Discovery of Novel 2, 5-Dioxoimidazolidine-Based P2X7 Receptor

Article pubs.acs.org/jmc

Discovery of Novel 2,5-Dioxoimidazolidine-Based P2X7 Receptor Antagonists as Constrained Analogues of KN62 Jin-Hee Park,†,▽ Ga-Eun Lee,‡,▽ So-Deok Lee,† Tran Thi Hien,∥ Sujin Kim,† Jin Won Yang,§ Joong-Heui Cho,† Hyojin Ko,† Sung-Chul Lim,⊥ Yoon-Gyoon Kim,@ Keon-Wook Kang,*,§ and Yong-Chul Kim*,† †

School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea Department of Pharmaceutical Industry, Korea Health Industry Development Institute(KHIDI), Chungcheongbuk-do 363-700, Republic of Korea § College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Republic of Korea ∥ College of Pharmacy and ⊥Department of Pathology, College of Medicine, Chosun University, Gwangju 501-759, Republic of Korea @ College of Pharmacy, Dankook University, Cheonan 330-714, Republic of Korea ‡

S Supporting Information *

ABSTRACT: Novel 2,5-dioxoimidazolidine-based conformationally constrained analogues of KN62 (1) were developed as P2X7 receptor (P2X7R) antagonists using a rigidification strategy of the tyrosine backbone of 1. SAR analysis of the 2,5-dioxoimidazolidine scaffold indicated that piperidine substitution at the N3 position and no substitution at N1 position were preferable. Further optimization of the substituents at the piperidine nitrogen and the spacer around the skeleton resulted in several superior antagonists to 1, including 1-adamantanecarbonyl analogue 21i (IC50 = 23 nM in ethidium uptake assay; IC50 = 14 nM in IL-1β ELISA assay) and (3-CF3-4-Cl)benzoyl analogue (−)-21w (54 nM in ethidium uptake assay; 9 nM in IL-1β ELISA assay), which was more potent than the corresponding (+) isomer. Compound 21w displayed potent inhibitory activity in an ex vivo model of LTP-induced pain signaling in the spinal cord and significant anti-inflammatory activity in in vivo models of carrageenan-induced paw edema and type II collagen-induced joint arthritis.

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INTRODUCTION Purinergic P2X receptors, cation-selective ion channels gated by extracellular ATP, are divided into seven receptor subtypes, P2X1-P2X7.1,2 According to a recent report regarding the crystal structure of the zP2X4 receptor, these receptors exist as a trimeric subunit, with each subunit consisting of two continuous transmembrane α-helices, an intracellular terminus, and a large extracellular domain rich with disulfide bonds.3 In particular, the P2X7 receptor (P2X7R), first identified from the rat brain in 1996,4 is distinct compared with the other P2X receptor subtypes because it has a considerably longer C-terminus, with an additional 100−200 amino acids.5 Brief exposure of P2X7R to the agonist ATP facilitates a transient current through the opening of cation channels, thereby triggering depolarization, homeostasis of Na+ and K+, and the massive influx of Ca2+.1,2,5 This process leads to activation of a diverse range of signal transduction pathways related to inflammatory signals, including phospholipase A2, phospholipase D, mitogen-activated protein kinase (MAPK), and the nuclear κB factor.6 Furthermore, repeated or sustained activation of the receptors of extracellular ATP induces large and nonspecific pore formation.5 The formation of an enlarged pore © 2015 American Chemical Society

allows the penetration of positively charged hydrophilic solutes, such as ethidium bromide and YO-Pro-1, which has a molecular mass of up to 900 Da.7 Pannexin-1, which acts as a plasma membrane hemichannel, was recently reported to be explicitly involved in the formation of the large pore of P2X7R.8,9 P2X7R is primarily expressed in hematopoietic-derived immune cells, including mast cells and macrophages, and in brain glial cells, including microglia and astrocytes.6,10 The most important role of P2X7R in the production of IL-1β in activated monocytes, macrophages, and microglia is part of a complex sequence of events. Initially, the activation of P2X7R triggers K+ efflux, leading to stimulation of the IL-1β-converting enzyme (caspase-1) in the NLRP3/NALP3 inflammasome, which converts pro-IL-1β to mature IL-1β.11−13 Interleukin-1β (IL-1β) is a cytokine protein belonging to the interleukin-1 (IL-1) family. IL-1, along with tumor-necrosis factor-α (TNF-α) and IL-6 is a major mediator of innate immune reactions that play central roles in the development of pathological conditions leading to inflammation and chronic pain.11,14,15 Received: February 28, 2014 Published: January 18, 2015 2114

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Journal of Medicinal Chemistry

Figure 1. Current P2X7 receptor antagonists.

of the 5-isoquinoline sulfonate with a substituted arylsulfonate at the para-position of the tyrosine phenyl ring (3), and the preparation of ring-constrained derivatives using 1,2,3,4tetrahydroisoquinoline (TIC) (4).29−34 Recently, a diverse series of P2X7R antagonists with enhanced druglike properties has been disclosed, including AstraZeneca’s adamantane amide (5),35 Pfizer’s 6-azauracil (6),36,37 Evotec’s pyridopyrimidine (7),38 Abott’s cyanoguanidine (8),39 and GSK’s pyroglutamic acid amide (9)40 analogues. Small-molecule P2X7R antagonists, such as AZD9056 (5), CE-224,535 (6), and EVT-401, have entered clinical trials for the treatment of inflammatory diseases, including rheumatoid arthritis (RA).25 Although 5 of AstraZeneca and 6 of Pfizer did not have sufficient efficacy in RA to proceed beyond phase II clinical trials,41,42 more novel P2X7 antagonists are in the drug development pipeline, including EVT-401, for which phase I clinical trials were completed in 2009.43 Our group has reported KN-62 analogues44 and protoberberine (10),45 pyrazolodiazepine,46 and 3,5dichloropyridine (11)47 derivatives as novel P2X7R antagonists. In this report, we describe the discovery of novel P2X7R antagonists based on the design of constrained structures of low-energy conformers of KN-62 through the introduction of a 2,5-dioxoimidazolidine scaffold as a rigidification strategy. We report extensive structure−activity relationships between the designed analogues regarding their activity as P2X7R antagonists and in the suppression of the release of IL-1β. In addition, we report the ex vivo antipain signaling and the in vivo anti-inflammatory activity of 21w, one of the representative compounds.

Therefore, the therapeutic regulation of inflammation through the suppression or blockade of the proinflammatory activities of IL-1 has been widely studied using various methods.12,16 For example, the recombinant form of IL-1 receptor antagonist (IL-1Ra), anakinra (Kineret, Amgen), has been used as a therapeutic agent in human patients with immune-related disease.15,17 Because P2X7R has been reported as a crucial regulator of both IL-1β maturation and externalization in immune cells,8,14,18 antagonizing this receptor has been recognized as another promising strategy for blocking the action of IL-1. This potential has been confirmed by P2X7R knockout studies in which the absence of P2X7R in macrophage and microglia was found to impair IL-1β secretion after ATP stimulation. Furthermore, mice lacking P2X7R have displayed suppressed nociceptive sensitivity in preclinical pain models, especially those for arthritis and neuropathic pain.19 In addition, the upregulated expression of P2X7R has been observed in human dorsal root ganglia and injured nerves from patients with chronic neuropathic pain.19 Recently, the activation of P2X7R-induced release of cathepsin B, a family of lysosomal proteases, in macrophages independent from the mechanism of IL-1 release was reported.20 Increased levels of cathepsin B in the synovial fluid of RA patients assist in the degradation of phagocytosed extracellular matrix protein, which is involved in bone and cartilage damage.21 Therefore, it has been suggested that P2X7R may be valuable as a therapeutic target to control inflammation and chronic pain in patients with RA or neuropathic pain. Several research groups and pharmaceutical companies are conducting research on new drugs targeting P2X7R (see Figure 1).22−27 KN62 (1) was one of the first generation antagonists of P2X7R discovered.28 Several attempts have been made to optimize analogues of 1, such as through the variation of substituents at the piperazine moiety (2), the replacement

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RESULTS AND DISCUSSION Design Strategy. Several research groups have conducted extensive SAR and pharmacological studies of 1, which contains

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Figure 2. (A) A low energy conformer of 1 (KN62) and distance analysis between key pharmacophores (in angstroms). (B) 2D structures of compound 19c and 1. (C) Superimposition of low energy conformers of 2,5-dioxoimidazolidine scaffold and compound 1. (D) Superimposition of low-energy conformers of compound 19c and 1.

Scheme 1. Synthesis of 2,5-Dioxoimidazolidine Derivatives (for R1 Substitutions)a

Reagents and conditions. (a) (i) KOCN, H2O, pyridine, 90 °C, 3 h; (ii) conc. HCl, AcOH, 120 °C, 1 h 30 min, 54%; (b) isoquinolinesulfonyl chloride·HCl, TEA, CH2Cl2, rt, 5 h, 80%; and (c) R1-OSO2Me, CsHCO3, DMF, 50 °C, 24 h, 44−81%.

a

L-tyrosine. In these investigations, 1 (hP2X7R; pIC50 = 7.9) has demonstrated the potent and specific antagonism of ATPinduced pro-inflammatory IL-1β release and the suppression of MAPK pathways, including p38 and ERK.48−50 Our strategy to design conformationally constrained derivatives of 1 was derived from the computational analysis of low-energy conformers by optimizing the geometric calculation in MOPAC using PM3 parameters in the CAChe program (BioMedCAChe version 5.0, FUJITSU, Beaverton, Oregon) (Figure 2). We calculated the distances between the hydroxy group, carboxyl group, and the α-nitrogen of the tyrosine moiety in a lowenergy conformation of 1, which were the primary sites for modification in this analog-based derivative (Figure 2A). On the basis of an analysis of the distance between these 3 points, we chose (S)-5-(4-hydroxybenzyl)imidazolidine-2,4-dione as a scaffold in order to restrict the free rotation of the α-nitrogen and the carboxyl group of the N-methyl tyrosine moiety of 1 (Figure 2B). This scaffold was expected to facilitate the introduction of substituents at key positions while maintaining

the triangular geometries delineated by each pair of these 3 points of 1 (Figure 2C). Thus, we designed compound 19c, in which the same isoquinolin-5-sulfonyl group of 1 was retained at the R1 position, the piperazine moiety of 1 was replaced with a substituted piperidinyl group at the R2 position, and an isoquinolin-5-yl methyl group replaced the isoquinolin5-sulfonyl group at the R3 position of 1. To support this basic idea, the low-energy conformers of 1 and compound 19c were superimposed on one another. As shown in Figure 2D, the overlap of the side chains was well-matched in 3dimensional space, suggesting that the 2,5-dioxoimidazolidine derivatives might act as rigidified derivatives of 1. As an initial trial of this strategy, we synthesized several derivatives through combined substitution of the isoquinolin-5-yl methyl group and the substituted piperidinyl group at the N1 or N3 position and evaluated their P2X7R antagonistic activity (Figure 2). Chemistry. The syntheses of the 2,5-dioxoimidazolidine derivatives (15, 18-19, 21−24, and 29) discussed in this 2116

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Journal of Medicinal Chemistry Scheme 2. Synthesis of 2,5-Dioxoimidazolidine Derivatives (for R2 and R1/R2 Substitution)a

a

Reagents and conditions. (a) isoquinolinesulfonyl chloride·HCl, TEA, CH2Cl2, rt, 5 h; (b) 20% TFA, CH2Cl2, rt, 3 h, 63% (overall yield for a, b); (c) R2CHO, NaBH(OAc)3, DCE, rt, 4 h, 63−80%; (d) KOCN, AcOH, rt, 5 h, 75−82%; and (e) R2-OSO2Me, CsHCO3, DMF, 50 °C, 24 h, 59−71%.

afforded the imidazolidine-2,4-dione derivatives 18a−18e in good yield. Finally, compounds 19a−19c were obtained by alkylation of 18a−18c using cesium bicarbonate. Scheme 3 shows the preparation of 4-substituted piperidinyl derivatives 20a, 21a−21x, 24a−24d, 4-piperidinyl methyl derivative 22a, and 4-piperidinyl ethyl derivatives 23a−23b. After Boc-deprotection of 18c−18e with 10% TFA in dichloromethane, 4-piperidinyl amine 20a−20c was acylated or alkylated to attach various groups, including aliphatic acyl groups and substituted benzoyl or benzyl groups, to yield 21a−21x, 22a, and 23a−23b. Furthermore, N-alkylation of 21w afforded compounds 24a−24d. Scheme 4 shows the synthesis of the phenyl isoquinoline-5sulfonate derivatives, which uses the same procedures as Scheme 2 except for the first step. Compound 25 was synthesized starting from (R)-methyl 2-amino-2-(4-hydroxyphenyl)acetate. To determine the enantiomeric purity of the compounds, we selected compound 20a and confirmed the structure of the corresponding Mosher amide by NMR analysis. Because reductive amination and harsh chemical conditions were employed in subsequent steps, we suspected that the synthesized derivatives may be racemized. The two singlet peaks for the methoxy group in the Mosher amide showed a ratio of 5:7 (Figure S1 of the Supporting Information). Therefore, the synthesized 2,5dioxoimidazolidine derivatives were confirmed to be racemates.

paper are summarized in Schemes 1−4. The 1-subsituted 4-((2,5-dioxoimidazolidin-4-yl)methyl)phenylisoquinoline-5sulfonates 15a and 15b were synthesized starting from O-benzylL-tyrosine (12, Scheme 1). The reaction of 12 with potassium cyanate yielded a urea intermediate that cyclized under acidic conditions at 90 °C; subsequent deprotection of the benzyl group yielded the 5-membered imidazolidine-2,4-dione 13.51 Isoquinoline-5-sulfonate 14 was prepared by reaction of 4-hydroxybenzyl compound 13 with 5-isoquinolinyl sulfonyl chloride. The regioselective N-alkylations to place substituents at the N1 position were easily performed under weak basic conditions in high yield due to its relatively acidic character compared to the amide NH at the N3 position, thus affording 15a−15b. Alkylation of the imide nitrogen at the N1 position occurred exclusively, as shown by the loss of the imide proton signal in the 1H NMR. Compounds 18a−18e and 19a−19c, with 4-substituted piperidinyl, hetero- or bulky aromatic moieties at R1 and R2, were obtained by a three- or four-step synthetic route (Scheme 2). Compound 16 was prepared using sulfonylation with 5isoquinoline sulfonyl chloride followed by Boc-deprotection under acidic conditions. Compounds 17a−17e were synthesized via reductive amination using the appropriate aldehyde or ketone in the presence of sodium triacetoxyborohydride. Next, cyclization of 17a−17e with potassium cyanate in acetic acid 2117

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Journal of Medicinal Chemistry Scheme 3. Synthesis of 2,5-Dioxoimidazolidine Derivatives (for the Variation of R3 and n)a

a Reagents and conditions. (a) 20% TFA, CH2Cl2, 0 °C, 1 h, 83−85%; (b) R-COCl, TEA, CH2Cl2, rt, 2 h, or R-COOH, EDC·HCl, TEA, CH2Cl2, rt, 2 h, or R-CHO, NaBH(OAc)3, DMF, rt, 2 h, 56−95%; and (c) R1I, K2CO3, DMF, rt, 6 h, 70−86%.

Scheme 4. Synthesis of 2,5-Dioxoimidazolidine Derivatives (for the Variation of Carbon Chain Length at N4 Position)a

a Reagents and conditions. (a) ditert-butyl dicarbonate, TEA, CH2Cl2, rt, 3 h, 100%; (b) isoquinoline-5-sufonyl chloride·HCl, TEA, CH2Cl2, rt, 4 h; (c) 20% TFA, CH2Cl2, rt, 2 h, 86% (overall yield for b, c); (d) 1-boc-4-piperidone, NaBH(OAc)3, DCE, rt, 4 h, 99%; (e) KOCN, AcOH, rt, 5 h; (f) 20% TFA, CH2Cl2, rt, 2 h, 76% (overall yield for e, f); and (g) R3COOH, EDC, TEA, CH2Cl2, rt, 1 h, 21−63%.

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Journal of Medicinal Chemistry Table 1. P2X7R Antagonistic Effects of 2,5-Dioxoimidazolidine Derivatives

compd

1 14 19a 19b 19c 15a 15b 18a 18b 18c 20b

R1

H 1-Boc-4-piperidinylmethyl isoquinolin-5-ylmethyl isoquinolin-5-ylmethyl isoquinolin-5-ylmethyl 1-Boc-4-piperidinylmethyl H H H H

% inhibition or IC50 (μM)a

R2

EtBr uptake in hP2X7-expressing HEK293 cellsb

IL-1β release in differentiated THP-1 cellsc

0.158 ± 0.018 NAd 12.0 ± 6.5 8.96 ± 1.8 1.97 ± 0.17 12.2 ± 1.7 7.72 ± 2.2 3.82 ± 0.97 1.82 ± 0.36 0.885 ± 0.023 NAd

0.143 ± 0.035 NDe 50 ± 4% 42 ± 6% 44 ± 1% 52 ± 1% 45 ± 1% 38 ± 2% 52 ± 4% 60 ± 2% NDe

H isoquinolin-5-ylmethyl 1-PhCH2-4-piperidinylmethyl 1-Boc-4-piperidinylmethyl H H isoquinolin-5-ylmethyl 1-PhCH2-4-piperidinylmethyl 1-Boc-4-piperidinylmethyl 4-piperidinylmethyl

a % Inhibition values at 1 μM were expressed as a percentage, relative to maximum release of IL-1β stimulated by BzATP only, and IC50 values were obtained from concentration−response curves. Data values are expressed as means ± SDs. All experiments were repeated at least 3−6 times. b Experiments were assessed in the ethidium+ accumulation assay using hP2X7-expressing HEK293 cells. cExperiments was evaluated with an ELISA for the detection of BzATP-activated IL-1β release in LPS/IFN-γ-differentiated human THP-1 cells. dNo activity at 10 μM. eNot determined.

