Design, Synthesis and Biological Evaluation of Benzothiazoles as Highly Potent ROCK Inhibitors Through Molecular Docking and Free Energy Calculations†

doi:10.7503/cjcu20170213

Abstract

Abstract:

Rock has been considered to provide a pharmacological strategy for preventing and treating multiple sclerosis, pulmonary hypertension, glaucoma, cardiovascular disease, erectile dysfunction and cancer. With 3 previously reported and benzothiazole-based ROCK inhibitors(1—3) as the research targets, the structure-activity relationship(SAR) was preliminary revealed from amino acid level by molecular docking after obtaining the stable ROCK2-ligand complexes in the binding pocket through molecular dynamic simulations. Then MM/GBSA free energy calculations of compounds 1—3 showed that there was good correlation between binding affinity(ΔG_bind) and inhibitory activities, and van der Waals interaction(ΔG_VDW) contributing to ΔG_bind most. And the key amino acids with outstanding contribution for high inhibition were obtained through free energy analysis. Finally, 3 series of benzothiazoles(D1—D10) were designed according to the results of molecular docking and free energy calculations. In the biological evaluation, compounds D1—D10 exhibited 2—105 nmol/L IC₅₀ values against ROCK2 and 11—288 nmol/L IC₅₀ values against ROCK1. Compounds D3—D5 exhibited higher metabolic stability than reported compounds 1 and 3 in human liver microsome studies. This work not only gave theoretical guidance for the design of highly potent ROCK, but also offered a series of highly active ROCK inhibitors with intellectual property right for fundamental research and application of ROCK.

Key words: ROCK2 inhibitor, Molecular docking, Molecular dynamics simulation, Free energy calculation, Benzothiazoles

CLC Number:

O626

TrendMD:

DUAN Yongbin, YIN Yan, MENG Fanli, ZHAO Lianhua, LIU Yukun, YUAN Zhe, FENG Yangbo. Design, Synthesis and Biological Evaluation of Benzothiazoles as Highly Potent ROCK Inhibitors Through Molecular Docking and Free Energy Calculations^†[J]. Chem. J. Chinese Universities, 2017, 38(9): 1568.

Figures/Tables 9

Fig.1 Structures of benzothiazole-based ROCK inhibitors 1—3

Scheme 1 Synthetic routes of compounds D1—D10Reagents and conditions: a. HATU, amine, DIEA, DMF, r. t. ; b. HOAc, TFA, 100 ℃; c. Pd(PPh3)4, K2CO3, 2-aminopyrid-in-4-ylboronic acid, dioxane/H2O, 95 ℃; d. saturated NaOH solution.

Table 1 Appearance, yields, melting points, HRMS and elemental analysis data of compounds D1—D10

Compd.	Appearance	Yield(%)	m.p./℃	HRMS(calcd.)[M+H]⁺	Elemental analysis(%, calcd.)
Compd.	Appearance	Yield(%)	m.p./℃	HRMS(calcd.)[M+H]⁺	C	H	O
D1	White solid	52%	204—205	308.0872(308.0858)	66.51(66.43)	4.36(4.26)	13.45(13.62)
D2	White solid	50%	214—215	338.0947(338.0963)	64.12(64.08)	4.36(4.48)	12.71(12.49)
D3	White solid	70%	234—235	337.1061(337.1072)	63.99(63.81)	4.36(4.28)	14.69(14.81)
D4	White solid	68%	248—249	395.0959(395.0978)	60.99(60.90)	3.96(3.83)	14.61(14.42)
D5	White solid	67%	281—282	407.1193(407.1178)	62.19(62.05)	4.69(4.46)	13.92(13.74)
D6	White solid	31%	238—239	515.2241(515.2229)	65.51(65.35)	5.66(5.88)	16.15(16.13)
D7	White solid	32%	244—245	489.2059(489.2073)	63.98(63.91)	5.53(5.78)	17.06(17.12)
D8	White solid	30%	223—224	544.2503(544.2495)	64.98(64.03)	6.83(6.62)	18.06(18.00)
D9	White solid	31%	216—217	529.2359(529.2386)	65.98(65.88)	6.02(6.10)	15.83(15.90)
D10	White solid	34%	254—255	503.2247(503.2229)	64.43(64.52)	5.97(6.02)	16.83(16.70)

