TW201733993A - Preparation of conjugates between docetaxel and muramyl dipeptide simplified substance, and anti-tumor effects - Google Patents

Abstract

The present invention relates to a compound represented by formula (I), chemical synthesis preparation of dual-functional conjugates capable of inhibiting tumors and tumor metastasis, and tumor resistance effects. In the present invention, multiple conjugates between docetaxel and muramyl dipeptide simplified substance are synthesized, and it is found in physical and chemical data and pharmacodynamical experiments that the conjugates have good druggability and has a better tumor resistance effect and a tumor metastasis resistance effect.

Description

Preparation and Antitumor Effect of Co-Inclusion of Docetaxel and Cell Wall Dipeptide

The invention mainly relates to a conjugate formed by docetaxel and a cell wall dipeptide derivative, a synthesis method thereof, and an application thereof in the treatment of cancer, and belongs to the technical field of medicine.

Malignant tumor is an important killer of human life and health. Tumor metastasis is one of the essential characteristics of malignant tumors, and it is also the most fundamental cause of cancer treatment failure. More than 80% of clinical cancer patients die from malignant tumor metastasis, and treat tumor metastasis. It is an important way to reduce the mortality of cancer. Controlling metastasis is the key factor determining the prognosis of cancer patients. It has attracted worldwide attention. Tumor metastasis is not only a part of the complex pathogenesis of tumors, but also a therapeutic problem that clinical medical workers hope to solve. Therefore, it is particularly urgent and crucial to seek drugs that inhibit tumor metastasis.

Taxanes, including paclitaxel and docetaxel, have the characteristics of low oral bioavailability. The main reason is that they are easily pumped out by the P-glycoprotein of the gastrointestinal epithelium, easily metabolized by cytochrome P450, and soluble in water. Poor sex. Since taxanes are still the first-line drugs in their therapeutic field, various studies surrounding such compounds are hot topics for drug chemists. Since the chemical conjugate of natural anti-tumor drugs and immunopotentiators has been proposed, it is hoped that the combination of chemotherapy and immunotherapy will find new progress in the search for drug molecules with anti-tumor metastasis.

The earlier patents that have been applied protect the two main classes of conjugates, one is the 2'-O-MTC conjugate formed by attaching the cell wall quinone dipeptide to the 2'-position of paclitaxel (see CN1712399A), but unfortunately The 2'-O-MTC conjugate was not able to display experimental results against tumor metastasis in experimental mice.

The other type is the MTC/MDC series conjugates formed by linking paclitaxel/docetaxel to the cell wall dipeptide derivative (see WO2011147330A1). The MTC/MDC series conjugates are used in tumor inhibition and tumor metastasis experiments in vitro. The performance must be corresponding, but due to its drug-forming problems, further drug use is subject to certain restrictions. 2'-O-MTC conjugate MTC/MDC series conjugate

Although the above series of compounds have contributed greatly to the field, in order to improve anticancer drugs, compounds having better antitumor metastasis activity and better drug properties have been sought, and research is continuing in the art.

The technical problem to be solved by the present invention is to provide a compound with good drug resistance against tumor and tumor metastasis to meet the needs of clinical application.

Another technical problem to be solved by the present invention is to provide a process for the preparation of such a compound.

Another technical problem to be solved by the present invention is to provide the use of such a compound for the preparation of a medicament for antitumor and tumor metastasis inhibition.

In order to solve the technical problem of the present invention, the following technical solutions are adopted: I wherein m is selected from a natural number from 0 to 1, ie m=0 or 1; n is selected from a natural number from 2 to 10, ie n = 2, 3, 4, 5, 6, 7, 8, 9 or 10 Preferably, n is selected from a natural number from 2 to 5, i.e., n = 2, 3, 4 or 5.

R _{1 is} selected from a substituted or unsubstituted five- to ten-membered aryl group, a substituted or unsubstituted heteroaryl group, and a substituted or unsubstituted methylene group; the substituted substituent is selected from the group consisting of a hydroxyl group, a mercapto group, a halogen, an amine. A nitro group, a cyano group, an aldehyde group, a C1-C6 alkyl group, a carboxyl group, a hydroxylamine group, a C2-C6 alkene group, a C1-C4 guanamine group, and a benzylamino group.

R _{2 is} selected from hydrogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, and the substituted substituent is selected from halogen.

The aryl group is preferably a 5- to 10-membered aryl group, and the heteroaryl group is preferably a 5- to 10-membered heteroaryl group.

The five-decade aryl group is preferably selected from the group consisting of a five-membered aryl group, a six-membered aryl group, and a ten-membered fused ring aryl group.

The five-membered aryl group is selected from .

The six-membered aryl group is selected from .

The ten-membered fused ring aryl group is selected from .

The heteroaryl group means an aromatic ring system of one or more hetero atoms, preferably having 1 to 4 hetero atoms, preferably selected from heteroaryl groups containing a hetero atom of N, O or S.

Preferred heteroaryl groups are selected from the group consisting of five to ten membered heteroaryl groups containing from 1 to 4 heteroatoms selected from N, O or S.

More preferred heteroaryl groups are selected from five-membered heterocyclic groups containing from 1 to 4 heteroatoms selected from N, O or S, and six-membered heterocyclic rings containing from 1 to 4 heteroatoms selected from N, O or S. A ten-membered fused heterocyclic group containing from 1 to 4 hetero atoms selected from N, O or S.

The five-membered heterocyclic group having 1 to 4 hetero atoms selected from N, O or S is selected from the group consisting of: .

The six-membered heterocyclic group having 1 to 4 hetero atoms selected from N, O or S is selected from the group consisting of: .

The ten-membered fused heterocyclic group having 1 to 4 hetero atoms selected from N, O or S is selected from the group consisting of: .

The C1-C6 alkyl group in the present invention means a straight-chain or branched alkane group having 1 to 6 carbon atoms, which may be selected from methyl, ethyl, n-propyl, isopropyl, cyclopropane. Base, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclopentyl, n-hexyl, cyclohexyl, dimethylpropyl, 2-methylbutyl, 2 , 2-dimethylbutyl, 2,3-dimethylbutyl.

The C1-C6 alkoxy group is selected from the group consisting of methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, and Pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy, methoxyethoxy, ethoxymethoxy, propoxymethoxy and propoxyethoxy.

The halogen is selected from the group consisting of fluorine, chlorine, bromine or iodine, and the C1-C4 guanamine group is selected from the group consisting of acetamino group, acrylamide, butylammonium or isobutylamino; C2-C6 The olefin group is selected from the group consisting of ethenyl, propenyl, butenyl, isobutenyl, 2-butenyl, pentenyl, isopentenyl, 2-pentenyl, hexenyl and isohexenyl.

The R 2 is hydrogen or hydrogen is replaced by a metal or non-metal cation to form a pharmaceutically acceptable salt, and the metal or non-metal cation is selected from the group consisting of Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Zn ^{2+ .} , Al ³⁺ , NH ₄ ⁺ .

The compounds of formula I include, but are not limited to, the compounds of formula IA: IA.

R _{1 is} as defined above.

The compound is selected from the group consisting of: , , , , , , , , , , , , with .

The present invention provides a general method for synthesizing conjugates of docetaxel and cell wall dipeptide derivatives as follows: 1. liquid phase synthesis of docetaxel 2'-O-alkaned diacid monoester; 2. solid phase synthesis cell a tick dipeptide derivative; 3. a liquid phase synthesis of a conjugate of docetaxel and a cell wall dipeptide derivative.

General Synthesis Step 1 : Synthesis of Docetaxel 2'-O- Alkane Diacid Monoester

Reaction reagents and conditions: succinic anhydride, TEA, THF, 0 ° C-rt, 4 h.

Operation steps: Docetaxel is dissolved in tetrahydrofuran, and a solution of triethylamine and alkane anhydride in tetrahydrofuran is added dropwise to the above reaction system under ice bath. After the completion of the dropwise addition, the reaction was carried out at room temperature for 4 hours, and the reaction was monitored by HPLC. The triethylamine in the reaction system was slowly neutralized with 1.0 M HCl in an ice bath, and the pH was adjusted to 3 to 5, and the resulting solution was reduced at 30 ° C. The mixture was concentrated to 1/3 volume by pressure, and then the concentrate was slowly added dropwise to 10 volumes of ice water to crystallize, and the solid was collected by filtration under reduced pressure; the obtained solid was beaten in ice water, filtered, washed three times with water, and dried at 30 ° C under vacuum. The target product was obtained as a white solid.

