Dolastatin 10 analogs

Abstract

Provided herein is a family of compounds that encompasses a range of structures afforded by biorthogonal "click chemistry" cyclization reactions, providing flexibility in the chemical and structural design conducive for forming conjugates of the compound with various functional agent, offering versatility in applications, including drug delivery, and other applications. Also provided are pharmaceutical compositions containing the compound or its conjugates, providing a basis for therapeutic use, as well as methods for treating medical conditions treatable with Dolastatin 10 using the disclosed compounds and / or the disclosed conjugates.

Description

[0001] DOLASTATIN 10 ANALOGS

[0002] RELATED APPLICATION

[0003] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 602,460 filed on November 24, 2023, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to Dolastatin 10 analogs, and more particularly, but not exclusively, to synthesis and uses of the Dolastatin 10 analogs and conjugates comprising the same.

[0006] Dolastatin 10 is a marine natural product isolated from the Indian Ocean sea hare Dollabella auricularia and from the marine cyanobacterium Symploca sp. VP642 from Palau. Being a small linear peptide molecule, Dolastatin 10 is considered a promising anti-cancer drug showing potency against breast and liver cancers, solid tumors and some types of leukemias. Preclinical research indicated potency in experimental antineoplastic and tubulin assembly systems.

[0007] Dolastatin 10

[0008] Dolastatin 10 is a mitotic inhibitor that acts on the microtubule assembly by interfering with tubulin formation and thereby disrupt cell division by mitosis and induces apoptosis and Bcl- 2 phosphorylation in several malignant cell types. It also noncompetitively inhibits binding of vincristine to tubulin (at a location known as the vinca / peptide region), while having been shown to bind to the RZX / MAY region. Dolastatin 10 is chemically related to symplostatin and analogues thereof, and the family of auristatins are potent synthetic analogs of Dolastatin 10. Dolastatin 10 has been shown to inhibit the growth of L1210 murine leukemia cells in culture, with a concordant rise in the mitotic index, and its IC50 value for cell growth was 0.5 nM. Comparable values for the other drugs were 0.5 nM for maytansine, 1 nM for rhizoxin, 20 nM for vinblastine, and 7 pM for phomopsin A. IC50 values were also obtained for the polymerization of purified tubulin in glutamate: 1.2 pM for Dolastatin 10, 1.4 pM for phomopsin A, 1.5 pM for vinblastine, 3.5 pM for maytansine, and 6.8 pM for rhizoxin. Dolastatin 10 and vinblastine were comparable in their effects on microtubule assembly dependent on microtubule associated proteins. Preliminary studies indicated that Dolastatin 10, like vinblastine, causes formation of a cold-stable tubulin aggregate at higher drug concentrations. Dolastatin 10 was shown to be the strongest inhibitor of the binding of radiolabeled vinblastine and vincristine to tubulin.

[0009] Bai, Ruoli, et al. [" Differential effects of active isomers, segments, and analogs of Dolastatin 10 on ligand interactions with tubulin: correlation with cytotoxicity", Biochemical pharmacology, 1993, 45(7), pp. 1503-1515] disclose the preparation of six chiral isomers, one tri- and one tetrapeptide segment, and one pentapeptide analog of Dolastatin 10, all of which differ little from Dolastatin 10 as inhibitors of tubulin polymerization, in order to examine the mechanism of action of Dolastatin 10.

[0010] Miyazaki, Koichi, et al. ["Synthesis and antitumor activity of novel Dolastatin 10 analogs" , Chemical and pharmaceutical bulletin, 1995, 43(10), pp. 1706-1718], disclose the synthesis and antitumor activity evaluation of a family of Dolastatin 10 analogs, each modified at one of the constituent amino acid derivatives.

[0011] Pettit, R.K. et al. ["Specific activities of Dolastatin 10 and peptide derivatives against Cryptococcus neoformans., Antimicrobial agents and chemotherapy, 1998, 42(11), pp. 2961- 2965], disclose the evaluation of the antifungal spectrum of Dolastatin 10 and four structural modifications thereof.

[0012] Maderna, Andreas, et al. ["Discovery of cytotoxic Dolastatin 10 analogues with N-terminal modifications" , Journal of medicinal chemistry, 2014, 57(24), pp. 10527-10543], disclose auristatins as synthetic analogues of the antineoplastic natural product Dolastatin 10, and reports their ultrapotent cytotoxic microtubule inhibitory activity and their use as payloads in antibodydrug conjugates (ADCs).

[0013] Shinya Y. et al. [“Synthesis and evaluation of novel Dolastatin 10 derivatives for versatile conjugations ', Bioorganic & Medicinal Chemistry, 2018, 26(8), pp. 1643-1652] disclose the design and synthesis of a series of Dolastatin 10 analogs useful as payloads for conjugated drugs. U.S. Patent Application Publication Nos. 20160068566, 20160083420, 20160068567, 20180141973, 20200010414, 20180222858, and U.S. Patent No. 10,351,593, disclose various aspects of derivatives and analogs of Dolastatin 10 or auristatins.

[0014] WO202 1 / 084532, by the present inventor, discloses versatile Dolastatin 10 analogs useful for preparing conjugates thereof with targeting, diagnostic, imaging and other moieties, as well as some exemplary conjugates and uses thereof.

[0015] Additional prior art documents include WO / 2003 / 008378, WO / 2014 / 174062, WO / 2014 / 174064, WO / 2014 / 174060, WO / 2016 / 192527 and WO / 2006 / 063707,

[0016] WO / 2006 / 063707.

[0017] Despite its promising characteristics, there are still great challenges and ongoing efforts in harnessing this agent for cancer treatment. Some of the challenges include toxicity and availability (supply). Dolastatin 10 has shown neurotoxicity in clinical trials, limiting its therapeutic potential, and sourcing Dolastatin 10 from its natural marine origin is challenging, while synthetic approaches have proven complex. Thus, while Dolastatin 10 holds promise as an antitumor agent, challenges such as toxicity and sourcing complexities still need to be addressed.

[0018] SUMMARY OF THE INVENTION

[0019] The present disclosure provides a novel class of Dolastatin 10 analog compounds represented by general Formula I, as well as methods for preparing and using these compounds. The compounds are characterized by a central core structure that is linked to various functional groups. These functional groups can be tailored to impart and / or append specific properties to the compounds, such as bioactivity, labeling ability, or diagnostic capability. The compounds and / or conjugates thereof with bioactive, labeling, or diagnostic agents can be used in a variety of medical applications, including the treatment of cancer.

[0020] According to an aspect of some embodiments of the present invention, there is provided a compound represented by general Formula I:

[0021]

[0022] A is represented by a general formula selected from the group consisting of:

[0023] Formula Al, Formula A2, and Formula A3;

[0024] Ri is a functional group;

[0025] R2 is selected from the group consisting of aryl, benzyl, heteroaryl, alkyl-aryl, and alkylene-aryl;

[0026] R3 is an alkyl or H;

[0027] D is a linear or branched C1-6 alkyl, a 1-6-mer PEG, or absent; and

[0028] Ra is selected from the group consisting of alkyl, aryl or H.

[0029] In some embodiments, Ri in Formula I is selected from the group consisting of an alkenyl, an alkynyl, a halo, a hydroxyl, a carbonyl, an aldehyde, a haloformyl (acyl halide), a carbonate ester, a carboxylate, a carboxyl, a carboalkoxy (ester), an alkoxy, a hydroperoxy, a peroxy, an ether, a hemiacetal, a hemiketal, an acetal, a ketal, an orthoester, a methylenedioxy, an orthocarbonate ester, a carboxylic anhydride, a carboxamide, an amine, a ketamine, an aldimine, an imide, an azide, an azo (diimide), a cyanate, an isocyanate, a nitrate, a nitrile, an isonitrile, a nitrosooxy, a nitro, a nitroso, an oxime, a pyridyl, a carbamate, a sulfhydryl, a sulfide, a disulfide, a sulfinyl, a sulfonyl, a sulfino, a sulfo, a sulfo-ester, a selenol, a selenide, a selenone, a thiocyanate, an isothiocyanate, a carbonothioyl, a carbonothioyl, a carbothioic S-acid, a carbothioic O-acid, a thiolester, a thionoester, a carbodithioic acid, a carbodithio, a phosphino, a phosphono, a phosphate, and a phosphate.

[0030] In some embodiments, Ri is selected from the group consisting of hydroxyl, amine, carboxyl, and amide. In some embodiments, D, when present, is selected from the group consisting of -CHz-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -CHCH3-, -C(CH3)2-, -(CH2)2- -CH2CHCH3-, -CH2C(CH3)2- -OCH2CH2O-, -(OCH2CH2)2-, -(OCH2CH2)3- -cyclic(CH2)3-

[0031] (corresponding to a cyclopropane moiety), -cyclic(CH2)3CH3- (corresponding to a methyl-cyclopropane moiety), and -(OCH2CH2)4- In some embodiments, D is a methylene group (-CH2-).

[0032] In some embodiments, R2 is selected from the group consisting of phenyl, benzyl, pyrrole, imidazole, oxazole, diazole, triazole, tetrazole, thiazole, pyridine, diazine, triazine, pyridomethylene and imidazolomethylene. In some embodiments, R2 is benzyl.

[0033] In some embodiments, R3 is methyl.

[0034] In some embodiments, A is represented by general Formula Al.

[0035] In some embodiments, Ri is hydroxyl, R2 is phenyl, R3 is methyl, D is a methylene group (-CH2-), and Ra is hydrogen.

[0036] In some embodiments, Ri is amine, R2 is phenyl, R3 is methyl, D is a methylene group (-CH2-), and Ra is hydrogen.

[0037] In some embodiments, Ri is carboxyl, R2 is phenyl, R3 is methyl, D is absent, and Ra is hydrogen.

[0038] In some embodiments, Ri could also include additional functional groups such as:

[0039] - Phosphonate esters (e.g. -PO(OR)2 where R is alkyl);

[0040] - Boronic acids (-B(0H)2) ;

[0041] - Silyl ethers (-Si(R)3 where R is alkyl);

[0042] - Alkyl halides (-CH2X where X is F, Cl, Br, or I);

[0043] - Alkyl tosylates (-CHzOTs); or

[0044] - Maleimides.

[0045] In some embodiments for R2, additional variations could include:

[0046] - Substituted phenyl groups (e.g. fluorophenyl, chlorophenyl, methoxyphenyl);

[0047] - Naphthyl;

[0048] - Biphenyl;

[0049] - Pyridyl;

[0050] - Quinolinyl; or

[0051] - Indolyl.

[0052] In some embodiments D could also include:

[0053] - Branched alkyl groups (e.g. -CH(CH3)CH2-, -C(CH3)2CH2-);

[0054] - Cycloalkyl groups (e.g. cyclopropyl, cyclobutyl, cyclopentyl); - Alkene groups (e g. -CH=CH-, -CH=CHCH2-); or

[0055] - Alkyne groups (e.g. -C=C-, -OCCH2-).

[0056] Additional variations for the linking moiety could include:

[0057] - Enzymatically cleavable peptide sequences (e.g. GFLG);

[0058] - pH-sensitive linkers (e.g. hydrazone, acetal);

[0059] - Photocleavable linkers (e.g. o-nitrobenzyl derivatives); or

[0060] - Redox-sensitive linkers (e.g. dithiols).

[0061] The functional moiety could also include:

[0062] - Aptamers;

[0063] - Small molecule ligands (e.g. folate, biotin);

[0064] - Cell-penetrating peptides;

[0065] - Nuclear localization sequences; or

[0066] - PEG chains of varying lengths.

[0067] For the synthesis, additional variations could include:

[0068] - Using different solid supports (e.g. Wang resin, Rink amide resin);

[0069] - Alternative coupling reagents (e.g. HBTU, PyBOP);

[0070] - Microwave-assisted synthesis; and / or

[0071] - Flow chemistry approaches.

[0072] The compound or conjugate could be formulated into various pharmaceutical compositions, such as:

[0073] - Liposomes;

[0074] - Nanoparticles (e.g. polymeric, gold);

[0075] - Hydrogels;

[0076] - Implants; and / or

[0077] - Transdermal patches.

[0078] According to another aspect of some embodiments of the present invention, there is provided a conjugate, which includes a moiety of the compound provided herein, a functional moiety (a moiety of a functional agent), and a linking moiety connecting the compound moiety and the functional moiety.

[0079] In some embodiments, the linking moiety that includes a biocleavable bond or group selected from the group consisting of an amide, an ester, a carbamate, a carbonate, a disulfide, a sulfonamide, an ether, a thioether, a valine-citrulline, a hydrazine and an oxyacrylate.

[0080] In some embodiments, the linking moiety that includes a disulfide. In some embodiments, the functional moiety is a moiety of a functional agent, including a moiety of a bioactive agent, a moiety of a labeling agent and / or a moiety of a diagnostic agent.

[0081] In some embodiments, the functional agent is selected from the group consisting of a peptide, a protein, an antibody, a biodegradable polymer, a targeting agent, a drug, a dye, a nanoparticle, a bead, a photodynamic therapy sensitizer, radiotherapy agent, a metal complex, an anti-cancer agent, an anti-proliferative agents, chemosensitizing agents, an anti-inflammatory agent, an antimicrobial agent, an anti-oxidant, a hormone, an anti-hypertensive agent, an antidiabetic agent, an immunosuppressant, an enzyme inhibitor, a neurotoxin and an opioid.

[0082] In some embodiments, the functional moiety is a moiety of an antibody.

[0083] According to another aspect of some embodiments of the present invention, there is provided a process of preparing the compound provided herein, which is effected by: forming an intermediate moiety corresponding to variable A in Formula I by means of a biorthogonal reaction on a solid support resin; and sequentially attaching BU-3, BU-4, Fmoc-Val and N,N-dimethyl Valin, respectively to the intermediate moiety by means of a solid-phase peptide synthesis (SPPS) reaction, and releasing the compound from the solid support resin, thereby obtaining the compound.

[0084] In some embodiments, the biorthogonal reaction is selected from the group consisting of: a copper-catalyzed azide-alkyne Huisgen 1,3-dipolar cycloaddition (CuAAC) reaction; a strain-promoted azide-alkyne cycloadditions (SPAACs) reaction; and a inverse electron demand Diels-Alder (IEDDA) reaction.

