WO2011019882A1

WO2011019882A1 - Nanostructure-beta-blocker conjugates

Info

Publication number: WO2011019882A1
Application number: PCT/US2010/045279
Authority: WO
Inventors: Lon J. Wilson; Ivana Romina Guadalupe Peralta
Original assignee: William Marsh Rice University
Priority date: 2009-08-12
Filing date: 2010-08-12
Publication date: 2011-02-17

Abstract

Nanostructure-beta-blocker conjugates are provided. Additionally, methods of synthesizing nanostructure beta-blocker conjugates and their use in the treatment and prevention of bone disease, such as osteoporosis, are also provided.

Description

NANOSTRUCTURE-BETA-BLOCKER CONJUGATES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/233,263, filed August 12, 2009, which is incorporated herein by reference.

BACKGROUND

Bone is the hard form of connective tissue that makes up most of the skeleton; it consists of an organic component, the cells and matrix, and an inorganic, or mineral component. Living bone is continuously recycled by the processes of bone formation and resorption. During an animal's growth years, bone formation exceeds resorption and the skeletal mass increases. In humans, bone mass reaches a peak between ages 20 and 30 years. After that time, the rate of formation and resorption stabilize the bone mass until age 35 to 40 years, at which time resorption begins to exceed formation, and the total mass slowly decreases. The process of bone turnover in adults is known as remodeling. Up to 15 percent of the total bone mass turns over every year in the remodeling process.

Two major cell types make up bone and are responsible for the remodeling process

(ossification): osteoclasts and osteoblasts. Osteoclasts absorb and remove mineralized bone, releasing calcium and phosphate. Osteoblasts assimilate calcium and phosphate to slowly produce crystals, or mineralized bone. As the mineralized bone accumulates and surrounds the osteoblast, that cell slows its activity and becomes an interior osteocyte.

Between the areas of osteoclastic and osteoblastic activity is a cement line containing bone matrix material, which delineates the zones of resorption and new bone formation. Bone formation takes place in areas where bone undergoes the greatest stress. Therefore, a bone that is underutilized, such as a leg that is immobilized, is prone to resorption.

Bone remodeling not only alters the architecture of the bone, it also enables the body to regulate the levels of calcium ions in the blood and interstitial fluid. These calcium levels must remain within a fairly narrow range in order to ensure the proper functioning of nerve transmission, the integrity and permeability of cellular membranes, and the ability of the blood to clot. Bone contains about 99 percent of the body's calcium. When fluid calcium levels fall too low, parathyroid hormone stimulates osteoclast activity (causing increased bone resorption) and the subsequent release of calcium into the bloodstream. When fluid calcium levels rise excessively, the hormone calcitonin inhibits resorption (acting against the parathyroid hormone), thereby restricting the release of calcium from the bones. It is necessary to have a healthy intake of calcium to maintain the body's calcium reserve; otherwise, the calcium levels in the body become dependent on the resorption of bone tissue. Vitamin D is also essential, as it makes possible the body's use of ingested calcium. Estrogen also inhibits bone resorption.

Many bone diseases are related to the composition and scale of bone tissue. For example, when a bone has much more bone tissue than average, it is termed osteosclerosis; when there is less, it is called osteopenia. If bone suffers from a lack of mineral content, it is called rickets in children and osteomalacia in adults. The afflicted bones become malleable and vulnerable to deformities. In children, this condition is often the result of vitamin D deficiency. Of all bone diseases, osteoporosis, a generalized osteopenia, is the most common. This disease primarily affects the aged and is more serious in women than men. Osteoporosis is responsible for many of the fractures encountered by the elderly. Another disease that often afflicts the elderly is Paget' s disease, characterized by bone deformity and calcium imbalance.

Likewise, bone cells can be killed by a lack of blood supply; this tissue death is termed necrosis or osteonecrosis. It can be brought on by injury, the blockage of an artery, circulatory problems, the administration of corticosteroid hormones for the treatment of another affliction, or by a disease of the metabolic system. Osteomyelitis refers to a bone infection, which can be acquired through an open wound, or from an infection elsewhere in the body. Tumors can also develop in bone tissue. Congenital bone diseases refer to abnormalities which are present at birth; some are genetically transferred but most occur due to problems during pregnancy or delivery. Lastly, bone fractures are the result of a force greater than the strength and resistance of the bone. Age and disease are factors that determine whether a given force will cause a fracture.

The conditions and factors listed above can cause undesired bone loss or a need to replace lost bone through enhanced bone growth. Hence, compositions that mitigate bone loss and/or encourage bone growth are the subject of ongoing research. SUMMARY

The present disclosure relates generally to nanostructure-beta-blocker conjugates. More particularly, in some embodiments, the present disclosure relates to nanostructure-beta-blocker conjugates, methods of their synthesis, and their use in the treatment and prevention of bone disease, such as osteoporosis.

In one embodiment, the present disclosure provides a composition comprising: a C_n carrier, wherein C_n refers to a fullerene moiety or nanotube comprising n carbon atoms, a bone targeting agent, and a beta-blocker.

