JP2003026657A - Compound for binding bio-active substance on bio-particle surface membrane, composition and method of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a compound for binding a bio-active substance such as a treating medicine or optionally a diagnostic agent with bio-compatible particles including living cells or viruses, and not-living carrier particles, a composition and a method of the same. SOLUTION: This compound for binding a therapeutically effective substance with lipid-containing bio-particles is characterized by formula (wherein, B is selected from a group consisting of a peptide, an enzyme and an antibody having CO and/or COOH functional groups and capable of binding with a spacer part; R, R1 are each a hydrocarbon substituting group equipped with 1-30 carbon atoms; R2 is a spacer component; X, X1 are same or different and each O, S, C(CH3 )2 or Se; Y is a bonding group selected from =CR8 -, and =CR8 -CR8 = CR8 -, Z is a substituting group selected from H, an alkyl, and OH; the alkyl group constituted by the above Z substituting group has a 1-4C; and further, A is a pharmaceutically permissible anion).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to the binding of bioactive agents, such as therapeutic agents, and optionally diagnostic agents, to biocompatible particles including viable cells and viruses, non-viable carrier particles. Of the compounds, compositions, and methods of. The invention also relates to the starting materials and intermediates used to prepare such compounds. The present invention further provides therapeutic agents, and optionally diagnostic agents, in vivo.
It relates to the use of compounds for site-selective delivery at o.

[0002]

The provision of therapeutic agents for the treatment of diseases and other pathological conditions can be accomplished by a variety of methods. They include oral, intravenous, subcutaneous, transdermal, intramuscular, or topical application. In the case of some therapeutic agents, the current delivery method is to provide a sufficient amount to the disease site without causing systemic side effects, or to treat the disease site for a sufficient period of time to produce the intended therapeutic effect. It is not possible to hold the substance well. Drugs that prevent or reduce the growth of pathological cell types are essential for the treatment and control of various diseases involving harmful or uncontrolled cell growth. However, antiproliferative substances, by definition, are toxic to certain cells. Systemic administration of these drugs is often not feasible because the dose required to control the diseased cell type can be toxic or lethal to normal cells in the patient. This difficulty can be avoided by administering the antiproliferative substance directly to the site of growth of harmful cells. Also,
In order to effectively control the growth of harmful cells while limiting migration and damage of normal cell types, a mechanism is needed to retain the antiproliferative drug at the site of disease. Specific diseases and conditions in which site-specific delivery and retention of antiproliferative substances are particularly effective are briefly described below. Each of these states
Including the particularly harmful proliferation of cell types, systemic therapeutic drug therapy for treatment has not yielded optimal results.

1. Reocclusion and Restenosis After Angioplasty Atherosclerotic lesions that limit or occlude coronary blood flow are the leading cause of death associated with coronary heart disease. Percutaneous transluminal coronary angioplasty (PTCA) or direct surgery with coronary artery bypass grafting has been used.
A major drawback of PTCA is the problem of vessel closure after angioplasty, which occurs both shortly after PTCA (acute reocclusion) and within a long period of time (restenosis). Restenosis after angioplasty is a reaction to the damage to the lining of the artery caused by angioplasty procedures. The exact mechanism is still under investigation, but generally involves the proliferation of smooth muscle cells in the intermediate layer of the artery and the migration of these cells towards the inner (intimal) layer where the cells continue to proliferate. Proliferation usually stops within the intima within 7-14 days after injury. Patients with symptomatic re-occlusion require a second PTCA or coronary artery bypass graft. A high proportion of patients who receive PTCA (30-50%) experience restenosis, which clearly limits the success of PTCA as a treatment for coronary artery disease. Attempts to prevent restenosis by pharmacological approaches usually require systemic administration of various drugs and are generally unsuccessful.

Some antiproliferative agents that are under active investigation for the prevention of restenosis are described in detail below. a. Heparin and heparin fragments Heparin is a highly anionic, heterogeneous glycosaminoglycan composed of extensively sulfated repeating disaccharide units of α-D-glucuronic acid and N-acetyl-D-glucosamine. . The primary therapeutic use of heparin is as an anticoagulant. However, heparin is also known to inhibit the growth of several different cell types, including vascular smooth muscle cells (SMC). Heparin fragments as small as tetrasaccharides have weak anticoagulant activity or no anticoagulant activity, but in vitro and in vivo
It has been found to have an antiproliferative effect on o. C
astellot et al. Cell Biol. 10,
2: 1979-1984 (1986). Heparin binds to the cell surface of SMC via a high affinity binding site and is taken up intracellularly. In addition, heparin can also bind to various artificial surfaces such as ionic bonded polypropylene with tridodecylmethylammonium chloride coating. Grode et al., Trans. Am. Soc. Art
if. Int. See Organs, 15: 1 (1969). There are no data on whether the antiproliferative effects of heparin are retained when bound to these substances.

B. Colchicine Colchicine is a naturally occurring alkaloid used in the therapeutic management of acute gouty arthritis. Colchicine
By interfering with mitotic spindle fiber formation and arresting cell division at metaphase, in vitro and in
Inhibits plant and animal cell division in vivo. The action of colchicine is similar to that of vinca alkaloids, vincristine and vinblastine. Daily administration of colchicine to rabbits with atherosclerotic iliac arteries has recently been reported to reduce the extent of restenosis observed on angiography 4 weeks after balloon injury.
Currier et al., Circulation, 80, 1
1-66 (1989). But in recent clinical studies,
Colchicine at 1 mg / day failed to reduce the incidence of restenosis in patients undergoing balloon angioplasty. C. Grines et al., Circulation, 8
4: II-365 (1991). Patients cannot tolerate doses above 1 mg / day due to toxicity limitations, but this dose results in human blood colchicine concentrations 10 to 20 times lower than in the rabbit study Is estimated.

C. Other Drugs Other drugs used in animal models that reduced thickening of the intimal muscle are as follows: (1) Angiotensin converting enzyme (ACE) inhibitor (J. Powell et al.
Science, 245: 186-188 (198
9); (2) Angiopeptin (C. Lunde
rgan et al., Am. J. Cardiol. , 17 (Su
ppl. B): 132B to 136B (1991)); C
yclosporin A (L. Jonasson et al.,
Proc. Natl. Acad. Sci. , 85:23
03-06 (1988)); (4) Goat anti-rabbit platelet-derived growth factor antibody (G. Ferns et al., Science).
e, 253: 1129 to 1132 (1991));
(5) Terbinafine (G. Nemecek,
J. Pharmacol. Exp. Thera. , 24
8: 1167-1174 (1989)); (6) Tra.
pidil (M. Liu et al., Circulation,
81: 1089-1093 (1990); and
(7) Interferon-gamma (G. Hansson
Et al., Circulation, 84: 1266-72.
(1991)). The use of systemic drug delivery, such as oral administration, to treat restenosis after angioplasty requires periodic administration at regular intervals, resulting in periodic fluctuations in therapeutic drug concentration at the disease site. Occurs. Moreover, over the 20-30 day period in which patients have to receive systemic administration of the drug, usually 99% or more of the ingested drug is processed by the drug in the liver or kidneys. This represents a possible serious side effect.

2. Rheumatoid arthritis Rheumatoid arthritis is a chronic disease characterized by chronic synovitis of the joints. Although there is currently no cure for rheumatoid arthritis, one of the accepted treatments is synovectomy with removal of inflamed soft tissue in the affected joint. N. Gschwen
d, Textbook of Rheumatolog
y, (eds. W. N. Kelly et al.), W. B. Sa
unders, pp. 1934-1961 (198
9). Synovectomy can be accomplished surgically, chemically, radiopharmaceutically. Surgical synovectomy has been shown to have a palliative effect on pain. However, in most cases, synovitis recurs within a few years after surgery. Moreover, surgical synovectomy can only be performed once for each joint, which is difficult for relatively small joints. Chemical synovectomy has been shown to be an effective alternative to surgical synovectomy, but its use is limited by the toxicity of drugs currently available for cartilage and bone. Radioisotopic synovectomy as an alternative to chemical synovectomy appears to suppress synovial proliferation. However, to the best of our knowledge, the currently used delivery systems require prolonged immobilization of the affected joints and systemic irradiation of the lymph nodes, spleen and liver with radioisotopes. The use of synovial ablation with radioisotopes is limited because of the need to use isotopes that are short-lived and difficult to market to reduce tolerances. Leakage of radioisotopes from sites of synovectomy is a primary consideration in the development of this method for the initial treatment of rheumatoid arthritis.

3. Ovarian Cancer Ovarian cancer is the fourth leading cause of cancer deaths in women.
Epithelial cancer accounts for approximately 80% to 90% of malignant diseases of the ovary. Epithelial cancer initially spreads to the surface or spreads lymphatically and spreads throughout the body. The most common type of spread outside the ovary is transperitoneal dissemination of cells spread from the surface of the primary tumor. The primary treatment of ovarian cancer is rarely cured, usually because cancer cells enter the peritoneal cavity early in the disease and cannot be surgically removed. External beam radiation therapy is also often used before and after surgery, but the single irradiation value is limited to the irradiation limit value of the abdominal organs (liver, kidney, stomach, intestine). Intraperitoneal instillation of radioactive colloidal gold or phosphorus to irradiate the peritoneal cavity has been used in the past. However, its use is declining due to uneven distribution of radiation and complications in the small intestine. Monoclonal antibodies have recently been tested as excipients for radioisotopes delivered intraperitoneally. To date, there are no approved monoclonal antibody conjugates for the treatment of ovarian cancer. Chemotherapy is the most common form of treatment for patients with advanced ovarian cancer. Drugs currently used against ovarian cancer can extend life expectancy by years. However, they show significant side effects at the recommended systemic doses.

4. Psoriasis Psoriasis is a common chronic skin disease that develops into uncontrolled growth of cutaneous keratinocytes, forming inflammation and ulcers. To date, there is no comprehensive cure for psoriasis. Current treatments for psoriasis include both systemic and topical treatments. Although both types of treatment are effective, they can cause significant side effects. Systemic treatment of psoriasis mainly involves the administration of corticosteroids and cytotoxic compounds such as methotrexate. Topical treatments include antibacterial or antifungal preparations, wood tar, exposure to sunlight or phototherapy with UV light, or application of topical steroids. Topical corticosteroids are more commonly used for psoriasis than any other physical treatment. However, conventional topical treatments suffer from the drawback of being easily scraped or washed away, impairing their long-term effectiveness. Moreover, topical use of corticosteroids tends to cause deleterious systemic accumulation by penetrating the peripheral blood vessels and from there to the systemic circulation, limiting their use. The administration of drug therapy that allows a higher concentration of the therapeutic agent to be maintained at the disease site for a long time without causing serious side effects has proven to be effective in treating the above-mentioned conditions. Ah

[0010]

Recent research efforts have focused on the use of specific biological interactions, such as receptor-ligand or antigen-antibody interactions, in the development of target-specific therapeutic or diagnostic agents. It was Other promising areas of research include target-specific cells, or vesicles (eg liposomes) containing suitable diagnostic and therapeutic agents. In particular, monoclonal antibodies have been used to provide target specificity to these cells or vesicles. Because this is a relatively new technology, such targeted drug therapy is not truly effective and reliable. In International Application No. PCT / US89 / 00087,
Various compounds, compositions, methods for their preparation, and binding of bioactive substances to the surface membrane of bioparticles such as cells or viruses without producing appreciable damaging effects on cell morphology or physiological function. The use of is described. The compound has the general formula R-B-R1, where B represents a bioactive substance, such as a therapeutic or diagnostic agent, and at least one of R and R1 provides the compound with a degree of lipophilicity to provide bioactivity. A hydrocarbon substituent selected to allow stable bonding of the particles to the surface membrane. Compositions containing the compounds described herein are constituted by suitable attachment means for stable attachment between the compound and the outer surface membrane of the bioparticle. These compositions are
In order to exert a predetermined site-specific effect in vivo by stably binding a compound to a selected bioparticle having an original or acquired affinity for the predetermined site,
By introducing the bioparticles in vivo, the bioparticles are used to carry the bioactive substance to a predetermined site so as to produce its intended effect.

[0011]

SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided a compound having the ability to attach therapeutically effective substances to lipid-containing bioparticles such as cells or viruses. The compound of the present invention can be added to a lipid component of a biocompatible particle containing a lipid in vivo or in vitro by making the compound sufficiently non-polar.
At least one hydrocarbon substituent selected for stable binding to either or both of a bioactive moiety comprised of a therapeutically active substance that is stably bound by a binding moiety . The compound optionally includes a spacer moiety that mediates a therapeutic effect that separates between the therapeutic agent and the binding moiety. Furthermore, the compounds of the present invention are characterized by having such a stable, but predictable, binding to the lipid component of the biomembrane. The compound has a surface film retention coefficient (MRC) of at least 90% and at least 3
It is sufficiently non-polar to provide 0% membrane binding stability. Furthermore, the compounds of the present invention are sufficient for use such that once the therapeutic agent is delivered directly to the site of choice, either by direct administration or by carrier, it remains there for the time being and in sufficient quantity to produce its intended effect. Need to be stable. Methods for determining membrane retention factor and membrane binding stability are described below.

The compounds described above according to the invention are used for the selective delivery of therapeutic agents and their retention at selected sites. According to a preferred aspect of the present invention, the therapeutic agent has an antiproliferative effect effective for the treatment of diseases involving cell proliferation or other pathological conditions. The antiproliferative drug can be composed of a radiotherapy substance or a chemotherapeutic substance. According to a preferred embodiment, the invention provides a compound in which the radiotherapeutic substance is composed of a chelating agent and a radioactive metal. In another preferred embodiment, the compound of the invention is
It is composed of chemotherapeutic substances such as heparin, hirudin and its derivatives, in addition to substances capable of interfering with the intracellular function of choice. According to another aspect of the invention, a chemotherapeutic agent can be releasably conjugated to a compound of the invention. These chemotherapeutic substances are, for example,
It can be selected from the group consisting of colchicine, vinca alkaloids, taxol and its derivatives, which exhibit their bio-effect only when released from the compound. These compounds interfere with tubulin synthetic polymerization, depolymerization, or decomposition and / or function, hereinafter referred to as the "tubulin" process. For example, acid cleaved colchicine derivatives are provided, to which colchicine analogs have been linked that are essentially inactive during conjugation to the binding moiety. The compound is supplied to the selected site and is finally taken up into the cell. Compound uptake is achieved by reducing the pH, which cleaves the colchicine component from the binding component. The released colchicine derivative can exert its intended biological effect of interfering with the tubulin process.

According to yet another aspect of the invention there is provided a pharmaceutical formulation composed of a compound of the invention which is compatible with biological media. According to another aspect of the invention,
Methods of using the various compounds of the present invention are provided that are comprised of therapeutically effective substances for use in the treatment of diseases or other pathological conditions, and optionally also include diagnostic agents. The compounds of the invention are preferably administered in vivo to retain them at the site of disease.
Administered directly by ivo supply. Alternatively, the compound can be bound to carrier particles that deliver the compound to the disease site. Direct in vivo delivery is particularly preferred for the treatment of conditions such as restenosis after angioplasty, chronic indirect rheumatism, ovarian cancer, psoriasis and is described in more detail below.

The present invention provides a number of distinct advantages over the compounds and methods currently available for delivering therapeutic agents to the site of disease. In particular, the compound of the present invention can be supplied to and retained at a selected site in the body by stably binding to the cellular structure of the site. The existing delivery methods are to provide a sufficient dose to the disease site without systemic side effects, or to hold the therapeutic substance at the disease site for a while to produce the desired therapeutic effect. It is impossible to hold. The compound of the present invention can achieve a therapeutically effective dose at the site of disease. For example, in the case of radiotherapy where the systemic distribution of radioisotopes is very detrimental, the compounds of the present invention allow stable binding and retention of the radiotherapeutic agent at the site of need, resulting in joint immobilization and extreme Suppress the systemic distribution of isotopes without the need for short-lived isotopes. Another significant advantage of the present invention is that the compounds can be adjusted to initially bind stably to the outer cell membrane and then be taken up intracellularly via the normal transmembrane process. This feature, together with the ability to tailor the compounds of the invention to be composed of releasably conjugated therapeutic agents,
Allows the delivery of potentially toxic substances into selected cells, where they can be activated to exert their therapeutic effect only on those cells and not on other cells in the body Is. This method includes antisense R
A supply of NA or DNA can also be included. Yet another advantage of the present invention is that the binding of the compounds described herein to cells and other biocompatible particles occurs essentially by lipophilicity. This is particularly important in the case of cells, since the cell membrane resides in individual protein moieties and not in the larger lipid moieties, reducing the chance of lipid binding interfering with important functional regions of the cell membrane. Is. Conventional methods that rely on binding of membrane proteins to cell receptors to deliver bioactive agents to cells often result in diminished functional capacity.

There are additional attendant advantages realized by binding to the lipid domain of cells. Since the lipid domain is composed of the majority of the cell surface area, it is possible to deploy large numbers of lipid-binding compounds, resulting in high concentrations of therapeutic agents within the plasma membrane. In addition, the compounds of the invention, by virtue of their lipophilic character, stably bind within membrane lipids and are therefore relatively insoluble in standard physiological salts. Therefore, once these compounds are bound to the membrane, they are effectively trapped therein and cannot be easily separated. As a result, cell leakage is minimized, thereby minimizing deleterious systemic effects.

[0016]

DETAILED DESCRIPTION OF THE INVENTION Definitions The following terms and expressions are defined as follows as a reference to the description of the present invention: Bio-active ingredient-The terms bio-active ingredient and bio-active agent refer here to the treatment of humans or animals,
Used to refer to a wide variety of substances that are useful for diagnosis, prevention, or other treatment. These terms include any substance capable of exerting a biological effect. 2. Biocompatible particle-as used herein, the term "biocompatible particle", in addition to viable entities, such as cells in vivo and in vitro, so long as it does not cause significant side effects upon administration in vivo. , Liposomes, lipoproteins and other non-viable entities. The expression "viable biocompatible particles with physiological function" refers here to viruses containing any viable cells or membranes. Moreover, as used herein, the term "cell" includes eukaryotic cells such as white blood cells, various tumor cells, and prokaryotic cells such as bacteria, and
Examples include Chinese hamster ovary cells, cultured mammalian cells such as yeast, and non-nuclear cells such as red blood cells, red blood cell ghosts and platelets. The detailed description of the invention below is
Living cells are described with particular reference. However, it should be understood that what is described with respect to cells generally applies to viruses that include membranes as well. Furthermore, in carrying out the site-selective delivery of the therapeutic agent according to the present invention, a non-viable entity is appropriately converted into a viable entity if it has a natural or acquired affinity for the intended delivery site. It needs to be understood that it can be replaced. For example, liposomes can be used as carriers for therapeutically active substances for delivery to the liver or spleen.

3. Diagnostic-in vivo or inv
detection of physiological conditions or conditions by in vitro tests,
A compound or substance that has the ability to facilitate measurement or analysis. The compounds of the present invention can serve a dual function as reporter molecules that are detectable from outside the body or in body fluids or biopsies obtained for in vitro analysis. 4. Chromophore-refers to a substance that is detectable by absorption of at least one selected wavelength of light, either visually or by using an instrument. 5. Therapeutically effective substance-refers to a substance having the ability to prevent, alleviate, treat or cure an abnormal or pathological condition in the body. These include maintaining, increasing, reducing physiological body function,
Included are substances that protect the organism by suppressing, killing, modifying or retaining the organism's microorganisms or antigens, as well as substances with the ability to limit or destroy. A therapeutically effective substance includes a drug or drug with therapeutic benefit.

6. Chemotherapeutic substance-refers to a therapeutically effective substance whose therapeutic effect results from the chemical characteristics of the substance.
Chemotherapeutic agents can include, for example, non-radioactive agents. They can include small molecules and more complex molecules such as lipids, carbohydrates, proteins or nucleic acids such as DNA or RNA. 7. Radiotherapy substance-refers to a therapeutically effective substance whose therapeutic effect is due to radioactivity. Suitable radiotherapeutic substances can be composed of radioisotopes. Preferably, the bioactive ingredient is 186Re, 90Y, 67Cu, 177.
It is composed of a chelating agent that forms a complex with various radioactive therapeutic nuclides such as Lu or 153Sm. 8. Antiproliferative drug-refers to a therapeutically effective substance with the ability to prevent, reduce or prevent the growth of cells.

II. Description of Compounds The compounds of the present invention are useful in a variety of pharmacotherapies that require the site-selective delivery of therapeutically effective substances.
According to a preferred embodiment of the present invention, the therapeutically effective substance is a radiotherapeutic substance or a chemotherapeutic substance antiproliferative drug. In another example, a compound of the invention may be comprised of a diagnostic agent such as a chromophore or radionuclide that allows for in vitro or in vivo tracking and / or detection of the compound of the invention. Therefore,
The compounds of the present invention can be composed of, for example, a therapeutically effective agent as a bioactive moiety and a detectable chromophore as a binding moiety. The diagnostic agents that make up the compounds of the present invention can be selected from among different types that can be detected by various analytical methods well known to those skilled in the art. Detectable fluorescent compounds are preferably cyanine dyes and derivatives thereof, including, for example, oxycarbocyanine, indocarbocyanine, thiocarbocyanine, or acridine dyes and derivatives thereof.
Other useful fluorescent compounds include, for example, styrylpyridine,
It includes xanthene, phenoxazine, phenothiazine, or diphenylhexatriene dyes and their derivatives.

