RU2817710C2 - Stable local drug levels for innate immunity agonists - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: group of inventions refers to pharmaceutics and can be used for treating cell proliferation disorders. Disclosed is a water-insoluble controlled-release pattern recognition receptor (PRRA) agonist or a pharmaceutically acceptable salt thereof, wherein said water-insoluble controlled-release PRRA releases one or more PRRAs, and at least 25% of the amount of PRRA remains locally in such tissue 3 days after interstitial administration, wherein the water-insoluble PRRA contains a carrier moiety Z, with which one or more fragments -L2-L1-D are conjugated, where: each -L2- is individually a C1-20 alkyl chain, which is optionally interrupted by one or more groups independently selected from -O-, -T- and -C(O)N(Ry1)-; each -L1- is individually a linker fragment with which -D is reversibly and covalently conjugated; each D is resiquimod; Z is a water-insoluble PEG hydrogel. Also disclosed is a pharmaceutical composition containing said water-insoluble PRRA and at least one excipient.

EFFECT: group of inventions provides effective treatment regimens with TLR agonists, higher local concentrations of PRRA for an extended period of time while maintaining low systemic concentrations of PRRA, which minimizes side effects.

11 cl, 17 ex

Description

The present invention relates to a water-insoluble controlled-release pattern recognition receptor agonist (“PRRA”) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising such a water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof for use in the treatment of a cell proliferation disorder wherein the water-insoluble controlled-release PRRA, a pharmaceutically acceptable salt or pharmaceutical composition thereof is administered via interstitial administration, and wherein at least 25% of the amount of PRRA remains locally in such tissue 3 days after administration; and related aspects.

Toll-like receptors (TLRs) are a family of evolutionarily conserved pathogen recognition receptors that play a critical role in the activation of both innate and adaptive immunity. To date, at least 13 different TLRs have been identified in mammals. TLR-1, -2, -4, -5 and -6 are located on the cell surface, while TLR-3, -7, -8 and -9 are located in endosomal compartments with their ligand-binding domains facing the lumen vesicles.

TLRs bind ligands derived from pathogens and malignant cells called pathogen-associated molecular patterns (PAMPs), which upon binding trigger the NF-KB and interferon response factor (IRF) pathways leading to the production of pro-inflammatory cytokines (e.g., IFN-α, IFN -β, IL-1β, IL-6, TNFα), chemokines (e.g. RANTES, MIP1α, MIP1β) and expression of immune stimulatory molecules (CD80, CD86, CD40) by dendritic cells (e.g. DC) and other antigen presenting cells such as e.g. macrophages. TLRs are critical for promoting DC maturation, antigen uptake and presentation, immune cell recruitment and differentiation of CD4 ⁺ T cells, and control of regulatory T (Treg) cells (Iwasaki & Medzhitov, Nat Immunol. 2004 Oct, 5(10): 987- 995).

There are many known ligands for each TLR, especially because small synthetic molecules that can activate TLRs are being actively developed and widely used for therapeutic purposes. For example, imiquimod and resiquimod, which can activate TLR-7 and TLR-7/8, respectively, have been extensively studied in preclinical and clinical studies for their antiviral and anticancer effects.

Depending on the therapeutic goals, TLR ligands have been administered through different routes, such as systemically, orally or intravenously, or locally, by topical cream, subcutaneous injection, or intratumoral injection. Efficacy, toxicity, bioavailability and other pharmacokinetic parameters vary greatly depending on the route of administration (Engel et al ., Expert Rev Clin Pharmacol. 2011 Mar, 4(2): 275-289).

The lack of clinical antitumor efficacy and tumor-centric immunological effects following systemic administration of TLR agonists may be due to failure to target the drug to its intended site of action. Because these drugs are designed to positively influence the immune response at the tumor site, systemic distribution may only serve to enhance global side effects due to systemic exposure of the active drug, while limiting the bioavailability of the active compound in the tumor environment, thereby precluding the possibility of reliable antitumor effect (Engel et al ., Expert Rev Clin Pharmacol. 2011 Mar, 4(2): 275-289).

Intratumoral injection of TLR agonists has been attempted using lipidation or various formulation techniques, including suspending the active drug in an oil medium, mixing with biomaterials, or conjugating with polymers to prolong exposure of a given TLR drug to tumor tissue. Dissemination of these soluble TLR agonists from the tumor can result in significant systemic exposure. In addition, frequent intratumoral administration of these compounds is necessary for long-term, continuous exposure of tumor tissue to TLR drugs, making effective TLR agonist therapy impractical or unfeasible for patients.

Although significant efforts have been made to develop new and improved TLR agonists that overcome one or more of the above-mentioned disadvantages, there remains a need to identify more effective TLR agonists. In addition, there remains a need to modify TLR agonist treatment regimens so that they overcome the shortcomings of prior art compounds and associated treatment methodologies, while also providing favorable antitumor responses and reducing adverse events associated with systemic exposure.

Thus, there is a need for more effective treatment.

The object of the present invention is to at least partially overcome the above-described disadvantages.

The object is achieved by means of a water-insoluble controlled-release pattern recognition receptor agonist (“PRRA”) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing such a water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof for use in the treatment of a cell proliferation disorder, wherein the water-insoluble controlled-release PRRA is administered via interstitial administration, and wherein at least 25% of the amount of PRRA remains locally in such tissue 3 days after administration.

According to another aspect, the present invention provides a water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof, wherein said water-insoluble controlled-release PRRA releases one or more PRRAs and wherein, following interstitial administration of said controlled-release pattern recognition receptor agonist or the same, pharmaceutically acceptable salt, the amount of PRRA remaining locally in such tissue after 3 days is at least 25%.

According to another aspect, the present invention relates to a pharmaceutical composition containing one or more water-insoluble controlled release PRRA or a pharmaceutically acceptable salt thereof according to the present invention.

According to another aspect, the present invention relates to a water-insoluble controlled release PRRA or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the present invention for use as a drug.

Surprisingly, it has been discovered that the water-insoluble controlled-release PRRA of the present invention can be used as a stand-alone immunotherapy agent (i.e., as a monoimmunotherapy agent), or, in another aspect, can be used in combination with other therapeutic agents, such that provides effective TLR agonist treatment regimens. In addition, the use of water-insoluble controlled-release PRRA provides higher local PRRA concentrations for an extended period of time while maintaining low systemic PRRA concentrations, thereby minimizing side effects.

In the context of the present invention, the terms used have the following meanings.

As used herein, the term “pattern recognition receptor agonist” (“PRRA”) refers to a molecule that binds to and activates one or more immune cell-associated receptors that recognize pathogen-associated molecular patterns (PAMPs), or molecular damage-associated patterns (DAMPs) leading to immune cell activation and/or inflammatory responses caused by pathogens or damage. PRRs are commonly expressed by cells of the innate immune system, such as monocytes, macrophages, dendritic cells (DCs), neutrophils and epithelial cells, as well as cells of the adaptive immune system.

In the context of the present invention, the term "controlled release pattern recognition receptor agonist" or "controlled release PRRA" refers to any conjugate that contains at least one pattern recognition receptor agonist, and of which at least one pattern recognition receptor agonist receptor (“PRRA”) is released with a half-life in vitro under physiological conditions (aqueous buffer, pH 7.4, 37°C) of at least 6 hours, such as at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 7 days, at least 10 days, at least 14 days, at least 21 days, at least one month, at least two months or at least 3 months.

In the context of the present invention, the terms "cytotoxic agent" and "chemotherapeutic agent" are used interchangeably and refer to compounds that are toxic to cells that prevent cell replication or growth, resulting in cell destruction/death. Examples of cytotoxic agents include chemotherapeutic agents and toxins, such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

In the context of the present invention, the terms "immune checkpoint inhibitor" and "immune checkpoint antagonist" are used interchangeably and refer to compounds that affect the function of or inhibit the binding of ligands that induce signaling through receptors expressed on the cell membrane that inhibit the function of inflammatory immune cells upon receptor activation. Such compounds may, for example, be biological compounds, for example antibodies, nanobodies, protels, anticalins or cyclic peptides or small molecule inhibitors.

As used herein, the term “immune checkpoint agonist” refers to compounds that directly or indirectly activate receptors expressed on the cell membrane that stimulate the function of inflammatory immune cells upon receptor activation.

As used herein, the terms “multispecific” and “multispecific drugs” refer to compounds that simultaneously bind to two or more different antigens and can mediate antagonistic, agonistic or specific antigen binding activity in a target dependent manner.

As used herein, the term “antibody-drug conjugate” (ADC) refers to compounds typically consisting of an antibody coupled to a biologically active cytotoxic payload, radiotherapy agent, or other drug designed to deliver cytotoxic agents to a tumor environment. ADCs are particularly effective in reducing tumor burden without significant systemic toxicity and may act to enhance the effectiveness of the immune response induced by checkpoint inhibitor antibodies.

In the context of the present invention, the term "radionucleotides" refers to radioactive isotopes that emit ionizing radiation leading to cell destruction/death. Radionucleotides conjugated to tumor-targeting carriers are called “targeted radionucleotide therapeutics.”

In the context of the present invention, the term “DNA damage repair inhibitor” refers to a drug that targets DNA damage repair elements, such as, for example, CHK1, CHK2, ATM, ATR and PARP. Some cancers are more susceptible to targeting these pathways due to existing mutations, such as patients with a BRCA1 mutation to PARP inhibitors due to the concept of synthetic lethality.

In the context of the present invention, the term "tumor metabolic inhibitor" refers to a compound that interferes with the function of one or more enzymes expressed in the tumor environment that produce metabolic intermediates that can inhibit the function of immune cells.

As used herein, the term “protein kinase inhibitor” refers to compounds that inhibit the activity of one or more protein kinases. Protein kinases are enzymes that phosphorylate proteins, which in turn can modulate protein function. It is understood that a protein kinase inhibitor can target more than one kinase, and any classification of protein kinase inhibitors used herein refers to the primary or best characterized target.

As used herein, the term “chemokine receptor and chemoattractant receptor agonist” refers to compounds that activate chemokine or chemoattractant receptors, a subset of G protein-coupled receptors or G protein-coupled-like receptors that are expressed on a wide variety of cells and primarily involved in the control of cell motility (chemotaxis or chemokinesis). These receptors may also be involved in processes unrelated to cell migration, such as angiogenesis, cell maturation, or inflammation.

As used herein, the term “cytokine receptor agonist” refers to soluble proteins that control the activation and proliferation of immune cells. Cytokines include, for example, interferons, interleukins, lymphokines and tumor necrosis factor.

In the context of the present invention, the term "death receptor agonist" refers to a molecule that is capable of inducing pro-apoptotic signaling through one or more death receptors, such as DR4 (TRAIL-R1) or DR5 (TRAIL-R2). The death receptor agonist may be selected from the group consisting of antibodies, death ligands, cytokines, vectors expressing a death receptor agonist, peptides, small molecule agonists, cells (eg, stem cells) expressing a death receptor agonist, and drugs that induce expression of the ligands of death.

In the context of the present invention, the term "antigen presenting cell" or "APC" refers to a cell, such as a macrophage, B cell or dendritic cell, that presents processed antigen peptides via MHC class II molecules to a T cell receptor. on CD4 T cells. APC can be identified by one skilled in the art using phenotypic methods such as flow cytometry. Phenotypic markers used to identify APC vary by species and tissue, but may include myeloid or dendritic cell surface markers (eg, CD11b, CD11c, CD14, CD16, CD33, CD34, Ly6C, Ly6G, GR-1, F4/ 80) or B cell surface markers (eg CD19, CD20, B220).

As used herein, the term "MHCII" refers to a class of major histocompatibility complex (MHC) molecules typically found only on antigen-presenting cells, such as myeloid cells, dendritic cells and B cells. MHCII presents processed antigenic peptides to the T cell receptor on CD4 T cells. MHCII expression can be measured by one skilled in the art using protein expression profiling techniques, such as flow cytometry. Changes in MHCII expression can be determined by analyzing changes in the mean MHCII fluorescence intensity signal or the percentage of cells positive for MHCII in a particular cell subpopulation of interest.

In the context of the present invention, the term "T cells" refers to a type of immune cell that plays a central role in the adaptive immune response. T cells are distinguished from other immune cells by the presence of the T cell receptor αβ or γδ (TCR) on their cell surface. T cells also express the CD3 protein complex, which is important for TCR signaling. αβ T cells can be divided into CD4, CD8, or CD4/CD8 double-negative subsets. Due to the high surface density of CD4 and CD8 on CD4+ and CD8+ T cells, CD4 and CD8 alone can often be used to identify CD4+ and CD8+ T cells, respectively. Once activated through TCR recognition of cognate antigen presented by MHC molecules, T cells can mature and divide to form effector T cells or memory T cells. Memory T cells are a subpopulation of T cells that have previously encountered and responded to their cognate antigen. Such T cells can recognize pathogenic antigens, such as antigens derived from bacteria or viruses, as well as antigens associated with cancer. One skilled in the art can identify T cells using phenotypic methods, such as flow cytometry. Phenotypic markers used to identify T cells are generally conserved across mammals and include CD3, TCRα, TCRβ, TCRδ, CD4, and CD8. Phenotypic markers used to identify memory T cells may vary depending on the type and tissue, but may include cell surface markers such as, for example, CD45RO, LY6C, CD44 and CD95.

In the context of the present invention, the term "intratissue administration" refers to the type of administration, for example local injection, of a drug into the tissue of interest, such as intratumoral, intramuscular, subdermal or subcutaneous injections or injections into or near normal tissue or diseased tissue or organ.

In the context of the present invention, the term "intratumoral administration" refers to a method of administration in which the drug is administered directly into the tumor tissue. The term "intratumoral administration" may, in certain embodiments, also refer to administration before or after resection in or on the tumor bed. When the tumor boundary is not clearly defined, it is also understood that intratumoral administration includes administration into tissue adjacent to tumor cells (“peritumoral administration”). Exemplary tumors for intratumoral administration are solid tumors and lymphomas, which are described in more detail elsewhere herein. Administration may occur by injection, including intramuscular and subcutaneous injections.

In the context of the present invention, the term "starting tissue" refers to a tissue sample taken from or adjacent to the area to be treated, prior to treatment. For example, a biopsy of the tissue to be treated may be taken immediately before treatment. It is understood that it is not always possible to take a control sample from the corresponding area before treatment, so the term "parent tissue" can also refer to untreated control tissue, which can be taken from a comparable location in the same animal or can be taken from a comparable location in a different animal the same type. It is understood that in general the term "animal" also includes a human and in certain embodiments means a mouse, rat, non-human primate and human and in certain embodiments means a mouse, rat, non-human primate or human.

In the context of the present invention, the term “antitumor activity” means the ability to inhibit tumor growth, i.e. suppression of tumor growth or tumor stasis, or the ability to cause a decrease in tumor size, i.e. tumor regression. In a certain embodiment, the term also refers to the ability to reduce the rate of tumor growth by at least 20%, such as by at least 25%, by at least 30%, by at least 35%, by at least 40% , by at least 45% or by at least 50%. Antitumor activity can be determined by comparing the mean relative tumor volumes between control and treatment conditions. Relative individual tumor volumes (individual RTVs) for day "x" can be calculated by dividing the absolute volume of an individual tumor on day "x" (T _x ) after the start of treatment by the absolute individual tumor volume of the same tumor on the day of start of treatment (T ₀ ) and multiplying by 100:

Antitumor activity in certain embodiments can be observed from 7 to 21 days after initiation of treatment. In certain embodiments, antitumor activity is observed 7 days after initiation of treatment. In certain embodiments, antitumor activity is observed 8 days after initiation of treatment. In certain embodiments, antitumor activity is observed 9 days after initiation of treatment. In certain embodiments, antitumor activity is observed 10 days after initiation of treatment. In certain embodiments, antitumor activity is observed 11 days after initiation of treatment. In certain embodiments, antitumor activity is observed 12 days after initiation of treatment. In certain embodiments, antitumor activity is observed 13 days after initiation of treatment. In certain embodiments, antitumor activity is observed 14 days after initiation of treatment. In certain embodiments, antitumor activity is observed 15 days after initiation of treatment. In certain embodiments, antitumor activity is observed 16 days after initiation of treatment. In certain embodiments, antitumor activity is observed 17 days after initiation of treatment. In certain embodiments, antitumor activity is observed 18 days after initiation of treatment. In certain embodiments, antitumor activity is observed 19 days after initiation of treatment. In certain embodiments, antitumor activity is observed 20 days after initiation of treatment. In certain embodiments, antitumor activity is observed 21 days after initiation of treatment. It is understood that these time points indicate the earliest time point at which antitumor activity is observed.

The tumor size, specified in mm ³ , can be measured physically by measuring the length (L), measured in mm, and width (W), measured in mm, of the tumor, which may include injected tumors and non-injected tumors. Tumor volume can be determined by methods such as, for example, ultrasound imaging, magnetic resonance imaging, computed tomography scanning, or approximately using the equation , where V denotes tumor volume. Tumor load, i.e. the total number of cancer cells in an individual can also be measured in the case of an experimental tumor model that expresses a reporter, for example, a luciferase enzyme or a fluorescent protein or other measurable protein or enzyme, by measuring the reporter element, i.e. luminescence or fluorescence, or expressed reporter protein or enzyme product as a measure of the total number of tumor cells present and the overall size of the tumor. The latter reporter models may be useful for tumors that are difficult to measure on animal surfaces (i.e., orthotopic tumors). It is understood that in general the term "animal" also includes a human and in a certain embodiment means a mouse, a rat, non-human primates and a human.

In the context of the present invention, the term "local inflammation" refers to inflammation that is limited to the area adjacent to the site of administration of the controlled release PRRA. The specific size of the area of inflammation will depend on the amount of agonist administered, the rate of diffusion into the tissue, the time at which the signal is measured after injection, the rate of drug uptake by neighboring cells, and the frequency of cells responding to the pattern recognition receptor in and around the treated area, but usually it can be detected at a distance of 2 times the radius ( r ) of the injection site in any direction, where r is the distance in centimeters (cm) calculated from the volume (V) of injected water-insoluble controlled-release PRRA in cubic centimeters ( ^cm3 ) in accordance with the spheroid equation . For example, if a given tissue is injected with 0.5 cm ³ of controlled release PRRA, a tissue sample weighing at least 0.025 g taken within 0.98 cm in any direction from the injection site shows a measurable inflammatory signal. In a volume 2 times greater than r , it is necessary to take tissue samples to determine the presence of a certain set of inflammatory markers. However, this does not mean that these inflammatory markers beyond a volume 2 times greater than r cannot be activated by at least 1.5 times. In general, the intensity of inflammation decreases with increasing distance from the injection site. However, one skilled in the art will appreciate that providing an outer boundary to such localized inflammation is not feasible since the extent of inflammation depends on various factors, such as the type of tumor. In any case, one skilled in the art will readily be able to distinguish local from systemic inflammation.

In the context of the present invention, the term "water insoluble" refers to a compound of which less than 1 g can be dissolved in one liter of water at 20°C to form a homogeneous solution. Accordingly, the term “water soluble” refers to a compound of which 1 g or more can be dissolved in one liter of water at 20°C to form a homogeneous solution.

In the context of the present invention, the term "drug" refers to a substance used in the treatment, cure, prevention or diagnosis of a disease or otherwise used to improve the physical or mental health of a patient. If a drug is conjugated to another moiety, the fragment of the resulting product derived from the drug is called a "drug fragment".

Any reference herein to a biological medicinal product, i.e. a drug produced, extracted from or semi-synthesized from biological sources, such as a protein drug, also covers biosimilar versions of the specified drug.

In the context of the present invention, the term "prodrug" refers to a drug moiety reversibly and covalently linked to a specialized protecting group through a reversible prodrug linker moiety, which is a linker moiety containing a reversible linkage to the drug moiety, and wherein the specialized protecting group modifies or eliminates undesirable properties in the parent molecule. It also involves enhancing the desired properties of the drug and suppressing undesirable properties. The dedicated non-toxic protecting group may also be referred to as a "carrier". A prodrug releases a reversibly and covalently bound drug moiety in the form of the corresponding drug. In other words, a prodrug is a conjugate containing a drug moiety that is covalently and reversibly conjugated to a carrier moiety via a reversible linker moiety, wherein the covalent and reversible conjugation of the carrier to the reversible linker moiety occurs either directly or through a spacer. A reversible linker may also be referred to as a "reversible prodrug linker". Such a conjugate may release the previously conjugated drug moiety in the form of a free drug, in which case the reversible linker or reversible prodrug linker is a traceless linker.

In the context of the present invention, the term "free form" of a drug means a drug in its unmodified pharmacologically active form.

In the context of the present invention, the term “ -electron para-donating heteroaromatic N-containing moiety” refers to a moiety that, upon cleavage of the bond between -D and -L ^1-, results in a drug DH, and where the drug moiety is -D, and likewise the corresponding DH contains at least one , such as one, two, three, four, five, six, seven, eight, nine or ten heteroaromatic nitrogen atoms that donate a π electron pair to the aromatic π system. Examples of chemical structures containing such heteroaromatic nitrogens that donate a π electron pair to an aromatic π system include, but are not limited to, pyrrole, pyrazole, imidazole, isoindazole, indole, indazole, purine, tetrazole, triazole, and carbazole. For example, in the imidazole ring below, the heteroaromatic nitrogen that donates a π electron pair to the aromatic π system is marked with a "#":

.

-electron para-donating heteroaromatic nitrogen atoms do not contain heteroaromatic nitrogen atoms that donate only one electron (i.e. not a pair of π electrons) to the aromatic π system, such as nitrogen, which is marked "§" in the above ring imidazole structure. A DH drug may exist in one or more tautomeric forms, such as with one hydrogen atom moving between at least two heteroaromatic nitrogen atoms. In all such cases, the linker moiety is covalently and reversibly attached to a heteroaromatic nitrogen, which donates a π electron pair to the aromatic π system.

In the context of the present invention, the term "spacer" refers to a fragment that connects at least two other fragments to each other.

In the context of the present invention, the term "reversible", "reversible", "degradable" or "degradable" in relation to the attachment of a first fragment to a second fragment means that the bond that connects the first and second fragments can be cleaved under physiological conditions, which are aqueous buffer at pH 7.4, 37°C, with a half-life of one day to three months, such as two days to two months, such as three days to one month. Such digestion, in certain embodiments, is non-enzymatic. Accordingly, the term “stable” with respect to the attachment of a first moiety to a second moiety means that the bond that connects said first and second moieties exhibits a half-life of greater than three months under physiological conditions.

As used herein, the term “reagent” means a chemical compound that contains at least one functional group to react with a functional group of another chemical compound or drug. It is understood that the drug containing the functional group is also a reagent.

As used herein, the term “fragment” means a portion of a molecule that is missing one or more atoms from the corresponding reactants. If, for example, a reagent of the formula “H-X-H” reacts with another reagent and becomes part of the reaction product, the corresponding fragment of the reaction product has the structure “H-X-“ or “-X-“, where each “-“ denotes addition to another fragment. Accordingly, the biologically active moiety, such as an antibiotic moiety, is released from the reversible bond as a drug, such as an antibiotic drug.

It is understood that if the chemical structure of a group of atoms is provided, and if that group of atoms is attached to two moieties or interrupts a moiety, said chemical structure can be attached to the two moieties in any orientation unless otherwise specified. For example, the fragment “-C(O)N(R ¹ )-” can be attached to two fragments or interrupt the fragment as either “-C(O)N(R ¹ )-” or “-N(R ¹ ) C(O)-.” Likewise, a fragment

can be attached to two fragments or interrupt a fragment, either , or how .

The term “substituted” as used herein means that one or more hydrogen atoms of a molecule or fragment are replaced by another atom or group of atoms, which are referred to as a “substituent”.

In the context of the present invention, the term “substituted” in certain embodiments refers to a moiety selected from the group consisting of halogen, -CN, -COOR ^x1 , -OR ^x1 , -C(O)R ^x1 , -C(O)N( R ^x1 R ^x1a ), -S(O) ₂ N(R ^x1 R ^x1a ), -S(O)N(R ^x1 R ^x1a ), -S(O) ₂ R ^x1 , -S(O)R ^x1 , -N(R ^x1 )S(O) ₂ N(R ^x1a R ^x1b ), -SR ^x1 , -N(R ^x1 R ^x1a ), -NO ₂ , -OC(O)R ^x1 , -N(R ^x1 ) C(O)R ^x1a , -N(R ^x1 )S(O) ₂ R ^x1a , -N(R ^x1 )S(O)R ^x1a , -N(R ^x1 )C(O)OR ^x1a , -N( R ^x1 )C(O)N(R ^x1a R ^x1b ), -OC(O)N(R ^x1 R ^x1a ), -T ⁰ , C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl, wherein -T ⁰ , C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted by one or more -R ^x2 which are the same or different, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T ⁰ -, -C(O)O-, -O-, -C(O)-, -C(O)N( R ^x3 )-, -S(O) ₂ N(R ^x3 )-, -S(O)N(R ^x3 )-, -S(O) ₂ -, -S(O)-, -N(R ^x3 )S(O) ₂ N(R ^x3a )-, -S-, -N(R ^x3 )-, -OC(OR ^x3 )(R ^x3a )-, -N(R ^x3 )C(O)N(R ^x3a )-, and -OC(O)N(R ^x3 )-,

-R ^x1 , -R ^x1a , -R ^x1b are independently selected from the group consisting of -H, -T ⁰ , C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl, where -T ⁰ , C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted by one or more -R ^x2 , which are the same or different, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _{2- 50} alkynyl optionally interrupted by one or more groups selected from the group consisting of -T ⁰ -, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^x3 )- , -S(O) ₂ N(R ^x3 )-, -S(O)N(R ^x3 )-, -S(O) ₂ -, -S(O)-, -N(R ^x3 )S(O ) ₂ N(R ^x3a )-, -S-, -N(R ^x3 )-, -OC(OR ^x3 )(R ^x3a )-, -N(R ^x3 )C(O)N(R ^x3a )-, and -OC(O)N(R ^x3 )-,

each ^T0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, _C3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, wherein each ^T0 is independently optionally substituted with one or more -R ^x2 which are the same or different,

each -R ^x2 is independently selected from the group consisting of halogen, -CN, oxo (=O), -COOR ^x4 , -OR ^x4 , -C(O)R ^x4 , -C(O)N(R ^x4 R ^x4a ) , -S(O) ₂ N(R ^x4 R ^x4a ), -S(O)N(R ^x4 R ^x4a ), -S(O) ₂ R ^x4 , -S(O)R ^x4 , -N(R ^x4 )S(O) ₂ N(R ^x4a R ^x4b ), -SR ^x4 , -N(R ^x4 R ^x4a ), -NO ₂ , -OC(O)R ^x4 , -N(R ^x4 )C(O)R ^x4a , -N(R ^x4 )S(O) ₂ R ^x4a , -N(R ^x4 )S(O)R ^x4a , -N(R ^x4 )C(O)OR ^x4a , -N(R ^x4 )C( O)N(R ^x4a R ^x4b ), -OC(O)N(R ^x4 R ^x4a ) and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different,

-R ^x3 , -R ^x3a , -R ^x4 , -R ^x4a , -R ^x4b are each independently selected from the group consisting of - H and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted by one or more halogens, which are the same or different.

In certain embodiments, a maximum of 6 - H atoms of an optionally substituted molecule are independently substituted with a substituent, e.g., 5 - H atoms are independently substituted with a substituent, 4 - H atoms are independently substituted with a substituent, 3 - H atoms are independently substituted with a substituent, 2 - H atoms are independently substituted with a substituent, or the 1 - H atom is replaced by a substituent.

As used herein, the term “hydrogel” means a hydrophilic or amphiphilic polymer network consisting of homopolymers or copolymers that is insoluble due to the presence of hydrophobic interactions, hydrogen bonds, ionic interactions and/or covalent chemical cross-links. Crosslinks provide structure and physical integrity to the network. In certain embodiments, the hydrogel is insoluble due to the presence of covalent chemical cross-links.

As used herein, the term “cross-linker” refers to a moiety that is a link between different hydrogel units, for example between two or more backbone moieties or between two or more strands of hyaluronic acid.

As used herein, the term “about” in combination with a numerical value is used to indicate a range within the interval and including a numerical value plus and minus not more than 25% of the specified numerical value, such as not more than plus or minus 20% of the specified value and such as no more than plus and minus 10% of the specified value. For example, the phrase “about 200” is used to indicate a range within and including 200 +/- 25%, i.e. in the range and including 150 - 250, preferably 200 +/-20%, i.e. in the range and including 160 - 240, even more preferably in the range and including 200 +/-10%, i.e. in the range and including 180-220. It is clear that the percentage given as “about 50%” does not mean “50% +/- 25%”, i.e. in the range and including 25 - 75%, but “about 50%” means in the range and including 37.5 - 62.5%, i.e. plus and minus 25% of the numerical value, which is 50.

As used herein, the term “polymer” means a molecule containing repeating structural units, i.e. monomers linked by chemical bonds in a linear, circular, branched, cross-linked or dendrimeric manner or a combination thereof, which may be of synthetic or biological origin or a combination of both. The monomers may be identical, in which case the polymer is a homopolymer, or they may be different, in which case the polymer is a heteropolymer. A heteropolymer may also be referred to as a "copolymer" and includes, for example, alternating copolymers in which different types of monomers alternate, periodic copolymers in which different types of monomers are arranged in a repeating sequence, random copolymers in which different types of monomers are arranged randomly, block copolymers, in which blocks of different homopolymers consisting of only one type of monomer are linked by a covalent bond, and gradient copolymers in which the composition of the different monomers gradually changes along the polymer chain. It will be understood that the polymer may also contain one or more other moieties, such as, for example, one or more functional groups. The term "polymer" also refers to a peptide or protein, even though the side chains of individual amino acid residues may be different. It is clear that covalently cross-linked polymers such as hydrogels cannot achieve meaningful molecular weight ranges.

As used herein, the term “polymer” means a reagent or fragment containing one or more polymers or polymer fragments. The polymeric reagent or fragment may optionally also contain one or more other fragments, which, in certain embodiments, are selected from the group consisting of:

• C _1-50 alkyl, C _2-50 alkenyl, C _2-50 alkynyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl ,

• branch points such as -CR<, >C< or -N<, and

• connections selected from a group consisting of

Where

dotted lines indicate attachment to the remainder of the fragment or reagent, and

-R, and -R ^{a are} independently selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2- methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl, and

wherein the moieties and bonds are optionally further substituted.

In certain embodiments, the polymeric reactant or moiety may also optionally contain one or more other moieties, which in certain embodiments are selected from the group consisting of:

• C _1-50 alkyl, C _2-50 alkenyl, C _2-50 alkynyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl , And

• connections selected from a group consisting of

Where

dotted lines indicate attachment to the remainder of the fragment or reagent, and

-R, and -R ^{a are} independently selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2- methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl, and

wherein the moieties and bonds are optionally further substituted.

One of ordinary skill in the art understands that the polymerization products obtained from a polymerization reaction do not all have the same molecular weight, but rather have a molecular weight distribution. Therefore, molecular weight ranges, molecular weights, number of monomers in a polymer, and number of monomers in a polymer ranges, as used herein, refer to number average molecular weight and average number of monomers, i.e. to the arithmetic average of the molecular weight of the polymer or polymer fragment and the arithmetic average of the number of monomers of the polymer or polymer fragment.

Accordingly, in a polymer fragment containing “x” monomer units, any integer given for “x” therefore corresponds to the arithmetic average of the number of monomers. Any range of integers given for “x” provides a range of integers within which the arithmetic average of the number of monomers lies. An integer for “x” given as “about x” means that the arithmetic averages of the monomer numbers lie in the integer range x +/- 25%, such as x +/-20 and, such as x +/- 10%.

As used herein, the term “number average molecular weight” means the usual arithmetic average of the molecular weights of individual polymers.

As used herein, the term “PEG-based” in relation to a moiety or reagent means that said moiety or reagent contains PEG. Preferably, the PEG fragment or reagent contains at least 10% (w/w) PEG, such as at least 20% (w/w) PEG, such as at least 30% (w/w) PEG. wt.) PEG, such as at least 40% (wt./wt.) PEG, such as at least 50% (wt./wt.) PEG, such as at least 60 (wt./wt.) PEG , such as at least 70% (w/w) PEG, such as at least 80% (w/w) PEG, such as at least 90% (w/w) PEG, such as for example at least 95% (w/w) PEG. The remaining percentages by weight of the PEG moiety or reagent are other moieties, such as those selected from the group consisting of:

• C _1-50 alkyl, C _2-50 alkenyl, C _2-50 alkynyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl , And

• Links selected from a group consisting of

Where

dotted lines indicate attachment to the remainder of the fragment or reagent, and

-R and -R ^{a are} independently selected from the group consisting of -H and C _1-6 alkyl, and

wherein the moieties and bonds are optionally further substituted.

The terms “poly(alkylene glycol) based”, “poly(propylene glycol) based” and “hyaluronic acid based” are used accordingly.

The term “interrupted” means that the fragment is inserted between two carbon atoms or - if the insertion is at one end of the fragment - between a carbon atom or heteroatom and a hydrogen atom.

As used herein, the term “C _1-4 alkyl,” alone or in combination, means a straight-chain or branched alkyl moiety having 1 to 4 carbon atoms. When present at the end of the molecule, examples of straight or branched C _1-4 alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. When two moieties of a molecule are linked by a C _1-4 alkyl, examples of such C _1-4 alkyl groups are -CH ₂ -, -CH ₂ -CH ₂ -, -CH(CH ₃ )-, -CH ₂ -CH ₂ -CH ₂ -, -CH(C ₂ H ₅ )-, -C(CH ₃ ) ₂ -. Each hydrogen of the C _1-4 alkyl carbon may optionally be replaced by a substituent as defined above. Optionally, the C _1-4 alkyl may be interrupted by one or more moieties, as defined below.

As used herein, the term “C_1-6 "alkyl" alone or in combination means a straight or branched alkyl moiety having from 1 to 6 carbon atoms. If present at the end of the molecule, examples of straight or branched C_1-6 alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl. When two fragments of a molecule are bonded C_1-6 alkyl group, examples of such C_1-6 alkyl groups are -CH₂-, -CH₂-CH₂-, -CH(CH₃)-, -CH₂-CH₂-CH₂-, -CH(C₂H₅)- and C(CH₃)₂-. Each hydrogen atom at C_1-6 carbon atom may optionally be replaced by a substituent as defined above. Optional, C_1-6 alkyl may be interrupted by one or more moieties, as defined below.

Accordingly, “C_1-10 alkyl", "C_1-20 alkyl" or "C_{1- 50} alkyl" means an alkyl chain having from 1 to 10, from 1 to 20 or from 1 to 50 carbon atoms, respectively, where each hydrogen atom at C_1-10, C_1-20 or C_1-50 carbon atom may optionally be replaced by a substituent as defined above. Optional, C_1-10 or C_1-50 alkyl may be interrupted by one or more moieties, as defined below.

As used herein, the term “C_2-6 "alkenyl" by itself or in combination means a straight-chain or branched hydrocarbon moiety containing at least one carbon-carbon double bond having from 2 to 6 carbon atoms. If present at the end of the molecule, examples are CH=CH₂, -CH=CH-CH₃, -CH₂-CH=CH₂, -CH=CHCH₂-CH₃ and -CH=CH-CH=CH₂. When two fragments of a molecule are bonded C_2-6 alkenyl group, then an example of such a C_2-6 alkenyl is -CH=CH-. Each hydrogen atom C_2-6 the alkenyl moiety may optionally be substituted with a substituent as defined above. Optional, C_2-6 alkenyl may be interrupted by one or more moieties, as defined below.

Accordingly, the terms “C_2-10 alkenyl", "C_2-20 alkenyl" or "C_2-50 "alkenyl" alone or in combination means a straight or branched hydrocarbon moiety containing at least one carbon-carbon double bond having from 2 to 10, from 2 to 20 or from 2 to 50 carbon atoms, respectively. Each hydrogen atom C_2-10 alkenyl, C_2-20 alkenyl or C_2-50 the alkenyl group may optionally be substituted with a substituent as defined above. Optional, C_2-10 alkenyl, C_2-20 alkenyl or C_2-50 alkenyl may be interrupted by one or more moieties, as defined below.

As used herein, the term “C _2-6 alkynyl,” alone or in combination, means a straight-chain or branched hydrocarbon moiety containing at least one carbon-carbon triple bond having from 2 to 6 carbon atoms. If present at the end of the molecule, examples are -C≡CH, -CH ₂ -C≡CH, CH ₂ -CH ₂ -C≡CH and CH ₂ -CC-CH ₃ . When two moieties of a molecule are linked by an alkynyl group, then an example is -C≡C-. Each hydrogen atom of the C _2-6 alkynyl group may optionally be replaced by a substituent as defined above. Optionally, one or more double bonds may be present. Optionally, the C _2-6 alkynyl may be interrupted by one or more moieties, as defined below.

Accordingly, as used herein, the terms “C _2-10 alkynyl,” “C _2-20 alkynyl,” and “C _2-50 alkynyl,” alone or in combination, mean a straight-chain or branched hydrocarbon moiety containing at least one a carbon-carbon triple bond having 2 to 10, 2 to 20, or 2 to 50 carbon atoms, respectively. Each hydrogen atom of a C _2-10 alkynyl, C _2-20 alkynyl or C _2-50 alkynyl group may optionally be replaced by a substituent as defined above. Optionally, one or more double bonds may be present. Optionally, C _2-10 alkynyl, C _2-20 alkynyl, or C _2-50 alkynyl may be interrupted by one or more moieties, as defined below.

As mentioned above, C₁₄ alkyl, C_1-6 alkyl, C_{1- 10} alkyl, C_1-20 alkyl, C_{1- 50} alkyl, C_2-6 alkenyl, C_2-10 alkenyl, C_2-20 alkenyl, C_2-50 alkenyl, C_2-6 alkynyl, C_2-10 alkynyl, C_2-20 alkenyl or C_2-50 the alkynyl may optionally be interrupted by one or more moieties, which are preferably selected from the group consisting of

Where

dotted lines indicate attachment to the remainder of the fragment or reagent, and

-R and -R ^{a are} independently selected from the group consisting of -H and C _1-6 alkyl.

As used herein, the term "C _3-10 cycloalkyl" means a cyclic alkyl chain having from 3 to 10 carbon atoms, which may be saturated or unsaturated, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. Each C _3-10 hydrogen atom of the cycloalkyl carbon may be replaced by a substituent as defined above. The term "C _3-10 cycloalkyl" also includes bridged bicycles such as norbornane or norbornene.

The term “8-30 membered carbopolycyclyl” or “8-30 membered carbopolycyclyl” means a cyclic moiety of two or more rings with 8 to 30 ring atoms, where two adjacent rings share at least one ring atom, and which may contain up to the maximum number of double bonds (aromatic or non-aromatic ring that is fully saturated, partially unsaturated or unsaturated). In one embodiment, an 8-30 membered carbopolycyclyl means a cyclic moiety of two, three, four or five rings. In another embodiment, the 8-30 membered carbopolycyclyl is a cyclic moiety of two, three or four rings.

As used herein, the term "3-10 membered heterocyclyl" or "3-10 membered heterocycle" means a ring with 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms, which may contain up to a maximum number of double bonds (an aromatic or non-aromatic ring that is fully saturated, partially unsaturated or unsaturated), wherein at least one ring atom to four ring atoms are replaced by a heteroatom selected from the group consisting of sulfur (including -S(O )-, -S(O) _2- ), oxygen and nitrogen (including =N(O)- ), and where the ring is connected to the remainder of the molecule through a carbon or nitrogen atom. Examples of 3-10 membered heterocycles include, but are not limited to, aziridine, oxirane, tyrane, azirine, oxirene, thiirene, azetidine, oxetane, thietan, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepane, azepine or homopiperazine. Each hydrogen atom of a 3-10 membered heterocyclyl or 3-10 membered heterocyclic group may be replaced by a substituent.

As used herein, the term "8- to 11-membered heterobicyclyl" or "8 to 11-membered heterobicycle" means a two-ring heterocyclic moiety with 8 to 11 ring atoms, where at least one ring atom is shared between two rings and which contains up to a maximum number of double bonds (an aromatic or non-aromatic ring that is fully saturated, partially unsaturated or unsaturated), wherein at least one ring atom to six ring atoms are replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O) _2- ), oxygen and nitrogen (including =N(O)- ), and where the ring is bonded to the remainder of the molecule through a carbon or nitrogen atom. Examples of 8-11 membered heterobicycles are indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, hydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline , benzazepine, purine or pteridine. The term 8-11 membered heterobicycle also includes two-ring spiro structures such as 1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles such as 8-azabicyclo[3.2.1]octane. Each hydrogen atom of an 8- to 11-membered heterobicyclyl or an 8 to 11-membered heterobicycle can be replaced by a substituent.

Similarly, the term “8-30 membered heteropolycyclyl” or “8-30 membered heteropolycycle” means a heterocyclic moiety of more than two rings with 8 to 30 ring atoms, such as three, four or five rings, where two adjacent rings are separated by at least one ring atom, and which may contain up to a maximum number of double bonds (an aromatic or non-aromatic ring that is fully saturated, partially unsaturated or unsaturated), where at least one ring atom to 10 ring atoms are replaced by a heteroatom, selected from the group consisting of sulfur (including -S(O)-, -S(O) _2- ), oxygen and nitrogen (including =N(O)- ), and wherein the ring is bonded to the remainder of the molecule via a carbon or nitrogen atom .

It is clear that the phrase “the pair R ^x /R ^y combines together with the atom to which they are attached to form a C _3-10 cycloalkyl or 3-10 membered heterocyclyl” refers to a fragment of the structure

Means that R ^x and R ^y form the following structure:

,

where R represents C _3-10 cycloalkyl or 3-10 membered heterocyclyl.

It is also clear that the phrase “the pair R ^x /R ^y joins together with the atom to which they are attached to form ring A” refers to a fragment of the structure

means that R ^x and R ^y form the following structure:

.

It is clear that the phrase “-R ¹ and the adjacent -R ² form a carbon-carbon bond, provided that n is selected from the group consisting of 1, 2, 3 and 4” with respect to a fragment of the structure:

Means that, for example, when n is 1, -R ¹ and the adjacent -R ² form the following structure:

,

and, if, for example, n is 2, R ¹ and the neighboring R ² form the following structure:

,

where the wavy bond means that -R ^1a and -R ^2a can be either on the same side of the double bond, i.e. in the cis configuration, or on opposite sides of the double bond, i.e. in the trans configuration, and where the term "adjacent" means that -R ¹ and -R ² are attached to carbon atoms that are located one after the other.

It is clear that the phrase “two adjacent -R ² form a carbon-carbon bond, provided that n is selected from the group consisting of 2, 3 and 4” in relation to a fragment of the structure:

Means that, for example, when n is 2, two adjacent -R ² form the following structure:

,

where the wavy bond means that each -R ^2a can be either on the same side of the double bond, i.e. in the cis configuration, or on opposite sides of the double bond, i.e. in the trans configuration, and where the term "adjacent" means that - R ² are attached to carbon atoms, which are located one after the other.

It is clear that the “N” in the phrase “ - electronic paranor heteroaromatic N” refers to nitrogen.

It is clear that the “N ⁺ ” in the phrases “electron-donating heteroaromatic N ⁺ -containing moiety” and “addition to N ⁺ in -D ⁺ ” refers to the positively charged nitrogen atom.

In the context of the present invention, the term "halogen" means fluorine, chlorine, bromine or iodine. In certain embodiments, the halogen is fluorine or chlorine.

In the context of the present invention, the term “alkali metal ion” refers to Na ⁺ , K ⁺ , Li ⁺ , Rb ⁺ and Cs ⁺ . In certain embodiments, “alkali metal ion” refers to Na ⁺ , K ⁺ and Li ^{+ .}

In the context of the present invention, the term “alkaline earth metal ion” refers to Mg ²⁺ , Ca ²⁺ , Sr ²⁺ and Ba ²⁺ . In certain embodiments, the alkaline earth metal ion is Mg ²⁺ or Ca ²⁺ .

As used herein, the term “functional group” means a group of atoms that can react with other groups of atoms. Exemplary functional groups are carboxylic acid, primary amine, secondary amine, tertiary amine, maleimide, thiol, sulfonic acid, carbonate, carbamate, hydroxyl, aldehyde, ketone, hydrazine, isocyanate, isothiocyanate, phosphoric acid, phosphonic acid, haloacetyl, alkyl halide, acryloyl , aryl fluoride, hydroxylamine, disulfide, sulfonamides, sulfuric acid, vinyl sulfone, vinyl ketone, diazoalkane, oxirane and aziridine.

In case the compounds of the present invention contain one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically acceptable salts. Thus, the compounds of the present invention containing acidic groups can be used in accordance with the present invention, for example, in the form of alkali metal salts, alkaline earth metal salts or ammonium salts. More specific examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as ethylamine, ethanolamine, triethanolamine, amino acids and quaternary ammonium salts such as tetrabutylammonium or cetyltrimethylammonium. Compounds of the present invention containing one or more basic groups, i.e. groups that can be protonated may be present and used according to the present invention in the form of their addition salts with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene sulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid , pivaloic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, trifluoroacetic acid and others acids known to those skilled in the art. Conversions of a basic group to a cation, such as alkylation of an amine group to produce a positively charged ammonium group and corresponding salt counterion, are known to those skilled in the art. If the compounds of the present invention simultaneously contain acidic and basic groups, the present invention also includes, in addition to the salt forms mentioned, internal salts or betaines (zwitterions). The corresponding salts can be prepared by conventional methods known to one skilled in the art, for example by contacting these compounds with an organic or inorganic acid or base in a solvent or dispersant, or by anionic or cationic exchange with other salts. The present invention also includes all salts of the compounds of the present invention which, due to their low physiological compatibility, are not directly suitable for use in pharmaceutical preparations, but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.

The term "pharmaceutically acceptable" means a substance that is not harmful when administered to a patient and, in certain embodiments, has been approved by a regulatory authority such as EMEA (Europe) and/or FDA (USA) and/or any other national regulatory authority for use in animals , such as in relation to a person.

As used herein, the term "excipient" refers to a diluent, excipient or carrier with which a therapeutic agent, such as a drug or prodrug, is administered. Such pharmaceutical excipient may be a sterile liquid such as water and oils, including liquids of petroleum, animal, vegetable or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred excipient when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are the preferred excipients when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol are preferably used as liquid excipients for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, milk powder, glycerin, propylene glycol, hyaluronic acid, water, ethanol and the like. The pharmaceutical composition may, if desired, also contain small amounts of wetting or emulsifying agents, pH buffering agents, such as, for example, acetate, succinate, tris, carbonate, phosphate, HEPES (4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, MES ( 2-(N-morpholine)ethanesulfonic acid) or may contain detergents such as Tween, poloxamers, poloxamines, CHAPS, Igepal, or amino acids such as, for example, glycine, lysine or histidine. Such pharmaceutical compositions may be in the form of solutions, suspensions, etc. emulsions, tablets, pills, capsules, powders, sustained release preparations and the like. The pharmaceutical composition may be in the form of a suppository, with conventional binders and excipients such as triglycerides. The oral preparation may contain conventional excipients such as pharmaceuticals. brands of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions contain a therapeutically effective amount of a drug or drug moiety, together with a suitable amount of an excipient, so as to provide a form suitable for administration to a patient. The composition must be appropriate for the route of administration.

As used herein, the term “peptide” refers to a chain of at least 2 and up to 50 amino acid monomer units, also referred to as “amino acid residues,” linked by peptide (amide) bonds. The amino acid monomers may be selected from the group consisting of proteinogenic amino acids and non-proteinogenic amino acids and may be D- or L-amino acids. The term "peptide" also includes peptidomimetics such as peptoids, beta-peptides, cyclic peptides and depsipeptides, and covers such peptidomimetic chains containing up to and including 50 monomeric fragments.

In the context of the present invention, the term "protein" refers to a chain consisting of more than 50 amino acid monomer fragments, which may also be referred to as "amino acid residues", linked by peptide bonds, in which preferably no more than 12,000 amino acid monomers linked by peptide bonds, such as no more than no more than 10,000 amino acid monomer fragments, no more than 8,000 amino acid monomer fragments, no more than 5,000 amino acid monomer fragments, or no more than 2,000 amino acid monomer fragments.

In general, the terms “comprises” or “comprising” also cover “consists of” or “consisting of”.

When a controlled release water-insoluble PRRA is administered interstitially, at least 25% of the amount of administered PRRA remains locally in such tissue after 3 days. The term “amount of administered PRRA” in that context refers to the total combined amount of free PRRA that has been released from the water-insoluble controlled-release PRRA and the PRRA still incorporated or covalently conjugated in the water-insoluble controlled-release PRRA.

In the context of the present invention, the term "local" refers to the area limited by the administered tissue or organ, in particular, the total volume around the injection site of controlled release PRRA within a 3-fold radius (r) from the injection site in any direction, where r is distance in centimeters (cm) calculated from the volume (V) of administered water-insoluble controlled-release PRRA in cubic centimeters (cm ³ ) according to the spheroid equation . For example, if 0.5 cm ³ of water-insoluble controlled release PRRA is injected into a given tissue, a sample aimed at capturing all of the injected material containing a total volume within 1.47 cm in any direction including the injection site will be measured for drug levels, i.e. amount of PRRA present.

Suitable measurements are known to one skilled in the art. To obtain the total amount of both free PRRA that has been released from the water-insoluble controlled-release PRRA and to measure the PRRA still incorporated or covalently conjugated in the water-insoluble controlled-release PRRA, the PRRA still needs to be released first. This can be done using suitable techniques, such as incubation under conditions that accelerate release, such as increasing temperature or changing pH. To separately measure free and conjugated PRRA in tissue, the tissue can be first weighed and then dissociated in a manner that does not disrupt the PRRA conjugate and allows free PRRA to be separated from the controlled release compound for measurement, and then, separately, PRRA can be released from the compound controlled release and measured.

In certain embodiments, PRRA is released from a water-insoluble controlled-release conjugate with a half-life under physiological conditions (aqueous buffer, pH 7.4, 37°C) of at least 3 days, such as at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 15 days, at least 17 days , at least 20 days or at least 25 days.

In certain embodiments, the water-insoluble controlled-release PRRA comprises a plurality of PRRA moieties covalently and reversibly conjugated to a carrier moiety, particularly to an insoluble carrier moiety.

At least 25% of the total amount of PRRA administered remains in such tissue after 3 days, such as at least 30%, at least 35%, at least 40%, or at least 45%. It is clear that the total amount of PRRA present in the tissue after 3 days does not exceed 100%. In certain embodiments, at least 25% of the total amount of PRRA administered remains in such tissue after 7 days, such as at least 30%, at least 35%, at least 40%, or at least 45%. In certain embodiments, at least 25% of the total amount of PRRA administered remains in such tissue after 10 days, such as at least 30%, at least 35%, at least 40%, or at least 45%. In certain embodiments, at least 25% of the total amount of PRRA administered remains in such tissue after 14 days, such as at least 30%, at least 35%, at least 40%, or at least 45%. In certain embodiments, at least 25% of the total amount of PRRA administered remains in such tissue after 21 days, such as at least 30%, at least 35%, at least 40%, or at least 45%. In certain embodiments, at least 25% of the total amount of PRRA administered remains in such tissue after 28 days, such as at least 30%, at least 35%, at least 40%, or at least 45%. In certain embodiments, at least 25% of the total amount of PRRA administered remains in such tissue after 35 days, such as at least 30%, at least 35%, at least 40%, or at least 45%. In certain embodiments, at least 25% of the total amount of PRRA administered remains in such tissue after 42 days, such as at least 30%, at least 35%, at least 40%, or at least 45%. In certain embodiments, at least 25% of the total amount of PRRA administered remains in such tissue after 49 days, such as at least 30%, at least 35%, at least 40%, or at least 45%. In certain embodiments, at least 25% of the total amount of PRRA administered remains in such tissue after 56 days, such as at least 30%, at least 35%, at least 40%, or at least 45%.

In certain embodiments, a water-insoluble controlled-release PRRA is used as the sole agent. In certain embodiments, the water-insoluble controlled-release PRRA is administered in combination with one or more additional drugs, wherein it or more additional drugs are administered either together with the water-insoluble controlled-release PRRA or in separate administration, wherein such separate administration occurs at time intervals ranging from 1 minute to 30 days either before or after administration of a water-insoluble controlled-release pattern recognition receptor agonist. In certain embodiments, the time difference is in the range of 1 minute to 1 hour. In certain embodiments, the time difference is in the range of 1 hour to 24 hours. In certain embodiments, the time difference is in the range of 2 days to 7 days. In certain embodiments, the time difference is in the range of 1 week to 2 weeks. In certain embodiments, the time difference is in the range of 2 weeks to one month.

If the interstitial administration is intratumoral administration, antitumor activity is observed in certain embodiments 7 days after such intratumoral administration of the controlled release PRRA. It is understood that such antitumor activity can only be observed in animals whose tumors have not previously been removed for drug level measurements, and that this requires the presence of at least a second comparable tumor in the same or a different animal 7 days after interstitial administration. In certain embodiments, such antitumor activity is observed 10 days after intratumoral administration of controlled release PRRA. In certain embodiments, such antitumor activity is observed 14 days after intratumoral administration of controlled release PRRA. In certain embodiments, such antitumor activity is observed 21 days after intratumoral administration of controlled release PRRA.

The tissue for interstitial administration may be selected from the group consisting of healthy or diseased tissues originating from lymphoid tissue, such as lymph node, tonsil, spleen and bone marrow; gastrointestinal tract, for example, salivary gland, oral mucosa, esophagus, stomach, duodenum, small intestine, colon and rectum; tissues of the genitourinary system, for example, fallopian tube, vagina, cervix, uterus, endometrium, ovaries, testes, prostate gland, epididymis and seminal vesicle; endocrine tissues, such as the thyroid gland, parathyroid gland and adrenal glands; tissues of the eye; tissues of the oral cavity; ear tissues; breast tissue; skin tissues; muscle tissues such as heart, skeletal and smooth muscles; lung tissues; liver tissues; heart tissues; vascular tissue; tissues of the central nervous system; tissues of the peripheral nervous system; spinal tissues; brain tissue; kidney tissues; bladder tissues; tissues of the nasopharynx; bronchus tissues; neck tissues; pancreatic tissues; gallbladder tissues; synovial tissues; cartilage tissues; connective tissue; fascia tissues; pleural tissues; adipose tissue; and peritoneal tissues.

In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased lymph node tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased colon tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased cervical tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased uterine tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased ovarian tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased prostate tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased breast tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased skin tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased lung tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased liver tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased brain tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased kidney tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased bladder tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased neck tissue. In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is healthy or diseased pancreatic tissue.

In certain embodiments, treatment of a cell proliferation disorder may, in addition to administering a water-insoluble controlled-release PRRA, also include administering at least one anticancer therapeutic agent, such as systemic immunotherapy. Examples of the at least one anticancer therapeutic agent are as disclosed elsewhere herein for one or more additional drugs that may, in certain embodiments, be present in the pharmaceutical composition of the present invention.

In certain embodiments, treatment with water-insoluble PRRA may be initiated before, concurrently with, or after surgical tumor removal or radiation therapy. In addition, such treatment may optionally be combined with at least one other anticancer therapeutic agent, such as systemic immunotherapy. Examples of the at least one anticancer therapeutic agent are as disclosed elsewhere herein for one or more additional drugs that may, in certain embodiments, be present in the pharmaceutical composition of the present invention. In certain embodiments, the insoluble PRRA is administered intratumorally before, concurrently with, or after combination with at least one systemic immunotherapy, prior to radiation therapy, or surgical removal of the injected tumor. In certain embodiments, the insoluble PRRA is administered intratumorally before, concurrently with, or after combination with at least one systemic immunotherapy, following radiation therapy, or surgical removal of the injected tumor. In certain embodiments, the insoluble PRRA is administered to the draining lymph nodes of the tumor before, simultaneously with, or after surgical removal of the tumor or radiation therapy. In certain embodiments, the insoluble PRRA is administered to the draining lymph nodes of a tumor before, concurrently with, or after a combination with at least one systemic immunotherapy and before, simultaneously with, or after surgical resection of the tumor or radiotherapy. In certain embodiments, the insoluble PRRA is administered intratumorally to metastatic tumors that may occur before or after surgical removal or radiotherapy of the primary tumor. In certain embodiments, insoluble PRRA is administered intratumorally to metastatic tumors that may occur before, concurrently with, or after combination with at least one systemic immunotherapy and before, concurrently with, or after surgical removal or radiotherapy of the primary tumor. In certain embodiments, at least one systemic therapy is administered prior to surgical removal of the tumor or radiotherapy, followed by intratumoral administration of water-insoluble PRRA. In certain embodiments, intratumoral administration of insoluble PRRA is performed first, followed by treatment in combination with at least one systemic therapy. In certain embodiments, at least one systemic therapy is administered prior to surgical removal of the tumor, followed by administration of water-insoluble PRRA into the tumor layer after surgery or by intratumoral administration into the tumor not removed by surgery.

In certain embodiments, interstitial administration may be a single, controlled-release injection of water-insoluble PRRA into tissue as described above. In certain embodiments, interstitial administration is accomplished through repeated interstitial administration. In certain embodiments, such repeated interstitial administration is performed into the same tissue at the same or different sites of administration within said tissue. In certain embodiments, repeat interstitial administration may be performed in a different tissue. Such other tissues could be, for example, other tumors. In the case of repeated interstitial administration, the time interval between two interstitial administrations can range from 1 minute to 28 weeks.

In certain embodiments, the tissue into which the water-insoluble controlled-release PRRA is administered is cancer tissue or one or more draining lymph nodes associated with cancer tissue. A suitable cancer may be selected from the group consisting of solid tumors and lymphomas.

In certain embodiments, intratumoral administration of water-insoluble controlled-release PRRA at dose X causes greater than 1.5-fold, such as greater than 1.5-fold, 1.7-fold, 2-fold, 2.2-fold, 2.5-fold, 3-fold. a 3.5-fold, 4-fold, or 5-fold increase in the percentage of antigen-presenting cells in tumor draining lymph nodes, 7 days after the indicated administration, compared with intratumoral administration of a dose of 0.5 to 1.5 X the corresponding free PRRA.

In certain embodiments, intratumoral administration of water-insoluble controlled-release PRRA at dose X causes greater than 1.5-fold, such as greater than 1.5-fold, 1.7-fold, 2-fold, 2.2-fold, 2.5-fold, 3-fold. a 3.5-fold, 4-fold, or 5-fold increase in MHCII expression on a subset of antigen-presenting cells in tumor draining lymph nodes, 7 days after the indicated administration, compared with intratumoral administration of a dose of 0.5 to 1.5 X the corresponding free PRRA.

In certain embodiments, interstitial administration of a water-insoluble controlled-release PRRA results in local inflammation. In one embodiment, the local inflammation is at least 1.5-fold, such as at least 1.7-fold, at least 2-fold, at least 2.2-fold, at least 2.5-fold, at least 3-fold , at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 7 times, at least 8-fold, at least 9-fold, or at least 10-fold increase in the levels of at least four proteins selected from the group consisting of TNFα, IL-1β, IL-10, IL-6, MCP- 1, MIP-1α, MIP-1β, MIP-2α, MIP-3α, IP-10, and KC, in certain embodiments selected from the group consisting of TNFα, IL-1β, IL-6, MCP-1, MIP- 1α, MIP-1β, MIP-2α, IP-10, and KC, and in certain embodiments selected from the group consisting of IL-1β, IL-6, MCP-1, MIP-1α, MIP-1β, MIP-2α , IP-10 and KC, compared with parent tissue, as measured 3 days after interstitial administration. This does not mean that local inflammation lasts only three days. In fact, local inflammation may last significantly longer, such as at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, etc. at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 20 days, at least 30 days, or longer. Accordingly, measurement of proteins selected from the group consisting of TNFα, IL-1β, IL-10, IL-6, MCP-1, MIP-1α, MIP-1β, MIP-2α, MIP-3α, IP-10 and KC , according to certain embodiments selected from the group consisting of TNFα, IL-1β, IL-6, MCP-1, MIP-1α, MIP-1β, MIP-2α, IP-10 and KC, and according to certain embodiments selected from group consisting of IL-1β, IL-6, MCP-1, MIP-1α, MIP-1β, MIP-2α, IP-10 and KC can also be performed at later time points, such as at 4 days after interstitial injection, at 5 days after interstitial injection, at 6 days after interstitial injection, at 7 days after interstitial injection, at 8 days after interstitial injection, at 9 days after interstitial injection, at 10 days after interstitial injection administration, at 11 days after interstitial administration, at 12 days after interstitial administration, at 13 days after interstitial administration, at 14 days after interstitial administration, at 20 days after interstitial administration, at 30 days after interstitial administration, or even later than 30 days after interstitial administration.

