CN104262457A - Water-soluble cancer targeted activation docetaxel derivatives and uses thereof - Google Patents
Water-soluble cancer targeted activation docetaxel derivatives and uses thereof Download PDFInfo
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Abstract
Water-soluble cancer targeted activation docetaxel derivatives and uses thereof are disclosed. The general formula of the docetaxel anti-tumor medicines is shown in the specification, wherein R1 is any amino acid selected from Ala, Thr, Val or Ile; R2 is any amino acid selected from Ala, Thr, Val or Asn; and n is any integer ranging from 1 to 150. The docetaxel derivatives have characteristics of good water solubility, specific tumor targeting properties and capability of promoting immunotherapy. Compared with docetaxel, efficacy of the docetaxel derivatives for inhibiting tumor growth and metastasis is largely improved, toxicity of the docetaxel derivatives is largely reduced, and the docetaxel derivatives have more efficient and safer antitumor functions and have a good application prospect.
Description
Technical field
The present invention relates to a kind of antitumour drug compounds, particularly, relate to a kind of antitumor drug preparation method and purposes of Docetaxel derivative of water-soluble targeted activation.
Background technology
Docetaxel (Docetaxel) is a kind of now widely used effective antitumour agent,
Be mainly used in the treatment of various noumenal tumour as ovarian cancer and mammary cancer, also have certain curative effect to lung cancer, large bowel cancer, melanoma, incidence cancer, lymphoma, brain tumor.But, apply this compound clinically because have serious toxic side effect (as bone marrow depression) and sensitivity response, and be limited its using dosage.Docetaxel has bone marrow toxicity, causes Neutrophilic granulocytopenia, has neurotoxicity and Cardiovascular Toxicity, and can cause anaphylaxis, overflows can to cause the inflammation of local, alopecia outside blood vessel, weakly even bring hepatotoxicity.Therefore, need to provide one can reduce Docetaxel toxicity, and drug use dosage can be improved and there is the targeting activation medicine of height drug effect as antineoplastic agent.The Docetaxel of water-soluble targeted activation provided by the invention is compared with Docetaxel, there is anti-tumor target tropism, special activation characteristic and the characteristic of immunological enhancement, the drug effect tool of Tumor suppression growth and transfer improves a lot, and the preparation of medicine changes, and toxicity reduces greatly, there is extraordinary application prospect.
Summary of the invention
The object of the invention is to provide the Docetaxel derivative that a kind of water-soluble cancer target activates, in this Docetaxel derivative, toxicity is reduced because Docetaxel combines appropriate group, simultaneously because linking group discharges Docetaxel in tumor locus gathering and specific activation, become more low toxicity and effective antitumour medicine.
In order to achieve the above object, the invention provides the antitumor drug of the Docetaxel that a kind of water-soluble cancer target activates, the structural formula of this compound is:
Wherein, R1 is Ala (L-Ala), any one amino acid in Thr (Threonine), Val (α-amino-isovaleric acid) or Ile (Isoleucine); R2 is any one amino acid in Ala, Thr, Val or Asn (aspartic acid), the arbitrary integer in n=1 ~ 150.
The antitumor drug of the Docetaxel that above-mentioned water-soluble cancer target activates, wherein, when n gets 1, R
1get Ala amino acid, R
2when getting Ala amino acid, to be the structural formula of S1, this S1 be this Docetaxel derivative:
The antitumor drug of the Docetaxel that above-mentioned water-soluble cancer target activates, wherein, when n gets 5, R
1get Ala amino acid, R
2when getting Ala amino acid, to be the structural formula of S2, this S2 be this Docetaxel derivative:
The antitumor drug of the Docetaxel that above-mentioned water-soluble cancer target activates, wherein, when n gets 11, R
1get Ala amino acid, R
2when getting Ala amino acid, to be the structural formula of S3, this S3 be this Docetaxel derivative:
The antitumor drug of the Docetaxel that above-mentioned water-soluble cancer target activates, wherein, when n gets 150, R
1get Ala amino acid, R
2when getting Ala amino acid, to be the structural formula of S4, this S4 be this Docetaxel derivative:
The Docetaxel derivative that the water-soluble cancer target of the present invention activates, wherein, it is that embodiment 10-24 obtains that described D51-7059 comprises compound S 10-S24 provided by the invention, and its n is 5, R
1and R
2structure is respectively following compounds (see table 1):
Table 1: compound S 10-S24
| Numbering | R 1 | R 2 | n |
| S10 | L-Ala | Threonine | 5 |
| S11 | L-Ala | α-amino-isovaleric acid | 5 |
| S12 | L-Ala | Asparagine | 5 |
| S13 | Threonine | L-Ala | 5 |
| S14 | Threonine | Threonine | 5 |
| S15 | Threonine | α-amino-isovaleric acid | 5 |
| S16 | Threonine | Asparagine | 5 |
| S17 | α-amino-isovaleric acid | L-Ala | 5 |
| S18 | α-amino-isovaleric acid | Threonine | 5 |
| S19 | α-amino-isovaleric acid | α-amino-isovaleric acid | 5 |
| S20 | α-amino-isovaleric acid | Asparagine | 5 |
| S21 | Isoleucine | L-Ala | 5 |
| S22 | Isoleucine | Threonine | 5 |
| S22 | Isoleucine | α-amino-isovaleric acid | 5 |
| S24 | Isoleucine | Asparagine | 5 |
The Docetaxel derivative that present invention also offers above-mentioned water-soluble cancer target activation prepares the purposes of antitumor drug.This antitumor drug can be used for the treatment of various cancers disease type, and described various cancers type comprises bladder cancer, the cancer of the brain, breast/mammary cancer, cervical cancer, colon-rectum, the esophageal carcinoma, kidney, liver cancer, lung cancer, nasopharyngeal carcinoma, carcinoma of the pancreas, prostate cancer, skin carcinoma, cancer of the stomach, uterus carcinoma, ovarian cancer, carcinoma of testis and leukemia etc.
