Boron-Based Drug Design.

Personal Account DOI: 10.1002/tcr.201402100

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Boron-Based Drug Design Hyun Seung Ban[a] and Hiroyuki Nakamura*[b] Biomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Republic of Korea) [b] Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503 (Japan), E-mail: [email protected]

[a]

Received: December 19, 2014 Published online: ■■

ABSTRACT: The use of the element boron, which is not generally observed in a living body, possesses a high potential for the discovery of new biological activity in pharmaceutical drug design. In this account, we describe our recent developments in boron-based drug design, including boronic acid containing protein tyrosine kinase inhibitors, proteasome inhibitors, and tubulin polymerization inhibitors, and ortho-carborane-containing proteasome activators, hypoxiainducible factor 1 inhibitors, and topoisomerase inhibitors. Furthermore, we applied a closododecaborate as a water-soluble moiety as well as a boron-10 source for the design of boron carriers in boron neutron capture therapy, such as boronated porphyrins and boron lipids for a liposomal boron delivery system. Keywords: boron, boronic acids, carboranes, drug design, inhibitors

1. Introduction The use of elements that are not generally observed in a living body possesses a high potential for the discovery of new biological activity in pharmaceutical drug design. Boron is one of these elements and is found as a micronutrient necessary for plant growth in some cases. Boron has atomic number 5 and a vacant p orbital that readily accepts electrons from a donor molecule to interconvert with ease between the neutral sp2 and the anionic sp3 hybridization states (Figure 1). If a boron atom can be logically introduced into a biologically active molecule at a position that is located close to a donor region in the target protein, interactions not only through conventional hydrogen bonds but also through covalent bonds to the target protein can be expected and these bimodal interactions would produce a potent biological activity (Figure 2). For this reason, various boronic acid containing enzyme inhibitors have been reported

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so far.[1–3] In 2003, bortezomib, a dipeptide boronic acid, was approved by the FDA for the clinical treatment of multiple myeloma in the United States (Figure 3).[4,5] This was a firstin-class proteasome inhibitor as well as the first boroncontaining drug on the market. The X-ray crystal structure of proteasome in complex with bortezomib suggests that bortezomib interacts with the 20S proteasome, including a covalent bond formation between the boronic acid moiety of bortezomib and the hydroxyl group of Thr-1 at the chymotrypsin-like activity of the 20S proteasome.[6] In 2014, tavaborole, a benzoxaborole, was approved by the FDA for the clinical treatment of onychomycosis of the toenail in adults (Figure 3).[7,8] The X-ray structural analysis of aminoacyltRNA synthetase in complex with tavaborole reveals that tavaborole forms a stable adduct with tRNALeu in the editing

© 2015 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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Fig. 1. Outline of boron-based drug design.

active site of the enzyme through the boron atom of tavaborole and the 2′- and 3′-oxygen atoms of the tRNA 3′-terminal adenosine (A76).[9] Furthermore, boron forms stable three-center twoelectron (3c2e) boron–hydrogen–boron bonds that produce a wide variety of stable boron clusters.[10] For instance, carboranes consisting of two carbon atoms and ten boron atoms have three isomers, ortho, meta, and para, and these boron clusters are highly hydrophobic. Therefore, they can be used as a hydrophobic pharmacophore.[11] In contrast, a carborane consisting of a carbon atom and eleven boron atoms and a dodecaborate consisting of twelve boron atoms are singly

and doubly negatively charged ionic boron clusters, respectively, and highly hydrophilic.[12] Therefore, these clusters can be used as a water-soluble function in the drug design. Indeed, based on these properties, several boron-cluster-containing compounds that possess unique biological activities have been developed. Endo and co-workers were the first to introduce carboranes as a hydrophobic pharmacophore in drug design. They used 1-phenyl-substituted carboranes as steroid analogues, where a carborane formally replaces the C and D rings.[13,14] Hawthorne and co-workers developed carboranecontaining analogues of the promising nonsteroidal antiinflammatory drug (NSAID) pharmaceuticals with decreased

Hyun Seung Ban received his Ph.D. in 2006 from the Graduate School of Pharmaceutical Sciences, Tohoku University, under the direction of Professor Kazuo Ohuchi. He then joined Professor Hiroyuki Nakamura’s group at Gakushuin University as an assistant professor during 2006–2011. He has been working at Korea Research Institute of Bioscience and Biotechnology (KRIBB) as a researcher since 2011. His research interests include cancer biology, medicinal chemistry, and chemical biology.

Hiroyuki Nakamura received his Ph.D. from Tohoku University under the supervision of Professor Yoshinori Yamamoto in 1996. He became an assistant professor at Kyushu University (1995–1997) and at Tohoku University (1997–2002). He worked as a visiting assistant professor at the University of Pittsburgh with Professor D. Curran (2000–2001). He was appointed as an associate professor at Gakushuin University in 2002 and promoted to professor in 2006. In 2013, he was appointed as professor at Tokyo Institute of Technology. He received the Chemical Society of Japan Award for Young Chemists in 1999 and the Award of the Japanese Society for Molecular Target Therapy of Cancer in 2007. His research interests include synthetic methodology, medicinal chemistry, chemical biology, and neutron capture therapy.

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Fig. 2. Interaction of a target protein with a carboxylic acid through hydrogen bonds (left) and with a boronic acid through both hydrogen and covalent bonds (right).

Fig. 3. Structures of boron-containing drugs on the market.

cyclooxygenase activity but the retention of similar efficacy as inhibitors of transthyretin dissociation.[15] Carboranecontaining nonsecosteroidal vitamin D receptor agonist[16] and carbonic anhydrase inhibitors[17] have been recently developed and their binding modes to the target enzymes were clarified by X-ray structural analysis. Metallacarboranes were also found to be specific and potent human immunodeficiency virus (HIV) protease inhibitors. According to X-ray structural analysis, two molecules of cobalt bis(1,2-dicarbollides) bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3′ subsites of HIV protease.[18] Another unique feature of boron is the neutron capture reaction. The nuclear reaction of the boron-10 isotope and a thermal neutron produces an alpha particle and recoils a lithium-7 atom bearing approximately 2.4 MeV. The alpha particle and lithium ion dissipate their kinetic energy before traveling one cell diameter, affording the potential for precise cell killing. If the compound containing the boron-10 isotope can be accumulated into tumor tissue selectively, tumor cells will be killed by neutron irradiation without effecting serious damage to normal tissues.[19] Boron neutron capture therapy

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(BNCT)[20,21] is based on this unique nuclear reaction of the boron-10 isotope. Two boron compounds have been used in BNCT, mercaptoundecahydrododecaborate (B12H11SH, BSH)[22] and l-para-boronophenylalanine (l-BPA).[23] BSH was the first boron compound that led to the successful treatment of malignant glioma, as reported by Hatanaka.[24] Accumulation of BSH into brain tumors is considered to be evidence of it passing through the blood–brain barrier.[25] l-BPA was first introduced into the clinical treatment of melanoma by Mishima[26] and it has been widely used for the treatment of not only melanoma but also malignant glioma[27,28] and head and neck cancer,[29] because it can be taken up selectively by tumor cells through an amino acid transporter.[30,31] Since 2012, accelerator-based BNCT[32] has been undergoing phase I clinical study for the treatment of brain tumor and head and neck cancer patients in Japan. Our strategy for the design of boron compounds is based on these unique properties unlike usual biologically active compounds. In this Personal Account, we describe boroncontaining biologically active compounds that have been recently synthesized in our laboratory.



