MXPA99007334A - Targeted therapeutic delivery of vitamin d compounds - Google Patents

Abstract

The present invention is directed to a conjugate which includes at least one vitamin D moiety thereof and at least one targeting molecule moiety to pharmaceutical compositions of the conjugate, and to methods for using the conjugate for target-specific delivery of vitamin D or analogs thereof to tissues in need thereof. When a particularly preferred form is administered to a patient, the targeting molecule component of the conjugate of this invention seeks out and binds to a tissue of interest, such as bone or tumor tissue, where the vitamin D has a therapeutic effect.

Description

DIRECTED THERAPEUTIC SUPPLY OF VITAMIN D COMPOUNDS REFERENCE TO RELATED REQUESTS This application claims the benefit of the priority date, under title 35 of the United States Code.

United Section 119 of the Provisional Application of the United States No. 60 / 038,364, filed on February 13, 1997.

DECLARATION RELATED TO THE INVESTIGATION OR DEVELOPMENT SPONSORED BY THE FEDERAL GOVERNMENT Not applicable BACKGROUND OF THE INVENTION This invention relates, in general, to the targeted therapeutic delivery of vitamin D compounds and, in particular, to the delivery to bone and tumor tissue. Very often the doctor faces a dilemma that can not be solved. To effectively obtain therapy against a disease, it is necessary to balance the devastation caused by the disease against the harmful effects conferred by the drugs used to treat it. Although therapy should represent a complete tolerance to the dose regimen, in absolute terms it often delineates a compromise position, thereby only performing an 'acceptable' level of treatment.In the absence of preventive measures to combat the onset of a disease, the Following alternative ideal is to design a drug that specifically recognizes the origin of the disorder and then correct it.This concept of supply of medication known as 'magic bullet' or site-specific is not a new concept. This concept is aimed at linking the action of drugs with events mediated by specific receptors. These concepts still serve as a primary foundation for the continued development of drugs and antibodies. The chemical modification of a drug, in some cases improves its specificity of target organ. For example, delivery systems directed to the brain based on the redox interconversion of the dihydropyridine-pyridinium salt have been developed for compounds such as estradiol and ethinylestradiol. Brewster et al., 31 J. Med. Chem. (1988) 244. The covalent coupling of some sugars with medicaments can improve their absorption by the liver. Ponpinom et al., In Receptor Mediated Targeting of Drugs, (Gregoriadis et al., Eds), NATO ASI series. Plenum Press, New York, 1983, p. 53. However, in many cases a specific carrier is needed that is designed to transport and deliver the drug to its target tissue. In these cases, the distribution characteristics of the drug are irrelevant, since the characteristics of the carrier determine whether the drug is delivered to the target cells. However, once delivered to the target cells, it will still be necessary to consider the distribution of the drug in place for the intracellular effect. Various particles have been proposed as drug delivery systems. Recent interest has focused on monoclonal antibodies and colloidal delivery systems such as liposomes and polymeric microspheres. See, for example, Davis et al. in Site-Specific Drug Delivery, (Tomlinson et al., eds.), John Wiley, New York, 1986, p. 63. Soluble molecules have also been proposed as drug carriers, including DNA, lecithins, poly-L-lysine, virosomes, insulin, dextran, HCG, and peptides and even cellular systems such as erythrocytes and fibroblasts. See, for example Poznansky et al., 36 Pharmacol. Rev. (1984) 277. Still others have suggested and tried the use of lipoproteins as carriers of drugs, especially low density lipoproteins. See, for example Counsell et al., 25 J. Med. Chem. (1982) 1115. Of course, the need for site-specific drug delivery is higher in pathological conditions such as cancer and severe viral infections. The unwanted effects of medications used in the treatment of neoplastic diseases are often very severe. Other rapidly growing tissues such as bone marrow and gastrointestinal delomorphic cells are affected by the antineoplastic drug, and often the therapy will have to be interrupted. The need for site-specific drug delivery is also high in bone conditions, particularly osteoporosis. With the recognition that osteoporosis occurs to some degree in all post-enopausal women, the specific site supply of bone agents to the mineralized bone matrix has been proposed. Certain agents are known for their characteristics of bone search or affinity with bone, and the binding of these bone-seeking agents with enzymes, steroids or hormones to provide a bone-specific drug delivery agent has been advanced. For example, it has been proposed to bind a bone-seeking agent, such as tetracycline, to a carbonic anhydrase inhibitor by a bridging agent in order to provide the compounds for the treatment of or prophylaxis of degenerative bone diseases. . See, the application of European Patent No. 201,057. In addition, it has been thought to bind a hormone, for example calcitonin or the insulin-like growth factor to an acid to inomethylene biphosphonic acid. See Japanese Patent Application No. 2104-593A. U.S. Patent No. 5,183,815 discloses that the binding of a steroid with an alkyl biphosphonic acid may have a localized therapeutic effect on the bone. The application of European Patent No. 0 512 844 Al describes the binding of a bone growth factor, such as transforming beta-growth factor to molecules that target the bone, such as tetracycline, calcitonin, bisphosphonates, acid polyaspartic, polyglutamic acid, aminophosphate, or estrogen to provide a local bone-building agent. It has long been established that vitamin D has an important biological function in the metabolism of bone and mineral. It is well known that vitamin D plays an important role in stimulating the absorption of calcium and regulating calcium metabolism. The discovery of the active forms of vitamin D (M.F. Holick et al., 68 Proc. Nati. Acad. Sci. USA, 803-804 (1971); G. Jones et al., 14 Biochemistry, 1250-1256 (1975) and the active analogues of vitamin D (MF Holick et al., Science 180, 190-191 (1973); HY Lam et al., Science 186, 1038 -1040 (1974)), caused great turmoil and speculation about the usefulness of these vitamin D compounds in the treatment of depletive bone disorders. Studies in animals, examining the effects of these active vitamin D compounds, suggest that these agents would be useful in restoring calcium balance. In addition, a first clinical study indicated that the administration of 0.5 μg / day of the 25-dihydroxyvitamin D3, the hormonally active form of vitamin D3 to a group of post-menopausal women improved the intestinal absorption of calcium as well as the balance of the calcium in women. On this basis, U.S. Patent No. 4,225,596 ("596 patent") describes and claims the use of 25-dihydroxyvitamin D 3 to increase calcium absorption and retention This use was also claimed in the same patent for, 25-dihydroxyvitamin D2, and la-dihydroxyvitamin D2, whose compounds, as taught by the patent, are 'obviously suitable and readily substitutable for 1,25-dihydroxycholecalciferol [la, 25-dihydroxyvitamin D3]. " The best indicator of the effectiveness of vitamin D compounds in the prevention or treatment of bone depletive disorders, however, is bone itself rather than calcium absorption or calcium balance. More recent clinical data indicate that, in the dose ranges shown in the '596 patent, la, 25-dihydroxyvitamin D has, at best, moderate efficacy in the prevention or restoration of mass loss bone or mineral content in bone (SM Ott and CH Chesnut, Ann Int. Med 110: 267-274 (1989); JC Gallagher et al., Ann Int. Med. 113: 649-655 (1990); J. Aloia et al., Amer. J. Med. 84: 401-408 (1988)). These clinical studies with the 25-dihydroxy itamine D3, and others carried out with the dihydroxyvitamin D (M. Shiraki et al., Endocrinol, Japan 32, 305-315 (1985)), indicate that the capacity of these two compounds Vitamin D to restore lost bone mass or bone mineral content is dose related. These studies also indicate, however, that in the dosage intervals necessary for the compounds to be truly effective, toxicity in the form of hypercalcemia and hypercalciuria becomes a major problem. Specifically, attempts to increase the amount of 25-dihydroxyvitamin D3 above 0.5 μg / day have often resulted in toxicity. At dosage levels below 0.5 μg / day no effects on bone mass or mineral content are observed. See, G.F. Jensen et al., Clin. Endocrinol 16, 515-524 (1982); C. Christiansen et al., Eur. J. Clin. Invest. 11, 305-309 (1981)). It was found that two μg / day of dihydroxyvitamin D3 is effective in increasing bone mass in patients with senile osteoporosis (O.H. Sorensen et al., Clin Endocrinol 7, 169S-175S (1977)). Data from clinical studies in Japan, a population that has low calcium intake, indicates that efficacy is found with la-dihydroxyvitamin D3 when administered at 1 μg / day (M. Shiraki et al., Endocrinol. Japan 32: 305-315 (1985), H. Ori o et al., Bone and Mineral 3, 47-52 (1987)). However, with 2 μg / day of la-dihydroxyvitamin D3 toxicity occurs in approximately 67% of patients, and with 1 μg / day this percentage is approximately 20%. In more recent years other functions of vitamin D have come to light. Nuclear receptors specific for 25-dihydroxyvitamin D3 have been found in cells of various organs not included in calcium homeostasis. For example Miller et al., 52 Cancer Res. (1992) 515-520, have shown specific, biologically active receptors for the 25-dihydroxyvitamin D3 in the human prostatic carcinoma cell line, LNCaP. More specifically, it has been reported that certain vitamin D compounds and analogs are potent inhibitors of malignant cell proliferation and inducers / stimulators of cell differentiation. For example, U.S. Patent No. 4,391,802 published by Suda et al discloses that the la-dihydroxyvitamin D compounds, specifically, 25-dihydroxyvitamin D3 and la-dihydroxyvitamin D3 possess potent antileukemic activity in the context of inducing the differentiation of malignant cells ( specifically, leukemia cells) for non-malignant macrophages (monocytes) and are useful in the treatment of leukemia. In another example, Skowronski et al., 136 Endocrinology (1995) 20-26, have reported antiproliferative and differentiation actions of the, 25-dihydroxyvitamin D3 and other vitamin D3 analogues in prostate cancer cell lines. Previous studies on proliferation, such as those mentioned above, focused exclusively on the compounds of vitamin D3. Although these compounds may in fact be highly effective in the differentiation of malignant cells in culture, their practical use in differentiation therapy as anticancer agents is severely limited due to their equally high potency as agents that affect the metabolism of cancer. At the levels necessary in vivo for effective use as anti-leukemic agents, these same compounds can induce markedly elevated and potentially dangerous blood calcium levels, by virtue of their inherent calcemic activity. That is, the clinical use of 25-dihydroxyvitamin D3 and other vitamin D3 analogues as anticancer agents is impeded, or severely limited, by the risk of hypercalcemia. This indicates a need for compounds with higher specific activity and action selectivity, ie vitamin D compounds with antiproliferative and differentiating effects but having less calcemic activity than the therapeutic amounts of the known compounds or vitamin D analogues. Almost nothing in the art it proposes materials or methods for the targeted delivery of vitamin D compounds to specific target tissue, for example bone or malignant sites, such as prostatic cancer cells.

