CN108774161B - Preparation and application of six PARP1 inhibitors - Google Patents

Preparation and application of six PARP1 inhibitors Download PDF

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CN108774161B
CN108774161B CN201810812259.4A CN201810812259A CN108774161B CN 108774161 B CN108774161 B CN 108774161B CN 201810812259 A CN201810812259 A CN 201810812259A CN 108774161 B CN108774161 B CN 108774161B
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史大永
王立军
郭传龙
李祥乾
江波
李晓伟
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Abstract

The invention relates to preparation of six novel PARP1 inhibitors, combination of the compounds and application of the compounds in antitumor and/or anticancer drugs, and also relates to a preparation method of the compounds, wherein the chemical structures of the compounds are as follows:

Description

Preparation and application of six PARP1 inhibitors
Technical Field
The invention relates to the technical field of medicines, in particular to six PARP1 inhibitors and pharmacological activity and pharmaceutical application thereof. The compound and the derivative thereof can be used for preventing and/or treating diseases such as tumors or cancers caused by various factors.
Background
Malignant tumor seriously affects human health, and is the second leading cause of human death, second only to cardiovascular and cerebrovascular diseases. According to the statistics of world health organization, the newly diagnosed tumor patients all over 1000 ten thousand each year in the world, and the total number of tumor deaths per year in the world reaches 700 thousand. With the increasing environmental pollution, the incidence of cancer is increasing year by year and is in a multiple situation, and world health organization 2014 reports that global cancer cases are predicted to be in a rapidly growing situation, from 1400 ten thousand in 2012, to 1900 ten thousand in 2025 year by year, to 2400 ten thousand in 2035, and the number of deaths is also increased from 600 ten thousand to 1000 ten thousand per year. In 2012, 307 new cancer patients are newly added in China and cause about 220 million people to die, which respectively account for 21.9% and 26.8% of the total amount of the world, and the cancer becomes the leading cause of death of urban and rural residents in China. The tumor not only seriously threatens the health of people and brings economic loss to patients and families of the patients, but also causes heavy burden to the nation and the society. The search for novel antitumor drugs with high efficiency, low toxicity, and a new action mechanism for selectively killing or inhibiting tumor cells has become an important direction for research and development of antitumor drugs.
The poly adenosine diphosphate ribose polymerase-1 (PARP-1) participates in the process of DNA repair and transcription regulation, can regulate the process of cell survival and death, is also a main transcription factor in the process of tumor generation and inflammation formation, and has become a hot target in the field of tumor treatment in recent years. The PARP-1 inhibitor has obvious curative effect on breast cancer and ovarian cancer with BRCA1/BRCA2 mutation, the main action mechanism is to aim at the function of PARP-1 participating in SSB/DSB repair, the PARP-1 catalytic activity is inhibited, the PARP-1 is captured on damaged DNA to play an anti-tumor role, the rucaparib of Clovis Oncology becomes the first drug of the drugs to obtain the prosperity, and at present, 3 PARP1 inhibitors are clinically applied as anti-tumor drugs.
The marine bromophenol compounds mainly refer to compounds which are obtained by separating marine animals, plants and microorganisms such as marine fungi, seaweed, sponge, ascidian, bryozoans and the like, contain one or more hydroxyl groups and bromine atoms in molecular structures to replace benzene rings, and are unique compounds from marine sources. At present, the activity screening of the bromophenol compounds is relatively deep, and a large number of lead compounds with good biological activity in the aspects of oxidation resistance, antibiosis, tumor resistance, thrombus resistance, blood sugar reduction, biological food refusal and the like are obtained [ Mar, Drugs 2011,9(7), 1273-; bioorganic chem.2015,60, 49-57; eur.j.med. chem.,2012,54,423-428. The potential application and development values of the compounds in the aspect of antitumor activity have also attracted great interest of researchers at home and abroad. Thiosemicarbazone compounds have various biological activities such as antivirus, antitumor, leprosy resistance, tuberculosis resistance, malaria resistance and the like, and the research on the synthesis and the biological activity of the compounds becomes a research hotspot of pharmaceutical chemistry (Environ solution.2015 Sep; 204:81-9, Sep Sci.2017 Jan; 40(2):514-523, Bioorg chem.2015 Jun; 60: 49-57).
