Design, Synthesis, Biological Evaluation, and Structure−Activity

4192 J. Med. Chem. 2009, 52, 4192–4199 DOI: 10.1021/jm9005093

Design, Synthesis, Biological Evaluation, and Structure-Activity Relationship (SAR) Discussion of Dipeptidyl Boronate Proteasome Inhibitors, Part I: Comprehensive Understanding of the SAR of r-Amino Acid Boronates Yongqiang Zhu,*,† Xin Zhao,† Xinrong Zhu,† Gang Wu,† Yuejie Li,† Yuheng Ma,† Yunxia Yuan,† Jie Yang,† Yang Hu,† Li Ai,† and Qingzhi Gao‡ †

Jiangsu Simcere Pharmaceutical Research Institute and Jiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, No. 699-18 Xuan Wu Avenue, Xuan Wu District, Nanjing 210042, PRC, and ‡Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency School of Pharmaceutical Science and Technology, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, PRC Received April 6, 2009

New series of dipeptidyl boronate inhibitors of 20S proteasome were designed and synthesized. The comprehensive understanding of the SAR was obtained by utilizing the variation of four substituents. From the screened compounds in enzyme, novel inhibitors 49 and 50 were identified to be highly potent druglike candidates with IC50 values of 1.2 and 1.6 nM, respectively, which showed better activities than the drug bortezomib on the market. Two hematologic human tumor cell lines, HL-60 and U266, were significantly sensitive to both candidates and showed nearly the same potency as the standard bortezomib with IC50 values less than 10 nM. But as for most of the eight human solid tumor cell lines, both candidates were more potent than the standard with the IC50 value range of 9.8-70 nM. The activity evaluation of the stereoisomers showed that changing R-isomers to S-isomers greatly reduced the potency and even induced inactivity.

Introduction The development and use of proteasome inhibitors have drawn wide attention in recent years both in fundamental and applied sciences.1 Especially, a dipeptidyl boronic acid bortezomib (PS-341, Figure 1) was approved by the FDA for the treatment of patients with relapsed and refractory multiple myeloma (MM, approved in 2003) with at least one prior line of therapy and for the treatment of mantle cell lymphoma (MCL, approved in 2006).2,3 This fact indicated that the proteasome was clinically validated as an effective target for cancer therapy. Three researchers won the 2004 Nobel Prize in chemistry for discovering how the ubiquitin-proteasome pathway (UPPa) regulated the degradation of intracellular proteins with extreme specificity as to target, time and space.4 The pathway plays a central role to recognize and degrade the misfolded and abnormal proteins in most mammalian cells.5 Such a process is very important in maintaining the biological homeostasis and regulation of different cellular processes such as cell differentiation, cell cycle control, antigen processing, and hormone metabolism.6,7 In this pathway, the 26S *To whom correspondence should be addressed. Phone: 86-2585566666-1726. Fax: 86-25-85566666-1835. E-mail: [email protected]. a Abbreviations: SAR, structure-activity relationship; IC50, inhibition constant; 3D-QSAR, three-dimensional quantitative structureactivity relationship; DCC, N,N0 -dicyclohexylcarbodiimide; HOBt, 1-hydroxybenzotriazole; EDC, 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide; MM, multiple myeloma; UPP, ubiquitin-proteasome pathway; MPC, multicatalytic proteinase complex; RP, regulatory particle; CT-L, chymotripsin-like activity; T-L, trypsin-like activity; PGPH, postglutamyl peptide hydrolysis activity; DIPEA, N,N-diisopropylethylamine.

pubs.acs.org/jmc

Published on Web 06/18/2009

proteasome was the main proteolytic component, which is found in all eukaryotic cells and consisted of the cylindershaped multicatalytic proteinase complex (MPC) 20S proteasome and regulatory particle (RP) 19S proteasome. The 19S proteasome locating at each end of the 20S proteasome is made up of 18 subunits, which controls the recognition, unfolding, and translocation of the protein substrates into the lumen of the 20S proteasome.8 X-ray crystallography of 26S proteasome revealed that the 20S proteasome is composed of 28 protein subunits arranged in four stack rings, with each ring comprising seven R- and β-type subunits, following an R1-7β1-7 stoichiometry.9,10 The two outer chambers are formed by R subunits, while the central chamber, containing the proteolytic active sites, is made up of β subunits. Three of the 14 β subunits are responsible for the postglutamyl peptide hydrolysis activity (PGPH, attributed to β1), trypsin-like activity (T-L, β2), and chymotripsin-like activity (CT-L, β5), respectively, and all these three active subunits hydrolyze the amide bond of protein substrates with the hydrophilic γ-hydroxyl group of the N-terminal threonine (Oγ-Thr1). Although bortezomib is a highly potent proteasome inhibitor, treatment with bortezomib resulted in some severe side effects such as neurologic and cardiovascular adverse effects, fatigue, nausea and vomiting, and diarrhea. Therefore, there remains a significant demand to develop new proteasome inhibitors with less toxicity and greater therapeutic index. Since bortezomib was launched, various reversible or irreversible proteasome inhibitors had been developed subsequently (Figure 1).1,7 Most proteasome inhibitors are short peptides harboring an electrophilic group at one end such as aldehyde (MG132, Figure 1), boronic acid (bortezomib, Figure 1), and vinyl sulfone (NLVS, Figure 1), all of which can form covalent r 2009 American Chemical Society

