Development of hydroxamate-based histone deacetylase inhibitors of bis-substituted aromatic amides with antitumor activities

Previously, we designed and synthesized a series of bis-substituted aromatic amide-based histone deacetylase (HDAC) inhibitors.

Abstract

Previously, we designed and synthesized a series of bis-substituted aromatic amide-based histone deacetylase (HDAC) inhibitors. In this study, we report the replacement of a bromine atom by different amides on the phenyl ring of the CAP region. Representative compounds 9d and 10k exhibited low nanomolar IC₅₀ values against HDAC1, which were ten times lower than that of the positive control SAHA. The IC₅₀ of 9d against the human A549 cancer cell line was 2.13 μM. Furthermore, 9d increased the acetylation of histones H3 and H4 in a dose-dependent manner. Moreover, 9d significantly arrested A549 cells at the G2/M phase and induced A549 cell apoptosis. Finally, molecular docking investigation rationalized the high potency of compound 9d.

1. Introduction

Epigenetic modification plays an important role in the treatment of cancers.1,2 Protein lysine acetylation, regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), can lead to changes in gene expression without modifying the gene sequence.3,4 HATs neutralize the positive charge of lysine residues by adding an acetyl group to the N-terminal tail of histone, which results in an open and transcriptionally active chromatin structure. In contrast, HDACs remove the acetyl group, leading to a closed chromatin structure and transcriptional repression.5,6 The overexpression of HDACs has been found in various cancers, which is associated with the down-regulation expression of onco-suppressor genes.7 Meanwhile, some non-histone proteins such as tubulin, HSP90, p53 and ER are also served as substrates of HDACs.8–10 Over the past decades, HDACs have been validated as practical targets for cancer treatments.11

To date, 18 HDAC isoforms have been found in humans and can be divided into classes I, II, III and IV. Classes I (1, 2, 3, and 8), II (4, 5, 6, 7, 9, and 10) and IV (11) HDACs are Zn²⁺-dependent enzymes, while class III HDACs (SIRT1–7) require NAD+ for their activities.12 Recent development of HDAC inhibitors (HDACis) mainly focuses on Zn²⁺-dependent HDACs. So far, five HDACis (SAHA,13 FK-228,14 PXD-101,15 LBH-589 (ref. 16) and chidamide17) (Fig. 1) have been approved by the US FDA for the treatment of cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL) or multiple myeloma (MM). A number of other inhibitors, such as 6 and 7, are also undergoing different stages of clinical investigations.11,17,18

Fig. 1. HDAC inhibitors approved or under clinical development.

The structures of most common HDACis usually comprise three parts: a zinc binding group (ZBG), a linker and a surface recognition group (CAP group).19,20 Recent structural modifications of HDACis mainly focused on the CAP and linker moieties to develop novel molecules with optimized activities and selectivities.21,22 In our previous study, we designed and synthesized a series of bis-substituted aromatic amide-based hydroxamic acid HDACis,23 among which, compound 8a displayed mild inhibition against nuclear extract HDAC. In this report, we aimed to seek more bis-substituted aromatic amide-based HDACis with better affinity and physicochemical properties based on the scaffold of 8a as shown in Fig. 2. According to the preliminary results of docking studies of 8a with HDAC1, we found that the meta-bromine in the A ring of the CAP region was in the cavity of the rim of the HDAC enzyme, so we replaced the bromine with different amides which could better occupy the cavity and a panel of new HDACis were synthesized and explored. Biological evaluations were also subsequently performed to examine their mechanism of action and in vitro antiproliferative activities in multiple cancer cell lines.

Fig. 2. Design strategy and general structures of the target compounds.

2. Results and discussion

2.1. Chemistry

The synthetic routes for compounds 9a–b, 9d–e and 10b–k are shown in Scheme 1. Compound 12 was coupled with different substituted amines in the presence of EDC and HOBt to generate 13a–k. Intermediates 14a–n were constructed from substituted anilines with 13a–k through reductive amination and were further condensed with pimelic acid anhydride in 1,4-dioxane to yield acids 15a–n. Subsequent esterification of 15a–n in methanol with catalytic amounts of thionyl chloride generated the corresponding esters 16a–n, which were finally stirred with freshly prepared hydroxylamine in methanol to afford hydroxamic acids 9a–b, 9d–e and 10b–k.

Scheme 1. Reagents and conditions: (a) RNH₂, EDC, HOBt, DMF; (b) ArNH₂, EtOH, 0 °C then to rt; (c) NaBH₄, 0 °C then to rt; (d) anhydride, 1,4-dioxane, reflux; (e) MeOH, cat. SOCl₂, reflux; (f) NH₂OH·HCl, KOH, MeOH.

The synthetic routes to compounds 9c, 10a and 10l are illustrated in Scheme 2. The important intermediates 20a and b were first synthesized via a similar procedure to that of 15a–n shown in Scheme 1. Compounds 21a and b were then obtained after reduction of the nitro group of 20 and were further reacted with different substituted carboxylic acids to furnish 22a–c containing the extra amide functionality. Finally, treatment of esters 22a–c with hydroxylamine gave the desired hydroxamic acid derivatives 9c, 10a and 10l.

Scheme 2. Reagents and conditions: (a) O-anisidine, EtOH, 0 °C then to rt; (b) NaBH₄, 0 °C then to rt; (c) anhydride, 1,4-dioxane, reflux; (d) MeOH, cat. SOCl₂, reflux; (e) Zn, CH₃COOH, EtOH/H₂O; (f) RCOOH, EDC, HOBt, DMF; (g) NH₂OH·HCl, KOH, MeOH.

2.2. HDAC inhibition assay

Initially, the effect of the amide group at the meta-position of the A ring was explored; compounds 9a–c and 10a–j with an arylalkyl amino carbonyl group at the meta-position of the A ring and a methoxy group at the ortho-position of the B ring were assayed against nuclear extract HDAC (mainly contains HDAC1 and 2) with 1 (SAHA) as a reference drug. The assay results were shown as inhibition rates as listed in Table 1. Compounds with mono-substitution and di-substitution on the nitrogen of amide (9a and 9b) showed mild inhibitory activities. Excitingly, when a phenyl group was inserted to the terminal alkyl, most 10-series compounds (10a–c, 10g–h and 10j) exhibited better activities than the 9-series compounds. In addition, the inversion of the amide bond seemed to show little influence on the HDAC inhibition (9avs.9c and 10avs.10b). Thus, the amide bond was retained in subsequent structural modifications. Analysis of compounds (10b–f) indicated that the carbon chain length between nitrogen and the benzene ring greatly affected the inhibitory potency. Compounds 10b and 10c containing one or two methylene bridges showed better activities than their analogues with a longer linker (10d–f). Furthermore, introduction of electron-withdrawing groups to the terminal phenyl ring was beneficial for maintaining the inhibitory activities (10g & 10hvs.10i). Particularly for the fluorine atom, compound 10j also displayed much better activity than 10d. Meanwhile, in consideration of the physicochemical properties of target compounds, the log P values of these derivatives were predicted through the ALOGPS 2.1 program (; http://146.107.217.178/lab/alogps/start.html). As shown in Table 1, reports suggested that compounds with log P between 1 and 3 could have good oral bioavailability.24 The results indicated that compound 9a may have the optimal absorption properties in this test.