The invention utilizes the principle of drug molecule hybrid design to couple and design anti-tumor active groups of bromophenol and thiosemicarbazide compounds into six compounds 1-6, and the prior art does not report the six compounds 1-6 provided by the invention and the application of the derivatives or pharmaceutical compositions thereof in preparing or treating drugs for diseases such as tumors or cancers caused by various factors.
Disclosure of Invention
The invention relates to the technical field of medicines, in particular to six PARP1 inhibitors, a preparation method thereof, and pharmacological activity and pharmaceutical application thereof. The compounds and derivatives thereof can be used for preventing and/or treating diseases such as tumors, cancers and the like caused by various factors.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention designs and synthesizes six PARP1 inhibitors, and the chemical structures of the inhibitors are shown as follows:
Figure BDA0001739459320000031
chemical names Chinese are respectively: 2,2' - (((butane-1, 4-diyldioxy) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (N-methylhydrazine-1-thioamide) (1), 2,2' - (((((propane-1, 3-diyldioxy) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (N- (3-morpholino) hydrazine-1-thioamide) (2), 2,2' - (((butane-1, 4-diyldioxy) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (N- (3-morpholino) hydrazine-1-thioamide) (3), 2,2' - (((hexane-1, 6-diyldioxy) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (N- (3-morpholinyl) hydrazine-1-thioamide) (4), 2,2' - (((((propane-1, 3-diyldioxy) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (hydrazine-1-thioamide) (5), 2,2' - (((((butane-1, 4-diylbio) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (hydrazine-1-thioamide) (6);
the English names are respectively: 2,2'- ((butane-1, 4-dimethyl) bis (oxy)) bis (3-methyl-5-methoxy-4, 1-phenylene) bis (methyl-idene)) bis (N-methyl-5-methoxy-4, 1-phenylene) bis (3-methyl-5-methyl-4, 1-phenylene) bis (methyl-3-methoxy-1-phenylene) bis (N- (3-methyl-1-phenoxy) bis (2,2' - ((butyl-1, 4-dimethyl) hydrazine-1-methyl-1-phenylene) (2), 2,2'- ((butyl-1, 4-dimethyl) bis (N- (3-methyl-1-phenylene) (2), 2,2' - ((butyl-1, 4-dimethyl) bis (3-methyl-5-phenyl-1-phenylene) (2,2 '-dimethyl-1, 4-dimethyl) bis (1-methyl-1-phenylene) (2,2' -dimethyl-1, 4-dimethyl) bis (3-methyl-1-phenylene) (2,2 '-dimethyl-1-phenylene) (2,2' -dimethyl-1-phenylene) (2, 2-dimethyl-1, 2-dimethyl-1-phenylene) (2-dimethyl-1, 2-phenylene), 6-diylbis (oxy)) bis (3-bromo-5-methoxy-4,1-phenylene) bis (methanoylene)) bis (N- (3-morpholinopropyl) hydrazine-1-carbothioamide) (4), 2,2'- ((propane-1, 3-dioxa) bis (3-bromo-5-methoxy-4,1-phenylene)) bis (methanoylene) bis (hydrazine-1-carbothioamide) (5), 2,2' - ((butane-1, 4-dioxa) bis (3-bromo-5-methoxy-4,1-phenylene)) bis (3-methoxy-5-methoxy-4, 1-phenylene)) bis (1-phenylene) (6-phenylene) bis (1-phenylene) (6)
One or a mixture of two or more of the six novel PARP1 inhibitors 1-6 has antitumor activity, and the inhibitor 1-6 referred to herein can be one of the chemical equivalents of the inhibitor 1-6 itself and a pharmaceutically acceptable salt of the inhibitor 1-6, but is not limited to the above chemical equivalents.
