Rocuronium bromide impurity molecule with anticancer activity and preparation method thereof

Abstract

The invention discloses rocuronium bromide impurity molecules with anticancer activity and a preparation method thereof, and the preparation method comprises the following steps: taking epiandrosterone I as a raw material, dissolving in a solvent I, adding a Dess Martin reagent, and reacting to obtain an intermediate II; dissolving the intermediate II in an acidic solvent II, adding bromine, and reacting to generate an intermediate III; dissolving the intermediate III in a solvent III, adding alkali and morpholine, and reacting to generate an intermediate IV; dissolving the intermediate IV in a solvent IV, adding triisobutyl potassium borohydride, and reacting at low temperature to generate a target product V. The preparation method comprises the following steps: by taking epiandrosterone as a raw material, oxidizing 3-site hydroxyl into carbonyl by using a Dess Martin reagent, brominating 2-site and 16-site by using bromine under an acidic condition, performing nucleophilic substitution with morpholine under an alkaline condition, and selectively reducing 3-site carbonyl into hydroxyl, thereby obtaining the target impurity with the molecular purity of more than 95%, and the quality and reliability of subsequent biological activity research and drug development are ensured.

Description

Technical Field

[0001] The invention relates to the technical field of drug synthesis, and in particular to a rocuronium bromide impurity molecule with anticancer activity and a preparation method thereof. Background Art

[0002] Cancer is a key contributor to the global disease burden.

[0003] In the synthesis process of drug API, the subsequent preparation production, and the storage, transportation and use of finished drugs, the drug impurities generated or introduced are the focus of drug quality research. These impurities will have a considerable impact on the efficacy and safety of the drug, and may even cause significant toxic side effects. Therefore, drug regulatory authorities in various countries have implemented a series of policies to control impurities generated in various links of drug production. Drug impurity research was carried out relatively early in my country, but the strict policy-guided impurity research has only been around for a decade.

[0004] Rocuronium is a deacetoxy analog of vecuronium, with a faster onset of action. It is an aminosteroid non-depolarizing neuromuscular blocker or muscle relaxant. It is currently used clinically in anesthesia to assist in endotracheal intubation and to relax skeletal muscles during surgery or mechanical ventilation. It was introduced in 1994 and has a rapid onset and a moderate duration of action. Rocuronium works by competing for cholinergic receptors at the motor endplate. This action can be antagonized by acetylcholinesterase inhibitors, such as neostigmine and idefenamide. Rocuronium works by competitive binding to nicotinic receptors. As a result, depolarization is prevented, calcium ions are not released, and muscle contraction does not occur. It is speculated that non-depolarizing agents bind to postganglionic ("curare") receptors and may therefore interfere with the flux of sodium and potassium, which is responsible for the depolarization and repolarization of membranes involved in muscle contraction. As mentioned above, the main function of drug impurities is to monitor the quality of drugs. Impurities with excessive content and cannot be eliminated must be subject to relevant toxicity studies according to regulations, but there are few studies on the biological activity of impurities. There are many factors that cause the formation of impurities, not just because of the inherent characteristics of chemical reactions. Light, moisture, oxidation and other factors also play a great role in the formation of impurities. Therefore, there are many impurities with special structures, which are often difficult to prepare under conventional chemical reaction conditions. Or because they are by-products of conventional reaction conditions, the amount that can be obtained is very small, and they are not used as objects of pharmacological activity research to screen related activities. The particularity of the structure of drug impurities and the scarcity of their amount make them a very attractive class of objects that can be studied as potential drug active lead compounds. Based on this, it is necessary to study the anticancer activity of related impurity molecules of rocuronium bromide. There are nearly 100 known impurity molecules of rocuronium bromide. Structure V is an impurity generated by the excessive reaction of rocuronium bromide intermediates with morpholine nucleophilic substitution. Its preparation has been reported in relevant literature, but there is no research related to its anticancer activity. The molecule was tested at 5mM and 10mM concentrations and showed moderate anticancer activity in HepG2, SW480 and U87 cancer cell lines compared with the control. This is the first time that rocuronium bromide, a muscle relaxant, has been found to have anticancer activity as an impurity molecule, which has the prospect of further subsequent drug chemical modification and development into a new anticancer drug. Summary of the invention

[0005] In view of the problems existing in the prior art, the present invention provides a rocuronium bromide impurity molecule with anti-cancer activity and a preparation method thereof.

