CN115073361B - 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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CN115073361B
CN115073361B CN202210833837.9A CN202210833837A CN115073361B CN 115073361 B CN115073361 B CN 115073361B CN 202210833837 A CN202210833837 A CN 202210833837A CN 115073361 B CN115073361 B CN 115073361B
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rosin
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diterpenoid
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CN115073361A (en
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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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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
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    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
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    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
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    • C07C2603/26—Phenanthrenes; Hydrogenated phenanthrenes

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a rosin diterpenoid compound, and a preparation method and application thereof. Experiments prove that the rosin diterpenoid compound has remarkable anti-proliferation and anti-migration activities on glioma cells, can achieve remarkable anti-glioma proliferation effect under the condition of low concentration, can remarkably inhibit migration of tumor cells, and has a good tumor treatment effect; in addition, the pKa of the rosin diterpenoid compound is in the range of 6-10.5, and the rosin diterpenoid compound has better blood brain barrier permeability and is beneficial to 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 medicine. More particularly relates to a rosin diterpenoid compound, 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, easy generation of drug resistance, poor prognosis and the like, the 5-year survival time of GBM patients is still less than 10% despite standardized treatment. In addition, because malignant gliomas have high mortality, the influence of tumor microenvironment and blood brain barrier on drug intake can exist in special parts such as cranial cavities, and many factors such as drug resistance, poor prognosis and the like exist, so that the research and development of anti-glioma drugs is long and difficult.
At present, the standardized treatment means for GBM is surgical excision combined with postoperative radiotherapy and chemotherapy, and gene therapy and immunotherapy are also adopted. Chemotherapeutic agents such as Temozolomide (TMZ), cisplatin, and carmustine are important clinical adjunctive therapeutic agents. However, even if standardized treatment is performed, almost all GBM patients relapse, because it is difficult for the surgeon to completely eliminate highly invasive growth of glioma cells without affecting normal brain function, and these cells have the characteristics of rapid proliferation, anti-apoptosis, and rapid metastasis, so postoperative relapse is one of the greatest challenges in treating GBM. Most GBM patients show insensitivity to radiotherapy and susceptibility to chemotherapy after recurrence. Due to drug resistance generated by chemotherapy, the median survival rate of GBM patients is about 15 months, and the treatment effect is not ideal. Therefore, development of a drug against brain glioma is necessary. How to effectively improve the blood brain barrier permeability of the medicine, reduce the toxic and side effects of the medicine and the like drives people to develop novel anti-glioma medicines.
The natural product has wide structural diversity and pharmacological multiple effects, is an important source for finding lead compounds, and has profound effects on drug development. For example, the application of eriodictyol as a medicament for treating glioma is disclosed in Chinese patent application, and experiments show that the eriodictyol can inhibit proliferation, migration and invasion of human glioma cells and promote apoptosis of human glioma cells. However, the technical scheme is only remained in the aspect of cell experiments, and medicines with good effects are still lacking in the treatment of glioma at present, so that the development of effective medicines for inhibiting glioma is still necessary.
Disclosure of Invention
The invention aims to overcome the defect and the defect of few medicaments for treating glioma in the prior art and provide a rosin diterpenoid compound.
The invention aims to provide a preparation method of the rosin diterpenoid compound.
The invention also aims to provide application of the rosin diterpenoid compound or the pharmaceutically acceptable salt thereof in preparing anti-glioma drugs.
The above object of the present invention is achieved by the following technical scheme:
the natural products Abietic Acid (AA) and dehydroabietic acid (dehydroabietic acid, DHAA) are tricyclic diterpenoid oxygen-containing compounds obtained from rosin or disproportionated rosin, the safety is high, and the inventor develops and obtains various compounds with anticancer effects on the basis of the structures of abietic acid and dehydroabietic acid.
A rosin diterpenoid compound having the structure of formula I or formula II:
r isWherein R is 1 Hydrogen or halogen; r is R 2 Is unsubstituted or takenSubstitute C 1～5 Alkoxy, said substitution C 1～5 The substituent of the alkoxy is phenyl, halogenated phenyl or heterocyclic aryl; r is R 3 Is unsubstituted or substituted amino, the substituent of the substituted amino is phenyl or C 1～5 Alkylphenyl, halophenyl; r is R 4 Is phenyl, C 1～5 Alkylphenyl, halophenyl; r is R 5 Is phenyl.
