Discovery of Indole-Containing Benzamide Derivatives as HDAC1 Inhibitors with In Vitro and In Vivo Antitumor Activities

Abstract

Targeting histone deacetylases (HDACs) has become an important focus in cancer inhibition. The pharmacophore of HDAC inhibitors (HDACis) reported so far is composed of three parts: a zinc-binding group (ZBG), a hydrophobic cavity-binding linker, and a surface-recognition cap interacting with HDAC surface located at the rim of active site cavity. This study aims to discover novel HDAC1 inhibitors with potent antitumor activities through modifying the cap and ZBG based on the structures of two marketed oral HDACis: chidamide and entinostat (MS-275). In this work, a series of benzamide derivatives were designed, synthesized, and evaluated for their antitumor activity. The structures of novel compounds were confirmed by ¹H NMR (nuclear magnetic resonance) and ESI-MS (electrospray ionization mass spectrometry), and all target compounds were tested in both HDAC1 enzymatic inhibitory activity and cellular antiproliferative activity. Our data showed that the potent compound 3j exhibited good HDAC1 enzyme inhibitory activity and high antitumor cell proliferation activity against a selected set of cancer cells (PC-3, HCT-116, HUT-78, Jurkat E6–1, A549, Colo205, and MCF-7 cells) with no observed effects on human normal cells. In particular, compound 3j inhibited HDAC1 over the other tested HDAC isoforms (HDAC2, HDAC6, and HDAC8). Encouraged by this, the safety characteristics, molecular docking, preliminary pharmacokinetic characteristics, and antitumor effect in vivo of compound 3j were further investigated. Our data showed that compound 3j demonstrated acceptable safety profiles and favorable oral pharmacokinetic properties. Moreover, compound 3j could bind well with HDAC1 and showed significant antitumor activity in a PC-3 tumor xenograft model in vivo, though not as potent as positive control entinostat (MS-275). In summary, 3j might have therapeutic potential for the treatment of human cancers.

Keywords

chidamide - benzamide - HDAC1 inhibitors - synthesis - antitumor

Introduction

Epigenetics is a study of a heritable change in gene function that culminates in a phenotypic change without altering DNA sequences.[1] It plays an important role in cellular and molecular regulatory processes.[2] [3] Epigenetic defects can lead to altered gene function and malignant cellular transformation and have been linked to the progression of several diseases, such as cancer.[4] [5] The reversible acetylation of histones, which is controlled by histone acetyltransferases and histone deacetylases (HDACs), is an important epigenetic modification implicated in cell proliferation and has been identified as a valuable target for anticancer drug.[6] [7] HDACs comprise a family of 18 genes, which are grouped into “classical HDACs” (Zn²⁺ for classes I, II, and IV) and sirtuins (NAD⁺ for class III) based on their catalytic mechanisms.[8] While the biological functions of many HDAC subtypes are still being defined, there is compelling evidence that class I HDACs (HDAC1, 2, 3, 8) are overexpressed in human cancers and therefore are viable targets for cancer therapeutics.[9] [10] [11] [12]

Currently, four HDAC inhibitors (HDACis), including vorinostat (SAHA), belinostat, panobinostat, and romidepsin, have been approved by Food and Drug Administration for the treatment of refractory or relapsed cutaneous and peripheral T cell lymphomas, or multiple myeloma ([Fig. 1]).[13] Unlike most “pan-HDACis” having an hydroxamic acid zinc-binding motif, HDACis containing an ortho-aminophenyl benzamide selectively inhibit HDACs 1–3. Specifically, entinostat (MS-275),[14] mocetinostat (MGCD0103), and chidamide (CS055) not only maintain potent enzymatic and cellular inhibition of HDACs activity in vitro but also display remarkable improvements in pharmacokinetic (PK) properties and antitumor efficacy in vivo. Besides, chidamide (4) was the first orally available benzamide HDACi developed and approved in China for peripheral T cell lymphoma treatment.[15]

Fig. 1 Currently approved HDACis for cancer treatment in clinical practice (chidamide was approved in China) and their representative pharmacophore model: cap, linker, and ZBG. HDACis, histone deacetylases inhibitors; ZBG, zinc-binding group.

The chemical structures of most HDACis reported so far can be divided into three functional parts,[16] [17] [18] consisting of a zinc-binding group (ZBG), a hydrophobic cavity-binding linker, and a surface-recognition cap interacting with HDAC surface located at the rim of active site cavity. The structural similarity of the existing class of benzamide HDACis suggested that rational isosteric modification of the surface-recognition domain or ZBG is feasible. The discovery of novel benzamide HDACis with high anticancer potency and good safety profiles is still a major activity of basic and clinical research.[19] [20] [21]

Inspired by our identification of a new HDACi for cancer treatment,[7] [22] cap and ZBG were modified based on the structures of chidamide and entinostat (MS-275) ([Fig. 2]), and a series of new benzamide HDAC1 inhibitors were designed and synthesized. Then, preliminary structure–activity relationship (SAR) analysis, antiproliferative activity, safety, and in vivo efficacy of these potent HDACis were further discussed.

