CN108299421B - Acylated derivative of resiquimod, preparation method and application - Google Patents
Acylated derivative of resiquimod, preparation method and application Download PDFInfo
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- CN108299421B CN108299421B CN201810088896.1A CN201810088896A CN108299421B CN 108299421 B CN108299421 B CN 108299421B CN 201810088896 A CN201810088896 A CN 201810088896A CN 108299421 B CN108299421 B CN 108299421B
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- C07—ORGANIC CHEMISTRY
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- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
Abstract
Hair brushThe invention relates to an acylated derivative of resiquimod and a preparation method and application thereof; the structural general formula of the acylated derivative of the resiquimod is shown as follows, wherein R1One selected from benzoyl, acetyl, formyl, phenylacetyl or hydrogen; r2One selected from benzoyl, formyl, hydrogen or acetyl. The resiquimod acylated derivative provided by the invention has good activity application on oral cancer cells and melanoma cells, and has good clinical application value. Has obvious effect in treating oral cancer and melanoma.
Description
Technical Field
The invention relates to the field of chemicals and chemical synthesis, in particular to an acylated derivative for resisting melanoma resiquimod and a preparation method thereof.
Background
Oral cancer is a serious, growing incidence of global malignant neoplastic disease. Because the high prevalence and incidence of oral cancer become a major public health problem, oral cancer is the sixth most common tumor in the world, 26.3 new cases of oral cancer occur every year, and the number of deaths is nearly 13 ten thousand. The western countries are regions with high incidence of oral cancer, and the infection rate of oral cancer diseases is gradually rising and receiving much attention. China is a large population country, has more people, and is very important for the prevention and treatment work of oral cancer and the development of effective medicines. At present, the clinical treatment methods for oral cancer mainly include surgical treatment, radiotherapy, chemotherapy and combination treatment of different methods. Despite the great progress in clinical diagnosis and treatment of oral cancer in recent years, the five-year survival rate of patients remains low.
Melanoma is a highly malignant tumor, has many skin, is easy to transfer in early stage, has high mortality rate and poor prognosis, and has poor treatment effect. 1/3 cases have relapse or distant metastasis after operation, metastatic melanoma has fast progress, is not sensitive to radiotherapy and chemotherapy, has poor prognosis and poses great threat to human health. Regarding the treatment of melanoma, biological immunotherapy has become a hot research topic in addition to surgical therapy, chemical drug therapy, and radiation therapy.
Resiquimod, also known as Resiquimod, is a member of the family of imidazoquinolines structurally related to Imiquimod (Imiquimod), is a modulator of immune responses and acts as an agonist at Toll-like receptors 7 and 8. Imiquimod is FDA approved for the treatment of many skin disorders, however, since this drug is only signaled through the Toll-like receptor 7(TLR7), it is clearly different from Resiquimod. Resiquimod is a more potent inducer of IFN-. alpha.TNF-. alpha.IL-1, IL-6, IL-8 and IL-12 than Imiquimod. Resiquimod has been shown to be 10-100 fold more potent than imiquimod in T cell activation, but has not been FDA approved for clinical use.
It has been shown that Resiquimod promotes cross-presentation of exogenous antigens and can efficiently induce CD8+ T cell-specific antigen responses in animal models. Resiquimod has been shown by in vivo experiments to have the ability to activate dendritic cells, including inducing local activation of immune cells, stimulating the production of pro-inflammatory cytokines, and enhancing antigen presentation by dendritic cells, thereby effectively activating cellular responses. In the lymphoma model, Resiquimod administered systemically diffusively has a persistent anti-tumor immune response. Resiquimod has been used in clinical trials to treat actinic keratosis, cutaneous T-cell lymphoma and herpes simplex virus, with scurvy. Resiquimod, as a vaccine adjuvant, is known in the art for the treatment of a variety of diseases, including metastatic melanoma, where the outcome of the treatment is uncertain. Resiquimod has been used as a vaccine adjuvant for NY-10ESO-1 protein vaccines for the treatment of melanoma.