Effective diagnostic agents further include ligands that facilitate detection, such as biotin or specific antibodies. Other effective diagnostic agents are chelating agents complexed with metals, which can be detected directly or indirectly. Suitable chelate-metal complexes are isotopes selected from the transition metal series with atomic numbers 21 to 49,
For example, it can be composed of indium 111 or technetium 99m. Such complexes can be bound to the cytoplasmic membrane of carrier cells and rendered radioactive to allow imaging with a gamma camera after delivery into the body. The chelating agent can further form a complex with the metal ion that is indirectly detectable, for example by producing a particular action at the site of interest. For example, complexes of paramagnetic elements can affect the relaxation times of nearby nuclei and can therefore be detected by magnetic resonance imaging (MRI). The chelate-metal complex is
Metal ions selected from the transition metal series with atomic numbers 21 to 29 or the lanthanide series with atomic numbers 59 to 66 are suitable for such applications.

If desired, compounds containing radioisotopes can be used in the various uses of the present invention.
Radioactive isotopes, such as 125I, 131I, 14C, 3H, 35S, or 75Se, which are abundant and naturally occurring non-radioactive forms of atoms in the hydrocarbon tails of bioactive components, chromophores or compounds. Can be replaced with Isotopes with non-zero spin states (eg 19F) can be introduced into the compounds of the invention such that their presence can be detected by MRI techniques. For therapeutic applications, the bioactive component can be composed of chelating agents of the type described above that are complexed with various therapeutic radionuclides such as 186Re, 90Y, or 67Cu. For therapeutic applications according to the present invention, further proteinaceous substances, including proteins, glycoproteins, riboproteins or peptides, can be linked to a hydrocarbon tail of suitable length via a suitable linking moiety. . Typical bio-acting proteinaceous substances are immunogens, toxins, hormones, enzymes,
Antigens, antibodies, and antibody fragments. Such therapeutically effective proteins are advantageously conjugated to lipophilic chromophores of the type described above, and due to the variable and predictable stability of binding to various bioparticles, including lipids illustrated below,
The resulting conjugate becomes prominent.

In other therapeutic applications, the bioactive component is composed of carbohydrates that allow the bound cells to move and change their circulating morphology within the cell body. One type of carbohydrate applicable in this manner includes sialic acid; the other type includes glycosaminoglycans. For example, a composition containing sialic acid can be applied to the plasma membrane of red blood cells to increase the amount of sialic acid on the membrane. Increasing the number of charged groups increases the lifespan of circulating red blood cells before they are cleared by the liver. The bioactive component can also be in the form of a tissue-specific receptor, or a ligand capable of binding to a receptor on cells within the target organ. Compounds containing such ligands, for example when bound to carrier cells, promote migration to specific organ sites. The compounds of the present invention can be delivered directly to a selected site within the body by a variety of methods, including injection, infusion, catheterization and topical application, among others. For example, autologous, allogeneic, or zeno
The compounds of the present invention can also be coupled to carrier biocompatible particles such as germic cells to facilitate the targeted delivery of bioactive agents. Unless otherwise specifically limited, the discussion set forth below refers to the binding of the compounds of the invention to viable cells, either by direct delivery to the disease site or adjustment of carrier excipients. One object of the present invention is to provide compounds capable of binding therapeutic agents or radioisotopes and of being dissolved in the fat bilayer which constitutes the outer membrane of cells. Delivery of therapeutic agents via the compounds of the present invention is such that when delivered directly to the site of the disease, these agents are maintained at high concentrations in cells in the area of the disease for extended periods of time, which cannot be achieved by other methods. To enable. Furthermore, the use of the compounds of the present invention allows the use of therapeutic agents which may be toxic when administered systemically.

The methods of action of the compounds of the invention on cells are diverse and depend on the following: (1) the type of cells to be attached; (2) the nature, length and quantity of the lipophilic tails. (3) the body part to be treated; (4) the nature of the bioactive ingredient; and (5) the mechanism by which the bioactive ingredient bound to the compound produces its action on the cell. The compounds of the invention deposit on cells and initially attach to the lipid bilayer outside the plasma membrane. Since the membrane components naturally communicate with the inside, these compounds are also taken up inside the cell. The rate at which this occurs depends on the type of individual cell, its growth state and its activity or degree of stimulation.

Some classes of anti-proliferative agents, such as radiotherapeutic agents, conjugated to the compounds of the present invention act whether they are located on the outer membrane or intracellularly. These agents emit radiation that damages the nucleus and, if the radiation is sufficiently transmitted, also inhibits the growth of surrounding cells. Drugs that must physically interact with intracellular components to suppress growth are only effective after the compound of the invention has been taken up into the cell. For extremely toxic drugs such as colchicine, other methods of action that can be conjugated to lipophilic components and bound to the outer membrane in an inactive form that is not toxic to cells Has been devised. Then, as the conjugate moves inward, a specially prepared bond (described in detail in the example below) allows release of the drug from the conjugate and exerts its antiproliferative effect. To

III. General formula Compounds within the scope of the present invention are given by the formula:

[Chemical 2] Here, B is a bio-active substance composed of therapeutically effective substances.
Represents components, R and R ₁Is hydrogen, alkyl, alkeni
Ru, alkynyl, alkaryl, or aralkyl groups
Represents a substituent independently selected from
Linear or branched, one or more non-polar
Desubstituted or substituted by a functional group, R and R₁
At least one of which is composed of hydrocarbon substituents
Length is effective in providing the compound with membrane binding capacity
R is equipped with a length_TwoRepresents the spacer component, and n is 0 or
Or 1 and L is a non-aromatic binding component when n = 0.
And B and R and R when n = 0 under the condition that
R between at least one of 1 and n = 1₂And R
And R₁Providing a stable bond with at least one of the
It represents the binding component to be provided.

The term as used herein "non-polar functional group", O- alkyl, S- alkyl, halogen, N (alkyl) _2, Se- alkyl, NO _2, CN, CO- alkyl, Si (alkyl) ₃ , O-Si (alkyl) ₃ , and the like. The binding component (L) is a saturated or unsaturated aliphatic linker or an alicyclic ring structure containing an aromatic ring, which is monocyclic, polycyclic, monocyclic, heterocyclic, soluble or insoluble. It is possible. Preferably, the binding component is a chromophore. The binding of the chromophore to the compounds of the invention facilitates compound tracking in vivo. Chromophores useful for this purpose include cyanine, acridine, pyridine, quinoline, xanthene, phenoxazine, phenothiazine, and diphenylhexatriene dyes and their derivatives.

A preferred class of compounds within the scope of the present invention is given by the formula:

[Chemical 3] Where B represents a bioactive substance, R and R₁Is water
Elementary, alkyl, alkenyl, alkynyl, alkaryl,
Alternatively, the substituents independently selected from the aralkyl group are represented.
The hydrocarbon chain has from 1 to 30 carbon atoms.
, Linear or branched, the substituent is
Is unsubstituted or substituted by further non-polar functional groups
And R_TwoRepresents the spacer component of the formula:-(R_Three)
_p-Q- (R _Four-Q ')_q-(R₅-Q '')_r-(R
₆-Q ""_s-(R₇Q '' '' ')_t, Where R_Three
Represents an aliphatic hydrocarbon, R_Four, R₅, R₆And R₇
Is an aliphatic, alicyclic or aromatic hydrocarbon, heterocyclic or
Is CH_TwoC (CO_TwoH) = CH among groups consisting of
Independently selected, Q and Q ', Q ", Q"', and
Q '' 'is amide, thiourea, hydrazone, acyl
Drazon, ketal, Ayatar, orthoester, d
Steal, anhydride, disulfide, urea, carbamic acid
Salt, imine, amine, ether, carbonate, thioete
, Sulfonamide, carbonyl, amidine and tri
Independently selected from functional linking groups composed of azine bonds
Q, Q ', Q ", Q"', Q "'"
Can further independently represent a valence bond, and can be an aliphatic
Or alicyclic hydrocarbon is 1 to 12 straight carbon sources
Aromatic hydrocarbons with 6 to 12 carbon atoms
N, p, q, r, s and t are each 0 or
Or 1. X and X1 have the same or different values.
O, S, C (CH_Three) 2 or Se
Y; = CR₈, = CR₈-CR₈= CR₈-, =
CR₈-CR₈= CR₈-CR₈= CR₈-Or
= CR₈-CR₈= CR₈-CR₈= CR₈-CR ₈=
CR₈Represents a linking group selected from-wherein R₈Is H,
CH_Three, CH_TwoCH _Three, CH_TwoCH_TwoCH_Three, Or
CH (CH_Three)_TwoZ is H, alkyl,
OH, -O-alkyl, COOH, CONH_Two, SO_Three
H, SO_TwoNH_Two, CONH-alkyl, CON- (a
Rukiru)_Two, NH-acyl, NH-alkyl, N (ar
kill)_Two, SH, S-alkyl, NO_Two, Halogen, S
i (alkyl)_Three, Or O-Si (alkyl)_Three,
Sn (alkyl) _Three, Or selected from Hg-halogen groups
An alkyl group composed of the Z substituent described above.
The group comprises 1 to 4 carbon atoms;
Represents a physically compatible anion. Highly stable organism
For applications requiring membrane binding, R and R of formula (II)
And one of R1 has at least 12 carbon atoms,
The sum of the linear carbon atoms of R and R1 is at least 23
You have to be individual.

The various spacer moieties (R ₂ ) are suitably present between the cyanine headgroup and the bioactive moiety, either on the headgroup and / or the bioactive moiety, according to known synthetic methods. It can be easily bonded via a functional group. For this purpose, a large number of different kinds of biofunction (homobiofunction and heterobiofunction) spacer reagents have been reported in the technical literature. Meth. En
z. , 91: 580-609 (1983). These spacer components differ depending on the type of reactive group, hydrophobicity, hydrophilicity, length, and whether the structure connecting the reactive group is cleavable or non-cleavable.

As mentioned above, the functional attachment of the spacer group is
Amide (-NHCO-), thiourea (-NHCSNH)
-), Hydrazone (-CH = NHN-), acylhydra
Zon (-CH = NHNCO-), ketal (-OC)
(Alkyl)_Two-O-), Ayatar (-O-CH (A
Alkyl) -O-), orthoester (-C (O-alk)
Le)_Two-O-), ester (-COO-), anhydride (-
COOCO-), disulfide (-SS-), urea
(-NHCONH-), carbamate (-NHCO ₂
-), Imine (-C = N-), amine (-NH-), d
Ether (-O-), carbonate (-O-CO₂−), Thioe
Ether (-S-), sulfonamide (-SO_Two-NH
-), Carbonyl (-CO-), and amidine (-N
HC = (NH⁺)-).

In a preferred embodiment for radiation therapy,
B especially represents a chelating agent which forms a complex with a radioactive metal such as rhenium or yttrium. In a preferred embodiment for chemotherapy, B represents heparin, hirudin, colchicine, vinblastine or analogs thereof, all of which are antiproliferative agents. In another preferred embodiment for chemotherapy B represents a peptide. Formula II above
It has been found that a derivative of a peptide known as substance P, which is contained in PG., Binds to erythrocytes in a stable manner and provides a prolongation of the therapeutic effect in the circulation as compared to the free, unconjugated peptide. It is expected that increased bioavailability may be achieved as well for other therapeutically active substances that, in unconjugated form, have a relatively short life in circulation. For certain applications of the compounds of this invention, such as combination therapy and combination diagnosis, Formula I
It is effective to use B as biotin.

III. Preparation of compounds of the invention 1. Lipophilic Functionalized Cyanines The compounds of formula (II) above are simply prepared from lipophilic cyanine precursors according to the following general formula:

[Chemical 4] Where R, R₁, X, X₁, Y, Z are the compounds of formula II
As defined above; further R_TwoIs the expression
Represents the spacer component of :-( R_Three)_p-(Q-R_Four)_q
-(Q'-R₅)_r-(Q "-R₆)_s-(Q ""
-R₇)_t-Where R_Three, R_Four, R₅, R₆, R₇,
Q, Q ', Q' ', Q' '', p, q, r, s, t are
As defined above for a compound of formula II;
W is amino (-NH_Two), Α-haloayatoamide
(-NH COCH₂-Hal) (hal = halogen
), Isothiocyanate (-NCS), halogen, a
Socyanate (-NCO), carboxyl (-COO
H), hydrazino (-NHNH₂), Acyl hydrazide
(-CONH-NH₂), Such as benzophenone
Ketone (-RCO), dithiopyridyl (-SS-C_FiveH_Five
N), hydrosulfur (-SH), aldehyde (-HCO), anhydrous
(-COOCO-alkyl), succinimide ester
(-COOC_FourH_FourNO₂), Hydroxy (-OH), sulfo
Nyl halide (-SO₂-Hal), imidoeste
Le (-C (= NH) OCH₂), Evoxide (-C₂H₃
O) maleimidyl (-NCOCH = CHCO) and
Zido (-N ₃) Group represents a reactive functional group selected from the group.

Suitable precursors for the preparation of the compounds of the present invention have a reactive amine (NH ₂ ) group as the substituent W in the above formula and can be prepared according to various synthetic methods, one of which is Is illustrated in Reaction Scheme 1 and discussed in detail in the examples below. According to Scheme 1, Gale and Wils
hire's method (Aust. J. Chem., 30: 6.
93 (1977)), 2,3,3- (3H) -trimethylindolenine (commercial product) is reacted with N-hydroxymethylphthalamide (commercial product) to form compound (1), which is further treated. Alcohol and 4-sulfonic acid chlorobenzene chloride from Sondermann
(Liebigs Ann. Chem., 749:
183-197 (1971)) to react with chlorobenzene alkyl 4-sulfonate to form intermediate (2). The phthalimido protecting group of (2) is removed by heating in concentrated hydrochloric acid according to the general procedure to produce compound (3), which is subsequently treated with methyl formate to produce (4). 2-methyl-benzoxazole (commercially available) or 2,3,3- (3
H) Prepare compound (5) by alkylation with either trimethylindolenine or chlorobenzene alkyl 4-sulfonate. Compound (5) is then reacted with N, N-diphenylformamidine (commercially available) to produce (6) using a method similar to that described in US Pat. No. 2,647,054. .
Then base (sodium acetate or triethylamine)
Intermediates (4) and (6) are mixed by stirring in alcohol in the presence of to produce (7). Dha
wan et al. (Orgn. Prep. and Pro
c. Int. , 7 (2) 85-88 (1975)) deprotects (7) to yield the amino-derived cyanine (8).

The aminocyanine (8) can be directly attached to a therapeutic agent containing appropriate functional groups (eg CO, COOH) to form a conjugate that aids in the delivery of site-specific drugs. In addition, aminocyanine (8) is a very versatile intermediate for the preparation of a wide range of other cyanine derivatives that can be conjugated with other bioactive components. Compound (8) is also an ideal molecule for use in reacting with a number of homo- and hetero-bifunctional spacer components to provide separation between the lipophilic cyanine component and the bioactive agent bound to it. Is. The resulting amine substituent on the cyanine derivative can be directly converted to other functional groups using well known reaction methods. For example, conversion to an isothiocyanic acid functional group is
The method of Costa et al. (J. of Lab. Compd
s. and Radiopharm. , 27 (9): 10
15 (1989)) by treatment with thiophosgene.

Amine group and 1,4-terephthalic acid di-N-
By reaction with hydroxysuccinimide ester,
Attachment of the succinimidyl functional group to the cyanine head group by an OOC-aryl-CONH-alkyl spacer moiety is provided. The resulting compound can be isolated and purified as a stable crystalline solid. Simple acylation of substituted amino groups in dimethylformamide with p-nitrophenyl iodoacetate (commercially available) provides a reactive iodo substituent attached to the cyanine head group by an alkyl-CONH-alkyl spacer moiety. . N-hydroxysuccinimidyl-3 (2-
Treatment of the amine group with pyridyldithio) propionic acid (commercially available) gives pyridyldithio functional groups and alkyl-
A compound with a CONH-alkyl spacer component is provided. Treatment of the amine group with γ-thiobutyrolactone provides a compound with a vulcanization functionality and an alkyl-OCNH-alkyl spacer moiety. Similarly, γ
-Treatment of amine groups with butyrolactone provides compounds with hydroxyl groups and alkyl-CONH-alkyl spacer moieties.

2. Protein / Peptide Lipophilic Cyanine Conjugates Lipophilic cyanine precursors prepared as generally described above can be coupled to proteins or peptides using a number of different synthetic methods to provide compounds of formula II. be able to. The binding of the protein or peptide to the lipophilic linker derivative as described above can be performed via the amine group (that is, the terminal α-amino group or the ε-amino group of lysine) existing on the protein or peptide. Alternatively, to attach the protein or peptide to the appropriate cyanine precursor, a carboxyl or thiol group (where cysteine residue is present)
Can be used. Suitable conditions for carrying out such coupling reactions are well known to those skilled in the art.

One method for conjugating the α-amino group of a polypeptide with a lipophilic cyanine precursor is described by Wet.
zel et al.'s method (Bioconjugate Che
m. 1, 114 (1990) is used. This is the formation of an iodoacetic acid amide derivative by acylation of the polypeptide with iodoacetic anhydride (commercially available) at pH 6, which is then prepared as described above to form a vulcanizate-derived lipophilic cyanine compound. And forming a conjugate through thioether bond formation with excellent stability. The conjugate via the ε-aliphatic amino group of lysine is such that only a limited fraction of the amine is deprotonated to become reactive by equilibrium,
It is best achieved at a pH above 8.5. This amino group must be highly reactive with some of the amino-reactive lipophilic linker compounds described above. For example, an isothiocyanic acid-derived linker compound is
It is highly stable under aqueous conditions and can react with the side chains of lysine to form conjugates linked via thiourea bonds.

The N-hydroxy-succinimidyl ester-derived linker compound described above is a particularly excellent reagent for reaction with lysine because the amide conjugate thus formed is very stable. This reaction is preferably carried out under anhydrous conditions in an organic solvent such as dimethylformamide, as hydrolysis of this special derivative under aqueous conditions is a competing side reaction. Formula I above
The reaction of conjugating the N-hydroxy-succinimidyl ester-derived linker of II with the amino group of the lysine residue at position 3 of the undeca peptide, substance P, is described in detail below. As on the C-terminal amino acid residue of the peptide or protein, the carboxylic acid group present on the side chain of the glutamic acid and aspartic acid residue is at a site capable of selective conjugation using the above amine-derived lipophilic cyanine compound. is there. This reaction is based on Hoare and Kos
Hland's method modified (J. Biol. Ch.
em. 242: 2447-2453 (1967), using a water-soluble carbodiimide such as 1-ethyl-3-dimethylamino-propylcarbodiimide (commercially available), or J. Biol. C
hem. 249: 5452 (1974) according to a modified method of Woodward's Reagent K, 2-
It can be carried out by using ethyl-5-phenyl-isoxazolium-3-sulfonate (commercially available product). The resulting conjugate is attached via a stable amino bond.

The binding of functionalized derivatives to the reactive groups of peptides or proteins essential for biological activity must be avoided, since inactive conjugates will always be formed. However, if the peptide component or protein can be released from the lipophilic cyanine derivative by a cleavable spacer component, this problem can be solved. Due to the lipophilicity of the compounds mentioned above, some of them are poorly soluble in typical aqueous buffer systems. Conjugation reactions that require aqueous conditions to maintain the drug's soluble or active conformation usually need to be performed in aqueous solvents modified with organic solvents. Solvents such as dimethylformamide, dimethylsulfoxide, acetonitrile, and alcohol can be mixed with water and are effective in solubilizing the lipophilic cyanine derivative to form the desired conjugate. The compounds of the invention can be purified by a variety of standard purification methods which utilize the size, charge or lipophilicity of the particular compound formed. A particularly effective purification method for therapeutic agents composed of peptides is Bohlen.
(Int. J. Rept. Prot. R.
esearch, 16: 306-10 (1980).

3. Biotinylated lipophilic cyanine The biotinylated lipophilic cyanine compound of the present invention comprises
Preferably it is given by:

[Chemical 5] Here, R, R ₁ and R ₈ are as defined above, and m takes a value of 0 to 6. Such biotinylated derivatives can be prepared by biotin reaction or biotin derivatives using the functionalized lipophilic cyanine compounds of formula III above. The biotin derivative used in this reaction is preferably given by the formula:

[Chemical 6] Where E represents the residue of the compound with a labile group which can be replaced by said reactive functional groups, and W and m are 0, 1, or 2.

For example, an amino-functionalized cyanine derivative (8) of the type prepared according to Reaction Scheme 1 was added to Hofm.
Ann, Finn, Kiso's method (J. Am. Che
m. Soc. , 100: 3585 (1987)), commercially available amine-reactive biotin derivatives, (+)-biotin 4-nitrophenyl ester, N-hydroxysuccinimidyl 6- (biotinamide) caproate and 6 ((6 ((Biotinoyl) -amino) hexanoyl) amino) caproic acid can be reacted with N-hydroxysuccinimimidyl ester to form the biotin-lipophilic cyanine conjugate of formula IV above, where each m = It is 0, 1, 2. These conjugates differ in the length of the spacer arm between the biotin component and the fat-binding component of the conjugate. The action of such spacer arms can be significant depending on the intended application of the biotinylated compound. Long spacer arms have been shown to have a beneficial effect on the ability to bind biotin conjugates to the "deep" biotin binding site in avidin.

Other types of biotin derivatives with reactive functional groups such as maleimide, α-iodoacetamide, hydrazino and amino are commercially available (Molecular).
rProbes, Eugene, OR, Handboo
k of Fluorescent Probes a
nd Research Reagents, 1989
˜91), and can be used for conjugation with various functionalized lipophilic linker compounds described above. Suitable reaction schemes will be apparent to those skilled in the art. Furthermore, a large number of different types of spacer moieties can be attached between the cyanine and biotin moieties by appropriate functional groups on both precursors. Reaction schemes for attachment of such spacers are well known to those skilled in the art.

