CN106674327A - Dolastatin 10 derivative and its preparation method and application - Google Patents

Abstract

The invention belongs to the technical field of medicine preparation, and relates to a dolastatin 10 derivative I, its preparation method and its application. Dolastatin 10 derivative I of the present invention; wherein, R ₁ , R ₂ and R ₃ are independently selected from hydrogen, nitro, halogen, trifluoromethyl or C ₁ -C ₆ alkoxy. The invention also discloses a preparation method of Dolastatin 10 derivative I and the application of Dolastatin 10 derivative I in the preparation of antitumor drugs. The Dolastatin 10 derivative I of the present invention is a novel compound with antitumor activity, which opens a new research direction for the development of anticancer drugs and expands the research scope of Dolastatin 10 derivative anticancer drugs.

Description

A kind of Dolastatin 10 derivative, its preparation method and application

technical field

The invention belongs to the technical field of medicine preparation, and relates to a Dolastatin 10 derivative, in particular to a Dolastatin 10 derivative I, its preparation method and its application.

Background technique

Dolastatin 10 has significant antitumor activity, but its application is limited due to strong toxicity and side effects. The compound leads to cell cycle arrest and apoptosis by inhibiting tubulin and microtubule-dependent GTP hydrolysis. Researchers at Pfizer studied the way in which dolastatin 10 binds to microtubules by synthesizing a series of analogs of dolastatin 10, and co-crystallized complexes showed that the compound binds to the junction of microtubule α,β-subunits, N-terminal Close to the vinblastine binding region, C-terminal close to the GTP/GDP exchange binding site. In addition to having microtubule inhibitory effects, studies have also found that dolastatin10 and its analogs also have anti-angiogenic effects. A series of C-terminal modification compounds represented by auristatin-PE and auristatin-PYE have reduced toxicity compared with dolastatin 10, but they have not been clinically used due to obvious side effects in clinical trials. The clinical application of dolastatin 10 and its synthetic analogues is limited. The research on dolastatin 10 has brought a breakthrough thanks to the research results of Miyazaki et al. They found that removal of one methyl group in the N-terminal dolavalin yielded only a slight decrease in the activity of the demethyl analog 6 compared to dolastatin 10. Using this achievement, Senter et al. designed ADCs by linking demethylated analogues to mAbs via a linker chain. Based on this design concept, ADCs with high activity and clinical use were discovered

Therefore, the significant antitumor activity of Dolastatin 10 derivatives and their compositions, and the development of them as new anticancer drugs or new drugs used in combination with other anticancer drugs will have a large market and will also generate greater social benefits and economic benefits.

Contents of the invention

The technical problem to be solved by the present invention is to provide a dolastatin 10 derivative I which is completely different from the prior art and has an anti-tumor effect, as well as its preparation method and application. The dolastatin 10 derivative I of the present invention is a novel compound with antitumor activity, which opens a new research direction for the development of anticancer drugs and expands the research scope of dolastatin 10 derivative anticancer drugs.

The present invention provides dolastatin 10 derivative I of the following formula;

Wherein, R ₁ is independently preferably selected from hydrogen, halogen or C ₁ -C ₆ alkoxy or alkyl. The C ₁ -C ₆ alkoxy or alkyl are preferably methoxy and isopropyl.

Among them, R ₂ is preferably selected from hydrogen, halogen or C ₁ -C ₆ alkoxy.

Said R ₁ , R ₂ and R ₃ are independently preferably selected from hydrogen or halogen. Said R ₁ , R ₂ and R ₃ are independently more preferably selected from fluorine or chlorine, and R ₁ , R ₂ and R ₃ are not hydrogen at the same time.

The dolastatin 10 derivative I is preferably as follows:

R1, R2, R3 are hydrogen;

Or, R1 is fluorine, R2, R3 are hydrogen;

Alternatively, R1 is hydrogen, R2 is fluorine and R3 is hydrogen;

Alternatively, R1 is hydrogen, R2 is chlorine and R3 is fluorine;

Alternatively, R1 is hydrogen, R2 is hydrogen and R3 is chlorine;

Alternatively, R1 is isopropyl, R2 is hydrogen and R3 is hydrogen;

Alternatively, R1 is methoxy, R2 is hydrogen and R3 is chlorine;

Alternatively, R1 is hydrogen, R2 is hydrogen and R3 is fluorine;

Alternatively, R1 is hydrogen, R2 is hydrogen and R3 is tert-butyl;

Alternatively, R1 is hydrogen, R2 is hydrogen and R3 is phenyl.

The present invention also provides a preparation method of dolastatin 10 derivative I,

Wherein, R ₁ , R ₂ and R ₃ are all as above.

Described condensation reaction can be this type of condensation reaction conventional in the art. The present invention particularly preferably follows reaction conditions:

The solvent is preferably a polar aprotic solvent. The polar aprotic solvent is preferably THF, pyridine, acetonitrile, dichloromethane, chloroform, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone and One or more of dimethyl sulfoxide.

The condensing agent can be a dehydrating agent commonly used in this type of reaction in the art, preferably dicyclohexylcarbodiimide (DCC), 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide Imine hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), N,N'-carbonyldiimidazole (CDI), O-(7-azabenzotriazol-1-yl)-N ,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), formazan One or more of base phosphonic anhydride, ethyl phosphonic anhydride, n-propyl phosphonic anhydride, propylphosphonic acid cyclic anhydride (T3P) and diethyl benzotriazolyl phosphate, more preferably 1-ethyl-3- [3-(Dimethylamino)propyl]carbodiimide hydrochloride and/or 1-hydroxybenzotriazole.

