CN108530518A - 10 analog of aplysiatoxin and its preparation method and application - Google Patents
10 analog of aplysiatoxin and its preparation method and application Download PDFInfo
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- CN108530518A CN108530518A CN201710122739.3A CN201710122739A CN108530518A CN 108530518 A CN108530518 A CN 108530518A CN 201710122739 A CN201710122739 A CN 201710122739A CN 108530518 A CN108530518 A CN 108530518A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/02—Linear peptides containing at least one abnormal peptide link
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention discloses a kind of 10 analogs of aplysiatoxin and preparation method thereof; using phenylalanine Hydrochloride as raw material; respectively by repeated deprotection and amide condensed reaction; obtain pentapeptide fragment compound; pentapeptide fragment compound is removed through methyl esters hydrolysis, protecting group again, and 10 analog of the aplysiatoxin is prepared.The preparation method of the present invention is at low cost, easy to operate, efficient.The compounds of this invention has better inhibition to cancer especially 116 human colon cancer cells of HCT, and has small toxicity, the high advantage of stability, and the anticancer drug for exploitation treatment colon cancer or other types cancer provides wide development space.
Description
Technical field
The present invention relates to biomedicine fields, are related to a kind of pentapeptide compound, more particularly to one kind has active anticancer
10 analog of aplysiatoxin and its preparation method and application.
Background technology
Aplysiatoxin 10 (dolastatin 10) is that the Pettit of the vertical university of State of Arizona, US teaches seminar from print
Spend a kind of Natural linear pentapeptide that separation and Extraction comes out in the sea mollusk Dolabella auricularia in ocean.Sea hare
Toxin 10 all has extremely strong rejection ability for kinds of tumor cells such as melanoma, Small Cell Lung Cancer, oophoroma etc..But due to
There are various side reactions in clinical studies in its too strong cytotoxicity, therefore fails to pass through clinical trial.People are logical
The structure activity study to aplysiatoxin 10 is crossed to carry out structural modification, it is desirable to find a smaller anticancer drug of toxicity.
The analog for being referred to as a series of aplysiatoxins 10 of " Auristatins " is designed to be synthesized in succession, wherein
Miyazaki has found that the methyl at the ends N- of aplysiatoxin 10 can be removed one without influencing its activity, which is named
For Auristatin D (MMAD)；Doronina, which is reported, replaces the thiazole ring at the ends C- in MMAD structures with carboxyl, obtains
Compound nomenclature is Auristatin F (MMAF), which is applied in antibody drug conjugates (ADCs), at present just
In clinical experimental stage.
When the ends N- of aplysiatoxin 10 are primary amine, then the tie point as drug molecule in ADCs and antibody is more advantageous to.
So far, a series of N- end structures modifications about aplysiatoxin 10 are all confined to remove one of methyl, same to time shift
Except two methyl make the ends N- of compound for after primary amine, it is found that its activity substantially reduces.
Invention content
The purpose of the present invention is on the architecture basics of MMAF, modify the ends N- of compound MMAD, provide a system
New 10 analog of aplysiatoxin is arranged, 10 analog of the aplysiatoxin has good antitumor activity, especially to HCT-116
Human colon cancer cell has preferable inhibitory activity, and small toxicity, stability high.The present invention also provides the aplysiatoxins
The preparation method of 10 analogs, using phenylalanine Hydrochloride as raw material, the deprotection Jing Guo four repetitions and amide condensed respectively
Reaction obtains pentapeptide fragment compound, and pentapeptide fragment compound is removed through methyl esters hydrolysis, protecting group again, and sea hare poison is prepared
Plain 10 analogs.This method is at low cost, easy to operate, efficient.
10 analog of aplysiatoxin proposed by the present invention, is made of, the n terminal amino acid of five amino acid five amino acid
Be for Valine, such as Valine, Serine, l-Alanine, L-phenylalanine, L-Leu, L-Aspartic acid,
Shown in structure such as following formula (1)：
Wherein, R is the alkyl of C1~C10, the phenyl that methylol, phenyl, C1~C10 replace, acetylamino, carboxyl, ammonia
Base, aminomethyl.
Preferably, R be isopropyl, methylol, methyl, benzyl, isobutyl group, acetylamino, carboxyl, amino, normal-butyl,
Phenyl, aminomethyl.
It is further preferred that R is isopropyl, methylol, methyl, benzyl, isobutyl group, acetylamino.
It is further preferred that 10 analog of the aplysiatoxin include as lower structure compound 1a, 1b, 1c, 1d, 1e and
1f：
The preparation method of 10 analog of aplysiatoxin proposed by the present invention, includes the following steps：
(1) synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe
I) synthesis of dipeptide fragment compound (3) N-Boc-Dap-Phe-OMe
In organic solvent, compound (2) N-Boc-Dap, phenylalanine methyl ester hydrochloride, DIPEA and DEPC carry out acyl
Amine condensation reaction obtains compound (3).
In step i), the organic solvent is selected from DMF, dichloromethane, tetrahydrofuran；Preferably, it is DMF.
In step i), the temperature of the amide condensed reaction is -5 DEG C -10 DEG C；Preferably, it is 0 DEG C.
In step i), the time of the amide condensed reaction is 3-5h；Preferably, it is 4h.
In step i), compound (2), phenylalanine methyl ester hydrochloride, DIPEA, DEPC molar ratio be 1：(1.0-
1.5)：(4.5-6.0)：(1.25-1.75)；Preferably, compound (2), phenylalanine methyl ester hydrochloride, DIPEA, DEPC rub
You are than being 1：1.2：5.0：1.5.
In step i), the dosage of the organic solvent is 8-13 times of volume of compound (2)；Preferably, it is compound (2)
10 times of volumes.
In step i), DIPEA is alkali, and DEPC is the condensing agent needed for amide condensed reaction.
Ii) the synthesis of tripeptide fragment compound (5) N-Boc-Dil-Dap-Phe-OMe：
In the first organic solvent, compound (3) and trifluoroacetic acid (TFA) carry out Boc protecting group elimination reactions, have reacted
Quan Hou adds the second organic solvent, compound (4), DIPEA and DEPC, carries out amide condensed reaction, obtains compound (5).
Step ii) in, first organic solvent is selected from dichloromethane, tetrahydrofuran, ethyl acetate；Preferably, it is two
Chloromethanes.
Step ii) in, second organic solvent is selected from DMF, dichloromethane, tetrahydrofuran；Preferably, it is DMF.
Step ii) in, the temperature of the Boc protecting groups elimination reaction is 0 DEG C -25 DEG C (room temperatures)；Preferably, it is 25 DEG C
(room temperature).
Step ii) in, the time of the Boc protecting groups elimination reaction is 5-8h；Preferably, it is 6h.
Step ii) in, the amide condensed temperature is -5 DEG C -10 DEG C；Preferably, it is 0 DEG C.
Step ii) in, the amide condensed time is 3-5h；Preferably, it is 4h.
Step ii) in, in Boc protecting group elimination reactions, the compound (3), TFA molar ratio be 1：(7.5-
12), it is preferable that compound (3), the molar ratio of TFA are 1：10.
Step ii) in, the dosage of first organic solvent is 4-7 times of volume of compound (3)；Preferably, it is chemical combination
5 times of volumes of object (3).