Preferably, said R 1 Hydrogen or halogen; r is R 2 Is unsubstituted or substituted C 1～3 Alkoxy, said substitution C 1～3 The substituent of the alkoxy is phenyl, halogenated phenyl or pyridine; r is R 3 Is unsubstituted or substituted amino, the substituent of the substituted amino is phenyl or C 1～3 Alkylphenyl, halophenyl; r is R 4 Is phenyl, C 1～3 Alkylphenyl, halophenyl; r is R 5 Is phenyl.
More preferably, the R 1 Hydrogen or halogen; r is R 2 Is unsubstituted or substituted methoxy, and the substituent of the substituted methoxy is phenyl, halogenated phenyl or pyridine; r is R 3 Is unsubstituted or substituted amino, and the substituent of the substituted amino is phenyl, tolyl or halogenated phenyl; r is R 4 Phenyl, tolyl, and halophenyl; r is R 5 Is phenyl.
Specifically, the R is
One of them.
For example, the rosin diterpenoid compound has any one of the following structures:
preferably, pharmaceutically acceptable salts obtained by reacting the rosin diterpenoid with an acid are also within the scope of the present 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 specifically comprises the following steps:
s1, taking AA as a starting material, carrying out acylation reaction under the action of an acylating agent 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 of the formula I;
s2, taking DHAA as a starting material, and carrying out an acylation reaction under the action of an acylating 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 of the formula I;
wherein R in R-H is as defined above.
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, 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 the 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 undergo an 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 pharmaceutically acceptable salt thereof in preparing anti-glioma drugs.
Preferably, the glioma is a glioma caused by abnormal expression of an epidermal growth factor receptor and/or a mutant thereof. More preferably, the gliomas include human glioma cells T98G, human brain astrocytoma cells U87MG, and human glial cells U251.
In addition, the invention also claims a pharmaceutical composition comprising one or more of the rosin diterpenoid compounds, pharmaceutically acceptable salts, hydrates, solvates, polymorphs, tautomers, stereoisomers or prodrugs thereof.
Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients and/or carriers.
Preferably, the auxiliary materials comprise 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, permeation promoters, pH modifiers, buffers, plasticizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, release agents, flocculants, anti-flocculants, filter aids, release retarders.
The pharmaceutical composition of the invention can be prepared into various dosage forms: the following formulations can be prepared, specifically, by classification of the dispersion system of the dosage forms: solution type, colloidal solution type, emulsion type, suspension type, gas dispersion type, microparticle dispersion type, and solid dispersion type; according to the morphological classification, the following dosage forms can be specifically prepared: liquid dosage forms (e.g., aromatic water, solution, injection, mixture, lotion, liniment, etc.), solid dosage forms (e.g., powder, pill, tablet, film, etc.), semisolid dosage forms (e.g., ointment, suppository, paste, etc.).
The invention has the following beneficial effects:
the invention provides a rosin diterpenoid compound, which has obvious antiproliferation and anti-migration activity on glioma cells, can achieve the effect of obviously resisting glioma proliferation under the condition of lower concentration, can obviously inhibit migration of tumor cells, and has a better tumor treatment effect; in addition, the pKa of the rosin diterpenoid compound is in the range of 6-10.5, and the rosin diterpenoid compound has better blood brain barrier permeability and is beneficial to entering the brain to exert the drug effect.
Drawings
FIG. 1 is a micrograph of T98G cells treated with a compound of the invention for 24h to inhibit T98G cell migration.
Fig. 2 is a graph of T98G cell mobility statistics for 24h inhibition of T98G cells treated with a compound of the invention, wherein data is from the mean of three random areas in each concentration group and compared to the control group, p <0.01.
