5.2
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
Corrigendum
Current Issue
Editorial
Erratum
Full Length Article
Full lenth article
Letter to Editor
Original Article
Research article
Retraction notice
Review
Review Article
SPECIAL ISSUE: ENVIRONMENTAL CHEMISTRY
5.3
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
Corrigendum
Current Issue
Editorial
Erratum
Full Length Article
Full lenth article
Letter to Editor
Original Article
Research article
Retraction notice
Review
Review Article
SPECIAL ISSUE: ENVIRONMENTAL CHEMISTRY
View/Download PDF

Translate this page into:

Original article
10 (
4
); 465-472
doi:
10.1016/j.arabjc.2015.10.007

Exploration of novel 5′(7′)-substituted-2′-oxospiro[1,3]dioxolane-2,3′-indoline-based N-hydroxypropenamides as histone deacetylase inhibitors and antitumor agents

, , , , , , , , ,
a Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Viet Nam
b School of Chemical Engineering, Hanoi University of Science and Technology, 1 Dai Co Viet, Hanoi, Viet Nam
c Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea
d College of Pharmacy, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea

⁎Corresponding authors. Tel.: +84 4 39330531; fax: +84 4 39332332. shan@chungbuk.ac.kr (Sang-Bae Han), namnh@hup.edu.vn (Nguyen-Hai Nam)

Received: , Accepted: ,
© The Author(s) 2015
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

A series of novel 5′(7′)-substituted-2′-oxospiro[1,3]dioxolane-2,3′-indoline-based N-hydroxypropenamides were designed, synthesized and evaluated for histone deacetylase (HDAC) inhibition and cytotoxicity. It was found that the compounds in this series displayed potent inhibitory effects against HDAC2 with IC50 values as low as 0.284 μM, almost comparable to that of SAHA (IC50, 0.265 μM), a positive control. In Western blot analysis, these compounds also exhibited noted inhibition toward histone deacetylation and this inhibition was found to correlate well with the cytotoxicity of the compounds in three human cancer cell lines. Docking studies indicated the compounds in this series bound to HDAC2 with high binding affinities (∼−9.8 kcal/mol) compared to SAHA (−7.4 kcal/mol).

Keywords

Dioxolane
Indoline
N-hydroxypropenamide
Histone deacetylase
Hydroxamic acid
1

1 Introduction

Since histone deacetylases (HDACs) are found to get involved in the regulation of cell-cycle progression and carcinogenic process (Ropero and Esteller, 2007; Archer and Hodint, 1999), these enzymes have become attractive targets for anticancer drug discovery. Currently, 18 mammalian HDACs have been identified and the HDACs can be divided into four main classes according to their homology to yeast HDACs (De Ruijter et al., 2003). Class I consists of HDAC 1, 2, 3, 8, class II consists of HDAC 4, 5, 6, 7, 9, 10 and class IV contains only one member, HDAC 11. All members of classes I, II and IV are known as “classical” HDACs and are Zn2+-dependent enzymes. The class III called Sirtuins (SIRT1–7) and the enzyme of this class are NAD+-dependent enzymes (Giannini et al., 2012).

Thousands of HDAC inhibitors with diverse structural features have been either isolated or synthesized. Many of these have been shown to exert very potent bioactivities, not only in terms of HDAC inhibition, but also in terms of cytotoxicity against human cancer cell lines. A dozen of compounds, such as suberoylanilide hydroxamic acid (SAHA, Vorinostat), LBH-589 (Panobinostat), MS-27-527 (Entinostat), PXD-01 (Belinostat), and romidepsin (Fig. 1), have been proved to be promising in both preclinical and clinical trials (Giannini et al., 2012; Kim et al., 2011). Two first HDAC inhibitors, including SAHA (suberoylanilide hydroxamic acid, trade name, Zolinza®) and romidepsin (tradename, Istodax®) (Fig. 1) have been approved by the US FDA in 2006 and 2009, respectively, to treat cutaneous T-cell lymphoma (Giannini et al. 2012; Kim et al., 2011). More recently, PXD-01 (Belinostat, trade name Beleodaq®) and LBH-589 (Panobinostat, trade name Farydak®) have also gained market approval in 2014 and 2015, respectively, to treat hematological malignancies and various solid cancer (Grasso, 2015).

Structures of selected HDAC inhibitors.
Figure 1
Structures of selected HDAC inhibitors.

Structurally, HDAC inhibitors can be classified into four chemical groups including hydroxamates, benzamides, carboxylates and cyclic peptides. Among these, the hydroxamates are still considered as the most potent HDAC inhibitors (Kim et al., 2011). In our previous papers (Nam et al., 2013; Nam et al., 2014; Oanh et al., 2011; Tung et al., 2013) we have reported several series of hydroxamic acids bearing benzothiazole, 5-substituted phenyl-1,3,4-thiadiazole or 3-substituted-2-oxoindoline (Fig. 2). These compounds were shown to be very potent HDAC inhibitors and exhibited promising anticancer effects. In continuity of our research, we have designed, synthesized and evaluated a series of hydroxamic acids containing 5′(7′)-substituted-2′-oxospiro[1,3]dioxolane-2,3′-indoline system. The present paper describes the results we obtained from this study.

