CN115073361A - Rosin diterpenoid compound and preparation method and application thereof - Google Patents

Rosin diterpenoid compound and preparation method and application thereof Download PDF

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CN115073361A
CN115073361A CN202210833837.9A CN202210833837A CN115073361A CN 115073361 A CN115073361 A CN 115073361A CN 202210833837 A CN202210833837 A CN 202210833837A CN 115073361 A CN115073361 A CN 115073361A
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rosin
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叶连宝
杨贤劲
区嘉怡
张富莉
金小宝
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Guangdong Pharmaceutical University
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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a rosin diterpenoid compound as well as a preparation method and application thereof. Experiments prove that the rosin diterpenoid compound has obvious antiproliferative and anti-migration activities on glioma cells, can achieve an obvious effect of resisting glioma proliferation under the condition of low concentration, can obviously inhibit the migration of tumor cells, and has a good tumor treatment effect; in addition, the pKa of the rosin diterpenoid compound is 6-10.5, so that the rosin diterpenoid compound has good blood brain barrier permeability and is favorable for entering the brain to exert the drug effect.

Description

Rosin diterpenoid compound and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines. More particularly relates to a rosin diterpenoid compound and a preparation method and application thereof.
Background
Gliomas are the most common primary malignant brain tumors, with glioblastoma multiforme (GBM) being the highest grade malignant glioma (WHO, grade IV), which develops from low-grade astrocytomas. Because GBM has the characteristics of invasive growth, BBB restriction, susceptibility to drug resistance, and poor prognosis, GBM patients have less than 10% of their 5-year survival despite standardized treatment. In addition, since the lethality rate of malignant glioma is high, many factors such as tumor microenvironment and blood brain barrier influence on drug intake, drug resistance, poor prognosis and the like exist in special parts such as cranial cavity, and the research and development of anti-glioma drugs is long and difficult.
Currently, the standardized treatment modalities for GBM are surgical resection combined with postoperative chemoradiotherapy, in addition to gene therapy and immunotherapy. Chemotherapeutic drugs such as Temozolomide (TMZ), cisplatin, and carmustine are important clinical adjunctive therapeutic drugs. However, even with standardized treatment, almost all patients with GBM will relapse, since it is difficult for surgeons to completely eliminate the highly invasive growing glioma cells without affecting normal brain function, and these cells have the characteristics of rapid proliferation rate, anti-apoptosis and rapid metastasis, and therefore, postoperative relapse is one of the biggest challenges in treating GBM. Most GBM patients after relapse appear insensitive to radiation therapy and susceptible to developing resistance to chemotherapy. Because of the drug resistance generated by chemotherapy, the median survival rate of GBM patients is about 15 months, and the treatment effect is not ideal. Therefore, it is necessary to develop an anti-glioma drug. The problems of how to effectively improve the blood brain barrier permeability of the drug, reduce the toxic and side effects of the drug and the like have always driven people to develop novel anti-glioma drugs.
The natural product has wide structural diversity and multiple pharmacology effects, is an important source for finding lead compounds, and has profound influence on drug development. As the application of the eriodictyol as a medicament for treating the brain glioma disclosed in the Chinese patent application, experiments show that the eriodictyol can inhibit the proliferation, migration and invasion of human brain glioma cells and promote the apoptosis of the human brain glioma cells. However, the technical scheme only stays at the level of cell experiments, and at present, drugs with good effects are still lacked in the treatment of glioma, so that more effective drugs for inhibiting glioma still need to be developed.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of few medicines for treating glioma in the prior art and providing a rosin diterpenoid compound.
The invention aims to provide a preparation method of the rosin diterpenoid compound.
The invention also aims to provide the application of the rosin diterpenoid compound or the pharmaceutically acceptable salt thereof in preparing anti-glioma medicaments.
The above purpose of the invention is realized by the following technical scheme:
the natural products Abietic Acid (AA) and dehydroabietic acid (DHAA) are tricyclic diterpene oxygen-containing compounds obtained from rosin or disproportionated rosin, the safety is high, and the inventor researches and develops various compounds with anticancer efficacy on the basis of the structures of abietic acid and dehydroabietic acid.
A rosin diterpenoid having the structure of formula I or formula II:
Figure BDA0003749367740000021
r is
Figure BDA0003749367740000022
Wherein R is 1 Is hydrogen or halogen; r 2 Is unsubstituted or substituted C 1～5 Alkoxy radical, said substituted C 1～5 The substituent of the alkoxy is phenyl, halogenated phenyl or heterocyclic aryl; r 3 Is unsubstituted or substituted amino, and the substituent of the substituted amino is phenyl or C 1～5 Alkylphenyl, halophenyl; r 4 Is phenyl, C 1～5 Alkylphenyl, halophenyl; r 5 Is phenyl.
Preferably, said R is 1 Is hydrogen or halogen; r 2 Is unsubstituted or substituted C 1～3 Alkoxy, said substituted C 1～3 The substituent of the alkoxy is phenyl, halogenated phenyl or pyridine; r 3 Is unsubstituted or substituted amino, and the substituent of the substituted amino is phenyl or C 1～3 Alkylphenyl, halophenyl; r 4 Is phenyl, C 1～3 Alkylphenyl, halophenyl; r 5 Is phenyl.
More preferably, said R 1 Is hydrogen or halogen; r 2 Is unsubstituted or substituted methoxyl, and the substituent of the substituted methoxyl is phenyl, halogenated phenyl or pyridine; r 3 Is unsubstituted or substituted amino, and the substituent of the substituted amino is phenyl, tolyl or halogenated phenyl; r 4 Phenyl, tolyl, halophenyl; r 5 Is phenyl.
Specifically, R is
Figure BDA0003749367740000031
Figure BDA0003749367740000032
Figure BDA0003749367740000033
To (3) is provided.
For example, the rosin diterpenoid compound has any one of the following structures:
Figure BDA0003749367740000034
Figure BDA0003749367740000041
Figure BDA0003749367740000051
preferably, the pharmaceutically acceptable salt obtained by reacting the rosin diterpenoid with acid is also in the protection scope of the invention. Wherein the acid is hydrochloric acid, nitric acid, sulfuric acid or trifluoroacetic acid, phosphoric acid, acetic acid or carbonic acid.
In addition, the invention also provides a preparation method of the rosin diterpenoid compound, which comprises the following steps:
Figure BDA0003749367740000052
s1, taking AA as an initial raw material, carrying out acylation reaction under the action of an acylation reagent and a polar organic solvent to obtain a compound 1, and carrying out amide condensation reaction with a compound R-H under the action of an acid-binding agent to obtain a compound shown in the formula I;
Figure BDA0003749367740000053
s2, taking DHAA as an initial raw material, and carrying out an acylation reaction under the action of an acylation reagent and a polar organic solvent to obtain a compound 2; then carrying out amide condensation reaction with a compound R-H under the action of an acid-binding agent to obtain a compound shown in the formula I;
wherein R in R-H is as defined above for R.
Further, the acylating reagent is selected from one or more of oxalyl chloride, thionyl chloride and dibenzoyl chloride.