[0085] According to another aspect of some embodiments of the present invention, there is provided a process of preparing the conjugate provided herein, which is effected by coupling the compound provided herein to the functional agent, thereby forming the linking moiety.

[0086] According to another aspect of some embodiments of the present invention, there is provided a process of preparing the conjugate provided herein, which is effected by: forming the functional moiety on a solid-support resin using solid-state peptide synthesis protocols, wherein the functional moiety that includes a linkable group, forming the compound starting from the linkable group, thereby forming the linking moiety on a solid-support resin using solid-state peptide synthesis protocols, and releasing the conjugate from the resin.

[0087] According to another aspect of some embodiments of the present invention, there is provided a pharmaceutical composition that includes the compound provided herein, and a pharmaceutically acceptable carrier. According to another aspect of some embodiments of the present invention, there is provided a pharmaceutical composition that includes the conjugate provided herein, and a pharmaceutically acceptable carrier.

[0088] According to another aspect of some embodiments of the present invention, there is provided a method of treating a medical condition treatable with Dolastatin 10 in a subject, which is effected by administering to the subject a therapeutically effective amount of the compound provided herein.

[0089] According to another aspect of some embodiments of the present invention, there is provided the compound provided herein, designated for use in treating a medical condition that is treatable with Dolastatin 10 in a subject, wherein the treatment is effected by administering to the subject a therapeutically effective amount of the compound provided herein.

[0090] According to another aspect of some embodiments of the present invention, there is provided a use of the compound provided herein in the preparation of a medicament useful in the treatment of a medical condition in a subject that is treatable with Dolastatin 10.

[0091] According to another aspect of some embodiments of the present invention, there is provided a method of treating a medical condition in a subject that is treatable with Dolastatin 10, which is effected by administering to the subject a therapeutically effective amount of the conjugate provided herein.

[0092] According to another aspect of some embodiments of the present invention, there is provided the conjugate provided herein designated for treating a medical condition in a subject that is treatable with Dolastatin 10, which is effected by administering to the subject a therapeutically effective amount of the conjugate provided herein.

[0093] According to another aspect of some embodiments of the present invention, there is provided a use of the conjugate provided herein in the preparation of a medicament useful in the treatment of a medical condition in a subject that is treatable with Dolastatin 10.

[0094] According to another aspect of some embodiments of the present invention, there is provided a compound as provided herein, for use in treating a medical condition in a subj ect, which is treatable with Dolastatin 10.

[0095] According to another aspect of some embodiments of the present invention, there is provided a conjugate as provided herein, for use in treating a medical condition in a subject, which is treatable with Dolastatin 10.

[0096] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0097] DESCRIPTION OF SOME SPECIFIC EMBODIMENTS OF THE INVENTION

[0098] The present invention, in some embodiments thereof, relates to Dolastatin 10 analogs, and more particularly, but not exclusively, to synthesis and uses of the Dolastatin 10 analogs and conjugates comprising the same.

[0099] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0100] As presented hereinabove, Dolastatin 10, a marine natural product derived from the sea hare Dolabella auricularia, has garnered significant attention as a potential anticancer agent due to its potent cytotoxic activity against a wide range of tumor cells. Despite its promising preclinical efficacy, utilization of Dolastatin 10 still faced several challenges in its development as a cancer therapeutic. In addition, it is difficult to link Dolastatin 10 to potential carriers due to lack of functional groups suitable for effective conjugation. Therefore, modified analogs known in the art are used for conjugation through modified N-terminus (typically, monomethyl Vai or alpha-amino isobutyric acid). To address these challenges, the present inventor explored various strategies to improve the usability of Dolastatin 10 as an anticancer drug.

[0101] While conceiving the present invention, the inventor contemplated a Dolastatin 10 analog that would allow the potent peptide to be conjugated to a bioactive, diagnostic or labeling moiety, such as an antibody, a nanoparticle, a protein, a biodegradable polymer, a peptide, and the likes, with minimal reduction of activity compared to the activity of the naturally occurring molecule. The inventor had further contemplated a modification that introduces a functional group to Dolastatin 10 - one which would allow conjugating a Dolastatin 10 analog to a bioactive, diagnostic or labeling moiety via a biocleavable linker. The inventor had further contemplated a modification that would be conducive to solid-state peptide synthesis methodologies that would open the possibility of rapid, effective and versatile high-throughput preparation and screening of series of analogs and conjugates thereof.

[0102] While reducing the present invention to practice, the inventor directed the abovementioned modification to the C-terminus of Dolastatin 10, focusing on “click chemistry” reactions, with particular emphasis on click reactions that lead to diverse family of compounds that can be synthesized also in a biorthogonal pathway.

[0103] The terms "bioorthogonal" and "biorthogonal" are used herein interchangeably to refer to a chemical reaction that occurs between two molecules without interfering with the normal biological processes of living cells. In the context of “click chemistry”, this is in contrast to traditional click chemistry reactions, which often require the use of metal catalysts that can be toxic to cells. In some embodiments of the present invention, Dolastatin 10 analogs are afforded by copper-free click chemistry, which is a type of bioorthogonal click chemistry that uses a strain- promoted alkyne-azide cycloaddition (SPAAC) instead of a copper catalyst. SPAAC reactions are faster and more efficient than traditional click chemistry reactions, and they do not require the use of toxic metals.

[0104] Dolastatin 10 analog compound:

[0105] According to an aspect of some embodiments of the present invention, there is provided a compound, also referred to herein as a “Dolastatin 10 analog”, which is represented by General Formula I: wherein:

[0106] A is represented by a general formula selected from the group consisting of:

[0107] Ri is a functional group; R2 is selected from the group consisting of aryl, benzyl, heteroaryl, alkyl-aryl, and alkylene-aryl;

[0108] R3 is hydrogen or an alkyl, such as methyl, ethyl and isopropyl; preferably R3 is methyl;

[0109] D is a linear or branched C1-6 alkyl, a 1-6-mer PEG, or absent; and

[0110] Ra is selected from the group consisting of alkyl, aryl or H.

[0111] The term "analog" as used herein may refer to a compound in which one or more atoms are replaced with a different atom or group of atoms. The term may also refer to compounds with an identity of atoms but of different isomeric configuration. Such isomers may be constitutional isomers, i.e., structural isomers having different bonding arrangements of their atoms or stereoisomers having identical bonding arrangements but different spatial arrangements of the constituent atoms. The term may also refer to a biological activity of the related compounds (analogous to each other), which may differ in intensity, or reactivity of the compounds in some aspects, but resemble each other in the essence of the biological activity, as in the case of the naturally occurring Dolastatin 10 and the compounds disclosed herein.

[0112] The compounds provided herein are advantageous also in the sense of their synthesis, which is conducive with solid-phase chemistry, allowing the rest of the compound, namely the Dolastatin moiety, to be constructed on the same solid support on which the A moiety is constructed and forming the C-terminus of the Dolastatin 10 analog.

[0113] The moiety represented by the letter “A” is afforded in a reaction known as, or similar to “click chemistry” reaction, namely a reaction in-which two molecular entities are joined to form of a substituted heterocyclic or a heteroaryl structure ring. According to some embodiments of the present invention, the compounds provided herein are characterized by comprising a heteroaryl / heterocyclic ring at the C-terminus of Dolastatin 10, which is afforded by a biorthogonal cycloaddition reaction, as presented hereinbelow.

[0114] For example, A moieties falling under General Formula Al are afforded by a “click” reaction between an azide group and an alkyne group, also known as the Huisgen azide-alkyne cycloaddition (HAAC) or the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). It is a versatile and efficient reaction that is widely used in organic synthesis and biological applications. The scheme below presents a typical copper-catalyzed azide-alkyne Huisgen 1,3-dipolar cycloaddition (CuAAC) reaction that can be used to form a moiety falling under General Formula Al, according to some embodiments of the present invention: alkyne azide

[0115] Alternatively, A moi eties falling under General Formula A2 are afforded by a “click” reaction between an azide group and an alkyne group within an aliphatic ring, are known as strain- promoted azide-alkyne cycloadditions (SPAACs). These reactions are similar to the copper(I)- catalyzed azide-alkyne cycloadditions (CuAACs), but they proceed without the need for a copper catalyst, making them biorthogonal by definition, which renders them more biocompatible and less toxic, which is important for biological applications. The scheme below presents a copper- free click chemistry, a bioorthogonal reaction as a variant of an azide-alkyne Huisgen cycloaddition, or copper-free azide-alkyne [3 + 2] cycloaddition (SPAAC) reaction that can be used to form a moiety falling under General Formula A2, according to some embodiments of the present invention: cyclooctyne azide

[0116] Further alternatively, A moieties falling under General Formula A3 are afforded by a reaction between a tetrazine moiety and a trans double bond in an aliphatic ring (e.g., transcyclooctene), is known as the inverse electron demand Diels-Alder (IEDDA) reaction, or a tetrazine ligation reaction. It is a type of cycloaddition reaction that proceeds in the opposite direction of a typical Diels-Alder reaction - while in a traditional Diels-Alder reaction, a diene reacts with a dienophile to form a six-membered cyclic compound; in an IEDDA reaction, a tetrazine, which is an electron-deficient diene, reacts with an electron-rich alkene or alkyne to form a cyclic moiety. The scheme below presents a typical [4+2] cycloaddition of trans-cyclooctene and tetrazines for ligations, which is a bioorthogonal reaction that can be used to form a moiety falling under General Formula A3, according to some embodiments of the present invention:

[0117] 4,5-dihydropyridazine 1 ,4-dihydropyridazine ligation product

[0118] Additional information pertaining to biorthogonal cycloaddition reactions is widely available, and one of ordinary skills in the art may find ample guidance in the literature, such as, for example, in Kang, K. et al. [“Tetrazine ligation for chemical proteomics’", Proteome Science, 2017, 15:15, DOI 10.1186 / sl2953-017-0121-5].

[0119] One of the key features of the compound provided herein is the functional group at position Ri, which is designed for conjugation of the compound to a functional moiety, as this term is defined hereinbelow. The purposeful design of the compound with a reactive functional group at the C-terminus of Dolastatin 10 analog provided herein, opens the possibility to link the analog to a targeting delivery agent, such as an antibody, without abolishing the Dolastatin 10 biological (e.g., anticancer) activity.

[0120] The term "functional group" as used herein, refers to an atom or a group of atoms that imparts a particular chemical function to a molecule bearing the same, such as amines, carboxylates, and the like. The phrase "reactive group", which is used interchangeably with the term “functional group", refers to a chemical group that is capable of undergoing a chemical reaction that typically leads to the formation a covalent bond. Chemical reactions that lead to a bond formation include, for example, condensations, nucleophilic and electrophilic addition reactions, nucleophilic and electrophilic substitutions, addition and elimination reactions, alkylation reactions, rearrangement reactions, cycloaddition reactions (such as the Diels-Alder's reaction, the 1,3-dipolar cycloaddition Huisgen reaction, and the similar "click reaction"), and any other known organic reactions that involve at least one reactive group and leads to a formation of at least one covalent bond.

[0121] Exemplary functional / reactive groups, contemplated as the context of embodiments of the present invention (e.g., Ri in Formula I) include, without limitation, an alkenyl, an alkynyl, a halo, a hydroxyl, a carbonyl, an aldehyde, a haloformyl (acyl halide), a carbonate ester, a carboxylate, a carboxyl, a carboalkoxy (ester), an alkoxy, a hydroperoxy, a peroxy, an ether, a hemiacetal, a hemiketal, an acetal, a ketal, an orthoester, a methylenedioxy, an orthocarbonate ester, a carboxylic anhydride, a carboxamide, an a amine, a ketamine, an aldimine, an imide, an azide, an azo (diimide), a cyanate, an isocyanate, a nitrate, a nitrile, an isonitrile, a nitrosooxy, a nitro, a nitroso, an oxime, a pyridyl, a carbamate, a sulfhydryl, a sulfide, a disulfide, a sulfinyl, a sulfonyl, a sulfino, a sulfo, a sulfo-ester, a thiocyanate, an isothiocyanate, a carbonothioyl, a carbonothioyl, a carbothioic S-acid, a carbothioic O-acid, a selenol (selenoalcohol; R-SeH), a selenide (selenoether; -RSeR-), a selenone (selenoketone; R2C=Se), a selenyl halide (R-Se-X), a selenoester (RCChSeR'), a diselenide (R-Se-Se-R), a selenoxide (RSeO), a thiolester, a thionoester, a carbodithioic acid, a carbodithio, a phosphino, a phosphono, a phosphate, and a phosphate, as these terms are known and widely used in the art. In some preferred embodiments, Ri is selected from the group consisting of hydroxyl, amine, carboxyl, and amide.

[0122] In some embodiments, the Ri in Formula I (the functional group) is a hydroxyl. Nonlimiting examples of chemical reactions that involve a reactive hydroxyl group that may be used to form of at least one covalent bond between two molecular entities, include, without limitation, nucleophilic substitution reactions (SN2), acid-catalyzed dehydration of alcohols, Williamson ether synthesis, epoxidation of alkenes, opening of epoxides with nucleophiles, and the likes.

[0123] Alternatively, in some embodiments, Ri in Formula I is an amine. Non-limiting examples of chemical reactions that involve a reactive primary or secondary amine group that may be used in the context of the present invention to form of at least one covalent bond between two molecular entities, include, without limitation, nucleophilic substitution reactions (SN2), amine-aldehyde condensation reactions, amine-aldehyde condensation reactions amine-acylation reactions, and the likes.

[0124] Further alternatively, in some embodiments, Ri in Formula I is a carb oxyl / carb oxy late. Non-limiting examples of chemical reactions that involve a carb oxyl / carb oxy late group that may be used in the context of the present invention to form of at least one covalent bond between two molecular entities, include, without limitation, nucleophilic acyl substitution (SN2) reactions, amide / peptide bond formation, esterification, saponification, decarboxylation and the likes.

[0125] In some embodiments, Ri in Formula I is an amide. Non-limiting examples of chemical reactions that involve an amide group that may be used in the context of the present invention to form of at least one covalent bond between two molecular entities, include, without limitation, nucleophilic acyl substitution (SN2) reactions, reactions of amides with alkyl halides, reactions of amides with acyl chlorides, amine-aldehyde condensation reactions, amide-acylation reactions, amine-alkylation reactions, quatemization and N-alkylation of amides, and the likes.

