Quinazolin-2,4-dione-Based Hydroxamic Acids as Selective Histone

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Quinazolin-2,4-dione-Based Hydroxamic Acids as Selective Histone Deacetylase-6 Inhibitors for Treatment of Non-Small-Cell Lung Cancer Chao-Wu Yu, Pei-Yun Hung, Hui-Ting Yang, Yi-Hsun Ho, Hsing-Yi Lai, Yi-Sheng Cheng, and Ji-Wang Chern J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01590 • Publication Date (Web): 10 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018

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Journal of Medicinal Chemistry

Quinazolin-2,4-dione-Based Hydroxamic Acids as Selective Histone Deacetylase-6 Inhibitors for Treatment of Non-Small-Cell Lung Cancer Chao-Wu Yu,‡, † Pei-Yun Hung,‡ Hui-Ting Yang,‡ Yi-Hsun Ho,‡ Hsing-Yi Lai,¶ Yi-Sheng Cheng,*, ¶

and Ji-Wang Chern*,†, §

†School

of Pharmacy, College of Medicine, and §Center for Innovative Therapeutics Discovery,

National Taiwan University, Taipei 10607, Taiwan ¶Department

of Life Science, Institute of Plant Biology, and Genome and Systems Biology

Degree Program, National Taiwan University, Taipei 10617, Taiwan ‡AnnJi

Pharmaceutical Co., Ltd., 18, Siyuan St., Taipei 10087, Taiwan

ABSTRACT We designed and synthesized quinazolin-2,4-one-based hydroxamic acids to serve as selective competitive inhibitors of histone deacetylase-6 (HDAC6). The most potent and selective compound, 3d (IC50, 4 nM, HDAC6; IC50 > 10 M, HDAC1) substantially increased acetylation of -tubulin instead of histones in the lung cancer cell line, LL2. Paclitaxel in combination with

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3d had a synergistic anticancer effect on reduction of programmed death-ligand 1 expression in LL/2 cells. When given orally, 3d was mainly found to locate in the liver and lungs, at a concentration 18- to 70-fold greater, respectively, than in plasma. As an orally active HDAC6 inhibitor, 3d (20 mg/kg) potentiated paclitaxel antitumor activity (percentage tumor growth inhibition, 67.5%) in a xenograft syngeneic non-small-cell lung cancer mouse model.

INTRODUCTION Histone deacetylase 6 (HDAC6) is the most studied member of the HDAC family, but, unlike what its name implies, HDAC6 deacetylates -tubulin, HSP90, cortactin, and peroxiredoxin and not histones.1,2 A HDAC6-knockout mouse develops normally with no obvious phenotype aberrations, whereas other types of HDAC-knockout mice display severe defects.3 Consequently, inhibitors of HDAC6 have been proposed to be more appropriate targets for drug development because the panHDAC inhibitors that have been developed to date have proven to possess undesirable side effects.4,5 Compounds that inhibit HDAC6 have been shown to decrease the negative effects associated with disease models including autoimmune disorders, Alzheimer’s disease, and cancer.6–8 Selective positive effects on HDAC6 by siRNA or HDAC6 inhibitors include inhibition of tumorigenesis, inhibition of metastasis , and increased sensitivity of tumors to other anticancer agents.9–11 Several inhibitors selective for HDAC6 have been developed to treat cancers including an inhibitor that bears a substituted urea linker with anti-melanoma activity.8 Another inhibitor, ACY-1215, is being tested clinically in combination with bortezomib, a proteasome inhibitor, for multiple myeloma.12 In addition, data has accumulated suggesting that prostate cancer may be treated with the HDAC6 inhibitor, tubacin, alone or in combination with a cytotoxic agent.11