Table 2 1H NMR data of compounds D1—D10

Compd.	¹H NMR(400 MHz, DMSO-d₆), δ
D1	9.68(s, 1H, NH), 8.46—8.45(m, 1H), 8.20—8.19(m, 1H), 8.09—8.07(m, 1H), 7.90—7.89(m, 1H), 7.65—7.52(m, 4H), 7.32—7.23(m, 2H), 4.02(s, 2H, CH₃)
D2	8.43(s, 1H, NH), 8.09—8.02(m, 2H), 7.89—7.84(m, 1H), 7.32—7.24(m, 2H), 6.93—6.86(m, 4H), 4.00(s, 2H, CH₂), 3.75(s, 3H, CH₃)
D3	12.94(s, 1H, NH), 12.63(s, 1H, NH), 8.22(s, 1H), 8.14—8.11(m, 1H), 7.74—7.72(m, 2H), 7.64—7.57(m, 1H), 7.16—7.08(m, 2H), 6.89—6.86(m, 1H), 6.80—6.78(m, 1H), 4.48—4.46(m, 1H, CH₂), 4.14—4.10(m, 1H, CH₂), 3.25—3.23(m, 1H, CH), 3.11—3.01(m, 2H, CH₂)
D3	12.94(s, 1H, NH), 12.63(s, 1H, NH), 8.22(s, 1H), 8.14—8.11(m, 1H), 7.74—7.72(m, 2H), 7.64—7.57(m, 1H), 7.16—7.08(m, 2H), 6.89—6.86(m, 1H), 6.80—6.78(m, 1H), 4.48—4.46(m, 1H, CH₂), 4.14—4.10(m, 1H, CH₂), 3.25—3.23(m, 1H, CH), 3.11—3.01(m, 2H, CH₂)
D4	12.63(s, 1H, NH), 8.22(s, 1H, NH), 8.21—8.05(m, 2H), 7.74—7.68(m, 2H), 7.05—7.02(m, 1H), 6.96—6.91(m, 2H), 6.82—6.79(m, 1H), 4.44(dd, J=10.8, 2.4 Hz, 1H, CH₂), 4.13(dd, J=10.8, 8.4 Hz, 1H, CH₂), 3.25—3.23(m, 1H, CH), 3.07—3.02(m, 2H, CH₂)
D5	12.61(s, 1H, NH), 8.25(s, 1H, NH), 8.22—8.21(m, 1H), 8.13—8.10(m, 2H), 7.74—7.68(m, 2H), 6.74—6.67(m, 3H), 4.40(dd, J=10.8, 2.0 Hz, 1H, CH₂), 4.06(dd, J=10.8, 8.8 Hz, 1H, CH₂), 3.69(s, 3H, OCH₃), 3.09—2.96(m, 3H, CH, CH₂)
D6	9.35(s, 1H, NH), 8.74—8.73(m, 1H, NH), 8.44—8.43(m, 1H), 8.19—8.07(m, 3H), 7.88—7.80(m, 2H), 7.55—7.54(m, 2H), 4.80(s, 2H, CH₂), 3.58—3.56(m, 5H, CH, CH₂), 3.34—3.30(m, 2H, CH₂), 3.10—3.06(m, 2H, CH₂), 1.85—1.84(m, 4H, CH₂), 0.79—0.69(m, 4H, CH₂)
D7	9.19(s, 1H, NH), 8.65—8.64(m, 1H), 8.37—8.36(m, 1H), 8.13—8.12(m, 1H), 8.02—8.00(m, 1H), 7.81—7.73(m, 3H), 7.48—7.47(m, 2H), 4.76(s, 2H), 3.62—3.61(m, 1H), 3.53—3.52(m, 2H), 3.20—3.10(m, 2H), 2.78(s, 3H), 2.77(s, 3H), 1.82—1.79(m, 2H), 0.83—0.79(m, 2H)
D8	9.17(s, 1H, NH), 8.6—8.58(m, 1H), 8.33—8.31(m, 1H), 8.10—8.08(m, 1H), 8.05—8.03(m, 1H), 7.85—7.76(m, 3H), 7.45—7.43(m, 2H), 4.72(s, 2H, CH₂), 3.62—3.61(m, 1H, CH), 3.53—3.52(m, 6H, CH₂), 3.20—3.10(m, 6H, CH₂), 2.78(s, 3H, CH₃), 1.80—1.79(m, 2H, CH₂), 0.87—0.84(m, 2H, CH₂)
D9	9.32(s, 1H, NH), 8.71—8.69(m, 1H), 8.47—8.45(m, 1H), 8.10—8.07(m, 3H), 7.88—7.64(m, 4H), 4.81(s, 2H, CH₂), 3.65—3.63(m, 1H, CH), 3.58—3.56(m, 2H, CH₂), 3.34—3.30(m, 2H, CH₂), 3.10—3.06(m, 2H, CH₂), 2.01—1.99(m, 2H, CH₂), 1.85—1.84(m, 4H, CH₂), 0.79—0.69(m, 6H, CH₂)
D10	9.10(s, 1H, NH), 8.78—8.79(m, 1H), 8.34—8.30(m, 1H), 8.10—7.98(m, 2H), 7.85—7.74(m, 3H), 7.55—7.47(m, 2H), 4.75(s, 2H, CH₂), 3.67—3.65(m, 1H, CH), 3.60—3.58(m, 2H, CH₂), 3.56—3.54(m, 2H, CH₂), 3.19—3.08(m, 2H, CH₂), 2.80(s, 3H, CH₃), 2.78(s, 3H, CH₃), 1.80—1.78(m, 2H, CH₂), 1.45—1.41(m, 2H, CH₂), 0.84—0.83(m, 2H, CH₂)