Table 2. Effects of Carbon Chain Length Adjacent to N3 Position of 2,5-Dioxoimidazolidine and R3 Substitutions in P2X7R Antagonistic Activity

To enable further studies, the two stereoisomers of compound (±)-21w were separated to afford (+)-21w and (−)-21w using a chiral HPLC column (Figure S2 of the Supporting Information). SAR of 2,5-Dioxoimidazolidine Derivatives as P2X7R Antagonists. To explore the structure−activity relationship (SAR) of the series of novel-substituted 4-((2,5-dioxoimidazolidin-4-yl)methyl)phenylisoquinoline-5-sulfonates, the compounds were evaluated using an assay of ethidium ion accumulation stimulated by 2′(3′)-O-(4-benzoylbenzoyl)-ATP (BzATP) in human HEK293 cells stably expressed with recombinant human P2X7R. A known P2X7R antagonist, 1, was used as a positive control for comparison (Tables 1−4). Compound 14, with no substitution on the imidazole skeleton, displayed no activity as a P2X7R antagonist. Therefore, we initially investigated the appropriate combinations of substituents at the R1 and R2 positions of the imidazolidine-2,4dione scaffold to confirm our rationale with respect to the design of conformationally constrained analogues of 1 (Table 1). Compound 19c, which has isoquinoline-5-ylmethyl and 1-Boc4-piperidinylmethyl groups at the R1 and R2 positions, respectively, was 6-fold more potent against P2X7R compared with 19a, the regioisomer of 19c. Thus, the substitution patterns of 19c may be preferred in order to adopt the biologically active conformation of 1, although compound 19c displayed approximately 12-fold weaker activity than the parent compound 1. Replacement of the Boc group of 19c with a benzyl group (19b), similar to 1, was not beneficial. Interestingly, when we tested analogues with single substitutions at the N1 or N3 positions, R2 substitutions were clearly more favorable than R1 substitutions (15a vs 18a and 15b vs 18c) for hP2X7R antagonistic activity. Notably, compound 18c, with a Boc piperidine group at the R1 position, showed increased antagonistic activity with an IC50 in the submicromolar range and demonstrated a 4-fold increase in potency over compound 18a, which was substituted with an isoquinoline group. Thus, the isoquinoline sulfonamide group of 1 does not appear to be critical pharmacophore with our designed skeleton for P2X7R


compd

n

R3

EtBr uptake in hP2X7expressing HEK293 cellsb

1 18e 21a 21b 18c 18b 22a 18d 23a 23b

0 0 0 1 1 1 2 2 2

Boc PhCH2 PhCO Boc PhCH2 PhCO Boc PhCH2 PhCO

0.158 ± 0.018 0.355 ± 0.006 1.62 ± 0.19 0.574 ± 0.055 0.885 ± 0.023 1.82 ± 0.36 0.789 ± 0.074 1.01 ± 0.11 5.73 ± 1.1 4.99 ± 0.15

IL-1β release in differentiated THP-1 cellsc 0.143 ± 0.035 59 ± 2% NDd 60 ± 2% 60 ± 2% 52 ± 4% 59 ± 2% 54 ± 3% NDd 33 ± 1%

% Inhibition values at 1 μM were expressed as a percentage, relative to maximum release of IL-1β stimulated by BzATP only, and IC50 values were obtained from concentration−response curves. Data values are expressed as means ± SDs. All experiments were repeated at least 3−6 times. bExperiments were assessed in the ethidium+ accumulation assay using hP2X7-expressing HEK293 cells. cExperiments was evaluated with an ELISA for the detection of BzATPactivated IL-1β release in LPS/IFN-γ-differentiated human THP-1 cells. dNot determined. a

antagonism. Compounds 18b and 20b, with a benzyl substituent and no substitution, respectively, at the position of the Boc group, showed 50% lower antagonistic activity and no activity, respectively. These results suggest that the 2119

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Journal of Medicinal Chemistry Table 3. Effects of Aliphatic Acyl Groups at R3 Position in P2X7R Antagonistic Activity

Table 4. Effects of Substituted Benzoyl Groups at R3 Position in P2X7R Antagonistic Activity



compd 1 20a 21c 21d 21e 21f 21g 21h 21i 21j 21k 21l 21m 21n 21o

R3 H CH3CH2CO (CH3)2CHCO (CH3)3CO CH3(CH2)2CO (CH3)2CHCH2CO c-C6H11CO 1-adamantylCO 1-adamantylCH2 1-adamantylCH2CO 3-Br-1-adamatnylCO (3,5-diMe)-1adamantylCO 3- noradamantylCO 2-norbornanylCH2CO

EtBr uptake in hP2X7-expressing HEK293 cellsb


0.158 ± 0.018 NAd 11.7 ± 1.9 7.22 ± 1.1 5.65 ± 0.80 8.77 ± 0.54 4.88 ± 0.71 0.133 ± 0.013 0.023 ± 0.009 2.29 ± 0.25 0.084 ± 0.012 0.030 ± 0.005 0.099 ± 0.017

0.143 ± 0.035 NDe 48 ± 6% 33 ± 3% 49 ± 3% 37 ± 1% 45 ± 1% 0.080 ± 0.013 0.014 ± 0.002 49 ± 1% 0.015 ± 0.004 0.027 ± 0.001 0.081 ± 0.016

0.069 ± 0.013 0.109 ± 0.004

0.049 ± 0.019 0.029 ± 0.004

compd

R3

1 21b 21p 21q 21r 21s 21t 21u 21v 21w 21x

PhCO (3,4-(MeO)2)PhCO (2-Cl)PhCO (3-Cl)PhCO (4-Cl)PhCO (4-F)PhCO (3,4-diF)PhCO (3,4-diCl)PhCO (3-CF3-4-Cl)PhCO (2-F-4-Cl)PhCO

IL-1β release in EtBr uptake in hP2X7- differentiated THP-1 cellsc expressing HEK293 cellsb 0.158 0.574 3.76 0.666 0.220 0.133 0.913 0.217 0.073 0.070 0.423

± ± ± ± ± ± ± ± ± ± ±

0.018 0.055 0.80 0.068 0.026 0.026 0.086 0.015 0.011 0.008 0.024

0.143 ± 0.035 60 ± 3% 52 ± 9% 68 ± 2% 0.313 ± 0.045 0.192 ± 0.019 NDd 0.081 ± 0.003 0.072 ± 0.016 0.050 ± 0.008 0.253 ± 0.016


% Inhibition values at 1 μM were expressed as a percentage, relative to maximum release of IL-1β stimulated by BzATP only, and IC50 values were obtained from concentration−response curves. Data values are expressed as means ± SDs. All experiments were repeated at least 3−6 times. bExperiments were assessed in the ethidium+ accumulation assay using hP2X7-expressing HEK293 cells. cExperiments was evaluated with an ELISA for the detection of BzATPactivated IL-1β release in LPS/IFN-γ-differentiated human THP-1 cells. dNo activity at 10 μM. eNot determined. a

(Table 3). Of this series of acyclic aliphatic acyl derivatives, compounds with branched alkyl groups (21d, 21e, and 21g) were relatively more potent than those with linear alkyl groups (21c and 21f). Therefore, we introduced mono- or polycycloalkylcarbonyl groups (21h−21o) at the R3 position and obtained compounds with dramatically increased potencies in the low nanomolar range. For example, the substitution of a cyclohexanoyl (c-C6H11CO) group (21h) at the R3 position increased the antagonistic activity of P2X7R more than 30-fold (IC50 = 133 nM) compared with the compounds with acyclic alkyl groups. Moreover, substitutions with polycycloalkylcarbonyl groups further increased the antagonist activities, with IC50 values in the range of 20−100 nM. In particular, a 1-adamantylcarbonyl group (21i) at the R3 position resulted in the most potent antagonist for P2X7R (IC50 = 23 nM), which was approximately 5-fold more potent than reference compound 1. Additional polycycloalkylcarbonyl derivatives (21k−21o) were tested to optimize the R3 group. Except for 21l, which has a 3-bromo-adamantylcarbonyl group at the R3 position, all other derivatives, including those with 3,5-dimethyladamantylcarbonyl (21m), 1-adamantylmethylcarbonyl (21k), noradamantylcarbonyl (21n), and norboranylmethylcarbonyl (21o) groups, exhibited a 2- to 3-fold decrease in antagonistic activity compared to compound 21i, suggesting that the hydrophobic pocket of the receptor should be tightly regulated for optimum antagonistic activity of the polycycloalkyl carbonyl derivatives. The importance of acyl substitutions at the R3 position was confirmed once again by testing compound 21j, which

substitution on the piperidine nitrogen should be pivotal for P2X7R antagonism. Next, we examined substitution on the 4-piperidinyl moiety (R3 position) and the effects of the length of the carbon linker between the piperidinyl group and the N3 position of the 2,5dioxoimidazolidine (Table 2). With regard to the effects of substitutions at the R3 position of the 4-piperidinyl moiety, P2X7R antagonistic activity increased in the following order: benzyl- < benzoyl- < Boc-. Therefore, we expected that acyl substitution at the 4-piperidinyl moiety would be preferred over aryl alkyl substitutions for antagonistic activity (21a vs 21b, 18b vs 22a, and 23a vs 23b). Furthermore, aliphatic hydrophobic groups may be preferred instead of aromatic hydrophobic groups at the R3 position of the 4-piperidinyl moiety (18e vs 21a−21b; 18c vs 18b, 22a; 18d vs 23a−23b). With regard to the optimum length of the alkyl spacer between the 4-piperidinyl moiety and the N3 position (from n = 2 to n = 0), analogues directly substituted with the 4-piperidinyl group (n = 0) at the N3 position (18e, 21a, and 21b) afforded the best P2X7R antagonistic activity. Thus, we chose the 2,5dioxoimidazolidine skeleton as a new lead structure for further optimization of P2X7R antagonism. Because compound 20a exhibited no antagonistic activity at P2X7R up to 10 μM, various aliphatic acyl groups (R3 groups) were substituted on the 4-piperidinyl moiety of compound 20a 2120

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and 21w, respectively, exhibited the best IL-1β inhibitory activities, with IC50 values in the range of 50−80 nM. Although substitution at the R1 position was not appropriate for antagonism, as shown in Table 1, we examined the effect of alkyl substitution at the R1 position. As shown in Table 5, the

exhibited dramatically decreased antagonistic activity for P2X7R. Most of the compounds with micromolar and high submicromolar IC50 values in the ethidium accumulation assay (e.g., 15a−15b, 18a−18d, 19a−19c, 21a−21g, 21j, 21p, 22a, and 23a−23b) exhibited a relatively low inhibitory effect on the release of IL-1β, ranging from 30 to 68% inhibition at 1 μM. However, compounds with low nanomolar and subnanomolar activities in the ethidium accumulation assay exhibited more than 70% inhibition of IL-1β release. Furthermore, compounds such as 21k, 21l, 21n, 21o, and 21w, which showed greater than 95% inhibition values against the release of IL-1β by BzATP at 1 μM, were further evaluated by obtaining full concentration−response curves to compare the IC50 values. As a result, the inhibitory effect of the above 2,5-dioxoimidazolidine derivatives on the release of IL-1β exhibited patterns similar to those observed in the ethidium accumulation assay. In the experiments for inhibitory activity against BzATPinduced IL-1β release in LPS-primed THP-1 cells, all of the polycycloalkylacyl-substituted analogues in Table 3 displayed two-digit nanomolar IC50 values. The most potent P2X7R antagonists in this series, 21i and 21l, exhibited parallel inhibitory activity against BzATP-induced IL-1β release in LPS-primed THP-1 cells, with IC50 values of 14 and 27 nM, respectively. Interestingly, 21k and 21o, which are both weaker P2X7R antagonists, also displayed potent inhibitory activity in the IL-1β ELISA assay, with IC50 values of 15 and 29 nM, respectively. Next, we investigated the effect of substituents on the benzoyl group at the R3 position, as shown in Table 4. Overall, meta- and/or para-substitutions with halides were more favorable than substitutions with electron-donating groups or ortho-substitution with halides. Thus, compounds with monoor dihalogen-substituted benzoyl groups at the R3 position (21q−21x) exhibited IC50 values in the 10−8 to 10−7 M range, whereas 21p, which has a 3,4-dimethoxybenzoyl group at the R3 position, exhibited significantly decreased antagonistic activity, with an IC50 value of 3.76 μM. In the SAR analysis of halogen substitutions on the benzoyl moiety, the profile of P2X7R antagonistic activity with chlorination displayed the following pattern: ortho- (21q) < meta- (21r) < para- (21s, IC50 = 133 nM). In the case of fluorine substitution, the parafluorobenzoyl analogue, 21t, was 5-fold less potent than the corresponding chlorinated compound, 21s, suggesting that the size of the substituent on the benzoyl moiety has a significant effect on P2X7R antagonism. Additionally, we investigated disubstituted (ortho, meta, para) benzoyl derivatives (21u−21x) at the R3 position. Among these derivatives, 3,4-disubstitution (21u−21w) resulted in synergistically enhanced antagonist activities, with IC50 values of 217, 73, and 70 nM, respectively, compared with the corresponding derivatives monosubstituted at the para position (21s, 21t). The antagonistic activity decreased by more than 3-fold with 2fluoro-4-chloro substitution (21x). Overall, the SAR of the substituents on the benzoyl moiety at the R3 position suggests that the electronic effects, relative size, and positions of the substitutions may play important roles in P2X7R antagonistic activity. In addition, the inhibitory activities of mono- and dihalogensubstituted benzoyl groups at the R3 position against IL-1β release through the activation of P2X7R were well-correlated with the results of an ethidium accumulation assay (Table 4). In particular, the 3,4-dichloro and 3-CF3-4-chloro derivatives 21v

Table 5. Effects of R1 Substitutions in P2X7R Antagonistic Activity


compd

R1

1 21w 24a 24b 24c 24d

H CH3 CH3CH2 CH3CH2CH2 (CH3)2CH



± ± ± ± ± ±

0.143 ± 0.035 0.050 ± 0.008 NDd NDd NDd NDd

0.158 0.070 0.542 0.847 1.67 3.24

0.018 0.008 0.069 0.121 0.50 0.74

a % Inhibition values at 1 μM were expressed as a percentage, relative to maximum release of IL-1β stimulated by BzATP only, and IC50 values were obtained from concentration−response curves. Data values are expressed as means ± SDs. All experiments were repeated at least 3−6 times. bExperiments were assessed in the ethidium+ accumulation assay using hP2X7-expressing HEK293 cells. cExperiments was evaluated with an ELISA for the detection of BzATP-activated IL-1β release in LPS/IFN-γ-differentiated human THP-1 cells. dNot determined.

size of the substituent at the R1 position of the derivatives (24a−24d) influenced the antagonistic activity. Compound 24a, which has a methyl substituent, was well-tolerated, with an IC50 of 542 nM, whereas compounds 24b−24d displayed gradually reduced antagonistic activities as the substituents increased in size. For the derivatives with an aromatic ring directly linked to a 2,5-dioxoimidazolidine skeleton, such as compound 29a−29c, P2X7R antagonism was completely absent despite the combination of the best substituents at the R3 position (Table 6). This result suggests that the spacer between the aromatic ring and the 2,5-dioxoimidazolidine skeleton, as is the case with 21i, 21k, and 21w, is critical for antagonistic activity. Effect of the Enantiomers (+)-21w and (−)-21w on the P2X7R Antagonism. A series of 2,5-dioxoimidazolidine derivatives were synthesized and tested as racemic mixtures. To investigate the stereochemical preference of 2,5-dioxoimidazolidine derivatives for P2X7R antagonism, the chiral separation of a representative compound, 21w, was conducted using a chiral HPLC column and afforded two enantiomers, (+)-21w and (−)-21w, which were subsequently tested in two biological assay systems. The (−) enantiomer of 21w exhibited 1.4- and 5.5-fold higher inhibitory effects than racemic 21w, whereas the (+) enantiomer of 21w was 2.4- and 2-fold less active in the ethidium accumulation assay and IL-1β release assay, respectively 2121

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receptors; the activities of 21w were measured in two-electrode voltage clamping (TEVC) experiments on Xenopus oocytes with overexpression of mP2X1 or hP2X3 receptors by microinjection of the corresponding capped ribonucleic acids (cRNA). As a result, 10 μM 21w exhibited only a 5−15% reduction of the 2 μM ATP-induced ion current of either the P2X1 or P2X3 receptor, suggesting that the compound was highly selective for P2X7R vs P2X1R and P2X3R. Detailed data are provided in Figure S3 of the Supporting Information. Effect of Compound 21w at Rodent P2X7 Receptor. We further assessed whether 21w inhibits rodent P2X7 receptor using J774.A1, a murine monocyte/macrophage cell line. IL-1β secretion was 170-fold increased by ATP stimulus in LPS-primed J774.A1 cells. Although relative potency is obviously lower than human THP-1 monocytes, compound 21w concentration-dependently suppressed IL-1β secretion in J774.A1 cells, and the IC50 value was determined as 11.8 μM (Figure 3). 5 was used as a reference compound, which was

Table 6. Effects of Carbon Chain Length Adjacent to 4 Position of 2,5-Dioxoimidazoldine in P2X7R Antagonistic Activity


compd

R3

1 29a 29b 29c

1-adamantylCO 1-adamantylCH2CO (3-CF3−4-Cl)PhCO


IL-1β release in differentiated THP1 cellsc

± ± ± ±

0.143 ± 0.035 NDd NDd NDd

0.158 48 46 30

0.018 11% 10% 10%


Table 7. Effects of Enantiomers of Compound 21w in P2X7R Antagonistic Activitya Figure 3. Inhibition of IL-1β secretion by compound 21w in J774A.1 mouse macrophages. J774A.1 macrophages were preincubated with 1 μg/mL LPS for 5 h and further treated with compound 21w, AZD9056 (5), and ATP for 30 min. IL-1β secretion was analyzed in culture supernatants. Data represent means SD with 6 different samples (significant versus control, **p < 0.01; significant versus ATPtreated group, #p < 0.05; ##p < 0.01).