Still further, the acid-binding agent is selected from one or more of triethylamine, pyridine and N, N-diisopropylethylamine.
Further, the polar organic solvent is selected from one or more of N, N-dimethylformamide and dichloromethane.
Preferably, the equivalent ratio of the AA or DHAA to the acylating agent is 1: 1.5-1.8; the acylation reaction is carried out at the reaction temperature of 0 ℃ to room temperature to generate the compound 1 or the compound 2.
Preferably, the compound 1 or the compound 2 and the compound R-H have amide condensation reaction, the reaction temperature is room temperature, and the reaction time is 16-18H.
In addition, the invention also provides application of the rosin diterpenoid compound or the pharmaceutically acceptable salt thereof in preparing anti-glioma medicines.
Preferably, the glioma is a glioma caused by abnormal expression of epidermal growth factor receptor and/or mutants thereof. More preferably, the glioma includes human glioma cell T98G, human brain astrocytoma cell U87MG and human glial cell U251.
In addition, the invention also claims a pharmaceutical composition containing one or more of the rosin diterpenoid compound, the pharmaceutically acceptable salt, the hydrate, the solvate, the polymorph, the tautomer, the stereoisomer or the prodrug thereof.
Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant and/or carrier.
Preferably, the auxiliary material comprises at least one of the following substances: solvents, propellants, solubilizers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-chelating agents, integration agents, penetration enhancers, pH regulators, buffers, plasticizers, solubilizing agents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, release agents, flocculants and deflocculants, filter aids, release retardants.
The pharmaceutical composition of the invention can be prepared into various dosage forms: the following preparations can be prepared according to the dispersion system of the dosage form: solution type, colloidal solution type, emulsion type, suspension type, gas dispersion type, fine particle dispersion type, solid dispersion type; according to the morphological classification, the traditional Chinese medicine can be prepared into the following dosage forms: liquid dosage forms (such as aromatic water agent, solution, injection, mixture, lotion, liniment, etc.), solid dosage forms (such as powder, pill, tablet, pellicle, etc.), and semisolid dosage forms (such as ointment, suppository, paste, etc.).
The invention has the following beneficial effects:
experiments prove that the rosin diterpenoid compound has remarkable antiproliferative and anti-migration activities on glioma cells, can achieve a remarkable glioma proliferation resisting effect under the condition of a low concentration, can remarkably inhibit the migration of tumor cells, and has a good tumor treatment effect; in addition, the pKa of the rosin diterpenoid compound is 6-10.5, so that the rosin diterpenoid compound has good blood brain barrier permeability and is favorable for entering the brain to exert the drug effect.
Drawings
FIG. 1 is a micrograph of T98G cells treated with a compound of the present invention to inhibit migration of T98G cells for 24 h.
Figure 2 is a statistical plot of the 24h inhibition of T98G cell mobility of compounds of the invention treated T98G cells, where the data are from the mean of three random regions in each concentration group, and compared to the control group,. p < 0.01.
FIG. 3 is a pH-VKOH (. mu.L) graph of the compounds of the present invention.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1: synthesis of (1R,4aR,4bR,10aR) -7-isopropyl-1, 4 a-dimethyl-N- (4- (pyridin-2-ylmethoxy) phenyl) -1,2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A1)
1. Synthesis of 4- (pyridine-2-methoxy) aniline (1a)
Figure BDA0003749367740000071
Adding 4-aminophenol (1g, 9.16mmol) to a solvent of N, N-dimethylformamide (10mL) to dissolve the 4-aminophenol sufficiently, adding benzaldehyde (1.16g, 10.91mmol) and stirring at normal temperature for 20 min; to the solvent were then added 2- (chloromethyl) pyridine hydrochloride (0.80g, 4.87mmol) and potassium carbonate (5.05g, 36.53mmol) in this order, and reacted at 50 ℃ for 18 h; after the reaction is finished, the mixture is filtered, filter residues are discarded, filtrate is kept, the pH value of the filtrate is adjusted to 1-2 by hydrochloric acid (2M), the filtrate is extracted by ethyl acetate and water, an organic phase is collected, the collected organic phase is dried and filtered, and after the solvent is evaporated under reduced pressure, the organic phase is purified by silica gel column chromatography, and a mixed solvent (v: v ═ 1:5) of ethyl acetate and petroleum ether is used as a mobile phase for elution, so that a brown solid compound 1a is obtained, and the yield is 52%.
1H NMR(400MHz,DMSO-d6)δ8.55(dt,J＝4.8,1.3Hz,1H),7.80(td,J＝7.7,1.8Hz,1H),7.48(d,J＝7.8Hz,1H),7.36–7.25(m,1H),6.79–6.69(m,2H),6.58–6.46(m,2H),5.02(s,2H),4.68(s,2H).
2. Synthesis of (1R,4aR,4bR,10aR) -7-isopropyl-1, 4 a-dimethyl-N- (4- (pyridin-2-ylmethoxy) phenyl) -1,2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A1)
Figure BDA0003749367740000081
Under the ice-bath condition, oxalyl chloride (0.63g, 4.97mmol) is slowly dropped into a dichloromethane (25mL) mixed solution containing abietic acid (1g, 3.31mmol), 1-2 drops of N, N-dimethylformamide are further dropped, and the mixture is turned to room temperature and reacted for 4 hours under the anhydrous condition; after the substrate completely reacts, evaporating the solvent under reduced pressure to obtain an orange-yellow oily solution containing acyl chloride, and adding anhydrous dichloromethane (10 mL); subsequently, a solution containing acid chloride was added dropwise to a mixed solution of compound 1a (0.31mg, 1.55mmol) and triethylamine (TEA,0.27g, 2.64mmol) after reacting for 10min at normal temperature, and the reaction was carried out for 18h at normal temperature under anhydrous conditions; after the reaction is finished, extracting for many times, washing the collected organic layer with saturated saline solution, drying with anhydrous sodium sulfate, performing reduced pressure rotary evaporation to obtain a solid crude product, and purifying by using silica gel column chromatography to obtain a brown solid compound A1 with the yield of 36%, mp: 131.5-133.2 ℃.
1H NMR(400MHz,DMSO-d6)δ9.11(d,J＝61.3Hz,1H),8.57(ddd,J＝4.8,1.8,1.0Hz,1H),7.82(td,J＝7.7,1.8Hz,1H),7.56–7.38(m,3H),7.33(ddd,J＝7.5,4.8,1.2Hz,1H),7.01–6.90(m,2H),5.78–5.69(m,1H),5.38–5.27(m,1H),5.13(s,2H),2.19(p,J＝6.7Hz,1H),2.09–1.95(m,3H),1.86–1.75(m,3H),1.54(td,J＝9.7,4.5Hz,3H),1.32–1.20(m,6H),1.19–1.11(m,3H),0.96(dd,J＝6.8,1.7Hz,5H),0.78(s,3H).ESI-MS m/z:485.3[M+H]+,C32H40N2O2.