Step ii) in, in amide condensed reaction, when compound (3) molal quantity used in Boc protecting group elimination reactions
When being 1, the DIPEA, DEPC, compound (4) molal quantity be respectively (4.5-6.0), (1.25-1.75), (1.0-1.25)；
Preferably, the DIPEA, DEPC, compound (4) molal quantity be respectively 5.0,1.5,1.1.
Step ii) in, the dosage of second organic solvent is 8-13 times of volume of compound (3)；Preferably, it is chemical combination
10 times of volumes of object (3).
Step ii) in, TFA is used for the removing of Boc protecting groups, and DIPEA is alkali, and DEPC is the contracting needed for amide condensed reaction
Mixture.
Iii) the synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe
In the first organic solvent, the elimination reaction of compound (5) and trifluoroacetic acid (TFA) progress Boc protecting groups, reaction
After completely, the second organic solvent, N-Boc-L- valines, DIPEA and bromo three (dimethylamino) phosphorus hexafluorophosphoric acid are added
Salt (Brop), carries out amide condensed reaction, obtains compound (6).
Step iii) in, first organic solvent is selected from dichloromethane, tetrahydrofuran, ethyl acetate；Preferably, it is two
Chloromethanes.
Step iii) in, second organic solvent is selected from dry methylene chloride, acetonitrile, tetrahydrofuran；Preferably, it is dry
Dry dichloromethane.
Step iii) in, the temperature of the Boc protecting groups elimination reaction is 0 DEG C -25 DEG C；Preferably, it is 25 DEG C (room temperatures).
Step iii) in, the time of the Boc protecting groups elimination reaction is 5-8h；Preferably, it is 6h.
Step iii) in, the temperature of the amide condensed reaction is 0 DEG C -25 DEG C；Preferably, it is 25 DEG C (room temperatures).
Step iii) in, the time of the amide condensed reaction is 15-48h；Preferably, to be protected from light 24 hours.
Step iii) in, it is preferable that before carrying out amide condensed reaction, each substance is added in ice-water bath, then heats up
Amide condensed reaction is carried out again to suitable temperature.
Step iii) in, in the elimination reaction of Boc protecting groups, the compound (5), TFA molar ratio be 1：(7.5-
12)；Preferably, the compound (5), TFA molar ratio be 1：10.
Step iii) in, the dosage of first organic solvent is 4-7 times of volume of compound (5)；Preferably, it is chemical combination
5 times of volumes of object (5).
Step iii) in, in amide condensed reaction, the compound (5), DIPEA, Brop, N-Boc-L- valine
Molar ratio is 1：(2.5-4.5)：(1.25-1.75)：(1.2-3.0)；Preferably, the compound (5), DIPEA, Brop, N-
The molar ratio of Boc-L- valines is 1：3.6：1.5：2.5.
Step iii) in, the dosage of second organic solvent is 8-13 times of volume of compound (5)；Preferably, it is change
Close 10 times of volumes of object (5).
Step iii) in, TFA is used for the removing of Boc protecting groups, and DIPEA is alkali, and Brop is needed for amide condensed reaction
Condensing agent.
Shown in reaction process following reaction formula (I)：
(2) synthesis of pentapeptide fragment compound (7).
In the first organic solvent, the elimination reaction of compound (6) and trifluoroacetic acid (TFA) progress Boc protecting groups, reaction
After completely, the second organic solvent, N-Boc-L- valines, DIPEA and DEPC are added, amide condensed reaction is carried out, is changed
Close object (7)；
Shown in reaction process following reaction formula (II)：
In step (2), first organic solvent is selected from dichloromethane, tetrahydrofuran, ethyl acetate；Preferably, it is two
Chloromethanes.
In step (2), second organic solvent is selected from dry DMF, acetonitrile, tetrahydrofuran；Preferably, it is dry DMF.
In step (2), the temperature of the Boc protecting groups elimination reaction is 0 DEG C -25 DEG C；Preferably, it is 25 DEG C (room temperatures).
In step (2), the time of the Boc protecting groups elimination reaction is 5-8h；Preferably, it is 6h.
In step (2), the amide condensed temperature is -5 DEG C -10 DEG C；Preferably, it is 0 DEG C.
In step (2), the amide condensed time is 3-5h；Preferably, it is 4h.
In step (2), it is preferable that each substance is added in ice-water bath, then carries out amide condensed reaction again.
In step (2), in the elimination reaction of Boc protecting groups, the compound (6), TFA molar ratio be 1：(7.5-
12), it is preferable that the molar ratio of inventory is compound (6), the molar ratio of TFA is=1：10.
In step (2), the dosage of first organic solvent is 4-7 times of volume of compound (6)；Preferably, it is chemical combination
5 times of volumes of object (6).
In step (2), in amide condensed reaction, the compound (6), DIPEA, DEPC, N-Boc-L- valine
Molar ratio is 1：(4.5-6.0)：(1.25-1.75)：(2.0-4.5)；Preferably, the compound (6), DIPEA, DEPC, N-
The molar ratio of Boc-L- valines is 1：5.0：1.5：3.0.
In step (2), the dosage of second organic solvent is 8-13 times of volume of compound (6)；Preferably, it is chemical combination
10 times of volumes of object (6).
In step (2), TFA is used for the removing of Boc protecting groups, and DIPEA is alkali, and DEPC is the contracting needed for amide condensed reaction
Mixture.
(3) hydrolysis of ester group of pentapeptide fragment compound (7) and N-Boc protecting group elimination reactions
In the first organic solvent, compound (7) and LiOH.H2O carries out hydrolysis of ester group reaction, obtains compound (7) first
Carboxylate after ester hydrolysis；In a second organic solvent, the carboxylate after compound (7) methyl esters hydrolysis and trifluoroacetic acid into
Row Boc protecting group elimination reactions obtain 10 analog compounds of the formula (1) aplysiatoxin.
Specifically, in the first organic solvent, compound (7) and LiOH.H2O carries out hydrolysis of ester group reaction (methyl esters water
Solution), then solvent under reduced pressure is spin-dried for, obtain white solid 1 (carboxylate after being hydrolyzed for compound (7) methyl esters)；By the white
Solid 1 is dissolved with solution, adjusts pH, has white solid 2 (free carboxy acid after being hydrolyzed for compound (7) methyl esters) to be precipitated, mistake
Filter, it is dry, obtain the carboxylate after the hydrolysis of compound (7) methyl esters；Carboxylate after compound (7) methyl esters is hydrolyzed is dissolved in second
In organic solvent and trifluoroacetic acid carries out Boc protecting group elimination reactions, obtains 10 analog chemical combination of the formula (1) aplysiatoxin
Object.
Obtained white solid 1 is the carboxylate form of compound (7), and white solid 2 is the carboxylic acid to dissociate.It operates in this way
Purpose be directly to be precipitated solid by adjusting pH, then achieve that the separation of product and impurity by filtering, avoid column layer
The use of analysis.
Shown in reaction process following reaction formula (III)：
In step (3), first organic solvent is selected from methanol, ethyl alcohol, isopropanol；Preferably, it is methanol.
In step (3), second organic solvent is selected from dichloromethane, tetrahydrofuran, ethyl acetate；Preferably, it is two
Chloromethanes.
In step (3), the solution for making white solid 1 dissolve is selected from hydrochloric acid, the methanol of 2N；Preferably, it is the salt of 2N
Acid.
In step (3), the pH is 3-7, it is preferable that is 5-6.
In step (3), the temperature of the hydrolysis of ester group reaction is 15 DEG C -25 DEG C；Preferably, it is 25 DEG C (room temperatures).