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2. Boron-Based Drug Design 2.1. Tyrosine Kinase Inhibitors Protein tyrosine kinases (PTKs) play a fundamental role in the cell cycle, cell migration, cell metabolism and many other substantial cell functions. The uncontrolled activation of PTKs is often associated with uncontrolled cell growth and tumor progression; thus, these kinases have been investigated as potential targets for cancer therapy.[33] In the last decade, many inhibitors of PTKs have been discovered and several of them have been approved for clinical treatment of cancer patients. Gefitinib[34,35] and erlotinib[36,37] are inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase and have been approved for non-small-cell lung cancer (NSCLC) therapy. Lapatinib is a dual reversible ErbB inhibitor with potent activity toward EGFR and HER2 kinases, and has been approved for breast cancer therapy.[38] Although the therapeutic response to these inhibitors can persist for as long as two to three years, most patients who show recurrence ultimately develop acquired resistance to these agents mainly due to a mutation, T790M, within the EGFR kinase domain. In this regard, irreversible EGFR inhibitors have attracted attention in recent years because of their potential to inhibit the activity of EGFR tyrosine kinase with the T790M mutation.[39] Various irreversible EGFR inhibitors have been investigated, including afatinib,[40] which was approved for the clinical treatment of metastatic NSCLC in 2013. Our idea is the introduction of a boronic acid moiety into the framework of tyrosine kinase inhibitors, expecting a new stable interaction between a boron atom of the inhibitor and a donor region of the tyrosine kinase pocket through a covalent bond. We first focused on the active pharmacophore of lavendustin A (indicated in bold in Figure 4), first isolated

Fig. 4. Structures of the active pharmacophore of lavendustin A and its boron analogues.

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from a butyl acetate extraction of Streptomyces griseolavendus culture filtrate and composed of two aromatic rings linked by a methyleneamino chain.[41,42] We synthesized a series of aminoboronic acid analogues 1–3 by reductive amination from the corresponding anilines and aldehydes in the presence of NaCNBH3 in MeOH, and their inhibitory activities against EGFR, vascular endothelial growth factor receptor-1 (VEGFR1/Flt-1) protein tyrosine kinases, and various protein kinases, PKA, PKC, PTK, and eEF2K, were evaluated. Although compound 1 did not show significant kinase inhibition at 100 μM concentration, compounds 2 and 3 selectively inhibited EGFR and VEGFR-1 tyrosine kinases, respectively, at 1.0 μM concentration.[43] We next focused on the 4-anilinoquinazoline framework, a common structure observed in gefitinib and erlotinib. According to the X-ray structural analysis of EGFR tyrosine kinase in complex with erlotinib, the thiol of Cys-797 and the carboxylate of Asp-800 are in fact located near the methoxyethoxy group substituted at the 6-position of erlotinib,[44] as shown in Figure 5. Furthermore, the hydroxyl of Thr-790 is located near the aniline group. We decided to introduce a boron moiety at the 6-position of the quinazoline framework and the aniline group to form a stable interaction through covalent bonds a, b, or c. We synthesized various boron-conjugated 4anilinoquinazolines as shown in Figure 6.[45] The inhibitory activity of the boron-conjugated 4-anilinoquinazolines against EGFR, HER2, Flt-1 (VEGFR-1 tyrosine kinase catalytic domain), and KDR (VEGFR-2 tyrosine kinase catalytic domain) tyrosine kinases was determined. As shown in Table 1, most of the boronic acid containing 4-anilinoquinazolines selectively suppressed the activity of EGFR tyrosine kinase without affecting the activity of HER2, Flt-1 or KDR kinases. Their IC50 values against EGFR tyrosine kinase were lower than 1 μM. These results indicate that the conjugation of a

Fig. 5. The X-ray structural analysis of EGFR tyrosine kinase in complex with erlotinib (gray) and the design of boronic acid containing 4-anilinoquinazolines.



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Fig. 6. Structures of boronic acid containing 4-anilinoquinazolines.

Table 1. Effects of the boron-conjugated 4-anilinoquinazolines on tyrosine kinase activity of EGFR, HER2, Flt-1, and KDR.

Compound 4 5 6 7 8 9 10 Tarceva

Inhibition at 1 μM (%) / IC50 (μM)[a] EGFR HER2 Flt-1 KDR 55 / 0.64 ± 0.02 53 / 0.58 ± 0.01 63 / 0.27 ± 0.02 65 / 0.20 ± 0.02 45 / nd 20 / nd 55 / 0.85 ± 0.03 82 / 0.06 ± 0.07

– / nd[b] – / nd – / nd – / nd – / nd – / nd – / nd – / nd

– / nd – / nd – / nd – / nd – / nd – / nd – / nd – / nd

11 / nd 6 / nd – / nd – / nd – / nd – / nd – / nd 40 / nd

[a] The drug concentrations required to inhibit the phosphorylation of the poly(Glu:Tyr) substrate by 50% (IC50) were determined from semilogarithmic dose–response plots, and results represent mean ± s.d. of triplicate samples. [b] –, no inhibitory effect at 1 μM; nd, not determined.

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boron moiety to the 6-position of 4-anilinoquinazolines has a selective inhibitory activity toward EGFR kinase. Among the boronic acid conjugated 4-anilinoquinazolines (4–10) synthesized, compounds 8 and 9 weakly inhibited EGFR tyrosine kinase (45 and 20%, respectively) at 1 μM. Compounds 6 and 10 showed more potent inhibitory activity compared to their ester derivatives 4, 5, and 9. Since the boron moiety may be located in the hydrophilic region surrounded by Cys-797 and Asp-800 in the binding formation to EGFR kinase, the boronic acids are preferable to the corresponding boron esters due to their steric and hydrophilic properties. In contrast, boronic acid 8 showed less potent inhibition because the linker between the boronic acid and the quinazoline ring may be too short to occupy the hydrophilic region. Interestingly, the prolonged inhibition ability of boronic acid 10 toward EGFR tyrosine kinase was investigated by experiment using A431 exposed to the compounds and washed prior to the assay. Indeed, boronic acid 10 inhibited EGFR tyrosine kinase activity in a concentration- and timedependent manner, suggesting that this compound irreversibly suppresses EGFR tyrosine kinase. Our quantum mechanical



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docking simulation revealed that the boronic acid moiety of 10 formed a covalent B–O bond with Asp-800 in addition to hydrogen bonds with Asp-800 and Cys-797, which may cause the prolonged inhibition of EGFR tyrosine kinase by 10.[45] Next, we found that the selective inhibition of EGFR and VEGFR tyrosine kinases was controlled by a boronic acid substituent at the 6-position (11 and 12) or aniline ring (13 and 14) of the 4-anilinoquinazoline framework (Figure 7). The inhibitory activity of the boron-conjugated 4-anilinoquinazolines 11–14 against EGFR, HER2, Flt-1, and KDR tyrosine kinases is shown in Table 2.[46] The boronconjugated 4-anilinoquinazolines 11 and 12, which have a boronate ester or a boronic acid group substituted at the C-6 position of the quinazoline framework, selectively suppressed EGFR tyrosine kinase activity without inhibiting HER2, Flt-1 or KDR kinases, and their IC50 values against EGFR tyrosine kinase were in the range of 0.46–0.80 μM. On the contrary, compounds 13 and 14, which have a boronic acid group substituted at the aniline ring of the quinazoline, displayed selective inhibition toward KDR tyrosine kinase. In particular, a boronic acid group substituted at the para position of the aniline, such as in compounds 14a–c, leads to potent and significant inhibitory activity toward KDR. The IC50 values of 14a and 14b were 0.036 and 0.037 μM, respectively, which are similar to that of the known KDR inhibitor AAL993[47] (IC50 = 0.014 μM). 2.2. Tubulin Binders Combretastatin A-4 (CA-4) and its phosphate prodrug CA4P exhibit antimitotic activity in various human cancer cell lines

Table 2. Effects of the boron-conjugated 4-anilinoquinazolines on tyrosine kinase activity of EGFR, HER2, Flt-1, and KDR.