Table 1. Inhibitory activity of compounds 9a–c and 10a–j on the HDAC nuclear extract at 0.5 μM.

Compd.	R1	Inhibition%	log P
8a	Br-	21%	4.05
9a	CH3CH2NHCO-	20%	2.75
9b	(CH3CH2)2NCO-	20%	3.44
9c	CH3CH2CONH-	22%	3.18
10a	PhCH2CONH-	37%	3.98
10b	PhCH2NHCO-	38%	3.74
10c	Ph(CH2)2NHCO-	38%	4.21
10d	Ph(CH2)4NHCO-	15%	5.06
10e	Ph(CH2)5NHCO-	16%	5.60
10f	Ph(CH2)6NHCO-	4%	6.07
10g	4-F–Ph(CH2)2NHCO-	39%	4.12
10h	4-Cl–Ph(CH2)2NHCO-	36%	4.71
10i	4-OCH3Ph(CH2)2NHCO-	27%	4.47
10j	4-F–Ph(CH2)4NHCO-	34%	5.12
SAHA	—	46%	1.88

According to the results of our previous study, compounds with electron-donating groups at the para-position of the B ring appeared to be optimal for increasing the activity.23 Therefore, in light of the physicochemical properties and the aforementioned activity results derived from the substituent exploration of the A ring, two compounds (9d and 10k) were further synthesized and evaluated, and a dramatic increase of potency was observed for both compounds, which could further confirm the conclusion that compounds with para-substituents are superior to their corresponding ortho-position derivatives. However, when the methoxyl of 9d was replaced with hydroxyl, the resulting compound 9e exhibited a decrease in inhibitory activity, which testified the importance of the methoxy group for inhibitors. Meanwhile, compound 10l, the “inverse amide” version of amide 10k, exhibited slightly decreased activity (Table 2).

Table 2. Inhibitory activity of compounds 9d–e and 10k on the HDAC nuclear extract at 0.5 μM.

Compd.	R1	R2	Inhibition%	log P
9d	CH3CH2 NHCO-	4′-OCH3	51%	2.85
9e	CH3CH2 NHCO-	4′-OH	21%	2.53
10k	4-F–Ph(CH2)2NHCO-	4′-OCH3	53%	4.16
10l	4-F–Ph(CH2)2CONH-	4′-OCH3	49%	4.52
SAHA	—	—	46%	1.88

Next, 9d and 10k with the best nuclear extract HDAC inhibitory rates were further evaluated against HDAC1. Results (Table 3) revealed that the two compounds showed significant HDAC1 inhibitory activities, being >10 fold potent than SAHA.

Table 3. Inhibition activity of tested compounds on HDAC1.

Compd.	IC50 (nM)
9d	2.2 ± 0.13
10k	2.3 ± 0.16
SAHA	25.0 ± 1.23

The in vitro anti-proliferative activities of 9d and 10k against three human tumor cell lines MDA-MB-231, MCF-7 and A549 were then evaluated using the SRB assay. As shown in Table 4, it was found that A549 cells were more sensitive to compounds 9d and 10k, as compared to the reference drug SAHA. Compound 9d showed the strongest growth inhibition toward A549 cells with an IC₅₀ value of 2.13 μM close to that (2.93 μM) of SAHA.

Table 4. Anti-proliferative activities of representative compounds against tumor cells ^a .

Compd.	IC50 (μM)
	MDA-MB-231	MCF-7	A549
9d	6.87 ± 0.84	6.96 ± 0.84	2.13 ± 0.33
10k	7.24 ± 0.87	7.17 ± 0.86	4.05 ± 0.61
SAHA	4.66 ± 0.67	6.17 ± 0.56	2.93 ± 0.47

^aThe inhibitory effects of individual compounds on the proliferation of cancer cell lines were determined by the SRB assay. The data are expressed as the mean ± SD of three independent experiments.

Moreover, to evaluate whether target compounds 9d and 10k show selectivity between cancer and non-cancer cells, their in vitro cytotoxicity was further tested against three normal cell lines: human breast epithelial cells (MCF-10A and MCF-10F) and human lung epithelial cells (Beas-2B). As shown in Table 5, the results indicated that both compounds displayed no obvious toxicities against the three human normal cell lines, and compound 9d behaved even better than SAHA.

Table 5. Anti-proliferative activities of representative compounds against normal cells.

Compd.	IC50 (μM)
	MCF-10A	MCF-10F	Beas-2B
9d	>100	>100	>100
10k	>50	>50	>20
SAHA	>50	>50	>50

2.3. Colony formation assay

Since compound 9d showed the best antiproliferative activity against the A549 cancer cell line among the investigated ones, we further evaluated its inhibitory behavior via a colony formation assay, with SAHA as the positive control. As depicted in Fig. 3, compound 9d resulted in significant inhibition of the colony formation more potently than SAHA. The results suggested that 9d could at least partly inhibit the growth and development of A549 cells.

Fig. 3. Compound 9d inhibited the colony formation of A549 cells. After treatment with different concentrations of 9d and SAHA in 6-well plates for 7 days, the cells were fixed with methanol and stained with 1% crystal violet and the number of cell clones was counted. Experiments were carried out in triplicate and repeated three times. Representative photographs from three independent experiments are shown. The data are expressed as means ± SD of three separate experiments. p values are for one-way analysis of variance (ANOVA). **p < 0.01, ***p < 0.001 vs. control.

2.4. Anti-migration activity

Investigating the effect of novel HDAC inhibitors against cancer cell migration is valuable for the treatment of metastatic or advanced cancers. Based on the previous results that the bis-substituted aromatic amides showed potent activities against tumor cell migration,23 compound 9d was then evaluated for its anti-metastatic effects via a wound-healing migration assay. As depicted in Fig. 4, 9d could decrease the migration of A549 cells in a dose-dependent manner, with higher potency than SAHA.

Fig. 4. Compound 9d inhibited the wound healing of A549 cells. After a wound was made, the cells were treated with different concentrations of 9d and SAHA for 24 h. Representative photographs from three independent experiments are shown. The data are expressed as means ± SD of three separate experiments. p values are for one-way analysis of variance (ANOVA). *p < 0.05, **p < 0.01 vs. control.

2.5. Western blot analysis

Considering its remarkable HDAC1 inhibitory potency, compound 9d was subjected to western blot analysis by an immunoblotting assay. A549 cells were incubated with SAHA and compound 9d (1.0, 2.5 and 5.0 μM). As shown in Fig. 5, 9d could dramatically increase the levels of both acetylated histones H3 and H4 in a dose-dependent manner, which was consistent with its HDAC1 inhibition activity. Compound 9d displayed more potent activity to induce histone acetylation than SAHA.