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Scheme 1. General Synthesis of N-Terminal Protected Amino Acids 3a-ua

Figure 1. Structures of currently developed proteasome inhibitors.

bonds with the catalytic Oγ-Thr1 in the three catalytic sites. Some nonpeptidic molecules such as NPI-0052 (in clinical phase I, Figure 1), lactacystin (Figure 1), and omuralide (Figure 1) inhibit the proteasome also by forming covalent adducts.11 We previously reported three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of tripeptide aldehyde proteasome inhibitors12 and subsequently carried out studies on the synthesis, biological evaluation, and SAR discussion of tripeptide boronate and boronic acid proteasome inhibitors,13 and as a continuation of our research work, we recently reported the 3D-QSAR studies of dipeptidyl boronates synthesized by Millennium Pharmaceuticals, Inc.14 However, in the compound libraries, only the substituents at P1, P2, and P4 positions were varied, which could not offer enough information to fully understand the interaction mode between the dipeptidyl boronates and 20S proteasome without the important information about P3 position. What is more, the influence of stereoisomers on the activity of 20S proteasome had never been elucidated. To obtain comprehensive understanding of the effect of variations at P1, P2, P3, and P4 positions on the activity and to summarize an accurate and detailed SAR for the next cycle of design, a large number of new dipeptidyl boronate inhibitors had been designed and synthesized. After biological evaluation of these compounds for β5 subunit of 20S proteasome, comprehensive SAR analysis was carried out on the substituents at P1, P2, P3, and P4 positions and the importance of variations at P3 position was elucidated for the first time. At the same time, the influence of the stereoisomers on the binding of 20S proteasome was also discussed. Finally, the cytotoxic activities of the two most potent inhibitors were assayed on 10 human tumor cell lines to evaluate their potentials as antitumor agents. From the screened compounds, these two candidates were selected and being evaluated in vivo. Results and Discussion Chemistry. The intermediates of N-terminal protected acids 3 were prepared as depicted in Scheme 1. From the natural or unnatural amino acids, methyl esters 2 were obtained using standard peptide synthesis procedures with

a Reagents and conditions: (i) P1COOH, DCC, HOBt, NMM, THF, 0 °C. (ii) (1) 2 N NaOH, acetone, 0 °C; (2) 2 N HCl, ethyl acetate, 0 °C.

N,N0 -dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) as catalytic coupling agents.15 After saponification and acidification, various aicds 3 were gained in high yields and no isomers were detected by chiral HPLC analysis. Whenever possible, commercially available protected amino acids 3r-u were used. The amino boronates 11a-f hydrochloride were the key intermediates in the total synthetic route, which were prepared following procedures reported in the literature (Scheme 2).16,17 The highly optical (þ)-pinanediol 5a and (-)-pinanediol 5b were synthesized according to the reported methods18,19 starting from 98% e.e. (þ)-R-pinene and 80% e.e. (-)-R-pinene, respectively. The two isomers were obtained in 96.5% and 92.9% yields after simple distillation in vacuo, respectively. The dimethyl dichloromethylboronate 7 was prepared by addition of trimethoxyborane to the solution of (dichloromethyl)lithium in THF generated in situ at -100 °C. Transesterification of compound 7 with chiral diols 5 gave dichlorosubstituted 8a-f with the chiral pinanediols as the stereodirected groups in high yields after column chromatography. In the presence of anhydrous zinc chloride as catalyst, monochlorosubstituted boronates 9a-f were obtained with the reaction of 8 and Grignard reagents P3CH2MgBr. It is noteworthy to point out that the bromide anion in the Grignard reagents should be employed instead of chloride in order to decrease the racemation.20 Treatment of monochlorosubstituted boronates 9a-f with lithium bis (trimethylsilyl)amide produced protected amines 10a-f that underwent facile deprotection in dry ethereal HCl solution to generate the desired amino boronates 11a-f as the hydrochloride salts. General synthetic pathway to obtain the dipeptidyl boronates and boronic acids 12-53 (listed in Table 1) was presented in Scheme 3. Coupling of amino boronates 11a-f with various acids 3 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC 3 HCl) and

