Discovery of Selective Histone Deacetylase 6 Inhibitors Using the

Article pubs.acs.org/jmc

Discovery of Selective Histone Deacetylase 6 Inhibitors Using the Quinazoline as the Cap for the Treatment of Cancer Zhuang Yang,†,‡,# Taijin Wang,‡,# Fang Wang,‡,# Ting Niu,§ Zhuowei Liu,∥ Xiaoxin Chen,∥ Chaofeng Long,∥ Minghai Tang,‡ Dong Cao,‡ Xiaoyan Wang,‡ Wei Xiang,‡ Yuyao Yi,§ Liang Ma,‡ Jingsong You,*,† and Lijuan Chen*,†,‡ †

State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy and Cancer Center, College of Chemistry, Sichuan University, Chengdu, 610064, China ‡ State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy and Cancer Center, West China Hospital of Sichuan University, Chengdu, 610041, China § Department of Hematology and Research Laboratory of Hematology, West China Hospital of Sichuan University, Chengdu, 610041, China ∥ Guangdong Zhongsheng Pharmaceutical Co., Ltd., Dongguan, Guangdong 523325, China S Supporting Information *

ABSTRACT: Novel selective histone deacetylase 6 (HDAC6) inhibitors using the quinazoline as the cap were designed, synthesized, and evaluated for HDAC enzymatic assays. NHydroxy-4-(2-methoxy-5-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)butanamide, 23bb, was the most potent selective inhibitor for HDAC6 with an IC50 of 17 nM and showed 25-fold and 200-fold selectivity relative to HDAC1 and HDAC8, respectively. In vitro, 23bb presented low nanomolar antiproliferative effects against panel of cancer cell lines. Western blot analysis further confirmed that 23bb increased acetylation level of α-tubulin in vitro. 23bb has a good pharmacokinetic profile with oral bioavailability of 47.0% in rats. In in vivo efficacy evaluations of colorectal HCT116, acute myelocytic leukemia MV4-11, and B cell lymphoma Romas xenografts, 23bb more effectively inhibited the tumor growth than SAHA even at a 4-fold reduced dose or ACY-1215 at the same dose. Our results indicated that 23bb is a potent oral anticancer candidate for selective HDAC6 inhibitor and deserves further investigation.

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icity.17,19−22 In addition, most of the current HDAC inhibitors show a lack of visible efficacy against solid tumors,5,13 which does really limit their application for the treatment of broad spectrum of cancer. To avoid the side effects and achieve the potency against solid tumors, an increasing number of investigations are focusing on the development of isotypeselective HDAC inhibitors, especially those targeting the isoform of HDAC6.9,10,23−37 HDAC6, which is expressed primarily in the cytoplasm, removes the acetyl group from lysine residues in a number of non-histone substrates, including α-tubulin, Hsp90.38−40 In contrast to the lethal effect of HDAC1−3 genetic ablation, it has been reported that mice with HDAC6-knocked out are viable.26,41 These results confirmed that HDAC6 selective inhibitors may have fewer side effects than pan-HDAC inhibitors or HDAC1−3 isoform selective inhibitors. The discovery of tubacin, a first reported selective HDAC6 inhibitor,

INTRODUCTION As one of the epigenetic targets, histone deacetylases (HDACs), which are responsible for deacetylation of lysine residues in histone and non-histone substrates,1 have recently emerged as an important target in the development of anticancer agents. 2−6 The 18 isoforms of HDAC are categorized into four groups: class I (HDACs 1, 2, 3, and 8), class II (class IIa (HDACs 4, 5, 7, and 9) and class IIb (HDACs 6 and 10)), and class IV (HDAC11) HDACs are all zincdependent deacetylases that are mechanistically distinct from NAD+-dependent class III HDACs.7−9 During past investigations on HDAC inhibition, a number of inhibitors have been reported, and some of them were licensed or at various stages of clinical evaluation (Figure 1).10,11 Vorinostat (SAHA),12−15 romidepsin (FK-228),13 belinostat (PXD101),16,17 and panobinostat (LBH-589)18 have gained FDA approvals for the treatment of cutaneous T-cell lymphoma, Tcell lymphoma, and multiple myeloma. However, most of them are class I selective (FK-228, PXD-101) or pan-HDAC inhibitors (SAHA, LBH-589). These nonselective or partially selective HDAC inhibitors usually lead to undesirable biological responses, such as fatigue, nausea/vomiting, and cardiotox© XXXX American Chemical Society

Special Issue: Epigenetics Received: June 20, 2015

A

DOI: 10.1021/acs.jmedchem.5b01342 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Examples of HDAC inhibitors.

Figure 2. Binding site of HDAC6 homology model and design of novel inhibitors selective for HDAC6.

rim differs greatly between the three isozymes. In order to identify the subtle difference, HDAC8, HDAC6 homology model and optimized HDAC1 were aligned by PyMol-1.5.3. The classic structure of HDAC inhibitors consists of a zinc binding group (ZBG) that chelates the active site Zn2+ ion, a linker, and a surface recognition cap group that interacts with the amino acid residues present at the surface of the HDAC. From the results of residue alignment, we found that among three protein structures, the residues of ZBG and linker region have a high degree of consistency and similar conformation. However, the forms and conformations of amino acids have a big difference in the cap region. This is thought to be due to the flexibility of loops (loop 1, Ser80-Tyr86; loop 2, Glu202Phe207 in HDAC6 model) (Supporting Information Table 1). Through comparing residues of cap region in HDAC1, HDAC6, and HDAC8 structures, we found that Phe82 (loop 1) and Met198 (loop 2) are critical for forming the surface groove which is composed of the following residues: Phe136, Ser84, Phe82, Met198, Phe196. But the residues of similar function are absent in HDAC1 and HDAC8 structures (Figure 2 and Supporting Information Figure S1). And literature surveys also indicated that the cap group may contribute an

which was identified in 2003 during a high-throughput screen of 7392 compounds, has prompted numerous investigations toward the development of HDAC6inhibitors, such as tubastain A, HPOB,14 and ACY-1215.42,43 Especially, ACY-1215 is currently in phase II clinical trials to treat multiple myeloma.44 Such promising results drew our attention to develop novel, selective, and highly effective small molecule inhibitors of HDAC6.

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RESULTS AND DISCUSSION Initial Molecular Docking Studies. The lack of crystal structure led to more difficulty in the designing of HDAC6 selective inhibitors than the pan-HDAC inhibitors and HDAC1−3-selective inhibitors. Intrigued by designing highly selective inhibitors of HDAC6 isoform, we performed the protein structure alignment of HDAC1 (PDB code 4BKX), HDAC6 (homology model), and HDAC8 (PDB code 2V5W). For HDAC6, a homology model was generated by multiplethread alignments, as described by Yang Zhang’s research group through a Web server (I-TASSER).45 Analysis of the Zn ion binding pockets of HDAC1, HDAC6, and HDAC8 revealed that, while the active pocket is relatively conserved, the channel B



Article

Scheme 1. Synthesis of 7a−da

Reagents and conditions: (a) NaH, MOMCl, DMF, 0 °C to rt; (b) H2, Pd/C (10%), EtOH, rt; (c) (i) (CH2O)n, MeONa, MeOH, rt; (ii) NaBH4, reflux; (d) (i) 4-chloro-2-methylquinazoline, cat. conc HCl, (Me)2CHOH, rt; (ii) EtOAc, HCl (g), rt; (e) Br(CH2)nCOOEt, Cs2CO3, CH3CN, reflux; (g) 50% NH2OH aq, NaOH, CH2Cl2/MeOH (1:2), rt. a


a

Reagents and conditions: (a) (i) 4-chloroquinazoline, cat. conc HCl, (Me)2CHOH, rt; (ii) EtOAc, HCl (g), rt; (b) Br(CH2)nCOOEt, Cs2CO3, CH3CN, reflux; (c) 50% NH2OH aq, NaOH, CH2Cl2/MeOH (1:2), rt.


a

Reagents and conditions: (a) guanidine carbonate, t-BuOH, t-BuOK; (b) POCl3, reflux; (c) (i) 4, cat. conc HCl, (Me)2CHOH, rt; (ii) EtOAc, HCl (g), rt; (d) Br(CH2)nCOOEt, Cs2CO3, CH3CN, reflux; (e) 50% NH2OH aq, NaOH, CH2Cl2/MeOH (1:2), rt.

shown in Scheme 1. Commercially available p-nitrophenol (1) was the starting material for the synthesis. The phenolic hydroxyl group was first protected by chloromethyl methyl ether (MOMCl) to give 2. Then reduction of 2 led to 3, which was further N-methylated by reacting with paraformaldehyde in MeONa−MeOH solution and reduction by NaBH4 sequentially. The resulting aniline 4 was coupled with 4-chloro-2methylquinazoline, giving the intermediate compound 5. Then we used the Br(CH2)nCOOEt (n = 1, 3−5) to react with 5 to obtain compounds 6a−d, which had different length alkyl chains. Those compounds were directly converted to hydroxamic acid compounds 7a−d by NH2OH.

important function to the selective activity. Hence, a series of fragments, which comfortably occupy the surface groove and form robust interaction, were designed to target this area. By molecular docking results and the feasibility analysis of synthesis (Supporting Information Figure S2 and S3), we chose 2-methylquinazoline as starting point, which is found in many bioactive compounds,28,46−48 and has been used in the research of HDAC inhibitor with some exciting results yielded.28 Chemistry. At the beginning of our research, we synthesized a series compounds with 2-methylquinazoline as the capping groups. The synthetic route to compounds 7a−d is C



Article

Scheme 4. Synthesis of 23aa−ae and 23ba−bea

Reagents and conditions: (a) NaH, MOMCl, DMF, 0 °C to rt; (b) H2, Pd/C (10%), EtOH, rt; (c) (i) (CH2O)n, MeONa, MeOH, rt; (ii) NaBH4, reflux; (d) (i) 4-chloro-2-methylquinazoline, cat. conc HCl, (Me)2CHOH, rt; (ii) EtOAc, HCl (g), rt; (e) Br(CH2)nCOOEt, Cs2CO3, CH3CN, reflux; (g) 50% NH2OH aq, NaOH, CH2Cl2/MeOH (1:2), rt. a

Scheme 5. Synthesis of 27a

a

Reagents and conditions: (a) Br(CH2)nCOOEt, Cs2CO3, CH3CN, reflux; (b) H2, Pd/C (10%), EtOH, rt; (c) 4-chloro-2-methylquinazoline, cat. conc HCl, (Me)2CHOH, rt; (d) 50% NH2OH aq, NaOH, CH2Cl2/MeOH (1:2), rt.