Disclosure of Invention
Based on the background technology, the invention provides an acylated derivative of Resiquimod (Resiquimod) for resisting melanoma, which is characterized in that the structure general formula is as follows:
wherein R is1One selected from benzoyl, acetyl, formyl, phenylacetyl or hydrogen; r2One selected from benzoyl, formyl, hydrogen or acetyl.
Further provided by the invention is R in the acylated derivative of the resiquimod1Is hydrogen, R2Is acetyl.
The structural formula is as follows:
the acylated derivatives of resiquimod are preferably prepared by the following preparation method:
the preparation method comprises the following steps:
1) dissolving resiquimod in an organic solvent at the temperature of-3-2 ℃, respectively adding triethylamine and 4-dimethylaminopyridine, and then slowly dropwise adding an acetyl chloride solution to obtain a mixed solution;
2) stirring the mixed solution at the temperature of 20-30 ℃ until a new spot appears on a TLC spot plate in TLC detection, and quenching the reaction to obtain the compound;
preferably, the organic solvent is chloroform or tetrahydrofuran, preferably chloroform.
Preferably, the molar ratio of the resiquimod to the acetyl chloride is 0.1-0.5: 1, preferably 0.3: 1;
preferably, every 1mL of the organic solvent is dissolved in 0.3-0.7 mmol of benzoyl chloride in the benzoyl chloride solution; more preferably, benzoyl chloride is dissolved in 0.5mmol of said benzoyl chloride solution per 1mL of said organic solvent.
The invention further provides that in any of the above preparation methods, the molar ratio of resiquimod to triethylamine is 0.1-0.5: 1, preferably 0.3: 1;
the triethylamine is used as a catalyst.
The invention further provides that any of the above preparation methods further comprises purification, specifically: after quenching reaction, extracting the reaction liquid for 2-4 times by using ethyl acetate, taking an organic phase, drying, and removing the organic phase by rotary evaporation; eluting with silica gel column chromatography, concentrating, and drying;
preferably, the elution is performed with ethyl acetate: and petroleum ether is performed in a gradient of 1: 4-2: 1.
The Resiquimod (Resiquimod) acylated derivative provided by the invention has good activity on oral cancer cells and melanoma cells, and has strong selectivity and good clinical application value. Has obvious effect in being applied to the medicines for treating oral cancer and melanoma.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The developing solvent in TLC detection in the following examples is specifically: ethyl acetate: petroleum ether is 2: 1; wherein the iodine jar develops color.
Example 1
This example provides an acylated derivative of Resiquimod (B), having the structure:
the acylated derivative of Resiquimod was prepared using a process comprising the steps of:
1) under the ice-bath condition, 47.2mg (0.15mMol, mw ═ 314) of resiquimod is dissolved in 10ml of chloroform, 51mg of triethylamine and 2mg of 4-dimethylaminopyridine are respectively added to the resiquimod, and then a chloroform solution of acetyl chloride is slowly dripped to obtain a mixed solution;
wherein 40mg of acetyl chloride in the chloroform solution of acetyl chloride is dissolved in 1ml of chloroform;
2) stirring the mixed solution at the temperature of 25 ℃ until a new spot appears on a TLC spot plate in TLC detection, and adding ice water to quench the reaction;
3) extracting the reaction solution of the quenching reaction with 10ml of ethyl acetate for 3 times, and taking an organic phase after liquid separation; drying the mixture by adopting anhydrous sodium sulfate, and then removing an organic phase by rotary evaporation; eluting with silica gel column chromatography, concentrating, and drying;
wherein the elution is performed with ethyl acetate: and petroleum ether is performed in a gradient of 1: 4-2: 1.
This example produced 21mg of Resiquimod acylated derivative (mw 356.42) in 39.3% yield.
Example 2
Oral squamous carcinoma cell SCC-15 and human tongue cancer cell CAL-27 were cultured and passaged (frozen if necessary) in RPMI 1640 complete culture medium containing 10% fetal bovine serum, 100 ten thousand units of penicillin and 80 ten thousand units of streptomycin at 37 ℃ under 5% CO2 and saturated humidity. Taking two strains of oral squamous carcinoma cell SCC-15 and human tongue squamous cell CAL-27 in logarithmic growth phase, digesting with trypsin, collecting suspension, centrifuging (1000r, 4min), discarding supernatant, adding culture medium, counting, diluting to 5 × 104 cells/ml, adding into 96-well plate, and adding 100ul per well.
