CN1074911A - The material of biological action is bonded to compound, composition and method on the biomone facial mask - Google Patents
The material of biological action is bonded to compound, composition and method on the biomone facial mask Download PDFInfo
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- CN1074911A CN1074911A CN92115176A CN92115176A CN1074911A CN 1074911 A CN1074911 A CN 1074911A CN 92115176 A CN92115176 A CN 92115176A CN 92115176 A CN92115176 A CN 92115176A CN 1074911 A CN1074911 A CN 1074911A
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Abstract
The invention provides the compound with ability on the lipoid that medical active substance is bonded to, these lipoid are included in the physiologically acceptable particle.These compounds comprise a biological action part, this part includes medical active substance, and connect with at least one selected hydrocarbon substituent by a coupling part, so that this compound has and enough nonpolarly gives compound with handle in conjunction with the ability of lipoid.Like this, compound of the present invention can be used for reconnaissance and carry medical preparation, and is detained at this chosen point.
Also provide the method for using all cpds of the present invention to carry out disease treatment or the conditioning of other pathology, for example: provide to be used for the treatment of: the restenosis after (1) angioplasty; (2) rheumatic arthritis; (3) tumor cell proliferation; (4) psoriasic method.
Description
The application is the US189192 of on May 2nd, 1988 application, and title continues for the part of the application of " material of biological action is bonded to compound, composition and method on the biomone facial mask ".
The invention relates to the material with biological action, as therapeutical agent, also can be that diagnostic reagent is bonded to the compound on the physiologically acceptable particle, composition and method; Biocompatible particle not only comprises viable cell and virus, but also comprises inviable carrier particle.The invention still further relates to the starting material and the intermediate that are used for this compounds preparation.The invention still further relates to this compounds and be used for the treatment of agent, also can be diagnostic reagent, in vivo the application of position selectivity release.
The release that is used for the therapeutical agent of disease and the treatment of other pathologic condition can be finished by various means.These means comprise: oral administration, intravenous administration, subcutaneous administration, through percutaneous drug delivery, intramuscular administration or topical application.For some therapeutical agent, more existing delivery modes, or when the side effect that does not have whole body, just can not discharge enough dosage to disease location, or can not make the treatment product stop the enough time to produce the predetermined treatment effect at disease location.
If prevent or reduce the outgrowth drug main of pathological cells to comprising the treatment and the control of various diseases of not wishing or not having control hyperplasia.But what limited to is that these anti-proliferative agents are deleterious to some cell type certainly.Because it is poisonous or deadly to patient's normal cell possibility to control the required dosage of ill cell, therefore can not often give these medicines capapie.Can not wish that the hyperplasia position prevents the generation of this difficulty by anti-proliferative agent is administered directly to.These anti-proliferative agents also need to keep a course at disease location, do not wish proliferation of cells so that they can be controlled effectively, prevent Normocellular migration and damage simultaneously.
Discharge and keep especially effectively that these specific diseases and situation are summarized as follows at privileged site for anti-proliferative agent.Each of these situations all relates to does not specificly wish proliferation of cells, and the whole body administration that is used for the pharmacological agent of their treatments can not get optimum.
1. the obturation again and the restenosis of postangioplasty
Limiting or block the atherosclerotic infringement of crown blood flow, is the major cause of relevant coronary heart disease death.By coronary angioplasty (PTCA) or coronary artery bypass grafting, used the direct intervention method through the skin transillumination.
Use the main difficulty of PTCA method to be, be not only after following the PTCA method closely (serious inaccessible), and be the closed-ended question of (restenosis) postangioplasty vascular for a long period of time.
The restenosis of postangioplasty is the reaction that is damaged by the artery inwall that angioplasty operation causes.Although mechanism still among positive exploration, it has been generally acknowledged that accurately, seem to relate to the hyperplasia of artery middle layer smooth muscle cell, these cell migrations subsequently are to interior (inner membrance) layer, and cell continues hyperplasia there.Normally in the 7-14 after damage days, in inner membrance, stop hyperplasia.
There is again the patient of inaccessible symptom to need further PTCA or coronary artery bypass grafting.Since the carrying out of so big percentage (30～50%) the patient of PTCA to experience restenosis, therefore very clear, will be restricted as the success of the PTCA of coronary artery disease methods of treatment.
By the pharmacology means prevent restenosis attempt generally include being administered systemically of various preparations, and generally speaking be unsuccessful.
Severally be used to prevent just will being described below in more detail of restenosis at the anti-proliferative agent of active research.
A. heparin and heparin fragment
The inhomogeneous glycosaminoglycan of reinforcing yin essence ionic that heparin is made up of the disaccharides repeating unit of thorough Sulfated α-D-glyconic acid and N-ethanoyl-D-glycosamine.The main therapeutic action of heparin is as anti-coagulant.Yet known heparin can also suppress to comprise the growth of some kinds of dissimilar cells of vascular smooth muscle cell (SMC).Have been found that have only very weak or without any the little heparin fragment as tetrose of blood coagulation resisting function in glass test tube and have anti-proliferative activity in vivo.See people's such as Castellot J.Cell Biol., 102:1979～1984(1986).
Heparin is bonded to the SMC cell surface by the combining site of high affinity, and is absorbed in the cell.By being coated with three (dodecyl) ammonio methacrylate bonded ion, heparin can also be bonded on the various artificial surfaces, as polysiloxane, and Mylar
, Dacron
, on polycarbonate, polyethylene and the polypropylene.See people's such as Grode Trans.Am.Soc.Artif.Int.Organs, 15:1(1969).
When heparin was bonded on these materials, the anti-proliferative activity as for whether also keeping heparin did not obtain any data.
B. colchicine
Colchicine is to be used for the treatment of the naturally occurring alkaloid of controlling serious urarthritis.By stoping the formation of mitotic shuttle shape fiber, colchicine can be at glass test tube and is stoped the division of animal and plant cells in vivo, thereby, stoped the cell fission in mid-term.This effect of colchicine is similar to vinca alkaloids, vincristine(VCR) and vincaleucoblastine.Recently, existing reporting fed colchicine every day to the rabbit that atherosis iliac artery is arranged, and carries out vasography and observe around inflatable damage back the, reduced the restenosis degree.See people such as Currier, Circulation, 80,11-66(1989).
Yet, find that in nearest clinical study for the patient who carried out the inflatable angioplasty, the colchicine of 1mg can not reduce the incidence of vascular restenosis every day.See people such as C.Grines, Circulation, 84: II-365(1991).Because toxic restriction, patient is impatient at the dosage more than 1mg/ days, estimates that the patient dosage of human institute be that the amount ratio of colchicine in blood is hanging down 10～20 times to the amount in the research of rabbit.
Other preparation of c
Other preparation that has used in animal experiment and endomysium thickness is reduced is: and angiotensin-conerting enzyme (ACE) inhibitor (people such as J.Powell, Science, 245:186-188(1989); (2) people such as Angiopeptin(C.Lundergan, Am.J.Cardiol, 17(Suppl.B): 132B-136B(1991); (3) people such as Cyclosporin A(L.Jonasson, Proc.Natl.Acad.Sci., 85:2303-06(1988)); (4) the growth hormone antibody that obtains of goat antirabbit thrombocyte (people such as G.Ferns, Science, 253:1129-1132(1991); (5) people such as Terbinafine(G.NemeceK, J.Pharmacol.Exp.Thera., 248:1167-1174(1989)); (6) people such as Trapidil(M.Liu, Circulation, 81:1089-1093(1990); (7) interferon-(people such as G.Hansson, Circulation 84:1266-72(1991)).
Use the drug release of whole body, the restenosis for the treatment of postangioplasty as oral administration comprises: with the periodic administration in fixed gap, periodically change the concentration of therapeutical agent subsequently at disease location.And, when patient must accept the whole body administration, usually must be through 20-30 days, the absorption medicine greater than 99% is handled through liver and kidney, and this depends on into medicine.This shows the chance that serious adverse reaction is arranged.
2. rheumatic arthritis
Rheumatic arthritis is a kind of chronic disease, it is characterized by chronic arthrosynovitis.Though without any the cure method of rheumatic arthritis, the therapy that a kind of approval is arranged is a villusectomy at present, this surgical blanking comprises the soft tissue of removing inflammation in the influenced joint.N.Gschwend, Textbook of Rheumatology, people such as (compile) W.N.Kelly, W.B.Saunders pp.1934-1961(1989).Villusectomy can adopt surgery, chemistry or radiopharmaceuticals to finish.The villusectomy that has proved surgery has the effect of alleviating to pain.Yet in most cases, after the several years, synovitis will recur once more in surgical operation.Moreover, can only carry out the villusectomy of a surgery in each joint, and be difficult to undergo surgery at relatively little joint.For the villusectomy of surgery, shown that the villusectomy of chemistry is a kind of effective alternative method, still, its application is limited by the toxicity to cartilage and bone of commercial preparation now.
For the villusectomy of chemistry,, can think the hyperplasia that has suppressed synovial membrane as the radioisotope synovectomy of the available method of another kind.Yet, radioisotope synovectomy is restricted, this be because, with regard to known situation, so far employed release system requires affected joint secular fixing, or requiring extremely lack work-ing life, inexecutable isotropic substance on the market is the release of the radio isotope whole body of acceptable value to be reduced to lymphoglandula, spleen or liver.Be used for the exploitation of this method of rheumatic arthritis early treatment, radioisotopic sewing is topmost problem on the synovectomy position.
3. ovarian cancer
Ovarian cancer is arranged the 4th of the woman cancer cause of the death.Epithelial cancer will cause about 80%～90% malignant ovarian.Epithelial cancer mainly is to spread all over whole body by exfoliation or lymph.The cell transperitoneal distribution of the outer modal dispersed form of ovary for coming off from the primary tumor surface.
The surgical operation that is used for the main therapy of ovarian cancer seldom can be cured fully, this be because cancer cells usually morbidity just enter peritoneal cavity in early days, can not remove with surgical operation.The radiography of outside ray also is often to use at the surgery perioperatively, yet irradiation dose is limited the radiating limited amount by abdomen organ's (liver, kidney, stomach and intestines).Past attempts is used the colloidal gold or the phosphorus of intraperitoneal injection radioactivity, with the radiation peritoneal cavity.Yet its use reduces, and this is because the uneven distribution and the enteral complication of irradiation.Monoclonal antibody is the current carrier that radio isotope is discharged as at intraperitoneal of testing.Up to now, still get the nod and can be used in the treatment of ovarian cancer without any a kind of these monoclonal antibodies.
Chemotherapy is to ovarian cancer patient's the most frequently used therapeutic modality in advance.Can make patient prolong several years life-spans to the employed medicine of ovarian cancer now.Yet when the systemic administration amount of suggestion, they very big side effect will occur.
4. psoriasis
Psoriasis is a kind of common chronic dermatosis, and it can develop into the no control growth of skin Keratinocytes, produces inflammation and ulcer.So far also can not get being used for psoriasic corresponding healing way.
Current be used for curing psoriasis have whole body with partial.Although two kinds of methods of treatment all are that effectively they all will produce a series of deleterious side effects.Psoriasic whole body therapeutic mainly comprises: the administration of virose compound of corticosteroid and pair cell such as methotrexate.Topical therapeutic comprises: antibacterium or antifungal preparation, tar, use are exposed to the optical treatment of sunlight or UV-light, or topical application steroid class.For psoriasis, the local corticosteroid of using is more than any other therapeutic modality.Yet topical therapeutic has the shortcoming of being wiped or washing off easily, thereby has damaged their long-term effect.The topical application of corticosteroid also is subjected to the restriction of following situation: they are tending towards infiltrating in the surrounding blood vessel, and enter subsequently in the normal circulation, cause undesirable whole body accumulation.
Allow the therapeutical agent of bigger concentration and retention and do not have the administering mode of the pharmacotherapy of a series of side effects at disease location, can prove that be useful when handling above-mentioned situation.
In the exploitation of the treatment of specific objective or diagnostic reagent, recent research work has concentrated in the interaction of using particular organisms, for example, and the interaction of acceptor-ligand or Ag-Ab.Another promising research field comprises: the cell or the capsule (for example, liposome) that contain the specific objective of an amount of diagnosis or therapeutical agent.Specifically, monoclonal antibody has been used for giving these cells or capsule with target signature.Because this is a quite new technology, therefore, these class methods of drug therapy that target is arranged also are not real useful or reliable.
In International Patent Application PCT/US89/00087, a series of compounds have been described, composition, their preparation method and will be attached to application on biomone (as cell or the virus) facial mask in conjunction with the material of biological action, this application can pair cell morphology or physiological function produce obvious deleterious effects.Said compound general formula is R-B-R
1, wherein B represents the biological action material, for example, and treatment or diagnostic reagent, and R and R at least
1One of the hydrocarbon substituent for selecting so that give can be stably with biomone facial mask bonded compound with lipotropy.Comprising the firm compound compositions utilization of describing is used for preparing at the compatible binding medium of compound and the intermembranous stable bond of biomone outside surface.For the predetermined effect of privileged site performance in vivo, utilize these compositions, they are by stably being used for the selectivity organism particle of predetermined position in conjunction with this compound to having intrinsic or acquired avidity, and with in this biomone introducing body, whereby, this biomone is loaded onto predetermined position with the biological action material, to produce its predetermined effect.
According to an aspect of the present invention, provide and had the compound of ability of combined treatment active substance to the lipid that contains biomone such as cell or virus.Compound of the present invention comprises: the biological action part, and comprise by the therapeutic active substance on the stable hydrocarbon substituent that is connected at least one selection in connection portion; So that it is enough nonpolar that described compound is had, can in vivo or in glass test tube, stably be bonded on the fat part that contains lipid physiologically acceptable particle.As requiring to transmit therapeutic activity, described compound can also comprise the spacer part, separates to constitute between therapeutant and connection portion.
The compounds of this invention is further characterized in that, it is indefinite but predictable to contain, the stablizer that partly combines with the microbial film lipid.For surface film retention (MRC) with respect at least 90% and at least 30% the film combination stability, described compound is enough nonpolar.Compound of the present invention in use also must be enough to stable, that is to say, in case after by direct administration or by the carrier administration therapeutical agent being delivered to the selectivity position, it can keep the time and the amount that be enough to produce its predetermined action at there.Details are as follows for the step of measurement film retention and film combination stability.
According to the present invention, above-claimed cpd is used as the therapeutical agent that carry at the selectivity position, and keeps at selection part.According to a preferred aspect of the present invention, this therapeutical agent has anti-proliferative effect, and is useful to the disease that relates to hyperplasia or the treatment of other pathologic condition.This anti-proliferative agent can comprise radiotherapy material or chemotherapy material.According to preferred concrete example, the invention provides some compounds like this, wherein, the radiotherapy material contains sequestrant and radioactive metal.In another preferred concrete example, compound of the present invention contains the chemotherapy material, as heparin, r-hirudin and their derivative, and the preparation that can influence the inner cell function of selecting.
According to a further aspect in the invention, the chemotherapy material can discharge the ground conjugation to compound of the present invention.For example, can select this class chemotherapy material from colchicine, vinca alkaloids, taxol and their derivative, these materials are only in the biological action that just demonstrates them when compound discharges.The synthetic combination of these compounds affect tubulins separates or degraded, and/or function, and these are referred to as " tubulin processing " in the concentrated area below.For example, provide a sour fissionable colchicine derivative, wherein, as long as it keeps conjugation to the connection portion, the colchicine analogue that is included in wherein is passivation basically.This compound is sent to selection part, and finally enters cell.Finish the absorption of this compound by reducing pH value, this will make the colchicine part produce division from the connection portion.The colchicine analogue that discharges can be brought into play its predetermined biological action, and this may influence the processing of tubulin.
According to another aspect of the invention, provide the pharmaceutical preparation that is contained in the The compounds of this invention in the compatible Biomedia.
Also have on the other hand according to the present invention, provide in the treatment of disease or other pathologic condition, the using method of all cpds of the present invention, compound comprises therapeutic active substance and not necessarily, comprises diagnostic reagent.
In order to keep at disease location, compound of the present invention is preferably by directly transporting administration in vivo.Other method is, compound can be bonded to be suitable for directly this compound being delivered on the carrier granule of disease location.For various pathologic conditions, as the restenosis of postangioplasty, rheumatic arthritis, ovarian cancer and psoriasis treatment particularly preferably are in the body and directly carry, and this is more detailed description below.
Compare with the present available Compounds and methods for that is used for therapeutical agent is delivered to disease location, the present invention has many tangible advantages.Be that by in stable the combining of selection part and cellularstructure, compound of the present invention can discharge and be retained in intravital selection part the most significantly.More existing delivery modes, or when the side effect that does not have whole body, just can not discharge enough dosage to disease location, or can not make the treatment product keep therapeutic action time and the amount that is enough to produce hope at disease location.Compound of the present invention can reach the effective dosage of treatment at disease location.For example, under radiocurable situation, the utmost point does not wish that the whole body of emitting isotope distributes, compound of the present invention can make the stable combination of radiotherapy material and keep at required position, and can limit isotopic whole body and distribute, and do not need to carry out arthrodesis or work-ing life extremely short isotropic substance.
Another significant advantage of the present invention is, can prepare compound like this, and promptly they stably combine with epicyte at the very start, and open processing by normal film subsequently, are absorbed in the cell.This characteristics connection is with the preparation ability that contains the The compounds of this invention that can discharge the conjugation therapeutical agent, make the genotoxic potential material can deliver to the cell interior of selection, their can become activation and independent these cells are brought into play its therapeutic action at there, and other cell of health is not worked.The conveying of anti-meaning RNA or DNA also can be finished with this method.
Another advantage of the present invention is, it mainly is that avidity by lipid produces that compound described herein is bonded on cell and other physiologically acceptable particle.This is particularly important with regard to cell because the combination in lipid will reduce being positioned on the discontinuous protein portion not in the jammer meeting of the vital role domain structure of the cytolemma in more wide lipid zone.Dependence is bonded on membranin and the cell receptor to carry the biological action material often to cause the ability to function that weakens to the previous method on the cell.
According to the combination in the cytolipin zone, also can obtain the benefit of some associations.This is because the lipid zone has comprised most cell surfaces, therefore just the therapeutical agent of more lipid binding compounds and bigger concentration like this can be inserted in the membrane plasmapheresis.And because the lipophilic characteristic of The compounds of this invention, they mix in the membrane lipid with being stabilized, so they are undissolved relatively in normal physiology salt.Therefore, in case after these chemical combination were bonded on the film, they are just collected effectively stayed the there, and can not separate easily.Therefore, oozing out from cell is reduced to minimum, thus, makes undesirable general action reduce to minimum.
With reference to accompanying drawing herein.
Fig. 1 shows the comparison test result of the vascular response curve of local injection Substance P and injection Substance P of the present invention-lipophilic cyanine conjugated body.Upper graph is about Substance P, and following graphic representation is about conjugated body.
Fig. 2 is presented at the antagonist that exists and do not have Substance P, (D-Pro
2, D-Trp
7.9During)-SP, the dosage-response relation curve of every kind of Substance P and Substance P of the present invention-oleophylic cyanine conjugated body.
Fig. 3 is a series of fluorescence/frequency histogram, and point is wherein represented independently cell; When from the former distance that increases each point of lighting of X-axis, represent to increase ruddiness brightness (LFL2 signal), otherwise, when from the former distance that increases each point of lighting of y axle, represent to increase green glow brightness (LFL1 signal).
Fig. 4 is with the function measurement of time, represents the graphic representation of Substance P of the present invention in vivo-lipophilic cyanine dyes conjugated body to the red blood cell combination stability.
Fig. 5 be radioiodine (
125I) bar graph of the lipophilic cyanine of mark degree of keeping on four kinds of dissimilar artificial surfaces (silicon rubber (SIL), polycarbonate (PC), polyvinyl chloride (PVC) and polyethylene (PE)).The striped post represent to be incorporated in at the beginning each lip-deep radioiodine (
125I) the lipophilic cyanine amount of mark; Closely knit post is illustrated in the continuous 6 hours amounts that keep behind the hemoperfusion.With the numerical value of paired column combination be the reservation percentage ratio of compound bonded original vol.
Fig. 6 shows the data that obtained by experiment, wherein, for the fibrinous reproductive performance that is suppressed at external thrombin generation by normal concentration, has tested the carrier cell with anti-coagulant lipophilic cyanine conjugated body mark.The concentration rational curve that is used for non-conjugated anti-coagulant is provided with making comparisons.The percent inhibition (y axle) of record zymoplasm response is as the function of the carrier cell number (going up the x axle, logarithmic scale) of the function of being tested compound volumetric molar concentration (following x axle, logarithmic scale) and each sample.
The speech of following definitions and phrase are used for the present invention to be described as a reference:
1. biological action part-term biological action part is used for representing to be used for various different material of treatment, diagnosis, prevention or other treatment of the mankind or animal with the biological action material interchangeably at this.These materials comprise any material that can bring into play biological action.
2. the term " biocompatible particle " of biocompatible particle-as used herein not only comprise in vivo with glass test tube in viable entity cell for example, but also comprise inviable entity, as liposome and lipoprotein, when needing only administration in vivo, they can not cause serious negative interaction.
Word " can play the viable physiologically acceptable particle of physiological action " and be used for the virus representing any viable cell or contain film at this.And, comprising at this used term " cell ": prokaryote, as bacterium; And eukaryotic cells, as white cell, various oncocytes; With the mammalian cell of cultivating, Chinese hamster ovary cell for example, yeast; And cytode, as red blood cell, red blood cell ghost and thrombocyte.Detailed description of the present invention will be described below according to specific life somatocyte.Yet, should be understood that described cells involved also is general to containing film virus.What it is also to be understood that is, according to the present invention, is carrying out during the therapeutical agent selection part carries, and inviable entity can suitably substitute with viable entity, as long as they are intrinsic or obtained avidity for the delivery site of being scheduled to.For example, liposome can be as the carrier that will be delivered to the therapeutic active substance on liver or the kidney.
3. diagnostic reagent-refer in vivo or in the glass test tube test can be convenient to the compound or the material of physiological conditions or state-detection, measurement or analysis.In use, compound of the present invention can be used as informational molecule and plays difunctional effect, can be in vitro detection, or can in glass test tube, analyze the body fluid or the living tissue that obtain and detect.
4. color base-refer to by absorbing the optical wavelength of at least one selection, with the naked eye or can detected material with instrument.
5. therapeutic active substance-refer to can prevent, relaxes, treats or cure the material of the unusual or pathologic condition of live body.These materials comprise: can keep, increase, reduce, limit or destroy the material of live body physiological action, and by suppress, kill, modification or keep microorganism or the material of its antigen protection live body.Therapeutic active substance comprises the medicine of medicament or treatment usefulness.
6. chemotherapy material-refer to its therapeutic action is the therapeutic active substance that the chemical property by this material produces.The chemotherapy material can comprise: for example cold medicament.They can comprise: small molecules; Or as the bigger complex molecule of lipid, carbohydrate; Protein; Or nucleic acid, as DNA or RNA.
7. radiation treatment material-the refer to therapeutic active substance that its therapeutic action is produced by its radioactivity.The radiation treatment material that is suitable for can comprise the radio isotope atom.This biological action part preferably contains and various radiotherapy nucleic, as
186Re,
90Y,
67Cu,
177Lu or
153The sequestrant of Sm chelating.
8. anti-proliferative agent-the refer to therapeutic active substance that can suppress, reduce or prevent hyperplasia.
Compound of the present invention can be used for various pharmacotherapy, and wherein, desirable is that therapeutic active substance is carried selection part.According to preferred concrete example of the present invention, this therapeutic active substance is an anti-proliferative agent, and it can be radiotherapy or chemotherapy material.In alternative another concrete example, compound of the present invention can comprise diagnostic reagent, and as color base or radionuclide, it can be to carrying out spike and/or detection at glass test tube or compound of the present invention in vivo.Therefore, compound of the present invention can comprise for example as the detected color base of connection portion with as biological action therapeutic active substance partly.
The diagnostic reagent that constitutes The compounds of this invention can be selected from: all kinds of materials that can detect by the known various analytical procedures of those of skill in the art in the prior art.Detectable fluorescent chemicals is cyanine dyes and their derivative preferably, for example comprises the oxo carbocyanine, indoles carbocyanine, thiono cyanine or acridine dye and their derivative.Other useful fluorescent chemicals comprises: for example, and stibazole, xanthene, phenoxazine, thiodiphenylamine or DPH dyestuff and their derivative.
Useful diagnostic reagent can also comprise the ligand of being convenient to detect, as vitamin H or specific antibody.Other useful diagnostic reagent is what can directly or indirectly detect, with the sequestrant of metal complex.The complex compound of suitable chelated mineral can comprise: be selected from atom and make up the number from the isotropic substance of the transition metal series of 21-49, for example indium-111 or technetium-99m.This compounds can be bonded on the cell membrane plasmapheresis of carrier cell, give them,, use γ-Kamera can make it imaging so that after in sending into body with radioactivity.But the chelating material can also with the complexing of metal ion of indirect detection, test example is as being undertaken by some the effect reason that produces at interested position.The complex compound of paramagnetic element for example, can influence the fall time near the nuclear place, and this can detect by magnetic response image (MRI).The chelating metal complex that contains the metal ion that is selected from the lanthanon that transition metal series that its ordination number is 21-29 or its ordination number be 59-66 is applicable to this purpose.
If desired, in various application of the present invention, also can use the compound that contains radioactive atom.In order to have more but the naturally occurring atom of inactivity form at the biological action of this compound part, color base or hydrocarbon afterbody, radio isotope as
125I,
131I,
14C,
3H,
35S or
75Se can be substituted.Isotropic substance with nonzero spin state (for example
19F) also can introduce in the compound of the present invention, so that make their existence to detect with MRI technology.
For treatment is used, the biological action part can comprise the above-mentioned type and with various radiotherapy nucleic as
186Re,
90Y or
67The sequestrant of Cu complexing.
For treatment according to the present invention is used, comprise that the proteic substance of protein, glycoprotein, lipoprotein or peptide also can be coupled to the hydrocarbon afterbody of suitable chain length by suitable connection section.The proteic substance of representational biological action is an immunogen, toxin, hormone, enzyme, antigen, antibody and antibody fragment.The best conjugation of this class therapeutic activity protein and by containing the different of lipid biomone but predictable combination stability carries out mark to final conjugated body with various, will be carried out to the oleophylic color base of above-mentioned type for example hereinafter.
