CN1781932A - Adriamycin derivative and its preparing method and use - Google Patents
Adriamycin derivative and its preparing method and use Download PDFInfo
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- CN1781932A CN1781932A CN 200410081387 CN200410081387A CN1781932A CN 1781932 A CN1781932 A CN 1781932A CN 200410081387 CN200410081387 CN 200410081387 CN 200410081387 A CN200410081387 A CN 200410081387A CN 1781932 A CN1781932 A CN 1781932A
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Abstract
The present invention provides one kind of adriamycin derivative in the structure as shown. The modified adriamycin derivative, pharmacological experiment shows, has obvious antitumor activity, as well as cytotoxicity lower than that of adriamycin, obvious targeting property, stable quality and high controllability.
Description
Technical field
The present invention relates to a kind of derivative of Zorubicin.
Technical background
Zorubicin has stronger antitumor action, because of both having contained fat-soluble anthracene nucleus aglucon in its structure, water miscible gentle brown sugar amine is arranged again; And there are acidic phenol hydroxyl and alkalescence amino.As non-specific anticancer chemotherapeutic agent of a kind of cycle, Zorubicin all has effect to each phase cell, but the most responsive in early days to the S phase, the M phase takes second place, and to G
1, S and G
2There is retarding action phase.Its mechanism of action is to directly act on DNA, inserts the duplex chain of DNA, and the latter is untied, and changes the template property of DNA, has both suppressed DNA thereby suppress archaeal dna polymerase, and it is synthetic also to suppress RNA.In addition, the Zorubicin tool forms the function of super oxygen base free radical, and the effect of special destruction membrane structure and function is arranged.
But Zorubicin has than serious adverse effects, shows following several respects:
1. bone marrow depression: be the major side effects of Zorubicin.White corpuscle dropped to lower-most point on 10th～14 after medication, return to normal level mostly in three weeks gradually, and anaemia and thrombopenia are generally not serious.
2. cardiac toxic: can occur and cross the property ECG change, showing as supraventricular tachycardia, premature ventricular beat and ST-T changes, generally do not influence treatment, carrying out property of retardance cardiac muscle pathology can appear in small number of patients, show as acute congestive heart failure, closely related with integral dose, appear at total amount＞400mg/m mostly
2The patient, these situations can take place and the no abnormal sign of routine electrocardiogram suddenly once in a while, the heart change that Zorubicin causes had more after the present drug withdrawal 1～6 month, cardiac toxic can increase the weight of because of the combined utilization other drug.
3. digestive tract reaction: show as appetite stimulator, feel sick, vomiting, also oral mucosa erythema, ulcer and esophagitis, gastritis can be arranged.
Though Zorubicin has a lot of untoward reactions, because Zorubicin has very strong antitumour activity, still as the main medicine for the treatment of cancer, the Zorubicin derivative of a kind of high-efficiency low-toxicity of necessary searching.Owing to after only in cancer cells, being decomposed anti-tumor activity is arranged, utilize a lot of that the asparagine restriction endonuclease (legumain) that contains in the cancer cells studies by cancer cells Mid-Heaven Gate winter acid amides restriction endonuclease; Many domestic and international research units carry out a large amount of structural modifications to Ah enzyme's element, effect is preferably people such as Liucheng at Cancer Research (63,2957-2964, June 1,2003) a last compound, utilize and contain a large amount of and specific asparagine restriction endonuclease (legumain) in the cancer cells, design a kind of target compound, connect the tetrapeptide that contains asparagine on the amino of Zorubicin, this compound can reduce the toxicity of Zorubicin greatly, but this compound is insoluble in the aqueous solution, can't make injection, and amino protecting group is very unstable, is easy to be hydrolyzed in the normal cell tissue, does not have actual application value; Therefore; be necessary at the unsettled defective of amino protecting group; design new compound; this compound is stable in the normal cell tissue, facile hydrolysis not, and do not have toxicity; and possesses target; after arriving cancer cells, amino protecting group is downcut by asparagine, the anti-tumor activity of performance Zorubicin.
Summary of the invention
Technical scheme of the present invention is at the unsettled defective of the plain amino protecting group of above-mentioned Ah enzyme, and the Zorubicin derivative is provided; Another technical scheme of the present invention has provided the preparation method and the purposes of this Zorubicin derivative.
The invention provides a kind of Zorubicin derivative, its structural formula is:
Wherein, R1 is β-alanyl or L-alanyl; R2 is carbonyl or alkylsulfonyl or phosphoryl; R3 is hydrogen atom, C1-C6 alkyl or alkenyl, replacement or does not replace C7-C10 aryl, replacement or unsubstituted C3-C8 cycloalkyl or cycloalkenyl group, replacement or do not replace C7-C12 aralkyl or thiazolinyl; R4 be respectively in hydrogen atom, hydroxyl or carboxyl identical or inequality, sulfonic group, phosphate, amino, the halogen atom substituting group one or more etc., X can replace the number of hydrogen atoms order on the R3.