FIG. 3 is a graph of pH-VKOH (. Mu.L) for compounds of the invention.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1: synthesis of (1R, 4aR,4bR,10 aR) -7-isopropyl-1, 4 a-dimethyl-N- (4- (pyridin-2-ylmethoxy) phenyl) -1,2,3, 4a,4b,5,6,10 a-decahydro phenanthrene-1-carboxamide (A1)
1. Synthesis of 4- (pyridine-2-methoxy) aniline (1 a)
4-aminophenol (1 g,9.16 mmol) was added to a solvent of N, N-dimethylformamide (10 mL) to be sufficiently dissolved, and then benzaldehyde (1.16 g,10.91 mmol) was added thereto and stirred at room temperature for 20 minutes; 2- (chloromethyl) pyridine hydrochloride (0.80 g,4.87 mmol) and potassium carbonate (5.05 g,36.53 mmol) were then added sequentially to the solvent, and reacted at 50℃for 18 hours; after the reaction, the mixture was filtered, the filter residue was discarded to leave a filtrate, the pH of the filtrate was adjusted to 1 to 2 with hydrochloric acid (2M), extraction was performed with ethyl acetate and water, an organic phase was collected, the collected organic phase was dried, filtered and the solvent was distilled off under reduced pressure, and then purification was performed by silica gel column chromatography, eluting with a mixed solvent of ethyl acetate and petroleum ether (v: v=1:5) as a mobile phase, to give a brown solid compound 1a in a yield of 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,10 aR) -7-isopropyl-1, 4 a-dimethyl-N- (4- (pyridin-2-ylmethoxy) phenyl) -1,2,3, 4a,4b,5,6,10 a-decahydro phenanthrene-1-carboxamide (A1)
Oxalyl chloride (0.63 g,4.97 mmol) was slowly added dropwise to a mixed solution of rosin acid (1 g,3.31 mmol) in dichloromethane (25 mL) under ice bath conditions, followed by 1-2 drops of N, N-dimethylformamide, and the mixture was allowed to react at room temperature under anhydrous conditions for 4 hours; after the substrate was completely reacted, the solvent was distilled off under reduced pressure to give an acid chloride-containing solution as an orange-yellow oil, and anhydrous methylene chloride (10 mL) was added; subsequently, to the mixture after reacting compound 1a (0.31 mg,1.55 mmol) with triethylamine (TEA, 0.27g,2.64 mmol) at room temperature for 10 minutes, a solution containing an acid chloride was added dropwise, and the reaction was carried out at room temperature for 18 hours under anhydrous conditions; after the reaction is completed, the organic layer is extracted for a plurality of times, the collected organic layer is washed by saturated saline water, dried by anhydrous sodium sulfate and distilled under reduced pressure to obtain a solid crude product, and the solid crude product is purified by silica gel column chromatography to obtain a brown solid compound A1, the yield is 36 percent, and 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,10 aR) -N- (3-chloro-4- (pyridin-2-ylmethoxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,4b,5,6,10 a-decahydro-phenanthrene-1-carboxamide (A2)
1. Synthesis of 3-chloro-4- (pyridine-2-methoxy) aniline (2 a)
Referring to the procedure for compound 1a, compound 2a was prepared 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,10 aR) -N- (3-chloro-4- (pyridin-2-ylmethoxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,4b,5,6,10 a-decahydro phenanthrene-1-carboxamide (A2)
Synthesis of Compound A2 the synthesis of reference Compound A1 gave Compound A2 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,10 aR) -N- (3-chloro-4- ((3-fluorobenzyl) oxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,4b,5,6,10 a-decahydro-phenanthrene-1-carboxamide (A3)
1. Synthesis of 3-chloro-4- (3-fluorobenzyloxy) aniline (3 a)
