CN112125916A

CN112125916A - Novel small molecule inhibitors of insulin-like growth factor-1receptor and uses thereof

Info

Publication number: CN112125916A
Application number: CN202011040572.4A
Authority: CN
Inventors: 王英杰; 康博
Original assignee: Hangzhou Renzhe Biotechnology Co ltd
Current assignee: Zhejiang University ZJU
Priority date: 2020-09-28
Filing date: 2020-09-28
Publication date: 2020-12-25
Also published as: CN114315848B; CN114276362A; CN114315848A; CN114276362B

Abstract

The invention provides a novel small molecule inhibitor of an insulin-like growth factor-1receptor and application thereof, wherein the novel small molecule inhibitor is a derivative of picropodophyllin, wherein a fluorine atom is used for replacing a hydrogen atom at the left side 2 of the picropodophyllin, so that the capability of molecules for penetrating through a blood brain barrier is improved; meanwhile, deuterium atoms are used for replacing hydrogen atoms, so that the half-life period of molecules in a body can be effectively prolonged. Compared with the existing picropodophyllotoxin in clinical test stage, the small molecule inhibitor of the invention has higher blood brain barrier permeability and longer half-life in vivo while maintaining the action mechanism and biochemical characteristics. When used alone, the compound can effectively inhibit the proliferation and brain metastasis of various tumors, and can be synergistically combined with other anti-cancer drugs, so that the proliferation of tumor cells is effectively inhibited, and the life cycle is remarkably prolonged.

Description

Novel small molecule inhibitors of insulin-like growth factor-1receptor and uses thereof

Technical Field

The present invention relates to novel compounds and the use of a novel substituted picropodophyllotoxin derivative for inhibiting the insulin-like growth factor-1receptor IGF-1R for the treatment of IGF-1R dependent diseases, particularly cancer.

Background

The development of primary nervous system malignancies, which refer to glial-derived brain tumors, including astrocytic, oligodendroglial, and ependymal tumors (Muir CS, Storm et al, Cancer Surv,19-20: 369-Dawley 392, 1994; Louis DN et al, Acta Neuropathy, 131: 803-Dawley-packay, 2016), is the most common type of primary tumor, is increasing year by year. Chemotherapy, represented by temozolomide, is the current gold standard for the treatment of high-grade gliomas, but only marginally extends patient survival. More effective targeted drug therapy has not been successful in gliomas to date because most targeted drugs cannot penetrate the blood brain barrier. It has been reported in the literature that Picropodophyllin (PPP) inhibits the proliferation of human glioma cells inoculated into the brain of rats by inhibiting the insulin-like growth factor-1receptor IGF-1R, suggesting that it may have the property of penetrating the blood-brain barrier (Yin S, et al., Neuro-Oncology,12:19-27,2010).

The picropodophyllotoxin is a cyclolignan compound and has the following structure:

picropodophyllin is considered to be a specific insulin-like growth factor-1receptor (IGF-1R) tyrosine kinase inhibitor and is used in the treatment of IGF-1R-induced diseases including various cancers, arteriosclerosis, psoriasis, restenosis following coronary angioplasty (WO02/102804), type 2 diabetes, nephropathy, retinopathy, glaucoma, thyroid eye disease (WO 2007/097707), rheumatoid arthritis, ulcers, multiple sclerosis, alzheimer's disease, asthma, eczema, post-transplant rejection (WO 2009/157858). Podophyllotoxins inhibit tumor cell growth and promote apoptosis by blocking IGF-1R-mediated intracellular signaling pathways (Girnita A, et al, Cancer Res, 64:236-242, 2004). The expression level of IGF-1R in various tumor cells is obviously increased, so that the picropodophyllin can target and inhibit the proliferation of tumor cells, and has less toxic and side effects on normal cells.

Although picropodophyllotoxin has high specificity and small toxic and side effects, our studies in mice show that it is metabolized rapidly in vivo and does not readily penetrate the blood brain barrier, perhaps being an important reason for its poor efficacy in human clinical trials. Therefore, there is a need to find compounds that are slower metabolizing in vivo, more potent in vivo, and more readily cross the blood-brain barrier for the treatment of IGF-1R dependent diseases, especially gliomas and cancers that are prone to brain metastases.

Disclosure of Invention

The technical problem to be solved by the invention is to design and synthesize a novel substituted picropodophyllotoxin derivative, prolong the half-life period of an IGF-1R inhibitor in vivo, improve the capability of the IGF-1R inhibitor to penetrate through a blood brain barrier and enhance the antitumor effect of the IGF-1R inhibitor.

In order to solve the technical problems, the invention provides the following technical scheme:

the substituted picropodophyllotoxin derivative provided by the invention has a structure shown in a general formula I:

and optical isomers thereof or salts thereof (including alkali metal salts, alkaline earth metal salts, acid addition salts, base addition salts and alkylated salts) acceptable as antitumor agents, wherein:

r is selected from F, -OH and CH₃CO-、CH₃COO-、CH₃CH₂COO-、CH₃CH₂CH₂COO-or substituted pyrazole ring derivatives are shown as general formula II:

in the formula:

b is

Any one of the above.

R₃Selected from H, C₁-C₄Any of alkyl, halogenated C₁-C₄Any one of alkyl groups, C₁-C₄Any of alkoxy, halogenated C₁-C₄Any one of alkoxy, unsubstituted or substituted five or six memberedA meta aryl group;

R₄selected from H, halogen, C₁-C₃Any of alkyl, halogenated C₁-C₃Any one of alkyl substituted at the 3 or 4 position of the pyrazole ring;

R₅、R₆selected from H, halogen, nitro, amino, cyano, C₁-C₄Any of alkyl, halogenated C₁-C₄Any one of alkyl groups, C₁-C₄Any of alkoxy, halogenated C₁-C₄Any one of alkoxy, R₅And R₆Synergistic or independent substitution at different positions of the intermediate five-membered ring of formula II;

x is selected from O, S, N (R)_h) Wherein R is_hSelected from H, C₁-C₅Any of alkyl, halogenated C₁-C₅Any one of alkyl groups, C₁-C₅Any of alkoxy, halogenated C₁-C₅Any one of alkoxy groups;

l is selected from C and N.

Wherein, the connecting position of the substituted pyrazole ring derivative group shown in the general formula II can be R₃、R₄、R₅、R₆And optionally a carbon atom in B.

Preferably, at least one hydrogen present in the compound of formula I is replaced by deuterium.

As a preferred scheme, the small-molecule inhibitor is a compound with a structure shown in general formula III and optical isomers thereof or salts which can be accepted by the anti-tumor agent:

the invention also provides the use of the small molecule inhibitor for preparing a medicament for preventing and treating different types of cancers, such as malignant melanoma; primary neuroectodermal tumors; gliomas, such as malignant gliomas and astrocytomas; lung cancer; prostate cancer; breast cancer; myeloproliferative and lymphoproliferative diseases, such as leukemia, lymphoma; tumors of the digestive tract, such as gastric cancer, colorectal cancer, liver cancer, and pancreatic cancer; gynecological cancers, such as ovarian cancer and cervical cancer. In view of the good blood brain barrier permeability of the compound, the compound is particularly suitable for treating tumors formed after the primary tumors are transferred to the brain.

In the case of tumors that are not completely IGF-1R dependent, the compounds of the present invention may potentiate the effects of other anticancer drugs. Thus, the invention also relates to the combination therapy of small molecule inhibitors with other anti-cancer drugs (including but not limited to chemotherapeutic drugs, targeted drugs, antibody drugs, nano-drugs, proteolytic targeting chimeras, nucleic acid drugs, etc.) or therapeutic approaches (e.g., surgical resection, radiation therapy, etc.).

