CN1391487A - Methods for treating therapy-resistant tumors - Google Patents

Methods for treating therapy-resistant tumors Download PDF

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CN1391487A
CN1391487A CN00813015A CN00813015A CN1391487A CN 1391487 A CN1391487 A CN 1391487A CN 00813015 A CN00813015 A CN 00813015A CN 00813015 A CN00813015 A CN 00813015A CN 1391487 A CN1391487 A CN 1391487A
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cell
prodrug
kinase
infectosome
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H·M·谢帕德
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Celmed Oncology USA Inc
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Celmed Oncology USA Inc
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • 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
    • A61K47/556—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 enzyme catalyzed therapeutic agent [ECTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Abstract

This invention provides a method for selectively inhibiting an infectious agent or a cell infected by an infectious agent by contacting the infectious agent or the cell infected with the agent with a prodrug that is selectively converted to a toxin by an activating enzyme expressed by the infectious agent. The activating enzyme is selective for the enzyme expressed by the infectious agent as compared to the same or similar enzyme expressed by the host cell or other infectious agents. The activating agent is not inhibited nor inactivated by the prodrug. Screens for identifying prodrugs are also provided herein.

Description

Enzymatic resisting-the treatment of infection agent
CROSS-REFERENCE TO RELATED APPLICATIONS
According to 35U.S.C. § 119 (e), the application requires the U.S. Provisional Application No. of the registration number No.60/145364 of the registration number No.60/153101 of JIUYUE in 1999 application on the 9th and application on July 22nd, 1999, and its disclosure is introduced into as reference disclosed by the invention.
Technical field
The treatment field that the present invention relates to catch more particularly, relates to compositions and the method that treatment is had the infectious disease of resistance for the treatment of.
Background of invention
The disclosure text from start to finish, by first author and date, the patent No. or each document of publication number incorporated by reference.Can see total bibliography of each list of references in this description.The disclosure of these publications is introduced in the disclosure text as a reference to describe the situation of this area involved in the present invention in more detail.
Infectious disease is a main health care problem to the resistance of chemotherapy and antibiotic therapy.A lot of Drug resistance are enzyme mediations in infectious disease.Typically, the enzyme that infectosome (infectious agent) is expressed changes chemotherapeutant or antibiotic fast, thereby destroys its therapeutic activity.In infectious disease, the influence that the amplification of beta-lactamase is expressed accounts for more than 1/3rd of all beta-Lactam antibiotic resistance separators (Felmingham and Washington (1999) chemotherapy magazine (J.Chemother) 11 supplementary issue 1:5-12), comprises most of resistance hemophilus influenzas (Haemophilis influenza) (upper respiratory tract infection) and morazella catarrhalis (Moraxella catarrhalis) (otitis media).In addition, natural existence have the gene of resistance to various dissimilar antibiosis, and generally gets more and more in infected organism population.Recently, carry simultaneously the infectosome that multiple antibiosis is have a gene of resistance and cause that traditional antibiotic therapy produces difficulty.The applicant has found to develop the treatment new way of the enzyme that targeting characterized well, and described enzyme is expressed by infectosome.This technology is different with previous and historical approach to the infectious disease treatment, and is referred to as " ECTA ", promptly enzymatic therapeutic agent.
Of the present invention open
The invention provides the method that selectivity suppresses the propagation of the cell that infectosome or infectosome infect.The infectosome that is fit to treat with method of the present invention is expressed the selective activation prodrug or prodrug is converted into the activated material of toxin.Described enzyme is not by deactivation of substrate prodrug compound or inhibition.Thereby this method requires to make the substrate compounds selectivity of described cell or described material contact effective dose to suppress infectosome, the propagation of cell or intracellular infectosome.
The present invention also provides a kind of method of screening prodrug, and the kinase that wherein said prodrug is expressed by infectosome is selectively converted to toxin in cell.Cells contacting candidate's prodrug and mensuration kinase that screening requires infectosome or infectosome are infected are the activation of toxicant with the prodrug activation.Perhaps, the inhibitory action of the propagation of the cell that infects by monitoring infectosome or described infectosome or growth is measured the activation of prodrug.
Accompanying drawing is briefly described
Fig. 1 illustrates the mechanism of action of ECTA prodrug of the present invention.
Fig. 2 is the fluorescence-causing substance that obtains is cultivated in explanation from bromoethyl 2 '-deoxyuridine monophosphate (BVdUMP) and recombined human thymidylate synthetase (rHuTS) figure.The incubation of BVdUMP and thymidylate synthetase causes the time of fluorescence-causing substance and enzyme dependency to produce.The rHuTS of BVdUMP and specified amount in 30 ℃ of following incubations (material and method), has still saved N5, the N10-methylene tetrahydrofolate from reaction in the standard reaction mixture.The numeral adjacent with each data and curves is referring to the TS enzyme unit.
Fig. 3 explanation is BVdUMP and deoxyuridine monophosphate (dUVP) competition in rHuTS.There is not (triangle) and having under (square) 20 μ M BVdUMP the external catalytic reaction of thymidylate synthetase of carrying out dUMP is converted into dTMP.The concentration range of dUMP is 10-100 μ M, N5, and N10-methylene tetrahydrofolate concentration is 140 μ M, enzyme concentration is 0.1 μ M.By measuring A 340Increase measure enzymatic activity.
Fig. 4 is the structure of product of the vitro reactions of the catalytic BVdUMP of rHuTS.Structure I and II and the mass ion that does not have to identify in the reactant mixture of cell match.
Fig. 5 is the activated mechanism of NB1011 of inferring.NB1011 one enters cell surely and be converted into BVdUMP before reacting with TS.Inferring the structure that produces after TS transforms is the acid of outer shroud pyrimidine nucleoside monophosphate.These chemical compounds can pair cell has cytotoxicity by various mechanism, comprises disturbing nucleotide and nucleic acid metabolism.
Fig. 6 explanation is to the detection of BVdUMP in the H630R10 cell of handling with NB1011.With 100 μ M NB1011 the H630R10 cell was handled 5 days, the liquid chromatography/mass spectrometry of carrying out then describing in material beneath and the method is analyzed.
Fig. 7 proves that NB1011 can be at external irreversible deactivation TS.Active effect is reversible to NB1011 fully to TS in the intact cell.Described in materials and methods, by from 5-[ 3H] BrdU release [ 3H] 2O measures TS activity in the complete RKO cell.By changing fresh culture, cultivated 60 minutes down at 37 ℃, repeat this process then, from cell, wash out NB1011.Contrast is carried out identical washing procedure with the cell of not handling.
Fig. 8 illustrates the interior TS expression of cell with bolster enlightening this (Tomudex) or NB1011 screening of the SDS PAGE Western blotting estimation of carrying out with anti-thymidylate synthetase antibody and tubulin.Swimming lane 1 expression MCF7 cell, drug screening of no use; The MCF7 cell that swimming lane 2 expressions are screened with 2 μ M tomudex; Swimming lane 3 is represented the MCF7 cell as swimming lane 2, but uses NB1011 to carry out follow-up screening as selective reagent before; Swimming lane 4 is represented the MCF7 cell as swimming lane 2, pass through under the Tomudex not having before.
Implement method of the present invention
Except as otherwise noted, enforcement of the present invention well known to a person skilled in the art molecular biology with application, microbiology, the routine techniques of cytobiology and recombinant DNA.Referring to for example Sambrook, Fritsch and Maniatis, molecular cloning: laboratory manual (MOLECULARCLONING:A LABORATORY MANUAL) second edition (1989): molecular biology current methods (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (F.M.Ausubel etc. write (1987)); Enzymology method (METHODS IN ENZYMOLOGY) series (AcademicPress.Inc.): PCR2: practical approach (PCR2:A PRACTICALAPPROACH) (M.J.MacPherson, B.D.Hames and G.R.Taylor write (1995)) and animal cell culture (ANIMAL CELL CULTURE) (R.I.Freshney writes (1987)).
As in description and claims, using, unless outside clear herein the indicating, " one " and " this " of singulative comprise most forms.For example, term " cell " comprises the plural number of cell, comprises its mixture.
Term " comprises " and means compositions and method comprises indicated key element, but do not get rid of other.When being used for definitions section compound and method, " basically by ... form " mean do not comprise any to significant basically other key element of combination.Therefore, the compositions of being made up of some key elements does not basically as defined herein get rid of to come for example phosphate buffer of self-separation and purification process and pharmaceutical acceptable carrier, the trace impurity of antiseptic etc." by ... form " should refer to not comprise the key element of Duo and be used to use the basic method steps of compositions of the present invention than the trace key element of other composition.These each self-defining embodiments of conversion term within the scope of the invention.
" infectosome " or " pathogen " is to be pathogenic organism to the cell of its infection or organism.The example of pathogenic organisms body includes but not limited to antibacterial, parasite, virus or yeast.The example of virus includes but not limited to herpesvirus, Varicella zoster, hepatitis C virus and EpsteinBarr virus.Parasitic example comprises Bu Shi trypanosoma (T.brucei), Ke Shi trichomonacide (T.cruzi) and plasmodium Plasmodium falcipurum.The example of antibacterial includes but not limited to all Gram-negatives and gram-positive bacterium, staphylococcus particularly, Enterococcus, mycoplasma (Myoplasma sp.), large intestine Chinese mugwort Xi Shi Bacillus, Rhodopseudomonas, eisseria (Nisseria sp.), and wherein preferred pathogen is those (referring to Murray, BE etc. " antibiotic resistant " are (1997) Adv.Int.Med.42:339-367 (AntibioticResistance)) that conventional antibiotic produced resistance.
" compositions " means active substance and inert (but for example detectable or label or pharmaceutical acceptable carrier) or other chemical compound of active (for example adjuvant) or the combination of composition.
" pharmaceutical composition " means and comprises that compositions is fit to is external, in the body or the active substance used of diagnosis ex vivo or treatment and the combination of inertia or active carrier.
As used herein, term " pharmaceutical acceptable carrier " comprises any standard pharmaceutical carrier, phosphate buffer for example, water, and emulsion, for example oil/water or water/oily emulsion and various types of wetting agent.Said composition also comprises stabilizing agent and antiseptic.Carrier, the example of stabilizing agent and adjuvant be referring to Martin, REMINGTON ' S PHARM.SCI. the 15th edition (Mack Publ.Co., Easton (1975)).
" effective dose " is the amount that is enough to obtain favourable or desired effects.Can one or many give with, use or effective dose is used in administration.
" experimenter ", " individuality " or " patient " exchanges use here, refers to vertebrates, preferred mammal, more preferably people.Mammal includes but not limited to Mus, monkey, people, farm-animals, motion animal and house pet.
" contrast " is other experimenter or the sample that uses in experiment, is used to contrast purpose.Contrast can be " positive " or " feminine gender " contrast.For example, experiment purpose is when measuring the concerning of the expression of change of gene and certain particular type pathogen, general preferred use positive control (carry this change and show this feature that catches symptom the experimenter or from experimenter's sample), and negative control (do not have the expression of this change and this disease Clinical symptoms the experimenter or from experimenter's sample).
As used herein, term " kinase " refers to the enzyme that the pathogen in its nature or the natural surroundings is expressed.It is applied enzyme or other material that activates prodrug in order to distinguish.
As used herein, term " pathological cells ", " target cell " and " test cell " comprises and is characterised in that the cell that has kinase.Pathogenic infection causes the expression that kinase takes place as mentioned above.Pathogen is expressed or the enzyme in the infection cell that the target thing is provided for this treatment includes but not limited to thymidylate synthetase and dihydrofolate reductase.Listed other example below.
Therapeutic Method
On the one hand, the present invention relates to suppress the propagation of the cell that infectosome or this infectosome infect or the method for growth, this method is by making the cells contacting substrate prodrug of infectosome or infection, and the kinase that described prodrug is expressed by infectosome optionally is converted into toxin in cell.Method and composition of the present invention is used for preferred growth or the propagation that suppresses to express or comprise the cell of kinase, and described cell for example is a microbial cell, the cell of virus infected cell or other pathogenic infection.Do not require the overexpression of enzyme, because specificity is relevant to the species specificity of the kinase of pathogen expression with prodrug.Kinase can also can be can't help host cell expression by host cell expression.But, even the enzyme of himself version of cellular expression also can screen prodrug on this basis, to compare with the enzyme version of host cell expression, the version of the enzyme that its preferential infected body surface reaches activates.Kinase can be mutant version (Hooker etc. (1996) Journal of Virologies (J.Virol.) 70 (11): 8010-8018) wild type or that prior art is treated the generation resistance.Example as the kinase of the selectivity target thing of prodrug of the present invention and method includes but not limited to thymidylate synthetase (TS), dihydrofolate reductase (DHFR) and beta-lactamase kinase.
Utilize activation thymidylate synthetase and the expression in human tumor cells thereof to describe notion of the present invention in detail.But it is illustrative using TS, does not think that claims only are confined to the system at TS.Thymidylate synthetase is used as the target thing here, kinase, because the height of its 26S Proteasome Structure and Function characterizes (Carreras and Santi (1995) Anne.Rev.Biochem64:721-726), it is by the fact (comparative example is as being appointed as the enzyme family of glutathione-S-transferase (GST)) of term single gene rather than gene cluster coding.In addition, the TS overexpression is by chemotherapeutical acquired resistance is caused.Similarly, in one embodiment, the result that kinase can be used as the resistance of previous treatment is expressed.
Other targeted activation enzyme includes but not limited to viral reverse transcriptase and protease.The example of the virus of encoding such enzymes includes but not limited to retrovirus (HIV-1 for example, two kinds of enzymes, referring to TurnerB.G. and Summers M.F. (1999) molecular biology magazine (J.Mol.Biol.) 285:1-32), picorna virus (hepatitis A virus (HAV) for example, and hepatitis C virus (KwongA.D. etc. (1999) antiviral research (Antiviral Res.) 41:67-84) Wang Q.M. (1999) Prog.DrugRes.52:197-219).Success (the summary of Shafer R.W. and Vuitton D.A. (1999) of using anti-HIV 1 reverse transcriptase and protease inhibitor to obtain clinically in early days, Biomed.Pharamcother.53:73-86), be affected owing to developing immunity to drugs, this mainly is because ((1999) acquired immune deficiency syndrome (AIDS) magazine (J.Acquir.Immune Defic.Syndr.) 21:203-208 such as Catucci M. that the sudden change of encoding viral enzyme causes; (1999) european journal of biological chemistry (Eur.J.Biochem) 263:238-44 such as MahalingamB.; And Palmer, S etc. (1999) acquired immune deficiency syndrome (AIDS) (AIDS) 13 (6): 661-667).In current clinical development, high drug-resistance HIV-1 clinical isolates has cross tolerance with a lot of anti-retroviral virus compounds.Hooker etc. (1996) above.In these Drug resistance situations, viral enzyme keeps their catalytic activity, because the mutant of this enzyme keeps the structure in the wild-type activity site of this enzyme.Specific design prodrug of the present invention and this avtive spot interact and are converted into toxin by this interaction.Therefore, drug-resistant virus infects being referred to as the substrate prodrug sensitivity of ECTA chemical compound, and described ECTA chemical compound needs kinase to produce toxin in infection cell.NB1011 is an example of such chemical compound, is oriented to the TS that mammal and people's cell and pathogen are expressed.
In one embodiment, described prodrug is the chemical compound with structure of more detailed description definition here.The term prodrug refers to the parent of active therapeutic agent.Perfectly prodrug is to be inert prodrug on the pharmacology before the mechanism that is supposed to activates.The prodrug scheme is meant has the toxic Therapeutic Method of potential orientation to focus, thereby avoids general toxicity.A lot of explorations have been carried out at this purpose.Mead etc. (1996) cancer research (CancerRes.) 26:2374-2379 and Nichol and Hakala (1966) biochemical pharmacology (Biochem.Pharmacol.) 15:1621-1623 have reported and have attempted the first time of the prodrug that is used for treatment of cancer.The guiding principle of this trial is to be the leukemic directed overexpression dihydrofolate reductase of methotrexate-resistance.Because the rising of dihydrofolate reductase may be converted into homofolic acid the metabolic poison to thymidylate synthetase in methotrexate-resistance tumor cell, expect to have self toxicity of tumor cell.The rational antitumous effect of finding homofolic acid afterwards is not owing to metabolic activation, and more may be because to (Livingston etc. (1968) biochemistrys (Biochem.) 7 (8): 2814-2818) of the inhibitory action in the folic acid transporte to cells.Having gathered prodrug-sample of the cracking tumor-selective target thing that is used to treat in the following table 1 attempts.These discussions have been obscured for one or several of following exercise question: a) suitably locate kinase and/or lack the tumor-selective of targeting enzymes; B) system of the activated prodrug toxicity that distributes and obtain; What and c) realization needed prevents by the activated substrates enzymes specificity of the enzyme except that the enzyme of targeting.For example, glutathion-s-transferring enzyme (GST) prodrug that Morgan etc. (1998) cancer researches (CancerRes.) 58:2568-2575 describes, to GST be " specific " simultaneously, just do not activated, and can be activated by GST-A1-1 by the GST-P1-I of tumor overexpression.This causes unsuitable medicine activation and potential toxicity.More gone through such argumentation in the incorporated by reference document that in table 1 above, provides.Activate described prodrug and before used kinase not activate the prodrug of target enzyme of the suitable character of this cell in only having in suitable target cell specificity by the exploitation targeting, method and composition disclosed by the invention has been avoided the shortcoming of prior art prodrug.