coupled with 4-chloro-2-methylquinazoline, and the resulting compound 26 was converted to hydroxamic acid compound 27. Biological Evaluation. In Vitro HDAC Isoform-Selectivity of the Compounds. The synthesized compounds were evaluated for the inhibitory activities on the HDAC1, HDAC6, and HDAC8 isoforms and SAHA, ACY-1215 as positive controls. The influence of various quinazoline analogues caps on HDAC6 is summarized in Table 1. Compounds 7a−d, which used the 2-methylquinazoline’s capping group and various chain length linkers with different lengths at the C-4 position, had an efficiently selective inhibition activity of HDAC6. The most potent compound 7a, with IC50 value of 8.6 nM on HDAC6, displayed the best selectivity of HDAC6 versus HDAC1 (20-fold) and HDAC8 (137-fold). The selective inhibitory activity of HDAC6 further decreased with an increase in carbon chain length, and the IC50 values of 7b (n = 3), 7c (n = 4), and 7d (n = 5) on HDAC6 were 196, 57, and 34 nM, respectively. Additionally, the selectivity toward HDAC6 versus HDAC1 and HDAC8 was also decreased. The same results were also observed in the classes with quinazoline (10a−d) and 2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine (16a−d) as the capping group. In the two classes, the short carbon chain length exhibited low nanomolar inhibition (10a and 16a) on HDAC6 and good selectivity

To search the structure and relationship analysis (SAR) of cap, we also synthesized two kinds of HDAC inhibitors which used different 2-methylquinazoline analogues as the capping group. The first one was quinazoline without substituent group at C-2 position (Scheme 2). The synthetic route was similar to Scheme 1, which using 4-chloroquinazoline replaced 4-chloro2-methylquinazoline to couple with 4, giving 10a−d. Scheme 3 shows the preparation of another quinazoline derivatives using a five fat ring that replaced the phenyl ring of quinazoline. 13, which was synthesized by 11 cyclizing with guanidine carbonate under the action of t-BuOK and chlorinated with POCl3, reacted with 4 and the subsequent reactions were same as Scheme 1, ging compounds 16a−d. Then we focused on the linker region. As shown in Scheme 4, the m-nitrophenol (17a) and 2-methoxy-5-nitrophenol (17b) were used as the starting materials, and the following reactions were similar as in Scheme 1. Eventually, we got the compounds 23aa−ad and 23ba−bd. On the basis of the most potent compound 23bb, we also synthesized the compound 27 to evaluate the function of Nmethyl group, and the synthesis is shown in Scheme 5. 17b was reacted with ethyl 4-bromobutyrate, giving 24, which was further hydrogenated by hydrogen. Then compound 25 was D



Article

Table 1. Enzyme Inhibition Data for 7a−e, 10a−e, and 16a−e

IC50 a (nM)

selective fold

compd

n

HDAC1

HDAC6

HDAC8

HDAC1/6

HDAC8/6

7a 7b 7c 7d 10a 10b 10c 10d 16a 16b 16c 16d SAHA ACY-1215

1 3 4 5 1 3 4 5 1 3 4 5

172 ± 30 84 ± 14 204 ± 23 272 ± 36 242 ± 27 76 ± 9 203 ± 6 176 ± 47 264 ± 55 195 ± 35 212 ± 32 120 ± 24 11 ± 1 38 ± 5

8.6 ± 1 196 ± 32 57 ± 7 34 ± 7 14 ± 1 34 ± 4 49 ± 7 19 ± 5 17 ± 3 40 ± 3 63 ± 11 20 ± 2 15 ± 3 9±2

1181 ± 53 1465 ± 60 1698 ± 189 1510 ± 323 1422 ± 79 2457 ± 20 1326 ± 2 1058 ± 207 2974 ± 627 6190 ± 876 1987 ± 101 1003 ± 176 172 ± 45 254 ± 57

20.0 0.4 3.6 8.0 17.3 2.2 4.1 9.3 15.5 4.9 3.4 6.0 0.7 4.2

137.3 7.5 29.8 44.4 101.6 72.3 27.1 55.7 174.9 154.8 31.5 50.2 11.5 28.2

Compounds were tested in the 10-dose IC50 mode in duplicate with 3-fold serial dilutions starting at 10 μM. The IC50 values are the mean of at least two experiments. a

Table 2. Enzyme Inhibition Data for 23aa−ad and 23ba−bd

IC50 a (nM)

selective fold

comp

n

HDAC1

HDAC6

HDAC8

23aa 23ab 23ac 23ad 23ba 23bb 23bc 23bd 27 SAHA ACY-1215

1 3 4 5 1 3 4 5

>1000 >1000 306 ± 90 73 ± 2 >1000 422 ± 1 253 ± 23 160 ± 3 >1000 11 ± 1 38 ± 5

>1000 >1000 >1000 >1000 >1000 17 ± 2 111 ± 30 23 ± 5 >1000 15 ± 3 9±2

>1000 >1000 >1000 >1000 >1000 3398 ± 487 5189 ± 648 1531 ± 78 >1000 172 ± 45 254 ± 57

HDAC1/6

HDAC8/6

24.8 2.3 7.0

199.9 46.7 66.6

0.7 4.2

11.5 28.2

a Compounds were tested in the 10-dose IC50 mode in duplicate with 3-fold serial dilutions starting at 10 μM. The IC50 values are the mean of at least two experiments.

toward to HDAC1 and HDAC8. Interestingly, compounds 7d, 10d, 16d with a carbon chain length of five carbons (n = 5) also showed low nanomolar inhibition on HDAC6 but decreased selectivity on HDAC1 and HDAC8. These results suggested that the length of the hydroxamic acid side chain is important for the activity and selectivity of HDAC6. When the carbon chain was at the C-4 position, the optimal length of the carbon

chain linker should be one carbon for the activity and selectivity of HDAC6. Uniquely, compound 16b, which used a five-fatty ring to replace the phenyl ring of quinazoline and a threecarbon linker, also showed 155-fold selectivity of HDAC6 versus HDAC8. All the tested compounds exhibited HDAC6 inhibitory activity and selectivity comparable to or superior to that of SAHA and ACY-1215, demonstrating that the strategy E



Article

of introducing quinazoline analogues as the cap group is successful. To investigate the position of side chain on the HDAC6 activity, we changed the hydroxamic acid side chain from C-4 to C-3 position and with (23ba−bd) or without methoxyl group (23aa−ad) at the C-4 position. As shown in Table 2, the activities of compounds 23aa−ad on HDAC6 were all decreased with various carbon chain lengths, and the IC50 values of 23aa−ad on HDAC6 were above 1 μM. In contrast, 23bb−bd with methoxyl group at the C-4 position dramatically increased the activity and selectivity on HDAC6. 23bb−bd significantly inhibited the HDAC6, especially compound 23bb, which had achieved 25-fold selectivity to HDAC1 and 200-fold selectivity to HDAC8 with an IC50 value of 17 nM on HDAC6. These results indicated that the substitute on C-4 was necessary to keep the activity and selectivity on HDAC6 and introduction of a methoxyl group to the C-4 position was a successful strategy for the selectivity of HDAC6. Interestingly, 23ba, with one carbon chain and compound 27, where the methyl was replaced by hydrogen, completely lost their activity of HDAC1, -6, and -8 with IC50 values above 1 μM. Compound 23bb, with three-carbon chain length, was the best one for the selectivity and activity on HDAC6. In comparison to 7a, 23bb increased two carbon chain lengths at the C-3 position, and its maintained activity on HDAC6 may contribute to the change of the carbon chain from the C-4 to C-3 position. Then 7a, 10a, 16a, and 23bb, which showed over 15-fold selectivity of HDAC6 versus HDAC1 and 100-fold selectivity of HDAC6 versus HDAC8, were evaluated in tubulin acetylation (Tub-Ac)27,39,49 and H3 acetylation (Ac-H3) as a surrogate for cellular HDAC6 inhibition. As shown in Table 3, compounds

Table 4. HDAC Inhibition Activity of Compound 23bb IC50 a (nM)

compd 7a 10a 16a 23bb

Tub-Ac 26.2 49.4 50.8 58.9

± ± ± ±

2 6 5 6

Ac-H3

A375

HeLa

213 ± 11 432 ± 27 2851 ± 54 45 ± 7

171 ± 20 379 ± 36 2217 ± 62 49 ± 20

HDAC1 HDAC2 HDAC3 HDAC8

422 ± 1 386 ± 7 439 ± 9 3398 ± 487

HDAC6 HDAC10 HDAC11

ACY-1215

LBH-589

Class I HDACs 38 ± 5 95 ± 9 135 ± 27 254 ± 57 Class IIa HDACs >10000 >10000 >10000 >10000 >10000 >10000 >10000 >10000 Class IIb HDACs 17 ± 2 9±2 1176 ± 168 194 ± 54 Class IV HDAC >10000 >10000

1 3 2 5

± ± ± ±

0.1 0.05 0.1 0.4

SAHA 11 ± 1 35 ± 11 30 ± 5 172 ± 45

338 ± 17 190 ± 35 4354 ± 267 888 ± 26

>10000 >10000 >10000 >10000

4 ± 0.2 4±1

15 ± 3 170 ± 10

4112 ± 84

>10000

a

Compounds were tested in the 10-dose IC50 mode in duplicate with 3-fold serial dilutions starting at 10 μM. The IC50 values are the mean of at least two experiments.