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Although LBH589 is a pan-HDAC inhibitor, it is therapeutically responsive to LNCap and PC-3 prostate cancer cells by inhibiting HDAC6.13 HDAC6, as a microtubule deacetylase, is crucial to the regulation of EGFR trafficking and EGFR degradation in lung cancer cells.14 Combined treatment of ACY-1215 and sorafenib, a tyrosine kinase inhibitor, overcomes HDAC6-mediated resistance in non-small-cell lung cancer cells.15 Remarkably, Nexturastat A, a potent inhibitor of HDAC6 having immunotherapeutic activity, downregulated protein programmed death-ligand 1 (PD-L1) expression by deactivating the STAT3 pathway.16 These studies highlight how HDAC6 inhibitors can regulate multiple pathways involved in carcinogenesis, tumor growth, and metastases. We have reported a series of quinazolin-4-one-based inhibitors selective for HDAC6.7 For the study reported herein, we designed and synthesized a new set of quinazoline-2,4-dione-based HDAC6 inhibitors with scaffold hopping of substituents on our previously developed quinazoline4-one derivatives (Figure 1). The hydroxamic acid and benzyl groups in the previously constructed derivatives maintained their original positions as a zinc-binding group and a linker, respectively. The benzyl-containing linker was moved to the N-1 position of the quinazoline-2,4-dione core, which is a more central location. In addition, the phenyl rings of the quinazoline-2,4-dione core were substituted, one at a time, with various functional groups to explore the HDAC inhibitory profiles of the resulting compounds. Selected compounds, which showed good inhibitory activity were then tested for cytotoxicity against lung cancer cells.


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O

O N

N HN HO

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Et

scaffold hopping

R' N

R N

O H N

O

OH

O Quinazoline-2,4-dione

Quinazoline-4-one

Figure 1. Design of HDAC6-selective inhibitors by scaffold hopping RESULTS AND DISCUSSION Chemistry. The syntheses of 3a−c began with the substituted anthranilic acids 1a−c (Scheme 1). Compounds 1a−c were first coupled with phenyl chloroformate, then amide coupled with phenethylamine, and cyclized under basic conditions to afford the intermediates 2a–c, which were alkylated with ethyl 4-(bromomethyl)benzoate using NaH in an ice bath to obtain the corresponding ester intermediates. These esters were transformed into hydroxamic acids via hydrolysis, amide coupling, and reductive deprotection (3c) or via deprotection by BBr3 (3a and 3b). The synthesis of 3d and 3f began with a ring opening of isatoic anhydride with a phenethylamine at the C-4 position. Then, the product was coupled with phenyl chloroformate and cyclized under basic conditions to obtain 2d, which was then alkylated with ethyl 4(bromomethyl)benzoate or ethyl 3-(bromomethyl)benzoate to afford the corresponding esters. These esters were transformed into the hydroxamic acid derivatives 3d and 3f. 3-Methoxy aniline was coupled with phenyl chloroformate and then transformed into the di-substituted urea, which was ortho-carbonylated through palladium-catalyzed C-H activation17 and cyclized under basic conditions to afford 2e. Compound 3e was synthesized from 2e in a manner similar way as above mentioned.


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In addition, we individually modified positions 6 and 7 of the quinazolin-di-one or replaced the phenyl group with a pyridine of the quinazolin-di-one core (Schemes 2 and 3, respectively). Compounds 1d, 1e, and 1f were each coupled with phenethylamine and then cyclized with ethyl chloroformate under basic conditions to give 2f, 2g, and 2h, respectively. These intermediates were the subjected to conditions similar to those shown in Scheme 1 to afford 3g, 3h, and 3i, respectively. Intermediate 2b was alkylated with ethyl 4-(bromomethyl)benzoate to give the ester 4. The chloro group of 4 was converted into a hydroxyl, methyl, or cyclopropyl by palladium catalysis under Buchwald-Hartwig condition, Negishi, or Suzuki reaction to afford 5a, 5b, or 5c, respectively,18,19 and these derivatives were then converted into the hydroxamic acids 3j, 3k, or 3l, respectively (Scheme 3). To explore the structure-activity relationship of substituents on the phenyl ring of the phenethyl group, the fluoro or methoxy-substituted compounds 3m–3r were synthesized under conditions similar to those of 3d and 3a (Scheme 4). The phenyl group of the linker or cap was also replaced with thiophene to generate 3s, 3t, or 3u, respectively (Scheme 5). The benzyl group was removed by BBr3 as the final step because a Pd-catalyzed reduction did not give the desire product and might have been caused by thiophene poisoning. Scheme 1. Syntheses of Compounds 3a-3ha


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Scheme 1a O O N H

NH2

MeO

a, d, e, f 6 5

3

g, h, i

O OH

R

NH2

1a, R = 5-Cl 1b, R = 4-Cl 1c, R = 4-F 1d, R = 5-F 1e, R = 4-CF3

5

a, b, c or b, h, f (for 2f, 2g)