In another aspect of the present invention, described prodrug does not have toxicity basically to the cell that not have normally infection.This aspect has further promoted the selectivity of prodrug and has improved total safety of treatment.Described prodrug can the selectivity cell killing, because have only the cell of infection to provide the toxic metabolite of the prodrug of effective dose to suppress pathogen or by the propagation of the cell of pathogenic infection.In other words, the optimum efficiency of prodrug of the present invention is relevant with the source of kinase.For example, the disclosed research of from following table 2, summing up (Barr (1983) journal of biological chemistry (J.Biol.Chem.) 258 (22): 13637-13631), do not reckon with that NB1011 likens to more effective to the substrate of microorganism TS as the substrate to people TS.
Can use several different methods to determine whether to meet the purpose of Therapeutic Method.This includes but not limited to that RT-PCR analyzes growth inhibited research (alamar blue test) and plaque assay.These methods are known and here describe.
The applicant finds that also the cell of crossing with the substrate prodrugs therapy can be converted into the previous phenotype of suitably treating by routine treatment.With TS as an example, the applicant proves that the tumor cell of handling with 5-FU becomes to drug resistant.At this moment, handle cell with NB1011.But subgroup survives and becomes to be had resistance to NB1011 obtains sensitivity (referring to table 9 and Fig. 8) to 5-FU once more.Therefore, the invention provides said method, wherein the another kind of anti-infective of effective dose is used with substrate prodrug of the present invention.On the one hand, second kind or the third activating agent are that pathogen is to its medicine that develops immunity to drugs in advance.Described other activating agent can be when using the substrate prodrug or administration subsequently.
The present invention further provides, compared with the cell that does not have to infect, the prodrug of the kinase selective conversion of infected body or pathogen generation or expression, described cell for example is a zooblast, mammalian cell, or people's cell.The applicant finds several enforcements preferred precursor medicine of the present invention.Describe in the structure of these chemical compounds and the synthetic method materials and methods below.
As used herein, term " contact " comprises external, exsomatize and body in use prodrug.When vivo medicine-feeding, the experimenter is used the effective dose prodrug.As used herein, term " experimenter " meaning comprises all suitable animal models, mice for example, rat, rabbit, monkey.Also comprise administration to people patient.
Another aspect of the present invention is by a kind of prodrug of experimenter's administering therapeutic effective dose being treated the experimenter who has infected pathogen, being selectively converted to toxin in the kinase cell that described prodrug is defined here.Described enzyme needn't overexpression.On the other hand, give with described substrate prodrug in, before or after use at least a other therapeutic agent of effective dose jointly.
When to experimenter mice for example, when rat or people patient use described prodrug, this prodrug can be added in the pharmaceutical acceptable carrier and to administration of experimenter's whole body or topical.
With dose, perhaps continous way or batch (-type) carry out vivo medicine-feeding in whole therapeutic process.The method of measuring effective means and application dosage is known and along with the compositions that is used for the treatment of for those skilled in the art, the purpose of treatment, and the target cell of treatment is with the experimenter of treatment and different.Undertaken in single or divided doses by dosage level and mode that the treatment doctor selects.Can see proper dosage form and the method for using described material below.
Pharmaceutical composition can be oral, intranasal, and parenteral or by the anapnotherapy administration, and can be tablet, lozenge, granule, capsule, pill, ampoule, suppository or aerosol form.They also can be active component in the moisture or suspension in the aqueous diluent not, solution and emulsion form, syrup, granule or powder agent form.Except chemical compound of the present invention, described pharmaceutical composition can also contain other medicines reactive compound or multiple chemical compound of the present invention.
More particularly, the chemical compound of structural formula of the present invention also refers to do active component here, can use by any suitable way to be used for the treatment of, comprise oral, rectum, nose, the part (comprises percutaneous, aerosol, contain agent and Sublingual), vagina, parenteral (comprises subcutaneous, intramuscular, intravenous and Intradermal) and lung in.It is also understood that optimization approach will be along with the disease of receiver's situation and age and treatment and different.
Generally speaking, for the proper dosage of each above-claimed cpd be every day per kilogram receiver body weight about 1 to about 100 nanogram ranges, preferably every day, per kilogram of body weight about 1 was to about 50 nanogram ranges, and most preferably every day, per kilogram of body weight about 1 was to about 25 nanogram ranges.Except as otherwise noted, all reactive compound weight is calculated for its salt or its ester parent compound with structural formula of the present invention, and weight will increase in proportion.Preferably, the dosage of expectation was used twice, three time with suitable interval in last one day, and four times, five times, six times or more times sub-doses.Can use these sub-doses with unit dosage form, for example the per unit dosage form contains about 1 to about 100 milligrams, and preferably approximately 1 to greater than about 25 milligrams, and most preferably about 5 to greater than about 25 milligrams active component.The dosage that is appreciated that chemical compound of the present invention and compositions can depend on disease type and the order of severity and stage, and can be different with patient's difference along with the patient.Determine that optimal dose relates generally to the balance of the useful level of treatment of the present invention and any risk or disadvantageous side effect.
Ideal situation is to use the maximum concentration that described prodrug is implemented in the affected area reactive compound.For example, randomly perhaps oral in saline by the described prodrug of intravenous injection, for example, as the tablet that contains active component, capsule or syrup are realized.By can keep the blood levels of the prodrug expected in the continuous diseased tissue of infusing with the active component that therapeutic dose is provided.The application of operative combination relates to provides therapeutic combination, and it respectively treats chemical compound or method than independent use may need various medicines than low dosage, thereby reduces side effect.
It is another aspect of the present invention that prodrug described herein is combined with above-mentioned other treatment.For example, prodrug described herein preferably be not that the medicine that prodrug of the present invention institute brings into play their toxic action through approach combines by utilizing.
Although it is possible using described prodrug composition separately, it preferably exists with the pharmaceutical preparation that contains at least a active component as defined above and one or more its carriers and randomly other treatment agent.From with the compatibility of other composition of preparation with the patient is not had on the meaning of infringement, various carriers must be " acceptable ".
Preparation comprise be fit to oral, rectum, nose, local (comprise percutaneous, contain agent and Sublingual), vagina, those of parenteral (comprise subcutaneous, intramuscular, intravenous and Intradermal) and feeding drug into pulmones.Preparation can exist with unit dosage form easily and can prepare with the known any method of pharmaceutical field.Such method comprises mixes active component with the carrier that constitutes one or more auxiliary elements.Generally speaking, the solid carrier by equably and nearly mixed active composition and liquid-carrier or porphyrize or both prepare preparation, then, if desired, with formed product.
Being fit to oral preparation of the present invention can exist with discrete unit, capsule for example, and cachet or tablet contain the active component of scheduled volume separately; As powder agent or granule; As solution or the suspension in moisture or not liquid, aqueous; Perhaps as oil-in-water liquid emulsion or Water-In-Oil liquid emulsion.Active component also can be with bolus, and electuary or paste exist.
By randomly suppressing or moldedly can preparing tablet with one or more auxiliary elements.Can prepare tabletting by compacting active component in suitable machine, wherein active component is free-flowing form, for example is powder agent or granule, randomly with binding agent (polyvinylpyrrolidone for example, gelatin, hydroxypropyl emthylcellulose), lubricant, inert diluent, antiseptic, disintegrating agent (Explotab for example, crospolyvinylpyrrolidone, cross-linking sodium carboxymethyl cellulose), surfactant or dispersant.Prepare the mold pressing tablet by mold pressing in suitable machine with the mixture of the moistening powder compounds of inert liquid diluent.Tablet is coating or indentation and can be mixed with can be slowly or the dosage form of controlled release active component wherein randomly, for example, uses the hydroxypropyl emthylcellulose of different proportion that the release profiles of expectation is provided.Tablet can randomly carry out enteric coating, is provided at the part of intestinal rather than release under one's belt.
Be adapted at that the preparation of topical comprises lozenge in the mouth, it contains active component in flavoring substrate, normally sucrose and arabic gum or Tragacanth; Pastille, it is at inert base for example gelatin and glycerol, or contains active component in sucrose and the arabic gum; And collutory, it contains active component in suitable liquid-carrier.
Pharmaceutical composition according to topical of the present invention can be mixed with ointment, ointment, suspension, lotion, powder agent, solution, paste, gel, spray, aerosol or grease.Perhaps, preparation can comprise that transdermal pastes or dressing, for example is soaked with active component and one or more excipient chosen wantonly or the binder or the sticking Gypsum Fibrosum of diluent.
For eye or other outside organization for example mouthful and skin, preferably with the part of containing active component with ointment or emulsifiable paste, amount with active component is, for example, about 0.075 to about 20%w/w, preferably approximately 0.2 to about 25%w/w, most preferably about 0.5 to about 10%w/w amount administered formulation.When being mixed with ointment, prodrug can use with paraffin or the not miscible substrate of water.Perhaps, can in emulsifiable paste, use oil-in-water emulsifiable paste matrix preparation prodrug composition.
If desired, the water of emulsifiable paste matrix can comprise, for example, at least about polyhydroxy-alcohol of 30%w/w promptly has the alcohol of two or more hydroxyls, propylene glycol for example, butane-1,3-glycol, mannitol, Sorbitol, glycerol and Polyethylene Glycol and composition thereof.Topical preparation preferably includes and strengthens the prodrug composition by skin or the Absorption of other involved area or the chemical compound of penetration.The example of such epidermis penetration enhancers comprises dimethyl sulfoxine and related analogs.
The oil phase of emulsion of the present invention can constitute with known composition with known method.This can only contain emulsifying agent (being also referred to as emulsifying agent emulgent) mutually, preferably contains at least a emulsifying agent and fat or oil or fat and both mixture of oil.Preferably, contain hydrophilic emulsifier and lipophilic emulsifier, the latter is as stabilizing agent.Also preferably contain oil ﹠ fat both.Emulsifying agent constitutes the material that is referred to as the emulsifying wax being with or without under the situation of stabilizing agent, and this wax is made the material that is referred to as the emulsifying ointment base with oil and/or fat, and it forms the oily decentralized photo of cream preparation.
The emulsifying agent and the emulsion stabilizer that are adapted at using in the preparation of the present invention comprise Tween60, Span80, cetostearyl alcohol, tetradecyl alchohol, glyceryl monostearate and sodium lauryl sulfate.
Be used for the suitable oil of preparation or the fatty cosmetic character of selection, because the dissolubility of reactive compound in most of oil that may use is very low in the medicine emulsification preparation to realize expecting.Therefore but ointment should preferably not have greasyly do not have color and product flush away, and it has suitable persistency, avoids from test tube or other container leakage.Can use one of straight or branched-or dialkyl, two dissidents, two acid esters for example, stearic acid isocetyl ester, the coco-nut oil fatty acid propylene glycol diesters, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, Palmic acid 2 one Octyl Nitrites or be called the mixture of Crodamol CAP branched ester, back three kinds is preferred ester.These can use separately or properties of combination is as requested used.Perhaps, can use high-melting-point lipid, for example paraffinum molle alba and/or liquid paraffin or other mineral oil.
Suitable preparation to the eye topical also comprises puts drops in one's eyes, and wherein active component is dissolved in or is suspended in the suitable carriers, and carrier is especially for the aqueous solvent of prodrug composition.Described prodrug composition preferably exists with such preparation, and wherein concentration is about 0.5 to about 20%, advantageously about 0.5 to about 10%, particularly about 1.5%w/w.
The preparation that is used for rectally can be used as suppository and exists, and suitable substrate is arranged, and comprises for example cocoa butter or salicylate.
The preparation that is fit to vagina administration can be with suppository, tampon, and emulsifiable paste, gel, paste, foam or spray exist, and contain suitable carriers well known in the art except the prodrug composition.
Wherein carrier is that the preparation that solid is fit to nasal administration comprises having for example about 20 coarse powder to about 500 micrometer range granularities, and its mode administration to suck with strength is promptly by sucking fast by nasal meatus from the container of the Sheng powder of being close to nose.Wherein carrier is the appropriate formulation that is used for the liquid of administration, nasal spray for example, nasal drop or comprise the aqueous solution or the oil solution of prodrug composition by the preparation of aerosol apparatus aerosol drug delivery.
The preparation that is fit to parenteral comprises and moisture and water-free etc. oozes sterile solution for injection, and it can contain antioxidant, buffer, antibacterial and the isoosmotic solute of blood that makes preparation and receiver to be administered; (it is designed to make the form of described targeting compounds blood constituent or one or more organs for moisture and water-free sterilization suspension (it can contain suspending agent and thickening agent) and liposome or other micronize system.Preparation can be divided in the container of unit dose or multiple dose sealing, for example ampoule and little phial, and can be kept under lyophilization (lyophilizing) condition, before using, require only to add sterilized liquid carrier, for example water for injection.Can be from the sterile powder agent of previous description type, granule and preparation tablets i.e. the injection and the suspension of usefulness.
Preferred unit dose formulations is the above-mentioned daily dose or the unit of containing of prodrug composition, those of day sub-doses, perhaps its suitable equal portions.
Should be appreciated that except the top composition of mentioning especially, preparation of the present invention dosage form as requested can contain other this area conventional substances, the preparation that for example is fit to oral administration can contain and resembles sweeting agent, other reagent that thickening agent and correctives are such.
The prodrug of structural formula of the present invention and compositions also can exist with the veterinary drug preparation form, and it can be for example by the preparation of this area conventional method.
Material of the present invention and compositions can be used for preparing medicine and be used for the treatment of people and other animal by for example using at the active component of pharmaceutical composition according to conventional methods.
Filler test
The present invention further provides a kind of by the cell of expressing kinase being provided and making this cells contacting candidate prodrug by being that kinase is converted into the method that toxin screens described prodrug with described prodrug.At least a this enzyme of test cellular expression pathogen text (wild type or saltant) and another kind of test cell on from the cell sample of host organisms, it can or can not express self text of this enzyme.The kinase of measuring the pathogen generation then is converted into prodrug the transformation of toxicant.As used herein, the test cell can be to have infected pathogen or transformed the prokaryotic cell or the eukaryotic cell of expressing described kinase.For example, the prokaryote bacillus coli of endogenous expression endocellular enzyme TS is not proper host cell or target cell.Perhaps, test cell can be from the isolating infection cell of experimenter, has perhaps infected the cultured cell of pathogen.Cell can have contrast homologue (lacking the target enzyme), perhaps in another embodiment, is the homologue (comprising suitable target enzyme species) through genetic modification differential expression target enzyme or enzyme.Can use more than one enzyme each host cell of transduceing respectively, make drug candidate compare to another kind of enzyme or from the effect of the corresponding enzyme of another species with it simultaneously the effect of target enzyme.
In another embodiment, the 3rd target cell is used as positive control, because it accepts the chemical compound of effective dose, chemical compound for example given below, they are effective prodrug.
In another embodiment, cell transformed is the NIH 3T3 cell (ATCC that transforms of Ras-for example, 10801University BLVD., Manassas, VA20110-2209, U.S.) handle to make through genetic engineering and express interested target enzyme from the clone cDNA variable and the recruitment of the described enzyme of encoding.Transfection is to use method well known in the art and the above transient transfection or the permanent transfection of the method described such as Sambrook.The suitable carriers of inserting described cDNA is from Stratagene, LaJolla, and CA and other suppliers are buied.Use the monoclonal or the polyclonal antibody of the anti-described enzyme that produces in advance to be used for immune detection,, can monitor the expression of enzyme in each cells transfected system by the immunoblotting and the immunoassay of cellular lysate.Copy number by importing to the expression cassette in the cell or by utilizing different promoteres can regulate expression.Also can be according to Carreras and Santi (1995), above, or method described below detects the enzymatic determination of amount of the enzyme of expression.
The test cell cultivates and is used to the biological activity of determination test prodrug on little porous plate.For the object of the invention, successful drug candidate will suppress the growth of pathogen or kill it and still do not injure the control cells type.
