Field of I nvention:

This invention relates to lipid based drug delivery system and particularly to novel modified bile acid conjugated drug nanoparticles. Such drug may include related diagnostic compounds. Process for preparation of such nanoparticles as well as their use in manufacturing anti-proliferative drug or diagnostic compounds. Background of the I nvention:

With the modern concept and various tools of drug design, various molecules have been synthesized having potent therapeutic action. However a large number of these new chemical entities have high molecular weight, poor aqueous solubility and also have high membrane permeability. These characteristics limit the bioavailability of many important drugs. The low solubility of these compounds leads to low dissolution, limited absorption in the body, limited bioavailability, and also high inter-and intra-subject variability as well as lack of dose proportionality.

One of these group of NCEs includes anti-proliferative drugs, which are the agents which block the acute/chronic proliferation of the cells such as in cancerous carcinoma, adenoma, fibroma, sarcoma, lymphoma etc.; in disorders like degenerative or atrophy conditions; inflammatory/auto immune conditions; cerebrovascular/neurodegenerative conditions etc. Many of the modern anticancer drugs such as taxanes, camptothecin and its analogues, vinca alkaloids, podophyllins, tretinoinefc. are hydrophobic and are water insoluble. A major problem in the delivery of such hydrophobic anti-proliferative drug molecules was hypersensitivity reactions, hematological problems, hepatotoxicity due to which sensitive patients may succumb to death. On the other hand many water soluble anticancer drugs such as doxorubicin, cisplatin derivatives, cyclophosphamide etc. are also absorbed weakly from intestinal epithelium; and due to their water solubility, intravenous administration of such drugs is more preferred. Because of high water solubility and small molecular size, they will be easily excreted from the body resulting in poor plasma drug concentrations.

Lipid based drug delivery systems for hydrophobic drugs uses parenteral (intravenous/intramuscular/subcutaneous) injections consists of suspension or emulsion based lipid vehicles. Pharmaceutical scientists formulated these potential anticancer drugs in some surfactant vehicles such as tween-80 (polysorbate-80) or Cremophor to deliver them via intravenous routes. Though lipid based drug delivery system is a technique for formulating orally adm inistrable drug delivery vehicle, none has been reported to effectively formulate an orally administrable anti-proliferative and particularly anti-cancer drugs and diagnostic compounds containing hydrophobic agents. Commercially available parenteral formulations of the hydrophobic drugs (eg: paclitaxel and docetaxel) use high amounts of surfactants, which upon intravenous administration lead to a spectrum of systemic toxicities including fatal hypersensitivity reactions. Existing drug delivery systems in market and delivery systems disclosed in many publications based on the physical encapsulation strategy such as phospholipid drug conjugate as liposomal formulation are having a low drug encapsulation capacity and high excipient to drug ratio as well low stability in biomedia leading to low bioavailability. Also physical encapsulation of drugs in the carrier, often leads to an initial burst release of drug in the circulation. Therefore there is a need for development of the effective parenteral injection formulations as well as other forms of administrable anti-proliferative drugs delivery system with steady bio-availability.

Objects of the I nvention:

The primary object of this invention is to develop different structural classes of lipid-drug conjugates, which can self-assemble to nanoparticles for effective administration.

It is yet another object of the invention to provide modified lipid based drug conjugate materials.

It is a further object of the invention to utilize the self-assembly properties of the covalent lipid-drug conjugate or covalent lipid-fluorophore/dye conjugate nanoparticles for preparation of effective drug delivery system. It is another object of the invention to provide a formulation of anti-proliferative drugs and marker compounds with increased and steady bio-availability.

It is another object of the invention to utilize the self-assembly property of the modified lipid-drug or lipid-fluorophore/dye conjugates for formulation of different structural assemblies of the drug delivery system such as micelles, vesicular formulations (liposomes), emulsions, solid lipid nanoparticles, and complex nanoparticles systems, when they formulated as such (neat lipid) or with other pharmaceutical excipients.

It is yet another object of the invention to provide effective method of production of different nanoparticles/ co-nanoparticles from the modified lipid conjugates by instrumental/manual methods such as nanoprecipitation method, emulsification method, thin film formation by solvent evaporation method, salting out method, dialysis method, extrusion through membrane filters, high pressure homogenization method, supercritical fluid technology, lyophilization method, microfluidization method, nanoelectro spray method, spray drying method etc.

It is yet further object of the present invention to provide the method of mixing of two or more different modified lipid-drug conjugates in certain ratio to prepare co- nanoform ulations of therapeutic class of molecules as well as mixture of the drug and imaging agent co-nanoformulation that would allow the real time regression of tumors avoiding the invasive procedures.

The above and the other objects of the invention will be clear from the detailed description of the invention.

Summary of the I nvention :

Accordingly the present invention provides a modified bile acid-drug/imaging agent conjugate nanoparticle which can be used as an effective drug delivery system through different routes.

According to the invention, bile acid-drug conjugate is modified with a suitable head group for higher biocompatibility, stability and bioavailability of the drug.

Brief Description of the Accompanying Drawings:

Figure 1 is characterization of lithocholic acid-docetaxel phospholipid conjugate nanoparticles (DTX-PC NP's, NMs 1) by (a) dynamic light scattering and (b) transmission electron microscopy. Figure 2 is characterization of polyethyleneglycol modified lithocholic acid-docetaxel conjugate nanoparticles (DTX-PEG NP's, NMs 2) by (a) dynamic light scattering and (b) transmission electron microscopy. Figure 3 shows the cytotoxicity assay of docetaxel (DTX) and polyethyleneglycol modified lithocholic acid-docetaxel conjugate nanoparticles (DTX-PEG NP's, NMs 2) in three breast cancer cell lines MCF-7, MDA-MB-231 , and 4T1 by tryphan blue exclusion method.

Figure 4 is graphical representation of the comparative efficiency data of DTX-PEG NP's (NMs 2) vis-a-vis DTX-TS.

Detailed Description of the Invention:

With the above objects in mind, the inventors have come up with novel modified lipid-drug conjugate nanoparticles suitable for effective delivery of anti proliferative drugs.

For the sake of brevity the modified lipid-drug conjugate nanoparticles of the present invention are described with the example of bile acid as the lipid carrier throughout the description hereinafter. It should be understood that the detailed description and specific examples, while indicating embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art. One skilled in the art, based upon the definitions herein, may utilize the present invention to its fullest extent. The following specific embodiments are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Except as defined herein, all the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention relates.

Unless otherwise indicated, the definitions various terms apply to the terms as they are used throughout the specification and the appended claims, either individually or as part of a larger group. These definitions should not be interpreted in the literal sense as they are not general definitions, and are relevant only for this application. The singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. For instance, the terms "a", "an" and "the" refers to "one or more" when used in the subject specification, including the claims. Thus, for example, reference to "a compound" may include a plurality of such compounds, or reference to "a disease" or "a disorder" includes a plurality of diseases or disorders.

Also, use of "(s)" as part of a term, includes reference to the term singly or in plurality. The term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise.

A symbol ( - ) is used to indicate a point of attachment to the atom, for example - COOH is attached through the carbon atom.

The term "independently" when used in the context of selection of substituents for a variable, it means that where more than one substituent is independently selected from a number of possible substituents, those substituents can be the same or different. Furthermore, unless stated otherwise, the alkyl, aryl, heteroaryl etc groups can be unsubstituted or substituted with one or more groups.

To achieve the above objectives, chemical modifications in the drug and carrier has been affected to overcome the physicochem ical problems and toxicological effects caused by the drug and carrier. It is found that covalent conjugation of drugs to the carrier lipids enhances the lipid to drug ratio in the formulation, thereby better drug encapsulating efficiency can be achieved. To achieve this carrier must have polar functional groups in order to participate in chemical reactions with drugs for covalent attachment. Moreover chemical conjugation reduces the burst release of drug from formulation. The use of polarized carrier functional group is important because chemistry of commercially available lipids and carriers do not encourage for the covalent conjugation of drugs.

To prepare such a class of compound bile acids as lipid carrier is used. Bile acids offer excellent chemistry to chemically conjugate the drugs and functional moieties. According to the invention the drug is first suitably modified with/without linker so as to chemically conjugate with the modified bile acid carrier. Then the bile acid lipid molecule is modified by covalent attachment of suitable head group.

The inventors have found that modification of bile acid drung conjugate with a suitable head group facilitates stable nanoparticle formation and helps in moelcular self assembly and better pharmacokinetics and lesser toxicity.

The choice of head group is critical in this matter as these head groups in addition to the above need to be pharmaceutically acceptable and biocompatible and also should have the property to enhance the circulation of drug in the body of the patient. The present invention is discloses a modified covalent lipid drug conjugate in nanoparticle form and also the formation of self-assembled nanoparticles from these conjugates in different isotonic aqueous media and their applications in various forms of solid cancers. The present invention also discloses the use of combinations of such drug-conjugated nanoparticles or co-nanoparticles in different forms and stages of cancers.

The bile acid conjugated anti-proliferative drug nanoparticles of the present invention can be represented by general formula (I) R-L1-BA-L2-D

(I)

or pharmaceutically acceptable salts thereof; wherein:

R is natural, synthetic or modified phospholipid head group, polyethylene glycol or poloxamer class of polyols, or other compatible pharmaceutically acceptable head group; BA is sterol structural class compound, natural and synthetic bile acids and bile salts;

D is drug molecules, enzyme/protein inhibitor molecules, imaging ligands, dyes, fluorophores and near-infrared dyes;

L1 and L2 represent linkers or spacers and are independently selected from covalent bond, carbon-carbon bond, aromatic, aliphatic, alicyclic, small polymeric linkers including functional groups that covalently conjugate the bile acid moiety and drug or polyethylene glycol moiety or another group.

Preferably the drug and other compounds include anticancer drug of taxol group such as paclitaxel, docetaxel, carbazitaxel; anthracyclines such as doxorubicin, daunorubicin, dactinomycin, idarubicin, valrubicin, epirubicin, pirarubicin, mitoxantrone; platins such as cisplatin, carboplatin, nidaplatin, oxaliplatin, satraplatin; natural product drugs such as camptothecin, topotecan, irinotecan, podophyllotoxin, etoposide, teniposide, vincristine, vinblastine, vinorelbine, mitomycin, bleomycin; synthetic drugs such as melphalan, chlorambucil, thiotepa; anti metabolites such as cladribine, clofarabine, cytarabine, fludarabaine, methotrexate, pemetrexed, gemcitabine, fluorouracil, floxuridine, capecitabine, azacitidine, mercaptopurine, thioguanine, hydroxyurea, pentostatin. inhibitors such as bortezomib, bosutinib, cabozantinib, carfilzomib, imatinib, steroidal ant i- inflmmatory agents such as dexamethasone, betamethasone, triamcinolone acetonide, nonsteroidal anti-inflammatory agents such as aspirin, paracetamol, diclofenac, flubiprofen, indomethacin, naproxen, celecoxib, rofecoxib; anti-angiogenic drugs such as TNP-470, combretastatin-A4-Phosphate, bevacizumab, sorafenib, sunitinib, pazopanib, evrolimus, covalent attachment of dyes or fluorophores such as NBD, FITC, cyanine5, cyanine 5.5, Alexafluor, Cy5, radio contrast imaging agents such as gadolinium-DOTA conjugates, near- infrared dyes such as cyanine dyes, cy dyes etc.

L1 and L2 represent linkers or spacers and are independently selected from covalent bond, carbon-carbon bond, aromatic, aliphatic, alicyclic, small polymeric linkers including functional groups that covalently conjugates the bile acid moiety and drug or polyethylene glycol or phospholipid moiety.

Preferably linker and linker L ₂ represent a covalent bond between natural phospholipid head group and bile acid or its salt, organic functional groups such as ester, amide, carbonate, carbamate, phosphate, phosphonate, phosphoramidate, amine, urea, thiourea, sulfonamide, ether, thioether, sulfoxide, sulfone, thioester, thioamide, disulfide, oxime, o- acyloxime, o-acyloxyalkyloxime, o-carbamoyloxime, small organic linker molecules including aliphatic polar molecules, polar aromatic compounds and heterocyclic molecules. Linker L2 may more preferably be independently selected from ethylene glycol, ethanolamine, p- aminobenzoic acid, p-azidobenzoic acid and triazole. In an embodiment, the bile acid conjugated anti-proliferative drug nanoparticles of general formula (I) can take the form;

R _! -L1-BA-L2-D wherein is selected from natural phospholipid head groups such as phosphocholine, phosphoethanolam ine, phosphoserine, phosphoinositol, phosphatidyl glycerol, phosphatidic acid, synthetic phospholipid head groups such as sugar modified, amino acid modified, nucleoside base modified phospholipid head groups, aromatic or heteroaromatic ring containing cyclic, small or long-straight or branched chain hydrocarbons containing phospholipid head groups.

BA is a compound selected from sterol structural class compound, natural and synthetic bile acids and bile salts.

D is selected from anti-proliferative drug molecules; various enzyme/protein inhibitor molecules used as anticancer agents, imaging ligands, dyes, fluorophores or near-infrared dyes. and L1 and L2 represent linkers or spacers and are independently selected from aromatic, aliphatic, alicyclic, small polymeric linkers including functional groups that covalently conjugates the bile acid moiety and drug or phospholipid moiety.

In another embodiment, the bile acid conjugated anti-proliferative drug nanoparticles of general formula (I) can take the form;

R ₂ -L1-BA-L2-D

Wherein R ₂ is selected from polyethylene glycol moiety or poloxomer class of polyols having varying molecular weight or chain length containing free ends with functional groups or one end free functional group and another end capped with any functional ligand such as methyl ether or ester or amide or targeting ligands to cancer cells.

Preferably the targeting ligands to cancer cells are folic acid, RGD peptide, antibody, TAT peptide, sugar molecule, NGR, PSMA and aptamers.

BA is a compound selected from sterol structural class compound, natural and synthetic bile acids or bile salts. D is selected from anti-proliferative drug molecules, various enzyme/protein inhibitor molecules used as anticancer agents, imaging ligands, dyes, fluorophores or near-infrared dyes. L1 and L2 represent linkers or spacers and are independently selected from aromatic, aliphatic, alicyclic, small polymeric linkers including functional groups that covalently conjugates the bile acid moiety and drug or polyethylene glycol moiety.

In another embodiment, the bile acid conjugated anti-proliferative drug nanoparticles of general formula (I) can take the form; wherein R ₃ is selected from other compatible pharmaceutically acceptable head group like sugar such as glucose, galactose, manose, trehalose, cellobiose or any other monosaccharide, disachharide, trisachharide or polysaccharide, natural or unnatural amino acid, small or large peptide, antibody, alkyl chains, fatty acid chains, aromatic groups like benzyl, naphthyl or charged quaternary head groups like pyridine, morpholine, pyrolidine, amine etc.

Molecular self-assembly implies the spontaneous organization of the molecular entities into complex hierarchical structures that are primarily driven by non-covalent interactions. When amphiphilic molecules introduced into selective polar media, they self-assemble at the interface as well as in the bulk intend to solvate their hydrophobic regions in the polar media. Around particular concentration known as "critical aggregation concentration" these will attain supra molecular structures in the bulk of the media. These supramolecular forms attain different structural assembly such as micelles, vesicles, emulsions etc. These supramolecular structures possess different physico-chemical properties than the amphiphilic molecules. These supramolecular assemblies can encapsulate either the hydrophilic or hydrophobic drug molecules to deliver the drugs at target sites.

The lipid based formulation approaches to either encapsulate or conjugate the drugs in different nanoparticle forms. Phospholipid-drug conjugates or PEGylated lipid-drug conjugates of general formula (I) forms supramolecular nanoparticles independently as well as when they are co-formulated with each other in isotonic aqueous media. These lipid-drug conjugate molecules could also form nanoparticles when they are co-formulated with hydrophilic/hydrophobic drug owing to their encapsulating capacity.

Bile acids are modified with either phospholipid head group, PEGylated macromolecule or other compatible pharmaceutically acceptable group or plurality of them followed by drug conjugation that enhances the self-assembling ability and biological efficacy.

The self-assembly properties of the covalent lipid-drug conjugate or covalent li id- fluorophore/dye conjugate molecules mentioned in general formula (I) is augmented in isotonic aqueous media such as saline, 0.9% sodium chloride, 5% dextrose, Phosphate buffered saline, HEPES, 10% sucrose solution etc.

The self-assembly of these lipid-drug or lipid-fluorophore/dye conjugates leads to different structural assemblies such as micelles, vesicular formulations (liposomes), emulsions, solid lipid nanoparticles, and complex nanoparticles systems, when they are formulated as such (neat lipid) or with other pharmaceutical excipients.

Co-formulating agents such as synthetic bile acid based PEGylated lipids, synthetic PEGylatedphospholipids, excipients such as fatty acids, fatty alcohols, stabilizers such as fatty acids, fatty alcohols, surfactants such ascryo/lyoprotectants such as glucose, sucrose, trehalose, mannose can be used in order to get better stability of the final nanoformulation in isotonic aqueous fluids.

It is also possible to combine two different lipid-drug conjugates to form co- nanoform ulation. For example mixture of PEGylated bile acid-drug conjugate with Phospholipid head containing bile acid drug conjugates in certain ratio to prepare co- nanoform ulations. These co-nanoformulations are not restricted to therapeutic class of molecules but could also be a mixture of the drug and imaging agent co-nanoformulation that would allow the real time regression of tumors avoiding the invasive procedures. The concept can be extended to co-nanoformulations by the use of such neat nanoparticle systems or co-nanoparticle systems in semi-solid dosage forms such as hydrogels, creams, and ointments for therapeutic purposes. It is possible to encapsulate one or more hydrophilic/hydrophobic antiproliferative /augmented therapeutic drug molecules in self-assembled nanoformulations.

These self-assembled nanoformulations mentioned in the general formula (I) have high capacity of encapsulation/conjugation of drug over the other conventional encapsulation in commercial and other formulations.

Presently commercial formulations of docetaxel consist of solubilized forms of docetaxel (which is not a nanoformulation) in a surfactant, which also consists maximum of the drug in the solution. For a comparison, other forms of nanoformulations such as liposomal formulation of doxorubicin (Doxil®, Myocet®, Daunoxome®) was used. In case of a liposomal formulation, the amount of drugs and lipids used for the formulation development varies with amounts in the final formulation as it involves multiple steps where mass reduction can occur. In contrast the nanoparticle preparation of the present invention happens in a single and simple step that causes no mass loss.

Specific non-limiting examples of the compounds of the present invention include but not limited to phospholipid modifiedlithocholic acid-docetaxel conjugate nanoparticles, phospholipid modified deoxycholic acid-docetaxel conjugate nanoparticles, phospholipid modified cholic acid-docetaxel conjugate nanoparticles, polyethyleneglycol modified lithocholic acid-docetaxel conjugate nanoparticles, phospholipid modified lithocholic acid- tamoxifen conjugate nanoparticles, phospholipid modified lithocholic acid-NBD conjugate nanoparticles, phospholipid modified deoxycholic acid-NBD conjugate nanoparticles, phospholipid modified cholic acid-NBD conjugate nanoparticles, benzylated modified lithocholic acid-doxorubicin conjugate nanoparticles and peptide modified lithocholic acid- tamoxifen conjugate nanoparticles.

Different nanoparticles/ co-nanoparticles from the lipid conjugates of formula (I) can be prepared by instrumental/manual methods such as nanoprecipitation method, emulsification method, thin film formation by solvent evaporation method, salting out method, dialysis method, extrusion through membrane filters, high pressure homogenization method, supercritical fluid technology, lyophilization method, m icrofluidization method, nanoelectro spray method, spray drying method etc.

The general process of preparation of these nanoparticles consists of drop wise addition of measured quantity of phospholipid-drug conjugates or PEGylated -lipid drug conjugates either alone or in combination in polarorganic solvents such as ethanol, THF or a mixture thereof to isotonic aqueous media at room temperature. Evaporation of the organic solvent on rotary vacuum evaporator yields colloidal nanoparticles formed from spontaneous self- assembly of the conjugates in isotonic aqueous media. General process of preparation may also involve the thin film formation of phospholipid-drug conjugates or bile acid-drug conjugates or PEGylated-lipid drug conjugates alone or in mixture on evaporation of organic solvent; followed by its hydration. Hydrated compounds were then vortexed or sonicated to make nanoparticles. In general the various nano formulations of the modified bile acid-drug conjugates can be prepared with the conjugates individually or with plurality of such conjugates. Further both the above can also be prepared in combination of any drug/imaging agent, excipients, stabilisers and surfactants in isotonic aqueous fluids, in order to get better stability of the final nano formulation.

Specific non limiting examples include conjugates of general formula (I) alone or co- formulated with any excipient such as lipids, fatty acids, fatty alcohols, stabilizers such as fatty acids, fatty alcohols, peptides, polymers, saccharides, surfactants such ascryo/lyoprotectants such as glucose, sucrose, trehalose, mannose in isotonic aqueous fluids.

Nanoem ulsions of phospholipid modified lithocholic acid-docetaxel conjugate in carboxymethylcellulose and/or polysorbate-80. Phospholipid modified lithocholic acid-docetaxel conjugate and polyethyleneglycol modified lithocholic acid conjugate nanoparticles in a suitable ratio.

Benzylated modified lithocholic acid-doxorubicin conjugate or any drug molecule and polyethyleneglycol modified lithocholic acid conjugate nanoparticles in a suitable ratio. Further, conanoformualtion of drug and imaging agent carrying conjugates such as phospholipid modified lithocholic acid-nitrobenzoxadiazole conjugate and polyethyleneglycol modified lithocholic acid conjugate nanoparticles in a suitable ratio is also possible. The present invention is exemplified with some non limiting preparatory examples.

Pharmaceutical basis for designing such synthetic chemistry is to use the principles of molecular self-assembly and to develop different nanoformulations for the docetaxel with covalent conjugation chemistry with endogenous bile acid lipid carriers. Similarly, conjugation of doxorubicin to bile acids helps them to get self-assembled in PEGylated bile acid amphiphiles and make nanomicelles.

As pharmaceutical scientists focused on replacement of the tween-80 and the ethanol from the components of taxotere® to reduce the toxicological profiles associated with the formulating agents, a polysorbate-80 and ethanol free self-assembled nanoformulation is developed to avoid the carrier associated toxicity as well as with an aim to improve the pharmacokinetic profiles and anti-tumor activities. In case of Doxorubicin formulation, existing formulation entrap the doxorubicin in the aqueous compartment of liposomes.

Preparatory Example 1

Synthesis of Bile acid DTX conjugates (1-3) (Scheme 1):

In following example, the lipid (BA) part is lithocholic acid (LCA), deoxycholic acid (DCA), or cholic acid (CA); and the drug conjugated to the BA part is docetaxel via a p-azidobenzoic acid linker with a triazole spacer by click chemistry.

1 : LCA-DTX F¾i = OH; R ₂ = H; R ₃

2: DCA-DTX F¾i = OH; R ₂ = H; R ₃ 3: CA-DTX R-i = OH; R ₂ = OH; R ₃

Scheme 1 Preparation of compound Docetaxel-benzoate ester linker of formula (102): To a solution of 4-azidobenzoic acid (0.163 g, 1 mmol.) and HBTU (1.137 g, 3 mmol., 3 equiv.) in dichloromethane (10 mL); DIPEA (0.87 mL) was added. After continuous stirring under inert condition for 20 min; solution of docetaxel (101) (0.807 g., 1 mmol., 1 equiv.) in dichloromethane (5 mL) was added to it. The reaction was stirred at room temperature for 24 h. The reaction was then quenched using distilled water (40 mL). Reaction mixture was then diluted with dichloromethane (50 mL), and washed with brine solution (2 X 30 mL). Trace amount of water present was removed using anhydrous sodium sulfate. Organic layer was then concentrated using rotary evaporator. Crude product was purified by silica gel combiflash column chromatography by using petroleum ether and ethyl acetate (65:25) to give colorless solid of formula (102). Yield (82 %); ¹ H NMR (400 MHz, CD ₃ OD): δ 0.88-0.92 (1 H, m), 1.14 (3 H, s) , 1.24-1.44 (18 H, m), 1.77-2.2 (18 H, m), 2.2-2.4 (2 H, m), 2.45 (3 H, s), 2.58-2.63 (1 H, m), 2.90 (1 H, s) , 2.98 (1 H, s), 3.36 (1 H, s) , 3.96 (1 H, d, J= 6.8 Hz), 4.14 (2 H, q, J= 7.2 Hz), 4.21 (1 H, d, J= 8.4 Hz), 4.27-4.3 (2 H, m), 4.98 (1 H, d, J = 9.2 Hz), 5.24 (1 H, s) , 5.32 (1 H, s), 5.46 (1 H, d, J= 9.6 Hz), 5.55 (2 H, brs), 5.70 (1 H, d, J= 6.8 Hz), 6.25 (1 H, t, J= 9.6 Hz), 7.09 (2 H, d, J= 8.4 Hz), 7.28-7.43 (5 H, m), 7.53 (1 H, t, J= 7.6 Hz), 7.64 (1 H, t, J= 7.6 Hz), 8.00 (1 H, d, J= 8.4 Hz), 8.12 (1 H, d, J = 7.6 Hz). HRMS (ESI-MS): m/ z {C ₅₀ H _5e N ₄ O ₁₅ ) ⁺ calculated 952.3742; found 953.4952 (M+H) ⁺ , 953.4952 (M+Na) ⁺ , 991.4423 (M+K) ⁺ .

General synthesis of compounds ( 104a-c) : Bile acid (103a- 103c) (4g, 9.79 mmol) , N- hydroxybenzotriazole (1.455g, 10.76 mmol), and EDC-HCI (2.064g, 10.76 mmol) were dissolved in 40 mL of anhydrous DMF/DCM (1:3); and DIPEA (3.58 mL, 20.56 mmol) was added to it. Reaction mixture was stirred at room temperature for 15 minutes; and propargyl amine (0.94 mL, 16.15 mmol) was added drop wise followed by stirring at room temperaturefor 12 h. Solvents were removed under vacuum; and reaction mixture was diluted with dichloromethane (250 mL). Organic layer was then washed with saturated NaHC0 ₃ solution (3 X 20 mL), and brine (2 X 20 mL). Organic phase was dried over anhydrous Na ₂ S0 ₄ , and solvent was evaporated under vacuum. Residue was purified by column chromatography on silica gel (230-400 mesh) using CH ₂ CI ₂ /MeOH as eluent to give a colorless solid (2.8 g). Compounds were characterized by ¹ H NMR spectroscopy .

General synthesis of Bile acid Docetaxel conjugate (1-3): To a solution of compound (104a-104c) (100 mg, lequiv.) and DTX-N ₃ (102, 299 mg, 1.3 equiv.) in methanol; sodium ascorbate (72 mg 1.5 equiv.) was added, and reaction mixture was stirred for 5 min.Then copper sulfate (30 mg 0.5 equiv.) in 1 ml water was added to solution and reaction mixture was stirred for 12 hours at room temperature. After the reaction completion, the reaction mixture was passed through celite. Organic solvent was removed under vacuo and compounds were isolated by silica gel (230-400mesh) combi flash chromatography using ethyl Acetate and Petroleum Ether (90:10) for LCA-DTX (1), ethyl acetate methanol (90:4) for DCA-DTX (2) and ethyl acetate methanol (90:10) for CA-DTX (3) . All compounds were characterized by 1 H NMR spectroscopy.

LCA-DTX (1) ( ¹ H NMR, 400MHz, DMSO-d ₆ ) <5: 0.52 (s, 3H), 0.84-2.32 (m, steroid, boc), 3.68(d, J= 7.2Hz, 1H), 4.00-4.07 (m, 3H), 4.38-4.48 (m, 3H), 4.91-4.94 (m, 2H), 5.02 (d, J= 6.8Hz, 1H), 5.11 (s, 1H), 5.33-5.55 (m, 1H), 5.40-5.42 (d, J= 8Hz, 1H), 5.85-5.90 (m, 1H), 7.19-7.23 (m, 1H), 7.40-7.48 (m, 4H) 7.56-7.60 (m, 2H), 7.65-7.70 (m, 1H), 7.96- 7.98 (m, 2H), 8.10-8.15 (m, 3H), 8.28-8.30 (m, 2H), 8.39-8.42 (t, J= 12Hz, 1H), 8.81 (s, 1H).

DCA-DTX (2) ( ¹ H NMR, 400MHz, DMSO-d ₆ ) <5: 0.51 (s, 3H), 0.81 -2.32(m , steroid, boc), 3.68 (d, J= 6Hz, 1H), 3.75 (d, J= 3Hz, 1H), 4.02-4.13 (m, 3H), 4.17 (d, J= 4Hz, 1 H) 4.39 (d, J= 5Hz, 2H) 4.45-4.48 (m, 2H), 4.91-4.95 (m, 2H), 5.03 (d, J= 7Hz, 1H), 5.11 (s,1H), 5.32-5.35 (m, 2H), 5.42 (d, J= 7Hz, 1 H) 5.87 (t, J= 8Hz, 1H), 7.21 (t, J= 7Hz, 1 H) 7.41- 7.48 (m, 4H), 7.57-7.61 (m, 2H), 7.66-7.70 (m, 1H), 7.97 (d, J= 8Hz, 2H), 8.11-8.16 (m, 2H), 8.29 (d, J= 8Hz, 2H), 8.40 (t, J= 6Hz, 1H), 8.82(s, 1 H)

CA-DTX (3) ( ¹ H NMR, 400MHz, DMSO-d ₆ ) «5: 0.50 (s, 3H), 0.77-2.32 (m, steroid, boc), 3.58 (s, 1H), 3.68 (d, J= 7H, 1H), 3.98-4.08 (m, 6H), 4.31 (s, 1H), 4.39 (d, J= 5Hz, 2H), 4.48 (s, 1H), 5.32-5.35 (m, 2H), 5.42 (d, J= 8Hz,1H), 5.87 (t, J= 9H, 1H), 7.13-7.14 (m, 1H), 7.38-7.48 (m, 4H), 7.57-7.61 (m, 2H), 7.66-7.70 (m, 1H), 7.97 (d, J= 7Hz, 2H), 8.11-8.16 (m, 3H), 8.29 (d, J= 8Hz, 2H), 8.41 (t, J= 5H, 1H), 8.82(s, 1H).

Preparatory Example 2

Synthesis of Bile Acid-PEG Conjugates (4-6) (Scheme 2) :

In the following example, the lipid (BA) part is lithocholic acid (LCA), deoxycholic acid (DCA), or cholic acid (CA); and the polyethyleneglycol is conjugated to BA.

Scheme 2

Synthesis of Bile Acid PEG2000 Conjugate (4-6) conjugates: To a solution of bile acid (103a-103c; 1 equiv.) and mPEG-NH ₂ (mol weight -2000) in anhydrous dichloromethane, DIPEA (2 equiv.) was added; followed by HBTU (1.5 equiv.). Reaction mixture wasstirred at room temperature for 24 hours. After the completion of the reaction, organic solvents were removed under reduced vacuum, extracted with DCM and washed with brine solution. The organic layer was dried over sodium sulphate and isolated at 5-10% MeOH in DCM. LCA-PEG ₂₀ oo(4) ( ¹ H NMR, 400MHz, CDCI ₃ ) «5: 0.64 (s, 3H), 0.74-2.11 (m, steroid), 2.19- 2.38 (m, 9 H), 3.89 (s, 4H), 3.44-3.48 (m, 4H), 3.54-3.56 (m, 6H), 3.65-3.83 (m, OCH ₂ CH ₂ 0), 4.21-4.22 (m, 1H), 6.25(s, 1H). DCA-PEG ₂₀ oo(5) ( ¹ H NMR, 400MHz, CDCI ₃ ) <5: 0.67 (s, 3H), 0.86 (s, 3H), 0.97(d, J= 8Hz, 3H), 0.99-1.9 (m, steroid), 2.05-2.13 (m, 1H), 2.21-2.28 (m, 1H), 3.38(s, 3H), 3.42- 3.47(m, 4H), 3.53-3.56 (m, 6 H), 3.64-3.65 (m, OCH ₂ CH ₂ 0), 3.80-3.82 (m, 1H), 3.97 (s, 1H), 6.31(s, 1H).

CA-PEG ₂₀ oo(6) ( ¹ H NMR, 400MHz, CDCI ₃ ) <5: 0.68(s, 3H), 0.73-2.42 (m, steroid), 3.37 (s, 3 H), 3.43-3.47 (m, 4H), 3.53-3.55 (m, 5H), 3.64-3.65 (m, OCH ₂ CH ₂ 0), 3.80-3.84 (m, 3H), 3.97 (s, 1H), 6.39 (s, 1H).

Preparatory Example 3

In following example, the lipid (BA) part is lithocholic acid (LCA), and the drug conjugated to the BA part is doxorubicin via a carbamate linker.

Synthesis of compound 106a: To a solution of compound 103a (1 equiv.) and DBU (1.5 equiv.) in anhydrous DMF; benzylbromide was added dropwise at room temperature. Reaction mixture was stirred at room temperature for 12 h. After completion of reaction, mixture was diluted with ethyl acetate, washed 4-5 times with excess brine solution, and organic layer was dried over sodium sulfate. Compound was purified by silica gel column chromatography using ethyl acetate and petroleum ether as eluent.

Synthesis of compound 107a: To a solution of compound 106a (1 equiv.) and DMAP (lequiv.) in anhydrous dichloromethane; p-nitrophenylchloroformate (1.2 equiv.) in 2ml_ DCM was added dropwise at 0 ^S C. Reaction mixture was stirred at room temperature for 8h; and after reaction completion, compound was purified with column chromatography using ethyl acetate and petroleum ether.

Synthesis of LCA-OBn-DOX (7): Doxorubicin hydrochloride (1 equiv.) and triethyamine (2 equiv.) in DMSO were stirred at room temperature for 20 min. Compound 107a (1.5 equiv.) in anhydrous DCM was then added dropwise and reaction was allowed to stir at room temperature for 48h. Reaction mixture was then diluted with ethyl acetate and washed with 3x 200ml_ brine solution. Red colored crystalline solid was isolated by silica gel combi-flash chromatography using ethyl acetate: pet ether (8:2) as eluent. Final compound was characterized by ¹ H NMR spectroscopy. Yield 20%; ( ¹ H NMR, 400MHz, CDCI ₃ ) <5: 0.60 (s, 3H), 0.72-1.94 (m, steroid), 2.14-2.42 (m, 4H), 2.98-3.05 (m, 2H), 3.28(d, J= 18Hz, 1H), 3.6 (s, 1H), 3.84 (s, 1H), 4.07 (s, 3H), 4.13-4.14 (m, 1H), 4.49-4.58 (m 2H), 4.76 (s, 2H), 4.95 (d, J= 8Hz, 1H), 5.07-5.14 (m, 2H), 5.29 (s, 1H), 5.50 (s, 1H), 7.30-7.40 (m, 6H), 7.78 (t, J= 8Hz, 1H), 8.03 (d, J= 8Hz, 1H), 13.25 (s, 1H), 13.97 (s, 1H).

Preparatory Example-4:

In the following example, Lithocholic acid phospholipid conjugated docetaxel in which docetaxel is attached to lithocholic acid derived phospholipid that has been synthesized according to Scheme 4. Docetaxel-benzoate ester linker of formula (102) is conjugated lithocholic acid phosphocholine of formula (105a) using click chemistry to give LCA-DTX-PC of formula (8). The molecule is characterized using ¹ H and ³¹ P NMR spectroscopy, HRMS mass spectrometry, and purity is confirmed with by reverse phase high-pressure liquid chromatography.

Synthesis of Phospholipid-Docetaxel Conjugate (8) (Scheme 4)

Scheme 4

Preparation of compound lithocholic acid phosphocholine of formula (105a): To the solution of compound (104a) (1.5 g, 3.6 mmol.) and triethylamine (0.753 ml_, 5.4 mmol., 1.5 equiv.) in THF (30 ml_); 2-chloro-1 ,3,2-dioxaphospholane-2-oxide (0.496 ml_, 5.4 mmol., 1.5 equiv.) was added drop wise for 20 minutes at 0 °C and stirred at room temperature for 8 h. The solution was filtered through celite to remove the NEt ₃ .HCI salt and the filtrate is evaporated to dryness. After complete removal of solvent, the residue was dissolved in acetonitrile (10 ml_) and transferred to a pressure tube. Trimethylamine gas was passed into pressure tube for 10 minutes at -15 °C with vigorous stirring. Then the reaction mixture was allowed to reflux for 48h. Solvent was evaporated in vacuo and crude product was purified by C ₁₈ silica gel (Redi-Sep columns) combi-flash column chromatography using water-acetonitrile system as eluent to give colorless solid of formula (105a). Yield (78%); ¹ H-NMR (400 MHz, CD ₃ OD): δ 0.69 (3 H, s), 0.95-1.94 (m), 2.016 (1 H, d, J= 12 Hz), 2.07-2.15 (1 H, m), 2.21-2.28 (1 H, m), 2.26 (1 H, s), 3.22 (9 H, s) , 3.63 (2 H, t, J= 4.4 Hz), 3.93 (2 H, d, J= 2.4 Hz), 4.01-4.13 (1 H, m), 4.25 (2 H, s). ³¹ P-NMR (162 MHz, CD ₃ OD): -0.92. HRMS (ESI): ml z ((^Hss^OsPT calculated 578.3849; found 579.4554 (M+H) ⁺ , 601.4399 (M+Na) ⁺ .

Preparation of Compound Docetaxel conjugated lithocholic acid phosphocholine of formula (8): To the solution of compound of formula (105a) (578.3 mg, 1 mmol.) and azido derivative of docetaxel (102) (1.047 g, 1.1 mmol., 1.1 equiv.) in 15 ml_ of 2:2:1 DCM: EtOH:H ₂ 0 solution;sodium ascorbate (198 mg, 1 mmol., 1 equiv.) and copper sulfate (24.9 mg, 0.1 mmol., 0.1 equiv. )was added. The reaction was stirred vigorously overnight at room temperature. After completion of reaction, all solvents were removed in vacuo and then the resultant crude product was purified by Ci ₈ silica gel (Redi-Sep columns) combi- flash column chromatography using water-acetonitrile system as eluent to give colorless solid of formula (8). Yield (82 %); ¹ H-NMR (400 MHz, CD ₃ OD): δ 0.50 (3 H, s), 0.84-2.0 (m), 2.14-2.47 (8 H, m), 3.18 (9 H, s), 3.59 (2 H, s), 3.87 (1 H, s), 4.05-4.21 (6 H, m), 4.48 (2 H, s), 4.58 (2 H, s), 4.9-4.98 (1 H, m), 5.26 (1 H, s) , 5.42-5.45 (2 H, m), 5.59 (1 H, d, J= 6.4 Hz), 6.07 (1 H, m), 7.21-7.60 (8 H, m), 7.97-8.06 (4 H, m), 8.27 (2 H, d, J = 7.6 Hz), 8.50 (1 H, s). ³¹ P-NMR (162 MHz, CD ₃ OD): -1.62. HRMS (ESI): ml z (C ₈ 2H ₁₁₁ N ₆ O ₂₀ P) ⁺ calculated 1530.7591; found 1530.9395 (M) ⁺ , 1531.4422 (M+H) ⁺ , 1553.9321 (M+Na) ⁺ .

Similarly, Docetaxel conjugated dicholic acid phosphocholine conjugate and Docetaxel conjugated cholic acid phosphocholine conjugate are also prepared and tested.

Preparatory Example-5:

In the following Exam pie, PEGylated lithocholic acid conjugated docetaxel of formula (9); docetaxel is attached to PEGylated bile acid with an ester linkage as shown in Scheme 2. Docetaxel-azidobe is benzoate ester was conjugated with PEGylated lithocholic acid using click chemistry to give LCA-DTX-MPEG12. The molecule is characterized using ¹ H NMR, HRMS mass spectrometry; and purity was confirmed by reverse phase high-pressure liquid chromatography.

Synthesis of PEGylated-Docetaxel Conjugate (9) (Scheme 5)

Scheme 5

Preparation of compound of formula (108a): To a solution of lithocholic acid propargyl amine (104a) (1 equiv.) and pyridine (0.33 mL, 4.5 mmol., 1.5 equiv.) in THF (20 ml_);a solution of p-nitrophenylchloroformate (0.907 g, 4.5 mmol., 1.5 equiv.) in THF (5 mL) was added. The reaction mixture was stirred for 24 h. After complete removal of solvent in vacuo, the residue was dissolved in ethyl acetate (150 mL) and washed with brine (3 X 50 ml_). Then combined ethyl acetate fractions dried over anhydrous sodium sulphate and concentrated in vacuo to give viscous oil. The crude product was purified by silica gel (230- 400 mesh) combi-flash column chromatography to get colorless solid of formula (108a). Yield (80 %); ¹ H-NMR (400 MHz, CDCI ₃ ): δ 0.67 (3 H, s), 0.88-2.15 (m), 2.25-2.32 (2 H, m), 4.08 (2 H, s), 4.72-4.78 (1 H, m), 5.6 (1 H, s), 7.40 (2 H, d, J= 8.8 Hz), 8.30 (2 H, d, J= 8.8 Hz). HRMS (ESI): ml z {C _{3 e} H ₂ O _e y calculated 578.3356; found 579.3455 (M+H) ⁺ , 601.3892 (M+Na) ⁺ .

Preparation of compound of formula (109a) (PEGylated lithocholic acid): To solution of compound of formula (108a) (750 mg, 1.296 mmol., 1 equiv.) and pyridine (0.104 ml_, 1.296 mmol., 1 equiv.) in dichloromethane (10 ml_);a solution of amino derivative of mono methoxy-PEG^was added (0.652 g, 1.166 mmol, 0.9 equiv.) in dichloromethane (5 ml_). The reaction mixture was stirred for 12 h. After completion of reaction, reaction mixture was diluted with dichloromethane (100 ml_) and washed with brine (3 X 30 ml_). Dichloromethane fractions were dried over anhydrous sodium sulfate and concentrated in vacuo to give viscous oil. The crude product was purified by silica gel (230- 400 mesh) combi-flash column chromatography using dichloromethane-methanol to get colorless viscous oil of formula (109a). Yield (88 %); ¹ H-NMR (400 MHz, CDCI ₃ ): δ 0.63 (3 H, s), 0.91 (6 H, s), 0.98-2.3 (m), 3.34-3.37 (5 H, m), 3.5-3.57 (4 H, m), 3.62-3.65 (41 H, m), 4.04 (2 H, s), 4.54-4.64 (1 H, m), 5.21 (1 H, brs), 5.68 (1 H, brs). HRMS (ESI): ml ^' z (C ₅ 3H ₉ 4N ₂ 0 ₁₅ ) ⁺ calculated 998.6654; found 1021.778 (M+Na) ⁺ , 1037.7515 (M+K) ⁺ .