4. The synthesis of lipophilic tributyltin, which serves to prepare lipophilic cyanine radiohalogenated cyanines substituted with radioisotopes and which can be advantageously used in the method of the present invention, is shown in Reaction Scheme 2 and in the examples below. Describe in detail. According to Reaction Scheme 2, Blak
ie et al. (J. Chem. Soc., 313 (192).
4)) and Moreau et al. (Eur. J. Med. Che.
m. Chim. Ther. , 9, (3): 274-28
0 (1974)), 5-iodo-2 prepared from iodoaniline (commercially available) using the method described by
3,3-Trimethyl- (3H) -indolenine (9)
Alkylated with alkyl-4-chlorobenzenesulfonate (prepared as previously described) (1
0) is generated. Treatment of (10) with N, N-diphenylformamidine in acetic anhydride gives the vinyl intermediate (11). The intermediates (11) and (5) (the latter prepared as previously described) are then combined by stirring in ethanol in the presence of sodium acetate. The reaction mixture is quenched with silver acetate and sodium chloride is added to give indocyanine (12). Then the catalyst, tetrakis- (triphenylphosphine) palladium (0)
Of Azizian et al. (J. Organomet. Chem., 215: 49) comprising heating (12) with bis- (tri-n-butyltin) in the presence of
~ 58 (1981)), the compound (13) can be prepared from (12). Following this, the tributylstannyl derivative (13) is modified under mild conditions, eg by a modification of the method of Wilbur et al. (J. Nu.
c. Med. , 30: 216-226 (1989). In addition, the method of Weiss et al. (J. Labeled Cm
pds. & Radiopharmaceutical
s, XXVI: 109-10 (1989), a modified method can be used to achieve the introduction of radioactive halogen into (12).

Another lipophilic cyanine derivative that can be used to introduce the radioactive halogen is Sn (Bu) 3
An analog of (13) in which the group is replaced with -HgX, where X represents halogen. The ring position of the halogen substituent of compound (12) can, of course, be changed to a suitably chosen starting material. For example, Bassignan
a's method (Spectrochemica Act
a. , 19 (11), 1885 (1963)) can be used to provide the corresponding 6-iodo derivatives.

5. Cleavable Colchicine-Lipophilic Cyanine Conjugates Colchicine-cyanine conjugates with cleavable bonds by acids can be prepared by selecting an appropriately functionalized active colchicine derivative (Bros.
si, J.M. Med. Chem. , 33: 2311, 23
19 (1990)), via a bond such as cis-aconityl, acetal, orthoester, ester, ketal, anhydride, or hydrazone, to a suitably functionalized lipophilic cyanine derivative. You can combine it. Linking via the cis-aconityl bond is accomplished by the method described by Shen et al. (Biochem, Biophvs. Res. Com.
mun. , 102, 3: 1048-1054 (198
It can be realized by using 1). This method involves coupling the free amino derivative of a drug (eg, desayatylcolchicine) and the amino form of a lipophilic cyanine with anhydrous cis-aconityl (commercially available).

Coupling via ayatal, orthoester, or ketal linkages is accomplished by the method described by Srinvasachar et al. (Biochemistry,
No. 28, 2501-2509 (1989)) can be used with modification to achieve this with appropriately functionalized colchicine and cyanine derivatives. Binding via a hydrazone bond is performed by the method described by Laguzza et al. (J. Med. Chem., 32: 548-.
555 (1989)) with a modification. This method involves coupling the aldehyde form of the drug with the hydrazide form of the lipophilic cyanine.
The synthesis of cleavable colchicine-cyanine conjugates according to the present invention is shown in Reaction Scheme 3 and further described in the examples below.

According to scheme 3, an amino-functionalized cyanine (8) is coupled with the monomethyl ester of glutaric acid (commercially available) to form the methyl ester derivative (14), which is treated with hydrazine in methanol. A hydrazino derivative (15) is provided. The colchicine component was prepared by reacting with deayatyl colchicine (commercially available) to produce an acid intermediate, which was then activated in situ by addition of carbonylimidizole to form an acylimidazole, which was tetrabutylhydrogen. Reduction with ammonium borohydride provides the alcohol (17). Oxidation of (17) with pyridinium chlorochromate gives 7-N- (5
-Oxopentanoyl) deayatylcolchicine (18)
Which is then coupled with a hydrazino derivative (15) to give a conjugate (19) in which the colchicine and cyanine moieties are linked via an acid cleavable acylhydrazone bond. 7-N- produced as an intermediate in Scheme 3
(5-oxopentanoyl) deacetylcolchicine
It is a new compound that forms part of the present invention. If desired, methylthio, or other chalcogen containing groups, can be replaced with methoxy groups at position 10 of the colchicine core.

Furthermore, deacetylthiocolchicine (Sh
ian et al. Pharma. Sci. , 64, 646
~ Adjusted as described by 648 (1975))
From the above, using the same reaction course as the preparation of the aldehyde (18) shown in Scheme 3, the more effective 7-N-
(5-oxopentanoyl) deayatylthiocolchicine analogs can be made. Furthermore, this derivative of thiocolchicine can also be bound to the hydrazino derivative (15) using the same coupling conditions to provide an acid cleavable conjugate. The kinetics of release of the colchicine analog from the conjugate can be modified by altering the type of hydrazone bond between the colchicine and cyanine moieties. For example, the binding of antibody-drug conjugates via a sulfonylphenylhydrazone and a phenylhydrazone bond provided a slower release kinetics than the corresponding acylhydrazone derivative Muelle.
r et al. (Bioconjugate Che
m. , 1: 325-330 (1990)).

6. Heparin-Lipophilic Cyanine Conjugates A method for synthesizing heparin-cyanine conjugates bound by a stable carbamate bond and useful in the present invention is shown in Reaction Scheme 4 and described in detail in the Examples below. According to Scheme 4, amino-functionalized cyanine (8) was added to Ecke
rt et al. (Angew Chem. Int. Ed.
Enql. , 26 (9): 894-95 (1987)).
According to the procedure, it is converted into a highly reactive isocyanate derivative (20) by treatment with triphosgene. The isocyanate derivative (20) was prepared without further separation or purification by the method of Dong ("Heparinized and fragmented polyurethaneurea surface with hydrophilic spacer groups", Dissertation, Utah University, 19
90) modified and used to react immediately with sodium heparin in a mixed solvent of dimethylformamide / formamide, with the hydroxyl group of heparin via carbamate bond formation (or via an amino group via urea bond formation). Heparin covalently bound to cyanine-
A lipophilic cyanine conjugate (21) is provided. Additionally, carbon spacer arms can be attached between the heparin and cyanine moieties by reactions well known to those skilled in the art using the commercially available reagent N-Boc-6 aminocaproic acid N-hydroxysuccinimide ester.

Since heparin is equipped with a large number of free hydroxyl groups, it is of course possible to modify the large number of cyanine groups per heparin molecule by controlling the stoichiometry of the reagents in the reaction. If necessary, the heparin-lipophilic cyanine conjugate can be purified from free heparin by hydrophobic interaction chromatography. Alternatively, the method described by Kin et al. (NonthrombogenicBioactive) may be used.
Surface Annals, New York
Academy of Sciences, p. 116
~ 130) is modified to use heparin-
The cyanine conjugate can be adjusted. This method
Conjugating the carboxylic acid group of heparin to the amino functionalized cyanine using a carbodiimide reagent. Evert et al. Determined that up to 20% of the carboxylic acid groups of heparin were induced without loss of bioactivity (Biomaterials: Interfaci.
al Phenomenon and Applicat
ion, Adv. Chem. Ser. 99, Ameri
can Chem. Soc. , Washington,
D. C. (1982).

7. Radiometal Complex-Lipophilic Cyanine Conjugate A method for synthesizing a radiometal complex-lipophilic cyanine conjugate is shown in Reaction Scheme 5, where the metal is a group consisting of rhenium, indium, copper, or palladium. Select one from. According to Scheme 5, Baid
oo and Lever (Tetrahedon Let)
t. , 31, 40, 5701-5704 (1990)).
Reacting a bifunctional chelating agent (22), prepared as described by, with an amino-functionalized cyanine (8),
This produces the derivative (23). Next, the compound (23) is reacted with a metal solution in an appropriate oxidation state to form a metal complex (24). Scheme 5 depicts the structure of the neutral metal complex obtained when the metal used is rhenium. The exact structure and charge of the complex over time depends on the metal used and the exact structure of the bifunctional chelate chosen.

Scheme 6 shows a method for preparing a radiometal complex lipophilic cyanine conjugate when the metal is selected from rare earth metals. A difunctionalized polyaminocarboxylic acid salt chelate having the structure of (25) is disclosed in European Patent Application No. 0.
It can be adjusted according to the method described in 353450 A1. According to Scheme 6, the chelate (25)
Is reacted with an amino-functionalized cyanine (8) at pH 9-9.5 to form a compound (26) in which the chelate and the cyanine component are attached via a stable thiourea bond. Compound (26) is then reacted with a solution of a radiometal salt in a suitable buffer system, again according to the method described in the above mentioned European patent application, to give the final radiometal complex-cyanine conjugate (27). To generate. M used in scheme 6 diagram
(PA-DOTA) is a radioactive metal complex composed of a polyamino-carboxylate chelate and a part of the spacer component (-(CH2) 2-C6H4-NH-) of the intermediate (26) shown in Scheme 6. Point to. The three-dimensional structure of the radioactive metal complex component of compound 27 is described by Spinlet et al. (Inorg).
en. Chem. 23: 4278-4283 (198).
It is expected to be similar to that described by 4).

Other types of bifunctional polyaminocarboxylic acid salt chelates are known and can be attached to the functionalized cyanine by other reactions well known to those skilled in the art. See, for example, Sundberge et al. Me
d. Chem. , 17: 1304 (1974).
Other tetradentate chelates containing nitrogen and tetradentate chelates containing sulfur are known and can be attached to appropriately functionalized cyanines by reactions well known to those skilled in the art. For example, Ras et al. Am. Che
m. Soc. , 112: 5798-5804 (198).
See 0).

IV. Binding Compounds of the Invention that Bind to Biocompatible Particles The large number of linear carbons on the hydrocarbon tails substituted on the compounds of the invention achieve the desired degree of stable binding between the compound and the surface membrane of the bioparticle. This is an important factor in doing so. In order to achieve stable attachment of compounds with a single hydrocarbon tail, eg acridine derivatives, the number of linear carbons should be 23 or higher. Experience with cyanine derivatives prepared according to the present invention includes:
For compounds with two or more hydrocarbon tails, one of the tails must have a linear portion that is at least 12 carbons long and the total number of linear carbon atoms in the hydrocarbon tail must be at least 23. It indicates that there is. Depending on the structure of the compound, leakage or conversion from one cell to another usually does not occur. In general, the longer the hydrocarbon tail, the higher the lipophilicity. However, a hydrocarbon tail with more than 30 linear carbon atoms may not be soluble in the same solvent as the bioactive component and the agent used to provide the hydrocarbon tail, This can be problematic because the chemistry for binding to the bioactive component becomes very difficult. Therefore, the length of the hydrocarbon tail may be substantially constrained by the chemistry of the binding moieties to which the tail is attached.

Structural differences in the binding moieties to which the "tail" binds also have a significant effect on membrane-bound stability. Positively charged binding moieties such as cyanine, styrylpyridine, xanthene, phenoxazine, phenothiazine, or diphenylhexatriene dyes and their derivatives are
It may contribute to the binding and retention of the compound on the negatively charged membrane. Neutral or negatively charged binding moieties may also help achieve controlled release from biomembranes. A stable bond between the binding component, the hydrocarbon tail, and the spacer or bioactive component may, in some cases, require a therapeutically effective substance to realize the therapeutic benefits provided by the invention. It can be essential for the site-selective retention for a long time and required amount.

The binding component (L) provides the compound of the present invention with the required stability so that the compound is present in the selected delivery site for a time and in an amount sufficient to achieve the desired therapeutic benefit. Have to choose to do. To this end, the binding component of the compound of formula I above has n = 0.
Provides a stable bond between the bioactive component (B) and at least one of R and R1, when n = 1.
In that case, a stable bond must be provided between the spacer component (R ₂ ) and at least _one of R and R ₁ . Compounds with the requisite binding stability conferred by the linking group can be determined based on retention time in the case of direct administration of the therapeutically effective lipophilic conjugate, or can be conjugated to the disease site. When the coalescence is supplied via a carrier, it can be determined based on the time of circulation. That is, the retention or circulation time of the therapeutically effective lipophilic conjugate of the invention must be greater than the retention or circulation time of the therapeutically active ingredient in unconjugated form.

In increasing the retention or circulation time is an important factor in achieving the therapeutic benefit provided by the present invention, a sufficient amount of the compound to provide the desired therapeutic benefit is sufficient for the disease. Presence or accumulation at the site is also an important factor, which also depends on the stability of the binding of the compounds of the invention. The determination of the holding time or circulation time can be carried out in various ways well known to the person skilled in the art, in particular as illustrated in Examples 9, 10 and 12 below. The determination of the amount of therapeutically effective lipophilic conjugate of the invention required to produce the sought-for effect is not in any particular way, as is often the case for therapeutic agents.
This is necessarily due to the diversity of therapeutically effective substances used in the practice of the present invention, the numerous disease states that can be treated thereby, and the varying conditions of the patient being treated. Absent. Therefore, the response of patients undergoing treatment according to the invention is monitored periodically to determine whether the abundance or accumulation of therapeutically active substances at the disease site is sufficient to produce the desired therapeutic effect. There is a need to.

The compounds of the present invention can be made to bind to the outer membrane of viable cells or other biocompatible particles without any initial detrimental effect on viability, Alternatively, it can be engineered to exert an immediate or delayed cytostatic or cytotoxic effect. To determine the extent of cytotoxicity by a cytotoxic bioactive component, for example, cells are exposed to a compound of the invention at various concentrations, including compounds that are not conjugated to the cytotoxin or zero concentration. . The cells are then exposed to trypan blue or propidium iodide (F. Celada et al., Proc. Natl. Aca.
d. Sci. 57: 630 (1967)). These dyes are normally cleared from living cells and penetrate only the membrane of dead cells. After an appropriate culture time, the cells are examined by a microscope or a flow cytometer to measure the percentage of stained cells (mortality).

The binding of the compounds of the invention to carrier cells also has no appreciable detrimental effect on the function of the cell, which is important for its ability to carry out target delivery as a carrier. For example, it may be important for the carrier cells to properly divide for the performance of certain applications. On the other hand, the compound used may alter some of the functions such that it has no effect on the ability to split or other abilities required of the cell for its intended application. Therefore, such compounds can be considered to have no appreciable deleterious effect on the functioning of the cells for the purposes of the present invention. See US Pat. No. 4,859,584, Slezak and Horan, Bl, for methods of measuring the effects on cell function that may be of importance to the practice of the present invention produced by the compounds of the present invention.
Wood, 74: 2172-77 (1989) and J. Im
munol. Meth. 117: 205-14 (19
89). Two criteria must be met when a cell binding means is selected to bind the compound of the invention in a reproducible manner to the plasma membrane of a target or carrier cell without adversely affecting the desired cell function. is there.
The cell binding means is isotonic to the biocompatible particles that bind the (i) compound and does not cause contraction or swelling, and does not damage the cells. 260 to 340 mOsmol), and (ii) the compound of the present invention must be soluble so that it can be bound at a constant concentration within the plasma membrane of cells. Solubility time course experiments (US Pat. No. 4,783,401, M. Melnicoff et al.
J. Leuk. Biok. , 43: 387-397 (1
988)) is only partially water soluble, such that compounds that act to stably bind to the plasma membrane may precipitate when dissolved in ionic solutions (eg phosphate buffered saline, media etc.). Decrease the binding of compounds to the plasma membrane, with a tendency to form either simple micelles or aggregates.
Alternatively, it has been shown to result in undesirable irregular binding.

Similar experimental methods for radioisotope compounds can be applied and the stability of the compounds can be measured using beta or gamma counters. In all cases, the amount of the compound of the invention in the supernatant of said isotonic solution at each time point is not suitable for labeled cells by comparison with a sample of such compound using ethanol as solvent. But is useful for the maximum solubility of the compound (overall). In determining the appropriate concentration of a compound of the invention that binds to the plasma membrane of a cell, several factors must be considered, including the desired effect produced by the compound, the type of cell that binds the compound. Generally, the first goal is to bind as much therapeutic agent as possible within the cell membrane. This can be accomplished by delivering the therapeutic compound directly to the site of the disease or by binding the therapeutic compound to cells ex vivo and reintroducing the modified cells in vivo. The number of carrier cells needed to administer a relatively low dose, or to reach a desired location and exert a desired effect, by maximizing the binding of the therapeutic agent into the plasma membrane of the cell. Can be reduced. In the case of cell-mediated delivery of therapeutic agents, the amount of compound bound intracellularly is such that there is no negative change in carrier cells in terms of cell viability or the ability of cells to move to their intended location. It only needs to be increased to a degree.

The compounds of the present invention enhance serum and other fats.
Carrier cells or other biocompatible particles in the absence of containing substances
Apply to children. Separate cells from the body or from culture
Remove and wash free of serum. Isoosmotic pressure adjustment
In order to form a composition containing the drug, they are usually
In addition to the on-solution, a suitable concentration of the compound of the invention (10 ^-Five
From 10^-7Suspend with M). The binding of the compound to the cell is
Usually completed within 10 minutes, with autologous or heterologous serum
The binding reaction is stopped by the addition. Next, the serum
Wash cells in medium containing (5-10% v / v) and
Place in culture or inject into recipient as appropriate.
It Methods of binding compounds of the type described here to cells are:
It is described in detail in US Pat. No. 4,783,401.
, The entire disclosure of which is fully described here,
Incorporated herein by reference.

Another cell binding method involves suspending the compound of the invention in saline to form micelles. The cells are then placed in the suspension and only phagocytic cells (eg monocytes, macrophages, neutrophils) are preferentially labeled. In this way, the compounds of the invention can be selectively directed to phagocytic cells. M. Melnico
ff et al. LeukocyteBiol. , 43: 3
87-97 (1988). Representative applications of the compounds, compositions, methods of the invention are described below with respect to particular drug therapies.

V. Uses of the Compounds of the Invention A. General treatment 1. Application to isotope therapy Radiation therapy to the disease site is possible because it has the ability to bind to the chelate ion that emits radioactive high linear energy transfer (LET) in the fat component of biocompatible particles including fat. The compounds of the present invention can be used to provide The chelated compound of the invention and a suitable radioactive ion (eg, 67Cu, 90Y, 186Re,
The cells are labeled with a compound of the invention by first forming a stable complex of the α-emitter), separating the complex and performing the conventional cell binding procedure described above. Tumor infiltrating lymphocytes (TIL) are isolated from the primary lesion, expanded in IL-2, conjugated to radiotherapeutic agents as described above, and injected intravenously. Labeled cells track the site of metastatic disease and emit radiation that kills metastatic tumor cells, thereby increasing the therapeutic effect of TIL and possibly reducing the number of cells required to achieve disease alleviation . Similarly, other types of cells that migrate to metastatic sites can be used to deliver local radiation therapy.

2. Targeting Cells by Binding Specific Proteins to Cell Membranes In another embodiment, the compounds of the present invention include protein-like substances containing proteins, glycoproteins, lipoproteins, or peptides in their interior as bioactive components. Combine as. These compounds were bound to cells as described above, in addition to embedding the hydrocarbon chains of said compound within the plasma membrane,
This places the protein on the surface of specific cell types. The above method is carried out by binding a monoclonal antibody against human fibrin to the surface of carrier cells such as red blood cells,
It can be used to feed fibrin clot sites.

Similar methods can be used to deliver monoclonal antibodies of human cell surface tumor antigens to the tumor site via carrier cells such as monocytes or lymphocytes. Tissue plasminogen activator can be similarly delivered by application to the surface of carrier cells (eg red blood cells). If desired, the above treatments can be used in combination. Thus, a monoclonal antibody that binds to human fibrin can be attached to the surface of cells (eg erythrocytes) which in turn is further attached to fibrinolytic compounds (eg tPA, stopkinase, urokinase). Monoclonal antibodies allow the binding of carrier cells to fibrin and subsequent delivery of a number of therapeutic fibrinolytic compounds.

These therapeutically effective proteins can be conjugated to lipophilic chromophores according to the general procedure described above. The techniques described above attach a variety of other therapeutically effective proteins or peptides to suitable biocompatible particles such as cells, viruses, liposomes or LDLs, thereby bioavailability of proteinaceous substances in the circulation. Can be used to greatly stretch. The protein-bound biocompatible particles are radioactive imaging compounds,
Alternatively, magnetic resonance imaging compounds can be used and labeled with isotopes as described above. The resulting bioparticles can be infused into a patient, thereby migrating cells to the disease site and imaged using standard gamma scintigraphy or nuclear imaging to assess the efficacy of therapeutic agents.

3. Conjugation of proteins to cells for vaccines In another application of the invention, an antigen, which may be a protein, glycoprotein, lipoprotein or peptide, against which protective antibody production is desired, is optionally conjugated. It contains groups and is used as a bioactive component to bind to the surface of cells (eg red blood cells, monocytes). The cells so modified are injected in the presence and absence of the antigenic booster.
The time interval of infusion depends on the nature of the antigen, but generally 10 ⁶ cells can be infused at each time interval not less than 2 weeks. The antibody concentration against the antigen is monitored by standard Elisa method. Cellular immunity can be measured by determining proliferation or cytotoxic response to immunized cells.

