2020 Volume 68 Issue 5 Pages 466-472
Histone deacetylases (HDACs) as attractive targets in many diseases therapies has been studied extensively, and its application in cancer research is the most important. Here, we developed a series of derivatives containing natural 2,5-diketopiperazine (DKP) skeleton. Several compounds exhibited distinct HDAC1 inhibitory activities, in particular 2a (IC50 = 405 nM). The selectivity profile for representative 2a indicated that this series of compounds had a preference for HDAC1–3. Additionally, 2a showed the best growth inhibitory activities against K562 and HL-60 tumor cell line with IC50 values of 4.23 and 4.16 µM, respectively. This work may lay the foundation for developing DKP-based HDAC inhibitors as a potential anticancer agent.
Histone lysine acetylation is an epigenetic marker associated with gene transcriptional activation and repression.1) Aberrant acetylation of histone proteins and suppression of gene are closely associated with precancerous or malignant states. Histone deacetylases (HDACs), which remove acetyl groups from lysine residues located in the amino radical terminal tails of core histones, recently have emerged as important targets for tumor therapy.2–5) The HDAC family containing 18 isozymes is categorized into class I (HDACs 1, 2, 3, and 8), class IIa (HDACs 4, 5, 7, and 9), class IIb (HDACs 6 and 10), and class IV (HDAC 11). Classes I, II, and IV HDACs are all zinc-dependent deacetylases that are mechanistically distinct from the class III isoforms, named sirtuins (sirtuins 1–7), requiring nicotine adenine dinucleotide as a cofactor.6–8) Class I HDACs are ubiquitos in all tissues and are key participators in epigenetic processes.9–12) They are highly expressed in various tumor cell lines and sustain malignant growth.
To data, a number of HDAC inhibitors (HDACis) have entered clinical trials, and several of them have been approved for use in cancer treatment. An aromatous cap, linker, and zinc binding group (ZBG) are typical characteristics of the HDACis13) (Fig. 1). Nonselective hydroxamic acid inhibitors, such as vorinostat,14) have been approved for the treatment of cutaneous T-cell lymphoma. Natural Romidepsin15) and belinostat16) both gained U.S. Food and Drug Administration approvals for the treatment of peripheral T-cell lymphoma. Panobinostat was approved for the treatment of multiple myeloma.17) The benzamide inhibitor chidamide, which was reported to be a class I selective HDACI,18,19) was approved for treating recurrent and refractory peripheral T-cell lymphoma by China in 2015.
Natural products have a profound impact on human health, and they serve as powerful starting materials to generate drug substances with novel therapeutic utility.20) Our previous work incorporated the natural 2,5-diketopiperazine (DKP) scaffold and synthesized a series of potent and highly selective HDAC6 inhibitors.21) Especially, 21b, with an IC50 of 0.73 nM against HDAC6, showed 144–10941-fold selectivity over other isozymes (Fig. 2). However, in cytotoxic assay, most of these DKPs did not show good antiproliferative activities as expected. It was difficult to dissect whether awful antiproliferative effects were caused cellular uptake or stability of these DKPs. It is worth noting that only inhibiting HDAC6 may not kill tumor cells.22–24) Considering that the class I HDACs, especially HDAC1, was widely implicated in both transcriptional repression and closely related to the tumor, developing DKP derivatives targeting class I HDACs may improve the antiproliferative activity and drug efficacy. In this paper, we replaced hydroxamic acid group with o-phenylenediamine as ZBG and synthesized a series of 1,3-disubstituded DKP-based benzamide HDAC1–3 inhibitors. Subsequently, structure–activity relationship (SAR) study and molecular simulation were performed.
Compounds 1a–e were synthesized according to our previous procedure.21) Then, amidation of 1a–e with various substituted benzene-1,2-diamine through 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) gave target compounds 2a–k, as described in Chart 1.