Example 2 Synthesis of (1R,4aR,4bR,10aR) -N- (3-chloro-4- (pyridin-2-ylmethoxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A2)
1. Synthesis of 3-chloro-4- (pyridine-2-methoxy) aniline (2a)
Figure BDA0003749367740000091
Compound 2a was prepared according to the procedure for compound 1a to give compound 2a as a gray solid in 54% yield.
1H NMR(400MHz,Chloroform-d)δ8.56(ddd,J＝4.9,1.7,0.9Hz,1H),7.73(td,J＝7.7,1.8Hz,1H),7.67–7.61(m,1H),7.21(ddd,J＝7.2,5.0,1.2Hz,1H),6.84–6.74(m,2H),6.51(dd,J＝8.7,2.8Hz,1H),5.18(s,2H).
2. Synthesis of (1R,4aR,4bR,10aR) -N- (3-chloro-4- (pyridin-2-ylmethoxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A2)
Figure BDA0003749367740000092
Synthesis of compound a2 referring to the synthesis of compound a1, compound a2 was obtained as a pale yellow solid in 31% yield, mp: 71.3-73.5 ℃.
1H NMR(400MHz,DMSO-d6)δ9.26(d,J＝60.0Hz,1H),8.58(d,J＝4.9Hz,1H),7.86(t,J＝7.7Hz,1H),7.78(d,J＝2.6Hz,1H),7.59–7.45(m,2H),7.38–7.32(m,1H),7.18(dd,J＝13.2,8.7Hz,1H),5.71(s,1H),5.31(d,J＝5.2Hz,1H),5.23(s,2H),2.24–2.17(m,1H),2.08–1.95(m,3H),1.81(t,J＝12.7Hz,4H),1.53(d,J＝10.9Hz,4H),1.26–1.22(m,5H),1.16(d,J＝3.4Hz,2H),0.97(d,J＝6.8Hz,5H),0.84(dt,J＝9.9,5.3Hz,3H).ESI-MS m/z:519.91[M+H]+,C32H39ClN2O2.
Example 3 Synthesis of (1R,4aR,4bR,10aR) -N- (3-chloro-4- ((3-fluorobenzyl) oxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A3)
1. Synthesis of 3-chloro-4- (3-fluorobenzyloxy) aniline (3a)
Figure BDA0003749367740000101
To N, N-dimethylformamide (20mL) were added 2-chloro-4-nitrophenol (1g, 5.76mmol), potassium carbonate (0.80g, 5.76mmol), and 3-fluorobenzyl bromide (1.10g, 5.82mmol), and the mixture was stirred at 85 ℃ for 3 h; then, the mixture was extracted with ethyl acetate and water, and the organic layer was collected and washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to give 2-chloro-1- ((3-fluorobenzyl) oxy) -4-nitrobenzene as a pale yellow solid;
to a solution of 2-chloro-1- ((3-fluorobenzyl) oxy) -4-nitrobenzene (1g,3.56mmol) and reduced iron (0.60g,10.68mmol) in ethanol (15mL) was added a solution of calcium chloride (0.42g, 3.56mmol, 1.4mL water), the mixture was stirred at 85 ℃ for 8h, then the solid was removed by filtration and the solvent was removed by rotary evaporation under reduced pressure, the resulting solid was subjected to column chromatography on silica gel with eluent ethyl acetate and n-hexane mixed solvent (v: v ═ 1:3) to give compound 3a as a yellow solid in 42% yield.
1H NMR(400MHz,DMSO-d6)δ7.42(p,J＝6.7,5.7Hz,1H),7.25(dd,J＝12.1,5.3Hz,2H),7.14(td,J＝8.7,3.0Hz,1H),6.90(dd,J＝9.1,4.8Hz,1H),6.72–6.59(m,1H),6.46(dt,J＝5.9,2.9Hz,1H),5.02(d,J＝5.2Hz,2H),4.95(s,2H).
2. Synthesis of (1R,4aR,4bR,10aR) -N- (3-chloro-4- ((3-fluorobenzyl) oxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A3)
Figure BDA0003749367740000102
After synthesis of compound 3a, synthesis of compound A3 referring to the synthesis of compound a1, white solid compound A3 was obtained in 25% yield, mp: 68.7-70.6 ℃.
1H NMR(400MHz,DMSO-d6)δ9.26(d,J＝53.0Hz,1H),7.77(t,J＝2.8Hz,1H),7.55–7.41(m,2H),7.33–7.24(m,2H),7.21–7.12(m,2H),5.71(d,J＝1.9Hz,1H),5.36–5.28(m,1H),5.19(s,2H),2.19(p,J＝6.8Hz,1H),2.04(dt,J＝17.9,4.6Hz,2H),1.98–1.86(m,2H),1.86–1.73(m,3H),1.68(d,J＝18.7Hz,1H),1.61–1.46(m,3H),1.28–1.11(m,7H),0.97(dd,J＝6.8,1.6Hz,5H),0.88–0.70(m,3H).ESI-MS m/z:536.2[M+H]+,C33H39ClFNO2.
Example 4 Synthesis of ((1R,4aR,4bR,10aR) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrin-1-yl) (4-phenylpiperazin-1-yl) methanone (A4)
Figure BDA0003749367740000111
Synthesis of compound a4 referring to the synthesis of compound a1, compound a4 was obtained as a white solid in 16% yield, mp: 104.2-106.5 ℃.
1H NMR(400MHz,DMSO-d6)δ7.22(t,J＝7.7Hz,2H),6.93(t,J＝7.6Hz,2H),6.81(q,J＝9.6,7.3Hz,1H),5.73(d,J＝18.4Hz,2H),4.01–3.55(m,4H),3.14–3.00(m,3H),2.94(s,1H),2.32–2.09(m,2H),2.04(t,J＝9.2Hz,1H),1.74(dt,J＝42.4,13.8Hz,5H),1.61–1.48(m,2H),1.37–1.07(m,10H),0.96(d,J＝6.9Hz,4H),0.89–0.63(m,3H).ESI-MS m/z:447.1[M+H]+,C30H42N2O.
Example 5 Synthesis of (1R,4aR,4bR,10aR) -7-isopropyl-1, 4 a-dimethyl-N- (6- (phenylamino) pyrimidin-4-yl) -1,2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A5)
1. Synthesis of N4-phenylpyrimidine-4, 6-diamine (4a)
Figure BDA0003749367740000112
To isopropanol (30mL) containing 4-amino-6-chloropyrimidine (3g, 23.16mmol) and aniline (3.26g, 34.97mmol), hydrochloric acid (1mL) was added dropwise and reacted at 80 ℃ for 4 h; after complete reaction, quenching the reaction by using saturated sodium bicarbonate, extracting by using ethyl acetate and water, washing an organic phase by using saturated sodium chloride, drying by using anhydrous sodium sulfate, carrying out reduced pressure rotary evaporation to obtain a large amount of solid, and finally carrying out silica gel column chromatography by using a mixed solvent (v: v ═ 3:1) of petroleum ether and ethyl acetate as a flowing relative mixture to obtain a white solid compound 4a after purification, wherein the yield is 66%.