MCP-1 is also known as CCL2, MIP-1α is also known as CCL3, MIP-1β is also known as CCL4, MIP-2α is also known as MIP-2 and CXCL2, MIP-3α is also known as CCL20, IP-10 is also known as CXCL10 and KC are also known as GROα and CXCL1. CCL5 is also known as RANTES. CSF-2 is also known as GM-CSF. CCL8 is also known as MCP-2.

It is clear that TNFα, IL-1β, IL-10, IL-6, MCP-1, MIP-1α, MIP-1β, MIP-2α, MIP-3α, IP-10 and KC are human proteins, and what if insoluble in water controlled release PRRA is administered to a non-human species, protein levels of the corresponding homologous proteins must be measured.

Protein levels can be measured by methods known to one skilled in the art. One method comprises the step of taking a sample of at least 0.025 g of tissue, such as at least 0.025 g, at least 0.05 g, at least 0.075 g, at least 0.1 g of tissue, from an area that is within two times the radius ( r ) from the injection site in any direction, where r is the distance in centimeters (cm) calculated from the volume (V) of the injected conjugate of the present invention in cubic centimeters (cm ³ ) according to the spheroid equation . Protein can be isolated from such a sample using standard methods known to those skilled in the art, such as disruption/homogenization of the tissue sample and cell lysis for protein analysis. Levels of at least four proteins selected from the group consisting of TNFα, IL-1β, IL-10, IL-6, MCP-1, MIP-1α, MIP-1β, MIP-2α, MIP-3α, IP-10 and KC, according to certain embodiments selected from the group consisting of TNFα, IL-1β, IL-6, MCP-1, MIP-1α, MIP-1β, MIP-2α, IP-10 and KC, and according to certain embodiments selected from the group consisting of IL-1β, IL-6, MCP-1, MIP-1α, MIP-1β, MIP-2α, IP-10 and KC, then measured from such a protein sample using standard methods known to those skilled in the art. this field of technology, such as enzyme-linked immunosorbent assay (ELISA).

In another embodiment, the local inflammation is at least 1.5-fold, such as at least 1.8-fold, at least 2-fold, at least 2.2-fold, at least 2.5-fold, at least 2.7-fold multiple, at least 3x, at least 3.5x, at least 4x, at least 4.5x, at least 5x, at least 5.5x, at least 6x at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold increase in the expression levels of at least four mRNAs selected from the group consisting of TNFα , IL1A , IL1B , IL10 , IL6 , IL12B , CCL2 , CCL8 , CCL3 , CCL4 , CXCL2 , CCL20 , CSF2 , pan-IFNA subtype members, IFNB1 , IL18 , CCL5 , CXCL10 , and CXCL1 , in certain embodiments selected from the group consisting of TNF , IL1B , IL10 , IL6 , CCL2 , CCL3 , CCL4 , CXCL2 , CSF2 , IL18 , CCL5 , CXCL10 , and CXCL1, and in certain embodiments selected from the group consisting of TNF , IL1B , IL6 , CCL2 , CCL3 , CCL4 , CXCL2 , CCL5 , CXCL10 , and CXCL1 , compared to parental tissue, as measured 3 days after interstitial administration. This does not mean that local inflammation lasts only three days. In fact, local inflammation may last significantly longer, such as for at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days , at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 20 days, at least 30 days or longer. Accordingly, measuring the expression levels of at least four mRNAs selected from the group consisting of TNF , IL1A , IL1B , IL10 , IL6 , IL12B , CCL2 , CCL8 , CCL3 , CCL4 , CXCL2 , CCL20 , CSF2 , pan-IFNA subtype members, IFNB1 , IL18 , CCL5 , CXCL10 , and CXCL1 , according to certain embodiments selected from the group consisting of TNF , IL1B , IL10 , IL6 , CCL2 , CCL3 , CCL4 , CXCL2 , CSF2 , IL18 , CCL5 , CXCL10 , and CXCL1, and according to certain embodiments selected from the group consisting of TNF , IL1B , IL6 , CCL2 , CCL3 , CCL4 , CXCL2 , CCL5 , CXCL10 and CXCL1 , can also be performed at later time points, such as at 4 days after interstitial administration, at 5 days after interstitial injection, at 6 days after interstitial injection, at 7 days after interstitial injection, at 8 days after interstitial injection, at 9 days after interstitial injection, at 10 days after interstitial injection, at 11 days after interstitial injection administration, at 12 days after interstitial administration, at 13 days after interstitial administration, at 14 days after interstitial administration, at 20 days after interstitial administration, at 30 days after interstitial administration, or even later than 30 days after interstitial administration.

It is clear that TNF , IL1A , IL1B , IL10 , IL6 , IL12B , CCL2 , CCL8 , CCL3 , CCL4 , CXCL2 , CCL20 , CSF2 , pan- IFNA subtype members, IFNB1 , IL18 , CCL5 , CXCL10 and CXCL1 are human genes, and if a water-insoluble controlled-release pattern recognition receptor agonist is administered to a non-human species, and the expression of the corresponding homologous genes in the non-human species is measured. For the mouse, the corresponding homologs are Tnf , Il1a , Il1b , Il10 , Il6 , Il12b , Ccl2 , Ccl8 , Ccl3 , Ccl4 , Cxcl2 , Ccl20 , Csf2 , Ifna (many subtype members), Ifnb1 , Il18 , Ccl5 , Cxcl10 and Cxcl1 .

Local inflammation mRNA levels can be measured by methods known to one skilled in the art. One method comprises the step of taking a sample of at least 0.025 g of tissue, such as at least 0.025 g, at least 0.05 g, at least 0.075 g, at least 0.1 g of tissue, from an area that is within two times the radius ( r ) from the injection site in any direction, where r is the distance in centimeters (cm) calculated from the volume (V) of the injected conjugate in cubic centimeters ( ^cm3 ) according to the spheroid equation . Total RNA is isolated from such a sample using standard methods known to one skilled in the art, such as by homogenizing/disrupting the tissue sample and lysing the cells for RNA analysis. Expression levels of at least four mRNAs selected from the group consisting of TNF , IL1A , IL1B , IL10 , IL6 , IL12B , CCL2 , CCL8 , CCL3 , CCL4 , CXCL2 , CCL20 , CSF2 , pan-IFNA subtype members, IFNB1 , IL18 , CCL5 , CXCL10 , and CXCL1 , according to certain embodiments selected from the group consisting of TNF , IL1B , IL10 , IL6 , CCL2 , CCL3 , CCL4 , CXCL2 , CSF2 , IL18 , CCL5 , CXCL10 , and CXCL1, and according to certain embodiments selected from the group , consisting of TNF , IL1B , IL6 , CCL2 , CCL3 , CCL4 , CXCL2 , CCL5 , CXCL10 and CXCL1 , then measured in such an RNA sample using standard methods known to one skilled in the art, for example, for example, using quantitative PCR in real time (qPCR).

The cancer may be selected from the group consisting of lip and oral cavity cancer, oral cavity cancer, liver/hepatocellular cancer, primary liver cancer, lung cancer, lymphoma, malignant mesothelioma, malignant thymoma, skin cancer, intraocular melanoma, metastatic squamous cell carcinoma of the neck with primary latency, multiple endocrine neoplasia syndrome in children, fungoid mycosis, cancer of the paranasal sinuses and nasal cavity, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, pheochromoblastoma, pituitary tumor, adrenocortical carcinoma, associated with AIDS malignant neoplasms, anal cancer, bile duct cancer, bladder cancer, brain and nervous system cancer, breast cancer, bronchial adenoma/carcinoid, carcinoid tumor of the gastrointestinal tract, carcinoma, colorectal cancer, endometrial cancer, esophageal cancer , extracranial germ cell tumor, extragonadal gonocytoma, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), kidney/renal cancer cell cancer, laryngeal cancer, pleuropulmonary blastoma, prostate cancer, transitional cell cancer of the renal pelvis and ureter, retinoblastoma, salivary gland cancer, sarcoma, Sezary syndrome, small intestine cancer, genitourinary cancer, malignant thymoma, thyroid cancer, Wilms tumor and cholangiocarcinoma.

In certain embodiments, the cancer is liver/hepatocellular cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is lymphoma. In certain embodiments, the cancer is a malignant thymoma. In certain embodiments, the cancer is skin cancer. In certain embodiments, the cancer is metastatic squamous cell carcinoma of the neck with primary latency. In certain embodiments, the cancer is neuroblastoma. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is bile duct cancer. In certain embodiments, the cancer is bladder cancer. In certain embodiments, the cancer is cancer of the brain and nervous system. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is a carcinoid tumor of the gastrointestinal tract. In certain embodiments, the cancer is a carcinoma. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is extrahepatic bile duct cancer. In certain embodiments, the cancer is gallbladder cancer. In certain embodiments, the cancer is gastric (stomach) cancer. In certain embodiments, the cancer is head and neck cancer. In certain embodiments, the cancer is kidney/renal cell carcinoma. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is a sarcoma. In certain embodiments, the cancer is small bowel cancer. In certain embodiments, the cancer is a cancer of the genitourinary system.

Examples of lung cancer are non-small cell lung cancer and small cell lung cancer. In certain embodiments, the cancer is non-small cell lung cancer. In a certain embodiment, the cancer is small cell lung cancer.

Examples of lymphoma include AIDS-associated lymphoma, primary central nervous system lymphoma, T-cell lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, Hodgkin's lymphoma of pregnancy, non-Hodgkin's lymphoma, follicular lymphoma, marginal zone lymphoma, diffuse large B-cell lymphoma , non-Hodgkin's lymphoma during pregnancy and angioimmunoblastic lymphoma. In certain embodiments, the cancer is cutaneous T-cell lymphoma.

Examples of skin cancer are melanoma and Merkel cell carcinoma. In a certain embodiment, the cancer is skin cancer. In a certain embodiment, the cancer is Merkel cell carcinoma.

Ovarian cancer may be, for example, epithelial cancer, gonocytoma or borderline ovarian tumor. In certain embodiments, the cancer is an epithelial cancer. In certain embodiments, the cancer is a gonocytoma. In certain embodiments, the cancer is a borderline ovarian tumor.

Pancreatic cancer may be, for example, an exocrine tumor/adenocarcinoma, pancreatic endocrine tumor (PET) or neuroendocrine tumor (NET). In certain embodiments, the cancer is an exocrine tumor/adenocarcinoma. In certain embodiments, the tumor is a pancreatic endocrine tumor. In certain embodiments, the cancer is a neuroendocrine tumor

Examples of cancers of the brain and nervous system are medulloblastoma, such as childhood medulloblastoma, astrocytoma, ependymoma, neuroectodermal tumors, neuroma, meningioma, eosinophilic adenoma and glioma. In a certain embodiment, the cancer is medulloblastoma. In certain embodiments, the cancer is childhood medulloblastoma. In certain embodiments, the cancer is an astrocytoma. In certain embodiments, the cancer is a neuroectodermal tumor. In certain embodiments, the tumor is a neuroma. In certain embodiments, the cancer is a meningioma. In certain embodiments, the cancer is an eosinophilic adenoma. In certain embodiments, the cancer is a glioma.

The astrocytoma may be selected from the group consisting of giant cell glioblastoma, glioblastoma, secondary glioblastoma, primary adult glioblastoma, primary childhood glioblastoma, oligodendroglial tumor, oligodendroglioma, anaplastic oligodendroglioma, oligoastrocytic tumor, oligoastrocytoma, anaplastic oligoastrocytoma oma, anaplastic astrocytoma, piloid astrocytoma, subependymal giant cell astrocytoma , diffuse astrocytoma, pleomorphic xanthoastrocytoma and cerebellar astrocytoma.

Examples of neuroectodermal tumor are primitive neuroectodermal tumor of the pineal gland and supratentorial primitive neuroectodermal tumor.

The ependymoma may be selected from the group consisting of subependymoma, ependymoma, myxopapillary ependymoma, and anaplastic ependymoma.

Meningioma can be an atypical meningioma or an anaplastic meningioma.

The glioma may be selected from the group consisting of glioblastoma multiforme, paraganglioma, suprantentorial primordial neuroectodermal tumor (sPNET), brainstem glioma, pediatric brainstem glioma, hypothalamic and optic pathway glioma, pediatric hypothalamic and optic pathway glioma, and malignant glioma

Examples of breast cancer are breast cancer during pregnancy, triple negative breast cancer, ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), tubular breast carcinoma, medullary breast carcinoma, mucoid breast carcinoma, papillary carcinoma breast, cribriform breast carcinoma, invasive lobular carcinoma (ILC), inflammatory breast cancer, lobular carcinoma in situ (LCIS), male breast cancer, Paget's disease of the nipple, phyllodes tumor of the breast and metastatic breast cancer. In certain embodiments, the cancer is breast cancer during pregnancy. In certain embodiments, the cancer is triple negative breast cancer. In certain embodiments, the cancer is ductal carcinoma in situ. In certain embodiments, the cancer is invasive ductal carcinoma. In certain embodiments, the cancer is tubular carcinoma of the breast. In certain embodiments, the cancer is medullary carcinoma of the breast. In certain embodiments, the cancer is mucoid carcinoma of the breast. In certain embodiments, the cancer is papillary breast carcinoma. In certain embodiments, the cancer is cribrous carcinoma of the breast. In certain embodiments, the cancer is invasive lobular carcinoma. In certain embodiments, the cancer is inflammatory breast cancer. In certain embodiments, the cancer is lobular carcinoma in situ. In certain embodiments, the cancer is male breast cancer. In certain embodiments, the cancer is Paget's disease of the nipple. In certain embodiments, the cancer is a phyllodes tumor of the breast. In certain embodiments, the cancer is metastatic breast cancer.

Examples of carcinoma are neuroendocrine carcinoma, adrenocortical carcinoma and islet cell carcinoma. In certain embodiments, the cancer is neuroendocrine carcinoma. In certain embodiments, the cancer is adrenocortical carcinoma. In certain embodiments, the cancer is islet cell carcinoma.

Examples of colorectal cancer are colon cancer and rectal cancer. In certain embodiments, the cancer is colon cancer. In certain embodiments, the cancer is rectal cancer.

The sarcoma may be selected from the group consisting of Kaposi's sarcoma, osteosarcoma/malignant fibrous histiocytoma of bone, soft tissue sarcoma, Young family tumors/sarcomas, rhabdomyosarcoma, clear cell sarcoma of the tendon sheath, central chondrosarcoma, central and periosteal chondroma, fibrosarcoma and uterine sarcoma. In certain embodiments, the cancer may be Kaposi's sarcoma. In certain embodiments, the cancer may be osteosarcoma/malignant fibrous histiocytoma of bone. In certain embodiments, the cancer may be a soft tissue sarcoma. In certain embodiments, the cancer may be Young's family tumors/sarcomas. In certain embodiments, the cancer may be rhabdomyosarcoma. In certain embodiments, the cancer may be a clear cell sarcoma of the tendon sheath. In certain embodiments, the cancer may be central chondrosarcoma. In certain embodiments, the cancer may be central and periosteal chondroma. In certain embodiments, the cancer may be a fibrosarcoma. In certain embodiments, the cancer may be a uterine sarcoma.

Examples of genitourinary cancers include testicular cancer, urethral cancer, vaginal cancer, cervical cancer, penile cancer and vulvar cancer. In certain embodiments, the cancer may be testicular cancer. In certain embodiments, the cancer may be urethral cancer. In certain embodiments, the cancer may be vaginal cancer. In certain embodiments, the cancer may be cervical cancer. In certain embodiments, the cancer may be penile cancer. In certain embodiments, the cancer may be vulvar cancer.

A water-insoluble, controlled-release PRRA releases one or more PRRAs. Such a PRRA may be selected from the group consisting of Toll-like receptor agonists, NOD-like receptors, RIG-I-like receptors, cytosolic DNA sensors, STING and aromatic hydrocarbon receptors (AhR).

In certain embodiments, PRRA is a Toll-like receptor agonist. In certain embodiments, PRRA is a NOD-like receptor. In certain embodiments, PRRA is a RIG-I-like receptor. In certain embodiments, PRRA is a cytosolic DNA sensor. In certain embodiments, PRRA is STING. In certain embodiments, the PRRA is an AhR.

Toll-like receptor agonists may be selected from the group consisting of TLR1/2 agonists, such as peptidoglycans, lipoproteins, Pam3CSK4, amplifier, SLP-AMPLIVANT, HESPECTA, ISA101 and ISA201, TLR2 agonists, such as LAM-MS, LPS -PG, LTA- BS, LTA-SA, PGN- BS, PGN- EB, PGN- EK, PGN-SA, CL429, FSL -1, Pam2CSK4, Pam3CSK4, zymosan, CBLB612, SV- 283, ISA204, SMP105, killed by heating Listeria monocytogenes , TLR3 agonists such as poly(A:U), poly(I:C) (poly-ICLC), rintatolimod, apoxime, IPH3102, poly-ICR, PRV300, RGCL2, RGIC.1, Riboxim (RGC100 , RGIC100), Riboxol (RGIC50) and Riboxon, TLR4 agonists such as lipopolysaccharide (LPS), neoceptin-3, glucopyranosyl lipid adjuvant (GLA), GLA-SE, G100, GLA-AF, Clinical Center Reference Endotoxin (CCRE), monophosphorylated lipid A, grass MATA MPL, PEPA10, ONT-10 (PET-Lipid A, oncothyroid), G-305, ALD046, CRX527, CRX675 (RC527, RC590), GSK1795091, OM197MPAC, OM294DP and SAR439794, TLR2/4 agonists, such as lipid A, OM174 and PGN007, TLR5 agonists such as flagellin, entolimod, mobilan, protectan CBLB501, TLR6/2 agonists such as diacylated lipoproteins, diacylated lipopeptides, FSL-1, MALP-2 and CBLB613 agonists TLR7, such as CL264, CL307, imiquimod (R837), TMX-101, TMX-201, TMX-202, TMX-302, gardikquimod, S-27609, 851, UC-IV150, 852A (3M-001, PF- 04878691), loxoribine, polyuridylic acid, GSK2245035, GS-9620, RO6864018 (ANA773, RG7795), RO7020531, isoribine, AN0331, ANA245, ANA971, ANA975, DSP0509, DSP3025 (AZD8 848), GS986, MBS2, MBS5, RG7863 (RO6870868) , sotirimod, SZU101 and TQA3334, TLR8 agonists such as ssPolyUridine, ssRNA40, TL8-506, XG-1-236, VTX-2337 (motolimod), VTX-1463, TMX-302, VTX-763, DN1508052 and GS9688, TLR7/8 agonists, such as CL075, CL097, poly(dT), resiquimod (R-848, VML600, S28463), MEDI9197 (3M-052), NKTR262, DV1001, IMO4200, IPH3201 and VTX1463, TLR9 agonists, such as , CpG DNA, CpG ODN, lefitolimod (MGN1703), SD-101, QbG10, CYT003, CYT003-QbG10, DUK-CpG-001, CpG-7909 (PF-3512676), GNKG168, EMD 1201081, IMO-2125, IMO- 2055, CpG10104, AZD1419, AST008, IMO2134, MGN1706, IRS 954, 1018 ISS, actilon (CPG10101), ATP00001, AVE0675, AVE7279, CMP001, DIMS0001, DIMS9022, DIMS9059, DV230, DV281, EnanDIM, heplisav (V270), kappaproct (DIMS0150), NJP834, NPI503, SAR21609 and Tolamba, and TLR7/9 agonists such as DV1179.

In certain embodiments, PRRA is a TLR1/2 agonist. In certain embodiments, PRRA is a TLR2 agonist. In certain embodiments, PRRA is a TLR3 agonist. In certain embodiments, PRRA is a TLR4 agonist. In certain embodiments, PRRA is a TLR2/4 agonist. In certain embodiments, PRRA is a TLR5 agonist. In certain embodiments, PRRA is a TLR6/2 agonist. In certain embodiments, PRRA is a TLR7 agonist. In certain embodiments, PRRA is a TLR8 agonist. In certain embodiments, PRRA is a TLR7/8 agonist. In certain embodiments, PRRA is a TLR9 agonist. In certain embodiments, PRRA is a TLR7/9 agonist.

Examples of CpG ODNs are ODN 1585, ODN 2216, ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN BW006, ODN D-SL01, ODN 2395, ODN M362 and ODN D-SL03.

NOD-like receptors can be selected from the group consisting of NOD1 agonists, such as C12-iE-DAP, C14-Tri-LAN-Gly, iE-DAP, iE-Lys, and Tri-DAP, and NOD2 agonists, such as for example, L18-MDP, MDP, M-TriLYS, murabutide and N-glycolyl-MDP.

RIG-I-like receptors can be selected from the group consisting of 3p-hpRNA, 5'ppp-DsRNA, 5'ppp RNA (M8), 5'OH kink RNA (CBS-13-BPS), 5'PPP SLR , KIN100, KIN 101, KIN1000, KIN1400, KIN1408, KIN1409, KIN1148, KIN131A, poly(dA:dT), SB9200, RGT100 and Hiltonol.

Cytosolic DNA sensors can be selected from the group consisting of cGAS agonists, dsDNA-EC, G3-YSD, HSV-60, ISD, ODN TTAGGG (A151), poly(dG:dC) and VACV-70.

STING can be selected from the group consisting of MK-1454, ADU-S100 (MIW815), 2'3'-CGAMP, 3'3'-CGAMP, c-di-AMP, c-di-GMP, cAIMP (CL592) , cAIMP difluoro (CL614), cAIM(PS) ₂ difluoro (Rp/Sp) (CL656), 2'2'-CGAMP, 2'3'-CGAM(PS)2 (Rp/Sp), 3'3'- CGAM fluorinated, c-di-AMP fluorinated, 2'3'-C-di-AMP, 2'3'-C-di-AM(PS)2 (Rp,Rp), c-di-GMP fluorinated, 2'3'-C-di-GMP, c-di-IMP, c-di-UMP and DMXAA (vadimezan, ASA404).

The aromatic hydrocarbon receptor (AhR) may be selected from the group consisting of FICZ, ITE and L-cunurenine.

In certain embodiments, the at least one PRRA is imiquimod. In certain embodiments, the at least one PRRA is resiquimod. In certain embodiments, the at least one PRRA is SD-101. In certain embodiments, the at least one PRRA is CMP001.

In certain embodiments, the water-insoluble controlled-release PRRA releases only one type of PRRA, i.e. all released PRRAs are identical. In certain embodiments, the water-insoluble controlled-release PRRA releases more than one type of PRRA, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of PRRA.

According to certain embodiments, the PRRA insoluble in water with a controlled release contains two types of PRRA, such as resylumads and nivolumab, resylumod and pembroralizumab, resetxolizumabs, resylcime and Abelumab, resylumod and durvalumab, resurns and ipilimumabs, reservice and tremorumamabs, residents Quimod and Trustuzumab, resiquimod and cetuximab, resiquimod and margetuximab, resiquimod and one of the CD47 or SIRPα blockers described herein, imiquimod and nivolumab, imiquimod and pembrolizumab, imiquimod and atezolizumab, imiquimod and avelumab, imiquimod and durvalumab, imiquimod and ipilimumab, imiquimod and tremelimumab, imiquimod and trastuzumab, imiquimod and cetuximab, imiquimod and margetuximab, imiquimod and one of the CD47 or SIRPα blockers described herein, SD101 and nivolumab, SD101 and pembrolizumab, SD101 and atezolizumab, SD101 and avelumab, SD101 and durvalumab, SD101 and ipilimum b, SD101 and tremelimumab, SD101 and trastuzumab, SD101 and cetuximab, SD101 and margetuximab, SD101 and one of the CD47 or SIRPα blockers described herein, CMP001 and nivolumab, CMP001 and pembrolizumab, CMP001 and atezolizumab, CMP001 and avelumab, CMP001 and durval umab, CMP001 and ipilimumab, CMP001 and tremelimumab, CMP001 and trastuzumab, CMP001 and cetuximab, CMP001 and margetuximab, CMP001 and one of the CD47 or SIRPα blockers described herein, MK-1454 and nivolumab, MK-1454 and pembrolizumab, MK-1454 and atezolizumab , MK -1454 and avelumab, MK-1454 and durvalumab, MK-1454 and ipilimumab, MK-1454 and tremelimumab, MK-1454 and trastuzumab, MK-1454 and cetuximab, MK-1454 and margetuximab, MK-1454 and one of the CD47 blockers, or SIRPα described herein, ADU-S100 and nivolumab, ADU-S100 and pembrolizumab, ADU-S100 and atezolizumab, ADU-S100 and avelumab, ADU-S100 and durvalumab, ADU-S100 and ipilimumab, ADU-S100 and tremelimumab, ADU- S100 and trastuzumab, ADU-S100 and cetuximab, ADU-S100 and margetuximab, ADU-S100 and one of the CD47 or SIRPα blockers described herein, 2'3'-CGAMP and nivolumab, 2'3'-CGAMP and pembrolizumab, 2 '3'-CGAMP and atezolizumab, 2'3'-CGAMP and avelumab, 2'3'-CGAMP and durvalumab, 2'3'-CGAMP and ipilimumab, 2'3'-CGAMP and tremelimumab, 2'3'-CGAMP and trastuzumab, 2'3'-CGAMP and cetuximab, 2'3'-CGAMP and margetuximab, or 2'3'-CGAMP and one of the CD47 or SIRPα blockers described herein.

In certain embodiments, at least some of the water-insoluble controlled-release PRRA is imiquimod, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% , about 80%, about 90% or 100%, i.e. all PRRA fragments present. In certain embodiments, at least some of the water-insoluble controlled-release PRRA is resiquimod, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% , about 80%, about 90% or 100%, i.e. all PRRA fragments present. In certain embodiments, at least some of the water insoluble controlled release PRRA is SD-101, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or 100%, i.e. all PRRA fragments present. In certain embodiments, at least some of the controlled release water insoluble PRRA is CMP001, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70% , about 80%, about 90% or 100%, i.e. all PRRA fragments present.

In certain embodiments of PRRA, controlled release release from water insoluble PRRA occurs with a release half-life under physiological conditions (aqueous buffer, pH 7.4, 37°C) of at least 3 days, such as at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 15 days, at least 17 days, at least 20 days or at least 25 days.

In certain embodiments, the water-insoluble controlled-release PRRA comprises a plurality of PRRA moieties covalently and reversibly conjugated to a carrier moiety, particularly an insoluble carrier moiety.

In certain embodiments, such a carrier comprises a polymer. In certain embodiments, the polymer is selected from the group consisting of 2-methacryloyl-oxyethyl phosphoylcholines, poly(acrylic acids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amides), poly(amidoamines) , poly(amino acids), poly(anhydrides), poly(aspartamides), poly(butyric acids), poly(glycolic acids), polybutylene terephthalates, poly(caprolactones), poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters), poly(ethylenes), poly(ethylene glycols), poly(ethylene oxides), poly(ethyl phosphates), poly(ethyl oxazolines), poly(glycolic acids), poly(hydroxyethyl acrylates), poly(hydroxyethyl oxazolines), poly (hydroxypropyl methacrylates), poly(hydroxypropyloxazolines), poly(iminocarbonates), poly(lactic acids), poly(copolymers of lactic and glycolic acids), poly(methacrylamides), poly(methacrylates), poly(methyloxazolines), poly(organophosphazenes), poly(orthoesters), poly(oxazolines), poly(propylene glycols), poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinylamines), poly(vinylmethyl ethers), poly(vinylpyrrolidones), silicones , celluloses, carbomethylcellulose, hydroxypropyl methylcellulose, chitins, chitosans, dextrans, dextrins, gelatins, hyaluronic acids and derivatives, functionalized hyaluronic acids, mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethyl starches and other polymers based on carbohydrates, xylans and their copolymers.

In certain embodiments, the carrier is a hydrogel. Such a hydrogel may be cleavable or may be non-cleavable, i.e. stable. In certain embodiments, such a hydrogel is cleavable. In certain embodiments, such a hydrogel is non-cleavable. In certain embodiments, the hydrogel comprises a polymer selected from the group consisting of 2-methacryloyl-oxyethyl phosphoylcholines, poly(acrylic acids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amides), poly( amidoamines), poly(amino acids), poly(anhydrides), poly(aspartamides), poly(butyric acids), poly(glycolic acids), polybutylene terephthalates, poly(caprolactones), poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamides) ), poly(esters), poly(ethylenes), poly(ethylene glycols), poly(ethylene oxides), poly(ethyl phosphates), poly(ethyl oxazolines), poly(glycolic acids), poly(hydroxyethyl acrylates), poly(hydroxyethyl oxazolines) , poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines), poly(iminocarbonates), poly(lactic acids), poly(copolymers of lactic and glycolic acids), poly(methacrylamides), poly(methacrylates), poly(methyloxazolines), poly(organophosphazenes) ), poly(orthoesters), poly(oxazolines), poly(propylene glycols), poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinylamines), poly(vinylmethyl ethers), poly(vinylpyrrolidones) , silicones, celluloses, carbomethylcelluloses, hydroxypropyl methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins, hyaluronic acids and derivatives, functionalized hyaluronic acids, mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethyl starches and other carbohydrate-based polymers, xylans and their copolymers.

In certain embodiments, the carrier is a hydrogel. In certain embodiments, one or more PRRAs are covalently and reversibly conjugated to such a hydrogel carrier. Such a hydrogel may be cleavable or may be non-cleavable, i.e. stable. In certain embodiments, such a hydrogel is cleavable. In certain embodiments, such a hydrogel is non-cleavable.

In certain embodiments, the hydrogel carrier is a PEG-based hydrogel or a hyaluronic acid-based hydrogel. In certain embodiments, the hydrogel carrier is a PEG-based hydrogel. Such a PEG-based hydrogel may be cleavable or may be non-cleavable, i.e. stable. In certain embodiments, such a PEG-based hydrogel is cleavable. In certain embodiments, such a PEG-based hydrogel is non-cleavable. In certain embodiments, the hydrogel carrier is a hyaluronic acid hydrogel. Such a hyaluronic acid-based hydrogel may be cleavable or non-cleavable, i.e. stable. In certain embodiments, such a hyaluronic acid hydrogel is cleavable. In certain embodiments, such a hyaluronic acid hydrogel is non-degradable.

In certain embodiments, the controlled release water insoluble PRRA is a conjugate, wherein said conjugate is water insoluble and contains a carrier moiety Z to which one or more -L ² -L ¹ -D moieties are conjugated, wherein

each -L ² - individually represents a chemical bond or spacer fragment;

each -L ^1- is individually a linker moiety to which -D is reversibly and covalently conjugated, and

each -D is individually a pattern recognition receptor agonist.

In certain embodiments, the water-insoluble controlled-release PRRA is a conjugate, wherein said conjugate is water-insoluble and contains a carrier moiety Z to which one or more -L ² -L ¹ -DL ¹ -L ² - moieties are conjugated, Where

each -L ² - individually represents a chemical bond or spacer fragment and is conjugated to Z;

each -L ^1- is individually a linker moiety to which -D is reversibly and covalently conjugated, and

each -D is individually a pattern recognition receptor agonist.

It is clear that according to this embodiment -L ² -L ¹ -DL ¹ -L ² - is connected at both ends thereof to Z.

One or more -L ² -L ^1- D moieties are covalently conjugated to Z. In certain embodiments, one or more -L ² -L ^1- D moieties are stably conjugated to Z, i.e. the connection between Z and -L ² - is a stable connection. If Z is a hydrogel, it is understood that the number of -L ² -L ^1- D moieties conjugated to such a hydrogel support is too large to be specified.

-D may be selected from the group consisting of Toll-like receptor (TLR) agonists, NOD-like receptors (NLR), RIG-I-like receptors, cytosolic DNA sensors, STING and aromatic hydrocarbon receptors (AhR).

In certain embodiments, -D is a Toll-like receptor agonist. In certain embodiments, -D is a NOD-like receptor. In certain embodiments, -D is a RIG-I-like receptor. In certain embodiments, -D is a cytosolic DNA sensor. In certain embodiments, -D is STING. In certain embodiments, -D is an aromatic hydrocarbon receptor.

If -D is a Toll-like receptor agonist, such Toll-like receptor agonists may be selected from the group consisting of TLR1/2 agonists, such as peptidoglycans, lipoproteins, Pam3CSK4, Amplivant, SLP-AMPLIVANT, HESPECTA, ISA101 and ISA201, TLR2 agonists, such as LAM-MS, LPS-PG, LTA-BS, LTA-SA, PGN-BS, PGN-EB, PGN-EK, PGN-SA, CL429, FSL-1, Pam2CSK4, Pam3CSK4, zymosan, CBLB612 , SV-283, ISA204, SMP105, heat-killed Listeria monocytogenes , TLR3 agonists such as poly(A:U), poly(I:C) (poly-ICLC), rintatolimod, apoxime, IPH3102, poly-ICR, PRV300, RGCL2, RGIC.1, Riboxim (RGC100, RGIC100), Riboxol (RGIC50) and Riboxon, TLR4 agonists such as lipopolysaccharides (LPS), neoceptin-3, glucopyranosyl lipid adjuvant (GLA), GLA-SE, G100, GLA-AF, clinical center reference endotoxin (CCRE), monophosphorylated lipid A, grass MATA MPL, PEPA10, ONT-10 (PET-lipid A, oncothyroid), G- 305, ALD046, CRX527, CRX675 (RC527, RC590), GSK1795091, OM197MPAC, OM294DP and SAR439794, TLR2/4 agonists, such as lipid A, OM174 and PGN007, TLR5 agonists, such as flagellin, entolimod, Mobilan, protectan CBLB501, TLR6/2 agonists, such as diacylated lipoproteins, diacylated lipopeptides, FSL-1, MALP- 2 and CBLB613, TLR7 agonists, such as CL264, CL307, imiquimod (R837), TMX-101, TMX-201, TMX-202, TMX302, gardiquimod, S-27609, 851, UC-IV150, 852A (3M-001, PF-04878691), loxoribine, polyuridylic acid, GSK2245035, GS-9620, RO6864018 (ANA773, RG7795), RO7020531, isatoribine, AN0331, ANA245, ANA971, ANA975, DSP0509, DSP3025 ( AZD8848), GS986, MBS2, MBS5, RG7863 ( RO6870868), sotirimod, SZU101 and TQA3334, TLR8 agonists such as ssPolyUridine, ssRNA40, TL8-506, XG-1-236, VTX-2337 (motolimod), VTX-1463, VTX378, VTX763, DN1508052 and GS9688 , TLR7 agonists/ 8, such as CL075, CL097, poly(dT), resiquimod (R-848, VML600, S28463), MEDI9197 (3M-052), NKTR262, DV1001, IMO4200, IPH3201 and VTX1463, TLR9 agonists, such as CpG DNA, CpG ODN, lefitolimod (MGN1703), SD-101, QbG10, CYT003, CYT003-QbG10, DUK-CpG-001, CpG-7909 (PF-3512676), GNKG168, EMD 1201081, IMO-2125, IMO-2055, 04, AZD1419 , AST008, IMO2134, MGN1706, IRS 954, 1018 ISS, actilon (CPG10101), ATP00001, AVE0675, AVE7279, CMP001, DIMS0001, DIMS9022, DIMS9054, DIMS9059, DV230, DV281, anDIM, heplisav (V270), cappaproct (DIMS0150), NJP834, NPI503, SAR21609 and Tolamba, and TLR7/9 agonists such as DV1179.

In certain embodiments, -D is a TLR1/2 agonist. In certain embodiments, -D is a TLR2 agonist. In certain embodiments, -D is a TLR3 agonist. In certain embodiments, -D is a TLR4 agonist. In certain embodiments, -D is a TLR2/4 agonist. In certain embodiments, -D is a TLR5 agonist. In a certain embodiment, -D is a TLR6/2 agonist. In certain embodiments, -D is a TLR7 agonist. In certain embodiments, -D is a TLR8 agonist. In certain embodiments, -D is a TLR7/8 agonist. In certain embodiments, -D is a TLR9 agonist.

Examples of CpG ODNs are ODN 1585, ODN 2216, ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN BW006, ODN D-SL01, ODN 2395, ODN M362 and ODN D-SL03.

In certain embodiments, -D is imiquimod. In certain embodiments, -D is resiquimod. In certain embodiments, -D is SD-101. In certain embodiments, -D is CMP001.

In certain embodiments, at least some of the -D moieties of the conjugate are imiquimod, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or 100%, i.e. all -D fragments present in the conjugate. In certain embodiments, at least some of the -D moieties of the conjugate are resiquimod, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or 100%, i.e. all -D fragments present in the conjugate. In certain embodiments, at least some of the -D moieties of the conjugate are SD-101, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80 %, about 90% or 100%, i.e. all -D fragments present in the conjugate. In certain embodiments, at least some of the -D moieties of the conjugate are CMP001, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or 100%, i.e. all -D fragments present in the conjugate.

If -D is a NOD-like receptor, the NOD-like receptor may be selected from the group consisting of NOD1 agonists, such as C12-iE-DAP, C14-Tri-LAN-Gly, iE-DAP, iE-Lys and Tri-DAP, and NOD2 agonists such as L18-MDP, MDP, M-TriLYS, murabutide and N-glycolyl-MDP.

In certain embodiments, -D is a NOD1 agonist. In certain embodiments, -D is a NOD2 agonist.

If -D is a RIG-I-like receptor, such RIG-I-like receptor may be selected from the group consisting of 3p-hpRNA, 5'ppp-DsRNA, 5'ppp RNA (M8), 5'OH RNA with kink (CBS -13-BPS), 5'PPP SLR, KIN100, KIN 101, KIN1000, KIN1400, KIN1408, KIN1409, KIN1148, KIN131A, poly(dA:dT), SB9200, RGT100 and Giltonol.

If -D is a cytosolic DNA sensor, the cytosolic DNA sensor may be selected from the group consisting of cGAS agonists, dsDNA-EC, G3-YSD, HSV-60, ISD, ODN TTAGGG (A151), poly(dG:dC) and VACV-70.

If -D is a STING, such STING may be selected from the group consisting of MK-1454, ADU-S100 (MIW815), 2'3'-CGAMP, 3'3'-CGAMP, c-di-AMP, c- di-GMP, cAIMP (CL592), cAIMP difluoro (CL614), cAIM(PS)2 difluoro (Rp/Sp) (CL656), 2'2'-CGAMP, 2'3'-CGAM(PS)2 (Rp/ Sp), 3'3'-CGAM fluorinated, c-di-AMP fluorinated, 2'3'-C-di-AMP, 2'3'-C-di-AM(PS)2 (Rp,Rp), c - di-GMP fluorinated, 2'3'-C-di-GMP, c-di-IMP, c-di-UMP and DMXAA (vadimezan, ASA404).

In certain embodiments, -D is MK-1454. In certain embodiments, -D is an ADU-S100 (MIW815). In certain embodiments, -D is 2'3'-CGAMP.

If -D is an aromatic hydrocarbon receptor (AhR), the AhR may be selected from the group consisting of FICZ, ITE and L-kynurenine.

In certain embodiments, the conjugate contains only one type of fragment -D, i.e. all fragments of the -D conjugate are identical. In certain embodiments, the conjugate contains more than one type of -D, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 different types of -D. If a conjugate contains more than one -D type, all -D moieties may be conjugated to the same -L ^1- type, or may be conjugated to different -L ^1- types, i.e. the first type -D can be conjugated to the first type -L ^1- , the second type -D can be conjugated to the second type -L ^1- , and so on. In certain embodiments, all fragments -L ^1- are of the same type, i.e. have the same structure. Alternatively, individual -D fragments of the same type can be conjugated to different types of -L ^1- fragment. The use of different -L ^1- fragments allows the release of conjugated drug fragments -D with different release kinetics. For example, the first linker moiety -L ^1- may have a short half-life and thus release the drug within a shorter time after administration to the patient than the second linker moiety -L ^1- which may have a longer half-life. The use of different fragments -L ^1- with different half-lives allows you to optimize the dosage regimen of one or more drugs.

The -L ^1- moiety is conjugated to -D via a functional group -D, wherein the functional group, in certain embodiments, is selected from the group consisting of carboxylic acid, primary amine, secondary amine, thiol, sulfonic acid, carbonate, carbamate, hydroxyl, aldehyde, ketone, hydrazine, isothiocyanate, phosphoric acid, phosphonic acid, acryloyl, hydroxylamine, sulfate, vinyl sulfone, vinyl ketone, diazoalkane, guanidine, aziridine, amide, imide, imine, urea, amidine, guanidine, sulfonamide, phosphonamide, forforamide, hydrazide and selenol. In certain embodiments, -L ^1- is conjugated to -D through a functional group -D selected from the group consisting of carboxylic acid, primary amine, secondary amine, thiol, sulfonic acid, carbonate, carbamate, hydroxyl, aldehyde, ketone, hydrazine, isothiocyanate, phosphoric acid, phosphonic acid, acryloyl, hydroxylamine, sulfate, vinyl sulfone, vinyl ketone, diazoalkane, guanidine and aziridine. In certain embodiments, -L ^1- is conjugated to -D through a -D functional group selected from the group consisting of hydroxyl, primary amine, secondary amine, thiol, sulfonic acid, carbonate, carbamate, hydroxyl, aldehyde, amidine, and carboxylic acid.

In certain embodiments, -L ^1- is conjugated to -D via the hydroxyl group of -D.

In certain embodiments, -L ^1- is conjugated to -D via a primary amine group -D.

In certain embodiments, -L ^1- is conjugated to -D via a secondary amine group -D.

In certain embodiments, -L ^1- is conjugated to -D via a carboxylic acid group -D.

In certain embodiments, -L ^1- is conjugated to -D via the amidine group -D.

If -D is resiquimod, -L ^{1- is} , in certain embodiments, conjugated to -D via its aromatic amine, i.e. amine functional group marked with an asterisk

.

If -D is imiquimod, -L ¹ - in certain embodiments is conjugated to -D via its aromatic amine, i.e. the amine functional group marked with an asterisk.

.

In certain embodiments, cleavage of the bond between -D and -L ¹ - occurs with a half-life under physiological conditions (aqueous buffer, pH 7.4, 37°C) of at least 3 days, such as at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 15 days, at least 17 days, at least 20 days or at least 25 days.

Fragment -L ^1- can be connected to -D through any type of connection, provided that it is reversible. In certain embodiments, -L ^1- is connected to -D through a bond selected from the group consisting of amide, ester, carbamate, acetal, aminal, imine, oxime, hydrazone, disulfide, acylguanidine, acylamidine, carbonate, phosphate, sulfate, urea, hydrazide, thioester, thiophosphate, thiosulfate, sulfonamide, sulfoamidine, sulfaguanidine, phosphoamide, phosphoamidine, phosphoguanidine, phosphoamidine, phosphonamidine, phosphoguanidine, phosphonate, borate and imide. In certain embodiments, -L ^1- is connected to -D through a bond selected from the group consisting of amide, ester, carbonate, carbamate, acetal, aminal, imine, oxime, hydrazone, disulfide, acrylamidine, and acylguanidine. In certain embodiments, -L ^1- is connected to -D via a bond selected from the group consisting of amide, ester, carbonate, acrylamide, and carbonate. It is understood that some of these bonds may be irreversible in themselves, but that in the present invention the adjacent groups present on -L ^1- make these bonds reversible.

In certain embodiments, -L ^1- is connected to -D via an ester bond.

In certain embodiments, -L ^1- is connected to -D via a carbonate bond.

In certain embodiments, -L ^1- is connected to -D via an acrylamidine bond.

In certain embodiments, -L ^1- is connected to -D via a carbamate bond.

In certain embodiments, -L ^1- is connected to -D via an amide bond.

If -D is resiquimod, the linkage between -D and -L ^1- in certain embodiments occurs via an amide bond, where the aromatic amine functionality of -D forms an amide bond with the carbonyl (-(C=O)- ) in -L ^{1 -}

,

where the dotted line shows the attachment to the remaining part -L ^1- .

When -D is imiquimod, the linkage between -D and -L ^1- in certain embodiments occurs via an amide bond, where the aromatic amine functionality of -D forms an amide bond with the carbonyl (-(C=O)- ) in -L ^{1 -}

,

where the dotted line shows the attachment to the remaining part -L ^1- .

The -L ^1- fragment is a linker fragment from which -D is released in its free form, i.e. generally in the form of D-H or D-OH. Such moieties are also known as “prodrug linkers” or “reversible prodrug linkers” and are known in the art, such as the reversible linker moieties described in WO 2005/099768 A2, WO 2006/136586 A2, WO 2011/089216 A1, WO 2013 /024053 A1, WO 2011/012722 A1, WO 2011/089214 A1, WO 2011/089215 A1, WO 2013/024052 A1 and WO 2013/160340 A1, which are incorporated herein by reference.

According to one embodiment, -L ^1- have the structure as disclosed in WO 2009/095479 A2. Accordingly, in certain embodiments, fragment -L ^1- has formula (II):

,

where the dotted line shows the addition to the -D nitrogen atom through the formation of an amide bond,

-X- represents -C(R ⁴ R ^4a )-, -N(R ⁴ )-, -O-, -C(R ⁴ R ^4a )-C(R ⁵ R ^5a )-, -C(R ⁵ R ^5a )-C(R ⁴ R ^4a )-, -C(R ⁴ R ^4a )-N(R ⁶ )-, -N(R ⁶ )-C(R ⁴ R ^4a )-, -C(R ⁴ R ^4a )-O-, -OC(R ⁴ R ^4a )-, or -C(R ⁷ R ^7a )-,

X ¹ represents C or S(O),

-X ^2- is -C(R ⁸ R ^8a )- or -C(R ⁸ R ^8a )-C(R ⁹ R ^9a )-,

=X ³ represents =O, =S or =N-CN,

-R ¹ , -R ^1a , -R ² , -R ^2a , -R ⁴ , -R ^4a , -R ⁵ , -R ^5a , -R ⁶ , -R ⁸ , -R ^8a , -R ⁹ , -R ^9a are independently selected from the group consisting of - H, and C _1-6 alkyl,

-R ³ , -R ^3a are independently selected from the group consisting of -H, and C _1-6 alkyl, with the proviso that if one or both of -R ³ , -R ^3a are other than -H, they are combined with N, to which they are attached via an sp ^3- hybridized carbon atom,

-R ⁷ is -N(R ¹⁰ R ^10a ) or -NR ¹⁰ -(C=O)-R ¹¹ ,

-R ^7a , -R ¹⁰ , -R ^10a , -R ¹¹ independently of each other represent -H or C _1-6 alkyl,

optionally one or more of the pairs -R ^1a /-R ^4a , -R ^1a /-R ^5a , -R ^1a /-R ^7a , -R ^4a /-R ^5a , -R ^8a /-R ^9a form a chemical bond,

optionally one or more of the pairs -R ¹ /-R ^1a , -R ² /-R ^2a , -R ⁴ /-R ^4a , -R ⁵ /-R ^5a , -R ⁸ /-R ^8a ,

-R ⁹ /-R ^9a are joined together with the atom to which they are attached to form a C _3-10 cycloalkyl or 3-10 membered heterocyclyl,

optionally one or more of the pairs -R ¹ /-R ⁴ , -R ¹ /-R ⁵ , -R ¹ /-R ⁶ , -R ¹ /-R ^7a , -R ⁴ /-R ⁵ , -R ⁴ / -R ⁶ , -R ⁸ /-R ⁹ , -R ² /-R ³ are joined together with the atoms to which they are attached to form ring A,

optionally, R ³ /R ^3a are joined together with the nitrogen atom to which they are attached to form a 3-10 membered heterocycle,

A is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl, and

wherein -L ^1- is substituted by at least one -L ² -, and where -L ^1- is optionally further substituted, provided that the hydrogen indicated by an asterisk in formula (II) is not replaced by -L ^2- or a substituent.

Preferably -L ^1- of formula (II) is replaced by one moiety -L ² -.

In one embodiment, -L ^1- of formula (II) is not further substituted.

It is clear that if -R ³ /-R ^3a of formula (II) combine together with the nitrogen atom to which they are attached to form a 3-10 membered heterocycle, only such 3-10 membered heterocycles can be formed in which the atoms , directly connected to a nitrogen atom are SP ^3- hybridized carbon atoms. In other words, such a 3-10-membered heterocycle formed by -R ³ /-R ^3a together with the nitrogen atom to which they are connected has the following structure:

,

Where

the dotted line shows the connection to the rest of -L ^1- ,

the ring contains from 3 to 10 atoms, including at least one nitrogen atom, and

R ^# and R ^## represent an SP ^3- hybridized carbon atom.

It is also understood that the 3-10 membered heterocycle may be further substituted.

Exemplary embodiments of suitable 3-10 membered heterocycles formed by -R ³ /-R ^3a of formula (II) together with the nitrogen atom to which they are attached are the following:

Where

the dotted line indicates attachment to the rest of the molecule, and

-R is selected from the group consisting of -H and C _1-6 alkyl.

-L ^1- of formula (II) may optionally be further substituted. In general, any substituent can be used as long as the cleavage principle is not affected, i.e. the hydrogen marked with an asterisk in formula (II) is not replaced, and the nitrogen of the fragment

formula (II) remains part of the primary, secondary or tertiary amine, i.e. -R ³ and R ^3a independently represent -H or are connected to -N< via an SP ^{3 -} hybridized carbon atom.

In one embodiment, -R ¹ or -R ^1a of formula (II) is substituted with -L ² -. In another embodiment, -R ² or -R ^2a of formula (II) is substituted with -L ² -. In another embodiment, -R ³ or -R ^3a of formula (II) is substituted with -L ² -. In another embodiment, -R ⁴ of formula (II) is substituted with -L ² -. According to another embodiment, -R ⁵ or -R ^5a of formula (II) is substituted with -L ² -. In another embodiment, -R ⁶ of formula (II) is substituted with -L ² -. According to another embodiment, -R ⁷ or -R ^7a of formula (II) is substituted with -L ² -. According to another embodiment, -R ⁸ or -R ^8a of formula (II) is substituted with -L ² -. In another embodiment, -R ⁹ or -R ^9a of formula (II) is substituted with -L ² -. According to another embodiment, -R ¹⁰ or -R ^10a of formula (II) is substituted with -L ² -. In another embodiment, -R ¹¹ of formula (II) is substituted with -L ² -.

In certain embodiments, -L ^1- has the structure as disclosed in WO 2016/020373 A1. Accordingly, in certain embodiments, fragment -L ^1- has formula (III):

(III),

Where

the dotted line indicates the addition of a primary or secondary amine or hydroxyl moiety -D via amide or ester bond formation, respectively.

-R ¹ , -R ^1a , -R ² , -R ^2a , -R ³ , and -R ^3a are independently selected from the group consisting of - H, -C(R ⁸ R ^8a R ^8b ), -C (=O)R ⁸ , -C≡N, -C(=NR ⁸ )R ^8a , -CR ⁸ (=CR ^8a R ^8b ),

-C≡CR ⁸ and -T,

-R ⁴ , -R ⁵ , and -R ^5a are independently selected from the group consisting of -H, -C(R ⁹ R ^9a R ^9b ) and -T,

a1 and a2 independently represent 0 or 1,

each -R ⁶ , -R ^6a , -R ⁷ , -R ^7a , -R ⁸ , -R ^8a , -R ^8b , -R ⁹ , -R ^9a , -R ^9b are independently selected from the group consisting of - H, halogen, -CN, -COOR ¹⁰ , -OR ¹⁰ , -C(O)R ¹⁰ , -C(O)N(R ¹⁰ R ^10a ), -S(O) ₂ N(R ¹⁰ R ^10a ) , -S(O)N(R ¹⁰ R ^10a ), -S(O) ₂ R ¹⁰ , -S(O)R ¹⁰ , -N(R ¹⁰ )S(O) ₂ N(R ^10a R ^10b ), -SR ¹⁰ , -N(R ¹⁰ R ^10a ), -NO ₂ , -OC(O)R ¹⁰ , -N(R ¹⁰ )C(O)R ^10a , -N(R ¹⁰ )S(O) ₂ R ^10a , -N(R ¹⁰ )S(O)R ^10a , -N(R ¹⁰ )C(O)OR ^10a , -N(R ¹⁰ )C(O)N(R ^10a R ^10b ), -OC(O )N(R ¹⁰ R ^10a ), -T, C _1-20 alkyl, C _2-20 alkenyl and C _2-20 alkynyl, where -T, C _1-20 alkyl, C _2-20 alkenyl, and C _{2- 20} alkynyl is optionally substituted with one or more -R ¹¹ , which are the same or different, and wherein C _1-20 alkyl, C _2-20 alkenyl, and C _2-20 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ¹² )-, -S(O) ₂ N(R ¹² )-, -S (O)N(R ¹² )-, -S(O) ₂ -, -S(O)-, -N(R ¹² )S(O) ₂ N(R ^12a )-, -S-, -N( R ¹² )-, -OC(OR ¹² )(R ^12a )-, -N(R ¹² )C(O)N(R ^12a )-, and -OC(O)N(R ¹² )-,

-R ¹⁰ , -R ^10a , -R ^10b are each independently selected from the group consisting of -H, -T, C _1-20 alkyl, C _2-20 alkenyl and C _2-20 alkynyl, where -T, C _{1- 20} alkyl, C _2-20 alkenyl, and C _2-20 alkynyl are optionally substituted by one or more -R ¹¹ that are the same or different, and wherein C _1-20 alkyl, C _2-20 alkenyl, and C _2-20 alkynyl are optional interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ¹² )-, -S( O) ₂ N(R ¹² )-, -S(O)N(R ¹² )-, -S(O) ₂ -, -S(O)-, -N(R ¹² )S(O) ₂ N( R ^12a )-, -S-, -N(R ¹² )-, -OC(OR ¹² )(R ^12a )-, -N(R ¹² )C(O)N(R ^12a )-, and -OC( O)N(R ¹² )-,

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, wherein each T is independently optionally substituted one or more -R ¹¹ which are the same or different,

each -R ¹¹ is independently selected from halogen, -CN, oxo (=O), -COOR ¹³ , -OR ¹³ , -C(O)R ¹³ , -C(O)N(R ¹³ R ^13a ), -S(O) ₂ N(R ¹³ R ^13a ), -S(O)N(R ¹³ R ^13a ), -S(O) ₂ R ¹³ , -S(O)R ¹³ , -N(R ¹³ ) S(O) ₂ N(R ^13a R ^13b ), -SR ¹³ , -N(R ¹³ R ^13a ), -NO ₂ , -OC(O)R ¹³ , -N(R ¹³ )C(O)R ^13a , -N(R ¹³ )S(O) ₂ R ^13a , -N(R ¹³ )S(O)R ^13a , -N(R ¹³ )C(O)OR ^13a , -N(R ¹³ )C(O )N(R ^13a R ^13b ), -OC(O)N(R ¹³ R ^13a ) and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different,

-R ¹² , -R ^12a , -R ¹³ , -R ^13a , -R ^13b are each independently selected from the group consisting of - H and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted by one or more halogens, which are the same or different

optionally one or more of the pairs -R ¹ /-R ^1a , -R ² /-R ^2a , -R ³ /-R ^3a , -R ⁶ /-R ^6a , -R ⁷ /-R ^7a are connected together with an atom, to which they are attached, to form a C _3-10 cycloalkyl or 3-10 membered heterocyclyl,

optionally one or more of the pairs -R ¹ / -R ² , -R ¹ / -R ³ , -R ¹ / -R ⁴ , -R ¹ / -R ⁵ , -R ¹ / -R ⁶ , -R ¹ / -R ⁷ , -R ² /-R ³ , -R ² /-R ⁴ , -R ² /-R ⁵ , -R ² /-R ⁶ , -R ² /-R ⁷ , -R ³ /-R ⁴ , -R ³ /-R ⁵ , -R ³ /-R ⁶ , -R ³ /-R ⁷ , -R ⁴ /-R ⁵ , -R ⁴ /-R ⁶ , -R ⁴ /-R ⁷ , -R ⁵ /-R ⁶ , -R ⁵ /-R ⁷ , -R ⁶ /-R ⁷ are joined together with the atoms to which they are attached to form ring A,

A is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl,

where -L ^1- is substituted by at least one -L ² -, and where -L ^1- is optionally further substituted.

The optional additional substituents -L ^1- of formula (III) preferably have the meaning as described above.

Preferably -L ^1- of formula (III) is replaced by one moiety -L ² -.

In one embodiment, -L ^1- of formula (III) is not further substituted.

In another embodiment, -L ^1- has the structure as disclosed in EP1536334B1, WO2009/009712A1, WO2008/034122A1, WO2009/143412A2, WO2011/082368A2 and US8618124B2, which are incorporated herein by reference.

In another embodiment, -L ^1- has the structure as disclosed in US8946405B2 and US8754190B2, which are incorporated herein by reference. Accordingly, in certain embodiments -L ^1- has formula (IV):

(IV),

Where

the dotted line shows the addition of -D through a moiety functional group -D selected from the group consisting of -OH, -SH and -NH ₂ .

m represents 0 or 1,

at least one or both of -R ¹ and -R ² is/are independently selected from the group consisting of -CN, -NO ₂ , optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -C(O)R ³ , -S(O)R ³ , -S(O) ₂ R ³ and -SR ⁴ ,

one and only one of -R ¹ and -R ² is selected from the group consisting of - H, optionally substituted alkyl, optionally substituted arylalkyl and optionally substituted heteroarylalkyl,

-R ³ is selected from the group consisting of -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR ⁹ and -N(R ⁹ ) ₂ .

-R ⁴ is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl and optionally substituted heteroarylalkyl,

each -R ⁵ is independently selected from the group consisting of - H, optionally substituted alkyl, optionally substituted alkenylalkyl, optionally substituted alkynylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl and optionally substituted heteroarylalkyl,

-R ⁹ is selected from the group consisting of -H and optionally substituted alkyl,

- Y- is missing and -X- is -O- or -S-, or

- Y- is -N(Q)CH ₂ -, and -X- is -O-,

Q is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl and optionally substituted heteroarylalkyl,

optionally, -R ¹ and -R ² can be joined to form a 3-8 membered ring, and

optionally, both -R ⁹ together with the nitrogen atom to which they are attached form a heterocyclic ring,

where -L ^1- is substituted by at least one -L ² -, and where -L ^1- is optionally further substituted.

Only in the context of formula (IV) the terms used have the following meanings:

The term “alkyl” as used herein includes linear, branched or cyclic saturated hydrocarbon groups having from 1 to 8 carbon atoms, or in some embodiments of the present invention from 1 to 6 or 1 to 4 carbon atoms.

The term “alkoxy” includes alkyl groups attached to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy and the like.

The term “alkenyl” includes non-aromatic unsaturated hydrocarbons with carbon-carbon double bonds.

The term “alkynyl” includes non-aromatic unsaturated hydrocarbons with carbon-carbon triple bonds.

The term “aryl” includes aromatic hydrocarbon groups having from 6 to 18 carbon atoms, preferably from 6 to 10 carbon atoms, including groups such as phenyl, naphthyl and anthracene. The term “heteroaryl” includes aromatic rings containing from 3 to 15 carbon atoms, containing at least one N, O or S atom, preferably from 3 to 7 carbon atoms, containing at least one N, O or S atom, including groups , such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isooxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl and the like.

In some cases, alkenyl, alkynyl, aryl or heteroaryl moieties may be linked to the remainder of the molecule via an alkylene bond. Under these circumstances, the substituent will be referred to as alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl, indicating that the alkylene moiety is between the alkenyl, alkynyl, aryl, or heteroaryl and the molecule to which the alkenyl, alkynyl, aryl, or heteroaryl is bonded.

The term “halogen” includes bromine, fluorine, chlorine and iodine.

The term “heterocyclic ring” refers to a 4 to 8 membered aromatic or non-aromatic ring containing from 3 to 7 carbon atoms and at least one N, O, or S atom. Examples are piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine and tetrahydrofuranyl, as well as the exemplary groups given above for the term “heteroaryl”.

