Design and Synthesis of Dual-Action Inhibitors Targeting Histone Deacetylases and Hmg-Coa Reductase For Cancer Treatment

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Article
Design and Synthesis of Dual-action Inhibitors Targeting Histone
Deacetylases and HMG-CoA Reductase for Cancer Treatment
Jhih-Bin Chen, Ting-Rong Chern, Tzu-Tang Wei, Ching-Chow Chen, Jung-Hsin Lin, and Jim-Min Fang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400179b • Publication Date (Web): 09 Apr 2013
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Page 1 of 50 Journal of Medicinal Chemistry
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4 Design and Synthesis of Dual-action Inhibitors Targeting Histone Deacetylases and
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HMG-CoA Reductase for Cancer Treatment
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14 Jhih-Bin Chen,†,# Ting-Rong Chern,‡,# Tzu-Tang Wei,¶,# Ching-Chow Chen,¶ Jung-Hsin
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17 Lin,*,‡ and Jim-Min Fang*,†
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23 Department of Chemistry, National Taiwan University, Taipei 106, Taiwan.
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‡
26 School of Pharmacy, National Taiwan University, Taipei 100, Taiwan.
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¶
29 Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 100,
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32 Taiwan.
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38 #
These authors contributed equally to this work.
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ACS Paragon Plus Environment

Journal of Medicinal Chemistry Page 2 of 50
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4 ABSTRACT
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7 A series of dual-action compounds were designed to target histone deacetylase (HDAC) and
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3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) by having a hydroxamate group
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14 essential for chelation with the zinc ion in the active site of HDAC and the key structural
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17 elements of statin for binding with both proteins. In our study, the statin hydroxamic acids
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20 prepared by fused strategy are most promising in cancer treatments. These compounds
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23 showed potent inhibitory activities against HDACs and HMGR with IC50 values in nanomolar
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26 range. These compounds also effectively reduced the HMGR activity as well as promoted the
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29 acetylations of histone and tubulin in cancer cells, but were not toxic to normal cells.
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4 INTRODUCTION
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7 Cancer cells may be developed from inherited defects or acquired damages of DNA. Cancer is
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a highly complex multi-genetic disease involving multiple cross-talks between signaling
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14 networks. Using drug-cocktails that combine multiple anti-cancer agents working in different
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17 mechanisms has been a standard treatment of cancers to avoid drug resistance.1–4
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20 Histone acetyltransferases (HATs) and histone deacetylases (HDACs) have recently
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23 emerged as important targets for cancer therapy. Acetylation of the lysine residues on histone
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26 H3 and H4 leads to a loose and active chromatin, which allows access of various transcription
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29 factors to the promoters of target genes. In contrast, deacetylation of the lysine residues
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32 results in a highly compact and transcriptionally inactive chromatin.5 The levels of histone
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35 acetylation and deacetylation are regulated by HATs and HDACs, respectively.6 HDAC
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38 overexpression has been found in a variety of human cancers, including myeloid neoplasia
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41 and solid tumors.7 The association of HDACs with oncogenic DNA-binding fusion proteins
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and other repressive transcription factors constitutively suppresses specific tumor suppressor
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genes.8 Therefore, HDACs represent a rational target for cancer treatment. Several HDAC
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51 inhibitors (HDACi’s) are currently under clinical trials on either monotherapy or combination
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54 therapy for cancer treatment.9, 10 HDACi’s are categorized into four groups: short-chain fatty
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57 acids, hydroxamic acids, cyclic tetrapeptides, and benzamides.8 Among them, the
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60 hydroxamate-containing HDACi’s trichostatin A (TSA) and suberoylanilide hydroxamic acid

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4 (SAHA, Figure 1A) exhibit the most potent efficacy.
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7 In another aspect, statins have recently been shown to be effective for cancer prevention
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in observational, preclinical, and certain randomized controlled studies.11 Statins, such as
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14 lovastatin (Figure 1B) and atorvastatin, are known to reduce serum cholesterol levels through
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17 competitive inhibition at 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). The
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20 HMGR inhibitors (HMGRi’s) are effectively used to decrease the incidence of cardiovascular
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23 and cerebrovascular disorders, and to prevent cardiovascular disease (CVD).12 Statins possess
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26 an established record of safety and efficacy in human CVD prevention.
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29 It has been reported that the combination use of anticancer agents with statins may reduce
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32 side effects to attain better treatment of cancers.13 Furthermore, the in vitro experiment using
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35 combination of HDACi and HMGRi has shown the synergistic induction of apoptosis of
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38 HeLa cells.15 The underlying synergistic mechanism has been proposed that the
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41 down-regulation of geranylgeranyltransferase (GGTase)-I β subunit, caused by HDACi (TSA
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in that study), enhances the depletion of mevastatin-induced geranylgeranylated RhoA.14
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However, the direct HDAC inhibition by statins15 could have also made significant
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51 contributions to this synergism.
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54 Given the aforementioned evidence, we conceived that concurrent inhibition of HDAC
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57 and HMGR would be a promising approach for cancer treatment. However, using
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60 multi-component drug-cocktails for therapeutics has some drawbacks, such as complex

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4 pharmacokinetics, unpredictable drug–drug interaction, and formulation problems due to
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7 different solubilities of individual drugs.16, 17 Alternatively, to design a single compound that
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simultaneously modulates multiple targets, dubbed designed multiple ligand (DML), has
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14 become an emerging paradigm for drug discovery.16, 18 DMLs constructed by incorporation of
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17 HDACi’s into other active agents targeting inosine monophosphate dehydrogenase,19, 20
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20 nuclear vitamin D receptor,21 tyrosine kinase receptor22, 23 or topoisomerase II24 have been
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23 tested in cancer treatments.25
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26 In this study, we designed the dual-action anticancer agents 6a, 6b, 10, and 12–15 by the
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29 knowledge- and structure-based approaches to target both HDAC and HMGR (Figure
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32 1C–E).17, 26
The rationale for the dual-inhibitor design was originated from our previous
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35 modeling work,16 which showed that lovastatin could fit the adjacent transient pocket about 4
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38 Å away from the active site of HDAC. We first designed the HMGR–HDAC dual inhibitors
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41 6a,b by direct connection of lovastatin with a triazole-linked SAHA27 (Figure 1C). According
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to the structural information,28 the hydroxamate group plays an essential role in chelation of
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zinc ion for HDAC inhibition, whereas the hydrophobic moiety of SAHA can be replaced by
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51 lovastatin fragment to accommodate with the deep pocket of HDAC. In the meantime,
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54 compounds 6a,b still retain the HMG-like structure to assure their potency to HMGR.29 These
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57 conjugated molecules were predicted to hold the key interactions with the two enzymes to
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60 exert the desired biological functions. To reduce the molecular weight of dual inhibitor, we

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4 further prepared a merged molecule 10 (Figure 1D), in which the two essential
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7 pharmacophoric elements, hydroxamic acid and hydrophobic moiety, are linked by a short
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aliphatic chain. We finally realized that the aliphatic chains in SAHA and lovastatin could be
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14 overlaid (Figure 1E), so that the fused molecules could act as HMGR–HDAC dual-targeting
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17 inhibitors. We report herein the synthesis, molecular modeling and biological activities of
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20 these conjugated, merged and hybrid HMGR–HDAC inhibitors.
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25 (A) HDAC inhibitors: (B) HMGR inhibitor:
26  OH
H HO O HO
27 N OH
N O HO O
28 O
H O O
29 H
Suberoylanilide hydroxamic acid (SAHA) O O
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H H
31 OH
N N
32 H
N N
33 SAHA analog Lovastatin (1, lactone form) Biologically active form
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(C) Designed conjugated molecules, e.g., 6a and 6b: (D) Designed merged molecules, e.g. 10:
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36 HO O Spacer HO O
Zn2+ binder
37 O O Spacer
O Zn2+ binder O O
38 H H
OH
39 O N O O N N
H H N H H H
40 N N
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5 HN OH
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Hydrophobic region Hydrophobic region
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(E) Designed hybrid HMGRHDAC inhibitors, e.g., 12:
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H
46 N OH
N O
47 H Spacer Zn2+ binder
O SAHA
48 O
OH OH O
49 H
OH
O N
50 H
51 O
OH OH O
52 H Hydrophobic region
53 OH Hybrid HMGRHDAC inhibitor
54 Lovastatin (active form)
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57 Figure 1. Designed dual inhibitors for HMG-CoA reductase (HMGR) and histone deacetylase
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60 (HDAC). (A) Structure of a representative HMGR inhibitor. (B) Structures of representative

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4 HDAC inhibitors. (C) Design of lovastatin derivative bearing hydroxamic acid group by
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conjugation strategy via click reaction. (D) Design of lovastatin derivative bearing
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hydroxamic acid group by merged strategy using a short aliphatic spacer. (E) Design of
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14 lovastatin hydroxamic acid by fused strategy to share a common 6-carbon unit from SAHA
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17 and lovastatin. The hydroxamic acid (red) is a surrogate of the carboxylate group in lovastatin,
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20 and acts as a zinc chelation group for HDAC inhibition. The HMG-like moiety (blue)
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23 provides the appropriate interactions with HMGR and HDAC.
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29 RESULTS AND DISCUSSION
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32 Chemistry. To synthesize the conjugated compounds 6a,b (Scheme 1), lovastatin in the
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35 lactone form was treated with KOH in H2O/MeOH to open the lactone ring and to cleave the
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38 ester bond. The lactone ring was regenerated by treatment with HCl (6 M) for 4.5 h at room
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41 temperature without causing side reactions due to elimination of water molecules. A bulky
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tetrabutyldimethylsilyl (TBS) group was selectively introduced to protect the less hindered
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hydroxyl group, giving intermediate 2. The subsequent treatment of 2 with p-nitrophenyl
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51 chloroformate in the presence of pyridine and 4-dimethylaminopyridine (DMAP) afforded the
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54 carbonate 3. The substitution reactions of 3 with 4-ethynylaniline and 4-ethynylbenzylamine
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57 were carried out to provide the carbamates 4a and 4b in 66% and 90% yields, respectively.
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60 Compounds 4a and 4b were then subjected to the Cu+-catalyzed 1,3-dipolar cycloadditions