General synthesis step 2 : Synthesis of cell wall dipeptide derivatives

The method utilizes various hydroxy resins, such as king resin (loading 0.83mmol/g) as solid phase carrier, and introduces Fmoc-L-Lys(Boc)-COOH and Fmoc-D-iso into the resin through the solid phase synthesis strategy of the peptide. -Gln-COOH, Fmoc-L-Ala-COOH and various organic carboxylic acids. After completion of the condensation reaction, various cell wall dipeptide derivatives are obtained by sufficiently washing the resin, removing the Fmoc protecting group, and cracking the resin. The various deuteration processes in the reaction are conventional guanamine condensation reactions, by adding an excess of reagents (amino acids or organic carboxylic acids) and potent condensing agents (such as DIC, DCC, HATU, HBTU, BOP, PyBOP, etc.) The various condensation reactions are completed.

Specific synthetic route: .

Reagents and conditions: (a) Fmoc-Lys(Boc)-OH, HOBt, DMAP, DIC, DCM, rt, 12h; (b) Ac2O, pyridine, DMAP, DCM, rt, 3h; (c) 20% piperidine /DMF; rt, 1h; (d) Fmoc-D-iso-Gln-OH, HOBt, DIC, DMF rt, 12h; (e) Fmoc-Ala-OH, HOBt, DIC, DMF, rt, 8h; RCOOH, HOBt, DIC, DMF, rt, 8h; (g) 90% TFA/H2O, rt, 2h.

Steps:

(a) Add King Resin (loading 0.83 mmol/g, 1.0 eq.), Fmoc-Lys(Boc)-OH (2.0 eq.), HOBt (2.0 eq.) and DMAP (0.05 eq.) to The solid phase reactor was vacuumed for 1 hour, and after adding anhydrous dichloromethane, the mixture was stirred for 0.5 hour, and then an activator DIC (2.0 eq.) was added thereto, and reacted at room temperature for 12 hours to introduce Fmoc-Lys (Boc) into the resin. -OH. The reaction solution was drained under reduced pressure, and the mixture was washed three times with N,N-dimethylformamide and methylene chloride, and dried, and applied directly to the next step.

(b) DCM, acetic anhydride (5.0 eq.), pyridine (5.0 eq.) and DMAP (0.05 eq.) were sequentially added to the reactor; the mixture was capped at room temperature for 3 hours, and the reaction solution was dried under reduced pressure. N,N-dimethylformamide and dichloromethane (1.5 L of the resin was washed 6 times, the reaction solution was drained, and the resin was washed three times with N,N-dimethylformamide and dichloromethane. , drained, used directly in the next step.

(c) adding 20% by volume of piperidine/N,N-dimethylformamide solution to remove the Fmoc protecting group of the amino acid. After reacting for 1 hour, the reaction solution is drained and N, N is used successively. The resin was thoroughly washed 6 times with dimethylformamide and dichloromethane, drained and used directly in the next step.

(d) Add Fmoc-D-iso-Gln-OH (2.0 eq.), HOBt (2.0 eq.) and N,N-dimethylformamide to the reactor and stir for 5 min until the system is homogeneous. The activator DIC (2.0 eq.) was introduced into the resin to introduce Fmoc-D-iso-Gln-OH at room temperature. After 12 hours of reaction, a small amount of resin was taken for ninhydrin detection. The resin did not appear blue, and the reaction was negative. After completion, the reaction solution was drained, and the resin was washed three times with N,N-dimethylformamide and dichloromethane, and dried, and used directly for the next step.

(e) Add Fmoc-Ala-OH (2.0 eq.), HOBt (2.0 eq.) and N,N-dimethylformamide solvent to the reactor, stir for 5 min until the system is homogeneous, then add activator DIC (2.0 eq.), Fmoc-Ala-OH was introduced into the resin at room temperature. After reacting for 8 hours, a small amount of resin was taken for ninhydrin detection. The resin did not appear blue, and the reaction was complete, indicating that the reaction was complete, and the reaction solution was drained. The resin was washed three times with N,N-dimethylformamide and dichloromethane, dried, and used directly in the next step.

(f) The organic acid RCOOH (1.5 eq.) and HOBt (1.5 eq.) and N,N-dimethylformamide were sequentially added to the reactor, stirred uniformly, and then DIC (1.5 eq.) was added. Warm reaction, introducing organic acid into the resin, after reacting for 8 hours, a small amount of resin was taken for ninhydrin detection, the resin did not appear blue, negative, indicating that the reaction was complete, and the reaction solution was drained, followed by N,N-dimethyl The methotrexate and dichloromethane were washed 3 times each and drained.

(g) 90% by volume of trifluoroacetic acid/water solution was added, cleavage at room temperature for 2 hours, filtration, and the resin was washed three times with dichloromethane, and the filtrate was combined with a lysate and evaporated to dryness under reduced pressure. Under ice-cooling conditions, a large amount of anhydrous methyl tert-butyl ether was added to the residue, and the mixture was stirred and evaporated. After filtration, the solid was washed three times with anhydrous methyl tert-butyl ether and dried under vacuum at 30 ° C to afford crude product.

General synthesis step 3 : Conjugate Synthesis of Docetaxel and Cell Wall Dipeptide Derivatives

Reagents and conditions: (a) HOSu, EDC.HCl, DMSO, rt, 12h; (b) NMM, DMSO, rt, 12h.

Procedure: (a) Add docetaxel 2'-O-alkyl diacid monoester (1.0 eq.), HOSu (1.1 eq.), EDC.HCl (1.1 eq.) and DMSO to the reactor. The reaction was stirred at room temperature for 12 h. The results of HPLC monitoring showed that the purity of docetaxel was less than 5% and the reaction was complete and used directly in the next reaction.

The cell wall dipeptide derivative oligopeptide (1.0 eq.) was added to another reactor, DMSO was added, and the mixture was stirred until dissolved at room temperature, then N-methylmorphine (5.0 eq.) was added, and the mixture was stirred at room temperature for 5 min. Then, the reaction solution of the above prepared N-hydroxysuccinimide active ester was slowly added dropwise to the reaction system, and reacted at room temperature for 12 hours, and monitored by HPLC to show an active ester intermediate of N-hydroxysuccinimide. A purity of less than 5% is considered complete. The reaction system was cooled to 0-4 ° C, and the N-methylmorphine in the reaction system was first neutralized with a 1 M aqueous hydrochloric acid solution, and the pH of the reaction system was adjusted to 3 to 5 with a 0.1 M aqueous hydrochloric acid solution. Then, the neutralized reaction solution was slowly added dropwise to 10 volumes of 0-4 ° C ice water to crystallize; the target crude product was obtained by filtration under reduced pressure; the crude product was washed three times with ice water and dried under vacuum at 30 ° C to obtain Target crude. The crude product was purified by HPLC to obtain the conjugate of the target docetaxel and the cell wall dipeptide derivative.

The alkane dianhydride of the present invention is selected from the group consisting of C4-C12 alkane dianhydrides.

The preparation method of the conjugate in the invention is mild, the reaction time is short, and the yield is stable, which is advantageous for the synthesis of the compound library by using, for example, combinatorial chemistry, and the method for synthesizing the compound library by the combined chemical method also belongs to the invention. The scope.

Among them, each abbreviation is defined as follows, and each reagent can be obtained commercially:

Another object of the present invention is to relate to the use of the conjugate in a medicament (agent) or prevention (agent) for treating and preventing various tumors, and various cancers caused thereby. The tumor is selected from the group consisting of melanoma, gastric cancer, lung cancer, preferably non-small cell lung cancer, breast cancer, breast cancer metastasis, renal cancer, liver cancer, oral epithelial cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, colon Cancer, colon cancer metastasis, brain cancer, preferably brain cancer caused by glioma.

Another object of the present invention is to provide a pharmaceutical composition comprising the conjugate compound or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers. The carrier includes, but is not limited to, various excipients suitable for pharmaceutical preparations, which can be administered to a mammal, particularly a cancer patient, via the gastrointestinal or parenteral route. For example, gastrointestinal administration includes various fillers which may be tablets or granules, such as microcrystalline cellulose.

