CN115504993A

CN115504993A - Light-activated Mcl-1 inhibitor prodrug and synthesis method and application thereof

Info

Publication number: CN115504993A
Application number: CN202211195578.8A
Authority: CN
Inventors: 卢幸; 蓝剑锋; 邬慧贤; 孙梦丹
Original assignee: Affiliated Hospital of Guilin Medical University
Current assignee: Affiliated Hospital of Guilin Medical University
Priority date: 2022-09-28
Filing date: 2022-09-28
Publication date: 2022-12-23

Abstract

The invention discloses a light-activated Mcl-1 inhibitor prodrug, a synthesis method and application thereof. The photo-activated Mcl-1 inhibitor prodrug is prepared by reacting a compound PPG (namely 7-diethylamino-4-hydroxymethyl-coumarin) and a selective Mcl-1 inhibitor S63845 in an organic solvent by adding a catalyst DMAP and a condensing agent under the heating condition or without heating. The test result of the applicant shows that the prodrug has stronger light-activated anti-tumor activity, shows extremely high inhibition rate on various tumor cells under the condition of 405nm light, has better potential medicinal value, and is expected to be used for preparing various anti-tumor medicaments or prodrugs.

Description

Light-activated Mcl-1 inhibitor prodrug and synthesis method and application thereof

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a photo-activated Mcl-1 inhibitor prodrug and a synthesis method and application thereof.

Background

The Mcl-1 (myeloid leukemia-1) protein, as a member of the Bcl-2 family of anti-apoptotic proteins, plays an important role in the development of cancer. Researchers have determined that Mcl-1 protein is essential for the growth of various hematological and solid tumors by methods such as gene knockout, RNA interference, and the like, including: acute myelogenous leukemia, breast cancer, multiple myeloma, non-small cell lung cancer, and the like. Meanwhile, researchers also find that Mcl-1 is closely related to the development of drug resistance. Various antitumor drugs (cisplatin, veratrole, vincristine, paclitaxel and the like) can cause the up-regulation of Mcl-1 protein, so that the drug resistance is generated. Therefore, the Mcl-1 protein is an important tumor therapy target.

Researchers have developed a variety of small molecule inhibitors for Mcl-1 for the treatment of tumors, where S63845 is a potent and selective Mcl-1 inhibitor with the structure shown below:

s63845 has a good inhibition effect on various tumor cells, but has a large toxic and side effect, and the problem needs to be solved urgently.

On the other hand, photoactivation inhibitor prodrugs are a class of molecules that can be activated by light, and their structures are usually linked to the active molecule by a Photodissociation Protecting Group (PPGs). The photoactivation inhibitor prodrug is inactive, but under the irradiation of light with specific wavelength, PPGs are dissociated to release active molecules. By activating the photoactivation inhibitor prodrug at specific sites of light, the side effects of the treatment are reduced. However, compounds in which not any photodissociation protecting group is attached to any active molecule at some site are characterized as photo-activated prodrugs; even if the dissociation of the light-operated dissociation protective group can be realized, the active molecular structure of the obtained compound is not damaged after the dissociation of the light-operated dissociation protective group; nor can it be determined whether the prodrug molecule itself is safe. Therefore, there is a difficulty in developing a prodrug of a photoactivation inhibitor. At present, no report related to the activation of Mcl-1 inhibitor prodrug by light is found, and no report related to a molecule formed by connecting 7-diethylamino-4-hydroxymethyl-coumarin to S63845 is found.

Disclosure of Invention

The invention aims to provide a light-activated Mcl-1 inhibitor prodrug with a novel structure, and a synthesis method and application thereof.

In order to solve the technical problem, the invention adopts the following technical scheme:

the photo-activated Mcl-1 inhibitor prodrug is formed by a selective Mcl-1 inhibitor S63845 and coumarin light-controlled dissociation protecting groups (7-diethylamino-4-hydroxymethyl-coumarin), and particularly is a photo-activated Mcl-1 inhibitor prodrug with a structure shown in the following formula (I):

the preparation method of the photo-activated Mcl-1 inhibitor prodrug comprises the following steps: dissolving a compound PPG (namely 7-diethylamino-4-hydroxymethyl-coumarin) shown in the formula (II) and a selective Mcl-1 inhibitor S63845 in an organic solvent, adding a catalyst and a condensing agent, and reacting under heating or non-heating conditions to obtain a target compound crude product; wherein, the catalyst is DMAP;

