Dc-rhoin analogs, methods of making and using the same

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to four kinds of DC-Rhoin analogues, which comprise at least two of a benzoheterocycle, a double bond in a heterocycle and an alpha, beta-unsaturated ketone structure of the double bond and a sulfonyl group, and a preparation method thereof is as follows: 3-acetylinde and triethylamine are sequentially added into a reaction bottle containing 1,2-dichloroethane, the reactants are added under ice bath, after normal temperature reaction, EtOAc and HCl are added for extraction; the organic phase is treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure and separated and purified through a silica gel column, and the DC-Rhoin analogue is obtained. The DC-Rhoin analogue can be used as a lead compound for preparing a RhoA inhibitor.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical chemistry, and in particular relates to a DC-Rhoin analogue and a preparation method and application thereof. Background Art

[0002] Anti-tumor metastasis is currently a hot topic and a challenge in anti-tumor drug research. As a key protein mediating tumor cell migration and invasion, RhoA protein is a highly promising anti-tumor drug target. However, due to the "undruggable" nature of RhoA protein, the development of RhoA protein inhibitors is difficult. Most existing RhoA pathway inhibitors do not directly target RhoA protein (Table 1). Rhosin, a small molecule inhibitor that directly targets RhoA protein, was designed and synthesized in 2012 and subsequently used as a positive control in general RhoA inhibitor research. However, Rhosin has a low binding affinity to RhoA protein and can only effectively inhibit RhoA activity in cells at concentrations exceeding 30µM. New, highly active RhoA inhibitors remain to be developed.

[0003] After years of research, the applicant team discovered a new pocket, called "Clock," on the RhoA protein and successfully developed a small molecule RhoA inhibitor, DC-Rhoin, that covalently binds to this pocket. DC-Rhoin has demonstrated activity in inhibiting tumor cell metastasis and proliferation. The discovery of DC-Rhoin, a small molecule covalent inhibitor of RhoA, provides a foundation for further research into RhoA protein inhibitors.

[0004] Table 1 RhoA pathway inhibitors and their target proteins

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a DC-Rhoin analogue and a preparation method and application thereof, so as to find a new compound core targeting RhoA from the DC-Rhoin analogue.

[0007] According to one object of the present invention, a DC-Rhoin analog is provided, which is a compound represented by formula (I), formula (II), formula (III) or formula (IV), or a medically acceptable soluble salt formed by a compound represented by formula (I), formula (II), formula (III) or formula (IV).

[0008]

[0009] It should be noted that the “medically acceptable soluble salts formed by the compounds” in the present invention may be: (1) medically acceptable soluble salts formed by the compounds with inorganic acids, such as hydrochloric acid, sulfuric acid, nitric acid, etc., which have the pharmacological activity of the corresponding compounds; (2) medically acceptable soluble salts formed by the compounds with organic acids, such as formic acid, methanesulfonic acid, etc., which have the pharmacological activity of the corresponding compounds.

[0010] According to another object of the present invention, a method for preparing the above-mentioned DC-Rhoin analog is provided, comprising the following steps: adding 3-acetylindole and triethylamine in sequence to a reaction flask containing 1,2-dichloroethane, adding the reactants under an ice bath, reacting at room temperature, and then adding EtOAc and HCl for extraction; treating the organic phase with saturated brine and anhydrous sodium sulfate, concentrating under reduced pressure, and separating and purifying by silica gel column chromatography to obtain the DC-Rhoin analog; wherein the reactant is one of dansyl chloride, 4-trifluoromethylbenzenesulfonyl chloride, carbazole-N-carbonyl chloride, and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl chloride.

[0011] According to another object of the present invention, there is provided a use of the above DC-Rhoin analogs as a lead compound for preparing RhoA inhibitors. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the synthesis and structural overview of DC-Rhoins analogs.

[0013] Figure 2 The structures, biomembrane interference experimental spectra and equilibrium dissociation constant KD of DC-Rhoins analogs f1, f4 and Rhosin. DETAILED DESCRIPTION

[0014] The present invention will be further described in detail below in conjunction with specific embodiments and accompanying drawings. Unless otherwise specified, the following raw materials are all commercially available.

[0015] 1. Synthesis and Characterization of DC-Rhoin Analogs

[0016] During the design process of DC-Rhoin analogs, some features of DC-Rhoin were retained, and covalent inhibitors of RhoA were sought in the new core. Among them, some features of DC-Rhoin were retained, including benzoheterocycles, double bonds within heterocycles, and at least two of the α,β-unsaturated ketone structure and sulfone group where the double bond is located. The synthesis and structure of DC-Rhoin analogs are shown in Figure 2. Figure 1 As shown, the synthesis method of specific analogues will be introduced in detail below.