IC50 (nM)b compd

EtBr uptake in hP2X7-expressing HEK293 cellsc

IL-1β release in differentiated THP-1 cellsd

(±)-21w (+)-21w (−)-21w

70 ± 8 168 ± 17 54 ± 10

50 ± 8 98 ± 23 9±2

a

Chiral separation of each enantiomer of compound 21w on a CHIRALPACK IA using the reversed-phase mode. Column: 4.6 × 250 mm; mobile phase: ethanol; flow: 0.5 mL/min. bIC50 values were obtained from concentration−response curves. Data values are expressed as means ± SDs. All experiments were repeated at least 3−6 times. cExperiments were assessed in the ethidium+ accumulation assay using hP2X7-expressing HEK293 cells. dExperiments was evaluated with an ELISA for the detection of BzATP-activated IL-1β release in LPS/IFN-γ-differentiated human THP-1 cells.

known as a low affinity rodent P2X7 receptor antagonist (see Figure 1). However, 5 did not inhibit ATP-stimulated IL-1β release up to 60 μM in the rodent macrophage cells. Ex Vivo Antipain Signaling Activity of Compound 21w. P2X7R is abundantly expressed in microglia and has been proposed to play a pivotal role in the cross-talk between microglia and neurons.52 Microglial P2X7R and its downstream signaling pathways contribute to the induction of spinal longterm potentiation (LTP).53 The antipain signaling effects of the 2,5-dioxoimidazolidine derivative, with pregabalin as a positive control, were assessed in the neuropathic rat dorsal horn.54 Pregabalin is an anticonvulsant drug used to treat neuropathic pain and partial seizures. Compound 21w, which contains a (3-CF3-4-Cl)benzoyl moiety, was selected for animal experiments due to its solubility and high potency in both assay systems (IC50 in EtBr uptake: 70 nM; IC50 in IL-1β release: 50 nM) compared with other derivatives. Compound 21w reduced ex vivo pain-related LTP signaling by 76% at 20 μM, whereas pregabalin exhibited only a 31% reduction at the same concentration, suggesting that 2,5dioxoimidazolidine derivatives may be potential therapeutic agents for the treatment of neuropathic pain. In Vivo Anti-inflammatory Activity of Compound 21w. P2X7R may play a major role in the regulation of processes related to inflammation,55 as demonstrated in in vivo inflammatory

(Table 7). Thus, the absolute stereochemistry of 2,5dioxoimidazolidine derivatives is important for P2X7R antagonistic activity. To determine the absolute configuration of an enantiomer of compound 21w by XRD (X-ray diffraction), we attempted to obtain crystals of each enantiomer of 21w as well as derivatives of 21w such as N-alkylated compounds at R1 position with various groups (e.g., benzyl group, methyl group, and chiral auxiliary group) or N-methylated compounds at the 5-isoquinoline group. Unfortunately, we had failed all the trials due to poor crystal formation. All schemes, solubility information, and HPLC data of derivatives were shown in Synthesis S2 and Figure S5 of the Supporting Information. Selectivity Evaluation of 21w at mP2X1 and hP2X3 receptors using a TEVC Assay. From these derivatives, a representative P2X7R antagonist, compound 21w, was further evaluated for its selectivity against the mP2X1 and hP2X3 2122

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Journal of Medicinal Chemistry animal models.19,55 The anti-inflammatory effect of compound 21w, the potent and selective hP2X7R antagonist, was evaluated in carrageenan-induced paw edema in SD rats. The paw edema volume was increased after carrageenan injection, and the maximal increase occurred at 6 h (Figure 4A). The paw edema

mouse and rat models, we checked pharmacokinetics profile of compound 21w in rats (Table 9). The absolute bioavailability of compound 21w after intraperitoneal injection was 86%. The plasma concentrations of compound 21w declined with mean terminal half-life of 77.2 min after intravenous injection. After intraperitoneal injection of compound 21w (10 mg/kg) in rats, maximal plasma concentration of compound 21w (Cmax) was 400 ± 200 ng/mL at 1.5 h. Considering in vitro IC50 value of compound 21w [50 nM in IL-1β release assay (THP-1 human monocytes) and 11.8 μM (J774,A1 mouse monocytes), respectively], the plasma concentration may not be enough to completely inhibit P2X7R in the animal study. We speculate that lower plasma concentration than the in vitro IC50 value (11.8 μM) in mouse IL-1β assay might be efficaciously enough for the blocking of P2X7R in arthritic tissues, considering that the analysis of in vitro IL-1β release was performed in supraphysiological concentration of ATP (5 mM), and compound 21w is a ATP-competitive antagonist of P2X7R (Figure S4 of the Supporting Information).

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CONCLUSIONS In conclusion, this report describes the identification of a 2,5dioxoimidazolidine class of novel potent P2X7R antagonists designed from conformationally constrained analogues of 1 (KN62). The results of the SAR studies for P2X7R antagonism are summarized as follows: (1) substitution at the N1 position of 2,5-dioxoimidazolidine is unfavored, (2) acyl substitution at the N3 position of the piperidine moiety is essential (Table 1), (3) a short chain length between the 2,5-dioxoimidazolidine and piperidine moieties is crucial for achieving high potency (18c, 18d, and 18e), and (4) hydrophobic and bulky aliphatic groups are preferred as acyl substituents on the piperidine. Compounds containing an adamantyl group (21i, 21k, 21l, and 21m) exhibited superior antagonistic effects compared to other derivatives. A representative optimized compound, 21w, exhibited not only low-nanomolar IC50 values in P2X7R antagonism and the inhibition of IL-1β release but also significant in vivo or ex vivo efficacy in animal models of inflammation, pain, and rheumatoid arthritis.

Figure 4. Anti-inflammatory effect of compound 21w (10 mg/kg, i.p.) on (A) time- and (B) dose-dependent increase in rat paw volume following carrageenin injection (0.1 mL/paw, 1% suspended in saline). Data represent mean ± SD (n = 4, significant as compared to control, *p < 0.05).

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volume of the 21w-treated group was significantly decreased compared to the control. Rats intraperitoneally injected with 10 mg/kg 21w also exhibited significant suppression of hind paw swelling 3−6 h after carrageenan injection (Figure 4B). We then assessed the antiarthritic effect of 21w in CIIinduced arthritis in DBA/1J mice. Histopathological RA grades were estimated by the severity of inflammation, fibrosis, damage to the articular cartilage and bone, and ankylosis in the ankle joints. Increased histological signs of damage were observed in the CII-induced arthritis group. However, these features were ameliorated by 21w treatment (Figure 5A). Injection of 21w (50 mg/kg) greatly suppressed the histological damage and decreased the cumulative arthritis injury scores compared with the vehicle-treated CII-RA group (Table 8). To verify whether 21w inhibits the production of proinflammatory mediators, we further determined the COX-2 and iNOS protein levels in ankle tissues. Compound 21w (25 and 50 mg/kg) reduced the protein expression of COX-2 and iNOS in the RA tissues (Figure 5B). These results suggest that the novel P2X7R antagonists could be utilized in anti-inflammatory treatment. In Vivo PK Study of Compound 21w. Because we determined in vivo antiarthritis effects of compound 21w by using

EXPERIMENTAL SECTION

Chemistry. 1H and 13C NMR spectra were determined with a JEOL JNM-LA 300WB spectrometer at 300 MHz or JEOL JNM-ECX 400P spectrometer at 400 MHz, and spectra were taken in CDCl3, DMSO-d6 or CD3OD. Unless otherwise noted, chemical shifts (δ) are expressed as ppm downfield from internal tetramethylsilane. Optical rotations were obtained on a ATAGO AP-100 polarimeter. Specific rotations ([α]D25) are reported in deg/dm, and the concentration (c) is given as g/100 mL in the specified solvent. Mass spectroscopy was carried out on electrospray, and high-resolution mass spectra (m/z) were recorded on a FAB. High-resolution mass analysis was performed at Korea Basic Science Institute (Daegu). The purity of all final products was determined by HPLC (at least 95% purity unless otherwise noted, Table S1 of the Supporting Information). The determination of purity was performed on a Shimadzu SCL-10A VP HPLC system using a Shimadzu Shim-pack C18 analytical column (250 × 4.6 mm, 5 mm, 100 Å) in linear gradient solvent systems. Solvent system was H2O:CH3CN = 60:40 to 10:90 over 30 min at a flow rate = 1 mL/min. Peaks were detected at 254 nm. Synthesized starting materials were prepared according to the Supporting Information. The other starting materials were purchased from Aldrich or Fluka. 5 (AZD9056) was synthesized according to the report from AstraZeneca.56 (L)-5-(4-Hydroxybenzyl)imidazolidine-2,4-dione (13). O-Benzyl(L)-tyrosine (12) (400 mg, 1.47 mmol) and potassium cyanate (299 mg, 2123

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Figure 5. Effects of compound 21w on the CII-induced arthritis (CIA). (A) H&E staining results. Control (salline/vehicle) group: arrow heads indicate intact articular cartilage. CII-treated (CIA/vehicle) group: arrow heads indicate severe loss of articular cartilage with subchondral bone destruction; CII with 50 mg/kg 21w-treated group: arrow heads indicate intact articular cartilage. Chronic inflammation and bone damage were not found in this sample. (B) Effect of compound 21w on the expression of COX-2 and iNOS in the ankle tissues. Each lane was loaded with 20 μg of the tissue lysates. The results were confirmed by two repeated experiments. The solution was kept at 120 °C for 1 h 30 min. The precipitate product was filtered and washed with cold water and dried under reduced pressure to give 1351(164 mg, 54.1% yield). 1H NMR (DMSO-d6, 300 MHz): δ 10.36 (1H, s), 9.23 (1H, s), 7.85 (1H, s), 6.93 (2H, d, J = 8.8 Hz), 6.61 (2H, d, J = 8.8 Hz), 4.21 (1H, t, J = 4.8 Hz), 2.76 (2H, d, J = 4.4 Hz); MS (ESI): [M]+ = 207.1. (L)-4-((2,5-Dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5sulfonate (14). To a suspension of NaH (87.3 mg of 60% oil suspension, 2.18 mmol) in dried THF (15 mL) was added 13 (150 mg, 0.73 mmol). After 10 min, the isoquinoline-5-sulfonyl chloride·HCl (480 mg, 1.82 mmol) in dried DCM was added. This mixture was stirred for 5 h at room temperature and then concentrated under vacuum. The residue was taken up in solution of saturated aq. NH4Cl and extracted with ethyl acetate. The combined extracts were dried over sodium sulfate, filtered, and evaporated. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 60:1) to give 14 (232 mg, 80.2% yield). 1H NMR (DMSO-d6, 400 MHz): δ 10.41(1H, br s), 9.56 (1H, s), 8.82 (1H, d, J = 6.0 Hz), 8.59 (1H, d, J = 8.4 Hz), 8.37 (1H, d, J = 6.0 Hz), 8.27 (1H, d, J = 8 Hz), 7.81−7.77 (2H, m), 7.08 (2H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.24 (1H, t, J = 4.8 Hz), 2.86 (1H, dd, J = 14.4, 4.8 Hz), 2.80 (1H, dd, J = 14.4, 5.2 Hz); HRMS (FAB) [M + H]+ (C19H16N3O5S): calcd. 398.0811, found 398.0814; purity 97.3%. (L)-4-((1-(Isoquinolin-5-ylmethyl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (15a). To a solution of 14 (95 mg, 0.24 mmol) in dried DMF (10 mL) in the presence of CsHCO3 (185 mg, 0.96 mmol) was added isoquinoline-5-ylmethyl methanesulfonate (114 mg, 0.48 mmol). The mixture was stirred overnight at 50 °C and then concentrated under vacuum. The residue was taken up in solution of saturated aq. NH4Cl and extracted with

Table 8. Effect of Compound 21w Treatments on Pathological Indices in CII-Induced RAa group

chronic inflammation

abcess formation

fibrosis

periarticular erosion

control CII +21w

0 3 0.25 ± 0.50*

0 2.67 ± 0.58 0.25 ± 0.50*

0 2.67 ± 0.58 0.50 ± 0.58*

0 2.67 ± 0.58 0*

Data represent the means ± SD of 3−4 samples (significant as compared to CII-treated group, *p < 0.05). All scores were semiquantitatively indexed based on a scale of 0−3 (0: negative, 1+: mild, 2+: moderate, 3+: severe).

a

Table 9. Pharmacokinetic Parameters of Compound 21w after i.p. Administration (10 mg/kg) and i.v. Administration (20 mg/kg) to Rats (n = 3) parameters

I.P. (n = 3)

I.V. (n = 3)

AUClast (μg min/mL) Cmax (ng/mL) t1/2 (min) F (%)

91.0 ± 45.8 400 ± 200 129 ± 41.0 86.0

212 ± 45.2 77.2 ± 8.8

3.69 mmol) were added to a solution of water (15 mL) and pyridine (15 mL). The mixture was heated to 90 °C for 3 h, cooled to room temperature, and pyridine was washed out with chloroform (4 × 30 mL). Glacial acetic acid (15 mL) and 6 N HCl (15 mL) were added to the aqueous phase, and the solution was refluxed for 1.5 h. 2124

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d, J = 8.4 Hz), 4.19 (1H, d, J = 13.2 Hz), 3.95 (1H, d, J = 13.2 Hz), 3.65 (3H, s), 3.50 (1H, dd, J = 7.6, 6.0 Hz), 2.91 (1H, dd, J = 14.0, 6.0 Hz), 2.83 (1H, dd, J = 14.0, 7.6 Hz). MS (ESI): [M]+ = 528.0. (±)-Methyl 2-((1-benzylpiperidin-4-yl)methylamino)-3-(4-(isoquinolin5-ylsulfonyloxy)phenyl)propanoate (17b). It was synthesized from 16 and 1-benzylpiperidine-4-carbaldehyde following the procedure described for 17a. Yield 62.7%. 1H NMR (CDCl3, 400 MHz): δ 9.39 (1H, s), 8.81 (1H, d, J = 6.4 Hz), 8.55 (1H, d, J = 6.4 Hz), 8.25− 8.21 (2H, m), 7.61 (1H, t, J = 7.6 Hz), 7.32−7.27 (5H, m), 7.01 (2H, d, J = 8.8 Hz), 6.77 (2H, d, J = 8.4 Hz), 3.57 (3H, s), 3.46 (2H, s), 3.34 (1H, t, J = 6.8 Hz), 2.84−2.79 (4H, m), 2.43 (1H, dd, J = 11.6, 6.8 Hz), 2.24 (1H, dd, J = 11.6, 6.8 Hz), 1.90−1.10 (7H, m). MS (ESI): [M]+ = 574.0. (±)-tert-Butyl 4-((3-(4-(isoquinolin-5-ylsulfonyloxy)phenyl)-1methoxy-1-oxopropan-2-ylamino)methyl)piperidine-1-carboxylate (17c). It was synthesized from 16 and tert-butyl 4-formylpiperidine-1carboxylate following the procedure described for 17a. Yield 65.7%. 1H NMR (CDCl3, 400 MHz): δ 9.42 (1H, s), 8.81 (1H, d, J = 6.0 Hz), 8.55 (1H, d, J = 6.0 Hz), 8.29−8.26 (2H, m), 7.66 (1H, t, J = 7.6 Hz), 7.02 (2H, d, J = 8.8 Hz), 6.79 (2H, d, J = 8.8 Hz), 4.04 (2H, m), 3.58 (3H, s), 3.42 (1H, t, J = 6.8 Hz), 2.83 (2H, m), 2.62 (2H, m), 2.44 (1H, dd, J = 11.2, 6.8 Hz), 2.24 (1H, dd, J = 11.2, 6.8 Hz), 1.75−1.08 (3H, m), 1.43 (9H, s), 0.99 (2H, m). MS (ESI): [M]+ = 584.2. (±)-tert-Butyl 4-(2-(3-(4-(isoquinolin-5-ylsulfonyloxy)phenyl)-1methoxy-1-oxopropan-2-ylamino)ethyl)piperidine-1-carboxylate (17d). It was synthesized from 16 and tert-butyl 4-(2-oxoethyl)piperidine-1-carboxylate following the procedure described for 17a. Yield 66.7%. 1H NMR (CDCl3, 400 MHz): δ 9.42 (1H, s), 8.81 (1H, d, J = 6.0 Hz), 8.54 (1H, d, J = 6.0 Hz), 8.29−8.27 (2H, m), 7.66 (1H, t, J = 7.6 Hz), 7.02 (2H, d, J = 8.4 Hz), 6.79 (2H, d, J = 8.4 Hz), 4.09− 3.94 (2H, m), 3.57 (3H, s), 3.40−3.37 (1H, m), 2.83−2.81 (2H, m), 2.83−2.81 (2H, m), 2.59−2.55 (3H, m), 2.42−2.41 (2H, m), 1.42 (9H, s), 1.39−1.30 (3H, m), 1.07 (2H, q, J = 11.6 Hz). MS (ESI): [M]+ = 584.0. (±)-tert-Butyl 4-(3-(4-(isoquinolin-5-ylsulfonyloxy)phenyl)-1methoxy-1-oxopropan-2-ylamino)piperidine-1-carboxylate (17e). It was synthesized from 16 and tert-butyl 4-oxopiperidine-1carboxylate following the procedure described for 17a. Yield 74.5%. 1 H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.82 (1H, d, J = 6.4 Hz), 8.56 (1H, d, J = 6.0 Hz), 8.29−8.24 (2H, m), 7.66 (1H, t, J = 7.6 Hz), 7.01 (2H, d, J = 8.4 Hz), 6.77 (2H, d, J = 8.4 Hz), 3.85 (2H, m), 3.59 (3H, s), 3.47 (2H, t, J = 7.2 Hz), 2.82 (4H, m), 2.47 (1H, m), 1.59 (4H, m), 1.43 (9H, s). MS (ESI): [M]+ = 569.9. (±)-4-((3-(Isoquinolin-5-ylmethyl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (18a). To a solution of 17a (300 mg, 0.57 mmol) in acetic acid (15 mL) was added potassium cyanate (231 mg, 2.84 mmol), and the resulting mixture was allowed to stir for 5 h at room temperature. The crude reaction was concentrated under vacuum with azeotropic removal of acetic acid with toluene, and the residue was diluted with saturated aq. NaHCO3 (20 mL). The aqueous mixture was extracted with ethyl acetate (3 × 20 mL), and the combined organic extracts were dried over sodium sulfate and concentrated under vacuum. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 35:1) to give 18a (234 mg, 76.3% yield). 1H NMR (CDCl3, 400 MHz): δ 9.42 (1H, s), 9.29 (1H, s), 8.81 (1H, d, J = 6.0 Hz), 8.58 (1H, d, J = 6.0 Hz), 8.53 (1H, d, J = 6.0 Hz), 8.30−8.27 (2H, m), 8.00 (1H, d, J = 8.4 Hz), 7.81 (1H, d, J = 6.0 Hz), 7.67 (1H, t, J = 8.0 Hz), 7.56 (1H, t, J = 8.0 Hz), 7.36 (1H, d, J = 7.2 Hz), 6.96 (2H, d, J = 8.8 Hz), 6.80 (2H, d, J = 8.4 Hz), 5.48 (1H, d, J = 14.8 Hz), 4.37 (1H, d, J = 15.2 Hz), 3.78 (1H, t, J = 4.8 Hz), 3.05 (2H, d, J = 4.8 Hz). 13C NMR (CDCl3) δ 195.8, 175.3, 172.3, 166.6, 162.9, 160.9, 149.8, 148.4, 147.8, 145.3, 144.3, 143.5, 142.08, 141.6, 139.0, 138.0, 133.6, 127.6, 125.9, 56.4, 33.6, 23.8. HRMS (FAB) [M + H]+ (C29H23N4O5S): calcd. 539.1389, found 539.1391; purity 99.3%. (±)-4-((3-((1-Benzylpiperidin-4-yl)methyl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (18b). It was synthesized from 17e following the procedure described for 18a. Yield 74.7%. 1H NMR (CDCl3, 400 MHz) δ 9.39 (1H, s), 8.79 (1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 5.6 Hz), 8.27 (1H, d, J = 8.4 Hz), 8.24 (1H, d, J = 7.2 Hz), 7.65 (1H, t, J = 8.0 Hz), 7.32−7.22 (5H, m), 6.98 (2H, d, J = 8.4 Hz), 6.78