in the above-mentioned production method, in order to increase the yield of the objective compound, it is preferable to carry out the reaction under the protection of an atmosphere such as nitrogen or an inert atmosphere (argon, helium, etc.), or under the protection from light. When the reaction is carried out under the conditions of atmosphere protection and light protection, the yield can be further improved; the test results of the applicant show that when the reaction is carried out under the conditions of atmosphere protection and light shielding, the reaction yield can be improved by 10-20% or even higher compared with the reaction yield which is not carried out under the conditions of atmosphere protection and light shielding.

In the above preparation method, the organic solvent may be one or a combination of two or more selected from dichloromethane, chloroform and carbon tetrachloride, preferably dichloromethane. The amount of the organic solvent to be used may be determined as needed, and is usually 20 to 50mL based on 1mmol of S63845.

In the above production method, the amounts of the catalyst and the condensing agent may be determined as needed, and in the present application, the amount of the catalyst is preferably 0.01 to 1 time the amount of the S63845 substance, and the amount of the condensing agent is preferably 1 to 5 times the amount of the S63845 substance. The condensing agent may be conventionally selected in the prior art, and in the present application, a carbodiimide-based condensing agent or the like is preferable, and the carbodiimide-based condensing agent is more preferably one or a combination of two or more selected from EDC HCl (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride), DCC (dicyclohexylcarbodiimide), and DIC (diisopropylcarbodiimide).

In the above production method, the reaction is preferably carried out without heating, and more preferably carried out under ice-bath conditions and then at room temperature. For the time of reaction, detection was followed by thin layer chromatography until the reaction was complete. When the reaction is carried out under the ice-bath condition and then is carried out under the normal temperature condition, the reaction time under the ice-bath condition is usually controlled to be 10-60 min, and then the reaction time when the temperature is raised to the normal temperature is preferably controlled to be 2-24 h.

The crude product of the target compound obtained by the above preparation method can be purified by conventional purification methods to increase the purity of the target compound. The crude target compound is usually subjected to silica gel column chromatography to obtain the purified target compound. When the crude target compound is purified by silica gel column chromatography, a mixed solvent consisting of dichloromethane and methanol is used as an eluent, wherein dichloromethane can be replaced by ethyl acetate, and methanol can be replaced by ethanol. In the composition of the mixed solvent, the volume ratio of dichloromethane (or ethyl acetate) to methanol (or ethanol) is 100:1 to 10:1, preferably 40:1 to 10:1, more preferably 10:1. in order to reduce the burden of the silica gel column, the crude product of the target compound can be subjected to impurity removal and then subjected to silica gel column chromatography. The impurity removal operation specifically comprises the following steps: the crude target compound is diluted with the organic solvent during the reaction, then washed with dilute hydrochloric acid (preferably 0.5-4 mol/L) and/or saturated sodium carbonate solution and/or saturated saline solution, the organic phase is collected, dried with magnesium sulfate, then the solvent is removed by evaporation, and the residue is subjected to silica gel column chromatography.

The photo-activated Mcl-1 inhibitor prodrug can be prepared by another method, and specifically comprises the following steps: reacting selective Mcl-1 inhibitor S63845 and oxalyl chloride in a mixture of DMF and the organic solvent, and evaporating the solvent to obtain a residue; and (3) placing the obtained residue and a PPG compound in the organic solvent for reaction, and adding a saturated sodium bicarbonate solution to quench the reaction after the reaction is finished to obtain a crude product of the target compound. The crude target compound can be purified in the same manner as described above to obtain the purified target compound.

The invention also discloses application of the photo-activated Mcl-1 inhibitor prodrug in preparation of anti-tumor drugs or prodrugs.

The present invention further comprises a pharmaceutical composition comprising a therapeutically effective amount of a prodrug of a light-activated Mcl-1 inhibitor according to claim 1 in combination with a pharmaceutically acceptable carrier or excipient.

Compared with the prior art, the invention provides a light-activated Mcl-1 inhibitor prodrug which is formed by covalently linking coumarin PPG and S63845 and has a novel structure, and a synthetic method and application thereof. The applicant researches the light activation inhibition effect of the prodrug on various tumor cell strains, and the result shows that the prodrug has stronger light activation anti-tumor activity, shows extremely high inhibition rate on various tumor cells under the condition of 405nm illumination, has better potential medicinal value, and is expected to be used for preparing various anti-tumor medicaments or prodrugs.