BRIEF SUMMARY OF THE INVENTION The present invention provides conjugates of vitamin D compounds and a leader molecule with a capacity for site-specific delivery of vitamin D. Specific conjugates include a therapeutic bone conjugate and an anti-tumor conjugate. The present invention also provides pharmaceutical formulations of this conjugate and methods for the site-specific delivery of a vitamin D portion of the conjugate to a tissue of interest in a patient. The present invention provides, surprisingly, the means useful for the delivery of the vitamin D compounds, analogs or derivatives in tissues of interest (ie, to a target tissue). The conjugates of the present invention contain at least a portion of vitamin D associated with a target molecule portion that has an affinity for a target tissue. In an embodiment of the invention. The conjugate includes at least a portion of vitamin D associated with the portion of the target molecule by means of at least one linking group, such as a link between the vitamin D portion and the target molecule portion, a bifunctional linker. , or other connectors such as biotin-avidin binding. In another embodiment, the conjugate includes a target molecule portion with an affinity for the tissue of interest, associated with at least a portion of vitamin D and with a second therapeutic agent other than vitamin D. The pharmaceutical compositions of the present invention include a conjugate of at least a portion of vitamin D associated with at least a portion of the target molecule, and a suitable pharmaceutically acceptable carrier. In another embodiment, the present invention provides a method for site-specific delivery of vitamin D or a vitamin D analogue, a method that includes administering to a patient a therapeutically effective dose of a conjugate in a carrier. pharmaceutically acceptable, wherein the conjugate has at least a portion of vitamin D associated with at least one target molecule by a linker, and wherein the target molecule has an affinity for a tissue of interest. The method is designed to effect site-specific delivery of the vitamin D portion in the tissue of interest to the patient. The conjugate, the pharmaceutical composition and the method of this invention allow the specific delivery of the vitamin D target in a specific tissue of a patient. By specifically targeting and supplying a vitamin D compound as part of the conjugates of this invention, it is possible to effect delivery of the compound to the target tissue by administering relatively small amounts of the conjugate to a patient, as compared to supplying the same amount of the compound by administering the compound itself. By reducing the amount of vitamin D compound administered, the present invention significantly reduces the risk of hypercalcemia and other side effects of the administration of vitamin D compounds or analogs to patients, using conventional administration means. Other advantages and a more complete appreciation of the specific adaptations, the variations in the compositions and the physical attributes will be obtained with an examination of the following detailed description of the preferred embodiments, taken together with the drawings. It is expressly understood that the drawings herein are for purposes of illustration and description only, and are not proposed as a definition of the limits of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS The preferred exemplary embodiment of the present invention will be described in conjunction with the accompanying drawings, in which like designations refer to like elements, in which: Figure 1 illustrates a reaction scheme for the preparation of a conjugate of the, 24-dihydroxyvitamin D2 ('la, 24- (OH) 2D2") and aminoalkyl-1, 1-bisphosphonate bound to C-24 of the vitamin D portion; Figures 2A and 2B illustrate a reaction scheme for the preparation of a conjugate of the 24- (OH) 2D2 and aminoalkyl-1, 1-bisphosphonate bound to C-1 of the vitamin D portion; Figure 3 illustrates a reaction scheme for the preparation of a conjugate of the 24- (OH) 2D2 and aminoalkyl-1, 1-bisphosphonate bound to the C-3 of the vitamin D portion; Figures 4A and 4B illustrate a reaction scheme for the preparation of a conjugate of the 25-dihydroxyvitamin D3 (la, 25- (OH) 2D3) and aminoalkyl-1, 1-bisphosphonate bound to the C-1 of the portion of vitamin D; Figure 5 illustrates a reaction scheme for the preparation of a conjugate of the 25- (OH) 2D3 and aminoalkyl-1, 1-bisphosphonate bound to the C-3 of the vitamin D portion; Figure 6 illustrates a reaction scheme for the preparation of a conjugate of the 25- (OH) 2D3 and aminoalkyl-1, 1-bisphosphonate bound to the C-25 of the vitamin D portion; And Figure 7 is a schematic diagram for the preparation of a vitamin D conjugate and a monoclonal antibody, using a biotin-avidin linker.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of vitamin D formulations in targeted applications. However, the present invention is more specifically adapted to the use in the specific provision of vitamin D site in bone and tumor cells. Accordingly, the present invention will now be described in detail with reference to these efforts. However, those skilled in the art will appreciate that this description of the invention is understood as exemplary only and should not be seen as limiting the full scope thereof.