In step (3), the time of the hydrolysis of ester group reaction is 2-5h；Preferably, it is 3h.
In step (3), the temperature of the Boc protecting groups elimination reaction is 0 DEG C -25 DEG C；Preferably, it is 25 DEG C (room temperatures).
In step (3), the time of the Boc protecting groups elimination reaction is 3-7h；Preferably, it is 5 hours.
In step (3), compound (7), LiOHH2O, the molar ratio of trifluoroacetic acid is 1：(1.5-4.0)：(8.0-
12.0)；Preferably, compound (7), LiOHH2O, the molar ratio of trifluoroacetic acid is 1：3.0：10.0.
In step (3), the dosage of first organic solvent is 4-7 times of volume of compound (7)；Preferably, it is chemical combination
5 times of volumes of object (7).
In step (3), the dosage of second organic solvent is 4-7 times of volume of compound (7)；Preferably, it is chemical combination
5 times of volumes of object (7).
In step (3), TFA is used for the removing of Boc protecting groups, LiOHH2O is hydrolyzed for methyl esters.
In a specific embodiment mode, the preparation method of 10 analog of aplysiatoxin of the present invention, synthesis step
It is as follows：
(1) synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe.
I) synthesis of dipeptide fragment compound (3) N-Boc-Dap-Phe-OMe
Compound (2) is dissolved in the DMF of 8-10 times of volume, sequentially add phenylalanine methyl ester hydrochloride and
DIPEA, ice-water bath cooling.Then, the DMF that DEPC is dissolved in 3 times of volumes is weighed, is added dropwise in above-mentioned reaction solution in 0 DEG C, adding makes
System maintains ice-water bath to react 3-5 hours.Water quenching is added to go out reaction, organic phase is washed in ethyl acetate extraction, dry, filtering, filtrate
It is concentrated under reduced pressure, obtains the crude product of compound (3), crude product passes through column chromatographic isolation and purification (ethyl acetate：Petroleum ether=1：
1) compound (3), is obtained.
The molar ratio of above-mentioned inventory is compound (2)：Phenylalanine methyl ester hydrochloride：DIPEA：DEPC=1：(1.0-
1.5)：(4.5-6.0)：(1.25-1.75), it is preferable that the molar ratio of inventory is compound (2)：Phenyalanine methyl ester hydrochloric acid
Salt：DIPEA：DEPC=1：1.2：5.0：1.5.
Ii) the synthesis of tripeptide fragment compound (5) N-Boc-Dil-Dap-Phe-OMe：
Compound (3) is dissolved in the dichloromethane of 5-6 times of volume, trifluoroacetic acid (TFA) is added, is stirred in room temperature
It mixes, until TLC display reactions are completed.It is concentrated under reduced pressure, obtained residue is dissolved in the drying DMF of 8-10 times of volume, then
Sequentially add compound (4) and DIPEA, ice-water bath cooling.Then, the DMF that DEPC is dissolved in 3 times of volumes is weighed, is added dropwise in 0 DEG C
In above-mentioned reaction solution, adding makes system that ice-water bath be maintained to react 2-5 hours.Water quenching is added to go out reaction, ethyl acetate extraction, washing has
Machine phase, dry, filtering, filtrate decompression concentration obtain the crude product of compound (5), crude product passes through column chromatographic isolation and purification (second
Acetoacetic ester：Petroleum ether=1：1) compound (5), is obtained.
In Boc protecting group elimination reactions, the molar ratio of above-mentioned inventory is compound (3)：TFA=1：(7.5-12),
Preferably, the molar ratio of inventory is compound (3)：TFA=1：10.
It is described when compound (3) molal quantity used in Boc protecting group elimination reactions is 1 in amide condensed reaction
DIPEA, DEPC, compound (4) molal quantity be respectively (4.5-6.0)：(1.25-1.75)：(1.0-1.25)；Preferably, institute
State DIPEA, DEPC, the molal quantity of compound (4) is respectively 5.0：1.5：1.1.
Iii) the synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe：
Compound (5) is dissolved in the dichloromethane of 5-6 times of volume, trifluoroacetic acid (TFA) is added, is stirred in room temperature
It mixes, until TLC display reactions are completed.It is concentrated under reduced pressure, obtained residue is dissolved in the dry methylene chloride of 8-10 times of volume
In, sequentially add N-Boc-L- valines and DIPEA, ice-water bath cooling.Then, bromo three (dimethylamino) phosphorus six is weighed
Fluorophosphate (Brop) is directly added into reaction solution, is directly added into above-mentioned reaction solution in 0 DEG C, and adding, which makes system be warmed to room temperature, is protected from light
Reaction 24 hours.Water, dichloromethane extraction is added to wash organic phase, dry, filtering, filtrate decompression concentration obtains compound (6)
Crude product, crude product passes through column chromatographic isolation and purification (ethyl acetate：Petroleum ether=2：1) compound (6), is obtained.
The molar ratio of above-mentioned inventory is compound (5)：TFA=1：(7.5-12), it is preferable that the molar ratio of inventory is
Compound (5)：TFA=1：10.
The molar ratio of above-mentioned inventory is compound (5)：DIPEA：Brop：Valine=1 N-Boc-L-：(2.5-4.5)：
(1.25-1.75)：(1.2-3.0), it is preferable that the molar ratio of inventory is compound (5)：DIPEA：Brop：N-Boc-L- figured silk fabrics
Propylhomoserin=1：3.6：1.5：2.5.
Shown in reaction process such as reaction equation (I ')：
(2) synthesis of pentapeptide fragment compound (7).
Compound (6) is dissolved in the dichloromethane of 5-6 times of volume, trifluoroacetic acid (TFA) is added, is stirred in room temperature
It mixes, until TLC display reactions are completed.It is concentrated under reduced pressure, obtained residue is dissolved in the drying DMF of 8-10 times of volume, then
Sequentially add N-Boc-L- valines and DIPEA, ice-water bath cooling.Then, the DMF that DEPC is dissolved in 3 times of volumes is weighed, in 0 DEG C
It is added dropwise in above-mentioned reaction solution, adding makes system that ice-water bath be maintained to react 3-6 hours.Water quenching is added to go out reaction, ethyl acetate extraction,
Organic phase is washed, dry, filtering, filtrate decompression concentration obtains the crude product of compound (7), crude product passes through column chromatography for separation
Purify (ethyl acetate：Petroleum ether=1：1) compound (7), is obtained.
The molar ratio of above-mentioned inventory is compound (6)：TFA=1：(7.5-12), it is preferable that the molar ratio of inventory is
Compound (6)：TFA=1：10.
The molar ratio of above-mentioned inventory is compound (6)：DIPEA：DEPC：Valine=1 N-Boc-L-：(4.5-6.0)：
(1.25-1.75)：(2.0-4.5), it is preferable that the molar ratio of inventory is compound (6)：DIPEA：DEPC：N-Boc-L- figured silk fabrics
Propylhomoserin=1：5.0：1.5：3.0.
Shown in reaction process such as reaction equation (II ')：
(3) hydrolysis of ester group of pentapeptide fragment compound (7) and N-Boc protecting group elimination reactions
Compound (7) is dissolved in 5-6 times of volumes methanol, then weighs LiOH.H2O is added in above-mentioned solution, is stirred at room temperature 3
Hour.Then decompression rotation removes solvent, obtains white solid, adds water that solid is made to dissolve, then with the hydrochloric acid conditioning solution PH of 2N is 5-
6, there is white solid precipitation, filter, dries solid.Obtained solid is dissolved in 5-6 times of volumes methylene chloride, trifluoro is added
Acetic acid is stirred at room temperature 5 hours, and decompression rotation removes solvent, obtained crude product preparation HPLC (SB-C18 columns, 5 μm, 9.4 ×
250mm, 40%CH3CN/60%H2O it) isolates and purifies, obtains white powder final product 1.