4. Alteration of migration morphology by modification of cell surface In other applications of this approach, compounds of the invention can be used to bind sialic acid or glycosaminoglycans to the plasma membrane of cells. Specific compounds are placed in the isotonic medium as described above. For example, placing red blood cells in solution results in the binding of the compound to the plasma membrane. The reaction is stopped by the addition of serum, after which the cells are washed with saline containing medium and are ready for injection. Erythrocytes migrate in the circulation and, between immature cells, have large amounts of sialic acid on their surface. As red blood cells grow, the amount of sialic acid per cell decreases, allowing spleen and liver macrophages to recognize red blood cell membrane antigens, thereby removing them from the circulation. By appropriately increasing the amount of sialic acid bound within the membrane of red blood cells, the life span of red blood cells in the circulation can be extended. The ability to prolong the life of red blood cells can be beneficial to transplant or anemia patients. When a bone marrow transplant patient receives a transplant, it takes several weeks before they can produce their own red blood cells. By using this procedure to extend the life of their own red blood cells, patients can receive multiple bone marrow transplants, if necessary, without causing anemia. In an individual with anemia, anemia may result from a reduced lifespan of red blood cells or a reduced rate of red blood cell production. In both cases, prolonging the life of red blood cells reduces anemia.

5. Providing Photodynamic Compounds for Therapeutic Action Photodynamic therapy to cure cancer is an area of intense research (Proceedings of
SPIE-The International So
ciency for Optical Engineer
ring; Volume 847, "New Dire
actions in Photodynamic Th
easy "Douglas C. Neckers, E
editor; (October 1987)). Many of these compounds are phthalocyanines or hematoporphyrins. All absorb light in the region of 600 to 800 nm, and in the process generate oxygen in an excited state.

Using the methodology of the present invention, lipophilic derivatives of compounds are made and then dissolved in isotonic solutions. Tumor-selective cells (eg TIL) are labeled with these compounds and the cells are injected into the patient. Tumor-selective cells then migrate to the micrometastase sites. Within 48 hours, the patient is exposed to intense light in the region where the photodynamic molecule absorbs, and the excited oxygen that is generated kills the tumor cells. In addition, the carrier cells are also killed causing inflammation, which causes more immune cells to collect to eliminate the dead cells and increase toxicity to the tumor. With this method of providing photodynamic effects, carrier cells need to selectively accumulate more therapeutic agent at the tumor site. In some cases, direct application of a compound of the invention to tumor deposition within a body cavity (eg, the pleural cavity) aids in site retention and allows for more effective intracavitary radiation therapy during surgery.

B. Treatment of specific diseases or pathological conditions by direct supply of therapeutically effective substances 1. Reocclusion and Restenosis after Angioplasty It was experimentally shown that the compounds of the invention can be retained not only on artificial surfaces but also on local vascular sites, even in the presence of continuous blood flow ( See Examples 8 and 12 below).
In addition, the ability to retain a biological effect in compounds of the present invention composed of therapeutically effective substances is in vitro.
o and in vivo (see Examples 4, 13 below).
And 15). Studies of animal model systems have revealed that the primary cell proliferation and migration that causes restenosis after angioplasty occurs within approximately 7-21 days after angioplasty. Therefore, in order to prevent this occurrence, it is necessary to retain an antiproliferative drug in smooth muscle cells of arteries repaired until 7 to 10 days after angioplasty. A compound of the invention made up of a suitable anti-proliferative drug can be delivered to the injured vessel wall during angioplasty and the non-proliferative drug conjugated to the compound retained by the injured cell. Thus, large doses of antiproliferative drugs can be provided directly to the injured cells and, by using the compounds of the invention, they are retained at the injured site for a longer period of time than in the case of systemic drug delivery. be able to. For example, in angioplasty, direct deposition of an antiproliferative drug via a drug delivery catheter allows the drug to be bound to the cell membrane at the site of angioplasty, whereas all unbound drug is
It flows from the artery during surgery. Does the drug bound to resting cells remain in the outer membrane when the catheter is removed?
Alternatively, it moves through the interstitial space to reach a deep cell layer. When these cells become active or in a growth state, the compound of the present invention moves into the cells as the membrane components move inward. Once inside the cell, it becomes possible to exert its anti-proliferative effect. Therefore, the compound of the present invention composed of a suitable antiproliferative drug is mainly supplied to the disease site,
The amount of drug processed at one time in the liver or kidneys can be minimized to reduce the occurrence of serious side effects.

In a preferred embodiment, the compounds of the present invention effective in the treatment of restenosis after angioplasty include heparin,
It is composed of antiproliferative drugs such as hirudin, colchicine, vinca alkaloids, taxol and its derivatives. Application of heparin to smooth muscle cells in culture, or administration to animals following arterial injury, results in reduced growth and thickening of the endomysium and reduced cell proliferation. Compounds of the present invention composed of heparin may be retained on the outer cell membrane of the inner arterial wall rather than being taken up inside these cells by hydrophobic interactions with one or more cell wall components. Composed. In contrast, anti-proliferative drugs such as colchicine, which interfere with the tubulin process, need to be taken up intracellularly in order to exert their anti-proliferative effect. In this example, it is preferred to synthesize the compounds of the invention so that they can be rapidly internalized into the medial wall arterial cells. In a preferred embodiment, colchicine is composed of the bioactive component of the compounds of the invention as an acid cleavable conjugate. Colchicine is inactive in its conjugate form. However, uptake of the compound into intracellular acid vesicles causes release of the drug from the compound, thereby activating it. Thus, active colchicine is delivered intracellularly at its site of action, blocking the tubulin process at that site and thereby blocking cell division.

Particularly preferred for use in the present invention are acid cleavable compounds, including colchicine, as provided by the formula:

[Chemical 7]

Other effective colchicine-containing compounds are given by the formula:

[Chemical 8]

Other antiproliferative agents contemplated for use in the practice of the present invention include the following drugs: angiotensin converting enzyme (ACE) inhibitors, angiopeptin, cyclosporin A, calcium channel blockers, goat- Anti-Rabbit Platelet-Derived Growth Factor Antibody, Terbinafine,
Heavy anions for the binding of Trapidil, interferon gamma, and cationic growth factors.

2. Rheumatoid arthritis The compounds of the invention are particularly suitable for the treatment of rheumatoid arthritis. They provide a means to perform chemical or radiation synovectomy to allow the retention of large amounts of therapeutic agents in joints without significant systemic release to the lymph nodes, spleen or liver. provide. A great advantage over any existing delivery system is that the compounds of the invention are delivered uniformly to the tissue in need of treatment and retained in these cells. In the case of the radiotherapeutic compound of the present invention, the radioactivity released from the compound causes a therapeutic effect on the cells of the synovium. Essentially all compounds in the body were found in the treated joints, and about 70% of the infused compounds remained there on day 6 as detailed in the examples below. This local, long-term retention reduces the amount of radioisotope required for each radiosynovectomy and side effects caused by conventional exposure to systemic exposure to radioisotopes released from joints. Lower.

Particularly preferred radiotherapeutic compounds of the present invention for radiation synovectomy can be synthesized by coupling to a suitable radioisotope, as exemplified below. For example, (1) a chelate containing nitrogen and sulfur, or (2)
A complex is composed of either a chelate containing nitrogen or oxygen and a radioactive metal. The radioisotope should be selected from the group consisting of radioactive halogen, copper, yttrium, rhodium, palladium, indium, iodine, samarium, gadolinium, holmium, erbium, ytterbium, lutetium, rhenium, gold, or combinations thereof. You can

Preferred compounds for use in the present invention are given by the formula:

[Chemical 9] Where L, R, R ₁ and R ₂ are as defined in formula I above; Z represents H or a metal coordination position;

[Chemical 10] Where each R'is independently a hydrogen atom, or an alkyl group, preferably a lower alkyl group, or a substituted lower alkyl, wherein the substituents can be any ester and R '' and R '. '' Is independently a hydrogen atom or an alkyl group, m and n can each be 0 or 1, and M is a radioactive metal selected from the group consisting of rhenium, indium, copper, and palladium. Represent

In a preferred embodiment, the compound of formula VIII above is given by the formula:

[Chemical 11]

[Chemical 12] Where R and R ₁ are hydrocarbon substituents having from 1 to about 30 carbon atoms; X and X ₁ can be the same or different and are O, S, C (CH ₃ ). _Two
Alternatively, it represents Se; A represents a pharmaceutically acceptable anion; Z represents H or a metal coordination position;

[Chemical 13] Wherein each R'independently represents a hydrogen atom or an alkyl group, preferably a lower alkyl group, or a substituted lower alkyl group, the substituents of which can be any ester, R "and R"'Is independently a hydrogen atom or an alkyl group, and m and n can be 0 or 1, respectively; M represents a radioactive metal selected from the group consisting of rhenium, indium, copper and palladium.

Other particularly preferred compounds for use in the present invention are those compounds of the formula:

[Chemical 14] Here, M represents a radiotherapy substance such as rhenium, indium, copper or palladium.

Other effective compounds are given by:

[Chemical 15] Where R and R ₁ are hydrocarbon substituents having 1 to about 30 carbon atoms; X and X 1 can be the same or different and are O, S, C (CH ₃ ) ₂ Or represents Se; A represents a pharmaceutically acceptable anion; M represents copper, technetium, rhodium, palladium, indium, samarium, gadolinium, holmium, erbium, ytterbium, lutetium, rhenium, yttrium, gold, Represents a radiotherapeutic substance selected from the group consisting of erbium, holmium or combinations thereof; n is 2, 3 or 4;
m is 1 or 2; p is 1 to 6.

3. The compounds of the present invention comprised of ovarian cancer chemotherapeutic agents or radiotherapeutic agents allow for high delivery of these drugs directly to the site of ovarian tumor cell growth. In addition, the therapeutic agent is retained in the peritoneal cavity for an extended period of time, thereby preventing tumor cell dissemination. In addition, this can be achieved without causing significant side effects associated with administration of these drugs at high concentrations by the systemic supply system. The compounds of the invention can be delivered intraperitoneally using the Tenckhoff catheter as a post-surgical treatment, or can be delivered as an adjunct treatment during re-lateral laparotomy and surgery. The compounds of the present invention containing acid cleavable colchicine are also effective in the antiproliferative treatment of ovarian tumor cells, as previously described. As mentioned above, these molecules are expected to remain in a non-toxic form on the outer membrane of cells. However, the chemotherapeutic agent can exert its anti-proliferative effect if the compound is taken up into cells where the chemotherapeutic agent is detached from the rest of the compound.

4. The compounds of the present invention composed of psoriasis corticosteroids can be effectively used in the treatment of psoriasis. They provide bulk drug retention at the site of psoriatic lesions, thereby
Enhances the effectiveness of drugs that reduce the proliferation of keratinocytes and immune cells. Furthermore, retention of the compounds of the invention at the site of the lesion prevents penetration of potentially toxic compounds into the circulatory system. This results in the clinical benefit of eliminating the need to stop treatment due to high serum levels of antiproliferative drug after a series of applications over two weeks. According to another application of the invention, platelets or low density lipoprotein (LD) for the early detection of atherosclerosis
In L) the site of atherosclerotic plaque deposits can be located. Platelets are separated from an individual's blood using standard gradient methods, then labeled with indium or technetium as an optional diagnostic component and reinjected intravenously. Suitable methods for binding compounds of the type described herein to platelets are described in US Pat. No. 4,762,701. Within the next 48 hours, radiolabeled platelets accumulate at the site of plaque formation on the arterial wall and gamma emission can be detected at that site using a gamma camera.

Similarly, by standard ultracentrifugation, L
The DL is purified and labeled with a compound of the invention, taking advantage of the pronounced fat content and binding affinity of the compound of the invention for the fat region of biocompatible particles. These radiolabeled LD
L accumulates in the atherosclerotic plaque concentrated area after reinfusion,
Allows detection by nuclear imaging. It is also known that monocytes accumulate in atherosclerotic plaques and can therefore be used to detect their formation; their only limitation is
It is possible to purify a suitable number of monocytes for radiolabelling. Furthermore, it is known that platelets accumulate at thrombotic sites (eg coronary artery thrombosis, deep vein thrombosis, endovascular grafts) and acute rejection sites after organ transplantation. Therefore, autologous platelets separated by standard methods and radiolabeled using the compounds and methods of the present invention, also in combination with drug therapy, allow non-invasive diagnosis of such disease processes.

In addition, chelates can be used to bind radiometal ions, but in the compounds of formula I above, the radioisotope may be a molecule such as a radioactive iodine, carbon, nitrogen, sulfur, phosphorus or yalen atom. It is also possible to produce fluorescent and non-fluorescent compounds as constituents of. Compounds that emit γ-rays of sufficient energy using radiolabeled compounds of the invention can be detected using gamma scintigraphy. If the isotope is a low-energy impervious beta-emitting element, the compound can be used for research using standard beta-counting methods. The biotinylated lipophilic compound of the present invention can act as a multipurpose reagent. For example, these compounds can be used to rapidly attach normally non-adherent cells to selected surfaces. this is,
This is important for assays that require immobilized cells, ie assays that monitor single cells in time. If the cell population is labeled with the biotinylated compounds of the invention, the resulting labeled cells are brought into contact with the surface that binds to streptavidin and the cells are rapidly attached to the surface. The analysis of cells can be started immediately. Fluorescence coupled with biotinylated compounds provides a convenient means of visually monitoring cells during an experiment.

Furthermore, the compound labeled with fluorescent cells can be used for monitoring the growing cells and measuring the growth rate by diluting the fluorescence. (US Patent No. 4,85
9, 584). In this method, when the fluorescence of the labeled compound is reduced to autofluorescence, the sensitivity decreases in 5 to 8 doubling time (depending on the cell type). Streptavidin conjugated with a fluorescent dye can be coupled to, for example, biotinylated cyanine as described herein to achieve fluorescence amplification. By this method, labeled cells can be confirmed even after the fluorescence of the dye is reduced to autofluorescence. If higher sensitivity is required, radiolabeled streptavidin can be attached to the biotinylated compounds of the invention and autoradiography can be performed to identify labeled cells. Another application of biotinylated compounds of the invention is protein binding. For some large proteins, Formula II above
As shown in I, it is impossible to bind the protein to cells by covalently binding the protein to the lipophilic compound of the present invention. In such cases, another binding mechanism is the avidin-biotin binding pair. For this purpose, biotinylated compounds of the invention are used to label target cells and large proteins are conjugated to avidin, or a suitable derivative of avidin, eg streptavidin. The protein is then stably bound to the cells by exposing the biotinylated cells to the avidin-protein conjugate.

C. Pharmaceutical Formulations Pharmaceutical formulations composed of the compounds of the invention are convenient for administration in combination with a salt-free isotonic solution, or a compatible biological medium such as a pharmaceutically acceptable liquid excipient. Can be created as The latter includes various inert oils, for example vegetable oils such as olive oil and peanut oil, or highly produced mineral oils. The concentration of active ingredient in the chosen medium will depend on the nature of the compound and the disease or condition being treated. It is particularly advantageous to compose the pharmaceutical preparation in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete pharmaceutical formulation units adapted for the patient to be treated. Each dosage unit should contain a quantity of active ingredient calculated to produce the desired therapeutic effect in association with the carrier of the chosen drug. Methods of determining appropriate dosage units for arresting cell proliferation or treating other pathological conditions in certain classes of patients are well known to those skilled in the art. For example, in the case of radiation synovectomy, approximately 5 mCI of ⁹⁰ Y (half-life 2.7 days, beta energy 2.2 MeV) or 300 mCi of "" Dy (half-life 2.3) administered in various colloidal forms. Time, beta energy-1.3 MeV) showed clinical efficacy.P. Lee, J. Rh.
eumatol. , 9: 165-167 (1982);
C. Sledge et al., Clin. Orthop. , 18
2: 37-40 (1984). Using standard dosimetry, approximately 0.05 μmol of the appropriate compound adjusted for specific activity of 200 mCi / μmol (eg Reaction Scheme 5
About 10 mCi provided by injection of compound 23)
It was estimated that a similar therapeutic effect could be obtained with a dose of 186 Re (half-life 3.7 days, beta energy 0.98 MeV). Other radiotherapeutic isotopes are known in the art. W. Volkert et al. Nuc
l. Med. 32: 174-185 (1991). In addition, other compounds of the invention are also considered effective in delivering effective dosages of these drugs.

In radiotherapy of tumors, a dose of 80 Gy was shown as a sterilization dose for radiotherapy of solidified tumors when supplied with monoclonal antibodies.
J. Humm, J. Nucl. Med, 27: 1490
~ 1497 (1986). Binding and internalization to cell surface membranes are expected to enhance the effectiveness of these treatments. J. Humm, J. Nucl. Med, 31:
75-83 (1990). The dose of 80 Gy is 200
Provided by injection of about 0.8 nmole of Compound 23 adjusted with mCi / μmol of specific activity. Further, it is expected that this compound and other compounds of the invention will be available for delivery of other radiotherapeutic isotopes known in the art. W. Volkert et al., Supra. . As with other types of anticancer radiotherapeutic agents, the compounds of the present invention can be directly delivered into tumor tissues, body cavities containing scattered tumors, or into blood vessels supplying tumors. C. Hoefnagel, Anti-Cancer
Drugs, 2: 107-132 (1991).

To demonstrate that a sufficient amount of the compound of the invention can be delivered to the pathophysiological site in the treatment of restenosis following angioplasty, Grines et al., Circul.
, 84: II-365 (1991), administered a therapeutic dose of human colchicine at 1 mg / day, but failed to prevent restenosis and failed to prevent 2 ng / ml or 5 nM (mw colchicine = 399). It can be mentioned that a maximum plasma concentration of 4) was produced. B
ochner et al., Handbook of Clini
cal Pharmacology, Little, B
row and Co. , Boston (1983),
pp. 151-152. 0.2 mg / kg / day, assuming (i) similar colchicine absorption and distribution by rabbits; (ii) rabbits of average 3 kg body weight, and (iii) average humans of 70 kg body weight. The dose is Currier et al., Circulation, 80: I.
Used by I-66 (1989), it was successful in preventing restenosis in rabbits at plasma concentrations of 28 ng / ml or 70 nM. Therefore, the colchicine concentration in rabbits showed that the concentration to prevent restenosis was 14 times the concentration achieved in clinical trials. The compounds of the present invention are 100 μM in the above-described compatible binding media, ie at concentrations of 1 shown to be effective in animal studies.
It can be adjusted up to 400 times and can be delivered directly to the arterial wall using a catheter during angioplasty.

The pharmaceutical preparation of the present invention is preferably administered by injection, intraperitoneal infusion, or catheterization.
In addition, oral administration or other administration methods such as aerosol administration may be effective in some cases. The pharmaceutical formulation can be administered at appropriate intervals. Due to the nature of the compounds of this invention, repeated administrations may not be necessary.
Determination of frequency of administration of pharmaceutical formulations is well known to those of ordinary skill in the relevant medical arts. In all cases, the appropriate time between any individual cases will usually depend on the condition of the patient and the type of pathological condition being treated. The following examples are provided to describe the invention in further detail. These examples are intended to illustrate certain aspects of the present invention and cannot be considered a limitation of the present invention.

[0090]

EXAMPLES Example 1 Determination of Membrane Retention Coefficient Membrane retention coefficient (MRC) provides information on how well a given compound is retained in the plasma membrane of cells and is described below. Determine as you did. Generation of erythrocyte ghosts used as model membranes was performed by centrifuging whole blood at 300 xg for 15 minutes to remove plasma and resuspending the cell pellet with 0.83% (w / v) ammonium chloride. Done by. 10,00
Pellet the ghost from ammonium chloride by centrifugation at 0xg for 10 minutes. This ammonium chloride wash treatment is repeated at least 5 times to achieve complete release of hemoglobin from the cells. Ghosts of the compound of interest at concentrations that allow for instrumental analysis or detection of labeled ghosts by fluorescence microscopy, and at the same concentrations used to label cells for the individual applications described above. Label. For the following measurements,
A stock solution of the compound in question was prepared with 2 × 10 ³ molar ethanol and a working dilution of the compound was prepared with isotonic sucrose (52 g / 500 ml distilled water).
After incubating the ghost for 10 minutes with a working dilution of the compound at a concentration of about 1 × 10 ⁹ ghost / ml, the sample was centrifuged at 10,000 × g to pellet the ghost and stained from the sample. The solution was aspirated. The labeled ghosts were resuspended in 1 ml of phosphate buffered saline containing 10% bovine serum (PBS-FBS). 20 μl in triplicate
An aliquot was removed from each sample to determine the amount of total compound present. Samples were centrifuged as above and triplicate 20 μl aliquots were removed from the supernatant for quantitative determination of total unbound compound present.

After sampling, the supernatant is aspirated,
Red blood cell ghost pellet with 1.0 ml PBS-FB
Resuspended in S and resampled as above. This method was repeated at least 6 times and monitoring was performed after 24 hours or more to allow detection of rapidly released compounds and even slower released compounds. For the determination of the amount of compound present in each sample, 20 μl aliquots were extracted into 3.0 ml n-butanol by stirring. Samples were centrifuged at 3000 xg to remove membrane debris and the butanol fraction was analyzed for compound concentration. Fluorescent compounds were analyzed in this manner using the maximum excitation and emission wavelengths of the particular compound analyzed to determine the fluorescence units of each sample. Radiolabeled compounds do not require butanol extraction and can be analyzed directly using a beta or gamma counter.

By measuring the amount of the compound present in each sample as described above, M at each time point after washing or fixing was measured.
Allows RC to be calculated. The value is given by the following formula: represents a _((C T -C ^{_s) /} C T * 100 where _{C T} is a unit determined by the method used for analysis of compound content (compound present in the total sample), C
_s represents the amount of compound present in the supernatant at a particular time point. Comparison of MRC values defines the criteria for identifying the compounds of the present invention, which criteria are: 1) an MRC value measured at each wash step of at least about 90; and 2) at least The difference in the percentage of MRC values during 24 hours is less than about 10%.