When the ring system is optionally substituted, suitable substituents are selected from the group consisting of alkyl, alkenyl, alkynyl or an additional ring, where each is optionally further substituted. Optional substituents in any group, including those above, include halo, nitro, cyano, OR, -SR, -NR ₂ , -OCOR, -NRCOR, -COOR, -CONR ₂ , -SOR, -SO ₂ R, -SONR ₂ . -SO ₂ NR ₂ wherein each R is independently alkyl, alkenyl, alkynyl, aryl or heteroaryl, or two R groups taken together with the atoms to which they are attached form a ring.

Preferably -L ^1- of formula (IV) is replaced by one moiety -L ² -.

In another embodiment, -L ^1- has the structure as disclosed in WO2013/036857A1, which is incorporated herein by reference. Accordingly, in certain embodiments -L ^1- has formula (V):

(V),

Where

the dotted line shows the addition of -D via the amine functional group of the -D moiety,

-R ¹ is selected from the group consisting of optionally substituted C _{1 -} C ₆ linear, branched or cyclic alkyl, optionally substituted aryl, optionally substituted heteroaryl, alkoxy and -NR ⁵ ₂ ,

-R ² is selected from the group consisting of -H, optionally substituted C _1- C ₆ alkyl, optionally substituted aryl and optionally substituted heteroaryl,

-R ³ is selected from the group consisting of -H, optionally substituted C _1- C ₆ alkyl, optionally substituted aryl and optionally substituted heteroaryl,

-R ⁴ is selected from the group consisting of -H, optionally substituted C _1- C ₆ alkyl, optionally substituted aryl and optionally substituted heteroaryl,

each -R ⁵ is independently selected from the group consisting of - H, optionally substituted C _1- C ₆ alkyl, optionally substituted aryl and optionally substituted heteroaryl, or two -R ⁵ moieties together may be cycloalkyl or cycloheteroalkyl,

where -L ^1- is substituted by at least one -L ² -, and where -L ^1- is optionally further substituted.

Only in the context of formula (V) the terms used have the following meanings:

“Alkyl”, “alkenyl” and “alkynyl” include linear, branched or cyclic hydrocarbon groups having from 1 to 8 carbon atoms or 1 to 6 carbon atoms or 1 to 4 carbon atoms, where alkyl is a saturated hydrocarbon, alkenyl includes one or more than carbon-carbon double bonds, and alkynyl contains one or more carbon-carbon triple bonds. Unless otherwise stated, they contain 1-6 carbon atoms.

“Aryl” includes aromatic hydrocarbon groups having from 6 to 18 carbon atoms, preferably 6 to 10 carbon atoms, including groups such as phenyl, naphthyl and anthracene. “Heteroaryl” includes aromatic rings containing 3-15 carbon atoms containing at least one N, O or S atom, preferably 3-7 carbon atoms containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isooxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl and the like.

The term “substituted” means an alkyl, alkenyl, alkynyl, aryl or heteroaryl group containing one or more substituent groups in place of one or more hydrogen atoms. Substituents may generally be selected from halogen, including F, Cl, Br and I, lower alkyl, including linear, branched and cyclic, lower haloalkyl, including fluoroalkyl, chloroalkyl, bromoalkyl and iodoalkyl, OH, lower alkoxy, including linear, branched and cyclic, SH, lower alkylthio, including linear, branched and cyclic, amino, alkylamino, dialkylamino, silyl, including alkylsilyl, alkoxysilyl and arylsilyl, nitro, cyano, carbonyl, carboxylic acid, carboxylic acid ester, carboxylic amide, aminocarbonyl, aminoacyl, carbamate, urea, thiocarbamate, thiourea, ketone, sulfone, sulfonamide, aryl, including phenyl, naphthyl and anthracenyl, heteroaryl, including 5-membered heteroaryls, including pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole , triazole, oxadiazole and tetrazole, 6-membered heteroaryls including pyridine, pyrimidine, pyrazine, and fused heteroaryls including benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole and benzisothiazole.

Preferably -L ^1- of formula (V) is replaced by one moiety -L ² -.

In another embodiment, -L ^1- has the structure as disclosed in US7585837B2, which is incorporated herein by reference. Accordingly, in certain embodiments -L ^1- has formula (VI):

(VI),

Where

the dotted line shows the addition of -D via the amine functional group of the -D moiety,

R ¹ and R ² are independently selected from the group consisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, -SO ₃ H, -SO ₂ NHR ⁵ , amino, ammonium, carboxyl, PO ₃ H ₂ and OPO ₃ H ₂ ,

R ³ , R ⁴ and R ⁵ are independently selected from the group consisting of hydrogen, alkyl and aryl,

where -L ^1- is substituted by at least one -L ² -, and where -L ^1- is optionally further substituted.

Suitable substituents for formula (VI) include alkyl (such as C _1-6 alkyl), alkenyl (such as C _2-6 alkenyl), alkynyl (such as C _2-6 alkynyl), aryl (such as phenyl), heteroalkyl moieties , heteroalkenyl, heteroalkynyl, heteroaryl (such as an aromatic 4-7 membered heterocycle) or halogen.

Only in the context of formula (VI) the terms used have the following meanings:

The terms “alkyl”, “alkoxy”, “alkoxyalkyl”, “aryl”, “alkaryl” and “aralkyl” mean alkyl radicals containing 1-8, preferably 1-4 carbon atoms, for example methyl, ethyl, propyl, isopropyl and butyl, and aryl radicals containing 6-10 carbon atoms, for example, phenyl and naphthyl. The term “halogen” includes bromine, fluorine, chlorine and iodine.

Preferably -L ^1- of formula (VI) is replaced by one moiety -L ² -.

In another embodiment, -L ^1- has the structure as disclosed in WO2002/089789A1, which is incorporated herein by reference. Accordingly, in certain embodiments -L ^1- has formula (VII):

*
(VII),

Where

the dotted line shows the addition of -D via the amine functional group of the -D moiety,

L ₁ represents a bifunctional linking group,

Y ₁ and Y ₂ are independently O, S or NR ⁷

R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are independently selected from the group consisting of hydrogen, C _1-6 alkyls, C _3-12 branched alkyls, C _3-8 cycloalkyls, C _1-6 substituted alkyls, C _3-8 substituted cycloalkyls, aryls, substituted aryls, aralkyls, C _1-6 heteroalkyls, substituted C _1-6 heteroalkyls, C _1-6 alkoxy, phenoxy and C _1-6 heteroalkoxy,

Ar is a moiety which, when included in formula (VII), forms a multisubstituted aromatic hydrocarbon or a multisubstituted heterocyclic group,

X represents a chemical bond or moiety that is actively transferred into the target cell, a hydrophobic moiety, or a combination thereof,

y represents 0 or 1,

where -L ^1- is substituted by at least one -L ² -, and where -L ^1- is optionally further substituted.

Only in the context of formula (VII) the terms used have the following meanings:

The term “alkyl” is to be understood to include, for example, straight-chain, branched, substituted C _1-12 alkyls, including alkoxy, C _3-8 cycloalkyl or substituted cycloalkyl, etc.

The term “substitution” is to be understood to include the addition or substitution of one or more atoms contained in a functional group, or compounds with one or more different atoms.

Substituted alkyls include carboxyalkyls, aminoalkyls, dialkylamines, hydroxyalkyls and mercaptoalkyls, substituted cycloalkyls include moieties such as 4-chlorocyclohexyl, aryls include moieties such as naphthyl, substituted aryls include moieties such as 3-bromophenyl, aralkyls include moieties such as eg toluyl, heteroalkyls include moieties such as ethylthiophene, substituted heteroalkyls include moieties such as 3-methoxythiophene, alkoxy includes moieties such as methoxy, and phenoxy includes moieties such as 3-nitrophenoxy. Halo-, as must be understood, includes fluorine, chlorine, iodine and bromine.

Preferably -L ^1- of formula (VII) is replaced by one moiety -L ² -.

In certain embodiments, -L ^1- contains a substructure of formula (VIII)

(VIII),

Where

the dotted line marked with an asterisk shows the addition to the -D nitrogen atom through the formation of an amide bond,

the unmarked dotted line shows the attachment to the remainder of -L ^1- , and

where -L ^1- is substituted by at least one -L ² -, and where -L ^1- is optionally further substituted.

Preferably -L ^1- of formula (VIII) is replaced by one moiety -L ² -.

In one embodiment, -L ^1- of formula (VIII) is not further substituted.

In certain embodiments, -L ^1- contains a substructure of formula (IX)

(IX),

Where

the dotted line marked with an asterisk shows the addition to the -D nitrogen atom through the formation of a carbamate bond,

the unmarked dotted line shows the attachment to the remainder of -L ^1- , and

where -L ^1- is substituted by at least one -L ² -, and where -L ^1- is optionally further substituted.

Preferably -L ^1- of formula (IX) is replaced by one moiety -L ² -.

In one embodiment, -L ^1- of formula (IX) is not further substituted.

In certain embodiments, -L ^1- has formula (IX-a):

(IX-a),

Where

the dotted line marked with an asterisk shows attachment to the -D nitrogen atom, and the unmarked dotted line shows attachment to -L ² -.

n represents 0, 1, 2, 3 or 4,

=Y ₁ , =Y ₅ are independently selected from the group consisting of =O and =S,

- Y _2- selected from the group consisting of -O- and -S-,

- Y _3- selected from the group consisting of -O- and -S-,

- Y ₄ - selected from the group consisting of -O-, -NR ⁵ - and -C(R ⁶ R ^6a )-,

-R ³ , -R ⁵ , -R ⁶ , -R ^6a are independently selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert- butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl,

-R ⁴ is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl , 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl,

- W- is selected from the group consisting of C _1-20 alkyl, optionally interrupted by one or more groups selected from the group consisting of C _3-10 cycloalkyl, 8-30 membered carbopolycyclyl, 3-10 membered heterocyclyl, -C(O)-, -C(O)N(R ⁷ )-, -O-, -S- and -N(R ⁷ )-,

-Nu is a nucleophile selected from the group consisting of -N(R ⁷ R ^7a ), -N(R ⁷ OH), -N(R ⁷ )-N(R ^7a R ^7b ), -S(R ⁷ ) ,-COOH,

- Ar- selected from the group consisting of

Where

dotted lines indicate attachment to the remaining part -L ^1- ,

- Z ^1- is selected from the group consisting of -O-, -S- and -N(R ⁷ )-, and

- Z ^2- is -N(R ⁷ )-, and

-R ⁷ , -R ^7a , -R ^7b are independently selected from the group consisting of - H, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl,

where -L ^1- is optionally further substituted.

In one embodiment, -L ^1- of formula (IX-a) is not further substituted.

In certain embodiments, -L ^1- has formula (IX- b):

(IX-b),

Where

the dotted line marked with an asterisk shows attachment to the -D nitrogen atom, and the unmarked dotted line shows attachment to -L ² -.

n represents 0, 1, 2, 3 or 4,

=Y _1, =Y ₅ are independently selected from the group consisting of =O and =S,

- Y _2- selected from the group consisting of -O- and -S-,

- Y _3- selected from the group consisting of -O- and -S-,

- Y ₄ - selected from the group consisting of -O-, -NR ⁵ - and -C(R ⁶ R ^6a )-,

-R ² , -R ³ , -R ⁵ , -R ⁶ , -R ^6a are independently selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec- butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl,

-R ⁴ is selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl , 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl,

- W- is selected from the group consisting of C _1-20 alkyl, optionally interrupted by one or more groups selected from the group consisting of C _3-10 cycloalkyl, 8-30 membered carbopolycyclyl, 3-10 membered heterocyclyl, -C(O)-, -C(O)N(R ⁷ )-, -O-, -S- and -N(R ⁷ )-,

-Nu is a nucleophile selected from the group consisting of -N(R ⁷ R ^7a ), -N(R ⁷ OH), -N(R ⁷ )-N(R ^7a R ^7b ), -S(R ⁷ ) , -COOH,

- Ar- selected from the group consisting of

Where

dotted lines indicate attachment to the remaining part -L ^1- ,

- Z ^1- is selected from the group consisting of -O-, -S- and -N(R ⁷ )-, and

- Z ^2- is -N(R ⁷ )-, and

-R ⁷ , -R ^7a , -R ^7b are independently selected from the group consisting of - H, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl,

where -L ^1- is optionally further substituted.

In one embodiment, -L ^1- of formula (IX-b) is not further substituted.

In certain embodiments, -L ^1- has the formula (X)

(X),

Where the dotted line shows the addition of the nitrogen atom of the amine functional group -D,

=X ¹ is selected from the group consisting of =O, =S and =N,

-X ^2- is selected from the group consisting of -O-, -S- and -N-,

-R is _C1-50 alkyl, wherein _C1-50 alkyl is optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O) -, -C(O)N(R ^z1 )-, -S(O) ₂ N(R ^z1 )-, -S(O)N(R ^z1 )-, -S(O) ₂ -, -S( O)-, -N(R ^z1 )S(O) ₂ N(R ^z1a )-, -S-, -N(R ^z1 )-, -OC(OR ^z1 )(R ^z1a )-, -N(R ^z1 )C(O)N(R ^z1a )- and -OC(O)N(R ^z1 )-, and wherein the C _1-50 alkyl is optionally substituted with one or more -R ^z2 ,

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl and 8 -30 membered heteropolycyclyl, wherein each T is independently optionally substituted with one or more -R ^z2 which are the same or different,

each -R ^z2 is independently selected from the group consisting of halogen, -CN, oxo (=O), -COOR ^z3 , -OR ^z3 , -C(O)R ^z3 , -C(O)N(R ^z3 R ^z3a ) , -S(O) ₂ N(R ^z3 R ^z3a ), -S(O)N(R ^z3 R ^z3a ), -S(O) ₂ R ^z3 , -S(O)R ^z3 , -N(R ^z3 )S(O) ₂ N(R ^z3a R ^z3b ), -SR ^z3 , -N(R ^z3 R ^z3a ), -NO ₂ , -OC(O)R ^z3 , -N(R ^z3 )C(O)R ^z3a , -N(R ^z3 )S(O) ₂ R ^z3a , -N(R ^z3 )S(O)R ^z3a , -N(R ^z3 )C(O)OR ^z3a , -N(R ^z3 )C( O)N(R ^z3a R ^z3b ), -OC(O)N(R ^z3 R ^z3a ), and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different, and

-R ^z1 , -R ^z1a , -R ^z3 , -R ^z3a and -R ^z3b are each independently selected from the group consisting of - H, and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different,

where -L ^1- is substituted by at least one -L ² -, and where -L ^1- is optionally further substituted.

In certain embodiments, -L ^1- is replaced by one -L ² -.

In one embodiment, -L ^1- of formula (X) is not further substituted.

In certain embodiments, =X ¹ of formula (X) is selected from the group consisting of =N and =O. In certain embodiments, =X ¹ of formula (X) is =N. In certain embodiments, =X ¹ of formula (X) is =O.

In certain embodiments, -X ^2- of formula (X) is selected from the group consisting of -N- and -O-. In certain embodiments, -X ^2- of formula (X) is -N-. In certain embodiments, -X ^2- of formula (X) is -O-.

In certain embodiments, =X ¹ of formula (X) is =N, and -X ^2- of formula (X) is -O-. In certain embodiments, =X ¹ of formula (X) is =O, and -X ^2- of formula (X) is -N-. In a certain embodiment, =X ¹ of formula (X) is =N, and -X ^2- of formula (X) is -N-. In certain embodiments, =X ¹ of formula (X) is =O, and -X ^2- of formula (X) is -O-.

In certain embodiments, -R of formula (X) is C _1-20 alkyl, wherein C _1-20 alkyl is optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, - O-, -C(O)-, -C(O)N(R ^z1 )-, -S(O) ₂ N(R ^z1 )-, -S(O)N(R ^z1 )-, -S( O) ₂ -, -S(O)-, -S-, -N(R ^z1 )-, -OC(OR ^z1 )(R ^z1a )-, -N(R ^z1 )C(O)N(R ^z1a )- and -OC(O)N(R ^z1 )-, and wherein the C _1-20 alkyl is optionally substituted with one or more -R ^z2 ,

-R ^z1 and -R ^z1a are each independently selected from the group consisting of -H and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different,

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, wherein each T is independently optionally substituted by one or more -R ^z2 which are the same or different,

each -R ^z2 is independently selected from the group consisting of halogen and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted by one or more halogens that are the same or different.

In certain embodiments, a portion of formula (X) is selected from the group consisting of formulas (X-1), (X-2), (X-3), (X-4), (X-5), (X-6 ), (X-7), (X-8), (X-9), (X-10), (X-11) and (X-12)

(X -1), (X-2), (X-3), (X-4), (X-5), (X-6), (X-7), (X-8), (X-9), (X-10), (X -11) and (X -12),

Where

the dotted line marked with an asterisk indicates the addition of an amine functional group -D to the nitrogen atom,

the unmarked dotted line shows the attachment to -L ² -,

-R ¹ is selected from the group consisting of - H, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl,

-R ² and -R ^2a are independently selected from the group consisting of - H, halogen, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl,

n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25,

m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25,

o represents an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10,

p is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and

q is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25.

In certain embodiments, n in the formula (X -1), (X-2), (X-3), (X-4), (X-5), (X-6), (X-7), ( X-8), (X-9), or (X-12) is 1. In certain embodiments, n in the formula (X-1), (X-2), (X-3), (X-4) , (X-5), (X-6), (X-7), (X-8), (X-9), or (X-12) is 2. In certain embodiments, n in the formula (X - 1), (X- 2), (X- 3), (X- 4), (X- 5), (X-6), (X- 7), (X- 8), (X- 9) or (X -12) is 3. In certain embodiments, n in the formula (X -1), (X-2), (X-3), (X-4), (X-5), (X- 6), (X-7), (X-8), (X-9), or (X-12) is 4. In certain embodiments, n in the formula (X-1), (X-2), ( X-3), (X-4), (X-5), (X-6), (X-7), (X-8), (X-9) or (X-12) represents 5. In certain embodiments, n in the formula (X -1), (X-2), (X-3), (X-4), (X-5), (X-6), (X-7), ( X-8), (X-9), or (X-12) is 6. In certain embodiments, n in the formula (X-1), (X-2), (X-3), (X-4) , (X-5), (X-6), (X-7), (X-8), (X-9), or (X-12) is 7. In certain embodiments, n in the formula (X - 1), (X- 2), (X- 3), (X- 4), (X- 5), (X-6), (X- 7), (X- 8), (X- 9) or (X -12) is 8. In certain embodiments, n in the formula (X -1), (X-2), (X-3), (X-4), (X-5), (X- 6), (X-7), (X-8), (X-9), or (X-12) is 9. In certain embodiments, n in the formula (X-1), (X-2), ( X-3), (X-4), (X-5), (X-6), (X-7), (X-8), (X-9) or (X-12) represents 10.

In certain embodiments, m in formula (X-8), (X-9), or (X-12) is 1. In certain embodiments, m in formula (X-8), (X-9), or (X - 12) is 2. In certain embodiments, m in formula (X-8), (X-9), or (X-12) is 3. In certain embodiments, m in formula (X-8), (X- 9) or (X -12) is 4. In certain embodiments, m in formula (X- 8), (X- 9), or (X -12) is 5. In certain embodiments, m in formula (X- 8), (X-9) or (X -12) is 6. In certain embodiments, m in the formula (X-8), (X-9) or (X -12) is 7. In certain embodiments m in formula (X-8), (X-9), or (X-12) is 8. In certain embodiments, m in formula (X-8), (X-9), or (X-12) is 9. In certain embodiments, m in formula (X-8), (X-9), or (X-12) is 10.

In certain embodiments, o in formula (X-10) or (X -11) is 0. In certain embodiments, o in formula (X-10) or (X -11) is 1. In certain embodiments, o in formula (X-10) or (X -11) is 2. In certain embodiments, o in formula (X-10) or (X -11) is 3. In certain embodiments, o in formula (X-10) or (X -11) is 4. In certain embodiments, o in formula (X-10) or (X -11) is 5. In certain embodiments, o in formula (X-10) or (X -11) is 6. In certain embodiments, o in formula (X-10) or (X -11) is 7. In certain embodiments, o in formula (X-10) or (X -11) is 8. In certain embodiments In embodiments, o in formula (X-10) or (X -11) is 9. In certain embodiments, o in formula (X-10) or (X -11) is 10.

In certain embodiments, p in formula (X-10) or (X -11) is 0. In certain embodiments, p in formula (X-10) or (X -11) is 1. In certain embodiments, p in formula (X-10) or (X -11) is 2. In certain embodiments, p in formula (X-10) or (X -11) is 3. In certain embodiments, p in formula (X-10) or (X -11) is 4. In certain embodiments, p in formula (X-10) or (X -11) is 5. In certain embodiments, p in formula (X-10) or (X -11) is 6. In certain embodiments, p in formula (X-10) or (X -11) is 7. In certain embodiments, p in formula (X-10) or (X -11) is 8. In certain embodiments In embodiments, p in formula (X-10) or (X -11) is 9. In certain embodiments, p in formula (X-10) or (X -11) is 10.

In certain embodiments, q in formula (X -11) is 1. In certain embodiments, q in formula (X -11) is 2. In certain embodiments, q in formula (X -11) is 3. In certain embodiments In certain embodiments, q in formula (X -11) is 4. In certain embodiments, q in formula (X -11) is 5. In certain embodiments, q in formula (X -11) is 6. In certain embodiments q in formula (X -11) is 7. In certain embodiments, q in formula (X -11) is 8. In certain embodiments, q in formula (X -11) is 9. In certain embodiments, q in formula (X -11) represents 10.

In certain embodiments, -R ¹ has the formula (X-5), (X-6), (X-7), (X-8), (X-9), (X-10), (X-11), or (X -12) is - H. In certain embodiments -R ¹ of the formula (X- 5), (X-6), (X-7), (X-8), (X-9), (X -10), (X -11) or (X -12) is C _1-10 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n -pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl or 3,3-dimethylpropyl. In certain embodiments, -R ¹ has the formula (X-5), (X-6), (X-7), (X-8), (X-9), (X-10), (X-11), or (X -12) is C _2-10 alkenyl. In certain embodiments, -R ¹ has the formula (X-5), (X-6), (X-7), (X-8), (X-9), (X-10), (X-11), or (X -12) is C _2-10 alkynyl.

In certain embodiments, -R ² of formula (X-10) or (X -11) is - H. In certain embodiments, -R ² of formula (X-10) or (X -11) is a halogen, such as fluorine or chlorine. In certain embodiments, -R ² of formula (X-10) or (X -11) is C _{1- 10} alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert -butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl or 3,3-dimethylpropyl. In certain embodiments, -R ² of formula (X-10) or (X -11) is C _2-10 alkenyl, such as C ₂ alkenyl, C ₃ alkenyl, C ₄ alkenyl, C ₅ alkenyl, or C ₆ alkenyl. In certain embodiments, -R ² of formula (X-10) or (X -11) is C _2-10 alkynyl, such as C ₂ alkynyl, C ₃ alkynyl, C ₄ alkynyl, C ₅ alkynyl, or C ₆ alkynyl.

In certain embodiments, -R ^2a of formula (X-10) or (X -11) is - H. In certain embodiments, -R ^2a of formula (X-10) or (X -11) is halogen. In certain embodiments, -R ^2a of formula (X-10) or (X-11) is C _1-10 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert -butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl or 3,3-dimethylpropyl. In certain embodiments, -R ^2a of formula (X-10) or (X-11) is C _2-10 alkenyl, such as C ₂ alkenyl, C ₃ alkenyl, C ₄ alkenyl, C ₅ alkenyl, or C ₆ alkenyl. In certain embodiments, -R ^2a of formula (X-10) or (X-11) is C _2-10 alkynyl, such as C ₂ alkynyl, C ₃ alkynyl, C ₄ alkynyl, C ₅ alkynyl, or C ₆ alkynyl.

In certain embodiments, at least one of -R ² and -R ^2a of formula (X-10) and (X-11) is not - H.

In certain embodiments, -L ^1- has the formula (X -1). In certain embodiments, -L ^1- has the formula (X -1) with n = 1. In certain embodiments, -L ^1- has the formula (X -1) with n = 2. In certain embodiments, -L ^1- has the formula (X -1) with n = 3. In certain embodiments, -L ^1- has the formula (X -1) with n = 4. In certain embodiments, -L ^1- has the formula (X -1) with n = 5.

In certain embodiments, -L ^1- has the formula (X- 2). In certain embodiments, -L ^1- has the formula (X- 2) with n = 1. In certain embodiments, -L ^1- has the formula (X- 2) with n = 2. In certain embodiments, -L ^1- has the formula (X- 2) with n = 3. In certain embodiments, -L ^1- has the formula (X- 2) with n = 4. In certain embodiments, -L ^1- has the formula (X- 2) with n = 5.

In certain embodiments, -L ^1- has the formula (X- 3). In certain embodiments, -L ^1- has the formula (X- 3) with n = 1. In certain embodiments, -L ^1- has the formula (X- 3) with n = 2. In certain embodiments, -L ^1- has the formula (X- 3) with n = 3. In certain embodiments, -L ^1- has the formula (X- 3) with n = 4. In certain embodiments, -L ^1- has the formula (X- 3) with n = 5.

In certain embodiments, -L ^1- has the formula (X- 4). In certain embodiments, -L ^1- has the formula (X- 4) with n = 1. In certain embodiments, -L ^1- has the formula (X- 4) with n = 2. In certain embodiments, -L ^1- has the formula (X- 4) with n = 3. In certain embodiments, -L ^1- has the formula (X- 4) with n = 4. In certain embodiments, -L ^1- has the formula (X- 4) with n = 5.

In certain embodiments, -L ^1- has the formula (X- 5). In certain embodiments, -L ^1- has the formula (X- 5), and -R ¹ is - H. In certain embodiments, -L ^1- has the formula (X- 5), and -R ¹ is methyl. In certain embodiments, -L ^1- has the formula (X- 5) and -R ¹ is ethyl. In certain embodiments, -L ^1- has the formula (X- 5), and n is 1. In certain embodiments, -L ^1- has the formula (X- 5), and n is 2. In certain embodiments, -L ^1- has the formula (X- 5), and n is 3. In certain embodiments, -L ^1- has the formula (X- 5), -R ¹ is - H, and n is 1. In certain embodiments -L ^1- has the formula (X- 5), -R ¹ is - H, and n is 2. In certain embodiments, -L ^1- has the formula (X- 5), -R ¹ is - H, and n is 3. In certain embodiments, -L ^1- has the formula (X- 5), -R ¹ is methyl, and n is 1. In certain embodiments, -L ^1- has the formula (X- 5) , -R ¹ is methyl, and n is 2. In certain embodiments, -L ^1- has the formula (X- 5), -R ¹ is methyl, and n is 3.

In certain embodiments, -L ^1- has formula (X-6). In certain embodiments, -L ^1- has the formula (X-6) and -R ¹ is -H. In certain embodiments, -L ^1- has the formula (X-6) and -R ¹ is methyl. In certain embodiments, -L ^1- has formula (X-6) and -R ¹ is ethyl. In certain embodiments, -L ^1- has the formula (X-6), and n is 1. In certain embodiments, -L ^1- has the formula (X-6), and n is 2. In certain embodiments, -L ^1- has the formula (X-6), and n is 3. In certain embodiments, -L ^1- has the formula (X-6), -R ¹ is - H, and n is 1. In certain embodiments -L ^1- has the formula (X-6), -R ¹ represents - H, and n represents 2. In certain embodiments, -L ^1- has the formula (X-6), -R ¹ represents - H, and n is 3. In certain embodiments, -L ^1- has the formula (X-6), -R ¹ is methyl, and n is 1. In certain embodiments, -L ^1- has the formula (X-6) , -R ¹ is methyl, and n is 2. In certain embodiments, -L ^1- has formula (X-6), -R ¹ is methyl, and n is 3.

In certain embodiments, -L ^1- has the formula (X- 7). In certain embodiments, -L ^1- has the formula (X- 7), and -R ¹ is - H. In certain embodiments, -L ^1- has the formula (X- 7), and -R ¹ is methyl. In certain embodiments, -L ^1- has the formula (X- 7) and -R ¹ is ethyl. In certain embodiments, -L ^1- has the formula (X- 7), and n is 1. In certain embodiments, -L ^1- has the formula (X- 7), and n is 2. In certain embodiments, -L ^1- has the formula (X- 7), and n is 3. In certain embodiments, -L ^1- has the formula (X- 7), -R ¹ is - H, and n is 1. In certain embodiments -L ^1- has the formula (X- 7), -R ¹ is - H, and n is 2. In certain embodiments, -L ^1- has the formula (X- 7), -R ¹ is - H, and n is 3. In certain embodiments, -L ^1- has the formula (X- 7), -R ¹ is methyl, and n is 1. In certain embodiments, -L ^1- has the formula (X- 7) , -R ¹ is methyl, and n is 2. In certain embodiments, -L ^1- has the formula (X- 7), -R ¹ is methyl, and n is 3.

In certain embodiments, -L ^1- has the formula (X- 8). In certain embodiments, -L ^1- has the formula (X- 8), and -R ¹ is - H. In certain embodiments, -L ^1- has the formula (X- 8), and -R ¹ is methyl. In certain embodiments, -L ^1- has the formula (X- 8) and -R ¹ is ethyl. In certain embodiments, -L ^1- has the formula (X- 8), and n is 1. In certain embodiments, -L ^1- has the formula (X- 8), and n is 2. In certain embodiments, -L ^1- has the formula (X- 8), and n is 3. In certain embodiments, -L ^1- has the formula (X- 8), and m is 1. In certain embodiments, -L ^1- has the formula (X - 8), and m is 2. In certain embodiments, -L ^1- has the formula (X- 8), and m is 3. In certain embodiments, -L ^1- has the formula (X- 8), -R ¹ is - H, n is 1, and m is 1. In certain embodiments, -L ^1- has the formula (X- 8), -R ¹ is - H, n is 1, and m is 2. In certain embodiments, -L ^1- has the formula (X- 8), -R ¹ is - H, n is 1, and m is 3. In certain embodiments, -L ^1- has the formula (X- 8), -R ¹ is -H, n is 2, and m is 1. In certain embodiments, -L ^1- has the formula (X- 8), -R ¹ is - H, n is 2 , and m is 2. In certain embodiments, -L ^1- has the formula (X- 8), -R ¹ is - H, n is 2, and m is 3. In certain embodiments, -L ^1- has the formula (X- 8), -R ¹ is - H, n is 3, and m is 1. In certain embodiments, -L ^1- has the formula (X- 8), -R ¹ is - H , n is 3, and m is 2. In certain embodiments, -L ^1- has the formula (X- 8), -R ¹ is -H, n is 3, and m is 3.

In certain embodiments, -L ^1- has the formula (X- 9). In certain embodiments, -L ^1- has the formula (X- 9), and -R ¹ is - H. In certain embodiments, -L ^1- has the formula (X- 9), and -R ¹ is methyl. In certain embodiments, -L ^1- has the formula (X- 9) and -R ¹ is ethyl. In certain embodiments, -L ^1- has the formula (X- 9), and n is 1. In certain embodiments, -L ^1- has the formula (X- 9), and n is 2. In certain embodiments, -L ^1- has the formula (X- 9), and n is 3. In certain embodiments, -L ^1- has the formula (X- 9), and m is 1. In certain embodiments, -L ^1- has the formula (X - 9), and m is 2. In certain embodiments, -L ^1- has the formula (X- 9), and m is 3. In certain embodiments, -L ^1- has the formula (X- 9), -R ¹ is - H, n is 1, and m is 1. In certain embodiments, -L ^1- has the formula (X- 9), -R ¹ is - H, n is 1, and m is 2. In certain embodiments, -L ^1- has the formula (X- 9), -R ¹ is - H, n is 1, and m is 3. In certain embodiments, -L ^1- has the formula (X- 9), -R ¹ is -H, n is 2, and m is 1. In certain embodiments, -L ^1- has the formula (X- 9), -R ¹ is - H, n is 2 , and m is 2. In certain embodiments, -L ^1- has the formula (X- 9), -R ¹ is - H, n is 2, and m is 3. In certain embodiments, -L ^1- has the formula (X- 9), -R ¹ is - H, n is 3, and m is 1. In certain embodiments, -L ^1- has the formula (X- 9), -R ¹ is - H , n is 3, and m is 2. In certain embodiments, -L ^1- has the formula (X- 9), -R ¹ is -H, n is 3, and m is 3.

In certain embodiments, -L ^1- has the formula (X-10). In certain embodiments, -R ¹ of formula (X-10) is - H. In certain embodiments, o in formula (X-10) is 0. In certain embodiments, o in formula (X-10) is 1. In certain embodiments, o in formula (X-10) is 2. In certain embodiments, o in formula (X-10) is 3. In certain embodiments, p in formula (X-10) is 0. In certain embodiments In certain embodiments, p in formula (X-10) is 1. In certain embodiments, p in formula (X-10) is 2. In certain embodiments, p in formula (X-10) is 3. In certain embodiments -R ² of formula (X-10) is - H. In certain embodiments, -R ² of formula (X-10) is a halogen, such as fluorine. In certain embodiments, -R ² of formula (X-10) is methyl. In certain embodiments, -R ² of formula (X-10) is ethyl. In certain embodiments, -R ² of formula (X-10) is n-propyl . In certain embodiments, -R ² of formula (X-10) is isopropyl. In certain embodiments, -R ² of formula (X-10) is 2-methylpropyl. In certain embodiments, -R ² of formula (X-10) is 2-methylpropyl. In certain embodiments, -R ² of formula (X-10) is 1-methylpropyl. In certain embodiments, -R ^2a of formula (X-10) is -H. In certain embodiments, both -R ² and -R ^2a of formula (X-10) are methyl. In certain embodiments, -R ² of formula (X-10) is fluorine, and -R ^2a of formula (X-10) is - H. In certain embodiments, -R ² of formula (X-10) is isopropyl, and -R ^2a of formula (X-10) is - H. In certain embodiments, -R ² of formula (X-10) is 2-methylpropyl, and -R ^2a of formula (X-10) is - H.

In certain embodiments, -L ^1- has the formula (X -11). In certain embodiments, -R ¹ of formula (X -11) is - H. In certain embodiments, -R ¹ of formula (X -11) is methyl. In certain embodiments, -R ¹ of formula (X -11) is ethyl. In certain embodiments, o in formula (X -11) is 0. In certain embodiments, o in formula (X -11) is 1. In certain embodiments, o in formula (X -11) is 2. In certain embodiments In certain embodiments, p in formula (X -11) is 0. In certain embodiments, p in formula (X -11) is 1. In certain embodiments, p in formula (X -11) is 2. In certain embodiments -R ² of formula (X -11) is -H. In certain embodiments, -R ² of formula (X -11) is a halogen, such as fluorine. In certain embodiments, -R ² of formula (X -11) is methyl. In certain embodiments, -R ² of formula (X -11) is ethyl. In certain embodiments, -R ² of formula (X -11) is n-propyl . In certain embodiments, -R ² of formula (X -11) is isopropyl. In certain embodiments, -R ² of formula (X -11) is 2-methylpropyl. In certain embodiments, -R ² of formula (X -11) is 2-methylpropyl. In certain embodiments, -R ² of formula (X -11) is 1-methylpropyl. In certain embodiments, -R ^2a of formula (X -11) is -H. In certain embodiments, both -R ² and -R ^2a of formula (X -11) are methyl. In certain embodiments, -R ² of formula (X -11) is fluorine, and -R ^2a of formula (X -11) is - H. In certain embodiments, -R ² of formula (X -11) is isopropyl, and -R ^2a of formula (X -11) is - H. In certain embodiments, -R ² of formula (X -11) is 2-methylpropyl, and -R ^2a of formula (X -11) is - H. In certain embodiments In certain embodiments, q in formula (X -11) is 1. In certain embodiments, q in formula (X -11) is 2. In certain embodiments, q in formula (X -11) is 3.

In certain embodiments, -L ^1- has the formula (X -12). In certain embodiments, L ^1- has the formula (X -12) and n is 1. In certain embodiments, L ^1- has the formula (X -12) and n is 2. In certain embodiments, L ^1- has formula (X -12), and n is 3. In certain embodiments, L ^1- has the formula (X -12), and m is 1. In certain embodiments, L ^1- has the formula (X -12), and m is 2. In certain embodiments, L ^1- has the formula (X -12) and m is 3. In certain embodiments, L ^1- has the formula (X -12) and both n and m are 1. According to In certain embodiments, L ^1- has the formula (X -12), and -R ¹ represents - H. In certain embodiments, L ^1- has the formula (X -12), and -R ¹ represents methyl. In certain embodiments, L ^1- has the formula (X -12) and -R ¹ is ethyl.

In certain embodiments, -L ^1- is selected from the group consisting of

(X-a1), (X-a2), (X-a3), (X-a4), (X-a5), (X-a6), (X-a7), (X-a8), (X-a9), (X-a10), (X-a11), (X-a12), (X-a13), (X-a14), (X-a15), (X-a16), (X-a17), (X-a18), (X-a19), (X-a20), (X-a21), (X-a22), (X-a23), (X-a24), (X-a25), (X-a26), (X-a27), (X-a28), (X-a29), (X-a30), (X-a31), (X-a32), (X-a33), (X-a34), (X-a35), (X-a36), (X-a37), (X-a38), (X-a39), (X-a40), (X-a41), (X-a42), (X-a43), (X-a44), (X-a45), (X-a46), (X-a47), (X-a48), (X-a49), (X-a50), (X-a51), (X-a52), (X-a53), (X-a54), (X-a55), (X-a56), (X-a57), (X-a58), (X-a59), (X-a60), (X-a61), (X-a62), (X-a63), (X-a64), (X-a65), (X-a66), (X-a67), (X-a68), (X-a69), (X-a70), (X-a71), (X-a72), (X-a73), (X-a74), (X-a75), (X-a76), (X-a77) and (X-a78),

Where

the dotted line marked with an asterisk indicates the addition of an amine functional group -D to the nitrogen atom, and

the unlabeled dotted line shows attachment to -L ² -.

In certain embodiments, -L ^1- has the formula (X-a1). In certain embodiments, -L ^1- has the formula (X-a2). In certain embodiments, -L ^1- has the formula (X-a3). In certain embodiments, -L ^1- has the formula (X-a4). In certain embodiments, -L ^1- has the formula (X-a5). In certain embodiments, -L ^1- has the formula (X-a6). In certain embodiments, -L ^1- has the formula (X-a7). In certain embodiments, -L ^1- has the formula (X-a8). In certain embodiments, -L ^1- has the formula (X-a9). In certain embodiments, -L ^1- has the formula (X-a10). In certain embodiments, -L ^1- has the formula (X-a11). In certain embodiments, -L ^1- has the formula (X-a12). In certain embodiments, -L ^1- has the formula (X-a13). In certain embodiments, -L ^1- has the formula (X-a14). In certain embodiments, -L ^1- has the formula (X-a15). In certain embodiments, -L ^1- has the formula (X-a16). In certain embodiments, -L ^1- has the formula (X-a17). In certain embodiments, -L ^1- has the formula (X-a18). In certain embodiments, -L ^1- has the formula (X-a19). In certain embodiments, -L ^1- has the formula (X-a20). In certain embodiments, -L ^1- has the formula (X-a21). In certain embodiments, -L ^1- has the formula (X-a22). In certain embodiments, -L ^1- has the formula (X-a23). In certain embodiments, -L ^1- has the formula (X- 24). In certain embodiments, -L ^1- has the formula (X-a25). In certain embodiments, -L ^1- has the formula (X-a26). In certain embodiments, -L ^1- has the formula (X-a27). In certain embodiments, -L ^1- has the formula (X-a28). In certain embodiments, -L ^1- has the formula (X-a29). In certain embodiments, -L ^1- has the formula (X-a30). In certain embodiments, -L ^1- has the formula (X-a31). In certain embodiments, -L ^1- has the formula (X-a32). In certain embodiments, -L ^1- has the formula (X-a33). In certain embodiments, -L ^1- has the formula (X-a34). In certain embodiments, -L ^1- has the formula (X-a35). In certain embodiments, -L ^1- has the formula (X-a36). In certain embodiments, -L ^1- has the formula (X-a37). In certain embodiments, -L ^1- has the formula (X-a38). In certain embodiments, -L ^1- has the formula (X-a39). In certain embodiments, -L ^1- has the formula (X-a40). In certain embodiments, -L ^1- has the formula (X-a41). In certain embodiments, -L ^1- has the formula (X-a42). In certain embodiments, -L ^1- has the formula (X-a43). In certain embodiments, -L ^1- has the formula (X-a44). In certain embodiments, -L ^1- has the formula (X-a45). In certain embodiments, -L ^1- has the formula (X-a46). In certain embodiments, -L ^1- has the formula (X-a47). In certain embodiments, -L ^1- has the formula (X-a48). In certain embodiments, -L ^1- has the formula (X-a49). In certain embodiments, -L ^1- has the formula (X-a50). In certain embodiments, -L ^1- has the formula (X-a51). In certain embodiments, -L ^1- has the formula (X-a52). In certain embodiments, -L ^1- has the formula (X-a53). In certain embodiments, -L ^1- has the formula (X-a54). In certain embodiments, -L ^1- has the formula (X-a55). In certain embodiments, -L ^1- has the formula (X-a56). In certain embodiments, -L ^1- has the formula (X-a57). In certain embodiments, -L ^1- has the formula (X-a58). In certain embodiments, -L ^1- has the formula (X-a59). In certain embodiments, -L ^1- has the formula (X-a60). In certain embodiments, -L ^1- has the formula (X-a61). In certain embodiments, -L ^1- has the formula (X-a62). In certain embodiments, -L ^1- has the formula (X-a63). In certain embodiments, -L ^1- has the formula (X-a64). In certain embodiments, -L ^1- has the formula (X-a65). In certain embodiments, -L ^1- has the formula (X-a66). In certain embodiments, -L ^1- has the formula (X-a67). In certain embodiments, -L ^1- has the formula (X-a68). In certain embodiments, -L ^1- has the formula (X-a69). In certain embodiments, -L ^1- has the formula (X-a70). In certain embodiments, -L ^1- has the formula (X-a71). In certain embodiments, -L ^1- has the formula (X-a72). In certain embodiments, -L ^1- has the formula (X-a73). In certain embodiments, -L ^1- has the formula (X-a74). In certain embodiments, -L ^1- has the formula (X-a75). In certain embodiments, -L ^1- has the formula (X-a76). In certain embodiments, -L ^1- has the formula (X-a77). In certain embodiments, -L ^1- has the formula (X-a78).

In certain embodiments, the half-life, i.e. the time at which half of all -D fragments are released from -L ^1- is independent of pH, particularly independent for pH in the range from about 6.8 to about 7.4. Such pH-independent release is preferred since the pH of tumor tissue can vary and such pH-independence allows for more uniform and thus more predictable drug release.

Surprisingly, it was found that the L ¹ fragments of formula (X-a11) and (X-a12) have a half-life that is independent of pH over the pH range of 6.8 to 7.4.

In certain embodiments, the -L ^1- D moiety has the formula (X- b1)

(X- b1),

where the dotted line shows the attachment -L ² -.

In certain embodiments, the -L ^1- D moiety has the formula (X- b2)

(X-b2),

where the dotted line shows the attachment -L ² -.

In certain embodiments, the -L ^1- D moiety has the formula (X- b3)

(X-b3),

where the dotted line shows the attachment -L ² -.

In certain embodiments, the fragment -L ^1- D has the following structure

(X-b4),

where the dotted line shows the attachment -L ² -.

In certain embodiments, the -L ^1- D moiety has the formula (X-b5)

(X-b5),

where the dotted line shows the attachment -L ² -.

In certain embodiments, the -L ^1- D moiety has the formula (X-b6)

(X- b6),

where the dotted line shows the attachment -L ² -.

In certain embodiments, the -L ^1- D moiety has the formula (X-b7)

(X-b7),

where the dotted line shows the attachment -L ² -.

In certain embodiments, the -L ^1- D moiety has the formula (X- b8)

(X-b8),

where the dotted line shows the attachment -L ² -.

In certain embodiments, -L ^1- has formula (XI)

(XI),

Where

the dotted line shows the connection to - electronic paranor heteroaromatic N fragment -D,

n is an integer selected from the group consisting of 0, 1, 2, 3 and 4,

=X ¹ is selected from the group consisting of =O, =S and =N(R ⁴ ),

-X ^2- is selected from the group consisting of -O-, -S-, -N(R ⁵ )- and -C(R ⁶ )(R ^6a )-,

-X ^3- selected from the group consisting of , , ,

-C(R ¹⁰ )(R ^10a )-, -C(R ¹¹ )(R ^11a )-C(R ¹² )(R ^12a )-, -O- and -C(O)-,

-R¹, -R^1a, -R⁶, -R^6a, -R¹⁰, -R^10a, -R^eleven, -R^11a, -R¹², -R^12a and each of -R², and -R^2a independently selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl, where C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl optionally substituted with one or more -R¹³, which are the same or different, and where C_1-6alkyl, C_2-6 alkenyl and C_2-6 alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R¹⁴)-, -S(O)₂N(R¹⁴)-, -S(O)N(R¹⁴)-, -S(O)₂-, -S(O)-, -N(R¹⁴)S(O)₂N(R^14a)-, -S-, -N(R¹⁴)-, -OC(OR¹⁴)(R^14a)-, -N(R¹⁴)C(O)N(R^14a)- and -OC(O)N(R¹⁴)-,

-R³, -R⁴, -R⁵, -R⁷, -R⁸, and -R⁹ independently selected from the group consisting of -H, -T, -CN, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl, where C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl optionally substituted with one or more -R¹³, which are the same or different, and where C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R¹⁴)-, -S(O)₂N(R¹⁴)-, -S(O)N(R¹⁴)-, -S(O)₂-, -S(O)-, -N(R¹⁴)S(O)₂N(R^14a)-, -S-, -N(R¹⁴)-, -OC(OR¹⁴)(R^14a)-, -N(R¹⁴)C(O)N(R^14a)- and -OC(O)N(R¹⁴)-,

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, wherein each T is independently optionally substituted by one or more -R ¹³ which are the same or different,

where -R ¹³ is selected from the group consisting of -H, -NO ₂ , -OCH ₃ , -CN, -N(R ¹⁴ )(R ^14a ), -OH, -C(O)OH and C _1-6 alkyl wherein the C _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different,

where -R ¹⁴ and -R ^14a are independently selected from the group consisting of - H and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted by one or more halogens that are the same or different,

optionally one or more of the pairs -R ¹ /-R ^1a , -R ² /-R ^2a , two adjacent R ² , -R ⁶ /-R ^6a , -R ¹⁰ /-R ^10a , -R ¹¹ /-R ^11a , and -R ¹² /-R ^12a are joined together with the atom to which they are attached to form a C _3-10 cycloalkyl, 3-10 membered heterocyclyl or 8-11 membered heterobicyclyl,

optionally one or more of the pairs -R ¹ / -R ² , -R ¹ / -R ⁵ , -R ¹ / -R ⁶ , -R ¹ / -R ⁹ , -R ¹ / -R ¹⁰ , -R ³ / -R ^6a , -R ⁴ /-R ⁵ , -R ^4a /-R ⁵ , -R ⁴ /-R ⁶ , -R ⁵ /-R ¹⁰ , -R ⁶ /-R ¹⁰ , and -R4a/-R ⁶ are joined together with the atoms to which they are attached to form a ring - A-,

where - A- is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl,

optionally, -R ¹ and the adjacent -R ² form a carbon-carbon bond, provided that n is selected from the group consisting of 1, 2, 3 and 4,

optionally, two adjacent -R ² form a carbon-carbon bond, provided that n is selected from the group consisting of 2, 3 and 4,

provided that if -X ^2- is -N(R ⁵ )-, -X ^3- is selected from the group consisting of , And , and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XI) is 5, 6 or 7 atoms, and, if present, a carbon-carbon double bond formed between -R ¹ and -R ² or two adjacent -R ² , is in a cis configuration, and

where -L ^1- is substituted with -L ² -, and where -L ^1- is optionally further substituted.

It is clear that two adjacent -R ² in formula (XI) can only exist if n is at least 2.

The expression “distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk” is intended to refer to the total number of atoms at the shortest distance between the nitrogen and carbon atoms marked with an asterisk, and also includes nitrogen and carbon marked with an asterisk. For example, in the structure below, n is 1 and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk is 5:

And in the structure below, n is 2, -R ¹ and -R ^1a form a cyclohexal, and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk is 6:

.

Optional additional substituents -L ^1- of formula (XI) are as described herein.

In certain embodiments, -L ^1- of formula (XI) is not further substituted.

In certain embodiments, =X ¹ of formula (XI) is =O. In certain embodiments, =X ¹ of formula (XI) is =S. In certain embodiments, =X ¹ of formula (XI) is =N(R ⁴ ).

In certain embodiments, -X ^2- of formula (XI) is -O-. In certain embodiments, -X ^2- of formula (XI) is -S-. In certain embodiments, -X ^2- of formula (XI) is -N(R ⁵ )-. In certain embodiments, -X ^2- of formula (XI) is -C(R ⁶ )(R ^6a )-.

In certain embodiments, -X ^3- of formula (XI) is .

In certain embodiments, -X ^3- of formula (XI) is .

In certain embodiments, -X ^3- of formula (XI) is .

In certain embodiments, -X ^3- of formula (XI) is -C(R ¹⁰ )(R ^10a )-. In certain embodiments, -X ^3- of formula (XI) is -C(R ¹¹ )(R ^11a )-C(R ¹² )(R ^12a )-. In certain embodiments, -X ^3- of formula (XI) is -O-. In certain embodiments, -X ^3- of formula (XI) is -C(O)-.

In certain embodiments, -X ^2- of formula (XI) is -N(R ⁵ )-, -X ^3- is , and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XI) is 5 atoms.

In certain embodiments, -X ^2- of formula (XI) is -N(R ⁵ )-, -X ^3- is , and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XI) is 6 atoms.

In certain embodiments, -X ^2- of formula (XI) is -N(R ⁵ )-, -X ^3- is , and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XI) is 7 atoms.

In certain embodiments, -X ^2- of formula (XI) is -N(R ⁵ )-, -X ^3- is , and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XI) is 5 atoms.

In certain embodiments, -X ^2- of formula (XI) is -N(R ⁵ )-, -X ^3- is , and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XI) is 6 atoms.

In certain embodiments, -X ^2- of formula (XI) is -N(R ⁵ )-, -X ^3- is , and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XI) is 7 atoms.

In certain embodiments, -X ^2- of formula (XI) is -N(R ⁵ )-, -X ^3- is , and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XI) is 5 atoms.

In certain embodiments, -X ^2- of formula (XI) is -N(R ⁵ )-, -X ^3- is , and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XI) is 6 atoms.

In certain embodiments, -X ^2- of formula (XI) is -N(R ⁵ )-, -X ^3- is , and the distance between the nitrogen atom marked with an asterisk and the carbon atom marked with an asterisk in formula (XI) is 7 atoms.

According to certain embodiments of -R¹, -R^1a, -R⁶, -R^6a, -R¹⁰, -R^10a, -R^eleven, -R^11a, -R¹², -R^12a and each of -R², and -R^2a formula (XI) are independently selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl.

According to certain embodiments of -R¹ formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R¹ formula (XI) is selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R¹ formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R¹ formula (XI) is selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl According to certain embodiments of -R¹ formula (XI) is - H. In certain embodiments - R¹ formula (XI) is -C(O)OH. According to certain embodiments of -R¹ formula (XI) is halogen. According to certain embodiments of -R¹ formula (XI) is - F. In certain embodiments - R¹ formula (XI) is -CN. According to certain embodiments of -R¹ formula (XI) is -OH. According to certain embodiments of -R¹ formula (XI) represents C_1-6 alkyl According to certain embodiments of -R¹ formula (XI) represents C_2-6 alkenyl According to certain embodiments of -R¹ formula (XI) represents C_2-6 alkynyl. According to certain embodiments of -R¹ formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

According to certain embodiments of -R^1a formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^1a formula (XI) is selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^1a formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^1a formula (XI) is selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl. According to certain embodiments of -R^1a formula (XI) is - H. In certain embodiments - R^1a formula (XI) is -C(O)OH. According to certain embodiments of -R^1a formula (XI) is halogen. According to certain embodiments of -R^1a formula (XI) is - F. In certain embodiments - R^1a formula (XI) is -CN. According to certain embodiments of -R^1a formula (XI) is -OH. According to certain embodiments of -R^1a formula (XI) represents C_1-6 alkyl According to certain embodiments of -R^1a formula (XI) represents C_2-6 alkenyl According to certain embodiments of -R^1a formula (XI) represents C_2-6 alkynyl. According to certain embodiments of -R^1a formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

According to certain embodiments of -R⁶ formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R⁶ formula (XI) is selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R⁶ formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R⁶ formula (XI) is selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl According to certain embodiments of -R⁶ formula (XI) is - H. In certain embodiments - R⁶ formula (XI) is -C(O)OH. According to certain embodiments of -R⁶ formula (XI) is halogen. According to certain embodiments of -R⁶ formula (XI) is - F. In certain embodiments - R⁶ formula (XI) is -CN. According to certain embodiments of -R⁶ formula (XI) is -OH. According to certain embodiments of -R⁶ formula (XI) represents C_1-6 alkyl According to certain embodiments of -R⁶ formula (XI) represents C_2-6 alkenyl. According to certain embodiments of -R⁶ formula (XI) represents C_2-6 alkynyl. According to certain embodiments of -R⁶ formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

According to certain embodiments of -R^6a formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^6a formula (XI) is selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^6a formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^6a formula (XI) is selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl According to certain embodiments of -R^6a formula (XI) is - H. In certain embodiments - R^6a formula (XI) is -C(O)OH. According to certain embodiments of -R^6a formula (XI) is halogen. According to certain embodiments of -R^6a formula (XI) is - F. In certain embodiments - R^6a formula (XI) is -CN. According to certain embodiments of -R^6a formula (XI) is -OH. According to certain embodiments of -R^6a formula (XI) represents C_1-6 alkyl According to certain embodiments of -R^6a formula (XI) represents C_2-6 alkenyl According to certain embodiments of -R^6a formula (XI) represents C_2-6 alkynyl. According to certain embodiments of -R^6a formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

According to certain embodiments of -R¹⁰ formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R¹⁰ formula (XI) is selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R¹⁰ formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R¹⁰ formula (XI) is selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl According to certain embodiments of -R¹⁰ formula (XI) is - H. In certain embodiments - R¹⁰ formula (XI) is -C(O)OH. According to certain embodiments of -R¹⁰ formula (XI) is halogen. According to certain embodiments of -R¹⁰ formula (XI) is - F. In certain embodiments - R¹⁰ formula (XI) is -CN. According to certain embodiments of -R¹⁰ formula (XI) is -OH. According to certain embodiments of -R¹⁰ formula (XI) represents C_1-6 alkyl According to certain embodiments of -R¹⁰ formula (XI) represents C_2-6 alkenyl According to certain embodiments of -R¹⁰ formula (XI) represents C_2-6 alkynyl. According to certain embodiments of -R¹⁰ formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

According to certain embodiments of -R^10a formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^10a formula (XI) is selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^10a formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^10a formula (XI) is selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl According to certain embodiments of -R^10a formula (XI) is - H. In certain embodiments - R^10a formula (XI) is -C(O)OH. According to certain embodiments of -R^10a formula (XI) is halogen. According to certain embodiments of -R^10a formula (XI) is - F. In certain embodiments - R^10a formula (XI) is -CN. According to certain embodiments of -R^10a formula (XI) is -OH. According to certain embodiments of -R^10a formula (XI) represents C_1-6 alkyl According to certain embodiments of -R^10a formula (XI) represents C_2-6 alkenyl According to certain embodiments of -R^10a formula (XI) represents C_2-6 alkynyl. According to certain embodiments of -R^10a formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

According to certain embodiments of -R^eleven formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^eleven formula (XI) is selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^eleven formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^eleven formula (XI) is selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl According to certain embodiments of -R^eleven formula (XI) is - H. In certain embodiments - R^eleven formula (XI) is -C(O)OH. According to certain embodiments of -R^eleven formula (XI) is halogen. According to certain embodiments of -R^eleven formula (XI) is - F. In certain embodiments - R^eleven formula (XI) is -CN. According to certain embodiments of -R^eleven formula (XI) is -OH. According to certain embodiments of -R^eleven formula (XI) represents C_1-6 alkyl According to certain embodiments of -R^eleven formula (XI) represents C_2-6 alkenyl. According to certain embodiments of -R^eleven formula (XI) represents C_2-6 alkynyl. According to certain embodiments of -R^eleven formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

According to certain embodiments of -R^11a formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^11a formula (XI) is selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^11a formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^11a formula (XI) is selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl According to certain embodiments of -R^11a formula (XI) is - H. In certain embodiments - R^11a formula (XI) is -C(O)OH. According to certain embodiments of -R^11a formula (XI) is halogen. According to certain embodiments of -R^11a formula (XI) is - F. In certain embodiments - R^11a formula (XI) is -CN. According to certain embodiments of -R^11a formula (XI) is -OH. According to certain embodiments of -R^11a formula (XI) represents C_1-6 alkyl According to certain embodiments of -R^11a formula (XI) represents C_2-6 alkenyl According to certain embodiments of -R^11a formula (XI) represents C_2-6 alkynyl. According to certain embodiments of -R^11a formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

According to certain embodiments of -R¹² formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R¹² formula (XI) is selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R¹² formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R¹² formula (XI) is selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl According to certain embodiments of -R¹² formula (XI) is - H. In certain embodiments - R¹² formula (XI) is -C(O)OH. According to certain embodiments of -R¹² formula (XI) is halogen. According to certain embodiments of -R¹² formula (XI) is - F. In certain embodiments - R¹² formula (XI) is -CN. According to certain embodiments of -R¹² formula (XI) is -OH. According to certain embodiments of -R¹² formula (XI) represents C_1-6 alkyl According to certain embodiments of -R¹² formula (XI) represents C_2-6 alkenyl. According to certain embodiments of -R¹² formula (XI) represents C_2-6 alkynyl. According to certain embodiments of -R¹² formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

According to certain embodiments of -R^12a formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^12a formula (XI) is selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^12a formula (XI) is selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^12a formula (XI) is selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl According to certain embodiments of -R^12a formula (XI) is - H. In certain embodiments - R^12a formula (XI) is -C(O)OH. According to certain embodiments of -R^12a formula (XI) is halogen. According to certain embodiments of -R^12a formula (XI) is - F. In certain embodiments - R^12a formula (XI) is -CN. According to certain embodiments of -R^12a formula (XI) is -OH. According to certain embodiments of -R^12a formula (XI) represents C_1-6 alkyl According to certain embodiments of -R^12a formula (XI) represents C_2-6 alkenyl According to certain embodiments of -R^12a formula (XI) represents C_2-6 alkynyl. According to certain embodiments of -R^12a formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

In certain embodiments, each of -R² formula (XI) is independently selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. In certain embodiments, each of -R² formula (XI) is independently selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. In certain embodiments, each of -R² formula (XI) is independently selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. In certain embodiments, each of -R² formula (XI) is independently selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl In certain embodiments, each of -R² of formula (XI) is - H. In certain embodiments, each of -R² formula (XI) is -C(O)OH. In certain embodiments, each of -R² formula (XI) is halogen. In certain embodiments, each of -R² of formula (XI) is - F. In certain embodiments, each of -R² formula (XI) is -CN. In certain embodiments, each of -R² formula (XI) is -OH. In certain embodiments, each of -R² formula (XI) represents C_1-6 alkyl In certain embodiments, each of -R² formula (XI) represents C_2-6 alkenyl In certain embodiments, each of -R² formula (XI) represents C_2-6 alkynyl. In certain embodiments, each of -R² formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

In certain embodiments, each of -R^2a formula (XI) is independently selected from the group consisting of -H, -C(O)OH, halogen, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. In certain embodiments, each of -R^2a formula (XI) is independently selected from the group consisting of -H, -C(O)OH, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. In certain embodiments, each of -R^2a formula (XI) is independently selected from the group consisting of -H, -C(O)OH, halogen, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. In certain embodiments, each of -R^2a formula (XI) is independently selected from the group consisting of -H, -C(O)OH, -OH and C_1-6alkyl In certain embodiments, each of -R^2a of formula (XI) is - H. In certain embodiments, each of -R^2a formula (XI) is -C(O)OH. In certain embodiments, each of -R^2a formula (XI) is halogen. In certain embodiments, each of -R^2a formula (XI) is - F. In certain embodiments, each of -R^2a formula (XI) is -CN. In certain embodiments, each of -R^2a formula (XI) is -OH. In certain embodiments, each of -R^2a formula (XI) represents C_1-6 alkyl In certain embodiments, each of -R^2a formula (XI) represents C_2-6 alkenyl In certain embodiments, each of -R^2a formula (XI) represents C_2-6 alkynyl. In certain embodiments, each of -R^2a formula (XI) is selected from the group consisting of - H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, 1,1-dimethylpropyl, 2,2 -dimethylpropyl, 3-methylbutyl, 1-methylbutyl and 1-ethylpropyl.