The invention particularly relates to application of a small molecule inhibitor in preparation of a medicament for treating brain glioma.

The terms to which the present invention relates are described below:

the term "aryl" as used herein refers to an all-carbon monocyclic or fused polycyclic group of 5 to 12 carbon atoms having a completely conjugated pi-electron system. Non-limiting examples of aromatic rings are: benzene, naphthalene and anthracene rings. The aromatic ring may be unsubstituted or substituted. The substituents of the aromatic ring are selected from halogen, nitro, amino, C₁-C₆Alkyl radical, C₁-C₆Alkoxy, halo C₁-C₆Alkyl, halo C₁-C₆Alkoxy radical, C₃-C₆Cycloalkyl, halo C₃-C₆A cycloalkyl group.

The term "cycloalkyl" as used herein refers to a saturated monocyclic carbocyclic ring having 3 to 6 carbon atoms, unless a different number of atoms is indicated. "cycloalkyl" includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be unsubstituted or substituted. The substitution of cycloalkyl groups may optionally be substituted on any available carbon with one or more substituents selected from alkoxy, halogen, and haloalkyl, such as perfluoroalkyl.

The term "alkoxy" as used herein refers to an-O-alkyl group, wherein alkyl is as defined above. Examples of "alkoxy" as used herein include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy and tert-butoxy. Alkoxy groups may be unsubstituted or substituted. Alkoxy groups may be optionally substituted one or more times with halo, such as trifluoromethoxy.

The term "halogen" as used herein denotes fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

The invention has the following technical advantages:

1. novel substituted picropodophyllotoxin derivative structures;

2. the small molecule inhibitor provided by the invention has good blood brain barrier permeability.

3. The small molecule inhibitor related to the invention has longer half-life in the body.

Drawings

FIG. 1 is a graph showing the results of in vitro drug effect assays for compound 10 and its chiral separation compounds;

FIG. 2 is a graph of Western blot detection results collected after U87 tumor microspheres are treated for 24 hours by using a compound 7 and a compound 11, and the effects of the compound 7 and the compound 11 on a target protein IGF1R and a downstream signaling protein AKT are detected;

FIG. 3 is a Western blot assay of mouse brain tissue samples taken 3 hours after administration by gavage at 100mg/kg to test the permeability of the drug to the blood-brain barrier of the mouse;

FIG. 4 is a graph showing the results of experiments on the dynamic changes of the concentration of the drug in the plasma (upper) and brain tissue (lower) of mice at different time periods after administration, wherein the administration mode is gavage at a dose of 50mg/kg, and the stability of the drug in the plasma and brain tissue of mice is tested;

FIG. 5 is a graph of the body weight change of mice after administration of different doses, showing a short-term toxicity test of the drug to the mice;

FIG. 6 is a graph showing the results of the inhibition of proliferation of U87-Luc glioma cells inoculated in situ in the brain of nude mice by Compound 11 alone and in combination with temozolomide (left) and the effect on survival of mice (right);

FIG. 7 is a graph showing the effect of Compound 11 on the inhibition of brain metastasis of MDA-MB-231-Luc breast cancer cells inoculated into nude mice;

FIG. 8 is a graph showing the results of the inhibition of LLC lung cancer cell proliferation in immunized normal mice (left) and the effect on survival in mice (right) of Compound 11 alone and in combination with PD-1 antibody.

Detailed Description

The following examples are provided to illustrate the applicability of the present invention, and it will be understood by those skilled in the art that various modifications and substitutions can be made to the corresponding technical features according to the teachings of the prior art, and still fall within the scope of the present invention as claimed.

Example 1, relatively- (5R,5aS,8aR,9R) -2, 2-difluoro-9-hydroxy-5- (3,4, 5-trimethoxyphenyl) -5,8,8a, 9-tetrahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-6 (5aH) -one (Compound 1)

Step 1), synthesis of 4-vinyldihydrofuran-2 (3H) -one (intermediate 1.1):

to a mixture of 2-butene-1, 4-diol (206.4g, 2.34mol, 1.0eq) and triethyl orthoacetate (546.7g, 3.4mol, 1.4eq) was added catalytic hydroquinone (54.00g, 0.49mol, 0.2eq) and the mixture was heated to 120 ℃. The ethanol was continuously distilled off until no more ethanol was produced, the reaction temperature was raised to 150 ℃ and the reaction mixture was stirred for 48 hours. Intermediate 1.1 was collected by vacuum distillation (70-75 ℃, 2-3mmHg) as a colorless oil (170.0g, 65% yield).¹HNMR(400MHz,CDCl₃)(ppm)2.36(dd,J＝17.4Hz,8.7Hz,1H),2.69(dd,J＝17.7Hz,8.4Hz,1H),3.19-3.29(m,1H),4.01-4.14(m,1H),4.43-4.47(m,1H),5.17-5.23(m,2H),5.75-5.84(m,1H)。

Step 2), Synthesis of the corresponding- (3S,4R) -3- (hydroxy (3,4, 5-trimethoxyphenyl) methyl) -4-vinyldihydrofuran-2 (3H) -one (intermediate 1.2)

Intermediate 1.1(170.0g, 1.52mol, 1.0eq) was mixed with tetrahydrofuran (1500ml), stirred at-78 ℃ under nitrogen protection, and di-dropwise addedLithium isopropylamide (2.0M, 834ml, 1.67mol, 1.1eq) and the reaction mixture was stirred for 30 min. Under the same conditions, a mixed solution of 3,4, 5-trimethoxybenzaldehyde (327.6g, 1.67mol, 1.1eq) and tetrahydrofuran (1500ml) was added dropwise, stirred for another 3 hours, and then gradually heated to ambient temperature overnight. The reaction mixture was cooled to-78 ℃ with saturated NH₄And (4) quenching by Cl. The thus-produced mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by silica gel column chromatography (200-300 silica gel, petroleum ether: ethyl acetate 5/1-1/1) to give intermediate 1.2(190.1g, yield 41%) as a pale yellow solid.¹HNMR(400MHz,CDCl₃)(ppm)2.75-2.80(m,1H),2.91-2.96(m,0.5H),3.26-3.31(m,0.5H),3.84(s,3H),3.89(s,6H),3.92(t,1H),4.30-4.40(m,1H),4.86-4.93(m,2H),6.59(s,1H),6.61(s,1H)。

Step 3), synthesis of the compound relative to- (3R,4R) -3- ((R) - (2, 2-difluoro-6-hydroxybenzo [ d ] [1,3] dioxa-5-yl) (3,4, 5-trimethoxyphenyl) methyl) -4-vinyldihydrofuran-2 (3H) -one (intermediate 1.3):

intermediate 1.2(106.7g,0.41mol,1.0eq), 5-phenolic hydroxy-2, 2-difluoro-1, 3-benzodioxazole (107.6g,0.61mol,1.5eq) and dichloromethane (2000ml) were mixed and stirred, FeCl was added₃(34.06g,0.20mol,0.5eq), the reaction mixture was heated to 40 ℃ for 2-3 h, using saturated NaHCO₃Quench and extract the aqueous phase further with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate and purified by silica gel column chromatography (200-300 silica gel, petroleum ether: ethyl acetate 5/1-1/1) to give intermediate 1.3(153.4g, 45% yield) as a beige solid.¹HNMR(400MHz,CDCl₃)(ppm)3.02-3.06(m,1H),3.19-3.21(m,1H),3.83(s,6H),3.88(s,3H),4.04-4.07(m,1H),4.25-4.29(m,1H),4.73-4.75(d,J＝4.8Hz,1H),5.14-5.22(m,2H),5.55(brs,1H),5.80-5.84(m,1H),6.51-6.59(m,2H),6.82-6.96(m,1H),7.29(s,1H)。