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4 (click reactions) with 7-azidoheptanoic acid to give practically pure triazole products 5a and
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5b, which were isolated simply by extraction with EtOAc. The acids 5a and 5b were activated
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by treatment with ethyl chloroformate to form mixed anhydrides, which were reacted in situ
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14 with hydroxylamine to give hydroxamates 6a and 6b in modest yields.
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20 Scheme 1. Synthesis of lovastatin derivatives 6a and 6b bearing hydroxamic acid group by
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23 conjugation strategy.a
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25 HO O TBSO O TBSO O
26 O O O NO2
O O
27 H
iiii
H
iv
H
v
28 H
O
H
OH
H
O O
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31 1 2 3
32 Lovastatin (lactone form)
33 TBSO O TBSO O
34 O
O
35 H
O vi H
O
36 O N n O N n
H H H H
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N O
38 N N
5 OH
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4a, n = 0 5a, n = 0
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41 TBSO O
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O
43 vii O
H
44 O N
45 H H n
O
46 N
N N
47 5 HN OH
48 6a, n = 0
6b, n = 1
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51 Reagents and conditions: (i) KOH, H2O/MeOH, reflux, 8 h; (ii) 6 M HCl, rt, 4.5 h; (iii)
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54 TBSCl, imidazole, CH2Cl2, rt, 5.5 h; 58% for 3 steps; (iv) p-nitrophenyl chloroformate,
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57 DMAP, pyridine, rt, 15 h; 81%; (v) 4-ethynylaniline or 4-ethynylbenzylamine, DMAP,
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60 pyridine, rt, 3–21 h; 66% for 4a; 90% for 4b; (vi) 7-azidoheptanoic acid, CuSO4·(H2O)5,

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4 sodium ascorbate, t-BuOH/H2O, 60 oC, 18 h; (vii) ClCO2Et, Et3N, THF, 0 oC, 10 min; (viii)
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NH2OH·HCl, KOH, THF/MeOH, 0 oC, 15 min; 37% for 6a from 4a; 53% for 6b from 4b.
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14 In another approach (Scheme 2), carbonate 3 was reacted with the allyl ester of
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17 7-aminoheptanoic acid (7) to afford carbamate 8. The allyl group in 8 was removed by the
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20 catalysis of palladium. The acid intermediate 9 was activated with ethyl chloroformate and
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23 reacted with hydroxylamine to give hydroxamate 10.
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29 Scheme 2. Synthesis of lovastatin derivative 10 bearing a hydroxamic acid group by merged
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32 strategy.a
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34 TBSO O
35 3 O
36 O O
+ i H ii
37 O
O N
5
O
H H
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NH2 O
39 5
40 7
8
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42 TBSO O TBSO O
43 O O
O O O O
44 H iii, iv H
OH
45 H
O N
5
OH O N N
5 H
H H H
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48 10
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51 Reagents and conditions: (i) DMAP, pyridine, rt, 2 h; 70%; (ii) Pd(PPh3)4, PPh3, Et3N,
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54 HCOOH, THF, rt, 3 h; (iii) ClCO2Et, Et3N, THF, 0 oC, 10 min; (iv) NH2OH·HCl, KOH,
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57 THF/MeOH, 0 oC, 15 min; 38% for 10 from 8.
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60

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7 As the commercially available lovastatin is in the lactone form, we attempted to explore
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its direct coupling reaction with hydroxylamine to afford lovastatin hydroxamic acid
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14 (lova-HA) without involvement of tedious protection–deprotection procedures.30 In our initial
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17 attempt, hydroxylamine was freshly prepared by neutralization of the hydrochloric salt with a
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20 base (KOH or NaOMe) in MeOH, and used as the nucleophile to react with lovastatin (1 in
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23 the lactone form) in anhydrous THF/MeOH in the presence of Et3N or DMAP at room
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26 temperature. Though the desired product of lova-HA (12) was observed by MS, 1H and 13
C
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29 NMR spectral analyses, the reaction was complicated by recovery of lovastatin (in the acid
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32 form) and formation of the methyl ester. To reduce the side reactions, Lewis acid was tested to
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35 activate the lactone moiety of lovastatin while retain the nucleophilicity of hydroxylamine
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38 (method A in Scheme 3). Among the examined Lewis acids (LiCl, MgBr2, ZnCl2 and CeCl3),
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41 using MgBr2 (2 equiv) along with NaHCO3 (8 equiv) for in situ neutralization of
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hydroxylamine hydrochloride (8.5 equiv) in THF/MeOH turned out to be a superior method
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for conversion of lovastatin (in lactone form) to 12 (79% yield). By similar procedures
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54 successfully converted to their corresponding hydroxamic acids 13 (simva-HA), 14
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57 (atorva-HA) and 15 (rosuva-HA) in 48%, 56% and 51% yields, respectively. The ester
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60 functionality and C=C double bonds existing in lovastatin, simvastatin and rosuvastatin were
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4 unchanged under such mild reaction conditions of our method, whereas these functional
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7 groups might not be retained on acid-catalyzed hydrolysis or hydrogenation involved in the
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previously reported protection–deprotection procedures.30 In these cases, only modest yields
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14 of the desired statin hydroxamic acids were obtained due to the competitive formation of the
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17 related methyl esters in MeOH solution. To reduce the formation of statin methyl esters in
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20 MeOH solution, we further investigated the substitution reactions of statin lactones with 50%
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23 hydroxylamine aqueous solution (method B in Scheme 3). To our satisfaction, high yields
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26 (88–95%) of statin hydroxamic acids 12–15 were obtained by treatment of the THF solutions
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29 of statin lactones with 50% aqueous hydroxylamine (5 equiv) for a short reaction time (1 h) at
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32 ambient temperature.
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4 Scheme 3. Synthesis of statin hydroxamic acids 12–15.
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7 HO O
8 O
O
9 H
10 O
H
R
11 1, R = H
12 11, R = Me
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14 Method A: Method B:
15 NH2OH-HCl, NaHCO3, MgBr2 NH2OH, THF/H2O, rt, 1 h.
16 THF/MeOH, rt, 22 h.
17 HO
O
18 HN OH
19 O OH
20 O
21 H
R
22 12, R = H
13, R = Me
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24 O HO
O
HO
25 HN OH
26 HN OH
OH
OH
27 F
28 F
29 N
N N
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PhHN
31 Ph
Me
N
SO2Me
O
32 14 15
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38 Molecular Modeling. In this work, we modeled the structures of class I HDACs (in
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41 particular HDACs 1 and 2) to focus on the design of HMGR–HDAC dual-targeting
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compounds for applications in cancer treatment, though inhibition of HDAC6 is reported to
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be a promising strategy for treatment of some neurodegenerative diseases.31 HDAC1 and
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51 HDAC2 are homologs with high sequence identify and similarity, especially for the active site
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54 residues (Figure s1 in Supporting Information (SI)). There were no HDAC1/2 crystal
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57 structures available when we launched the design project. To simplify the design process, we
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60 mainly used the homology modeled HDAC1 as our primary protein structure of design. The
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4 RMSD of catalytic residues between our homology modeled HDAC1 and the HDAC2 crystal
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7 structure (3MAX)32 published in 2010 was only 0.921Å , justifying our result of homology
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modeling (Figure s2 in SI).
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14 Figures 2 and 3 illustrate the predicted binding modes and the detailed protein–inhibitor
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17 interactions of compound 12 with HDAC1 and HMGR, respectively. The molecular modeling
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20 results for compounds 13–15 are collected in SI (Figure s3). These statin hydroxamic acids all
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23 show the 3,5,N-trihydroxyheptamide moiety consistently inserts into the catalytic outer-tunnel
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26 of HDAC. The hydroxamate group also chelates a zinc ion, which is important for the
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29 catalytic process of HDAC. In addition, the 3,5-dihydroxyl groups also contribute to the
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32 hydrogen-bonding interactions with the residues in the catalytic tunnel. In contrast to the
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35 previously developed HDAC inhibitors that simply contain aliphatic chains to exert
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38 hydrophobic interactions with the catalytic tunnel of HDAC, our results of molecular
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41 modeling reveal that placing polar substituents at proper locations of ligand, e.g., the OH
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groups at C-3 and C-5 positions in 12, can still maintain the binding affinity to HDAC
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inhibitors. Thus, statin hydroxamates bearing 3,5-dihydroxyl substituents on the alkyl chain
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51 may become better HDAC inhibitors.
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54 In another aspect, hydroxamic acid is considered as a bioisostere of carboxylic acid.
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57 Similar to the crystal structures of statin–HMGR complexes (Figure s4 in SI),29 our molecular
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60 docking studies also indicate that the hydroxamate group in compounds 12–15 contributes to
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4 substantial hydrogen bondings with at least two residues of Lys A735, Ser B684, and Lys
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7 B692 in HMGR, wherein A and B represent A and B chains of the protein. Thus, compounds
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12–15 are also predicted to exhibit reasonable affinity to HMGR. Our alignment analysis
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14 using various binding poses of compound 12 further indicates that the binding pockets of
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17 HMGR and HDAC1 are dissimilar while the highly flexible nature of compound 12 still fits
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20 into each active site with different conformations (Figure s5 in SI).
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43 Figure 2. Predicted binding mode and receptor–ligand interaction diagrams of compound 12
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46 on homology modeled HDAC1. The structural template, HDLP structure, was obtained from
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49 Protein Data Bank (PDB ID: 3CSR). Amino acid residues within 4.5 Å of ligand are
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52 presented in the two-dimensional interaction diagram. The blue circle on the ligand represents
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55 its exposure to the solvent. The larger circle indicates more exposure of the ligand to the
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58 solvent. The green and magenta dashed lines represent hydrogen bonding and metal chelation,
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respectively.
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29 on HMGR. The illustration is similar to that described in Figure 2. The letters A and B before
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32 the residue numbers represent the A and B chains, respectively, in the PDB entry of HMGR
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35 (PDB ID: 1HW9).
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41 HMGR and HDAC Inhibition. We first used in vitro enzymatic assays to examine the
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inhibition of synthetic compounds on HMGR and HDAC activities. As shown in Table 1,
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compounds 6a, 6b, 10, and 12–15 all inhibited HMGR activity with IC50 values similar to
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51 statins in nanomolar range. However, SAHA had no inhibition on HMGR activity even at a
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54 dose of 10 μM. Compounds 6a, 6b, 10, and 12–15 appreciably inhibited HDAC1 (class I),
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57 HDAC2 (class I) and HDAC6 (class II) activities with IC50 values in nanomolar range.
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60 However, lovastatin and atorvastatin inhibited HDACs at a concentration of more than 10 μM.
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4 These experiments clearly indicated that compounds 6a, 6b, 10, and 12–15 acted as potent
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7 dual functional inhibitors against HMGR and HDACs.
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14 Table 1. Inhibitory activities (IC50) against HMGR and HDACs.
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17 IC50 (nM)a
18 compound
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20 HMGR HDAC1 HDAC2 HDAC6
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23 lovastatin 29.5 ± 3.5 11911 ± 681 25933 ± 651 16285 ± 1575
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26 atorvastatin 12.9 ± 1.3 11619 ± 382 22547 ± 1618 14466 ± 567
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29 SAHA >10,000 20.9 ± 7.1 100.9 ± 10.0 19.4 ± 6.0
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32 6a 36.5 ± 5.3 159.0 ± 8.4 463.3 ± 28.0 127.4 ± 21.5
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35 6b 53.8 ± 5.2 124.7 ± 6.3 881.8 ± 9.7 34.0 ± 4.3
36
37
38 10 54.1 ± 2.1 122.0 ± 12.7 657.7 ± 21.1 139.7 ± 5.7
39
40
41
12 16.8 ± 1.9 64.8 ± 5.4 468.3 ± 27.2 51.0 ± 6.1
42
43
44
45
13 13.1 ± 1.5 63.4 ± 4.5 414.2 ± 15.5 69.5 ± 4.1
46
47
48 14 12.3 ± 2.7 122.9 ± 9.5 467.2 ± 19.0 86.6 ± 6.0
49
50
51 15 43.7 ± 1.6 125.2 ± 7.1 600.1 ± 34.0 133.3 ± 5.5
52
53 a
54 Data are shown as mean ± SD of three experiments.
55
56
57
58
59
60 To further evaluate their inhibition on HMGR in lung cancer cells, compounds 6a, 6b, 10,
16