The base can be an organic base and/or an inorganic base. The organic base is preferably one or more of pyridine, triethylamine, N,N-dimethylaminopyridine and diisopropylethylamine (DIEA).

The reaction temperature of the condensation reaction is preferably -15°C to 100°C, more preferably 0°C to 30°C

The progress of the condensation reaction can be monitored by TLC or HPLC. Generally, the end point of the reaction is when the compound II disappears. Preferably, the reaction ends after 1 to 120 hours, and more preferably, the reaction ends after 24 to 48 hours.

After the condensation reaction is finished, a post-treatment process may also be included to further purify the obtained dolastatin 10 derivative I. The post-treatment process can be a conventional post-treatment process in the art, preferably comprising the following steps:

The present invention also provides the application of dolastatin 10 in the preparation of antitumor drugs.

On the basis of not violating common knowledge in this field, above-mentioned each preferred condition, can combine arbitrarily, obtain each preferred example of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progress effect of the present invention is that: the present invention provides a dolastatin 10 derivative I which is completely different from the prior art and has an anti-tumor effect, which provides a new direction for the research and development of podophyllin-derived anticancer drugs.

detailed description

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions. Compounds involved in the embodiments of the present invention:

Example 1 (S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-( (1R,2R)-1-methoxy-2-methyl-3-oxo-3-(3-phe nylazetidin-1-yl)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl Preparation of )-N,3-dim ethylbutanamide 1

A: tert-butyl 3-phenylazetidine-1-carboxylate

Compound 5 (400mg, 1.0mmol, 1.00eq) and phenylboronic acid (244mg, 2.0mmol, 2.00eq) were dissolved in dry 1,2-dioxane, Cs2CO3 (650mg, 2.0mmol, 2.00eq) was added, 110 React at ℃ for 24 hours; return to room temperature, add dichloromethane, wash with saturated sodium bicarbonate solution, separate the organic layer, wash with saturated brine, dry with anhydrous sodium sulfate, filter and evaporate to dryness, ethyl acetate/petroleum ether (1:10) Column chromatography was carried out as the eluent to obtain 180 mg of colorless oily substance 1A, with a yield of 77%;

B: tert-butyl(S)-2-((1R,2R)-1-methoxy-2-methyl-3-oxo-3-(3-phenylazetidin-1-yl)propyl)pyrrolidine-1-carboxylate

Compound 1A (22mg, 0.1mmol) was dissolved in dichloromethane (1mL), and 1mL of trifluoroacetic acid was added. After reacting at room temperature for half an hour, the solvent was distilled off, and the oil pump was dried for two hours, dissolved in dry THF, and diisopropylethyl Amine (40uL, 0.27mmol, 3.00eq), pyAop (70mg, 0.14mmol, 1.5eq), compound 8 (25mg, 0.1mmol, 1.00eq), react at room temperature for 18 hours. The solvent was evaporated to dryness, and ethyl acetate/petroleum ether (1:1) was used as the eluent for column chromatography to obtain 30 mg of colorless oily substance 1B, with a yield of 81%

Preparation of (S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-3-methoxy-1-((S)-2-((1R ,2R)-1-methoxy-2-methyl-3-oxo-3-(3-phe nylazetidin-1-yl)propyl)pyrrolidin-1-yl)-5-methyl-1-oxoheptan-4-yl)- N,3-dim ethylbutanamide

Compound 1B (30mg, 0.07mmol, 1.00eq) and compound 11 (34mg, 0.07mmol, 1.00eq) were respectively dissolved in dichloromethane, deprotected with trifluoroacetic acid, evaporated to dryness, and dried by oil pump; dissolved in dry tetrahydrofuran , add diisopropylethylamine (30uL, 0.21mmol, 3.00eq), pyAOP (54mg, 0.11mmol, 1.5eq), and react at room temperature for 24 hours. The solvent was evaporated to dryness, and column chromatography was performed with dichloromethane/methanol (40:1-20:1) as the eluent to obtain 23 mg of the compound with a yield of 43%.

¹ H NMR (400MHz, CDCl ₃ ) 7.40-7.36(m, 2H), 7.31-7.27(m, 3H), 6.93(d, J=9.1Hz, 1H), [4.87(t, J=7.2Hz) and 4.79(t, J=7.8Hz), 1H], 4.66-4.59(m, 1H), 4.54-4.46(m, 1H), 4.45-4.40(m, 1H), 4.37-4.32(m, 1H), 4.28 -4.23(m,1H),4.21-3.77(m,11H),[3.43(s)and 3.41(s),3H],[3.36(s)and 3.35(s),3H],[3.34(s) and 3.30(s), 3H], 3.14(d, J=6.2Hz, 1H), 3.03-3.02(m, 2H), 2.63-0.79(m, 36H). ¹³ C NMR(150MHz, CDCl ₃ ) 174.4, 147.1,173.8,173.7,173.1,171.2,170.2,169.9,161.8,161.7,160.1,129.2,129.1,129.1,129.0,128.8,128.4,128.2,128.1,127.9,127.7,127.7,124.5,124.4,116.0,115.8, 115.7, 115.6, 115.5, 115.5, 86.4, 86.2, 82.6, 82.4, 78.3, 77.8, 76.0, 61.9, 61.8, 60.5, 60.4, 59.4, 59.2, 59.0, 58.1, 57.9, 57.8, 56.5, 56.4, 56.2, 5 55.8,53.8,53.6,53.4,53.2,47.7,47.5,46.6,46.5,42.6,39.4,38.8,38.8,37.7,37.4,35.8,33.2,33.1,32.3,31.8,30.9,28.4,27.8,27.7,27.3, MS 714.0 (M+H ) ⁺ -50.200 (MeOH, 0.50).