Further, R1 is β-alanyl or L-alanyl; R2 is a carbonyl; R3 is hydrogen atom, C1-C6 alkyl or alkenyl, replacement or does not replace C7-C10 aryl, replacement or unsubstituted C3-C8 cycloalkyl or cycloalkenyl group, replacement or do not replace C7-C12 aralkyl or thiazolinyl; R4 be respectively in hydrogen atom, hydroxyl or carboxyl identical or inequality, sulfonic group, phosphate, amino, the halogen atom substituting group one or more etc., X can replace the number of hydrogen atoms order on the R3.
Further, R1 is β-alanyl or L-alanyl; R2 is a carbonyl; R3 is an ethyl; R4 be respectively in hydrogen atom, hydroxyl or carboxyl identical or inequality, sulfonic group, phosphate, amino, the halogen atom substituting group one or more etc., X is 5.
Preferentially, R1 is β-alanyl or L-alanyl; R2 is a carbonyl; R3 is an ethyl; R4 is a carboxyl.
The invention provides succinyl β-alanyl L-alanyl L-asparagine acyl L-leucyl Zorubicin and salt or hydrate.
The invention provides succinyl L-alanyl L-alanyl L-asparagine acyl L-leucyl Zorubicin and salt or hydrate.
The present invention also provides the preparation method of this Zorubicin derivative, comprises the steps:
The tetrapeptide of anamorphic zone amido protecting at first; carboxyl on this tetrapeptide again with the amino condensation of Zorubicin, obtain the tetrapeptide Zorubicin of amido protecting, then remove the amino protecting group on the peptide; use all kinds of acid and amino group condensation then, obtain the present invention and get the Zorubicin derivative.
The present invention also provides the purposes of Zorubicin derivative in the medicine of preparation treatment cancer.
The invention provides a kind of pharmaceutical composition for the treatment of cancer, it comprises as the Zorubicin compound of activeconstituents and pharmaceutically acceptable carrier thereof.
Prove that by pharmacodynamics test Zorubicin derivative of the present invention is the compound with high-efficiency low-toxicity of antitumour activity, and have tangible target.
The present invention adds alpha-non-natural amino acid by changing the plain amino protecting group of Ah enzyme in the peptide chain, increased water-solublely, improves bioavailability; And increased the stability of compound, can improve cancer cells kind drug concentrations, significantly reduced side effect, had tangible target, drug effect is significantly increased than Zorubicin; Pass through chemically modified simultaneously, the stability of compound improves greatly, is difficult for being hydrolyzed.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
The embodiment of form is described in further detail foregoing of the present invention again by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Specific implementation method
Embodiment 1 SUC-L-ALA-L-ALA-L-ASN-LEU-DOX's (SD-1) is synthetic
(succinyl L-alanyl L-alanyl L-asparagine acyl L-leucyl Zorubicin) (SD-1)
1, BOC-L-ALA-L-ALA-L-ASN-L-LEU-OH's is synthetic
(tertbutyloxycarbonyl)-L-alanyl-L-alanyl-L-asparagine acyl-L-leucine)
The preparation of a, Boc-L-ASN-LEU-OBZL
Boc-L-ASN (tertbutyloxycarbonyl-altheine) 23.2g (100mmol), L-LEU-OBZL (L-leucine benzyl ester) 24.3g (110mmol), HOBT (1-hydroxyl benzotriazole) 14.8g (110mmol), THF (tetrahydrofuran (THF)) 300ml, dissolving together, the frozen water cooling adds DCC (N, the N`-dicyclohexyl carbodiimide) 22.7g (110mmol), reaction 4hr removes by filter DCU (N, N`-dicyclohexylurea (DCU)), decompression steams THF, add ethyl acetate 200ml, use the pickling decon, dry concentrating, the product 39g of recrystallization in ethyl acetate, yield 90%.Product structure obtains mass spectrum and confirms M
+436.1, be not further purified, be directly used in next step reaction.
The preparation of b, Boc-L-ALA-L-ASN-LEU-OBZL
Claim previous step product B oc-L-ASN-LEU-OBZL34.8g (80mmol), be dissolved in 1N HCL (hydrogenchloride) ethyl acetate 200ml, reaction 1hr, concentrating under reduced pressure gets solid, is dissolved in THF 100ml, add NMM (4-methylmorpholine) and be neutralized to weakly alkaline, add BOC-L-ALA (tert-butoxycarbonyl-l-alanine) 16.6g (88mmol), HOBT10.8g (80mmol), THF100ml, the frozen water cooling, add DCC 18.1g, reaction 4hr removes by filter DCU, decompression steams THF, add ethyl acetate 200ml, use the pickling decon, dry concentrating, the product 32g of recrystallization in ethyl acetate, yield 80%.Product structure obtains mass spectrum and confirms M
+507.1, be not further purified, be directly used in next step reaction.