1
2
3
4 and 12–15 at 1–50 μM were applied to cells for 24 h, and the HMGR activity in whole cell
5
6
7
lysates was measured. Compounds 6a, 6b, 10, and 12–15 effectively reduced HMGR activity
8
9
10
11
in a dose-dependent manner with IC50 values similar to lovastatin in nanomolar range (Table
12
13
14 2). However, SAHA did not affect HMGR at any dose.
15
16
17
18
19
20 Table 2. Inhibition on the HMGR activity in A549 lung cancer cells.
21
22
23 compound IC50 (μM)a
24
25
26 lovastatin 19.8 ± 2.2
27
28
29 SAHA NDb
30
31
32 6a 22.3 ± 5.9
33
34
35 6b 16.1 ± 4.8
36
37
38 10 13.2 ± 5.1
39
40
41
12 15.9 ± 0.8
42
43
44
45
13 7.9 ± 1.4
46
47
48 14 5.7 ± 1.2a
49
50
51 15 14.9 ± 1.7
52
53 a
54 Data are shown as mean ± SD of three experiments. b ND: not determined (>10 μM) due to
55
56
57 high toxicity of SAHA to cells.
58
59
60
17

1
2
3
4 Intracellular histone acetylation status is a direct marker of class I HDAC inhibition,
5
6
7
whereas α-tubulin is a substrate of HDAC6 (class II). As shown in Figure 4, compounds 6a,
8
9
10
11
6b, and 12–15 promoted histone and tubulin acetylations in a dose-dependent manner. SAHA
12
13
14 and lovastatin were included for comparison.
15
16
17
18
19
20 (A)
21
22
23
24
25
26
27
28
29
30
31
32
33
34 (B)
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Figure 4. Effect of hydroxamate compounds on acetylation of histone H3 and tubulin in A549
49
50
51 lung cancer cell lines. A549 cells were treated with indicated doses of drugs for 24 h: (A)
52
53
54 conjugated compounds 6a and 6b; (B) hybrid compounds 12–15. SAHA (5 μM) and
55
56
57 lovastatin (30 or 50 μM) were used for comparison. Total protein lysates were subjected to
58
59
60
18

1
2
3
4 Western blotting using antibodies specific for acetyl-histone H3, acetyl-tubulin or actin.
5
6
7 Equivalent protein loading with similar level of β-actin was applied.
8
9
10
11
12
13
14 Cell Growth Inhibition. Cell viability assays were performed to determine the
15
16
17 cytotoxicity and specificity of compounds 12–14 (Table 3). A549 human lung cancer cells,
18
19
20 MEF normal mouse fibroblast cells, and HS68 normal human fibroblast cells were treated
21
22
23 with different doses of drug for 72 h, and the IC50 values were evaluated. SAHA was toxic to
24
25
26 both cancer and normal cells without specificity. The selectivity index of statins for cancer
27
28
29 and normal cells was low. Compounds 12–14 significantly induced cytotoxicity in the cancer
30
31
32 cells (IC50 < 20 μM), but were not toxic to the normal cells at 100 μM. Our results indicate
33
34
35 that compounds 12–14 have a potential to be developed as safer drugs than statins and SAHA
36
37
38 in cancer treatment and other therapeutic uses.
39
40
41
42
43
44
Table 3. Inhibition on the growth of cancer and normal cells.
45
46
47
48 IC50 (μM)a S.I.b
49 compound
50
51 A549a MEFa HS68a A549/MEF A549/HS68
52
53
54 lovastatin 11.4 ± 6.3 35.0 ± 5.9 23.2 ± 3.5 3.1 2.0
55
56
57 simvastatin 16.3 ± 0.1 36.7 ± 4.4 26.4 ± 2.1 2.3 1.6
58
59
60 atorvastatin 8.7 ± 1.1 30.7 ± 3.2 22.7 ± 2.0 3.5 2.6
19

1
2
3
4 SAHA 4.5 ± 0.8 4.4 ± 1.4 4.6 ± 0.7 1.0 1.0
5
6
7 18.2 ± 3.4 > 100 > 100 > 5.6 > 5.6
12
8
9
10
11
13 20.0 ± 3.1 > 100 > 100 > 5.0 > 5.0
12
13
14 14 17.5 ± 4.7 > 100 > 100 > 5.7 > 5.7
15
16 a
17 Cells were treated with indicated doses of test compounds for 72 h, and the cell viability was
18
19
20 measured by MTT assay. Data are shown as mean ± SD of three experiments. A549: human
21
22
23 lung cancer cells; MEF: normal mouse fibroblast cells; HS68: normal human fibroblast cells.
24
25
b
26 Selectivity index: the ratio of IC50 on cancer cell to IC50 on normal cell.
27
28
29
30
31
32 CONCLUSION
33
34
35 HDAC has been a validated target for cancer therapy. Though a potent HDAC inhibitor
36
37
38 SAHA has been approved for treatment of cutaneous T cell lymphoma, the high toxicity of
39
40
41 SAHA is a serious concern. In contrast, statins have shown safety and efficacy in preventing
42
43
44
45
human cardiovascular diseases. The recent studies also indicate that statins may have
46
47
48 beneficial effects in prevention and treatment of cancers. We thus conceive a new therapeutic
49
50
51 approach for cancer treatment by concurrent inhibition of HDAC and HMGR. We have
52
53
54 successfully synthesized a series of dual-action compounds to target HDAC and HMGR by
55
56
57 having a hydroxamate group essential for chelation with the zinc ion in the active site of
58
59
60 HDAC and the key structural elements of statin for binding with both proteins. In addition to
20

1
2
3
4 using hydroxamic acid as a surrogate of carboxylic acid, we also found that the aliphatic
5
6
7
chains in SAHA and statin could be overlaid. Thus, the hybrid molecules 12–15 exhibited
8
9
10
11
high inhibitory activities against both HDAC and HMGR. Our cell-based assays also showed
12
13
14 that these statin hydroxamic acids effectively reduced the HMGR activity and promoted the
15
16
17 acetylations of histone and tubulin in cancer cells (IC50 < 20 μM), but were not toxic to
18
19
20 normal cells at the concentration as high as 100 μM.
21
22
23 The in vivo experiments, pharmacokinetics and metabolic studies of the HDAC–HMGR
24
25
26 dual-action inhibitors are currently under investigation. Our results indicated that oral
27
28
29 administration of compound 12 could prevent and treat azoxymethane (AOM)/dextran
30
31
32 sulphate sodium (DSS)-induced colitis-associated colorectal cancer in mice. Treatment with
33
34
35 compound 12 did not show any toxicity by biochemical examinations and hematoxylin and
36
37
38 eosin (H&E) staining of various organs (manuscript in preparation). The pharmacokinetics of
39
40
41 compound 12 has been evaluated in healthy rats through intravenous or oral administration.
42
43
44
We need to stress that it is still possible to find more efficient dual-action compounds by
45
46
47
48
taking other possible combinations of different HMGR and HDAC inhibitors. For example,
49
50
51 using different zinc binding groups may provide a higher affinity toward the target proteins
52
53
54 and thus render a better therapeutic efficiency. On the other hand, changing the cap region in
55
56
57 the dual-action compounds may achieve better selectivity to differentiate HDAC subtypes. In
58
59
60 summary, we have demonstrated the first example of dual-action inhibitors targeting HDAC
21

