Synthesis and biological evaluation of C-4 substituted phenoxazine-bearing hydroxamic acids with potent class II histone deacetylase inhibitory activities

Abstract

Class II histone deacetylases (HDACs) are considered as potential targets to treat Alzheimer’s disease (AD). Previously, C-3 substituted phenothiazine-containing compounds with class II HDAC-inhibiting activities was found to promote neurite outgrowth. This study replaced phenothiazine moiety with phenoxazine that contains many C-3 and C-4 substituents. Some resulting compounds bearing the C-4 substituent on a phenoxazine ring displayed potent class II HDAC inhibitory activities. Structure-activity relationship (SAR) of these compounds that inhibited HDAC isoenzymes was disclosed. Molecular modelling analysis demonstrates that the potent activities of C-4 substituted compounds probably arise from π-π stacked interactions between these compounds and class IIa HDAC enzymes. One of these, compound 7d exhibited the most potent class II HDAC inhibition (IC₅₀= 3–870 nM). Notably, it protected neuron cells from H₂O₂-induced neuron damage at sub-μM concentrations, but with no significant cytotoxicity. These findings show that compound 7d is a lead compound for further development of anti-neurodegenerative agents.

Keywords:

• Histone deacetylase (HDAC)
• phenoxazine
• structure-activity relationship (SAR)
• neuron cells

Introduction

Due to the rapid increase in the elderly population globally, age-related neurodegenerative diseases such as Alzheimer’s disease (AD), and Parkinson’s disease (PD) are attracting increasing attention¹. AD accounts for 60% to 80% of cases of dementia in aged patients². The global prevalence of AD, which is gradually increasing, is estimated to reach 152.8 million in 2050. In 2021, AD was the fourth most common cause of death for elderly people in the US. Accordingly, AD imposes a heavy burden on health and welfare systems and economic development. AD patients typically have symptoms from mild cognitive impairment in the early stages to severe memory impairment in the late stages^1,3. To date, only three classes of FDA-approved drugs treat AD. They include acetylcholine esterase (AChE) inhibitors – rivastigmine, galantamine, and donepezil, N-methyl-D-aspartate (NMDA) receptor antagonist – memantine, and amyloid beta (Aβ)-targeting antibody – aducanumab^4,5. These five drugs only alleviate some symptoms of AD, but without slowing its progression. The need for an effective therapeutic treatment for AD remains unmet.

Histone deacetylases (HDACs) that are recognised as epigenetic enzymes can regulate gene expression, but without modifying the gene sequence. HDACs catalyse the removal of the acetyl group from lysine residue on histone, forming the condensed nucleosome bead structure that inhibits gene expression^6,7. HDACs also catalyse the acetylation of some nonhistone proteins such as tubulin, HSP90, and P53^8–10. These established findings show that HDACs are associated with a wide spectrum of cellular events. HDACs, which contain 18 isoform enzymes, are divided into four classes depending on their sequence homology, cofactor, and intracellular distribution. Class I, II, and class IV HDACs have Zn as a cofactor in their catalytic domain, whereas class III HDACs are NAD⁺-dependent. Of these Zn-dependent HDAC isoform enzymes, class IIa HDACs have a weak but measurable deacetylase activity, and class IIb HDACs are able to deacetylate histone and non-histone proteins. Additionally, class II HDACs can be subdivided into class IIa HDACs (4, 5, 7, and 9); and class IIb HDACs (6 and 10)¹¹.

Increasing evidence demonstrates that some of these class II HDAC isoforms are involved in learning, memory formation, and neuroplasticity^9,12,13. Studies reveal that neurodegenerative diseased tissues have greater expression of class II HDAC isozymes than normal counterpart tissues. For instance, the overexpression of HDAC4 is implicated with neurotoxicity and neurodegeneration in murine neuron cells⁷. The levels of HDAC5 in the frontal cortex of AD patients exceed those of normal individuals¹⁴. The overexpression of HDAC7 reduces β-catenin activity, downregulating Wnt/β-catenin signalling that is related to AD progression^15,16. The elevation of HDAC9 is found to inhibit the gene expression of choline acetyltransferase (ChAT), reducing the levels of the neurotransmitter acetylcholine (ACh), which is critical for memory formation and CNS functions. However, the inhibition of HDAC9 promotes ACh synthesis as well as neuron dendrite outgrowth¹⁷. Hyperphosphorylated tau protein causes the formation of intracellular neurofibrillary tangles (NFTs) that are associated with synaptic dysfunction and neuron impairment¹⁰. HDAC6 inhibition enhances tau protein acetylation, suppressing the phosphorylation of tau⁸. In addition to causing tauopathy in AD, HDAC6 inhibition attenuates Aβ aggregation ¹⁸and alleviates cognitive impairment in an in-vivo AD mouse model⁹. HDAC6 levels are significantly elevated in the hippocampus and the cortex of the AD patient's brain¹⁹. Notably, the class IIb HDAC6 inhibitors tubastatin A and ACY1215 demonstrated in-vitro as well as in-vivo anti-AD activity²⁰. Class IIa HDAC inhibitor MC1568 exhibited activity in a neurodegenerative disorder animal model¹². Class II HDAC inhibitor W2 not only inhibits the aggregation of Aβ, but also improves cognition in AD mice²¹. These established findings suggest that class II HDACs are potential targets in the treatment of neurodegenerative diseases, especially AD.

Owing to the role of class II HDACs implicated with AD as mentioned, we sought to develop potent class II HDAC inhibitors against AD. The reported HDAC inhibitors can be structurally divided into three moieties, which include a zinc-binding group (ZBG), a hydrophobic carbon linker, and a capping group also in hydrophobic nature. Previously, we have incorporated various commercially available phenothiazines, which have different C-3 substituents, as the cap group into ZBG hydroxamic acid using a benzyl linker ²² (Scheme 1). Several resulting compounds exhibited more potent class II HDAC-inhibiting activities than did the clinically used HDAC inhibitor SAHA. In addition to exhibiting potent enzyme inhibitory activity, they can protect neuron cells from H₂O₂-induced neuron injury, but with no significant cytotoxicity. Despite their potent class II HDAC inhibition, the investigation of SAR of C-3 substituted phenothiazine compounds is only limited.