Structures of some benzothiazole-, 5-substituted phenyl-1,3,4-thiadiazole- and 2-oxoindoline-based hydroxamic acids.
Figure 2
Structures of some benzothiazole-, 5-substituted phenyl-1,3,4-thiadiazole- and 2-oxoindoline-based hydroxamic acids.

2

2 Experimental

2.1

2.1 Materials and reagents

All the reagents and solvents used for the syntheses were purchased from Aldrich or Fluka Chemical Corp. (Milwaukee, WI, USA) or Merck and used without any further purification. The Fluoregenic HDAC2 Assay Kit used for enzymatic HDAC assay was obtained commercially from BPS Bioscience, Inc.

2.2

2.2 Physicochemical measurements

Melting points were obtained on a Gallenkamp Melting Point Apparatus (LabMerchant, London, United Kingdom) and are uncorrected. Infrared (IR) spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer (PerkinElmer, Inc., USA) and an Agilent 660 FTIR (Agilent Technologies, Inc., USA) using KBr disk method. Electrospray ionization (ESI-MS) spectra were obtained on a LTQ Orbitrap XL (Thermo Scientific) and an Agilent 6460 Triple Quad LC/MS instrument spectrometer. Nuclear magnetic resonance (NMR) spectra were recorded using tetramethylsilane as an internal standard on a Bucker 500 MHz spectrometer with DMSO-d6 used as a solvent unless otherwise indicated. Thin-layer chromatography (TLC) was performed on Whatman® 250 μm Silica Gel GF254 Uniplates and visualized under UV light at 254 nm.

2.3

2.3 General procedure for preparation of (E)-N-hydroxy-3-(4-((2′-oxospiro[[1,3]dioxolane-2,3′-indoline]-1′-yl)methyl)phenyl)propenamide derivatives

2.3.1

2.3.1 Synthesis of (E)-N-hydroxy-3-(4-((2′-oxospiro[[1,3]dioxolane-2,3′-indoline]-1′-yl)methyl)phenyl)propenamide (4a)

2.3.1.1
2.3.1.1 Synthesis of spiro[[1,3]dioxolane-2,3′-indoline]-2′-one (2a)

A solution of isatin (1a, 147 mg, 1 mmol), ethane-1,2-diol (0.56 mL, 620 mg, 10 mmol) and p-toluenesulfonic acid (p-TsOH, 1 mmol) in 3 mL of toluene was refluxed for 4 h. After the reaction completed, the crude reaction mixture was extracted with toluene and washed with water. The organic layer was dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure to furnish the desired compound 2a as slightly yellow solid in 86% yield.

2.3.1.2
2.3.1.2 Synthesis of (E)-methyl 3-(4-((2′-oxospiro[[1,3]dioxolane-2,3′-indoline]-1′-yl)methyl)phenyl)propenoate (3a)

To a solution of compound 2a (190 mg, 1 mmol) in anhydrous dimethylformamide (DMF, 2 mL) were added K2CO3 (207 mg, 1.5 mmol) and KI (20 mg, a catalytic amount). The resulting mixture was continuously stirred at room temperature for 30 min. Then, (E)-methyl 3-(4-bromomethyl)phenyl)propenoate (255 mg, 1 mmol) dissolved in anhydrous DMF (1 mL) was added dropwise into the reaction mixture, which was again stirred at room temperature for further 24 h. The reaction mixture was finally poured into 30 mL of ice-water. The precipitates formed were filtered and washed with water, then dried at 60 °C for 24 h to afford the desired compound 3a as yellow solid in 84% yield.

2.3.1.3
2.3.1.3 Synthesis of (E)-N-hydroxy-3-(4-((2′-oxospiro[[1,3]dioxolane-2,3′-indoline]-1′-yl)methyl)phenyl)propenamide (4a)

The intermediate 3a (190 mg, 0.5 mmol) and NH2OH.HCl (700 mg, 10 mmol) were dissolved in MeOH (4 mL) in a round bottom flask. The solution was cooled to −5 °C. A solution of NaOH (800 mg, 20 mmol) in 1.5 ml of H2O was also cooled to 0–5 °C and added dropwise into the reaction flask. The mixed solution was stirred at −5 °C until the starting material was consumed completely. The reaction mixture was acidified with HCl 5% solution to form precipitates. The crude product was filtered, dried and recrystallized from MeOH/H2O to give the target compound 4a as white solid in 40% yield. Mp. 267.2–268.7 °C; Rf = 0.52 (DCM/MeOH = 9/1); IR (KBr, cm−1): 3211.1 (NH), 3060.1 (C—H, arene), 2925.5, 2854.7 (C—H, CH2), 1727.9, 1666.1 (C⚌O), 1618.5, 1488.7, 1468.9 (C⚌C); ESI-MS (m/z): 388.9 [M + Na]+, 367.0 [M + H]+; 1H NMR (500 MHz, DMSO-d6, ppm): δ 10.78 (1H, s, NH), 9.10 (1H, s, OH), 7.54 (2H, d, J = 8.0 Hz, H-2′, H-6′), 7.42 (1H, d, J = 15.75 Hz, H-2), 7.39 (1H, d, J = 7.5 Hz, H-4′′), 7.35 (1H, d, J = 7.5 Hz, H-6′′), 7.31 (2H, d, J = 8.0 Hz, H-3′, H-5′), 7.08 (1H, t, J = 7.5 Hz, H-5′′), 6.95 (1H, d, J = 8.0 Hz, H-7′′), 6.43 (1H, d, J = 15.75 Hz, H-3), 4.85 (2H, s, H-7′), 4.43–4.40 (2H, m, H-8′′a, H-9′′a), 4.32–4.30 (2H, m, H-8′′b, H-9′′b); 13C NMR (125 MHz, DMSO-d6, ppm): 172.77, 162.75, 143.13, 137.87, 137.24, 134.25, 131.73, 127.96, 127.78, 124.83, 124.14, 123.32, 119.24, 109.52, 101.57, 65.73, 42.20. Anal. Calcd. For C20H18N2O5 (366.12): C, 65.67; H, 4.95; N, 7.65. Found: C, 65.71; H, 4.97; N, 7.64.