The preparation of c, Boc-L-ALA-L-ALA-L-ASN-LEU-OBZL
Claim previous step product B oc-L-ALA-L-ASN-LEU-OBZL 30.3g (60mmol), be dissolved in 1N HCL ethyl acetate 200ml, reaction 1hr, concentrating under reduced pressure gets solid, is dissolved in THF100ml, add NMM and be neutralized to weakly alkaline, add BOC-L-ALA12.5g (66mmol), HOBT8.1g (60mmol), THF100ml, the frozen water cooling, add DCC13.6g, reaction 4hr removes by filter DCU, decompression steams THF, add ethyl acetate 200ml, use the pickling decon, dry concentrating, the product 29.4g of recrystallization in ethyl acetate, yield 85%.Product structure obtains mass spectrum and confirms M
+578.1, be not further purified, be directly used in next step reaction.
D, Boc-L-ALA-L-ALA-L-ASN-LEU-OH are synthetic
Claim previous step product B oc-L-ALA-L-ALA-L-ASN-LEU-OBZL24.4g (50mmol) to be dissolved among the 500MLTHF, add 5% palladium carbon, 2 grams, feed hydrogen, room temperature reaction spends the night, and removes by filter palladium carbon, and concentrating under reduced pressure gets product 19 grams, yield 97%.Products obtained therefrom is not further purified, and can be directly used in next step reaction, can be used as the raw material of preparation SUC-L-ALA-L-ALA-L-ASN-LEU-DOX (SD-1) yet.
2, Fmoc-L-ALA-L-ALA-L-ASN-LEU-OH's is synthetic
Claim Boc-L-ALA-L-ALA-L-ASN-LEU-OH 14.6g (30mmol), add the 40ml trifluoroacetic acid, 0 ℃ is stirred 30min, after reacting completely, decompression steams trifluoroacetic acid (TFA), use the 150ml water dissolution, transfer pH value 8-9, claim Fmoc-osu (fluorenylmethyloxycarbonyl succinimide ester) 11.1g (33mmol) to be dissolved among the 50mlTHF by NaCO3, slowly add in the reaction solution, and control pH value be about 8, room temperature reaction 2 hours is after reacting completely, adjust pH about 4, the product that filtration is separated out, it is inferior to give a baby a bath on the third day after its birth with ether 50ml, drying.Product structure obtains mass spectrum and confirms M
+610.1, be not further purified, be directly used in preparation SUC-L-ALA-L-ALA-L-ASN-LEU-DOX (SD-1).
3, SUC-L-ALA-L-ALA-L-ASN-LEU-DOX's (SD-1) is synthetic
Synthetic method one
(1) Boc-L-ALA-L-ALA-L-ASN-LEU-DOX is synthetic
Boc-L-ALA-L-ALA-L-ASN-LEU-OH (1.1g, 2.2mmol), Doxorubicin.HCL (Lipodox) (1.0g, 1.7mmol) be dissolved in DMF (N, dinethylformamide) 100ml adds NMM0.4ml, is cooled to 0 ℃, add HBTU (benzotriazole-N, N, N ', N ',-tetramethyl-urea phosphofluoric acid ester) 1.1g, room temperature reaction 2 hours is poured in the 500ml water, fully stirs, filter, get the red-brown powder, it is inferior to give a baby a bath on the third day after its birth with the 50ml ether again, drying under reduced pressure, get product 1.45g, yield 84%.Product molecular formula C48H64N6018 calculates molecular weight 1012.4, and structure obtains mass spectrum and confirms M
-1011.3, be not further purified, be directly used in next step reaction.
(2) SUC-L-ALA-L-ALA-L-ASN-LEU-DOX's is synthetic
Claim Boc-L-ALA-L-ALA-L-ASN-LEU-DOX 1g (1mmol) to be dissolved in the 10ml methylene dichloride, add trifluoroacetic acid 1ml (13mmol), 0 ℃ was reacted 0.5 hour.Be cooled to-10 ℃ with cryosel, add NMM2.9ml (26mmol), controlled temperature adds Succinic anhydried 5.0g (50mmol) below-10 ℃, rises to room temperature reaction gradually 2 hours.Add ether 100ml and stir fully, filtering-depositing gets product 530mg with chromatographic silica gel chloroform/methanol (5/1) purifying, yield 52%, and product molecular formula C47H60N6019 calculates molecular weight 1012.2.Mass spectrum is confirmed as M+NA 1035.3, proves to obtain desired structure.