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Scheme 2. General Synthesis of Amino Boronates 11a-f Hydrochloridea

a Reagents and conditions: (i) OsO4, t-butyl alcohol, trimethylamine N-oxide dihydrate, pyridine, water, reflux, 24 h; (ii) THF, -100 °C; (iii) B(OCH3)3, -100 °C to room temp; (iv) 5a or 5b, THF, room temp; (v) P3CH2MgBr for 9a-e, (CH3)2CHCH2MgBr for 9f, Et2O, anhydrous ZnCl2, -78 °C to room temp; (vi) LiN(SiMe3)2, THF, -78 °C to room temp; (vii) petroleum ether, dry HCl in Et2O, -78 °C to room temp.

HOBt afforded the dipeptidyl boronates 12-47, and the boronate was transesterificated with isobutylboronic acid under acid conditions to give boronic acids. It had been reported that the boronic esters of this nature had the same activity and specificity as their undeprotected counterparts,21 and our previous experimental data also supported it.13 So in this work, we decided to leave the pinanediol group on the boronic ester at the onset of our 20S proteasome assay screening. Only some interesting boronic esters such as 14, 16, and 34 were selected to remove the pinanediol group to prepare boronic acids 48, 49, and 50 for further development. At the same time, to investigate the influence of stereoisomers on the biological activities, boronic acids 51, 52, and 53 were also prepared. Biology. The capacities of dipeptidyl boronates and boronic acids to inhibit the CT-L activity of 20S human proteasome were assayed using appropriate fluorogenic substrates (Table 1). The marketed bortezomib was used as standard. To fully understand SAR of dipeptidyl boronates and boronic acids, various substituents on the P1, P2, P3, and P4 positions were screened. Table 1 summarized the biological results. First, to investigate the influence of substituents at P4 position on the potency, bortezomib and its pinanediol protected boronate 12 were prepared. The biological data

indicated that both compounds nearly showed equal activities (IC50: 1.92 nM vs 1.90 nM). The same cases were also accounted by compounds 16 (1.12 nM) and 49 (1.20 nM), 34 (0.76 nM) and 50 (1.60 nM), as well as 14 (3.21 nM) and 48 (4.37 nM). This result is consistent with the reported one,21 so it is reasonable to screen in vitro such kind of inhibitors without removing the protecting pinanediol group. Second, variations at P1 position were carried out to elucidate the SAR of this position. Aromatic groups at P1 position such as pyrazinyl (12, 1.92 nM) and 5,6,7,8-tetrahedronaphthyl (34, 0.76 nM) were favored to the biological activities. However, addition of an alkyl methyl in this position (39, 2.34 nM) led to a minor loss of potency. All these facts suggested that negative and aromatic substituents were essential to improve the activity of inhibitors. To obtain more potent compounds for drug development, other tetrahedronaphthyl groups at P1 position, such as racemized 1,2,3,4-tetrahedronaphthyl and its two isomers (S)-1,2,3,4-tetrahedronaphthyl and (R)-1,2,3,4-tetrahedronaphthyl, were screened. Among these isomers, the S-one (16, 1.12 nM) showed the most potent inhibitory activity and the R-one (17, 5.17 nM) gave the worst result, and the racemized one (15, 4.18 nM) fell in the middle. However, replacement of negative groups with positive and bulky Boc

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Table 1. Structures of Compounds 12-53, Bortezomib, and Their Inhibition of Human 20S Proteasome

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Table 1. Continued

a

IC50 value obtained for bortezomib under our experimental conditions.

Scheme 3. General Synthesis of Dipeptidyl Pinanediol Boronates and Boronic Acidsa

a Reagents and conditions: (i) EDC 3 HCl, HOBt, DIPEA, CH2Cl2, 15 °C to room temp; (ii) isobutylboronic acid, 2 N HCl, MeOH, hexane.