The compound of the present invention has the following advantages: 1. The compound of the present invention has significantly better solubility and hydrophilicity, can greatly reduce or avoid the use of the dissolution aid in the research of the pharmaceutical preparation, and has significantly better drug-forming properties and anti-allergic drugs. Sex. 2. The compound of the present invention has a good therapeutic effect on a disease caused by abnormal expression of a gene, such as a tumor. 3. The compound of the present invention has a function of suppressing tumor metastasis while suppressing tumor.

The various aspects and features of the present invention are specifically illustrated below by preferred embodiments of conjugate synthesis and biology of docetaxel and cell wall dipeptide (MDP) derivatives. Those skilled in the art will appreciate that the examples are for illustrative purposes only and are not intended to limit the scope of the invention. The scope of protection of the present invention is limited only by the scope of the patent application. Various modifications and improvements can be made to the various aspects of the invention without departing from the scope of the invention, and such modifications and improvements are also within the scope of the invention.

In addition, it should be noted that the various materials and reagents used in the following examples are all materials and reagents commonly used in the art, unless otherwise specified, and can be obtained by conventional commercial means; the intermediates used can be obtained through conventional commercial routes. It is obtained or prepared by a known method; the methods used are all conventional methods well known to those skilled in the art.

Chemical example

Example 1: Solid phase synthesis of the cell wall dipeptide derivative MDA-1 (general step 2)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.59 (1H, br.s), 8.47 (1H, d, J = 6.8 Hz), 8.24 (1H, d, J = 8.1 Hz), 8.11 (1H , d, J = 7.8 Hz), 7.76 (1H, dd, J = 8.8, 6.2 Hz), 7.73-7.63 (3H, m), 7.55 (1H, dd, J = 8.8, 2.6Hz), 7.37-7.27 ( 2H, m), 7.13 (1H, s), 6.78 (1H, d, J = 15.7 Hz), 4.42 (1H, q, J = 6.9 Hz), 4.15 (2H, m), 2.77 (2H, m), 2.16 (2H, t, J = 8.0 Hz), 1.97 (1H, m), 1.71 (2H, m), 1.60-1.45 (3H, m), 1.40-1.20 (6H, m). HR-MS(ESI- TOF) m/z: Calcd for C ₂₃ H ₃₂ N ₅ O ₆ FCl [M-CF ₃ COO] ⁺ 528.2020; Found 528.2023.

Example 2: Solid phase synthesis of the cell wall dipeptide derivative MDA-2 (general step 2)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.58 (1H, br.s), 8.38 (1H, d, J = 6.8 Hz), 8.21 (1H, d, J = 8.1 Hz), 8.10 (1H , d, J = 7.8 Hz), 7.70 (3H, s), 7.59 (2H, d, J = 8.3 Hz), 7.48 (2H, d, J = 8.3 Hz), 7.40 (1H, d, J = 15.8 Hz) ), 7.31 (1H, s), 7.11 (1H, s), 6.77 (1H, d, J = 15.8 Hz), 4.40 (1H, q, J = 6.7 Hz), 4.15 (2H, m), 2.76 (2H , m), 2.16 (2H, t, J = 8.0Hz), 1.96 (1H, m), 1.71 (2H, m), 1.60-1.45 (3H, m), 1.40-1.20 (6H, m). HR- MS (ESI-TOF) m/z: Calcd for C ₂₃ H ₃₃ N ₅ O ₆ Cl [M-CF ₃ COO] ⁺ 510.2114; Found 510.2116.

Example 3: Solid phase synthesis of the cell wall dipeptide derivative MDA-3 (general step 2)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.58 (1H, br.s), 8.54 (1H, d, J = 6.1 Hz), 8.25 (1H, d, J = 7.7 Hz), 8.12 (1H , d, J = 7.3 Hz), 7.96 (1H, s), 7.85-7.60 (4H, m), 7.53 (1H, d, J = 10.5 Hz), 7.44 (1H, d, J = 15.9 Hz), 7.40 -7.30 (2H, m), 7.13 (1H, s), 6.87 (1H, d, J = 15.9 Hz), 4.40 (1H, q, J = 6.7 Hz), 4.15 (2H, m), 2.76 (2H, m), 2.16 (2H, t , J = 8.0 Hz), 1.96 (1H, m), 1.71 (2H, m), 1.60-1.45 (3H, m), 1.40-1.20 (6H, m). HR-MS (ESI-TOF) m/z: Calcd for C ₂₃ H ₃₂ N ₅ O ₆ FCl [M-CF ₃ COO] ⁺ 528.2020; Found 528.1924.

Example 4: Solid phase synthesis of the cell wall dipeptide derivative MDA-4 (general step 2)

HR-MS (ESI-TOF) m/z: Calcd for C ₂₄ H ₃₆ N ₅ O ₆ [M-CF ₃ COO] ⁺ 490.2660; Found 490.2666.

Example 5: Solid phase synthesis of the cell wall dipeptide derivative MDA-5 (general step 2)

HR-MS (ESI-TOF) m/z: Calcd for C ₂₄ H ₃₃ N ₆ O ₆ [M-CF ₃ COO] ⁺ 501.2456; Found 501.2446.

Example 6: Solid phase synthesis of the cell wall dipeptide derivative MDA-6 (general step 2)

HR-MS (ESI-TOF) m/z: Calcd for C ₂₁ H ₃₂ N ₅ O ₆ S [M-CF ₃ COO] ⁺ 482.2068; Found 482.2053.

Example 7: Synthesis of S-01 (general synthesis step 3)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.49 (1H, br.s), 8.45 (1H, d, J = 6.7 Hz), 8.25 (1H, d, J = 8.0 Hz), 8.09 (1H , d, J = 7.5 Hz), 7.99 (2H, d, J = 7.5 Hz), 7.90-7.80 (2H, m), 7.80-7.70 (2H, m), 7.70-7.60 (3H, m), 7.54 ( 1H, dd, J = 2.2, 8.7 Hz), 7.45-7.30 (6H, m), 7.18 (1H, t, J = 6.7 Hz), 7.11 (1H, s), 6.79 (1H, d, J = 15.7 Hz ), 5.79 (1H, t, J = 8.2 Hz), 5.41 (1H, d, J = 6.9 Hz), 5.15-4.85 (6H, m), 4.50-4.38 (2H, m), 4.20-4.10 (2H, m), 4.10-3.95 (3H, m), 3.64 (1H, d, J = 6.5 Hz), 3.02 (2H, m), 2.70-2.55 (2H, m), 2.38 (2H, t, J = 6.9 Hz ), 2.30-2.05 (6H, m), 1.98 (1H, m), 1.85 (1H, m), 1.78-1.60 (6H, m), 1.60-1.47 (5H, m), 1.45-1.20 (16H, m ), 0.99 (6H, s). HR-MS (ESI-TOF) m/z: Calcd for C ₇₀ H ₈₆ N ₆ O ₂₂ FClNa [M+Na] ⁺ 1439.5365; Found 1439.5358.

Example 8: Synthesis of S-01-Na sodium salt

S-01 (283 mg, 0.2 mmol) was dissolved in 50 mL of a mixed solvent of acetonitrile / water = 1 / 1 (V / V), then the reaction flask was placed in a low temperature reactor at -10 ° C, stirred for 5 min. To the above solution, 100 mL of a 0.002 mmol/mL aqueous NaOH solution was added dropwise, and after completion of the dropwise addition, the resulting solution was quickly reacted at -20 ° C for 4 hours; and lyophilized to obtain 290 mg of a white powder solid.

¹ H-NMR (500 MHz, DMSO- d ₆ ): 8.45 (1H, d, J = 6.7 Hz), 8.25 (1H, d, J = 8.0 Hz), 8.09 (1H, d, J = 7.5 Hz), 7.99 (2H, d, J = 7.5 Hz), 7.90-7.80 (2H, m), 7.80-7.70 (2H, m), 7.70-7.60 (3H, m), 7.54 (1H, dd, J = 2.2, 8.7 Hz), 7.45-7.30 (6H, m), 7.18 (1H, t, J = 6.7 Hz), 7.11 (1H, s), 6.79 (1H, d, J = 15.7 Hz), 5.79 (1H, t, J = 8.2 Hz), 5.41 (1H, d, J = 6.9 Hz), 5.15-4.85 (6H, m), 4.50-4.38 (2H, m), 4.20-4.10 (2H, m), 4.10-3.95 (3H, m), 3.64 (1H, d, J = 6.5 Hz), 3.02 (2H, m), 2.70-2.55 (2H, m), 2.38 (2H, t, J = 6.9 Hz), 2.30-2.05 (6H, m ), 1.98 (1H, m), 1.85 (1H, m), 1.78-1.60 (6H, m), 1.60-1.47 (5H, m), 1.45-1.20 (16H, m), 0.99 (6H, s). HR-MS (ESI-TOF) m/z: Calcd for C ₇₀ H ₈₅ N ₆ O ₂₂ FCl [M-Na] ^- 1415.5395; Found 1415.5380.