Synthetic method two
(1) Fmoc-L-ALA-L-ALA-L-ASN-LEU-DOX is synthetic
Fmoc-L-ALA-L-ALA-L-ASN-LEU-OH (1.34g, 2.2mmol), (1.0g 1.7mmol) is dissolved in DMF100ml to Doxorubicin.HCL, add NMM0.4ml, be cooled to 0 ℃, add HBTU benzotriazole-N, N, N ', N ' ,-tetramethyl-urea phosphofluoric acid ester 1.1g, room temperature reaction 2 hours is poured in the 500ml water, fully stirs, filter, get the red-brown powder, it is inferior to give a baby a bath on the third day after its birth with the 50ml ether again, drying under reduced pressure gets product 1.68g, yield 87%.Product structure obtains mass spectrum and confirms M-1133.2, and FT-IR (infrared spectra) analyzes OH (hydroxyl) absorption peak and 1668cm-1C=O (carbonyl) absorption peak with 3410cm-1, proves that tetrapeptide reacts in Zorubicin.Be not further purified, be directly used in next step reaction.
(2) SUC-L-ALA-L-ALA-L-ASN-LEU-DOX's is synthetic
Claim Fmoc-L-ALA-L-ALA-L-ASN-LEU-DOX 1.3g (1mmol) to be dissolved among the 10mlDMF, add piperazine and decide 2ml, room temperature reaction 5 minutes.Be cooled to-10 ℃ with cryosel, add Succinic anhydried 5.0g (50mmol), rose to room temperature reaction gradually 2 hours.Add ether 100ml and stir fully, filtering-depositing gets product 650mg, yield 63% with chromatographic silica gel chloroform/methanol (5/1) purifying.Product molecular formula C47H60N6019 calculates molecular weight 1012.2, and mass spectrum is confirmed as M+NA1035.3, and FT-IR (infrared spectra) analyzes carboxylic OH (hydroxyl) absorption peak and 1657cm-1 carboxylic C=O (carbonyl) absorption peak with 3328cm-1, proves to obtain desired structure.
Embodiment 2, SUC-β-ALA-L-ALA-L-ASN-LEU-DOX's (SD-2) is synthetic
(succinyl β-alanyl L-alanyl L-asparagine acyl L-leucyl Zorubicin) (SD-2)
1, BOC-β-ALA-L-ALA-L-ASN-L-LEU-OH's is synthetic
The preparation of a, Boc-L-ASN-LEU-OBZL
Boc-L-ASN23.2g (100mmol), L-LEU-OBZL24.3g (110mmol), HOBT14.8g (110mmol), THF300ml, dissolving together, frozen water cooling, add DCC22.7g (110mmol), reaction 4hr removes by filter DCU, decompression steams THF, add ethyl acetate 200ml, use the pickling decon, dry concentrating, the product 39g of recrystallization in ethyl acetate, yield 90%.Product structure obtains mass spectrum and confirms M
+436.1, be not further purified, be directly used in next step reaction.
The preparation of b, Boc-L-ALA-L-ASN-LEU-OBZL
Claim previous step product B oc-L-ASN-LEU-OBZL34.8g (80mmol), be dissolved in 1NHCL ethyl acetate 200ml, reaction 1hr, concentrating under reduced pressure gets solid, is dissolved in THF100ml, add NMM and be neutralized to weakly alkaline, add BOC-L-ALA16.6g (88mmol), HOBT10.8g (80mmol), THF100ml, the frozen water cooling, add DCC18.1g, reaction 4hr removes by filter DCU, decompression steams THF, add ethyl acetate 200ml, use the pickling decon, dry concentrating, the product 32g of recrystallization in ethyl acetate, yield 80%.Product structure obtains mass spectrum and confirms M
+507.1, be not further purified, be directly used in next step reaction.
The preparation of c, Boc-β-ALA-L-ALA-L-ASN-LEU-OBZL
Claim previous step product B oc-L-ALA-L-ASN-LEU-OBZL30.3g (60mmol), be dissolved in 1N HCL ethyl acetate 200ml, reaction 1hr, concentrating under reduced pressure gets solid, is dissolved in THF100ml, add NMM and be neutralized to weakly alkaline, add BOC-β-ALA 12.5g (66mmol), HOBT8.1g (60mmol), THF100ml, the frozen water cooling, add DCC13.6g, reaction 4hr removes by filter DCU, decompression steams THF, add ethyl acetate 200ml, use the pickling decon, dry concentrating, the product 29.4g of recrystallization in ethyl acetate, yield 85%.Product structure obtains mass spectrum and confirms M
+578.1, be not further purified, be directly used in next step reaction.
D, Boc-β-ALA-L-ALA-L-ASN-LEU-OH are synthetic
Claim previous step product B oc-β-ALA-L-ALA-L-ASN-LEU-OBZL 24.4g (50mmol) to be dissolved among the 500mlTHF, add 5% palladium carbon, 2 grams, feed hydrogen, room temperature reaction spends the night, and removes by filter palladium carbon, and concentrating under reduced pressure gets product 19 grams, yield 97%.Be not further purified, can be directly used in next step reaction, can be used as the raw material of preparation SUC-β-ALA-L-ALA-L-ASN-LEU-DOX (SD-1) yet.