O

O

6

j, k, l, 6 m or n (for 3a, 3b) R

N

R 8

N H

O

2a, R = 6-Cl 2b, R = 7-Cl 2c, R = 7-F 2d, R = H 2e, R = 7-OMe 2f, R = 6-F 2g, R = 7-CF3

5

O N

8

N

O

p

o m

R1

3a, R = 6-Cl , R1=p-CONHOH 3b, R = 7-Cl , R1=p-CONHOH 3c, R = 7-F , R1=p-CONHOH 3d, R = H , R1=p-CONHOH 3e, R = 7-OMe , R1=p-CONHOH 3f, R = H , R1=m-CONHOH 3g, R = 6-F , R1=p-CONHOH 3h, R = 7-CF3 , R1=p-CONHOH

a

Reagents and conditions: (a) PhOCOCl, 1 N NaOH, dioxane, ice bath, 1 h; (b) EDCI, HOBt,

PhCH2CH2NH2, DMF, rt, 4.5 h; (c) TEA, DMF, W 150 W, 20 min; (d) PhCH2CH2NH2, TEA, DMF/MeOH (1:1), W 100 W, 20 min; (e) Pd(MeCN)2(OTf)2, BQ, p-TSAH2O, CO (1 atm), THF/MeOH (1:1), rt, 5 h; (f) NaOH (2N), THF, 80 °C, 2 h; (g) PhCH2CH2NH2, DMAC, W 250 W, reflux, 6 min; (h) EtOCOCl, THF, reflux, 3.5 h; (i) NaOH (2 N), EtOH, 100 C, 1 h; (j) i) NaH, DMF, ice bath, 1.5 h; ii) 4-COOEtC6H4CH2Br or 3-COOEtC6H4CH2Br, 15 h; (k) LiOH (2.5 N), THF/MeOH (5:1), rt, 4 h; (l) NH2OBnHCl, EDCI, HOBt, TEA, DMF, 4 h; (m) Pd/C, (10% w/w), H2 (1 atm), MeOH/THF (3:1), rt, 5 h; (n) BBr3 (1 M in DCM), DCM, ice bath 1 h. HDAC Inhibition Profile. We first screened the compounds that contained substitutions on the phenyl of the quinazolin-di-one core (Table 1). Compound 3d, with an non-functionalized quinazolin-di-one core, was the best inhibitor of HDAC6 (4 nM) and had the most selectivity over


6

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HDAC1 (2790-fold). Introduction of a chloro (3a) or fluoro (3g) at the C-6 position was detrimental to HDAC6 inhibition. Compared with 3g, shifting the fluoro to C-7, which afforded 3c, significantly increased inhibition of HDAC 6 and HDAC8. However, enlargement of the C-7 group in 3e, 3h, 3k, and 3l did not improve HDAC6 inhibitory activity. Regarding the electronic effect, the electron-withdrawing group in 3h substantially reduced HDAC6 inhibition to 636 nM and selectivity over HDAC1 (13-fold) compared with the electron-donating substituents in 3e and 3j. Compounds 3k and 3l, containing a methyl and a cyclopropyl, respectively, had generally comparable HDAC-inhibition profiles. The data shown in Table 1 indicated that the core size of the quinazolinone cap determined if it could properly be accommodated at the rim of the HDAC6 active site, with an increase in bulkiness being harmful for HDAC6 inhibition. Consequently, 3d was used as the lead compound for further modification. We next synthesized 3m-3r to examine how substituents on the phenyl ring of the phenethyl group affected activity (Table 2). Introduction of fluoro at the para (3m) or meta (3n) position did not increase potency or selectivity. Adding a methoxy group at the para (3o) or ortho (3p) position also did not improve activity. However, substitutions at the C-7-postition more negatively affected potency and selectivity. We assumed that the flexible phenethyl appendage might protrude into the solvent away from the HDAC6 active site. Compound 3q, with a para hydroxyl group, was more selective for HDAC6 compared with HDAC1 (1816-fold) and HDAC8 (169-fold). In addition to SAR on phenyl group, orientation of zinc binding group and introduction of heterocycles on 3d was further investigated (Table 3). First, we moved the hydroxamic acid group from the para position to the meta position of the phenyl to give 3f. Intriguingly, the meta-oriented hydroxamic acid abolished the inhibition of HDAC6 inhibition and gave an inhibition profile