ethyl acetate. The combined extracts were dried over sodium sulfate, filtered, and evaporated. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 75:1) to give 15a (56.3 mg, 43.7% yield). 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 9.23 (1H, s), 8.83 (1H, d, J = 6.0 Hz), 8.56−8.52 (2H, m), 8.29 (1H, d, J = 8.0 Hz), 8.23 (1H, d, J = 7.2 Hz), 8.06 (1H, d, J = 6.0 Hz), 7.92 (1H, d, J = 8.0 Hz), 7.65 (1H, t, J = 8.0 Hz), 7.59 (1H, d, J = 7.2 Hz), 7.52 (1H, t, J = 7.6 Hz), 6.95 (2H, d, J = 8.8 Hz), 6.65 (2H, d, J = 8.4 Hz), 5.50 (1H, s), 5.00 (1H, d, J = 14.8 Hz), 4.95 (1H, d, J = 14.8 Hz), 4.23 (1H, dd, J = 7.6, 4.0 Hz), 3.11 (1H, dd, J = 14.4, 4.0 Hz), 2.83 (1H, dd, J = 14.4, 7.6 Hz). 13C NMR (CDCl3): δ 199.2, 177.6, 175.9, 170.8, 164.6, 152.4, 150.4, 149.8, 144.4, 144.1, 139.2, 138.0, 135.2, 133.6, 129.2, 126.7, 122.2, 58.4, 29.9, 25.5. HRMS (FAB) [M + H]+ (C29H23N4O5S): calcd. 539.1389, found 539.1393; purity 95.3%. (L)-tert-Butyl 4-((4-(4-(isoquinolin-5-ylsulfonyloxy)benzyl)-2,5dioxoimidazolidin-1-yl)methyl)piperidine-1-carboxylate (15b). It was synthesized from 14 following the procedure described for 15a. Yield 80.6%. 1H NMR (CDCl3, 400 MHz): δ 9.42 (1H, s), 8.82 (1H, d, J = 6.0 Hz), 8.54 (1H, d, J = 6.0 Hz), 8.31−8.28 (2H, m), 8.30 (1H, d, J = 8.0 Hz), 7.68 (1H, t, J = 8.0 Hz), 7.07 (2H, d, J = 8.4 Hz), 6.86 (2H, d, J = 8.4 Hz), 5.25 (NH, s), 4.20 (1H, dd, J = 8.0, 4.0 Hz), 4.04− 4.02 (2H, m), 3.32 (1H, dd, J = 13.2, 7.2 Hz), 3.27 (1H, dd, J = 13.2, 7.2 Hz), 3.17 (1H, dd, J = 14.0, 4.0 Hz), 2.85 (1H, dd, J = 14.0, 8.0 Hz), 2.58−2.53 (2H, m), 1.76−1.68 (1H, m), 1.43 (9H, s), 1.35−1.24 (2H, m), 1.10−1.00 (2H, m). 13C NMR (CDCl3) δ 195.6, 176.72, 174.0, 172.4, 166.6, 163.3, 149.8, 149.3, 145.3, 144.3, 143.7, 138.0, 133.6, 127.5, 80.0, 52.9, 35.4, 32.0, 27.0, 24.3, 17.8, 16.3. HRMS (FAB) [M + H]+ (C30H35N4O7S): calcd. 595.2226, found 595.2223; purity 96.9%. (L)-Methyl 2-amino-3-(4-(isoquinolin-5-ylsulfonyloxy)phenyl)propanoate (16). Compound 16 was obtained through a two-step process. First, to a suspension of NaH (406 mg of 60% oil suspension, 10.2 mmol) in dried THF (50 mL) was added Boc-(L)-tyrosine methyl ester (1 g, 3.39 mmol). After 10 min, the isoquinolinesulfonyl chloride·HCl (2.24 g, 8.47 mmol) dissolved in dried DCM was added. This mixture was stirred for 5 h at room temperature and then concentrated under vacuum. The residue was taken up in solution of saturated aq. NH4Cl and extracted with ethyl acetate. The combined extracts were dried over sodium sulfate, filtered, and evaporated. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 80:1). Next, to a 0.08 M solution of protected amide product (1.5 g, 3.08 mmol) in dichloromethane at 0 °C was added TFA (8 mL), and the resulting mixture was allowed to stir for 3 h at room temperature. The crude reaction was concentrated under vacuum with azeotropic removal of TFA with hexane, and the residue was diluted with saturated aq. NaHCO3 (50 mL). The aqueous mixture was extracted with dichloromethane (3 × 50 mL), and the combined organic extracts were dried over sodium sulfate and concentrated under vacuum. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 60:1) to give 16 (977 mg, 62.7% two-step overall yield). 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.81 (1H, d, J = 6.0 Hz), 8.54 (1H, d, J = 6.0 Hz), 8.29−8.27 (2H, m), 7.66 (1H, t, J = 7.6 Hz), 7.04 (2H, d, J = 8.8 Hz), 6.81 (2H, d, J = 8.8 Hz), 3.65 (3H, s), 3.64 (1H, dd, J = 7.6, 5.2 Hz), 2.98 (1H, dd, J = 13.6, 5.2 Hz), 2.79 (1H, dd, J = 13.6, 7.6 Hz). MS (ESI): [M]+ = 387.0. (±)-Methyl 2-(isoquinolin-5-ylmethylamino)-3-(4-(isoquinolin-5ylsulfonyloxy)phenyl)propanoate (17a). A solution of 16 (550 mg, 1.42 mmol) and isoquinoline-5-carboxaldehyde (224 mg, 1.42 mmol) in dried DCE (30 mL) was treated with sodium triacetoxyborohydride (754 mg, 3.56 mmol). After 4 h, the resulting mixture was treated with 10% aq potassium hydroxide (40 mL). The organic phase was extracted with dichloromethane (3 × 20 mL), and combined organic phases were dried over sodium sulfate and concentrated under vacuum. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 40:1) to give 17a (602 mg, 80.2% yield). 1H NMR (CDCl3, 400 MHz): δ 9.40 (1H, s), 9.23 (1H, s), 8.81 (1H, d, J = 6.4 Hz), 8.55 (1H, d, J = 6.4 Hz), 8.47 (1H, d, J = 6.0 Hz), 8.28−8.25 (2H, m), 7.88 (1H, d, J = 7.6 Hz), 7.77 (1H, d, J = 6.0 Hz), 7.63 (1H, t, J = 7.6 Hz), 7.52−7.25 (2H, m), 7.00 (2H, d, J = 8.0 Hz), 6.77 (2H, 2125

DOI: 10.1021/jm500324g J. Med. Chem. 2015, 58, 2114−2134

Article

Journal of Medicinal Chemistry (2H, d, J = 8.8 Hz), 4.20 (1H, t, J = 4.4 Hz), 3.57−3.50 (3H, m), 3.06 (2H, d, J = 4.4 Hz), 2.88 (2H, d, J = 10.4 Hz), 2.73 (1H, dd, J = 13.6, 6.0 Hz), 1.93−1.91 (4H, m), 1.50−1.48 (2H, m), 1.27−1.24 (2H, m). 13 C NMR (CDCl3): δ 195.9, 175.1, 172.3, 166.6, 163.0, 153.1, 149.7, 149.5, 147.8, 145.3, 144.1, 143.5, 142.2, 141.6, 141.0, 139.6, 138.0, 133.5, 127.6, 59.5, 57.7, 46.8, 38.3, 23.8, 23.2, 18.1, 17.7. HRMS (FAB) [M + H]+ (C32H33N4O5S): calcd. 585.2172, found 585.2174; purity 95.1%. (±)-tert-Butyl 4-((5-(4-(isoquinolin-5-ylsulfonyloxy)benzyl)-2,4dioxoimidazolidin-1-yl)methyl)piperidine-1-carboxylate (18c). It was synthesized from 17c following the procedure described for 18a. Yield 82.1%. 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.0 Hz), 8.53 (1H, d, J = 6.8 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.26 (1H, d, J = 7.6 Hz), 7.66 (1H, t, J = 8.0 Hz), 7.04 (5H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.20−4.18 (2H, m), 4.09−4.06 (2H, m), 3.56 (1H, dd, J = 14.4, 9.2 Hz), 3.08−3.06 (2H, d, J = 4.8 Hz), 2.75 (1H, dd, J = 14.0, 5.6 Hz), 2.64−2.59 (2H, m), 1.88−1.64 (2H, m), 1.42 (9H, s), 1.13 (2H, qd, J = 12.4, 4.4 Hz). 13C NMR (CDCl3): δ 172.1, 155.7, 154.6, 153.2, 148.6, 145.7, 135.2, 135.0, 133.6, 131.6, 130.7, 130.1, 128.6, 125.8, 122.2, 117.5, 79.6, 61.4, 45.9, 34.7, 34.0, 29.4, 28.4. HRMS (FAB) [M + H]+ (C30H35N4O7S): calcd. 595.2226, found 595.2223; purity 97.0%. (±)-tert-Butyl 4-(2-(5-(4-(isoquinolin-5-ylsulfonyloxy)benzyl)-2,4dioxoimidazolidin-1-yl)ethyl)piperidine-1-carboxylate (18d). It was synthesized from 17d following the procedure described for 18a. Yield 75.8%. 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 6.0 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.26 (1H, d, J = 7.6 Hz), 7.67 (2H, t, J = 7.6 Hz), 7.00 (2H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.20−4.17 (1H, m), 4.13−4.01 (2H, m), 3.74− 3.69 (1H, m) 3.06−3.04 (2H, m), 2.90−2.83 (1H, m), 2.66−2.58 (2H, m), 1.66−1.54 (4H, m), 1.43 (9H, s), 1.26 (1H, t, J = 7.2 Hz), 1.12− 0.98 (2H, m). 13C NMR (CDCl3): δ 195.9, 175.0, 174.2, 172.3, 166.5, 163.0, 146.8, 149.5, 147.9, 145.3, 144.1, 143.6, 141.6, 138.0, 133.5, 127.6, 80.0, 56.4, 28.4, 23.4, 23.0, 22.6, 20.8, 20.2, 16.3. HRMS (FAB) [M + H]+ (C31H37N4O7S): calcd. 609.2383, found 609.2381; purity 99.4%. (±)-tert-Butyl 4-(5-(4-(isoquinolin-5-ylsulfonyloxy)benzyl)-2,4dioxoimidazolidin-1-yl)piperidine-1-carboxylate (18e). It was synthesized from 17b following the procedure described for 18a. Yield 80.9%. 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.0 Hz), 8.51 (1H, d, J = 6.0 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.26 (1H, d, J = 7.6 Hz), 7.66 (1H, t, J = 7.6 Hz), 7.00 (2H, d, J = 8.4 Hz), 6.79 (2H, d, J = 8.4 Hz), 4.19 (1H, t, J = 4.4 Hz), 3.74 (1H, m), 3.05 (2H, m), 2.89 (1H, m), 2.62 (2H, m), 1.43 (9H, s), 1.26 (2H, t, J = 7.2 Hz), 1.10 (2H, m). 13C NMR (CDCl3): δ 199.7, 174.1, 172.4, 165.9, 163.1, 152.0, 149.6, 149.4, 145.4, 143.8, 141.7, 137.9, 132.9, 127.7, 80.1, 55.2, 46.9, 45.5, 30.0, 20.0, 16.3. HRMS (FAB) [M + H]+ (C29H33N4O7S): calcd. 581.2070, found 581.2072; purity 97.0%. (±)-tert-Butyl 4-((3-(isoquinolin-5-ylmethyl)-4-(4-(isoquinolin-5ylsulfonyloxy)benzyl)-2,5-dioxoimidazolidin-1-yl)methyl)piperidine1-carboxylate (19a). To a solution of 18a (100 mg, 0.19 mmol) in dried DMF in the presence of CsHCO3 (103 mg, 0.74 mmol) was added 1-boc-4-piperidinylmethyl methanesulfonate (109 mg, 0.37 mmol). The mixture was stirred overnight at 50 °C and then concentrated under vacuum. The residue was taken up in solution of saturated aq NH4Cl and extracted with ethyl acetate. The combined extracts were dried over sodium sulfate, filtered, and evaporated. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 75:1) to give 19a (96.6 mg, 70.7% yield). 1H NMR (CDCl3, 400 MHz): δ 9.43 (1H, s), 9.29 (1H, s), 8.83 (1H, d, J = 6.0 Hz), 8.55 (2H, t, J = 6.4 Hz), 8.31−8.28 (2H, m), 8.00 (1H, d, J = 8.4 Hz), 7.80 (1H, d, J = 6.0 Hz), 7.67 (1H, t, J = 8.0 Hz), 7.56 (1H, t, J = 8.0 Hz), 7.37 (1H, d, J = 6.8 Hz), 6.96 (2H, d, J = 8.8 Hz), 6.82 (2H, d, J = 8.4 Hz), 5.57 (1H, d, J = 15.2 Hz), 4.41 (1H, d, J = 15.2 Hz), 3.98−3.96 (2H, m), 3.68 (1H, t, J = 4.8 Hz), 3.25 (1H, dd, J = 14.0, 7.6 Hz), 3.20 (1H, dd, J = 14.0, 7.6 Hz), 3.08 (1H, dd, J = 14.8, 4.8 Hz), 3.03 (1H, dd, J = 14.8, 4.8 Hz), 2.55 (2H, q, J = 13.2 Hz), 1.44 (9H, s), 1.30−0.91 (3H, m). 13C NMR (CDCl3): δ 195.1, 176.1, 174.0, 172.5, 166.7, 163.3, 161.1, 149.8, 149.2, 148.3, 147.6, 145.4, 144.6, 143.8, 143.3, 142.1, 139.0, 137.9, 133.4, 127.5, 125.8,