2.3.2

2.3.2 Synthesis of (E)-N-hydroxy-3-(4-((5′-fluoro-2′-oxospiro[[1,3]dioxolane-2,3′-indoline]-1′-yl)methyl)phenyl)propenamide (4b)

The target compound 4b was synthesized according to the general procedures (Scheme 1) and the crude product was purified by crystallization from MeOH/H2O. Slight yellow solid; Yield: 64%; Mp. 260.5–261.3 °C; Rf = 0.41 (DCM/MeOH = 9/1); IR (KBr, cm−1): 3544.4 (OH), 3187.2 (NH), 3059.9 (C—H, arene), 2903.8 (C—H, CH2), 1716.4, 1670.8 (C⚌O), 1631.7, 1513.4, 1493.2 (C⚌C); ESI-MS (m/z): 383.1 [M-H]−; 1H NMR (500 MHz, DMSO-d6, ppm): 10.76 (1H, s, NH), 9.01 (1H, s, OH), 7.54 (2H, d, J = 8.0 Hz, H-2′, H-6′), 7.42 (1H, d, J = 15.75 Hz, H-2), 7.37 (1H, dd, J = 7.5 Hz, J′ = 2.5 Hz, H-4′′), 7.31 (2H, d, J = 8.0 Hz, H-3′, H-5′), 7.22 (1H, td, J = 9.0 Hz, J′ = 2.5 Hz, H-6′′), 6.98 (1H, dd, J = 8.5 Hz, 4.0 Hz, H-7′′), 6.42 (1H, d, J = 15.75 Hz, H-3), 4.87 (2H, s, H-7′), 4.41–4.37 (2H, m, H-8′′a, H-9′′a), 4.36–4.31 30 (2H, m, H-8′′b, H-9′′b); 13C NMR (125 MHz, DMSO-d6, ppm): 172.55, 162.60, 157.81, 139.12, 137.71, 136.94, 134.24, 127.89, 127.71, 126.08, 126.02, 119.24, 118.00, 117.81, 112.76, 112.56, 111.18, 111.12, 101.23, 65.90, 42.25. Anal. Calcd. For C20H17FN2O5 (384.01): C, 62.50; H, 4.46; N, 7.29. Found: C, 62.53; H, 4.48; N, 7.26.

The synthetic route of the target compounds. Reagents and conditions: (i) ethane-1,2-diol, toluene, p-TsOH, rfx; (ii) (E)-methyl 3-(4-bromomethyl)phenyl)acrylate, K2CO3, KI, DMF, rt; (iii) NH2OH.HCl, NaOH, MeOH, −5 °C.
Scheme 1
The synthetic route of the target compounds. Reagents and conditions: (i) ethane-1,2-diol, toluene, p-TsOH, rfx; (ii) (E)-methyl 3-(4-bromomethyl)phenyl)acrylate, K2CO3, KI, DMF, rt; (iii) NH2OH.HCl, NaOH, MeOH, −5 °C.

2.3.3

2.3.3 Synthesis of (E)-N-hydroxy-3-(4-((5′-chloro-2′-oxospiro[[1,3]dioxolane-2,3′-indoline]-1′-yl)methyl)phenyl)propenamide (4c)

The target compound 4c was synthesized according to the general procedures (Scheme 1) and the crude product was purified by crystallization from MeOH/H2O. Yellow solid; Yield: 65%; Mp: 255.7–256.2 °C, Rf = 0.44 (DCM/MeOH = 9/1), IR (KBr, cm−1): 3588.1 (OH), 3280.4 (NH), 3003.6 (C—H, arene), 2905.5 (C—H, CH2), 1743.8, 1705.6 (C⚌O), 1660.8, 1488.0 (C⚌C); ESI-MS (m/z): 399.1 [M-H]−; 1H NMR (500 MHz, DMSO-d6, ppm): 10.78 (1H, s, NH), 9.05 (1H, s, OH), 7.55 (2H, d, J = 8.5 Hz, H-2′, H-6′), 7.54 (1H, s, H-4′′), 7.44 (1H, d, J = 7.0 Hz, H-6′′), 7.42 (1H, d, J = 15.5 Hz, H-2), 7.31 (2H, d, J = 8.5 Hz, H-3′, H-5′), 7.02 (1H, d, J = 8.5 Hz, H-7′′), 6.43 (1H, d, J = 15.5 Hz, H-3), 4.88 (2H, s, H-7′), 4.41–4.39 (2H, m, H-8′′a, H-9′′a), 4.37–4.35 (2H, m, H-8′′b, H-9′′b); 13C NMR (125 MHz, DMSO-d6, ppm): 172.82, 163.10, 142.34, 138.21, 137.34, 134.76, 131.92, 129.14, 128.43, 128.20, 128.12, 127.99, 126.84, 125.43, 119.75, 112.12, 102.60, 66.49, 42.76. Anal. Calcd. For C20H17ClN2O5 (400.08): C, 59.93; H, 4.28; N, 6.99. Found: C, 59.97; H, 4.25; N, 6.97.