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slightly selective for HDAC8, which suggested that the catalytic site of HDAC6 is not as flexible as that of HDAC8 and does not allow for a conformational change.20 Second, replacing the phenyl ring of the core structure with a pyridine to produced 3i reduced the activity toward HDAC6 around sevenfold but did not affect activity against HDAC1. However, 3u with thiophene ring strengthen its interaction with HDAC8 comparing to 3i. Finally, by replacing the phenyl linker with 2,4disubstituted thiophene to give 3t which possesses a similar inhibitory profile as 3f, that might be due to their comparable orientation of hydroxamic acid group. Remarkably, 3s with 2,5disubstituted thiophene linker reduced the selectivity for HDAC6 over HDAC1 and HDAC8 (1147- and ~fivefold, respectively) provided a dual HDAC6/8 inhibitor. Scheme 2. Synthesis of Compound 3ia Scheme 2a O

O OH N

NH2 1f

a, b, c

O d, e, f, g

N N

N H

O

N N

N

O

2h

H N 3i

aReagents

OH

O

and conditions: (a) PhCH2CH2NH2, EDCI, HOBt, DMF, rt, 4.5 h; (b) EtOCOCl, THF,

reflux 3.5 h; (c) NaOH (2 N), EtOH, 100 °C, 1 h; (d) i) NaH, DMF, ice bath, 1.5 h; ii) 4COOEtC6H4CH2Br, 15 h; (e) LiOH (2.5N), THF/MeOH (5:1), rt, 4 h; (f) NH2OBn·HCl, EDCI, HOBt, TEA, DMF, 4 h; (g) Pd/C, (10% w/w), H2 (1 atm), MeOH/THF (3:1), rt, 5 h. Scheme 3. Syntheses of Compounds 3j-3la


8

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Scheme 3a

a

N N H

Cl

O

O

O

N N

Cl

O

R

O

2b

N

O OEt

OEt 4

O

O

N e, f, g

N

b or c or d

R

N

O 5a, R = OH 5b, R = CH3 5c, R = CH(CH2)2

O H N

OH

O 3j, R = OH 3k, R = CH3 3l, R = CH(CH2)2

aReagents

and conditions: (a) i) NaH, DMF, ice bath, 1.5 h; ii) 4-COOEtC6H4CH2Br, 15 h; (b)

Pd(OAc)2, XPhos, Cs2CO3, DMF/H2O (10:1), W, reflux, 30 min; (c) (CH3)2Zn, Pd2(dba)3, [(tBu)3PH]BF4, THF, W, 120 °C, 30 min; (d) (CH2)2CHBF3K, Pd(OAc)2, XPhos, Cs2CO3, THF/H2O (10:1), 80 °C, 24 h; (e) LiOH (2.5N), THF/MeOH (5:1), rt, 4 h; (f) NH2OBn．HCl, EDCI, HOBt, TEA, DMF, 4 h; (g) Pd/C, (10% w/w), H2 (1 atm), MeOH/THF (3:1), rt, 5–8 h.

Scheme 4. Syntheses of Compounds 3m-3ra


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Scheme 4a O

O a, b, c

O N H

O

R' N

N H

O d, e, f, g N

O

O H N

2i, R' = p-F 2j, R' = m-F 2k, R' = p-OCH3 2l, R' = o-OCH3 2m,R' = m-CF3

OH

O

2k

aReagents

R' N

d, e, f, h

3m, R' = p-F 3n, R' = m-F 3o, R' = p-OCH3 3p, R' = o-OCH3 3q, R' = p-OH 3r, R' = m-CF3

and conditions: (a) PhCH2CH2NH2, DMAC, W 250 W, reflux, 6 min; (b) EtOCOCl,

THF, reflux 3.5 h; (c) NaOH (2 N), EtOH, 100 °C, 1 h; (d) i) NaH, DMF, ice bath, 1.5 h; ii) 4COOEtC6H4CH2Br, 15 h; (e) LiOH (2.5 N), THF/MeOH (5:1), rt, 4 h; (f) NH2OBn·HCl, EDCI, HOBt, TEA, DMF, 4 h; (g) Pd/C, (10% w/w), H2 (1 atm), MeOH/THF (3:1), rt, 5 h; (h) BBr3 (1 M in DCM), DCM, ice bath 1 h.