In another embodiment, the present disclosure provides a method comprising administering a therapeutically effective amount of a composition comprising a C_n carrier, wherein C_n refers to a fullerene moiety or nanotube comprising n carbon atoms, a bone targeting agent, and a beta-blocker to a mammal.

In yet another embodiment, the present disclosure provides a method comprising providing a C_n carrier, wherein C_n refers to a fullerene moiety or nanotube comprising n carbon atoms, and allowing a bone targeting agent and a beta-blocker to react with the carrier so as to form a nanostructure-beta-blocker conjugate.

The features and advantages of the present disclosure will be readily apparent to those skilled in the art upon a reading of the description of the embodiments that follows.

DRAWINGS

A more complete understanding of this disclosure may be acquired by referring to the following description taken in combination with the accompanying figures in which:

FIGURE 1 shows the chemical structures of certain beta-blockers, where * designates the chiral center.

FIGURE 2 shows the synthesis and Chemical Structure of levobunolol, where * designates the chiral center.

FIGURE 3 shows the two dimensional structure of a C₆₀-bisphosphonate-beta-blocker conjugate, according to one embodiment.

FIGURE 4 shows the synthesis of a model C₆₀ hydrazone compound (3). (a) acetone (b) C₆₀, CBr₄, DBU, toluene:CH₂Cl₂ (2:1).

FIGURE 5 shows the Synthesis of tert-bxxty\ 3-[(4-hydrazinyl-4-oxobutyl)amino]-3- oxopropanoate (5). (a) Et₃N, DCC, CH₂Cl₂, 88%; (b) anhydrous N₂H₄, methanol, 99%.

FIGURE 6 shows the synthesis of a second model C₆₀ hydrazone compound (7). (a) ethanol, 21%; (b) C₆₀, CBr₄, DBU, toluene;CH₂Cl₂(2:l).

FIGURE 7 shows the protection of levobunolol (8).(a) BoC₂O, DIPEA, t-BuOH:H₂0 (10:1).

FIGURE 8 shows the synthesis of a C₆₀-beta-blocker conjugate (11), according to one embodiment, (a) anhydrous N₂H₄, methanol, (b) protected levobunolol, 2-methoxy-ethanol, (c) CBr₄, DBU, toluene; CH₂C1₂(2:1).

FIGURE 9 shows the synthesis of a C₆o-Bisphosphonate-Beta-blocker Conjugate (13), according to one embodiment, (a) CBr₄, DBU, toluene;CH₂Cl₂(2:l), (b) Si(CH₃)₃I, CCl₄, H₂O, NaOH, TFA, NaCl. Only one possible geometrical isomer of 12 is shown.

FIGURE 10 is an image depicting the MALDI-TOF MS of 12.

While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the figures and are herein described in more detail. It should be understood, however, that the description of specific example embodiments is not intended to limit the disclosure to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as defined by the appended claims.

DESCRIPTION

The present disclosure relates generally to nanostructure-beta-blocker conjugates. More particularly, in some embodiments, the present disclosure relates to nanostructure-beta-blocker conjugates, methods of their synthesis, and their use in the treatment and prevention of osteoporosis.

It has been previously reported that a link exists between leptin, a hormone that regulates appetite and metabolism, and bone remodeling. Likewise, it has been demonstrated that the sympathetic nervous system of mice mediates bone resorption through β₂-adrenergic receptors on bone cells. Thus, it was hypothesized that blocking the sympathetic nervous system from interaction with β receptors could prevent or treat osteoporosis.

Beta-adrenergic antagonists or beta-blockers comprise a group of drugs that are mostly used to treat cardiovascular disorders such as hypertension, cardiac arrhythmia, or ischemic heart disease. They possess a natural high degree of enantioselectivity in binding to β (βi and/or β₂) receptors, and they competitively inhibit the beta effects of endogenous catecholamines, which are "fight-or-flight" hormones (adrenaline, noradrenaline and dopamine) that the adrenal glands release in response to stress. However, because beta-blockers are so active, they cannot be administered systemically for the treatment of osteoporosis. Thus, for beta-blockers to be used as a therapy for osteoporosis or other bone disorders, they should be targeted and delivered mainly to bone to minimize cardiovascular side effects. Accordingly, in some embodiments, the present disclosure provides a nanostructure-beta-blocker conjugate that may provide a targeted therapy for bone disease, such as osteoporosis.

In one embodiment, the present disclosure provides a composition comprising: a C_n carrier, wherein C_n refers to a fullerene moiety or nanotube comprising n carbon atoms, a bone targeting agent, and a beta-blocker. As used herein, a C_n carrier refers to a fullerene moiety comprising n carbon atoms or a nanotube moiety comprising at least n carbon atoms. In some embodiments, a C_n carrier may act as an ideal scaffold for targeted beta-blocker delivery because it is a biologically stable molecule that is non-toxic, non-immunogenic and efficiently cleared in mammals when it is properly derivatized.