Can directly add candidate's prodrug in the cell culture medium or the specific part of a kind of pair cell surface receptor of coupling in advance adds culture medium then.It is well known in the art that cell-specific send the coupling method of passing, referring to for example U.S. Patent No. 5459127; 5264618; With disclosed patent specification W091/17424 (on November 14th, 1991 is open).The leaving group of candidate's prodrug can be used for example tritium detectability labelling.Then target cell or culture medium are measured from the amount of the labelling of candidate's prodrug release.Perhaps, by using Lasic, D.D. the method described of (1996) nature (Nature) 380:561-562 is packaged into prodrug in the liposome or according to Lewis, periodical (Proc.Natl.Acad.Sci.USA) 93:3176-3181 of institute of J.G. etc. (1996) NAS is described to be taken in cytofectin combination can enhancing cell.
Should be understood that, although statement clearly always can further not changed each embodiment by the chemical compound (chemical compound for example given below (verified its is potential prodrug)) of accepting effective dose in contrast by different target cells is provided.
Here the activating agent that also provides this method to identify.
In one embodiment, according to following materials and methods and the description in the experimental section, provide test to the prodrug effect by born of the same parents' intracellular metabolite product of analyzing prodrug; The result provides in Fig. 4.In this embodiment, prodrug comprises a detectable label, can prodrug be converted in the noxious substance process its monitoring at kinase.In another embodiment, the candidate prodrug is by the detectability labelling, for example with fluorescent labeling or radiosiotope.On the other hand, the two or more at least same atoms of the detectable label different isotope of bromine atoms for example.In this embodiment, the mass spectrum of people by product can detect prodrug and be modified to toxic by-products.A method that realizes this test is to utilize mass spectrum by more detailed description hereinafter, and the result provides in Fig. 6.
Utilize above-mentioned screening, people also can be from several prodrug of sample prescreen available from experimenter (for example people patient).People can utilize this screening to determine the most effective substrate prodrug and at each pathogen and experimenter's treatment.
I. materials and methods
A. synthetic method
Method (Dyer etc. (1991) nucleic acid chemistry: improved and new synthetic method, method and technology, Townsend etc. (writing) John Wiley﹠amp by Dyer etc.; Sons, Inc., New York, pp.79-83) preparation (E)-5-(2-bromo vinyl)-2 '-BrdU (BVdU).Under vacuum under 75 ℃ dry respectively this chemical compound and (Fisher/Acros) 5-fluoro-2 '-BrdU (5FdU) of buying, before using, use P 2O 5Dry.(CA) enterprising conduct is used to have the Merck silica gel-60 of fluorescence indicator as adsorbent to chromatography for HarrisonResearch, Palo Alto at the Chromatotron instrument.Standard chemical phosphorylation preparation (E)-5-(2-bromo vinyl)-2 '-BrdU 5 '-a phosphoric acid (" BVdUMP ") by BVdU.
On Varian Associates Gemini spectrometer,, use six deuterates-dimethyl sulfoxine (C in the record NMR 1H NMR of 300MHz place spectrum 2H 3) 2SO solution.Represent chemical shift relatively with mark tetramethyl monosilane object of reference in the d=0.0ppm.At 75MHz place record 13C NMR spectrum is represented chemical shift relatively with mark five deuterates-dimethyl sulfoxine in the d=39.5ppm.On the Bruker photometer at 202MHz place record 31P NMR spectrum is with d=0.0ppm external standard 85%H 2O/15%H 3PO 4, the volume/volume ratio is represented chemical shift relatively.
Be prepared as follows NB1011 ((E)-5-(2-bromo vinyl)-2 '-deoxidation-5 '-uridnine base phenyl L-alanyl phosphoramidate (BVdU-PA, " NB1011 ")).Argon drips phenyl L-methoxy propyl aminoacyl phosphoro chloride (phosphorochloridate) (McGuigan etc. (1996) down; pharmaceutical chemistry magazine (J.Med.Chem.) 39:1748-1753) (15; 350 milligrams; 1.26 mM) handle in 2 milliliters of dry DMF (420 milligrams of BVdU; 1.26 mM) and (103 milligrams of imidazoles; 1.51 solution mM), and reactant mixture stirred 24 hours under 23 ℃ of following argon.By on silica gel, using 10%MeOH/90%CH 2Cl 2, volume/volume is carried out TLC as eluant, from starting material (R f=0.53) produces converted product (R f=0.70), but just to a certain degree (about 15%), therefore add other imidazoles (52 milligrams, 0.75 mM) and phosphoro chloride reagent (8,175 milligrams, 0.63 mM), and with this mixture restir 24 hours under 23 ℃ of following argon.By the TLC monitoring, conversion ratio is brought up to about 30% degree.Handle the progress that does not at all promote reaction with other phosphoro chloride and imidazoles below.By rotary evaporation is reduced to 0.75 ml volumes with solution under 40 ℃ of following vacuum approximating, add isopyknic dichloromethane then, solution is directly imposed on dry 4 millimeters silica gel plates.At this moment, if remove most of residual DMF then help following separation by this plate being put into vacuum desiccator 30 minutes.Use 250 milliliters of dichloromethane (coming residual reagent of eluting and DMF), then with 10% methanol/90% dichloromethane, volume/volume (coming eluted product eluting starting material afterwards), carry out radial chromatography, obtain 144 milligrams of (20%) converted products and 294 milligrams of starting materials, based on the starting material that does not have to reclaim, obtain the converted product of 67% productive rate.By 1H NMR detects whether have impurity imidazoles (d=7.65 and 7.01) or DMF (d=7.95,2.89 and 2.73), carries out other radial chromatography purification.In this way, obtain chemical compound 3, TLC and 1It is 98% that H NMR detects purity, is the approaching molar mixture that waits of phosphorus atoms center base diastereomer, is oil/glue or foam-powder type: 1H NMR ((C 2H 3) 2SO) d=11.4 (bs can with 2H 2The O exchange, 1, N3H), 8.28 (vacation-t, 1, H6), 7.35 (vacation-t, 2, o-Ph), 7.31 (d, 1, vinyls 1H), 7.20 (vacation-t, 3, m-and p-Ph), 6.89 (d, 1, vinyl 2H), 6.19 (t, 1, H1 '), 6.08 (t, can with 2H 2O exchange, 1, alanyl NH), 5.45 (bs, can with 2H 2The O exchange, 1, O3 1H), 4.32 (m, 1, H3 '), 4.22 (m, 2,5 ' CH2), 3.97 (m, 1, H4 '), 3.86 (t, 1, alanyl CH), 6.58 (two s, 3, CO 2Me), 2.15 (m, 2,2 ' CH2), 1.23 (vacation-t, 3, alanyl CH3), J vinyl CH-alanyl NH～6Hz. analyze with 1H/1H COSY 2D NMR and determine that spectrum shifts. 13C NMR (( 2H 3O) 2SO)) d=173.7 and 173.6 (alanyl CO 2), 162.1 and 161.6 (C2), 150.61; 150.5 (ipso-Ph), 149.2 (C4), 139.4 and 139.2 (C6); 129.8 with 129.6 (M-Ph), 124.7 (P-Ph), 120.3; 120.2 (o-Ph), 107.1 (vinyl C1), 87.5 (vinyl C2); (84.8 C4 '); (83.8 C1 '), 70.1 (C3 '), 66.1 (C5 '); (51.9 alanyl OMe); 49.7 (alanyl α-H), 29.5 (C2 '), 19.6 (alanyl α-Me); 3JP-C4 '=7.8; 2JP-C5 '=4.4,2JP-ipso-Ph=6.5Hz.31P NMRd=3.99,3.69 low resolution DCI (NH 3), mass spectrum: 593/591
(MNH 4 +)，576/574(MH +)。
Just for convenience, the general structure of the illustrational prodrug that uses in the inventive method is divided into the I group and II organizes.
General the synthesizing of I group chemical compound
The L and the D isomer of I group chemical compound have following structure:
Figure A0081301500231
In the superincumbent structural formula, (5-position) R 1Be or comprise leaving group, it is to have and the molecular size and the electrophilic chemical individual that adapt from separating of pyrimidine ring by kinase (for example thymidylate synthetase), and it has the ability that suppresses described material or cell proliferation when pyrimidine ring discharges by enzyme.
In the superincumbent structural formula, Q is a sugar, is carbocyclic ring or non-ring compound, or it comprises and be selected from glycosyl, sulfo-glycosyl, the phosphate ester of sheltering or the phosphoramidic acid ester derivant of the chemical individual of carbon ring group and its derivant.The example of glycosyl includes but not limited to monosaccharide ring-type glycosyl, for example from oxetanes (4-unit cyclohexanol), and furanose (5-unit cyclohexanol), and deutero-those groups of pyranose (6-unit cyclohexanol).The example of furanose comprises furan Soviet Union-glycosyl (from threose, a kind of 4 carbon atom sugar); Furan erythrose base (from erythrose, a kind of 4 carbon sugar); Ribofuranosyl (from ribose, a kind of 5 carbon sugar); Arabinofuranosyl (also is referred to as the arabinofuranosyl base; From arabinose, a kind of 5 carbon sugar); Furyl xylose base (from xylose, a kind of 5 carbon sugar) and furan lysol glycosyl (from lyxose, a kind of 5 carbon sugar).The example of glycosyl derivatives comprises " deoxidation ", " ketone " and " dehydrogenation " derivant and substitutive derivative.The example of sulfo-glycosyl comprises the sulfur analogs of above-mentioned glycosyl, wherein becomes epoxy atom to be replaced by sulphur atom.The example of carbon ring group comprises and can further have one or more substituent groups the C of (for example-OH group) 4Carbocyclic ring, C 5Carbocyclic ring, and C 6Carbocyclic ring.
In one embodiment, Q is the β-D-ribofuranosyl of following formula
Figure A0081301500241
R wherein 7Connect and be selected from H at 5 ' of furan, phosphate ester of sheltering or phosphoramidate and derivant thereof, and R wherein 2And R 3Be identical or different and be independently-H or-OH.
In some embodiments, R 1Can comprise alkenyl, promptly (CH=CH) n-R 4, wherein n is 0 or the integer of 1-10, R 4Be halogen atom (I for example -, Br -, Cl -), or CN -Or hydrargyrum -R wherein 2Be H and R 3Be-OH; Perhaps R wherein 2Be OH and R 3Be H; R wherein 2And R 3Be H; Perhaps R wherein 2And R 3Be OH.On the other hand, R 4Be or comprise and be selected from H, halogen atom, alkyl, alkenyl, alkynyl, hydroxyl ,-O-alkyl ,-O-aryl ,-O-heteroaryl ,-S-alkyl ,-S-aryl, cyanide, cyanate, and thiocyanic ester, vinyl halides base, halo mercuri ,-S-heteroaryl ,-NH 2,-NH-alkyl ,-N (alkyl) 2,-NHCHO ,-NHOH ,-NHO-alkyl, NH 2CONHO-, and NHNH 2Group.In these embodiments, others comprise: R wherein 2And R 3Be H; R wherein 2Be OH and R 3Be H; R wherein 2Be H and R 3Be OH; Perhaps R wherein 2And R 3Be OH.
R 1The bit substituent preferred embodiment is the group that can have pi-allyl to exchange.
On the other hand, described candidate therapeutic agent is the chemical compound of following formula: Wherein n is 0 or the integer of 1-10; Wherein A is a phosphorus derivant, or the chemical compound of following formula: Wherein Q as above defines, and on the other hand, the candidate therapeutic agent is the chemical compound of following formula
Figure A0081301500251
Wherein R=2 '-deoxidation-5-uridnine base, m is 0 or 1, and n is the integer of 0-10.
Under suitable situation, chemical compound can be their enantiomer, and diastereomer or stereoisomer form comprise, for example D-or L-configuration, and can be any spatial chemistry conformation comprise, for example α-or β-anomer form.
Can realize the synthetic of pyrimidine nucleoside one phosphoric acid that pyrimidine nucleoside that above-mentioned 5-replaces and 5-replace by means commonly known in the art.For example, at Li 2PdCl 4Exist and use halogenated alkyl compounds, halogenated acetic acids ester or halogenated olefine to handle 5-chloro mercuri-2 '-BrdU down, cause forming the 5-alkyl respectively 5-acyl group or 5-ketone alkenyl derivative by the organic palladium intermediate.Another example that the C5-of pyrimidine nucleoside and nucleotide modifies is by handling then at Li with mercuric acetate 2PdCl 4Exist the styrene that adds styrene or ring-replacements down form C5-instead-styryl derivative.Bigge etc. (1980) American Chemical Society's proceedings (J.Am.Chem.Soc.) 102 (6): 2033-2038.Made pyrimidine dideoxyribonucleotide triphosphate 5 derivatizations of hydrargyrum in 3 hours by handling with the mercuric acetate in the acetate buffer down at pyrimidine ring at 50 ℃.Dale etc. (1973) PNAS 70 (8): 238-2242.Estimate that such processing also is effective to modifying Monophosphate; Perhaps, adorned triguaiacyl phosphate can Enzymatic transformation be the Monophosphate of modifying, for example, and by following the purification Monophosphate with alkali phosphatase control treatment.Molecule performance is similar to hydrargyrum but the other parts with organic or inorganic of preferred pharmaceutical property can be substituted.About the conventional method of the synthetic pyrimidine that replaces referring to for example U.S. Patent No. 4247544; 4267171; With 4948882; With (1981) organic chemistry magazines (J.Org.Chem.) 46 (7) such as Bergstrom: the 1432-1441 said method also can be applied to comprise except ribose or 2 '-deoxyribose (2 '-3 '-dideoxy ribose for example, arabinose, furanose, lyxose, pentasaccharides, six sugar, seven sugar and pyranose) outside the pyrimidine nucleoside that replaces of the 5-of sugar and the derivant of nucleotide synthetic.An example of 5-bit substituent is the vinyl halides base, E-5-(2-bromo vinyl)-2 ' deoxyuridylic acid for example, Barr etc. (1983) journal of biological chemistry (J.Biol.Chem.) 258 (22): 13627-13631 and biochemistry (Biochem.) 22:1696-1703.
Perhaps, from Glen Research, Sterling, VA (USA), Sigma-Aldrich Corporation, St.Louis, MO (USA), Moravek Biochemicals, Inc., Brea, CA (USA), ICN, Costa Mesa, CA (USA) and New England Nuclear, Boston, MA (USA) buys 5-bromouracil deoxyribose, idoxuridine and one phosphoric acid derivatives.Use is from Glen Research, Sterling, VA (USA) and ICN, Costa Mesa, the reagent that CA (USA) buys is by zymogenesis, the Monophosphate that the 5-bromouracil deoxyribose buied and idoxuridine can be converted into them with chemical method or Enzymology method.These halogen atom derivants can be with other substituent group combination results new and more effective antimetabolite.
General the synthesizing of II group chemical compound
In this embodiment, the present invention relates to by activated four compounds of enzyme (for example TS).Each class is by the organization definition of the uracil base of uracil base that exists or modification.They are ECTA chemical compounds, and wherein: I) base is the furyl-pyrimidone derivatives of uracil; II) base is a 6-furo uracil; And III) base is uracil derivative and the IV that the 4-hydrazone replaces) base is uracil.Synthetic 5 chemical compounds that replaced by the toxicity leaving group of the uracil derivative of use uracil or modification connect by the chain at 5-position electron orbit, and comprise the spacer groups part between electron orbit and the toxicity leaving group.The ECTA chemical compound comprises " Q ", and it can be not by phosphorylated, 5 ' Monophosphate, 5 '-di-phosphate ester, the comparable derivant of (shelter ") interchangeable carbohydrate part of BrdU or R7 position of or 5 '-protection and as described below.The deoxyuridine monophosphate derivant that the 5-of protection replaces is wherein to seal those of phosphoric acid part by connecting suitable chemoproection base.The protection of the deoxyuridine monophosphate derivant that 5-replaces can improve dissolubility, helps Premeabilisation of cells, helps by blood brain barrier, and prevents the effect of cell or the outer phosphatase of born of the same parents, and it may cause losing of phosphate groups in addition.In another embodiment, use uracil or the uridine derivatives that 5-replaces to having the active cell of nucleoside kinase, wherein uracil/the uridine derivatives of 5-replacement is converted into the uridine monophosphate derivant that 5-replaces.Also can modify uridine derivatives to improve their dissolubility, cell-penetrating and/or the ability of crossing blood brain barrier.
Thymidylate synthetase can discharge the substituent group (" leaving group ") that is connected with the 5-position of pyrimidine ring to the effect of the uridine monophosphate derivant that 5-replaces.The substituent group that discharges then can be inherently or react with another kind of cell component after, work as the inhibitor of toxin or cell proliferation.