According to certain embodiments of -R³, -R⁴, -R⁵, -R⁷, -R⁸, and -R⁹ formulas (XI) are independently selected from the group consisting of -H, -T, -CN, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R³, -R⁴, -R⁵, -R⁷, -R⁸, and -R⁹ formulas (XI) are independently selected from the group consisting of -H, -T, -CN, C_1-6 alkyl and C_2-6 alkenyl. According to certain embodiments of -R³, -R⁴, -R⁵, -R⁷, -R⁸, and -R⁹ formulas (XI) are independently selected from the group consisting of -H, -T, -CN and C_1-6 alkyl According to certain embodiments of -R³, -R⁴, -R⁵, -R⁷, -R⁸, and -R⁹ formulas (XI) are independently selected from the group consisting of -H, -T and C_1-6 alkyl According to certain embodiments of -R³, -R⁴, -R⁵, -R⁷, -R⁸, and -R⁹ formulas (XI) are independently selected from the group consisting of - H and C_1-6 alkyl

According to certain embodiments of -R³ formula (XI) is selected from the group consisting of -H, -T, -CN, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R³ formula (XI) is - H. In certain embodiments - R³ formula (XI) is -T. According to certain embodiments of -R³ formula (XI) is -CN. According to certain embodiments of -R³ formula (XI) represents C_1-6 alkyl According to certain embodiments of -R³ formula (XI) represents C_2-6 alkenyl. According to certain embodiments of -R³ formula (XI) represents C_2-6 alkynyl.

According to certain embodiments of -R⁴ formula (XI) is selected from the group consisting of -H, -T, -CN, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R⁴ formula (XI) is - H. In certain embodiments - R⁴ formula (XI) is -T. According to certain embodiments of -R⁴ formula (XI) is -CN. According to certain embodiments of -R⁴ formula (XI) represents C_1-6 alkyl According to certain embodiments of -R⁴ formula (XI) represents C_2-6 alkenyl. According to certain embodiments of -R⁴ formula (XI) represents C_2-6 alkynyl

According to certain embodiments of -R⁵ formula (XI) is selected from the group consisting of -H, -T, -CN, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R⁵ formula (XI) is - H. In certain embodiments - R⁵ formula (XI) is -T. According to certain embodiments of -R⁵ formula (XI) is -CN. According to certain embodiments of -R⁵ formula (XI) represents C_1-6 alkyl According to certain embodiments of -R⁵ formula (XI) represents C_2-6 alkenyl According to certain embodiments of -R⁵ formula (XI) represents C_2-6 alkynyl.

According to certain embodiments of -R⁷ formula (XI) is selected from the group consisting of -H, -T, -CN, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R⁷ formula (XI) is - H. In certain embodiments - R⁷ formula (XI) is -T. According to certain embodiments of -R⁷ formula (XI) is -CN. According to certain embodiments of -R⁷ formula (XI) represents C_1-6 alkyl According to certain embodiments of -R⁷ formula (XI) represents C_2-6 alkenyl. According to certain embodiments of -R⁷ formula (XI) represents C_2-6 alkynyl.

According to certain embodiments of -R⁸ formula (XI) is selected from the group consisting of -H, -T, -CN, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R⁸ formula (XI) is - H. In certain embodiments - R⁸ formula (XI) is -T. According to certain embodiments of -R⁸ formula (XI) is -CN. According to certain embodiments of -R⁸ formula (XI) represents C_1-6 alkyl According to certain embodiments of -R⁸ formula (XI) represents C_2-6 alkenyl. According to certain embodiments of -R⁸ formula (XI) represents C_2-6 alkynyl.

According to certain embodiments of -R⁹ formula (XI) is selected from the group consisting of -H, -T, -CN, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R⁹ formula (XI) is - H. In certain embodiments - R⁹ formula (XI) is -T. According to certain embodiments of -R⁹ formula (XI) is -CN. According to certain embodiments of -R⁹ formula (XI) represents C_1-6 alkyl According to certain embodiments of -R⁹ formula (XI) represents C_2-6 alkenyl. According to certain embodiments of -R⁹ formula (XI) represents C_2-6 alkynyl.

In certain embodiments, T in formula (XI) is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, _C3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl. In certain embodiments, T in formula (XI) is phenyl. In certain embodiments, T in formula (XI) is naphthyl. In certain embodiments, T in formula (XI) is indenyl. In certain embodiments, T in formula (XI) is indanyl. In certain embodiments, T in formula (XI) is tetralinyl. In certain embodiments, T in formula (XI) is C _3-10 cycloalkyl. In certain embodiments, T in formula (XI) is a 3-10 membered heterocyclyl. In certain embodiments, T in formula (XI) is an 8- to 11-membered heterobicyclyl.

In certain embodiments, T in formula (XI) is substituted with one or more -R ¹³ that are the same or different.

In certain embodiments, T in formula (XI) is replaced by a single -R ¹³ .

In certain embodiments, T in formula (XI) is not substituted with -R ¹³ .

In certain embodiments, -R ¹³ of formula (XI) is selected from the group consisting of -H, -NO ₂ , -OCH ₃ , -CN, -N(R ¹⁴ )(R ^14a ), -OH, -C(O) OH and C _1-6 alkyl.

In certain embodiments, -R ¹³ of formula (XI) is -H. In certain embodiments, -R ¹³ of formula (XI) is -NO ₂ . In certain embodiments, -R ¹³ of formula (XI) is -OCH ₃ . In certain embodiments, -R ¹³ of formula (XI) is -CN. In certain embodiments, -R ¹³ of formula (XI) is -N(R ¹⁴ )(R ^14a ). In certain embodiments, -R ¹³ of formula (XI) is -OH. In certain embodiments, -R ¹³ of formula (XI) is -C(O)OH. In certain embodiments, -R ¹³ of formula (XI) is C _1-6 alkyl.

In certain embodiments, -R ¹⁴ and -R ^14a of formula (XI) are independently selected from the group consisting of -H and C _1-6 alkyl. In certain embodiments, -R ¹⁴ of formula (XI) is - H. In certain embodiments, -R ¹⁴ of formula (XI) is C _1-6 alkyl. In certain embodiments, -R ^14a of formula (XI) is - H. In certain embodiments, -R ^14a of formula (XI) is C _1-6 alkyl.

In certain embodiments, n in formula (XI) is selected from the group consisting of 0, 1, 2, and 3. In certain embodiments, n in formula (XI) is selected from the group consisting of 0, 1, and 2. In certain embodiments n in formula (XI) is selected from the group consisting of 0 and 1. In certain embodiments, n in formula (XI) is 0. In certain embodiments, n in formula (I) is 1. In certain embodiments, n in formula (XI) is 2. In certain embodiments, n in formula (I) is 3. In certain embodiments, n in formula (XI) is 4.

In certain embodiments, -L ^1- of formula (XI) is connected to -D through a bond selected from the group consisting of amide, carbamate, dithiocarbamate, O-thiocarbamate, S-thiocarbamate, urea, thiourea, thioamide, amidine and guanidine. It is understood that some of these bonds may be irreversible in themselves, but that in the present invention, adjacent groups present in -L ^1- , such as amide, primary amine, secondary amine and tertiary amine, make these bonds reversible.

In certain embodiments, -L ^1- of formula (XI) is conjugated to -D via an amide bond, i.e. =X ¹ is =O, and -X ^2- is -C(R ⁶ )(R ^6a )-.

In certain embodiments, -L ^1- of formula (XI) is conjugated to -D via a carbamate bond, i.e. =X ¹ represents =O, and -X ^2- represents -O-.

In certain embodiments, -L ^1- of formula (XI) is conjugated to -D via a dithiocarbamate bond, i.e. =X ¹ represents =S, and -X ^2- represents -S-.

In certain embodiments, -L ^1- of formula (XI) is conjugated to -D via an O-thiocarbamate bond, i.e. =X ¹ represents =S, and -X ^2- represents -O-.

In certain embodiments, -L ^1- of formula (XI) is conjugated to -D via an S-thiocarbamate bond, i.e. =X ¹ represents =O, and -X ^2- represents -S-.

In certain embodiments, -L ^1- of formula (XI) is conjugated to -D via a urea bond, i.e. =X ¹ represents =O, and -X ^2- represents -N(R ⁵ )-.

In certain embodiments, -L ^1- of formula (XI) is conjugated to -D via a thiourea bond, i.e. =X ¹ is =S, and -X ^2- is -N(R ⁵ )-.

In certain embodiments, -L ^1- of formula (XI) is conjugated to -D via a thioamide bond, i.e. =X ¹ is =S, and -X ^2- is -C(R ⁶ )(R ^6a )-.

In certain embodiments, -L ^1- of formula (XI) is conjugated to -D via an amidine bond, i.e. =X ¹ is =N(R ⁴ ), and -X ^2- is -C(R ⁶ )(R ^6a )-.

In certain embodiments, -L ^1- of formula (XI) is conjugated to -D via a guanidine bond, i.e. =X ¹ is =N(R ⁴ ), and -X ^2- is -N(R ⁵ )-.

In certain embodiments, -L ^1- has the formula (XI´):

(XI´),

where the dotted line shows the connection to - electronic paranor heteroaromatic N in -D, and

-R ¹ , -R ^1a , -R ³ , and -R ⁴ have the meaning as defined for formula (XI).

In certain embodiments, -R ¹ and -R ^1a of formula (XI´) are both - H.

In certain embodiments, -R ¹ of formula (XI') is -H, and -R ^1a of formula (XI') is C _1-6 alkyl.

In certain embodiments, -R ³ of formula (XI') is C _1-6 alkyl.

In certain embodiments, -R ⁴ of formula (XI') is methyl.

In certain embodiments, -R ⁴ of formula (XI') is ethyl.

In certain embodiments, -L ^1- has formula (XII)

(XII),

Where

the dotted line marked with an asterisk shows the attachment to -L ² -,

The dotted line not marked with an asterisk indicates the connection to - electronic paranor heteroaromatic N in -D,

- Y- is selected from the group consisting of -N(R ³ )-, -O- and -S-,

-R¹, -R² and -R³ independently selected from the group consisting of -H, -T, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl, where C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl optionally substituted with one or more -R⁴, which are the same or different, and where C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R⁵)-, -S(O)₂N(R⁵)-, -S(O)N(R⁵)-, -S(O)₂-, -S(O)-, -N(R⁵)S(O)₂N(R^5a)-, -S-, -N(R⁵), -OC(OR⁵)(R^5a)-, -N(R⁵)C(O)N(R^5a)- and -OC(O)N(R⁵)-,

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, wherein each T is independently optionally substituted by one or more -R ⁴ which are the same or different,

wherein -R ⁴ , -R ⁵ , and -R ^5a are independently selected from the group consisting of - H and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different, and

where -L ^1- is substituted with -L ² -, and where -L ^1- is optionally further substituted.

Optional additional substituents -L ^1- of formula (XII) are as described herein.

In certain embodiments, -L ^1- of formula (XII) is further unsubstituted.

In certain embodiments, -Y- of formula (XII) is -N(R ³ )-.

In certain embodiments, -Y- of formula (XII) is -O-.

In certain embodiments, -Y- of formula (XII) is -S-.

According to certain embodiments of -R¹, -R², and -R³ formulas (XII) are independently selected from the group consisting of -H, -T, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl.

According to certain embodiments of -R¹ formula (XII) is independently selected from the group consisting of -H, -T, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R¹ formula (XII) is - H. In certain embodiments - R¹ formula (XII) is -T. According to certain embodiments of -R¹ formula (XII) represents C_1-6 alkyl According to certain embodiments of -R¹ formula (XII) represents C_2-6 alkenyl. According to certain embodiments of -R¹ formula (XII) represents C_2-6 alkynyl.

According to certain embodiments of -R² formula (XII) is independently selected from the group consisting of -H, -T, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R² formula (XII) is - H. In certain embodiments - R² formula (XII) is -T. According to certain embodiments of -R² formula (XII) represents C_1-6 alkyl According to certain embodiments of -R² formula (XII) represents C_2-6 alkenyl. According to certain embodiments of -R² formula (XII) represents C_2-6 alkynyl.

According to certain embodiments of -R³ formula (XII) is independently selected from the group consisting of -H, -T, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R³ formula (XII) is - H. In certain embodiments - R³ formula (XII) is -T. According to certain embodiments of -R³ formula (XII) represents C_1-6 alkyl According to certain embodiments of -R³ formula (XII) represents C_2-6 alkenyl. According to certain embodiments of -R³ formula (XII) represents C_2-6 alkynyl.

In certain embodiments, T in formula (XII) is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, _C3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl. In certain embodiments, T in formula (XII) is phenyl. In certain embodiments, T in formula (XII) is naphthyl. In certain embodiments, T in formula (XII) is indenyl. In certain embodiments, T in formula (XII) is indanyl. In certain embodiments, T in formula (XII) is tetralinyl. In certain embodiments, T in formula (XII) is C _3-10 cycloalkyl. In certain embodiments, T in formula (XII) is a 3-10 membered heterocyclyl. In certain embodiments, T in formula (XII) is an 8- to 11-membered heterobicyclyl.

In certain embodiments, T in formula (XII) is substituted with one or more -R ⁴ .

In certain embodiments, T in formula (XII) is replaced by one -R ⁴ .

In certain embodiments, T in formula (XII) is not substituted with -R ⁴ .

In certain embodiments, -R ⁴ , -R ⁵ and -R ^5a of formula (XII) are independently selected from the group consisting of - H and C _1-6 alkyl.

In certain embodiments, -R ⁴ of formula (XII) is selected from the group consisting of - H and C _1-6 alkyl. In certain embodiments, -R ⁴ of formula (XII) is - H. In certain embodiments, -R ⁴ of formula (XII) is C _1-6 alkyl.

In certain embodiments, -R ⁵ of formula (XII) is selected from the group consisting of - H and C _1-6 alkyl. In certain embodiments, -R ⁵ of formula (XII) is - H. In certain embodiments, -R ⁵ of formula (XII) is C _1-6 alkyl.

In certain embodiments, -R ^5a of formula (XII) is selected from the group consisting of -H and C _1-6 alkyl. In certain embodiments, -R ^5a of formula (XII) is - H. In certain embodiments, -R ^5a of formula (XII) is C _1-6 alkyl.

In certain embodiments, -L ^1- of formula (XII) is connected to -D via a geminal linkage.

In certain embodiments, -L ^1- of formula (XII) is connected to -D via an aminal linkage.

In certain embodiments, -L ^1- of formula (XII) is connected to -D via a hemitioaminal bond.

Drugable fragment L1 D which when bound to L1 contains an electron-donating heteroaromatic N ⁺ moiety or quaternary ammonium cation and becomes a -D ⁺ fragment upon binding to L1 has formula (XIII)

A fragment -L ^1- suitable for D drugs, which when bound to -L ^1- contains an electron-donating heteroaromatic N ⁺ moiety or quaternary ammonium cation and becomes a fragment -D ⁺ when bound to -L ^1- has the formula (XIII )

(XIII)

Where

the dotted line marked with an asterisk shows the attachment to -L ² -, the unstarred dotted line shows the attachment to N ⁺ in -D ⁺ ,

- Y ^# - selected from the group consisting of -N(R ^#3 )-, -O- and -S-,

-R^#1, -R^#2, and -R^#3 independently selected from the group consisting of -H, -T^#, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl, where C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl optionally substituted with one or more -R^#4, which are the same or different, and where C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of -T^#-, -C(O)O-, -O-, -C(O)-, -C(O)N(R^#5)-, -S(O)₂N(R^#5)-, -S(O)N(R^#5)-, -S(O)₂-, -S(O)-, -N(R^#5)S(O)₂N(R^#5a)-, -S-, -N(R^#5), -OC(OR^#5)(R^#5a)-, -N(R^#5)C(O)N(R^#5a)- and -OC(O)N(R^#5)-,

each T ^# is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, _C3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, wherein each T ^# is independently optionally substituted with one or more -R ^#4 that are the same or different, and

wherein -R ^#4 , -R ^#5 , and -R ^#5a are independently selected from the group consisting of -H and _C1-6 alkyl, wherein the _C1-6 alkyl is optionally substituted with one or more halogens that are the same or different , And

each -L ^1- is substituted with -L ^2- and optionally further substituted.

It will be understood that in certain embodiments -D⁺ may contain both electron-donating heteroaromatic N⁺, and a quaternary ammonium cation, and similarly the corresponding D may contain both an electron-donating heteroaromatic N and a tertiary amine. It is also clear that if D is conjugated to -L^1-, then -D⁺And -L^1-form a quaternary ammonium cation, which may have a counterion. Examples of counteranions include, but are not limited to, chloride, bromide, acetate, bicarbonate, sulfate, bisulfate, nitrate, carbonate, alkylsulfonate, arylsulfonate, and phosphate.

Such drug moiety -D ⁺ contains at least one, such as one, two, three, four, five, six, seven, eight, nine or ten, electron-donating heteroaromatic N ⁺ or quaternary ammonium cations, and likewise a corresponding released drug D contains at least one, such as one, two, three, four, five, six, seven, eight, nine or ten, electron-donating heteroaromatic N or tertiary amines. Examples of chemical structures involving heteroaromatic nitrogens i.e. N ⁺ or N that is an electron donor for the aromatic π system includes, but is not limited to, pyridine, pyridazine, pyrimidine, quinoline, quinazoline, quinoxaline, pyrazole, imidazole, isoindazole, indazole, purine, tetrazole, triazole and triazine. For example, in the imidazole ring below is heteroaromatic nitrogen, which is a one-electron donor for the aromatic - systems marked “§”:

.

Such electron-donating heteroaromatic nitrogen atoms do not contain heteroaromatic nitrogen atoms that donate one electron pair (i.e., more than one electron) to the aromatic π system, such as, for example, nitrogen, which is marked with a “#” in the above imidazole ring structure . Drug D may exist in one or more tautomeric forms, for example, with one hydrogen atom moving between at least two heteroaromatic nitrogen atoms. In all such cases, the linker moiety is covalently and reversibly attached to the heteroaromatic nitrogen, which is the electron donor for the aromatic π system.

In certain embodiments, -Y ^# - of formula (XIII) is -N(R ^#3 )-. In certain embodiments, -Y ^# - of formula (XI) is -O-. In certain embodiments, -Y ^# - of formula (XI) is -S-.

According to certain embodiments of -R^#1, -R^#2, and -R^#3 formulas (XIII) are independently selected from the group consisting of -H, -T^#, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl.

According to certain embodiments of -R^#1 formula (XIII) is independently selected from the group consisting of -H, -T^#, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^#1 formula (XIII) is - H. In certain embodiments - R^#1 formula (XIII) represents -T^#. According to certain embodiments of -R^#1 formula (XI) represents C_1-6 alkyl According to certain embodiments of -R^#1 formula (XIII) represents C_2-6 alkenyl. According to certain embodiments of -R^#1 formula (XIII) represents C_2-6 alkynyl.

According to certain embodiments of -R^#2 formula (XIII) is independently selected from the group consisting of -H, -T^#, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^#2 formula (XI) is - H. In certain embodiments - R² formula (XIII) represents -T^#. According to certain embodiments of -R^#2 formula (XI) represents C_1-6 alkyl According to certain embodiments of -R^#2 formula (XIII) represents C_2-6 alkenyl. According to certain embodiments of -R^#2 formula (XIII) represents C_2-6 alkynyl.

In certain embodiments, -R^#3 formula (XIII) is independently selected from the group consisting of -H, -T^#, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R^#3 formula (XIII) is - H. In certain embodiments - R^#3 formula (XIII) represents -T^#. In certain embodiments, -R^#3 represents C_1-6 alkyl According to certain embodiments of -R^#3 formula (XIII) represents C_2-6 alkenyl. According to certain embodiments of -R^#3 formula (XIII) represents C_2-6 alkynyl.

In certain embodiments, the T ^# of formula (XIII) is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, _C3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl. In certain embodiments, T ^# of formula (XIII) is phenyl. In certain embodiments, T ^# of formula (XIII) is naphthyl. In certain embodiments, T ^# of formula (XIII) is indenyl. In certain embodiments, T ^# of formula (XIII) is indanyl. In certain embodiments, T ^# of formula (XIII) is tetralinyl. In certain embodiments, T ^# of formula (XIII) is C _3-10 cycloalkyl. In certain embodiments, T ^# of formula (XIII) is a 3-10 membered heterocyclyl. In certain embodiments, T ^# of formula (XIII) is an 8- to 11-membered heterobicyclyl. In certain embodiments, T ^# of formula (XIII) is substituted with one or more -R ⁴ .

In certain embodiments, the T ^# of formula (XIII) is replaced by a single -R ⁴ .

In certain embodiments, T ^# of formula (XIII) is not substituted with -R ⁴ .

In certain embodiments, -R ^#4 , -R ^#5 , and -R ^#5a of formula (XIII) are independently selected from the group consisting of -H and _C1-6 alkyl.

In certain embodiments, -R ^#4 of formula (XIII) is selected from the group consisting of -H and C _1-6 alkyl. In certain embodiments, -R ^#4 of formula (XIII) is - H. In certain embodiments, -R ^#4 of formula (XIII) is C _1-6 alkyl.

In certain embodiments, -R ^#5 of formula (XIII) is selected from the group consisting of -H and C _1-6 alkyl. In certain embodiments, ^-R5 of formula (XIII) is -H. In certain embodiments, -R ^#5 of formula (XIII) is _C1-6 alkyl.

In certain embodiments, -R ^#5a of formula (XIII) is selected from the group consisting of -H and C _1-6 alkyl. In certain embodiments, -R ^#5a of formula (XIII) is - H. In certain embodiments, -R ^#5a of formula (XIII) is C _1-6 alkyl.

A fragment -L ^1- suitable for D drugs, which when bound to -L ^1- contains an electron-donating heteroaromatic N ⁺ moiety or quaternary ammonium cation and becomes a fragment -D ⁺ when bound to -L ^1- has the formula (XIV )

(XIV)

Where

the dotted line shows the addition to N ⁺ of -D ⁺ ,

t is selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6,

- A- is a ring selected from the group consisting of monocyclic or bicyclic aryl and heteroaryl, provided that - A- is connected to - Y and -C(R ¹ )(R ^1a )- through carbon atoms, wherein said monocyclic or bicyclic aryl or heteroaryl is optionally substituted by one or more -R ² which are the same or different,

-R¹, -R^1a and every -R²independently selected from the group consisting of -H, -C(O)OH, -halogen, -NO₂, -CN, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl, where C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl optionally substituted with one or more -R³, which are the same or different, and where C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R⁴)-, -S(O)₂N(R⁴)-, -S(O)N(R⁴)-, -S(O)₂-, -S(O)-, -N(R⁴)S(O)₂N(R^4a)-, -S-, -N(R⁴)-, -OC(OR⁴)(R^4a)-, -N(R⁴)C(O)N(R^4a)- and -OC(O)N(R⁴)-,

each -T- is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, wherein each -T- independently is optional substituted with one or more -R ³ which are the same or different,

where -R ³ is selected from the group consisting of -H, -NO ₂ , -OCH ₃ , -CN, -N(R ⁴ )(R ^4a ), -OH, -C(O)OH and C _1-6 alkyl wherein the C _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different,

where -R ⁴ and -R ^4a are independently selected from the group consisting of - H and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different,

- Y is selected from the group consisting of:

, , , , , , , , , , and peptidyl fragment,

Where

the dotted line marked with an asterisk shows the connection to -A-,

-Nu is a nucleophile,

- Y ^1- is selected from the group consisting of -O-, -C(R ¹⁰ )(R ^10a )-, -N(R ¹¹ )- and -S-,

=Y ² is selected from the group consisting of =O, =S and =N(R ¹² ),

-Y ^3- is selected from the group consisting of -O-, -S- and -N(R ¹³ ),

- E- selected from the group consisting of C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl and -Q-, where C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl optionally substituted with one or more -R¹⁴, which are the same or different,

-R⁵, -R⁶, each -R⁷, -R⁸, -R⁹, -R¹⁰, -R^10a, -R^eleven, -R¹², and -R¹³ independently selected from a group consisting of C_1-20 alkyl, C_2-20 alkenyl, C_2-20 alkynyl and - Q, where C_1-20alkyl, C_2-20 alkenyl and C_2-20 alkynyl optionally substituted with one or more -R¹⁴, which are the same or different, and where C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of Q, -C(O)O-, -O-, -C(O)-, -C(O)N(R¹⁵)-, -S(O)₂N(R¹⁵), -S(O)N(R¹⁵)-, -S(O)₂-, -S(O)-, -N(R¹⁵)S(O)₂N(R^15a)-, -S-, -N(R¹⁵)-, -OC(OR¹⁵)R^15a-, -N(R¹⁵)C(O)N(R^15a)- and -OC(O)N(R¹⁵)-,

each Q is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, wherein each Q is independently optionally substituted by one or more -R ¹⁴ which are the same or different,

wherein -R ¹⁴ , -R ¹⁵ , and -R ^15a are independently selected from the group consisting of - H and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different, and

each -L ^1- is substituted with -L ^2- and optionally further substituted.

It will be understood that in certain embodiments -D⁺ may contain both electron-donating heteroaromatic N⁺, and a quaternary ammonium cation, and similarly the corresponding D may contain both an electron-donating heteroaromatic N and a tertiary amine. It is also clear that if D is conjugated to -L^1-, then -D⁺And -L^1-form a quaternary ammonium cation, which may have a counterion. Examples of counteranions include, but are not limited to, chloride, bromide, acetate, bicarbonate, sulfate, bisulfate, nitrate, carbonate, alkylsulfonate, arylsulfonate, and phosphate.

Optional additional substituents -L ^1- of formula (XIV) are as described herein.

In certain embodiments, -L ^1- of formula (XIV) is not further substituted.

Such drug moiety -D ⁺ contains at least one, such as one, two, three, four, five, six, seven, eight, nine or ten, electron-donating heteroaromatic N ⁺ or quaternary ammonium cations, and likewise a corresponding released drug D contains at least one, such as one, two, three, four, five, six, seven, eight, nine or ten, electron-donating heteroaromatic N or tertiary amines. Examples of chemical structures involving heteroaromatic nitrogens i.e. N ⁺ or N that is an electron donor for the aromatic π system includes, but is not limited to, pyridine, pyridazine, pyrimidine, quinoline, quinazoline, quinoxaline, pyrazole, imidazole, isoindazole, indazole, purine, tetrazole, triazole and triazine. For example, in the imidazole ring below is heteroaromatic nitrogen, which is a one-electron donor for the aromatic - systems marked “§”:

.

Such electron-donating heteroaromatic nitrogen atoms do not contain heteroaromatic nitrogen atoms that donate one electron pair (i.e., more than one electron) to the aromatic π system, such as, for example, nitrogen, which is marked with a “#” in the above imidazole ring structure . Drug D may exist in one or more tautomeric forms, for example, with one hydrogen atom moving between at least two heteroaromatic nitrogen atoms. In all such cases, the linker moiety is covalently and reversibly attached to the heteroaromatic nitrogen, which is the electron donor for the aromatic π system.

In the context of the present invention, the term "monocyclic or bicyclic aryl" means an aromatic hydrocarbon ring system, which may be monocyclic or bicyclic, where the monocyclic aryl ring consists of at least 5 ring carbon atoms and may contain up to 10 ring carbon atoms, and where the bicylic aryl ring the ring consists of at least 8 ring carbon atoms and may include up to 12 ring carbon atoms. Each hydrogen atom of a monocyclic or bicyclic aryl may be replaced by a substituent as defined below.

In the context of the present invention, the term "monocyclic or bicyclic heteroaryl" means a monocyclic aromatic ring system, which may contain from 2 to 6 ring carbon atoms and from 1 to 3 ring heteroatoms, or a bicyclic aromatic ring system, which may contain from 3 to 9 ring atoms carbon and 1 to 5 ring heteroatoms such as nitrogen, oxygen and sulfur. Examples of monocyclic or bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzothiophenyl, furanyl, imidazolyl, indolyl, azaindolyl, azabenzimidazolyl, benzoxazolyl, benzthiazolyl, benzthiadiazolyl, benzotriazolyl, tetrazinyl, tetrazolyl, isothiazolyl, oxazolyl, isoxazolyl, pyrazinyl, pyrazolyl, zinyl, pyridinyl, pyrimidinyl , pyrrolyl, quinolinyl, quinazolinyl, quinoxalinyl, triazolyl, thiazolyl and thiophenyl. Each hydrogen atom of a monocyclic or bicyclic heteroaryl may be replaced by a substituent as defined below.

In the context of the present invention, the term "nucleophile" refers to a reagent or functional group that forms a bond with its reaction partner, i.e. electrophile, by donating both bonding electrons.

In certain embodiments, T in Formula (XIV) is 0. In certain embodiments, T in Formula (XIV) is 1. In certain embodiments, T in Formula (XIV) is 2. In certain embodiments, T in Formula (XIV) is 2. In certain embodiments, T in Formula (XIV) is 2. XIV) is 3. In certain embodiments, T in formula (XIV) is 4. In certain embodiments, T in formula (XIV) is 5. In certain embodiments, T in formula (XIV) is 6.

In certain embodiments, - A- of formula (XIV) is a ring selected from the group consisting of monocyclic or bicyclic aryl and heteroaryl. In certain embodiments, -A- of formula (XIV) is substituted with one or more -R ² that are the same or different. In certain embodiments, -A- of formula (XIV) is not substituted with -R ² . In certain embodiments, - A- of formula (XIV) is selected from the group consisting of:

where each V is independently selected from the group consisting of O, S and N.

According to certain embodiments of -R¹, -R^1a and every -R² formula (XIV) are independently selected from the group consisting of -H, -C(O)OH, -halogen, -CN, -NO₂, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. According to certain embodiments of -R¹ formula (XIV) is - H. In certain embodiments - R¹ formula (XIV) is -C(O)OH. According to certain embodiments of -R¹ formula (XIV) represents - halogen. According to certain embodiments of -R¹ formula (XIV) is - F. In certain embodiments - R¹ formula (XIV) is -CN. According to certain embodiments of -R¹ formula (XIV) represents -NO₂. According to certain embodiments of -R¹ formula (XIV) is -OH. According to certain embodiments of -R¹ formula (XIV) represents C_1-6 alkyl According to certain embodiments of -R¹ formula (XIV) represents C_2-6 alkenyl According to certain embodiments of -R¹ represents C_2-6 alkynyl. According to certain embodiments of -R^1a formula (XIV) is - H. In certain embodiments - R^1a formula (XIV) is -C(O)OH. According to certain embodiments of -R^1a formula (XIV) represents - halogen. According to certain embodiments of -R^1a formula (XIV) is - F. In certain embodiments - R^1a formula (XIV) is -CN. According to certain embodiments of -R^1a formula (XIV) represents -NO₂. According to certain embodiments of -R^1a formula (XIV) is -OH. According to certain embodiments of -R^1a formula (XIV) represents C_1-6 alkyl According to certain embodiments of -R^1a formula (XIV) represents C_2-6 alkenyl According to certain embodiments of -R^1a formula (XIV) represents C_2-6 alkynyl.

In certain embodiments, each of -R² formula (XIV) is independently selected from the group consisting of -H, -C(O)OH, -halogen, -CN, -NO₂, -OH, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl. In certain embodiments, each of -R² formula (XIV) is - H. In certain embodiments, each of -R² formula (XIV) is -C(O)OH. In certain embodiments, each of -R² formula (XIV) represents - halogen. In certain embodiments, each of -R² formula (XIV) is - F. In certain embodiments, each of -R² formula (XIV) is -CN. In certain embodiments, each of -R² formula (XIV) represents -NO₂. In certain embodiments, each of -R² formula (XIV) is -OH. In certain embodiments, each of -R² formula (XIV) represents C_1-6 alkyl In certain embodiments, each of -R² formula (XIV) represents C_2-6 alkenyl. In certain embodiments, each of -R² formula (XIV) represents C_2-6 alkynyl.

In certain embodiments, T in formula (XIV) is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, _C3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl. In certain embodiments, T in formula (XIV) is phenyl. In certain embodiments, T in formula (XIV) is naphthyl. In certain embodiments, T in formula (XIV) is indenyl. In certain embodiments, T in formula (XIV) is indanyl. In certain embodiments, T in formula (XIV) is tetralinyl. In certain embodiments, T in formula (XIV) is C _3-10 cycloalkyl. In certain embodiments, T in formula (XIV) is a 3-10 membered heterocyclyl. In certain embodiments, T in formula (XIV) is an 8- to 11-membered heterobicyclyl.

In certain embodiments, T in formula (XIV) is substituted with one or more -R ³ that are the same or different. In certain embodiments, T in formula (XIV) is replaced by a single -R ³ . In certain embodiments, T in formula (XIV) is not substituted with -R ³ .

In certain embodiments, -R ³ of formula (XIV) is selected from the group consisting of -H, -NO ₂ , -OCH ₃ , -CN, -N(R ⁴ )(R ^4a ), -OH, -C(O) OH and C _1-6 alkyl. In certain embodiments, -R ³ of formula (XIV) is -H. In certain embodiments, -R ³ of formula (XIV) is -NO ₂ . In certain embodiments, -R ³ of formula (XIV) is -OCH ₃ . In certain embodiments, -R ³ of formula (XIV) is -CN. In certain embodiments, -R ³ of formula (XIV) is -N(R ⁴ )(R ^4a ). In certain embodiments, -R ³ of formula (XIV) is -OH. In certain embodiments, -R ³ of formula (XIV) is -C(O)OH. In certain embodiments, -R ³ of formula (XIV) is C _1-6 alkyl. In certain embodiments, -R ⁴ and -R ^4a of formula (XIV) are independently selected from the group consisting of - H and C _1-6 alkyl. In certain embodiments, -R ⁴ of formula (XIV) is - H. In certain embodiments, -R ⁴ is C _1-6 alkyl. In certain embodiments, -R ^4a of formula (XIV) is - H. In certain embodiments, -R ^4a of formula (XIV) is C _1-6 alkyl.

In certain embodiments, Y of formula (XIV) is

,

where -Nu, -E, -Y ^1- , =Y ² and -Y ^3- have the meanings as defined herein, and the dotted line marked with an asterisk indicates the addition to -A- of formula (XIV).

In certain embodiments, -Nu of formula (XIV) is a nucleophile selected from the group consisting of a primary, secondary, tertiary amine and amide. In certain embodiments, -Nu of formula (XIV) is a primary amine. In certain embodiments, -Nu of formula (XIV) is a secondary amine. In certain embodiments, -Nu of formula (XIV) is a tertiary amine. In certain embodiments, -Nu of formula (XIV) is an amide.

In certain embodiments, -Y ^1- of formula (XIV) is selected from the group consisting of -O-, -C(R ¹⁰ )(R ^10a )-, -N(R ¹¹ )- and -S-. In certain embodiments, -Y ^1- of formula (XIV) is -O-. In certain embodiments, -Y ^1- of formula (XIV) is -C(R ¹⁰ )(R ^10a )-. In certain embodiments, -Y ^1- of formula (XIV) is -N(R ¹¹ )-. In certain embodiments, -Y ^1- is -S-.

In certain embodiments, =Y ² of formula (XIV) is selected from the group consisting of =O, =S, and =N(R ¹² ). In certain embodiments, =Y ² of formula (XIV) is =O. In certain embodiments, =Y ² of formula (XIV) is =S. In certain embodiments, =Y ² of formula (XIV) is =N(R ¹² ).

In certain embodiments, -Y ^3- of formula (XIV) is selected from the group consisting of -O-, -S-, and -N(R ¹³ ). In certain embodiments, -Y ^3- of formula (XIV) is -O-. In certain embodiments, -Y ^3- of formula (XIV) is -S-. In certain embodiments, -Y ^3- of formula (XIV) is -N(R ¹³ ).

In certain embodiments, -Y ^1- of formula (XIV) is -N(R ¹¹ )-, =Y ² of formula (XIV) is =O, and -Y ^3- is -O-.

In certain embodiments, -Y ^1- of formula (XIV) is -N(R ¹¹ )-, =Y ² of formula (XIV) is =O, -Y ^3- of formula (XIV) is -O- and -Nu formula (XIV) is -N(CH ₃ ) ₂ .

In certain embodiments, - E- of formula (XIV) is selected from the group consisting of C_1-6 alkyl, C_2-6 alkenyl, C_2-6 alkynyl and -Q-. In certain embodiments, E- of formula (XIV) is C_1-6 alkyl In certain embodiments, E- of formula (XIV) is C_2-6 alkenyl. In certain embodiments, E- of formula (XIV) is C_2-6 alkynyl. In certain embodiments, - E- of formula (XIV) is - Q-.

In certain embodiments, q in formula (XIV) is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl. In certain embodiments, q in formula (XIV) is phenyl. In certain embodiments, q in formula (XIV) is naphthyl. In certain embodiments, q in formula (XIV) is indenyl. In certain embodiments, q in formula (XIV) is indanyl. In certain embodiments, q in formula (XIV) is tetralinyl. In certain embodiments, q in formula (XIV) is C _3-10 cycloalkyl. In certain embodiments, q in formula (XIV) is a 3-10 membered heterocyclyl. In certain embodiments, q in formula (XIV) is an 8- to 11-membered heterobicyclyl. In certain embodiments, q in formula (XIV) is substituted with one or more -R ¹⁴ . In certain embodiments, q in formula (XIV) is not substituted with -R ¹⁴ .

According to certain embodiments of -R⁵, -R⁶, each -R⁷, -R⁸, -R⁹, -R¹⁰, -R^10a, -R^eleven, -R¹², and -R¹³ formulas (XIV) are independently selected from the group consisting of C_1-20 alkyl, C_2-20 alkenyl, C_2-20 alkynyl and - Q.

According to certain embodiments of -R⁵ formula (XIV) represents C_1-20 alkyl According to certain embodiments of -R⁵ formula (XIV) represents C_2-20 alkenyl. According to certain embodiments of -R⁵ formula (XIV) represents C_2-20 alkynyl. According to certain embodiments of -R⁵ formula (XIV) represents - Q.

According to certain embodiments of -R⁶ formula (XIV) represents C_1-20 alkyl According to certain embodiments of -R⁶ formula (XIV) represents C_2-20 alkenyl. According to certain embodiments of -R⁶ formula (XIV) represents C_2-20 alkynyl. According to certain embodiments of -R⁶ represents - Q.

In certain embodiments, each of -R⁷ formula (XIV) is independently selected from the group consisting of C_1-20 alkyl, C_2-20 alkenyl, C_2-20 alkynyl and -Q. In certain embodiments, each of -R⁷ formula (XIV) represents C_1-20 alkyl In certain embodiments, each of -R⁷ formula (XIV) represents C_2-20 alkenyl. In certain embodiments, each of -R⁷ formula (XIV) represents C_2-20 alkynyl. In certain embodiments, each of -R⁷ formula (XIV) represents - Q.

According to certain embodiments of -R⁸ formula (XIV) represents C_1-20 alkyl According to certain embodiments of -R⁸ formula (XIV) represents C_2-20 alkenyl. According to certain embodiments of -R⁸ formula (XIV) represents C_2-20 alkynyl. According to certain embodiments of -R⁸ formula (XIV) represents - Q.

According to certain embodiments of -R⁹ formula (XIV) represents C_1-20 alkyl According to certain embodiments of -R⁹ formula (XIV) represents C_2-20 alkenyl. According to certain embodiments of -R⁹ formula (XIV) represents C_2-20 alkynyl. According to certain embodiments of -R⁹ formula (XIV) represents - Q.

According to certain embodiments of -R¹⁰ formula (XIV) represents C_1-20 alkyl According to certain embodiments of -R¹⁰ formula (XIV) represents C_2-20 alkenyl. According to certain embodiments of -R¹⁰ formula (XIV) represents C_2-20 alkynyl. According to certain embodiments of -R¹⁰ formula (XIV) represents - Q.

According to certain embodiments of -R^10a formula (XIV) represents C_1-20 alkyl According to certain embodiments of -R^10a formula (XIV) represents C_2-20 alkenyl. According to certain embodiments of -R^10a formula (XIV) represents C_2-20 alkynyl. According to certain embodiments of -R^10a formula (XIV) represents - Q.

According to certain embodiments of -R^eleven formula (XIV) represents C_1-20 alkyl According to certain embodiments of -R^eleven formula (XIV) represents C_2-20 alkenyl According to certain embodiments of -R^eleven formula (XIV) represents C_2-20 alkynyl. According to certain embodiments of -R^eleven formula (XIV) represents - Q.

According to certain embodiments of -R¹² formula (XIV) represents C_1-20 alkyl According to certain embodiments of -R¹² formula (XIV) represents C_2-20 alkenyl. According to certain embodiments of -R¹² formula (XIV) represents C_2-20 alkynyl. According to certain embodiments of -R¹² formula (XIV) represents - Q.

According to certain embodiments of -R¹³ formula (XIV) represents C_1-20 alkyl According to certain embodiments of -R¹³ formula (XIV) represents C_2-20 alkenyl. According to certain embodiments of -R¹³ formula (XIV) represents C_2-20 alkynyl. According to certain embodiments of -R¹³ formula (XIV) represents - Q.

In certain embodiments, -R ¹⁴ , -R ¹⁵ , and -R ^15a of formula (XIV) are selected from the group consisting of - H and C _1-6 alkyl.

In certain embodiments, -R ¹⁴ of formula (XIV) is - H. In certain embodiments, -R ¹⁴ of formula (XIV) is C _1-6 alkyl.

In certain embodiments, -R ¹⁵ of formula (XIV) is - H. In certain embodiments, -R ¹⁵ of formula (XIV) is C _1-6 alkyl.

In certain embodiments, -R ^15a of formula (XIV) is - H. In certain embodiments, -R ^15a of formula (XIV) is C _1-6 alkyl.

In certain embodiments, Y of formula (XIV) is

where -R ⁵ has the meanings as defined above and the dotted line marked with an asterisk indicates the attachment to -A-.

In certain embodiments, Y of formula (XIV) is

where -R ⁶ has the meanings as defined above and the dotted line marked with an asterisk indicates the attachment to -A-.

In certain embodiments, -R ⁶ of formula (XIV) has formula (XIVa):

(XIVa),

where - Y ⁴ - is selected from the group consisting of C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, which are optionally substituted by one or more -R ¹⁸ , which are the same or different,

-R¹⁶, and -R¹⁷ independently selected from the group consisting of - H, C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl, where C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl optionally substituted with one or more -R¹⁸, which are the same or different, and where C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of -A´-, -C(O)O-, -O-, -C(O)-, -C(O)N(R¹⁹)-, -S(O)₂N(R¹⁹), -S(O)N(R¹⁹)-, -S(O)₂-, -S(O)-, -N(R¹⁹)S(O)₂N(R^19a)-, -S-, -N(R¹⁹)-, -OC(OR¹⁹)R^19a-, -N(R¹⁹)C(O)N(R^19a)-, -OC(O)N(R¹⁹)- and -N(R¹⁹)C(NH)N(R^19a)-,

each A' is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, wherein each A' is independently optionally substituted with one or more -R ¹⁸ which are the same or different,

wherein -R ¹⁸ , -R ¹⁹ and -R ^19a are independently selected from the group consisting of - H and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different, and

where the dotted line marked with an asterisk shows the attachment to the remaining part - Y.

In certain embodiments, Y ⁴ of formula (XIVa) is selected from the group consisting of C _3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl. In certain embodiments, Y ⁴ of formula (XIVa) is C _3-10 cycloalkyl. In certain embodiments, Y ⁴ of formula (XIVa) is a 3-10 membered heterocyclyl. In certain embodiments, Y ⁴ of formula (XIVa) is an 8-11 membered heterobicyclyl. In certain embodiments, -Y ⁴ - of formula (XIVa) is substituted with one or more -R ¹⁸ that are the same or different. In certain embodiments, -Y ⁴ of formula (XIVa) is not substituted with -R ¹⁸ .

According to certain embodiments of -R¹⁶, and -R¹⁷ formulas (XIVa) are selected from the group consisting of C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl. According to certain embodiments of -R¹⁶ formula (XIVa) represents C_{1- 10} alkyl According to certain embodiments of -R¹⁶ formula (XIVa) represents C_2-10 alkenyl According to certain embodiments of -R¹⁶ formula (XIVa) represents C_2-10 alkynyl. According to certain embodiments of -R¹⁷ formula (XIVa) represents C_{1- 10} alkyl According to certain embodiments of -R¹⁷ formula (XIVa) represents C_2-10 alkenyl. According to certain embodiments of -R¹⁷ formula (XIVa) represents C_2-10 alkynyl.

In certain embodiments, A' of formula (XIVa) is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl. In certain embodiments, A' of formula (XIVa) is phenyl. In certain embodiments, A' of formula (XIVa) is naphthyl. In certain embodiments, A' of formula (XIVa) is indenyl. In certain embodiments, A' of formula (XIVa) is indanyl. In certain embodiments, A' of formula (XIVa) is tetralinyl. In certain embodiments, A' of formula (XIVa) is C _3-10 cycloalkyl. In certain embodiments, A' of formula (XIVa) is a 3-10 membered heterocyclyl. In certain embodiments, A' of formula (XIVa) is an 8- to 11-membered heterobicyclyl.

In certain embodiments, A' of formula (XIVa) is substituted with one or more -R ¹⁸ that are the same or different. In certain embodiments, A' of formula (XIVa) is not substituted with -R ¹⁸ .

In certain embodiments, -R ¹⁸ , -R ¹⁹ , and -R ^19a of formula (XIVa) are selected from the group consisting of - H and C _1-6 alkyl.

In certain embodiments, -R ¹⁸ of formula (XIVa) is - H. In certain embodiments, -R ¹⁸ of formula (XIVa) is C _1-6 alkyl. In certain embodiments, -R ¹⁹ of formula (XIVa) is - H. In certain embodiments, -R ¹⁹ of formula (XIVa) is C _1-6 alkyl. In certain embodiments, -R ^19a of formula (XIVa) is - H. In certain embodiments, -R ^19a of formula (XIVa) is C _1-6 alkyl.

In certain embodiments, -R ⁶ of formula (XIV) has formula (XIVb):

(XIVb),

where - Y⁵- selected from the group consisting of - Q´-, C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl, where C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl optionally substituted with one or more -R²³, which are the same or different, and where C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of -Q´-, -C(O)O-, -O-, -C(O)-, -C(O)N(R²⁴)-, -S(O)₂N(R²⁴), -S(O)N(R²⁴)-, -S(O)₂-, -S(O)-, -N(R²⁴)S(O)₂N(R^24a)-, -S-, -N(R²⁴)-, -OC(OR²⁴)R^24a-, -N(R²⁴)C(O)N(R^24a)-, -OC(O)N(R²⁴)- and -N(R²⁴)C(NH)N(R^24a)-,

-R²⁰, -R²¹, -R^21a, and -R²² independently selected from the group consisting of - H, C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl, where C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl optionally substituted with one or more -R²³ which are the same or different, and where C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of -Q´-, -C(O)O-, -O-, -C(O)-, -C(O)N(R²⁴)-, -S(O)₂N(R²⁴), -S(O)N(R²⁴)-, -S(O)₂-, -S(O)-, -N(R²⁴)S(O)₂N(R^24a)-, -S-, -N(R²⁴)-, -OC(OR²⁴)R^24a-, -N(R²⁴)C(O)N(R^24a)-, -OC(O)N(R²⁴)- and -N(R²⁴)C(NH)N(R^24a)-,

each Q' is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, wherein each Q' is independently optionally substituted with one or more -R ²³ which are the same or different,

wherein -R ²³ , -R ²⁴ , and -R ^24a are independently selected from the group consisting of - H and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different,

optionally, the pair -R ²¹ /-R ^21a are joined together with the atoms to which they are attached to form a C _3-10 cycloalkyl, 3-10 membered heterocyclyl or 8-11 membered heterobicyclyl, and

where the dotted line marked with an asterisk shows the attachment to the remaining part - Y.

According to certain embodiments - Y⁵- formula (XIVb) is selected from the group consisting of - Q´-, C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl. According to certain embodiments - Y⁵- formula (XIVb) represents - Q´-. According to certain embodiments - Y⁵- formula (XIVb) represents C_{1- 10} alkyl According to certain embodiments - Y⁵- formula (XIVb) represents C_2-10 alkenyl. According to certain embodiments - Y⁵- formula (XIVb) represents C_2-10 alkynyl.

In certain embodiments, Q' of formula (XIVb) is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl. In certain embodiments, Q' of formula (XIVb) is phenyl. In certain embodiments, Q' of formula (XIVb) is naphthyl. In certain embodiments, Q' of formula (XIVb) is indenyl. In certain embodiments, Q' of formula (XIVb) is indanyl. In certain embodiments, Q' of formula (XIVb) is C _3-10 cycloalkyl. In certain embodiments, Q' of formula (XIVb) is a 3-10 membered heterocyclyl. In certain embodiments, Q' of formula (XIVb) is an 8- to 11-membered heterobicyclyl. In certain embodiments, Q' of formula (XIVb) is substituted with one or more -R ²³ which are the same or different. In certain embodiments, Q' of formula (XIVb) is not substituted with -R ²³ .

According to certain embodiments of -R²⁰, -R²¹, -R^21a, and -R²² formulas (XIVb) are selected from the group consisting of - H, C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl. According to certain embodiments of -R²⁰ formula (XIVb) is - H. In certain embodiments - R²⁰ formula (XIVb) represents C_{1- 10} alkyl According to certain embodiments of -R²⁰ formula (XIVb) represents C_2-10 alkenyl According to certain embodiments of -R²⁰ formula (XIVb) represents C_2-10 alkynyl. According to certain embodiments of -R²¹ formula (XIVb) is - H. In certain embodiments - R²¹ formula (XIVb) represents C_{1- 10} alkyl According to certain embodiments of -R²¹ formula (XIVb) represents C_2-10 alkenyl According to certain embodiments of -R²¹ formula (XIVb) represents C_2-10 alkynyl According to certain embodiments of -R^21a formula (XIVb) is - H. In certain embodiments - R^21a formula (XIVb) represents C_{1- 10} alkyl According to certain embodiments of -R^21a formula (XIVb) represents C_2-10 alkenyl According to certain embodiments of -R^21a formula (XIVb) represents C_2-10 alkynyl. According to certain embodiments of -R²² formula (XIVb) is - H. In certain embodiments - R²² formula (XIVb) represents C_{1- 10} alkyl According to certain embodiments of -R²² formula (XIVb) represents C_2-10 alkenyl According to certain embodiments of -R²² formula (XIVb) represents C_2-10 alkynyl.

In certain embodiments, -R ²³ , -R ²⁴ , and -R ^24a of formula (XIVb) are selected from the group consisting of - H and C _1-6 alkyl. In certain embodiments, -R ²³ of formula (XIVb) is - H. In certain embodiments, -R ²³ of formula (XIVb) is C _1-6 alkyl. In certain embodiments, -R ²⁴ of formula (XIVb) is - H. In certain embodiments, -R ²⁴ of formula (XIVb) is C _1-6 alkyl. In certain embodiments, -R ^24a of formula (XIVb) is - H. In certain embodiments, -R ^24a of formula (XIVb) is C _1-6 alkyl.

In certain embodiments, the pair -R ²¹ /-R ^21a of formula (XIVb) are combined together with the atoms to which they are attached to form a C _3-10 cycloalkyl.

In certain embodiments, -R ⁶ of formula (XIVb) has formula (XIVc):

(XIVc),

Where

-R²⁵, -R²⁶, -R^26a, and -R²⁷ independently selected from the group consisting of - H, C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl, where C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl optionally substituted with one or more -R²⁸, which are the same or different, and where C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of -Q*-, -C(O)O-, -O-, -C(O)-, -C(O)N(R²⁹)-, -S(O)₂N(R²⁹), -S(O)N(R²⁹)-, -S(O)₂-, -S(O)-, -N(R²⁹)S(O)₂N(R^29a)-, -S-, -N(R²⁹)-, -OC(OR²⁹)R^29a-, -N(R²⁹)C(O)N(R^29a)-, -OC(O)N(R²⁹)- and -N(R²⁹)C(NH)N(R^29a)-,

each Q* is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, wherein each Q* is independently optionally substituted with one or more -R ²⁸ which are the same or different,

wherein -R ²⁸ , -R ²⁹ , and -R ^29a are independently selected from the group consisting of - H and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different,

optionally, the pair -R ²⁶ /-R ^26a are joined together with the atoms to which they are attached to form a C _3-10 cycloalkyl, 3-10 membered heterocyclyl or 8-11 membered heterobicyclyl, and

where the dotted line marked with an asterisk shows the attachment to the remaining part - Y.

According to certain embodiments of -R²⁵, -R²⁶, -R^26a, and -R²⁷ formulas (XIVc) are selected from the group consisting of - H, C_{1- 10} alkyl, C_2-10 alkenyl and C_2-10 alkynyl. According to certain embodiments of -R²⁵ formula (XIVc) is - H. In certain embodiments - R²⁵ formula (XIVc) represents C_{1- 10} alkyl According to certain embodiments of -R²⁵ formula (XIVc) represents C_2-10 alkenyl According to certain embodiments of -R²⁵ formula (XIVc) represents C_2-10 alkynyl. According to certain embodiments of -R²⁶ formula (XIVc) is - H. In certain embodiments - R²⁶ formula (XIVc) represents C_{1- 10} alkyl According to certain embodiments of -R²⁶ formula (XIVc) represents C_2-10 alkenyl According to certain embodiments of -R²⁶ formula (XIVc) represents C_2-10 alkynyl. According to certain embodiments of -R^26a formula (XIVc) is - H. In certain embodiments - R^26a formula (XIVc) represents C_{1- 10} alkyl According to certain embodiments of -R^26a formula (XIVc) represents C_2-10 alkenyl According to certain embodiments of -R^26a formula (XIVc) represents C_2-10 alkynyl. According to certain embodiments of -R²⁷ formula (XIVc) is - H. In certain embodiments - R²⁷ formula (XIVc) represents C_{1- 10} alkyl According to certain embodiments of -R²⁷ formula (XIVc) represents C_2-10 alkenyl According to certain embodiments of -R²⁷ formula (XIVc) represents C_2-10 alkynyl.

In certain embodiments, Q* of formula (XIVc) is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl. In certain embodiments, Q* of formula (XIVc) is phenyl. In certain embodiments, Q* of formula (XIVc) is naphthyl. In certain embodiments, Q* of formula (XIVc) is indenyl. In certain embodiments, Q* of formula (XIVc) is indanyl. In certain embodiments, Q* of formula (XIVc) is tetralinyl. In certain embodiments, Q* of formula (XIVc) is C _3-10 cycloalkyl. In certain embodiments, Q* of formula (XIVc) is a 3-10 membered heterocyclyl. In certain embodiments, Q* of formula (XIVc) is an 8- to 11-membered heterobicyclyl. In certain embodiments, Q* of formula (XIVc) is substituted with one or more -R ²⁸ that are the same or different. In certain embodiments, Q* of formula (XIVc) is not substituted with -R ²⁸ .

In certain embodiments, -R ²⁸ , -R ²⁹ and -R ^29a of formula (XIVc) are selected from the group consisting of - H and C _1-6 alkyl. In certain embodiments, -R ²⁸ of formula (XIVc) is - H. In certain embodiments, -R ²⁸ of formula (XIVc) is C _1-6 alkyl. In certain embodiments, -R ²⁹ of formula (XIVc) is - H. In certain embodiments, -R ²⁹ of formula (XIVc) is C _1-6 alkyl. In certain embodiments, -R ^29a of formula (XIVc) is - H. In certain embodiments, -R ^29a of formula (XIVc) is C _1-6 alkyl.

In certain embodiments, the pair -R ²⁶ /-R ^26a of formula (XIVc) are combined together with the atoms to which they are attached to form a C _3-10 cycloalkyl. In certain embodiments, the pair -R ²⁶ /-R ^26a of formula (XIVc) are combined together with the atoms to which they are attached to form cyclobutyl.

In certain embodiments, Y of formula (XIV) is

, where each -R ⁷ has the meanings as defined above, and the dotted line, marked with an asterisk, shows the attachment to -A-. It is understood that in this case, the release of drug D may be caused by an enzyme such as a phosphatase.

In certain embodiments, Y of formula (XIV) is

, where the dotted line marked with an asterisk shows the attachment to -A-.

In certain embodiments, Y of formula (XIV) is

, where the dotted line marked with an asterisk shows the attachment to -A-.

In certain embodiments, Y of formula (XIV) is

where -R ⁸ has the meanings as defined above and the dotted line marked with an asterisk indicates the attachment to -A-.

In certain embodiments, Y of formula (XIV) is

where -R ⁹ has the meanings as defined above and the dotted line marked with an asterisk indicates the attachment to -A-. It is understood that in this case the release of drug D may be caused by an enzyme such as sulfatase.

In certain embodiments, Y of formula (XIV) is

, where the dotted line marked with an asterisk shows the attachment to -A-. It is understood that in this case, the release of drug D can be caused by an enzyme such as - galactosidase.

In certain embodiments, Y of formula (XIV) is

, where the dotted line marked with an asterisk shows the attachment to -A-. It is understood that in this case, the release of drug D can be caused by an enzyme such as - glucuronidase.

In certain embodiments, Y of formula (XIV) is

, where the dotted line marked with an asterisk shows the attachment to -A-. It is understood that in this case, the release of drug D can be caused by an enzyme such as - glucuronidase.

In certain embodiments, Y of formula (XIV) is a peptidyl moiety.

It will be understood that if - Y of formula (XIV) is a peptidyl moiety, then the release of drug D can be triggered by an enzyme such as a protease. In certain embodiments, the protease is selected from the group consisting of cathepsin B and cathepsin K. In certain embodiments, the protease is cathepsin B. In certain embodiments, the protease is cathepsin K.

In certain embodiments, Y of formula (XIV) is a peptidyl moiety, such as a dipeptidyl, tripeptidyl, tetrapeptidyl, pentapeptidyl, or hecaspeptidyl moiety. In certain embodiments, Y of formula (XIV) is a dipeptidyl moiety. In certain embodiments, Y of formula (XIV) is a tripeptidyl moiety. In certain embodiments, Y of formula (XIV) is a tetrapeptidyl moiety. In certain embodiments, Y of formula (XIV) is a pentapeptidyl moiety. In certain embodiments, Y of formula (XIV) is a hexapeptidyl moiety.

In certain embodiments, Y of formula (XIV) is a peptidyl moiety selected from the group consisting of:

, And

where the dotted line marked with an asterisk shows the attachment to -A-.

In certain embodiments, Y of formula (XIV) is

.

In certain embodiments, Y of formula (XIV) is

.

In certain embodiments, Y of formula (XIV) is

.

In certain embodiments, one hydrogen provided by -R ^1a of formula (XIV) is replaced by -L ^2- and -L ^1- has formula (XIV'):

(XIV')

Where

the unstarred dotted line shows attachment to N ⁺ of the -D ⁺ fragment, the dotted line marked with an asterisk shows attachment to -L ² -, and

-R ¹ , -Ar-, -Y, R ² and t have the meanings as in formula (XIV).

In certain embodiments, one hydrogen provided by -R ² of formula (XIV) is replaced by -L ^2- and -L ^1- has formula (XIV''):

(XIV'')

Where

the dotted line not marked with an asterisk shows the attachment to N ⁺ of the -D ⁺ fragment, the dotted line marked with an asterisk shows the attachment to -L ² -,

-R ¹ , - Ar-, - Y and R ² have the meanings as in formula (XIV), and

t' is selected from the group consisting of 0, 1, 2, 3, 4 and 5.