The present invention is characterized by an ability to target vitamin D compounds using vitamin D conjugates and a leader molecule having an affinity for a tissue of interest to a specific site. For example, a vitamin D conjugate and an agent with affinity to bone is designed to transport and deliver vitamin D into bone. These attributes are obtained through a novel combination of physical and chemical characteristics. The following description of the invention, the steps of the process are carried out at room temperature and atmospheric pressure, unless otherwise specified. As used herein, the term "leader molecule" refers to a molecule that binds to or influences the metabolism of the tissue of interest, For example, agents that target bone may include bone-seeking molecules such as tetracycline, calcitonin, bisphosphonates, chelators, phosphates, polyaspartic acid, polyglutamic acid, aminophosphoazores, peptides known to be associated with the bone mineral phase such as osteonectin, bone sialoprotein and osteopontin, proteins with bone mineral binding domains, and the like The molecules that target the bone can also include molecules that in themselves affect the rates of resorption and bone formation, such as bisphosphonates, estrogens and other steroids, such as dihydroepiandrosterone (DHEA). they may also possess therapeutic properties of bone growth and / or may give rise to an effect synergistic or additive with the vitamin D compound and on resorption or bone formation. Skin-seeking molecules include certain chelates ion metal ion amino acid; Prostate-seeking molecules include certain steroids such as DHEA. Tumor-seeking agents include certain antibodies. As used herein, the terms "tissue of interest" or "target tissue" are meant to refer to a desired objective or site in the body for the treatment or placement of a vitamin D or analog compound. The term "treatment" or "treatment" is understood to refer to the repair, prevention, alleviation, improvement, prophylaxis of a diseased or defective tissue of interest, as well as the inhibition of abnormal growth, such as hyperproliferation of cells , and the promotion of differentiation of cells. When used herein, the term "therapeutic agent" refers to a material that has or presents healing powers when administered to or delivered to a tissue of interest.The term "bone therapeutic agent" is used herein to refer to a specific type of therapeutic agent, one that alleviates bone diseases or disorders when delivered or administered to bone. Examples of the bone therapeutic agents include vitamin D compounds, conjugated estrogens or their equivalents, antiestrogens, calcitonin, bisphosphonates, calcium supplements, covalamin, pertussis toxin, boron and other bone growth factors such as the factor of transforming growth beta, activin or morphogenic bone protein. The conjugates according to the present invention include at least one vitamin D compound, analog, component or portion (herein designated as 'D') associated with at least a portion of the target molecule (herein designated as * t "). ) and includes those represented by the formula (I): (D) m * (T) n (I) where n and m represent integers of 1 or greater; and * indicates that the portion of the target molecule (T) is associated with the vitamin D compound, the analog, component or portion (D). It is understood that, as used herein, the term "vitamin D" includes all compounds that have conventional structure of vitamin D, rings A, C and D and side chain in C-17 as well as compounds previtamin D which are the thermal isomers of their corresponding vitamin D forms, in which the basic structures can be substituted, unsubstituted or modified, for example, the 19-nor compounds.The vitamin D portion is also understood to maintain its biological effectiveness in the conjugated, for example, its beneficial effect with respect to bone, or in the case of previtamin D, isomerized to its corresponding D form, having biological effectiveness.As used herein, the terms' associated with 'or' association "is meant to refer to the binding or binding of a component of the conjugate (e.g. vitamin D portion) or vitamin D portion and the linker to another component of the conjugate, e.g. target molecule or the target molecule and the linker, by covalent bonding, hydrogen bonding, metal bonding, van der Wall forces, ionic bonding coulombic forces, hydrophobic or hydrophilic forces, adsorption or absorption, chelate-like association or any combination thereof . The term "portion" and "component" used in connection with vitamin D or D, or in connection with the portion of the target molecule or T, is understood to refer to the D molecule or target molecule in the conjugated forms described herein. , that is, after the association occurs. The association between vitamin D analog and the target molecule can occur at any position in the analogous molecule of vitamin D depending on the functionality of the target molecule. For example, a bisphosphonate or amide may be suitably linked at the positions on the vitamin D compound or the analogous molecule of vitamin D with a hydroxyl group, such as at C-1, C-3, C- 24, C-25. The compounds of vitamin D and functional analogs in the present invention are suitably represented by formula (II): wherein R1 is H or OH; Z represents a straight-chain or branched, saturated or unsaturated, substituted or unsubstituted Ci-Cis hydrocarbon group; And it is a group = CH2; and t is 0 or 1, so that, when t is 0, the compound of the formula (II) is a 19-nor compound. Preferably, Z is a side chain represented by the formula (IIIA): (IIIA) where m is 0 or 1; R5 is H or OH; R6 and R7 are independently H, OH, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower O-acyl, O-aromatic alkyl, lower cycloalkyl or, taken together with the carbon to which they are attached (ie, C-25) ), form a cyclohydrocarbon ring of C3-Cs; and Q is -C = C -, - G = C-, or R \ ^ - ^ where n is 0 or an integer of 1 a (~ C-) n 7, R3 is CH3 or H and R4 is H or OH. For example Z includes a side chain cholesterol or ergosterol represented by the formula (IIIB): wherein R8 and R9 each are H or, taken together form a double bond between C-22 and C-23, and R3 is CH3 or H; R4 and R5 are independently H or OH; and R6 and R7 are independently H, OH, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower O-acyl, aromatic O-acyl, lower cycloalkyl or, taken together with the carbon to which they are attached (ie, C- 25) form a cyclocarbon ring of C3-Cs. Also included as compounds of vitamin D within the scope of the present invention are the compounds previtamin D, preferably the hydroxyprevitamin D which may include the common side chains of cholesterol and ergosterol which may optionally be substituted, preferably hydroxy substituted, by example in C-24 or C-25. The compounds of previtamin D are the thermal isomers of the corresponding vitamin D compounds, for example la-hydroxyprevitamin D3, is the thermal isomer of la-hydroxyvitamin D3, and exists in thermal equilibrium therewith. Preferred among the target molecules are those which, when used as a component of the conjugate of the formula (I), give rise to at least a portion of the conjugate, specifically, the vitamin D component of the conjugate, being delivered to a desired target ( for example, a directed cell, a directed organ, a tumor, etc.). Preferred target molecules include the chemical functionalities that exhibit the specificity for the target, hormones (for example biological response modifiers) and antibodies, preferably monoclonal or polyclonal antibodies, or antibody fragments having the specificity of the required target. Included among the preferred target molecules that have the specific affinity for bone are the bisphosphonates, tetracycline, polymalonates and dihydroepiandrosterone. The conjugates of the formula (I) are prepared under conditions (such as specific pH, temperature and salt concentrations) that are not detrimental to the specific conjugate components and in the presence of a suitable solvent when required. To control the pH, a buffer or the addition of an appropriate acid or base is used. The reaction conditions will depend on the type of association (*) that is to be formed between the vitamin D compound or the analogue (D) and the portion of the target molecule (T) to produce the conjugate. The molar ratio of T: D in the conjugates of formula (I) is preferably 1: 1. Also within the scope of the present invention are those conjugates in which D is associated with T through the linker (herein designated as 'G' between D and T), these preferred conjugates are represented by the formula (IV): [ (T) n - (G ') f] g * [(G ") h - (D) m] k (IV) where each Gf represents the same or different linking group, each G" represents the same linking group or different; gyk each individually represents an integer of 1 or greater, f and h each represents an integer of 0 or greater individually - indicates a link in cases where a connector group is present; n and m are as defined above in the present; and * indicates that the component of the target molecule is associated with the vitamin D component by the G 'or G "linker or by both connectors, in both cases where both are present. the conjugate or, when D is a previtamin D, the isomers for its corresponding vitamin D form with biological effectiveness In those cases where foh is O (ygyk are 1), the conjugate of formula (IV) is simply represented by the formula (V): Dm- G- Tn (V) In these cases, G is a bifunctional linker, for example, polyglutamic acid or polyaspartic acid, or a linking group formed by the modification of D and / or T and with the subsequent link formation Suitable linking groups for use in the formation of the conjugates of the formula (IV) are those which link the portion of the vitamin D with the target molecule portion without significantly deteriorating the biological effectiveness of the vitamin D, and without significantly deteriorating the affinity of the target molecule component of the conjugate. The linkers form a link between the portion of the target molecule and the vitamin D portion of the conjugate, a link that must be of sufficient stability to preserve intact, at least until the conjugate is delivered to the region near the target. Under some circumstances, vitamin D delivered to the target region as part of the conjugate is more effective after at least one linker that unites the portion of the target molecule and the vitamin D portion is unfolded in these cases, at least this connector is preferably unfolded once the conjugate is supplied close to the target. In other words, by splitting the connectors in these cases it is possible to avoid steric hindrance between the vitamin D and the portion of the target molecule, which exists in some of the conjugates of the present invention, where the steric hindrance reduces the effectiveness of vitamin D of 1 conjugate. As an illustrated embodiment, the conjugate of the present invention includes an agent effective in the treatment of bone disorders, ie, the conjugate includes a portion of vitamin D and a bone-seeking agent. Bisphosphonates ('BP') are preferred among the agents that have an affinity for bone. (also known as diphosphonates). For example, a specific bisphosphonate portion that can operate suitably in the present invention is represented by formula (VI): wherein R2 is H or OH, Y is NH, 0 or NR8, wherein R8 is H or C? -C alkyl, and n is an integer from 1 to 4. A therapeutic conjugate for bone, particularly preferred, wherein T is BP, is represented by the formula (VII): D-BP (VII) wherein BP is conveniently linked in, for example, the position C-1, C-3, C-17, C-24 or C- 25 of the D-portion. Specific therapeutic bone conjugates suitable for use in the present invention, wherein T is a bisphosphonate include the conjugates of the formula (VIII): wherein R1 is H or OH; R2 is H or OH; X is 0 or S; Y is NH, O or NR where R is H or C? -C4 alkyl; n is 1-4; and pharmaceutically acceptable salts thereof, ie, the bisphosphonate is linked to the vitamin D portion in C-17. Conjugates of the formula (IX) are also provided: wherein R1 is H or OH; R2 is H or OH; R3 is CH3 or H; R4 is H or OH, X is O or S; Y is NH, O or NR, where R is H or C? -C alkyl; n is an integer from 1 to 4; R8 and R9 each are H or taken together form a double bond between C-22 and C-23; and the pharmaceutically acceptable salts thereof, i.e., the bisphosphonate of unites at the C-25 position of the vitamin D portion. Conjugates of the formula (X) are also provided: wherein R1 is H or OH; R ^ is H or OH; RJ is CH3 or H; R4 is H or OH, X is O or S; Y is NH, O or NR where R is H or C? -C4 alkyl; R5 is H or OH; n is an integer from 1 to 4; R8 and R9 are each H or taken together form a double bond between C-22 and C-23; and the pharmaceutically acceptable salts thereof, that is, the bisphosphonate is bound to the vitamin D portion is C-3. Conjugates of the formula are also provided (XI): wherein R2- is H or OH; R3 is CH3 or H; R4 is H or OH, X is O or S; Y is NH, O or NR where R is H or C? -C4 alkyl; R5 is H or OH, n is an integer from 1 to 4, R8 and R9 each are H or, taken together form a double bond between C-22 and C-23; and the pharmaceutically acceptable salts thereof, i.e., the bisphosphonate bond is in the C-1 portion of the vitamin D. It will be noted that, in general, the bond between the bisphosphonate portion and the vitamin D portion is through a hydroxyl in vitamin D where the hydroxyl becomes a group: X II-o-c-Y and it binds to the amine or hydroxy group, that is, Y of the bisphosphonate to form a carbamate or carbonate type bond. X may be 0 or S. For example, a hydroxyl group may be contained in the structure of vitamin D at C-1, C-3, C-24, C-25 and conjugation may be performed at any hydroxyl position but, for convenience in one of the previous ones. The synthesis of the conjugates of the formula (I), where T is a bisphosphonate, is obtained according to the scheme shown in Figures 1-6. In general terms, the synthesis includes the conversion of a hydroxyl to vitamin D in a haloformate (for example a chloroformate) or thioformate group with subsequent reaction with the appropriate amino or hydroxyl group of the bisphosphonate to form a carbamate, thiocarbamate, carbonate or thiocarbonate. If R2 is hydroxy or the vitamin D compound contains 1 or more hydroxyl groups in addition to the desired hydroxyl group, these can be protected by conventional hydroxy protecting groups, such as benzyl, silyloxyl, etc. before the reaction that converts the desired hydroxyl into a haloformate or thioformate group. Specifically, in the illustrated embodiment, the initial vitamin D compound or the analogue (when appropriate, protected in the hydroxyl) is reacted with phosgene to form a chloroformate, the chloroformate is reacted with an aminobutyl-1,1-bisphosphonate to form a carbamate bond. Then, any of the protected hydroxyls is deprotected. Figure 1 is an illustrative scheme for the synthesis of a conjugate of the, 24- (OH) 2D2-aminoalkyl and 1,1-bisphosphonate. As can be seen in Figure 1, the initial material, 24- (OH) 2D2 is protected by the silyloxyl groups at positions C-1 and C-3. The protected compound D (1) is reacted with phosgene in toluene to form the chloroformate (2). The chloroformate (2) is reacted with tetraisopropyl 4-aminobutyl-l, 1-bisphosphonate (i.e., with the protected hydroxyl) in dichloromethane, and the reaction mixture is purified by flash chromatography to produce the protected conjugate (4). The protected conjugate (4) is reacted with tetrahydroform (THF) [sic] and tetrabutylammonium fluoride (TBAF) to deprotect the hydroxyls of C-1 and C-3 to produce the tetraisopropylester of the conjugate (5). The tetraisopropyl ester (5) is hydrolyzed with trimethyl silyl bromide to form the structure of the conjugated bisphosphonic acid (6). The compound (6) and the 9-acetylanthracene in methanol are irradiated, filtered, concentrated and lyophilized to produce the conjugate (7). The synthesis illustrated in Figures 2-6 are described in detail in the section of the examples, below. Also, preferred among the conjugates of Formula (1) are those in which T is of DHEA. The common steroidal structure of DHEA has a 17-keto group and a 1-hydroxy group. Any of these groups can be conveniently linked at the hydroxylated positions of vitamin D, for example, by ether or ester linkages. The reactions for these bonds are well known.