1H NMR(400MHz,DMSO-d6)δ8.86(s,1H),8.03(s,1H),7.60–7.45(m,2H),7.33–7.19(m,2H),7.02–6.83(m,1H),6.32(s,2H),5.78(d,J＝1.0Hz,1H).
2. Synthesis of (1R,4aR,4bR,10aR) -7-isopropyl-1, 4 a-dimethyl-N- (6- (phenylamino) pyrimidin-4-yl) -1,2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A5)
Figure BDA0003749367740000121
Synthesis of compound a5 referring to the synthesis of compound a1, compound a5 was obtained as a pale yellow solid in 29% yield, mp: 99.2-102.4 ℃.
1H NMR(400MHz,DMSO-d6)δ10.11–9.67(m,1H),9.65–9.37(m,1H),8.35(dd,J＝28.4,10.8Hz,1H),7.58(ddd,J＝31.2,21.6,12.8Hz,3H),7.41–7.17(m,2H),7.06–6.87(m,1H),5.69(dd,J＝26.7,12.1Hz,1H),5.27(d,J＝25.0Hz,1H),2.23–2.08(m,2H),1.96(d,J＝23.8Hz,2H),1.65–1.43(m,6H),1.19(dt,J＝34.8,11.5Hz,8H),0.93(dt,J＝20.2,7.0Hz,6H),0.81–0.71(m,3H).ESI-MS m/z:471.4[M+H]+,C30H38N4O.
Example 6 Synthesis of (1R,4aR,4bR,10aR) -N- (6-aminopyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-N-phenyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A6)
Figure BDA0003749367740000122
Referring to example 5 synthesis of compound 4a, compound a6 synthesis referring to the synthesis of compound a1, compound a6 was obtained as a yellow-orange solid in 22% yield, mp: 131.1-133.1 ℃.
1H NMR(400MHz,DMSO-d6)δ8.45–8.13(m,1H),7.61(d,J＝18.7Hz,1H),7.41(t,J＝7.7Hz,2H),7.31(t,J＝7.3Hz,2H),7.13(dd,J＝12.5,7.3Hz,2H),6.83(d,J＝10.2Hz,1H),5.85(d,J＝12.3Hz,1H),5.74(d,J＝12.4Hz,1H),2.36–2.13(m,4H),2.07–2.01(m,2H),1.77(q,J＝12.2,8.7Hz,4H),1.69–1.61(m,2H),1.46(d,J＝13.3Hz,3H),1.25(d,J＝9.5Hz,3H),1.07–0.93(m,8H),0.75(s,1H).ESI-MS m/z:471.4[M+H]+,C30H38N4O.
Example 7 Synthesis of (1R,4aR,4bR,10aR) -7-isopropyl-1, 4 a-dimethyl-N- (6- (p-toluylamino)) pyrimidin-4-yl) -1,2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A7)
1、N 4 Synthesis of (p-tolyl) pyrimidine-4, 6-diamine (5a)
Figure BDA0003749367740000131
Synthesis of compound 5a was obtained as a white solid in 61% yield according to the method for synthesizing compound 4 a.
1H NMR(400MHz,DMSO-d6)δ8.73(s,1H),8.00(d,J＝1.0Hz,1H),7.40–7.31(m,2H),7.12–7.02(m,2H),6.27(s,2H),5.73(d,J＝1.0Hz,1H),2.24(s,3H).
2. Synthesis of (1R,4aR,4bR,10aR) -7-isopropyl-1, 4 a-dimethyl-N- (6- (p-toluidino)) pyrimidin-4-yl) -1,2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A7)
Figure BDA0003749367740000132
Synthesis of compound a7 referring to the synthesis of compound a1, compound a7 was obtained as a white solid in 27% yield, mp: 99.8-101.6 ℃.
1H NMR(400MHz,DMSO-d6)δ9.74(s,1H),9.42(s,1H),8.34(d,J＝1.0Hz,1H),7.60–7.41(m,3H),7.17–7.04(m,2H),5.73(d,J＝16.3Hz,2H),2.26(s,3H),1.99–1.91(m,3H),1.82–1.71(m,5H),1.54(d,J＝11.5Hz,4H),1.25–1.22(m,5H),0.97(dd,J＝6.8,1.7Hz,10H).ESI-MS m/z:485.4[M+H]+,C31H40N4O.
Example 8 Synthesis of (1R,4aR,4bR,10aR) -N- (6-aminopyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-N- (p-tolyl) -1,2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A8)
Figure BDA0003749367740000141
Referring to example 7 synthesis of compound 5a, compound A8 synthesis reference compound a1 synthesis gave compound A8 as a white solid in 21% yield, mp: 86.5-88.4 ℃.
1H NMR(400MHz,DMSO-d6)δ8.15(d,J＝1.0Hz,1H),7.30–7.07(m,3H),7.05–6.96(m,2H),6.77(s,1H),5.79(d,J＝1.0Hz,1H),5.72(s,1H),5.34(s,1H),2.30(s,3H),2.01(d,J＝7.1Hz,3H),1.76(t,J＝13.7Hz,2H),1.44(s,2H),1.27–1.20(m,10H),1.03–0.93(m,10H).ESI-MS m/z:485.2[M+H]+,C31H40N4O.
Example 9 Synthesis of (1R,4aR,4bR,10aR) -N- (6- ((4-fluorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A9)
1. Synthesis of N4- (4-fluorophenyl) pyrimidine-4, 6-diamine (6a)
Figure BDA0003749367740000142
Synthesis of compound 6a was obtained as a gray solid in 63% yield, according to the method for synthesizing compound 4 a.
1H NMR(400MHz,DMSO-d6)δ8.87(s,1H),8.02(d,J＝1.0Hz,1H),7.57–7.47(m,2H),7.16–7.05(m,2H),6.32(s,2H),5.71(d,J＝1.1Hz,1H).
2. Synthesis of (1R,4aR,4bR,10aR) -N- (6- ((4-fluorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A9)
Figure BDA0003749367740000151
Synthesis of compound a9 referring to the synthesis of compound a1, compound a9 was obtained as a white solid in 25% yield, mp: 102.6-104.4 ℃.
1H NMR(400MHz,DMSO-d6)δ9.79(s,1H),9.56(s,1H),8.36(s,1H),7.63(dd,J＝9.1,4.9Hz,2H),7.53(d,J＝1.1Hz,1H),7.15(t,J＝8.9Hz,2H),5.75(s,1H),5.71(s,1H),2.19(dd,J＝11.5,5.1Hz,2H),2.09–1.92(m,6H),1.77(t,J＝11.6Hz,3H),1.54(d,J＝8.1Hz,3H),1.24(s,4H),0.97(dd,J＝6.8,1.6Hz,9H).ESI-MS m/z:489.3[M+H]+,C30H37FN4O.
Example 10 Synthesis of (1R,4aR,4bR,10aR) -N- (6-aminopyrimidin-4-yl) -N- (4-fluorophenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A10)
Figure BDA0003749367740000152
Referring to example 9 synthesis of compound 6a, compound a10 synthesis reference compound a1 synthesis gave compound a10 as a white solid in 20% yield, mp: 112.8-114.6 ℃.