[0006] In order to achieve the above object, the present invention provides a rocuronium bromide impurity molecule with anticancer activity, and its structural formula V is:

[0007]

[0008] The present invention also provides a method for preparing a rocuronium bromide impurity molecule having anticancer activity, comprising the following steps:

[0009] Step S1: Take epiandrosterone I as a raw material and dissolve it in solvent 1, add Dess-Martin reagent, and react to obtain intermediate II. The reaction equation is as follows:

[0010]

[0011] Step S2: Dissolve the intermediate II in an acidic solvent II, add bromine, and react to generate the intermediate III. The reaction equation is as follows:

[0012]

[0013] Step S3: Dissolve the intermediate III in solvent three, add a base and morpholine, and react to generate the intermediate IV. The reaction equation is as follows:

[0014]

[0015] Step S4: Dissolve the intermediate IV in solvent IV, add potassium triisobutylborohydride, and react at low temperature to generate the target product V. The reaction equation is as follows:

[0016]

[0017] Preferably, the molar ratio of epiandrosterone I to Dess-Martin reagent in step S1 is 1:1.2 to 1:7.

[0018] Preferably, the solvent one in step S1 is selected from one or more of dichloromethane, methanol, and ethyl acetate; the reaction temperature in step S1 ranges from 0°C to 25°C; the reaction time is 6h-24h; more preferably, the molar ratio of epiandrosterone I to Dess-Martin reagent in step S1 is 1:3.5; the solvent one is preferably dichloromethane; the reaction temperature in step S1 is 25°C, and the reaction time is 12-18h.

[0019] Preferably, the molar ratio of intermediate II to bromine in step S2 is 1:2.5 to 1:3.

[0020] Preferably, the second solvent in step S2 is selected from one of aqueous solutions of formic acid, acetic acid and hydrobromic acid; the reaction temperature in step S2 is 0°C to 25°C, and the reaction time is 3-6h; more preferably, the molar ratio of intermediate II to bromine in step S2 is 1:2.5; the second solvent is preferably acetic acid; the reaction temperature in step S2 is 0°C; and the reaction time is 3h.

[0021] Preferably, the molar ratio of the intermediate III to the base in step S3 is 1:2.5 to 1:4.

[0022] Preferably, the solvent three in step S3 is selected from one of dichloromethane, tetrahydrofuran and acetonitrile; the reaction temperature in step S3 is 0°C to 50°C, and the reaction time is 4-9h;

[0023] More preferably, the molar ratio of intermediate III to base in step S3 is 1:2.5; the solvent III is preferably acetonitrile; the reaction temperature in step S3 is 50° C., and the reaction time is 5 h.

[0024] Preferably, the molar ratio of intermediate IV to potassium triisobutylborohydride in step S4 is 1:1.1 to 1:2.

[0025] Preferably, the solvent in step S4 is selected from one of dichloromethane, ethyl acetate, isopropyl ether and tetrahydrofuran; the reaction temperature in step S4 is -78°C to 0°C, and the reaction time is 3-9h;

[0026] More preferably, the molar ratio of intermediate IV to potassium triisobutylborohydride in step S4 is 1:1.1; the solvent is preferably tetrahydrofuran; the reaction temperature in step S4 is -78°C, and the reaction time is 6h.

[0027] The technical solution of the present invention has the following beneficial effects:

[0028] The invention provides a new rocuronium bromide impurity molecule, which has not been reported to have anticancer activity in existing studies, and provides a new lead compound for the field of anticancer drug research.

