Sonodynamic therapy agents

Abstract

Disclosed herein are activatable inactive sonosensitizer compounds, referred to as locked sonosensitizer compounds, designed with a detachable stimuli-responsive tether acting as a trigger or safety, as these compounds restore their inherent sonosensitization activity upon the detachment of the tether. The invention further encompasses pharmaceutical compositions comprising the same, offering a strategic approach to sonodynamic therapy, whereas the compounds and compositions are formulated to be administered and applied under favorable conditions, enhancing their efficacy in the context of therapeutic applications, since the therapeutic methods presented herein are more versatile and efficient compared to presently known SDT methods due to the controlled activation of the sonosensitizer.

Description

[0001] SONODYNAMIC THERAPY AGENTS

[0002] RELATED APPLICATION / S

[0003] This application claims the benefit of priority of U.S. Provisional Patent Application Nos. 63 / 627,125 filed on January 31, 2024 and 63 / 668,238 filed on July 7, 2024, 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 sonodynamic therapy, and more particularly, but not exclusively, to activatable locked sonosensitizers and methods of using the same for sonodynamic therapy.

[0006] Malignancies and infections are two of the leading causes of death worldwide. Malignancies, also known as cancer, are diseases in which cells grow uncontrollably and spread to other parts of the body. Infections are caused by microorganisms such as bacteria, viruses, and fungi. In recent years, there has been growing interest in the use of sonodynamic therapy (SDT) for the treatment of malignancies and infections.

[0007] Sonodynamic Therapy (SDT) is a minimally invasive medical approach that leverages the power of sound waves to trigger therapeutic effects within the human body. Much like Photodynamic Therapy (PDT) exploits light to activate a potent substance, SDT exploits sound wave energy as the activating agent to initiate beneficial physiological responses. The principle underlying SDT is the activation of a sonosensitizers, a biologically active compound that possesses the unique ability to absorb sound waves at specific frequencies. This interaction becomes particularly significant when the sonosensitizer encounters low-intensity ultrasound (US) waves, whose frequencies match those absorbed by the sonosensitizer. The outcome of this interaction is the generation of Reactive Oxygen Species (ROS), including singlet oxygen, through a sonochemical reaction.

[0008] When considering SDT over PDT , it is typically due to the penetration depth of ultrasound into the body that is much greater than that of light (10-30 for ultrasound cm vs 2-4 cm for light / near-IR), making SDT more effective than PDT for various conditions, especially for malignancies that are located deep within the tissue.

[0009] Presently known sonosensitizers belong to the dye classes as photosensitizers (specifically, long-wavelength porphyrins, phthalocyanines, or xanthenes), but only a few photosensitizers were successfully employed with ultrasound excitation. Importantly, possibility to employ any photosensitizer as a sonosensitizer is not confirmed and not obvious. To date, SDT is therefore considerably less developed method than PDT. Currently available sonosensitizers and photosensitizers (collectively refer to herein as "sensitizers") suffer from two main caveats, which are associated with significant treatment sideeffects: (i) specificity: targeting of sensitizers to abnormal cells is insufficient; and (ii) delivery: upon intravenous administration sensitizers may temporarily or permanently accumulate in normal (non-targeted) cells and organs, where they can be activated accidentally by ultrasound and / or light irradiation, thus damaging healthy tissue.

[0010] While the targeting specificity issue can potentially be resolved by tethering the sensitizers with target- specific carriers such as antibodies, avoiding the accidental irradiation of sensitizers in healthy tissues is far more challenging.

[0011] A notable gap in SDT research revolves around the limited availability of activatable sonosensitizers - compounds that can be selectively activated by specific ultrasound frequencies. The success of SDT hinges on the interplay between a sonosensitizer and ultrasound waves. Several activatable photosensitizers have been developed based on BODIPY-cyanines (boron- dipyrromethene linked to tetramethylindo(di)-carbocyanines), xanthenes, and xanthene-cyanines (notably, porphyrins and phthalocyanines cannot function as activatable dyes), but these have not been developed as sonosensitizers. Oxonols, another class of dyes with high light absorption and brightness in the long-wavelength region, which are known but rarely used as permanently fluorescent reporters have not yet been investigated as either activatable or non-activatable sonosensitizers or photosensitizers, and methods for synthesizing activatable oxonols have not yet been developed. Thus, to date, activatable polymethines (the dye class to which cyanines, xanthene-cyanines, and oxonols belong) have not been investigated as sonosensitizers. The sonodynamic capability of only two non-activatable cyanines that are not oxonols has been reported: Indocyanine Green (ICG) and IR780. Hence, there are currently no activatable sonosensitizers of any dye class.

[0012] Another problem associated with the use of sonosensitizers in SDT pertains to the concentration of the sonosensitizer within the target tissue, that increases following administration and then decreases due to metabolic activity. Therefore, to minimize the side-effects and increase the efficacy of both SDT and PDT, the time in which the sensitizer should be irradiated by ultrasound or light after administration must be assessed and thereafter optimized. While realtime fluorescence imaging can be used for such optimization, activatable dyes (sensitizers) are non-fluorescent in their "Off" forms and it is not possible to monitor their distribution (transport and accumulation) in the body using fluorescence.

[0013] 5- Aminolevulinic acid (5-ALA) has been investigated in the context of SDT. In traditional photodynamic therapy (PDT), 5-ALA is used as a prodrug to induce the production of a photosensitive compound called protoporphyrin IX (PpIX) in specific cells. When activated by light, PpIX generates reactive oxygen species (ROS) that selectively destroy targeted cells. In a similar manner, 5-ALA has been explored in sonodynamic therapy, where instead of light, ultrasound waves are used to activate the photosensitizer. The process involves administering 5- ALA to a patient, allowing it to accumulate in target cells. Subsequently, low-frequency ultrasound waves are applied to the area of interest. These ultrasound waves induce mechanical effects and create microbubbles around the cells containing the accumulated 5-ALA. The interaction between the ultrasound waves and the microbubbles triggers the production of ROS, leading to cell damage and death.

[0014] CN113925965A discloses a targeted sonosensitizer compound for sonodynamic therapy and application thereof, wherein the targeted sonosensitizer compound comprises a marker and a sonosensitizer, and the marker and the sonosensitizer are simultaneously grafted to corresponding target molecules so as to realize specific identification of the target molecules; after irradiation with sound waves, the sonosensitizer undergoes a sonochemical reaction to produce reactive oxygen species that are cytotoxic, thereby causing damage to cells or tissues.

[0015] WO2019036249A1 provides methods for treating diseased cells in a subject using sonodynamic therapy (SDT), comprising administering to the subject a sonosensitizer composition comprising IRDye® 700DX, and thereafter applying an ultrasonic wave to the diseased cell, wherein the sonosensitizer composition associates with the diseased cell.

[0016] Poplinger, D. et al. [" Ratiometric Fluorescence Monitoring of Antibody -Guided Drug Delivery to Cancer Cells”, Bioconjugate Chem., 2021, 32(8), 1641-1651] disclose a dual-dye TDD system, Cy5s-Ab-Flu-Aza, comprising the switchable fluorescein-based dye (Flu) linked to the anticancer drug azatoxin (Aza), reference pentamethine cyanine dye (Cy5s), and Her2-specific humanized monoclonal Trastuzumab (Herceptin) antibody.

[0017] Rozovsky, A. et al. [“Theranostic system for ratiometric fluorescence monitoring of peptide-guided targeted drug delivery”, RSC Advances, 2019, 9, pp. 32656-32664] disclose a TDD system comprising turn-on NIR fluorescent reporter attached to an anticancer drug and targeting peptide. The system provides both targeted drug delivery and NIR fluorescence monitoring of drug release events in target tissue. In this TDD system, a carboxyderivatized xanthene-cyanine dye (XCy) is attached to an anticancer drug chlorambucil (CLB) via a hydrolytically cleavable ester linker and coupled to a targeting peptide octreotide amide (OCTA), which is specific to somatostatin receptors SSTR-2 and STTR-5 overexpressed on many tumor cells. This OCTA-G-XCy-CLB conjugate exhibits no detectable fluorescence, while upon the hydrolytic cleavage of the ester linker a bright NIR fluorescence appears at about 710 nm signaling the drug release.

[0018] Thankarajan, E. et al. [“Antibody guided activatable NIR photosensitizing system for fluorescently monitored photodynamic therapy with reduced side effects”, Journal of Controlled Release, 2022, 343, 506-517] disclose an antibody-guided, activatable photosensitizing system, Ab-mI2XCy-Ac, where the trastuzumab (Ab) is linked to the non-active (not phototoxic and not fluorescent) dye, mI2XCy-Ac, that contains the hydroxyl group protected by acetyl (Ac). This targeting, non-photo-active conjugate was shown to be safely (without detectable side-effects) delivered to the targeted tumor, where it is activated by the esterase-mediated acetyl group cleavage and effectively treats the tumor upon NIR light irradiation.

[0019] Kaushik, R. et al. [“Near-Infrared Probes for Biothiols ( Cysteine, Homocysteine, and Glutathione): A Comprehensive Review”, ACS Omega, 2023, 8, 98-126] review near-infrared (NIR) organic probes designed to detect and quantify the important biological molecules cysteine, homocysteine, and glutathione (collectively known as biothiols) in living systems. These biothiols play crucial roles in various physiological processes, and imbalances in their concentrations can lead to diverse health problems. NIR probes offer significant advantages over traditional methods for biothiol detection due to their deep tissue penetration, reduced photodamage, and ease of use. The review comprehensively details various NIR probes reported in the literature, discussing their sensing mechanisms, photophysical properties, limitations, and potential applications in bioimaging. By comparing and contrasting these probes, the article aims to provide a valuable resource for researchers seeking suitable tools to study and understand the roles of biothiols in living organisms.

[0020] SUMMARY OF THE INVENTION

[0021] The present invention provides a library of novel compounds defined by a common trait: their switchable sonosensitization potential, achieved through a chemical switch. The innovative compounds, identified as "locked sonosensitizer compounds" in this disclosure, have been meticulously engineered to incorporate optional pay load and targeting functionalities. This strategic design imparts a high degree of efficacy, positioning these compounds as potent cytotoxic agents. Their adaptability and versatility make them particularly valuable for the treatment of medical conditions wherein controlled cytotoxic effects are a therapeutic requirement. This novel class of compounds presents a promising avenue for addressing a spectrum of medical conditions by leveraging their precise and controlled cytotoxic capabilities.

[0022] Specifically, the invention revolves around an inventive concept of tethering a cleavable chemical group to a hydroxyl, thiohydroxyl or amino group positioned either at the edge or within the conjugated 7t-system of the dye core of a sonosensitizer compound, thereby rendering this hydroxyl, thiohydroxyl or amino group into a “safety switch”. This controlled and reversible modification effectively renders the sonosensitizer inactive until its activation is desired.

[0023] Thus, according to an aspect of some embodiments of the present invention, there is provided a compound characterized by a general formula, each individually selected from the group consisting of:

[0024] and any hydrate, solvate, ester, or pharmaceutically acceptable salt thereof, wherein:

[0025] SRT is a biocleavable linking moiety;

[0026] T is O, S, or NRT, RTis H, alkyl, aryl, or amine; n is an integer ranging 1-5; each of RH1-RH3is independently H, Br, I, aryl-bromide, or aryl-iodide, provided that at least one of RH1-RH3is Br, I, aryl-bromide, or aryl-iodide; each of W’-W3is independently O, S, Se, C=O, CRARB, CRA=CRB, or NRC, wherein each of RA-RCis independently H, alkyl, aryl, halo, -Rs-S03H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RCmay form a ring; each of RN1-RN4is independently hydrogen, a substitute or unsubstituted alkyl, substitute or unsubstituted aryl, an aryl substituted with Br or I, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs- NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide;

[0027] Rsis a spacer moiety selected from -(CH2-)1-8 and -(OCH2CH2-)I-4; and each of R1-R17 is independently selected from the group consisting of H, a substitute or unsubstituted alkyl, a substitute or unsubstituted aryl, fused aryl (benzo), halo, haloalkyl, -CF3, -CO2H, -CO2Me, -SO3H, -ON, -NO2, -NH2, NHMe, -N(Me)2, carboxamide, and -OMe, or any two adjacent R1-R17 form a ring; with the proviso that the compound is not one of the compounds presented in Table 1 below.

[0028] In some or all embodiments, the biocleavable linking moiety is selected from the group consisting of amide, carbamate, carbonate, carboxylate, acetal, glucoside, aldimine, aminal, aminoacetal, acryl, boronic acid, boronic ester, cycloalkene, cyclohexene, disulfide, sulfate, epoxide, ester, heteroalicyclic, heteroaryl, hydrazide, hydrazone, imide, imine, ketal, ketimine, lactam, lactone, methyleneamine, oxime, peroxo, phosphate, phosphateester, semicarbazone, alkyl silyl, aryl silyl, thioacetal, thiohydrazide, thioketal, triazine, and triazole. In some or all embodiments, the biocleavable linking moiety is a self-immolative linker selected from the group consisting of a glutathione-cleavable self-immolative linker, a cathepsin B-cleavable self-immolative linker, galactosidase responsive self-immolative linker, peroxide-responsive self-immolative linker glucuronidase responsive self-immolative linker, sulfatase responsive self-immolative linker, and a fluoride-responsive self-immolative linker, as these terms are known in the art and / or defined herein.

[0029] In some or all embodiments, the self-immolative linker is selected from the group consisting of: wherein: the wiggled line marks the tether bond position; each of R18-R21 is independently H, methyl, ethyl, cyclopropyl or cyclobutyl ;

[0030] R22 is selected from H, -OH, -NH2, -NHMe, -NMe2, -NMe3+, -PO4H2, -PO2(OMe)2, -CO2H, -CO(CH2)2CO2H, -CO(CH2)3CO2H, -CO2Me, -SO3H, alkyl, alkoxy, haloalkyl, -(OCH2CH2-)I-3, -CO2-D, -CONH-D, -CONMe-D, -OCO-NH-D,

[0031] -OCO2-D, and -OCO-NMe-D; and

[0032] D is a functional moiety selected from the group consisting of a bioactive agent, an imaging agent, a labeling agent, and / or a diagnostic agent.

[0033] In some or all embodiments, the compound comprises at least one iodine atom.

[0034] In some or all embodiments, the compound further comprising a solubilizing group selected from -SO3H and -N+R3, wherein R is independently selected from hydrogen and any substituent, such as, for a non-limiting example, a C1-6 alkyl.

[0035] In some or all embodiments, the solubilizing group is directly attached to an aromatic system of the compound.

[0036] In some or all embodiments, the solubilizing group is attached to the compound via a spacer.

[0037] In some or all embodiments the compound further comprising a carboxylic function for binding to a carrier moiety.

[0038] In some or all embodiments, the compound is conjugated to a carrier moiety.

[0039] In some or all embodiments, the carrier moiety is an antibody.

[0040] In some or all embodiments, the drug-carrier ratio is optimized to avoid aggregation of the conjugate.

[0041] In some or all embodiments, the compound is designed for use in targeted sonodynamic therapy.

[0042] In some or all embodiments, the compound further includes a functional moiety attached thereto via the biocleavable linking moiety (the SRT), the functional moiety is selected from the group consisting of a moiety of a bioactive agent, a moiety of an imaging agent, a moiety of a labeling agent, and / or a moiety of a diagnostic agent.

[0043] In some or all embodiments, the bioactive moiety is of a bioactive agent which is a drug, and in some preferred embodiments, the drug is an anticancer drug

[0044] In some or all embodiments, the imaging agent, the labeling agent, and / or the diagnostic agent include contrast agents, biomarkers, radioactive tracers, fluorescent probes and molecular imaging agents.

[0045] In some or all embodiments, the compound is conjugated to a targeting moiety, as this terms is known in the art. In some or all embodiments, the targeting moiety includes ligands of targeted receptors, antibodies that target specific antigens, aptamers that target specific nucleic acid sequences, peptides and vitamins that target specific receptors or cells, and nanoparticles modified with targeting moieties.

[0046] In some or all embodiments, the targeting moiety is selected from the group consisting of a nanoparticle, a peptide, a protein, an antibody or a fragment thereof, a porphyrin, a hormone, a saccharide, a polysaccharide, an antigens, a hapten, a DNA fragment, a folate, a polymeric micelle, a liposome, a lipoprotein-based drug carrier, a dendrimer, siRNA, an oligonucleotide, an RNA fragment, and a receptor ligand.

[0047] In some or all embodiments, the targeting moiety is an antibody or a fragment thereof.

[0048] In some or all embodiments, the compound the compounds provided herein is intended for use in the treatment of a medical condition in a subject, and / or as an active ingredient in a pharmaceutical composition / medicament.

[0049] According to another aspect of some embodiments of the present invention, there is provided a pharmaceutical composition which includes, as an active ingredient, at least one of the compounds provided herein, and a pharmaceutically acceptable carrier.

[0050] In some or all embodiments, the pharmaceutical composition is intended for use in the treatment of a medical condition in a subject.

[0051] According to another aspect of some embodiments of the present invention, there is provided a method of treating a medical condition in a subject in need thereof, which is effected by administering to the subject a therapeutically effective amount the compound provided herein, and exposing at least a part of the subject to ultrasound energy.

[0052] In some or all embodiments, the method is effected by exposing the subject using a focused ultrasound device, which is focused on the at least a part of the subject.

[0053] In some or all embodiments, the method is effected by exposing the whole body of the subject, or any part or parts thereof.

[0054] In some or all embodiments, the method is effected by using ultrasound energy at a frequency of 1.0-2.0 MHz and an intensity of 0.5-10 W / cm2.

[0055] In some or all embodiments of any aspect of the present invention, the medical condition is one that is treatable by inducing at least one cytotoxic effect in at least one cell.

[0056] In some or all embodiments of any aspect of the present invention, the medical condition is any type of cancer, an autoimmune disease, a viral infection, a transplant rejection and a neurodegenerative disease, arthritis, including juvenile rheumatoid arthritis, psoriatic arthritis, rheumatoid arthritis, systemic lupus erythematosus, as well as conditions such as steroid-resistant polymyositis, dermatomyositis, Wegener’s granulomatosis, polyarteritis, and some forms of vasculitis.

[0057] DESCRIPTION OF EMBODIMENTS OF THE INVENTION

[0058] The present invention, in some embodiments thereof, relates to sonodynamic therapy, and more particularly, but not exclusively, to activatable locked sonosensitizers and methods of using the same for sonodynamic therapy.

[0059] 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 disclosure is meant to encompass other embodiments or of being practiced or carried out in various ways.

[0060] As discussed hereinabove, sonodynamic therapy (SDT) represents an emerging approach that offers the possibility of non-invasively eradicating solid tumors in a site-directed manner. It involves the sensitization of target tissues with a non-toxic sensitizing chemical agent and subsequent exposure of the sensitized tissues to relatively low-intensity ultrasound. Essentially, both aspects (the sensitization and ultrasound exposure) are harmless, and cytotoxic events occur when both are combined. Due to the significant depth that ultrasound penetrates tissue, the approach provides an advantage over similar alternative approaches, such as photodynamic therapy (PDT), in which less penetrating light is employed to provide the cytotoxic effect in sensitized tissues. This suggests that sonodynamic therapy may find wider clinical application, particularly for the non-invasive treatment of less accessible lesions.

[0061] Early SDT-based approaches employed many of the sensitizers used in PDT, although the manner in which ultrasound activates the sensitizer differs from activation events in PDT, and indeed, activatable sonosensitizers, though conceptually powerful, are relatively scarce. These specialized compounds are required to possess the ability to remain inactive until exposed to specific ultrasound frequencies, ensuring precise activation only within the desired treatment region. This selectivity is vital to avoid collateral damage to healthy tissues and enhance the therapeutic efficacy of SDT.

[0062] The present inventors have recently demonstrated that, in targeted-drug-delivery (TDD) applications, combining an activatable PDT dye with an additional, permanently fluorescent (non- switchable) reference dye enables the fluorescence monitoring of the drug distribution; moreover, by using ratiometric measurements of two signals originating from the activatable dye and reference dyes, this approach enables the quantification of the drug-release events in the target. Such ratiometric monitoring has never been realized in SDT applications. Another problem associated with the use of sonosensitizers in SDT arises lack of activatable sonosensitizers that have been combined with chemotherapeutic drugs to achieve a combined (synergistic) SDT and chemotherapeutic effect. In addition, activatable SDT agents have never been combined with activatable PDT agents, although synergistic effect of the conventional (non-activatable) SDT and PDT agents were reported.

[0063] To substantially expand the range of available therapeutic agents and improve the efficacy and safety of sonodynamic treatment, the present inventors have developed locked sonosensitizers, which enables the targeted delivery thereof, followed by their safe activation at the target sites.

[0064] Locked Sonosensitizer Compounds:

[0065] As used herein, the term “sonosensitizer” refers to a dye molecule capable of converting ultrasound energy into a desired biological effect, primarily through sonochemical reactions or mechanical interactions, enabling applications in therapeutic domains like sonodynamic therapy and diagnostic fields like contrast enhancement. In the context of some aspects and embodiments of the present invention, the sonosensitizer is capable of absorbing ultrasound energy and exerting a cytotoxic (cell killing) effect under physiological conditions.

[0066] In the context of a photo- and a sonosensitizer compound, the term “sensitization activity” refers to the biological impact afforded upon irradiation of the compound, typically leading to cell death that results, inter alia, from generation of reactive oxygen species (ROS) upon irradiation of the compound. In the context of interaction with ultrasound energy, the activity of a sonosensitizer, such as the compound provided herein, can be defined from two perspectives: energy conversion and biological impact, commonly referred to herein as sonosensitization activity. The energy conversion of a sonosensitizer efficiently absorbs acoustic energy within the therapeutic ultrasound range (typically 20 kHz - 10 MHz). This absorbed energy triggers sonochemical reactions within the surrounding environment, leading to generation of ROS and free radicals in general through mechanisms like cavitation, singlet oxygen production, and electron transfer. In addition, sonosensitization activity may include induction of mechanical effects such as tissue heating (hyperthermia) and cellular disruption through cavitation bubbles. The biological impact of sonosensitizers includes the generated ROS or mechanical effects lead to specific cellular and tissue responses, depending on the application. In sonodynamic therapy (SDT), ROS cause oxidative stress-induced cell death in targeted malignant cells. Tissue ablation is caused by hyperthermia or cavitation that can be used for controlled ablation of unwanted tissues. In the context of the present invention sonosensitizers are used also for drug delivery, whereas ultrasound and sonosensitization can enhance the permeability of cell membranes, facilitating drug uptake.

[0067] In broad terms, a dye molecule which can serve as a sonosensitizer, according to some embodiments of the present invention, include cationic, anionic or zwitterionic (neutral) polymethine dye, or any cyanine dye, oxonol dye, cyanino-oxonol dye or any hemioxonol dye, as these terms are known in the art, and illustrated by the general structures presented below.