[0126] In some embodiments, the functional group (Ri) is selected to be suitable for form a linking moiety connecting the compound provided herein, namely a moiety of a Dolastatin 10 analog, and a moiety of a functional molecular entity, as this term is defined and exemplified hereinbelow, such as, for example, a bioactive agent, a diagnostic agent or a labeling agent. The compound presented herein may exhibit a spacer moiety separating the ring portion of A in Formula I from the functional group, referred to herein as D. In some embodiments, D is a linear or branched Ci-6 alkyl, a Ci-6 alkenyl, a Ci-6 alkynyl, a 1-6-mer PEG, or absent. A 1-6-mer PEG refers to a short poly (ethylene glycol) chain having 1-6 -(CH2-O)- monomers. In some preferred embodiments, D is a methylene group (-CH2-).

[0127] In some embodiments the linear or branched C1-6 alkyl is selected from the group consisting of -CH2-, -CHCH3-, -C(CH3)2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -CH2CHCH3- -(CH2)5-, -(CH2)6-, -(CH(CH3))CH2-, -cyclic(CH2)3- -cyclic(CH2)3CH3- and

[0128] -CH2C(CH3)2-

[0129] According to some embodiments of the present invention, D is, without limitation, -CH2-0-, -(CH2)2-O-, -(CH2)3-O-, -OCH2CH2O-, -(OCH2CH2)2-, -(OCH2CH2)3-, -(OCH2CH2)4-, -CH=CH-CH=CH-, -C=C-C=C-, -CH2CH(OH)CH2- -CH2-O-CH2-, -CH2-O-CH2-O-, -(CH2)2-O-(CH2)2-, -(CH2)2-O-(CH2)2-O-, -CH2-mC6H4-CH2-, -CH2-mC6H4-CH2-O-, -CH2- / 2C6H4-CH2-, -CH2- / 2C6H4-CH2-O-, -CH2-NHCO-

[0130] -C6H4-NHCO-, -CH2-O-CH2-, -cyclic(CH2)3- -cyclic(CH2)3CH3- and -CH=CH-CH2-NH-(CH2)2- Another feature characterizing the compounds provided herein is R2, which is selected from the group consisting of aryl, as this term is defined hereinbelow, heteroaryl, as this term is defined hereinbelow, alkyl-aryl, which is defined as an aryl connected to the compound via a short alkyl (Cl -4) spacer, and alkylene-aryl, which is defined as an aryl connected to the compound via a short alkenyl (Cl -4) spacer. In some preferred embodiments, R2 is benzyl. According to some preferred embodiments, A in General Formula I is represented by

[0131] General Formula Al.

[0132] Exemplary compounds encompassed within the scope of the present invention, according to some embodiments thereof, include without limitation the compounds (Dolastatin 10 analogs) presented in Table 1 below.

[0133] Table 1

[0134]

[0135]

[0136]

[0137]

[0138] Conjugates ofDolastatin 10 analog:

[0139] The compounds (Dolastatin 10 analogs) provided herein can be used as a stand-alone bioactive agent, or be tethered to a functional agent to form a conjugate; such conjugate is designed to exert activity by both sides thereof, namely the activity exerted by the Dolastatin 10 analog and the activity exerted by the functional agent. The conjugate can be designed to stay as a conjugate (intact), or disassociate (cleave) into at least the two parts comprising the same.

[0140] Thus, according to yet another aspect of some embodiments of the present invention, there is provided a conjugate of the compound provided herein, which includes: a moiety of the compound, according to some embodiments of the present invention, a functional moiety (a moiety of a functional agent), and a linking moiety connecting the compound moiety via Ri (the functional group denoted in Formula I), and the functional moiety.

[0141] In the context of the present disclosure, the term “moiety” is used to denote a portion of a molecule, which may be a functional group, or describe a portion of a molecule with multiple functional groups which share common structural aspects. In some embodiments, the term “moiety” refers to the major part of a molecule being attached to another molecule or a functional group. In some embodiments, the moiety substantially exhibits the same biologic activity of the molecule. In some embodiments, the moiety can generate the molecule upon cleavage from a conjugate comprising the same. In the context of embodiments of the present invention, the term “moiety” is used similarly to the term “residue” in the context of amino acids in a polypeptide.

[0142] As used herein, the term “linking moiety” describes a chemical moiety (a group of atoms or a covalent bond) that links two chemical moieties via one or more covalent bonds. A linking moiety may include atoms that form a part of one or both of the chemical moieties it links, and / or include atoms that do not form a part of one or both of the chemical moieties it links. For example, a peptide bond (amide) linking moiety that links two moieties includes at least a nitrogen atom and a hydrogen atom from one moiety and at least a carboxyl of the other moiety. In general, the linking moiety can be formed during a chemical reaction, such that by reacting two or more functional / reactive groups, the linking moiety is formed as a new chemical entity which can comprise a bond (between two atoms), or one or more bonded atoms. Alternatively, the linking moiety can be a chemical moiety comprising two or more reactive groups to which the reactive groups of other compounds can be attached, either directly or indirectly.

[0143] In the context of some embodiments of the present invention, the term “linking moiety” is synonymous with the term “cleavable linker”, meaning that the linking moiety is selected or designed to break under certain conditions, or at certain locations in the treated subject. The term “biocleavable linker”, for example, refers to a cleavable linker that, inter alia, cleaves under physiological conditions.

[0144] The positions at which the elements of the conjugate are linked to one-another are generally selected such that once cleaved off the conjugate, any or at least some of the moieties stemming from the cleavage substantially exhibit their original biological activity (mechanism of biological activity), or at least some thereof. According to some embodiments of the present invention, the linking moiety is formed such that the biological activity of the elements of the conjugate remains substantially the same as the biological activity of the standalone elements, while still in the conjugated form. In some embodiments, the functional moiety of the conjugate can be regarded as a prodrug as long as it is bound to the conjugate that regains its original functional activity once cleaved-off the conjugate.

[0145] Representative examples of reactive (functional) groups that may be used to form a cleavable linker include, without limitation, acyl halide, aldehyde, alkoxy, alkyne, amide, amine, aryloxy, azide, aziridine, azo, carbamate, carbonyl, carboxyl, carboxylate, cyano, diene, dienophile, epoxy, guanidine, guanyl, halide, hydrazide, hydrazine, hydroxy, hydroxylamine, imino, isocyanate, isothiocyanate, maleimide, N-hydroxycuccinimide, carboxylic acid halide, alkyl halide, nitro, phosphate, phosphonate, sulfinyl, sulfonamide, sulfonate, thioalkoxy, thioaryloxy, thiocarbamate, thiocarbonyl, thiohydroxy, thiourea and urea, as these terms are defined hereinafter.

[0146] According some embodiments of the present invention, various elements of the conjugate presented herein are attached to one or more linking moieties via spacer moieties. As used herein, the phrase “spacer moiety” describes a chemical moiety that typically extends between two chemical moieties and is attached to each of the chemical moieties via covalent bonds. The spacer moiety may be linear or cyclic, be branched or unbranched, rigid or flexible, hydrophobic or hydrophilic. A spacer moiety may be similar in properties and composition to variable D in Formula I, but it is not necessarily the same moiety, namely D may serve as a spacer between the compound moiety and the functional moiety.

[0147] The nature of the spacer moieties can be regarded as having an effect on two aspects, the synthetic aspect, namely the influence of the spacer moieties on the process of preparing the conjugates presented herein, and the influence of the spacer moieties on the biology activity of the conjugates in terms of drug-release profile(s), biological activity, bioavailability and other ADME- Tox considerations.

[0148] According to some embodiments of the present invention, the spacer moieties are selected such that they allow and / or promote the conjugation reaction between various elements of the conjugates presented herein, and reduce the probability for the formation of side-products due to undesired reactions. Such traits can be selected for in terms of spacer's length, flexibility, structure and specific chemical reactivity or lack thereof. Spacer moieties with fewer reactive groups will present a simpler synthetic challenge, requiring less protection / deprotection steps and affording higher chemical yields. For example, saturated and linear alkyls of 1-10, or 1-5 carbon atoms, having one reactive group at the end atom for conjugation with a corresponding reactive group, would afford substantially higher yield and fewer side products. Similarly, a spacer moiety based on one or two chained benzyl rings would also lead to an efficient conjugation reaction.

[0149] According to some embodiments of the present invention, the spacer moieties are selected such that they provide favorable cleavage conditions, as these are discussed hereinbelow. For example, a spacer may alter the accessibility of an enzyme to the linking moiety, thereby allowing the enzyme to cleave the linkage between the bioactive agent and the conjugate. In some embodiments, a spacer moiety can be regarded as forming a part of a linking moiety.

[0150] According to some embodiments of the present invention, the spacer moieties include, without limitation, -CH2-, -CH2-O-, -(CH2)2-, -(CH2)2-O-, -(CH2)3-, -(CH2)3-O-, -(CH2)4-, -(CH2)5- , -(CH2)6-, -(CH(CH3))-CH2-, -CH=CH-CH=CH-, -C=C-C=C-, -CH2CH(OH)CH2-, -CH2-O-CH2- , -CH2-O-CH2-O-, -(CH2)2-O-(CH2)2-, -(CH2)2-O-(CH2)2-O-, -CH2-mC6H4-CH2-, -CH2-mC6H4- CH2-O-, -CH2- / 2C6H4-CH2-, -CH2- / 2C6H4-CH2-O-, -CH2-NHCO-, -C6H4-NHCO-, -CH2-O-CH2- and -CH=CH-CH2-NH-(CH2)2-.

[0151] Examples of linking moieties, according to some embodiments of the present invention, include without limitation, acetal, aldimine, amide, aminal, aminoacetal, carbamate, carbonate, carboxylate, cycloalkene, cyclohexene, disulfide, ester, heteroalicyclic, heteroaryl, hydrazone, imide, imine, ketal, ketimine, lactam, lactone, oxime, phosphate ester, semicarbazone, thioacetal, thioketal, triazine, triazole, and the like. Other linking moieties are defined hereinbelow, and further other linking moieties are contemplated within the scope of the term as used herein. According to some embodiments, the cleavable linker, or labile linking moiety, is selected from the group consisting of an amide, an ester, a carbamate, a carbonate, a disulfide, a sulfonamide, an ether, a thioether, a valine-citrulline, a hydrazine and an oxyacrylate. In some preferred embodiments, the linking moiety comprises a disulfide.

[0152] Acetal / ketal Carbonate Ester Phosphate ester

[0153] Di sulfide Sulfonamide Ether Thioether

[0154] Valine-Citrulline Oxy aery 1 ate Hy drazi ne

[0155] Definitions of specific functional groups, chemical terms, and general terms used throughout the specification are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, 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, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987.

[0156] As used herein, the terms “amine” or “amino”, describe both a -NR’R” end group and a -NR'- linking moiety, wherein R’ and R" are each independently hydrogen, alkyl, cycloalkyl, aryl, as these terms are defined hereinbelow. Herein throughout, the phrase "end group" describes a chemical group that is attached to one compound (a substituent; a reactive group; a functional group etc.), while the term “linking moiety” describes a group that is attached to two compounds and links therebetween.

[0157] The amine group can therefore be a primary amine, where both R’ and R” are hydrogen, a secondary amine, where R’ is hydrogen and R” is alkyl, cycloalkyl or aryl, or a tertiary amine, where each of R’ and R” is independently alkyl, cycloalkyl or aryl.

[0158] Alternatively, R' and R" can each independently be hydrogen, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azide (azido), sulfonamide, carbonyl, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine, as these terms are defined herein.

[0159] The term "alkyl" describes a saturated aliphatic hydrocarbon including straight chain (unbranched) and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms. Whenever a numerical range; e.g., "1-20", is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkyl is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted. Substituted alkyl may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azide (azido), sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N- amide, guanyl, guanidine and hydrazine.

[0160] The alkyl group can be an end group, as this phrase is defined hereinabove, wherein it is attached to a single adjacent atom, or a linking moiety, as this phrase is defined hereinabove, which connects two or more moieties via at least two carbons in its chain. When an alkyl is a linking moiety, it is also referred to herein as “alkylene”, e.g., methylene, ethylene, propylene, etc.

[0161] The term "alkenyl" describes an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl may be substituted or unsubstituted by one or more substituents, as described for alkyl hereinabove. The terms "alkynyl" or "alkyne", as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.

[0162] The term "cycloalkyl" describes an all-carbon monocyclic or fused ring (i.e., rings that share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system. The cycloalkyl group may be substituted or unsubstituted. Substituted cycloalkyl may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azido, sulfonamide, C- carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine. The cycloalkyl group can be an end group, as this phrase is defined hereinabove, wherein it is attached to a single adjacent atom, or a linking moiety, as this phrase is defined hereinabove, connecting two or more moieties at two or more positions thereof.

[0163] The term "heteroalicyclic" describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. The heteroalicyclic may be substituted or unsubstituted. Substituted heteroalicyclic may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azido, sulfonamide, C -carb oxy late, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide, N- amide, guanyl, guanidine and hydrazine. The heteroalicyclic group can be an end group, as this phrase is defined hereinabove, where it is attached to a single adjacent atom, or a linking moiety, as this phrase is defined hereinabove, connecting two or more moieties at two or more positions thereof. Representative examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the like.

[0164] The term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi-electron system. The aryl group may be substituted or unsubstituted. Substituted aryl may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azido, sulfonamide, C -carb oxy late, O-carboxylate,

[0165] N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C-amide, N- amide, guanyl, guanidine and hydrazine. The aryl group can be an end group, as this term is defined hereinabove, wherein it is attached to a single adjacent atom, or a linking moiety, as this term is defined hereinabove, connecting two or more moieties at two or more positions thereof. Preferably, the aryl is a substituted or unsubstituted phenyl.

[0166] The term "heteroaryl" describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted. Substituted heteroaryl may have one or more substituents, whereby each substituent group can independently be, for example, hydroxyalkyl, trihaloalkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, azido, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, O-carbamate, N-carbamate, C-amide, N-amide, guanyl, guanidine and hydrazine. The heteroaryl group can be an end group, as this phrase is defined hereinabove, where it is attached to a single adjacent atom, or a linking moiety, as this phrase is defined hereinabove, connecting two or more moieties at two or more positions thereof. Representative examples are pyridine, pyrrole, oxazole, indole, purine and the like.