Scheme 1. Design of potent class II HDAC inhibitor 7d from investigating structure-activity relationship (SAR) of phenothiazine-based hydroxamates

Phenoxazine is a scaffold commonly found in agents that exhibit anticancer, sedative, antiepileptic, tranquillizing, spasmolytic, antitubercular, anthelminthic and^23,24, in particular, neuroprotective activities²⁵. Phenoxazine exhibits potent antioxidant activity in the range of nM concentrations²⁶. It displays a stronger protective effect against MPP⁺-induced neurotoxicity than does phenothiazine²⁶. Furthermore, the molecular modelling analysis that unsubstituted phenoxazine-containing compound 7a bound to HDAC6 demonstrated a small pocket near the phenoxazine ring of compound 7a as shown in Fig. 1S. We suggest the substituents, such as functional groups capable of hydrogen bond formation (amide, hydroxyimidamide, and nitrile) and hydrophobic moieties added to the C-4 position of the phenoxazine ring, potentially provide the additional interactions to residues F680 and L749 in the small pocket of HDAC6 protein (Figure. 2S). The C-4 substitution may further enhance the enzyme-inhibiting activity, which was supported by C-4 hydroxyimidamide-substituted phenoxazine 7d with greater activity than unsubstituted phenoxazine 7a. In addition, we previously verified compound LH4f, which has C-3 amide on phenothiazine, exhibited potent class II HDAC inhibition. Due to the various properties of phenoxazine as well as the substituent observations of phenoxazine ring as described above, in this investigation, the phenothiazine moiety of C-3 substituted phenothiazine series compounds LH4a-g is replaced with their ring bioisostere phenoxazine that contain various C-3 and C-4 substituents using different hydrogen bond formation groups and hydrophobic aryl moieties (Scheme 1). This effort reveals the structure-activity relationship (SAR) of phenothiazine-derived phenylhydroxamates, which inhibit different HDAC isoform enzymes, thus, yielding potential agents for targeting neurological disorders. Among the resulting compounds, a potent class II HDAC inhibitor 7d was identified to protect neuron cells from H₂O₂-induced neurotoxicity, but with slight cytotoxicity. These findings suggest that this compound can serve as a lead compound for further improvement of the biological activity targeting AD.

Materials and methods

General procedure

NMR spectra (¹H- and ¹³C- NMR) were obtained using the Bruker Fourier 300 and AVIII 500 spectrometer with standard plus programs. The chemical shifts were presented in parts per million (ppm, δ) with TMS as an internal standard. The MS data were measured on a Finnigan Mat TSQ-7000 mass spectrometer (ESIMS and HRESIMS). HPLC was performed using a C18 column (250 mm x 4.6 mm, Waters) and an L-2130 pump (Hitachi, Ibaraki, Japan). Column chromatography was performed on silica gel (70–230 mesh, Merck, Darmstadt, Germany). The TLC analysis was performed on silica gel plates (KG60-F254, Merck). A microplate spectrophotometer Victor 2X (Perkin Elmer Fremont, CA, United States) was used for fluorometric analysis; an Envision 2104 Multilabel Reader (Perkin Elmer, CA, United States) was used for luminescence analysis, and a Sunrise microplate reader (TECAN, Männedorf, Switzerland) was used for absorbance analysis. Unless otherwise mentioned, all chemicals and materials were used as received from commercial suppliers without any purification. Anhydrous dichloromethane was distilled from calcium hydride under N₂.

N-Hydroxy-4-[(10H-phenoxazin-10-yl)methyl]benzamide (7a)

To a solution of compound 6a (100 mg, 0.30 mmol) in MeOH-THF (1: 1, 8 ml) was added the mixture of 50%NH₂OH_(aq) (2 ml) and NaOH (60 mg, 1.50 mmol) at 0 °C. The reaction mixture was warmed to RT and the resulting solution was stirred for 2h. The reaction mixture was diluted with distd-H₂O and neutralised to pH 7–8 at 0 °C to give precipitate. The solid was washed with EtOAc and distd-H₂O to give compound 9²⁷ (89 mg, 89%) as a yellow solid. Mp 213–215 °C; ¹H-NMR (Acetone-d₆, 300 MHz): δ 10.71 (s, 1H), 8.11 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 6.72 (m, 6H), 6.46 (d, J = 7.5 Hz, 2H), 4.96 (s, 2H). ¹³C-NMR (Acetone-d₆, 125 MHz): δ 145.7, 141.5, 134.4, 130.9, 128.3, 127.2, 124.8, 122.2, 116.0, 113.3, 48.6; HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₀H₁₇O₃N₂, 333.1234, found 333.1226.

Synthesis of compounds 7b-7c

To a solution of compound 6b (400 mg, 1.12 mmol) in MeOH-THF (1:1, 32 ml) was added the mixture of 50%NH₂OH_(aq) (8 ml) and NaOH (224 mg, 5.60 mmol) at 0 °C. The mixture was warmed to RT and stirred for 6 h. The reaction solution was diluted with distd-H₂O and neutralised to pH 7–8 at 0 °C and the aqueous layer was extracted with EtOAc (50 ml x 3). The organic layer was washed with distd-H₂O and dried over Na₂SO₄, filtered and the solvent was removed in vacuo. The residue was purified by silica gel column chromatography to give compounds 7b (163 mg, 50%), and 7c (31 mg, 10%).

N-Hydroxy-4-[(2-N'-hydroxycarbamimidoyl-10H-phenoxazin-10-yl)methyl]benzamide (7b)

A light yellow solid; Mp 180–183 °C; ¹H-NMR (DMSO-d₆, 300 MHz): δ 11.13 (s, 1H), 9.46 (s, 1H), 9.05 (s, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 8.2 Hz, 1H), 6.72 (m, 4H), 6.39 (d, J = 7.4 Hz, 1H), 5.64 (s, 2H), 4.93 (s, 2H); ¹³C-NMR (DMSO-d₆, 125 MHz): δ 164.3, 150.4, 144.8, 143.9, 139.7, 132.9, 132.8, 131.8, 129.3, 127.5, 126.2, 124.3, 121.5, 118.7, 115.3, 114.8, 112.6, 109.3, 47.2; HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₁H₁₉O₄N₄, 391.1401, found 391.1400.