Step 4), synthesis of relative-6- ((R) - ((3R,4R) -2-oxo-4-vinyltetrahydrofuran-3-yl) (3,4, 5-trimethoxyphenyl) methyl) -2, 2-difluorobenzo [ d ] [1,3] dioxa-5-yl trifluoromethanesulfonate (intermediate 1.4):

in the middle of continuous stirringTriethylamine (52.62g,0.52mol,2.0eq) was added to 1.3(120.0g,0.26mol,1.0eq) and methylene chloride (1500ml), and trifluoromethanesulfonic anhydride (110.0g,0.39mol,1.5eq) was added dropwise at a temperature of 10 ℃ and the mixture was stirred at the same temperature for 30 minutes. Saturated NaHCO for reaction₃Quench and extract the aqueous phase further with dichloromethane. The combined organic phases were washed successively with 2N HCl and brine, dried over anhydrous sodium sulfate and purified by silica gel column chromatography (200-300 silica gel, petroleum ether: ethyl acetate 6/1-3/1) to give intermediate 1.4(120.0g, yield 61%) as a white solid.¹HNMR(400MHz,CDCl₃)(ppm)2.97-3.06(m,1H),3.13-3.17(m,1H),3.84(s,6H),3.85(s,3H),4.03-4.08(m,1H),4.37-4.41(m,1H),4.64-4.66(d,J＝8.3Hz,1H),5.08-5.20(dt,2H),5.71-5.80(m,1H),6.58(s,2H),7.04(s,1H),7.23(s,1H)。

Step 5), synthesis of relative- (5R,5aR,8aS) -2, 2-difluoro-9-methylene-5- (3,4, 5-trimethoxyphenyl) -5,8,8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxa-6 (5aH) -one (intermediate 1.5):

to a continuously stirred mixture of intermediate 1.4(120.0g,0.20mol,1.0eq) and acetonitrile (1500ml) was added triphenylphosphine (15.83g,60.0mmol,0.3eq), K₂CO₃(82.93g,0.60mol,3.0 eq.) and Pd (OAc)₂(4.49g,20.0mmol,10 mol%) was heated to 80 ℃ for 20 hours. The reaction was filtered and the precipitate was eluted with dichloromethane. The combined organic phases were concentrated to dryness and purified by column chromatography on silica gel (200-300 silica gel, petroleum ether: ethyl acetate 5/1-1/1) to afford intermediate 1.5(76.13g, yield 61%) as a white solid.¹HNMR(400MHz,CDCl₃)(ppm)3.31-3.34(dd,1H),3.65-3.68(t,1H),3.76(s,6H),3.84(s,3H),4.21-4.24(dd,1H),4.57-4.59(m,2H),5.21-5.22(d,1),5.57(s,1H),6.31(s,2H),6.95(s,1H),7.27(s,1H)。

Step 6) Synthesis of comparative- (5aR,8aR,9R) -2, 2-difluoro-9- (3,4, 5-trimethoxyphenyl) -5a,6,8a, 9-tetrahydrofuran [3',4':6,7] naphtho [2,3-d ] [1,3] dioxo-5, 8-dione (intermediate 1.6):

OsO was added to intermediate 1.5(79.04g,0.18mol,1.0eq), 4-methylmorpholine N-oxide (168.7g,0.72mol,3.0eq) and dichloromethane (1200ml) with constant stirring₄(4.00g,157mmol,8 mol%) the mixture was stirred further at ambient temperature for 12 hours. Adding solid NaIO in batches into reactants₄(77.00g,0.36mol,2.0eq), stirring was continued for 1 hour. The reaction was incubated with saturated Na in an ice bath₂S₂O₃(300ml) quench and the aqueous phase is further extracted with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate and purified by silica gel column chromatography (200-300 silica gel, petroleum ether: ethyl acetate 5/1-1/1) to give intermediate 1.6(48.80g, yield 60%) as a white solid.¹HNMR(400MHz,CDCl₃)(ppm)3.37-3.42(m,2H),3.78(s,6H),3.82(s,3H),4.38-4.42(m,1H),4.79-4.80(d,J＝2.8Hz,2H),6.22(s,2H),7.02(s,1H),7.80(s,1H)。

Step 7) Synthesis of comparative- (5R,5aS,8aR,9R) -2, 2-difluoro-9-hydroxy-5- (3,4, 5-trimethoxyphenyl) -5,8,8a, 9-tetrahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-6 (5aH) -one (Compound 1):

intermediate 1.6(43.30g,98.9mmol,1.0eq) was mixed with diethyl ether (1500ml), stirred at-78 ℃ under nitrogen protection, LiAl (OtBu) was added dropwise₃(200ml,197mmol,2.0eq) and then gradually heated to ambient temperature overnight. The reaction was quenched with 2N HCl in an ice bath and the aqueous phase was further extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The residue was recrystallized from ethyl acetate to give compound 1(33.10g, yield 74%) as a white solid.¹HNMR(400MHz,CDCl₃)(ppm)7.38(s,1H),6.53(s,1H),6.47(s,2H),4.67(d,J＝0.9Hz,1H),4.55-4.43(m,2H),4.03(d,J＝6.4Hz,1H),3.89(s,3H),3.85(s,6H),3.26(q,1H),2.96(d,J＝6.9Hz,1H),2.66(m,1H)。

Example 2, relatively- (5R,5aS,8aR,9R) -2, 2-difluoro-8-oxa-9- (3,4, 5-trimethoxyphenyl) -5,5a,6,8,8a, 9-hexahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-5-yl acetate (Compound 2)

Compound 1(1.15g,2.55mmol,1.0eq) and dichloromethane (30ml) were mixed with constant stirringThen Et is added₃N (770mg,7.6mmol,3.0eq) and DMAP (310mg,2.55mmol,1.0eq), then AcCl (400mg,5.1mmol,2.0eq) was added under ice bath and the mixture was stirred at ambient temperature for 12 h. Dropwise addition of LiAl (OtBu)₃(200ml,197mmol,2.0eq) and then gradually heated to ambient temperature overnight. Saturated NH for reaction₄The Cl was quenched and the aqueous phase was further extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The residue was recrystallized from petroleum ether/ethyl acetate to give compound 2(810mg, yield 65%) as a white solid.¹HNMR(400MHz,CDCl₃)(ppm)7.02(s,1H),6.76(s,1H),6.41(s,2H),5.81(d,J＝6.3Hz,1H),4.48-4.34(m,3H),3.87(s,3H),3.84(s,6H),3.32(d,1H),3.01(m,1H),2.12(s,3H)。

Example 3, relatively- (5R,5aS,8aR,9R) -2,2, 9-trifluoro-5- (3,4, 5-trimethoxyphenyl) -5,8,8a, 9-tetrahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-6 (5aH) -one (Compound 3)