[0167] The term “alkaryl” describes an alkyl, as defined herein, which is substituted by one or more aryl or heteroaryl groups. An example of alkaryl is benzyl.

[0168] The term "amine-oxide” describes a -N(OR’)(R”) or a -N(OR')- group, where R’ and R” are as defined herein. This term refers to a -N(OR')(R") group in cases where the amine-oxide is an end group, as this phrase is defined hereinabove, and to a -N(OR')- group in cases where the amine-oxime is an end group, as this phrase is defined hereinabove.

[0169] As used herein, the term “acyl” refers to a group having the general formula -C(=O)R’, -C(=O)OR’, -C(=O)-O-C(=O)R’, -C(=O)SR’, -C(=O)N(R’)2, -C(=S)R’, -C(= S)N(R’)2, and -C(=S)S(R’), -C(=NR’)R”, -C(=NR’)OR”, -C(=NR’)SR”, and -C(=NR’)N(R”)2, wherein R’ and R” are each independently hydrogen, halo, substituted or unsubstituted hydroxyl, substituted or unsubstituted thiol, substituted or unsubstituted amine, substituted or unsubstituted acyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic, cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic, cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkyl, cyclic or acyclic, substituted or unsubstituted, branched or unbranched alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or di-aliphaticamino, mono- or di-heteroaliphaticamino, mono- or di-alkylamino, mono- or di-heteroalkylamino, mono- or diarylamino, or mono- or di-heteroarylamino; or two RX1groups taken together form a 5- to 6- membered heterocyclic ring. Exemplary acyl groups include aldehydes (-CHO), carboxylic acids (-CO2H), ketones, acyl halides, esters, amides, imines, carbonates, carbamates, and ureas. Acyl substituents include, but are not limited to, any of the substituents described herein, that result in the formation of a stable moiety (e.g., aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl, acyl, oxo, imino, thioxo, cyano, isocyano, amino, azido, nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino, alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

[0170] As used herein, the term “aliphatic” or “aliphatic group” denotes an optionally substituted hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (“carbocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-12 carbon atoms (C1-12). In some embodiments, aliphatic groups contain 1-6 carbon atoms (Ci-e). In some embodiments, aliphatic groups contain 1-4 carbon atoms (C1-4), and in yet other embodiments, aliphatic groups contain 1-3 carbon atoms (C1-3). Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0171] As used herein, the terms “heteroaliphatic” or “heteroaliphatic group”, denote an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, heteroaliphatic groups contain 1-6 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups.

[0172] The term “halo” describes fluorine, chlorine, bromine or iodine substituent.

[0173] The term "halide" describes an anion of a halogen atom, namely F", Cl" Br" and I".

[0174] The term “haloalkyl” describes an alkyl group as defined above, further substituted by one or more halide.

[0175] The term “sulfate” describes a -O-S(=O)2-OR’ end group, as this term is defined hereinabove, or an -O-S(=O)2-O- linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.

[0176] The term “thiosulfate” describes a -O-S(=S)(=O)-OR’ end group or a -O-S(=S)(=O)-O- linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.

[0177] The term “sulfite” describes an -O-S(=O)-O-R’ end group or a -O-S(=O)-O- group linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.

[0178] The term “thiosulfite” describes a -O-S(=S)-O-R’ end group or an -O-S(=S)-O- group linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.

[0179] The term “sulfinate” or “sulfinyl” describes a -S(=O)-OR’ end group or an -S(=O)-O- group linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.

[0180] The terms “solfoxide” or “sulfinyl” describe a -S(=O)R’ end group or an -S(=O)- linking moiety, as these phrases are defined hereinabove, where R’ is as defined hereinabove.

[0181] The term "sulfonate” or “sulfonyl” describes a -S(=O)2-R’ end group or an -S(=O)2- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0182] The term “S-sulfonamide” describes a -S(=0)2-NR’R” end group or a -S(=O)2-NR’- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0183] The term "N-sulfonamide" describes an R’ S(=0)2-NR”- end group or a -S(=O)2-NR’- linking moiety, as these phrases are defined hereinabove, where R’ and R’ ’ are as defined herein.

[0184] The term “disulfide” refers to a -S-SR’ end group or a -S-S- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0185] The term “phosphate” describes an -0-P(=0)2(0R’) end or reactive group or a -O-P(=O)2(O)- linking moiety, as these phrases are defined hereinabove, with R’ as defined herein. The term “phosphonate” describes a -P(=O)(OR’)(OR”) end or reactive group or a -P(=O)(OR’)(O)- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0186] The term “thiophosphonate” describes a -P(=S)(OR’)(OR”) end group or a -P(=S)(OR’)(O)- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0187] The term "carbonyl" or "carbonate" as used herein, describes a -C(=O)-R’ end group or a -C(=O)- linking moiety, as these phrases are defined hereinabove, with R’ as defined herein.

[0188] The term "thiocarbonyl" as used herein, describes a -C(=S)-R’ end group or a -C(=S)- linking moiety, as these phrases are defined hereinabove, with R’ as defined herein.

[0189] The term “oxo” as used herein, described a =0 end group.

[0190] The term “thioxo” as used herein, described a =S end group.

[0191] The term “oxime” describes a =N-0H end group or a =N-O- linking moiety, as these phrases are defined hereinabove.

[0192] The term “hydroxyl” describes a -OH group.

[0193] As used herein, the term “aldehyde” refers to an -C(=0)-H group.

[0194] The term “acyl halide” describes a -(C=O)R"" group wherein R"" is halo, as defined hereinabove.

[0195] The term “alkoxy” as used herein describes an -O-alkyl, an -O-cycloalkyl, as defined hereinabove. The ether group -O- is also a possible linking moiety.

[0196] The term "aryloxy" describes both an -O-aryl and an -O-heteroaryl group, as defined herein.

[0197] The term “disulfide” as used herein describes an -S-S- linking moiety, which in some cases forms between two thiohydroxyl groups.

[0198] The terms “thio”, "sulfhydryl" or "thiohydroxyl" as used herein describe an -SH group.

[0199] The term "thioalkoxy" or “thioether” describes both a -S-alkyl group, and a -S-cycloalkyl group, as defined herein. The thioether group -S- is also a possible linking moiety.

[0200] The term "thioaryloxy" describes both a -S-aryl and a -S-heteroaryl group, as defined herein. The thioarylether group -S-aryl- is also a possible linking moiety.

[0201] The term "cyano" or “nitrile” describes a -C=N group.

[0202] The term “isocyanate” describes an -N=C=0 group.

[0203] The term "nitro" describes an -NO2 group.

[0204] The term “carboxylate” or "ester", as used herein encompasses C-carboxylate and O- carboxylate. The term “C -carb oxy late” describes a -C(=O)-OR’ end group or a -C(=O)-O- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0205] The term “O-carboxylate” describes a -OC(=O)R’ end group or a -OC(=O)- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0206] The term “thiocarboxylate” as used herein encompasses “C-thiocarboxylate and O- thiocarb oxy late.

[0207] The term “C-thiocarboxylate” describes a -C(=S)-OR’ end group or a -C(=S)-O- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0208] The term “O-thiocarboxylate” describes a -OC(=S)R’ end group or a -OC(=S)- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0209] The term “carbamate” as used herein encompasses N-carbamate and O-carbamate.

[0210] The term “N-carbamate” describes an R”OC(=O)-NR’- end group or a -OC(=O)-NR’- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0211] The term “O-carbamate” describes an -OC(=O)-NR’R” end group or an -OC(=O)- NR’- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0212] The term “thiocarbamate” as used herein encompasses N-thiocarbamate and O- thiocarbamate.

[0213] The term “O-thiocarbamate” describes a -OC(=S)-NR’R” end group or a -OC(=S)-NR’- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0214] The term “N-thiocarbamate” describes an R”OC(=S)NR’- end group or a -OC(=S)NR’- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0215] The term “dithiocarbamate” as used herein encompasses N-dithiocarbamate and S- dithiocarbamate.

[0216] The term “S-dithiocarbamate” describes a -SC(=S)-NR’R” end group or a -SC(=S)NR’- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0217] The term “N-dithiocarbamate” describes an R”SC(=S)NR’- end group or a -SC(=S)NR’- linking moiety, as these phrases are defined hereinabove, with R’ and R” as defined herein.

[0218] The term "urea", which is also referred to herein as “ureido”, describes a -NR’C(=O)- NR”R”’ end group or a -NR’C(=O)-NR”- linking moiety, as these phrases are defined hereinabove, where R’ and R” are as defined herein and R'" is as defined herein for R' and R". The term “thiourea”, which is also referred to herein as “thioureido”, describes a -NR’- C(=S)-NR”R”’ end group or a -NR’-C(=S)-NR”- linking moiety, with R’, R” and R’” as defined herein.

[0219] The term “amide” as used herein encompasses C-amide and N-amide.

[0220] The term “C-amide” describes a -C(=O)-NR’R” end group or a -C(=O)-NR’- linking moiety, as these phrases are defined hereinabove, where R’ and R” are as defined herein.

[0221] The term “N-amide” describes a R’C(=O)-NR”- end group or a R’C(=O)-N- linking moiety, as these phrases are defined hereinabove, where R’ and R” are as defined herein.

[0222] The term “imine”, which is also referred to in the art interchangeably as “Schiff-base”, describes a -N=CR'- linking moiety, with R' as defined herein or hydrogen. As is well known in the art, Schiff bases are typically formed by reacting an aldehyde or a ketone and an amine- containing moiety such as amine, hydrazine, hydrazide and the like, as these terms are defined herein. The term “aldimine" refers to a -CH=N- imine which is derived from an aldehyde. The term “ketimine" refers to a -CR-N- imine which is derived from a ketone.

[0223] The term “hydrazone" refers to a -R'C=N-NR”- linking moiety, wherein R’ and R” are as defined herein.

[0224] The term “semicarbazone" refers to a linking moiety which forms in a condensation reaction between an aldehyde or ketone and semi carb azide. A semicarbazone linking moiety stemming from a ketone is a -R'C=NNR"C(=O)NR"'-, and a linking moiety stemming from an aldehyde is a -CR'=NNR"C(=O)NR"'-, wherein R’ and R” are as defined herein and R'" or as defined for R’ .

[0225] As used herein, the term "lactone" refers to a cyclic ester, namely the intra-condensation product of an alcohol group -OH and a carboxylic acid group -COOH in the same molecule.

[0226] As used herein, the term "lactam" refers to a cyclic amide, as this term is defined herein. A lactam with two carbon atoms beside the carbonyl and four ring atoms in total is referred to as a P-lactam, a lactam with three carbon atoms beside the carbonyl and five ring atoms in total is referred to as a y-lactam, a lactam with four carbon atoms beside the carbonyl and six ring atoms in total is referred to as a 6-lactam, and so on.

[0227] The term “guanyl” describes a R’R”NC(=N)- end group or a -R’NC(=N)- linking moiety, as these phrases are defined hereinabove, where R’ and R” are as defined herein.

[0228] The term “guanidine” describes a -R’NC(=N)-NR”R”’ end group or a - R’NC(=N)- NR”- linking moiety, as these phrases are defined hereinabove, where R’, R" and R'" are as defined herein. The term “hydrazine” describes a -NR’-NR”R”’ end group or a -NR’ -NR”- linking moiety, as these phrases are defined hereinabove, with R’, R”, and R'" as defined herein.

[0229] As used herein, the term “hydrazide” describes a -C(=O)-NR’-NR”R”’ end group or a - C(=O)-NR’-NR”- linking moiety, as these phrases are defined hereinabove, where R’, R” and R’” are as defined herein.

[0230] The term "hydroxylamine", as used herein, refers to either a -NHOH group or a -ONH2.

[0231] As used herein, the terms “azo” or “diazo” describe a -N=N-R’ end group or a -N=N- linking moiety, as these phrases are defined hereinabove, where R’ is as defined herein.

[0232] As used herein, the term “azido” described a -N=N+=N" (-N3) end group.

[0233] The term “triazine" refers to a heterocyclic ring, analogous to the six-membered benzene ring but with three carbons replaced by nitrogen atoms. The three isomers of triazine are distinguished from each other by the positions of their nitrogen atoms, and are referred to as 1,2,3- triazine, 1,2,4-triazine, and 1,3,5-triazine. Other aromatic nitrogen heterocycles include pyridines with 1 ring nitrogen atom, diazines with 2 nitrogen atoms in the ring and tetrazines with 4 ring nitrogen atoms.

[0234] The term "triazole" refers to either one of a pair of isomeric chemical compounds with molecular formula C2H3N3, having a five-membered ring of two carbon atoms and three nitrogen atoms, namely 1,2,3-triazoles and 1,2,4-triazoles.

[0235] The term “aziridine", as used herein, refers to a reactive group which is a three membered heterocycle with one amine group and two methylene groups, having a molecular formula of - C2H3NH.

[0236] As used herein, the term “thiohydrazide” describes a -C(=S)-NR’-NR”R”’ end group or a -C(=S)-NR’-NR”- linking moiety, as these phrases are defined hereinabove, where R’, R” and R’” are as defined herein.

[0237] As used herein, the term “methyleneamine” describes an -NR’-CH2-CH=CR”R’” end group or a -NR’-CH2-CH=CR”- linking moiety, as these phrases are defined hereinabove, where R’, R” and R’” are as defined herein.

[0238] The term "diene", as used herein, refers to a -CR'=CR"-CR"'=CR""- group, wherein R’ as defined hereinabove, and R", R'" and R"" are as defined for R'.

[0239] The term "dienophile", as used herein, refers to a reactive group that reacts with a diene, typically in a Diels-Alder reaction mechanism, hence a dienophile is typically a double bond or an alkenyl.

[0240] The term “epoxy", as used herein, refers to a reactive group which is a three membered heterocycle with one oxygen and two methylene groups, having a molecular formula of -C2H3O. According to some embodiments of the present invention, some linking moieties result from a reaction between two reactive groups. Alternatively, a desired linking moiety is first generated and a bioactive agent and / or a spacer moiety are attached thereto.