To N, N-dimethylformamide (20 mL) were added 2-chloro-4-nitrophenol (1 g,5.76 mmol), potassium carbonate (0.80 g,5.76 mmol) and 3-fluorobenzyl bromide (1.10 g,5.82 mmol), and the mixture was stirred at 85℃for 3h; the mixture was then extracted with ethyl acetate and water, and the organic layer was collected and washed with saturated brine, and the solvent was distilled off under reduced pressure over anhydrous sodium sulfate 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 (1 g,3.56 mmol) and reduced iron (0.60 g,10.68 mmol) in ethanol (15 mL) was added a solution of calcium chloride (0.42 g,3.56mmol,1.4mL water), the mixture was stirred at 85 ℃ for 8h, then the solid was filtered off, the solvent was removed by rotary evaporation under reduced pressure, and the resulting solid was purified using silica gel column chromatography 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,10 aR) -N- (3-chloro-4- ((3-fluorobenzyl) oxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,4b,5,6,10 a-decahydro phenanthrene-1-carboxamide (A3)
After synthesis of compound 3a, synthesis of compound A3 referred to the synthesis of compound A1 gave compound A3 as a white solid 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,10 aR) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,4b,5,6,10 a-decahydro-phenanthren-1-yl) (4-phenylpiperazin-1-yl) methanone (A4)
Synthesis of Compound A4 reference compound A1 Synthesis method gave Compound A4 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,10 aR) -7-isopropyl-1, 4 a-dimethyl-N- (6- (phenylamino) pyrimidin-4-yl) -1,2,3, 4a,4b,5,6,10 a-decahydro-phenanthrene-1-carboxamide (A5)
1. Synthesis of N4-phenylpyrimidine-4, 6-diamine (4 a)
To isopropyl alcohol (30 mL) containing 4-amino-6-chloropyrimidine (3 g,23.16 mmol) and aniline (3.26 g,34.97 mmol) was added dropwise hydrochloric acid (1 mL), and reacted at 80℃for 4h; after complete reaction, quenching the reaction with saturated sodium bicarbonate, extracting with ethyl acetate and water, washing the organic phase with saturated sodium chloride, drying with anhydrous sodium sulfate, and spin-evaporating under reduced pressure to obtain a large amount of solids, finally preparing petroleum ether and ethyl acetate mixed solvent (v: v=3:1) as flowing relative mixture, and performing silica gel column chromatography, purifying to obtain white solid compound 4a with a yield of 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,10 aR) -7-isopropyl-1, 4 a-dimethyl-N- (6- (phenylamino) pyrimidin-4-yl) -1,2,3, 4a,4b,5,6,10 a-decahydro phenanthrene-1-carboxamide (A5)
Synthesis of Compound A5 reference compound A1 Synthesis method gave Compound A5 as a pale yellow solid with a yield of 29%, 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,10 aR) -N- (6-aminopyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-N-phenyl-1, 2,3, 4a,4b,5,6,10 a-decahydro-phenanthrene-1-carboxamide (A6)
Reference example 5 synthesis of compound 4a, synthesis of compound A6 reference compound A1, gave compound A6 as an orange-yellow 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,10 aR) -7-isopropyl-1, 4 a-dimethyl-N- (6- (p-toluylamino)) pyrimidin-4-yl) -1,2,3, 4a,4b,5,6,10 a-decahydro-phenanthrene-1-carboxamide (A7)
1、N 4 Synthesis of- (p-tolyl) pyrimidine-4, 6-diamine (5 a)
Synthesis of Compound 5a the synthesis of reference Compound 4a gave Compound 5a as a white solid in 61% yield.
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,10 aR) -7-isopropyl-1, 4 a-dimethyl-N- (6- (p-toluylamino)) pyrimidin-4-yl) -1,2,3, 4a,4b,5,6,10 a-decahydro phenanthrene-1-carboxamide (A7)
Synthesis of Compound A7 reference compound A1 was synthesized to give Compound A7 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,10 aR) -N- (6-aminopyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-N- (p-tolyl) -1,2,3, 4a,4b,5,6,10 a-decahydro-phenanthrene-1-carboxamide (A8)
Reference example 7 synthesis of compound 5a, synthesis of compound A8 reference compound A1, 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,10 aR) -N- (6- ((4-fluorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,4b,5,6,10 a-decahydro phenanthrene-1-carboxamide (A9)
1. Synthesis of N4- (4-fluorophenyl) pyrimidine-4, 6-diamine (6 a)
Synthesis of Compound 6a the synthesis of reference Compound 4a gave Compound 6a as a gray solid in 63% yield.
