Design, synthesis, and biological evaluation of novel carbazole derivatives as potent DNMT1 inhibitors with reasonable PK properties

Figures & data

Figure 1. The approved or clinically investigated DNMT inhibitors.

Scheme 1. Reagents and conditions: (a) NaO^tBu, Pd(OAc)₂, [HP^tBu₃][BF₄], 4-bromofluorobenzene, toluene, reflux, 4 h; (b) Pd(OAc)₂, [HP^tBu₃][BF₄], NaOtBu, 1,4-dioxane, reflux, 18 h; (c) Epichlorohydrin, KOH, DMF, r.t. (d) Amines, EtOH, 60 °C; (e) 3-fluoro-9H-carbazole (a3) or 3,6-difluoro-9H-carbazole (b3), KOH, Na₂SO₄, acetone, r.t.; (f) Epichlorohydrin, KOH, Na₂SO₄, acetone, r.t.; (g) Amines, isopropanol, 60 °C.

Figure 2. Schematic showing design for binding DNMT1 pharmacophore.

Table 1. Biochemical assay results for DC_05 and DC_517 analogues against DNMT1 catalytic activity.

Cpd.	Scaffold	Substituent	^aInh% at 50 μM	^aInh% at 100 μM	^bIC50 (μM)
WK-1	I		82	100	28
WK-2			55	66	–
WK-3			71	79	27
WK-4			56	60	–
WK-5			59	65	–
WK-6			68	79	–
WK-7			67	83	9.6
WK-8			16	58	–
WK-9			25	53	–
WK-10			5.1	−9.0	–
WK-11			41	50	–
WK-12			99	94	17
WK-13			82	90	19
WK-14			79	84	6.0
WK-15			75	82	7.7
WK-16			27	46	–
WK-17			73	79	–
WK-18			70	87	10
WK-19			78	93	9.2
WK-20	II		68	77	–
WK-21			39	34	–
DC_05			43	88	52
WK-22	III		99	100	4.9
WK-23			99	100	5.0
WK-24			2.3	2.6	–
WK-25			100	100	–
WK-26			8.7	−16	–
WK-27			100	100	–
WK-28			91	91	–
WK-29			85	80	–
WK-30			–	–	–
DC_517			96	98	2.3

^aMeasured by EpiQuik DNA methyltransferase (DNMT) activity/inhibitor assay kit.

^bMeasured by H³-labeled radioactive methylation assay.

Figure 3. Enzymatic selectivity of preliminarily screened compounds (50 μM).

Table 2. The PK properties of selected compound WK-22, WK-27, WK-23, and DC_517

Cpd.	Administration route and dosage	T1/2 (h)	Tmax (h)	AUC(0–t) (h*ng/mL)	F (%)
WK-22	i.v. (2 mg/kg)	12.2 ± 0.8	0.1 ± 0.0	867.2 ± 15.9	27.0 ± 4.6
	p.o. (10 mg/kg)	8.8 ± 1.3	7.3 ± 1.2	1172.5 ± 197.3
WK-27	i.v. (2 mg/kg)	11.5 ± 1.2	0.1 ± 0.0	427.2 ± 60.0	1.8 ± 1.9
	p.o. (10 mg/kg)	3.9 ± 2.5	2.7 ± 1.2	28.5 ± 40.0
WK-23	i.v. (2 mg/kg)	7.9 ± 1.0	0.08 ± 0.0	1064.9 ± 121.2	37.1 ± 1.7
	p.o. (10 mg/kg)	6.5 ± 0.3	6.7 ± 1.2	1973.9 ± 91.1
DC_517	i.v. (2 mg/kg)	6.7 ± 0.2	0.08 ± 0.0	1022.7 ± 60.9	38.7 ± 2.9
	p.o. (10 mg/kg)	6.7 ± 0.9	6.0 ± 0.0	2079.5 ± 112.8

Data were shown as mean ± SD (n = 3).

Figure 4. Effect of WK-23 and DC_517 on the viability of human colon cell lines and human lung cell lines. WK-23 has stronger concentration dependence and time dependence than DC_517, which inhibited the viability of HCT116 and A549 cells in the cell viability assay. Significance between groups was analysed by one-way analysis of variance (ANOVA) using IBM SPSS software. *p < 0.05.

Table 3. Derivatives’ Inh % at 50 μM on HCT116 and A549 cell lines

Cpd.	HCT116			A549
	24 h	48 h	72 h	24 h	48 h	72 h
WK-1	90.9	88.0	94.9	88.2	78.6	87.6
WK-12	77.7	88.8	96.7	59.9	80.1	84.7
WK-13	–	86.3	93.2	–	69.2	84.1
WK-19	5.6	59.4	88.3	7.9	68.4	79.9
WK-22	77.6	91.8	97.4	64.3	98.0	95.7
WK-23	87.7	92.9	97.5	75.9	58.1	93.6

Values are the means of at least two independent experiments.

Figure 5. Putative binding mode of WK-23 with DNMT1 (PDB code 4WXX). The protein (white) is shown as a cartoon. The compound (deep salmon) and the side chain of F1145, E1168, W1170, and R1574 were shown in sticks (blue).