Preparation of compound of formula (9) (Docetaxel conjugated PEGylated lithocholic acid): To the solution of compound (109a) (600 mg, 0.6 mmol.) and azido derivative of docetaxel (102) (0.628 g, 0.66 mmol., 1.1 equiv.) in 15 ml_ of 2:2:1 DCM/EtOH/H ₂ 0 solution; sodium ascorbate (139.7 mg, 0.66 mmol, 1.1 equiv) and copper sulfate (14.98 mg, 0.06 mmol., 0.1 equiv.) were added. The reaction was stirred vigorously overnight at room temperature. After completion of reaction, all solvents were removed in vacuo and then the resultant crude product was purified by combi-flash column chromatography using dichloromethane-methanol as eluent to give colorless solid of formula (9). Yield (90 %); ¹ H-NMR (400 MHz, CDCI ₃ ): δ 0.60 (3 H, s), 0.86-2.3 (m), 2.45 (3 H, s), 2.57-2.58 (1 H, m), 3.34-2.37 (5 H, m), 3.54 (5 H, d, J= 5.2 Hz), 3.64 (43 H, d, J = 4.4 Hz), 3.93 (1 H, d, J= 6.4 Hz), 4.08-4.32 (5 H, m), 4.57 (3 H, d, J= 6 Hz), 4.97 (1 H, d, J= 8.8 Hz), 5.23-5.29 (2 H, m), 5.56 (3 H, d, J= 19.6 Hz), 5.67 (1 H, d, J= 6.8 Hz), 6.22 (1 H, br s), 6.38 (1 H, br s), 7.289 (1 H, s) , 7.36-7.42 (4 H, m), 7.50 (2 H, t, J= 7.6 Hz), 7.62 (1 H, t, J= 7.2 Hz), 7.84 (2 H, d, J= 8.4 Hz), 8.119 (5H, t, J= 8.4 Hz). HRMS (ESI): m/z (C ₁₀ 3H ₁₅₀ N ₆ 03o) ⁺ calculated 1951.0396; found 1974.0393 (M+Na) ⁺ .

Preparatory Example 6

In the following example, Lithocholic acid conjugated tamoxifen, where tamoxifen was attached with lihocholic acid phospholipid to give LCA-Tam-PC of formula (10). The molecule is characterized using ¹ H and ³¹ P NMR spectroscopy, HRMS mass spectrometry and purity is confirmed with by reverse phase high-pressure liquid chromatography.

Synthesis of Phospholipid-Tamoxifen Conjugate (10) (Scheme 6)

Scheme 6 Preparation of Compound of formula (10) (Tamoxifen conjugated lithocholic acid phosphocholine) : To the solution of tamoxifen derivative of lithocholic acid (110a) (715 mg, 1 mmol.) and triethylamine (0.28 ml_, 2 mmol, 2 equiv.) in THF (20 ml_); 2-chloro- 1 ,3,2-dioxaphospholane-2-oxide (0.184 m L, 2 mmol., 2 equiv.) was added drop wise for 15 minutes at 0 °C and then stirred at room temperature for 10 h. Solution was filtered through celite to remove the NEt ₃ .HCI salt and the filtrate is evaporated to dryness. After complete removal of solvent, the residue (111a) was dissolved in acetonitrile (10 ml_) and transferred to a pressure tube. Trimethylamine gas was passed into pressure tube for 10 minutes at -15 °C with vigorous stirring. Reaction mixture was allowed to reflux for 48h. Then the solvent evaporated in vacuo and crude product was purified by C ₁₈ silica gel (Redi- Sep Columns) combi-flash column chromatography using water-acetonitrile as eluent. Yield (78%); ¹ H-NMR (400 MHz, CDCI ₃ ): δ 0.62 (3 H, s), 0.89-2.31 (m), 2.45 (2 H, d, J = 7.2 Hz), 2.92 (1 H, s), 3.06 (2 H, s) , 3.34 (9 H, s) , 3.63 (2 H, d, J= 4.8 Hz), 3.75 (2 H, s) , 3.92-3.97 (2 H, m), 4.04 (1 H, brs), 4.25-4.39 (7 H, m), 6.51 (2 H, d, J= 8 Hz), 6.76 (2 H, d, J= 7.6 Hz), 7.1-7.35 (10 H, m). ³¹ P-NMR (162 MHz, CDCI ₃ ): -1.54. HRMS (ESI): m/z (C ₅ 4H77N ₂ 0 ₆ P) ⁺ calculated 880.5519; found 881.5612 (M+H) ⁺ , 903.5417 (M+Na) ⁺ . Preparatory Example 7

Synthesis of Bile Acid- PEG Conjugates (11) (Scheme 7) :

In the following example, the lipid (BA) part is lithocholic acid (LCA), and Polyethyleneglycol is conjugated to 3'-hydroxyl terminal of bile acid. For synthesis of amphiphile LCA-OBn- PEG12 (11); LCA derivative (107a) was reacted with MPEG12-amine yielding PEGylated lithocholate, LCA-OBn-PEG( 11 ) that was characterized by ¹ H-NMR. ¹ H NMR, 400MHz, CDCI ₃ ) δ: 0.61 (s, 3H), 0.85-0.90 (m, 6H), 1.00-1.96 (m, steroid), 2.25-2.31 (m, 1H), 2.36-2.40 (m, 1H), 3.36-3.38 (m, 6H), 3.46-3.56 (m, 6H), 3.63-3.65 (m, 50H, OCH2CH20), 4.59 (s, 1H), 5.07-5.15 (m, 2H),5.30 (s, 1H), 7.32-7.35 (m, 5H).

107a 11

Scheme 7

Preparatory Example 8

In the following example, bile acid (Lithocholic acid) conjugated with imaging agent Nitrobenzoxadiazole(NBD, a fluorophore) in which imaging agent attached to bile acid by click chemistry.

Preparation of compound of formula 12a (Bile acid conjugated to imaging agent) (Scheme 8): To a solution of compound (104a) (100 mg, lequiv.) and NBD-N ₃ (1.3 equiv.) in methanol; sodium ascorbate (1.5 equiv.) was added, and stirred for 5 min. Then copper sulfate (0.5 equiv.) in 1 mL water was added to solution and reaction mixture was stirred for 12 hours at room temperature. After the reaction completion, organic solvent removed under vacuo and compound was isolated by silica gel (230-400mesh) combi flash chromatography using 95:5 DCM: methanol as eluent. The molecules are characterized using ¹ H NMR spectroscopy .

( _12a ) LCA-NBD

Scheme 8

LCA-NBD (12a): ( ¹ H-NMR 400 MHz, DMSO-d ₆ ): δ 0.57 (3 H, s), 0.83-2.08 (m, steroid), 3.95 (s, 2H), 4.21 (d, J = 6Hz, 2H), 4.45 (d, J = 4Hz, 1H), 4.67 (t, J = 6Hz, 2H), 6.34 (d, J = 8Hz, 1H), 7.93 (s, 1H), 8.25 (t, J = 6Hz, 1H), 8.48 (d, J = 8Hz, 1H), 9.44 (s, 1H).

Preparatory Example 9

In the following example, phospholipid modified bile acid (Lithocholic acid) conjugated with imaging agent Nitrobenzoxadiazole(NBD, a fluorophore) in which imaging agent attached to bile acid derived phospholipid by click chemistry.

Preparation of compound of formula 13a (Bile acid phospholipid conjugates with Imaging agent) (Scheme 9):

Scheme 9

Synthesis of lithocholic acid NBD Phosphocholine derivative (13a): To a solution of compound (105a) (lequiv.) and NBD-N ₃ (1.3 equiv.) in Methanol; Sodium Ascorbate (1.5 equiv.) was added, stirred for 5 min. Then copper sulfate (0.5 equiv.) in 1 ml_ water was added to solution and reaction mixture was stirred for 12 hours at room temperature. After the reaction completion, organic solvent removed under vacuo and compounds was isolated by reverse phase C-18 siliica gel combi flash chromatography using ACN-water (30:70) as eluent. The molecules was characterized using ¹ H NMR and ³¹ P NMR spectroscopy. LCA- NBD-PC (13a) (Where R ₂ and R ₃ = H) ( ¹ H NMR, 400MHz, MeOH-d ₄ ) <5: 0.66 (s, 3H), 0.93 (d, J = 6Hz, 3H), 0.96 (s, 3H), 1.04-2.31 (m, steroid), 3.24 (s, 9H), 3.63-3.66 (m, 2H), 4.09-4.15 (m, 4H), 4.26-4.27 (m, 2H), 4.36 (s, 2H), 4.77 (t, J= 6Hz, 2H), 6.23 (d, J= 8Hz, 1H), 7.86 (s, 1H), 8.48 (d, J = 8Hz, 1H). ³¹ P-NMR (162 MHz, MeOH-d ₄ ) : -0.82. Preparatory Example 10

In the following example of tamoxifen conjugated lithocholic acid dipeptide, tamoxifen is conjugated to lithocholic acid and then derivatized with dipeptide.