2 Fmoc-β-ALA-L-ALA-L-ASN-LEU-OH's is synthetic
Claim Boc-β-ALA-L-ALA-L-ASN-LEU-OH14.6g (30mmol), add the 40ml trifluoroacetic acid, 0 ℃ is stirred 30min, and after reacting completely, decompression steams trifluoroacetic acid, uses the 150ml water dissolution, by NaCO
3Adjust pH 8-9 claims Fmoc-osu 11.1g (33mmol) to be dissolved among the 50mlTHF, slowly add in the reaction solution, and control pH value is about 8, and room temperature reaction 2 hours, after reacting completely, adjust pH is about 4, filters the product of separating out, and it is inferior to give a baby a bath on the third day after its birth with ether 50ml, drying.Product structure obtains mass spectrum and confirms M
+610.1, be not further purified, be directly used in preparation SUC-β-ALA-L-ALA-L-ASN-LEU-DOX (SD-1).
3 SUC-β-ALA-L-ALA-L-ASN-LEU-DOX's is synthetic
Synthetic method one
(1) Boc-β-ALA-L-ALA-L-ASN-LEU-DOX is synthetic
Boc-β-ALA-L-ALA-L-ASN-LEU-OH (1.1g, 2.2mmol), Doxorubicin.HCL (1.0g, 1.7mmol) be dissolved in DMF100ml, add NMM0.4ml, be cooled to 0 ℃, add HBTU1.1g, room temperature reaction 2 hours is poured in the 500ml water, fully stir, filter, get the red-brown powder, it is inferior to give a baby a bath on the third day after its birth with the 50ml ether again, drying under reduced pressure gets product 1.45g, yield 84%.Product structure obtains mass spectrum and confirms M
-1011.2, be not further purified, be directly used in next step reaction.
(2) SUC-β-ALA-L-ALA-L-ASN-LEU-DOX's is synthetic
Claim Boc-β-ALA-L-ALA-L-ASN-LEU-DOX 1g (1mmol) to be dissolved in the 10ml methylene dichloride, add trifluoroacetic acid 1ml (13mmol), 0 ℃ was reacted 0.5 hour.Be cooled to-10 ℃ with cryosel, add NMM2.9ml (26mmol), controlled temperature adds Succinic anhydried 5.0g (50mmol) below-10 ℃, rises to room temperature reaction gradually 2 hours.Add ether 100ml and stir fully, filtering-depositing gets product 530mg with chromatographic silica gel chloroform/methanol (5/1) purifying, yield 52%, and product molecular formula C47H60N6019 calculates molecular weight 1012.2 product structures and obtains mass spectrum affirmation M
-1011.2.
Synthetic method two
(1) Fmoc-β-ALA-L-ALA-L-ASN-LEU-DOX is synthetic
Fmoc-β-ALA-L-ALA-L-ASN-LEU-OH (1.34g, 2.2mmol), Doxorubicin.HCL (1.0g, 1.7mmol) be dissolved in DMF100ml, add NMM0.4ml, be cooled to 0 ℃, add HBTU1.1g, room temperature reaction 2 hours is poured in the 500ml water, fully stir, filter, get the red-brown powder, it is inferior to give a baby a bath on the third day after its birth with the 50ml ether again, drying under reduced pressure gets product 1.68g, yield 87%.Product structure obtains mass spectrum and confirms M-1133.2, proves the two condensation reaction.Be not further purified, be directly used in next step reaction.
(2) SUC-β-ALA-L-ALA-L-ASN-LEU-DOX's is synthetic
Claim Fmoc-β-ALA-L-ALA-L-ASN-LEU-DOX 1.3g (1mmol) to be dissolved among the 10mlDMF, add piperazine and decide 2ml, room temperature reaction 5 minutes.Be cooled to-10 ℃ with cryosel, add Succinic anhydried 5.0g (50mmol), rose to room temperature reaction gradually 2 hours.Add ether 100ml and stir fully, filtering-depositing gets product 650mg, yield 63% with chromatographic silica gel chloroform/methanol (5/1) purifying.Product molecular formula C47H60N6019 calculates molecular weight 1012.2 product structures and obtains mass spectrum affirmation M
-1011.2; Have carbonyl 1658cm-1 and hydroxyl 3382cm-1 absorption peak in the tangible carboxyl among the FT-IR (infrared spectra), prove to obtain desired structure.