[0093]

[Chemical 16]

The data presented in Table I below is one experimental result.
The result is an example of MRC measurement. In Table I, the tables A to C
The compound is a compound of formula XII above, wherein
X and X in compound₁Is C (CH₃)₂Represents Z and
Z1 represents H, and further R / R ₁Is C-5 / C-5 (compound
A), C-10 / C-10 (compound B), C-14 /
Represents C-14 (compound C); compounds represented by D to K are the same
And a compound of formula XIII, wherein Z and Z1 are
Represents H, R / R₁Is C-14 / C-3 (Compound D),
C-18 / C-3 (Compound E), C-20 (3, 7, 1
1,15-Tetramethylhexadecyl) / C-3 (Compound
Product F), C-22 / C-3 (Compound G), C-20 / C
-3 (Compound H), C-18 / C-8 (Compound I), C
-18 / C-5 (Compound J), C-22 / C-3 (Compound
Compound K); and the compound represented by L—N is the above formula X
A compound of IV, wherein Z and Z₁Respectively
N (CH (at ring positions 3 and 6)₃)₂Represents Z
₂Represents H, R represents C-22 (compound L), C-18
(Compound M) and C-26 (Compound N) are shown. Compound K
So the anion is chloride and all the rest
In the compound, the anion is iodide.

[0095]

[Table 1]

The MRC values given in Table I above show excellent correlation with the results obtained from intracellular compound conversion analysis, as already described in the above-mentioned international application PCT / US89 / 00087. .

Example 2 Determination of Membrane Binding Stability The free energy of hydrocarbon chain conversion from the aqueous phase (eg extracellular medium) to the liquid hydrocarbon phase (eg hydrocarbon interior of biological membranes) is determined by the degree of branching, It is known to depend on the degree of unsaturation and the number of methylene groups in the hydrocarbon chain. The free energy of the methylene-hydrocarbon interaction is
Found in hydrocarbons in solvents containing non-polar hydrocarbons with a minimum of methylene groups closest to aqueous mediation, followed by increased methylene groups, and hydrocarbon groups of four or more carbons inside the lipid of the membrane. Is almost equal to what is given. Thus, a hydrocarbon tail (singular or plural, symmetrical or asymmetrical) composed of a polar head group and a linear four or more carbons, such as the compounds of the invention.
The number of carbon equivalents that give approximately equal binding energies when completely immersed in a hydrocarbon solvent can be calculated for all structures, including: Carbonized equivalents = 0 +. 25+. 5+. 75 + n-4 (+)
0+. 25+. 5+. 75 + m-4 (+) ... where n is equal to the number of linear hydrocarbons in the first tail and m is
Equal to the number of linear hydrocarbons in the second tail.

The existence of a correlation between the carbon equivalent of the compound of the invention and its membrane retention coefficient (MRC) was determined experimentally, as described in Example 1 above. For example,
Compounds of the invention with more than 18-19 carbon equivalents have MRC's of 90 or more and show minimal conversion between labeled and unlabeled cells in intracellular compound conversion assays (see above). reference). Therefore, these compounds are also expected to show excellent stability in binding to cells labeled in vivo. But surprisingly this is not the case. In fact, some of the compounds of the invention exhibiting different MRCs show that several compounds of the invention differ by only 10% in vivo.
Shows a fold different reduction rate.

In various practical applications of the compounds and methods of the present invention, it is important that the stability of the bond between the compound and the biofilm in vivo can be assessed using several predictive criteria. For example, in applications such as delivery of therapeutic radionuclides to the tumor site, loss of the compound can cause the radionuclide to produce toxic effects on non-tumor sites, requiring very stable binding to the membrane. . on the other hand,
For applications requiring controlled therapeutic drug delivery, more rapid loss of compound from biological membranes may be desirable. Therefore, an analysis that measures MBS under conditions that approximate those found in vivo is described below.

In performing this analysis, serum albumin (5%), which approximates physiological concentrations, is used. further,
Many of the compounds of the present invention have low solubility in saline containing 5% albumin, thus limiting the volume of "physiological"
A closed system with liquid achieves an equilibrium between the solubility in the membrane and the solubility in the surrounding medium within 24 hours. Analysis of MBS was performed by suspending a reduced number of labeled membrane ghosts in saline containing a fixed volume of albumin to fully approximate the effect of high volume liquids exposing labeled cells in vivo. To do. At 24 hours, the total amount of label present is determined by sampling a well-mixed suspension; the membrane ghosts are then pelleted by centrifugation and the amount of label released in the supernatant is determined. Measured and expressed as percentage of total label. The retention rate was plotted against the number of ghosts per 1 ml of the suspension, and 5 × 10 7 ghosts / ml and 4 × 10 8 ghosts / ml were plotted.
Measure MBS in the lower part of the curve between ml. In this analysis, the compound showing infinite membrane binding stability was 3.5 × 10 5.
There are 10 MBS and the results are expressed as a ratio to this maximum MBS.

The following table lists the data obtained from the MRC measurements described in Example 1 above and the MBS measurements of this example for representative samples of the compounds of the invention. Compounds represented by the O-T is a compound of formula XII, wherein X and X ₁ represents a C (CH _{3) 2,} Z and Z ₁ represents H, R / R ₁ is, C-12 / C-10 (Compound O), C-22 / C-12 (Compound P), C-14
/ C-3 (Compound Q), C-14 / C-14 (Compound R), C-16 / C-16 (Compound S), C-22 / C
It represents -14 (Compound T).

[0102]

[Table 2] * Slezek and Horan, Blood 74: 217
2-77, the half-life of depletion of dye from rabbit erythrocytes (± standard error of the mean) after labeling and reinjection in vivo, determined as described in (1990).

From the above table, the MR
It can be seen that compounds having almost the same C but having different binding stability to biological membranes can be identified in vivo. In various applications of the invention, the additional advantage of using MBS analysis to select compounds with appropriate binding characteristics is:
Whereas the effect of changes in headgroup structure on membrane binding stability can be confirmed, this can only be done poorly by MRC analysis. As can be seen in Table III below, the length of the hydrocarbon tail (expressed as a carbon equivalent for comparison of symmetrical and asymmetrical structures) and the head group structure together are related to membrane-bound stability. The effect of the headgroup is more pronounced for low to moderate carbon equivalent numbers.
Therefore, the same effect should be sought for other types of head groups with various functional groups useful for the application of the present invention (for example, radiometal chelates, proteins, peptides, radionuclides, and the like). And the equilibrium between the action of the head group and the carbon equivalent required to reach the desired membrane-bound stability can be estimated. Compounds with at least about 30% or more MBS are expected to be beneficial in the application of the invention of the type described herein. Furthermore, it can also be observed that the substituents on the head group can have a significant effect on the membrane binding stability even if the number of carbon equivalents is kept constant. As seen in Table III below, the AC and AD compounds differ only in the head group substituents, but each MBS is very different.

In Table III, the compounds represented by U, W and Y are
A compound of formula XIII above, wherein X of each compound
And X₁Represents O, Z and Z₁Represents H, R / R
₁Is C-22 / C-30 (compound U), C-14 / C
-14 (Compound W), C-18 / C-18 (Compound Y)
Further, a compound represented by V, X, AA is also represented by the formula XII
A compound of X and X₁Represents S, and Z
Z1 represents H, R / R ₁Is C-22 / C-3 (compound
V), C-14 / C-14 (Compound X), C-18 / C
It represents -18 (compound AA). Represented by AB, AC, AD
The compound is of formula XII above, wherein X and
And X₁Is (CH₃)₂Represents Z₁Is H, then R / R₁
Is C-14 / C-22 (compound AB), C-14 / C
-3 (compounds AC and AD). In compound AB, Z
Is -CH₂Represents -NHOCH. In compound AC, Z is
Represents H. In compound AD, Z is non-trivial as shown below.
Represents a cleavable colchicine derivative.

[0105]

[Chemical 17]

Many of the symmetrical dyes shown in Table III have M
olecular Probes, Inc. , Euge
It can be obtained as a product from ne, OR.

[Table 3]

Example 3 Preparation of Compounds a. 5-Aminomethyl-1′-docosanyl-1-tetradecyl-3,3,3 ′, 3′-tetramethylindo-carbocyanine iodide The title compound was prepared as described above according to Reaction Scheme 1. In the following description, the numbers in parentheses refer to the correspondingly numbered reagents shown in Reaction Scheme 1. The product obtained is given by the formula of compound 8, where X and X ₁ represent C (CH ₃ ) ₂ , R / R ₁ represents C ₁₄ H ₂₉ / C ₂₂ H ₄₅ and A represents I. 5- (N-phthalimidoaminomethyl) -2,3,3- (3H) -trimethylindolenine (1) was added to Gale et al., Aust. J. Chem. Three
0: 693 (1977) was modified and adjusted.
2,3,3- (3H) -trimethylindolenine (2
3.85 g, 0.15 mol, Aldrich), 15
It was dissolved in 0 ml concentrated sulfuric acid. Next, the flask was placed in an ice bath and N-hydroxymethylphthalimide (26.
55 g, 0.15 mol, Fluka) was added portionwise over 30 minutes. The ice bath was removed and the solution was stirred at room temperature for 5 days. The reaction mixture was then poured onto 200 g of crushed ice and pour with 50% NaOH solution, keeping the temperature below 35 ° C. by adding ice as needed.
The H was adjusted to 9.0. The resulting precipitate was collected by filtration, washed with distilled water and dried at elevated pressure overnight. The crude product was recrystallized from methylene chloride / hexane to give 5- (N-phthalimidoaminomethyl-2,
Generate 3,3- (3H) -trimethylindolenine (1) (30 g, 63%).

Docosanyl 4-chlorobenzenesulfonate was prepared using the method described in PCT / US89 / 00087. The tetradecyl 4-chlorobenzene sulfonate was prepared in a similar manner. 5- (N-phthalimido-aminomethyl) -2,3,3- (3H) -trimethylindolenine (6.36 g, 2 mmol),
Tetradecyl-4-chlorobenzene sulfonate (7.
62 g, 2 mmol) and combine them at 130 ° C for 2
Heated for hours. The reaction mixture is then cooled to room temperature and the crude product is purified from pure 5- (N-phthalimidoaminomethyl) -1-tetradecyl-2,3,3- (3H) -trimethylindolenium 4-chlorobenzenesulfonic acid. Salt (2) (10.23 g) 72%), m.p. p. Recrystallize from ethyl acetate to yield = 141 ° C.

2,3,3-Trimethyl- (3H) -indolenine (6.26 g, 0.04 mol Aldric
h) and n-docosanyl-4-chlorobenzenesulfonate (20.02 g, 0.04 mol) were heated together at 140 ° C. for 3 hours with stirring. The reaction mixture was then cooled to room temperature so that it became a waxy solid. Then dissolve the solid in ethanol (250 ml) and
1 of saturated KI solution was added and the solution was stirred for 30 minutes. 1 liter of cold water was added and stirring was continued for another 15 minutes. The resulting precipitate was collected, washed twice with distilled water and dried under high pressure overnight. The crude material was recrystallized from methylene chloride / hexane to give pure 1-docosanyl-2,3,3- (3H) trimethyl-indolenium iodide (5) (14.5 g, 61%), m.p. p. = 1
Generated 07-110 ° C. 1-docosanil-2,3,
3- (3H) -Trimethylindolenium iodide (8.94 g) 0.015 mol), N, N-diphenylformamidine (2.94 g) 0.015 mol, Ald
rich) and acetic anhydride (60 ml) in a round bottom flask fitted with a condenser, purge the flask with argon,
Then, the cooler was connected to the drying tube. The flask was placed in a preheated (160 ° C.) oil bath and refluxed for 60 minutes. The flask was then removed from the oil bath and cooled to room temperature. It was then transferred to a 1 liter Erlenmeyer flask and diluted with ethanol (60 ml) followed by 60 ml saturated KI solution and the mixture was stirred for 30 minutes. Cold water (800 ml) was added and stirring was continued for another 15 minutes. The precipitated product was collected by filtration, washed with distilled water, dried under high pressure overnight, and 2- (β-acetonylidvinyl) -1-docosanyl-3,3- (3H) dimethylindolenium. Iodide (6) (10.52 g, 9
5%), m.p. p. = 98-100 ° C. The raw product was used without further production.

5- (N-phthalimidoaminomethyl)-
1-Tetradecyl-2,3,3- (3H) -trimethylindolenium 4-chlorobenzene-sulfonate (2) (14.1 g, 20 mmol) was dissolved in 300 ml concentrated HCl. Slowly add 1
The mixture was heated to 15 ° C (be careful of bubbling) and refluxed for 22:00. The mixture was then cooled to room temperature and placed in an ice bath. The pH was adjusted to 9.0 with ammonium hydroxide (30%), keeping the temperature between 15 and 20 ° C. Then, dilute the solution with distilled water to double the volume,
It was extracted with methylene chloride (3 x 200 ml). Combine the methylene chloride extracts, dry over magnesium sulfate and concentrate to give a yellow oil (3) (6.9 g, 90%) 5-
Aminomethyl-3,3-dimethyl-2-methylene-1-
This produced tetradecyl-indoline. 5-Aminomethyl-3,3-dimethyl-2-methylene-1-tetradecyl-indoline (3) (14.88 g, 38.75 mm)
ol) in methyl formate (75 ml),
Heat to reflux (55 ° C.) under argon for 24 hours. The solution was then cooled to room temperature and the methyl formate was evaporated. The residue was recrystallized from hexane to give 5- (N
-Formylaminomethyl) -3,3dimethyl-2-methylene-1-tetradecyl-indoline (4) (10.8
5 g, 68%).

2- (β-acetone ylide vinyl) -1-
Docosanyl-3,3- (3H) -dimethylindolenium iodide (6) (740 mg, 1 mmol) and 5-
(N-formylaminomethyl) -3,3-dimethyl-2
-Methylene-1-tetradecyl-indoline (4) (3
30 mg, 0.8 mmol) and anhydrous sodium acetate (150 mg, 1.8 mmol) were dissolved in isopropanol, and the mixture was stirred at room temperature for 24 hours. After that, the solution was transferred to a 250 ml Erlenmeyer flask, and ethanol (20
ml) and saturated KI solution (20 ml) and the mixture was stirred for 30 minutes. The product was precipitated by adding 100 ml cold water and the resulting solution was added to 15
Stir for minutes. The precipitate was collected by filtration, washed with distilled water and dried under high pressure overnight. Raw product (870
mg) was divided into two groups, each of which was subjected to flash column chromatography (silica gel, 1 in methylene chloride).
0% isopropanol) to give pure 1 '
-Docosanil-5 (N-formylaminomethyl) -1-
This produced tetradecyl-3,3,3 ', 3' tetramethylindocarbocyanine iodide (7) (414 mg, 52%).

100 ml of concentrated HCl: methanol solution (prepared by mixing 11 ml of concentrated HCl and 120 ml of methanol) was added to 1'-docosanyl-5- (N-formylaminomethyl) -1-tetradecyl-3,3, 3 ',
3'-Tetramethylindocarbocyanine iodide (7) (250 mg) was added and the solution was cooled to 16-2 at room temperature.
Stir for 4 hours. After that, the solution is ice water (100 ml)
It was diluted with and cooled in an ice bath and saturated sodium bicarbonate solution was added slowly to adjust the pH to 7.5-8.0. The aqueous phase is then extracted with methylene chloride (2 × 100 ml), the combined organic phases are dried over sodium sulphate, filtered, concentrated (Buchi bath temperature <30 ° C.) and then dried under high pressure to give the product. (8) (240 mg,
98%).

B. 2- [3- (2,3-dihydro-3,
3-dimethyl-5-aminomethyl-1-tetradecyl-
(2H) -Indole-2-ylidene) -1-propenyl] -1-docosanyl-benzoxazolium iodide Further the title compound was prepared according to Reaction Scheme 1. In the following description, the numbers in parentheses indicate the corresponding numbers of reagents shown in Reaction Scheme 1. The resulting product has the formula of compound (8), where X is C
(CH ₃ ) ₂ , X ₁ is oxygen, and R / R ₁ is C ₁₄ H ₂₉ / C
₂₂ H ₄₅ , A represents iodide. 2-Methylbenzoxazole prepared as described above (2.65 g, 19.
9 mmol, Aldrich) and docosanyl-4-chlorobenzenesulfonate (10.0 g, 19.9 mmo).
l) stirred solution at 160-170 ° C. (oil bath temperature)
Heated for 6 hours. The reaction mixture was then cooled to room temperature and the resulting solid mass was recrystallized from methylene chloride to give pure 1-docosanyl-2-methylbenzoxazolium-4-chlorobenzenesulfonate (5).
(7.3 g, 58%), m.p. p. = 124-125 ° C produced.

1-Docosanyl-2-methyl-benzoxazolium-4-chlorobenzenesulfonate (5)
(1.5 g, 2.36 mmol), N) N'-diphenyl-formamidine (0.462 g, 2.36 mmo).
l, Aldrich) and acetic anhydride (7 ml) in a stirred solution in an oil bath (heated to 160 ° C in advance).
Reflux for 0 minutes. After cooling to room temperature, the mixture was diluted with absolute ethanol (15 ml), then saturated potassium iodide solution (10 ml) and stirred for 30 minutes. Then water (150 ml) was added and the precipitated product was collected by filtration, washed with water and dried under high pressure overnight. The dried raw product was recrystallized from ethyl acetate to give pure 2
-([Beta] -Acetone ylidevinyl) -1-docosanyl-benzoxazolium iodide (6) (1.51 g, 90
%), M. p. = 67-68 ° C. 3a above
2- (β-acetone ylide vinyl)-
1-docosanyl-benzoxazolium iodide (6)
(1.10 g, 1.53 mmol), 5- (N-formylaminomethyl) -1-tetradecyl-3,3-dimethyl-2-methylene iodide (4) (63 mgs, 1.5
3 mmol), triethylamine (0.5 ml) and ethanol (25 ml) were heated to reflux for 1 hour. The solution was then cooled to room temperature, transferred to an Erlenmeyer flask and diluted with ethanol (40 ml) and saturated KI solution.
The mixture was then stirred for 30 minutes, 200 ml of cold water was added and the solution was extracted with methylene chloride. The methylene chloride extracts were combined, dried over magnesium sulphate, filtered and concentrated to give the raw product. This product was subjected to flash column chromatography (silica gel,
Purified with 5% methanol in methylene chloride), 2-
[3- (2,3-dihydro-3,3-dimethyl-5-
(N-formylaminomethyl) -1-tetradecyl-
(2H) -Indole-2-ylidene) -1-propenyl] -1-docosanyl-benzoxazolium iodide (7) (305 mg, 20%) was produced.

25 ml of concentrated HCL: methanol solution prepared as in a) above was added to 2- [3- (2,3-dihydro-3,3-dimethyl-5- (N-formylaminomethyl) -1- Tetradecyl- (2H) -indole-2
-Ylidene) -1-propenyl] -1-docosanyl-benzoxazolium iodide (7) (50 mg),
The solution was stirred at room temperature for 16-24 hours. Then dilute the solution with ice water (30 ml) and cool in an ice bath,
The pH was adjusted to 7.5-8.0 by the slow addition of saturated sodium bicarbonate solution. The aqueous phase was then extracted with methylene chloride (2 × 50 ml), the combined organic phases were dried with sodium sulfate, filtered and concentrated (Buch bath temperature 0-5 ° C.), then dried under high pressure to make the product. (8) (48 mgs, 99%).

C. 5-Aminomethyl-1'-docosanyl-1-tetradecyl-3,3,3 ', 3'-tetramethylindocarbocyanine iodide terephthaloyl N-hydroxyl succinimide ester derivative at room temperature under argon atmosphere using a cannula tube , Di-terephthalic acid in dry tetrahydrofuran (30 ml)
A stirred solution of N-hydroxysuccinimide ester (100 mgs, 0.278 mmol) (prepared by reacting terephthaloyl chloride with N-hydroxysuccinimide) was prepared in tetrahydrofuran (10 ml) as in Example 3a above. Aminomethyl-
1'-docosanyl-1-tetradecyl-3,3,3 ',
A solution of 3'-tetramethylindocarbocyanine iodide was added. The resulting solution was stirred for 2 hours then concentrated on a Buchi. The resulting raw product was purified by flash column chromatography (silica gel, 5% methanol in methylene chloride) to provide the title compound (101 mg, 34%).

D. Material P-5-lipophilic cyanine conjugate Material P (21 mg, 0.013 mmol) and a stirred solution of the product of Example 3c above (30 mg, 0.024 mmol) in dry dimethylformamide (10 ml). , Triethylamine (6
0 μl) and the resulting solution at 4 ° C at 0-2 ° C.
Stir for hours. After that, trifluoroacetic acid (100μ
The reaction product was quenched by addition of 1), the solution was transferred to a 250 ml flask, diluted with water (80 ml) and lyophilized overnight. The resulting sample was purified by passing through a column of octadecyl silica gel,
Elute unconjugated peptide first with 80: 20: 1 (methanol: water: trifluoroacetic acid), then 100: 2: 1.
The desired product was eluted with (methanol: water: trifluoroacetic acid). Fractions containing the peptide-lipophilic cyanine conjugate were combined, concentrated on a Buchi, and the residue was lyophilized from water (50 ml) to yield a pure violet powdered conjugate (14 mgs, 40 mg). %). The purity by hplc is 90% or more, and it is 0.0 from the released substance P.
It was 2% smaller. The conjugate thus obtained is given by the following structural formula (using the conventional three letter code to indicate the amino acid sequence of substance P):

[Chemical 18]

Since both the peptide and the cyanine reporter are linked to the spacer component via an amino bond, each of the resulting conjugates should be relatively stable in vivo.