Fig. 5. Western blot analysis of 9d on the effects of acetylated histone levels in A549 cancer cells. A549 cells were treated with different concentrations of 9d or SAHA for 24 h. Cell lysates were lysed, collected and immunoblotted using western blotting. The relative levels of each protein compared to control β-actin were determined by densimetric scanning. Representative photographs from three independent experiments are shown. The data are expressed as means ± SD of three separate experiments. p values are for one-way analysis of variance (ANOVA). **p < 0.01, ***p < 0.001 vs. control.

2.6. Cell cycle analysis

Cell cycle analysis of the most potent HDACi 9d was carried out in A549 cancer cells. As shown in Fig. 6, in comparison to the control group, compound 9d arrested A549 cells mainly in the G2/M phase (40.3% at 5 μM for 9d), which was obviously more potent than SAHA (24.97% at 5 μM). We also evaluated the effect of 9d treatment on cell-cycle-regulatory proteins, CDK1 and cyclin B, which were involved in the G2–M transition.25 The results in Fig. 6 displayed that 9d treatment decreased the CDK1 and cyclin B1 protein levels.

Fig. 6. The effects of 9d on cell cycle progression in A549 cells. (A) A549 cells were treated with a vehicle (control), SAHA, or different doses of 9d and stained with PI, followed by flow cytometry analysis. Representative photographs from three independent experiments are shown. (B) Representative histograms. Data are expressed as means ± SD of the percentages of A549 cells from three independent experiments. p values are for one-way analysis of variance (ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. (C) The whole cell lysates were analyzed for the detection of cyclin B and CDK1 using western blotting. β-Actin was used as an equal loading control. Representative photographs from three independent experiments are shown. (D) Quantitative analysis. The relative levels of CDK1 and cyclin B used to control β-actin were determined by densimetric scanning. The data are expressed as means ± SD of three separate experiments. p values are for one-way analysis of variance (ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. The blots are representative of three independent experiments.

2.7. Cell apoptosis analysis

To further determine whether the antiproliferative effects of compound 9d were associated with enhanced apoptosis of cancer cells, we carried out Annexin V-PE/7-AAD staining and a flow cytometry assay, and the percentages of apoptotic cells were determined. A549 cancer cells were incubated with different concentrations of 9d or SAHA for 72 h. As shown in Fig. 7, 9d treated A549 cells exhibited a dose-dependent increase of apoptosis by 30.03%, 56.44%, and 81.45% at 1.0 μM, 2.5 μM, and 5.0 μM, respectively. The 81.45% induction of A549 cell apoptosis with 5.0 μM 9d was higher than that with 5.0 μM SAHA (75.64% apoptotic cells). We also examined the cleavage states of caspase-3 and PARP (Fig. 7C and D). It was revealed that 9d resulted in more cleavage of both PARP and caspase-3 than SAHA. Taken together, these results proved that 9d treatment induced apoptosis associated with cleavage of caspase 3 and PARP in A549 cells.

Fig. 7. The effects of 9d on cell apoptosis in A549 cells. (A) Flow cytometry analysis. Representative photographs from three independent experiments are shown. (B) Quantitative analysis of apoptotic cells. Data are expressed as means ± S.D. of the percentages of apoptotic cells from three independent experiments. p values are for one-way analysis of variance (ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. (C) A549 cells were incubated with, or without, 9d and SAHA at the indicated concentrations for 48 h and the levels of protein expression were detected using specific antibodies. Data shown are representative images of each protein for three separate experiments. (D) Quantitative analysis of cleaved caspase 3 and cleaved PARP. The relative levels of each protein compared to control β-actin were determined by densimetric scanning. Data are expressed as means ± SD from three separate experiments. p values are for one-way analysis of variance (ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. The blots are representative of three independent experiments.

2.8. Docking studies

Docking simulation was performed for the selected compound 9d which was docked into HDAC1. Besides chelating with Zn²⁺, the hydroxamic acid group of 9d formed two hydrogen bonds with His140 and His178, respectively (Fig. 8). Moreover, hydrogen bonding interaction was also observed between the amide group of 9d and Leu271. In addition, the para-methoxy group of 9d docked into the hydrophobic pocket of the surface region of HDAC1, which could preliminarily explain the better inhibitory activity of 9d than other analogues.

Fig. 8. Proposed binding mode of compound 9d with HDAC1 (PDB ; 4BKX). (A) Molecular surface of the HDAC1 binding pocket with 9d. (B) 9d interacting with the active site of HDAC1.

3. Conclusion

In conclusion, a series of new bis-substituted aromatic amide-based hydroxamic acid HDACis were designed, synthesized and evaluated for their antitumor activities. Two analogues 9d and 10k exhibited improved potency against HDAC1 compared with SAHA. Moreover, compound 9d showed modest in vitro antiproliferative effects towards three different types of cancer cells, and increased the levels of acetylated histones H3 and H4 in a dose-dependent manner in A549 cells. Finally, a molecular modeling study was also performed to assess the potential binding ability of 9d to HDAC1. The present work afforded new HDACis that could be further investigated as promising anticancer drug leads.

4. Experimental section

4.1. Chemistry: general methods

All reagents and solvents were purchased from Adamas-Beta Ltd., Aladdin-Reagents Inc., or J&K Inc., and used without further purification. All reactions were carried out using standard techniques and were monitored by TLC. ¹H and ¹³C NMR spectra were recorded on Bruker 300 or 600 MHz instruments. The chemical shifts (δ) were reported in parts per million (ppm) and coupling constants (J) were reported in Hz. High resolution mass spectra were measured using a Bruker MicroTOF-Q II LCMS instrument operating in electrospray ionization (ESI) mode. HPLC analyses were carried out on an Agilent Technologies 1260 series, using the following conditions: Eclipse XDB C18 column, 5 μm, 4.6 × 150 mm, column temperature 40 °C; solvent A: water; solvent B: methanol; gradient of 40–70% B (0–5 min), 70–90% B (5–10 min), 90–40% B (10–15 min); flow rate of 1.5 mL min^–1. The purities of all compounds for biological tests were ≥95%.

4.2. General procedure for the preparation of target compounds

4.2.1. N ¹-(3-(Ethylcarbamoyl)benzyl)-N⁷-hydroxy-N¹-(2-methoxy-phenyl)heptanediamide (9a)

To a solution of hydroxyl amine hydrochloride (945 mg, 13.6 mmol) in 10 mL MeOH was added KOH (763 mg, 13.6 mmol). Then the reaction mixture was stirred at 40 °C for 10 min and cooled to 0 °C and filtered. Compound methyl 7-((3-(ethylcarbamoyl)benzyl)(2-methoxyphenyl)amino)-7-oxoheptanoate (300 mg, 0.68 mmol) was added to the filtrate followed by KOH (76.3 mg, 1.36 mmol) at room temperature for 30 min. The solvent was removed and extracted with EtOAc. The organic layer was washed with saturated NH₄Cl aqueous solution and brine and dried over Na₂SO₄. The obtained residue was finally purified by column chromatography [eluted with EtOAc followed by 10 : 1 CH₂Cl₂–MeOH] to give compound 9a as a colorless oil (179 mg, 59.5% yield).