Synthesizing of embodiment 3 β-other derivatives of ALA-L-ALA-L-ASN-LEU-DOX
Based on β-ALA-L-ALA-L-ASN-LEU-DOX, can be easily acyl chlorides and amino reaction by various acid, the condensation reaction of amino and various acid, and acid anhydrides and amino reaction, the reaction of active ester and amino etc., the derivative of synthetic various β-ALA-L-ALA-L-ASN-LEU-DOX, comprise various natural and non-natural derivatives, various carboxylic acids, sulfonic acid, phosphoric acid analog derivative, these reaction pair synthetic works person is easy to realize, in the present invention with regard to plain statement.
Claim β-ALA-L-ALA-L-ASN-LEU-DOX 0.912g (1mmol), BocGlu (obut) (tertbutyloxycarbonyl glutamic acid tert-butyl) 0.33g (1.1mmol) is if in the reaction flask; adding DMF5ml; the frozen water cooling is if HBTU0.42g (1.1mmol) reacted 2 hours; pour in the water and precipitate; filter, with ether washing, drying; behind 1NHCL ethyl acetate solution deprotection, get product G lu-β-ALA-L-ALA-L-ASN-LEU-DOX
Claim that β-ALA-L-ALA-L-ASN-LEU-DOX 0.912g (1mmol) is dissolved among the pyridine 5ml, be cooled to 0 ℃ and add Tosyl chloride (Tosl-cl) 0.2lg (1.1mmol), reacted 2 hours, add in the entry, use ethyl acetate extraction, purifying, the dry product Tosl-β-ALA-L-ALA-L-ASN-LEU-DOX that gets
The preparation that embodiment 4 SD-1 SD-2 make
With SD-1, SD-2 vacuum-drying obtains orange red loose block or powder, and by the aseptically process that gas sterilization is carried out strictness, it is aseptic subpackaged to use the ampere bottle of having sterilized in advance to carry out at sterilisable chamber, gets powder injection.
With SD-1, SD-2 vacuum-drying obtains orange red loose block or powder is dissolved in the water for injection at sterilisable chamber, carries out packing, and lyophilize gets powder injection.
The method of embodiment 5 SD-1 SD-2 assays and the scope of content.SD-1, SD-2 standard substance adopt preparation HPLC (high performance liquid phase) to obtain 99.9% sample (Agilent HPLC, C-4 post, 100mm * 40mm, 25ml/ minute, 50% methyl alcohol/50% water, wavelength 254nm), SD-1, the SD-2 sample adopts analysis mode HPLC (Alltech HPLC, C-18,150mm * 4.6mm, 1ml/ minute, 30% second cyanogen/70% water, wavelength 254nm), purity is at 98.0%--98.5%.
Below prove beneficial effect of the present invention by pharmacodynamics test.
Test example 1 SD-1, SD-2 is subjected to reagent intravenous administration LD
50Mensuration
Test objective: by measuring the LD of mouse tail vein injection administration
50, the acute toxicity of understanding preparation of the present invention.
Material:
Medicine: SD-1, SD-2 injection liquid, red transparence liquid.Being diluted to respective concentration with injection physiological saline during test uses.
Animal: one-level ICR mouse, body weight 18-20g, male and female half and half.
Method and result:
(1) trial test
24 of ICR mouse, male and female half and half, body weight 18-20g is divided into 4 groups at random by body weight, and 6 every group, male and female half and half.Press 5mg/kg, 50mg/kg, the disposable tail vein injection administration of 500mg/kg, 5000mg/kg, administration volume 0.1ml/kg observed 7 continuously, the record death condition, and mortality ratio is respectively 0%, 0%, 100%, 100% as a result; 24 mouse again, the grouping situation is the same, carries out revision test by 50mg/kg, 100mg/kg, 200mg/kg, 400mg/kg dosage, records the about 400mg/kg of Dm, the about 100mg/kg of Dn.
(2) official test
50 of ICR mouse, male and female half and half, body weight 18-20g is divided into 5 groups at random by body weight, and 10 every group, male and female half and half.By between 0.8 group than (being respectively 320mg/kg, 256mg/kg, 205mg/kg, 164mg/kg, 131mg/kg) disposable tail vein injection administration, administration volume 0.1ml/kg observed 7 continuously, the record death condition, carry out statistical treatment with the Bliss method, the results are shown in Table 1.
