Angiogenesis and anti-leukaemia activity of novel indole derivatives as potent colchicine binding site inhibitors

Figures & data

Figure 1. Representative anti-angiogenic agents binding to tubulin colchicine binding site.

Figure 2. Identification of hit compound (DYT-1) from our chemical library and further optimizations to discover lead compound (29e).

Scheme 1. Reagents and conditions: (i) EtOH, 40% NaOH, 0 °C, 30 min; room temperature, 4 h.

Scheme 2. Reagents and conditions: (i) EtOH, 40% NaOH, 0 °C, 30 min; room temperature, 4 h; (ii) NaH, different haloalkanes, THF, 0 °C to room temperature.

Table 1. Tubulin inhibitory activities and antiangiogenic activities in Zebrafish of chalcone derivatives with variation of the LHS

Cpd.	R	Tubulin^a(μM)	Zebrafish^a(μM)
6		8.5 ± 1.2	27.8 ± 0.9
7		18.6 ± 0.5	29.5 ± 1.5
8		45.7 ± 2.3	68.6 ± 2.1
9		20.8 ± 1.4	>100
CA-4		9.0 ± 1.7	ND^b

^aEach compound was tested in triplicate; the data are presented as the mean ± SD.

^bND: not detected because of its strong toxicity.

Table 2. Tubulin inhibitory activities and antiangiogenic activities in Zebrafish of chalcone derivatives with variation of both LHS and RHS.

Cpd.	R^1	R^2	Tubulin^a(μM)	Zebrafish^a(μM)
23a	H	-CH3	>100	–^b
23b	H	-CH2CH3	>100	–
23c	H	-CH2CH2CH3	>100	–
23d	H	-CH2-Ph	68.6 ± 3.2	–
23e	H	-SO2-Ph	>100	–
24a	−3-Cl	-CH3	>100	–
24b	−3-Cl	-CH2CH3	>100	–
24c	−3-Cl	-CH2CH2CH3	>100	–
24d	−3-Cl	-CH2-Ph	>100	–
24e	−3-Cl	-SO2-Ph	92.3 ± 2.4	–
25a	−4-Cl	-CH3	>100	–
25b	−4-Cl	-CH2CH3	>100	–
25c	−4-Cl	-CH2CH2CH3	>100	–
25d	−4-Cl	-CH2-Ph	>100	–
25e	−4-Cl	-SO2-Ph	>100	–
26a	−4-OCH3	-CH3	85.7 ± 2.5	–
26b	−4-OCH3	-CH2CH3	78.8 ± 1.3	–
26c	−4-OCH3	-CH2CH2CH3	72.5 ± 1.7	–
26d	−4-OCH3	-CH2-Ph	75.8 ± 2.6	–
26e	−4-OCH3	-SO2-Ph	58.4 ± 1.7	35.6 ± 3.2
27a	−3-OCH3	-CH3	>100	–
27b	−3-OCH3	-CH2CH3	>100	–
27c	−3-OCH3	-CH2CH2CH3	52.1 ± 2.9	18.6 ± 3.2
27d	−3-OCH3	-CH2-Ph	>100	–
27e	−3-OCH3	-SO2-Ph	87.2 ± 1.4	–
28a	−4-CF3	-CH3	>100	–
28b	−4-CF3	-CH2CH3	>100	–
28c	−4-CF3	-CH2CH2CH3	26.8 ± 4.6
28d	−4-CF3	-CH2-Ph	>100	–
28e	−4-CF3	-SO2-Ph	10.4 ± 0.7	5.8 ± 2.1
29a	−3,4,5-OCH3	-CH3	49.9 ± 8.5	15.7 ± 3.2
29b	−3,4,5-OCH3	-CH2CH3	>100	–
29c	−3,4,5-OCH3	-CH2CH2CH3	>100	–
29d	−3,4,5-OCH3	-CH2-Ph	24.8 ± 2.1	6.7 ± 0.5
29e	−3,4,5-OCH3	-SO2-Ph	4.8 ± 0.7	3.4 ± 0.3

^aEach compound was tested in triplicate; the data are presented as the mean ± SD.

^bNot tested.

Table 3. IC₅₀ Values of 29e and CA-4 against various leukaemia cell lines

Tumour type	Cell line	IC50^a(μM)
		29e	CA-4
leukaemia, AML	MV4-11	0.25 ± 0.02	0.12 ± 0.03
leukaemia, APL	HL60	0.18 ± 0.002	0.26 ± 0.05
leukaemia, CML	K562	0.09 ± 0.001	0.27 ± 0.01
leukaemia, AML	THP-1	0.37 ± 0.03	0.08 ± 0.01
leukaemia, ALL	CCRF-CEM	0.84 ± 0.08	0.71 ± 0.11
leukaemia, ALL	Jurkat	1.22 ± 0.12	0.19 ± 0.02
leukaemia, ALL	HuT 78	0.26 ± 0.11	0.24 ± 0.04

^aEach compound was tested in triplicate; the data are presented as the mean ± SD.

Figure 3. Compound 29e induced apoptosis K562 cells. (A) Apoptosis ratio detection by flow cytometry assays for 48 h. (B) The quantitative analysis of apoptotic rate at early and advanced stages of K562 cells. (C) Western blot analysis of the apoptosis related proteins. (D) The quantitative analysis of the protein levels. The data was presented as the mean ± SD of three independent tests.

Figure 4. Compound 29e bind to the colchicine site of tubulin and inhibit the microtubule polymerisation. (A) 2 D model of the interaction between compound 29e and the amino acid residues of tubulin colchicine site. (B) 3 D model of the binding position of compound 29e. (C) RMSD tendency of two systems (29e, colchicine) versus time in the 60 ns MD simulation. (D) Effect of compound 29e on tubulin binding of [³H] colchicine. (E) Compound 29e affected microtubule assembly in vitro. (F) The effects of 29e on the organisation of cellular microtubule network of K562 cells.

Figure 5. Compound 29e showed antivascular activity in vitro. (A) The wound-healing assay was used to evaluate the migration of HUVEC cells, and images were captured at 0 h and 48 h after treatments with 29e. (B) The invasion suppressing effects of 29e against HUVECs cells by Transwell assay. (C) Typical images depicting tubule formation of HUVEC cells by treatments with 29e for 6 h. (D) Quantitative analysis of the migration ability of HUVEC tubule formation.

Figure 6. Anti-angiogenic effect of 29e evaluated by zebrafish embryos assay. (A)Inhibitory effect of 29e on the angiogenesis of transgenic zebrafish. (B) Histogram showed the numbers of zebrafish ISVs per field under confocal microscopy.

Figure 7. Inhibitory effects of 29e on the proliferation and metastasis of K562 cells in zebrafish xenograft models. CMTPX labelled K562 cells (red) were microinjected into zebrafish embryos, and indicated concentration of 29e were added. White solid arrows indicate disseminated cells.

Table 4. Physicochemical parameters and metabolic stability of compound 29e

MW^a	PSA^a	cLogD^a	Solubility^b(μg/mL)		Degradation half-life (min)		In Vitro Clint (µL/min/mg protein)
			pH 2.0	pH 6.5	Human	Mouse	Human	Mouse
477.5	83.85	3.7	25–50	75–150	35	60	16	77

^aCalculated using ChemAxon JChem software; ^bKinetic solubility determined by nephelometry (SolpH).