Also desirable among the conjugates of Formula (I) are those in which T is a metal ion, M. Metal ions are known to reach multiple tissue sites, for example, the strontium ion in bone. The conjugates can take the form of direct complexes between a portion of vitamin D and the metal ion, wherein the portion has a terminal group with a negative charge, for example with one or more carboxyl groups at C-24, C-25, etc., in general DX. The conjugate can be represented by the formula (XII): D-X-.

D-x- "(ii) wherein X is a negatively charged group, such as carboxyl, -C02, and M is a divalent metal ion. Otherwise, the conjugate can be of the form of the formula (V) wherein DIM are associated by a linker, for example an amino acid. For example, it has been described that the metal ion-amino acid chelates are capable of directing the supply to a tissue site. See, for example, U.S. Patent Nos. 4,863,898; 4,176,564 and 4,172,072, each of which is incorporated herein by reference. For example, magnesium-lysine chelates have been targeted to bone; Zinc and methionine have been targeted to the skin. These chelates are of the form: (AA) * M * (AA) where M is a metal ion and AA is an amino acid residue, (eg, lysine, arginine, etc.), and * indicates an association of the amino acid residue ( AA) with the metallic ion (M). In the conjugates of the present invention, the amino acid of the metal-amino acid chelate is linked to the vitamin D portion through the amide bond shown above in the case where T is bisphosphonate. The conjugates are represented by the formula (XIII): D - (AA) * M (XIII) wherein D is a portion of vitamin D having a group capable of forming a bond with an amino acid, for example, a hydroxy group; (AA) is the amino acid residue and M is a metal ion, preferably a divalent ion such as Sr2 +, Zn2 +, Mg2 +, Fe2 +, Cu2 +, Mn2 +, Ca2 +, Cu2 +, Cr2 +, or Mo2 +. More specifically, the conjugates are provided in the form represented by the formula (XIV): O H 11 I D-0-C-N- (AA) * (XIV) where D, M, AA and * are as defined. Also preferred among the conjugates of the formula (I) are those where T is an antibody. Antibodies or antibody fragment that can be used in these preferred conjugates can be prepared by well known techniques. An example of suitable antibodies are immunoglobulins, such as IgG, IgA, IgD and IgE. High specificity monoclonal antibodies can be produced by well-known hybridization techniques. See, for example, Kohier et al., 245 Nature (1975) 495-497 and 6 Eur. J. Immunol. (1976) (511-519, which are incorporated herein by reference.) These antibodies can usually have a highly specific reactivity.The polyclonal antibodies are also suitable for use as the target component molecule of the conjugate.However, when the portion of the molecule target is an antibody, it is more preferable that it be a monoclonal antibody (Mab) .The selected monoclonal antibodies are highly specific for a single epitope, making the monoclonal antibodies particularly useful as the components of the leader molecule of the conjugates of this invention. Conjugates of vitamin D and monoclonal antibodies can be targeted to specific sites within a target region, such as specific vitamin D receptors on the surface of cancerous tissue or bone tissue. production of monoclonal or polyclonal antibodies for anti- Specific genes, such as antibodies to selected target tissue or even to specific target proteins are well known. See, for example, Molecular Cloning, 2nd ed., Sambrook et al., Eds., Cold Spring Harbor Lab. Press 1989, § 18.3 et seq. Polyclonal antibodies can also be used and produced in a more economical way than monoclonal antibodies. In but, polyclonal antibodies are less specific leader molecules. However, it is possible to produce large amounts of monoclonal antibodies suitable for use in the conjugates of the present invention by tissue culture (e.g., a hybridoma cell line).