HDACs (HDAC4, -5, -7, -9, -11), 23bb did not show appreciable inhibition at 10 μM. The HDAC6 selectivity of compound 23bb is superior to ACY-1215, LBH-589, and SAHA. Although the ACY-1215 is known as a selective HDAC6 inhibitor, the selective indexes of HDAC6 with class I isoforms were lower than 23bb. The results indicate that 23bb is a potential candidate for further study as a selective HDAC6 inhibitor. In Vitro Antiproliferative Activities of 23bb. The antiproliferative activities against 11 kinds of hematological tumors (myelomaU266, RPMI8226 cells, human leukemia MV4-11, K562 cells, and human B cell lymphoma Ramos cells) or solid tumors (ovarian cancer A2780s, SKOV-3 cells, breast cancer SKBR3 cells, liver cancer HepG2 cells, lung cancer H460, A549 cells, cervical cancer HeLa cells and colon cancer HCT116, HT29 cells) cell lines of the compound 23bb were evaluated by MTT, and the SAHA and ACY-1215 were as positive control. Compound 23bb showed significant antiproliferative potential with the IC50 values ranging from 14 to 104 nM in these tumor cell lines. In contrast, the selective inhibitor ACY-1215 was weakly active to all the cell lines, with IC50 values above 5 μM. SAHA was weakly active to the solid tumor cell lines, such as A2780s, SKOV-3, SKBR3, HepG2, H460, A549, HCT116, and HT29, with IC50 values above 1 μM. It turned out that the antiproliferative activities of 23bb were better than ACY-1215 and SAHA (Table 5), and 23bb could be used in the therapy of solid tumors. Selective Upregulation of the Aceylation Level of αTubulin. The effects of 23bb, ACY-1215, LBH-589, and SAHA on the acetylation level of histone H3 (a known substrate for HDACs 1, 2, and 3) and α-tubulin (a known substrate for HDAC6), the biomarkers of HDAC inhibition,50 in HCT116 and MV4-11 cells were measured by Western blot. As shown in Figure 3, in agreement with the relative potency in HDAC6 inhibition and Tub-Ac assays, 23bb and ACY-1215 induced Acα-tubulin in a concentration-dependent manner and could upgrade the Ac-α-tubulin level at 10 nM, while increase of histone acetylation was only observed at high concentration of 1000 nM. LBH-589 and SAHA, the nonselective pan-HDAC

IC50 b (nM)

456.5 ± 24 1260 ± 187 1767 ± 302 1741 ± 156

23bb

HDAC4 HDAC5 HDAC7 HDAC9

Table 3. Tubulin Acetylation and H3 Acetylation Induction and Antiproliferative Activities against A375 and HeLa Cells of Selected Compounds EC50 a (nM)

isoform

a

EC50 value values of tubulin acetylation and Ac-H3 are based on ELISA experiments run in duplicate in HeLa cells. bIC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SEM from the dose−response curves of at least three independent experiments.

showed strong Tub-Ac activities and low Ac-H3 induction in cellular assays, which were in line with the in vitro HDAC inhibitory activities. We then evaluated the antiproliferative activity on human malignant melanoma A375 cells and cervical cancer HeLa cells, 23bb showed the most potent activities with IC50 values of 50 and 49 nM on A375 and HeLa cells, respectively. Since 23bb showed both potent antiproliferative activity and selectivity of HDAC6, it was further determined for the activity on the other HDAC isoforms. As summarized in Table 4, compound 23bb was potent at 400 nM against HDAC1−3 and at micromolar level against HDAC8 and HDAC10. 23bb showed 25-fold and 200-fold selectivity of HDAC6 relative to the inhibition of HDAC1 and HDAC8 in comparison to 4-fold and 21-fold of ACY-1215. With regard to class IIa and class IV F



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Docking Results. The molecular docking with compound 23bb in HDAC6 was performed, and the results are shown Figure 4. The hydroxamic tail of compound 23bb forms key Hbonds with Tyr298 and Glu255. These hydrogen bond forces are critical for stabilizing inhibitors in a specific pose chelating with Zn ion. More importantly, the quinazoline of compound 23bb occupies the cap region and comfortably locks into the surface groove. The ring of quinazoline could form π−π stacking interaction with residues of Phe136 and Phe82, hydrophobic interaction with residues of Ile85, Met198, and Gly135. Additionally, the nitrogen atom of quinazoline could form H-bond with residues of Gly135. And yet in the same docking procedures in HDAC1 and HDAC8, compound 23bb could not enough occupy the cap region and form the similar interaction (Supporting Information Figure S4). And this shows good agreement with the results of biological activity. Pharmacokinetic Studies. 23bb (dissolved in ddH2O for intravenous administration and 0.5% CMC-Na aqueous solution for oral dosing) was administered to SD rats intravenously (iv) at 12 mg/kg body weight and orally at 12 mg/kg body weight. Blood samples were taken, and the plasma was analyzed for concentration of 23bb using an LC−MS/MS system. As shown in Table 6, low clearance and long terminal half-life are observed in 23bb. The oral bioavailability of 23bb is excellent in rats and the bioavailability was up to 47.0%,

Table 5. Activities of 23bb and SAHA against various tumor cell Lines IC50 a (nM) tumor type multiple myeloma leukemia B cell lymphoma ovarian breast liver lung cervical cancer colon

cell line

23bb

U266

14 ± 6

RPMI8226 MV4-11 K562 Ramos

15 60 63 71

A2780s SKOV-3 SKBR3 HepG2 H460 A549 HeLa HCT116 HT29

46 ± 11 50 ± 8 22 ± 8 41 ± 1 55 ± 13 104 ± 29 49 ± 20 35 ± 21 72 ± 37

± ± ± ±

4 32 18 31

ACY-1215

SAHA

>5000

581 ± 96

1468 ± 310 >5000 >5000 >5000

423 ± 54 85 ± 19 444 ± 43 >1000

>5000 >5000 >5000 >5000 >5000 >5000 >5000 >5000 >5000

>1000 >1000 >1000 >1000 >1000 >1000 711 ± 245 >1000 >1000

a

IC50 = compound concentration required to inhibit tumor cell proliferation by 50%. Data are expressed as the mean ± SEM from the dose−response curves of at least three independent experiments.

inhibitors, increased both Ac-H3 and Ac-α-tubulin levels at low concentration.

Figure 3. Western blot analysis of acetylated α-tubulin, acetylated histone H3 inHCT116, and MV4-11cell lines after 6 h of treatment with compound 23bb, ACY-1215, LBH-589, and SAHA at 10, 100, 1000, 10000 nM. GAPDH was used as a loading control. G



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Antitumor Activity in Vivo. We first evaluated the in vivo efficacy of 23bb using a HCT116 xenograft Balb/c nude mouse model and a MV4-11 xenograft NOD/SCID mouse model, with SAHA administrated for comparison. The administration, dosing schedules, and results are presented in Table 7. As displayed in Table 7, 23bb reduced the tumor growth in both the hematological tumor MV4-11 xenograft model and solid tumor HCT116 xenograft model, and the tumor inhibitory effects were superior to positive drug SAHA. The significant antitumor activities were observed by intravenous administration of 23bb at 50 mg/kg on MV4-11 and HCT116 xenograft models. The growth of MV4-11 and HCT116 cancer cell xenografts was suppressed by 55.0% and 76.3% (percent of tumor mass change [TGI] values) after iv administration of 23bb at 50 mg/kg. In contrast, SAHA had no inhibitory activity at the same dose on the MV4-11 xenograft model and only showed 36.6% tumor growth inhibition on HCT116 xenograft model. We also established the HCT116 xenograft model to investigate the antitumor activity of oral administration of 23bb. As displayed in Table 7, the TGI value of oral administration of 23bb (25 mg/kg) on HCT116 xenograft model was 60.4%, which was superior to the SAHA group (100 mg/kg, 59.2%). Additionally, the body weight decrease is acceptable and no other adverse effects were observed upon treatment with 23bb (Figure 5). Since LBH589 is a new pan-inhibitor of HDAC approved by FDA for the treatment of hematologic cancer, we further established a Ramos xenograft NOD/SCID female B cell lymphoma model to compare the in vivo activity of 23bb with ACY1215 and LBH589. As shown in Table 7, 23bb inhibited the tumor growth dose-dependently; the TGI values were 22.5% and 58.79% at 40 and 80 mg/kg, respectively, by oral administration. In contrast, ACY-1215 had no effect at 40 mg/ kg. Although LBH-589 at ip 10 mg/kg administration caused the tumor reduction with the TGI values of 16.6%, the toxicity (body weight of mice decreased and three of eight mice died during experimental period) of LBH 589 was observed. Those results indicated that 23bb is more effective and safe in contrast to ACY-1215 and LBH-589, suggesting that 23bb could be used as a novel potent compound for further research on the therapy of both hematological tumor and solid tumor.

Figure 4. Compound 23bb docked into HDAC6 (homology model).

Table 6. Pharmacokinetic Parameters Tested in Vivo 23bb route

iv

po

Na dose (mg/kg) CL (L h−1 kg−1)b Vss (L/kg)c AUC0−inf (μg/L·h)d Cmax (μg/L)e T1/2 (h)f F (%)g

6 12 7.008 61.263 2434.117 2213.217 7.658

6 12 12.877 154.811 1242.234 238.133 9.62 47.0

a

Numbers of rats. bSystemic clearance. cVolume of distribution following intravenous dosing. dArea under the curve following intravenous dosing, integrated drug concentration with respect to time and integrated drug concentration with respect to time following oral dosing. eMaximum plasma concentration following intravenous dosing. fplasma half-life. gPercent oral bioavailability.

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CONCLUSIONS To develop novel HDAC6 selective inhibitors with a quinazoline cap group, a series of hydroxamic acid analogues were prepared and evaluated for bioactivity in vitro and in vivo.

suggesting that 23bb is suitable both for iv and oral dosing as an anticancer agent. Table 7. Summary of Tumor Growth Inhibition administration tumor model MV4-11 HCT116 HCT116 Ramos

compd 23bb SAHA 23bb SAHA 23bb SAHA 23bb ACY-1215 LBH-589

a

dose (mg/kg) 50 50 50 50 25 100 40 80 40 10

toxicity a

route

body weight change (%)

death

antitumor activity, tumor mass change (%)

12 12 9 9 8 8 6 6 6 6

iv ip iv ip po po po po po ip

−3.8

0/6 0/6 0/7 0/7 0/7 0/7 0/8 1/8 0/8 3/8

55.0 0 76.3 36.6 60.4 59.2 22.5 58.79 −6.4 16.6

schedule Q2D Q2D Q2D Q2D Q2D Q2D Q2D Q2D Q2D Q2D

× × × × × × × × × ×

−5.7 −3.0 −3.8 −6.1

−1.9

Q2D, every 2 days. H



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Figure 5. Inhibitory effect of 23bb on the xenograft models: (A) 23bb inhibited tumor growth on the MV4-11 xenograft model; (B) 23bb inhibited tumor growth on the HCT116 xenograft model; (C) 23bb inhibited tumor growth on the Ramos xenograft model.

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The inhibitory activities on the HDAC1, HDAC6, and HDAC8 isoforms of the synthesized compounds and a SAR analysis revealed that the strategy successfully achieved the goal to find a novel selective and highly efficient small molecule inhibitor of HDAC6. The most potent compound 23bb displayed at least 22.7-fold selectivity for HDAC6 over the other isoforms with an IC50 value of 17 nM. The antiproliferation assay revealed that the IC50 values of 23bb ranged from 14 to 104 nM against 11 diverse cancer cell lines, representing both the hematological and solid models. Subsequently, the Western blot analysis further confirmed the selectivity profile of compound 23bb. 23bb, with the oral bioavailability of 47.0% in rats, showed substantial antitumor activity in both solid and hematologic tumor models. 23bb caused tumor growth inhibition of 55.0% in human AML MV4-11 xenograft model and 76.3%, 60.4% in human colorectal HCT116 xenograft model after iv treatment with 50 mg/kg and po treatment with 25 mg/kg, respectively, which was more efficient than SAHA groups (0 for MV4-11 xenograft models and 36.6%, 59.2% for HCT116 xenograft models, 50 mg/kg, ip, and 100 mg/kg, po, respectively). In a Ramos xenograft NOD/SCID female B cell lymphoma model, 23bb exhibited superior inhibitory activity on tumor growth to ACY-1215 and LBH-589, suggesting that 23bb is a potential and promising oral anticancer agent with selective HDAC6 inhibitory properties.