Table 2 1H NMR data of compounds D1—D10

Compd.	¹H NMR(400 MHz, DMSO-d₆), δ
D1	9.68(s, 1H, NH), 8.46—8.45(m, 1H), 8.20—8.19(m, 1H), 8.09—8.07(m, 1H), 7.90—7.89(m, 1H), 7.65—7.52(m, 4H), 7.32—7.23(m, 2H), 4.02(s, 2H, CH₃)
D2	8.43(s, 1H, NH), 8.09—8.02(m, 2H), 7.89—7.84(m, 1H), 7.32—7.24(m, 2H), 6.93—6.86(m, 4H), 4.00(s, 2H, CH₂), 3.75(s, 3H, CH₃)
D3	12.94(s, 1H, NH), 12.63(s, 1H, NH), 8.22(s, 1H), 8.14—8.11(m, 1H), 7.74—7.72(m, 2H), 7.64—7.57(m, 1H), 7.16—7.08(m, 2H), 6.89—6.86(m, 1H), 6.80—6.78(m, 1H), 4.48—4.46(m, 1H, CH₂), 4.14—4.10(m, 1H, CH₂), 3.25—3.23(m, 1H, CH), 3.11—3.01(m, 2H, CH₂)
D3	12.94(s, 1H, NH), 12.63(s, 1H, NH), 8.22(s, 1H), 8.14—8.11(m, 1H), 7.74—7.72(m, 2H), 7.64—7.57(m, 1H), 7.16—7.08(m, 2H), 6.89—6.86(m, 1H), 6.80—6.78(m, 1H), 4.48—4.46(m, 1H, CH₂), 4.14—4.10(m, 1H, CH₂), 3.25—3.23(m, 1H, CH), 3.11—3.01(m, 2H, CH₂)
D4	12.63(s, 1H, NH), 8.22(s, 1H, NH), 8.21—8.05(m, 2H), 7.74—7.68(m, 2H), 7.05—7.02(m, 1H), 6.96—6.91(m, 2H), 6.82—6.79(m, 1H), 4.44(dd, J=10.8, 2.4 Hz, 1H, CH₂), 4.13(dd, J=10.8, 8.4 Hz, 1H, CH₂), 3.25—3.23(m, 1H, CH), 3.07—3.02(m, 2H, CH₂)
D5	12.61(s, 1H, NH), 8.25(s, 1H, NH), 8.22—8.21(m, 1H), 8.13—8.10(m, 2H), 7.74—7.68(m, 2H), 6.74—6.67(m, 3H), 4.40(dd, J=10.8, 2.0 Hz, 1H, CH₂), 4.06(dd, J=10.8, 8.8 Hz, 1H, CH₂), 3.69(s, 3H, OCH₃), 3.09—2.96(m, 3H, CH, CH₂)
D6	9.35(s, 1H, NH), 8.74—8.73(m, 1H, NH), 8.44—8.43(m, 1H), 8.19—8.07(m, 3H), 7.88—7.80(m, 2H), 7.55—7.54(m, 2H), 4.80(s, 2H, CH₂), 3.58—3.56(m, 5H, CH, CH₂), 3.34—3.30(m, 2H, CH₂), 3.10—3.06(m, 2H, CH₂), 1.85—1.84(m, 4H, CH₂), 0.79—0.69(m, 4H, CH₂)
D7	9.19(s, 1H, NH), 8.65—8.64(m, 1H), 8.37—8.36(m, 1H), 8.13—8.12(m, 1H), 8.02—8.00(m, 1H), 7.81—7.73(m, 3H), 7.48—7.47(m, 2H), 4.76(s, 2H), 3.62—3.61(m, 1H), 3.53—3.52(m, 2H), 3.20—3.10(m, 2H), 2.78(s, 3H), 2.77(s, 3H), 1.82—1.79(m, 2H), 0.83—0.79(m, 2H)
D8	9.17(s, 1H, NH), 8.6—8.58(m, 1H), 8.33—8.31(m, 1H), 8.10—8.08(m, 1H), 8.05—8.03(m, 1H), 7.85—7.76(m, 3H), 7.45—7.43(m, 2H), 4.72(s, 2H, CH₂), 3.62—3.61(m, 1H, CH), 3.53—3.52(m, 6H, CH₂), 3.20—3.10(m, 6H, CH₂), 2.78(s, 3H, CH₃), 1.80—1.79(m, 2H, CH₂), 0.87—0.84(m, 2H, CH₂)
D9	9.32(s, 1H, NH), 8.71—8.69(m, 1H), 8.47—8.45(m, 1H), 8.10—8.07(m, 3H), 7.88—7.64(m, 4H), 4.81(s, 2H, CH₂), 3.65—3.63(m, 1H, CH), 3.58—3.56(m, 2H, CH₂), 3.34—3.30(m, 2H, CH₂), 3.10—3.06(m, 2H, CH₂), 2.01—1.99(m, 2H, CH₂), 1.85—1.84(m, 4H, CH₂), 0.79—0.69(m, 6H, CH₂)
D10	9.10(s, 1H, NH), 8.78—8.79(m, 1H), 8.34—8.30(m, 1H), 8.10—7.98(m, 2H), 7.85—7.74(m, 3H), 7.55—7.47(m, 2H), 4.75(s, 2H, CH₂), 3.67—3.65(m, 1H, CH), 3.60—3.58(m, 2H, CH₂), 3.56—3.54(m, 2H, CH₂), 3.19—3.08(m, 2H, CH₂), 2.80(s, 3H, CH₃), 2.78(s, 3H, CH₃), 1.80—1.78(m, 2H, CH₂), 1.45—1.41(m, 2H, CH₂), 0.84—0.83(m, 2H, CH₂)