Example 9: Synthesis of S-01-Ca calcium salt

S-01 (283 mg, 0.2 mmol) was dissolved in 20 mL of a mixed solvent of acetonitrile / water = 1 / 1 (v / v), then the reaction flask was placed in a low temperature reactor at 0 ° C, stirred for 5 min, To the above solution, 20 mL of 0.01 mmol/mL Ca(OH) ₂ aqueous solution was added dropwise. After the addition was completed, the obtained solution was quickly poured into 80 mL of ice water, and the temperature was lowered to -20 ° C for 4 hours; lyophilized to obtain white. Powder solid 260 mg.

¹ H-NMR (500 MHz, DMSO- d ₆ ): 8.45 (1H, d, J = 6.7 Hz), 8.25 (1H, d, J = 8.0 Hz), 8.09 (1H, d, J = 7.5 Hz), 7.99 (2H, d, J = 7.5 Hz), 7.90-7.80 (2H, m), 7.80-7.70 (2H, m), 7.70-7.60 (3H, m), 7.54 (1H, dd, J = 2.2, 8.7 Hz), 7.45-7.30 (6H, m), 7.18 (1H, t, J = 6.7Hz), 7.11 (1H, s), 6.79 (1H, d, J = 15.7 Hz), 5.79 (1H, t, J = 8.2 Hz), 5.41 (1H, d, J = 6.9 Hz), 5.15-4.85 (6H, m), 4.50-4.38 (2H, m), 4.20-4.10 (2H, m), 4.10-3.95 (3H, m), 3.64 (1H, d, J = 6.5 Hz), 3.02 (2H, m), 2.70-2.55 (2H, m), 2.38 (2H, t, J = 6.9Hz), 2.30-2.05 (6H, m ), 1.98 (1H, m), 1.85 (1H, m), 1.78-1.60 (6H, m), 1.60-1.47 (5H, m), 1.45-1.20 (16H, m), 0.99 (6H, s). HR-MS (ESI-TOF) m/z: Calcd for C ₇₀ H ₈₅ N ₆ O ₂₂ FCl [M-1/2Ca] ^- 1415.5395; Found 1415.5386.

Example 10: Synthesis of S-01-Me methyl ester

Reagents and conditions: (a) SOCl2, MeOH, 0 °C-rt, 12h; (b) HOSu, EDC.HCl, DMSO, rt, 12h; (c) NMM, DMSO, rt, 12h.

Reaction operation: (a) MDA-1 (6.0 g, 9.4 mmol) was dissolved in 80 mL of methanol under ice bath, stirred for 5 min, and dichlorohydrazine (0.75 mL, 10.37 mmol) was slowly added dropwise to the above reactor. After the dropwise addition was completed, the reaction system was allowed to rise to room temperature, and the reaction was carried out for 12 hours. The solvent was evaporated to dryness <RTI ID=0.0></RTI> to <RTI ID=0.0></RTI> to <RTI ID=0.0></RTI> </RTI> <RTIgt; For the steps (b) and (c), see General Synthesis Step 3. Finally, 4.5 g of pure S-01-Me was obtained in a yield of 34% (three-step yield).

¹ H-NMR (500 MHz, DMSO- d ₆ ): 8.42 (1H, d, J = 6.8 Hz), 8.20 (2H, t, J = 7.8 Hz), 7.98 (2H, d, J = 7.5 Hz), 7.90-7.60 (7H, m), 7.53 (1H, dd, J = 2.6, 8.8 Hz), 7.45-7.25 (6H, m), 7.18 (1H, t, J = 6.8 Hz), 7.09 (1H, s) , 6.78 (1H, d, J = 15.7 Hz), 5.79 (1H, t, J = 8.3Hz), 5.40 (1H, d, J = 7.1 Hz), 5.09 (3H, s), 4.98 (1H, d, J = 7.1 Hz), 4.90 (2H, d, J = 10.4 Hz), 4.50-4.35 (2H, m), 4.25-4.10 (2H, m), 4.10-3.95 (3H, m), 3.64 (1H, d , J = 7.0 Hz), 3.61 (3H, s), 3.01 (2H, q, J = 5.9 Hz), 2.70-2.55 (2H, m), 2.38 (2H, t, J = 7.1 Hz), 2.35-2.20 (4H, m), 2.17 (2H, t, J = 8.0 Hz), 1.98 (1H, m), 1.85 (1H, m), 1.78-1.60 (6H, m), 1.60-1.55 (2H, m), . 1.52 (3H, s), 1.45-1.20 (16H, m), 0.99 (6H, s) HR-MS (ESI-TOF) m / z: Calcd for C 71 H 87 N 6 O 22 FCl [MH] - 1429.5551; Found 1429.5549.

Example 11: Synthesis of S-02 (general synthesis step 3)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.42 (1H, br.s), 8.48 (1H, d, J = 5.2 Hz), 8.21 (1H, d, J = 7.3 Hz), 8.06 (1H , d, J = 6.7 Hz), 7.96 (2H, d, J = 6.6Hz), 7.90-7.75 (2H, m), 7.75-7.55 (4H, m), 7.51 (1H, d, J = 10.3 Hz) , 7.45-7.20 (6H, m), 7.20-7.00 (2H, m), 6.83 (1H, d, J = 16.0 Hz), 5.76 (1H, m), 5.37 (1H, d, J = 6.0Hz), 5.15-4.80 (6H, m), 4.50-4.30 (2H, m), 4.20-3.90 (5H, m), 3.61 (1H, m), 2.98 (2H, m), 2.70-2.55 (2H, m), 2.40-2.30 (2H, m), 2.30-2.05 (6H, m), 1.94 (1H, m), 1.80 (1H, m), 1.75-1.57 (6H, m), 1.57-1.43 (5H, m), . 1.42-1.15 (16H, m), 0.95 (6H, s) HR-MS (ESI-TOF) m / z: Calcd for C 70 H 86 N 6 O 22 FClNa [m + Na] + 1439.5365; Found 1439.5360.

Example 12: Synthesis of S-03 (general synthesis step 3)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.45 (1H, br.s), 8.28 (1H, d, J = 6.6 Hz), 8.18 (1H, d, J = 7.8 Hz), 8.06 (1H , d, J = 7.6 Hz), 7.96 (2H, d, J = 7.5 Hz), 7.90-7.75 (2H, m), 7.70 (1H, t, J = 7.1 Hz), 7.63 (2H, t, J = 7.3 Hz), 7.45-7.25 (8H, m), 7.20 (2H, d, J = 7.6 Hz), 7.16 (1H, t, J = 6.6 Hz), 7.08 (1H, s), 6.67 (1H, d, J = 15.7Hz), 5.76 (1H, t, J = 8.1 Hz), 5.38 (1H, d, J = 6.8 Hz), 5.06 (3H, s), 4.99 (1H, d, J = 6.8Hz), 4.95 -4.80 (2H, m), 4.45-4.30 (2H, m), 4.20-4.05 (2H, m), 4.05-3.90 (3H, m), 3.62 (1H, d, J = 6.3Hz), 2.98 (2H , m), 2.70-2.55 (2H, m), 2.36 (2H, t, J = 6.7Hz), 2.30 (3H, s), 2.26-2.05 (6H, m), 1.94 (1H, m), 1.82 ( 1H, m), 1.75-1.57 (6H, m), 1.57-1.43 (5H, m), 1.42-1.15 (16H, m), 0.95 (6H, s). HR-MS(ESI-TOF) m/z Calcd for C ₇₁ H ₉₁ N ₆ O ₂₂ [M+H] ⁺ 1379.6186; Found 1379.6180.