The L of chemical compound of the present invention and D isomer are selected from the chemical compound with following structural formula: In the said structure formula, R 1Have following formula:
Figure A0081301500272
In the said structure formula, R 2Be or comprise two valence orbit groups.In one embodiment, R 2Be or comprise a unsaturated or how unsaturated electron orbit that effect is to make electronics from the pyrimidine ring conduction and pass to leaving group R 1, prerequisite is that n can be zero in I group chemical compound.In one embodiment, R 2Be to be selected from undersaturated alkyl; The aromatic hydrocarbyl that comprises one or more unsaturated alkyls; With the heteroaryl that comprises one or more unsaturated alkyls.
In one embodiment, R 2Be to have the unsaturated alkyl that is selected from following structural formula:
Figure A0081301500281
In one embodiment, R 2And R 3Form together and be selected from following structure: In one embodiment, R 2Be to have the aromatic hydrocarbyl that is selected from following structure: In one embodiment, R 2Be to have the heteroaryl that is selected from following structure:
Figure A0081301500284
Wherein J is a hetero atom, for example-O-,-S-, or-Se-, or hetero atom, for example-NH-or-NR ALK, R wherein ALKBe to have the straight or branched alkyl of 1-10 carbon atom or have the cycloalkyl of 3-10 carbon atom.
In the superincumbent structural formula, R 3Be bivalence spacer groups part, also be referred to as basic unit at interval.In one embodiment, R 3Be to have the bivalence spacer groups part that is selected from following structure:
Figure A0081301500291
R wherein 5Be identical or different, and be cycloalkyl or the R that has the straight or branched alkyl of 1-10 carbon atom or have 3-10 carbon atom independently 5Be halogen atom (F, Cl, Br, I).
In one embodiment, R 3Be to have the bivalence spacer groups part that is selected from following structure:
Figure A0081301500292
In one embodiment, R 3Be to have the bivalence spacer groups part that is selected from following structure:
R 3
Figure A0081301500301
In the superincumbent structural formula, n is the integer of 0-10, and m is 0 or 1.In one embodiment, n is 0 or the integer of 1-10, and m is 1.In one embodiment, n be 0 and m be 0.In one embodiment, work as R 7Be-during H, then n is not 0.In one embodiment, work as R 7Be-during H, then m is not 0.In one embodiment, work as R 7Be-during H, then n be not 0 and m be not 0.In one embodiment, work as R 7Be-during H, R 4Be not halogen atom (promptly-F ,-Cl ,-Br ,-I).In one embodiment, work as R 7Be-H and m are 0 o'clock, R then 4Be not halogen atom (promptly-F ,-Cl ,-Br ,-I).In one embodiment, work as R 7Be-H and m be 0 and n be 0 o'clock, R then 2Be not halogen atom (promptly-F ,-Cl ,-Br ,-I).
In the superincumbent structural formula, R 4Be to send out group malicious.As used herein, term " send out malicious group " refers to be or comprise the part of the leaving group that is chemical individual, described chemical individual has and consistent molecular size and the electrophilicity of part that proposes from pyrimidine ring by kinase, and it has the ability that suppresses cell proliferation or cell killing when the pyrimidine ring enzymatic discharges.
In one embodiment, send out group malicious and be or be included in the leaving group that the endocellular enzyme of overexpression in the cell activates or discharges.In one embodiment, R 4Be or comprise and have the group that is selected from following structure:
Figure A0081301500302
Figure A0081301500311
Wherein, X is-Cl ,-Br ,-I or other potential leaving group (include but not limited to-CN ,-OCN and-SCN); Y is identical or different in each position, and be independently-H or-F; Z is identical or different, and be independently-O-or-S-, R 8And R 9Be low alkyl group, R 10Be H or CH 3
In one embodiment, R 4Be or comprise and be selected from following chemical individual :-Br ,-I ,-O-alkyl ,-O-aryl ,-O-heteroaryl ,-S-alkyl ,-S-aryl ,-S-heteroaryl ,-CN ,-OCN ,-SCN ,-NH 2,-NH-alkyl ,-N (alkyl) 2,-NHCHO ,-NHOH ,-NHO-alkyl, NH 2CONHO-, NHNH 2,-N 3And cis-platinum derivative, for example
Figure A0081301500321
In the superincumbent structural formula, Q is or comprises the functional bonded group of supporting prodrug and enzyme (for example TS or TK).In one embodiment, Q is selected from following structure:
R wherein 6Be identical or different and be independently-H, F ,-OH ,-OC (=O) CH 3, or the hydroxyl of other protection (includes but not limited to benzoyl ,-COC 6H 5, and toluyl ,-COC 6H 4CH 3); R 7Be connected with 5 ' of Q and be hydrogen atom, phosphate radical, phosphodiester group, phosphoramidate, perhaps other phosphorus-containing groups.
In one embodiment, R 7Be from the deutero-phosphoramidic acid ester group of aminoacid (comprising for example 20 kinds of naturally occurring aminoacid).In one embodiment, R 7Be from the deutero-phosphoramidic acid ester group of alanine.In one embodiment, R 7Be or comprise group with following structure:
Figure A0081301500331
Above-mentioned group with and preparation method thereof be described in (1996) pharmaceutical chemistry magazine (J.Med.Chem.) 39:1748-1753 such as (1993) pharmaceutical chemistry magazine (J.Med.Chem.) 36:1048-1052 such as McGuigan and McGuigan.
In one embodiment, R 7Be from the deutero-phosphoramidic acid ester group of tryptophan.In one embodiment, R 7Be or comprise group with following structure:
Figure A0081301500332
Above-mentioned group and preparation method thereof is described in (1996) pharmaceutical chemistry magazine (J.Med.Chem.) 39:4569-4575 such as Abraham.
In one embodiment, R 7It is bound phosphate groups.In one embodiment, R 7Be or comprise and have the group that is selected from following structure:
Figure A0081301500333
Above two groups first and preparation method thereof be described in (1989) biochemical pharmacology (Biochem.Pharmacol.) 38:3193-3198 such as Freed; Sastry etc. (1992) molecular medicine is learned (Mol.Pharmacol.) 41:4411-445; (1995) pharmaceutical chemistry magazine (J.Med.Chem.) 38:448-495 such as Arquhar etc. (1994) pharmaceutical chemistry magazine (J.Med.Chem.) 37:3902-3909 and Farquhar.Above second of two groups and preparation method thereof be described in (1996) pharmaceutical chemistry magazines (J.Med.Chem.) 39 such as (1996) pharmaceutical chemistry magazine (J.Med.Chem.) 39:1981 such as Valette and Benzaria, p.4958.
In one embodiment, R 7Be or comprise and have the group (wherein R is an aromatic substituent) that is selected from following structure:
Above two groups first and preparation method thereof to be described in Meier etc. (1997) biological organic. medicine. chemical wall bulletin (Bioorg.Med.Chem.Lett.) 7:1577; Meier etc. (1997) are biological organic. medicine. and chemical wall bulletin (Bioorg.Med.Chem.Lett.) 7:99; With (1997) international antiviral news (International Antiviral News) 5:183 such as Meier.Above second of two groups and preparation method thereof be described in (1997) biochemical pharmacology (Biochem.Pharmacol.) 53:1815 such as Hostetler; With Hostetler etc., disclosed international patent application No.WO96/40088 (1996).
In one embodiment, R 7Form cyclic group with Q.Such embodiment and preparation method thereof (wherein DMTr is 4,4 '-dimethoxytrityl, Boc is a tertbutyloxycarbonyl, DCC is 1,3-dicyclohexyl carbodiimide, and 4-DMAP is a 4-dimethylaminopyridine) is described below:
Figure A0081301500351
In one embodiment, chemical compound can be any enantiomer, and diastereomer or stereoisomer form comprise, D-form or L-configuration, and α-anomer or β-anomer form.
In one embodiment, chemical compound can be a salt form, or the protection or prodrug form, perhaps its combination, for example as salt, ether or ester.
In another embodiment, utilize the method that describes below that said structure is further modified to make and have D2EHDTPA two aziridine replacement di(2-ethylhexyl)phosphate aziridine group.
Can finish the synthetic of above-mentioned new 5-substituted pyrimidines by means commonly known in the art and according to above-mentioned.
Perhaps, from Glen Research, Sterling, vA (USA), Sigma-Aldrich Corporation, St.Louis, MO (USA), Moravek Biochemicals, Inc., Brea, CA (USA), ICN, Costa Mesa, CA (USA) and New England Nuclear, Boston, MA (USA) buys 5-bromouracil deoxyribose, idoxuridine and one phosphoric acid derivatives.Use is from Glen Research, Sterling, VA (USA) and ICN, Costa Mesa, the reagent that CA (USA) buys can be with the 5-bromouracil deoxyribose of buying with chemical method or Enzymology method by zymogenesis, and idoxuridine is converted into their Monophosphate.These halogen atom derivants can be with other substituent group combination results new and more effective antimetabolite.
The structure at 5-position place is referred to as chain, because their connect leaving group of inferring (send out malicious group) and heterocycle.By with Cys residue reacting activation heterocycle the time, a negative charge conducts to chain from the 6-position of uracil in the activation site of for example people TS.5 '-one phosphorylation chemical compound about (BVdDU), Barr etc. (1983) biochemistrys (Biochenistry) 22:1696-1703 has described its mechanism, about (E)-5-(3,3,3-three fluoro-1-acrylic)-2 '-BrdU (TFPe-dUrd), Wataya etc. (1979) pharmaceutical chemistry magazines (J.Med.Chem.) 22:339-340; Santi (1980) pharmaceutical chemistry magazine (J.Med.Chem.) 23:103-111; With (1984) pharmaceutical chemistry magazine (J.Med.Chem.) 27:279-284 such as Bergstrom its mechanism has been described.
Chain between toxin and the dNMP " base at interval " must be undersaturated, and it can conduct toxin-unstable negative charge that the TS-Cys sulfydryl connects supply like this.In the obtainable a lot of unsaturated organo-functional groups of this purpose, vinyl, pi-allyl and propargyl are simple, and be little and synthetic easily.The unitary advantage of vinyl and pi-allyl is that they can be prepared into one of two kinds of geometric isomer forms that can not transform mutually.Therefore, they can be as activating " probe " that prodrug is regulated in the site by TS.On the one hand, the propargyl unit has the symmetric advantage of column type, and the catalytic toxin of TS-discharges the orientation that does not depend on about dUMP uracil ring from the chain of the type like this, and vinyl is the same with the situation of pi-allyl molecule.
Adopt two kinds of diverse ways to design nucleotide of the present invention-based precursor medicine.A kind of structure based on BVDU one phosphoric acid, and be characterised in that leaving group/toxin directly connects the end of C5 place (many) the vinyl substituted base of dUMP.This is the vinyl chain method.Another kind of structure based on TFPe-dUMP, and be similar to first kind, but there is MU (methylene unit) to separate leaving group/toxin and unsaturated unit, therefore comprise pi-allyl or propargyl unit.This is a pi-allyl chain method.
The activated mechanism of the propargyl form of pi-allyl chain method has precedent ((1983) such as Barr etc. (1981) pharmaceutical chemistry magazine (J.Med.Chem.) 24:1385-1388 and Barr above) in the interaction of 5-acetenyl-2 '-BrdU 5 '-a phosphoric acid (EdUMP) and 5-(3-hydroxyl-1-propinyl)-2 ' BrdU 5 '-a phosphoric acid (HOPdUMP) and TS.EdUMP is the potential inhibitor (Ki=0.1TM) of TS, and may be at the material of avtive spot formation based on allene.HOPdUMP (Ki=3.0TM) shows unusual inhibition kinetics.This may be because at the material of avtive spot formation based on cumulene.
Having synthesized structure recently is similar to 5 of the 5-alkane alkylene of the common intermediate of vinyl and pi-allyl chain method mechanism, 6-dihydrouracil (Anglada etc. (1996) heterocyclic chemistry (J.Hetercyclic Chem.) 33 (4): 1259).These show high electrophilicity.It is the convention that adds the suitable TS of water catalytic regeneration that they and ethanol synthesis generate 5-(ethoxyl methyl) uracil.Recently, the follow up study proves the C5 methylene intermediate (Barett etc. (1998) American Chemical Society's proceedings (J.Am.Chem.Soc.) 120:449-450) that existence produces by TS.
ECTA chemical compound synthetic that has the propargyl chain
Purpose is synthetic have propargyl and the allylic 2 ' BrdU that carries alcohol.A lot of these chemical compounds and close derivant thereof have been reported in the document, some even study related with TS.For example, 5-alkynyl-dUMP, comprise that 5-(3-methoxyl group-1-propinyl) and 5-(3-hydroxyl-1-propinyl) have been verified as TS inhibitor (Barr etc. (1981) pharmaceutical chemistry magazines (J.Med.Chem.) 24:1385-1388), and wherein some have been proved to be and import TS-and lack (Balzarini etc. (1985) FEBS Lett373 (1): 41-4) among the DNA of cancerous cell.
In the presence of palladium catalyst, after 5-hydrargyrum (Ruth etc. (1978) organic chemistry magazines (J.Org.Chem.) 43:2870-2876) and 5-ioduria glycosides (Robins etc. (1981) tetrahedron wall bulletin (Tetrahedron Lett) 22:421-424) easily have the uridnine of C5 chain with alkene and alkynes condensation generation.The more frequent use of one approach (Robins etc. (1982) Canadian Journal of Chemistries (Can.J.Chem.) 60:554-557; Asakura (1988) tetrahedron wall bulletin (TetrahedronLett) 29:2855-2858; And Asakura (1990) organic chemistry magazine (J.Org.Chem.) 55:4928-4933). realized 5-iodo-2 '-BrdU and the t-butyldimethylsilyl propargyl ether ((1983) pharmaceutical chemistry magazine (J.Med.Chem.) 26:661-666 such as (1998) J.Med.Soc.Perk.Trans.1:1131-1138 such as Graham and De Clercq) of protection; With methyl propargyl ether (Tolstikov etc. (1997) nucleoside nucleotide (Nucleoside Nucleotides) 16:215-225), even and the high yield condensation of propargyl ethanol ((1993) tetrahedron wall bulletin (Tetrahedron Lett) 34:5549-5552 such as proceedings (J.Am.Chem.Soc.) 117:10434-10442 of Chaudhuri etc. (1995) American Chemical Society and Goodwin) itself. DIBAL-H reduction by methacrylic acid group also can increase the 3-hydroxyl-1-propinyl substituent group ( Cho etc. ( 1994 ) tetrahedron wall bulletin ( Tetrahedron Lett ) 25:1149-1152 ) that imports by back one reaction； itself identical Heck reaction generation from using BVDU synthetic.The catalytic reaction of these palladiums is that versatility like this makes them can be used to very long and complicated chain and 5-the iodo-2 '-BrdU ( Livak etc. ( 1992 ) nucleic acids research ( Nucleic AcidsRes. ) 20:4831-4837 and Hobbs ( 1989 ) organic chemistry magazine ( J.Org.Chem. ) 54:3420-3422 ) based on propargyl of function of condensation.By producing with Undiar catalyst member hydrogenation propargyl parent based on ( Z )-allylic chain ( Robins ( 1983 ) organic chemistry magazine ( J.Org.Chem. ) 5 ( 11 ) : 3546-3548 ) and ( Barr ( 1983 ) journal of biological chemistry ( J.Biol.Chem. ) 258 ( 22 ) : 13627-13631 and biochemistry ( Biochem. ) 22:1696-1703 ) , and the Heck coupling of pass through ( E )-tributyl stannyl ethylene prepares based on ( E )-allylic chemical compound best ( Crisp ( 1989 ) synthesising communication ( Synth.Commun. ) 19:2117-2123 ) .
Strict with literature method, preparation has the 2 '-BrdU of 3 ', 5 '-two-O-protection of t-butyldimethylsilyl propargyl ether, and (Graham etc. (1998) above; And reduce that it is converted into that part of (Barr etc. (1983) above) of (Z)-allyl ether accordingly (1983) pharmaceutical chemistry magazine (J.Med.Chem.) 26:661-666 such as De Clercq).Because the removal of the TBAF-of TBDMS group mediation produces oxygen anion, it can the original position functionalization, and the propargyl of these TBDMS-protections-and (Z)-pi-allyl chain nucleoside will have the parent easily of the target thing of sending out group malicious as some.About the nucleoside of band (E)-allyl alcohol, the document Heck coupling by (E)-tributyl stannyl ethylene prepares known O-THP trtrahydropyranyl ether derivant (Crisp (1989) above).