EXPERIMENTAL SECTION

Chemistry. All the chemical solvents and reagents, which were analytically pure without further purification, were commercially available. TLC was performed on 0.20 mm silica gel 60 F254 plates (Qingdao Haiyang Chemical, China). 1H NMR and 13C NMR spectra were on a Bruker Avance 400 spectrometer (Bruker Company, Germany), using TMS as an internal standard. Chemical shifts were given in ppm (parts per million). Mass spectra were recorded on QTOF Priemier mass spectrometer (Micromass, Manchester, U.K.). The purity of each compound (>95%) was determined on an Waters e2695 series LC system (column, Xtimate C18, 4.6 mm × 150 mm, 5 μm; mobile phase, methanol (90%)/H2O (10%) to methanol (20%)/ H2O (80%); flow rate, 1.0 mL/min; UV wavelength, 254−400 nm; temperature, 25 °C; injection volume, 10 μL). General Procedures of Method A for the Synthesis of 2, 18a, and 18b. Phenol (200 mmol) was dissolved in DMF (500 mL), and the resulting solution was cooled to 0 °C. 60% NaH (15.36 g, 400 mmol, 2 equiv) was added in portions. After 0.5 h, the MOMCl (30.4 mL, 400 mmol, 2 equiv) was added dropwise. The reaction mixture was moved to room temperature and monitored by TLC (petroleum ether/ethyl acetate, 2:1). When the reaction was completed, the resulting solution was poured into water (5 L), affording a yellow solid. This solid was collected by filtration and purified by recrystallization from EtOH to give the title compounds as yellow acicular crystal. General Procedures of Method B for the Synthesis of 3, 19a, 19b, and 25. The nitro compound was dissolved in EtOH (500 mL), and 10% Pd/C (5%) was added. Then the mixture was stirred at room I



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temperature under H2. When the reaction was finished, the mixture was filtered by Celite and washed by ethyl acetate. The filtrate was concentrated in vacuo to give the title compounds as red solid, used in the next step without further purification. General Procedures of Method C for the Synthesis of 4, 20a, and 20b. An amount of 500 mL of MeOH was added into a 1 L bottle and cooled to 0 °C. Na (13.8 g, 600 mmol, 5 equiv) was added in portions. The resulting mixture was moved to room temperature. When Na was dissolved, aniline (120 mmol, 1 equiv) and paraformaldehyde (5 g, 168 mmol, 1.4 equiv) were added. The mixture was stirred overnight. Then NaBH4 (4.54 g, 120 mmol, 1 equiv) was added, and the resulting solution was reflux for 2 h and concentrated in vacuo. An amount of 1 L of NaOH (1 N) was added to the residue, affording a white solid. This solid was collected by filtration and dried to give the title compound. General Procedures of Method D for the Synthesis of 5, 8, 14, 21a, 21b, and 26. (i) Quinazoline analogues (85 mmol, 1 equiv) and N-methylaniline (85 mmol, 1 equiv) were added to 500 mL of (Me)2CHOH. Then 1 mL of concentrated HCl was added to the mixture and stirred at room temperature. The reaction generated a lot of yellow solid precipitation, which was collected by filtration and basified by saturated NaHCO3 aqueous solution. The resulting mixture was extracted with ethyl acetate (3 × 200 mL). The organic layer was collected without further treatment. (ii) The previously collected organic layer was placed into a three-neck bottle. Then HCl gas was passed into the solvent, which was stirred at room temperature. The reaction was monitored by TLC (petroleum ether/ethyl acetate, 1:2). With the reaction going on, a lot of yellow solid was formed. When the reaction was finished, the mixture was filtered. The filter cake was added into saturated NaHCO3 aqueous solution. The resulting mixture was filtered again, giving a white solid which was recrystallized with EtOH to give the title compound as white solid. 4-(Methyl(2-methylquinazolin-4-yl)amino)phenol (5). 5 was obtained from compound 4 and 4-chloro-2-methylquinazoline as described for method D. 1H NMR (400 MHz, DMSO-d6) δ: 9.71 (s, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.62 (t, J = 7.1 Hz, 1H), 7.12 (d, J = 8.6 Hz, 2H), 7.08 (t, J = 7.2 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 3.52 (s, 3H), 2.62 (s, 3H). 4-(Methyl(quinazolin-4-yl)amino)phenol (8). 8 was obtained from compound 4 and 4-chloroquinazoline as described for method D. 1 H NMR (400 MHz, DMSO-d6) δ: 10.04 (s, 1H), 8.91 (d, J = 1.8 Hz, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H), 7.28 (t, J = 8.4 Hz, 1H), 7.24 (d, J = 7.3 Hz, 2H), 6.91 (d, J = 8.3 Hz, 3H), 3.64 (d, J = 1.1 Hz, 3H). 4-(Methyl(2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)amino)phenol (14). 14 was obtained from compound 4 and compound 13 as described for method D. 1H NMR (400 MHz, DMSO-d6) δ: 9.58 (s, 1H), 7.10−7.00 (m, 2H), 6.80−6.72 (m, 2H), 3.32 (s, 3H), 2.58 (t, J = 7.7 Hz, 2H), 2.40 (s, 3H), 1.79 (t, J = 7.2 Hz, 2H), 1.68−1.59 (m, 2H). 3-(Methyl(2-methylquinazolin-4-yl)amino)phenol (21a). 21a was obtained from compound 20a and 4-chloro-2-methylquinazoline as described for method D. 1H NMR (400 MHz, DMSO-d6) δ: 9.67 (s, 1H), 8.53 (d, J = 7.8 Hz, 1H), 7.85−7.77 (m, 2H), 7.70 (d, J = 7.4 Hz, 1H), 7.62−7.52 (m, 2H), 7.29 (t, J = 8.2 Hz, 1H), 6.80−6.75 (m, 1H), 3.42 (s, 3H), 2.53 (s, 3H). 2-Methoxy-5-(methyl(2-methylquinazolin-4-yl)amino)phenol (21b). 21b was obtained from compound 20b and 4-chloro2-methylquinazoline as described for method D. 21 g, 84% yield two steps. 1H NMR (400 MHz, DMSO-d6) δ: 9.31 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.59 (ddd, J = 8.3, 6.1, 2.0 Hz, 1H), 7.13−7.03 (m, 2H), 6.94 (dd, J = 7.1, 1.9 Hz, 1H), 6.64 (dd, J = 7.4, 2.2 Hz, 2H), 3.79 (s, 3H), 3.47 (s, 3H), 2.58 (s, 3H). Ethyl 4-(2-Methoxy-5-((2-methylquinazolin-4-yl)amino)phenoxy)butanoate (26). 26 was obtained from compound 25 and 4-chloro-2-methylquinazoline as described for method D step (i): 85.3% yield. 1H NMR (400 MHz, DMSO-d6) δ: 9.56 (s, 1H), 8.47 (d, J = 8.2 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 7.67 (d, J = 8.2 Hz, 2H), 7.53 (t, J = 7.5 Hz, 1H), 7.43 (dd, J = 8.7, 2.2 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 4.07 (q, J = 7.1 Hz, 2H), 4.02 (t, J = 6.4 Hz, 2H), 3.77 (s, 3H),