Example 13: Synthesis of S-04 (general synthesis step 3)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.54 (1H, br.s), 8.99 (1H, d, J = 5.2 Hz), 8.64 (1H, d, J = 6.9 Hz), 8.45 (1H , d, J = 5.2 Hz), 8.30-8.10 (4H, m), 8.03 (2H, d, J = 5.2 Hz), 7.98-7.85 (3H, m), 7.85-7.65 (4H, m), 5.84 ( 1H, m), 5.46 (1H, d, J = 6.0Hz), 5.25-4.90 (6H, m), 4.70 (1H, m), 4.50 (1H, s), 4.30-4.00 (5H, m), 3.69 (1H, d, J = 6.8 Hz), 3.07 (2H, m), 2.80-2.65 (2H, m), 2.50-2.35 (2H, m), 2.35-2.15 (6H, m), 2.03 (1H, m ), 1.90 (1H, m), 1.75-1.57 (6H, m), 1.57-1.43 (5H, m), 1.42-1.15 (16H, m), 1.03 (6H, s). HR-MS(ESI-TOF m/z: Calcd for C ₇₁ H ₈₇ N ₇ O ₂₂ Na [M+Na] ⁺ 1412.5802; Found 1412.5794.

Example 14: Synthesis of S-05 (general synthesis step 3)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.47 (1H, br.s), 8.35 (1H, d, J = 5.1 Hz), 8.19 (1H, d, J = 7.1 Hz), 8.07 (1H , d, J = 6.8 Hz), 7.99 (2H, d, J = 6.6 Hz), 7.90-7.80 (1H, m), 7.78-7.50 (5H, m), 7.50-7.33 (5H, m), 7.30 ( 1H, s), 7.20-7.05 (3H, m), 6.50 (1H, d, J = 15.6 Hz), 5.79 (1H, t, J = 8.1 Hz), 5.40 (1H, d, J = 6.8 Hz), 5.06 (3H, s), 4.99 (1H, d, J = 6.8 Hz), 4.95-4.80 (2H, m), 4.50-4.30 (2H, m), 4.20-4.10 (2H, m), 4.10-3.90 ( 3H, m), 3.63 (1H, d, J = 6.3 Hz), 3.01 (2H, s), 2.70-2.55 (2H, m), 2.38 (2H, t, J = 6.7 Hz), 2.30-2.05 (6H , m), 1.96 (1H, m), 1.84 (1H, m), 1.75-1.57 (6H, m), 1.57-1.43 (5H, m), 1.42-1.15 (16H, m), 0.98 (6H, s HR-MS (ESI-TOF) m/z: Calcd for C ₆₈ H ₈₅ N ₆ O ₂₂ S [MH] ^- 1369.5443; Found 1369.5445.

Example 15: Synthesis of S-06 (general synthesis step 3)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.48 (1H, br.s), 8.98 (1H, d, J = 7.0 Hz), 8.25-8.15 (2H, m), 8.10 (1H, d, J = 7.4 Hz), 7.98 (2H, d, J = 7.3 Hz), 7.93-7.80 (3H, m), 7.75-7.60 (4H, m), 7.45-7.30 (4H, m), 7.27 (1H, s ), 7.18 (1H, t, J = 4.8 Hz), 7.11 (1H, s), 5.78 (1H, t, J = 8.1 Hz), 5.40 (1H, d, J = 7.0Hz), 5.08 (3H, s ), 5.01 (1H, d, J = 6.4 Hz), 4.95-4.80 (2H, m), 4.50-4.30 (2H, m), 4.19 (1H, q, J = 5.4 Hz), 4.11 (1H, q, J = 5.4 Hz), 4.08-3.95 (3H, m), 3.63 (1H, d, J = 6.6 Hz), 3.01 (2H, s), 2.70-2.55 (2H, m), 2.38 (2H, t, J = 6.8 Hz), 2.30-2.05 (6H, m), 1.95 (1H, m), 1.83 (1H, m), 1.78-1.60 (6H, m), 1.60-1.43 (5H, m), 1.42-1.15 ( 16H, m), 0.98 (6H, s). HR-MS (ESI-TOF) m/z: Calcd for C ₆₈ H ₈₃ ClN ₇ O ₂₄ [MH] ^- 1416.5183; Found 1416.5167.

Example 16: Synthesis of S-07 (general synthesis step 3)

^{1 H-NMR (500MHz, DMSO-} d 6): 12.49 (1H, br.s), 8.48 (1H, d, J = 6.7 Hz), 8.24 (1H, d, J = 8.1 Hz), 8.09 (1H, d, J = 7.6 Hz), 7.99 (2H, d, J = 7.4 Hz), 7.93-7.80 (2H, m), 7.77-7.60 (4H, m), 7.50-7.30 (7H, m), 7.18 (2H , t, J = 7.2 Hz), 7.12 (1H, s), 6.82 (1H, d, J = 16.0 Hz), 5.78 (1H, t, J = 8.6 Hz), 5.40 (1H, d, J = 7.0 Hz ), 5.15-4.80 (6H, m), 4.50-4.35 (2H, m), 4.20-3.95 (5H, m), 3.63 (1H, d, J = 6.7 Hz), 3.01 (2H, m), 2.70- 2.55 (2H, m), 2.38 (2H, t, J = 7.0 Hz), 2.30-2.05 (6H, m), 1.98 (1H, m), 1.84 (1H, m), 1.78-1.60 (6H, m) , 1.60-1.45 (5H, m), 1.40-1.20 (16H, m), 0.98 (6H, s). HR-MS(ESI-TOF) m/z: Calcd for C ₇₀ H ₈₅ N ₆ O ₂₂ F ₂ [MH] ^- 1399.5690; Found 1399.5688.

Example 17: Synthesis of S-08 (general synthesis step 3)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.49 (1H, br.s), 8.34 (1H, d, J = 6.8 Hz), 8.23 (1H, d, J = 8.1 Hz), 8.09 (1H , d, J = 7.5Hz), 7.98(2H, d, J = 7.4Hz), 7.91-7.80(2H, m), 7.75-7.60(4H, m), 7.55-7.28(8H, m), 7.18( 1H, t, J = 7.1Hz), 7.11(1H, s), 6.75(1H, d, J = 15.8Hz), 5.78(1H, t, J = 8.6Hz), 5.40(1H, d, J = 7.1 Hz), 5.15-4.85(6H, m), 4.50-4.35(2H, m), 4.20-3.95(5H, m), 3.63(1H, d, J = 7.0Hz), 3.00(2H, m), 2.70 -2.55(2H, m), 2.37(2H, t, J = 7.0Hz), 2.30-2.05(6H, m), 1.98(1H, m), 1.83(1H, m), 1.78-1.60(6H, m ), 1.60-1.45 (5H, m), 1.40-1.20 (16H, m), 0.98 (6H, s).

HR-MS (ESI-TOF) m/z: Calcd for C ₇₀ H ₈₅ N ₆ O ₂₂ F ₂ [MH] ^- 1399.5690; Found 1399.5692.

Example 18: Synthesis of S-09 (general synthesis step 3)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.49 (1H, br.s), 8.35 (1H, d, J = 6.8 Hz), 8.24 (1H, d, J = 8.0 Hz), 8.10-7.95 (3H, m), 7.90-7.80(2H, m), 7.78-7.60(4H, m), 7.60-7.30(9H, m), 7.17(1H, t, J = 7.1Hz), 7.10(1H, s ), 6.83(1H, d, J = 15.9Hz), 5.78(1H, t, J = 8.6Hz), 5.40(1H, d, J = 7.0Hz), 5.15-4.85(6H, m), 4.50-4.35 (2H, m), 4.20-3.95(5H, m), 3.63(1H, d, J = 7.0Hz), 3.00(2H, m), 2.70-2.55(2H, m), 2.38(2H, t, J = 7.1 Hz), 2.30-2.05 (6H, m), 1.98 (1H, m), 1.82 (1H, m), 1.78-1.60 (6H, m), 1.60-1.45 (5H, m), 1.40-1.20 ( 16H, m), 0.98 (6H , s) .HR-MS (ESI-TOF) m / z: Calcd for C 70 H 87 N 6 O 22 FK [m + K] + 1421.5495; Found 1421.5627.