Examples of suitable C_n carriers for use in conjunction with the compositions of the present disclosure include, but are not limited to, buckminsterfullerenes, gadofullerenes, single walled carbon nanotubes (SWNTs), and ultra-short carbon nanotubes (US-tubes). Buckminsterfullerenes, also known as fullerenes or more colloquially, buckyballs, are closed- cage molecules consisting essentially of sp²-hybridized carbons. Fullerenes are the third form of pure carbon, in addition to diamond and graphite. Typically, fullerenes are arranged in hexagons, pentagons, or both. Most known fullerenes have 12 pentagons and varying numbers of hexagons, depending on the size of the molecule. Common fullerenes include C₆₀ and C₇₀ (e.g. n=60 or n=70), although fullerenes comprising up to about 400 carbon atoms are also known.

SWNTs, also known as single walled tubular fullerenes, are cylindrical molecules consisting essentially of sp² hybridized carbons. In defining the size and conformation of single- walled carbon nanotubes, the system of nomenclature described by Dresselhaus et al., Science of Fullerenes and Carbon Nanotubes, Ch. 19. will be used. Single walled tubular fullerenes are distinguished from each other by a double index (x,y), where x and y are integers that describe how to cut a single strip of hexagonal graphite such that its edges join seamlessly when the strip is wrapped onto the surface of a cylinder. When x=y, the resultant tube is said to be of the "armchair" or (x,x) type, since when the tube is cut perpendicularly to the tube axis, only the sides of the hexagons are exposed and their pattern around the periphery of the tube edge resembles the arm and seat of an arm chair repeated n times. When y=0, the resultant tube is said to be of the "zig-zag" or (x,0) type, since when the tube is cut perpendicular to the tube axis, the edge is a zig zag pattern. Where x≠y and y≠O, the resulting tube has chirality. The electronic properties of the nanotube are dependent on the conformation, for example, arm-chair tubes are metallic and have extremely high electrical conductivity. Other tube types are metallic, semi-metals, or semiconductors, depending on their conformation. Regardless of tube type, all SWNTs have extremely high thermal conductivity and tensile strength. The SWNT may be a cylinder with two open ends, a cylinder with one closed end, or a cylinder with two closed ends. Generally, an end of an SWNT can be closed by a hemifullerene, e.g. a (10,10) carbon nanotube can be closed by a 30-carbon hemifullerene. If the SWNT has one or two open ends, the open ends can have any valences unfilled by carbon-carbon bonds within the single wall carbon nanotube filled by bonds with hydrogen, hydroxyl groups, carboxyl groups, or other groups. SWNTs can also be cut into ultra-short pieces, thereby forming US-tubes. As used herein, the term "US-tubes" refers to ultra short carbon nanotubes with lengths from about 20 nm to about 100 nm.

A C_n carrier can be substituted or unsubstituted. By "substituted" it is meant that a group of one or more atoms is covalently linked to one or more atoms of a C_n carrier. Generally, in situ Bingel chemistry may be used to substitute a C_n carrier with appropriate groups to form the targeted nanostructures of the present disclosure. Examples of groups suitable for use include, but are not limited to, malonate groups, serinol malonates, groups derived from malonates, serinol groups, carboxylic acid, polyethyleneglycol (PEG), and the like. In one embodiment, a C_n carrier is substituted with one or more water-solubilizing groups. Water-solubilizing groups are polar groups (that is, groups having a net dipole moment) that render the generally hydrophobic fullerene core soluble in water. The addition of such groups allow for greater biocompatibility of a C_n carrier. Generally, a C_n carrier may contain from 1 to 4 addends. A C_n carrier can be substituted with any water solubilizing group to allow for sufficient water solubility and biocompatibility, but the spectroscopic properties of the C_n carrier should not be compromised. In certain embodiments, a C_n carrier may be further substituted with either a thiol (-SH) or an amine (-NH₂) group.

As mentioned above, compositions of the present disclosure further comprise a bone targeting agent. One example of a suitable bone targeting agent may include, but is not limited to, a bisphosphonate. Bisphosphonates are compounds generally characterized by two C-P bonds. Non-limiting examples of suitable bisphosphonates may include etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, zoledronate and a combination thereof.

While not bound to any particular theory, it is currently believed that all bisphosphonates generally act in a similar manner on bone: they bind permanently to mineralized bone surfaces and inhibit subsequent osteoclastic activity, namely the removal of bone during the process of bone remodeling. Because they reduce the amount of bone tissue degraded during the remodeling cycle, they are sometimes referred to as "antiresorptive agents." The application of bisphosphonates usually reduces bone loss and, correspondingly, the risk of broken bones, and sometime increases bone mass.

As a group, the bisphosphonates may offer several advantages over estrogens in treating osteoporosis. They are bone-tissue specific, have minimal side effects (e.g., nausea, abdominal pain and loose bowel movements), cause no known risk of carcinogenesis, and have antiresorptive efficacy that is equivalent to or greater than estrogens. Furthermore, there is some evidence that the use of bisphosphonates can cause a reduction in incident vertebral fractures.