The Docetaxel derivative that above-mentioned water-soluble cancer target activates prepares the purposes of antitumor drug, and wherein, this antitumor drug can also be used for immunotherapy.
The Docetaxel derivative that above-mentioned water-soluble cancer target activates prepares the purposes of antitumor drug, and wherein, this antitumor drug prevents after can also being used for tumor operation and treats metastases.
The Docetaxel derivative that above-mentioned water-soluble cancer target activates prepares the purposes of antitumor drug, and wherein, this antitumor drug can be used for the anticancer adjuvant drug use in radiation treatment.
The Docetaxel derivative of water-soluble targeted activation provided by the invention, experimental design thinking derives from the complex compound by the different mode connected of a large amount of compound experiments design preparation, then by connecting complex compound to 2 on Docetaxel or 7 (being namely connected to by complex compound respectively on the OH of in Docetaxel molecular formula the 7th or the 2nd), when being existed by tumor tissues or l-asparagine endopeptidase (Legumain), the size of activation efficiency carries out the screening of Docetaxel efficiency again, and screen gained compound successively at R1, R2, and the restraining effect of n to tumour when getting different value, finally obtain toxicity to reduce, the Docetaxel derivative of the water-soluble targeted activation that release efficiency is high.Wherein the structure of the Docetaxel of water-soluble targeted activation is synthesized first for us and is reported.The structure of this compound different piece has an immense impact on to functions such as the target of the Docetaxel of water-soluble targeted activation, activation, stable, toxicity and drug effects.The Docetaxel derivative of water-soluble targeted activation can by the toxicity of complicated chemical connecting sealed Docetaxel, and make effectively to be activated by l-asparagine endopeptidase in its tumour cell in tumor tissues or tumor-associated macrophages, thus the release toxicity of target, reach the effect for the treatment of tumour.Obtain tumor-targeting, it is relevant to the structure activity relationship of whole compound entirety for closing toxicity with efficient activation.By R1, R2, and the screening study of n finds R1, R2, and the selection of n also has certain structure activity relationship, but can find out that the result for the treatment of that n preferred span obtains when being 1 ~ 150 is almost identical from experiment.The Docetaxel of water-soluble targeted activation effectively can reduce the toxicity of Docetaxel, but adds the effect for the treatment of tumour, defines strong structure activity relationship.
The Docetaxel derivative that water-soluble cancer target provided by the invention activates is also based on following discovery:
(1) the Docetaxel derivative of water-soluble targeted activation has gathering at tumor locus, is detained and activation effect, has the characteristic of target tumor microenvironment;
(2) the Docetaxel derivative of water-soluble targeted activation by the activation of tumor tissue specificity or fracture, can generate Docetaxel;
(3) the Docetaxel derivative of internal metabolism Water In The Experiment dissolubility targeted activation does not activate in blood in vitro, has the characteristic of long-term blood stability and normal tissue organ low toxicity;
(4) toxicity of the Docetaxel derivative of water-soluble targeted activation is compared Docetaxel and is greatly reduced;
(5) the coupled position of the both sides group of compound small peptide, and Docetaxel directly determines the activation release of medicine and the solvability of medicine, and stability and validity are all closely related; If medicine cannot activate, medicine is the medicine of a no cytotoxicity, can not have curative effect;
(6) the Docetaxel derivative of water-soluble targeted activation activates in kinds of tumors, adds deliquescent change, directly can change the situation of Docetaxel tumour indication restriction, directly become broad spectrum medicine;
(7) length of the Docetaxel derivative of water-soluble targeted activation also has different impacts, namely the activation of medicine on overall medicine of n different lengths has impact, along with n value increases, Activation Activity slightly declines, and increase Deng a mole drug quality number for metering, but also make the drug metabolism transformation period increase because n value increases, therefore overall drug effect does not reduce.Find through experiment, be selected from the scope of 1 ~ 150 at n, the result for the treatment of that Docetaxel derivative can obtain does not have considerable change.
(8) time in theory due to Nasopharyngeal neoplasms, the protease hydrolysis activities such as the aspartic acid that this Docetaxel derivative is corresponding raise, so it has special curative effect to metastases treatment.
Tumor-associated macrophages (M2 type) is different from monocyte and inflammatory type scavenger cell (M1 type) confirms that mark is exactly the expression of l-asparagine endopeptidase.The cytokine induction monocyte transformation of tumors secrete is tumor-associated macrophages, the immunosuppression that tumor-associated macrophages can stimulate generation strong and directly help tumor cell invasion and transfer.Find in our research that tumor microenvironment release property Docetaxel has killing tumor cells associated macrophages, weaken the immunological enhancement of immunosuppressant cytokine and promotion toxicity cd8 cell in microenvironment.Wherein, the more important thing is, tumor microenvironment release property Docetaxel only activates at tumor by local, be different from classic chemotherapy medicine and can damage overall immunity system, tumor microenvironment release property Docetaxel and PD-1 (Programmed death 1 in our experiment, programmed death-1) suppress antibody (PDL1-antibody, commercially available, think the drug candidate with immunotherapeutic effects at present) there is strong synergistic therapeutic action, the drawback that immunotherapy is difficult to be combined with chemotherapeutics can be solved.
The D51-7059 of water-soluble targeted activation of the present invention is preparing the application in antitumor drug, and described medicine is the pharmaceutical composition be made up of the D51-7059 of water-soluble targeted activation and pharmaceutical excipient.