N-Hydroxy-4-[(2-carbamoyl-10H-phenothiazin-10-yl)methyl]benzamide (7c)

A yellow solid; Mp 187–190 °C; ¹H-NMR (DMSO-d₆, 300 MHz): δ 11.15 (s, 1H), 9.01 (s, 1H), 7.83 (d, J = 8.1 Hz, 1H), 7.76 (1H, s), 7.71 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.23 (dd, J = 1.5, 8.1 Hz, 1H), 7.16 (s, 1H), 6.96 (s, 1H), 6.72 (m, 3H), 6.42 (d, J = 7.7 Hz, 1H), 4.95 (s, 2H). ¹³C-NMR (DMSO-d₆, 125 MHz): δ 167.2, 146.6, 143.7, 139.7, 133.0, 132.6, 131.8, 130.1, 127.5, 126.3, 126.1, 124.5, 121.6, 121.3, 115.4, 114.8, 112.7, 111.4, 47.0; HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₁H₁₈O₄N₃, 376.1292, found 376.1290.

Synthesis of compounds 7d-7e

Following the procedure as described for compounds 7b-7c, the reaction of compound 6c (400 mg, 1.12 mmol), 50%NH₂OH_(aq) (8 ml) and NaOH (224 mg, 5.60 mmol) in MeOH-THF (1:1, 32 ml) gave compounds 7d (106 mg, 32%), and 7e (33 mg, 10%).

N-Hydroxy-4-[(3-N'-hydroxycarbamimidoyl-10H-phenoxazin-10-yl)methyl]benzamide (7d)

A yellow solid. Mp 156–159 °C; ¹H-NMR (CD₃OD, 300 MHz): δ 7.72 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.00 (d, J = 8.3 Hz, 1H), 6.97 (s, 1H), 6.70 (m, 3H), 6.40 (m, 2H). ¹³C-NMR (DMSO-d₆, 125 MHz): δ 164.2, 150.1, 144.0, 143.7, 139.9, 133.8, 132.7, 131.8, 129.0, 128.4, 127.5, 126.5, 126.4, 124.3, 121.8, 121.3, 115.4, 112.6, 112.2, 111.9, 46.8; HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₁H₁₉O₄N₄, 391.1401, found 391.1399.

N-Hydroxy-4-[(3-carbamoyl-10H-phenoxazin-10-yl)methyl]benzamide (7e)

A light yellow solid; Mp 204–207 °C; ¹H-NMR (DMSO-d₆, 300 MHz): δ 11.15 (s, 1H), 9.06 (s, 1H), 7.74 (s, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 7.29 (dd, J = 1.9, 8.3 Hz, 1H), 7.20 (d, J = 1.9 Hz, 1H), 7.15 (s, 1H), 6.74 (m, 3H), 6.51 (s, 1H), 6.49 (s,1H), 4.96 (s, 2H); ¹³C-NMR (DMSO-d₆, 125 MHZ): δ 166.9. 144.1, 143.6, 139.6, 136.0, 132.3, 131.8, 127.5, 127.0, 126.4, 124.4, 124.3, 122.2, 115.4, 114.3, 112.9, 111.8, 46.9; HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₁H₁₈O₄N₃, 376.1292, found 376.1289.

N-Hydroxy-4-{[2–(4-benzoylphenylcarbamoyl)-10H-phenoxazin-10-yl]methyl}nbenzamide (12a)

Following the procedure as described for compound 7a, the reaction of compound 11a (100 mg, 0.18 mmol), 50%NH₂OH_(aq) (2 ml), and NaOH (36 mg, 0.90 mmol) gave compound 12a (20 mg, 20%) as a white solid. Mp 160–163 °C; ¹H-NMR (DMSO-d₆, 300 MHz): δ 11.17 (s, 1H), 10.34 (s, 1H), 9.04 (s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.6 Hz, 2H), 7.70 (m, 5H), 7.63 (d, J = 7.2 Hz, 1H), 7.53 (m, 2H), 7.41 (m, 3H), 6.98 (s, 1H), 6.85 (d, J = 8.2 Hz, 1H), 6.75 (m, 3H), 6.48 (d, J = 7.6 Hz, 1H), 5.00 (s, 2H). ¹³C-NMR (DMSO-d₆, 125 MHz): δ 194.9, 165.2, 147.4, 143.7, 143.4, 139.7, 137.6, 133.4, 132.6, 132.5, 131.8, 131.1, 130.2, 129.5, 128.7, 127.6, 126.4, 124.8, 121.9, 121.8, 119.7, 115.5, 115.0, 112.9, 111.8, 30.8. HR-ESI-MS m/z: [M + H]⁺ calcd for C₃₄H₂₆O₅N₃, 556.1867, found 556.1858.

N-Hydroxy-4-[(2-phenylcarbamoylphenoxazin-10-yl)methyl]benzamide (12b)

Following the procedure as described for compound 7a, the reaction of compound 11b (100 mg, 0.22 mmol), 50%NH₂OH_(aq) (2 ml), and NaOH (44 mg, 1.10 mmol) gave compound 12b (41 mg, 41%) as a white solid. Mp 215–218 °C; ¹H-NMR (Acetone-d₆, 300 MHz): δ 10.72 (s, 1H), 9.29 (s, 1H), 8.13 (s, 1H), 7.82 (d, J = 7.4 Hz, 2H), 7.70 (d, J = 7.0 Hz, 2H), 7.48 (d, J = 7.4 Hz, 2H), 7.37 (d, J = 8.0 Hz, 1H), 7.28 (t, J = 7.0 Hz, 2H), 7.10 (s, 1H), 7.05 (t, J = 7.3 Hz, 1H), 6.74 (m, 4H), 6.50 (d, J = 7.3 Hz, 1H), 5.05 (s, 2H). ¹³C-NMR (DMSO-d₆): δ 164.9, 147.3, 144.1, 140.0, 139.5, 133.7, 133.0, 132.3, 131.0, 129.0, 127.9, 126.8, 126.6, 125.0, 124.1, 123.8, 122.1, 121.7, 120.9, 115.8, 115.2, 113.2, 112.0, 47.5. HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₇H₂₂O₄N₃, 452.1605, found 452.1600.