1
2
3
4 and HMGR as a promising approach to cancer therapy.
5
6
7
8
9
10
11
EXPERIMENTAL SECTION
12
13
14 Materials and Methods. All the reagents were commercially available and used without
15
16
17 further purification unless indicated otherwise. All solvents were anhydrous grade unless
18
19
20 indicated otherwise. Dichloromethane was distilled from CaH2; triethylamine was distilled
21
22
23 from MgSO4. Lovastatin and simvastatin were obtained from Lotus Pharmaceutical Company
24
25
26 (Nantou, Taiwan). Atorvastatin was obtained from Synpac Kingdom Pharmaceutical
27
28
29 Company (Taipei, Taiwan). SAHA was purchased from Merck (Frankfurter Sparkasse,
30
31
32 Germany). Anti-acetylhistone H3 antibody was purchased from Millipore (Bedford, MA,
33
34
35 USA). Anti-β-actin antibody was purchased from Gene Tex (Irvine, CA, USA). HDAC
36
37
38 Fluorimetric Assay/Drug Discovery Kit (AK-500) was purchased from Biomol (Plymouth
39
40
41 Meeting, PA, USA). HMG-CoA reductase activity kit (CS-1090), and anti-acetyltubulin
42
43
44
antibody were purchased from Sigma (St. Louis, MO, USA).
45
46
47
48
All non-aqueous reactions were carried out in oven-dried glassware under a slight
49
50
51 positive pressure of argon unless otherwise noted. Reactions were magnetically stirred and
52
53
54 monitored by thin-layer chromatography on silica gel. Analytical thin layer chromatography
55
56
57 (TLC) was performed on 0.25 mm silica gel 60 F254 plates. Compounds were visualized by
58
59
60 UV, or using p-anisaldehyde, ninhydrin, phosphomolybdic acid, KMnO4 or I2 as visualizing
22

1
2
3
4 agent. Flash chromatography was carried out on columns packed with silica gel 60
5
6
7 (0.040–0.063 mm particle sizes). High-performance liquid chromatography (HPLC) was
8
9
10
11
performed on Agilent 1100 Series instrument equipped with a degasser, Quat pump, and UV
12
13
14 detector.
15
16
17 Melting points were recorded on an Electrothermal MEL-TEMP 1101D melting point
18
19
20 apparatus and were not corrected. Optical rotations were measured on digital polarimeter of
21
22
23 Japan JASCO Co. DIP-1000; [α]D values are given in units of 10–1 deg cm2 g–1. Infrared (IR)
24
25
26 spectra were recorded on a Nicolet Magna 550-II FT-IR spectrometer. Nuclear magnetic
27
28
29 resonance (NMR) spectra were obtained on Varian Unity Plus-400 (400 MHz) or Bruker
30
31
32 Avance-III 400 MHz NMR spectrometers. Chemical shifts (δ) were recorded in parts per
33
34
35 million (ppm) relative to δH 7.24/ δC 77.0 (central line of t) for CHCl3/CDCl3 or δH 0.00/ δC
36
37
38 77.0 (central line of t) for TMS/CDCl3, δH 3.31/ δC 49.0 for CH3OH/CD3OD, and δH 2.50
39
40
41 39.5 (m) for (CH3)2SO/(CD3)2SO. The splitting patterns are reported as s (singlet), d
C
42
43
44
(doublet), t (triplet), q (quartet), m (multiplet) and br (broad). Coupling constants (J) are given
45
46
47
48
in hertz. The ESI–MS experiments were conducted on a Bruker Daltonics BioTOF III
49
50
51 high-resolution mass spectrometer.
52
53
54 General Compound Characterization. New compounds were characterized by their
55
56
57 physical and spectroscopic properties (mp, TLC, [α], IR, ESI–MS, 1H and 13
C NMR). Purity
58
59
60 of synthetic compounds 6a, 6b, 10, and 12–15 was assessed to be  95% by HPLC analysis
23

1
2
3
4 (Agilent HP-1100) on an HC-C18 column (250 mm × 4.6 mm i.d., 5 μm particle size) using
5
6
7 gradient elution of aqueous CH3CN for 20–30 min at a flow rate of 1 mL/min with detection
8
9
10
11
at 254 nm wavelength.
12
13
14 Homology Modeling. The sequence alignment between human HDAC1 and histone
15
16
17 deacetylase-like protein (HDLP) was determined by the multiple sequence alignment between
18
19
20 human class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8) and HDLP using the
21
22
23 ClastalW module with BLOSUM scoring matrix in Discovery Studio 2.55 (Accelrys, Inc. San
24
25
26 Diego, CA, USA). The homology model structure was generated and optimized by
27
28
29 MODELLER in Discovery Studio 2.55 (Accelrys, Inc. San Diego, CA, USA). The structural
30
31
32 template of the homology modeling of HDAC1 and HDAC2, the HDLP structure was
33
34
35 obtained from Protein Data Bank (PDB ID: 3CSR). The one with the lowest energy score was
36
37
38 selected as the final model.
39
40
41 Molecular Docking. The crystal structure of HMG-CoA reductase was taken from
42
43
44
Protein Databank (PDB 1HW9). The structure of HDAC1 was prepared from the homology
45
46
47
48
modeling described above. All non-protein molecules were deleted except the zinc ion of the
49
50
51 HDAC complex. The receptor is prepared by AutoDock 4 suite.33 The grid box size was
52
53
54 adjusted to cover the original ligand and its surrounding binding pocket residues with 48 × 40
55
56
57 × 40 in HDAC1 and 40 × 40 × 52 in HMG-CoA reductase with grid spacing of 0.375Å .
58
59
60 The 2D ligand structures were prepared using ChemDraw Ultra 12 and the 3D structures were
24

1
2
3
4 generated by OpenBabel 2.3.1.34 The molecular docking simulations were performed by
5
6
7 AutoDock4, with the recent reparameterized scoring function (AutoDock4RAP).35 It has been
8
9
10
11
shown that the native ligand of HDAC can be reproduced with the new scoring function and a
12
13
14 new divide-and-conquer docking approach. 36 The binding pose with lowest score in each case
15
16
17 is selected to represent the predicted binding mode. The 3D and 2D protein–ligand interaction
18
19
20 plots were presented using ICM Browser version 3.7-2d (Molsoft L.L.C., San Diego, USA)
21
22
23 and Molecular Operating Environment version 2009.10 (Chemical Computing Group,
24
25
26 Montreal, Canada), respectively.
27
28
29 Cell Lines and Culture Conditions. A549 human lung carcinoma cells from American
30
31
32 Type Culture Collection (ATCC, Manassas, VA) were cultured in Dulbecco’s Modified
33
34
35 Eagle’s Medium (DMEM). MEF normal mouse fibroblast cells and HS68 normal human
36
37
38 fibroblast cells were cultured in RPMI 1640. All media were supplemented with 10% FBS,
39
40
41 100 U/mL penicillin G and 100 mg/mL streptomycin sulfate. Cells were maintained in a
42
43
44
humidified incubator containing 5% CO2 in air. Cells were subcultured by trypsinization in
45
46
47
48
laminar flow when grew about 80% in the culture dishes.
49
50
51 HDAC Activity Assay. The HDAC activity was performed using the HDAC fluorimetric
52
53
54 activity assay kit (BIOMOL, Plymouth Meeting, PA, USA) according to the manufacturer’s
55
56
57 instructions. Briefly, recombinant proteins of HDAC1, HDAC2 or HDAC6 were incubated
58
59
60 with test compounds, and HDAC reaction was initiated by addition of Fluor-de-Lys substrate.
25

1
2
3
4 Samples were incubated for 10 min at room temperature, followed by adding developer to
5
6
7 stop the reaction. Fluorescence was measured by fluorimetric reader with excitation at 360 nm
8
9
10
11
and emission at 460 nm. The HDAC activity was expressed as arbitrary fluorescence units
12
13
14 (AFU). The HDAC activity was calculated as a percentage of activity compared with the
15
16
17 control group. The 50% of inhibition concentration (IC50) values for the test compounds were
18
19
20 calculated using SigmaPlot software.
21
22
23 HMG-CoA Reductase Activity Assay. The HMGR activity was performed using the
24
25
26 HMGR assay kit according to the manufacturer’s instructions (Sigma–Aldrich, St. Louis, MO,
27
28
29 USA). Briefly, recombinant HMGR protein incubated with lovastatin or test compounds for
30
31
32 10 min at room temperature, or total cell lysate from A549 treated with lovastatin or test
33
34
35 compounds was mixed with HMG-CoA and NADPH, and incubated for 5 min at 37 oC. The
36
37
38 absorbance at 340 nm was measured. The 50% of inhibition concentration (IC50) values for
39
40
41 the test compounds were calculated using SigmaPlot software.
42
43
44
Western Blot Analysis. Following treatment with test compounds, cells were lysed on ice.
45
46
47
48
Total cell lysates were prepared and subjected to SDS–PAGE using adequate percentage
49
50
51 polyacrylamide gels. Immunoblotting was performed using specific antibodies to evaluate the
52
53
54 expression of different proteins.
55
56
57 Cell Proliferation Assay. Cells were seeded at 3000 cells/well in 96-well plates and
58
59
60 maintained for 14–16 h. Cells were treated with DMSO or various concentrations of test
26

1
2
3
4 compounds for 72 h, and then washed with PBS twice. A medium containing
5
6
7 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 0.5 mg/mL) was added,
8
9
10
11
and the cells were incubated for 4 h at 37 oC under 5% CO2. During this period, cells having
12
13
14 functional succinate dehydrogenase in mitochondria would convert MTT to formazan. The
15
16
17 medium was replaced with 200 μL of DMSO for 30 min at room temperature, and the 96-well
18
19
20 plate was read by an ELISA reader at 570 nm to get the values of absorbance density. The
21
22
23 IC50 values were calculated using SigmaPlot software.
24
25
26 General Methods for Preparation of Statin Hydroxamates. Method A. To a solution of
27
28
29 lovastatin (1, in lactone form, 0.74 mmol) and MgBr2 (1.48 mmol) in anhydrous THF/MeOH
30
31
32 (7:3, 3 mL) was added hydroxylamine hydrochloride (6.3 mmol) and sodium bicarbonate (5.9
33
34
35 mmol). The mixture was stirred at ambient temperature for 22 h, and concentrated under
36
37
38 reduced pressure. The residue was extracted with EtOAc and brine. The organic phase was
39
40
41 dried over MgSO4, filtered, concentrated, and purified by flash chromatography (silica gel,
42
43
44
CH2Cl2/MeOH (15:1)) to give the desired statin hydroxamic acid.
45
46
47
48
Method B. To a solution of statin (in lactone form, 0.25 mmol) in THF (469 μL) was
49
50
51 added 50% aqueous hydroxylamine (1.24 mmol, 76 μL). The mixture was stirred at ambient
52
53
54 temperature for 1 h, and then concentrated under reduced pressure. The residue was purified
55
56
57 by flash chromatography (silica gel, CH2Cl2/MeOH (15:1)) to give the desired statin
58
59
60 hydroxamic acid.
27