The LD of table 1 SD-1 medicine mouse mainline administration
50
| Group | Dosage (mg/kg) | Animal (only) | Death toll (only) | Mortality ratio (%) | LD 50(mg/kg) |
| 1 | 320 | 10 | 9 | 90％ | 215.29 (95% fiducial limit: 186.18～248.95) |
| 2 | 256 | 10 | 6 | 60％ | |
| 3 | 205 | 10 | 5 | 50％ | |
| 4 | 164 | 10 | 2 | 20％ | |
| 5 | 131 | 10 | 1 | 10％ |
The LD of table 2 SD-2 medicine mouse mainline administration
50
| Group | Dosage (mg/kg) | Animal (only) | Death toll (only) | Mortality ratio (%) | LD 50(mg/kg) |
| 1 | 320 | 10 | 8 | 80％ | 228.30 (95% fiducial limit: 193.58～269.25) |
| 2 | 256 | 10 | 6 | 60％ | |
| 3 | 205 | 10 | 4 | 40％ | |
| 4 | 164 | 10 | 2 | 20％ | |
| 5 | 131 | 10 | 1 | 10％ |
Result and discussion: the LD50 value is the important index of sign drug toxicity intensity, table 1, SD-1 shown in the table 2, the value of SD-2LD50 is higher than Zorubicin LD50 (20mg/Kg) (medicinal application and toxicity data, Zhang Guiqing, Lv Ailing etc., press of Henan Medical Univ. far away, 1999.4, p472), show SD-1, two kinds of compound acute toxicities of SD-2 are than the obvious reduction of Zorubicin.
Test example 2 SD-1, SD-2 is at intravital drug distribution of mice with tumor and drug efficacy study
Purpose:, observe the distribution situation of medicine and the intensity of antitumor action thereof by to the tumor-bearing mice intravenously administrable.
Method and result:
One, pharmacodynamic study
1, animal: C57BL/6 mouse, age in 6-8 week, male or female (with a collection of experiment unanimity).
2, the making of model
1) cultivation of lung carcinoma cell: with frozen LEWIS lung carcinoma cell thaw the back use the 1640+10% calf serum, 37 ℃, 5%CO
2Under the condition, cultivate go down to posterity 3-4 time (going down to posterity once in per 3 days).
2) making of knurl source animal: the lung carcinoma cell 10 that will go down to posterity and cultivate
6/ inoculated with subcutaneous injections treats that to the mouse oxter knurl bulk-growth is to 1-2cm (about 14 days).Aseptic operation takes off the knurl body, shreds and homogenate, and the centrifuging and taking supernatant is continued to employ.
3) every mouse oxter of the supernatant liquor that obtains inoculated with subcutaneous injections 0.3ml, about experiment mice that can get the lotus knurl in 7 days is standby.
3, antitumor test
1) animal model for tumour: by on the tumor-bearing mice that obtains, treat that tumour grows to about 0.5cm random packet.
2) grouping: test is divided into lotus knurl animal pattern control group, lotus knurl animal pattern positive drug treatment control group (Zorubicin), lotus knurl animal pattern SD-1, SD-2 medication therapy groups.10 of every treated animals.
3) grouping and dosage
| Group | Animal | Dosage mg/kg | Concentration mg/ml | Route of administration | Administration volume ml/kg | The clinical multiple of people |
| SD-1 group SD-2 group Zorubicin contrast model control group | 10 10 10 10 | 20 20 10 - | 2.0 2.0 1.0 physiological saline | Tail vein tail vein tail vein tail vein | 10 10 10 10 | 10 10 10 - |
4) testing sequence: press SD-1, the approach of SD-2 clinical application, lotus knurl animal pattern SD-1, SD-2 medication therapy groups administration weekly twice, totally two weeks.Positive drug treatment control group (Zorubicin) is pressed the clinical treatment dosed administration, and lotus knurl animal pattern control group is only given isopyknic physiological saline.Measured the size of a tumour in per 3 days.After the administration 24 hours the last time, put to death each treated animal, the line of apsides with each animal knurl body of trip kind of calliper by formula calculates tumor size.
5) observe and testing index: according to the volume size data of each group knurl body, model administration group and model control group are relatively calculated the tumour inhibiting rate % of each dose drug, take statistics and learn processing, and with the positive controls comparative effectiveness.
6) test-results sees Table 3
Table 3 SD-1, SD-2 are subjected to the influence of reagent to tumor-bearing mice
| Group | Animal (only) | Knurl body size (mm 3) | Tumour inhibiting rate (%) | ||
| 9 days | 15 days | 9 days | 15 days | ||
| SD-1 group SD-2 group Zorubicin contrast model control group | 10 10 10 10 | 18±2.33 13±2.02 133±10.17 228±16.85 | 17±2.88 14±1.97 200±20.04 335±28.66 | 92.11 94.30 41.67 | 94.93 95.82 40.30 |
The result shows: SD-1, SD-2 obviously have tumor inhibition effect, compare with Zorubicin to have significantly improved the tumor suppression effect.
The site of action of test example 3 medicines of the present invention distributes and studies
1, the preparation of knurl mouse is the same.
2, drug distribution is measured 90 of tumor-bearing mices, is divided into 3 groups, and 30 every group, 2 groups is the medicine group, disposable tail vein injection SD-1, SD-2 medicine 12mg/kg; 1 group is control group, disposable tail vein injection Zorubicin 6mg/kg, 1,4,12 hour each group is broken cervical vertebra respectively and is put to death 10 mouse after administration, get knurl body, the heart, liver, nephridial tissue, add after the 50% ethanolic soln homogenate centrifugal with the hydrochloric acid of the 0.5M of 20 times of volumes, get supernatant liquor and prepare sample, the fluorescence spectrophotometry detection method detects the medicament contg in each tissue.The results are shown in Table 4.