2.48 (d, J = 5.7 Hz, 5H), 2.02 (p, J = 6.8 Hz, 2H), 1.18 (t, J = 7.1 Hz, 3H). General Procedures of Method E for the Synthesis of 6a−e, 9a−e, 15a−e, 22aa−ae, 22ba−be, and 24. The phenol intermediate (1 mmol, 1 equiv) and Cs2CO3 (652 mg, 2 mmol, 2 equiv) were added into 5 mL of CH3CN, and the resulting mixture was heated at reflux for 0.5 h. Then the bromo-fatty acid ethyl ester (2 mmol, 2 equiv) was added dropwise. After 2 h, the solvent was filtered through Celite. The filtrate was concentrated in vacuo to yield a yellow oily product, without further purification. Ethyl 2-(4-(Methyl(2-methylquinazolin-4-yl)amino)phenoxy)acetate (6a). 6a was obtained from compound 5 and ethyl bromoacetate as described for method E. 1H NMR (400 MHz, CDCl3) δ: 7.74 (d, J = 8.3 Hz, 1H), 7.51 (ddd, J = 8.3, 6.7, 1.6 Hz, 1H), 7.10 (d, J = 8.9 Hz, 2H), 7.03−6.93 (m, 2H), 6.91 (d, J = 8.9 Hz, 2H), 4.63 (s, 2H), 4.28 (q, J = 7.1 Hz, 2H), 3.58 (s, 3H), 2.72 (s, 3H), 1.30 (t, J = 7.1 Hz, 3H). Ethyl 4-(4-(Methyl(2-methylquinazolin-4-yl)amino)phenoxy)butanoate (6b). 6b was obtained from compound 5 and ethyl 4-bromobutyrate as described for method E. 1H NMR (400 MHz, CDCl3) δ: 7.74 (d, J = 8.3 Hz, 1H), 7.55−7.48 (m, 1H), 7.09 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 7.7 Hz, 1H), 6.97 (dd, J = 11.1, 4.1 Hz, 1H), 6.88 (d, J = 8.8 Hz, 2H), 4.16 (q, J = 7.2 Hz, 2H), 4.02 (t, J = 6.1 Hz, 2H), 3.57 (s, 3H), 2.72 (s, 3H), 2.53 (t, J = 7.3 Hz, 2H), 2.18− 2.08 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H). Ethyl 5-(4-(Methyl(2-methylquinazolin-4-yl)amino)phenoxy)pentanoate (6c). 6c was obtained from compound 5 and ethyl 5-bromovalerate as described for method E. 1H NMR (400 MHz, CDCl3) δ: 7.73 (d, J = 8.3 Hz, 1H), 7.55−7.47 (m, 1H), 7.09 (d, J = 8.8 Hz, 2H), 7.02 (d, J = 7.7 Hz, 1H), 6.99−6.93 (m, 1H), 6.88 (d, J = 8.8 Hz, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.98 (s, 2H), 3.57 (s, 3H), 2.71 (s, 3H), 2.40 (t, J = 6.6 Hz, 2H), 1.84 (s, 4H), 1.26 (t, J = 7.1 Hz, 3H). Ethyl 6-(4-(Methyl(2-methylquinazolin-4-yl)amino)phenoxy)hexanoate (6d). 6d was obtained from compound 5 and ethyl 6-bromohexanoate as described for method E. 1H NMR (400 MHz, CDCl3) δ: 7.73 (d, J = 8.3 Hz, 1H), 7.50 (dd, J = 10.8, 4.1 Hz, 1H), 7.08 (d, J = 8.7 Hz, 2H), 7.01 (d, J = 8.0 Hz, 1H), 6.99−6.92 (m, 1H), 6.87 (d, J = 8.7 Hz, 2H), 4.13 (dd, J = 14.3, 7.1 Hz, 2H), 3.96 (t, J = 6.2 Hz, 2H), 3.57 (s, 3H), 2.71 (s, 3H), 2.34 (t, J = 7.4 Hz, 2H), 1.88−1.77 (m, 2H), 1.77−1.68 (m, 2H), 1.57−1.47 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H). Ethyl 2-(4-(Methyl(quinazolin-4-yl)amino)phenoxy)acetate (9a). 9a was obtained from compound 8 and ethyl bromoacetate as described for method E. 1H NMR (400 MHz, CDCl3) δ: 8.80 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.60−7.55 (m, 1H), 7.15−7.08 (m, 2H), 7.05 (d, J = 3.6 Hz, 2H), 6.94−6.87 (m, 2H), 4.67 (s, 2H), 4.15 (q, J = 7.2 Hz, 2H), 3.60 (s, 3H), 1.27 (t, J = 7.2 Hz, 3H). Ethyl 4-(4-(Methyl(quinazolin-4-yl)amino)phenoxy)butanoate (9b). 9b was obtained from compound 8 and ethyl 4bromobutyrate as described for method E. 1H NMR (400 MHz, CDCl3) δ: 8.80 (s, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.60−7.55 (m, 1H), 7.15−7.08 (m, 2H), 7.05 (d, J = 3.6 Hz, 2H), 6.94−6.87 (m, 2H), 4.16 (q, J = 7.2 Hz, 2H), 4.03 (t, J = 6.1 Hz, 2H), 3.60 (s, 3H), 2.54 (t, J = 7.2 Hz, 2H), 2.18−2.09 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H). Ethyl 5-(4-(Methyl(quinazolin-4-yl)amino)phenoxy)pentanoate (9c). 9c was obtained from compound 8 and ethyl 5bromovalerate as described for method E. 1H NMR (400 MHz, CDCl3) δ: 8.80 (s, 1H), 7.85 (d, J = 8.3 Hz, 1H), 7.62−7.54 (m, 1H), 7.15−7.08 (m, 2H), 7.05 (d, J = 3.9 Hz, 2H), 6.94−6.87 (m, 2H), 4.14 (q, J = 7.1 Hz, 2H), 4.00 (t, J = 5.6 Hz, 2H), 3.60 (s, 3H), 2.40 (t, J = 6.9 Hz, 2H), 1.90−1.80 (m, 4H), 1.27 (t, J = 7.1 Hz, 3H). Ethyl 6-(4-(Methyl(quinazolin-4-yl)amino)phenoxy)hexanoate (9d). 9d was obtained from compound 8 and ethyl 6bromohexanoate as described for method E. 1H NMR (400 MHz, CDCl3) δ: 8.80 (s, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.60−7.55 (m, 1H), 7.15−7.08 (m, 2H), 7.05 (d, J = 3.7 Hz, 2H), 6.93−6.87 (m, 2H), 4.14 (q, J = 7.1 Hz, 2H), 3.98 (t, J = 6.4 Hz, 2H), 3.60 (s, 3H), 2.35 (t, J = 7.4 Hz, 2H), 1.89−1.79 (m, 2H), 1.79−1.69 (m, 2H), 1.60−1.48 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H). J



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Ethyl 2-(4-(Methyl(2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)amino)phenoxy)acetate (15a). 15a was obtained from compound 14 and ethyl bromoacetate as described for method E. 1 H NMR (400 MHz, DMSO-d6) δ: 7.17 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.9 Hz, 2H), 4.81 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.35 (s, 3H), 2.60 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 1.77 (t, J = 7.2 Hz, 2H), 1.70− 1.59 (m, 2H), 1.21 (t, J = 7.1 Hz, 3H). Ethyl 4-(4-(Methyl(2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)amino)phenoxy)butanoate (15b). 15b was obtained from compound 14 and ethyl 4-bromobutyrate as described for method E. 1H NMR (400 MHz, DMSO-d6) δ: 7.15 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 4.06 (q, J = 7.1 Hz, 2H), 3.97 (t, J = 5.9 Hz, 2H), 3.34 (s, 3H), 2.60 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 2.35 (t, J = 7.0 Hz, 2H), 1.80−1.55 (m, 6H), 1.18 (t, J = 7.1 Hz, 3H). Ethyl 5-(4-(Methyl(2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)amino)phenoxy)pentanoate (15c). 15c was obtained from compound 14 and ethyl 5-bromovalerate as described for method E. 1H NMR (400 MHz, DMSO-d6) δ: 7.15 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 4.06 (q, J = 7.1 Hz, 2H), 3.98 (t, J = 5.9 Hz, 2H), 3.34 (s, 3H), 2.60 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 2.37 (t, J = 7.0 Hz, 2H), 1.83−1.59 (m, 8H), 1.18 (t, J = 7.1 Hz, 3H). Ethyl 6-(4-(Methyl(2-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl)amino)phenoxy)hexanoate (15d). 15d was obtained from compound 14 and ethyl 6-bromohexanoate as described for method E. 1H NMR (400 MHz, DMSO-d6) δ: 7.15 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H), 4.05 (q, J = 7.1 Hz, 2H), 3.96 (t, J = 6.4 Hz, 2H), 3.34 (s, 3H), 2.59 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 2.31 (t, J = 7.3 Hz, 2H), 1.78 (t, J = 7.2 Hz, 2H), 1.75−1.69 (m, 2H), 1.69−1.54 (m, 4H), 1.47−1.38 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). Ethyl 2-(3-(Methyl(2-methylquinazolin-4-yl)amino)phenoxy)acetate (22aa). 22aa was obtained from compound 21a and ethyl bromoacetate as described for method E. 1H NMR (400 MHz, DMSO-d6) δ: 7.71 (d, J = 8.2 Hz, 1H), 7.65 (t, J = 7.5 Hz, 1H), 7.34 (t, J = 8.1 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H), 6.93 (dd, J = 14.1, 8.0 Hz, 3H), 4.69 (s, 2H), 4.13 (q, J = 7.1 Hz, 2H), 3.31 (s, 3H), 2.54 (s, 3H), 1.19 (t, J = 7.0 Hz, 3H). Ethyl 4-(3-(Methyl(2-methylquinazolin-4-yl)amino)phenoxy)butanoate (22ab). 22ab was obtained from compound 21a and ethyl 4-bromobutyrate as described for method E. 1H NMR (400 MHz, DMSO-d6) δ: 7.67 (d, J = 8.1 Hz, 1H), 7.60 (t, J = 7.2 Hz, 1H), 7.31 (t, J = 8.1 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 7.01−6.97 (m, 1H), 6.94 (s, 1H), 6.88 (s, 1H), 6.82 (d, J = 8.0 Hz, 1H), 4.13 (t, J = 7.0 Hz, 2H), 4.01 (q, J = 7.1 Hz, 2H), 3.31 (s, 3H), 2.59 (s, 3H), 2.40 (t, J = 7.1 Hz, 2H), 1.98−1.89 (m, 2H), 1.14 (t, J = 7.1 Hz, 3H). Ethyl 5-(3-(Methyl(2-methylquinazolin-4-yl)amino)phenoxy)pentanoate (22ac). 22ac was obtained from compound 21a and ethyl 5-bromovalerate as described for method E. 1H NMR (400 MHz, DMSO-d6) δ: 7.66 (d, J = 8.2 Hz, 1H), 7.60 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 8.1 Hz, 1H), 7.06 (t, J = 7.4 Hz, 1H), 6.96 (t, J = 8.1 Hz, 2H), 6.86 (s, 1H), 6.80 (d, J = 7.8 Hz, 1H), 4.10 (t, J = 7.1 Hz, 2H), 4.04−3.97 (m, 2H), 3.31 (s, 3H), 2.59 (s, 3H), 2.36 (t, J = 7.1 Hz, 2H), 1.68 (dd, J = 14.1, 7.2 Hz, 2H), 1.60 (dd, J = 14.6, 7.4 Hz, 2H), 1.13 (t, J = 7.1 Hz, 3H). Ethyl 6-(3-(Methyl(2-methylquinazolin-4-yl)amino)phenoxy)hexanoate (22ad). 22ad was obtained from compound 21a and ethyl 6-bromohexanoate as described for method E. 1H NMR (400 MHz, DMSO-d6) δ: 7.66 (d, J = 8.2 Hz, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 8.1 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H), 6.96 (dd, J = 9.6, 3.9 Hz, 2H), 6.87 (s, 1H), 6.81 (d, J = 8.2 Hz, 1H), 4.04 (dq, J = 14.6, 7.2 Hz, 6H), 3.31 (s, 3H), 2.59 (s, 3H), 2.34−2.22 (m, 4H), 1.67 (dd, J = 14.4, 7.2 Hz, 2H), 1.59 (dd, J = 14.9, 7.5 Hz, 2H), 1.55−1.47 (m, 2H), 1.40 (dd, J = 14.1, 6.7 Hz, 2H), 1.16 (dt, J = 14.3, 7.2 Hz, 6H). Ethyl 2-(2-Methoxy-5-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)acetate (22ba). 22ba was obtained from compound 21b and ethyl bromoacetate as described for method E. 1H NMR (400 MHz, DMSO-d6) δ: 7.64 (d, J = 8.1 Hz, 1H), 7.58 (t, J = 7.4 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.02−6.96 (m, 2H), 6.92 (d, J = 2.2 Hz, 1H), 6.75 (dd, J = 8.5, 2.2 Hz, 1H), 4.74 (s, 2H), 4.01 (q, J = 7.1 Hz, 2H), 3.80 (s, 3H), 3.48 (s, 3H), 2.58 (s, 3H), 1.10 (t, J = 7.1 Hz, 3H).