The invention also provides application of the six novel PARP1 inhibitors 1-6 in preparation of medicines for preventing and/or treating tumors and/or cancers and other related diseases caused by various factors.
The six novel PARP1 inhibitors of the present invention, when used as a medicament, may be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90% of the compound of the present invention, and the balance pharmaceutically acceptable carriers and/or excipients.
The pharmaceutically acceptable carrier or excipient is one or more of solid, semi-solid and liquid diluents, fillers and pharmaceutical adjuvants. The pharmaceutical composition of the present invention is used in the form of a dose per unit body weight. The medicine of the invention can be administered by three forms of injection (intravenous injection and intramuscular injection), oral administration and external application.
The invention aims to provide six novel PARP1 inhibitors and a preparation method thereof, and the compounds and the derivatives thereof can prevent and/or treat diseases such as tumors and/or cancers caused by various factors, such as one or more than two of the tumors and/or cancer diseases such as lung cancer, liver cancer, breast cancer, ovarian cancer, prostate cancer and the like.
1. Synthesis and structural identification of compounds
The preparation method of the six novel PARP1 inhibitors 1-6 comprises the following steps:
Figure BDA0001739459320000051
step 1: preparation of intermediate alkoxy dibromophenol benzaldehyde (I-III):
mixing 5-bromovanillin (13.9g, 60mmol) and potassium carbonate (2.8g, 20mmol) at room temperature, adding 20mL of DMF, dissolving, stirring for 10min, dropwise adding 1, 3-dibromopropane or 1, 4-dibromobutane or 1, 6-dibromohexane (2mL, 20mmol) while stirring, heating at 80 ℃, condensing and refluxing for 10h, adding 500mL of water to precipitate white flocculent precipitate, filtering the precipitate, washing the precipitate with saturated sodium bicarbonate, and washing the precipitate with ethyl acetate to obtain an intermediate.
Step 2: preparation of Compounds 1-6:
weighing the intermediate compound I, II or III (2mmol) and the corresponding thiosemicarbazide (4.4mmol), mixing in 100mL of 95% ethanol, stirring for 10min, dropwise adding 1000 mu L of acetic acid while stirring, heating at 70 ℃, condensing and refluxing for 12h, separating out a white precipitate, carrying out suction filtration on the precipitate, washing the precipitate with ethanol, and drying to obtain the compound 1-6. The thiosemicarbazide is one or more than two of 4-methyl thiosemicarbazide, 4- [3- (4-morpholine) propyl ] -3-thiosemicarbazide and thiosemicarbazide.
2. The method for determining the inhibitory activity of the six compounds 1-6 of the invention on poly (A-bis (phosphoribosyl) transferase) (PARP1)
All reagents used in the experiment should be balanced to room temperature, and the reagents or samples need to be fully mixed when being prepared, so that foaming is avoided.
(1) Sample adding: respectively setting a blank hole, a standard hole and a sample hole to be measured. And adding 100 mu L of standard substance and sample diluent into the blank hole, respectively adding 100 mu L of standard substance or sample to be detected into the rest holes, uniformly mixing, and incubating for 90 minutes at 37 ℃.
(2) The liquid was discarded, spun off, 100. mu.L of biotinylated antibody working solution was added to each well, and incubated at 37 ℃ for 1 hour.
(3) The well liquid was discarded, spun dry, plate washed 3 times, soaked 1-2 minutes each time, approximately 350 μ L/well, spun dry and patted dry on absorbent paper.
(4) Add 100. mu.L of enzyme conjugate working solution (prepared within 15 minutes before use) to each well and incubate at 37 ℃ for 30 minutes.
(5) And (4) discarding liquid in the holes, spin-drying, and washing the plate for 5 times, wherein the method is the same as the step 3.
(6) Substrate solution (TMB) 90. mu.L was added to each well and incubated at 37 ℃ for about 15 minutes in the absence of light.
(7) The reaction was stopped by adding 50. mu.L of stop solution to each well.