Nanostructure-beta-blocker conjugates of the present disclosure further comprise a beta- blocker. In some embodiments, a suitable beta-blocker possesses at least one chiral center and at least one of the chiral carbon atoms on the alkyl side chain is directly attached to a hydroxyl group. Furthermore, in some embodiments, suitable beta-blockers have at least one aromatic ring structure attached to a side alkyl chain possessing a secondary hydroxyl and amine functional group. In some embodiments, the amine may have an isopropyl or bulkier substituent group which appears to favor the interaction with beta-receptors. In general, it is believed that the nature of the substituents on the aromatic ring determines whether the effect will be predominantly activation or blockage. Most of the beta-blockers (e.g., propranolol, metoprolol, atenolol, and pindolol) have one chiral center, and as a result, they are marketed as a racemate of two enantiomers; the exception is timolol which is marketed as an S-enantiomer.

Examples of suitable beta-blockers may include, but are not limited to, dichloroisoproterenol, propanolol, atenolol, pindolol, metoprolol, timolol, bunolol, levobunolol, isomers thereof and combinations thereof (Figure 1). Pharmacologically, beta-blockers are distinguished based on their selectivity for beta-receptors. The non-selective beta-blockers, including propranolol, pindolol, and timolol, block both βi- and β₂-adrenergic receptors. On the other hand, selective beta-blockers, including metoprolol and atenolol, have greater affinity for βi receptors.

In general, beta-blockers are non-stereoselectively absorbed from the gastrointestinal tract via passive diffusion. The lipophilic beta-blockers are eventually metabolized and therefore eliminated. However, the more hydrophilic beta-blockers are usually excreted unchanged in urine.

Levobunolol, (-)-5-[3-(tert-Butylamino)-2-hydroxypropoxy]-3,4-dihydro-l(2H)- naphthalenone, is the levorotatory and pharmacologically active isomer of bunolol. It is a potent non-selective beta-blocker which may be synthesized by the reaction of epichlorohydrin with 5- hydroxy- 1 -tetralone in the presence of NaOH (Figure 2). It is currently marketed as a hydrochloride ophthalmic solution for the treatment of glaucoma as AKBeta® and Betagan®.

One specific example of a nanostructure-beta-blocker conjugate of the present disclosure is shown in Figure 3 and comprises a C₆₀ fullerene, bisphosphonate, and levobunolol. The carboxyl group of the tetralone of levobunolol was reacted with a malonic hydrazide and then coupled to the C₆o-Bis(bisphosphonate) compound via a Bingel reaction.

Generally, the nanostructure-beta-blocker conjugates of the present disclosure may be synthesized using Bingel-Hirsch additions to the carbon nanostructure. Furthermore, in certain embodiments, the nanostructure-beta-blocker conjugates of the present disclosure may be used for treating a disease. For example, the nanostructure-beta-blocker conjugates of the present disclosure may be used for the treatment of, among other things, osteoporosis.

To facilitate a better understanding of the present disclosure, the following examples of certain aspects of some embodiments are given. In no way should the following examples be read to limit, or define, the entire scope of the disclosure. EXAMPLE 1

Materials and Methods

All compounds were reagent grade or better. The following reagents were used as received: C₆₀ (MER Corporation), anhydrous hydrazine (Aldrich), diethylmalonate (Aldrich), DBU (Aldrich), trifluoroacetic acid (Acros), methyl-4-aminobutyrate HCl (Fluka), 5-methoxy-l- tetralone (Aldrich), tert-butyl-methyl malonate (Aldrich),triethylamine (Acros), DCC (Sigma),

DIPEA (Alfa Aesar), levobunololΗCl (BalPharma) and di-tert-butyl dicarbonate (Fisher).

All solvents were of HPLC grade and purchased from Fisher. The following solvents were used as received: toluene, CHCl₃, acetone, H₂O, ethanol, methanol, 2-methoxy-ethanol, acetic acid, and cyclohexane. CH₂Cl₂ was pre-dried with CaCl₂ and freshly distilled over P₂O₅.

All procedures for rendering anhydrous materials were carried under dry N₂ atmosphere.

The N₂ (Trigas, prepurified) was purified by passing though a column containing R3-11 catalyst

(Chemical Dynamics Corp.) on vermiculite to remove trace O₂ followed by trace H₂O removal with a column of Drierite (CaSO₄). For anhydrous reactions, all glassware was dried at 200 ⁰C for at least 24 hours.

Flash chromatography was carried out using silica gel (70-230 mesh from EM science) after activation at 200 ⁰C for at least 24 hours. TLC analyses were carried out using Whatman 250 μm layer silica gel with fluorescent indicator on polyester backing (PE SIL G/UV). HPLC was performed on a Hitachi L-6200A Intelligent Pump HPLC system with a Hitachi Model L- 3000 UV-vis photodiode array detector using various columns as specified in the synthesis section. All samples were filtered with a 0.2 μm nylon syringe filter (Fisher) prior to injections.

All ¹H and ¹³C NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer. All NMR solvents were from Cambridge Isotope Laboratories and were used as received.

Mass spectra were obtained on a Bruker MS Reflex IV MALDI-TOF mass spectrometer. For the MALDI-TOF spectra, unless otherwise specified, trans-2-[3-(4-tert-Butylphenyl)-2- rnethyl-2-propenylidene]malononitrile (DCTB) was used as matrix. The analytes were dissolved in an appropriate solvent and mixed with the matrix before spotting it on the sample plate.