[0017] 1, 1-(1-((5-(dimethylamino)naphthalen-1-yl)sulfonyl)-1H -indol-3-yl)ethan-1-one / 1-(1-((5-(dimethylamino)naphthalen-1-yl)sulfonyl)-1 H -indol-3-yl)ethan-1-one (f1)

[0018]

[0019] 3-Acetylindole (1.26 mmol, 1.0 eq) and triethylamine (3.78 mmol, 3.0 eq) were added sequentially to a reaction flask containing 5 mL of 1,2-dichloroethane. Dansyl chloride (1.76 mmol, 1.4 eq) was then added under ice-cooling. After approximately 22 hours of reaction at room temperature, the mixture was extracted with 20 mL of EtOAc and an equal volume of 0.25 N HCl. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 15 / 85, v / v) to obtain 331.1 mg of product f1 as a yellow solid in an approximately 67.0% yield.

[0020] 1 HNMR (400MHz, CDCl3) δ 8.59 (dt, J =8.6, 1.2Hz,1H),8.44 (s, 1H), 8.34–8.31 (m, 1H), 8.31–8.30 (m, 1H), 8.28 (dt, J =8.8, 1.0Hz, 1H), 7.76–7.71 (m,1H), 7.56 (d, J =7.6Hz, 1H), 7.54 (d, J =7.5Hz, 1H), 7.33–7.26 (m, 2H), 7.15 (d, J =7.5Hz, 1H), 2.83 (s, 6H), 2.60 (s, 3H). 13 CNMR (101 MHz, CDCl3) δ 193.59,152.48, 135.06, 132.87, 132.85, 130.44, 130.15, 129.61, 129.39, 127.45,125.71, 124.81, 123.23, 123.19, 120.99, 117.65, 115.91, 113.08, 45.46, 27.97.ESI-HRMS calculated for C 22H 20 N2O3S [M + Na]+: 415.1087, found 415.1122.

[0021] 2. 1-(1-((4-(trifluoromethyl)phenyl)sulfonyl)-1 H -indol-3-yl)ethan-1-one / 1-(1-((4-(trifluoromethyl)phenyl)sulfonyl)-1 H -indol-3-yl)ethan-1-one (f2)

[0022]

[0023] 3-Acetylindole (1.26 mmol, 1.0 eq) and triethylamine (3.78 mmol, 3.0 eq) were added sequentially to a reaction flask containing 5 mL of 1,2-dichloroethane. 4-Trifluoromethylbenzenesulfonyl chloride (1.76 mmol, 1.4 eq) was then added under ice. After approximately 22 h of reaction at room temperature, the mixture was extracted with 20 mL of EtOAc and an equal volume of 0.25 N HCl. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 15 / 85, v / v) to yield 283.5 mg of product f2 as a white solid in an approximately 61.3% yield.

[0024] 1 HNMR (400 MHz, CDCl3) δ 8.37–8.32 (m, 1H), 8.19 (s, 1H), 8.09 (s,1H), 8.07 (s, 1H), 7.96–7.92 (m, 1H), 7.77 (s, 1H), 7.75 (s, 1H), 7.44–7.35(m, 2H), 2.58(s, 3H). 13 C NMR (101 MHz, CDCl3) δ 193.42, 141.05, 136.74,136.41, 136.08, 135.75, 135.01, 131.87, 127.75, 127.05, 127.02, 126.98,126.94, 126.34, 125.45, 124.22, 123.57, 122.59, 121.50, 113.02, 28.00. ESI-HRMS calculated for C 17 H 12F3NO3S [M + Na]+: 390.0382, found 390.0403.

[0025] 3.1-(1-(9 H -carbazole-9-carbonyl)-1 H -indol-3-yl)ethan-1-one / 1-(1-(9 H -carbazole-9-carbonyl)-1 H -indol-3-yl)ethan-1-one (f4)

[0026]

[0027] 3-Acetylindole (0.94 mmol, 1.0 eq) and triethylamine (1.32 mmol, 3.0 eq) were added sequentially to a reaction flask containing 5 mL of 1,2-dichloroethane. Carbazole-N-carbonyl chloride (2.83 mmol, 1.4 eq) was added under ice-cooling. After approximately 22 h of reaction at room temperature, 20 mL of EtOAc and an equal volume of HCl (3.00 mmol) were added for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95, v / v) to obtain 168.8 mg of product f4 as a white solid in an approximately 51.0% yield.

[0028] 1 HNMR (400 MHz, CDCl3) δ 8.46 (dt, J = 8.1, 1.0Hz, 1H), 8.10–8.04 (m,2H), 7.97 (s, 1H), 7.80 (dt, J = 8.0, 1.0Hz, 1H), 7.46–7.36 (m, 8H), 2.44 (s,3H). 13C NMR (101 MHz, CDCl3) δ 193.80, 148.79, 138.41, 135.80, 133.00,127.50, 127.47, 126.24, 126.11, 125.45, 124.22, 123.21, 121.99, 120.56,114.60, 114.42, 27.95. ESI-HRMS calculated for C 23 H 16 N2O2[M + Na]+: 375.1104, found 375.1126.

