide mapping of the human exon junction complex. Nat. Struct. Mol. Biol. 2012, 19, 1124−1131. (7) Le Hir, H.; Andersen, G. R. Structural insights into the exon junction complex. Curr. Opin. Struct. Biol. 2008, 18, 112−119. (8) Palacios, I. M.; Gatfield, D.; St Johnston, D.; Izaurralde, E. An eIF4AIII-containing complex required for mRNA localization and nonsense-mediated mRNA decay. Nature 2004, 427, 753−757. (9) Ferraiuolo, M. A.; Lee, C.-S.; Ler, L. W.; Hsu, J. L.; Costa-Mattioli, M.; Luo, M.-J.; Reed, R.; Sonenberg, N. A nuclear translation-like factor eIF4AIII is recruited to the mRNA during splicing and functions in nonsense-mediated decay. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 4118−4123. (10) Shibuya, T.; Tange, T. O.; Sonenberg, N.; Moore, M. J. eIF4AIII binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat. Struct. Mol. Biol. 2004, 11, 346−351. (11) Le Hir, H.; Séraphin, B. EJCs at the heart of translational control. Cell 2008, 133, 213−216. (12) Reed, R.; Hurt, E. A conserved mRNA export machinery coupled to pre-mRNA splicing. Cell 2002, 108, 523−531. (13) Michelle, L.; Cloutier, A.; Toutant, J.; Shkreta, L.; Thibault, P.; Durand, M.; Garneau, D.; Gendron, D.; Lapointe, E.; Couture, S.; Le Hir, H.; Klinck, R.; Elela, S. A.; Prinos, P.; Chabot, B. Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators. Mol. Cell. Biol. 2012, 32, 954−967. (14) Wang, Z.; Murigneux, V.; Le Hir, H. Transcriptome-wide modulation of splicing by the exon junction complex. Genome Biol. 2014, 15, 551. (15) Baker, K. E.; Parker, R. Nonsense-mediated mRNA decay: terminating erroneous gene expression. Curr. Opin. Cell Biol. 2004, 16, 293−299. (16) Keeling, K. M.; Bedwell, D. M. Suppression of nonsense mutations as a therapeutic approach to treat genetic diseases. Wiley Interdiscip. Rev. RNA 2011, 2, 837−852. (17) Martin, L.; Grigoryan, A.; Wang, D.; Wang, J.; Breda, L.; Rivella, S.; Cardozo, T.; Gardner, L. B. Identification and characterization of small molecules that inhibit nonsense mediated RNA decay and suppress nonsense p53 mutations. Cancer Res. 2014, 74, 3104−3113. (18) Pastor, F.; Kolonias, D.; Giangrande, P. H.; Gilboa, E. Induction of tumour immunity by targeted inhibition of nonsense-mediated mRNA decay. Nature 2010, 465, 227−230. (19) Popp, M. W.; Maquat, L. E. Attenuation of nonsense-mediated mRNA decay facilitates the response to chemotherapeutics. Nat. Commun. 2015, 6, 6632−6632.

AUTHOR INFORMATION

Corresponding Authors

*M.I.: phone, +81-466-32-1196; e-mail, masahiro.ito@takeda. com. *Y.I.: phone, +81-466-32-4112; e-mail, yasuhiro.imaeda@ takeda.com. ORCID

Masahiro Ito: 0000-0001-5965-535X Douglas R. Cary: 0000-0003-3940-6078 Yusuke Kamada: 0000-0003-3701-2075 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Yasumi Kumagai and Motoo Iida for their assistance with NMR spectroscopic experiments and analysis, Mitsuyoshi Nishitani for acquiring and determining X-ray crystal structures, and Yumi Imai and Haruyuki Nishida for supervising of structure determination. We also thank Natsumi Fujii, Chie Kushibe, and Katsuhiko Miwa for HPLC purification and analysis of compounds, Ryosuke Arai, Kouichi Iwanaga, and Izumi Nomura for conducting high-throughput synthesis. We thank Jyun-ichi Sakamoto for support of SPR analysis, Takashi Ito for preparation of proteins, and Shinji Moriyama, Shin-ichi Matsumoto, Shigeru Kondo, and Akihiko Naotsuka for conducting high-throughput screening. We express our appreciation to Nao Morishita, Satoshi Endo, Daisuke Nakata, Shinsuke Araki, Masato Yugami, Ryo Mizojiri, and Hironobu Maezaki for helpful discussions and advice. We thank Ryosuke Tokunoh, Tomohiko Suzaki, Yuuko Hitomi, Atsuo Baba, Hiroshi Nara, and Nobuo Cho for their cooperation in helping us execute this work. We thank Hiromichi Kimura, Daisuke Morishita, and Satoshi Kitazawa for the pioneering spirit they demonstrated in initiating the eIF4A3 project.

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ABBREVIATIONS USED Boc2O, di-tert-butyl dicarbonate; BRR2, bad response to refrigeration 2 (an ATP-dependent RNA helicase); CDCl3, deuterated chloroform; DBU, 1,8-diazabicyclo[5.4.0]undec-7ene; DHX29, DEAH-box helicase 29 (an ATP-dependent RNA helicase); DIPEA, N,N′-diisopropylethylamine; DME, 1,2dimethoxyethane; DMSO-d6, dimethyl sulfoxide-d6; EDC, 1(3-(dimethylamino)propyl)-3-ethylcarbodiimide; EDC·HCl, 1(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride; Et2NH, diethylamine; Et3N, triethylamine; EtOAc, ethyl acetate; EtOH, ethanol; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′tetramethyluronium hexafluorophosphate; HOBt, 1-hydroxybenzotriazole; Me3Al, trimethylaluminum; (Me3Al)2·DABCO, 3350



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Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active

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