Melting points were measured on a manual Melt-Temp^® apparatus.

FT-IR spectra of the neat compounds were collected on a Nicolet Avatar FT-IR spectrometer, and the acquired data was processed using Origin" 7.5.

Syntheses

For this example, the strategy for the synthesis of a nanostructure-bisphosphonate-beta- blocker conjugate, according to one embodiment, involved the synthesis of a malonohydrazide derivative and coupling it to levobunolol via a hydrazone bond, followed by the attachment to C₆₀. Since hydrazide compounds have not been previously coupled to C₆₀, two model compounds were first prepared. This synthetic sequence was designed to minimize the number of steps involving levobunolol due to its high cost.

As shown in Figure 4, the first synthesis involved the preparation of malonodihydrazide 1 according to Jung et. al and its reaction with acetone to form a hydrazone 2. The Bingel reaction of 2 with C₆₀ with in situ generation of the brominated intermediate gave compound 3. The Bingel reaction (nucleophilic cyclopropanation) was used since it was by far the simplest and most versatile method of C₆₀ derivatization. This reaction, like most fullerene derivatization procedures, afforded a mixture of mono, bis, tris a tetraaducts.

Once it was established that a hydrazone could be coupled to C₆₀ via a Bingel reaction, a second model compound reaction was designed. As shown in Figure 5, for this reaction, the ketone chosen had a tetralone group similar to the levobunolol molecule. Malonate 4 was formed by reacting mono-tert-butyl malonate and methyl-4-aminobutyrate HCl in the presence of DCC. After addition of dry hydrazine, hydrazide 5 was formed. As shown in Figure 6, 5-methoxy-l- tetralone was then coupled to 5 forming hydrazone 6.

The long arm provided by the aminobutyrate was first thought to provide better stability upon reaction with C₆₀ and to prevent levobunolol-HCl from reacting with C₆₀. However, the amide formation reaction was too slow and the side product, NJNP-dicyclohexylurea, kept precipitating. Therefore, it was decided that just a simple reaction of 9 with protected levobunolol 8 (Figure 7) to form the hydrazone 10, and the subsequent Bingel reaction with C₆₀ would finally afford the desired C₆₀-beta-blocker conjugate 11 (Figure 8).

The formation of the 11 gave rise to the second C₆₀ hydrazone derivative needed to finally form the sought-after C₆o-bisphosphonate-beta-blocker conjugate. Thus, when 10 was reacted with C₆o[C(P0₃iPr₂)₂]₂ in the presence of DBU, the protected C₆₀-bisphosphonate-beta- blocker conjugate 12 was finally obtained, while its water soluble form 13 was ultimately obtained by deprotection with Si(CH₃)₃l and TFA (Figure 9).

Spectroscopic and MALDI-TOF MS data for the C₆₀-bisphosphonate-beta-blocker conjugate are consistent with the assigned structure. The presence of the conjugate was verified by MALDI-TOF MS with the molecular ion peak at m/z =1949 (Figure 10).

All new compounds for the synthesis of the nanostructure-bisphosphonate-beta-blocker conjugate detailed above were fully characterized. The characterization data is at the end of the section for each compound. Previously prepared compounds were fully characterized and compared to the literature data. Propanedioic acid, 1,3-dihydrazide (1). In a typical synthesis, 0.41 mL (12.68 mmol) of anhydrous hydrazine was added to a methanol/H₂O solution of 1.02 g (3.64 mmol) of diethylmalonate and stirred at room temperature for two hours. The resulting white solid was collected by filtration and dried. Yield 0.47g (97%); mp 153-4 °C. MALDI-TOF MS calcd. 132.1. Found 154.8 [M + Na⁺].

Propanedioic acid, l,3-bis[2-(l-methylethylidene)hydrazide] (2). A suspension of 0.20 g (1.51 mmol) of 1 in 3.5 mL of acetone was refluxed for two hours. The solid compound was collected by filtration and washed with 5 mL of ethanol and dried in vacuum. Yield 0.315 g (98%); mp 163-5 ⁰C. MALDI-TOF MS calcd. 212.2. Found 286.4[M + NH₂Et₂ ⁺]. (matrix = α- Cyano-4-hydroxycinnamic acid diethylamine salt)

Propanedioic acid, l,3-bis[2-(l-methylethylidene)hydrazide] C₆₀ (3). C₆₀ (500 mg, 0.694 mmol) was dissolved in 900 mL of toluene: CH₂Cl₂ (2:1). 2 (589 mg, 2.78 mmol) was dissolved in 20 mL of CH₂Cl₂ and added together with CBr₄ (921 mg, 2.78 mmol) to the C₆₀ solution. DBU (158.24 mg, 2.78 mmol) in 30 mL of toluene was added to the solution over one hour. After stirring at room temperature for an additional two hours, the solvent was removed in vacuo. The crude reaction mixture was chromatographed on silica gel using toluene as eluent to remove unreacted C₆₀. The mono, bis, tris and tetra adducts obtained were not separated. MALDI-TOF MS calcd. for adducts 930.92, 1141.16, 1351.39 and 1561.62. Found 931.42, 1141.51, 1351.61 and 1562.7[M + H⁺].