N-Hydroxy-4-{[3–(4-benzoylphenyl)carbamoyl-phenoxazin-10-yl]methyl}benzamide (12c)

Following the procedure as described for compound 7a, the reaction of compound 11c (200 mg, 0.36 mmol), 50%NH₂OH_(aq) (4 ml), and NaOH (72 mg, 1.80 mmol) gave compound 12c (101 mg, 50%) as a white solid. Mp 158–161 °C; ¹H-NMR (Acetone-d₆, 300 MHz): δ 10.75 (s, 1H), 9.61 (s, 1H), 8.22 (s, 1H), 8.01 (d, J = 8.1 Hz, 2H), 7.77 (m, 6H), 7.63 (d, J = 7.3 Hz, 1H), 7.56 (d, J = 7.6 Hz, 2H), 7.52 (s, 1H), 7.46 (d, J = 7.6 Hz, 2H), 7.36 (s, 1H), 6,75 (m, 3H), 6.57 (m, 2H), 5.04 (s, 2H). ¹³C-NMR (Acetone-d₆, 125 MHz): δ 195.3, 165.2, 145.5, 145.3, 144.5, 140.8, 139.1, 137.9, 133.3, 133.1, 132.8, 131.9, 130.3, 129.2, 128.4, 127.3, 125.2, 125.1, 123.2, 119.9, 116.3, 115.1, 113.8, 112.6, 48.5; HR-ESI-MS m/z: [M + H]⁺ calcd for C₃₄H₂₆O₅N₃, 556.1867, found 556.1859.

N-Hydroxy-4-[(3-phenylcarbamoylphenoxazin-10-yl)methyl]benzamide (12d)

Following the procedure as described for compound 7a, the reaction of compound 11d (100 mg, 0.22 mmol), 50%NH₂OH_(aq)(2 ml), and NaOH (44 mg, 1.10 mmol) gave compound 12d (86 mg, 86%) as a yellow solid. Mp 216–219 °C; ¹H-NMR (Acetone-d₆, 300 MHz): δ 10.76 (s, 1H), 9.26 (s, 1H), 8.20 (s, 1H), 7.83 (m, 4H), 7.46 (d, J = 7.6 Hz, 2H), 7.43 (d, J = 1.9 Hz, 1H), 7.33 (m, 3H), 7.07 (t, J = 7.4 Hz, 1H), 6.75 (m, 3H), 6.57 (d, J = 8.4 Hz, 2H), 5.04 (s, 2H). ¹³C-NMR (DMSO-d₆, 125 MHz): δ 164.4, 144.4, 144.0, 139.9, 139.7, 136.7, 132.6, 132.3, 129.0, 127.9, 127.7, 126.7, 125.0, 124.7, 123.9, 122.6, 120.7, 115.8, 114.6, 113.4, 112.2, 47.2; HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₇H₂₂O₄N₃, 452.1605, found 452.1608.

N-Hydroxy-4-[(3-cyano-10H-phenoxazin-10-yl)methyl]benzamide (16)

To a solution of compound 15 (86 mg, 0.20 mmol) was added 1.25 N HCl in MeOH (5 ml). The resulting solution was stirred at RT for 2 h. The reaction mixture was diluted with distd-H₂O, neutralised to pH = 7, and extracted with CH₂Cl₂. The organic layer was dried over Na₂SO₄, filtered, and the solvent was removed in vacuo. The residue was purified by silica gel chromatography to give compound 16 (63 mg, 90%) as a white solid. Mp 178–181 °C; ¹H-NMR (DMSO-d₆, 300 MHz): δ 11.20 (s, 1H,), 9.07 (s, 1H), 7.70 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 7.20 (dd, J = 1.8, 8.4 Hz, 1H), 7.10 (d, J = 1.8 Hz, 1H), 6.75 (m, 3H), 6.54 (d, J = 8.4 Hz, 2H), 4.97 (s, 2H); ¹³C-NMR (DMSO-d₆, 125 MHZ): δ 144.3, 143.9, 139.1, 138.1, 131.9, 131.7, 129.9, 129.1, 128.5, 127.7, 126.4, 124.8, 123.1, 119.0, 117.8, 115.7, 113.5, 112.9, 102.6, 47.1; HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₁H₁₆O₃N₃, 358.1186, found 358.1186.

N-Hydroxy-4-[(3-bromo-10H-phenoxazin-10-yl)methyl]benzamide (18)

Following the procedure as described for compound 7a, the reaction of compound 17 (70 mg, 0.17 mmol), 50%NH₂OH_(aq)(0.7 ml), and NaOH (34 mg, 0.85 mmol) gave compound 18 (42 mg, 73%) as a light brown solid. Mp 133–136 °C; ¹H-NMR (DMSO-d₆, 300 MHz): δ 11.15 (s, 1H), 9.00 (s, 1H), 7.71 (d, J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 2.1 Hz, 1H), 6.90 (s, 1H), 6.75 (m, 3H), 6.49 (d, J = 7.6 Hz, 1H), 6.40 (d, J = 9.2 Hz, 1H), 4.96 (s, 2H); ¹³C-NMR (DMSO-d₆, 125 MHz): δ 164.5, 145.4, 144.1, 139.9, 133.2, 133.0, 132.2, 127.9, 127.0, 126.7, 124.9,122.1, 118.2,115.8,114.3,113.1, 112.1, 47.3; HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₀H₁₆O₂N₂Br, 411.0339, found 411.0338.

N-Hydroxy-4-[(3-phenyl-10H-phenoxazin-10-yl)methyl]benzamide (20)

Following the procedure as described for compound 7a, the reaction of compound 19 (130 mg, 0.32 mmol), 50%NH₂OH_(aq)(2 ml) and NaOH (64 mg, 1.50 mmol) gave compounds 20 (40 mg, 31%)as a white solid. Mp 188–191 °C; ¹H-NMR (DMSO-d₆, 300 MHz): δ 11.16 (s, 1H), 9.00 (s, 1H), 7.73 (d, J = 8.3 Hz, 2H), 7.58 (d, J = 8.3 Hz, 2H), 7.39 (m, 4H), 7.28 (t, J = 7.2 Hz, 1H), 7.08 (dd, J = 2.1, 8.3 Hz, 1H), 7.05 (d, J = 8.3 Hz, 1H), 6.74 (m, 4H), 6.56 (d, J = 8.3 Hz, 1H), 6.51 (d, J = 7.2 Hz, 1H), 4.97 (s, 2H); ¹³C-NMR (DMSO-d₆, 125 MHz): δ 144.9, 144.4, 139.3, 133.7, 133.2, 132.9, 129.3, 127.8, 127.4, 126.7, 126.2, 124.6, 122.5, 121.9, 115.7, 113.6, 113.2, 112.9, 47.3; HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₆H₂₁O₃N₂, 409.1547, found 409.1547.

Synthesis of compounds 23a-23b

Following the procedure as described for compound 7a, the reaction of compound 17 (700 mg, 1.96 mmol), 50%NH₂OH_(aq)(13 ml), and NaOH (392 mg, 9.20 mmol) gave compounds 23a (158 mg, 21%) and 23b (196 mg, 27%).