(8) The optical density (OD value) of each well was measured at a wavelength of 450nm using a microplate reader.
(9) The inhibition was calculated from the measured OD values.
3. The toxicity of the six compounds 1-6 of the invention to various tumor cells is determined
The cytotoxicity of the synthesized derivative on human tumor cells cultured in vitro is detected by adopting a currently common tetrazolium salt (MTT) method. Cell lines selected for in vitro anti-tumor experiments: human lung cancer cell a549 and human liver cancer cell HepG2, human normal cell strain: human umbilical vein endothelial cells HUVEC. The determination method comprises the following steps: taking cells in logarithmic growth phase, inoculating cell suspension into 96-well plate, and culturing at 37 deg.C and 100% relative humidity with 5% CO2And pre-culturing in an incubator with 95% air for 24h, and adding medicine. In addition, each concentration was set with a negative control (equal concentration of DMSO) and a blank background (no cells added), and each group was set with 3 replicate wells. The culture was continued for another 24h, then MTT solution was added to each well and after a further 4h of culture, the supernatant was carefully aspirated (suspension of cells, centrifugation was required before aspiration of the supernatant). Adding DMSO into each well, placing in a micro oscillator, shaking for 5min to dissolve the crystal completely, performing single-wavelength color comparison with an ELISA reader at 492nm, and determining OD value. Inhibition (%) - (experimental OD mean-blank OD mean)/(control OD mean-blank OD mean) x 100%, and IC was calculated50The value is obtained.
The invention has the following advantages:
the compounds 1-6 provided by the invention have strong inhibitory activity to PARP1, have strong inhibitory activity to various experimental tumor cell strains through an MTT method, and have low toxicity to normal cell human umbilical vein endothelial cells HUVEC, so that the compounds have good anti-tumor activity and good application prospect in the development of anti-tumor drugs. The invention carries out chemical synthesis preparation on six PARP1 inhibitors, has cheap and easily obtained starting materials, low preparation cost, high yield of target compounds and simple process operation, and has good industrialized production prospect.
Detailed Description
In order to better understand the essence of the present invention, the following examples are provided to illustrate the preparation method and pharmacological effects of the six PARP1 inhibitors 1-6 of the present invention, but the present invention is not limited by these examples.
Example 1: preparation of 2,2'- (((butane-1, 4-diyldioxy) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (N-methylhydrazine-1-thioamide) (2,2' - ((butyl-1, 4-diylbis (oxy)) bis (3-bromo-5-methoxy-4,1-phenylene)) bis (methanoylidine)), compound 1);
(1) weighing 5-bromovanillin (13.9g, 60mmol) and potassium carbonate (2.8g, 20mmol), adding into a 1L flask, adding 20mL of DMF, dissolving, stirring for 10min, dropwise adding 1, 4-dibromobutane (2mL, 20mmol) while stirring, heating at 80 deg.C, condensing, refluxing for 10h, adding 500mL of water, precipitating white flocculent precipitate, filtering the precipitate, washing the precipitate with saturated sodium bicarbonate, washing the precipitate with a small amount of ethyl acetate, and drying to obtain an intermediate compound II.