1
2
3
4 (4R,6R)-6-[2-((1S,2S,6R,8S,8aR)-8-Hydroxy-2,6-dimethyl-1,2,6,7,8,8a-hexahydronap
5
6
7
hthyl)ethyl]-4-tert-butyldimethylsilyloxy-3,4,5,6-tetrahydro-2H-pyran-2-one (2).
8
9
10
11
Lovastatin (1, 9.0 g, 22.3 mmol) was heated with potassium hydroxide (12.6 g, 224.5 mmol)
12
13
14 in H2O/MeOH (1:6, 63 mL) at refluxing for 8 h. After adding H2O (49.5 mL) to the mixture,
15
16
17 MeOH was removed under reduced pressure. To the residue were added H2O (180 mL),
18
19
20 CH2Cl2 (45 mL), and 6 M HCl aqueous solution until pH = 2. The mixture was stirred at room
21
22
23 temperature for 4.5 h, was and then neutralized with saturated NaHCO3 aqueous solution. The
24
25
26 mixture was extracted with CH2Cl2. The combined organic phase was dried over anhydrous
27
28
29 MgSO4, filtered and concentrated to give a deacylation product as orange oil. TLC (EtOAc) Rf
30
31
32 = 0.33.
33
34
35 The crude product was treated with tert-butyldimethylsilyl chloride (8.7 g, 57.9 mmol)
36
37
38 and imidazole (8.8 g, 129.1 mmol) in CH2Cl2 (82 mL) at room temperature for 5.5 h. The
39
40
41 mixture was concentrated under reduced pressure, and the residue was extracted with CH2Cl2
42
43
44
and H2O. The organic phase was dried over MgSO4, filtered, concentrated, and purified by
45
46
47
48
flash chromatography (silica gel, EtOAc/hexane (2:8) to EtOAc) to give the compound 2 (5.6
49
50
51 g, 58% overall yield from lovastatin). C25H42O4Si; white solid, mp 141.1–142.3 oC; TLC
52
53
54 (EtOAc/hexane (6:4)) Rf = 0.61; 1H NMR (CDCl3, 400 MHz) δ 5.98 (1 H, d, J = 9.6 Hz),
55
56
57 5.78–5.82 (1 H, m), 5.55 (1 H, br s), 4.66–4.70 (1 H, m), 4.29–4.30 (1 H, m), 4.23–4.25 (1 H,
58
59
60 m), 2.54–2.65 (2 H, m), 2.35–2.45 (2 H, m), 2.16–2.18 (1 H, m), 1.70–1.93 (7 H, m),
28

1
2
3
4 1.44–1.55 (2 H, m), 1.15 (3 H, d, J = 7.6 Hz), 0.88–0.91 (12 H, m), 0.08 (6 H, d, J = 1.6 Hz);
5
6
7 13
C NMR (CDCl3, 100 MHz) 170.4, 133.6, 131.3, 129.9, 128.4, 76.3, 65.1, 63.5, 39.2, 38.7,
8
9
10
11
36.8, 36.3, 35.7, 32.9, 30.7, 27.3, 25.6 (3 ×), 24.2, 23.7, 17.9, 13.9, –5.0 (2 ×).
12
13
14 (4R,6R)-6-(2-{(1S,2S,6R,8S,8aR)-8-[(p-Nitrophenoxy)carbonyloxy]-2,6-dimethyl-1,2,
15
16
17 6,7,8,8a-hexahydronaphthyl}ethyl)-4-tert-butyldimethylsilyloxy-3,4,5,6-tetrahydro-2H-p
18
19
20 yran-2-one (3). A mixture of alcohol 2 (5.0 g, 11.5 mmol), p-nitrophenyl chloroformate (16.2
21
22
23 g, 80.2 mmol) and DMAP (98.0 g, 80.2 mmol) was stirred in anhydrous pyridine (80.2 mL) at
24
25
26 room temperature for 15 h. Pyridine was removed under reduced pressure, and the residue
27
28
29 was extracted with CH2Cl2 and 1 M HCl aqueous solution. The combined organic phase was
30
31
32 washed with saturated NaHCO3 aqueous solution and brine, dried over MgSO4, filtered and
33
34
35 concentrated under reduced pressure. The residue was purified by flash chromatography
36
37
38 (silica gel, CH2Cl2/hexane (6:4 to 8:2)) to give the carbonate compound 3 (5.6 g, 81%).
39
40
41 C32H45NO8Si; white powder, mp 146.5–147.3 oC; [α]25D = +233.9 (EtOAc, c = 1.0); TLC
42
43
44
(EtOAc/hexane (2:8)) Rf = 0.24; IR νmax (neat) 2955, 2930, 2857, 2360, 1760, 1594, 1525,
45
46
47
48
1347, 1258, 1216, 1082 cm–1; 1H NMR (CDCl3, 400 MHz) δ 8.27 (2 H, d, J = 9.2 Hz), 7.40 (2
49
50
51 H, d, J = 8.8 Hz), 6.01 (1 H, d, J = 9.6 Hz), 5.79–5.83 (1 H, m), 5.57 (1 H, br s), 5.34 (1 H, br
52
53
54 s), 4.68–4.70 (1 H, m), 4.27–4.29 (1 H, m), 2.52–2.59 (3 H, m), 2.34–2.41 (2 H, m),
55
56
57 2.17–2.23 (1 H, m), 1.66–2.00 (6 H, m), 1.45–1.53 (2 H, m), 1.17 (3 H, d, J = 7.6 Hz), 0.93 (3
58
59
60 H, d, J = 6.8 Hz), 0.86 (9 H, s), 0.06 (6 H, d, J = 5.6 Hz); 13C NMR (CDCl3, 100 MHz) 170.2,
29

1
2
3
4 155.6, 152.3, 145.3, 133.2, 131.1, 129.3, 128.1, 125.2 (2 ×), 122.0 (2 ×), 75.4, 74.5, 63.6, 39.3,
5
6
7 37.5, 36.7, 36.1, 32.5, 32.2, 30.8, 27.3, 25.7 (3 ×), 23.5, 22.5, 17.9, 13.9, –4.9 (2 ×);
8
9
10
11
ESI–HRMS calcd. for C32H46NO8Si: 600.2993, found: m/z 600.3002 [M + H]+.
12
13
14
15
16
17 (4R,6R)-6-(2-{(1S,2S,6R,8S,8aR)-8-[(4-ethynylphenyl)carbamoyloxy]-2,6-dimethyl-1,2,6,
18
19
20 7,8,8a-hexahydronaphthyl}ethyl)-4-tert-butyldimethylsilyloxy-3,4,5,6-tetrahydro-2H-pyr
21
22
23 an-2-one (4a). A solution of carbonate 3 (1.5 g, 2.5 mmol), 4-ethynylaniline (2.1 g, 17.6
24
25
26 mmol) and DMAP (2.1 g, 17.6 mmol) in anhydrous pyridine (6.3 mL) was stirred at ambient
27
28
29 temperature for 21 h. Pyridine was removed under reduced pressure, and the residue was
30
31
32 extracted with CH2Cl2 and 1 M HCl aqueous solution. The combined organic phase was
33
34
35 washed with saturated NaHCO3 aqueous solution and brine, dried over MgSO4, filtered and
36
37
38 concentrated under reduced pressure. The residue was purified by flash chromatography
39
40
41 (silica gel, EtOAc/hexane (15:85)) to give the carbamate compound 4a (957 mg, 66%).
42
43
44
C34H47NO5Si; orange oil; [α]25D = +213.9 (EtOAc, c = 1.0); TLC (EtOAc/hexane (5:5)) Rf =
45
46
47
48
0.66; IR νmax (neat) 3299, 2955, 2858, 1734, 1591, 1523, 1313, 1255, 1219, 1082, 1047, 839
49
50
51 cm–1; 1H NMR (CDCl3, 400 MHz) δ 7.41 (4 H, s), 7.00 (1 H, s), 6.00 (1 H, d, J = 9.6 Hz),
52
53
54 5.79–5.83 (1 H, m), 5.56 (1 H, br s), 5.32 (1 H, br s), 4.62–4.63 (1 H, m), 4.18–4.20 (1 H, m),
55
56
57 3.01 (1 H, s), 2.46–2.52 (3 H, m), 2.29–2.39 (2 H, m), 2.17–2.21 (1 H, m), 1.53–1.96 (6 H, m),
58
59
60 1.39–1.43 (2 H, m), 1.10 (3 H, d, J = 7.6 Hz), 0.91 (3 H, d, J = 6.8 Hz), 0.86 (9 H, s), 0.05 (6
30