group dramatically decreased the potency, as evidenced by the loss of potency shown for compounds 41-45, the IC50 values of which were all greater than 10 nM. Substitutions at P2 position generated a distinct SAR (Table 1). Negative phenyl substituent (34, 0.76 nM) at P2 position was much more beneficial to the potency compared with positive methyl (33, 6.96 nM). However, with too much bulky aromatic groups at this position, the potency of these compounds decreased. This is indeed the case for inhibitor 35 (1.34 nM). Compared with compound 34, analogue 35 harboring a much steric naphthyl group nearly exhibited 2fold weaker potency, but as for alkyl substituents at P2 position, it is an exception for compound 32 (1.15 nM). Replacement of two smaller methyl groups of 33 with a much hindered isopropyl (32) improved the inhibition with 6-fold. Incorporation of hydroxyl groups in the 4-position of phenyl ring significantly resulted in diminished potency, which was demonstrated by compounds 41 and 42, presumably because hydrophilic characteristic of the hydroxyl group did not

match the hydrophobic residues of the β5 subunit.10,12 On the basis of these experimental results, it can be concluded that negative and aromatic groups with a proper volume were favored to the potency, which was consistent with our published theoretical results.14 The steric and electronic characteristics of the substituents at P3 position also greatly affected the potency and made a great contribution for SAR analysis, which had never been discussed in detail previously in the literature. Data from Table 1 offered the chance for us to summarize the SAR for this position. On the one hand, bulky groups contributed more to the activity than small ones. For example, steric isopropyl substituted compound 39 (2.34 nM) was 3-fold potent more than n-propyl substituted one (38, 8.46 nM). The different potency between compounds 16 and 18 also accounted for this. On the other hand, it seemed that positive alkyl groups instead of negative aromatic ones were favored to the potency. Both 16 and 18 were substituted by isopropyl and n-propyl group at P3 position, respectively, and displayed more potent activity than phenyl substituted 19. However, incorporation of an electrowithdrawing group in the para-position of phenyl ring at P3 position led to significant improvement of potency. Inhibitor 21 bearing a fluorine atom at 4-position of phenyl ring showed an IC50 value of 1.26 nM, removal of the fluorine atom of 21 to give 19 caused the inhibition to decrease more than 4-fold, and replacement of the negative fluorine atom with a positive methyl substituent in the 4-position of phenyl (20) even resulted in 6-fold loss in enzymatic potency. The isomers of a drug always result in different or even reverse effects such as the famous thalidomide event. So in this work, the corresponding isomers (51, 52, and 53) of compounds bortezomib, 49 and 50, were prepared and

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Table 2. Cellular Activities of Two Drug Candidates and Bortezomib (IC50 (SD), nM) compd

H460

SW-480

SKOV-3

A549

HELA

KB

HepG2

BGC

HL-60

U266

49

36 (3.0)

30 (2)

40 (2)

38.5 (3)

37.5 (2)

70 (2.8)

60 (7)

580 (9.5)

9.55 (0.62)

2.28 (0.18)

50

21.5 (2.6)

30 (1.5)

60 (2.3)

40.5 (4)

23.2 (2.5)

9.8 (1)

13.1 (1.8)

1210 (11.1)

9.25 (0.67)

2.95 (0.1)

bortezomib

115 (20)

50 (10)

130 (5)

255 (7)

92 (10)

59.5 (10)

37.5 (2)

880 (60)

5.50 (0.21)

2.45 (0.04)

biologically evaluated. Intriguingly, the results were surprising that the three isomers showed different influence on the enzymatic potency. Isomers 51 and 52 greatly reduced the IC50 values to 700 and 2290 nM, respectively, which indicated the chirality of these two compounds did not strictly match the active site of biological target 20S proteasome. However, the activity of inhibitor 53 (9.19 nM) and its isomer 50 (1.6 nM) was not significantly different from that of other two pairs. This is presumably due to the proper conjugation between the aromatic phenyl ring and carboxyl bond of this enantiomer. For drug development, the two most potent inhibitors (49 and 50) in enzyme were further tested in 10 human tumor cell lines to determine their effects on cancer cells. The 10 cell lines included two hematologic tumors, such as promyelocytic leukemia cell line HL-60, and multimyeloma cell line U266, and eight solid tumors, encompassing human large cell lung cancer cell line H460, human nonsmall cell lung cancer cell line A549, human colon carcinoma cell line SW480, human ovarian carcinoma SKOV-3, human epithelia carcinoma cell line HELA, oral cancer cell KB, human hepatocellular liver carcinoma cell line HepG2, and human gastric carcinoma cell line BGC-823. The cellular activities of these two compounds and bortezomib were displayed in Table 2. In general, two hematologic tumor cell lines were more sensitive to such kind of inhibitors than the eight solid tumors. All the three inhibitors (49, 50, and bortezomib) inhibited the two hematologic tumor cell lines at the same level with IC50 less than 10 nM, while inhibition of the eight solid tumors was almost more than 10 nM. However, a loss in cellular potency was observed in the solid BGC-823 tumor cell line, which was inhibited at more than 500 nM level by the three inhibitors. In comparison with bortezomib, both compounds 49 and 50 showed much higher activities against the solid tumors. These two candidates significantly inhibited seven solid tumor cell lines at the level of less than 100 nM. Especially for compound 50, it acted on the oral cancer cell KB at 9.8 nM level and inhibited HepG2 at 13.1 nM, which displayed that this compound had the potential for the treatment of some solid tumors, and now these two compounds were being evaluated in vivo for some hematologic and solid tumors. The results will be published later. Conclusion We report here a comprehensive understanding of SAR of a series of R-amino acids composed dipeptidyl boronates proteasome inhibitors for the first time. By varying different substituents at P1, P2, P3, and P4 positions of the backbone, a series of structurally novel compounds were synthesized and biologically evaluated against the 20S proteasome. Detailed SAR discussions indicated that substituents at P3 position were also essential for biological activities in addition to those at P1, P2, and P4 positions, which had never been investigated.