Fig. 2 The structures of entinostat, chidamide, and our design strategy of target compounds.

Results and Discussion

The synthetic routes of target compounds are shown in [Scheme 1]. First, different fragments, such as quinoline, naphthalene, indole, and thiophene, were inserted into the cap region to synthesize compounds 3a–3 g. After screening for HDAC1 inhibition, it was determined that the indole fragment in the cap region had the best activity. Then, compounds 3h–3o were synthesized with different substituents inserting into the 5 position of indole and the o-aminophenyl of ZBG modified. After the HDAC1 enzyme activity test, 3j was found to be the optimal compound. Specifically, different raw material acids 1a–1i and 4-(aminomethyl)benzoic acid were coupled in the presence of O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorophosphate (HBTU) to obtain key intermediates 2a–2i. The key intermediates 2a–2i are further condensed in the presence of HBTU with excess 1,2-phenylenediamine, pyridine-3,4-diamine, or 4-fluorobenzene-1,2-diamine to obtain target compounds 3a–3o, respectively. All target compounds were fully characterized by MS (mass spectrometry) and ¹H nuclear magnetic resonance (NMR) spectra.

Scheme 1 Synthesis of compounds 3a–3o. Reagent and conditions: (a) i. HBTU, TEA, r.t., NaOH (aq), 4 hours, CH₃CN; ii. HCl (aq), pH = 5–6; (b) HBTU, TEA, DMF, r.t., 6 hours. HBTU, O-(benzotriazol-1-yl)-N,N,N′,N′ -tetramethyluronium-hexafluorophosphate.

These target compounds in this study were initially tested for inhibition of recombinant human HDAC1 since our work and that of others have clearly shown that HDAC1 is widely implicated in both transcriptional repression and chromatin remodeling.[22] [23] [24]

First, we synthesized compounds 3a–3 g ([Table 1]) and discussed the effect of different fragments in the cap region on the inhibitory activity of HDAC1. It was found that when the indole fragment was inserted, compound 3 g (HDAC1 IC₅₀ = 0.803 µmol/L) had better enzyme inhibitory activity on HDAC1 than the positive control drug chidamide (HDAC1 IC₅₀ = 1.280 µmol/L). When other fragments like quinoline, naphthalene, and thiazole are connected, the inhibitory activity of HDAC1 is much lower than that of indole fragment derivatives. Next, compounds 3h–3o were synthesized with different substituents inserting into the 5-position of indole and the o-aminophenyl group of ZBG modified ([Table 1]). The HDAC1 inhibitory activity was best when ZBG is 1,2-phenylenediamine, rather than pyridine-3,4-diamine or 4-fluorobenzene-1,2-diamine, with the IC₅₀ value of 3 g < 3h < 3i, 3j < 3k < 3l, 3m < 3n < 3o. Compound 3j with the 5-position substituent of indole being fluorine (HDAC1 IC₅₀ = 0.330 μmol/L) has the best inhibitory activity against HDAC1, which exceeds the positive control drug MS-275 (HDAC1 IC₅₀ = 0.668 µmol/L).

Table 1 Human HDAC1 enzyme inhibitory activities of all target compounds

Compound	R	M	Y	HDAC1 IC50 (μmol/L)
3a		CH	–	9.401
3b		CH	–	11.67
3c		CH	–	12.21
3d		CH	–	18.43
3e		CH	–	5.432
3f		CH	–	4.298
3 g		CH	–	0.803
3h		C	F	4.211
3i		N	–	9.017
3j		CH	–	0.330
3k		C	F	3.647
3l		N	–	10.54
3m		CH	–	0.553
3n		C	F	4.298
3o		N	–	6.50
MS-275				0.668
Chidamide				1.280

Abbreviation: HDAC1, histone deacetylase 1.

Encouraged by its HDAC1 inhibitory profile, the preferred compound 3j was progressed to in vitro antiproliferative activity assay against a panel of human cancer cells (PC-3, HCT-116, HUT-78, Jurkat E6–1, A549, Colo205, and MCF-7) and human fetal lung fibroblast normal cell lines MRC-5 using CCK-8 assay. Data from [Table 2] demonstrated that 3j exhibited broad antiproliferative activities in most tested cancer cell lines with low IC₅₀ values, ranging from 0.0088 μmol/L (HUT-78) to 1.248 μmol/L (A549). Notably, compound 3j showed significant growth inhibition with IC₅₀ values of 0.1914, 0.09903, 0.0088, and 0.128 μmol/L against PC-3, HCT-116, HUT-78, and Jurkat e6–1 cells, which were superior to MS-275 (IC₅₀ values were 0.502, 0.3674, 0.5281, and 0.5614 μmol/L, respectively). Furthermore, the tested compound 3j showed weak inhibitory activity against MRC-5 cell lines (IC₅₀ > 100 μmol/L), which indicated their differential growth inhibitory activities toward human cancer cells with desirable selectivity over human normal cell MRC-5.