1H NMR(400MHz,DMSO-d6)δ8.17(d,J＝1.0Hz,1H),7.44–7.09(m,5H),6.84(s,2H),5.85(d,J＝1.0Hz,1H),5.73(d,J＝13.6Hz,1H),2.33–2.15(m,2H),1.98(dd,J＝24.5,14.1Hz,5H),1.85–1.70(m,3H),1.45(s,2H),1.24(d,J＝9.2Hz,3H),1.04–0.94(m,9H),0.75(s,3H).ESI-MS m/z:489.3[M+H]+,C30H37FN4O.
Example 11 Synthesis of (1R,4aR,4bR,10aR) -N- (6- ((4-chlorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A11)
1、N 4 Synthesis of- (4-chlorophenyl) pyrimidine-4, 6-diamine (7a)
Figure BDA0003749367740000161
Synthesis of Compound 7a was obtained as a pale yellow solid in 59% yield, according to the method for synthesizing Compound 4 a.
1H NMR(400MHz,DMSO-d6)δ9.03(s,1H),8.05(d,J＝1.0Hz,1H),7.69–7.53(m,2H),7.37–7.24(m,2H),6.38(d,J＝3.4Hz,1H),5.76(d,J＝1.0Hz,1H).
2. Synthesis of (1R,4aR,4bR,10aR) -N- (6- ((4-chlorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A11)
Figure BDA0003749367740000162
Synthesis of compound a11 referring to the synthesis of compound a1, compound a11 was obtained as a white solid in 28% yield, mp: 106.9-108.2 ℃.
1H NMR(400MHz,DMSO-d6)δ9.85(s,1H),9.70(d,J＝6.0Hz,1H),8.41(q,J＝2.2,1.7Hz,1H),7.79–7.52(m,3H),7.42–7.29(m,2H),5.71(s,1H),5.31(d,J＝5.0Hz,1H),2.26–2.13(m,2H),1.99(t,J＝7.6Hz,3H),1.91–1.84(m,1H),1.77(t,J＝11.6Hz,2H),1.60–1.50(m,3H),1.28–1.21(m,4H),1.18–1.07(m,3H),1.01–0.82(m,6H),0.77(s,3H).ESI-MS m/z:505.3[M+H]+,C30H37ClN4O.
Example 12 Synthesis of ((1R,4aR,4bR,10aR) -N- (6-aminopyrimidin-4-yl) -N- (4-chlorophenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,4b,5,6,10,10 a-decahydrophenanthrene-1-carboxamide (A12)
Figure BDA0003749367740000171
Referring to example 11 synthesis of compound 7a, compound a12 synthesis referring to the synthesis of compound a1, compound a12 was obtained as a white solid in 23% yield, mp: 130.9-132.7 ℃.
1H NMR(400MHz,DMSO-d6)δ8.20(d,J＝4.1Hz,1H),7.81–7.55(m,2H),7.46(d,J＝8.6Hz,1H),7.35(d,J＝9.5Hz,1H),7.13(d,J＝8.7Hz,2H),6.85(d,J＝9.4Hz,1H),5.90(d,J＝12.5Hz,1H),5.72(s,1H),2.35–2.14(m,3H),2.09–1.93(m,4H),1.77(q,J＝12.7,10.1Hz,4H),1.46(s,3H),1.30(s,1H),1.25(d,J＝10.1Hz,4H),1.07–0.94(m,8H).ESI-MS m/z:505.3[M+H]+,C30H37ClN4O.
Example 13 Synthesis of (1R,4aS,10aR) -7-isopropyl-1, 4 a-dimethyl-N- (4- (pyridin-2-ylmethoxy) phenyl) -1,2,3,4,4a,9,10,10 a-octahydrophenanthrene-1-carboxamide (B1)
Figure BDA0003749367740000172
Reference example 1 synthesis of compound 1a, synthesis of compound B1 reference method for the synthesis of compound a1 gave compound B1 as a white solid in 32% yield, mp: 76.3-77.7 ℃.
1H NMR(400MHz,DMSO-d6)δ9.19(s,1H),8.57(ddd,J＝4.9,1.8,0.9Hz,1H),7.82(td,J＝7.7,1.8Hz,1H),7.56–7.43(m,3H),7.33(ddd,J＝7.6,4.8,1.2Hz,1H),7.23–7.11(m,1H),7.04–6.91(m,3H),6.84(d,J＝2.0Hz,1H),5.14(s,2H),2.86–2.67(m,3H),2.29(d,J＝12.6Hz,1H),2.20(dd,J＝12.4,2.2Hz,1H),1.78(s,1H),1.66(d,J＝12.1Hz,1H),1.59–1.48(m,2H),1.39(s,1H),1.25(d,J＝6.5Hz,4H),1.20–1.11(m,10H).ESI-MS m/z:483.3[M+H]+,C32H38N2O2.
Example 14 Synthesis of (1R,4aS,10aR) -N- (3-chloro-4- (pyridin-2-ylmethoxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,9,10,10 a-octahydrophenanthrene-1-carboxamide (B2)
Figure BDA0003749367740000181
Referring to example 1 synthesis of compound 2a, synthesis of compound B2 reference to the synthesis of compound a1 gave compound B2 as a white solid in 34% yield, mp: 56.8-58.5 ℃.
1H NMR(400MHz,DMSO-d6)δ9.33(s,1H),8.58(d,J＝4.9Hz,1H),7.86(t,J＝6.8Hz,1H),7.79(d,J＝2.6Hz,1H),7.58–7.44(m,2H),7.35(dd,J＝7.5,4.8Hz,1H),7.18(dd,J＝13.6,8.6Hz,2H),6.98(d,J＝7.1Hz,1H),6.84(s,1H),5.23(s,2H),2.77(tt,J＝14.9,7.4Hz,2H),2.30(d,J＝12.4Hz,1H),2.23–2.15(m,1H),1.74(dt,J＝28.1,12.8Hz,4H),1.58–1.42(m,3H),1.24(d,J＝8.2Hz,5H),1.20–1.11(m,8H).ESI-MS m/z:517.0[M+H]+,C32H37ClN2O2.
Example 15 Synthesis of (1R,4aS,10aR) -N- (3-chloro-4- ((3-fluorobenzyl) oxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,9,10,10 a-octahydrophenanthrene-1-carboxamide (B3)
Figure BDA0003749367740000182
Referring to example 3 synthesis of compound 3a, synthesis of compound B3 referring to the synthesis of compound a1, compound B3 was obtained as a white solid in 28% yield, mp: 62.2 to 63.8 ℃.
1H NMR(400MHz,DMSO-d6)δ9.33(s,1H),7.79(d,J＝2.5Hz,1H),7.55–7.40(m,2H),7.28(t,J＝8.0Hz,2H),7.21–7.12(m,3H),6.97(dd,J＝8.2,2.0Hz,1H),6.83(s,1H),5.19(s,2H),2.84–2.68(m,4H),2.24(dd,J＝31.6,11.9Hz,2H),1.82–1.72(m,3H),1.65(d,J＝11.4Hz,1H),1.58–1.52(m,1H),1.47(d,J＝12.8Hz,1H),1.18–1.11(m,12H).ESI-MS m/z:534.0[M+H]+,C33H37ClFNO2.
