EA046205B1 - PHOSPHOLIPID-FLAVAGLIN CONJUGATES AND METHODS OF THEIR APPLICATION FOR TARGETED CANCER THERAPY - Google Patents

Description

Link to related applications

This application claims benefit from U.S. Provisional Patent Application No. 62/655,659, filed April 10, 2018, which is incorporated herein by reference in its entirety.

Field of technology to which the present invention relates

The present invention relates to phospholipid-flavaglin conjugates and methods for targeted cancer therapy.

Introduction

The effectiveness of most anticancer drugs when used in clinical settings is limited by their toxicity to all proliferating cells and/or their inability to exert their effects on all tumor cells. The development of new agents with unique mechanisms of action to provide improved targeting continues, however, many of these compounds still lack absolute tumor selectivity and continue to limit their therapeutic use due to off-target effects. Antibody-drug conjugates (ADCs) have been designed to bind to specific epitopes on the surface of tumor cells, and they have offered an alternative method for targeting tumor cells to reduce associated toxicities. Although highly selective, very few antibody-drug conjugates are therapeutically useful as they achieve moderate cellular uptake and limited cytolytic activity. More effective platforms for targeting tumors are required.

Brief Disclosure of the Present Invention

The invention relates to a conjugate according to formula II or a salt thereof

About СНз иI®

Z---Y---X--(CH ₂ )18-O-r-o-(CH ₂ ) ₂ -N-CH3

IΘI ^{0 CH} 3(II).

Another aspect of the present disclosure provides a composition comprising a conjugate as described in detail herein and a pharmaceutically acceptable carrier.

Another aspect of the present disclosure provides a method of treating cancer in a subject, the method comprising administering to the subject a conjugate as described in detail herein. Another aspect of the present disclosure provides a method of targeting a drug to a tumor or cancer cell in a subject, the method comprising administering to the subject a conjugate as described in detail herein.

In accordance with some embodiments, the flavaglin anticancer drug is localized or translocated to the cytoplasm or organelle of the tumor or cancer cell. In accordance with some embodiments, the conjugate or flavaglin anticancer drug is selective for cancer cells in a subject. In some embodiments, the conjugate or flavaglin anticancer drug is included in at least about 2-fold more tumor or cancer cells compared to healthy cells. In some embodiments, the cancer is selected from melanoma, brain cancer, lung cancer, adrenal cancer, liver cancer, kidney cancer, pancreatic cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, anal cancer duct, prostate cancer, ovarian cancer, breast cancer, cervical cancer, lymphoma, leukemia, myeloma, hematological malignancy, hepatocellular carcinoma, retinoblastoma, glioma, sarcoma, blastoma, squamous cell carcinoma and adenocarcinoma. In accordance with some embodiments, the subject is a human.

Other aspects and embodiments are contemplated by the present disclosure which will become apparent in view of the following detailed description and the accompanying drawings.

Brief description of drawings

Fig. 1A, 1B, 1C, 1D, 1E are images of tumor cells with lipid rafts noted. Tumor cells are characterized by a higher concentration of lipid rafts compared to normal cells. Fig. 1F is an image of normal fibroblasts and Caki-2 tumor cells using CLR 1501 (compound (1)). CLR 1501 localized at a high level in Caki-2 cells and at a minimal level in normal fibroblasts. Fig. 1G is an image of control A549 cells, and FIG. 1H is an image of A549 cells treated with methyl-beta-cyclodextrin to disrupt lipid rafts. Cells as in Fig. 1G, and in Fig. 1H was incubated with CLR 1501 (compound (1)), and disruption of most lipid rafts in A549 cells resulted in a 60% reduction in CLR 1501 uptake (compound (1)). Fig. 1I, 1J and 1K are images of PC3 cells incubated with CLR 1501 (compound (1)) and stained to visualize the endoplasmic reticulum (ER). CLR 1501 (compound (1)) localized to the ER in malignant cells but not in normal cells (not shown).

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Fig. 1L, 1M and 1N are images of PC3 cells incubated with CLR 1501 (compound (1)) and stained to visualize the nucleus and mitochondria. CLR 1501 (compound (1)) was localized in mitochondria.

Fig. 2 is an image of a colon tumor-bearing (HCT-116) mouse injected with CLR 1502 (compound (2)), showing the location of the tumor.

In fig. 3 is a plot of cytotoxicity versus concentration for the cytotoxic compound (FLV1) versus the PLE-conjugated cytotoxic compound (CLR 1865, Compound (8)) in A549 (human lung adenocarcinoma) cells or normal human skin fibroblasts (NHDF).

In fig. 4 is a plot of fold increase versus time for the uptake of PLE conjugate CLR 1852 (compound (9)) in A375 (human melanoma) and HEK293 (human embryonic kidney) cells.

In fig. 5 shows images of a mouse model of breast cancer showing the uptake of CLR 1502 (compound (2)) in vivo.

In fig. 6 shows images of myeloma cell lines showing uptake of CLR 1501 (compound (1)).

In fig. 7 shows images of cancer stem cells, normal brain tissue and normal stem cells showing the specific uptake of CLR 1501 (compound (1)) into cancer cells.

In fig. 8 is a plot of percent cytotoxicity versus concentration for CLR 1852 (compound (9)) versus FLV3 in A549 cells (human lung adenocarcinoma) and normal human skin fibroblasts (NHDF).

In fig. 9 is a plot of volume versus time for media compared to CLR 1852 (compound (9)) in the HCT 116 tumor model.

In fig. 10 is a graph of body weight versus time for media compared to CLR 1852 (compound (9)) in the HCT 116 tumor model.

In fig. 11 is a plot of concentration versus time for FLV3 detected in the cytosol (normalized to cytosolic volume) in the A375 (human melanoma) and A549 (human lung adenocarcinoma) cell lines.

Detailed Disclosure of the Present Invention

Disclosed herein are phospholipid compounds and phospholipid-flavaglin conjugates. Based on the fact that many animal and human tumors contain higher concentrations of naturally occurring lipid esters than normal tissues, phospholipid ester (PLE) molecules were developed. The PLE molecules detailed herein can be used as a tumor targeting platform for selective drug delivery to tumors and cancer cells.

In one aspect, the present disclosure relates to a phospholipid ester (PLE) according to Formula I or a salt thereof

O uN ₃

X-(CH ₂ ) _1e -O-^-O-(CH ₂ ) ₂ -N-CH ₃ IsI ^{0 CH} 3(I),

wherein X represents hydrogen, methyl or phenyl substituted with carboxyl.

In accordance with some embodiments, the PLE is selected from the following

Osn iI®

-PO-(CH ₂ ) ₂ -N-CH ₃

Ιθ ⁰ CH ₃ (3)i

CH3 Ιθ n ₃ s—(CH ₂ ) ₁₃ -OPQ-(CH ₂ ) ₂ -N-CH ₃

IΘ |

In accordance with some embodiments, the PLE further includes a detectable fragment attached thereto.

In accordance with a further aspect, the present disclosure relates to a composition comprising PLE, which is described in detail herein, and a carrier.

Another aspect of the present disclosure provides a connection selected from the following

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Another aspect of the present disclosure provides a composition containing at least one of these compounds and a carrier.

Another aspect of the present disclosure provides a conjugate corresponding to Formula II, or a salt thereof

is a linker containing a disulfide; and Z is a flavaglin anticancer drug. In accordance with some embodiments, the flavaglin anticancer drug comprises FLV-1, FLV-3, a derivative or analog thereof, or a combination thereof.

In accordance with some embodiments, the linker comprises the following

In accordance with some embodiments, the conjugate is selected from the following

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(8) and

(9) .

As described in detail herein, tissue distribution of PLE molecules was assessed in cells from more than 100 different tumors, including fresh human tumor samples. PLE molecules showed increased uptake in tumor tissue compared to normal tissue. PLE molecules can be conjugated to flavaglin molecules and their derivatives through linkers to form phospholipid-flavaglin conjugates.

1. Definitions.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art. In the event of a conflict, this document, including its definitions, will control. Preferred methods and materials are described below, however, methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications, patent applications, patents and other sources referenced herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting. The terms contain (contains), include (includes), has, has, may, contain (contains) and variations thereof, when used in the context of this document, are intended to be open transitional phrases, terms or words that are not exclude the possibility of additional actions or structures. Singular forms include corresponding plural forms unless the context clearly requires otherwise. This disclosure also provides other embodiments containing, consisting of, and consisting substantially of the embodiments or elements presented herein, whether expressly set forth or not.