Compound 11a 11b 11c 11d 12a 12b 12c 12d 13a 13b 13c 14a 14b 14c Tarceva AAL993

Inhibition at 1 μM (%) / IC50 (μM)[a] EGFR HER2 Flt-1 KDR 0.59 ± 0.03 0.57 ± 0.04 0.49 ± 0.08 0.52 ± 0.07 0.65 ± 0.02 0.80 ± 0.07 0.46 ± 0.05 0.51 ± 0.01 46 / nd 24 / nd 45 / nd 40 / nd 36 / nd 28 / nd 0.047 ± 0.003 30 / nd

31 / nd – / nd[b] – / nd – / nd 15 / nd – / nd – / nd – / nd – / nd – / nd – / nd – / nd – / nd – / nd – / nd – / nd

12 / nd 8 / nd – / nd – / nd 7 / nd – / nd – / nd – / nd – / nd – / nd 19 / nd 49 / nd 41 / nd – / nd – / nd – / nd

– / nd – / nd – / nd – / nd – / nd – / nd – / nd – / nd 0.39 ± 0.03 0.19 ± 0.02 0.18 ± 0.02 0.036 ± 0.006 0.037 ± 0.012 0.86 ± 0.12 38 / nd 0.014 ± 0.002

[a] The drug concentrations required to inhibit the phosphorylation of the poly(Glu:Tyr) substrate by 50% (IC50) were determined from semilogarithmic dose–response plots, and results represent mean ± s.d. of triplicate samples. [b] –, no inhibitory effect at 1 μM; nd, not determined.

via inhibition of tubulin polymerization.[48] To date, various combretastatin derivatives have been developed. AC-7739 contains the HCl salt of the amine instead of the hydroxyl group (Figure 8). We focused on the aromatic rings of combretastatin A-4 and designed boronic acid analogues. The inhibitory effects of boronic acid analogues on cell growth and tubulin polymerization are summarized in Table 3. Among the boronic acid analogues, meta-substituted boronic acids 17a–d were more effective at inhibiting B-16 cell growth than para-substituted boronic acids 16a and 16b. The inhibitory activity of compound 17c (6.3 nM) was similar to those of combretastatin A-4 (4.6 nM) and amide derivative 15a (8.0 nM). We next performed tubulin polymerization with purified tubulin from porcine brains. Among the boronic acid analogues, compounds 17c and 17d showed significant inhibitory activity with IC50 values of 22 and 21 μM, respectively, although their potency was lower than those of combretastatin A-4 (1.8 μM) and compound 15a (5.0 μM). The discrepancy between boronic acid 17c and combretastatin A-4 for the inhibition of cell growth and tubulin polymerization indicates that 17c may affect cellular processes other than suppression of tubulin polymerization.[49] 2.3. Proteasome Regulators

Fig. 7. Structures of boronic acid containing 4-anilinoquinazolines.

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The ubiquitin–proteasome system is a major proteolytic pathway and plays a pivotal role in cancer through regulation



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Fig. 8. Structures of combretastatin A-4 and boronic acid analogues.

Table 3. Inhibition of cell growth and tubulin polymerization by boronic acid analogues.

Compound

B-16 cell growth IC50 (μM)[a]

Tubulin polymerization IC50 (μM)[b]

15a 15b 15c 16a 16b 17a 17b 17c 17d Combretastatin A-4

0.0080 ± 0.0012 nd 160 ± 15 2.6 ± 0.14 12 ± 0.72 0.49 ± 0.019 1.8 ± 0.13 0.0063 ± 0.0015 0.019 ± 0.0032 0.0046 ± 0.0005

5.0 ± 0.21 100 ± 16 >100 >100 79 ± 7.0 >100 >100 22 ± 2.1 21 ± 2.6 1.8 ± 0.10

[a]

Growth inhibition in mouse B-16 melanoma cells determined by MTT assay. Tubulin polymerization was monitored by measuring the increase in absorbance values (350 nm), and the drug concentration needed for 50% inhibition of tubulin polymerization was determined. nd, not determined.

[b]

of protein degradation such as p53, p27, and Bax.[50] Furthermore, the proteasome pathway activates transcription factor NF-κB via degradation of IκBα. Uncontrolled regulation of NF-κB has been reported in multiple myeloma as well as in various cancer cells.[51] Therefore, inhibition of the proteasome is an attractive approach for anticancer therapy. Until now, various natural and synthetic inhibitors including a peptide epoxyketone (epoxomicin), peptide vinylsulfone (NLVS), peptide aldehyde (MG-132), and peptide boronate

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(PS-341, bortezomib) have been developed.[52] In 2003, PS-341 was approved by the FDA for the treatment of multiple myeloma. The X-ray co-crystal structure revealed that PS-341 binds to the N-terminal threonine residues of three catalytic active sites, β1, β2, and β5, in the proteasome through a B–O covalent linkage, which generates an sp3 hybridized orbital complex.[6] In 2000, Asai and co-workers reported that belactosin A and C isolated from Streptomyces sp. inhibited the chymotrypsin-like activity of the proteasome and suppressed proliferation of HeLa S3 cells.[53] The β-lactone moiety of belactosin covalently binds to the catalytic active site of the proteasome through a nucleophilic ring opening by the hydroxyl group of the N-terminal threonine residue.[54] We focused on the β-lactone moiety and reasoned that replacement with boronic acid would give candidate analogues of belactosins that act as reversible proteasome inhibitors. We therefore synthesized boron peptides as shown in Figure 9 and their biological activities were evaluated. The inhibitory activity of boron peptides against cell growth and proteasome activity is shown in Table 4. The boron peptide 18a significantly inhibited HeLa cell growth, and 18b–d and 19 gave moderate growth inhibition. However, 20a, 20b and 21a–c did not display the inhibitory activity. The boron peptides showed selective inhibitory activity against the proteasome chymotrypsin-like activity (β5). Among the boron peptides, 18a and 19 most potently inhibited the chymotrypsin-like activity, with IC50 values of 0.28 and 0.54 μM, respectively.[55]



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Fig. 9. Structures of boron peptides designed from belactosin analogues.

Table 4. Inhibition of cell growth and proteasome activity by boron peptides.

Compound 18a 18b 18c 18d 19 20a 20b 21a 21b 21c MG-132 PS-341

[b]

Cell growth inhibition 0.35 ± 0.02 2.35 ± 0.59 0.79 ± 0.03 0.66 ± 0.09 5.11 ± 0.31 >10 >10 >10 >10 >10 nd[d] 0.02 ± 0.01

IC50 (μM)[a] β5[c] 0.28 ± 0.04 0.84 ± 0.04 1.50 ± 0.42 1.47 ± 0.30 0.54 ± 0.14 2.74 ± 0.32 4.28 ± 0.08 4.51 ± 0.31 >10 4.12 ± 0.81 0.07 ± 0.03 0.02 ± 0.01

β1[c]

β2[c]

8.54 ± 1.97 9.84 ± 1.17 >10 >10 >10 >10 >10 >10 >10 >10 7.23 ± 1.16 0.68 ± 0.09

>10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10

[a] Concentration required to inhibit cell growth or proteasome activity by 50%. The values are mean ± s.d. of triplicate samples. [b]Growth inhibition in HeLa cells determined by MTT assay. [c]Inhibition of chymotrypsin-like (β5), caspase-like (β1) and trypsin-like (β2) activity of human 20S proteasome. [d]nd, not determined.

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carborane derivatives represent potential chemical probes for proteasome research. 2.4. HIF Inhibitors

Fig. 10. Structure of ortho-carborane derivatives.

On the other hand, proteasome activators have been reported to a lesser extent. Several types of molecules, including denaturing reagent sodium dodecyl sulfate, lipids, peptides, and fatty acids were shown to activate the proteasome.[56] Betulinic acid and its dimethylsuccinyl derivatives activate the chymotrypsin-like and caspase-like activities of 20S proteasome.[57] We discovered that ortho-carborane derivatives have the potential to increase the proteasome activities (Figure 10).[58] The effects of the carborane derivatives on the β1, β2, and β5 activities of 20S proteasome are summarized in Table 5. Among the carborane derivatives, 23c significantly induced β5 activity (358%) along with slight activation of β1 (176%) and β2 (160%), and inhibited HeLa cell growth with an IC50 value of 21.9 μM. Both compounds 24e and 24f also significantly induced β1 and β2 activity, but only 24f also showed a potent inhibitory effect on β5 activity (95.4% inhibition) and HeLa cell growth (IC50 = 20.3 μM). Since small-molecule proteasome activators have not yet been developed, these