In another treatment was used, biological action partly contains can change the carbohydrate that is bonded to intravital migration of cell and Recycle design.The one class carbohydrate that can be used for this method comprises sialic acid; The another kind of glycosaminoglycan that comprises.For example, contain sialic prescription and can be used for erythrocytic membrane plasmapheresis, to increase the quantity of sialic acid on this film.The quantity increase of the group of filling will be increased in discharges from liver the erythrocytic life-span in the preceding working cycle.
This biological action part can also be the ligand form, and it can be bonded on the acceptor of particular organization or be bonded on the acceptor of demarcating on the organ inner cell.The compound that contains this class ligand is in the time of for example on being incorporated in to carrier cell, with the transporting action that strengthens the certain organs position.
Can compound of the present invention directly be delivered to position selected in the body by various means, these means comprise: injection, perfusion, catheterization and topical application and some other means.Compound of the present invention also can be bonded to for example autogenous cell, on the compatible particle of carrier organism of different group cell of the same race or zenogenic cell, so that the calibration of biological action material is carried.Except as otherwise noted, the discussion that shows below refers to, and perhaps by directly being delivered to disease location, or in the preparation of vehicle excipients compound of the present invention is bonded on the viable cell.An object of the present invention is to provide medicine or radio isotope and can be connected to compound on it, and described compound is dissolved in the double-layer of lipoid that constitutes epicyte.When directly being delivered to disease location, the conveying of the therapeutant by The compounds of this invention is compared with other attainable conveying, will these materials can be kept with higher concentration and longer time on the cell of zone of action.And it may be that the use of virose therapeutical agent becomes possibility that the use of The compounds of this invention can make under whole body administration situation.
The mode of action of The compounds of this invention pair cell is different, and depends on: the cell category that (1) this compound connects; (2) character of lipophilic tail, length and quantity, (3) by the body part of being treated, the effect of the pair cell that the character of (4) biological action part and (5) produce by the course that biological action partly is connected on this compound.Compound of the present invention is deposited on the cell, and at the very start directly to the outer double-layer of lipoid of membrane plasmapheresis.Because the film component is transmission inwards naturally, so these compounds also will be brought in the cell.At the cell place, the speed that this situation takes place will depend on the kind of specific cells, its growth conditions with and activation or excitation values.
The anti-proliferative agent of some type as the radiotherapy material of conjugation to the The compounds of this invention, no matter they concentrate on the adventitia still in cell, all will be active.These anti-proliferative agents send the radiation of infringement nuclear, if this radiation is enough to penetrate, so, it also will suppress the growth of peripheral cell.Must interact to suppress the medicine of growth with intracellular component in fact, after compound of the present invention is absorbed into cell, just will be effective only.Also designed the another kind of mode of action, whereby, can be with the medicine of suitable poison such as colchicine conjugation to the lipophilic part, and be bonded on the adventitia with the form of passivation, this passivation form pair cell is avirulent.Then, when this conjugated body inwardly transmitted, the connection portion (with more detailed description in the example below) of a special preparation made this medicine come off from conjugated body, and brings into play its anti-proliferative effect.
The compound that falls into the scope of the invention is those compounds of following general formula:
Wherein B represents to contain the biological action part of effective therapeutant, R and R
1Expression is selected from the substituting group of hydrogen, alkyl, thiazolinyl, alkynyl, alkaryl or aralkyl independently, and its hydrocarbon chain is a straight or branched, and they be non-replacement or replace R and R at least by one or more nonpolar functional groups
1One of comprise hydrocarbon substituent, its chain length is for can give the film binding ability effectively to compound; R
2Represent a spacer part, n is 0 or 1, and when n=0, L is illustrated in B and R and R at least
1One of between constitute the connection portion of stable bond, when n=1, L is illustrated in R
2At least R and R
1One of between constitute the connection portion of stable bond, suppose that when n=0 L is the connection portion of non-aromatics.
" nonpolar functional group " refers to as oxygen-alkyl, sulphur-alkyl, halogen, N(alkyl in this employed word)
2, selenium-alkyl, NO
2, CN, CO-alkyl, Si(alkyl)
3, the O-Si(alkyl)
3Deng substituting group.
Connection portion (L) can be saturated or unsaturated aliphatic connection portion (linker), maybe can be to comprise alicyclic and ring texture aromatics, and these ring structures can be monocyclic, polycyclic, isocyclic, condensed or uncondensed.
The connection portion is a color base preferably.In The compounds of this invention, introduce color base and will help this compound of spike in vivo.The useful color base that is used for this purpose comprises: cyanine, acridine, pyridine, quinoline, xanthene, phenoxazine, thiodiphenylamine and diphenyl hexatriene dyestuff with and derivative.
The compound that falls into the preferred class of the scope of the invention is the compound of following structural formula:
Wherein B represents the biological action material, R and R
1Expression is selected from the substituting group of hydrogen, alkyl, thiazolinyl, alkynyl, alkaryl or aralkyl independently, its hydrocarbon chain has 1～about 30 carbon atoms, and be straight chain or side chain, said substituting group is non-replacement, or by the replacement of one or more nonpolar functional groups, R
2Be expressed as follows the spacer part of general formula:
-(R
3) p-Q-(R
4-Q ') q-(R
5-Q ") r-(R
6-Q
) s-(R
7-Q
) t, wherein R
3The expression aliphatic hydrocrbon, R
4, R
5, R
6And R
7Be independently selected from: aliphatic hydrocrbon, alicyclic hydrocarbon or aromatic hydrocarbon, heterogeneous ring compound or CH
2C(CO
2H)=CH, Q, Q ', Q ", Q
And Q
Be independently selected from: by acid amides, thiocarbamide, hydrazone, acylhydrazone, ketone acetal, acetal, ortho ester, ester, acid anhydrides, disulphide, urea, carbamate, imines, amine, ether, carbonic ether, thioether, sulphonamide, carbonyl, amidine and triazine connecting key, in addition, Q ', Q ", Q
, Q
, can represent to have C by valency bond independently
1-12The aliphatic series of Linear Carbon atom or clicyclic hydrocarbon and C
6-12Aromatic hydrocarbon; N, p, q, r, s and t can be 0 or 1 separately.
X and X
1Can be identical or different, and represent O, S, C(CH
3)
2Or selenium;
Y represents to be selected from=CR
8-,=CR
8-CR
8=CR
8-,=CR
8-CR
8=CR
8-CR
8=CR
8-or=CR
8-CR
8=CR
8-CR
8=CR
8-CR
8=CR
8-linking group, R wherein
8Be selected from H, CH
3, CH
2CH
3, CH
2CH
2CH
3Or CH(CH
3)
2;
Z represent to be selected from H, alkyl, OH ,-O-alkyl, COOH, CONH
2, SO
3H, SO
2NH
2, CONH-alkyl, CON-(alkyl)
2, NH-acyl group, NH-alkyl, N(alkyl)
2, SH, S-alkyl, NO
2, halogen, Si(alkyl)
3Or O-Si(alkyl)
3, tin (alkyl)
3Or the Hg-halogen, include C
1-4The substituting group of the substituent alkyl group of said Z, A represents biocompatible negatively charged ion.Use R or R in the structure formula II with respect to the microbial film bonded that requires high stable
1One of 12 carbon atoms must be arranged at least, at R and R
1Neutral line the total number of carbon atoms amounts to necessary 23 at least.
According to the synthetic route of knowing, can be by being present in or on headgroup, perhaps on the biological action part, or the suitable functional group that all has on both, between cyanine headgroup and biological action portion, introduce various spacers part (R expediently
2).The interval dose of dissimilar difunctionalitys (homotype difunctionality and special-shaped difunctionality) is reported at the technical literature that is used for such purposes in a large number.See Meth.Enz., 91:580-609(1983).According to the kind of reactive group, hydrophobicity or wetting ability, the structure of length and ligation group is a cleavable, still can not cracked, these spacers parts are with different.
As mentioned above, the sense of spacer connect can be acid amides (NHCO-), thiocarbamide (NHCSNH-), hydrazone (=NHN-), acylhydrazone (=NHNCO-), ketone acetal (O-C(alkyl)
2-O-), acetal (O-CH(alkyl)-O-), ortho ester (C(O-alkyl)
2-O-), ester (COO-), acid anhydrides (COOCO-), disulphide (S-S-), urea (NHCONH-), carbamate (NHCO
2-), imines (=N-), amine (NH-), ether (O-), carbonic ether (O-CO
2-), thioether (S-), sulphonamide (SO
2-NH-), carbonyl (CO-) and amidine (NHC=(NH
-)).
In radiotherapeutic preferred concrete example, B represents the sequestrant with complexings such as radioactive metal such as rhenium or yttriums.In chemotherapeutic preferred concrete example, B represents heparin, r-hirudin, colchicine, vincaleucoblastine or their analogue, and all these is an anti-proliferative agent.In chemotherapeutic another preference, B represents peptide.With free is that unconjugated peptide is compared, and has found that the peptide derivant that is called as Substance P that falls into said structure formula II can stably be bonded on the red corpuscle, and long therapeutic action is provided in circulation.Can expect when with unconjugated form, in circulation, to have short-life relatively other therapeutic activity thing and can obtain the enhanced bioavailability equally.
In some application of The compounds of this invention, for example combination therapy and diagnosis, the B in formula I is preferably vitamin H.
1. the functionalized cyanine of lipophilic
According to the lipophilic cyanine precursor of following general formula, can prepare the compound of said structure formula II easily:
Wherein R, R
1, X, X
1, the above-mentioned definition in Y, Z and the A reference configuration formula II compound; R
2The spacer part of structural formula below the expression:
-(R
3) p-(Q-R
4) q-(Q '-R
5) r-(Q " R
6) s-(Q
-R
7) t-, wherein R
3, R
4, R
5, R
6, R
7, Q, Q ', Q ", Q
, the above-mentioned definition in p, q, r, s and the t reference configuration formula II compound, W represents to be selected from amino (NH
2-), alpha-halogen kharophen (NH-COCH
2-hal) (hal=halogen), isothiocyanate (NCS), halogen, isocyanate (NCO), carboxyl (COOH), diazanyl (NHNH
2), hydrazides (CONH-NH
2), ketone (RCO), for example benzophenone, dithio pyridyl (ss-C
5H
5N), sulfydryl (SH), aldehyde (HCO), acid anhydrides (COOCO-alkyl), succinimide ester (COOC
4H
4NO
2), hydroxyl (OH), halosulfonyl groups (SO
2-halogen), imido-ester (C(=NH) OCH
3), Oxyranyle (C
2H
3O), dimaleoyl imino (NCOCH=CHCO) and azido-(N
3).
The suitable precursor that is used to prepare The compounds of this invention can be prepared according to different synthetic routes, the synthetic route of one of them is illustrated in the reaction scheme 1, and will go through in the example below, described precursor contains in the said structure formula reactive amines (NH as substituting group W
2) base.
According to scheme 1, according to Gale and Wilshire, Aust.J.Chem., step 30:693(1977), with 2,3,3-(3H)-tri-methyl indole false (available on the market) and N-methylol phthalamide (available on the market) reaction, obtain compound (1), with this compound and the reaction of alkyl 4-closilate, get intermediate (2) again, said sulphonate is to pass through Sondermann, Liebigs Ann.Chem., step 749:183-197(1971) makes according to corresponding pure and mild 4-chlorobenzene sulfonyl chloride.By in concentrated hydrochloric acid, heating, remove the benzene imide base protecting group in the intermediate (2), carry out subsequently alkalescence put in order compound (3), again this compound is handled with methyl-formiate, (4).Compound (5) be by or 2,3,3-(3H)-tri-methyl indole false or 2-methyl-benzoxazoles (available on the market) make with the alkylating of alkyl 4-closilate.Subsequently, by being similar to US2,647,054 methods of describing, with compound (5) and N, N-amitraz diphenylurea (available on the market) reaction, (6).Intermediate (4) and (6) can be coupled together by stirring in ethanol in the presence of alkali (sodium acetate or triethylamine) subsequently, get (7).By people such as Dhawan, Orgn.Prep.and Proc.Int.7(2): step 85-88(1975), the provide protection of going of (7) will obtain the cyanine (8) of amino derivatization.
This amino cyanine (8) can be connected directly to and contain appropriate functional group (for example, CO on therapeutical agent COOH), can be used for the conjugated body of privileged site drug conveying with production.In addition, for can conjugation the preparation of many other cyanine derivatives to other biological action part, this amino cyanine (8) is very general intermediate.And with regard to regard to the reaction of many homotypes or special-shaped difunctionality compartment, compound (8) is the ideal molecule, with lipophilic cyanine part be connected to it on the biological action material between constitute and separate.
Amine substituting group on the cyanine derivative that generates can convert other functional group to easy method with the reaction scheme of knowing.For example, according to people such as de Costa, J.of Lab.Compds.and Radiopharm., 27(9): step 1015(1989), the processing by with thiophosgene can realize the conversion of isothiocyanate functional group.
Amido and 1, the reaction of 4-terephthalic acid two-N-hydroxy-succinamide ester has formed by OOC-aryl-CONH-alkyl spacer and partly has been connected to the succinimido functional group on the cyanine headgroup.The compound that generates can separate and purify becomes stable crystalline solid.
The amino and the simple acylation of p-nitrophenyl iodo acetic ester (market is available) in dimethyl formamide that replace have formed by alkyl-CONH-alkyl spacer and partly have been coupled to active iodine on the cyanine headgroup for substituting group.
Amido and N-hydroxy-succinamide base-3(2-pyridine disulfide group) reaction of propionic salt (market is available) has formed the compound that pyridine disulfide group functional group and alkyl-CONH-alkyl spacer part are arranged.
The reaction of amido and γ-sulfo-butyrolactone has formed the compound that sulfhedryl functional group and alkyl-OCNH-alkyl spacer part are arranged.Similarly, the reaction of amido and gamma-butyrolactone has formed the compound that hydroxy functional group and alkyl-CONH-alkyl spacer part are arranged.
2. proteins/peptides lipophilic cyanine conjugated body
The lipophilic cyanine precursor that is generally preparation as mentioned above can be connected on protein or the peptide by many different synthetic routes, to obtain the compound of structural formula II.
The coupling to aforesaid those lipophilic connecting key derivatives for example of protein or peptide can be undertaken by the amido that is present on protein or the peptide (being the alpha-amino group or the epsilon-amino of Methionin).In addition, carboxyl or thiol (wherein having the halfcystine resistates) can be used for protein or peptide are coupled on the suitable cyanine precursor.The appropriate condition that is used to carry out such coupled reaction is known to those skilled in the art.
A kind of method that is used for the alpha-amino conjugation of lipophilic cyanine precursor and polypeptide is by people such as Wetzel, Bioconjugate Chem., and 1, the improvement of step 114(1990) is carried out.This is included in the acylation that pH is 6 o'clock polypeptide and iodo diacetyl oxide (market is available); to form the iodo-acetamide derivative; this derivative reacts with the sulfhedryl deutero-lipophilic cyanine compound for preparing as mentioned above subsequently, and forms conjugated body by the thioether bond with good stability.
The conjugation of the ε-aliphatic amino by Methionin is preferably in more than the pH8.5 to be carried out, with this understanding, when reaching balance, only have limited amount amine moiety right and wrong protonated with active.The active lipophilic of above-mentioned some amine is connected compound, and this amino must have high reactivity.For example, different sulfur hydrogen salt deutero-connecting key compound demonstrates high stability under aqueous conditions, and can react with the side chain of Methionin, forms the conjugated body that connects by the thiocarbamide chain.
With regard to regard to the Methionin reaction, above-mentioned N-hydroxyl-succinimido ester deutero-connecting key compound is good especially reagent, and this is because so the acid amides conjugated body that forms is very stable.This reaction is preferably carried out in as the organic solvent of dimethyl formamide under anhydrous condition, and this is because this specific hydrolysis of derivative under aqueous conditions is the side reaction of a competition.A kind of N-hydroxyl of said structure formula III-succinimido ester deutero-connecting key is reacted on the amino of the Methionin remnants of conjugation to ten peptide (Substance P) the 3rd position be will be discussed in more detail below.
Be present in the carboxylic acid group on the amino acid remnants on the side switch of L-glutamic acid and aspartic acid remnants and that be present in peptide or protein C-end, be to use above-mentioned amine deutero-lipophilic cyanine compound to select the possible position of conjugated.This reaction can or be passed through to use as the water miscible carbodiimide of reaction coupler, as 1-ethyl-3-dimethylamino-propyl carbodiimide diimine (market is available), according to Hoare and Koshland, J.Biol.Chem., the improvement of step 242:2447-2453(1967) is carried out, perhaps by using 2-ethyl-5-phenyl-isoxazoles-3-sulfonate (reagent K of Woodward, market is available) as reactive coupler, according at J.Biol.Chem., the improvement of the step of describing in 249:5452(1974) is carried out.The conjugated body that generates is to connect by stable amido linkage.
Must avoid functionalized derivative to be connected to and be mainly used on bioactive peptide or the activity of proteins group, this be because, this will produce the conjugated body of passivation usually.Yet if peptide or albumen mass-energy discharge from lipophilic cyanine derivative by the spacer part of cleavable, this problem just can be overcome.
Because the lipotropy of above-claimed cpd, therefore, some of them will be difficult to be dissolved in the water buffer system commonly used.Therefore, require aqueous conditions will have in the water-containing solvent of organic solvent modification, carry out with the conjugation reaction that keeps medicine dissolution or activity conformation.Solvent such as dimethyl formamide, dimethyl sulfoxide (DMSO), acetonitrile and alcohols and water are mixable, and can be used to dissolve lipophilic cyanine derivative, produce desirable conjugation.
Utilize the size of formed specific compound, electric charge or lipotropy by the purifying technique of various standards, can be purified compound of the present invention.For containing the useful especially method of purification of peptide therapeutics is people such as Bohlen, Int.J.Rept.Prot.Research, method 16:306-10(1980).
3. the lipophilic cyanine of biotinylation (Biotinylated)
Biotinylated lipophilic cyanine compound of the present invention preferably has following structural formula:
R wherein, R
1And R
8As the front definition, m is 0～6.Biotinylated derivative like this can make by the reaction of the functionalized lipophilic cyanine compound of vitamin H or biotin derivative and said structure formula III.The biotin derivative that is used for this reaction preferably has following structural formula:
Wherein E represents the remnants of the compound with labile state group that can replace by said active function groups W, m=0,1 or 2.
For example, the cyanine derivative (8) of the aminofunctional that makes according to reaction scheme 1, can be according to Hofmann, Finn and Kiso, J.Am.Chem.Soc., step 100:3585(1987), with the active biotin derivative of the available amine in market, (+)-vitamin H 4-nitrophenyl ester, N-hydroxy-succinamide base 6-(biotin acylamino) capronate and 6((6((biotin acyl)-amino) hexanoyl) amino) reaction of caproic acid N-hydroxy-succinamide base ester, to form the vitamin H-lipophilic cyanine conjugated body of said structure formula IV, wherein m is respectively 0,1 and 2.With regard to the length of the biotin moiety of conjugated body and lipid bound fraction interbody spacer arm, these conjugated bodies are different, depend on the purposes that this biotinylation compound is predetermined, and the effect of this spacerarm can be very important.Proved that long spacerarm has useful effect to the binding ability of biotin-conjugated body " degree of depth " combining site of vitamin H in avidin.
Band active function groups such as dimaleoyl imino, alpha-iodine is for acetamido, diazanyl and amino various other biotin derivative are available (the Molecular Probes in market, Eugene, OR, Handbook of Fluorescent Probes and Research Reagents 1989-91), and can be used to and various above-mentioned functionalized lipophilic connecting key compound couplings.Suitable reaction scheme will be known to those skilled in the art.
In addition, by the suitable functional group on cyanine and the biotin moiety precursor, can between them, introduce various dissimilar spacer parts.The reaction scheme of introducing these spacers is known by those of ordinary skill in the art.
4. the lipophilic cyanine that replaces of radio isotope
The synthetic of lipophilic tributyl tin intermediate listed in the reaction scheme 2, and will describe in detail in the example below; Said intermediate can be used for the preparation of radioactive halogen-labeled cyanine derivative, and this derivative can be advantageously used in the method for the present invention.
According to reaction scheme 2, with 5-iodo-2,3,3-trimethylammonium-(3H)-pseudo-indole (9) and alkyl-4-closilate (preparation as described above) carry out alkylating to form (10); Said pseudo-indole is by iodo aniline (market is available), uses people such as Blaikie, J.Chem.Soc., 313, people such as step described in (1924) and Moreau, Eur.J.Med.Chem.Chim.Ther., 9, (3): the step 274-280(1974) makes.Subsequently, (10) and N in diacetyl oxide, the reaction of N-amitraz diphenylurea has made vinyl intermediate (11).Then, in the presence of sodium acetate, intermediate (11) and (5) are linked together by in ethanol, stirring.Intermediate (5) prepares as described above.Use silver acetate, with sodium-chlor this mixture is carried out quenching subsequently, get iodocyanin (12).
Subsequently, can be by people such as H.Azizian, J.Organomet.Chem., step 215:49-58(1981) prepares compound (13) with (12).This step is included under the existence of catalyzer, four-(triphenylphosphine) palladium (O), with two (three-normal-butyl tin) heating (12).Then, can be under the condition of gentleness, by people such as for example Wilbur, J.Nuc.Med., the improvement of step 30:216-226(1989) is easily carried out radioactive halogen-labeled to tributyl stannyl derivative (13).By the solid phase exchange, use people such as Weiss, J.Labelled Cmpds.﹠amp; Radiopharmaceuticals, the change of step XXVI:109-10(1989) also can realize introducing radiohalogen to (12).
The another kind of lipophilic cyanine derivative that can be used for introducing radiohalogen is the analogue of (13), wherein usefulness-Hgx substituted-Sn(Bu)
3, X represents halogen.Certainly, by the suitable selection of raw material, can change the position of the ring of halogenic substituent in the compound (12).For example, according to people such as Bassignana, SpectrochemicaActa., 19(11), 6-iodo-2-methylbenzothiazole that step 1885(1963) makes can be used for forming corresponding 6-iodo derivative.
5. fissionable colchicine-lipophilic cyanine conjugated body
By suitably functionalized active colchicine derivative (as Brossi, J.Med.Chem., 33:2311, describe 2319(1990)) selection, and pass through as suitable rhizome of Chinese monkshood base, acetal, ortho ester, ester, ketone acetal, acid anhydrides or hydrazone key, be connected on the suitably functionalized lipophilic cyanine derivative, can make colchicine-cyanine conjugated body that band acid can be divided key.
Use people such as Shen, Biochem, Biophys.Res.Commun., 102,3:1048-1054(1981) the middle step of describing can obtain by the coupling along rhizome of Chinese monkshood base key.This step comprises the coupling along the lipophilic cyanine (market is available) of the amine-format of bird head base acid anhydrides of the free amine group derivative (as the deacetylate colchicine) of medicine and band.
Can use people such as Srinivasachar with suitable functionalized colchicine and cyanine derivative, Biochemistry, 28, the 2501-2509(1989) improvement of described step realizes by acetal the coupling of ortho ester or ketal linkage.
Can use people such as Laguzza, J.Med.Chem., 32:548-555(1989) described in the improvement of step, carry out coupling by the hydrazone key.This step comprises the coupling of the lipophilic cyanine of the medicine of aldehyde form and hydrazides form.
Be shown in the reaction scheme 3 according to fissionable colchicine of the present invention-the synthetic of cyanine conjugated body, and, will be described in detail in the example below.
According to reaction scheme 3, mono-methyl (market the is available) coupling of the cyanine of aminofunctional (8) and pentanedioic acid making mono-methyl derivative (14), when this derivative (14) is handled with hydrazine, will form diazanyl derivative (15) in methyl alcohol.
Colchicine partly is to make to form a sour intermediate with the Pyroglutaric acid reaction by the colchicine (market is available) with deacetylate; Subsequently, this intermediate is activated on the spot by adding Carbonylidiimidazole, to form acylimidazole; When this acylimidazole reduces with hydrogen boronation tetrabutylammonium, form alcohol (17).(17) and the oxidation of chloro chromic acid pyridine will produce 7-N-(5-oxo pentanoyl) deacetylate colchicine (18); subsequently; (18) make conjugated body (19) with diazanyl derivative (15) coupling; wherein, colchicine and cyanine partly carry out coupling by the fissionable acyl group hydrazone of acid key.The 7-N-(5-oxo pentanoyl of producing in scheme 3 as intermediate) the deacetylate colchicine is the compound that constitutes the novelty of a part of the present invention.If desired, the methoxyl group in the 10th position that colchicine is examined can replace with methylthio group or other group that contain chalcogen.
In addition; more effective 7-N-(5-oxo pentanoyl) deacetylate thio-colchicine analogue can adopt the same reaction program that is used to prepare aldehyde 18 in the scheme 3; by the deacetylate thio-colchicine (as people such as Shian; J.Pharma.Sci.; 64,646-648(1975) described in the preparation) make.The derivative of this thio-colchicine also can be connected to identical coupling condition on the diazanyl derivative (15), forms acid and can divide conjugated body.
The kinetics that colchicine analogue discharges from conjugated body also can be by hydrazone key type modification between colchicine and cyanine part is changed.For example, the antibody-drug conjugated body by alkylsulfonyl phenyl hydrazones and phenyl hydrazones coupling is by people such as Mueller, in Bioconjugate Chem., describes in 1:325-330(1990), so that the release dynamics slower than corresponding acylhydrazone derivatives to be provided.
6. heparin-lipophilic cyanine conjugated body
By stable amino-formate bond coupling, and the synthetic route that can be used for heparin of the present invention-cyanine conjugated body is shown in the reaction scheme 4, and will be explained in detail in the example below.
According to scheme 4, according to people such as Eckert, Angew Chem.Int.Ed.Engl., 26(9): step 894-95(1987), when handling with triphosgene, the cyanine of aminofunctional (8) will change into the isocyanate derivates (20) of high activity.This isocyanate derivates (20) is not further separated or purify, subsequently, according to Dong, " Heparinized Segmented Polyurethane Urea Surfaces with Hydrophilic Spacer Groups ", Dissertation, University of Utah, the improvement of 1990 step in the mixed solvent of dimethyl formamide/methane amide, is reacted it and heparin sodium immediately, to form heparin-lipophilic cyanine conjugated body (21), wherein, by its hydroxyl, by forming amino-formate bond (or by amino, by forming the urea key), heparin covalently is connected on the cyanine.Use the available reagent in market, N-Boc-6-hexosamine N-hydroxy-succinamide ester by to the known reaction of those skilled in the art, also can be introduced the carbon spacerarm between heparin and cyanine part.