[0241] Cleavable linker lability:

[0242] As used herein, the words "link", “linked”, "linkage" "linker", "bound", “coupled” or “attached”, are used interchangeably herein and refer to the presence of at least one covalent bond between species and moieties, unless specifically noted otherwise. The phrase "covalent bond", as used herein, refers to one or more pairs of electrons that are shared between atoms in a form of chemical bonding.

[0243] According to some embodiments of the present invention, the conjugate exhibits a linking moiety that is stable at physiological conditions, namely a linking moiety that does not disintegrate spontaneously or otherwise for the duration of exposure to the physiological environment in the bodily site. Such linking moiety is referred to herein a "biostable". Biostable linking moieties offer the advantage of an extended period of time at which the conjugate can exert its biological activity, up to the time it is secreted or otherwise removed from the bodily site. An exemplary biostable linking moiety is a triazole-based linking moiety. It is noted that biostability is also a relative term, meaning that a biostable linking moiety takes longer to break or requires certain cleavage conditions which hare less frequently encountered by the conjugate when present in physiological conditions.

[0244] According to some embodiments of the present invention, the linking moiety is a cleavable linker, or a biocleavable-linking moiety. In the context of some embodiments of the present invention, the linking moiety is a cleavable linker, or biocleavable linking moiety, which is selected so as to break and release the bioactive agent attached thereto at certain conditions, referred to herein as “drug-releasing conditions” or “cleavage conditions”. As used herein, the terms “biocleavable” and “biodegradable” are used interchangeably to refer to moieties that degrade (i.e., break and / or lose at least some of their covalent structure) under physiological or endosomal conditions. Biodegradable moieties are not necessarily hydrolytically degradable and may require enzymatic action to degrade.

[0245] As used herein, the terms “cleavable linker”, “biocleavable moiety” or “biodegradable moiety” describe a chemical moiety, which undergoes cleavage in a biological system such as, for example, the digestive system of an organism or a metabolic system in a living cell.

[0246] According to some embodiments of the present invention, the linking moiety is a photocleavable linker that cleaves upon light irradiation. In some embodiments, the cleavable linker is selected according to its susceptibility to certain enzymes that are likely to be present at the targeted bodily site or at any other bodily site where cleavage is intended, thereby defining the cleavage conditions.

[0247] Representative examples of biocleavable moieties include, without limitation, amides, carboxylates, carbamates, phosphates, hydrazides, thiohydrazides, disulfides, epoxides, peroxo and methyleneamines. Such moieties are typically subjected to enzymatic cleavages in a biological system, by enzymes such as, for example, hydrolases, amidases, kinases, peptidases, phospholipases, lipases, proteases, esterases, epoxide hydrolases, nitrilases, glycosidases and the like.

[0248] For example, hydrolases (EC number beginning with 3) catalyze hydrolysis of a chemical bond according to the general reaction scheme A-B + H2O — A-OH + B-H. Ester bonds are cleaved by sub-group of hydrolases known as esterases (EC number beginning with 3.1), which include nucleases, phosphodiesterases, lipases and phosphatases. Hydrolases having an EC number beginning with 3.4 are peptidases, which act on peptide bonds.

[0249] Additional information pertaining to enzymes, enzymatic reactions, and enzyme-linking moiety correlations can be found in various publically accessible sources, such as Bairoch A., “The ENZYME database in 200(J Nucleic Acids Res, 2000, 28, pp. 304-305.

[0250] Functional moiety:

[0251] As disclosed herein, the conjugate is designed to comprise a payload, or carry a releasable payload in the form of the Dolastatin 10 analog provided herein and a functional moiety, derived from a bioactive agent, a diagnostic agent or a labeling agent. In embodiments in which the moieties are of agents are different from one-another, the conjugate of the present invention provides for simultaneous, concerted or sequential release of the agents and can therefore be specifically advantageous in cases where the different agents confer a cumulative and / or a synergistic effect, or when one agent serves as the targeting agent for the other, bringing the payload to an intended target.

[0252] The term “functional moiety”, as used in the context of embodiments of the present invention, refer to a moiety of a bioactive, a diagnostic or a labeling agent, such as, without limitation, a targeting agent, a drug or a dye, which may take the form of an amino-acid, a peptide, a polypeptide, a protein, a polysaccharide, an antibody, a biopolymer, a nanoparticle, a bead, and the likes. The term “functional moiety” relates to the term “bioactive agent” while considering that a biologic activity, a diagnostic activity and / or an imaging activity, effected or exerted in vitro and / or in vivo, is generally regarded as a biologic activity. In the context of the present embodiments, the terms "bioactive agent", and "pharmaceutically active agent" are used interchangeably. In some embodiments the bioactive agent is a drug. In some preferred embodiments the bioactive agent is an antibody, constituting an antibody-drug conjugate (ADC).

[0253] As used herein, in some embodiments, the terms “functional moiety”, "bioactive agent" and “drug” refer to small molecules or large or small biomolecules / polymers that alter, inhibit, activate, or otherwise affect a biological mechanism or event. Bioactive agent that can be tethered to the conjugate, according to embodiments of the present invention, include, but are not limited to, anti-cancer substances for all types and stages of cancer and cancer treatments (chemotherapeutic, proliferative, acute, genetic, spontaneous etc.), anti-proliferative agents, photosensitizing agents, chemosensitizing agents, anti-inflammatory agents (including steroidal and non-steroidal anti-inflammatory agents and anti-pyretic agents), antimicrobial agents (including antibiotics, antiviral, antifungal, anti-parasite, anti-protozoan etc.), anti-oxidants, hormones, anti-hypertensive agents, anti-AIDS substances, anti-diabetic substances, immunosuppressants, enzyme inhibitors, neurotoxins, opioids, hypnotics, anti-histamines, lubricants, tranquilizers, anti-convulsants, muscle relaxants and anti-Parkinson substances, antipruritic agents, anti-spasmodics and muscle contractants including channel blockers, miotics and anti-cholinergics, anti-glaucoma compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, vitamins, vasodilating agents, inhibitors of DNA, RNA or protein synthesis, analgesics, anti-angiogenic factors, anti- secretory factors, anticoagulants and / or anti -thrombotic agents, anesthetics, ophthalmics, prostaglandins, anti-depressants, anti-psychotic substances, anti-emetics, radioactive agents and imaging agents. A more comprehensive listing of exemplary drugs suitable for use in the present invention may be found in “Pharmaceutical Substances: Syntheses, Patents, Applications” by Axel Kleemann and Jurgen Engel, Thieme Medical Publishing, 1999; the “Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals”, edited by Susan Budavari et al., CRC Press, 1996, and the United States Pharmacopeia-25 / National Formulary-20, published by the United States Pharmcopeial Convention, Inc., Rockville Md., 2001.

[0254] As used herein, the term “small molecule” refers to molecules, whether naturally-occurring or artificially created (e.g., via chemical synthesis), that have a relatively low molecular weight. Typically, small molecules are monomeric and have a molecular weight of less than about 1500 Da. Preferred small molecules are biologically active in that they produce a local or systemic effect in animals, preferably mammals, more preferably humans. In certain preferred embodiments, the small molecule is a drug. Preferably, though not necessarily, the drug is one that has already been deemed safe and effective for use by the appropriate governmental agency or body. For example, drugs for human use listed by the FDA under 21 C.F.R. §§330.5, 331 through 361, and 440 through 460; drugs for veterinary use listed by the FDA under 21 C.F.R. §§500 through 589, are all considered acceptable for use in accordance with the present invention.

[0255] Anti-cancer drugs that can be linked and controllably released from the conjugate according to some embodiments of the invention include, but are not limited to Chlorambucil; 3- (9-Acridinylamino)-5-(hydroxymethyl)aniline; Azatoxin; Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflomithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-nl; Interferon Alfa-n3; Interferon Beta- I a; Interferon Gamma- I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofuirin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinl eurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride. Additional antineoplastic agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's "The Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division).

[0256] Non-limiting examples of chemotherapeutic agents that can be efficiently delivered by the conjugates of the present invention, include amino containing chemotherapeutic agents such as camptothecin, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, anthracycline, mitomycin C, mitomycin A, 9-amino aminopertin, antinomy cin, N8-acetyl spermidine, l-(2- chloroethyl)-l,2-dimethanesulfonyl hydrazine, bleomycin, tally somucin, and derivatives thereof; hydroxy containing chemotherapeutic agents such as etoposide, irinotecan, topotecan, 9-amino camptothecin, paclitaxel, docetaxel, esperamycin, l,8-dihydroxy-bicyclo[7.3.1]trideca-4-ene-2,6- diyne-13-one, anguidine, morpholino-doxorubicin, vincristine and vinblastine, and derivatives thereof, sulfhydril containing chemotherapeutic agents and carboxyl containing chemotherapeutic agents. Additional chemotherapeutic agents include, without limitation, an alkylating agent such as a nitrogen mustard, an ethylenimine and a methylmelamine, an alkyl sulfonate, a nitrosourea, and a triazene; an antimetabolite such as a folic acid analog, a pyrimidine analog, and a purine analog; a natural product such as a vinca alkaloid, an epipodophyllotoxin, an antibiotic, an enzyme, a taxane, and a biological response modifier; miscellaneous agents such as a platinum coordination complex, an anthracenedione, an anthracycline, a substituted urea, a methyl hydrazine derivative, or an adrenocortical suppressant; or a hormone or an antagonist such as an adrenocorticosteroid, a progestin, an estrogen, an antiestrogen, an androgen, an antiandrogen, a gonadotropin-releasing hormone analog, bleomycin, doxorubicin, paclitaxel, 4-OH cyclophosphamide and cisplatinum.

[0257] Anti-inflammatory drugs that can be linked and controllably released from the conjugate according to some embodiments of the invention include, but are not limited to Alclofenac; Alclometasone Dipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide; Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac; Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen; Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide; Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate; Clobetasone Butyrate; Clopirac; Cloticasone Propionate; Cormethasone Acetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone; Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium; Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate; Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab; Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole; Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac; Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate; Flunixin; Flunixin Meglumine; Fluocortin Butyl; FluoromethoIone Acetate; Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate; Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; Halopredone Acetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol; Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole; Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen; Lofemizole Hydrochloride; Lomoxicam; Loteprednol Etabonate; Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate; Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate; Momiflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone; Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone; Paranyline Hydrochloride; Pentosan Polysulfate Sodium; Phenbutazone Sodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; Piroxicam Olamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone; Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex; Salnacedin; Salsalate; Sanguinarium Chloride; Seclazone; Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate; Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam; Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; Tolmetin Sodium; Triclonide; Triflumidate; Zidometacin; and Zomepirac Sodium.

[0258] Suitable antimicrobial agents, including antibacterial, antifungal, antiprotozoal and antiviral agents, for use in context of the present invention include, without limitation, beta-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin, tetracycline, erythromycin, amikacin, triclosan, doxycycline, capreomycin, chlorhexidine, chlortetracycline, oxytetracycline, clindamycin, ethambutol, metronidazole, pentamidine, gentamicin, kanamycin, lineomycin, methacycline, methenamine, minocycline, neomycin, netilmicin, streptomycin, tobramycin, and miconazole. Also included are tetracycline hydrochloride, farnesol, erythromycin estolate, erythromycin stearate (salt), amikacin sulfate, doxycycline hydrochloride, chlorhexidine gluconate, chlorhexidine hydrochloride, chlortetracycline hydrochloride, oxytetracycline hydrochloride, clindamycin hydrochloride, ethambutol hydrochloride, metronidazole hydrochloride, pentamidine hydrochloride, gentamicin sulfate, kanamycin sulfate, lineomycin hydrochloride, methacycline hydrochloride, methenamine hippurate, methenamine mandelate, minocycline hydrochloride, neomycin sulfate, netilmicin sulfate, paromomycin sulfate, streptomycin sulfate, tobramycin sulfate, miconazole hydrochloride, amanfadine hydrochloride, amanfadine sulfate, triclosan, octopirox, parachlorometa xylenol, nystatin, tolnaftate and clotrimazole and mixtures thereof.

[0259] Non-limiting examples of anti-oxidants that are usable in the context of the present invention include ascorbic acid (vitamin C) and its salts, ascorbyl esters of fatty acids, ascorbic acid derivatives (e.g., magnesium ascorbyl phosphate, sodium ascorbyl phosphate, ascorbyl sorbate), tocopherol (vitamin E), tocopherol sorbate, tocopherol acetate, other esters of tocopherol, butylated hydroxy benzoic acids and their salts, 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid (commercially available under the trade name TroloxR), gallic acid and its alkyl esters, especially propyl gallate, uric acid and its salts and alkyl esters, sorbic acid and its salts, lipoic acid, amines (e.g., N,N-diethylhydroxylamine, amino-guanidine), sulfhydryl compounds (e.g., glutathione), dihydroxy fumaric acid and its salts, lycine pidolate, arginine pilolate, nordihydroguaiaretic acid, bioflavonoids, curcumin, lysine, methionine, proline, superoxide dismutase, silymarin, tea extracts, grape skin / seed extracts, melanin, and rosemary extracts.

[0260] Non-limiting examples of vitamins usable in context of the present invention include vitamin A and its analogs and derivatives: retinol, retinal, retinyl palmitate, retinoic acid, tretinoin, iso-tretinoin (known collectively as retinoids), vitamin E (tocopherol and its derivatives), vitamin C (L-ascorbic acid and its esters and other derivatives), vitamin B3 (niacinamide and its derivatives), alpha hydroxy acids (such as glycolic acid, lactic acid, tartaric acid, malic acid, citric acid, etc.) and beta hydroxy acids (such as salicylic acid and the like).

[0261] Non-limiting examples of antihistamines usable in context of the present invention include chlorpheniramine, brompheniramine, dexchlorpheniramine, tripolidine, clemastine, diphenhydramine, promethazine, piperazines, piperidines, astemizole, loratadine and terfenadine.