¹H NMR (600 MHz, CD₃OD) δ 7.67–7.65 (m, 2H), 7.35–7.31 (m, 3H), 7.06 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 7.8 Hz, 1H), 6.88 (dd, J = 7.8, 7.8 Hz, 1H), 5.15 (d, J = 14.4 Hz, 1H), 4.54 (d, J = 14.4 Hz, 1H), 3.74 (s, 3H), 3.38 (q, J = 7.2 Hz, 2H), 2.11–2.04 (m, 2H), 2.03 (d, J = 7.2 Hz, 2H), 1.57–1.54 (m, 2H), 1.52–1.49 (m, 2H), 1.25–1.22 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, CD₃OD) δ 176.15, 172.81, 169.84, 156.45, 139.27, 135.85, 133.01, 131.34, 131.14, 131.00, 129.38, 128.72, 127.15, 121.98, 113.38, 56.08, 52.75, 35.82, 34.46, 33.55, 29.60, 26.38, 25.96, 14.90. HRMS (ESI) m/z calcd. for C₂₄H₃₁N₃O₅ [M + H]⁺ 442.2336, found 442.2331. HPLC purity: 95.2%, t_R = 4.7 min.

4.2.2. N ¹-(3-(Diethylcarbamoyl)benzyl)-N⁷-hydroxy-N¹-(2-methoxy-phenyl)heptanediamide (9b)

Compound 9b (35.9% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (300 MHz, CDCl₃) δ 10.33 (brs, 1H), 7.43 (s, 1H), 7.27–7.22 (m, 3H), 7.08 (d, J = 7.2 Hz, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.83 (dd, J = 8.4, 8.4 Hz, 1H), 6.74 (d, J = 8.4 Hz, 1H), 5.31 (d, J = 14.1 Hz, 1H), 4.28 (d, J = 14.1 Hz, 1H), 3.72 (s, 3H), 3.53–3.50 (m, 2H), 3.23–3.20 (m, 2H), 2.17 (t, J = 6.9 Hz, 2H), 2.02 (t, J = 6.9 Hz, 2H), 1.67–1.56 (s, 4H), 1.28–1.20 (s, 6H), 1.12–1.06 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 176.05, 173.37, 172.79, 156.46, 139.44, 138.00, 131.26, 131.16, 131.13, 130.98, 129.65, 127.67, 126.33, 121.97, 113.42, 56.13, 52.60, 44.92, 40.85, 34.45, 33.54, 29.61, 26.37, 26.00, 14.41, 13.06. HRMS (ESI) m/z calcd. for C₂₆H₃₅N₃O₅ [M + H]⁺ 470.2649, found 470.2647. HPLC purity: 95.4%, t_R = 6.0 min.

4.2.3. N ¹-(3-(Propionamido)benzyl)-N⁷-hydroxy-N¹-(2-methoxy-phenyl)heptanediamide (9c)

Compound 9c (16.6% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (300 MHz, DMSO-d6): δ 10.30 (br s, 1H), 9.81 (br s, 1H), 8.65–8.62 (m, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.40 (s, 1H), 7.33 (dd, J = 7. 5, 7.2 Hz, 1H), 7.17–7.10 (m, 2H), 6.98 (d, J = 7.5 Hz, 1H), 6.87 (dd, J = 7.5, 7.2 Hz, 1H), 6.77 (d, J = 7.2 Hz, 1H), 5.20 (d, J = 14.1 Hz, 1H), 4.12 (d, J = 14.1 Hz, 1H), 3.77 (s, 3H), 2.30 (q, J = 7.5 Hz, 2H), 1.87 (t, J = 7.2 Hz, 2H), 1.47–1.42 (m, 2H), 1.39–1.34 (m, 2H), 1.20–1.15 (m, 4H), 1.08 (t, J = 7.5 Hz, 3H). ¹³C NMR (150 MHz, CD₃OD) δ 176.02, 175.41, 172.82, 156.45, 139.96, 139.48, 131.43, 131.17, 131.04, 129.58, 125.42, 121.88, 121.37, 120.12, 113.34, 56.13, 52.72, 34.45, 33.54, 31.02, 29.57, 26.36, 25.98, 10.28. HRMS (ESI) m/z calcd. for C₂₄H₃₁N₃O₅ [M + H]⁺ 442.2336, found 442.2330. HPLC purity: 99.2%, t_R = 5.2 min.

4.2.4. N ¹-(3-(Ethylcarbamoyl)benzyl)-N⁷-hydroxy-N¹-(4-methoxy-phenyl)heptanediamide (9d)

Compound 9d (49.1% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (300 MHz, DMSO-d₆) δ 10.31 (brs, 1H), 8.65 (brs, 1H), 8.43 (t, J = 5.1 Hz), 7.68 (d, J = 7.5 Hz, 1H), 7.63 (s, 1H), 7.36 (dd, J = 7.5, 7.5 Hz, 1H), 9.29 (d, J = 7.5 Hz, 1H), 7.04 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 4.85 (s, 2H), 3.72 (s, 3H), 3.26 (q, J = 7.2 Hz, 2H), 2.05 (t, J = 7.5 Hz, 2H), 1.89 (t, J = 7.5 Hz, 2H), 1.50–1.45 (m, 2H), 1.41–1.36 (m, 2H), 1.19–1.15 (m, 2H), 1.10 (t, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, CD₃OD) δ 175.76, 172.81, 169.80, 160.86, 139.35, 136.10, 135.82, 132.78, 130.55, 129.66, 128.51, 127.22, 115.84, 55.94, 53.84, 35.84, 34.88, 33.54, 29.62, 26.37, 26.20, 14.89. HRMS (ESI) m/z calcd. for C₂₄H₃₁N₃O₅ [M + H]⁺ 442.2336, found 442.2342. HPLC purity: 99.1%, t_R = 4.6 min.

4.2.5. N ¹-(3-(Ethylcarbamoyl)benzyl)-N⁷-hydroxy-N¹-(4-hydroxy-phenyl)heptanediamide (9e)

Compound 9e (58.5% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (600 MHz, CD₃OD) δ 7.69 (d, J = 7.2 Hz, 1H), 7.65 (s, 1H), 7.38 (dd, J = 7.8, 7.8 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 6.87 (d, J = 9.0 Hz, 2H), 6.74 (d, J = 9.0 Hz, 2H), 3.64 (s, 2H), 3.40 (q, J = 7.2 Hz, 2H), 2.14 (t, J = 7.8 Hz, 2H), 2.04 (d, J = 7.8 Hz, 2H), 1.60–1.57 (m, 2H), 1.54–1.51 (m, 2H), 1.27–1.24 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H). ¹³C NMR (150 MHz, CD₃OD) δ 175.89, 172.82, 170.06, 158.66, 139.41, 136.06, 134.69, 132.81, 130.48, 129.64, 128.50, 127.20, 117.15, 53.87, 52.08, 34.84, 33.54, 29.63, 26.39, 26.23, 14.88. HRMS (ESI) m/z calcd. for C₂₃H₂₉N₃O₅ [M + H]⁺ 428.2180, found 428.2177. HPLC purity: 95.6%, t_R = 4.4 min.