Certainly, by the control to the reagent stoichiometric quantity, can change the quantity of cyanine group in each heparin molecule in reaction, this is because heparin has many effective free hydroxyl groups.If necessary, can from unbound heparin, heparin-lipophilic cyanine conjugated body be purified by hydrophobic interaction chromatography method.
In addition, can be with people such as Kin, in Nonthrombogenic Bioactive Surface Annals, New York Academy of Sciences, the improvement of the step of describing among the p116-130, preparation heparin-cyanine conjugated body.This step comprises uses carbon imide reagent, the carboxylic acid group that will be on heparin and the cyanine coupling of aminofunctional.People such as Ebert are in Biomaterials:Interfacial Phenomenon and Application, Adv.Chem.Ser.99, American Chem.Soc., Washington, D.C.(1982) definite in, reaching 20% carboxyl on heparin can be derived, and does not lose biological activity.
7. radiometal complex-lipophilic cyanine conjugated body
The synthetic route that is used for radiometal complex-lipophilic cyanine conjugated body is shown in reaction scheme 5, and metal wherein is selected from rhenium, indium, copper or palladium.According to scheme 5, with of cyanine (8) reaction of two sense sequestrants (22), to make derivative (23) with aminofunctional, wherein said sequestrant such as Baidoo and Lever are in Tetrahedron Lett., 31,40,5701-5704(1990) middle being prepared of describing.Subsequently with the metallic solution reaction of compound (23), to make metal complexes (24) with the suitable oxidizing attitude.When used metal was rhenium, scheme 5 was expressed the structure of gained neutral metal title complex.Certainly, the accurate structure of this title complex and electric charge depend on the accurate structure of used metal and selected bifunctional chelants.
The route of preparation radiometal complex-lipophilic cyanine conjugated body is shown in the reaction scheme 6, and metal wherein is selected from rare earth metal.
The Bifunctionalized polyaminocarboxylic acid ester sequestrant of structure (25) can be according to EP0353450A
1Described in step preparation.According to scheme 6, sequestrant (25) is in cyanine (8) reaction of PH9～9.5 and aminofunctional, and to make compound (26), wherein, sequestrant and cyanine partly are by stable thiocarbamide key coupling.Subsequently, apply for described step according to above-mentioned EP equally, with the solution reaction of this compound (26) and the radioactive metal salt in suitable buffer system, to make final radiometal complex-cyanine conjugated body (27).
The M(PA-DOTA that is used to the scheme of illustrating 6) refers to radiometal complex.Described title complex comprises poly-amino-carboxylicesters sequestrant (Chelator) and part the compartment ((CH in the scheme of being shown in 6 intermediates (26)
2)
2-C
6H
4-NH-).Can expect, the three-dimensional structure of the radiometal complex of compound (27) part will with people such as Spinlet in Inorgn.Chem., the 23:4278-4283(1984) structural similitude described in.
The Bifunctionalized polyaminocarboxylic acid ester sequestrant of other type is known, and can be coupled on the functionalized cyanine by known other reactive mode of those skilled in the art.For example, can be referring to people such as Sundberge, J.Med.Chem., 17:1304(1974).
Other nitrogenous tetradentate chelator and sulfur-bearing tetradentate chelator are known, and can be coupled on the suitable functionalized cyanine by the known reactive mode of those skilled in the art.For example, can be referring to people such as Ras, J.Am.Chem.Soc., 112:5798-5804(1980).
IV. compound of the present invention is bonded on the biocompatible particle
Linear Carbon atomicity on The compounds of this invention in the substituted hydrocarbon tail is the important factor that obtains the stable bond of desired degree between compound and biomone facial mask.In order to obtain to have the stable bond of single hydrocarbon tail compound, acridine derivatives for example, the Linear Carbon atomicity is necessary for 23 or bigger.The experience of cyanine derivative prepared in accordance with the present invention shows that in the compound that two or more hydrocarbon tails are arranged, the hydrocarbon tail of one of them must have the lineal measure of at least 12 carbon, is at least 23 at the sum of all hydrocarbon tail neutral line carbon atoms.According to the structure of compound, leakage or transfer from a cell to another cell do not take place usually.Usually, the hydrocarbon tail is long more, and lipotropy is just high more.Yet, contain the hydrocarbon tail that surpasses 30 Linear Carbon atoms and may propose a problem, this be because, biological action part and the reactant that is used to provide the hydrocarbon tail may be insoluble in the same solvent, this will make hydrocarbon tail chemistry be connected to biological action and partly become very difficult.Therefore, according to will with the chemical property of hydrocarbon tail bonded connection portion, can there be the qualification of a reality in the length of hydrocarbon tail.
The structure modify of the connection portion that is connected with the hydrocarbon tail also will have great influence to the degree of film combination stability.The connection portion of positively charged, for example cyanine, stibazole, xanthene, Phenazoxine, thiodiphenylamine or diphenyl hexatriene dyestuff and their derivative can help compound in electronegative film introducing and keep.Neutral or electronegative connection portion also can be used for obtaining the release controlled from microbial film.
As the case may be, the stable bond between connection portion, hydrocarbon tail and compartment or the biological action part is absolutely necessary for keeping reaching treatment benefit time necessary provided by the invention and amount of selection part of therapeutic active substance acquisition.
Must select connection portion (L) like this,, thereby make these compounds be enough to obtain desirable treatment benefit in time and the amount that the delivery site of selecting exists so that give compound of the present invention with desired stability.For this reason, the connection portion in the said structure formula I compound must provide biological action part (B) and R and R at least when n=0
1Both one of stable bond, and compartment (R
2) with R and R at least when n=1
1Both one of stable bond.By the compound that linking group is given, under situation therapeutic activity, the direct administration of lipophilic conjugated body, can determine according to the time of keeping with necessary combination stability; Conjugated body being delivered under the situation of disease location, can determine according to cycling time by carrier.That is to say, therapeutic activity of the present invention, the time of keeping of lipophilic conjugated body or cycling time must be longer than the time of keeping or the cycling time of the therapeutic activity component of non-conjugated form.
Though time of keeping that increases or cycling time are the important factors that obtains treatment benefit provided by the invention, but existing is another factor of equal importance that depends on the The compounds of this invention combination stability equally, just exists or the amount of the compound of accumulation is enough to provide desirable treatment benefit at disease location.
The known the whole bag of tricks of definite available those skilled in the art of keeping time or cycling time carries out, and enumerates among the embodiment 9.10 that this will be below and 12 etc.Determine (precipitation is to utilize the situation of therapeutical agent) to the amount of the therapeutic activity of the present invention of the effect that requires to produce hope, lipophilic conjugated body is not subjected to the qualification of ad hoc approach.Owing to can be used for the difference of the therapeutic active substance in the present invention practice, can be with the numerous disease situation of therapeutic active substance treatment and the volatility of the patient of receiving treatment, this also must be so.Therefore,, must periodically monitor, exist or the therapeutic active substance amount that accumulates is enough to produce desirable treatment benefit at disease location determining the patient's that receives treatment according to the present invention reaction.
Can design compound of the present invention, on the adventitia with the adventitia that is bonded to the cell of to survive or other physiologically acceptable particle, and viability not had the inherent deleterious effect.Perhaps they can be designed to bring into play immediately or postpone performance and suppress cell or cytotoxic effect.Because the cytotoxicity content of cytotoxic biological action part for example, can comprise the compound of zero-dose with cellular exposure to various concentration, and there be not the compound of conjugation to the cytotoxic agent in order to measure.Subsequently, with these cellular exposure to people such as Trypan Blue or Propidiumiodide(F.Celada, Proc.Natl.Acad.Sci., 57:630(1967)).These dyestuffs are usually by lived cellular rejection, and can only permeate the film of dead cell.Behind suitable incubation time, check these cells with microscope or by the hemocyte flowmeter counter, and measure the percentage ratio (percentage mortality ratio) of staining cell.
With compound of the present invention be bonded on the carrier cell also must pair cell function do not produce any tangible deleterious effect, this is crucial to them as the ability that carrier carries out targeted delivery.For example, the orderly separation of carrier cell is important to its use in given application.On the other hand, when the branch ionization potential of the cell that expection is used or other desired performance of cell did not influence, used compound can change some function.Therefore, can think that this compounds does not have tangible deleterious effect to the function that is used for cell of the present invention.Be used for producing by The compounds of this invention, exist the measuring method of effect of the cell function of potential importance to be described in US4859584 to putting into practice the present invention, the Blood of Slezak and Horan, 74:2172-77(1989) and J.Immunol.Meth., 117:205-14(1989) in.
For on the membrane plasmapheresis that can repeatedly compound of the present invention be bonded to target or carrier cell and the desirable function of pair cell does not produce deleterious effect, in the selection of cell binding medium, must satisfy two standards.ⅰ): it must be isotonic down in isotonic concentration (the about 260-340mos mole of mammalian cell) that this cell binding medium is wanted bonded physiologically acceptable particle for The compounds of this invention, so that these cells are not produced shrinkage or swelling and possible infringement; ⅱ): make compound of the present invention solubilisate in such a way, promptly they can be introduced in the membrane plasmapheresis of cell with constant concentration.The experiment of dissolution time process (people such as US4783401 and M.Melnicoff, J.Leuk.Biol., 43:387-397(1988)) showing, only is that part is water-soluble and in order to stably to be bonded to the compound on the membrane plasmapheresis, tend to form micelle, perhaps form aggregation; When solubilisate in the anion solutions (for example phosphatic buffer saline, developing medium etc.), described micelle or aggregation can be precipitated come out, and this will cause this compound that the introducing of membrane plasmapheresis is reduced, or produce undesirably inconsistent introducing.
For the radio isotope compound, but experimental procedure like the application class, and also the stability of this compound can be passed through β-or γ-counter and measures.At all occasions, at each time point, will the amount of the The compounds of this invention in the said isosmoticity solution supernatant liquid with compare with such compound sample of ethanol as solvent; Make marks although alcohol solvent is not suitable for pair cell, it is enough to make this compound to reach maxima solubility (total).
In order to be identified for being bonded to the proper concn of the The compounds of this invention on the cell membrane plasmapheresis, must consider several factors, this comprises the effect of the expection that is produced by compound and the type of the cell that compound connects etc.Usually, major objective is as much as possible therapeutical agent to be introduced cytolemma.This can be by treating that compound directly is delivered to disease location or by treating that compound is bonded on the cell in vitro and the cell of this modification being introduced in the body realized again.By making the therapeutical agent of introducing the cell membrane plasmapheresis reach maximum value, can carry out administration by relatively low dosage, maybe can require to reach and wish that the position is to bring into play the less carrier cell of the effect of wishing.By under the situation of cell delivery therapeutical agent, for viability that migrates to the cell of wishing the position and capacity, the compound amount that is introduced into cell need only increase to and not observe the negative such value that substitutes in carrier cell.
Do not exist under serum and other light wood material, be applied to compound of the present invention on the carrier cell or on other the physiologically acceptable particle.Cell is taken out or takes from culture from health, and wash to serum-free.Their are suspended forming a composition, and said composition comprises that the isoosmotic pressure that is insoluble to anion solutions usually adjusts the compound of the present invention (10 of agent and proper concn
-5-10
-7M).Usually, in 10 minutes, finish the combination of compound to cell, and, can add autologous serum or foreign sera and stop this association reaction.Then, the described cell of washing in containing serum medium (5-10%V/V), and, place culture or injection to give the blood recipient them according to application.
The integrating step of the compound pair cell of said type further is described in above-mentioned US4783401 in detail, and its whole disclosure is introduced specification sheets of the present invention as a reference, and is the same just like writing out fully at this.
Another cell combined process comprises, The compounds of this invention is suspended in the salt solution, so that form micelle.Subsequently described cell is put into the suspension of generation, cytophagous cell (for example, monocyte, scavenger cell and neutrophilic leukocyte) will preferentially become mark.Like this, just cytophagous cell can be aimed in compound selective of the present invention ground.See people's such as M.Melnicoff J.Leukocyte Biol., 43:387-97(1988).
Now, with reference to specific drug therapy, the representational application of compound of the present invention, composition and method is described.
V. the using method of The compounds of this invention
A. Chang Yong methods of treatment
1. the application of isotope therapy
Since compound of the present invention have the ability that can be introduced in the lipid component that contains lipid physiologically acceptable particle and with radioactive, and the high linear ability that can change (LET) radiating ion chelating of emission, therefore, they can be used for radiation-therapy is sent to disease location.By at first with chelate compound of the present invention with suitably isotopic ion is (for example
67Cu,
90Y,
186Re, alpha emitter) form stable complex, separate title complex then, and with compound of the present invention cell is marked according to above-mentioned cell integrating step commonly used.
The lymphocyte (TIL) that knurl is soaked into can separate from chancre, expands in IL-2, is incorporated in to as mentioned above the radiation treatment material and in intravenous injection.The Cellular tracking of mark shifts the position of disease, and concurrent radiation of shooting the oncocyte of dead transfer to death will increase the treatment validity to TIL thus, perhaps can also reduce the acquisition disease desired cell count that disappears.Similarly, also can use other class cell that migrates to metastasis site, be used for the conveying of partial radiation therapy.
2. by particular proteins being bonded to the mark of the cell of cytolemma
In another concrete example, compound of the present invention is introduced in the proteinic material that comprises as biological action protein, glycoprotein, lipoprotein or peptide partly.As mentioned above, these compounds are incorporated in on the cell, and thereon, the hydrocarbon chain of said compound will embed in the membrane plasmapheresis, thus, protein be placed on the surface of specific cell type.
Step recited above can be used for and will be connected to the surface that human fibrinous monoclonal antibody is bonded to carrier cell, for example, is used to be delivered to the erythrocytic surface at fibrin clot position.
The monoclonal antibody that a kind of similar method can be used for being connected to human fiber surface tumor antigen is delivered to the knurl position by carrier, carrier for example: monocyte or lymphocyte.By to surperficial the applying of carrier cell (for example red corpuscle), similarly can carry soft tissue proplasmin activator.
If desired, above-mentioned therapy can be used in combination.Therefore, be bonded on the surface that human fibrinous monoclonal antibody can be bonded to cell (for example red corpuscle), this cell subsequently also with fibrinolytic compound (for example tPA, streptokinase, urokinase) combination.This monoclonal anti physical efficiency is bonded on the scleroproein carrier cell, and, at the fibrinolytic compound of carrying a large amount of treatments in conjunction with the back.
According to above-mentioned preparation process commonly used, these therapeutic activity protein can conjugation to the lipophilic color base.
Above-mentioned technology also can be used for various other therapeutic activity protein or peptides are introduced in the suitable physiologically acceptable particle, these biomones such as cell, virus, liposome or low-density lipoprotein (LDLs), thus, in working cycle, the biological effectiveness of proteinaceous substances will be prolonged substantially.
As mentioned above, also can carry out isotopic labeling to the physiologically acceptable particle of protein bound with radioactivity imaging compound or magnetic response imaging compound.Final biomone can be to patient injection, whereby with cell migration to disease location, this disease location can utilize the γ-scintigraphy of standard or nucleon imaging to carry out imaging, with the effect of appraisal therapeutical agent.
3. protein is coupled to the cell that is used for vaccine
In another application of the invention, as the biological action part, be not necessarily to comprise the connection base that is used to be bonded to cell (for example red corpuscle, monocyte) surface with immunogen.For immunogen, hope can produce the antibody of protectiveness, and it can be protein, glycoprotein, lipoprotein or peptide.Then, existing or not existing under the situation of adjuvant, the cell of modification like this is injected.The timed interval of injection will be depended on immunogenic character, but being no less than under the situation in two weeks at interval, can inject 10 usually at every turn
6Individual cell.
Monitor with the Elisa method of standard with respect to antigenic antibody amount, intracellular immune amount can be measured the hyperplasia or the toxic reaction of pair cell of immunocyte by definite.
4. make the migration pattern change by the modified cell surface
In the Another Application of this technology, use compound of the present invention, sialic acid or glycosaminoglycan can be bonded on the membrane plasmapheresis of cell.As mentioned above, specific compound is placed the isoosmotic pressure medium, for example, red corpuscle is placed this solution, the result is that this compound is bonded on the membrane plasmapheresis.By adding serum this reaction is stopped, then, these cells of washing are ready for injection in containing the salt solution of medium.
Red corpuscle as immature cell circulates through this, and a large amount of sialic acids is arranged on their surfaces.Along with this is erythrocytic aging, will reduce sialic amount on each cell, this makes the scavenger cell of spleen and liver can discern erythrocyte membrane antigen, whereby, they is removed from circulation.Amount by suitable increase sialic acid introducing erythrocyte membrane can increase the life-span of red corpuscle in circulation.The ability that increases red blood cell life span may be useful for transplant patient or anaemia patient.When the bone marrow transplant patient accepts to transplant, must be after several weeks, they could make themselves red blood cell.By using this technology that prolongs their Autoerythrocyte life-span, if desired, patient can carry out the transplanting of several times marrow under the situation that does not have the anaemia outbreak.
Under anaemia patient's situation, anaemia can be caused by shortening of red blood cell life span, or cause by producing reducing of red corpuscle speed.In both cases, increasing the erythrocytic life-span will reduce anaemia.
5. be used for the treatment of the conveying of the photodynamic compound of effect
The photodynamic therapy that is used for cancer therapy is a field (Proceedings of SPIE-The International Society for Optical Engineering who conscientiously explores; Volume 847, and " New Directions in Photodynamic Therapy ", Douglas C.Neckers edits (1987.10)).These compounds of great majority are phthalocyanines or haematoporphyrin (hematoporphrin) class.All absorb light are the 600-800nm scope in treating processes, and produce the oxygen of excited state.
Use methodology of the present invention, produce the lipophilic derivative of this compound, and be dissolved in the isotonic solution.Select cell (for example, TIL) to mark, and give patient injection with these compounds to knurl with these cells.Subsequently, these knurls select cell migration to the micrometastasis position.In 48 hours, patient is exposed in the high-strength light zone, absorbs at this area light power molecular, and the excited state oxygen of generation will kill oncocyte.And carrier cell also will be killed, and this will produce an inflammation, and more thus immunocyte compiles, and to remove dead cell, increases the toxicity to knurl.In this method that photodynamic action is carried, carrier cell is responsible for optionally accumulating more therapeutical agent at the knurl position.In some cases, The compounds of this invention directly is applied on the knurl settling in the body cavity (as pleural space), can helps to make and when surgical operation, carry out more effective intracavitary therapy the keeping of this position.
B. by specific disease or pathologic condition are treated in the direct conveying of therapeutic active substance
1. the obturation again and the restenosis of postangioplasty
Experiment shows that compound of the present invention can be retained in partial vascular site and on artificial surfaces, even (face embodiment 8 and 12 as follows) keeps in the presence of blood flow continuously.In addition, show that at glass test tube and in vivo, biological activated energy is retained in (face embodiment 4,13 and 15 as follows) in the compound of the present invention that contains therapeutic active substance.
Zooscopy shows that the main effect of hyperplasia and migration is to cause the generation of the restenosis of postangioplasty in the about 7-21 of postangioplasty days.Thereby antiproliferative pharmaceutical must be retained in the artery of repairerment as many as 7～10 days at postangioplasty by smooth muscle cell, to prevent the generation of these effects.The compound of the present invention that contains suitable anti-proliferative agent can be delivered to the tube wall of damage during angioplasty, and conjugation to the anti-proliferative agent of this compound can be retained by the cell of damage.Therefore, can directly give the anti-proliferative drug of higher dosage, and this medicine can keep the longer time by the application of The compounds of this invention in affected area than carrying by systemic drug to affected cell.For example, in angioplasty, the direct deposition of the anti-proliferative agent by drug delivery pipe can make this medicine be combined on the cytolemma of angioplasty operating station, and anyly in the angioplasty operation does not have the bonded medicine to wash from artery.Take out drug delivery pipe, the medicine that has been bonded on the resting cell will be retained in the adventitia, or arrive darker cellular layer through the gap.If those cells enter activity or growth conditions, compound of the present invention will inwardly transmit with membrane component and move in the cell.In case enter in the cell, they just can bring into play its anti-proliferative effect.Therefore, the compound of the present invention that comprises suitable anti-proliferative agent mainly can be delivered to disease location, and the time in office, the medication amount of being handled by liver or kidney is reduced to minimum, and this will reduce the chance of the serious negative interaction of generation.
In preferred concrete example, the compound of the present invention that can be used for treating postangioplasty restenosis comprises anti-proliferative agent, as, heparin, r-hirudin, colchicine, vinca alkaloids, taxol and their derivative.Behind arterial injury, heparin is applied to smooth muscle cell in the culture, or by animals administer, the result makes cell reduce growth, reduces endomysium and thickens and hyperplasia.The The compounds of this invention that contains heparin is preferably made such structure, and they are by being retained on the adventitia of intra-arterial parietal cell with the hydrophobic interaction of one or more cell walls components, rather than are absorbed into the inside of those cells.On the contrary, the anti-proliferative agent as colchicine must be absorbed by cell, to bring into play their anti-proliferative effect; Colchicine influences microtubule cell prominent (tubulin processes).In this case, preferably come to synthesize The compounds of this invention like this, even they can arrive the arterial cell of inwall more quickly.In preferred concrete example, colchicine comprises the biological action part that can divide the The compounds of this invention of conjugated body as acid.This colchicine is passivation in its conjugate form.Yet when this compound is absorbed in the cell sour capsule, said preparation will discharge from compound, make it activation thus.So active colchicine is sent to its reactive site in the cell, and it is prominent to suppress the microtubule cell therein, thereby suppresses the division of cell.
Being used for the particularly preferred compound of the present invention is the compound that contains sour fissionable following structural formula of colchicine:
The another kind of useful compound structure that contains colchicine is as follows:
Other anti-proliferative agent of preparing to use in the present invention's practice comprises: Zinc metallopeptidase Zace1 (ACE) inhibitor, angiogenic peptide (angiope ptin), Sandimmune, calcium channel blocker, by growth hormone antibody deutero-goat-antirabbit thrombocyte, Terbinafine and Trapidil are used for positively charged ion growth hormone bonded interferon-and polyanion.
2. rheumatic arthritis
Treatment compound of the present invention is specially adapted to the treatment of rheumatic arthritis.They provide carries out chemistry or the more such means of radiation synovectomy, and make and close a large amount of therapeutical agent of energy-conservation reservation, and to lymphoglandula, the not significant release comprehensively of spleen or liver.A major advantage all better than the delivery system of all existence is that compound of the present invention is delivered to that sheet tissue that needs treatment equably, and is retained in those cells.With regard to radiotherapy compound of the present invention, produced therapeutic action to the synovial cell from the radioactivity of this compound emission.As embodiment more detailed description below, find nearly all compound in vivo all by in the joint for the treatment of, and after 6 days, still keep about 70% of injection compound in the joint.This partial and lasting keeping will reduce the required radio isotope amount of each step of radiation synovectomy, and will reduce the side effect of using traditional remedies that systemic exposure is caused in the radio isotope that is discharged by the joint.
The particularly preferred radiotherapy compound of the present invention that is used to radiate synovectomy can followingly synthesize for example, to introduce suitable radio isotope.For example, the sequestrant of available or (1) nitrogenous and sulphur, or (2) nitrogenous and sequestrant oxygen carries out complexing to radioactive metal.Radio isotope can be selected from radiohalogen, copper, yttrium, rhodium, palladium, indium, iodine, samarium, gadolinium, holmium, erbium, ytterbium, lutetium, rhenium, gold or their combination.
Being used for preferred compound of the present invention is the compound with following structural formula:
Wherein each R ' is hydrogen atom or alkyl independently, low alkyl group preferably, or the low alkyl group that replaces, substituting group any ester wherein, R " and R
Be that hydrogen atom or alkyl, m and n can be zero or 1 separately independently, M represents to be selected from the radioactive metal of rhenium, indium, copper and palladium.
In preferred concrete example, the compound of said structure formula VIII has following structural formula:
Wherein R and R
1For having C
1-30Hydrocarbon substituent; X and X
1Can be identical or different, and represent O, S, C(CH
3)
2Or Se; A represents receptible negatively charged ion on the pharmacology; Z represents the coordination position of hydrogen or metal; R '=R " or
Wherein each R ' is hydrogen atom or alkyl independently, the low alkyl group of low alkyl group or replacement preferably, and wherein, substituting group is any ester, R " and R
Be hydrogen atom or alkyl independently, m and n can be zero or 1 separately; M represents to be selected from the radioactive metal of rhenium, indium, copper and palladium.
The compound that another is used for particularly preferred compound of the present invention is following structure:
Wherein, M represents the radiation treatment material as rhenium, indium, copper or palladium.
Another useful compound is the compound of following structure:
Wherein R and R
1Be C
1-30Hydrocarbon substituent, X and X
1Can be identical or different, and represent O, S, C(CH
3)
2Or Se; A represents acceptable negatively charged ion on the pharmacology; M represents to be selected from copper, technetium, rhodium, palladium, indium, samarium, gadolinium, holmium, erbium, ytterbium, lutetium, rhenium, yttrium, gold, or the radiation treatment material of their composition; N is 2,3 or 4; M is 1 or 2; P is 1～6.
3. ovarian cancer
The compound of the present invention that contains chemotherapeutic or radiotherapy dose can make those therapeutical agents with high density, directly be delivered to oophoroma hyperplasia position.And these therapeutical agents will keep long period of time in peritoneal cavity, therefore, stop the diffusion of oncocyte.In addition, this mode can be finished under the apparent side effect of not carrying this therapeutical agent of high density to follow by whole body.
Compound of the present invention can be after surgical operation, by carrying at intraperitoneal as the Tenckhoff conduit of treatment, or when surgical operation as adjuvant therapy, by second vision slit (second-look laparotomy) of laparotomy, carry at intraperitoneal.