Table 2 The antiproliferative activities of compound 3j and entinostat (MS-275)

Compound	IC50 (μmol/L)
	PC-3	HCT-116	Hut-78	Jurkat E6–1	A549	Colo205	MCF-7	MRC-5
3j	0.1914	0.09903	0.0088	0.128	1.248	0.451	1.021	>100
MS-275	0.502	0.3674	0.5281	0.5614	2.278	1.09	2.718	>100

To evaluate the potency and subtype selectivity, compound 3j was further chosen for in vitro assays against part of HDAC enzyme subtypes, including HDAC enzymes of class I (HDAC1, 2, and 8) and class IIb (HDAC6). As the result shown in [Table 3], compound 3j showed good selectivity against HDAC1 over HDAC2 (IC₅₀ values against HDAC2 was 8.4 μmol/L), and was inactive against both HDAC8 (IC₅₀ > 10 μmol/L) and HDAC6 (IC₅₀ > 10 μmol/L). In vitro cardiovascular safety was assessed in a patch-clamp hERG K⁺ channel screen ([Table 3]). Our data showed that compound 3j was inactive in hERG binding assay with a IC₅₀ >30 μmol/L (reducing incidence of QT interval prolongation). As is apparent from the data in [Table 3], compound 3j exhibited low toxicity to ICR mice with maximum tolerance dose value over 3,000 mg/kg by oral administration.

Table 3 IC₅₀ value for the inhibition of HDAC enzyme subtypes, automated patch-clamp assay for hERG activity, and the MTD value in ICR mice

Compound	IC50 (μmol/L)				hERG IC50 (μmol/L)[a]	MTD (mg/kg)
	HDAC1	HDAC2	HDAC6	HDAC8
3j	0.33	8.4	>10	>10	>30	>3,000

Abbreviations: HDAC, histone deacetylase; MTD, maximum tolerance dose.

^a hERG patch clamp screen was performed as described in Dubin et al.[25] IC₅₀ values represent the concentration to inhibit 50% of hERG current. Numbers represent IC₅₀ values generated from 3-point concentration response relationships in duplicate.

To gain insights into the possible interactions of 3j with the active site of HDAC1 (PDB code: 4BKX), the molecular docking was performed. Consistent with the biochemical results, compound 3j could bind well with HDAC1 ([Fig. 3]). The amino group of compound 3j could chelate the Zn²⁺ very well (1.94 Å to the nitrogen atom in aniline). Besides, two amino groups of amides formed two hydrogen-bonding interaction with ASP-99 and GLY-149, respectively. The benzene ring in the middle formed π–π stacking with PHE150 and HIE178. Collectively, the predicted molecular docking analysis further supports the experimental results that compound 3j has tight contacts with HDAC1.

Fig. 3 Predicted binding modes of compound 3j (carbon in pink) with HDAC1 (PDB entry: 4BKX). Hydrogen bonds are depicted as yellow lines and π–π stacking are depicted as cyan lines.

Meanwhile, the PK parameters of compound 3j were measured in male Sprague–Dawley (SD) rats after single intravenous (iv; 5 mg/kg) and oral (po; 25 mg/kg) administration. When SD rats were administrated with 3j (25 mg/kg, po), and a C _max of 1,950.7 ng/mL was obtained at 1.50 hours. The compound was well cleared (CL = 2.34 L/h/kg) in rats, and the elimination half-life was 4.38 hours. Compound 3j was also well distributed (Vz = 14.64 L/kg) and had a moderate oral bioavailability (F = 29.5%) in rats ([Table 4]).

Table 4 Pharmacokinetic parameters of compound 3j

Parameter	25 mg/kg (po)	5 mg/kg (iv)
AUC(0-t) (ng/mL × h)	10,567.0	7,160.2
AUC(0-∞) (ng/mL × h)	10,741.8	7,160.8
MRT(0-t) (h)	4.61	0.96
V Z (L/kg)	14.64	0.93
CL (L/h/kg)	2.34	0.71
t 1/2 (h)	4.38	0.91
T max (h)	1.50
C max (ng/mL)	1,950.7	7,455.3
F (%)	29.5

The antitumor activity of compound 3j in a mouse xenograft model (PC-3, prostate cancer) was further assessed using MS-275 as a positive control. The mice were given daily intragastric (ig) doses of 12.5, 25, and 50 mg/kg of compound 3j for 21 days, then the relative tumor volume was recorded from day 24 to day 45 since the treatment ([Fig. 4]). Our data suggested that the mice were tolerant at a dose of 50 mg/kg (ig) and the ratio of tumor volume (day 45) in treated versus control mice (T/C) was 23.38%. In the same dose group of MS-275, the T/C was 12.97%. The tested mice did not experience significant weight loss (data not shown). It can be seen that compound 3j shows significant antitumor activity in the PC-3 tumor xenograft model in vivo, though not as potent as positive control entinostat (MS-275).