4.2.6. N ¹-(3-(2-Phenylacetamido)benzyl)-N⁷-hydroxy-N¹-(2-methoxy-phenyl)heptanediamide (10a)

Compound 10a (41.2% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (300 MHz, DMSO-d₆): δ 10.31 (br s, 1H), 10.14 (br s, 1H), 8.66 (br s, 1H), 7.52 (d, J = 7.2 Hz, 1H), 7.42–7.41 (m, 1H), 7.34–7.31 (m, 4H), 7.28 (d, J = 7.2 Hz, 1H), 7.25–7.22 (m, 1H), 7.16 (dd, J = 7.8, 7.5 Hz, 1H), 7.10 (d, J = 7.5 Hz, 1H), 6.96 (d, J = 7.8 Hz, 1H), 6.88 (dd, J = 7. 5, 7.5 Hz, 1H), 6.81 (d, J = 7.5 Hz, 1H), 5.21 (d, J = 14.7 Hz, 1H), 4.15 (d, J = 14.7 Hz, 1H), 3.75 (s, 3H), 3.62 (s, 2H), 1.95–1.90 (m, 2H), 1.87 (t, J = 7.5 Hz, 2H), 1.45 (t, J = 7.5 Hz, 2H), 1.37 (t, J = 7.5 Hz, 2H), 1.12–1.08 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 176.03, 172.82, 172.34, 156.43, 139.82, 139.50, 136.86, 131.40, 131.12, 131.02, 130.14, 129.98, 129.60, 127.95, 125.67, 121.89, 121.50, 120.23, 113.31, 56.09, 52.73, 44.70, 34.42, 33.52, 29.55, 26.34, 25.96. HRMS (ESI) m/z calcd. for C₂₉H₃₃N₃O₅ [M + H]⁺ 504.2493, found 504.2488. HPLC purity: 99.5%, t_R = 6.7 min.

4.2.7. N ¹-(3-(Benzylcarbamoyl)benzyl)-N⁷-hydroxy-N¹-(2-methoxy-phenyl)heptanediamide (10b)

Compound 10b (37.2% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (600 MHz, CD₃OD) δ 7.73–7.69 (m, 1H), 7.68 (s, 1H), 7.35–7.34 (m, 2H), 7.33–7.29 (m, 5H), 7.26–7.24 (m, 1H), 7.04 (d, J = 7.8 Hz, 1H), 6.95 (d, J = 7.8 Hz, 1H), 6.88 (dd, J = 7.8, 7.8 Hz, 1H), 5.12 (d, J = 14.4 Hz, 1H), 4.57 (d, J = 14.4 Hz, 1H), 4.55 (s, 2H), 3.71 (s, 3H), 2.11–2.05 (m, 2H), 2.02 (t, J = 7.8 Hz, 2H), 1.57–1.52 (m, 2H), 1.51–1.48 (m, 2H), 1.24–1.20 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 176.14, 172.80, 169.94, 156.44, 140.21, 139.29, 135.63, 133.20, 131.33, 131.13, 130.98, 129.54, 129.45, 128.84, 128.52, 128.19, 127.30, 121.99, 113.36, 56.06, 52.77, 44.48, 34.45, 33.54, 29.59, 26.37, 25.95. HRMS (ESI) m/z calcd. for C₂₉H₃₃N₃O₅ [M + H]⁺ 504.2493, found 504.2511. HPLC purity: 96.9%, t_R = 6.5 min.

4.2.8. N ¹-Hydroxy-N⁷-(2-methoxyphenyl)-N⁷-(3-(phenethyl-carbamoyl)benzyl)heptanediamide (10c)

Compound 10c (35.6% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (300 MHz, DMSO-d₆) δ 10.32 (s, 1H), 8.66 (s, 1H), 8.52 (t, J = 5.7 Hz, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 7.8 Hz, 2H), 7.31–7.28 (m, 2H), 7.24 (d, J = 9.0 Hz, 2H), 7.21–7.19 (m, 2H), 7.10 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 7.5 Hz, 1H), 6.87 (dd, J = 7.5, 7.5 Hz, 1H), 5.17 (d, J = 14.7 Hz, 1H), 4.34 (d, J = 14.7 Hz, 1H), 3.74 (s, 3H), 3.49–3.43 (m, 2H), 2.82 (t, J = 7.5 Hz, 2H), 2.05–1.94 (m, 2H), 1.87 (t, J = 7.5 Hz, 2H), 1.48–1.41 (m, 2H), 1.39–1.33 (m, 2H), 1.13–1.07 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 178.31, 176.63, 175.97, 172.82, 169.54, 161.12, 135.98, 133.80, 133.40, 130.70, 130.54, 130.37, 130.06, 128.91, 127.01, 126.84, 124.26, 116.14, 115.97, 56.00, 49.85, 46.27, 34.66, 34.46, 33.51, 29.53, 26.36, 26.00. HRMS (ESI) m/z calcd. for C₃₀H₃₅N₃O₅ [M + H]⁺ 518.2649, found 518.2655. HPLC purity: 96.7%, t_R = 6.9 min.

4.2.9. N ¹-Hydroxy-N⁷-(2-methoxyphenyl)-N⁷-(3-((4-phenylbutyl)carbamoyl)benzyl)heptanediamide (10d)

Compound 10d (34.6% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (600 MHz, CD₃OD) δ 7.67–7.64 (m, 1H), 7.62 (s, 1H), 7.34–7.29 (m, 3H), 7.24 (dd, J = 7.8, 7.8 Hz, 2H), 7.18 (d, J = 7.2 Hz, 2H), 7.14 (dd, J = 7.2, 7.2 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 7.8 Hz, 1H), 6.87 (dd, J = 7.8, 7.8 Hz, 1H), 5.13 (d, J = 14.4 Hz, 1H), 4.55 (d, J = 14.4 Hz, 1H), 3.72 (s, 3H), 3.38 (t, J = 7.2 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 2.10–2.06 (m, 2H), 2.03 (t, J = 7.2 Hz, 2H), 1.70–1.67 (m, 2H), 1.65–1.61 (m, 2H), 1.56–1.53 (m, 2H), 1.52–1.49 (m, 2H), 1.24–1.19 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 176.13, 172.81, 170.01, 156.45, 143.61, 139.24, 135.85, 133.04, 131.32, 131.13, 130.99, 129.45, 129.39, 129.33, 128.73, 127.18, 126.77, 121.98, 113.36, 56.08, 52.74, 40.76, 36.52, 34.46, 33.56, 30.08, 30.06, 29.60, 26.38, 25.96. HRMS (ESI) m/z calcd. for C₃₂H₃₉N₃O₅ [M + H]⁺ 546.2962, found 546.2972. HPLC purity: 96.1%, t_R = 7.8 min.