2.3.4

2.3.4 Synthesis of (E)-N-hydroxy-3-(4-((5′-bromo-2′-oxospiro[[1,3]dioxolane-2,3′-indoline]-1′-yl)methyl)phenyl)propenamide (4d)

The target compound 4d was synthesized according to the general procedures (Scheme 1) and the crude product was purified by crystallization from MeOH/H2O. Yellow solid; Yield: 70%; Mp: 257.6–258.5 °C; Rf = 0.54 (DCM/MeOH = 9/1), IR (KBr, cm−1): 3216.1 (NH), 3050.7 (C—H, arene), 2973.4, 2855.4 (C—H, CH2), 1731.6, 1660.8 (C⚌O), 1612.6, 1515.0, 1485.1 (C⚌C); ESI-MS (m/z): 443.3 [M-H]+; 1H NMR (500 MHz, DMSO-d6, ppm): 10.78 (1H, s, NH), 9.05 (1H, s, OH), 7.64 (1H, s, H-4′′), 7.56 (1H, t, J = 9.5 Hz, H-6′′), 7.55 (2H, d, J = 8.5 Hz, H-2′, H-6′), 7.43 (1H, d, J = 15.75 Hz, H-2), 7.30 (2H, d, J = 8.5 Hz, H-3′, H-5′), 6.96 (1H, d, J = 8.5 Hz, H-7′′), 6.43 (1H, d, J = 15.75 Hz, H-3), 4.88 (2H, s, H-7′), 4.41–4.38 (2H, m, H-8′′a, H-9′′a), 4.37–4.33 (2H, m, H-8′′b, H-9′′b); 13C NMR (125 MHz, DMSO-d6, ppm): 172.69, 163.10, 143.89, 142.78, 138.21, 137.31, 134.78, 129.14, 128.43, 128.19, 128.14, 127.14, 119.75, 115.60, 112.60, 101.55, 66.51, 42.74. Anal. Calcd. For C20H17BrN2O5 (444.03): C, 59.93; H, 4.28; N, 6.99. Found: C, 59.97; H, 4.25; N, 6.97.

2.3.5

2.3.5 Synthesis of (E)-N-hydroxy-3-(4-((5′-methyl-2′-oxospiro[[1,3]dioxolane-2,3′-indoline]-1′-yl)methyl)phenyl)propenamide (4e)

The target compound 4e was synthesized according to the general procedures (Scheme 1) and the crude product was purified by crystallization from MeOH/H2O. White solid; Yield: 62%; Mp: 247.3–248.8 °C; Rf = 0.56 (DCM/MeOH = 9/1); IR (KBr, cm−1): 3473.5 (OH), 3208.7 (NH), 3028.5 (C—H, arene), 2920.8 (C—H, CH2), 1708.0 1670.2 (C⚌O), 1629.8 1497.8 (C⚌C); ESI-MS (m/z): 379.2 [M-H]−; 1H NMR (500 MHz, DMSO-d6, ppm): 10.80 (1H, s, NH), 9.05 (1H, s, OH), 7.53 (2H, d, J = 8.0 Hz, H-2′, H-6′), 7.42 (1H, d, J = 15.5 Hz, H-2), 7.29 (2H, d, J = 8.0 Hz, H-3′, H-5′), 7.22 (1H, s, H-4′′), 7.14 (1H, d, J = 8.0 Hz, H-7′′), 6.82 (1H, d, J = 8.0 Hz, H-6′′), 6.43 (1H, d, J = 15.5 Hz, H-3), 4.82 (2H, s, H-7′), 4.41–4.37 (2H, m, H-8′′a, H-9′′a), 4.32–4.28 (2H, m, H-8′′b, H-9′′b), 2.24 (3H, s, CH3); 13C NMR (125 MHz, DMSO-d6, ppm): 172.77, 163.45, 140.70, 137.33, 134.22, 132.64, 131.79, 127.95, 127.75, 127.67, 125.43, 124.18, 119.21, 109.75, 101.76, 65.74, 42.22, 20.42. Anal. Calcd. For C21H20N2O5 (380.14): C, 66.31; H, 5.30; N, 7.36. Found: C, 66.35; H, 5.27; N, 7.34.