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,10 aR) -N- (6- ((4-fluorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,4b,5,6,10 a-decahydro phenanthrene-1-carboxamide (A9)
Synthesis of Compound A9 reference compound A1 was synthesized to give Compound A9 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,10 aR) -N- (6-aminopyrimidin-4-yl) -N- (4-fluorophenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,4b,5,6,10 a-decahydro phenanthrene-1-carboxamide (A10)
Reference example 9 synthesis of compound 6a, synthesis of compound a10 reference compound A1, 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,10 aR) -N- (6- ((4-chlorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,4b,5,6,10 a-decahydro-phenanthrene-1-carboxamide (A11)
1、N 4 Synthesis of- (4-chlorophenyl) pyrimidine-4, 6-diamine (7 a)
Synthesis of Compound 7a the synthesis of reference Compound 4a gave Compound 7a as a pale yellow solid in 59% yield.
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,10 aR) -N- (6- ((4-chlorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,4b,5,6,10 a-decahydro phenanthrene-1-carboxamide (A11)
Synthesis of Compound A11 reference compound A1 was synthesized to give Compound A11 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,10 aR) -N- (6-aminopyrimidin-4-yl) -N- (4-chlorophenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,4b,5,6,10 a-decahydro-phenanthrene-1-carboxamide) (A12)
Reference example 11 synthesis of compound 7a the synthesis of compound a12 was followed by the synthesis of reference compound A1 to give compound a12 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,10 aR) -7-isopropyl-1, 4 a-dimethyl-N- (4- (pyridin-2-ylmethoxy) phenyl) -1,2,3, 4a,9,10 a-octahydrophenanthrene-1-carboxamide (B1)
Reference example 1 synthesis of compound 1a after synthesis of compound B1 reference compound A1 synthesis procedure 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,10 aR) -N- (3-chloro-4- (pyridin-2-ylmethoxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,9,10 a-octahydrophenanthrene-1-carboxamide (B2)
Reference example 1 synthesis of compound 2a followed by synthesis of compound B2 reference 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,10 aR) -N- (3-chloro-4- ((3-fluorobenzyl) oxy) phenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,9,10 a-octahydrophenanthrene-1-carboxamide (B3)
Reference example 3 synthesis of compound 3a followed by synthesis of compound B3 reference compound A1 gave compound B3 as a white solid in 28% yield, mp:62.2-63.8 ℃.
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 (B4) methanone (1R, 4aS,10 aR) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,9,10 a-octahydrophenanthren-1-yl) (4-phenylpiperazin-1-yl)
Synthesis of Compound B4 reference compound A4 was synthesized to give Compound B4 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,10 aR) -7-isopropyl-1, 4 a-dimethyl-N- (6- (phenylamino) pyrimidin-4-yl) -1,2,3, 4a,9,10 a-octahydrophenanthrene-1-carboxamide (B5)
Reference example 5 synthesis of compound 4a followed by synthesis of compound B5 reference compound A1 gave compound B5 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,10 aR) -N- (6-aminopyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-N-phenyl-1, 2,3, 4a,9,10 a-octahydrophenanthrene-1-carboxamide (B6)
Reference example 5 synthesis of compound 4a followed by synthesis of compound B6 reference compound A1 gave compound B6 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,10 aR) -N- (6-aminopyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-N-phenyl-1, 2,3, 4a,9,10 a-octahydrophenanthrene-1-carboxamide (B7)
Reference example 7 synthesis of compound 5a, synthesis of compound B7 reference 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,10 aR) -N- (6-aminopyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-N- (p-tolyl) -1,2,3, 4a,9,10 a-octahydrophenanthrene-1-carboxamide (B8)
Reference example 7 synthesis of compound 5a followed by synthesis of compound B7 reference 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,10 aR) -N- (6- ((4-fluorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,9,10 a-octahydrophenanthrene-1-carboxamide (B9)
Reference example 9 synthesis of compound 6a, synthesis of compound B9 reference compound A1, gave compound B9 as a white solid in 31%, 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,10 aR) -N- (6-aminopyrimidin-4-yl) -N- (4-fluorophenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,9,10 a-octahydrophenanthrene-1-carboxamide (B10)
Reference example 9 synthesis of compound 6a followed by synthesis of compound B10 reference 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,10 aR) -N- (6- ((4-chlorophenyl) amino) pyrimidin-4-yl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,9,10 a-octahydrophenanthrene-1-carboxamide (B11)
Reference example 11 synthesis of compound 7a followed by synthesis of compound B11 reference 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,10 aR) -N- (6-aminopyrimidin-4-yl) -N- (4-chlorophenyl) -7-isopropyl-1, 4 a-dimethyl-1, 2,3, 4a,9,10 a-octahydrophenanthrene-1-carboxamide (B12)
Reference example 11 synthesis of compound 7a followed by synthesis of compound B12 reference 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.