Example 2 (S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R )-3-(3-(2-fluorophenyl)azetidin-1-yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan- Preparation of 4-yl)-N,3-dimethylbutanamide (2)

Preparation process is with embodiment 1;

¹ H NMR (400 MHz, CDCl3) δ7.41–7.31(m,1H),7.25–7.20(m,1H),7.16(dt,J＝8.7,4.4 Hz,1H),7.04(m,1H), 4.33(t, J=8.1 Hz, 2H), 4.08–4.02(m, 2H), 4.01–3.95(m, 1H), 1.46(s, 9H).

1H NMR (400 MHz, Chloroform-d) δ7.27-7.10(m,1H),6.96-6.94(m,1H),6.83(d,J=1.7 Hz,1H),4.50-4.43(m,1H) ,4.35-4.31(m,1H),4.27-4.24(m,1H),4.19-4.08(m,2H),3.97-3.82(m,3H),3.81(s,3H),3.78-3.74(m, 1H),3.26-3.22(m,1H),1.95(s,3H),1.92-1.68(m,4H),1.52(s,3H),1.44(d,J=7.8 Hz,3H).

¹³ C NMR (150 MHz, Chloroform-d) δ174.4, 154.6, 129.0, 128.1, 128.0, 128.0, 124.5, 115.8, 84.2, 84.1, 82.1, 79.9, 79.1, 61.2, 60.8, 59.5, 59.3, 58.9, 55.4, 53. ,53.3,47.0,46.7,39.2,38.4,28.7,28.6,27.8,27.3,27.4,26.2,26.1,25.7,24.7,24.6,24.3,14.7,14.6,13.8.

¹ H NMR (400 MHz, CDCl ₃ ) 7.36-7.28(m, 1H), 7.09-6.95(m, 4H), [4.87(t, J=7.5 Hz) and 4.79(t, J=7.8 Hz), 1H ],4.66-4.60(m,1H),4.57-4.50(m,1H),4.45-4.41(m,1H),4.38-4.34(m,1H),4.27-4.22(m,13H),[3.43( s) and 3.41(s), 3H], [3.36(s) and 3.35(s), 3H], [3.33(s) and 3.29(s), 3H], 2.63-0.79(m, 34H); ^13C NMR (150 MHz, CDCl ₃ ) δ174.6, 174.3, 173.9, 173.3, 171.7, 170.4, 169.9, 169.5, 142.1, 141.91, 141.3, 129.1, 129.1, 128.9, 127.5, 127.3, 126.8, 126.57, 126.7, 86.3,82.7,82.6,77.3,77.1,76.9,62.00,60.6,60.5,59.53,59.34,59.1,58.3,58.1,58.0,57.9,57.8,57.6,57.6,55.94,55.2,55.15,55.04,34.8, 53.8,47.8,47.6,46.8,46.7,46.7,45.9,43.00,39.71,39.0,37.9,37.64,36.03,33.59,33.44,33.3,33.3,33.2,33.1,32.5,32.06,31.09,278,26.8,27 26.18, 25.88, 25.28, 25.1, 24.8, 24.5, 23.7, 22.8, 20.30, 19.9, 19.8, 19.7, 18.1, 17.92, 16.03, 15.64, 14.9, 14.2, 13.8, 13.6, 11.1, 10.9, 10.9MS, 10.1 (M+H)+ -35.200 (MeOH, 0.50).

Example 3

(S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3 -(3-(3-fluorophenyl)azetidin-1-yl)-1-methoxy-2-methyl-3-oxopr opyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4- Preparation of yl)-N,3-dimethylbu tanamide(3)

Preparation process is with embodiment 1;

1H NMR (400MHz, CDCl3) 8.63(s, 1H), 7.36-6.99(m, 5H), [4.88-4.84(m) and 4.80-4.74(m), 1H], 4.70-4.63(m, 1H), 4.58-4.54(m,1H),4.50-4.34(m,2H),4.29-3.68(m,10H),3.53-3.45(m,2H),[3.43(s)and 3.39(s),3H], [3.36(s)and3.35(s),3H],[3.12(s)and 3.02(s),3H],2.78-0.80(m,34H)

¹³ C NMR (150MHz, CDCl ₃ ) δ173.1, 169.4, 150.4, 122.0, 82.5, 78.1, 77.3, 77.1, 76.9, 61.9, 60.5, 59.6, 59.4, 59.1, 58.4, 58.2, 58.0, 56.7, 54.4, 54.3, 53.9 ,53.6,47.8,47.6,46.8,45.9,42.6,42.1,39.8,39.5,37.6,33.6,33.3,32.6,32.5,32.0,31.5,31.0,30.3,29.8,27.0,26.5,26.1,25.8,25.1,24.8 ,24.5,23.7,20.1,19.8,19.6,18.2,18.1,17.6,16.0,13.6,13.4,11.8,11.1,10.9 -41.400 (MeOH, 0.50).