Example 19: Synthesis of S-10 (general synthesis step 3)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.48 (1H, br.s), 9.85 (1H, s), 8.20 (2H, dd, J = 11.3, 7.6 Hz), 8.08 (1H, d, J = 7.8 Hz), 7.98 (2H, d, J = 7.4 Hz), 7.90-7.80 (2H, m), 7.72 (1H, t, J = 7.2 Hz), 7.65 (2H, t, J = 7.4 Hz) , 7.45-7.25(8H, m), 7.18(1H, t, J = 7.0Hz), 7.10(1H, s), 6.79(2H, d, J = 8.5Hz), 6.52(1H, d, J = 15.8 Hz), 5.78 (1H, t, J = 8.6Hz), 5.40(1H, d, J = 7.1Hz), 5.15-4.85(6H, m), 4.50-4.35(2H, m), 4.20-3.95(5H , m), 3.63(1H, d, J = 6.8Hz), 3.01(2H, m), 2.70-2.55(2H, m), 2.37(2H, t, J = 7.1Hz), 2.30-2.05(6H, m), 1.99(1H, m), 1.82(1H, m), 1.78-1.60(6H, m), 1.60-1.45(5H, m), 1.40-1.20(16H, m), 0.98(6H, s) .HR-MS (ESI-TOF) m/z: Calcd for C ₇₀ H ₈₈ N ₆ O ₂₃ Na [M+Na] ⁺ 1403.5799; Found 1403.5795.

Example 20: Synthesis of S-11 (general synthesis step 3)

¹ H-NMR (500 MHz, DMSO- d ₆ ): 12.49 (1H, br.s), 8.75 (1H, s), 8.56 (1H, d, J = 4.1 Hz), 8.42 (1H, d, J = 6.8 Hz), 8.25 (1H, d, J = 8.0 Hz), 8.09 (1H, d, J = 6.9 Hz), 7.98 (3H, d, J = 7.7 Hz), 7.93-7.80 (2H, m), 7.80 -7.60(3H, m), 7.50-7.30(7H, m), 7.18(1H, d, J = 6.6Hz), 7.12(1H, s), 6.87(1H, d, J = 16.0Hz), 5.78( 1H, t, J = 8.6Hz), 5.40(1H, d, J = 6.9Hz), 5.15-4.85(6H, m), 4.50-4.35(2H, m), 4.20-3.95(5H, m), 3.63 (1H, d, J = 6.8Hz), 3.01(2H, m), 2.70-2.55(2H, m), 2.37(2H, t, J = 6.8Hz), 2.30-2.05(6H, m), 1.96( 1H, m), 1.81(1H, m), 1.78-1.60(6H, m), 1.60-1.45(5H, m), 1.40-1.20(16H, m), 0.98(6H, s).HR-MS( ESI-TOF) m/z: Calcd for C ₆₉ H ₈₆ N ₇ O ₂₂ [MH] ^- 1364.5831; Found 1364.5820.

Example 21: Synthesis of S-12 (general synthesis step 3)

¹ H-NMR (300 MHz, DMSO- d ₆ ): 12.44 (1H, br.s), 8.62 (1H, d, J = 4.0 Hz), 8.57 (1H, d, J = 6.7 Hz), 8.21 (1H , d, J = 8.2Hz), 8.09(1H, d, J = 7.7Hz), 7.99(2H, d, J = 7.1Hz), 7.93-7.80(3H, m), 7.78-7.62(3H, m) , 7.58(1H, d, J = 7.8Hz), 7.50-7.28(7H, m), 7.16(3H, m), 5.78(1H, t, J = 8.5Hz), 5.40(1H, d, J = 7.1 Hz), 5.15-4.85(6H, m), 4.50-4.35(2H, m), 4.20-3.95(5H, m), 3.63(1H, d, J = 7.1Hz), 3.01(2H, m), 2.70 -2.55(2H, m), 2.38(2H, t, J = 7.0Hz), 2.30-2.05(6H, m), 1.98(1H, m), 1.82(1H, m), 1.78-1.60(6H, m ), 1.60-1.45 (5H, m), 1.40-1.20 (16H, m), 0.98 (6H, s). HR-MS (ESI-TOF) m/z: Calcd for C ₆₉ H ₈₆ N ₇ O ₂₂ [ MH] ^- 1364.5831; Found 1364.5826.

The compound S-01 disclosed in the present invention and the MDC-405 reported in the literature are operated according to the pharmacopoeia solubility standard, and the test results show that as the pH value of the buffer increases, the solubility gradually increases, and when the pH value is 6.5 or more, S The solubility of -01 reaches the dissolution level, exhibits better hydrophilicity, and thus exhibits markedly superior drug-forming properties. See Table 1 for the test results. 【Table 1】 Remarks: Very soluble: 1g solute dissolved in less than 1ml solvent; soluble: 1g solute dissolved in 1~10ml solvent; Dissolved: 1g solute dissolved in 10~30ml solvent; slightly soluble: 1g solute in 30~100ml Dissolved in solvent; slightly soluble: 1g solute dissolved in 100~1000ml solvent; very slightly dissolved: 1g solute dissolved in 1000~10000ml solvent; almost insoluble or insoluble means that 1g solute can not be completely dissolved in 10000ml solvent. Biological Example In Vitro Activity Test Section

Example 22

First, experimental cells : NCI-H460, human large cell lung cancer cells; A549, human non-small cell lung cancer cells.

2. Experimental materials 2.1 Cell culture related materials and reagents 2.2 96-well plate 2.3 Dispenser 2.4 SRB kit 2.4.1 Trimethylamine-based methane (Trizma base): V900483-500G, Lot#WXBB4482V, PCode: 101421913 2.4 .2 Trichloroacetic acid (TCA): T9159-500G, Lot#BCBL5964V, PCode: 10146647 2.4.3 Sulforhodamine B (SRB): 341737-1G, Lot #20223EAV, PCode: 1001890567 2.5 Absorbent paper 2.6 Draining gun and pipette head 2.7 Sterilizing water 2.8 Dimethyl hydrazine DIMETHYL SULPHOXIDE (DMSO): D2438, Lot RNBD1974, Exp: 04/2016 2.9 Acetic acid: 338826-500ML, Lot#SHBD0354V, PCode: 1001616171

Third, the experimental method

3.1 cell activation The water bath was warmed to 37 ° C, preheated in 10% FBS and 1% penicillin-streptomycin in RPMI-1640 medium at 37 °C; the centrifuge was turned on to reduce the temperature to 4 °C; Take about 5 ml of 10% FBS and 1% penicillin-streptomycin in RPMI-1640 medium in a safety cabinet into a 15 ml centrifuge tube for use; The frozen cells were taken out from the liquid nitrogen tank into a measuring cup containing 37 ° C water, and the frozen tube was shaken in a 37 ° C water bath to dissolve about 90%-95% of the frozen solution; Move the cell-containing cryopreservation solution to Centrifuge in a centrifuge tube at 1000 rpm/min for 5 min; The supernatant was removed, and RPMI-1640 medium supplemented with about 1 ml of 10% FBS and 1% penicillin-streptomycin was shaken and mixed, and transferred to RPMI-1640 medium containing 4 ml of 10% FBS and 1% penicillin-streptomycin. 25 cm ^{2 in the} flask; Incubate in an incubator at 37 ° C, 5% CO 2 , and 100% relative humidity.

3.2 cell passage Turn on the centrifuge to reduce the temperature to 4 °C; remove the medium in the flask from the extraction cabinet, and rinse the flask 1 or 2 times with PBS; Add 1 ml of 0.25% trypsin-EDTA and digest at 37 °C; Digestion until a small amount of cells were observed to shed, about 2.5 min, the digestion was terminated by adding 10 ml of 10% FBS and 1% penicillin-streptomycin in RPMI-1640 medium, and the cells were dispersed and transferred to a 15 ml centrifuge tube; 1000 rpm/min, 4 ° C, centrifugation for 5 min; The supernatant was removed, and RPMI-1640 medium supplemented with 1 ml of 10% FBS and 1% penicillin-streptomycin was shaken and passaged 1:3 to 75 cm2; Incubate in an incubator at 37 ° C, 5% CO 2 , and 100% relative humidity.

3.3 SRB Kit and reagent preparation

Formulated in 10 mM Tris base (ddH ₂ O): 0.1211 g of trishydroxymethylaminomethane was accurately weighed and made up to 100 ml by adding ddH2O.

Preparation of 0.4% SRB (1% acetic acid): Weigh accurately 0.4 g of SRB, and add 1% acetic acid to make up to 100 ml.

1% Acetic acid: Accurately measure 5 ml of glacial acetic acid, and add ddH ₂ O to make up to 500 ml.

