基于分子对接和自由能计算的高活性苯并噻唑类ROCK抑制剂的设计、 合成和生物学评价

doi:10.7503/cjcu20170213

摘要/Abstract

摘要：

以3个已报道的苯并噻唑类Rho关联含卷曲螺旋蛋白激酶(ROCK)抑制剂(化合物1~3)为研究对象, 经分子动力学模拟获得其在ROCK2蛋白结合口袋中的稳定结合构象, 通过分子对接结果从氨基酸角度初步揭示了此类抑制剂的结构-活性关系(SAR); 然后, 对这3个抑制剂进行MM/GBSA结合自由能(ΔG_bind)研究, 结合自由能计算可知ΔG_bind与化合物活性之间具有良好的相关性, 且范德华作用能(ΔG_VDW)对ΔG_bind的贡献最大. 通过自由能分解获得了对于高活性抑制剂具有重要影响的关键残基. 最后, 根据分子对接和自由能研究结果设计并合成了3类新型苯并噻唑类似物(D1~D10). 生物学评价结果表明, 这10个化合物分别具有11~288 nmol/L(ROCK1)和2~105 nmol/L(ROCK2)的抑制活性. 其中, 化合物D3~D5在人肝微粒体代谢研究中展现出比已报道化合物更高的代谢稳定性. 本研究不仅为高活性ROCK抑制剂的设计提供了理论指导, 也为ROCK的应用研究提供了一系列结构新颖的高活性抑制剂.

关键词: ROCK抑制剂, 分子对接, 分子动力学模拟, 自由能计算, 苯并噻唑类似物

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

中图分类号:

O626

TrendMD:

段永斌, 殷燕, 孟凡丽, 赵连花, 刘玉坤, 袁哲, 冯阳波. 基于分子对接和自由能计算的高活性苯并噻唑类ROCK抑制剂的设计、合成和生物学评价. 高等学校化学学报, 2017, 38(9): 1568.

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^†. Chem. J. Chinese Universities, 2017, 38(9): 1568.

图/表 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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