Example 4

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-(3-chloro-4-fluorophenyl)azetid in-1- yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-meth yl-1-oxoheptan-4-yl)-2-((S)-2-( Preparation of dimethylamino)-3-methylbutanamido)-N,3-di methylbutanamide (4)

Preparation process is with embodiment 1;

1H NMR (400MHz, Chloroform-d) δ7.39–7.34(m,1H),7.21–7.15(m,1H),7.12(t,J=7.8Hz,1H),4.33(t,J=8.7Hz, 2H),3.95–3.87(m,2H),3.73–3.63(m,1H),1.47(s,9H);13C NMR(151MHz,Chloroform-d)δ156.4,139.5,129.1,126.6,126.6,117.3,117.0 ,116.8,114.8,80.0,56.6,32.8,29.8,28.5.

1H NMR (400MHz, Chloroform-d) δ7.36–7.31(m,1H),7.15–7.11(m,2H),4.58-4.55(m,1H),4.44-4.33(m,1H),4.19-3.57 (m,6H),3.45(s,3H),3.27-3.25(m,1H),2.50-2.41(m,1H),1.98-1.74(m,4H),1.52(s,9H),1.46(d ,J＝6.8Hz,3H).

13C NMR (151MHz, DMSO-d6) δ173.3, 156.9, 155.3, 153.3, 140.2, 129.1, 128.8, 127.6, 127.5, 127.4, 127.4, 119.5, 117.1, 117.0, 116.9, 116.8, 83.4, 80.8.7, 82 .

1H NMR (400MHz, CDCl3) 7.37-7.35(m,2H),7.16-7.10(m,2H),[4.88-4.87(m)and 4.79-4.77(m),1H],4.64-4.60(m,1H ),4.52-4.46(m,1H),4.48-4.41(m,1H),4.35-4.33(m,1H),4.21-4.19(m,2H),4.18-4.01(m,3H),3.89-3.76 (,2H),[3.49(s)and 3.43(s),3H],[3.39(s)and 3.35(s),3H],[3.13(s)and 3.02(s),3H],2.63-0.79 (m,36H);13C NMR(151MHz,DMSO-d6)δ175.5,174.5,174.3,173.9,173.2,171.3,170.0,158.1,156.4,138.8,129.9,129.8,129.0,128.9,128.8,128.7,1266.2 ,121.3,117.0,116.9,116.8,86.3,86.1,82.5,82.3,61.8,60.6,59.5,59.2,59.0,58.2,58.1,57.8,57.7,57.4,57.1,55.9,55.2,55.0,54.8,54.6,5 ,47.8,46.7,42.7,39.5,38.8,37.6,35.9,33.4,32.5,32.4,29.7,29.5,29.2,27.6,27.2,26.3,26.0,25.7,25.4,25.0,24.4,22.6,20.8,20.0,19.5 , 18.2, 15.8, 14.1, 10.9; 13C NMR (151MHz, CDCl3) δ174.5, 174.1, 173.9, 173.7, 173.3, 171.3, 170.3, 170.2, 169.5, 158.1, 134.0, 133.9, 133.7, 133.0, 1278.7 127.25,127.15,120.7,120.6,111.5,111.4,111.3,111.3,86.3,82.5,82.4,78.9,78.15,77.37,77.16,76.9,76.23,61.9,61.9,60.8,50.7,54,8.2,59 57.9, 57.8, 57.6, 57.0, 56.3, 56.1, 55.9, 55.80, 55.7 ,54.1,53.8,53.0,52.9,52.8,52.7,47.9,,46.8,46.7,45.9,42.8,39.48,39.10,39.03,38.94,37.7,37.6,35.9,33.5,33.1,33.0,32.5,32.0,31.0, 29.8,29.0,28.9,28.6,27.8,26.3,26.1,25.8,25.6,25.1,25.0,24.8,24.5,23.7,20.8,20.2,20.1,19.9,19.8,19.6,18.1,18.0,17.9,17.9,17.9, 16.7,16.5,16.2,15.6,14.9,14.7,14.1,12.4,11.0,10.8,10.8,10.4.MS 76.0(M+H)+ -37.600 (MeOH, 0.50).

Example 5

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-(4-chlorophenyl)azetidin-1-yl)-1- methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxo Preparation of heptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylb utanamide (5)

Preparation process is with embodiment 1;

1H NMR (400MHz, Chloroform-d) δ7.23(d, J=8.1Hz, 2H), 7.15(d, J=8.1Hz, 2H), 4.25(t, J=8.7Hz, 2H), 3.85(t ,J＝8.6,5.8Hz,2H),3.64-3.57(m,1H),1.39(s,9H)

1H NMR (400MHz, CDCl3) 7.33-7.32(m, 2H), 7.24-7.22(m, 3H), 4.58-4.53(m, 1H), 4.45-4.40(m, 1H), 4.36-4.34(m, 1H ),4.20-3.78(m,6H),3.46(s,3H),3.33-3.25(m,1H),2.50-2.39(m,1H),1.95-1.71(m,4H),1.52(s,9H ), 1.45 (d, J=6.8Hz, 3H); ¹³ C NMR (151MHz, Chloroform-d) δ174.6, 154.5, 133.1, 129.0, 128.2, 128.0, 84.3, 84.1, 82.6, 79.9, 79.1, 61.1, 60.8, 59.5, 59.3, 58.8, 57.6, 57.6, 55.9, 54.9, 54.8, 47.0, 46.7, 39.6, 39.3, 38.6, 38.5, 32.2, 29.7, 28.7, 28.6, 26.3, 24.2, 14.6.