In certain embodiments, T' in formula (XIV'') is 0. In certain embodiments, T' in formula (XIV'') is 1. In certain embodiments, T' in formula (XIV'') is 2 In certain embodiments, T' in formula (XIV'') is 3. In certain embodiments, T' in formula (XIV'') is 4. In certain embodiments, T' in formula (XIV'') is 5.

In certain embodiments, -L ^1- has formula (XV):

(XV),

Where

the dotted line shows the addition of a primary or secondary amine -D to the nitrogen atom,

v is selected from the group consisting of 0 or 1,

-X ^1- is selected from the group consisting of -C(R ⁸ )(R ^8a )-, -N(R ⁹ )- and -O-,

=X ² is selected from the group consisting of =O and =N(R ¹⁰ ),

-X ³ is selected from the group consisting of -O, -S and -Se,

each p is independently selected from the group consisting of 0 or 1, provided that at most one p is 0,

-R ⁶ , -R ^6a , -R ¹⁰ are independently selected from the group consisting of -H, -C(R ¹¹ )(R ^11a )(R ^11b ) and -T,

-R ⁹ is selected from the group consisting of -C(R ¹¹ )(R ^11a )(R ^11b ) and -T,

-R¹, -R^1a, -R², -R^2a, -R³, -R^3a, -R⁴, -R^4a, -R⁵, -R^5a, -R⁷, -R⁸ -R^8a, -R^eleven, -R^11a and -R^11b independently selected from the group consisting of -H, halogen, -CN, -C(O)OR¹², -OR¹², -C(O)R¹², -C(O)N(R¹²)(R^12a), -S(O)₂N(R¹²)(R^12a), -S(O)N(R¹²)(R^12a), -S(O)₂R¹², -S(O)R¹², -N(R¹²)S(O)₂N(R^12a)(R^12b), -SR¹², -NO₂, -N(R¹²)C(O)OR^12a, -N(R¹²)C(O)N(R^12a)(R^12b), -OC(O)N(R¹²)(R^12a), -T, C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl, where C_1-6 alkyl, C_2-6 alkenyl and C_2-6 alkynyl optionally substituted with one or more -R¹³, which are the same or different, and where C_1-6alkyl, C_2-6 alkenyl and C_2-6 alkynyl groups are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R¹⁴)-, -S(O)₂N(R¹⁴)-, -S(O)N(R¹⁴)-, -S(O)₂-, -S(O)-, -N(R¹⁴)S(O)₂N(R^14a)-, -S-, -N(R¹⁴)-, -OC(OR¹⁴)(R^14a)-, -N(R¹⁴)C(O)N(R^14a)- and -OC(O)N(R¹⁴)-,

-R ¹² , -R ^12a , -R ^12b are independently selected from the group consisting of -H, -T, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl, where -T, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl are optionally substituted by one or more -R ¹³ which are the same or different, and wherein C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ¹⁴ )-, -S(O) ₂ N(R ¹⁴ )-, -S(O)N(R ¹⁴ )-, -S(O) ₂ -, -S(O)-, -N(R ¹⁴ )S(O) ₂ N(R ^14a )-, -S-, -N(R ¹⁴ )-, -OC(OR ¹⁴ )(R ^14a )-, -N(R ¹⁴ )C(O)N(R ^14a )- and -OC(O)N (R ¹⁴ )-,

wherein each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, wherein each T is independently optionally substituted with one or more than -R ¹³ , which are the same or different,

-R ¹³ is selected from the group consisting of halogen, -CN, oxo, -C(O)OR ¹⁵ , -OR ¹⁵ , -C(O)R ¹⁵ , -C(O)N(R ¹⁵ )(R ^15a ) , -S(O) ₂ N(R ¹⁵ )(R ^15a ), -S(O)N(R ¹⁵ )(R ^15a ), -S(O) ₂ R ¹⁵ , -S(O)R ¹⁵ , - N(R ¹⁵ )S(O) ₂ N(R ^15a )(R ^15b ), -SR ¹⁵ , -N(R ¹⁵ )(R ^15a ), -NO ₂ , -OC(O)R ¹⁵ , -N( R ¹⁵ )C(O)R ^15a , -N(R ¹⁵ )S(O) ₂ R ^15a , -N(R ¹⁵ )S(O)R ^15a , -N(R ¹⁵ )C(O)OR ^15a , -N(R ¹⁵ )C(O)N(R ^15a )(R ^15b ), -OC(O)N(R ¹⁵ )(R ^15a ) and C _1-6 alkyl, where C _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different,

wherein -R ¹⁴ , -R ^14a , -R ¹⁵ , -R ^15a , and -R ^15b are independently selected from the group consisting of - H and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different,

optionally one or more of the pairs -R ¹ /-R ^1a , -R ² /-R ^2a , -R ³ /-R ^3a , -R ⁴ /-R ^4a , -R ⁵ /-R ^5a or -R ⁸ / -R ^8a are joined together with the atom to which they are attached to form a C _3-10 cycloalkyl, 3-10 membered heterocyclyl or 8-11 membered heterobicyclyl,

optionally one or more of the pairs -R ¹ /-R ² , -R ¹ /-R ⁸ , -R ¹ /-R ⁹ , -R ² /-R ⁹ or -R ² /-R ¹⁰ are connected together with atoms, to which they are attached, forming a ring - A-,

where - A- is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl,

optionally one or more of the pairs -R ³ /-R ⁶ , -R ⁴ /-R ⁶ , -R ⁵ /-R ⁶ , -R ⁶ /-R ^6a or -R ⁶ /-R ⁷ form together with atoms, to which they are attached, the ring is A´-,

where - A´- is selected from the group consisting of 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, and

where -L ^1- is substituted by at least one -L ² -, and where -L ^1- is optionally further substituted.

The optional additional substituents -L ^1- of formula (XV) preferably have the meanings as described above.

Preferably -L ^1- of formula (XV) is replaced by one moiety -L ² -.

In one embodiment, -L ^1- of formula (XV) is not further substituted.

In the conjugates of the present invention, -L ^2- represents a chemical bond or spacer moiety. In certain embodiments, -L ^2- does not contain a reversible bond, i.e. all bonds in -L ^2- are stable bonds. In certain embodiments, -L ^1- is connected to -L ^2- via a stable connection. In certain embodiments, -L ^2- is connected to -Z via a stable connection.

In certain embodiments, -L ^2- is a chemical bond.

In certain embodiments, -L ^2- i is a spacer moiety.

In certain embodiments, -L ^2- is a spacer moiety selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y1 )-, -S(O) ₂ N(R ^y1 )-, -S(O)N(R ^y1 )-, -S(O) ₂ -, -S(O)-, -N(R ^y1 ) S(O) ₂ N(R ^y1a )-, -S-, -N(R ^y1 )-, -OC(OR ^y1 )(R ^y1a )-, -N(R ^y1 )C(O)N(R ^y1a )-, -OC(O)N(R ^y1 )-, C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl, where -T-, C _{1- 50} alkyl, C _2-50 alkenyl, and C _2-50 alkynyl are optionally substituted by one or more -R ^y2 , which are the same or different, and wherein C _1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl are optionally interrupted by one or more groups selected from group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y3 )-, -S(O) ₂ N(R ^y3 ) -, -S(O)N(R ^y3 )-, -S(O) ₂ -, -S(O)-, -N(R ^y3 )S(O) ₂ N(R ^y3a )-, -S- , -N(R ^y3 )-, -OC(OR ^y3 )(R ^y3a )-, -N(R ^y3 )C(O)N(R ^y3a )-, and -OC(O)N(R ^y3 )- ,

-R ^y1 , and -R ^y1a are independently selected from the group consisting of -H, -T, C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl, where -T, C _1-50 alkyl, _C2-50 alkenyl, and _C2-50 alkynyl are optionally substituted by one or more ^-Ry2 , which are the same or different, and wherein _C1-50 alkyl, _C2-50 alkenyl, and _C2-50 alkynyl are optional interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y4 )-, -S( O) ₂ N(R ^y4 )-, -S(O)N(R ^y4 )-, -S(O) ₂ -, -S(O)-, -N(R ^y4 )S(O) ₂ N( R ^y4a )-, -S-, -N(R ^y4 )-, -OC(OR ^y4 )(R ^y4a )-, -N(R ^y4 )C(O)N(R ^y4a )-, and -OC( O)N(R ^y4 )-,

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl and 8 -30-membered heteropolycyclyl, wherein each T is independently optionally substituted with one or more -R ^y2 , which are the same or different,

each -R ^y2 is independently selected from the group consisting of halogen, -CN, oxo (=O), -COOR ^y5 , -OR ^y5 , -C(O)R ^y5 , -C(O)N(R ^y5 R ^y5a ) , -S(O) ₂ N(R ^y5 R ^y5a ), -S(O)N(R ^y5 R ^y5a ), -S(O) ₂ R ^y5 , -S(O)R ^y5 , -N(R ^y5 )S(O) ₂ N(R ^y5a R ^y5b ), -SR ^y5 , -N(R ^y5 R ^y5a ), -NO ₂ , -OC(O)R ^y5 , -N(R ^y5 )C(O)R ^y5a , -N(R ^y5 )S(O) ₂ R ^y5a , -N(R ^y5 )S(O)R ^y5a , -N(R ^y5 )C(O)OR ^y5a , -N(R ^y5 )C( O)N(R ^y5a R ^y5b ), -OC(O)N(R ^y5 R ^y5a ) and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different, and

-R ^y3 , -R ^y3a , -R ^y4 , -R ^y4a , -R ^y5 , -R ^y5a , and -R ^y5b are each independently selected from the group consisting of - H and C _1-6 alkyl, wherein C _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different.

In certain embodiments, -L ^2- is a spacer moiety selected from -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y1 )-, -S(O) ₂ N(R ^y1 )-, -S(O)N(R ^y1 )-, -S(O) ₂ -, -S(O)-, -N(R ^y1 )S(O) ₂ N(R ^y1a )-, -S-, -N(R ^y1 )-, -OC(OR ^y1 )(R ^y1a )-, -N(R ^y1 )C(O)N(R ^y1a )-, - OC(O)N(R ^y1 )-, C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl, where -T-, C _1-20 alkyl, C _2-20 alkenyl, and C _{2- 20} alkynyl is optionally substituted with one or more -R ^y2 , which are the same or different, and wherein C _1-20 alkyl, C _2-20 alkenyl, and C _2-20 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y3 )-, -S(O) ₂ N(R ^y3 )-, -S (O)N(R ^y3 )-, -S(O) ₂ -, -S(O)-, -N(R ^y3 )S(O) ₂ N(R ^y3a )-, -S-, -N( R ^y3 )-, -OC(OR ^y3 )(R ^y3a )-, -N(R ^y3 )C(O)N(R ^y3a )- and -OC(O)N(R ^y3 )-,

-R ^y1 , and -R ^y1a are independently selected from the group consisting of -H, -T, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl, where -T, C _1-10 alkyl, _C2-10 alkenyl, and _C2-10 alkynyl are optionally substituted by one or more ^-Ry2 , which are the same or different, and wherein _C1-10 alkyl, _C2-10 alkenyl, and _C2-10 alkynyl are optional interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y4 )-, -S( O) ₂ N(R ^y4 )-, -S(O)N(R ^y4 )-, -S(O) ₂ -, -S(O)-, -N(R ^y4 )S(O) ₂ N( R ^y4a )-, -S-, -N(R ^y4 )-, -OC(OR ^y4 )(R ^y4a )-, -N(R ^y4 )C(O)N(R ^y4a )-, and -OC( O)N(R ^y4 )-,

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl and 8 -30-membered heteropolycyclyl, wherein each T is independently optionally substituted with one or more -R ^y2 , which are the same or different,

-R ^y2 is selected from the group consisting of halogen, -CN, oxo (=O), -COOR ^y5 , -OR ^y5 , -C(O)R ^y5 , -C(O)N(R ^y5 R ^y5a ), - S(O) ₂ N(R ^y5 R ^y5a ), -S(O)N(R ^y5 R ^y5a ), -S(O) ₂ R ^y5 , -S(O)R ^y5 , -N(R ^y5 )S (O) ₂ N(R ^y5a R ^y5b ), -SR ^y5 , -N(R ^y5 R ^y5a ), -NO ₂ , -OC(O)R ^y5 , -N(R ^y5 )C(O)R ^y5a , -N(R ^y5 )S(O) ₂ R ^y5a , -N(R ^y5 )S(O)R ^y5a , -N(R ^y5 )C(O)OR ^y5a , -N(R ^y5 )C(O) N(R ^y5a R ^y5b ), -OC(O)N(R ^y5 R ^y5a ), and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different, and

-R ^y3 , -R ^y3a , -R ^y4 , -R ^y4a , -R ^y5 , -R ^y5a , and -R ^y5b are each independently selected from the group consisting of - H and C _1-6 alkyl, where C The _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different.

In certain embodiments, -L ^2- is a spacer moiety selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y1 )-, -S(O) ₂ N(R ^y1 )-, -S(O)N(R ^y1 )-, -S(O) ₂ -, -S(O)-, -N(R ^y1 ) S(O) ₂ N(R ^y1a )-, -S-, -N(R ^y1 )-, -OC(OR ^y1 )(R ^y1a )-, -N(R ^y1 )C(O)N(R ^y1a )-, -OC(O)N(R ^y1 )-, C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl, where -T-, C _{1- 50} alkyl, C _2-50 alkenyl, and C _2-50 alkynyl are optionally substituted by one or more -R ^y2 , which are the same or different, and wherein C _1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl are optionally interrupted by one or more groups selected from group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y3 )-, -S(O) ₂ N(R ^y3 ) -, -S(O)N(R ^y3 )-, -S(O) ₂ -, -S(O)-, -N(R ^y3 )S(O) ₂ N(R ^y3a )-, -S- , -N(R ^y3 )-, -OC(OR ^y3 )(R ^y3a )-, -N(R ^y3 )C(O)N(R ^y3a )-, and -OC(O)N(R ^y3 )- ,

-R ^y1 , and -R ^y1a are independently selected from the group consisting of -H, -T, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl,

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl and 8 -30-membered heteropolycyclyl,

each -R ^y2 is independently selected from the group consisting of halogen and C _1-6 alkyl, and

-R ^y3 , -R ^y3a , -R ^y4 , -R ^y4a , -R ^y5 , -R ^y5a , and -R ^y5b are each independently selected from the group consisting of - H and C _1-6 alkyl, where C The _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different.

In certain embodiments, -L ^2- is a C _1-20 alkyl chain that is optionally interrupted by one or more groups independently selected from -O-, -T-, and -C(O)N(R ^y1 )-, and wherein The C _1-20 alkyl chain is optionally substituted with one or more groups independently selected from -OH, -T and -C(O)N(R ^y6 R ^y6a ), where -R ^y1 , -R ^y6 , -R ^y6a are independently selected from group consisting of H and C _1-4 alkyl, and where T is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl and 8-30 membered heteropolycyclyl.

In certain embodiments, -L ^2- has a molecular weight ranging from 14 g/mol to 750 g/mol.

In certain embodiments, -L ^2- comprises a fragment selected from

.

In certain embodiments, -L ^2- has a length of from 1 to 20 atoms.

In the context of the present invention, the term "chain length" in relation to the fragment -L ^2- refers to -L ^1- and -Z.

In certain embodiments, -L ^2- has the formula (A -1)

(A -1),

Where

the dotted line marked with an asterisk shows the connection to -L ^1- ,

unmarked dotted line shows attachment to Z,

r is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10,

s selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10,

t is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10,

u is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10,

v is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and

-R ¹ is selected from the group consisting of -H, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl.

In certain embodiments, r of formula (A -1) is 1. In certain embodiments, r of formula (A -1) is 2. In certain embodiments, r of formula (A -1) is 3. In certain embodiments, r of formula (A -1) is 4. In certain embodiments, r of formula (A -1) is 5. In certain embodiments, r of formula (A -1) is 6. In certain embodiments, r of formula (A -1 ) is 7. In certain embodiments, r of formula (A -1) is 8. In certain embodiments, r of formula (A -1) is 9. In certain embodiments, r of formula (A -1) is 10.

In certain embodiments, s of formula (A -1) is 1. In certain embodiments, s of formula (A -1) is 2. In certain embodiments, s of formula (A -1) is 3. In certain embodiments, s of formula (A -1) is 4. In certain embodiments, s of formula (A -1) is 5. In certain embodiments, s of formula (A -1) is 6. In certain embodiments, s of formula (A -1 ) is 7. In certain embodiments, s of formula (A -1) is 8. In certain embodiments, s of formula (A -1) is 9. In certain embodiments, s of formula (A -1) is 10.

In certain embodiments, t in formula (A -1) is 1. In certain embodiments, t in formula (A -1) is 2. In certain embodiments, t in formula (A -1) is 3. In certain embodiments In certain embodiments, t in formula (A -1) is 4. In certain embodiments, t in formula (A -1) is 5. In certain embodiments, t in formula (A -1) is 6. In certain embodiments t in formula (A -1) is 7. In certain embodiments, t in formula (A -1) is 8. In certain embodiments, t in formula (A -1) is 9. In certain embodiments, t in formula (A -1) represents 10.

In certain embodiments, u of formula (A -1) is 1. In certain embodiments, u of formula (A -1) is 2. In certain embodiments, u of formula (A -1) is 3. In certain embodiments, u of formula (A -1) is 4. In certain embodiments, u of formula (A -1) is 5. In certain embodiments, u of formula (A -1) is 6. In certain embodiments, u of formula (A -1) ) is 7. In certain embodiments, u of formula (A -1) is 8. In certain embodiments, u of formula (A -1) is 9. In certain embodiments, u of formula (A -1) is 10.

In certain embodiments, v of formula (A -1) is 1. In certain embodiments, v of formula (A -1) is 2. In certain embodiments, v of formula (A -1) is 3. In certain embodiments, v of formula (A -1) is 4. In certain embodiments, v of formula (A -1) is 5. In certain embodiments, v of formula (A -1) is 6. In certain embodiments, v of formula (A -1 ) is 7. In certain embodiments, v of formula (A -1) is 8. In certain embodiments, v of formula (A -1) is 9. In certain embodiments, v of formula (A -1) is 10.

In certain embodiments, -R ¹ of formula (A -1) is - H. In certain embodiments, -R ¹ of formula (A -1) is methyl. In certain embodiments, -R ¹ of formula (A -1) is ethyl. In certain embodiments, -R ¹ of formula (A -1) is n-propyl. In certain embodiments, -R ¹ of formula (A -1) is isopropyl. In certain embodiments, -R ¹ of formula (A -1) is n-butyl. In certain embodiments, -R ¹ of formula (A -1) is isobutyl. In certain embodiments, -R ¹ of formula (A -1) is sec-butyl. In certain embodiments, -R ¹ of formula (A -1) is tert-butyl. In certain embodiments, -R ¹ of formula (A -1) is n-pentyl. In certain embodiments, -R ¹ of formula (A -1) is 2-methylbutyl. In certain embodiments, -R ¹ of formula (A -1) is 2,2-dimethylpropyl. In certain embodiments, -R ¹ of formula (A -1) is n-hexyl. In certain embodiments, -R ¹ of formula (A -1) is 2-methylpentyl. In certain embodiments, -R ¹ of formula (A -1) is 3-methylpentyl. In certain embodiments, -R ¹ of formula (A -1) is 2,2-dimethylbutyl. In certain embodiments, -R ¹ of formula (A -1) is 2,3-dimethylbutyl. In certain embodiments, -R ¹ of formula (A -1) is 3,3-dimethylpropyl.

In certain embodiments, r of formula (A -1) is 1, s of formula (A -1) is 2, T in formula (A -1) is 2, u of formula (A -1) is 1, v of formula (A -1) is 2, and -R ¹ of formula (A -1) is -H.

In certain embodiments, r of formula (A -1) is 1, s of formula (A -1) is 2, T in formula (A -1) is 3, u of formula (A -1) is 1, v of formula (A -1) is 2, and -R ¹ of formula (A -1) is -H.

In certain embodiments, r of formula (A -1) is 1, s of formula (A -1) is 2, T in formula (A -1) is 4, u of formula (A -1) is 1, v of formula (A -1) is 2, and -R ¹ of formula (A -1) is -H.

In certain embodiments, r of formula (A -1) is 1, s of formula (A -1) is 2, T in formula (A -1) is 5, u of formula (A -1) is 1, v of formula (A -1) is 2, and -R ¹ of formula (A -1) is -H.

In certain embodiments, Z comprises a polymer.

In certain embodiments, Z is non-cleavable. In certain embodiments, Z is cleavable. Cleavable fragment Z causes the carrier fragment to cleave over time, which may be useful in certain applications.

In certain embodiments, Z is a hydrogel.

In certain embodiments, such Z comprises a polymer selected from the group consisting of 2-methacryloyloxyethyl phosphoylcholines, poly(acrylic acids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amides), poly (amidoamines), poly(amino acids), poly(anhydrides), poly(aspartamides), poly(butyric acids), poly(glycolic acids), polybutylene terephthalates, poly(caprolactones), poly(carbonates), poly(cyanoacrylates), poly( dimethylacrylamides), poly(esters), poly(ethylenes), poly(ethylene glycols), poly(ethylene oxides), poly(ethyl phosphates), poly(ethyl oxazolines), poly(glycolic acids), poly(hydroxyethyl acrylates), poly(hydroxyethyl oxazolines ), poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines), poly(iminocarbonates), poly(lactic acids), poly(copolymers of lactic and glycolic acids), poly(methacrylamides), poly(methacrylates), poly(methyloxazolines), poly( organophosphazenes), poly(orthoesters), poly(oxazolines), poly(propylene glycols), poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinylamines), poly(vinylmethyl ethers), poly(vinylpyrrolidones) ), silicones, celluloses, carbomethylcelluloses, hydroxypropyl methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins, hyaluronic acids and derivatives, functionalized hyaluronic acids, mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethyl starches and other carbohydrate-based polymers, xylans and their copolymers.

In certain embodiments, Z is a poly(alkylene glycol) hydrogel, such as a poly(propylene glycol) hydrogel or a poly(ethylene glycol) hydrogel or a hyaluronic acid hydrogel.

In certain embodiments, Z is a PEG-based hydrogel. Suitable hydrogels are known in the art. Examples are disclosed in WO2006/003014, WO2011/012715 and WO2014/056926, which are incorporated herein by reference.

In certain embodiments, such a PEG-based hydrogel contains a plurality of backbone moieties that are cross-linked by -CL ^p - cross-linking moieties. Optionally, there is a spacer fragment -SP ^1- between the main chain fragment and the cross-linking fragment. In certain embodiments, such spacer moiety -SP ^1- has the meanings as defined above for -L ² -.

In certain embodiments, the backbone fragment has a molecular weight ranging from 1 kDa to 20 kDa.

In certain embodiments, the backbone moiety has the formula (pA)

B*-(A- Hyp) _x (pA),

Where

B* represents the branching core,

A is a PEG based polymer,

Hyp is a branched fragment,

x represents an integer from 3 to 16,

and wherein each backbone fragment is connected to one or more crosslinking moieties and one or more -L ² - moieties, wherein the crosslinking moieties and -L ^{2 -} moieties are connected to Hyp, either directly or through a spacer moiety.

In certain embodiments, B* of formula (pA) is selected from the group consisting of polyalcohol moieties and polyamine moieties. In certain embodiments, B* of formula (pA) is a polyalcohol moiety. In certain embodiments, B* of formula (pA) is a polyamine moiety.

In certain embodiments, the B* polyalcohol moieties of formula (pA) are selected from the group consisting of pentaerythritol moieties, tripentaerythritol moieties, hexaglycerol moieties, sucrose moieties, sorbitol moieties, fructose moieties, mannitol moieties, and glucose moieties. In certain embodiments, B* of formula (pA) is a pentaerythritol moiety, i.e. formula fragment

, where the dotted lines show the attachment to -A-.

In certain embodiments, the polyamine moieties for B* of formula (pA) are selected from the group consisting of an ornithine moiety, a diaminobutyric acid moiety, a trilysine moiety, a tetralysine moiety, a pentalysine moiety, a hexalysine moiety, a heptalysine moiety, an octalysine moiety, a nonalysine moiety, a decalysine moiety, an undecalysin fragment, a dodecalysin fragment, a tridecalysin fragment, a tetradecalysin fragment and a pentadecalysin fragment. In certain embodiments, B* of formula (pA) is selected from the group consisting of an ornithine moiety, a diaminobutyric acid moiety, and a trilysine moiety.

The main chain moiety of formula (pA) may consist of the same or different PEG-based moieties -A- and each moiety -A- may be selected independently. In certain embodiments, all -A- moieties present in the backbone moiety of formula (pA) have the same structure. It will be understood that the phrase “have the same structure” with respect to polymer moieties, such as the PEG-A- based polymer, means that the number of polymer monomers, such as the number of ethylene glycol moieties, may vary depending on the polydisperse nature of the polymers. In certain embodiments, the number of monomer units does not vary by more than a factor of 2 among all -A hydrogel moieties.

In certain embodiments, each A- formula (pA) has a molecular weight ranging from 0.3 kDa to 40 kDa, for example, from 0.4 to 30 kDa, from 0.4 to 25 kDa, from 0.4 to 20 kDa, from 0.4 to 15 kDa, from 0.4 to 10 kDa or from 0.4 to 5 kDa. In certain embodiments, each -A- has a molecular weight of 0.4 to 5 kDa. In certain embodiments, -A- has a molecular weight of about 0.5 kDa. In certain embodiments, -A- has a molecular weight of about 1 kDa. In certain embodiments, -A- has a molecular weight of about 2 kDa. In certain embodiments, -A- has a molecular weight of about 3 kDa. In certain embodiments, -A- has a molecular weight of about 5 kDa.

In certain embodiments, - A- formula (pA) has formula (pB-i)

-(CH ₂ ) _n1 (OCH ₂ CH ₂ ) _n X-(pB- i),

Where

n1 represents 1 or 2,

n is an integer in the range from 3 to 250, such as 5 to 200, such as 8 to 150 or 10 to 100, and

X represents the chemical bond or bond that covalently links A and Hyp.

In certain embodiments, - A- formula (pA) has formula (pB- ii)

-(CH ₂ ) _n1 (OCH ₂ CH ₂ ) _n -(CH ₂ ) _n2 X-(pB- ii),

Where

n1 represents 1 or 2,

n is an integer in the range from 3 to 250, such as 5 to 200, such as 8 to 150 or 10 to 100,

n2 is 0 or 1, and

X represents the chemical bond or bond that covalently links A and Hyp.

In certain embodiments, - A- formula (pA) has formula (pB- i')

(pB-i'),

Where

the dotted line marked with an asterisk shows the connection to B*,

the unmarked dotted line shows the attachment to - Hyp, and

n3 is an integer ranging from 10 to 50.

In certain embodiments, n3 of formula (pB-i') is 25. In certain embodiments, n3 of formula (pB-i') is 26. In certain embodiments, n3 of formula (pB-i') is 27. In certain embodiments In certain embodiments, n3 of formula (pB-i') is 28. In certain embodiments, n3 of formula (pB-i') is 29. In certain embodiments, n3 of formula (pB-i') is 30.

In certain embodiments, the B*-(A) ₄ moiety has the formula (pB-a)

(pB-a),

Where

dotted lines indicate attachment to Hyp, and

each n3 is independently an integer selected from 10 to 50.

In certain embodiments, n3 of formula (pB-a) is 25. In certain embodiments, n3 of formula (pB-a) is 26. In certain embodiments, n3 of formula (pB-a) is 27. In certain embodiments, n3 of formula (B-a) is 28. In certain embodiments, n3 of formula (pB-a) is 29. In certain embodiments, n3 of formula (pB-a) is 30.

The backbone moiety of the formula (pA) may be composed of the same or different -Hyp dendritic moieties, and where each -Hyp may be independently selected. In certain embodiments, all - Hyp moieties present in the backbone fragment of formula (pA) have the same structure.

In certain embodiments, each Hyp of formula (pA) has a molecular weight ranging from 0.3 kDa to 5 kDa.

In certain embodiments, Hyp is selected from the group consisting of the formula moiety (pHyp-i)

(pHyp-i),

Where

the dotted line marked with an asterisk shows the connection to -A-,

unmarked dotted lines indicate attachment to the spacer moiety -SP ^1- , cross-linker moiety -CL ^p - or -L ² -, and

p2, p3 and p4 are the same or different and each independently represents an integer from 1 to 5,

formula fragment (pHyp-ii)

(pHyp-ii),

Where

the dotted line marked with an asterisk shows the connection to -A-,

unmarked dotted lines indicate attachment to the spacer fragment -SP ^1- , linking fragments -CL ^p - or to -L ² -, and

p5 -P11 are the same or different and each independently represents an integer from 1 to 5,

formula fragment (pHyp-iii)

(pHyp-iii),

Where

the dotted line marked with an asterisk shows the connection to -A-,

unmarked dotted lines indicate attachment to the spacer fragment -SP ^1- , linking fragments -CL ^p - or to -L ² -, and

p12 - p26 are the same or different and each independently represents an integer from 1 to 5,

formula fragment (pHyp-iv)

(pHyp-iv),

Where

the dotted line marked with an asterisk shows the connection to -A-,

unmarked dotted lines indicate attachment to the spacer fragment -SP ^1- , linking fragments -CL ^p - or to -L ² -, and

p27 - p28 are the same or different and each independently represents an integer from 1 to 5, and

q represents an integer from 1 to 8,

where the fragments (pHyp- i) -(pHyp- iv) at each chiral center can have either an R- or S- configuration.

In certain embodiments, the chiral center of the moiety (pHyp-i), (pHyp-ii), (pHyp-iii), or (pHyp-iv) all have the same configuration. In certain embodiments, the chiral center of the moiety (pHyp-i), (pHyp-ii), (pHyp-iii), or (pHyp-iv) is all R-configured. In certain embodiments, the chiral center of the moiety (pHyp-i), (pHyp-ii), (pHyp-iii), or (pHyp-iv) is all in the S configuration.

In certain embodiments, p2, p3, and p4 of the formula (pHyp-i) are 4.

In certain embodiments, p5 -P11 of formula (pHyp-ii) is 4.

In certain embodiments, p12-P26 of formula (pHyp-iii) is 4.

In certain embodiments, q in formula (pHyp-iv) is 2 or 6. In certain embodiments, q in formula (pHyp-iv) is 6.

In certain embodiments, p27 and p28 of formula (pHyp-iv) are 4.

In certain embodiments, Hyp of formula (pA) comprises a branched polypeptide fragment.

In certain embodiments, Hyp of formula (pA) contains a lysine moiety. In certain embodiments, each Hyp of formula (pA) is independently selected from the group consisting of a trilysine moiety, a tetralysine moiety, a pentalysine moiety, a hexalysine moiety, a heptalysine moiety, an octalysine moiety, a nonalysine moiety, a decalysine moiety, an undecalysine moiety, a dodecalysine moiety, and a tridecalysine moiety. , tetradecalysin fragment, pentadecalysin fragment, hexadecalysin fragment, heptadecalysin fragment, octadecalysin fragment and nonadecalysin fragment.

In certain embodiments, Hyp contains 3 lysine moieties. In certain embodiments, Hyp contains 7 lysine moieties. H In certain embodiments, Hyp contains 15 lysine moieties. In certain embodiments, Hyp contains heptalisinyl.

In certain embodiments, formula x (pA) is 3. In certain embodiments, formula x (pA) is 4. In certain embodiments, formula x (pA) is 6. In certain embodiments, formula x (pA) is 8.

In certain embodiments, the backbone moiety has the formula (pC1)

(pC1),

Where

dotted lines indicate attachment to spacer moiety -SP ^1- , cross-linker moiety -CL ^p - or -L ² -, and

n is in the range from 10 to 40.

In certain embodiments, n in formula (pC1) is about 28.

In certain embodiments, the backbone moiety has the formula (pC2)

(pC2),

Where

dotted lines indicate attachment to spacer moiety -SP ^1- , cross-linker moiety -CL ^p - or -L ² -, and

n is in the range from 10 to 40.

In certain embodiments, there is no spacer moiety -SP ^1- present between the backbone moiety and the crosslinker moiety -CL ^p -, i.e. -CL ^p - directly related to - Hyp.

The cross-linking moiety of the -CL ^p - PEG-based hydrogel in certain embodiments is based on poly(alkylene glycol) (PAG). In certain embodiments, the cross-linking moiety is based on poly(propylene glycol). In certain embodiments, the cross-linking moiety -CL ^p - is PEG-based.

In certain embodiments, such a PAG -CL ^p - cross-linking moiety has the formula (pD)

(pD),

Where

dotted lines indicate attachment to the main chain fragment or to the spacer fragment -SP ^1- ,

- Y ^1- has the formula

,

where the dotted line marked with an asterisk shows the connection to -D ^1- and the unmarked dotted line shows the connection to -D ^2- ,

- Y ^2- has the formula

,

where the dotted line marked with an asterisk shows the attachment to -D ⁴ - and the unmarked dotted line shows the attachment to -D ³ -,

- E ^1- has the formula

,

where the dotted line marked with an asterisk shows attachment to -(C=O)- and the unmarked dotted line shows attachment to -O-,

- E ^2- has the formula

,

where the dotted line marked with an asterisk shows attachment to -G ^1- and the unmarked dotted line shows attachment to -(C=O)-,

- G ^1- has the formula

,

where the dotted line marked with an asterisk shows the attachment to -O- and the unmarked dotted line shows the attachment to ^-E2- ,

- G ^2- has the formula

,

where the dotted line marked with an asterisk shows attachment to -O- and the unmarked dotted line shows attachment to -(C=O)-,

- G ^3- has the formula

,

where the dotted line marked with an asterisk shows attachment to -O- and the unmarked dotted line shows attachment to -(C=O)-,

-D ^1- , -D ² -, -D ³ -, -D ⁴ -, -D ⁵ - and -D ⁶ - are identical or different and each is independently selected from the group consisting of -O-, - NR ^11- , -N ⁺ R ¹² R ^12a -, -S-, -(S=O)-, -(S(O) ₂ )-, -C(O)-, -P(O)R ¹³ - , -P(O)(OR ¹³ ) and -CR ¹⁴ R ^14a -,

-R ¹ , -R ^1a , -R ² , -R ^2a , -R ³ , -R ^3a , -R ⁴ , -R ^4a , -R ⁵ , -R ^5a , -R ⁶ , -R ^6a , -R ⁷ , -R ^7a , -R ⁸ , -R ^8a , -R ⁹ ,
-R ^9a , -R ¹⁰ , -R ^10a , -R ¹¹ , -R ¹² , -R ^12a , -R ¹³ , -R ¹⁴ , and -R ^14a are identical or different and are each independently selected from the group, consisting of - H and C _1-6 alkyl,

optionally one or more of the pairs -R ¹ /-R ^1a , -R ² /-R ^2a , -R ³ /-R ^3a , -R ⁴ /-R ^4a , -R ¹ /-R ² , -R ³ / -R ⁴ , -R ^1a /-R ^2a , -R ^3a /-R ^4a , -R ¹² /-R ^12a and -R ¹⁴ /-R ^14a form a chemical bond or are joined together with the atom to which they are attached, with forming a C _3-8 cycloalkyl, or to form a ring A, or joined together with the atom to which they are attached, to form a 4-7 membered heterocyclyl or 8-11 membered heterobicyclyl or adamantyl,

A is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl and tetralinyl,

r1, r2, r5, r6, r13, r14, r15 and r16 are independently 0 or 1,

r3, r4, r7, r8, r9, r10, r11, r12 are independently 0, 1, 2, 3, or 4,

r17, r18, r19, r20, r21 and r22 independently represent 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

s1, s2, s4, s5 are independently 1, 2, 3, 4, 5 or 6, and

s3 is in the range from 1 to 900.

In certain embodiments, s3 is in the range from 1 to 500. In certain embodiments, s3 is in the range from 1 to 200.

In certain embodiments, r1 of formula (pD) is 0. In certain embodiments, r1 of formula (pD) is 1. In certain embodiments, r2 of formula (pD) is 0. In certain embodiments, r2 of formula (pD) is 1. In certain embodiments, r5 of formula (pD) is 0. In certain embodiments, r5 of formula (pD) is 1.

In certain embodiments, r1, r2, r5, and r6 of formula (pD) are 0.

In certain embodiments, r6 of formula (pD) is 0. In certain embodiments, r6 of formula (pD) is 1. In certain embodiments, r13 of formula (pD) is 0. In certain embodiments, r13 of formula (pD) is 1. In certain embodiments, r14 of formula (pD) is 0. In certain embodiments, r14 of formula (pD) is 1. In certain embodiments, r15 of formula (pD) is 0. In certain embodiments, r15 of formula (pD) is 1. In certain embodiments, r16 of formula (pD) is 0. In certain embodiments, r16 of formula (pD) is 1.

In certain embodiments, r3 of formula (pD) is 1. In certain embodiments, r3 of formula (pD) is 2. In certain embodiments, r4 of formula (pD) is 1. In certain embodiments, r4 of formula (pD) is 2. In certain embodiments, r3 and r4 of formula (pD) are both 1. In certain embodiments, r3 and r4 of formula (pD) are both 2. In certain embodiments, r3 and r4 of formula (pD) are both 3.

In certain embodiments, r7 of formula (pD) is 0. In certain embodiments, r7 of formula (pD) is 1. In certain embodiments, r7 of formula (pD) is 2. In certain embodiments, r8 of formula (pD) is 0. In certain embodiments, r8 of formula (pD) is 1. In certain embodiments, r8 of formula (pD) is 2. In certain embodiments, r9 of formula (pD) is 0. In certain embodiments, r9 of formula (pD) is 1. In certain embodiments, r9 of formula (pD) is 2. In certain embodiments, r10 of formula (pD) is 0. In certain embodiments, r10 of formula (pD) is 1. In certain embodiments, r10 of formula ( pD) is 2. In certain embodiments, r11 of formula (pD) is 0. In certain embodiments, r11 of formula (pD) is 1. In certain embodiments, r11 of formula (pD) is 2. In certain embodiments, r12 formula (pD) is 0. In certain embodiments, r12 of formula (pD) is 1. In certain embodiments, r12 of formula (pD) is 2.

In certain embodiments, r17 of formula (pD) is 1. In certain embodiments, r18 of formula (pD) is 1. In certain embodiments, r19 of formula (pD) is 1. In certain embodiments, r20 of formula (pD) is 1. In certain embodiments, r21 of formula (pD) is 1.

In certain embodiments, s1 of formula (pD) is 1. In certain embodiments, s1 of formula (pD) is 2. In certain embodiments, s2 of formula (pD) is 1. In certain embodiments, s2 of formula (pD) is 2. In certain embodiments, s4 of formula (pD) is 1. In certain embodiments, s4 of formula (pD) is 2.

In certain embodiments, the s3 of the formula (pD) is in the range from 5 to 500. In certain embodiments, the s3 of the formula (pD) is in the range from 10 to 250. In certain embodiments, the s3 of the formula (pD) is in the range from 12 to 150 In certain embodiments, the formula s3 (pD) is in the range from 15 to 100. In certain embodiments, the formula s3 (pD) is in the range from 18 to 75. In certain embodiments, the formula s3 (pD) is in the range from 20 to 75. 50.

In certain embodiments, -R ¹ of formula (pD) is - H. In certain embodiments, -R ¹ of formula (pD) is methyl. In certain embodiments, -R ¹ of formula (pD) is ethyl. In certain embodiments, -R ^1a of formula (pD) is - H. In certain embodiments, -R ^1a of formula (pD) is methyl. In certain embodiments, -R ^1a of formula (pD) is ethyl. In certain embodiments, -R ² of formula (pD) is - H. In certain embodiments, -R ² of formula (pD) is methyl. In certain embodiments, -R ² of formula (pD) is ethyl. In certain embodiments, -R ^2a of formula (pD) is - H. In certain embodiments, -R ^2a of formula (pD) is methyl. In certain embodiments, -R ^2a of formula (pD) is ethyl. In certain embodiments, -R ³ of formula (pD) is - H. In certain embodiments, -R ³ of formula (pD) is methyl. In certain embodiments, -R ³ of formula (pD) is ethyl. In certain embodiments, -R ^3a of formula (pD) is - H. In certain embodiments, -R ^3a of formula (pD) is methyl. In certain embodiments, -R ^3a of formula (pD) is ethyl. In certain embodiments, -R ⁴ of formula (pD) is - H. In certain embodiments, -R ⁴ of formula (pD) is methyl. In certain embodiments, -R ⁴ of formula (pD) is methyl. In certain embodiments, -R ^4a of formula (pD) is - H. In certain embodiments, -R ^4a of formula (pD) is methyl. In certain embodiments, -R ^4a of formula (pD) is ethyl. In certain embodiments, -R ⁵ of formula (pD) is - H. In certain embodiments, -R ⁵ of formula (pD) is methyl. In certain embodiments, -R ⁵ of formula (pD) is ethyl. In certain embodiments, -R ^5a of formula (pD) is - H. In certain embodiments, -R ^5a of formula (pD) is methyl. In certain embodiments, -R ^5a of formula (pD) is ethyl. In certain embodiments, -R ⁶ of formula (pD) is - H. In certain embodiments, -R ⁶ of formula (pD) is methyl. In certain embodiments, -R ⁶ of formula (pD) is ethyl. In certain embodiments, -R ^6a of formula (pD) is - H. In certain embodiments, -R ^6a of formula (pD) is methyl. In certain embodiments, -R ^6a of formula (pD) is ethyl. In certain embodiments, -R ⁷ of formula (pD) is - H. In certain embodiments, -R ⁷ of formula (pD) is methyl. In certain embodiments, -R ⁷ of formula (pD) is ethyl. In certain embodiments, -R ⁸ of formula (pD) is - H. In certain embodiments, -R ⁸ of formula (pD) is methyl. In certain embodiments, -R ⁸ of formula (pD) is ethyl. In certain embodiments, -R ^8a of formula (pD) is - H. In certain embodiments, -R ^8a of formula (pD) is methyl. In certain embodiments, -R ^8a of formula (pD) is ethyl. In certain embodiments, -R ⁹ of formula (pD) is - H. In certain embodiments, -R ⁹ of formula (pD) is methyl. In certain embodiments, -R ⁹ of formula (pD) is ethyl. In certain embodiments, -R ^9a of formula (pD) is - H. In certain embodiments, -R ^9a of formula (pD) is methyl. In certain embodiments, -R ^9a of formula (pD) is ethyl. In certain embodiments, -R ^9a of formula (pD) is - H. In certain embodiments, -R ^9a of formula (pD) is methyl. In certain embodiments, -R ^9a of formula (pD) is ethyl. In certain embodiments, -R ¹⁰ of formula (pD) is - H. In certain embodiments, -R ¹⁰ of formula (pD) is methyl. In certain embodiments, -R ¹⁰ of formula (pD) is ethyl. In certain embodiments, -R ^10a of formula (pD) is - H. In certain embodiments, -R ^10a of formula (pD) is methyl. In certain embodiments, -R ^10a of formula (pD) is ethyl. In certain embodiments, -R ¹¹ of formula (pD) is - H. In certain embodiments, -R ¹¹ of formula (pD) is methyl. In certain embodiments, -R ¹¹ of formula (pD) is ethyl. In certain embodiments, -R ¹² of formula (pD) is - H. In certain embodiments, -R ¹² of formula (pD) is methyl. In certain embodiments, -R ¹² of formula (pD) is ethyl. In certain embodiments, -R ^12a of formula (pD) is - H. In certain embodiments, -R ^12a of formula (pD) is methyl. In certain embodiments, -R ^12a of formula (pD) is ethyl. In certain embodiments, -R ¹³ of formula (pD) is - H. In certain embodiments, -R ¹³ of formula (pD) is methyl. In certain embodiments, -R ¹³ of formula (pD) is ethyl. In certain embodiments, -R ¹⁴ of formula (pD) is - H. In certain embodiments, -R ¹⁴ of formula (pD) is methyl. In certain embodiments, -R ¹⁴ of formula (pD) is ethyl. In certain embodiments, -R ^14a of formula (pD) is - H. In certain embodiments, -R ^14a of formula (pD) is methyl. In certain embodiments, -R ^14a of formula (pD) is ethyl.

In certain embodiments, -D ^1- of formula (pD) is -O-. In certain embodiments, -D ^1- of formula (pD) is -NR ^11- . In certain embodiments, -D ^1- of formula (pD) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ^1- of formula (pD) is -S-. In certain embodiments, -D ^1- of formula (pD) is -(S=O). In certain embodiments, -D ^1- of formula (pD) is -(S(O) ₂ )-. In certain embodiments, -D ^1- of formula (pD) is -C(O)-. In certain embodiments, -D ^1- of formula (pD) is -P(O)R ¹³ -. In certain embodiments, -D ^1- of formula (pD) is -P(O)(OR ¹³ )-. In certain embodiments, -D ^1- of formula (pD) is -CR ¹⁴ R ^14a -.

In certain embodiments, -D ^2- of formula (pD) is -O-. In certain embodiments, -D ^2- of formula (pD) is -NR ^11- . In certain embodiments, -D ^2- of formula (pD) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ^2- of formula (pD) is -S-. In certain embodiments, -D ^2- of formula (pD) is -(S=O). In certain embodiments, -D ^2- of formula (pD) is -(S(O) ₂ )-. In certain embodiments, -D ^2- of formula (pD) is -C(O)-. In certain embodiments, -D ^2- of formula (pD) is -P(O)R ¹³ -. In certain embodiments, -D ^2- of formula (pD) is -P(O)(OR ¹³ )-. In certain embodiments, -D ^2- of formula (pD) is -CR ¹⁴ R ^14a -.

In certain embodiments, -D ^3- of formula (pD) is -O-. In certain embodiments, -D ^3- of formula (pD) is -NR ^11- . In certain embodiments, -D ^3- of formula (pD) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ^3- of formula (pD) is -S-. In certain embodiments, -D ^3- of formula (pD) is -(S=O). In certain embodiments, -D ^3- of formula (pD) is -(S(O) ₂ )-. In certain embodiments, -D ^3- of formula (pD) is -C(O)-. In certain embodiments, -D ^3- of formula (pD) is -P(O)R ¹³ -. In certain embodiments, -D ^3- of formula (pD) is -P(O)(OR ¹³ )-. In certain embodiments, -D ^3- of formula (pD) is -CR ¹⁴ R ^14a -.

In certain embodiments, -D ⁴ - of formula (pD) is -O-. In certain embodiments, -D ⁴ - of formula (pD) is -NR ^11- . In certain embodiments, -D ⁴ - of formula (pD) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ⁴ - of formula (pD) is -S-. In certain embodiments, -D ⁴ - of formula (pD) is -(S=O). In certain embodiments, -D ⁴ - of formula (pD) is -(S(O) ₂ )-. In certain embodiments, -D ⁴ - of formula (pD) is -C(O)-. In certain embodiments, -D ⁴ - of formula (pD) is -P(O)R ¹³ -. In certain embodiments, -D ⁴ - of formula (pD) is -P(O)(OR ¹³ )-. In certain embodiments, -D ⁴ - of formula (pD) is -CR ¹⁴ R ^14a -.

In certain embodiments, -D ⁵ - of formula (pD) is -O-. In certain embodiments, -D ⁵ - of formula (pD) is -NR ^11- . In certain embodiments, -D ⁵ - of formula (pD) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ⁵ - of formula (pD) is -S-. In certain embodiments, -D ⁵ - of formula (pD) is -(S=O)-. In certain embodiments, -D ⁵ - of formula (pD) is -(S(O) ₂ )-. In certain embodiments, -D ⁵ - of formula (pD) is -C(O)-. In certain embodiments, -D ⁵ - of formula (pD) is -P(O)R ¹³ -. In certain embodiments, -D ⁵ - of formula (pD) is -P(O)(OR ¹³ )-. In certain embodiments, -D ⁵ - of formula (pD) is -CR ¹⁴ R ^14a -.

In certain embodiments, -D ⁶ - of formula (pD) is -O-. In certain embodiments, -D ⁶ - of formula (pD) is -NR ^11- . In certain embodiments, -D ⁶ - of formula (pD) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ⁶ - of formula (pD) is -S-. In certain embodiments, -D ⁶ - of formula (pD) is -(S=O). In certain embodiments, -D ⁶ - of formula (pD) is -(S(O) ₂ )-. In certain embodiments, -D ⁶ - of formula (pD) is -C(O)-. In certain embodiments, -D ⁶ - of formula (pD) is -P(O)R ¹³ -. In certain embodiments, -D ⁶ - of formula (pD) is -P(O)(OR ¹³ )-. In certain embodiments, -D ⁶ - of formula (pD) is -CR ¹⁴ R ^14a -.

In one embodiment, -CL ^p - has the formula (pE)

(pE),

Where

dotted lines marked with an asterisk indicate the connection point of the upper and lower substructure,

unmarked dotted lines indicate attachment to a backbone fragment or spacer fragment -SP ^1- ,

-R ^b1 , -R ^b1a , -R ^b2 , -R ^b2a , -R ^b3 , -R ^b3a , -R ^b4 , -R ^b4a , -R ^b5 , -R ^b5a , -R ^b6 , and -R ^b6 independently selected from the group consisting of - H and C _1-6 alkyl,

c1, c2, c3, c4, c5 and c6 are independently selected from the group consisting of 1, 2, 3, 4, 5 and 6,

d is an integer ranging from 2 to 250.

In certain embodiments, formula d (pE) is in the range from 3 to 200. In certain embodiments, formula d (pE) is in the range from 4 to 150. In certain embodiments, formula d (pE) is in the range from 5 to 100 In certain embodiments, formula d (pE) is between 10 and 50. In certain embodiments, formula d (pE) is between 15 and 30. In certain embodiments, formula d (pE) is about 23.

In certain embodiments, -R ^b1 and -R ^b1a of formula (pE) are - H. In certain embodiments -R ^b1 and -R ^b1a of formula (pE) are - H. In certain embodiments -R ^b2 and -R ^b2a of the formula (pE) is - H. In certain embodiments, -R ^b3 and -R ^b3a of the formula (pE) are - H. In certain embodiments, -R ^b4 and -R ^b4a of the formula (pE) are - H. In certain embodiments, -R ^b5 and -R ^b5a of formula (pE) are - H. In certain embodiments, -R ^b6 and -R ^b6a of formula (pE) are - H.

In certain embodiments, -R ^b1 , -R ^b1a , -R ^b2 , -R ^b2a , -R ^b3 , -R ^b3a , -R ^b4 , -R ^b4a , -R ^b5 , -R ^b5a , -R ^b6 , and -R ^b6 formulas (pE) all represent - H.

In certain embodiments, c1 of formula (pE) is 1. In certain embodiments, c1 of formula (pE) is 2. In certain embodiments, c1 of formula (pE) is 3. In certain embodiments, c1 of formula (pE) is 4. In certain embodiments, c1 of formula (pE) is 5. In certain embodiments, c1 of formula (pE) is 6.

In certain embodiments, c2 of formula (pE) is 1. In certain embodiments, c2 of formula (pE) is 2. In certain embodiments, c2 of formula (pE) is 3. In certain embodiments, c2 of formula (pE) is 4. In certain embodiments, c2 of formula (pE) is 5. In certain embodiments, c2 of formula (pE) is 6.

In certain embodiments, c3 of formula (pE) is 1. In certain embodiments, c3 of formula (pE) is 2. In certain embodiments, c3 of formula (pE) is 3. In certain embodiments, c3 of formula (pE) is 4. In certain embodiments, c3 of formula (pE) is 5. In certain embodiments, c3 of formula (pE) is 6.

In certain embodiments, c4 of formula (pE) is 1. In certain embodiments, c4 of formula (pE) is 2. In certain embodiments, c4 of formula (pE) is 3. In certain embodiments, c4 of formula (pE) is 4. In certain embodiments, c4 of formula (pE) is 5. In certain embodiments, c4 of formula (pE) is 6.

In certain embodiments, c5 of formula (pE) is 1. In certain embodiments, c5 of formula (pE) is 2. In certain embodiments, c5 of formula (pE) is 3. In certain embodiments, c5 of formula (pE) is 4. In certain embodiments, c5 of formula (pE) is 5. In certain embodiments, c5 of formula (pE) is 6.

In certain embodiments, c6 of formula (pE) is 1. In certain embodiments, c6 of formula (pE) is 2. In certain embodiments, c6 of formula (pE) is 3. In certain embodiments, c6 of formula (pE) is 4. In certain embodiments, c6 of formula (pE) is 5. In certain embodiments, c6 of formula (pE) is 6.

In certain embodiments, the cross-linking moiety -CL ^p - has the formula (pE- i)

-(pE-i),

Where

dotted lines indicate attachment to the main chain fragment or to the spacer fragment -SP ^1- .

In certain embodiments, Z is a hyaluronic acid hydrogel. Such hyaluronic acid-based hydrogels are known in the art, such as from WO2018/175788A1, which is incorporated herein by reference.

If - Z is a hyaluronic acid hydrogel, the conjugate of the present invention is, in certain embodiments, a conjugate comprising cross-linked hyaluronic acid strands to which a plurality of drug moieties are covalently and reversibly conjugated, wherein the conjugate contains a plurality of linked units selected from the group, consisting of

,

And ,

Where

the unmarked dotted line shows the point of attachment to an adjacent unit on the dotted line marked #, or to a hydrogen,

the dotted line marked # indicates the point of attachment to an adjacent unit on the unmarked dotted line or to a hydroxyl,

the dotted line marked § shows the connection point between at least two Z ³ links via the fragment -CL-,

-D, -L ^1- and -L ² each have the meanings as defined above,

each -CL- independently represents a fragment connecting at least two Z ³ units, and where there is at least one cleavable bond in a direct connection between two carbon atoms, marked *, connected by the -CL- fragment,

each -SP- is independently absent or represents a spacer fragment,

each -R ^a1 is independently selected from the group consisting of - H, C _1-4 alkyl, ammonium ion, tetrabutyl ammonium ion, cetylmethyl ammonium ion, alkali metal ion and alkaline earth metal ion,

each -R ^a2 is independently selected from the group consisting of -H and C _1-10 alkyl,

Where

all Z ¹ units present in the conjugate can be the same or different,

all Z ² units present in the conjugate can be the same or different,

all Z ³ units present in the conjugate can be the same or different,

at least one Z ³ unit is present on a hyaluronic acid strand that is connected to at least one Z ³ unit on another hyaluronic acid strand, and

the conjugate contains at least one fragment-L ² -L ^1- D.

The presence of at least one cleavable bond between the carbon atom marked * of the first Z ³ fragment and a direct connection with the carbon atom marked * of the second Z ³ fragment ensures that, after cleavage of all such cleavable bonds, the hyaluronic acid strands present in said conjugate are no longer cross-linked, allowing the hyaluronic acid network to break down.

It is clear that if the cleavable bond is located in a ring structure present in the direct connection of the carbon atom marked * of the first fragment Z ³ and the carbon atom marked Z ³ fragment, such a cleavable bond is insufficient for complete cleavage, and, accordingly, there are one or more additional cleavable bonds present in the direct connection of the carbon atom marked * of the first Z ³ fragment and the carbon atom marked of the Z ³ fragment.

It is clear that the phrase “the dotted line marked § indicates the connection point between at least two Z ³ units through the fragment -CL-” refers to the following structure

if -CL-, for example, is connected to two fragments of Z ³ , where two fragments of Z ³ are connected in the position indicated by §, through the fragment -CL-.

It is clear that a three-dimensionally cross-linked hydrogel cannot be formed if all the hyaluronic acid strands of the present conjugate contain only one Z ³ unit, which is connected to one Z ³ unit on another hyaluronic acid strand. However, if the first Z ³ link is connected to more than one Z ³ link on another thread, i.e. if -CL- is branched, such a first Z ³ unit may be cross-linked with two or more other Z ³ units on two or more different hyaluronic acid strands. Accordingly, the number of Z ³ units per hyaluronic acid strand required for a cross-linked hyaluronic acid hydrogel depends on the degree of branching -CL-. In certain embodiments, at least 30% of all hyaluronic acid strands present in the conjugate are connected to at least two other hyaluronic acid strands. It is clear that it is sufficient if the remaining hyaluronic acid threads are connected to only one other hyaluronic acid thread.

It is clear that such a hydrogel also contains many reacted and unreacted units and the presence of such fragments cannot be avoided. In certain embodiments, the sum of such multiple reacted and unreacted units is constitutes no more than 25% of the total number of units present in the conjugate, such as no more than 10%, such as no more than 15%, or such as no more than 10%.

It is further understood that in addition to the Z ¹ , Z ² and Z ³ units, partially reacted and unreacted units, the conjugate may also contain units that may result from cleavage of a reversible bond between -D and -L ^{1 -} or one or more cleavable bonds present in a direct connection between any two carbon atoms, marked *, connected to fragments -CL-, i.e. units as a result of cleavage of the conjugate.

In certain embodiments, each strand present in the conjugates of the present invention contains at least 20 units, such as 20 to 2500 units, 25 to 2200 units, 50 to 2000 units, 75 to 100 units, 75 up to 100 links, from 80 to 560 links, from 100 to 250 links, from 200 to 800 links, from 20 to 1000, from 60 to 1000, from 60 to 400 or from 200 to 600 links.

In certain embodiments, the -CL- moieties present in the conjugates of the present invention have different structures. In certain embodiments, the -CL- moieties present in the conjugates of the present invention have the same structure.

In general, any fragment that connects at least two other fragments is suitable for use as a -CL- fragment, which may also be referred to as a “cross-linking fragment”.

The at least two Z ³ units that are linked via the -CL- moiety can either be located on the same hyaluronic acid strand or on different hyaluronic acid strands.

The fragment -CL- can be linear or branched. In certain embodiments, -CL- is linear. In certain embodiments, -CL- is branched.

In certain embodiments, -CL- connects two Z ³ units. In certain embodiments, -CL- connects three Z ³ units. In certain embodiments, -CL- connects four Z ³ units. In certain embodiments, -CL- connects five Z ³ units. In certain embodiments, -CL- connects six Z ³ units. In certain embodiments, -CL- connects seven Z ³ units. In certain embodiments, -CL- connects eight Z ³ units. In certain embodiments, -CL connects nine Z ³ units.

If -CL- connects two Z ³ links, -CL- can be linear or branched. If -CL- connects more than two Z ³ units, -CL- is branched.

The branched fragment -CL- contains at least one branch point from which at least three branches emerge, which may also be referred to as “arms”. Such a branch point may be selected from the group consisting of

,

Where

the dotted lines indicate the attachment to the shoulder, and

-R ^B is selected from the group consisting of -H, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl, wherein C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl are optionally substituted one or more -R ^B1 which are the same or different, and wherein C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl are optionally interrupted by -C(O)O-, -O-, -C(O )-, -C(O)N(R ^B2 )-, -S(O) ₂ N(R ^B2 )-, -S(O)N(R ^B2 )-, -S(O) ₂ -, -S (O)-, -N(R ^B2 )S(O) ₂ N(R ^B2a )-, -S-, -N(R ^B2 )-, -OC(OR ^B2 )(R ^B2a )-, -N( R ^B2 )C(O)N(R ^B2a )- and -OC(O)N(R ^B2 )-, where -R ^B1 , -R ^B2 and -R ^B2a are selected from - H, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl.

In certain embodiments, -R ^B is selected from the group consisting of - H, methyl, and ethyl.

The branched fragment -CL- may contain multiple branch points, such as 1, 2, 3, 4, 5, 6, 7 or more branch points, which may be the same or different.

If a fragment -CL- connects three Z ³ units, such fragment -CL- contains at least one branch point from which at least three arms extend.

If a -CL- fragment connects four Z ³ units, such a -CL- fragment may contain one branch point from which four arms emerge. However, other geometries are also possible, such as at least two branch points from which at least three arms extend. The greater the number of connected Z ³ links, the greater the number of possible geometries.

According to the first embodiment, at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, or such as at least 95% of the number of hyaluronic acid conjugate strands of the present invention contain at least one Z ² moiety and at least one Z ³ moiety. According to this embodiment, Z ² and Z ³ units can be found in essentially all hyaluronic acid strands present in the conjugates of the present invention.