Where numerical ranges are listed herein, each number in between is specifically provided for with the same degree of precision. For example, in the case of the range 6-9, the numbers 7 and 8 are provided in addition to 6 and 9, and in the case of the range 6.0-7.0, 6.0, 6.1, 6.2, 6.3, 6 are specifically provided. 4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0.

The term approximately, as used in the context of this document in relation to one or more values of interest, refers to a value that is similar to the stated target value. In accordance with certain aspects, the term approximately refers to a range of values that are within the range of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less in either direction (more or less) from the stated target value, unless otherwise stated or unless otherwise obvious from the context (unless such number would exceed 100% of the possible value).

Definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, ^75th Ed., inside cover, and specific functional groups are generally identified as described in those references. In addition, general principles of organic chemistry as well as specific functional moieties and reactivity are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, ^7th Edition, John Wiley & Sons, Inc., New York, 2013; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989;

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Carruthers, Some Modern Methods of Organic Synthesis, ^3rd Edition, Cambridge University Press, Cambridge, 1987; and the entire contents of each of these sources are incorporated herein by reference.

The terms alkoxy or alkoxy as used herein refer to an alkyl group, as defined herein, attached to the parent molecular moiety via an oxygen atom. Typical examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy and t-butoxy.

The term alkyl, as used herein, means a straight or branched saturated hydrocarbon chain containing from 1 to 20 carbon atoms. The terms lower alkyl or C _1-6 alkyl mean a straight or branched saturated chain hydrocarbon containing from 1 to 6 carbon atoms. The term C _1-4 alkyl means a straight or branched chain hydrocarbon containing from 1 to 4 carbon atoms. The term C _1-3 alkyl means a straight or branched chain hydrocarbon containing from 1 to 3 carbon atoms. Typical examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, npentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2, 2-dimethylpentyl, 2,3-dimethylpentyl, nheptyl, n-octyl, n-nonyl and n-decyl. The term alkenyl, as used herein, means an unsaturated hydrocarbon chain containing from 2 to 20 carbon atoms and at least one carbon-carbon double bond.

The term alkynyl, as used herein, means an unsaturated hydrocarbon chain containing from 2 to 20 carbon atoms and at least one carbon-carbon triple bond.

The term alkoxyalkyl, as used herein, refers to an alkoxy group, as defined herein, attached to the parent molecular moiety via an alkylene group, as defined herein.

The term arylalkyl, as used herein, refers to an aryl group, as defined herein, attached to the parent molecular moiety via an alkylene group, as defined herein.

The term alkylamino, as used herein, means at least one alkyl group, as defined herein, attached to the parent molecular moiety via an amino group, as defined herein.

The term alkylene, as used herein, refers to a divalent group derived from a straight or branched chain hydrocarbon with 1-10 carbon atoms, for example 2-5 carbon atoms. Typical examples of alkylene include, but are not limited to, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2- and -CH2CH2CH2CH2CH2-.

The term amide as used herein means -C(O)NR- or -NRC(O)-, wherein R may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl or heteroalkyl.

The term aminoalkyl, as used herein, means at least one amino group, as defined herein, attached to the parent molecular moiety via an alkylene group, as defined herein.

The term amino as used herein means -NR _x R _y , wherein R _x and R _y may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl or heteroalkyl. In the case of an aminoalkyl group or any other moiety in which an amino joins two other moieties together, the amino may be -NR _x- in which R _x may be hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocycle, alkenyl or heteroalkyl.

The term aryl as used herein refers to an aromatic group, such as a phenyl group, or a bicyclic fused ring system. An example of bicyclic fused ring systems is a phenyl group attached to the parent molecular moiety and fused to a cycloalkyl group as defined herein, a phenyl group, a heteroaryl group as defined herein, or a heterocycle as defined herein. Typical examples of aryl include, but are not limited to, indolyl, naphthyl, phenyl, quinolinyl and tetrahydroquinolinyl. The term arylalkyl refers to alkyl, as defined herein, substituted with an aryl radical.

Arylene refers to an aryl, as defined herein, having two monovalent radical centers obtained by removing two hydrogen atoms from two carbon atoms in the parent aryl. Typical arylene radicals include, but are not limited to, phenylene and naphthylene. Arilalkylene refers to an arylalkyl, as defined herein, having two monovalent radical centers obtained by removing one hydrogen atom from the aryl radical and the other hydrogen atom being removed from the alkyl radical of the group.

The term carboxyl as used herein means a carboxylic acid or -COOH.

The term cycloalkyl means a monovalent saturated hydrocarbon ring or bicyclic

- 5 046205 group. Cycloalkyl groups have zero heteroatoms and zero double bonds. Cycloalkyl groups are monocyclic or are fused, spiro- or bridged bicyclic ring systems. Monocyclic cycloalkyl groups contain 3-10 carbon atoms, preferably 4-7 carbon atoms, and more preferably 5-6 carbon atoms in the ring. Bicyclic cycloalkyl groups contain 8-12 carbon atoms, preferably 9-10 carbon atoms in the ring. Cycloalkyl groups may be saturated or unsaturated. Cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. The term cycloalkenyl as used herein means a non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having from 5 to 10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl and cycloheptenyl.

The term cycloalkynyl, as used herein, means a monocyclic or polycyclic ring system containing at least one carbon-carbon triple bond and preferably having from 5 to 10 carbon atoms per ring or more than 10 carbon atoms per ring. The term haloalkyl, as used herein, means an alkyl group, as defined herein, in which one, two, three, four, five, six, seven or eight hydrogen atoms are replaced by a halogen. Typical examples of haloalkyl include, but are not limited to, 2-fluoroethyl, 2,2,2-trifluoroethyl, trifluoromethyl, difluoromethyl, pentafluoroethyl, and trifluoropropyl such as 3,3,3-trifluoropropyl.

The terms halogen or halide as used in this document mean Cl, Br, I or F.

The term heteroalkyl, as used herein, means an alkyl group as defined herein in which at least one of the carbons in the alkyl group is replaced by a heteroatom such as oxygen, nitrogen and sulfur. Typical examples of heteroalkyls include, but are not limited to, alkyl ethers, secondary and tertiary alkyl amines, amides and alkyl sulfides.

The term heteroaryl, as used herein, refers to an aromatic monocyclic ring or an aromatic bicyclic ring system containing at least one heteroatom independently selected from the group consisting of N, O and S. Aromatic monocyclic rings are five- or six-membered rings containing at least one heteroatom independently selected from the group consisting of N, O and S. Five-membered aromatic monocyclic rings have two double bonds, and six-membered aromatic monocyclic rings have three double bonds. An example of bicyclic heteroaryl groups is a monocyclic heteroaryl ring attached to the parent molecular moiety and fused to a monocyclic cycloalkyl group as defined herein, a monocyclic aryl group as defined herein, a monocyclic heteroaryl group as defined herein, or a monocyclic heterocycle , which is defined in this document. Typical examples of heteroaryl include, but are not limited to, indolyl, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, pyrrolyl, benzopyrazolyl, 1. 2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzoxadiazolyl, benzothienyl, benzofuranyl, isobenzofuranyl, furanyl, oxazolyl, isoxazolyl, purinyl, isoindolyl, quinoxalinyl, indazolyl, quinazolinyl, 1,2,4-triazinyl, 1,3,5-triazinyl, isoquinolinyl, quinolinyl, 6,7-dihydro- 1,3-benzothiazolyl, imidazo[1,2-a]pyridinyl, naphthyridinyl, pyridoimidazolyl, thiazolo[D-^pyridin-Ayl, thiazolo[5,4d]pyrimidin-2-yl. The terms heterocycle, or heterocyclic, or heterocyclyl, as used herein, mean monocyclic heterocycle, bicyclic heterocycle (heterobicyclic), or tricyclic heterocycle. A monocyclic heterocycle is a three-, four-, five-, six-, seven-, or eight-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. The three- or four-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N and S. A five-membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. A six-membered ring contains zero, one or two double bonds and one, two or three heteroatoms selected from the group consisting of O, N and S. Seven- and eight-membered rings contain zero, one, two or three double bonds and one, two or three heteroatoms, selected from the group consisting of O, N and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3dithiolanyl, 1,3 -dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl , tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl , thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1