Determination of anti-proliferative Activity of the Compounds of example 25 against glioma cells
The experimental method comprises the following steps:
the antiproliferative activity of rosin diterpene derivatives A1-A12 and B1-B12 on human glioma cells T98G, human brain astrocyte tumor cells U87MG and human glial cells U251 is explored by adopting a CCK-8 method. The CCK-8 method (Cell Counting Kit-8) is currently one of the most common standard methods for detecting in vitro cell proliferation and toxicity. The experiment uses Fluorouracil (5-fluorous cil, 5-FU), carmustine (Carmustine, BCUN) and Temozolomide (Temozolomide, TMZ) as positive controls to obtain half-inhibitory concentrations (IC 50 values) of different compounds on three human brain glioma cells, and the results are shown in Table 1.
Table 1 antiproliferative activity of target compounds against various human brain gliomas (IC 50, μM)
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N.d. =not detected, indicating that IC was not detected 50 。
As can be seen from the table, the broad-spectrum antitumor drugs 5-FU and BCUN showed no antiproliferative activity on both T98G, U MG and U251 of the three glioma cells; compared with the IC50 values of positive control TMZ and lead compounds Abietic Acid (AA) and dehydroabietic acid (DHAA), the derivatives obtained by the condensation reaction of the rosin diterpene mother nucleus and secondary amine on N4-phenylpyrimidine-4, 6-diamine intermediates containing different substituents show better antiproliferative activity on three human brain glioma cells, and the derivatives obtained by the combination of the mother nucleus and primary amine on the intermediates have little influence on tested cells. Furthermore, among such derivatives containing 4, 6-disubstituted aminopyrimidine fragments, B-series DHAA derivatives have a stronger antitumor activity than a-series AA derivatives.
For T98G cells, the antiproliferative activity of DHAA derivatives B6, B8, B10 and B12 (IC 50 values 22.17-71.09 μm) was far better than the positive drug TMZ (ic50=1477 μm), which was nearly 70-fold different from the most active compound B6 (ic50=22.17 μm); in addition, the AA derivatives A6, A8, A12 to A14 also showed relatively good activity on T98G cells (IC 50 values of 102.40 to 235.70. Mu.M), but were weaker than the corresponding DHAA derivatives. For U251 cells, compounds numbered 6, 8, 10, 12 of both a and B series showed strong proliferation inhibitory activity (IC 50 values 11.47-22.56 μm), with most of the B series derivatives being better active than the a series derivatives; compound B8 showed most remarkable antitumor activity against U251 cells, with an IC50 of 11.47 μm, which was approximately 28-fold stronger than TMZ (ic50= 322.60 μm); furthermore, the simple modified compounds a13 and a14 also showed better activity towards U251 (IC 50<100 μm). For U87MG cells, the target compounds A8, a10, a14, B6, B8, B10 and B12 all showed good anti-cell proliferation activity (IC 50 values of 99.54-295.60 μm), with compound B10 (IC 50=99.54 μm) having the strongest activity, far superior to the activity of TMZ (IC 50>1600 μm). Through comprehensive comparison, the compounds B6, B8, B10 and B12 show strong antiproliferative activity on three human brain glioma cells tested, 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 were grown at 5X 10 5 The density of the individual/well is inoculated in a 6-well plate and placed in a cell incubator for culturing for 24 hours; preparing medicinal liquid containing compounds (0, 5, 10, 20 and 40 μm) with different concentrations; scraping scratches were performed on the monolayer of cells with a 200 μl pipette tip and the original medium was aspirated, washed with PBS to remove the dropped cells, and the wash was repeated twice; adding prepared chemical liquid containing different concentrations; at intervals of 0h and 24h, cell migration was observed and photographed in a plurality of random areas (at least three) per well using an inverted microscope. This example was conducted on behalf of compound B12, with the remaining compounds having similar results.