From the highly potent compounds, two candidates 49 and 50 were further tested against 10 human main cancer cell lines. Both inhibitors exhibited IC50 values less than 10 nM activities with the same magnitudes as the marketed drug bortezomib against two hematologic cancer cell lines. For seven solid tumors, the two candidates possessed IC50 values ranging from 9.8 to 70 nM and showed better activities than the drug bortezomib, which indicated their potentials also for use as antisolid tumor agents. At the same time, the activity difference of the stereoisomers was also investigated in this study. It was found that changing R-isomers to S-isomers greatly reduced the potency and even induced inactivity. Nowadays, the in vivo biological investigation as well as the safety evaluation of the two candidates was being conducted both in solid and hematologic tumor models and the results will be reported in the near future. Experimental Section Chemistry. Commercially available reagents were used directly without any purification unless otherwise stated. Absolutely anhydrous solvents were obtained with the proper methods introduced in the literature. Yields refer to chromatographically unless otherwise stated. Reactions were monitored by thin-layer chromatography carried out on silica gel aluminum sheets (60F-254) and RP-18 F254s using UV light as a visualizing agent, 15% ethanolic phosphomolybdic acid, and heat or ninhydrin and heat as developing agent. Column chromatography was performed on 200-300 mesh silica gel and ODS C-18 column. Analytical reverse phase HPLC was run using a Kromasil 100-5C18, 4.6 mm  250 mm column eluting with a mixture of methanol and water containing 0.02% triethylamine and 0.03% trifluoacetic acid. HPLC showed purity of all the final products was greater than 95%. Melting points were obtained on an YRT-3 melting point apparatus and were uncorrected. 1H NMR and 13C NMR spectra were recorded at room temperature on a Bruker Avance 300 or Avance 500 spectrometers. Chemical shifts were reported in ppm (δ units), and tetramethylsilane (TMS) was used as internal reference. Coupling constants (J) are expressed in hertz. The following abbreviations were used to designate the multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet, dd = doublet of doublets. Mass spectra were obtained using Agilent LC-MS (1956B) instruments in electrospray positive and negative ionization modes. High-resolution mass spectra were recorded on a ZAB-HS instrument using an electrospray source (ESI). The synthesis of the target compounds were shown below. A typical procedure for preparation of dipeptide boronates of 13-47 was exemplified by the synthesis of compound 12. Preparation of 48-53 was employed the similar method of bortezomib. For description of general methods and preparation of the other key intermediates 3a-q, 5a, 5b, 8a, 8b, 9a-f, and 11a-f, and structural characterization data for compounds 14-50, see Supporting Information. N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-Hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-3-methylbutyl]amino]carbonyl]-2-phenyl]-2-pyrazincarboxamide (12). To a cooled solution (-5 °C) of pyrazin-L-phenyl alanine (3a) (0.27 g, 1.00 mmol) dissolved in anhydrous CH2Cl2 (50 mL) was added