Compound 1(700mg,1.60mmol,1.0eq) and dichloromethane (20ml) were mixed with constant stirring, diethylaminosulfur trifluoride (520mg,3.20mmol,2.0eq) was added dropwise, and the mixture was stirred at ambient temperature for 12 hours. Saturated NaHCO for reaction₃Quench and continue stirring for 30 minutes. After separation of the organic phase, the aqueous phase is further extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (200-300 silica gel, dichloromethane: methanol 100/1-50/1) to give compound 3(130mg, yield 18%) as a white solid.¹HNMR(400MHz,CDCl₃)(ppm)7.27(s,1H),6.66(s,1H),6.44(s,2H),5.49-5.34(d,J＝50.8Hz,1H),4.60-4.47(m,2H),4.20(d,J＝5.0Hz,1H),3.88(s,3H),3.84(s,6H),3.28(m,1H),3.01(m,1H)。

Example 4, relatively- (5R,5aS,8aR,9R) -2, 2-difluoro-9-methoxy-5- (3,4, 5-trimethoxyphenyl) -5,8,8a, 9-tetrahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-6 (5aH) -one (Compound 4)

Compound 1(600mg,1.33mmol,1.0eq) and dichloromethane (15ml) were mixed with constant stirring, trimethyloxonium tetrafluoroborate (246mg,1.66mmol,1.2eq) was added and the mixture was stirred at ambient temperature for 20 h. The reaction was quenched with saturated NaHCO3 and stirred for an additional 30 minutes. After separation of the organic phase, the aqueous phase is further extracted with dichloromethane. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The residue was purified by silica gel column chromatography (200-300 silica gel, dichloromethane: methanol 100/1-50/1) to give compound 4(65mg, yield 11%) as a white solid. 1HNMR (400MHz, CDCl3) (ppm)7.16(s,1H),6.74(s,1H),6.41(s,2H),4.45-4.36(m,3H),4.34(d, J ═ 4.3Hz,1H),3.88(s,3H),3.83(s,6H),3.42(d,1H),3.31(s,3H),3.14(m, 1H).

Example 5, relatively- (5R,5aS,8aR,9R) -2, 2-difluoro-9-hydroxy-5- (3,4, 5-trimethoxyphenyl) -5,8,8a, 9-tetrahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-6 (5aH) -one-9-deutero (Compound 5)

Intermediate 1.6(400mg,0.89mmol,1.0eq) and methanol (10ml) were mixed with constant stirring, NaBD4(37.5mg,0.89mmol,1.0eq) was added portionwise and the mixture was stirred for 4 hours. The reaction was quenched with water, concentrated and dried. The residue was diluted with water and extracted with ethyl acetate. The combined organic phases were washed with saturated brine and dried over anhydrous sodium sulfate. The residue was recrystallized from petroleum ether/ethyl acetate to give compound 5(140mg, yield 35%) as a white solid. 1HNMR (400MHz, CDCl3) (ppm)7.38(s,1H),6.57(s,1H),6.47(s,2H),4.66(d, J ═ 9.8Hz,1H),4.47(m,1H),4.06(d, J ═ 6.2Hz,1H),3.89(s,3H),3.87(s,6H),3.27(d,1H),2.68(m,1H),2.44(s, 1H).

Example 6, (5R,5aS,8aR,9R) -2, 2-difluoro-9-hydroxy-5- (3,4, 5-trimethoxyphenyl) -5,8,8a, 9-tetrahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-6 (5aH) -one (Compound 6)

The compound 6 is obtained by chiral separation from the compound 1 by using a chiral column chromatography method. The main parameters of chiral column chromatography are as follows: instruments water SFC 200; a chromatographic column of Daicel Chiralcel AD,250 × 50mm I.D.,10 μm; the mobile phase A is CO2, and the mobile phase B is isopropanol; gradient B20%; the flow rate is 150 mL/min; back pressure of 100 bar; the column temperature is 38 ℃; the wavelength is 220 nm; the circulation time is 6.5 min; sample separation, compound is dissolved in methanol of 600 ml; the injection volume is 3ml each time.¹HNMR(400MHz,CDCl₃)(ppm)7.38(s,1H),6.53(s,1H),6.47(s,2H),4.67(d,J＝0.9Hz,1H),4.55-4.43(m,2H),4.03(d,J＝6.4Hz,1H),3.89(s,3H),3.85(s,6H),3.26(q,1H),2.96(d,J＝6.9Hz,1H),2.66(m,1H)；e.e.＝98.56％；[α]＝11.94°(MeOH,c＝0.88g/100ml)。

Example 7, (5R,5aS,8aR,9R) -2, 2-difluoro-9-hydroxy-5- (3,4, 5-trimethoxyphenyl) -5,8,8a, 9-tetrahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-6 (5aH) -one-9-deutero (Compound 7)

The compound 7 is obtained by chiral separation from the compound 5 by using a chiral column chromatography method. The main parameters of chiral column chromatography are as follows: instruments water SFC 200; a chromatographic column of Daicel Chiralcel AD,250 × 50mm I.D.,10 μm; the mobile phase A is CO2, and the mobile phase B is isopropanol; gradient B20%; the flow rate is 150 mL/min; back pressure of 100 bar; the column temperature is 38 ℃; the wavelength is 220 nm; the circulation time is 6.5 min; sample separation, compound is dissolved in methanol of 600 ml; the injection volume is 3ml each time.¹HNMR(400MHz,CDCl₃)(ppm)7.38(s,1H),6.57(s,1H),6.47(s,2H),4.66(d,J＝9.8Hz,1H),4.47(m,1H),4.06(d,J＝6.2Hz,1H),3.89(s,3H),3.87(s,6H),3.27(d,1H),2.68(m,1H),2.44(s,1H)。

Example 8, relatively- (5R,5aS,8aR,9R) -2, 2-methyl-9-hydroxy-5- (3,4, 5-trimethoxy-phenyl) -5,8,8a, 9-tetrahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-6 (5aH) -one (Compound 8)

Compound 8 was synthesized in a similar manner to the synthesis of compound 1 in example 1, using 5-phenolic hydroxy-2, 2-methyl-1, 3-benzodioxazole as the starting material.¹HNMR(400MHz,CDCl₃)(ppm)6.95(s,1H),6.48(s,2H),6.30(s,1H),4.43-4.53(m,3H),4.12(d,J＝5.2Hz,1H),3.87(s,3H),3.84(s,6H),3.25(dd,J＝5.2Hz,9.3Hz,1H),2.74-2.80(m,1H),2.18(brs,1H),1.68(s,3H),1.65(s,3H)。

Example 9, relatively- (5R,5aS,8aR,9R) -2, 2-difluoro-9-hydroxy-5- (3,4, 5-trimethoxy-phenyl) -5,8,8a, 9-tetrahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-6 (5aH) -one-5, 9-dideutero (Compound 9)

Compound 9 was synthesized in a similar manner to the synthesis of compound 5 in example 5, starting from 3,4, 5-trimethoxybenzaldehyde-d-formyl.¹HNMR(400MHz,CDCl₃)(ppm)7.38(s,1H),6.56(s,1H),6.47(s,2H),4.65(d,J＝9.8Hz,1H),4.47(dd,J＝6.1Hz,9.7Hz,1H),3.90(s,3H),3.86(s,6H),3.26(d,J＝9.4Hz,1H),2.66-2.70(m,1H),2.52(brs,1H)。

Example 10, p- (5R,5aS,8aR,9R) -2, 2-difluoro-9-hydroxy-5- (3,4, 5-tris (deuterated methoxy-d 3) phenyl) -5,8,8a, 9-tetrahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-6 (5aH) -one-9-deutero (Compound 10)