Hypoxia-inducible factors (HIF) are heterodimeric (α and β) transcription factors that regulate tumor angiogenesis and progression.[59] Under aerobic conditions, HIF-1α undergoes ubiquitination and proteasomal degradation, whereas under hypoxic conditions, HIF-1α is stabilized and dimerized with HIF-1β. The dimer binds to hypoxia-responsive elements (HREs) in promoters of various genes involved in angiogenesis, iron metabolism, glucose metabolism, metastasis, and cell proliferation/survival. The overexpression of HIF-1α has been observed in various cancers,[60,61] including brain, breast, cervical, esophageal, and ovarian cancers correlated with treatment failure and mortality, as a result of intratumoral hypoxia and genetic alterations affecting key oncogenes and tumor suppressor genes. Therefore, drugs targeting HIF-1 could represent a novel approach to cancer therapy.[62,63] LW6, a phenoxyacetanilide derivative, inhibits HIF-1α transcriptional activity by selectively inhibiting the hypoxiainduced accumulation of cellular HIF-1α protein.[64,65] We focused on the structure of LW6, and synthesized boroncontaining phenoxyacetanilide derivatives as shown in Figure 11.[66] We examined the effect of the newly synthesized boroncontaining phenoxyacetanilide derivatives on the hypoxiainduced transcriptional activity of HIF-1 in HeLa cells stably expressing the HRE-luciferase reporter gene. The results are summarized in Table 6. The pinacol esters 25a–c and boronic acids 26a–c did not inhibit the transcriptional activity of HIF-1. Bulky substituents at the para position of the benzene ring enhanced the HIF-1 inhibitory activity. Among the compounds

Table 5. Proteasome activation and cell growth inhibition of carborane derivatives. Compound[a]

β1 (%)[b]

β2 (%)[b]

β5 (%)[b]

GI50 (μM)[c]

22 23a 23b 23c 24a 24b 24c 24d 24e 24f Betulinic acid

138 ± 6.4 102 ± 5.6 116 ± 18.6 176 ± 1.8 106 ± 12.7 67.1 ± 20.9 111 ± 7.3 97.1 ± 17.3 350 ± 28.4 181 ± 7.7 609.5 ± 24.0

94.0 ± 1.8 76.0 ± 0.8 95.2 ± 0.1 160 ± 2.5 73.7 ± 1.2 81.1 ± 0.5 102 ± 7.0 70.0 ± 3.8 205 ± 26.5 373 ± 49.8 522.8 ± 24.9

103 ± 7.8 124 ± 1.9 106 ± 4.4 358 ± 57.9 91.5 ± 0.8 133 ± 41.4 124 ± 7.2 145 ± 8.5 87.6 ± 3.5 4.5 ± 1.0 545.2 ± 39.6

>100 17.1 ± 3.7 >100 21.9 ± 1.4 56.2 ± 1.9 23.4 ± 0.1 >100 85.7 ± 12.1 >100 20.3 ± 0.8 15.9 ± 0.4

Proteasome activities were measured at 10 μM. [b]Chymotrypsin-like (β5), caspase-like (β1) and trypsin-like (β2) activity of human 20S proteasome. Concentration required to inhibit HeLa cell growth by 50%. The values are mean ± s.d. of triplicate samples.

[a] [c]

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Fig. 11. Structures of HIF inhibitor LW6 and boron-containing phenoxyacetanilide derivatives.

Table 6. Inhibition of HIF-1 transcriptional activity and cell growth.

Compound

HRE-Luc IC50 (μM)[a]

Cell growth inhibition GI50 (μM)[b]

25a 25b 25c 25d 25e 25f 25g 26a 26b 26c 26d 26e 26f 26g LW6

90.6 ± 2.67 91.8 ± 8.24 >100 70.4 ± 1.88 35.3 ± 1.64 3.1 ± 0.15 1.7 ± 0.58 >100 >100 >100 84.8 ± 5.53 40.3 ± 6.77 4.6 ± 0.86 0.7 ± 0.24 3.1 ± 0.07

17.7 ± 0.66 8.6 ± 0.46 5.8 ± 0.82 13.4 ± 0.53 12.1 ± 0.67 16.0 ± 0.47 52.4 ± 2.49 25.7 ± 0.21 14.9 ± 0.23 85.9 ± 0.73 29.2 ± 6.73 21.3 ± 0.40 15.2 ± 1.13 16.2 ± 0.70 51.6 ± 0.89

[a]

HeLa cells stably transfected with HRE-Luc were incubated for 12 h with or without drugs under normoxic or hypoxic conditions. Luciferase assay was performed and the drug concentration required to inhibit the relative light units by 50% (IC50) was determined from semilogarithmic dose–response plots, and results represent the mean ± s.d. of triplicate samples. [b]The drug concentrations required to inhibit the HeLa cell growth by 50% (GI50) were determined.

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Fig. 12. Effect of carboranyl phenoxyacetanilide 26g on hypoxia-induced accumulation of HIF-1α protein and expression of VEGF mRNA in HeLa cells. (A) The levels of each protein were detected by immunoblot analysis with HIF-1α- or HIF-1β-specific antibodies. (B) HIF-1α, VEGF, and GAPDH mRNA expression was detected by RT-PCR.

synthesized, ortho-carborane substituents 25g and 26g showed more potent HIF-1 inhibitory activity than LW6, and the IC50 values were 1.7 and 0.7 μM, respectively. In addition, there was no relationship between HIF-1 inhibitory activity and cell growth inhibition of the compounds synthesized. The inhibition of HIF-1 transcriptional activity by carboranyl phenoxyacetanilide 26g was mediated by suppression of the hypoxia-induced accumulation of HIF-1α protein (Figure 12A). Furthermore, 26g suppressed the hypoxiainduced levels of VEGF mRNA without affecting HIF-1α mRNA expression (Figure 12B). We clarified the mechanism of action of 26g in HIF inhibition. Based on a chemical biology approach, we designed



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Fig. 13. Fluorescence imaging of target protein bound to the chemical probe. (A) Structures of multifunctional probes of 26g (GN26361). (B) HeLa cell lysate was irradiated for 30 min at 360 nm with various concentrations (30, 100, and 300 μM) of each probe. The conjugation of probe and Alexa Fluor 488 azide was performed by click reaction. (C) Recombinant HSP60 (1 μM) was incubated with or without 26g, and then treated with ATP (1 μM). After incubation for 30 min at 37°C, ATP content was determined by luciferase/luciferin reaction.

and synthesized multifunctional chemical probes of 26g substituted with benzophenone for covalent binding with a target protein through photoaffinity labeling and an acetylene moiety for conjugation with an azide-linked fluorophore through the click reaction (Figure 13A). Using these probes, a specific protein bound to the probes was visualized by direct in-gel fluorescence detection and identified as heat shock protein (HSP) 60 by mass spectrometry (Figure 13B). Moreover, 26g (GN26361) significantly inhibited HSP60 chaperone activity at the same inhibitory concentrations for transcriptional activity of HIF-1 and accumulation of HIF-1α protein (Figure 13C). From these results, we clarified an association between HSP60 and HIF-1α.[67] We further designed and synthesized variously substituted ortho-carboranyl phenoxyacetanilides and evaluated their inhibitory activity against HIF activation and HSP60 chaperone activity (Table 7). Among compounds 27a–27e with no substituent at R3 of the ortho-carborane, compounds with a hydroxyl group at R2 (27b and 27c) showed inhibitory activity of HIF similar to LW6. Interestingly, the inhibitory activity was significantly increased when substituents were introduced at the R3 position (27f–m). Furthermore, the introduction of a boronic acid at the R1 position of the aniline ring (27k–m)

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enhanced the inhibitory activity, with IC50 values of 1.4– 1.9 μM (Table 7). The inhibitory effects of the compounds on HSP60 chaperone activity were tested by determination of malate dehydrogenase (MDH) refolding.[68] As shown in Figure 14, compound 26g and compound 27l more potently suppressed HSP60 chaperone activity than the known HSP60 inhibitor ETB (IC50 = 10.9 μM), with IC50 values of 2.5 and 6.8 μM, respectively.[69] Recently, HSP60 has been focused on as a target for anticancer therapy and a biomarker for diagnosis.[70,71] Manassantins are natural products isolated from Saururus cernuus L (Saururaceae) that have potent inhibitory activity against HIF-1 transcriptional activation.[72] Manassantin A consists of a core tetrasubstituted syn,anti,syn-tetrahydrofuran unit with C2 symmetric side chains (Figure 15). We focused on the tetrahydrofuran scaffold in manassantin A, and designed ortho- and meta-carborane-containing derivatives (Figure 15). Among the carboranes synthesized, compounds 28, 29, and 33 significantly suppressed HIF-1 transcriptional activity, with IC50 values of 3.2, 2.2, and 5.1 μM, respectively (Table 8). The meta-carborane framework showed more potent inhibitory activity than the ortho-carborane framework; that is, compound 28 (3.2 μM) versus 29 (2.2 μM) or compound 30



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Table 7. Inhibition of HIF-1 transcriptional activity and HeLa cell growth.