Utilize two step literature methods (Phelps etc. (1980) pharmaceutical chemistry magazine (J.Med.Chem.) 23:1229-1232 and Hsiao and Bardos (1981) pharmaceutical chemistry magazine (J.Med.Chem.) 24:887-889), propargyl and (E) and (Z)-allyl alcohol is converted into their corresponding two aziridine phosphoramidates or sulfo-amino phosphate esters, and the TS of such 5 '-mononucleotide handles and discharges the active metabolite (Dirven etc. (1995) cancer research (Cancer Res.) 55:1701-1706) that suppresses cell drug TEPA or sulfo-TEPA respectively.
Figure A0081301500391
Synthetic two-aziridine-1-base-phosphinic acid 3-[2-BrdU-5-yl]-third-2-alkynes ester (" TEPA ") and passing through 1H NMR analyzes and obtains following result: 1H NMR ((CD 3) 2SO) because noise and complexity.Marked feature: δ 8.28 (D, 1, H6), 6.10 (vacation-t, 1, H1 '), 5.26 (m, can with D 2O exchange, 1,3 '-OH), 5.13 (m, can with D 2The O exchange, 1,5 '-OH), 4.81 (q or dd, 2, propargyl-CH 2), 4.24 (m, 1, H3 '), 3.57 (m, 2,5 '-CH 2), 2.15-2.0 (m, 8, aziridine-CH 2).
Synthetic two-aziridine-1-base-thiophosphinic acid 3-[2-BrdU-5-yl]-third-2-alkynes ester (" sulfo-TEPA ") and passing through 1H NMR analyzes and obtains following result: 1H NMR ((CD 3) 2SO) because noise and complexity.Marked feature: δ 8.29 (D, 1, H6), 6.10 (vacation-t, 1, H1 '), 5.22 (m, can with D 2O exchange, 1,3 '-OH), 5.10 (m, can with D 2The O exchange, 1,5 '-OH), 4.88 (q or dd, 2, propargyl-CH 2), 4.31 (m, 1, H3 '), 3.52 (m, 2,5 '-CH 2), 2.15-2.0 (m, 8, aziridine-CH 2).
Synthesizing of furo pyrimidone
There is 2 '-BrdU of C5 propargyl-alcohol to begin synthetic furo pyrimidone from synthetic.Form the furo pyrimidinone compound from above-mentioned O-THP trtrahydropyranyl ether derivant then.The reaction of the oxygen bonding that second carbon by propargyl is connected with the C4 position of pyrimidine ring is synthesized, and obtains fluorescence furo pyrimidone, and it separates from reactant mixture easily.Such chemical compound provides the other concrete electron orbit that passes through, at interval the basis of the synthetic ECTA chemical compound of the various combinations of base and toxicity leaving group.
Under the condition that these fluorescent chemicalses of known promotion generate (Barr etc. (1983) above); by condensation 2 '; 3 '-two-O-is right-toluyl or 2 '; 3 '-two-O-acetyl group-5-iodo-2 '-BrdU and 1-(THP trtrahydropyranyl oxygen base)-2-propine (Jones and Mann (1953) American Chemical Society's proceedings (J.Am.Chem.Soc.) 75:4048-4052); preparation furo [2,3-d] pyrimidone nucleoside.Alkali-catalytic elimination sugar blocking group obtains the bicyclic nucleoside of 6-(Pentamethylene oxide .-2-base oxygen ylmethyl)-replace, itself or same procedure regioselectivity ground 5 '-phosphoramidic acid esterification of carrying out standard acidic THP base hydrolysis (TFA in the dichloromethane) or using by preparation BvdU-PA and 5FUdR-PA.After the phosphoramidic acid esterification, can remove the THP group by acidolysis.
3-(2-deoxidation-β-D-ribofuranosyl)-6-(Pentamethylene oxide .-2-base oxygen ylmethyl) furo [2,3-d] pyrimidines-2 (3H)-ketone 1H NMR ((CD 3) 2SO) δ 8.28 (s, 1, H4), 6.74 (s, 1, H5), 6.16 (vacation-t, 1, H1 '), 5.27 (d, can with D 2O exchange, 1,3 '-OH), 5.12 (m, can with D 2The O exchange, 1,5 '-OH), 4.72 (m, 1, THP-H2), 4.56 (q, 2, CH 2OTHP), 3.92 (m, 1, H4 '), 3.64 (m, 2,5 '-CH 2), 2.40 (m, 1, H2 ' a), 2.03 (m, 1, H2 ' b), 1.68 and 1.50 (m, 8, THP). low resolution mass spectrum (DCI-NH 3) on bis-TMS derivative, m/z323 (B+TMS+H +), 511 (MH +), 583 (M+TMS +).
3-(2-deoxidation-β-D-ribofuranosyl)-6-(methylol) furo [2,3-d] pyrimidines-2 (3H)-ketone 1H NMR ((CD 3) 2SO) δ 12.0 (bs, 1, OH), 8.24 (s, 1, H4), 6.53 (s, 1, H5), 5.51 (vacation-t, 1, H1 '), 4.42 (m, 2, CH 2OH) low resolution mass spectrum (DCI-NH 3), m/z167 (B+2H +), 184 (B+NH4 +).
1-[6-(Pentamethylene oxide .-2-base oxygen ylmethyl) furo [2,3-d] pyrimidines-2 (3H)-ketone-3-yl]-2-deoxidation-β-D-ribofuranose-5-base phenyl methoxyl group-L-alanyl phosphoramidate. 1HNMR ((CD 3) 2SO) owing to have diastereomer and complexity.Marked feature: δ 8.62 and 8.59 (each s, each 1, H4), 7.4-7.1 (m, 5; PhO), 6.61 and 6.60 (each s, each 1, H5); (6.25 m, 1, H1 '), 4.56 (q; 2, propargyl CH2), 3.56 and 3.54 (each s, each 3; CO2Me), 2.0 (m, 1, H2 ' is b); 1.22 (m, 3, alanyl-α-Me), low resolution mass spectrum (DCI-NH 3), m/z167 (B+2H +), 184 (B+H ++ NH4+-THP).
1-[6-(methylol) furo [2,3-d] pyrimidines-2 (3H)-ketone-3-yl]-2-deoxidation-β-D-ribofuranose-5-base phenyl methoxyl group-L-alanyl phosphoramidate. 1H NMR (CDCl 3) 2SO is owing to exist diastereomer and complexity.Marked feature: δ 8.5 (s, 1, H4), 7.4-7.1 (m, 5, PhO), 6.36 and 6.30 (each s, each1, H5), and 6.23 (m, 1, H1 '), 3.67 and 3.65 (each s, each 3, CO 2Me), 2.69 (m, 1, H2 ' a), 2.1 (m, 1, H2 ' b), 1.35 (m, 3, alanyl-α-Me), low resolution mass spectrum (DCI-NH 3), m/z 525 (MH +), 595 (MNH 4 +).
The 4-nitrobenzophenone ether derivant for preparing 5-(3-hydroxyl-1-propinyl)-2 '-BrdU according to standard ether synthetic method as follows.
Figure A0081301500421
5-[3-(4-nitrophenoxy)-1-propinyl]-2 '-BrdU.Under the hydrogen, with (696 milligrams of 4-nitrophenols, 5 mMs), (787 milligrams of triphenylphosphines, 3 mMs) and (590 liters of diisopropyl azo-2-carboxylic acids, 3 mMs) handle dry in advance 5-(3-hydroxyl-1-propinyl)-2 '-BrdU (" nucleic acid compound 39; 5-iodine is to the uracil base and the nucleoside of the effective conversion and the deutero-5-replacement of 5-alkynes " (Barr etc. (1983) above) (565 milligrams excessively among 40 milliliters of anhydrous THF, 2 mMs), and reactant mixture is heated down up to the solution clarification at 60 ℃, and then heated 1 hour.Make this mixture be cooled to 23 ℃, make it be evaporated to SiO then 2Upward and by the use ethanol/methylene carry out chromatogram purification, obtain the ether products of the expectation of 107 milligrams (13%): Mp 112-118 ℃. 1H NMR ((CD 3) 2SO) δ 11.65 (s, can with D 2The O exchange, 1 ,-NH), 8.29 (s, 1, H6), 2.24 (d, j=9.3Hz, 2, m-ArH), 7.23 (d, j=9.2Hz, 2, o-ArH), 6.09 (vacation-t, 1, H1 '), 5.17 (s, 2, propargyl-CH 2), 4.22 (m, 1, H3 '), 3.80 (m, 1, H4 '), 3.59 (m, 2,5 '-CH 2), 2.13 (vacation-t, 2,2 '-CH 2).Low resolution mass spectrum (DCI-NH on per-trimethyl silyl formed material 3), m/z 547[M (TMS) 2H +], 565[M (TMS) 2NH 4 +], 620[M (TMS) 3H +]
TS ECTA chemical compound based on furo-pyrimidone.As what above-mentioned synthetic TEPA and sulfo-TEPA derivant were explained, make toxicity R with making toxicity leaving group and hydroxyl on the C5 propinyl uridnine chemical compound be connected employed similar approach 4Leaving group connects furan-2 methylol.Be to it is evident that various toxicity leaving group is a theme of the present invention for those skilled in the art.In addition, also required R 2The length of electron orbit composition and composition and to R 3The modification of the composition of base at interval.
TS ECTA chemical compound based on furo-pyrimidone also can be made up of " Q " part of different modifying.2 '-BrdU that a lot of 5-replace is not the substrate of people TK, and still interesting is to find that 5-(4-hydroxyl-ethyl acetylene base)-2 '-BrdU is an exception (Barr etc. (1981) above).Therefore, expect that the nucleoside that group sent out malicious by some bands also has suitable TK substrate activity.Therefore, the ECTA chemical compound can have the freedom 5 '-hydroxyl that connects with different glycosyls, 5 '-Monophosphate, or 5 '-phosphoramidic acid ester group.By make 2-deoxidation-3 '-hydroxyl in the presence of the HCl scavenger, 5 '-hydroxyl does not have the nucleotide of protection and the reaction of chloro phosphate ester to realize the new method that such phosphoramidate chemical compound is synthetic.In a preferred embodiment, described chloro phosphate ester comprises the phosphorus substituent group, its from aminoacid for example alanine derive.For example, described chloro phosphate ester can be phenyl-L-methoxy propyl propylhomoserin chloro phosphate ester.
Chemical compound based on C6 floxuridine and C4 hydrazone.In the normal TS with dUMP reacts, the two keys of pyrimidine C5-C6 are added neutral mercaptan carry out exothermic reaction (every mole of 3-9 kilocalorie; Referring to summary Les etc. (1998) biomolecular structure and kinetics (Biomolecular Structure andDynamics) 15 (4): 703-715).By active people TS cysteine (with the cys-198 homology of L.casci), help forming the different substituents (comprising fluorine atom) of 6 TS active hydrogens of sulfydryl key with enzyme.Such substituent group of other position in pyrimidine ring also can help the reaction between substrate and the TS.For example, the 4-hydrazone on the uracil replaces the reaction that (Les and Rhode (1998) biomolecular structure and kinetics (Biomolecular Structure and Dynamics) 15 (4): 703-715 is described) helps mercaptan and TS.Nucleotide-the mercaptan that obtains (TS) intermediate is reset in such a way makes to be important by the passive release this point of finishing the nucleotide of change of hydrolysis.
Figure A0081301500431
Do not report in the past at C6 position introducing fluorine atom, but can be according to Krajewskas and Shugar (1982) biochemical pharmacology (Biochem.Pharmacol.) 31 (6): 1097-102 synthesizes synthetic description, above-mentioned document description several 6 uracil that replace and uridine analogs synthetic.
The replacement that helps chemical reaction of the C4 position of pyrimidine bases is well known to a person skilled in the art.The example of document description comprises (1999) Farmaco 54 (1-2): 83-89 such as Wallis; Negishi etc. (1996) nucleic acid seminar 35 (the 23rd nucleic acid chemistry seminar) 137-138; Barbato etc. (1991) nucleoside nucleotides (NucleosideNucleotides) 10 (4): 853-66; Barbato etc. (1989) nucleoside nucleotide (Nucleoside Nucleotides) 8 (4): 515-528; With (1999) pharmaceutical chemistry magazine (J.Med.Chem.) 42 (12): 2064-2086 such as Holy.These synthetic technologys also can make up metalepsis, (Pluta etc. (1999) Boll.Chim.Farm.138 (1): 30-33) or at the C2 and C4 position (Zeid etc. (1999) nucleoside nucleotide (Nucleoside Nucleotides) 18 (1): 95-111) of pyrimidine ring for example in the C4 of pyrimidine ring and C5 position.
In one embodiment of the invention, add different electron orbits, at interval the synthetic ECTA chemical compound of base section and toxicity leaving group by pyrimidine to C6 fluoro-uridnine base or the modification of C4 hydrazone.Above the method for the synthetic description of 2-BrdU base ECTA chemical compound also is applicable to the synthetic of such molecule.
The derivant of the chemical compound of B.I group and II group
The salt of above-claimed cpd disclosed herein, ester and ether are also within the scope of the invention.The salt of prodrug of the present invention can be derived from inorganic or organic bronsted lowry acids and bases bronsted lowry.The example of acid comprises hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic, lactic acid, salicylic acid, succinic acid, p-methyl benzenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethyl sulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid and benzenesulfonic acid.Other acid, oxalic acid for example although itself be that pharmacy is unacceptable, can accepted to use in the preparation of salt of intermediate of acid-addition salts as obtaining chemical compound of the present invention and pharmacy thereof.The example of alkali comprises alkali metal (for example sodium) hydroxide, alkaline-earth metal (for example magnesium) hydroxide, ammonia and formula NW 4 +Chemical compound, wherein W is C 1-4Alkyl.
The example of salt comprises: acetate, adipate, alginate, aspartate, benzoate, benzene sulfonate, disulfate, butyrate, citrate, camphorate, camsilate, cyclopentane propionate, digluconate, lauryl sulfate, esilate, fumarate, the fluco enanthate, glycerophosphate, Hemisulphate, enanthate, caproate, hydrochlorate, hydrobromate, hydriodate, the 2-isethionate, lactate, maleate, mesylate, 2-naphthalene sulfonate, nicotinate, oxalates, palmitate, pectate, peroxydisulfate, phenylpropionic acid salt, picrate, glutarate, propionate, succinate, tartrate, thiocyanate, toluene fulfonate and ten-alkyl salt.Other example of salt comprises and suitable cation Na for example +, NH 4 +, and NW 4 +(wherein W is C 1-4Alkyl) anion of bonded chemical compound of the present invention.
For therapeutic use, the salt of chemical compound of the present invention is that pharmacy is acceptable.But, can accept also can find in the preparation of chemical compound and the purification that in pharmacy non-pharmacy can accept the purposes of the salt of bronsted lowry acids and bases bronsted lowry.
The prodrug of identifying by method of the present invention or the ester of chemical compound comprise by esterification 2 '-, 3 '-and/or the carboxylate that obtains of 5 '-hydroxyl (promptly-O-C (=OR)), wherein R is selected from (1) straight or branched alkyl (n-pro-pyl for example, the tert-butyl group or normal-butyl), alkoxyalkyl (for example methoxy), aralkyl (for example benzyl), aromatic yloxy yl alkyl (for example phenoxymethyl), aryl is (for example randomly by for example halogen atom, C 1-4Alkyl or C 1-4Alkoxyl or the amino phenyl that replaces); (2) sulphonic acid ester, for example alkyl sulphonyl (for example mesyl) or aralkyl sulfonyl; (3) amino-acid ester (for example L-valyl or L-isoleucyl-); (4) phosphate ester and (5)-, two-or triguaiacyl phosphate.By for example C 1-20Alcohol or its response derivative, perhaps by 2,3-two-(C 6-24) the further esterification phosphate ester of acylglycerol.In these esters, except as otherwise noted, the moieties of existence advantageously contains 1-18 carbon atom, a particularly 1-6 carbon atom, more particularly 1-4 carbon atom.Any cycloalkyl moiety that exists in such ester advantageously contains 3-6 carbon atom.Any aryl moiety that exists in such ester advantageously comprises phenyl.Those that the example of furan lyxose prodrug derivatives of the present invention comprises for example the chemoproection hydroxyl (the O-acetyl group is for example arranged), for example 2 '-O-acetyl group-furan lysol glycosyl; 3 '-O-acetyl group-furan lysol glycosyl; 5 '-O-acetyl group-furan lysol glycosyl; 2 ', 3 '-two-O-acetyl group-furan lysol glycosyl and 2 ', 3 ', 5 '-three-O-acetyl group-furan lysol glycosyl.
The ester of chemical compound of the present invention comprises methyl ester, ethyl ester, propyl ester, butyl ester, isobutyl ester and secondary butyl ester.