Compound 10 starting from 3,4, 5-trimethoxybenzaldehyde-d 9 was neutralized as in example 5Compound 5 was synthesized in a similar manner.¹HNMR(400MHz,CDCl₃)(ppm)7.38(s,1H),6.55(s,1H),6.46(s,2H),4.64(dd,J＝1.2Hz,9.8Hz,1H),4.45(dd,J＝6.0Hz,9.8Hz,1H),4.04(d,J＝6.3Hz,1H),3.26(dd,J＝6.4Hz,9.3Hz,1H),2.78(s,1H),2.66(dd,J＝6.0Hz,8.5Hz,1H)。

Example 11, (5R,5aS,8aR,9R) -2, 2-difluoro-9-hydroxy-5- (3,4, 5-tris (deuterated methoxy-d 3) phenyl) -5,8,8a, 9-tetrahydrofuro [3',4':6,7] naphtho [2,3-d ] [1,3] -dioxa-6 (5aH) -one-9-deutero (Compound 11)

The compound 11 is obtained by chiral separation from the compound 10 by using a chiral column chromatography. The main parameters of chiral column chromatography are as follows: instruments water SFC 200; a chromatographic column of Daicel Chiralcel AD,250 × 50mm I.D.,10 μm; the mobile phase A is CO2, and the mobile phase B is isopropanol; gradient B20%; the flow rate is 150 mL/min; back pressure of 100 bar; the column temperature is 38 ℃; the wavelength is 220 nm; the circulation time is 6.5 min; sample separation, compound is dissolved in methanol of 600 ml; the injection volume is 3ml each time.¹HNMR(400MHz,CDCl₃)(ppm)7.38(s,1H),6.55(s,1H),6.46(s,2H),4.64(dd,J＝1.2Hz,9.8Hz,1H),4.45(dd,J＝6.0Hz,9.8Hz,1H),4.04(d,J＝6.3Hz,1H),3.26(dd,J＝6.4Hz,9.3Hz,1H),2.78(s,1H),2.66(dd,J＝6.0Hz,8.5Hz,1H)。

The general design idea of the invention is to take picropodophyllotoxin (PPP) molecules as basic skeletons and replace hydrogen atoms at the left side 2 with fluorine atoms, thereby improving the ability of the molecules to penetrate the blood brain barrier; deuterium atoms replace hydrogen atoms at positions 4, and the half-life period of the molecule in the body is prolonged. In this connection, compound 11 (designated PB-020) is the final product of interest in the present invention, and compound 6 (designated PB-004) which performs only fluorine atom substitution and compound 7 (designated PB-016) which performs fluorine atom substitution with one deuterium atom substitution are the most important structural analogues. These 3 compounds are all chiral molecules, the corresponding chiral enantiomers of which are biologically inactive.

Biological activity detection of novel substituted picropodophyllotoxin derivatives

The following biological activity tests mainly compare the drug effects of compound 11 (named PB-020) and picropodophyllotoxin (PPP) on human glioma cells cultured in vitro and the indexes of the drug effects, such as the metabolic kinetics, the blood brain barrier permeability, the anti-tumor drug effects and the safety and the like of the drug effects in mice, and in part of the tests, compound 6 (named PB-004) and compound 7 (named PB-016) are used as controls.

In vitro pharmacodynamic test of compound

1. The test steps are as follows:

1) cell source: human brain astrocytoma/glioma cells U-87MG (abbreviated as U87) were purchased from cell bank of Chinese academy of sciences (Shanghai), cat # TCTU 138; human glioma cell DBTRG-05MG (DBTRG for short) is from American tissue culture Bank ATCC, Cat. number CRL-2020; human glioma cells KNS-81 are from AcceGen Biotech, cat # ABC-TC 0535.

2) Cell culture and passage: the above cells were all cultured adherently in a medium containing complete medium [ DMEM-containing high-sugar basic medium (# SH30243.01B, Hyclone) with 10% FBS (#1101-500, Shanghai prairie) and Penicilin-Streptomyces double antibody (# SV30010, Hyclone) at the final concentration]6cm petri dish (#430166, Corning) or T75 flask (#3276, Corning), the petri dish (flask) was placed at 37 ℃ with 5% CO₂And, saturated humidity cell culture box (#3111, Thermo Fisher Scientific). During passage, the medium was aspirated, washed 2 times with PBS phosphate buffered saline (# GNM-10944, Ginuo Hangzhou), and then a suitable amount of 0.25% trypsin-0.02% EDTA (#25200-072, Gibco) was added, and the culture dish (bottle) was shaken to cover the cells uniformly and observed under a phase contrast microscope. When most cells retract and become round and fall off by gentle shaking, complete medium with twice of the volume of pancreatin is quickly added for termination, and the cells are gently blown into single cells. The cell suspension was transferred to a centrifuge tube of appropriate size and centrifuged at 800rpm for 5 min. Discard the supernatant, resuspend the cell pellet with fresh complete culture, blow back into single cells, subculture to new dishes (flasks) at a ratio of 1:3-1:6 and replenish the complete medium. Standing at 37 deg.C for 5% CO₂Cultured in a cell culture box.

3) Cell dosing treatment: will be provided withEach of the cells was digested, counted, inoculated at a density of 5000 cells/200. mu.l of culture medium into each well of a 96-well cell culture plate (#3988, Corning), and placed at 37 ℃ with 5% CO₂The cells are cultured in the cell culture box for 24 hours to be fully attached to the wall. Complete cultures containing the serially diluted test compounds (3 replicate wells per compound concentration) and DMSO (# D5879, Sigma-Aldrich) solvent control were then replaced with the original cultures and incubated for a further 72 h.

4) Pharmacodynamic assay and statistics: cells treated with the above compounds were first subjected to morphological observation and photographic recording using an inverted phase contrast microscope (X71, Olympus). Then, the original culture solution was replaced with phenol red-free culture solution containing CCK-8 detection reagent (# E606335, Shanghai Biotech), and the culture was continued in an incubator for 2 hours. The absorbance (OD) of OD450nm was then measured on a multifunctional microplate reader (168-. The cell survival rate (cell survival rate) or cell growth rate (cell growth rate) after treatment of the cells with the test compound is calculated by the following formula: survival rate is the OD value of the drug adding group/the OD value of the control group multiplied by 100 percent; the inhibition rate (growth inhibition rate) of the compound on cell proliferation is calculated by the formula: the inhibition rate is (control group OD value-addition group OD value)/control group OD value multiplied by 100%. Further, IC50 was calculated for each compound in SPSS based on the inhibition values.

2. And (3) test results:

fig. 1 shows the results of in vitro pharmacodynamic assays of compound 10 and its chiral compound, which can identify compound 11 as the enantiomer of compound 10 that exerts anti-glioma efficacy.

The in vitro pharmacodynamic comparative statistics of PB-020 and PPP, PB-004, PB-016 are shown in Table 1

TABLE 1 in vitro pharmacodynamic assay results for Compound 11 and analogs thereof

Note: data are mean ± standard deviation of the values of IC50 from 3 independent experiments,

all 3 human glioma cell lines are cultured adherent.

PPP: picropodophyllotoxin; PB-004: compound 6; PB-016: chemical combination

An object 7; PB-020: compound 11

The results show that the compound 11(PB-020) and the analogues thereof have the proliferation inhibition effect close to PPP on human glioma cell lines cultured adherently in vitro, and the IC50 is about 1 mu M.

Secondly, the verification of the action mechanism of the compound

1. The test steps are as follows:

1) cell source: human brain astrocytoma/glioma cells U-87MG (abbreviated as U87) was purchased from cell bank of Chinese academy of sciences (Shanghai), cat # TCTU 138.