The conjugates of the present invention produced using these leader molecules antibodies can be directed against, for example, cells, organs, tumors, differentiation and other cell membrane antigens, polynucleic acids such as DNA and RNA or any biologically active molecule. Antibodies to the vitamin D receptors of target cells of interest may be suitable for use in the conjugates of this invention. For those conjugates according to the present invention, when T is a monoclonal antibody, the T * D association is suitably made by a 'G' linker, for example, a biotin-avidin bond represented by G '-G ", using the biotin-avidin methodologies known in the art. This conjugate based on a linker, for example, vitamin D-biotin-avidin-antibody, is suitably represented as: [D- G '] * [G "- T] A schematic diagram for the coupling of the antibodies with the compounds of Vitamin D or analogues using, for example, the biotin-avidin conjugates is provided in Figure 7. With respect to Figure 7, avidin possesses a high affinity for coenzyme biotin.This is a strong, non-covalent interaction that has been exploited for the conjugation of antibodies with different compounds Biotin or avidin is suitably coupled to any vitamin D compound or antibody component As such, different schemes are possible for the binding of vitamin D compounds and antibodies. For example, biotin binds properly with the antibody to form a biotinylated antibody complex, while avidin binds appropriately with the vitamin D compound for form an avidin-vitamin D complex. The two complexes are subsequently reacted to form an antibody-biotin-avidin-vitamin D conjugate. The vitamin D-biotin-avidin-antibody conjugate is formed in a similar manner as shown in the Figure 7. It may be desirable to conjugate hormones or other agents (designated as? ") To the conjugates of formula (I), to form a bifunctional conjugate as represented by formula (XV). (D) m * (T) "* (A) p (XV) wherein A represents a therapeutic agent other than vitamin D and p is an integer of 1 or greater, D, T, m and n are as defined herein , with the proviso that D and A maintain their biological effectiveness, and * indicates that the portion of the target molecule is associated with the vitamin D portion and with the therapeutic agent different from vitamin D. For example, a bifunctional conjugate of Formula (XV) is one that has the ability to supply vitamin D in bone as well as another osteogenic agent such as an estrogen. Thus, included within the scope of the present invention are conjugates of formula (XV) which are therapeutic conjugates of bone, wherein A is a hormone or other agents that are known to ameliorate bone diseases or disorders. These agents may include conjugated estrogens or their equivalents, anti-estrogens, calcitonin, bisphosphonates, calcium supplements, cobalamin, pertussis toxin, boron, DHEA and other bone growth factors such as transforming growth factor beta, activin or protein. morphogenic of bone. Also provided in the present invention are conjugates of the formula (XV) which are antiproliferative conjugates, wherein A is a cytotoxic agent. These agents include stromustene, phosphate, prednimustine, cisplatin, S-fluorouracil, melphalan, hiroxyurea, mitomycin, idarubicin, methotrexate, adriamycin and daunomycin. Included within the scope of the present invention are all possible enantiomers of any conjugate of the invention that presents optical isomers and all possible geometric isomers due to a cis-trans configuration in the double bonds. In addition, all pharmaceutically acceptable salts of the compounds described herein, such as the sodium, potassium, lithium ammonium salts of the compounds, and the like. The magnitude of a prophylactic or therapeutic dose of the conjugates, according to the invention, will vary with the nature or severity of the condition being treated and with the specific composition and its route of administration. In general, the daily dose range for use in bone diseases is within the range of about 0.025 nmol / kg of body weight to about 2.5 nmol / kg. The daily dose for the treatment of hyperproliferative diseases, such as cancers, is in the range of about 0.025 nmol / kg to about 5 nmol / kg of body weight. The conjugates of the formula (I) are useful as active compounds in pharmaceutical compositions with reduced side effects and low toxicity in comparison with the known analogs of the active forms of vitamin D3. The pharmacologically active conjugates of this invention can be processed according to conventional methods of the pharmacy to produce medicinal agents for administration in patients, for example, mammals including humans. Any suitable administration route can be employed to provide an effective dose of the conjugate. For example, it is possible to employ oral, rectal, topical, parenteral, intravenous, intramuscular, subcutaneous, ocular, nasal, buccal, and the like. The pharmaceutical compositions of the present invention include the conjugate of the present invention as an active ingredient or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier and, as an option, other therapeutic ingredients. The compositions are those suitable for the different routes of administration described herein, although the most appropriate route in any case will depend on the nature and severity of the condition being treated and on the nature of the active ingredient. These for convenience are presented in unit dosage form. Suitable pharmaceutically acceptable carriers for use in the composition or method of the present invention include, but are not limited to, water, saline solutions, alcohols, gum arabic, vegetable oils (e.g., corn oil, seed oil). cotton, peanut oil, olive oil, coconut oil), fish liver oils, oily esters such as polysorbate 80, polyethylene glycols, gelatin, carbohydrates (for example lactose, amylose or cotton), magnesium stearate, talc, acid silicic acid, viscous paraffin, monoglycerides and diglycerides of fatty acids, fatty acid esters of pentaerythritol, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like. The pharmaceutical preparations can be sterilized and, if desired, mixed with auxiliary agents, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, dyes and flavorings. In addition, the conjugate of the present invention can be coated or protected to prevent immunogenicity or reticuloendothelial response (RES) by, for example, the liver. Agents that can be used for this purpose include polyethylene glycol (PEG) and others known in the art. In another aspect, the present invention is a method of site-specific delivery of a portion of vitamin D to a tissue of interest in a patient, which consists of: (1) providing a conjugate of a portion of vitamin D and at least one target molecule portion in a pharmaceutically acceptable carrier; and (2) administering a therapeutically effective dose of the conjugate of the present invention described herein. However, the conjugate is preferably supplied in one of the following forms, depending on the specific administration method. For parenteral application, particularly suitable are injectable, sterile solutions, preferably in oil or aqueous solution, as well as suspensions, emulsions or implants, including suppositories. The ampoules are convenient unit dosages. For enteric administration, particularly suitable are tablets, capsules, liquids, drops, suppositories, pills, powders or capsules. A syrup, elixir, or the like can be used if a sweetened vehicle is desired. Due to their ease of administration, tablets and capsules represent the most advantageous oral dosage unit. For rectal administration, the conjugates are formed in a pharmaceutical composition containing a suppository base such as cocoa butter or other triglycerides. To prolong shelf life, the composition advantageously includes an antioxidant, such as ascorbic acid, butylated hydroxyanisole or hydroquinone. Suitable topical formulations include transdermal devices, aerosols, creams, ointments, lotions, dusts and the like. Oral administration of the pharmaceutical compositions of the present invention is preferred. The daily dosage of the compounds according to this invention, in general, is about 0.025 to about 2.5 nmol / kg, preferably about 0.025 to about 1 nmol / kg. In general, the conjugates of this invention are administered by unit dosage forms in a pharmaceutically acceptable carrier. For the treatment of hyperproliferative diseases such as cancers, the enteric dosage of the conjugates of the formula (I) is from about 1 mmol to about 100 nmol per unit dosage; for bone diseases, approximately 0.5 nmol to 50 nmol per unit dose. In addition, those skilled in the art will also appreciate that these doses can be encapsulated in time release systems, for example, sustained release, delayed or targeted systems such as a delivery system by liposomes, polysaccharides that have a release mechanism slow, salistic implants [sic] or other polymers or microspheres, as well as those where the active ingredient is suitably protected with one or more coatings that can be degraded differently, for example, by microencapsulation, enteric coating, multiple coatings, and so on, and these means effect the continuous dosing of the compositions contained therein. For example, an enteric coating is one that resists disintegration in gastric juice. It is also possible to freeze the active ingredient by freezing and use the lyophilizate obtained, for example, for the preparation of products for injection. Furthermore, it will be appreciated that the preferred, actual amounts of the active compound in a specific case will vary according to the effectiveness of the specific compound being used, the specific formulations formulated, the mode of application and the specific site and organism concerned. For example, the specific dose for a particular patient will depend on age, sex, body weight, general health status, diet, time and mode of administration, rate of excretion and on medications used in combination and the severity of the disorder specific to which the therapy is applied. The doses for a certain host can be determined using conventional considerations, for example, by customary comparison of the differential activities of the compounds and of a known agent, such as by means of a suitable conventional pharmacological protocol. The present invention is further explained by the following examples that should not be considered as limiting the scope of the present invention.Example 1: Synthesis of la- (OH) -24-aminoalkyl-l, 1- bisphosphonate-D2 conjugate (7). Reference is made to the reaction scheme of Figure 1. A solution of known alcohol (1) (1.0 g, 1.52 mmol) in toluene (5 ml) is added to 22 ml of a 12.5% solution of phosgene in toluene, and The solution is stirred at room temperature for 20 hours. The reaction mixture is concentrated under reduced pressure to provide the chloroformate (2). Pyridine (0.18 ml, 2.2 mmol) is added to a solution of (2) (1.1 g, 1.53 mmol) and tetraisopropyl 4-aminobutyl-l, 1-bisphosphonate 3 (see, Saari et al., Patent United States No. 5,183,815) (0.92 g, 2.2 mmol) in CH2C12 (12 ml), and the mixture is stirred at room temperature for 3 days. The reaction mixture is concentrated under reduced pressure, and the residue is purified by flash chromatography on silica gel using methanol / chloroform as the eluent, to produce compound (4). A solution of compound (4) (0.80 g, 0.74 mmol) in tetrahydrofuran (THF) (10 ml) and tetrabutylammonium fluoride (TBAF) (2.2 ml, of a 1.0 M THF solution, 2.2 mmol) is stirred at room temperature for 24 hours. The reaction mixture is diluted with water (30 ml) and extracted with CH2C12 (3 x 40 ml). The combined CH2Cl2 fractions are dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel using methanol / chloroform as eluent to produce the tetraisopropylester compound (5). Trimethylsilyl bromide (0.39 ml, 2.92 mmol) is added to a solution of the compound tetraisopropyl ester (5) (0.5 g, 0.58 mmol) in CH2C12 (6 ml), and the mixture is stirred at room temperature for 24 hours in an atmosphere inert. The reaction mixture is concentrated under reduced pressure and the residue is diluted with water (15 ml). The mixture is filtered and the filtrate is lyophilized to provide the compound - (6). A solution of compound (6) (250 mg, 0.36 mmol) and 9-acetylanthracene (21 mg) in methanol (93 ml) is placed in a 500 ml ACE reactor and the solution purged with nitrogen for 15 minutes. The reaction mixture is cooled to 0 ° C and irradiated with a 400W Hanovia lamp filtered through uranyl glass for 2 hours. The reaction mixture is filtered and the filtrate is concentrated under reduced pressure. The residue is diluted with water (10 ml). The mixture is filtered and the filtrate is lyophilized to provide the above-titled conjugate (7).