In another embodiment, substrate may not be that chemistry is relevant with pyrimidine or folic acid, but is really on the basis of the known parameters that rational drug designs synthetic (referring to (1996) pharmaceutical chemistry magazine (J.Med.Chem.) 39:4825 such as Dunn).
Measure product (for example wherein product is the antimetabolic product of the bromo vinyl derivant of dUMP) according to the method that the embodiment that provides below or (Barr etc. (1983) above) describe.
C. the RT-PCR of Pi Pei normal structure and tumor tissues analyzes
Transcript level by human TS in the normal colonic tissue that utilizes contrary RT-PCR amplification quantitative assay colon cancer tissue and coupling.The following oligonucleotide primers that is designed for amplification human TS and beta-actin: thymidylate synthetase has adopted primer 5 '-GGGCAGATCCAACACATCC-3 ' (corresponding to the base 208-226 of thymidylate synthetase cDNA sequence, Genbank registration number No.X02308), antisense primer 5 '-GGTCAACTCCCTGTCCTGAA-3 ' (corresponding to base 564-583), beta-actin has adopted primer 5 '-GCCAACACAGTGCTGTCTG-3 ' (corresponding to the base 2643-2661 of beta-actin gene order, Genbank registration number No.M10277) and antisense primer 5 ' CTCCTGCTTGCTGATCCAC-3 ' (corresponding to base 2937-2955).
From Cooperative Human Tissue Network (CHTN, WesternDivision, Cleveland, OH) normal structure that obtains the human colon carcinoma tissue and mate.According to supplier's explanation, (available from Boehringer MannheinCorp., Indianapolis IN) separates total RNA to use the pure separation agent of Tri.Pollute in order to detect possible DNA, the primer that is designed for the amplification beta-actin is crossed over 5 connections of exon 4/ intron 5/ exon.The genomic DNA template produces 313bp beta-actin fragment, and the cDNA template produces the 210bp product.
(Gibco/BRL, Gaithersburg MD) carry out reverse transcription in system to use SuperScript to increase in advance.According to supplier's explanation, in the buffer of 20 microlitre volumes, carry out reverse transcription reaction with the total RNA of 3 micrograms.
Carry out the PCR reaction in the volume of 96 microlitres, this volume contains the cDNA mixture of 5 microlitres from reverse transcription reaction, 3mM MgCl 2, 50mM KCl, 20mM Tris-Cl, pH8.4, the various dNTP of 0.2mM, 0.3 μ M thymidylate synthetase have justice and antisense primer and 5 Taq of unit archaeal dna polymerases (available from Promega, Madison, WI).This reactant mixture then carries out following 9 circulations 94 ℃ of following incubations 3 minutes: 94 ℃ of incubations 1 minute, and 58 ℃ of incubations 1 minute, 72 ℃ of incubations are 1 minute then.After 9 circulations, add 4 microlitre people beta-actin primers, reach the final concentration of 0.2 μ M, make the end reaction volume reach 100 microlitres.Proceed PCR reaction and carry out altogether 30 times, 32 times or 34 circulations are then 72 ℃ of incubations 7 minutes.
By electrophoresis on 2% agarose gel then with SYBR Gold nucleic acid gel dyestuff (available from Molecular probes, Eugene, OR) dyeing separates 10 microlitre PCR products.The quantitative assay result shows that the amplification of thymidylate synthetase and B-actin is linear between 30 and 34 circulations.Quantitative assay is proofreaied and correct corresponding to the DNA swimming band of thymidylate synthetase and by molecular dynamics method (Molecular Dynamics storm) and is the value with respect to μ-actin.The quantitative expression water-glass is shown the ratio between TS and the beta-actin.This test also is used for detecting the pathogen of mammalian cell, as described in (1999) J.Hepatol-30:965-969 as described in Nagata etc.
D. cell line and transfection
In PRMI 1640 culture medium that are supplemented with 10% hyclone, cultivate the HT1080 cell, and with the transfection of GFP-TS expression vector.After 48 hours, with cells transfected with trypsin treatment (tripsinized) and in the culture medium that contains 750 mcg/ml G418 subculture.Screening after 2 weeks with G418, is that the basis will survive cell divide with the luciferase expression.Select a clone (called after TSH/HT1080) of higher luciferase expression and a clone (called after TSL/HT1080) of low luciferase expression, and in the cell line that increases.With the stable HT1080 cell of pEGFP-C3 transfection in contrast.
The structure of E.GFP-TS expression vector
Primer below using obtains the cDNA fragment of the conserved region (aminoacid 23-313) of coding human TS by pcr amplification: adopted primer is arranged, 5 '-CGGAAGCTTGAGCCGCGTCCGCCGCA-3 ' and antisense primer, 5 '-GAAGGTACCCTAAACAGCCATTTCCA-3 '.Meet frame with the GFP sequence cDNA is cloned into mammalian expression vector pEGFP-C3 (Clontech Laboratories.Inc., PaloAlto, HindIII CA) and KpnI site.Confirm that by dna sequencing cDNA inserts.
F. western blot analysis
In being supplemented with the RPMI1640 culture medium of 10% hyclone, cultivate human normal cell line and cancerous cell.Cell grows in 100 millimeters culture dishs and converges and dissolving in 0.5 milliliter of RIPA buffer (50mM Tris-HCl, pH7.5,150mM NaCl, 0.5%Triton X-100,0.1%SDS, 0.5% deoxycholic acid, sodium salt and protease inhibitor).(available from Pierce, Rockford IL) measures protein concentration by using BCA-200 protein determination test kit.Separate 15 microgram gross proteins by 12%SDS-PAGE from each cell strain/be.Isolating protein transduction moves on on the pvdf membrane, and then sheep anti-mice the Ig two anti-(available from Amersham, Britain) that is connected with horseradish peroxidase with human TS monoclonal one anti-(NeoMarkers, Fremont, CA preparation) carries out immunoblotting.Use ECLplus test kit (Amersham) to measure immunoreactivity.Quantitative assay is proofreaied and correct corresponding to the swimming band of thymidylate synthetase and by the relative tubulin of molecular dynamics Storm.The quantitative expression water-glass is shown the value with respect to cell strain CCD18co.
G. pass through from dUMP- 3The tritium of H discharges measures the TS activity
The density of cell dull and stereotyped upper berth flat board to 30000 cell/plate in 24 holes and incubation made and adhere to dull and stereotyped frosting in 16 hours.
Before thymidylate synthetase is measured, replace culture medium with the hyclone of RPMI+10% dialysis.Add 0.5 μ Ci 5-[to each hole 3H] BrdU, do not add CO 2Following dull and stereotyped 37 ℃ of following incubations 60 minutes.By active carbon under the room temperature (10%) absorption 5-[in 1xPBS 3H] BrdU measured in 5 minutes [ 3H] discharge.With after centrifugal 5 minutes of the 13000RPM by liquid scintillation counting measure in the supernatant [ 3H] amount.
H. growth inhibited research
Index growthing cell is transferred in the flat tissue culturing plate in 384-hole.All cell types are paved into flat board, and in 25 microlitre complete mediums (RPMI 1640+10% hyclone+antibiotic/mould resistant), density is 500 cells in every hole.After 24 hours (the 0th day), it is 10 that triplicate adding contains dosage range To 10
Figure A0081301500482
Complete medium 25 microlitres of the experimental compound of M.The contact medicine time is 120 hours (the 5th day), measures the growth inhibited effect then.In each hole, add 5 microlitre oxidation-reduction indicator alamarBlue (10%v/v).After 37 ℃ of incubations 4 hours, 535nm excite with 595nm emission under monitor fluorescence.
Concentration is used the Hill equation to relative fluorescence unit (RFU) mapping, and sigmoid curve coincide.The IC of the inflection point indication of curve 50Be the concentration that growth was suppressed 50% o'clock.
Same growth inhibited and cytotoxicity test also can be used for measuring the cytotoxicity of the kinase of infected as described in the present application material coding from the toxin of ECTA release.As mentioned above, such infected material includes but not limited to Mycobacterium, Chlamydia sp., Dermacentroxenus and Pheumocystis sp.pathogenic Enterococcus, moraxella, haemophilus, and staphylococcus.Can utilize bacterium colony to generate test comes on the assay plate or the cytotoxicity (Miller of metabolic ECTA chemical compound on the outer pathogen antibacterial of born of the same parents or other pathogen in the fluid medium, J.H.A antibacterial heredity short-term process: the experimental implementation handbook of bacillus coli and Related Bacteria (Short Course in Bacterial Genetics:A Laboratory Manualand Hardbook for E.Coli and Related Bacteria), cold spring port publishing house (ColdSpring Harbor Press) (1992)).
The Cytotoxic Tomudex of I.NB1011 suppresses
MCF7-TDX is transferred to 384 hole test boards, 500 cells in the 25 microlitre complete mediums of every hole.After 24 hours (the 0th day), double add contain by the NB1011 of 1mM series doubling dilution and discontinuous concentration (0,1,10,100,25 microlitre complete mediums of the mixture of Tomudex 1000nM).Contact is 120 hours (the 5th day) the medicine time, then as in growth inhibited research above, describe with the effect of alamarBlue mensuration growth inhibited.
J. enzyme preparation
In order to add amino terminal His labelling.Use NdeI
Figure A0081301500491
SacI inserts the site, with clone human TS plasmid pBCHTS sub-clone to bacillus coli BL21 (DE3)/pET-28a (+) (Novagen).In bacillus coli, express enzyme by inducing, and pass through at Ni with IPTG 2+Affinitive layer purification on the His Bind metal-chelating resin (Novagen).Post Ni 2+His Bind metal-chelating resin column 20mM Tris pH7.9,5mM imidazoles, 0.5M NaCl washing; With 20mM Tris pH7.9,60mM imidazoles, 0.5M NaCl eluting thymidylate synthetase activity.
K. enzyme is tested and kinetic determination
By 40mM Tris pH7.5,25mM MgCl 2, 1mM EDTA, the 25mM mercaptoethanol, 125MdUMP and the indicated N5 of 65 μ M carry out thymidylate synthetase and measure in 96 hole Costar UV lamella lucidas in the 200 microlitre reaction volumes that the N10-methylene tetrahydrofolate constitutes.By tetrahydrofolic acid (Sigma) directly is dissolved in 0.2M Tris pH7.5, the 0.5M-mercaptoethanol prepares the tetrahydrofolic acid stock solution; Store stock solution down at-80 ℃.By adding 12 microlitres, 3.8% formaldehyde to 1 milliliter of 0.65mM tetrahydrofolic acid solution and preparing N5, N10-methylene tetrahydrofolate in 5 minutes at 37 ℃ of following incubations.N5, N10-methylene tetrahydrofolate are kept on ice and use in 2 hours after preparation.
In microtitration plate at the bottom of 96 hole Dynex MicrofluorB1ack " U " types, in containing the thymidylate synthetase reaction of 125M BVdUMP, 200ml measures the conversion that BVdUMP is converted into fluorescence-causing substance by thymidylate synthetase with 340nm excitation wavelength and 595nm emission wavelength.Measure fluorescence with Tecan Spectrafluor Plus exometer.
Use above-mentioned enzymatic determination condition that human TS substrate dUMP and BVdUMP are measured enzyme kinetics constant (K mAnd V Max).By measure to the A of dUMP reaction 340Increase and to the A of BVdUMP reaction 294Reduce and measure the enzyme reaction initial rate.From kinetic constant K mAnd V MaxCalculate enzyme catalysis efficient (K Cat/ K m).
L. liquid chromatography/mass spectrometry
Under the room temperature with PBS with cell washing three times, freeze/thaw dissolving in 5 milliliters of PBS then.With centrifugal 10 minutes of cell extract, be adsorbed in Sep PakC with 10KRPM then 18And with 10 milliliters of PBS washings.With 1 ml distilled water eluting BVdUMP.Linear gradient by using 0.1% formic acid-0.1% formic acid/95% acetonitrile is at C 18Analyze the LC/MS sample by reversed phase chromatography on the post.Carry out mass spectral analysis with three grades of level Four spectrometers of Micromass Quattro II.
M. the reverse of resistance
The source and the feature (Drake etc. (1996)-biochemical pharmacology (Biochem.Pharmacol.) 51 (10): 1349-1355) of human breast carcinoma MCF7 TDX cell line had before been described.In brief, by being exposed to the TDX that the concentration of 2.0 μ M at the most progressively increases progressively continuously, to the Drug resistance of MCF-7 breast cancer cell in-vitro screening to Tomudex.But do not have TDX the culture medium that 50 μ M NB1011 are arranged by continuously parent MCF7 TDX cell line being exposed to, the screening of antagonism property of medicine subbreed is to the Drug resistance of NBl011, wherein NB1011 concentration approximately than in the parent MCF7 TDX cell line to the IC of NB1011 50High about 16 times.Significant initial cell kills after the effect, the Drug resistance bacterium colony occurs, forms the monolayer that grows vigorously.Respectively by Western blotting and alamar Blue cytotoxic assay, the MCF7 TDX/1011 cell line that obtains is measured 5-FU, TS protein level and the IC of TDX and NB1011 according to the description in " materials and methods " 50
II. experiment
The vitro reactions of A.BVdUMP and human TS
1. with rHuTS BVdUMP is carried out acellular processing and produce fluorescence-causing substance.
Lactobacillus casei (L.casei) TS has produced potential reactive intermediate (Barr etc. (1983) above) to the acellular processing proof of BVdUMP.Because this reason expects that TS causes the irreversible deactivation of people TS (Balzarini (1987) molecular medicine (Mol.Pharmacol.) 32 (3): 410-6) to the processing of BVdUMP.DeClercq, (Balzarini (1987) is above for Balzarini and colleague thereof; Balzarini (1993) journal of biological chemistry (J.Biol.Chem.) 268 (a): 6332-7; Balzarini (1995) FEBS Lett373 (1): such notion is supported in the experiment of the molecular basis that 41-4) carries out, in case promptly BVDU is converted into Monophosphate (by the herpesvirus Adenosine kinase) in cell, then its in this process in conjunction with and deactivation HuTS.But, the never abundant real reaction that characterizes remarkable TS and BVdUMP.Santi and colleague (Barr (1983) above) have used antibacterial TS to prove from the BVdUMP+TS reaction for their work and have produced product, DeClercq and colleague thereof have used cell and cellular lysate, and (Balzarini (1987) above not have the people TS of purification; Balzarini (1993) above; Balzarini (1995) above).
Because the China invites the person has studied the interaction of the recombined human TS (rHuTS) of BVdUMP and purification repeatedly to can specificity interested by the generation of the activated treatment substrate of TS.When BVdUMP and rHuTS in the standard reaction mixture during incubation, reaction causes the generation (Fig. 2) of fluorescence-causing substance.The reduction of initial BVdUMP concentration is followed in the increase of fluorescence time dependence.Part has characterized the product that obtains, and showing is exocyclic pyrimidine nucleoside acid derivative (seeing below).
This result is wonderful, causes that previous result supports such notion, i.e. TS and BVdUMP reaction should deactivation people TS enzyme (Balzarini etc. (1987), (1993), (1995), above).Because carry out in the above-mentioned cell free system that is reflected at the composition with purification, the composition that medium provides in the possible born of the same parents causes in NB1011 is converted into cell TS inactivation after free nucleotide one phosphoric acid.This content is described in more detail hereinafter.
2.dUMP and the kinetics of BVdUMP and rHuTS relatively
Lactobacillus casei TS is used in the work of report such as previous Barr (1983), and (Balzarini (1995) above; Balzarini (1987) above; Balzarini (1993) is above), use cell and cellular lysate, whether the reaction of not knowing BVdUMP and people TS causes the irreversible inactivation of this enzyme.For clear and definite this problem, there is or do not exist the kinetics of having measured BVdUMP and rHuTS under the dUMP.
Competitive inhibitory effect is the most identical with the reaction of not deactivation of BVdUMP TS enzyme wherein.In order to help further to clarify this situation, in order to measure than wherein carrying out rHuTS that kinetics contingent any deactivation of longer time wants and the incubation (Fig. 4) of BVdUMP.
Even these data show after incubation rHuTS and BVdUMP 20 hours,, there is not or almost do not have the enzyme deactivation situation to occur according to what measure as the conversion rate of the THF DHP dUMP of substrate.This result with in cell, preferably in the cell of overexpression TS, the TS of overexpression matches the prospective ability that BVdUMP is converted into the cytotoxicity metabolite, does not make this enzyme deactivation at last.