Methyl 4-[(3-tert-butoxy-3-oxopropanoyl)amino]butyrate (4). 5 g (42.68 mmol) of methyl 4-aminobutyrate hydrochloride, 6.41 g (40.02 mmol) of mono-tert-Butyl malonate and 4.17 g (41.3 mmol) of triethylamine were dissolved in IL Of CH₂Cl₂ and cooled to 0⁰C. 10.99 g (53.3 mmol) of DCC were added and the mixture was stirred overnight letting the reaction reach room temperature. A few drops of acetic acid were added to precipitate all the N₉N¹- dicyclohexylurea and then the solution was filtered. The solvent was evaporated in vacuo to leave an impure yellow oil. For further purification, 50 mL of cyclohexane was added and left to rest for two hours in the refrigerator. The precipitate formed was filtered off, the solvent removed in vacuo and the remaining oil was flash chromatographed on silica gel using CH₂Cl₂ as eluent. The solvent was removed again in vacuo to give a clear oil as product. Yield 6.72 g (88 %). FT-IR (cm^"1) υ 3293 (s, N-H stretch), 2935 (m, C-H), 2862 (s, C-H), 1732 (s, ester C=O), 1651 (s, amide C=O), 1552 (s, N-H bend), 1437 (m, CH₂ bend), 1367 (s, CH₃ bend), 1253 (s, C-N stretch), 1145 (d, ester C-O)-¹H NMR (CDCl₃) δ 1.47 (s, 9H), 1.85 (p, 2H), 2.36 (t, 2H), 3.22 (s, 2H), 3.31 (q, 2H), 3.68 (s, 3H). MALDI-TOF MS calcd. 259. 29. Found 259.07 [M + H⁺]. Tert-butyl 3-[(4-hydrazinyl-4-oxobutyI)amino]-3-oxopropanoate (5). 2 g (7.71 mmol) of 4 were dissolved in 15 mL of methanol and 0.375 niL (11.95 mmol) of anhydrous hydrazine was added. The mixture was stirred at 50⁰C for two days while the progress of the reaction was monitored on H-NMR. The solvent was evaporated in vacuo giving a white solid. Yield: 1.98 g (99%) ¹H NMR (CDCl₃) δ 1.47 (s, 9H), 1.86 (p, 2H), 2.19 (t, 2H), 3.23 (s, 2H), 3.32 (q, 2H). MALDI-TOF MS calcd. 259. 29. Found 259.07 [M + H⁺].

J^^-butyl 3-({4-[(2Z)-2-(5-methoxy-3,4-dihydronaphthalen-l(2H)-ylidene) hydrazinyI]-4-oxobutyl}amino)-3-oxopropanoate (6). 728 mg (2.80 mmol) of 5 were dissolved in ethanol and 544 mg (3.09mmol) of 5-methoxy-l-tetralone were added. The mixture was allowed to stir for two days at 50 °C. The solvent was evaporated in vacuo and the hydrazone was recrystallized from methanol as a white solid. Yield: 245 mg (21%) FT-IR (cm^"1) υ 3379 (s, N-H stretch), 2947 (m, C-H), 2867 (s, C-H), 1718 (s, ester C=O), 1662 (s, amide C=O), 1575 (s, C=N), 1523 (s, N-H bend), 1464 (m, CH₂ bend), 1346 (s, CH₃ bend), 1257 (s, C-N stretch), 1137 (d, ester C-O), 1037 (s, N-N). ¹H NMR (CDCl₃) δ 1.47 (s, 9H), 1.86 (p, 2H), 2.19 (t, 2H), 3.20 (s, 2H), 3.38 (q, 2H), 3.84 (s, 3H). ¹³C NMR (CDCl₃) δ 21.97, 24.20, 27.01, 28.00, 30.26, 37.05, 39.20, 42.39, 55.54, 82.52, 101.94, 110.40, 116.58, 126.65, 128.88, 133.11, 147.16, 156.55, 165.57, 168.82, 175.12. MALDI-TOF MS calcd. 417.49. Found 417.03 [M + H⁺].

r^-butyI 3-({4-[(22)-2-(5-methoxy-3,4-dihydronaphthalen-l(2H)- ylidene)hydrazinyl]-4-oxobutyl}amino)-3-oxopropanoate C₆₀ (7). C₆₀ (100 mg, 0.139 mmol) was dissolved in 100 mL of toluene: CH₂Cl₂ (2:1). Then 6 (58 mg, 0.139 mmol), CBr₄ (46.1 mg, 0.139 mmol), and DBU (21.2 mg, 0.139 mmol) were added. After stirring at room temperature for two hours, the reaction mixture was filtered and the solvent was removed in vacuo. The crude reaction mixture was chromatographed on silica gel using CH₂Cl₂ methanol (20:1) to give mono and bis adducts. MALDI-TOF MS calcd. 1136.12 and 1551.64. Found 1136.84 and 1554.52 [M + H⁺].