4.2.10. N ¹-Hydroxy-N⁷-(2-methoxyphenyl)-N⁷-(3-((5-phenylpentyl)carbamoyl)benzyl)heptanediamide (10e)

Compound 10e (64.8% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (600 MHz, CD₃OD) δ 7.65–7.63 (m, 1H), 7.62 (s, 1H), 7.36–7.30 (m, 3H), 7.22 (dd, J = 7.8, 7.2 Hz, 2H), 7.16 (d, J = 7.2 Hz, 2H), 7.12 (dd, J = 7.8, 7.2 Hz, 1H), 7.05 (dd, J = 8.4 Hz, 1H), 6.94 (d, J = 7.8 Hz, 1H), 6.88 (dd, J = 7.8, 7.8 Hz, 1H), 5.15 (d, J = 15.0 Hz, 1H), 4.54 (d, J = 15.0 Hz, 1H), 3.74 (s, 3H), 3.34 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 7.8 Hz, 2H), 2.11–2.04 (m, 2H), 2.02 (t, J = 7.2 Hz, 2H), 1.69–1.64 (m, 2H), 1.63–1.60 (m, 2H), 1.58–1.55 (m, 2H), 1.52–1.48 (m, 2H), 1.42–1.37 (m, 2H), 1.24–1.20 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 176.14, 172.78, 170.01, 156.46, 143.76, 139.26, 135.89, 133.00, 131.35, 131.14, 131.00, 129.43, 129.38, 129.27, 128.73, 127.17, 126.67, 121.98, 113.38, 56.09, 52.75, 40.91, 36.76, 34.47, 33.54, 32.37, 30.27, 29.60, 27.55, 26.38, 25.96. HRMS (ESI) m/z calcd. for C₃₃H₄₁N₃O₅ [M + H]⁺ 560.3119, found 560.3118. HPLC purity: 95.4%, t_R = 8.2 min.

4.2.11. N ¹-Hydroxy-N⁷-(2-methoxyphenyl)-N⁷-(3-((6-phenylhexyl)-carbamoyl)benzyl)heptanediamide (10f)

Compound 10f (43.6% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (300 MHz, DMSO-d₆): δ 10.31 (br s, 1H), 8.65 (br s, 1H), 8.37 (t, J = 5.1 Hz, 1H), 7.66 (d, J = 7.2 Hz, 1H), 7.62 (s, 1H), 7.32 (dd, J = 7.5, 7.5 Hz, 2H), 7.27–7.24 (m, 3H), 7.19–7.15 (m, 3H), 7.09 (d, J = 7.2 Hz, 1H), 6.95 (d, J = 7.5 Hz, 1H), 6.87 (dd, J = 7.5, 7.5 Hz, 1H), 5.18 (d, J = 14.7 Hz, 1H), 4.35 (d, J = 14.7 Hz, 1H), 3.75 (s, 3H), 3.21 (q, J = 6.9 Hz, 2H), 2.59 (t, J = 7.8 Hz, 2H), 2.02–1.97 (m, 2H), 1.88 (t, J = 7.5 Hz, 2H), 1.55 (t, J = 7.8 Hz, 2H), 1.52–1.48 (m, 4H), 1.46–1.38 (m, 6H), 1.13–1.10 (m, 2H). ¹³C NMR (151 MHz, CD₃OD) δ 176.14, 172.80, 170.00, 156.45, 143.90, 139.26, 135.89, 133.02, 131.33, 131.13, 130.99, 129.40, 129.26, 128.73, 127.22, 127.18, 126.64, 121.97, 113.37, 56.08, 52.74, 40.99, 36.83, 34.46, 33.55, 32.69, 30.42, 30.00, 29.60, 27.91, 26.37, 25.96. HRMS (ESI) m/z calcd. for C₃₄H₄₃N₃O₅ [M + H]⁺ 574.3275, found 574.3287. HPLC purity: 97.4%, t_R = 9.5 min.

4.2.12. N ¹-(3-((4-Fluorophenethyl)carbamoyl)benzyl)-N⁷-hydroxy-N¹-(2-methoxyphenyl)heptanediamide (10g)

Compound 10g (74.4% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (300 MHz, DMSO-d₆): δ 10.29 (br s, 1H), 8.63 (br s, 1H), 8.47 (t, J = 5.7 Hz, 1H), 7.64–7.61 (m, 2H), 7.32 (d, J = 7.5 Hz, 1H), 7.31–7.28 (m, 2H), 7.26–7.23 (m, 2H), 7.12–7.07 (m, 3H), 6.94 (d, J = 7.5 Hz, 1H), 6.87 (dd, J = 7.5, 7.5 Hz, 1H), 5.14 (d, J = 15.0 Hz, 1H), 4.33 (d, J = 15.0 Hz, 1H), 3.73 (s, 3H), 3.47–3.42 (m, 2H), 2.81 (t, J = 7.8 Hz, 2H), 2.02–1.97 (m, 2H), 1.87 (t, J = 7.2 Hz, 2H), 1.47–1.42 (m, 2H), 1.38–1.34 (m, 2H), 1.13–1.08 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 176.14, 172.83, 170.01, 163.05 (d, J = 240.5 Hz), 156.46, 139.31, 136.55 (d, J = 3.3 Hz), 135.76, 133.11, 131.55 (d, J = 7.8 Hz), 131.33, 131.15, 130.99, 129.41, 128.70, 127.15, 121.99, 116.11 (d, J = 21.5 Hz), 113.39, 56.08, 52.73, 42.57, 35.67, 34.46, 33.56, 29.61, 26.38, 25.96. HRMS (ESI) m/z calcd. for C₃₀H₃₄FN₃O₅ [M + H]⁺ 536.2555, found 536.2563. HPLC purity: 96.6%, t_R = 6.9 min.