R1

R2

R3


CO2Me CO2Me CO2Et COPh CO2H CO2Et CO2Et CO2Et CO2Et CO2H B(OH)2 B(OH)2 B(OH)2 B(OH)2

H OH OH H H OH OH OH OH OH OH OH OH OH

H H H H H Me Et i-Bu n-Bu n-Bu Me Et i-Bu H

>100 31 ± 0.4 19 ± 2.0 69 ± 3.8 >100 2.2 ± 0.2 1.9 ± 0.4 2.1 ± 0.1 25 ± 4 5.7 ± 2.4 1.9 ± 0.2 1.4 ± 0.2 1.8 ± 0.3 2.2 ± 0.2

Compound 27a 27b 27c 27d 27e 27f 27g 27h 27i 27j 27k 27l 27m 26g (GN26361) [a]

HRE reporter gene assay in HeLa cells. The results represent the mean ± s.d.

(17 μM) versus 33 (5.1 μM). Furthermore, hydroxyl groups at the benzyl position are required for HIF-1 inhibition. In parallel to HIF inhibition, the carboranes inhibited HeLa cell growth.[73] Another study on the application of boron clusters was the replacement of the cis geometry of the stilbene framework by

ortho-carborane in combretastatin A4. As shown in Figure 16, we designed and synthesized ortho-carborane analogues of combretastatin A4. The significant inhibition of HIF-1 transcriptional activity was induced by all synthesized compounds (Table 9). Among them, compounds 36a, 36b, 36d, and 36e more potently suppressed HIF-1 transcriptional activity than HIF inhibitor YC-1, and their IC50 values were 0.4–1.2 μM. Furthermore, by using an ortho-carborane analogue as a chemical probe, we clarified that tubulin was a primary target molecule.[74] 2.5. Topoisomerase Inhibitors Topoisomerase is an enzyme that alters the supercoiling of double-stranded DNA by transiently cutting one or both strands of the DNA.[75] Inhibition of topoisomerase activity causes permanent DNA damage and apoptosis.[76] Therefore, topoisomerase is considered as an attractive target for chemotherapeutic agents. Various topoisomerase inhibitors including camptothecin, doxorubicin, and etoposide have been developed.[77,78] 1,3,5-Triazines are nitrogen-containing cyclic compounds with thermal and chemical stability.[79] Compounds bearing 1,3,5-triazine moieties exhibit various biological activities, such as antitumor,[80] antiprotozoal,[81] antimalarial,[82] and antiviral activities.[83] The alkylating agent hexamethylmelamine (HMM), its analogue trimelamol and PI3K inhibitor ZSTK474 have been developed as antitumor agents (Figure 17). We focused on the 1,3,5-triazine scaffold as a template for the development of BNCT agents, and synthesized ortho-carborane-containing 1,3,5-trizaines.[84,85] Among the compounds synthesized, the cell growth inhibition profile of 37 (TAZ-6) was found to be similar to that of the

Fig. 14. Inhibition of HSP60 activity by ETB, 26g (GN26361) and 27l. HSP60 chaperone activity was determined using MDH as substrate. The IC50 values were calculated for ETB (10.9 μM), 26g (2.46 μM), and 27l (6.80 μM).

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Fig. 15. Structures of manassantin A and carborane-containing derivatives.

Table 8. Effects of carboranes on hypoxia-induced HIF-1 transcriptional activity in HeLa cells.

Compound


Cell growth GI50 (μM)[b]

28 29 30 31 32 33 34 35 YC-1

3.2 ± 1.1 2.2 ± 1.6 17 ± 1.5 >100 84 ± 8.4 5.1 ± 0.6 50 ± 9.1 76 ± 12.0 1.5 ± 0.7

3.8 ± 0.03 7.5 ± 0.3 14.3 ± 0.4 >100 41.7 ± 1.5 13.8 ± 0.7 >100 >100 nd

[a] HRE reporter gene assay in HeLa cells. not determined.

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[b]

HeLa cell growth inhibition; nd,

analogues

Fig. 16. Structures analogues.

of

combretastatin


A4

and

ortho-carborane


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Table 9. Inhibition of HIF-1 transcriptional activity by ortho-carborane analogues.

Compound 36a 36b 36c 36d [a]

essential for the selective inhibition of topoisomerase I and II activity.[86]


Compound

HRE-Luc IC50 (μM)

0.6 1.2 5.1 0.8

36e 36f 36g YC-1

0.4 5.5 4.6 1.5

HRE reporter gene assay in HeLa cells.

topoisomerase II inhibitor ICRF-193 by COMPARE analysis of a panel of 39 human cancer cell lines (JFCR39) with a correlation coefficient (r) value of 0.724 (Figure 17). Based on these results, we further designed and synthesized additional carboranyl triazines (Figure 18) and evaluated their biological activity. Among the compounds synthesized, compounds 38b and 38c showed the most potent cell growth inhibitory activity and their GI50 values were 1.8 and 1.5 μM, respectively (Table 10). Other carboranyl triazines moderately inhibited HeLa cell growth. Furthermore, the inhibitory effects of the carboranyl triazines on topoisomerase I and II were examined. Compounds 38c, 39b, 39c, 39f, and 39j selectively inhibited topoisomerase I activity, and compounds 38a and 38d selectively inhibited topoisomerase II activity. These results suggest that the substituents on the ortho-carborane are

2.6. Boron Cluster Conjugated Porphyrins Photodynamic therapy (PDT) is an emerging therapeutic method for cancer treatment and requires three components: photosensitizer, light and oxygen. Accumulation of the photosensitizer into the tumor followed by illumination with specific light results in reactive-oxygen-induced cancer cell death.[87] Porphyrin-based PDT agents such as hematoporphyrin and Photofrin have been developed. Because of their high tumor selectivity and low cytotoxicity, porphyrin compounds have been utilized not only for PDT but also for BNCT. Among various boronated porphyrins that have been synthesized so far, the tetrakis-carborane carboxylate ester of 2,4-bis(α,βdihydroxyethyl)deuteroporphyrin IX (BOPP)[88] was the only compound to be employed in a phase I clinical study. However, this study failed due to toxic side effects, mainly caused by thrombocytopenia.[89] We considered that these toxic side effects were probably due to the lipophilic carborane clusters in the molecule. Therefore, we designed closo-dodecaborateconjugated water-soluble protoporphyrin derivatives, as shown in Figure 19. The levels of intracellular accumulation of the synthesized boronated porphyrins were determined, as shown in Figure 20. Compounds 41c, 41d, and 42 showed a fourfold increase in boron accumulation relative to other boronated porphyrins

Fig. 17. Structures of antitumor agent 1,3,5-triazines, carboranyl triazine 37 (TAZ-6), and topoisomerase II inhibitor ICRF-193. r means the correlation coefficient between 37 (TAZ-6) and ICRF-193 determined by a panel of 39 human cancer cell lines (JFCR39).

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Fig. 18. Structures of ortho-carborane-conjugated 1,3,5-triazines.

Table 10. Inhibitory activity of ortho-carborane-conjugated 1,3,5-trizaines toward cell growth and topoisomerase I/II.