1H NMR (400MHz, CDCl3) 7.36-7.31(m, 2H), 7.24-7.20(m, 2H), 7.06(brs, 1H), [4.86(t, J=7.2Hz) and 4.78(t, J=7.8 Hz), 1H], 4.67-4.60(m, 1H), 4.56-4.50(m, 1H), 4.43(t, J=9.5Hz, 1H), 4.34(t, J=9.5Hz, 1H), 4.21- 3.74(m,11H),[3.43(s)and 3.41(s),3H],[3.36(s)and 3.35(s),3H],[3.13(s)and 3.02(s), 3H],2.63 -0.79(m,36H); ¹³ C NMR(150MHz,CDCl ₃ )δ174.6,174.3,174.0,173.3,171.6,170.3,169.9,140.6,140.3,140.0,139.8,133.3,133.0,129.29,129.10,1289. .0,87.1,86.4,86.2,82.6,77.3,77.1,76.9,76.41,61.9,60.5,59.36,59.1,58.4,58.0,57.73,57.5,55.2,54.9,53.9,47.6,45.9,42.3,40.1,39.7 . ,13.713.110.9,10.4MS 770.1(M+Na)+; -33.400 (MeOH, 0.50).

Example 6

(S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3 -(3-(2-isopropylphenyl)azetidin-1-yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4- Preparation of yl)-N,3-dimethyl lbutanamide(6)

Preparation process is with embodiment 1;

¹ H NMR (400MHz, CDCl3) 7.65(d, J=7.6Hz, 2H), 7.20(d, J=7.5Hz, 2H), 7.07(brs, 1H), [4.88-4.84(m) and 4.80-4.75 (m), 1H], 4.58-4.54(m, 1H), 4.50-4.32(m, 3H), 4.21-3.76(m, 10H), 3.52-3.46(m, 2H), [3.43(s) and 3.41 (s),3H],3.39-3.36(m,2H),[3.33(s)and 3.28(s),3H],[3.13(s)and 3.09(s),3H],3.03-0.79(m, 35H); ¹³ C NMR (150MHz, CDCl ₃ ) δ174.6, 174.3, 174.0, 173.3, 171.6, 169.9, 140.6, 140.0, 139.8, 133.0, 129.29, 129.10, 128.2, 87.1, 86.4, 86.2, 82.6, 7 76.9,76.41,61.9,60.5,59.36,59.1,58.4,58.0,57.73,57.5,55.2,54.9,53.9,47.6,45.9,42.3,40.1,39.7,39.2,38.9,37.8,36.0,33.3,32.9,32 (M+H)+ -30.400 (MeOH, 0.50).

Example 7

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-(4-chloro-2-methoxyphenyl)aze tidin-1- yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-m ethyl-1-oxoheptan-4-yl)-2-((S)-2-( Preparation of dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamide (7)

Preparation process is with embodiment 1;

1H NMR (400MHz, Chloroform-d) δ6.94(dd, J=8.1,1.8Hz,1H),6.83(d,J=1.7Hz,1H),4.25(t,J=8.4Hz,2H),4.04 –3.86(m,3H),3.80(s,3H),1.45(s,9H).

1H NMR (400MHz, Chloroform-d) δ7.27-7.10(m,1H),6.96-6.94(m,1H),6.83(d,J=1.7Hz,1H),4.50-4.43(m,1H), 4.35-4.31(m,1H),4.27-4.24(m,1H),4.19-4.08(m,2H),3.97-3.82(m,3H),3.81(s,3H),3.78-3.74(m,1H ),3.26-3.22(m,1H),1.95(s,3H),1.92-1.68(m,4H),1.52(s,3H),1.44(d,J=7.8Hz,3H).

13C NMR(151MHz,Chloroform-d)δ174.41,158.12,154.65,133.83,128.15,128.02,120.67,111.42,84.21,84.10,82.59,79.96,79.22,61.30,60.91,59.55,59.41,59.01,58.90,56.06,55.74 ,55.70,52.95,52.79,52.72,47.11,46.79,39.15,38.66,28.79,28.76,26.33,26.12,25.87,24.63,24.31,14.76,14.61,13.88.

¹ H NMR (400MHz, CDCl ₃ ) 7.15-6.84(m, 4H), [4.88-4.87(m) and 4.79-4.77(m), 1H], 4.54-4.50(m, 1H), 4.45-4.41( m,1H),4.35-4.33(m,1H),4.35-4.33(m,1H),4.23-4.21(m,1H),4.18-4.13(m,4H),3.99-3.76(m,6H), [3.44(s)and 3.43(s),3H],[3.35(s)and 3.33(s),3H],[3.31(s)and 3.29(s),3H],2.63-0.80(m,39H) ;