Example 16 Synthesis of (((1R,4aS,10aR) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,9,10,10 a-octahydrophenanthren-1-yl) (4-phenylpiperazin-1-yl)) methanone (B4)
Figure BDA0003749367740000191
Synthesis of compound B4 referring to the synthesis of compound a4, compound B4 was obtained as a white solid in 35% yield, mp: 124.0-125.8 ℃.
1H NMR(400MHz,DMSO-d6)δ7.20(dt,J＝15.4,8.0Hz,3H),6.95(dd,J＝16.3,8.2Hz,3H),6.85(s,1H),6.80(t,J＝7.3Hz,1H),3.81–3.61(m,4H),3.09(s,4H),2.78(t,J＝7.1Hz,2H),2.27(d,J＝12.8Hz,1H),2.19(d,J＝11.3Hz,1H),1.85–1.58(m,5H),1.47(d,J＝10.4Hz,1H),1.40–1.04(m,14H).ESI-MS m/z:445.1[M+H]+,C30H40N2O.
Example 17 Synthesis of (1R,4aS,10aR) -7-isopropyl-1, 4 a-dimethyl-N- (6- (phenylamino) pyrimidin-4-yl) -1,2,3,4,4a,9,10,10 a-octahydrophenanthrene-1-carboxamide (B5)
Figure BDA0003749367740000192
Referring to example 5 synthesis of compound 4a, compound B5 synthesis referring to the synthesis of compound a1, compound B5 was obtained as a white solid in 26% yield, mp: 94.8-96.3 ℃.
1H NMR(400MHz,DMSO-d6)δ9.95(d,J＝48.2Hz,1H),9.55(s,1H),8.38(dd,J＝6.5,1.0Hz,1H),7.74–7.54(m,3H),7.30(dd,J＝8.6,7.3Hz,2H),7.17(dd,J＝13.9,8.2Hz,1H),6.99(ddd,J＝8.6,6.2,1.8Hz,2H),6.82(dd,J＝20.4,2.0Hz,1H),2.87–2.70(m,3H),2.34(dd,J＝12.5,2.1Hz,1H),1.88–1.80(m,2H),1.79–1.71(m,2H),1.65(d,J＝9.7Hz,2H),1.60(s,1H),1.57(s,10H),1.19–1.10(m,12H).ESI-MS m/z:469.1[M+H]+,C30H36N4O.
Example 18 Synthesis of (1R,4aS,10aR) -N- (6-aminopyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-N-phenyl-1, 2,3,4,4a,9,10,10 a-octahydrophenanthrene-1-carboxamide (B6)
Figure BDA0003749367740000201
Referring to example 5 synthesis of compound 4a, compound B6 synthesis referring to the synthesis of compound a1, compound B6 was obtained as a white solid in 21% yield, mp: 74.7-76.4 ℃.
1H NMR(400MHz,DMSO-d6)δ8.18(d,J＝6.6Hz,1H),7.41(t,J＝7.6Hz,2H),7.30(t,J＝7.4Hz,1H),7.18–7.07(m,3H),6.96(dd,J＝8.2,2.0Hz,1H),6.83(s,3H),5.86(s,1H),2.77(ddt,J＝19.5,12.8,5.9Hz,2H),2.36–2.20(m,1H),1.99(s,2H),1.91(s,1H),1.84(s,1H),1.75(dd,J＝9.5,5.3Hz,1H),1.66(t,J＝8.0Hz,1H),1.62–1.51(m,2H),1.50–1.35(m,2H),1.26(d,J＝25.4Hz,1H),1.20–1.09(m,8H),1.02(s,2H).ESI-MS m/z:469.2[M+H]+,C30H36N4O.
Example 19 Synthesis of (1R,4aS,10aR) -N- (6-aminopyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-N-phenyl-1, 2,3,4,4a,9,10,10 a-octahydrophenanthrene-1-carboxamide (B7)
Figure BDA0003749367740000211
Referring to example 7 synthesis of compound 5a, synthesis of compound B7 reference to the synthesis of compound a1 gave compound B7 as a white solid in 25% yield, mp: 87.5-89.2 ℃.
1H NMR(400MHz,DMSO-d6)δ9.96(s,1H),9.44(s,1H),8.34(t,J＝1.2Hz,1H),7.57(d,J＝1.2Hz,1H),7.50(dd,J＝8.3,4.1Hz,2H),7.18(d,J＝8.2Hz,1H),7.11(d,J＝8.3Hz,2H),6.98(dd,J＝8.1,2.0Hz,1H),6.84(d,J＝2.0Hz,1H),2.26(s,3H),1.81–1.69(m,2H),1.67–1.54(m,4H),1.26–1.21(m,8H),1.15(d,J＝6.6Hz,10H).ESI-MS m/z:483.3[M+H]+,C31H38N4O.
Example 20 Synthesis of (1R,4aS,10aR) -N- (6-aminopyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-N- (p-tolyl) -1,2,3,4,4a,9,10,10 a-octahydrophenanthrene-1-carboxamide (B8)
Figure BDA0003749367740000212
Referring to example 7 synthesis of compound 5a, synthesis of compound B7 reference to the synthesis of compound a1 gave compound B8 as a white solid in 23% yield, mp: 139.7-141.5 ℃.
1H NMR(400MHz,DMSO-d6)δ8.15(dd,J＝7.0,1.1Hz,1H),7.43–7.24(m,1H),7.21(dd,J＝8.4,2.8Hz,2H),7.12(d,J＝8.2Hz,1H),7.07–6.90(m,3H),6.83(d,J＝1.9Hz,1H),6.77(d,J＝6.4Hz,1H),5.80(dd,J＝11.9,1.0Hz,1H),2.77(tt,J＝14.8,6.7Hz,2H),2.30(s,4H),1.98(d,J＝8.2Hz,1H),1.83(d,J＝8.2Hz,1H),1.74(t,J＝4.7Hz,1H),1.69–1.61(m,2H),1.60(d,J＝5.4Hz,1H),1.52(d,J＝5.6Hz,1H),1.46(s,1H),1.30(s,2H),1.24(d,J＝9.1Hz,5H),1.14(s,2H),1.11(s,2H),1.02(s,2H).ESI-MS m/z:483.4[M+H]+,C31H38N4O.
Example 21 Synthesis of (1R,4aS,10aR) -N- (6- ((4-fluorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,9,10,10 a-octahydrophenanthrene-1-carboxamide (B9)
Figure BDA0003749367740000221
Referring to example 9 synthesis of compound 6a, synthesis of compound B9 reference to the synthesis of compound a1 gave compound B9 as a white solid in 31% yield, mp: 85.1-87.3 ℃.