[0029] High-purity preparation: The preparation method of the present invention uses epiandrosterone as a raw material, first oxidizes the 3-hydroxyl group to a carbonyl group using Dess-Martin reagent, and then bromines the 2- and 16-positions with bromine under acidic conditions. Then, under alkaline conditions, nucleophilic substitution occurs with morpholine, and then the 3-carbonyl group is selectively reduced to a hydroxyl group, thereby obtaining the target impurity molecule. The target product obtained by the preparation method has a purity of more than 95% as determined by HPLC, ensuring the quality and reliability of subsequent biological activity research and drug development.

[0030] Anticancer activity: The present invention uses the MTT method to show that the molecule exhibits moderate anticancer activity in three cancer cell lines, HepG2, SW480 and U87, at two concentrations of 5mM and 10mM, compared with the control, showing potential anticancer effects. The anticancer treatment is used for the treatment of prostate cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, etc.

[0031] Potential for medicinal chemical modification: The structure of the impurity molecule provides the possibility for further medicinal chemical modification, which will help develop new anti-cancer drugs that are more effective and selective.

[0032] Clinical application prospects: Rocuronium bromide is a known muscle blocker or muscle relaxant. The discovery of the anti-cancer activity of its impurity molecules provides a new perspective for clinical application and helps to develop drugs with dual effects, that is, muscle relaxants and anti-cancer effects at the same time.

[0033] Public health significance: In view of the high incidence and mortality of cancer worldwide, the rocuronium bromide impurity molecule of the present invention and its potential anti-cancer application are of great significance for reducing the burden of public health.

[0034] Simplified process flow: The preparation method provided by the present invention has reasonable process design, simple post-processing, and is easy for large-scale production, which is conducive to reducing production costs and improving efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A process flow chart for preparing the rocuronium impurity structure V provided by the present invention;

[0036] Figure 2 The HPLC spectrum of the rocuronium impurity structure V provided by the present invention;

[0037] Figure 3 A mass spectrum of the rocuronium impurity structure V provided by the present invention;

[0038] Figure 4 The NMR spectrum of the rocuronium impurity structure V provided by the present invention;

[0039] Figure 5 This is a diagram showing the cancer cell inhibition effect of the rocuronium impurity structure V provided by the present invention using the MTT method. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0041] Refer to the figure

[0042] Embodiment 1:

[0043]

[0044] Epiandrosterone I (3.5 g, 12.1 mmol) was added to 50 mL of dichloromethane to dissolve, and then Dess-Martin reagent (17.96 g, 42.4 mmol) was added, and the reaction was stirred at room temperature for 12 to 18 h. When TLC showed that the reaction was complete, the solvent was evaporated under reduced pressure, and the residue was purified on a silica gel column with petroleum ether and ethyl acetate as the mobile phase to obtain an off-white solid intermediate II (3.32 g, 95%).

[0045]

[0046] Add intermediate II (3.32g, 11.5mmol) to a 100mL three-necked reaction bottle, add acetic acid (50mL). After cooling to 0°C, add bromine (4.6g, 28.8mmol) dropwise; after addition, react at the same temperature for about 3h. TLC shows that the reaction is complete, add ice water (50mL) to dilute, then add an appropriate amount of 10% sodium thiosulfate solution to neutralize the remaining bromine in the reaction, extract with dichloromethane (50mLX3), combine the organic phases, wash the organic phases with water and saturated brine respectively, dry the organic phase with anhydrous sodium sulfate, spin dry the solvent, and purify the residue on a silica gel column with ethyl acetate and petroleum ether system to obtain a dark brown solid intermediate III (3.64g, 71%).