[0068] Cyanine dyes Oxonol dyes Cyanino-oxonol dyes

[0069] The sonosensitization properties of any give polymethine dye depend on its chemical structure, the presence of particular elements in the structure, and the particular arrangement of atoms and bonds within the dye molecule. While there are many factors that govern sonosensitization properties and thus sonosensitization activity, some selection rules can be apply in order to generalize and predict whether a given polymethine dye possess the capacity to exhibit the desired sonosensitization activity. To guide the user of this disclosure, provided herein are some selection rules related to the sonosensitization activity of polymethine dyes, as found in the literature:

[0070] Conjugation Length and rigidity: a longer conjugated 7t-systems tend to enhance sonosensitization activity. Longer conjugated 7t-systems generally exhibit red-shifted absorption and emission spectra, potentially overlapping with the ground state absorption of target molecules for efficient energy transfer in sonosensitization. Rigid 7t-systems minimize vibrational relaxation pathways, favoring intersystem crossing and triplet state lifetimes for effective sonoluminescence or singlet oxygen generation. Example: Cyanine dyes with extended ^-conjugation and cyclic structures show good sonosensitization activity. In the context of the present invention, the conjugated 7t-system includes at least five atoms, such as in -A-A=A-A=A- or -A=A-A=A-A=, with A being a carbon, nitrogen, oxygen and / or sulfur atom.

[0071] Heavy Atom Effect: the presence of at least one heavy atom substituent, such as a heavy halogen (e.g., Br, I, Cl), in, along or at the flanks of the conjugated system can improve sonosensitization properties. The presence of heavy halo substituents along the conjugated n- system enhances intersystem crossing (ISC) efficiency, promoting triplet excited state formation crucial for sonosensitization. Example: iodinated xanthene-cyanine dye exhibiting enhanced sonosensitization. Electron Donor-Acceptor System: incorporating an electron donor-acceptor system within the dye molecule can enhance sonosensitization activity. Push-pull substitution patterns create intramolecular charge transfer (ICT) states, influencing ISC rates and singlet-triplet energy gaps. Electron-donating groups (e.g., -NR2, -OCH3) on one end and electron- withdrawing groups (e.g., -NO2, -CN) on the other end of the 7t-system can tune the excited state properties for efficient sonosensitization. Example: Rhodamine B with dimethylamino and carboxylic acid substituents exhibits efficient singlet-triplet energy conversion for sonosensitization.

[0072] Solubility: dyes with appropriate solubility in the target medium (e.g., biological tissues) may exhibit better sonosensitization. Polar solvents can stabilize charge-separated states and affect ISC rates, influencing sonosensitization efficiency. Protic solvents may quench triplet excited states through protonation, reducing sonosensitization activity. Optimizing solvent polarity should be considered when tailoring the performance of specific polymethine dyes in sonosensitization applications. Aggregation due to inadequate solubility of polymethine dyes can lead to excimer (short for “excited dimer”) formation and energy transfer quenching, reducing sonosensitization efficiency. Strategies to prevent aggregation and self-quenching, such as using bulky substituents or controlling dye concentration, can be incorporated into the molecular design for optimizing sonosensitization activity.

[0073] Substituent Effects: certain functional groups or substituents may positively or negatively influence sonosensitization activity.

[0074] Aromaticity: aromatic systems within the polymethine dye structure may contribute to improved sonosensitization.

[0075] Steric Effects: avoiding bulky substituents that may hinder efficient interaction with ultrasound waves.

[0076] Electronic Structure: the electronic structure, including the distribution of electron density, plays a crucial role in sonosensitization.

[0077] Isomerism: the isomeric form of the polymethine dye can impact sonosensitization properties.

[0078] Photo- and thermal stability: dyes that are photostable and thermostable may demonstrate better performance as sonosensitizers.

[0079] UV-Vis Absorption Band: The UV-Vis and near-infrared (NIR) absorption band of polymethine dyes are affected by the heteroaromatics and linkage groups, which can also influence their sonosensitization properties.

[0080] Symmetry Rule: According to the selection rule, the lowest-energy ground state transition of a polymethine is two-photon forbidden due to the symmetry. Absorption and Emission Wavelengths: matching the absorption and emission wavelengths to the ultrasound frequency range of interest is essential. pH Sensitivity: pH-sensitive dyes may exhibit variable sonosensitization activity under different environmental conditions.

[0081] In the context of the Heavy Atom Effect, this effect can be achieved by introducing one or more heavy atoms as a substituent on one or more of the aromatic rings of the compound, as presented and demonstrated herein throughout. Alternatively or in addition, the Heavy Atom Effect can be achieved by replacing one or more of the ring carbons of the compound with an atom heavier than carbon, such as selenium or sulfur (see, for example, Wx-W3in Formula I).

[0082] The dye or the dye moiety, according to some embodiments of the present invention, is a polymethine dye that is capable of generating ROS upon irradiation. Such polymethine dyes are characterized by sharp and intense absorption bands and narrow emission bands with high extinction coefficients. These characteristics typically manifest in the red and near-infrared region.

[0083] The present disclosure also encompasses a locked methylthioninium chloride, more commonly known as methylene blue, which is a member of the phenothiazine dye family. Phenothiazines, encompassed herein as a family of lockable (fitted with SRT) dye molecules are organic compounds with a characteristic tricyclic structure consisting of a central benzene ring fused to a thiazine ring on one side and another aromatic ring (usually another benzene ring) on the opposite side. Methylthioninium dyes are characterized by the presence of a sulfur atom and a quaternary nitrogen atom in the molecule. These features give them their characteristic blue color and their ability to act as redox indicators and photosensitizers. They can also be used as antiseptic and staining agents. Some examples of methylthioninium dyes, which are contemplated within the scope of the present invention as lockable dye molecules include, methylene blue (methylthioninium chloride), toluidine blue (toluithioninium chloride), and azure A ( azure A chloride).

[0084] In structural terms, the locked sonosensitizer compound provided herein comprises a dye moiety in the form of a polymethine dye, which is characterized by a conjugated chain of (2n + 1) sp2-hybridized carbon atoms that connect a terminal 7t-electron-accepting (^-electron withdrawing) group with a terminal 7t-electron-donating group. This structure is responsible for the dye’s ability to absorb and emit light in the red and near-infrared region. The polymethine dye moiety, according to some embodiments of the present invention, has an odd number (2n + 3) of 7t-centers and an even number (2n + 4) of 7t-electrons in the conjugated chain, where n equals the number of methine (-CR=CR-) groups, such as vinylene groups -CH=CH-. This configuration contributes to the dye’s photo and thermal stability, as well as its strong fluorescent emission in organic solvents. The terminal groups of the polymethine dye moiety contain nitrogen or oxygen atoms that play a crucial role in the dye’ s ability to produce reactive oxygen species (ROS) upon irradiation.

[0085] These rules for selecting a ROS -generating dye are general guidelines and may vary based on the specific application and experimental conditions. The user of the present invention would appreciate the need to tailor the chemical structure of polymethine dyes to optimize their sonosensitization properties for targeted applications. The user of the present invention may find ample guidance in the literature for selecting a polymethine dye that is capable of generating ROS upon irradiation; for example, in Dereje, D.M. et al. [“Polymethine dyes for PDT: recent advances and perspectives to drive future applications” , Photochem Photobiol Sci, 2022, 21, 397-419, doi: 10.1007 / s43630-022-00175-6] .

[0086] According to an aspect of some embodiments of the present invention, there is provided a compound in the form of a locked sonosensitizer, which is a sonosensitizer derivative exhibiting a labile functional group referred to herein as a labile linking moiety, which is attached to the sonosensitizer at a location that disrupts 7t-system of the sonosensitizer, thus inhibiting any sensitization activity including sonosensitization activity, at least until the labile linking moiety is detached therefrom (a detachable stimuli-responsive tether). The compound, according to embodiments of the present invention, comprises a polymethine or phenothiazine dye moiety corresponding to a polymethine or phenothiazine dye capable of exerting sensitization activity upon irradiation, and a biocleavable linking moiety attached thereto, wherein the labile linking moiety is attached to the polymethine dye moiety at a location that disrupts the conjugated it- system of the polymethine dye moiety, thereby substantially inactivating said sensitization activity, whereas the sensitization activity is substantially regained upon detachment of the labile linking moiety.

[0087] The locked state is reversed and activity is restored upon moiety removal. Unlocked sonosensitizers are electronically active and susceptible to excitation, analogous to a "safety off' state. Locked states resemble "safety on," preventing excitation. Other terms for locked / unlocked states include deactivated / activated, disabled / enabled, and inactive / active. More elaborately, the term “locked”, as used herein in the context of a dye molecule, such as a sonosensitizer compound (e.g., as in “a locked sonosensitizer compound”), refers to a derivative of the dye molecule that comprises a labile linking moiety at certain positions in the molecule, which disrupts the delocalized 7t-system that is responsible for the sensitization activity of the dye, thereby abolishing sensitization activity by interrupting conjugation within the 7t-system; since the lock is afforded by a labile linking moiety, the sensitization activity is be regained upon detachment of the labile linking moiety. A locked sonosensitizer compound cannot be electronically excited by external radiation, at least in the ultrasound (US) range, as long as the labile linking moiety is attached thereto, and thus is analogous to an armament in a “safety on” state, whereas a sonosensitizer compound without, or after detachment of the labile linking moiety, is ready for electronic excitation, and thus is analogous to the sonosensitizer compound in a “safety off’ state, ready for triggering by ultrasound irradiation. Other terms that may be used to describe a locked sonosensitizer compound include “deactivated”, “secured”, “disabled”, “safeguarded” and even “bolstered”. Inversely, a sonosensitizer compound that does not have the abovementioned labile linking moiety is electronically active and can exert sensitization activity, and thus may be referred to as “unlocked”, “activated”, “enabled”, “unsecured”, “armed”, “charged”, “live”, “engaged” and even “hot”.

[0088] According to some embodiments of the present invention, a locked sonosensitizer comprises a dye moiety that comprises a labile linking moiety, referred to herein as a “Stimuli- Responsive Tether” or a “Sonosensitizer Release Trigger” (SRT), therefore the term "locked" in the context of a dye molecule (e.g., a sonosensitizer) refers to a derivative of the dye featuring a stimuli-responsive tether that disrupts the conjugated 7t-system of the dye, thereby abolishing sensitization activity. Removing a detachable SRT reverses this "lock," regaining activity; thus locked / unlocked corresponds respectively to active / inactive states.

[0089] In certain embodiments of the current invention, the Stimuli-Responsive Tether or Sonosensitizer Release Trigger (SRT) undergoes cleavage in response to alterations in biological or physiological conditions experienced or encountered by the locked sonosensitizer. This occurs, for instance, when the locked sonosensitizer compound provided herein is internalized by a cell and exposed to enzymes within that cell. In the context of a pharmaceutical agent, wherein the presently disclosed locked sonosensitizer compound serves as an active ingredient in a pharmaceutical composition, the SRT demonstrates sensitivity to the specific biological and physiological conditions found within a predefined targeted cell, tissue, or organ. Outstanding from previously known sonosensitizers, it remains non-responsive to the biological and physiological conditions present outside the predefined targeted cell, tissue, or organ in the subject's body, thereby allowing advantageous treatment conditions, such as dosage control, fullbody expose to ultrasound, and timing thereof.

[0090] The present inventors have identified at least one structural feature that is common to almost all known sonosensitizer compounds, and that is a hydroxyl, thiohydroxyl or amino group in one of the edges of, or branching from within the conjugated 7t-system of the dye scaffold, and the gist of the present invention is harnessing this hydroxyl, thiohydroxyl or amino group to act as the “safety switch” of sonosensitization activity of the compound. In other words, the invention is directed at tethering a liable chemical group to a hydroxyl, thiohydroxyl or amino group found along the conjugated 7t-system of the dye scaffold, thereby rendering the sonosensitizer inactive in a controllable reversible manner.

[0091] Families of locked sonosensitizer compounds:

[0092] Embodiments of the present invention encompass compounds, which share common technical features, as described hereinabove, yet can be divided into several families based on the core skeleton of the compound. Thus, according to an aspect of some embodiments of the present invention, there is provided a compound, which is a locked sonosensitizer as defined hereinabove, characterized by a general formula, each formula is independently and individually selected from the group consisting of: and any enantiomer, ion, solvate, hydrate, ester (e.g., methyl, acetyl), and / or pharmaceutically acceptable salt thereof, wherein: SRT is a detachable stimuli-responsive tether, or biocleavable linking moiety, as defined hereinbelow, the presence of which is accountable for preventing the dye from responding to ultrasound;

[0093] T is O, S, or NRT, RTis H, alkyl, aryl, or amine; n is an integer ranging 1-5; each of RH1-RH3is independently H, Br, I, aryl-bromide, or aryl-iodide, provided that at least one of RH1-RH3is Br, I, aryl-bromide, or aryl-iodide; each of W’-W3is independently O, S, Se, C=O, CRARB, CRA=CRB, or NRC, wherein each of RA-RCis independently H, alkyl, aryl, halo, -Rs-SO3H, -RS-CO2H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RCmay form a ring; each of RN1-RN4is independently hydrogen, a substitute or unsubstituted alkyl, substitute or unsubstituted aryl, an aryl substituted with Br or I, -Rs-SO3H, -RS-CO2H, -RS-NH2, -Rs- NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide;

[0094] Rsis a spacer moiety selected from -(CH2-)i-8 and -(OCH2CH2-)I-4; and each of R1-R17 is independently selected from the group consisting of H, a substitute or unsubstituted alkyl, a substitute or unsubstituted aryl, fused aryl (benzo), halo, haloalkyl, -CF3, -CO2H, -CO2Me, -SO3H, - C=N, -NO2, -NH2, NHMe, -N(Me)2, carboxamide, and -OMe, or any two adjacent R1-R17 form a ring; with the proviso that the compound is not one of the compounds presented in Table 1, including any enantiomer, ion, solvate, hydrate, ester, and / or pharmaceutically acceptable salt thereof.

[0095] The general formulae contain ring substituents that are presented with bonds ending inside the ring, meaning that the position of the substituent can be any one of the available positions on the ring.

[0096] It is noted herein that the present disclosure refers to each and every family of the above compounds individually, namely to each of Formulae I-XVIII individually.

[0097] According to some embodiments of the present invention, the compound is represented by Formula I, as presented hereinabove.

[0098] According to some embodiments of the present invention, the compound is represented by Formula II, as presented hereinabove.

[0099] According to some embodiments of the present invention, the compound is represented by Formula III, as presented hereinabove.

[0100] According to some embodiments of the present invention, the compound is represented by Formula IV, as presented hereinabove.

[0101] According to some embodiments of the present invention, the compound is represented by Formula V, as presented hereinabove.

[0102] According to some embodiments of the present invention, the compound is represented by Formula VI, as presented hereinabove.

[0103] According to some embodiments of the present invention, the compound is represented by Formula VII, as presented hereinabove.

[0104] According to some embodiments of the present invention, the compound is represented by Formula VIII, as presented hereinabove.

[0105] According to some embodiments of the present invention, the compound is represented by Formula IX, as presented hereinabove.

[0106] According to some embodiments of the present invention, the compound is represented by Formula X, as presented hereinabove.

[0107] According to some embodiments of the present invention, the compound is represented by Formula XI, as presented hereinabove.

[0108] According to some embodiments of the present invention, the compound is represented by Formula XII, as presented hereinabove. According to some embodiments of the present invention, the compound is represented by Formula XIII, as presented hereinabove.

[0109] According to some embodiments of the present invention, the compound is represented by Formula XIV, as presented hereinabove.

[0110] According to some embodiments of the present invention, the compound is represented by Formula XV, as presented hereinabove.

[0111] According to some embodiments of the present invention, the compound is represented by Formula XVI, as presented hereinabove.

[0112] According to some embodiments of the present invention, the compound is represented by Formula XVII, as presented hereinabove.

[0113] According to some embodiments of the present invention, the compound is represented by Formula XVIII, as presented hereinabove.

[0114] According to some embodiments, in each of the above families, namely each of Formulae I-XVII, T is O, which turns into hydroxyl upon cleavage of the SRT.

[0115] According to some embodiments, in Formula XVIII, T is O, which turns into hydroxyl containing heterocycle upon cleavage of the SRT.

[0116] According to some embodiments, in each of the above families, namely each of Formulae I-XVIII, at least one, at least two or all of RH1-RH3is I.

[0117] According to some embodiments, in each of the above families, namely each of Formulae I-XVIII, at least one, at least one or all of Wx-W3is C(Me)2 or C=O.

[0118] According to some embodiments, in each of the above families, namely each of Formulae I-XVIII, at least one, at least two or all

[0119] According to some embodiments, in each of the above families, namely each of Formulae I-XVIII, R1-R17 is hydrogen.

[0120] Compound familied in alternative embodiments:

[0121] In some preferred embodiments pertaining to compounds represented by Formula I:

[0122] Formula I wherein:

[0123] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH2is independently H, Br, I, provided that at least one of RH1-RH2is Br or I; more preferably, at least one of RH1and RH2is I; each of Wx-W2is independently O, S, Se, CRARB, or NRC, wherein each of RA-RCis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, each of W1and W2is CRARB, and each of RAand RBis Me (W1= W2= CMe2), or RAis Me and RBis (CH2)3SO3H or (CH2)4CO2H (W1, W2= C(Me)(CH2)3SO3H or C(Me)(CH2)4CO2H); each of RN1-RN2is independently hydrogen, methyl, ethyl, -Rs-SO3H, -Rs-C02H, -Rs- NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide; more preferably, each

[0124] Rsis a spacer moiety selected from each of Ri and R13 is hydrogen, at least two adjacent R1-R13 form a fused aryl ring (benzo) and each of the remaining R1-R13 is H; more preferably, each of R1-R13 is H.

[0125] In some preferred embodiments pertaining to compounds represented by Formula II:

[0126] Formula II wherein:

[0127] T is O, S, NH or NMe; more preferably, T is O;

[0128] RH1is independently H, Br, I, provided that RH1is at least one of Br or I; more preferably, at least one of RH1is I;

[0129] W1is independently O, S, Se, CRARB, or NRC, wherein each of RA-RCis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-maleimide and -Rs- succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, W1is CRARB, and each of RAand RBis Me (W1= CMe2), or RAis Me and RBis (CH2)3SO3H or (CH2)4CO2H (W1= C(Me)(CH2)3SO3H or C(Me)(CH2)4CO2H) each of RN1-RN2is independently hydrogen, alkyl, aryl, -Rs-SO3H, -Rs-C02H, -Rs- NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide; more preferably, each Rsis a spacer moiety selected from -(CH2-)i-8 and -(OCH2CH2-) 1-4; and each of R1-R3 is independently H, Br, I, at least two adjacent R1-R3 and R5-R6 form a fused aryl ring (benzo), R4 is H, methyl, aryl, and each of the remaining Ri-Re is H; more preferably, each of Ri is I, R2-R3 and R5-R6 is H, R4 is Me.

[0130] In some preferred embodiments pertaining to compounds represented by Formula III:

[0131] Formula III wherein:

[0132] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH2is independently H, Br, I, provided that at least one of RH1-RH2is Br or I; more preferably, at least one of RH1is H, and RH2is I; each of Wx-W2is independently O, S, Se, CRARB, or NRC, wherein each of RA-RCis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, each of W1= O, and W2is CRARB, and each of RAand RBis Me (W1= W2= CMe2) or RAis Me and RBis (CH2)3SO3H or (CH2)4CO2H (W2= C(Me)(CH2)3SO3H or C(Me)(CH2)4CO2H)

[0133] RN1is independently hydrogen, methyl, ethyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs- NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs- succinimide; more preferably, RN1is (CH3)3SO3H or (CH3)4CO2H

[0134] Rsis a spacer moiety selected from -(CH2-)i-8 and -(OCH2CH2-)I-4; and each of R9-R11 is independently H, Br, or I, at least two adjacent R1-R2 R7-R11 form a fused aryl ring (benzo) and each of the remaining R1-R11 is H; more preferably, each of R1-R10 is H, Rn is I.

[0135] In some preferred embodiments pertaining to compounds represented by Formula IV :

[0136] Formula IV wherein:

[0137] T is O, S, NH or NMe; more preferably, T is O;

[0138] RH1is independently H, Br, I, provided that at least one of RH1is Br or I; more preferably, at least RH1is I;

[0139] W1is independently CRARB, wherein each of RA-RBis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-maleimide and -Rs- succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, each of W1is CRARB, and each of RAand RBis H or Me (W1= CH2 or CMe2). each of R1-R2 is H Br, or I, or two adjacent R1-R2 form a fused aryl ring (benzo); more preferably, each of R1-R2 is H.

[0140] In some preferred embodiments pertaining to compounds represented by Formula V : wherein:

[0141] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH3is independently H, Br, I, provided that at least one of RH1-RH3is Br or I; more preferably, at least one of RH1-RH2is H and RH3is I;

[0142] W1is independently O, S, Se, CRARB, or NRC, wherein each of RA-RCis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-maleimide and -Rs- succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, W1is CRARB, and each of RAand RBis Me (W1= W2= CMe2), or RAis Me and RBis (CH2)3SO3H or (CH2)4CO2H (W1, W2= C(Me)(CH2)3SO3H or C(Me)(CH2)4CO2H); each of RN1-RN2is independently hydrogen, methyl, ethyl, -Rs-SO3H, -Rs-C02H, -Rs- NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide; more preferably, each of RN1and RN2is (CH2)3SO3H or (CH2)4CO2H

[0143] Rsis a spacer moiety selected from -(CH2-)i-8 and -(OCH2CH2-)I-4; and

[0144] Each of Re, R7, R10-R12 is independently H, Br or I, at least two adjacent R1-R12 form a fused aryl ring (benzo) and each of the remaining R1-R12 is H; more preferably, each of R1-R11 is H and R12 is I.

[0145] In some preferred embodiments pertaining to compounds represented by Formula VI:

[0146] Formula VI wherein:

[0147] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH2is independently H, substituted and non-substituted alkyl, substituted and non-substituted aryl, Br, I, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-maleimide and -Rs- succinimide, provided that at least one of RH1-RH2is bromobenzene, iodobenzen, Br or I; more preferably, at least one of RH1-RH2is iodobenzen; each of RN1-RN2is independently hydrogen, substituted and non-substituted alkyl, substituted and non-substituted aryl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs-NMe3+, -Rs- azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide; more preferably, each of RN1and RN2is (CH2)3SO3H, (CH2)4CO2H or para-Ph sulfonate (p-SO3H-C6H4-).

[0148] Rsis a spacer moiety selected from -(CH2-)i-8 and -(OCH2CH2-)I-4; and

[0149] Ri -R3 is H, Me, Et, Ph, two adjacent R2-R3 form a fused aryl ring (benzo) and the remaining Ri is H, more preferably, each of R1-R3 is H.

[0150] In some preferred embodiments pertaining to compounds represented by Formula VII: wherein:

[0151] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH3is independently H, Br, I, provided that at least one of RH1-RH3is Br or I; more preferably, RH2is H and RH1and RH3is I; each of RN1-RN2is independently hydrogen, alkyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs- NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs- succinimide; more preferably, each of RN1and RN2is (CH2)3SO3H or (CH2)4CO2H

[0152] Rsis a spacer moiety selected from -(CH2-)i-8 and -(OCH2CH2-)I-4; and

[0153] R1-R12 is H, Br or I, at least two adjacent R1-R12 form a fused aryl ring (benzo) and each of the remaining R1-R12 is H; more preferably, each of R1-R12 is H. In some preferred embodiments pertaining to compounds represented by

[0154] Formula VIII: wherein:

[0155] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH3is independently H, Br, I, provided that at least one of RH1-RH3is Br or I; more preferably, at least one of RH1-RH3is I; and each of the remaining R1-R15 is H; more preferably, each of R1-R15 is H.