Fig. 4 The RTV curve of each group of animals during the administration period. RTV was measured by calculating the ratio of V _t/V _initial, where V _t is the tumor volume at each measurement, and V _initial is the tumor volume measured at the time of group administration. RTV, relative tumor volume.

Conclusion

In summary, we designed and synthesized a series of new benzamide HDAC1 inhibitors based on the structure of chidamide and MS-275. Our preliminary SAR analysis showed that the introduction of indole fragments into the cap region had a better inhibitory activity on HDAC1 than chidamide. The most promising compound 3j exhibited more potent activities in both HDAC1 enzymatic inhibitory activity and cellular antiproliferative activity in comparation to MS-275. Compound 3j did not possess observed effects on normal human cells and the hERG potassium channel, and selectively inhibited HDAC1 over the other tested HDAC isoforms. Molecular docking suggested good binding of 3j with HDAC1. Meanwhile, compound 3j had good PK properties and significantly inhibited tumor growth in an PC-3 prostate cancer mouse xenograft model study. It can be seen that compound 3j is a safe and effective HDAC1 inhibitor and may be a potential compound for further cancer treatment.

Experimental Section

Instruments and Reagents

All starting materials were obtained from commercial sources and were analytically pure. The ¹H NMR spectra were recorded on Bruker Avance 400 or 600 spectrometers (Bruker Company, Germany) using TMS as an internal standard and DMSO-d ₆ as solvents. The mass spectra were recorded on an Esquire 3000 LC-MS mass spectrometer. The melting point was measured with a WRS-2A microcomputer melting point apparatus (Shanghai Yidian Physical Optical Instrument Co., Ltd.) without the calibration of thermometer. Absorbance was measured using a SpectraMax M5 Microplate Reader (Molecular Devices). Thin-layer chromatography analyses were performed on silica gel plates GF254 (Qingdao Haiyang Chemical, China). Column chromatography separations were performed on silica gel 200–300 mesh.

General Synthetic Procedure of 2a–2i

Acid 1a–1i (34.65 mmol) and HBTU (40.88 mmol) were added into acetonitrile (20 mL) and stirred evenly to form a paste. After 20 minutes, triethylamine (TEA) (87.31 mmol) was added. The reaction solution was cooled, and continued to stir at room temperature (r.t.) for 2 hours. To the reaction solution was added dropwise a solution of p-methylaminobenzoic acid (5.34 g, 35.34 mmol) in aqueous NaOH solution (1.41 g, 35.34 mmol in 20 mL of water). The resulting mixture was stirred at r.t. overnight. The next day, the ice water was cooled, filtered out the insoluble impurities, and the pH value was adjusted to 5–6 with dilute hydrochloric acid to precipitate a solid. The suspension was stirred for 3 hours and filtered to obtain a condensation product, which was washed with water, and filtered to obtain the intermediates 2a–2i.

General Synthetic Procedure of 3a–3o

A mixture of intermediates 2a–2i (7.02 mmol) and HBTU (7.37 mmol) in 10 mL of DMF was stirred uniformly for 15 minutes followed by the addition of TEA (33.34 mmol), and the mixture was stirred at r.t. for 2 hours. Then, benzene-1,2-diamine, 4-fluorobenzene-1,2-diamine or pyridine-3,4-diamine (8.35 mmol) was added. The reaction solution was stirred at r.t. overnight, then slowly dropped into 50 mL of water to precipitate a solid. The suspension was stirred for 3 hours and filtered to obtain the target compounds 3a–3o.

N- (4-((2-aminophenyl)carbamoyl)benzyl)quinoline-6-carboxamide (3a): mp 192.0–192.9°C. ESI-MS (m/z): calcd. for C₂₄H₂₀N₄O₂ [M + H]⁺ 397.1586; found 397.10. ¹H NMR (600 MHz, DMSO-d ₆) δ 9.64 (s, 1H), 9.39 (t, J = 6.0 Hz, 1H), 9.00 (dd, J = 4.2, 1.7 Hz, 1H), 8.59 (d, J = 2.0 Hz, 1H), 8.50 (dd, J = 8.3, 1.8 Hz, 1H), 8.24 (dd, J = 8.8, 2.0 Hz, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.97 (d, J = 8.0 Hz, 2H), 7.63 (dd, J = 8.3, 4.2 Hz, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 7.8 Hz, 1H), 6.98 (td, J = 7.6, 1.5 Hz, 1H), 6.79 (dd, J = 8.0, 1.4 Hz, 1H), 6.60 (td, J = 7.4, 1.4 Hz, 1H), 4.90 (s, 2H), 4.63 (d, J = 5.9 Hz, 2H).