Accordingly, the conjugate according to this first embodiment comprises cross-linked hyaluronic acid strands to which a plurality of drug moieties are covalently and reversibly conjugated, wherein the conjugate comprises a plurality of linked units selected from the group consisting of

,

And ,

Where

the unmarked dotted line shows the point of attachment to an adjacent unit on the dotted line marked #, or to a hydrogen,

the dotted line marked # indicates the point of attachment to an adjacent unit on the unmarked dotted line or to a hydroxyl,

the dotted line marked § shows the connection point between at least two Z ³ links via the fragment -CL-,

-D, -L ^1- and -L ² each have the meanings as defined above,

Where

all Z ¹ units present in the conjugate can be the same or different,

all Z ² units present in the conjugate can be the same or different,

all Z ³ units present in the conjugate can be the same or different,

the number of Z ¹ units is in the range from 1% to 98% of the total number of units present in the conjugate,

the number of Z ² units is in the range from 1% to 98% of the total number of units present in the conjugate, provided that at least one Z ² unit is present in the conjugate,

the number of Z ³ units is in the range from 1% to 97% of the total number of units present in the conjugate, provided that at least one Z ³ unit is present per strand, and

wherein at least 70% of all hyaluronic acid strands contain at least one Z ² fragment and at least one Z ³ fragment.

In the conjugate according to the first embodiment, the number of Z ² units is in the range of 1 to 70% based on all units present in the conjugate, such as 2 to 15%, 2 to 10%, 16 to 39, 40 up to 65%, or from 50 to 60% based on all units present in the conjugate.

In the conjugate according to this first embodiment, the number of Z ³ units is in the range of 1 to 30% based on all units present in the conjugate, such as 2 to 5%, 5 to 20%, 10 to 18%, or from 14 to 18% based on all units present in the conjugate.

In the conjugate according to this first embodiment, the number of Z ¹ units is in the range of 10 to 97% based on all units present in the conjugate, such as 20 to 40%, such as 25 to 35%, such as from 41 to 95%, such as from 45 to 90%, such as from 50 to 70%, based on all units present in the conjugate.

Each cleavable bond present in the direct connection between the two carbon atoms marked * connected by the -CL- fragment may be different, or all such cleavable bonds present in the conjugate may be the same.

Each direct connection between two carbon atoms, marked *, connected by a -CL- fragment may have the same or a different number of cleavable bonds.

In certain embodiments, the number of cleavable bonds present in the conjugate of the present invention between all combinations of two carbon atoms marked * connected by the -CL- moiety is the same, and all such cleavable bonds have the same structure.

According to the first embodiment, the at least one cleavable bond present in the direct connection between the two carbon atoms marked * connected by the -CL- moiety may be selected from the group consisting of ester, carbonate, sulfate, phosphate, carbamate and amide bonds . It is clear that carbamates and amides themselves are not reversible, and that in this context, neighboring groups make these bonds reversible. In certain embodiments, there is one cleavable bond selected from the group consisting of ester, carbonate, sulfate, phosphate, carbamate, and amide bonds in a direct connection between two carbon atoms, marked *, connected by a -CL- moiety. In certain embodiments, there are two cleavable bonds selected from the group consisting of ester, carbonate, sulfate, phosphate, carbamate, and amide bonds in a direct connection between two carbon atoms, marked *, connected by a -CL- moiety, wherein the cleavable bonds may be same or different. In certain embodiments, there are three cleavable bonds selected from the group consisting of ester, carbonate, sulfate, phosphate, carbamate, and amide bonds in a direct connection between two carbon atoms, marked *, connected by a -CL- moiety, wherein the cleavable bonds may be same or different. In certain embodiments, there are four cleavable bonds selected from the group consisting of ester, carbonate, sulfate, phosphate, carbamate, and amide bonds in a direct connection between two carbon atoms, marked *, connected by a -CL- moiety, wherein the cleavable bonds may be same or different. In certain embodiments, there are five cleavable bonds selected from the group consisting of ester, carbonate, sulfate, phosphate, carbamate, and amide bonds in a direct connection between two carbon atoms, marked *, connected by a -CL- moiety, where the cleavable bonds may be same or different. In certain embodiments, there are six cleavable bonds selected from the group consisting of ester, carbonate, sulfate, phosphate, carbamate, and amide bonds in a direct connection between two carbon atoms, marked *, connected by a -CL- moiety, where the cleavable bonds may be same or different. It will be understood that if more than two Z ³ units are connected by -CL-, there are more than two carbons marked * that are connected, and thus there is more than one shortest connection with at least one cleavable bond. Each shortest connection may have the same or a different number of cleavable bonds.

In certain embodiments, at least one cleavable bond, such as one, two, three, four, five, six cleavable bonds, is located within -CL-.

In certain embodiments, the at least one cleavable bond present in the direct bond between the two carbon atoms marked * connected by the -CL- moiety is a single ester bond. In other embodiments, the at least one cleavable bond is two ester bonds. In other embodiments, the at least one cleavable bond is three ester bonds. In other embodiments, the at least one cleavable bond is four ester bonds. In other embodiments, the at least one cleavable bond is five ester bonds. In other embodiments, the at least one cleavable bond is six ester bonds.

In certain embodiments, the at least one cleavable bond present in the direct connection between the two carbon atoms marked * connected by the -CL- moiety is a single carbonate bond. In other embodiments, the at least one cleavable bond is two carbonate bonds. In other embodiments, the at least one cleavable bond is three carbonate bonds. In other embodiments, the at least one cleavable bond is four carbonate bonds. In other embodiments, the at least one cleavable bond is five carbonate bonds. In other embodiments, the at least one cleavable bond is six carbonate bonds.

In certain embodiments, the at least one cleavable bond present in the direct bond between the two carbon atoms marked * connected by the -CL- moiety is a single phosphate bond. In other embodiments, the at least one cleavable bond is two phosphate bonds. In other embodiments, the at least one cleavable bond is three phosphate bonds. In other embodiments, the at least one cleavable bond is four phosphate bonds. In other embodiments, the at least one cleavable bond is five phosphate bonds. In other embodiments, the at least one cleavable bond is six phosphate bonds.

In certain embodiments, the at least one cleavable bond present in the direct bond between the two carbon atoms marked * connected by the -CL- moiety is one sulfate bond. In other embodiments, the at least one cleavable bond is two sulfate bonds. In other embodiments, the at least one cleavable bond is three sulfate bonds. In other embodiments, the at least one cleavable bond is four sulfate bonds. In other embodiments, the at least one cleavable bond is five sulfate bonds. According to other options for sulfate bonds.

In certain embodiments, the at least one cleavable bond present in the direct bond between the two carbon atoms marked * connected by the -CL- moiety is a single carbamate bond. In other embodiments, the at least one cleavable bond is two carbamate bonds. In other embodiments, the at least one cleavable bond is three carbamate bonds. In other embodiments, the at least one cleavable bond is four carbamate bonds. In other embodiments, the at least one cleavable bond is five carbamate bonds. In other embodiments, the at least one cleavable bond is six carbamate bonds.

In certain embodiments, the at least one cleavable bond present in the direct bond between the two carbon atoms marked * connected by the -CL- moiety is a single amide bond. In other embodiments, the at least one cleavable bond is two amide bonds. In other embodiments, the at least one cleavable bond is three amide bonds. In other embodiments, the at least one cleavable bond is four amide bonds. In other embodiments, the at least one cleavable bond is five amide bonds. In other embodiments, the at least one cleavable bond is six amide bonds.

It has been found that a high degree of derivatization of the disaccharide units of hyaluronic acid, meaning that the number of Z ¹ units is less than 80% based on all units present in the conjugate, prevents certain hyaluronidases from degrading the hydrogel. As a result, less cleavage by hyaluronidases occurs and chemical cleavage of the bond being cleaved becomes more relevant. This makes the conjugate cleavage more predictable. The reason for this is that the level of enzymes, such as hyaluronidase for example, shows inter-patient variability and can vary between different injection sites, while chemical degradation is predominantly dependent on temperature and pH, which are more stable parameters, and thus , chemical degradation tends to be more predictable.

In some embodiments, -CL- is a C _{1- 50} alkyl that is optionally interrupted by one or more atoms or groups selected from the group consisting of -T-, -C(O)O-, -O-, -C( O)-, -C(O)N(R ^c1 )-, -S(O) ₂ -, -S(O)-, -S-, -N(R ^c1 )-, -OC(OR ^c1 )( R ^c1a )- and -OC(O)N(R ^c1 )-,

where -T- is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl and 8-11 membered heterobicyclyl, and

-R ^c1 , and -R ^c1a are selected from the group consisting of -H and C _1-6 alkyl. H

In certain embodiments, -CL- is a fragment of Formula (A)

(A),

Where

- Y ^1- has the formula

,

where the dotted line marked with an asterisk shows the connection to -D ^1- and the unmarked dotted line shows the connection to -D ^2- ,

- Y ^2- has the formula

,

where the dotted line marked with an asterisk shows the attachment to -D ⁴ - and the unmarked dotted line shows the attachment to -D ³ -,

- E ^1- has the formula

,

where the dotted line marked with an asterisk shows attachment to -(C=O)- and the unmarked dotted line shows attachment to -O-,

- E ^2- has the formula

,

where the dotted line marked with an asterisk shows attachment to -G ^1- and the unmarked dotted line shows attachment to -(C=O)-,

- G ^1- has the formula

,

where the dotted line marked with an asterisk shows the attachment to -O- and the unmarked dotted line shows the attachment to ^-E2- ,

- G ^2- has the formula

,

where the dotted line marked with an asterisk shows attachment to -O- and the unmarked dotted line shows attachment to -(C=O)-,

- G ^3- has the formula

(C-vii), ,

where the dotted line marked with an asterisk shows attachment to -O- and the unmarked dotted line shows attachment to -(C=O)-,

-D ^1- , -D ² -, -D ³ -, -D ⁴ -, -D ⁵ -, -D ⁶ - and -D ⁷ - are identical or different and each is independently selected from the group consisting of -O-, -NR ^11- , -N ⁺ R ¹² R ^12a -, -S-, -(S=O)-, -(S(O) ₂ ), -C(O)-, -P(O )R ¹³ and -CR ¹⁴ R ^14a -,

-R ¹ , -R ^1a , -R ² , -R ^2a , -R ³ , -R ^3a , -R ⁴ , -R ^4a , -R ⁵ , -R ^5a , -R ⁶ , -R ^6a , -R ⁷ , -R ^7a , -R ⁸ , -R ^8a , -R ⁹ , -R ^9a , -R ¹⁰ , -R ^10a , -R ¹¹ , -R ¹² , -R ^12a , -R ¹³ , -R ¹⁴ , and -R ^14a are identical or different and are each independently selected from the group consisting of - H and C _1-6 alkyl,

optionally one or more of the pairs -R ¹ /-R ^1a , -R ² /-R ^2a , -R ³ /-R ^3a , -R ⁴ /-R ^4a , -R ¹ /-R ² , -R ³ / -R ⁴ , -R ^1a /-R ^2a , -R ^3a /-R ^4a , -R ¹² /-R ^12a , and -R ¹⁴ /-R ^14a form a chemical bond or are joined together with the atom to which they are attached, to form a C _3-8 cycloalkyl or to form ring A or are joined together with the atom to which they are attached to form a 4-7 membered heterocyclyl or 8-11 membered heterobicyclyl or adamantyl,

A is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl and tetralinyl,

r1, r2, r5, r6, r13, r14, r15 and r16 are independently 0 or 1,

r3, r4, r7, r8, r9, r10, r11, r12 are independently 0, 1, 2, 3, or 4,

r17, r18, r19, r20, r21 and r22 are independently 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and

s1, s2, s4, s5 independently represent 1, 2, 3, 4, 5 or 6.

s3 is in the range from 1 to 200, preferably from 1 to 100 and more preferably from 1 to 50

In certain embodiments, r1 of formula (A) is 0. In certain embodiments, r1 of formula (A) is 1. In certain embodiments, r2 of formula (A) is 0. In certain embodiments, r2 of formula (A) is 1. In certain embodiments, r5 of formula (A) is 0. In certain embodiments, r5 of formula (A) is 1. In certain embodiments, r6 of formula (A) is 0. In certain embodiments, r6 of formula (A) is 1. In certain embodiments, r13 of formula (A) is 0. In certain embodiments, r13 of formula (A) is 1. In certain embodiments, r14 of formula (A) is 0. In certain embodiments, r14 of formula ( A) is 1. In certain embodiments, r15 of formula (A) is 0. In certain embodiments, r15 of formula (A) is 1. In certain embodiments, r16 of formula (A) is 0. In certain embodiments, r16 formula (A) is 1.

In certain embodiments, r3 of formula (A) is 0. In certain embodiments, r3 of formula (A) is 1. In certain embodiments, r4 of formula (A) is 0. In certain embodiments, r4 of formula (A) is 1. In certain embodiments, r3 of formula (A) and r4 of formula (A) are both 0.

In certain embodiments, r7 of formula (A) is 0. In certain embodiments, r7 of formula (A) is 1. In certain embodiments, r7 of formula (A) is 2. In certain embodiments, r8 of formula (A) is 0. In certain embodiments, r8 of formula (A) is 1. In certain embodiments, r8 of formula (A) of formula (A) is 2. In certain embodiments, r9 of formula (A) is 0. In certain embodiments, r9 of formula (A) is 1. In certain embodiments, r9 of formula (A) is 2. In certain embodiments, r10 of formula (A) is 0. In certain embodiments, r10 of formula (A) is 1. In certain embodiments According to certain embodiments, r10 of formula (A) is 2. In certain embodiments, r11 of formula (A) is 0. In certain embodiments, r11 of formula (A) is 1. In certain embodiments, r11 of formula (A) is 2. According to In certain embodiments, r12 of formula (A) is 0. In certain embodiments, r12 of formula (A) is 1. In certain embodiments, r12 of formula (A) is 2.

In certain embodiments, r17 of formula (A) is 1. In certain embodiments, r18 of formula (A) is 1. In certain embodiments, r19 of formula (A) is 1. In certain embodiments, r20 of formula (A) is 1. In certain embodiments, r21 of formula (A) is 1.

In certain embodiments, s1 of formula (A) is 1. In certain embodiments, s1 of formula (A) is 2. In certain embodiments, s2 of formula (A) is 1. In certain embodiments, s2 of formula (A) is 2. In certain embodiments, s4 of formula (A) is 1. In certain embodiments, s4 of formula (A) is 2.

In certain embodiments, the s3 of formula (A) is in the range from 1 to 100. In certain embodiments, the s3 of formula (A) is in the range from 1 to 75. In certain embodiments, the s3 of formula (A) is in the range from 2 to 50 In certain embodiments, the s3 of formula (A) is in the range from 2 to 40. In certain embodiments, the s3 of formula (A) is in the range from 3 to 30. In certain embodiments, the s3 of formula (A) is about 3.

In certain embodiments, -R ¹ of formula (A) is - H. In certain embodiments, -R ¹ of formula (A) is methyl. In certain embodiments, -R ¹ of formula (A) is ethyl. In certain embodiments, -R ^1a of formula (A) is - H. In certain embodiments, -R ^1a of formula (A) is methyl. In certain embodiments, -R ^1a of formula (A) is ethyl. In certain embodiments, -R ² of formula (A) is - H. In certain embodiments, -R ² of formula (A) is methyl. In certain embodiments, -R ² of formula (A) is ethyl. In certain embodiments, -R ^2a of formula (A) is - H. In certain embodiments, -R ^2a of formula (A) is methyl. In certain embodiments, -R ^2a of formula (A) is ethyl. In certain embodiments, -R ³ of formula (A) is - H. In certain embodiments, -R ³ of formula (A) is methyl. In certain embodiments, -R ³ of formula (A) is ethyl. In certain embodiments, -R ^3a of formula (A) is - H. In certain embodiments, -R ^3a of formula (A) is methyl. In certain embodiments, -R ^3a of formula (A) is ethyl. In certain embodiments, -R ⁴ of formula (A) is - H. In certain embodiments, -R ⁴ of formula (A) is methyl. In certain embodiments, -R ⁴ of formula (A) is methyl. In certain embodiments, -R ^4a of formula (A) is - H. In certain embodiments, -R ^4a of formula (A) is methyl. In certain embodiments, -R ^4a of formula (A) is ethyl. In certain embodiments, -R ⁵ of formula (A) is - H. In certain embodiments, -R ⁵ of formula (A) is methyl. In certain embodiments, -R ⁵ of formula (A) is ethyl. In certain embodiments, -R ^5a of formula (A) is - H. In certain embodiments, -R ^5a of formula (A) is methyl. In certain embodiments, -R ^5a of formula (A) is ethyl. In certain embodiments, -R ⁶ of formula (A) is - H. In certain embodiments, -R ⁶ of formula (A) is methyl. In certain embodiments, -R ⁶ of formula (A) is ethyl. In certain embodiments, -R ^6a of formula (A) is - H. In certain embodiments, -R ^6a of formula (A) is methyl. In certain embodiments, -R ^6a of formula (A) is ethyl. In certain embodiments, -R ⁷ of formula (A) is - H. In certain embodiments, -R ⁷ of formula (A) is methyl. In certain embodiments, -R ⁷ of formula (A) is ethyl. In certain embodiments, -R ⁸ of formula (A) is - H. In certain embodiments, -R ⁸ of formula (A) is methyl. In certain embodiments, -R ⁸ of formula (A) is ethyl. In certain embodiments, -R ^8a of formula (A) is - H. In certain embodiments, -R ^8a of formula (A) is methyl. In certain embodiments, -R ^8a of formula (A) is ethyl. In certain embodiments, -R ⁹ of formula (A) is - H. In certain embodiments, -R ⁹ of formula (A) is methyl. In certain embodiments, -R ⁹ of formula (A) is ethyl. In certain embodiments, -R ^9a of formula (A) is - H. In certain embodiments, -R ^9a of formula (A) is methyl. In certain embodiments, -R ^9a of formula (A) is ethyl. In certain embodiments, -R ^9a of formula (A) is - H. In certain embodiments, -R ^9a of formula (A) is methyl. In certain embodiments, -R ^9a of formula (A) is ethyl. In certain embodiments, -R ¹⁰ of formula (A) is - H. In certain embodiments, -R ¹⁰ of formula (A) is methyl. In certain embodiments, -R ¹⁰ of formula (A) is ethyl. In certain embodiments, -R ^10a of formula (A) is - H. In certain embodiments, -R ^10a of formula (A) is methyl. In certain embodiments, -R ^10a of formula (A) is ethyl. In certain embodiments, -R ¹¹ of formula (A) is - H. In certain embodiments, -R ¹¹ of formula (A) is methyl. In certain embodiments, -R ¹¹ of formula (A) is ethyl. In certain embodiments, -R ¹² of formula (A) is - H. In certain embodiments, -R ¹² of formula (A) is methyl. In certain embodiments, -R ¹² of formula (A) is ethyl. In certain embodiments, -R ^12a of formula (A) is - H. In certain embodiments, -R ^12a of formula (A) is methyl. In certain embodiments, -R ^12a of formula (A) is ethyl. In certain embodiments, -R ¹³ of formula (A) is - H. In certain embodiments, -R ¹³ of formula (A) is methyl. In certain embodiments, -R ¹³ of formula (A) is ethyl. In certain embodiments, -R ¹⁴ of formula (A) is - H. In certain embodiments, -R ¹⁴ of formula (A) is methyl. In certain embodiments, -R ¹⁴ of formula (A) is ethyl. In certain embodiments, -R ^14a of formula (A) is - H. In certain embodiments, -R ^14a of formula (A) is methyl. In certain embodiments, -R ^14a of formula (A) is ethyl.

In certain embodiments, -D ^1- of formula (A) is -O-. In certain embodiments, -D ^1- of formula (A) is -NR ^11- . In certain embodiments, -D ^1- of formula (A) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ^1- of formula (A) is -S-. In certain embodiments, -D ^1- of formula (A) is -(S=O). In certain embodiments, -D ^1- of formula (A) is -(S(O) ₂ )-. In certain embodiments, -D ^1- of formula (A) is -C(O)-. In certain embodiments, -D ^1- of formula (A) is -P(O)R ¹³ -. In certain embodiments, -D ^1- of formula (A) is -P(O)(OR ¹³ )-. In certain embodiments, -D ^1- of formula (A) is -CR ¹⁴ R ^14a -.

In certain embodiments, -D ^2- of formula (A) is -O-. In certain embodiments, -D ^2- of formula (A) is -NR ^11- . In certain embodiments, -D ^2- of formula (A) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ^2- of formula (A) is -S-. In certain embodiments, -D ^2- of formula (A) is -(S=O). In certain embodiments, -D ^2- of formula (A) is -(S(O) ₂ )-. In certain embodiments, -D ^2- of formula (A) is -C(O)-. In certain embodiments, -D ^2- of formula (A) is -P(O)R ¹³ -. In certain embodiments, -D ^2- of formula (A) is -P(O)(OR ¹³ )-. In certain embodiments, -D ^2- of formula (A) is -CR ¹⁴ R ^14a -.

In certain embodiments, -D ^3- of formula (A) is -O-. In certain embodiments, -D ^3- of formula (A) is -NR ^11- . In certain embodiments, -D ^3- of formula (A) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ^3- of formula (A) is -S-. In certain embodiments, -D ^3- of formula (A) is -(S=O). In certain embodiments, -D ^3- of formula (A) is -(S(O) ₂ )-. In certain embodiments, -D ^3- of formula (A) is -C(O)-. In certain embodiments, -D ^3- of formula (A) is -P(O)R ¹³ -. In certain embodiments, -D ^3- of formula (A) is -P(O)(OR ¹³ )-. In certain embodiments, -D ^3- of formula (A) is -CR ¹⁴ R ^14a -.

In certain embodiments, -D ⁴ - of formula (A) is -O-. In certain embodiments, -D ⁴ - of formula (A) is -NR ^11- . In certain embodiments, -D ⁴ - of formula (A) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ⁴ - of formula (A) is -S-. In certain embodiments, -D ⁴ - of formula (A) is -(S=O). In certain embodiments, -D ⁴ - of formula (A) is -(S(O) ₂ )-. In certain embodiments, -D ⁴ - of formula (A) is -C(O)-. In certain embodiments, -D ⁴ - of formula (A) is -P(O)R ¹³ -. In certain embodiments, -D ⁴ - of formula (A) is -P(O)(OR ¹³ )-. In certain embodiments, -D ⁴ - of formula (A) is -CR ¹⁴ R ^14a -.

In certain embodiments, -D ⁵ - of formula (A) is -O-. In certain embodiments, -D ⁵ - of formula (A) is -NR ^11- . In certain embodiments, -D ⁵ - of formula (A) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ⁵ - of formula (A) is -S-. In certain embodiments, -D ⁵ - of formula (A) is -(S=O)-. In certain embodiments, -D ⁵ - of formula (A) is -(S(O) ₂ )-. In certain embodiments, -D ⁵ - of formula (A) is -C(O)-. In certain embodiments, -D ⁵ - of formula (A) is -P(O)R ¹³ -. In certain embodiments, -D ⁵ - of formula (A) is -P(O)(OR ¹³ )-. In certain embodiments, -D ⁵ - of formula (A) is -CR ¹⁴ R ^14a -.

In certain embodiments, -D ⁶ - of formula (A) is -O-. In certain embodiments, -D ⁶ - of formula (A) is -NR ^11- . In certain embodiments, -D ⁶ - of formula (A) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ⁶ - of formula (A) is -S-. In certain embodiments, -D ⁶ - of formula (A) is -(S=O). In certain embodiments, -D ⁶ - of formula (A) is -(S(O) ₂ )-. In certain embodiments, -D ⁶ - of formula (A) is -C(O)-. In certain embodiments, -D ⁶ - of formula (A) is -P(O)R ¹³ -. In certain embodiments, -D ⁶ - of formula (A) is -P(O)(OR ¹³ )-. In certain embodiments, -D ⁶ - of formula (A) is -CR ¹⁴ R ^14a -.

In certain embodiments, -D ⁷ - of formula (A) is -O-. In certain embodiments, -D ⁷ - of formula (A) is -NR ^11- . In certain embodiments, -D ⁷ - of formula (A) is -N ⁺ R ¹² R ^12a -. In certain embodiments, -D ⁷ - of formula (A) is -S-. In certain embodiments, -D ⁷ - of formula (A) is -(S=O). In certain embodiments, -D ⁷ - of formula (A) is -(S(O) ₂ )-. In certain embodiments, -D ⁷ - of formula (A) is -C(O)-. In certain embodiments, -D ⁷ - of formula (A) is -P(O)R ¹³ -. In certain embodiments, -D ⁷ - of formula (A) is -P(O)(OR ¹³ )-. In certain embodiments, -D ⁷ - of formula (A) is -CR ¹⁴ R ^14a -.

In certain embodiments, -CL- has formula (B)

(B),

Where

a1 and a2 are independently selected from the group consisting of a1 and a2 are independently selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14, and

b is an integer ranging from 1 to 50.

In certain embodiments, a1 and a2 of formula (B) are different. In certain embodiments, a1 and a2 of formula (B) are the same.

In certain embodiments, a1 of formula (B) is 1. In certain embodiments, a1 of formula (B) is 2. In certain embodiments, a1 of formula (B) is 3. In certain embodiments, a1 of formula (B) is 4. In certain embodiments, a1 of formula (B) is 5. In certain embodiments, a1 of formula (B) is 6. In certain embodiments, a1 of formula (B) is 7. In certain embodiments, a1 of formula (B) is 8. In certain embodiments, a1 of formula (B) is 9. In certain embodiments, a1 of formula (B) is 10.

In certain embodiments, a2 of formula (B) is 1. In certain embodiments, a2 of formula (B) is 2. In certain embodiments, a2 of formula (B) is 3. In certain embodiments, a2 of formula (B) is 4. In certain embodiments, a2 of formula (B) is 5. In certain embodiments, a2 of formula (B) is 6. In certain embodiments, a2 of formula (B) is 7. In certain embodiments, a2 of formula (B) is 8. In certain embodiments, a2 of formula (B) is 9. In certain embodiments, a2 of formula (B) is 10.

In certain embodiments, b of formula (B) is in the range from 1 to 500. In certain embodiments, b of formula (B) is in the range from 2 to 250. In certain embodiments, b of formula (B) is in the range from 3 to 100 In certain embodiments, b of formula (B) is in the range from 3 to 50. In certain embodiments, b in formula (B) is in the range from 3 to 25. In certain embodiments, b in formula (B) is 3. In certain embodiments, b in formula (B) is in the range 3. In embodiments, b of formula (B) is 25.

In certain embodiments, -CL- has the formula (B-i)

(B-i).

In certain embodiments, -CL- has formula (C)

(C),

Where

a1 and a2 are independently selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14,

b is an integer ranging from 1 to 50, and

-R ¹¹ is selected from the group consisting of -H and C _1-6 alkyl.

In certain embodiments, a1 and a2 of formula (C) are different. In certain embodiments, a1 and a2 of formula (B) are the same.

In certain embodiments, a1 of formula (C) is 1. In certain embodiments, a1 of formula (C) is 2. In certain embodiments, a1 of formula (C) is 3. In certain embodiments, a1 of formula (C) is 4. In certain embodiments, a1 of formula (C) is 5. In certain embodiments, a1 of formula (C) is 6. In certain embodiments, a1 of formula (C) is 7. In certain embodiments, a1 of formula (C) is 8. In certain embodiments, a1 of formula (C) is 9. In certain embodiments, a1 of formula (C) is 10.

In certain embodiments, a2 of formula (C) is 1. In certain embodiments, a2 of formula (C) is 2. In certain embodiments, a2 of formula (C) is 3. In certain embodiments, a2 of formula (C) is 4. In certain embodiments, a2 of formula (C) is 5. In certain embodiments, a2 of formula (C) is 6. In certain embodiments, a2 of formula (C) is 7. In certain embodiments, a2 of formula (C) is 8. In certain embodiments, a2 of formula (C) is 9. In certain embodiments, a2 of formula (C) is 10.

In certain embodiments, b of formula (C) is in the range from 1 to 500. In certain embodiments, b of formula (C) is in the range from 2 to 250. In certain embodiments, b of formula (C) is in the range from 3 to 100 In certain embodiments, b of formula (C) is in the range from 3 to 50. In certain embodiments, b in formula (C) is in the range from 3 to 25. In certain embodiments, b in formula (C) is 3. In certain embodiments, b in formula (C) is in the range 3. In embodiments b of formula (C) is 25.

In certain embodiments, -R ¹¹ of formula (C) is - H. In certain embodiments, -R ¹¹ of formula (C) is methyl. In certain embodiments, -R ¹¹ of formula (C) is ethyl. In certain embodiments, -R ¹¹ of formula (C) is n-propyl. In certain embodiments, -R ¹¹ of formula (C) is isopropyl. In certain embodiments, -R ¹¹ of formula (C) is n-butyl. In certain embodiments, -R ¹¹ of formula (C) is isobutyl. In certain embodiments, -R ¹¹ of formula (C) is sec-butyl. In certain embodiments, -R ¹¹ of formula (C) is tert-butyl. In certain embodiments, -R ¹¹ of formula (C) is n-pentyl. In certain embodiments, -R ¹¹ of formula (C) is 2-methylbutyl. In certain embodiments, -R ¹¹ of formula (C) is 2,2-dimethylpropyl. In certain embodiments, -R ¹¹ of formula (C) is n-hexyl. In certain embodiments, -R ¹¹ of formula (C) is 2-methylpentyl. In certain embodiments, -R ¹¹ of formula (C) is 3-methylpentyl. In certain embodiments, -R ¹¹ of formula (C) is 2,2-dimethylbutyl. In certain embodiments, -R ¹¹ of formula (C) is 2,3-dimethylbutyl. In certain embodiments, -R ¹¹ of formula (C) is 3,3-dimethylpropyl.

In certain embodiments, -CL- has the formula (C-i)

(C-i).

According to a second embodiment, the fragment -CL- is selected from the group consisting of

(C-i), (C-ii),

Where

each dotted line shows an attachment to link Z ³ , and

-L ^1- , -L ^2- and -D have the meanings as defined for Z ² .

It is understood that in formula (C-i) two drug functional groups are conjugated to one -L ^1- moiety each, and that in formula (C-ii) three drug functional groups are conjugated to one -L ^1- moiety each. The -CL- fragment of formula (C-i) connects two Z ³ fragments and the -CL- fragment of formula (C- ii) connects three Z ³ fragments, which can be on the same or different strands of hyaluronic acid. In this embodiment, -CL- contains at least two cleavable bonds if -CL- has the formula (C- i) or at least three cleavable bonds if -CL- has the formula (C- ii), namely cleavable bonds , which connect D with fragment -L ^1- . The conjugate may contain only -CL- fragments of formula (C-i), may contain only -CL- fragments of formula (C-ii), or may contain only -CL- fragments of formula (C-i) and formula (C-ii).

Accordingly, the conjugate according to this second embodiment comprises cross-linked hyaluronic acid strands to which a plurality of drug moieties are covalently and reversibly conjugated, wherein the conjugate comprises a plurality of linked units selected from the group consisting of

,

And ,

Where

the unmarked dotted line shows the point of attachment to an adjacent unit on the dotted line marked #, or to a hydrogen,

the dotted line marked # indicates the point of attachment to an adjacent unit on the unmarked dotted line or to a hydroxyl,

the dotted line marked § shows the connection point between at least two Z ³ links via the fragment -CL-,

each -CL- contains at least one cleavable bond between two carbon atoms, marked *, connected by a fragment -CL-, and each -CL- is independently selected from the group consisting of formula (C-i) and (C-ii)

(C-i), (C-ii),

Where

dotted lines show connection to link Z ³ ,

-D, -L ^1- , -L ^2- , -SP-, -R ^a1 and -R ^a2 have the meanings as defined for Z ¹ , Z ² and Z ³ .

Where

all Z ¹ units present in the conjugate can be the same or different,

all Z ² units present in the conjugate can be the same or different,

all Z ³ units present in the conjugate can be the same or different,

the number of Z ¹ units is in the range from 1% to 98% of the total number of units present in the conjugate,

the number of Z ² units is in the range from 0% to 98% of the total number of units present in the conjugate,

the number of Z ³ units is in the range from 1% to 97% of the total number of units present in the conjugate, provided that at least one Z ³ unit is present on the thread, which is connected to at least one Z ³ unit on the other thread hyaluronic acid.

It is understood that such a hydrogel according to the second embodiment also contains a plurality of reacted and unreacted units, and the presence of such units cannot be avoided. In certain embodiments, the sum of such multiple reacted and unreacted units is constitutes no more than 25% of the total number of units present in the conjugate, such as no more than 10%, such as no more than 15%, or such as no more than 10%.

In the conjugate according to this second embodiment, the number of Z ² units is in the range from 0 to 70% based on all units present in the conjugate, such as from 2 to 15%, from 2 to 10%, from 16 to 39, from 40 to 65%, or from 50 to 60% based on all units present in the conjugate.

In the conjugate according to this second embodiment, the number of Z ³ units is in the range of 1 to 30% based on all units present in the conjugate, such as 2 to 5%, 5 to 20%, 10 to 18%, or from 14 to 18% based on all units present in the conjugate.

In the conjugate according to this second embodiment, the number of Z ¹ units is in the range of 10 to 97% based on all units present in the conjugate, such as 20 to 40%, such as 25 to 35%, such as from 41 to 95%, such as from 45 to 90%, such as from 50 to 70%, based on all units present in the conjugate.

More specific embodiments for -D, -L ^1- , -L ² -, -SP-, -R ^a1 and -R ^a2 according to the second embodiment are as described herein.

According to a third embodiment, the fragment -CL- is a fragment

(D-i),

Where

each dotted line shows an attachment to the Z ³ link.

It will be understood that the fragment -CL- of formula (D-i) contains at least one branch point, where the branch point may be selected from the group consisting of

,

Where

the dotted lines indicate the attachment to the shoulder, and

-R ^B is selected from the group consisting of -H, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl, wherein C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl are optionally substituted one or more -R ^B1 which are the same or different, and wherein C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl are optionally interrupted by -C(O)O-, -O-, -C(O )-, -C(O)N(R ^B2 )-, -S(O) ₂ N(R ^B2 )-, -S(O)N(R ^B2 )-, -S(O) ₂ -, -S (O)-, -N(R ^B2 )S(O) ₂ N(R ^B2a )-, -S-, -N(R ^B2 )-, -OC(OR ^B2 )(R ^B2a )-, -N( R ^B2 )C(O)N(R ^B2a )- and -OC(O)N(R ^B2 )-, where -R ^B1 , -R ^B2 , and -R ^B2a are selected from - H, C _1-6 alkyl, C _2-6 alkenyl and C _2-6 alkynyl.

In certain embodiments, -R ^B is selected from the group consisting of - H, methyl, and ethyl.

Accordingly, the conjugate according to the third embodiment comprises cross-linked hyaluronic acid strands to which a plurality of drug moieties are covalently and reversibly conjugated, wherein the conjugate comprises a plurality of linked units selected from the group consisting of

,

And ,

Where

the unmarked dotted line shows the point of attachment to an adjacent unit on the dotted line marked #, or to a hydrogen,

the dotted line marked # indicates the point of attachment to an adjacent unit on the unmarked dotted line or to a hydroxyl,

the dotted line marked § shows the connection point between at least two Z ³ links via the fragment -CL-,

each -CL- contains at least one cleavable bond between two carbon atoms, marked *, connected by the fragment -CL-, and each -CL- independently has the formula (D- i)

(D-i),

Where

dotted lines show connection to link Z ³ ,

-D, -L ^1- , -L ^2- , -SP-, -R ^a1 , and -R ^a2 have the meanings as defined for Z ¹ , Z ² and Z ³ .

Where

all Z ¹ units present in the conjugate can be the same or different,

all Z ² units present in the conjugate can be the same or different,

all Z ³ units present in the conjugate can be the same or different,

the number of Z ¹ units is in the range from 1% to 99% of the total number of units present in the conjugate,

the number of Z ² units is in the range from 0% to 98% of the total number of units present in the conjugate, and

the number of Z ³ units ranges from 1% to 97% of the total number of units present in the conjugate, provided that at least one Z ³ unit is present per strand.

It is understood that such a hydrogel according to the third embodiment also contains a plurality of reacted and unreacted units, and the presence of such units cannot be avoided. In certain embodiments, the sum of such multiple reacted and unreacted units is constitutes no more than 25% of the total number of units present in the conjugate, such as no more than 10%, such as no more than 15%, or such as no more than 10%.

In the conjugate according to this third embodiment, the number of Z ² units is in the range of 0 to 70% based on all units present in the conjugate, such as 2 to 15%, 2 to 10%, 16 to 39, 40 to 65%, or from 50 to 60% based on all units present in the conjugate.

In the conjugate according to this third embodiment, the number of Z ³ units is in the range of 1 to 30% based on all units present in the conjugate, such as 2 to 5%, 5 to 20%, 10 to 18%, or from 14 to 18% based on all units present in the conjugate.

In the conjugate according to this third embodiment, the number of Z ¹ units is in the range of 10 to 97% based on all units present in the conjugate, such as 20 to 40%, such as 25 to 35%, such as from 41 to 95%, such as from 45 to 90%, such as from 50 to 70%, based on all units present in the conjugate.

According to this third embodiment, -CL- contains a moiety -L ² -L ^1- D, so the presence of Z ² units is optional according to this embodiment. According to a certain embodiment, the Z ² units are not present according to the third embodiment. In certain embodiments, the conjugate of the third embodiment also contains Z ² units. The presence of Z ² units may have the effect that if a high drug loading is desired, which in this embodiment also means a high degree of cross-linking, an undesirable high degree of cross-linking can be avoided by the presence of Z ² units.

More specific embodiments for -D, -L ^1- , -L ² -, -SP-, -R ^{a1, and -Ra2} of the second embodiment are as described herein.

-SP- missing or a spacer fragment. In certain embodiments, -SP- does not contain a reversible bond, i.e. all bonds in -SP- are stable bonds.

In certain embodiments, -SP- is absent.

In certain embodiments, -SP- is a spacer moiety.

In certain embodiments, -SP- does not contain a cleavable bond, i.e. all -SP- bonds are stable bonds. In certain embodiments, at least one cleavable bond in a direct connection between two carbon atoms, marked * by the connected moiety -CL-, is provided by -SP-.

In certain embodiments, -SP- is a spacer moiety selected from the group consisting of -T-, C _{1- 50} alkyl, C _2-50 alkenyl, and C _2-50 alkynyl, wherein -T-, C _{1- 50} alkyl , C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted by one or more -R ^y2 , which are the same or different, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y3 )-, -S(O) ₂ N(R ^y3 )-, -S(O)N(R ^y3 )-, -S(O) ₂ -, -S(O)-, -N(R ^y3 )S(O) ₂ N(R ^y3a ) -, -S-, -N(R ^y3 )-, -OC(OR ^y3 )(R ^y3a )-, -N(R ^y3 )C(O)N(R ^y3a )- and -OC(O)N( R ^y3 )-,

-R ^y1 , and -R ^y1a are independently selected from the group consisting of -H, -T, C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl, where -T, C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted by one or more -R ^y2 which are the same or different, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y4 )-, -S(O) ₂ N(R ^y4 )-, -S(O)N(R ^y4 )-, -S(O) ₂ -, -S(O)-, -N(R ^y4 )S(O) ₂ N(R ^y4a )-, -S-, -N(R ^y4 )-, -OC(OR ^y4 )(R ^y4a )-, -N(R ^y4 )C(O)N(R ^y4a )- and -OC(O)N (R ^y4 )-,

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl and 8 -30-membered heteropolycyclyl, wherein each T is independently optionally substituted with one or more -R ^y2 , which are the same or different,

each -R ^y2 is independently selected from the group consisting of halogen, -CN, oxo (=O), -COOR ^y5 , -OR ^y5 , -C(O)R ^y5 , -C(O)N(R ^y5 R ^y5a ) , -S(O) ₂ N(R ^y5 R ^y5a ), -S(O)N(R ^y5 R ^y5a ), -S(O) ₂ R ^y5 , -S(O)R ^y5 , -N(R ^y5 )S(O) ₂ N(R ^y5a R ^y5b ), -SR ^y5 , -N(R ^y5 R ^y5a ), -NO ₂ , -OC(O)R ^y5 , -N(R ^y5 )C(O)R ^y5a , -N(R ^y5 )S(O) ₂ R ^y5a , -N(R ^y5 )S(O)R ^y5a , -N(R ^y5 )C(O)OR ^y5a , -N(R ^y5 )C( O)N(R ^y5a R ^y5b ), -OC(O)N(R ^y5 R ^y5a ) and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens that are the same or different, and

-R ^y3 , -R ^y3a , -R ^y4 , -R ^y4a , -R ^y5 , -R ^y5a , and -R ^y5b are each independently selected from the group consisting of -H and C _1-6 alkyl, where C _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different.

In certain embodiments, -SP- is a spacer moiety selected from the group consisting of -T-, C _1-50 alkyl, C _2-50 alkenyl, and C _2-50 alkynyl, wherein -T-, C _1-20 alkyl , C _2-20 alkenyl and C _2-20 alkynyl are optionally substituted by one or more -R ^y2 , which are the same or different, and wherein C _1-20 alkyl, C _2-20 alkenyl and C _2-20 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y3 )-, -S(O) ₂ N(R ^y3 )-, -S(O)N(R ^y3 )-, -S(O) ₂ -, -S(O)-, -N(R ^y3 )S(O) ₂ N(R ^y3a ) -, -S-, -N(R ^y3 )-, -OC(OR ^y3 )(R ^y3a )-, -N(R ^y3 )C(O)N(R ^y3a )- and -OC(O)N( R ^y3 )-,

-R ^y1 and -R ^y1a are independently selected from the group consisting of -H, -T, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl, where -T, C _1-10 alkyl , C _2-10 alkenyl and C _2-10 alkynyl are optionally substituted by one or more -R ^y2 , which are the same or different, and wherein C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y4 )-, -S(O) ₂ N(R ^y4 )-, -S(O)N(R ^y4 )-, -S(O) ₂ -, -S(O)-, -N(R ^y4 )S(O) ₂ N(R ^y4a ) -, -S-, -N(R ^y4 )-, -OC(OR ^y4 )(R ^y4a )-, -N(R ^y4 )C(O)N(R ^y4a )- and -OC(O)N( R ^y4 )-,

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl and 8 -30-membered heteropolycyclyl, wherein each T is independently optionally substituted with one or more -R ^y2 , which are the same or different,

-R ^y2 is selected from the group consisting of halogen, -CN, oxo (=O), -COOR ^y5 , -OR ^y5 , -C(O)R ^y5 , -C(O)N(R ^y5 R ^y5a ), - S(O) ₂ N(R ^y5 R ^y5a ), -S(O)N(R ^y5 R ^y5a ), -S(O) ₂ R ^y5 , -S(O)R ^y5 , -N(R ^y5 )S (O) ₂ N(R ^y5a R ^y5b ), -SR ^y5 , -N(R ^y5 R ^y5a ), -NO ₂ , -OC(O)R ^y5 , -N(R ^y5 )C(O)R ^y5a , -N(R ^y5 )S(O) ₂ R ^y5a , -N(R ^y5 )S(O)R ^y5a , -N(R ^y5 )C(O)OR ^y5a , -N(R ^y5 )C(O) N(R ^y5a R ^y5b ), -OC(O)N(R ^y5 R ^y5a ) and C _1-6 alkyl, wherein the C _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different, and

-R ^y3 , -R ^y3a , -R ^y4 , -R ^y4a , -R ^y5 , -R ^y5a , and -R ^y5b are each independently selected from the group consisting of -H and C _1-6 alkyl, where C The _1-6 alkyl is optionally substituted with one or more halogens, which are the same or different.

In certain embodiments, -SP- is a spacer moiety selected from the group consisting of -T-, C _{1- 50} alkyl, C _2-50 alkenyl, and C _2-50 alkynyl, wherein -T-, C _{1- 50} alkyl , C _2-50 alkenyl and C _2-50 alkynyl are optionally substituted by one or more -R ^y2 , which are the same or different, and wherein C _1-50 alkyl, C _2-50 alkenyl and C _2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R ^y3 )-, -S(O) ₂ N(R ^y3 )-, -S(O)N(R ^y3 )-, -S(O) ₂ -, -S(O)-, -N(R ^y3 )S(O) ₂ N(R ^y3a ) -, -S-, -N(R ^y3 )-, -OC(OR ^y3 )(R ^y3a )-, -N(R ^y3 )C(O)N(R ^y3a )- and -OC(O)N( R ^y3 )-,

-R ^y1 , and -R ^y1a are independently selected from the group consisting of -H, -T, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl,

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl and 8 -30-membered heteropolycyclyl,

each -R ^y2 is independently selected from the group consisting of halogen and C _1-6 alkyl, and

-R ^y3 , -R ^y3a , -R ^y4 , -R ^y4a , -R ^y5 , -R ^y5a and -R ^y5b are each independently selected from the group consisting of -H and C _1-6 alkyl, where C ₁ The _-6 alkyl is optionally substituted with one or more halogens, which are the same or different.

In certain embodiments, -SP- is a C _1-20 alkyl chain that is optionally interrupted by one or more groups independently selected from -O-, -T-, -N(R ^y3 )- and -C(O)N( R ^y1 )-, and wherein the C _1-20 alkyl chain is optionally substituted with one or more groups independently selected from -OH, -T, -N(R ^y3 )- and -C(O)N(R ^y6 R ^y6a ), where -R ^y1 , -R ^y6 , -R ^y6a are independently selected from the group consisting of H and C _1-4 alkyl, where T is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl , 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl and 8-30 membered heteropolycyclyl, provided that -SP- is attached to -X ^0E - and -X ^0F - through the carbon atom -SP-.

In certain embodiments, -SP- has a molecular weight ranging from 14 g/mol to 750 g/mol.

In certain embodiments, -SP- has a chain length ranging from 1 to 20 atoms.

In certain embodiments, all fragments of the -SP- conjugate are identical.

In certain embodiments, -SP- is C _1-10 alkyl. In certain embodiments, -SP- is C ₁ alkyl. In certain embodiments, -SP- is C ₂ alkyl. In certain embodiments, -SP- is C ₃ alkyl. In certain embodiments, -SP- is C ₄ alkyl. In certain embodiments, -SP- is C ₅ alkyl. In certain embodiments, -SP- is C ₆ alkyl. In certain embodiments, -SP- is C ₇ alkyl. In certain embodiments, -SP- is _C8 alkyl. In certain embodiments, -SP- is C ₉ alkyl. In certain embodiments, -SP- is C ₁₀ alkyl.

According to another aspect, the present invention relates to a pharmaceutical composition comprising one or more water-insoluble controlled release PRRAs according to the present invention and at least one excipient. According to a certain embodiment, the pharmaceutical composition is in the form of a suspension. In certain embodiments, the pharmaceutical composition is a dry composition.

Such a pharmaceutical composition may also contain one or more additional drugs. Such one or more additional drugs may be selected from the group consisting of cytotoxic/chemotherapeutic agents, immune checkpoint inhibitors or antagonists, immune checkpoint agonists, multispecific drugs, antibody-drug conjugates (ADCs), radionucleotides, or targeted radionucleotide therapeutics. agents, DNA damage repair inhibitors, tumor metabolism inhibitors, pattern recognition receptor agonists, protein kinase inhibitors, chemokine and chemoattractant receptor agonists, chemokine and chemokine receptor antagonists, cytokine receptor agonists, death receptor agonists, CD47 or SIRPα antagonists, oncolytic drugs, transformative signal proteins, epigenetic modifiers, tumor peptides or tumor vaccines, heat shock protein (HSP) inhibitors, proteolytic enzymes, ubiquitin and proteasome inhibitors, adhesion molecule antagonists, and hormones, including hormonal peptides and synthetic hormones.

In certain embodiments, the one or more additional drugs are a cytotoxic/chemotherapeutic agent. In certain embodiments, the one or more additional drugs are an immune checkpoint inhibitor or antagonist. In certain embodiments, the one or more additional drugs are a multispecific drug. In certain embodiments, the one or more additional drugs are an antibody-drug conjugate (ADC). In certain embodiments, the one or more additional drugs are a radionucleotide or a targeted radionucleotide therapeutic. In certain embodiments, the one or more additional drugs are a DNA damage repair inhibitor. In certain embodiments, the one or more additional drugs are a tumor metabolism inhibitor. In certain embodiments, the one or more additional drugs are a pattern recognition receptor agonist. In certain embodiments, the one or more additional drugs are a protein kinase inhibitor. In certain embodiments, the one or more additional drugs are a chemokine and chemoattractant receptor agonist. In certain embodiments, the one or more additional drugs are a chemokine and chemoattractant receptor antagonist. In certain embodiments, the one or more additional drugs are a cytokine receptor agonist. In certain embodiments, the one or more additional drugs are a death receptor agonist. In certain embodiments, the one or more additional drugs are a CD47 antagonist. In certain embodiments, the one or more additional drugs are a SIRPα antagonist. In certain embodiments, the one or more additional drugs are an oncolytic drug. In certain embodiments, the one or more additional drugs are a signal transducing protein. In certain embodiments, the one or more additional drugs are an epigenetic modifier. In certain embodiments, the one or more additional drugs are a tumor peptide or a tumor vaccine. In certain embodiments, one or more additional drugs is a heat shock protein (HSP) inhibitor. In certain embodiments, the one or more additional drugs are a proteolytic enzyme. In certain embodiments, the one or more additional drugs are a ubiquitin and a proteasome inhibitor. In certain embodiments, the one or more additional drugs are an adhesion molecule antagonist. In certain embodiments, the one or more additional drugs are a hormone, including hormonal peptides and synthetic hormones.

The cytotoxic or chemotherapeutic agent may be selected from the group consisting of alkylating agents, antimetabolites, microtubule-targeting agents, topoisomerase inhibitors, cytotoxic antibiotics, auristatins, enediines, lexitropsins, duocarmycins, cyclopropylpyrroloindoles, puromycin, dolastatins, maytansine derivatives, alkylsulfonates, triazenes and piperazines.

The alkylating agent may be selected from the group consisting of nitrogen mustards, such as mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan, nitrosoureas, such as N-nitroso-N-methylurea, carmustine, lomustine, semustine, fotemustine and streptozotocin , tetrazines such as dacarbazine, mitozolomide and temozolomide, ethylenimines such as altretamine, aziridines such as thiotepa, mitomycin and diaziquoin, cisplatin and its derivatives such as cisplatin, carboplatin, oxaliplatin, and non-classical alkylating agents such as for example, procarbazine and hexamethylmelamine.

The antimetabolite may be selected from the group consisting of antifolates, such as methotrexate and pemetrexed, fluoropyrimidines, such as fluorouracil and capecitabine, deoxynucleoside analogues, such as cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine and pentostatin , and thiopurines, such as thioguanine and mercaptopurine.

The microtubule-targeting agent may be selected from the group consisting of vinca alkaloids, such as vincristine, vinblastine, vinorelbine, vindesine and vinflunine, taxanes, such as paclitaxel and docetaxel, podophyllotoxins and derivatives thereof, such as podophyllotoxin, etoposide and teniposide, stilbenoid phenol and its derivatives, such as sibrestat (CA4P), and BNC105.

The topoisomerase inhibitor may be selected from the group consisting of topoisomerase I inhibitors, such as irinotecan, topotecan and camptothecin, and topoisomerase II inhibitors, such as etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbaron and aclarubicin.

The cytotoxic antibiotic may be selected from the group consisting of anthracyclines, such as doxorubicin, daunorubicin, epirubicin and idarubicin, pirarubicin, aclarubicin, bleomycin, mitomycin C, mitoxantrone, actinomycin, dactinomycin, adriamycin, mithramycin and tirapazamine.

Auristatin may be selected from the group consisting of monomethyl auristatin E (mmAE) and monomethyl auristatin F (mmAF).

Endiyne may be selected from the group consisting of neocarzinostatin, lidamycin (C-1027), calicheamicins, esperamycins, dinemycins and golfomycin A.

The maytansine derivative may be selected from the group consisting of ansamitocin, mertansine (emtansine, DM1) and ravtansine (soravtansine, DM14).

The immune checkpoint inhibitor or antagonist may be selected from the group consisting of CTLA-4 (cytotoxic T lymphocyte associated protein 4) inhibitors, such as ipilimumab, tremelimumab, MK-1308, FPT155, PRS010, BMS-986249, BPI- 002, CBT509, JS007, ONC392, TE1254, IBI310, BR02001, CG0161, KN044, PBI5D3H5, BCD145, ADU1604, AGEN1884, AGEN1181, CS1002 and CP675206, PD-1 (programmed death) inhibitors 1), such as pembrolizumab, nivolumab, pidilizumab , AMP-224, BMS-936559, cemiplimab and PDR001, PD-L1 (programmed cell death protein 1) inhibitors such as MDX-1105, MEDI4736, atezolizumab, avelumab, BMS-936559 and durvalumab, PD-L2 (ligand programmed death 2), KIR (killer cell immunoglobulin-like receptor) inhibitors such as lirilumab (IPH2102) and IPH2101, B7-H3 inhibitors such as MGA271, B7-H4 inhibitors such as FPA150, BTLA inhibitors ( attenuator of B- and T-lymphocytes), LAG3 inhibitors (lymphocyte activation gene 3), such as IMP321 (eftilagimod alfa), relatlimab, MK-4280, AVA017, BI754111, ENUM006, GSK2831781, INCAGN2385, LAG3Ig, LAG525, REGN3767, m016 , Sym022, TSR033, TSR075 and XmAb22841, TIM-3 inhibitors (T-cell immunoglobulin and mucin-domain containing 3), such as LY3321367, MBG453, and TSR-022, VISTA inhibitors (V-domain Ig suppressor of T cell activation ), such as JNJ-61610588, ILT2/LILRB1 inhibitors (Ig-like transcript 2/leukocyte Ig-like receptor 1), ILT3/LILRB4 inhibitor (Ig-like transcript 3/leukocyte Ig-like receptor 4), ILT4 inhibitors/ LILRB2 (Ig-like transcript 4/leukocyte Ig-like receptor 2), such as MK-4830, TIGIT inhibitors (T cell immunoreceptor with Ig and ITIM domains), such as MK-7684, PTZ-201, RG6058 and COM902 , NKG2A inhibitors, such as IPH-2201, and PVRIG inhibitors, such as COM701.

One example of a CTLA-4 inhibitor is an anti-CTLA4 conjugate or a pharmaceutically acceptable salt thereof, wherein said conjugate contains a plurality of anti-CTLA4 moieties -D _CTLA4 covalently conjugated through at least one moiety -L ^{1 -} L ^2- to a polymeric moiety Z, where -L ^{1- is} covalently and reversibly conjugated to -D _CTLA4 , and -L ^2- is covalently conjugated to Z, and where -L ^1- is a linker moiety, and -L ^2- is a chemical bond or spacer moiety, wherein the moieties -L ^1- , -L ^2- and Z are as described herein for the conjugate of the present invention. In certain embodiments, -D _CTLA4 is selected from the group consisting of _Fc wild-type anti-CTLA4 antibodies, Fc enhanced for effector function/FcγR binding anti-CTLA4 antibodies, anti-CTLA4 antibodies typically active in the tumor microenvironment, anti-CTLA4 small molecules, CTLA4 antagonistic fusion proteins, anti-CTLA4 anticalins, anti-CTLA4 nanobodies and anti-CTLA4 multispecific biological compounds based on antibodies, scFV or other formats. In certain embodiments, -D _CTLA4 is ipilimumab. In certain embodiments, -D _CTLA4 is tremelimumab. In certain embodiments, the anti-CTLA4 conjugate has the following structure:

,

Where

the dotted line marked with an asterisk indicates attachment to the -D amine nitrogen _{of CTLA4} , specifically the amine nitrogen of ipilimumab, and

the unmarked dotted line indicates attachment to Z, such as a hydrogel, in particular a cross-linked hyaluronic acid-based hydrogel.

It is understood that a plurality of -D _CTLA4 -L ^1- L ^2- moieties are connected to Z if Z is a hydrogel, such as a cross-linked hyaluronic acid hydrogel.

In certain embodiments, the nitrogen of the amine functional group -D _{of CTLA4} , and in particular of ipilimumab, is an amine or a lysine residue. In certain embodiments, the nitrogen of the amine functional group -D _{of CTLA4} , and in particular of ipilimumab, is the N-terminal amine.

In certain embodiments, the one or more additional drugs are a CTLA4 inhibitor, as described above.

The immune checkpoint agonist may be selected from the group consisting of CD27 agonists, such as recombinant CD70, such as HERA-CD27L, and varlilumab (CDX-1127), CD28 agonists, such as recombinant CD80, recombinant CD86, TGN1412 and FPT155, CD40 agonists such as recombinant CD40L, CP-870,893, decatuzumab (SGN-40), Chi Lob 7/4, ADC-1013 and CDX1140, 4 -1BB agonists (CD137) such as recombinant 4 -1BBL, urelumab, utomilumab and ATOR-1017, OX40 agonists such as recombinant OX40L, MEDI0562, GSK3174998, MOXR0916 and PF-04548600, GITR agonists such as recombinant GITRL, TRX518, MEDI1873, INCAGN01876, M K-1248, MK-4166, GWN323 and BMS-986156, and ICOS agonists such as recombinant ICOSL, JTX-2011 and GSK3359609.

The multispecific drug may be selected from the group consisting of biologics and small molecule immune checkpoint inhibitors. Examples of biologics are multispecific immune checkpoint inhibitors, such as lipocalin CD137/HER2, PD1/LAG3, FS118, XmAb22841 and XmAb20717, and multispecific immune checkpoint agonists. Such multispecific immune checkpoint agonists may be selected from the group consisting of Ig superfamily agonists, such as ALPN-202, TNF superfamily agonists, such as ATOR-1015, ATOR-1144, ALG.APV-527, lipocalin/PRS- 343, PRS344/ONC0055, FAP-CD40 DARPin, MP0310 DARPin, FAP-0X40 DARPin, EGFR-CD40 DARPin, EGFR41BB/CD137 DARPin, EGFR-0X40/DARFPin, HER2-CD40 DARPin, HER2-41BB/CD137 DARPin , HER2-0X40 DARPin, FIBRONECTIN ED-B-CD40 DARPin, FIBRONECTIN ED-B- 41BB/CD137 and FIBRONECTIN ED-B-0X40 DARPin, CD3 multispecific agonists, such as blinatumomab, solitomab, MEDI-565, ertumaxomab, anti-HER2/CD3 1Fab - immunoglobulin G TDB, GBR 1302, MGD009, MGD007, EGFRBi, EGFR-CD Probody, RG7802, PF-06863135, PF-06671008, MOR209/ES414, AMG212/BAY2010112 and CD3-5T4, and CD16 multispecific agonists, like 1633 BiKE, 161533 TriKE, OXS-3550, OXS-C3550, AFM13 and AFM24.

An example of a small molecule immune checkpoint inhibitor is CA-327 (TIM3/PD-L1 antagonist).

The antibody-drug conjugate may be selected from the group consisting of ADCs targeting hematopoietic cancers, such as gemtuzumab ozogamicin, brentuximab vedotin, inotuzumab ozogamicin, SAR3419, BT062, SGN-CD19A, IMGN529, MDX-1203, polatuzumab vedotin (RG759 6 ), pinatuzumab vedotin (RG7593), RG7598, milatuzumab-doxorubicin and OXS-1550, and ADCs targeting solid tumor antigens, such as trastuzumab emtansine, glembatumomab vedotin, SAR56658, AMG-172, AMG-595, BAY-94- 9343, BIIB015, vorsetuzumab mafodotin (SGN-75), ABT-414, ASG-414, ASG-ASG-5ME, enfortumab vedotin (ASG-22ME), ASG-16M8F, IMGN853, indusatumab vedotin (MLN-0264), vadortuzumab vedotin (RG7450), sofituzumab vedotin (RG7458), lifastuzumab vedotin (RG7599), RG7600, DEDN6526A (RG7636), PSMA TTC, 1095 from Progenics Pharmaceuticals, lorvotuzumab mertansine, lorvotuzumab emtansine, IMMU -130, sakituzumab govitecan (IMMU -13 2), PF - 06263507 and MEDI0641.

The radionucleotides may be selected from the group consisting of β-emitters, such as ¹⁷⁷ Lutetium, ¹⁶⁶ holmium, ¹⁸⁶ rhenium, ¹⁸⁸ rhenium, ⁶⁷ copper, ¹⁴⁹ promethium, ¹⁹⁹ gold, ⁷⁷ bromine, ¹⁵³ samarium, ¹⁰⁵ rhodium, ⁸⁹ strontium, ⁹⁰ yttrium, ¹³¹ iodine, α-emitters such as ²¹³ bismuth, ²²³ radium, ²²⁵ actinium, ²¹¹ astatine, and Auger electron emitters such as ⁷⁷ bromine, ¹¹¹ iodine, ¹²³ iodine and ¹²⁵ iodine.