2.3.6

2.3.6 Synthesis of (E)-N-hydroxy-3-(4-((5′-methoxy-2′-oxospiro[[1,3]dioxolane-2,3′-indoline]-1′-yl)methyl)phenyl)propenamide (4f)

The targeted compound 4f was synthesized according to the general procedure (Scheme 1) and the crude product was purified by crystallization from MeOH/H2O. White solid; Yield: 45%; Mp: 245.3–246.5 °C; Rf = 0.59 (DCM/MeOH = 9/1); IR (KBr, cm−1): 2959.5, 2839.2 (C—H, CH2), 1722.9 (C⚌O), 1633.3, 1608.4 1487.2 (C⚌C); ESI-MS (m/z): 397.3 [M-H]−; 1H NMR (500 MHz, DMSO-d6, ppm): 10.77 (1H, s, NH), 8.99 (1H, s, OH), 7.57 (2H, d, J = 7.5 Hz, H-2′, H-6′), 7.39 (1H, d, J = 15.5 Hz, H-2), 7.31 (2H, d, J = 7.5 Hz, H-3′, H-5′), 7.25 (1H, s, H-4′′), 7.11 (1H, d, J = 7.5 Hz, H-7′′), 6.89 (1H, d, J = 7.5 Hz, H-6′′), 6.47 (1H, d, J = 15.5 Hz, H-3), 4.87 (2H, s, H-7′), 4.43–4.39 (2H, m, H-8′′a, H-9′′a), 4.34–4.30 (2H, m, H-8′′b, H-9′′b), 3.79 (3H, s, OCH3); 13C NMR (125 MHz, DMSO-d6, ppm): 171.89, 162.46, 149.92, 138.41, 133.28, 132.99, 132.06, 128.79, 128.98, 127.63, 125.68, 124.34, 120.35, 110.06, 102.63, 65.74, 55.34, 43.67; Anal. Calcd. For C21H20N2O6 (396.13): C, 63.63; H, 5.09; N, 7.07. Found: C, 63.66; H, 5.11; N, 7.09.

2.3.7

2.3.7 Synthesis of (E)-N-hydroxy-3-(4-((7′-chloro-2′-oxospiro[[1,3]dioxolane-2,3′-indoline]-1′-yl)methyl)phenyl)propenamide (4g)

The targeted compound 4g was synthesized according to the general procedure (Scheme 1) and the crude product was purified by crystallization from MeOH/H2O. Slight yellow solid; Yield: 67%; Mp: 254.8–256.2 °C; Rf = 0.46 (DCM/MeOH = 9/1); IR (KBr, cm−1): 3300.6 (OH), 3220.6 (NH), 3089.7 (C—H, arene), 2971.3, 2903.5 (C—H, CH2), 1734.9, 1684.6 (C⚌O), 1617.4, 1514.4 (C⚌C); ESI-MS (m/z): 399.2 [M-H]−; 1H NMR (500 MHz, DMSO-d6, ppm): 7.54 (2H, d, J = 8.0 Hz, H-2′, H-6′), 7.45 (1H, d, J = 7.0 Hz, H-6′′), 7.42 (1H, d, J = 15.75 Hz, H-2), 7.40 (1H, d, J = 7.5 Hz, H-4′′), 7.19 (2H, d, J = 8.0 Hz, H-3′, H-5′), 7.15 (1H, t, J = 8.0 Hz, H-5′′), 6.43 (1H, d, J = 15.75 Hz, H-3), 5,21 (2H, s, H-7′), 4.44–4.39 (2H, m, H-8′′a, H-9′′a), 4.39–4.35 (2H, m, H-8′′b, H-9′′b); 13C NMR (125 MHz, DMSO-d6, ppm): 173.45, 162.34, 138.88, 138.36, 133.84, 127.85, 127.43, 126.33, 124.95, 124.02, 119.05, 114.89, 100.46, 65.94, 43.77; Anal. Calcd. For C20H17ClN2O5 (400.08): C, 59.93; H, 4.28; N, 6.99. Found: C, 59.95; H, 4.27; N, 7.04.

2.4

2.4 Biological evaluation

2.4.1

2.4.1 Western Blot analysis

The human cancer line SW620 was lysed by treating in RIPA buffer (50 mM Tris–Cl [pH 8.0], 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride) then extracted total protein. The lysates determined the concentration of protein using a Bio-Rad protein assay kit (Bio-Rad Laboratories Inc.). Samples were separated on SDS-polypropenamide gels and transferred to nitrocellulose membranes. The membranes were incubated with blocking buffer (Tris-buffered saline containing 0.2% Tween-20 and 3% nonfat dried milk) and probed with the primary antibodies against acetyl-histone-H3, -H4 and GAPDH. After washing, membranes were reprobed with horseradish peroxidase-conjugated secondary antibodies. An enhanced chemiluminescent protein (ELC) detection system was used to perform detection of Western blot assay.

2.4.2

2.4.2 Fluorogenic HDAC2 assay

The enzymatic HDAC2 assay was based on the HADC2 enzyme and the Fluorogenic HDAC2 Assay Kit (BPS Bioscience), carried out according to the supplier’s instruction. Briefly, HDAC2 enzymes were incubated with vehicle or assayed samples at different concentrations for 30 min at 37 °C in the presence of an HDAC fluorimetric substrate. The HDAC assay developer was added, and the fluorescence was measured using VICTOR3 (PerkinElmer, Waltham, MA, USA) with excitation at 360 nm and 460 nm. The measured activities were subtracted by the vehicle-treated control enzyme activities and IC50 values were calculated using GraphPad Prism (GraphPad Software, San Diego, CA, USA).