N -(4-((2-aminophenyl)carbamoyl)benzyl)quinoline-3-carboxamide (3b): mp 191.9–196.5°C. ESI-MS (m/z): calcd. for C₂₄H₂₀N₄O₂ [M + H]⁺ 397.1586; found 397.10. ¹H NMR (600 MHz, DMSO-d ₆) δ 9.65 (s, 1H), 9.49 (t, J = 6.0 Hz, 1H), 9.35 (d, J = 2.3 Hz, 1H), 8.91 (d, J = 2.4 Hz, 1H), 8.12 (t, J = 8.8 Hz, 2H), 7.98 (d, J = 7.8 Hz, 2H), 7.89 (ddd, J = 8.3, 6.8, 1.5 Hz, 1H), 7.73–7.70 (m, 1H), 7.52 (d, J = 7.9 Hz, 2H), 7.18 (d, J = 7.8 Hz, 1H), 6.98 (td, J = 7.6, 1.5 Hz, 1H), 6.80–6.78 (m, 1H), 6.61 (t, J = 7.1 Hz, 1H), 4.90 (s, 2H), 4.65 (d, J = 5.9 Hz, 2H).

N -(4-((2-aminophenyl)carbamoyl)benzyl)-2-naphthamide (3c): mp 193.1–195.6°C. ESI-MS (m/z): calcd. for C₂₅H₂₁N₃O₂ [M + H]⁺ 396.1634; found 396.1731. ¹H NMR (400 M Hz, DMSO-d ₆) δ 9.56 (s, 1H), 9.22 (t, J = 6.0 Hz, 1H), 8.52 (s, 1H), 8.02 (m, 6H), 7.62 (m, 2H), 7.49 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 7.6 Hz, 1H), 6.97 (t, J = 7.2 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.60 (t, J = 7.2 Hz, 1H), 4.84 (s, 2H), 4.61 (d, J = 6.0 Hz, 2H).

N -(4-((2-aminophenyl)carbamoyl)benzyl)-1-naphthamide (3d): mp 241.6–243.3°C, ESI-MS (m/z): calcd. for C₂₅H₂₁N₃O₂ [M + H]⁺ 396.1634; found 396.1729. ¹H NMR (400 M Hz, DMSO-d ₆) δ 9.61 (s, 1H), 9.11 (t, J = 6.0 Hz, 1H), 8.22 (t, J = 5.2 Hz, 1H), 8.00 (m, 4H), 7.67 (dd, J = 1.2 Hz, 8.0 Hz, 1H), 7.57 (m, 5H), 7.19 (d, J = 7.2 Hz, 1H), 6.98 (dt, J = 1.2 Hz, 8.0 Hz, 1H), 6.80 (dd, J = 1.2 Hz, 8.0 Hz, 1H), 6.62 (dt, J = 1.2 Hz, 8.0 Hz, 1H), 4.84 (s, 2H), 4.62 (d, J = 6.0 Hz, 2H).

N -(4-((2-aminophenyl)carbamoyl)benzyl)benzofuran-2-carboxamide (3e): mp 159.2–160.4°C. ESI-MS (m/z): calcd. for C₂₃H₁₉N₃O₃ [M + H]⁺ 386.4230; found 386.9851. ¹H NMR (400 M Hz, DMSO-d ₆) δ 9.58 (s, 1H), 9.31 (s, 1H), 7.91 (m, 2H), 7.65 (d, J = 8.0 Hz, 1H), 7.57 (d, J = 2.4 Hz, 1H), 7.45 (m, 4H), 7.34 (t, J = 7.6 Hz, 1H), 7.16 (d, J = 7.6 Hz, 1H), 6.95 (dt, J = 1.2 Hz, 8.0 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.59 (dt, J = 1.2 Hz, 8.0 Hz, 1H), 4.81 (s, 2H), 4.54 (d, J = 7.2 Hz, 2H).

N -(4-((2-aminophenyl)carbamoyl)benzyl)benzo[ b ]thiophene-2-carboxamide (3f): mp 159.3–162.0°C. ESI-MS (m/z): calcd. for C₂₃H₁₉N₃O₂S [M + H]⁺ 402.1198; found 402.1301. ¹H NMR (400 M Hz, DMSO-d ₆) δ 9.65 (s, 1H), 9.44 (t, J = 8.0 Hz, 1H), 8.17 (s, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.97 (d, J = 8.0 Hz, 2H), 7.45 (m, 4H), 7.17 (d, J = 8.0 Hz, 1H), 6.97 (t, J = 8.0 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.60 (t, J = 8.0 Hz, 1H), 4.90 (s, 2H), 4.58 (d, J = 8.0 Hz, 2H).