1H NMR(400MHz,DMSO-d6)δ10.15–9.72(m,1H),9.58(s,1H),8.37(dt,J＝6.6,1.2Hz,1H),7.66(ddd,J＝8.7,5.0,3.4Hz,2H),7.56(dt,J＝7.1,1.2Hz,1H),7.21–7.11(m,3H),6.97(td,J＝8.3,2.1Hz,1H),6.84(s,1H),2.77(ddq,J＝13.7,7.1,4.0,2.4Hz,3H),2.37–2.29(m,1H),1.88–1.83(m,1H),1.76–1.70(m,2H),1.68–1.59(m,4H),1.57(d,J＝3.3Hz,1H),1.15(d,J＝6.8Hz,12H).ESI-MS m/z:487.3[M+H]+,C30H35FN4O.
Example 22 Synthesis of (1R,4aS,10aR) -N- (6-aminopyrimidin-4-yl) -N- (4-fluorophenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,9,10,10 a-octahydrophenanthrene-1-carboxamide (B10)
Figure BDA0003749367740000222
Referring to example 9 synthesis of compound 6a, synthesis of compound B10 reference to the synthesis of compound a1 gave compound B10 as a white solid in 22% yield, mp: 128.8-130.5 ℃.
1H NMR(400MHz,DMSO-d6)δ8.37(d,J＝5.3Hz,1H),7.69(s,1H),7.35(t,J＝5.8Hz,5H),7.12(d,J＝8.2Hz,1H),6.96(dd,J＝8.2,1.9Hz,1H),6.82(d,J＝1.9Hz,1H),5.80(d,J＝14.7Hz,1H),1.98(s,2H),1.91(s,1H),1.86–1.56(m,9H),1.20–1.08(m,12H).ESI-MS m/z:487.2[M+H]+,C30H35FN4O.
Example 23 Synthesis of (1R,4aS,10aR) -N- (6- ((4-chlorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,9,10,10 a-octahydrophenanthrene-1-carboxamide (B11)
Figure BDA0003749367740000231
Referring to example 11 synthesis of compound 7a, synthesis of compound B11 reference to the synthesis of compound a1 gave compound B11 as a white solid in 28% yield, mp: 91.3-93.3 ℃.
1H NMR(400MHz,DMSO-d6)δ10.07(s,1H),9.71(s,1H),8.40(s,1H),7.76–7.66(m,2H),7.62(s,1H),7.40–7.29(m,2H),7.18(d,J＝8.2Hz,1H),6.98(d,J＝8.1Hz,1H),6.84(s,1H),2.78(dh,J＝14.5,7.7,7.1Hz,3H),2.34(d,J＝12.3Hz,1H),2.21(d,J＝12.2Hz,1H),1.85(d,J＝11.4Hz,1H),1.74(t,J＝9.2Hz,1H),1.62(dd,J＝22.5,11.2Hz,3H),1.24(d,J＝9.9Hz,4H),1.15(d,J＝7.1Hz,10H).ESI-MS m/z:503.2[M+H]+,C30H35ClN4O.
Example 24 Synthesis of (1R,4aS,10aR) -N- (6-aminopyrimidin-4-yl) -N- (4-chlorophenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3,4,4a,9,10,10 a-octahydrophenanthrene-1-carboxamide (B12)
Figure BDA0003749367740000241
Referring to example 11 synthesis of compound 7a, synthesis of compound B12 reference to the synthesis of compound a1 gave compound B12 as a white solid in 24% yield, mp: 88.2-89.7 ℃.
1H NMR(400MHz,DMSO-d6)δ8.19(d,J＝6.6Hz,1H),7.46(td,J＝5.9,2.6Hz,2H),7.21–7.08(m,3H),6.96(dd,J＝8.2,2.0Hz,1H),6.88(d,J＝6.1Hz,2H),6.84(d,J＝2.0Hz,1H),5.89(d,J＝11.3Hz,1H),2.79(dtd,J＝25.7,13.6,11.5,7.4Hz,2H),2.35–2.21(m,1H),2.10–1.97(m,2H),1.91(s,1H),1.84(s,1H),1.76–1.56(m,5H),1.49(d,J＝12.9Hz,2H),1.32–1.21(m,2H),1.21–1.08(m,8H).ESI-MS m/z:503.2[M+H]+,C30H35ClN4O.
EXAMPLE 25 determination of the antiproliferative Activity of Compounds on glioma cells
The experimental method comprises the following steps:
the anti-proliferation activity of rosin diterpene derivatives A1-A12 and B1-B12 on human glioma cells T98G, human brain astrocytoma cells U87MG and human glial cells U251 is researched by adopting a CCK-8 method. At present, the CCK-8 method (Cell Counting Kit-8) is one of the most common standard methods for detecting in vitro Cell proliferation and toxicity experiments. The results of experiments using Fluorouracil (5-Fluorouracil,5-FU), Carmustine (BCUN) and Temozolomide (TMZ) as positive controls were shown in Table 1, giving the half inhibitory concentrations (IC50 values) of different compounds against three human brain glioma cells.
TABLE 1 antiproliferative activity of the target compounds against various human brain gliomas (IC50, μ M)
Figure BDA0003749367740000242
Figure BDA0003749367740000251
Figure BDA0003749367740000261
N.D.＝not detected indicates that no IC was detected 50 。
As can be seen from the table, the broad-spectrum antitumor drugs 5-FU and BCUN do not show antiproliferative activity on three glioma cells T98G, U87MG and U251; compared with the IC50 value of positive control TMZ, lead compounds Abietic Acid (AA) and dehydroabietic acid (DHAA), the derivative obtained by condensation reaction of rosin diterpene parent nucleus and secondary amine on N4-phenylpyrimidine-4, 6-diamine intermediate containing different substituents shows better antiproliferative activity on the three human brain glioma cells, and the derivative obtained by combining the parent nucleus and the primary amine on the intermediate hardly influences the tested cells. In addition, in the derivatives containing the 4, 6-disubstituted aminopyrimidine fragments, the B-series DHAA derivatives have stronger antitumor activity than the A-series AA derivatives.
For T98G cells, the antiproliferative activity (IC50 value is 22.17-71.09 mu M) of DHAA derivatives B6, B8, B10 and B12 is far better than that of positive drug TMZ (IC50 is 1477 mu M), and compared with compound B6 with the best activity (IC50 is 22.17 mu M), the difference of TMZ is nearly 70 times; in addition, AA derivatives A6, A8 and A12-A14 show relatively good activity on T98G cells (IC50 value is 102.40-235.70 mu M), but the activity is weaker than that of corresponding DHAA derivatives. For U251 cells, the compounds with A series numbers 6, 8, 10 and 12 and B series numbers 12 show strong proliferation inhibition activity (IC50 value is 11.47-22.56 mu M), wherein the activity of most B series derivatives is better than that of A series derivatives; the anti-tumor activity of the compound B8 on U251 cells is the most prominent, and the IC50 of the compound B8 is 11.47 mu M and is 28 times stronger than that of TMZ (IC50 is 322.60 mu M); in addition, the simply modified compounds a13 and a14 also showed better activity on U251 (IC50<100 μ M). For U87MG cells, the target compounds A8, A10, A14, B6, B8, B10 and B12 all show good anti-cell proliferation activity (the IC50 value is 99.54-295.60 mu M), wherein the activity of the compound B10(IC50 is 99.54 mu M) is strongest and is far better than that of TMZ (IC50>1600 mu M). Through comprehensive comparison, the compounds B6, B8, B10 and B12 all show stronger antiproliferative activity on three tested human glioma cells, and have dose dependence in a certain concentration range.