TCA (50%) (ddH ₂ O): Weigh accurately 50 g of TCA and add ddH ₂ O to make up to 100 ml.

3.4 Cell seeding (collecting cells, plating)

H460 cells or A549 were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin. Logarithmic growth phase cells were shaken with 5%-1640 complete medium, the cell density was adjusted to 7.5*10 ⁴ cells/mL, 100 μL/well, seeded in 96-well plates, and the culture plates were placed in an incubator for 24 hours.

3.5 Drug (compound configuration)

3.5.1 Method for configuring the storage compound storage solution

3.5.2 Preparation method of the test compound storage solution:

The 20Mm stock solution is diluted ten times to obtain 2*10 ^-3 M dilution solution. Similarly, it is diluted to 2*10 ^-4 M, 2*10 ^-5 M, 2*10 ^-6 M, 2*10 ^{- 7} M, 2*10 ^-8 M, 2*10 ^-9 M dilution, protected from light, stored at 4 °C.

3.6 Parallel control group cell fixation

3.6.1 50% TCA placed at 4 ° C for 1 h

3.6.2 Add 25 μL/well TCA to the parallel control plate and place at 4 °C for 1 h.

3.6.3 Wash 5 times with deionized water and dry naturally.

3.7 Cell administration

3.7.1 Vehicle control group: Take 4 μL of DMSO, add 796 μL of 5%-1640 medium, 100 μL/well/plate.

3.7.2 Test group: Take 2 μL of each concentration solution and add 398 μL of 5%-1640 complete medium to obtain 10 ^-4 M, 10 ^-5 M, 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, 100 μL/well/plate, sequentially loaded.

3.8 Administration group Fixed (drug treatment 48h Rear)

3.8.1 50% TCA placed at 4 ° C for 1 h

3.8.2 Add 50 μL/well TCA to the test panel and place it at 4 °C for 1 h.

3.8.3 Wash 5 times with deionized water, dry naturally

3.9 Cell staining

3.9.1 Add 100 μL/well 0.4% SRB to the plate and let stand for 10 min at room temperature.

3.9.2 Wash 1% acetic acid 5 times and dry naturally (remove unbound SRB)

3.10 Detection

Add 150 μL/well 10 mM Tris lye to the plate, shake for 5 min, and measure OD at 515 nm.

3.11 Data processing method

The OD value of the parallel control group was expressed as Tz, the OD value of the vehicle control group was expressed as C, and the OD value of the test administration group was expressed as Ti:

If Ti ≥ Tz, it means that the cells still grow after dosing.

Percentage growth = [(Ti-Tz) / (C-Tz)] × 100

If Ti <Tz, it means the cells are killed after dosing.

Percentage growth = [(Ti-Tz) / Tz] × 100

The curve was fitted with software GraphPad and the GI50 was calculated.

The preferred compounds disclosed in the present invention, the results of experiments on A549 and H460 human tumor cell lines, indicate that the growth inhibition 50% value (GI50 value) of this type of conjugate has a smaller GI50 value relative to MDC-405, It is predicted to have a better anti-tumor effect, and the experimental results are shown in the attached Table 2. [Table 2] GI50 in vitro test results Note: E means ×10 ⁿ , for example, 5.06E-08 means 5.06 × 10 ^-8 .

In vivo activity test

Example twenty three Using mouse breast cancer 4T1 Lung metastasis model:

(1) Cell culture and tumor inoculation: 4T1 cells were cultured in 1640 medium (Gibco) containing 10% fetal calf serum (Hyclone Corp, USA), 1% glutamine and 1% cyan-streptomycin. 4T1 cells in the logarithmic growth phase were collected and the cell concentration was adjusted to 1.5 × 10 ⁶ /mL. Female BALB/c mice were inoculated into 4T1 cells in the 4th mammary fat pad, and the inoculation volume was 0.1 mL, that is, 1.5×10 ⁵ /piece.

4T1 breast cancer cells were inoculated into the mammary fat pad of BALB/c mice, and D0 was set on the day of inoculation. Group 4 was administered on the 4th day after inoculation. Four groups were set up in the experiment: Solvent control group (blank control group), S-01 10mg/kg group, DTX (Docetaxel) 5.7 mg/kg group, MDC-405 10 mg/kg group. with It was a positive control group.

10 animals per group. Mice were administered once a week in the tail vein for 4 consecutive weeks. Animal body weight and tumor volume were monitored during the administration. The body weight of the animal was weighed every 2 to 3 days, and the long and short diameters of the breast tumor were measured with a vernier caliper. The tumor size was calculated by the formula: (1/2) × long diameter × (short diameter) ² . On the 28th day after the tumor was received (D28), the experiment was performed. After the blood was collected from the eyeball, the mice were sacrificed by cervical dislocation. The breast tumor was weighed and the lung weight was calculated. The number of metastatic nodules on the lung surface was calculated. The tumor weight test results are shown in Fig. 1A, and the experimental results of the number of lung surface metastasis nodules are shown in Fig. 1B.

As shown in Figures 1A and 1B, S-01, MDC-405, and docetaxel significantly inhibited breast tumor weight and number of pulmonary surface metastasis nodules relative to the blank control group, and more importantly, S-01 was more than the positive control group. There was a significant difference in the inhibition of paclitaxel, and there was no significant difference in the inhibition of MDC-405 relative to docetaxel. It can be seen that S-01 has better tumor suppressor and metastasis effects than MDC-405 and docetaxel. .

(2) Cell culture and tumor inoculation: 4T1 cells were cultured in 1640 medium (Gibco) containing 10% fetal calf serum (Hyclone Corp, USA), 1% glutamine and 1% cyan-streptomycin. 4T1 cells in the logarithmic growth phase were collected and the cell concentration was adjusted to 1.5 × 10 ⁶ /mL. Female BALB/c mice were inoculated with 4T1 cells in the 4th mammary fat pad, and the inoculation volume was 0.1 mL, that is, 1.5×10 5 /piece.

Grouping and administration: 4T1 breast cancer cells were inoculated into the mammary fat pad of BALB/c mice, and D0 was set on the day of inoculation, and grouped on the 4th day after inoculation. Five groups were set up in the experiment: Solvent control group (blank control group), S-01 10mg/kg group, DTX (Docetaxel) 5.7 mg/kg group, DTX (5.7 mg/kg) + MDA-1 (4.53 mg/kg) group, DTX (5.7 mg/kg) + MDA-1-linker (4.43 mg/kg) group. , with It was a positive control group.

10 animals per group. Mice were administered once a week in the tail vein for 4 consecutive weeks. Animal body weight and tumor volume were monitored during the administration. The body weight of the animal was weighed every 2 to 3 days, and the long and short diameters of the breast tumor were measured with a vernier caliper. The tumor size was calculated by the formula: (1/2) × long diameter × (short diameter) ² . On the 28th day after the tumor was received (D28), the experiment was performed. After the blood was collected from the eyeball, the mice were sacrificed by cervical dislocation. The breast tumor was weighed and the lung weight was calculated. The number of metastatic nodules on the lung surface was calculated. The results of the tumor weight experiment are shown in Fig. 2A, and the experimental results of the number of lung surface metastasis nodules are shown in Fig. 2B.

As shown in Figure 2A, S-01, docetaxel, docetaxel + MDA-1, and docetaxel + MDA-1-linker significantly inhibited breast tumor weight increase relative to the blank control group, and more importantly, S- 01 was significantly different from the positive control group with docetaxel, docetaxel + MDA-1 and docetaxel + MDA-1-linker, indicating that S-01 has better tumor inhibition than the positive control group. effect.

As shown in Figure 2B, S-01, docetaxel, docetaxel + MDA-1, and docetaxel + MDA-1-linker significantly inhibited the increase in the number of pulmonary surface metastases relative to the blank control group, and more importantly, The inhibition of S-01 was significantly different from that of the positive control group, and the inhibitory effect of docetaxel + MDA-1 and docetaxel + MDA-1-linker increased by 27.9% and 18.6%, respectively, indicating that S- 01 has a better inhibitory effect on cancer metastasis than the positive control group.

Cell culture and tumor inoculation: BALB/c (nu/nu) mice, female, SPF grade, 4-5 weeks old, purchased from Guangdong Experimental Animal Center, certificate number: NO.44007200013511, mice were raised in Shenzhen Xinli SPF Animal Laboratory of Innovative Drug Research Center of Technology Center of Thai Pharmaceutical Co., Ltd. The tumor strain used in the experiment was presented by Professor Liu Gang of the Chinese Academy of Medical Sciences. The tumor strain was MDA-MB-231 breast cancer, which was activated in the laboratory for the third generation after passage.