Protected levobunolol (8). A solution of di-tert-butyldicarbonate (100 mg, 0.46 mmol) in 3 mL of t-BuOH:H₂O (10:1) was added to a solution of levobunolol-HCl (150 mg, 0.46 mmol) and DIPEA (59.3 mg, 0.46 mmol) in 3 mL of t-BuOH:H₂O (10:1) and stirred for 24 hours at room temperature. Then the solution was poured onto 60 mL of water and extracted with hexanes (3 x 30 mL). The combined organic layers were dried over Na₂SO₄. The mixture was filtered and the filtrate was concentrated in vacuo. Further purification was performed by flash chromatography on silica gel using hexanes:diethyl ether (1 :1) as eluent to give the final product N-Boc-levobunolol as a white powder. Yield: 72 mg (35 %). FT-IR (cm^"1) υ 3359 (br, O-H stretch), 2976 (m, C-H), 2935 (s, C-H), 1740 (s, ester C=O), 1683 (s, amide C=O), 1471 (s, CH₂ bend), 1367 (s, CH₃ bend), 1262 (s, C-N stretch), 1162 (d, ester C-O). ¹H NMR (CDCl₃) δ 1.44 (s, 9H), 1.52 (s, 9H), 2.11 (dddd, 2H), 2.63 (ddd, 2H), 2.89 (d, 2H), 2.96 (ddd, 2H), 3.73 (tt, IH), 4.01 (d, 2H), 7.04 (dd, IH), 7.27 (dd, IH), 7.67 (dd, IH). ¹³C NMR (CDCl₃) δ 22.48, 22.89, 28.55, 29.94, 38.74, 48.85, 56.13, 70.43, 72.34, 81.22, 115.28, 119.21, 136.88, 133.67, 152.88, 155.68, 198.78. MALDI-TOF MS calcd. 391.50. Found 414.13 [M + Na⁺].

Tert-butyl 3-hydrazinyl-3-oxopropanoate (9). 2 g of tert-butyl methyl malonate were dissolved in methanol and 0.396 mL (12.63 mmol) of anhydrous hydrazine was added. The mixture was stirred at 50 °C, and after eight hours the solvent was removed in vacuo to leave a white powder. Yield: 1.96 mg (98 %). FT-IR (cm^"1) υ 3300 (d, N-H stretch), 3201 (s, N-H stretch), 2979 (m, C-H), 2933 (s, C-H), 1728 (s, ester C=O), 1637 (s, amide C=O), 1525 (s, N-H bend), 1346 (s, CH₃ bend), 1307 (s, C-N stretch), 1147 (d, ester C-O), 1053 (s, N-N). ¹H NMR (CDCl₃) 6 1.47 (s, 3H), 3.26 (s, 2H). ¹³C NMR (CDCl₃) δ 27.98, 41.13, 82.81, 166.34, 168.05. MALDI-TOF MS calcd. 174.19. Found 198.9 [M + Na⁺].

Protected levobunolol malonate (10). To 72 mg (0.184 mmol) of 8 in 15 mL of 2- methoxy-ethanol was added 35 mg (0.202 mmol) of 9. The solution was stirred overnight in an oil bath at 50 ⁰C, the solvent was removed in vacuo and the solid was purified by flash chromatography on silica gel using CH₂Cl₂ methanol (20:1) as eluent to give a white powder as the final product. Yield 65.3 mg (64.8 %). FT-IR (cm^"1) υ 3398 (br, O-H stretch), 3197 (br, N-H stretch), 2974 (m, C-H), 2931 (s, C-H), 1734 (s, ester C=O), 1674 (s, amide C=O), 1575 (s, N-H bend), 1456 (s, CH₂ bend), 1365 (s, CH₃ bend), 1255 (s, C-N stretch), 1132 (d, ester C-O), 1037 (s, N-N). ¹H NMR (CDCl₃) δ 1.43 (s, 9H), 1.44 (s, 9H), 1.51 (s, 9H), 1.92 (dddd, 2H), 2.59 (ddd, 2H), 2.74 (d, 2H), 3.62 (ddd, 2H), 3.69 (s, 2H), 3.92 (dddd, 2H), 3.98 (m, IH), 6.83 (dd, IH), 7.16 (dd, IH), 7.73 (dd, IH). ¹³C NMR (CDCl₃) 20.99, 21.97, 24.53, 28.00, 28.56, 29.92, 41.25, 42.81, 48.89, 56.07, 70.28, 72.33, 80.97, 81.56, 111.27, 117.37, 126.48, 128.80, 133.13, 147.95, 155.42, 158.53, 166.86, 169.65. MALDI-TOF MS calcd. 547.68. Found 573.12 [M + Na⁺].

Protected levobunolol malonate C_6O (H)- C₆₀ (86 mg, 0.1 19 mmol) was dissolved in 100 mL of toluene: CH₂Cl₂ (2:1), and then 10 (65.3mg, 0.119 mmol), CBr₄ (39.5 mg, 0.119 mmol), and DBU (18 mg, 0.119 mmol) were added. After stirring at room temperature for two hours, the reaction mixture was filtered and the solvent was removed in vacuo. The crude reaction mixture was chromatographed on silica gel using CH₂Cl₂:methanol (20:1) as eluent to give a mono-adduct. MALDI-TOF MS calcd. 1266.30. Found 1265.52 [M + H⁺].