[0047]

[0048] The intermediate III (3.64 g, 8.2 mmol) was added to a 100 mL three-necked reaction bottle, and dry acetonitrile (50 mL) was added, followed by potassium carbonate (2.83 g, 20.5 mmol); the reaction was carried out at 50 ° C for about 5 h. When TLC showed that the reaction was complete, ice water (50 mL) was added for dilution, and then extracted with dichloromethane (50 mL×3). The organic phases were combined, and the organic phases were washed with water and saturated brine, respectively. The organic phase was dried over anhydrous sodium sulfate, the solvent was spin-dried, and the residue was purified on a silica gel column with ethyl acetate and petroleum ether system to obtain a light brown intermediate IV (3.27 g, 87%).

[0049]

[0050] Add intermediate IV (3.27g, 7.1mmol) to a 100mL three-necked reaction bottle, add dry tetrahydrofuran (50mL), cool to -78°C; then add potassium triisobutylborohydride (1N in THF, 7.8ml) dropwise; react at the same temperature for about 6h, and when TLC shows that the reaction is complete, add an appropriate amount of methanol to quench the reaction, and then add ice water (50mL) to dilute the reaction system; extract with dichloromethane (50mLX3), combine the organic phases, wash the organic phases with water and saturated brine respectively, dry the organic phase with anhydrous sodium sulfate, spin dry the solvent, and purify the residue on a silica gel column with ethyl acetate and petroleum ether to obtain a white solid V (2.58g, 79%). The spectral data of the target product V: ESI-LCMS (m / z): 461.17 (M+H) + , HPLC: 99.30%, 1 H NMR (400MHz, CDCl3) δ3.88 (dd, J = 14.7, 7.9Hz, 1H), 3.71 (m, 7H), 3.29 (s, 1H), 3.05-2.91 (m, 1H), 2. 79-2.37(m,9H),2.02(dd,J=15.6,8.6Hz,1H),1.81(m,3H),1.59-1.07(m,13H),1.04-0.69(m,8H).

[0051] Performance test: HepG2, SW480 and U87 cells: MTT assay to detect cell viability:

[0052] Before each experiment, HepG2, SW480 and U87 cells were cultured with phenol red-free RPMI-1640 medium and activated carbon-treated FBS for 2 days; 1000 HepG2, SW480 and U87 cells were inoculated in each well of a 96-well plate and cultured overnight to allow them to adhere to the wall; all compounds were dissolved in DMSO, and the control group was treated with DMSO, administered at one thousandth, and incubated for 120 hours; 20 μL MTT reagent (5 mg / mL, prepared in PBS) was added to each well, incubated for 4 hours, the culture medium was discarded, and 200 μL DMSO solution was added, and the shaker was shaken for about 10 minutes to fully dissolve the formazan crystals, and the absorbance was measured at 490 nm; cell survival rate: cell survival rate (%) = (OD drug group OD blank group) / (OD control group OD blank group) × 100%, and the results are shown in Figure 5 , the results are shown in Figure 5 ,in Figure 5 12 is the impurity structure V of rocuronium bromide. After testing, the rocuronium impurity structure V molecule showed moderate anti-cancer activity in three cancer cell lines, HepG2, SW480 and U87, at two concentrations of 5mM and 10mM, compared with the control.

[0053] It can be seen from the above examples that the present invention provides a new rocuronium bromide impurity molecule, which has not been reported to have anti-cancer activity in existing studies, and provides a new lead compound for the field of anti-cancer drug research.

[0054] High-purity preparation: The preparation method of the present invention uses epiandrosterone as a raw material, first oxidizes the 3-hydroxyl group to a carbonyl group using Dess-Martin reagent, and then bromines the 2- and 16-positions with bromine under acidic conditions. Then, under alkaline conditions, nucleophilic substitution occurs with morpholine, and then the 3-carbonyl group is selectively reduced to a hydroxyl group, thereby obtaining the target impurity molecule. The purity of the target product obtained by the preparation method can reach more than 95%, ensuring the quality and reliability of subsequent biological activity research and drug development.

[0055] Anticancer activity: The present invention uses the MTT method to show that the molecule exhibits moderate anticancer activity in three cancer cell lines, HepG2, SW480 and U87, at two concentrations of 5mM and 10mM, compared with the control, showing potential anticancer effects. The anticancer treatment is used for the treatment of prostate cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, etc.