1
2
3
4 H, d, J = 3.6 Hz); 13C NMR (CDCl3, 100 MHz) 170.6, 153.1, 138.8, 133.4, 132.9 (2 ×), 131.9,
5
6
7 129.6, 128.1, 118.0 (2 ×), 116.4, 83.5, 76.3, 75.6, 69.6, 63.5, 39.2, 37.3, 36.6, 36.3, 32.5, 32.4,
8
9
10
11
30.8, 27.4, 25.6 (3 ×), 23.4, 22.6, 17.9, 13.9, –5.0 (2 ×); ESI–HRMS (negative mode) calcd.
12
13
14 for C34H46NO5Si: 576.3145, found: m/z 576.3131 [M – H]–.
15
16
17 (4R,6R)-6-(2-{(1S,2S,6R,8S,8aR)-8-[(4-Ethynylbenzyl)carbamoyloxy]-2,6-dimethyl-1,
18
19
20 2,6,7,8,8a-hexahydronaphthyl}ethyl)-4-tert-butyldimethylsilyloxy-3,4,5,6-tetrahydro-2H-
21
22
23 pyran-2-one (4b). A solution of carbonate 3 (500 mg, 0.8 mmol), 4-ethynylbenzylamine
24
25
26 hydrochloride (419 mg, 2.5 mmol) and DMAP (712 mg, 5.8 mmol) in anhydrous pyridine (2
27
28
29 mL) was stirred at ambient temperature for 3 h. Pyridine was removed under reduced pressure,
30
31
32 and the residue was extracted with CH2Cl2 and 1 M HCl aqueous solution. The combined
33
34
35 organic phase was washed with saturated NaHCO3 aqueous solution and brine, dried over
36
37
38 MgSO4, filtered and concentrated under reduced pressure. The residue was purified by flash
39
40
41 chromatography (silica gel; EtOAc/hexane (2:8)) to give the carbamate compound 4b (444
42
43
44
mg, 90%). C35H49NO5Si; colorless oil; [α]25D = +217.6 (EtOAc, c = 1.0); TLC (EtOAc/hexane
45
46
47
48
(5:5)) Rf = 0.57; IR νmax (neat) 3306, 2955, 2829, 2857, 1727, 1509, 1461, 1359, 1259, 1081,
49
50
51 1044, 1016, 924, 837, 778 cm–1; 1H NMR (CDCl3, 400 MHz) δ 7.42 (2 H, d, J = 8.0 Hz), 7.22
52
53
54 (2 H, d, J = 8.0 Hz), 5.97 (1 H, d, J = 9.6 Hz), 5.75–5.79 (1 H, m), 5.52 (1 H, br s), 5.31 (1 H,
55
56
57 br s), 5.22 (1 H, br s), 4.61 (1 H, br s), 4.30–4.41 (2 H, m), 4.23 (1 H, br s), 3.05 (1 H, s), 2.54
58
59
60 (2 H, br s), 2.43 (1 H, br s), 2.12–2.36 (3 H, m), 1.78–1.90 (4 H, m), 1.24–1.54 (4 H, m), 1.09
31

1
2
3
4 (3 H, d, J = 7.2 Hz), 0.87–0.91 (12 H, m), 0.07 (6 H, d, J = 2.8 Hz); 13C NMR (CDCl3, 100
5
6
7 MHz) 170.5, 156.4, 140.0, 133.3, 132.2 (2 ×), 131.9, 129.6, 128.2, 127.2 (2 ×), 120.8, 83.3,
8
9
10
11
76.7, 75.5, 68.9, 63.6, 44.4, 39.3, 37.3, 36.5, 36.2, 32.6, 30.9, 27.4, 25.6 (3 ×), 23.2, 22.6, 17.9,
12
13
14 13.9, –4.9 (2 ×); ESI–HRMS calcd. for C35H50NO5Si: 592.3458, found: m/z 592.3458 [M +
15
16
17 H]+.
18
19
20
21
22
23 (4R,6R)-6-(2-{(1S,2S,6R,8S,8aR)-8-[(4-{1-[7-(Hydroxyamino)-7-oxoheptyl]-1H-1,2,3-triaz
24
25
26 ol-4-yl}phenyl)carbamoyloxy]-2,6-dimethyl-1,2,6,7,8,8a-hexahydronaphthyl}ethyl)-4-tert
27
28
29 -butyldimethylsilyloxy-3,4,5,6-tetrahydro-2H-pyran-2-one (6a). A mixture of alkynyl
30
31
32 compound 4a (827 mg, 1.4 mmol), CuSO4•5H2O (71 mg, 0.29 mmol), sodium ascorbate (168
33
34
35 mg, 0.85 mmol) and 7-azidoheptanoic acid (245 mg, 1.4 mmol) in H2O/t-BuOH (1:1, 19 mL)
36
37
38 was stirred at 60 oC for 18 h. The mixture was concentrated under reduced pressure, and the
39
40
41 residue was extracted with EtOAc and H2O. The organic phase was dried over MgSO4,
42
43
44
filtered, and concentrated to give a practically pure 1,3-cycloaddition product 5a.
45
46
47
48
C41H60N4O7Si; colorless oil; TLC (EtOAc/hexane (7:3)) Rf = 0.11.
49
50
51 The above-prepared carboxylic acid 5a was treated with ethyl chloroformate (0.4 mL, 4.3
52
53
54 mmol) and Et3N (0.8 mL, 5.7 mmol) in anhydrous THF (4.3 mL) at 0 oC for 10 min. A
55
56
57 solution of hydroxylamine, freshly prepared by neutralization of hydroxylamine
58
59
60 hydrochloride (497 mg, 7.2 mmol) with KOH (360 mg, 6.4 mmol) in anhydrous MeOH (2
32

1
2
3
4 mL), was added. The mixture was stirred at 0 oC for another 15 min, and concentrated under
5
6
7 reduced pressure. The residue was extracted with EtOAc and H2O. The organic phase was
8
9
10
11
dried over MgSO4, filtered, and concentrated to yield an pale orange oil, which was purified
12
13
14 by flash chromatography (silica gel, CH2Cl2/MeOH (15:1 to 9:1)) to give hydroxamic acid 6a
15
16
17 (404 mg, 37% overall yield from 4a). The purity of 6a was 98% as shown by HPLC analysis
18
19
20 on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 μm), tR = 17.9 min (gradients of 55–100%
21
22
23 aqueous CH3CN in 30 min). C41H61N5O7Si; colorless oil; [α]24D = +129.1 (EtOAc, c = 1.0);
24
25
26 IR νmax (neat) 3287, 2929, 2857, 1734, 1662, 1596, 1531, 1460, 1359, 1313, 1221, 1081, 1047,
27
28
29 837, 779 cm–1; 1H NMR (CDCl3, 400 MHz) δ 7.73 (3 H, m), 7.50 (2 H, d, J = 7.2 Hz), 7.12 (1
30
31
32 H, br s), 6.00 (1 H, d, J = 9.6 Hz), 5.79–5.83 (1 H, m), 5.56 (1 H, br s), 5.33 (1 H, br s), 4.64
33
34
35 (1 H, br s), 4.35 (2 H, br s), 4.19 (1 H, br s), 2.30–2.50 (5 H, m), 2.12–2.20 (3 H, m),
36
37
38 1.26–1.96 (16 H, m), 1.11 (3 H, d, J = 7.6 Hz), 0.91 (3 H, d, J = 7.2 Hz), 0.85 (9 H, s), 0.04 (6
39
40
41 H, s); 13
C NMR (CDCl3, 100 MHz) 170.8, 153.4, 147.5, 138.3, 133.5, 131.9, 129.8, 128.2,
42
43
44
126.4 (2 ×), 125.4, 119.2, 118.8 (2 ×), 77.2, 75.8, 69.6, 63.6, 50.0, 39.3, 37.4, 36.5, 36.4, 32.6,
45
46
47
48
32.4, 30.9, 29.8, 27.8, 27.5, 25.7 (3 ×), 25.5, 24.8, 23.4, 22.7, 17.9, 14.0, –4.9 (2 ×);
49
50
51 ESI–HRMS calcd. for C41H62N5O7Si: 764.4419, found: m/z 764.4423 [M + H]+.
52
53
54
55
56
57 (4R,6R)-6-(2-{(1S,2S,6R,8S,8aR)-8-[(4-{1-[7-(Hydroxyamino)-7-oxoheptyl]-1H-1,2,3-triaz
58
59
60 ol-4-yl}benzyl)carbamoyloxy]-2,6-dimethyl-1,2,6,7,8,8a-hexahydronaphthyl}ethyl)-4-tert
33

1
2
3
4 -butyldimethylsilyloxy-3,4,5,6-tetrahydro-2H-pyran-2-one (6b). By a procedure similar to
5
6
7
that for 6a, the 1,3-dipolar cycloaddition product 5b obtained from alkyne 4b (734 mg, 1.2
8
9
10
11
mmol) and 7-azidoheptanoic acid (202 mg, 1.2 mmol) was activated with ethyl chloroformate
12
13
14 and reacted with hydroxylamine (6.2 mmol) in MeOH (1.7 mL) to give hydroxamic acid 6b
15
16
17 (511 mg, 53% overall yield from 4b). The purity of product 6b was 98% as shown by HPLC
18
19
20 analysis on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 μm), tR = 17.4 min (gradients of
21
22
23 55–100% aqueous CH3CN in 30 min). C42H63N5O7Si; colorless oil; [α]23D = +171.9 (EtOAc, c
24
25
26 = 1.0); IR νmax (neat) 3291, 2952, 2829, 2857, 1719, 1668, 1519, 1460, 1360, 1259, 1080,
27
28
29 1044, 836, 778 cm–1; 1H NMR (CDCl3, 400 MHz) δ 7.76 (3 H, m), 7.31 (2 H, d, J = 7.6 Hz),
30
31
32 5.98 (1 H, d, J = 9.6 Hz), 5.76–5.80 (1 H, m), 5.53 (1 H, br s), 5.35 (1 H, br s), 5.27 (1 H, br
33
34
35 s), 4.59–4.61 (1 H, m), 4.30–4.47 (4 H, m), 4.17 (1 H, br s), 2.09–2.46 (8 H, m), 1.19–1.94
36
37
38 13
(16 H, m), 1.11 (3 H, d, J = 7.2 Hz), 0.86–0.90 (12 H, m), 0.05 (6 H, s); C NMR (CDCl3,
39
40
41 100 MHz) 171.0, 156.5, 147.3, 139.2, 133.3, 131.9, 129.8, 129.5, 128.2 (2 ×), 127.6, 125.8 (2
42
43
44
×), 119.8, 77.2, 76.0, 68.8, 63.5, 50.1, 44.4, 39.2, 37.3, 36.5, 36.0, 32.7, 31.0, 29.8, 29.7, 27.8,
45
46
47
48
27.5, 25.7 (3 ×), 25.6, 24.8, 23.1, 22.7, 17.9, 14.0, –4.9 (2 ×); ESI–HRMS calcd. for
49
50
51 C42H64N5O7Si: 778.4575, found: m/z 778.4584 [M + H]+.
52
53
54
55
56
57 (4R,6R)-6-[2-((1S,2S,6R,8S,8aR)-8-{[7-(Allyloxy)-7-oxoheptyl]carbamoyloxy}-2,6-dimeth
58
59
60 yl-1,2,6,7,8,8a-hexahydronaphthyl)ethyl]-4-tert-butyldimethylsilyloxy-3,4,5,6-tetrahydro
34