2) Culturing tumor microspheres: u87 cells were cultured adherently in complete medium [ DMEM-containing high-sugar basal medium (# SH30243.01B, Hyclone) with 10% final concentration of FBS (#1101-500, Shanghai prairie) and Penicilin-Streptomyces double antibody (# SV30010, Hyclone)]In a 6cm petri dish (#430166, Corning) or a T75 flask (#3276, Corning), the petri dish (flask) was incubated in a cell incubator (#3111, Thermo Fisher Scientific) at 37 ℃, 5% CO2, saturated humidity. U-87MG cells in logarithmic growth were trypsinized, harvested and resuspended in serum-free DMEM. Counting by trypan blue staining method with cell counter (the viable cell ratio is above 90%), and adding 10⁵The cells were seeded in 6cm dishes and 5ml of serum-free stem cell culture medium (EGF 20 ng/ml; bFGF 20 ng/ml; LIF 10 ng/ml; B271: 50; PS 1:100) was cultured in a cell incubator at 37 ℃ and saturated humidity, 5% CO 2. The formation of typical tumor microspheres can be observed after 3-5 days of culture with 1ml of fresh serum-free stem cell culture medium every 3 days.

3) Cell dosing treatment: after the U-87MG tumor microspheres are formed, replacing the original culture solution with the gradient diluted fresh culture solution containing various compounds to be detected and DMSO solvent control, and continuously culturing for 1-3 days.

4) IGF1R signaling pathway biomarker detection: collecting the suspension of U-87MG tumor microspheres treated by the compound for 1-3 days, centrifuging, removing the supernatant culture solution by suction, washing with ice-bath precooled PBS for 2 times, and sucking the liquid. Add 200. mu.l of cell lysate (# P0013, Byunyan day), shake vigorously for 30s, stand on ice for 5min, repeat 3 times. Cell lysis sample is centrifuged at 13000rpm and 4 ℃ for 6min, the supernatant is mixed with 4 XLaemmli loading buffer solution (# 161-. 4-15% pre-gradient gel (456-8084, Bio-Rad) was loaded into the electrophoresis chamber and sufficient 1 XSDS-PAGE running buffer was added. Mu.l of the above cell lysate protein sample was added using a 20. mu.l pipette. Covering the electrophoresis tank with a cover, switching on a power supply, performing electrophoresis at 80V for about 30min, adjusting the voltage to 120V when bromophenol blue in the sample is pressed into a thin line at the boundary line of the concentrated gel and the separation gel, and adjusting the electrophoresis duration according to the sizes of the target protein and the reference protein bands. Pouring the precooled 1 Xmembrane-transferring buffer solution into a container with a proper size, assembling a sandwich structure of a foam pad-filter paper-glue-PVDF membrane-filter paper-foam pad according to the instruction, and filling the sandwich structure into an electrophoresis tank. Adding ice blocks and carrying out ice bath on the whole electrophoresis tank, and connecting a power supply to carry out membrane conversion at 250mA for 2 h. PVDF membrane (IPVH00010, Millipore) was blocked in 5% skimmed milk powder formulated from 1 XTBST for 1h at room temperature. Then, incubation (1 h incubation at room temperature respectively) of different antibodies (anti-pIGF1R-Y1135/Y1136, #3024S, CST; anti-pAKT-T308, #13038, CST; anti-pAKT-S473, #4060, CST; anti-pIRS1-S302, #2384, CST; anti-GAPDH [ HRP ], # A00191-40, GenScript) and secondary antibodies (anti-Mouse IgG-linked antibody, #7076, CST; anti-Rabbit IgG-linked antibody, #7074, CST) with membrane washing (1 × TBST membrane washing 3 times, 5min each) was performed in sequence. Finally, the PVDF membrane was placed in the middle of the plastic membrane, added to the ECL membrane for reaction for 3min, covered with another layer of plastic membrane, and exposed in a full-automatic chemiluminescence/fluorescence image analysis system (5200-Multi, Nature).

2. And (3) test results:

the results are shown in fig. 2, compound 11(PB-020) and compound 7(PB-016) both have dose-dependent inhibitory effects on a series of IGF1R signaling pathway biomarkers in U87 tumor microspheres, suggesting that the drug targets are IGFIR, and they both inhibit U87 cell proliferation efficiently by inhibiting IGF1R downstream effector molecules. Of particular note are: PB-020 also significantly reduces the protein content of its target protein IGF1R (total IGF1R, first row), possibly resulting from its promotion of IGF1R endocytosis and degradation.

Third, compound crossing blood brain barrier test (biomarker detection)

1. The test steps are as follows:

1) the experimental animal source is as follows: SPF grade male BALB/c nude mice were ordered from Shanghai Spirochai with week age of 6 to 8 weeks.

2) And (3) administration treatment of experimental animals: test compounds were dissolved in DMSO (# D5879, Sigma-Aldrich) and prepared as 50mg/ml stock solutions. The test compound stock was diluted with PBS and mice were gavaged at a dose of 100mg/kg, and the experiment was repeated with 3 mice per test compound. Fasting was overnight before dosing.

3) Preparation of a detection sample: after 3 hours of administration, mice were sacrificed by cervical dislocation, the right brains were removed, 800 μ l of precooled cell lysate (# P0013, picnic, protease inhibitor #78444, Thermo FisherScientific were added freshly), homogenized thoroughly on ice, centrifuged at 12000rpm for 10 minutes, and the supernatants were removed. The protein concentration of the sample was measured by BCA method, and a corresponding amount of 4 × Laemmli loading buffer (# 161-.

4) IGF1R signaling pathway biomarker detection: 4-15% pre-gradient gel (456-8084, Bio-Rad) was loaded into the electrophoresis chamber and sufficient 1 XSDS-PAGE running buffer was added. Mu.l of the above cell lysate protein sample was added using a 20. mu.l pipette. Covering the electrophoresis tank with a cover, switching on a power supply, performing electrophoresis at 80V for about 30min, adjusting the voltage to 120V when bromophenol blue in the sample is pressed into a thin line at the boundary line of the concentrated gel and the separation gel, and adjusting the electrophoresis duration according to the sizes of the target protein and the reference protein bands. Pouring the precooled 1 Xmembrane-transferring buffer solution into a container with a proper size, assembling a sandwich structure of a foam pad-filter paper-glue-PVDF membrane-filter paper-foam pad according to the instruction, and filling the sandwich structure into an electrophoresis tank. Adding ice blocks and carrying out ice bath on the whole electrophoresis tank, and connecting a power supply to carry out membrane conversion at 250mA for 2 h. PVDF membrane (IPVH00010, Millipore) was blocked in 5% skimmed milk powder formulated from 1 XTBST for 1h at room temperature. Then, incubation (incubation for 1h at room temperature) of different antibodies (anti-pIGF1R-Y1135/Y1136, #3024S, CST; anti-pAKT-T308, #13038, CST; anti-pAKT-S473, #4060, CST; anti-GAPDH [ HRP ], # A00191-40, GenScript) and secondary antibodies (anti-Mouse IgG HRP-linked antibody, #7076, CST; anti-Rabbit IgG HRP-linked antibody, #7074, CST) with washing membrane (1 × TBST washing membrane 3 times, 5min each) was performed in sequence. Finally, the PVDF membrane was placed in the middle of the plastic membrane, added to the ECL membrane for reaction for 3min, covered with another layer of plastic membrane, and exposed in a full-automatic chemiluminescence/fluorescence image analysis system (5200-Multi, Nature).