Ceo-Bisphosphonate-Beta-Blocker Conjugate (12). C₆₀[C(PO₃iPr₂)₂]2 (50 mg, 0.0357 mmol) was dissolved in 100 mL of toluene: CH₂Cl₂ (2:1), and then 10 (19.5 mg, 0.036 mmol), CBr₄ (11.8 mg, 0.036 mmol), and DBU (5.5 mg, 0.036 mmol) were added. After stirring at room temperature for two hours, the reaction mixture was filtered and the solvent was removed in vacuo. MALDI-TOF MS calcd. 1950.91. Found 1949.97 [M + H⁺].

Hydrolysis and Deprotection of C_δo-Bisphosphonate-Beta-Blocker Conjugate (13). 19.5 mg (0.01 mmol) of 12 were treated with a 1.5 excess of trimethylsilyl iodide (24 mg, 0.12 mmol) in dry CCl₄ at 45 ⁰C for one hour. Transformation to the diphosphonic acid was achieved by treating with excess H2O for one hour. Later, the aqueous layer was extracted and rotoevaporated. The resulting compound was deprotected with trifluoro acetic acid and finally made into a sodium salt by treatment with NaOH.

Therefore, the present disclosure is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this disclosure as illustrated, in part, by the appended claims.

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Claims

CLAIMS What is claimed is:

1. A composition comprising:

a C_n carrier, wherein C_n refers to a fullerene moiety or nanotube comprising n carbon atoms,

a bone targeting agent, and

a beta-blocker.

2. The composition of claim 1 wherein the bone targeting agent is chemically bonded to the C_n carrier.

3. The composition of claim 1 wherein the beta-blocker is chemically bonded to the C_n carrier.

4. The composition of claim 1 wherein the C_n carrier is a buckminsterfullerene, single walled carbon nanotube, or an ultra-short carbon nanotube.

5. The composition of claim 1 wherein the C_n carrier is a buckminsterfullerene.

6. The composition of claim 1 wherein the bone targeting agent comprises a bisphosphonate.

7. The composition of claim 1 wherein the bisphosphonate comprises at least one bisphosphonate selected from the group consisting of: etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, zoledronate and a combination thereof.

8. The composition of claim 1 wherein the beta-blocker comprises at least one beta- blocker selected from the group consisting of: dichloroisoproterenol, propanolol, atenolol, pindolol, metoprolol, timolol, bunolol, levobunolol, an isomer thereof and a combination thereof.

9. The composition of claim wherein the beta-blocker comprises levobunolol.

10. A method comprising:

administering a therapeutically effective amount of a composition comprising a C_n carrier, wherein C_n refers to a fullerene moiety or nanotube comprising n carbon atoms, a bone targeting agent, and a beta-blocker to a mammal.

1 1. The method of claim 10 wherein the mammal has osteoporosis.

12. The method of claim 10 wherein the bone targeting agent is chemically bonded to the C_n carrier.

13. The method of claim 10 wherein the beta-blocker is chemically bonded to the C_n carrier.

14. The method of claim 10 wherein the C_n carrier is a buckminsterfullerene, single walled carbon nanotube, or an ultra-short carbon nanotube.

15. The method of claim 10 wherein the C_n carrier is a buckminsterfullerene.

16. The method of claim 10 wherein the bone targeting agent comprises a

bisphosphonate.

17. The method of claim 10 wherein the bisphosphonate comprises at least one bisphosphonate selected from the group consisting of: etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, zoledronate and a combination thereof.

18. The method of claim 10 wherein the beta-blocker comprises at least one beta- blocker selected from the group consisting of: dichloroisoproterenol, propanolol, atenolol, pindolol, metoprolol, timolol, bunolol, levobunolol, an isomer thereof and a combination thereof.

19. The method of claim 10 wherein the beta-blocker comprises levobunolol.

20. A method comprising:

providing a C_n carrier, wherein C_n refers to a fullerene moiety or nanotube comprising n carbon atoms, and

allowing a bone targeting agent and a beta-blocker to react with the carrier so as to form a nanostructure-beta-blocker conjugate.

21. The method of claim 20 wherein the C_n carrier is a buckminsterfullerene, single walled carbon nanotube, or an ultra-short carbon nanotube.

22. The method of claim 20 wherein the C_n carrier is a buckminsterfullerene.

23. The method of claim 20 wherein the bone targeting agent comprises a

bisphosphonate.

24. The method of claim 20 wherein the bisphosphonate comprises at least one bisphosphonate selected from the group consisting of: etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, zoledronate and a combination thereof.

25. The method of claim 20 wherein the beta-blocker comprises at least one beta- blocker selected from the group consisting of: dichloroisoproterenol, propanolol, atenolol, pindolol, metoprolol, timolol, bunolol, levobunolol, an isomer thereof and a combination thereof.

26. The method of claim 20 wherein the beta-blocker comprises levobunolol.