Example 2: Synthesis of 1-aminoalkyl-l, l-bisphosphonate-24- (OH) -D2 (16) Reference is made to the reaction scheme depicted in Figures 2A and 2B. To a solution of compound (1) (2.0 g, 3.04 mmol) and N, N-diisopropylethylamine (0.78 g, 6.1 mmol) in CH2C12 (25 mL) is added chloromethyl methyl ether (0.29 g, 3.6 mmol). The resulting reaction mixture is stirred at 0 ° C for 1 hour, then at room temperature for 7 hours, before dilution with water (30 ml). The separated aqueous phase is extracted with CH2Cl2 (3 x 25 ml), and the combined organic phases are dried over anhydrous sodium sulfate, filtered and the filtrate concentrated in vacuo. The residue is purified by flash chromatography on silica gel to provide compound (8). A solution of compound (8) (1.99 g, 2.84 mmol) in THF (38 ml) and TBAF (8.4 ml of a 1.0 M THF solution) is stirred at room temperature for 24 hours. The reaction mixture is diluted with water (100 ml) and extracted with CH2C12 (3 x 100 ml). The combined CH2C12 phases are dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel using methanol / chloroform as eluent to produce the diol compound (9). To a solution of the diol (9) (1.25 g, 2.64 mmol), N, N-dimethylformamide (20 ml) and imidazole (0.54 g, 7.93 mmol) is added tert-butyldimethylsilyl chloride (0.40 g).2.64 mmol). The reaction mixture is stirred at room temperature for 6 hours, before dilution with water (60 ml) and extraction with CH2C12 (3 x 70 ml). The combined organic phases are washed with brine (50 ml), dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel to provide two separate products. These are identified in Figure 2A as alcohol (10) and alcohol (eleven) . As illustrated in the upper part of Figure 2B, a solution of alcohol (10) (0.5 g, 0.85 mmol) in toluene (2.5 ml) is added to 12.3 ml of a 12.5% solution of phosgene in toluene and the solution it is stirred at room temperature for 20 hours. The reaction mixture is concentrated under reduced pressure to provide chloroformate (12) Pyridine (0.09 ml, 1.1 mmol) is added to a solution of chloroformate (12) (0.5 g, 0.77 mmol) and 4-aminobutyl-1, tetraisopropyl bisphosphonate. (3) (0.46 g, 1.1 mmol) in CH2C12 (6 mL) and the mixture is stirred at room temperature for 3 days. The reaction mixture is concentrated under reduced pressure and the residue is purified by flash chromatography on silica gel using methanol / chloroform as the eluent to produce compound (13). A solution of compound (13) (0.50 g, 0.49 mmol) THF (7 ml) and TBAF (0.74 ml) of a 1.0 M THF solution, 0.74 mmol) is stirred at room temperature for 24 hours. The reaction mixture is diluted with water (20 ml) and extracted with CH2C1 (3 x 30 ml). The combined CHC12 fractions are dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel using methanol / chloroform as the eluent to provide compound (14). • Trimethylsilyl bromide (0.26 ml, 1.96 mmol) is added to a solution of tetraisopropylester (14) (0.35 g, 0.39 mmol) in CH2C12 (4 ml) and the mixture is stirred at room temperature for 24 hours under an inert atmosphere. The reaction mixture is concentrated under reduced pressure and the residue is diluted with water (15 ml), and methanol (3 ml) and stirred for 8 hours. The mixture is filtered and the filtrate is lyophilized to provide the compound (15). A solution of compound (15) (0.21 g, 0.31 mmol) and 9-acetylanthracene (18 mg) in methanol (80 ml) is placed in a photoreactor, and the solution is purged with nitrogen for 15 minutes. The reaction mixture is cooled to 0 ° C and irradiated with a 400W Hanovia lamp filtered through uranyl glass for 2 hours. The reaction mixture is filtered, the filtrate is concentrated under reduced pressure. The residue is diluted with water (10 ml). The mixture is filtered and the filtrate is lyophilized to provide the conjugate of the above title (16).

Example 3: Synthesis of the, 24- (OH) 2-3-aminoalkyl-l, 1-bisphosphonate-D2 (21). Reference is made to Figure 3. A solution of alcohol (11) (0.5 g, 0.85 mmol) in toluene (2.5 ml) is added to 12.3 ml of a 12.5% solution of phosgene in toluene, and the solution is stirred at room temperature for 20 hours. The reaction mixture is concentrated under reduced pressure to provide chloroformate (17). Pyridine (0.81 ml, 1 mmol) is added to a solution of (17) (0.45 g, 0.69 mmol) and tetraisopropyl 4-aminobutyl-l, 1-bisphosphonate (0.41 g, 1 mmol) in CH2C12 (5 mL), and the mixture is stirred at room temperature for 3 days. The reaction mixture is concentrated under reduced pressure, and the residue is purified by flash chromatography on silica gel using methanol / chloroform as the eluent, to provide compound (18). A solution of compound (18) (0.47 g, 0.46 mmol) in THF (6.5 ml) and TBAF (0.7 ml, of a 1.0 M THF solution, 0.7 mmol) is stirred at room temperature for 24 hours. The reaction mixture is diluted with water (20 ml) and extracted with CH2C12 (3 x 30 ml). The combined CH2C12 fractions are dried over sodium sulfate. Anhydride is filtered and the filtrate is concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel using methanol / chloroform as the eluent to produce the compound (19). Trimethylsilyl bromide (0.23 ml, 1.74 mmol) is added to a solution of tetraisopropyl ester (19) (0.31 g, 0.34 mmol) in CH 2 Cl 2 (4 ml), and the mixture is stirred at room temperature for 24 hours under one atmosphere inert. The reaction mixture is concentrated under reduced pressure and the residue is diluted with water (15 ml) and methanol (3 ml) and stirred for 8 hours. The mixture is filtered and the filtrate is lyophilized to provide the compound (20). A solution of the compound (20) (0.21 g, 0.31 mmol) and 9-acetylanthracene (18 mg) in methanol (80 ml) is placed in a 500 ml ACE photoreactor and the solution purged with nitrogen for 15 minutes. The reaction mixture is cooled to 0 ° C and irradiated with a 400W Hanovia lamp filtered through uranyl glass for 2 hours. The reaction mixture is filtered and the filtrate is concentrated under reduced pressure. The residue is diluted with water (10 ml). The mixture is filtered and the filtrate is lyophilized to provide the conjugate of the previous title (21).