4.2.13. N ¹-(3-((4-Chlorophenethyl)carbamoyl)benzyl)-N⁷-hydroxy-N¹-(2-methoxyphenyl)heptanediamide (10h)

Compound 10h (62.5% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (600 MHz, CD₃OD) δ 7.63–7.61 (m, 1H), 7.59 (s, 1H), 7.34–7.30 (m, 3H), 7.27 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 7.8 Hz, 1H), 6.88 (dd, J = 8.4, 8.4 Hz, 1H), 5.13 (d, J = 14.4 Hz, 1H), 4.54 (d, J = 14.4 Hz, 1H), 3.72 (s, 3H), 3.56 (t, J = 7.8 Hz, 2H), 2.89 (t, J = 7.2 Hz, 2H), 2.13–2.04 (m, 2H), 2.03 (t, J = 7.2 Hz, 2H), 1.59–1.54 (m, 2H), 1.52–1.48 (m, 2H), 1.25–1.20 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 176.12, 172.81, 170.04, 156.46, 139.44, 139.31, 135.72, 133.16, 133.12, 131.54, 131.32, 131.15, 130.98, 129.52, 129.41, 128.69, 127.16, 121.99, 113.39, 56.08, 52.72, 42.31, 35.80, 34.46, 33.56, 29.61, 26.38, 25.96. HRMS (ESI) m/z calcd. for C₃₀H₃₄ClN₃O₅ [M + H]⁺ 552.2260, found 552.2256. HPLC purity: 97.0%, t_R = 7.5 min.

4.2.14. N ¹-Hydroxy-N⁷-(3-((4methoxyphenethyl)carbamoyl)benzyl)-N⁷-(2-methoxyphenyl)heptanediamide (10i)

Compound 10i (37.3% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (600 MHz, CD₃OD) δ 7.63–7.62 (m, 1H), 7.60 (s, 1H), 7.34–7.32 (m, 3H), 7.15 (d, J = 9.0 Hz, 2H), 7.05 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 7.8 Hz, 1H), 6.88 (dd, J = 7.8, 7.8 Hz, 1H), 6.84 (d, J = 8.4 Hz, 2H), 5.14 (d, J = 14.4 Hz, 1H), 4.54 (d, J = 14.4 Hz, 1H), 3.76 (s, 3H), 3.72 (s, 3H), 3.53 (t, J = 7.8 Hz, 2H), 2.83 (t, J = 7.2 Hz, 2H), 2.12–2.07 (m, 2H), 2.03 (t, J = 7.2 Hz, 2H), 1.58–1.55 (m, 2H), 1.53–1.50 (m, 2H), 1.25–1.20 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 176.13, 172.80, 170.02, 159.76, 156.46, 139.28, 135.84, 133.06, 132.54, 131.33, 131.14, 130.99, 130.82, 129.39, 128.69, 127.16, 121.99, 114.92, 113.39, 56.08, 55.65, 52.73, 42.82, 35.66, 34.47, 33.56, 29.61, 26.39, 25.96. HRMS (ESI) m/z calcd. for C₃₁H₃₇N₃O₆ [M + H]⁺ 548.2755, found 548.2756. HPLC purity: 95.3%, t_R = 6.7 min.

4.2.15. N ¹-(3-((4-(4-Fluorophenyl)butyl)carbamoyl)benzyl)-N⁷-hydroxy-N¹-(2-methoxyphenyl)heptanediamide (10j)

Compound 10j (63.2% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (300 MHz, DMSO-d₆): δ 10.30 (br s, 1H), 8.64 (br s, 1H), 8.39 (t, J = 5.1 Hz, 1H), 7.65 (d, J = 6.9 Hz, 1H), 7.62 (s, 1H), 7.33–7.30 (m, 3H), 7.21 (dd, J = 7.5, 7.5 Hz, 2H), 7.10–7.06 (m, 3H), 6.94 (d, J = 7.5 Hz, 1H), 6.84 (dd, J = 7.5, 7.5 Hz, 1H), 5.15 (d, J = 14.7 Hz, 1H), 4.32 (d, J = 14.7 Hz, 1H), 3.73 (s, 3H), 3.25 (q, J = 7.2 Hz, 2H), 2.58 (t, J = 7.2 Hz, 2H), 2.10–1.97 (m, 2H), 1.87 (t, J = 7.2 Hz, 2H), 1.55–1.50 (m, 4H), 1.40–1.32 (m, 4H), 1.16–1.11 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 176.14, 172.82, 170.02, 162.69 (d, J = 240.5 Hz), 156.45, 139.51 (d, J = 3.0 Hz), 139.27, 135.85, 133.04, 131.34, 131.14, 131.01 (d, J = 6.7 Hz), 129.41, 128.73, 127.71, 121.98, 115.84 (d, J = 21.0 Hz), 113.37, 56.08, 52.75, 40.71, 35.62, 34.46, 33.56, 30.11, 30.00, 29.60, 26.38, 25.96. HRMS (ESI) m/z calcd. for C₃₂H₃₈FN₃O₅ [M + H]⁺ 564.2868, found 564.2882. HPLC purity: 95.9%, t_R = 7.7 min.

4.2.16. N ¹-(3-((4-Fluorophenethyl)carbamoyl)benzyl)-N⁷-hydroxy-N¹-(4-methoxyphenyl)heptanediamide (10k)

Compound 10k (48.7% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (300 MHz, DMSO-d₆): δ 10.33 (br s, 1H), 8.67 (br s, 1H), 8.53 (t, J = 5.4 Hz, 1H), 7.65 (d, J = 7.5 Hz, 1H), 7.61 (s, 1H), 7.36 (dd, J = 7. 5, 7.5 Hz, 1H), 7.31–7.23 (m, 3H), 7.10 (d, J = 9.0 Hz, 2H), 7.04 (d, J = 9.0 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 4.85 (s, 2H), 3.72 (s, 3H), 3.47–3.40 (m, 2H), 2.80 (t, J = 7.5 Hz, 2H), 2.03 (t, J = 7.2 Hz, 2H), 1.88 (t, J = 7.2 Hz, 2H), 1.49–1.45 (m, 2H), 1.40–1.35 (m, 2H), 1.12–1.09 (m, 2H). ¹³C NMR (150 MHz, CD₃OD) δ 175.73, 172.43, 169.98, 163.03 (d, J = 241 Hz), 160.84, 139.38, 136.54 (d, J = 3.2 Hz), 136.00, 135.80, 132.86, 131.55 (d, J = 8.0 Hz), 130.54, 129.70, 128.45, 127.23, 116.05 (d, J = 21.2 Hz), 115.84, 55.94, 53.83, 42.60, 35.67, 34.88, 33.42, 29.62, 26.38, 26.20. HRMS (ESI) m/z calcd. for C₃₀H₃₄FN₃O₅ [M + H]⁺ 536.2555, found 536.2556. HPLC purity: 97.2%, t_R = 8.6 min.