3.BVdUMP the sign with the TS reaction: cofactor and inhibitor
The reaction that characterizes the most fully of TS is the conversion of dUMP to dTMP.This reaction comprises that with N5 the methylene of N10-methylene tetrahydrofolate (THF) is converted into the C-5 position of dUMP (CarrerasCW (1995) above).Cofactor (THF) is depended in this reaction, and by the uridylic acid analogies, 5F-dUMP suppresses, and 5F-dUMP causes the generation of stable compound and lost enzymatic activity with enzyme reaction the time.The inhibitor that the active second kind of quilt of TS characterizes fully is Tomudex, Tomudex has occupied the folic acid binding site of TS homodimer, prevent the combination of THF, and blocking-up TS is at intracellular activity (Drake (1996) biochemical pharmacology (Biochem.Pharmacol.) 51 (10): 1349-1355 and Touroutoglou and Pazdur (1996) Clinical Cancer Research (Clin.Cancer Res.) 2 (2): 227-243).As characterizing the preliminary part of making great efforts of rHuTS and BVdUMP reaction, 5F-dUMP has been compared in the reaction of enzyme and dUMP and BVdUMP, the effect of Tomudex and cofactor.These experimental results show that (table 1), and are similar with the situation of dUMP, and 5F-dUMP can prevent the conversion of BVdUMP to fluorescence-causing substance.In addition, Tomudex also can prevent to form product from dUMP and BVdUMP.But, matching with lactobacillus casei report (Barr etc. (1983) are above) early, BVdUMP does not need THF to the conversion of fluorescence-causing substance.In addition, the data in the table 1 also prove the generation of THF stimulation fluorescence-causing substance in the reaction of BVdUMP and rHuTS.From early stage report THF this reaction there not be not this result of expectability of the data (Barr etc. (1983) above) that influence, and potential important probability is described, i.e. cofactor, or cofactor antagonist, weevil acyl tetrahydrofolic acid can not be regulated the reaction of BVdUMP and people TS.
The Michaelis-Menton dynamic analysis of this reaction proof dUMP is met the competitive inhibitory effect of expection form to the inhibitory action of BVdUMP, coincide with nucleotide as the substrate of rHuTS.
As described in following table 2, before the data with lactobacillus casei TS report showed that BVdUMP was that (Barr etc. (1983) above for the substrate littler 385 times than dUMP effect; Santi D.V. (1980) above).Here report experimental results show that this situation is different fully when the personnel selection enzyme.For rHuTS, it is 60 times that the relative catalytic efficiency of dUMP is compared with BVdUMP.This representative is compared the raising of catalytic efficiency greater than 6.4 times with endogenous substrate.The efficient that previous result with lactobacillus casei TS causes estimating the desmoenzyme reaction is too low for NB1011 not to be effectively to treat substrate, because it is had to and a large amount of endogenous dUMP competes.Here Bao Dao discovery, promptly people's enzyme has the BVdUMP transformation efficiency greater than 6.4 times raising, is an important factor using NB1011.People's enzyme and lactobacillus casei enzyme compare the efficient of the raising of BVdUMP application and determine that also the species specificity substrate is possible and can designs.Reported ability (Stout (1999) biochemistry of comparing with people TS (Biochemistry) 38 (5): (1999) pharmaceutical chemistry magazines (J.Med.Chem.) 42 (12) such as 1607-17 and Costi: 2112-2124) that passes through to suppress isodynamic enzyme recently in conjunction with species specificity district specificity on the surface of lactobacillus casei.The specificity difference relevant with the avtive spot of the TS that is made up of proteinic topnotch conserved region (Carreras (1995) above) is wonderful and had not before reported.
The product of the acellular enzymatic reaction by mass spectral analysis rHuTS and BVdUMP.Molecular structure I that Fig. 5 provides and II have the quality that the quality of molecular ion matches in the TS reactant mixture after the rHuTS incubation with BVdUMP and purification.Can be used for understanding the final mechanism of the effect of NB1011 to the knowledge of this product.In addition, this information can be used for designing new chemotherapeutant, because the TS-BVdUMP product itself has the potential probability as chemotherapeutant.
4.NB1011 in tumor cell, be converted into Monophosphate
According to the bonded requirement in advance of TS, NB1011 is converted into the Monophosphate form from phosphoramidate in cell.Fig. 5 explanation is for the approach of inferring of the phosphate ester of not sheltering of NB1011, its with TS combine and to the conversion of toxic metabolite.
In order to determine whether this committed step takes place advantageously in the conversion, utilized the uncommon character of bromine atoms, promptly it exists in the isotope form of occurring in nature with two kinds of equal abundance.This situation will be measured the bromated Monophosphate of bag to the mass spectral analysis of BVdUMP expectation mass ion (411 and 413 dalton) by concentrating.With H630 R10 tumor cell (it expresses high-caliber TS) and 100 μ M NB1011 incubations.According to the extract for preparing the cellular lysate of handling described in the method.Use Mass Spectrometer Method, then carry out initial purification with liquid chromatograph according to the formation that not have the nucleotide mass ion (it has the identical holdup time on reversed phase chromatography) protected of BVdUMP.
NB1011 in these results (Fig. 6) and the activated pathway after the first step matches.
The sign of the cytotoxic activity of B.NB1011
1. tumor/Normocellular screening
As the bioactive beginning step that characterizes NB1011, normal and tumor cell type is tested susceptiveness to NB1011 and 5-fluorouracil to a large amount of series in alarmar blue test.
Measure according to the description in the method above.With to Normocellular average IC 50With average IC to tumor cell 50The ratio calculation therapeutic index.All mensuration is carried out three times at least.
These digital proofs NB1011 meets the basic purpose of design to TS ECTA chemical compound, and promptly to compare the effectiveness of tumor cell higher with the normal cell type.In a word, and normal cell is compared, to high about 2 times of the cytotoxicity of tumor cell, and 5FU is bigger 3 times than its toxicity to tumor cell to Normocellular toxicity.Therefore, compare with 5FU, total advantage point of NB1011 is that the NB1011 therapeutic index improves (2) * (3)=6 times.It is the activity of screening to normal cell and tumor cell type that screening has the rigorous method of just treating exponential chemotherapeutant.Do not use this method in the field of finding new cancer treatment drugs always.For example, screening is screening technique (Curt (1996) Oncologist 1 (3): part II-III) of National Cancer Institute (National Cancer Institute) to the new candidate compound of normal cell type.
2. NB1011 does not make the TS inactivation in the body
The above results indicates the BVdUMP that produces from NB1011 in the cell can not deactivation TS during it is converted into product.But cell free system is different with medium in the born of the same parents.In order further to explain this problem, carry out active mensuration based on cell to TS.In these experiments to cell culture medium add external source 5-( 3H) (Carreras and Santi (1995) are above in the release of BrdU and monitoring tritiated water; Roberts (1966) biochemistry (Bioehem.) 5 (11) 3546-3548).Fig. 8 illustrate that the existence of NB1011 has reduced in the cell culture medium [ 3H] 2O from 5-( 3H) rate of release of dUMP.In order to determine that this is the irreversible inhibiting result of TS, in fresh culture, reclaim the cell that NB1011-handled, measure the TS activity then.The cell that reclaims in not having the culture medium of NB1011 has the TS activity with the untreated cell par.This result supports such hypothesis, and promptly NB1011 can reversible deactivation TS enzyme after experience born of the same parents internal procedure.
Whether adopt other approach to understand NB1011 mainly may the interference cell growth by deactivation TS.The research is based on the adenosine rescue of the cell of TS-sealing.The cell of Tomudex or 5FdUMP (then handling with 5FdUrd) blocking-up can not prepare dTMP by de novo synthesis.Because this reason, they have only by scavenger mechanism and could survive.One of important scavenger mechanism is interests born of the same parents extracellular adenosines.The adenosine that mixes by target cell can be converted into dTMP by thymidine kinase usually, and it is synthetic to continue to carry out DNA like this.Other approach that uses the external source adenosine was also described.If the active important mechanism of NB1011 is the inhibition by endogenous TS, then when adding adenosine, the pair cell culture medium should alleviate cytotoxicity.For this experiment, they replenish the ability of recovering from these reagent to the sensitivity of Tomudex and 5FdUrd with by adenosine to the screening of various kinds of cell system.Use normal colonic chrotoplast, CCD18co, because they are to NB1011,5FUdR and Tomudex have the sensitivity that can measure.According to (Patterson etc. (1998) cancer researches (CancerRes.) 58:2737-2740), be with or without under the 10 μ M adenosines, experimentize, measure cytotoxicity but be to use alamar blue to measure (referring to materials and methods).The result shows from 15 times of (IC of Tomudex rescue 50Become 95nM from 6.5nM), save greater than 590 times of (IC from 5FudR 50From becoming greater than 5.9 μ M less than 0.01 μ M), do not exist the adenosine end to edge down and hang down the 223 μ M that become under the existence 10 μ M adenosines.
3. to the dependency between the cytotoxicity of the TS level of tumor cell line and NB1011 mediation
Utilize several method to establish the cytotoxicity that TS participates in the NB1011-mediation: to check that 1) NB1011 expresses 5FU-resistance, the activity that tumor cell is compared to normal colon cell and high TS; 2) the TS transfection produces clone's derivant in the tumor cell background, but it expresses different closely similar with TS basically; With 3) use the specific inhibitor of TS, Tomudex reduces TS activity in the cell.
When the cytotoxicity analysis that begins NB1011 and 5FUdR-mediation, to normal colon endothelial cell types of CCD18co and H630R 10,5FU Drug resistance colon tumor cell system compares (Copur etc. (1995) biochemical pharmacologies (Biochem.Pharm) 49 (10): 1419-1426).This has recorded the cytotoxicity to normal cell (CCD18co), and the cytotoxicity that records the drug-resistant tumors cell (H630R 10) to overexpression TS.This is important, because the current chemotherapy thing of significant limitation is their cytotoxicities to normal structure.Table 4 has provided the result.
This experimental results show that compares 5FUdR to high about 18 times of the toxicity of normal colon cell (CCD18co) with 5FU-resistance H630R 10 tumor cells.Should negative therapeutic index be current chemotherapeutical main limitation, promptly it be to the toxicity of normal structure.Also reported so negative therapeutic index (Smith etc. (1985) J.Natl.Cancer Inst.74 (2): (1990) cancer researches (Cancer Res.) 50 (10) such as 341-7 and Smith: 2943-2948) for amycin.But opposite with 5FUdR, compare (IC with normal colonic chrotoplast 50Greater than 2500 μ M), antagonism property of medicine H630R 10 cell (IC 50=216.7 μ M), NB1011 has the activity that improves more than 11 times.This results suggest: 1) activity of NB1011 is more remarkable to high TS expressing tumor cell; With 2) compare with 5FUdR, use TS ECTA chemical compound can realize that therapeutic index improves (18) * (11)=198 times altogether.
4.HT1080 the overexpression of TS improves their sensitivity to NB1011 in the tumor cell.
The activation of NB1011 needs several steps.These comprise that cell-penetrating is converted into nucleotide one phosphoric acid, in conjunction with TS and the toxicity metabolism followed.The accurate mechanism of cell-penetrating and conversion is also not definite fully.Cell enters and may depend in part on nucleoside transporting mechanism ((1998) biochemistry such as Cass and cytobiology (Biochem.Cell Biol.) 76 (5): 761-70).Similarly, the phosphoramidate process that is converted into Monophosphate has been used not too definite mechanism (Abraham etc. (1996) pharmaceutical chemistry magazines (J.Med.Chem.) 8:39 (23): 4589-4575) of definition.
These results are tangible especially, because their proofs, under very uniform genetic background, the indication of the TS level of raising is to the enhanced sensitivity of NB1011.In addition, data also prove the resistance of the TS level indication of raising to fluorinated pyrimidine, and (Copur etc. (1995) above for result and bibliographical information; Banerjee etc. (1998) cancer research (Cancer Res.) 58:4292-4296) matches.
5.NB1011-the Cytotoxic inhibitor of mediation
Tomudex is the chemotherapeutant that mainly works by the inhibitory action to TS.If NB1011 shows cytotoxicity by the TS enzyme, then should reduce the cytotoxicity of NB1011-mediation to the inhibition of TS with Tomudex.In order directly to test this hypothesis, compare overexpression TS11 Tomudex-resistance MCF7 cell doubly with parent MCF7 cell line and in the presence of the TDX of cumulative concentration, contact NB1011.
Cell paved plate and according to the chemical compound of describing the contact prescribed concentration in the top materials and methods chapters and sections.
Data show, use specific inhibitor Tomudex that the blocking-up of TS is caused Cytotoxic up to about 25 times inhibition to the NB1011-mediation.These results support such notion, promptly produce the NB1011 activity by TS from its metabolite.
In order further to characterize born of the same parents' intracellular metabolite of NB1011, with formyl tetrahydrofolic acid (LV; 5-formoxyl tetrahydrofolic acid) carries out group practices.This experiment is initiated, because we find that THF stimulates the generation of fluorescence-causing substance in the acellular reaction of BVdUMP and rHuTS.Suppose if fluorescence-causing substance is relevant with the cytotoxic effect of NB1011, then also strengthen the cytotoxic effect of NB1011-mediation in culture medium by level in the THF born of the same parents that the LV raising is provided.Astoundingly, according to what measure in alarmarblue test, compare with independent NB1011, in the presence of 3 μ M LV, NB1011 reduces more than 90% the activity of H630R10 cell line.The active true prompting NB1011 of LV destruction NB1011 that replenishes the folic acid total amount of minimizing in the born of the same parents may be partly owing to working by reducing these total amounts.Perhaps, LV (or metabolite) may by with TS mutual interference and directly impact the metabolism of BVdUMP mutually.
Whether directly impact the reaction of BVdUMP and TS in order to disclose LV, react ,+/-THF and BVdUMP, perhaps use THF+dUMP and+/-methotrexate (MTX), LV or Tomudex (TDX).
These results's (table 6) are wonderful both ways: 1) although the fluorescence-causing substance that produces from BVdUMP in the presence of THF increases, the speed that the base consumption (BVdUMP) of use has taken place in the presence of this cofactor reduces; With 2) in the presence of cofactor, all three kinds of experimental compounds (MTX, TDX and LV) suppress the rHuTS+BVdUMP reaction greatly.In all cases, this inhibitory action than in the dUMP+rHuTS reaction or do not having in the presence of the THF to see in the reaction with BVdUMP more remarkable.
The above results proof LV, MTX and TDX be to the inhibitory action of BVdUMP+TS reaction and other, and this acts on cofactor (THF) and exists more significantly down, and The above results points out other chemotherapeutant can regulate the NB1011 activity.Importantly, save the cell that NB1011-handled easily, be similar to LV rescue from MTX by LV is provided.Under the MTX situation, the folic acid total amount takes place in the born of the same parents by replenishing in the LV rescue, and the folic acid total amount reduces by the MTX inhibitory action of dihydrofolate reductase and TS in the born of the same parents.If the folic acid that is reduced between the reaction period at BVdUMP and TS reduces in cell, then other chemical compound that reduces thymidine in the born of the same parents or purine nucleotides total amount by different mechanism when use, can provide with NB1011 add with or collaborative anti-cell effect.The example of such chemical compound (Dorr and Von Hoff (1994) are above) comprises Ismipur, thioguanine and 2 '-deoxycoformycin, and all these disturbs purine metabolism.The inhibitory action blocking-up pyrimidine biosynthesis of the orotidine decarboxylase of AzGR-mediation is like this by thymidine level in the born of the same parents in the mechanism reduction cell different with NB1011.
The pharmacogenomics of C.TS ECTA
1.TS and the comparison of HER2
A current discovery and an important aspect developing new Therapeutic Method are to identify the patient of most probable aitiogenic to treating (selection of positive drug genome).Take the lead one of the medicine of cutting edge of a knife or a sword position of this field is Herceptin, is used for treating the breast carcinoma of overexpression HER2 proto-oncogene now.Use the early time data of anti--HER2 antibody to show that tumor cell and Normocellular activity to selecting at random are little.But, if the tumor cell line of selecting HER2 to express at least 4 times of raisings then can proof be compared tangible activity and be resisted-HER2 antibody (Shepard etc. (1991) are above) with the tumor cell of the HER2 gene outcome of normal cell or the low amount of expression; (Lewis (1993) Cancer ImmuralImmunother37 (4): 255-63).
The cell line result that Fig. 2 provides can point out the other similarity between TS and the HER2/NEU system.This similarity is that each has similar overexpression requirement (about 4 times), its indication TS and HER2/NEU overexpression patient disease more serious (Johnston etc. (1994) J.Clin.Oncol.12:2640-2647).
2.NB1011 anti-5FU and Tomudex-resistance colon and breast cancer cell line are activated.