In sum, the Docetaxel derivative that water-soluble cancer target provided by the invention activates has anti-tumor target tropism, special Activation Activity and the characteristic of immunological enhancement, relative to Docetaxel, the drug effect tool of the growth of its Tumor suppression and transfer improves a lot, and the toxicity of medicine reduces greatly, there is extraordinary application prospect.Up to now, the Docetaxel of the water-soluble targeted activation not yet having patent and bibliographical information identical with us, the Docetaxel medicine of not similar to us water-soluble targeted activation is used for the treatment of the effective ways of human tumor.
Embodiment
The Docetaxel derivative that water-soluble cancer target provided by the invention activates, the structural formula of this Docetaxel derivative is:
Wherein, R1 is Ala (L-Ala), any one amino acid in Thr (Threonine), Val (α-amino-isovaleric acid) or Ile (Isoleucine); R2 is any one amino acid in Ala, Thr, Val or Asn (aspartic acid), the arbitrary integer in n=1 ~ 150.
When n gets 1, R
1get Ala amino acid, R
2when getting Ala amino acid, to be the structural formula of S1, this S1 be this Docetaxel derivative:
When n gets 5, R
1get Ala amino acid, R
2when getting Ala amino acid, to be the structural formula of S2, this S2 be this Docetaxel derivative:
When n gets 11, R
1get Ala amino acid, R
2when getting Ala amino acid, to be the structural formula of S3, this S3 be this Docetaxel derivative:
When n gets 150, R
1get Ala amino acid, R
2when getting Ala amino acid, to be the structural formula of S4, this S4 be this Docetaxel derivative:
Below in conjunction with embodiment, technical scheme of the present invention is described further.
The synthesis of the Docetaxel S1 of the water-soluble targeted activation of embodiment 1
1. the synthesis of two (2-methoxy ethoxy ethanoyl)-1B ethyl esters (I)
By 2-(2-methoxy ethoxy) acetic acid (161mg, 1.2mmol) be dissolved in N, in dinethylformamide (10mL), after ice bath cooling, 2-(7-azo benzotriazole)-N, N, N is added ' under stirring, N '-tetramethyl-urea phosphofluoric acid ester (462mg, 1.2mmol), N, N-diisopropyl ethyl amine (313mg, 2.4mmol) and 1B ethyl ester dihydrochloride (100mg, 0.4mmol), finish, stirred overnight at room temperature, remove solvent under reduced pressure, crude product obtains I (128mg, yield: 77.8%) through reverse phase preparative column purifying.
2. the synthesis of two (2-methoxy ethoxy ethanoyl)-1B (II)
By two (2-methoxy ethoxy ethanoyl)-1B ethyl ester I (122mg; 0.3mmol) be dissolved in tetrahydrofuran (THF) (15mL); lithium hydroxide (39mg is dripped at being cooled to 0 DEG C; 0.9mmol) the aqueous solution (5mL), stirring at room temperature reacts 2 hours.Adjust pH to 2 with concentrated hydrochloric acid under ice bath cooling, steam except freeze-drying after tetrahydrofuran (THF), (112mg, yield: 99%) are not purifiedly directly used in next step to obtain crude product II.
3. the synthesis of two (2-methoxy ethoxy ethanoyl)-L-Lys-L-Ala-L-Ala-L-Asn (Trt)-to aminobenzyl alcohol (III)
By two (2-methoxy ethoxy ethanoyl)-1B (112mg; 0.3mmol) be dissolved in N; in dinethylformamide (10mL); 3-(the adjacent acyloxy of diethoxy)-1 is dripped at being cooled to 0 DEG C; 2; 3-phentriazine-4-ketone (109mg; 0.36mmol), L-Ala-L-Ala-L-Asn (Trt)-to aminobenzyl alcohol (188mg; 0.3mmol) and N; N-diisopropyl ethyl amine (117mg; 0.9mmol), finish, room temperature for overnight.Remove solvent under reduced pressure, crude product obtains III (159mg, yield: 54.0%) through reverse phase preparative column purifying.
4. the synthesis of two (2-methoxy ethoxy ethanoyl)-L-Lys-L-Ala-L-Ala-L-Asn (Trt)-PAB p-nitrophenyl carbonate (IV)
Two (2-methoxy ethoxy ethanoyl)-L-Lys-L-Ala-L-Ala-L-Asn (Trt)-to aminobenzyl alcohol (167mg is added in there-necked flask; 0.17mmol) be dissolved in tetrahydrofuran (THF) (10mL); p-nitrophenyl chloroformate ester (73mg is dripped at being cooled to 0 DEG C; 0.36mmol) with pyridine (39mg, 0.50mmol).Room temperature for overnight, removes solvent under reduced pressure, and crude product obtains IV (153mg, yield: 78.5%) through reverse phase preparative column purifying.
5. the synthesis of two (2-methoxy ethoxy ethanoyl)-L-Lys-L-Ala-L-Ala-L-Asn-PAB p-nitrophenyl carbonate (V)
By two (2-methoxy ethoxy ethanoyl)-L-Lys-L-Ala-L-Ala-L-Asn (Trt)-PAB p-nitrophenyl carbonate (IV) (100mg; 0.087mmol) be dissolved in trifluoroacetic acid (1mL); instillation two is dripped; drain with oil pump immediately; crude product V (80mg), be not purifiedly directly used in next step.