As previously mentioned, the compound sour fissionable of the present invention that contains colchicine is treated the anti-hyperplasia that also can be used for the oophoroma cell.As mentioned above, such molecule will be retained on the adventitia of cell with nontoxic form.Yet when this compound was absorbed in the cell, chemotherapeutic agent was from the nubbin division of compound, and this chemotherapy material can be brought into play its anti-proliferative activity.
4. psoriasis
The compound of the present invention of corticosteroid-containing can be used to help psoriasis treatment.They can provide more medicine to keep at the position of psoriasis infringement, therefore, aspect the hyperplasia that reduces angle albuminous cell and immunocyte, have strengthened the effectiveness of medicine.In addition, keeping of damaging part The compounds of this invention, will stop and to infiltrate the recycle system by deleterious compound.This will produce clinical benefit, and wherein, behind two all continuous uses, this therapy does not need to interrupt owing to the high serum-concentration of anti-proliferative drug.
According to other application of the present invention, thrombocyte or low-density lipoprotein (LDL) can be used for the spike at the calm position of the atherosclerotic atherosclerotic plaque of early detection.Thrombocyte can be isolated from individual's blood with the gradient technique of standard, marks with indium or technetium as not necessarily diagnosis part then, and injects to intravenously again.In above-mentioned US4762701, provide compound with described type to be bonded to suitable method on the thrombocyte.In 48 hours after injection, radiolabeled thrombocyte will form the position accumulation at the arterial wall spot, use γ photography function to detect the gamma-radiation that spot forms the position.
Similarly, LDL also can purify with the super centrifugation technique of standard, and relies on its high lipid content and with respect to the bonding force of the The compounds of this invention in physiologically acceptable particle fat zone, mark with compound of the present invention.These radiolabeled LDL will in the accumulation of atherosclerotic plaque formation place, make and can survey by this imaging after inject again.Know that also monocyte also accumulates at the atherosclerotic plaque place, thereby, also can be used for the detection that monocyte forms.Can expect, their restriction just is used for the monocytic validity of radiolabeled an amount of purification.
Know that also thrombocyte is the serious hindrance position accumulation after thrombosis position (for example, crown formation thrombus, dark vein form grafting in thrombus, the blood vessel) and organ transplantation also.Therefore, when combining, isolating and utilize the radiolabeled non-invasive diagnosis that this lysis also will be provided from the intrinsic thrombocyte of body of Compounds and methods for of the present invention by standard method with pharmacotherapy.
In addition, use sequestrant can be bonded on the radioactive metal ion, also can make the fluorescence or the non-fluorescent chemicals of said structure formula I compound simultaneously, wherein the radio isotope atom, as, radioiodine, carbon, nitrogen, sulphur, phosphorus or selenium atom are the integral parts of this molecule.To the compound that uses emitting isotope ground mark of the present invention can the enough gamma-radiations of radiation can compound, available γ-scintillography is surveyed.If the isotropic substance nonpenetrating beta emitter that is low energy so, uses the beta counting technology of standard, this compound can be used for research and use.
Biotinylated lipophilic compound of the present invention can play the multi-usage reagent.For example, such compound can be used to make common nonadherent cell promptly to adhere on the surface of selection.This is for the analysis that requires immobilized cell, and it is important promptly being used for the full-time monitoring of individual cells.If cell is totally marked with biotinylated compound of the present invention, and the cell of final mark contacted with streptavidin bonded surface, these cells will promptly adhere to this surface.The analysis of this cell can begin immediately.Also provide the easy means of with the naked eye monitoring cell at experimental session with biotinylated compound bonded fluorescence.
In addition, aforementionedly show that by the dilution of fluorescence, the compound of fluorocyte mark can be used for monitoring the growth of cell and measure growth rate (US4859584).After 5-8 the doubling time (depending on cell type), when the fluorescence of tagged compound reduces to automatic fluorescent value, this technology will lose susceptibility.By fluorescence dye conjugated streptavidin is bonded on the cyanine of biological example elementization described herein, can realize amplification to fluorescence.By this method, even after color base fluorescence has been reduced to automatic fluorescent value, cell that still can discernable marks.If also need further susceptibility, the streptavidin of radiation-mark can be bonded on the biotinylated compound of the present invention, can carry out radioautography then, with the cell of discernable marks.
The Another application of the biotinylated compound of the present invention is the combination in protein.For some macro-molecular protein, perhaps can not by with this protein covalent linkage be bonded to lipophilic compound of the present invention (represented) and go up and this protein is connected on the cell as above-mentioned structural formula III.In this case, another kind of coupling mechanism is that avidin-vitamin H is in conjunction with right.For this purpose, target cell can be marked with the biotinylated compound of the present invention, and this macro-molecular protein is by conjugation to avidin or on the suitable derivative such as streptavidin of avidin.Subsequently with biotinylated cellular exposure to avidin-protein conjugation body, the result be protein stabilization be bonded on the cell.
C. pharmaceutical preparation
With respect to acceptable liquid excipient administration on physiologically acceptable medium such as salt-free isotonic solution or the pharmacology, the pharmaceutical preparation that contains The compounds of this invention can be prepared easily.Vehicle comprises various inert oils, for example, and vegetables oil such as sweet oil or peanut oil, or height purified mineral oil.According to the character of compound and disease or the pathologic condition that will treat, the concentration of active batching is with different in selected medium.
Even for the ease of administration and dosage, be useful especially with the form compounding pharmaceutical preparation of dose unit.Refer to the in fact dispersive pharmaceutical preparation unit that is applicable to that patient treats at this used dosage unit form.Every dose unit must comprise the active batching of calculated amount, combines to produce the therapeutic action of hope with the pharmaceutical carrier of selecting.In the patient of given type, the measuring method that is used for inhibition of cell proliferation or is used for the suitable dose unit of other pathologic condition treatment is known to those skilled in the art.For example, in the radiation villusectomy, with about 5 millicuries (mci) of various colloidal form administrations
90The Y(transformation period, 2.7 days, β can, 2.2MeV) or 300mci
165The Dy(transformation period, 2.3 hours, the β energy, dosage 1.3MeV) has shown that clinical is effective.See P.Lee, J.Rheumatol., 9:165-167(1982); People such as C.Sledge, Clin.Orthop., 182:37-40(1984).Utilize the dose measurement of standard to calculate, the about 10mci dosage of estimated service life
1863.7 days Re(transformation period, β energy 0.98MeV) will obtain same therapeutic action, this dosage will provide by about 0.05 micromole (μ moles) suitable compound (for example compound 23 of reaction scheme 5) of injection with the given activity preparation of 200mci/ μ mole.Other radiation treatment isotropic substance also is known in the prior art.See people such as W.Volkert, J.Nucl.Med., 32:174-185(1991).Expection, other compound of the present invention also can be used for carrying this class therapeutical agent of significant quantity.
In the radiotreatment of knurl, when carrying by monoclonal antibody, for the solid knurl, the dosage of having advised 80Gy is radiotherapeutic sterilizing dose.See J.Humm, J.Nucl.Med, 27:1490-1497(1986).Expection is bonded to the cell facial mask and makes it internalization, will strengthen the effectiveness of this therapy.See Humm J.L., J.Nucl.Med., 31:75-83(1990).Compound 23 by the given activity with 200mci/ μ mole of injecting about 0.8 nanomole (nmoles) prepares will provide the dosage of 80Gy.Can expect that also this compound of the present invention and other compound can be used for carrying other radiocurable isotropic substance well known in the prior art.See people such as W.Volkert, Supra..The same with the anticancer radiopharmaceuticals of other type, compound of the present invention can be delivered directly in the tumor tissue, be conveyed in the body cavity of the knurl that contains diffusion, or it is medium to be conveyed into the blood vessel of supplying this knurl.See C.Hoefnagel, Anti-Cancer Drugs, 2:107-132(1991).
In the treatment of restenosis of postangioplasty, the compound of the present invention of q.s can be delivered to the pathologic, physiologic position in order to show, can note by people such as Grines, Circulation, 84: the colchicine of everyone 1mg/ days therapeutic dose of administration of II-365(1991), this can not prevent restenosis, causes the plasma peak concentration of 2 nanogram(ng)s (ng)/milliliter (ml) or 5 nanometers (colchicine molecular weight=399.4).See people such as Bochner, Handbook of Clinical Pharmacology, Little, Brown and Co., Boston(1983), pp.151-152.Suppose: (ⅰ) in rabbit the absorption of colchicine with distribute identical; (ⅱ) the rabbit mean body weight is 3kg; (ⅲ) body weight is 70kg for each person, presses people such as Currier, Circulation, and 80: 0.2mg/kg days the dosage that II-66(1989) is used, will successfully prevent restenosis, the plasma concentration of corresponding rabbit is 28mg/ml or 70nM.Therefore, prevent that the shown colchicine concentration in rabbit of restenosis from being 14 times of the concentration that obtains of clinical trial.Compound of the present invention can be in above-mentioned compatible binding medium high to 100 μ M promptly 1400 times shown in zooscopy be to be prepared in the effective concentration, and can in the angiopoiesis process, directly be delivered on the arterial wall by conduit.
Pharmaceutical preparation of the present invention is preferably by injection, and intraperitoneal perfusion or conduit insert and carry out administration.Other administering mode also may be effectively, as oral administration in some cases, or aerosolization effect administration.
This pharmaceutical preparation can carry out administration with proper spacing.Because the character of The compounds of this invention, repeat administration is seemingly unwanted.The method of determining the pharmaceutical preparation administration frequency is known the those of skill in the art of corresponding medical field.Under any circumstance, the appropriate gap in any specific occasion all will depend on the patient's that will treat the situation and the type of pharmacology situation usually.
Now provide following embodiment, with more detailed description the present invention.These examples are just illustrated some aspect of the present invention, and will not be interpreted as limitation of the present invention by any way.
The mensuration of film retention
Film retention (MRC) provides about given compound and keep how good information in the cell membrane plasmapheresis, and it is measured as described below.
By with whole blood centrifugal 15 minutes, remove blood plasma, and in 0.83%(W/V in 300 * g) ammonium chloride in resuspending cell ball, finished production as the red blood cell ghost of replica.By centrifugal 10 minutes, these ghosts are made ball by ammonium chloride at 10000 * g.The minimum carrying out washing treatment that repeats this ammonium chloride 5 times discharges fully to guarantee the oxyphorase in the cell.With above-claimed cpd ghost is marked under the identical concentration of the concentration of cell of the above-mentioned application-specific of mark with being used in the concentration that can survey the ghost of mark with instrumental analysis or fluorescence microscopy method.Consider mensuration hereinafter, 2 * 10
-3Volumetric molar concentration prepares the storing solution of above-claimed cpd in ethanol, and prepares the operation with dilution of this compound in isoosmotic pressure sucrose (52g/500ml distilled water).Ghost in the operation with dilution of compound with about 1 * 10
9After the concentration of ghost/ml is hatched 10 minutes, sample is carried out centrifugation in 10000 * g, so that ghost is made ball, and from sample, extract staining fluid out.With the ghost resuspending of mark in the salt-water liquid of the phosphate buffered that contains 10% foetal calf serum (PBS-FBS).From each sample, take out the aliquots containig of three part of 20 μ l, be used to measure the total amount that has compound.As mentioned above sample is carried out centrifugation, and from supernatant liquid, take out the aliquots containig of three part of 20 μ l, be used for measuring quantitatively the amount that does not have the bonded compound of existence.
After the sampling, extract supernatant liquid out, and with red corpuscle ghost ball resuspending in the PBS-FBS of 1.0ml, carry out a test again as mentioned above.Repeat this process at least 6 times so that the detection of the compound of snap-out release, and after being equal to or greater than time of 24 hours this process of monitoring so that the more detection of the compound put of slow release.For the compound amount that exists in each sample is measured,, 20 μ l aliquots containigs are extracted in the 3.0ml propyl carbinol by shaking.With these samples of 3000 * g centrifugation, removing the striping chip, and the butanols fraction is carried out the analysis of compound concentration.Utilize to want the peak value of analyzed specific compound to excite and radiation wavelength, analysis of fluorescence compound by this way is to measure the unit and fluorescence unit in each sample.Radiolabeled compound does not require the butanols extraction, and can directly analyze with β or γ calculating instrument.
The mensuration of the compound amount that exists in aforesaid each sample is convenient to the calculating of the MRC of each washing or set time point.Obtain the MRC value by following formula:
（C
T-C
S）/C
＊
T100
C wherein
TBe illustrated in the compound amount (utilizing the unit that measures by the method that is used for analysis of compounds) that exists in the whole sample, C
SThe compound amount that exists in the supernatant liquid sample of expression particular point in time.The judging criterion of The compounds of this invention has been determined in the contrast of MRC value, and these standards are: 1) the MRC value of each washing step mensuration must have about 90 value, 2 at least) must be through the percent difference between at least 24 hours MRC value less than about 10%.
The data that provide in the following table I are the results that obtained by an experiment, and are used as the example that MRC measures.In the table I, the compound of representing as A-C has said structure formula XII, wherein X and X in each compound
1Expression C(CH
3)
2, Z and Z
1Expression H, R/R
1Expression C-5/C-5(compd A), C-10/C-10(compd B) and the C-14/C-14(Compound C); The compound of representing as D-K also has structural formula X III, wherein Z and Z
1Expression hydrogen, R/R
1Expression C-14/C-3(Compound D), C-20(3 C-18/C-3(compd E),, 7,11,15-tetramethyl-hexadecyl)/the C-3(compound F 17-hydroxy-corticosterone), C-22/C-3(compound G), the C-20/C-3(compound H), the C-18/C-8(Compound I), C-18/C-5(compound J) and the C-22/C-3(compound K); The compound of representing as L-N has said structure formula X IV, wherein Z and Z
1All represent N(CH
3)
2(at 3 and 6 that encircle), Z
2The expression H, R represents the C-22(compound L), C-18(compound M) and the C-26(compound N).In compound K, negatively charged ion is a muriate, and in all remaining compounds, negatively charged ion is iodide.
Listed MRC value is with as described above described in International Application PCT/US89/00087 in the last table I, and the result who is obtained by cell internalizing compound transfer analysis compares the consistence that demonstrates excellence.
The mensuration of film combination stability
Known, from containing water (for example extracellular medium) depends on hydrocarbon chain to the hydrocarbon chain transfer free energy of liquid hydrocarbon phase (the hydrocarbon inside of biological example film) the degree of branching, degree of unsaturation and methylene radix.The free energy of methylene radical hydrocarbon phase mutual effect is minimum for the methylene radical of the most close water termination, and increase with methylene radical successively, final approximately with in containing the solvent of non-polar hydrocarbon, 4 of alkyl or more a plurality of carbon enter the free energy that the hydrocarbon of membrane lipid inside finds and equate.Therefore, might calculate any structure, as compound of the present invention, it is (single or multiple that it contains polarity headgroup and the linear hydrocarbons tail be made up of 4 or more a plurality of carbon, symmetrical or asymmetric), when being dipped in the hydrocarbon solvent fully, the carbon equivalent number will be similar to and equal the bonded free energy, and will be as described below:
The 0+0.25+0.5+0.75+m-4(+ of carbon equivalent=0+0.25+0.5+0.75+n-4(+)) ..., wherein n equals first hydrocarbon tail neutral line hydrocarbon number, and m equals the second hydrocarbon tail neutral line hydrocarbon number, or the like.
Measure like that by the foregoing description 1 is described, sample plot records, exists dependency between the carbon equivalent of The compounds of this invention and their the film retention (MRC).For example, have carbon equivalent and have 90 MRC or bigger greater than the The compounds of this invention of 18-19, and, during with cell internalizing compound transfer analysis (seeing above-mentioned), demonstrate minimum transfer with unlabelled iuntercellular at mark.Therefore, can expect that these compounds also will demonstrate in vivo the cell bonded satisfactory stability with mark.Surprisingly, situation is not always like this.In fact, demonstrate MRC only 10% different of the present invention some compound exhibits go out the rate of loss that differs 10 times in vivo.
Some that utilize combination stability between compound and microbial film in vivo and predictability measured, and is important for the various practical applications that can assess The compounds of this invention and method.For example, for the application that is delivered to the knurl position as radiotherapy nuclear, may require very stably to be bonded on the film, because the toxic action that the loss of compound will cause radioactive nuleus to produce at non-knurl position.On the other hand, for the application that relates to control therapeutical agent rate of release, it may be desirable losing compound more quickly from the microbial film.Therefore, will be described in hereinafter with body in measure the test of MBS under the proximate condition.
Carry out in the process in this test, use the serum albumin (5%) of approximate physiological concentration.In addition, because chemical compound lot of the present invention is a low-solubility in containing 5% albuminous salt solution, so, in the enclosed system of " physiology " liquid that contains defined volume, the solvability in 24 hours in film and reach balance between the solvability in the medium around.For the effect of the cellular exposure that more is similar to mark in vivo, by at the film ghost of the mark that contains suspension reduction in the albumin salt solution of fixed volume and carry out the test of MBS in wherein big liquid volume.In the time of 24 hours, carry out the mark total amount that sampling and measuring exists by suspension to good mixing, then, the film ghost is made ball, and measure the labelled amount that is released in the supernatant liquid, and represent with the percentage ratio of total mark by centrifugation.Ghost number according to every milliliter of suspension is mapped to the percentage retention, and mensuration MBS is 5 * 10
7Ghost/ml and 4 * 10
8Area under a curve between ghost/ml.In this test, the compound that demonstrates unlimited film combination stability will provide 3.5 * 10
10MBS, this result will be expressed as the percentage ratio of this maximum MBS.
Following table listed respectively according to the representational sample of The compounds of this invention as top embodiment 1 described MRC determination data and present embodiment MBS determination data.The compound of representing with O-T has the structural formula XII, wherein X and X
1Expression C(CH
3)
2, Z and Z
1Expression H, R/R
1Expression C-12/C-10(compound 0), C-16/C-16(compound S C-14/C-14(compound R C-22/C-12(Compound P), C-14/C-3(compound Q))) and C-22/C-14(compound T).
The table II
MRC
#MBS (film the most in vivo
The % of the big value of compound carbon equivalent (24 hours time)) transformation period (my god
*)
P 19 65±2.0 48±3.1 1.4
Q 20.25 85±0.6 41±5.0 10±0.7
R 23 87±0.6 55±1.7 36
S 27 87±1.2 79±3.7 58
T 31 97±0.1 85±1.7 130±17
*As at Slezak and Horan, Blood.74:2172-77 measures described in (1990), at mark and after injection is determined again in vivo, by transformation period of rabbit red blood cell dye loss (±: the intermediate value of standard error).
As seen from the above table, MBS test makes MRC much at one but in vivo become possibility with the differentiation of the different compound of microbial film bonded stability.
Utilize the MBS test and Selection to be used for the additional benefits that the various application of the present invention have the compound of suitable binding characteristic to be, it can discern the influence of headgroup structural changes to the film combination stability, and the MRC test is almost powerless to this.As the following table III as can be seen, hydrocarbon tail length (be expressed as and make symmetry and the correlated carbon equivalent of unsymmetrical structure energy) and headgroup structure all have remarkable influence to the film combination stability, and when being low to moderate the carbon equivalent intermediate value, the influence of headgroup is more remarkable.Therefore, for be used for the present invention practice with the headgroup of other type of various functional groups (for example, the radioactive metal sequestrant, protein, peptide, radioactive nuleus etc.), can record identical influence, and can estimate in headgroup influence and need reach balance between the carbon equivalent of film combination stability of hope.MBS at least about 30% or the expection of bigger compound can be used in the application of the present invention of following type.Also can be observed, constant even the carbon equivalent number keeps, the substituting group on the headgroup also has remarkable influence to the film combination stability.Can find out that as the following table III compd A C and AD are only different on the headgroup substituting group, yet their MBS values separately differ widely but.
In the table III, the compound of representing with U, W and Y has said structure formula X III, wherein X and X in each compound
1Expression O, Z and Z
1Expression H, R/R
1Expression C-22/C-3(compound U), C-14/C-14(compound W) and C-18/C-18(compound Y); The compound of representing with V, X and AA has the structural formula XII, wherein X and X
1Expression S, Z and Z
1Expression H, R/R
1Expression C-22/C-3(compound V), C-14/C-14(compounds X) and C-18/C-18(compd A A).
The compound of representing with AB, AC and AD has said structure formula XII, wherein X and X
1Expression (CH
3)
2, Z
1Expression H, R/R
1Represent C-14/C-22(compd A B) and C-14/C-3(compd A C, AD).In compd A B, Z represents-CH
2-NHOCH.In compd A C, Z represents H.In compd A D, shown in Z is expressed as follows can not the splitted colchicine derivative.
The many symmetrical dyestuff of identifying in table I-III is from Molecular Probes Inc., Eugene, and OR obtains.
The table III
Compound headgroup type carbon equivalent MBS
Q indoles carbocyanine
(Indocarbocyanine) 20.25 42±5.7
U Evil carbocyanine
(Oxacarbocyanine) 20.25 47±3.1
V thiophene carbocyanine
(Thiacarbocyanine) 20.25 64±3.4
R indoles carbocyanine 23 55 ± 1.6
W Evil carbocyanine 23 77 ± 1.1
X thiophene carbocyanine 23 91 ± 1.3
T indoles carbocyanine 31 85 ± 1.7
Y Evil carbocyanine 31 93 ± 1.0
AA thiophene carbocyanine 31 94 ± 1.5
AB indoles carbocyanine 31 91 ± 0.5
AC indoles carbocyanine 12.25 10 ± 2.1
AD indoles carbocyanine 12.25 28 ± 1.0
Example 3
The preparation of compound
A) the 5-aminomethyl-1,2 '-docosyl-1-tetradecyl-3,3,3 ', 3 '-preparation of tetramethyl-indoles carbocyanine iodide
According to above-mentioned response diagram 1 preparation motif compound.In narrating below, the reactant of the corresponding numeral that the numeral that provides in the bracket shows in response diagram 1.Products therefrom has the chemical formula of compound 8, wherein X and X
1Expression C(CH
3)
2And R/R
1Expression C
14H
29/ C
22H
45And A represents I.
By people such as Gale, Aust.J.Chem., the preparation 5-(N-phthalimido amino methyl of improving one's methods 30:693(1977))-2,3,3 ,-(3H)-tri-methyl indole false (1).With 2,3,3-(3H)-(23.85g, 0.15mol Aldrich) are dissolved in the 150ml vitriol oil tri-methyl indole false.Then flask place ice bath and in 30 minutes, add in batches N-hydroxymethyl phthalic imidine (26.55g, 0.15mol, Fluka).Shift out ice bath and with solution stirring at room 5 days.Reaction mixture is poured in the 200g trash ice then, with 50%NaOH solution pH value is transferred to 9.0 when keeping temperature to be lower than 35 ℃ by (when needing) on the rocks.Filter and collect the throw out that generates, with distilling washing and dried overnight under high vacuum.Recrystallize goes out crude product from dichloromethane/hexane, generates 5-(N-phthalimido amino methyl)-2,3,3-(3H)-tri-methyl indole false (1) (30g, 63%).
Prepare docosyl-4-closilate with method described in the PCT/US89/00087.
Prepare tetradecyl-4-closilate with similar method.With 5-(N-phthalimido amino methyl)-2,3,3-(3H)-tri-methyl indole false (6.36g, 2mmol) and tetradecyl-4-closilate (7.62g 2mmol) mixes, and one coexists 130 ℃ and heated 2 hours.Reaction mixture cool to room temperature then, crude product recrystallize from ethyl acetate goes out, and generates pure 5-(N-phthalimido amino methyl)-1-tetradecyl-2,3,3-(3H)-and tri-methyl indole false 4-closilate (2) (10.23g, 72%), m.p.=141 ℃.
When stirring with 2,3,3-trimethylammonium-(3H)-pseudo-indole (6.26g, 0.04mol, Aldrich) and n-docosane base-4-closilate (20.02g 0.04mol) one coexists 140 ℃ and heated 3 hours.The reaction mixture cool to room temperature generates cured shape solid then.Then solid is dissolved in the ethanol (250ml), adds the saturated KI solution of 200ml, this solution stirring 30 minutes.Add 1 liter of cold water and continue and stirred 15 minutes.Collect and generate throw out, with distillation washing twice, a dry night under high vacuum.Recrystallize goes out thick material from methylene dichloride/hexane, generates pure 1-docosyl-2,3,3-(3H)-tri-methyl indole false iodide (5) (14.5g, 61%), m.p.=107-110 ℃.
With 1-docosyl-2,3,3-(3H)-tri-methyl indole false iodide (8.94g, 0.015mol), N, the N-amitraz diphenylurea (2.94g, 0.015mol, Aldrich) and acetic anhydride (60ml) place the round-bottomed flask that condenser is housed, flask argon purification, condenser dress drying tube then.Flask is put into pre-warmed oil bath (160 ℃) and refluxed 60 minutes.Remove flask and cool to room temperature from oil bath then, transfer in 1 liter of conical flask and with ethanol (60ml) and reach the then saturated KI solution dilution of 60ml, mixture stirred 30 minutes.Add cold water (800ml) and continue again and stirred 15 minutes.The filtration collecting precipitation produces, with the distillation washing and under high vacuum a dry night, generate 2-(β-Acetanilide vinyl)-1-docosyl-3,3-(3H) dimethyl pseudo-indole iodide (6) (10.52g, 95%), m.p.=98～100 ℃.Use the crude product that is not further purified.
With 5-(N-phthalimido amino methyl)-1-tetradecyl-2,3,3-(3H)-tri-methyl indole false, (14.1g 20mmol) is dissolved in the 300ml concentrated hydrochloric acid 4-closilate (2).Solution slowly is heated to 115 ℃ (careful, as can to spume) and refluxed 22 hours.After this mixture cool to room temperature and place ice bath.When maintaining the temperature between 15 and 20 ℃, the pH value is transferred to 9.0 with ammonium hydroxide (30%).Then with distilled water with solution dilution to the twice of its volume, and with dichloromethane extraction (3 * 200ml).Mix dichloromethane extraction liquid,, concentrate and obtain yellow oily 5-amino methyl-3,3-dimethyl-2-methylene radical-1-tetradecyl indoline (3) (6.9g, 90%) through dried over mgso.
With 5-amino methyl-3,3-dimethyl-2-methylene radical-1-tetradecyl indoline (14.88g 38.75mmol) is dissolved in the methyl-formiate (75ml), and under argon gas reflux (55 ℃) 24 hours.Solution cool to room temperature and steam methyl-formiate then.Residuum recrystallize from hexane goes out, and generates 5-(N-formamido group methyl)-3,3-dimethyl-2-methylene radical-1-tetradecyl indoline (4) (10.85g, 68%).