- 6 046205 dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl and tritianyl. A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a bridged monocyclic heterocyclic ring system in which two non-contiguous the atoms in the ring are linked by an alkylene bridge of 1,2,3 or 4 carbon atoms or by an alkylene bridge of two, two, three or four carbon atoms. Typical examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3-dihydrobenzofuranyl, 2,3-dihydrobenzothienyl, 2,3-dihydroisoquinoline, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[ 2.2.1]hept-2-yl), 2,3-dihydro-N-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl and tetrahydroisoquinolinyl. An example of tricyclic heterocycles is a bicyclic heterocycle fused with a phenyl group, or a bicyclic heterocycle fused with a monocyclic cycloalkyl, or a bicyclic heterocycle fused with a monocyclic cycloalkenyl, or a bicyclic heterocycle fused with a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms in the bicyclic ring are linked by an alkylene bridge of 1, 2, 3 or 4 carbon atoms or by an alkylene bridge of two, three or four carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-N-N-methanocyclopentaVDfuran, hexahydro-N-1,4-methanocyclopenta[c]furan, azaadamantane (1-azatricyclo[3.3.1.1 ^{3 ,7} ]decane) and oxa-adamantane (2-oxatricyclo[3.3.1.1 ^3,7 ]decane). Monocyclic, bicyclic and tricyclic heterocycles are connected to the parent molecular fragment through any carbon atom or any nitrogen atom contained in the rings, and may be unsubstituted or substituted.

The term heteroarylalkyl, as used herein, refers to a heteroaryl group, as defined herein, attached to the parent molecular moiety via an alkylene group, as defined herein.

The term heterocyclylalkyl, as used herein, refers to a heterocyclic group, as defined herein, attached to the parent molecular moiety via an alkylene group, as defined herein.

The terms hydroxyl or hydroxy, as used herein, mean an -OH group.

The term hydroxyalkyl, as used herein, means at least one -OH group attached to the parent molecular moiety via an alkylene group, as defined herein. In some cases, the number of carbon atoms in a hydrocarbon substituent (eg, alkyl or cycloalkyl) is designated by the prefix Cx-y-, with x representing the minimum number and y representing the maximum number of carbon atoms in the substituent. Thus, for example, C^alkyl refers to an alkyl substituent containing from 1 to 3 carbon atoms.

The term substituted refers to a group that may be further substituted by one or more substituent groups other than hydrogen. Substituent groups include, but are not limited to, halogen, =O(oxo), =S(thioxo), cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl , heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfonylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl , -COOH, ketone, amide, carbamate and acyl.

The term bH=l· means a single bond (—) or a double bond (=) or a triple bond (^=).

In the case of the compounds described herein, the groups and their substituents can be selected in accordance with the permissible valency of the atoms and substituents so that the selected groups and substituents result in a stable compound, for example, which does not undergo spontaneous transformation, such as by rearrangement, cyclization, elimination, etc. The terms administration or administration as used herein refer to providing, contacting and/or delivering a compound or conjugate via any suitable route to achieve the desired effect. These compounds or conjugates can be administered to a subject in several ways, including, but not limited to, orally, ophthalmic, nasally, intravenously, topically, aerosol, suppository, etc., and can be used in combination. As used herein, cancer may include any cell or tissue derived from a tumor, neoplasm, cancer cells, precancerous tissue, cell line, malignancy, or any other source of cells that has the potential to spread and grow to an unlimited extent. Cancer cells can be obtained from naturally occurring sources or can be created artificially. Cancer cells may also be capable of invading other tissues and metastasizing. Cancer cells additionally

- 7 046205 include any malignant cells that grow into other tissues and/or metastasize. One or more cancer cells within an organism may also be referred to as a cancer, tumor, neoplasm, tumor growth, malignancy, or any other term used in the art to describe cells in a cancerous state. Cancer may include, for example, melanoma, brain cancer, lung cancer, adrenal cancer, liver cancer, kidney cancer, pancreatic cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, anal cancer, prostate cancer, ovarian cancer, breast cancer, cervical cancer, lymphoma, leukemia, myeloma, hematological malignancy, hepatocellular carcinoma, retinoblastoma, glioma, sarcoma, blastoma, squamous cell carcinoma and adenocarcinoma.

The terms control, reference level, and benchmark are used interchangeably in this document. The reference level may be a predetermined value or range that is used as a reference point against which a measured result is evaluated. A control group in the context of this document refers to a group of control subjects. The predetermined level may represent a cutoff value relative to a control group. The predetermined level may be the average of the control group. Boundary values (or predefined boundary values) can be determined using the adaptive forecasting model (AIM) technique. Breakpoints (or predefined cutoff values) can be determined by analyzing the sensitivity versus false positive rate (ROC) curve in biological samples from a patient population. ROC analysis, which is generally known in the field of biology, is a determination of the ability of an analysis to distinguish one condition from another. A description of the ROC analysis is presented in PJ. Heagerty et al. (Biometrics 2000, 56, 337-44), the disclosure of which is hereby incorporated by reference in its entirety. Alternatively, cutoff values can be determined using quartile analysis of biological samples from a cohort of patients. For example, a cutoff value may be determined by selecting a value that corresponds to any value in the range of the 25th to 75th percentile, preferably a value that corresponds to the 25th percentile, the 50th percentile, or the 75th percentile, and more preferably , 15th percentile. Such statistical analyzes can be performed using any method known in the art, and they can be implemented in any number of commercially available software packages (for example, from Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station, Texas, USA; SAS Institute Inc., Cary, North Carolina, USA). Healthy levels or normal levels or ranges for a target or for protein activity can be determined in accordance with standard practice. The control may be a subject whose disease condition is known or a sample from it. The subject may be healthy, diseased, diseased before treatment, diseased during treatment, diseased after treatment, or healthy after treatment, or a combination of these conditions, or the sample may be a sample from a subject who is healthy, diseased, diseased before treatment, diseased during treatment, sick after treatment, or healthy after treatment, or may be characterized by a combination of these conditions. The term normal subject as used in the context of this document means a healthy subject, i.e. a subject who does not have clinical signs or symptoms of the disease. The normal subject undergoes clinical evaluation for otherwise undetectable signs or symptoms of disease, which evaluation may include a routine physical examination and/or laboratory testing. In accordance with some embodiments, the control is a healthy control. In accordance with some embodiments, the control comprises a cancerous tumor.

The terms effective dose, or therapeutic dose, or therapeutically effective amount, or effective amount, as used herein, mean an amount sufficient to provide a therapeutic effect, or a dose of drug effective for periods of time necessary to achieve the desired therapeutic effect. The effective dose can be determined by one skilled in the art and may vary depending on factors such as the disease state, age, sex and weight of the individual, route of administration, stage and severity of the disease, general health of the subject, the judgment of the attending physician and ability drug to provide the desired response in an individual. A therapeutically effective amount is also an amount at which the therapeutically beneficial effects are greater than any toxic or harmful effects of the substance. A prophylactically effective amount refers to an amount effective to achieve the desired prophylactic result at the required dosages and for the required periods of time. Generally, since the prophylactic dosage is administered to subjects pre-disease or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The terms inhibit or inhibition mean that activity is reduced or prevented in the presence of an inhibitor, as opposed to the absence of an inhibitor. The term inhibiro

- 8 046205 refers to the reduction or down-regulation of a process or the removal of a stimulus to a process, resulting in the absence or minimization of expression or activity of a biological marker or polypeptide. Inhibition can be direct or indirect. Inhibition may be specific, that is, the inhibitor inhibits a biological marker or polypeptide and not others.

A sample or test sample as used herein can mean any sample in which the presence and/or level of a compound or target is to be detected or determined. Samples may include liquids, solutions, emulsions, mixtures or suspensions. The samples may include a medical sample. Samples may include any biological fluid or tissue such as blood, whole blood, blood fractions such as plasma and serum, peripheral blood mononuclear cells (PBMC), muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal discharge, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, vomit, feces, lung tissue, peripheral blood mononuclear cells, total leukocytes, lymph node cells, spleen cells, palatal cells tonsils, cancer cells, tumor cells, bile, digestive juices, skin or combinations thereof. In accordance with some embodiments, the sample contains an aliquot. In other embodiments, the sample comprises a biological fluid. Samples can be obtained using any means known in the art. The sample may be used directly after collection from the patient, or it may be subjected to pre-treatment, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, etc., to modify the properties of the sample in some way, as discussed herein, or otherwise as known in the art. Samples may be obtained before diagnosis, before treatment, during treatment, after treatment, or after diagnosis, or at several of these points.