E. 2- [3- (2,3-dihydro-3,
3-Dimethyl-5-(+)-biotinamide methyl-1
-Tetradecyl- (2H) -indole-2-ylidene) -1-propenyl] -1-docosanyl-benzoxazolium iodide 2- [3- prepared as described in Example 3d above.
(2,3-Dihydro-3,3-dimethyl-5-aminomethyl-1-tetradecyl- (2H) -indole-2-
Iridin) -1-propenyl] -1-docosanyl-benzoxazolium iodide (53 mg, 0.055 mmo
The solution of l) was cooled in an ice bath under argon gas in dimethylformamide. To this solution, (+)-biotin 4-nitrophenyl ester (23 mg, 0.63 m
mol, Aldrich) was added, then the imidazole (16 mg, 0.23 mmol, Aldrich) and the solution were stirred in the ice bath for 1 h and left at room temperature overnight. The reaction mixture was then concentrated under high pressure in a Buchi and the residue was flash chromatographed (silica gel, 7.5% methanol in methylene chloride then 10% methanol in methylene chloride) to yield the title compound (22 mg. , 36%).

F. 5- {6- (6-(+)-biotinoylamidehexanamide) -hexanamidemethyl}-
1'-docosanyl-1-tetradecyl-3,3,3 ',
3′-Tetramethylindocarbocyanine iodide A method similar to that described in Example 3e above was used again for the following aliquots of reagent: prepared as above 5
-Aminomethyl-1'-docosanyl-1-tetradecyl-3,3,3 ', 3'-tetramethylindocarbocyanine iodide (90 mgs, 0.09 mmol), 6
[(6- (Biotinoyl) amino) hexanoylamino] caproic acid N-hydroxy-succinimidyl ester (55 mg, 0.097 mmol, (Molecu
lar Probes) and dimethylformamide (2
0ml) and imidazole (20mgs, 0.24mmo
l). Flash chromatography (silica gel, 10% methanol in methylene chloride) produced the title compound (27.5 mgs, 21%).

Compound 2- [3- (2,3-dihydro-
3,3-Dimethyl-5- (6-{(+)-biotinamide} -hexamidomethyl) -1-tetradecyl- (2
H) -Indole-2-ylidene) -1-propenyl]
-1-docosanyl-benzoxazolium and 2- [3-
(2,3-dihydro-3,3-dimethyl-5- (6-
(6 (+)-Biotinoylamide hexamido) -hexamidomethyl) -1-tetradecyl- (2H) -indole-2-ylidene) -1-propenyl] -1-docosanyl-benzoxazolium salt is N-hydroxysuccinimidyl 6- (biotinamide) caproate, 6
[6-((Biotinoyl) amino) hexanoylamino] caproic acid N-hydroxysuccinimidyl ester was prepared in the same manner (Molecular).
r Probes).

G. Nn-docosanyl-N'-n-teto
Ladecyl-5-tributylstannyl-3,3,3 ', 3'
-Tetramethylindo-carbocyanine chloride The title compound was synthesized according to Reaction Scheme 2. Got
The obtained product is given by the formula of compound (13), and X and
And X₁Is C (CH₃)₂Represents R / R₁Is C_{twenty two}H ₄₅/ C₁₄
H₂₉And A represents Cl. 4-Indopheni
Luhydrazine was prepared according to Blakie et al. Chem. S
oc. 313: 296 (1924).
Arranged Sodium nitrate dissolved in water (100 ml)
(16.56 g) 0.24 mol, Aldrich)
4-iodoaniline (43.9) in ice water (600 ml)
g, 0.20 mol, Aldrich) and cooled to 0-2 ° C
Within 45 minutes in the solution with concentrated hydrochloric acid (200 ml)
Was added drop by drop. The reaction mixture is further stirred at 0-2 ° C.
For 30 minutes, and then the temperature of the reaction mixture is 0 to 2 ° C.
While maintaining between c. Salt in HCl (150 ml)
Tin (II) iodide (151.68 g, 0.8 mol, Ald
rich) was added drop wise over 90 minutes.
After this addition, warm the resulting solution to room temperature.
And stirred for 3 hours. Then separated from the solution yellow
The solid was collected by filtration and placed in ice water (800 ml).
And adjust the pH to 10 with 25% aqueous potassium hydroxide.
It was The resulting solid was collected by filtration and washed with a little water.
Washed and dried under vacuum. Then the product is
Put in Ruen (400ml) to remove insoluble impurities
Filtered. Hexane (1200 ml) was added,
While cooling in the refrigerator, the separated yellow needles are filtered.
So collect, wash with hexane (100 ml), high vacuum
Dried under pure 4-iodophenyl hydrazine
(23.36 g, 50%), m.p. p. = 94 ° C produced
It was

Moreau et al., Eur. J. Med. C
hem. Chim. Ther. , 9 (3): 274-2.
80 (1974) modified to include 5-iodo-2,
The 3,3-trimethyl- (3H) -indolenine (9) was prepared. 4-Indophenyl-hydrazine (23.3
6 g, 0.0998 mol) and ethanol (100 m
2-methylbutanone in 1) (8.59 g, 0.099
(8 mol) was refluxed for 3 hours. Then, ethanol (1
Concentrated sulfuric acid (9.92 g, 0.
0998 mol) was added drop-wise over 1 hour and the resulting solution was refluxed for a further 3 hours. After cooling to room temperature, the precipitated solid was removed by filtration, the filtrate was concentrated to 80 ml and then poured into ice water. The aqueous solution was then extracted with methylene chloride and the combined organic phases were dried over magnesium sulfate, filtered and concentrated to yield the raw product (26.9g, 95%). Pure product 5-iodo-2,3,3-trimethyl- (3H)
-Indolenine (9) (16.7g, 59.0%)
b. p. Obtained after vacuum distillation at 81-88 ° C and 0.03 mmHg.

5-Iodo-2,3,3-trimethyl-
(3H) -Indolenine (2.85 g, 0.01 mol)
And n-docosanyl-4-chlorobenzenesulfonic acid (5.55 g, 0.01 mol) were heated to 130 ° C (oil bath temperature) with continuous stirring for 4 hours. The cooled reaction mixture was then recrystallized from ethyl acetate and
-N-docosanyl-5-iodo-2,3,3-trimethylindolinium 4-chlorobenzenesulfonate (1
0) (4.62 g, 59%) m.p. p. = 118 ° C tannin crystals were provided. 2,3,3-trimethyl- (3
H) -Indolenine (6.36 g) 0.04 mol, Al
Both drich) and n-tetradecyl-4-chlorobenzene-sulfonate (15.52 g, 0.04 mol) were heated at 130 to 135 ° C (oil bath temperature) for 3 hours with continuous stirring. The raw sample was then dissolved in ethanol (200 ml) and then stirred with saturated potassium iodide solution (50 ml) for 30 minutes. Cold water (5
00 ml) was added and the precipitate was collected by filtration and washed thoroughly with cold water. The dried raw material was crystallized from ethyl acetate and the collected crystals were washed thoroughly with ether and dried to give N-tetradecyl-2,3,3-trimethylindolinium iodide (5) (12.8 g, 66.8
%), M. p. It produced 97 ° C.

N-docosanyl-5-iodo-2,3,3
-Trimethylindolinium 4-chlorobenzenesulfonate (2.36 g, 3.0 mmol), N, N'-diphenylformamidine (0.59 g, 3.0 mmo
l, Aldrich) and 50 ml of acetic anhydride (20 ml)
Place in a ml round bottom flask and place it in a reflux condenser and stir bar under an atmosphere of argon. Then, this flask was put into an oil bath previously heated to a constant temperature of 170 ° C., and the mixture was refluxed for 60 minutes. Then, the reaction flask was cooled to room temperature and transferred to a 500 ml Erlenmeyer flask. Then, the flask was put in an ice bath and saturated potassium iodide solution was added. After stirring for 15 minutes,
Cold water (250 ml) was added and the mixture was stirred for a further 15 minutes. The precipitated product, Nn-docosanyl-5-iodo-2- (β-ayatonilide vinyl) -3,3-dimethylindolinium iodide (11) (2.37 g, 9
1%) m. p. = 170 ° C. (decomp) was collected by filtration and dried.

N-docosanyl-5-iodo-2- (β-
Acetonilide vinyl) -3,3-dimethylindolinium iodide (511 mgs) 0.59 mmol) and N
-Tetradecyl-2,3,3-trimethylindolinium iodide (233 mgs, 0.47 mmol) and anhydrous sodium acetate (4
8 mgs, 0.59 mmol, Aldrich) was stirred at room temperature for 24 hours. The deep red mixture was then transferred to an Erlenmeyer flask and diluted with ethanol (25ml). Then silver acetate (492mgs) dissolved in water (25ml)
2.95 mmol, Aldrich) was added and the solution was stirred for 15 minutes. Next, ethanol (25 ml) and saturated sodium chloride solution were added, and the mixture was continuously stirred for 15 minutes. After that, the solution was transferred to a separating funnel, and water (200 m
It was diluted with l) and extracted with methylene chloride (2 x 100 ml). The organic phase is dried over anhydrous magnesium sulfate,
Filtration and evaporation provided the raw product. The raw product was purified by flash chromatography (silica gel, 5% methanol in methylene chloride first, followed by 8% methanol in methylene chloride) and N-docosanyl-
N'-tetradecyl-5-iodo-3,3,3 ', 3'-
Tetramethyl indocarbocyanine chloride (12) (2
72 mg, 58%).

N-docosanyl-N'-tetradecyl-5
-Iodo-3,3,3 ', 3'-tetramethylindocarbocyanine chloride (12) (200 mg, 0.2 mmo
Degassing was carried out by dissolving l) in dry toluene (15 ml, freshly distilled from calcium hydroxide) and bubbling the resulting solution through an argon atmosphere. Next, Bis- (n-tributyltin) (0.237
ml, 0.47 mmol, Aldrich) was added with a syringe and tetrakis- (triphenylphosphine) palladium (0) (2.34 mgs, 2.0 μmol, Ald.
rich) was added. The resulting solution is 4 under argon.
Refluxed for 8 hours. The toluene was then removed under vacuum and the residue was subjected to flash chromatography (silica gel,
Performing 5% methanol in methylene chloride) gave the title compound (13) (71 mg, 31%). Found
M +, 1122C ₇₁ H ₁₂₃ N ₂ Sn requires
1122. Compound (13) is described by Wilbur et al. N
ucl. Med. , 30: 216-226 (1989).
Is a versatile intermediate for attachment to radioactive halogen atoms using the method described by.

H.¹⁸⁶Re-chelate lipophilic cyan
Conjugate The title compound was prepared according to Reaction Scheme 5 shown above.
It was adjusted. The reference numbers used in this reaction refer to those in Reaction Scheme 5.
Corresponds to numbers. The compound obtained is of the formula 24
Given by X and X₁Is C (CH₃)₂And R /
R₁Is C₁₄H₂₉/ C_{twenty two}H₄₅And A represents I
It Bifunctional chelate, 400 μL of tetrahydro
Compound 22 (84.5 m dissolved in furan (THF)
g, 0.291 mmol) (8) (66.8 mg)
0.066 mmol) in a pear-shaped flask.
I got it. The solution was stirred at room temperature. Thin layer chromatograph
I (TCL; silica, 90:10 CH_TwoCl _Two: Me
OH) continued the reaction. Hexa 4 hours later
The reaction mixture is diluted with hexane and 75:25 THF:
Make a hexane solution and use short-path silica gel (20 g).
I put it in rum. Eluent is a shade of bromocresol green
50:50 of excess compound 22 until sexual reaction
Elute with Sun: EtOAc. 90:10 solvent composition
CH₂C1₂: Change to MeOH and elute compound 22
It was The solvent was evaporated under reduced pressure to give 65.0 mg of compound
Product 23 was produced.

High magnetic field ¹ H nmr (300 MHz)
Showed a downfield shift of the benzylmethylene protons from 3.9 ppm to 4.5 ppm, as previously seen with the formyl protected compound 8. Relative integration of signals at 4.5 ppm and 8.4 ppm
integration) showed a binding ratio of 1: 1.
The product, compound 23, was converted to the HCl salt by addition of HCl gas / ethanol solution. A solution of oxalic acid in ethanol was added as an antioxidant, the solution was evaporated and the residual solid was stored under nitrogen. 0.1N NaOH
(0.181 mCi / nmol) 60 n in 107 μl
mol Na ¹⁸⁶ ReO ₄ in 150 μl of conversion chelate solution (200 mg citric acid, 40 mg SnCl ₂ H ₂
O and 20 mg of gentisic acid were dissolved in ₂ mL of H ₂ O), followed by 43 μL of 0.1 M NaOH. The solution is vortexed for 1 minute and stirred at 55 ° C for 10 minutes.
Heated for minutes. Ethanol (0.6gmL) was added to the reaction mixture, followed by compound 23 (0.8mg, 0.69μ).
300 pL of ethanol solution of (mol) was added. The solution was vortexed for 15 seconds and heating continued for 1 hour. The reaction mixture was loaded onto a semiprep HPLC column (Waters).
Novapak, 7.5 x 300 mm), A:
Gradient B from 50:50 to 100% B, flow rate 4.0.
Elute at mL / min for over 30 minutes. (Solvent A
Is 0.1% trifluoroacetic acid (TFA) and 0.05%
75:25 H2O: CH3CN with gentisic acid. Solvent B, 20:80 containing 0.1% of TFA and 0.05% gentisic acid CH ₃ CN: THF). The fraction eluted for about 20 minutes was confirmed as the fraction containing the title compound.

The above fractions were mixed with Solvent 1 (0.02 in H ₂ O).
Dilute 1: 1 with 5% gentisic acid) 1 and first add 10
Sep-Pa washed with mL of solvent 2 (0.05% gentisic acid in EtOH) followed by 10 mL of solvent 1.
It was placed in a k column (C-18, Waters 36805). Column is 10 mL solvent 1, 150 μL solvent 2
, And 300 μL of solvent 2. The solvent was evaporated by a stream of N _{2 to} a volume of 10 μL and then diluted with EtOH to 40 μL. It was Specific activity is
186 ± 2 against the theoretical value of 181 mCi / μmol
It was 1 mCi / μmol. Product recovery is based on total Re
It was less than 9% of -186. The radioactive purity was measured by HPLC after 1 day and was found to be less than 94%. Most void volume (void v) of impurities (~ 5%)
and was considered to be free perrhenate.

I. 7-N (5-oxopentanoyl) deacetylcortisine lipophilic cyanine hydrazone conjugate and acid cleavability measurement (a) Preparation The title compound was prepared according to the general method described in Reaction Scheme 3 above. . The reference numbers here correspond to the numbers shown in Reaction Scheme 3. The product obtained is given by the formula of compound 19, wherein X and X ₁ = C (C
_{H 3) 2, R / R} 1 = C 14 H 29 / C 22 H 45, A represents Cl. 5-aminomethyl-1′-docosanyl-1-tetradecyl-3,3,3 ′, 3′-tetramethylindocarbocyanine iodide (Compound 8) was used, where R was C ₁₄ H ₂₉ ; R ₁
Was C ₂₂ H ₄₅ ; X and X ₁ = C (CH ₃ ) ₂ was prepared using the general method of Reaction Scheme 1.

A stirred solution of monomethyl glutarate (0.14 ml, 1.1 mmol) in dimethylformamide (15 ml, distilled from lithium aluminum hydroxide).
Aldrich) with N-hydroxysuccinimide (126.5 mg, 1.1 mmol, Aldrich)
Was added at room temperature. The mixture was cooled in an ice bath and dicyclohexylcarbodiimide (226.6 mg, 1.1
mmol) was added. The reaction mixture was gradually warmed to room temperature and stirred for a further 3 hours. Then, the reaction mixture was freshly prepared Compound 8 (700 mg, 0.7 mmo
l) and transfer the reaction mixture to room temperature for 3 hours at room temperature.
I kept stirring at 0:00. The dimethylformamide was then removed under high pressure and the resulting raw product was diluted with absolute ethanol (250 ml) and water (250 ml). Then silver acetate (434 mg, 2.6 mmol) was added and stirred for 30 minutes, then saturated sodium chloride solution (100 ml) was added and the mixture was stirred for another 30 minutes. The aqueous layer was extracted with methylene chloride (4 x 100 ml), the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. 'The residue was flash chromatographed (silica gel, eluting with 2.5% in methylene chloride followed by 10% methanol) to give 5- [N-
(Monomethylglutaryl) -aminomethyl] -1'-docosanyl-1-tetradecyl-3,3,3 ', 3'-tetramethylindocarbocyanine chloride (14) (520
mg, 63%).

The obtained 5- [N- (monomethylglutaryl) -aminomethyl] -1'-docosanyl-1-tetradecyl-3,3,3 ', 3'-tetramethyl-indocarbocyanine chloride (520 mg) , 0.44 mmol) in anhydrous ethanol was added slowly to anhydrous hydrazine (5 ml, Aldrich) at room temperature. The reaction mixture was continuously stirred for 2 hours, then
Concentrate by rotary evaporation and flash to the residue.
Chromatography (silica gel, 10 in methylene chloride
%, Followed by 30% methanol).
[N- (monohydrazino) glutaryl-aminomethyl]
-1'-docosanyl-1-tetradecyl-3,3,3 ',
3'-Tetramethyl-indocarbocyanine chloride (1
5) (110 mg, 27%) was produced. Glutaric anhydride (111.2 mg, 0.975 mmol, Aldri
ch) at room temperature with stirring deacetylcolchicine (16) (0.2902 g) 0.8 in methylene chloride (5 ml).
1 mmol, Molecular Probes) and the reaction mixture was continuously stirred at room temperature for 2 hours.
The methylene chloride was then removed by rotary evaporation and the raw product was dried under high vacuum to yield 7- (N-glutaryl) deayatylcolchicine (0.397g, 100%) as a solid.

Next, carbonyldiimidazole (0.1
97 g, 1.22 mmol, Aldrich) was stirred in a solution of 7- (N-glutaryl) deayatylcolchicine (0.397 g, 0.83 mmol) in dimethylformamide (5 mL, freshly distilled from lithium aluminum hydride). To the reaction mixture at room temperature and the reaction mixture was kept stirring at room temperature for 2 hours. A precipitate formed in the solution within this time. Dimethylformamide was removed by vacuum distillation and the residue was dissolved in methylene chloride (5 mL). Tetrabutylammonium borohydride (250 mg, 0.972 mmol, Aldric
h) was added and the reaction mixture was stirred for 3 hours. It was then poured into water (50 mL), the organic layer was separated and the aqueous layer was re-extracted with methylene chloride (2 x 25 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. Flash chromatography (silica gel, 10% methanol in methylene chloride) on the raw product.
Was carried out and 7-N- (5-hydroxylpentanoyl)
Deacetylcolchicine (17) (72 mg, 25%) was produced.

7-N- (5 in methylene chloride (5 mL)
-Hydroxylpentanoyl) deacetylcolchicine (72 mg, 0.16 mmol) solution at room temperature with pyridinium chlorochromate (41.3 mg, 0.19 mm
ol, Aldrich) was added and the mixture was stirred for 2 hours. Then it was poured into water (50 mL), the organic layer was separated and the aqueous layer was re-extracted with methylene chloride (2 x 25 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated. Fla for the residue obtained
sh chromatography (silica gel, 1 in methylene chloride
0% methanol) to give 7-N- (5-oxopentanoyl) deacetylcolchicine (18) (35 mg,
49%) as an oil.

5- [N in absolute ethanol (20 ml)
-Glutaryl (monohydrazino) -aminomethyl]-
1'-docosanyl-1-tetradecyl-3,3,3 ',
3'-Tetramethylindocarbocyanine chloride solution (110 mg, 0.12 mmol) was added to 7-N- (5-
Oxopentanoyl) deayatyl colchicine (40m
g, 0.088 mmol) and the reaction mixture was stirred for 20 hours. The ethanol was then removed by rotary evaporation and flash was applied to the residue.
Chromatography (silica gel, 10% in methylene chloride
Methanol) to provide the title compound.
(19) (26.5 mg, 27%). 20 of this compound
Accumulation of 0 MHz proton NMR shows a binding ratio of 1: 1 and corresponds to the expected M ⁺ of the product ion C ₉₀ H ₁₃₅ N ₆ O ₈ in fast atom collision mass spectroscopy (Grillol / thiogrillol complex). And showed M ⁺ = 1429. In addition, the product was characterized by reverse phase high pressure liquid chromatography (HPLC) according to the conditions described below, with a retention time of 55 minutes. 350 nm
It was found to be 98% pure using UV detection in. Less than 0.30% free 7-N- (5-oxopentanoyl) deayatylcolchicine (retention time = 1
2 min) was detected in this product.

The HPLC system used was W600E.
Gradient controller, U6K injector and Model
Weight with 990 photodiode array detector
It consists of a rsModel 600E solvent feeder. Chromatographic conditions are as follows: Column: Waters Nova-Pak (phenyl, 4
μm, 3.9 mm × 15 cm); Mobile phase: solvent A = water: methanol: acetonitrile: PIC A reagent (water) (395: 25: 80: 4), solvent B = water: methanol: acetonitrile: PIC A reagent ( Water) (2
25: 25: 250: 4), solvent C = methanol: water:
PICA reagent (water) (490: 15: 4). Gradient conditions: 100% (A) to 60%: 40% over 20 minutes
(A: B), then 100% (C) in 10 minutes or more,
100% (C) in another 40 minutes. Flow rate: 2 mL / min. Detection: 240-575 nm photodiode array.

(B) Measurement of acid cleavability The acid hydrolysis rate of the hydrazone conjugate (19) producing 7-N- (5-oxopentanoyl) deayatylcolchicine was investigated by HPLC at three different pHs. did. The buffer is Gomori's "Methods"
in Enzymology ", 16: 138 (195
It adjusted according to the method described in 5). HPLC conditions and retention times were the same as above. 7-N- (5
The detection limit of -oxopentanoyl) deacetylcolchicine was about 100 ng at 350 nm. 50p
1 hydrazone conjugate solution (1 mg / ml methanol)
To the desired pH of citrate phosphate buffer (200 μ
The compound solution to be investigated was prepared in addition to the screw-capped vial containing 1). Close the glass bottle and run the HPLC (3
The solution was analyzed by (detection at 50 nm) for remaining conjugate (19), and 7-N- (5-oxopentanoyl) deacetylcolchicine formed after an additional 24 and 48 hours. Each hplc injection was 200 μl.