[0156] In some preferred embodiments pertaining to compounds represented by Formula IX: wherein:

[0157] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH3is independently H, Br, I, provided that at least one of RH1-RH3is Br or I; more preferably, at least one of RH1-RH3is H and RH2is I; W1is independently O, S, Se, CRARB, or NRC, wherein each of RA-RCis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -RS-CO2H, -RS-NH2, -Rs-NMe2, -Rs-NMe3+, -Rs- maleimide and -Rs-succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, each of W1is CRARB, and each of RAand RBis Me (W1= W2= CMe2), or RAis Me and RBis (CH2)3SO3H or (CH2)4CO2H (W1, W2= C(Me)(CH2)3SO3H or C(Me)(CH2)4CO2H); each of RN1-RN3is independently hydrogen, -Rs-SO3H, -RS-CO2H, -RS-NH2, -Rs- NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs- succinimide; more preferably, each of RN1and RN2is (CH2)3SO3H and ad RN3(CH2)4CO2H

[0158] Rsis a spacer moiety selected from -(CH2-)I-S and -(OCH2CH2-)I-4; and

[0159] Each of R1-R17 is H, Br or I, at least two adjacent RI-R3and R6-R17 form a fused aryl ring (benzo) and each of the remaining R1-R17 is H; more preferably, each of R1-R17 is H.

[0160] In some preferred embodiments pertaining to compounds represented by Formula X: wherein:

[0161] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH3is independently H, Br, I, provided that at least one of RH1-RH3is Br or I; more preferably, at least one of RH1is H and RH2-RH3is I; each of Wx-W2is independently O, S, Se, CRARB, or NRC, wherein each of RA-RCis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -RS-CO2H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, each of Wx-W2is CRARB, and each of RAand RBis Me (W1= W2= CMe2), or RAis Me and RBis (CH2)3SO3H or (CH2)4CO2H (W1, W2= C(Me)(CH2)3SO3H or C(Me)(CH2)4CO2H); each of RN1-RN3is independently hydrogen, methyl, ethyl, -Rs-SO3H, -RS-CO2H, -Rs- NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide; more preferably, each of RN1and RN2is (CH2)3SO3H and ad RN3(CH2)4CO2H Rsis a spacer moiety selected from -(CH2-)i-8 and -(OCH2CH2-)M; and

[0162] Each of R1-R16 is H, Br or I, at least two adjacent R1-R4 and R7-R16 form a fused aryl ring (benzo) and each of the remaining R1-R16 is H; more preferably, each of R1-R16 is H.

[0163] In some preferred embodiments pertaining to compounds represented by Formula XI:

[0164] Formula XI wherein:

[0165] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH3is independently H, Br, I, provided that at least one of RH1-RH3is Br or I; more preferably, at least one of RH1-RH3is I; each of Wx-W3is independently O, S, Se, CRARB, or NRC, wherein each of RA-RCis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, each of Wx-W3is CRARB, and each of RAand RBis Me (W1= W2= CMe2), or RAis Me and RBis (CH2)3SO3H or (CH2)4CO2H (W1, W2= C(Me)(CH2)3SO3H or C(Me)(CH2)4CO2H); each of RN1-RN3is independently hydrogen, methyl, ethyl, -Rs-SO3H, -Rs-C02H, -Rs- NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide; more preferably, e

[0166] Rsis a spacer moiety selected from -(CH2-)i-8 and -(OCH2CH2-)I-4; and

[0167] Each of R1-R15 is H, Br or I, at least two adjacent R1-R5 and Rs-Ris form a fused aryl ring (benzo) and each of the remaining R1-R15 is H; more preferably, each of R1-R15 is H.

[0168] In some preferred embodiments pertaining to compounds represented by Formula XII:

[0169] Formula XII wherein:

[0170] T is O, S, NH or NMe; more preferably, T is O;

[0171] RH1is independently H, Br, I, provided that at least one of RH1is Br or I; more preferably, at least RH1is I; each of R1-R2 is H, Br or I, R3is H, substituted and non-substituted alkyl, substituted and non-substituted aryl, or at least two adjacent R1-R2 form a fused aryl ring (benzo), more preferably, each of R2-R3 is H; Ri is I.

[0172] In some preferred embodiments pertaining to compounds represented by Formula XIII:

[0173] Formula XIII wherein:

[0174] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH2is independently H, Br, I, provided that at least one of RH1-RH2is Br or I; more preferably, at least one of RH1is I, and RH2is H; each of Wx-W2is independently O, S, Se, CRARB, or NRC, wherein each of RA-RCis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, each of W1and W2is CRARB, and each of RAand RBis Me (W1= W2= CMe2), or RAis Me and RBis (CH2)3SO3H or (CH2)4CO2H (W1, W2= C(Me)(CH2)3SO3H or C(Me)(CH2)4CO2H);

[0175] RN1is independently hydrogen, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide; more preferably, RN1is (CH2)3SO3H or (CH2)4CO2H

[0176] Rsis a spacer moiety selected from -(CH2-)i-8 and -(OCH2CH2-)I-4; and

[0177] Each of R1-R2 is H, Br or I, each of R3-R4 is at least two adjacent fused aryl ring (benzo) and each of the remaining R1-R4 is H; more preferably, each of R1-R4 is H.

[0178] In some preferred embodiments pertaining to compounds represented by Formula XIV :

[0179] Formula XIV wherein:

[0180] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH2is independently H, Br, I, provided that at least one of RH1-RH2is Br or I; more preferably, at least one of RH1is I, and RH2is H;

[0181] W1is independently O, S, Se, CRARB, or NRC, wherein each of RA-RCis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-maleimide and -Rs- succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, W1is CRARB, and each of RAand RBis Me (W1= W2= CMe2), or RAis Me and RBis (CH2)3SO3H or (CH2)4CO2H (W1, W2= C(Me)(CH2)3SO3H or C(Me)(CH2)4CO2H);

[0182] RN1is independently hydrogen, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide; more preferably, RN1is (CH2)3SO3H or (CH2)4CO2H

[0183] Rsis a spacer moiety selected from each of R1-R3 is H, Br or I, each of R1-R5 is at least two adjacent fused aryl ring (benzo) and each of the remaining 1 -Re is H; more preferably, each of Ri-Re is H.

[0184] In some preferred embodiments pertaining to compounds represented by Formula XV : wherein:

[0185] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH3is independently H, Br, I, provided that at least one of RH1-RH3is Br or I; more preferably, RH1-RH2is I, and RH3is H; each of Wx-W2is independently O, S, Se, CRARB, or NRC, wherein each of RA-RCis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, each of W1and W2is CRARB, and each of RAand RBis Me (W1= W2= CMe2), or RAis Me and RBis (CH2)3SO3H or (CH2)4CO2H (W1, W2= C(Me)(CH2)3SO3H or C(Me)(CH2)4CO2H); each of RN1-RN2is independently hydrogen, methyl, ethyl, -Rs-S03H, -Rs- CO2H, -RS-NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs- succinimide; more preferably, each of

[0186] Rsis a spacer moiety selected from -(CH2-)i-8 and -(OCH2CH2-)I-4; and each of R1-R14 is H, Br or I, at least two adjacent form a fused aryl ring (benzo) and each of the remaining R1-R14 is H; more preferably, each of R1-R14 is H.

[0187] In some preferred embodiments pertaining to compounds represented by Formula XVI:

[0188] Formula XVI wherein:

[0189] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH3is independently H, Br, I, provided that at least one of RH1-RH3is Br or I; more preferably, RH1and RH2is I, and RH3is H; each of Wx-W2is independently O, S, Se, CRARB, or NRC, wherein each of RA-RCis independently H, methyl, ethyl, iso-propyl, -Rs-SO3H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RCmay form a cyclopropyl or cyclobutyl rings; more preferably, each of W1and W2is CRARB, and each of RAand RBis Me (W1= W2= CMe2), or RAis Me and RBis (CH2)3SO3H or (CH2)4CO2H (W1, W2= C(Me)(CH2)3SO3H or C(Me)(CH2)4CO2H); each of RN1-RN2is independently hydrogen, methyl, ethyl, -Rs-SO3H, -Rs-C02H, -Rs- NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide; more preferably, each

[0190] Rsis a spacer moiety selected from -(CH2-)i-8 and -(OCH2CH2-)I-4; and each of R1-R12 is H, Br or I, at least two adjacent R1-R12 form a fused aryl ring (benzo) and each of the remaining R1-R12 is H; more preferably, each of R1-R12 is H.

[0191] In some preferred embodiments pertaining to compounds represented by Formula XVII:

[0192]

[0193] Formula XVII wherein:

[0194] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH3is independently H, Br, I, provided that at least one of RH1-RH3is Br or I; more preferably, RH1and RH2is I, and RH3is H; each of W1is independently O, S, Se, SiRARB, CRARB, or NRC, wherein each of RA-RCis independently methyl, ethyl, iso-propyl, -Rs-S03H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RBmay form a cyclopropyl or cyclobutyl rings; more preferably, W1is S; W2is C=O, SO2, CRARB, and each of RAand RBis H (W2= CH2), or Me (W2= CMe2); more preferably W2is C=O or CH2; each of RN1-RN4is independently hydrogen, Me, Et, Propyl isopropyl; -Rs-S03H, -Rs- CO2H, -RS-NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs- succinimide; more preferably, each of

[0195] Rsis a spacer moiety selected from each of R1-R7 is H, Br or I , at least two adjacent R1-R7 form a fused aryl ring (benzo) and each of the remaining R1-R7 is H; more preferably each of Ri, R2, Re, R7 are H or I.

[0196] In some preferred embodiments pertaining to compounds represented by Formula XVIII:

[0197] Formula XVIII wherein:

[0198] T is O, S, NH or NMe; more preferably, T is O; each of RH1-RH2is independently H, Br, I, provided that at least one of RH1-RH2is Br or I; more preferably, RH1is H or I, and RH2is I; each of W1is independently O, S, Se, SiRARB, CRARB, or NRC, wherein each of RA- Rcis independently H, methyl, ethyl, iso-propyl, -Rs-S03H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RBmay form a cyclopropyl or cyclobutyl rings; more preferably, W1is S; W2is C=O, SO2, more preferably W2is C=O; each of RN1-RN4is independently hydrogen, Me, Et, Propyl isopropyl; -Rs-S03H, -Rs- CO2H, -RS-NH2, -Rs-NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs- each of R1-R4 is H, Br or I, at least two adjacent R1-R4 form a fused aryl ring (benzo) and each of the remaining R1-R4 is H; more preferably, each of R1-R4 is H or I.

[0199] Unlocked compound familied:

[0200] General Formulae I'-XVIII' present the sonosensitizer compound in its unlocked (“safety- off’) active configuration, wherein the abovementioned “safety switch” position, represented by “T”, is restored or removed once the SRT cleaved off (detached), (formulae I- XVII) for example, or turns into detached cyclic monothiocarbonate or S -thiocarbamate systems (formula XVIII), whereas all other variables are as presented for formulae I-XVIII hereinabove. For simplicity, general formulae I'-XVIII' present a sub-set of each family, wherein non-functional substituents (R1-R17) are shown as hydrogens (not shown), the heavy halo substituent(s) is positioned without limitation in one optional position, and T is positioned without limitation in one optional position and selected to be oxygen while noting that “T” can be also sulfur or nitrogen:

[0201] Formula I’, Formula II’,

[0202]

[0203] The present invention relates to novel dye compounds optimized for targeted sonodynamic therapy (SDT), with emphasis on the incorporation of heavyhalogenand their effects on molecular properties and therapeutic efficacy. Sonodynamic therapy is an emerging treatment modality that utilizes ultrasound energy to activate photosensitizing compounds, generating reactive oxygen species (ROS) for therapeutic effects. The efficacy of SDT can be significantly enhanced by the incorporation of heavy atoms, particularly halogens, into the dye molecules used as sonosensitizers. According to some or all embodiments, the dye moiety of the compound provided herein includes at least one heavy halo substituent (bromine or iodine) on an aryl group which forms part of the complete (uninterrupted) conjugated 7t-system of the dye. In reference to halogen substituents, halogens are often categorized by their "mass" or atomic weight, whereas heavier halogens like bromine (Br) and iodine (I) are considered "heavy" compared to lighter ones like chlorine (Cl) and fluorine (F). The introduction of heavy atoms, particularly iodine, into organic dye molecules improves the efficacy of SDT in pathogens and cancer cells. This enhanced efficacy is attributed to the increasing probability of spin-orbit coupling, resulting in elevated rates of reactive species generation. Heavy halides affect a molecule's properties differently due to their larger size and polarizability, influencing the dye's reactivity to energy in the ultrasound range.

[0204] Halogens, being highly electronegative compared to carbon, create polar bonds with a partial positive charge on the carbon atom and a partial negative charge on the halogen. This polarity affects bond strength and introduces an inductive effect, where the electron- withdrawing nature of halogens extends a negative charge influence through the carbon chain, weakening adjacent bonds and affecting nearby functional groups. When attached to a molecule with a conjugated 7t-system, halogens influence the electron distribution through the mesomeric effect. The lone pair electrons on the halogen can participate in resonance, delocalizing electron density across the conjugated system. This can stabilize the molecule and affect its reactivity. The mesomeric effect of halogen substituents further affects stabilization or destabilization, depending on the halogen's position, weight, and the overall electronic structure of the 7t-system.

[0205] The size and electronegativity differences between heavy halogens (Br, I) and light halogens (F, Cl) lead to distinct influences on steric hindrance, inductive effect, and mesomeric effect. Although all halogens are electron- withdrawing, the inductive effect is stronger for heavier halogens due to their lower electronegativity difference compared to carbon. Heavier halogens have larger and more polarizable lone pairs, which can lead to greater delocalization of electron density through resonance, potentially affecting the molecule's stability and reactivity.

[0206] For successful design and implementation of targeted SDT with dyes bearing heavy atoms, several crucial parameters need to be optimized: the number of heavy (iodine) atoms in the dye, introduction of solubilizing groups (-SO3H, -N+Rs) in the dye, either directly on the aromatic system or via a spacer, solubility of the carrier, and drug-carrier ratio (DCR) to avoid aggregation of the conjugate. The introduction of heavy atoms increases the hydrophobicity of the dye, potentially reducing solubility and sono-activity. This can be addressed by conjugating the iodinated dye to a carrier, such as an antibody, where the solubility is influenced by the carrier itself. However, excessive conjugation can lead to undesired aggregation, harming sono-activity. To mitigate these issues, solubilizing polar / charged groups like sulfonates and quaternary amines can be introduced to iodinated dyes. While this complicates synthesis and may reduce sensitizing potential, it offers a balance between solubility and efficacy. The presence of carboxylic functions enables further binding of these dyes to various carriers without significantly affecting the dye's solubility in general.

[0207] In some or all embodiments, the compound provided herein comprises at least one iodine or bromine atom. Herein and throughout, the description should be interpreted with iodine and bromine being interchangeable and commutatively replaceable. The incorporation of iodine atoms into the compound structure enhances its efficacy in sonodynamic therapy by increasing the probability of spin-orbit coupling, which results in elevated rates of reactive species generation. The presence of iodine atoms also influences the compound's reactivity to ultrasound energy due to their larger size and polarizability compared to lighter halogens.

[0208] The compound may further comprise a solubilizing group selected from -SO3H and / or - N+R3, wherein R is independently selected from hydrogen and C1-6 alkyl. These solubilizing groups are introduced to mitigate the increased hydrophobicity caused by the incorporation of heavy atoms, such as iodine. The addition of these polar or charged groups helps to maintain the compound's solubility and sono-activity, striking a balance between the desired heavy atom effect and the need for adequate solubility in physiological environments.

[0209] In some or all embodiments, the solubilizing group is directly attached to an aromatic moiety / system of the compound. This direct attachment allows for a more compact molecular structure and may influence the electronic properties of the aromatic system, potentially affecting the compound's overall reactivity and stability. The proximity of the solubilizing group to the aromatic system can also impact the compound's interaction with the surrounding environment and its ability to generate reactive species upon ultrasound activation.

[0210] Alternatively, the solubilizing group may be attached to the compound via a spacer. The use of a spacer provides greater flexibility in the molecular design, allowing for optimization of the compound's properties. The spacer can help to reduce steric hindrance, potentially improving the compound's interaction with its target or carrier while maintaining the solubilizing effect of the polar or charged group. The length and nature of the spacer can be tailored to achieve the desired balance between solubility and sono-activity.

[0211] As used herein, the term "spacer" refers to a chemical moiety that connects two or more moieties, functional groups or structural elements within a molecule. The spacer may be a linear or branched chain comprising 1 to 6 carbon atoms, and may include alkyl, alkenyl, or alkynyl groups. The spacer may be substituted or unsubstituted. Substituents may include, but are not limited to, halogen, hydroxyl, amino, carboxyl, or alkoxy groups. The carbon chain of the spacer may be interrupted by one or more heteroatoms selected from nitrogen (N), oxygen (O), and sulfur (S). In some embodiments, the spacer may include cyclic structures, such as cycloalkyl or heterocyclic groups. The spacer serves to provide distance, flexibility, or specific spatial orientation between the connected groups, and may influence the overall properties of the molecule, including but not limited to solubility, reactivity, and biological activity. For the purposes of this disclosure, the term "spacer" encompasses all variations and combinations of the aforementioned structural features, unless explicitly stated otherwise.

[0212] The compound may further comprise a carboxylic function for binding to a carrier. The presence of this carboxylic group enables the compound to be readily conjugated to various carriers, such as proteins, antibodies, or nanoparticles. This feature is crucial for targeted delivery of the compound to specific tissues or cell types, enhancing the selectivity and efficacy of the sonodynamic therapy while minimizing potential side effects.

[0213] In certain or all embodiments, the compound is conjugated to a carrier. This conjugation allows for targeted delivery of the compound to specific sites in the body, improving its therapeutic index. The carrier can be selected based on its ability to recognize and bind to specific cell surface markers or to accumulate in particular tissues, thereby concentrating the compound at the desired site of action for sonodynamic therapy.

[0214] The carrier used for conjugation may be an antibody. Antibodies offer highly specific targeting capabilities due to their ability to recognize and bind to specific antigens. By conjugating the compound to an antibody, it can be selectively delivered to cells or tissues expressing the target antigen, maximizing the therapeutic effect at the desired site while minimizing exposure to healthy tissues.

[0215] The drug-carrier ratio is optimized to avoid aggregation of the conjugate. This optimization is crucial to maintain the sono-activity of the compound and ensure its effective delivery to the target site. Excessive conjugation can lead to undesired aggregation, which may reduce the compound's ability to generate reactive species upon ultrasound activation. By carefully controlling the drug-carrier ratio, the conjugate's solubility, stability, and therapeutic efficacy can be maximized.

[0216] The compound is designed for use in targeted sonodynamic therapy. This design consideration encompasses all aspects of the compound's structure and properties, including the incorporation of heavy atoms, the addition of solubilizing groups, and the ability to conjugate to carriers. The compound is specifically engineered to generate reactive oxygen species upon activation by ultrasound energy, making it suitable for localized treatment of various conditions, including cancer and microbial infections, with minimal damage to surrounding healthy tissues.

[0217] By carefully optimizing the above parameters, this invention provides a framework for designing highly effective heavy atom-bearing dye conjugates for targeted sonodynamic therapy, balancing the enhanced reactive species generation with suitable solubility and aggregation characteristics.

[0218] Excluded compounds:

[0219] Excluded from the scope of the present invention are the molecules presented in Table 1 below.

[0220] Table 1

[0221]

[0222] Stimuli-Responsive Tether:

[0223] The locked sonosensitizer compound provided herein is locked in the sense that it is not able to interact with radiation energy, and in particular with ultrasound radiation, as long as its conjugated π-system is interrupted reversibly. The purposeful and reversible locking of the sonosensitizer afforded through a strategic bond rearrangement, namely this 7t-system interruption can be rectified (reversed) by bond rearrangement afforded by dissociation of one or more covalent bonds in the locked sonosensitizer; this dissociation releases a part of the compound, which is referred to herein as a “stimuli-responsive tether”, “sonosensitizer release trigger”, or SRT. In the context of the present invention, the term “stimuli-responsive tether” is synonymous with the term “biocleavable linking moiety”, representing a subset of the more general group of “labile linking moieties”.

[0224] It is noted that in some embodiments of the present invention, the SRT may be a terminal group, namely a labile chemical moiety or labile functional group that does not link or tether other moieties to the dye moiety, yet still acts as a trigger for sensitization activity; thus, the terms “labile linking moieties” and “biocleavable linking moiety” are meant to encompass these embodiments as well, wherein upon bond rearrangement in the SRT, the sonosensitizer undergoes locked to unlocked transition.

[0225] In the context of the present invention, a "labile linking moiety" refers to a chemical group, a moiety or structure within a molecule that is susceptible to cleavage or modification under certain conditions. The term "labile" indicates that this linking moiety is inherently unstable or reactive under certain conditions, and it can undergo chemical transformations, such as hydrolysis or other cleavage reactions, under specific environmental or chemical circumstances. In the context of the present invention, the incorporation of labile linking moieties is an intentional and strategic molecular design. According to embodiments of the present invention, a labile linking moiety serves to facilitate controlled release of a functional molecular entity from the compounds disclosed herein, enabling site-specific dissociation, triggered by a specific chemical event. The labile nature of the linking moiety allows for the selective and controlled manipulation of the molecule under predetermined conditions. The choice of a labile linking moiety depends on the desired application and the specific chemical reactions or processes targeted for the molecule's behavior. Labile biocleavable linking moieties are commonly employed in drug delivery systems, prodrugs, and other chemical constructs where controlled reactivity or release is essential.

[0226] The term “biocleavage” refers to a biochemical reaction that causes the linking moiety to break / dissociate. In the context of embodiments of the present invention, biocleavage is typically mediated by biomolecules (e.g., enzymes, RNA and the likes) in an organism, an organ, or a cell thereof, whereas each such mediator is active or more active under certain conditions, including location in the organism (cell, tissue, organ), temperature, pH, ionic strength, light and other reaction effectors, as these are known in the art. Linking moieties having different biocleavage condition allow a differential cleavage at different locations in the subject, and / or different times, and / or under otherwise different physiological conditions.

[0227] A linking moiety that is stable at physiological conditions, namely the linking moiety does not disintegrate for the duration of exposure to the physiological environment in the bodily site, is referred to herein a "biostable linking moiety". 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 molecular structure when present in physiological conditions.

[0228] In some embodiments of the present invention, the linking moieties in the compound provided herein are all biocleavable linking moieties. In the context of some embodiments of the present invention, biocleavable linking moieties are selected so as to break and unlock the sonosensitization capabilities of the dye, or release a payload under certain conditions, referred to herein as “biocleavage conditions” or stimuli.