[0262] Representative examples of hormones include, without limitation, methyltestosterone, androsterone, androsterone acetate, androsterone propionate, androsterone benzoate, androsteronediol, androsteronedi ol-3 -acetate, androsteronediol- 17-acetate, androsteronediol 3-17- diacetate, androsteronediol-17-benzoate, androsteronedione, androstenedione, androstenediol, dehydroepiandrosterone, sodium dehydroepiandrosterone sulfate, dromostanolone, dromostanolone propionate, ethylestrenol, fluoxymesterone, nandrolone phenpropionate, nandrolone decanoate, nandrolone furylpropionate, nandrolone cyclohexane-propionate, nandrolone benzoate, nandrolone cyclohexanecarboxylate, androsteronediol-3-acetate-l-7- benzoate, oxandrolone, oxymetholone, stanozolol, testosterone, testosterone decanoate, 4- dihydrotestosterone, 5a-dihydrotestosterone, testolactone, 17a-m ethyl- 19-nortestosterone and pharmaceutically acceptable esters and salts thereof, and combinations of any of the foregoing.

[0263] Non-limiting examples of analgesic agents that can be efficiently delivered by the conjugates of the present invention, include acetaminophen, alfentanil hydrochloride, aminobenzoate potassium, aminobenzoate sodium, anidoxime, anileridine, anileridine hydrochloride, anilopam hydrochloride, anirolac, antipyrine, aspirin, benoxaprofen, benzydamine hydrochloride, bicifadine hydrochloride, brifentanil hydrochloride, bromadoline maleate, bromfenac sodium, buprenorphine hydrochloride, butacetin, butixirate, butorphanol, butorphanol tartrate, carbamazepine, carbaspirin calcium, carbiphene hydrochloride, carfentanil citrate, ciprefadol succinate, ciramadol, ciramadol hydrochloride, clonixeril, clonixin, codeine, codeine phosphate, codeine sulfate, conorphone hydrochloride, cyclazocine, dexoxadrol hydrochloride, dexpemedolac, dezocine, diflunisal, dihydrocodeine bitartrate, dimefadane, dipyrone, doxpicomine hydrochloride, drinidene, enadoline hydrochloride, epirizole, ergotamine tartrate, ethoxazene hydrochloride, etofenamate, eugenol, fenoprofen, fenoprofen calcium, fentanyl citrate, floctafenine, flufenisal, flunixin, flunixin meglumine, flupirtine maleate, fluproquazone, fluradoline hydrochloride, flurbiprofen, hydromorphone hydrochloride, ibufenac, indoprofen, ketazocine, ketorfanol, ketorolac tromethamine, letimide hydrochloride, levomethadyl acetate, levomethadyl acetate hydrochloride, levonantradol hydrochloride, levorphanol tartrate, lofemizole hydrochloride, lofentanil oxalate, lorcinadol, lornoxicam, magnesium salicylate, mefenamic acid, menabitan hydrochloride, meperidine hydrochloride, meptazinol hydrochloride, methadone hydrochloride, methadyl acetate, methopholine, methotrimeprazine, metkephamid acetate, mimbane hydrochloride, mirfentanil hydrochloride, molinazone, morphine sulfate, moxazocine, nabitan hydrochloride, nalbuphine hydrochloride, nalmexone hydrochloride, namoxyrate, nantradol hydrochloride, naproxen, naproxen sodium, naproxol, nefopam hydrochloride, nexeridine hydrochloride, noracymethadol hydrochloride, ocfentanil hydrochloride, octazamide, olvanil, oxetorone fumarate, oxycodone, oxycodone hydrochloride, oxycodone terephthalate, oxymorphone hydrochloride, pemedolac, pentamorphone, pentazocine, pentazocine hydrochloride, pentazocine lactate, phenazopyridine hydrochloride, phenyramidol hydrochloride, picenadol hydrochloride, pinadoline, pirfenidone, piroxicam olamine, pravadoline maleate, prodilidine hydrochloride, profadol hydrochloride, propiram fumarate, propoxyphene hydrochloride, propoxyphene napsylate, proxazole, proxazole citrate, proxorphan tartrate, pyrroliphene hydrochloride, remifentanil hydrochloride, salcolex, salethamide maleate, salicylamide, salicylate meglumine, salsalate, sodium salicylate, spiradoline mesylate, sufentanil, sufentanil citrate, talmetacin, talniflumate, talosalate, tazadolene succinate, tebufelone, tetrydamine, tifurac sodium, tilidine hydrochloride, tiopinac, tonazocine mesylate, tramadol hydrochloride, trefentanil hydrochloride, trolamine, veradoline hydrochloride, verilopam hydrochloride, volazocine, xorphanol mesylate, xylazine hydrochloride, zenazocine mesylate, zomepirac sodium and zucapsaicin.

[0264] Non-limiting examples of photosensitizers include photofrin, photoporphyrin, benzoporphyrin, tookad, antrin, purlytin, foscan, and halogenated dyes disclosed, e.g. in U.S. Patent Nos 9,572,881, 9,040,721, 8,962,797, 8,748,446 and EP2850061.

[0265] According to some preferred embodiments of the present invention, the conjugate includes a somatostatin analog as a functional moiety. In other preferred embodiments, the conjugate includes a Dolastatin 10 analog linked via a -CH2- on its thiazole moiety, and a octreotide moiety, conjugated via a disulfide linking moiety. Exemplary conjugates, according to some embodiments of the present invention, include without limitation conjugate 8b, conjugate 8c, or conjugate 8d, which have been prepared and their biological activity characterized and analysed, as demonstrated in the Examples section that follows below.

[0266] Targeting moiety:

[0267] In some embodiments, the functional moiety is a moiety of a bioactive agent which exhibits an affinity to a specific biologic / chemical target, and therefore it is referred to herein as a targeting moiety. As used herein, the term "targeting moiety" describes a molecular entity that exhibits an affinity to a desired molecular or bodily site (e.g., particular organ, cells, tissues, organelles, biopolymers, receptors or the likes). In some embodiments, a targeting moiety is specific to certain targets. The target is typically a biomolecule that occurs at a higher concentration or exclusively at the targeted bodily site. In some embodiments, the targeting moiety is a biomolecule or a derivative thereof that has a specific and relatively high affinity to the target.

[0268] Targeting moieties are often employed as the bimolecular carrier in order to direct a drug to specific structures in the body or sites of physiological functions. According to some embodiments, a targeting moiety is a compound with structure or site specific reactivity.

[0269] Exemplary targeting agents include, without limitation, peptides, proteins, porphyrins, hormones, antigens, haptens, antibodies and fragments thereof, DNA fragments, RNA fragments and analogs and derivatives thereof, and any receptor ligands that bind to receptors that are expressed specifically or more abundantly at the targeted bodily sites.

[0270] As used herein, the term “biomolecule” refers to molecules (e.g., polypeptides, amino acids, polynucleotides, nucleotides, polysaccharides, sugars, lipids, nucleoproteins, glycoproteins, lipoproteins, steroids, metabolites, etc.) whether naturally-occurring or artificially created (e.g., by synthetic or recombinant methods) that are commonly found in cells and tissues. Specific classes of biomolecules include, but are not limited to, enzymes, receptors, neurotransmitters, hormones, cytokines, cell response modifiers such as growth factors and chemotactic factors, antibodies, vaccines, haptens, toxins, interferons, ribozymes, anti-sense agents, plasmids, DNA, and RNA.

[0271] In some embodiments, a targeting moiety comprises a cell-internalizing moiety, such that the molecular structure can more readily penetrate a targeted cell. Exemplary cell-internalizing moieties include, without limitation, positively charges (at physiological environment) moieties such as guanidines and amines, and moieties containing same (e.g., arginine and lysine).

[0272] In some embodiments, the targeting moiety exhibits a specific affinity to cancerous cells and neoplastic tissues. Such targeting moieties may be used to target the molecular structure presented herein, thereby delivering anticancerous bioactive agents, according to some embodiments of the present invention, to cancerous cells and tissues. The result is an enhanced effect and an improved exposure of the cancerous cells and neoplastic tissues to the anticancerous bioactive agent, preferably accompanied by reduced exposure of non-cancerous cells to the anticancerous bioactive agents.

[0273] A class of compounds that is suitable as targeting moieties, according to some embodiments of the present invention, are short peptides and peptide analogs, generally referred to herein as peptidomimetic compounds, that display more favorable pharmacological properties than their prototype native peptides. The native peptide itself, the pharmacological properties of which have been optimized, generally serves as a lead for the development of these peptidomimetics. In general, a small number of amino acids (usually four to eight) are responsible for the biological activity (recognition and binding; targeting) of a peptide ligand (targeting moiety) by a receptor (target). Once this biologically active site is determined, a lead structure for development of peptidomimetic can be optimized, for example by molecular modeling programs. U.S. Patent Nos. 5,811,392, 6,407,059 and 7,084,244, which are incorporated herein by reference in their entirety, describe the preparation and use of a class of cyclic peptidomimetic targeting moieties, which can be used in the context of some embodiments of the present invention.

[0274] Peptide nucleic acid (PNA) constitute an exemplary class of targeting moiety that may be used in the context of some embodiments of the present invention. U.S. Patent No. 6,395,474, which is incorporated herein by reference in its entirety, describes PNA as an analogue of DNA in which the phosphodiester backbone of DNA is replaced with a pseudo-peptide such as N-(2- amino-ethyl)-glycine. Methylenecarbonyl linkers attach DNA, RNA, or synthetic nucleobases to the polyamide backbone. PNA, obeying Watson-Crick hydrogen bonding rules, mimics the behavior of DNA and RNA by binding to complementary nucleic acid sequences such as those found in DNA, RNA, and other PNAs. An exemplary molecular structure utilizing PNA, according to some embodiments of the present invention, may bind, for example, to a specific mutated nucleic acid sequence found in the DNA of a cancerous tumor.

[0275] One example of a class of targeting moieties, which can be used advantageously in the context of embodiments of the present invention, is the family of tumor-targeting moieties that bind selectively to avp3 and avp5 integrins, referred to herein as the RGD (Arg-Gly-Asp) family [Arap, W. et al., Science, 1998, 279(5349):377-80], Short peptides and peptidomimetic analogs, which are based on the RGD motif and exhibit is biological binding activity, can be used as targeting moieties in a molecular structure, according to some embodiments of the present invention, to inhibit the growth and possibly eradicate tumors in the treatment of cancer.

[0276] Additional targeting moieties, which can be used effectively in the context of the molecular structures presented herein for treating cancer, are described in the literature [e.g., “Novel Oncology Therapeutics: Targeted Drug Delivery for Cancer '', Journal of Drug Delivery, Vol. 2013, 2013],

[0277] Non-limiting examples of targeting moieties which are useful in the context of some embodiments of the present invention include octreotide (OCT), lanreotide, pasireotide, vapreotide, cilengitide analog c(RGDfK), and luteinizing Hormone-Releasing Hormone (LHRH), bombesin, and arginine-glycine-aspartic acid (RGD).

[0278] Advantageous process of preparation:

[0279] According to embodiments of the present invention, the compound provided herein, which is a Dolastatin 10 analog, can be prepared using standard and / or modified solid-state peptide synthesis (SSPS) techniques and reagents, as demonstrated in the Examples section that follows. In addition to the compound, some of the functional moieties contemplated above and demonstrated below, can also be prepared by SSPS techniques and reagents, as also demonstrated in the Examples section that follows.

[0280] The use of SSPS allows diversification of the product, namely allows relatively simple exploration of analogs space aiming at discovering new compounds with improved bioactivity and ADME-Tox properties, and optimization thereof. For example, since the conjugate, according to some embodiments of the present invention, may comprise a number of naturally occurring and / or synthetic amino-acid residues, as well as other conjugate elements that are built into the structure by SSPS, such as the linker, it would be relatively simple to form a family of analogous conjugates using standard or high-throughput SSPS techniques.

[0281] Hence, according to some embodiments of the present invention, there is provided a process for producing the compound provided herein, using SSPS approach. The process may include a step of attaching a first peptide unit of the compound to a solid support resin, and thereafter adding the next peptide units sequentially before releasing the resulting compound from the resin. The first peptide unit of the compound provided herein to be attached the resin may be an amine-protected variant of propargyl amine than subsequently undergoes Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to afford first triazole building unit onto resin. The following peptide units to be coupled to the first unit may be an amine-protected derivative of (2R,3R)-3- methoxy-2-methyl-3-((S)-pyrrolidin-2-yl)propanoic acid, followed by an amine-protected derivative of (3R,4S,5S)-3-methoxy-5-methyl-4-(methylamino)heptanoic acid, followed by an amine-protected derivative of Val-OH, followed by dimethyl-Val to afford DT-NH2 after cleaving the nascent peptide from the resin.

[0282] In embodiments wherein the conjugate comprises a peptide-like functional moiety, the same approach h may be applied, starting from constructing the functional moiety or from the Dolastatin 10 analog side, and working the way down the sequence to afford a complete conjugate.

[0283] The linker moiety may also be part of the SSPS process, or alternatively, the two major moiety of the conjugate may be synthesized separately, by SSPS or otherwise, and thereafter be coupled to form a linking moiety.

[0284] Medical conditions:

[0285] The compound presented herein can be used to treat any medical condition that is treatable by Dolastatin 10, by administration of a therapeutically effective amount of the compound presented herein to a subject in need thereof. The analog, according to some embodiments of the present invention, can also be used to prepare a pharmaceutical composition comprising the same and a pharmaceutically acceptable carrier and other optional ingredients. Thus, the compound presented herein can be used as an active ingredient in a method of treating any medical condition that is treatable by Dolastatin 10, by administering a therapeutically effective amount thereof to the subject in need thereof.

[0286] The conjugate presented herein can also be used to treat any medical condition that is treatable by administration of a bioactive agent (drug) with Dolastatin 10 or an analog thereof, according to some embodiments of the present invention. According to some embodiments of the present invention, it is advantageous to use the conjugate to treat medical conditions, which are treatable by administration of a combination of drugs. In some embodiments, the medical condition includes an autoimmune disease, a genetic disease, a degenerative disease, a psychiatric or mental disease or condition. In some embodiments, the medical condition includes a peptic ulcer disease, Alzheimer’s disease, rheumatoid arthritis, post-traumatic stress disorder, Crohn's disease, tuberculosis, leprosy, malaria and HIV / AIDS. According to some embodiments, the degenerative disease includes Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS), a.k.a., Lou Gehrig's Disease, Osteoarthritis, Atherosclerosis, Cancer, Charcot Marie Tooth Disease (CMT), Chronic Obstructive Pulmonary Disease (COPD), Chronic traumatic encephalopathy, Diabetes, Ehlers-Danlos Syndrome, Essential tremor, Friedreich's ataxia, Leg Disease, Huntington's Disease, Inflammatory Bowel Disease (IBD), Keratoconus, Keratoglobus, Macular degeneration, Marfan's Syndrome, Multiple sclerosis, Multiple system atrophy, Muscular dystrophy, Niemann Pick disease, Osteoporosis, Parkinson's Disease, Progressive supranuclear palsy, Prostatitis, Retinitis Pigmentosa, Rheumatoid Arthritis, and Tay-Sachs Disease.