Table 4 SD-1, the SD-2 medicine is in the intravital distribution of tumor-bearing mice (ug/g)
| Group | 4h | 12h | ||||||
| The knurl body | The heart | Liver | Kidney | The knurl body | The heart | Liver | Kidney | |
| SD-1 group SD-2 group control group | 470±20.88 580±27.43 40±6.25 | 5±0.71 4±0.44 15±3.72 | 13±1.44 10±1.06 20±2.66 | 17±2.93 16±2.69 35±4.17 | 450±21.32 550±25.64 35±4.44 | 3±0.46 2±0.46 13±3.39 | 7±1.10 7±1.34 15±3.06 | 13±2.63 12±2.11 30±2.88 |
The result shows, SD-1, behind the SD-2 intravenously administrable, improve greatly at cancerous tissue Chinese traditional medicine concentration ratio Zorubicin, and medicine summation in other organ-tissues of animal significantly reduces, and especially cardiac muscular tissue's Chinese traditional medicine distributes and reduces obviously, and medicine has tangible target, this is to improving curative effect, and it is highly beneficial to reduce toxic side effect.
In sum, the present invention synthesizes the modified compound of two kinds of Ah enzyme's elements, and all has tangible tumor promotion by two kinds of compounds of pharmacological evaluation proof, father's medicine Ah enzyme element than them has lower cytotoxicity, the two also has tangible target, and steady quality, and controllability is strong.
Claims (10)
1, a kind of Zorubicin derivative, its structural formula is:
Wherein, R1 is β-alanyl or L-alanyl; R2 is carbonyl or alkylsulfonyl or phosphoryl; R3 is C1-C6 alkyl or alkenyl, replacement or does not replace C7-C10 aryl, replacement or unsubstituted C3-C8 cycloalkyl or cycloalkenyl group, replacement or do not replace C7-C12 aralkyl or thiazolinyl; R4 is one or more in hydrogen atom, hydroxyl or carboxyl identical or inequality, sulfonic group, phosphate, amino, the halogen atom, and x can replace the hydrogen atom number on the R3.
2, Zorubicin derivative according to claim 1 is characterized in that: R1 is β-alanyl or L-alanyl; R2 is a carbonyl; R3 is the C1-C6 alkyl or alkenyl, replaces or do not replace the C7-C10 aryl, replaces or unsubstituted C3-C8 cycloalkyl or cycloalkenyl group, replaces or do not replace C7-C12 aralkyl or thiazolinyl; R4 is one or more in hydrogen atom, hydroxyl or carboxyl identical or inequality, sulfonic group, phosphate, amino, the halogen atom, and x can replace the number of hydrogen atoms order on the R3.
3, Zorubicin derivative according to claim 2 is characterized in that: R1 is β-alanyl or L-alanyl; R2 is a carbonyl; R3 is an ethyl; R4 is one or more in hydrogen atom, hydroxyl or carboxyl identical or inequality, sulfonic group, phosphate, amino, the halogen atom, and x is 5.
4, Zorubicin derivative according to claim 3 is characterized in that: R1 is β-alanyl or L-alanyl; R2 is a carbonyl; R3 is an ethyl; R4 is a carboxyl.
5, Zorubicin derivative according to claim 4, it is characterized in that: R1 is β-alanyl, described Zorubicin derivative is: succinyl β-alanyl L-alanyl L-asparagine acyl L-leucyl Zorubicin and salt or hydrate.
6, according to the described Zorubicin derivative of claim, it is characterized in that: R1 is the L-alanyl, and described Zorubicin derivative is: succinyl L-alanyl L-alanyl L-asparagine acyl L-leucyl Zorubicin and salt or hydrate.
7, a kind of method for preparing the described Zorubicin derivative of claim 1 comprises the steps:
The tetrapeptide of anamorphic zone amido protecting at first, the amino condensation of the carboxyl of this tetrapeptide and Zorubicin obtains the tetrapeptide Zorubicin of amido protecting, then removes the amino protecting group on the peptide, uses all kinds of acid and amino group condensation then, obtains the present invention and gets the Zorubicin derivative.
8, the purposes of the described Zorubicin derivative of claim 1 in the medicine of preparation treatment cancer.
9, a kind of pharmaceutical composition for the treatment of cancer, it comprises the preparation that forms as the described compound of the claim 1 of activeconstituents and pharmaceutically acceptable preparing carriers thereof.
10, the pharmaceutical composition of treatment cancer according to claim 9 is characterized in that: described preparation is an injection formulations.
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