Grouping and administration: tumor-bearing animals with good tumor growth and good general condition were selected and sacrificed by cervical dislocation. The tumor pieces were taken out under aseptic conditions, and the tumor pieces were cut into 2-3 mm diameter pieces with a scalpel, and the trocars were inoculated subcutaneously in the nude mice. The tumors grew naturally and were randomized after the tumor volume was as long as 100 mm ³ . Let the group administration date be D0. There are six groups in the test, which are: Solvent control group, SAL-0101 5 mg/kg group, SAL-0101 10 mg/kg group, DTX 2.85 mg/kg group, DTX (2.85mg/kg) + MDA-1 (2.26 mg/kg) group, DTX (2.85 mg/kg) + MDA-1-linker (2.21 mg/kg) group.

Eight animals in each group were dosed starting from the animal's body weight from now on. Each group of animals was administered intravenously once a week for 3 times.

The tumor length, short diameter and body weight of the animals were measured every 2-3 days during the administration. The tumor volume was calculated by the formula: (1/2) × long diameter × (short diameter) 2, and the experiment was terminated on the 18th day after grouping (D18). . At the end of the experiment, the animals were sacrificed by cervical dislocation, the tumor was removed, the tumor weight was calculated, and the tumor growth inhibition rate was calculated. Tumor volume (TV) and relative tumor volume (RTV) were calculated. The statistical significance of the differences in tumor weight, tumor volume, RTV and other indicators between the groups was compared by t test.

Calculated as follows:

Tumor inhibition rate (%) = (average tumor weight of vehicle control group - mean tumor weight of treatment group / mean tumor weight of vehicle control group) × 100

Tumor volume (TV) = (length × width ² ) / 2

Relative tumor volume (RTV) = Vt / Vo (where Vo is the TV measured at the time of sub-cage administration, and Vt is the TV at each subsequent measurement.)

The evaluation index of antitumor activity was relative tumor proliferation rate T/C (%), T/C (%) = (treatment group (T) RTV / vehicle control group (C) RTV) × 10

Efficacy evaluation criteria: T/C (%) > 40 was ineffective; T / C (%) ≤ 40, and statistically treated P < 0.05 was effective.

The results of the tumor volume experiment are shown in Fig. 3A, and the results of the tumor weight experiment are shown in Fig. 3B.

As shown in Figures 3A and 3B, S-01, docetaxel, docetaxel + MDA-1, and docetaxel + MDA-1-linker significantly inhibited tumor volume and tumor weight, which is more important than the blank control group. The inhibition of S-01 was significantly different from the positive control group of docetaxel, docetaxel + MDA-1 and docetaxel + MDA-1-linker, indicating that S-01 has a positive relative to the positive control group. Better inhibition of tumors.

Through the same experiment, it was found that other preferred compounds of the present invention have the similar antitumor effect and antitumor metastasis effect of S-01, but are most excellent in S-01, and it can be inferred that the preferred compounds of the present invention are relative to MDC. -405 and docetaxel, have better anti-tumor effect and anti-tumor effect.

Note that in the above experiment, the structure of MDA-1-linker is as follows: .

Figure 1 is a comparison of the SST, MDC-405 and docetaxel mouse breast cancer 4T1 lung metastasis model test, wherein 1A is a comparison of breast tumor weight, 1B is a comparison of the number of lung surface metastasis nodules, of which 10mg S-01 and 10 mg MDC-405, with 5.7 mg docetaxel as the molar. 2 is a comparison result of a 4T1 lung metastasis model of breast cancer of S-01, docetaxel, docetaxel + MDA-1 and docetaxel + MDA-1-linker mice, wherein 2A is a breast tumor weight comparison As a result, 2B is a comparison of the number of lung surface metastatic nodules, in which 10 mg of S-01 and 5.7 mg of docetaxel are equal molars. Figure 3 is a graph showing the inhibitory effect of S-01, docetaxel, docetaxel + MDA-1 and docetaxel + MDA-1-linker on human breast cancer MDA-MB-231 xenograft tumor in nude mice, wherein Figure 3A shows the tumor volume comparison results, and Figure 3B shows the tumor weight comparison results, wherein DTX represents docetaxel, 5 mg S-01 and 2.85 mg docetaxel are isomolar.

Claims (11)

a compound of formula I and a pharmaceutically acceptable salt thereof Wherein m = 0 or 1; n = 2, 3, 4, 5, 6, 7, 8, 9, or 10; R _{1 is} selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted Or an unsubstituted C1-C6 alkoxy group, the substituted substituent being selected from the group consisting of a hydroxyl group, a thiol group, a halogen group, an amine group, a nitro group, a cyano group, an aldehyde group, a C1-C6 alkyl group, a carboxyl group, a hydroxylamine group, C2-C6 olefinic group, C1-C4 guanamine group; R _{2 is} selected from hydrogen, substituted or unsubstituted C1-C6 alkyl group, substituted or unsubstituted C1-C6 alkoxy group, said substituted The substituent is selected from halogen. The compound according to claim 1, wherein the aryl group is selected from the group consisting of a penta-decavalent aryl group and the pharmaceutically acceptable salt thereof is selected from the group consisting of a penta-decavalent heteroaryl group. The compound according to claim 2, wherein the penta-decagonal aryl group is selected from the group consisting of a five-membered aryl group, a six-membered aryl group, a nine-membered fused ring aryl group, and a pharmaceutically acceptable salt thereof. a fused aromatic aryl group, wherein the five-membered aryl group is selected from The six-membered aryl group is selected from The nine-membered fused ring aryl group is selected from the group consisting of , The ten-membered fused ring aryl group is selected from The five-tenth heteroaryl group is selected from a five-membered heterocyclic group containing 1 to 4 hetero atoms selected from N, O or S, and 1 to 4 hetero atoms selected from N, O or S. a six-membered heterocyclic group, a ten-membered fused heterocyclic group having 1 to 4 hetero atoms selected from N, O or S; and said five or more heteroatoms selected from N, O or S The metacyclic heterocyclic group is selected from: The six-membered heterocyclic group having 1 to 4 hetero atoms selected from N, O or S is selected from the group consisting of: The ten-membered fused heterocyclic group having 1 to 4 hetero atoms selected from N, O or S is selected from the group consisting of: . The compound according to any one of claims 1 to 3, wherein the C1-C6 alkyl group is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, and a pharmaceutically acceptable salt thereof. Cyclopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, n-pentyl, cyclopentyl, n-hexyl, cyclohexyl, dimethylpropyl, 2-methylbutyl 2,2-dimethylbutyl and 2,3-dimethylbutyl; the C1-C6 alkoxy group is selected from the group consisting of methoxy, ethoxy, n-propoxy, isopropoxy, n-Butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy, isohexyloxy, methoxyethoxy, ethoxymethoxy Base, propoxy methoxy and propoxy ethoxy. The compound according to any one of claims 1 to 4, wherein the halogen is selected from the group consisting of fluorine, chlorine, bromine or iodine; and the olefin group of the C2-C6 is ethylene. Base, propenyl, butenyl, isobutenyl, 2-butenyl, pentenyl, isopentenyl, 2-pentenyl, hexenyl and isohexenyl; said C1-C4 guanamine It is an acetamino group, a propylamine group, a butylammonium group or an isobutylammonium group. The compound according to any one of claims 1 to 5, wherein the R ₂ is hydrogen or the hydrogen is replaced by a metal or a non-metal cation to form a pharmaceutically acceptable salt, and the pharmaceutically acceptable salt thereof. The metal or non-metal cation is selected from the group consisting of Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Al ³⁺ and NH ₄ ⁺ . The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound of formula I includes, but is not limited to, a compound of formula IA: IA. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of , , , , , , , , , , , , with . A use of a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for preventing or treating various tumors. The use according to claim 9, wherein the tumor is selected from the group consisting of melanoma, gastric cancer, lung cancer, preferably non-small cell lung cancer, breast cancer, breast cancer metastasis, renal cancer, liver cancer, oral epithelial cancer, Cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, colon cancer, colon cancer metastasis, brain cancer, preferably brain cancer caused by glioma. A pharmaceutical composition comprising the compound of any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.