Drawings

FIG. 1 shows the NMR spectrum of the final product obtained in example 1 of the present invention.

FIG. 2 shows the NMR spectrum of the final product obtained in example 1 of the present invention.

FIG. 3 is a high resolution mass spectrum of the final product obtained in example 1 of the present invention.

Detailed Description

In order to better explain the technical solution of the present invention, the present invention is further described in detail with reference to the following examples, but the embodiments of the present invention are not limited thereto.

In the following examples and experimental examples, PPG represents the compound PPG (i.e. 7-diethylamino-4-hydroxymethyl-coumarin), S63845 represents an abbreviation of selective Mcl-1 inhibitor S63845, and PPG-S63845 represents a photo-activated Mcl-1 inhibitor prodrug according to the present invention.

Example 1

1) 82.9mg (0.1 mmol) of S63845, 29.6mg (0.12 mmol) of PPG and 5ml of dichloromethane are placed in a 50ml round-bottom flask, 37mg of EDC & HCl (0.19 mmol) and 5mg of DMAP (0.04 mmol) are added, the reaction device is placed in an ice-water bath under the conditions of light protection and nitrogen protection for reaction for 1h, and then the reaction is continued for 16h at room temperature;

2) After the reaction is finished, 15ml of dichloromethane is added, the mixture is washed three times by using 0.5mol/L dilute hydrochloric acid solution (10 ml), then the mixture is washed twice by using saturated sodium bicarbonate solution (10 ml), and then the mixture is washed once by using saturated saline solution (10 ml), an organic phase is dried by using anhydrous magnesium sulfate, and a crude product is obtained after the solvent is evaporated;

3) The crude product obtained in the previous step was purified by silica gel column chromatography (eluting solvent is dichloromethane: methanol =10:1, volume ratio) to yield the product as a pale yellow solid (98.3 mg, 93% yield).

The product obtained in this example was subjected to nuclear magnetic hydrogen spectroscopy, carbon spectroscopy and high resolution mass spectrometry, and the spectrograms are shown in fig. 1, fig. 2 and fig. 3, respectively. Thus, the light yellow solid obtained in the example can be determined to be the photo-activated Mcl-1 inhibitor prodrug PPG-S63845.

Example 2

1) 82.9mg (0.1 mmol) of S63845, 24.7mg (0.1 mmol) of PPG and 5ml of chloroform were placed in a 50ml round-bottom flask, 19.5mg of EDC & HCl (0.1 mmol) and 5mg of DMAP (0.04 mmol) were added, and the reaction apparatus was placed in an ice-water bath under protection of light and nitrogen for reaction for 1h, followed by further reaction at room temperature for 22h;

2) After the reaction is finished, adding 15ml of trichloromethane, washing the trichloromethane three times by using 0.5mol/L dilute hydrochloric acid solution (10 ml), then washing the trichloromethane two times by using saturated sodium bicarbonate solution (10 ml), washing the trichloromethane once by using saturated saline solution (10 ml), drying an organic phase by using anhydrous magnesium sulfate, and evaporating the solvent to obtain a crude product;

3) The crude product obtained in the previous step was purified by column chromatography on silica gel (eluting solvent is dichloromethane: methanol =10:1, vol) to yield the product as a pale yellow solid (79.3 mg, 75% yield).

The product obtained in the example is analyzed by nuclear magnetic hydrogen spectrum, carbon spectrum and high-resolution mass spectrum, and is determined to be the photo-activated Mcl-1 inhibitor prodrug PPG-S63845.

Example 3

1) 82.9mg (0.1 mmol) of S63845, 29.6mg (0.12 mmol) of PPG and 3ml of dichloromethane are placed in a 50ml round-bottom flask, 20.7mg of DCC (0.1 mmol) and 5mg of DMAP (0.04 mmol) are added, and the reaction device is stirred for reaction for 18 hours at room temperature under the conditions of light protection and nitrogen protection;

2) After the reaction is finished, 15ml of dichloromethane is added, then saturated sodium bicarbonate solution (10 ml) is used for washing twice, an organic phase is dried by anhydrous magnesium sulfate, and a crude product is obtained after the solvent is distilled off;

3) The crude product obtained in the previous step was purified by column chromatography on silica gel (eluting solvent is dichloromethane: methanol =10:1, volume ratio) to yield the product as a pale yellow solid (86.7 mg, 82% yield).