(2) 0.2mmol of intermediate compound II (100mg) and 0.44mmol of 4-methyl thiosemicarbazide (46.3mg) are weighed and placed in a 100mL reaction bottle, 10mL of 95% ethanol is added, stirring is carried out for 10min, then 100 mu L of acetic acid is dripped, heating, condensing and refluxing are carried out for 12h at 70 ℃, white precipitate is separated out, suction filtration is carried out, the precipitate is washed by ethanol, and drying is carried out to obtain a white solid compound 1, wherein the yield is 85.2%.1H-NMR(500MHz，DMSO-d6)δ：11.52(s，2H，CH)，8.54(m，2H，NH)， 7.93(s，2H，NH)，7.68(s，2H，ArH)，7.38(s，2H，ArH)，4.01(s，4H，OCH2)， 3.86(s，6H，OCH3)，3.01(d，J＝4.5Hz，6H，NCH3)，1.90(s，4H，CH2)；13C NMR(125MHz，DMSO-d6)δ：178.1，154.0，146.3，140.5，131.9，123.2， 117.9，111.7，72.9，56.8，31.3，26.7。
Example 2: 2,2'- ((propane-1, 3-diyldioxy) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (N- (3-morpholinyl) hydrazine-1-thioamide) (2,2' - ((propane-1,3-diylbis (oxy)) bis (3-bromo-5-methoxy-4,1-phenylene)) bis (methyleneidene)) bis (N- (3-morpholinopropyl) hydrazine-1-carbothioamide), Compound 2)
Compound 2 was prepared in a similar manner to Compound 1 except that 1, 4-dibromobutane was replaced with 1, 3-dibromopropane to prepare intermediate Compound I, and 4-methylaminothio was replaced with 4- [3- (4-morpholine) propyl ] sulfide]-3-thiosemicarbazide, compound 2 was prepared as a white solid in 83.2% yield. 1H-NMR (500MHz, DMSO-d6)δ：11.50(s， 2H，CH)，8.56(t，J＝6Hz，2H，NH)，7.94(s，2H，NH)，7.68(s，2H，ArH)， 7.35(d，J＝1.5Hz，2H，ArH)，4.19(t，J＝6.5Hz，4H，OCH2)，3.85(s，6H， OCH3)，3.59(dd，4H，CH2)，3.54(s，8H，CH2)，2.31(m，10H，CH2)，2.12(m， 2H，CH2)，1.74(m，4H，CH2)；13C NMR(125MHz，DMSO-d6)δ：177.3， 153.8，146.3，140.7，131.8，123.0，117.8，112.1，70.6，66.6，56.8，56.4， 53.8，42.6，31.1，26.2。
Example 3: 2,2'- ((butane-1, 4-diyldioxy) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (N- (3-morpholinyl) hydrazine-1-thioamide) (2,2' - ((butane-1,4-diylbis (oxy)) bis (3-bromo-5-methoxy-4,1-phenylene)) bis (methanoylene)) bis (N- (3-morpholinopropyl) hydrazine-1-carbothioamide), Compound 3)
Compound 3 was prepared in a similar manner to Compound 1, as in example 1, to give intermediate Compound II, which was then substituted with 4-methylaminothio to 4- [3- (4-morpholine) propyl]-3-thiosemicarbazide, obtained in 84.6% yield as a white solid compound 3. 1H-NMR (500)MHz， DMSO-d6)δ：11.49(s，2H，CH)，8.55(t，J＝6Hz，2H，NH)，7.94(s，2H， NH)，7.68(s，2H，ArH)，7.35(s，2H，ArH)，4.02(s，4H，OCH2)，3.86(s， 6H，OCH3)，3.59(dd，4H，CH2)，3.54(s，6H，CH2)，2.31(m，10H，CH2)， 1.90(s，4H，CH2)，1.74(m，4H，CH2)；13C NMR(125MHz，DMSO-d6)δ： 177.3，154.0，146.3，140.7，131.8，122.9，118.0，112.0，72.9，66.6，56.8， 56.4，53.8，42.6，26.7，26.2。
Example 4: 2,2'- ((hexane-1, 6-diyldioxy) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (N- (3-morpholinyl) hydrazine-1-thioamide) (2,2' - ((hexane-1,6-diyl (oxy)) bis (3-bromo-5-methoxy-4,1-phenylene)) bis (methyleneidene)) bis (N- (3-morpholinopropyl) hydrazine-1-carbothioamide), Compound 4)