[0229] As used herein, the terms “biocleavable linking moiety” or “stimuli-responsive tether” (SRT) 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. In some embodiments, biocleavable linking moieties are selected according to their susceptibility (sensitivity) 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.

[0230] According to some embodiments of the present invention, the labile linking moiety, or biocleavable linking moiety, is a stimuli-responsive tether (SRT), namely a detachable moiety that undergoes cleavage (detachment) when exposed to certain stimuli, such as a change in physical conditions (temperature, electrostatic field, magnetic field, electromagnetic field etc.), a change in chemical conditions (pH, ionic strength, anions, free radicals etc.), and a change in biological / physiological conditions (exposure to enzyme catalysis, membrane potential / gradient etc.). The terms “biocleavable linking moiety” and “detachable linking moiety” are used herein interchangeably.

[0231] According to some embodiments of the present invention, the SRT is cleaved upon change in biological / physiological conditions that the locked sonosensitizer experiences or exposed to, for example, when being taken-in by a cell and exposed to enzymes within the cell. In the context of a pharmaceutical agent, and as an active ingredient in a pharmaceutical composition, the presently disclosed locked sonosensitizer compound, the SRT is sensitive to biological / physiological conditions that are present in a predefined targeted cell / tissue / organ, and is not sensitive to the biological / physiological conditions that are present in the rest of the subject, not in the predefined targeted cell / tissue / organ.

[0232] A stimuli-responsive tether essentially functions as a specialized type of a labile linking moiety, and according to some embodiments of the present invention, a detachable biocleavable linking moiety. Thus, a "stimuli-responsive tether," according to some embodiments of the present invention, is a detachable moiety designed to undergo cleavage in response to specific stimuli. This type of linking moiety is intentionally engineered to be sensitive to particular environmental cues, triggering its cleavage and consequent reinstatement of the sonosensitization activity of the dye moiety of the compound, and optionally the release of a functional agent, e.g., in the form of a bioactive agent. The term "stimuli-responsive" indicates that the cleavage and detachment event is activated by external factors or changes in the surroundings. These stimuli can encompass a variety of physical, chemical and biological / physiological conditions, or other biochemical factors, triggering the cleavage of the tether. In other words, a stimuli-responsive tether (SRT) is a labile linking moiety with the capacity to undergo cleavage in response to specific external stimuli, offering a controlled and triggered release of the tethered functional group and / or structural modification. In the context of the present invention, this design has a practical application in the form of rendering the dye moiety of the compound electronically active, and optionally using the compound as an effective drug delivery and controlled-release system.

[0233] Representative examples of biocleavable moieties that can serve as SRTs in the context of the locked sonosensitizer compounds provided herein, include, without limitation, amides (peptide bonds), acetals, boronic acids, boronic esters, ketals carboxylates, carbamates, phosphates, hydrazides, thiohydrazides, disulfides, epoxides, peroxo, methyleneamines, glucuronide, β- galactose, alkyl silyls and aryl silyls. 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.

[0234] 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.

[0235] Additional information pertaining to enzymes, enzymatic reactions, and enzyme-linking moiety correlations can be found in various publicly accessible sources, such as Bairoch A., “The ENZYME database in 2000”, Nucleic Acids Res, 2000, 28, pp. 304-305.

[0236] In some embodiments, certain linking moieties are selected to be more labile than other linking moieties present in the molecular structure. By “more labile”, it is meant that some of the linking moieties have a higher tendency to break at given cleavage conditions compared to other linking moieties. In some embodiments, the linking moieties are selected according to a certain lability hierarchy that allows the design of a particular drug-releasing profile, wherein the order and the rate of drug release is controllable according to the lability hierarchy. In the context of some embodiment of the invention, the more labile linking moieties, higher in the lability hierarchy will break first and at a higher rate than those lower in the lability hierarchy.

[0237] Biocleavable linking moieties, or SRTs, according to some embodiments of the present invention, include without limitation, amide, ester, ether, carbonate, carbamate, disulfide, thiocarbamate, sulfonamide, boronic acid, boronate, glucuronide, β-galactose, silyl and phosphate.

[0238] Additional non-limiting examples of biocleavable linking moieties that can serve as SRTs in the context of a locked sonosensitizer compound, according to some embodiments of the present invention, include, amide, acryl, carbamate, carbonate, lactone, lactam, carboxylate, ester, cycloalkene, cyclohexene, heteroalicyclic, heteroaryl, triazine, triazole, disulfide, imine, imide, oxime, aldimine, ketimine, hydrazone, semicarbazone, acetal, ketal, glucoside bond, aminal, aminoacetal, thioacetal, thioketal, phosphate ester, 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.

[0239] According to some embodiments, the linking moiety is selected from the group consisting

[0240]

[0241] In some embodiments of the present invention, the SRT is not amide, namely in some embodiments, amide is excluded by proviso from the SRT position. Self-immolative linker:

[0242] In some embodiments, the SRT is a self-immolative linker.

[0243] As used herein, the term “self-immolative linker” refers to a specialized type of chemical bonds, chemical moieties, structures or sequences within molecules that are designed and / or selected to undergo spontaneous and controlled degradation or decomposition under specific conditions, leading to the release of attached functional groups or substances. According to embodiments of the present invention, a "self-immolative linker" is a covalent assembly that connects a chemical moiety to another molecular entity. This covalent assembly is designed to undergo a sequential cleavage of two chemical bonds or more in response to a specific stimulus. The cleavage results in the release of the attached chemical moiety and a small molecule, often a benign leaving group. The term "self-immolative" signifies the ability of the linker to self-cleave, thereby releasing the attached chemical moiety. According to some embodiments, the design of the self-immolative linker is such that the cleavage of an initial bond triggers a cascade of intramolecular reactions leading to the complete disassembly of the linker. A person skilled in the art would appreciate the meaning of the term "self-immolative linker" and find it useful in identifying such moieties. Furthermore, a person skilled in the art would understand how to incorporate and use self-immolative linkers in the context of the present invention to achieve controlled release of the chemical moiety under specific conditions. It should be noted that the specific structure and composition of the self-immolative linker can vary depending on the desired application, the nature of the chemical moiety to be released, and the specific stimulus for cleavage. The design and synthesis of self-immolative linkers are within the purview of a person skilled in the art.

[0244] In the context of drug delivery systems or prodrugs, self-immolative linkers are often employed to facilitate the controlled release of active compounds at the target site, hence, in the context of embodiments of the present invention, the term "self-immolative linker" refers to a type of a labile linking moiety within the presently disclosed locked sonosensitizer compound, that has the ability to undergo spontaneous cleavage or degradation under certain conditions, whereas the term "self-immolative" implies that the labile linking moiety contains elements that, once stimulated, lead to a cascade of intramolecular reactions resulting in the release of a functional group or a fragment from the parent compound. In other words, a self-immolative linker is designed to be inherently unstable under specific environmental or chemical conditions. Upon exposure to these conditions, the linker undergoes a series of chemical transformations, ultimately leading to the cleavage of the molecule at the linker site. This controlled degradation is often utilized in drug delivery systems, prodrugs, or other molecular constructs where a triggered release of a payload or functional group is desired at a particular location and / or time.

[0245] Generally, according to embodiments of the present invention, the self-immolative linker is designed and / or selected to undergo a programmed, irreversible disassembly upon specific stimuli. This self-destruction process acts like a domino effect, breaking down the linker molecule one bond at a time, ultimately releasing the attached functional groups. According to some embodiments of the present invention, a self-immolative linker may comprise several functional groups and biocleavable moieties, and be tethered to the dye via spacer moieties and also be terminated with any type of functional group. A terminal group on a self-immolative linker SRT or an inherent part of the self-immolative linker, may be used to tether another functional moiety, such as a bioactive agent, a targeting moiety, and / or any type of payload that is designed to be released from the compound upon cleavage of the self-immolative linker.

[0246] Following below is a non-limiting list of self-immolative linkers, contemplated within the scope of some embodiments of the present invention, categorized by the enzyme or biochemical mechanism responsible for their cleavage:

[0247] Esterase-Cleavable Linkers: Acetate -based linkers that are cleaved by esterases to trigger the breakdown of the entire linker; and

[0248] Benzoate-based linkers: Specifically designed to be cleaved by esterases in the liver.

[0249] Phosphatase-Cleavable Linkers:

[0250] Phosphate esters: These linkers are cleaved by phosphatases, leading to a self- immolation that releases the drug.

[0251] Protease-Cleavable Linkers:

[0252] Peptide linkers: Composed of specific amino acid sequences that are targets for proteases like cathepsin B, which can be found in tumor tissues; and

[0253] Arginine-rich linkers: Targeted by trypsin-like proteases, commonly used in targeted cancer therapies.

[0254] Redox-Responsive Linkers:

[0255] Disulfide / diselenide linkers: Cleaved by reduction reactions mediated by glutathione, which is abundant inside cells but low in blood plasma, aiding selective release within cells. pH-Responsive Linkers:

[0256] Hydrazone linkers: These are stable at neutral pH but cleave at the acidic pH found in tumor environments or endosomes; and

[0257] Ketal linkers: Cleaved in acidic conditions, useful for release in the stomach or other acidic environments.

[0258] Enzyme-Specific Trigger Linkers: β-Galactosidase-cleavable linkers: Utilized for drug release in senescence tissues where β- galactosidase is overexpressed. β-Glucuronidase-cleavable linkers: Utilized for drug release in cancer tissues where β-glucuronidase is overexpressed; and

[0259] Nitroreductase-activated linkers: Cleaved in hypoxic conditions typical of solid tumors.

[0260] The person skilled in the art of organic chemistry and biochemistry would be able to find ample guidance in the literature when selecting or designing a self-immolative linker to serve as an SRT, that would suit the particular purpose of the compound provided herein. For example, a review article by Gavriel, A.G et al. [“Recent advances in self-immolative linkers and their applications in polymeric reporting systems”, Polym. Chem., 2022, 13, 3188-3269], the contents of which are incorporated herein by reference in its entirety, provides useful practical information that can be used to design the SRT of the compound provided herein. Other sources of information regarding self-immolative linkers that are relevant in the context of designing the compound provided herein include Blencowe, C.A. et al. [“Self-immolative linkers in polymeric delivery systems”, Polym. Chem., 2011, 2, 773-790], Tranoy-Opalinski, I. et al. [“Design ofSelf- Immolative Linkers for Tumour-Activated Prodrug Therapy”, Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry - Anti-Cancer Agents, 2008, 8(6), 618- 637(20)], Deng, Z. et al. [“Disulfide-Based Self-Immolative Linkers and Functional Bioconjugates for Biological Applications” , Macromol. Rapid Commun., 2019, 1900531], Bargh, J.D., et al. [" Sulfatase-cleavable linkers for antibody-drug conjugates" , Chem. Sci., 2020, 11, 2375-2380], Ashman, N. et al. [" Peroxide-cleavable linkers for antibody-drug conjugates" , Chem. Commun., 2023, 59, 1841-1844] and Gonzaga, R.V. et al. [“Perspectives About Self-immolative Drug Delivery Systems”, Journal of Pharmaceutical Sciences, 2020, 109, 3262-3281].

[0261] A glutathione-cleavable self-immolative linker:

[0262] In some embodiments of the present invention, the SRT in the sonosensitizer compound provided herein comprises a glutathione-cleavable self-immolative linker. Disulfide bonds (R-S- S-R) exists broadly in natural organisms, and owing to its reversibility, disulfide and diselenide bonds can be reduced to free thiols or selenols respectively in the presence of reducing agents such as glutathione (GSH), which is the most abundant intracellular thiol-containing small molecule. In living organisms, GSH and its oxidized form, glutathione disulfide (GSSG), in conjunction with redox enzymes such as thioredoxin (Trx), thioredoxin reductase (TrxR), and glutathione reductase, constitute the major intracellular redox couples, which are of crucial importance to regulate intracellular redox homeostasis and protect organisms from oxidative stress. Notably, the GSH concentration drastically increases from about 2-10 pm in blood and other body fluids to —1—10 mm within the cytosol. More importantly, GSH has been recognized as a biomarker of tumor development and elevated GSH levels were found inside tumor mass (about 100-fold).

[0263] Exemplary glutathione-cleavable self-immolative linkers that can serve as an SRT in the context of the locked sonosensitizer compound provided herein include, without limitation, disulfide linkers such as 5-[(N-maleoylamino)(3-carboxyphenyl)(sulfanyl)methyl]pentanoic acid (MCPS), selenophene-based MCPS wherein the central sulfur atom in MCPS is replaced with selenium, N-(5-nitro-2-pyridyl)carbamoyl-cysteamine (NPCC), Bis-(y-glutamyl) cystamine (BGC); spirocyclic carbonates such as glycol-linked spirocyclic carbonates (GLSCs), valproic acid-based spirocyclic carbonates (VACs); aryl N-sulfone amides such as 3,4-Dihydroxyphenyl- N-(4-hydroxyphenyl)sulfonylamide (DHPDS), p-Aminophenylsulfonylvaline (PASV), and selenadiazole amides that mimic the N-sulfone amide structure with a selenium-nitrogen double bond; dibenzyl telluroxide linkers such as 4,4'- Di(telluromethyl)biphenyl and 1,2-Ethanediyl bis(telluromethyl) bis(benzamide); selenocysteine-containing peptide linkers; diselenide bridge linkers; tellurium-containing oxime carbonates; selenol esters; and other examples such as 5-((2- nitrophenyl)oxycarbonyl)thioethyl sulfamate (NPES), β-thioesters, silyl esters, vinyl sulfide linkers, and oxime carbonates.

[0264] In the context of some embodiments of the present invention, the SRT is a glutathione- cleavable self-immolative linker, which is exemplified, without limitation, by the following non- limiting exemplary moieties:

[0265] A cathepsin B-cleavable self-immolative linker:

[0266] In some embodiments of the present invention, the SRT in the sonosensitizer compound provided herein comprises a cathepsin B-cleavable self-immolative linker. Briefly, cathepsin B is a lysosomal enzyme found in various tissues, including tumors. Correspondingly, cathepsin B- cleavable self-immolative linkers are designed to be stable outside the cell yet rapidly dissociate upon cleavage by cathepsin B within the cell. This cleavage liberates a "payload" molecule, which could be a bioactive agent (e.g., a drug), imaging agent, or other functional entity. The self- immolative process involves a chain reaction triggered by the cathepsin B cleavage, ensuring complete release of the payload.

[0267] Some examples of cathepsin B-cleavable self-immolative linkers include, without limitation, Val-Cit, Val-Cit-PABA, Vai-Ala, Val-Ala-PABA, Ala-Ala-Leu, Phe-Lys, Gly-Phe- Leu-Gly, Nle-Lys-Arg, Arg- Arg, Phe-Arg and Gly-Phe-Gly peptide linkers. It is noted that in some cases the self immolative process occurs at the PABA moiety, whereas the peptide sequence serves as the recognition part that after cleavage of peptide bond releases PABA that spontaneously releases payload.

[0268] All of the aforementioned short cathepsin B-cleavable peptides may also feature a terminal spacer moiety bridging between the peptide and the linked entity. Suitable spacer moieties which are relevant in the context of all types of SRTs of the locked sonosensitizer compounds provided herein (not only cathepsin B-cleavable SRTs), include alkyl chains such as methylene, ethylene, etc.; polyethylene glycol (PEG) chains which are versatile spacers offering water solubility, increased linker stability, and controlled drug release profiles through their variable chain lengths; aromatic spacers which are rigid scaffolds like phenylene or biphenylene that can influence linker conformation and impact cleavage kinetics; peptide spacers which are short peptide sequences, typically di- or tripeptides, which provide additional functionalities like biocompatibility, specific interactions with target sites, and controlled cleavage by other enzymes; amide-based spacers, such as simple alkyl or aromatic amides that offer flexibility and potential for incorporating targeting moieties or cleavable functionalities; heterocycle-containing spacers such as the cyclic backbones like piperidine, morpholine, or tetrahydrofuran that alter linker rigidity, solubility, and potentially interact with specific biological targets. Other noteworthy examples of spacers include carbonate spacers for controlled cleavage in specific environments like acidic conditions, ester spacers that are susceptible to hydrolysis in the cell, potentially influenced by endogenous enzymes, and disulfide spacers which are cleaved by glutathione, introducing an additional trigger mechanism for drug release.

[0269] Non-limiting examples of short peptide-based cathepsin B-cleavable self-immolative linker featuring a spacer moiety include Val-Cit-p ABA-PEG (pAB or PABA refers to paraaminobenzyl), wherein the Val-Cit dipeptide is cleaved by cathepsin B, followed by release of an optional payload attached to the pABA unit through the PEG spacer; Gly-Phe-Leu-Cit-pABC- PEG (pABC refers to para-aminobenzoic acid) spacer being a longer linker that incorporates additional amino acids for increased spacer length and potentially altered release kinetics; Dab(Cbz)-Cit-pABC-PEG spacer, which utilizes a Dab residue with Cbz protection for increased stability and controlled release through the PEG spacer.

[0270] Non-limiting examples of heterocyclic-based cathepsin B-cleavable self-immolative linker featuring a spacer moiety include spirocyclical oxazolidinone linkers with alkyl spacers, which combine a cathepsin B-cleavable oxazolidinone ring with different alkyl chains for varying spacer lengths and properties; pyrrolidine-based linkers with PEG spacers wherein the pyrrolidine ring acts as the cleavable unit, attached to the dye or payload via a PEG spacer for controlled release; 1,2,3-triazole-based linkers with aromatic spacers wherein the triazole ring is susceptible to cathepsin B, while the aromatic spacer provides rigidity and potentially influences release kinetics.

[0271] Additional non-limiting examples of cathepsin B-cleavable self-immolative linker with spacers include citronellol-based linkers with alkyl spacers that utilize a cis-acetal bond sensitive to cathepsin B, connected to the dye or payload through various alkyl spacer lengths; proarginine- based linkers with peptide spacers wherein the proarginine residue is cleaved by cathepsin B, releasing the attached dye or payload via a short peptide spacer; hydrazone-based linkers with PEG spacers wherein the hydrazone bond undergoes enzymatic cleavage, with the PEG spacer ensuring controlled release of the drug molecule. This linker type is typically fitted with a paraaminobenzoic acid spacer that is designed to ensure that the cathepsin-mediated cleavage is unimpeded by the pay load. The pABC spacer undergoes a 1,6-elimination reaction to release the drug payload tracelessly following the initial enzymatic cleavage.

[0272] In some preferred embodiments, the cathepsin B -cleavable self-immolative linker is a dipeptide or a tripeptide fitted with an optional spacer, such as p-aminobenzyl that is afforded by using p-aminobenzyloxycarbonlyl (pABA or pABC) as a building block. The dipeptide is preferably valine-citrulline (Val-Cit) dipeptide linker that has been utilized in ADCs clinically, and displays an excellent balance between plasma stability and intracellular protease cleavage. It is one of the most commonly used cleavable linkers in ADCs; phenylalanine-lysine (Phe-Lys) dipeptide linker that shows greater systemic stability with rapid enzymatic drug release in the target cell; and valine-alanine (Vai- Ala) dipeptide linker.

[0273] The following are non-limiting exemplary moieties of cathepsin B-cleavable self- immolative linkers featuring a dipeptide and a p-aminobenzyl spacer, whereas the wiggled line marks the dye tether position, and Ac is acetyl: wherein R' is an optional functional group that can be used to tether a functional moiety (bioactive agent, drug, payload, targeting moiety, etc.) to the dye via the SRT. A glucuronidase-cleavable self- immolative linker:

[0274] In some embodiments of the present invention, the SRT in the sonosensitizer compound provided herein comprises a glucuronidase responsive self-immolative linker. Glucuronidase is an enzyme found in various tissues, including tumors, that catalyzes the hydrolysis of glucuronides, also known as glucuronosides, which belong to the glycosides family. Glucuronides are substances produced by linking glucuronic acid to another substance via a glycosidic bond. Glucuronidase responsive self-immolative linkers are designed to be stable outside the cell but rapidly dissociate upon cleavage by glucuronidase within the target environment. This cleavage liberates a "payload" molecule, which could be a bioactive agent, imaging agent, or other functional entity.

[0275] Some examples of glucuronidase-cleavable self-immolative linkers include, without limitation, β-D-galactose, β-D-glucose, β-D-glucuronide, β-maltose, 4-nitrobenzyl ether, aryl sulfonate ester, O-glucuronide, and hydrazide; β-D-glucuronidoalkyl derivatives such as (β-D- glucuronido)alkyl phenyl acetates, β-D-glucuronidoalkyl carbamates, N-Acetyl-(β-D- glucuronido)alkyl-3-hydroxytetrahydrofuran, N-Boc-(β-D-glucuronido)alkyl-3- aminotetrahydrofuran, 7-hydroxycoumarin-β-D-glucuronides, 3-hydroxycoumarin-β-D- glucuronides; tetrahydrofuran-based linkers such as N-acetyl-(β-D-glucuronido)alkyl-3- hydroxytetrahydrofuran, N-Boc-(β-D-glucuronido)alkyl-3-aminotetrahydrofuran; coumarin- based linkers such as 7-hydroxycoumarin-β-D-glucuronides, 3-hydroxycoumarin-β-D- glucuronides; and other examples such as triazole-based linkers, iminosydnone-based linkers, spirocyclic scaffolds with β-D-glucuronide side chains and oxime bonds with β-D-glucuronide substituents.

[0276] The following are non-limiting exemplary moieties of β-glucuronidase-cleavable self- immolative linkers, whereas the wiggled line marks the dye tether position:

[0277] A galactosidase-cleavable self-immolative linker: In some embodiments of the present invention, the SRT in the sonosensitizer compound provided herein comprises a galactosidase responsive self-immolative linker. A "galactosidase-cleavable self-immolative linker" is designed to be cleaved specifically by the enzyme β-galactosidase. Upon enzyme interaction, the linker initiates a self-immolation process that cleaves and optionally releases a drug pay load attached to it. This type of linker is particularly useful in targeting cells or tissues where β-galactosidase is overexpressed or externally applied.

[0278] Examples of such linkers are used in the development of prodrugs for targeted cancer therapy, where the drug is released only after the linker is cleaved by the enzyme, enhancing the drug's efficacy and reducing side effects [Sheyi, R. et al., “Linkers: An Assurance for Controlled Delivery ofAntibody-Drug Conjugate” , Pharmaceuti.cs, 2022, 14(2), 396]. The following are nonlimiting exemplary moieties of galactosidase-cleavable self-immolative linkers, whereas the wiggled line marks the dye tether position: wherein R' is an optional functional group that can be used to tether a functional moiety (bioactive agent, drug, payload, targeting moiety, etc.) to the dye, such as a prop- 1-yne group which is conducive to click chemistry conjugation.