Because NB1011 has the active anticancer of prediction, it is compared with other chemotherapeutant with regard to safety is important.When it is compared with Tomudex, further emphasize in treatment for cancer, to use NB1011, Tomudex is a kind of chemotherapeutant, as 5FU, it often is used to treat colon cancer and breast carcinoma and other malignant tumor.
The result shows that for normal cell (CCD18co), NB1011 is littler more than 10 times than TDX toxicity, and to MCF7-TDX resistance tumor cell, it reaches more than 30 times than TDX is more effective.Obtained similar results for other TDX-resistance tumor cell line.To Normocellular low-level toxicity with to TDX RThe high toxicity support of tumor cell is used NB1011 to the drug resistant cancer of overexpression TS.
3. according to from dUMP- 3The mensuration that the H tritium discharges, NB1011 more relies on the TS protein level than TS activity.
Use four types test to come TS level in characterize cells and the tissue.Technology (Johnston (1994) J.Clin.Oncol.12:2640-2647 that the most frequently used is based on antibody; Johnston and Allegra (1995) cancer research (Cancer Res.)), but also measured RT-PCR in various researchs, 5FdUMP-combination and tritium discharge (van Laar (1996) Clinical Cancer Research (Clin.Cancer Res) 2 (8): 1327-33; VanTriest (1999) Clin.Cancer.Res.5 (3): 643-54; Jackman (1995) otolaryngology record event (Ann.Oncol.) 6 (9): 871-81; Larsson (1996) Acta.Oncol.35 (4): 469-72; Komaki (1995) breast cancer research treatment (Breast CancerRes.Treat.) 35 (2): 157-62; Mulder (1994) anticancer research (AnticancerRes.) 14 (6B): 2677-80)
For characterize cells system, we concentrate carry out Western blotting and from 3The tritium of H-dUMP discharges.Selecting these tests, because antibody-detection is usually used in clinical sample, and is directly the measuring of catalytic activity of TS in the cell from the tritium that the tritium of the BrdU of labelling discharges.
According to cultured cell described in the method and sign.The TS expression is with respect to CCD18co, normal colon endothelial cell line.Described in method, it is the subtracting background value that tritium discharges.ND=fails to detect the value that is higher than background.
The data analysis that provides in the table 7 show the TS protein level and to the relation between the sensitivity of NB1011 than TS activity (from 3The tritium of H-dUMP discharges) and NB1011 sensitivity between relation closer.In the parental cell line and drug-resistant tumors cell line of each cover coupling, the Drug resistance derivant respectively has the more TS protein than the parent, also has the sensitivity to NB1011 of raising.But when carrying out identical comparison about the TS activity, parental cell line usually has comparable, perhaps bigger TS activity, and more insensitive to the cytotoxicity of NB1011-mediation.
These results take place by the combination of a lot of different mechanism or mechanism, may be 3H-dUMP may be subjected to the restriction of some composition to the conversion of dUMP (with following tritium to discharge), may be the cofactor availability.But, because the conversion of BVdUMP does not rely on cofactor, so even the reaction of itself and TS therein cofactor be also to be successive in limited such cell culture medium.This discovery is important, because do not attempt the active typical tritium of TS is discharged the result of test as the TS substrate of therapeutic agent, in described test, the drug-resistant tumors cell that discovery worsens most has lower TS activity than their parent.These results support such prompting in addition, promptly detect the patient who selects to carry out TS ECTA treatment simply on the basis of TS level by antibody staining.
Table 1. The prodrug scheme relatively
Technology Abbreviation Explanation The main reference document
Metabolic activation No Folacin by ' lethal synthetic ' is to the conversion of toxin Mead etc. (1996) above
The prodrugs therapy of antibody control ADEPT Antibody-multienzyme complex is in conjunction with tumor-selective antigen.Use prodrug and when it runs into the enzyme of antibodies, be activated. Syrigos and Epenetos (1999) anticancer research (Anticancer Res.) 19 (1A): 605-13
The prodrugs therapy of Gene Handling GDEPT The gene of coding kinase is transduceed in the big T cell Connors and Knox (1995) stem cell (StemCells) 13:501-511
The prodrugs therapy of enzyme control EDEPT Use prodrug, it is activated by the exoenzyme that just exists at tumor sites place high level. Breistol etc., (1998) European cancer magazine 34, (19): 1602-1606 and Bosslet etc., (1998) cancer research 58:1195-1201
The activated cytotoxin of tumor " TAC " Glutathion-activated the prodrug of s-transferring enzyme Morgan etc. (1998) above
Enzymatic treatment activates ECTA As tumor inhibitor gene forfeiture and the enzyme activation prodrug by the overexpression of results of screening in the chemotherapy body As disclosed herein
Table 2 The comparison of antibacterial and rHuTS kinetic parameter
Kinetic constant Lactobacillus casei rHuTS
?dUMP K m 3.0μM 7.7μM
K cat 6.4s -1 0.2s
K cat/K m 2.1×10 6M -1s -1 2.6×10 4M -1s -1
K i(of?BVdUMP) 0.6μM 4.5μM
?BVdUMP K m 3.3μM 16μM
K cat 0.018s -1 0.0067s -1
K cat/K m 5.6×10 3M -1s -1 4.2×10 3M -1s -1
K i(of?dUMP) 2.0μM 17.5μM
Relative catalytic efficiency (dUMP is to BVdUMP) 385-doubly 60-doubly
According to carrying out the enzyme kinetics experiment described in the method.Data about lactobacillus casei (Lactobacillus casei) derive from (1983) such as Barr above.According to above preparing rHuTS described in the materials and methods. Table 3 Tomudex and 5-FdUMP are to the inhibitory action of rHuTS reaction
Substrate+cofactor There is not inhibitor Tomusde×(500nM) 5-FdUMP(500nM)
BVdUMP±THF ?109±16 ?RFU/min ?(100％) 67±3 (61％) 44±2 (40％)
BVdUMP-THF ?75±11 ?(100％) 34±3 (45％) 93±13 (129％)
dUMP+THF ?1500±20 ?nmoles/min ?(100％) 690±40 (46％) 290±70 (19％)
Tomudex and 5-FdUMP are to the inhibitory action of rHuTS reaction.Be reflected at 30 ℃ of following incubations according to the thymidylate synthetase that will contain enzyme inhibitor or cofactor described in the materials and methods, for the BVdUMP reaction, excite and the 595nm emission increase of relative fluorescence unit down by measuring 340nm, perhaps, for the dUMP reaction, measure A 340Increase, measure the initial rate of enzyme reaction. Table 4 pair normally with the cytotoxicity of the NB1011 of the relative 5FU of tumor cell line
Normal cell ???????IC 50(μM) Tumor cell ??????????IC 50(μM)
??NB101.1 ????5FU ??NB101.1 ????5FU
????CCD1800 (colon) ????562 ????2.0 ????H630R10 (colon) ????65 ????41.6
????DET551 (skin) ????262 ????0.8 ????HT1080 (colon) ????449 ????0.8
????NHDF (skin) ????359 ????0.8 ????COLO320 (colon) ????401 ????1.5
????H527 (skin) ????273 ????1.6 ????COLO205 (colon) ????105 ????1.3
????W138 (lung) ????335 ????1.0 ????SW620 (colon) ????374 ????4.6
????MRC9 (lung) ????303 ????1.1 ????SKCO1 (colon) ????184 ????1.4
????NHLF (lung) ????139 ????0.9 ????HCTC (colon) ????280 ????2.8
????NHA (brain) ????839 ????0.9 ????MCF7 (mammary gland) ????141 ????1.0
????NHOST (bone) ????642 ????4.7 ????MDAMB361 (mammary gland) ????365 ????5.0
????NPRSC (prostate) ????369 ????1.7 ????MDAMB468 (mammary gland) ????172 ????4.4
????NHEPF (liver) ????2085 ????1.7 ????SW527 (mammary gland) ????431 ????4.3
????NCIH520 (lung) ????135 ????0.6
Meansigma methods ????561 ????1.6 ????SKLU1 (lung) ????270 ????7.9
????SOAS2 (bone) ????232 ????1.4
????PANC1 (pancreas) ????492 ????1.9
????SKOV3 (ovary) ????484 ????3.0
????PC3 (prostate) ????184 ????0.9
????HEPG2 (liver) ????704 ????22.8
????SKHEP1 (liver) ????247 ????1.7
????A431 (skin) ????266 ????0.2
????MCIxc (brain) ????61. ????1.2
Meansigma methods ????288 ????5.3
Table 4, continuous
????NB101.1 ?5FU
Therapeutic index (N/T) ????1.95 ?0.30
The pair cell analysis is to the reaction of NB1011 or 5FU in alamar blue test (method).All tests are carried out three times at least.Standard deviation is less than 0%.With IC 50(meansigma methods of all cells type) and IC 50The ratio of (meansigma methodss of all tumor cell lines) calculates therapeutic index.Table 5 pair genetic engineering is handled the NB1011 cytotoxicity with the cell line of expressing HuTS
Cell line The TS level ??????????????IC 50
(％) * NB1011 (μM) FUDR (μM) ?5-FU (μM) TDX (μM)
?C/HT1080 ?100 ?320 ?＜0.1 ?1.0 ?3.6
?TSL/HT1080 ?409 ?196 ?2.2 ?1.7 ?24
?TSL/HT1080 ?702 ?0.8 ?3.1 ?3.5 ?153
Meet frame with GFP and will encode the cDNA sub-clone of rHuTS in carrier pEGFP-C3.With this construct transfection in the HT1080 cell and in order to obtain clone that stably express merges rHuTS with G418 (750 μ g/ milliliter) screening.According to serving as each cell of basis clone with high luciferase expression or low luciferase expression described in the method.By using western blot analysis to measure * TS level, with expression with respect to the value representation quantitative assay of cell strain CCD18co.
Table 6 Tomudex suppresses the cytotoxicity of NR1011 mediation
[Tomudex] (nM) 0nM ?1nM ?10nM ?100nM ?1000nM
NB1011IC 50(μM) 5.7 ?25.5 ?87.7 ?140.3 ?103.0
The protection multiple 1 ?4.5x ?15.4x ?24.6x ?18.1x
Described in method, carry out Tomudex rescue test (alamar blue) with TDX-resistance MCF7 breast cancer cell.To be with or without the IC that adds TDX 50Ratio calculate " protection multiple ". The influence of table 7 folic acid inhibitor
Inhibitor BVdUMP, and THF BVdUMP，w/o?THF DUMP, and THF
None ?100％ 138％ 100％
MTX ?10％ 24％ 31％
LV ?17％ 97％ 77％
TDX ?0％ 25％ 18％
Described in method, use rHuTS to carry out acellular test, merge suitable substrate and other composition.Add MTX (140 μ M), LV (140 μ M) or TDX (5 μ M) estimate their inhibition activity.The application of substrate (perhaps BVdUMP or THF) is used as measuring of reaction rate.The activity that numeral is residual.
Table 8 discharges with tritium to be compared, and NB1011 is active more relevant with TS protein
Cell line Drug screening Albumen Tritium discharges NB1011- IC 50
?H630 Do not have ?288 ?3206 ?414
Colon cancer ??5FU ?2350 ?1840 ?65
??TDX ?671 ?3980 ?2.3
?RKO Do not have ?142 ?4920 ?136
Colon cancer ??TDX ?279 ?1625 ?28
?MCF7 Do not have ?178 ?5185 ?327
Breast carcinoma ??TDX ?1980 ?875 ?2.8
?NIS1 Do not have ?197 ?12.565 ?494
??5FU ?1241 ?ND ?204
The MDF7 TDX cell that table 9 is selected is to the resistance ratio of NB1011
More responsive to 5-fluorouracil and Tomudex
IC 50(micromole) *Relative TS
5-FU Tomudex NB1011 protein level
MCF7?????????????10-?????.026-???????291-????????1X-
MCF7?TDX?????????32??????＞10????????2???????????11X
MCF7?TDX/1011????2???????.041????????240?????????4X
*=according to result by the alamar blue measurements determination described in the materials and methods.
TDX＝Tomudex；1011＝NB1011。
The discussion of front and embodiment are just in order to describe claims of the present invention in detail.It will be apparent to one skilled in the art that not exceeding the spirit and scope of the present invention can carry out various changes to embodiment and claims.

Claims (20)

1. plant the method that selectivity suppresses infectosome propagation, wherein said infectosome is expressed kinase, and wherein said kinase is by the deactivation of substrate prodrug compound, is selectively converted to the substrate compounds that the toxin selectivity suppresses infectosome propagation thereby this method comprises the kinase that passes through of the cells contacting effective dose that makes infectosome or infected by described infectosome in cell.
2. the process of claim 1 wherein that the substrate prodrug is following structure L or D chemical compound
R wherein 1Be or comprise leaving group, described leaving group is to have and the molecular size and the electrophilic chemical individual that adapt from separating of pyrimidine ring by kinase, and it is had the ability that suppresses described infectosome or cell proliferation when pyrimidine ring discharges by kinase;
Wherein Q is selected from sugar, carbocyclic compound, the part of non-ring compound and the phosphate ester of sheltering or phosphoramidic acid ester derivant.
3. sharp 1 the method that requires, wherein said chemical compound has following structure:
R wherein 1Be the following formula group:
Figure A0081301500031
Prerequisite is in the Compound I, and n can be 0,
R 2Be to be selected from two following valence orbit groups:
Undersaturated alkyl;
The aromatic hydrocarbyl that comprises one or more unsaturated alkyls;
The heteroaryl that comprises one or more unsaturated alkyls;
R 3Be to be selected from following bivalence group at interval:
Figure A0081301500032
R 5Can be identical or different, and be straight or branched alkyl independently, or have the cycloalkyl of 3-10 carbon atom with 1-10 carbon atom, or halogen atom (F, Cl, Br, I);
N is the integer of 0-10;
M is 0 or 1;
R 4Be to be selected from following to send out group malicious:
R 8And R 9Be low alkyl group and R 10Be H or CH 3
X is-Cl ,-Br, and-I, perhaps other possible leaving group, prerequisite is to work as R 7Be-H and m are 0 o'clock, R then 4Be not halogen atom, perhaps when m be 0 and n when being 0, then R 4It or not halogen atom;
Y is independently-H or-F;
Z is independently-O-or-S-;
Q is selected from following group:
R 6Be-H-OH ,-OC (=O) CH independently 3, the hydroxyl of F or other protection; And
R 7Be hydrogen, bound phosphate groups, phosphodiester group or phosphoramidic acid ester group;
Wherein said chemical compound can be any enantiomer, and diastereomer, or stereoisomer form comprise, D-form, L-configuration, α-anomer, and β-anomer form.
4. the process of claim 1 wherein that described infectosome is selected from antibacterial, parasite, virus and yeast.
5. each method of claim 1-4, wherein said kinase is a thymidylate synthetase.
6. each method of claim 1-4, wherein said kinase is selected from thymidylate synthetase, beta-lactamase, virus protease, dihydrofolate reductase or viral reverse transcriptase.
7. each method of claim 1-4, wherein said contact is external, exsomatize and body in.
8. the process of claim 1 wherein that described contact is in vivo.
9. each method of claim 1-4 further comprises second kind of activating agent of the inhibition infectosome propagation that makes infectosome or cells contacting effective dose.
10. the kinase that infected body surface reaches is selectively converted to the screening technique of the prodrug of toxin, the described prodrug of not deactivation of wherein said prodrug comprises the cells contacting of the infectosome infection that makes candidate's prodrug and infectosome or express kinase and measures this infectosome or the inhibition of proliferation effect of the cell that infectosome infects.
11. the method for claim 10 further comprises making the cell that does not have normally to infect contact and measure this candidate's prodrug to Normocellular growth or inhibition of proliferation effect with candidate's prodrug.
12. the method for claim 10, wherein said kinase are the thymidylate synthetase that described infectosome is expressed.
13. each method of claim 1-4, wherein said kinase is selected from thymidylate synthetase, beta-lactamase, virus protease, dihydrofolate reductase or viral reverse transcriptase.
14. each method of claim 10-13, wherein said detection comprises the born of the same parents' intracellular metabolite product by the described candidate prodrug of mass spectral analysis.
15. each method of claim 10-13, but wherein said candidate material comprises detectable.
16. the method for claim 15, but wherein said detectable is a kind of fluorescent labeling.
17. the method for claim 1 or 10, wherein said kinase is a wild-type enzyme.
18. the method for claim 1 or 10, wherein said kinase are the mutants of described enzyme.
19. the method for claim 18, wherein said kinase are the mutants that treatment is had resistance.
20. the method for claim 18, wherein said kinase are to show the mutant that 3 '-axido-3 '-deoxyribosylthymine (AZT) is had the HIV-1 reverse transcriptase of resistance.
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