The molar ratio of above-mentioned inventory is compound (7)：LiOH·H2O：Trifluoroacetic acid=1：(1.5-4.0)：(8.0-
12.0), it is preferable that the molar ratio of inventory is compound (7)：LiOH·H2O：Trifluoroacetic acid=1：3.0：10.0.
Shown in reaction process such as reaction equation (III ')：
Shown in the reaction mechanism such as formula (a) of the present invention, as amide condensed dose, DEPC first is formed DEPC with substrate carboxylic acid
Mixed acid anhydride obtains activated intermediate by carboxylic acid activated, and then the amine of nucleophilicity removes the attack activated intermediate, last phosphoric acid again
Ester leaves away to form amido bond, to which 10 analog of formula (1) aplysiatoxin be prepared.
The invention also provides 10 analog application in preparations of anti-tumor drugs of aplysiatoxin shown in formula (1).Wherein,
The tumour includes colon cancer, breast cancer, Small Cell Lung Cancer；Preferably, the tumour is colon cancer；It is further preferred that knot
Colon-cancer cell is HCT-116.
The beneficial effects of the present invention are the present invention is former using different amino acid substitutions on the architecture basics of MMAF
Some N, N- dimethyl valine structural unit, synthesis is a series of to obtain new 10 analog of aplysiatoxin.What the present invention designed
This kind of new 10 analog of aplysiatoxin, modifies the ends N- of aplysiatoxin 10, is expanded by original secondary amine or tertiary amine
Primary amine is arrived.10 analog of formula (1) aplysiatoxin has good antitumor activity, especially for HCT-116 human colon carcinomas
Cell has preferable rejection ability, IC50Between 0.25~11.13 μM, four compounds 1a, 1c, 1d and 1e therein
Better rejection ability is shown than control compound MMAF；10 analog of formula (1) aplysiatoxin also has small toxicity, stability
High feature.The preparation method of 10 analog of aplysiatoxin of the present invention, it is at low cost, it is easy to operate, it is efficient.The sea hare of the present invention
The N- end structures of 10 analog of toxin are primary amine, are more advantageous to as drug molecule and connector in antibody drug conjugates (ADCs)
Connection site, provide advantage for more other kinds of ADCs researchs.
Specific implementation mode
In conjunction with following specific examples, the present invention is described in further detail, and of the invention protects content not limit to
In following embodiment.Without departing from the spirit and scope of the invention, those skilled in the art it is conceivable that variation and excellent
Point is all included in the present invention, and using appended claims as protection domain.Implement the present invention process, condition,
Reagent, experimental method etc. are among the general principles and common general knowledge in the art, this hair in addition to the following content specially referred to
It is bright that content is not particularly limited.
The preparation of 1 aplysiatoxin of embodiment, 10 analog
(1) synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe
I) synthesis of dipeptide fragment compound (3)
Compound (2) (2.87g, 10mmol) is dissolved in DMF (30mL), phenyalanine methyl ester hydrochloric acid is sequentially added
Salt (2.58g, 12mmol) and DIPEA (5.16g, 40mmol), ice-water bath cooling.Then, DEPC (2.45g, 15mmol) is weighed
It is dissolved in DMF (5mL), is added dropwise in above-mentioned reaction solution in 0 DEG C, adding makes system that ice-water bath be maintained to react 5 hours.Water quenching is added to go out instead
It answers, organic phase is washed in ethyl acetate extraction, dry, filtering, and filtrate decompression concentration obtains the crude product of compound (3), thick to produce
Object passes through column chromatographic isolation and purification (ethyl acetate：Petroleum ether=1：1) compound (3) (4.03g, 90%), is obtained.
1H NMR(400MHz,CDCl3) δ 7.36-7.03 (m, 5H), 4.93-4.66 (m, 1H), 3.77 (t, J=31.2Hz,
4H), 3.55 (d, J=5.0Hz, 1H), 3.37 (s, 3H), 3.17 (dd, J=14.0,5.2Hz, 2H), 3.10-2.97 (m, 1H),
2.34 (dd, J=31.4,6.3Hz, 1H), 1.76 (s, 2H), 1.61 (d, J=25.7Hz, 2H), 1.49 (s, 9H), 1.16 (d, J
=7.0Hz, 3H).
Ii) the synthesis of tripeptide fragment compound (5)
Compound (3) (3.58g, 8mmol) is dissolved in dichloromethane (30mL), add trifluoroacetic acid (9.12g,
80mmol), it is stirred at room temperature, until TLC display reactions are completed.It is concentrated under reduced pressure, obtained residue is dissolved in dry DMF
In (35mL), compound (4) (2.67g, 8.8mmol) and DIPEA (5.16g, 40mmol), ice-water bath cooling are sequentially added.
Then, it weighs DEPC (1.96g, 12mmol) and is dissolved in (5mL) DMF, be added dropwise in above-mentioned reaction solution in 0 DEG C, adding makes system tie up
Ice-water bath is held to react 3 hours.Water quenching is added to go out reaction, organic phase is washed in ethyl acetate extraction, dry, filtering, and filtrate decompression is dense
Contracting, obtains the crude product of compound (5), crude product passes through column chromatographic isolation and purification (ethyl acetate：Petroleum ether=1：1) it, obtains
Compound (5) (4.31g, 85%).
1H NMR(400MHz,CDCl3)δ7.33–7.17(m,5H),4.96–4.71(m,1H),4.17–4.05(m,4H),
3.89 (s, 1H), 3.78-3.67 (m, 3H), 3.47-3.40 (m, 1H), 3.40-3.31 (m, 6H), [3.19 (d, J=5.3Hz)
And3.16 (d, J=4.9Hz), 1H], 3.12-3.00 (m, 1H), 2.78-2.63 (m, 2H), 2.49-2.30 (m, 2H), 2.23
(s, 1H), 1.93 (d, J=7.5Hz, 1H), 1.87-1.60 (m, 4H), 1.46 (d, J=6.6Hz, 9H), 1.34 (td, J=
7.1,1.0Hz, 4H), 1.17 (d, J=7.0Hz, 3H), 1.01-0.85 (m, 6H).
Iii) the synthesis of tetrapeptide fragment compound (6)
Compound (5) (4.11g, 6.5mmol) is dissolved in dichloromethane (30mL), trifluoroacetic acid is added
(7.41g, 65mmol), is stirred at room temperature, until TLC display reactions are completed.It is concentrated under reduced pressure, obtained residue is dissolved in dry
In dry dichloromethane (40mL), sequentially add N-Boc-L- valines (3.47g, 16mmol) and DIPEA (3.02g,
23.4mmol), ice-water bath cools down.Then, weigh (dimethylamino) the phosphorus hexafluorophosphate of bromo three (Brop) (3.78g,
It 9.75mmol) is directly added into reaction solution, is directly added into above-mentioned reaction solution in 0 DEG C, adding, which makes system be warmed to room temperature, is protected from light
24 hours.Water, dichloromethane extraction is added to wash organic phase, dry, filtering, filtrate decompression concentration obtains the thick of compound (6)
Product, crude product pass through column chromatographic isolation and purification (ethyl acetate：Petroleum ether=2：1), obtain compound (6) (3.38g,
71%).