2.4.3

2.4.3 In vitro cytotoxicity assay

In vitro cytotoxicity assay was determined by the sulforhodamine B (SRB) method as described in the literature (Longo-Sorbello et al., 2006; Skehan et al., 1990). Three human cancer cell lines including SW620 (colon cancer), PC3 (prostate cancer), and AsPC-1 (pancreatic cancer) cell lines were obtained from a cancer cell bank at the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Cells were seeded into 96-well plates at density of 9 × 103 cells/well, incubated overnight and treated with samples for 48 h. All compounds were dissolved in dimethyl sulfoxide (DMSO). The IC50 values were calculated according to the Probit method (Wu et al., 1992). The values reported of these compounds are averages of three separate determinations.

2.4.4

2.4.4 Docking

AutoDock Vina program (The Scripps Research Institute, CA, USA) (Trott and Olson, 2010) was used in the docking studies. The initial structure HDAC2 (Lauffer et al., 2013) (complexed with SAHA) was obtained from the Protein Data Bank (PDB) (PDB ID: 4LXZ) and coordinates for the compounds were generated using the GlycoBioChem PRODRG2 Server (http://davapc1.bioch.dundee.ac.uk/prodrg/) (Schuttelkopf and van Aalten, 2004). The grid maps for docking studies were centered on the SAHA binding site and comprised 26 × 26 × 22 points with 1.0 Å spacing after SAHA was removed from the complex structure, as described previously. AutoDock Vina program was run with eight-way multithreading and the other parameters were default settings in AutoDock Vina program.

3

3 Results and discussion

Compounds 4a–g were reached straightforward by a three-step synthetic pathway. The first step involved refluxing the corresponding isatin or isatin derivatives with excess of ethane-1,2-diol in the presence of a catalytic amount of p-TsOH to give the intermediates 2a–g in high yields. Nucleophilic substitution of 2a–g with (E)-methyl 3-(4-bromomethyl)phenyl)propenoate proceeded smoothly in the presence of K2CO3 in anhydrous DMF. The conversion was faster and more efficient when a catalytic KI was added. The final step, a nucleophilic acyl substitution reaction, proceeded more efficiently under basic conditions and this step turned out to be the most troublesome since under these conditions the hydrolysis of the ester functional group always competed with the substitution by hydroxylamine. Therefore, low temperature and short reaction time needed to be appropriately controlled. The yields of the final steps were moderate (around 40%).

All target compounds were unambiguously determined by careful spectroscopic analysis. 1H NMR spectra of the compounds showed a typical singlet at around 4.90 ppm, which was interpreted for two protons and attributable for a methylene group (H-7′) (Fig. 3). Two olefinic protons (H-2, H-3) appeared at around 6.40 and 7.30 ppm and coupled to each other with J values of approximately 16.0 Hz, corresponding to trans configuration. Two multiplets appeared at around 4.30–4.40 ppm were typical of four protons (H-8′′, H-9′′) from dioxolane moiety. 13C NMR spectral data of the compounds were in good accordance with their structures, noting that C-2′ and C-6′, C-3′ and C-5′, and C-8′′ and C-9′′ appeared overlapped, respectively, in their spectra.

Numbering for the target structures used in spectral assignments.
Figure 3
Numbering for the target structures used in spectral assignments.

The synthesized compounds were first evaluated for their inhibitory effects on HDAC2 and cytotoxicity against three human cancer cell lines, including SW620 (colon cancer), PC3 (prostate cancer), and AsPC-1 (pancreatic cancer). HDAC2 enzyme was chosen in this first line biological evaluation since it is one the key players involved in different types of cancer (Ropero and Esteller, 2007). The results showed that all compounds in the series 4a–g potently inhibited HDAC2 with IC50 values as low as 0.284 μM, almost comparable to that of SAHA (IC50, 0.265 μM) a positive control (Table 1). These HDAC2 inhibitory effects were also comparable to those of a series of 3-oxime-2-oxoindoline-based N-hydroxypropenamides reported by our group recently (Dung et al., 2015). The cytotoxicity of these compounds, however, was less potent in comparison with SAHA. The IC50 values of these compounds in three assayed cancer cell lines were generally higher than those of SAHA (Table 2). Low water solubility of these compounds (as manifested by logP values, Table 2), leading to lower cellular penetration, among other factors, might be explainable for this disparity between enzyme inhibitory potency and cytotoxicity. It seemed that most of the compounds were more cytotoxic toward colon cancer cells (SW620) than prostate (PC-3) and pancreatic (AsPC-1) cancer cells. Substitution at position 5 on the indoline ring seemed to be more favorable for both HDAC2 inhibition and cytotoxicity, as manifested by the IC50 values of compound 4c vs. 4g (Table 2). These results have also been observed in the series of 3-oxime-2-oxoindoline-based N-hydroxypropenamides (Dung et al., 2015).