Compound 38a 38b 38c 38d 39a 39b 39c 39d 39e 39f 39g 39h 39i 39j 39k 39l 40a 40b 40c 40d 37 (TAZ-6) Camptothecin Etoposide

Growth inhibition (GI50/μM)[a]

Topo I inhibition[b]

Topo II inhibition[b]

9.2 ± 1.8 1.8 ± 2.1 1.5 ± 0.5 37.1 ± 12.8 14.2 ± 2.1 14.1 ± 3.7 9.6 ± 0.5 14.7 ± 2.4 40.9 ± 1.5 51.0 ± 7.4 35.1 ± 5.5 16.7 ± 0.4 17.5 ± 0.4 40.1 ± 2.2 >100 15.2 ± 0.2 16.1 ± 5.8 16.8 ± 2.8 30.2 ± 15.8 32.8 ± 5.5 >100 2.5.[91] In order to achieve a sufficient concentration of 10B atoms in a tumor, the liposomal drug delivery system (DDS) has attracted much interest recently.[92] Liposomes are spherical vesicles composed of a unilamellar lipid bilayer and are able to transport their contents to various tumors in a manner that is essentially independent of their contents by the enhanced permeability and retention (EPR) effect.[93] We focused on the structure of phospholipids that consist of a hydrophilic head moiety and two long lipophilic alkyl tails. We designed boron lipids that contain an ionic boron cluster as a hydrophilic head conjugated with two long alkyl chains as shown in Figure 21. We were the first to synthesize nido-carborane lipid 43, which formed stable vesicles as confirmed by transmission electron micrographs.[94] Although the transferrin-conjugated liposomes containing boron lipid 43 accumulated in tumors in tumor-bearing mice, acute toxicity was observed at a dose of 14 mg [10B]/kg.[95] Therefore, we synthesized closo-dodecaborate lipids 44–49, which have different lengths of alkyl tails.[96] The closododecaborate-conjugated cholesterols 50 were also synthesized to be embedded into the liposomal membrane.[97] The liposomes prepared from these boron lipids did not show acute toxicity at a dose of at least 30 mg [10B]/kg.[98] We

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also synthesized fluorescence-labeled closo-dodecaborate lipid 51.[99] The maximum emission wavelength of the 51-labeled liposomes appeared at 531 nm. A preliminary in vivo imaging study of tumor-bearing mice revealed that the 51-labeled liposomes were delivered to the tumor tissue but not distributed to hypoxic regions. These results encouraged us to prepare a BSHencapsulating closo-dodecaborate lipid liposome as a highboron-content liposome.[100] This liposome displayed excellent boron delivery efficacy to tumors: boron concentrations reached 174, 93, and 32 ppm at doses of 50, 30, and 15 mg [10B]/kg, respectively. A significant antitumor effect was observed in mice injected with this liposome, even at the dose of 15 mg [10B]/kg; the tumor completely disappeared three weeks after thermal neutron irradiation. Furthermore, we found that the countercations of the closo-dodecaborates affected both the encapsulation and formation of liposomes. Indeed, the use of spermidinium (spd) as the countercation resulted not only in the formation of high-boron-content liposome solutions but also in efficient boron delivery to tumors.[101] A remarkable antitumor effect was observed in the tumor-bearing mice treated with spd-[10BSH]-encapsulating liposomes at a dose of 30 mg [10B]/kg; 100% of the mice survived up to 100 days after BNCT (Figure 22).

3. Summary and Outlook In conclusion, we have synthesized various boron compounds. Not only boronic acids but also various boron clusters, including carboranes, hydrophobic boron clusters, and closododecaborates, hydrophilic boron clusters, have been demonstrated as alternative possible functions for pharmaceutical drug design. In the last decade, two boron compounds, bortezomib and tavaborole, have appeared on the market for the clinical treatment of multiple myeloma and onychomycosis of the toenail, respectively. The approval of these drugs has sparked a renewed interest in the investigation of boron compounds as drug candidates for a wide range of diseases. In this



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Fig. 22. Survival curve of tumor-bearing mice after thermal neutron irradiation. The irradiation was performed 36 h after injection of closo-dodecaborates (—, spd-BSH: 30 mg [10B]/kg; ○, spd-BSH: 15 mg [10B]/kg; ◊, spd10 B12H11NH3: 15 mg [10B]/kg) for 50 min (1.3–2.2 × 1012 neutrons/cm2). ×, cold control; ●, hot control. Mice were sacrificed when their tumor volumes reached ∼3000 mm3.

regard, boron-based drug design will take on an increasingly important role for further development in medicinal chemistry.

REFERENCES

Fig. 21. Design of boron lipids.

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[1] L. Ciani, S. Ristori, Expert Opin. Drug Discovery 2012, 7, 1017–1027. [2] B. C. Das, P. Thapa, R. Karki, C. Schinke, S. Das, S. Kambhampati, S. K. Banerjee, P. Van Veldhuizen, A. Verma, L. M. Weiss, T. Evans, Future Med. Chem. 2013, 5, 653–676. [3] P. C. Trippier, C. McGuigan, MedChemComm 2010, 1, 183– 198. [4] P. G. Richardson, T. Hideshima, K. C. Anderson, Cancer Control 2003, 10, 361–369. [5] J. Adams, M. Behnke, S. W. Chen, A. A. Cruickshank, L. R. Dick, L. Grenier, J. M. Klunder, Y. T. Ma, L. Plamondon, R. L. Stein, Bioorg. Med. Chem. Lett. 1998, 8, 333–338. [6] M. Groll, C. R. Berkers, H. L. Ploegh, H. Ovaa, Structure 2006, 14, 451–456. [7] A. K. Gupta, D. Daigle, Expert Rev. Anti-Infect. Ther. 2014, 12, 735–742. [8] S. J. Baker, Y. K. Zhang, T. Akama, A. Lau, H. Zhou, V. Hernandez, W. Mao, M. R. Alley, V. Sanders, J. J. Plattner, J. Med. Chem. 2006, 49, 4447–4450. [9] F. L. Rock, W. Mao, A. Yaremchuk, M. Tukalo, T. Crepin, H. Zhou, Y. K. Zhang, V. Hernandez, T. Akama, S. J. Baker, J. J. Plattner, L. Shapiro, S. A. Martinis, S. J. Benkovic, S. Cusack, M. R. Alley, Science 2007, 316, 1759–1761. [10] R. N. Grimes, Carboranes, 2nd edition, Academic Press, Oxford, 2011, pp. 7–20. [11] M. Scholz, E. Hey-Hawkins, Chem. Rev. 2011, 111, 7035– 7062. [12] R. N. Grimes, Carboranes, 2nd edition, Academic Press, Oxford, 2011, pp. 267–299.