80.1, 54.5, 35.9, 34.0, 24.2, 23.4, 17.4, 16.3. HRMS (FAB) [M + H]+ (C40H42N5O7S): calcd. 736.2805, found 736.2807; purity 98.1%. (±)-4-((3-((1-Benzylpiperidin-4-yl)methyl)-1-(isoquinolin-5-ylmethyl)2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (19b). It was synthesized from 18e and isoquinolin-5-ylmethyl methanesulfonate following the procedure described for 19a. Yield 59.1%. 1H NMR (CDCl3, 400 MHz): δ 10.32 (NH, s), 9.41 (1H, s), 9.20 (1H, s), 8.85 (1H, d, J = 6.0 Hz), 8.50 (2H, d, J = 6.0 Hz), 8.28 (1H, d, J = 8.4 Hz), 8.16 (1H, d, J = 7.2 Hz), 7.97 (1H, d, J = 6.0 Hz), 7.87 (1H, d, J = 7.6 Hz), 7.62 (1H, t, J = 8.0 Hz), 7.52 (2H, m), 7.44 (5H, m), 6.71 (2H, d, J = 8.0 Hz), 6.35 (2H, d, J = 8.4 Hz), 4.83 (2H, qd, J = 14.8 Hz), 4.20 (1H, t, J = 4.0 Hz), 3.69 (4H, m), 3.01 (4H, m), 1.97 (2H, m), 1.76 (4H, m), 1.25 (2H, m). 13C NMR (CDCl3): δ 195.9, 192.2, 185.9, 177.8, 176.4, 174.8, 172.5, 163.3, 153.6, 151.7, 149.6, 149.2, 146.8, 145.7, 144.9, 144.0, 143.4, 142.2, 141.1, 138.9, 137.9, 137.2, 136.4, 132.9, 129.2, 60.6, 57.4, 50.6, 48.0, 46.5, 42.0, 37.8, 34.1. HRMS (FAB) [M + H]+ (C42H40N5O5S): calcd. 726.2750, found 726.2753; purity 97.4%. (±)-tert-Butyl 4-((3-(Isoquinolin-5-ylmethyl)-5-(4-(isoquinolin-5ylsulfonyloxy)benzyl)-2,4-dioxoimidazolidin-1-yl)methyl)piperidine1-carboxylate (19c). It was synthesized from 18c and isoquinolin-5ylmethyl methanesulfonate following the procedure described for 19a. Yield 68.3%; 1H NMR (CDCl3, 400 MHz): δ 9.42 (1H, s), 9.02 (1H, s), 8.87 (1H, d, J = 6.0 Hz), 8.52 (2H, d, J = 6.0 Hz), 8.27 (1H, d, J = 8.4 Hz), 8.16 (1H, d, J = 7.6 Hz), 7.99 (1H, d, J = 5.6 Hz), 7.87 (1H, d, J = 7.6 Hz), 7.62 (1H, t, J = 8.0 Hz), 7.44 (2H, m), 6.72 (2H, d, J = 8.4 Hz), 6.36 (2H, d, J = 8.4 Hz), 4.91 (2H, qd, J = 14.8 Hz), 4.15 (2H, m), 3.63 (1H, m), 3.00 (2H, m), 2.81 (1H, d, J = 5.6 Hz), 2.62 (2H, m), 1.70 (4H, m), 1.43 (9H, s), 1.15 (2H, qd, J = 12.4 Hz). 13C NMR (CDCl3): δ 195.0, 175.7, 174.0, 172.4, 172.1, 166.1, 163.2, 160.1, 149.6, 149.2, 148.0, 146.7, 145.8, 145.2, 143.9, 143.6, 143.3, 141.6, 141.2, 138.9, 137.9, 132.9, 127.6, 126.3, 80.3, 55.8, 38.5, 29.7, 24.4, 22.7, 18.0, 17.6, 16.2. HRMS (FAB) [M + H]+ (C40H42N5O7S): calcd. 736.2805, found 736.2803; purity 95.9%. (±)-4-((2,5-Dioxo-3-(piperidin-4-yl)imidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (20a). To a 0.08 M solution of 18e (1g, 1.72 mmol) in dichloromethane at 0 °C was added TFA (5 mL), and the resulting mixture was allowed to stir at 0 °C for 1 h. The crude reaction was concentrated under vacuum with azeotropic removal of TFA with hexane, and the residue was diluted with saturated aq. NaHCO3 (50 mL). The aqueous mixture was extracted with dichloromethane (3 × 50 mL), and the combined organic extracts were dried over sodium sulfate and concentrated under vacuum. The residue was purified by flash column chromatography (silica gel, CHCl3:MeOH:ammonia = 95:4.5:0.5) to give 20a (705 mg, 85.2% yield): 1H NMR (DMSO-d6, 400 MHz) δ 9.56 (1H, s), 8.82 (1H, d, J = 6.0 Hz), 8.59 (1H, d, J = 8.0 Hz), 8.36 (1H, d, J = 6.0 Hz), 8.25 (1H, d, J = 7.6 Hz), 7.80 (1H, t, J = 8.0 Hz), 7.08 (2H, d, J = 7.6 Hz), 6.79 (2H, d, J = 7.2 Hz), 4.34−4.32 (1H, m), 3.13 (1H, s), 3.07−3.00 (1H, m), 2.98 (2H, dd, J = 15.2, 4.8 Hz), 2.91−2.81 (2H, m), 2.26− 2.14 (2H, m), 1.86−1.77 (1H, q, J = 12.0 Hz), 1.59−1.55 (2H, m), 1.37−1.34 (1H, m). 13C NMR (CDCl3): δ 173.6, 156.3, 153.9, 147.6, 145.9, 136.2, 135.3, 131.3, 130.6, 128.7, 128.4, 126.5, 121.4, 116.5, 72.3, 60.2, 29.0, 22.1, 19.4. HRMS (FAB) [M + H]+ (C24H25N4O5S): calcd. 481.1546, found 481.1543; purity 95.9%. (±)-4-((2,5-Dioxo-3-(piperidin-4-ylmethyl)imidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (20b). It was synthesized from 18c following the procedure described for 20a. Yield 83.7%. 1H NMR (DMSO-d6, 400 MHz): δ 9.56 (1H, s), 8.82 (1H, d, J = 6.0 Hz), 8.60 (1H, d, J = 8.0 Hz), 8.35 (1H, d, J = 6.0 Hz), 8.26 (1H, d, J = 7.6 Hz), 7.81 (1H, t, J = 8.0 Hz), 7.06 (2H, d, J = 7.6 Hz), 6.79 (1H, d, J = 7.2 Hz), 4.19−4.16 (1H, m), 3.56−3.49 (1H, m), 3.07−3.00 (3H, m), 2.74 (1H, dd, J = 14.8, 5.2 Hz), 2.55−2.48 (2H, m), 1.97−1.94 (1H, m), 1.53−1.38 (4H, m), 1.18−1.07 (2H, m). MS (ESI): [M]+ = 494.7. (±)-4-((2,5-Dioxo-3-(2-(piperidin-4-yl)ethyl)imidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (20c). It was synthesized from 18d following the procedure described for 20a. Yield 82.5%. 1H NMR (DMSO-d6, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 6.0 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.26 (1H, d, J = 7.1 Hz), 7.67 (2H, t, J = 7.6 Hz), 7.00 (2H, d, J = 8.4 Hz), 6.80 (2H, d, 2126

DOI: 10.1021/jm500324g J. Med. Chem. 2015, 58, 2114−2134

Article

Journal of Medicinal Chemistry J = 8.4 Hz), 4.195 (1H, t, J = 4.4 Hz), 4.13 (2H, m), 3.74 (1H, m), 3.05 (2H, s), 2.90 (1H, m), 2.62 (2H, m), 1.66 (4H, m), 1.43 (9H, s), 1.31 (1H, t, J = 7.2 Hz), 1.10 (2H, m); MS (ESI): [M]+ = 508.8. (±)-4-((3-(1-Benzylpiperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21a). A solution of 20a (50.0 mg, 0.10 mmol) and benzaldehyde (12.7 μL, 0.12 mmol) in dried DMF (7 mL) was treated with sodium triacetoxyborohydride (44.1 mg, 0.21 mmol). After 2 h, the resulting mixture was treated with 10% aq potassium hydroxide (10 mL). The organic phase was extracted with dichloromethane (3 × 10 mL), and combined organic phases were dried over sodium sulfate and concentrated under vacuum. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 40:1) to give 21a (41.8 mg, 70.4% yield). 1H NMR (CDCl3, 400 MHz): δ 9.39 (1H, s), 8.79 (1H, d, J = 6.4 Hz), 8.51 (1H, d, J = 6.0 Hz), 8.27 (1H, d, J = 8.4 Hz), 8.24 (1H, d, J = 7.2 Hz), 7.64 (H=1, t, J = 8.0 Hz), 7.32 (5H, m), 6.98 (2H, d, J = 8.4 Hz), 6.78 (2H, d, J = 8.8 Hz), 4.19 (1H, d, J = 4.4 Hz), 3.56 (1H, m), 3.05 (2H, d, J = 4.4 Hz), 2.87 (2H, d, J = 10.0 Hz), 2.72 (1H, dd, J = 13.6 Hz), 1.93 (3H, m), 1.50 (2H, m), 1.30 (2H, m). 13C NMR (CDCl3): δ 196.1, 175.7, 172.2, 166.5, 162.9, 149.7, 149.6, 147.8, 146.8, 145.4, 144.5, 143.6, 143.1, 142.5, 141.2, 140.1, 138.1, 133.2, 127.7, 58.6, 56.3, 46.3, 25.3, 18.7, 16.8. HRMS (FAB) [M + H]+ (C31H31N4O5S): calcd. 571.2016, found 571.2019; purity 96.2%. (±)-4-((3-(1-Benzoylpiperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21b). To a solution of 20a (50.0 mg, 0.10 mmol) and benzoic acid (25.4 mg, 0.21 mmol) in dried DCM (7 mL) was triethylamine (21.8 μL, 0.16 mmol) followed by EDC·HCl (29.9 mg, 0.16 mmol). The resulting mixture was allowed to stir for 2 h. The reaction mixture was diluted with ethyl acetate and washed with aq NaHCO3 (3 × 10 mL) and 1 N HCl (3 × 10 mL). The solution was dried over sodium sulfate, filtered, and evaporated. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 75:1) to give 21b (42.6 mg, 70.0% yield). 1H NMR (CDCl3, 400 MHz): δ 9.40 (1H, s), 8.79 (1H, d, J = 5.6 Hz), 8.52 (1H, d, J = 5.6 Hz), 8.27 (NH, s), 8.25 (2H, d, J = 8.4 Hz), 7.65 (1H, t, J = 8.0 Hz), 7.42−7.40 (5H, m), 7.04 (2H, d, J = 8.4 Hz), 6.81 (2H, d, J = 8.4 Hz), 4.81−4.79 (1H, m), 4.21−4.19 (1H, m), 3.84−3.81 (1H, m), 3.47−3.42 (1H, m), 3.15 (1H, qd, J = 14.8, 4.4 Hz), 2.95− 2.52 (2H, m), 1.89−1.49 (4H, m). 13C NMR (CDCl3): δ 195.7, 194.7, 175.4, 172.3, 166.6, 162.9, 150.0, 149.8, 149.4, 148.0, 145.3, 144.3, 143.6, 143.3, 141.4, 139.4, 138.1, 133.5, 127.6. HRMS (FAB) [M + H]+ (C31H29O6N4S): calcd. 585.1808, found 585.1810; purity 99.6%. (±)-4-((2,5-Dioxo-3-(1-propionylpiperidin-4-yl)imidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21c). To a solution of 20a (50 mg, 0.10 mmol) and triethylamine (0.10 mmol, 1 equiv) in dried DCM (7 mL) was added propinyl chloride (10.9 μL, 0.12 mmol). The mixture was stirred for 2 h at room temperature. The residue was taken up in solution of saturated aq NH4Cl and extracted with dichloromethane. The combined extracts were dried over sodium sulfate, filtered, and evaporated. The residue was purified by silica gel column chromatography (CHCl3:MeOH = 40:1) to give 21c (50.1 mg, 89.7% yield). 1H NMR (CDCl3, 400 MHz): δ 9.40 (1H, s), 8.79 (1H, d, J = 5.2 Hz), 8.56 (1H, s), 8.51 (1H, d, J = 6.0 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.24 (1H, d, J = 7.2 Hz), 7.66 (1H, t, J = 7.6 Hz), 7.03 (2H, d, J = 8.8 Hz), 6.80 (2H, d, J = 8.8 Hz), 4.75−4.69 (1H, m), 4.19 (1H, dd, J = 9.6, 5.2 Hz), 3.91−3.88 (1H, m), 3.47−3.41 (1H, m), 3.14−2.83 (3H, m), 2.41−2.38 (1H, m), 2.35 (2H, q, J = 7.6 Hz), 2.02−1.57 (4H, m), 1.13 (3H, t, J = 7.6 Hz). 13C NMR (CDCl3): δ 196.2, 196.0, 175.9, 172.3, 166.5, 162.9, 149.8, 149.5, 148.0, 145.3, 144.3, 143.5, 141.6, 138.1, 133.4, 127.6, 57.1, 46.6, 36.7, 32.2, 25.5, 19.0, 18.2, 13.8. HRMS (FAB) [M + H]+ (C27H29N4O6S): calcd. 537.1808, found 537.1808; purity 99.2%. (±)-4-((3-(1-Isobutyrylpiperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21d). It was synthesized from 20a and isobutyryl chloride following the procedure described for 21c. Yield 92.3%. 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.4 Hz), 8.52 (1H, d, J = 6.0 Hz), 8.30 (1H, d, J = 8.4 Hz), 8.25 (1H, d, J = 7.2 Hz), 8.06 (NH, s), 7.66 (1H, t, J = 8.0 Hz), 7.03 (2H, d, J = 8.8 Hz), 6.81 (2H, d, J = 8.4 Hz), 4.78−4.72 (1H, m), 4.18 (1H, s), 4.01−3.97 (1H, m), 3.46 (1H, s), 3.14 (1H, dd, J = 15.2,

4.8 Hz), 3.05−3.01 (1H, m), 2.97 (1H, q, J = 12.4 Hz), 2.79−2.73 (1H, m), 2.41−2.28 (1H, m), 2.00−1.69 (4H, m), 1.11 (6H, s). 13C NMR (CDCl3): δ 200.0, 195.8, 175.3, 172.3, 166.6, 163.0, 149.8, 149.4, 147.9, 145.3, 144.3, 143.5, 141.6, 138.1, 133.4, 133.4, 127.6, 57.0, 46.7, 36.6, 32.3, 25.4, 18.6, 5.1. HRMS (FAB) [M + H]+ (C28H31N4O6S): calcd. 551.1964, found 551.1963; purity 98.1%. (±)-4-((2,5-Dioxo-3-(1-pivaloylpiperidin-4-yl)imidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21e). It was synthesized from 20a and pivaloyl chloride following the procedure described for 21c. Yield 94.6%. 1H NMR (CDCl3, 400 MHz): δ 9.40 (1H, s), 8.79 (1H, d, J = 6.0 Hz), 8.51 (1H, d, J = 6.4 Hz), 8.29 (2H, d, J = 8.0 Hz), 8.25 (2H, d, J = 7.2 Hz), 7.67 (1H, t, J = 8.0 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.54−4.46 (2H, m), 4.19 (1H, t, J = 4.4 Hz), 3.53−3.47 (1H, m), 3.15 (2H, qd, J = 10.0 Hz), 2.66 (2H, bt, J = 12.0 Hz), 1.98 (1H, qd, J = 12.0, 3.2 Hz), 1.80−1.63 (3H, m), 1.25 (9H, s). 13C NMR (CDCl3): δ 201.1, 196.2, 175.8, 172.4, 166.4, 163.1, 149.8, 149.4, 148.2, 145.2, 144.3, 143.4, 141.6, 138.1, 133.3, 127.5, 56.7, 46.5, 36.6, 36.3, 29.1, 25.5, 18.6, 18.0, 16.2. HRMS (FAB) [M + H]+ (C29H33N4O6S): calcd. 565.2121, found 565.2125; purity 96.6%. (±)-4-((3-(1-Butyrylpiperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21f). It was synthesized from 20a and butyryl chloride following the procedure described for 21c. Yield 83.0%. 1H NMR (CDCl3, 400 MHz): δ 9.40 (1H, s), 8.78 (1H, d, J = 6.0 Hz), 8.51 (1H, d, J = 6.0 Hz), 8.45 (NH, s), 8.29 (1H, d, J = 8.0 Hz), 8.25 (1H, d, J = 7.2 Hz), 7.66 (1H, t, J = 8.0 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.76−4.69 (1H, m), 4.18−4.17 (1H, m), 3.92−3.89 (1H, m), 3.47−3.44 (1H, m), 3.13−3.01 (2H, m), 2.94 (1H, q, J = 13.6 Hz), 2.40 (1H, q, J = 13.2 Hz), 2.30 (2H, t, J = 7.6 Hz), 2.00−1.58 (6H, m), 0.96 (3H, t, J = 7.2 Hz). 13C NMR (CDCl3): δ 195.9, 195.1, 175.6, 172.3, 166.6, 162.9, 149.8, 149.4, 148.0, 145.3, 144.3, 143.5, 141.6, 138.1, 133.4, 127.6, 57.1, 46.6, 44.2, 36.9, 32.1, 25.6, 24.7, 19.1, 18.3, 4.1. HRMS (FAB) [M + H]+ (C28H31N4O6S): calcd. 551.1964, found 551.1962; purity 96.8%. (±)-4-((3-(1-(3-Methylbutanoyl)piperidin-4-yl)-2,5-dioxoimidazolidin4-yl)methyl)phenyl isoquinoline-5-sulfonate (21g). It was synthesized from 20a and 3-methylbutanoyl chloride following the procedure described for 21c. Yield 74.4%. 1H NMR (CDCl3, 400 MHz): δ 9.40 (1H, s), 8.79 (1H, d, J = 5.6 Hz), 8.51 (1H, d, J = 6.0 Hz), 8.46 (NH, s), 8.29 (1H, d, J = 8.0 Hz), 8.25 (1H, d, J = 7.6 Hz), 7.66 (1H, t, J = 7.6 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.77 (1H, t, J = 13.2 Hz), 4.18−4.17 (1H, m), 3.94−3.91 (1H, m), 3.48− 3.41 (1H, m), 3.13−3.00 (2H, m), 2.94 (1H, q, J = 13.2 Hz), 2.40− 2.27 (1H, m), 2.19 (2H, d, J = 6.4 Hz), 2.11−2.04 (1H, m), 1.96−1.58 (4H, m), 0.95 (6H, d, J = 6.4 Hz). 13C NMR (CDCl3): δ 196.1, 194.5, 175.7, 172.3, 166.5, 162.9, 149.8, 149.5, 148.0, 145.3, 144.3, 143.5, 141.6, 138.1, 133.4, 127.6, 57.1, 46.6, 37.2, 33.2, 32.1, 25.5, 19.2, 18.4, 17.9, 17.4, 12.9, 9.2. HRMS (FAB) [M + H]+ (C29H33N4O6S): calcd. 565.2121, found 565.2123; purity 99.2%. (±)-4-((3-(1-(Cyclohexanecarbonyl)piperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21h). It was synthesized from 20a and cyclohexanecarboxylic acid following the procedure described for 21b. Yield 78.0%. 1H NMR (CDCl3, 400 MHz): δ 9.40 (1H, s), 8.79 (1H, d, J = 5.6 Hz), 8.51 (1H, d, J = 5.6 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.24 (1H, d, J = 7.6 Hz), 7.66 (1H, t, J = 8.4 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.79 (2H, d, J = 8.4 Hz), 4.75− 4.69 (1H, m), 4.18 (1H, br s), 3.98−3.95 (1H, m), 3.49−3.43 (1H, m), 3.13 (2H, qd, J = 15.2, 3.6 Hz), 2.93 (1H, q, J = 12.0 Hz), 2.45− 2.39 (1H, m), 2.35−2.22 (1H, m), 2.05−1.57 (8H, m), 1.49−1.37 (2H, m), 1.26−1.22 (4H, m). 13C NMR (CDCl3): δ 199.0, 195.9, 175.4, 172.3, 166.6, 163.0, 149.8, 149.4, 148.0, 145.3, 144.3, 143.6, 141.6, 138.1, 133.4, 127.6, 57.0, 46.7, 36.6, 32.2, 31.3, 25.5, 17.6, 13.0. HRMS (FAB) [M + H]+ (C31H35N4O6S): calcd. 591.2277, found 591.2276; purity 96.4%. (±)-4-((3-(1-Adamantanecarbonylpiperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21i). It was synthesized from 20a and 1-adamantanecarbonyl chloride following the procedure described for 21c. Yield 78.8%. 1H NMR (CDCl3, 400 MHz) δ 9.40 (1H, s), 8.78 (1H, d, J = 5.6 Hz), 8.51 (2H, d, J = 6.0 Hz), 8.29 (1H, d, J = 8.0 Hz), 8.23 (1H, d, J = 7.6 Hz), 7.66 (1H, t, J = 8.0 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.79 (2H, d, J = 8.0 Hz), 2127