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HOBt (0.16 g, 1.20 mmol). After 20 min, the temperature of the reaction system was cooled to -15 °C and EDC 3 HCl (0.19 g, 1.00 mmol) was added. Finally the precooled (0 °C) mixture of the known pinanediol boronate amino hydrochloride (11a) (0.30 g, 1.00 mmol) and DIPEA (0.26 mL, 1.48 mmol) in anhydrous CH2Cl2 (10 mL) was poured. The mixture stirred at -15 °C for 1 h and at room temperature for 2 h, finally quenched with water. The aqueous phase was extracted with CH2Cl2 (3  100 mL). Combined organic phase was washed with 10% citric acid, 5% NaHCO3, and brine, dried over anhydrous Na2SO4, filtered, and evaporated to provide crude product. ODS column chromatography using acetonitrile/H2O (3:1) afforded 0.43 g (79.0%) of 12 as a glassy solid. 1H NMR (CDCl3, 500 MHz) δ 0.82-0.85 (-CH3, m, 9H), 1.16-1.51 (-CH3, -CH2, -CH, m, 10H), 1.771.92 (-CH2, -CH, m, 2H), 1.95-2.04 (-CH, m, 1H), 2.16-2.21 (-CH2, m, 1H), 2.29-2.35 (-CH2, m, 1H), 3.12-3.26 (-CH2, -CH, m, 3H), 4.25-4.31 (-CH, m, 1H), 4.78-4.83 (-CH, m, 1H), 5.78-5.94 (-CONH, m, 1H), 7.19-7.31 (-Ph, m, 5H), 8.38 (-CONH, t, J=8.9 Hz, 1H), 8.52-8.54 (Pyz, m, 1H), 8.74 (Pyz, t, J=1.6 Hz, 1H), 9.34-9.35 (Pyz, m, 1H). 13C NMR (CDCl3, 125 MHz) δ 22.00, 22.84, 24.00, 25.48, 26.26, 27.09, 28.59, 35.44, 35.50 (br), 38.21, 38.58, 39.52, 39.91, 51.38, 54.34, 77.94, 85.97, 126.94, 128.58, 129.45, 136.61, 142.70, 144.13, 144.30, 147.37, 162.80, 170.23. MS (ESI) m/z 519.2 [M þ H]þ. HRMS [M þ Na]þ calcd, 541.2962; found, 541.2974. N-[(1S)-1-[[[(1R)-1-[(3aS,4S,6S,7aR)-Hexahydro-3a,5,5-trimethyl-4,6-methano-1,3,2-benzodioxaborol-2-yl]-2-(4-methylphenyl)ethyl]amino]carbonyl]-2-phenyl]-2-pyrazincarboxamide (13). 1 H NMR (CDCl3, 500 MHz) δ 0.83-0.85 (-CH3, m, 3H), 1.15-1.23 (-CH2, m, 1H), 1.27-1.29 (-CH3, m, 3H), 1.371.40 (-CH3, m, 3H), 1.81-1.90 (-CH2, -CH, m, 2H), 1.99-2.03 (-CH, m, 1H), 2.15-2.16 (-CH2, m, 1H), 2.22-2.23 (-CH3, m, 3H), 2.31-2.34 (-CH2, m, 1H), 2.62-2.73 (-CH2, m, 1H), 2.87-2.90 (-CH2, m, 1H), 3.15-3.32 (-CH2, -CH, m, 3H), 4.29-4.31 (-CH, m, 1H), 4.76-4.78 (-CH, m, 1H), 5.82-5.98 (-CONH, m, 1H), 6.84-6.93 (-Ph, m, 4H), 7.18-7.28 (-Ph, m, 5H), 8.22-8.28 (-CONH, m, 1H), 8.51 (-Pyz, t, J=8.0 Hz, 1H), 8.74 (-Pyz, s, 1H), 9.30 (-Pyz, d, J=17.5 Hz, 1H). 13C NMR (CDCl3, 125 MHz) δ 20.98, 24.06, 26.26, 27.17, 28.59, 35.44, 36.05, 38.42, 38.60, 39.34, 51.56, 54.23, 78.08, 86.11, 127.11, 128.72, 128.98, 129.19, 129.49, 129.64, 136.02, 136.45, 142.78, 144.46, 147.50, 162.82, 171.32. MS (ESI) m/z 565.3 [M - H]-. HRMS [MþNa]þ calcd, 589.2963; found, 589.2940. [(1R)-1-[[(2S)-3-Phenyl-2-[(pyrazin-2-carbonyl)amino]-1-oxopropyl]amino]-3-methylbutyl]boronic Acid (Bortezomib). To the solution of 12 (0.30 g, 0.58 mmol) and 2-methylpropylboronic acid (0.30 g, 1.68 mmol) dissolved in methanol (5 mL) and hexane (10 mL) was added 1 N HCl (1.5 mL). The reaction was stirred at room temperature for 18 h. The methanolic phase was washed with hexane (3  10 mL), and the hexane layer was extracted with methanol (315 mL). The combined methanolic layers were evaporated in vacuo, and the residue was dissolved in CH2Cl2 (10 mL). The solution was washed with 5% NaHCO3 (10 mL), and the organic layer was dried over anhydrous Na2SO4, evaporated, and purified with chromatography (CH3OH:CHCl3 = 1:20) to obtain 123 mg (56.4% yield) of a white foam solid. HPLC indicates a purity of 99.4 area %. 1H NMR (CDCl3, 500 MHz) δ 0.69-0.80 (-CH3, m, 3H), 1.111.15 (-CH, m, 1H), 1.23-1.25 (-CH2, m, 2H), 1.79 (-OH, br, 2H), 3.13-3.15 (-CH, m, 1H), 3.19-3.28 (-CH2, m, 2H), 5.20 (-CH, q, J=7.9 Hz, 1H), 7.22-7.29 (-Ph, m, 5H), 7.38 (-CONH, br, 1H), 8.29 (-CONH, d, J = 8.6 Hz, 1H), 8.50 (-Pyz, m, 1H), 8.73 (-Pyz, d, J=2.4 Hz, 1H), 9.19 (-Pyz, d, J=1.3 Hz, 1H). 13C NMR (CDCl3, 125 MHz) δ 22.16, 22.98, 25.35, 38.12, 39.66, 53.08, 127.11, 128.79, 129.45, 135.86, 142.72, 143.83, 144.36, 147.56, 162.90. MS (ESI) m/z 407.2 [M þ Na]þ. HRMS [M þ Na þ 2CH2]þ calcd, 435.2189; found, 435.2147. [(1S)-1-[[(2S)-3-Phenyl-2-[(pyrazin-2-carbonyl)amino]-1-oxopropyl]amino]-3-methylbutyl]boronic Acid (51). HPLC indicates a purity of 97.5 area %. 1H NMR (CD3OD, 500 MHz)