2. And (3) test results:

the results are shown in fig. 3, and compound 11 and compound 7 orally administered to mice down-regulated key biomarkers in IGF1R signaling pathways, such as phosphorylated IGF1R/AKT, in brain tissue within 3 hours, suggesting that both can cross the blood brain barrier of mice. PPP, however, failed to effectively down-regulate key biomarkers in the IGF1R signaling pathway, suggesting that it cannot effectively cross the blood brain barrier in mice.

Fourthly, pharmacokinetics of the Compound

1. The test steps are as follows:

1) the experimental animal source is as follows: nude mice of male or female BALB/c strain were ordered from shanghai slaike laboratory animals llc with a week age of 7 weeks.

2) And (3) administration treatment of experimental animals: test compounds were dissolved in DMSO (# D5879, Sigma-Aldrich) and prepared as 50mg/ml stock solutions. Test compounds were finally dissolved in 10% DMSO + 90% corn oil (# C8267-500ML, Sigma-Aldrich) mice were gavaged at a dose of 50 mg/kg.

3) Preparation of a detection sample: after the mice are respectively dosed for 1,3,6,12 or 24 hours, injecting 4% chloral hydrate aqueous solution into the abdominal cavity according to the dosage of 250 mul/20 g body weight, anesthetizing the mice, taking 300 mul of blood from carotid artery to a heparin tube, mixing uniformly, centrifuging at 4000rpm5 minutes, taking 200 mul of supernatant, adding 600 mul of acetonitrile, mixing uniformly and ultrasonically for 20 minutes, centrifuging at 12000rpm for 10 minutes, standing the supernatant overnight at 4 ℃, centrifuging at 12000rpm for 10 minutes, and taking the supernatant as a blood plasma detection sample; taking about 0.3-0.5g of whole brain, quickly rinsing with precooled PBS 3 times, adding 1ml of acetonitrile for homogenate (PRIMA handheld homogenizer PB100, 35000rpm, 1 minute), carrying out ultrasonic treatment for 20 minutes, centrifuging at 12000rpm for 10 minutes, standing the supernatant overnight at 4 ℃, centrifuging at 12000rpm for 10 minutes, and taking the supernatant as a brain tissue detection sample.

4) And (4) HPLC detection: the "plasma test sample" and the "brain tissue test sample" were each subjected to standard control, HPLC mobile phase blank control, and the like, and the content of the test compound in the sample was analyzed by a high performance liquid chromatograph (Hitachi Chromaster-5430 detector, Hitachi Chromaster-5310 column incubator, Hitachi Chromaster-5210 autosampler, Hitachi Chromaster-5110 pump).

2. And (3) test results:

the results are shown in FIG. 4, where compound 11(PB-020) and compound 7(PB-016) both have much higher peak concentrations in plasma and brain tissue than PPP, suggesting that PPP is metabolized faster in mice and hardly penetrates the blood-brain barrier, probably because PPP is almost metabolized before it can penetrate the blood-brain barrier. The peak concentration of compound 11 was again significantly higher in both plasma and brain tissue than compound 7. In plasma and brain tissue, compound 7 peaked 1 hour after dosing, while compound 11 peaked 3 hours after dosing. Thus, pharmacokinetic results suggest that compound 11 is metabolized more slowly in both plasma and brain tissue than compound 7 and is therefore more stable.

Fifth, evaluation of short-term safety of Compounds

BALB/c male nude mice were gavaged with four doses of compound 11(PB-020) at 50,100,200,400mg/kg, administered 1 time per day, weighed on

days

2,4,6,8, respectively, and observed for morphology and behavior, using DMSO as a control.

The results are shown in fig. 5, and within 8 days after the gavage administration, no weight loss and obvious morphological and behavioral abnormalities were observed in all the dose groups of mice, suggesting that compound 11 has better safety. The following in vivo pharmacodynamic tests in mice were performed using two lower doses of compound 11, 50mg/kg and 100 mg/kg.

Sixthly, compound in vivo pharmacodynamic test

(I) inhibition of compound 11 single drug and compound 11+ temozolomide on proliferation of human glioma cells inoculated in situ to brain of nude mice

1. The test steps are as follows:

1) modeling and grouping: 40 female BALB/c strain nude mice of 10 weeks old are selected, and 5 μ l of 3x 10-containing solution is injected into cranial cavity of each mouse⁵A suspension of U87-Luc cells (Cat. # BW124577, Perkin Elmer) was recovered for several days and then examined for fluorescence intensity of the inoculated U87-Luc cells by in vivo imaging to determine proliferation. Based on the fluorescence intensity of U87-Luc cells, successfully inoculated mice were randomly divided into 4 groups of 8 mice each for administration experiments.

2) Administration: vehicle group, each mouse was gavaged with 200. mu.l of 10% DMSO + 90% corn oil (# C8267-500ML, Sigma-Aldrich) 1 time a day for 14-19 consecutive days (until the mice died); PB-020 group: PB-020 (Compound 11) was dissolved in 10% DMSO + 90% corn oil and gavaged at a dose of 50mg/kg in a total volume of 200 μ l 1 time a day for 17-21 days (until death of the mice); TMZ group: dissolving TMZ (temozolomide) in PBS, and performing intraperitoneal injection on the mice at a dose of 20mg/kg and a total volume of 200 mu l, wherein the administration is performed 1 time per day for 5 days continuously; PB-020+ TMZ group: the single medicine is respectively administrated according to the administration mode and the dosage.

3) And (3) evaluating the drug effect: after the administration of the mouse is started, measuring the fluorescence intensity of the inoculated U87-Luc cells by using a living body imaging method every 3 days so as to judge the proliferation condition of the U87-Luc cells in the brain of the mouse and the inhibition degree of the drug to be detected on the proliferation; weighing and recording the weight of the mice every day, and suspending administration when the weight of the mice is reduced by more than 10% in one day; observing and recording the morphology and behavior characteristics of the mice every day; the mice were recorded for death and survival curves were developed.

2. And (3) test results:

as shown in FIG. 6, compound 11(PB-020) and TMZ both effectively inhibited the rapid proliferation of U87 cells in the mouse brain within 1 week of administration. Over 1 week, compound 11 had significantly reduced potency, while TMZ continued to be effective and was able to reduce the volume of the transplanted tumor to a small extent. Within 10 days of administration, the combination of compound 11 and TMZ showed more significant effect than single agent, and after more than 10 days, the effect of TMZ was close to that of single agent. Compound 11 was effective in prolonging survival of transplanted tumor mice compared to the control group, and p was 0.02. In this experimental model, since TMZ has excellent drug efficacy alone, the drug efficacy of compound 11 in combination therewith is difficult to be effectively exhibited.

Inhibition of transfer of human-derived breast cancer cells into brain by Compound 11 inoculated into nude mice

1. The test steps are as follows:

1) modeling and grouping: 24 female Balb/c nude mice of 5 weeks old are bred adaptively for 5-7 days, and the experiment is started. Recovering MDA-MB-231-Luc cells, and performing amplification culture. After the number of cells meets the experimental requirements, digesting and collecting the cells, mixing the cells into PBS to prepare single cell suspension, and adjusting the cell density to be 2 multiplied by 10⁶Ml, used for experiments. Mice were anesthetized with avertin and cells were injected in the left ventricle. The cell inoculum size was 100. mu.l/cell. D-Luciferin was intraperitoneally injected after inoculation, IVIS examined the inoculation effect, and 20 were selected for the experiment. And returning to the IVC system for continuous feeding after the mice revive. Live imaging was performed 14 days after inoculation and mice were randomized into 2 groups of 8 mice each, depending on imaging effect.