Compound 4 was prepared in a similar manner to Compound 1 except that 1, 4-dibromobutane was replaced with 1, 6-dibromohexane to prepare intermediate Compound III, and 4-methylaminothio was replaced with 4- [3- (4-morpholine) propyl ] sulfide]-3-thiosemicarbazide, compound 4 was prepared as a white solid in 83.2% yield.1H NMR(DMSO-d6,500MHz,ppm):δ11.48(s, 2H),8.54(overlap,4H),7.94(s,2H),7.67(s,2H),7.34(s,2H),3.95(t,4H,J＝6.5 Hz),3.86(s,6H),3.53-3.61(overlap,12H),2.29-2.33(overlap,12H), 1.70-1.77(overlap,8H),1.50(m,4H)；13C NMR(DMSO-d6,125MHz,ppm):δ177.3(2C),154.0(2C),146.5(2C),140.7(2C),131.8(2C),123.0(2C),117.9 (2C),112.0(2C),73.2(2C),66.6(4C),56.8(2C),56.4(2C),53.8(4C),42.6(2C), 30.0(2C),26.2(2C),25.6(2C)；
Example 5:2, 2'- ((propane-1, 3-diyldioxy) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (hydrazine-1-thioamide) (2,2' - ((propane-1,3-diylbis (oxy)) bis (3-bromo-5-methoxy-4,1-phenylene)) bis (methyleneidene)) bis (hydrazine-1-carbothioamide, Compound 5)
The preparation method of the compound 5 is similar to that of the compound 1,it is different from example 1 in that intermediate compound I was produced by replacing 1, 4-dibromobutane as a raw material with 1, 3-dibromopropane, and then replacing 4-methylaminothio with thiosemicarbazide to produce compound 5 as a white solid in a yield of 91.0%. 1H-NMR (500MHz, DMSO-d)6)δ：11.46(s，2H，CH)，8.23(s，2H，NH)， 8.16(s，2H，NH)，7.92(s，2H，NH)，7.63(s，2H，ArH)，7.47(s，2H，ArH)，4.18(t，J＝6.5Hz，4H，OCH2)，3.84(s，6H，OCH3)，2.11(m，2H，CH2)；13C NMR(125MHz，DMSO-d6)δ：178.4，153.9，146.4，140.9，131.9，123.9， 117.6，111.1，70.6，56.8，31.1。
Example 6: 2,2'- ((butane-1, 4-diyldioxy) bis (3-bromo-5-methoxy-4-phenyl)) dimethylene) bis (hydrazine-1-thioamide) (2,2' - ((butane-1,4-diylbis (oxy)) bis (3-bromo-5-methoxy-4,1-phenylene)) bis (methanoylidine)) bis (hydrazine-1-carbothioamide, Compound 6)
Compound 6 was prepared in a similar manner to Compound 1, using the same procedure as in example 1 to give intermediate Compound II, which was then converted from 4-methylaminothio to thiosemicarbazide, to give Compound 6 as a white solid in 90.5% yield. 1H-NMR (500MHz, DMSO-d)6)δ：11.45(s， 2H，CH)，8.22(s，2H，NH)，8.16(s，2H，NH)，7.92(s，2H，NH)，7.60(s， 2H，ArH)，7.47(s，2H，ArH)，4.00(s，4H，OCH2)，3.85(s，6H，OCH3)，1.98(s，4H，CH2)；13C NMR(125MHz，DMSO-d6)δ：：178.4，154.1，146.4， 140.9，131.9，123.9，117.7，111.0，72.9，56.8，26.7。
Example 7: determination of inhibitory Activity of Compound 1-6 against PolyA-diphospho-ribotransferase (PARP1)
All reagents used in the experiment should be balanced to room temperature, and the reagents or samples need to be fully mixed when being prepared, so that foaming is avoided.
(1) Sample adding: respectively setting a blank hole, a standard hole and a sample hole to be measured. And adding 100 mu L of standard substance and sample diluent into the blank hole, respectively adding 100 mu L of standard substance or sample to be detected into the rest holes, uniformly mixing, and incubating for 90 minutes at 37 ℃.
(2) The liquid was discarded, spun off, 100. mu.L of biotinylated antibody working solution was added to each well, and incubated at 37 ℃ for 1 hour.
(3) The well liquid was discarded, spun dry, plate washed 3 times, soaked 1-2 minutes each time, approximately 350 μ L/well, spun dry and patted dry on absorbent paper.