[0279] A peroxide-responsive linker: In some embodiments of the present invention, the SRT in the sonosensitizer compound provided herein comprises a peroxide -responsive self-immolative linker. A "peroxide-cleavable self-immolative linker" is a type of chemical bond used, for example, in antibody-drug conjugates (ADCs), that is cleaved specifically in the presence of hydrogen peroxide (H2O2) - a reactive oxygen species (ROS) that is often found at higher levels in cancer cells compared to normal cells. This self-immolative linker is designed to exploit the elevated ROS levels in cancer cells, allowing the selective release of the drug at the target site, thereby minimizing off-target effects. The linker typically includes a boronic acid or ester moiety that undergoes a chemical reaction when exposed to hydrogen peroxide, leading to the breakdown of the linker and the release of the drug. Examples of peroxide-cleavable self-immolative linkers include, without limitation, arylboronic acid linkers, which are designed to rapidly cleave in the presence of hydrogen peroxide, but remain stable in plasma. Such linkers have been described in conjunction with pay loads like 7-amino 4-methyl coumarin (AMC) for experimental studies [Ashman, N. et al. [" Peroxide-cleavable linkers for antibody-drug conjugates" , Chem. Commun., 2023, 59, 1841-1844]. These linkers show enhanced fluorescence upon cleavage, providing a visual confirmation of cleavage under oxidative conditions. The following are non-limiting exemplary moieties of peroxide-cleavable self- immolative linkers, whereas the wiggled line marks the dye tether position: wherein R' / R” is an optional alkyl, cycloalkyl, aryl or functional group that can be used to tether a functional moiety (bioactive agent, drug, payload, targeting moiety, etc.) to the dye, such as a prop-l-yne group which is conducive to click chemistry conjugation.

[0280] Additional self-immolative linkers which are useful in the context of the present invention, include:

[0281] Sulfatase-cleavable linkers (Arylsulfate linkers): These linkers are designed to be cleaved by lysosomal sulfatase enzymes. Upon enzymatic cleavage of the sulfate group, a subsequent spontaneous chemical reaction (1,6-elimination) releases the drug payload completely. This mechanism was shown to be effective with payloads such as monomethyl auristatin E (MMAE), where the cytotoxicity assays demonstrated that arylsulfate linkers provided a more stable and efficient release mechanism compared to traditional dipeptide linkers.

[0282] The following are non-limiting exemplary moieties of sulf atase-cleav able linkers, whereas the wiggled line marks the dye tether position: wherein R' may be a functional group that can be used to tether a functional moiety (bioactive agent, drug, payload, targeting moiety, etc.) to the dye, such as a prop-l-yne group which is conducive to click chemistry conjugation. More information pertaining to sulfatase-cleavable linkers can be found, e.g., in Bargh, J.D., et al. [" Sulfatase-cleavable linkers for antibody-drug conjugates" , Chem. Sci., 2020, 11, 2375-2380].

[0283] Fluoride-cleavable linkers (Silyl linkers):

[0284] According to some embodiments, in the context of general Formulae I- XVIII, the SRT is selected from the group consisting of:

[0285] wherein: each of R18-R21 is independently H, Me or Et;

[0286] R22 is selected from H, -OH, -NH2, -NHMe, -NMe2, -NMe3+, -PO4H2, -PO2(OMe)2, -

[0287] CO2H, -CO2Me, -SO3H, alkyl, alkoxy, haloalkyl, -(OCH2CH2-)I-3, -CO2-D, -CONH-D, -CONMe- D, -OCO-NH-D, -OCO2-D, and -OCO-NMe-D; and

[0288] D is an optional is a functional moiety, such as a bioactive agent (e.g., a drug), an imaging agent, a labeling agent, and / or a diagnostic agent moiety. Dual-action drug-delivery compounds:

[0289] According to some embodiments of the present invention, the compound provided herein carries a payload in the form of a releasable functional agent, attached to the compound via a liable linking moiety. The liable linking tethers the releasable functional agent at any suitable location in the compound, such as, for example the SRT. When in the tethered form, while attached to the compound, the functional agent is referred to herein as a functional moiety.

[0290] The term “agent” is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. The terms “bioactive agent”, “therapeutic agent”, “drug” and “payload” are used herein interchangeably and refer to an agent (a substance) that is capable of modulating a biological process and / or has biological activity. In some embodiments, the term “bioactive agent” refers to a drug or a therapeutic agent. An anticancer drug is an exemplary bioactive agent or therapeutic agent.

[0291] The term “chemotherapeutic agent”, “anticancer drug” or “anticancer agent”, is used herein to refer to all agents that are effective in treating cancer regardless of mechanism of action. Inhibition of metastasis or angiogenesis is frequently a property of a chemotherapeutic agent. Chemotherapeutic agents include antibodies, biological molecules, and small molecules, and encompass sonosensitizers, as described herein. A chemotherapeutic agent may be a cytotoxic or cytostatic agent. The term “cytostatic agent” refers to an agent that inhibits or suppresses cell growth and / or multiplication of cells. The term “cytotoxic agent” refers to a substance that causes cell death primarily by interfering with a cell's expression activity and / or functioning.

[0292] According to some embodiments of the present invention, the SRT is a labile linking moiety, which is also used to conjugate a functional agent, such as a bioactive agent, such as a drug, an imaging / labeling agent, such as a dye, and / or a diagnostic agent, to the locked sonosensitizer. In such embodiments, the cleavage event not only activates the sonosensitizer (switch to “safety off’ state), but also releases the functional agent from the compound, allowing it to exert it intended sonosensitization effect.

[0293] The term “functional moiety”, as used in the context of embodiments of the present invention, refer to a moiety of a functional agent (e.g., a bioactive agent, a diagnostic agent or a labeling agent), such as, without limitation, a drug or a dye, which may take the form of a small molecule, an amino-acid, a peptide, a polypeptide, a protein, a polysaccharide, and the likes. The term “functional moiety” relates to the term “functional agent” while considering that the “function” is 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.

[0294] In some embodiments, the term “functional agent” refers to a "bioactive agent" such as a pharmaceutical drug, whereas the term "bioactive moiety" refer to a moiety of the corresponding bioactive agent. According to some embodiments, the term "bioactive agent" refers to small molecules and large or small biomolecules / polymers that alter, inhibit, activate, or otherwise affect a biological mechanism, process or event. Bioactive agent that can be tethered to the compound, 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 antiinflammatory 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-cholinergic s, anti-glaucoma compounds, modulators of cell-extracellular matrix interactions including cell growth inhibitors and antiadhesion 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, antiemetics, 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.

[0295] 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.

[0296] 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; Eflornithine 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; Prednimu stine; 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; Vinleurosine 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).

[0297] 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, antinomycin, 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.

[0298] 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; Difhmisal; 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.

[0299] 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.

[0300] 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.

[0301] 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).

[0302] 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.

[0303] Representative examples of hormones include, without limitation, methyltestosterone, androsterone, androsterone acetate, androsterone propionate, androsterone benzoate, androsteronediol, androsteronediol-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-methyl-19-nortestosterone and pharmaceutically acceptable esters and salts thereof, and combinations of any of the foregoing.

[0304] 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, lomoxicam, 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.

[0305] 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.

[0306] In some embodiments, the bioactive moiety is of a bioactive agent which is a drug, and in some preferred embodiments, an anticancer drug. According to some preferred embodiments, the anticancer drug includes, without limitation, the following bioactive agents: wherein the wiggled line represents an optional point of attachment of the moiety on a bioactive agent (e.g., a drug) to the SRT. It is noted that the proposed point of attachment depicted in the schemes above should not be taken as limiting, since other attachments points can also be utilized.

[0307] In some embodiments, the functional agent is a bioactive agent, preferably an anticancer drug, and more preferably a member of the dolastatins family, which is a group of linear and cyclic, natural or synthetic peptides and peptidomimetic analogues thereof. In some embodiments, the functional agent is the anticancer drug doxorubicin, and any derivative or analogues thereof.

[0308] In some embodiments, the functional agent is another sensitization agent.

[0309] In some embodiments, the term “functional agent” refers to a "diagnostic agent" such as a biomarker, whereas the term "diagnostic moiety" refer to a moiety of the corresponding diagnostic agent. According to some embodiments, the term " diagnostic agent" refers to contrast agents such as, without limitation, iodine-based contrast agents (e.g., iodixanol), and gadolinium-based contrast agents (e.g., gadopentetate dimeglumine); biomarkers such as, without limitation, pro state- specific antigen (PSA), cardiac troponins (troponin T and troponin I), C-reactive protein (CRP), and CA-125 for monitoring ovarian cancer; radioactive tracers such as, without limitation, fluorodeoxyglucose ([18F]FDG), "Tc complexes, and131I containing molecules; fluorescent probes such as, without limitation, green fluorescent protein (GFP), fluorescein isothiocyanate (FITC), and rhodamine-based dyes; and molecular imaging agents such as, without limitation, technetium-99m sestamibi, and radiolabeled somatostatin analogs.

[0310] In some embodiments, the functional agent is another dye moiety serving as a bioactive agent, an imaging agent and / or a diagnostic agent.

[0311] Targeting moiety:

[0312] The locked sonosensitizer compound, as described herein, can also be designed to specifically target certain cells or tissues within a living organism, such as a patient undergoing treatment. This specificity is achieved by optionally attaching a targeting moiety to the compound, forming a conjugate that can accumulate in (internalization) or near the targeted cells or tissues. In some embodiments, this targeting moiety, conjugated to the locked sonosensitizers, facilitates a higher concentration of the locked sonosensitizer at the targeted site compared to non-targeted sites. This selective accumulation of the compound, which takes place before ultrasound energy is applied, significantly reduces the adverse side effects associated with the cytotoxicity of the unlocked sonosensitizer and any optional functional agents attached thereto. Furthermore, the SRT is chosen specifically for its ability to cleave under conditions that are more prevalent at the targeted site than at non-targeted sites. This selective cleavage unlocks the sonosensitizer and potentially releases a payload of functional agents at the targeted site at a higher rate compared to non-targeted sites, further enhancing the treatment’s effectiveness.

[0313] As used herein, the term “targeting moiety” refers to a molecular entity that exhibits an affinity to a specific bodily site (e.g., particular organ, cells and / or tissues) by virtue of binding to a particular structural arrangement of functional groups that characterized the target, such as for example, a pharmacophore. Hence, as used herein, the term “targeting moiety” refers to an optional molecular component (a moiety) of the compound provided herein, that possesses an affinity for a specific site within the body, which is often established through the recognition and binding to particular structural features or functional groups characteristic of the target site, which may include specific cells, organs, or tissues. The concept of a pharmacophore, a molecular framework that is recognized by a biological target, is commonly employed in drug design and molecular recognition. 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.

[0314] Exemplary targeting moieties which are contemplated within the scope of the present invention include, without limitation, nanoparticles, peptides, proteins, porphyrins, hormones, saccharides, polysaccharides, biopolymers, antigens, haptens, antibodies and fragments thereof, DNA fragments, folates, polymeric micelles, liposomes, lipoprotein-based drug carriers, dendrimers, siRNA, oligonucleotides, 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.

[0315] 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.

[0316] 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). 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.

[0317] 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.

[0318] 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.

[0319] 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 avPs 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.

[0320] 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],

[0321] 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).

[0322] One of the most employed targeting moieties in the field are antibodies, constituting the discipline of antibody-drag conjugates (ADC). ADCs are engineered bioconjugates comprising antibodies (Abs), oftentimes monoclonal Abs (mAbs), that are selectively linked to potent cytotoxic drugs (payload) via chemical linkers. ADCs have revolutionized cancer therapy by combining the exquisite targeting power of monoclonal antibodies with the potent cell-killing abilities of cytotoxic agents. This targeted approach delivers the cytotoxic agent pay load directly to tumor cells, minimizing harm to healthy tissues. ADCs are particularly suited for treating cancers that express specific surface antigens, like HER2 -positive breast cancer and CD30-positive lymphoma. Upon binding to the target antigen, the ADC is internalized by the tumor cell, triggering release of the drug and potent tumor cell death. This targeted delivery significantly improves patient outcomes by increasing efficacy and reducing off-target toxicities compared to traditional chemotherapy.

[0323] ADCs represent an emerging therapeutic discipline with applications extending beyond traditional cancer therapy. While initially developed for cancer treatment, the versatility of ADCs has led to exploration in various therapeutic domains. In infectious diseases, according to some embodiments of the present invention, ADCs, as well as compounds featuring other targeting moieties, are contemplated as targeted agents against pathogenic microorganisms, leveraging the targeting moiety’s specificity to recognize and neutralize infectious agents while delivering a payload of antimicrobial drugs. Additionally, ADCs, as well as compounds featuring other targeting moieties that are not related to antibodies, are emerging as promising candidates in autoimmune disorders, where precise targeting of immune cells responsible for aberrant responses can be achieved, minimizing systemic side effects.

[0324] The skilled artisan would appreciate the abundance of guidance pertaining to conjugation of the presently disclosed locked sonosensitizer compound to a targeting moiety, such as provided in, for example, Tsuchikama, K. and An, Z., “Antibody-drug conjugates: recent advances in conjugation and linker chemistries” , Protein Cell, 2018, 9, pp. 33-46; Kesireddy, M., et al., “A Review of the Current FDA-Approved Antibody-Drug Conjugates: Landmark Clinical Trials and Indications” , Pharm Med, 2023; and Riccardi, F. et al., “A comprehensive overview on antibody - drug conjugates: from the conceptualization to cancer therapy”, Front Pharmacol., 2023, 18; 14: 1274088, the contents of which is incorporated herein by reference in their entirety. These reviews provide a wealth of information on the latest advances in ADC chemistry, including the design and synthesis of linkers, the selection of payloads, and the conjugation techniques used to attach the payload to the antibody. These reviews also discuss the clinical applications of ADCs, their efficacy in treating various types of cancer, and the challenges and future directions in this field, all of which would be appreciated by a person of ordinary skills in the relevant art.

[0325] In some embodiments of the present invention, one locked sonosensitizer compound, according to embodiments of the present invention, is optionally conjugated to one targeting moiety, constituting a 1:1 conjugate. In some embodiments, more than one locked sonosensitizer compound is attached to one targeting moiety. In some embodiments, the tethering of the compound to the targeting moiety is effected via a liable linking moiety, such as, for example an SRT. For example, in some embodiments of the present invention, wherein the targeting moiety is an antibody, the large molecular size and the variety of functional groups available for tethering naturally present in an antibody molecule, allows multiple copies of a locked sonosensitizer as provided herein to be attached to a single Ab molecule, thereby amplifying its therapeutic effect.

[0326] Targeting Moiety Conjugation:

[0327] Traditional chemotherapy drugs, as well as sonosensitizer, can be highly effective at destroying cancer cells, but in general their specificity for cancer tissue is extremely low, and many healthy cells are killed as well. Conjugation of the locked sonosensitizer compound provided herein to a targeting moiety (TM), is achieved via various functional groups that are available, selected or purposely generated on one or both sides of the conjugation, as these conjugations are known in the art. In the context of the present invention, the locked sonosensitizer compound provided herein is conjugated to a TM similarly to other drug-carrier conjugates known in the art, such as antibody-drug conjugates (ADCs); in the context of the present invention, the ADC is a conjugate between an antibody and the locked sonosensitizer compound provided herein. In the context of the present invention, the locked sonosensitizer compound is the payload and the TM.

[0328] It is noted that some of the below-provided information pertaining to TM-compound conjugation is relevant also in the context of drug-compound conjugation, namely the optional attachment of a functional agent to the SRT on the locked sonosensitizer compound provided herein.

[0329] One of the most ubiquitous conjugation methods is conjugation via thiols, which employs the thiols of interchain cysteine residues in protein-type TMs, such as monoclonal antibodies (mAb). Provided that the TM exhibits multiple available thiol groups, this mode allows the conjugation of multiple copies of the compound provided herein to a single TM. For example, in an immunoglobulin G1 (IgGl), a subclass of Immunoglobulin G (IgG) that is the most common type of antibody found in blood circulation, there are four interchain disulfide bonds that can be used as potential conjugation sites. The four interchain disulfide bonds can be reduced, resulting in eight thiol groups that are available for conjugating.

[0330] Another approach for said conjugation is the generation of a maleimide functional group on cysteine residues in the TM. Classically, cysteine residues can be modified through addition of thiols to electrophiles such as maleimides. The conjugate can be achieved by reducing the disulfide bonds on the TM followed by Cys alkylation by maleimides.

[0331] Disulfide-thiol exchange is another commonly used approach in protein-drug conjugation, effected by forming a new disulfide bond between the compound and the TM. Disulfide-thiol exchange is a widely used process in the formation of ADCs. In the context of some embodiments of the present invention, disulfide-thiol exchange involves the reaction of a disulfide bond in the antibody with a thiol-containing locked sonosensitizer compound. This reaction results in the formation of a new disulfide bond between the antibody and the locked sonosensitizer compound, effectively linking the two. The disulfide linkage approach could also be applied to amine- functionalized locked sonosensitizer compound. This process is essentially a combination of two direct displacement events, with sulfur atoms acting as nucleophile, electrophile, and leaving group.

[0332] To avoid the maleimide instability issues, a heterobifunctional reagent, sodium 4-((4- (cyanoethynyl)benzoyl)oxy)-2,3,5,6-tetrafluorobenzenesulfonate (CBTF), can be used for amine- to-thiol coupling. Briefly, CBTF is a heterobifunctional reagent developed for amine-to-thiol coupling in the context of ADCs conjugation. CBTF comprises a 3 -arylpropionitrile (APN) function in replacement of maleimide and allows for the preparation of remarkably stable conjugates. The coupling can be performed with high selectivity in biological medium using mild reaction conditions. CBTF readily reacts with organic amines in basic conditions to produce APN- probes suitable for cysteine conjugation and labeling.

[0333] Disulfide Re-Bridging is another strategy in the formation of ADCs, which is relevant in the context of TM conjugation to the locked sonosensitizer compound provided herein. This process involves the reduction of the interchain disulfides of a TM, e.g., an IgGl antibody, followed by reaction with a bis-reactive reagent. This approach has been successful for the synthesis of ADCs bearing two and four pay loads. A recent advancement in this field is the development of TetraDVP linkers, which simultaneously re-bridge all four interchain disulfides of an IgGl antibody. This results in ADCs loaded with a single warhead, expanding the repertoire of available drug loading and enabling the synthesis of more finely tuned therapeutics.

[0334] Forming an amide bond as a conjugation linking moiety is one of the most useful approaches in the context of the present invention. Amines could typically be acylated by carboxyl via some familiar activating reagents, such as N-hydroxysuccinimide (NHS), 2-Succinimido- 1,1,3,3-tetra-methyluronium tetrafluoroborate (TSTU), and Benzotriazol-l-yl- oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP). Amines on the TM side can react with the carboxyl groups present or derived on the locked sonosensitizer side in the effect of the NHS to afford conjugates. Amines of lysines are commonly used for conjugation because lysine residues are usually exposed on the surface of the TM, therefore easily accessible. For example, antibodies contain up to 80 lysine residues, and as a result, conjugation through lysine residues inevitably leads to twofold heterogeneity: (1) different number of payloads per TM; and (2) TMs with the same number of payloads attached at different sites. The heterogeneity with respect to DARs can be restricted to a certain extent by adjusting the stoichiometry of payload and TM used in the reaction; and with respect to site-specificity, the heterogeneity can be limited by the chemical accessibility of reactive groups.

[0335] Amines on the locked sonosensitizer compound could react with hydroxyls that derived from the linkers in the effect of phosgene, 4-nitrophenyl chloroformate, etc. and form a carbamate containing compound-linkers, which can then be coupled to TMs.

[0336] Similar to amines, alcohols can react with chloroformates to form carbonates. A conjugation approach based on alcohols reacting with chloroformates to form carbonates can be employed in the context of TM-compound and functional agent-SRT conjugation. Chloroformates are reactive esters where the alcohol part of the ester is chloroform. When an alcohol reacts with a chloroformate, it results in the formation of a carbonate ester. The carbonate linkage is stable under physiological conditions, but can be cleaved in the slightly acidic environment of the endosome following internalization by the target cell, thereby releasing the payload. For example, conjugation of 7-ethyl-10-hydroxycamptothecin (SN-38) derivatives to hMN-14, a humanized anti-CEACAM5 mAb, via a carbonate bond.

[0337] According to some embodiments of the present invention, a conjugation approach based on the formation of a carbamate link can be employed to tether a TM to the locked sonosensitizer compound provided herein. This conjugation approach typically involves the reaction of an alcohol group present in the TM or the compound with a chloroformate to form a carbamate. The carbamate linkage is stable under physiological conditions, but can be cleaved in the slightly acidic environment of the endosome following internalization by the target cell, thereby releasing the payload. This method provides a stable and cleavable linker system for ADCs, allowing for the controlled release of the compound within the target cell.

[0338] According to some embodiments of the present invention, a conjugation approach based on the formation of an ester link can be employed to tether a TM to the locked sonosensitizer compound provided herein. For example, Paclitaxel-monoclonal antibody (PTX-mAb) conjugates use polyethylene glycol (PEG) as a spacer-linker, wherein the drug paclitaxel is attached to the linker through an ester bond using glutarate (GL) or succinate (SX). The resulting PTX-L- Lys[(PEG12)3-PEG4]-PEG6-CO2NHS (where L = GL or SX) is then conjugated to C225, an antiepidermal growth factor receptor (anti-EGFR) monoclonal antibody. This results in completely soluble conjugates that can deliver significant doses of drugs to tumor cells, whereas the ester bond used in this approach is stable under physiological conditions but can be cleaved in the slightly acidic environment of the endosome following internalization by the target cell, thereby releasing the payload.

[0339] Conjugation via aldehydes is another method contemplated by the inventors for linking the compound provided herein to a TM. Formylglycine-generating enzymes (FGEs), which recognize and modify a short CXPXR (where X is any amino acid) sequence, can be used to modify the cysteine residues of protein-type TMs to aldehyde-containing formylglycine (Fgly) residues. This method was applied to generate site-specific ADCs via incorporating cytotoxic drugs into monoclonal antibodies with a formylglycine

[0340] Click-chemistry reactions can be used, according to some embodiments of the present invention, to tether various components of the conjugates provided herein. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) have been used to construct ADCs, wherein alkyne and azides were introduced to the corresponding members of the conjugate. For example, an azide-containing reagent, sodium (difluoroalkylazido)sulfinate (DAAS-Na), allowed azide groups to be linked to hetero aromatics, and the products could then be attached to monoclonal antibodies by click reactions. DAAS-Na was used in the heteroarene functionalization reaction, in which ZnCh and TsOH H2O were acid additives and tBuOOH was an oxidant. The resulting azide-linked drugs could react with a dibenzylcyclooctyne (DBCO) containing antibody through a CuAAC reaction. In addition, click reactions with terminal alkynes is effected by attachment of azide-functionalized porphyrins to a TM (e.g., mAb).

