CN1087642A - Be used to discharge the method for cytotoxic agents and its component - Google Patents

Be used to discharge the method for cytotoxic agents and its component Download PDF

Info

Publication number
CN1087642A
CN1087642A CN93109871A CN93109871A CN1087642A CN 1087642 A CN1087642 A CN 1087642A CN 93109871 A CN93109871 A CN 93109871A CN 93109871 A CN93109871 A CN 93109871A CN 1087642 A CN1087642 A CN 1087642A
Authority
CN
China
Prior art keywords
ethanoyl
phenyl
group
amino
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN93109871A
Other languages
Chinese (zh)
Inventor
V·J·陈
L·N·容海姆
D·L·迈耶
T·A·谢泼德
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hybritech Inc
Original Assignee
Hybritech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hybritech Inc filed Critical Hybritech Inc
Publication of CN1087642A publication Critical patent/CN1087642A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/552—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being an antibiotic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6899—Antibody-Directed Enzyme Prodrug Therapy [ADEPT]

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Nanotechnology (AREA)
  • Molecular Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Medical Informatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to treat the method for tumor disease, this method comprises:
A) to the antibody-enzyme conjugates of infection host administering therapeutic significant quantity; Then
B) to the substrate-cytotoxic agents of infection host administering therapeutic significant quantity, wherein substrate is the substrate for enzyme.
The present invention also discloses antibody-enzyme conjugates and substrate-cytotoxic agents compound.

Description

Be used to discharge the method for cytotoxic agents and its component
For many years, the many cytotoxic drugs that are used for the treatment of tumor disease have been developed.Therefore the main drawback of the medicine that all these are useful is to exist indiscriminate toxicity for patient's all cells, has limited the dosage of this medicine, thereby and has limited its effectiveness.Cytotoxic agents acts on the position of tumor disease with the potential form.Therefore, reducing aspect the undesirable side effect maybe may suppress this medicine, the invention provides the advantage of mark.Owing to further reduced the result of harmful side effect, take cytotoxic agents by increasing dosage, make the medicine on the tumor locus produce higher concentration.
Be on the one hand, the invention provides the method that is used for the treatment of tumor disease, its method comprises:
A) to the antibody-enzyme conjugates of infection host administering therapeutic significant quantity; Then
B) to the substrate-cytotoxic agents of infection host administering therapeutic significant quantity, wherein substrate is the substrate for enzyme; Wherein describe in detail in the definition below of antibody-enzyme conjugates and substrate-cytotoxic agents.
On the other hand, the present invention includes antibody-enzyme conjugates and the substrate-cytotoxic agents that describes in detail in the definition below.
Fig. 1 has described in isolating each organ by the organ weight, the bio distribution of representing with injected dose % of anti-KS I/4-beta-lactam enzyme conjugates in bare mouse.
Fig. 2 has described the identical measurement of the Fig. 1 that represents with injected dose in each organ.
Fig. 3 has illustrated by being combined in of the prodrug compound of anti-CEA-beta-lactam enzyme conjugates and embodiment 11 and has suppressed the T380 growth of tumor in the bare mouse.Treatment was taken medicine in 3 processes through 3 weeks.Each process comprises 35 μ g binding substancess, and after 72 hours, 4 per daily doses are 1mg prodrug/Kg body weight.
Fig. 4 describes except being that the method for 0.25mg prodrug/Kg body weight is taken the prodrug with 4 per daily doses, has also described the similar restraining effect of bonded about the identical combination thing prodrug of T380 tumour as Fig. 3.
Fig. 5 is that the precursor by anti-KSI/4 antibody-beta-lactam enzyme conjugates or anti-CEA-beta-lactam binding substances and embodiment 11 combines relatively suppress the LS174T growth of tumor in bare mouse.
Fig. 6 has described in nude mouse the restraining effect that the prodrug for the anti-TAG72-beta-lactam enzyme conjugates of LS174T tumour and embodiment 11 combines.
Fig. 7 is the comparison about the effect of the dosage of the binding substances that suppresses the T380 tumor growth that the prodrug by anti-CEA-beta-lactam enzyme conjugates of the present invention and embodiment 11 combines.
An aspect of of the present present invention is a formula
Antibody-enzyme I
The enzyme antibody binding substances
Wherein:
Enzyme and formula
Substrate-cytotoxic agents II
Compound (this compound is described below) reaction, separate from cytotoxic agents to cause substrate; And antibody and target tissue carry out complexing.
The antigen complexing mutually of antibody of the present invention and one or more target cells.Target cell is the part of tumor tissues (or benign or pernicious).The cell that the example of this class target cell comprises psorosis, many squamous cell carcinomas, adenocarcinoma cell, small cell carcinoma cell, glyoma cell, melonoma cell, renal cell carcinoma cell, transitional cell cancer cells, sarcoma cell, the tumor vascular cell of support and lymph sample tumour is leukemia and lymphoma for example.
Antibody can be selected from any kind of that comprises IgG, IgA, IgM, IgE and IgD or the immunoglobulin (Ig) of group.The kind of origin is not crucial, as long as antibody and target cell complexing.
In the art, monoclonal antibody is that to be used for the present invention most preferred.Yet, outside polyclonal antibody also is not precluded within.Novel antibody is a chimeric antibody, and it prepares by the recombinant chou technology in testing laboratory, and said recombinant chou technology allows to express the DNA that modifies, this dna encoding the district of conjugated antigen of any required antibody, any other amino acid needed order of also having encoded.Therefore, chimeric antibody (wherein a part is derived by a kind of species, and another part is derived by different plant species) can obtain and can be used for the present invention.
In addition, the origin of antibody and character are not crucial, as long as its aims at the target cell that will treat, and itself to the patient be do not have toxic.Those those of ordinary skill can easily prepare the enzyme conjugates with candidate antibody, and they are estimated.How to select this antibody-like to go through below.
Think, correctly select the fragment of antibody to have the effect identical with antibody.Therefore, in enforcement of the present invention, the fragment of antibody is F(ab ' for example) 2, F(ab ') and the antibody (V of single area HDistrict or dAbs) and F(ab ' particularly) fragment (discerning and the cell bonded antigen that will treat) can be the same useful with complete antibody.(dAbs is described in Nature by people such as Ward E., and 341, p.544, in 1989; Be described in Proc.Natl.Acad.Sci. by people such as Orlandi R., (USA) 86, p.3833, in 1989.）
A large amount of antibody all can be applicable to the present invention for the immunologist.Useful in addition antibody is disclosed in the publication of each relevant magazine.In enforcement of the present invention, can not and the also complete unnecessary table look-up that provides applicable antibody.The immunologist of ordinary skill and chemist fully can be from for example American Type Culture Collection (ATCC) (Rockville, Maryland, U.S.A), and Linscott ' s Directory of Immunological and Biological Reagents(is announced by Linscott ' s Directory, 40 Glen Drive, Mill Valley, California, select antibody in the source of products catalogue U.S.A., 94941).Therefore, those skilled in the art to be chosen on this target cell in fact anti-any antigenic antibody be simple thing.
Another kind method is, the necessary hybridoma that is used to produce this antibody-like can pass through ATCC or Northern Regional Research Laboratories(NRRL) (U.S.Department of Agriculture, Peroia, Illinois U.S.A) obtains with other clone collection.
Some present known antibody are significant especially for the present invention, and a kind of preferred specific antibody is the L/IC that is produced by ATCC hybridoma HB9682.Another kind is preferably produced by ATCC hybridoma HB9620.The suitable carcinomebryonic antigen complexing of above-mentioned antibody (being called CEM231.6.7) and tumor cells expression by several types.Recently, chimeric antibody CEM231.6.7 is described in Application No. 07/165,856 and 07/272, and the 577(applying date is respectively on March 3rd, 1988 and on November 17th, 1988).This antibody also is specified in people's such as Beidler C.B. J.Immunology(141, pp.4053-4060,1988) in.The antibody that reacts with MAT65 T-cell marker is T-101 antibody.This T-101 antibody is produced by ATCC hybridoma #CRL8023, and is described in U.S. Patent number 4,675, in 386.
Another kind of significant antibody is KSI/4, and it at first is disclosed in Cancer Research(44 by people such as Varki, PP.681-686,1984) in.Some plasmids of coded sequence that comprise the not same district of monoclonal antibody KSI/4 are present on the settling, and can be obtained by Northern Regional Research Laborotory.This plasmid can use by those those skilled in the art, so that produce chimeric antibody with the recombinant chou method, this antibodies is on the antigen that has highdensity cell surface on the adenocarcinoma cell.The structure of this antibody-like is specified in Application No. 07/184, and the 522(applying date is on April 21st, 1988) in.Following plasmid relates to KSI/4.
The plasmid PGKC2310 that is separated by E.coli K12 MM294/PGKC2310, NRRLB-18356 is the coded sequence of light chain, signal peptide and light chain bonded and 5 ' and 3 ' the do not translate coded sequence in district.
By the plasmid PG2A52 that separates among E.coli K12 MM294/PG2A52, the NRRL B-18357 is the coded sequence of heavy chain, signal peptide and heavy chain bonded and 5 ' and 3 ' the do not translate coded sequence in district.
The plasmid CHKC2-6 that is separated by E.coli K12 DH5/CHKC2-6, NRRL B-18358 is the coded sequence of variable region of light chain, the order of the coding of the light chain constant region of signal peptide and light chain bonded coded sequence and people's IgG.
The plasmid CHKC2-18 that is separated by E.coli K12 DH5/CHKC2-18, NRRL B-18359 is the coded sequence of derivative variable region of light chain, the order of the coding of the light chain constant region of signal peptide and light chain bonded coded sequence and people's IgG.
The plasmid CH2A5 that is separated by E.coli K12 MM294/CH2A5, NRRL B-18360 is the coded sequence of variable region of heavy chain, the order of the coding of the heavy chain constant region of signal peptide and heavy chain bonded coded sequence and people's IgG1.
The plasmid CH2A5IG2 that is separated by E.coli K12 DH5/CH2A5IG2, NRRL B-18361 is the coded sequence of variable region of heavy chain, the order of the coding of the heavy chain constant region of signal peptide and heavy chain bonded coded sequence and people's IgG2.
The plasmid CH2A5IG3 that is separated by E.coli K12 DH5/CH2A5IG3, NRRL B-18362 is the coded sequence of variable region of heavy chain, the order of the coding of the heavy chain constant region of signal peptide and heavy chain bonded coded sequence and people's IgG3.
The plasmid CH2AIG4 that is separated by E.coli K12 DH5/CH2AIG4, NRRL B-18363 is the coded sequence of variable region of heavy chain, the order of the coding of the heavy chain constant region of signal peptide and heavy chain bonded coded sequence and people's IgG4.
The antibody 5E 9C11 that is produced by the ATCC hybridoma discerns the TfR of being expressed by many tumours.By National Cancer Institute(NCI) antibody recognition that is called CC49 that the obtains TAG-72 antigen of expressing people such as (, Cancer Research, 48, PP.4588-4596,1988) Muraro R. by mammary cancer and colorectal carcinoma.Antibody ZCE025 also is applicable to the present invention.This antibody is at first by Jean-Pierre Mach and his group (for example, people such as Mach J.P., Int.J.Cancer, 33, PP.643-649,1984; With people such as Patt Y., Cancer Bull., 40, PP.218-221,1988) describe.Be called BRE3 with the antigen reactive in addition useful antibody that exists on many cancers, and be useful people such as (, Hybridoma, 9, PP.221-235,1990) Peterson J.A. in enforcement of the present invention.Up to the present be used for useful antibody of the present invention in addition and also discern KSI/4 antigen, and be called as 007B.This antibody is obtained by Eli Lillyand Company, Lilly Corporate Center, Indianapolis, Indiana 46285.
The enzyme that is used for this binding substances is the enzyme of identification substrate, and when it is incorporated on the said cytotoxic agents, it also will discern the sort of substrate.In addition, in case said enzyme is incorporated on this antibody the essential activity that keeps it of this enzyme.
For antibody, can not and also there is no need to list the table look-up that is used for all enzymes of the present invention.Many suitable examples can obtain from the market, for example from Sigma Chemical Company, st.Louis, Missouri; Boehringer Mannheim Company, Indianapolis, Indiana; Cal Biochem, San Diego, California etc. obtain.The method of various separation and enzyme purification all can find in as this class reference such as Methods In Enzymology, those skilled in the art recognize that also the fragment of the enzyme of the catalytic activity of the enzyme that is kept perfectly and specificity (being similar to active and specific any catalyzer such as catalytic antibody) also is applicable to the present invention.
Can be used for a kind of in two kinds of main type reaction of enzyme catalysis of the present invention.First type reaction is that substrate-cytotoxic agents compound is become the reaction of its two components part by splitting therein.For the activity of the enzyme of substrate must be because the existence of cytotoxic agents and being without damage basically.The preferred enzyme that is used for this type reaction be the existence to cytotoxic agents be insensitive and with this cytotoxic agents structure-irrelevant.This fermentoid always allows specific binding substances to be used for the combination of multiple substrate-cytotoxic agents.The example of this class (insensitive) enzyme comprises isozyme, some carboxypeptidase (for example enzyme of Pseudomonas sP.), the D-aminopeptidase (people such as Asano Y. of beta-galactosidase enzymes, various alkaline phosphatases, J.Biol.Chem., 264, PP.14233-14239,1989; People such as Asano Y., Biochem.Biophys.Res.Comm., 162, PP.470-474,1989; People such as Asano Y., Biochem., 31, PP.2316-2328,1992), pyroglutamic acid ester aminopeptidase and various β-Nei Xiananmei.These enzymes can be discerned galactosyl as substrate, phosphate-based, some simple amino acid, 1-pyroglutamic acid ester, d-amino acid amide and cephalosporin compound respectively, and prepare these substrates by combined any other molecule thereon of these substrates.Institute's inherent is in these required characteristics of enzyme, enzyme only promptly is connected the characteristic of the functional group reactions of substrate and cytotoxic agents with a kind of specific functional group, and described cytotoxic agents is such to cause its available different functional groups of any number that enzyme influences that is not subjected to be replaced.Beta-lactam is the best example with this specific character.Contain oxygen desulfurization cynnematin and 1-carboxamide desulfurization cynnematin is not formed by the functional group of enzyme damage by some as penicillin, cynnematin, cynnematin sulfoxide, the 1-of substrate.Said β-Nei Xiananmei only with the reaction of the beta-lactam nucleus of these substrate compounds.Because the result of this reaction, 3 on the substrate '-substituting group usually splits from the rest part of cynnematin ring.
By this enzymatic second type reaction is the substance metabolism reaction, in other words is the reaction that enzyme becomes this precursor conversion cytotoxic agents.Therefore, for such enzyme, said precursor at first will play the effect of substrate, play cytotoxic agents then.The such bonded example of substrate-enzyme compound is trichothecin (trichothecenes), a kind of saxitoxin or amanitine.
After having taken cytotoxic agents to the patient who shelters with the form of substrate-cytotoxic agents compound, irrelevant with enzymatic reaction type, and the purpose of enzyme is identical, promptly produces cytotoxic agents (and being in high density on the site of antibody-enzyme conjugates) in vivo.Can be used for enzyme of the present invention and comprise various β-Nei Xiananmeis, irrelevant with the kind of microorganism, said enzyme can be separated from the carboxypeptidase G2 of the isozyme of pyroglutamic acid ester aminopeptidase, beta-galactosidase enzymes, D-aminopeptidase, various alkaline phosphatases, various carboxypeptidase such as Pseudomonas sp. etc.
The most useful enzyme is can repressed those enzymes by ad hoc fashion, and this inhibition is to be undertaken by the material that does not suppress other enzymes undiscovered usually and discovery in vivo in vivo.The useful especially example of this zymoid is a β-Nei Xiananmei, and it can be suppressed by various β-Nei Xiananmei resistance penicillin and cynnematin such as cloxacillin.The advantage that this penicillin and cynnematin have usually is that they have been used for human body and have obtained proof.In addition, because the reason of the molecule of lower molecular weight expects that they will penetrate organ very soon, are causing undesirable effect at this precursor medicine/antibody-binding substances medicine.
The method of enzyme and antibodies is known in the art.Referring to, for example can buy the listed protein-protein coupling reagent of (from Pierce Chemical Company) on the market.Be used for that the various functional groups of enzyme-antibodies normally are not determined, and it is unessential for the objective of the invention is.What all needed is that this antibody keeps the immunoreactive essential part of its unbound state.Equally, for desmoenzyme, what all needed is that it keeps the active essential part from its unbound state.At last, binding substances and the new substrate-cytotoxic agents that forms must prove practical pharmacokinetics in infected host.The reagent that can be used for enzyme and antibodies comprises SMCC, sulfo--SMCC, SPDP, 2-imino-mercaptan alkane, MBS, sulfo--MBS, SMPB, sulfo--SMPB, SIAB, sulfo--SIAB, GMBS, EMCS, N; N '-two (the amino propionyl of 3-maleimide)-2-hydroxyl-1,3-propylene diamine, N-succinyl monobromo-acetic acid ester (Sigma Chemical Company), BMME(Boehringer Mannheim) etc.Owing to depend in the specific functional group applicatory of the reaction on antibody and the enzyme, selected antibody fragment with in zymophore and antibody binding site or near the reactivity of the group of zymophore and antibody binding site, these reagent can be used for combination.Also can use reagent crosslinked between two amino, for example DMA or DSS(Pierce Chemical Company).Some enzyme and antibody can have amino, sulfydryl or hydroxyl respectively in their reactive site or joint portion.Therefore; these active function groups they with as above the example of the reagent listed can at first make it to shelter [according to reference (Pierce Chemical Company Catolog and Schmuel and Shaltiel before combining by protecting group such as citraconic anhydride, DTNB and DNFB; Biochem.and Biophys Res.Comm.; 29; PP.178-183,1967) method].Pierce Chemical Company Catalog has listed the reference of describing many application of mentioned reagent, and the use of any residue reagent can be found in this academic documents.
Same wish but optionally be that suppose that enzyme is not present in the person, substrate is not present in the human body yet.Present technique field those of ordinary skill can see easily that if enzyme or substrate are present in the human body, so, the chance that discharges cytotoxic agents in the zone except that target tissue site will increase.
Although any combination of aforesaid antibody and enzyme all can be satisfied the present invention, preferred binding substances is that given combination technology is homologous binding substances as far as possible.Different with complete antibody is F(ab ') fragment can be by near the carboxyl terminal and leave the thiol that antigen-binding site exists and derive through given zone, and this fragment is preferred in the present invention.Other suitable methods are to prepare more homologous binding substances.A kind of European Patent Application No. 243929,359428 and 360433(Drs.Offord and Rose of being disclosed in these class methods) and document (Fisch I., Kunzig G., Rose K., and Offord R., Site-Specific Modification of a Fragment of a Chimeric Monoclonal Antibody Using Reverse Proteolysis, Bioconj, Chem., 3, PP.147-153,1992) in.Other method comprises functional moiety's the vegetative propagation of gene of codase and antibody molecule and fusion, with these gene fusion together and express the protein that merges in the host cell that is fit to.This class fused protein is produced out.(European Patent Application No. 392745AZ).In addition, binding substances should not carry out metabolism degraded (degratory) organ such as liver and spleen, and this is a kind ofly can prevent enough being combined in cumulative interacts on the target tissue.Yet on the other hand, preferably, after this binding substances had been had an opportunity to the target tissue location, binding substances is very fast to be discharged from blood.Two aspects of these pharmacokinetic properties will reduce the chance that produces cytotoxic agents on the position except that target tissue.Help the performance of these pharmacokinetic properties by having low-molecular-weight binding substances, this is a kind of by antibody fragment F(ab ' particularly) characteristic supplied with of fragment.Along identical thinking, because the reason identical with antibody fragment, the enzyme with lower molecular weight (as 50,000 or lower) is preferred.
The preferred embodiment of said antibody-enzyme conjugates comprises:
A. aforesaid binding substances, wherein enzyme is β-Nei Xiananmei, beta-galactosidase enzymes, pyroglutamic acid ester aminopeptidase or D-aminopeptidase in addition;
The binding substances of B.A, wherein enzyme is a β-Nei Xiananmei in addition;
The binding substances of C.B, the wherein in addition antigen complexing of antibody and single expression, or be present in the antigen complexing of malignant cell with supernatural abundance;
The binding substances of D.C, wherein antibody and carcinomebryonic antigen complexing in addition;
The binding substances of E.C, wherein in addition antibody with the complexing of KSI/4 antigen;
The binding substances of F.C, wherein antibody and the complexing of TAG-72 antigen in addition;
The binding substances of G.C, itself and the complexing of MAT-65 antigen;
The binding substances of H.D, wherein antibody is called as ZCE025 in addition;
The binding substances of I.D, wherein antibody is called as CEM231.6.7 in addition;
The binding substances of J.C, wherein antibody is called as T101 in addition;
The binding substances of K.C, wherein antibody is called as CC49 in addition;
The binding substances of L.C, wherein antibody is called as 007B in addition;
The binding substances of M.C, D, E, F, G, H, I, J, K or L, wherein antibody is F(ab ' in addition) fragment, and enzyme is β-Nei Xiananmei (from Enterobacter cloacae).
The other aspect of the present invention is a formula II compound
Substrate-cytotoxic agents II
In the formula:
Substrate is the substrate for enzyme or its active fragments, wherein the reaction of said enzyme-to-substrate-cytotoxic drug causes the chemical separation of this substrate and cytotoxic agents, and, by the ether that forms by the suitable active group on this substrate, thioether group, ester group, amido, amino, hydrazide group, carbonate group, carbamate groups, the thiocarbamate base, the azepine carbamate groups, thioester substrate, thio acylamino or thiocarbonic acid SOH ester group and the hydroxyl on cytotoxic agents, thioether group, amino, amido, hydrazide group, carboxyl or carbamate groups combine substrate and cytotoxic agents.Another kind method is that substrate can be the precursor of cytotoxic agents.
Term " cytotoxic agents " refers to treating optimum or malignant tumor is useful compound.Usually, this class medicine comprise alkylating agent, antiproliferative, in conjunction with tubulin agent, common cytotoxin etc.The preferred compound of this class is mustargen agent, anti-folic acid, nucleoside analog, catharanthus alkaloid, anthracycline, mitomycin, bleomycin, born of the same parents' poison nucleosides, pteridine section medicine, podophyllotoxin, sulfonylurea (described on May 20th, 1987 disclosed European Patent Application No. 222,475) and lower molecular weight toxin such as trichothecin and colchicine.Those useful especially compounds comprise; for example, Zorubicin, daunomycin, aminopterin-induced syndrome, methotrexate, taxol, methopterin, dichloro methotrexate, ametycin, porfiromycin, 5-fluor-uracil, Ismipur, cytosine(Cyt), Arabinoside, podophyllotoxin, Zuyeyidal, melphalan, vincaleucoblastine, vincristine(VCR), deacetylation vincaleucoblastine hydrazides, west, Changchun are fixed, vindesine, virosine, trichothecin, Deacetylcolchicine etc.Certainly, common experienced chemist can make unessential modification chemically to described preferred and general compound, so that make their reaction easier.
Certainly, can prepare preferred substrate-cytotoxic agents compound by preferred cytotoxic agents.The preferred embodiment of formula II compound is listed below.In following enumerating, every kind of new preferred embodiment all has all definition and it is about the restriction to above-mentioned example.Think that equally the enzyme that relates in this manual also refers to its active fragments.Therefore, the present invention's preferred embodiment in this respect comprises:
A. formula II compound, wherein substrate is the substrate for β-Nei Xiananmei, L-pyroglutamic acid ester aminopeptidase, beta-galactosidase enzymes or D-aminopeptidase; (for example, the substrate for β-Nei Xiananmei can be that penicillin, penem, carboxylic benzyl, penem (Carbapenem), cynnematin, cynnematin sulfoxide, 1-carboxamide desulfurization cynnematin (1-Carbadethi acephalosporin) or 1-contain oxygen desulfurization cynnematin; )
B. the compound of example A, wherein substrate is:
ⅰ) the cynnematin of following formula:
Figure 931098718_IMG10
Zz is 0 or 1 in the formula, R 1For by C 1To C 30Alkyl is derived and next acyl group, R 2Be hydrogen, organic or inorganic positively charged ion, carboxyl-protecting group, or the base of the unsettled ester formation of nontoxic metabolism;
ⅱ) L-pyroglutamic acid ester;
ⅲ) β-D-semi-lactosi;
ⅳ) D-amino acid, particularly D-L-Ala;
C. the compound of example B, wherein substrate is the cynnematin of following formula:
D. the compound of example C, R in the formula 1Be:
ⅰ) C 1To C 4Alkyl, C 1To C 6The alkyl, the C that replace 1To C 4Alkoxyl group, C 1To C 4The group of alkylthio or following formula:
Figure 931098718_IMG13
Or the carboxy derivatives of amino and/or its protection of protection;
ⅱ) the group of following formula:
Figure 931098718_IMG14
Figure 931098718_IMG15
Each R in the formula 5, R 6And R 7Be alone hydroxyl, nitro, amino, the protection of hydrogen, halogen, hydroxyl, protection amino, amine salt, cyano group, trifluoromethyl, aminomethyl, protection aminomethyl, N-(methyl or ethylsulfonyl) amino, C 1To C 6Alkyl or C 1To C 4Alkoxyl group; R 4Carboxyl, phenyl carboxylicesters, (5-indanyl) carboxylicesters, sulfonic acid, sulfonate, azido-, halogen or C for the amino of the hydroxyl of hydroxyl, protection, methanoyl, amino, protection, amine salt, carboxyl, carboxylate salt, protection 1To C 6Alkyl; R 6Be C 1To C 4Alkoxyl group; Z is oxygen or sulphur; N is 0,1,2 or 3; With m be 0 or 1;
ⅲ) the group of following formula:
Figure 931098718_IMG16
R in the formula 9Be heterocycle; R 10Carboxyl, phenyl carboxylicesters, (5-indanyl) carboxylicesters, sulfonic acid, sulfonate, azido-, halogen or C for the amino of the hydroxyl of hydroxyl, protection, methanoyl, amino, protection, amine salt, carboxyl, carboxylate salt, protection 1To C 6Alkyl; N is 0,1,2 or 3; With z be oxygen or sulphur.
E. the compound of example D, cytotoxic agents is a following formula in the formula:
R in the formula 12Be amino or hydroxyl;
R 13Be hydrogen or methyl;
R 14Be hydrogen, fluorine, chlorine, bromine, iodine;
R 15Part for hydroxyl or formation carboxylate salt;
Figure 931098718_IMG18
R in the formula 17Be amino, C 1-C 3Alkylamino; Two (C 1-C 3) alkyl) amino or C 4-C 6Polymethylene amino;
Figure 931098718_IMG19
Figure 931098718_IMG20
R in the formula 19Be hydrogen or methyl;
R 20Be methyl or thienyl;
Figure 931098718_IMG21
R in the formula 21Be H, CH 3Or CHO; Work as R 23And R 24When choosing separately, R 24Be H, R 22And R 23One of be ethyl, and another is H or OH; Work as R 23And R 24The carbon that is connected with them is together the time, and they form the oxyethane ring, and this moment R 22Be ethyl; R 25Be hydrogen, (C 1-C 3Alkyl)-(C that CO-or chlorine replace 1-C 3Alkyl)-CO; P is 0 or 1; R 26Be a key ,-(C 2-C 4Alkyl) X, or require P be 1 and this group itself link group on the carbonyl oxygen base again; X is-O-,-S-or-NH-;
Figure 931098718_IMG22
Figure 931098718_IMG23
R in the formula 28Be hydrogen, methyl, bromine, fluorine, chlorine or iodine;
R 29For-OR 18Or-NHR 18;
R 30Be hydrogen, bromine, chlorine or iodine;
Figure 931098718_IMG24
Wherein A be-O-,-NCH 3-,-CH 2-,-CH 2CH 2-or-CH 2O-;
D is-CH 2-or-O-;
R 31Be hydrogen or halogen;
R 32Be halogen or trifluoromethyl;
Figure 931098718_IMG25
5 FU 5 fluorouracil or deacetylate colchicine
In the preferred in the above formula, formula III compounds represented anthracycline compound; The formula IV is represented the methotrexate compounds; The formula V is represented mitomycin; The formula VI is represented bleomycin; The formula VII is represented melphalan; The formula VIII is represented Ismipur; The formula IX is represented cytarabin; The formula X is represented podophyllin; The formula XI is represented the Vinca medicine; On behalf of Difluoronucleosides and formula X III and formula X IV, the formula XII represent the sulfonylurea compound.
F. the compound of example E, wherein R 1Be ethanoyl, propionyl, different propionyl, valeryl, butyryl radicals, the methoxyl methyl ethanoyl, phenylacetyl, the phenoxy group ethanoyl, the 2-(aminomethyl) phenylacetyl, 2-phenyl-2-hydroxyacetyl, 2-phenyl-2-(sodium sulfonate group) ethanoyl, 2-phenyl-2-carboxyl ethanoyl, the 2-(4-hydroxy phenyl)-2-carboxyl ethanoyl, 2-phenyl-2-glycyl, the 2-(4-hydroxy phenyl)-the 2-glycyl, the 2-(3-(N-(methanesulfonamido)) phenyl)-the 2-glycyl, 2-phenyl-2-(5-(2, the 3-indanyl) ethanoyl carboxylic acid ester groups), 2-phenyl-2-(phenyl carboxylic acid ester groups) ethanoyl, 2-phenyl-2-azido-ethanoyl, 2-phenoxy group propionyl, 2,5-dimethoxy benzoyl, the 2-(methanoyl)-the 2-phenylacetyl, 2-(2-oxyethyl group naphthalene-1-yl) ethanoyl, 2-(naphthalene-1-yl)-the 2-glycyl, 2-(naphthalene-2-yl)-the 2-glycyl, 2-(2,5-dichlorobenzene sulfenyl) ethanoyl, 2-(3,4-dichlorobenzene sulfenyl) ethanoyl, the 2-(1-tetrazyl) ethanoyl, 2-(N-(3,5-dichloropyridine-4-oxygen base)) ethanoyl, 2-(2-aminothiazole-4-yl) ethanoyl, the 2-(2-thienyl) ethanoyl, 2-(4-pyridine sulfenyl) ethanoyl, 2-(N-methyl-4-pyridine sulfenyl) ethanoyl, 2-(2-amino-4-phenyl thiazole-5-yl) ethanoyl, 2-(3-hydroxyl-4-carboxyl isothiazole-5-base sulfenyl) ethanoyl, 3-phenyl-5-methyl-isoxazole base-3-formyl radical, the 3-(2-chloro-phenyl-)-5-methyl-isoxazole base-3-formyl radical, 3-(2, the 5-dichlorophenyl)-5-methyl-isoxazole base-3-formyl radical, 3-(2-fluoro-5-chloro-phenyl-)-5-methyl-isoxazole base-3-formyl radical, the 2-(2-thienyl)-the 2-glycyl, the 2-(2-thienyl)-and the 2-(carboxylic acid sodium salt) ethanoyl, the 2-(N-(4-pyridine)) ethanoyl, the 2-(2-benzothienyl) ethanoyl, the 2-(3-benzothienyl) ethanoyl, the 2-(2-benzofuryl) ethanoyl and 2-(3-benzofuryl) ethanoyl;
G. the compound of example F, wherein cytotoxic agents is methotrexate, 5 FU 5 fluorouracil, deacetylate vincaleucoblastine aminoothyl mercaptan, deacetylate vincaleucoblastine hydrazide group carboxy moiety, 7-(carboxyamino) deacetylate colchicine part or Zorubicin;
H. the compound of example G, wherein R 1Being 2-(thiophene-2-yl) ethanoyl, ZZ be zero;
I. the compound of example H, wherein cytotoxic agents is a methotrexate;
J. the compound of example I, wherein methotrexate is linked on the cynnematin substrate by its carboxylicesters base key;
K. the compound of example H, its born of the same parents' poison is a 5 FU 5 fluorouracil;
L. the compound of example K, wherein 5 FU 5 fluorouracil is linked on the cynnematin substrate by its N-1 nitrogen-atoms key;
M. the compound of example H, wherein cytotoxic agents is a deacetylate vincaleucoblastine aminoothyl mercaptan;
N. the compound of example M, wherein deacetylate vincaleucoblastine aminoothyl mercaptan is linked the cynnematin part by the mercaptan base key of its aminoothyl mercaptan part;
O. the compound of example H, wherein cytotoxic agents is a deacetylate vincaleucoblastine hydrazide group carboxy moiety;
P. the compound of example O, wherein deacetylate vincaleucoblastine part is linked on the cynnematin substrate by the Sauerstoffatom key of hydrazide group carboxyl;
Q. the compound of example G, wherein R 1Be 2-(thiophene-2-yl) ethanoyl, ZZ is 1;
R. the compound of example Q, wherein cytotoxic agents is the 7-(carboxyamino) deacetylate colchicine part;
S. the compound of example R, wherein 7-(carboxyamino) the deacetylate colchicine is partly by the 7-(carboxyamino) the Sauerstoffatom key of group links on the cynnematin substrate;
T. the compound of example Q, wherein cytotoxic agents is a deacetylate vincaleucoblastine aminoothyl mercaptan;
U. the compound of example T, wherein cytotoxic agents is a deacetylate vincaleucoblastine aminoothyl mercaptan;
V. the compound of example Q, wherein cytotoxic agents is a deacetylate vincaleucoblastine hydrazide group carboxy moiety;
IV. the compound of example V, wherein deacetylate vincaleucoblastine hydrazides carboxy moiety is linked on the cynnematin part by the Sauerstoffatom key of hydrazide group carboxyl;
X. the compound of example Q, wherein cytotoxic agents is a Zorubicin;
Y. the compound of instance X, wherein the Zorubicin part is linked the 3-(ketonic oxygen methylene of cynnematin sulfoxide group by the nitrogen-atoms key of amino saccharides) carbonyl carbon on;
Z. the compound of example G, wherein R 1Be the phenoxy group ethanoyl, ZZ is 1;
AA. the compound of example Z, wherein cytotoxic agents is a deacetylate vincaleucoblastine hydrazide group carboxy moiety;
BB. the compound of example AA, wherein deacetylate vincaleucoblastine hydrazide group carboxy moiety is linked on the cynnematin substrate by the Sauerstoffatom key of amino carboxyl.
CC. through the example I compound of BB, R wherein 2Be hydrogen, sodium cation, or potassium cationic.
Certainly, formula II compound also can be used as solvate and hydrate existence.Therefore, these compounds can with as the water of hydration or with one or several or the molecular crystal of the parent solution solvents of umber arbitrarily.Solvate and hydrate are included within the scope of the invention.
Similarly, formula II compound can be used as the acceptable salt existence of medicine.These compounds had both comprised acid functional groups such as carboxyl and sulfonic salt, also comprised the salt of basic functional group such as amino.This class salt comprises that organic and inorganic cation described below adds basic group and the following acid salt by acid-base reaction formation, described acid has: hydrochloric acid, sulfuric acid, phosphoric acid, acetate, succsinic acid, citric acid, lactic acid, toxilic acid, fumaric acid, palmitinic acid, cholic acid, Pamoic, tetrahydroxyadipic acid, D-L-glutamic acid, d-dextrocamphoric acid, pentanedioic acid, phthalic acid, tartrate, lauric acid, stearic acid, Whitfield's ointment, methylsulfonic acid, Phenylsulfonic acid, Sorbic Acid, picric acid, phenylformic acid, styracin, or the like.
Used term R in above-mentioned preferred embodiment 1The representative " by C 1To C 30The acyl group of carboxylic acid derivatives " be meant and linked on the C-6 amino of penicillin by key; on the C-7 amino of cynnematin, 1-sulfoxide cynnematin, the assorted cynnematin of 1-oxa-desulfuration or 1-formamyl cynnematin, and the acyl group on the C-3 amino of monocycle beta-lactam (as azthreonam series)." by C 1To C 30The acyl group of carboxylic acid derivatives " can be separated arbitrarily by heteroatoms.The example of this class acyl group can be referring to " the Cephalosporins and Penicillins; Chemistry and Biology " of document as being edited by Edwin W.Flynn; Academic Press; New York 1972 and " the Chemistry and Biology of β-lactam Antibiotics " Vols.1,2 and 3 that edits by Robert B.Morin and Marvin Gorman; Academic Press, New York 1982.
At R 1On the example of acyl group can also be referring to following patent literature: people such as Yoshioka M., United States Patent (USP) 4,478,997(1984 issued October 23); People such as Belleau B.R., United States Patent (USP) 4,172,199(1979 issued October 23); People such as Kamiya T., United States Patent (USP) 4,472,300(1984 issued September 18) (particularly hurdle, 25 hurdle to 36), they all are introduced into this paper as a reference." by C 1To C 30The acyl group of carboxylic acid derivatives " other examples can be referring to people's such as Koster United States Patent (USP) 4,478,749(1984 issued October 23).
Term " organic or inorganic positively charged ion " refers to the counter ion of the carboxylate anion of carboxylate salt.Counter ion are selected from basic metal and alkaline-earth metal, as lithium, sodium, potassium, barium and calcium; Ammonium; Reach organic cation such as dibenzyl ammonium, hexadecyldimethyl benzyl ammonium, 2-hydroxyethyl ammonium, two (2-hydroxyethyl) ammonium, styroyl benzyl amine, dibenzyl ethylene ammonium, or the like.Other positively charged ions that top term comprises comprise PROCAINE HCL, PHARMA GRADE, quinine and the N-methylglucosamine of protonated form, and the basic aminoacids of protonated form is as glycine, ornithine, Histidine, phenylglycocoll, Methionin and arginine.In addition, this term is preferably by any zwitterionic form of carboxylic acid with the amino instantaneous compound that forms.
Used term " carboxyl-protecting group " is meant and is generally used for protecting the carboxylic acid group and makes one of ester derivative of the carboxylic acid group who carries out on other functional groups that are reflected at compound in specification sheets.These carboxylic acid protecting groups' example comprises the 2-nitrobenzyl; the 4-nitrobenzyl; the 4-methoxy-benzyl; 3; the 4-dimethoxy-benzyl; 2; the 4-dimethoxy-benzyl; 2; 4; 6-trimethoxy benzyl; 2; 4; the 6-trimethyl benzyl; the pentamethyl-benzyl; 3; 4-methylene radical dioxy base benzyl; diphenyl-methyl; 4; 4 '-the dimethoxy diphenyl-methyl; 2; 2 '; 4,4 '-the tetramethoxy diphenyl-methyl; the tertiary butyl; tert-pentyl; trityl; 4-methoxyl group trityl, 4; 4 '-dimethoxytrityl; 4; 4 ', 4 " trimethoxy trityl, 2-phenyl third-2-base; trimethyl silyl; t-butyldimethylsilyl; phenacyl; 2; 2,2-three chloroethyls; β (trimethyl silyl) ethyl; β-((di-n-butyl) methyl-silicane base) ethyl; the p-toluenesulfonyl ethyl; 4-nitrobenzyl alkylsulfonyl ethyl; allyl group; cinnamyl; 1-(trimethyl silyl methyl) third-1-alkene-groups such as 3-base.The kind of used carboxyl-protecting group and non-key as long as the deutero-carboxylic acid is stable for the condition at other locational subsequent reactions of cynnematin molecule, and can be removed and saboteur's rest part not in due course.Preferred carboxylic acid protecting group is an allyl group.The similar carbonyl-protection base that is used for cynnematin, penicillin and peptide field also can be used for protecting the carboxyl substituent of instantaneous cynnematin substrate.Other examples of these groups are referring to Haslam E " Protective Groups in Organic Chemistry ", McOmie H.G.W., Ed., Plenum Press, New York.N.Y.1973, the 5th chapter and Greene T.W. " Protective Groups in Organic Synthesis ", John Wiley ﹠amp; Sons, New York, N.Y.1981, the 5th chapter.Relational language is " carboxyl of protection ", and it is meant by the monobasic carboxyl of top carboxyl-protecting group.
Term " the unsettled one-tenth ester group of nontoxic metabolism " is meant those biological activity ester-formins.They bring out the effect that blood concentration increases and prolongs corresponding no esterification formalization compound.This class ester group comprises the lower alkoxy methyl, for example methoxymethyl, ethoxyl methyl, isopropoxy methyl etc.; α-(C 1To C 4) alkoxyethyl, for example methoxy ethyl, ethoxyethyl group, propoxy-ethyl, isopropoxy ethyl etc.; 2-oxo-1,3-Dioxol-4-yl methyl, as 5-methyl-2-oxo-1,3-Dioxol-4-yl methyl, 5-phenyl-2-oxo-1,3-Dioxol-4-yl methyl etc.; C 1To C 3The alkylthio methyl is as methylthiomethyl, ethylmercapto group methyl, iprotiazem ylmethyl etc.; The acyloxy methyl is as oxy acid methyl neopentyl, α-acetoxy-methyl etc.; Ethoxycarbonyl-1-methyl; Alpha-acyloxy-alpha-substitution methyl is as α-acetoxyl group ethyl; 2-benzo [C] furanonyl or 5,6-dimethyl-2-benzo [C] furanonyl; 1-(C 1To C 4Alkoxycarbonyloxy) second-1-base is as 1-(ethoxycarbonyl-oxygen base) second-1-base; And 1-(C 1-C 4Alkyl amino carbonyl oxy) second-1-base is as 1-(amino-carbonyl oxygen base) second-1-base.
Should give definition with several terms that top example D uses.Therefore, term " C 1-C 6Alkyl " represent group such as methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, sec-butyl, the tertiary butyl, amyl group, tert-pentyl, hexyl or the like.
Term " C 1-C 6The alkyl that replaces " represent by the above-mentioned C of one or two following substituting group replacement 1-C 6Alkyl.Described substituting group has: the amino of the hydroxyl of halogen, hydroxyl, protection, amino, protection, C 1-C 7The carboxyl of acyloxy, nitro, carboxyl, protection, formamyl, carbamoyloxy, cyano group, methylsulfonyl amino or C 1-C 4Alkoxyl group.The alkyl that replaces can replace once or twice with identical or different substituting group.
Term " C used herein 1-C 4Alkoxyl group " represent groups such as group such as methoxyl group, oxyethyl group, positive propoxy, isopropoxy, n-butoxy, tert.-butoxy.
Used term " amino of protection " is meant the amino that is replaced by an amino protecting group commonly used in the definition above; as tertbutyloxycarbonyl (t-BOC), carbobenzoxy-(Cbz), 4-methoxyl group benzyloxy carbonyl, 2; 2, amino protecting groups such as 2-trichloro-ethoxycarbonyl, trimethyl silyl.The character of this class amino protecting group is also non-key, as long as the amido functional group of protection is stable under following reaction conditions.
Term " hydroxyl of protection " is meant that be stable under the reaction conditions of the subsequent step of synthetic cynnematin substrate compounds, but is easy to any group of cracked afterwards.This class group comprises methanoyl, chloroethene acyloxy, diphenyl-methyl oxygen base, trityl oxygen base, trimethyl silyl, or the like.
In the definition in front, hydroxyl, amino and carboxyl-protecting group are not limited fully.The effect of this class group is a protective reaction functional group in the process of the required product of preparation, is removed under the prerequisite of saboteur's rest part not then.Many these class protecting groups are known in the art, the use of other groups is equally applicable to method of the present invention and compound, group is considered to suit described in following document: McOmie J.F.W., " Protective Groups in Organic Chemistry ", Plenum Press, 1973 and Greene T.W., " Protective Groups in Organic Synthesis ", John Wiley ﹠amp; Sons, New York, N.Y.1981.Therefore, state that for " blocking group " mentioned in this specification sheets it does not have novelty or creativeness.
Term " heterocycle " when with term " by C 1To C 30The acyl group of carboxylic acid derivatives " be meant the ring in people's such as Durckheimer W. the United States Patent (USP) 4,278,793 when using together.Specific examples is a ring unsubstituted or that replace, as tetrazyl, oxazolyl, isoxazolyl, thienyl, furyl, thiazolyl, thiadiazolyl group, isothiazolyl, pyridyl, 4-pyriconyl, pyridine, benzothienyl, benzofuryl or indyl.Certainly, these examples can replace with the substituting group described in 4,278, No. 793 patents of people such as Durckheimer.
A third aspect of the present invention is the methods of treatment of treatment tumor disease, and this method comprises:
1) take the antibody-enzyme conjugates compound of the formula I of treatment significant quantity for the host who infects; Then
2) take the substrate-cytotoxic agents of the above-mentioned formula II of treatment significant quantity for the host who infects.
Should be appreciated that term " treatment effectively " will be the amount that is enough to cause some target death of neoplastic cells when it relates to substrate-cytotoxic agents, it can also refer to use this medicine for tumor disease is alleviated, and perhaps uses this medicine with the amount that is enough to prevent usually.
Usually, term " is treated effectively " specific purposes that are meant at the moment and is provided the substrate-cytotoxic agents of same amount at least with respect to cytotoxic agents itself.Can provide relatively large substrate-cytotoxic agents in the present invention, because the originally connected substrate of the toxicity of cytotoxic agents is sheltered, but subsequently at its point of release by with abzyme binding substances reaction and demasked, so just, alleviated the side effect to patient, this side effect is relevant with the amount of cytotoxic agents itself usually.
The effective dosage of the treatment of antibody-enzyme conjugates is the amount that discharges 1 to 500 preferred 1 to 50mg antibody (or its part) to the host who infects.
List the preferred embodiment of the inventive method below.About compound of the present invention aspect, below listed embodiment comprised the restriction and the definition of the embodiment that it is related to.The example of the preferred embodiment of the inventive method comprises:
A. method, wherein substrate is the substrate to β-Nei Xiananmei, Pyrrolidonecarboxylic acid aminopeptidase, beta-galactosidase enzymes or D-aminopeptidase, and antibody-enzyme conjugates compound, wherein enzyme is β-Nei Xiananmei, Pyrrolidonecarboxylic acid aminopeptidase, beta-galactosidase enzymes or D-aminopeptidase;
The B.A method, wherein enzyme is a β-Nei Xiananmei;
The C.B method, wherein antibody be present in antigen complexing on the malignant cell individually or with supernatural abundance;
D. the method for example C, wherein substrate is the cynnematin of following formula:
Figure 931098718_IMG26
Wherein ZZ is 0 or 1; R 1For by C 1To C 30Alkyl deutero-acyl group, R 2Be hydrogen, organic or inorganic positively charged ion, carboxyl-protecting group or the nontoxic unsettled one-tenth ester group of metabolism; As for defined those terms of the preferred embodiment B of substrate of the present invention-cytotoxic agents aspect;
E. the method for example D, wherein antibody and carcinomebryonic antigen complexing;
F. the method for example D, wherein antibody and the complexing of KS1/4 antigen;
G. the method for example D, wherein antibody and the complexing of TAG-72 antigen;
H. the method for example D, wherein antibody and the complexing of MAT65 antigen;
I. the method for example E, wherein R 1With the R among the preferred embodiment D of formula II compound 1Identical;
J. the method for example I, wherein cytotoxic agents is a listed general formula compound among the preferred embodiment E of formula II compound;
K. the method for example J, wherein R 1With the R among the preferred embodiment F of formula II compound 1Identical;
L. the method for example K, wherein cytotoxic agents is methotrexate, 5 FU 5 fluorouracil, deacetylate vincaleucoblastine aminoothyl mercaptan, deacetylate vincaleucoblastine aminocarboxylic base section, 7-(carboxyamino) deacetylate colchicine part; Or Zorubicin;
M. the method for example L, the wherein R of cynnematin substrate 2Be 2-(thiophene-2-yl) ethanoyl, ZZ is 0;
N. the method for example M, wherein antibody is called as CEM231.6.7;
O. the method for example N, wherein cytotoxic agents is a methotrexate;
P. the method for example O, wherein methotrexate is linked on the cynnematin substrate by the carboxyl key;
Q. the method for example N, wherein cytotoxic agents is a 5 FU 5 fluorouracil;
R. the method for example Q, wherein 5 FU 5 fluorouracil is linked on the cynnematin substrate by its N-1 nitrogen-atoms key;
S. the method for example N, wherein cytotoxic agents is a deacetylate vincaleucoblastine aminoothyl mercaptan;
T. the method for example S, wherein deacetylate vincaleucoblastine aminoothyl mercaptan is linked on the cynnematin part by the mercaptan base key of aminoothyl mercaptan;
U. the methods of treatment of example N, wherein cytotoxic agents is a deacetylate vincaleucoblastine aminocarboxylic base section.
V. the methods of treatment of example U, wherein deacetylate vincaleucoblastine hydrazide group carboxy moiety is linked on the cynnematin substrate by the Sauerstoffatom key of hydrazide group carboxyl.
W. the methods of treatment of example L, wherein R 1Be 2-(thiophene-2-yl) ethanoyl, ZZ is 1.
AA. the methods of treatment of instance X, wherein cytotoxic agents is the 7-(carboxyamino) deacetylate colchicine part.
BB. the methods of treatment of example AA, wherein cytotoxic agents is the 7-(carboxyamino) deacetylate colchicine part.
CC. the methods of treatment of instance X, wherein cytotoxic agents is a deacetylate vincaleucoblastine aminoothyl mercaptan.
DD. the methods of treatment of example CC, wherein deacetylate vincaleucoblastine aminoothyl mercaptan is linked on the cynnematin substrate by the mercaptan base key.
EE. the methods of treatment of instance X, wherein cytotoxic agents is a deacetylate vincaleucoblastine hydrazide group carboxy moiety.
FF. the methods of treatment of example EE, wherein deacetylate vincaleucoblastine hydrazide group carboxy moiety is linked on the cynnematin substrate by the Sauerstoffatom key of hydrazide group carboxyl.
GG. the methods of treatment of example W, wherein cytotoxic agents is a Zorubicin.
HH. the methods of treatment of embodiment GG, wherein Zorubicin is linked on the cynnematin substrate by the nitrogen-atoms key of amino saccharides.
II. the methods of treatment of example L, wherein R 1Be (phenoxy group) ethanoyl, ZZ is 1.
JJ. the methods of treatment of example II, wherein cytotoxic agents is a deacetylate vincaleucoblastine hydrazide group carboxy moiety.
KK. the methods of treatment of example JJ, wherein deacetylate vincaleucoblastine hydrazide group carboxy moiety is linked on the cynnematin substrate by the Sauerstoffatom key of hydrazide group carboxyl.
LL. the method for example N to KK, wherein antibody is F(ab ') part.
Substrate of the present invention-cytotoxic agents compound can be synthetic by various known methods.Therefore, under Sn 1 or Sn 2 conditions, the nucleophilic group of substrate can be used for replacing the leavings group at the electrophilic center place of cytotoxic agents, or vice versa.May disturb substrate and the carboxyl on the cytotoxic agents, amino, thiol group or the hydroxyl of the reaction of required functional group before reaction, to shelter, after reaction, go protection then with protecting group.The use of suitable protecting group is described in the works of McOmie above-mentioned and Greene in its elsewhere.Desired to cytotoxic agents compound of the present invention is that after the effect that is subjected to enzyme was with the substrate chemical separation, cytotoxic agents should be the effective antitumour agent, and the derivatization of substrate and cytotoxic agents can greatly not reduce the reactivity of substrate and enzyme.Preferably substrate-cytotoxic agents compound is sheltered the toxicity of cytotoxic agents, or cytotoxicity is not more than unsubstituted cytotoxic agents Cytotoxic 70%.
Concerning the inventive method, preferably enzyme has suitable catalytic rate.Because enzyme will run into the insufficient substrate of concentration (with substrate-cytotoxic agents compound form), so particularly importantly the enzyme-substrate binding substances has high Kcat/Km ratio.Therefore, preferably select to have at least 1 * 10 6M -1S -1The enzyme of Kcat/Km ratio.When enzyme is attached on the antibody in enzyme preferred this identical ratio.Similarly, preferred antibody has high affinity to the antigen on the target tumour cell, and preferred about 1 * 10 6(liter/mole) or higher.Preferably bonded antibody keeps it unconjugated active 80% in addition, therefore when in conjunction with the time preferably have 60% immunoreactivity.
It is that standard method by the protein chemistry field is finished that enzyme is attached on the antibody.Also can prepare binding substances by above-mentioned Offord and Rose method, or by gene fusion produce fusion rotein (european patent application 392745, open day is October 17 nineteen ninety; People such as Neuberger M.S., Nature, 312, P.604,1984; People such as Seehaus T., Gene, 114, P.1,1992).Can use different difunctionality reagent and with difunctionality reagent.Therefore, can they can have been bought from the market with MBS, Sulfo-MBS, SMCC, Sulfo-SMCC, SIAB, SPDP, Sulfo-SMPB, DIDS, DFDNB, BMH() wait and prepare binding substances of the present invention.The bonded purifying that produces can be finished with suitable known chromatographic process such as gel infiltration or ion exchange method.Like this, among the present invention the method for enzyme and antibodies just without any novelty.Preferably regulate stoichiometry and other conditions of association reaction (and purifying), so that produce 1: 1 antibody: enzyme conjugates, but the binding substances that has disproportionately relatively large arbitrary composition in binding substances includes in the present invention.
After having selected antibody and enzyme, those skilled in the art must determine to be to use complete antibody also to be to use its part.The method for preparing part, for example be specified in people such as M.Brennan, Science, 229 with stomach en-or papoid, PP.81-83,1985 and people such as M.Glennie, J.Immunology, 139, PP.2367-2375, in 1987, will no longer discuss, but will should be mentioned that F(ab ' this this paper) be the preferred form of antibody of the present invention.
Isolate after the enzyme with above-mentioned standard, it is attached on the aforesaid suitable antibody, the purifying binding substances is used the gel electrophoresis analysis binding substances.In addition, high pressure liquid chromatography (HPLC) also can be used for analyzing binding substances.
Measure the enzymic activity of binding substances again, use key to link substrate on the molecule specifically, and the spectrophotometric that molecule can be surveyed when giving substrate (or this molecule itself) fracture changes, thereby measure Kcat and K.This mensuration will show whether keeping its activity through enzyme after the integrating step.Next parameter to be measured is the external immunoreactivity of binding substances.This measurement is normally at first carried out radio-labeling with binding substances and (is for example used 125I), with target antigen coating solid surface (for example, little plastic bead or titer plate cave).Whether the antibody moiety that this mensuration will detect binding substances has unacceptably been changed in integrating step.As the Kcat and the K that measure binding substances with substrate-cytotoxic agents MThe time just finished the first step, whether the component of promptly observing the inventive method has played effect in the presence of target antigen.(discharging the free cytotoxic agents by enzyme can be shown by color atlas).Study binding substances then to determine whether it can concentrate on the tumour in vivo.Finishing normally of this research injects radiolabeled binding substances to the mouse that has tumour, and described tumour has been expressed target antigen, then mouse put to death and detects the radioactive amount that is present in tumour and the various organ.This research will show that binding substances is to concentrate on tumour, or unacceptably target is penetrated anosis organ, in addition or because it is as the state of foreign protein and by metabolism.
When determining binding substances is when concentrating on tumour, must show the binding substances fracture that substrate-cytotoxic agents will be concentrated.Therefore, or with binding substances or only use enzyme (in the same old way) that the target antigen of express cell is suspended and cultivate.With the cell resuspending handled like this in the damping fluid of another amount, then with the substrate cultivation that contains the twice compound, so that take place to absorb in pairs at the different wave length place of visible spectrum before the cracking and afterwards.Whether the supernatant liquor of measuring these suspension then shows time and the concentration dependence to the amount of the binding substances that adds with the suspension of determining to contain binding substances.Like this, if to show this dependency in the same old way, the condition of some except that binding substances will cause cracking so.Suppose that component of the present invention demonstrates concentrated binding substances in the test in front and determine to cause substrate-cytotoxic agents cracked reagent, then should in experimental animal such as mouse, measure the serum kinetics of binding substances, after with the binding substances administration, whether reach when can take substrate-cytotoxic agents to infected host so that determine.Preferably binding substances demonstrates short serum half-life, so as can be in reasonable time after with the binding substances administration (as 72 hours) with substrate-cytotoxic agents molecule administration.When finding that the bonded serum half-life is acceptable, just can measure the enzymic activity that concentrates on experimental animal such as the intravital binding substances of mouse.Take to the mouse that has the target tumour binding substances, its tumour is put to death and excised to the process time enough with mouse after binding substances is concentrated.With tumour chopping and with the substrate cultivation of product pigment.Measure the mark of enzymic activity of the supernatant liquor of these suspension.
If the component of the inventive method has successfully overcome all these obstacles, just can measure the vitro cytotoxicity of this component.
Then target cell is suspended and cultivation with binding substances.Afterwards the cell of handling is also cultivated with substrate-cytotoxic agents resuspending.Regulate the cell growth by absorbing the radiolabeled nutrition that is added to subsequently in the developing medium.The final step of estimating the inventive method is an effectiveness of estimating component in naked mouse tumor model research.
Another aspect of the present invention is the pharmaceutical composition of the antibody-enzyme conjugates of formula I and the substrate of formula II-cytotoxic agents compound.Particularly these pharmaceutical compositions can be used for treating tumor disease of the present invention, and comprise the formula I binding substances or the formula II compound of appropriate carriers and treatment significant quantity.
For liquid preparations for oral administration (for example tablet and capsule), term " appropriate carriers " is meant vehicle commonly used, as tackiness agent, for example syrup, gum arabic, gelatin, Sorbitol Powder, tragacanth gum, polyvinylpyrrolidone (Povidone), methylcellulose gum, ethyl cellulose, Xylo-Mucine, Vltra tears, sucrose and starch; Weighting agent or carrier, for example W-Gum, gelatin, lactose, sucrose, Microcrystalline Cellulose, kaolin, mannitol, phosphoric acid-hydrogen calcium, sodium-chlor and alginic acid; Disintegrating agent such as croscarmellose sodium, Microcrystalline Cellulose, W-Gum, sodium starch glycollate, alginic acid and variable wetting agent such as sodium lauryl sulphate; And lubricant, as Magnesium Stearate and other metallic stearates, stearic acid, siloxane fluid, talcum powder, wax oil and colloid silicon.Can also use seasonings such as peppermint, wintergreen oil, cherry seasonings etc., preferably add tinting material so that the formulation appearance is more beautiful, or help to confirm this product.Tablet can also be used the methods known in the art dressing.
Pharmaceutical composition of the present invention can also be an oral liquid, they or a) moisture or oily suspension, solution, emulsion or syrup; Or b) dried powder that reconstitutes of used water or another suitable medium before use.When using together with this oral liquid, term " appropriate carriers " is meant conventional additive, as suspension agent, for example Prunus amygdalus oil, fractionated Oleum Cocois, grease of Sorbitol Powder, syrup, methylcellulose gum, glucose/syrup, gelatin, Natvosol, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible oil for example, propylene glycol or ethanol; Sanitas such as methyl p-hydroxybenzoate or propyl ester, or Sorbic Acid.
Pharmaceutical composition also can be used for intravenous application (IV).Particularly water-soluble shape binding substances or substrate-cytotoxic agents can be dissolved in a kind of intravenous fluid commonly used, and by the infusion administration.When using together with the composition that is used for the purposes IV, term " appropriate carriers " is meant fluid such as physiological saline, Ringer's solution or 5% glucose solution.
For the intramuscular preparation, can prepare the sterile preparation of the suitable salt form of binding substances or substrate-cytotoxic agents compound (for example, hydrochloride or sodium salt) with " appropriate carriers ".The example of this sterile preparation is or is dissolved in the pharmaceutical diluents (for example water for injection, physiological saline, 5% glucose) or is suspended in suitable salt form in water base or the acceptable oil base of medicine (for example long chain fatty acid ester such as ethyl oleate).
Partial composition can be prepared as hydrophobic or hydrophilic group with " appropriate carriers ".This class matrix comprises ointment, missible oil or washing lotion.
The medication composition of binding substances and substrate-cytotoxic agents compound can be formulated in the feed or drinking-water of farming animal.In addition, can as long or fast release matrix binding substances or substrate-cytotoxic agents compound be mixed with preparation in the breast with " appropriate carriers ".
Also antibody-the enzyme conjugates of formula I and the substrate of formula II-cytotoxic agents compound can be formulated in sterile vials, comprise in the ampoule with the aseptic plastic capsule of membranous outlet or sterile seal with unit dose shape.Binding substances or compound (or corresponding acceptable salt of medicine) can be dried powder or crystallization or lyophilized form.The amount of the binding substances of per unit dosage or substrate-cytotoxic agents compound can be about 5 milligrams of extremely about 10 grams.
Following experiment and method are used to further specify the present invention.Unless otherwise noted, used abbreviation has the meaning that is usually directed to them in the association area below.N.m.r. spectrum is made by General Electric G.E.-300 instrument.Infrared spectra obtains by the Perkin-Elmer281 instrument.UV spectrum obtains (except embodiment 7 and method 6 to 13, wherein the U.V. spectrum obtains by Hewlett-Packard 8451A instrument) by Cary 118 instruments.Fast atom bombardment MS obtains by VG ZAB-3 instrument.
Method 1
7-β-(2-(thiophene-2-yl) kharophen)-3-acetoxyl group methylene radical-3-cephem-4-carboxylic acid allyl ester
With 7-β-(2-(thiophene-2-yl) kharophen)-3-(acetoxyl group methylene radical)-3-cephem-4-carboxylic acid sodium (11.0g) is dissolved in 1: the mixture of 1DMF and water.Add allyl bromide 98 (3.6g), reaction mixture was stirred 48 hours under the room temperature nitrogen atmosphere.Use 0.1N hydrochloric acid diluted reaction mixture then with ethyl acetate.Separate organic layer and through dried over mgso, filter, vacuum concentration obtains orange.This oily matter is carried out purification by flash chromatography with eluent ethyl acetate on the 3 inch posts that 100-200 order activated silica gel carrier is housed.Contain the product fraction through placement by some and form white crystal.Filter and collect these crystal, use 1: 1 chloroform then: the ether recrystallization is also dry, obtains the 5.5g title product.NMR：
（300MHz，CDCl 3）：δ7.25（d，1，J＝7）;7.0（m，2）;6.27（br.d，1，J＝9），6.0-5.8（m，1）;5.83（dd，1，J＝5.9）;5.4-5.2（m，2）;5.06（1/2 ABq，1，J＝14）;4.96（d，1，J＝5）;4.81（1/2 ABq，1，J＝14）;4.74（br.d，2，J＝6）;3.85（s，2）;3.55（1/2 ABq，1，J＝18）;3.33（1/2 ABq，1，J＝18）;2.06（s，3）.
Method 2
7 β-(2-(thiophene-2-yl) kharophen)-the 3-(iodomethyl)-3-cephem-4-carboxylic acid allyl ester
With 7 β-(2-(thiophene-2-yl) kharophen)-3-acetoxy-methyl-3-cephem-4-carboxylic acid allyl ester (5.01g 11.5mmol) is dissolved in the methylene dichloride (55ml), add TMSI(3.27ml), gained solution was stirring at room 70 minutes.Solution is diluted with ethyl acetate, use 10% ice-cold Sulfothiorine, saturated sodium bicarbonate aqueous solution and salt water washing then successively.Solution is through dried over sodium sulfate, and vacuum concentration obtains 4.6g(productive rate 79%) title product, for having bisque foam: NMR:
（300MHz，CDCl3）：δ7.25（d，1，J＝7）;7.0（m，2）;6.42（d，1，J＝9）;6.0-5.85（m，1）;5.78（dd，1，J＝5，9）;5.4-5.2（m，2）;4.95（d，1，J＝5）;4.74（d，2，J＝6）;4.38（ABq，2，J＝10）;3.85（s，2）;3.72（1/2 ABq，1，J＝18）;3.44（1/2 ABq，1，J＝18）.
Embodiment 1
7 β-(2-(thiophene-2-yl) kharophen)-and 3-((methotrexate-γ-ester) methylene radical)-3-cephem-4-allyl ester
With 7 β-(2-(thiophene-2-yl) kharophen)-the 3-(iodomethyl)-3-cephem-4-carboxylic acid allyl ester (4.6g, 9.1mmol), methotrexate-γ-ester (4.6g, 9.1mmol, Sigma), DMF(50ml) and sodium bicarbonate (1.54g 18.25mmol) mixes.Solution stirring at room 18 hours, is added drop-wise in acetate (4ml) water (400ml) solution then.Collect the precipitation and the vacuum-drying that produce, obtain about 7g powder.With powder by silicagel column 15% methyl alcohol: the chloroformic solution wash-out of 2% acetate carries out purification by flash chromatography, obtains about 2.1g orange solid.Solid is passed through the flash chromatography on silica gel purifying, with gradient is 10% methyl alcohol: the chloroformic solution wash-out of chloroformic solution to 2% acetate of 1% acetate, use the chloroformic solution wash-out of 15% methyl alcohol and 2% acetate then, obtain 1.06g title product (Rf=0.5 is with the chloroformic solution of 2%HOAc, 15%MeOH):
IR:(KBr): 1782,1730,1607,1562,1513cm -1.FABMS: calculated value C 37H 38N 10O 9S 2Na=853.2162.
The λ of measured value=853.2106.UV:(EtOH) Max=370(ε=3290); 297(ε=12000); 260(ε=13300) .NMR:(300MHz, DMSO-d 6): δ 9.42(bd, 1, J=8); 9.13(d, 1, J=9); 8.52(s, 1), 7.45-6.7(m, 5); 6.6(bs, 2); 5.88(m, 1, allyl group CH); 5.39(dd, 1, J=5,8); 5.35-4.55(m, 7) and 4.7(s, 2) stack; 4.16(m, 1); 3.70(s, 2); 3.50(ABq, 2, J=10); 3.2(s, 3); 2.1(m, 2); 1.9(m, 2).
Embodiment 2
7 β-(2-(thiophene-2-yl) kharophen)-and 3-((methotrexate-γ-ester) methylene radical)-3-cephem-4-carboxylic acid
With acid chloride (II) (3mg, 0.013mmol) and triphenylphosphine (14mg 0.053mmol) mixes in ethyl acetate (0.5ml).With 7 β-(2-(thiophene-2-yl) kharophen)-3-((methotrexate ester) methylene radical)-3-cephem-4-carboxylic acid allyl ester (130mg, 0.15mmol) be suspended in 1: 1: 1 ethyl acetate: methyl alcohol: in the acetate mixture, add triethyl-silicane (0.1ml) afterwards.The gained reaction mixture is stirred 16 hours vacuum concentration then.Enriched material water (15ml) dilution is by adding NaHCO 3PH value of solution is adjusted to 7.Gained solution and ethyl acetate layering are also stirred mixture 1 hour.Filter, be separated, water is with other ethyl acetate extraction, lyophilize then.
Cryodesiccated solid is at C 18Process medium pressure liquid chromatography purifying on the reversed-phase column.This post is first with 15% acetonitrile solution balance.Then lyophilized products is contained on the post, uses earlier 30% acetonitrile: 1% acetic acid aqueous solution (600ml) wash-out; Use 40% acetonitrile then: 1% acetic acid aqueous solution wash-out.The fraction that is contained in the peak of 4.8 minutes wash-outs is merged (Watars C 18μ bondpdk analytical column, with 30% acetonitrile, 1%HOAc, water elution, the 2ml/ branch), lyophilize obtains title product:
NMR:(300MHz, DMSO-d 6): δ 9.0(d, 1, J=9); 8.52(s, 1); 7.7(d, 2, J=10); 7.64(br.s, 2); 7.30(m, 1); 6.88(m, 2); 6.75(d, 2, J=10); 6.55(br.s, 2); 5.42(dd, 1, J=4.5,9); 4.94(d, 1, J=11.6,1/2 ABq); 4.89(d, 1, J=4.5); 4.80(d, 1, J=11.5,1/2 ABq); 4.72(s, 2); 4.06(m, 1); 3.60(s, 2); 3.15(s, 3); 2.1-1.7(m, 4) and .IR:(KBr) 1763,1757,1606,1559cm -1.FABMS: measured value=791.2037
Embodiment 3
7 β-(2-(thiophene-2-yl) kharophen)-and the 3-((N-1-(5-Fluracil) base) methylene radical)-3-cephem-4-carboxylic acid allyl ester
With 7 β-(2-(thiophene-2-yl) kharophen)-3-(acetoxyl group methylene radical)-3-cephem-4-carboxylic acid allyl ester (1.0g) is dissolved in the methylene dichloride (5ml), in solution, add then trimethyl silyl iodine (0.65ml, 5mmol).Dark brown solution was stirred 90 minutes under the room temperature nitrogen atmosphere.Reaction mixture dilutes with ethyl acetate, uses cold 1N Sulfothiorine, saturated sodium bicarbonate aqueous solution and salt water washing then successively.Layering is also used the dried over sodium sulfate organic layer, with its vacuum concentration.
(0.30g 2.3mmol) is dissolved in the dry DMF, then triethylamine (0.40ml, 2.3mmol), above-mentioned 3-(iodo methylene radical) cephem compounds is dissolved among the DMF, and is added in the 5 FU 5 fluorouracil solution with 5 FU 5 fluorouracil.With the solution that obtains at stirring at room 2 hours vacuum concentration then.Enriched material dilutes with cold 1N HCl and ethyl acetate.Tell ethyl acetate layer,, obtain the 0.70g brown solid with dried over mgso and vacuum concentration.It is passed through the flash chromatography on silica gel purifying, with the dichloromethane solution wash-out of 5% methyl alcohol.
NMR:(300MHz, CDCl 3), 10.10(br.s, 1), 7.81(d, 1, J=5); 7.20(m, 1); 7.05(d, 1, J=7); 6.92(m, 2), 6.0-5.8(m, 2); 5.40-5.25(m, 2); 4.95(d, 1, J=5); 4.85(d, 1, J=9); 4.75(d, 2, J=5); 4.41(d, 1, J=9); 3.80(s, 2); 3.45(d, 1, J=12); 3.30(d, 1, J=12) .IR:(chloroform): 1792,1720,1704,1671,1507cm-1; UV:(EtOH); λ Max: 272(ε=14,100), 238(ε=12,900) .FABMS:M+1=507.
Embodiment 4
7-β-(2-(thiophene-2-yl) kharophen)-and the 3-((N-1-(5-Fluracil) base) methylene radical)-3-cephem-4-carboxylic acid
With four [triphenylphosphine] palladium [O] (8mg, 0.006mmol) and triphenylphosphine (3mg 0.01mmol) mixes in 1: 1 methylene dichloride and ethyl acetate mixture (2ml).With 7-β-(2-(thiophene-2-yl) kharophen)-the 3-((N-1-(5-Fluracil) base) methylene radical)-3-cephem-4-carboxylic acid allyl ester (0.112g, 0.221mmol) also be dissolved in 1: 1 methylene dichloride and the ethyl acetate mixture, at room temperature the gained brown solution is added in the solution that contains catalyzer.Adding 2 ethyl hexanoic acid sodium in this solution (0.040g, 0.243mmol).With gained solution stirring 1.5 hours, precipitation separation was by adding acetone earlier in reaction mixture, then mixture being transferred in the centrifuge tube.After centrifugal supernatant liquor is strained, cream-coloured precipitation is washed with ether, and is dry in vacuum desiccator.Beige solid becomes brown in the drying process.Brown solid is water-soluble, with ether washing and lyophilize, obtain the 85mg pale solid.This solid through reversed-phase HPLC (Waters Associates C 18 posts) with 15% acetonitrile/1% acetic acid/water wash-out.Merge the fraction and the lyophilize that contain product, obtain the 15mg title product:
NMR:(DMSO-d 6, 300MHz): δ 9.08(d, 1, J=8.3); 8.24(bd, 1, J=5.8); 7.30(m, 1); 6.88(m, 2); 5.59(dd, 1, J=4.9 and 3.4); 4.97(d, 1, J=4.9); 4.72(d, 1, J=14.6); 4.23(d, 1, J=14.6); 3.68(s, 2); 3.50(d, 1, J=14.6); (by fuzzy bimodal of the water among the DMSO) .IR:(CHCl at other of 3.3 δ places 3) 1782,1695,1666cm -1.UV:(ethanol): λ Max=271nm(ε=10,800), 237(ε=10,200) .FABMS: measured value=467.0489.
Method 3
N-tertbutyloxycarbonyl-2-aminoothyl mercaptan
(9.9g 0.08mol) is suspended in the acetonitrile (about 25ml), adds two (sec.-propyl) amine (14.9ml), in adition process mixture is remained on 0 ℃ with 2-aminoothyl mercaptan hydrochloride under nitrogen atmosphere.0 ℃ keep 10 minutes after, add dimethyl dicarbonate butyl ester (20.0ml), the gained mixture stirred 15 minutes at 0 ℃, then room temperature restir 45 minutes.With the reaction mixture vacuum concentration, add 40: 60 ethyl acetate: hexanes mixtures.The white solid of Sheng Chenging passes through filtering separation like this.Filtrate by 7 o'clock silicagel columns with 40: 60 ethyl acetate: the hexanes mixtures wash-out carries out purification by flash chromatography.Merge the fraction that contains product, vacuum concentration obtains the 15g title product, is colorless oil.NMR：（300MHz，CDCl 3）：4.92（bs，1，NH）;3.28（q，2，J＝6.3）;2.62（q，2，J＝6.9）;1.42（s，9）;1.33（t，1，J＝8.5）。
Method 4
7-β-(2-(thiophene-2-yl) kharophen)-the 3-((N-(t-butoxycarbonyl amino)-the 2-ethyl thioether) methylene radical)-3-cephem-4-carboxylic acid
With 7-β-(2-(thiophene-2-yl) kharophen)-3-(acetoxyl group methylene radical)-3-cephem-4-carboxylic acid sodium (2.0g, 5.05mmol) in distilled water, stir, add potassiumiodide (8g) and N-tertbutyloxycarbonyl-2-aminoothyl mercaptan (0.87g) then.Mixture slowly is heated to 65 ℃, kept 2 hours, kept again 2 hours at 70 ℃ then in this temperature.With the reaction mixture cooling, under agitation in the mixture of impouring frozen water and ethyl acetate.In this mixture, add 1N hydrochloric acid pH is transferred to 2.Collect ethyl acetate layer, use dried over mgso, filter, vacuum concentration obtains the 2g title product, is white solid.
NMR:(300 MHz, DMSO-d 6): δ 9.10(d, NH, J=9); 7.30(m, 1); 6.88(m, 2); 5.59(m, 1); 5.05(d, 1, J=5); 3.71(s, 2); 3.60(m, 4); 3.26(bs, 2); 3.02(m, 2); 2.45(m, 2, with the DMSO signal overlap).
Method 5
7-β-(2-(thiophene-2-yl) kharophen)-the 3-(((1-t-butoxycarbonyl amino)-the 2-ethyl thioether) methylene radical)-3-cephem-4-carboxylic acid allyl ester
With 7-β-(2-(thiophene-2-yl) kharophen)-the 3-(((1-(t-butoxycarbonyl amino)-2-ethyl sulfonyl) methylene radical)-3-cephem-4-carboxylate salt (0.50g, 0.975mmol), allyl bromide 98 (0.25ml, 2.92mmol) and sodium bicarbonate (0.4g) in 5ml DMF, mix, under the room temperature nitrogen atmosphere, stir and spend the night.Reaction mixture dilutes with ethyl acetate and 0.1N hydrochloric acid.Tell organic layer, through dried over mgso, filter, vacuum concentration obtains the 0.5g yellow oil.With this oily matter by 6 inch silicagel columns with 1: 1 ethyl acetate: the hexane wash-out carries out purification by flash chromatography, merges the fraction that contains product, and vacuum concentration obtains the 200mg title product.
NMR：（CDCl 3，300MHz）：δ7.20（m，1）;6.92（m，2）;5.90（m，1）;5.75（dd，1，J＝5，9）;5.30（m，2）;4.98（d，1，J＝5）;4.68（d，2）;3.80（s，2）;3.55（q，2，J＝12）;3.4-3.1（m，4）;2.6（t，2，J＝5）;1.40（s，9）.
IR：（KBr）1771，1756，1714，1680，1670，1531cm-1.FDMS：M+1＝554.
Embodiment 5
7-β-((2-thiophene-2-yl) kharophen)-3-(((1 deacetylate vincaleucoblastine) amino)-and the 2-ethyl thioether) methylene radical)-3-cephem-4-carboxylic acid allyl ester
With 7-β-(2-(thiophene-2-yl) kharophen)-the 3-(((1-(t-butoxycarbonyl amino)-the 2-ethyl thioether) methylene radical)-3-cephem-4-carboxylic acid allyl ester (0.46g, 0.823mmol) be dissolved in the methylene dichloride (5ml), and under nitrogen atmosphere, be cooled to 0 ℃.(pure sample, 2ml), the gained mixture stirred 1 hour to add trifluoroacetic acid.With the reaction mixture vacuum concentration,, use CHCl then with dilution with toluene and vacuum concentration twice 3Dilution and vacuum concentration twice obtain de-protected aminocompound.
De-protected aminocompound is dissolved in methylene dichloride (3ml) and solution is cooled to 0 ℃.(0.3ml) is added in the mixture with N-methylmorpholine, slowly adds the dichloromethane solution (5ml) of deacetylate vincaleucoblastine trinitride (pressing United States Patent (USP) 4,203, the described method preparation in 898 embodiment, 5,20 hurdles) then.The gained mixture stirred 30 minutes at 0 ℃, reaction mixture was concentrated to 1/2 of its original volume, then in stirred overnight at room temperature.The reaction mixture dilute with water, layering, the organic layer dried over sodium sulfate is filtered vacuum concentration.Enriched material is by the flash chromatography on silica gel purifying, with the dichloromethane solution wash-out of 4% methyl alcohol.Merge the fraction that contains product and also concentrate, obtain the 100mg title product.
NMR:(CDCl 3): δ (mixture of △-2 and △-3 isomers) 9.68(s, 1); 9.58(s, 1); 8.60(d, 1, J=9); 8.05(s, 1); 7.55(d, 1, J=9); 7.25-7.10(m, 6); 7.0(m, 2); 6.90(s, 1); 6.60(s, 1); 6.10(s, 1); 6.0-5.6(m, 5), 5.60(s, 1); 5.40-5.3(m, 2); 4.8-4.6(m, 3); 3.86(s, 2); 3.80(s, 3); 3.75(s, 2); 3.62(s, 3); 2.80(s, 3); 0.90(two eclipsed triplets, 6);
Remaining proton is the mixture of 4.0-1.0 multiplet.UV:(ethanol) λ Max265nm(absorbancy=0.6609), 214(absorbancy=1.8358).FABMS：M+1＝1190.4720。
Embodiment 6
7-β-(2-(thiophene-2-yl) kharophen)-and 3-(((1-(deacetylate vincaleucoblastine) amino)-the 2-ethyl thioether) methylene radical)-3-cephem-4-carboxylic acid.
Under nitrogen atmosphere, with four [triphenylphosphine] palladium [O] (5.8mg) and triphenylphosphine (1.8mg) be dissolved in 1: 1 ethyl acetate: in the hexane.In this solution, add 7-β-(2-(thiophene-2-yl) kharophen)-3-(((1-(deacetylate vincaleucoblastine) amino)-the 2-ethyl thioether) methylene radical)-3-cephem-4-carboxylic acid allyl ester (0.20g; 0.168mmol); add then triethyl-silicane (40.3 μ l, d=0.728).Reaction mixture at room temperature stirs.After 1 hour, add Glacial acetic acid (2), with mixture restir 1 hour.In reaction mixture, add ether and make the product precipitation, reaction mixture is centrifugal to collect the cream-coloured precipitation (85mg) that produces.This precipitation, with the 10ml/min wash-out, detects at 254nm with 30% acetonitrile, 0.2% aqueous formic acid with Waters C-18 column chromatography purifying by reversed-phase HPLC.Merge the fraction that contains product and also concentrate, obtain 30mg white solid title product.
NMR:(DMSO-d 6, 300MHz): δ 9.30(s, 1); 9.0(d, 1, J=9); 8.45(m, 1); 8.17(br.s, 1); 7.9-7.6(m, 3); 7.31(m, 2); 7.20(d, 1, J=9); 7.0-6.8(m, 4); 6.39(s, 1); 6.14(s, 1); 5.7-5.5(m, 2); 5.40(dd, 1, J=5,9); 4.92(d, 1, J=5); 3.90(s, 1); 3.70(s, 2); 3.67(s, 3); 3.49(s, 3); 2.67(s, 3); 0.74(t, 3, J=7); 0.68(t, 3, J=7); Remaining proton is several multiplets of 4.9-1.1
IR:(KBr) 1768,1764,1648,1630,1616cm-1; UV:(ethanol) λ Max=267nm(ε=22,900), 215(ε=53,800); FABMS:1150.44435.
Embodiment 7
Antibody (F(ab ')) enzyme conjugates is synthetic
The salt brine solution of isotonic phosphate buffer preparation Sulfo-SMCC(Pierce Chemical Company), its pH 7.2(PBS), the about 35mM of concentration.Using 0.3M Na 2CO 3With afterwards, measure the vacuum concn of this solution in the absorbancy of 260nm measurement aliquots containig before the hydrolysis by using molar extinction coefficient 8000.
According to Cartwright and Waley method (Biochem.J., 221, PP 505-512,1984) purifying derives from Enterobacter cloacae(and is obtained by Sigma Chemical Company) the β-Nei Xiananmei (" β-L ") of P99 bacterial strain, then by 50mM Sodium Tetraborate, 100mM sodium-chlor, pH 8.0(BBS) dialysis and storage.Adding Nonidet P-40(Sigma Chemical Company) after 0.01%, use Centiprep TMConcentrating instrument (Amicon Corporation, Danvers, Massachusetts) will contain the proteic liquor capacity of 20mg and be concentrated to about 10mg/ml, its precise concentrations of absorbance measuring (dullness 1mg/ml=1.4 by 280nm, Cartwright and Waley, Biochem.J., 26, PP.5329-5337(1984)).
The Sulfo-SMCC solution (～35 μ l) that will contain 2.0 molar equivalents is added in β-L solution, uses 0.3M Na 2CO 3PH is transferred to 8.0.The solution of Jiao Baning was not placed 1 hour in room temperature.Make reaction mixture pass through 50ml P6DG(Bio-Rad Laboratories then, Richmond, California) post is with 50mM ammonium citrate, 1mM DTPA, the 100mM NaCl wash-out of pH6.2.
The protein that will comprise spike (detecting at 280nm) is collected in the fraction, measures its concentration by measuring this solution aliquots containig in the absorbancy of 280nm.The maleimide amine content of protein soln is by the excessive halfcystine reaction of aliquots containig and twice (10 minutes, room temperature) measures, detect remaining halfcystine with DTNB: the sulfo-nitrobenzoic acid of release is in molar extinction coefficient=13,600 of 412nm.Under described reaction conditions, the molar average ratio that obtains maleimide/β-L is～0.9.Deutero-protein used within 2 hours.
According to method known in the art and that describe by people such as people such as above-mentioned Breman and Glemmie, by gastric pepsin digestion then with the F(ab ' of halfcystine reduction preparation anti-carcinoembryonic-antigen (CEA) antibody CEM231) part.Measure free mercaptan/F(ab ' by the aliquots containig of measuring protein soln in the absorbancy (dullness of 1mg/ml solution=1.4) of 280nm) ratio, and measure concentrations of mercaptans with DTNB.General mercaptan/F(ab ') average specific is in 2.0 to 2.5 scopes.
Make the β-L and the CEM231F(ab ' of the maleimide of deriving of molar equivalent) each with the concentration of 1-5mg/ml at 50mM ammonium citrate, 1mM DTPA, 100mM NaCl(pH6.2) in stirring reaction 1 hour not at room temperature.Reaction adds excessive N-ethyl maleimide when finishing stop reaction.
Then reaction mixture directly is added in the 500ml Sephadex G-150 Superfine post, uses the BBS buffer solution elution.Determine the product fraction with PAGE, thereby obtain the pure substance of molecular weight～90,000.Then the product of mixing fraction is concentrated to～1.5mg/mL, sterile filtration is stored in the bottle of aseptic barrier film covering.
Method 6
The mensuration of the enzymic activity of β-Nei Xiananmei
Lower part A shows, the quantitative assay of enzymic activity is with cepovenin (Sizma Chemical Company) or PADAC(Cal-Biochem) to make substrate, in the Hewlett-Packard 8451A of the thermostatic container carriage that stirring is housed spectrophotometer, respectively 264 or 570nm carry out under detecting.In general test, with the experimental concentration that under significant wavelength, is enough to provide the 0.5-1.0 absorbancy substrate is added to and suitably is being generally 7.2 at suitable PH(in the damping fluid (PBS, HEPES etc.)) be equilibrated in 37 ℃ the reference solution of stirring of 5nM P-9.9E.cloacae β-Nei Xiananmei (" β-L ").(β-L enzyme (also being " penicillinase ") can buy (for example Sigma Chemical Company) from the market, and can be obtained by literature method.）。Serve as at interval with the variation of spectrophotometric determination absorbancy with 5 seconds or longer time then.
Part B has illustrated the mensuration of β-L-CEM231 binding substances to the enzymic activity of the compound of embodiment 11).
Part A
K to β-L enzyme and binding substances MAnd V MaxMensuration
The preparation of cepovenin storage liquid (13mM) is that the 5.5mg cepovenin is dissolved in 1.0ml 10mM sodium phosphate, 150mM sodium-chlor, pH7.2(PBS) in.Pass through A 280The storage liquid of measuring P-99 E.cloacae β-Nei Xiananmei (" β-L ") is 1.3 μ M β-L solution, 7.7 these solution of μ L is added in the cuvette that contains PBS stirring, equilibrium temperature, so that final volume reaches 2.0ml.With the spectrophotometric meter calibrating.Adding substrate storage liquid then makes experimental concentration reach 5 to 130 μ M.Make spectrophotometer wait for for 5 seconds for substrate mixing first, write down absorbancy 2 minutes per 10 seconds at 264nm then.
The result is converted into absorbancy by initial value changes, and (BBN Software Products Company, Cambridge Massachusetts) map with the RS/3 program.Inspection to figure shows that then initial rate is identical greater than 50 μ M for concentration of substrate, and initial rate can be by the estimation of the multiple before 25 seconds.(gather the absorbancy reading per 5 seconds one time in subsequent measurements.) △-OD value after 20 seconds that will react is converted to △-[substrate]/second, and at double reciprocal plot: 1/V is to 1/[S] go up and be associated with concentration of substrate (wherein V=△ [substrate]/second), therefrom with its limit of error of being correlated with mensuration K MAnd V MaxAccording to relational expression: Kcat=V Max/ [E] calculating K cat(by 5 second the 264nm of time point place absorbancy, calculate concentration of substrate with the molar extinction coefficient 8,540 of cepovenin.）
Other two groups of β-L-CEM231 binding substances is similarly tested.To β-L and two groups of binding substances K MValue is respectively 4.8+/-0.1,4.4+/0.3 and 5.2+/-0.4 μ M; Kcat is respectively 54+/-0.7,35+/-1.4 and 49+/-2.8 second -1
Part B
To cultivate at 37 ℃ of compounds at the 1.5ml PBS damping fluid (pH7.4) in the container that stirred with the embodiment 11 of different concns.In each situation, be that β-Nei Xiananmei-CEM231 binding substances of 0.11nM makes the reaction beginning, and make it be incubated for 90 seconds by adding concentration.To 0.5ml by 34% acetonitrile and 200mM KH 2PO 4Damping fluid (is used H 3PO 4Adjusting pH3) adds the 0.5ml reaction mixture in the solution of forming, make the sample quenching.
The reduction of concentration is to measure by being averaged at the last HPLC injection liquid to two parts of multiple quenching samples of 0.46 * 15cm C-18 reversed-phase column (detecting at 266nm) before the cynnematin.Flow rate is per minute 1ml, carries out the isoconcentration wash-out with above-mentioned 34% acetonitrile damping fluid.
The data of gathering
Minute μ M prodrug μ M/min 1/C 1/V
1
2
3
4
5 6.960 0.482 0.144 2.037
6 8.000 0.563 1.125 1.776
7 8.700 0.592 1.115 1.689
8 10.000 0.691 0.100 1.448
9 10.440 0.751 0.096 1.331
10 12.000 0.858 0.083 1.166
11 12.180 0.888 0.082 1.127
12 13.920 0.879 0.072 1.138
13 14.000 0.898 0.071 1.113
14 15.660 1.020 0.064 0.980
15 16.000 1.069 0.062 0.935
16 17.400 1.120 0.057 0.892
17 18.000 1.238 0.056 0.808
18 20.000 1.152 0.050 0.868
19 25.000 1.481 0.040 0.675
20 26.100 1.756 0.038 0.569
21 30.000 1.687 0.033 0.593
22 34.800 2.358 0.029 0.424
23 40.000 2.191 0.025 0.456
24 43.500 2.824 0.023 0.354
25 50.000 2.950 0.020 0.339
k m＝155±42μM
k cat＝1721±467sec-1
k Cat/ k m=11.1 ± 42sec -1μ M -1Compound to embodiment 11
Method 7
Immunoreactive experiment in conjunction with the CEM231 of β-Nei Xiananmei
Iodization: at room temperature at Biorad Enzymobeads(BioRad Laboratories) and 25 μ l, 1% β-D-glucose in the presence of, in 0.2M sodium phosphate (pH7.1), with 200 μ Ci 125I is handled the 10 μ g binding substancess 20 minutes of 1 μ g/ μ L.Mixture 50 μ l PBS solution (pH7.2) quenchings that contain 0.1% sodiumazide and 0.35mg/mL Sodium Pyrosulfite.Add carrier with the 100 μ l PBS solution forms that contain 0.1% sodiumazide, 0.1% gelatin and 1% sodium iodide.
Using purified mixture on PBS, 0.1% sodiumazide, the 0.1% gelatin solution equilibrated 5mL Sephadex G-25 post, with the PBS(pH7.5 that contains 0.1% sodiumazide) with the 0.5ml fraction it is eluted in the pipe that contains 100 μ l, 0.1% gelatin solution.With ISODATA 20/20 Series Gamma Counter (RIA Data Systems, Rclling Meadows Illinois) carries out radiocounting to fraction, radioactive first peak is the binding substances of mark.
Immunoreactivity:
With carcinomebryonic antigen (CEA) or with irrelevant albumen (be used for non-specific binding in the same old way) preparation polystyrene bead, be to be that 10% horse serum of every pearl 150ng immerses polystyrene bead in the desirable proteins solution with concentration.Iodinating binding substances is diluted to about 200cpm/ μ l 10% horse serum.The pearl of per three antigen positives and antigen negative is placed in the independent gamma ray counting tube, and washing is handled with the binding substances of 200 μ l marks.With pearl 37 ℃ of incubated overnight.Measure the grand total in each pipe; Supernatant liquor is transferred in the pipe of second pearl that contains same type.At last, with all pearl washings, measure the fraction of counting on each pearl.Be calculated as follows immunoreactivity: A ++ B +(1-A +), be calculated as follows non-specific binding: A -+ B -(1-A -), A wherein +And B +Be to be connected in fraction on the antigen positive pearl, A in first and second circulations respectively -And B -Be to be connected in fraction on the antigen negative pearl in first and second circulations respectively.
The immunoreactivity of the β that records-L-CEM231 binding substances is about 80%, and this is the F(ab ' with unmodified CEM231) the part income value compares and obtains.Non-specific binding does not have owing to increasing with combining of β-Nei Xiananmei yet.
Method 8
By handle with β-Nei Xiananmei by 3 of cynnematin '-methylene radical discharges substituent experiment
Discharging substituting group (cytotoxic agents) by cynnematin 3 ' position can illustrate by color atlas (particularly HPLC) analysis to reaction mixture, perhaps illustrates by the spectrophotometric analysis to d/d functional group.If (detection method is not instantaneous, by inhibition such as the Cloxacillin Sodium (Sigma Chemical Company) (is 5 μ M to its effective concentration of Cloxacillin Sodium) that adds effective concentration enzyme is failed.
For example, the model system of the inventive method adopts by 7-β-(2-(thiophene-2-yl) kharophen)-3-((2-amino-ethyl-1-thioether) methylene radical)-3-cephem-4-carboxylic acid deaminize sulfur alcohol.Carry out common enzyme analysis (pressing the 264nm absorbancy), but, under pH8.0, carry out in the 100mM NaCl damping fluid at 50mM Tris.The mercaptan that quantitative assay discharges in follow-up test, this is by with 250mM DTNB(disulfide group nitrobenzoic acid) be included in the reaction mixture, and the absorbancy of observation 412nm changes.With the molar extinction coefficient 8,540 of 264nm absorbancy and the molar extinction coefficient 13,600 of 412nm absorbancy, can compare the speed (reaction of mercaptan and DTNB is instantaneous under these conditions) of two reactions.Result's figure line shows, because the cracking of beta-lactam enzyme catalysis cynnematin discharges mercaptan, but rate of release is slower than catalytic rate.
In order to illustrate by 7-β-(2-(thiophene-2-yl) kharophen)-the 3-((N-1-(5-Fluracil) base) methylene radical)-3-cephem-4-carboxylic acid (Ceph-5-FU) sloughs 5 FU 5 fluorouracil, (the 1mg cynnematin is at 2ml 20%N for preparation cynnematin storage liquid, in N-N,N-DIMETHYLACETAMIDE and the pH7.0HEPES damping fluid, ultimate density 500 μ g/ml).The storage liquid of preparation P-99E.cloacae β-Nei Xiananmei.In the storage liquid (90 μ l) of cynnematin substrate, add beta-lactam enzyme solution storage liquid (10 μ l).By HPLC(on Waters AssociatesC18 μ bondapak post) detection reaction, with 25% acetonitrile/1% acetic acid aqueous solution wash-out (detecting absorbancy) at 254nm.The retention time of 5 FU 5 fluorouracil is 1.9 minutes, and the retention time of cynnematin-(5 FU 5 fluorouracil) compound is 5.3 minutes.After mixing about 30 seconds, the aliquots containig of 5 μ l reaction mixtures is expelled to HPLC.The HPLC spike shows that cynnematin is consumed, and retention time to have occurred be 4.3 minutes new compound, and a small amount of retention time is 1.9 minutes 5 FU 5 fluorouracil.The mixing of two kinds of storage liquid after 21 minutes, in the compound minimizing of 4.3 minutes wash-outs, and is observed a large amount of compounds at 1.9 minutes wash-outs.To store liquid and mix after 36 minutes, the integration by peak area shows that the amount at the compound of 4.3 minutes wash-outs is further reduced, and correspondingly is increased in the amount of the compound of 1.9 minutes wash-outs.
Therefore, enzyme causes that cynnematin-(5 FU 5 fluorouracil) substrate-cytotoxic agents is converted into cytotoxic agents.
Method 9
The tumor-localizing of iodate β-L-CEM231 binding substances
As described, use immunoassay 125I mark binding substances.Get 12 bare mouse, in mouse, the LS174T tumour cell is carried out subcutaneous injection, and make growth of tumour cell, be injected at 50 μ g mark binding substancess among the 100 μ l BBS to the tail vein of these mouse again to about 0.4g size.Respectively 6 mouse were killed, dissect at 4 and 24 hours, and measure the content of radioactive substance in each organ.The result is with percentage ratio (%) expression of injected dose/gm tissue.
The result:
Blood bone heart kidney liver lung muscle skin splenic tumor GI
4 hours
15.8 2.4 4.3 7.4 3.7 8.1 1.3 3.8 4.2 13.9 3.1
24 hours
1.5 0.2 0.4 0.7 0.6 1.0 0.1 0.8 0.4 7.5 0.7
These results show that binding substances determines preferentially to accumulate in the tumour, and do not have unsuitable accumulation in critical organ.The result also shows and binding substances can be removed rapidly from serum, and this point is highly significant from substrate-cytotoxic agents to the viewpoint that the cytotoxic agents that is specific to a certain object changes.These results and similar from the observed result of mouse hybrid antibody of approximately same molecular weight, and do not demonstrate the proteinic improper metabolism therapy of carrying disease germs.
Method 10
Enzymatic determination with antigen presentation cell bonded binding substances
In order to show that the beta-lactam enzymic activity can be attached on the tumour cell by the β-L-CEM231 binding substances of preparation, at Autopow medium (GIBCO, Grand Island, New York) prepared the single-cell suspension liquid that CEA expresses LS174T cell and the negative MOLT4 cell of CEA in.The density of two kinds of suspension is 2 * 10 7Cell/ml; The LS174T surviving rate is 23%, and the MOLT4 surviving rate is 80%.Suspension is placed in the ice standby.
With 10 or 50pmol binding substances or unconjugated β-Nei Xiananmei culturing cell (1ml) 1 or 10 minute.Eccentric cell then, abandoning supernatant makes it be suspended in the damping fluid recentrifuge again, make cell suspension contain PADAC(Cal-Biochem then) the 1.0ml damping fluid in, PADAC(spectrophotometry in the damping fluid) be about the known concentration of 30 μ M.After 10 minutes, eccentric cell is measured PADAC concentration in the supernatant liquor with substrate cultivation.The result is to cultivate percentage ratio (%) expression of the remaining original specific absorption in back (the absorbing wavelength 570nm place at the PADAC peak).
The result:
The conjugate content binding time is cultivated the remaining %OD 570 in back
pmol/ml min
The LS174T cell
B-LCEM231 10 10 46
B-LCEM231 50 10 23
B-L 10 10 95
B-L 50 10 87
B-LCEM231 10 1 62
B-LCEM231 50 1 36
The MOLT4 cell
B-LCEM231 10 10 94
B-LCEM231 50 10 93
These results show that the beta-lactam enzymic activity can particular form be combined on the antigen-positive cell, and cell does not have such activity when having binding substances, in conjunction with the catalytic activity that does not hinder enzyme, and in conjunction with changing with the variation of concentration in time.
Method 11
The dynamic (dynamical) PADAC of the serum of β-L-CEM231 binding substances measures
Will be at 50mM Sodium Tetraborate, 100mM NaCl(BBS) in β-L CEM231 binding substances (50 μ g) inject every (totally 18) not with the tail vein of the bare mouse of tumour.1,24,48,72,96 and 120 hour is three mouse bloodletting at every turn after injection, and the serum of collecting them is also freezing.Adopt standard test condition to measure the β-L activity of serum sample.The serum of proper amt is diluted to 2.0ml with PBS, and also will carries out the variation of necessity for the detection of active minute.The 20ml EtOH solution that uses PADAC(dissolving 2.4mg/ml to make), thereby avoid interference from serum protein as substrate.
By observing the linear portion of definite specific absorption-time curve, adopt linear regression (RS/3, BBN software product company) to use these points to match.The slope that obtains from the binding substances of concentration known under these collinear slopes and the same terms is compared, and dilution is proofreaied and correct, thereby calculate the binding substances concentration in the serum.
The result:
The serum μ l μ g β-LCEM231/mL serum of time-sloped mensuration
hr x10 4
1 27.0 10 20
24 13.3 100 1.0
48 2.2 50 0.33
72 4.3 200 0.16
96 5.3 600 0.06
120 5.2 600 0.06
These results show do not have the beta-lactam enzymic activity in the normal serums of these mouse, and the conjugate content in the serum descends very fast, so substrate-cytotoxic agents treatment can begin after the time (48 hours) quite short behind injection (in bare mouse) binding substances.When having enough fast serum clearance rate among the mankind, also can obtain similar experimental result.To compare by the content of enzyme method conjugate content of determining and the iodate binding substances of determining by the radiation detection method (after injection, measuring in 4 and 24 hours), find that enzyme can not be inactivated or suppress (referring to method 9) in serum.
Method 12
Be combined in the beta-lactam enzymic activity on the antigen presentation tumor tissues in vivo
LS174T or MOLT4 cell are implanted bare mouse, make these cells grow up to the tumour of 0.5～1.0gm, the 50 μ g β-L-CEM231 binding substancess that will dilute in 100 μ l BBS inject the tail vein of these mouse then.Behind the injection twenty four hours, tumor resection and is cut into～block of 100mg.
With the tumor mass chopping, use 1ml PBS water culture 10 minutes then, in this 1ml PBS solution, added 10 μ l by 1mg PADAC being dissolved in the stock solution that 400 μ l 100%EtOH make before cultivating.The accurate concentration spectrophotometer method (ε of PADAC in test soln 570nm=48,000) determines.(10 minutes, 1000xg), supernatant liquid detected with spectrophotometer centrifugal then sample.Because supernatant liquid is still very muddy, can not quantitative assay, but in the LS174T tumor sample, clearly observe colour-change from purplish red to orange.With Photographic technique outcome record is got off.In taking from the LS174T tumor biopsy of two different bare mouse, obtained same positive result, and obtained negative test from the MOLT4 tumor biopsy of taking from two different bare mouse.
Method 13
Vitro cytotoxicity is measured
Target cell (antigen positive or antigen negative) is suspended in 75% with the concentration of 200,000 cells lacks leucine EBSS-MEM(GIBCO)+foetal calf serum (the FBS)+gentamicin (GIBCO) of 10% dialysis in.The aliquots containig 0.2ml of this cell suspending liquid inoculation in the dish of 96 caves, and at 37 ℃, 5%CO 2Following overnight incubation.Add antibody-enzyme conjugates in supernatant liquor, adding to ultimate density is that the unconjugated enzyme of 25 μ g/ml(, unconjugated antibody are not considered).Cultivate and take out supernatant liquor after 1 hour, and with these cells of medium rinsing once.Replace original medium with the same media that contains substrate-cytotoxic agents then, the concentration of substrate-cytotoxic agents in medium is between 0.001 and 10 μ g/ml.At 37 ℃, 5%CO 2Under the condition with substrate-cytotoxic agents compound culturing cell 3-48 hour.Cultivate adding 4 μ Ci in the cave to every 3The leucic medium of H-.Leucine pair cell with mark was cultivated 24 hours, gathered then.By the absorbed labelled leucine of liquid flashing counting measuring.(all sample person replications three times.) result drops to the 50%(ID of correlative value with the leucine that is introduced into 50) time substrate-cytotoxic agents concentration represent.
An example as above-mentioned general method; measured 7-β-(2-(thiophene-2-yl) kharophen)-3-((((1-(deacetylate vincaleucoblastine) amino)-the 2-ethyl thioether) methylene radical)-3-cephem-4-carboxylic acid (" compound ") is to the LS174T(ATCC of band CEA) cytotoxicity of tumour cell; this tumour cell can use β-L-CEM231 binding substances (" binding substances ") to handle, and maybe handles without this binding substances.So, at 37 ℃, 5%CO 2Under the condition, suitable cultivation cave is exposed to the β that concentration is 25 μ g/ml-L-CEM231 binding substances reaches 1 hour.Rinsing cell then, and it is suspended in the medium that contains above-claimed cpd.24 hours this constantly, wash these suitable cultivation caves, and add the 0.2ml fresh medium finally to finish 48 hours cultivation.Adopt above-mentioned standard method to carry out remaining experiment.
This result of experiment is as follows:
The compound treatment time:
With following mass treatment cell 1: 6 hours 48 hours
1) compound and binding substances: 0.265 0.001
2) has only compound: 2.154＜0.001
3) has only cytotoxic agents 2: 0.260＜0.001
1These numerical value are ID 50(μ g/ml)
2Deacetylate vincaleucoblastine aminoothyl mercaptan
These data show, well below the toxicity of cytotoxic agents (from then on compound derives) to tumour cell, but when cell was handled with binding substances earlier, the toxicity of compound had increased compound significantly to the toxicity of tumour cell.
Method 14
7-β-(2-(phenoxy group) kharophen)-and 3-((p-nitrophenyl carbonate group) methylene radical)-3-cephem-4-carboxylic acid tertiary butyl ester
7-β-(2-(phenoxy group kharophen)-3-(hydroxyl methylene radical)-(0.50g 1.15mmol) is dissolved in dry tetrahydrofuran (5ml) to 3-cephem-4-carboxylic acid tertiary butyl ester, and the solution that obtains is cooled to 0 ℃ under nitrogen atmosphere.(0.35g, 1.72mmol), (DMAP 2mg), slowly adds 2 at last, 6-lutidine (0.20ml) to add Dimethylamino pyridine then to add (p-nitrophenyl) chloro-formic ester in this cooling solution.The mixture that obtains has produced a large amount of precipitations; This mixture is placed in ice bath to spend the night.Add some THF again, and precipitate with spatula is broken.Filter this mixture, on silica gel, carry out purification by flash chromatography then, obtain 0.40g title product: NMR with 1: 1 mixture wash-out of methylene dichloride and ethyl acetate:
（CDCl 3，DMSO-d 6300MHZ）;δ8.01（d，2，J＝8.9）;7.75（d，1，J＝10.2）;7.25-7.00（m，3）;6.80-6.62（m，4）;5.86（dd，1，J＝4.9，10.2）;5.28（d，1，J＝13.3）;4.67（d，1，J＝13.3）;4.56（d，1，J＝4.6）;4.33（s，2）;3.65（d，1，J＝18.6）;3.32（d，1，J＝18.5）;1.30（s，9）;IR：（CHCl 3）3020，1807，1771，1725，1696cm -1;UV：（EtOH）， λ max392（ε＝1100），268（ε＝13，500）;FABMS：（M+H）602，546，363.
Embodiment 8
7-β-(2-(phenoxy group) kharophen)-and 3-((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen methylene)-3-cephem-4-carboxylic acid tertiary butyl ester
Deacetylate vincaleucoblastine hydrazides vitriol (900mg) is dissolved in sodium bicarbonate aqueous solution, and then has just made the free alkali of above-mentioned vitriol with this solution of dichloromethane extraction.Organic phase obtains white solid through dried over sodium sulfate, filtration and concentrating under reduced pressure.This free alkali is dissolved in dry pyridine (6ml), and is added to 7-β-(2-(phenoxy group) kharophen)-the 3-(O-(p-nitrophenyl) carbonate group) methylene radical-3-cephem-4-carboxylic acid tertiary butyl ester (0.40g, 0.666mmol) in.Flask after reinforced washs with another part (2ml) pyridine, and washing soln is added in the reaction mixture.With small amount of N, N-diisopropylethylamine (2) adds reaction mixture, and the mixture that obtains stirs under nitrogen and room temperature and spends the night.Reaction mixture dilutes with ethyl acetate, then concentrating under reduced pressure.Enriched material carries out purification by flash chromatography on silica gel, obtain the 0.54g title product with 90/10 methylene chloride wash-out:
NMR:(CDCl 3, 300mHz): δ 10.0(s, 1); 8.70(s, 1); 8.02(s, 1); 7.93(d, 1, J=12); 7.50(d, 1, J=12), 7.38-6.90(m, 10); 6.10(s) and 6.12(m, 1) overlapping; 5.80(d, 1, J=12); 5.65(d, 1, J=12); 4.38(s, 2); 4.02(d, 1, J=12); 3.75(s, 3); 3.58(s, 3); 2.8(brs, 3); 1.52(s, 9); 0.90(brs, 6); Remaining is the multiplet of 4.0-1.1ppm scope;
IR:(CHCl 3) 1802,1721,1696,1515cm -1; UV:(EtOH), λ Max=267(ε=23,800), 214(ε=53,000); FABMS: calculated value .(M+H)=1231.53853. measured value=1231.54230.
Embodiment 9
7-β-(2-(phenoxy group) kharophen)-and 3-((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen methylene)-3-cephem-4-carboxylic acid trifluoroacetate
With 7-β-(2-(phenoxy group) kharophen)-3-((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen methylene)-3-cephem-4-carboxylic acid tertiary butyl ester (123mg; 0.1mmol) be dissolved in methylene dichloride (3ml); this solution is cooled to 0 ℃; add triethyl silicane (1ml), add trifluoroacetic acid (1ml) again.The solution stirring that obtains 30 minutes removes ice bath to its triethyl silicane and trifluoroacetic acid that adds same volume again, and reaction mixture at room temperature stirred 3 hours.Mixture under reduced pressure concentrates, and with cold acetonitrile (3 times of volumes) dilution, under reduced pressure concentrates again.Add tetracol phenixin, mixture concentrating under reduced pressure once more obtains the 120mg title product:
NMR:(DMSO-d 6, 300MHz): δ 9.76(br.s, 1); 9.46(br.s, 1); 9.41(br.s, 1); 8.14(d, 1, J=9.3); 7.47(d, 1, J=7.9Hz); 7.35-7.21(m, 4); 7.18-6.80(m, 6); 6.70(s, 1); 6.30(s, 1); 6.00(dd, 1, J=4.8,9.5); 5.75(s, 2); 5.15(d, 1, J=12); 4.92(d, 1, J=4.8); 4.6(s, overlapping with the broad peak of water);
3.72(s, 3), 3.53(s, 3); 2.79(br.s, 3); 0.82(t, 3, J=7); 0.70(t, 3, J=7); Remaining is the multiplet of 4.0-1.1ppm scope; IR:(KBr) 1788,1720,1685,1677cm -1; UV:(EtOH), λ Max=311(ε=3800), 268(ε=18,500), 215(ε=45,100); FABMS: calculated value (M+H)=1175.47593. measured value=1175.47600.
Method 15
7-β-(2-(thiophene-2-yl) kharophen)-the 3-(methylol)-2-cephem-4-carboxylic acid benzhydryl ester
Press Kukolja S., J.Med.Chem., P.1114, the 1970 described 7-β that obtain-(2-(thiophene-2-yl) kharophen)-the 3-(methylol)-(5.0g 14.1mmol) is dissolved in 1/1 mixture of acetone and acetonitrile 2-cephem-4-carboxylic acid.Drip diphenyl diazomethane (2.7g, acetonitrile solution 13.9mmol) to this solution.The reaction mixture that obtains at room temperature stirred 1 hour, then concentrating under reduced pressure.Acetonitrile is added in the enriched material, collects the beige solid shape title product (4.4g) that obtains by filtering:
NMR:(CDCl 3, 300MHz), 7.40-7.23(m, 11); 7.02-6.95(m, 2); 6.88(s, 1); 6.45(d, 1, J=7.6); 6.25(s, 1); 5.61(dd, 1, J=5,8); 5.20(d, 1, J=5); 5.15(s, 1); 4.10(q, 2, J=8); 3.82(s, 2); IR:(CHCl 3) 1778cm -1; 1743,1683; EA: calculated value C=62.29, H=4.64, N=5.38. measured value: C=62.07, H=4.73, N=5.51.
Method 16
7-β-(2-(thiophene-2-yl) kharophen)-3-(hydroxyl methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid benzhydryl ester
With 7-β-(2-(thiophene-2-yl) kharophen)-3-(hydroxyl methylene radical)-(2.0g 3.85mmol) is dissolved in 4/1 mixture (125ml) of methylene dichloride/Virahol 2-cephem-4-carboxylic acid benzhydryl ester, and the solution that obtains is cooled to-10 ℃.The metachloroperbenzoic acid (1.21g) that will be dissolved in the Virahol (25ml) was added drop-wise in the above-mentioned cooling solution in the clock time at 30 minutes.The reaction mixture vaporising under vacuum that obtains adds benzene to doing, and makes the mixture vaporising under vacuum to doing again.Add ethyl acetate in solid, filter and collect brown solid, drying obtains 1.5g title product: NMR:(300MHz, CDCl 3+ DMSO-d 6): δ 7.4-7.0(m, 11); 6.80(m, 2); 6.70(s, 1); 5.82(dd, 1, J=4.6,9.4); 4.42(d, 1, J=4.7); 4.37(d, 1, J=15.0); 4.18(d, 1, J=15.0); 3.84(d, 1, J=19.2); 3.67(s, 2), 3.22(d, 1, overlapping with water; (not identifying OH and NH); UV:(EtOH), λ Max=258nm(ε=6540); IR:(KBr) 1654cm -1, 1721,1755,1773,1785; EA: theoretical value C=60.43, H=4.51, N=5.22. measured value C 60.57, H 4.37, and N 5.01; FABMS:M+H=537.
Method 17
7-β-(2-(thiophene-2-yl) kharophen)-and the 3-(O-(p-nitrophenyl) carbonate group) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid benzhydryl ester
7-β-(2-(thiophene-2-yl) kharophen)-the 3-(methylol)-(1.0g 1.87mmol) is partly dissolved in the dry THF (20ml) 3-cephem-1-β-sulfoxide-4-carboxylic acid benzhydryl ester, under nitrogen this mixture is cooled to 0 ℃.(0.49g 2.43mmol), adds Dimethylamino pyridine (5mg) again, adds 2 at last, 6-lutidine (0.28ml) to add (p-nitrophenyl) chloro-formic ester earlier to this cooling solution.The solution that obtains stirred 30 minutes down at 0 ℃, again stirring at room 1 hour.Filtering mixt, the solid that leaches washs with ethyl acetate, and filtrate decompression concentrates.The solid that obtains carries out purification by flash chromatography on silica gel, obtain the colourless title product of 0.32g: NMR with 60/40 mixture wash-out of dichloromethane/ethyl acetate:
(300MHz, DMSO-d 6): δ 8.49(d, 1, J=8.3Hz); 8.27(d, 2, J=9.2); 7.5-7.2(m, 13); 6.92(m, 3); 5.94(dd, 1, J=4.9,8.2); 5.25(d, 1, J=13.0); 4.93(d, 1, J=4.4); 4.85(d, 1, J=13.0); 4.04(d, 1, J=18.7); 3.88(d, 1, J=15.3); 3.78(d, 1, J=15.3); 3.66(d, 1, J=18.7); UV:(EtOH) 265nm(ε=3570); IR:(KBr) 1720cm -1, 1765,1658; EA: theoretical value C=58.20, H=3.88, N=5.99. measured value: C=58.48, H=4.02, N=5.90.
Embodiment 10
7-β-(2-(thiophene-2-yl) kharophen)-and 3-((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen base) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid benzhydryl ester
Press the free alkali of the foregoing description 8 described preparation deacetylate vincaleucoblastine hydrazides vitriol (0.50g).This free alkali is dissolved in dry pyridine (10ml), and be added to 7-β-(2-(thiophene-2-yl) kharophen)-the 3-(O-(p-nitrophenyl) carbonate group) methylene radical)-3-cephem-1-sulfoxide-4-carboxylic acid benzhydryl ester (0.32g, 0.456mmol) in.Add N, behind the N-diisopropylethylamine (2), in nitrogen atmosphere and this mixture of stirring at room.The reaction mixture that obtains stirs under nitrogen and room temperature and spends the night, then with the ethyl acetate dilution, and concentrating under reduced pressure.The solid that obtains like this carries out purification by flash chromatography on silica gel, obtain 0.34g pale solid shape title product: NMR with 90/10 mixture wash-out of methylene chloride:
(CDCl 3, 300MHz): δ 9.91(s, 1); 8.66(br.s, 1); 8.01(s, 1); 7.53-6.86(m, 23); 6.55(s, 1); 6.08(s, 1); 6.05(dd, 1, J=4.7,9.8); 5.8-5.6(m, 2); 5.3-5.2(m, 1); 4.85(br.s, 1); 4.47(d, 1, J=4.4); 3.83(s, 2); 3.74(s, 3); 3.57(s, 3); 0.87(t, 6, J=7.2); Remaining is the multiplet of covering of 4.1-1.1ppm scope; UV:(EtOH), λ Max=268nm(ε=25200); IR:(CHCl 3) 1690cm -1, 1730,1803; FABMS: calculated value (M+H) C 71H 79N 8O 14S 2=1331.51569. measured value=1331.51224.
Embodiment 11
7-β-(2-(thiophene-2-yl) kharophen)-and 3-((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen base) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid trifluoroacetate
7-β-(2-(thiophene-2-yl) kharophen)-and 3-((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen base) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid diphenyl methyl ester (105mg; 0.00789mmol) be dissolved in methylene dichloride (3ml); this solution is cooled to 0 ℃ under nitrogen; add triethyl silicane (0.5ml) to solution then, add trifluoroacetic acid (0.5ml) again.After 15 minutes, solution is with dilution in acetonitrile and concentrating under reduced pressure, once more with dilution in acetonitrile and concentrating under reduced pressure.The solid that obtains is dissolved in chloroform earlier, is dissolved in ether again, all carries out concentrating under reduced pressure after each dissolving.The solid that obtains in a vacuum with room temperature under drying obtain the 100mg title product:
NMR：（300MHz，DMSOd 6）：δ9.76（s，1）;9.46（s，1）;9.40（s，1）;8.43（d，1，J＝8.1）;7.47（d，1，J＝8.0），7.4-6.9（m，16）;6.68（s，1）;6.28（s，1）;5.80（dd，1，J＝5.8）;
With 5.75(s, 1) overlapping; ; 5.16(d, 1, J=12.7); 4.85(m, 1); 4.64(d, 1, J=13.2); 3.88(s, 2); 3.78(s, 2); 3.71(s, 3); 3.53(s, 3); 2.78(br.s, 3); 0.80(t, 3, J=7); 0.70(t, 3, J=7); Remaining is the multiplet UV:(EtOH of 4.3-1.0ppm scope), λ Max=266nm(ε=19,300), 214(ε=49,800); FABMS: calculated value (M+H) C 58H 69N 8O 14S 2=1165.43744.
Measured value=1165.43290.
Embodiment 12
7-β-(2-(thiophene-2-yl) kharophen)-and 3-(((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen base) methylene radical)-3-cephem-4-carboxylic acid diphenyl methyl ester
With 7-β-(2-(thiophene-2-yl) kharophen)-3-(((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen base) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid benzhydryl ester (0.24g; 0.180mmol) and tin protochloride (0.10g; 0.451mmol) be dissolved in dimethyl formamide (DMF; 3ml); this solution cools off with ice bath, and slowly adds Acetyl Chloride 98Min. (0.32ml) with syringe to it.The reaction mixture that obtains stirred 2 minutes under the ice bath cooling, at room temperature stirred 20 minutes again.In reaction mixture impouring cold water (20ml), the pH value of water is adjusted to 8 with cold sodium bicarbonate aqueous solution.The mixture ethyl acetate extraction that obtains, the organic phase dried over sodium sulfate is filtered, and reduction vaporization is to doing.The solid that obtains carries out purification by flash chromatography on silica gel, obtain the 47mg title product with 90/10 mixture wash-out of methylene chloride:
NMR：（300MHz，CDCl 3）：δ10.30（s，1）;9.90（s，1）;9.20（s，1）;8.70（s，1）;8.02（d，2，J＝8Hz）;7.6-6.8（m，25）;6.58（br.s，1）;6.05（s，1）;5.83（dd，1，J＝5.8）;5.8-5.7（m，2）;5.12（d，1，J＝13）;4.92（d，1，J＝5）;4.10（d，1，J＝8）;3.78（s，2）;3.72（s，3）;3.55（s，3）;2.77（s，3）;0.88（t，6，J＝7）;
Remaining is the multiplet .FABMS that covers of 4.0-1.1ppm scope: calculated value C 71H 79N 8O 13S 2=1315.52077. measured value=1315.51781.
Embodiment 13
7-β-(2-(thiophene-2-yl) kharophen)-and 3-(((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen base) methylene radical)-3-cephem-4-carboxylic acid, trifluoroacetate
7-β-(2-(thiophene-2-yl) kharophen)-and 3-(((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen base) methylene radical)-3-cephem-4-carboxylic acid benzhydryl ester (46mg; 0.0035mmol) be dissolved in methylene dichloride (2ml); the solution that obtains is cooled to 0 ℃ under nitrogen; add triethyl silicane (0.5ml) to this solution, and then add trifluoroacetic acid (0.5ml).The reaction mixture that obtains stirred 30 minutes with 0 ℃ under nitrogen, added cold acetonitrile to it, and made the solution vaporising under vacuum to doing.Repeat this adding acetonitrile, be evaporated to dried step, make solid be dissolved in ether then, and vaporising under vacuum is to doing, remaining pale solid (the heavy 42mg in dry back), i.e. title product: NMR:(300MHz, DMSO-d 6): δ 9.78(s, 1); 9.46(s, 1); 9.35(s, 1); 9.10(d, 1, J=8); 7.47(d, 1, J=8); 7.4-6.8(m, 16); 6.68(s, 1); 6.29(s, 1); 5.75(s, 1); 5.65(dd, 1, J=5.8); 5.2-4.9(m, 2); 3.85(s, 2), 3.72(s, 3); 3.50(s, 3); 2.75(br.s, 3); 0.80(t, 3, J=7); 0.65(t, 3, J=7); Remaining is the multiplet of 4.2-1.0ppm scope; FABMS: calculated value C 58H 69N 8O 13S 2=1149.44252.
Measured value 1149.44007.
Method 18
7-β-(2-(thiophene-2-yl) kharophen)-3-((1-(tert-butoxycarbonyl amino)-the 2-ethyl thioether) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid allyl ester
7-β-(2-(thiophene-2-yl) kharophen)-3-((1-tert-butoxycarbonyl amino)-the 2-ethyl thioether) methylene radical)-3-cephem-4-carboxylic acid allyl ester (1.0g, 1.81mmol) being dissolved in methylene dichloride (20ml), this solution is cooled to-78 ℃ under nitrogen.The drips of solution that metachloroperbenzoic acid (0.37g) is dissolved in methylene dichloride (20ml) gained is added in the refrigerated cynnematin solution.This reaction mixture stirred 15 minutes under nitrogen, made it be warming up to-10 ℃ with ice/brine bath then.After 15 minutes, make the reaction mixture vaporising under vacuum to doing.The solid that obtains carries out purification by flash chromatography on silica gel, obtain the 0.60g title product with the dichloromethane solution wash-out that contains 2% methyl alcohol:
NMR:(DMSO-d 6, 300MHz): δ 8.40(d, 1, J=8.3Hz); 7.32(m, 1); 6.90(m, 2); 6.80(t, 1, J=4.8); 6.0-5.8(m, 1); 5.78(dd, 1, J=4.8,8.3); 5.34(d, 1, J=17.2); 5.21(d, 1, J=10.5); 4.91(d, 1, J=4.4); 4.71(d, 1, J=5.4); 4.0-3.4(m, 6); 3.0(m, 2); 2.5(m, 2), 1.32(s, 9); 13C(DMSO-d 6, 270MHz) 28.19; 30.68; 32.70; 35.67; 46.42; 58.05; 66.06; 66.54; 77.69; 118.76; 122.93; 123.10; 125.04; 126.44; 126.66; 131.78; 136.78; 155.50; 160.65; 164.12; 170.00; UV:(EtOH); 271(ε=8,330), 234(ε=11,000); IR:(CHCl 3) 1801,1728,1700,1696cm -1; FABMS:M+1=570; EA: calculated value: C 50.60, H 5.48, and N 7.38. measured value: C 50.83, and H 5.55, N 7.32.
Embodiment 14
7-β-(2-(thiophene-2-yl) kharophen)-and 3-(((1-deacetylate vincaleucoblastine) amino)-the 2-ethyl thioether) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid allyl ester
Repeat the step of embodiment 5, but with raw material cynnematin-1-β-sulfoxide (0.34g, 0.598mmol) the cynnematin thioether of replacement embodiment 5 of method 18.And, also use other reagent of number of columns down:
Deacetylate vincaleucoblastine hydrazides (0.46g, 0.60mmol);
N-methylmorpholine (0.20ml, 1.8mmol);
Sodium Nitrite (0.083g, 1.2mmol).
For this embodiment, reactant stirs under room temperature and nitrogen and spends the night, rather than stirs 30 minutes at 0 ℃ resembling among the embodiment 5.Product carries out purification by flash chromatography on silica gel, obtain the title product of 0.30g yellow solid shape with the methylene chloride solvent gradient elution of 95/5-90/10:
NMR:(CDCl 3, 300MHz): δ 9.48(s, 1); 8.01(s, 1); 7.5-6.9(m, 12); 6.52(s, 1); 6.02(s, 1); 5.95(dd, 1, J=4.2,9.5Hz); With 5.92(m, 1) overlapping;
5.70(s, 2); 5.35(d, 1, J=17.1); 5.25(d, 1, J=7.5); 4.73(br.s, 2); 4.62(d, 1, J=4.2); 3.71(s, 2); 3.67(s, 3); 3.66(s, 3); 3.55(s, 3); 2.73(s, 3); 0.88(t, 3, J=7); With 0.85(t, 3, J=7) overlapping; Remaining is the various multiplets of 4.1-1.1ppm scope;
UV（EtOH）， λ max＝268（ε＝22，600），214（ε＝57，000）;IR：（CHCl 3）1799，1730，1669，1615cm -1;
FABMS: calculated value: C 62H 76N 7O 12S 3=1206.47139.
Measured value=1206.47138.
Embodiment 15
7-β-(2-(thiophene-2-yl) kharophen)-and 3-(((1-deacetylate vincaleucoblastine) amino)-the 2-ethyl thioether) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid
7-β-(2-(thiophene-2-yl) kharophen)-and 3-(((1-(deacetylate vincaleucoblastine) amino)-the 2-ethyl thioether) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid allyl ester (0.30g; 0.249mmol) be dissolved in methylene dichloride (3ml); this solution is added to four [triphenylphosphine] palladium (O) (11.4mg; 0.0099mmol) and triphenylphosphine (2.6mg is in methylene dichloride 0.0099mmol) (1ml) solution.The reaction mixture that obtains stirred 10 minutes under room temperature and nitrogen, added triethyl silicane (4) and Glacial acetic acid (2) then.Then reaction mixture stirs under room temperature and nitrogen and spends the night.Add palladium, phosphine, silane and the acetate reagent of the same quantity of another part to reaction mixture, and restir 3 hours.Add ether, adopt centrifuging to collect the solid that obtains.The dry pale solid of collecting obtains the 183mg title product under vacuum and room temperature.The part of this product (100mg) is used HPLC(C18,50 * 350 posts, 1000psi, 50ml/min, with 15% acetonitrile/1% formic acid/84% water elution 10 minutes, become 30% acetonitrile/69% water/1% formic acid at 18 fens clock time internal solvents in the linear gradient mode, be eluted to purifying with the latter then and finish) purifying.
NMR：（DMSO-d 6，300MHz）：δ9.40（s，1）;8.32（d，1，J＝8Hz）;8.0-7.6（m，4）;7.4-7.2（m，4）;7.0-6.8（m，4）;6.39（s，1）;6.15（s，1）;5.75-5.50（m，3）;4.81（d，1，J＝4）;3.75（s，2）;3.65（s，3）;3.50（s，3）;2.62（s，3）;0.75（t，3，J＝7）;0.65（t，3，J＝7）;
Remaining is the multiplet of 4.0-1.1ppm scope; UV:(EtOH), λ Max=266(ε=25,600), 215ws(ε=59,200); IR:(CHCl 3) 1792,1623,1616,1602cm -1; FABMS: calculated value: C 59H 72N 7O 12S 3=1166.44009. measured value=1166.43436.
Method 19
7-β-(2-(thiophene-2-yl) kharophen)-and the 3-(((O-(p-nitrophenyl) carbonate group) methylene radical)-2-cephem-4-carboxylic acid diphenyl methyl ester
7-β-(2-(thiophene-2-yl) kharophen)-the 3-(methylol)-2-cephem-4-carboxylic acid benzhydryl ester (3.0g, 5.77mmol) be dissolved in dry tetrahydrofuran (THF, 5ml), this solution is cooled to 0 ℃ under nitrogen, add then (p-nitrophenyl) chloro-formic ester (1.74g, 8.65mmol) and Dimethylamino pyridine (2mg).Drip 2 to this reaction mixture, (1.0ml, 8.65mmol), reactant stirs under nitrogen and spends the night the 6-lutidine then, makes it slowly be warming up to 23 ℃.By the gravity filtration reaction mixture, and under vacuum concentrated filtrate.Enriched material carries out purification by flash chromatography on silica gel, obtain the title product of 2.9g white foam shape with 95/5 dichloromethane/ethyl acetate wash-out:
NMR：（CDCl 3，300MHz）：δ8.28（d，2，J＝9.1）;7.42-7.28（m，13）;7.05-7.00（m，2）;6.92（s，1）;6.55（s，1）;6.35（d，1，J＝8.7）;
5.65（dd，1，J＝4.0，8.7Hz）;5.24（d，1，J＝4.0）;5.21（S，1）;
4.82(d, 1, J=12.4); 4.72(d, 1, J=12.5); 3.88(s, 2); IR:(CHCl 3) 1777,1749,1686,1528cm -1; UV:(EtOH), λ Max=244(ε=16,800); FABMS: calculated value: (M+H)=686.12669.
Measured value=686.12451.
Embodiment 16
7-β-(2-(thiophene-2-yl) kharophen)-and 3-((deacetylate colchicine-7-amino) carbonyl oxygen base) methylene radical)-3-cephem-4-carboxylic acid benzhydryl ester
According to Raffauf, J.Amer.Chem.Soc.P.5292, the method for describing in 1953 prepare the deacetylate colchicine (70mg, 0.196mmol).The deacetylate colchicine that will as above make under nitrogen and room temperature is dissolved in dry pyridine; and this solution is added to 7-β-(2-(thiophene-2-yl) kharophen)-the 3-((O-(p-nitrophenyl) carbonate group) methylene radical)-2-cephem-4-carboxylic acid benzhydryl ester (130mg; 0.196mmol) in; then add N again, N diisopropylethylamine (1).Reaction mixture stirs under nitrogen and room temperature and spends the night.Use the ethyl acetate diluted reaction mixture, then vacuum concentration.Repeat dilution/enrichment step.Enriched material carries out purification by flash chromatography on silica gel, obtain the title product of 0.17g yellow oily with the dichloromethane solution wash-out that contains 10% methyl alcohol:
NMR:(CDCl 3, 300MHz), δ 2: δ 3Mix at 1: 1;
8.78（br.s，1）;8.10（d，1，J＝6）;7.6-7.1（m，13）;7.0-6.8（m，4）;6.52（s，1）;6.38（s，1）;5.8（dd，1，J＝4.8）;5.7（dd，1，J＝6）;5.55（dd，1，J＝4，8）;5.30（d，1，J＝6）;5.16（d，1，J＝4）;5.10（s，1）;4.95（d，1，J＝12）;4.90（d，1，J＝4）;4.65（d，1，J＝12）;4.4-4.2（m，1）;3.92（s，3）;3.90（s，3）;3.88（s，3）;3.60（s，3）;3.42（d，1，J＝12）;3.23（d，1，J＝12）;2.58-2.45（m，1）;2.40-2.18（m，2）;UV：（EtOH）341（ε＝81，600），219（ε＝214，000），202（ε＝311，000）;FABMS：（M+H）904.
Embodiment 17
7-β-(2-(thiophene-2-yl) kharophen)-and 3-(((deacetylate colchicine-7-amino) carbonyl oxygen base) methylene radical-3-cephem-1-β-sulfoxide-4-carboxylic acid benzhydryl ester
7-β-(2-(thiophene-2-yl) kharophen)-and 3-(((deacetylate colchicine-7-amino) carbonyl oxygen base) methylene radical-2; 3-cephem-4 carboxylic acid benzhydryl ester (0.17g; 0.188mmol) be dissolved in methylene dichloride, and make this solution under nitrogen, be cooled to 0 ℃.(0.038g 0.188mmol) is dissolved in methylene dichloride (2ml) to metachloroperbenzoic acid, and this solution slowly is added in the cynnematin solution.Reaction mixture stirred 1 hour under 0 ℃ and nitrogen, and vaporising under vacuum is to doing then.The solid that obtains carries out purification by flash chromatography on silica gel, obtain the 70mg title product with 90/10 methylene chloride/methanol mixture wash-out:
NMR：（CDCl 3，300MHz）：δ8.08（d，1，J＝6.2Hz）;7.48-7.10（m，13）;
7.0-6.8(m, 4); 6.51(s, 1), 6.0(m, 1); 5.15(d, 1, J=13); 4.62(d, 1, J=13); 4.45(d, 1, J=4); 4.32(m, 1); 3.95(s, 2); 3.92(s, 3); 3.90(s, 3); 3.82(s, 3); 3.60(s, 3); With 3.6(d, 1, J=13) overlapping; ; 3.15(d, 1, J=13); 2.50-2.18(m, 3); 1.78(m, 1); UV:(EtOH), λ Max=346(ε=14,500); FABMS: calculated value: (M+H)=920.25228. measured value=920.25448.
Embodiment 18
7-β-(2-(thiophene-2-yl) kharophen)-and 3-(((deacetylate colchicine-7-amino) carbonyl oxygen base) methylene radical-3-cephem-1-β-sulfoxide-4-carboxylic acid
7-β-(2-(thiophene-2-yl) kharophen)-and 3-(((deacetylate colchicine-7-amino) carbonyl oxygen base) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid benzhydryl ester (70mg) is dissolved in methylene dichloride (3ml), and is cooled to 0 ℃ under nitrogen.Add triethyl silicane (1ml) to this cooling solution, and then add trifluoroacetic acid (1ml).The reaction mixture that obtains stirred 30 minutes under 0 ℃ and nitrogen.Reaction mixture is with cold dilution in acetonitrile, and vaporising under vacuum is to doing.Add cold acetonitrile in residuum, the solution that obtains reduction vaporization is once more extremely done.Residuum is dissolved in ether, and the gained solution decompression is evaporated to the dried yellow solid that obtains.After the drying, carried out purifying according to former method and obtain 70mg title product: NMR:(DMSO-d 6, 300MHz); 8.37(d, 1, J=8.4); 8.13(d, 1,7.9); 8.06(d, 1, J=9.1); 7.38-6.80(m, 6); 6.70(s, 1); 5.74(dd, 1, J=4,8); 5.07(d, 1, J=13.2); 4.82(d, 1, J=4); 4.42(d, 1, J=13.2); 4.05(m, 1); 3.88(s, 2); 3.82(s, 3); 3.77(s, 3); 3.73(s, 3); 3.46(s, 3); Two proton 2.58-2.40(m that cover are arranged, 1); 2.20-1.90(m, 2); 1.83-1.70(m, 1); IR:(KBr) 1788,1721,1693,1592cm -1; UV:(EtOH), λ Max=345(ε=14,200), 240(ε=33,000); FABMS: calculated value: (M+H)=754.17403. measured value=754.17598.
Method 20
Preparation 7-β-(2-(thiophene-2-yl) kharophen)-the 3-(methylol)-2-cephem-4-carboxylic acid allyl ester
7-β-(2-(thiophene-2-yl) kharophen)-the 3-(methylol)-2-cephem-4-carboxylic acid (17.5g, 49.5mmol) be dissolved in 250ml DMF and 150ml diox, add sodium bicarbonate (4.5g to this solution, 53.6mmol), then add again allyl bromide 98 (5.9ml, 68.3mmol).The mixture heating up that obtains was refluxed 1 hour.Reaction mixture is cooled to room temperature, and distributes in ethyl acetate (500ml) and salt solution (500ml).Organic phase salt solution and water washing 6 to 7 times (400ml), dry (sodium sulfate) then, concentrating under reduced pressure obtains 10.2g dark-brown buttery title product.Thick productive rate: 52%.This crude product need not be further purified promptly and can be used in next step.But also can obtain analyzing the analytic sample of usefulness as follows: 0.84g crude product oil is dissolved in 1: 1 ethyl acetate/dichloromethane of 5ml, adds hexane (50ml) until there being yellow solid to form.After the filtration, filtrate obtains the title compound (0.04g) of white solid through placement.
IR:(CHCl 3): 1779,1750,1684,1510cm -; EA:C 17H 18N 2O 5S 2: calculated value: C=51.76, H=4.50, N=7.10. measured value: C=51.50, H=4.56, N=6.93.UV:(EtOH) ε Max=234(ε=14,384), 201(ε=15.766); NMR:(300MHz, CDCl 3): δ 7.27(m, 1); 7.00(m, 2); 6.38(d, 1, J=9); 6.28(s, 1); 5.9(m, 1); 5.65(d of d, 1, J=4.9); 5.3(m, 3); 5.08(s, 1); 4.66(d, 2, J=6); 4.20(q, 2, J=13); 3.85(s, 2).
Method 21
Preparation 7-β-(2-(thiophene-2-yl) kharophen)-and 3-((p-nitrophenyl carbonate group) methylene radical-3-cephem-4-carboxylic acid allyl ester and corresponding 2-cephem isomer
7-β-(2-(thiophene-2-yl) kharophen)-the 3-(methylol)-(10.2g 25.8mmol) is dissolved in 50ml THF to 2-cephem-4-carboxylic acid allyl ester, and this solution is cooled off in ice-water bath.Add lutidine (4.8ml, 41.3mmol), then add again the p-nitrophenyl chloro-formic ester (8.50g, 41.3mmol) and Dimethylamino pyridine (0.10g, 0.8mmol).Make reaction mixture be warmed to room temperature, and stirred 0.5 hour.The filtering reaction thing is collected pale precipitation, and concentrating under reduced pressure filtrate.The residuum that obtains obtains the gelationus title compound of dark yellow (5.66g, productive rate 39%) by purification by flash chromatography with 5% ethyl acetate/95% methylene dichloride wash-out.This material mainly is β-2 isomer.It need not be further purified promptly and can be used in next step.By flash chromatography (5% ethyl acetate/CH 2Cl 2) purifying can obtain analytic sample, the 0.28g yellow jelly can provide β-2 isomer of 0.08g water white transparency oily.IR：
(CHCl 3): 1779,1730,1685,1529,1510cm -1: EA:C 24H 21N 3O 9S 2: calculated value: C=51.51, H=3.78, N=7.51, S=11.46. measured value: C=51.72, H=3.88, N=7.27, S=11.22.UV:(EtOH): ε Max=239(ε=17,365), 201(ε=24,226); NMR:(300MHz, CDCl 3): ε Max=8.28(d, 2, J=9); 7.38(d, 2, J=9); 7.25(m, 1); 7.01(m, 2); 6.56(s, 1); 6.32(m, 1, J=8); 5.91(m, 1); 5.68(d of d, 1, J=4.9); 5.33(m, 3); 5.09(s, 1); 4.92(d, 1, J=12); 4.77(d, 1, J=12); 4.68(d, 2, J=7); 3.86(s, 2).
Embodiment 19
Preparation 7-β-(2-(thiophene-2-yl) kharophen)-and 3-((p-nitrophenyl carbonate group) methylene radical-3-cephem-1-β-sulfoxide-4-carboxylic acid allyl ester
7-β-(2-(thiophene-2-yl) kharophen)-and 3-((p-nitrophenyl carbonate group) (5.66g 10.1mmol) is dissolved in 225ml CH with corresponding β-2 mixture of isomers to methylene radical-3-cephem-4-carboxylic acid allyl ester 2Cl 2, the solution that obtains cools off in ice-water bath.To this solution add metachloroperbenzoic acid (3.23g, 55%, 10.3mmol is dissolved in other 50ml CH 2Cl 2In) solution, the adding of peroxybenzoic acid solution is finished in 15 minutes in the dropping mode.The solution that merges was placed 10 minutes in room temperature again.The concentrating under reduced pressure reaction mixture obtains brown oil, obtains 4.24g(productive rate 74% with methyl alcohol development) title compound, light brown solid.
IR:(CHCl 3): 1808,1772,1733,1689,1530cm -1MS:(FAB): M+=576.EA:C 24H 21N 3O 10S 2: calculated value: C=50.08, H=3.68, N=7.30. measured value: C=50.02, H=3.63, N=7.04.UV:(EtOH): λ Max=400(ε=688), 332(ε=2,394), 325(ε=2,353) and, 270(ε=17,094), 239(ε=14,438) and, 201(ε=28,842) .NMR:(300MHz, DMSO-d 6): λ=8.48(d, 1, J=8); 8.29(d, 2, J=9); 7.52(d, 2, J=9); 7.33(m, 1); 6.92(m, 2); 5.9(m, 2); 5.2-5.41(m, 3); 4.91(d, 1, J=4); 4.87(d, 1, J=13); 4.74(d, 2, J=5); 4.04(d, 1, J=18); 3.82(AB q, 2, J=15); 3.66(d, 1, J=18).
Embodiment 20
Preparation 7-β-(2-(thiophene-2-yl) kharophen)-and 3-O-[3 '-N-urea groups] Zorubicin] methylene radical-3-cephem-1-β-sulfoxide-4-carboxylic acid allyl ester
Zorubicin-HCl(0.87g, 1.5mmol) at (5.0ml, Aldrich Sure-Seal) makes slurries among the DMF, add diisopropylethylamine (0.3ml to these slurries, with ball shape KOH drying, 0.25mmol), the mixture to gained adds 7-β-(2-(thiophene-2-yl) kharophen then)-3-((p-nitrophenyl carbonate group) and methylene radical-3-cephem-1-β-sulfoxide-4-carboxylic acid allyl ester (0.87g, 1.51mmol).Make the reaction lucifuge, and stirred 2.5 hours.Add ether to reactant, be settled out the 1.29g red solid.This material need not promptly can be used among the embodiment 21 by purifying.Analytic sample can obtain like this: with red solidify (130mg) of a part by Chromatotron, on 1 micron column plate with 6% methyl alcohol/94%CH 2Cl 2Wash-out obtains the required product of 29mg, red solid.Go up classification at C-18 reverse-phase chromatographic column (Waters C18-bondpack) again, with 35%/35%/30%MeOH/ acetonitrile/water+0.5% ammonium acetate wash-out, the retention time of required product retention time @3.6 minute (flow velocity=2ml/min).When with above-mentioned HPLC condition purifying, obtained purity and be 90% title product.
IR:(CHCl 3): 1806,1724,1700,1610,1582,1509cm -1; EA: calculated value: C=55.15, H=4.63, N=4.29. measured value C=55.07, H=4.51, N=4.52.UV:(EtOH): ε Max=532(ε=4,274), 496(ε=7,857), 480(ε=7,839); 349(ε=1,467), 251(ε=21,091), 234(ε=30,361); NMR:(300MHz, DMSO d 6): δ 13.9(s, 1); 13.2(s, 1); 8.35, (d, 1, J=8); 7.84, (m=2); 7.56, (m, 1); 7.31, (d, 1, J=4); 6.90, (m, 3); 5.9-5.75, (m, 1) and 5.76, dd, 1, J=5.9) overlapping; ; 5.32, (m, 1); 5.16, (m, 2); 5.02, (d, 1, J=13); 4.8, (m, 2); 4.65, (m, 2); 4.53, (bs, 2); 4.44, (d, 1, J=13); 4.10, (d, 1, J=5); 3.92, (s, 3); 3.88-3.70, (m, 4); 3.62, (m, 1); 3.5-3.1, (m, 2) and 3.28, s, 2) and overlapping; ; 2.83, (AB q, 2, J=18); 2.2-2.0, (m, 2); 1.76, (m, 1); 1.40, (m, 1); 1.08(d, 3, J=6).
Embodiment 21
Preparation 7-β-(2-(thiophene-2-yl) kharophen)-and 3-O-[3 '-N-urea groups] Zorubicin] methylene radical-3-cephem-1-β-sulfoxide-4-carboxylic acid
Method A
Acid chloride (3.5mg, 0.016mmol) and triphenylphosphine (22mg 0.084mmol) is dissolved in ethyl acetate (0.1ml), this mixture about 2 minutes with sonic treatment.7-β-(2-(thiophene-2-yl) kharophen)-and 3-O-[3 '-N-urea groups] Zorubicin] (0.11g 0.11mmol) is dissolved in by 5.5ml EtOAc, 2.5ml MeOH, 3ml CH methylene radical-3-cephem-1-β-sulfoxide-4-carboxylic acid allyl ester in addition 2Cl 2In the mixed solvent of forming with 80 μ l acetate, this solution is added in the palladium solution with pipette, then add again triethyl silicane (0.02ml, 0.13mmol).Make the reaction mixture lucifuge, and at room temperature stirred 4 hours.Make reaction mixture be concentrated into red oil (0.25g).This crude product oil (54mg) of a part is dissolved in DMSO(1.5ml), inject 22 * 250mm RSIL Phenyl and prepare HPLC post (Alltech Associates), with 35%CH 3The mixture wash-out (flow velocity 8ml/min, Jian Ceqi @480nm) of CN/0.05%TFA in water 12 minutes is again with 45%CH 3The mixture wash-out of CN/0.05% TFA in water 18 minutes, the operation 30 minutes after again with 60%CH 3The mixture wash-out of CN/0.05%TFA in water.The chromatographic effluent that lyophilize contains product obtains the title product of 26mg red solid shape.
IR:1780cm -1.MS:(ESI): 962.1(MNa +) .MS:HRMS:(FAB): calculated value:
C 42H 41N 3O 18S 2Li(MLi +)=946.1987. measured value=946.1955.NMR:(DMSO-d 6): δ 14.0, (s, 1); 13.2, (s, 1); 8.33, (d, 1, J=8); 7.91, (d, 2, J=4); 7.64, (t, 1, J=5); 7.35, (m, 1); 6.91, (m, 3); 5.71, (dd, 1, J=5,8); 5.43, (s, 1); 5.19, (m, 1); 5.05, (m, 1); 4.92, (m, 1); 4.82, (m, 2); 4.70, (d, 1, J=5); 4.55, (d, 2, J=6) with 4.46, (m, 1) is overlapping; 4.12, (d, 1, J=9); 3.97, (s.3);
3.80, (ABq, 2, J=15; HOD disturbs the spectrum of 3.4-3.0);
2.96，（m，2）;2.12，（m，2）;1.82，（m，1）;1.43，（m，1）;
1.09，（d，3，J＝6）.
Method B
With acid chloride (0.04g, 0.18mmol) and triphenylphosphine (0.2g 0.76mmol) mixes, and makes slurries in the 1ml ethyl acetate.This mixture is used sonic treatment 1 minute, has generated white precipitate.Add ethyl acetate (50ml), methyl alcohol (25ml) and acetate (0.75ml) to this precipitation.Adding is according to allyl group 7-β-(2-(thiophene-2-yl) kharophen of the method preparation identical with embodiment 20)-3-O-[3 '-N-urea groups] Zorubicin] methylene radical-3-cephem-1-β-sulfoxide-4-carboxylicesters (1.36g, 1.39mmol), add 125ml 10%MeOH/90%CH simultaneously 2Cl 2To improve solvability.Add again triethyl silicane (0.35ml, 2.20mmol).After 4 hours, a part has only been finished in reaction.Acid chloride, triphenylphosphine and the triethyl silicane of adding and aforementioned same quantity, and the reactant stirring is spent the night.The concentrating under reduced pressure reaction mixture obtains the title compound of 1.4g red solid shape.Use reversed-phase HPLC short run ground this material of purifying, two 25 * 100mm pillars (Waters μ bondapak C-18 post) are linked in sequence together, with 30% acetonitrile/70% water+0.1%TFA wash-out, flow velocity is 8ml/min, detects output at the 480nm place.After 25 minutes, moving phase is changed to 40% acetonitrile/60% water+0.1%TFA.Under these conditions, wash-out has gone out title compound about 55 minutes the time.The chromatographic effluent of lyophilize purity assay＞95% can obtain title compound.It is 95% title compound that the 76mg crude product can provide 24mg purity.
Method 22
Rat blood is learned experimental record
The purpose of following method is the toxicity of comparison substrate-cytotoxic agents and the toxicity of independent cytotoxic agents.
1. the 0th day: give 25 male Fischer 344 rat (@100g) bloodletting in advance (eye blood), and carry out intravenous injection by as follows:
The A group: 5 rat+comparison vehicle: 0.5ml, 10mM HEPES, 150mg NaCl contains 10% alcoholic acid PH7.1 damping fluid.
B group: 5 rat+1mg/Kg 4-deacetylate vincaleucoblastine-3-hydrazides (deacetylate vincaleucoblastine hydrazides).1mg deacetylate vincaleucoblastine hydrazides is dissolved in the 0.5ml dehydrated alcohol, adds 4.5ml HEPES-NaCl damping fluid again.Give every rat injection 0.5ml.
C group: 5 rat+1mg/Kg LY262758(7-β-2-(phenoxy group kharophens)-3-(((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen base) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid, trifluoroacetate) (Vinca content, 58% Vinca).Dissolve 2mg as stated above, and injection 0.5ml.
D group: 5 rat+1mg/Kg LY266494, (7-β-(2-(thiophene-2-yl) kharophen)-3-(((deacetylate vincaleucoblastine hydrazide group) methylene radical carbonyl oxygen base))-3-cephem-4-carboxylic acid, hydrochloride (55.8% Vinca).Dissolving 2mg, injection 0.5ml.
E group: 5 rat+1mg/Kg embodiment 17 compound 7-β-(2-(thiophene-2-yl) kharophens)-3-(((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen base) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid, trifluoroacetate.Dissolving 2mg, injection 0.5ml.
All rats are all by the mark identification of piercing one's ears, and bloodletting in the 3rd and the 7th day after injection, are used to measure white cell (WBC).Provided raw data below.Compare with every other group, the rat of handling with deacetylate vincaleucoblastine hydrazides reduces at most at the 3rd day WBC.WBC value the 7th day each group can not be distinguished (and the numerical value be lower than bloodletting in advance before, this may be the cause owing to the different Instrument measuring WBC of use).
Rat blood is learned experimental data
Numerical value * 1000=WBC ' s
Experimental group the 0th day the 3rd day the 7th day
Comparative group #1 rat 13.2 14.2 6.0
#2 rat 12.4 16.4 9.3
#3 rat 13.1 13.7 6.8
#4 rat 10.4 10.3 10.1
#5 rat 10.1 13.1
Average 11.8 13.5 8.1
+/-SD 1.5 2.2 2.0
DAVLB-Hyd #1 rat 17.4 * * 5.0
#2 rat 10.9 4.4 2.8
#3 rat 10.2 9.0 19.8
#4 rat 12.7 5.4 * *
#5 rat 17.6 10.6 * *
Average 13.8 7.4 9.2
+/-SD 3.5 2.9 9.2
LY262758 #1 rat 13.3 13.3 14.0
#2 rat 14.3 14.6 13.0
#3 rat 9.4 15.0 12.5
#4 rat 10.2 16.3 9.0
#5 rat 19.7 14.7 7.7
Average 13.4 14.8 11.2
+/-SD 4.1 1.1 2.7
LY266494 #1 rat 15.6 19.2 8.3
#2 rat 16.5 16.2 6.8
#3 rat 14.3 14.3 11.4
#4 rat * * 13.3 13.2
#5 rat 16.0 12.1 20.1
Average 15.6 15.0 12.0
+/-SD 0.9 2.8 5.2
LY266070 #1 rat 14.1 15.9 7.0
#2 rat 15.0 12.8 14.5
#3 rat 9.9 15.1 9.6
#4 rat 9.9 14.5 0.6
#5 rat 16.2 11.3 7.1
Average 13.0 13.9 9.6
+/-SD 2.9 1.9 3.0
The * sample is formed clot-can not measure the WBC number.
Method 23
Use bare mouse model to estimate prodrug/antibody-enzyme conjugates system in vivo
Following method is used for determining the dosage of original observed when prodrug is had toxicity.And, the prodrug maximal dose when also having determined to observe anti-tumor activity.
1. the 0th day: with 1 * 10 7LS174T tumour cell (ATCC) be implanted subcutaneously 30 bare mouse (CHARLES RIVER, 25G);
2. by the standby prodrug treatment solution of following preparation:
With 7-β-(2-(thiophene-2-yl) kharophen)-3-(((deacetylate vincaleucoblastine hydrazide group) carbonyl oxygen base) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid, trifluoroacetate are water-soluble; add sodium chloride solution (9%); obtain waiting and ooze (0.9%) salt brine solution, this solution contains the prodrug of 2mg/ml concentration.Before using the solution that obtains is carried out filter-sterilized, and injection as early as possible after the prodrug preparation.
A group: every injected in mice 0.25ml salt solution
B group: the 1.7ml stock solution is put into bottle, every injected in mice 0.25ml.
C group: 0.625ml stock solution+0.775ml salt solution;
Injection 0.2ml
D group: 0.31ml stock solution+1.09ml salt solution;
Injection 0.2ml
E group: 0.15ml stock solution+1.25ml salt solution;
Injection 0.2ml
F group: 0.075ml stock solution+1.325ml salt solution;
Injection 0.2ml
3. at the 3rd, 4,5,6 day bare mouse is carried out intravenous injection by following:
A group: 5 injected in mice 0.25ml salt solution
B group: 5 injected in mice 20mg/Kg ceph-Vinca prodrugs
C group: 5 injected in mice 10mg/Kg prodrugs
D group: 5 injected in mice 5mg/Kg prodrugs
E group: 5 injected in mice 2.5mg/Kg prodrugs
F group: 5 injected in mice 1.25mg/Kg prodrugs
4. behind the implantation tumour cell, measured tumour on the the 14th, 21,28 day.
5. the result is as follows:
Dosage suppresses the tumor weight of active (%) death toll/altogether
Mg/Kg average mg S.D.
A group contrast-0/5 787
B group 20.0-5/5
C organizes 10.0 94 1/5 50+/-64
D organizes 5.0 33 0/5 529+/-778
E organizes 2.5 30 2/5 552+/-233
F organizes 1.25 47 0/5 414+/-387
B group dosage has toxicity.The toxic C group of tool dosage does not show that independent prodrug has anti-tumor activity at this dosage.Can infer from these results, optimum effective dose is generally in 1 to 8mg/Kg scope.
Method 24
Spike and biodistribution research
Anti-KS 1/4(is 007B)-bio distribution of beta-lactam enzyme conjugates is by using 111Indium mark binding substances is measured.Binding substances earlier with isothiocyano benzyl DTPA reaction, carry out mark according to the method for having established people such as (, Anal.Biochem., 142, PP.68-78,1984) Meares C. again.Mark binding substances (20 μ g and every mouse of 10 μ Ci) is injected the tail vein of the bare mouse of band LS174T tumour.Mouse is divided into 6 one group, respectively kills one group on the 4th, 24,48,72 and 120 hour behind the injection binding substances.The method of killing mouse, sample collection, counting and data processing once had narration people such as (, Can.Res., 43, PP.5347-5355,1983) Halpern S.E. in the past.By in out of the count bare female mice (Charles River Breeding Laboratories, Wilmington, flank portion subcutaneous vaccination 1 * 10 Massachusetts) 7Individual cell implantation tumour.In 9 days, LS174T tumor growth to the mean size of implanting with this method is 0.2gm.These results of study are reflected among Fig. 1 and Fig. 2.The result shows that binding substances concentrates on the tumour, and it promptly (promptly in 72 hours) from serum, remove.
Method 25
The growth of ADC treatment back T380 clone's tumour in bare mouse
At the 0th day, by the T380 tumour being implanted in the mouse body at the subcutaneous vaccination 0.1ml of flank portion of out of the count female bare mouse tumour slurries.In 14 days, tumour is confirmed as growing to about 0.3gm size, and the size of tumour is by the bisecting compasses measurement with by formula V=0.5 * L * W 2Calculate to determine, wherein L and W be respectively the longest limit of tumour and with the length on perpendicular limit, this limit.All injections all are to inject tail vein.In Fig. 3,4 researchs of describing,, also measured the body weight of mouse as toxic a kind of expression.
Every group of 8 mouse to be treated all injection in the 14th, 21 and 28 day anti--CEA(is CEM231)-β-Nei Xiananmei (35 μ g) binding substances, irrelevant antibody (being CHA255)-β-Nei Xiananmei (35 μ g) binding substances or salt solution.After these injections, inject beginning in 72 hours behind the binding substances certainly, also to carry out four injections of the preceding drug compound of embodiment 11.Once a day, injection volume is 1mg/Kg(Fig. 3) or 0.25mg/Kg(Fig. 4).In the comparative group of this experiment, prodrug injection replaces with salt solution or with the deacetylate vincaleucoblastine hydrazides of preceding pharmaceutical quantities equimolar amount, and the latter's injected dose is 0.6mg/Kg(Fig. 3) or 0.15mg/Kg(Fig. 4).
Data point among Fig. 3 and 4 is represented the following result of present method: filled symbols is represented experiment (binding substances/prodrug that tumour is exclusive) group; Hollow title is represented control group.Other titles are explained as follows among the figure: hollow garden: salt solution/salt solution; Hollow triangle: salt solution/prodrug; Open squares: salt solution/medicine; Open diamonds: irrelevant antibody-beta-lactam enzyme conjugates/prodrug; Hollow arrow: injection binding substances or salt solution; Filled arrows: in injection prodrug, medicine or the brinish 4 days first day.For experimental group, tumor regression, mouse demonstrates long disease-free state.Control group only demonstrates being suppressed of tumor growth, does not demonstrate tumor regression, and demonstrates secular tumour stability.
Method 26
The growth of ADC treatment back LS174T clone's tumour in bare mouse
Implant the LS174T tumour by method 24, and measure its growing state by method 25.At the 9th, 16 and 23 day, with respectively being 35 μ g anti-KS 1/4(007B)-beta-lactam enzyme conjugates, anti--CEA(CEM231)-beta-lactam enzyme conjugates, irrelevant antibody (CHA255)-beta-lactam enzyme conjugates or salt solution give mouse (10 of saline control groups; 5 of treatment groups) injection.In above-mentioned injection back 96 hours, also to carry out a prodrug (embodiment 11 compounds) (4mg/Kg), medicine (deacetylate vincaleucoblastine hydrazides) (2.3mg/Kg) or saline injection.Measured tumor size and be depicted among Fig. 5.As for the T380 tumour shown in Fig. 3, only observe tumor regression, and all do not observe tumor regression at other any one control groups in tumour specificity treatment group.
Data point among Fig. 5 is represented the following result of this method: solid garden: anti-KS 1/4-beta-lactam enzyme conjugates/prodrug; Filled squares: anti--CEA-beta-lactam enzyme conjugates/prodrug; Hollow garden: salt solution/salt solution; Hollow triangle: salt solution/prodrug; Open squares: salt solution/medicine; Open diamonds: irrelevant antibody-beta-lactam enzyme conjugates/prodrug; Hollow arrow: injection binding substances or salt solution; Filled arrows: injection prodrug, medicine or salt solution.Also compare experiment, whether brought reinforced effects with the non-exclusive synergy between check binding substances and the medicine with anti-KS1/4-beta-lactam binding substances/medicine, anti--CEA-beta-lactam enzyme conjugates/medicine and irrelevant antibody-beta-lactam enzyme conjugates/medicine.Do not observe difference (data do not show in the drawings) from salt solution/medicine.
Measured the body weight of mouse.The mouse body weight loss of accepting the treatment of this dosage free drug is very big, and this drug dose and referral have caused dead mouse in other experiments.In the mouse group of accepting anti--CEA-beta-lactam enzyme conjugates/prodrug treatment, only measure more a spot of body weight loss, and in accepting the mouse group of anti-KS 1/4-β-Nei Xiananmei conjugates for therapy, do not measure body weight loss (data do not show in the drawings).
Method 27
ADC treatment back LS174T growth of tumor
The method here is identical with method 26, just will make tumour look bigger before the treatment beginning in present method, and the binding substances that uses be anti--TAG-72(CC49)-β-Nei Xiananmei.Even for bigger tumour with as the TAG-72 of target antigen, just the treatment of tumour specificity has caused tumor regression, and other any contrasts treatments all do not cause tumor regression.
Data point among Fig. 6 is represented the following result of this method: solid garden: anti--TAG-72-beta-lactam enzyme conjugates/prodrug; Hollow garden: salt solution/salt solution; Hollow triangle: salt solution/prodrug; Open squares: salt solution/medicine; Open diamonds: irrelevant antibody (CHA255)-beta-lactam enzyme conjugates/prodrug; Hollow arrow: injection binding substances or salt solution; Filled arrows: injection prodrug, medicine or salt solution.
Method 28
Binding substances dosage is to suppressing the influence of tumor growth
In general following method is exactly the method for method 25, but has following exception.Mouse was accepted 35 μ g(filled squares at the 14th, 21 and 28 day), the solid garden of 14 μ g() or 3.5 μ g(black triangles) anti--CEA(CEM231)-treatment of beta-lactam enzyme conjugates, after 72 hours, use prodrug again with 1mg/Kg/ days dosage treatment 4 days.The 4th experimental group only accepted the treatment of 35 μ g anti-CEA-beta-lactam enzyme conjugates (solid diamond) at the 14th day, then resemble to treat with prodrug the referrals of other mouse.Data point among Fig. 5 is represented the following result of this method: hollow garden: salt solution/salt solution; Open squares: salt solution/prodrug; Hollow arrow: injection binding substances or salt solution; Filled arrows: in injection prodrug, medicine or the brinish 4 days first day.The result shows that optimum medicine efficacy perhaps is the continuity of depending on the result of treatment of binding substances dosage, rather than the size of this result of treatment.The binding substances that the result was further illustrated in each course of treatment has all been made contribution to the continuity of response.
Method 29
Preparation diphenyl methyl 7-β-(2-(thiophene-2-yl) kharophen)-and the 3-(O-(p-nitrophenyl) carbonate group) methylene radical)-2-cephem-4-carboxylicesters
To 7-β-(2-(thiophene-2-yl) kharophen)-3-methylol-2-cephem-4-carboxylic acid diphenyl methyl ester (3.0g, 5.77mmol) dry THF (5ml) solution (0 ℃) add Dimethylamino pyridine (2mg), and then adding p-nitrophenyl chloro-formic ester (1.74g, 8.65mmol).Drip drying 2, the 6-lutidine (1.0ml, 8.65mmol), place to spend the night and make it be warmed to room temperature by the solution that obtains.Remove by filter some insolubless, concentrating under reduced pressure filtrate.Residuum is through the purification by flash chromatography (CH that contains 5%EtOAc 2Cl 2Be eluent) obtain 2.9g(productive rate 74%) title compound of white foam shape.
NMR:(CDCl 3) δ 8.28(d, 2, J=9.1); 7.42-7.28(m, 13); 7.05-7.0(m, 2); 6.92(s, 1); 6.55(s, 1); 6.35(d, 1, J=8.7); 5.65(dd, 1, J=4,8.7); 5.24(d, 1, J=4); 5.21(s, 1); 4.82(d, 1, J=12); 4.72(d, 1, J=12); 3.88(s, 2). 13C NMR:(CDCl 3) δ 169.99,165.90,164.17,155.26,152.04,145.46,138.76,138.69,134.59,128.84,128.77,128.60,128.47,127.90,127.55,127.14,126.79,126.07,125.30,124.64,121.72,117.93,79.49,69.90,60.45,53.2,50.14,37.07.IR(CHCl 3) 1777,1749,1686,1528cm -1.UV:(EtOH) λ Max=244(ε=16,800) .FABMS: calculated value: C 34H 27N 3O 9S 2: C=59.55; H=3.97; N=6.13. measured value: C=59.34; H=4.05; N=5.93.
Method 30
Preparation 7-β-(2-(thiophene-2-yl) kharophen)-and the 3-(O-(p-nitrophenyl) carbonate group) methylene radical)-3-cephem-1-β-sulfoxide-4-carboxylic acid diphenyl methyl ester
To diphenyl methyl 7-β-(2-(thiophene-2-yl) kharophen)-the 3-(O-(p-nitrophenyl) carbonate group) methylene radical)-2-cephem-1-4-carboxylicesters (2.5g, CH 3.65mmol) 2Cl 2(40ml) solution (0 ℃) adds the CH of 55% metachloroperbenzoic acid (1.1g, 3.65mmol, equivalent) 2Cl 2(10ml) solution.Maintenance is after 1 hour down at 0 ℃, and TLC(contains the CH of 20%EtOAc 2Cl 2) show that raw material is exhausted.Removal of solvent under reduced pressure, residuum is through the flash chromatography (CH that contains 20%EtOAc 2Cl 2) purifying obtains 2.2g(productive rate 86%) title compound of white solid.
NMR:(DMSO-d 6): δ 8.49(d, 1, J=8); 8.27(d, 2, J=9); 7.5-7.2(m, 13); 6.92(m, 3); 5.94(dd, 1, J=4.5,8); 5.25(d, 1, J=13); 4.93(d, 1, J=4.5); 4.85(d, 1, J=13); 4.04(d, 1, J=18.7); 3.88(d, 1, J=15.3); 3.78(d, 1, J=15.3); 3.66(d, 1, J=18.7) .IR:(KBr) 1765,1720,1658cm -1.UV:(EtOH) ε Max=265(ε=3570). calculated value: C 34H 27N 3O 10S 2: C=58.20; H=3.88 N=5.99. measured value: C=58.48; H=4.08; N=5.90.

Claims (24)

1, the compound of following formula:
Figure 931098718_IMG1
Zz is 0 or 1 in the formula, R 1For by C 1To C 30Alkyl is derived and next acyl group, R 2Be hydrogen, organic or inorganic positively charged ion, carboxyl-protecting group, or the base of the unsettled ester formation of nontoxic metabolism; With cytotoxic agents in the formula be the compound of following formula:
Figure 931098718_IMG2
2, the compound of claim 1, wherein R 1Be:
ⅰ) C 1To C 4Alkyl, C 1To C 6The alkyl, the C that replace 1To C 4Alkoxyl group, C 1To C 4The group of alkylthio or following formula:
Figure 931098718_IMG3
Or the carboxy derivatives of amino and/or its protection of protection:
ⅱ) the group of following formula:
Figure 931098718_IMG4
Each R in the formula 5, R 6And R 7Be alone hydroxyl, nitro, amino, the protection of hydrogen, halogen, hydroxyl, protection amino, amine salt, cyano group, trifluoromethyl, aminomethyl, protection aminomethyl, N-(methyl or ethylsulfonyl) amino, C 1To C 6Alkyl or C 1To C 4Alkoxyl group; R 4Carboxyl, phenyl carboxylicesters, (5-indanyl) carboxylicesters, sulfonic acid, sulfonate, azido-, halogen or C for the amino of the hydroxyl of hydroxyl, protection, methanoyl, amino, protection, amine salt, carboxyl, carboxylate salt, protection 1To C 6Alkyl; R 6Be C 1To C 4Alkoxyl group; Z is oxygen or sulphur; N is 0,1,2 or 3; With m be 0 or 1;
ⅲ) the group of following formula:
Figure 931098718_IMG5
R in the formula 9Be heterocycle; R 10Carboxyl, phenyl carboxylicesters, (5-indanyl) carboxylicesters, sulfonic acid, sulfonate, azido-, halogen or C for the amino of the hydroxyl of hydroxyl, protection, methanoyl, amino, protection, amine salt, carboxyl, carboxylate salt, protection 1To C 6Alkyl; N is 0,1,2 or 3; With z be oxygen or sulphur.
3, the compound of claim 2, wherein R 1Be ethanoyl; propionyl; butyryl radicals; different propionyl; valeryl; methoxymethyl; phenyl acetyl; the phenoxy group ethanoyl; the 2-(aminomethyl) phenyl acetyl; 2-phenyl-2-hydroxyacetyl; 2-phenyl-2-(sodium sulfonate) ethanoyl; 2-phenyl-2-carboxyl ethanoyl; the 2-(4-hydroxy phenyl)-2-carboxyl ethanoyl; 2-phenyl-2-glycyl; the 2-(4-hydroxy phenyl)-the 2-aminoacetal; 2-the 3-[N-(methanesulfonamido)] phenyl }-the 2-glycyl; 2-phenyl-2-(5-indanyl carboxylicesters) ethanoyl; 2-phenyl-2-(phenyl carboxylicesters) ethanoyl; 2-phenyl-2-azido-ethanoyl; 2-phenoxy group propionyl; 2; 5-dimethoxy benzoyl; the 2-(methanoyl)-the 2-phenyl acetyl; 2-(2-oxyethyl group naphthalene-1-yl) ethanoyl; 2-(naphthalene-1-yl)-the 2-glycyl; 2-(naphthalene-2-yl)-the 2-glycyl; 2-(2; 5-dichlorophenyl sulfo-) ethanoyl; 2-(3; 4-dichlorophenyl sulfo-) ethanoyl; the 2-(1-tetrazyl) ethanoyl; 2-[N-(3; 5-dichloropyridine-4-oxygen base)] ethanoyl; 2-(2-aminothiazole-4-yl) ethanoyl; the 2-(2-thienyl) ethanoyl; the 2-(4-pyridylthio) ethanoyl; 2-(N-methyl-4-pyridine sulfo-) ethanoyl; 2-(2-amino-4-phenyl thiazole-5-yl) ethanoyl; 2-(3-hydroxyl-4-carboxyl isothiazole-5-base sulfo-) ethanoyl; 3-phenyl-5-methyl-isoxazole base-3-formyl radical; the 3-(2-chloro-phenyl-)-5-methyl-isoxazole base-3-formyl radical; 3-(2, the 5-dichlorophenyl)-5-methyl-isoxazole base-3-formyl radical; 3-(2-fluoro-5-chloro-phenyl-)-5-methyl-isoxazole base-3-formyl radical; the 2-(2-thienyl)-the 2-glycyl; the 2-(2-thienyl)-and the 2-(carboxylic acid sodium) ethanoyl; the 2-[N-(4-pyridine)] ethanoyl; the 2-(2-benzothienyl) ethanoyl; the 2-(3-benzothienyl) ethanoyl; the 2-(2-benzofuryl) ethanoyl and 2-(3-benzofuryl) ethanoyl.
4, the compound of claim 3, wherein R 1Be 2-(thiophene-2-yl) ethanoyl, and ZZ is 0.
5, the compound of claim 4, wherein Zorubicin part by link 3 of cynnematin '-nitrogen of amino saccharides on the carbonyl of carbamate groups is attached on the cynnematin.
6, the compound of claim 3, wherein R 1Be 2-(thiophene-2-yl) ethanoyl, and ZZ is 1.
7, the compound of claim 6, wherein Zorubicin by link 3 of cynnematin '-nitrogen of amino saccharides on the carbonyl of carbonate group is attached on the cynnematin.
8, the compound of claim 3, wherein R 1Be the phenoxy group ethanoyl, and ZZ is 1.
9, the compound of claim 8, wherein Zorubicin by link 3 of cynnematin '-nitrogen of amino saccharides on the carbonyl of carbonate group is attached on the cynnematin.
10, the method that is used for the treatment of tumor disease, this method comprises:
A) use the antibody-enzyme conjugates of the treatment significant quantity of following formula,
Antibody-(β-Nei Xiananmei)
In the formula:
The compound of enzyme and claim 1 reaction, and causing separating of cytotoxic agents and cynnematin, antibody and antigen complexing by the target cell tissue expression;
B) to the substrate-cytotoxic agents of the claim 1 of infection host administering therapeutic significant quantity, wherein substrate is the substrate for enzyme.
11, the methods of treatment of claim 10, wherein antibody and carcinomebryonic antigen complexing.
12, the methods of treatment of claim 11, wherein antibody and the complexing of KS1/4 antigen.
13, the methods of treatment of claim 12, wherein antibody and the complexing of TAG-72 antigen.
14, the methods of treatment of claim 13, wherein antibody and the complexing of MAT-65 antigen.
15, the methods of treatment of claim 14, wherein R 1For:
ⅰ) C 1To C 4Alkyl, C 1To C 6The alkyl, the C that replace 1To C 4Alkoxyl group, C 1To C 4Alkylthio, or the group of following formula:
Figure 931098718_IMG6
Or the carboxy derivatives of amino and/or its protection of protection;
ⅱ) following formula group:
Figure 931098718_IMG7
Figure 931098718_IMG8
Each R in the formula 5, R 6And R 7Be alone hydroxyl, nitro, amino, the protection of hydrogen, halogen, hydroxyl, protection amino, amine salt, cyano group, trifluoromethyl, aminomethyl, protection aminomethyl, N-(methyl or ethylsulfonyl) amino, C 1To C 6Alkyl or C 1To C 4Alkoxyl group; R 4Carboxyl, phenyl carboxylicesters, (5-indanyl) carboxylicesters, sulfonic acid, sulfonate, azido-, halogen or C for the amino of the hydroxyl of hydroxyl, protection, methanoyl, amino, protection, amine salt, carboxyl, carboxylate salt, protection 1To C 6Alkyl; R 6Be C 1To C 4Alkoxyl group; Z is oxygen or sulphur; N is 0,1,2 or 3; With m be 0 or 1;
ⅲ) the group of following formula:
Figure 931098718_IMG9
R in the formula 9Be heterocycle; R 10Carboxyl, phenyl carboxylicesters, (5-indanyl) carboxylicesters, sulfonic acid, sulfonate, azido-, halogen or C for the amino of the hydroxyl of hydroxyl, protection, methanoyl, amino, protection, amine salt, carboxyl, carboxylate salt, protection 1To C 6Alkyl; N is 0,1,2 or 3; With z be oxygen or sulphur.
16, the methods of treatment of claim 15, wherein R 1Be ethanoyl; propionyl; butyryl radicals; different propionyl; valeryl; methoxymethyl; phenyl acetyl; the phenoxy group ethanoyl; the 2-(aminomethyl) phenyl acetyl; 2-phenyl-2-hydroxyacetyl; 2-phenyl-2-(sodium sulfonate) ethanoyl; 2-phenyl-2-carboxyl ethanoyl; the 2-(4-hydroxy phenyl)-2-carboxyl ethanoyl; 2-phenyl-2-glycyl; the 2-(4-hydroxy phenyl)-the 2-aminoacetal; 2-the 3-[N-(methanesulfonamido)] phenyl }-the 2-glycyl; 2-phenyl-2-(5-indanyl carboxylicesters) ethanoyl; 2-phenyl-2-(phenyl carboxylicesters) ethanoyl; 2-phenyl-2-azido-ethanoyl; 2-phenoxy group propionyl; 2; 5-dimethoxy benzoyl; the 2-(methanoyl)-the 2-phenyl acetyl; 2-(2-oxyethyl group naphthalene-1-yl) ethanoyl; 2-(naphthalene-1-yl)-the 2-glycyl; 2-(naphthalene-2-yl)-the 2-glycyl; 2-(2; 5-dichlorophenyl sulfo-) ethanoyl; 2-(3; 4-dichlorophenyl sulfo-) ethanoyl; the 2-(1-tetrazyl) ethanoyl; 2-[N-(3; 5-dichloropyridine-4-oxygen base)] ethanoyl; 2-(2-aminothiazole-4-yl) ethanoyl; the 2-(2-thienyl) ethanoyl; the 2-(4-pyridylthio) ethanoyl; 2-(N-methyl-4-pyridine sulfo-) ethanoyl; 2-(2-amino-4-phenyl thiazole-5-yl) ethanoyl; 2-(3-hydroxyl-4-carboxyl isothiazole-5-base sulfo-) ethanoyl; 3-phenyl-5-methyl-isoxazole base-3-formyl radical; the 3-(2-chloro-phenyl-)-5-methyl-isoxazole base-3-formyl radical; 3-(2, the 5-dichlorophenyl)-5-methyl-isoxazole base-3-formyl radical; 3-(2-fluoro-5-chloro-phenyl-)-5-methyl-isoxazole base-3-formyl radical; the 2-(2-thienyl)-the 2-glycyl; the 2-(2-thienyl)-and the 2-(carboxylic acid sodium) ethanoyl; the 2-[N-(4-pyridine)] ethanoyl; the 2-(2-benzothienyl) ethanoyl; the 2-(3-benzothienyl) ethanoyl; the 2-(2-benzofuryl) ethanoyl and 2-(3-benzofuryl) ethanoyl.
17, the methods of treatment of claim 16, the wherein R in the cynnematin substrate 1Be 2-(thiophene-2-yl) ethanoyl, and ZZ is 0.
18, the methods of treatment of claim 17, wherein antibody is CEM231.6.7.
19, the methods of treatment of claim 18, wherein the cytotoxic agents Zorubicin by link 3 of cynnematin '-nitrogen of amino saccharides on the carbonyl of carbonate group is attached on the cynnematin.
20, the methods of treatment of claim 16, wherein R 1Be 2-(thiophene-2-yl) ethanoyl, and ZZ is 1.
21, the methods of treatment of claim 20, wherein antibody is called as CEM231.6.7.
22, the methods of treatment of claim 21, wherein antibody is called as CC 49.
23, the methods of treatment of claim 22, wherein antibody is called as ZCE025.
24, the methods of treatment of claim 23, wherein Zorubicin by link 3 of cynnematin '-nitrogen of amino saccharides on the carbonyl of carbonate group is attached on the cynnematin.
CN93109871A 1992-07-06 1993-07-06 Be used to discharge the method for cytotoxic agents and its component Pending CN1087642A (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US90992492A 1992-07-06 1992-07-06
US909,924 1992-07-06

Publications (1)

Publication Number Publication Date
CN1087642A true CN1087642A (en) 1994-06-08

Family

ID=25428053

Family Applications (1)

Application Number Title Priority Date Filing Date
CN93109871A Pending CN1087642A (en) 1992-07-06 1993-07-06 Be used to discharge the method for cytotoxic agents and its component

Country Status (6)

Country Link
CN (1) CN1087642A (en)
AU (1) AU4663493A (en)
IL (1) IL106228A0 (en)
MX (1) MX9304056A (en)
WO (1) WO1994001137A1 (en)
ZA (1) ZA934825B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025061A1 (en) * 1993-04-23 1994-11-10 Glyko, Inc. Methods and compositions for treating diseases with carbohydrate modifying enzymes
US5928888A (en) * 1996-09-26 1999-07-27 Aurora Biosciences Corporation Methods and compositions for sensitive and rapid, functional identification of genomic polynucleotides and secondary screening capabilities
US7767792B2 (en) 2004-02-20 2010-08-03 Ludwig Institute For Cancer Research Ltd. Antibodies to EGF receptor epitope peptides

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4975278A (en) * 1988-02-26 1990-12-04 Bristol-Myers Company Antibody-enzyme conjugates in combination with prodrugs for the delivery of cytotoxic agents to tumor cells
KR0185967B1 (en) * 1989-01-23 1999-05-01 에프. 쥐. 엠. 헤르만스; 에이. 쥐. 제이. 베르미렌 Site-specific in-vivo activation of therapeutic drugs
AU633867B2 (en) * 1989-02-02 1993-02-11 Eli Lilly And Company Delivery of cytotoxic agents
AU649275B2 (en) * 1990-11-06 1994-05-19 Bristol-Myers Squibb Company Prodrugs for beta-lactamase and uses thereof

Also Published As

Publication number Publication date
WO1994001137A1 (en) 1994-01-20
ZA934825B (en) 1995-01-05
MX9304056A (en) 1994-05-31
AU4663493A (en) 1994-01-31
IL106228A0 (en) 1993-11-15

Similar Documents

Publication Publication Date Title
JP6955042B2 (en) Uniform antibody drug conjugate by enzymatic method
WO2020090979A1 (en) Compound comprising substance with affinity for antibody, cleavage site and reactive group, or salt thereof
CN105636612B (en) Antibody-drug conjugates and methods of use and treatment
CN110526933B (en) Biotin variants, streptavidin mutants and use thereof
US8664407B2 (en) Chemical linkers with single amino acids and conjugates thereof
EP0392745B1 (en) Immunoconjugates and prodrugs and their use in association for drug delivery
Schneider et al. Recent progress in transglutaminase-mediated assembly of antibody-drug conjugates
KR102076348B1 (en) Human arginase and site-directed pegylated human arginase and use thereof
CN114395050A (en) Amatoxin-antibody conjugates
CN1044095A (en) Macrocyclic bifunctional chelants, its title complex and their antibody conjugates
CN1826331A (en) Pi-3 kinase inhibitor prodrugs
CN1511044A (en) Elogated and multiple spacers in activatible prodrugs
CN1956722A (en) Improved cytotoxic agents comprising new maytansinoids
CN101312748A (en) Antibody-drug conjugates and methods of use
CN1074911A (en) The material of biological action is bonded to compound, composition and method on the biomone facial mask
CN1272841A (en) Novel lactam metalloprotease inhibitors
CN1171054A (en) Compound
CN1058595A (en) Thioalkylthio cephalosporin derivatives
JPH02247164A (en) Method for introducing cytotoxic agent
CN1087642A (en) Be used to discharge the method for cytotoxic agents and its component
US20240123079A1 (en) Immunomodulatory antibody-drug conjugates
CN1193278A (en) Chemical compounds
CN1668759A (en) Method of searching substane having antidiabetic activity
US20230149558A1 (en) Duocarmycin derivative and use thereof
CN1206231C (en) 7-acylamino-3-heteroarylthio-3-cephem carboxylic and prodrugs thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C01 Deemed withdrawal of patent application (patent law 1993)
WD01 Invention patent application deemed withdrawn after publication