The results of hydrolysis at pH 4.21, 5.74, 7.35 are summarized below. pH 4.21: 2
After 4 hours, 78% of (19) was cut (1
8) was produced and 100% was cleaved after 48 hours. p
H5.74: After 24 hours, 45% of (19) was cleaved to produce (18) and after 48 hours 100% was cleaved. pH 7.35: After 24 hours, (1
36% of 9) was cleaved to produce (18) and after 48 hours 77% was cleaved. The only free colchicine hydrolysis product of the hydrazone conjugate detected by HPLC was considered to be the aldehyde of compound (18).

(J) Heparin Lipophilic Cyanine Conjugate The title compound was prepared according to the general procedure described in Reaction Scheme 4 above. This reference number corresponds to the number in Reaction Scheme 4. The product obtained is compound 2
With an equation of the kind represented by 1 and X and X ₁ = C
(CH ₃ ) ₂ , R / R ₁ = C ₁₄ H ₂₉ / C ₂₂ H ₄₅ , A = Cl
Is. Compound 8 was prepared as described above. Freshly prepared compound 8 (18.0 mg, 18.0 pmol)
In an NMR tube with deuterated chloroform (0.5 m
1, Aldrich). Then triethylamine (5 μl, 0.036 mmol), followed by triphosgene (1.95 mg, 6.7 μmol, Aldri).
ch) was added and the mixture was shaken for 2 minutes. Proton NMR was then performed to show the shift from 3.95 in the benzylic protons to 4.95 as seen in compound (8) and the product 5-methylene isocyanate-1'-docosanyl-1- It showed the formation of tetradecyl-3,3,3 ', 3'-tetramethylindocarbocyanine chloride (20).

A solution of the isocyanate salt in deuterated chloroform (20) was concentrated by rotary distillation and the residue was dissolved in dimethylformamide (4 ml, dried and distilled) and stirred rapidly. A solution of heparin in formamide (9.4 mg) was added to the rapidly stirred solution.
/ Ml, 2 ml, 0.0016 mmol) was added, the flask was further capped, and the mixture was stirred at room temperature for 20 hours. The insoluble material was then removed by filtration and the filtrate was concentrated by rotary distillation under high vacuum at 50 ° C. The residue is washed with water (20
ml) and methylene chloride. The aqueous layer was separated and washed again with methylene chloride. Next, the aqueous solution was added to a dialysis bag (Spectra / Por membrane MWCO: 10).
00), dialyzed against water and then lyophilized to yield a pink solid (24.3 mg). This material was considered to be a mixture of compound 21 and unreacted heparin without further purification. It was used for biological evaluation (see Example 14).

Example 4 Biological Effects of Conjugating Therapeutically Effective Proteins to Lipophilic Molecules The in vivo pharmacology of the compounds produced in Example 3d above was described in U.S. Pat. Forstermann et al. P
armacol. Exp. Ther. 234: 10
55-61 (1987),
Blood was perfused on the hindlimb specimens of rabbits. Blood was removed from the cannulated carotid artery of an artificially ventilated rabbit anesthetized with pentobarbital and passed through a medical grade silastic tube using a constant speed roller pump to a cannulated femoral artery.
Terminal perfusion pressure (mmHg) in the femoral artery bed was measured by passing a perfusion tube so that a micro pressure transducer (Millar) was located just ahead of the femoral catheter tip. First, the pump speed was adjusted so that the terminal perfusion pressure was close to the systemic mean arterial pressure, after which blood flow was held constant for the remainder of each experiment. The resistance variation then changes according to the terminal perfusion pressure as described by Ohm's law, as follows: vascular resistance = perfusion pressure / blood flow, thus by keeping blood flow constant during each experiment. As the decrease in pressure means vasodilation and the increase in pressure means vasoconstriction, changes in peripheral perfusion pressure are directly reflected in vascular resistance. Therefore, vascular biology
Direct infusion into the perfusion system can be used to analyze the pharmacology of substances that are believed to affect vascular smooth muscle tone in vivo.

FIG. 1 shows the qualitative identity of the in vivo anti-b response of substance P and the conjugate of example 3d. Both substance P and the conjugate produce a short-term, dose-related reduction in hindlimb perfusion pressure when injected locally into the hindlimb preparation of perfused rabbits. The response of these dilators is
In full agreement with the known vascular pharmacology of substance P. For example, J. Beny et al. Physiol. 398:
277-89 (1988).

FIG. As seen in 2, there is a quantitative difference between the reaction of substance P and the conjugate. The mean dose threshold for substance P requires that a reduction in perfusion pressure of approximately 0.003 pmoles should occur, whereas the maximum reduction in pressure is achieved in the dose range of 3-10 pmoles. The minimum dose of conjugate required to cause a reduction in perfusion pressure is greater than that for substance P, 1-10
pmoles, the largest decrease is 1000 pmole
It is achieved at the time of s. The ED50 values calculated for substance P and the conjugate by the logical function are 0.13 and 34 pmoles, respectively, indicating that substance P is 250 times more potent. However, both substances showed parallel dose-response curve slopes consistent with normal reaptamer interactions. Furthermore, FIG. 2 also shows the dose-response relationship between the substance P and the conjugate at the time of infusion of [D-PRO ² , D-Trp ^7,9 ] ^-substance P which is a known substance P antagonist. It has been reported that this synthetic peptide at position 3 with the D amino acid antagonizes the neuronal, behavioral and vascular actions of endogenous substance P and exogenously administered substance P. The decrease in perfusion pressure for both substance P and the conjugate, as analyzed by a parallel shift of the dose-response curves of both compounds to the right in the presence of the antagonist, [DP
RO ² , D-Trp ^7,9 ] ^-suppressed by substance P.
The magnitude of the rightward shift of the dose-response curve in the presence of the antagonist is similar for substance P and the conjugate, further suggesting that these two substances act at a common receptor site. . Fig. The data given in 2 are based on a study involving 3 rabbits and are the mean (± S) of the perfusion pressure from maximum response to substance P in the absence of antagonist.
EM) expressed as a rate of change.

In addition to the above, it was experimentally confirmed that administration of the corresponding unconjugated lipophilic cyanines at concentrations of 1 and 10 nmoles showed no vasorelaxant effect. Furthermore, 1,10,100 pmol of β-adrenergic receptor agonist isoproterenol
Administration at concentrations of es also resulted in a dose-related decrease in perfusion pressure, which was a SP antagonist [D-PR
It is not antagonized by O ² , D-Trp ^7,9 ] -SP, and [D-PRO ² , D-Tr for the SP receptor]
p ^7,9 ] -SP specificity was demonstrated. The conjugate of Example 3d (100 uM) was easily bound to washed rabbit erythrocytes suspended in phosphate-buffered saline containing 1 mM EDTA and 0.5% rabbit serum albumin, both ex Cultured in vivo. The fluorescent component of the conjugate allows for rapid detection in RBC using flow cytometry. Infusion of conjugate-labeled rabbit RBCs into the hind limb of rabbits results in a reduction in perfusion pressure, the magnitude of which is related to the number of cells infused. Another experiment is 10 ⁶
It was shown that the infusion of -10 ⁹ RBCs significantly reduced the perfusion pressure of the rabbit hindlimb preparation. When the RBC sample labeled with the conjugate was separated into a supernatant fraction and a cell fraction, and the suspension was resuspended in the above RBC buffer and reinjected, the supernatant fraction was converted into the original cell suspension organism. It shows that the conjugate has the ability to diffuse RBCs, including most of the biological activity. By repeating the washing of RBC, a supernatant fraction having a vasodilatory effect is produced,
It suggests a prolongation of the release capacity of the conjugate.

From the experiments described above, the substance P lipophilic cyanine conjugate of Example 3d above has a vasodilatory effect qualitatively similar to that of the natural substance P, and such an action is obtained.
It is understood that it can be antagonized by known antagonists of the receptor. The results of these experiments tend to show that the interaction between substance P and its receptor is maintained regardless of its chemical conjugation with the lipophilic revotor molecule.

Example 5 Determination of the effect of cell binding on the structure and biological interaction of lipophilic cyanine-derived substance P The substance P component of the conjugate of Example 3d is structurally unaffected,
To determine that it is recognizable on the surface of peripheral red blood cells (RBC), 2 ml of human RBC (10 ⁶ cells / m 2
l) was labeled with the conjugate (10 μM; red fluorescence) and then investigated using flow cytometry. The result of this investigation is shown in FIG. 3A-C. Specifically, untreated RBCs and conjugate-labeled RBCs were analyzed to determine: The conjugate is bound on the cell surface (Fig.
3A comparison). 2. 2. non-specific binding of goat anti-rabbit antibody conjugated with fluoryaine isothiocyanate (FITC) to conjugate-labeled RBC or unlabeled cells (comparison of Fig. 3B), or 3. The rabbit anti-substance P antibody + FITC anti-rabbit antibody specifically binds to the conjugate-labeled RBC (comparison of FIG. 3C).

FIG. In 3A, background autofluorescence (cell populations located near the origin of the green (LFL1) and red (LFL2) axes) was examined in unstained RBCs.
, But the conjugate-labeled cells showed an increase in red fluorescence signal compared to unlabeled cells. Fig. In 3B, F
ig. Treatment of the conjugate-labeled cells with goat anti-rabbit antibody stained with fluorescein resulted in minimal non-RBC uptake by the antibody, as evidenced by a small increase in the green fluorescence signal (LFL1) compared to the 3A results. It was shown that only specific binding was performed. Fig. 3C is an unlabeled RBC or a conjugate-labeled RBC that was indirectly fluorescently labeled as a primary reagent using a rabbit anti-substance P antibody, and then stained with goat anti-rabbit antibody as a secondary reagent.
The fluorescence histogram of is shown. In the conjugate labeled cells, significant amounts of green and red fluorescence were detectable on the cells (L
The FL1 signal is shown in FIG. 3B), indicating specific binding to the cell-bound conjugate. This increase in fluorescence was not observed in RBCs not labeled with the conjugate. This method thus demonstrates that substance P was antigenically "recognized" on the surface of RBCs by the anti-substance P antibody, confirming the presence and maintenance of the structure of substance P on erythrocytes.

Example 6 In Vivo Stability Measurement of the Substance P-Lipophilic Cyanine Conjugate on Red Blood Cells In vivo on red blood cells labeled with the conjugate of Example 3d above.
To investigate the characteristics in vivo, 0.5 ml of fresh anticoagulated blood obtained from each of the two rabbits was labeled with the conjugate (final concentration 10 μM) and recollected in the same rabbit from which the blood was collected. Injected. After labeling with the conjugate, prior to injection, the aliquots of labeled cells were examined using flow cytometry to ensure proper labeling. Following administration of the labeled cells in vivo, 30 minutes,
Blood samples were taken at days 1, 2, 3, 4, and 7 and examined for cell fluorescence using flow cytometry. The result of this survey is shown in FIG. As shown in the graph of No. 4, from the graph, it is shown that the conjugates from the labeled cells are in vivo.
It can be seen that the separation at <tb> occurs at a relatively slow rate. The fluorescence of the labeled cells (logarithmic fluorescence intensity) declined with a half-life of 5-6 days, but the life span of the natural substance P in circulation was in minutes. Therefore,
From the above experimental results, it becomes clear that the labeling of bioparticles with lipophilic molecules with therapeutic agents provides an innovative delivery vehicle for parenteral administration of therapeutic agents.

Example 7 Effect of Iodination on Dye Binding, Cell Viability and Membrane Bound Stability Addition of an iodine atom to a compound of this compound does not substantially affect its cell labeling properties. In order to demonstrate, in vitro uptake of compounds, viability of cells after labeling, and membrane retention measurements were performed on the following compounds:

[Chemical 19]

Compounds T, AA, according to the general method described in Reaction Scheme 1 above and described in Example 3 above.
AB can be adjusted. Slezak and Hora
n, Blood, 74: 2172-2177 (198).
Recovery and viability were calculated at 5, 10 and 20 μM labeling concentrations and at 5 or 10 μM labeling concentrations for compounds AA and AB as described in 9). However, compounds AA and AB reduced viability and recovery to 70-80% at a labeling concentration of 20 μM.

The number of compound molecules bound per cell was determined for cells labeled with 10 pM compound. Remove aliquots of 10 6 cells (10 ul) from washed cells after final resuspension and counting, 250
Compounds were extracted from cell membranes with a mixture of μl of 100% ethanol and fluorescent microplate area.
The fluorescence intensity of 200 μl of extract was measured using a combination of derFluoroskan II and a filter. Made in 100% ethanol, Fluorosk
The compound concentration of each extract was determined from the calibration curve of known compound concentration measured with anII. next,
The number of molecules of compound per cell was calculated from the known extract concentration, total volume of extract, and known number of extracted cells. The calculated value of Compound T is (4.0 ± 0.4) × 10.
⁶ , Compound AA is (1.3 ± 0.2) × 10 ⁶ , Compound A
B was (2.5 ± 0.7) × 10 ⁶ .

Relative membrane retention was measured using an erythrocyte membrane ghost prepared by NH ₄ Cl lysis (Slez
ak and Horan, Blood, supra). Ghosts were labeled with 10 μM compound for 5 minutes at 4 × 10 ⁸ / ml at room temperature using the method described above. But PB
Post-label washes were performed using 1 mM EDTA containing S and 5% BSA. After the final washing, 4 × 10 ⁸ /
The ghost was resuspended in PBS + EDTA + 5% BSA in ml and incubated at 37 ° C for 24 hours. 50 μl aliquots doubled at the 24 hour time point were a) a well-mixed suspension of ghosts (for determination of all compounds present), and b) ghosts pelleted at 12,500 xg for 15 minutes, rest From the supernatant (for measurement of unbound compound present). Aliquots were extracted with a mixture of 200 μl of 100% ethanol and the fluorescence intensity of each extract was determined by fluorescence microplate area.
It was measured using a derFluoroskan II. The percentage of retained compound was calculated by the following formula: Total Compound-Unbound Compound x100 Total Compound Calculated Value is (91.9 ± 2.9) for Compound T.
%, Compound AA is (97.7 ± 0.2)%, compound AB
Was found to be (96.8 ± 1.3)%.

Based on the above-mentioned experiments, it was concluded that the iodinated compounds of the present invention exhibit the same or better membrane retention properties than the non-iodinated compounds. Binding into the red blood cell membrane is
Although somewhat lower than equivalent concentrations of non-iodinated compounds, this difference is large enough to change their effectiveness in the above-mentioned types of cell labeling applications, as expected due to their large molecular size and molecular weight. I don't think so.

Example 8 Retention on Artificial Surfaces To analyze the ability of the compounds of the invention to be retained on artificial surfaces, radioactive iodine ( ¹²⁵
I) conjugated lipophilic cyanine (compound 1 above)
2, reaction scheme 2, example 3g) was applied to medical silast
It was placed in contact with the luminal surface of a short piece of several different types of plastic tubing, including ic (SIL), polycarbonate (PC), polyvinyl chloride (PVC), and polyethylene (PE). The compound was allowed to stay in contact with the tube for 10 minutes, after which it was removed and the tube gently flushed with PBS. After performing a baseline radioactivity measurement of the labeled tubes, the fragments were connected to a closed tube circuit containing approximately 30 ml of fresh anti-coagulated human blood. The circuit of tubing was passed through a roller pump and blood was circulated for 6 hours. After 6 hours, the tube fragment was removed, PBS was gently flushed to remove any attached blood, and the radioactivity was measured. The result of this experiment is shown in FIG.
5 schematically shows. The length of the tube segment, the diameter of the compound and the compound of the present invention to which the radioactive iodine was bound, so that the compound / mm ² bound to the luminal surface / mm ² (x-axis of the bar graph) can be represented by a picogram. The specific activity was recorded. Fig. 5 indicates that at the start, the compound with the highest amount of radioiodine bound to the PVC tubing and the least compound bound to PE. It was PVC (97.6 ± 2.1% of the radioactivity initially retained) that showed the maximum retention in the tube in contact with blood after 6 hours,
Retention on PC was minimal (56.0 ± 10.4% of radioactivity initially retained). However, for all pipes the average retention is over 50%, with PVC 100
%, Demonstrating that the compounds of the invention have the ability to remain on artificial surfaces for extended periods of time, even in contact with biological fluids. Thus, the compounds of the present invention are
It can be effective in retaining bioactive substances on artificial surfaces.

Example 9 Retention of 186Re-Chelating Lipophilic Cyanine Conjugate and Na186ReO4 in vivo after Intratumoral Injection To remove excess gentisic acid and avoid the need for pH adjustment prior to in vivo administration. The 186Re chelating lipophilic cyanine conjugate was prepared essentially according to the method described in Example 3h, except SepPak was washed with about 10 mL of distilled water prior to elution of the product with ethanol. 24). After evaporation in vacuo, the concentrated stock solution is diluted with ethyl alcohol to about 25μ.
L final volume (compound 2 at a concentration of about 500 μM
4; about 123 mCi / μmol specific activity). in v
Preparation of Compound 24 containing approximately 5-6 μCi ¹⁸⁶ Re and Na ¹⁸⁶ ReO ₄ for ivo injection was performed as follows. Na ¹⁸⁶ ReO ₄ (NEZ301, 0.1N NaO
(In H) was diluted with 300 mOsM of sterile Dulbecco's phosphate buffered saline, pH was checked to be 7.0 using pH paper, and the modifier was then re-filtered through a 0.22 μm filter. Sterilized. Sterilize Compound 24 300
Diluted with mOsM glucose. A sterile 50 pL Hamilton syringe was loaded with 5.0 μL of Na ¹⁸⁶ ReO 4 or Compound 24 modifier using the sterilization method. Separate aliquots were taken to provide a counting standard for each adjustment and to serve as a standard for whole body rooster image collapse correction and biodistribution measurements.

Retention of the above modifiers after intratumoral injection was evaluated as follows. By intradermal injection into the right hind limb of female C57B1 / 6 mice 5-6 days prior to intratumoral injection, using an inoculum of approximately 1 × 10 ⁶ viable tumor cells suspended in sterile Hank's buffered saline. A grown MC38 adenocarcinoma nodule was introduced. The animals were reanesthetized on day 0, the right hind limb was sterilized with an alcohol swab and 5.0 μ of compound 24
L aliquots or Na ¹⁸⁶ ReO ₄ were injected directly into the tumor nodules with a diameter of approximately 5 mm. The infusion was performed for approximately 10 seconds and the needle was held in place for 5-10 seconds after the infusion was completed to allow any back pressure to dissipate and minimize leakage at the infusion site. Immediately after infusion (within 10-15 minutes) and daily for 4 days using an energy window of 140 ± 20 keV, GE Starcam3.
Animals and metrics were roosterized using the 00 system. Two
After completion of imaging on day 4 and day 4, various organs were collected and weighed at the expense of animal groups and cpm / gm was measured to assess the biodistribution of ¹⁸⁶ Re.

The biodistribution of ¹⁸⁶ Re on day 4 of selected organs is shown in Table IV, A. The results are expressed as the ratio of the recovery from specific organs to the total number of injections after collapse correction. Since organs and tumors differ significantly in size, the relative concentrations of ¹⁸⁶ Re found in each organ are compared in Table IVB. The results represent the ratio of the number of injections found in a 1 g tissue mass with collapse correction. Whole-body scintigraphic images were analyzed by identifying the region of interest (ROI) corresponding to the tumor or whole body; the number of each region of interest was then determined as a function of time after injection. Localization of markers within the tumor was determined by the number of tumor ROIs and systemic ROIs.
Was calculated as the ratio of the quantities (columns 2 and 3 of Table V). The retention of the label in the body was calculated as the ratio of the quantity of whole body ROI to the quantity of reference ROI (Table 2, 4th column and 5th column).
Column). The data in Tables IV and V are from 18 tumor sites.
It is shown that the magnitude of 6Re leakage is greatly reduced by administration of the radionuclide in the form of compound 24. Furthermore, these data show good agreement between the two different methods used to measure intratumoral retention (whole body gamma scintigraphy and direct excitation).

[0159]

[Table 4] ¹ mean ± standard deviation, n = 3 per group, except n = 2 of ¹⁸⁶ Re-PKH113 group after 2 days

[0160]

[Table 5] ¹ Mean ± standard deviation (difference less than 5%, statistically 0 and identical) n = 6 is after ² 0-2 days, n = 5 in after n = ³ 3 0-2 days after 3-4 days, N = 3 after 3-4 days

Example 10 ¹⁸⁶ Re-chelating lipophilic cyanine conjugate and Na ¹⁸⁶
In vivo retention after intra-articular injection of ReO ₄ Two properties of the compounds of the present invention are important in achieving site-selective administration and retention, as well as therapeutic efficacy: a) unbound therapeutic agents Strength of retention at the site of application compared to, and b) its distribution morphology after injection at the site (heterogeneous and homogeneous).
These properties were analyzed based on the delivery of rhenium-containing compounds to the synovial lining in the joint cavity using fluorescent or radiolabeled compounds. ¹⁸⁶ Re-chelate lipophilic cyanine conjugate (Compound 24 of Reaction Scheme 5) Example 3h
The concentrated stock solution was made up in ethyl alcohol to a final volume of about 40 pL as described in. in vivo
For injection, the modifier of Compound 24 and Na ¹⁸⁶ ReO ₄ were prepared as follows. Na ¹⁸⁶ ReO ₄ (NEZ3
01, 0.1 N in NaOH) with 300 mOsM of sterile Dulbecco's Phosphate Buffered Saline and confirm the pH to 7.0 using pH paper, then add the modifier to 0. Re-sterilized through a .22 μm filter.
Compound 24 was diluted with sterilized 300 mOsM glucose and pH was adjusted to 7.0 by addition of sterilized 0.1 N NaOH (no buffer present in glucose diluent and gentisic acid present in final regulator). However, it requires the fact that an unadjusted pH of 1-2 is produced). 1. With a sterile 25G x 5/8 inch needle
A 0 mL tuberculin syringe was weighed and approximately 0.1 mL of Na ¹⁸⁶ ReO ₄ or a modifier of Compound 24 was placed using the sterilization method and reweighed to determine the weight prior to injection. Furthermore, in order to obtain the cpm / μL of each adjusting agent, an aliquot was taken out.