Table 1 HDAC2 inhibition and cytotoxicity of compounds 4a–g against some human cancer cell lines. .
Cpd. code R Molecular weight Log Pa HDAC2 inhibition (IC50,b μM) Cytotoxicity (IC50,b μM)/cell linec
SW620 AsPC-1 PC-3
4a H 366.37 2.36 0.308 3.60 18.45 18.89
4b 5-F 384.36 2.51 0.284 3.30 7.28 11.50
4c 5-Cl 400.81 2.91 0.891 3.42 22.35 17.44
4d 5-Br 445.26 3.18 0.460 3.05 6.83 7.30
4e 5-CH3 380.39 2.84 0.358 3.44 7.47 12.25
4f 5-OCH3 396.39 2.23 0.870 4.62 27.25 22.70
4g 7-Cl 400.81 2.91 1.779 38.35 69.51 >100
SAHAd 264.32 1.44 0.265 1.44 7.04 5.30
a Calculated by ChemDraw 9.0 software.
b The concentration (μM) of compounds that produces a 50% reduction in enzyme activity or cell growth, the numbers represent the averaged results from triplicate experiments with deviation of less than 10%.
c Cell lines: SW620, colon cancer; PC3, prostate cancer; AsPC-1, pancreatic cancer.
d SAHA, suberoylanilide hydroxamic acid, a positive control.
Table 2 Binding affinities of compounds 4a–g toward HDAC2. .
Cpd. code R Molecular weight Log Pa Binding Affinity (kcal/mol)
4a H 366.37 2.36 −9.8
4b 5-F 384.36 2.51 −9.9
4c 5-Cl 400.81 2.91 −9.9
4d 5-Br 445.26 3.18 −9.9
4e 5-CH3 380.39 2.84 −9.7
4f 5-OCH3 396.39 2.23 −9.9
4g 7-Cl 400.81 2.91 −9.6
SAHAb 264.32 1.44 −7.4
a Calculated by ChemDraw 9.0 software.
b SAHA: suberoylanilide hydroxamic acid.

When analyzed by Western Blot assay, it was clearly observed that the inhibition of histones (H3 and H4) deacetylation by the compounds was very well correlated with cytotoxicity. For example, the levels of acetyl-histone-H3 and -H4 in total cell lysates in the presence of compound 4g were not visible (Fig. 4). Compound 4g was the least cytotoxic in the series (Table 1).

Effects of the compounds 4a–g on histone acetylation in SW620 cells. Cells were treated with compounds or SAHA at 3 μg/mL for 24 h. Levels of acetyl-histone-H3 and -H4 in total cell lysates were determined by Western immunoblot analysis.
Figure 4
Effects of the compounds 4a–g on histone acetylation in SW620 cells. Cells were treated with compounds or SAHA at 3 μg/mL for 24 h. Levels of acetyl-histone-H3 and -H4 in total cell lysates were determined by Western immunoblot analysis.

To draw some insights into the interaction of these compounds with HDAC, we have implemented docking experiments using the active site of HDAC. Since histone-H3 and histone-H4 deacetylation has been shown to be principally regulated by HDAC2 and HDAC3 (Pelzel et al., 2010), we decided to select the structure of HDAC2 in complex with SAHA as a docking template. The crystal structure of HDAC2 in complex with SAHA (PDB ID: 4LXZ) has been reported recently by Lauffer and co-workers (Lauffer et al., 2013). We executed control docking experiments with SAHA to the crystal structures of HDAC2 using AutoDock Vina program (Trott and Olson, 2010) after SAHA was removed from the complex structure, as described previously (Oanh et al., 2011; Nam et al., 2013). It was found from docking experiments that all the compounds synthesized were located in the active site with binding affinities much higher than that of SAHA, as manifested by stabilization energies ranging from −9.9 to −9.6 kcal/mol, much lower than that of SAHA (−7.4 kcal/mol) (Table 2). It was found from the docking experiments that, a zinc ion (gray sphere) was coordinated by three residues of HDAC2, including Asp181, His183 and Asp269 (Fig. 5). All compounds in series 4a–g were shown to have very similar binding modes and these compounds interacted with the zinc ion in a similar manner as SAHA did. Their orientation and binding affinity appeared to be insignificantly different. For all compounds, it was found that a central benzene ring linking the indoline and N-hydroxypropenamide moieties was tightly stacked between Phe155 and Phe210 residues of the enzymes (Fig. 5) and this stacking interaction could be the key factor attributing to the high binding affinities of the compounds with HDAC2.

Stereo-view presentations of the actual binding poses of SAHA and simulated docking poses of compounds 4a–g to HDAC2. SAHA is represented as a bold stick model with carbon, nitrogen, and oxygen atoms in pink, blue and red, respectively. Compounds 4a–g are presented as a stick model. The most important parts for the enzyme for interaction of these compounds were shown as a stick model with carbon, nitrogen, and oxygen colored as gray, blue and red, respectively. Zn2+ ion is shown as a bright gray sphere.
Figure 5
Stereo-view presentations of the actual binding poses of SAHA and simulated docking poses of compounds 4a–g to HDAC2. SAHA is represented as a bold stick model with carbon, nitrogen, and oxygen atoms in pink, blue and red, respectively. Compounds 4a–g are presented as a stick model. The most important parts for the enzyme for interaction of these compounds were shown as a stick model with carbon, nitrogen, and oxygen colored as gray, blue and red, respectively. Zn2+ ion is shown as a bright gray sphere.

It was found from these docking experiments that the indoline part insignificantly interacted with the enzymes. Subsequently, very little variance in the binding affinities among the compounds with different substituted groups was observed. These values were almost similar between the compounds within the series (4a–g). Thus, these docking results presently could not explain for the 2 to 3-fold difference between the IC50 value of compound 4g and that of other compounds in the HDAC2 enzyme inhibition assay (Table 1). Similarly, it was also not possible to explain a 7-fold difference in the IC50 value of compound 4g in comparison with that of SAHA. To fully explain this arbitrary, a more detailed docking protocol might need to be designed and executed.