The term specificity as used in this document refers to the number of true negatives divided by the number of true negatives plus the number of false positives, where specificity (spec) may be within the range 0<spec<1. Therefore, a method that has both a sensitivity and specificity of one or 100% is preferred.

By specifically binding, it is generally meant that a compound or conjugate binds to a target when it binds to the target more readily than it would bind to a random unbound target. Subject, as used herein, may mean a mammal that desires or needs the compounds or methods described herein. The subject may be a human or a non-human animal. The subject may be a mammal. The mammal may be a primate or a non-primate mammal. The mammal may be a primate such as a human; a non-primate mammal such as, for example, dog, cat, horse, cow, pig, mouse, rat, camel, llama, goat, rabbit, sheep, hamster and guinea pig; or a non-human primate such as, for example, a monkey, chimpanzee, gorilla, orangutan and gibbon. The subject may be of any age or developmental stage, such as an adult, adolescent, or infant. The subject may be male or female. In accordance with some embodiments, the subject has a particular genetic marker.

As used herein, the term toxic refers to the amount of a chemical molecule, agent, or substance that will be harmful to a subject or cause any adverse effect. The term non-toxic refers to a substance that is characterized by a relatively low degree to which it can cause harm to a subject. Cytotoxic refers to a chemical molecule, medium, or substance that is toxic to cells. Toxicity can refer to effects on an entire organism, such as an animal, bacterium, plant, or other entity as defined herein, as well as effects on a structure of the body, such as a cell (cytotoxicity) or an organ (organotoxicity), such as the liver (hepatotoxicity). A basic concept in toxicology is that effects are dose-dependent; even water can cause water intoxication when taken in large enough doses, while even for a very toxic substance such as snake venom, there is a dose below which there is no detectable toxic effect. A composition or compound that is relatively non-toxic may provide a wider range of subjects to which the composition or compound can be safely administered without significant safety concerns or risks.

The terms treating, receiving treatment, or administering treatment, as used herein, refer to a therapeutic intervention in which the goal is to slow down (alleviate) an undesirable physiological condition, disorder, or disease, or to produce beneficial or desired clinical results. In the context of the present invention, beneficial or desired clinical results include, without limitation, relief

- 9 046205 symptoms; reduction in the severity of a condition, disorder or disease; stabilization (i.e., no worsening) of a condition, disorder, or disease; delaying the onset or slowing the progression of a condition, disorder or disease; relief of a condition, disorder or disease state and remission (either partial or complete), either detectable or undetectable, or aggravation or improvement of the condition, disorder or disease. Treatment also involves prolonging life compared to what would have been expected without receiving treatment. Treating or administering a treatment, in relation to protecting a subject from a disease, means containing, suppressing, reducing or completely eliminating a disease. Prevention of disease involves administering a composition of the present invention to a subject before the onset of disease. Disease control involves administering a composition of the present invention to a subject after the induction of the disease but before its clinical manifestation. Suppression or amelioration of a disease involves administering a composition of the present invention to a subject after clinical manifestation of the disease. The disease may be cancer.

2. Phospholipid esters.

Phospholipid ester (PLE) molecules are presented herein. PLE may correspond to Formula I or be a salt thereof

O CH ₃

X-(CH ₂ ) _1E -O—p—O-(CH ₂ ) ₂ -N^-CH ₃

Jo I wherein X represents hydrogen, methyl or phenyl substituted with carboxyl. In accordance with some embodiments, the PLE is selected from the following

About SNz

II I®

H ₃ s—(CH _g ) _1E -O-R-O-(CH2) ₂ -M-CH ₃

I© I

About CHz (6).

The PLE may be conjugated to a detectable moiety (also called a reporter or tag), such as, for example, a fluorescent molecule, a chemiluminescent molecule, a radioisotope label, a magnetic label, an infrared detectable molecule, or a combination thereof. Magnetic tags are labeling molecules that, when sufficiently bound to a magnetic proximity sensor, can be detected by the magnetic proximity sensor and cause the magnetic proximity sensor to emit a signal. Magnetic tags may include one or more materials selected from paramagnetic, superparamagnetic, ferromagnetic, antiferromagnetic materials, combinations thereof, and the like. Fluorescent tags are labeling fragments that can be detected using a fluorescence detector. Suitable fluorescent molecules (fluorophores) include, but are not limited to, fluorescein, fluorescein isothiocyanate, carboxyfluorescein succinimidyl esters, fluorescein succinimidyl esters, fluorescein dichlorotriazine 5-isomer, locked carboxyfluorescein-alanine carboxamide, Oregon Green 488, ore rut green 514; Lucifer yellow, acridine orange, rhodamine, tetramethylrhodamine, Texas red, propidium iodide, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazoylcarbocyanine iodide), tetrabromorhodamine 123, rhodamine 6G , TMRM (tetramethylrhodamine methyl ester), TMRE (tetramethylrhodamine ethyl ester), tetramethylrosamine, rhodamine B and 4-dimethylaminotetramethylrosamine, green fluorescent protein, blue-shifted green fluorescent protein, blue-shifted green fluorescent protein spectrum, green fluorescent protein with a fluorescence shift to the red region of the spectrum, green fluorescent protein with a fluorescence shift to the yellow region of the spectrum, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid; acridine and derivatives such as acridine, acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-1sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate; N(4-anilino-1-naphthyl)maleimide; anthranilamide; 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-5-indacene-3propionic acid BODIPY; cascade blue; brilliant yellow; coumarin and derivatives: coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120), 7-amino-4-trifluoromethylcoumarin (coumarin 151); cyanine dyes; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5',5dibromopyrogallol-sulfonaphthalein (bromopyrogallol red); 7-diethylamino-3-(4'isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5(dimethylamino)naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); 4-dimethylaminophenylazophenyl-4'

- 10 046205 isothiocyanate (DABITC); eosin and derivatives: eosin, eosin isothiocyanate, erythrosine and derivatives: erythrosin B, erythrosine, isothiocyanate; ethidium; fluorescein and derivatives: 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)amino-1-fluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6carboxyfluorescein ( JOE), fluorescein, fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; malachite green isothiocyanate; 4-methylumbelli-feronorthocresolphthalein; nitrotyrosine; pararosaniline; phenol red; B-phycoerythrin; o-phthalic dialdehyde; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl-1-pyrene; butyrate quantum dots; reactive red 4 (Cibacron™ brilliant red 3B-A), rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxy-rhodamine (R6G), lissamine rhodamine B sulfonyl chloride-rhodamine (Rhod), rhodamine B, rhodamine 123 , rhodamine X-isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas red); HHNa,Na-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethylrhodamine; tetramethylrhodamine isothiocyanate (TRITC); riboflavin; 5-(2'aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid; CAL Fluor Orange 560; chelated terbium derivatives; Su 3; Su 5; Su 5.5; Su 7; IRD 700; IRD 800; La Jolla Blue; phthalocyanine and naphthalocyanine, coumarins and related dyes, xanthene dyes such as rhodoles, resorufins, bimanes, acridines, isoindoles, dansyl dyes, aminophthalic acid hydrazides such as luminol and isoluminol derivatives, aminophthalimides, aminonaphthalimides, aminobenzofurans, aminoquinolines, dicyanohydroquino us, fluorescent complexes europium and terbium; their combinations, etc. The detectable fragment may be covalently linked to PLE or may be connected to PLE by a cleavable linker. For example, the labeled PLE can be selected from the following (CH ₂ } ₁₃ -0·

CLR 1501 (1) and

CLR 1502 (2)

The above compound (1) is PLE (3) in which the fluorescent moiety, BODIPY, is stably associated with PLE and may also be named CLR 1501. The above compound (2) is PLE (3) in which the molecule detected in near-infrared, IR-775, is stably bound to PLE and may also be called CLR 1502. Compounds (1) and (2) may also be referred to as phospholipid-drug conjugate (PDC).

PLE or a conjugate thereof may be specific for a tumor or cancer cell. Once administered to a subject, PLE or its conjugate may localize to a tumor or cancer cell. PLE or its conjugate may be more actively incorporated into a tumor or cancer cell than into a healthy cell. PLE or a conjugate thereof may be included at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold. multiple, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20-fold, at least about 25-fold, at least about 30-fold greater number of tumor or cancer cells compared to healthy cells.