N-Hydroxy-3-[(3-N'-hydroxycarbamimidoyl-10H-phenoxazin-10-yl)methyl]benzamide (23a)

As a white solid; Mp 149–152 °C; ¹H-NMR (DMSO-d₆, 300 MHz): δ 11.12 (s, 1H), 9.47 (s, 1H), 7.61 (m, 1H), 7.42 (s, 1H), 7.40 (s, 1H), 7.06 (dd, J = 2.0, 8.4 Hz, 1H), 6.99 (d, J = 2.0 Hz, 1H), 6.71 (m, 3H), 6.48 (t, J = 8.4 Hz, 1H), 5.65 (s, 2H), 4.93 (s, 2H); ¹³C-NMR (DMSO-d₆, 125 MHz): δ 150.4, 144.4, 144.1, 137.4, 134.1, 133.8, 133.1, 129.3, 129.1, 126.9, 125.8, 125.6, 124.6, 122.0, 121.6, 115.6, 113.0, 112.5, 112.2, 47.4; HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₁H₁₉O₄N₄, 391.1401, found 391.1399.

N-Hydroxy-3-[(3-carbamoyl-10H-phenothiazin-10-yl)methyl]benzamide (23b)

As a white solid; Mp 148–151 °C; ¹H-NMR (DMSO-d₆, 300 MHz): δ 11.22 (s, 1H), 8.99 (s, 1H), 7.61 (m, 1H), 7.42 (s, 1H), 7.40 (s, 1H), 7.06 (dd, J = 2.0, 8.4 Hz, 1H), 6.99 (d, J = 2.0 Hz, 1H), 6.71 (m, 3H), 6.48 (t, J = 8.4 Hz, 1H), 5.65 (s, 2H), 4.93 (s, 2H); ¹³C-NMR (DMSO-d₆, 125 MHz): δ167.1, 164.5, 144.4, 143.9, 137.2, 136.3, 133.8, 132.8, 129.3, 129.2, 127.4, 125.9, 125.5, 124.6, 124.5, 122.4, 115.7, 114.5, 113.2, 112.1, 47.4; HR-ESI-MS m/z: [M + H]⁺ calcd for C₂₁H₁₈O₄N₃, 376.1292, found 376.1290.

HDAC activity assay

The HDAC activity assay was performed as described previously²⁸. Enzymes, inhibitors, and substrates were diluted with HDAC buffer (15 mM Tris-HCl pH 8.1, 0.25 mM EDTA, 250 mM NaCl, 10% v/v glycerol). Briefly, 10 μL diluted HDAC, i.e., HeLa nuclear extract (Enzo), HDAC 4, HDAC6 (BPS), HDAC7 (BPS), HDAC9 (BPS), and 50 μL test compound solution at varying concentrations were added to each well of a 96-well microtiter plate and pre-incubated at 37 °C for 5 min. The enzymatic reaction was started by the addition of 40 μL substrate, which was i.e., Boc-Lys(Ac)-AMC (BPS) for HeLa nuclear extract and HDAC6; Boc-Lys(TFA)-AMC (BPS) for HDAC4; and 50040 (BPS) for HDAC7, −9 in HDAC buffer. After incubation at 37 °C for 30 min, the reaction was terminated by the addition of 100 μL trypsin solution (comprising 10 mg/mL trypsin in 50 mM Tris-HCl at pH 8, 100 mM NaCl, 2 mM SAHA). After incubation at 37 °C for a further 20 min, fluorescence was measured (excitation λ = 355 nm, emission λ = 460 nm). To calculate IC₅₀ values, the fluorescence in wells without the test compound (0.1% DMSO, negative control) was set at 100% enzymatic activity and the fluorescence in wells with 2 mM SAHA (positive control) was set at 0% enzymatic activity. All experiments were carried out in triplicate.

Cell culture

SH-SY5Y cells were kindly provided by Professor Ying-Chen Yang (National Ilan University) and cultured in DMEM/F12 (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated foetal bovine serum and penicillin (100 U/ml)/streptomycin (100 μg/ml) (Invitrogen, Carlsbad, CA, USA) at 37 °C in an atmosphere of 5% CO₂.

Cell viability assay

Cell viability was assessed using the alamar blue (7-hydroxy-10-oxidophenoxazin-10-ium-3-one) assay in six replicates as described previously²⁹. Briefly, 5 × 10³ cells/well were seeded in 96-well flat-bottomed plates; 24 h later, cells were treated with compounds or DMSO as control. After 24 h of incubation, the medium was removed, 100 µL medium containing 10 μL of alamar blue was added, and cells were incubated in the 5% CO₂ incubator at 37 °C for 4 h. Absorbance at 570 nm was determined by Synergy HT microplate reader. The results were averaged over three different independent experiments. The cell viability was also evaluated by trypan blue exclusion test.

Western blot analysis

Western blot analysis was performed as previously described³⁰. Cells were lysed in 1X Laemmli Sample Buffer (60 mM Tris (pH 6.8), 2% SDS, and 10% glycerol). Equal protein amounts were separated by SDS-PAGE. Then the proteins were transferred onto a nitrocellulose membrane. The membranes were blocked with 5% skim milk and the indicated proteins were then probed with specific primary antibodies, followed by detection with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch) with enhanced chemiluminescence (ECL) substrates (Bio-Rad).

H₂O₂-Induced neurotoxicity assay

H₂O₂-induced neurotoxicity on SH-SY5Y cells was used to evaluate the neuroprotective effect²⁹. Briefly, a density of 2 × 10⁴ SH-SY5Y cells was seeded per well in 96-well plates. After 24 h of incubation, the medium was replaced with serum-free medium containing H₂O₂ at a concentration of 500 μM and treated with different concentrations of compounds. SH-SY5Y cells were also cultured without any treatment as a control group and results were indicated by percentage of control. After 3 h, cell viability was performed by MTT assay. The cells were treated with MTT (5 mg/ml in PBS) for 4 h at 37 °C. The formazan crystals were generated by viable mitochondrial succinate dehydrogenase from MTT. The supernatant was then aspirated off and the formazan crystals were dissolved with DMSO. Absorbance at 570 nm was determined by the Tecan Sunrise microplate reader.

Molecular docking simulation

The molecular docking software LeadIT³¹was used to perform the docking analysis. The crystal structures of HDAC6 (PDB ID: 5EDU) and HDAC7 (PDB ID: 3ZNR) were obtained from the Protein Data Bank³². The ligand binding was driven by a hybrid enthalpy/entropy approach. The active site was determined as 10 Å from the co-crystal ligand. The maximum number of solutions per iteration and fragmentation was set to 300. The other docking parameters were used with the default settings. Figures were produced in 3D using the software Pymol³³.