¹³ C NMR (150MHz, DMSO-d6) δ175.5, 174.5, 174.3, 173.9, 173.2, 171.3, 170.0, 158.1, 156.4, 138.8, 129.9, 129.8, 129.0, 128.9, 128.8, 128.7, 126.5, 121.0.3, 116.9, 116.8, 86.3, 86.1, 82.5, 82.3, 61.8, 60.6, 59.5, 59.2, 59.0, 58.2, 58.1, 57.8, 57.7, 57.4, 57.1, 55.9, 55.2, 55.0, 54.8, 54.6, 53.9, 47.8, 46.7 42.7,39.5,38.8,37.6,35.9,33.4,32.5,32.4,29.7,29.5,29.2,27.6,27.2,26.3,26.0,25.7,25.4,25.0,24.4,22.6,20.8,20.0,19.5,18.2,15.8, 14.1, 10.9; 13C NMR (151MHz, CDCl3) δ174.5, 174.1, 173.9, 173.7, 173.3, 171.3, 170.3, 170.2, 169.5, 158.1, 134.0, 133.9, 133.7, 133.1, 128.1, 128.0, 127.1, 1277 ,120.6,111.5,111.4,111.3,111.3,86.3,82.5,82.4,78.9,78.15,77.37,77.16,76.9,76.23,61.9,61.9,60.8,60.7,59.6,59.4,59.1,58.2,57.5,75 ,57.0,56.3,56.1,55.9,55.80,55.7,54.1,53.8,53.0,52.9,52.8,52.7,47.9,,46.8,46.7,45.9,42.8,39.48,39.10,39.03,38.94,37.7,37,6,35.9 33.5,33.1,33.0,32.5,32.0,31.0,29.8,29.0,28.9,28.6,27.8,26.3,26.1,25.8,25.6,25.1,25.0,24.8,24.5,23.7,20.8,20.2,20.1,19.9,19.8, 19.6, 18.1, 18.0, 17.9, 17.9, 17.9, 16.7, 16.5, 16.2, 15.6, 14.9, 14.7, 14.1 ,12.4,11.0,10.8,10.8,10.4.MS 778.0(M+H)+ -42.955 (MeOH, 0.44).

Example 8

(S)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N-((3R,4S,5S)-1-((S)-2-(( 1R,2R)-3-(3-(4-fluorophenyl)azetidin-1-yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1 -oxoheptan-4-yl)-N,3-dimethylbutana mide (8) preparation

¹ H NMR (400MHz, CDCl ₃ ) 7.15-6.84(m, 4H), [4.88-4.87(m) and 4.79-4.77(m), 1H], 4.54-4.50(m, 1H), 4.45-4.41( m,1H),4.35-4.33(m,1H),4.35-4.33(m,1H),4.23-4.21(m,1H),4.18-4.13(m,4H),3.99-3.76(m,6H), [3.44(s)and 3.43(s),3H],[3.35(s)and 3.33(s),3H],[3.31(s)and 3.29(s),3H],2.63-0.80(m,39H) ;

¹³ C NMR (150MHz, DMSO-d6) δ175.5, 174.5, 174.3, 173.9, 173.2, 171.3, 170.0, 158.1, 156.4, 138.8, 129.9, 129.8, 129.0, 128.9, 128.8, 128.7, 126.5, 121.0.3, 116.9, 116.8, 86.3, 86.1, 82.5, 82.3, 61.8, 60.6, 59.5, 59.2, 59.0, 58.2, 58.1, 57.8, 57.7, 57.4, 57.1, 55.9, 55.2, 55.0, 54.8, 54.6, 53.9, 47.8, 46.7 42.7,39.5,38.8,37.6,35.9,33.4,32.5,32.4,29.7,29.5,29.2,27.6,27.2,26.3,26.0,25.7,25.4,25.0,24.4,22.6,20.8,20.0,19.5,18.2,15.8, 14.1, 10.9; ¹³ C NMR (150MHz, CDCl3) δ174.5, 174.1, 173.9, 173.7, 173.3, 171.3, 170.3, 170.2, 169.5, 158.1, 134.0, 133.9, 133.7, 133.1, 128.1, 128.1, 25, 127 120.7, 120.6, 111.5, 111.4, 111.3, 111.3, 86.3, 82.5, 82.4, 78.9, 78.15, 77.37, 77.16, 76.9, 76.23, 61.9, 61.9, 60.8, 60.7, 59.6, 59.4, 59.1, 59.2, 75 57.6,57.0,56.3,56.1,55.9,55.80,55.7,54.1,53.8,53.0,52.9,52.8,52.7,47.9,46.8,46.7,45.9,42.8,39.48,39.10,39.03,38.954,37.7,37.6 ,33.5,33.1,33.0,32.5,32.0,31.0,29.8,29.0,28.9,28.6,27.8,26.3,26.1,25.8,25.6,25.1,25.0,24.8,24.5,23.7,20.8,20.2,20.1,19.9,19.8 ,19.6,18.1,18.0,17.9,17.9,17.9,16.7,16.5,16.2,15.6,14.9,14.7,14 .1,12.4,11.0,10.8,10.8,10.4.MS 778.0(M+H)+ -40.955 (MeOH, 0.44).

Example 9

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-(4-(tert-butyl)phenyl)azetidin-1 -yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)-2-( Preparation of dimethylamino)-3-methylbutanamido)-N,3-dimet hylbutanamide (9)

1H NMR (400MHz, Chloroform-d) δ7.38(d, J=8.3Hz, 2H), 7.25(d, J=8.1Hz, 2H), 4.31(t, J=8.6Hz, 2H), 4.10–3.87 (m,2H),3.72(m,1H),1.47(s,9H),1.32(s,9H).

1H NMR (400MHz, Chloroform-d) δ7.38(d, J=8.3Hz, 2H), 7.25(d, J=8.1Hz, 2H), 4.55-4.51(m, 1H), 4.43-4.32(m, 1H),4.12-3.76(m,8H),3.46(s,3H),2.48-2.39(m,1H),1.98-1.74(m,4H),1.52(s,9H),1.32(s,9H) ,1.24(d,J=6.4Hz,3H).