(4) Add 100. mu.L of enzyme conjugate working solution (prepared within 15 minutes before use) to each well and incubate at 37 ℃ for 30 minutes.
(5) And (4) discarding liquid in the holes, spin-drying, and washing the plate for 5 times, wherein the method is the same as the step 3.
(6) Substrate solution (TMB) 90. mu.L was added to each well and incubated at 37 ℃ for about 15 minutes in the absence of light.
(7) The reaction was stopped by adding 50. mu.L of stop solution to each well.
(8) The optical density (OD value) of each well was measured at a wavelength of 450nm using a microplate reader.
(9) Calculated by substituting the measured OD values into the standard curve.
Table 1: data sheet for PARP inhibition activity of Compounds 1-6 at 100nM concentration
Compound (I) Inhibition ratio (100%)
1 68.4
2 54.3
3 56.1
4 55.4
5 62.2
6 49.6
Example 8: determination of tumor cell proliferation inhibitory Activity of Compounds 1-6
The cytotoxicity of the synthesized compound on human tumor cells cultured in vitro is detected by adopting a currently common tetrazolium salt (MTT) method. Cell lines selected for in vitro anti-tumor experiments: human lung cancer cell A549, human liver cancer cell HepG2, and human umbilical vein endothelial cell HUVEC. The determination method comprises the following steps: taking cells in logarithmic growth phase, inoculating cell suspension into 96-well plate to make the number of cells per well be 3X 103The cells were pre-cultured at 37 ℃ and 100% relative humidity in an incubator containing 5% CO2 by volume and 95% air for 24 hours, and then dosed. In addition, 1.25, 2.5, 5.0, 10.0, 20.0 micrograms/ml of compound 1-6, each concentration with negative control (equal concentration DMSO) and blank background (no cells), each group in 3 duplicate wells. The culture was continued for another 24h, then 20 μ l of 5 mg/ml MTT solution was added to each well and after a further 4h of culture, the supernatant was carefully aspirated (suspension of cells, centrifugation was required before aspiration of the supernatant). Adding 150 microliters of DMSO (dimethyl sulfoxide) into each hole, placing the hole in a micro oscillator, shaking for 5min to completely dissolve crystals, carrying out single-wavelength color comparison by using a microplate reader at 492nm, and determining the OD value. Inhibition (%) - (experimental OD mean-blank OD mean)/(control OD mean-blank OD mean) x 100%, and IC was calculated using SPSS17.0 software50Values (table 2).
Table 2: data sheet for tumor cell proliferation inhibitory Activity of Compounds 1-6 (IC)50,ug/mL)
Figure BDA0001739459320000131
Experimental results show that the compounds 1-6 provided by the invention have strong inhibitory activity to PARP1 at a concentration of 100nM, the compounds 1-6 have good anti-tumor activity, have strong inhibitory activity to various experimental tumor cell lines in vitro, and have low toxicity to normal human umbilical vein endothelial cells HUVEC.
Comparative example 9:
the following compounds were tested for PARP1 inhibitory activity and tumor cell proliferation inhibitory activity by the same assay as compounds 1-6, and it was found that the comparative compound showed very weak inhibitory activity (5.2%) against PARP1 at 100nM concentration and no proliferation inhibitory activity against the tumor cell lines tested.
Figure BDA0001739459320000141
Example 10: preparation of compound injection
Dissolving compounds 1-6 with small amount of DMSO (weight ratio of 1: 0.1-1: 0.5, here 1: 0.4), adding water for injection (weight ratio of 1:20-1:200, here 1:200), fine filtering, bottling, and sterilizing to obtain injection.
Example 11: preparation of compound powder injection
Dissolving compounds 1-6 with small amount of DMSO (weight ratio: 1: 0.1-1: 0.5, herein 1: 0.5), respectively, dissolving in sterile water for injection (weight ratio: 1:20-1: 60, herein 1:60), stirring for dissolving, filtering with sterile suction filter funnel, sterile fine filtering, packaging in ampoule, freeze drying at low temperature, and sterile melt sealing to obtain powder for injection.