[0341] The skilled artisan would appreciate the abundance of information and guidance in the field of ADC preparation that is useful in the formation of conjugates of the locked sonosensitizer compounds provided herein with an antibody, as can be found, for example, in the review articles by Watanabe, T. et al. [“Scale-Up Synthesis of Site-Specific Antibody-Drug Conjugates Using AJ1CAP Second-Generation Technology” , Org. Process Res. Dev., 2023, 27(6), pp. 1136-1143; and “Gram-Scale Synthesis of Site-Specific Antibody-Drug Conjugates Using AJICAP Second- Generation Technology” , 2023], Su, Z. et al. ["Antibody-drug conjugates: Recent advances in linker chemistry" , Acta Pharmaceutica Sinica B, 2021, 11(12), pp. 3889-3907] and Zhao, P. et al. [" Recent advances of antibody drug conjugates for clinical applications", Acta Pharmaceutica Sinica B, 2020, 10(9), pp. 1589-1600],

[0342] Exemplary locked sonosensitizer compounds:

[0343] The compounds exemplified below presents, without limitation, some embodiments of the present invention. It is noted that the core of the compound is a locked sonosensitizer, which includes a dye moiety and an SRT attached thereto, whereas a targeting moiety (TM), a bioactive moiety, a payload, or any other functional moiety, are optional and are not shown in some of the examples provided below. wherein:

[0344] Dye - a member of the Formula III family;

[0345] SRT - cysteine-responsive acryl (for monitoring endogenous cysteine levels); and

[0346] TM - Ab; an antibody as an exemplary targeting moiety attached to the dye moiety via a succinimide linking moiety attached at the RN1position.

[0347] wherein:

[0348] Dye - a member of the Formula III family; SRT - a fluoride-responsive alkyl silyl (R',R", R'" = each independently a C1-5 alkyl); and

[0349] TM - Ab; an antibody as an exemplary targeting moiety attached to the dye moiety via a succinimide linking moiety attached at the RN1position. wherein:

[0350] Dye - a member of the Formula III family;

[0351] SRT - a cathepsin B-cleavable self-immolative linker (R’ = H or acetyl); and

[0352] TM - Ab; an antibody as an exemplary targeting moiety attached to the dye moiety via a succinimide linking moiety attached at the RN1position. wherein:

[0353] Dye - a member of the Formula III family;

[0354] SRT - a glutathione-cleavable self-immolative linker (sulfur or selenium);

[0355] TM - Ab; an antibody as an exemplary targeting moiety attached to the dye via a succinimide linking moiety attached at the RN1position. wherein:

[0356] Dye - a member of the Formula II family;

[0357] SRT - a glutathione-cleavable self-immolative linker; and

[0358] TM - Ab; an antibody as an exemplary targeting moiety attached at the RN1position. wherein:

[0359] Dye - a member of the Formula III family;

[0360] SRT - a β-glucuronidase-cleavable self-immolative linker;

[0361] TM - Ab; an antibody as an exemplary targeting moiety attached to the dye via a succinimide linking moiety attached at the RN1position. wherein:

[0362] Dye - a member of the Formula II family;

[0363] SRT - a β-glucuronidase-cleavable self-immolative linker;

[0364] TM - Ab; an antibody as an exemplary targeting moiety attached at the RN1position.

[0365]

[0366] Dye - a member of the Formula II family; SRT - a cathepsin B-cleavable self-immolative linker;

[0367] TM - Ab; an antibody as an exemplary targeting moiety attached at the RN1position. wherein:

[0368] Dye - a member of the Formula VI family;

[0369] SRT - a glutathione-cleavable self-immolative linker;

[0370] TM - Ab; an antibody as an exemplary targeting moiety.

[0371] wherein:

[0372] Dye - two moieties of a member of the Formula III family (one may be seen as a functional moiety in the form of another dye moiety serving as an bioactive agent);

[0373] SRT - a glutathione-cleavable self-immolative linker;

[0374] TM - Ab; an antibody as an exemplary targeting moiety attached at the RN1position.

[0375] Dye -a member of the Formula III;

[0376] SRT - a glutathione-cleavable self-immolative linker;

[0377] TM - Ab; an antibody as an exemplary targeting moiety attached at the RN1position; and Drug (a payload) - DA-3 (a NH2-Dolastatin 10 derivative)

[0378] wherein:

[0379] Dye -a member of the Formula III; SRT - a glutathione-cleavable self-immolative linker;

[0380] TM - Ab; an antibody as an exemplary targeting moiety attached at the RN1position; and Drug (a payload) - SN-38, an antineoplastic drug (7-ethyl-10-hydroxycamptothecin). wherein:

[0381] Dye -a member of the Formula III;

[0382] SRT - a glutathione-cleavable self-immolative linker;

[0383] TM - Ab; an antibody as an exemplary targeting moiety attached at the RN1position; and Drug (a pay load) - Dolastatinol.

[0384] wherein:

[0385] Dye -a member of the Formula III; SRT - a glutathione-cleavable self-immolative linker;

[0386] TM - Ab; an antibody as an exemplary targeting moiety attached at the RN1position; and Drug (a bioactive agent, a payload) - Bendamustine. wherein:

[0387] Dye -a member of the Formula III;

[0388] SRT - a glutathione-cleavable self-immolative linker;

[0389] TM - Ab; an antibody as an exemplary targeting moiety attached at the RN1position; and Drug (a bioactive agent, a payload) - DA-5 (a NMe-Dolastatin 10 derivative).

[0390]

[0391] Compound 25 wherein:

[0392] Dye -a member of the Formula XII;

[0393] SRT - a Cathepsin B -cleavable self-immolative linker;

[0394] R’ = H or acetyl;

[0395] Carboxyl group in Formula XII is used to form an amide connection with a linking moiety that tethers the dye to a targeting moiety or a bioactive agent (pay load);

[0396] TM - Ab; an antibody as an exemplary targeting moiety attached via linking moiety to a maleimide moiety.

[0397] Dye

[0398] Compound 26 wherein:

[0399] Dye -a member of the Formula XII

[0400] SRT - a glutathione-cleavable self-immolative linker;

[0401] Carboxyl group in Formula XII is used to form an amide connection with a linking moiety that tethers the dye to a targeting moiety or a bioactive agent (pay load);

[0402] TM - Ab; an antibody as an exemplary targeting moiety attached via linking moiety. wherein:

[0403] Dye -a member of the Formula XVII;

[0404] SRT - a Cathepsin B -cleavable self-immolative linker;

[0405] TM - Ab; an antibody as an exemplary targeting moiety. wherein:

[0406] Dye -a member of the Formula XVII;

[0407] SRT - a glutathione-cleavable self-immolative linker;

[0408] TM - Ab; an antibody as an exemplary targeting moiety.

[0409] Dye -a member of the Formula I;

[0410] SRT - a glutathione-cleavable self-immolative linker;

[0411] T =0;

[0412] RH1, RH2= I;

[0413] W1 , W2= CMe2;

[0414] R1-R13 = H;

[0415] RNIRN2 =(CH2)3SO3H

[0416] SRT, RN1and RN2may each independently serve as optional targeting moiety or payload attachment positions. wherein:

[0417] Dye -a member of the Formula I;

[0418] SRT - a β-glucuronidase-cleavable self-immolative linker;

[0419] T =0;

[0420] RH1= H, RH2= I;

[0421] W1 , W2= CMe2;

[0422] Ri = SO3H; R2-Ri3 = H;

[0423] RN1= (CH2)3SO3H;

[0424] RN2= (CH2)3-maleimide;

[0425] RN1and RN2may each independently serve as optional targeting moiety or payload attachment positions. wherein:

[0426] Dye -a member of the Formula I;

[0427] SRT - a cathepsin B-cleavable self-immolative linker;

[0428] T =0;

[0429] RH1= H, RH2= I;

[0430] Wi , W2= CMe2;

[0431] Ri = SO3H;

[0432] R2-Ri3 = H;

[0433] RN1= (CH2)3SO3H;

[0434] RN2= (CH2)4CO2H;

[0435] SRT, RN1and RN2may each independently serve as optional targeting moiety or payload attachment positions.

[0436] Dye -a member of the Formula I;

[0437] SRT - a peroxide-cleavable self-immolative linker;

[0438] T =0;

[0439] RH1= H, RH2= I;

[0440] W1, W2= CMe2;

[0441] Ri = SO3H;

[0442] R2-Ri3 = H;

[0443] RN1= (CH2)3SO3H;

[0444] RN2= (CH2)3-maleimide;

[0445] RN1and RN2may each independently serve as optional targeting moiety or payload attachment positions. wherein:

[0446] Dye -a member of the Formula I;

[0447] SRT - a β-galactosidase-cleavable self-immolative linker;

[0448] T =0;

[0449] RH1= H, RH2= I;

[0450] W1 , W2= CMe2; Ri = SO3H;

[0451] R2-Ri3 = H;RN1= (CH2)3SO3H;

[0452] RN2= (CH2)3-maleimide;

[0453] RN1and RN2may each independently serve as optional targeting moiety or payload attachment positions.

[0454] Compound 34 wherein:

[0455] Dye -a member of the Formula III;

[0456] SRT - a glutathione-cleavable self-immolative linker;

[0457] T =NH;

[0458] RH1= H, RH2= I;

[0459] Wi, W2= CMe2;

[0460] R1-R10 = H;

[0461] Rn = I;

[0462] RN1= (CH2)4-maleimide;

[0463] RN1may serve as optional targeting moiety or pay load attachment positions.

[0464] Compound 35 wherein:

[0465] Dye -a member of the Formula III;

[0466] SRT - a cathepsin B-cleavable self-immolative linker;

[0467] T =0;

[0468] RH1= H, RH2= I;

[0469] Wi = O, W2= CMe2;

[0470] Ri-Rio = H;

[0471] Rn = I;

[0472] RN1= (CH2)5CO2H;

[0473] RN1may serve as optional targeting moiety or pay load attachment positions. wherein:

[0474] Dye -a member of the Formula III;

[0475] SRT - a glutathione-cleavable self-immolative linker;

[0476] T =0;

[0477] RH1= H, RH2= I;

[0478] Wi = O, W2= CMe2;

[0479] R1-R10 = H, R11 = I;

[0480] RN1= (CH2)3SO3H;

[0481] SRT and RN1may each independently serve as optional targeting moiety or payload attachment positions. wherein:

[0482] Dye -a member of the Formula XII; SRT - a glutathione-cleavable self-immolative linker;

[0483] T =0;

[0484] RH1, RH2= I;

[0485] R1-R2 = H;

[0486] Carboxyl group in Formula XII may be used to tether the dye to a targeting moiety or a bioactive agent (pay load). wherein: Dye -a member of the Formula XII;

[0487] SRT - a cathepsin B-cleavable self-immolative linker;

[0488] T =0;

[0489] RH1= I, RH2= H;

[0490] R1-R2 = H; Carboxyl group in Formula XII may be used to tether the dye to a targeting moiety or a bioactive agent (pay load).

[0491] wherein:

[0492] Dye -a member of the Formula XV ; SRT - a glutathione-cleavable self-immolative linker;

[0493] T =0;

[0494] RH1= H, RH2= I;

[0495] Ri = SO3H, R2-R13 = H, R14= I;

[0496] RN1= (CH2)3SO3H, RN2= (CH2)4CO2H; RN1and / or RN2may each independently be used to tether the dye to a targeting moiety or a bioactive agent (pay load). wherein:

[0497] Dye -a member of the Formula XV ;

[0498] SRT - a glutathione-cleavable self-immolative linker;

[0499] T =0;

[0500] RH1, RH2= I; Ri-Ru = H;

[0501] RN1= (CH2)3SO3H, RN2= (CH2)4CO2H;

[0502] RN1and / or RN2may each independently be used to tether the dye to a targeting moiety or a bioactive agent (pay load). wherein:

[0503] Dye -a member of the Formula XV ; SRT - a peroxide-cleavable self-immolative linker;

[0504] T =0;

[0505] RH1, RH2= I;

[0506] Ri-Ru = H;

[0507] RN1= (CH2)3SO3H, RN2= (CH2)4CO2H; RN1and / or RN2may each independently be used to tether the dye to a targeting moiety or a bioactive agent (pay load).

[0508] wherein:

[0509] Dye -a member of the Formula XVI; SRT - a cathepsin B-cleavable self-immolative linker;

[0510] T =0;

[0511] RH1= I, RH2= H;

[0512] Ri-Rn = H;

[0513] R12 = SO3H RN1= (CH2)3SO3H, RN2= (CH2)4-melamide;

[0514] RN1and / or RN2may each independently be used to tether the dye to a targeting moiety or a bioactive agent (pay load).

[0515] Compound 43 wherein:

[0516] Dye -a member of the Formula XVII;

[0517] SRT - a glutathione-cleavable self-immolative linker; T =0;

[0518] RH1= I, RH2, RH3= H;

[0519] Wi = S, W2= CH2;

[0520] R1-R7 = H;

[0521] RN1-RN4= CH3;

[0522] SRT may be used to tether the dye to a targeting moiety or a bioactive agent (pay load).

[0523] Compound 44 wherein:

[0524] Dye -a member of the Formula XVII;

[0525] SRT - a cathepsin B-cleavable self-immolative linker;

[0526] T =NH;

[0527] RH1= I, RH2, RH3= H;

[0528] Wi = S, W2= CH2;

[0529] R1-R7 = H;

[0530] RN1-RN4=(CH2)3CO2H;

[0531] SRT and RN1-RN4may each independently be used to tether the dye to a targeting moiety or a bioactive agent (pay load).

[0532] Compound 45 wherein:

[0533] Dye -a member of the Formula XVII;

[0534] SRT - a glutathione-cleavable self-immolative linker;

[0535] T =0;

[0536] RH1= I, RH2, RH3= H;

[0537] Wi = S, W2= CH2;

[0538] R1-R7 = H;

[0539] RN1-RN3 = CH3, RN4= (CH2)3CO2H;

[0540] SRT and RN4may each independently be used to tether the dye to a targeting moiety or a bioactive agent (pay load).

[0541] Compound 46 wherein:

[0542] Dye - a molecule similar to members of Formula XVIII,

[0543] SRT - a glutathione-cleavable self-immolative linker connects to a carbonyl;

[0544] T =NH;

[0545] RH1, RH2= I;

[0546] Wi = S; W2= carbonyl

[0547] R1-R4 = H;

[0548] RN1-RN4= CH3;

[0549] SRT may be used to tether the dye to a targeting moiety or a bioactive agent (payload).

[0550] Compound 47 wherein:

[0551] Dye - a molecule similar to members of Formula XVIII, SRT - a glutathione-cleavable self-immolative linker connects to a carbonyl;

[0552] T =0;

[0553] RH1= I, RH2= H;

[0554] Wi = S; W2 = carbonyl

[0555] R1-R4 = H;

[0556] RN1-RN4= CH3.

[0557] SRT may be used to tether the dye to a targeting moiety or a bioactive agent (payload). Compound 48 wherein:

[0558] Dye -a member of the Formula III;

[0559] SRT - a cathepsin B-cleavable self-immolative linker with mono succinamide spacer;

[0560] T =0; RH1= H, RH2= I;

[0561] Wi = O, W2= CMe2;

[0562] R1-R10 = H;

[0563] Rn = I;

[0564] RN1= (CH2)3SO3H;

[0565] RN1may serve as optional targeting moiety or pay load attachment positions.

[0566] Compound 49 wherein:

[0567] Dye - a molecule similar to members of Formula XVIII,

[0568] SRT - a glutathione-cleavable self-immolative linker connects to a carbonyl;

[0569] T =0;

[0570] RH1= I, RH2= I;

[0571] Wi = S; W2= carbonyl

[0572] RI, R3= I

[0573] R2,R4= H;

[0574] RN1-RN4= CH3.

[0575] SRT may be used to tether the dye to a targeting moiety or a bioactive agent (payload).

[0576]

[0577] Compound 50 wherein:

[0578] Dye - a molecule similar to members of Formula XVIII,

[0579] SRT - a glutathione-cleavable self-immolative linker connects to a carbonyl;

[0580] T =0;

[0581] RH1, RH2= I;

[0582] Wi = S; W2= carbonyl

[0583] R1-R4 = H;

[0584] RN1-RN4= CH3;

[0585] SRT may be used to tether the dye to a targeting moiety or a bioactive agent (payload).

[0586] Compound 51 wherein:

[0587] Dye -a member of the Formula XVII;

[0588] SRT - a cathepsin B-cleavable self-immolative linker with mono succinamide spacer;

[0589] T =NH;

[0590] RH1, RH2= I, , RH3= H;

[0591] Wi = S, W2= CH2;

[0592] R1-R7 = H;RN1-RN4= CH3;

[0593] SRT and RN1-RN4may each independently be used to tether the dye to a targeting moiety or a bioactive agent (pay load).

[0594] Compound 52 wherein:

[0595] Dye -a member of the Formula I;

[0596] SRT - a glutathione-cleavable self-immolative linker with monosuccinimide spacer;

[0597] T =0;

[0598] RH1, RH2= I;

[0599] Wi, W2= CMe2;

[0600] R1-R13 = H;

[0601] RNIRN2 =(CH2)3SO3H

[0602] SRT, RN1and RN2may each independently serve as optional targeting moiety or payload attachment positions.

[0603] Use of the compound provided herein:

[0604] Sonodynamic therapy (SDT), as well as PDT, is a treatment modality that utilizes ultrasound (or light) and a sensitizing agent to kill targeted cells. The purpose of the compound provided herein, in a therapeutic, diagnostic or research context, is to cause a cytotoxic effect “cell death”, or necrosis in a controllable manner. In some embodiments, the cytotoxic effect is typically a direct cytotoxic effect, namely it is effected by the sonosensitization activity of the unlocked compound, and in some embodiments, the combination of the sonosensitization activity and the that of a bioactive agent attached thereto. In the context of embodiments of the present invention, direct cytotoxic effects include induction of apoptosis, necrosis, and / or physical destruction of cells. This feat is achieved by contacting cells, tissue, specimen, subject or any other biologic sample with the compound, followed by detachment of the SRT, and exposure of the biologic sample now containing the compound to ultrasound (US) radiation (energy); said exposure is also referred to as “sonication”. Since the compound is a locked sonosensitizer, the sonication can be effected before, during and after detachment of the SRT. Other than in vivo conditions, the compound may also be used in / for in vitro, ex vivo and nonbiologic purposes that require controlled activation of a sonosensitizer.

[0605] It is noted herein that some sonosensitizers, including some of the compounds provided herewith, may also be sensitive to electromagnetic radiation, which is a different type of energy than ultrasound, namely some of the compounds provided herein may serve also in photodynamic therapy (PDT). Use of such compounds having dual sensitivity to light at certain wavelengths as well as to ultrasound energy, may follow a modified protocol that accounts for the light sensitivity and either avoid uncontrolled exposure to light, and / or harnesses this sensitivity as part of a method of treatment. Thus, according to an aspect of some embodiments of the present invention, the compounds provided herein are suitable for use as locked photosensitizers in photodynamic therapy, locked photosensitizers in photodynamic therapy. Alternatively, according to an aspect of some embodiments of the present invention, there is provided a method of treating a medical condition in a subject in need thereof, which is effected by administering to the subject a therapeutically effective amount a compound as provided herein, and exposing at least a part of the subject to light.

[0606] Cell death (necrosis) or cell deterioration is the eventual result of the direct cytotoxic effect delivered by the compound provided herein upon unlocking and exposure to ultrasound energy. The optionally added releasable functional agent provides additional therapeutic effect (double warhead) that may be synergistic, and the optionally added targeting moiety provides specificity, lowering adverse effects and increasing efficacy.

[0607] While cancer- specific antibodies have dominated the field of targeted drug delivery, various other targeting moieties can effectively guide cytotoxic agents to specific cell types, thereby also expanding the scope of treatable medical conditions beyond cancer. Medical conditions, which treatable by the locked sonosensitizers provided herein, where targeting specific cell populations can be a valuable therapeutic approach, include, without limitation:

[0608] Cancer: selective destruction of cancerous cells is a core principle in various cancer therapies, including chemotherapy, radiation therapy, and targeted therapies. These approaches aim to eliminate malignant cells while minimizing harm to healthy tissues;

[0609] Autoimmune diseases: in conditions like rheumatoid arthritis or lupus, the immune system mistakenly attacks healthy cells. Treatment often involves suppressing the overactive immune response and preventing it from targeting healthy cells; Viral infections: certain viruses replicate within host cells, hijacking their machinery. Antiviral medications may work by inhibiting viral replication or inducing infected cells to undergo programmed cell death, thus limiting viral spread;

[0610] Transplant rejection: when receiving an organ transplant, the recipient’s immune system may recognize the new organ as foreign and attempt to attack it. Immunosuppressive medications aim to prevent this rejection by targeting specific immune cells involved in the attack; and

[0611] Neurodegenerative diseases: some neurodegenerative diseases involve the progressive death of specific neuronal populations. Research is exploring potential neuroprotective therapies that may prevent or slow down this cell death.

[0612] In some embodiments of the present invention, the medical condition is associated with malignant cells and tumors, collectively referred to herein as cancer. 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.

[0613] 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 compound provided herein, 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.

[0614] 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.

[0615] Angiogenesis inhibitors are currently investigated for their use in cancer treatment and to date, one anti- angiogene tic 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.

[0616] In some embodiments, the functional moiety of the compound provided herein, is a targeting moiety that is responsible for the higher concentration (accumulation) of the compound at the targeted bodily site before it had been exposed to ultrasound energy, compared to nontargeted bodily sites, thereby reducing the adverse side effects associated with the cytotoxicity effected by the sonosensitization activity and / or of the anti-cancer drugs releasably attached thereto. In addition, the linking moieties attached the anti-cancer drugs to the compound provided herein 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.

[0617] 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, non-melanoma 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.

[0618] While traditionally used for treating cancers and other conditions, there's growing interest in the potential application of sensitizers (SDT and PDT agents) for autoimmune diseases. SDT / PDT offers a promising approach for autoimmune diseases by leveraging its targeted action. A sensitizing agent selectively accumulates in immune cells involved in the autoimmune response. When exposed to specific stimulus (ultrasound / light), the sensitizer generates reactive oxygen species (ROS) that damage and kill these targeted cells. This approach has the potential to be more specific than traditional immunosuppressive drugs, which can affect the entire immune system and lead to side effects. Additionally, SDT / PDT might not only eliminate target cells but also modulate the overall immune response, potentially leading to longer-lasting effects. According to some embodiments of the present invention, sensitizers with high selectivity for specific immune cell types are provided. Some potential applications include targeting immune cells in inflamed joints for rheumatoid arthritis, treating skin lesions in psoriasis, and targeting specific immune cells involved in systemic inflammation of lupus.

[0619] According to some embodiments, the autoimmune disease, which may be treated by controlled and targeted cytotoxic agent, 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 102ulfoxidel02el02s (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 Polyangiitis (GPA).

[0620] According to some embodiments, some degenerative diseases may be treated by controlled and targeted cytotoxic agent against axons. Such medical conditions include 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.

[0621] 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.

[0622] Medical conditions associated with a pathogenic microorganism, which may be treated by controlled and targeted cytotoxic agent, 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.

[0623] Non-limiting examples of pathogenic fungi, which may be treated by controlled and targeted cytotoxic agent, include genus Absidicr. 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 carrionii ; genus Coccidioides : Coccidioides immitis ; genus Cryptococcus : Cryptococcus neoformans ; genus Cunninghamella : Cunninghamella sp. ; genus Epidermophyton : Epidermophyton floccosum ; genus Exophiala : Exophiala dermatitidis ; genus Filobasidiella : Filobasidiella neoformans ; genus Fonsecaea : Fonsecaea pedrosoi ; genus Fusarium : Fusarium solani ; genus Geotrichum : Geotrichum candidum ; genus Histoplasma : Histoplasma capsulatum ; 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, which may be treated by controlled and targeted cytotoxic agent, 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, Eisteria 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.