[0056] Potential for medicinal chemical modification: The structure of the impurity molecule provides the possibility for further medicinal chemical modification, which will help develop new anti-cancer drugs that are more effective and selective.

[0057] Clinical application prospects: Rocuronium bromide is a known muscle blocker or muscle relaxant. The discovery of the anti-cancer activity of its impurity molecules provides a new perspective for clinical application and helps to develop drugs with dual effects, that is, muscle relaxants and anti-cancer effects at the same time.

[0058] Public health significance: In view of the high incidence and mortality of cancer worldwide, the rocuronium bromide impurity molecule of the present invention and its potential anti-cancer application are of great significance for reducing the burden of public health.

[0059] Simplified process flow: The preparation method provided by the present invention has reasonable process design, simple post-processing, and is easy for large-scale production, which is conducive to reducing production costs and improving efficiency.

[0060] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A rocuronium bromide impurity molecule with anticancer activity, characterized in that: Its structural formula V is:

2. A method for preparing a rocuronium bromide impurity molecule having anticancer activity, characterized in that: The steps include: Step S1: Take epiandrosterone I as a raw material and dissolve it in solvent 1, add Dess-Martin reagent, and react to obtain intermediate II. The reaction equation is as follows: Step S2: Dissolve the intermediate II in an acidic solvent II, add bromine, and react to generate the intermediate III. The reaction equation is as follows: Step S3: Dissolve the intermediate III in solvent three, add a base and morpholine, and react to generate the intermediate IV. The reaction equation is as follows: Step S4: Dissolve the intermediate IV in solvent IV, add potassium triisobutylborohydride, and react at low temperature to generate the target product V. The reaction equation is as follows:

3. The method for preparing the rocuronium bromide impurity molecule with anticancer activity according to claim 2, characterized in that, The molar ratio of epiandrosterone I to Dess-Martin reagent in step S1 is 1:1.2 to 1:

7.

4. The method for preparing the rocuronium bromide impurity molecule with anticancer activity according to claim 3, characterized in that: The solvent in step S1 is selected from one or more of dichloromethane, methanol, and ethyl acetate; the reaction temperature in step S1 ranges from 0°C to 25°C; and the reaction time is 6h-24h.

5. The method for preparing the rocuronium bromide impurity molecule with anticancer activity according to claim 1, characterized in that: The molar ratio of intermediate II to bromine in step S2 is 1:2.5 to 1:

3.

6. The method for preparing the rocuronium bromide impurity molecule with anticancer activity according to claim 5, characterized in that: The second solvent in step S2 is selected from one of aqueous solutions of formic acid, acetic acid and hydrobromic acid; the reaction temperature in step S2 is 0°C to 25°C, and the reaction time is 3-6h.

7. The method for preparing the rocuronium bromide impurity molecule with anticancer activity according to claim 1, characterized in that: The molar ratio of the intermediate III to the base in step S3 is 1:2.5 to 1:

4.

8. The method for preparing the rocuronium bromide impurity molecule with anticancer activity according to claim 7, characterized in that: The solvent three in step S3 is selected from one of dichloromethane, tetrahydrofuran and acetonitrile; the reaction temperature in step S3 is 0° C. to 50° C., and the reaction time is 4-9 hours.

9. The method for preparing the rocuronium bromide impurity molecule with anticancer activity according to claim 1, characterized in that: The molar ratio of the intermediate IV to potassium triisobutylborohydride in step S4 is 1:1.1 to 1:

2.

10. The method for preparing the rocuronium bromide impurity molecule with anticancer activity according to claim 9, characterized in that: The solvent in step S4 is selected from one of dichloromethane, ethyl acetate, isopropyl ether and tetrahydrofuran; the reaction temperature in step S4 is -78°C to 0°C, and the reaction time is 3-9h.

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