[0287] According to some embodiments, the autoimmune disease includes Acute Disseminated Encephalomyelitis (ADEM), Acute necrotizing hemorrhagic leukoencephalitis, Addison’s disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti- GBM / Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (AIED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease, Autoimmune urticaria, Axonal & neuronal neuropathies, Balo disease, Behcet’s disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid / benign mucosal pemphigoid, Crohn’s disease, Cogans syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic’s disease (neuromyelitis optica), Discoid lupus, Dressier’s syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis (GPA) (formerly called Wegener’s Granulomatosis), Graves’ disease, Guillain-Barre syndrome, Hashimoto’s encephalitis, Hashimoto’s thyroiditis, Hemolytic anemia, Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (SLE), Lyme disease, chronic, Meniere’s disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic’s), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Reiter’s syndrome, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren’s syndrome, Sperm & testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac’s syndrome, Sympathetic ophthalmia, Takayasu’s arteritis, Temporal arteritis / Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, Type 1 diabetes, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vesiculobullous dermatosis, Vitiligo and Wegener’s granulomatosis (now termed Granulomatosis with Poly angiitis (GPA).

[0288] In some embodiments of the present invention, the medical condition is associated with an infection caused by a pathogenic microorganism, including a viral infection, a bacterial infection, a yeast infection, a fungal infection, a protozoan infection, a parasite-related infection and the like.

[0289] Medical conditions associated with a pathogenic microorganism include, without limitation, actinomycosis, anthrax, aspergillosis, bacteremia, bacterial, bacterial skin diseases, bartonella infections, botulism, brucellosis, burkholderia infections, Campylobacter infections, candidiasis, cat-scratch disease, chlamydia infections, cholera, Clostridium infections, coccidioidomycosis, cryptococcosis, dermatomycoses, dermatomycoses, diphtheria, ehrlichiosis, epidemic louse borne typhus, Escherichia coli infections, fusobacterium infections, gangrene, general infections, general mycoses, gram-negative bacterial infections, Gram-positive bacterial infections, histoplasmosis, impetigo, klebsiella infections, legionellosis, leprosy, leptospirosis, listeria infections, lyme disease, maduromycosis, melioidosis, mycobacterium infections, mycoplasma infections, necrotizing fasciitis, nocardia infections, onychomycosis, ornithosis, pneumococcal infections, pneumonia, pseudomonas infections, Q fever, rat-bite fever, relapsing fever, rheumatic fever, rickettsia infections, Rocky-mountain spotted fever, salmonella infections, scarlet fever, scrub typhus, sepsis, sexually transmitted bacterial diseases, staphylococcal infections, streptococcal infections, surgical site infection, tetanus, tick-borne diseases, tuberculosis, tularemia, typhoid fever, urinary tract infection, vibrio infections, yaws, yersinia infections, Yersinia pestis plague, zoonoses and zygomycosis.

[0290] Non-limiting examples of pathogenic fungi include genus Absidia: Absidia corymbifera: genus Ajellomyces: Ajellomyces capsulatus, Ajellomyces dermatitidis,' genus Arthroderma: Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii,' genus Aspergillus: Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, genus Blastomyces: Blastomyces dermatitidis,' genus Candida: Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida pelliculosa,' genus Cladophialophora: Cladophialophora carrioniv, genus Coccidioides: Coccidioides immitis,' genus Cryptococcus: Cryptococcus neoformans,' genus Cunninghamella: Cunninghamella sp.,' genus Epidermophyton: Epidermophyton floccosunv, genus Exophiala: Exophiala dermatitidis,' genus Filobasidiella: Filobasidiella neoformans,' genus Fonsecaea: Fonsecaea pedrosoi,' genus Fusarium: Fusarium solani,' genus Geotrichum: Geotrichum candidum,' genus Histoplasma: Histoplasma capsulatunr, genus Hortaea: Hortaea werneckii,' genus Issatschenkia: Issatschenkia orientalis,' genus Madurella: Madurella grisae,' genus Malassezia: Malassezia furfur, Malassezia globosa, Malassezia obtusa, Malassezia pachydermatis, Malassezia restricta, Malassezia slooffiae, Malassezia sympodialis,' genus Microsporum: Microsporum canis, Microsporum fulvum, Microsporum gypseum,' genus Mucor: Mucor circinelloides,' genus Nectria: Nectria haematococca,' genus Paecilomyces: Paecilomyces variotii,' genus Paracoccidioides: Paracoccidioides brasiliensis,' genus Penicillium: Penicillium marneffei,' genus Pichia, Pichia anomala, Pichia guilliermondii,' genus Pneumocystis: Pneumocystis carinii,' genus Pseudallescheria: Pseudallescheria boydii,' genus Rhizopus: Rhizopus oryzae,' genus Rhodotorula: Rhodotorula rubra,' genus Scedosporium: Scedosporium apiospermum,' genus Schizophyllum: Schizophyllum commune,' genus Sporothrix: Sporothrix schenckii,' genus Trichophyton: Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton verrucosum, Trichophyton violaceum,' and genus Trichosporon: Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin, Trichosporon mucoides. Non-limiting examples of other pathogenic microorganism include Acanthamoeba and other free-living amoebae, Aeromonas hydrophila, Anisakis and related worms, Ascaris lumbricoides, Bacillus cercus, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cryptosporidium parvum, Cyclospora cayetanensis, Diphyllobothrium, Entamoeba histolytica, Eustrongylides, Giardia lamblia, Listeria monocytogenes, Nanophyetus, Plesiomonas shigelloides, Salmonella, Shigella, Staphylococcus aureus, Streptococcus, Trichuris trichiura, Vibrio cholerae, Vibrio parahaemolyticus , Vibrio vulnificus and other vibrios, Yersinia enterocolitica and Yersinia pseudotuberculosis.

[0291] Cancer treatment and chemotherapy:

[0292] In some embodiments of the present invention, the medical condition is associated with malignant cells and tumors, collectively referred to herein as cancer.

[0293] To date, chemotherapy remains the most common and most frequently used in cancer treatment, alone or in combination with other therapies. Currently available anticancer chemotherapies act by affecting specific molecular targets in proliferating cancer cells, leading to inhibition of essential intracellular processes such as DNA transcription, synthesis and replication.

[0294] Unfortunately, anticancerous drugs are highly toxic, as they are designed to kill mammalian cells, and are therefore harmful also to normal proliferating cells resulting in debilitating and even lethal side effects. Some of these adverse effects are gastrointestinal toxicity, nausea, vomiting, and diarrhea when the epithelial lining of the intestine is affected. Other side effects include alopecia, when the hair follicles are attacked, bone marrow suppression and neutropenia due to toxicity of hematopoietic precursors. Therefore, the effectiveness of currently used anticancerous drugs is dose-limited due to their toxicity to normal rapidly growing cells. The use of a conjugate according to embodiments of the present invention, can optimize the balance between the desired anticancer activity of certain anticancer drugs and their adverse side effects, by quantitative determination of the actual amount of drug released in the targeted cells.

[0295] One of the contemporary approaches in the fight against cancer is engineering of molecular targeted drugs that permeate cancer cells and specifically modulate activity of molecules that belong to signal-transduction pathways. These targets include products of frequently mutated oncogenes, such as k-Ras and other proteins that belong to tyrosine kinase signal transduction pathways. For example, Imatinib (Gleevec®), is the first such drug, approved for treatment of chronic myelogenous leukemia (CML). Imatinib blocks the activity of non-receptor tyrosine kinase BCR-Abl oncogene, present in 95 % of patients with CML. Imatinib was found to be effective in the treatment of CML and certain tumors of the digestive tract. Nevertheless, as others, this new compound is not completely specific to its target; therefore side effects emerge, including severe congestive cardiac failure, pulmonary tuberculosis, liver toxicity, sweet syndrome (acute febrile neutrophilic dermatosis), leukocytosis, dermal edemas, nausea, rash and musculoskeletal pain.

[0296] Angiogenesis inhibitors are currently investigated for their use in cancer treatment and to date, one anti-angiogenetic drug, Bevacizumab (Avastin®), was approved for the treatment of solid tumors in combination with standard chemotherapy. However, as in all chemotherapeutic drugs, Bevacizumab causes a number of adverse side effects such as hypertension, blood clots, neutropenia, neuropathy, proteinuria and bowel perforation.

[0297] In some embodiments, the functional moiety of the conjugates presented herein, is a targeting moiety that is responsible for the higher concentration of the conjugate at the targeted bodily site compared to non-targeted bodily sites, thereby reducing the adverse side effects associated with the toxicity of the anti-cancer drugs attached thereto. In addition, the linking moieties attached the anti-cancer drugs to the conjugate are selected such that they cleave in conditions that are present at the targeted site more so than in non-targeted sites, thereby releasing the payload of drugs at the targeted site at a higher rate compared to non-targeted sites.

[0298] In the context of some embodiments of the present invention, the term “cancer” refers, but not limited to acute lymphoblastic, acute lymphoblastic leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendix cancer, basal-cell carcinoma, bladder cancer, brain cancer, brainstem glioma, breast cancer, bronchial adenomas / carcinoids, Burkitt's lymphoma, carcinoid tumor, cerebellar or cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondrosarcoma, chronic lymphocytic or chronic lymphocytic leukemia, chronic myelogenous or chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial uterine cancer, ependymoma, esophageal cancer, Ewing's sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic tumor, glioma of the brain stem, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, Islet cell carcinoma, Kaposi sarcoma, laryngeal cancer, leukaemia, lip and oral cavity cancer, liposarcoma, lymphoma, male breast cancer, malignant mesothelioma, medulloblastoma, melanoma, Merkel cell skin carcinoma, mesothelioma, metastatic squamous neck cancer, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic / myeloproliferative diseases, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, nonmelanoma skin cancer, non-small cell lung cancer, oligodendroglioma, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma, ovarian cancer, ovarian germ cell tumor, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian low malignant potential tumor, pancreatic cancer, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary adenoma, plasma cell neoplasia, pleuropulmonary blastoma, primary carcinoma, primary central nervous system lymphoma, primary liver cancer, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sezary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, supratentorial primitive neuroectodermal tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom macroglobulinemia and Wilms tumor.

[0299] Definitions:

[0300] As used herein the term “about” or “approximately,” refers to ±10 %. For example, the term “about 100 units” encompasses the value 100 units, as well as the values 90 units, 91 units, 92 units, 93 units, 94 units, 95 units, 96 units, 97 units, 98 units, 98 units, 99 units, 100 units, 101 units, 102 units, 103 units, 104 units, 105 units, 106 units, 107 units, 108 units, 109 units, and 110 units.

[0301] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to"; namely, as used herein, these terms are intended to be open-ended and not limiting. They indicate that the presence of the listed elements does not preclude the inclusion of additional, unrecited elements or method steps.

[0302] The term “consisting of’ means “including and limited to”.

[0303] The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0304] The phrase “one or more” as used herein includes one, two, three, or more of the described elements or components and does not exclude any combinations or sub-combinations thereof. The terms “preferred” or “preferably” indicate an example or embodiment that is more suitable or favorable under certain circumstances, but these terms are not intended to limit the scope of the invention or to suggest that other variations are excluded.

[0305] As used herein, the phrase “selected from the group consisting of’ includes all members of the recited group, each member of the recited group, and all possible combinations. For example, selected from the group consisting of A, B, and C, includes A, only, as well as B, only, as well as C, only, as well as A and B, as well as A and C, as well as B and C, and as well as A, B, and C.

[0306] The term “substantially,” when used in reference to a characteristic or parameter, means that the characteristic or parameter need not be absolute but is close enough to the specified value or condition so as to achieve the intended purpose or effect.

[0307] As used herein, the phrases "substantially devoid of' and / or "essentially devoid of in the context of a certain substance, refer to a composition that is totally devoid of this substance or includes less than about 5, 1, 0.5 or 0.1 percent of the substance by total weight or volume of the composition. Alternatively, the phrases "substantially devoid of and / or "essentially devoid of in the context of a process, a method, a property or a characteristic, refer to a process, a composition, a structure or an article that is totally devoid of a certain process / method step, or a certain property or a certain characteristic, or a process / method wherein the certain process / method step is effected at less than about 5, 1, 0.5 or 0.1 percent compared to a given standard process / method, or property or a characteristic characterized by less than about 5, 1, 0.5 or 0.1 percent of the property or characteristic, compared to a given standard. Further alternatively, the terms "substantially" and / or "essentially " in the context of a characterizing property, means that the characterizing property is expressed to at least 99 %, at least 95 %, at least 90 % of its full or complete expression. For example, the phrase “the particles are maintained substantially in a non-contacting, spaced-apart arrangement” should be read as “at least 99 % of the particles are maintained in a non-contacting, spaced-apart arrangement”.

[0308] As used herein, the phrases "substantially devoid of and / or "essentially devoid of in the context of a certain substance, refer to a composition that is totally devoid of this substance or includes less than about 5, 1, 0.5 or 0.1 percent of the substance by total weight or volume of the composition. Alternatively, the phrases "substantially devoid of and / or "essentially devoid of in the context of a process, a method, a property or a characteristic, refer to a process, a composition, a structure or an article that is totally devoid of a certain process / method step, or a certain property or a certain characteristic, or a process / method wherein the certain process / method step is effected at less than about 5, 1, 0.5 or 0.1 percent compared to a given standard process / method, or property or a characteristic characterized by less than about 5, 1, 0.5 or 0.1 percent of the property or characteristic, compared to a given standard.

[0309] When applied to an original property, or a desired property, or an afforded property of an object or a composition, the term “substantially maintaining”, as used herein, means that the property has not change by more than 20 %, 10 % or more than 5 % in the processed object or composition.