The product obtained in this example was analyzed by nuclear magnetic hydrogen spectrum, carbon spectrum and high resolution mass spectrum, and was identified as photo-activated Mcl-1 inhibitor prodrug PPG-S63845 according to the present invention.

Example 4

1) 82.9mg (0.1 mmol) of S63845, 29.6mg (0.12 mmol) of PPG and 3ml of dichloromethane were taken and placed in a 50ml round-bottom flask, 20.7mg of DCC (0.1 mmol) and 5mg of DMAP (0.04 mmol) were added, and the reaction apparatus was stirred at room temperature for 18 hours (without protection from light and nitrogen);

3) The crude product obtained in the previous step was purified by column chromatography on silica gel (eluting solvent is dichloromethane: methanol =10:1, volume ratio) to yield the product as a pale yellow solid (31.7 mg, 30% yield).

Example 5

1) A50 ml round bottom flask was charged with 82.9mg (0.1 mmol) S63845, added with 0.2mmol oxalyl chloride, 1 drop of DMF and 5ml dichloromethane, and reacted at room temperature for 4h under nitrogen protection;

2) After the solvent was evaporated to dryness under reduced pressure, 5ml of methylene chloride, 24.7mg (0.1 mmol) of PPG, 0.1mmol of triethylamine and 5mg of DMAP (0.04 mmol) were added thereto, and the mixture was stirred at room temperature for reaction for 10 hours;

3) After the reaction is finished, adding 10ml of saturated sodium bicarbonate solution to quench the reaction, adding ethyl acetate to extract for three times (10 ml), collecting an organic phase, drying the organic phase by using anhydrous magnesium sulfate, and evaporating to remove the solvent to obtain a crude product;

4) The crude product obtained in the previous step was purified by silica gel column chromatography (eluting solvent is dichloromethane: methanol =10:1, vol) to yield the product as a pale yellow solid (56.0 mg, 53% yield).

Experimental example 1: test for light activated prodrug Properties of PPG-S63845

To fully illustrate that PPG-S63845 of the present invention is a photo-activated Mcl-1 inhibitor prodrug, applicants examined its affinity for Mcl-1 under dark conditions and 405nm light conditions by Surface Plasmon Resonance (SPR) experiments.

1. Coupling Mcl-1 protein to CM5 chip

In the experiment, firstly, a BIAcore amino coupling kit is used for coupling Mcl-1 protein on the surface of a CM5 chip: a1 mg/ml solution of Mcl-1 protein was dissolved in a 10mM solution of sodium acetate (pH = 5.5) in a coupling amount of 7000Ru.

2. Treatment of compound solutions

The PPG-S63845 solution was left to stand under dark conditions or 405nm light conditions for 20min, respectively.

3. Affinity assay

The treated compound solution was added to a BIAcore T-200 instrument and passed over the chip surface at a flow rate of 20. Mu.l/min, and the data was recorded.

4. Fitting data

Affinity values were obtained by fitting the data to the 1 binding model using BIAcore T-200 self data (as shown in Table 1 below).

Table 1: affinity K of PPG-S63845 to Mcl-1 under non-light conditions and after light treatment _D (nM)

From the SPR test result, after the PPG-S63845 is subjected to light treatment, the affinity to the Mcl-1 is improved by 325 times compared with that under the condition of no light, and the effect is very obvious. Therefore, PPG-S63845 is a good photo-activated Mcl-1 inhibitor prodrug and has good application prospect.

Experimental example 2: in vitro antitumor Activity test of PPG-S63845

In order to fully illustrate the application of the PPG-S63845 in the preparation of medicines, the applicant tests the inhibition effect of the PPG-S63845 on various tumor cells under dark conditions and 405nm light conditions through an in vitro anti-tumor activity experiment.

1. Cell lines and cell cultures

The experiment selects 5 cell strains of human myeloma cell H929, human lung cancer cell H23, human hepatoma cell strain Hep G2, human cervical cell HeLa and human normal hepatocyte HL-7702.