N -(4-((2-aminophenyl)carbamoyl)benzyl)-1 H -indole-2-carboxamide (3 g): mp 233.7–234.7°C ESI-MS (m/z): calcd. for C₂₃H₂₀N₄O₂ [M + H]⁺ 386.1586; found 385.1761. ¹H NMR (400 M Hz, DMSO-d ₆) δ 11.47 (s, 1H), 9.51 (s, 1H), 8.98 (t, J = 6.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 1H), 7.41 (m, 4H), 7.13 (m, 3H), 6.99 (dt, J = 0.8 Hz, 8.0 Hz, 1H), 6.73 (dd, J = 1.2 Hz, 7.6 Hz, 1H), 6.55 (dt, J = 1.2 Hz, 7.6 Hz, 1H), 4.77 (s, 2H), 4.53 (d, J = 6.4 Hz, 2H).

N -(4-((2-amino-4-fluorophenyl)carbamoyl)benzyl)-1 H -indole-2-carboxamide (3h): mp 224.5–225.5°C. ESI-MS (m/z): calcd. for C₂₃H₁₉FN₄O₂ [M + H]⁺ 403.1492; found 403.1524. ¹H NMR (400 M Hz, DMSO-d ₆) δ 11.47 (s, 1H), 9.45 (s, 1H), 8.98 (t, J = 6.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.0 Hz, 1H), 7.40 (m, 3H), 7.13 (m, 2H), 7.06 (dt, J = 2.4 Hz, 8.8 Hz, 1H), 6.98 (dt, J = 0.8 Hz, 8.0 Hz, 1H), 6.49 (dd, J = 2.8 Hz, 11.2 Hz, 1H), 6.30 (dt, J = 2.8 Hz, 8.8 Hz, 1H), 5.09 (s, 2H), 4.53 (d, J = 6.0 Hz, 2H).

N -(4-((3-aminopyridin-4-yl)carbamoyl)benzyl)-1H-indole-2-carboxamide (3i): mp 210.6–213.6°C. ESI-MS (m/z): calcd. for C₂₂H₁₉N₅O₂ [M + H]⁺ 386.1539; found 386.1643. ¹H NMR (400 M Hz, DMSO-d ₆) δ 11.47 (s, 1H), 9.60 (s, 1H), 8.99 (t, J = 6.0 Hz, 1H), 8.04 (s, 1H), 7.88 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 5.2 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.40 (m, 4H), 7.13 (t, J = 8.0 Hz, 2H), 6.98 (t, J = 7.2 Hz, 1H), 5.05 (s, 2H), 4.53 (d, J = 6.4 Hz, 2H).

N -(4-((2-aminophenyl)carbamoyl)benzyl)-5-fluoro-1 H -indole-2-carboxamide (3j): mp 244.7–246.6°C. ESI-MS (m/z): calcd. for C₂₃H₁₉FN₄O₂ [M + H]⁺ 403.1492; found 403.1586. ¹H NMR (400 M Hz, DMSO-d ₆) δ 11.65 (s, 1H), 9.56 (s, 1H), 9.08 (t, J = 6.0 Hz, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.47 (m, 4H), 7.18 (m, 2H), 7.05 (dd, J = 2.8 Hz, 8.4 Hz, 1H), 6.95 (dt, J = 1.2 Hz, 7.6 Hz, 1H), 6.77 (dd, J = 0.8 Hz, 8.0 Hz, 1H), 6.59 (dt, J = 1.2 Hz, 7.6 Hz, 1H), 4.83 (s, 2H), 4.58 (d, J = 6.0 Hz, 2H).

N -(4-((2-amino-4-fluorophenyl)carbamoyl)benzyl)-5-fluoro-1 H -indole-2-carboxamide (3k): mp > 250°C. ESI-MS (m/z): calcd. for C₂₃H₁₈F₂N₄O₂ [M + H]⁺ 421.1398; found 421.1476. ¹H NMR (400 M Hz, DMSO-d ₆) δ 11.63 (s, 1H), 9.51 (s, 1H), 9.09 (t, J = 5.6 Hz, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.43 (m, 4H), 7.17 (s, 1H), 7.04 (dt, J = 2.4 Hz, 9.6 Hz, 1H), 6.35 (dt, J = 2.0 Hz, 8.4 Hz, 1H), 6.55 (dd, J = 2.8 Hz, 10.8 Hz, 1H), 6.35 (dt, J = 2.8 Hz, 8.4 Hz, 1H), 5.13 (s, 2H), 4.57 (d, J = 6.0 Hz, 2H).