The retention of the above modifiers after intra-articular injection was evaluated as follows. Female New Zealand White rabbits weighing 3-4 kg were anesthetized with ketamine and xylazine, the knee area was carefully shaved and disinfected with a solution of iodine. Using the sterilization method, flex the knee about 120 ° to temporarily move the patella laterally, insert the needle centrally through the skin into the joint space, and add about 0.1 mL of compound 24 (3 Head animal) or Na
¹⁸⁶ ReO ₄ modifier (2 animals) was injected, the needle was removed and the patella was returned to its normal position. Re-weigh the syringe,
The difference between the weight before and after injection was calculated (the density of the injectate was 1.
0 g / mL), cpm / μL values obtained by counting aliquots of known volume for each modifier were multiplied by the measured volume to inject Compound 24 or Na ¹⁸⁶ ReO _4.
The exact volume and action of was calculated. A known volume of blood (about 1 mL) was removed from the central ear artery within 5-10 minutes after intra-articular injection. Animals were placed in cages where urine and feces could be collected independently and observed for 6 days. Blood was drawn each day and all urine excreted in the last 24 hours was collected and collected in a Packard Cobra Mode.
The l5003 gamma counter was used to determine the cpm / mL of aliquots of known volume of blood and urine. Total blood production was estimated based on 5.5% of total body weight and blood cpm / mL; total urine output was calculated based on 24-hour urine volume and urine and cpm / mL. No significant amount of stool was detected. G on days 0, 1, 4, and 6
E's Starcam 300 system for about 120-15
Gamma scintigraphy was performed using an energy window of 0 kEV. On day 6, animals were sacrificed after blood and urine samples were collected, various organs were collected and measured,
Cpm / mL was measured for evaluation of the biodistribution of ¹⁸⁶ Re.

Table VI shows circulating concentrations of Compound 24 and Na ¹⁸⁶ ReO ₄ in blood and the amount excreted in urine over a period of 6 days after the infusion. The distribution of ¹⁸⁶ Re on day 6 of selected organs is shown in Table VII. All results in Tables VI and VII are expressed as a ratio of collapse to injection volume corrected for collapse. The data in Tables VI and VII show that the magnitude of ¹⁸⁶ Re leakage from the intra-articular space measured by blood concentration, urine output, and accumulation in other organs resulted in administration of radionuclide in the form of Compound 24. It shows that it has been greatly reduced by. In addition, knee retention was assessed by measuring the abundance of specific regions of interest (knee and / or whole body) from gamma scintigraphy images and is shown in Table VIII. These results also show that compound 24
Shows a significant improvement in retention when the radionuclide is administered in the form of.

[0164]

[Table 6] ¹ Adjust as in example 3h above ² Not applicable

[0165]

[Table 7] ¹ Lung, heart, spleen, gallbladder, thyroid and lymph nodes

[0166]

[Table 8] ¹ Collapse correction value; <5% difference is not statistically different from 0

Example 11 Distribution of lipophilic cyanine conjugates in synovium after intra-articular injection To assess homogeneity or heterogeneity of binding by synovial cells after intra-articular injection, pH 7.0 at 300 mOsM.
Approximately 0.1 mL of a 10 μM iodide salt solution of Compound T (see Example 2 above) in glucose of 10 mg was injected into the knee as described in Example 10. One hour later, the animals were sacrificed and both the infused and uninjected knee joints were dissected. Cross-section of whole joints was not feasible because demineralization was necessary to dissect the thin section and the demineralizing medium removed the compound of the invention from the tissue. Therefore, the infrapatellar fat pad that underlies the patella and bounds the synovial cavity was carefully removed, snap frozen on dry ice and stored at -80 ° C until frozen sections were prepared. Thick (10μ) cryostat sections were cut, attached to glass slides and analyzed by light and fluorescence microscopy. Light micrographs of fat pad sections (knees with and without injection) show synovial cells
A thin, dark linear layering along the inner edge of the section on the surface of the fat pad facing the joint space was observed when examined at 100x magnification. (The synovial layer is only 1-2 cells thick in normal joints, but is thickened by hyperproliferation in arthritic joints).
The rest of the tissue was mainly composed of large adipocytes. Illumination of the sections of the injected knee under blue excitation light shows that the presence of bright and relatively uniform labels is essentially confined to the synovial cell layer, while the adipocyte area is Alternatively, uninjected knees showed only very dark green autofluorescence.

Example 12 Retention of lipophilic cyanine conjugates at intravascular sites An experiment was conducted to determine if the compounds of the present invention remained in the intravascular site once applied, in which experiment
A 125 I-substituted lipophilic cyanine prepared as described in Example 2 (Compound 12, Reaction Scheme 2) was applied to the luminal surface of the femoral artery of anesthetized rabbits. The femoral artery was surgically isolated and the small mesial collateral was cannulated with a short PE10 tube. A 1 cm portion of the artery around the side branch was occluded and 20-50 μl of a solution containing 125I-substituted Compound 12 was administered through the cannula to the occluded area and allowed to remain for 5-10 minutes. The 125 I-substituted compound was then removed along the cannula to permanently join the side branch, remove the femoral artery occlusion to restore blood flow, and close the femoral incision. Next, Ludiu
m Model 2200 Scaler Ratem
eter and Model44-17 2 inch cryst
With al, ¹²⁵ I radioactivity was observed at selected times over the next 3 weeks in a window optimized for ¹²⁵ I detection. Graphical analysis of the resulting data shows that in the two-phase release process, 34.0 ± 10.9% (approximate sem, n = 4) of the applied substance (radioactivity) was blocked in the The initial release half-life was 0.77 ± 0.29 days. However, after the first 24 hours, the remaining material was released very slowly with a half-life of release of 15.24 ± 2.89 days. Thus, due to the slow release from the site of intravascular application, a significant amount of 125 I-substituted compound could be detected at least 3 weeks after application.

Example 13 In Vitro Antiproliferative Effect of Colchicine Lipophilic Cyanine Conjugates on In Vitro To investigate the antiproliferative effect of the compounds of the invention, they proliferate as myogenic cells and are phenotypically smooth muscle cells. In vitro studies were performed using A10 cells and a clonal cell line derived from embryonic rat thoracic aorta that grows into cells similar to those in B. Kimes and B. Brandt, Expt.
l. Cell Res. 98: 349 (1976). Usually, 2500-10,000 A10 with 12 flat plates
Cells / cm2 were inoculated into Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Calf Serum (FCS) and 3
It was attached and stabilized at 7 ° C. for 24 hours. next,
Adjust the test substance to the appropriate cell-binding medium at the required concentration,
After aspirating the plating medium, it was placed in individual baths at 37 ° C. for only 10 minutes. The treatment was then aspirated, the bath was washed 4 times with untreated medium containing medium and placed in medium containing DMEM with 10% untreated FCS for the duration of the study. Aspirating three tanks at different times after applying the treatment,
The aspirate was saved and the bath was treated with 0.25 ml of 0.25% trypsin to remove attached cells. The enzyme action of trypsin on the cells causes the excess protein (1.75 m
1 DMEM + 10% FCS) was stopped, the aspirate was returned to the bath and the resulting aliquot of cell suspension was removed from the Coulter Counter (Mod).
El ZM and Sampling Stand II). The cell number is c in the growth area (surface area of the tank bottom).
Normalized to the number of cells per m ² .

As shown in Table IX below, compounds of the invention (Compound 19, Reaction Scheme 3, Example 3i, here "
Proliferation after 7 days in cell culture treated with colchicine conjugate ") was only 5.2% of maximum growth treated with vehicle. Significant cell binding fluorescence was seen (flow). (Determined by haemocytometry) showed the presence of binding of the colchicine conjugate to A10 cells, while the presence of cells treated with the same initial concentration of unconjugated colchicine showed
It was only half the cell density of vehicle-treated cells, showing an approximately 10-fold increase in cell number over that obtained with the compounds of the invention.
Thus, the compounds of the present invention allow significant retention of the anti-proliferative effect of the parent compound in A10 cells despite exposure for only 10 minutes, much greater than unconjugated colchicine applied in the same way. It showed an antiproliferative effect.

[0171]

[Table 9] ¹ value is mean ± standard deviation, p = 0.05 from n = 3 replicate ² vehicle group, p <0.05 from ³ colchicine

Example 14 Binding of Anticoagulant Lipophilic Cyanine Conjugates to Biological Membranes In order to evaluate whether the anticoagulant compounds of the present invention can be stably bound to biological membranes, the above example. In vitro with rabbit erythrocyte ghosts as described in 1.
Research was conducted. 2 × 10 ⁸ ghost / ml,
(1) Induced lipophilic cyanine without anticoagulant (reaction scheme 1, compound 3 of example 3a), (2) Anticoagulant lipophilic cyanine conjugate (reaction scheme 4, compound 21 of example 3j), Or (3) with fluorescent isothiocyanate-labeled heparin (FITC heparin), 10 μM, 20 μ
Labeled at concentrations of M and 20 μM, respectively. The ghost is then washed further, 1 mM EDTA and 5% BS
It was placed in phosphate buffered saline containing A at 37 ° C for 24 hours. The samples are agitated and 50 μl aliquots removed from each sample and placed in a 200 microtiter plate incubator.
Placed in μl of Triton X-100 ^R® (meaning total suspension fluorescence). The original sample was centrifuged for 10 minutes and a sample of 50 μl of ghost supernatant was removed and placed in 200 μl of Triton X-100 ^R (meaning free fluorescence in liquid phase only). The fluorescence of all samples was measured with a fluorimetric microtiter plate reader. Bound fluorescence was determined by subtracting free fluorescence from total after correction for background from unstained ghosts; bound and free fluorescence divided by total fluorescence and multiplied by 100 to give the ratio of free and bound fluorescence. Respectively asked.

The results, shown in Table X below, show that the anticoagulant conjugates of the invention are 9% of the compounds of the invention that are free of anticoagulant.
After 24 hours, 90.94% was bound to the ghost's membrane, compared to 4.69% retention, indicating excellent membrane retention. FITC heparin gave only 37.63% fluorescence which remained after 24 hours, resulting in poor retention. This value was considered to be an overestimation of the actual retention rate of FITC heparin because the fluorescence signal of FITC heparin on the ghost was slightly above the background fluorescence intensity, and was calculated from these values. The accuracy of binding and liberation remains questionable. However, the data below clearly show that the compounds of the invention, unlike fluorescent heparin, are well retained on biological membranes.

[0174]

[Table 10] * Numerical values of fluorescent compounds are obtained by repeating mean value ± sem, n = 3.

Example 15 Anticoagulant Retention Properties of Anticoagulant-Lipophilic Cyanine Conjugates When Bound to Biomembrane Surfaces The anticoagulant properties of the compounds of the invention are retained when coated onto biomembrane surfaces. In vi to see if
A tro study was performed to analyze the ability of the compounds of the invention to block the coagulation enzyme thrombin. Rabbit red blood cell ghosts were first labeled with 10 μM anticoagulant-lipophilic cyanine conjugate (reaction scheme 4, compound 21 of Example 3j), washed 4 times with PBS containing 0.1% BSA, then To various amounts of ghost / ml. Compound 21 on the ghost was placed by placing a known amount of ghost in a microtiter plate and comparing the observed fluorescence with the fluorescence produced by a known standard concentration of compound.
Was analyzed. Next, Krtenansky and Ma
o, FEBS Let. 211: 10 (1987), an activity assay was performed by a method similar to that reported in 100 μl human plasma diluted 1:10 in phosphate buffered saline (PBS) buffer, 50 μl PBS containing various concentrations of labeled ghost or thrombin blocker, and a fixed amount (0.5 nM)
50 μl of PBS containing the above thrombin was added to the microtiter plate culture tank and the spectrophotometric plate culture reader (B
Change the absorbance at 405 nm with io-Tek) to 6
Recorded over 0 minutes. The data obtained from the tanks treated with the blocking agent were expressed as the blocking rate in the absorbance range obtained in the absence of the blocking agent. Data obtained from a representative experiment are
g. 6 shows. EC ₅₀ values of 1.03 nM and 21.23n obtained by three-variable nonlinear logic regression of heparin (●) and the anticoagulant conjugate of the present invention (◯), respectively.
M (@ 1.43 × 10 ⁷ ghost / 200 μl), when applied to biological membranes, the compounds of the invention are 20 times less effective than the parent compound as thrombin inhibitors, but to a lesser extent It is shown that. However, this inhibition is bound to all membranes, since the serially diluted ghost-free supernatants analyzed simultaneously (⋄) have no thrombin inhibition. Thus, the compounds of the invention, even when bound to biological membranes, still provide a potent antithrombin effect.

Various aspects of the invention have been described and illustrated above with respect to certain preferred embodiments, namely, the synthesis and use of chemotherapeutic and radiotherapeutic antiproliferative compounds.
However, numerous other embodiments will be apparent to those of skill in the art. For example, other chemo- and radiotherapy conjugates can be synthesized and used for the treatment or analysis of various disease or pathological conditions. Additionally, the compounds and methods of the invention can be used to develop site-selective delivery and retention of other types of drugs such as antibacterial, antifungal, or anti-inflammatory drugs. Therefore, the present invention is not limited to the embodiments specifically described and illustrated, but can be modified and modified within the scope of the following claims.

[0177]

[Chemical 20]

[0178]

[Chemical 21]

[0179]

[Chemical formula 22]

[0180]

[Chemical formula 23]

[0181]

[Chemical formula 24]

[0182]

[Chemical 25]

[Brief description of drawings]

FIG. 1 shows the test results comparing the vascular reaction of locally injected substance P with the substance P-lipophilic cyanine conjugate of the present invention. The upper graph is for substance P and the lower graph is for the conjugate. (Fig. 1A, Fig. 1B)

FIG. 2 is a graph showing a dose-response relationship between a substance P and a substance P-lipophilic cyanine conjugate of the present invention, showing a substance P antagonist [D-Pro ² , D-Trp], respectively.
⁷⁹ ] -in the absence and presence of SP. (Fig.
2)

FIG. 3 shows a series of fluorescence / frequency histograms, where points represent individual cells; increasing distance of points from the origin of the x-axis represents increasing red fluorescence intensity (LFL2 signal),
Increasing the distance of the point from the origin of the y-axis is due to the green fluorescence intensity (LF
L1 signal). (Fig. 3A-1, Fi
g. 3A-2)

FIG. 4 shows a series of fluorescence / frequency histograms, where points represent individual cells; increasing distance of points from the origin of the x-axis represents increasing red fluorescence intensity (LFL2 signal),
Increasing the distance of the point from the origin of the y-axis is due to the green fluorescence intensity (LF
L1 signal). (Fig. 3B-1, Fi
g. 3B-2)

FIG. 5 shows a series of fluorescence / frequency histograms where dots represent individual cells; increasing distance of points from the origin of the x-axis represents increasing red fluorescence intensity (LFL2 signal),
Increasing the distance of the point from the origin of the y-axis is due to the green fluorescence intensity (LF
L1 signal). (Fig. 3C-1, Fi
g. 3C-2)

FIG. 6 is a graph showing the stability of in vivo erythrocyte binding of the substance P lipophilic cyanine dye conjugate of the present invention as a function of time. (Fig.
4)

FIG. 7. Four different types of artificial surfaces of lipophilic cyanine bound to radioactive iodine ( ¹²⁵ I), namely si
Lastic rubber (SIL), Polycarbonate (P
C), polyvinyl chloride (PVC), polyethylene (P
8 is a bar graph showing the degree of retention in E). The dotted bars are the radioactive iodine ( ¹²⁵ I) bound to each surface first.
Represents the amount of lipophilic cyanine bound to; the black bar represents the amount that remained after 6 hours of continuous blood perfusion. The number associated with the pair of bars is the retention of the first aliquot of bound compound. (Fig. 5)

FIG. 8 shows data from an experiment testing the ability of carrier cells labeled with an anticoagulant lipophilic cyanine conjugate to inhibit the in vitro generation of fibrin produced by a reference concentration of thrombin. The concentration response of unconjugated anticoagulant is provided for comparison. The percent inhibition of the thrombin response (y-axis) was recorded as a function of the molar concentration of the compound tested (lower x-axis; log scale), the number of carrier cells per test specimen (upper x-axis; log scale). (F
ig. 6)

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Brian Dee Gray             19003, Pennsylvania, United States,             Ardmore, Apartment A, Hay             Verford Road 2307 (72) Inventor David E. Troutner             19460, Pennsylvania, United States of America,             Phoenixville, Black Powdered             Live 1210 (72) Inventor Catherine A. Muirhead             19380, Pennsylvania, United States of America,             West Chester, Cass Warrend             Live 226 (72) Inventor Chia En Lin             19401, Pennsylvania, United States of America,             Norristown, Chain Street 718 (72) Inventor Kamleshkuma A. Shes             19335, Pennsylvania, United States,             Downingtown, Carlson Way 34 (72) Inventor Jeshou Yu             19403, Pennsylvania, United States,             Jeffersonville, Plowshare Low             Do 106 (72) Inventor Susan G. Lever             21209, Maryland, United States of America             Baltimore, Greenberry Road 1931 (72) Inventor Kwamena E. Baidu             21207 Maryland, United States,             Baltimore, Camberwell Court             2515 (72) Inventor Bruce Dee Jensen             19426, Pennsylvania, United States of America,             Collegeville, Forge Road 204 (72) Inventor Sue Ellen Slesak             19335, Pennsylvania, United States,             Downingtown, Valley View Rain             209 F-term (reference) 4C076 CC41 EE59                 4C204 AB03 DB02 EB03 FB03 GB13

Claims (5)

[Claims] 1. A lipid-containing bioparticle containing a therapeutically effective substance.
A compound of the formula: [Chemical 1] Here, B has a CO and / or COOH functional group.
Peptides that can bind to the spacer moiety, enzymes and
Selected from the group consisting of antibodies; R and R₁From 1
A hydrocarbon substituent having 30 carbon atoms; R₂
Represents the spacer component of the formula:-(R_Three)_p-Q-
(R_Four-Q ')_q-(R_Five-Q '') r- (R₆-Q '' ')_S−
(R₇Q '' '')_t , Where R_ThreeRepresents an aliphatic hydrocarbon
And R_Four, R₅, R₆And R₇Is aliphatic, alicyclic or
Is aromatic hydrocarbon, heterocyclic or CH₂C (CO₂H)
Independently selected from the group consisting of ═CH,
Q ′, Q ″, Q ′ ″, and Q ″ ″ are amides (—NHCO
-), Thiourea (-NHCSNH-), hydrazone (-
CH = NHN-), acylhydrazone (-CH = NHN
CO-), ketal (-O-C (alkyl)_Two-O
-), Acetal (-O-CH (alkyl) -O-),
Orthoester (-C- (O-alkyl)_Two-O-),
Ester (-COO-), anhydride (-COOCO-),
Disulfide (-SS-), urea (-NHCONH
-), Carbamate (-NHCO_Two-), Imine (-C
= N-), amine (-NH-), ether (-O-),
Carbonate (-O-CO_Two-), Thioether (-S
-), Sulfonamide (-SO_Two-NH-), carbony
(-CO-), amidine (-NHC = (NH⁺−)) Conclusion
Independently selected from the functional linking groups composed of
R; Q, Q ', Q' ', Q' '', Q '' '' are independent
Can represent a valence bond; said aliphatic hydrocarbon is one
To 12 straight carbon atoms; said aromatic hydrocarbon
The element comprises 6 to 12 carbon atoms; n is 1;
p, q, r, s and t are each 0 or 1;
X and X₁Can have the same or different values,
O, S, C (CH_Three)_TwoOr Se is represented; Y is = C
R₈-, = CR₈-CR₈= CR₈-, = CR₈-CR₈= C
R₈-CR₈= CR₈-Or = CR₈-CR₈= CR₈
-CR₈= CR₈-CR₈= CR₈A linking group selected from
Represents, where R₈Is H, CH₃, CH₂CH₃, CH₂CH₂
CH₃, Or CH (CH₃)₂Choose from; Z is
H, alkyl, OH, -O-alkyl, COOH, CO
NH₂, SO₃H, SO₂NH₂, SH, S-alkyl, C
ONH-alkyl, CON- (alkyl) ₂, NH-a
Sil, NH-alkyl, N (alkyl)₂, NO₂,Halo
Gen, Si (alkyl)₃, O-Si (alkyl)₃, S
n (alkyl)₃, Or selected from -Hg-halogen groups
An alkyl group composed of the Z substituent described above.
The group has from 1 to 4 carbon atoms;
A chemically acceptable anion;
Compound 2. A R or one compound of claim 1, further comprising at least 12 linear carbon atoms of R _1, when adjusting the total linear carbon atoms in R and R _1, At least 90% of a detectable amount of the compound is labeled with one or more lipids of the population at any time during 24 hours after labeling the population of one lipid-containing bioparticle with the compound of claim 1. A compound according to claim 1 which is tightly bound to the containing bioparticle. 3. R ₂ is composed of a cleavable bond, B
2. A compound according to claim 1, characterized in that is released from said compound under releasably bound to said compound under predetermined conditions causing cleavage of said cleavable bond. 4. A compound according to claim 3, characterized in that B is able to exert its intended biological effect when conjugated to the compound and released from the compound. 5. A delivery vehicle for the compound of claim 1 which is composed of biocompatible particles comprising a lipid component to which the compound is attached.