Ethyl 4-(2-Methoxy-5-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)butanoate (22bb). 22bb was obtained from compound 21b and ethyl 4-bromobutyrate as described for method E. 1H NMR (400 MHz, DMSO-d6) δ: 7.63 (d, J = 8.1 Hz, 1H), 7.57 (t, J = 7.5 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 8.2 Hz, 1H), 6.96 (d, J = 3.7 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 6.72 (dd, J = 8.5, 2.4 Hz, 1H), 4.04 (q, J = 7.1 Hz, 2H), 3.90 (t, J = 6.4 Hz, 2H), 3.78 (s, 3H), 3.50 (s, 3H), 2.58 (s, 3H), 2.39 (t, J = 7.3 Hz, 2H), 1.88 (p, J = 6.8 Hz, 2H), 1.16 (t, J = 7.1 Hz, 3H). Ethyl 5-(2-Methoxy-5-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)pentanoate (22bc). 22bc was obtained from compound 21b and ethyl 5-bromovalerate as described for method E. 1 H NMR (400 MHz, DMSO-d6) δ: 7.64 (d, J = 8.2 Hz, 1H), 7.57 (t, J = 7.5 Hz, 1H), 7.06 (t, J = 7.2 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.97−6.90 (m, 2H), 6.70 (dd, J = 8.5, 2.3 Hz, 1H), 4.04 (q, J = 7.1 Hz, 3H), 3.88 (t, J = 5.8 Hz, 2H), 3.77 (s, 3H), 3.50 (s, 3H), 2.58 (s, 3H), 2.31 (t, J = 6.9 Hz, 2H), 1.67−1.59 (m, 4H), 1.17 (t, J = 7.1 Hz, 3H). Ethyl 6-(2-Methoxy-5-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)hexanoate (22bd). 22bd was obtained from compound 21b and ethyl 6-bromohexanoate as described for method E. 1H NMR (400 MHz, DMSO-d6) δ: 7.64 (d, J = 8.1 Hz, 1H), 7.57 (t, J = 6.8 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H), 6.94 (d, J = 8.8 Hz, 2H), 6.70 (dd, J = 8.5, 2.4 Hz, 1H), 4.04 (q, J = 7.1 Hz, 3H), 3.86 (t, J = 6.5 Hz, 2H), 3.77 (s, 3H), 3.50 (s, 3H), 2.58 (s, 3H), 2.27 (t, J = 7.3 Hz, 4H), 1.65−1.57 (m, 2H), 1.57−1.47 (m, 2H), 1.40−1.28 (m, 2H), 1.17 (t, J = 7.1 Hz, 3H). Ethyl 4-(2-Methoxy-5-nitrophenoxy)butanoate (24). 24 was obtained from compound 17b and ethyl 4-bromobutyrate as described for method E. 1H NMR (400 MHz, DMSO-d6) δ: 9.55 (s, 1H), 8.47 (d, J = 8.0 Hz, 1H), 7.78 (t, J = 7.1 Hz, 1H), 7.70−7.64 (m, 2H), 7.53 (t, J = 7.5 Hz, 1H), 7.43 (dd, J = 8.7, 2.4 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 4.07 (q, J = 7.1 Hz, 2H), 4.01 (t, J = 6.4 Hz, 2H), 3.77 (s, 3H), 2.50−2.45 (m, 5H), 2.07−1.97 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H). General Procedures of Method F for the Synthesis of Hydroxamic Acid Derivatives. The ester intermediate (1 mmol, 1 equiv) was dissolved in CH2Cl2 and methanol (1:2, 9 mL). The resulting solution was cooled to 0 °C, then hydroxamic (50 wt % in water, 1 mL, 30 mmol, 30 equiv) and NaOH (0.4 g, 10 mmol, 10 equiv) were added. At the temperature, the reaction was stirred for 1 h. The solvent was then removed under reduced pressure, and the obtained solid was dissolved in water, which was adjusted to pH 7−8 by acetic acid and extracted with ethyl acetate (3 × 50 mL). The organic layer was collected and dried over anhydrous MgSO4. After the removal of MgSO4 by filtration, the filtrate was concentrated in vacuo to yield a solid product which recrystallized with EtOH to give the title compound. N-Hydroxy-2-(4-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)acetamide (7a). 7a was obtained from compound 6a as described for method F: mp 156−158 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.86 (s, 1H), 9.00 (s, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.59 (t, J = 7.4 Hz, 1H), 7.21 (d, J = 8.8 Hz, 2H), 7.06 (t, J = 7.2 Hz, 1H), 7.00 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.6 Hz, 1H), 4.49 (s, 2H), 3.50 (s, 3H), 2.59 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 164.06, 162.33, 161.03, 156.05, 151.71, 141.57, 131.84, 127.50, 127.09, 125.64, 124.05, 116.02, 114.14, 66.03, 42.30, 26.12. MS (ESI, m/z): 339.16 [M + H]+. N-Hydroxy-4-(4-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)butanamide (7b). 7b was obtained from compound 6b as described for method F: mp 174−176 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.43 (s, 1H), 8.75 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.58 (t, J = 7.4 Hz, 1H), 7.18 (d, J = 8.6 Hz, 2H), 7.06 (t, J = 7.4 Hz, 1H), 6.97 (d, J = 8.3 Hz, 3H), 3.97 (t, J = 6.1 Hz, 2H), 3.49 (s, 3H), 2.59 (s, 3H), 2.14 (t, J = 7.2 Hz, 2H), 2.02−1.88 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 168.56, 162.32, 161.00, 156.85, 151.71, 140.97, 131.81, 127.48, 127.16, 125.65, 124.02, 115.78, 114.13, 67.07, 42.32, 28.66, 26.12, 24.75, 18.53. MS (ESI, m/z): 367.18 [M + H]+. N-Hydroxy-5-(4-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)pentanamide (7c). 7c was obtained from compound 6c as described for method F: mp 155−157 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.38 (s, 1H), 8.70 (s, 1H), 7.65 (d, J = 7.8 Hz, 1H), K



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J = 5.8 Hz, 2H), 3.34 (s, 3H), 2.59 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 1.98 (t, J = 6.9 Hz, 2H), 1.80 (t, J = 7.5 Hz, 2H), 1.70−1.60 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ: 172.38, 168.84, 164.44, 158.87, 157.10, 138.25, 128.71, 114.65, 113.48, 67.32, 39.41, 33.30, 31.86, 29.72, 28.19, 25.35, 21.03. MS (ESI, m/z): 357.10 [M + H]+. N-Hydroxy-5-(4-(methyl(2-methyl-6,7-dihydro-5Hcyclopenta[d]pyrimidin-4-yl)amino)phenoxy)pentanamide (16c). 16c was obtained from compound 15c as described for method F: mp 150−152 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.39 (s, 1H), 8.71 (s, 1H), 7.15 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.7 Hz, 2H), 3.97 (t, J = 5.8 Hz, 2H), 3.34 (s, 3H), 2.60 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 2.03 (t, J = 6.9 Hz, 2H), 1.78 (t, J = 7.2 Hz, 2H), 1.70−1.60 (m, 6H). 13 C NMR (101 MHz, DMSO-d6) δ: 172.39, 168.86, 164.48, 158.92, 157.06, 138.29, 128.70, 114.67, 113.44, 67.29, 39.44, 33.27, 31.88, 29.71, 28.16, 25.45, 21.76, 21.18. MS (ESI, m/z): 371.12 [M + H]+. N-Hydroxy-6-(4-(methyl(2-methyl-6,7-dihydro-5Hcyclopenta[d]pyrimidin-4-yl)amino)phenoxy)hexanamide (16d). 16d was obtained from compound 15d as described for method F: mp 157−159 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.36 (s, 1H), 8.68 (s, 1H), 7.15 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 3.96 (t, J = 6.3 Hz, 2H), 3.34 (s, 3H), 2.59 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 1.99−1.94 (m, 2H), 1.78 (t, J = 7.2 Hz, 2H), 1.72 (dd, J = 14.1, 7.1 Hz, 2H), 1.64 (dt, J = 15.0, 7.4 Hz, 2H), 1.56 (dt, J = 14.9, 7.3 Hz, 2H), 1.39 (dt, J = 14.5, 7.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 172.37, 168.96, 164.48, 158.92, 157.09, 138.26, 128.70, 114.65, 113.42, 67.56, 39.43, 33.26, 32.19, 29.70, 28.40, 25.45, 25.15, 24.89, 21.17. MS (ESI, m/z): 385.19 [M + H]+. N-Hydroxy-2-(3-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)acetamide (23aa). 23aawas obtained from compound 22aa as described for method F: mp 97−99 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.38 (s, 1H), 8.70 (s, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.65−7.58 (m, 1H), 7.33 (t, J = 8.1 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 7.05−7.00 (m, 2H), 6.88 (s, 1H), 6.84 (d, J = 7.8 Hz, 1H),4.21(s, 2H), 3.47 (s, 3H), 2.57 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 165.32, 162.74, 161.41, 152.25, 148.80, 148.43, 141.31, 132.33, 127.84, 126.11, 124.87, 119.47, 114.76, 113.92, 112.84, 56.29, 42.69, 26.51. MS (ESI, m/z): 339.04 [M + H]+. N-Hydroxy-4-(3-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)butanamide (23ab). 23ab was obtained from compound 22ab as described for method F: mp 84−86 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.38 (s, 1H), 8.70 (s, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.65−7.58 (m, 1H), 7.33 (t, J = 8.1 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 7.01−6.93 (m, 2H), 6.88 (s, 1H), 6.84 (d, J = 7.8 Hz, 1H), 4.09 (t,J = 7.2 Hz, 2H), 3.31 (s, 3H), 2.61 (s, 3H), 2.06 (t, J = 7.3 Hz, 2H), 1.94− 1.83 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 169.10, 162.71, 161.13, 158.36, 147.89, 132.74, 131.23, 127.53, 124.75, 119.75, 114.72, 114.59, 114.42, 94.39, 56.11, 53.14, 30.30, 26.41, 23.19. MS (ESI, m/ z): 337.14 [M + H]+. N-Hydroxy-5-(3-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)pentanamide (23ac). 23ac was obtained from compound 22ac as described for method F: mp 82−84 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.37 (s, 1H), 8.67 (s, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.63−7.57 (m, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.09−7.04 (m, 1H), 6.99−6.92 (m, 2H), 6.85 (s, 1H), 6.80 (d, J = 8.0 Hz, 1H), 4.13−4.04 (m, 2H), 3.34 (s, 3H), 2.59 (s, 3H), 1.99 (t, J = 7.2 Hz, 2H), 1.64 (d, J = 6.8 Hz, 2H), 1.56 (d, J = 7.5 Hz, 2H). 13C NMR (101 MHz, DMSOd6) δ: 162.88, 161.12, 158.33, 152.45, 148.19, 132.50, 131.16, 128.04, 126.09, 124.57, 119.59, 114.85, 114.36, 114.22, 94.39, 56.08, 53.15, 32.54, 26.76, 26.62, 23.27. MS (ESI, m/z): 381.37 [M + H]+. N-Hydroxy-6-(3-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)hexanamide (23ad). 23ad was obtained from compound 22ad as described for method F: mp 71−73 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.29 (s, 1H), 8.73 (s, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.59 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 8.1 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.99−6.92 (m, 2H), 6.86 (s, 1H), 6.81 (d, J = 7.9 Hz, 1H), 4.11− 4.02 (m, 2H), 3.30 (s, 3H), 2.59 (s, 3H), 1.95 (t, J = 7.3 Hz, 2H), 1.70−1.62 (m, 2H), 1.53 (dd, J = 14.8, 7.4 Hz, 2H), 1.31 (dd, J = 14.5, 7.5 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 169.47, 162.89, 161.06, 158.34, 152.45, 148.18, 132.46, 131.15, 128.02, 126.10, 124.51,