Example 4: Synthesis of α-aminoalkyl-1, l-bisphosphonate-25 (0H) -D3 (32) Reference is made to the reaction scheme depicted in Figures 4A and 4B. A known ketone solution (22) (3.1 g, 11.1 mmol) (see, Baggiolini et al., 51 J. Org. Chem. (1986), 3098-3108), incorporated herein by reference, 3, 4- dihydro-2H-pyran (1.52 ml, 16-7 mmol), and pyridinium p-toluenesulfonate (0.1 g, 0.4 mmol), are dissolved in CH2C12 (50 ml) and stirred at room temperature for 24 hours. The reaction mixture is washed with water (30 ml), and the organic phase is dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel to produce the ketone compound (23). A solution of the known phosphine oxide (24) (1.35 g, 2.32 mmol) in 35 ml of anhydrous THF is cooled to -78 ° C and treated with m-butyllithium (1.45 ml of a 1.6 M solution in hexane), drop a drop. The anion solution is stirred during minutes at -78 ° C before the addition of the ketone (23) (0.57 g, 1.56 mmol) dissolved in anhydrous THF (10 ml) during -15 minutes. The reaction mixture is stirred for 2 hours at -78 ° C, then it is diluted with 2N sodium potassium tartrate (6 ml) and 2N potassium bicarbonate (6 ml) the solution is warmed to room temperature and extracted with acetate of ethyl (4 x 25 ml). The combined organic fractions are washed with brine (30 ml), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel using ethyl acetate / hexane as eluent to provide compound (25). A solution of compound (25) (0.95 h, 1.3 mmol) in THF (17 ml) and TBAF (3.9 ml of a 1.0 M THF solution, 3.9 mmol) is stirred at room temperature for 24 hours. The reaction mixture is diluted with water (50 ml) and extracted with CH2C12 (3 x 60 ml). The combined CH2Cl2 fractions are dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel using methanol / chloroform as eluent to provide the diol compound (26). To a solution of the compound diol (26) (0.55 g, 1.1 mmol), N, N-dimethylformamide (8 ml) and imidazole (0.255 g, 3.3 mmol) was added tert-butyldimethylsilyl chloride (0.166 g, 1.1 mmol). The reaction mixture is stirred at room temperature for 6 hours, before dilution with water (30 ml) and extraction with CH2Cl2 (3 x 40 ml). The combined organic phases are washed with brine (30 ml), and dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel to provide two separate products. These are identified, in the lower part of Figure 4A as alcohol (27) and alcohol (28). A solution of alcohol (27) (0.25 g, 0.41 mmol) in toluene (3 ml) is added to 6 ml of a 12.5% solution of phosgene in toluene and the reaction mixture is stirred at room temperature for 20 hours. The reaction mixture is concentrated under reduced pressure to provide chloroformate (29), as shown in the upper part of Figure 4B. Pyridine (0.047 mL, 0.57 mmol) is added to a solution of chloroformate (29) (0.27 g, 0.4 mmol) and 4-aminobutyl-1,1-bisphosphonate tetraisopropyl (0.24 g, 1.57 mmol) in CH2C12 (4 mL). and the mixture is stirred at room temperature for 3 days. The reaction mixture is concentrated under reduced pressure and the residue is purified by flash chromatography on silica gel using methanol / chloroform as eluent to produce compound (30). A solution of compound (30) (0.36 g, ml, 0.35 mmol) THF (5 ml) and TBAF (0.52 ml) of a solution of 1.0 M THF, 0.52 mmol) is stirred at room temperature for 24 hours. The reaction mixture is diluted with water (15 ml) and extracted with CH2C12 (3 x 25 ml). The combined fractions of CH2C12 are dried over sodium sulfate anhydride, they are filtered and concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel using methanol / chloroform as the eluent to provide the compound (31). Tetramethylsilyl bromide (0.20 ml, 1.54 mmol) is added to a solution of tetraisopropylester (31) (0.28 g, 0.30 mmol) in CH2C12 (4 ml) and the mixture is stirred at room temperature for 24 hours under an inert atmosphere. The reaction mixture is concentrated under reduced pressure and the residue is diluted with water (15 ml), and methanol (3 ml) and stirred for 0.5 hour. The mixture is filtered and the filtrate is lyophilized to provide the conjugate of the previous title (32).

Example 5: Synthesis of la-25 (OH) 2-3-aminoalkyl-1, 1-bisphosphonate-D3 (36). Reference is made to Figure 5. A solution of alcohol (28) (0.26 g, 0.43 mmol) in toluene (3 ml) is added to 6.2 ml of a 12.5% solution of phosgene in toluene, and the reaction is stirred at room temperature. environment for 20 hours.

The reaction mixture is concentrated under reduced pressure to provide chloroformate (33). Pyridine (0.049 ml, 0.59 mmol) is added to a solution of chloroformate (33) (0.28 g, 0.42 mmol) and 4-aminobutyl-1,1-bisphosphonate tetraisopropyl (0.5 g, 0.59 mmol) in CH2C1 (5 ml) , and the mixture is stirred at room temperature for 3 days. The reaction mixture is concentrated under reduced pressure, and the residue is purified by flash chromatography on silica gel using methanol / chloroform as eluent, to produce compound (34). A solution of compound (34) (0.37 g, 0.36 mmol) in THF (5 ml) and TBAF (0.54 ml, of a 1.0 M THF solution, 0.54 mmol) is stirred at room temperature for 24 hours. The reaction mixture is diluted with water (15 ml) and extracted with CH2C12 (3 x 25 ml). The combined CH2C12 fractions are dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel using methanol / chloroform as eluent to produce tetraisopropylester (35). Trimethylsilyl bromide (0.21 ml, 1.60 mmol) is added to a solution of tetraisopropyl ester (35) (0.29 g, 0.31 mmol) in CH 2 Cl 2 (5 ml), and the mixture is stirred at room temperature for 24 hours under an inert atmosphere . The reaction mixture is concentrated under reduced pressure and the residue is diluted with water (15 ml) and methanol (3 ml) and stirred for 0.5 hour. The mixture is filtered and the filtrate is lyophilized to provide the conjugate of the previous title (36).

Example 6: Synthesis of la- (OH) -25-aminoalkyl-1, 1-bisphosphonate-D3 (41). Reference is made to Figure 6. A solution of ether (25) (1.31 g, 1.8 mmol) in methanol (10 ml) and water (2 ml) is added pyridinium p-toluensulfonate hydrate (0.034 g, 0.18 mmol). The reaction mixture is stirred at room temperature for 1 hour, diluted with water (20 ml) and extracted with CH2C12 (3 x 30 ml). The combined organic phases are dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue is purified by flash chromatography on silica gel using methanol / chloroform as eluent to provide alcohol (37) A solution of alcohol (37) (1.09 g, 1.7 mmol) in toluene (9 ml) is added to 25 ml of a 12.5% solution of phosgene in toluene, and the reaction is stirred at room temperature for 20 hours. The reaction mixture is concentrated under reduced pressure to provide chloroformate (38) Pyridine (0.19 ml, 2.32 mmol) is added to a solution of chloroformate (38), (1.17 g, 1.65 mmol) and tetraisopropyl 4-aminobutyl-1, 1-bisphosphonate (0.98 g, 2.32 mmol) in CH2C12 (20 ml ), and the mixture is stirred at room temperature for 3 days. The reaction mixture is concentrated under reduced pressure and the residue is purified by flash chromatography on silica gel, using methanol / chloroform as eluent to produce alcohol (39).