6. the synthesis of two (2-methoxy ethoxy ethanoyl)-L-Ala-L-Ala-L-Asn-PAB Docetaxel (S1)
By two (2-methoxy ethoxy ethanoyl)-L-Lys-L-Ala-L-Ala-L-Asn-PAB p-nitrophenyl carbonate (1176mg; 1.3mmol) with Docetaxel (1293mg; 1.6mmol) with dry N; dinethylformamide (20mL) dissolves; DMAP (318mg is added at being cooled to 0 DEG C; 2.6mmol), stirred overnight at room temperature.Poured into by reaction solution in methylene dichloride, merge organic phase, washing, anhydrous sodium sulfate drying, rotary evaporation is except desolventizing, and crude product obtains target product S1 (511mg, yield: 25%) through reverse phase preparative column purifying.The corresponding mass-to-charge ratio of mass spectrum (MS) detected result S1 is respectively 1573, and to obtain molecular weight 1573.69 corresponding with Structure Calculation
The synthesis of S2, S3, S4, with reference to the synthesis of S1, distinguishes the difference of the molecular weight being only the alkoxyl group substituted acetic acid that the synthesis of the 1st step uses.The synthesis of S2 substitutes 2-(2-methoxy ethoxy) acetic acid with 3,6,9,12,15,18-six oxa-nonadecanoic acid; The synthesis of S3 substitutes 2-(2-methoxy ethoxy) acetic acid with 3,6,9,12,15,18,21,24,27,30,33,36-ten dioxa three margaric acid; The synthesis of S4 substitutes 2-(2-methoxy ethoxy) acetic acid with polyoxy heteroatom fatty acid; The corresponding mass-to-charge ratio of mass spectrum (MS) detected result S2, S3 is respectively 1926,2454, and to obtain molecular weight 1926.11 and 2454.74 corresponding with Structure Calculation.Substance assistant laser desorpted ionized/flight time mass spectrum (MALDI-TOF-MS) detects S4 molecular weight and is about 14964, and to obtain molecular weight 14964.56 corresponding, as shown in table 2 with Structure Calculation.
Table 2: the proterties of compound S 1-S4, mass spectrum and fluorometric investigation result
Embodiment 10 ~ 24
Synthetic method is close with embodiment S1, and when just amino acid connects, raw material difference is as shown in table 3 below.By the R of correspondence
1amino acid and R
2amino acid is dissolved in DMF, adds condensing agent (as 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate) reaction respectively, in 0 DEG C-25 DEG C reaction 0.5-2h, then adds asparagine; , in 0 DEG C-25 DEG C reaction 2-24h.Reaction solution through after by purification process, obtaining 3 peptides, is that synthetic mesophase replaces preparation S10-S24 compound by tripeptides by the method substitute for Al a-Ala-Asn in embodiment 1.Mass spectrum (MS) detected result confirms that S10-S24 compound molecular weight is successively as following table, and the molecular weight predicted with Structure Calculation is consistent.
Table 3: the proterties of compound S 10-S24, mass spectrum and fluorometric investigation result
In the Docetaxel of the water-soluble targeted activation of embodiment 2, the different group of compound is to the tremendous influence of the formulation protocol of medicine
The S1 of synthesis, S2, S3 and S4 and various control compound, respectively through vacuum-drying, by gaseous sterilization aseptically process, carry out packing at sterilisable chamber.Before experimentation on animals, S1, S2, S3 and S4 dissolve with solvent 1 (water for injection) or solvent 2 (30% alcohol, 70% water for injection) in sterilisable chamber, then are diluted to desired concn with water for injection; And control compounds (C1, C2, C3, C4, C5, C6) does not meet the formulation requirements of medicine, as shown in table 4 below.Docetaxel is water insoluble, improved Docetaxel dissolution characteristics generation great variety, and the solubleness in water increases, and the formulation protocol of change to medicine of dissolution characteristics has tremendous influence.Compared with traditional water-fast Docetaxel medicine, S1, S2, S3 and S4 can produce the pharmaceutical preparation of solubility, injected dose and curative effect can be improved, avoid as Docetaxel uses supersensitivity auxiliary material Viscotrol C, be that medicine can the huge advance of application, the Docetaxel indicating water-soluble targeted activation has extraordinary novelty and application prospect.
Table 4: adopt screening of medicaments dissolubility test, the disappearance of approximate heterogeneity or Docetaxel 7 or 2 connections in control compound (that is, respectively group 2 is linked to 2 of Docetaxel or 7-OH on) pharmaceutical preparation solvability is affected.
| Compound | Solvent 1 | Solvent 2 |
| C1:AAN-group 2 Note 2-Docetaxel (2 connections) | Insoluble | Insoluble |
| C2: group 1 Note 1-AANL-Docetaxel (2 connections) | Insoluble | Insoluble |
| C3:AAN-Docetaxel (2 connections) | Insoluble | Insoluble |
| C4: group 1 Note 1-AAN-group 2 Note 2-Docetaxel (7 connections) | Insoluble | Insoluble |
| C5: group 1 Note 1-AANL-group 2 Note 2-Docetaxel (2 connections) | Insoluble | Solvable |
| C6: group 1 Note 1-AANK-group 2 Note 2-Docetaxel (2 connections) | Insoluble | Insoluble |
| S1 | Insoluble | Solvable |
| S2 | Solvable | Solvable |
| S3 | Solvable | Solvable |
| S4 | Solvable | Solvable |
Note 1: the structure of group 1 is
Note 2: the structure of group 2 is
the group 1 hereinafter related to is identical therewith with group 2;
Wherein, AAN, AANL, AANK in table 1 be the amino acid whose junction symbol of representation compound Small Peptides respectively, and A represents Ala, N and represents Asn, L and represent Leu, K and represent Lys.
The Docetaxel derivative of water-soluble targeted activation provided by the invention, experimental design thinking derives from the complex compound by the different mode connected of a large amount of compound experiments design preparation, then by connecting complex compound to 2 on Docetaxel or 7 (being namely connected to by complex compound respectively on the OH of in Docetaxel molecular formula the 7th or the 2nd), when being existed by tumor tissues or l-asparagine endopeptidase, the size of activation efficiency carries out the screening of Docetaxel efficiency again, and screen gained compound successively at R1, R2, and the restraining effect of n to tumour when getting different value. the activation site of tumor tissue specificity is the junction between AAN and group 2, after activating fracture, group 2 can from release, and discharge Docetaxel further.Because the activity center of l-asparagine endopeptidase is positioned at the bottom of ball vesicular invaginations, cleavage site needs close to activity center, and at this moment complex compound becomes extremely important to cleavage site steric hindrance of whether having living space.