7.58 (t, J = 7.5 Hz, 1H), 7.18 (d, J = 8.8 Hz, 2H), 7.06 (t, J = 7.1 Hz, 1H), 6.97 (d, J = 8.8 Hz, 3H), 3.98 (t, J = 5.8 Hz, 2H), 3.49 (s, 3H), 2.59 (s, 3H), 2.03 (t, J = 6.9 Hz, 2H), 1.75−1.60 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ: 168.87, 162.32, 160.99, 156.96, 151.70, 140.88, 131.79, 127.47, 127.14, 125.65, 124.01, 115.73, 114.13, 67.33, 42.32, 31.87, 28.13, 26.11, 21.75. MS (ESI, m/z): 381.19 [M + H]+. N-Hydroxy-6-(4-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)hexanamide (7d). 7d was obtained from compound 6d as described for method F: mp 113−115 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.36 (s, 1H), 8.70 (s, 1H), 7.65 (d, J = 7.6 Hz, 1H), 7.62−7.55 (m, 1H), 7.17 (d, J = 8.8 Hz, 2H), 7.06 (t, J = 7.1 Hz, 1H), 6.97 (d, J = 8.8 Hz, 3H), 3.96 (t, J = 6.4 Hz, 2H), 3.49 (s, 3H), 2.58 (s, 3H), 1.98 (t, J = 7.2 Hz, 2H), 1.79−1.65 (m, 2H), 1.62−1.50 (m, 2H), 1.45−1.32 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 169.01, 161.68, 160.83, 157.40, 139.98, 132.55, 127.22, 125.89, 124.57, 115.84, 113.60, 67.67, 42.67, 32.18, 28.35, 25.12, 24.87. MS (ESI, m/z): 395.34 [M + H]+. N-Hydroxy-2-(4-(methyl(quinazolin-4-yl)amino)phenoxy)acetamide (10a). 10a was obtained from compound 9a as described for method F: mp 172−170 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.40 (s, 1H), 8.71 (s, 2H), 7.74 (d, J = 7.9 Hz, 1H), 7.69−7.65 (m, 1H), 7.20 (d, J = 8.8 Hz, 2H), 7.18−7.10 (m, 1H), 7.00 (d, J = 9.0 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 4.45 (s, 2H), 3.51 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 131.84, 127.50, 127.09, 125.64, 124.05, 116.02, 66.03, 42.30, 26.12. MS (ESI, m/z): 339.16 [M + H]+. N-Hydroxy-4-(4-(methyl(quinazolin-4-yl)amino)phenoxy)butanamide (10b). 10b was obtained from compound 9b as described for method F: mp 187−189 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.43 (s, 1H), 8.71 (s, 2H), 7.74 (d, J = 7.9 Hz, 1H), 7.67−7.61 (m, 1H), 7.20 (d, J = 8.8 Hz, 2H), 7.18−7.11 (m, 1H), 7.01 (d, J = 8.9 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 3.98 (t, J = 6.3 Hz, 2H), 3.51 (s, 3H), 2.14 (t, J = 7.4 Hz, 2H), 1.99−1.90 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 168.78, 160.09, 157.02, 153.95, 151.07, 140.68, 131.91, 128.25, 127.22, 125.75, 124.90, 115.98, 115.87, 67.14, 42.46, 27.17. MS (ESI, m/z): 325.26 [M + H]+. N-Hydroxy-5-(4-(methyl(quinazolin-4-yl)amino)phenoxy)pentanamide (10c). 10c was obtained from compound 9c as described for method F: mp 176−178 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.38 (s, 1H), 8.71 (s, 2H), 7.74 (d, J = 8.1 Hz, 1H), 7.68−7.60 (m, 1H), 7.20 (d, J = 8.8 Hz, 2H), 7.17−7.11 (m, 1H), 7.02 (d, J = 9.0 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 3.98 (t, J = 5.9 Hz, 2H), 3.50 (s, 3H), 2.03 (t, J = 6.9 Hz, 2H), 1.77−1.58 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ: 168.88, 160.89, 157.09, 153.98, 151.07, 140.70, 131.96, 128.05, 127.18, 125.73, 124.91, 115.98, 115.78, 67.35, 42.46, 31.88, 28.13, 21.76. MS (ESI, m/z): 367.19 [M + H]+. N-Hydroxy-6-(4-(methyl(quinazolin-4-yl)amino)phenoxy)hexanamide (10d). 10d was obtained from compound 9d as described for method F: mp 176−178 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.35 (s, 1H), 8.71 (s, 2H), 7.74 (d, J = 7.8 Hz, 1H), 7.67−7.61 (m, 1H), 7.19 (d, J = 8.9 Hz, 2H), 7.17−7.11 (m, 1H), 7.01 (d, J = 8.8 Hz, 1H), 6.98 (d, J = 8.9 Hz, 2H), 3.97 (t, J = 6.4 Hz, 2H), 3.50 (s, 3H), 1.98 (t, J = 7.3 Hz, 2H), 1.78−1.65 (m, 2H), 1.63−1.49 (m, 2H), 1.44−1.35 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 168.99, 160.88, 157.12, 153.98, 151.07, 140.67, 131.95, 128.04, 127.17, 125.73, 124.89, 115.98, 115.76, 67.62, 42.45, 32.20, 28.37, 25.14, 24.88. MS (ESI, m/z): 381.18 [M + H]+. N-Hydroxy-2-(4-(methyl(2-methyl-6,7-dihydro-5Hcyclopenta[d]pyrimidin-4-yl)amino)phenoxy)acetamide (16a). 16a was obtained from compound 15a as described for method F: mp 173−175 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.86 (s, 1H), 9.00 (s, 1H), 7.18 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 4.48 (s, 2H), 3.34 (s, 3H), 2.60 (t, J = 7.7 Hz, 2H), 2.41 (s, 3H), 1.78 (t, J = 7.1 Hz, 2H), 1.71−1.59 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 172.48, 164.50, 164.10, 158.89, 156.15, 138.97, 128.62, 114.97, 113.51, 66.03, 39.43, 33.27, 29.76, 25.47, 21.21. MS (ESI, m/z): 329.05 [M + H]+. N-Hydroxy-4-(4-(methyl(2-methyl-6,7-dihydro-5Hcyclopenta[d]pyrimidin-4-yl)amino)phenoxy)butanamide (16b). 16b was obtained from compound 15b as described for method F: mp 166−168 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.37 (s, 1H), 8.70 (s, 1H), 7.15 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 8.5 Hz, 2H), 3.97 (t, L



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4BKX), we used the crystal structure reported by Millard et al.53 and ELM2-SANT fragment was deleted. For the above HDAC1 structure and HDAC6 model to be exploited for protein structure alignment and molecular docking, the FG-MD method was employed to improve the local geometry, the torsion angle, and the hydrogen-binding networks.54 Molecular docking was carried out on the HDAC8, HDAC1, and HDAC6 model by Autodock4Zn.55 HDAC Enzymes Inhibition Assays. In vitro HDACs inhibition assay was conducted utilizing 4-amino-7-methylcoumarin (AMC) labeled Ac-peptide (Ac-peptide-AMC) substrates, a service provided by Chempartner Company (Shanghai, China). In detail, upon deacetylation of the substrate, the release of AMC was promoted in the existence of trypsin. The compounds, diluted to the indicated concentrations, were mixed with full-length recombinant HDAC enzymes (BPS Biosciences), trypsin, as well as Ac-peptide-AMC substrates and incubated at room temperature for 1 h. The fluorescence intensity was measured with excitation at 355 nm wavelength and emission at 460 nm wavelength. The inhibition rates of the tested groups were calculated via comparison with the DMSO (vehicle) treated group. α-Tubulin and Histone H3 Acetylation Cytoblot Assay. The cytoblot assays were conducted generally as described.27,49 The antiacetylated tubulin antibody, antiacetylated histone H3 antibody (Santa Cruz), and horseradish peroxidase (HRP) conjugated secondary antibodies (Jackson) were used. HeLa cells were seeded into opaque 96-well plates at a density of 5000 cells per well. After cell attachment, the compounds were added at various indicated concentrations and incubated for 6 h. The cells were fixed by 3.7% paraformaldehyde in ice cold TBS at 4 °C for 1 h. After removal of the fixing solution, the cells were permeabilized via addition of −20 °C MeOH (5 min incubation at 4 °C). Subsequently, the wells were washed with 3% nonfat dry milk in TBS. The antibodies were added, and the plates were incubated for 4 h at 4 °C. Finally, the wells were washed thrice by TBS following addition of enhanced chemiluminescence reagent. After a short incubation for 5 min, the luminescence was recorded by a Spectramax M5 microtiter plate luminometer. The EC50 values were calculated by curve fitting with the software GraphPad Prism 5.0. Antiproliferative Assays. A375, A2780s, SKBR3, HepG2, HeLa, HCT116, A549, and SKOV-3 cells were cultured in DMEM (Gibco, Milano, Italy). RPMI8226, K562, H460, HT29, and Ramos cells were cultured in RPMI-1640 medium (Gibco, Milano, Italy). MV4-11 cells were cultured in IMDM (Gibco, Milano, Italy). All media contained 10% fetal bovine serum (FBS) (Invitrogen, Milano, Italy), 100 units/ mL penicillin (Gibco, Milano, Italy), and 100 μg/mL streptomycin (Gibco, Milano, Italy). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. Cells in logarithmic phase were seeded into 96-well culture plates at densities of 3000−5000 cells per well and subsequently treated with various concentrations of compounds for 72 h in final volumes of 200 μL. Upon end point, 20 μL of MTT (5 mg/ mL) was added to each well, and the cells were incubated for an additional 1−3 h. After carefully removal of the medium, the precipitates were dissolved in 150 μL of DMSO via mechanically shaking, and then absorbance values at a wavelength of 570 nm were taken on a spectrophotometer (Molecular Devices, Sunnyvale, USA). IC50 values were calculated using percentage of growth versus untreated control. Western Blotting. The cells were treated by the indicated ways before collection. After washing by PBS 2 times, the cells were resuspended in RIPA lysis buffer (Beyotime Co.). After 30 min of incubation on ice, the lysates were collected by centrifuging at 12 000g for 15 min at 4 °C. The protein concentration was measured. Equivalent samples (20 μg of protein) were subjected to 15% SDS− PAGE, and then the proteins were transferred onto activated PVDF membranes. After blocking by 5% nonfat milk for 1 h at room temperature, the membranes were incubated with the indicated primary antibodies and subsequently probed by the appropriate secondary antibodies conjugated to horseradish peroxidase. Immunoreactive bands were visualized using enhanced chemiluminescence (Millipore, USA).