[0624] The compound presented herein can therefore be used as an active ingredient in a variety of pharmaceutical compositions, and in the preparation of a variety of medicaments. Accordingly, there is provided a pharmaceutical composition that includes, as an active ingredient, the compound, according to embodiments of the present invention, and a pharmaceutically acceptable carrier. Similarly, there is provided a use of the compound, according to embodiments of the present invention, in the preparation of a medicament. According to some embodiments of the present invention, the compound, the pharmaceutical composition or the medicament comprising the same, are used to treat various medical conditions, as described in details hereinabove.

[0625] A “pharmaceutical composition” refers to a preparation which is in such form as to permit administration and subsequently provide the intended biological activity of the active ingredient(s) and / or to achieve a therapeutic effect, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The pharmaceutical composition may be sterile.

[0626] “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, for use in animals, and more particularly in humans. For example, the term “a pharmaceutically acceptable salt” may include, without limitation, hydrochloride (e.g., cetirizine dihydrochloride), sulfate (e.g., atorvastatin calcium sulfate), mesylate (e.g., imatinib mesylate), acetate (e.g., prednisolone acetate), citrate (e.g., tamoxifen citrate), tartrate (e.g., albuterol sulfate), maleate (e.g., enalapril maleate), phosphate (e.g., oseltamivir phosphate), lactate (e.g., ropinirole hydrochloride), and besylate (e.g., amlodipine besylate).

[0627] A “pharmaceutically acceptable carrier” or a “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like. An “effective amount” of, e.g., a locked sonosensitizer compound, as disclosed herein, is an amount sufficient to perform treatment, or a specifically stated purpose, for example to produce a therapeutic effect after administration, such as, for example, a reduction in tumor growth rate or tumor volume, a reduction in a symptom of cancer, or some other indicia of treatment efficacy. An effective amount can be determined in a routine manner in relation to the stated purpose. The term “therapeutically effective amount” refers to an amount of the herein-provided compound effective to treat a medical condition (a disease or disorder) in a subject. In the case of cancer, a therapeutically effective amount of a compound can reduce the number of cancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and / or relieve one or more symptoms. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0628] As used herein, “to treat” or “therapeutic” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and / or a lessening of side effects which are the byproducts of an alternative therapeutic modality. As is readily appreciated in the art, full eradication of disease is encompassed but not required for a treatment act. Each of the terms “treatment”, “treating”, and “treat”, as used herein, refers to the administration of the compounds provided herein or a composition / medicament comprising the same, to a subject, e.g., a patient. The treatment can be to cure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder, e.g., a cancer. In some embodiments, in addition to treating a subject with a condition, a composition disclosed herein can also be provided prophylactically to prevent or reduce the likelihood of developing that condition.

[0629] Methods of treatment:

[0630] In the context of some embodiments of the present invention, one of the objectives of using the compound provided herein is to induce a therapeutic cytotoxic effect to targeted cells with minimal adverse effects, while treating medical conditions that are treatable by selective induction of at least one direct cytotoxic effect. This direct cytotoxic effect may involve various mechanisms, including the generation of reactive oxygen species (ROS), delivering a cytotoxic functional agent, and other cellular stress responses that lead to cell death. The term “cytotoxic effect”, as used herein, refers to the combined effect of the compound provided herein as a result of introducing the compound into a cell, unlocking (activating) the compound by biologic or chemical processes, and exposure of the cell to ultrasound energy. A cytotoxic effect is a result of administering the compound provided herein to a subject, and exposing the subject or parts thereof to ultrasound energy.

[0631] The terms “subject” and “patient” are used interchangeably herein to refer to any animal, such as any mammal, including but not limited to, humans, non-human primates, rodents, and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0632] In the field of sonodynamic therapy (SDT), researchers often assess the therapeutic efficacy by examining the cytotoxic effects induced in the treated cells. This evaluation includes observing changes in cell morphology, viability, and other cellular responses, whereas the focus of SDT and one of the objectives of the present invention, is on achieving a controlled and selective cytotoxic effect in the targeted cells while minimizing damage to healthy surrounding tissues.

[0633] As used herein, the term “sonosensitizer”, “sonodynamic therapy agent”, and “ultrasoundsusceptibility modification agent” are synonymous, and refer to the compound provided herein in its “unlocked”, “activated”, or “safety off’ state. An effective amount of one or more SDT agent of this invention together with an effective amount of ultrasound energy, when administered to cells in a subject such as diseased cells and cells that cause disease, is cytotoxic to those cells. In the absence of said effective amount of sonodynamic therapy agent, the effective amount of ultrasound energy is substantially non-cytotoxic to any cells, and in the absence of said effective amount of ultrasound energy, the effective amount of the sonodynamic therapy agent is substantially non-cytotoxic the cells. In other words, referring to a sonosensitization effect, administering the compound provided herein under its effective amount, the ultrasound energy will not exert a cytotoxic effect, and the compound will not harm cells, and on the other hand, using ultrasound energy under the effective parameters (frequency and amplitude; power), the sonodynamic therapy agent will also not harm the cells. The aforementioned statement refers only to the sonosensitization effect and disregards a cytotoxic effect that may be rendered by the functional agent once released from the compound.

[0634] The term “cytotoxic”, as used herein, is meant to include the terms “cytopathogenic”, “cytolytic”, “cytocidic”, “cytoclastic”, and “cytostatic”, as well as other descriptors which relate to the onset or potentiation or initiation of cell death, cell fragmentation, cell rupture, cell dormancy, or change in one or more cellular function to effect a change from a diseased to a nondiseased state, including from infection to non-infection, relative to the subject of treatment.

[0635] In the treatment method for a medical condition necessitating selective cell death, a sonosensitizer is employed with precision and technical parameters. Initially, the compound provided herein, a sonosensitizer characterized by its reactivity when exposed to ultrasound, is administered to a subject. The compound is designed to selectively accumulate within the target cells or tissues associated with the specific medical condition.

[0636] Following administration, the sonosensitizer undergoes sufficient localization within the targeted area, facilitated by its specific affinity for the targeted cells or tissues. Once optimal accumulation / localization is achieved, focused ultrasound waves are applied externally, with careful consideration given to technical parameters such as frequency, intensity, and duration. These parameters are tuned to ensure a controlled and effective response from the sonosensitizer.

[0637] The interaction between the ultrasound waves and the sonosensitizer triggers a cascade of photochemical reactions, leading to the generation of reactive oxygen species (ROS) within the targeted cells. The dose of sonosensitizer administered, along with the energy parameters of the ultrasound waves, is precisely calculated to induce therapeutic cell death selectively in the affected cells while minimizing impact on healthy surrounding tissues.

[0638] In addition to the sonosensitization effect, in some embodiments of the present invention, the compound further carries a releasable payload in the form of a bioactive agent that exerts additional therapeutic effect, including cytotoxicity. The releasable bioactive agent may be released as a result of cleavage of the SRT in the compound, or as a result of another chemical reaction or biological interaction in the targeted cell.

[0639] The timing of ultrasound application is strategically coordinated with the optimal concentration of the compound (the sonosensitizer) within the target, ensuring the therapeutic effect is maximized. Continuous monitoring of the treatment process is optional, employing imaging techniques or other monitoring tools to assess the localized response and adjust parameters as needed during the procedure.

[0640] The treatment regimen involves a systematic approach to dosing, timing, and monitoring, creating a comprehensive strategy for achieving selective cell death. This method leverages the synergistic effects of ultrasound, the sonosensitizer and the optional payload, introducing a nuanced and technical dimension to the targeted treatment of specific medical conditions requiring localized cellular intervention. As research in this field advances, refining these technical parameters is well within the capabilities of a person of ordinary skills in the art of SDT.

[0641] The effective amount of the compound provided herein, also referred to as the dose, is determined based on various factors including the type and severity of the medical condition, the size and location of the target cells, and the individual characteristics of the subject, such as age, weight, and overall health status. The dose is sufficient to cause selective death of the target cells upon application of the ultrasound energy, but is not so high as to cause unacceptable side effects. The ultrasound energy applied to the subject has specific parameters, including amplitude, frequency, and power, which are selected to activate the compound provided herein and cause death of the target cells. The amplitude and frequency of the ultrasound energy are selected based on the specific sonosensitizer used, the type and location of the target cells, and the depth of penetration required. The power of the ultrasound energy is sufficient to activate the locked sonosensitizer compound provided herein and cause cell death, but is not so high as to cause damage to surrounding healthy tissues.

[0642] Setting the timing of the administration of the locked sonosensitizer compound provided herein and the application of the ultrasound energy is also within the capacity of a skilled artisan in the art of SDT. The locked sonosensitizer compound provided herein is administered to the subject and allowed to selectively accumulate in the target cells. The ultrasound energy is then applied at a time when the concentration of the sonosensitizer in the target cells is optimal (e.g., at its peak), thereby maximizing the effectiveness of the treatment.

[0643] The treatment regimen may include multiple cycles of administration of the locked sonosensitizer compound provided herein and application of the ultrasound energy, depending on the type and severity of the medical condition and the response of the subject to the treatment. The progress of the treatment is monitored using suitable methods, such as imaging techniques, to assess the extent of cell death and the effect on the medical condition.

[0644] The present invention therefore provides a method for selectively killing cells in a subject, which is particularly useful for treating medical conditions that require targeted cell death, such as cancer. The method is advantageous in that it allows for targeted cell death with minimal damage to surrounding healthy tissues, and can be tailored to the individual characteristics of the subject and the specific requirements of the medical condition. The method is further advantageous in that it can be monitored and adjusted in real time to optimize the effectiveness of the treatment.

[0645] Thus, according to an aspect the invention, there is provided a method of treating a medical condition in a subject in need thereof (e.g., a patient) by sonodynamic therapy, which is effected by administering the compound provided herein to the subject, and exposed the compound in the subject to ultrasound energy so as to achieve a cytotoxic effect at a site (e.g., a tumor site) therein.

[0646] The method employs a sonosensitizer chosen for its selective affinity towards the target cell population and efficient conversion of ultrasound energy into desired biological effects.

[0647] The sonosensitizer is administered to the patient via a suitable route, such as, but not limited to intravenous injection, topical application, or direct intratumoral injection, ensuring delivery to the target tissue. Optimal dose is determined based on factors like sonosensitizer properties, target tissue characteristics, and desired treatment outcomes. Preclinical and clinical studies establish safety and efficacy profiles for specific dose ranges.

[0648] Ultrasound energy of appropriate frequency and amplitude is applied to the target region following sonosensitizer administration. The chosen frequency ensures adequate penetration depth and minimal off-target effects. Common therapeutic ultrasound frequencies range from 20 kHz to 10 MHz, often tailored to the specific sonosensitizer and tissue properties. Energy output (power) is carefully controlled to achieve desired sonosensitization within the target tissue without exceeding safety thresholds for surrounding healthy tissue.

[0649] The treatment timing considers the pharmacokinetics of the locked sonosensitizer compound provided herein, and its accumulation within the target cells. Ultrasound application typically occurs after sufficient sonosensitizer uptake has been achieved. Treatment duration and potential repeated administrations are determined based on the desired extent of cell death and response assessment. Imaging techniques like MRI or ultrasound monitoring can be employed to visualize treatment progress and assess target tissue response.

[0650] The specific parameters of sonosensitizer selection, dose, ultrasound frequency, amplitude, and power, as well as treatment timing and duration, are optimized through preclinical and clinical studies. Individualized treatment regimens are then established based on patient characteristics, disease severity, and desired therapeutic outcomes.

[0651] The present invention provides sonodynamic therapy (SDT) agents that contribute to the development of SDT, which will significantly improve both the safety and efficacy of current SDT practices. In some of the current SDT therapeutic modalities, sonosensitizers are administered intravenously to reach abnormal cells (e.g., cancers or bacteria) and then, upon external ultrasound irradiation, which can reach deep into the body, produce cytotoxic species that kill these cells. Replacing currently available sonosensitizers with the herein-provided targeted activatable sonodynamic therapeutic (TASDT) agents, namely the locked sonosensitizer compounds according to some embodiments of the present invention, will increase both the efficacy of the treatment (by facilitating the highly selective accumulation of the sonosensitizer within the targeted cells) and its safety (by generating the cytotoxic species only inside abnormal cells). These effects, in turn, should altogether prevent the photosensitivity associated with SDT, dramatically reducing the generation of cytotoxic species in non-targeted tissues, and enable a distributed, potentially whole -body ultrasound irradiation (instead of the focused irradiation needed to reduce side effects in traditional SDT), which is crucial for treating advanced cancers and systemic infectious diseases. I l l

[0652] Importantly, the herein-provided TASDT platform is highly versatile, capable of accommodating a wide range of sonosensitizers, carriers, stimulus-responsive tethers, payloads, drugs, and fluorescent reference dyes. As such, the provisions of the present invention greatly advances the currently available SDT and boost the development of novel, more efficient, and safer tools for treating a wider variety of cancers, and microbial, fungal, and viral diseases, which could be specifically targeted by replacing the carrier with other antibodies, peptides, nanoparticles, etc. The herein-provided proof-of-principle of the TASDT approach can thus pave the way to further pre-clinical evaluations and, later, to clinical application prospects.

[0653] General definitions:

[0654] In the context of embodiments of the present invention, the term “imaging agent” refers to an substance that can be introduced into the body (medicine) or a sample (chemistry / biochemistry), and being able to interacts with specific tissues, organs, or molecules (depending on the application), allowing for their visualization or detection using imaging techniques (e.g., X-ray, MRI, fluorescence microscopy).

[0655] The term “labeling agent”, similar to the term imaging agent, is a molecule introduced into a system (body, cell, or attached to another molecule) in medicine, chemistry, and biochemistry. However, unlike imaging agents that directly interact with the target for visualization, labeling agents bind specifically to the target of interest. This binding allows the target to be indirectly detected through the properties of the attached labeling agent, which can then be visualized using various imaging techniques. Essentially, it acts like a tag that helps identify and track a specific molecule or structure within a complex environment.

[0656] In the context of the present invention, the terms “imaging agent”, “labeling agent”, and “diagnostic agent” are all intertwined in their roles for investigative purposes. All three involve introducing a substance into a body (medicine) or a sample (chemistry / biochemistry). Imaging agents directly interact with the target, allowing for its visualization using techniques like X-ray or MRI. Labeling agents, on the other hand, specifically bind to the target molecule, acting like a tag for indirect detection through the properties of the attached agent. Finally, the broader term “diagnostic agent” encompasses both imaging and labeling agents. They can either directly visualize abnormalities or trigger a measurable response upon interaction with the target, ultimately aiding in diagnosis. Exemplary diagnostic agents include:

[0657] Contrast agents that are used in imaging techniques like X-ray, CT scans, and MRIs to enhance the contrast between different tissues and organs, aiding in visualizing abnormalities or specific structures, (e.g., Barium sulfate for X-ray imaging of the digestive system, Gadolinium contrast for MRI scans) Radiopharmaceuticals, which are radioactive materials that are used in nuclear medicine for imaging and diagnosis. They accumulate in specific tissues or organs based on their function, allowing for visualization of abnormalities or function assessment, (e.g., FDG-PET scan for cancer diagnosis, Technetium-99m for bone scans)

[0658] Immunoassays reagents that utilize antibodies labeled with enzymes, fluorescent molecules, or radioactive isotopes to detect specific antigens (foreign substances) in blood or other samples. Used for diagnosing infections, autoimmune diseases, and allergies, (e.g., ELISA tests for HIV or pregnancy)

[0659] Biosensors, which are devices that combine biological recognition elements (enzymes, antibodies) with signal transducers to detect specific molecules in a sample. Can provide real-time results for glucose monitoring, blood gas analysis, or detecting bacterial infections.

[0660] Genetic probes, which are short pieces (oligomers) of DNA or RNA used to identify specific genetic mutations or sequences associated with diseases. Used in prenatal testing, diagnosing genetic disorders, and targeted cancer therapies.

[0661] Endogenous biomarkers, which are typically naturally occurring molecules in the body whose altered levels can indicate disease states. Examples include blood sugar levels for diabetes diagnosis, cholesterol levels for heart disease risk assessment, and troponin for heart muscle damage.

[0662] Exogenous contrast agents that are similar to contrast agents but used for specific functions beyond imaging. For example, ultrasound contrast agents improve visualization during ultrasound procedures.

[0663] Dyes and stains typically used in histology and cytology to differentiate different cell types and visualize abnormalities in tissues. Examples include hematoxylin and eosin (H&E) stain for routine tissue examination and fluorescent dyes for specific cell types.

[0664] 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. The term “solvate” refers to a complex of variable stoichiometry (e.g., di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by a solute (the compound as described herein) and a solvent, whereby the solvent does not interfere with the biological activity of the solute. Suitable solvents include, for example, ethanol, acetic acid and the like.

[0665] The term “hydrate” refers to a solvate, as defined hereinabove, where the solvent is water.

[0666] 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.

[0667] 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.

[0668] 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, 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.

[0669] 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, sulfonyl, 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. For example, an alkyl substituted with an amine group is an alkyl-amine, an alkyl substituted with a sulfonyl group is an alkyl- sulfonyl, and an alkyl substituted with a phosphonate group is an alkyl-phosphonate.

[0670] 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.

[0671] 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.

[0672] 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.

[0673] 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 7t-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.

[0674] 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 7t-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-carboxylate, 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. The term “aryl” describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms; also referred to as “fused aryl”) groups having a completely conjugated 7t-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- carboxylate, O-carboxylate, 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 phenyl.

[0675] In some embodiments represented by general formulae, an aryl-bromide substituent or an aryl-iodide substituent is an aryl substituent having at least one bromide or at least one iodide attached thereto.

[0676] 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 7t-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.

[0677] 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.

[0678] 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.

[0679] 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, hetero aryloxy, 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, hetero aryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy, and the like, each of which may or may not be further substituted).

[0680] 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. In some embodiments, aliphatic groups contain 1-6 carbon atoms. In some embodiments, aliphatic groups contain 1-4 carbon atoms, and in yet other embodiments aliphatic groups contain 1-3 carbon atoms. 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.

[0681] 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.

[0682] The term “halo” describes fluorine (-F), chlorine (-C1), bromine (-Br) or iodine (-1) substituent.

[0683] The term “halide” describes an anion of a halogen atom, namely F“, Cl’ Br and T.

[0684] The term “haloalkyl” describes an alkyl group as defined above, further substituted by one or more halo, or wherein all hydrogen atoms are replaced by halo.

[0685] 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.

[0686] 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.

[0687] 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.

[0688] The term “117ulfoxidel l7e” 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.

[0689] 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.

[0690] The terms “117ulfoxide” 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. 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.

[0691] 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.

[0692] The term “N- sulfonamide” describes an R’S(=O)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.

[0693] 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.

[0694] The term “phosphate” describes an -O-P(=O)2(OR’) end or reactive group or a -O-P(=O)2(O)- linking moiety, as these phrases are defined hereinabove, with R’ as defined herein.

[0695] 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.

[0696] 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.

[0697] 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.

[0698] 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.

[0699] The term “oxo” as used herein, described a =0 end group.

[0700] The term “thioxo” as used herein, described a =S end group.

[0701] The term “oxime” describes a =N-0H end group or a =N-0- linking moiety, as these phrases are defined hereinabove.

[0702] The term “hydroxyl” describes a -OH group.

[0703] As used herein, the term “aldehyde” refers to an -C(=0)-H group.

[0704] The term “acyl halide” describes a -(C=0)R”” group wherein R”” is halo, as defined hereinabove.

[0705] 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.

[0706] The term “aryloxy” describes both an -O-aryl and an -O-heteroaryl group, as defined herein. The term “disulfide” as used herein describes an -S-S- linking moiety, which in some cases forms between two thiohydroxyl groups.

[0707] The terms “thio”, “sulfhydryl” or “thiohydroxyl” as used herein describe an -SH group.

[0708] 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.

[0709] 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.

[0710] The term “cyano” or “nitrile” describes a -C=N group.

[0711] The term “isocyanate” describes an -N=C=O group.

[0712] The term “nitro” describes an -NO2 group.

[0713] The term “carboxylate” or “ester”, as used herein encompasses C-carboxylate and O- carboxylate.

[0714] The term “C-carboxylate” 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.

[0715] 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.

[0716] The term “thiocarboxylate” as used herein encompasses “C-thiocarboxylate and O- thiocarboxylate.

[0717] 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.

[0718] 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.

[0719] The term “carbamate” as used herein encompasses N-carbamate and O-carbamate.

[0720] 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.

[0721] 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.

[0722] The term “thiocarbamate” as used herein encompasses N-thiocarbamate and O- thiocarbamate.

[0723] 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.

[0724] 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. The term “dithiocarbamate” as used herein encompasses N-di thiocarbamate and S- dithiocarbamate.

[0725] 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.

[0726] 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.

[0727] 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”.

[0728] 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.

[0729] The term “amide” as used herein encompasses C-amide and N-amide.

[0730] 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.

[0731] 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.

[0732] 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.

[0733] The term “hydrazone” refers to a -R’C=N-NR”- linking moiety, wherein R’ and R” are as defined herein.

[0734] The term “semicarbazone” refers to a linking moiety which forms in a condensation reaction between an aldehyde or ketone and semicarbazide. 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’ .

[0735] 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. 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 β-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.

[0736] 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.

[0737] 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.

[0738] 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.

[0739] 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.

[0740] The term “hydroxylamine”, as used herein, refers to either a -NHOH group or a -ONH2.

[0741] 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.

[0742] As used herein, the term “azido” described a -N=N+=N“ (-N3) end group.

[0743] 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.

[0744] 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.

[0745] 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.

[0746] 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. 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.

[0747] 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’.

[0748] 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.

[0749] 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.

[0750] The term “alkyl silyls”, as used herein, refers to -Si(R)s, wherein R is a C(i-4) alkyl, such as, for example, trimethylsilyl (TMS; Si(CHs)3), and triethylsilyl (TES; SiCCFhCHs ).

[0751] The term “aryl silyls”, as used herein, refers to -Si(R)3 , wherein R is an aryl, such as, for example, triphenylsilyl (TPS), trifluoromethylphenylsilyl (TFMPS), diphenylmethylsilyl (DPMS) and naphthylmethylsilyl (NMS).

[0752] The term “boronic acid”, as used herein, refers to a -B(0H)2.

[0753] The term “boronic ester”, as used herein, refers to a -B(0R)2, wherein R is as defined herein, or joint to form a ring.

[0754] 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.

[0755] As used herein the term “about” refers to ± 10 %. For example, the term “about 100 pm” encompasses the value 100 pm, as well as the values 90 pm, 91 pm, 92 pm, 93 pm, 94 pm, 95 pm, 96 pm, 97 pm, 98 pm, 98 pm, 99 pm, 100 pm, 101 pm, 102 pm, 103 pm, 104 pm, 105 pm, 106 pm, 107 pm, 108 pm, 109 pm, and 110 pm.

[0756] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0757] The term “consisting of’ means “including and limited to”.

[0758] 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.

[0759] 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.

[0760] 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.