DOI: 10.1021/jm500324g J. Med. Chem. 2015, 58, 2114−2134

Article


(CDCl3, 400 MHz): δ 9.40 (1H, s), 8.79 (1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 5.6 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.24 (1H, d, J = 7.2 Hz), 7.66 (1H, t, J = 8.4 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.19−4.17 (1H, m), 3.53−3.47 (1H, m), 3.14 (2H, qd, J = 15.2, 4.4 Hz), 2.73−2.70 (1H, m), 2.61−2.53 (2H, m), 2.29 (2H, br s), 2.05−2.02 (2H, m), 1.98−1.78 (8H, m), 1.68−1.55 (4H, m). 13C NMR (CDCl3): δ 200.1, 195.9, 175.5, 172.3, 166.6, 162.9, 149.8, 149.5, 148.0, 145.3, 144.3, 143.5, 141.6, 138.1, 133.4, 127.6, 56.9, 46.6, 46.8, 41.1, 35.5, 34.1, 27.2, 25.5, 24.6, 18.7, 18.1. HRMS (FAB) [M + H]+ (C34H37N4O6S): calcd. 629.2434, found 629.2431; purity 100%. (±)-4-((3-(1-(2-Norbornanylacetyl)piperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21o). It was synthesized from 20a and 2-norbornane acetic acid following the procedure described for 21b. Yield 87.2%. 1H NMR (CDCl3, 400 MHz): δ 9.40 (1H, s), 8.79 (1H, d, J = 6.4 Hz), 8.51 (1H, d, J = 6.0 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.24 (1H, d, J = 8.0 Hz), 7.66 (1H, t, J = 8.0 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.8 Hz), 4.75 (1H, t, J = 12.4 Hz), 4.18 (1H, d, J = 4.0 Hz), 3.93 (1H, d, J = 13.2 Hz), 3.47−3.42 (1H, m), 3.14 (2H, q, J = 14.8 Hz), 2.93 (1H, q, J = 12.8 Hz), 2.39−2.10 (4H, m), 1.99−1.41 (8H, m), 1.29−1.03 (5H, m). 13C NMR (CDCl3): δ 196.1, 194.5, 175.7, 172.3, 166.5, 162.9, 149.8, 149.5, 148.0, 145.3, 144.3, 143.5, 141.6, 138.1, 133.41, 127.6, 57.1, 46.6, 37.0, 32.2, 30.8, 28.9, 28.3, 26.7, 25.5, 24.9, 19.2, 18.4, 17.4, 16.4. HRMS (FAB) [M + H]+ (C33H37N4O6S): calcd. 617.2434, found 617.2432; purity 96.7%. (±)-4-((3-(1-(3,4-Dimethoxybenzoyl)piperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21p). It was synthesized from 20a and 3,4-dimethoxybenzoic acid following the procedure described for 21b. Yield 70.8%. 1H NMR (CDCl3, 400 MHz): δ 9.39 (1H, s), 8.79 (1H, d, J = 6.0 Hz), 8.51 (1H, d, J = 6.0 Hz), 8.28 (1H, d, J = 8.0 Hz), 8.26 (1H, d, J = 7.6 Hz), 7.66 (1H, t, J = 7.6 Hz), 7.04 (2H, d, J = 8.8 Hz), 4.21−4.19 (1H, m), 3.90 (6H, d, J = 4.8 Hz), 3.47−3.40 (1H, m), 3.15 (2H, qd, J = 15.2, 4.4 Hz), 2.75− 2.68 (2H, m), 2.06−2.00 (2H, m), 1.94−1.82 (2H, m), 1.63−1.60 (2H, m). 13C NMR (CDCl3): δ 195.8, 193.8, 175.4, 172.3, 168.9, 167.0, 166.6, 163.0, 149.8, 149.4, 148.0, 145.3, 144.3, 143.6, 141.6, 140.2, 138.1, 133.5, 130.8, 127.6, 119.4, 118.8, 57.2, 50.8, 46.8, 31.8, 25.5, 18.1. HRMS (FAB) [M + H]+ (C33H33N4O8S): calcd. 645.2019, found 645.2021; purity 98.6%. (±)-4-((3-(1-(2-Chlorobenzoyl)piperidin-4-yl)-2,5-dioxoimidazolidin4-yl)methyl)phenyl isoquinoline-5-sulfonate (21q). It was synthesized from 20a and 2-chlorobenzoyl chloride following the procedure described for 21c. Yield 71.2%. 1H NMR (CDCl3, 400 MHz): δ 9.37 (1H, s), 8.79 (1H, d, J = 6.0 Hz), 8.51 (1H, d, J = 5.6 Hz), 8.27 (2H, t, J = 7.2 Hz), 7.67 (1H, t, J = 7.6 Hz), 7.42 (3H, m), 7.04 (2H, d, J = 7.0 Hz), 6.79 (2H, d, J = 7.2 Hz), 4.93 (1H, m), 4.19 (1H, t, J = 4.8 Hz), 3.71 (1H, m), 3.50 (1H, t, J = 13.6 Hz), 3.14 (4H, m), 2.01 (4H, m). 13 C NMR (CDCl3): δ 195.4, 189.4, 175.0, 172.3, 166.6, 163.0, 149.8, 149.4, 148.0, 147.7, 145.3, 144.3, 143.8, 143.0, 142.8, 141.6, 140.4, 139.9, 138.1, 133.7, 127.6, 56.5, 46.8, 39.0, 32.2, 25.6, 18.6. HRMS (FAB) [M + H]+ (C31H28ClN4O6S): calcd. 619.1418, found 619.1415; purity 96.4%. (±)-4-((3-(1-(3-Chlorobenzoyl)piperidin-4-yl)-2,5-dioxoimidazolidin4-yl)methyl)phenyl isoquinoline-5-sulfonate (21r). It was synthesized from 20a and 3-chlorobenzoyl chloride following the procedure described for 21c. Yield 75.9%. 1H NMR (CDCl3, 400 MHz): δ 9.39 (1H, s), 8.78 (1H, d, J = 5.6 Hz), 8.54 (NH, s), 8.50 (1H, d, J = 5.6 Hz), 8.28 (1H, d, J = 8.0 Hz), 8.25 (1H, d, J = 7.6 Hz), 7.65 (1H, t, J = 7.6 Hz), 7.41−7.32 (3H, m), 7.03 (2H, d, J = 7.2 Hz), 6.81 (2H, d, J = 7.2 Hz), 4.82−4.70 (1H, m), 4.20 (1H, t, J = 4.8 Hz), 3.77−3.71 (1H, m), 3.41 (1H, t, J = 12.0 Hz), 3.15 (2H, qd, J = 15.2, 4.0 Hz), 2.94− 2.78 (1H, m), 2.69−2.54 (1H, m), 2.07−1.97 (1H, m), 1.88−1.54 (4H, m). 13C NMR (CDCl3): δ 197.7, 188.4, 181.0, 175.9, 172.4, 166.7, 150.6, 149.4, 147.9, 146.9, 144.3, 143.4, 141.8, 141.4, 139.6, 138.0, 137.0, 133.6, 127.6, 57.4, 46.1, 23.0, 19.4, 18.2. HRMS (FAB) [M + H]+ (C31H28ClN4O6S): calcd. 619.1418, found 619.1421; purity 98.6%. (±)-4-((3-(1-(4-Chlorobenzoyl)piperidin-4-yl)-2,5-dioxoimidazolidin4-yl)methyl)phenyl isoquinoline-5-sulfonate (21s). It was synthesized from 20a and 4-chlorobenzoyl chloride following the procedure described

4.63−4.56 (2H, m), 4.18−4.16 (1H, m), 3.54−3.48 (1H, m), 3.13 (2H, qd, J = 14.8, 4.0 Hz), 2.63−2.57 (2H, m), 2.02 (3H, s), 1.95 (6H, s), 1.83−1.63 (10H, m). 13C NMR (CDCl3) δ 200.5, 196.0, 175.5, 172.3, 166.6, 163.0, 149.8, 149.5, 148.0, 145.3, 144.3, 143.5, 138.1, 133.4, 127.6, 56.9, 46.7, 36.9, 32.9, 29.6, 26.4, 25.6, 19.0, 18.2, 16.3. HRMS (FAB) [M + H]+ (C35H39O6N4S): calcd. 643.2590, found 643.2593; purity 96.5%. (±)-4-((3-(1-Adamantylmethylpiperidin-4-yl)-2,5-dioxoimidazolidin4-yl)methyl)phenyl isoquinoline-5-sulfonate (21j). It was synthesized from 20a and 1-adamantanecarbaldehyde following the procedure described for 21a. Yield 72.4%. 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 6.4 Hz), 8.28 (1H, d, J = 8.0 Hz), 8.23 (1H, d, J = 7.6 Hz), 7.99 (NH, s), 7.67 (1H, t, J = 8.4 Hz), 7.05 (2H, d, J = 8.4 Hz), 6.83 (2H, d, J = 8.4 Hz), 4.53 (1H, t, J = 10.8 Hz), 4.20 (1H, q, J = 4.4 Hz), 3.69−3.63 (1H, m), 3.47 (1H, t, J = 11.6 Hz), 3.14−2.89 (3H, m), 2.48−2.35 (1H, m), 2.02−1.63 (10H, s). 13C NMR (CDCl3): δ 195.1, 174.8, 172.4, 166.7, 163.1, 149.8, 149.4, 147.9, 145.3, 144.3, 143.7, 138.0, 133.6, 127.6, 57.2, 46.6, 37.0, 36.0, 35.6, 29.4, 27.7, 25.5, 19.8, 19.4, 19.1, 18.5, 17.9, 17.2, 16.9. HRMS (FAB) [M + H]+ (C35H41N4O5S): calcd. 629.2797, found 629.2802; purity 99.3%. (±)-4-((3-(1-Adamantaylacetylpiperidin-4-yl)-2,5-dioxoimidazolidin4-yl)methyl)phenyl isoquinoline-5-sulfonate (21k). It was synthesized from 20a and 1-adamantane acetic acid following the procedure described for 21b. Yield 70.2%. 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 6.0 Hz), 8.30 (1H, d, J = 8.8 Hz), 8.26 (1H, d, J = 7.2 Hz), 8.01 (NH, s), 7.67 (1H, t, J = 7.6 Hz), 7.03 (2H, d, J = 8.8 Hz), 6.80 (2H, d, J = 8.4 Hz), 4.83−4.77 (1H, m), 4.20−4.16 (1H, m), 4.04−4.00 (1H, m), 3.50−3.46 (1H, m), 3.15 (1H, dd, J = 14.8, 4.0 Hz), 3.05−2.87 (2H, m), 2.41−2.28 (1H, m), 2.21 (1H, d, J = 13.6 Hz), 2.10 (1H, d, J = 13.6 Hz), 1.95 (3H, s), 1.89−1.73 (4H, m), 1.70−1.56 (12H, m). 13C NMR (CDCl3): δ 195.7, 192.9, 172.3, 166.6, 163.0, 149.8, 149.4, 145.3, 144.4, 143.6, 142.0, 141.6, 141.0, 138.1, 133.5, 127.6, 55.9, 46.5, 38.6, 34.3, 32.0, 26.6, 22.8, 18.4, 17.5, 16.6, 15.3; HRMS (FAB) [M + H]+ (C36H41N4O6S): calcd. 657.2747, found 657.2744; purity 99.3%. (±)-4-((3-((3-Bromo-1-adamantylcarbonyl)piperidin-4-yl)-2,5dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21l). It was synthesized from 20a and 3-bromo adamantane-1carboxylic acid following the procedure described for 21b. Yield 55.6%. 1 H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.4 Hz), 8.52 (1H, d, J = 6.4 Hz), 8.31 (2H, d, J = 8.4 Hz), 8.26 (2H, d, J = 7.6 Hz), 7.80 (NH, br s), 7.67 (1H, t, J = 8.0 Hz), 7.04 (2H, d, J = 8.4 Hz), 6.82 (2H, d, J = 8.8 Hz), 4.57−4.50 (2H, m), 4.20−4.17 (1H, m), 3.51−3.43 (1H, m), 3.14 (2H, qd, J = 14.8, 4.4 Hz), 2.67−2.60 (2H, m), 2.53 (2H, br s), 2.35−2.27 (4H, m), 2.22 (2H, br s), 1.96 (4H, br s), 1.84−1.54 (6H, m). 13C NMR (CDCl3): δ 197.5, 195.4, 175.0, 172.4, 166.6, 163.0, 149.8, 149.4, 147.9, 145.3, 144.3, 143.6, 138.0, 133.6, 127.6, 61.1, 57.1, 46.7, 43.3, 40.9, 38.3, 37.0, 27.4, 25.6, 23.9, 20.8, 18.7. HRMS (FAB) [M + H]+ (C35H38BrN4O6S): calcd. 723.1681, found 723.1755; purity 98.5%. (±)-4-((3-(3,5-Dimethyladamantanecarbonylpiperidin-4-yl)-2,5dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21m). It was synthesized from 20a and 3,5-dimethyl adamantane carboxylic acid following the procedure described for 21b. Yield 76.0%. 1 H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 6.0 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.25 (1H, d, J = 7.6 Hz), 7.91 (NH, s), 7.66 (1H, t, J = 8.0 Hz), 7.03 (2H, d, J = 7.6 Hz), 6.80 (2H, d, J = 7.6 Hz), 4.61−4.54 (1H, m), 4.19−4.17 (1H, m), 3.53−3.47 (1H, m), 3.14 (2H, qd, J = 14.0, 4.0 Hz), 2.63−2.57 (2H, m), 2.11 (1H, br s), 1.91 (1H, q, J = 11.2 Hz), 1.77 (2H, br s), 1.67− 1.51 (7H, m), 1.34 (4H, br s), 1.18−1.10 (2H, m), 0.83 (6H, s). 13C NMR (CDCl3): δ 200.2, 195.7, 175.1, 172.3, 166.6, 163.0, 149.8, 149.5, 148.0, 145.3, 144.3, 143.6, 138.1, 133.5, 127.7, 56.9, 46.8, 44.0, 37.4, 36.9, 35.3, 34.2, 27.8, 25.6, 19.8, 19.1, 18.2, 17.6. HRMS (FAB) [M + H]+ (C37H43N4O6S): calcd. 671.2903, found 671.2900; purity 96.3%. (±)-4-((3-(1-(3-Noradamantylcarbonyl)piperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21n). It was synthesized from 20a and 3-noradamantane carboxylic acid following the procedure described for 21b. Yield 84.5%. 1H NMR 2128