Reagents and conditions: HATU, N,N-diisopropylethylamine (DIPEA), N,N-dimethylformamide (DMF), various substituted o-phenylenediamine, room temperature (r.t.), 6 h
We tested all prepared compounds for their inhibitory ability toward HDAC1, using chidamide as the positive control. As shown in Table 1, IC50 results illustrated that the S stereoisomer 2a bearing an indolyl, with IC50 value of 405 nM, exhibited the best inhibitory activity against HDAC1. The potency of compound 2c (IC50 = 1.50 µM) using phenyl capping group decreased on HDAC1 inhibition. Besides, the stereochemistry seemed to have obvious impact on the effect. The (S)-isomers showed better activity than the (R)-isomers, as exemplified by 2a vs. 2i and 2c vs. 2g. Notably, compounds 2e and 2f with the DKP scaffold attached to the 3-position of the phenyl linker lost their enzymatic potency. The HDAC1 inhibitory activities were markedly influenced by different substituents on the phenyl of the ZBG group. Compound 2b (IC50 = 13.8 µM), with a fluorine atom on the para-position of the amide, exhibited an almost 30-fold reduction on the HDAC1 activity than that of 2a. This SAR was consistent with results we previously reported.25) The enzymatic results of 2c vs. 2d and 2g vs. 2h also confirmed this conclusion. When chlorine or methyl substituent was introduced on the same position, 2j and 2k lost enzymatic activities.
 |
a)IC50 values for enzymatic inhibition of HDAC1 enzyme. We ran experiments in duplicate, standard deviation (S.D.) <15%. Assays were performed by Reaction Biology Corporation (Malvern, PA, U.S.A.). b)NA: no activity.
To further ascertain the selectivity of these DKPs, the most potent compound 2a was selected and tested against other class I HDACs (HDAC2, 3, 8) and HDAC6 (class II), using chidamide as the positive. As shown in Table 2, 2a inhibited HDAC2 and HDAC3 with IC50 values of 0.557 µM and 0.740 µM, respectively, and a relatively weaker inhibition of HDAC8 was observed (IC50 = 1.370 µM). However, 2a had no activity against HDAC6.
| Compound | IC50a) | ||||
|---|---|---|---|---|---|
| HDAC1 | HDAC2 | HDAC3 | HDAC6 | HDAC8 | |
| 2a | 0.405 | 0.557 | 0.740 | NAb) | 1.370 |
| Chidamide | 0.310 | 0.492 | 0.280 | NA | 0.933 |
a) IC50 values for enzymatic inhibition of HDAC enzymes. We ran experiments in duplicate, S.D. <15%. Assays were performed by Reaction Biology Corporation (Malvern, PA, U.S.A.). b) NA: no inhibition activity.
Acetylation of histone H3 is an important epigenetic marker for HDAC1-3 inhibition. Western blot assay was performed to further validate the HDAC1 selectivity of the DKPs. HCT116 cells were treated with compound 2a and chidamide at 1, 5 and 10 µM for 24 h to measure the acetylation status of H3 acetylation (Fig. 3). As expected, we observed a dose-dependent increase in the level of Ac-H3 when the cells were treated with compound 2a.
To further understanding of the interaction between these inhibitors and protein and guiding the SARs, we docked the representative compounds 2a, 2b, 2c and 2i in the active site of HDAC1 (PDB code: 5ICN) using Cdocker software, revealing excellent shape complementarity between ligands and the binding pocket (Fig. 4). As described in Fig. 4A, 2a exhibits a bidentate benzamide -Zn2+ coordination with a Zn2+···N separation of 2.37 Å and a Zn2+···O (C=O) separation of 2.77 Å. And the amino of the benzamide ZBG group in 2a formed two key hydrogen (H) bonds with residues of Asp176 and His140, and the H atom of amide also generated an H-bond interaction with carbonyl of Gly149. These H-bond forces positioned inhibitors in a specific conformation to efficiently chelate with the zinc atom. Moreover, the phenyl linkage of 2a could form a stacking π–π interaction with residues of Phe150 and Phe205. Additionally, an indolyl ring occupied the surface groove and came into close contact with hydrophobic residues at the rim region. In Fig. 4B, 2b only retained one H-bond with Gly149, and the fluorine atom on the para-position of amide made the molecule deviate from the zinc ion with Zn2+···N and Zn2+···O separations of 3.23 Å and 3.35 Å, respectively. The Steric hindrance may play an important role near the specific binding site (at the para-position of amide), and this conclusion could also be confirmed by the worse enzymatic activities of 2j and 2k with larger substituents (-Cl and -Me). As seen in Figs. 4C and 4D, 2c and 2i displayed similar binding modes with 2a. Among these three compounds, 2a showed the best interaction energy value of −45.25 kj/mol, and this result was consistent with their enzymatic inhibitions.
The H-bonding interactions with residues were labeled in gray. Zinc ion was labeled in light gray. (B) Docking model of 2b (gray) in HDAC1. (c) Docking model of 2c (gray) in HDAC1. (d) Docking model of 2i (light gray) in HDAC1. For clarity, the HDAC1 binding site is shown as a surface representation in gray.
On the basis of the enzyme results shown in Table 1, we chose several representative compounds 2a, 2c, 2d, and 2i to evaluate antiproliferative activities against human leukemia cell lines HL-60 and K562. Besides, 21b as a potent selective HDAC6 inhibitor (IC50 = 0.73 nM) was also tested for comparison. As shown in Table 3, the trend of the antiproliferative activities of these DKPs were consistent with their enzymatic activities. Compound 2a which had the best HDAC1 inhibition demonstrated the most potent cytotoxicity, with IC50 values of 4.23 µM and 10.74 µM against K562 and HL-60, respectively. Notably, all the four HDAC1-targeted DKPs exhibited superior antiproliferative activities to that of 21b.
| Compound | K562 | HL-60 | Compound | K562 | HL-60 |
|---|---|---|---|---|---|
| 2a | 4.23 | 4.16 | 2i | 7.39 | 6.75 |
| 2c | 9.88 | 10.74 | 21b | 46.5 | 39.2 |
| 2d | 23.02 | 22.64 | Chidamide | 4.25 | 3.68 |
a) IC50 values are averages of three independent experiments, S.D. <10%.
In summary, we designed and synthesized a series of DKP-based class I HDACs-targeted inhibitors. As expected, most of these compounds exhibited distinct inhibitory activities against HDAC1. Compound 2a showed the greatest HDAC1 activity with an IC50 value of 405 nM, and exhibited an excellent selective inhibition on HDAC1–3. We performed molecular docking of 2a, 2b, 2c and 2i, and the results suggested that these compounds could bind well with the active site of HDAC1. In vitro, 2a showed a comparable antiproliferative activity to chidamide. The activities difference between 2a and 21b further indicated that HDAC1 and HDAC6 performed different roles in cancer. And this study might provide a reference for follow-up development of potent HDACis.
All of the starting materials were obtained commercially and were used without further purification. All of the reported yields were for isolated products and were not optimized. Melting points were determined in open capillaries on a WRS-1A digital melting point apparatus (Shenguang). 1H-NMR spectra was recorded in dimethyl sulfoxide (DMSO)-d6 on a Bruker DRX-500 (500 MHz) using TMS as internal standard. 13C-NMR spectra was recorded in DMSO-d6 on a Bruker DRX-500 (126 MHz) using TMS as internal standard. The chemical shifts were reported in ppm (δ) and coupling constants (J) values were given in Hertz (Hz). Mass spectra were obtained from Agilent 1100 LC/MSD (Agilent) or Q-tof micro MS (Micromass) and the high-resolution (HR) electrospray ionization-time of flight (ESI-TOF)-MS was recorded on Agilent 6224 A (TOF) LC/MS. The purity of all tested compounds was established by HPLC to be >95.0%. HPLC analysis was performed at room temperature (r.t.) using an Agilent Eclipse XDB-C18 (250 × 4.6 mm) and 30% MeOH/H2O as a mobile phase and plotted at 254 nm.
(S)-4-((3-((1H-indol-3-yl)methyl)-2,5-dioxopiperazin-1-yl)methyl)-N-(2-aminophenyl)benzamide (2a)To a stirred mixture of 1a (100 mg, 0.265 mmol), HATU (93.6 mg, 0.292 mmol) and DIPEA (0.184 mL, 1.06 mmol) in 10 mL of DMF was added benzene-1,2-diamine (28.66 mg, 0.265 mmol) at 0°C. After stirring at r.t. for 4 h, the reaction mixture was diluted with saturated sodium chloride (50 mL) and extracted with EtOAc (50 mL × 3). The combined organic extracts were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The product was obtained by chromatography on a silica gel column (70.0 mg, 56.5%). mp: 145–147°C. 1H-NMR (500 MHz, DMSO-d6) δ: 10.98 (s, 1H), 9.60 (s, 1H), 8.38 (s, 1H), 7.82 (d, J = 7.9 Hz, 2H), 7.57 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.19 (d, J = 7.4 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H), 7.07 (d, J = 1.7 Hz, 1H), 7.05–6.94 (m, 4H), 6.80 (d, J = 7.2 Hz, 1H), 6.62 (t, J = 7.3 Hz, 1H), 4.92 (s, 2H), 4.35 (dd, J = 34.5, 16.4 Hz, 3H), 3.45–3.38 (m, 2H), 3.08 (dd, J = 14.4, 4.0 Hz, 1H), 2.76 (d, J = 17.1 Hz, 1H). 13C-NMR (126 MHz, DMSO-d6) δ: 166.69, 165.60, 165.17, 143.54, 139.73, 136.52, 134.24, 128.45, 127.99, 127.93, 127.09, 126.95, 125.20, 123.84, 121.54, 119.22, 119.03, 116.78, 116.63, 111.82, 108.60, 56.18, 49.01, 48.49, 30.09. HR-MS (ESI, m/z): Calcd for: 468.2030. (C27H26N5O3+, [M + H]+). Found: 468.2033.
(S)-4-((3-((1H-indol-3-yl)methyl)-2,5-dioxopiperazin-1-yl)methyl)-N-(2-amino-4-fluorophenyl) Benza-mide (2b)Using the synthetic method for 2a, compound 1a and 4-fluorobenzene-1,2-diamine gave 2b as a white solid, 61.2% yield. mp: 198–200°C. 1H-NMR (500 MHz, DMSO-d6) δ: 10.99 (s, 1H), 9.54 (s, 1H), 8.39 (s, 1H), 7.82 (d, J = 7.9 Hz, 2H), 7.57 (d, J = 7.9, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.12 (dd, J = 14.8, 7.7 Hz, 2H), 7.07 (d, J = 1.6 Hz, 1H), 7.02 (t, J = 7.7 Hz, 3H), 6.56 (dd, J = 11.2, 2.6 Hz, 1H), 6.45–6.32 (m, 1H), 5.25 (s, 2H), 4.39 (d, J = 15.0 Hz, 1H), 4.31 (d, J = 14.9 Hz, 2H), 4.06 (m, 1H), 3.43–3.38 (m, 1H), 3.08 (dd, J = 14.4, 4.1 Hz, 1H), 2.75 (d, J = 17.1 Hz, 1H). 13C-NMR (126 MHz, DMSO-d6) δ: 166.69, 165.85, 165.17, 160.58, 145.90, 139.75, 136.51, 134.10, 128.97, 128.47, 127.97, 127.92, 125.21, 121.54, 119.76, 119.21, 119.02, 111.82, 108.58, 102.53, 101.96, 56.17, 48.98, 48.17, 30.09. HR-MS (ESI, m/z): Calcd for: 486.1936. (C27H25FN5O3+, [M + H]+). Found: 486.1937.
(S)-N-(2-Aminophenyl)-4-((3-benzyl-2,5-dioxopiperazin-1-yl)methyl)benzamide (2c)Using the synthetic method for 2a, compound 1b and benzene-1, 2-diamine gave 2c as a white solid, 75.3% yield. mp: 172–174°C. 1H-NMR (500 MHz, DMSO-d6) δ: 9.70 (s, 1H), 8.40 (s, 1H), 7.96 (d, J = 6.8 Hz, 2H), 7.31–7.18 (m, 6H), 7.14 (d, J = 6.3 Hz, 2H), 7.01 (t, J = 6.9 Hz, 1H), 6.83 (d, J = 7.1 Hz, 1H), 6.64 (t, J = 6.7, 1H), 4.94 (s, 2H), 4.66 (d, J = 14.8 Hz, 1H), 4.33 (d, J = 6.1 Hz, 2H), 3.54 (d, J = 17.2 Hz, 1H), 3.21 (d, J = 13.3 Hz, 1H), 3.06–2.89 (m, 1H), 2.80 (d, J = 17.2 Hz, 1H). 13C-NMR (126 MHz, DMSO-d6) δ: 166.04, 165.51, 165.28, 143.66, 139.69, 136.11, 134.32, 130.57, 128.62, 128.45, 127.32, 127.23, 127.01, 123.77, 116.75, 116.61, 56.06, 48.94, 48.67, 39.53. HR-MS (ESI, m/z): Calcd for: 429.1921. (C25H25N4O3+, [M + H]+). Found: 429.1923.
(S)-N-(2-Amino-4-fluorophenyl)-4-((3-benzyl-2,5-dioxopiperazin-1-yl)methyl)benzamide (2d)Using the synthetic method for 2a, compound 1b and 4-fluorobenzene-1,2-diamine gave 2d as a white solid, 66.8% yield. mp: 175–177°C. 1H-NMR (500 MHz, DMSO-d6) δ: 9.62 (s, 1H), 8.40 (s, 1H), 7.95 (d, J = 6.1 Hz, 2H), 7.33–7.19 (m, 5H), 7.15 (s, 3H), 6.58 (d, J = 10.9 Hz, 1H), 6.39 (s, 1H), 5.26 (s, 2H), 4.65 (d, J = 14.7 Hz, 1H), 4.34 (d, J = 12.9 Hz, 2H), 3.53 (d, J = 17.2 Hz, 1H), 3.21 (d, J = 13.1 Hz, 1H), 2.96 (d, J = 13.0 Hz, 1H), 2.80 (d, J = 17.2 Hz, 1H). 13C-NMR (126 MHz, DMSO-d6) δ: 166.03, 165.78, 165.27, 160.60, 146.00, 139.72, 136.12, 134.20, 130.56, 129.08, 128.62, 128.48, 128.40, 127.33, 119.72, 102.52, 101.94, 56.06, 48.94, 48.66, 39.53. HR-MS (ESI, m/z): Calcd for: 447.1827. (C25H24FN4O3+, [M + H]+). Found: 447.1828.
(S)-N-(2-Aminophenyl)-3-((3-benzyl-2,5-dioxopiperazin-1-yl)methyl)benzamide (2e)Using the synthetic method for 2a, compound 1c and benzene-1,2-diamine gave 2e as a white solid, 56.3% yield. mp: 174–176°C. 1H-NMR (500 MHz, DMSO-d6) δ: 9.74 (s, 1H), 8.37 (s, 1H), 7.94 (d, J = 7.5 Hz, 1H), 7.86 (s, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.34 (d, J = 7.4 Hz, 1H), 7.28–7.12 (m, 4H), 7.09 (d, J = 7.1 Hz, 2H), 7.01 (t, J = 7.3 Hz, 1H), 6.82 (d, J = 7.8 Hz, 1H), 6.63 (t, J = 7.3 Hz, 1H), 4.93 (s, 2H), 4.66 (d, J = 14.4 Hz, 1H), 4.31 (d, J = 14.1, 2H), 3.53 (d, J = 17.2, 1H), 3.19 (dd, J = 13.4, 3.6 Hz, 1H), 2.94 (d, J = 4.9 Hz, 1H), 2.70 (d, J = 17.3 Hz, 1H). 13C-NMR (126 MHz, DMSO-d6) δ: 166.00, 165.70, 165.39, 143.69, 136.50, 135.98, 131.81, 130.53, 128.94, 128.59, 128.54, 127.42, 127.24, 127.21, 127.05, 123.73, 116.76, 116.63, 60.25, 56.02, 48.82, 36.27. HR-MS (ESI, m/z): Calcd for: 429.1921. (C25H25N4O3+, [M + H]+). Found: 429.1923.
(S)-N-(2-amino-4-fluorophenyl)-3-((3-benzyl-2,5-dioxopiperazin-1-yl)methyl)benzamide (2f)Using the synthetic method for 2a, compound 1c and 4-fluorobenzene-1,2-diamine gave 2f as a white solid, 71.6% yield. mp: 123–125°C. 1H-NMR (500 MHz, DMSO-d6) δ: 9.69 (s, 1H), 8.40 (s, 1H), 7.96 (d, J = 6.9 Hz, 2H), 7.35–7.18 (m, 6H), 7.14 (d, J = 6.3 Hz, 2H), 7.01 (t, J = 7.0 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.65 (d, J = 6.9 Hz, 1H), 4.93 (s, 2H), 4.66 (d, J = 14.7 Hz, 1H), 4.33 (d, J = 4.4 Hz, 2H), 3.54 (d, J = 17.2 Hz, 1H), 3.28–3.11 (m, 1H), 3.07–2.87 (m, 1H), 2.80 (d, J = 17.2 Hz, 1H). 13C-NMR (126 MHz, DMSO-d6) δ: 166.00, 165.39, 162.80, 160.63, 146.00, 136.47, 135.98, 135.28, 131.81, 130.53, 129.05, 128.92, 128.58, 128.54, 127.45, 127.24, 119.68, 102.57, 101.97, 60.25, 56.02, 48.82, 36.27. HR-MS (ESI, m/z): Calcd for: 447.1827. (C25H24FN4O3+, [M + H]+). Found: 447.1828.
(R)-N-(2-Aminophenyl)-4-((3-benzyl-2,5-dioxopiperazin-1-yl)methyl)benzamide (2g)Using the synthetic method for 2a, compound 1d and benzene-1,2-diamine gave 2g as a white solid, 85.7% yield. mp: 168–170°C. 1H-NMR (500 MHz, DMSO-d6) δ: 9.69 (s, 1H), 8.40 (s, 1H), 7.96 (d, J = 6.9 Hz, 2H), 7.35–7.18 (m, 6H), 7.14 (d, J = 6.3 Hz, 2H), 7.01 (t, J = 7.0 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.65 (d, J = 6.9 Hz, 1H), 4.93 (s, 2H), 4.66 (d, J = 14.7 Hz, 1H), 4.33 (d, J = 4.4 Hz, 2H), 3.54 (d, J = 17.2 Hz, 1H), 3.28–3.11 (m, 1H), 3.07–2.87 (m, 1H), 2.80 (d, J = 17.2 Hz, 1H). 13C-NMR (126 MHz, DMSO-d6) δ: 166.03, 165.51, 165.28, 143.66, 139.69, 136.11, 134.32, 130.57, 128.62, 128.45, 127.32, 127.23, 123.77, 116.74, 116.61, 56.06, 48.94, 48.67, 39.53. HR-MS (ESI, m/z): Calcd for: 429.1921. (C25H25N4O3+, [M + H]+). Found: 429.1923.
(R)-N-(2-Amino-4-fluorophenyl)-4-((3-benzyl-2,5-dioxopiperazin-1-yl)methyl)benzamide (2h)Using the synthetic method for 2a, compound 1d and 4-fluorobenzene-1, 2-diamine gave 2h as a white solid, 77.2% yield. mp: 155–157°C. 1H-NMR (500 MHz, DMSO-d6) δ: 9.69 (s, 1H), 8.40 (s, 1H), 7.96 (d, J = 6.9 Hz, 2H), 7.35–7.18 (m, 6H), 7.14 (d, J = 6.3 Hz, 2H), 7.01 (t, J = 7.0 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.65 (d, J = 6.9 Hz, 1H), 4.93 (s, 2H), 4.66 (d, J = 14.7 Hz, 1H), 4.33 (d, J = 4.4 Hz, 2H), 3.54 (d, J = 17.2 Hz, 1H), 3.28–3.11 (m, 1H), 3.07–2.87 (m, 1H), 2.80 (d, J = 17.2 Hz, 1H). 13C-NMR (126 MHz, DMSO-d6) δ: 166.04, 165.78, 165.26, 160.60, 145.98, 139.72, 136.13, 134.20, 130.56, 129.06, 128.61, 128.47, 128.40, 127.32, 119.73, 102.52, 101.94, 56.07, 48.94, 48.67, 39.54. HR-MS (ESI, m/z): Calcd for: 447.1827. (C25H24FN4O3+, [M + H]+). Found: 447.1828.
(R)-4-((3-((1H-Indol-3-yl)methyl)-2,5-dioxopiperazin-1-yl)methyl)-N-(2-aminophenyl)benzamide (2i)Using the synthetic method for 2a, compound 1e and benzene-1, 2-diamine gave 2i as a white solid, 74.6% yield. mp: 161–163°C. 1H-NMR (500 MHz, DMSO-d6) δ: 10.99 (s, 1H), 9.61 (s, 1H), 8.39 (d, 1H, J = 2.3 Hz), 7.82 (d, 2H, J = 8.0 Hz), 7.58 (d, 1H, J = 7.9 Hz), 7.40 (d, 1H, J = 8.1 Hz), 7.19 (d, 1H, J = 7.6 Hz), 7.12 (t, 1H, J = 7.5 Hz), 7.07 (d, 1H, J = 2.0 Hz), 7.01 (m, 4H), 6.81 (d, 1H, J = 7.3 Hz), 6.63 (d, 1H, J = 7.5 Hz), 4.92 (s, 2H), 4.47–4.20 (m, 3H), 3.44–3.37 (m, 2H), 3.08 (dd, 1H, J1 = 14.4 Hz, J2 = 4.3 Hz), 2.76 (d, 1H, J = 17.1 Hz). 13C-NMR (126 MHz, DMSO-d6) δ: 166.65, 165.55, 165.15, 143.58, 139.73, 136.48, 134.20, 128.44, 127.97, 127.90, 127.11, 126.96, 125.20, 123.76, 121.53, 119.21, 119.02, 116.71, 116.57, 111.81, 108.56, 56.15, 48.97, 48.42, 30.07.
(R)-4-((3-((1H-Indol-3-yl)methyl)-2,5-dioxopiperazin-1-yl)methyl)-N-(2-amino-4-chlorophenyl)benzamide (2j)Using the synthetic method for 2a, compound 1e and 4-chlorobenzene-1,2-diamine gave 2i as a white solid, 69.7% yield. mp: 168–170°C. 1H-NMR (500 MHz, DMSO-d6) δ: 10.99 (d, 1H, J = 1.3 Hz), 9.59 (s, 1H), 8.40 (s, 1H), 7.83 (d, 2H, J = 8.0 Hz), 7.59 (d, 1H, J = 7.9 Hz), 7.41 (d, 1H, J = 8.1 Hz), 7.20 (1H, J = 8.4 Hz), 7.12 (t, 1H, J = 7.4 Hz), 7.08 (d, 1H, J = 2.1 Hz), 7.03 (t, 3H, J = 7.8 Hz), 6.85 (d, 1H, J = 2.4 Hz), 6.63 (dd, 1H, J1 = 8.4 Hz, J2 = 2.3 Hz), 5.27 (s, 2H), 4.52–4.21 (m, 3H), 3.44–3.39 (m, 2H), 3.09 (dd, 1H, J1 = 14.4 Hz, J2 = 4.3 Hz), 2.76 (d, 1H, J = 17.1 Hz). 13C-NMR (126 MHz, DMSO-d6) δ: 166.67, 165.78, 165.16, 145.28, 142.41, 139.86, 136.49, 134.02, 130.93, 128.70, 128.51, 127.98, 127.91, 125.21, 122.45, 121.54, 119.22, 119.03, 115.87, 115.25, 111.82, 108.57, 56.16, 48.97, 48.44, 30.09.
(R)-4-((3-((1H-Indol-3-yl)methyl)-2,5-dioxopiperazin-1-yl)methyl)-N-(2-amino-4-methylphenyl)benzamide (2k)Using the synthetic method for 2a, compound 1e and 4-methylbenzene-1,2-diamine gave 2i as a white solid, 55.2% yield. mp: 165–167°C. 1H-NMR (500 MHz, DMSO-d6) δ: 10.98 (s, 1H), 9.53 (s, 1H), 8.38 (d, 1H, J = 2.1 Hz), 7.82 (d, 2H, J = 7.9 Hz), 7.57 (d, 1H, J = 7.9 Hz), 7.40 (d, 1H, J = 8.1 Hz), 7.12 (t, 1H, J = 7.5 Hz), 7.09–7.05 (m, 1H), 7.04 (d, 1H, J = 3.5 Hz), 7.01 (d, 3H, J = 8.2 Hz), 6.62 (s, 1H), 6.44 (d, 1H, J = 7.6 Hz), 4.83 (s, 2H), 4.35 (m, 3H), 3.43–3.37 (m, 2H), 3.08 (dd, 1H, J1 = 14.4 Hz, J2 = 4.3 Hz), 2.76 (d, 1H, J = 17.1 Hz), 2.22 (s, 3H). 13C-NMR (126 MHz, DMSO-d6) δ: 166.65, 165.52, 165.15, 143.38, 139.65, 136.48, 135.97, 134.26, 128.41, 127.96, 127.91, 127.02, 125.20, 121.53, 121.35, 119.21, 119.01, 117.56, 116.97, 111.81, 108.56, 56.15, 48.97, 48.43, 30.07, 21.33.
In Vitro HDAC Enzyme AssayThe inhibitory activity of HDAC isoforms were conducted by the Reaction Biology Corporation, Malvern, PA using HDAC fluorescent activity assay based on the unique Fluor de Lys™ substrate and developer combination. Compounds were dissolved in DMSO and tested in at least 10-dose IC50 mode with 3-fold serial dilution starting at 50 µM. For HDAC assays: Add 2× of HDAC enzyme into reaction plate except control wells (no enzyme), where buffer (50 mM Tris–HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl2) was added instead. Add inhibitors in 100% DMSO into the enzyme mixture via acoustic technology (Echo550; nanoliter range). Spin down and preincubate. Add 2× substrate mixture: Fluorogenic HDAC General Substrate: 50 µM, Arg-His-Lys-Lys(Ac). Spin and shake. Incubate for 2 h at 30°C with seal. Add developer with trichostatin A to stop the reaction and to generate fluorescent color. Carry out kinetic measurements for 1.5 h with Envision with 15 min interval (Ex/Em = 360/460 nm). Take end point reading for analysis after the development reaches plateau. GraphPad Prism 5.0 software was used to calculate the IC50 values for each compound.
Cell Culture and Cytotoxicity/Proliferation AssayCulture Medium and Culture Condition of Cell Lines. The cells were cultured in IMDM medium with 20% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin. All cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in air.
Cell Growth Inhibitory Assay. The protocol using Alamar blue reagent for antiproliferative activitives was as follows: 1. plated 100 µL cell suspension or completed medium into 96-well plate using a Matrix 12-channel pipettor. And filled residual wells with 200 µL PBS per well; 2. drugs were added to each well of 96-well plate; 3. placed plate into an incubator with corresponding culture condition for 72 h; 4. pipetted 22 µL Alamar blue solution (1 mM) into each well of 96-well plate; 5. returned plate to incubator and leave for 5–6 h; 6. shook plate for 10 s and recorded fluorescence at 530/590 nm.
Computational MethodsAll computational work was performed in Discovery Studio 3.0 software (BIOVIA, 5005 Wateridge Vista Drive, San Diego, CA92121 U.S.A.). Docking was conducted using cdocker based on the cocrystal of HDAC1 (PDB: 5ICN). HDAC1 was used as the receptor. The cavity occupied by a peptide inhibitor was selected as the ligand binding site. The docking sphere radius value based on the peptide inhibitor was default. Water molecules outside the binding pocket were excluded. The energy minimization for compound 2a, 2c and 2g were performed by Powell’s method for 1000 iterations using Tripos force field and with Gasteiger–Hückel charge. The other docking parameters were kept as default.
This work was supported by the National Natural Science Foundation of China (81903464, 21503168 and 31800031).
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