Chemistry

Phenoxazine compounds 7a-e were synthesised as described in Scheme 2. Coupling of compound 1 with compound 2 in the presence of NaH gave 3³⁴. Cyclisation of compound 3 using N-methyl-2-pyrrolidone (NMP) yielded 4b³⁵. Compounds 4a-b reacted with compound 5 to provide 6a-b, respectively. Compound 6a reacted with NH₂OH to produce 7a. However, under similar reaction conditions, compound 6b produced amide 7c and hydroxyimidamide 7b. The amide group of product 7c was formed by the basic hydrolysis of the nitrile group of compound 6a. The amide of 7c was further reacted with excess NH₂OH to produce hydroxyimidamide of 7b. Compound 1 was coupled with compound 2 using KOH to give 4c³⁵. Compound 4c reacted with compound 5 to provide 6c. Reaction of compound 6c with NH₂OH yielded 7d-e.

Scheme 2. Reagents and conditions: (a) NaH, DMF, 0 °C, 72 h; (b) NMP, toluene, 180 °C; (c) NaH, DMF, RT; (d) i) THF-MeOH, 0 °C; ii) 50%NH₂OH_(aq), NaOH, RT; (e) KOH, DMSO, 110 °C, 25 h.

Compounds 12a-d were synthesised as described in Scheme 3. Acid hydrolysis of phenoxazine 4b with C-3 nitrile and of phenoxazine 4c with C-4 nitrile gave acids 8a-b, respectively. Amide coupling of compounds 8a-b with aniline 9a and benzophenone amine 9b provided 10a-d. Compounds 10a-d reacted with compound 5 to produce 11a-d, respectively. Compounds 11a-d were converted to corresponding 12a-d using NH₂OH.

Scheme 3. Reagents and conditions: (a) 12 N HCl_(aq), AcOH, Δ; (b) EDC, DMAP, DCM, RT; (c) NaH, DMF, RT; (d) i) TH MeOH, 0 °C; ii) 50%NH₂OH_(aq), NaOH, RT.

Since the nitrile group is liable to undergo a reaction with the base, the synthetic route of nitrile-containing phenylhydroxamic acid 16 was modified using a mild condition as described in Scheme 4. Benzoic acid 13 was activated to form benzoyl chloride prior to the reaction with NH₂OTHP to produce 14. Compound 4c reacted with compound 14 to yield 15. THP deprotection of compound 15 generated phenylhydroxamate 16.

Scheme 4. Reagents and conditions: (a) i) (COCl)₂, DMF, CH₂Cl₂, RT; ii) NH₂OTHP, Et₃N, CH₂Cl₂, RT; (b) NaH, DMF, RT. (c)1.25 N HCl-MeOH, RT.

Compounds 18 and 20 were synthesised according to Scheme 5. Bromination of compound 6a using NBS gave 17. Compound 17 reacted with NH₂OH to provide 18. Suzuki coupling of 17 in the presence of Pd(PPh₃)₄ yielded 19. Compound 19 reacted with NH₂OH to generate 20.

Scheme 5. Reagents and conditions: (a) NBS, THF, -20 °C; (b) i) THF-MeOH, 0 °C ; ii) 50%NH₂OH_(aq), NaOH, RT; (c) Pd(PPh₃)₄, K₂CO₃, THF, EtOH, H₂O, N₂, Δ.

m-Phenoxazine phenylhydroxamic acids 23a-b were synthesised as described in Scheme 6. Compound 4c was reacted with compound 21 to produce 22. Compound 22 reacted with NH₂OH to form 23a-b. The Supplementary Material provides details of the synthesis, isolation, and characterisation of intermediates 3, 4b-c, 6a-c, 8a-b, 10a-d, 11a-d, 14–15, 17, 19, and 22, as well as the ¹H- and ¹³C-NMR, and HRMS spectra of compounds 7a-e, 12a-d, 16, 18, 20, and 23a-b. These compounds were determined to be at least 95% pure using HPLC (Supplementary Material).

Scheme 6. Reagents and conditions: (a) NaH, DMF, RT; (b) i) THF-MeOH, 0 °C ; ii) 50%NH₂OH_(aq), NaOH, RT

Results and discussion

HDAC enzyme inhibitory activity

The enzyme-inhibiting activities of compounds 7a-e, 12a-d, 16, 18, 20, and 23a-b against HeLa nuclear HDACs, which include class I HDACs (1, 2, 3), class IIa HDACs (4, 7, 9), and class IIb HDAC6, were evaluated (Table 1). SAHA, which inhibits a broad spectrum of HDAC isoform enzymes, is found to protect neuron cells against oxidative stress-mediated neuron damage³⁶. In addition to its in-vitro neuron protection, SAHA not only can alleviate age-related cognition impairment in mice³⁷, but also improves memory function in APP/PS1-induced AD mice³⁸. Therefore, SAHA was tested as a reference compound for comparison with synthesised compounds. The established results show that the five compounds 7b-e and 16 exhibited greater class II HDAC inhibition than SAHA, but with weaker class I HDACs inhibitory activities. Of these, compound 7d exhibited the highest activity. Notably, it not only inhibited HDACs 4, 7 and 9 at sub-μM levels, but also exhibited anti-HDAC6 activity with an IC₅₀ value as low as 3 nM. Compound 7d exhibited more potent inhibition against class II HDACs than unsubstituted compound 7a, suggesting that the C-4 hydroxyimidamide moiety on phenoxazine contributes greatly to the inhibition of these HDAC isoforms. Compounds 7d-e with the C-4 substituent on the phenoxazine ring exhibited stronger class IIa activities than counterpart compounds 7b-c with the C-3 substituent on the phenoxazine ring, suggesting that the C-4 substitution contributes positively to class IIa HDACs enzyme-inhibiting activities. This suggestion was supported by the observation that compounds 12c-d with C-4 arylamide exhibited more potent activities than corresponding compounds 12a-b with C-3 arylamide. Compound 12d had higher enzyme inhibition against tested HDACs, except HDAC7, than did compound 12c, suggesting that the extension of the aryl group weakens the enzyme inhibitory activity. Next, compounds 16, 18, and 20 were synthesised to investigate the relationship between C-4 substituent on phenoxazine and anti-HDAC effect. C-4 Nitrile-bearing compound 16 had less potent enzyme-inhibiting activities against these three classes of HDAC isoform enzymes than did 7d. Similarly, compound 18, which has the C-4 Br on phenoxazine, exhibited moderate enzyme inhibition for class I, IIa and IIb HDACs. Compound 20, which has the C-4 phenyl ring on phenoxazine, exhibited weaker activity towards most HDAC enzymes than did unsubstituted compound 7a, suggesting that the rigid phenyl ring in C-4 position reduces activity. Finally, meta-phenoxazine phenylhydroxamic acids 23a-b were synthesised. A test of enzyme inhibition indicated that these two compounds exhibited much lower HDAC-inhibiting activities than their para-substituted counterpart compounds 7d-e, suggesting that the para-phenoxazine moiety contribute greatly to the enzyme inhibition.