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THE CHEMICAL RECORD

[13] Y. Endo, T. Iijima, Y. Yamakoshi, M. Yamaguchi, H. Fukasawa, K. Shudo, J. Med. Chem. 1999, 42, 1501–1504. [14] Y. Endo, T. Iijima, Y. Yamakoshi, H. Fukasawa, C. Miyaura, M. Inada, A. Kubo, A. Itai, Chem. Biol. 2001, 8, 341– 355. [15] R. L. Julius, O. K. Farha, J. Chiang, L. J. Perry, M. F. Hawthorne, Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 4808– 4813. [16] S. Fujii, H. Masuno, Y. Taoda, A. Kano, A. Wongmayura, M. Nakabayashi, N. Ito, M. Shimizu, E. Kawachi, T. Hirano, Y. Endo, A. Tanatani, H. Kagechika, J. Am. Chem. Soc. 2011, 133, 20933–20941. [17] J. Brynda, P. Mader, V. Sicha, M. Fabry, K. Poncova, M. Bakardiev, B. Gruner, P. Cigler, P. Rezacova, Angew. Chem. Int. Ed. 2013, 52, 13760–13763. [18] P. Cigler, M. Kozisek, P. Rezacova, J. Brynda, Z. Otwinowski, J. Pokorna, J. Plesek, B. Gruner, L. Doleckova-Maresova, M. Masa, J. Sedlacek, J. Bodem, H. G. Krausslich, V. Kral, J. Konvalinka, Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 15394– 15399. [19] G. L. Locher, Am. J. Roentgenol. 1936, 36, 1–13. [20] R. F. Barth, J. A. Coderre, M. G. Vicente, T. E. Blue, Clin. Cancer Res. 2005, 11, 3987–4002. [21] T. Yamamoto, K. Nakai, A. Matsumura, Cancer Lett. 2008, 262, 143–152. [22] A. H. Soloway, H. Hatanaka, M. A. Davis, J. Med. Chem. 1967, 10, 714–717. [23] H. R. Snyder, A. J. Reedy, W. J. Lennarz, J. Am. Chem. Soc. 1958, 80, 835–838. [24] H. Hatanaka, J. Neurol. 1975, 209, 81–94. [25] T. Kageji, Y. Nakagawa, K. Kitamura, K. Matsumoto, H. Hatanaka, J. Neuro-Oncol. 1997, 33, 117–130. [26] Y. Mishima, M. Ichihashi, S. Hatta, C. Honda, K. Yamamura, T. Nakagawa, Pigm. Cell Res. 1989, 2, 226–234. [27] Y. Takahashi, Y. Imahori, K. Mineura, Clin. Cancer Res. 2003, 9, 5888–5895. [28] S.-I. Miyatake, Y. Tamura, S. Kawabata, K. Iida, T. Kuroiwa, K. Ono, Neurosurgery 2007, 61, 82–91. [29] I. Kato, K. Ono, Y. Sakurai, M. Ohmae, A. Maruhashi, Y. Imahori, M. Kirihata, M. Nakazawa, Y. Yura, Appl. Radiat. Isot. 2004, 61, 1069–1073. [30] A. Wittig, W. A. Sauerwein, J. A. Coderre, Radiat. Res. 2000, 153, 173–180. [31] G. W. Kabalka, M. L. Yao, Anti-Cancer Agents Med. Chem. 2006, 6, 111–125. [32] M. Suzuki, H. Tanaka, Y. Sakurai, G. Kashino, L. Yong, S. Masunaga, Y. Kinashi, T. Mitsumoto, S. Yajima, H. Tsutsui, T. Sato, A. Maruhashi, K. Ono, Radiother. Oncol. 2009, 92, 89–95. [33] P. Blume-Jensen, T. Hunter, Nature 2001, 411, 355–365. [34] A. E. Wakeling, S. P. Guy, J. R. Woodburn, S. E. Ashton, B. J. Curry, A. J. Barker, K. H. Gibson, Cancer Res. 2002, 62, 5749–5754. [35] M. Fukuoka, S. Yano, G. Giaccone, T. Tamura, K. Nakagawa, J.-Y. Douillard, Y. Nishiwaki, J. Vansteenkiste, S. Kudoh, D. Rischin, R. Eek, T. Horai, K. Noda, I. Takata, E. Smit, S.

Chem. Rec. 2015, ••, ••–••

[36]

[37]

[38] [39]

[40]

[41]

[42] [43] [44] [45] [46] [47]

[48] [49] [50] [51] [52] [53]

[54] [55] [56] [57]

Averbuch, A. Macleod, A. Feyereislova, R.-P. Dong, J. Baselga, J. Clin. Oncol. 2003, 21, 2237–2246. J. D. Moyer, E. G. Barbacci, K. K. Iwata, L. Arnold, B. Boman, A. Cunningham, C. DiOrio, J. Doty, M. J. Morin, M. P. Moyer, M. Neveu, V. A. Pollack, L. R. Pustilnik, M. M. Reynolds, D. Sloan, A. Theleman, P. Miller, Cancer Res. 1997, 57, 4838–4848. F. A. Shepherd, J. Rodrigues Pereira, T. Ciuleanu, E. H. Tan, V. Hirsh, S. Thongprasert, D. Campos, S. Maoleekoonpiroj, M. Smylie, R. Martins, M. van Kooten, M. Dediu, B. Findlay, D. Tu, D. Johnston, A. Bezjak, G. Clark, P. Santabarbara, L. Seymour, N. Engl. J. Med. 2005, 353, 123–132. M. H. Nelson, C. R. Dolder, Ann. Pharmacother. 2006, 40, 261–269. E. L. Kwak, R. Sordella, D. W. Bell, N. Godin-Heymann, R. A. Okimoto, B. W. Brannigan, P. L. Harris, D. R. Driscoll, P. Fidias, T. J. Lynch, S. K. Rabindran, J. P. McGinnis, A. Wissner, S. V. Sharma, K. J. Isselbacher, J. Settleman, D. A. Haber, Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7665–7670. D. Li, L. Ambrogio, T. Shimamura, S. Kubo, M. Takahashi, L. R. Chirieac, R. F. Padera, G. I. Shapiro, A. Baum, F. Himmelsbach, W. J. Rettig, M. Meyerson, F. Solca, H. Greulich, K. K. Wong, Oncogene 2008, 27, 4702–4711. T. Onoda, H. Iinuma, Y. Sasaki, M. Hamada, K. Isshiki, H. Naganawa, T. Takeuchi, K. Tatsuta, K. Umezawa, J. Nat. Prod. 1989, 52, 1252–1257. F. Mu, D. J. Lee, D. E. Pryor, E. Hamel, M. Cushman, J. Med. Chem. 2002, 45, 4774–4785. T. Asano, H. Nakamura, Y. Uehara, Y. Yamamoto, ChemBioChem 2004, 5, 483–490. J. Stamos, M. X. Sliwkowski, C. Eigenbrot, J. Biol. Chem 2002, 277, 46265. H. S. Ban, T. Usui, W. Nabeyama, H. Morita, K. Fukuzawa, H. Nakamura, Org. Biomol. Chem. 2009, 7, 4415–4427. H. Nakamura, R. Horikoshi, T. Usui, H. S. Ban, MedChemComm 2010, 1, 282–286. P. W. Manley, P. Furet, G. Bold, J. Bruggen, J. Mestan, T. Meyer, C. R. Schnell, J. Wood, M. Haberey, A. Huth, M. Kruger, A. Menrad, E. Ottow, D. Seidelmann, G. Siemeister, K. H. Thierauch, J. Med. Chem. 2002, 45, 5687–5693. M. A. Jordan, L. Wilson, Nat. Rev. Cancer 2004, 4, 253–265. H. Nakamura, H. Kuroda, H. Saito, R. Suzuki, T. Yamori, K. Maruyama, T. Haga, ChemMedChem 2006, 1, 729–740. D. Hoeller, I. Dikic, Nature 2009, 458, 438–444. J. Adams, Trends Mol. Med. 2002, 8, S49–S54. A. F. Kisselev, A. L. Goldberg, Chem. Biol. 2001, 8, 739–758. A. Asai, T. Tsujita, S. V. Sharma, Y. Yamashita, S. Akinaga, M. Funakoshi, H. Kobayashi, T. Mizukami, Biochem. Pharmacol. 2004, 67, 227–234. M. Groll, E. P. Balskus, E. N. Jacobsen, J. Am. Chem. Soc. 2008, 130, 14981–14983. H. Nakamura, M. Watanabe, H. S. Ban, W. Nabeyama, A. Asai, Bioorg. Med. Chem. Lett. 2009, 19, 3220–3224. L. Huang, C. H. Chen, Curr. Med. Chem. 2009, 16, 931–939. L. Huang, P. Ho, C. H. Chen, FEBS Lett. 2007, 581, 4955– 4959.