A solution of alcohol (39) (1.61 g, 1.5 mmol) in THF (20 ml) and TBAF (4.5 ml) of a 1.0 M THF solution, (4.5 mmol) is stirred at room temperature for 24 hours. The reaction mixture is diluted with water (60 ml) and extracted with CH2Cl2 (3 x 60 ml). The combined CH2Cl2 phases are dried over anhydrous sodium sulfate, filtered and the filtrate is concentrated under reduced pressure. The residue is purified by flash chromatography on silica gel using methanol / chloroform as eluent, to provide the tetraisopropyl ester compound (40). Trimethylsilyl bromide (0.75 ml, 5.68 mmol) is added to a solution of the compound tetraisopropylester (40) (0.93 g, 1.1 mmol) in CH 2 Cl (20 ml), and the mixture is stirred at room temperature for 24 hours under an inert atmosphere . The reaction mixture is concentrated under reduced pressure and the residue is diluted with water (30 ml). The mixture is filtered and the filtrate is lyophilized to provide the title compound above (41). In summary, the present invention provides vitamin D conjugates useful in targeted applications.

The conjugates include a portion of vitamin D and a portion of the leader molecule that has affinity for a tissue of interest. The conjugates are characterized by a site-specific localization capacity of vitamin D compounds, for example, a vitamin D conjugate and an agent with affinity in bone is designed to transport and deliver vitamin D into bone tissue. Although the present invention has now been described and exemplified with some specificity, those skilled in the art will appreciate that various modifications, including variations, additions and omissions can be made in what has been described.

Claims (40)

1. A conjugate that consists of at least a portion of vitamin D associated with a portion of the target molecule that has an affinity for a tissue of interest.

The conjugate of claim 1, wherein the molar ratio of the at least one portion of vitamin D to the at least one portion of the target molecule is 1: 1.

3. The conjugate of claim 1, wherein the vitamin D portion is associated with a target molecule portion by a linking group.

The conjugate of claim 3, wherein the linking group is a linking group formed by the modification of the vitamin D portion and the portion of the target molecule to form a link therebetween.

5. The conjugate of claim 3, wherein the linking group is a bifunctional linker.

The conjugate of claim 3, wherein the portion of the vitamin D is associated with the portion of the target molecule via the linking group and at least one additional linking group.

The conjugate of claim 1, wherein the portion of the target molecule is a bisphosphonate moiety.

8. The conjugate of claim 1, wherein the portion of the target molecule is a dihydroepiandrosterone moiety.

The conjugate of claim 7, wherein the bisphosphonate is linked to the vitamin D portion at a position of the vitamin D portion that is C-1, C-3, C-24 or C-25.

10. The conjugate of claim 1, wherein the portion of the target molecule is a metal ion.

11. The conjugate of claim 5, wherein the bifunctional linker is an amino acid chelated to the portion of the target molecule and linked to the vitamin D portion by an amide bond.

12. The conjugate of claim 10, wherein the metal ion is a divalent metal ion selected from the group consisting of Sr2 +, Zn2 +, Mg2 +, Fe2 [sic], Cu2 +, Mn2 +, Ca2 +, Cu2 +, Co2 +, Cr2 + or Mo2 +.

The conjugate of claim 1, wherein the portion of the target molecule is an antibody.

The conjugate of claim 13, wherein the portion of the antibody target molecule is associated with the vitamin D portion by a biotin-avidin bond, wherein the biotin is bound to the antibody and the avidin binds to the portion of the antibody. vitamin D.

15. The conjugate of claim 13, wherein the portion of the target molecule is a monoclonal antibody.

16. The conjugate of claim 13, wherein the portion of the target molecule is a polyclonal antibody.

17. The conjugate of claim 1 further comprises at least one therapeutic agent different from the portion of vitamin D conjugated thereto.

The conjugate of claim 17, wherein the therapeutic agent is a therapeutic agent for bone, selected from the group consisting of conjugated estrogens or their equivalents, antiestrogens, calcitonin, bisphosphonates, calcium supplements, cobalamin, pertussis toxin, boron , dihydroepiandrosterone, beta bone growth factor, transformant, activin and morphogenic bone protein.

The conjugate of claim 17, wherein the therapeutic agent is a cytotoxic agent selected from the group consisting of stromustene phosphate, prednimustine, cisplatin, S-fluorouracil, melphalan, hydroxyurea, mitomycin, idarubicin, methotrexate, adriamycin, and daunomycin.

20. A pharmaceutical composition containing: a conjugate that includes at least a portion of vitamin D associated with at least one target molecule portion that has affinity for a tissue of interest, and a suitable pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20 further comprises a differentially degradable coating that encapsulates the conjugate for delivery by release of the conjugate over time.

22. The pharmaceutical composition of claim 21, wherein the coating is an enteric coating.

23. A site-specific delivery method of a vitamin D portion at a site of interest to a patient, comprises the steps of: a) providing a conjugate that includes a vitamin D portion and a pharmaceutically acceptable carrier, the conjugate having at least one vitamin D portion associated with at least one target molecule portion, the target molecule portion having affinity for the tissue of interest, and b) administering a therapeutically effective dose of the conjugate to the patient.

The method of claim 23, wherein the conjugate is administered orally to a patient having a bone disease, from 0.5 nmol to 50 nmol per unit dose.

25. The method of claim 23, wherein the conjugate is administered orally to a patient for the treatment of hyperproliferative diseases, in about 1 nmol to about 100 nmol per unit dose.

26. The method of claim 23, wherein the conjugate is delivered to a tissue of interest after being administered to the patient.

The method of claim 26, wherein the portion of the target molecule is unfolded after the conjugate is delivered to the tissue of interest, thereby improving the effectiveness of the vitamin D portion of the conjugate.

28. A conjugate of formula (I) (D) a * (T) n (I) wherein each D represents a vitamin D portion; each T represents a target molecule portion; n and m represent integers of 1 or greater; and * indicates that the target molecule portion is associated with the vitamin D portion.

29. An antiproliferative composition comprising the conjugate of claim 28, wherein T is an agent that has an ability to search for a vitamin D receptor of a cancer cell.

30. The conjugate of claim 28, wherein T is a monoclonal, polyclonal antibody or fragment thereof, a metal ion, a bone-seeking agent or a tumor-seeking agent.

The conjugate of claim 30, wherein the bone targeting agent is a bisphosphonate, tetracycline, a polymalonate or dihydroepiandrosterone.

32. A conjugate of formula (IV): [(T) n - (G ') f] g * [(G ") h - (D) m] k (IV) wherein each G' represents a linking group; each G "represents the same connecting group or different as G '; each D represents a vitamin D portion; each T represents a target molecule portion; g, k, m and n represent integers of one or greater; and f and h represent integers of 0 or greater; -indicates a link in cases where a connector group is present; and * indicates that each D is associated with each T by the connector G 'or G "or by the connector G' and G" when both connectors are present.

33. The conjugate of claim 32, wherein G 'is biotin; G "is avidin and where * represents a biotin-avidin bond

34. The conjugate of formula (XV): (D) m * (T) n * (A) p (XV) where each D represents a portion vitamin D, each T represents a target molecule portion, each A represents a therapeutic agent in addition to vitamin D, m, n and p represent integers of 1 or greater, and * indicates that the target molecule portion is associated with the vitamin D portion and with the therapeutic agent other than vitamin D.

35. The conjugate of claim 34, wherein A is a cytotoxic agent or a therapeutic agent for bone

36. The conjugate of claim 35, wherein T is a bone-seeking agent.

37. A method of treatment for bone diseases in a human being, consists of administering to the person a therapeutically effective amount of the conjugate of claim 36.

38. The conjugate of claim 34, wherein T is a bone and A bone searching agent. It is a bone-seeking agent.

39. A method of treating bone diseases in a human being, consists in administering to the person a therapeutically effective amount of the conjugate of claim 38.

40. An antiproliferative composition containing the conjugate of claim 34, wherein T is an agent that has a capacity to search for a vitamin D receptor of a cancer cell, and A is a cytotoxic agent.