Fig.2 RMSD of the backbone Cα atoms vs. simulation time

Fig.3 Docking results of compounds 1—3 and the catalytic domain of ROCK2(A), (C) and (E): interaction of compounds 1—3 and ROCK2, respectively; (B), (D) and (F): the hydrophobic pocket of ROCK2 with compounds 1—3, respectively.

Table 3 Predicted binding free energies and individual energy components

Compd.	ΔE_VDW/ (kJ·mol^-1)	ΔG_SA/ (kJ·mol^-1)	ΔE_ELE/ (kJ·mol^-1)	ΔG_GB/ (kJ·mol^-1)	ΔG_bind/ (kJ·mol^-1)	IC₅₀/ (nmol·L^-1)
1	-181.4458	-21.8627	-152.2825	170.4642	-185.1268	11
2	-214.9761	-26.5596	-67.0682	140.4793	-168.1246	500
3	-226.3453	-26.0829	-141.0213	191.2943	-202.1552	0.9

Fig.4 Contribution of key residues to binding energy(A, C) and individual energy terms(B, D)

Table 4 Inhibitory activities and metabolic stabilities of newly designed inhibitors

Compd.	Structure	IC₅₀/(nmol·L^-1)		$t 12 *$ /min
Compd.	Structure	ROCK1		ROCK2
1		64	11	18
3		2	0.9	34
D1		72	44	60
D2		64	42	19
D3		72	14	45.9
D4		171	22	56.4
D5		288	105	91
D6		60	21	30
D7		95	35	12
D8		11	2	9
D9		58	37	13
D10		45	19	45

Table 4 Inhibitory activities and metabolic stabilities of newly designed inhibitors

Compd.	Structure	IC₅₀/(nmol·L^-1)		$t 12 *$ /min
Compd.	Structure	ROCK1		ROCK2
1		64	11	18
3		2	0.9	34
D1		72	44	60
D2		64	42	19
D3		72	14	45.9
D4		171	22	56.4
D5		288	105	91
D6		60	21	30
D7		95	35	12
D8		11	2	9
D9		58	37	13
D10		45	19	45

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