After overnight incubation, the stock culture was removed, and the experimental group was supplemented with Resiquimod and Resiquimod acylated derivative B (Resiquimod acylated derivative prepared in example 1) at final concentrations of 200ug/ml, 100ug/ml, 50ug/ml, 25ug/ml, and 10ug/ml, respectively, and the negative control group was supplemented with the complete culture. After 24 hours and 48 hours of reaction, removing original culture solution, adding 10ul of MTT reagent, incubating at 37 ℃ for 4 hours, taking out and removing supernatant, adding 100ul of DMSO, oscillating for 15 minutes on a micropore oscillator, measuring absorbance (OD value) at wavelength of 570nm by using an enzyme-linked reaction marker, recording results, calculating an average value, and calculating the proliferation inhibition rate. The test is repeated for more than 3 times, and the results are basically consistent.
Resiquimod and Resiquimod acylated derivative B were allowed to act on oral squamous carcinoma cells SCC-15 in the same 1 96-well plate according to the method described above. The two compounds have different degrees of inhibition on cell growth (see table 1), wherein the derivatives have the best effect, and the half inhibitory concentration IC50 of the two compounds is 169.55 mu g/ml and 17.59 mu g/ml respectively.
Resiquimod and Resiquimod acylated derivative B were allowed to act on human tongue cancer cells CAL-27 in the same 1-well 96-well plate according to the method described above. Three compounds have different degrees of inhibition on cell growth (see table 2), wherein the derivatives have the best effect, and the half inhibitory concentrations IC50 of the two compounds are respectively 0.92mg/ml and 27.09 mu g/ml.
TABLE 1 MTT assay for the Effect of 2 active Agents on SCC-15 cells
TABLE 2 MTT method for determining the Effect of 2 active Agents on CAL-27 cells
Example 3
Human melanoma cell A875 and malignant melanoma cell WM451Lu were cultured and passaged (frozen if necessary) in RPMI 1640 complete culture medium containing 10% fetal bovine serum, 100 ten thousand units of penicillin and 80 ten thousand units of streptomycin at 37 ℃ under a saturated humidity environment with 5% CO2, respectively. Taking two strains of human melanoma cells A875 and malignant melanoma cells WM451Lu in logarithmic growth phase, digesting with trypsin, collecting suspension, centrifuging (1000r for 4min), discarding supernatant, adding culture medium, counting cells, diluting to 5 × 104 cells/ml, and adding into 96-well plate with each well being 100 ul.
After overnight culture, the stock culture was removed, and the experimental group was supplemented with Resiquimod and Resiquimod acylated derivative B to a final concentration of 200ug/ml, 100ug/ml, 50ug/ml, 25ug/ml, 10ug/ml, respectively, and the negative control group was supplemented with the complete culture. Respectively acting for 48h, removing original culture solution, adding 10ul MTT reagent, incubating at 37 ℃ for 4h, taking out and removing supernatant, adding 100ul DMSO, oscillating for 15min on a micropore oscillator, measuring absorbance (OD value) at wavelength of 570nm by using an enzyme-linked reaction labeling instrument, recording results, calculating an average value, and calculating the proliferation inhibition rate. The test is repeated for more than 3 times, and the results are basically consistent.
Resiquimod, Resiquimod acylated derivative B was allowed to act on human melanoma cells A875 in the same 1 96-well plate as described above. Three compounds had different degrees of inhibition of cell growth (see Table 3), two of which were significantly more potent than Resiquimod, with half maximal inhibitory concentrations IC50 of 219.44. mu.g/ml, 16.88. mu.g/ml and 28.57. mu.g/ml for the three compounds.
Resiquimod, Resiquimod acylated derivative A and Resiquimod acylated derivative B were allowed to act on malignant melanoma cells WM451Lu on the same 1-well 96-well plate according to the method described above. Two compounds have different degrees of inhibition on cell growth (see Table 1), wherein the derivatives have more significant effects than Resiquimod, and the half inhibitory concentrations IC50 of the two compounds are 194.56 mu g/ml and 39.37 mu g/ml respectively.