Scheme 5. Syntheses of Compounds 3s-3ua


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Scheme 5a O

O a, b, c, d

N N H

O N

N

O

2d

N

O

3s

O

S

NH OH

OMe

1g

O a, b, c, d

N S

NH2

N H

O

3t

O e, f, g

O H N OH

S O

S

N

or

O

N S

N

O

2n

H N 3u

aReagents

OH

O

and conditions: (a) i) NaH, DMF, ice bath, 0.5 h; ii) methyl 5-

(bromomethyl)thiophene-2-carboxylate or methyl 4-(bromomethyl)thiophene-2-carboxylate or 4COOEtCH6H4CH2Br, 5 h; (b) LiOH (2.5 N), THF/MeOH (5:1), rt, 4 h; (c) NH2OBn·HCl, EDCI, HOBt, TEA, DMF, 4 h; (d) BBr3 (1 M in DCM), DCM, ice bath 1 h; (e) PhOCOCl, THF, W, 200 W, reflux, 10 min; (f) PhCH2CH2NH2, TEA, MeOH, W 100 W, 10 min; (g) NaOH (2 N), EtOH, 100 °C, 8 h. Table 1. HDAC-inhibition profiles of 3a–3e, 3g, 3h, and 3j–3l

5

O

6

N

R 8

N

O H N

OH

O


11


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HDAC inhibition, IC50 (nM) a Selectivity, Cpd

R

HDAC1

HDAC6

HDAC8

HDAC11 HDAC1/HDAC6

3a

6-Cl

18860

35

NDb

ND

539

3b

7-Cl

9540

22

1640

ND

434

3c

7-F

9410

8

95

15400

1176

3d

H

11160

4

309

13900

2790

3e

7-OCH3

12000

51

731

ND

235

3g

6-F

13410

57

1155

16740

235

3h

7-CF3

8021

636

1442

7392

13

3j

7-OH

8275

32

2751

5501

259

3k

7-CH3

10870

170

2521

52050

64

3l

7-CH(CH2)2

14500

168

3060

10500

86

aThe

assays were conducted by the Reaction Biology Corporation, Malvern, PA. Compounds

were tested in the 10-dose IC50 mode with threefold serial dilutions starting at 30 M. bND,

not determined.

Table 2. HDAC-inhibition profiles of 3d and 3m–3q O

R' N

N

O H N

OH

O


12

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R'

HDAC1

HDAC6

HDAC8

HDAC11 HDAC1/HDAC6

3d

H

11160

4

309

13900

2790

3m

p-F

10810

23

1049

14730

470

3n

m-F

13170

10

429

10660

1317

3o

p-OCH3

7374

20

1150

13850

369

3p

o-OCH3

10430

12

525

34370

869

3q

p-OH

9080

5

845

11290

1816

3r

m-CF3

12700

24

1760

ND

529

aThe


were tested in 10-dose IC50 mode with threefold serial dilutions starting at 30 M. ND, not determined. Table 3. HDAC-inhibition profiles of 3d, 3f, 3i, and 3r O

O

O N

N

O

N

N

O

3i

NH OH

N

O

3s

N S

O

N

O

H N OH

S

OH

O

O N

N

O H N

3f

O N

N

O

NH OH

S

O

3t

H N 3u

OH

O


HDAC1

HDAC6

HDAC8

HDAC11 HDAC1/HDAC6

3d

11160

4

309

13900

2790


13


aThe

Page 14 of 60

3f

>10000

4630

653

>10000

>2

3i

11150

29

1068

25050

384

3s

22940

20

97

19800

1147

3t

>10000

1194

1191

>10000

>8

3u

6655

15

169

5686

443


were tested in the 10-dose IC50 mode with threefold serial dilutions starting at 30 M.