With 2-(β-phenyl methyl ketone amido vinyl)-1-docosyl-3,3-(3H)-dimethyl pseudo-indole iodide (6) (740mg, 1mmol) with 5-(N-formamido group methyl)-3,3-dimethyl-2-methylene radical-1-tetradecyl indoline (4) (330mg, 0.8mmol) and anhydrous sodium acetate (150mg 1.8mmol) is dissolved in the Virahol and stirring at room 24 hours.Solution is sent into the 250ml conical flask then, and with ethanol (20ml) and KI saturated solution (20ml) dilution, mixture stirred 30 minutes.Be settled out product and stirred Generation Liquid 15 minutes by adding 100ml cold water.Filter the collecting precipitation thing, wash and a dry night under high vacuum with distillation.Crude product (870mg) is divided into two batches, each is by flash column chromatography (silica gel, 10% Virahol is in methylene dichloride) purifying, generate pure 1 '-docosyl-5-(N-formamido group methyl)-1-tetradecyl-3,3,3 ', 3 '-tetramethyl-indoles carbocyanine iodide (7) (414mg, 52%).
Concentrated hydrochloric acid with 100ml: methanol solution (by mixing the preparation of 11ml concentrated hydrochloric acid and 120ml methyl alcohol) is added to 1 '-docosyl-5-(N-formamido group methyl)-1-tetradecyl-3,3,3 ', 3 '-tetramethyl-indoles carbocyanine iodide (7) (250mg) in, solution was at stirring at room 16-24 hour.Solution is with frozen water (100ml) dilution then, cooling and transfer pH value to 7.5～8.0 by slow adding saturated sodium bicarbonate solution in ice bath.Then liquid phase with dichloromethane extraction (2 * 100ml), mix organic phase through dried over sodium sulfate, filtration, concentrate (Buchi bath temperature＜30 ℃), dry under high vacuum then, generate product (8) (240mg, 98%).
B) 2-(3-(2,3-dihydro-3,3-dimethyl-5-aminomethyl-1,2-tetradecyl-(2H)-indylidene-2-yl)-1-propenyl)-1-docosyl benzoxazole iodide
Also according to response diagram 1 preparation motif compound.Or in this narration, the reactant of the corresponding numeral that given numeral shows in response diagram 1 in the bracket.Products therefrom has the chemical formula of compound (8), and wherein X represents C(CH
3)
2, X represents oxygen, R/R
1Represent C
14H
29/ C
22H
45Represent iodide ion with A.
As mentioned above the 2-Jia base benzoxazole of preparation (2.65g, 19.9mmol, Aldrich) and docosyl-4-closilate (10.0g, stirred solution 19.9mmol) heated 6 hours at 160-170 ℃ (oil bath temperature).After this, the reaction mixture cool to room temperature, the solids that recrystallize go out to generate from methylene dichloride generates pure 1-docosyl-2-first base benzoxazole-4-closilate (5) (7.3g, 58%), m.p.=124～125 ℃.
1-docosyl-2-methyl-benzoxazoles-4-closilate (5) (1.5g, 2.36mmol), N, N '-amitraz diphenylurea (0.462g, 2.36mmol, Aldrich) and the stirred solution of acetic anhydride (7ml) in oil bath (being preheating to 160 ℃), refluxed 30 minutes.When cool to room temperature, mixture is with straight alcohol (15ml) and follow saturated solution of potassium iodide (10ml) dilution, and stirs 30 minutes, adds water (150ml) then and filters the collecting precipitation product, washes with water and a dry night under high vacuum.Recrystallize goes out the exsiccant crude product from ethyl acetate, generates pure 2-(β-Acetanilide-vinyl)-1-docosyl benzoxazole iodide (6) (1.51g, 90%), m.p.=67-68 ℃.
2-(β-Acetanilide vinyl)-1-docosyl benzoxazole iodide (6) (1.10g, 1.53mmol), 5-(N-formamido group methyl)-1-tetradecyl-3, (630mg, 1.53mmol) (as preparing among the above-mentioned 3a), triethylamine (0.5ml) and ethanol (25ml) reflux are 1 hour for 3-dimethyl-2-methylene radical indoline (4).The solution cool to room temperature is sent into conical flask and is used ethanol (40ml) and saturated KI solution (20ml) dilution then.This mixture stirs after 30 minutes and adds 200ml cold water, with this solution of dichloromethane extraction.Mix dichloromethane extraction liquid,, filter and concentrate, generate crude product through dried over mgso.This product is by flash column chromatography (silica gel, 5% methyl alcohol is in methylene dichloride) purifying, generate 2-(3-(2,3-dihydro-3,3-dimethyl-5-(N-formamido group methyl)-the 1-tetradecyl-(2H)-indylidene-2-yl)-the 1-propenyl)-1-docosyl benzoxazole iodide (7) (305mg, 20%).
Will be as the dense HCl of 25ml of preparation in above-mentioned (a): methanol solution be added to 2-(3-(2,3-dihydro-3,3-dimethyl-5-(N-formamido group methyl)-1-tetradecyl-(2H)-indylidene-2-yl)-the 1-propenyl)-1-docosyl benzoxazole iodide (7) (50mg) in, solution was at stirring at room 16-24 hour.Solution cools off in ice bath with frozen water (30ml) dilution then, transfers pH value to 7.5～8.0 by slow adding saturated sodium bicarbonate solution.Use then the dichloromethane extraction water (2 * 50ml), mix organic phase through dried over sodium sulfate, filtering and concentrating (0-5 ℃ of Buchi bath temperature), dry under high vacuum then, obtain product (8) (48mg, 99%).
(c) the 5-aminomethyl-1,2 '-docosyl-1-tetradecyl-3,3,3 ', 3 '-the terephthaloyl N-hydroxy-succinamide ester derivative of tetramethyl-indoles carbocyanine iodide.
Under room temperature and the argon gas atmosphere through the two-N-hydroxy-succinamide ester (100mg of sleeve pipe to the terephthalic acid in dry tetrahydrofuran (30ml), 0.278mmol) stirred solution of (by terephthalyl chloride and N-hydroxy-succinamide prepared in reaction) be added on 5-aminomethyl-1,2 in the tetrahydrofuran (THF) (10ml) '-docosyl-1-tetradecyl-3,3,3 ', 3 '-solution of tetramethyl-indoles carbocyanine iodide (as preparing among the top 3a).The solution stirring that generates 2 hours concentrates on Buchi then.The crude product that obtains is made motif compound (101mg, 34%) by flash column chromatography (silica gel, 5%, methyl alcohol is in methylene dichloride) purifying.
D) P-5-lipophilic cyanine conjugate
Under argon gas atmosphere in 0-2 ℃ to the Substance P (21mg in dry dimethyl formamide (10ml), 0.013mmol) and the product (30mg of above-mentioned routine 3c, 0.024mmole) stirred solution in add triethylamine (60 μ l), the solution that generates stirred 4 hours at 0-2 ℃, by adding trifluoroacetic acid (100 μ l) inhibited reaction, solution is sent into the 250ml flask, water (80ml) dilution and one night of freeze-drying.Resultant passes through the 18 alkyl silica gel column purification, and earlier with 80: 20: 1(methyl alcohol: water: trifluoroacetic acid) wash-out is used 100: 2: 1(methyl alcohol: water then to remove non-conjugated peptide: the product of wash-out needs trifluoroacetic acid).Mix the composition that contains peptide-lipophilic cyanine conjugate and concentrate on Buchi, residue (50ml) ice from water is done, and obtains red-violet colour powdered pure conjugate (14mg, 40%).High efficiency liquid chromatography (hplc) purity is greater than 90%, and educt P is lower than 0.02% simultaneously.The conjugate of gained has following structural formula (the amino acid order of wherein representing Substance P with three letter characters commonly used):
Because peptide and cyanine reporter group all are to be connected to the spacer groups part by amido linkage, therefore the conjugate that generates is metastable in vivo.
E) 2-(3-(2,3-dihydro-3,3-dimethyl-5-(+)-biotin amido group methyl isophthalic acid-tetradecyl-(2H)-indylidene-2-yl)-1-propenyl)-1-docosyl benzoxazole iodide
Under argon gas atmosphere, as the 2-(3-(2 in dimethyl formamide for preparing among the above-mentioned routine 3b, 3-dihydro-3,3-dimethyl-5-aminomethyl-1,2-tetradecyl-(2H)-indylidene-2-yl)-the 1-propenyl)-(53mg, stirred solution 0.055mmol) cools off in ice bath 1-docosyl benzoxazole iodide.Add in this solution (+)-vitamin H 4-nitro phenyl ester that (Aldrich), (Aldrich), solution stirred 1 hour for 16mg, 0.23mmol, then at one night of stirring at room to add imidazoles then for 23mg, 0.63mmol in ice bath.Reaction mixture is in concentrating on Buchi under the high vacuum, and residuum flash chromatography (silica gel, 7.5% methyl alcohol are in methylene dichloride, and 10% methyl alcohol is in methylene dichloride then) generates motif compound (22mg, 36%).
F) 5-6-(6-(+)-vitamin H acyl group amido hexanamido)-the hexanamido methyl-1 '-docosyl-1-tetradecyl-3,3,3 ', 3 '-tetramethyl-indoles carbocyanine iodide.
Re-use described reactant with quadrat method and following amount as above-mentioned routine 3e: as the 5-aminomethyl-1,2 of above-mentioned preparation '-docosyl-1-tetradecyl-3; 3; 3 '; 3 '-tetramethyl-indoles carbocyanine iodide (90mg; 0.09mmol) 6-(6-((vitamin H acyl group) amino) hexanamido caproic acid N-hydroxyl-succinimide ester (55mg, 0.097mmol, Molecular Probes); dimethyl formamide (20ml) and imidazoles (20mg, 0.24mmol).Flash chromatography (silica gel, 10% methyl alcohol is in methylene dichloride) generates motif compound (27.5mg, 21%).
Respectively by N-hydroxy-succinamide base 6-(biotin acylamino) capronate and 6-(6-((vitamin H acyl group) amino) hexanamido) the similar preparation compound 2-of caproic acid N-hydroxy-succinamide fat (available from Molecular Probes) (3-(2; 3-dihydro-3; 3-dimethyl-5-(6-(+)-biotin acylamino) the hexanamido methyl)-the 1-tetradecyl-(2H)-indylidene-2-yl)-1-propenyl-1-docosyl-benzoxazoles and 2-{ 3-(2; 3-dihydro-3, the 3-dimethyl-5-(6-(6(+)-and the biotin acylamino hexanamido) the hexanamido methyl)-the 1-tetradecyl-(2H)-indylidene-2-yl)-the 1-propenyl)-1-docosyl benzoxazole salt.
G) N-just-docosyl-N '-n-tetradecane base-5-tributyl stannyl-3,3,3 ', 3 '-tetramethyl-indoles carbocyanine muriate
Press response diagram 2 synthetic motif compounds, products therefrom has the chemical formula of compound (13), wherein X and X
1Represent C(CH
3)
2, R/R
1Represent C
22H
45/ C
14H
29Represent Cl with A.By people such as Blaikie at J.Chem.Soc.313; Method 296(1924) prepares 4-iodophenyl hydrazine.To be dissolved in the SODIUMNITRATE (16.56g in the water (100ml), 0.24mol, Aldrich) (43.9g, 0.20ml is in solution Aldrich) (being cooled to 0-2 ℃) to be added drop-wise to 4-Iodoaniline in frozen water (600ml) and concentrated hydrochloric acid (200ml) in 45 minutes.Reaction mixture stirred 30 minutes in 0-2 ℃ again, was keeping reaction mixture temperature in 0-2 ℃, will be at dense HCl(150ml in 90 minutes) in tin chloride (II) (151.68g, 10.8ml Aldrich) splash into.After having added, reaction mixture is raised to room temperature and stirred 3 hours.Filter then and collect isolated yellow solid from solution, place frozen water (800ml), transfer pH value to 10 with 25% potassium hydroxide aqueous solution.Filter and collect the solid that generates, with the less water washing, and dry under vacuum.Product places toluene (400ml) and removes by filter insoluble impurity then.Add hexane (1200ml),, it is filtered collection because of but having isolated yellow needle-like crystal in refrigerator and cooled, with hexane (100ml) washing, and dry under high vacuum, generate pure 4-iodophenyl hydrazine (23.36g, 50%), m.p.=94 ℃.
With people such as Moreau, at Eur.J.Med.Chem.Chim.Ther., 9(3); The preparation 5-iodo-2,3 of improving one's methods 274-280(1974), the 3-trimethylammonium-(3H)-pseudo-indole (9).Will the 4-iodophenyl hydrazine in the ethanol (100ml) (23.36g, 0.0998mol) and the 2-espeleton (8.59g, 0.0998mol) solution refluxed 3 hours.After this, (9.92g, 0.0998mol), the solution of generation refluxed 3 hours again to drip the vitriol oil that is dissolved in the ethanol (100ml) with 1 hour time.In cool to room temperature, remove by filter precipitated solid, filtrate is concentrated to 80ml, pours in the frozen water then.Use the dichloromethane extraction aqueous solution then, mix organic phase, filter and concentrate, generate crude product (26.9g, 95.0%) through dried over mgso.Obtain pure products 5-iodo-2,3 after the vacuum distilling, the 3-trimethylammonium-(3H)-pseudo-indole (9) (16.7g, 59.0%), b.p.81-88 ℃ (under 0.03mmHg).
In continuously stirring, 5-iodo-2,3, the 3-trimethylammonium-(3H)-(2.85g, 0.01mol) (5.55g is 0.01mol) 130 ℃ (oil bath temperature) heating 4 hours with n-docosane base-4-chlorobenzene iodate for pseudo-indole.Then refrigerative reaction mixture recrystallize from ethyl acetate goes out, and generates brown crystalline N-n-docosane base-5-iodo-2,3, m.p.=118 ℃ of 3-trimethylammonium indoline 4-chlorobenzene iodate (10) (4.62g, 59%).
In continuously stirring, 2,3, the 3-trimethylammonium-(3H)-(Aldrich) (15.52g is 0.04mol) together 130-135 ℃ (oil bath temperature) heating 3 hours with n-tetradecane base-4-chlorobenzene iodate for 6.36g, 0.04mol for pseudo-indole.Crude product is dissolved in the ethanol (200ml) then, and stirs 30 minutes with saturated solution of potassium iodide (50ml).Add cold water (500ml), filter the collecting precipitation thing, clean with icy.Crystallization goes out the exsiccant crude product from ethyl acetate, cleans the crystal and the drying of collecting with ether, generates N-tetradecyl-2,3,3-trimethylammonium indoline iodide (5) (12.8g, 66.8%), m.p.=97 ℃.
With N-docosyl-5-iodo-2,3,3-trimethylammonium indoline 4-closilate (2.36g, 3.0mmol), N, N '-amitraz diphenylurea (0.59g, 3.0mmol.Aldrich) and acetic anhydride (20ml) put into the 50ml round-bottomed flask, this flask is under the argon gas atmosphere and reflux exchanger is housed and agitator arm.This flask places oil bath then, and this oil bath has been preheating to 170 ℃ constant temperature, and mixture refluxed 60 minutes.The reaction flask cool to room temperature is sent in the 500ml conical flask then then.This bottle places ice bath and adds saturated solution of potassium iodide then.Stir after 15 minutes, add cold water (250ml), mixture restir 15 minutes.Filter and collect and drying precipitated product, obtain N-n-docosane base-5-iodo-2-(β-Acetanilide vinyl)-3,3-dimethyl indoline iodide (11) (2.37g, 91%), m.p.=170 ℃ (decomposition).
At room temperature with N-docosyl-5-iodo-2-(β-Acetanilide vinyl)-3,3-dimethyl indoline iodide (511mg, 0.59mmol), N-tetradecyl-2,3, and 3-trimethylammonium indoline iodide (233mg, 0.47mmol) and the anhydrous sodium acetate (48mg in dehydrated alcohol (15ml), 0.59mmol mixture Aldrich) stirred 24 hours.The scarlet reaction mixture is poured in the conical flask and with ethanol (25ml) and is diluted then.Add then the silver acetate be dissolved in the water (25ml) (492mg, 2.95mmol, Aldrich), solution stirring 15 minutes.Add ethanol (25ml) and saturated nacl aqueous solution then, continue to stir 15 minutes.Solution is sent in the separating funnel then, and water (200ml) dilutes and (2 * 100ml) extract with methylene dichloride.Through the anhydrous magnesium sulfate drying organic phase, filtration and evaporation are to generate crude product.With flash chromatography (silica gel, earlier 5% methyl alcohol in methylene dichloride back 8% methyl alcohol in methylene dichloride) the purifying crude product, generate N-docosyl-N '-tetradecyl-5-iodo-3,3,3 ', 3 '-tetramethyl-indoles carbocyanine muriate (12) (272mg, 58%).
With N-docosyl-N '-tetradecyl-5-iodo-3,3,3 ', 3 '-tetramethyl-indoles carbocyanine muriate (12) (200mg, 0.2mmol) be dissolved in the dry toluene (15ml newly steams from hydrolith), the solution of generation is by outgasing the argon gas bubbling.Add through syringe then two-(positive tributyl tin) (0.237ml, 0.47mmol.Aldrich) then add tetrakis triphenylphosphine palladium (0) (2.34mg, 2.0 μ mol, Aldrich).The solution that generates refluxed 48 hours under argon gas.Remove toluene then in a vacuum, residuum flash chromatography (silica gel, 5% methyl alcohol is in methylene dichloride) generates motif compound (13) (71mg, 31%), measured value M
+, 1122, C
71H
123N
2Sn theoretical value 1122.
Compound (13) is to use by people such as Wilbur at J.NuCl.Med, 30:216-226(1989) in the method for narration introduce the general intermediate of radioactive halogen atom.
H)
186Re-sequestrant lipophilic cyanine conjugate
According to shown in response diagram 5 preparation motif compounds, the note numeral that occurs in reaction is corresponding to those of reaction synoptic diagram 5.Products therefrom has the chemical formula of compound 24, wherein X and X
1Represent C(CH
3)
2, R
1/ R
2Represent C
14H
29/ C
22H
45Represent I with A.
(84.5mg 0.291mmol) is added to and contains (8) (66.8mg is in pyriform flask 0.066mmol) will to be dissolved in bifunctional chelants compound (22) in the 400 μ l tetrahydrofuran (THF)s (THF).Stir this solution at ambient temperature.By thin-layer chromatography (TLC; Silica, 90: 10 CH
2Cl
2: the MeOH) process of monitoring reaction.After 4 hours, reaction mixture generates 75: 25 THF with the hexane dilution: the solution of hexane also is added on short distance silica gel (20g) post.Excessive compound (22) was with 50: 50 hexanes: the EtOAc wash-out produces negative reaction up to elutriant and tetrabromo-mcresolsulfonphthalein.Solvent compositions becomes 90: 10CH
2Cl
2: MeOH and wash-out compound 22.The vapourisation under reduced pressure solvent generates 65.0mg compound 23.
High-Field
1Hnmr(300MHz) low chemical shift of demonstration benzyl methene proton is from 3.9ppm to 4.5ppm, seen in the compound 8 of formyl protection in front.4.5 and the relative integral of the signal at 8.4ppm place show 1: 1 coupling rate.
Make product (compound 23) be transformed into HCl salt by adding HCl gas/ethanolic soln.The ethanolic soln that adds oxalic acid is as oxidation inhibitor, evaporating solns, store solids residuum under nitrogen.
Will be at 107 μ 10.1N NaOH(0.181mci/nmol) in 60nmol Na
186ReO
4Be added to 150 μ l and shift chelate solution (200mg citric acid, 40mg SnCl
22H
2O and 20mg are dissolved in 2ml H
2Among the O 2, the 5-resorcylic acid) in, then add 43 μ l0.1M NaOH.Solution eddy current 1 minute and 55 ℃ the heating 10 minutes.Add ethanol (0.69ml) to reaction mixture, then add 300 μ l compound 23(0.8mg, 0.69 μ mol) ethanolic soln.Solution eddy current 15 seconds also continues heating 1 hour.
Reaction mixture is added to half preparation HPLC post (Waters Novapak, 7.5 * 300mm) in, divided with 50 with flow rate 4.0ml/ with 30 minutes: 50A: B is to the gradient elution of 100%B, and wherein solvent orange 2 A is to contain 0.1% trifluoroacetic acid (TFA) and 0.05%2,75 of 5-resorcylic acid: 25H
2O: CH
3CN, solvent B contain 0.1%TFA and 0.05%2,20 of 5-resorcylic acid: 80CH
3CN: THF).In the about 20 minutes cut of wash-out, find to contain the cut of motif compound.
Above-mentioned cut to use solvent 1(0.05%2 at 1: 1, the 5-resorcylic acid is in water) dilute and be added to Sep-Pak post (C-18, Waters 36805) on, this post is used 10ml solvent 2(0.05%2 earlier, and the 5-resorcylic acid is in ethanol) then with 1 washing of 10ml solvent.With 10ml solvent 1,150 μ l solvent 2 and 300 μ l solvents, 2 elution cylinders.Get this end cuts and make product.To about 10 μ l volumes, be diluted with ethanol to about 40 μ l with the nitrogen gas stream evaporating solvent then.By the concentration at the above-mentioned products therefrom of absorption measurement of 555nm is 252 μ M.Compare with theoretical value 181mci/ μ mol, peculiar activity is 186 ± 21mci/ μ mol.The recovery of product is a Re-186 total amount-9%.Surveying radioactive purity and find with HPLC after one day is about 94%.The major portion of finding impurity (about 5%) is in the gap, and supposition is the free perrhenate.
(i) .7-N(5-oxo pentanoyl) take off the mensuration of acetyl colchicine lipophilic cyanine hydrazone conjugate and acid-cleavability
(a) preparation
Prepare motif compound according to above-mentioned reaction general process shown in Figure 3.Wherein the note numeral is corresponding to reaction those in synoptic diagram 3.Products therefrom has the chemical formula of compound 19, wherein X and X
1=C(CH
3)
2And R/R
1=C
14H
29/ C
22H
45And A represents Cl.
With the general process of response diagram 1 prepare the 5-aminomethyl-1,2 '-docosyl-1-tetradecyl-3,3,3 ', 3 '-tetramethyl-indoles carbocyanine iodide (compound 8), wherein R is C
14H
29; R
1Be C
22H
45, X and X
1=C(CH
3)
2
At room temperature to the monomethyl glutarate in dimethyl formamide (15ml steams from lithium aluminium hydride) (0.14ml, 1.1mmol, stirred solution Aldrich) add N-hydroxy-succinamide (126.5mg, 1.1mmol, Aldrich).This mixture in ice bath, cool off and add dicyclohexyl carbodiimide (226.6mg, 1.1mmol).Reaction mixture was warming up to room temperature and restir 3 hours gradually.Thereafter, reaction mixture is sent into and is contained new preparation compound 8(700mg, and in flask 0.7mmol), reaction mixture keeps stirring 30 hours in room temperature.Remove dimethyl formamide then under high vacuum, the crude product of generation is dissolved in dehydrated alcohol (250ml) and the water (250ml).(434mg 2.6mmol), stirs after 30 minutes, adds saturated nacl aqueous solution (100ml), mixture restir 30 minutes to add silver acetate then.(4 * 100ml), the mixing organic layer filters also concentrated through anhydrous sodium sulfate drying with the dichloromethane extraction water layer.Residuum carries out flash chromatography (silica gel, first back again with 5%) with 10% methyl alcohol wash-out in methylene dichloride with 2.5% back, generate 5-(N-(monomethyl glutaryl)-amino methyl)-1 '-docosyl-1-tetradecyl-3,3,3 ', 3 '-tetramethyl-indoles carbocyanine muriate (14) (520mg, 63%).
At room temperature with anhydrous hydrazine (5ml, Aldrich) slowly be added in the stirred solution of reactant, this solution is by 5-(N-(monomethyl glutaryl)-amino methyl)-1 '-docosyl-1-tetradecyl-3,3,3 ', 3 '-tetramethyl-indoles carbocyanine muriate (520mg, 0.44mmol) and dehydrated alcohol (6ml) constitute.Reaction mixture keeps stirring 2 hours, concentrate by rotatory evaporator then, residuum carries out flash chromatography (silica gel, earlier with 10% back 30% methyl alcohol wash-out in methylene dichloride), generate 5-(the single diazanyl of N-() glutaryl amino methyl)-1 '-docosyl-1-tetradecyl-3,3,3 ', 3 '-tetramethyl-indoles carbocyanine muriate (15) (110mg, 27%).
At room temperature to taking off acetyl colchicine (16) (0.2902g, 0.81mmol, Molecular Probes) and add glutaric anhydride (111.2mg, 0.975mmol in the stirred solution in methylene dichloride (5ml), Aldrich), reaction mixture keeps stirring 2 hours in room temperature.Rotary evaporation is removed methylene dichloride then, and dry raw product under high vacuum generates solid 7-(N-glutaryl-) take off acetyl colchicine (0.397g, 100%).
Then at room temperature to the 7-(N-glutaryl-) take off acetyl colchicine (0.397g; 0.83mmol) at dimethyl formamide (5ml; by what newly steam in the lithium aluminium hydride) in stirred solution in add carbonyl dimidazoles (0.197g; 1.22mmol; Aldrich); reaction mixture at room temperature kept stirring 2 hours, formed precipitation in the meantime in solution.Dimethyl formamide is removed in vacuum-evaporation, and residuum is dissolved in the methylene dichloride (5ml).(Aldrich), the mixture of generation stirred 3 hours, poured into then in the water (50ml), was separated into organic layer for 250mg, 0.972mmol to add the TBuA borane to this solution.Water-bed layer dichloromethane extraction (2 * 25ml).Mix organic layer through anhydrous sodium sulfate drying, filter and concentrate.Thick product carries out flash chromatography (silica gel, 10% methyl alcohol is in methylene dichloride), generates 7-N-(5-hydroxyl pentanoyl) take off acetyl colchicine (17) (72mg, 25%).
