Novel HDAC/Tubulin Dual Inhibitor: Design, Synthesis and Docking Studies of α-Phthalimido-Chalcone Hybrids as Potential Anticancer Agents with Apoptosis-Inducing Activity

Figures & data

Figure 1 Examples of some approved HDACi.

Figure 2 Examples on some microtubule targeting agents with affinity for colchicine-binding site.

Figure 3 Diagram represents the design strategy of novel HDAC/tubulin inhibitors and the reported anticancer compounds CA-4 and entinostat.

Scheme 1 Synthesis of compounds 6 and 7: Reagents and conditions: (A) Zn dust, NaOH, CuSO₄; (B) KOH, Acetone; (C) DMF, reflux 6 hr, HCl; SOCl₂, reflux, 4 hr; (D) 4-Aminoacetophenone, CH₂Cl₂, TEA, 4 hr, rt; (E) Aromatic aldehyde, 20% NaOH, EtOH, 8–20 hr, rt.

Figure 4 X-ray structural analysis of compound 6. Ellipsoids represent 50% probability levels.

Table 1 IC₅₀ Values for Anticancer Activity of Compounds 6, 7a–7j and CA4 Using MTT Assay Against MCF-7, and HepG2 Cell Lines

Compound No.	IC50 (µM)^a
	MCF-7	Hep G2
6	17.59 ± 0.46	10.82 ± 0.63
7a	14.43 ± 0.51	12.93 ± 0.26
7b	4.11 ± 0.17	3.21 ± 0.17
7c	2.41 ± 0.11	1.87 ± 0.13
7d	11.31 ± 0.43	8.07 ± 0.39
7e	5.28 ± 0.25	8.47± 0.34
7f	9.78 ± 0.54	15.85± 0.71
7g	2.09 ± 0.19	1.75 ± 0.08
7h	5.44 ± 0.21	9.05 ± 0.33
7i	3.45 ± 0.27	1.96 ± 0.07
7j	1.88 ± 0.06	1.62 ± 0.02
CA-4^b	4.85 ± 0.18	7.32 ± 0.28

Notes:^aMean IC₅₀ value of three independent experiments. ^bCA-4: Positive reference.

Figure 5 (A) IC₅₀ values for anticancer activity of compounds 6, 7a–7j and CA4 using MTT assay against MCF-7 cell line. (B) IC₅₀ values for anticancer activity of compounds 6, 7a–7j and CA-4 using MTT assay against Hep G2 cell line.

Table 2 IC₅₀ Values for in vitro β-Tubulin Polymerization Inhibitory Activity of 7c, 7g, 7j, and CA-4 Using ELISA Assay

Compound No.	Tubulin IC50 (μM)^a
7c	3.68 ± 0.21
7g	3.27 ± 0.33
7j	2.32 ± 0.15
CA-4^b	2.62 ± 0.14

Notes:^aMean IC₅₀ value of three independent experiments. ^bCA-4: Positive reference.

Figure 6 IC₅₀ values for in vitro β-tubulin polymerization inhibitory activity of 7c, 7g, 7j and CA-4 using ELISA assay.

Table 3 IC₅₀ Values for in vitro HDAC1 & 2 Inhibitory Activity of Compounds 7c, 7g, 7j and Entinostat

Compound No.	HDAC1 IC50 (µM)	HDAC2 IC50 (µM)^a
7c	0.49 ± 0.03	0.85 ± 0.06
7g	0.32 ± 0.01	0.56 ± 0.03
7j	0.22 ± 0.01	0.43 ± 0.02
Entinostat^b	0.28 ± 0.02	0.55 ± 0.04

Notes:^aMean IC₅₀ value of three independent experiments. ^bEntinostat: Positive reference.

Figure 7 IC₅₀ values for in vitro HDAC1 and HDAC2 inhibitory activity of compounds 7c, 7g, 7j and entinostat.

Table 4 Cell Cycle Analysis of Compounds 7j, 7g, CA-4 and Entinostat Against MCF-7 Cell Line

Compound No.	DNA Content
	% G0-G1	% S	% G2/M	%Pre-G1	Comment
7j	27.58	24.32	48.1	23.52	PreG1 apoptosis and Cell growth arrest at G2/M
7g	31.52	23.94	44.54	24.63	PreG1 apoptosis and Cell growth arrest at G2/M
CA-4	27.43	26.72	45.85	29.03	PreG1 apoptosis and Cell growth arrest at G2/M
Entinostat	43.29	25.41	31.3	22.43	PreG1 apoptosis and Cell growth arrest at G2/M
Control	55.46	29.71	14.83	1.78	–

Figure 8 (A) Cell cycle analysis in MCF-7 cell line treated with 7j and 7g hybrids at their IC₅₀ (µM). (B) Cell cycle analysis and apoptosis effect in MCF-7 cell line treated with 7j, 7g, CA-4 and entinostat compounds.

Table 5 Results of Apoptotic Assay of 7j, 7g, CA-4 and Entinostat

Compound No.	Apoptosis			Necrosis
	Total	Early	Late
7j	23.52	6.75	14.72	2.05
7g	24.63	6.99	14.93	2.71
CA-4	29.03	4.92	21.65	2.46
Entinostat	22.43	8.67	10.68	3.08
Control	1.78	0.47	0.33	0.98

Figure 9 Flow cytometric analysis of Annexin V-FITC/PI induced by compounds 7j, 7g, CA-4, entinostat at their IC₅₀ (µM).

Table 6 Energy Binding Scores (Kcal/mol) for Compounds 7j, 7g, 7c and CA4 with the Colchicine Binding Site

Compound No.	Energy Binding Scores
7j	−9.10
7g	−8.87
7c	−7.20
CA4	−6.76

Figure 10 Docking and binding pattern of compounds 7j, 7g, 7c and CA-4 showing interactions with different amino acid residues found in the active site of colchicine binding site in tubulin protein (PDB code: 1SA0). (A) 3D structure of compound 7j (yellow) (B) 2D structure of compound 7j. (C) 3D structure of compound 7g (yellow) (D) 2D structure of compound 7g. (E) 3D structure of compound 7c (yellow) (F) 2D structure of compound 7c. (G) 3D structure of reference compound CA-4 (yellow) (H) 2D structure of reference compound CA-4.

Table 7 Energy Binding Scores (Kcal/mol) for Compounds 7j, 7g, 7c and Entinostat at the Active Site of HDAC2

Compound No.	Energy Binding Scores
7j	−9.01
7g	−8.43
7c	−8.39
Entinostat	−7.67

Figure 11 Docking and binding pattern of compounds 7j, 7g, 7c and CA-4 showing interactions with different amino acid residues found in HDAC2 active site (PDB code: 4LXZ). (A) 3D structure of compound 7j (yellow) (B) 2D structure of compound 7j. (C) 3D structure of compound 7g (yellow) (D) 2D structure of compound 7g. (E) 3D structure of compound 7c (yellow) (F) 2D structure of compound 7c. (G) 3D structure of entinostat reference compound (yellow) (H) 2D structure of entinostat reference compound.