1H NMR(400MHz,CDCl3) δ 7.25-7.09 (m, 5H), 4.91-4.52 (m, 2H), 4.33 (dd, J=9.2,
6.8Hz, 1H), 4.25-3.93 (m, 2H), 3.89-3.76 (m, 1H), [3.69 (d, J=13.2Hz) and3.63 (d, J=
6.3Hz), 3H], 3.40-3.33 (m, 1H), 3.30-3.24 (m, 5H), 3.14-2.98 (m, 2H), 2.95 (d, J=12.2Hz,
2H),2.44–2.20(m,3H),2.00–1.80(m,3H),1.79–1.54(m,4H),[1.35(s)and1.34(s),9H],
1.13–1.08(m,3H),1.01–0.77(m,15H)；
(2) synthesis of pentapeptide fragment compound (7)
The synthesis of (2-1) compound (7) a
Compound (6) (146mg, 0.2mmol) is dissolved in dichloromethane (1.5mL), trifluoroacetic acid (TFA) is added
(228mg, 2mmol), is stirred at room temperature, until TLC display reactions are completed.It is concentrated under reduced pressure, obtained residue is dissolved in dry
In dry DMF (2.5mL), N-Boc-L- valines (43.4mg, 0.6mmol) and DIPEA (129mg, 1mmol) are sequentially added,
Ice-water bath cools down.Then, it weighs DEPC (19mg, 0.3mmol) and is dissolved in DMF (0.5mL), be added dropwise in above-mentioned reaction solution in 0 DEG C,
Adding makes system that ice-water bath be maintained to react 3 hours.Water quenching is added to go out reaction, organic phase is washed in ethyl acetate extraction, dry, filtering,
Filtrate decompression concentrates, and obtains the crude product of compound (7) a, crude product passes through column chromatographic isolation and purification (dichloromethane：Methanol=
30：1) compound (7) a (135mg, 81%), is obtained.
1H NMR(400MHz,CDCl3) δ 7.33-7.15 (m, 5H), 7.09 (d, J=6.8Hz, 1H), 7.01 (d, J=
7.1Hz,1H),6.72–6.63(m,1H),5.18–5.01(m,1H),4.94–4.85(m,1H),4.80–4.68(m,2H),
4.23-4.09 (m, 2H), 4.02-3.91 (m, 1H), 3.87 (ddd, J=13.8,7.4,2.2Hz, 1H), 3.81-3.75 (m,
1H), 3.70 (d, J=5.7Hz, 2H), 3.47-3.38 (m, 1H), 3.35 (dd, J=13.4,7.5Hz, 6H), 3.21-3.07
(m, 2H), 3.02 (d, J=8.5Hz, 2H), 2.51-2.30 (m, 3H), 2.14-1.66 (m, 8H), 1.44 (s, 9H), 1.38-
1.32(m,2H),1.20–1.15(m,3H),1.04–0.78(m,20H)；
The synthesis of (2-2) compound (7) b
Compound (7) b, as the synthetic operation step of compound (7) a, by the N-Boc-L- figured silk fabrics in compound (7) a
Propylhomoserin replaces can be obtained with N-Boc-L- serines, yield 87%.
1H NMR(400MHz,CDCl3)δ7.33–7.14(m,5H),4.90–4.60(m,3H),4.23–4.11(m,3H),
3.90 (dd, J=21.0,7.4Hz, 2H), 3.79-3.69 (m, 3H), 3.62-3.52 (m, 1H), 3.48-3.26 (m, 8H),
3.23-3.10 (m, 2H), 3.04 (dt, J=30.4,13.3Hz, 3H), 2.60-2.25 (m, 5H), 2.10-1.88 (m, 3H),
1.87-1.62 (m, 4H), 1.44 (s, 9H), 1.16 (d, J=7.0Hz, 3H), 1.07-0.80 (m, 14H).
The synthesis of (2-3) compound (7) c
Compound (7) c, as the synthetic operation step of compound (7) a, by the N-Boc-L- figured silk fabrics in compound (7) a
Propylhomoserin replaces can be obtained with N-Boc-L- alanine, yield 46%.
1H NMR(400MHz,CDCl3)δ7.25–7.09(m,5H),5.12–5.02(m,1H),4.85–4.79(m,1H),
4.67 (dt, J=20.9,9.9Hz, 2H), 4.18-4.07 (m, 3H), 3.81 (d, J=7.6Hz, 1H), [3.70 (d, J=
8.1Hz) and3.63 (d, J=5.9Hz), 3H], 3.40-3.24 (m, 8H), 3.12-2.87 (m, 5H), 2.43-2.18 (m,
4H), 2.01-1.83 (m, 3H), 1.80-1.72 (m, 1H), 1.70-1.55 (m, 3H), 1.36 (s, 10H), 1.09 (d, J=
7.0Hz,3H),0.98–0.72(m,16H)。
The synthesis of (2-4) compound (7) d
Compound (7) d, as the synthetic operation step of compound (7) a, by the N-Boc-L- figured silk fabrics in compound (7) a
Propylhomoserin replaces can be obtained with N-Boc-L- phenylalanines, yield 64%.
1HNMR(400MHz,CDCl3)δ7.31–7.13(m,10H),5.04–4.83(m,2H),4.79–4.68(m,2H),
4.38 (s, 1H), 4.16 (s, 1H), 3.88 (dd, J=7.6,1.7Hz, 1H), 3.77 (d, J=14.9,1H), 3.70 (d, J=
8.9,2H), 3.50-3.22 (m, 8H), 3.20-2.93 (m, 6H), 2.48-2.25 (m, 3H), 2.16 (ddd, J=20.2,
15.0,7.4Hz, 2H), 1.99 (ddd, J=19.0,12.3,7.4Hz, 3H), 1.91-1.54 (m, 5H), 1.39 (s, 9H),
1.16 (d, J=7.0Hz, 3H), 1.03-0.93 (m, 6H), 0.93-0.81 (m, 8H).
The synthesis of (2-5) compound (7) e
Compound (7) e, as the synthetic operation step of compound (7) a, by the N-Boc-L- figured silk fabrics in compound (7) a
Propylhomoserin replaces can be obtained with N-Boc-L- leucines, yield 65%.
1H NMR(400MHz,CDCl3)δ7.26–7.10(m,5H),5.09–4.94(m,1H),4.90–4.57(m,3H),
4.09 (d, J=13.8Hz, 2H), 3.91 (d, J=6.7Hz, 1H), 3.84-3.77 (m, 1H), 3.71 (d, J=9.4Hz, 1H),
3.63 (d, J=6.2Hz, 2H), 3.40-3.20 (m, 8H), 3.16-2.89 (m, 5H), 2.46-2.14 (m, 4H), 2.03-1.80
(m, 3H), 1.80-1.56 (m, 5H), 1.36 (s, 9H), 1.09 (d, J=7.0Hz, 3H), 0.99-0.69 (m, 22H).
The synthesis of (2-6) compound (7) f
Compound (7) f, as the synthetic operation step of compound (7) a, by the N-Boc-L- figured silk fabrics in compound (7) a
Propylhomoserin replaces can be obtained with N-Boc-L- aspartic acids, yield 65%.