At room temperature to 7-N-(5-hydroxyl pentanoyl) take off acetyl colchicine (72mg; 0.16mmol) the solution in methylene dichloride (5ml) in add Pyridinium chlorochromate on silica gel (41.3mg; 0.19mmol; Aldrich); mixture stirred 2 hours; pour into then in the water (50ml), isolate organic layer, water-bed layer dichloromethane extraction (2 * 25ml).Mix organic layer through anhydrous sodium sulfate drying, filter and concentrate.The residuum that generates carries out flash chromatography (silica gel, 10% methyl alcohol is in methylene dichloride), generates oily 7-N-(5-oxo pentanoyl) take off acetyl colchicine (18) (35mg, 49%).
To containing 7-N-(5-oxo pentanoyl) take off acetyl colchicine (40mg; 0.088mmol) flask add 5-(N-glutaryl (single diazanyl) amino methyl)-1 '-docosyl-1-tetradecyl-3; 3; 3 '; 3 '-tetramethyl-indoles carbocyanine muriate (110mg; 0.12mmol) solution in ethanol (20ml), reaction mixture stirred 20 hours.Remove ethanol by rotary evaporation then, residuum carries out flash chromatography (silica gel, 10% methyl alcohol is in methylene dichloride), generates motif compound (19) (26.5mg, 27%).1: 1 the coupling rate of integral representation of the 200MHz proton N MR of this compound, fast atom bombardment(FAB) mass spectrum assay method (glycerine/thioglycerol matrix) shows M
+=1429 corresponding to product ion C
90H
135N
6O
8Predetermined M
+(HPLC) further characterizes this product by following condition by reversed-phase high pressure liquid chromatography, and has retention time 55 minutes.Survey to find that at 350nm purity is 98% with UV, in the detection of this product, find to be lower than 0.30% free 7-N-(5-oxo pentanoyl) take off acetyl colchicine (retention time=12 minute).
Used HPLC system comprises Waters Model 600E solvent delivery system, U6K syringe and the Model 990 light diode array detectors of band W600E gradient controller.Chromatographic condition is as follows: cylinder: Waters Nova-Pak(phenyl, 4 μ m, 3.9mmX 15cm); Moving phase: solvent orange 2 A=water: methyl alcohol: acetonitrile: PICA reagent (Waters) (395: 25: 250: 4); Solvent B=water: methyl alcohol: acetonitrile: PICA reagent (225: 25: 250: 4); Solvent C=methyl alcohol: water: PICA reagent (490: 15: 4).Gradient condition: 100%(A) to 60%: 40%(A:, arrive 100%(c then), follow 100%(c with 10 minutes B) with 20 minutes) with 40 minutes.Flow rate: 2ml/ branch.Survey: from the 240-575nm light diode array.
(b). the mensuration of sour cleavability
Generate 7-N-(5-oxo pentanoyl by HPLC in the acid hydrolysis of three different PH research hydrazone conjugates (19)) take off the ratio of acetyl colchicine.
According to Gomori " Methods in Enzymology ", 16:138(1955) described method prepares buffered soln.HPLC condition and retention time are same as described above.Measure 7-N-(5-oxo pentanoyl) sensing range of taking off the acetyl colchicine is at the about 100ng of 350nm.
In the nut phial (1ml) that contains predesigned pH value Citrate trianion phosphate buffered saline buffer 200 μ l, prepare the compound solution that to study by adding 50 μ l hydrazone conjugate solution (1mg/ml methyl alcohol).Phial keeps sealing, is surveyed by HPLC(350nm) reservation of the conjugate (19) of analytical solution and at the 7-N-(5-oxo pentanoyl of 24 hours and generation in 48 hours) take off the acetyl colchicine.Each HPLC injects 200 μ l.
Be summarized as follows in PH4.21,5.74 and 7.35 results of hydrolysis.
78% (19) decompose generation (18) after PH4.21:24 hour, and 100% decomposes after 48 hours.
45% (19) decompose generation (18) after PH5.74:24 hour, and 100% decomposes after 48 hours.
36% (19) decompose generation (18) after PH7.35:24 hour, and 77% decomposes after 48 hours.
Survey by HPLC, only the free colchicine hydrolysate of hydrazone conjugate is the aldehyde of expection, compound 18.
J) heparin lipophilic cyanine conjugate
The general method shown in Figure 4 according to above-mentioned reaction prepares motif compound.Wherein the note numeral is corresponding at shown in the response diagram 4 those.Products therefrom has the chemical formula by compound 21 representative types, wherein X and X
1=C(CH
3)
2, R/R
1=C
14H
29/ C
22H
45And A=Cl.
Prepare compound 8 as mentioned above.
With the compound 8(18.0mg of new system, 18.0 μ mol) be dissolved in deuterate chloroform in the NMR pipe (0.5ml, Aldrich) in.(5 μ l, 0.036mmol), (Aldrich), this mixture shook two minutes for 1.95mg, 6.7 μ mol then to add triphosgene to add triethylamine.Carry out proton N MR, be presented in the benzylic protons from 3.95 to 4.95 displacement seen in compound (8), prove product 5-isocyanato-methylene radical-1 '-docosyl-1-tetradecyl-3,3,3 ', 3 '-generation of tetramethyl-indoles carbocyanine muriate (20).By the deuterate chloroformic solution of the concentrated isocyanic ester (20) of rotary evaporation, residuum is dissolved in the dimethyl formamide (4ml, dry and distilled) and stirring rapidly.(0.0016mmol), flask covers cap and at room temperature stirred 20 hours for 9.4mg/ml, 2ml to add the formamide soln of heparin to this solution that stirs rapidly.Remove by filter insolubles, under high vacuum in 50 ℃ by the rotary evaporation concentrated filtrate.Residuum is added in the mixture of water (20ml) and methylene dichloride (20ml).Isolate water layer and wash once more with methylene dichloride.The aqueous solution places dialysis bag (spetra/Por membrane MWCO: 1000) and to the water dialysis, freeze-drying generates pink solid (24.3mg) then then.This material no longer is further purified and be sure of is the mixture of compound 21 and unreacted heparin.Original state is as biological assessment (seeing example 14).
Example 4
With the biological effect of medical active protein conjugation to the lipophilic molecule
Being similar to people such as U.Forstermann at J.Pharmacol.EXP.Ther, 234, the body inner recipient pharmacology of the made compound of above-mentioned routine 3d is studied in 1055-61(1987) the rabbit blood hind leg perfusion test described in.Blood is from the rabbit of the artificial respiration of Sodital anesthesia, and takes out from the carotid artery of cannulate, passes constant speed roller pump and directly import the femoral artery of cannulate in the medical grade silicone rubber tube.Measure the distally Ppa pulmonary artery pressure (with mmHg) of femoral artery bed by extending into minute-pressure umformer (Millar) that in the intrusion pipe and position just exceeded strand catheter tip.Beginning is adjusted pump speed to making the distally Ppa pulmonary artery pressure near system's mean arterial blood pressure, and the remainder in each experiment keeps blood constant thereafter.Then, be similar to the described method of Ohm's law, the variation of resistance directly is directly proportional with the distally injection pressure, like this:
Vascular resistance=(injection pressure)/(blood flow)
Therefore, by keeping blood flow constant at each experimental session, the variation in the injection pressure of distally directly reflects vascular resistance, thus pressure reduce to show vasorelaxation, and the increase of pressure shows vasoconstriction.Like this can be by being injected directly in the perfusion loop, estimate to confirm the material biology and the pharmacology of generation effect on the vascular smooth muscle elasticity in vivo.
In itself similar of the response curve that Fig. 1 is illustrated in substance in vivo P and routine 3d conjugate.When Substance P and conjugate are expelled to the perfusion rabbit hind leg when the part, on the hind leg injection pressure, all produce the significantly reduction relative with dosage.The response of these dilators meets the known blood vessel drug eluting of Substance P fully to be learned, and for example sees people such as J.Beny at J.Physiol, the 398:277-89(1988) example in.
In Fig. 2 as can be seen, there is quantitative difference in the response between Substance P and conjugate.Requiring the average gauge dosage of the Substance P of generation perfusion pressure drop is about 0.003pmol, and the dosage range of maximum pressure drop is 3-10pmol.Requirement causes that the minimum dose of the conjugate of perfusion pressure drop is higher than Substance P, is 1-10pmol, and reaches maximum pressure drop at 1000pmol.Logarithmic function by Substance P and conjugate calculates ED
50Value is respectively 0.13 and 34pmol, shows greater than 250 times of Substance P abilities.Yet the dose response curve with parallel slope that two materials show meets the interaction of common acceptor.Fig. 2 has also represented at Substance P, (D-Pro
2, D-Trp
7.9In the infusion process of the known antagonist of)-Substance P, the dose response relation of Substance P and conjugate.The effect of neuron, behavior and the blood vessel of this Substance P of being reported that former material P and exogenesis give in the amino acid whose synthetic peptide of 3 bit strip D-has been resisted.When antagonism exists, (D-Pro
2, D-Trp
7.9) the Substance P reduction that stops Ppa pulmonary artery pressure to produce because of Substance P and conjugate, determined as parallel to the right drift in two compound dosage response curves.The amount of the dose response curve drift to the right of Substance P and conjugate is similar when antagonist exists, and then can suppose that these two kinds of materials are to act on the common acceptor site.Listed data are based on the test that comprises three rabbits among Fig. 2, and be illustrated in do not have antagonism exist down corresponding to the peak response value of Substance P on injection pressure average (± SEM) change percentage.
Except above-mentioned, measuring go out with 1 and 10nmol concentration take accordingly not that conjugation lipophilic cyanine does not have the blood vessel relexation.β-adrenoceptor stimulant, isoproterenol with 1,10,100pmol concentration takes the injection pressure that also produces relative dosage and reduces, but it is not subjected to SP antagonist (D-Pro
2, D-Trp
7.9The antagonism of)-SP shows (D-Pro
2, D-Trp
7.9)-SP is to the characteristic of SP acceptor.
When the two during in external common cultivation, routine 3d(100 μ M) conjugate can be readily incorporated on the washing rabbit red blood cell that is suspended in the phosphate buffered salt solution that contains 1mM EDTA and 0.5% albumin rabbit serum.The fluorescence part of conjugate can make it measure detection soon by wandering cells on RBCs.When injecting the rabbit RBCs of conjugate mark in rabbit hind leg, reducing appears in injection pressure, and its amount is relevant with injected cell count.And experiment shows 10
6-10
9RBCs injection cause that injection pressure reduces in rabbit hind leg.When the sample separation of conjugate mark became supernatant liquor part and cell part, it should be by resuspending in above-mentioned RBCs damping fluid, and then injects.Supernatant liquor partly contains the biological activity significant part of organic cell suspending liquid, illustrates that conjugate can ooze out from RBCs.The RBCs that is partly produced by supernatant liquor that repetitive scrubbing has vasodilator activity eliminates the delay release performance from the conjugate of RBCs.
Can recognize that from top experiment the Substance P lipophilic cyanine conjugate of routine 3d has the ability of vasodilator activity, its similar performance is in natural material P, and should activity can be by the antagonist neutralization of Substance P acceptor.These experimental results are tending towards showing, the interaction of Substance P and acceptor is protected and is repelled with the chemical conjugation of lipophilic informational molecule.
Example 5
Measurement is by structurally the cell keying action and the biological interaction of lipophilic cyanine deutero-Substance P
For test whether the Substance P component of the conjugate of routine 3d structurally do not change and whether recognize on the surface of spherical erythrocyte (RBCs) usefulness conjugate (10 μ m, red fluorescence) mark 2ml people RBCs(10
6Cell/ml), then by the wandering cells measurement Research.This result of study is shown among Fig. 3 A-C.Especially, analyze the RBCs of be untreated RBCs and conjugate mark, whether to measure:
1. on cell surface, be combined with conjugate (Fig. 3 A relatively).
Conjugation on non-specific that be bonded to the conjugate mark or the unlabelled cell of the fluorescein isothiocyanate of goat anti-rabbit antibodies (FITC) (table 3B relatively), or
3. the anti-rabbit antibodies specific of the anti-Substance P antibody+FITC of rabbit is bonded to the RBCs last (table 3C relatively) of conjugate mark.
In Fig. 3 A, the detection of unlabelled RBCs only represents that (cell population is located at green (LFL to spontaneous fluorescein background
1) and red (LFL
2) the close section start of axle), and the cell thing of conjugate mark has shown the increase of red fluorescent signal when with the unlabeled cells comparison.
In Fig. 3 B, the labeled cell conjugate shows that with the cell of the conjugate mark that the fluorescence goat anti-rabbit antibodies is handled this antibody only has on the minimum non-specific RBCs of being bonded to, this can by with Fig. 3 A in result green fluorescence signal (LFL relatively the time
1) a small amount of increase prove.
Fig. 3 C represents unmarked RBCs or it is made first reactant with the anti-Substance P antibody of rabbit and makes the second reactant indirect fluorescent mark with the fluorescence goat anti-rabbit antibodies then with the RBCs(of conjugate mark) the fluorescence histogram.In the cell of conjugate mark, on cell, may detect the green and red fluorescence (LFL of significant quantity
1Signal increases with respect to Fig. 3 B), illustrate with cell that conjugate is connected on the specificity combination.Not using increases with then not to be noted this fluorescence of the RBCs of conjugate mark.Therefore, this technical specification, but recognize at the lip-deep Substance P of RBCs by anti-Substance P antibody antigen ground, prove the existence and the reservation of Substance P structure on red corpuscle.
Example 6
The body internal stability of Substance P lipophilic cyanine conjugate is measured on erythrocyte
Research is with the erythrocyte character in vivo of above-mentioned routine 3d conjugate mark, and 0.5ml with conjugate mark (final concn is 10 μ M), injects back the same rabbit of getting blood from each fresh anticoagulant blood in two rabbits then.Behind the conjugate mark but before injection, measure, measure the aliquot of labeled cell, to guarantee enough marks by wandering cells.Labeled cell is applied in the body then, at 30 minutes and the 1st, 2,3,4,7 day blood sampling, and carries out the mensuration of fluorocyte by the wandering cells measurement.
This result of study is shown in the figure of Fig. 4, and therefrom as can be seen, the decomposition in vivo of the conjugate of labeled cell takes place slowly relatively.Compare with minute cycle life of natural material p, the fluorescence of labeled cell (Log fluorescence intensity) reduced with 5-6 days transformation period.
Therefore, demonstrated by above-mentioned experimental result, biomone is labeled as the conveying vehicle that the parenteral administration therapeutical agent provides innovation with the lipophilic molecule that is loaded with therapeutical agent.
Example 7
The influence of the iodization on dyeing combination, cells survival and film bonding stability.
For check iodine atom is added to the performance that does not influence its labeled cell on the The compounds of this invention substantially, to the external test that following compound carries out that compound absorbs, cells survival and film keep behind the mark:
Prepare compound T, AA and AB by above-mentioned response diagram 1 and example 3 described general methods.
Reclaim and survivability (pressing Slezak and Horan at Blood, 74:2172-2177, the method calculating described in 1989) with the label concentration of 5,10 and 20 μ M and the 5 or 10 μ M concentration determinations of compd A A and AB.Survivability and recovery are reduced to 70～80% concerning the compd A A of 20 μ M label concentration and AB.
To molecule number with each cell bonded compound of raji cell assay Raji of the compound mark of 10 μ M.After final resuspending and counting, from washed cell, get 10
6The aliquot of cell (10 μ l) extracts compound by mixing from cytolemma with 250 μ l100% ethanol, uses the fluorescence intensity of the multiple measurement 200 μ l extracts of Fluoroskan little dish reader of II fluorescence and strainer.By with known compound in 100% ethanol concentration and the working curve that on the Fluoroskan II, measures measure compound concentrations in each extract.Calculate the compound molecule quantity of each cell then by the dose known amounts of total extract volume of the concentration of known extract and extraction cell.Calculated value is (4.0 ± 0.4) * 10 for compound T
6, compd A A is (1.3 ± 0.2) * 10
6With compd A B be (2.5 ± 0.7) * 10
6
Use is by NH
4(Slezak and Horan, Blood Supra) measure relative film and keep the erythrocyte membrane ghost of Cl dissolving preparation.Use aforesaid method at room temperature with 4 * 10
8/ ml was with the compound mark ghost of 10 μ M 5 minutes.Carry out washing behind the mark with the PBS that contains 1mMEDTA and 5%BSA.After the final washing, with 4 * 10
8/ ml is the resuspending ghost in PBS+EDTA+5%BSA, and 37 ℃ of hatchings 24 hours.After 24 hours, from a) mixed uniformly ghost suspension (the total compound amount that exists with measurement) and b) at ghost with 12, after 500 * g one-tenth bead 15 minutes, (to measure the not amount of binding compounds) gets double 50 μ l aliquot in the supernatant liquor that keeps, with containing 200 μ l100% alcoholic acid mixture extraction aliquot, measure the fluorescence intensity of each extract with Fluoroskan II fluorescence microplate reader.The percentage composition of compound that following calculating keeps:
(total compound-no bonding compound)/(total compound) * 100
Find that calculated value is that compound T is (91.9 ± 2.9) %, compd A A is that (97.7 ± 0.2) % and compd A B are (96.8 ± 1.3) %.
Based on above-mentioned experiment, can calculate the film retention that iodinated compounds of the present invention presents and be equal to or higher than not iodinating compound.It is somewhat low with combining of erythrocyte membrane although compare with the not iodinating compound of equivalent concentration, can assert than macromole size and weight according to it, does not expect that this difference is enough to change use that its is used the above-mentioned type cell marking.
Example 8
The reservation on artificiality surface
For estimating the ability that The compounds of this invention is retained in the artificiality surface, radioiodine (
125I) changing the solution of the lipophilic cyanine compound 12 of response diagram 2 gained (among the above-mentioned routine 3g by) in 100% ethanol contacts with the chamber of the short segment of dissimilar plastics tubings is surperficial, plastics comprise medical grade silicon rubber (SIL), polycarbonate (PC), polyvinyl chloride (PVC) and polyethylene (PE).Allow compound remove then and with PBS washpipe lightly with guaranteeing to hold to contact in 10 minutes.After the baseline radioactivity of finishing the mark pipe was measured, pipeline section contacted with the long closing pipe line that contains the fresh anti-condensation human blood of about 30ml.Pipeline is through a roller pump and made circulation of blood 6 hours.Disconnected pipeline section when finishing, and washed gently to remove adherent blood and to calculate radioactivity in 6 hours with PBS.This experimental result is illustrated among Fig. 5.Write down the given activity of length of pipe section, diameter and radioiodine compound of the present invention, be expressed as the picogram/mm of radioiodide
2Tubular cavity surface (X-axis of bar graph).Fig. 5 shows that the initial bonded radioiodine of pvc pipe compound is maximum, and the PE bonded is minimum.Contact with blood that to keep after 6 hours maximum be that the initial radioactivity of PVC(97.6 ± 2.1% is retained on pipe), and the reservation amount of PC the poorest (56.0 ± 10.4% initial radioactivity is retained).Yet the tubulate average reservation amount of institute surpasses 50%, and PVC although compound of the present invention is described to be contacted with biological fluid, but still has the ability that keeps a long period on the artificiality surface near 100%.Therefore, compound of the present invention is used in and keeps biological passivation on the artificiality surface.
Example 9
After in tumour, injecting,
186Re sequestrant lipophilic cyanine conjugate reservation and Na in vivo
186ReO
4Comparison
Substantially by the described method preparation of routine 3h
186Re sequestrant lipophilic cyanine conjugate (compound 24 of response diagram 5), different is before with the ethanol elution product, SepPak is with about 10ml distilled water wash, and is excessive 2 to remove, 5-resorcylic acid, and the needs of adjusting pH value before avoiding using in vivo.After the vacuum-evaporation, make concentrating raw material soln in ethanol reach the final volume (concentration of compound 24 is about 500 μ M, the about 123mci/ μ of given activity mol) of about 25 μ l.Preparation compound as described below 24 and contain the 5-6 μ ci that has an appointment
186Re is used for the Na of injection in the body
186ReO
4In aseptic 300mOSM Dulbecco phosphate buffered saline (PBS), dilute Na
186ReO
4(NEZ301 is in 0.1N NaOH) adjusts pH value up to being 7.0 with PH test paper check pH value, and the gained preparation is sterilized by 0.22 μ m strainer again then.Diluted compounds 24 in the aseptic glucose of 300mOSM.Use Aseptic technique with 5.0 μ l Na
186ReO
4Or compound 24 preparations are packed in the aseptic 50 μ l Hamilton syringes.Separating aliquot provides base of calculation and is used as whole imaging and the sample of the attenuation correction that bio distribution is measured to give each sample preparation.
Evaluation as described below is injected into the reservation of the above-mentioned sample preparation in back in the tumour.Injection is preceding 5 to 6 days in the tumour, uses and has an appointment 1 * 10
6The tumour cell of living is suspended in the inoculum in the aseptic Hank buffer salt solution, and corium injection on female C57B1/6 mouse right hind is so that the growth of beginning MC38 gland cancer knurl.In the time of 0 day, animal is anaesthetized again, with alcohol swab sterilization right hind and with the compound 24 or the Na of 5.0 μ l aliquot
186ReO
4Directly inject the tumour of about 5mm diameter.With about 10 second time injection, after injection was finished, pin kept in the original place 5 to 10 seconds so that in the diffusion of injection position back pressure with reduce to leak again.Injection back (10 to 15 minutes) at once to animal and base of calculation imaging, after this uses the energy window of 140 ± 20KeV to carry out day by day with GE Starcam300 system 4 days.After finishing imaging in the 2nd day and the 4th day, the kill animals group is also collected various organs, weighs, for estimating
186The bio distribution of Re and measure cpm/gm.
Select the 4th day organ
186Re distributes and is shown among the table IV A, and the result reclaims the percentage of injecting total amount in the certain organs after being expressed as correction for attenuation.Because organ is obviously different dimensionally with tumour, observes in each organ
186The comparison of Re relative concentration is in table IV B.The result is expressed as the percentage of injection volume, and injection volume can be by finding out in the 1 gram body piece in cell tissue behind the correction for attenuation.Analyze whole spark photograph image by setting up corresponding to tumour or whole survey region (ROI).The function of time after the quantity of each survey region is confirmed as injecting.Concentrating by the total of the tumour ROI ratio to the total (2 and 3 hurdles in the table V) of whole ROI of mark calculated in tumour.The reservation percentage of body internal labeling is calculated by the ratio of whole ROI total to reference ROI total (4 and 5 hurdles in the table 2).
The data declaration of table IV and V can significantly reduce by using radionuclide with the form of compound 24
186Re is the leakiness of tumor injection position internally.These data illustrate that also the reservation of using two kinds of diverse ways (whole γ flash photographic method and directly cutting) to measure in tumour obtains good consistence.
Example 10
After the intra-articular injection
186Re sequestrant lipophilic cyanine conjugate reservation and Na in vivo
186ReO
4Comparison
Except its medicine function, two performances of compound of the present invention are important in the realization bit selecting is used and kept:
A) the reservation level of application position when comparing with unconjugated therapeutical agent;
B) the scope of its distribution of injection back, a position (inhomogeneous with homogeneous phase than).
Use fluorescence or radio-labeled compound, detect these performances according to containing in the synovia lining that rhenium compound is transported to joint cavity.
As preparation as described in the routine 3h
186Re sequestrant lipophilic cyanine conjugate (compound 24 of response diagram 5).Making and concentrating the raw material final volume is about 40 μ l in ethanol.The compound 24 and the Na that are used for injection in the body
186ReO
4Following being prepared of sample preparation.In aseptic 300mOSM Dulbecco ' s phosphate-buffered saline, dilute Na
186ReO
4(NEZ301 is at 0.1N NaOH), adjusting pH value is 7.0 with PH detection paper pH value, then it is sterilized by 0.22 μ m strainer again.In disinfectant 300moSM glucose diluted compounds 24 and add aseptic 0.1N NaOH transfer pH value to 7.0(because practical situation are not have buffer reagent to exist in the glucose thinner, and 2, the 5-resorcylic acid is present in the last sample preparation, is 1-2 and cause unadjusted pH value).Aseptic 1.0ml tuberculin syringe (band 25G * 5/8 inch syringe needle) is weighed, with Aseptic technique about 0.1ml Na that packs into
186ReO
4Or compound 24 sample preparations, weigh again to measure preform injection liquid weight.Take out aliquot to measure the cpm/ μ l of each sample preparation.The reservation of above-mentioned sample preparation after the following evaluation intra-articular injection.With ketamine and the heavy female New Zealand white rabbit of xylazine (xylazine) anesthesia 3-4kg, carefully scrape the Mao Bingyong iodine solution and clean the knee district.Use Aseptic technique, with about 120 ° of knee bend, the of short duration Patella that is displaced sideways passes skin and is inserted into the joint space with in the middle of the pin, injects 24(3 animal of about 0.1ml compound) or Na
186ReO
4Syringe needle is extracted in the sample preparation of (2 animals), and Patella returns to the normal position.The compound 24 or the Na of injection
186ReO
4Accurate volume and active by following definite: by the syringe of weighing again, calculate the front and back difference of injection liquid weight, (density of supposing injection liquid is 1.0g/ml) and be that the measurement volumes of μ l multiply by cpm/ μ l value with unit, this value is to be obtained by the aliquot of calculating each sample preparation known volume.After the intra-articular injection within 5-10 minute therefrom the auricle artery get the blood (approximately 1ml) of known volume.Then animal is placed in the cage, this cage can be collected urine and ight soil respectively and monitor 6 days.Get blood every day and collect excretory urine in preceding 24 hours, the aliquot blood and the urine of known volume are measured cpm/ml with Packard Cobra Model 5003 gamma counters.Calculate the total blood cell number that produces based on the blood volume of TBW 5.5% and the cpm/ml of blood; Cpm/ml based on 24 hours urine volumes and urine calculates total urine number.In ight soil, do not detect significant figure.Use GE Starcam300 system to carry out the γ spark photograph at the 0th, 1,4 and 6 day with about 120-150kev energy window.At the 6th day, after collection blood and the urine sample, kill animals was also collected each organ, weighed, and was to estimate
186The bio distribution of Re is measured cpm/gm.