DOI: 10.1021/jm500324g J. Med. Chem. 2015, 58, 2114−2134

Article

Journal of Medicinal Chemistry for 21c. Yield 77.6%. 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.78 (1H, d, J = 5.6 Hz), 8.51 (1H, d, J = 5.2 Hz), 8.40 (NH, s), 8.29 (2H, t, J = 7.2 Hz), 7.67 (1H, t, J = 8.0 Hz), 7.39 (2H, d, J = 8.0 Hz), 7.35 (2H, d, J = 8.0 Hz), 7.04 (2H, d, J = 8.4 Hz), 6.81 (2H, d, J = 8.0 Hz), 4.78−4.75 (1H, m), 4.20−4.18 (1H, m), 3.81−3.76 (1H, m), 3.43 (1H, br t, J = 11.2 Hz), 3.14 (2H, qd, J = 14.4, 4.4 Hz), 2.86−2.79 (1H, m), 2.75−2.58 (1H, m), 2.07−1.97 (1H, m), 1.86−1.56 (4H, m). 13 C NMR (CDCl3): δ 195.3, 192.5, 174.9, 172.4, 166.6, 163.1, 149.8, 149.4, 147.9, 147.9, 145.3, 144.3, 143.6, 141.8, 141.4, 138.0, 133.6, 127.6, 57.4, 46.7, 30.1, 25.6, 21.1. HRMS (FAB) [M + H]+ (C31H28ClN4O6S): calcd. 619.1418, found 619.1415; purity 96.2%. (±)-4-((3-(1-(4-Fluorobenzoyl)piperidin-4-yl)-2,5-dioxoimidazolidin4-yl)methyl)phenyl isoquinoline-5-sulfonate (21t). It was synthesized from 20a and 4-fluorobenzoyl chloride following the procedure described for 21c. Yield 72.7%. 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 5.6 Hz), 8.52 (1H, d, J = 5.6 Hz), 8.29 (2H, t, J = 6.8 Hz), 8.11 (1H, t, J = 7.2 Hz), 7.67 (1H, t, J = 7.6 Hz), 7.43 (2H, t, J = 7.6 Hz), 7.14 (2H, q, J = 8.4 Hz), 7.05 (1H, d, J = 8.4 Hz), 6.83 (1H, d, J = 8.4 Hz), 4.87−4.60 (1H, m), 4.21−4.19 (1H, m), 3.65−3.63 (1H, m), 3.45−3.39 (1H, m), 3.16 (2H, qd, J = 14.8, 4.8 Hz), 2.76−2.61 (2H, m), 2.02−1.98 (2H, m), 1.81−1.67 (2H, m). 13C NMR (CDCl3): δ 171.7, 169.5, 164.4, 162.4, 155.5, 153.3, 148.7, 145.8, 135.2, 134.9, 133.5, 131.8, 131.6, 130.7, 130.2, 129.2, 129.2, 128.7, 125.8, 122.3, 117.4, 115.7, 115.5, 50.9, 45.7, 34.9, 34.0, 29.7; HRMS (FAB) [M + H]+ (C31H28FN4O6S): calcd. 603.1714, found 603.1711; purity 97.6%. (±)-4-((3-(1-(3,4-Difluorobenzoyl)piperidin-4-yl)-2,5-dioxoimidazolidin4-yl)methyl)phenyl isoquinoline-5-sulfonate (21u). It was synthesized from 20a and 3,4-difluorobenzoyl chloride following the procedure described for 21c. Yield 72.7%. 1H NMR (CDCl3, 400 MHz): δ 9.42 (1H, s), 8.79 (1H, d, J = 5.6 Hz), 8.52 (1H, d, J = 6.4 Hz), 8.31 (2H, t, J = 8.4 Hz), 7.76 (NH, s), 7.69 (1H, t, J = 8.0 Hz), 7.24−7.16 (3H, m), 7.05 (2H, d, J = 8.4 Hz), 6.83 (2H, d, J = 8.8 Hz), 4.87−4.60 (1H, m), 4.21−4.16 (1H, m), 3.96−3.73 (1H, m), 3.43 (1H, t, J = 12.0 Hz), 3.15 (2H, qd, J = 14.4, 4.4 Hz), 2.91−2.66 (2H, m), 2.08−2.06 (2H, m), 1.88−1.78 (2H, m). 13C NMR (CDCl3): δ 195.8, 190.9, 175.5, 172.3, 166.5, 162.9, 149.9, 149.4, 148.0, 147.7, 146.4, 145.9, 145.3, 144.2, 143.5, 141.6, 138.1, 135.2, 134.8, 133.6, 128.0, 126.9, 57.4, 47.8, 33.1, 25.5, 23.3. HRMS (FAB) [M + H]+ (C31H27F2N4O6S): calcd. 621.1619, found 621.1621; purity 97.4%. (±)-4-((3-(1-(3,4-Dichlorobenzoyl)piperidin-4-yl)-2,5-dioxoimidazolidin4-yl)methyl)phenyl isoquinoline-5-sulfonate (21v). It was synthesized from 20a and 3,4-dichlorobenzoic acid following the procedure described for 21b. Yield 70.3%. 1H NMR (CDCl3, 400 MHz): δ 9.40 (1H, s), 8.79 (1H, d, J = 6.4 Hz), 8.51 (1H, d, J = 6.4 Hz), 8.29 (1H, d, J = 7.6 Hz), 7.66 (1H, t, J = 8.0 Hz), 7.49 (2H, d, J = 8.0 Hz), 7.24 (2H, dd, J = 8.4 Hz), 7.04 (2H, d, J = 8.4 Hz), 6.82 (2H, d, J = 8.4 Hz), 4.85 (2H, m), 4.20 (1H, t, J = 4.8 Hz), 3.41 (1H, m), 3.14 (2H, qd, J = 6.4 Hz), 2.07 (2H, m), 1.98 (2H, m), 1.64 (2H, m). 13C NMR (CDCl3): δ 195.2, 190.7, 174.8, 172.4, 166.7, 163.1, 149.8, 149.7, 149.4, 148.0, 147.1, 145.4, 144.2, 142.2, 138.6, 138.3, 133.6, 127.6, 57.5, 46.7, 25.6. HRMS (FAB) [M + H]+ (C31H27Cl2N4O6S): calcd. 653.1028, found 653.1026; purity 97.0%. (±)-4-((3-(1-(4-Chloro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21w). It was synthesized from 20a and 4-chloro-3-(trifluoromethyl)benzoic acid following the procedure described for 21b. The crude product was purified using preparative HPLC to yield racemic compound (±)-21w. Yield 75.3%. 1H NMR (CDCl3, 400 MHz): δ 9.39 (1H, s), 8.78 (1H, d, J = 6.0 Hz), 8.50 (1H, d, J = 5.6 Hz), 8.38 (NH, s), 8.29 (1H, d, J = 8.4 Hz), 8.26 (1H, d, J = 7.2 Hz), 7.74 (1H, s), 7.66 (1H, t, J = 8.0 Hz), 7.56 (1H, d, J = 8.4 Hz), 7.51 (1H, d, J = 8.0 Hz), 7.04 (2H, d, J = 8.4 Hz), 6.82 (2H, d, J = 8.0 Hz), 4.80 (1H, m), 4.20 (1H, t, J = 4.8 Hz), 3.74 (1H, m), 3.40 (1H, t, J = 12.0 Hz), 3.14 (2H, m), 2.94 (2H, m), 2.16 (4H, m). 13C NMR (CDCl3): δ 195.8, 190.6, 175.5, 172.3, 166.5, 162.9, 149.9, 149.4, 148.5, 148.0, 145.4, 144.9, 144.2, 143.6, 142.1, 141.7, 139.0, 138.0, 135.3, 133.6, 132.0, 127.6, 57.5, 46.5, 25.5. HRMS (FAB) [M + H]+ (C32H27ClF3N4O6S): calcd. 687.1292, found 687.1292; purity 95.4%.

Twenty milligrams (±)-21w was applied in chiral HPLC with an analytical Chiralpak IA column eluted with ethanol as the mobile phase. The first-eluting enantiomer, tR 19.24 min, was collected and evaporated to afford (+)-21w (2.3 mg, 11.5%). HRMS (FAB) [M + H]+ (C32H26ClF3N4O6S): calcd. 687.1292, found 687.1292. NMR equal to (±)-21w. [α26 D ] = +7.75 (c 0.26, DMF). The secondeluting enantiomer, tR 23.82 min, was collected and evaporated to afford (−)-21w (3.2 mg, 16.0%). HRMS (FAB) [M + H]+ (C32H26ClF3N4O6S): calcd. 687.1292, found 687.1292. [αD26] = −7.75° (c 0.26, DMF). NMR in agreement with (±)-21w. (±)-4-((3-(1-(4-Chloro-2-fluorobenzoyl)piperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (21x). It was synthesized from 20a and 4-chloro-2-fluorobenzoic acid following the procedure described for 21b. Yield 82.8%. 1H NMR (CDCl3, 400 MHz): δ 9.40 (1H, s), 8.77 (1H, d, J = 5.6 Hz), 8.50 (1H, s), 8.41 (1H, s), 8.30 (1H, d, J = 7.2 Hz), 8.25 (1H, d, J = 7.6 Hz), 7.68 (1H, q, J = 4.0 Hz), 7.36 (1H, t, J = 7.6 Hz), 7.22 (1H, d, J = 8.0 Hz), 7.14 (1H, d, J = 6.4 Hz), 7.04 (2H, d, J = 8.0 Hz), 6.81 (2H, t, J = 6.4 Hz), 4.84 (1H, br t, J = 12.8 Hz), 4.19 (1H, s), 3.59 (1H, d, J = 5.6 Hz), 3.53−3.33 (1H, m), 3.14 (2H, q, J = 14.0 Hz), 3.025 (1H, q, J = 12.0 Hz), 2.64−2.53 (1H, m), 2.07−1.59 (5H, m). 13C NMR (CDCl3): δ 195.7, 186.0, 179.8, 176.6, 175.4, 172.3, 166.6, 163.0, 151.7, 149.8, 149.4, 147.9, 145.3, 144.3, 143.5, 141.6, 138.0, 137.5, 133.7, 127.6, 126.7, 57.0, 46.3, 38.9, 32.6, 17.3. HRMS (FAB) [M + H]+ (C31H27ClFN4O6S): calcd. 637.1324, found 637.1320; purity 99.4%. (±)-4-((3-((1-Benzoylpiperidin-4-yl)methyl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (22a). It was synthesized from 20b and benzoic acid following the procedure described for 21b. 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 5.6 Hz), 8.29 (1H, d, J = 8.4 Hz), 8.26 (1H, d, J = 7.6 Hz), 7.67 (1H, t, J = 8.0 Hz), 7.40−7.37 (5H, m), 7.01 (2H, d, J = 8.4 Hz), 6.82 (2H, d, J = 8.4 Hz), 4.70−4.66 (1H, m), 4.22−4.19 (1H, m), 3.76−3.72 (1H, m), 3.59−3.54 (1H, m), 3.09 (2H, d, J = 6.4 Hz), 2.94−2.85 (1H, m), 2.81 (2H, dd, J = 14.0, 5.6 Hz), 1.82−1.70 (2H, m), 1.26−1.10 (3H, m). 13C NMR (CDCl3): δ 171.8, 170.4, 155,5, 153.3, 148.6, 145.8, 135.8, 135.2, 134.9, 133.5, 131.6, 130.7, 130.2, 129.7, 128.7, 128.5, 126.8, 125.8, 122.3, 117.4, 45.8, 41.7, 34.9, 34.0, 29.3. HRMS (FAB) [M + H]+ (C32H31N4O6S): calcd. 599.1964, found 599.1962; purity 99.2%. (±)-4-((3-(2-(1-Benzylpiperidin-4-yl)ethyl)-2,5-dioxoimidazolidin4-yl)methyl)phenyl isoquinoline-5-sulfonate (23a). It was synthesized from 20c and benzaldehyde following the procedure described for 21a. Yield 84.3%. 1H NMR (CDCl3, 400 MHz): δ 9.39 (1H, s), 8.79 (1H, d, J = 6.0 Hz), 8.50 (1H, d, J = 5.6 Hz), 8.28 (2H, t, J = 8.0 Hz), 7.66 (1H, t, J = 8.0 Hz), 7.39 (5H, m), 7.00 (2H, d, J = 8.0 Hz), 6.80 (2H, d, J = 8.8 Hz), 4.19 (1H, t, J = 4.0 Hz), 3.74 (2H, m), 3.05 (2H, d, J = 4.0 Hz), 2.95 (2H, m), 1.85 (4H, m), 1.41 (2H, m), 1.26 (1H, t, J = 7.2 Hz), 1.20 (2H, m). 13C NMR (CDCl3): δ 196.0, 175.0, 172.3, 166.5, 163.0, 149.8, 149.5, 148.0, 145.3, 144.1, 143.6, 142.9, 141.6, 141.2, 140.3, 138.1, 133.5, 127.6, 59.1, 56.4, 47.1, 28.5, 23.4, 22.8, 21.8, 20.1. HRMS (FAB) [M + H]+ (C33H35N4O5S): calcd. 599.2328, found 599.2332; purity 98.8%. (±)-4-((3-(2-(1-Benzoylpiperidin-4-yl)ethyl)-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5-sulfonate (23b). It was synthesized from 20c and benzoic acid following the procedure described for 21b. Yield 84.3%. 1H NMR (CDCl3, 400 MHz): δ 9.39 (1H, s), 8.79 (1H, d, J = 6.0 Hz), 8.50 (1H, d, J = 5.6 Hz), 8.28 (2H, t, J = 8.0 Hz), 7.66 (1H, t, J = 8.0 Hz), 7.39 (5H, m), 7.00 (2H, d, J = 8.0 Hz), 6.80 (2H, d, J = 8.8 Hz), 4.19 (1H, t, J = 4.0 Hz), 3.74 (2H, m), 3.05 (2H, d, J = 4.0 Hz), 2.95 (2H, m), 1.85 (4H, m), 1.41 (2H, m), 1.26 (1H, t, J = 7.2 Hz), 1.20 (2H, m). 13C NMR (CDCl3): δ 196.1, 193.7, 175.2, 172.3, 166.5, 162.9, 150.8, 149.8, 149.4, 147.9, 145.3, 144.1, 143.5, 142.7, 141.6, 141.3, 139.2, 138.1, 133.5, 127.6, 56.4, 40.4, 33.5, 28.3, 23.4, 22.9, 20.9. HRMS (FAB) [M + H]+ (C33H33N4O6S): calcd. 613.2121, found 613.2123; purity 98.0%. (±)-4-((3-(1-(4-Chloro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)1-methyl-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5sulfonate (24a). To a solution of 21w (10 mg, 0.015 mmol) in dried DMF in the presence of K2CO3 (6.22 mg, 0.045 mmol) was added iodomethane (1.4 μL, 0.023 mmol). After the mixture was stirred for 2129

DOI: 10.1021/jm500324g J. Med. Chem. 2015, 58, 2114−2134

Article

Journal of Medicinal Chemistry 6 h at room temperature, the solvent was removed under vacuum. The residue was taken up in solution of saturated aq NH4Cl and extracted with ethyl acetate. The combined extracts were dried over sodium sulfate, filtered, and evaporated. The residue was purified by silica gel column chromatography (hexane:ethyl acetate = 1:2) to give 24a (8.9 mg, 84.6% yield). 1H NMR (CD3OD, 400 MHz): δ 9.54 (1H, s), 8.81 (1H, d, J = 8.0 Hz), 8.57 (1H, d, J = 8.0 Hz), 8.35 (1H, d, J = 6.0 Hz), 8.23 (1H, dd, J = 7.6 Hz), 7.81 (1H, s), 7.80 (1H, d, J = 9.2 Hz), 7.76 (1H, t, J = 8.0 Hz), 7.70 (1H, d, J = 8.0 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.76 (2H, d, J = 8.4 Hz), 4.50−4.41 (1H, m), 4.08−4.04 (1H, m), 3.56−3.41 (2H, m), 3.02−2.97 (2H, m), 2.68−2.60 (1H, m), 2.56 (3H, s), 2.01−1.82 (2H, m), 1.80−1.60 (2H, m), 1.50−1.33 (1H, m). ESI [M + H]+: 701.32; purity 98.1%. (±)-4-((3-(1-(4-Chloro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)1-ethyl-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline-5sulfonate (24b). It was synthesized from 21w and idoethane following the procedure described for 24a. Yield 85.8%. 1H NMR (CD3OD, 400 MHz): δ 9.54 (1H, s), 8.82 (1H, d, J = 6.4 Hz), 8.57 (1H, d, J = 8.0 Hz), 8.34 (1H, d, J = 6.0 Hz), 8.22 (1H, dd, J = 7.6 Hz), 7.82 (1H, s), 7.80 (1H, d, J = 7.6 Hz), 7.78 (1H, t, J = 7.0 Hz), 7.71 (1H, d, J = 8.0 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.78 (2H, d, J = 8.4 Hz), 4.58−4.38 (1H, m), 4.13−4.05 (1H, m), 3.70−3.57 (1H, m), 3.52−3.40 (1H, m), 3.11 (2H, s), 3.09−2.92 (2H, m), 2.75−2.59 (1H, m), 2.05−1.86 (2H, m), 1.80−1.60 (2H, m), 1.54−1.44 (1H, m), 0.57 (3H, t, J = 5.2 Hz). ESI [M + H]+: 715.35; purity 95.8%. (±)-4-((3-(1-(4-Chloro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)2,5-dioxo-1-propylimidazolidin-4-yl)methyl)phenyl isoquinoline-5sulfonate (24c). It was synthesized from 21w and idoethane following the procedure described for 24a. Yield 71.3%. 1H NMR (CD3OD, 400 MHz): δ 9.45 (1H, s), 8.74 (1H, d, J = 8.0 Hz), 8.55 (1H, d, J = 8.0 Hz), 8.51−8.43 (1H, m), 8.27 (1H, d, J = 8.0 Hz), 7.84 (1H, s), 7.73 (1H, d, J = 7.2 Hz), 7.71 (1H, t, J = 7.2 Hz), 7.67 (1H, d, J = 8.0 Hz), 7.06 (2H, d, J = 8.4 Hz), 6.78 (2H, d, J = 8.4 Hz), 4.73−4.58 (1H, m), 4.35 (1H, s), 3.72−3.58 (1H, m), 3.65−3.50 (2H, m), 3.11 (2H, d, J = 4.8 Hz), 2.85−2.71 (1H, m), 2.20−2.07 (1H, m), 2.01− 1.90 (1H, m), 1.69−1.50 (1H, m), 1.34−1.17 (2H, m), 1.26 (2H, d, J = 6.8 Hz), 0.93−0.80 (1H, m), 0.62 (3H, t, J = 7.2 Hz). ESI [M + H]+: 730.76; purity 97.2%. (±)-4-((3-(1-(4-Chloro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)1-isopropyl-2,5-dioxoimidazolidin-4-yl)methyl)phenyl isoquinoline5-sulfonate (24d). It was synthesized from 21w and idoethane following the procedure described for 24a. Yield 69.5%. 1H NMR (CD3OD, 400 MHz): δ 9.44 (1H, s), 8.74 (1H, d, J = 6.0 Hz), 8.55 (1H, d, J = 5.6 Hz), 8.48 (1H, s), 8.25 (1H, d, J = 7.2 Hz), 7.85 (1H, s), 7.73 (1H, d, J = 7.2 Hz), 7.19 (1H, t, J = 7.2 Hz), 7.66 (1H, d, J = 8.0 Hz), 7.04 (2H, d, J = 8.4 Hz), 6.77 (2H, d, J = 8.4 Hz), 4.77−4.55 (1H, m), 4.27 (1H, s), 4.02−3.90 (1H, m), 3.74−3.53 (2H, m), 3.09 (2H, d, J = 4.4 Hz), 2.04−2.65 (1H, m), 2.21−2.05 (1H, m), 1.69− 1.48 (1H, m), 1.45−1.21 (1H, s), 1.08 (3H, d, J = 6.8 Hz), 1.01 (3H, d, J = 6.4 Hz). ESI [M + H]+: 730.76; purity 97.4%. (R)-Methyl 2-((tert-butoxycarbonyl)amino)-2-(4-hydroxyphenyl)acetate (25). (R)-Methyl 2-amino-2-(4-hydroxyphenyl)acetate (400 mg, 2.21 mmol) and di-tert-butyl dicarbonate (530 mg, 2.43 mmol) and TEA (677 μL, 4.86 mmol) were added to dried DCM (30 mL). The solution was stirred at room temperature. After 3 h, the residue was taken up in saturated aq NH4Cl and extracted with ethyl acetate. Combined organic layer was dried over sodium sulfate, and the product was concentrated under reduced pressure. Without purification, this product was used to perform the next reaction (591 mg, 100% yield). 1H NMR (DMSO-d6, 400 MHz): δ 7.13 (2H, d, J = 8.4 Hz), 6.68 (2H, d, J = 8.4 Hz), 5.02 (2H, d, J = 8.0 Hz), 3.55 (2H, s), 1.34 (1H, s). ESI [M + H]+: 281.79. (R)-Methyl 2-amino-2-(4-((isoquinolin-5-ylsulfonyl)oxy)phenyl)acetate (26). It was synthesized from 25 following the procedure described for 16. Yield 86.3% (two-step overall). 1H NMR (CDCl3, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.4 Hz), 8.53 (1H, d, J = 6.0 Hz), 8.29 (1H, d, J = 8.0 Hz), 8.27 (1H, d, J = 8.0 Hz), 7.67 (1H, t, J = 8.0 Hz), 7.25 (2H, d, J = 8.8 Hz), 6.86 (2H, d, J = 8.8 Hz), 4.53 (1H, s), 3.66 (3H, s). ESI [M + H]+: 372.55. (±)-tert-Butyl 4-((1-(4-((isoquinolin-5-ylsulfonyl)oxy)phenyl)-2methoxy-2-oxoethyl)amino)piperidine-1-carboxylate (27). It was