1H NMR (400MHz, CDCl3) 7.36-7.31(m, 2H), 7.24-7.20(m, 2H), 7.06(brs, 1H), [4.86(t, J=7.2Hz) and 4.78(t, J=7.8 Hz), 1H], 4.67-4.60(m, 1H), 4.56-4.50(m, 1H), 4.43(t, J=9.5Hz, 1H), 4.34(t, J=9.5Hz, 1H), 4.21- 3.74(m,11H),[3.43(s)and 3.41(s),3H],[3.36(s)and 3.35(s),3H],[3.13(s)and 3.02(s),3H],2.63 -0.79(m,36H); ¹³ C NMR(150MHz,CDCl ₃ )δ174.6,174.3,174.0,173.3,171.6,170.3,169.9,140.6,140.3,140.0,139.8,133.3,133.0,129.29,129.10.2,129 .0,87.1,86.4,86.2,82.6,77.3,77.1,76.9,76.41,61.9,60.5,59.36,59.1,58.4,58.0,57.73,57.5,55.2,54.9,53.9,47.6,45.9,42.3,40.1,39.7 . , 13.713.110.9, 10.4; MS 770.1 (M+Na)+; -33.400 (MeOH, 0.50).

Example 10

(S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(3-([1,1'-biphenyl]-4-yl) azetidin-1-yl)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-2-((S)- Preparation of 2-(dimethylamino)-3-methylbutanamido)-N,3-dimeth ylbutanamide(10)

1H NMR (400MHz, CDCl3) 7.58(d, J=7.8Hz, 4H), 7.44(t, J=7.5Hz, 2H), 7.40-7.33(m, 3H), 4.61-4.56(m, 1H), 4.48 -4.37(m,1H),4.23-4.06(m,2H),3.91-3.78(m,3H),3.46(s,3H),3.28-3.26(m,1H),2.49-2.42(m,1H) ,2.08-1.92(m,6H),1.53(s,9H),1.25(t,J=6.4Hz,3H);13C NMR(150MHz,Chloroform-d)δ156.58,141.42,140.83,140.10,128.93,128.88, 127.57, 127.45, 127.36, 127.29, 127.17, 79.74, 33.37, 28.58.

1H NMR (400MHz, CDCl3) 7.58 (d, J = 8.4Hz, 4H), 7.43 (t, J = 7.5Hz, 2H), 7.37-7.35 (m, 3H), 6.94 (brs, 1H), [4.89- 4.85(m) and 4.80-4.76(m),1H], 4.69-4.62(m,1H),4.58-4.52(m,1H),4.48-4.46(m,1H),4.42-4.35(m,1H),4.30-4.27(m,1H),4.22-4.19(m ,4H),[3.43(s)and 3.42(s),3H],[3.36(s)and3.34(s),3H],[3.15(s)and 3.02(s),3H],2.78-0.81 (m,42H);13C NMR(400MHz,CDCl3)13C NMR(150MHz,Chloroform-d)δ156.58,141.42,140.83,140.10,128.93,128.88,127.57,127.45,127.36,127.29,1257.17,79.374,28

¹ H NMR (400MHz, CDCl ₃ ) 7.58(d, J=7.8Hz, 4H), 7.44(t, J=7.5Hz, 2H), 7.40-7.33(m, 3H), 4.36(t, J=8.6Hz , 2H), 4.02 (J=8.6Hz, 2H), 3.81-3.75 (m, 1H), 1.48 (s, 3H); ¹³ C NMR (400MHz, CDCl ₃ ) 13C NMR (150MHz, Chloroform-d) δ156. 58,141.42,140.83,140.10,128.93,128.88,127.57,127.45,127.36,127.29,127.17,79.74,52.9,52.8,52.7,47.9,,46.8,46.7,45.9,42.8,39.48,39.10,39.03,38.94,37.7,37.6 ,35.9,33.5,33.1,33.0,32.5,32.0,31.0,29.8,29.0,28.9,28.6,27.8,26.3,26.1,25.8,25.6,25.1,25.0,24.8,24.5,23.7,20.8,20.2,20.1,19.9 , 19.8, 19.6; MS 790.0 (M+H)+; -23.636 (MeOH, 0.11).

Example 11

In vitro tumor cell proliferation inhibitory activity screening

Different tumor cell lines were selected to screen the synthesized 10 compounds for anti-tumor activity in vitro.

Experimental Materials

Cell lines: SU-DHL-6, MCF-7, HCT 116, HeLa;

Culture medium: DMEM cell culture medium (GIBCO): containing 10% inactivated newborn calf serum (Shanghai Saida Biological Pharmaceutical Co., Ltd.); L-glutamine (imported packaging, SANGON); sodium pyruvate; GIBCO Double antibody, for KB cells;

1640 cell culture medium: containing 10% inactivated newborn calf serum, GIBCO double antibody, used for K562, L1210 cells;

IMDM cell culture medium: containing 20% inactivated fetal bovine serum, GIBCO double antibody; used for HL-60 cells;

Drug preparation method: Sample preparation: Dissolve the sample in dimethyl sulfoxide to obtain a solution with a concentration of 10 mg/ml. Then use phosphate buffer saline (PBS) for gradient dilution to obtain diluted samples with concentrations of 1000 μg/ml, 100 μg/ml, 10 μg/ml, 1 μg/ml, 0.1 μg/ml, and 0.01 μg/ml;

Tumor cell culture in vitro

1. For pelleted cells, use steps 3-7, and for suspension cells, use steps 4-7;

2. Take the cells out of the liquid nitrogen, thaw them quickly in a 37°C water bath, transfer the cells to a 10ml sterile centrifuge tube in a sterile operating table, add 6ml of the corresponding cell culture medium, and centrifuge at 1000 rpm for 5 minutes;