Targeted radionucleotide therapeutics may be selected from the group consisting of Zevalin ( ⁹⁰ Y-ibritumomab tiuxetan), Bexar ( ¹³¹ I-tositumomab), Oncolim ( ¹³¹ I-Lym 1), Lymphocide ( ⁹⁰ Y-epratuzumab), Cotard ( ¹³¹ I -ChTNN -1/B), labetuzumab ( ⁹⁰ Y or ¹³¹ I-CEA), teragin ( ⁹⁰ Y- pemtumomab), licartin ( ¹³¹ I-metuximab), radretumab ( ¹³¹ I-L19) PAM4 ( ⁹⁰ Y-clivatuzumab tetraxetane) , xofigo ( ²²³ Ra dichloride), ( ¹⁷⁷ Lu-DOTA-Tyr ^3- octreotate) and ¹³¹ I-MIBG.

The DNA damage repair inhibitor may be selected from the group consisting of poly(ADP-ribose) polymerase (PARP) inhibitors, such as olaparib, rucaparib, niraparib, veliparib, CEP 9722 and E7016, CHK1/CHK2 dual inhibitors, such as AZD7762 , V158411, CBP501 and XL844, CHK1 selective inhibitors, such as PF477736, MK8776/SCH900776, CCT244747, CCT245737, LY2603618, LY2606368/prexasertib, AB-IsoG, ARRY575, CBP93872, ESP01, GDC0425, SAR020106, SRA737, V158411 and VER250840, CHK2 inhibitors such as CCT241533 and PV1019, ATM inhibitors such as AZD0156, AZD1390, KU55933, M3541 and SX-RDS1, ATR inhibitors such as AZD6738, BAY1895344, M4344 and M6620 (VX-97 0), and DNA-PK inhibitors, such as M3814.

The tumor metabolism inhibitor may be selected from the group consisting of adenosine pathway inhibitors, tryptophan metabolism inhibitors and arginine pathway inhibitors.

Examples of adenosine pathway inhibitors are A2AR (adenosine A2A receptor) inhibitors, such as ATL-444, istradefylline (KW-6002), MSX-3, preladenant (SCH-420,814), SCH-58261, SCH412,348, SCH-442,416, ST-1535, caffeine, VER-6623, VER-6947, VER-7835, vipadenant (BIIB-014), ZM-241,385, PBF-509 and V81444, CD73 inhibitors such as IPH53 and SRF373, and CD39 inhibitors such as for example IPH52.

Examples of tryptophan metabolism inhibitors are IDO inhibitors, such as indoximod (NLG8189), epacadostat, navoximod, BMS-986205 and MK-7162, TDO inhibitors, such as 680C91, and dual IDO/TDO inhibitors.

Examples of arginine pathway inhibitors are arginase inhibitors, such as INCB001158.

The pattern recognition agonist may be selected from the group consisting of Toll-like receptor, NOD-like receptor, RIG-I-like receptor, cytosolic DNA sensor, STING, and aromatic hydrocarbon receptor (AhR) agonists.

Toll-like receptor agonists may be selected from the group consisting of TLR1/2 agonists, such as peptidoglycans, lipoproteins, Pam3CSK4, amplifier, SLP-AMPLIVANT, HESPECTA, ISA101 and ISA201, TLR2 agonists, such as LAM-MS, LPS -PG, LTA- BS, LTA-SA, PGN- BS, PGN- EB, PGN- EK, PGN-SA, CL429, FSL -1, Pam2CSK4, Pam3CSK4, zymosan, CBLB612, SV- 283, ISA204, SMP105, killed by heating Listeria monocytogenes , TLR3 agonists such as poly(A:U), poly(I:C) (poly-ICLC), rintatolimod, apoxime, IPH3102, poly-ICR, PRV300, RGCL2, RGIC.1, Riboxim (RGC100 , RGIC100), Riboxol (RGIC50) and Riboxon, TLR4 agonists such as lipopolysaccharide (LPS), neoceptin-3, glucopyranosyl lipid adjuvant (GLA), GLA-SE, G100, GLA-AF, Clinical Center Reference Endotoxin (CCRE), monophosphorylated lipid A, grass MATA MPL, PEPA10, ONT-10 (PET-Lipid A, oncothyroid), G-305, ALD046, CRX527, CRX675 (RC527, RC590), GSK1795091, OM197MPAC, OM294DP and SAR439794, TLR2/4 agonists, such as lipid A, OM174 and PGN007, TLR5 agonists such as flagellin, entolimod, mobilan, protectan CBLB501, TLR6/2 agonists such as diacylated lipoproteins, diacylated lipopeptides, FSL-1, MALP-2 and CBLB613 agonists TLR7, such as CL264, CL307, imiquimod (R837), TMX-101, TMX-201, TMX-202, TMX-302, gardikquimod, S-27609, 851, UC-IV150, 852A (3M-001, PF- 04878691), loxoribine, polyuridylic acid, GSK2245035, GS-9620, RO6864018 (ANA773, RG7795), RO7020531, isoribine, AN0331, ANA245, ANA971, ANA975, DSP0509, DSP3025 (AZD8 848), GS986, MBS2, MBS5, RG7863 (RO6870868) , sotirimod, SZU101 and TQA3334, TLR8 agonists such as ssPolyUridine, ssRNA40, TL8-506, XG-1-236, VTX-2337 (motolimod), VTX-1463, TMX-302, VTX-763, DN1508052 and GS9688, TLR7/8 agonists, such as CL075, CL097, poly(dT), resiquimod (R-848, VML600, S28463), MEDI9197 (3M-052), NKTR262, DV1001, IMO4200, IPH3201 and VTX1463, TLR9 agonists, such as , CpG DNA, CpG ODN, lefitolimod (MGN1703), SD-101, QbG10, CYT003, CYT003-QbG10, DUK-CpG-001, CpG-7909 (PF-3512676), GNKG168, EMD 1201081, IMO-2125, IMO- 2055, CpG10104, AZD1419, AST008, IMO2134, MGN1706, IRS 954, 1018 ISS, actilon (CPG10101), ATP00001, AVE0675, AVE7279, CMP001, DIMS0001, DIMS9022, DIMS9059, DV230, DV281, EnanDIM, heplisav (V270), kappaproct (DIMS0150), NJP834, NPI503, SAR21609 and Tolamba, and TLR7/9 agonists such as DV1179.

Examples of CpG ODNs are ODN 1585, ODN 2216, ODN 2336, ODN 1668, ODN 1826, ODN 2006, ODN 2007, ODN BW006, ODN D-SL01, ODN 2395, ODN M362 and ODN D-SL03.

NOD-like receptors may be selected from the group consisting of NOD1 agonists, such as C12-iE-DAP, C14-Tri-LAN-Gly, iE-DAP, iE-Lys, and Tri-DAP, and NOD2 agonists, such as for example, L18-MDP, MDP, M-TriLYS, murabutide and N-glycolyl-MDP.

RIG-I-like receptors can be selected from the group consisting of 3p-hpRNA, 5'ppp-DsRNA, 5'ppp RNA (M8), 5'OH kink RNA (CBS-13-BPS), 5'PPP SLR , KIN100, KIN 101, KIN1000, KIN1400, KIN1408, KIN1409, KIN1148, KIN131A, poly(dA:dT), SB9200, RGT100 and Hiltonol.

Cytosolic DNA sensors can be selected from the group consisting of cGAS agonists, dsDNA-EC, G3-YSD, HSV-60, ISD, ODN TTAGGG (A151), poly(dG:dC) and VACV-70.

STING can be selected from the group consisting of MK-1454, ADU-S100 (MIW815), 2'3'-CGAMP, 3'3'-CGAMP, c-di-AMP, c-di-GMP, cAIMP (CL592) , cAIMP difluoro (CL614), cAIM(PS) ₂ difluoro (Rp/Sp) (CL656), 2'2'-CGAMP, 2'3'-CGAM(PS)2 (Rp/Sp), 3'3'- CGAM fluorinated, c-di-AMP fluorinated, 2'3'-C-di-AMP, 2'3'-C-di-AM(PS)2 (Rp,Rp), c-di-GMP fluorinated, 2'3'-C-di-GMP, c-di-IMP, c-di-UMP and DMXAA (vadimezan, ASA404).

The aromatic hydrocarbon receptor (AhR) may be selected from the group consisting of FICZ, ITE and L-cunurenine.

The protein kinase inhibitor may be selected from the group consisting of receptor tyrosine kinase inhibitors, intracellular kinase inhibitors, cyclin-dependent kinase inhibitors, phosphoinositide 3-kinase inhibitors, mitogen-activated protein kinase inhibitors, nuclear factor kappa-β kinase (IKK) inhibitors, and Wee-1 inhibitors. .

Examples of receptor tyrosine kinase inhibitors are EGF receptor inhibitors, such as afatinib, cetuximab, erlotinib, gefitinib, pertuzumab and margetuximab, VEGF receptor inhibitors, such as axitinib, lenvatinib, pegaptanib and linifanib (ABT-869), C-KIT receptor inhibitors, such as CDX0158 (KTN0158), ERBB2 (HER2) inhibitors, such as Herceptin (trastuzumab), ERBB3 receptor inhibitors, such as CDX3379 (MEDI3379, KTN3379) and AZD8931 (sapitinib), FGF receptor inhibitors, such as erdafitinib, inhibitors AXL receptor, such as BGB324 (BGB 324, R 428, R428, bemcentinib) and SLC391, and MET receptor inhibitors, such as CGEN241.

Examples of intracellular kinase inhibitors include Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib, acalabrutinib, GS-4059, spebrutinib, BGB-3111, HM71224, zanubrutinib, ARQ531, BI-BTK1 and vecabrutinib, spleen tyrosine kinase inhibitors such as fostamatin b, inhibitors Bcr-Abl tyrosine kinases, for example, imatinib and nilotinib, Janus kinase inhibitors, for example, ruxolitinib, tofacitinib and fedratinib, and multispecific tyrosine kinase inhibitors, for example, bosutinib, crizotinib, cabozantinib, dasatinib, entrectinib, lapatinib, mubritinib, pazopanib, , sunitinib , SU6656 and vandetanib.

One example of a tyrosine kinase inhibitor is a tyrosine kinase inhibitor (“TKI”) conjugate or a pharmaceutically acceptable salt thereof, wherein said conjugate comprises a plurality of TKI -D _TKI moieties covalently conjugated through at least one -L ^{1 -} L ^{2 -} moiety to a polymeric moiety Z, where -L ¹ is covalently and reversibly conjugated to -D _TKI and -L ^2- is covalently conjugated to Z, and where -L ^1- is a linker moiety and -L ^2- is a chemical bond or spacer moiety, where the moieties are -L ^1- , -L ^2- and Z are as described herein for the conjugate of the present invention. In certain embodiments, the -D _TKI is selected from the group consisting of receptor tyrosine kinase inhibitors, intracellular kinase inhibitors, cyclin-dependent kinase inhibitors, phosphoinositide 3-kinase (PI3K) inhibitors, mitogen-activated protein kinase inhibitors, nuclear factor kappa-β kinase inhibitors (IKK) and Wee -1 inhibitors. In certain embodiments, the -D _TKI is axitinib. In certain embodiments, the -D _TKI is lenvatinib. In certain embodiments, the -D _TKI is pegaptanib. In certain embodiments, the -D _TKI is linifanib.

In certain embodiments, the TKI conjugate has the following structure

,

Where

the dotted line shows the attachment of Z, such as PEG hydrogel or hyaluronic acid hydrogel.

In certain embodiments, the TKI conjugate has the following structure

,

Where

the dotted line shows the attachment of Z, such as a PEG-based hydrogel or a hyaluronic acid-based hydrogel.

In certain embodiments, the TKI conjugate has the following structure

,

Where

the dotted line shows the attachment of Z, such as a PEG-based hydrogel or a hyaluronic acid-based hydrogel.

In certain embodiments, the TKI conjugate has the following structure

,

Where

the dotted line shows the attachment of Z, such as PEG hydrogel or hyaluronic acid hydrogel.

Examples of cyclin-dependent kinase inhibitors are ribociclib, palbociclib, abemaciclib, trilaciclib, purvalanol A, olomucin II and MK-7965.

Examples of phosphoinositide 3-kinase inhibitors are IPI549, GDc-0326, pictilisib, serabelisib, IC-87114, AMG319, seletalisib, idealisib and CUDC907.

Examples of mitogen-activated protein kinase inhibitors include Ras/farnesyl transferase inhibitors such as tipirafinib and LB42708, Raf inhibitors such as regorafenib, encorafenib, vemurafenib, dabrafenib, sorafenib, PLX-4720, GDC-0879, AZ628, lifirafenib, and RO5126766, MEK inhibitors such as cobimetinib, trametinib, binimetinib, selumetinib, pimasertib, refametinib and PD0325901, ERK inhibitors such as MK-8353, GDC-0994, ulixertinib and SCH772984.

Examples of nuclear factor kappa-β kinase (IKK) inhibitors are BPI-003 and AS602868.

An example of a Wee -1 inhibitor is adavosertib.

The chemokine receptor and chemoattractant receptor agonist may be selected from the group consisting of CXC chemokine receptors, CC chemokine receptors, C chemokine receptors, CX3C chemokine receptors, and chemoattractant receptors.

The CXC chemokine receptor may be selected from the group consisting of CXCR1 agonists, such as recombinant CXCL8 and recombinant CXCL6, CXCR2 agonists, such as recombinant CXCL8, recombinant CXCL1, recombinant CXCL2, recombinant CXCL3, recombinant CXCL5, recombinant CXCL6, 145 and SB251353, CXCR3 agonists, such as recombinant CXCL9, recombinant CXCL10, recombinant CXCL11 and recombinant CXCL4, CXCR4 agonists, such as recombinant CXCL12, ATI2341, CTCE0214, CTCE0324 and NNZ4921, CXCR5 agonists, e.g. recombinant CXCL13, CXCR6 agonists like for example, recombinant CXCL16, and CXCL7 agonists, such as recombinant CXCL11.

The CC chemokine receptor may be selected from the group consisting of CCR1 agonists, such as recombinant CCL3, ECI301, recombinant CCL4, recombinant CCL5, recombinant CCL6, recombinant CCL8, recombinant CCL9/10, recombinant CCL14, recombinant CCL15, recombinant CCL16, ny CCL23 , PB103, PB105 and MPIF1, CCR2 agonists such as recombinant CCL2, recombinant CCL8, recombinant CCL16, PB103 and PB105, CCR3 agonists such as recombinant CCL11, recombinant CCL26, recombinant CCL7, recombinant CCL13, recombinant C CL15, recombinant CCL24, recombinant CCL5, recombinant CCL28 and recombinant CCL18, CCR4 agonists such as recombinant CCL3, ECI301, recombinant CCL5, recombinant CCL17 and recombinant CCL22, CCR5 agonists such as recombinant CCL3, ECI301, recombinant CCL5, recombinant C CL8, recombinant CCL11, recombinant CCL13, recombinant CCL14, recombinant CCL16, PB103 and PB105, CCR6 agonists such as recombinant CCL20, CCR7 agonists such as recombinant CCL19 and recombinant CCL21, CCR8 agonists such as recombinant CCL1, recombinant CCL16, PB103 and PB105, agonists CCR9, such as recombinant CCL25, CCR10 agonists, such as recombinant CCL27 and recombinant CCL28, and CCR11 agonists, such as recombinant CCL19, recombinant CCL21, and recombinant CCL25.

Chemokine receptor C may be an XCR1 agonist, such as recombinant XCL1 or recombinant XCL2.

CX3C chemokine receptors may be a CX3CR1 agonist, such as recombinant CX3CL1.

Chemoattractant receptors may be selected from the group consisting of formyl peptide receptor agonists, such as N-formyl peptides, N-formylmethionine leucyl phenylalanine, enfuvirtide, T21/DP107, Annexin A1, Ac2-26 and Ac9-25, C5a receptor agonists , and chemokine-like receptor 1 agonists, such as chemerin.

Chemokine antagonists may be selected from the group consisting of CXCL chemokine inhibitors, such as UNBS5162, CXCL8 inhibitors, such as BMS986253 and PA620, CXCL10 inhibitors, such as TM110, eldelumab and NI0801, CXCL12 inhibitors, such as NOX-A12 and JVS100, CXCL13 inhibitors such as VX5, CCL2 inhibitors such as PA508, ABN912, AF2838, BN83250, BN83470, C243, CGEN54, CNTO888, NOXE36, VT224 and SSR150106, CCL5 inhibitors such as HGS1025 and NI 0701, inhibitors CCL2/CCL5, such as BKTP46, CCL5/FMLP receptor inhibitors, such as RAP160, CCL11 inhibitors, such as bertilimumab and RAP701, CCL5/CXCL4 inhibitors, such as CT2008 and CT2009, CCL20 inhibitors, such as GSK3050002, and CX3CL1 inhibitors, such as ketmolimab.

Chemokine receptor antagonists may be selected from the group consisting of CXCR1 inhibitors, such as repertaxin, CCX832, FX68 and KB03, CXCR2 inhibitors, such as AZD5069, AZD5122, AZD8309, GSK1325756, GSK1325756H, PS291822, 5 and SB656933, CXCR1/CXCR2 inhibitors, such as DF1970, DF2156A, DF2162, DF2755A, reparixin, SX576, SX682, PACG31P, AZD4721 and PA401, CXCR3 inhibitors, CXCR4 inhibitors such as BL8040, CXCR4/E-selectin inhibitors such as GMI1359, CXCR6 inhibitors such as CCX5224, CCR1 inhibitors, such as AZD4818, BAY865047, BMS817399, CCX354, CCX634, CCX9588, CP481715, MLN3701, MLN3897, PS031291, PS375179 and PS386113, CCR2 inhibitors, such as AZD2423, 0, BMS741672, CCX140, CCX598, CCX872, CCX915, CNTX6970, INCB3284, INCB3344, INCB8696, JNJ17166864, JNJ27141491, MK0812, OPLCCL2LPM, PF4136309, serocion, STIB0201, STIB0211, STIB0221, STIB0232, STIB0234, TAK202, 26, CCR2/CCR5 inhibitors, such as PF04634817, RAP103 and TBR652, CCR2/CCR5 inhibitors/ CCR8, such as RAP310, CCR3 inhibitors, such as ASM8, AXP1275, BMS639623, CM101, DPC168, GW766994, GW824575, MT0814, OPLCCL11LPM and QAP642, CCR4 inhibitors, such as AT008, AZD2098, CCX6239, X193, FLX475, GBV3019, GSK2239633, IC487892 and poteligo, CCR5 inhibitors, such as 5P12-RANTES, AZD5672, AZD8566, CMPD167, ESN196, GSK706769, GW873140, HGS004, INCB15050, INCB9471, L872, microbicide, PF232798 PRO140 , RAP101, SAR113244, SCH350634, SCH351125, SCH417690, Selzentry , TAK779, TBR220, TD0232 and VX286, CCR5/CXCR4 inhibitors such as AMD887, ND401 and SP01A, CCR6 inhibitors such as CCX507, CCX9664 and STIB100X, CCR6 inhibitors such as CCX025, CCX507, CCX807, MLN31, 26, POL7085, traficet-EN, CXCR3 inhibitors, such as AMG487, AT010, STIA120X, CXCR4 inhibitors, such as AD114, AD214, ALX0651, ALX40-4C, AMD070, AT007, AT009, BKT170, BMS936564, selixafor, CTCE9908, , GSK812397, KRH2731, KRH3140, LY2510924, LY2624587, Mozobil, OPLCXCL12LPM, PF06747143, POL6326, Q122, Revixil, TG0054, USL311, X4P001 and X4P002, and CXCR7 inhibitors such as CCX650 and CCX662.

Cytokine receptor agonists may be selected from the group consisting of mRNA, DNA or plasmids encoding the genes IL-2, IL-15, IL-7, IL-10, IL-12, IL-21, IFNα 1-17, IFNβ, IFNγ, IL-18, IL-27, TNFα, GM-CSF, FLT3L and TRAIL and recombinant proteins such as IL-2/IL-15 β/γ receptor agonists, IL-10 receptor agonists, IL-12 receptor agonists , IL-18 receptor agonists, IL-21 receptor agonists, IL-7 receptor agonists, IFNα/β receptor agonists, IFNγ receptor agonists, FLT3 receptor agonists and TNFα receptor agonists.

Examples of IL-2/IL-15 β/γ receptor agonists are recombinant IL-2, recombinant IL-15, ALKS4230, ALT803, APN301, MDNA109, NKTR214, RG7461, RG7813, AM0015, NIZ985, NKTR255, RTX-212, SO- C101, XmAb24306, L19-IL2, THOR-707 and PB101.

In certain embodiments, the IL-2 agonist is as described in WO2019/185705A1, which is incorporated herein by reference in its entirety. Specifically, the IL-2 agonist according to certain embodiments is a conjugate comprising the IL-2 protein of SEQ ID NO:1

PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTCMLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFSQSIIST LT,

wherein the cysteine sulfur at position 37 in SEQ ID NO:1 is conjugated to a moiety of formula (2)

(2),

where the dotted line indicates the addition of said sulfur, and

n is about 113 or about 226,

and where is the amine nitrogen of the side chain of any of the lysine residues, i.e. one of the lysine residues selected from the group consisting of lysine residues at positions 7, 8, 31, 34, 42, 47, 48, 53, 63, 75 and 96 in SEQ ID NO: 1, conjugated to a moiety of formula (3)

(3),

where the dotted line indicates the attachment of the indicated side chain nitrogen of the indicated lysine residue, and

p1, p2, p3 and p4 are independently an integer in the range from 200 to 250.

In a certain embodiment, the IL-2 protein sequence differs by at least one amino acid from the sequence of SEQ ID NO: 1, such as by one amino acid, two amino acids, three amino acids, four amino acids, or five amino acids.

In certain embodiments, n in formula (2) is 113. In certain embodiments, n in formula (2) is 226.

In certain embodiments, p1, p2, p3, and p4 are independently an integer in the range of 220 to 240. In certain embodiments, p1, p2, p3, and p4 are the same integer.

Examples of IL-12 receptor agonists include AM0012, AS1409, dodequine, HemaMax, LipoVIL12, MSB0010360N and NHS-IL12.

An example of an IL-18 receptor agonist is SB485232.

An example of an IL-21 receptor agonist is BMS982470 (denenicokin).

Examples of IL-7 receptor agonist are CYT107, CYT99007 and GX-I7.

Examples of TNFα receptor agonist are L19-TNFα, Aurimune, Beromune, BreMel/TNFα, Fibromune, Refnot and TNFPEG20.

Death receptor agonists may be selected from the group consisting of TRAILR1/DR4 agonists, such as AMG951 (dulanermin), APG350, APG880, HGSETR1 (mapatumumab) and SL231, and TRAILR2/DR5 agonists, such as AMG655, DS8273, HGSETR2 ( lexatumumab), HGSTR2J, IDD004/GEN1029, INBRX109, LBY135, MEDI3039, PRO95780, RG7386 and TAS266.

CD47 antagonists may be selected from the group consisting of ALX148, CC-90002, Hu5F9G4, SRF231, TI061, TTI-621, TTI-622, AO176, IBI188, IMC002 and LYN00301.

An example of a SIRPα antagonist is FSI89.

Examples of oncolytic drugs are CAVATAK, BCG, Mobilan, TG4010, Pexa-Vec (JX-594), JX-900, JX-929 and JX-970.

Examples of signal transducing proteins are Fn14-TRAIL (KAHR101), CTLA4-FasL (KAHR102), PD1-41BBL (DSP 105), PD1-CD70 (DSP 106) and SIRPα-41BBL (DSP 107).

Epigenetic modifiers may be selected from the group consisting of DNA methyltransferase inhibitors, lysine-specific demethylase 1 inhibitors, Zeste homolog 2 inhibitors, bromodomain extra-terminal motif (BET) protein inhibitors, such as GSK525762, and histone deacetylase (HDAC) inhibitors, such as , beleodaq, SNDX275 and CKD-M808.

Examples of tumor peptides/tumor vaccines are NY-ESO, WT1, MART-1, IO102 and PF-06753512.

Examples of heat shock protein (HSP) inhibitors are HSP90 inhibitors, such as PF-04929113 (SNX-5422).

Examples of proteolytic enzymes are recombinant hyaluronidase, such as rHuPH20 and PEGPH20.

Ubiquitin and proteasome inhibitors may be selected from the group consisting of ubiquitin-specific protease (USP) inhibitors, for example P005091, 20S proteasome inhibitors, such as bortesimibe, carfilzomib, ixazomib, oprosomib, delanzomib and celastrol, and immunoproteasome inhibitors, such as ,ONX-0914.

The adhesion molecule antagonists may be selected from the group consisting of β2 integrin antagonists, such as PGG imprim, and selectin antagonists.

The hormones may be selected from the group consisting of hormone receptor agonists and hormone receptor antagonists.

An example of a hormone receptor agonist is the somatostatin receptor agonists, such as somatostatin, lanreotide, octreotide, FX125L, FX141L and FX87L.

Examples of hormone receptor antagonists are antiandrogens, antiestrogens, and antiprogestogens. Examples of antiandrogens are steroidal antiandrogens, such as cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, oxendolone and osaterone acetate, non-steroidal antiandrogens, such as flutamide, bicalutamide, nilutamide, topilutamide, enzalutamide and apalutamide, androgen synthesis inhibitors, for example , ketoconazole, abiraterone acetate, seviteronel, aminoglutethimide, finasteride, dutasteride, epristeride and alfatradiol. Examples of antiestrogens are selective estrogen receptor modulators (SERMs), such as tamoxifen, clomiphene, fareston and raloxifene, silent ER antagonists and selective estrogen receptor degraders (SERD), such as fulvestrant, aromatase inhibitors, such as anastrozole, letrozole, exemestane , vorozole, formestane and fadrozole, and antigonadotropins such as testosterone, progestogens and GnRH analogues. Examples of antiprogestogens are mifepristone, lilopristone and onapristone.

In certain embodiments, the water-insoluble controlled-release PRRA releases resiquimod, i.e. one type -D is resiquimod, and one or more additional drugs of the pharmaceutical composition is nivolumab. In certain embodiments, the water-insoluble controlled-release PRRA releases resiquimod, i.e. one type -D is resiquimod, and one or more additional drugs is pembrolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases resiquimod, i.e. one type -D is resiquimod, and one or more additional drugs is atezolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases resiquimod, i.e. one type -D is resiquimod, and one or more additional drugs is avelumab. In certain embodiments, the water-insoluble controlled-release PRRA releases resiquimod, i.e. one type -D is resiquimod, and one or more additional drugs is durvalumab. In certain embodiments, the water-insoluble controlled-release PRRA releases resiquimod, i.e. one type -D is resiquimod, and one or more additional drugs is ipilimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases resiquimod, i.e. one type -D is resiquimod, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases resiquimod, i.e. one type -D is resiquimod, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases resiquimod, i.e. one type -D is resiquimod, and one or more additional drugs is cetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases resiquimod, i.e. one type -D is resiquimod, and one or more additional drugs is margetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases resiquimod, i.e. one type -D is resiquimod, and one or more additional drugs is one of the CD47 or SIRPα blockers described above. It is understood that the conjugates may not only release resiquimod or contain -D moieties in the form of resiquimod, but may also contain one or more other types of -D.

In certain embodiments, the water-insoluble controlled-release PRRA releases imiquimod, i.e. one type -D is imiquimod, and one or more additional drugs of the pharmaceutical composition is nivolumab. In certain embodiments, the water-insoluble controlled-release PRRA releases imiquimod, i.e. one type -D is imiquimod, and one or more additional drugs is pembrolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases imiquimod, i.e. one type -D is imiquimod, and one or more additional drugs is atezolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases imiquimod, i.e. one type -D is imiquimod, and one or more additional drugs is avelumab. In certain embodiments, the water-insoluble controlled-release PRRA releases imiquimod, i.e. one type -D is imiquimod, and one or more additional drugs is durvalumab. In certain embodiments, the water-insoluble controlled-release PRRA releases imiquimod, i.e. one type -D is imiquimod, and one or more additional drugs is ipilimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases imiquimod, i.e. one type -D is imiquimod, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases imiquimod, i.e. one type -D is imiquimod, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases imiquimod, i.e. one type -D is imiquimod, and one or more additional drugs is cetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases imiquimod, i.e. one type -D is imiquimod, and one or more additional drugs is margetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases imiquimod, i.e. one type -D is imiquimod, and one or more additional drugs is one of the CD47 or SIRPα blockers described above. It is understood that the conjugates may not only release imiquimod or contain -D moieties in the form of imiquimod, but may also contain one or more other types of -D.

In certain embodiments, the water-insoluble controlled-release PRRA releases SD-101, i.e. one type -D is SD-101, and one or more additional drugs of the pharmaceutical composition is nivolumab. In certain embodiments, the water-insoluble controlled-release PRRA releases SD-101, i.e. one type -D is SD-101, and one or more additional drugs is pembrolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases SD-101, i.e. one type -D is SD-101, and one or more additional drugs is atezolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases SD-101, i.e. one type -D is SD-101, and one or more additional drugs is avelumab. In certain embodiments, the water-insoluble controlled-release PRRA releases SD-101, i.e. one type -D is SD-101, and one or more additional drugs is durvalumab. In certain embodiments, the water-insoluble controlled-release PRRA releases SD-101, i.e. one type -D is SD-101, and one or more additional drugs is ipilimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases SD-101, i.e. one type -D is SD-101, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases SD-101, i.e. one type -D is SD-101, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases SD-101, i.e. one type -D is SD-101, and one or more additional drugs is cetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases SD-101, i.e. one type -D is SD-101, and one or more additional drugs is margetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases SD-101, i.e. one type -D is SD-101, and one or more additional drugs is one of the CD47 or SIRPα blockers described above. It is understood that the conjugates may not only release SD-101 or contain -D moieties in the form of SD-101, but may also contain one or more other types of -D.

In certain embodiments, the water-insoluble controlled-release PRRA releases CMP001, i.e. one type -D is CMP001, and one or more additional drugs of the pharmaceutical composition is nivolumab. In certain embodiments, the water-insoluble controlled-release PRRA releases CMP001, i.e. one type -D is CMP001, and one or more additional drugs is pembrolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases CMP001, i.e. one type -D is CMP001, and one or more additional drugs is atezolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases CMP001, i.e. one type -D is CMP001, and one or more additional drugs is avelumab. In certain embodiments, the water-insoluble controlled-release PRRA releases CMP001, i.e. one type -D is CMP001, and one or more additional drugs is durvalumab. In certain embodiments, the water-insoluble controlled-release PRRA releases CMP001, i.e. one type -D is CMP001, and one or more additional drugs is ipilimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases CMP001, i.e. one type -D is CMP001, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases CMP001, i.e. one type -D is CMP001, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases CMP001, i.e. one type -D is CMP001, and one or more additional drugs is cetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases CMP001, i.e. one type -D is CMP001, and one or more additional drugs is margetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases CMP001, i.e. one type -D is CMP001, and one or more additional drugs is one of the CD47 or SIRPα blockers described above. It is understood that the conjugates may not only release CMP001 or contain -D fragments in the form of CMP001, but may also contain one or more other types of -D.

In certain embodiments, the water-insoluble controlled-release PRRA releases MK-1454, i.e. one type -D is MK-1454, and one or more additional drugs of the pharmaceutical composition is nivolumab. In certain embodiments, the water-insoluble controlled-release PRRA releases MK-1454, i.e. one type -D is MK-1454, and one or more additional drugs is pembrolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases MK-1454, i.e. one type -D is MK-1454, and one or more additional drugs is atezolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases MK-1454, i.e. one type -D is MK-1454, and one or more additional drugs is avelumab. In certain embodiments, the water-insoluble controlled-release PRRA releases MK-1454, i.e. one type -D is MK-1454, and one or more additional drugs is durvalumab. In certain embodiments, the water-insoluble controlled-release PRRA releases MK-1454, i.e. one type -D is MK-1454, and one or more additional drugs is ipilimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases MK-1454, i.e. one type -D is MK-1454, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases MK-1454, i.e. one type -D is MK-1454, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases MK-1454, i.e. one type -D is MK-1454, and one or more additional drugs is cetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases MK-1454, i.e. one type -D is MK-1454, and one or more additional drugs is margetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases MK-1454, i.e. one type -D is MK-1454, and one or more additional drugs is one of the CD47 or SIRPα blockers described above. It is understood that the conjugates may not only release MK-1454 or contain -D moieties in the form of MK-1454, but may also contain one or more other types of -D.

In certain embodiments, the water-insoluble controlled release PRRA releases ADU-S100, i.e. one type -D is ADU-S100, and one or more additional drugs of the pharmaceutical composition is nivolumab. In certain embodiments, the water-insoluble controlled release PRRA releases ADU-S100, i.e. one type -D is ADU-S100, and one or more additional drugs is pembrolizumab. In certain embodiments, the water-insoluble controlled release PRRA releases ADU-S100, i.e. one type -D is ADU-S100, and one or more additional drugs is atezolizumab. In certain embodiments, the water-insoluble controlled release PRRA releases ADU-S100, i.e. one type -D is ADU-S100, and one or more additional drugs is avelumab. In certain embodiments, the water-insoluble controlled release PRRA releases ADU-S100, i.e. one type -D is ADU-S100, and one or more additional drugs of the pharmaceutical composition is durvalumab. In certain embodiments, the water-insoluble controlled release PRRA releases ADU-S100, i.e. one type -D is ADU-S100, and one or more additional drugs is ipilimumab. In certain embodiments, the water-insoluble controlled release PRRA releases ADU-S100, i.e. one type -D is ADU-S100, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled release PRRA releases ADU-S100, i.e. one type -D is ADU-S100, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled release PRRA releases ADU-S100, i.e. one type -D is ADU-S100, and one or more additional drugs is cetuximab. In certain embodiments, the water-insoluble controlled release PRRA releases ADU-S100, i.e. one type -D is ADU-S100, and one or more additional drugs is margetuximab. In certain embodiments, the water-insoluble controlled release PRRA releases ADU-S100, i.e. one type -D is ADU-S100, and one or more additional drugs is one of the CD47 or SIRPα blockers described above. It is understood that the conjugates may not only release ADU-S100 or contain -D moieties in the form of ADU-S100, but may also contain one or more other types of -D.

In certain embodiments, the water-insoluble controlled-release PRRA releases 2'3'-cGAMP, i.e. one type -D is 2'3'-cGAMP, and one or more additional drugs of the pharmaceutical composition is nivolumab. In certain embodiments, the water-insoluble controlled-release PRRA releases 2'3'-cGAMP, i.e. one type -D is 2'3'-cGAMP, and one or more additional drugs is pembrolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases 2'3'-cGAMP, i.e. one type -D is 2'3'-cGAMP, and one or more additional drugs is atezolizumab. In certain embodiments, the water-insoluble controlled-release PRRA releases 2'3'-cGAMP, i.e. one type -D is 2'3'-cGAMP, and one or more additional drugs is avelumab. In certain embodiments, the water-insoluble controlled release PRRA releases 2'3'-cGAMP, i.e. one type -D is 2'3'-cGAMP, and one or more additional drugs is durvalumab. In certain embodiments, the water-insoluble controlled-release PRRA releases 2'3'-cGAMP, i.e. one type -D is 2'3'-cGAMP, and one or more additional drugs is ipilimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases 2'3'-cGAMP, i.e. one type -D is 2'3'-cGAMP, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases 2'3'-cGAMP, i.e. one type -D is 2'3'-cGAMP, and one or more additional drugs is tremelimumab. In certain embodiments, the water-insoluble controlled-release PRRA releases 2'3'-cGAMP, i.e. one type -D is 2'3'-cGAMP, and one or more additional drugs is cetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases 2'3'-cGAMP, i.e. one type -D is 2'3'-cGAMP, and one or more additional drugs is margetuximab. In certain embodiments, the water-insoluble controlled-release PRRA releases 2'3'-cGAMP, i.e. one type -D is 2'3'-cGAMP, and one or more additional drugs is one of the CD47 or SIRPα blockers described above. It is understood that the conjugates may not only release 2'3'-cGAMP or contain -D moieties in the form of 2'3'-cGAMP, but may also contain one or more other types of -D.

In another aspect, the present invention relates to a method of treating a mammalian patient in need of treatment of one or more diseases that can be treated with a PRRA drug, comprising the step of administering to said patient in need thereof a therapeutically effective amount of a water-insoluble, controlled-release PRRA or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing a water-insoluble controlled release PRRA according to the present invention.

In certain embodiments, one or more diseases that can be treated with a PRRA drug are cell proliferation disorders. Examples of such cell proliferation disorders are as described herein. In particular, a cell proliferation disorder that can be treated with the water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the water-insoluble controlled-release PRRA according to the present invention is cancer. Such cancer may be selected from the group consisting of liquid tumors, solid tumors and lymphomas.

The lymphatic tissue tumor may be leukemia or myeloid neoplasm, such as chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, lymphoblastic leukemia, myeloid leukemia, plasma cell leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia h (AML), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), post-MPN AML, post-MDS AML, del(5q)-associated high-risk MDS or AML, blast phase chronic myeloblastic leukemia, multiple myeloma, myelodysplastic syndrome, chronic myeloproliferative disorders, plasma cell tumor and Waldenström's macroglobulinemia.

The solid tumor or lymphoma may be selected from the group consisting of lip and oral cavity cancer, oral cavity cancer, liver/hepatocellular cancer, primary liver cancer, lung cancer, lymphoma, malignant mesothelioma, malignant thymoma, skin cancer, intraocular melanoma, metastatic squamous cell carcinoma of the neck with primary latency, multiple endocrine neoplasia syndrome in children, fungoid mycosis, cancer of the paranasal sinuses and nasal cavity, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, pheochromoblastoma, pituitary tumor, adrenocortical carcinoma, AIDS-related malignancies, anal cancer, bile duct cancer, bladder cancer, brain and nervous system cancer, breast cancer, bronchial adenoma/carcinoid, carcinoid tumor of the gastrointestinal tract, carcinoma, colorectal cancer, endometrial cancer , esophageal cancer, extracranial germ cell tumor, extragonadal gonocytoma, extrahepatic bile duct cancer, gallbladder cancer, gastric (stomach) cancer, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), kidney cancer /renal cell cancer, laryngeal cancer, pleuropulmonary blastoma, prostate cancer, transitional cell cancer of the renal pelvis and ureter, retinoblastoma, salivary gland cancer, sarcoma, Sézary syndrome, small intestine cancer, genitourinary cancer, malignant thymoma, thyroid cancer , Wilms tumor and cholangiocarcinoma.

In certain embodiments, the cancer is liver/hepatocellular cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is lymphoma. In certain embodiments, the cancer is a malignant thymoma. In certain embodiments, the cancer is skin cancer. In certain embodiments, the cancer is metastatic squamous cell carcinoma of the neck with primary latency. In certain embodiments, the cancer is neuroblastoma. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is bile duct cancer. In certain embodiments, the cancer is bladder cancer. In certain embodiments, the cancer is cancer of the brain and nervous system. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is a carcinoid tumor of the gastrointestinal tract. In certain embodiments, the cancer is a carcinoma. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is extrahepatic bile duct cancer. In certain embodiments, the cancer is gallbladder cancer. In certain embodiments, the cancer is gastric (stomach) cancer. In certain embodiments, the cancer is head and neck cancer. In certain embodiments, the cancer is kidney/renal cell carcinoma. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is a sarcoma. In certain embodiments, the cancer is small bowel cancer. In certain embodiments, the cancer is a cancer of the genitourinary system.

Examples of lung cancer are non-small cell lung cancer and small cell lung cancer. In certain embodiments, the cancer is non-small cell lung cancer. In a certain embodiment, the cancer is small cell lung cancer.

Examples of lymphoma include AIDS-associated lymphoma, primary central nervous system lymphoma, T-cell lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, Hodgkin's lymphoma of pregnancy, non-Hodgkin's lymphoma, non-Hodgkin's lymphoma of pregnancy, and angioimmunoblastic lymphoma.

Examples of skin cancer are melanoma and Merkel cell carcinoma. In a certain embodiment, the cancer is skin cancer. In a certain embodiment, the cancer is Merkel cell carcinoma.

Ovarian cancer may be, for example, epithelial cancer, gonocytoma or borderline ovarian tumor. In certain embodiments, the cancer is an epithelial cancer. In certain embodiments, the cancer is a gonocytoma. In certain embodiments, the cancer is a borderline ovarian tumor.

Pancreatic cancer may be, for example, an exocrine tumor/adenocarcinoma, pancreatic endocrine tumor (PET) or neuroendocrine tumor (NET). In certain embodiments, the cancer is an exocrine tumor/adenocarcinoma. In certain embodiments, the tumor is a pancreatic endocrine tumor. In certain embodiments, the cancer is a neuroendocrine tumor.

Examples of cancers of the brain and nervous system are medulloblastoma, such as childhood medulloblastoma, astrocytoma, ependymoma, neuroectodermal tumors, neuroma, meningioma, eosinophilic adenoma and glioma. In a certain embodiment, the cancer is medulloblastoma. In certain embodiments, the cancer is childhood medulloblastoma. In certain embodiments, the cancer is an astrocytoma. In certain embodiments, the cancer is a neuroectodermal tumor. In certain embodiments, the tumor is a neuroma. In certain embodiments, the cancer is a meningioma. In certain embodiments, the cancer is an eosinophilic adenoma. In certain embodiments, the cancer is a glioma.

The astrocytoma may be selected from the group consisting of giant cell glioblastoma, glioblastoma, secondary glioblastoma, primary adult glioblastoma, primary childhood glioblastoma, oligodendroglial tumor, oligodendroglioma, anaplastic oligodendroglioma, oligoastrocytic tumor, oligoastrocytoma, anaplastic oligoastrocytoma oma, anaplastic astrocytoma, piloid astrocytoma, subependymal giant cell astrocytoma , diffuse astrocytoma, pleomorphic xanthoastrocytoma and cerebellar astrocytoma.

Examples of neuroectodermal tumor are primitive neuroectodermal tumor of the pineal gland and supratentorial primitive neuroectodermal tumor.

The ependymoma may be selected from the group consisting of subependymoma, ependymoma, myxopapillary ependymoma, and anaplastic ependymoma.

Meningioma can be an atypical meningioma or an anaplastic meningioma.

The glioma may be selected from the group consisting of glioblastoma multiforme, paraganglioma, suprantentorial primordial neuroectodermal tumor (sPNET), brainstem glioma, pediatric brainstem glioma, hypothalamic and optic pathway glioma, pediatric hypothalamic and optic pathway glioma, and malignant glioma

Examples of breast cancer are breast cancer during pregnancy, triple negative breast cancer, ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), tubular breast carcinoma, medullary breast carcinoma, mucoid breast carcinoma, papillary carcinoma breast, cribriform breast carcinoma, invasive lobular carcinoma (ILC), inflammatory breast cancer, lobular carcinoma in situ (LCIS), male breast cancer, Paget's disease of the nipple, phyllodes tumor of the breast and metastatic breast cancer. In certain embodiments, the cancer is breast cancer during pregnancy. In certain embodiments, the cancer is triple negative breast cancer. In certain embodiments, the cancer is ductal carcinoma in situ. In certain embodiments, the cancer is invasive ductal carcinoma. In certain embodiments, the cancer is tubular carcinoma of the breast. In certain embodiments, the cancer is medullary carcinoma of the breast. In certain embodiments, the cancer is mucoid carcinoma of the breast. In certain embodiments, the cancer is papillary breast carcinoma. In certain embodiments, the cancer is cribrous carcinoma of the breast. In certain embodiments, the cancer is invasive lobular carcinoma. In certain embodiments, the cancer is inflammatory breast cancer. In certain embodiments, the cancer is lobular carcinoma in situ. In certain embodiments, the cancer is male breast cancer. In certain embodiments, the cancer is Paget's disease of the nipple. In certain embodiments, the cancer is a phyllodes tumor of the breast. In certain embodiments, the cancer is metastatic breast cancer.

Examples of carcinoma are neuroendocrine carcinoma, adrenocortical carcinoma and islet cell carcinoma. In certain embodiments, the cancer is neuroendocrine carcinoma. In certain embodiments, the cancer is adrenocortical carcinoma. In certain embodiments, the cancer is islet cell carcinoma.

Examples of colorectal cancer are colon cancer and rectal cancer. In certain embodiments, the cancer is colon cancer. In certain embodiments, the cancer is rectal cancer.

The sarcoma may be selected from the group consisting of Kaposi's sarcoma, osteosarcoma/malignant fibrous histiocytoma of bone, soft tissue sarcoma, Young family tumors/sarcomas, rhabdomyosarcoma, clear cell sarcoma of the tendon sheath, central chondrosarcoma, central and periosteal chondroma, fibrosarcoma and uterine sarcoma. In certain embodiments, the cancer may be Kaposi's sarcoma. In certain embodiments, the cancer may be osteosarcoma/malignant fibrous histiocytoma of bone. In certain embodiments, the cancer may be a soft tissue sarcoma. In certain embodiments, the cancer may be Young's family tumors/sarcomas. In certain embodiments, the cancer may be rhabdomyosarcoma. In certain embodiments, the cancer may be a clear cell sarcoma of the tendon sheath. In certain embodiments, the cancer may be central chondrosarcoma. In certain embodiments, the cancer may be central and periosteal chondroma. In certain embodiments, the cancer may be a fibrosarcoma. In certain embodiments, the cancer may be a uterine sarcoma.

Examples of genitourinary cancers include testicular cancer, urethral cancer, vaginal cancer, cervical cancer, penile cancer and vulvar cancer. In certain embodiments, the cancer may be testicular cancer. In certain embodiments, the cancer may be urethral cancer. In certain embodiments, the cancer may be vaginal cancer. In certain embodiments, the cancer may be cervical cancer. In certain embodiments, the cancer may be penile cancer. In certain embodiments, the cancer may be vulvar cancer.

In certain embodiments, the mammalian patient is selected from the group consisting of mouse, rat, non-human primate, and human. In a certain embodiment, the mammalian patient is a human patient.

In another aspect, the present invention relates to a method of treating a mammalian patient in need of treatment of one or more diseases that can be treated with a PRRA drug, comprising the step of administering to said patient in need thereof a therapeutically effective amount of a water-insoluble, controlled-release PRRA or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising a water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof according to the present invention, and in addition one or more other drug molecules. It is understood that one or more additional drug molecules can be administered in the form of a pharmaceutically acceptable salt or in the form of a pharmaceutical composition containing such one or more additional drug molecules.

In a certain embodiment, treatment of the cell proliferation disorder is performed in a patient being treated with at least one additional drug or therapy selected from the group consisting of anti-PD1 and anti-PDL1 compounds, other immune checkpoint antagonist therapies, pattern agonist compounds, recognition receptor, immune agonist therapies, oncolytic viral therapy, cancer vaccination, immunostimulatory cytokines, kinase inhibitors, transcription factor inhibitors, DNA repair inhibitors, cell therapy, chemotherapy, radiation therapy and surgery. Specific embodiments for these drug classes are as described herein.

Such at least one additional drug can be administered to the patient before, simultaneously with, or after administration of the water-insoluble controlled-release PRRA. In certain embodiments, at least one additional drug may be administered to the patient prior to administration of the water-insoluble controlled-release PRRA. In certain embodiments, at least one additional drug may be administered to a patient concurrently with administration of the water-insoluble controlled-release PRRA. In certain embodiments, at least one additional drug may be administered to the patient following administration of the water-insoluble controlled-release PRRA.

Said one or more additional drug molecules may be administered to said patient before, together with, or after administration of the water-insoluble controlled-release PRRA and a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the water-insoluble controlled-release PRRA of the present invention. If one or more additional drug molecules are administered together with a water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing a water-insoluble controlled-release PRRA according to the present invention, said one or more additional drug molecules may either be present in the same preparation, such as a pharmaceutical composition, or may be present in another preparation.

In certain embodiments, the one or more additional drugs are IL-2. In certain embodiments, said IL-2 is administered systemically. It is understood that such an IL-2 drug may be administered in the form of free or unmodified IL-2 or in a controlled release form of IL-2. In certain embodiments, such IL-2 drug is administered in the form of free or unmodified IL-2. In certain embodiments In embodiments, such IL-2 drug is administered or in a controlled release form of IL-2. Embodiments of such IL-2 are as described herein.

In certain embodiments, intratumoral administration of a water-insoluble controlled-release PRRA and systemic administration of IL-2 causes a greater than 1.5-fold, such as greater than 2-fold, 3-fold, 4-fold, or 5-fold increase in the percentage of subpopulations antigen-presenting cells in tumor draining lymph nodes, 7 days after this administration, compared with intratumoral administration of an equimolar amount of the same water-insoluble controlled-release PRRA alone or an equimolar amount of the same IL-2 alone. It is understood that IL-2 may be in the form of free or unmodified IL-2 or in the form of controlled release IL-2. Administration of PRRA and IL-2 can occur simultaneously or sequentially by administering either first followed by the latter.

In certain embodiments, the plasma protein level of at least one cytokine selected from the group consisting of IL-6, CCL2, and IL-10 is greater than a 10-fold decrease in the maximum plasma protein level within 24 hours after interstitial administration of an insoluble in water controlled-release PRRA, compared with an equimolar dose of the corresponding free PRRA when administered interstitially.

For example, if the amount of conjugate administered to an animal is 50 nmol of PRRA, as would be measured if all of the PRRA were released from the vehicle, then the equivalent dose of free PRRA would also be 50 nmol. Fold differences in cytokine levels are calculated using the following equation:

_,

Where

"Maximum plasma cytokine with free PRRA" is the highest plasma concentration of one of the cytokines measured during the 24-hour period after intrastitial administration of free PRRA to the first group of animals and

"Maximum plasma cytokine during conjugate administration" is the highest plasma concentration of the same cytokine measured over a 24-hour period following intracellular administration of a conjugate of the present invention to a second group of animals.

In general, the term “animal” also includes a human and, in a certain embodiment, means a mouse, a rat, a non-human primate, and a human.

It is understood that the terms "first group of animals" and "second group of animals" may, in a particular embodiment, refer to the same individuals, provided that there is a period of time between the two administrations sufficient for complete clearance of the PRRA and the conjugate. If the second group of animals includes different individuals than the first group of animals, such individuals of the second group are comparable to the first group of animals in all basic parameters, such as species, breed, sex or age.

In one embodiment, the at least one cytokine is IL-6. In another embodiment, the at least one cytokine is CCL2. In another embodiment, the at least one cytokine is IL-10. In another embodiment, the at least one cytokine is IL-6 and CCL2. In another embodiment, the at least one cytokine is CCL2 and IL-10. In another embodiment, the at least one cytokine is IL-6 and IL-10. In another embodiment, the at least one cytokine is IL-6, CCL2 and IL-10.

Protein levels can be measured by taking plasma samples prior to interstitial administration and at various time points, for example, at 3, 4, 5, 6, 7 or 8 time points, within 24 hours after interstitial administration and then determining the protein levels of at least one cytokine. Suitable methods for quantifying protein levels are known to one skilled in the art, such as using an enzyme-linked immunosorbent assay (ELISA). Data points are plotted and maximum protein levels are determined over 24 hours.

The maximum plasma protein level of at least one cytokine is greater than 10-fold, such as greater than 12-fold, greater than 15-fold, greater than 20-fold, greater than 30-fold, greater than 50-fold, or a greater than 100-fold reduction following interstitial administration of a conjugate of the present invention, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention, compared with interstitial administration of an equivalent molar dose of the corresponding free PRRA.

In certain embodiments, the one or more additional drugs are TKIs. In certain embodiments, said TKI is administered systemically. It is understood that such a TKI drug may be administered in the form of a free or unmodified TKI or in the form of a controlled release TKI. In certain embodiments, such TKI drug is administered in the form of a free or unmodified TKI. In certain embodiments, such a TKI drug is administered in a controlled release form of the TKI. Embodiments of such a TKI are described herein.

In certain embodiments, the one or more additional drugs are a CTLA-4 inhibitor. In certain embodiments, said CTLA-4 inhibitor is administered systemically. It is understood that such a CTLA-4 inhibitor drug can be administered in the form of a free or unmodified CTLA-4 inhibitor or in the form of a controlled release of a CTLA-4 inhibitor. In certain embodiments, such a CTLA-4 inhibitor drug is administered in the form of a free or unmodified CTLA-4 inhibitor. In certain embodiments, such a CTLA-4 inhibitor drug is administered in a controlled release form of the CTLA-4 inhibitor. Embodiments of such a CTLA-4 inhibitor are described herein.

In certain embodiments, intratumoral administration of a water-insoluble controlled-release PRRA and systemic administration of IL-2 causes a greater than 1.5-fold, such as greater than 2-fold, 3-fold, 4-fold, or 5-fold increase in the percentage of cells CD8 T in tumor draining lymph nodes, 7 days after this administration, compared with intratumoral administration of an equimolar amount of the same water-insoluble controlled-release PRRA alone. It is understood that IL-2 may be in the form of free or unmodified IL-2 or in the form of controlled release IL-2. Administration of PRRA and IL-2 can occur simultaneously or sequentially by administering either first followed by the latter.

In certain embodiments, intratumoral administration of water-insoluble controlled-release PRRA and systemic administration of IL-2 causes greater than 1.25-fold, such as greater than 1.5-fold, 1.7-fold, 2-fold, 2.2-fold, 2.5-fold , 3-fold, 3.5-fold, 4-fold, or 5-fold increase in the expression of CD8 T cell memory markers in tumor draining lymph nodes, 7 days after the indicated administration, compared with intratumoral administration of an equimolar amount of the same water-insoluble PRRA with controlled released separately. It is understood that IL-2 may be in the form of free or unmodified IL-2 or in the form of controlled release IL-2. Administration of PRRA and IL-2 can occur simultaneously or sequentially by administering either first followed by the latter.

In certain embodiments, intratumoral administration of a water-insoluble controlled-release PRRA of the present invention and systemic administration of IL-2 causes greater than 1.5-fold, such as greater than 1.8-fold, 2-fold, 2.5-fold, 2.8-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, or 5-fold increase in the percentage of CD8 T cells in peripheral blood 4 days after the indicated administration compared with treatment or vehicle alone or compared with treatment with intratumoral injection of insoluble in PRRA controlled release water alone. It is understood that IL-2 may be in the form of free or unmodified IL-2 or in the form of controlled release IL-2. Administration of the water-insoluble controlled-release PRRA of the present invention and IL-2 may occur simultaneously or sequentially by administering either first followed by the latter.

In certain embodiments, intratumoral administration of a water-insoluble controlled-release PRRA and systemic administration of IL-2 causes greater than 1.5-fold, such as greater than 1.5-fold, 1.7-fold, 2-fold, 2.2-fold, 2.5-fold , 3-fold, 3.5-fold, 4-fold, or 5-fold reduction in the percentage of CD4 T cells in tumor draining lymph nodes, 7 days after the indicated administration, compared with intratumoral administration of an equimolar amount of the same water-insoluble controlled-release PRRA alone or an equimolar amount of IL-2 alone. It is understood that IL-2 may be in the form of free or unmodified IL-2 or in the form of controlled release IL-2. Administration of PRRA and IL-2 can occur simultaneously or sequentially by administering either first followed by the latter.

Materials and methods

Chemical compounds

All materials were obtained from commercial suppliers unless otherwise noted.

Purification by RP-HPLC:

Preparative RP-HPLC purification was performed using a Waters 600 controller with a 2487 dual absorption detector or an Agilent Infinity 1260 preparative system using a Waters XBridge BEH300 Prep C18 10 µm, 150 x 30 mm column as the stationary phase. Products were detected at 215 nm or 320 nm.

Flash chromatography:

Flash chromatography purification was performed on an Isolera One system or an Isolera Four system from Biotage AB, Sweden, using Biotage KP-Sil silica cartridges. Products were detected at 215 nm, 254 nm or 280 nm.

Cleaning with RP-LPLC:

Purification by low pressure RP chromatography was carried out on an Isolera One system or an Isolera Four system from Biotage AB, Sweden, using Biotage SNAP C18 cartridges. Products were detected at 215 nm.

Analytical methods

UPLC-MS Analytical Analysis:

Analytical ultra performance LC (UPLC)-MS was performed on a Waters Acquity or Agilent 1290 Infinity II system equipped with a Waters BEH300 C18 column (2.1 x 50 mm, 1.7 µm particle size, or 2.1 x 100 mm, particle size 1 .7 µm), solvent A: water containing 0.05% TFA (v/v), solvent B: acetonitrile containing 0.04% TFA (v/v), connected to Waters Micromass ZQ or associated with Agilent Single Quad MS system

Determination of amine content on PEG hydrogel beads:

The amino group content of the PEG hydrogel was determined by conjugating the Fmoc amino acid to the free amino groups of the hydrogel and subsequent Fmoc determination as described in Gude, M., J. Ryf, et al. ( 2002 ) Letters in Peptide Science 9 (4): 203-206.

Determination of the content of conjugated resiquimod in hydrogel suspensions

The resiquimod content of the hydrogel suspension was determined by incubating a sample of the hydrogel suspension with an equal volume of 1 M NaOH at 37°C for 16-20 hours. After pH adjustment with 1M HCl, resiquimod content was determined by HPLC (detection at 320 nm) from a calibration curve derived from at least 4 different calibration standards.

Example 1: Synthesis of Linker Reagent 6

Stage 1:

In a 250 mL round bottom flask, dissolved 3,6,9-trioxoundecanedioic acid (9.45 g, 29.79 mmol, 10.01 equiv.) and glycine benzyl ester hydrochloride (600.00 mg, 2.98 mmol, 1 .00 eq.) in anhydrous dichloromethane (50.00 ml). HOSu (858.20 mg, 7.46 mmol, 2.51 eq) and EDC (1.15 g, 5.98 mmol, 2.01 eq) were added to produce a cloudy mixture that became clear after adding DIPEA (4.16 ml, 23.80 mmol, 8.00 eq). The solution was stirred at room temperature for 3.5 hours.

The solvent was evaporated and the residue was dissolved in acetonitrile/water 1:1 (v/v, 0.1% TFA, 10 ml). The crude product was purified by RP-LPLC using a gradient of (10-35%) acetonitrile (0.1% TFA) in water (0.1% TFA). Product fractions were pooled and lyophilized.

Yield: 1.07 g (97.36%) colorless oil

m/z = 370.40 [M+H] ⁺

Stage 2:

Compound 1 (525.30 mg, 1.42 mmol, 1.00 eq.) and PyBOP (740.08 mg, 1.42 mmol, 1.00 eq.) were dissolved in anhydrous DMF (5.00 ml). β-Alanine tert-butyl ester hydrochloride (258.35 mg, 1.42 mmol, 1.00 eq.) and DIPEA (496.77 μL, 2.84 mmol, 2.00 eq.) were added sequentially, and the solution was stirred at room temperature for 4.5 h. The reaction was quenched by adding 1 N HCl (2.2 ml). The mixture was diluted with DCM (100 ml) and washed with 0.1 N HCl (3x50 ml), saturated aqueous NaHCO ₃ (3x50 ml) and saline (50 ml). The organic phase was dried over Na ₂ SO ₄ , filtered and the solvent was evaporated. The crude product thus obtained was purified by flash silica chromatography using a gradient (10-100%) of acetonitrile in DCM. The product fractions were combined, concentrated under reduced pressure and dried in vacuo.

Yield: 495.10 mg (70.11%) colorless oil

m/z = 497.49 [M+H] ⁺

Stage 3:

Compound 2 (495.10 mg, 1.00 mmol, 1.00 eq.) was dissolved in anhydrous THF (10.00 ml). Palladium on activated carbon (10 wt.%, 21.22 mg, 0.20 mmol, 0.20 eq.) was added to the solution, and the reaction mixture was stirred at room temperature under a hydrogen atmosphere for 1 h. The reaction mixture was filtered, the volatile compounds were evaporated under reduced pressure and the residue was dried in vacuum. 354 mg of residue was purified by preparative RP-HPLC using a gradient of (0-50%) acetonitrile (0.1% TFA) in water (0.1% TFA). Product fractions were pooled and lyophilized.