N -(4-((3-aminopyridin-4-yl)carbamoyl)benzyl)-5-fluoro-1 H -indole-2-carboxamide (3l): mp > 250°C. ESI-MS (m/z): calcd. for C₂₂H₁₈FN₅O₂ [M + H]⁺ 404.1445; found 404.1524. ¹H NMR (400 M Hz, DMSO-d ₆) δ 11.65 (s, 1H), 9.65 (s, 1H), 9.10 (t, J = 6.0 Hz, 1H), 8.10 (s, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.79 (d, J = 5.2 Hz, 1H), 7.47 (m, 5H), 7.18 (s, 1H), 7.04 (dt, J = 1.2 Hz, 9.6 Hz, 1H), 5.11 (s, 2H), 4.59 (d, J = 6.0 Hz, 2H).

N -(4-((2-aminophenyl)carbamoyl)benzyl)-5-methoxy-1 H -indole-2-carboxamide (3m): mp 231.3–233.0°C. ESI-MS (m/z): calcd. for C₂₄H₂₂N₄O₃ [M + H]⁺ 415.1692; found 415.1769. ¹H NMR (400 M Hz, DMSO-d ₆) δ 11.38 (s, 1H), 9.56 (s, 1H), 8.99 (t, J = 6.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.10 (m, 2H), 6.97 (dt, J = 1.2 Hz, 7.6 Hz, 1H), 6.85 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 6.78 (dd, J = 1.2 Hz, 7.6 Hz, 1H), 6.60 (dt, J = 1.2 Hz, 7.6 Hz, 1H), 4.83 (s, 2H), 4.58 (d, J = 6.0 Hz, 2H), 3.77 (s, 3H).

N -(4-((2-amino-4-fluorophenyl)carbamoyl)benzyl)-5-methoxy-1 H -indole-2-carboxamide (3n): mp 226.9–227.2°C. ESI-MS (m/z): calcd. for C₂₄H₂₁FN₄O₃ [M + H]⁺ 433.1598; found 433.1680. ¹H NMR (400 M Hz, DMSO-d ₆) δ 11.38 (s, 1H), 9.50 (s, 1H), 8.98 (t, J = 6.0 Hz, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H), 7.35 (s, 1H), 7.13 (m, 3H), 6.84 (dd, J = 2.4 Hz, 8.8 Hz, 1H), 6.55 (dd, J = 3.2 Hz, 11.2 Hz, 1H), 6.35 (td, J = 2.8 Hz, 8.4 Hz, 1H), 5.15 (s, 2H), 4.58 (d, J = 6.0 Hz, 2H), 3.77 (s, 3H).

N -(4-((3-aminopyridin-4-yl)carbamoyl)benzyl)-5-methoxy-1 H -indole-2-carboxamide (3o): mp 229.7–230.6°C. ESI-MS (m/z): calcd. for C₂₃H₂₁N₅O₃ [M + H]⁺ 416.1644; found 416.1706. ¹H NMR (400 M Hz, DMSO-d ₆) δ 11.38 (s, 1H), 9.65 (s, 1H), 8.99 (t, J = 5.6 Hz, 1H), 8.11 (s, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.80 (d, J = 5.2 Hz, 1H), 7.49 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 5.2 Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H), 7.10 (s, 2H), 6.85 (dd, J = 2.0 Hz, 6.8 Hz, 1H), 5.11 (s, 2H), 4.59 (d, J = 6.0 Hz, 2H), 3.77 (s, 3H).

In Vitro Testing of HDAC1, HDAC2, HDAC6, and HDAC8

A 500 μmol/L of the tested compound was diluted with fourfold serial dilution to 0 μmol/L with DMSO. Then, to the optiPlate-96 black microplates (Perkinelmer, Cat: 6005270) was added 11.5 μL assay buffer (BPS, Cat: 50031) and 2.5 μL of compounds with different diluted concentrations at r.t. For negative control and blank control wells, 11.5 μL assay buffer were added, respectively, and for positive control wells, 11.5 μL assay buffer and 2.5 μL MS-275 were added. All determinations were performed in duplicate. A 6 μL of human recombinant HDAC1 (BPS, Cat: 50051), human recombinant HDAC1 (BPS, Cat: 50062), or human recombinant HDAC6 (BPS, Cat: 50006) was added respectively to microplates and incubated for 10 minutes. Then, 2.5 μL BSA was added to each reaction well, followed by the addition of 2.5 μL HDAC substrate (BPS, Cat: 50037). The mixture was incubated at 37°C for 45 minutes, then added the stop solution (HDAC developer [BPS, Cat: 50030], 25 µL). After incubation at 25°C for 15 minutes, the fluorescence was measured on SpectraMax M5 (Molecular Devices) with an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

A commercial HDAC8 fluorimetric drug discovery kit [Fluor de Lys(R)-HDAC8, BMLKI178] was used to perform HDAC8 activity assay according to the manufacturer's instructions. The enzyme was incubated for 90 minutes at 37°C, with a substrate concentration of 50 µmol/L and increasing concentrations of inhibitors. Measurement was performed as described for HDAC1/2/6.