Example 26 Compounds inhibit T98G cell migration assay
The experimental method comprises the following steps: T98G cells at 5X 10 5 Inoculating the seeds in a 6-well plate at the density of each hole, and placing the plates in a cell culture box for culturing for 24 hours; preparing liquid medicine containing compounds (0, 5, 10, 20 and 40 mu M) with different concentrations; scratching was performed on a monolayer of cells with a 200 μ L pipette tip and the original medium was aspirated off, washed with PBS to remove the dropped cells, and the washing was repeated twice; adding prepared compound liquid medicines with different concentrations; at intervals of 0h and 24h, cell migration was observed and photographed in multiple random areas (at least three) per well using an inverted microscope. This example was tested on compound B12 as representative, and the results were similar for the remaining compounds.
As can be seen from fig. 1 and fig. 2, compared with the vehicle control group, the administration group can significantly inhibit the migration of T98G cells after 24h, and the scratch healing area is reduced with the increase of the administration concentration, which indicates that compound B12 inhibits the migration of T98G cells in a concentration and time-dependent manner.
EXAMPLE 27 pKa value determination of representative Compounds
The experimental method comprises the following steps: preparing a representative compound drug mother liquor (acetonitrile, 1mM, 5mL), an acetonitrile aqueous solution (acetonitrile V: water ═ 3:7, 50mL), a KCl solution (water, 0.15M, 10mL), a KOH solution (acetonitrile V: water ═ 3:7, 60mM, 10mL), and an HCl solution (acetonitrile V: water ═ 3:7, 100mM, 10mL) for later use; adding 10mL of the prepared acetonitrile aqueous solution into a 15mL beaker, measuring the pH value of the acetonitrile aqueous solution by using a pH meter, and recording after the value is stable; adding 1mL of mother liquor of the medicine into the solution, measuring and recording the pH, and soaking the probe in absolute ethyl alcohol for 30 min; taking 10mL of acetonitrile aqueous solution again to a 25mL beaker, measuring the pH value of the acetonitrile aqueous solution, adding 1mL of KCl solution, and measuring the pH value after uniformly stirring; adjusting the pH value to 2.0 by using 100mM HCl solution, adding 1mL of medicine mother liquor, after the solution is fully and uniformly stirred, slowly titrating the solution by using 60mM KOH solution, and recording the change of the pH value; experimental data were processed and plotted by Origin 2021 software. The test was carried out using compounds B6, B8, B10, B12 as representatives, and the results were similar for the remaining compounds.
The results are shown in table 2 and fig. 3, and it can be seen that the pKa of representative compounds B6, B8, B10 and B12 is 7.17-7.35, and all within ideal parameter ranges, according to the Lipinski's principle, the pKa of a small molecule compound capable of improving BBB permeability needs to be within the range of 6< pKa <10.5, and the possibility that a compound with pKa <8 can be a P-glycoprotein (P-gp) substrate is extremely low, which indicates that the target compounds may have good blood brain barrier permeability to enter the brain to exert efficacy.
Table 2 pKa of representative compounds
Compounds B6 B8 B10 B12
pKa 7.35 7.34 7.35 7.17
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. A rosin diterpenoid having the structure of formula I or formula II:
Figure FDA0003749367730000011
r is
Figure FDA0003749367730000012
Wherein R is 1 Is hydrogen or halogen; r 2 Is unsubstituted or substituted C 1～5 Alkoxy radical, said substituted C 1～5 The substituent of the alkoxy is phenyl, halogenated phenyl or heterocyclic aryl; r is 3 Is unsubstituted or substituted amino, and the substituent of the substituted amino is phenyl or C 1～5 Alkylphenyl, halophenyl; r 4 Is phenyl, C 1～5 Alkylphenyl, halophenyl; r 5 Is phenyl.
2. The rosin diterpenoid compound according to claim 1, wherein R is 1 Is hydrogen or halogen; r 2 Is unsubstituted or substituted C 1～3 Alkoxy radical, said substituted C 1～3 The substituent of the alkoxy is phenyl, halogenated phenyl or pyridine; r 3 Is unsubstituted or substituted amino, and the substituent of the substituted amino is phenyl or C 1～3 Alkylphenyl, halophenyl; r 4 Is phenyl, C 1～3 Alkylphenyl, halophenyl; r is 5 Is phenyl.
3. The rosin diterpenoid compound according to claim 2, wherein R is 1 Is hydrogen or halogen; r 2 Is unsubstituted or substituted methoxyl, and the substituent of the substituted methoxyl is phenyl, halogenated phenyl or pyridine; r 3 Is unsubstituted or substituted amino, and the substituent of the substituted amino is phenyl, tolyl or halogenated phenyl; r 4 Phenyl, tolyl, halophenyl; r 5 Is phenyl.
4. According to claimThe rosin diterpenoid compound according to claim 3, wherein R is
Figure FDA0003749367730000021
Figure FDA0003749367730000022
One kind of (1).
5. The preparation method of the rosin diterpenoid compound according to any one of claims 1 to 4, which is characterized by comprising the following steps:
Figure FDA0003749367730000023
s1, taking AA as an initial raw material, carrying out an acylation reaction under the action of an acylation reagent and a polar organic solvent to obtain a compound 1, and carrying out an amide condensation reaction with a compound R-H under the action of an acid-binding agent to obtain a compound shown in the formula I;
Figure FDA0003749367730000024
s2, taking DHAA as an initial raw material, and carrying out an acylation reaction under the action of an acylation reagent and a polar organic solvent to obtain a compound 2; then carrying out amide condensation reaction with a compound R-H under the action of an acid-binding agent to obtain a compound shown in the formula I;
wherein R in R-H is as defined in any one of claims 1 to 4.
6. The method according to claim 5, wherein the acylating agent is selected from one or more of oxalyl chloride, thionyl chloride and dibenzoyl chloride.
7. The preparation method of claim 5, wherein the acid scavenger is selected from one or more of triethylamine, pyridine, and N, N-diisopropylethylamine.
8. The method according to claim 5, wherein the polar organic solvent is one or more selected from the group consisting of N, N-dimethylformamide and dichloromethane.
9. Use of the rosin diterpenoid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 for preparing an anti-glioma medicament.
10. A pharmaceutical composition comprising one or more of the rosin diterpenoid compounds, their pharmaceutically acceptable salts, hydrates, solvates, polymorphs, tautomers, stereoisomers or prodrugs thereof according to any one of claims 1 to 4.
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Citations (2)

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US10064837B1 (en) * 2018-02-15 2018-09-04 King Saud University Abietic acid derivatives as anti-tumor agents
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