[0761] 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.

[0762] 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.

[0763] 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.

[0764] 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.

[0765] 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.

[0766] 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.

[0767] 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.

[0768] 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.

[0769] It is expected that during the life of a patent maturing from this application many relevant locked sonosensitizer compounds will be developed and the scope of the phrase “locked sonosensitizer compound” is intended to include all such new technologies a priori.

[0770] 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.

[0771] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0772] 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.

[0773] EXAMPLE 1

[0774] Synthesis of an exemplary cyanine-based sonosensitizer

[0775] Synthesis of the maleimide-modified cyanine-based sonosensitizer (unlocked form) and its corresponding (locked form) is briefly presented hereinbelow.

[0776] (E')-l-(6-((2-(2,5-dioxo-2,5-dihydro-l / Z-pyrrol-l-yl)ethyl)amino)-6-oxohexyl)-2-(2-(6- hydroxy-2,3-dihydro-l / Z-xanthen-4-yl)vinyl)-4,6-diiodo-3,3-dimethyl-3 / Z-indol-l-ium (mhXCy), a maleimide-modified cyanine-based sonosensitizers in the “safety off’ unlocked sonosensitizer form was prepared according to the procedure provided in Thankarajan, E. el al. [Journal of Controlled Release, 2022, 343, 506-517].

[0777] The SRT moiety belonging to the cathepsin B -cleavable self-immolative linkers family, consisting of an exemplary p-aminobenzyl spacer derived from p-aminobenzylbromide (pABBr) positioned between an exemplary cathepsin B-cleavable dipeptide, Val-Cit, and the dye, was tethered thereto as presented in Scheme 1 below to afford the exemplary locked sonosensitizer (E)-2-(2-(6-((4-(2-(2-acetamido-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)-2,3- dihydro-lH-xanthen-4-yl)vinyl)-l-(6-((2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethyl)amino)-6- oxohexyl)-4,6-diiodo-3,3-dimethyl-3H-indol-l-ium (mI2XCy-CatB l).

[0778] Scheme 1

[0779] In the locked form, the dye’s conjugated system electronic mechanism is disabled, preventing accidental sonosensitization activity, namely non-intentional interaction with ultrasound energy and the resulting generation of reactive oxygen species (ROS). EXAMPLE 2

[0780] Synthesis of exemplary oxonol-based sonosensitizers

[0781] The dye 3-(4-((Z)-4-((2E,4E)-5-(5-hydroxy-3-methyl-l-(4-sulfocyclohexa-l,5-dien-l-yl)- lH-pyrazol-4-yl)penta-2,4-dien-l-ylidene)-l-(4-iodophenyl)-5-oxo-4,5-dihydro-lH-pyrazol-3- yl)phenyl)propanoic acid, abbreviated herein to Oxi, has been, modified to exhibit a maleimide functionality (mOxl) for optional tethering to, e.g., a targeting moiety such as a monoclonal antibody, and further locked with a glutathione-cleavable self-immolative linker serving as the SRT to afford 4-(5-(((2-((2-(dimethylamino)ethyl)disulfaneyl)ethyl)carbamoyl)oxy)-4- ((lE,3E,5Z)-5-(3-(4-(3-(2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)-3-oxopropyl)phenyl)- 1 -(4-iodophenyl)-5-oxo- 1 ,5-dihydro-4H-pyrazol-4-ylidene)penta- 1 ,3 -dien- 1 -yl)-3 -methyl- 1 H- pyrazol-l-yl)cyclohexa-2,4-diene-l-sulfonic acid , abbreviated herein to mOxl-SSl.

[0782] Briefly, as presented in Scheme 2 below, AICI3 (3.73 g, 28.09 mol, 5 equiv.) was added by small portions, under nitrogen atmosphere and stirring at 0 °C, to a solution of ethyl 3-chloro-3- oxopropanoate (1, 0.76 g, 0.6 ml, 5.618 mmol, 1 equiv.) and ethyl 3-phenylpropanoate (2, 1 g, 5.618 mmol, 1 ml, 1 equiv.) in 50 mL of DCM. After stirring for 4 h at r.t., the reaction mixture was poured in ice and the resulting mixture was kept under stirring for 30 minutes. Thereafter, the organic phase was separated, washed with water, dried over sodium sulfate, filtered, and the solvent removed by distillation under vacuum to give ethyl 3-(4-(3-ethoxy-3-oxopropyl)phenyl)- 3-oxopropanoate (Ketl, a yellow oil) in 80% yield.

[0783] Scheme 2

[0784] Ethyl 3-chloro-3-oxopropanoate Ethyl 3-phenylpropanoate

[0785] Ethyl 3-(4-(3-ethoxy-3- oxopropyl)phenyl)-3-oxopropanoate

[0786] Thereafter, as presented in Scheme 3 below, a mixture of 3-(4-(3-ethoxy-3- oxopropyl)phenyl)-3-oxopropanoate (Ketl, 0.5 g, 1.152 mmol, 1 equiv.) was added to (4-iodophenyl)hydrazine (3, 0.27 g, 1.152 mmol, 1 equiv.) in 15 mL AcOH and refluxed for 24 h. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure and purified on a preparative HPLC to give pyrazolone Pyrl at 80% yield. Then, the pyrazolone Pyrl was treated with LiOH (2 equiv.) for 2 h at r.t. and washed with HC1 to give 3- (4-(3-(4-iodophenyl)-5-oxo-4,5-dihydro-l / Z-pyrazol-l-yl)phenyl)propanoic acid

[0787] (carboxylated pyrazolone Pyr2) in 70% yield.

[0788] A mixture of carboxylated pyrazolone Pyr2 (0.5 g, 1.152 mmol, 1 equiv.), N-((1E,2E,4E)- 5-(phenylamino)penta-2,4-dien-l-ylidene)benzeneaminium chloride (4, 0.110 g, 0.384 mmol, 0.3 equiv.) and TEA (0.8 mL,5.76 mmol, 5 equiv.) in methanol (15 mL) was stirred at r.t. for 30 min to form derivatized pyrazolone 5. The structure of 5 was confirmed by LCMS. Then, 4-(5-hydroxy- 3-methyl-l / / -pyrazol-lyl)benzenesulfonic acid (pyrazole Pyr3, 0.292.6 g, 1.152 mmol, 1 equiv.) was added and the reaction mixture was stirred for 30 min at r.t. The reaction was monitored by LCMS. The reaction mixture was concentrated under reduced pressure and purified on a preparative HPLC to give Oxi in a 32% yield.

[0789] A carboxylic group in Oxi was modified to exhibit a maleimide functionality by treatment with DCC and HOBT to yield mOxl; and then the dye was modified to exhibit glutathione- cleavable self-immolative linker by pre-activating (heating) mOxl with o-nitrophenyl chloroformate (1 h, rt) in dichloromethane (DCM) in the presence of N,N-dimethyl cysteamine and DIE A to form the locked dye mOxl-SSl.

[0790] Scheme 4 presents the full synthesis of the maleimide-modified oxonol-based sonosensitizers mOxl (unlocked form) and the exemplary locked sonosensitizer 1-SS-mOxl, according to some embodiments of the present invention. Scheme 4 Following the same rationale and synthetic flow, another exemplary oxonol-based sonosensitizer, m0x2-SeSel, exhibiting a diselenide-type SRT (a glutathione-cleavable self- immolative linker), has been prepared, as illustrated in Scheme 5 below. Scheme 5

[0791] Following the above synthetic strategy, some exemplary oxonol-based sonosensitizer, m0x3-GlcA, exhibiting a glucuronic acid (GlcA)-type or glucuronides-type SRT (a glucuronidase responsive self-immolative linker), have been prepared, as described and illustrated in Scheme 6 below. Reducing sugars may be directly converted into the corresponding para-nitrophenyl (pNP) glycosides using 2-chloro-l,3-dimethylimidazolinium chloride (DMC), para-nitrophenol, and a suitable base in aqueous solution. The reaction is stereoselective for sugars with either a hydroxyl or an acetamido group at position 2, yielding the 1,2-trans pNP glycosides [Xin Qiu and Antony J. Fairbanks, “Direct Synthesis of para-Nitrophenyl Glycosides from Reducing Sugars in Water”, Organic Letters, 2020 22(6), pp. 2490-2493, DOI: 10(dot)1021 / acs(dot)orglett(dot)0c00728]. The synthesis was carried out according to Scheme 6 presented below, illustrating the preparation of four locked sonosensitizer compounds, exhibiting β-D-galactose, β-D-glucose, β- D-glucuronide, and β-maltose SRTs, and affording l-(6-((2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)ethyl)amino)-6-oxohexyl)-5-iodo-3,3-dimethyl-2-((lE,3E,5E)-6-(3-methyl-l-(4- sulfophenyl)-5-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2- yl)oxy)-lH-pyrazol-4-yl)hexa-l,3,5-trien-l-yl)-3H-indol-l-ium (m0x3-Gala), l-(6-((2-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethyl)amino)-6-oxohexyl)-5-iodo-3,3-dimethyl-2- ((lE,3E,5E)-6-(3-methyl-l-(4-sulfophenyl)-5-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)-lH-pyrazol-4-yl)hexa-l,3,5-trien-l-yl)-3H- indol- 1-ium (m0x3-Glus), 2-((lE,3E,5E)-6-(5-(((2S,3R,4S,5S,6S)-6-carboxy-3,4,5- trihydroxytetrahydro-2H-pyran-2-yl)oxy)-3-methyl-l-(4-sulfophenyl)-lH-pyrazol-4-yl)hexa- l,3,5-trien-l-yl)-l-(6-((2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethyl)amino)-6-oxohexyl)-5- iodo-3,3-dimethyl-3H-indol- 1-ium (m0x3-Gluc), and 2-((lE,3E,5E)-6-(5-(((2S,3R,4R,5S,6R)- 3,4-dihydroxy-6-(hydroxymethyl)-5-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6- (hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-3-methyl-l-(4- sulfophenyl)-lH-pyrazol-4-yl)hexa-l,3,5-trien-l-yl)-l-(6-((2-(2,5-dioxo-2,5-dihydro-lH-pyrrol- l-yl)ethyl)amino)-6-oxohexyl)-5-iodo-3,3-dimethyl-3H-indol- 1-ium (m0x3-Malt), respectively.

[0792] Scheme 6

[0793] EXAMPLE 3 Synthesis of an exemplary oxonol-based sonosensitizer

[0794] Synthesis of the maleimide-modified oxonol-based sonosensitizer (unlocked form) and its corresponding (locked form), pre-modified to exhibit a maleimide functionality for conjugation with a target moiety, and featuring a cathepsin B-cleavable self-immolative linker, is briefly presented hereinbelow in Scheme 7 below. Scheme 7

[0795] As can be seen in Scheme 7, the locked sonosensitizer compound 2-((lE,3E,5E)-6-(5-((4- (2-(2-acetamido-3-methylbutanamido)propanamido)benzyl)oxy)-3-(4-iodophenyl)-l-(4- sulfophenyl)-lH-pyrazol-4-yl)hexa-l,3,5-trien-l-yl)-l-(6-((2-(2,5-dioxo-2,5-dihydro-lH-pyrrol- l-yl)ethyl)amino)-6-oxohexyl)-5-iodo-3,3-dimethyl-3H-indol- 1-ium (mOx4-CatB2), featuring a maleimide functionality for TM conjugation, and the cathepsin B -cleavable self-immolative linker Val-Ala-pAB.

[0796] EXAMPLE 4

[0797] Attaching an anticancer drug to a sonosensitizer

[0798] The synthesis of an exemplary compound, according to some embodiments of the present invention, comprising an oxonol-based sonosensitizer, a glutathione-cleavable self-immolative linker, and an anticancer drug, is presented in Scheme 8 below. Scheme 8

[0799] 4-(5-(((2-((2-(((((S)-4,l l-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-lH- pyrano [3 ',4' : 6,7] indolizino [ 1 ,2-b] quinolin-9- yl)oxy)carbonyl)amino)ethyl)disulfaneyl)ethyl)carbamoyl)oxy)-4-((lE,3E,5Z)-5-(3-(4-(3-(2-

[0800] (2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethoxy)-3-oxopropyl)phenyl)-l-(4-iodophenyl)-5-oxo-

[0801] 1 ,5-dihydro-4H-pyrazol-4-ylidene)penta- 1 ,3 -dien- 1 -yl)-3 -methyl- 1 H-pyrazol- 1 -yl)cy clohexa-

[0802] 2,4-diene-l -sulfonic acid (mOxl-SS-SN-38) is an exemplary locked sonosensitizer compound that exhibits a maleimide functionality for TM conjugation and the payload of the anticancer drug 7- ethyl- 10-hydroxycamptothecin (SN-38). EXAMPLE 5

[0803] Attaching a targeting moiety drug to a sonosensitizer

[0804] The synthesis of an exemplary compound, according to some embodiments of the present invention, comprising an oxonol-based sonosensitizer, a glutathione-cleavable self-immolative linker, and an anticancer drug, is presented in Scheme 9 below.

[0805] As can be seen in Scheme 9, mOx4-CatB2, presented in Example 3, Scheme 7 hereinabove, is conjugated to an antibody via the maleimide functionality.

[0806] EXAMPLE 6

[0807] Targeted activatable sonodynamic therapeutic procedure

[0808] Following is an exemplary targeted activatable sonodynamic therapeutic (TASDT) procedure, exemplified on mice with a sonosensitizer belonging to the Formula III family (EXCy in the schemes below), locked by a cathepsin B self-immolative SRT (PABA-Cit-Val in the schemes below) and conjugated to trastuzumab (Ab in the schemes below), representing an embodiment of the present invention. The exemplary locked sonosensitizer, Ab-LXCy-P ABACI it- Vai is depicted in the scheme below:

[0809] Ab-l2XCy-PABA-Cit-Val

[0810] Mice Preparation

[0811] All experiments with mice are carried out in compliance with the Israel Council on Animal Care regulations and are approved by the Animal Care Committee of Ariel University (Authorization number IL- 179-06- 19).

[0812] The control molecule is the photosensitizer Ab-XCy, having the same dye scaffold and the same conjugation to the same antibody as Ab-LXCy-PABA-Cit-Val, but lacking iodine substituents and a self-immolative SRT.

[0813] Ab-XCy

[0814] Eighteen six- week-old athymic Balb / c female nude mice (Harlan Labs, Nes Ziona, Israel) are subcutaneously inoculated on the dorsal right side with human breast cancer cell line BT-474 (1x10 cells in PBS into nu / nu mice, 100 |aL per mouse) and tumors allowed to establish until the tumor sizes reached around 42 mm3(16 days). Thereafter, the tumor-bearing mice are randomly separated into the six groups (3 mice per group). Group 1 is used as the control for tumor growth and background autofluorescence in imaging experiments. Group 2 is used to evaluate solely ultrasound effect. Groups 3 (SDT) and 4 (SDT + PDT) are intravenously (IV) administered (tail) with Ab-hXCy-PABA-Cit-Val (100 pg in 200 pL PBS) and groups 5 (SDT) and 6 (SDT + PDT) are IV administered with control photosensitizer Ab-XCy, having the same dye scaffold but without iodine substituents and with the SRT, the same conjugation to the same antibody as Ab-FXCy-PABA-Cit-Val. at the same dosage to evaluate sonoimmunotherapeutic and combined sonoimmunotherapeutic and photoimmunotherapeutic effect of iodinated activatable sonosensitizer Ab-FXCy-PABA-Cit-Val.

[0815] Anesthesia

[0816] Before imaging and / or photo / sono dynamic treatment, mice are anesthetized by an intraperitoneal injection of Ketamine and Xylazine combination. Dose: Make up 0.5 mL Ketamine + 0.25 mL Xylazine + 4.25 mL of water for injection. Dose rate is 0.1 mL / 10 g mouse.

[0817] Animal Imaging

[0818] Imaging is carried out using multispectral fluorescence in vivo imaging system IVIS Spectrum in vivo imaging system. For imaging acquisition mice are anesthetized, and the imaging is performed after the conjugate administering at certain time intervals of 6 h and 24 h postinjection. The images are obtained in the white light mode and fluorescence channel: excitation filter 710+30 nm, emission filter 780+20 nm. The data processing is performed by IVIS living image software.

[0819] PDT and SDT Experiment

[0820] BT-474 tumor-bearing mice are established as described above until the tumor volumes reached approximately 42 mm3. The mice are randomly divided into the six groups. Group 1 is used as the control for tumor growth and background autofluorescence in imaging experiments. Group 2 is used to evaluate solely ultrasound effect. Groups 3 and 4 are administered in the tail vein (IV) with Ab-2IXCy-PABA-Cit-Val and groups 5 and 6 with Ab-XCy (100 pg in 200 pL PBS). Accumulation of the conjugates is monitored by fluorescence imaging at 6 h and 24 h. After 24h the mice from groups 3 and 4 (injected with Ab-2IXCy-PABA-Cit-Val) demonstrated specific accumulation in the tumor and reached maximum fluorescent intensity. Mice from groups 3-6 are anesthetized and subjected to ultrasound (SDT) (f= 1 MHz, P = 0.7 W, 100 mW / cm2, 1 min irradiation / 1 min rest, 5 times, ultrasound dose = 30 J / cm2). Additionally, groups 4 and 6 are subjected to NIR light irradiation (PDT) (730 nm, 30 W LED, 70 mW / cm2, 15 min, light dose 63 J / cm2). Then the tumor sizes are measured in vivo over time by using the hands-on digital caliper method; and the tumor volumes are calculated according to the equation: Tumor volume (mm3) = 0.5 x Tumor Length x Tumor Width2

[0821] The results are expected to show elimination of the tumor in groups 3 and 4 as opposed to groups 5 and 6. No augmented efficacy is expected in combined SDT and PDT therapy vs SDT alone. 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. It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is 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. 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 characterized by a general formula individually selected from the group consisting of:Formula VII, Formula VIII,Formula XV, Formula XVI,and any hydrate, solvate, ester, or pharmaceutically acceptable salt thereof, wherein:SRT is a biocleavable linking moiety;T is O, S, or NRT, RTis H, alkyl, aryl, or amine; n is an integer ranging 1-5; each of RH1-RH3is independently H, Br, I, aryl-bromide, or aryl-iodide, provided that at least one of RH1-RH3is Br, I, aryl-bromide, or aryl-iodide; each of W’-W3is independently O, S, Se, C=O, CRARB, CRA=CRB, or NRC, wherein each of RA-RCis independently H, alkyl, aryl, halo, -Rs-S03H, -Rs-C02H, -RS-NH2, -Rs-NMe2, -Rs- NMe3+, -Rs-maleimide and -Rs-succinimide, or any adjacent RA-RCmay form a ring; each of RN1-RN4is independently hydrogen, a substitute or unsubstituted alkyl, substitute or unsubstituted aryl, an aryl substituted with Br or I, -Rs-S03H, -Rs-C02H, -RS-NH2, -Rs- NMe2, -Rs-NMe3+, -Rs-azide, -Rs-phosphonate, -Rs-maleimide and -Rs-succinimide;Rsis a spacer moiety selected from -(CEh-ji-s and -(OCH2CH2-)I-4; and each of R1-R17 is independently selected from the group consisting of H, a substitute or unsubstituted alkyl, a substitute or unsubstituted aryl, fused aryl (benzo), halo, haloalkyl, -CF3, -CO2H, -CO2Me, -SO3H, -ON, -NO2, -NH2, NHMe, -N(Me)2, carboxamide, and -OMe, or any two adjacent R1-R17 form a ring; with the proviso that the compound is not presented in Table 1.

2. The compound of claim 1, wherein said biocleavable linking moiety is selected from the group consisting of amide, carbamate, carbonate, carboxylate, acetal, glucoside, aldimine, aminal, aminoacetal, acryl, boronic acid, boronic ester, cycloalkene, cyclohexene, disulfide, sulfate, epoxide, ester, heteroalicyclic, heteroaryl, hydrazide, hydrazone, imide, imine, ketal, ketimine, lactam, lactone, methyleneamine, oxime, peroxo, phosphate, phosphateester, semicarbazone, alkyl silyl, aryl silyl, thioacetal, thiohydrazide, thioketal, triazine, and triazole.

3. The compound of claim 2, wherein said biocleavable linking moiety is a self- immolative linker selected from the group consisting of a glutathione-cleavable self-immolative linker, a cathepsin B-cleavable self-immolative linker, galactosidase responsive self-immolative linker, peroxide-responsive self-immolative linker glucuronidase responsive self-immolative linker, sulfatase responsive self-immolative linker, and a fluoride-responsive self-immolative linker.

4. The compound of claim 3, wherein said self-immolative linker is selected from the group consisting of:wherein: each of R18-R21 is independently H, methyl, ethyl, cyclopropyl or cyclobutyl ;R22 is selected from H, -OH, -NH2, -NHMe, -NMe2, -NMe3+, -PO4H2, -PO2(OMe)2, - CO2H, -CO(CH2)2CO2H, -CO(CH2)3CO2H, -CO2Me, -SO3H, alkyl, alkoxy, haloalkyl, -(OCH2CH2-)I-3, -CO2-D, -CONH-D, -CONMe-D, -OCO-NH-D, -OCO2-D, and -OCO- NMe-D; andD is a functional moiety selected from the group consisting of a bioactive agent, an imaging agent, a labeling agent, and / or a diagnostic agent.

5. The compound of any one of claims 1-4, further comprising a functional moiety attached thereto via said biocleavable linking moiety, said functional moiety is selected from the group consisting of a bioactive agent, an imaging agent, a labeling agent, and / or a diagnostic agent.

6. The compound of any one of claims 1-5, conjugated to a targeting moiety.

7. The compound of claim 6, wherein said targeting moiety is selected from the group consisting of a nanoparticle, a peptide, a protein, an antibody or a fragment thereof, a porphyrin, a hormone, a saccharide, a polysaccharide, an antigens, a hapten, a DNA fragment, a folate, a polymeric micelle, a liposome, a lipoprotein-based drug carrier, a dendrimer, siRNA, an oligonucleotide, an RNA fragment, and a receptor ligand.

8. The compound of claim 7, wherein said targeting moiety is an antibody or a fragment thereof.

9. A pharmaceutical composition comprising, as an active ingredient, the compound of any one of claims 1-8, and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 9, for use in the treatment of a medical condition in a subject.

11. The compound of any one of claims 1-8, for use in the treatment of a medical condition in a subject.

12. A method of treating a medical condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount the compound of any one of claims 1-8, and exposing at least a part of the subject to ultrasound energy.

13. The method of claim 12, wherein said exposing is effected using a focused ultrasound device focused on said at least a part of the subject.

14. The method of claim 12, wherein said exposing is effected on a whole body of the subject, or a part thereof.

15. The method of any one of claims 12-14, wherein said exposing is effected at a frequency of 1.0-2.0 MHz and an intensity of 0.5-10 W / cm2.

16. The composition of claim 10, the compound of claim 11, and the method of claim 12, wherein said medical condition is treatable by induction of at least one cytotoxic effect.

17. The composition, compound and method of claim 16, wherein said medical condition is selected from the group consisting of cancer, an autoimmune disease, a viral infection, a transplant rejection and a neurodegenerative disease.

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