1
2
3
4 -2H-pyran-2-one (8). By a procedure similar to that for 4a, carbonate 3 (354 mg, 0.6 mmol)
5
6
7
was treated with allyl 7-aminohepanoate (7) hydrochloric salt (393 mg, 0.78 mmol) and
8
9
10
11
DMAP (505 mg, 4.1 mmol) in anhydrous pyridine (1.5 mL) at ambient temperature for 2 h to
12
13
14 give carbamate 8 (267 mg, 70% yield). C36H59NO7Si; colorless oil; [α]23D = +174.5 (EtOAc, c
15
16
17 = 1.0); TLC (EtOAc/hexane (1:1)) Rf = 0.57; IR νmax (neat) 3369, 2930, 2857, 1742, 1520,
18
19
20 1462, 1339, 1253, 1082, 926, 837, 778 cm–1; 1H NMR (CDCl3, 400 MHz) δ 5.98 (1 H, d, J =
21
22
23 9.6 Hz), 5.88–5.95 (1 H, m), 5.76–5.80 (1 H, m), 5.52 (1 H, br s), 5.32 (1 H, d, J = 17.2 Hz),
24
25
26 5.23 (1 H, d, J = 10.4 Hz), 5.18 (1 H, br s), 4.79 (1 H, br s), 4.63–4.66 (1 H, m), 4.57 (2 H, d,
27
28
29 J = 5.6 Hz), 4.28–4.29 (1 H, m), 3.20–3.23 (1 H, m), 3.05–3.10 (1 H, m), 2.56–2.63 (2 H, m),
30
31
32 2.09–2.42 (6 H, m), 1.26–1.88 (16 H, m), 1.08 (3 H, d, J = 7.2 Hz), 0.88–0.90 (12 H, m), 0.08
33
34
35 (6 H, s); 13C NMR (CDCl3, 100 MHz) 173.2, 171.5, 156.3, 133.3, 132.2, 132.0, 129.6, 128.2,
36
37
38 118.0, 75.8, 68.4, 64.9, 63.6, 40.7, 39.3, 37.3, 36.6, 36.3, 34.0, 32.6, 32.5, 30.9, 29.7, 28.6,
39
40
41 27.4, 26.2, 25.6 (3 ×), 24.7, 22.5, 17.9, 13.9, –5.0 (2 ×); ESI–HRMS calcd. for C36H60NO7Si:
42
43
44
646.4139, found: m/z 646.4142 [M + H]+.
45
46
47
48
49
50
51 (4R,6R)-6-[2-((1S,2S,6R,8S,8aR)-8-{[7-(Hydroxyamino)-7-oxoheptyl]carbamoyloxy}-2,6-
52
53
54 dimethyl-1,2,6,7,8,8a-hexahydronaphthyl)ethyl]-4-tert-butyldimethylsilyloxy-3,4,5,6-tetr
55
56
57 ahydro-2H-pyran-2-one (10). A mixture of allyl ester 8 (268 mg, 0.4 mmol),
58
59
60 tetrakis(triphenylphosphine)palladium (48 mg, 0.04 mmol), triphenylphosphine (22 mg, 0.08
35

1
2
3
4 mmol), triethylamine (0.17 mL, 1.2 mmol) and formic acid (0.047 mL, 1.2 mmol) in degassed
5
6
7 THF (2 mL) was stirred at ambient temperature for 3 h. The mixture was concentrated under
8
9
10
11
reduced pressure. CH2Cl2 and H2O were added, and the mixture was acidified with 1 M HCl
12
13
14 aqueous solution to pH = 2. The mixture was extracted with CH2Cl2. The combined organic
15
16
17 phase was dried over MgSO4, filtered, concentrated and separated by flash chromatography
18
19
20 (silica gel, CH2Cl2/MeOH (17:1)) to give a carboxylic acid product 9 as colorless oil.
21
22
23 By a procedure similar to that for 6a, the above-prepared carboxylic acid 9 was activated
24
25
26 with ethyl chloroformate and reacted with hydroxylamine (2.1 mmol) in MeOH (0.6 mL) to
27
28
29 give hydroxamic acid 10 (98 mg, 38% overall yield from 8). The purity of product 10 was
30
31
32 97% as shown by HPLC analysis on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 μm), tR =
33
34
35 15.2 min (gradients of 60–100% aqueous CH3CN in 30 min). C33H56N2O7Si; colorless oil;
36
37
38 [α]26D = +132.2 (EtOAc, c = 1.0); IR νmax (neat) 3288, 2929, 2857, 1713, 1522, 1462, 1359,
39
40
41 1255, 1081, 1046, 837, 778 cm–1; 1H NMR (CDCl3, 400 MHz) δ 5.98 (1 H, d, J = 9.6 Hz),
42
43
44
5.76–5.80 (1 H, m), 5.52 (1 H, br s), 5.19 (1 H, br s), 4.88 (1 H, br s), 4.68 (1 H, br s), 4.30 (1
45
46
47
48
H, br s), 3.15–3.16 (2 H, m), 2.53–2.66 (2 H, m), 2.08–2.42 (6 H, m), 1.26–1.89 (16 H, m),
49
50
51 1.08 (3 H, d, J = 7.2 Hz), 0.89 (12 H, m), 0.08 (6 H, s); 13C NMR (CDCl3, 100 MHz) 171.1,
52
53
54 171.0, 156.5, 133.2, 131.9, 129.6, 128.2, 77.2, 68.4, 63.4, 40.5, 39.2, 37.3, 36.2, 36.1, 32.5,
55
56
57 32.3, 30.9, 29.5, 28.2, 27.3, 25.9, 25.6 (3 ×), 25.0, 23.0, 22.5, 17.8, 13.8, –5.0 (2 ×);
58
59
60 ESI–HRMS calcd. for C33H57N2O7Si: 621.3935, found: m/z 621.3922 [M + H]+.
36

1
2
3
4
5
6
7
(3R,5R)-7-{(1S,2S,6R,8S,8aR)-Hexahydro-2,6-dimethyl-8-[2-methylbutyrloxy]naphthale
8
9
10
11
nyl}-3,5-dihydroxy-N-hydroxyheptanamide (12). Lovastatin (1, in lactone form) was
12
13
14 converted to the corresponding hydroxamic acid 12 in 79% yield by general method A and
15
16
17 92% yield by general method B. The purity of 12 was 96% as shown by HPLC analysis on an
18
19
20 HC-C18 column (Agilent, 4.6 × 250 mm, 5 μm), tR = 14.1 min (gradients of 30–80% aqueous
21
22
23 CH3CN in 30 min). C24H39NO6; colorless oil; [α]24D = +208.1 (EtOAc, c = 1.0); TLC
24
25
26 (CH2Cl2/MeOH (9:1)) Rf = 0.32; IR νmax (neat) 3323, 3017, 2963, 2932, 2872, 1725, 1659,
27
28
29 1459, 1382, 1264, 1191, 1115, 1081, 1016, 975, 860 cm–1; 1H NMR (CDCl3, 400 MHz) δ 5.98
30
31
32 (1 H, d, J = 9.6 Hz), 5.76–5.80 (1 H, m), 5.51 (1 H, br s), 5.41 (1 H, br s), 4.27 (1 H, br s),
33
34
35 3.76 (1 H, br s), 2.24–2.43 (6 H, m), 1.93 (2 H, br s), 1.59–1.68 (5 H, m), 1.42–1.47 (2 H, m),
36
37
38 13
1.26 (2 H, br s), 1.06–1.11 (6 H, m), 0.87 (6 H, br s); C NMR (CDCl3, 100 MHz) 177.4,
39
40
41 169.7, 133.4, 131.8, 129.3, 128.1, 71.5, 68.4, 68.2, 43.0, 41.5, 40.6, 37.3, 36.6, 34.9, 32.7,
42
43
44
30.6, 27.4, 26.8, 24.4, 22.8, 16.2, 13.8, 11.6; ESI–HRMS (negative mode) calcd. for
45
46
47
48
C24H38NO6: 436.2699, found: m/z 436.2697 [M – H]–.
49
50
51
52
53
54 (3R,5R)-7-{(1S,2S,6R,8S,8aR)-Hexahydro-2,6-dimethyl-8-[2-dimethylbutyrloxy]naphthal
55
56
57 enyl}-3,5-dihydroxy-N-hydroxyheptanamide (13). Simvastatin (11, in lactone form) was
58
59
60 converted to the corresponding hydroxamic acid 13 in 48% yield by general method A and
37

1
2
3
4 95% yield by general method B. The purity of product 13 was 98% as shown by HPLC on an
5
6
7 HC-C18 column (Agilent, 4.6 × 250 mm, 5 μm), tR = 20.6 min (gradients of 30–80% aqueous
8
9
10
11
CH3CN in 30 min). C25H41NO6; colorless oil; [α]24D = +194.1 (EtOAc, c = 1.0); TLC
12
13
15
16
17 1539, 1461, 1261, 1162, 1125, 1058, 975, 860 cm–1; 1H NMR (CDCl3, 400 MHz) δ 5.98 (1 H,
18
19
20 d, J = 9.6 Hz), 5.76–5.79 (1 H, m), 5.49 (1 H, br s), 5.44 (1 H, br s), 4.22 (1 H, br s), 3.77 (1
21
22
23 H, br s), 2.22–2.44 (6 H, m), 1.99 (1 H, dd, J = 13.2, 8.0 Hz), 1.85–1.89 (1 H, m), 1.50–1.58
24
25
13
26 (7 H, m), 1.26 (2 H, br s), 1.09–1.12 (9 H, m), 0.80–0.87 (6 H, m); C NMR (CDCl3, 100
27
28
29 MHz) 178.5, 169.7, 133.2, 131.7, 129.3, 128.2, 71.4, 68.4, 68.3, 43.0, 40.6, 37.5, 36.6, 34.9,
30
31
32 33.0, 32.8, 30.6, 29.6, 27.2, 24.7, 24.6, 24.4, 23.0, 13.8, 9.2; ESI–HRMS (negative mode)
33
34
35 calcd. for C25H40NO6: 450.2856, found: m/z 450.2854 [M – H]–.
36
37
38 (3R,5R)-7-[2-(4-Fluorophenyl)-5-isopropyl-3-phenyl-4-phenylcarbamoyl
39
40
41 pyrrol-1-yl]-3,5-dihydroxy-N-hydroxyheptanamide (14). Atorvastatin (in lactone form)
42
43
44
was converted to the corresponding hydroxamic acid 14 in 56% yield by general method A
45
46
47
48
and 88% yield by general method B. The purity of product 14 was 95% as shown by HPLC
49
50
51 on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 μm), tR = 14.8 min (gradients of 30–100%
52
53
54 aqueous CH3CN in 30 min). C33H36FN3O5; colorless oil; [α]26D = –1.3 (EtOAc, c = 1.0); TLC
55
56
58
59
60 1595, 1527, 1508, 1436, 1314, 1241, 1223, 1157, 1108, 1078, 1046, 843, 753, 692 cm–1; 1H
38