2) Administration: in the Vehicle group, each mouse was gavaged with 100. mu.l of 10% DMSO + 90% corn oil 1 time a day for 30 consecutive days; PB-020 group: PB-020 (Compound 11) was dissolved in 10% DMSO + 90% corn oil and gavage was performed on mice at a dose of 50mg/kg in a total volume of 100. mu.l 1 time a day for 30 consecutive days.

3) And (3) evaluating the drug effect: after the mice begin to be dosed, living body imaging is carried out on

days

0,5,16,24 and 30, and the fluorescence intensity of MDA-MB-231-Luc cells inoculated in the brains of the mice is measured, so that the transfer condition of the MDA-MB-231-Luc cells to the brains of the mice and the inhibition degree of the compound 11 on the transfer/proliferation of the MDA-MB-231-Luc cells are judged; weighing and recording the weight of the mice every day, and suspending administration when the weight of the mice is reduced by more than 10% in one day; the morphology and behavior characteristics of the mice were recorded with daily observation.

2. And (3) test results:

as shown in FIG. 7, MDA-MB-231-Luc cells transferred to the brain of mice proliferated relatively slowly, and reached the rapid proliferation stage 24 days after administration. In a rapid proliferation period of 24 days to 30 days after administration, the compound 11(PB-020) can remarkably inhibit MDA-MB-231-Luc cell proliferation. Since the transfer model has a very low lethality, a survival curve cannot be prepared.

(III) inhibitory Effect of Compound 11 in combination with PD-1 antibody on proliferation of Lung cancer cells in Normal immunized mice

1. The test steps are as follows:

1) modeling and grouping: 32 5-week-old C57BL/6 females were immunized against normal mice and were acclimatized for 1 week for experimental use. The LLC lung cancer cells of the mouse are cultured conventionally, and are prepared into the LLC lung cancer cell with the density of 1x10 after meeting the experimental requirements⁷A single cell suspension in ml for use. LLC cells were inoculated into the right underarm of C57BL/6 mice and returned to continuous feeding. When the tumor grows to 50-150mm³At the time, the mice were randomly divided into 4 groups of 8 mice each.

2) Administration: vehicle group, each mouse was gavaged with 200. mu.l of 10% DMSO + 90% corn oil (# C8267-500ML, Sigma-Aldrich) 1 time per day, and the administration was continued until the mice died; PB-020 group: PB-020 (Compound 11) was dissolved in 10% DMSO + 90% corn oil and gavaged at 50mg/kg in a total volume of 200 μ l 1 time a day until death; PD1 group: mu.g anti-mouse PD-1(RMP1-14, Bio X Cell) was dissolved in 100. mu.l PBS and intraperitoneally injected 1 time a day and 1 time a 3 day per mouse; PB-020+ PD1 group: the single medicine is respectively administrated according to the administration mode and the dosage.

3) And (3) evaluating the drug effect: after the mice began to dose, tumor volume was followed 2 times a week; weighing and recording the weight of the mice every day, and suspending administration when the weight of the mice is reduced by more than 10% in one day; observing and recording the morphology and behavior characteristics of the mice every day; mice were recorded for death time with tumor volumes exceeding 2000mm³And (3) determining that the mice die according to the ethical requirements of the animals until all the mice in the Vehicle group die, and drawing a survival curve of the mice.

2. And (3) test results:

the result is shown in figure 8, the compound 11(PB-020) single drug can effectively inhibit the proliferation of the murine LLC lung cancer cells in an immune normal mouse, and the effect is slightly better than that of the PD-1 antibody single drug; the compound 11 and the PD-1 antibody have a certain synergistic effect, can effectively inhibit LLC cell proliferation, and obviously prolongs the survival period of mice.

In conclusion, the invention designs and synthesizes a novel small molecule inhibitor compound 11(PB-020) targeting insulin-like growth factor-1receptor and a series of analogues thereof. Compared with the existing picropodophyllotoxin in clinical test stage, the compound 11 has higher blood brain barrier permeability and longer in vivo half-life while maintaining the action mechanism and biochemical characteristics. When used alone, the compound 11 can effectively inhibit the proliferation and brain metastasis of a plurality of nude mouse transplantable tumors (particularly glioma); the compound 11 and temozolomide are used together, so that the proliferation of in-situ transplantation glioma cells can be effectively inhibited, and the survival period of a mouse is remarkably prolonged; the compound 11 and the PD-1 antibody have certain synergistic effect, can effectively inhibit the multiplication of LLC cells in an immune normal mouse body, and obviously prolongs the survival time of the mouse.

Finally, it should be noted that the above-mentioned list is only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims

1. A novel small molecule inhibitor of an insulin-like growth factor-1receptor is characterized in that the small molecule inhibitor is a compound with a structure shown in a general formula I and an optical isomer thereof or an antitumor acceptable salt thereof:

wherein R is selected from F, -OH and CH₃CO-、CH₃COO-、CH₃CH₂COO-、CH₃CH₂CH₂COO-or substituted pyrazole ring derivative groups, e.g.Represented by the general formula II:

in the formula:

b is

Any one of the above.

R₃Selected from H, C₁-C₄Any of alkyl, halogenated C₁-C₄Any one of alkyl groups, C₁-C₄Any of alkoxy, halogenated C₁-C₄Any one of alkoxy, unsubstituted or substituted five-or six-membered aryl.

R₄Selected from H, halogen, C₁-C₃Any of alkyl, halogenated C₁-C₃Any of the alkyl groups, substituted at the 3 or 4 position of the pyrazole ring.

R₅、R₆Selected from H, halogen, nitro, amino, cyano, C₁-C₄Any of alkyl, halogenated C₁-C₄Any one of alkyl groups, C₁-C₄Any of alkoxy, halogenated C₁-C₄Any one of alkoxy, R₅And R₆And (3) synergistic or independent substitution is carried out on different positions of the intermediate five-membered ring in the general formula II.

X is selected from O, S, N (R)_h) Wherein R is_hSelected from H, C₁-C₅Any of alkyl, halogenated C₁-C₅Any one of alkyl groups, C₁-C₅Any of alkoxy, halogenated C₁-C₅Any of alkoxy groups.

L is selected from C and N.

2. The novel small molecule inhibitor according to claim 1, wherein at least one deuterium atom is present in the compound of formula i.

3. The novel small-molecule inhibitor according to claim 1, which is characterized in that the small-molecule inhibitor is a compound having a structure shown in general formula III and an optical isomer thereof or an antitumor-acceptable salt thereof:

4. the novel small molecule inhibitor according to claim 1, wherein said antineoplastic acceptable salt is an alkali metal salt, an alkaline earth metal salt, an acid addition salt, a base addition salt, or an alkylated salt.

5. Use of a novel small molecule inhibitor of the insulin-like growth factor-1receptor according to any one of claims 1 to 4 for the preparation of a medicament for the prevention and treatment of cancer.

6. The use according to claim 5, wherein the cancer is an IGF-1R dependent disease, including malignant melanoma, primary neuroectodermal tumors, glioma, lung cancer, prostate cancer, breast cancer, myeloproliferative and lymphoproliferative diseases, tumors of the digestive tract, gynecological cancers and the like.

7. Use according to claim 6, wherein the glioma is a glioblastoma or astrocytoma. Myeloproliferative and lymphoproliferative disorders are leukemia and lymphoma; the digestive tract tumor is gastric cancer, colorectal cancer, etc.

8. The use of claim 5, wherein the medicament further comprises other anticancer drugs, including chemotherapeutic drugs, targeted drugs, antibody drugs, nano-drugs, proteolytic targeting chimeras, nucleic acid drugs, and the like.