The inhibition rates were calculated by the slope of the linear reaction process of each well as follows: %Inhibition ≡ (Mean (Max) − Sample Signal)/(Mean (Max) − Mean (Min)) × 100. The IC₅₀ value of each compound's enzyme inhibitory activity was obtained by fitting the dose–response curve through GraphPad Prism 5 software.

CCK-8 Assay for Cell Proliferative Assessment

Cells were cultured in medium to log phase and then digested, centrifuged, and resuspended in fresh medium. Then, they were seeded into 96-well plates (Corning Costar, Cat: 3599, 104 cells/well) and incubated overnight at 37°C under 5% CO₂ atmosphere to make them reattach. Subsequently, cells were treated with the target compounds at decreasing concentrations (10 concentrations from a concentration of 100 μmol/L with threefold serial dilution) for another 24 hours. The absorbance values at 450 nm (optical density: 450) were recorded by SpectraMax M5 Microplate Reader (Molecular Devices). There are two repeated wells for each concentration. The IC₅₀ value was defined as a concentration that caused 50% loss of cell viability, which was calculated by Origin 7.5 software.

Molecular Modeling Study

The docking study was conducted in Glide module of Schrodinger Maestro. PDB entry 4BKX was downloaded from Protein Data Bank for molecular docking. In Protein Preparation Wizard, the HDAC1 protein was prepared by the process of removing waters and adding hydrogens. The most important of resulting structure was refined in the force field of OPLS3 with the hydrogen only. Then, the Receptor Grid Preparation was defined according to the position of the zinc ion. Compound 3j generated all possible combinations at the target pH 7.0 ± 2.0 in the force field of OPLS3 in LigPrep module. Ligand docking parameter used was set default. Molecular docking result was generated using PyMol (http://pymol.sourceforge.net/).

hERG Inhibitory Activity Assay

The whole-cell recordings were conducted using Automated Qpatch (Sophion).[26] Cells were voltage clamped at a holding potential of −80 mV. The hERG current was activated by depolarizing at + 20 mV for 5 seconds, after which the voltage was taken back to −50 mV for 5 seconds to remove the inactivation and observe the deactivating hERG tail current. The voltage stimulation was applied per 15 seconds. Cells were treated with different concentrations of compound solutions (from low to high concentration) for 2 minutes and 10 μmol/L cisapride was applied at the end of perfusion of compound solution. Each concentration was tested on at least three cells.

Acute Toxicity Assay

ICR mice (18–22 g) were purchased from SLRC Laboratory Animal Inc., Shanghai, China. A total of 10 mice (five males, five females) were used in this study. Compound 3j was administered orally to mice at a single dose of 3,000 mg/kg, respectively. Mouse death was monitored daily up to 14 days of treatment. All animals were euthanized and necropsied for gross lesion examination for possible damage to the heart, liver, and kidneys.

Pharmacokinetics Study

Compound 3j was dissolved and vortexed in 5% DMSO, 10% Tween 80, and 75% physiological saline for a concentration of 0.2 mg/mL and 1 mg/mL. The male SD rats were housed in a room with controlled temperature and humidity and allowed free access to food and water. The rats were split into iv injection group (5 mg/kg) and ig administration group (25 mg/kg) before starting treatment. Rat blood was collected at indicated time points up to 12 hours, and plasma samples were analyzed using an Agilent 1200 HPLC system coupled with Agilent 6410B triple quadruple mass spectrometer. A solution of 0.05 N HCl in saline was used as the vehicle for both iv and po dosing.

In Vivo Antitumor Activity Assay

BALB/C nude mice were subcutaneously inoculated with PC-3 tumor cells to establish a PC-3 nude mouse xenograft model. After 24 days, the average tumor volume was ∼207 mm³. Tumor-bearing mice were randomly divided into five groups: vehicle control, MS-275, and 3j (12.5, 25, 50 mg/kg) groups. All groups were administered drugs (ig, 10 mL/kg) once every 2 days for 21 successive days. Mice in vehicle control were given blank solvent (3% ethanol, 10% HCl [pH 0.75], 20% solutol HS 15, 40% PEG400, and acetate buffer [pH 4.0]). Animal state was observed and recorded regularly every day; if the animal dies, the animal is grossly dissected, and the internal organs are visually observed for any abnormality and recorded. After 45 days, the animals were sacrificed, and the tumor mass was removed, weighed, and photographed. Tumor weight and tumor inhibition rate were calculated. The tumor volume (V) was calculated as follows: V = [length (mm) × width² (mm²)]/2.

Conflict of Interest

The authors declare no conflicts of interests.

Ethics Statement

The present study was approved by the animal ethics committee and abides by the relevant agreements of China State Institute of Pharmaceutical Industry, Shanghai, People's Republic of China.

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Publikationsverlauf

Eingereicht: 21. Dezember 2021

Angenommen: 21. April 2022

Artikel online veröffentlicht:
30. Juni 2022