synthesized from 26 following the procedure described for 17. Yield 98.5%. 1H NMR (DMSO-d6, 400 MHz): δ 9.41 (1H, s), 8.80 (1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 6.0 Hz), 8.31 (1H, d, J = 7.2 Hz), 8.30 (1H, d, J = 8.0 Hz), 7.67 (1H, t, J = 8.0 Hz), 7.24 (2H, d, J = 8.8 Hz), 6.87 (2H, d, J = 8.8 Hz), 4.41 (1H, s), 3.83−3.83 (4H, m), 3.65 (3H, s), 3.04−2.98 (1H, m), 2.71−2.67 (2H, m), 2.49−2.22 (2H,m), 1.42 (9H, s); ESI[M + H]+: 555.60. (±)-4-(2,5-Dioxo-3-(piperidin-4-yl)imidazolidin-4-yl)phenyl isoquinoline-5-sulfonate (28). It was synthesized from 27 following the procedure described for 18 and 20. Yield 75.9% (two-step overall). 1 H NMR (DMSO-d6, 400 MHz): δ 9.56 (1H, s), 8.81 (1H, d, J = 6.0 Hz), 8.60 (1H, d, J = 8 Hz), 8.33 (1H, d, J = 6.4 Hz), 8.32 (1H, d, J = 7.6 Hz), 7.82 (1H, t, J = 8.4 Hz), 7.26 (2H, d, J = 8.4 Hz), 6.93 (2H, d, J = 8.4 Hz), 3.60−3.42 (1H, m), 2.87 (1H, d, J = 12.4 Hz), 2.75 (1H, d, J = 11.6 Hz), 1.56−1.40 (2H, m), 1.26 (2H, d, J = 10.4 Hz), 0.87 (2H, d, J = 4.0 Hz). ESI[M + H]+: 466.64. (±)-4-(3-(1-(Adamantane-1-carbonyl)piperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)phenyl isoquinoline-5-sulfonate (29a). It was synthesized from 28 following the procedure described for 21. Yield 39.6%: 1H NMR (CD3OD, 400 MHz): δ 9.46 (1H, s), 8.75 (1H, d, J = 6.0 Hz), 8.56 (1H, d, J = 6.4 Hz), 8.52 (1H, d, J = 8.4 Hz), 8.32 (1H, d, J = 7.2 Hz), 7.80 (1H, t, J = 7.6 Hz), 7.25 (1H, d, J = 6.8 Hz), 6.92 (2H, d, J = 6.8 Hz), 5.09 (1H, s), 4.55 (1H, d, J = 13.6 Hz), 4.41 (1H, d, J = 13.2 Hz), 3.87−3.79 (1H, m), 2.72−2.61 (2H, m), 1.98− 1.72 (15H, m), 1.56 (1H, d, J = 10.0 Hz), 1.35−1.17 (1H, m), 1.06− 0.96 (1H, m). ESI[M + H]+: 628.53; purity 98.0%. (±)-4-(3-(1-(2-(Adamantan-1-yl)acetyl)piperidin-4-yl)-2,5-dioxoimidazolidin-4-yl)phenyl isoquinoline-5-sulfonate (29b). It was synthesized from 28 and idoethane following the procedure described for 21. Yield 21.2%. 1H NMR (CDCl3, 400 MHz): δ 9.42 (1H, s), 8.82 (1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 6.0 Hz), 8.31 (1H, d, J = 6.0 Hz), 8.29 (1H, d, J = 4.0 Hz), 7.70 (1H, t, J = 8.0 Hz), 7.22 (1H, d, J = 6.8 Hz), 6.98 (2H, d, J = 6.8 Hz), 5.33 (1H, s), 4.76 (1H, d, J = 13.2 Hz), 4.65 (1H, d, J = 13.2 Hz), 4.01−3.82 (4H, m), 3.00−2.85 (4H, m), 2.03 (2H, s), 1.73−1.46 (15H, m). ESI[M + H]+: 641.88; purity 98.1%. (±)-4-(3-(1-(4-Chloro-3-(trifluoromethyl)benzoyl)piperidin-4-yl)2,5-dioxoimidazolidin-4-yl)phenyl isoquinoline-5-sulfonate (29c). It was synthesized from 28 and idoethane following the procedure described for 21. Yield 62.6%. 1H NMR (DMSO-d6, 400 MHz): δ 9.42 (1H, s), 8.82 (1H, d, J = 6.0 Hz), 8.52 (1H, d, J = 6.0 Hz), 8.33 (1H, d, J = 6.0 Hz), 8.31 (1H, d, J = 6.4 Hz), 7.70 (1H, t, J = 8.0 Hz), 7.64 (1H, s), 7.55 (1H, d, J = 8.0 Hz), 7.42 (1H, s), 7.22 (2H, d, J = 8.8 Hz), 7.00 (2H, d, J = 8.8 Hz), 4.86 (1H, s), 4.78−4.54 (1H, m), 3.95− 3.78 (1H, m), 3.75−3.45 (1H, m), 3.10−2.70 (2H, m), 2.75−2.57 (2H, m), 1.91−1.69 (2H, m). ESI [M + H]+: 674.01. Purity 97.5%. Superimposing of the Compounds. To superimpose the compounds and analyze their conformations, a computational analysis was performed using the CAChe program (BioMedCAChe Version 5.0, CAChe Scientific, Inc.). Each compound was built, subjected to Beautify/Comprehensive, and saved as a ‘.cdw’ file. The saved structure was calculated using Augmented MM2 parameters, which enable minimization calculations for square planar, trigonal bipyramidal, and octahedral atoms. After calculating the potential energy of the structure, Mechanics adjusts the atomic positions and calculates the potential energy of the new structure. This procedure is an iterative process that continues until the energy change between iterations is within a specified limit. The structure obtained by Mechanics was calculated using MOPAC 2002 with PM3 parameters, which determines the bond strengths, atomic hybridizations, partial charges, orbitals from the positions of the atoms, and the net charge, to search for the lowest energy conformers by comparing their heats of formation (HF). Separation of Two Enantiomers and Measurement of Optical Rotation. Each enantiomer (+)21w and (−)21w was obtained by an analytical chiral HPLC using a Chiralpak IA column (250 mm × 4.6 mm) eluted with ethanol as the mobile phase (retention times, tR, of 19.24 and 23.82 min, flow rate of 0.5 mL/min). To investigate the specific rotation of the separated enantiomers, (+)21w and (−)21w, the optical rotation was determined using a 2130

DOI: 10.1021/jm500324g J. Med. Chem. 2015, 58, 2114−2134

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Journal of Medicinal Chemistry polarimeter (Atago Automatic Polarimeter AP-100). The compounds were dissolved in dimethylformamide (c 0.26). The first eluted enantiomer had a value of [α26 D ] = +7.75, and the second enantiomer had a value of [α26 D ] = −7.75. Biological Assays. Measurement of Ethidium Bromide Accumulation in hP2X7-Expressing HEK293 Cells. We used a stable HEK293 cell line expressing human P2X7R; the line has been described previously.57 Briefly, HEK293 cells were transfected, using Lipofectamine, with a pcDNA3.1 vector-based plasmid harboring hP2X7R (Invitrogen), according to the manufacturer’s instructions. HEK293 cells stably transfected with the human P2X7R were maintained in a humidified 5% CO2 atmosphere at 37 °C in DMEM supplemented with 10% (v/v) FBS, 2 mM L-glutamine, and antibiotics (50 U/mL penicillin and 50 μg/mL streptomycin). BzATP-induced pore formation was determined using a fluorescence plate reader by measuring the cellular uptake of ethidium in hP2X7-expressing HEK293 cells, as described previously.58 hP2X7expressing HEK293 cells were rinsed once in the appropriate assay buffer, which was also removed before performing the assay additions. All studies were performed at room temperature, and the final assay volume was 100 μL. The cells were resuspended at 2.5 × 106 cells/mL in an assay buffer consisting (in millimolar) of HEPES 10, N-methyl-Dglucamine 5, KCl 5.6, D-glucose 10, and CaCl2 0.5 (pH 7.4) and supplemented with either 280 mM sucrose or 140 mM NaCl. To calculate the IC50 values of the 2,5-dioxoimidazolidine derivatives and the reference compound 1, these compounds were added to cells at different doses with BzATP but without preincubation. After incubation for 2 h in a humidified 5% CO2 atmosphere, ethidium dye uptake was observed by measuring the fluorescence with a BioTek Instrument FL600 fluorescence plate reader (excitation wavelength of 530 nm and emission wavelength of 590 nm). The results are expressed as percentages relative to the maximum accumulation of ethidium bromide when stimulated with BzATP only, and the IC50 values were calculated using nonlinear regression analysis (i.e., percentage accumulation of ethidium bromide vs compound concentration). Enzyme-Linked Immunosorbent Assay (ELISA) of Human IL1β in Differentiated THP-1 Cells. The human monocytic cell line THP-1 was maintained in suspension culture at a density of 5 × 104 to 1 × 106 cells/mL in RPMI 1640 medium supplemented with 10% (v/v) FBS, 2 mM L-glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL). THP-1 cells were incubated at 37 °C in a humidified atmosphere under 5% (v/v) CO2. New cell line stocks were thawed and cultured every 8 weeks. All studies were performed at room temperature, and the final assay volume was 150 μL. Human monocytic leukemia THP-1 cells were plated onto 96-well plates to 1.5 × 105 cells/150 μL/well. To differentiate THP-1 cells into macrophages, 25 ng/mL LPS and 10 ng/mL IFN-γ were added to the cells for 4 h. Differentiated cells were incubated with various concentrations of 2,5-dioxoimidazolidine derivatives, and the reference compound 1 at 37 °C for 30 min and then with 1 mM BzATP at 37 °C for an additional 30 min. Next, the culture media were collected by centrifugation at 1000 rpm for 5 min, and the supernatants were stored at −70 °C. The level of IL-1β in the media was determined by ELISA using antihuman IL-1β antibody as a capture antibody and a biotinylated antihuman IL-1β antibody as a detection antibody (BD Biosciences). Recombinant human IL-1β was used as a standard (BD Biosciences). ELISA was performed as described in the manufacturer’s instructions. These results are expressed as a percentage of the maximal BzATP in terms of the absolute amount of IL-1β released, and the IC50 values were calculated using nonlinear regression analysis (i.e., percentage IL-1β release vs compound concentration). A basal level of IL-1β was subtracted from the data in the normalization calculations. ELISA of Mouse IL-1β in J774.A1 Cells. J774 A.1 macrophages were seeded at 3.0 × 105 cells/well in a 48-well dish. The cells were preincubated with 1 μg/mL LPS (E. coli 0111:B4; Sigma-Aldrich, St. Louis, MO) for 5 h, and further, the cells were washed with serum-free medium and incubated with compound 21w, 5 and ATP (catalog no. A6419, Sigma-Aldrich, St. Louis, MO) for 30 min. IL-1β secretion

were analyzed in culture supernatants by IL-1β (mouse) ELISA kit (ENZO, Farmingdale, NY). Long-Term Potentiation (LTP)-Induced Neuropathic Pain. The LTP induction model for the antipain effect was designed ex vivo, according to the reported method.53 Live spinal cords were prepared in the rats by laminectomy. Spinal cords were sliced 400 μm thick and stained using voltage sensitive dye (Di-4-ANEPPS). The suction electrode was used to generate the electric pulse. In the dorsal horn neuron, the change of the optical signal was recorded before and after the induction of LTP. The 2 Hz (0.5 ms pulse)-induced LTP was retained for 2 min. The compound was administered after LTP induction, and the antipain activity of the compound was recorded. Carrageenan-Induced Paw Edema. Sprague−Dawley (SD) male rats (150 ± 5 g, Joong-Ang Experimental Animals Company, Seoul, Korea) were used. Edema was induced in the left hind paw of the rats by subcutaneous injection of 0.1 mL of 1% carrageenan into the footpad. The paw volume of each rat was measured before a carrageenan injection using a plethysmometer (UGO Basil, Italy). The compound was dissolved in 50% water, 40% PEG400, 5% Tween 80, and 5% ethanol and intraperitoneally injected at 1 h before carrageenan injection. The relative paw edema increase was the percent increase compared to the initial value of paw edema volume before carrageenan injection. Type II Collagen (CII)-Induced Arthritis and Pathological Assessment. The institutional animal care and utilization committee of Chosun University approved all of the animal procedures used in this study. Male DBA/1J mice (Joong-Ang Experimental Animals Company, Seoul, Korea), age 8 weeks, were used. Bovine CII was dissolved in 0.1 M acetic acid and emulsified in an equal volume of complete Freund’s adjuvant. The mice were immunized intradermally at the base of the tail with 100 μL of an emulsion-containing 150 μg CII. On day 21, the mice were boosted intradermally with 100 μg CII and monitored for arthritis development for 10 days. The compound was dissolved in solubilization solvent (PEG400, Tween 80, ethanol, and sterile water) and intraperitoneally injected from day 22 every other day (4 injections). The mice were sacrificed on day 10 after the second CII booster. The left hind limbs, including the paws and ankles, were dissected, fixed immediately for 12 h in 10% neutralizing formaldehyde, decalcified in Calci-Clear Rapid (National Diagnostics, Atlanta, GA) for 12 h, and embedded in paraffin. Tissue sections (4 μm) were mounted on common slides for staining with hematoxylin and eosin. A certified pathologist scored the samples in a blinded fashion. The data are expressed as the mean chronic inflammation, fibrosis, articular cartilage damage, synovial proliferation, bone damage, and ankylosis scores. All scores were semiquantitatively indexed based on a scale of 0−3.59 COX-2 and iNOS Immunoblotting. Ankle tissue homogenates were fractionated by 10% gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. The membranes were subsequently incubated with primary antibodies (COX-2 and iNOS) and then with horseradish peroxidase-conjugated secondary antibodies. Finally, the membranes were developed using either 5-bromo-4chloro-3-indoylphosphate and nitroblue tetrazolium or an ECL chemiluminescence detection kit. Intravenous PK Study. Compound 21w dissolved in PEG400 (40%), EtOH (5%), Tween80 (5%), and distilled water (50%) at doses of 20 mg/kg was administered over 1 min via the jugular vein of each rat. Approximately 0.15 mL aliquot of blood was collected via the carotid artery at 0 (to serve as a control), 1 (at the end of the injection), 5, 15, 30, 60, 90, 120, 180, 240, 360, 480, and 1440 min after the start of the intravenous injection of compound 21w. Approximately 0.3 mL aliquot of heparinized NaCl-injectable solution (50 units/mL) was used to flush the cannula immediately after each blood sampling to prevent blood clotting. Blood samples were immediately centrifuged, and an aliquot of each plasma sample was stored at −70 °C freezer until used for the HPLC analysis of compound 21w. Intraperitoneal PK Study. Compound 21w (the same solution used in the intravenous study) at doses of 10 mg/kg was administered intraperitoneally to rats. The blood sampling times were 0 (to serve as a control), 5, 15, 30, 60, 90, 120, 180, 240, 360, 480, and 1440 min 2131

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after intraperitoneal administration of compound 21w. The other procedures were similar to those of the intravenous study.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Detailed information in Section A: the synthesis of starting materials (30−35); 1H spectra of the mosher ester compound, 21b; chiral phase HPLC separation of (±)-21w by analytical HPLC system; selectivity data for compound 21w; competitive antagonism of 21w; and analytical data of the HPLC analysis of final compounds for purity. Section B: the synthesis of derivatives of 21w (36−40) for the crystallization; and chiral phase HPLC separation of (±)-21w by semipreparative HPLC system. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: 82-62-970-2502. Fax: 82-62970-2484. *E-mail: [email protected]. Author Contributions ▽

J.-H.P. and G.-E.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. W. Stuhmer and Dr. F. Soto in the Max-Plank Institute for generously providing the cDNA of the hP2X3 receptor. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (Grant NRF-2014R1A2A1A11052300).

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ABBREVIATIONS: ATP, adenosine-5′-triphosphate Boc, tert-butoxycarbonyl BzATP, 2′(3′)-O-(4-benzoylbenzoyl)-ATP DCE, 1,2-dichloroethane DCM, dichloromethane DMEM, Dulbecco’s Modified Eagle Medium DMF, dimethylformamide EDC, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide ELISA, enzyme-linked immunosorbent assay ERK1/2, extracellular regulated kinases 1/2 FAB, fast atom bombardment FBS, fetal bovine serum HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC, high-pressure liquid chromatography HRMS, high-resolution mass spectrometry IFN-γ, interferon-γ IL-1β, Interleukin-1β KN62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine LPS, lipopolysaccharide LTP, long-term potentiation MAPK, mitogen-activated protein kinase P2X7R, P2X7 receptor RPMI 1640, Roswell Park Memorial Institute 1640 medium SAR, structure−activity relationship TEA, triethylamine TFA, trifluoroacetic acid 2132

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published ASAP on February 23, 2015. Corrections were made to Tables 2 and 6, and the revised version was reposted on February 26, 2015.

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DOI: 10.1021/jm500324g J. Med. Chem. 2015, 58, 2114−2134

Discovery of Novel 2, 5-Dioxoimidazolidine-Based P2X7 Receptor

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