[0310] The term “exemplary” is used herein to mean “serving as an example, instance or illustration”. Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and / or to exclude the incorporation of features from other embodiments.

[0311] The words “optionally” or “alternatively” are used herein to mean “is provided in some embodiments and not provided in other embodiments”. Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.

[0312] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0313] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0314] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0315] As used herein the terms “process” and "method" refer to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, material, mechanical, computational and digital arts.

[0316] Terms used in the singular form shall also include the plural, and vice versa, unless context clearly indicates otherwise. Furthermore, words of any gender include all genders and are intended to cover all corresponding terms.

[0317] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0318] It is expected that during the life of a patent maturing from this application Dolastatin 10 analogs as provided herein, and conjugates comprising the same, will be developed and the scope of the term “Dolastatin 10 analog” is intended to include all such new technologies a priori.

[0319] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0320] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental and / or calculated support in the following examples.

[0321] EXAMPLES

[0322] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0323] EXAMPLE 1

[0324] Synthesis of Dolastatin analog DT-NH2

[0325] DT-NH2, an exemplary Dolastatin 10 analog according to some embodiments of the present invention (see, Table 1 hereinabove), was synthesized on solid support (Cl-Trt resin), performed by in situ generation of triazole core. The procedure started by loading the alkyne chain propargyl amine on 100 mg of Cl-Trt resin (100-200 mesh, 0.7 -0.8 mmol / g loading; DCM:DMF 1 :1), and subsequently react (9H-fluoren-9-yl)methyl (S)-(l-azido-3-phenylpropan-2- yl)carbamate (BU 2), following the procedure proposed by Sureshbabu, V. V. et al. [Protein Pept. Lett., 17(4), pp. 499-506] under Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to afford the resin-loaded triazole tether after deprotection (Fmoc removal).

[0326] Thereafter, CuBr (0.126 mmol), Na-Ascorbate (0.378 mmol), DIPEA (1.26 mmol) and 2,6- Lutidine (1.26 mmol) were added to DMF, and loaded to resin. The mixture was shaken for 8 hours, and after completion of the reaction the solvent was removed by filtration and further washings with DCM (3x3 mL). The Fmoc protecting group was removed by treatment with 20 % piperidine in DMF (2 x 15 min, 5 mL each) and subsequent washing (2 x CH2C12, 2 x DMF, 5 mL each).

[0327] BU-2 BU-3 BU-4

[0328] A solution of corresponding amino acid (0.25 mmol) and HATU, 95 mg (0.25 mmol), in dry DMF (4 mL) was added in a jacketed fritted peptide vessel to the preloaded resin from the previous step. After addition of DIEA (0.09 mL; 0.52 mmol), the mixture was shaken for 2 hours. After completion of the coupling reaction, the solvent was removed by filtration and further washings with DCM (3x3 mL).

[0329] The following amino acids were sequentially coupled to the emerging compound on the resin using the following procedure: BU-3 (DolaPro), then BU-4 (Dolalle), then Fmoc-Val and finally N,N-dimethyl Valin.

[0330] For the cleavage of the peptide from the resin, the resin was washed before cleavage with 2 x CH2CI2 (5 mL each), dried in air and transferred to a vial for cleavage. The cleavage carried out in cold solution of TFA / TIP / DCM (2.5:2.5:95) (4 mL). After shaking for 1 hour, the solution was collected, and the resin washed with cold TFA (1 mL). After combining the TFA solutions, the solvent evaporated under N2 stream, and the residue was purified on preparative HPLC (AcCN / H2O).

[0331] Scheme 1 below presents that solid-phase peptide synthesis (SPPS) of DT-NH2 (IUPAC: (2S)-N-((4S,5S)-l-((2S)-2-((lS)-3-(((R)-l-(4-(aminomethyl)-lH-l,2,3-triazol-l-yl)-3- phenylpropan-2-yl)amino)-l-methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-3-methoxy-5- methyl- l-oxoheptan-4-yl)-2-((S)-2-(dimethylamino)-3-methylbutanamido)-N, 3- dimethylbutanamide).

[0332] Scheme 3 wherein: a. (i) NH2-CH2-C=CH, Py / DMF (1 : 1), room temperature; 48 hours; b. (i) BU-2, CuBr, Sodium ascorbate, 2,6-Lutidine, DMF, DIPEA, room temperature, 8 hours; (ii) 20 % Piperidine in DMF; c. Fmoc DolaPro (BU-3), HATU, DIEA, DMF, room temperature; (ii) 20 % Piperidine in DMF; d. Fmoc Dolalle (BU-4), HATU, DIEA, DMF, room temperature; (ii) 20 % Piperidine in DMF; e. Fmoc-Val-OH, HATU, DIEA, DMF, room temperature; (ii) 20 % Piperidine in DMF; f. (Me)2Val-OH, HATU, DIEA, DMF, room temperature.; g. 2.5:2.5:95 TFA / TIS / DCM, 0 °C then room temperature, 30 minutes.

[0333] EXAMPLE 2

[0334] Activity ofDT-Nl

[0335] The exemplary Dolastatin 10 analog, DT-NH2, was subjected to an in vitro assay to test its cytotoxicity towards two cancerous cell lines: PANC-1 (malignant pancreatic cell line)) and H1299 (human non-small cell lung carcinoma cell line), MDA-MB-231 (triple negative breast cancer cell line) and PC-3 (prostate cancer cell line). The study was design to assess the activity of a Dolastatin 10 analog, according to some embodiments of the present invention, compared to other Dolastatin 10 derivative known in the art, Dolastatinol (Dolas-Ol; see, WO2021 / 084532, by the present inventor).

[0336] Briefly, PANC-1, H1299, MDA-MB-231and PC-3 cells were exposed to increasing doses of Dolas-Ol and DT-NH2 and at the end of 8 days incubation, metabolic activities of cells were measured using the XTT assay kit. All samples contained DMSO at final concentration <0.05 %. Cells cultures were initiated in microplate wells at a concentration of 2-4xl04cells / well and the cells were allowed to adhere for 24 hours incubation period. At this time the cells were washed, fresh medium containing different concentrations of the tested substances were added and the cells were incubated for additional 8 days. The medium was then removed, all the wells were washed with PBS and cultured for 24 hrs in fresh medium without drugs. The cells were washed again and given a fresh medium containing the XTT reagent and re-incubated for 2-4 h. Absorbance in the wells was measured with a TECAN Infinite M200 ELISA reader at both 480 and 680 nm e, the latter being the background absorbance. The absorbencies were normalized to the absorbance of cells grown in medium and solvent. All the tests were repeated three times in quadruplicates.

[0337] The results are presented in Table 2 in IC50 values in nM.

[0338] Table 2

[0339] As can be seen in Table 2, the analog designed according to the concept presented herein (see, Formula I), appears to be the most active namely exhibit the highest cytotoxicity against cancerous cells.

[0340] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0341] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

[0342] In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A compound represented by general Formula I:wherein:A is represented by a general formula selected from the group consisting of:Formula Al , Formula A2, and Formula A3 ;Ri is a functional group;R2 is selected from the group consisting of aryl, benzyl, heteroaryl, alkyl-aryl, and alkylene-aryl;R3 is an alkyl or H;D is a linear or branched C1-6 alkyl, a 1-6-mer PEG, or absent; andRa is selected from the group consisting of alkyl, aryl or H.

2. The compound of claim 1, wherein Rl is selected from the group consisting of an alkenyl, an alkynyl, a halo, a hydroxyl, a carbonyl, an aldehyde, a haloformyl (acyl halide), a carbonate ester, a carboxylate, a carboxyl, a carboalkoxy (ester), an alkoxy, a hydroperoxy, a peroxy, an ether, a hemiacetal, a hemiketal, an acetal, a ketal, an orthoester, a methylenedioxy, an orthocarbonate ester, a carboxylic anhydride, a carboxamide, an amine, alkyl amine, dialkyl amine trialkyl ammonium, a ketamine, an aldimine, an imide, an azide, an azo (diimide), a cyanate, an isocyanate, a nitrate, a nitrile, an isonitrile, a nitrosooxy, a nitro, a nitroso, an oxime, a pyridyl, a carbamate, a sulfhydryl, a sulfide, a disulfide, a sulfinyl, a sulfonyl, a sulfino, a sulfo, a sulfo-ester,a selenol, a selenide, a selenone, a thiocyanate, an isothiocyanate, a carbonothioyl, a carbonothioyl, a carbothioic S-acid, a carbothioic O-acid, a thiolester, a thionoester, a carbodithioic acid, a carbodithio, a phosphino, a phosphono, a phosphate, and a phosphate.

3. The compound of claim 2, wherein said Ri is selected from the group consisting of hydroxyl, amine, methyl amine, dimethylamine, thiol, carboxyl, and amide.

4. The compound of any one of claims 1-3, wherein D, when present, is selected from the group consisting of -CH2-, -CHCH3-, -C(CH3)2-, -(CH2)2-, -CH2CHCH3-, -CH2C(CH3)2- , -OCH2CH2O-, -(OCH2CH2)2-, -(OCH2CH2)3-, -(CH2)3-, -(CH2)3CH3-, and -(OCH2CH2)4-.

5. The compound of claim 4, wherein D is a methylene group (-CH2-).

6. The compound of any one of claims 1-5, wherein R2is selected from the group consisting of phenyl, benzyl, pyrrole, imidazole, oxazole, diazole, triazole, tetrazole, thiazole, pyridine, diazine, triazine, pyridomethylene and imidazolomethylene.

7. The compound of claim 5, wherein R2is benzyl.

8. The compound of any one of claims 1-7, wherein R3is methyl.

9. The compound of claim 1, wherein A is represented by general Formula Al .

10. The compound of claim 8, wherein:Ri is hydroxyl;R2is benzyl;R3is methyl;D is a methylene group (-CH2-); andRa is hydrogen.

11. The compound of claim 8, wherein:Ri is amine;R2is benzyl;R3is methyl;D is a methylene group (-CH2-); and Ra is hydrogen.

12. The compound of claim 8, wherein:Ri is carboxyl;R2 is benzyl;R3 is methyl;D is absent; andRa is hydrogen.

13. A compound selected from the group consisting of:

14. A conjugate, comprising: a moiety of the compound according to any one of claims 1-13, a moiety of a functional agent, and a linking moiety connecting said compound and said functional agent.

15. The conjugate claim 14, wherein said linking moiety comprises a biocleavable bond or selected from the group consisting of an amide, an ester, a carbamate, a carbonate, a disulfide, a sulfonamide, an ether, a thioether, a valine-citrulline, a hydrazine and an oxyacrylate.

16. The conjugate of claim 15, wherein said linking moiety comprises a disulfide.

17. The conjugate any one of claims 14-16, wherein said functional agent selected from the group consisting of a bioactive agent, a labeling agent and / or a diagnostic agent.

18. The conjugate any one of claims 14-16, wherein said functional agent is selected from the group consisting of a peptide, a protein, an antibody, a biodegradable polymer, a targeting agent, a drug, a dye, a nanoparticle, a bead, a photodynamic therapy sensitizer, radiotherapy agent, a metal complex, an anti-cancer agent, an anti-proliferative agents, chemosensitizing agents, an anti-inflammatory agent, an antimicrobial agent, an anti-oxidant, a hormone, an anti-hypertensive agent, an anti-diabetic agent, an immunosuppressant, an enzyme inhibitor, a neurotoxin and an opioid.

19. The conjugate any one of claims 14-16, wherein said functional agent is an antibody.

20. A process of preparing the compound of any one of claims 1-13, comprising: forming an intermediate moiety corresponding to variable A in Formula I by means of a biorthogonal reaction on a solid support resin; and sequentially attaching BU-3, BU-4, Fmoc-Val and N,N-dimethyl Valin, respectively to said intermediate moiety by means of a solid-phase peptide synthesis (SPPS) reaction, and releasing the compound from said solid support resin, thereby obtaining the compound.

21. The process of claim 20, wherein said biorthogonal reaction is selected from the group consisting of: a copper-catalyzed azide-alkyne Huisgen 1,3-dipolar cycloaddition (CuAAC) reaction; a strain-promoted azide-alkyne cycloadditions (SPAACs) reaction; and a inverse electron demand Diels-Alder (IEDDA) reaction.

22. A process of preparing the conjugate of any one of claims 14-19, comprising coupling the compound of any one of claims 1-13 to said functional moiety, thereby forming said linking moiety.

23. A process of preparing the conjugate of any one of claims 14-19, comprising, forming said functional moiety on a solid-support resin using solid-state peptide synthesis protocols, wherein said functional moiety comprises a linkable group, forming said compound starting from said linkable group, thereby forming said linking moiety on a solid-support resin using solid-state peptide synthesis protocols, and releasing the conjugate from said resin.

24. A pharmaceutical composition comprising the compound of any one of claims 1- 13, and a pharmaceutically acceptable carrier.

25. A pharmaceutical composition comprising the conjugate of any one of claims 14- 19, and a pharmaceutically acceptable carrier.

26. A method of treating a medical condition treatable with Dolastatin 10 in a subject, comprising administering to the subject a therapeutically effective amount of the compound of any one of claims 1-13.

27. A compound of any one of claims 1-13, for use in the treatment of a medical condition that is treatable with Dolastatin 10 in a subject, said treatment comprises administering to the subject a therapeutically effective amount of the compound.

28. A compound of any one of claims 1-13 for use in the preparation of a medicament useful in the treatment of a medical condition treatable with Dolastatin 10 in a subject.

29. A method of treating a medical condition treatable with Dolastatin 10 in a subject, comprising administering to the subject a therapeutically effective amount of the conjugate of any one of claims 14-18.

30. A conjugate of any one of claims 14-18, for use in the treatment of a medical condition that is treatable with Dolastatin 10 in a subject, said treatment comprises administering to the subject a therapeutically effective amount of the conjugate.

31. A conjugate of any one of claims 14-18, for use in the preparation of a medicament useful in the treatment of a medical condition treatable with Dolastatin 10 in a subject.

32. A compound according to any one of claims 1-13, for use in treating a medical condition treatable with Dolastatin 10 in a subject.

33. A conjugate according to any one of claims 14-18, for use in treating a medical condition treatable with Dolastatin 10 in a subject.

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