Synthesis of conjugate 14:

Synthesis of compound 112a: Lithocholic acid (3 mmol), DMAP (3.6 mmol), and TAMNHMe (3.6 mmol) dissolved in dry DCM and stirred at 25 °C for 15 min. HBTU (6 mmol), was added to reaction mixture in a lot and reaction allow to stir for 48h at 25°C. After completion of reaction, crude mixture diluted with dichloromethane and washed two times with saturated NaHC0 ₃ solution and dilute HCI. Organic layer dried over anhydrous sodium sulphate and concentrated on vacuo. Crude product purified with flash column chromatography using ethyl acetate and petroleum ether as eluents. Yield 85%,

(14) LCA-Tam-Gly-Gly

Scheme 10

Preparation of compound with formula 14 (Tamoxifen conjugated lithocholic acid dipeptide) (Scheme 10): Compound 112a (1 equiv), DMAP (1.2 equiv), and Boc-Gly-Gly- OH (2 equiv.) were dissolved in dry DCM and stirred at 25 °C for 15 min. Coupling agent (DCC, 1.5 equiv.) was then added to reaction mixture and reaction was allowed to stir for 24h at 25 °C. After completion of reaction, mixture was diluted with dichloromethane, and washed two times with saturated NaHC0 ₃ solution and 1 N HCI. Organic layer was dried over anhydrous sodium sulphate and concentrated on vacuo. Crude product was purified with flash column chromatography using ethyl acetate and petroleum ether as eluents. Resultant product was dissolved in Dichloromethane (5ml_), and 4M HCI in Dioxane (4 ml_) was added and stirred at 0 °C for 2h. After completion of reaction, solvent was reduced at rotavapor and product was precipitated by addition of diethyl ether. Resultant precipitate (compound 14) was washed twice with diethyl ether and dried under vacuum . Yield 70% , ¹ H NMR (400 MHz, DMSO-d ₆ ) & 8.88 (bs, 1H), 8.21 (bs, 3H), 7.39-7.25 (m, 2H), 7.20-7.07 (m, 7H), 7.03-6.91 (m, 2H), 6.82 (d, J = 6.9 Hz, 1H), 6.74-6.70 (m, 1H), 6.59-6.57 (m, 1H), 4.65 (s, 1H), 4.14-3.89 (m, 4H), 3.69-3.52 (m, 4H), 3.06-2.79 (m, 3H), 2.45-2.34 (m, 3H), 1.92-0.74 (m, 35H, steroid), 0.59 (s, 3H).

Preparatory Example 11 Nano-emulsion preparation for molecules of formula 1 :

Nano-emulsion were prepared by addition of molecule (1) (0.1-2.0 mg) in 50 μΙ_ of polysorbate-80 in 2 mL centrifuge tube, vortexing it for 5-10 mins followed by addition of 50 μί of ethanol (100%). Vortexing of resulting solution for 5 mins formed the clear solution that was diluted by addition of 900 μί of 0.5% Carboxymethylcellulose (CMC) and mixed together by vortexing. The above formulation was tested for its stability in gastric condition by incubating it in stimulated gastric fluid (SGF) with pepsin for 2 hours. SGF was prepared according to USP specifications (United States Pharmacopeia) where 0.2 g of sodium chloride was dissolved in 50 mLMilli-Q water in 100 mL conical flask; and 0.7 mLof HCI was added to adjust the pH of the solution to 1.2. Then 4.0 mg of pepsin (250U/mg) was dissolved with gentle shaking, and the volume made up to 100 mL with Milli-Q water. SGF (5 ml) was taken in 50 mL round bottom flask on constant stirring and heating at 37 ^S C. To this solution, 500 μί of nano-emulsion (1mg/mL) was added; and after regular intervals of 30 min, 1 hour, and 6-hour time point; 150 μί of test solution was taken out and 450 μί of stop solution (chilled methanol) was added to it. The solution was vortexed and centrifuged at 12000 rpm for 10 mins. Supernatant was taken and stored at -20 ^s Cand later run on HPLC to quantify the drug. These molecules made stable emulsions in SGF.

Preparatory Example 12 Nanom iceller formation from molecules of formula 4 and 7:

Stock solutions of molecules 4 and 7 were prepared in chloroform. Both the solutions were mixed in different ratios in 5 mL glass vial. Chloroform was evaporated under N ₂ gas while rotating the solution and a thin film was created. Residual chloroform was removed by keeping the vials in speed-vac. The film was re-suspended in 1 mL of 1X PBS and subjected to sonication for 10-15 min. The resulting solution was passed through 0.22 m syringe filter. Encapsulation efficiency (% EE) was 30.8 %, 14.9 %, 1.7 % for 8:1 , 4:1 , and 2:1 ratio of 4 and 7 molecules respectively; and dynamic light scattering measurements showed mean diameter (Z-av) of -127.3 nm, 156 nm, 148.3 nm for 8:1, 4:1 and 2:1 respectively. The stability of the different ratios of nanoparticles prepared was monitored in PBS at pH 7.4 through the measurement of diameter of nanoparticles; and these nanoparticles were stable for more than a week.

Preparatory Example 13 Nanoformulations of Phospholipid head containing bile acid drug conjugate (NMs 1 , DTX PC NPs)

In the following example, the nanomiceller aggregate formation of molecules with formula 8 and 11 is presented. These nanomicelles were then investigated in detail for their anticancer activities.

Nanomiceller formation from molecules of formula 8 and 11 (NMs 1) :To a 5% (w/v) dextrose solution in a round bottom flask employed with magnetic bead and stirring at 1500 rpm; 200 microliters of tetrahydrofuran solution of 1:1 mixture of two components PEGylated lithocholic acid derivative (Formula 11 , 9.56 mg/mL) and lithocholic acid- docetaxel phospholipid conjugate (Formula 8, 9.56 mg/mL) were injected drop wise over a period of 10 min. After complete addition, cloudy turbid solution wasformed due to ouzo effect. Then the added tetrahydrofuran solvent was completely removed by rotary evaporation. Formation of colloidal nanomicelles (NMs 1) of -30 nm hydrodynamic radius wereconfirmed by dynamic light scattering.

Figure 1 shows characterization resulting nanomiceller aggregates NMs 1 (a) dynamic light scattering and (b) transmission electron microscopy. These show the formation of colloidal nanomicelles of -30 nm hydrodynamic radius. Preparatory Example 14

Nanoformulations of PEGylated bile acid drug conjugate (NMs 2, DTX-PEG NP's)

In the following example, the nanomiceller aggregate formation of molecules with formula 9 is presented. Nanomiceller formation from molecules of formula 9: To a 5% (w/v) dextrose solution in a round bottom flask employed with magnetic bead and stirring at 1500 rpm, 200 microliters of tetrahydrofuran solution of PEGylatedlithocholic acid-docetaxel conjugate (Formula 9, 6.15 mg/mL) was injected drop wise over a period of 10 min. After complete addition, cloudy turbid solution was formed due to ouzo effect. Then the added tetrahydrofuran solvent was completely removed by rotary evaporation. Formation of colloidal nanomicelles of -80 nm hydrodynamic radius was observed which was confirmed by dynamic light scattering as shown in Figure 2. Characterization of PEGylated lithocholic acid-docetaxel conjugate nanomicelles (NMs 2) was also done by Transmission Electron Microscopy and is shown in Figure 2. Figure 2 shows characterization of PEGylated lithocholic acid-docetaxel conjugate nanomicelles (DTX-PEG NP's, NMs 2) by (a) dynamic light scattering and (b) transmission electron microscopy shows the formation of colloidal nanomicelles of -80 nm hydrodynamic radius. Preparatory Example 15

In the following examples, the anticancer activity studies related to NMs 1 and NMs 2 formed in example 13 and 14 are presented. Anticancer activities of NMs 1 (DTX-PC NPs) and NMs 2 (DTX-PEG NP's)

Cell culture: MCF-7, MDA-MB-231 , and 4T1 cells were maintained as monolayers for experiments. 4T1 cells were cultured in RPMI-1640 media; and MCF-7, and MDA- MB-231 were cultured in DMEM containing 10% (w/v) fetal bovine serum, penicillin (100 g/mL), streptomycin (100 U/mL), gentamycin (45 g/mL) at 37 °C in a humidified atmosphere with 5% C0 ₂ . Subcultures were made by trypsinization and reseeded for experiments.