As a result, referring to fig. 1 and 2, it can be seen that the administration group significantly inhibited T98G cell migration after 24 hours as compared with the vehicle control group, and the scratch healing area decreased with the increase of the administration concentration, which suggests that compound B12 inhibited T98G cell migration in a concentration and time-dependent manner.
EXAMPLE 27 representative Compound pKa value determination
The experimental method comprises the following steps: representative compound drug stock solutions (acetonitrile, 1mM,5 mL), acetonitrile in water (V acetonitrile: V water=3:7, 50 mL), KCl solution (water, 0.15M,10 mL), KOH solution (V acetonitrile: V water=3:7, 60mM,10 mL), HCl solution (V acetonitrile: V water=3:7, 100mM,10 mL) were prepared for use; 10mL of the acetonitrile aqueous solution prepared above was added to a 15mL beaker, the pH was measured by using a pH meter, and the pH was recorded after the value was stable; adding 1mL of medicine mother liquor into the solution, measuring and recording pH, and soaking the probe in absolute ethyl alcohol for 30min; re-taking 10mL of acetonitrile water solution to a 25mL beaker, measuring the pH value of the solution, adding 1mL of KCl solution, uniformly stirring the solution, and measuring the pH value; adjusting the pH to 2.0 by using 100mM HCl solution, adding 1mL of drug mother solution, slowly titrating the solution by using 60mM KOH solution after the solution is fully and uniformly stirred, and recording the change of the pH value; experimental data were processed and plotted by Origin 2021 software. The test was performed on the representative of compounds B6, B8, B10, B12, with similar results for the remaining compounds.
As a result, referring to table 2 and fig. 3, it can be seen that the pKa of representative compounds B6, B8, B10 and B12 is 7.17 to 7.35, all within the ideal parameters, and the pKa of the small molecule compound capable of improving BBB permeability is required to be within the range of 6< pKa <10.5 according to the Lipinski's fifth principle, while the probability that the compound with pKa <8 can become P-glycoprotein (P-gp) substrate is extremely small, indicating that these target compounds may have better 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 examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (7)

1. A rosin diterpenoid compound, characterized in that the rosin diterpenoid compound has a structure of formula I or formula II:
when the rosin diterpenoid compound has a structure shown as a formula I, R is
The rosin IIWhen the terpenoid has the structure of formula II, R is
2. The preparation method of the rosin diterpenoid compound according to claim 1, which is characterized by comprising the following steps:
s1, taking AA as a starting material, carrying out acylation reaction under the action of an acylating agent 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 of the formula I;
s2, taking DHAA as a starting material, and carrying out an acylation reaction under the action of an acylating 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 of a formula II;
wherein R in R-H is as defined in claim 1.
3. The preparation method according to claim 2, wherein the acylating agent is one or more selected from oxalyl chloride, thionyl chloride, and dibenzoyl chloride.
4. The preparation method according to claim 2, wherein the acid binding agent is one or more selected from triethylamine, pyridine, and N, N-diisopropylethylamine.
5. The preparation method according to claim 2, wherein the polar organic solvent is one or more selected from the group consisting of N, N-dimethylformamide and methylene chloride.
6. The use of a rosin diterpenoid compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-glioma drug.
7. A pharmaceutical composition comprising the rosin diterpenoid compound of claim 1, one or more of its pharmaceutically acceptable salts, tautomers, or stereoisomers.
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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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US10064837B1 (en) * 2018-02-15 2018-09-04 King Saud University Abietic acid derivatives as anti-tumor agents
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