Example 12: preparation of compound powder
The compound 1-6 is added with excipient (Tween 80: propylene glycol: cyclodextrin: lactose: 1:2:4:12) according to the weight ratio of the compound to the excipient of 9:1, and then the mixture is prepared into powder.
Example 13: preparation of Compound tablets
The compounds 1 to 6 were added to the excipient (hydroxypropylmethylcellulose E5: microcrystalline cellulose MCC102: magnesium stearate (8% povidone K30) ═ 15:15:2:0.1) at a weight ratio of 5:1, respectively, and granulated and tableted.
Example 14: preparation of compound oral liquid
Adding the compounds 1-6 into distilled water containing 20% simple syrup and 0.1% sodium benzoate, and making into oral liquid with concentration of 15 μ g/mL by conventional oral liquid preparation method.
Example 15: preparation of Compound Capsule
The compounds 1 to 6 were mixed with excipients (pharmaceutical starch: glucose: hydrolyzed gelatin: glycine: 30:10:1:1) at a weight ratio of 5:1, respectively, and made into capsules.
Example 16: preparation of Compound Capsule
The compounds 1 to 6 were mixed with excipients (pharmaceutical starch: glucose: hydrolyzed gelatin: glycine: 30:10:1:1) at a weight ratio of 3:1, respectively, and made into capsules.

Claims (6)

1. A compound useful as a PARP1 inhibitor, characterized by: the compound has the following structural formula:
Figure 921368DEST_PATH_IMAGE001
。
2. an antitumor agent characterized by:
the active ingredients of the antitumor drug are one or more than two of six compounds and pharmaceutically acceptable salts of the six compounds as claimed in claim 1; the application of the medicine taking one or more than two of the six compounds and the pharmaceutically acceptable salts of the six compounds as active ingredients in the preparation of one or more than two medicines for preventing and/or treating tumor-related diseases and symptoms.
3. Antitumor drug according to claim 2, characterized in that:
the anti-tumor medicine is tablets, capsules, oral liquid, granules, pills or injection.
4. Antitumor drug according to claim 2, characterized in that: the tumor-related diseases and symptoms refer to lung cancer, liver cancer, breast cancer, ovarian cancer and prostate cancer.
5. A medicament for the prevention and/or treatment of one or more of tumor-related diseases and symptoms, characterized in that: the pharmaceutical composition contains 0.1-99% of one or more of the six compounds and the pharmaceutically acceptable salts of the six compounds as described in claim 1, and the balance is pharmaceutically acceptable pharmaceutical carriers and/or excipients.
6. A process for the preparation of a compound of claim 1 useful as a PARP1 inhibitor, wherein:
Figure 369667DEST_PATH_IMAGE002
step 1: preparation of intermediate alkoxy dibromophenol benzaldehyde (I-III):
mixing 5-bromovanillin and potassium carbonate at room temperature, adding the mixture into a flask, adding 20mL of DMF for dissolving, stirring for 10min, dropwise adding 1, 3-dibromopropane or 1, 4-dibromobutane or 1, 6-dibromohexane while stirring, heating at 80 ℃, condensing and refluxing for 10h, adding 500mL of water, separating out white flocculent precipitate, filtering the precipitate, washing the precipitate with saturated sodium bicarbonate, and washing the precipitate with ethyl acetate to obtain an intermediate compound I, II or III;
step 2: preparation of Compounds 1-6:
weighing an intermediate compound I, II or III and corresponding thiosemicarbazide, mixing in 100mL of 95% ethanol, stirring for 10min, dropwise adding 1000 muL of acetic acid while stirring, heating at 70 ℃, condensing and refluxing for 12h, separating out a white precipitate, carrying out suction filtration on the precipitate, washing the precipitate with ethanol, and drying to obtain a compound 1-6;
the corresponding thiosemicarbazide is 4-methyl thiosemicarbazide, 4- [3- (4-morpholine) propyl ] -3-thiosemicarbazide or thiosemicarbazide.
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