Cytotoxicity of NMs 1 and NMs 2 in different human (MCF-7, MDA-MB-231) and murine breast cancer cell line (4T1) was studiedby tryphan blue exclusion assay method. Typically, in this method different cell lines were seeded at a density of 0.5 X 10 ⁵ cellsin 1 ml_ media per well in a 12-well plate. After 24 h ensuring attachment of cells, media were removed, and cells were treated with 1 ml_ fresh media containing different concentrations of docetaxel and NMs 1. After 48 h of incubation, the media with dead cells was collected from different wells in different microcentrifuge tubes; the remaining attached cells were then washed with DPBS twice (2 X 1 ml_) and pooled in respective microcentrifuge tubes. Finally, the remaining attached cells were harvested using 100 μΙ_ trypsin-EDTA, and collected using media. Pellet was collected by centrifuging the media containing dead and live cells at 5000 rpm. The pellet was then washed twice (2 X 1 ml_) with cold DPBS and finally re-suspended in 0.5 ml_ DPBS. One part of 0.4% trypan blue solution was mixed with one part cell suspension and the mixture was incubated for 3 min. at room temperature. Live and dead cells were counted using haemocytometer. Percentage of live cells was plotted against the respective concentration of either DTX or NMs 1 or NMs 2. In vitro efficacy of NMs 1 or NMs 2 revealed that NMs land NMs 2are cytotoxic to the breast cancer cells similar to parent DTX (Figure 3) .

Animal experiments: All the animal experiments were done with the approval of Institutional Animal Ethical Committee (IAEC), Nil, New Delhi. First the maximum tolerable dose of NMs 1 upon intravenous administration was estimated; and then antitumor activities NMs 1 were compared with parent tween solubilized docetaxel (DTX-TS) in 4T1 tumor model. The MTD after administration of single as well as multiple doses of DTX-TS and NMs 1 at different escalating doses was estimated. Multiple (5) injections of 0, 5, 10, 20 mg/Kg or a single injection 0, 40, 80, 160 mg/Kg DTX-TS and NMs 1 (with equivalent DTX dose) were administered; and mice were observed for general health status (health ruffled hair, reduction of body weight by 10-15%, lethargy, toxicity, survival) for period of one week post doses.

A dose dependent decrease in body weight of mice receiving DTX-TS formulation was observed. Mice receiving 5mg/Kg dose of DTX-TS were initially reduced their weight but after treatment schedules; they recovered. The groups receiving 10 mg/Kg and 20 mg/Kg DTX-TS drastically reduced their weight by 10 and 20% respectively. Mortality in 10 mg/Kg and 20 mg/Kg DTX-TS dose-receiving mice was observed. In contrast, all mice injected with NMs 1 survived without any change in body weights compared to the control. No peripheral ascites/lesions on the tails of the mice treated with NMs 1 was observed. Toxicology studies in Sprague dawley rats: Sprague-Dawley rats (about 6 weeks) were randomized in to three groups (n= 3 per group). Two groups of Rats were anaesthetized with ketamine xylazine mixture, and administered intravenously with single dose of 10 mg/Kg of DTX-TS or equivalent doses of NMs 1 ; with third group used as control. Post 24 hours of treatment, blood samples were collected from retro-orbital sinus in ACD- microcentrifuge tube under mild anesthesia. Whole blood and isolated plasma samples were analyzed for the toxicity markers. Hematological parameters such as neutrophils and platelets are affected by the treatment of DTX-TS whereas these parameters were normal when equivalent doses of NMs 1 are administered. Similarly, plasma biochemistry shows liver biomarkers are highly affected by the treatment of DTX-TS whereas the parameters were normal upon administration of equivalent doses of NMs 1.

Similar toxicity studies in murine, rabbit and Primate model systems were performed; and observed that NMs 1 are much less toxic than DTX-TS formulation. Pharmacokinetics of NMs 1 in Sprague-Dawley rat models: Female Sprague-Dawley rats (about 6 weeks) were randomized into 3 groups (n= 3 per group). Rats were anaesthetized with ketamine xylazine mixture and then administered a single intravenous dose (5 mg/kg) of DTX-TS or NMs 1 (DTX equivalent). At various time points, serial bleeds were collected in ACD-m icrocentrifuge tube through the orbital sinus under mild anaesthesia at 0.08, 0.25, 0.5, 1, 2, 4, 6, 12, 24 h after intravenous injection. Plasma was separated from the samples and stored under -80 °C until analysis. Drug was then extracted from 1 ΟΟμί of plasma samples with the addition of 100μΙ_ of extracting solvent (typically for DTX- TS, the extracting solvent is Tetrahydrofuran (THF); for NMs 1 , extracting solvent is Tetrahydrofuran: Ethanol (THF:EtOH) (3:1)). Samples were then centrifuged at 10,000 rpm for 5 min after 2 min of vortex, and 5 min sonication cycles. Supernatant extract was taken, and extraction cycles were repeatedfor at least two times; andfinally, supernatant samples were pooled for each time point. Blank plasma samples were spiked with known amount of docetaxel or DTX conjugate (molecule 8) and simultaneously extracted using the above mentioned procedure to determine the extraction efficiency of the drug and NMs from the spiked plasma samples. Samples were analyzed by Waters HPLC system equipped with C18 TSK gel column (S0098) 4.6 X 25 mm (5μηη) with UV detection parameters for DTX-TS at 230 nm and for NMs 1 at 270 nm. Samples were transferred into snap capped vials and loaded in to HPLC auto sampler. A HPLC isocratic 10 minute run program was set using 50:50 mixture of solvent-A (37.5 : 60 : 2.5 : 0.1 (v/v) Water : MeOH : THF : Ammonium hydroxide) and Solvent-B (acetonitrile) at room temperature at a flow rate of 1 mL/min. We performed same pharmacokinetic experiments in mice and primates. We observed multifold increase in mean C ₀ and mean AUC with reduced clearance (CL) values on NMs 1 treatment as compared to DTX-TS. Therefore, NMs possessed a higher mean circulatory time, half-life, AUC, and reduced clearance of DTX-PC NPs (NMs 1) as compared to DTX-TS in Balb/c mice, SD rats, and Primates. In vivo antitumor activity (Figure 4) : The anti-tumor potency of NMs 2 ( DTX-PEG NP's) in subcutaneous 4T1 Murine breast cancer model was investigated, and compared with DTX- TS. Murine cancer cells (4T1) were injected into mice. After mice develop 75 mm ³ average tumor sizes, the mice were randomized into 4 groups a) vehicle control group (5% glucose, i.v.), b) Plain NMs (20 mg/kg, i.v.), c) DTX-TS (5 mg/Kg, i.v.) and d) NMs 2(DTX-PEG NP's)(5 mg/kg, i.v.) injected group. All the formulations were given intravenously through lateral tail vein. Alternate day doses (a total of 10 doses) were administered during the study period. Tumor volume was measured every alternate day with a digital caliper using the formula Lx B ² /2 followed by continuous monitoring of the body weight, and general health status of the mice. Study was continued till the last mouse was found live in control groups. We observed no significant change in the tumor volume between the control (5% glucose) and plain nanoparticles. Treatment with NMs 2(DTX-PEG NP's)showed significant reduction in the tumor volume by > 50% as compared to control (5% glucose) and plain nanoparticles. General health status of mice was also monitored and it was found that DTX- TS treated mice lost weight significantly compared to the controls and NMs 2 (DTX-PEG NP's). DTX-TS causes severe dose dependent acute hypersensitivity reactions and lethality to mice depending on the severity of acuteness of the allergic reaction. The median survival for the mice of control, neat nanoparticles, DTX-TS and NMs 2confirm that NMs 2 (DTX- PEG NP's) showed better anti-tumor activity with much better survival of the mice.

The drug conjugates nanoparticles of the present invention can be delivered through all the routes of administration, preferentially intravenous, bolus or intravenous infusion in i.v. fluids or intra tumoral route or oral administrations etc.

The nanoparticles and co-nanoparticles of the present invention maintains high circulation drug levels compared to that of the naive drugs.

The drug conjugate nanoparticle formulations can be used in the treatment of pathological conditions associated with abnormal cell proliferation. Such abnormal proliferative pathological conditions include but not limited to the abnormal cellular proliferation of malignant or non-malignant cells in various tissues and/or organs, including, not limited to muscle, bone and /or conjunctive tissues; the skin, brain, lungs and sexual organs; the lymphatic and /or renal system; mammary cells and /or blood cells; the liver, digestive system, and pancreas; and the thyroid and/or adrenal glands. These pathological conditions can also include psoriasis, poly cystic kidney diseases (PKD) and rheumatoid arthritis; solid tumors; ovarian, breast, brain, prostate, colon, stomach, kidney, and/or testicular cancer, Kaposi's sarcoma; cholangiocarcinoma; choriocarcinoma; neuroblastoma; Wilm'stumor, Hodgkin's disease; melanomas; multiple myelomas; chronic lymphocytic leukemias; and acute or chronic granulocytic lymphomas.