Zhu et al.

δ 0.85-0.89 (-CH3, m, 6H), 1.58-1.59 (-CH2, m, 2H), 1.831.89 (-CH, m, 1H), 2.69-2.72 (-CH2, m, 2H), 3.12-3.23 (-CH, m, 1H), 5.08 (-CH, t, J= 7.4 Hz, 1H), 7.20-7.23 (-Ph, m, 1H), 7.26-7.31 (-Ph, m, 4H), 8.68 (-Pyz, dd, J1 = 1.5 Hz, J2=0.96 Hz, 1H), 8.78 (-Pyz, d, J=2.5 Hz, 1H), 9.16 (-Pyz, d, J = 1.4 Hz, 1H). 13C NMR (CD3OD, 125 MHz) δ 22.45, 23.60, 26.90, 31.99, 38.46, 45.87, 52.73, 128.28, 129.73, 130.51, 137.29, 144.83, 144.88, 145.75, 148.87, 165.31, 176.99. MS (ESI) m/z 435.1 [M þ Na þ 2CH2]þ. HRMS [M þ Naþ 2CH2]þ calcd, 435.2189; found, 435.2141. [(1S)-1-[[(2S)-3-Phenyl-2-[((S)-(-)-1,2,3,4-tetrahedro-naphthoic1-carbonyl)amino]-1-oxopropyl]amino]-3-methylbutyl]boronic Acid (52). HPLC indicates a purity of 98.0 area %. 1H NMR (CD3OD, 500 MHz) δ 0.88-0.89 (-CH3, m, 6H), 1.28-1.29 (-CH, m, 1H), 1.32-1.36 (-CH2, m, 2H), 1.60-1.64 (-CH2, m, 2H), 1.81-1.88 (-CH2, -OH, m, 3H), 2.01 (-OH, br, 1H), 2.66-2.72 (-CH2, m, 2H), 2.76-2.77 (-CH, m, 1H), 3.01 (-CH2, dd, J1=9.1 Hz, J2= 4.7 Hz, 1H), 3.16 (-CH2, dd, J1=6.5 Hz, J2=7.3 Hz, 1H), 3.683.70 (-CH, m, 1H), 4.91 (-CH, dd, J1 = 6.5 Hz, J2=2.4 Hz, 1H), 6.96 (-Ph, d, J=7.6 Hz, 1H), 7.04-7.10 (-Ph, m, 3H), 7.23-7.26 (-Ph, m, 3H), 7.29-7.30 (-Ph, m, 2H). 13C NMR (CD3OD, 125 MHz) δ 22.13, 22.34, 23.71, 26.99, 28.72, 30.17, 30.74, 38.46, 40.86, 44.92, 54.78, 126.39, 126.99, 127.85, 128.19, 129.68, 130.06, 130.41, 135.22, 137.37, 138.84, 177.42, 178.15. MS (ESI) m/z 459.1 [M þ Na]þ. HRMS [M þ Na þ 2CH2]þ calcd, 487.2754; found, 487.2671. [(1S)-1-[[(2S)-3-Phenyl-2-[(5,6,7,8-tetrahedro-naphthoic-1-carbonyl)amino]-1-oxopropyl]amino]-3-methylbutyl]boronic Acid (53). HPLC indicates a purity of 98.7 area %. 1H NMR (CD3OD, 500 MHz) δ 0.90-0.93 (-CH3, m, 6H), 1.29-1.32 (-CH, m, 1H), 1.35-1.40 (-CH2, m, 2H), 1.65-1.69 (-CH2, m, 2H), 1.72-1.75 (-CH2, m, 2H), 2.41-2.45 (-CH2, m, 1H), 2.56-2.59 (-CH2, m, 1H), 2.70-2.76 (-CH2, -CH, m, 1H), 3.04 (-CH2, dd, J1=9.6 Hz, J2=4.2 Hz, 1H), 3.22 (-CH2, dd, J1=7.6 Hz, J2=6.2 Hz, 1H), 5.06 (-CH, dd, J1=6.3 Hz, J2= 3.3 Hz, 1H), 6.97 (-Ph, d, J=7.2 Hz, 1H), 7.05-7.11 (-Ph, m, 2H), 7.26-7.27 (-Ph, m, 1H), 7.30-7.32 (-Ph, m, 4H). 13C NMR (CD3OD, 125 MHz) δ 22.37, 23.85, 24.08, 26.00, 27.02, 27.42, 30.65, 38.30, 40.92, 52.56, 54.77, 125.30, 126.27, 128.20, 129.67, 130.50, 131.94, 135.64, 137.38, 137.65, 139.10, 173.34, 177.50. MS (ESI) m/z 437.1 [MþH]þ. HRMS [M þ Na þ 2CH2]þ calcd, 487.2754; found, 487.2649. Enzyme and Inhibition Asssays. 20S proteasome activity assay kit was purchased from Chemicon (Chemicon, USA). Other reagents and solvents were purchased from commercial sources. In brief, substrates and compounds were previously dissolved in DMSO, with the final solvent concentration kept constant at 3% (v/v). The reaction buffers were (pH 7.5) 20 mM Tris, 1 mM DTT, 10% glycerol, 0.02% (w/v) DS for CT-L activities. Proteasome activity was determined by monitoring the hydrolysis of the fluorogenic substrate, Suc-Leu-Leu-Val-Tyr-AMC (λexc = 360 and λexc = 465 nm for AMC substrates), reacting for 1 h at 37 °C in the presence of untreated (control) or proteasome that had been incubated with different concentration of test compounds. Fluorescence was measured using an Infinite M200 microplate reader (Tecan, Austria). Cell Culture and Cytotoxicity Assays. HL-60 (promyelocytic leukemia cell line), HELA (human epithelia carcinoma cell line), and U266 (multi myeloma cell line) human cell lines were obtained from the American Type Culture Collection (Manassas, VA). H460 (human large cell lung cancer cell line), A549 (human nonsmall cell lung cancer cell line), SW-480 (human colon carcinoma cell line), SKOV-3 (human ovarian carcinoma), KB (oral cancer cell), HepG2 (human hepatocellular liver carcinoma cell line), and BGC-823 (human gastric carcinoma cell line) cell lines were obtained from China Pharmaceutical University. HL60 cell was cultured in IMDM supplemented with 20% fetal bovine serum at 37 °C in 5% CO2. BGC-823, KB, NCI-H460, HepG2, and SW-480 cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. U266 cell