3. Add 3.0mL Trypsin-EDTA solution, observe under a microscope until the layers are separated;

4. Add 8.0mL growth medium and carefully remove the cells;

5. Add 6ml of the corresponding cell culture medium to a sterile centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant; the next day, take out the cells from the incubator, discard the DMEM cell culture medium in the cell bottle, and add PBS ( pH 7.4) 2-3ml shake and wash, pour off the PBS solution and repeat the wash again, add 3-5 drops of 0.25% trypsin solution to the culture bottle, shake evenly, cover and place in a 37°C cell culture incubator for about 3 minutes , observed under the microscope that the cells were detached from the wall of the culture flask, added 2ml of DMEM cell culture medium, blown with a dropper to make the cells completely detach from the flask wall, then transferred to two clean culture flasks, added 5-6ml of DMEM cell culture medium and blown Evenly, place in a cell culture incubator at 37°C; take out 1-2ml suspension of suspended cells, and transfer them to 2 clean culture flasks respectively, add 5-6ml of 1640 cell culture medium to K562 and L1210, add IMDM cell culture medium to HL-60, Place in a 37°C cell culture incubator;

6. The next day, repeat step ③. During the whole culture process, the adherent cells are not allowed to grow too densely, and the suspension cells always maintain the logarithmic growth phase;

Inhibitory effect of 14 compounds on different tumor strains measured by MTT assay

Harvest the cells in the logarithmic growth phase, count and adjust the concentration to 20×10 ⁴ /mL, add 90 μL of cell suspension to each well of a 96-well plate, add 10 μL/well of the drug after proportional dilution, calculate the final concentration, and mix well for 37 ℃, 5% CO ₂ incubator for 48 hours, add 5mg/ml MTT (PBS) solution 20 μ L/well, 37 ℃, 5% CO ₂ incubator for 4 hours, add solution (10% SDS , 5% isobutanol, 0.02M HCl) 100 μL/well, 37° C., 5% CO ₂ Place overnight in an incubator, observe the complete dissolution of the precipitate in each well the next day, then use a microplate reader to measure the OD value at 570 nm.

Regression was performed and _IC50 was calculated.

Claims (10)

1. there is the derivative Is of Dolastatin 10 of below formula,

Wherein, R1, R2, R3 are independently selected from hydrogen, phenyl ring, halogen or C₁～C₆Alkoxyl.

2. derivative Is of Dolastatin as claimed in claim 1 10, it is characterised in that：R₁And R₂It is independent Ground is selected from hydrogen, C₁～C₆Alkyl or C₁～C₆Alkoxyl；R₃Selected from hydrogen, halogen or C₁～C₆Alkane Epoxide.

3. derivative Is of Dolastatin as claimed in claim 1 10, it is characterised in that：Described Dolastatin In 10 derivative Is,

R1, R2, R3 are hydrogen；

Or, R1 is fluorine, R2, R3 are hydrogen；

Or, R1 is hydrogen, R2 is fluorine and R3 is hydrogen；

Or, R1 is hydrogen, R2 is chlorine and R3 is fluorine；

Or, R1 is hydrogen, R2 is hydrogen and R3 is chlorine；

Or, R1 is isopropyl, R2 is fluorine and R3 is hydrogen；

Or, R1 is methoxyl group, R2 is hydrogen and R3 is chlorine；

Or, R1 is hydrogen, R2 is hydrogen and R3 is fluorine；

Or, R1 is hydrogen, R2 is hydrogen and R3 is the tert-butyl group；

Or, R1 is hydrogen, R2 is hydrogen and R3 is phenyl.

4. a kind of preparation method of the derivative Is of Dolastatin 10 as described in any one of claims 1 to 3, It is characterized in that：Including step：By document route prepare compound 6,6 remove Boc protection after with Compound 8 carries out being condensed to yield compound 9；9 remove Boc and the tert-butyl group is laggard respectively with compound 11 Row condensation；

5. preparation method as claimed in claim 4, it is characterised in that：Solvent employed in it is tetrahydrochysene Furans, pyridine, acetonitrile, dichloromethane, chloroform, N,N-dimethylformamide, N, N- dimethyl One or more in acetamide, 1-METHYLPYRROLIDONE and dimethyl sulfoxide.

6. preparation method as claimed in claim 4, it is characterised in that：Condensing agent employed in it is two Carbodicyclo hexylimide, 1- ethyl -3- [3- (dimethylamino) propyl group] carbodiimide hydrochloride, 1- hydroxy benzos Triazole, N, N'- carbonyl dimidazoles, O- (7- azepine benzo triazol-1-yls)-N, N, N ', N '-tetramethylurea hexafluoro Phosphate, BTA-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester, methylphosphine acid anhydrides, ethyl phosphine One kind or many in acid anhydrides, n-propyl phosphonic acid anhydrides, propyl-phosphine acid cyclic acid anhydride and BTA base diethyl phosphate Kind；The consumption of described condensing agent is 1.1～2 molar equivalents.

7. preparation method as claimed in claim 4, it is characterised in that：Alkali employed in it be pyridine, Triethylamine, N, one or more in TMSDMA N dimethylamine yl pyridines and diisopropylethylamine.

8. preparation method as claimed in claim 6, it is characterised in that：The reaction temperature of described condensation reaction Spend for -15 DEG C～100 DEG C.

9. the preparation method of the derivative Is of Dolastatin as claimed in claim 6 10, it is characterised in that：

The reaction temperature of described condensation reaction is -15 DEG C～30 DEG C.

10. the derivative Is of Dolastatin 10 described in any one of claims 1 to 3 are in antineoplastic is prepared Purposes.