By the result of screening experiment, we infer the connection of group 2, effectively can avoid directly connecting that Docetaxel brings is sterically hindered, and do not affect the close of l-asparagine endopeptidase.And the structure activity relationship of group 1 can increase the polarity of cleavage site, make more water miscible proteolytic enzyme easier close to restriction enzyme site, and increase cutting efficiency.Be connected to Docetaxel the 2nd and also obviously decrease Docetaxel to the sterically hindered of proteolytic enzyme and the more overall hydrophilic polar group that exposes, increase cutting efficiency and water-soluble.
Embodiment 3 S1, the method for S2, S3 and S4 assay and content range.
S1, S2, S3 and S4 use analysis mode HPLC (Agilent 1220 (Agilent1220series), 5 μm, C8 post, 4.6mm ID × 250mm, moving phase is 0 ~ 95% acetonitrile (ACN), and purity is at 95%-99%).
In the Docetaxel of the water-soluble targeted activation of embodiment 4, the not isoplastic connection of compound and Docetaxel to activate at tumor tissues medicine and have different impacts.
Mutual structure activity relationship between Docetaxel with the compound group be connected determines at the target of tissue site and activation effect.In our experiment, the proteolytic enzyme in the tumor tissues homogenate of 100 microgram acidifyings at 37 DEG C of temperature adds the compound of 1 mg/ml, and tumor tissues homogenate can impel release Docetaxel.By HPLC can the reduction of detection compound and the increase of Docetaxel and comparison of tumor tissue to the activation efficiency of medicine, be connected in the compound of screening by the current compound of screening discovery and have the highest activation efficiency, the activation simultaneously in different tumor type also illustrate that the broad spectrum (see table 5) of pharmacological agent.The specific compound simultaneously produced in comparative compound screening process, analyzes the plan activation efficiency in same tissue, is connected in the compound of screening has the highest activation efficiency by screening discovery compound S 1, S2, S3, S4 of the present invention.(see table 6).
The activation of table 5:S1, S2, S3, S4 measures (%), the S1 in different tumor tissues homogenate, and the compound of S2, S3, S4 activates ratio
Table 6: screen the change or Docetaxel 7 or 2 impacts connected MDA-MB231 tumour activation medicine that are similar to heterogeneity in control compounds
| Compound | Activation efficiency (%) |
| C1:AAN-group 2-taxol (2 connections) | 17.5 |
| C2: group 1-AAN-taxol (2 connections) | 46.6 |
| C3:AAN-taxol (2 connections) | 38.5 |
| C4: group 1-AAN-group 2-taxol (7 connections) | 16.3 |
| C5: group 1-AANL-group 2-taxol (2 connections) | 67.4 |
| C6: group 1-AANK-group 2-taxol (2 connections) | 56.6 |
| S1: group 1-AANL-group 2-taxol (2 connections) | 93.4 |
| S2 | 91.6 |
| S3 | 90.6 |
| S4 | 88.5 |
As seen from Table 6, the activation efficiency of Docetaxel 2 connection is far away higher than 7 connections.
The above results illustrates: the not isoplastic connection of D51-7059 of the water-soluble targeted activation of the present invention and taxol to activate at tumor tissues medicine has different impacts.Mutual structure activity relationship between taxol with the compound group be connected determines at the target of tissue site and activation effect.S1, S2, S3, the S4 activation of (10 kinds of different tumours) in different tumor type describes the broad spectrum (table 5) of drug activation.The specific compound simultaneously produced in comparative compound screening process, analyze the activation efficiency in same human breast carcinoma MDA-MB435 tumor tissues, experiment proves S1, S2, each group of S3, S4 compound is selected to be relative higher activation efficiency (table 6).
Embodiment 5 is by the mensuration of reagent intravenous administration maximum tolerated dose (MTD)
Test objective: by measuring mouse vein medication MTD experiment, understand the acute toxicity of this new drug preparation.
Medicine: S1, S2, S3 and S4 injection liquid, during test with normal saline dilution to respective concentration.
Animal: one-level bar match (BALB/C) mouse, body weight 19-21g is female entirely.
Method and result: tested BALB/C mice 210, body weight 19-21g, is divided into 21 groups at random by body weight, often organizes 10.As shown in table 7, by the injection of 0mg/kg, 125mg/kg, 150mg/kg, 175mg/kg, 200mg/kg disposable vein S1, S2, S3 and S4 injection liquid, and, carry out the controlled trial of physiological saline group, the injection of Docetaxel group, administration volume 0.2ml.Continuous Observation 17 days, observe whether animal occurs that piloerection is established every day, the tarnish that tousles, lethargic sleep, hunch, radical response etc., record body weight and death condition.Carry out complete blood count at the 3rd, 5,14 day blood-sample withdrawal, dissected animal at the 14th day and take heart, liver, kidney, lung, spleen, pancreas HE to dye observation.
Table 7: S1, S2, S3 and S4 injection liquid that test mice accepts various dose respectively contrasts with the mortality results of physiological saline, Docetaxol injection
Result and discussion: inject S1, S2, the mouse group of S3 and S4 injection liquid, when 175mg/kg dosage, animal does not occur that piloerection is established, the tarnish that tousles, lethargic sleep, hunch, radical response and death condition, the MTD value of S1 and S2 injection liquid as shown in table 7 is about 150mg/kg, much larger than the MTD value 25mg/kg of Docetaxel, by the important references index that reagent intravenous administration maximum tolerated dose is drug toxicity, show that the toxicity ratio Docetaxel of the Docetaxel that targeted activation discharges significantly reduces.