All tumor cell lines were cultured in RPMI-1640 medium containing 10wt% calf blood, 100U/mL penicillin and 100U/mL streptomycin, and cultured in a CO2 incubator containing 5% by volume at 37 ℃; the human normal cell line was cultured in DMEM containing 10wt% calf blood, 100U/mL penicillin and 100U/mL streptomycin.

2. Preparation of test Compounds

PPG-S63845 used is the product obtained in the embodiment 1 of the invention, is obtained by column chromatography purification, has the purity of more than or equal to 95 percent, and is diluted into 2 concentration gradients in turn by a DMSO stock solution (the concentration is 0.01 mol/L) through RMPI1640 culture medium,

respectively 1,0.01 mu mol/L, wherein the final concentration of the cosolvent DMSO is less than or equal to 1 percent. PPG-S63845 was first tested for inhibition under dark conditions and 405nm light conditions.

3. Cell growth inhibition experiment (CCK 8 method)

(1) Taking tumor cells in logarithmic growth phase, digesting with trypsin, preparing a cell suspension with the concentration of 5000/mL by using a culture solution containing 10% calf serum, inoculating 190 mu L of the cell suspension into a 96-well culture plate, and enabling the cell density to be detected to be 1000-10000 per well (filling a marginal hole with sterile PBS);

(2)5％CO ₂ incubating for 24h at 37 ℃ until a cell monolayer is paved on the bottom of each well, adding 10 mu L of medicine with a certain concentration gradient into each well, and arranging 4 compound wells in each concentration gradient;

(3) The treatment was carried out for 20min under light and dark conditions at 405nm, respectively, according to the groups.

(4)5％CO ₂ Incubating for 6h at 37 ℃, and observing under an inverted microscope;

(5) Adding 10 mu L of CCK8 solution into each hole, and continuously culturing for 4h;

(6) Terminating the culture, uniformly mixing by using an oscillator, and detecting the optical density value of each hole with the detection wavelength of 450 nm;

(7) The number of living cells was judged from the measured optical density values (OD values), and the larger the OD value, the stronger the cell activity.

Using the formula:

and (4) calculating the inhibition rate of the compound on the growth of tumor cells. The test results are shown in tables 2 and 3 below.

Table 2: growth inhibition rate (%) of PPG-S63845 on different tumor cell strains under no illumination condition

Table 3: growth inhibition rate (%) of PPG-S63845 on different tumor cell strains under 405nm lighting condition

From the results of in vitro anti-tumor activity tests, the PPG-S63845 shows high inhibition rate to various tumor cells under the condition of 405nm light, and the inhibition rate is low under the condition of no light, which indicates that the PPG-S63845 has light-activated anti-tumor activity. In addition, PPG-S63845 has low toxicity to normal cells under the condition of illumination, has good potential medicinal value, and is expected to be used for preparing various antitumor drugs or prodrugs.

Claims

1. A photo-activated Mcl-1 inhibitor prodrug having the structure shown in formula (I):

2. the process for preparing a prodrug of a photoactivated Mcl-1 inhibitor as claimed in claim 1, wherein the compound PPG of formula (II) and optionally the Mcl-1 inhibitor S63845 are dissolved in an organic solvent, and then a catalyst and a condensing agent are added to react with or without heating to obtain the crude target compound; wherein, the catalyst is DMAP;

3. the method according to claim 2, wherein the reaction is carried out under an atmosphere.

4. The process according to claim 2 or 3, wherein the reaction is carried out under protection from light.

5. The method according to claim 2 or 3, wherein the organic solvent is one or a combination of two or more selected from the group consisting of dichloromethane, chloroform and carbon tetrachloride.

6. The production method according to claim 2 or 3, wherein the condensing agent is a carbodiimide-based condensing agent.

7. The process according to claim 2 or 3, wherein the reaction is carried out under ice-bath conditions and then at room temperature.

8. The process according to claim 2 or 3, further comprising a step of purifying the crude target compound obtained.

9. Use of a photo-activated Mcl-1 inhibitor prodrug according to claim 1 for the preparation of an anti-tumor drug or for the preparation of a photo-activated Mcl-1 inhibitor prodrug.

10. A pharmaceutical composition comprising a therapeutically effective amount of a pro-drug of a light-activated Mcl-1 inhibitor according to claim 1.