TABLE 3 MTT assay for the Effect of 2 active Agents on 2 melanoma cells
Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (12)
1. An acylated derivative of resiquimod, which is characterized by the following structural general formula:
wherein R is1Is hydrogen, R2Is acetyl.
2. Process for the preparation of acylated derivatives of resiquimod according to claim 1, characterized in that it comprises the following steps:
1) dissolving resiquimod in an organic solvent at the temperature of-3-2 ℃, respectively adding triethylamine and 4-dimethylaminopyridine, and then slowly dropwise adding an acetyl chloride solution to obtain a mixed solution;
2) and stirring the mixed solution at the temperature of 20-30 ℃ until a new spot appears on a TLC spot plate in TLC detection, and quenching the reaction to obtain the compound.
3. The method according to claim 2, wherein the organic solvent is chloroform or tetrahydrofuran.
4. The preparation method of claim 2, wherein the molar ratio of the resiquimod to the acetyl chloride is 0.1-0.5: 1.
5. The preparation method according to claim 4, wherein the molar ratio of the resiquimod to the acetyl chloride is 0.3: 1.
6. The method according to any one of claims 2 to 5, wherein the acetyl chloride is dissolved in the acetyl chloride solution in an amount of 0.3 to 0.7mmol per 1mL of the organic solvent.
7. The method according to claim 6, wherein acetyl chloride is dissolved in 0.5mmol of the acetyl chloride solution per 1mL of the organic solvent.
8. The preparation method of any one of claims 2 to 5, wherein the mass ratio of the resiquimod to the triethylamine is 0.1-0.5: 1.
9. The preparation method of claim 8, wherein the mass ratio of the resiquimod to the triethylamine is 0.3: 1.
10. The preparation method according to any one of claims 2 to 5, further comprising purification, in particular: after quenching reaction, extracting the reaction liquid for 2-4 times by using ethyl acetate, taking an organic phase, drying, and removing the organic phase by rotary evaporation; eluting with silica gel column chromatography, concentrating, and drying.
11. The method of claim 10, wherein the elution is performed with ethyl acetate: petroleum ether = gradient of 1:4 to 2: 1.
12. Use of acylated derivatives of resiquimod according to claim 1 or acylated derivatives of resiquimod prepared by the preparation method according to any one of claims 2 to 11 for the preparation of medicaments for treating oral cancer and melanoma.
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| US20080187511A1 (en) * | 2006-10-25 | 2008-08-07 | University Of Pittsburgh-Of The Commonwealth | Ointment for cancer treatment |
| CN101300254A (en) * | 2005-09-09 | 2008-11-05 | 科利制药集团公司 | Amide and carbamate derivatives of n-{2-[4-amino-2- (ethoxymethyl)-1h-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}methanesulfonamide and methods |
| CN106232599A (en) * | 2014-04-22 | 2016-12-14 | 豪夫迈·罗氏有限公司 | 4 aminooimidazoles quinoline compound |
| CN106432226A (en) * | 2007-01-31 | 2017-02-22 | 于崇曦 | Prodrug of 1H-imidazo[4,5-c]quinoline-4-amine and related compound |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101300254A (en) * | 2005-09-09 | 2008-11-05 | 科利制药集团公司 | Amide and carbamate derivatives of n-{2-[4-amino-2- (ethoxymethyl)-1h-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}methanesulfonamide and methods |
| US20080187511A1 (en) * | 2006-10-25 | 2008-08-07 | University Of Pittsburgh-Of The Commonwealth | Ointment for cancer treatment |
| CN106432226A (en) * | 2007-01-31 | 2017-02-22 | 于崇曦 | Prodrug of 1H-imidazo[4,5-c]quinoline-4-amine and related compound |
| CN106232599A (en) * | 2014-04-22 | 2016-12-14 | 豪夫迈·罗氏有限公司 | 4 aminooimidazoles quinoline compound |
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Inventor after: Huang Linfang Inventor after: Li Benhan Inventor after: Cao Yu Inventor after: Ding Jiaji Inventor before: Huang Linfang Inventor before: Li Benhan Inventor before: Cao Yu Inventor before: Ding Jiaji Inventor before: Xu Xiaowei Inventor before: Liu Shujing |