3. Phospholipid-flavaglin conjugates.

- 11 046205

PLE can be conjugated via a linker to a flavaglin compound to form a phospholipid-flavaglin conjugate (also called a PLE-flavaglin conjugate). The conjugate may correspond to Formula II or be a salt thereof

is a linker containing a disulfide; and Z is a flavaglin anticancer drug. The Formula II conjugate may also be referred to as a phospholipid drug conjugate (PDC).

Flavaglins are a family of natural products found in plants of the genus Aglaia (Meliaceae). Flavaglins have a cyclopenta-benzofuran backbone. Flavaglins may exhibit potent insecticidal, antifungal, anti-inflammatory, neuroprotective, cardioprotective, and anticancer activities. Flavaglins may enhance the effectiveness of chemotherapy and/or mitigate the adverse effects of chemotherapy on the heart. Flavaglins may include, for example, FLV1, FLV3, a derivative or analogue thereof, or a combination thereof.

In some embodiments, flavaglin comprises FLV1 or a salt thereof

FLV1 (4).

In some embodiments, flavaglin contains FLV3 or a salt thereof

The flavaglin compound can exist as a stereoisomer in which asymmetric or chiral centers are present. The stereoisomer is an R- or S-form depending on the configuration of the substituents relative to the chiral carbon atom. The terms R and S used in the context of this document represent configurations as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, in Pure Appl. Chem., 1976, 45, 13-30. Various stereoisomers and mixtures thereof are provided by the present disclosure and are specifically included within the scope of the present invention. Stereoisomers include enantiomers and diastereomers, as well as mixtures of enantiomers or diastereomers. Individual stereoisomers of compounds can be obtained by synthesis from commercially available starting materials that contain asymmetric or chiral centers, or by preparing racemic mixtures, following resolution methods well known to one skilled in the art. Examples of these separation methods are (1) attaching a mixture of enantiomers to a chiral auxiliary, separating the resulting mixture of diastereomers by recrystallization or chromatography, and optionally separating the optically pure product from the auxiliary, as described in Furniss, Hannaford, Smith, and Tatchell, Vogel's Textbook of Practical Organic Chemistry, 5th edition (1989), Longman Scientific & Technical, Essex CM20 2JE, England, or (2) direct

- 12 046205 separation of a mixture of optical enantiomers on columns for chiral chromatography, or (3) fractional recrystallization methods. It should be understood that the flavaglin compound may have tautomeric forms as well as geometric isomers, and that these also constitute embodiments of the present disclosure. The linker may be a cleavable linker, such as a disulfide, and is specifically designed to deliver flavaglin to a tumor or cancer cell. In accordance with some embodiments, the linker contains a disulfide. In accordance with some embodiments, the linker comprises the following

N ₃ with sn _{3 (7)}

In accordance with some embodiments, the phospholipid-flavaglin conjugate is selected from the following:

CLR 1852 (with FLV3) (9)

The present disclosure also includes an isotope-labeled compound, such as isotope-labeled PLE, isotope-labeled flavaglin, isotope-labeled linker, or isotope-labeled phospholipid-flavaglin conjugate. An isotopically labeled compound is identical to the compounds detailed herein, except for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes suitable for inclusion in the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as, but not limited to, ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁸ O, ¹⁷ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl, respectively. Replacement with heavier isotopes such as deuterium, i.e. ^2H may provide certain therapeutic benefits resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances. The compound may include positronic isotopes for medical imaging and positron emission tomography (PET) studies to determine receptor distribution. Suitable positron-active isotopes that can be included in the compounds are 11C, ^13N , ^15O and ^18F . Isotopically labeled compounds can generally be prepared by conventional techniques known to those skilled in the art or by methods similar those described in the accompanying examples, using the appropriate isotope-labeled reagent in place of the non-isotope-labeled reagent.

The disclosed PLE or phospholipid-flavaglin conjugate may exist as pharmaceutically acceptable salts. The term pharmaceutically acceptable salt refers to salts or zwitterions of compounds that are soluble or dispersible in water or oil, suitable for the treatment of disorders without undue toxicity, irritation and allergic reaction commensurate with a reasonable benefit/risk ratio and effective for their intended use. Salts can be obtained during the final isolation and purification of the compounds or separately by reacting the amino group of the compounds with a suitable acid. For example, the compound can be dissolved in a suitable solvent, such as, but not limited to, methanol and water, and treated with at least one equivalent of an acid like hydrochloric acid. The resulting salt can be precipitated and isolated by filtration and dried under reduced pressure. Alternatively, the solvent and excess acid can be removed under reduced pressure to ensure

- 13 046205 salt. Typical salts include acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, isethionate, fumarate, lactate, maleate, methanesulfonate , naphthylene sulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, oxalate, maleate, pivalate, propionate, succinate, tartrate, thiochloroacetate, trifluoroacetate, glutamate, paratoluenesulfonate, undecanoate, hydrochloric acid salt, hydrobromic acid salt, salt sulfuric acid, phosphoric acid salt, etc. The amino groups of the compounds can also be quaternized with alkyl chlorides, bromides and iodides such as methyl, ethyl, propyl, isopropyl, butyl, lauryl, myristyl, stearyl and the like. Base addition salts can be prepared during the final isolation and purification of the disclosed compounds by reacting a carboxyl group with a suitable base, such as the hydroxide, carbonate or bicarbonate of a metal cation such as lithium, sodium, potassium, calcium, magnesium or aluminum, either primary, secondary or tertiary organic amine. Quaternary ammonium salts can be prepared such as those obtained from methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,Ndimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine, dibenzylamine, N.N-dibenzylphenethylamine. 1-ephenamine and N.N'-dibenzylethylenediamine. ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.

The phospholipid-flavaglin conjugate may be specific for a tumor or cancer cell. Once administered to a subject, the phospholipid-flavaglin conjugate may localize to the tumor or cancer cell. The phospholipid-flavaglin conjugate can localize or translocate into the cytoplasm or organelle of a tumor or cancer cell. The phospholipid-flavaglin conjugate may be more actively incorporated into a tumor or cancer cell than into a healthy cell. The phospholipid-flavaglin conjugate may be incorporated at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold , at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20-fold, at least about 25-fold, at least about 30-fold greater number of tumor or cancer cells compared to healthy cells.

Flavaglin may be cleaved from PLE, such as due to in vivo cleavage following administration to a subject. Flavaglin can be localized or translocated into the cytoplasm or organelle of a tumor or cancer cell. Flavaglin may be more actively incorporated into a tumor or cancer cell than into a healthy cell. Flavaglin may be included at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25-fold, at least about 30-fold more tumor or cancer cells compared to healthy cells.

A. Synthesis.

PLE can be prepared by synthesis in accordance with Example 1. Alternatively, PLE, which is described in detail herein, can be prepared by synthesis using methods known to one skilled in the art. Flavaglin can be produced by synthesis in accordance with Example 1. Alternatively, flavaglin, which is described in detail herein, can be obtained by synthesis using methods known to one skilled in the art. Flavaglins are also commercially available, for example from Haoyuan Chemexpress Co. (Shanghai, China). The PLE-flavaglin conjugate can be prepared by synthesis in accordance with Example 1. Alternatively, the PLE-flavaglin conjugate, which is described in detail herein, can be prepared by synthesis using methods known to one skilled in the art.

4. Pharmaceutical compositions.

PLE and phospholipid-flavaglin conjugates, which are described in detail herein, can be formulated into pharmaceutical compositions in accordance with standard techniques well known to those skilled in the pharmaceutical field. The composition may contain a compound (such as PLE or a phospholipid-flavaglin conjugate) and a pharmaceutically acceptable carrier. The term pharmaceutically acceptable carrier as used herein means a non-toxic, inert solid, semi-liquid or liquid excipient, diluent, encapsulating material or formulation aid of any type.

The route by which the disclosed PLE and phospholipid-flavaglin conjugates are administered and the form of the composition will dictate the type of carrier used. The pharmaceutical composition may be present in a number of forms suitable, for example, for systemic administration (e.g. oral, rec

- 14 046205 oral, sublingual, buccal, implant, intranasal, intravaginal, transdermal, intravenous, intraarterial, intratumoral, intraperitoneal or parenteral) or local administration (e.g. dermal, pulmonary, nasal, auricular, ocular, in delivery systems based on liposomes or by iontophoresis). In accordance with some embodiments, the pharmaceutical composition is for administration to the central nervous system of a subject. Methods and formulations can generally be found in Remington's Pharmaceutical Sciences, (Meade Publishing Co., Easton, Pa.). Pharmaceutical compositions generally must be sterile and stable under the conditions of manufacture and storage. All media in the compositions are optional.

Pharmaceutically acceptable carriers include, for example, diluents, lubricants, binders, disintegrating agents, coloring agents, flavoring agents, sweeteners, antioxidants, preservatives, lubricants, solvents, suspending agents, wetting agents, surfactants, emollients, propellant gases, wetting agents, powders, pH regulators and combinations thereof. Although the amounts of components in the compositions may vary depending on the preferred type of composition, typically systemic compositions may include from 0.01 to 50% of the compound (such as PLE or phospholipid-flavaglin conjugate) and from 50 to 99.99% to one or more media. Compositions for parenteral administration may generally include from 0.1 to 10% of the compound and from 90 to 99.9% of one or more carriers. Oral dosage forms may include, for example, at least about 5% or from about 25% to about 50% of the compound. Compositions in oral dosage form may include from about 50% to about 95% carriers or from about 50% to about 75% carriers. The amount of carrier used in conjunction with the disclosed compound is sufficient to provide a practical amount of the composition for administration at a unit dose of the compound. Methods and compositions for preparing dosage forms useful in the methods of the present invention are described in the following references: Modern Pharmaceutics, Chapters 9 and 10, Banker & Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms: Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms, 2nd Ed., (1976).

5. Introduction.

PLE or phospholipid-flavaglin conjugate or pharmaceutical compositions containing them can be administered to a subject. Such compositions containing a compound (such as PLE or a phospholipid-flavaglin conjugate) can be administered in dosages and by techniques that are well known to those skilled in the art of medicine, taking into account factors such as the age, sex, weight and condition of the particular subject and route of administration.

A compound (such as PLE or a phospholipid-flavaglin conjugate) can be administered for prophylactic or therapeutic purposes. When administered prophylactically, the compound may be administered in an amount sufficient to induce a response. For therapeutic applications, the compounds are administered to a subject in need thereof in an amount sufficient to provide a therapeutic effect. An amount sufficient to achieve this is defined as a therapeutically effective amount. Amounts effective for this use will depend, for example, on the particular schedule of administration of the compound composition, the route of administration, the stage and severity of the disease, the general health of the patient and the judgment of the attending physician. A therapeutically effective amount is also an amount at which the therapeutically beneficial effects outweigh any toxic or harmful effects of the compound. A prophylactically effective amount refers to an amount effective to achieve the desired prophylactic result at the required dosages and for the required periods of time. Generally, since the prophylactic dosage is administered to subjects pre-disease or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

For example, a therapeutically effective amount of a compound may be from about 1 mg/kg to about 1000 mg/kg, from about 5 mg/kg to about 950 mg/kg, from about 10 mg/kg to about 900 mg/kg, from about 15 mg/kg to about 850 mg/kg, from about 20 mg/kg to about 800 mg/kg, from about 25 mg/kg to about 750 mg/kg, from about 30 mg/kg to about 700 mg/kg, from about 35 mg/kg to about 650 mg/kg, about 40 mg/kg to about 600 mg/kg, about 45 mg/kg to about 550 mg/kg, about 50 mg/kg to about 500 mg/kg , from about 55 mg/kg to about 450 mg/kg, from about 60 mg/kg to about 400 mg/kg, from about 65 mg/kg to about 350 mg/kg, from about 70 mg/kg to about 300 mg /kg, from about 75 mg/kg to about 250 mg/kg, from about 80 mg/kg to about 200 mg/kg, from about 85 mg/kg to about 150 mg/kg, and from about 90 mg/kg to about 100 mg/kg.

The compound can be administered using methods well known in the art and described in Donnelly et al. (Ann. Rev. Immunol. 1997, 75, 617-648); Feigner et al. (U.S. Patent No. 5,580,859 issued

- 15 046205 December 3, 1996); Feigner (US Patent No. 5,703,055, issued December 30, 1997); and Carson et al. (U.S. Patent No. 5,679,647, issued Oct. 21, 1997), each of which is incorporated herein by reference in its entirety. The compound can form complexes with particles or granules, which can be administered to an individual, for example, using a needleless injector. One skilled in the art will recognize that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration. The compound can be delivered through various routes. Typical routes of delivery include parenteral administration, such as intradermal, intramuscular or subcutaneous delivery. Other routes include oral, intranasal, intravaginal, transdermal, intravenous, intraarterial, intratumoral, intraperitoneal and epidermal routes. In some embodiments, the compound is administered to the subject intravenously, intra-arterially, or intraperitoneally. In some embodiments, the compound is administered intravenously to a subject. In accordance with some embodiments, the compound is administered to the subject orally. In accordance with some embodiments, the compound is administered in a controlled release formulation. For example, the compound may be released into the bloodstream. In some embodiments, the compound may be released over a period of at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 1 week, at least about 1.5 weeks, at least about 2 weeks, at least about 2.5 weeks, at least about 3 .5 weeks, at least about 4 weeks, or at least about 1 month. The compound may be administered as a single dose, or episodic, or multiple doses. For example, the compound can be administered once every hour, 2, 4, 8, 12, 25, 36 hours, 2, 3, 4, 5, 6 days, 1, 2, 3 or 4 weeks.

6. Methods.

a. Methods of treating cancer in a subject.

Described herein are methods of treating cancer in a subject. The method may include administering to the subject a phospholipid-flavaglin conjugate, which is described in detail herein. In some embodiments, the cancer is selected from melanoma, brain cancer, lung cancer, adrenal cancer, liver cancer, kidney cancer, pancreatic cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, anal cancer duct, prostate cancer, ovarian cancer, breast cancer, cervical cancer, lymphoma, leukemia, myeloma, hematological malignancy, hepatocellular carcinoma, retinoblastoma, glioma, sarcoma, blastoma, squamous cell carcinoma and adenocarcinoma.

b. Methods of targeting a drug to a tumor or cancer cell in a subject.

Provided herein are methods of targeting a drug to a tumor or cancer cell in a subject. The method may include administering to the subject a phospholipid-flavaglin conjugate, which is described in detail herein. In some embodiments, the cancer is selected from melanoma, brain cancer, lung cancer, adrenal cancer, liver cancer, kidney cancer, pancreatic cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, anal cancer duct, prostate cancer, ovarian cancer, breast cancer, cervical cancer, lymphoma, leukemia, myeloma, hematological malignancy, hepatocellular carcinoma, retinoblastoma, glioma, sarcoma, blastoma, squamous cell carcinoma and adenocarcinoma.

- 16 046205

Examples

Example 1. Chemical synthesis.

PLE.

PLE was prepared by synthesis according to Scheme 1 below

Scheme 1

O

CLR1410

Flavaglins.

Flavaglins were available from a commercial supplier. FLV1 and FLV3 were purchased from Haoyuan Chemexpress Co. (Shanghai, China).

Alternatively, FLV1 and FLV3 were synthesized according to Scheme 2 below

Scheme 2

FLV-L

Code S.M. Weight Product Conditions Exit Notes 1 13 0.317 g FLV3 50°C, 10 hours 0.235 g (78%) corresponds to 'H NMR 2 eleven 0.170 g 12 from 0°С to rt, O/N -- reaction continues

PLE-flavaglin constructs. CLR 1852 (compound (9)) and CLR 1865 (compound (8)) were prepared by synthesis according to Scheme 3 below

- 17 046205

Scheme 3

Example 2. Presence of lipid rafts on tumor cells.

More than 100 cell lines were stained with cholera toxin subunit B, fixed with 4% formaldehyde, and stained with filipin III for 30 min. As shown in FIG. Ία-ID, almost every tumor type studied showed a high concentration of lipid rafts in the cell membrane (in 100 cell lines, fresh patient samples, etc.). As shown in FIG. 1E, A549 cells were cocultured with normal fibroblasts for 48 h and then stained with cholera toxin subunit B, fixed with 4% formaldehyde, and stained with filipin III for 30 min. These results demonstrated that tumor cells have a higher concentration of lipid rafts than normal cells.

Example 3 Selective uptake of PDC into tumor cells.

Normal fibroblasts and Caki-2 (human clear cell renal cell carcinoma) tumor cells were seeded and cocultured overnight (Fig. 1F). Cells were then incubated with 5 μM CLR 1501 (compound (1)) for 24 h at 37°C in complete medium. The next day, cells were washed and co-stained with nuclear dye (Hoescht 33342). CLR 1501 fluorescence was excited and then detected using a filter for Alexa-Fluor 488. CLR 1501 localized at high levels in Caki-2 cells and at minimal levels in normal fibroblasts.

Example 4 Disruption of lipid rafts reduced PDC uptake.

A549 cells were seeded overnight in individual wells. The next day, cells were either left untreated (Fig. 1G) or treated with methyl L-cyclodextrin (Fig. 1H), which has been shown to selectively disrupt lipid rafts. All cells were then incubated for 24 h with 5 μM CLR 1501 (compound (1)). Disruption of most lipid rafts in A549 cells resulted in a 60% reduction in CLR 1501 uptake (Fig. 1H) compared to untreated cells (Fig. 1G).

Example 5: PDCs are tracked in the endoplasmic reticulum.

Human prostate adenocarcinoma (PC3) cells were seeded overnight on microplate VI (Ibidi, Verona, WI, USA) and then incubated with 5 μM CLR 1501 (compound (1)) for 24 h at 37°C in full environment. After washing, cells were co-stained using ER-tracker® according to the protocol and imaged using a Nikon A1R confocal light microscope. CLR 1501 and ER were excited for fluorescence and detection was performed using Alexa-Fluor 488 using standard fluorescein filters. CLR 1501 colocalized with the ER in malignant (Fig. 1I-1K) but not in normal cells (not shown).

Example 6: PDCs are monitored in the endoplasmic reticulum.

The PC3 cell line (human prostate adenocarcinoma stage IV) was cultured overnight on microscopy slide VI (Ibidi, Verona, WI, USA). The next day, cells were incubated with 5 μM CLR 1501 (compound (1)) for 24 h at 37°C in complete medium. The next day, after washing with PBS, cells were co-stained with a nuclear dye (Hoechst 33342) and a mitochondrial marker (Mitotracker®) (Invitrogen, Carlsbad, CA, USA). Cells were observed using a Nikon AIR confocal microscope. CLR 1501 fluorescence was excited and detected using an Alexa filter

- 18 046205

Fluor 488, while nuclear dye and mitochondrial dye fluorescence were excited and detected using a DAPI filter and a Texas red filter, respectively. CLR 1501 was localized to mitochondria (Fig. 1L-1N).

Example 7 PDCs provide targeted delivery in vivo.

Colon tumor-bearing nude mice (HCT-116) were injected with 1 mg CLR 1502 (compound (2)) and imaged using the Pearl Infrared Imaging System. The different colors reflect the intensity of CLR 1502 over time. Approximately 5.5 hours after injection, the tumor still showed a red color (reflecting the highest prevalence of CLR 1502) (Fig. 2). Maximum distribution of CLR 1502 was obtained within 24 hours. Initial targeting was observed within 30 minutes (not shown).

Example 8 Cytotoxic PDCs Provide Targeting and Potentially Improved Therapeutic Index.

A549 cells (human lung adenocarcinoma) and normal human skin fibroblasts (NHDF) were seeded in 96-well dishes overnight. All cells were treated with increasing concentrations of either the parent cytotoxic compound alone (FLV1 or FLV3) or PDC (the parent cytotoxic compound conjugated to a PLE moiety using a cleavable linker, CLR 1865 (compound (8)) or CLR 1852 (compound (9)) ). The parent cytotoxic compound demonstrated activity against A549 cells that was almost equal to that against NHDF cells. However, PDC molecules showed selectivity for A549 cells (Fig. 3). The PDC molecules showed virtually no effect on NHDF cells up to the highest concentrations and showed activity essentially similar to that of the parent molecule in A549 cells. The difference between the cytotoxicity of the PDC molecule towards tumor cells and the cytotoxicity towards normal cells may indicate the potential to improve the therapeutic index of the parent molecules.

Example 9 Cytotoxic PDCs provide targeting.

The uptake of CLR 1852 (compound (9)) in A375 cells (human melanoma) and HEK293 cells (human embryonic kidney) was assessed. Cells were incubated with CLR 1852 (compound (9)) for 24 hours. It was shown that tumor cells in all cases had a 6-28-fold increase in PDC content compared to normal cells within 24 hours of treatment (Fig. 4).

The results from Examples 2-9 indicated that phospholipid ester molecules target tumor cells through lipid rafts. PDCs showed significant uptake into tumor cells compared to normal cells even in co-culture. Upon entry into tumor cells, PDCs were monitored in mitochondria and endoplasmic reticulum. In vivo, PDCs both targeted and rapidly accumulated in the tumor. Cytotoxic PDCs provided improved targeting and potential for improved safety.

Example 10: Uptake of PLE by Cells.

Various cancer cell lines were exposed in vitro and in vivo to fluorescently labeled PLE (CLR 1501, compound (1)). Uptake by tumor cells was measured continuously over 24 hours. The results are presented in table. 1 and Fig. 5, 6 and 7. The PLE compound was specific for tumors and cancer cells.

Fluorescently labeled PLE (CLR 1501, compound (1)) was injected into benign tissues and no uptake was observed (Table 2).

Table 1

Uptake of PLE by cells in vitro

Model tumor View Category Uptake into tumor 1 PC-3 prostate SCID mouse Adenocarcinoma Eat 2 A-549 lung (NSCLC) SCID mouse Adenocarcinoma Eat 3 NCI H-69 lung (oat cells) SCID mouse Adenocarcinoma Eat 4 H-295 adrenal gland SCID mouse Adenocarcinoma Eat 5 RL-251 adrenal gland SCID mouse Adenocarcinoma Eat 6 Colon-51 SCID mouse Adenocarcinoma of the colon and rectum Eat

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7 LSI80 colon SCID mouse Adenocarcinoma of the colon and rectum Eat 8 Colon DLDI SCID mouse Adenocarcinoma of the colon and rectum Eat 9 NT-29 colon SC1D mouse Adenocarcinoma of the colon and rectum Eat 10 LS-180 colon naked mouse Adenocarcinoma Eat AND A-375 melanoma naked mouse Adenocarcinoma Eat 12 NTV-77 ovary naked mouse Adenocarcinoma Eat 13 VHRS of the pancreas naked mouse Adenocarcinoma Eat 14 Sarai-1 pancreas naked mouse Adenocarcinoma Eat 15 renal cell Caki-2 "naked" mouse (orthotopic) Adenocarcinoma Eat 16 renal cell ACHN "naked" mouse (orthotopic) Adenocarcinoma Eat 17 SCC1 naked mouse Squamous cell carcinoma Eat 18 SCC6 naked mouse Adenocarcinoma Eat 19 LnCap prostate Mouse Adenocarcinoma Eat 20 LuCap prostate Mouse Adenocarcinoma Eat 21 MCF-7 breast Rat Adenocarcinoma Eat 22 4T1 breast Endogenous murine (orthotopic) Adenocarcinoma Eat

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23 MatLyLu prostate gland Rat Adenocarcinoma Eat 24 Walker-256 Rat Carcinosarcoma Eat 25 TRAMP prostate Endogenous murine Adenocarcinoma Eat 26 ST-26 colon SCID mouse Adenocarcinoma Eat 27 Mouse intestine min Endogenous murine Adenocarcinoma Eat 28 Melanoma Mouse Adenocarcinoma Eat 29 SCC breast Apc ^Min/+ mouse Squamous cell carcinoma Eat thirty AS of the mammary gland Ars ^M1p/+ mouse Adenocarcinoma Eat 31 Hepatocellular carcinoma Endogenous murine Adenocarcinoma Eat 32 Glioma L9 Rat xenograft Glioma Eat 33 Glioma C6 Rat xenograft Glioma Eat 34 Glioma CNS1 Rat xenograft Glioma Eat 35 Glioma RG2 Rat xenograft Glioma Eat 36 Retinoblastoma Endogenous murine Blastoma Eat 37 S-shus of the pancreas Endogenous murine Adenocarcinoma Eat 38 Kras pancreas Endogenous murine Adenocarcinoma Eat 39 Cervix Endogenous murine Adenocarcinoma Eat 40 Sarcoma (Meth-A) naked mouse Fibrosarcoma Eat 41 Esophagus Endogenous murine Adenocarcinoma Eat

Table 2 Uptake of CLR1404 into benign tissues

Model tumor View Category Uptake into tumor 1 Intestinal polyp Endogenous murine Adenoma (benign) No 2 Alveolar hyperplasia of the mammary gland Endogenous murine Hyperplasia (benign) No

Example 11. Activity of PLE-flavaglin conjugates in cancer cells.