1HNMR(400MHz,CDCl3) δ 7.45 (d, J=8.1Hz, 1H), 7.33-7.16 (m, 5H), 6.09 (d, J=
7.6Hz, 1H), 5.90 (s, 1H), 4.81-4.64 (m, 2H), 4.47 (s, 1H), 4.15 (s, 2H), 3.87 (dd, J=7.5,
1.6Hz, 1H), 3.77 (d, J=10.3Hz, 1H), 3.70 (d, J=4.8Hz, 2H), 3.49-3.25 (m, 8H), 3.22-2.92
(m, 5H), 2.64-2.30 (m, 5H), 2.10-1.88 (m, 3H), 1.88-1.60 (m, 4H), 1.43 (s, 9H), 1.16 (d, J=
7.0Hz,3H),1.05–0.77(m,15H)。
(3) removing of the hydrolysis of ester group and N-Boc protecting groups of pentapeptide fragment compound (7)
The synthesis of (3-1) compound (1) a
Compound (7) a (30mg, 0.06mmol) is dissolved in methanol (2mL), then weighs LiOH.H2O(5mg,
It 0.12mmol) is added in above-mentioned solution, is stirred at room temperature 3 hours.Then decompression rotation removes solvent, obtains white solid, adds water to make solid
Body dissolves, then with the hydrochloric acid conditioning solution PH of 2N is 5-6, there is white solid precipitation, filters, and dries solid.The solid that will be obtained
It is dissolved in dichloromethane (2.5mL), adds trifluoroacetic acid (68mg, 0.6mmol), be stirred at room temperature 5 hours, decompression rotation is except molten
Agent, and obtained crude product preparation HPLC (SB-C18 columns, 5 μm, 9.4 × 250mm, 40%CH3CN/60%H2O it) isolates and purifies,
Obtain white powder final product 1a (24mg, 93%).
1H NMR(400MHz,CD3OD)δ7.19–7.09(m,5H),4.71–4.51(m,2H),4.10–3.92(m,1H),
3.73-3.64 (m, 1H), 3.60-3.52 (m, 1H), 3.46-3.38 (m, 1H), 3.34-3.15 (m, 10H), 2.83 (dd, J=
24.8,11.5Hz,1H),2.39–2.33(m,2H),2.23–1.94(m,4H),1.84–1.59(m,3H),1.58–1.37(m,
2H), 1.37-1.15 (m, 4H), 1.09 (dd, J=11.1,6.8Hz, 3H), 1.02 (d, J=6.4Hz, 1H), 0.98-0.66
(m,18H)。
The synthesis of (3-2) compound (1) b
Compound (1) b, as the synthetic operation step of compound (1) a, yield 87%.
1H NMR(400MHz,CD3OD)δ7.34–7.09(m,5H),4.81–4.66(m,2H),4.17–3.86(m,3H),
3.78–3.62(m,2H),3.60–3.39(m,2H),3.36–3.28(m,8H),3.21(s,1H),3.18–3.05(m,2H),
2.95 (dd, J=25.3,14.9Hz, 1H), 2.57-2.09 (m, 4H), 1.96-1.78 (m, 3H), 1.73-1.38 (m, 3H),
1.32 (d, J=17.0Hz, 3H), 1.20 (d, J=8.7Hz, 3H), 1.13-0.84 (m, 13H).
The synthesis of (3-3) compound (1) c
Compound (1) c, as the synthetic operation step of compound (1) a, yield 94%.
1H NMR(400MHz,CD3OD) δ 7.33-7.13 (m, 5H), 4.77 (d, J=7.2Hz, 1H), 4.66 (dd, J=
17.4,8.2Hz, 1H), 4.18-3.96 (m, 2H), 3.67 (s, 1H), 3.59-3.40 (m, 2H), 3.34 (d, J=14.7Hz,
8H),3.23(s,2H),3.14(s,1H),3.02–2.83(m,1H),2.67–2.39(m,2H),2.38–2.00(m,4H),
1.95–1.72(m,3H),1.71–1.50(m,3H),1.50–1.43(m,3H),1.31(s,3H),1.24–1.15(m,3H),
1.14–0.84(m,14H)。
The synthesis of (3-4) compound (1) d
Compound (1) d, as the synthetic operation step of compound (1) a, yield 94%.
1H NMR(400MHz,CD3OD)δ7.45–6.96(m,10H),4.72–4.52(m,2H),4.13–3.91(m,2H),
3.54 (d, J=18.5Hz, 2H), 3.40 (d, J=14.5Hz, 1H), 3.34-3.00 (m, 13H), 2.88-2.73 (m, 2H),
2.40 (d, J=14.4Hz, 1H), 2.28-1.89 (m, 3H), 1.87-1.62 (m, 3H), 1.58-1.38 (m, 2H), 1.21 (d, J
=16.7Hz, 3H), 1.15-0.69 (m, 16H).
The synthesis of (3-5) compound (1) e
Compound (1) e, as the synthetic operation step of compound (1) a, yield 93%.
1H NMR(400MHz,CD3OD) δ 7.21-7.02 (m, 5H), 4.67 (d, J=8.2Hz, 1H), 4.54 (d, J=
8.9Hz, 1H), 4.10-3.92 (m, 1H), 3.91-3.80 (m, 1H), 3.76 (d, J=9.3Hz, 1H), 3.59-3.55 (m,
1H),3.48–3.38(m,1H),3.34–3.15(m,11H),3.11(s,2H),3.02(s,1H),2.89–2.76(m,1H),
2.48-2.28 (m, 2H), 2.24-1.90 (m, 3H), 1.84-1.64 (m, 3H), 1.63-1.47 (m, 4H), 1.20 (t, J=
7.0Hz, 3H), 1.09 (dd, J=13.9,6.7Hz, 3H), 1.03-0.72 (m, 20H).
The synthesis of (3-6) compound (1) f
Compound (1) f, as the synthetic operation step of compound (1) a, yield 82%.
1H NMR(400MHz,CD3OD) δ 7.21-6.99 (m, 5H), 4.67 (d, J=6.5Hz, 1H), 4.53 (d, J=
7.9Hz, 1H), 4.17 (td, J=9.4,3.8Hz, 1H), 4.01 (d, J=34.0Hz, 1H), 3.63-3.51 (m, 1H), 3.46-
3.37 (m, 1H), 3.33-3.29 (m, 1H), 3.23 (d, J=15.3Hz, 10H), 3.11 (s, 2H), 3.01 (s, 1H), 2.88-
2.71 (m, 2H), 2.63-2.52 (m, 1H), 2.38 (d, J=6.1Hz, 2H), 2.23-2.07 (m, 2H), 1.93 (d, J=
5.3Hz, 1H), 1.83-1.62 (m, 3H), 1.60-1.42 (m, 2H), 1.36-1.30 (m, 1H), 1.20 (d, J=7.2Hz,
3H), 1.08 (dd, J=17.1,6.6Hz, 3H), 1.01 (d, J=6.5Hz, 2H), 0.97-0.86 (m, 8H), 0.79 (dd, J=
13.7,6.8Hz,4H)。
The preparation of 2 aplysiatoxin of embodiment, 10 analog
(1) synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe
I) synthesis of dipeptide fragment compound (3)
Compound (2) (5.74g, 20mmol) is dissolved in DMF (60mL), phenyalanine methyl ester hydrochloric acid is sequentially added
Salt (5.17g, 24mmol) and DIPEA (15.48g, 120mmol), ice-water bath cooling.Then, weigh DEPC (4.90g,
It 30mmol) is dissolved in DMF (15mL), is added dropwise in above-mentioned reaction solution in 0 DEG C, adding makes system that ice-water bath be maintained to react 5 hours.Add
Water quenching is gone out reaction, and organic phase is washed in ethyl acetate extraction, dry, filtering, and filtrate decompression concentration obtains the thick production of compound (3)
Product, crude product pass through column chromatographic isolation and purification (ethyl acetate：Petroleum ether=1：1) compound (3) (8.24g, 92%), is obtained.