Table 1. IC₅₀^a values (μM) of enzyme inhibition of compounds 7a-e, 12a-d, 16, 18, 20, and 23a-b against class I, class IIa and class IIb HDACs.

Compound	HeLa nuclear HDACs	Class IIa			Class IIb
		HDAC4	HDAC7	HDAC9	HDAC6
7a	0.54 ± 0.05	4.32 ± 0.11	0.79 ± 0.03	2.20 ± 0.01	0.017 ± 0.003
7b	0.29 ± 0.01	4.47 ± 0.56	1.84 ± 0.32	2.54 ± 0.02	0.006 ± 0.000
7c	0.12 ± 0.01	6.41 ± 0.69	1.79 ± 0.09	3.8 ± 0.22	0.008 ± 0.001
7d	0.29 ± 0.02	0.87 ± 0.03	0.37 ± 0.03	0.58 ± 0.09	0.003 ± 0.000
7e	0.20 ± 0.01	1.41 ± 0.18	0.52 ± 0.01	1.04 ± 0.20	0.008 ± 0.000
12a	0.54 ± 0.00	6.82 ± 0.14	1.51 ± 0.15	>40	0.155 ± 0.011
12b	0.11 ± 0.02	3.66 ± 0.22	2.26 ± 0.01	3.49 ± 0.70	0.046 ± 0.002
12c	1.91 ± 0.76	2.07 ± 0.14	0.46 ± 0.02	15.18 ± 0.50	0.0748 ± 0.002
12d	0.24 ± 0.04	2.94 ± 0.52	1.58 ± 0.30	3.64 ± 0.06	0.0431 ± 0.003
16	0.87 ± 0.02	3.23 ± 0.59	1.22 ± 0.32	4.08 ± 0.20	0.020 ± 0.000
18	0.53 ± 0.01	3.18 ± 0.33	1.75 ± 0.01	1.56 ± 0.19	0.015 ± 0.000
20	5.71 ± 2.41	8.06 ± 0.21	5.12 ± 0.02	6.32 ± 2.06	0.023 ± 0.003
23a	15.00 ± 1.20	>40	21.70 ± 2.57	24.90 ± 5.97	0.797 ± 0.057
23b	>40	>40	23.15 ± 0.12	>40	0.820 ± 0.182
SAHA	0.03 ± 0.00	31.05 ± 1.84	34.63 ± 0.90	47.39 ± 8.33	0.016 ± 0.001

^aData are provided from three independent experiments.

Binding mode analyses of compounds 7b and 7d for HDAC protein

The molecular docking analysis was performed using the docking software LeadIT³¹ to further understand the interactions between compounds and proteins. The most potent compound 7d was docked into the binding site of HDAC7, which serves as a representative of class IIa HDAC isoforms. As shown in Figure 1(A), compound 7d occupies the binding site of HDAC7. Compound 7d contains a hydroxamic acid moiety that functions as the ZBG. Its hydroxamic acid moiety not only chelates with zinc but also forms a hydrogen bond with residues H669 and H670. The aryl linker of compound 7d extends into the tunnel of HDAC7 and generates a π–π T-shaped interaction with residue F679. The phenoxazine of compound 7d served as a cap group to create π–π stacked interactions with surface rim residue F738. Besides that, the C-4 hydroxyimidamide moiety forms three hydrogen bonds with residues N736, F738, and P809. These interactions facilitate compound 7d occupation within the HDAC7 binding site. Additionally, the counterpart compound 7b with the same hydroxyimidamide moiety at C-3 position (Figure 1(B)) is compared with compound 7d to elucidate the key interactions to HDAC7. Both compounds 7b and 7d consist of the same ZBG and linker that form similar interactions to HDAC7. However, the C-3 hydroxyimidamide moiety of compound 7b results in steric clashes and the phenoxazine ring cannot create π-π stacked interactions with surface rim residue F738 (Figure 1(C,D)). The loss of the π-π stacked interactions reduces the potency of HDAC7 inhibition activity of compound 7b. Taken together, these findings support that the C-3 and C-4 hydroxyimidamide on phenoxazine cap causes the difference between the HDAC7 inhibitory activities of compounds 7d and 7b.

Figure 1. Interactions analysis of compounds 7b and 7d in HDAC7 binding site. (A) Docking pose of compounds (A) 7d (blue-green) and (B) 7b (yellow) in HDAC7 (light pink). (C) Superimposed docking poses of compounds 7b and 7d in HDAC7. (D) The surface model of the superimposed docking poses for compounds 7b and 7d. The zinc ions are denoted as grey sphere. Chelating bonds are represented by red dash lines, while green dash lines indicate hydrogen bonds. Figures are created using PyMOL.

In addition to its potent anti-HDAC7 activity, compound 7d also exhibits excellent inhibitory activity towards HDAC6. Therefore, compound 7d was docked into the binding site of HDAC6 (Figure 2(A)). The molecular docking result demonstrates that the ZBG group of compound 7d chelates with zinc and generates two hydrogen bonds with residues H610 and G619. The linker of compound 7d spans the hydrophobic tunnel of HDAC6 and its phenyl ring facilitates π–π stacked interactions with residues F620 and F680 and π–π T-shaped interaction with residue H651. The cap of compound 7d has hydrophobic contacts with HDAC6 such as π–π T-shaped interaction with residue F620 and π-alkyl interactions with residue L749. In addition to the hydrophobic interactions, its C-4 hydroxyimidamide moiety on phenoxazine cap also forms two hydrogen bonds with residue L749. The interactions produced by compound 7d correlate its excellent HDAC6 inhibitory activity.