PLE-flavaglin conjugates CLR 1852 (compound (9)) and CLR 1865 (compound (8)) were introduced into cells of the A375 (human malignant melanoma), A549 (human lung adenocarcinoma), HCT 116 (human colon cancer) and NHDF ( normal human skin fibroblasts). IC ₅₀ was calculated. The results are presented in table. 3.

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Table 3

IC ₅₀ (µM/ml) in various cancer cell lines

Compound A375 A549 NST 116 NHDF Therapeutic index (TI) FLV3 0.015 0.09 0.009 0.008 0.89 CLR1852 0.9 0.02 0.06 7.2(18.3) 360 (915) CLR1865 9.75 0.67 1.28 7.8 (25) 11.64 (37.3)

The cytotoxicity of CLR 1852 was determined at various concentrations. As shown in FIG. 8, CLR 1852 showed a moderate loss of activity compared to FLV3 alone, possibly due to incomplete release. The stability of CLR 1865 (compound (8)), CLR 1852 (compound (9)) and FLV3 in blood plasma was assessed with propantheline as a control (Table 4). A small amount of the molecule was exposed to blood plasma, and the blood plasma was then analyzed using HPLC (high performance liquid chromatography) or MS (mass spectrometry) to determine whether the molecule had degraded. CLR 1865 and CLR 1852 showed excellent stability in human plasma. CLR 1852 was superior to CLR 1865 in mouse plasma. Only CLR 1865 was stable in mouse plasma for 3.3 hours. CLR 1852 was stable in mouse plasma for at least 7 hours.

Table 4 Stability in blood plasma

Compound Half-life in humans (min.) Half-life in mouse (min.) CLR1852 >400 >400 CLR1865 >400 199 Propantheline 54 85

The therapeutic index (TI) for CLR 1865 and CLR 1852 was assessed in mice (Table 5). In table Figure 5 shows the doses administered to the mice and the number of mice that remained alive after treatment (3/3 indicates that 3 out of 3 mice remained alive). Both CLR 1852 and CLR 1865 demonstrated excellent improvement in tolerability compared with FLV3 alone. CLR 1852 did not reach the maximum tolerated dose (MTD), possibly due to its solubility being limited by the higher dose. The MTD for CLR 1865 was 5 to 10 mg/kg. The in vivo therapeutic index was at least 25 for CLR 1852 and 12.5 for CLR 1865.

Table 5

Therapeutic index in vivo

Dose (mg/kg) FLV3 CLR 1852 CLR 1865 OD 3/3 3/3 3/3 0.25 3/3 3/3 3/3 0.5 LD 3/3 3/3 1.0 No data 3/3 3/3 5.0 No data 3/3 3/3 10.0 No data 3/3 2/3

The effectiveness of CLR 1852 in HCT 116 cells (human colon cancer) was assessed. CLR 1852 was introduced into HCT 116 cells in three doses of 1 mg/mg each. Compared to medium, CLR 1852 reduced tumor volume starting at approximately day 27 (Fig. 9). Compared to medium, CLR 1582 caused moderate weight loss, presumably due to toxicity (Fig. 10). CLR 1582 provided at least a sixfold increase in tolerability compared with FLV3 alone (data not shown).

CLR 1852 and FLV3 were separately introduced into A375 and A549 cells. FLV3 levels in cell lysate and growth medium were measured using LC/MS (liquid chromatography/mass spectrometry). As shown in FIG. 11, high levels of FLV3 were present in the cell lysate after 24 hours. Moderate levels of FLV3 were present in the growth medium after 24 hours. Intracellular FLV3 levels reached a plateau after 24 hours, while intracellular FLV3 levels continued to increase.

The foregoing description of specific aspects will be as complete as the description of other aspects when applying knowledge within the skill in the art, and can be easily modified and/or adapted to various applications of such specific aspects without undue experimentation, without departing from general idea

- 22 046205 of this disclosure. Accordingly, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects based on the information and guidance provided herein. It should be understood that statements or terminology herein are intended to be descriptive and not limiting, and therefore terminology or terminology herein should be interpreted by a qualified person in light of the information and guidelines.

The scope and scope of the present disclosure is not to be limited by any of the illustrative aspects described above, but is to be determined only in accordance with the following claims and their equivalents. All publications, patents, patent applications and/or other documents cited in this application are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication, patent, patent application and /or other document has been separately incorporated by reference.

For the sake of completeness, various aspects of the present invention are set forth in the following numbered paragraphs.

Item 1. Phospholipid ester (PLE) according to formula I or a salt thereof

About sn ₃

IX-(CH2) _la -OPO-(CH ₂ ) ₂ -N-CH ₃ ΙθI ° ^снз (I), where X represents hydrogen, methyl or phenyl substituted with carboxyl.

Clause 2. PLE according to clause 1, selected from the following „ OCH / \ and I®

NOOC—(U(CH ₂ ) ₁₈ -O-R-O-(CH ₂ ) ₂ -\-CH ₃ \ - / I©| ° CH ₃ (3)i

OSNZ

III® n ₃ s-(CH ₂ ) ₁₈ -o-p—o-(CH ₂ ) ₂ -N-SN ₃

I©

ABOUT

Clause 3. PLE according to clause 1 or clause 2, additionally containing a detectable fragment attached to it.

Clause 4. A composition containing PLE according to claim 1, or claim 2, or claim 3 and a carrier.

Item 5. Connection selected from the following

Clause 6. Composition containing the compound according to claim 5 and a carrier.

Clause 7. Conjugate according to formula II or a salt thereof

About sn ₃

Ζ---Υ---X--(CH ₂ ) _1a -O—p—O-(CH ₂ ) ₂ -N—CH ₃

Ιθ I

- 23 046205 where X represents

> or

or methylene or bond; Y is a disulfide containing linker; and Z is a flavaglin anticancer drug.

Claim 8. The conjugate according to claim 7, wherein the flavaglin anticancer drug contains FLV-1, FLV-3, a derivative or analogue thereof, or a combination thereof.

Claim 9. A conjugate according to any one of claims 7-8, wherein the linker contains the following

Claim 10. Conjugate according to any one of claims 7-9, selected from the following

Claim 11. A composition containing a conjugate according to any one of claims 7 to 10 and a pharmaceutically acceptable carrier.

Claim 12. A method of treating cancer in a subject, the method comprising administering to the subject a conjugate according to any one of claims 7-10.

Claim 13. A method of targeting a drug to a tumor or cancer cell in a subject, the method comprising administering to the subject a conjugate according to any one of claims 7-10.

Claim 14. The method according to any one of claims 12-13, wherein the flavaglin anticancer drug is localized or translocated to the cytoplasm or organelle of the tumor or cancer cell.

Clause 15. The method according to any one of claims 12-13, wherein the conjugate or flavaglin anticancer drug is selective for cancer cells in the subject.

Clause 16. The method according to any one of claims 12-13, wherein the conjugate or flavaglin anticancer drug is included in at least about 2 times more tumor or cancer cells compared to healthy cells.

Clause 17. The method according to any one of claims 12 to 16, wherein the cancer is selected from melanoma, brain cancer, lung cancer, adrenal cancer, liver cancer, kidney cancer, pancreatic cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, anal cancer, prostate cancer, ovarian cancer, breast cancer, cervical cancer, lymphoma, leukemia, myeloma, hematologic malignancy, hepatocellular carcinoma, retinoblastoma, glioma, sarcoma, blastoma, squamous cell carcinoma and adenocarcinoma.

Clause 18. The method according to any one of claims 12-17, wherein the subject is a human.

-

Claims (7)

CLAIM 1. Conjugate according to formula II or a salt thereof with X representing and or or methylene or bond; Y is a linker containing And Z is a flavaglin anticancer drug. 2. The conjugate according to claim 1, wherein the flavaglin anticancer drug contains FLV- 1, FLV-3 or a combination thereof, where FLV-1 and FLV-3 have formula (4) and (5) 3. Conjugate according to any one of claims 1, 2, selected from the following (9). 4. A pharmaceutical composition containing the conjugate according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. 5. A method of treating cancer in a subject, comprising administering to the subject a conjugate according to any one of claims 13. 6. A method of targeting a drug to a tumor or cancer cell in a subject, comprising administering to the subject a conjugate according to any one of claims 1 to 3. 7. Method according to any one of claims 5-6, wherein the flavaglin anticancer drug is -