Compound 24 and Na during 6 days after the injection
186ReO
4Cycle values in blood and the excretion in urine are shown in the table VI.In the 6th day selected organs
186The distribution of ReO is shown in the table VII.All results in table VI and VII are expressed as the percentage of the injection volume behind correction for attenuation.Data in table VI and the VII show, by using radionuclide to significantly reduce with the form of compound 24
186The degree that Re leaks from the intraarticular gap (as by blood value, drainage urine and cumulative measurement in other organ).As show as shown in the VIII, by γ spark photograph imaging measurement in the particular studies district amount of (knee and/or integral body) can further calculate reservation at knee.These results also show, when active nucleus is when using with the form of compound 24, have improved reservation widely.
After the intra-articular injection of table VI
186The percentage of the value injection volume of Re in blood and urine
| Injection back fate | In blood | In urine |
| 01234560 to 6 | Na 186ReO 4 186Re-Cpd.24 121?±?5 0.35?±?.06 .048?±.020 0.093±.047 .006±.009 0.12±.039 .004±.006 0.21±.022 .006±.008 0.12?±?.023 0?±?0 0.25?±?0.13 .002±.002 0.19±?0.071 NA NA | Na 186ReO 4 186Re-Cpd.24 NA 2 NA 75?±?6 3.1?±?1.7 9.9±.15 2.2?±?0.31 .67±.36 2.1?±?0.51 .70±.12 1.6?±?0.50 .17±.07 2.5?±?0.63 .28±.22 2.6?±?0.79 ; 87?±?5 14?±?2.0 |
As above-mentioned routine 3h in prepared,
2.HA=it is inapplicable
The table VII
After the intra-articular injection
186Re was 6 days bio distribution
The percentage of organ injection volume
Na
186ReO
4 186Re-Cpd.24
Liver .0005 ± .0007 0.16 ± .073
Blood .0015 ± .0021 .190 ± .070
1. the summation of lung, the heart, spleen, gall-bladder, Tiroidina and lymphatic node
The table VIII
After the intra-articular injection
186Re is in the reservation of knee
After the injection
Time is present in the integral part amount in the knee
1Be retained in the amount in the knee on the 0th day
1
Na
186ReO
4 186Re-Cpd.24 Na
186ReO
4 186Re-Cpd.24
30 minutes 11% 96 ± 2% 100% 100 ± 0%
1 day＜5% 103 ± 3%＜5% 98 ± 1%
4 days＜5% 108 ± 2%＜5% 83 ± 11%
6 days＜5% 114 ± 4%＜5% 68 ± 2%
1. correction for attenuation value,＜5% do not add up be different from 0 poor
Example 11
The synovia of lipophilic cyanine conjugate distributes behind the joint injection
Add up blended homogeneity and ununiformity by synovial fluid cell after the intra-articular injection, 10 μ M injection of solution of salt compounded of iodine example 2 on will seeing at the 0.1ml compound T(in the glucose of 300mosM PH7.0) are in the knee described in the example 10, after 1 hour, kill animals, the knee joint of dissecting injection and not injecting.The cross section sample preparation in whole joint is disabled, because demineralized requires to be cut into thin section and the demineralized solvent has been removed The compounds of this invention from cell tissue.Therefore, fat pad under the careful Chu Qu Patellar (under knee and form a Synovial cavity border), a freezing fritter in dry ice, and-80 ℃ of storages up to preparing freezing microtome section.Prepare thick (10 μ) cryostat section, adhere on the sheet glass, and by light and fluorescent microscope evaluation.
The light microgram of fat pad cross section (injection or the knee of not injecting) shows that (when when amplifying 100 times of observations) synovial fluid cell touches off the surface of fat pad in the face of the joint space with the thin black line along the cross section inner edge.(the synovia layer only is a 1-2 cells thick in general joint, but owing to too high hyperplasia in arthritic joint thickening).The remainder of tissue is mainly by the significant quantities of fat cellularity.With the irradiation explanation of blue exciting light by the cross section of injection knee, bright and relative uniform mark mainly is present in the synovial fluid cell layer, and the adipocyte zone is not injected the cross section of knee and only presented many dark green spontaneous fluorescence.
Example 12
The reservation of position lipophilic cyanine conjugate in blood vessel
Carry out application experiment one time, wherein as preparation as described in the above-mentioned example 2
125The lipophilic cyanine (compound 12 of response diagram 2) that I replaces is administered on the surface, chamber of anesthetized rabbit femoral artery, and whether compound of the present invention is retained on the endovascular position to measure.Operation separates femoral artery, and its contiguous side draws PE10 pipe sleeve short, and the 1cm artery part of propping up around side is contained 20-25 μ l by inaccessible
125The solution of I substitution compound 12 is administered to by sleeve pipe and blocks part and allow it keep 5-10 minute.Take out along sleeve pipe
125The compound that I replaces is pinioned side enduringly and is propped up, and the obstruction of removing femoral artery allows blood recover to flow involution thigh otch.Then with the band optimal detection
125The Ludium Model 2200Scaler Ratemeter of I window and Model 44-17 2 inch crystal three weeks thereafter with selected time monitoring
125The I radioactivity.
The graphicanalysis of result data provides a kind of two sections exclusive methods, and remaining in endarterial application after 24 hours is 34.0 ± 10.9%(mean value ± standard error of the mean, n=4) and 0.77 ± 0.29 day initial eliminating transformation period arranged.Yet after 24 hours, the eliminating that keeps material is very slow, and getting rid of the transformation period is 15.24 ± 2.89 days.Therefore, because application position is got rid of slowly in the blood vessel, may detect obvious amount after using at least 3 weeks
125The compound that I replaces.
Example 13
The anti-proliferative activity of colchicine lipophilic cyanine conjugate on external A10 cell
For measuring the anti-proliferative activity of The compounds of this invention, use A10 cell, the original clone clone that is derived from the thoracic aorta of mouse embryo to finish in vitro study, these two kinds of cells are as the sarcoplast hyperplasia and develop into the similar smooth muscle cell of phenotype (B.Kimes and B.Brandt, Exptl.Cell Res 98:349(1976)).Typically, be used in Dulbecco ' s Modified Eagles Medium(DMEM) in 2500-10000A10 cell/cm
2With 10% tire sura serum (FCS) inoculation, 12 cave plates, allow its adhesion and stablized 24 hours at 37 ℃.Then suitable in conjunction with cell medium in desired prepared at concentrations substances, behind the suction coated media, be added in each cave in only during 10 minutes at 37 ℃.The suction process thing is washed the cave 4 times with containing the medium that is untreated then, is placed on during studying then in the medium that is untreated that contains DMEM with 10%FCS.In each time after use handling, it is drawn in one group of three cave, the preservation aspirate, with 0.25ml0.25% trypsin treatment cave to discharge adherent cell.(1.75ml DMEM ± 10%FCS) stops the enzyme effect of trypsinase on cell by adding excess protein, aspirate turns back in the cave, with Coulter Counter(Model ZM with Sampling stand II) aliquot of the cell suspending liquid that generates of metering.The cell count standard is changed into the cell count of every square centimeter of growth area (surface-area at the bottom of the cave).
As shown in the following table IX, after 7 days,, have only 5.2% vehicle to handle and grow up to maximum in the hyperplasia explanation of the cell cultures of handling with The compounds of this invention (routine 3i, the compound 19 of response diagram 3 are referred to as " colchicine conjugate " here).There is the cell (measuring) that effectively connects fluorescence, illustrates that the colchicine conjugate is attached to the persistence on the A10 cell by flow cytometry.On the contrary, the cell of handling with the non-conjugated colchicine of identical initial concentration only exists with half of the cell density of the cell handled with vehicle, and the cell enlargement with compound gained of the present invention on cell quantity is about 10 times.Therefore, have only 10 minutes although expose to the open air, compound of the present invention allows to keep effectively anti-proliferative activity on the parent compound of A10 cell, and this shows and in kind uses non-conjugated colchicine to compare to have much bigger anti-proliferative effect.
Example 14
Antithrombotics lipophilic cyanine conjugate combines with biomembranous
Whether can stably be attached on the microbial film for evaluating anticoagulant compound of the present invention, the rabbit red blood cell ghost of describing with front example 1 (ghosts) carries out the research in glass test tube.Use (1) deutero-not contain the lipophilic cyanine (compound 7 of antithrombotics, reaction scheme 1, example 3a) or (2) antithrombotics lipophilic cyanine conjugate (compound 21, reaction scheme 4, routine 3j) or the heparin (FITC-heparin) of (3) fluorescein isothiocyanate mark respectively with the 10 μ concentration mark 2 * 10 of μ M and 20 μ M M.20
8Individual ghost/ml.Thoroughly wash ghost then, and in the phosphate buffered saline (PBS) that has 1mM EDTA and 5%BSA, placed 24 hours in 37 ℃.Stir sample, the sample aliquot of taking-up 50 μ l is placed in 200 μ l Triton X-100 in the titer plate cave from each sample
In (representing the fluorescence of all suspension), centrifugal original sample 10 minutes, the ghost supernatant liquor of 50 μ l samples is removed, and is placed on 200 μ lTriton X-100
In (expression only do not have fluorescence) at aqueous phase.On fluorometric assay titer plate reader, measure the fluorescence of all samples.Behind the background fluorescence of having revised unmarked ghost, non-blooming by from total, deducting, determine bonded fluorescence.The percentage ratio of no fluorescence and combined with fluorescent is by with combined with fluorescent and do not have fluorescence and multiply by 100 divided by total fluorescence respectively and determine.
Following Table X result displayed illustrates that anti-freezing conjugate of the present invention shows good film retention rate, and 90.94% fluorescence and ghost symphysis connect after 24 hours.By contrast, the The compounds of this invention that does not contain antithrombotics has 94.96% retention rate.The FITC-heparin only shows very poor retention rate, only is 37.63% fluorescence retention rate after 24 hours.This value is likely the overvaluation of the true retention rate of FITC-heparin, because, the only a little higher than background fluorescence level of the fluorescent signal of FITC-heparin on ghost, and in conjunction with percentile calculating and not have these values be insecure on precision.But following data have clearly illustrated that The compounds of this invention is retained on the microbial film well, and the fluorescence heparin can not.
The table X
Fluorescence intensity
Suspension supernatant liquor % does not have the % combination
1439.00 73.69 5.04 94.96
±33.55 ±2.53 ±0.06 ±0.06
Unlabelled 1.68 1.24
191.40 18.36 9.06 90.94
The heparin conjugate
±6.07 ±0.53 ±0.04 ±0.04
Unlabelled 1.64 1.166
0.599 0.517 62.37 37.63
The FITC-heparin
±0.005 ±0.011 ±1.29 ±1.29
Unlabelled 0.463 0.465
* the value of fluorescent chemicals provides with mean value ± standard error of the mean, the n=3 replication.
Example 15
The maintenance of the anti-freezing performance of antithrombotics when being attached to biofilm surface-lipophilic cyanine conjugate
For measure compound of the present invention on covering biofilm surface the time anti-freezing performance whether also keep, in glass test tube, study, with the ability of evaluation The compounds of this invention Trombin inhibiting, thrombus.The red blood cell ghost of rabbit is at first used antithrombotics-lipophilic cyanine conjugate (compound 21, reaction scheme 4, routine 3j) mark of 10 μ M, then with the PBS washing that contains 0.1%BSA 4 times, is diluted to the ghost/ml of various quantity then.Be placed in the titer plate cave by ghost, and observed fluorescence and the fluorescence that is produced by known standard concentration compound are compared, estimate the concentration of the compound 21 on ghost dose known amounts.Then with by Krstenansky and Mao at FEBS Let.211: 10(1987) similar fashion that goes up report is carried out activation analysis, wherein 1: the 10 human plasma diluent of 100 μ l in phosphate buffered saline (PBS) buffer reagent, PBS that 50 μ l contain various concentration mark ghosts or standard depressor of thrombus and 50 μ l contain quantitatively that the PBS of (0.5nM) thrombus is added in the titer plate cave, and the absorption that is recorded in the 405nm place with 60 minutes cycles on spectrophotometric plate reader (Bio-Tek) changes.Sample is divided into three parts, and is average then.Change with respect to the absorption of time data and to be drawn, and carry out measurement in area under curve by computer.The data that the cave of being handled by inhibitor obtains are expressed the inhibition per-cent of the absorption area that work obtains in the presence of not at inhibitor.Obtain data presentation in Fig. 6 by model experiment.3 parametrical nonlinearity numbers reason by heparin () and antithrombotics conjugate of the present invention (⊙) returns 1.03nM and the 21.23nM(that obtains respectively
1.43 * 10
7Ghost/200 μ l) EC
50Value shows, in the time of on being applied to microbial film, compound of the present invention is lower than 20 times as depressor of thrombus than the effectiveness of parent compound.But because analytical sample does not have thrombus to suppress active the supernatant liquor (◇) of the no ghost of a series of dilutions the time, this inhibition activity is the film of all connections.Like this, compound of the present invention still keeps effective anti thrombotic action, even on being attached to microbial film the time.
Below described and for example understood all respects of the present invention, be i.e. the anti-hyperplasia compound of chemotherapy and radiation treatment synthetic and using according to some preferred embodiments.But some other embodiment are tangible to those skilled in the art.For example, other chemotherapies and radiation treatment conjugate can synthesize and be used for the treatment of or estimate the conditioning of various morbid state or pathology.In addition, the chosen point that Compounds and methods for of the present invention also can be used for developing the medicine of other class is carried and is detained, for example: antibacterial agent, antimycotic agent, anti-inflammatory agent.Therefore, the invention is not restricted to special the description and illustrational embodiment, and can under the situation that does not break away from claim scope of the present invention, change or improve.
Claims (99)
1, a kind of material that is used for to medical functions to be arranged is attached to the compound on the class pledge that is contained in biomone, and the general formula of described compound is:
Wherein B represents a kind of biological action partly, and this partly comprises a kind of material that medical functions is arranged; R and R
1Expression is selected from the substituting group of hydrogen, alkyl, alkenyl, alkynyl, alkaryl, aralkyl respectively, and hydrocarbon chain wherein is linear or side chain, and described substituting group is unsubstituted or with the replacement of one or more nonpolar functional groups, R and R at least
1One of comprise a hydrocarbon substituent, the chain length of described hydrocarbon substituent can be given described compound lipoid bonded ability effectively; R
2Represent a compartment, n is 0 or 1; L represents a coupling part, when n=0, at B and R at least
1And provide stable connection between one of R, when n=1, at R
2With R and R at least
1One of between stable connection is provided, condition is when n=0, L is the coupling part of a non-aromatics.
2, compound as claimed in claim 1 comprises a compartment, and this part is suitable for described biological action part covalency is connected to described coupling part.
3, compound as claimed in claim 2, wherein R and R
1One of be alkyl with at least 12 Linear Carbon atoms, R and R
1In the Linear Carbon total atom number be at least 23.
4, compound as claimed in claim 2, wherein R and R
1One of have at least 12 Linear Carbon atoms, R and a R
1In the Linear Carbon total atom number be enough to give described compound at least 90% film retention rate.
5, compound as claimed in claim 2, wherein R and R
1One of have at least 12 Linear Carbon atoms, R and a R
1In the Linear Carbon total atom number be enough to give described compound at least 30% film combination stability.
6, compound as claimed in claim 2, wherein said coupling part are selected from cyanine, acridine, pyridine, quinoline, xanthene, phenoxazine, thiodiphenylamine and diphenyl hexatriene dyestuff and their derivative.
7, compound as claimed in claim 2, wherein R
2Comprise a scissionable bond, and described biological action part is bonded to releasedly on the described compound, under the predetermined condition that causes described scissionable bond fracture, discharges from described compound.
8, compound as claimed in claim 7, wherein said biological action part can be brought into play its predetermined biological action when described compound discharges.
9, compound as claimed in claim 7, wherein said biological action partly when conjugation to and can bring into play its predetermined biological action when breaking away from described compound.
10, compound as claimed in claim 1, wherein said biological action partly comprise a chemotherapy material.
11, as the compound of claim 10, wherein said chemotherapeutic includes a kind of anti-proliferative agent.
12, as the compound of claim 11, wherein said anti-proliferative agent can disturb mutually with the tubulin method.
13, as the compound of claim 11, wherein said anti-proliferative agent is selected from colchicine, Changchun joint alkaloid, taxol and their derivative.
14, as the compound of claim 11, wherein said chemotherapy material includes anti-coagulant.
15, as the compound of claim 14, wherein said anti-coagulant is selected from heparin and derivative thereof.
16, as the compound of claim 14, wherein said antithrombotics is selected from r-hirudin and derivative thereof.
17, compound as claimed in claim 1, wherein said biological action part in fact under physiological condition conjugation to described compound.
18, compound as claimed in claim 1, general formula is:
Wherein B represents the chemotherapy material, R and R
1Be to have from 1 hydrocarbon substituent to about 30 carbon atoms;
R
2The following compartment of expression general formula:
R wherein
3The expression aliphatic hydrocrbon, R
4, R
5, R
6And R
7Be selected from aliphatic series, alicyclic or aromatic hydrocarbon, heterocycle or CH respectively
2C(CO
2H)=CH, Q and Q ', Q ", Q
And Q
Be selected from the functional linkage group of such composition respectively: acid amides, thiocarbamide, hydrazone, acylhydrazone, ketone acetal, acetal, ortho ester, ester, acid anhydride, disulphide, urea, carbamate, imines, amine, ether, carbonic ether, thioether, sulphonamide, phosphinylidyne and amidine key, Q ', Q ", Q
, Q
Also can represent a valence link respectively; Said aliphatic hydrocarbon has from 1 to 12 Linear Carbon atom; Said aromatic hydrocarbons has from 6 to 12 carbon atoms; N, p, q, r, s and t each 0 or 1;
X and X
1Identical or different, and represent O, S, C(CH
3)
2Or Se;
Y represents a linking group, is selected from=CR
8-,=CR
8-CR
8=CR
8-,=CR
8-CR
8=CR
8-CR
8=CR
8-, or=CR
8-CR
8=CR
8-CR
8=CR
8-CR
8=CR
8-, R wherein
8Be selected from H, CH
3, CH
2CH
3, CH
2CH
2CH
3Or CH(CH
3)
2;
Z represents a substituting group, be selected from H, alkyl, OH ,-O-alkyl, COOH, CONH
2, SO
3H, SO
2NH
2, SH, S-alkyl, CONH-alkyl, CON-(alkyl)
2, NH-acyl group, NH-alkyl, N(alkyl)
2, NO
2Halogen, Si(alkyl)
3, the O-Si(alkyl)
3, the Sn(alkyl)
3Or-the Hg-halogen, the alkyl in described Z substituting group has from 1 to 4 carbon atom;
A represents the acceptable negatively charged ion of pharmacology.
19, as the compound of claim 18, comprise a radio isotope, be selected from radioactive hydrogen, carbon, nitrogen, halogen, sulphur, selenium or their mixture.
20, as the compound of claim 18, wherein said chemotherapy material is selected from immunogen, toxin, hormone, enzyme, antigen, antibody and antibody fragment.
21, as the compound of claim 18, general formula is:
22, as the compound of claim 18, general formula is:
23, as the compound of claim 18, general formula is:
24, as the compound of claim 18, general formula is:
25, compound as claimed in claim 2, general formula is:
Wherein B represents the radiotherapy material;
R and R
1Be to have from 1 hydrocarbon substituent to about 30 carbon atoms;
R
2The following compartment of expression general formula:
R wherein
3The expression aliphatic hydrocrbon, R
4, R
5, R
6And R
7Be selected from aliphatic series, alicyclic or aromatic hydrocarbon, heterocycle respectively;
Q and Q ', Q ", Q
And Q
Be selected from the functional linkage group of such composition respectively: acyl acid amides, thiocarbamide, acetal, ester, disulphide, urea, carbamate, amine, ether, thioether, sulphonamide, phosphinylidyne and amidine and key; Q ', Q ", Q
, Q
Also can represent a valence link respectively; Described aliphatic hydrocrbon has from 1 to 12 Linear Carbon atom; Described aromatic hydrocarbon has from 6 to 12 carbon atoms; N, p, q, r, s and t, each is 0 or 1 years old;
X and X
1Can be identical or different, and represent O, S, C(CH
3)
2Or Se;
Y represents a linking group, is selected from=CR
8-,=CR
8-CR
8=CR
8-,=CR
8-CR
8=CR
8-CR
8=CR
8-or=CR
8-CR
8=CR
8-CR
8=CR
8-CR
8=CR
8-, R wherein
8Be selected from H, CH
3, CH
2CH
3, CH
2CH
2CH
3Or CH(CH
3)
2;
Z represents a substituting group, be selected from H, alkyl, OH ,-O-alkyl, COOH, CONH
2, SO
3H, SO
2NH
2, SH, S-alkyl, CONH-alkyl, CON-(alkyl)
2, NH-acyl group, NH-alkyl, N(alkyl)
2, NO
2, halogen, Si(alkyl)
3, the O-Si(alkyl)
3, the Sn(alkyl)
3Or-the Hg-halogen, the alkyl that is included in the described Z substituting group has 1 to 4 carbon atom;
A represents the acceptable negatively charged ion of medicine.
26, as the compound of claim 25, comprise a radio isotope, be selected from radioactive hydrogen, carbon, nitrogen, halogen, sulphur, selenium or their mixture.
27, as the compound of claim 25, wherein said radiation treatment material comprises the sequestrant that cooperates with the radiotherapy nucleic.
28, as the compound of claim 25, general formula is:
Wherein R and R
1Be hydrocarbon substituent with from 1 to 30 carbon atom, X and X
1Identical or different, and represent O, S, C(CH
3)
2Or Se;
A represents the acceptable negatively charged ion of medicine,
M represents the radiation treatment material, is selected from copper, technetium, rhodium, palladium, indium, samarium, gallium, holmium, erbium, ytterbium, lutetium, rhenium, yttrium, gold, erbium, holmium or their mixture, and n is 2,3 or 4; M is 1 or 2, and p is 1 to 6.
29, it is the radiation treatment material of form that compound as claimed in claim 1, wherein said biological action compound comprise with sequestrant and radioactive metal.
30, as the compound of claim 29, comprise one with radioactive metal nitrogenous, that the sulfur-bearing sequestrant cooperates.
31, as the compound of claim 29, comprise one with nitrogenous, contain the radioactive metal that the oxygen sequestrant cooperates.
32, as the compound of claim 29, wherein said radiation treatment material comprises anti-proliferative agent.
33, as the compound of claim 29, comprise a radio isotope, be selected from radiohalogen, copper, yttrium, technetium, rhodium, palladium, indium, samarium, holmium, erbium, ytterbium, lutetium, rhenium, gold or their mixture.
34, as the compound of claim 29, general formula is:
Wherein L, R, R
1And R
2As claim 1 definition, Z represents H or a metal-complexing position;
Wherein each R ' is respectively H atom or alkyl, low alkyl group preferably, or the low alkyl group that replaces, but substituting group ester wherein, R " and R
Be respectively hydrogen atom or alkyl, m and n each 0 or 1; M represents radioactive metal, is selected from rhenium, indium, copper and palladium.
35, as the compound of claim 34, general formula is:
Wherein R and R
1Be to have from 1 hydrocarbon substituent, X and X to about 30 carbon atoms
1Identical or different, and represent O, S, C(CH
3)
2Or Se;
A represents the acceptable negatively charged ion of medicine;
Z represents H or metal-complexing position;
Wherein each R ' is respectively H atom or alkyl, preferably rudimentary low alkyl group, and wherein substituting group can be an ester, R " and R
Be respectively H atom or alkyl, each can be 0 or 1 for m and n; M represents radioactive metal, is selected from rhenium, indium, copper, palladium.
36, as the compound of claim 35, general formula is:
Wherein M is the radiation treatment material, is selected from rhenium, indium, copper, palladium.
37, compound as claimed in claim 1, it further comprises the diagnostic reagent that can detect in vivo.
38, a kind of pharmaceutical preparation comprises a kind of compound as claimed in claim 1 and compatible Biomedia.
39, as the pharmaceutical preparation of claim 38, wherein said compatible Biomedia is selected from salt-free isotonic solution and the acceptable liquid excipient of medicine.
40, as the composition of claim 39, wherein said medium has isotonicity between 260mos and 340mos, and to want the bonded biomone for this compound be isoosmotic.
41, a kind of compound, general formula is:
Wherein R and R
1Identical or different, and representative is selected from the substituting group of H, alkyl, alkenyl, alkynyl, alkaryl, aralkyl respectively, its hydrocarbon chain has from 1 to 30 C atom, and be linearity or side chain, described substituting group is unsubstituted or replaces with one or more nonpolar functional groups;
R
2The following compartment of expression general formula:
R wherein
3Represent an aliphatic hydrocrbon, R
4, R
5, R
6And R
7Be selected from aliphatic series, alicyclic ring or aromatic hydrocarbon or heterocycle or CH respectively
2C(CO
2H)=and CH, Q, Q ', Q " and Q
Be selected from function key group acid amides, thiocarbamide, hydrazone, acyl group hydrazone, ketone acetal, acetal, ortho ester, ester, acid anhydride, disulphide, urea, carbamate, imines, amine, ether, carbonic ether, thioether, sulphonamide, phosphinylidyne and the amidine key of following composition respectively; Q ', Q " and Q
Also can represent a valence link respectively, described aliphatic hydrocrbon has from 1 to 12 Linear Carbon atom, and described aromatic hydrocarbon has from 6 to 12 C atoms; Each can be 0 or 1 for p, q, r, s and t;
X and X
1Can be identical or different, and represent O, S, C(CH
3)
2Or Se;
Y represents a linking group, is selected from=CR
8-,=CR
8-CR
8=CR
8-,=CR
8-CR
8=CR
8-CR
8=CR
8-or=CR
8-CR
8=CR
8-CR
8=CR
8-CR
8=CR
8-, R wherein
8Be selected from H, CH
3, CH
2CH
3, CH
2CH
2CH
3Or CH(CH
3)
2;
Z represents a substituting group, is selected from H, alkyl, OH, O-alkyl, COOH, CONH
2, SO
3H, SO
2NH
2, SH, S-alkyl, CONH-alkyl, CON-(alkyl)
2, NH-acyl group, NH-alkyl, N(alkyl)
2, NO
2, halogen, Si(alkyl)
3, the O-Si(alkyl)
3, the Sn(alkyl)
3Or-the Hg-halogen, be contained in alkyl in the described Z substituting group and have carbon atom from 1-4;
W represents a reactive functional group, is selected from amino, α-Lu Daiyixianan, isothiocyanate, isocyanate, carboxyl, diazanyl, two thiopyridines bases, sulfydryl, aldehyde, acid anhydride, succinimido ester, ketone, halogen, hydroxyl, sulfuryl halide, imido-ester, epoxide, maleimide, hydrazide group (acylhydrazido) and azido-;
A represents negatively charged ion.