1
2
3
4 NMR (DMSO-d6, 400 MHz) δ 10.31 (1 H, br s), 9.77 (1 H, br s), 8.68 (1 H, br s), 7.50 (2 H, d,
5
6
7 J = 7.6 Hz), 7.18–7.24 (6 H, m), 7.07 (4 H, br s), 6.98–7.00 (2 H, m), 4.69 (1 H, br s), 4.60 (1
8
9
10
11
H, d, J = 4.0 Hz), 3.92–3.95 (1 H, m), 3.72–3.83 (2 H, m), 3.53 (1 H, br s), 3.21–3.25 (1 H,
12
13
14 m), 2.01 (2 H, d, J = 6.0 Hz), 1.63 (1 H, br s), 1.53 (1 H, br s), 1.28–1.38 (8 H, m); 13C NMR
15
16
17 (DMSO-d6, 100 MHz) 167.4, 166.1, 162.8, 160.3, 139.4, 135.9, 134.9, 133.4, 129.1 (2 ×),
18
19
20 128.7, 128.4 (2 ×), 127.6 (2 ×), 127.3, 125.3, 122.9, 120.6, 119.4 (2 ×), 117.5, 115.4, 115.2,
21
22
23 66.0, 65.6, 43.8, 40.9, 40.7, 25.6, 22.3 (2 ×); ESI–HRMS (negative mode) calcd. for
24
25
26 C33H35FN3O5: 572.2561, found: m/z 572.2562 [M – H]–.
27
28
29
30
31
32 (3R,5S,6E)-7-[4-(4-Fluorophenyl)-2-(N-methylmethanesulfonamido)-6-isopropyl-pyrimi
33
34
35 din-5-yl]-3,5-dihydroxy-N-hydroxyhept-6-enoic amide (15). Rosuvastatin (in lactone form)
36
37
38 was converted to the corresponding hydroxamic acid 15 in 51% yield by general method A
39
40
41 and 95% yield by general method B. The purity of product 15 was 97% as shown by HPLC
42
43
44
on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 μm), tR = 13.79 min (gradients of 25–80%
45
46
47
48
aqueous CH3CN in 30 min). C22H29FN4O6S; colorless oil; [α]24D = –1.1 (EtOAc, c = 1.0);
49
50
51 TLC (CH2Cl2/MeOH (9:1)) Rf = 0.35; IR νmax (neat) 3326, 2925, 2853, 1737, 1660, 1604,
52
53
54 1546, 1510, 1437, 1381, 1336, 1230, 1153, 1069, 965, 901, 845, 776 cm–1; 1H NMR
55
56
57 (DMSO-d6, 400 MHz) δ 10.32 (1 H, br s), 8.70 (1 H, br s), 7.71–7.74 (2 H, m), 7.30 (2 H, t, J
58
59
60 = 8.8 Hz), 6.50 (1 H, dd, J = 1.2, 16.2 Hz), 5.54 (1 H, dd, J = 5.6, 16.2 Hz), 4.94 (1 H, d, J =
39

1
2
3
4 4.4 Hz), 4.70 (1 H, d, J = 4.8 Hz), 4.19–4.22 (1 H, m), 3.88 (1 H, br s), 3.55 (3 H, s),
5
6
7 3.41–3.48 (4 H, m), 2.05 (2 H, d, J = 6.4 Hz), 1.48–1.56 (1 H, m), 1.37–1.43 (1 H, m), 1.22 (6
8
9
10
11
H, d, J = 6.8 Hz); 13C NMR (DMSO-d6, 100 MHz) 174.8, 167.9, 164.3, 163.3, 161.8, 157.3,
12
13
14 141.7, 134.9, 134.8, 132.6, 132.5, 122.2, 121.9, 115.6, 115.4, 69.1, 65.6, 44.6, 42.0, 41.2, 33.7,
15
16
17 31.7, 22.0 (2 ×); ESI–HRMS calcd. for C22H30FN4O6S: 497.1870, found: m/z 497.1873 [M
18
19
20 + H]+.
21
22
23
24
25
26 ASSOCIATED CONTENT
27
28
29 Supporting Information Available:
30
31
32 Figure s1–s7, synthetic procedures, 1H and 13
C NMR spectra of new compounds. This
33
34
35 material is available free of charge via the Internet at http://pubs.acs.org.
36
37
38
39
40
41 AUTHOR INFORMATION
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44
Corresponding Author
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48
*For J.-H.L.: phone, 8862-27823212 ext. 886; fax, 8862-27823060; E-mail, jlin@ntu.edu.tw.
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51 *For J.-M.F.: phone, 8862-33661663. fax, 8862-23637812. E-mail, jmfang@ntu.edu.tw.
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53
54
55
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57 Notes
58
59
60 The authors declare no competing financial interest.
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7
ACKNOWLEDGEMENTS
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We thank the National Science Council and Academia Sinica for financial support, as well as
12
13
14 the National Center for High Performance Computing for computing time and facilities.
15
16
17
18
19
20 ABBREVIATIONS USED
21
22
23 AFU, arbitrary fluorescence units; AOM, azoxymethane; ATCC, American Type Culture
24
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26 Collection; atorva, atorvastatin; CVD, cardiovascular disease; DMEM, Dulbecco’s Modified
27
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29 Eagle’s Medium; DML, designed multiple ligands; DSS, dextran sulphate sodium; GGTase,
30
31
32 geranylgeranyltransferase; H&E, hematoxylin and eosin; HAT, histone acetyltransferase;
33
34
35 HDACi, HDAC inhibitor; HDLP, histone deacetylase-like protein; HMG,
36
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38 3-hydroxy-3-methylglutaryl； HMGR, HMG-CoA reductase; HMGRi, HMGR inhibitor; HA,
39
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41 hydroxamic acid; lova, lovastatin; Lys, lysine; MTT,
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44
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; rosuva, rosuvastatin; SAHA,
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48
suberoylanilide hydroxamic acid; SD, standard deviation; Ser, serine; simva, simvastatin;
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51 TSA, trichostatin A.
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41

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Legends of Figures, Schemes, and Tables.
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Figure 1. Designed dual inhibitors for HMG-CoA reductase (HMGR) and histone deacetylase
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14 (HDAC). (A) Structure of a representative HMGR inhibitor. (B) Structures of
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17 representative HDAC inhibitors. (C) Design of lovastatin derivative bearing hydroxamic
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20 acid group by conjugation strategy via click reaction. (D) Design of lovastatin derivative
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23 bearing hydroxamic acid group by merged strategy using a short aliphatic spacer. (E)
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26 Design of lovastatin hydroxamic acid by fused strategy to share a common 6-carbon unit
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29 from SAHA and lovastatin. The hydroxamic acid (red) is a surrogate of the carboxylate
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32 group in lovastatin, and acts as a zinc chelation group for HDAC inhibition. The HMG-like
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35 moiety (blue) provides the appropriate interactions with HMGR and HDAC.
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41 on homology modeled HDAC1. The structural template, HDLP structure, was obtained
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from Protein Data Bank (PDB ID: 3CSR). Amino acid residues within 4.5 Å of ligand are
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presented in the two-dimensional interaction diagram. The blue circle on the ligand
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51 represents its exposure to the solvent. The larger circle indicates more exposure of the
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54 ligand to the solvent. The green and magenta dashed lines represent hydrogen bonding and
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57 metal chelation, respectively.
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47

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4 on HMGR. The illustration is similar to that described in Figure 2. The letters A and B
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7 before the residue numbers represent the A and B chains, respectively, in the PDB entry of
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HMGR (PDB ID: 1HW9).
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14 Figure 4. Effect of hydroxamate compounds on the acetylations of histone H3 and tubulin in
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17 A549 lung cancer cells. A549 cells were treated with indicated doses of drugs for 24 h: (A)
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20 conjugated compounds 6a and 6b; (B) hybrid compounds 12–15. SAHA (5 μM) and
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23 lovastatin (30 μM) were used as controls. Equivalent protein loading with similar level of
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26 β-actin was applied. Total protein lysates were subjected to Western blotting using
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29 antibodies specific for acetyl-histone H3, acetyl-tubulin or actin. SAHA (5 μM) and
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32 lovastatin (30 or 50 μM) were used for comparison. Total protein lysates were subjected to
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35 Western blotting using antibodies specific for acetyl-histone H3, acetyl-tubulin or actin.
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38 Equivalent protein loading with similar level of β-actin was applied.
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41 Scheme 1. Synthesis of lovastatin derivatives 6a and 6b bearing hydroxamic acid group by
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44
conjugation strategy.a
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48
Scheme 2. Synthesis of lovastatin derivative 10 bearing a hydroxamic acid group by merged
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51 strategy.a
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54 Scheme 3. Synthesis of statin hydroxamic acids 12–15.
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57 Table 1. Inhibitory activities (IC50) against HMGR and HDACs.
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60 Table 2. Inhibition on the HMGR activity in A549 lung cancer cells.
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4 Table 3. Inhibition on the growth of cancer and normal cells.
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Design and Synthesis of Dual-Action Inhibitors Targeting Histone Deacetylases and Hmg-Coa Reductase For Cancer Treatment

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