Embodiment 6 S1, the drug efficacy study of S2, S3 and S4 injection liquid in nude mice (nude mice)
Test objective: by the oncotherapy model of mouse, understands the antitumor drug effect of S1, S2, S3 and S4 medicine.
Medicine: S1, S2, S3 and S4 injection liquid and Docetaxol injection, during test with normal saline dilution to respective concentration.
Method and result:
1. animal: nude mice, 6-8 age in week is entirely female.
2. produce tumor model
1) human prostata cancer PC-3cells (cell) is from American Type Culture collection warehousing (American type culture collection, ATCC) buy, and the qualification of cell is carried out according to the specification sheets that ATCC provides, cell uses improves Iger (family name) substratum (abbreviation containing 10% foetal calf serum Da Erbaike (family name), DMEM nutrient solution) at 37 DEG C, cultivate under the carbon dioxide conditions of 5%.Within every 3 days, go down to posterity once, within cell was used in for 15 generations.
2) tumour produces, by 5 × 10
6panc-1 cell subcutaneous injection, to nude mice (nude mice) mouse back, is treated that tumour is grown to and is reached 100mm less
3random packet during left and right, begin treatment, with begin treatment same day for first day.
3) therapeutic process
According to S1, S2, S3 and S4 clinical application uses intravenous injection (i.e. IV injection), S1, S2, S3 and S4 use the metering 25 mgs/kg of dosage being less than 1/6MTD, Docetaxel medication therapy groups uses metering 8.3 mgs/kg of dosage of 1/3MTD, control group uses physiological saline, weekly administration, totally 4 weeks.
4) grouping is as shown in table 8 below with outcome measurement
Table 8:S1, S2, S3 and S4 medicine, Docetaxel and control group are to the effect of nude mice treatment tumour
5) result and discussion: as shown in Table 8, with etc. the Docetaxel medication therapy groups control group of volumetric molar concentration compare, at S1, the tumor growth inhibitory effect of S2, S3 and S4 treatment group is greatly improved.
Embodiment 7 S1, S2, S3 and S4 medicine is at the drug efficacy study of D121 tumour immunity model
Test objective: by D121 lung cancer tumor immune model treatment model, understand the antitumor drug effect of S1, S2, S3 and S4 medicine.
Animal: C57 mouse, 6-8 age in week is entirely female.
Produce tumor model:
1) D121 lung cancer tumor is bought from American Type Culture Collection ATCC, and cell uses and contains 10% foetal calf serum DMEM nutrient solution at 37 DEG C, cultivates under the carbon dioxide conditions of 5%.Within every 3 days, go down to posterity once, within cell was used in for 15 generations.
2) tumour immunity, mouse peritoneal injection 5 × 10
5through the D121 lung carcinoma cell (purchased from American Type culture collection institute) of radiation death, immunization 3 times, every minor tick 2 weeks.Oncocyte is injected after immunity terminates, and then administration, weekly administration, totally 4 weeks.
3) tumour produces: at the 32nd day, by 10
6the D121 lung cancer tumor cell subcutaneous injection of living to the C57 mouse back of tumour immunity, begin treatment when tumour grows to about 0.3 ~ 0.4cm.
4) tumour CD8+T cell analysis.Tumor tissues is through homogenate, filter to isolate individual cells in tumour, twice is washed with damping fluid, the antibody that leukocyte common antigen (LCA) CD45-PE and T lymphocyte antigen CD8-FITC marks combines in room temperature for 1 hour, cell washes twice with comprising 1% foetal calf serum phosphoric acid buffer PBS, then the ratio of T lymphocyte antigen (CD8) positive cell in flow cytometry analysis leukocyte common antigen (LCA) (CD45) positive cell is used, the rising of this ratio then shows T lymphocyte immune cell and increases, and the immunizing power for tumour indicated in animal body improves.
5) grouping is as shown in table 9 with outcome measurement.
The effect of table 9:S1, S2, S3 and S4 medicine, Docetaxel treatment group and control group tumor suppression and immuno-stimulating
6) result and discussion: as shown in Table 9, compare with other treatment control group with immunized controls group, S1, S2, S3 and S4 improves greatly in the result for the treatment of of C57 mouse, S1 and PDL1-antibody has good collaborative Promote immunity and synergistic therapeutic effect effect, can by improving the growth of immunosuppression tumour.
Embodiment 8 S1, the drug efficacy study of S2, S3 and S4 medicine in the metastasis models of BALB/C mice
Test objective: by the metastases treatment model of BALB/C mice, understand the antitumor drug effect of S1, S2 and S3 medicine.
Medicine: S1, S2, S3 and S4 injection liquid and Docetaxol injection, during test with normal saline dilution to respective concentration.
1. animal: BALB/C mice, 6-8 age in week is entirely female.
2. produce tumor model
1) 4T1cells buys from ATCC, and carries out the qualification of cell according to the specification sheets that ATCC provides, and cell uses and contains 10% foetal calf serum DMEM nutrient solution at 37 DEG C, cultivates under the carbon dioxide conditions of 5%.Within every 3 days, go down to posterity once, within cell was used in for 15 generations.
2) generation of metastases, by 10
6t1cells cell subcutaneous injection is to BALB/C mice back, random packet when tumour grows to about 1.5cm, Subcutaneous tumor is removed in operation, and start with pharmacological agent, mouse is put to death after anesthesia the 27th day time, take out whole lung, put into Bouin's solution (Bouin ' s solution) dyeing, under dissecting microscope, statistics transfers to the tumour quantity of lung.