[0029] 4. 1-(1-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-1 H -indol-3-yl)ethan-1-one / 1-(1-((2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-yl)sulfonyl)-1 H -indol-3-yl)ethan-1-one (f3)

[0030]

[0031] 3-Acetylindole (0.94 mmol, 1.0 eq) and triethylamine (1.32 mmol, 3.0 eq) were added sequentially to a reaction flask containing 5 mL of 1,2-dichloroethane. 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl chloride (2.83 mmol, 1.4 eq) was then added under ice. After approximately 22 h of reaction at room temperature, 20 mL of EtOAc and an equal volume of HCl (3.00 mmol) were added for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (ethyl acetate / petroleum ether = 5 / 95, v / v) to obtain 162.2 mg of product f3 as a white oil in an approximately 41.9% yield.

[0032] 1 HNMR (400 MHz, CDCl3) δ 8.36 (dt, J = 8.1, 1.1 Hz, 1H), 8.29 (s, 1H),7.33–7.28 (m, 1H), 7.24–7.22 (m, 1H), 7.22–7.20 (m, 1H), 3.01 (s, 2H), 2.58(s, 3H), 2.50 (s, 3H), 2.37 (s, 3H), 2.05 (s, 3H), 1.48 (s, 6H). 13C NMR (101MHz, CDCl3) δ 193.79, 161.66, 141.24, 135.48, 135.04, 132.94, 127.31, 126.20,125.83, 125.31, 124.40, 123.18, 119.47, 119.08, 112.23, 87.88, 43.03, 28.65,27.93, 19.43, 17.36, 12.62. ESI-HRMS calculated for C 23 H 25 NO4S [M + Na]+:434.1397, found 434.1420.

[0033] 2. Interaction experiment between DC-Rhoin analogs and RhoA protein

[0034] 1. Experimental methods

[0035] (1) Biotinylation of RhoA protein

[0036] Dissolve 100 μg of human RhoA recombinant protein (His) in 100 μL of PBS buffer. Add 0.83 μL of Genemore G-MM-IGT biotinylation reagent and incubate at room temperature in the dark for 1 hour. After the reaction, pass the mixture through a Genemore GMM-25 desalting column (PD MiniTrap G-25) to obtain the biotinylated RhoA protein. Determine the protein concentration using a BCA protein assay kit.

[0037] (2) Determination of compound-protein relative affinity by biomembrane interference experiment

[0038] A Super Streptavidin (SSA) sensor (ForteBio) was immersed in a protein solution, and biotinylated RhoA protein was immobilized onto the SSA sensor. To eliminate loose, nonspecifically bound proteins and establish a stable baseline, the sensor with protein was moved and immersed in a well containing pure assay buffer and equilibrated in the buffer for 10 minutes. Binding-dissociation cycles were performed by moving and dipping the sensor into wells containing a compound solution of a specific concentration, followed by dipping it into a well containing pure buffer.

[0039] The volume of each well in a 96-well plate was 200 μL. The buffer used was PBS containing 0.02% Tween and 0.5% DMSO. A DMSO control was included in all assays. The manufacturer's data analysis software was used to analyze the collected raw kinetic data using a double subtraction method (subtracting the DMSO control and the unimmobilized protein control) to obtain the equilibrium dissociation constant (KD) of the compound with the protein.

[0040] 2. Experimental results

[0041] Biomembrane interference experiments were performed on DC-Rhoin analogs and Rhosin (positive control). Among the data obtained, three compounds met the three criteria for data quality judgment: (1) the signal size showed a concentration-dependent response; (2) the signal separation was relatively good; and (3) the fitted curve was visually consistent with the measured curve. The results are shown in Figure 2. Figure 2 As shown, the KD values ​​of compounds f1 and f4 for Rhosin were 9.2, 7.3, and 8.5 μM, respectively. These results suggest that compounds f1 and f4 may serve as lead compounds with potential for development as RhoA inhibitors.

[0042] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A DC-Rhoin analogue, characterized in that A compound represented by formula (I) or formula (IV), or a medically acceptable soluble salt formed by a compound represented by formula (I) or formula (IV): 。 2. The method for preparing the DC-Rhoin analogue according to claim 1, characterized in that: When the DC-Rhoin analogue is a compound represented by formula (I), the steps are as follows: 3-Acetylindole and triethylamine were added sequentially to a reaction flask containing 1,2-dichloroethane, and dansyl chloride was added under ice bath. After reaction at room temperature, EtOAc and HCl were added for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain the product.

3. The method for preparing the DC-Rhoin analogue according to claim 1, characterized in that: When the DC-Rhoin analogue is a compound represented by formula (IV), the steps are as follows: 3-Acetylindole and triethylamine were added sequentially to a reaction flask containing 1,2-dichloroethane, and carbazole-N-carbonyl chloride was added under ice bath. After the reaction at room temperature, EtOAc and HCl were added for extraction. The organic phase was treated with saturated brine and anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by silica gel column chromatography to obtain the product.

4. Use of the DC-Rhoin analogue according to claim 1 in the preparation of a RhoA inhibitory compound.

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