Article

was cultured in RPMI 1640 supplemented with 15% fetal bovine serum at 37 °C in 5% CO2. A549 cell was cultured in F12-k supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. SKOV-3 cell was cultured in McCoy’s 5A supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. HELA cell was cultured in EMEM supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. A standard MTT assay was used to measure cell growth. In brief, a suspension of 3000 cells/150 μL of medium was added to each well of 96-well plates and allowed to grow. Twenty-four hours later, drugs prepared in medium at 10 different concentrations were added to the corresponding plates at a volume of 50 μL per well, and the plates were incubated for 72 h with drugs. Then 20 μL of a solution of 5 mg/mL MTT were added to each well and incubated for another 4 h at 37 °C. Plates were then centrifuged at 1000 rpm at 4 °C for 5 min, and the medium was carefully discarded. The formazan crystals were dissolved in 100 μL of DMSO, and absorbance was read on an Infinite M200 (Tecan, Austria) microplate reader at 540 nm. The result was expressed as the mean IC50 value, which is the average from at least three independent determinations.

Acknowledgment. We thank Prof. Luhua Lai, College of Chemistry and Molecular Engineering, State Key Laboratory for Structural Chemistry of Stable and Unstable Species, Peking University, for her encouragement and manuscript reading. Note Added after ASAP Publication. This manuscript was released ASAP on June 18, 2009 with an incomplete Table 1. The correct version was posted on June 24, 2009. Supporting Information Available: General and experimental procedures, characterization data for 3a-q, 5a, 5b, 8a, 8b, 9a-f, 11a-f, and 14-50. This material is available free of charge via the Internet at http://pubs.acs.org.

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14

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