Ii) the synthesis of tripeptide fragment compound (5)
Compound (3) (7.16g, 16mmol) is dissolved in dichloromethane (60mL), trifluoroacetic acid is added
(18.24g, 160mmol), is stirred at room temperature, until TLC display reactions are completed.It is concentrated under reduced pressure, obtained residue is dissolved in
In dry DMF (70mL), compound (4) (6.31g, 20.8mmol) and DIPEA (10.32g, 80mmol), ice are sequentially added
Water-bath cooling.Then, it weighs DEPC (3.91g, 24mmol) and is dissolved in (12mL) DMF, be added dropwise in above-mentioned reaction solution, add in 0 DEG C
It is complete to make system that ice-water bath be maintained to react 3 hours.Water quenching is added to go out reaction, organic phase is washed in ethyl acetate extraction, dry, is filtered, filter
Liquid is concentrated under reduced pressure, and obtains the crude product of compound (5), crude product passes through column chromatographic isolation and purification (ethyl acetate：Petroleum ether=1：
1) compound (5) (8.11g, 80%), is obtained.
Iii) the synthesis of tetrapeptide fragment compound (6)
Compound (5) (6.32g, 10mmol) is dissolved in dichloromethane (50mL), trifluoroacetic acid is added
(12.54g, 110mmol), is stirred at room temperature, until TLC display reactions are completed.It is concentrated under reduced pressure, obtained residue is dissolved in
In dry methylene chloride (60mL), sequentially add N-Boc-L- valines (4.34g, 20mmol) and DIPEA (6.45g,
50mmol), ice-water bath cools down.Then, weigh (dimethylamino) the phosphorus hexafluorophosphate of bromo three (Brop) (4.86g,
It 12.5mmol) is directly added into reaction solution, is directly added into above-mentioned reaction solution in 0 DEG C, adding, which makes system be warmed to room temperature, is protected from light
24 hours.Water, dichloromethane extraction is added to wash organic phase, dry, filtering, filtrate decompression concentration obtains the thick of compound (6)
Product, crude product pass through column chromatographic isolation and purification (ethyl acetate：Petroleum ether=2：1) compound (6) (4.1g, 56%), is obtained.
(2) synthesis of pentapeptide fragment compound (7)
The synthesis of (2-1) compound (7) a
Compound (6) (146mg, 0.2mmol) is dissolved in dichloromethane (1.5mL), trifluoroacetic acid (TFA) is added
(228mg, 2mmol), is stirred at room temperature, until TLC display reactions are completed.It is concentrated under reduced pressure, obtained residue is dissolved in dry
In dry DMF (2.5mL), N-Boc-L- valines (36.2mg, 0.5mmol) and DIPEA (129mg, 1mmol) are sequentially added,
Ice-water bath cools down.Then, it weighs DEPC (16.5mg, 0.26mmol) and is dissolved in DMF (0.5mL), above-mentioned reaction solution is added dropwise in 0 DEG C
In, adding makes system that ice-water bath be maintained to react 3 hours.Water quenching is added to go out reaction, organic phase is washed in ethyl acetate extraction, dry, mistake
Filter, filtrate decompression concentration, obtains the crude product of compound (7) a, crude product passes through column chromatographic isolation and purification (dichloromethane：Methanol
=30：1) compound (7) a (128mg, 77%), is obtained.
The synthesis of (2-2) compound (7) b
Compound (7) b, as the synthetic operation step of compound (7) a, by the N-Boc-L- figured silk fabrics in compound (7) a
Propylhomoserin replaces can be obtained with N-Boc-L- serines, yield 79%.
The synthesis of (2-3) compound (7) c
Compound (7) c, as the synthetic operation step of compound (7) a, by the N-Boc-L- figured silk fabrics in compound (7) a
Propylhomoserin replaces can be obtained with N-Boc-L- alanine, yield 56%.
The synthesis of (2-4) compound (7) d
Compound (7) d, as the synthetic operation step of compound (7) a, by the N-Boc-L- figured silk fabrics in compound (7) a
Propylhomoserin replaces can be obtained with N-Boc-L- phenylalanines, yield 58%.
The synthesis of (2-5) compound (7) e
Compound (7) e, as the synthetic operation step of compound (7) a, by the N-Boc-L- figured silk fabrics in compound (7) a
Propylhomoserin replaces can be obtained with N-Boc-L- leucines, yield 68%.
The synthesis of (2-6) compound (7) f
Compound (7) f, as the synthetic operation step of compound (7) a, by the N-Boc-L- figured silk fabrics in compound (7) a
Propylhomoserin replaces can be obtained with N-Boc-L- aspartic acids, yield 51%.
(3) removing of the hydrolysis of ester group and N-Boc protecting groups of pentapeptide fragment compound (7)
The synthesis of (3-1) compound (1) a
Compound (7) a (30mg, 0.06mmol) is dissolved in methanol (2mL), then weighs LiOH.H2O(4mg,
It 0.09mmol) is added in above-mentioned solution, is stirred at room temperature 3 hours.Then decompression rotation removes solvent, obtains white solid, adds water to make solid
Body dissolves, then with the hydrochloric acid conditioning solution PH of 2N is 5-6, there is white solid precipitation, filters, and dries solid.The solid that will be obtained
It is dissolved in dichloromethane (2.5mL), adds trifluoroacetic acid (68mg, 0.6mmol), be stirred at room temperature 5 hours, decompression rotation is except molten
Agent, and obtained crude product preparation HPLC (SB-C18 columns, 5 μm, 9.4 × 250mm, 40%CH3CN/60%H2O it) isolates and purifies,
Obtain white powder final product 1a (22.7mg, 88%).
The synthesis of (3-2) compound (1) b
Compound (1) b, as the synthetic operation step of compound (1) a, yield 81%.
The synthesis of (3-3) compound (1) c
Compound (1) c, as the synthetic operation step of compound (1) a, yield 92%.
The synthesis of (3-4) compound (1) d
Compound (1) d, as the synthetic operation step of compound (1) a, yield 98%.
The synthesis of (3-5) compound (1) e
Compound (1) e, as the synthetic operation step of compound (1) a, yield 85%.
The synthesis of (3-6) compound (1) f
Compound (1) f, as the synthetic operation step of compound (1) a, yield 87%.
3 antitumor activity of embodiment is tested
HCT-116 human colon cancer cells are inoculated in McCoy, s5A culture solutions (10% serum, 1% blueness-streptomysin) and
In 1640 culture medium.37 DEG C are placed in, 5%CO2In incubator, per 2-3 days, passage was primary, tested logarithmic growth phase cell.
CCK-8 methods measure growth inhibitory effect of the compound to HCT116 cells.
Logarithmic growth phase cell adjusts cell suspension to 2500~4000/ml with the fresh medium configured, takes
100 μ l (2000 cells/well) cell suspension inoculations are to 96 well culture plates.It is placed in 5%CO2, training is incubated overnight in 37 DEG C of incubators
After supporting, fresh cell medium is replaced, 200 μ l DMSO diluted concentration gradient drugs in equal volume are added per hole, are incubated altogether with cell
72h is educated, fresh cell medium is replaced, adds 100 μ l+10 μ l CCK-8 solution per hole, continues to be incubated 1-4 hours, terminates culture,
The absorbance of 450nm, the absorbance correction cell number of 620nm are detected with multi-function microplate reader (Molecular Devices M5)
Difference.
Untested compound (1a, 1b, 1c, 1d, 1e, 1f) is dissolved in DMSO, and is further diluted in culture solution.
DMSO ultimate densities are no more than 0.1% (v/v).Control group is the tumour cell that isometric DMSO is added；Blank group is acellular,
It is added in culture solution and isometric DMSO is added.In primary experiment, each experiment condition is all provided with 3 multiple holes.Calculate each concentration
The inhibiting rate of compounds on cell growth, calculation formula are：Inhibiting rate (%)={ 1- [(dosing group)-(blank group)]/[(control
Group)-(blank group)] × 100%, calculate IC with GraphPad Prim650(IC50Inhibit the medicine needed for 50% cell growth
Object concentration), testing result is as shown in table 1 below：
The antitumor activity of 1 10 analog of aplysiatoxin of the present invention of table
To sum up, biological activity test the experimental results showed that, compound 1a, 1b, 1c, 1d, 1e and 1f are to human colon cancer cell
All have significant inhibiting effect, IC50Between 0.25~11.13 μM；Compared with control compound MMAF, compound 1a, 1c,
1d and 1e inhibitory activity highers, there is the potentiality for developing into potential treatment drug.The present invention be exploitation treatment colon cancer or other
The anticancer drug of types of cancer provides wide development space.
Claims (14)
1. 10 analog of aplysiatoxin, which is characterized in that shown in its structure such as formula (1)：
Wherein, R be selected from C1~C10 alkyl, methylol, phenyl, C1~C10 substitution phenyl, acetylamino, carboxyl, amino,
Aminomethyl.
2. 10 analog of aplysiatoxin as described in claim 1, which is characterized in that 10 analog of the aplysiatoxin includes such as
Compound 1a, 1b, 1c, 1d, 1e and 1f of lower structure：
3. a kind of preparation method of 10 analog of aplysiatoxin, which is characterized in that the described method comprises the following steps：
(1) synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe
I) synthesis of dipeptide fragment compound (3) N-Boc-Dap-Phe-OMe
In organic solvent, compound (2) N-Boc-Dap, phenylalanine methyl ester hydrochloride, DIPEA and DEPC carry out amide contracting
Reaction is closed, compound (3) is obtained；
Ii) the synthesis of tripeptide fragment compound (5) N-Boc-Dil-Dap-Phe-OMe：
In the first organic solvent, compound (3) and trifluoroacetic acid carry out Boc protecting group elimination reactions, after the reaction was complete, then add
Enter the second organic solvent, compound (4), DIPEA and DEPC, carry out amide condensed reaction, obtains compound (5)；
Iii) the synthesis of tetrapeptide fragment compound (6) N-Boc-Val-Dil-Dap-Phe-OMe
In the first organic solvent, compound (5) and trifluoroacetic acid carry out Boc protecting group elimination reactions, after the reaction was complete, then add
Enter the second organic solvent, N-Boc-L- valines, DIPEA and bromo three (dimethylamino) phosphorus hexafluorophosphate, carries out amide
Condensation reaction obtains compound (6)；
Shown in reaction process following reaction formula (I)：
(2) synthesis of pentapeptide fragment compound (7)
In the first organic solvent, compound (6) and trifluoroacetic acid carry out Boc protecting group elimination reactions, after the reaction was complete, then add
Enter the second organic solvent, N-Boc-L- amino acid, DIPEA and DEPC, carry out amide condensed reaction, obtains pentapeptide fragment compound
(7)；Shown in reaction process following reaction formula (II)：
(3) hydrolysis of ester group of pentapeptide fragment compound (7) and N-Boc protecting group elimination reactions
In the first organic solvent, compound (7) and LiOH.H2O carries out hydrolysis of ester group reaction, obtains the hydrolysis of compound (7) methyl esters
Carboxylate afterwards；In a second organic solvent, the carboxylate after compound (7) the methyl esters hydrolysis and trifluoroacetic acid carry out Boc
Protecting group elimination reaction obtains 10 analog compounds of the formula (1) aplysiatoxin；Reaction process following reaction formula (III) institute
Show：
4. preparation method as claimed in claim 3, which is characterized in that in step i), the temperature of the reaction is -5 DEG C -10
℃；The time of the reaction is 3-5h.
5. preparation method as claimed in claim 3, which is characterized in that in step i), compound (2), phenyalanine methyl ester salt
Hydrochlorate, DIPEA, DEPC molar ratio be 1：(1.0-1.5)：(4.5-6.0)：(1.25-1.75).
6. preparation method as claimed in claim 3, which is characterized in that step ii) in, the Boc protecting groups elimination reaction
Temperature is 0 DEG C -25 DEG C；The temperature of the amide condensed reaction is -5 DEG C -10 DEG C.
7. preparation method as claimed in claim 3, which is characterized in that step ii) in, in Boc protecting group elimination reactions, institute
State compound (3), the molar ratio of trifluoroacetic acid is 1：(7.5-12)；In amide condensed reaction, when Boc protecting group elimination reactions
Middle compound (3) molal quantity used be 1 when, the DIPEA, DEPC, compound (4) molal quantity be respectively (4.5-6.0)：
(1.25-1.75)：(1.0-1.25).
8. preparation method as claimed in claim 3, which is characterized in that step iii) in, the Boc protecting groups elimination reaction
Temperature is 0 DEG C -25 DEG C；The temperature of the amide condensed reaction is 0 DEG C -25 DEG C.
9. preparation method as claimed in claim 3, which is characterized in that step iii) in, in Boc protecting group elimination reactions,
The compound (5), trifluoroacetic acid molar ratio be 1：(7.5-12)；In amide condensed reaction, the compound (5),
DIPEA, (dimethylamino) phosphorus of bromo three hexafluorophosphate, N-Boc-L- valines molar ratio be 1：(2.5-4.5)：
(1.25-1.75)：(1.2-3.0).
10. preparation method as claimed in claim 3, which is characterized in that in step (2), the Boc protecting groups elimination reaction
Temperature is 0 DEG C -25 DEG C；The temperature of the amide condensed reaction is -5 DEG C -10 DEG C.
11. preparation method as claimed in claim 3, which is characterized in that in step (2), in Boc protecting group elimination reactions,
The compound (6), trifluoroacetic acid molar ratio be 1：(7.5-12)；In amide condensed reaction, the compound (6),
The molar ratio of DIPEA, DEPC, N-Boc-L- valine is 1：(4.5-6.0)：(1.25-1.75)：(2.0-4.5).
12. preparation method as claimed in claim 3, which is characterized in that in step (3), the temperature of the hydrolysis of ester group reaction
It is 15 DEG C -25 DEG C；The temperature of the Boc protecting groups elimination reaction is 0 DEG C -25 DEG C.
13. preparation method as claimed in claim 3, which is characterized in that in step (3), compound (7), LiOHH2O, trifluoro
The molar ratio of acetic acid is 1：(1.5-4.0)：(8.0-12.0).
14. 10 analog of aplysiatoxin as described in claim 1 is preparing the application in treating anticancer drug.
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