119.69, 114.82, 114.42, 114.32, 94.40, 56.07, 53.33, 32.68, 26.61, 26.50, 25.34. MS (ESI, m/z): 395.22 [M + H]+. N-Hydroxy-2-(2-methoxy-5-(methyl(2-methylquinazolin-4yl)amino)phenoxy)acetamide (23ba). 23ba was obtained from compound 22ba as described for method F: mp 172−174 °C. 1H NMR (400 MHz, DMSO-d6) δ: 7.63 (d, J = 8.1 Hz, 1H), 7.57 (t, J = 6.5 Hz, 1H), 7.05 (d, J = 7.7 Hz, 2H), 7.00 (d, J = 8.1 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.63 (d, J = 8.1 Hz, 1H), 4.26 (s, 2H), 3.75 (s, 3H), 3.47 (s, 3H), 2.57 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ: 164.39, 162.81, 161.45, 152.09, 148.83, 148.26, 141.36, 132.33, 127.89, 126.17, 124.57, 119.51, 114.70, 113.22, 112.94, 67.50, 56.17, 42.75, 26.58. MS (ESI, m/z): 369.12 [M + H]+. N-Hydroxy-4-(2-methoxy-5-(methyl(2-methylquinazolin-4yl)amino)phenoxy)butanamide (23bb). 23bb was obtained from compound 22bb as described for method F: mp 161−163 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.41 (s, 1H), 8.71 (s, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H) 6.99 (d, J = 8.6 Hz, 2H), 6.94 (d, J = 8.6 Hz, 1H), 6.68 (dd, J = 8.4, 2.0 Hz, 1H), 3.88 (t, J = 6.2 Hz, 2H), 3.78 (s, 3H), 3.50 (s, 3H), 2.58 (s, 3H), 2.08 (t, J = 7.4 Hz, 2H), 1.94−1.81 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 168.57, 162.30, 160.89, 151.62, 148.88, 147.69, 140.99, 131.78, 127.40, 125.65, 123.99, 118.08, 114.21, 112.57, 111.49, 67.82, 55.67, 42.26, 28.64, 26.11, 24.66. MS (ESI, m/z): 397.12 [M + H]+. N-Hydroxy-5-(2-methoxy-5-(methyl(2-methylquinazolin-4yl)amino)phenoxy)pentanamide (23bc). 23bc was obtained from compound 22bc as described for method F: mp 172−174 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.35 (s, 1H), 8.69 (s, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 6.69 (dd, J = 8.5, 2.3 Hz, 1H), 3.88 (t, J = 5.6 Hz, 2H), 3.78 (s, 3H), 3.51 (s, 3H), 2.58 (s, 3H), 1.99−1.94 (m, 2H), 1.70−1.52 (m, 4H). 13 C NMR (101 MHz, DMSO-d6) δ: 168.84, 162.30, 160.90, 151.63, 148.99, 147.61, 140.99, 131.78, 127.40, 125.68, 123.97, 117.86, 114.21, 112.51, 111.26, 67.94, 55.66, 42.26, 31.86, 28.04, 26.11, 21.77. MS (ESI, m/z): 411.16 [M + H]+. N-Hydroxy-6-(2-methoxy-5-(methyl(2-methylquinazolin-4yl)amino)phenoxy)hexanamide (23bd). 23bd was obtained from compound 22bd as described for method F: mp 152−154 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.35 (s, 1H), 8.67 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.58 (t, J = 7.1 Hz, 1H), 7.06 (t, J = 7.4 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.94 (dd, J = 5.3, 3.0 Hz, 2H), 6.70 (dd, J = 8.4, 2.1 Hz, 1H), 3.85 (t, J = 6.4 Hz, 2H), 3.77 (s, 3H), 3.50 (s, 3H), 2.58 (s, 3H), 1.94 (t, J = 7.2 Hz, 2H), 1.67−1.56 (m, 2H), 1.56−1.44 (m, 2H), 1.37−1.26 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 168.91, 162.30, 160.89, 151.63, 148.98, 147.62, 140.97, 131.77, 127.38, 125.68, 123.94, 117.83, 114.20, 112.52, 111.29, 68.18, 55.65, 42.24, 32.17, 28.25, 26.11, 25.07, 24.85. MS (ESI, m/z): 425.18 [M + H]+. N-Hydroxy-4-(2-methoxy-5-((2-methylquinazolin-4-yl)amino)phenoxy)butanamide (27). 27 was obtained from compound 26 as described for method F: mp 189−191 °C. 1H NMR (400 MHz, DMSO-d6) δ: 10.45 (s, 1H), 9.55 (s, 1H), 8.72 (s, 1H), 8.48 (d, J = 8.1 Hz, 1H), 7.82−7.74 (m, 1H), 7.66 (dd, J = 7.9, 5.4 Hz, 2H), 7.57−7.50 (m, 1H), 7.45 (dd, J = 8.7, 2.3 Hz, 1H), 6.98 (d, J = 8.8 Hz, 1H), 3.99 (t, J = 6.3 Hz, 2H), 3.78 (s, 3H), 2.17 (t, J = 7.4 Hz, 2H), 2.05−1.94 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ: 169.09, 163.39, 157.96, 150.63, 147.92, 145.75, 133.30, 133.15, 127.65, 125.58, 123.13, 114.61, 113.70, 112.51, 108.99, 68.07, 56.30, 40.61, 40.40, 40.20, 39.99, 39.78, 39.57, 39.36, 29.25, 26.73, 25.38. MS (ESI, m/z): 383.27 [M + H]+. Biological Assay Methods. Homology Modeling and Molecular Simulation. HDAC6 has two catalytic domains (CDI and CDII), a ubiquitin binding domain, and a dynein binding domain.51 For HDAC6, a homology model was generated by multiplethread alignments, as described by Yang Zhang’s research group through a Web server (I-TASSER).45 In the process of building HDAC6 homology model, only the second catalytic subunit (CDII, Tyr485-Arg835) was generated, as it plays an essential role in catalytic function. Then the zinc ion, extracted from the crystal structure of HDAC8−substrate complex (PDB code 2V5W),52 was added into the corresponding site of HDAC6 structure. For HDAC1 (PDB code M



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In Vivo Pharmacokinetics Evaluation in Mice. A 2 mg/mL dosing solution was preparing by dissolving the appropriate amount of the compound in ddH2O for iv administration and 0.5% CMC-Na aqueous solution for oral dosing. SD rats, weighing 200−250 g each, were obtained from Beijing HFK Bioscience Co., Ltd.. Each tested compound was separately administered intravenously to a group of six rats per time point (12 mg/kg dose) by a bolus injection (12 mg/kg dose) to the tail vein or periorally. At time points 0 (prior to dosing), 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h after dosing, a blood sample was collected from each animal via cardiac puncture and stored in ice (0−4 °C). Plasma was separated from the blood by centrifugation (4000g for 15 min at 4 °C) and stored in a freezer at −80 °C. All samples were analyzed for the test compound by LC−MS/MS (Waters Acquity UPLC system; Waters Quattro Premier XE). Data were acquired via monitoring of multiple reactions. Plasma concentration data were analyzed by a standard noncompartmental method. Animal Tumor Models and Treatment. For the MV4-11 and Ramos xenograft models, MV4-11 and Ramos cells (107 cells in 100 μL of serum-free IMDM) were injected subcutaneously into the right flanks of 5- to 6-week-old female NOD/SCID mice. For the HCT116 xenograft, HCT116 cells (107 cells in 100 μL of serum-free DMEM) were injected subcutaneously into the right flanks of 5- to 6-week-old female Balb/c nude mice. When the size of the formed xenografts reached 100−150 mm3, the mice were randomly divided (6 mice per group in MV4-11 model, 8 mice per group in Ramos model, and 7 mice per group in HCT116 model) into control group and treated groups. The mice in the experimental groups received intravenous (iv) injection (50 mg/kg, dissolved in physiological saline containing 5% ethanol and 5% Cremophor EL) or oral administration (25 mg/kg, dissolved in physiological saline containing 5% ethanol and 5% Cremophor EL) of 23bb every 2 days. The mice in the vehicle group received iv injection or oral administration of equal amount of physiological saline containing 5% ethanol and 5% Cremophor EL. Those in the SAHA or LBH-589 or ACY-1215 groups (positive controls) received ip injection (50 mg/kg for SAHA and 10 mg/kg for LBH-589, dissolved in physiological saline containing 10% DMSO and 45% PEG400 to a concentration of 10 mg/mL) or oral administration (100 mg/kg for SAHA and 40 mg/kg for ACY-1215, dissolved in the same way described above) every 2 days. Tumor burden was measured every 2 days by a caliper. Tumor volume (TV) was calculated using the following formula: TV = length × width2 × 0.52. The day that treatment started was defined as day 0. At the end of the experiment, mice were sacrificed and tumors were collected and weighed. The animal studies were conducted in conformity with institutional guide for the care and use of laboratory animals, and all mouse protocols were approved by the Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China).

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors greatly appreciate the financial support from National Key Programs of China during the 12th Five-Year Plan Period (Grant 2012ZX09103101-009) and Guangdong Innovative Research Team Program (Grant 2011Y073).

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ABBREVIATIONS USED HDAC, histone deacetylase; ZBG, zinc-binding group; DMSO, dimethyl sulfoxide; SAHA, suberoylanilide hydroxamic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SAR, structure−activity relationship

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01342. Results of HDAC1, HDAC6, and HDAC8 structures comparisons and the binding energy of the comfortable fragments (PDF) Molecular formula strings (CSV)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*J.Y.: e-mail, [email protected]. *L.C.: phone, +86-28-85164063; fax, +86-28-85164060. e-mail, [email protected]. Author Contributions #

Z.Y., T.W., and F.W. contributed equally and are considered as co-first authors. N



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