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[58] H. S. Ban, H. Minegishi, K. Shimizu, M. Maruyama, Y. Yasui, H. Nakamura, ChemMedChem 2010, 5, 1236–1241. [59] G. L. Semenza, Nat. Rev. Cancer 2003, 3, 721–732. [60] P. Jaakkola, D. R. Mole, Y.-M. Tian, M. I. Wilson, J. Gielbert, S. J. Gaskell, A. V. Kriegsheim, H. F. Hebestreit, M. Mukherji, C. J. Schofield, P. H. Maxwell, C. W. Pugh, P. J. Ratcliffe, Science 2001, 292, 468–472. [61] M. Ivan, K. Kondo, H. Yang, W. Kim, J. Valiando, M. Ohh, A. Salic, J. M. Asara, W. S. Lane, W. G. Kaelin, Jr., Science 2001, 292, 464–468. [62] H. S. Ban, Y. Uto, H. Nakamura, Expert Opin. Ther. Pat. 2011, 21, 131–146. [63] Y. Xia, H. K. Choi, K. Lee, Eur. J. Med. Chem. 2012, 49, 24–40. [64] K. Lee, J. H. Lee, S. K. Boovanahalli, Y. Jin, M. Lee, X. Jin, J. H. Kim, Y.-S. Hong, J. J. Lee, J. Med. Chem. 2007, 50, 1675–1684. [65] K. Lee, H. S. Ban, R. Naik, Y. S. Hong, S. Son, B.-K. Kim, Y. Xia, K. B. Song, H.-S. Lee, M. Won, Angew. Chem. Int. Ed. 2013, 52, 10286–10289. [66] K. Shimizu, M. Maruyama, Y. Yasui, H. Minegishi, H. S. Ban, H. Nakamura, Bioorg. Med. Chem. Lett. 2010, 20, 1453–1456. [67] H. S. Ban, K. Shimizu, H. Minegishi, H. Nakamura, J. Am. Chem. Soc. 2010, 132, 11870–11871. [68] Y. Nagumo, H. Kakeya, M. Shoji, Y. Hayashi, N. Dohmae, H. Osada, Biochem. J. 2005, 387, 835–840. [69] H. Nakamura, Y. Yasui, M. Maruyama, H. Minegishi, H. S. Ban, S. Sato, Bioorg. Med. Chem. Lett. 2013, 23, 806–810. [70] F. Cappello, F. Angileri, E. C. de Macario, A. J. Macario, Curr. Pharm. Des. 2013, 19, 452–457. [71] F. Cappello, A. Marino Gammazza, A. Palumbo Piccionello, C. Campanella, A. Pace, E. Conway de Macario, A. J. Macario, Expert Opin. Ther. Targets 2014, 18, 185–208. [72] C. F. Hossain, Y. P. Kim, S. R. Baerson, L. Zhang, R. K. Bruick, K. A. Mohammed, A. K. Agarwal, D. G. Nagle, Y. D. Zhou, Biochem. Biophys. Res. Commun. 2005, 333, 1026–1033. [73] H. Minegishi, T. Matsukawa, H. Nakamura, ChemMedChem 2013, 8, 265–271. [74] H. Nakamura, L. Tasaki, D. Kanoh, S. Sato, H. S. Ban, Dalton Trans. 2014, 43, 4941–4944. [75] J. C. Wang, Nat. Rev. Mol. Cell Biol. 2002, 3, 430–440. [76] O. Sordet, Q. A. Khan, K. W. Kohn, Y. Pommier, Curr. Med. Chem.: Anti-Cancer Agents 2003, 3, 271–290. [77] D. B. Khadka, W.-J. Cho, Expert Opin. Ther. Pat. 2013, 23, 1033–1056. [78] C. Bailly, Chem. Rev. 2012, 112, 3611–3640. [79] G. Blotny, Tetrahedron 2006, 62, 9507–9522. [80] R. Menicagli, S. Samaritani, G. Signore, F. Vaglini, L. Dalla Via, J. Med. Chem. 2004, 47, 4649–4652. [81] A. Baliani, G. J. Bueno, M. L. Stewart, V. Yardley, R. Brun, M. P. Barrett, I. H. Gilbert, J. Med. Chem. 2005, 48, 5570–5579.

Chem. Rec. 2015, ••, ••–••

[82] S. Melato, D. Prosperi, P. Coghi, N. Basilico, D. Monti, ChemMedChem 2008, 3, 873–876. [83] Y.-Z. Xiong, F.-E. Chen, J. Balzarini, E. De Clercq, C. Pannecouque, Eur. J. Med. Chem. 2008, 43, 1230–1236. [84] C.-H. Lee, H.-G. Lim, J.-D. Lee, Y.-J. Lee, J. Ko, H. Nakamura, S. O. Kang, Appl. Organomet. Chem. 2003, 17, 539–548. [85] C.-H. Lee, G. F. Jin, J. H. Yoon, Y. J. Jung, J.-D. Lee, S. Cho, H. Nakamura, S. O. Kang, Tetrahedron Lett. 2008, 49, 159– 164. [86] H. Nakamura, A. Shoji, A. Takeuchi, H. S. Ban, J.-D. Lee, T. Yamori, S. O. Kang, Aust. J. Chem. 2011, 64, 1430–1437. [87] T. C. Zhu, J. C. Finlay, Med. Phys. 2008, 35, 3127–3136. [88] J. S. Hill, S. B. Kahl, A. H. Kaye, S. S. Stylli, M. S. Koo, M. F. Gonzales, N. J. Vardaxis, C. I. Johnson, Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 1785–1789. [89] M. A. Rosenthal, B. Kavar, J. S. Hill, D. J. Morgan, R. L. Nation, S. S. Stylli, R. L. Basser, S. Uren, H. Geldard, M. D. Green, S. B. Kahl, A. H. Kaye, J. Clin. Oncol. 2001, 19, 519–524. [90] M. E. El-Zaria, H. S. Ban, H. Nakamura, Chem. Eur. J. 2010, 16, 1543–1552. [91] A. H. Soloway, W. Tjarks, B. A. Barnum, F. G. Rong, R. F. Barth, I. M. Codogni, J. G. Wilson, Chem. Rev. 1998, 98, 1515–1562. [92] H. Nakamura, Methods Enzymol. 2009, 465, 179–208. [93] H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, J. Controlled Release 2000, 65, 271–284. [94] H. Nakamura, Y. Miyajima, T. Takei, S. Kasaoka, K. Maruyama, Chem. Commun. 2004, 1910–1911. [95] Y. Miyajima, H. Nakamura, Y. Kuwata, J. D. Lee, S. Masunaga, K. Ono, K. Maruyama, Bioconjugate Chem. 2006, 17, 1314–1320. [96] J.-D. Lee, M. Ueno, Y. Miyajima, H. Nakamura, Org. Lett. 2007, 9, 323–326. [97] H. Nakamura, M. Ueno, J. D. Lee, H. S. Ban, E. Justus, P. Fan, D. Gabel, Tetrahedron Lett. 2007, 48, 3151–3154. [98] M. Ueno, H. S. Ban, K. Nakai, R. Inomata, Y. Kaneda, A. Matsumura, H. Nakamura, Bioorg. Med. Chem. 2010, 18, 3059–3065. [99] H. Nakamura, N. Ueda, H. S. Ban, M. Ueno, S. Tachikawa, Org. Biomol. Chem. 2012, 10, 1374–1380. [100] H. Koganei, M. Ueno, S. Tachikawa, L. Tasaki, H. S. Ban, M. Suzuki, K. Shiraishi, K. Kawano, M. Yokoyama, Y. Maitani, K. Ono, H. Nakamura, Bioconjugate Chem. 2013, 24, 124–132. [101] S. Tachikawa, T. Miyoshi, H. Koganei, M. E. El-Zaria, C. Vinas, M. Suzuki, K. Ono, H. Nakamura, Chem. Commun. 2014, 50, 12325–12328.



Data-directed drug design.

Parachors in drug design.

PARP1 Inhibitors: antitumor drug design.

Design characteristics for drug nebulizers.

Computer-assisted rational drug design.

From ethnopharmacology to drug design.

Molecular dynamics in drug design.

Peptides and Peptidomimetics for Antimicrobial Drug Design.

Study design and the drug development process.

Drug design and development. A realistic appraisal.

Protein targets for structure-based drug design.

LIGAND-RECEPTOR INTERACTIONS AND DRUG DESIGN.

Volume of Distribution in Drug Design.

Computational Methods Applied to Rational Drug Design.

Improving data mining strategies for drug design.

Non-protein engineering: small drug design.

Isosterism and bioisosterism in drug design.

Steroid receptor structure and antihormone drug design.

Drug design from the cryptic inhibitor envelope.

Helicases, G4-DNAs, and drug design.

TRPV1: A Target for Rational Drug Design.

Rational drug design applied to myeloperoxidase inhibition.

Design and implementation of a laboratory-based drug design and synthesis advanced pharmacy practice experience.

Structural genomics for drug design against the pathogen Coxiella burnetii.