HDAC6 Kinetic Activity Study. To delineate the interaction between some of our synthesized compounds and HDAC6, the enzymatic activity of HDAC6 was measured in the presence of a substrate at different concentrations and with different concentrations of the inhibitors. In addition to full-length HDAC6, the activity of the catalytic domain 2 (CD2) from HDAC6 was studied (CD1 does not have catalytic active).21 The Ki values of the potential inhibitors were similar to that of tubastatin A, with 3d being the most potent (Table 4). The inhibition data were consistent with a competitive inhibition model for full-length HDAC6 and for CD2. The v values for CD2 were ~50% those of full-length HDAC6 for all inhibitors. The Km values for the substrate were of the same order of magnitude as were the Ki values for the quinazolinone derivatives. Table 4. HDAC6 and CD2 kinetic parameters in the presence of 3d, 3i, and 3p

Cpd

Enzyme

v (M·s–1)

Km (M)

Ki (nM)

3d

Full-length HDAC6

0.70 ± 0.03

16.7 ± 1.20

1.60 ± 0.40


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3i

Full-length HDAC6

0.77 ± 0.42

21.8 ± 8.30

3.89 ± 1.64

3p

Full-length HDAC6

1.19 ± 0.22

34.6 ± 4.20

2.10 ± 0.44

Tubastatin A

Full-length HDAC6

1.04 ± 0.35

29.1 ± 16.4

3.43 ± 1.06

3d

CD2

0.45 ± 0.03

29.8 ± 5.50

3.70 ± 0.50

3i

CD2

0.43 ± 0.02

35.3 ± 5.00

4.50 ± 0.50

3p

CD2

0.45 ± 0.04

32.5 ± 8.60

2.60 ± 0.50

The More Potent Inhibitors of Recombinant HDAC6 Also Decreased Lung Cancer Cell Viability In Vitro. A 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to characterize the cytotoxicities and IC50 values of some of the HDAC6-selective inhibitors (Table 5). Human lung cancer A549 cells, were treated with serial dilutions of the HDAC6 inhibitors at 37 °C for 72 h. Compound 3i had the greatest activity against the cells with an IC50 value of 2.8 M. Although, the cytotoxicities of the HDAC6 inhibitors were not as potent as that of the pan-HDAC inhibitor, suberanilohydroxamic acid (SAHA), it showed better safety without severe adverse effects than pan-HDAC inhibitors.12,22 Table 5. Cytotoxicities of (IC50) of selected potential and known HDAC6 inhibitors against A549 lung cancer cells.


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IC50a (M)

Cpd 3c

3.92 ± 0.61

3d

7.87 ± 0.70

3g

12.19 ± 0.39

3i

2.84 ± 0.02

3m

3.65 ± 0.06

3p

11.78 ± 0.15

3r

6.03 ± 0.43

3s

13.5 ± 0.07

SAHA

2.24 ± 0.07

ACY1215

aThe

Page 16 of 60

7.7 ± 0.12

IC50 values were the concentrations of the compounds that had a 50% cytotoxic effect on the

A549 cells.

Combined Cytotoxic Effect of Paclitaxel and 3d on Cultured Mouse Lung Cancer LL/2 Cells. As described above, our data indicated that the HDAC6-selective inhibitors potentially had useful anti-lung cancer activity. To analyze the combined effect of paclitaxel and 3d, we employed MTT assay for cell viability. Because the IC50 values for paclitaxel and 3d were 10 nM and 7.87 M, respectively, we performed the combination experiments with 10 nM paclitaxel and 4 M of 3d. Compound 3d (4 M) or paclitaxel (10 nM) when used separately killed between 10 and 20% of


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the LL/2 cells by day 3, whereas when used together, they killed 80 to 90% of the cells by day 3. To assess the combined efficacies of 3d or 3r with paclitaxel, LL/2 cells were treated with 3d or 3r separately and in combination with paclitaxel for 3 days. The combined treatment of paclitaxel and 3d or 3r resulted in significantly greater inhibition of LL/2 cell viability when compared with treatment of either inhibitor alone (Figures 2b and c). Further, increasing concentrations of 3d (010μM) and PAX were cultured in LL/2 cells for 72 hours and the viability was analyzed by MTT to determinate the combination effect. When 3d combined with PAX, the IC50 of 3d and PAX in LL/2 cells were found to decrease significantly. Synergism was evaluated by Chou-Talalay method and showed the combination index

Quinazolin-2,4-dione-Based Hydroxamic Acids as Selective Histone

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