42, as the compound of claim 41, comprise a radio isotope, be selected from radioactive hydrogen, carbon, nitrogen, halogen, sulphur, selenium or their mixture.
43, as the compound of claim 41, W-R wherein
2The combination of expression reactive functional group-spacer groups is selected from NH
2-CH
2, SCN-CH
2, SCN-aryl-HNSCHN-CH
2, 4,6-dichlorotriazine base-NH-CH
2, OCN-CH
2, succinimido-OOC-aryl-CONH-CH
2, ICH
2CONH-CH
2, (2-pyridyl)-S-S-(CH
2)
2-CONH-CH
2, HO(CH
2)
3CONHCH
2, HS(CH
2)
3C(NH) NHCH
2, HS(CH
2)
3CONHCH
2Or dimaleoyl imino-CH
2
44, as the compound of claim 41, general formula is:
45, as the compound of claim 41, be coupled on the derivative of vitamin H or vitamin H by described reactive functional group W.
46, as the compound of claim 45, the derivative of wherein said vitamin H is represented by following general formula:
Wherein E represents a kind of remnants of defeated troops of compound, and this compound has can be by described reactive functional group, W, the reactive functional group of replacement, and m=0,1 or 2.
47, the interaction by avidin-vitamin H is coupled on the proteinaceous substances that carries the avidin or derivatives thereof as the biotinylation compound of claim 45.
48, as the compound of claim 45, wherein said reactive functional group W is NH
2, R
2It is alkyl.
49, a kind of biotin derivative, general formula is:
Wherein R and R
1The expression substituting group is selected from H, alkyl, alkaryl, aralkyl, its hydrocarbon chain respectively and has from 1 to 30 C atom, and is linear or side chain, and described substituting group is unsubstituted or with one or more nonpolar functional groups replacements;
X and X
1Can be identical or different, and represent O, S, C(CH
3)
2Or Se;
R
3Represent H, methyl, ethyl, propyl group or sec.-propyl;
M is from 0 to 6 number;
A is a negatively charged ion.
50, as the compound of claim 49, wherein R and R
1One of have at least 12 Linear Carbon atoms, R and a R
1In the Linear Carbon atom add up at least 23.
51, according to the 2-(3-(2 of claim 49,3-dihydro-3, the 3-dimethyl-5-(+)-biotin amido group methyl isophthalic acid-tetradecyl-(2H)-indylidene-2-base (indol-2-yliden)-1-propyl group)-1-docosyl-benzoxazole halogenide.
52, according to the 2-(3-(2 of claim 49,3-dihydro-3,3-dimethyl-5-(6-{ (+)-biotin amido group }-hexanamide ylmethyl)-the 1-tetradecyl-(2H)-indylidene-2-base (indol-2-yliden))-the 1-propyl group)-1-docosyl-benzoxazole halogenide.
53, according to the 2-(3-(2 of claim 49,3-dihydro-3,3-dimethyl-5-(6-(6(+)-and biotin acyl amide group-hexanoyl amido) the hexanamide ylmethyl }-the 1-tetradecyl-(2H)-indylidene-2-base (indol-2-yliden))-the 1-propyl group)-1-docosyl-benzoxazole halogenide.
54, according to the 5-of claim 49 { 6-(6-(+) biotin acyl amide group hexanoyl amido amide ylmethyl }-1 '-docosyl-1-tetradecyl-3,3,3 ', 3 '-tetramethyl-indoles carbocyanine halogenide.
55, a kind of compound, general formula is:
Wherein R and R
1The expression substituting group is selected from H, alkyl, alkenyl, alkynyl, alkaryl or aralkyl respectively, and its hydrocarbon chain has from 1 to 30 C atom, and is linearity or side chain, and described substituting group is unsubstituted or with the replacement of one or more nonpolar functional groups;
X and X
1Identical or different, represent O, S, C(CH
3)
2Or Se;
Y represents a linking group, is selected from=CR
8-,=CR
8-CR
8=CR
8-,=CR
8-CR
8=CR
8-CR
8=CR
8-or=CR
8-CR
8=CR
8-CR
8=CR
8-CR
8=CR
8-, R wherein
8Be selected from H, CH
3, CH
2CH
3CH
2CH
2CH
3Or CH(CH
3)
2
Z and Z
1Identical or different, represent substituting group, be selected from H, OH ,-O-alkyl, NH
2, COOH, CONH
2, SO
3H, SO
2NH
2, SH, S-alkyl, CONH-alkyl, CON-(alkyl)
2, NH-acyl group, NH-alkyl, N(alkyl)
2, the Si(alkyl)
3, the O-Si(alkyl)
3, the Sn(alkyl)
3Or the Hg-halogen, be included in described Z and Z
1Alkyl in the substituting group has from 1 to 4 carbon atom, and condition is Z and Z
1Can not be H entirely;
A represents negatively charged ion.
56, as the compound of claim 55, wherein R or R
1One of have at least 12 Linear Carbon atoms, R and a R
1In the Linear Carbon total atom number be at least 23.
57, as the compound of claim 55, comprise a radio isotope, be selected from radioactivity H, C, N, halogen, S, Se or their mixture.
58, according to the compound of claim 53, general formula is:
59, the conveying vehicle that is used for medical active substance comprises the physiologically acceptable particle with lipoid composition, and the following compound of general formula is coupled on the lipoid composition:
Wherein B represents to comprise the biological action part of medical active substance; R and R
1Represent substituting group, be selected from hydrogen, alkyl, alkenyl, alkynyl, alkaryl or aralkyl respectively, its hydrocarbon chain is linearity or side chain, and described substituting group is unsubstituted or replaces with one or more nonpolar functional groups, at least R and R
1One of comprise a hydrocarbon substituent, the chain length of described hydrocarbon substituent is enough to giving described compound, R in conjunction with the ability of lipoid
2Represent a compartment, n is 0 or 1, and L representative connects partly, when n=0, at B and R and R at least
1One of between stable connection is provided, when n=1, at R
2With R and R at least
1One of between stable connection is provided, condition is when n=0, L is non-aromatics connection portion, what described compound can be stable is attached on the described lipoid composition.
60, as the medical active substance of claim 59 conveying vehicle, wherein R or R
1One of have at least 12 Linear Carbon atoms, R and a R
1In the Linear Carbon atom add up at least 23.
61, as the medical active substance of claim 59 conveying vehicle, wherein said physiologically acceptable particle is alive, and the tissue that contains film of physiological function is arranged.
62, as the medical active substance of claim 60 conveying vehicle, wherein said physiologically acceptable particle is selected from red blood corpuscle, white cell, thrombocyte.
63, as the medical active substance of claim 59 conveying vehicle, wherein said physiologically acceptable particle is a lipoprotein.
64, patient is given in medical active substance administration so that produce the method for the systematic treating effect that prolongs, described method comprises:
(ⅰ) be coupled to the following compound of a kind of general formula on the physiologically acceptable particle:
Wherein B represents a biological action part, and this part comprises medical active substance, R and R
1The expression substituting group is selected from hydrogen, alkyl, alkenyl, alkynyl, alkaryl or aralkyl respectively, and its hydrocarbon chain is linearity or side chain, and described substituting group is unsubstituted or with one or more nonpolar functional groups replacements, R and R at least
1One of comprise a hydrocarbon substituent, the chain length of described hydrocarbon substituent is effectively giving described compound in conjunction with the ability of lipoid.R
2Represent a compartment, n is 0 or 1, and L represents the connection portion, when n=0, at B and R and R at least
1One of between stable connection is provided, when n=1, at R
2With R and R at least
1One of between stable connection is provided, condition is when n=0, L is non-aromatics connection portion, described compound can stable bond to described physiologically acceptable particle, formed medical active delivery vehicle thus,
(ⅱ) described medical active delivery vehicle is incorporated in described patient's the recycle system.
65, as the method for claim 64, wherein R and R
1One of have at least 12 Linear Carbon atoms, R and a R
1In Linear Carbon total atom number altogether at least 23.
66, as the method for claim 65, wherein said physiologically acceptable particle is selected from red blood corpuscle, white cell, thrombocyte.
67, as the method for claim 66, wherein the physiologically acceptable particle that described compound is coupled on it derives from described patient.
68, as the method for claim 64, wherein said physiologically acceptable particle is a lipoprotein.
69, as the method for claim 64, the general formula that wherein is coupled to the described compound on the described physiologically acceptable particle is:
70, the method for medical active substance is used in reconnaissance, and the compound that general formula is following is transported to patient's intravital chosen point, and described select location comprises the physiologically acceptable particle that contains lipoid:
Wherein B represents to comprise the biological action part of medical active substance, R and R
1The expression substituting group is selected from hydrogen, alkyl, alkenyl, alkynyl, alkaryl or aralkyl respectively, and its hydrocarbon chain is linearity or side chain, and described substituting group is not to be substituted or with the replacement of one or more nonpolar functional groups, at least R and R
1One of comprise a hydrocarbon chain, the length of described hydrocarbon chain is effectively giving described compound, R in conjunction with the ability of lipoid
2Represent a compartment, n is 0 or 1, and L represents a connection portion, when n=0 at B and R and R at least
1One of between stable connection is provided, when n=1 at R
2At least R and R
1One of between provide stable and connect.
71, as the method for claim 70, wherein said compound is taken to comprise the pharmaceutical preparation of described compound in a compatible Biomedia.
72, as the method for claim 70, conjugation is to described compound releasedly for wherein said medical active substance, and described compound is carried under the condition that is causing described medical active substance to discharge in described position from the described compound.
73, as the method for claim 72, wherein said medical active substance only when described compound discharges, be only active.
74, in blood vessel, reduce the method for postangioplasty restenosis, comprise will be transported to as the compound of claim 11 stand as described in the blood vessel place of angioplasty, condition is that described thus compound stably is connected with the cell of described blood vessel, and exists with an amount of bringing into play anti-hyperplasia effect on described cell effectively.
75, as the method for claim 74, wherein said compound is carried to finish described angioplasty from used conduit.
76, in blood vessel, reduce the method for postangioplasty restenosis, comprise will be delivered to as the compound of claim 32 stand as described in the blood vessel place of angioplasty, condition is that described thus compound stably is connected with the cell of described blood vessel, and exists with an amount of bringing into play anti-hyperplasia effect on described cell effectively.
77, as the method for claim 76, wherein said compound is carried to finish described angioplasty from used conduit.
78, the method for treatment rheumatic arthritis, comprise and to be delivered to the film place of the synovia in joint as the compound of claim 11, condition is that described thus compound stably is connected with the cell of described Synovial membrane, and exists with an amount of bringing into play anti-hyperplasia effect on described cell effectively.
79, as the method for claim 78, wherein said compound passes through injection delivery.
80, the method for treatment rheumatic arthritis, comprise the film place that the compound of claim 32 is delivered to the synovia in joint, condition is that described thus compound stably is connected with the cell of the film of described synovia, and exists with an amount of bringing into play anti-hyperplasia effect on described cell effectively.
81, as the method for claim 80, wherein said compound passes through injection delivery.
82, reduce the method for tumor cell proliferation, the patient who comprises tumour as described in flowing to as the compound of claim 11, condition is that described thus compound stably is connected with cell in described tumor site, and exists with an amount of bringing into play anti-hyperplasia effect on described cell effectively.
83, as the method for claim 82, wherein said compound is carried to be selected from following method, (ⅰ) be expelled in described patient's the body cavity that has described tumour, (ⅱ) be expelled to the blood vessel neutralization (ⅲ) that blood is fed on the described tumour cell and be injected directly on the entity that comprises described tumour cell.
84, reduce the method for tumor cell proliferation, the compound that comprises claim 32 flows to the patient with described tumour, condition is that described thus compound stably is connected with cell in described tumor site, and exists with an amount of bringing into play anti-hyperplasia effect on described cell effectively.
85, as the method for claim 84, wherein said compound is carried to be selected from following method, (ⅰ) be expelled in described patient's the body cavity that has described tumour, (ⅱ) be expelled to and be supplied to the blood vessel neutralization (ⅲ) on the described tumour cell to be injected directly on the entity that comprises described tumour cell blood.
86, reduce the outgrowth method of ovarian tumor cell, the compound that comprises claim 11 is transported in patient's the body cavity that has described tumour, condition is that described thus compound stably is connected with cell at described tumor cell proliferation place, and exists with an amount of bringing into play anti-hyperplasia effect on described cell effectively.
87, as the method for claim 86, wherein said compound passes through injection delivery.
88, reduce the outgrowth method of ovarian tumor cell, comprise in the body cavity of conveying as the compound of claim 32 tumour as described in the existing of patient, condition is that described thus compound stably is connected with cell at described tumor cell proliferation place, and exists with an amount of bringing into play anti-hyperplasia effect on described cell effectively.
89, as the method for claim 88, wherein said compound passes through injection delivery.
90, treat psoriasic method, comprise on the cell in psoriasis affected part as described in will being transported to as the compound of claim 11, condition is that described thus compound stably is connected with described cell, and exists with an amount of bringing into play anti-hyperplasia effect on described cell effectively.
91, as the method for claim 90, wherein said compound is carried by topical application.
92, treat psoriasic method, comprise on the cell in psoriasis affected part as described in will being transported to as the compound of claim 32, condition is that described thus compound stably is connected with described cell, and exists with an amount of bringing into play anti-hyperplasia effect on described cell effectively.
93, as the method for claim 92, wherein said compound is carried by topical application.
94, chosen point is used the method for anti-proliferative agent, comprise and to be transported on the intravital chosen point of patient as the compound in the claim 7, condition is that anti-proliferative agent discharges from said compound thus, and this release can make described dose at described its anti-proliferative effect of some performance.
95, chosen point is used the method for anti-proliferative agent, comprises to be transported to as the compound in the claim 9 on the intravital chosen point of patient, and condition is that anti-proliferative agent can be at described its anti-proliferative effect of some performance when conjugation is to described compound thus.
96, a kind of compound, general formula is:
Wherein each R ' is respectively hydrogen atom or alkyl, preferably low alkyl group, or the low alkyl group that replaces, and wherein substituting group can be an ester, R " and R
Be respectively hydrogen atom or alkyl, the salt of ligand comprises single salt, for example haloid acid, oxalic acid and tartrate, and each m and n can respectively be 0 or 1.
97, as the compound of claim 96, general formula is as follows:
Wherein R and R
1Have from 1 hydrocarbon substituent, X and X to about 30 carbon atoms
1Identical or different, and represent O, S, C(CH
3)
2Or Se;
A represents the acceptable negatively charged ion of medicine;
Z represents H or unsettled thiol protecting group;
Wherein each R ' is respectively hydrogen atom or alkyl, the low alkyl group of low alkyl group or replacement preferably, and wherein substituting group can be an ester, R " and R
Be respectively hydrogen atom or alkyl, the salt of ligand comprises single salt for example haloid acid, oxalic acid and tartrate.And m and n can respectively be 0 or 1.
98, as the compound of claim 97, general formula is:
99, a kind of compound, general formula is:
Wherein X represents oxygen or sulphur.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US79893691A | 1991-11-27 | 1991-11-27 | |
| US798,936 | 1991-11-27 | ||
| US884,432 | 1992-05-15 | ||
| US07/884,432 US5667764A (en) | 1988-05-02 | 1992-05-15 | Compounds, compositions and methods for binding bio-affecting substances to surface membranes of bio-particles |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1074911A true CN1074911A (en) | 1993-08-04 |
Family
ID=27122052
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN92115176A Pending CN1074911A (en) | 1991-11-27 | 1992-11-26 | The material of biological action is bonded to compound, composition and method on the biomone facial mask |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0643706A1 (en) |
| JP (2) | JP3682974B2 (en) |
| CN (1) | CN1074911A (en) |
| AU (2) | AU3221993A (en) |
| CA (1) | CA2124329C (en) |
| IL (1) | IL103874A0 (en) |
| MX (1) | MX9206844A (en) |
| NZ (1) | NZ245271A (en) |
| PH (1) | PH31676A (en) |
| WO (1) | WO1993011120A1 (en) |
Cited By (1)
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|---|---|---|---|---|
| CN105745266A (en) * | 2013-11-26 | 2016-07-06 | 住友化学株式会社 | Rubber composition and vulcanization aid |
Families Citing this family (37)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69435342D1 (en) * | 1993-07-19 | 2011-05-05 | Angiotech Pharm Inc | Anti-angiogenic agents and methods of use |
| EP1118325B2 (en) * | 1993-07-29 | 2010-01-06 | The United States of America, represented by the Secretary, Department of Health and Human Services | Use of Paclitaxel and its derivatives in the manufacture of a medicament for treating restenosis. |
| US5480901A (en) * | 1994-10-07 | 1996-01-02 | Zynaxis, Inc. | Method for reducing unwanted cellular adhesions |
| US6492332B1 (en) | 1995-12-12 | 2002-12-10 | Omeros Corporation | Irrigation solution and methods for inhibition of tumor cell adhesion, pain and inflammation |
| US6004536A (en) * | 1995-11-14 | 1999-12-21 | Molecular Probes, Inc. | Lipophilic cyanine dyes with enchanced aqueous solubilty |
| WO1997024459A1 (en) * | 1995-12-29 | 1997-07-10 | Phanos Technologoes, Inc. | Method for reducing unwanted cellular adhesions |
| US5871436A (en) * | 1996-07-19 | 1999-02-16 | Advanced Cardiovascular Systems, Inc. | Radiation therapy method and device |
| US6495579B1 (en) | 1996-12-02 | 2002-12-17 | Angiotech Pharmaceuticals, Inc. | Method for treating multiple sclerosis |
| US6515016B2 (en) | 1996-12-02 | 2003-02-04 | Angiotech Pharmaceuticals, Inc. | Composition and methods of paclitaxel for treating psoriasis |
| JP2001521503A (en) * | 1997-03-31 | 2001-11-06 | ネオルックス コーポレイション | Therapeutic inhibitors of vascular smooth muscle cells |
| US6241762B1 (en) | 1998-03-30 | 2001-06-05 | Conor Medsystems, Inc. | Expandable medical device with ductile hinges |
| US20040254635A1 (en) | 1998-03-30 | 2004-12-16 | Shanley John F. | Expandable medical device for delivery of beneficial agent |
| US7208010B2 (en) | 2000-10-16 | 2007-04-24 | Conor Medsystems, Inc. | Expandable medical device for delivery of beneficial agent |
| US7045305B1 (en) * | 1998-04-08 | 2006-05-16 | The Regents Of The University Of California | Methods and reagents for targeting organic compounds to selected cellular locations |
| US6120847A (en) * | 1999-01-08 | 2000-09-19 | Scimed Life Systems, Inc. | Surface treatment method for stent coating |
| US6333347B1 (en) | 1999-01-29 | 2001-12-25 | Angiotech Pharmaceuticals & Advanced Research Tech | Intrapericardial delivery of anti-microtubule agents |
| US6419692B1 (en) | 1999-02-03 | 2002-07-16 | Scimed Life Systems, Inc. | Surface protection method for stents and balloon catheters for drug delivery |
| US6156373A (en) | 1999-05-03 | 2000-12-05 | Scimed Life Systems, Inc. | Medical device coating methods and devices |
| CA2387477A1 (en) * | 1999-08-23 | 2001-03-01 | Angiogene Inc. | Radioactively coated device and method of making same for preventing restenosis |
| CO5261573A1 (en) | 1999-11-19 | 2003-03-31 | Novartis Ag | DERIVATIVES OF BENZOXA AND BEZOTIAZOL, COMPOUND AND PHARMACEUTICAL COMPOSITION CONTAINING THEM AND PROCESS FOR THE PREPARATION OF THE MENTIONED COMPOSITION |
| IT1319168B1 (en) * | 2000-03-17 | 2003-09-26 | Indena Spa | ANTI-CANCER CONDENSATION DERIVATIVES, THEIR METHOD OF PREPARATION AND FORMULATIONS CONTAINING THEM. |
| ES2243556T3 (en) | 2000-10-16 | 2005-12-01 | Conor Medsystems, Inc. | EXPANDABLE MEDICAL DEVICE TO PROVIDE A BENEFICIAL AGENT. |
| DE10115740A1 (en) | 2001-03-26 | 2002-10-02 | Ulrich Speck | Preparation for restenosis prophylaxis |
| DE10244847A1 (en) | 2002-09-20 | 2004-04-01 | Ulrich Prof. Dr. Speck | Medical device for drug delivery |
| US7776529B2 (en) | 2003-12-05 | 2010-08-17 | Life Technologies Corporation | Methine-substituted cyanine dye compounds |
| AU2005307195A1 (en) | 2004-11-22 | 2006-05-26 | Ge Healthcare As | Contrast agents to target extracellular matrix |
| US9050393B2 (en) | 2005-02-08 | 2015-06-09 | Bruce N. Saffran | Medical devices and methods for modulation of physiology using device-based surface chemistry |
| JP5306811B2 (en) | 2005-05-11 | 2013-10-02 | ライフ テクノロジーズ コーポレーション | Fluorescent chemicals with high selectivity to double-stranded DNA and their use |
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| US9198968B2 (en) | 2008-09-15 | 2015-12-01 | The Spectranetics Corporation | Local delivery of water-soluble or water-insoluble therapeutic agents to the surface of body lumens |
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| US10525171B2 (en) | 2014-01-24 | 2020-01-07 | The Spectranetics Corporation | Coatings for medical devices |
| EP3101012A1 (en) | 2015-06-04 | 2016-12-07 | Bayer Pharma Aktiengesellschaft | New gadolinium chelate compounds for use in magnetic resonance imaging |
| CN110035996B (en) | 2016-11-28 | 2022-08-09 | 拜耳医药股份公司 | Novel highly relaxant gadolinium chelates for magnetic resonance imaging |
| KR20210095168A (en) | 2018-11-23 | 2021-07-30 | 바이엘 악티엔게젤샤프트 | Formulation of Contrast Media and Method for Preparing Same |
| JP7423888B2 (en) * | 2019-11-22 | 2024-01-30 | 株式会社同仁化学研究所 | Dye for staining lipid bilayer membranes and method for staining lipid bilayer membranes using the same |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1523965A (en) * | 1976-03-19 | 1978-09-06 | Ici Ltd | Pharmaceutical compositions containing steroids |
| EP0047470B1 (en) * | 1980-09-02 | 1985-04-24 | Fuji Photo Film Co., Ltd. | Method for immunochemical measurement |
| US4501728A (en) * | 1983-01-06 | 1985-02-26 | Technology Unlimited, Inc. | Masking of liposomes from RES recognition |
| US4751219A (en) * | 1985-02-05 | 1988-06-14 | Nederlandse Centrale Organisatie Voor Toegepast-Natuur-Wetenschappelijk Onderzoek | Synthetic glycolipides, a process for the preparation thereof and several uses for these synthetic glycolipides |
| US4783401A (en) * | 1986-10-31 | 1988-11-08 | Smithkline Beckman Corporation | Viable cell labelling |
| US4762701A (en) * | 1986-10-31 | 1988-08-09 | Smithkline Beckman Corporation | In vivo cellular tracking |
| US4906749A (en) * | 1987-06-30 | 1990-03-06 | Viomedics Inc. | Cyclic anhydride derivatives of chromophors |
| JPH03502327A (en) * | 1988-01-19 | 1991-05-30 | ボード・オブ・リージェンツ，ザ・ユニバーシティ・オブ・テキサス・システム | Ester form of 3'-diaminodoxorubicin, its liposome composition, and its use |
| EP0430968B1 (en) * | 1988-05-02 | 1996-11-20 | PHANOS TECHNOLOGIES, Inc. | Compounds, compositions and method for binding bio-affecting substances to surface membranes of bio-particles |
| US5149794A (en) * | 1990-11-01 | 1992-09-22 | State Of Oregon | Covalent lipid-drug conjugates for drug targeting |
-
1992
- 1992-11-24 AU AU32219/93A patent/AU3221993A/en not_active Abandoned
- 1992-11-24 JP JP51019093A patent/JP3682974B2/en not_active Expired - Lifetime
- 1992-11-24 CA CA002124329A patent/CA2124329C/en not_active Expired - Lifetime
- 1992-11-24 WO PCT/US1992/010076 patent/WO1993011120A1/en not_active Application Discontinuation
- 1992-11-24 EP EP93900600A patent/EP0643706A1/en not_active Withdrawn
- 1992-11-25 NZ NZ245271A patent/NZ245271A/en unknown
- 1992-11-25 IL IL103874A patent/IL103874A0/en unknown
- 1992-11-26 PH PH45327A patent/PH31676A/en unknown
- 1992-11-26 CN CN92115176A patent/CN1074911A/en active Pending
- 1992-11-27 MX MX9206844A patent/MX9206844A/en unknown
-
1997
- 1997-04-30 AU AU19914/97A patent/AU1991497A/en not_active Abandoned
-
2002
- 2002-07-01 JP JP2002192417A patent/JP4095847B2/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105745266A (en) * | 2013-11-26 | 2016-07-06 | 住友化学株式会社 | Rubber composition and vulcanization aid |
| TWI637992B (en) * | 2013-11-26 | 2018-10-11 | 住友化學股份有限公司 | Rubber composition and vulcanization aid |
| US10287419B2 (en) | 2013-11-26 | 2019-05-14 | Sumitomo Chemical Company, Limited | Rubber composition and vulcanization aid |
Also Published As
| Publication number | Publication date |
|---|---|
| IL103874A0 (en) | 1993-04-04 |
| CA2124329A1 (en) | 1993-06-10 |
| AU3221993A (en) | 1993-06-28 |
| NZ245271A (en) | 1996-03-26 |
| JP3682974B2 (en) | 2005-08-17 |
| EP0643706A4 (en) | 1994-12-27 |
| WO1993011120A1 (en) | 1993-06-10 |
| JPH08502719A (en) | 1996-03-26 |
| PH31676A (en) | 1999-01-18 |
| AU1991497A (en) | 1997-07-10 |
| JP4095847B2 (en) | 2008-06-04 |
| MX9206844A (en) | 1993-07-01 |
| EP0643706A1 (en) | 1995-03-22 |
| JP2003026657A (en) | 2003-01-29 |
| CA2124329C (en) | 2008-11-18 |
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