3) therapeutic process: according to S1, S2, S3 and S4 clinical application uses IV injection, S1, S2, S3 and S4 and all use the metering of 1/6MTD, 17.6 micro-/kilogram dosage that rub, Docetaxel medication therapy groups uses the micro-/kilogram dosage that rubs of metering 3 of 1/6MTD, and control group uses physiological saline, every three days single administrations, totally 4 times.
4) grouping is as shown in table 10 with outcome measurement
Table 10:S1, S2, S3 and S4 medicine, Docetaxel treatment group and control group are for the effect of nude mouse tumor metastasis inhibition
5) result and discussion: as shown in table 10, compares, at S1 with Docetaxel treatment group control group, S2, after S3 and S4 group intraperitoneal administration, be greatly improved in the metastases inhibition of BALB/C mice, illustrate that this type of medicine has good anti metastasis drug effect.
Embodiment 9 S1 injection liquid is at the drug efficacy study of many tumor models
Test objective: by many tumor models of mouse, understands the broad spectrum of the antitumor drug of S1.
Medicine: S1 injection liquid, during test with normal saline dilution to respective concentration.
Method and result:
1. animal: nude mice, 6-8 age in week is entirely female.
2. produce tumor model
1) corresponding cell is from American Type Culture collection warehousing (American type culture collection, ATCC) buy, and the qualification of cell is carried out according to the specification sheets that ATCC provides, cell uses improves Iger (family name) substratum (abbreviation containing 10% foetal calf serum Da Erbaike (family name), DMEM nutrient solution) at 37 DEG C, cultivate under the carbon dioxide conditions of 5%.Within every 3 days, go down to posterity once, within cell was used in for 15 generations.
2) tumour produces, by 5 × 10
6corresponding cell subcutaneous injection, to nude mice (nude mice) mouse back, is treated that tumour is grown to and is reached 100mm less
3random packet during left and right, begin treatment, with begin treatment same day for first day.
3) therapeutic process
Use IV injection according to S1 clinical application, S1 uses the micro-/kilogram dosage that rubs of the metering 17.6 of 1/6MTD, and control group uses physiological saline, weekly administration, totally 3 weeks.
4) grouping is as shown in table 11 below with outcome measurement
Table 11:S1 is in the result for the treatment of of many tumor models
5) result and discussion: as shown in table 11, S1 has good drug effect in kinds of tumors model, illustrates that medicine has become the anti-tumor medicine of a broad spectrum.
Some embodiments of the present invention (embodiment 10 ~ 24, synthetic method is close with embodiment S1) in, for the activation characteristic of the Docetaxel of the water-soluble targeted activation of different aminoacids structure, tumour inhibiting rate and suppress the rate of transform to be tested respectively, testing method and above-described embodiment 4,6,8 is identical, and test result is as shown in table 12:
Table 12: the activation characteristic of the Docetaxel of the water-soluble targeted activation of embodiment 10 ~ 24, tumour inhibiting rate and suppress rate of transform result
Result and discussion: as shown in table 12, the compound of embodiment 10 ~ 24 has certain Activation Activity and growth and metastasis of tumours inhibition, illustrate that we screen individual process and have the practical significance optimizing activation and curative effect, by the embodiment of above-mentioned preferred compound, will be appreciated that above-mentioned description should not be considered to limitation of the present invention.After those skilled in the art have read foregoing, for R of the present invention
1and R
2the amino acid of position is replaced and changed will be all apparent.
In some embodiments of the invention, also synthesize the docetaxel of other water-soluble targeted activation, wherein, the arbitrary integer in n=1-150, R
1for any one amino acid in Ala, Thr, Val or Ile; R
2for any one amino acid in Ala, Thr, Val or Asn; And done above-mentioned preparation (method is with embodiment 2), MTD test (method is with embodiment 5), experiment to the effectiveness study (method is with embodiment 6,7) of tumour, transfer curative effect (method is with embodiment 8) and many tumor efficiencies (method is with embodiment 8), and achieve the experimental result similar to S1-S4.The experiment proved that, within the scope of n=1-150, along with n increases, tumour inhibiting rate slightly declines.Along with n value increases, Activation Activity slightly declines, and increase Deng a mole drug quality number for metering, but also make the drug metabolism transformation period increase because n value increases, therefore overall drug effect is just slight declines, be selected from the scope of 1 ~ 150 at n, all can arrive the close technique effect of embodiment of the present invention S1-S4.
In sum, the present invention has synthesized the antitumor drug of the Docetaxel of water-soluble targeted activation, and to prove that compound has lower toxicity than Docetaxel by toxicity and the test of pesticide effectiveness, better formulation method, medicine simultaneously, immunotherapy and directly treat all drug effects and greatly improve, and there is treatment indication expand and to the specific notable feature of metastases, there is good actual therapeutic using value.
Although content of the present invention has done detailed introduction by above preferred embodiment, will be appreciated that above-mentioned description should not be considered to limitation of the present invention.After those skilled in the art have read foregoing, for multiple amendment of the present invention and substitute will be all apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (10)
1. a Docetaxel derivative for water-soluble cancer target activation, it is characterized in that, the structural formula of this Docetaxel derivative is:
Wherein, R
1for any one amino acid in Ala, Thr, Val or Ile; R
2for any one amino acid in Ala, Thr, Val or Asn; Arbitrary integer in n=1 ~ 150.
2. the Docetaxel derivative of water-soluble cancer target activation as claimed in claim 1, it is characterized in that, this Docetaxel derivative is compound S 1, wherein, n=1, R
1get Ala amino acid, R
2get Ala amino acid, the structural formula of this S1 is:
3. the Docetaxel derivative of water-soluble cancer target activation as claimed in claim 1, it is characterized in that, this Docetaxel derivative is compound S 2, wherein, n=5, R
1get Ala amino acid, R
2get Ala amino acid, the structural formula of this S2 is:
4. the Docetaxel derivative of water-soluble cancer target activation as claimed in claim 1, it is characterized in that, this Docetaxel derivative is compound S 3, wherein, n=11, R
1get Ala amino acid, R
2get Ala amino acid, the structural formula of this S3 is:
5. the Docetaxel derivative of water-soluble cancer target activation as claimed in claim 1, it is characterized in that, this Docetaxel derivative is compound S 4, wherein, n=150, R
1get Ala amino acid, R
2get Ala amino acid, the structural formula of this S4 is:
6. the D51-7059 of water-soluble targeted activation as claimed in claim 1, is characterized in that, the Docetaxel derivative that water-soluble cancer target activates is any one in S10 ~ S24.
7. one kind adopts the purposes of Docetaxel derivative for the preparation of antitumor drug of the water-soluble cancer target activation in claim 1-6 described in any one, it is characterized in that, this antitumor drug can be used for the treatment of various cancers disease type, and described various cancers type comprises bladder cancer, the cancer of the brain, breast/mammary cancer, cervical cancer, colon-rectum, the esophageal carcinoma, kidney, liver cancer, lung cancer, nasopharyngeal carcinoma, carcinoma of the pancreas, prostate cancer, skin carcinoma, cancer of the stomach, uterus carcinoma, ovarian cancer, carcinoma of testis and leukemia.
8. purposes as claimed in claim 7, is characterized in that, the Docetaxel derivative that described water-soluble cancer target activates is for the preparation of the antitumor drug used auxiliary in radiation treatment.
9. purposes as claimed in claim 7, is characterized in that, the Docetaxel derivative that described water-soluble cancer target activates is for the preparation of the Synergistic treatment medicine in immunotherapy and immunotherapy.
10. purposes as claimed in claim 7, is characterized in that, the Docetaxel derivative that described antitumor drug is activated by described water-soluble cancer target and the pharmaceutical composition that pharmaceutical excipient forms.
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| CN201410417885.5A CN104262457A (en) | 2014-08-22 | 2014-08-22 | Water-soluble cancer targeted activation docetaxel derivatives and uses thereof |
| CN201580044392.4A CN106715457B (en) | 2014-08-22 | 2015-08-21 | Small molecule targeting couplet specifically activated by tumor microenvironment and application thereof |
| CA2958495A CA2958495C (en) | 2014-08-22 | 2015-08-21 | Small molecule conjugates specifically activated in tumor microenvironment for targeting and use thereof |
| AU2015306574A AU2015306574B2 (en) | 2014-08-22 | 2015-08-21 | Specifically activated micromolecular target coupling body in tumor microenvironment and use thereof |
| EP15833275.9A EP3184540A4 (en) | 2014-08-22 | 2015-08-21 | Specifically activated micromolecular target coupling body in tumor microenvironment and use thereof |
| JP2017529129A JP6854759B2 (en) | 2014-08-22 | 2015-08-21 | Small molecule target conjugates specifically activated by the tumor microenvironment and their use |
| US15/505,861 US10682371B2 (en) | 2014-08-22 | 2015-08-21 | Small molecule conjugates specifically activated in tumor microenvironment for targeting and use thereof |
| PCT/CN2015/087746 WO2016026458A1 (en) | 2014-08-22 | 2015-08-21 | Specifically activated micromolecular target coupling body in tumor microenvironment and use thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016026458A1 (en) * | 2014-08-22 | 2016-02-25 | 亚飞（上海）生物医药科技有限公司 | Specifically activated micromolecular target coupling body in tumor microenvironment and use thereof |
| CN109422799A (en) * | 2017-08-22 | 2019-03-05 | 复旦大学 | Docetaxel anti-liver cancer and anti-targeted prodrug and its pharmaceutical usage |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101374856A (en) * | 2005-11-29 | 2009-02-25 | 斯克里普斯研究学院 | Inhibiting tumor cell invasion, metastasis and angiogenesis |
| CN103044521A (en) * | 2012-12-26 | 2013-04-17 | 亚飞（上海）生物医药科技有限公司 | Aspartase-targeted activated adriamycin derivative as well as preparation method and application thereof |
-
2014
- 2014-08-22 CN CN201410417885.5A patent/CN104262457A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101374856A (en) * | 2005-11-29 | 2009-02-25 | 斯克里普斯研究学院 | Inhibiting tumor cell invasion, metastasis and angiogenesis |
| CN103044521A (en) * | 2012-12-26 | 2013-04-17 | 亚飞（上海）生物医药科技有限公司 | Aspartase-targeted activated adriamycin derivative as well as preparation method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 何明磊 等: "合成分枝型聚乙二醇的简便新方法", 《高等学校化学学报》 * |
| 吴进: "聚乙二醇衍生物的合成及其对胰蛋白酶的化学修饰", 《中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016026458A1 (en) * | 2014-08-22 | 2016-02-25 | 亚飞（上海）生物医药科技有限公司 | Specifically activated micromolecular target coupling body in tumor microenvironment and use thereof |
| US10682371B2 (en) | 2014-08-22 | 2020-06-16 | Yafei Shanghai Biolog Medicine Science & Technology Co. Ltd. | Small molecule conjugates specifically activated in tumor microenvironment for targeting and use thereof |
| CN109422799A (en) * | 2017-08-22 | 2019-03-05 | 复旦大学 | Docetaxel anti-liver cancer and anti-targeted prodrug and its pharmaceutical usage |
| CN109422799B (en) * | 2017-08-22 | 2022-07-29 | 复旦大学 | Docetaxel liver cancer-resisting targeted prodrug and medicinal application thereof |
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