4

4 Conclusion

In conclusion we have designed, synthesized and evaluated a series of N-hydroxypropenamides containing 5′(7′)-substituted-2′-oxospiro[1,3]dioxolane-2,3′-indoline system. The compounds in this series displayed potent inhibitory effects against HDAC2 with IC50 values as low as 0.284 μM (compound 4b), almost comparable to those of SAHA (IC50, 0.265 μM), a positive control. In Western blot analysis, these compounds also exhibited noted inhibition toward histone deacetylation and this inhibition was found to correlate well with the cytotoxicity of the compounds in three human cancer cell lines. Docking studies indicated the compounds in this series bound to HDAC2 with high binding affinities (∼−9.8 kcal/mol) compared to SAHA (−7.4 kcal/mol). Thus, the 5′(7′)-substituted-2′-oxospiro[1,3]dioxolane-2,3′-indoline system could be used as replacement for further design of potential HDAC inhibitors.

Acknowledgment

We gratefully acknowledge the principal financial supports from the National Foundation for Science and Technology of Vietnam (NAFOSTED, Grant Number 104.01-2014.55). The biological study was supported by the Medical Research Center program (MRC, Grant Number 2008-0062275) and the docking study was supported by the Global Core Research Center (GCRC, Grant Number NRF-2011-0030001) from the National Research Foundation (NRF) of Korea.

References

  1. , , . Histone acetylation and cancer. Curr. Opin. Genet. Develop.. 1999;9:171.
    [Google Scholar]
  2. , , , , , . Histone deacetylases (HDACs): characterization of the classical HDAC family. Biochem. J.. 2003;370:737.
    [Google Scholar]
  3. Dung, D.T.M., Dung, P.T.P., Oanh, D.T.K., Hai, P.T., Huong, L.T.T., Loi, V.D., Hahn, H., Han, B.W., Kim, J., Han, S.B., Nam, N.H., 2015. Novel 3-substituted-2-oxoindoline-based N-hydroxypropenamides as histone deacetylase inhibitors and antitumor agents. Med. Chem (in press).
  4. , , , , . Histone deacetylase inhibitors in the treatment of cancer: overview and perspectives. Future Med. Chem.. 2012;4:1439.
    [Google Scholar]
  5. , , . Histone deacetylase inhibitors: molecular mechanisms of action and clinical trials as anti-cancer drugs. Am. J. Trans. Res.. 2011;3:166.
    [Google Scholar]
  6. , . Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. Nat. Med.. 2015;21:555.
    [Google Scholar]
  7. , , , , , , , , , , , , , , , , , , , . Histone deacetylase (HDAC) inhibitor kinetic rate constants correlate with cellular histone acetylation but not transcription and cell viability. J. Biol. Chem.. 2013;288:26926.
    [Google Scholar]
  8. , . Cytotoxicity and Cell Growth Assay. In: , ed. Cell Biology Vol vol. 1. (third ed.). Academic Press, USA: Elsevier; . p. :315-324.
    [Google Scholar]
  9. , , , , , , , , , , , . Novel isatin-based hydroxamic acids as histone deacetylase inhibitors and antitumor agents. Eur. J. Med. Chem.. 2013;70:477.
    [Google Scholar]
  10. , , , , , , , , , , . Synthesis, bioevaluation and docking study of 5-substitutedphenyl-1,3,4-thiadiazole-based hydroxamic acids as histone deacetylase inhibitors and antitumor agents. J. Enzyme Inhib. Med. Chem.. 2014;29:611.
    [Google Scholar]
  11. , , , , , , , , , , . Benzothiazole-containing hydroxamic acids as histone deacetylase inhibitors and antitumor agents. Bioorg. Med. Chem. Lett.. 2011;21:7509.
    [Google Scholar]
  12. , , , . Histone H4 deacetylation plays a critical role in early gene silencing during neuronal apoptosis. BMC Neurosci.. 2010;11:62.
    [Google Scholar]
  13. , , . The role of histone deacetylases (HDACs) in human cancer. Mol. Oncol.. 2007;1:19.
    [Google Scholar]
  14. , , . PRODRG: a tool for high-throughput crystallography of protein–ligand complexes. Acta Crystallogr. Sect. D, Biol. Cryst.. 2004;60:1355.
    [Google Scholar]
  15. , , , , , , , , , , . New colorimetric cytotoxicity assay for anticancer drug screening. J. Natl. Cancer Inst.. 1990;82:1107.
    [Google Scholar]
  16. , , . AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem.. 2010;31:455.
    [Google Scholar]
  17. , , , , , , , , , , . New benzothiazole/thiazole-containing hydroxamic acids as potent histone deacetylase inhibitors and antitumor agents. Med. Chem.. 2013;9:1051.
    [Google Scholar]
  18. , , , , , , , , , , , . Multidrug-resistant phenotype of disease-oriented panels of human tumor cell lines used for anticancer drug screening. Cancer Res.. 1992;52:3029.
    [Google Scholar]

Fulltext Views
839

PDF downloads
303
View/Download PDF
Download Citations
BibTeX
RIS
Show Sections

Suggested read for related articles: