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. 2017 Jun 28;8(38):63187-63207.
doi: 10.18632/oncotarget.18730. eCollection 2017 Sep 8.

Structure Based Design, Synthesis and Activity Studies of Small Hybrid Molecules as HDAC and G9a Dual Inhibitors

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Free PMC article

Structure Based Design, Synthesis and Activity Studies of Small Hybrid Molecules as HDAC and G9a Dual Inhibitors

Lanlan Zang et al. Oncotarget. .
Free PMC article

Abstract

Aberrant enzymatic activities or expression profiles of epigenetic regulations are therapeutic targets for cancers. Among these, histone 3 lysine 9 methylation (H3K9Me2) and global de-acetylation on histone proteins are associated with multiple cancer phenotypes including leukemia, prostatic carcinoma, hepatocellular carcinoma and pulmonary carcinoma. Here, we report the discovery of the first small molecule capable of acting as a dual inhibitor targeting both G9a and HDAC. Our structure based design, synthesis, and screening for the dual activity of the small molecules led to the discovery of compound 14 which displays promising inhibition of both G9a and HDAC in low micro-molar range in cell based assays.

Keywords: G9a inhibitors; HDAC inhibitors; dual inhibitors; epigenetics; pharmacophore.

Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no competing financial interest.

Figures

Figure 1
Figure 1. Examples for known HDAC inhibitors and G9a Inhibitors
Figure 2
Figure 2. Diagram for Drug design
(A) Design for Hybrid Molecules, (B) Structural representation of designed compounds (R = Linker + Hydroxamic acid).
Figure 3
Figure 3. Reagents and conditions for scheme 1
(i) 1-methylpiperidin-4-amine/1-isopropylpiperidin-4-amine, DIPEA, DMF, rt, 3 h, 80-86%, (ii) tert-butyl (2-aminoethyl)carbamate, DIPEA, 160°CMicrowave, 10 min, 60-66%, (iii) TFA/DCM, 3 h, (iv) MonomethylSuberate, EDCl, HOBt, 8 h, (v) 50% NH2OH in water, MeOH, 60°C, 8 h, 30-38% over two steps.
Figure 4
Figure 4. Reagents and conditions for scheme 2
(i) Urea, 200°C, 2 h, (ii) POCl3, reflux 16 h, 40% in two steps, (iii) 4-aminobenzylpiperidin/1-isopropylpiperidin-4-amine, DIPEA, DMF, rt, 3 h, 74% and 86%, (iv) tert-butyl (2-aminoethyl)carbamate, DIPEA, 160°C Microwave, 10 min, 64% and 68%, (v) TFA/DCM, 3 h, (vi) MonomethylSuberate, EDCl, HOBt, 8 h, 70% in two steps, (vii) 50% NH2OH in water, MeOH, 60°C, 8 h, 30-40%.
Figure 5
Figure 5. Reagents and conditions for scheme 3
(i) 4-aminobenzylpiperidin, DIPEA, DMF, rt, 3 h, 90%, (ii) 1-methyl-1,4-diazepane, DIPEA, 160°C Microwave, 10 min, 74%, (iii) EtOH, Pd/C, H2, 8 h, (iv) Monomethylsuberate/monomethylpimelate, EDCl, HOBt, 8 h, (v) 50% NH2OH in Water, MeOH, 60°C, 8 h, 44% and 45%, (vi) NHBoc-ethylinediamine, DIPEA, DMF, rt, 3 h, 78%, (vii) 1-methyl-1,4-diazepane, DIPEA, 160°C Microwave, 10 min, 69%, (viii) TFA/DCM, 8 h, (ix) Monomethylsuberate/ monomethylpimelate, EDCl, HOBt, 8 h, (x) 50% NH2OH in water, MeOH, 60°C, 8 h, 29%-36%.
Figure 6
Figure 6. Results of biological assays
(a) Methylation pattern observed via MALDI-TOF after incubating with inhibitor 14 and BIX-01294 for 30 min, (b) % ratio of the H3K9Me0, H3K9Me1 and H3K9Me2 after incubating 30 min with 14 and BIX-01294 versus no inhibitor, (c) In-Cell Western (ICW) assay of 14 and BIX-01294 in MDA-MB 231 cell lines, (d) Result of homogenous histone deacetylase assay of 14 alongside SAHA in K562 cell lines.
Figure 7
Figure 7. Molecular docking study results
(A) Predicted binding mode of compound 14 on HDAC8 (PDB ID: 1T67), (B) Predicted binding mode of compound 14 on G9a (PDB ID: 3FPD), (C) Binding model of active compound 14 (orange) as revealed from GLIDE docking in the HDAC8 catalytic site (PDB ID: 1T67). The green dashed lines represent hydrogen bonds. The turquoise sphere represents Zn cation and with a trigonal bipyramidal coordination geometry. The pink dashed lines indicates the two contacts between the ligand and Zn cation, with the mixed dashed lines representing interaction between Zn cation and amino acid residues. H-bond distances (Å) between heteroatoms of ligand and amino acid residues are as follows: Asp101 (1.90), His142 (2.02), His143 (3.64), Gly151 (3.68), Gly304 (3.00), Tyr306 (2.17), (D) Binding model of active compound 14 (orange) as revealed from GLIDE docking in the G9a catalytic site (PDB ID: 3FPD). The green dashed lines represent hydrogen bonds. H-bond distances (Å) between heteroatoms of ligand and amino acid residues are as follows: Asp1131 (1.66), Asp1135 (1.75, 1.81), Arg1137 (3.33), Glu1138 (3.98), Asp1140 (1.77), Arg1214 (2.68, 2.90).

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