4.2.17. N ¹-(3-(3-(4-Fluorophenyl)propanamido)benzyl)-N⁷-hydroxy-N¹-(4-methoxyphenyl)heptanediamide (10l)

Compound 10l (33.5% yield) was prepared according to the procedure described for the preparation of compound 9a. ¹H NMR (600 MHz, DMSO-d₆) δ 10.31 (brs, 1H), 9.88 (s, 1H), 8.65 (brs, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.37 (s, 1H), 7.28–7.25 (m, 2H), 7.17 (dd, J = 7.8, 7.8 Hz, 1H), 7.09 (dd, J = 8.4, 8.4 Hz, 2H), 7.06 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 6.79 (d, J = 7.2 Hz, 1H), 4.75 (s, 2H), 3.73 (s, 3H), 2.88 (t, J = 7.8 Hz, 2H), 2.59 (t, J = 7.2 Hz, 2H), 2.01 (t, J = 7.2 Hz, 2H), 1.89 (t, J = 7.2 Hz, 2H), 1.49–1.44 (m, 2H), 1.40–1.35 (m, 2H), 1.15–1.01 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 171.97, 170.27, 169.04, 160.69 (d, J = 239.8 Hz), 158.28, 139.19, 138.33, 137.31 (d, J = 3.0 Hz), 134.88, 130.02 (d, J = 7.8 Hz), 129.33, 128.59, 122.63, 118.38, 117.72, 114.96 (d, J = 20.8 Hz), 114.57, 55.24, 52.03, 38.00, 33.25, 32.13, 29.93, 28.21, 24.93, 24.68. HRMS (ESI) m/z calcd. for C₃₀H₃₄FN₃O₅ [M + H]⁺ 536.2555, found 536.2553. HPLC purity: 98.1%, t_R = 8.9 min.

4.3. HDAC1 inhibitory assay

The in vitro HDAC1 inhibitory activity was determined by the protease-coupled assay. Different concentrations of tested compounds were incubated with recombinant HDAC1 (BPS Biosciences, US) at room temperature for 15 min, followed by addition of trypsin as well as Ac-peptide-AMC substrates to initiate the reaction in Tris-based assay buffer. Fluorescent AMC released from the substrate was measured on a SynergyMx (BioTek, US) using filter sets with excitation = 355 nm and emission = 460 nm. IC₅₀ values were calculated using GraphPad Prism software (California, USA).

4.4. Biological evaluation

4.4.1. In vitro anti-proliferation assay

Cell lines A549, MDA-MB-231, and MCF-7 used in this study were obtained from the American Type Culture Collection. The cells were cultured in RPMI 1640 medium (HyClone, SH30809.01B) supplemented with 10% fetal bovine serum (FBS) (HyClone, SV30087.02) at 37 °C in a humidified atmosphere with 5% CO₂. Human breast epithelial cells (MCF-10A and MCF-10F) and human lung epithelial cells (Beas-2B) were obtained from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. And the cells were cultured in MEM (HyClone, SH30243.01B) supplemented with 10% fetal bovine serum (FBS) (Gibco, 10270) at 37 °C in a humidified atmosphere with 5% CO₂.

The SRB assay was performed according to the manufacturer's instruction (Sigma Aldrich). Tumor cells were seeded into 96-well plates at the appropriate cell densities during the experiment. After incubation for 24 h, the cells were treated with various concentrations of tested compounds for 48 h. Then the cells were fixed with 10% TCA for 1 h at 4 °C and washed with distilled water five times. The plates were allowed to air dry followed by dyeing with 0.4% SRB for 5 min at room temperature. After dyeing, the plates were washed with 1% acetic acid and allowed to air dry. 150 μL of 10 mM Tris-based solution was added to each well, and the absorption at 540 nm was measured using a microplate reader (TECAN). The IC₅₀ values were calculated using GraphPad prism 7.0. Three independent experiments were carried out in triplicate.

4.4.2. Colony formation assay

A549 cells were plated into 35 mm dishes (800 cells per dish) and treated with 9d or SAHA for one week which were replenished every 24 h. After the removal of culture media, the cells were washed with PBS three times prior to methanol fixation. The cells were then immediately stained using 1% crystal violet (Beyotime) for 15 min and washed with PBS once. Images were collected using a scanning apparatus (Canon, Japan). Experiments were carried out in triplicate and repeated three times.

4.4.3. Wound healing assay

1.5 × 10⁵ A549 cells were counted and plated in 6-well dishes. The cells were incubated overnight yielding confluent monolayers for wounding. Wounds were made using a pipette tip and photographs were taken immediately (0 h), 12 h and 24 h after wounding for A549 cells, respectively. The distance migrated by the cell monolayer to close the wounded area during this time period was measured via the Image J software (NIH, USA). Results were expressed as the percentage of wound closure—that is, the distance migrated initially (0 h) minus the distance migrated after 12 h or 24 h relative to the distance migrated initially (0 h). Experiments were carried out in triplicate and repeated three times.

4.4.4. Western blotting

After treatment with 9d for indicated times, the cells were harvested with RIPA buffer containing 150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris and cocktails of protease and phosphatase inhibitors for 10 min at room temperature and boiled for another 10 min. Equal amounts of total proteins (35 μg) underwent 15% SDS PAGE and were electroblotted onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% (w/v) non-fat dry milk in PBS-Tween 20 (PBST; 0.05%) for 1 h and incubated with primary antibodies (1 : 1000 in PBST) at 4 °C overnight. After three washings in PBST, the PVDF membrane was incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1 : 20 000) for 1 h at room temperature. The immunoreactive bands were developed with the ECL western blotting system.

β-Actin was used as the loading control. The relative quantity of proteins was analyzed via the Image J software (NIH, USA).

4.4.5. Cell cycle analysis

Cell cycle analysis was carried out by estimating DNA contents with flow cytometry. After incubation with the indicated doses of 9d for 24 h, A549 cells were fixed in 70% ice-cold ethanol, incubated overnight at –20 °C, stained with PI containing RNaseA solution for 30 min at 37 °C, and then analyzed by FACS.

4.4.6. Cell apoptosis analysis

Cell apoptosis analysis was performed by the Annexin V FITC/PI assay using an Annexin v-PE/7-AAD apoptosis detection kit (BD). Briefly, A549 cells (8 × 104/well) were treated with DMSO and compound 9d for 72 h. The cells were then harvested and stained with annexin binding buffer, Alexa Fluor 488 annexin and propidium iodide for 15 min in the dark. After staining, 400 μL of 1× annexin-building buffer was added, mixed gently and kept on ice. The samples were measured using an ACEA Biosciences NovoCyte flow cytometer.

4.5. Molecular docking studies

Molecular docking studies were carried out with Autodock-4.27. For the docking calculations, the HDAC1 crystal structure (PDB code: 4BKX) was retrieved from the Protein Data Bank (; www.pdb.org). For protein preparation, all the water molecules were removed from HDAC1, and Gasteiger partial charges were assigned to the selected compounds and enzyme atoms. The docking results were analyzed with the programs AutoDockTools, 27 DOCKRES and VMD.

4.6. Statistical analyses

All experiments were performed in duplicate and repeated at least three times. Each experimental value was expressed as the mean ± standard deviation (SD). Data were analyzed by Student's t-test between two groups and by one-way analysis of variance (ANOVA) followed by a Bonferroni test for multiple comparisons. These analyses were performed using GraphPad Prism software version 5.0 (GraphPad Software, Inc. CA). *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively.

Conflicts of interest

The authors declare that there are no conflicts of interest.