Figure 2. Interactions analysis of compounds 7b and 7d in HDAC6 binding site. (A) Docking pose of compounds 7d (blue-green) in HDAC6 (light blue). (B) Superimposed docking poses of compounds 7b (yellow) and 7d in HDAC6. The zinc ions are denoted as grey sphere. Chelating bonds are represented by red dash lines, while green dash lines indicate hydrogen bonds. Figures are created using PyMOL.

Compound 7b shows HDAC6 inhibitory activity comparable to compound 7d. The docking pose of compound 7b adopts a similar conformation to that of compound 7d (Figure 2(B)). Compound 7b displays similar polar and hydrophobic interactions compared to compound 7d. Unlike for HDAC7, the C-3 hydroxyimidamide moiety of compound 7b does not cause steric clashes in HDAC6 and creates two hydrogen bonds with residues H651 and F680. Overall, these results demonstrate the C-3 and C-4 hydroxyimidamide of the cap phenoxazine has slight effect on HDAC6 inhibitory activity.

Effects of compound 7d and SAHA on cell viability in human neuroblastoma SH-SY5Y cells

To verify its neuron toxicity, the cytotoxicity of compound 7d at different concentrations towards human neuroblastoma SH-SY5Y cells was evaluated (Figure 3). SH-SY5Y cells were used to mimic nerve cells. The experimental results show that compound 7d, even at concentrations up to 20 µM, had only a slight cytotoxic effect on SH-SY5Y cells. This compound was less cytotoxic than SAHA. In contrast, SAHA exhibited potent cytotoxicity towards SH-SY5Y cells with an IC₅₀ value of 2.42 μM. These findings suggest that compound 7d exhibited low neurotoxicity.

Figure 3. Effect of compound 7d and SAHA on viability of human neuroblastoma SH-SY5Y cells, as determined by alamar blue assay. All data are provided from three experiments, and each comprised six replicates. *p < 0.05, **p < 0.01, ***p < 0.001 vs control. Statistical analysis was performed using one way ANOVA, followed by a Bonferroni test.

Enhancement of histone and α-tubulin acetylation by compound 7d and SAHA

Acetyl-histone and -tubulin are biomarkers associated with class I HDACs and HDAC6 activity in cells, respectively³⁹. Inhibition of these two enzymes increased the levels of acetylation of histone and tubulin protein. The promotion by compound 7d at various concentrations of histone and α-tubulin acetylation in SH-SY5Y cells was evaluated (Figure 4) in the western blotting analysis. Compound 7d induced α-tubulin acetylation in a dose-dependent manner. At the low concentration (0.625 µM) of these tested compounds, the levels of α-tubulin acetylation that were induced by compound 7d exceed those induced by SAHA. Compound 7d, at a concentration as high as 10 µM, only slightly promoted the acetylation of histone H3. These findings are consistent with the data that showed compound 7d not only exhibited higher HDAC6 selectivity than class I HDACs but also exhibited greater HDAC6-inhibiting activity than that of SAHA (Table 1).

Figure 4. Compound 7d and SAHA induce histone H3 and tubulin acetylation in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were incubated with various concentrations of compound 7d and SAHA for 24 h.

Neuroprotection of compounds 7b and 7d against H₂O₂-induced toxicity

The production of excess reactive oxygen species (ROS) is found to be involved in the pathogenesis of various neurodegenerative disorders, including AD, Parkinson’s disease, and amyotrophic lateral sclerosis^40,41. Hydrogen peroxide (H₂O₂), a peroxide molecule of ROS, can cause oxidative stress, which induces cell apoptosis. The HDAC inhibitor Trichostatin A is shown to inhibit the formation of ROS stress granules⁴². Compounds 7b and 7d were investigated for the potential to protect human neuroblastoma SH-SY5Y cells from H₂O₂-induced neuron injury. First, SH-SY5Y cells were treated with various concentrations of H₂O₂ (Fig. 3S). The concentration of 500 μM of H₂O₂, which significantly reduced the cell viability to 30.2%, was used to produce neurotoxicity. Compounds 7b, 7d, and SAHA at various concentrations were tested in H₂O₂-treated SH-SY5Y cells. The results thus obtained demonstrate that compound 7d protected cells against H₂O₂-induced neurotoxicity in a dose-dependent manner (Figure 5). Moreover, compound 7b was found to exhibit neuroprotective activity, whereas its activity was weaker than that of compound 7d. The finding is likely due to the superior class IIa HDAC inhibition of compound 7d compared to that of compound 7b. Notably, compound 7d provided greater neuroprotection than SAHA. This compound at sub-μM concentrations (0.313–0.625 μM) significantly restored cell viability. These findings suggest that compound 7d exhibited protective activity against H₂O₂-induced toxicity.

Figure 5. Protection by compounds 7b, 7d and SAHA against H₂O₂-induced cytotoxicity in human neuroblastoma SH-SY5Y cells as determined by MTT assay. Compounds are tested at various concentrations and cell injury is induced by H₂O₂ (500 μM) in human neuroblastoma SH-SY5Y cells. All data are obtained from three experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. H₂O₂.

Conclusion

In conclusion, we previously developed a series of phenothiazine-based class II HDAC inhibitors LH4a-g with different C-3 substituents. We intend to investigate extensively the SAR of these compounds for targeting neurological disorders. In this study, the phenothiazine of LH4a-g was replaced with its ring bioisostere phenoxazine which contains various C-3 and C-4 substituents. Some resulting compounds that have the C-4 substituents on phenoxazine exhibited much greater class IIa HDAC-inhibiting activities than those that contain C-3 substituents. Comparisons of the molecular docking of these two series of compounds bound to class II HDACs reveal the differences between the binding modes of C-3 and C-4 substituted compounds. Of the potent C-4 substituted series, compound 7d exhibited the highest enzyme inhibitory activity against class II HDACs (4, 5, 6, 7, 9), but with weaker class I HDAC inhibition compared to SAHA. Compound 7d at sub-μM concentrations remarkably protected neuron cells against H₂O₂-induced neurotoxicity; however, it was only slightly toxic for cells. Overall, compound 7d, which exhibited potent class II HDAC inhibitory activities, is suggested to be a lead compound for structure optimisation. Further improvement of the biological activities of compound 7d against AD is being undertaken.

Acknowledgements

We gratefully acknowledge the support from the National Science and Technology Council (MOST111-2320-B-038–042-MY3 and MOST110-2320-B-038–024-MY3) in Taiwan.

Disclosure statement

The authors report no conflicts of interest.

Additional information

Funding

This work was supported by the Ministry of Science and Technology, Taiwan.