Yield: 307.00 mg colorless oil

m/z = 407.44 [M+H] ⁺

Step 4: Resiquimod binding

Resiquimod 4 (32.50 mg, 103.38 µmol, 1.00 equiv) was added to a solution of protected linker reagent 3 (76.00 mg, 186.99 µmol, 1.80 equiv) in anhydrous DMF (0.40 ml). PyBOP (98.00 mg, 188.32 µmol, 1.81 eq.) and DIPEA (160.00 µl, 918.58 µmol, 8.84 eq.) were added. After 18 h at room temperature, the reaction was quenched with AcOH (160 μl) and 2 ml of 30 mM phosphate buffer (pH 8.2) which contained 20% acetonitrile to obtain approximately 2.7 ml of crude product solution. The product was purified by preparative RP-HPLC using a gradient of (25-45%) acetonitrile in 30 mM sodium phosphate buffer (pH 8.2). The product fractions were combined and transferred to a separatory funnel. The aqueous phase was extracted with ethyl acetate (60 ml, 30 ml, 30 ml) and the combined organic phases were dried (MgSO ₄ ), filtered, concentrated under reduced pressure and dried in vacuo.

Yield: 61.4 mg (84%).

m/z = 703.65 [M+H] ⁺

Step 5: Removal of the protecting group

Compound 5 (64.00 mg, 0.09 mmol, 1.00 eq.) was dissolved in anhydrous dichloromethane (2.00 ml) and trifluoroacetic acid (2.00 ml). After 2 hours, the reaction solution was concentrated under reduced pressure. To the residue was added 1 ml of 30 mM phosphate buffer pH 8.2 containing 20% acetonitrile. The resulting emulsion was purified by preparative RP-HPLC using a gradient (5-50%) of acetonitrile in water. The combined fractions were lyophilized. The residue (43.7 mg, 74%) was dissolved in anhydrous DMF (2.18 ml) to give a solution containing 21.8 mg/ml.

Yield: 43.7 mg (74%)

m/z = 647.59 [M+H] ⁺

Example 2: Synthesis of PEG-based hydrogel beads containing free amino groups (0.075 mmol/g)

Step 1: Agent 7 Backbone Synthesis

Backbone reagent 7 was synthesized as an HCl salt using L-lysine building blocks similar to the previously described procedure (WO2013/053856, example 1, compound 1g therein):

Stage 2: Polymerization

A 250 ml bottom outlet cylindrical reactor, 60 mm in diameter, equipped with baffles, was charged with CithrolTM DPHS emulsion (0.4 g) in heptane (80 ml). The reactor contents were mixed with a paddle mixer with a diameter of 45 mm at 460 rpm at room temperature. A solution of PEG-disuccinimidyl glutarate, 1 kDa 8 (Innochemie, 4290 mg) and backbone reagent 7 (2000 mg) in DMSO (38.6 g) was added to the reactor and stirred for 10 min until an emulsion formed. TMEDA (8.9 ml) was added to effect polymerization, and the mixture was stirred at room temperature for 16 hours. While stirring, acetic acid (13.7 ml) was added. After 10 minutes, sodium chloride solution (15 wt.%, 100 ml) was added with stirring. After 10 min, the stirrer was stopped and the phases were allowed to separate. After 95 minutes, the aqueous phase containing the PEG hydrogel beads was discarded.

For size fractionation, the water-hydrogel suspension was diluted with ethanol (40 ml) and sieved on stainless steel sieves of 125, 100, 75, 63 and 50 μm (mesh size) with a diameter of 200 mm using a sieving machine for 15 min. Sifting amplitude 1.5 mm, liquid flow 250 ml/min. Water (4000 ml) was used as wetting liquid. Hydrogel beads were collected from sieves into 50 mL Falcon tubes using 20% ethanol in water. After centrifugation at 5000 rpm for 1 minute, the yield of the suspension was noted (see below). The fractions were processed. Washing by centrifugation at 5000 rpm, 1 min, was carried out 3 times with 0.1% AcOH, then with EtOH until no decrease in volume was observed. Fractions were transferred to individual syringes with a polyethylene filter and dried for 3 days at <1 mbar. The amine content of the hydrogel was determined from the dry material.

Outputs: 63 µm sieve fraction: ≈ 15 ml suspension, 1493 mg after drying sieve fraction 75 microns: ≈ 15 ml suspension, 1433 mg after drying Amine content: 0.075 mmol/g

Example 3: Synthesis of PEG hydrogel beads containing free amino groups (0.11-0.5 mmol/g)

Hydrogels from reagents 7 and 10 (see WO 2011/012715 Al, example 2, compound 2d ) were prepared according to the procedure. Described in WO 2011/012715 Al, example 3.

Hydrogel 11a was synthesized from 1398 mg of reagent 7 and 4473 mg of reagent 10 in 36.2 g of DMSO. The resulting amine loading was 0.151 mmol/g.

Hydrogel 11b was synthesized from 3.40 g of reagent 7 and 8.91 g of reagent 10 in 75.6 g of DMSO. The resulting amine loading was 0.296 mmol/g.

Example 4: Acetylation of hydrogels

Hydrogel 9 (3.184 g, 0.239 mmol) was filled into a 50 mL syringe equipped with a polyethylene frit and washed 3 times with a 1% (v/v) solution of DIPEA in anhydrous DMF. A solution of acetic anhydride (0.45 mL, 4.77 mmol, 20.00 eq.) and DIPEA (0.83 mL, 4.77 mmol, 20.00 eq.) in anhydrous DMF (38.18 mL) was drawn into a syringe. ), the syringe was closed with a sterile cap and shaken for 1 hour at 1000 rpm at room temperature. The solvent was removed, the syringe was washed 10 times with anhydrous DMF and 10 times with ethanol. The volume of the swollen hydrogel after ethanol removal was 11 ml. The resulting hydrogel was dried in vacuum. Under sterile conditions, Ac-9 hydrogel (2.98 g, 1.00 eq) was transferred into a 50 mL Falcon tube. Formulation buffer (30 mL) was added and the Falcon tube was shaken for 30 min on a shaker until a homogeneous suspension was formed.

In a similar procedure, hydrogel 11a was acetylated to produce Ac-11a , and hydrogel 11b was acetylated to produce Ac-11b .

Example 5: Loading Compound 6 onto Hydrogels

Under sterile conditions, hydrogel 9 (457.00 mg, 34.28 µmol, 1.00 eq.) was weighed into a 20 ml syringe equipped with a polyethylene frit. The hydrogel swelled when anhydrous DMF (1% DIPEA, 10 ml) was drawn into the syringe; the syringe was manually shaken for 1 min and the solvent was removed. This procedure was repeated three times. A solution of compound 6 in DMF (2.00 ml, 21.80 mg/ml, 67.42 µmol, 1.97 eq.) and DIPEA (35.82 µl, 205.66 µmol, 6.00 eq.) was mixed and was drawn into a syringe containing the hydrogel, followed by the addition of a solution of PyBOP (35.67 mg, 68.55 μmol, 2.00 eq.) in anhydrous DMF (1.00 ml). Air was drawn into the syringe to drain the cannula and frit. The syringe was shaken for 3.5 hours at room temperature. The solution was removed. The hydrogel was washed with DMF (10 × 10 ml), sterile pyrogen-free water (10 × 10 ml), and formulation buffer (10 × 10 ml). After the last washing step, about 10 ml of buffer was drawn into the syringe. The syringe was capped with a sterile stopper and incubated at 37°C for 1 h. The buffer was removed and the hydrogel was washed with formulation buffer (10 × 10 ml). The plunger was removed and the suspension was transferred to a 50 mL Falcon tube. The buffer supernatant was removed, resulting in a suspension with a final volume of approximately 6 ml. The content of resiquimod in the resulting hydrogel 12 was about 1.5 mg resiquimod eq/ml. Using a similar procedure, compound 6 was loaded onto hydrogel 11a to obtain hydrogel 12a with a resiquimod loading of approximately 1.9 mg resiquimod eq/ml.

Using a similar procedure, compound 6 was loaded onto hydrogel 11b to obtain hydrogel 12b with a resiquimod loading of approximately 4.9 mg resiquimod eq/ml.

Using a similar procedure, compound 6 was loaded onto hydrogel 11a to obtain hydrogel 12c with a resiquimod loading of approximately 2.7 mg resiquimod eq/ml.

Example 6: Dose adjustment

Under sterile conditions, Ac-9 hydrogel suspension (11.23 mL) was combined with Hydrogel 12 suspension (1.52 mg resiquimod eq/mL, 4.57 mL) in a sterile 50 mL Falcon tube. The combined hydrogel was homogenized by slowly shaking the Falcon tube for 5 minutes. The content of the resulting hydrogel suspension was 0.376 mg resiquimod eq/ml.

Using a similar procedure, the following suspensions of hydrogels were prepared from their acetylated and resiquimod-loaded components.

Compound Acetylated hydrogel Resiquimod hydrogel Resiquimod content in hydrogel suspension buffer 13a Ac-9 12 92 µg eq/ml PBST 13b Ac-11a 12a 119 µg eq/ml PBST 13c Ac-9 12 376 µg eq/ml PBST 13d Ac-11b 12b 103 µg eq/ml PTP 13e Ac-11b 12b 410 µg eq/ml PTP 13f Ac-11b 12b 1649 µg eq/ml PTP 13g Ac-11b 12b 4040 µg eq/ml PTP 13h Ac-11b 12b 4321 µg eq/ml PTP 13i Ac-11a 12c 226 µg eq/ml PTP

Example 7: loading of compound 6 onto a hydrogel followed by acetylation

Hydrogel 11a (200 mg, 0.03 mmol) was weighed into a 10 mL syringe equipped with a polyethylene frit. The hydrogel swelled when anhydrous DMF (1% DIPEA, 3 ml) was drawn into the syringe; the syringe was manually shaken for 1 min and the solvent was removed. This procedure was repeated three times.

A solution of compound 6 (7.76 mg, 12.0 µmol, 1.0 equiv), PyBOP (7.5 mg, 14.4 µmol, 1.2 equiv) and DIPEA (16.8 µl, 96 µmol, 8 eq.) in DMF (3 ml), and the suspension was shaken at room temperature overnight. After completion of the reaction, the hydrogel was washed with DMF (10 × 5 ml).

A solution of acetic anhydride (60 µl, 0.63 mmol) and DIPEA (110 µl, 0.63 mmol) in DMF (2.83 ml) was drawn into a syringe and the suspension was shaken at room temperature for 2 hours. The supernatant was removed and the hydrogel was washed with DMF (10×3 ml), water (10×3 ml), EtOH (10×3 ml) and dried in vacuum.

The resiquimod content of the resulting hydrogel 14 was 17.4 mg/g.

Example 8: Release of resiquimod from hydrogel 14

A suspension of hydrogel 14 (0.23% w/w) in phosphate buffer pH 7.4 was incubated at 37°C. Over a period of 33 days, supernatant samples were collected and resiquimod content was determined by UPLC using a calibration curve. Nonlinear regression analysis of the concentrations obtained gave a half-life of 15.3 days.

Example 9: Preparation of Polymeric Prodrug of IL-2 Mutein with Shifted Activity

Step 1: Preparation of cysteine protected mutein 15 IL-2

The IL-2 variant (mutein) was custom made and obtained from the supplier exteRNAl, where the proteins were expressed from E. coli using standard purification strategies known to one of ordinary skill in the art. The following proteins were prepared

15: PTSSSTKKTQ LQLEHLLLDL QMILNGINNY KNPKLTC*MLT FKFYMPKKAT ELKHLQCLEE ELKPLEEVLN LAQSKNFHLR PRDLISNINV IVLELKGSET TFMCEYADET ATIVEFLNRW ITFSQSIIST LT (SEQ ID NO:1, cysteine marked with “*” is connected to free cysteine through a disulfide connection)

Step 2: Preparation of polymeric prodrug of IL-2 mutein with shifted activity 16

23.2 mg of TCEP (tris(2-carboxethyl)phosphine hydrochloride) was dissolved in 1.62 ml of PBS (phosphate buffered saline), pH 7.4, to obtain a 50 mM solution. No pH adjustment was performed.

45.2 ml of 15, formulated at 1.8 mg/ml in PBS, 10% glycerol, pH about 9, was mixed with 13.6 ml of 0.5 M sodium phosphate, pH 7.4, then 710 μl of TCEP solution was added. The sample was incubated at ambient temperature for 30 min.

Then 5.5 ml of 5 mM 5 kDa PEG maleimide (Sunbright ME-050MA, CAS 883993-35-9, NOF Europe N.V., Grobbendonk, Belgium) in PBS, pH 7.4 (5 mol equiv.) was added to the reaction solution. . After incubation at ambient temperature for 10 min, the formation of conjugates was confirmed by analytical size exclusion chromatography.

The conjugation mixture buffer was exchanged to 100 mM borate, pH 9.0, using an Aekta system equipped with a HiPrep Desalting 26/10 column. The sample was incubated at 25°C overnight, then concentrated to 5.3 mg/ml using Amicon Ultra -15, Ultracel 3 K centrifugation filters (Merck Millipore). 0.847 g of 40 kDa mPEG linker reagent (as described in WO 2016079114, example 2) was dissolved in 9.75 ml of water, resulting in a stock solution with a concentration of 2.1 * 10 ^-3 mol/L. The solution was stored on ice.

A 12.9 mL protein solution was diluted to 4 mg/mL by adding 100 mM borate, pH 9.0, then 8.4 mL of cooled stock solution of 40 kDa mPEG linker reagent was added (corresponding to 4 mole equivalent of protein). The conjugation mixture was placed in a water bath at 14°C for 2 hours. The pH was shifted to pH 4 by adding 8.4 ml water and 33.5 ml 200 mM sodium acetate, pH 3.6, followed by incubation at 25°C overnight.

The conjugate with one single 40 kDa mPEG linker attached (monoconjugate) was isolated from the reaction mixture using HiScreen Capto MMC resin (column size: 0.77 × 10 cm) connected to an Aekta system. A flow rate of 1.2 mL/min and a linear gradient from 10 mM succinic acid, pH 5.5 to 80% 10 mM succinic acid, 1 M NaCl, pH 5.5 in 12 column volumes was used for all three runs. Fractions containing primarily monoconjugate were identified by analytical size exclusion chromatography. The salt concentration of each fraction was adjusted to 150 mM by adding 10 mM succinic acid, 1 M NaCl, pH 5.5, then the fractions were combined and concentrated to 2.8 mg/ml in Amicon Ultra -15, Ultracel 10 K filters (Merck Millipore).

The concentrated solution (8.1 ml) was diluted with 0.4 ml of 10 mM succinic acid, 150 mM NaCl, 1% Tween20, pH 5.5 and 14.4 ml of 10 mM succinic acid, 150 mM NaCl, 0.05% Tween20, pH 5.5 to a final concentration of 1 mg/ml . The final sample was filtered through a 0.22 µm PVDF filter membrane.

Example 10: In vivo PK study

Resiquimod and resiquimod-releasing hydrogels were administered subcutaneously to rats, and plasma resiquimod levels were monitored for 28 days. Resiquimod 4 was dissolved in 10 mM succinate, 90.0 mg/ml trehalose dihydrate, pH 5.0, at a concentration of 104 μg/ml. The hydrogels were suspended (about 6% w/v) in PBST buffer at pH 7.4. Male WISTAR rats (n = 3 per group) received a single subcutaneous injection of either resiquimod solution 4 or hydrogels 13a or 13b, each corresponding to a dose of 25 μg equiv. resiquimod. Blood samples were collected and used to obtain plasma for 28 days. Resiquimod concentrations in plasma samples were quantified using LC-MS/MS. Plasma concentration profiles were generated and analyzed using Phoenix WinNonlin software (Certara, Princeton, NJ, USA).

Results:

Maximum plasma concentrations, terminal half-life and calculated AUC are given below:

Compound C _max [pg/ml] t _1/2 AUC _{Pred- ∞} [h*pg/ml] 4 23100 1.5 h and 10 h (biphasic) 65400 13a 281 13.6 days 74700 13b 234 10.5 days 65900

Example 11: In Vivo Antitumor Efficacy

The study was performed on female BALB/C mice aged 6-11 weeks on the day of tumor inoculation. Mice were subcutaneously implanted with 3 × ¹⁰⁵ CT26 tumor cells in the left and right flanks. When the tumors to be injected had grown to a mean tumor volume of ~80 mm ³ , mice were randomly assigned to treatment groups (day 0). The day after randomization, animals received a single dose of 20 μg of resiquimod 4 (dissolved in 10 mM succinate, 90.0 mg/ml trehalose dihydrate, pH 5.0) or hydrogel 13c as a single intratumoral dose in an injection volume of 50 μl or a single intratumoral dose injection of 50 μl of Ac-9 suspension. Hydrogels were introduced as suspensions in PBST buffer. After initiation of treatment, antitumor efficacy was assessed by determining tumor volumes at various time points based on tumor size measurements with calipers. Tumor volumes were calculated using the formula

Tumor volume = (L×W²)×0.5

where L represents the length of the tumor, and W the width (both in mm). Mice were removed from the study as soon as the tumor became larger than 1500 mm ³ .

Results:

Absolute tumor volumes

Days after treatment Group 0 2 4 7 9 eleven 14 Ac-9 Medium (mm ³ ) 89.01 125.62 151.98 449.94 792.78 1065.45 1402.37 SEM (mm ³ ) 3.65 7.52 11.88 43.28 74.31 91.43 100.91 N 10 10 10 10 10 10 6 4 Medium (mm ³ ) 88.82 108.44 128.42 316.07 549.72 957.90 1220.96 SEM (mm ³ ) 3.48 5.34 8.69 49.35 56.21 125.30 137.76 N 10 10 10 10 10 10 8 13c Medium (mm ³ ) 88.96 123.60 141.09 215.87 305.38 , 378.04 , 609.37 , SEM (mm ³ ) 2.15 6.78 9.55 33.39 58.73 75.61 129.13 N 15 15 15 15 15 15 eleven

SEM = standard error of the mean, N = sample size, p<0.05 compared to Ac-9 , p<0.05 compared to 4 . Significance was determined by two-way analysis of variance followed by multiple comparisons using post hoc analysis with Tukey's HSD test.

Example 12: In vivo induction of cytokines

The study was performed on female BALB/C mice aged 6-11 weeks on the day of tumor inoculation. Mice were subcutaneously implanted with ^3x105 CT26 tumor cells in the left and right flanks. When tumors to be injected had grown to a mean tumor volume of ~105 mm ³ , mice were randomly assigned to treatment groups (day 0). The day after randomization, animals received a single dose of 20 μg of resiquimod 4 (dissolved in 10 mM succinate, 90.0 mg/ml trehalose dihydrate, pH 5.0) or hydrogel 13c as a single intratumoral dose in an injection volume of 50 μl or a single intratumoral dose injection of 50 μl of Ac-9 suspension. Hydrogels were introduced as suspensions in PBST buffer. K2 EDTA preserved blood samples were collected by retroorbital bleeding at various time points after drug administration, and plasma was isolated after centrifugation at 2000 × g for 5 minutes at 4°C and frozen. Plasma samples were stored at -80°C. Plasma was thawed and undiluted samples were assessed for cytokine levels using the 36-Plex Mouse ProcartaPlex™ Cytokine Panel 1A (ThermoFisher Scientific) according to the manufacturer's recommendations. Cytokines were measured using Bio-Plex 200 (BioRad) according to the kit instructions. For sample values below or at the lower limit of quantitation (LLOQ) of the assay, a value of 0.01 pg/mL was used instead to determine mean cytokine concentrations.

Results:

Plasma cytokine levels

IFNγ Hours after treatment Group 1 3 6 10 24 Ac-9 Average (pg/ml) 0.01 0.01 0.01 0.01 0.01 SEM (pg/ml) 0.00 0.00 0.00 0.00 0.00 N 3 3 3 3 3 4 Average (pg/ml) 4.04 15.49 , 40.72 , 28.19 , 2.38 SEM (pg/ml) 0.45 6.64 3.49 4.50 1.21 N 3 3 3 3 3 13c Average (pg/ml) 0.01 0.49 2.76 4.51 2.10 SEM (pg/ml) 0.00 0.48 1.42 0.87 0.37 N 3 3 3 3 3

SEM = standard error of the mean, N = sample size, p < 0.0002 compared to Ac-9 , p < 0.0002 compared to 13c . Significance was determined by two-way analysis of variance followed by multiple comparisons using post hoc analysis with Tukey's HSD test.

IL-6 Hours after treatment Group 1 3 6 10 24 Ac-9 Average (pg/ml) 40.31 25.68 4.86 14.93 57.67 SEM (pg/ml) 12.13 15.78 4.85 4.55 12.86 N 3 3 3 3 3 4 Average (pg/ml) 3618.87 , 1739.75 , 88.59 154.51 44.71 SEM (pg/ml) 146.11 360.03 16.66 69.39 28.80 N 3 3 3 3 3 13c Average (pg/ml) 52.18 141.69 80.47 71.58 270.01 SEM (pg/ml) 18.14 55.52 11.99 22.72 96.47 N 3 3 3 3 3

SEM = standard error of the mean, N = sample size, p<0.0001 compared to Ac-9 , p<0.0001 compared to 13c . Significance was determined by two-way analysis of variance followed by multiple comparisons using post hoc analysis with Tukey's HSD test.

CCL2/MCP-1 Hours after treatment Group 1 3 6 10 24 Ac-9 Average (pg/ml) 17.81 38.01 23.18 39.77 50.04 SEM (pg/ml) 10.94 10.74 1.02 8.97 19.42 N 3 3 3 3 3 4 Average (pg/ml) 357.83 4230.35 , 1039.17 , 847.07 92.58 SEM (pg/ml) 80.35 147.36 279.41 182.24 1.59 N 3 3 3 3 3 13c Average (pg/ml) 62.28 282.21 309.21 508.54 237.05 SEM (pg/ml) 21.46 114.89 108.22 71.83 6.31 N 3 3 3 3 3

SEM = standard error of the mean, N = sample size p < 0.0001 compared to Ac-9 , p < 0.0001 compared to 13c , p < 0.02 compared to Ac-9 . Significance was determined by two-way analysis of variance followed by multiple comparisons using post hoc analysis with Tukey's HSD test.

TNFα Hours after treatment Group 1 3 6 10 24 Ac-9 Average (pg/ml) 2.29 1.93 4.73 1.36 2.11 SEM (pg/ml) 2.28 1.92 0.25 1.35 2.10 N 3 3 3 3 3 4 Average (pg/ml) 830.84 136.25 35.49 43.32 9.74 SEM (pg/ml) 99.38 , 17.86 , 1.05 14.06 2.14 N 3 3 3 3 3 13c Average (pg/ml) 7.34 27.00 26.46 13.78 19.70 SEM (pg/ml) 0.80 4.31 8.56 3.21 0.99 N 3 3 3 3 3

SEM = standard error of the mean, N = sample size, p<0.004 compared to Ac-9 , p<0.02 compared to 13c . Significance was determined by two-way analysis of variance followed by multiple comparisons using post hoc analysis with Tukey's HSD test.

Example 13: In vivo dose escalation, tumor cytokine and chemokine profiling and tumor efficacy study

The study was conducted on female BALB/C mice aged 6-11 weeks on the day of tumor inoculation. Mice were implanted with 3 × ¹⁰⁵ CT26 tumor cells in the right flank. When tumors had grown to a mean tumor volume of ~115 mm ³ , mice were randomized to treatment groups (day 0). The day after randomization, animals received either 13g, 13f, 13e, or 13d as a single intratumoral dose in an injection volume of 50 μl or a single intratumoral injection of 50 μl of Ac-11b suspension. Hydrogels were introduced as suspensions in PTP buffer. After initiation of treatment, antitumor efficacy was assessed by determining tumor volumes at various time points based on tumor size measurements with calipers. Tumor volumes were calculated using the formula

Tumor volume = (L×W²)×0.5

where L is the length of the tumor and W is the width (both in mm). On the same day as tumor measurements, mice were weighed to determine absolute body weight. At specified time points (6 hours, 3 days and 7 days after the start of treatment), 2-3 mice per group were sacrificed and tumors were collected and frozen, and plasma was obtained after blood collection. Plasma was also obtained for all mice that were excluded from the study when the termination criteria were determined. Resiquimod concentrations in plasma samples were quantified using LC-MS/MS. Serum PK parameters for 13e- or 13d-treated animals were analyzed using a non-compartmental CA (NCA) approach using Phoenix 64 (version 8). Frozen tumors were cut into pieces approximately 0.3–0.8 mm in length, then mechanically homogenized using a mortar and pestle and kept frozen. To assess tumor protein cytokines and chemokines, an aliquot of homogenized tumor was lysed in 400 μl of ProcartaPlex cell lysis buffer (ThermoFisher Scientific) for every 50 μg of tissue. Samples were sonicated to facilitate tumor lysis. Lysates were centrifuged at 30,000 G for 20 min at 4°C, and supernatants were collected. Protein concentrations were measured using the Bio-Rad DC Protein Assay Kit (Bio-Rad) according to the manufacturer's recommendations. Samples were diluted in PBS to a protein concentration of 5.5 mg/mL. 25 μL of concentration-adjusted samples were then assessed for chemokine and cytokine levels using the 36-Plex Mouse ProcartaPlex Cytokine Panel 1A (ThermoFisher Scientific) according to the manufacturer's recommendations. Cytokines were measured on a Bio-Plex 200 instrument (Bio-Rad) according to the kit instructions. For sample values below or at the lower limit of quantitation (LLOQ) of the assay, a value of 0.01 pg/mL was used instead when determining the mean cytokine concentration. Fold changes were determined by dividing the mean cytokine concentration in treated samples by the mean cytokine concentration in Ac-11b-treated samples at each time point. To assess tumor cytokine and chemokine gene expression, RNA was isolated from an aliquot of homogenized tumor using the mirVana siRNA isolation kit (Ambion) according to the manufacturer's recommendations. After the first column washing step, DNA was digested on-column using an RNase-free DNase kit (Qiagen) according to the manufacturer's recommendations. RNA was eluted with RNase-free water. RNA concentration was measured using NanoDrop (ThermoFisher) and then adjusted to 215–250 ng/ml with RNase-free water. RNA quality was assessed using a Bioanalyzer (Agilent). High quality RNA integrity was confirmed (RIN from 6.5 to 10). 1 μg of RNA was reverse transcribed into cDNA using the M-MLV reverse transcriptase kit (ThermoFisher). Reverse transcription was performed using random primers, 10 mM dNTP mixture, and RNase inhibitor (Promega). Reverse transcription was performed with the following heat treatment steps: 65°C for 5 minutes, 4°C for 5 minutes, 25°C for 10 minutes, 4°C for 5 minutes, 37°C for 50 minutes, 42 °C for 10 minutes. 25 ng of cDNA was used for qPCR using the KAPA SYBR FAST qPCR Master Mix (2X) kit (Kapa Biosystems) according to the manufacturer's recommendations. The primers used for the qPCR reactions were as follows:

Gene Direct sequence Reverse sequence Ubb GTCTGAGGGGTGGCTATTAA
(SEQ ID NO:2) GCTTACCATGCAACAAAACC
(SEQ ID NO:3) Ccl2 CAGCTCTCTCTTCCTCCACC
(SEQ ID NO:4) TGGGATCATCTTGCTGGTGA
(SEQ ID NO:5) Ccl3 CCAGCAGGTGTCATTTTTCC
(SEQ ID NO:6) AGGCATTCAGTTCCAGGTCA
(SEQ ID NO:7) Ccl4 TCTGTGCTAACCCCAGTGAG
(SEQ ID NO:8) CTCTCCTGAAGTGGCTCCTC
(SEQ ID NO:9) Ccl5 TGCCAACCCAGAGAAGAAGT
(SEQ ID NO:10) AGATGCCCATTTTCCCAGGA
(SEQ ID NO:11) Csf2 CTGCGTAATGAGCCAGGAAC
(SEQ ID NO:12) TCCTCGTTTGTCTTCCGCT
(SEQ ID NO:13) Cxcl1 TTGTATGGTCAACACGCACG
(SEQ ID NO:14) ACGAGACCAGGAGAAAACAGG
(SEQ ID NO:15) Cxcl2 CTACATCCCACCCACACAGT
(SEQ ID NO:16) TGTTCTACTCTCCTCGGTGC
(SEQ ID NO:17) Cxcl10 GCCGTCATTTTTGCCTCAT
(SEQ ID NO:18) GATAGGCTCGCAGGGATGAT
(SEQ ID NO:19) Il1b ACTCATTGTGGCTGTGGAGA
(SEQ ID NO:20) TTGTTCATCTCGGAGCCTGT
(SEQ ID NO:21) Il6 TTCTTGGGACTGATGCTGGT
(SEQ ID NO:22) CAGGTCTGTTGGGAGTGGTA
(SEQ ID NO:23) Il10 ACCTGGTAGAAGTGATGCCC
(SEQ ID NO:24) AGGGTCTTCAGCTTCTCACC
(SEQ ID NO:25) Il18 GGACACTTTCTTGCTTGCCCA
(SEQ ID NO:26) ACCCTCCCCACCCTAACTTTG
(SEQ ID NO:27) Tnf TGAGGTCAATCTGCCCAAGT
(SEQ ID NO:28) GGGGTCAGAGTAAAGGGGTC
(SEQ ID NO:29)

Cycle threshold (CT) values were collected using a StepOnePlus real-time PCR system (Applied Biosystems Ubb was used as a control housekeeping gene. Data are presented as mean 2^ΔΔCT values for each treatment. 2^ΔΔCT values were calculated using the following formula:

2^ΔΔCT = 2^-(ΔCT(treated)-ΔCT(untreated))

ΔCT(Treatment) = CT(Treatment)-CT(HomeTreatment), where CT(Treatment) = CT of the sample gene of interest replicated in the treatment group at a given time point, and CT(HomeTreatment) = CT UBB of the housekeeping gene from one and the same replicate sample in the same treatment group at the same time point

ΔCT(untreated) = CT( Ac -11b )-CT( Ac -11b home), where CT( Ac -11b ) = average of CT values for 3 Ac -11b samples at the same time point, since CT(treated ) comparator and CT( Ac -11b home) = average of UBB home gene CT values for 3 Ac -11b samples at the same time point.

Results:

Absolute tumor volumes (mm ³ )

Days after treatment Group 0 3 6 8 10 Ac-11b Medium (mm ³ ) 113.58 354.04 516.60 885.98 852.10 SEM (mm ³ ) 6.66 34.52 61.18 101.23 81.08 N 17 14 eleven eleven 8 13d Medium (mm ³ ) 114.19 359.94 497.52 639.44 795.98 SEM (mm ³ ) 6.37 20.55 33.21 50.47 117.89 N 17 14 eleven eleven 8 13e Medium (mm ³ ) 114.03 319.01 367.39 518.55 587.11 SEM (mm ³ ) 6.17 29.95 48.16 68.48 79.87 N 17 14 eleven eleven 8 13f Medium (mm ³ ) 113.93 309.38 352.56 458.16 585.91 SEM (mm ³ ) 6.10 24.91 47.46 60.90 72.34 N 17 13 9 9 7 13g Medium (mm ³ ) 114.17 240.09 287.44 , 369.03 , 411.80 , SEM (mm ³ ) 5.67 20.63 52.27 73.29 94.97 N 17 14 10 9 7

SEM = standard error of the mean, N = sample size, p<0.01 compared to Ac -11b , p<0.02 compared to 13d . Significance was determined by two-way analysis of variance followed by multiple comparisons using post hoc analysis with Tukey's HSD test.

Absolute body weight (g)

Days after treatment Group 0 3 6 8 10 Ac-11b Average (g) 17.86 18.25 18.76 19.28 19.10 SEM (g) 0.29 0.30 0.42 0.46 0.50 N 17 14 eleven eleven 8 13d Average (g) 17.82 18.08 18.73 19.29 19.41 SEM (g) 0.27 0.26 0.30 0.30 0.42 N 17 14 eleven eleven 8 13e Average (g) 18.23 18.14 18.59 19.06 11/19 SEM (g) 0.28 0.30 0.35 0.33 0.45 N 17 14 eleven eleven 8 13f Average (g) 17.97 17.85 18.23 18.39 18.57 SEM (g) 0.30 0.29 0.40 0.41 0.47 N 17 13 10 9 7 13g Average (g) 18.04 17.39 18.34 19.02 18.96 SEM (g) 0.21 0.27 0.28 0.40 0.40 N 17 14 eleven 9 7

Concentration of resiquimod in plasma samples

Time (days) 0.25 3 7 9 12 14 16 Group Resiquimod (pg/ml) 13d Average 55.6 122 81.5 67.5 19.9 57 ND SD 8.1 59 23 NC NC 8.5 ND N 3 3 3 1 1 3 ND CV% 14.6 48.2 27.9 NC NC 14.9 ND 13e Average 216 383 319 160 155 ND 127 SD 75 230 63 NC 39 ND NC N 3 3 3 1 2 ND 1 CV% 34.9 59.7 19.7 NC 25.5 ND NC

SD = Standard Deviation, CV% = Coefficient of Variation, N = Sample Size, NC = Not Calculated, ND = Not Determined

Calculated parameters PK =

Dose Average Cmax (pg/ml) Average AUC (ng.h/ml) MRT (hours) 13d 122 (pg/ml) 35.4 287 13e 383 (pg/ml) 120.4 280

MRT: estimated mean residence time, estimated area under the plasma concentration-time profile, Cmax: estimated maximum plasma concentration

Cytokine levels in tumor lysate

CXCL1/GROα/KC Hours after treatment Group 6 72 168 Ac-11b Average (pg/ml) 332.60 640.50 727.60 SEM (pg/ml) 66.37 162.20 185.10 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (pg/ml) 1270.00 3624.00 3339.00 SEM (pg/ml) 360.70 752.10 492.00 Fold change from Ac -11b 3.82 5.66 4.59 N 3 3 3 13f Average (pg/ml) 875.40 3924.00 4538.00 SEM (pg/ml) 145.20 968.60 751.00 Fold change from Ac -11b 2.63 6.13 6.24 N 3 3 2 13g Average (pg/ml) ND 1419.00 2785.00 SEM (pg/ml) ND 335.40 989.40 Fold change from Ac -11b ND 2.22 3.83 N N.A. 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

IL-1β Hours after treatment Group 6 72 168 Ac-11b Average (pg/ml) 30.83 23.30 17.86 SEM (pg/ml) 1.31 0.15 1.47 Fold change compared to Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (pg/ml) 132.10 54.65 53.85 SEM (pg/ml) 35.22 10.35 3.67 Fold change from Ac -11b 4.28 2.35 3.02 N 3 3 3 13f Average (pg/ml) 119.80 64.03 69.12 SEM (pg/ml) 26.84 7.54 8.70 Fold change from Ac -11b 3.89 2.75 3.87 N 3 3 2 13g Average (pg/ml) ND 56.54 59.42 SEM (pg/ml) ND 10.20 16.10 Fold change from Ac -11b ND 2.43 3.33 N N.A. 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

IL-6 Hours after treatment Group 6 72 168 Ac-11b Average (pg/ml) 59.01 86.57 100.10 SEM (pg/ml) 1.71 11/19 18.59 Fold change compared to Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (pg/ml) 755.40 270.60 286.10 SEM (pg/ml) 301.20 51.04 41.06 Fold change from Ac -11b 12.80 3.13 2.86 N 3 3 3 13f Average (pg/ml) 744.10 523.60 82.92 SEM (pg/ml) 136.80 159.70 17.37 Fold change from Ac -11b 12.61 6.05 0.83 N 3 3 2 13g Average (pg/ml) ND 128.30 99.04 SEM (pg/ml) ND 31.05 13.27 Fold change from Ac -11b ND 1.48 0.99 N N.A. 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

CXCL10/IP-10 Hours after treatment Group 6 72 168 Ac-11b Average (pg/ml) 164.10 144.40 145.40 SEM (pg/ml) 1.28 18.04 15.02 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (pg/ml) 912.10 276.80 212.70 SEM (pg/ml) 81.34 59.89 11.13 Fold change from Ac -11b 5.56 1.92 1.46 N 3 3 3 13f Average (pg/ml) 950.00 390.20 197.80 SEM (pg/ml) 104.00 86.74 10.13 Fold change from Ac -11b 5.79 2.70 1.36 N 3 3 2 13g Average (pg/ml) ND 426.40 225.70 SEM (pg/ml) ND 43.68 54.55 Fold change from Ac -11b ND 2.95 1.55 N N.A. 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

CCL2/MCP-1 Hours after treatment Group 6 72 168 Ac-11b Average (pg/ml) 1682.00 1619.00 1763.00 SEM (pg/ml) 89.34 98.08 136.10 Fold change compared to Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (pg/ml) 3470.00 2738.00 2690.00 SEM (pg/ml) 533.60 185.80 260.40 Fold change compared to Ac -11b 2.06 1.69 1.53 N 3 3 3 13f Average (pg/ml) 3746.00 2891.00 2603.00 SEM (pg/ml) 767.80 93.48 74.97 Fold change compared to Ac -11b 2.23 1.79 1.48 N 3 3 2 13g Average (pg/ml) ND 2658.00 2457.00 SEM (pg/ml) ND 148.30 103.70 Fold change compared to Ac -11b ND 1.64 1.39 N N.A. 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

CCL3/MIP-1α Hours after treatment Group 6 72 168 Ac-11b Average (pg/ml) 475.30 525.20 518.50 SEM (pg/ml) 44.13 33.93 31.84 Fold change compared to Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (pg/ml) 1214.00 840.50 862.40 SEM (pg/ml) 214.40 111.00 45.95 Fold change compared to Ac -11b 2.55 1.60 1.66 N 3 3 3 13f Average (pg/ml) 1260.00 1209.00 1131.00 SEM (pg/ml) 244.50 214.30 118.40 Fold change compared to Ac -11b 2.65 2.30 2.18 N 3 3 2 13g Average (pg/ml) ND 1099.00 970.80 SEM (pg/ml) ND 62.99 212.50 Fold change from Ac -11b ND 2.09 1.87 N N.A. 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

CCL4/MIP-1β Hours after treatment Group 6 72 168 Ac-11b Average (pg/ml) 160.50 142.20 99.26 SEM (pg/ml) 10.87 24.60 13.86 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (pg/ml) 1105.00 339.00 265.80 SEM (pg/ml) 469.40 94.18 57.35 Fold change from Ac -11b 6.88 2.38 2.68 N 3 3 3 13f Average (pg/ml) 1031.00 432.00 458.50 SEM (pg/ml) 388.30 58.90 67.76 Fold change from Ac -11b 6.42 3.04 4.62 N 3 3 2 13g Average (pg/ml) ND 561.40 357.10 SEM (pg/ml) ND 106.60 138.10 Fold change from Ac -11b ND 3.95 3.60 N N.A. 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

CXCL2/MIP-2α Hours after treatment Group 6 72 168 Ac-11b Average (pg/ml) 188.00 548.30 1140.00 SEM (pg/ml) 56.39 124.40 172.90 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (pg/ml) 1122.00 1910.00 3144.00 SEM (pg/ml) 365.00 439.60 84.67 Fold change from Ac -11b 5.97 3.48 2.76 N 3 3 3 13f Average (pg/ml) 921.00 2471.00 2884.00 SEM (pg/ml) 299.70 564.80 285.20 Fold change from Ac -11b 4.90 4.51 2.53 N 3 3 2 13g Average (pg/ml) ND 2570.00 2723.00 SEM (pg/ml) ND 221.20 317.60 Fold change from Ac -11b ND 4.69 2.39 N N.A. 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

TNFα Hours after treatment Group 6 72 168 Ac-11b Average (pg/ml) 16.77 10.32 8.44 SEM (pg/ml) 2.88 0.61 0.91 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (pg/ml) 122.30 43.68 39.25 SEM (pg/ml) 17.09 3.91 4.16 Fold change from Ac -11b 7.29 4.23 4.65 N 3 3 3 13f Average (pg/ml) 139.70 102.60 68.57 SEM (pg/ml) 48.51 53.71 14.95 Fold change from Ac -11b 8.33 9.94 8.12 N 3 3 2 13g Average (pg/ml) ND 57.06 33.27 SEM (pg/ml) ND 3.84 12.87 Fold change from Ac -11b ND 5.53 3.94 N N.A. 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

Tumor gene expression

Ccl2 Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.02 1.00 1.01 SEM(2^ΔΔCT) 0.13 0.04 0.10 Fold change compared to Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 4.76 1.76 1.33 SEM(2^ΔΔCT) 2.33 0.40 0.12 Fold change from Ac -11b 4.68 1.76 1.32 N 3 3 3 13f Average (2^ΔΔCT) 5.12 1.43 1.44 SEM(2^ΔΔCT) 1.61 0.25 0.57 Fold change from Ac -11b 5.04 1.43 1.42 N 3 3 2 13g Average (2^ΔΔCT) 7.14 2.00 2.31 SEM(2^ΔΔCT) 1.67 0.11 0.27 Fold change from Ac -11b 7.02 2.00 2.28 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

Ccl3 Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.01 1.01 1.11 SEM(2^ΔΔCT) 0.12 0.08 0.37 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 9.81 2.78 1.17 SEM(2^ΔΔCT) 5.42 0.95 0.24 Fold change compared to Ac -11b 9.68 2.76 1.05 N 3 3 3 13f Average (2^ΔΔCT) 7.26 12.36 5.05 SEM(2^ΔΔCT) 2.88 10.11 1.43 Fold change from Ac -11b 7.17 12.27 4.54 N 3 3 2 13g Average (2^ΔΔCT) 8.64 5.15 7.74 SEM(2^ΔΔCT) 2.19 0.76 2.25 Fold change from Ac -11b 8.53 5.11 6.95 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

Ccl4 Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.01 1.04 1.09 SEM(2^ΔΔCT) 0.09 0.23 0.33 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 6.93 1.54 0.68 SEM(2^ΔΔCT) 3.49 0.42 0.10 Fold change from Ac -11b 6.88 1.47 0.62 N 3 3 3 13f Average (2^ΔΔCT) 5.42 5.49 2.37 SEM(2^ΔΔCT) 2.36 4.41 0.68 Fold change from Ac -11b 5.39 5.26 2.16 N 3 3 2 13g Average (2^ΔΔCT) 8.91 1.95 2.68 SEM(2^ΔΔCT) 2.37 0.24 0.75 Fold change from Ac -11b 8.85 1.87 2.45 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable

Ccl5 Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.01 1.09 1.08 SEM(2^ΔΔCT) 0.10 0.32 0.27 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 12.58 2.13 1.08 SEM(2^ΔΔCT) 7.87 0.89 0.20 Fold change from Ac -11b 12.46 1.96 1.00 N 3 3 3 13f Average (2^ΔΔCT) 15.64 7.50 3.92 SEM(2^ΔΔCT) 7.95 5.77 1.16 Fold change from Ac -11b 15.49 6.90 3.63 N 3 3 2 13g Average (2^ΔΔCT) 22.13 3.98 3.05 SEM(2^ΔΔCT) 5.66 0.68 1.10 Fold change from Ac -11b 21.91 3.66 2.82 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

Csf2 Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.00 1.31 1.32 SEM(2^ΔΔCT) 0.05 0.68 0.58 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 6.99 2.14 0.56 SEM(2^ΔΔCT) 4.27 0.56 0.17 Fold change from Ac -11b 6.97 1.64 0.43 N 3 3 3 13f Average (2^ΔΔCT) 5.27 2.61 0.23 SEM(2^ΔΔCT) 1.68 0.89 0.04 Fold change from Ac -11b 5.25 2.00 0.17 N 3 3 2 13g Average (2^ΔΔCT) 5.24 0.53 0.45 SEM(2^ΔΔCT) 0.43 0.07 0.02 Fold change from Ac -11b 5.22 0.41 0.34 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable

Cxcl1 Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.00 1.33 1.47 SEM(2^ΔΔCT) 0.03 0.71 0.64 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 2.00 5.28 1.03 SEM(2^ΔΔCT) 1.00 1.86 0.50 Fold change from Ac -11b 2.00 3.97 0.70 N 3 3 3 13f Average (2^ΔΔCT) 1.23 2.08 0.42 SEM(2^ΔΔCT) 0.30 0.96 0.02 Fold change compared to Ac -11b 1.23 1.57 0.28 N 3 3 2 13g Average (2^ΔΔCT) 3.74 0.70 0.77 SEM(2^ΔΔCT) 0.41 0.14 0.25 Fold change compared to Ac -11b 3.74 0.53 0.52 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

Cxcl2 Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.08 1.10 1.31 SEM(2^ΔΔCT) 0.28 0.33 0.64 Fold change compared to Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 4.94 4.00 1.03 SEM(2^ΔΔCT) 2.06 1.50 0.24 Fold change compared to Ac -11b 4.58 3.63 0.78 N 3 3 3 13f Average (2^ΔΔCT) 2.66 13.18 3.91 SEM(2^ΔΔCT) 0.61 10.73 1.26 Fold change compared to Ac -11b 2.47 11.98 2.98 N 3 3 2 13g Average (2^ΔΔCT) 3.33 4.12 8.49 SEM(2^ΔΔCT) 0.78 1.07 2.66 Fold change from Ac -11b 3.09 3.75 6.48 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

Cxcl10 Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.02 1.01 1.01 SEM(2^ΔΔCT) 0.15 0.09 0.11 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 29.23 3.24 2.09 SEM(2^ΔΔCT) 16.71 0.85 0.53 Fold change from Ac -11b 28.57 3.21 2.07 N 3 3 3 13f Average (2^ΔΔCT) 31.41 3.83 1.92 SEM(2^ΔΔCT) 13.05 1.05 0.31 Fold change from Ac -11b 30.70 3.81 1.90 N 3 3 2 13g Average (2^ΔΔCT) 32.96 2.15 2.29 SEM(2^ΔΔCT) 6.33 0.29 0.43 Fold change from Ac -11b 32.22 2.14 2.27 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

Il1b Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.02 1.00 1.10 SEM(2^ΔΔCT) 0.13 0.05 0.34 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 6.50 5.43 2.26 SEM(2^ΔΔCT) 4.23 2.10 0.54 Fold change from Ac -11b 6.39 5.41 2.06 N 3 3 3 13f Average (2^ΔΔCT) 2.82 6.79 0.27 SEM(2^ΔΔCT) 1.14 4.11 0.27 Fold change from Ac -11b 2.77 6.77 0.25 N 3 3 2 13g Average (2^ΔΔCT) 14.22 0.65 0.46 SEM(2^ΔΔCT) 2.71 0.26 0.11 Fold change from Ac -11b 13.98 0.65 0.41 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

Il6 Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.02 1.24 1.45 SEM(2^ΔΔCT) 0.16 0.58 0.70 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 6.25 2.77 1.31 SEM(2^ΔΔCT) 4.00 0.88 0.49 Fold change from Ac -11b 6.13 2.24 0.90 N 3 3 3 13f Average (2^ΔΔCT) 6.68 4.54 4.31 SEM(2^ΔΔCT) 1.98 1.41 3.78 Fold change from Ac -11b 6.55 3.67 2.98 N 3 3 2 13g Average (2^ΔΔCT) 5.05 0.98 0.60 SEM(2^ΔΔCT) 0.93 0.25 0.15 Fold change from Ac -11b 4.95 0.79 0.41 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable

Il10 Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.04 1.31 1.36 SEM(2^ΔΔCT) 0.20 0.67 0.57 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 1.58 0.81 1.29 SEM(2^ΔΔCT) 0.38 0.03 0.50 Fold change from Ac -11b 1.52 0.62 0.94 N 3 3 3 13f Average (2^ΔΔCT) 4.89 4.01 1.06 SEM(2^ΔΔCT) 0.54 1.57 0.45 Fold change from Ac -11b 4.70 3.06 0.78 N 3 3 2 13g Average (2^ΔΔCT) 1.69 2.06 0.72 SEM(2^ΔΔCT) 0.21 0.74 0.35 Fold change from Ac -11b 1.63 1.57 0.53 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

Il18 Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.01 1.15 1.21 SEM(2^ΔΔCT) 0.09 0.45 0.43 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 2.14 0.94 0.97 SEM(2^ΔΔCT) 0.56 0.14 0.31 Fold change from Ac -11b 2.12 0.82 0.80 N 3 3 3 13f Average (2^ΔΔCT) 4.45 3.27 0.53 SEM(2^ΔΔCT) 0.83 1.71 0.38 Fold change from Ac -11b 4.40 2.85 0.43 N 3 3 2 13g Average (2^ΔΔCT) 2.58 1.78 0.72 SEM(2^ΔΔCT) 0.47 0.92 0.19 Fold change from Ac -11b 2.56 1.55 0.59 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable, p<0.05 compared with Ac -11b at the same time point. Significance was determined by one-way analysis of variance followed by comparisons with Ac-11b- treated controls at each time point using Dunnett's multiple comparisons post hoc test.

Tnf Hours after treatment Group 6 72 168 Ac-11b Average (2^ΔΔCT) 1.03 1.18 1.20 SEM(2^ΔΔCT) 0.17 0.49 0.44 Fold change from Ac -11b 1.00 1.00 1.00 N 3 3 3 13e Average (2^ΔΔCT) 4.63 1.25 0.73 SEM(2^ΔΔCT) 2.75 0.28 0.15 Fold change from Ac -11b 4.51 1.06 0.61 N 3 3 3 13f Average (2^ΔΔCT) 3.33 3.87 2.19 SEM(2^ΔΔCT) 0.92 2.48 0.09 Fold change from Ac -11b 3.25 3.28 1.83 N 3 3 2 13g Average (2^ΔΔCT) 3.36 4.78 2.76 SEM(2^ΔΔCT) 0.57 1.40 1.40 Fold change from Ac -11b 3.27 4.05 2.30 N 3 3 2

SEM = standard error of the mean, N = sample size, ND = not determined, NA = not applicable

Example 14: In Vivo Antitumor Inhibitory Efficacy of Wild-Type IL-2 Combination and Repeat Tumor Study

The study was conducted on female BALB/C mice aged 6-8 weeks on the day of tumor inoculation. Mice were implanted with ^5x105 CT26 tumor cells in the left and right flanks. Once the right flank tumors had grown to a mean tumor volume of ~101 mm ³ , mice were randomized to treatment groups (day 0). On the same day of randomization, animals received 13h as a single intratumoral dose in an injection volume of 50 μl or a single intratumoral injection of 50 μl of Ac-11b suspension in the tumor of the right side. Hydrogels were introduced as a suspension in PTP buffer. Some groups received an additional 20 μg of human IL-2 (Peprotech, Rocky Hill, NJ) intraperitoneally (IP) twice daily for 5 days followed by a 3-day rest period, then an additional 20 μg of human IL-2 IP once a day. day for 5 additional days. After initiation of treatment, antitumor efficacy was assessed by determining tumor volumes at various time points based on tumor size measurements with calipers. Tumor volumes were calculated using the formula

Tumor volume = (L×W²)×0.5

where L represents the length of the tumor and W the width (both in mm).

3 of 7 mice treated with both 13h and human IL-2 experienced complete regression in both treated and untreated tumors and were re-implanted with 5 x 10 ⁵ CT26 tumor cells in their right anterior flank approximately 60 days after initial treatment. After reimplantation, mice were monitored for signs of tumor growth at the newly implanted site. Naïve female BALB/C mice were also implanted with the same tumor on the same day as the re-implanted mice as naïve control mice for comparison with normal tumor growth. Tumor growth was assessed by determining tumor volumes at various time points after implantation from tumor size measurements with calipers and calculated using the formula

Tumor volume = (L×W²)×0.5

where L is the length of the tumor and W is the width (both in mm). Tumor growth was not observed in mice treated with both 13h and human IL-2 ~60 days earlier at the end of the study period.

Results:

Absolute tumor volumes (mm ³ ) of tumors injected into the right side

Days after treatment Group 0 2 5 7 9 12 14 16 Ac-
11b Average
(mm ³ ) 101.57 222.96 390.09 676.66 975.86 1460.78 1836.12 2271.84 S.E.M.
(mm ³ ) 2.86 14.07 36.74 84.40 88.75 106.17 122.25 101.39 N 10 10 10 10 7 7 7 7 13h Average
(mm ³ ) 101.70 147.86 222.54 359.25 503.92 691.12 864.10 1354.10 S.E.M.
(mm ³ ) 3.09 5.89 18.62 38.62 63.80 101.80 135.92 149.33 N 10 10 10 10 7 7 7 7 Ac11b + human IL-2 Average
(mm ³ ) 101.79 144.70 210.24 311.11 413.93 539.45 659.38 856.24 , S.E.M.
(mm ³ ) 2.94 3.09 14.94 41.16 54.23 68.25 93.21 131.97 N 10 10 10 10 7 7 7 7 13h +
human IL-2 Average
(mm ³ ) 101.79 135.21 161.22 183.69 228.72 , 247.55 , , 255.14 , , 288.10 , , S.E.M.
(mm ³ ) 2.99 6.19 12.56 26.42 45.45 59.32 67.52 90.98

SEM = standard error of the mean, N = sample size, p<0.03 compared to Ac -11b , p<0.03 compared to 13h , p<0.02 compared with Ac -11b + human IL-2. Significance was determined by two-way analysis of variance followed by multiple comparisons using post hoc analysis with Tukey's HSD test.

Absolute tumor volumes (mm ³ ) of non-injected tumors of the right side

Days after treatment Group 0 2 5 7 9 12 14 16 Ac-
11b Medium (mm ³ ) 94.45 192.41 327.64 583.05 769.07 1192.25 1644.95 2223.11 SEM (mm ³ ) 4.84 19.38 38.05 89.15 86.70 118.05 137.96 166.70 N 10 10 10 10 7 7 7 7 13h Medium (mm ³ ) 98.95 144.19 220.17 432.34 631.58 948.54 1239.77 1854.24 SEM (mm ³ ) 4.08 5.30 21.21 43.94 63.59 97.86 136.23 187.27 N 10 10 10 10 7 7 7 7 Ac-
11b+
human
IL-2 Medium (mm ³ ) 99.01 136.38 199.08 313.92 420.65 542.96 , 718.21 , 995.12 , SEM (mm ³ ) 8.07 8.19 17.95 46.42 58.92 86.36 135.03 219.23 N 10 10 10 10 7 7 7 7 13h+
human
IL-2 Medium (mm ³ ) 92.48 121.70 139.10 177.79 296.62 , 347.49 , 411.71 , 484.22 , , SEM (mm ³ ) 4.47 9.59 13.12 34.35 82.23 101.27 139.26 165.58

SEM = standard error of the mean, N = sample size, p<0.002 compared to Ac -11b , p<0.04 compared to 13h , p<0.0003 compared with Ac -11b + human IL-2. Significance was determined by two-way analysis of variance followed by multiple comparisons using post hoc analysis with Tukey's HSD test.

Absolute tumor volumes (mm ³ ) in reimplanted and newly implanted mice

Days after CT26 implantation Group 0 3 7 10 14 17 21 24 Native control mice Medium (mm ³ ) 0 0 17.94 87.77 444.76 672.99 1622.95 2024.37 SEM (mm ³ ) 0 0 4.16 7.81 26.22 59.35 127.86 129.16 N 10 10 10 10 10 10 10 10 Re-implanted: 13h + human IL-2 Medium (mm ³ ) 0 13.61 0 0 0 0 0 0 SEM (mm ³ ) 0 13.61 0 0 0 0 0 0 N 3 3 3 3 3 3 3 3

SEM = standard error of the mean, N = sample size

Example 15: Flow Cytometric Profiling of Tumor Draining Immune Cells

The study was performed on female BALB/C mice aged 6-8 weeks on the day of tumor inoculation. Mice were implanted with 5×10 ⁵ CT26 tumor cells in the left and right flanks. Once the right flank tumors had grown to a mean tumor volume of ~101 mm ³ , mice were randomized to treatment groups (day 0). On the same day of randomization, animals received a single dose of 141 μg of resiquimod 4 (dissolved in 10 mM succinate, 90.0 mg/ml trehalose dihydrate, pH 5.0), 13h as a single intratumoral dose in an injection volume of 50 μl or a single intratumoral injection 50 µl of Ac-11b suspension in the tumor of the right side. Hydrogels were introduced as a suspension in PTP buffer. Some groups were additionally given 20 μg of human IL-2 (Peprotech, Rocky Hill, NJ) intraperitoneally (IP) twice daily for 5 days. Mice were sacrificed 7 days after randomization (D0). After killing, draining lymph nodes of the tumor were isolated from both sides and mechanically dissociated to obtain a suspension of single cells at a concentration of 1×10 ⁶ cells per sample. Cell suspensions were centrifuged at 300 g for 5 minutes. Supernatants were discarded and cells were resuspended in FACS buffer with 1 μg/ml Fc-Block and incubated at 4°C for 10 min in the dark. Surface marker antibody mixtures (antibody concentration: 10 μg/mL) in FACS buffer were added to each sample, and the samples were incubated in the dark at 4°C for 30 minutes. Cells were centrifuged at 300 g for 5 minutes and supernatants were discarded. Cells were washed and then resuspended in FACS buffer before cytometer collection.

Description of antibodies used for FACS profiling

Markers Fluorophore Clone Isotype CD45 BUV661 30-F11 rat IgG2b κ CD3 BUV395 17A2 rat IgG2b κ CD4 BV421 GK1.5 rat IgG2b κ CD8 PE-eFluor610 53-6.7 rat IgG2a κ CD335 BV605 29A1.4 rat IgG2a, κ I-A/I-E (MHCII) BB515 2G9 rat IgG2a κ Ly-6C APC HK1.4 rat IgG2c, κ L/D eFluor780 - -

After collection, FACS data were analyzed using FlowJo version 10.6.1. Compensation was digitally corrected using single antibody-stained beads. Samples with viability less than 90%, as determined by LiveDead cell staining, were excluded from analysis. Cells were identified using the following gating strategy:

1) Ly-6C ⁺ antigen-presenting cells: FSC- H/FSC- A Singlets/LiveDead ^- /CD45 ⁺ /CD3-/CD335 ^- /Ly-6C ⁺

2) Ly-6C ⁺ MHCII ⁺ antigen-presenting cells: FSC- H/FSC- A Singlets/LiveDead ^- /CD45 ⁺ /CD3-/CD335 ^- /Ly-6C ⁺ /IA/IE (MHCII) ⁺

3) CD8 ⁺ T cells: FSC- H/FSC- A Singlets/LiveDead ^- /CD45 ⁺ /CD3 ⁺ /CD8 single positive

4) Ly-6C ⁺ CD8 ⁺ T cells: FSC- H/FSC- A Singlets/LiveDead ^- /CD45 ⁺ /CD3 ⁺ /CD8 single positive/Ly-6C ⁺

Results:

Frequency of Ly-6C ⁺ antigen-presenting non-T cells

Tumor Group inserted, right side Not inserted, left side 4 Average (%) 2.5 7.8 SEM (%) 0.36 2.1 N 3 2 Ac-11b Average (%) 2.27 4.21 SEM (%) 0.40 0.68 N 3 2 13h Average (%) 15.43 , 25.23 SEM (%) 5.22 7.91 N 3 3 Ac-11b + human IL-2 Average (%) 3.49 7.69 SEM (%) 0.27 3.21 N 3 2 13h+ human IL-2 Average (%) 48.5 , , , 31 SEM (%) 1 4.34 N 2 3

SEM = standard error of the mean, N = sample size, Injected tumors: p<0.03 compared to Ac -11b , p<0.04 compared to 4 , p<0.05 compared to 13h, p<0.0001 compared with Ac -11b + human IL-2. Significance was determined by one-way analysis of variance followed by post hoc analysis using Tukey's multiple comparisons test.

Frequency of IA-IE (MHCII) ⁺ antigen-presenting cells for Ly-6C ⁺ antigen-presenting cells

Tumor Group Inserted, right side Not inserted, left side 4 Average (%) 69.63 35.05 SEM (%) 4.06 6.15 N 3 2 Ac-11b Average (%) 70.5 24.8 SEM (%) 10.91 1.4 N 3 2 13h Average (%) 94.77 95.6 SEM (%) 4.48 3.01 N 3 3 Ac-11b + human IL-2 Average (%) 66.3 45.25 SEM (%) 1.47 29.45 N 3 2 13h+ human IL- 2 Average (%) 95.75 77.4 SEM (%) 2.75 16.69 N 2 3

SEM = standard error of the mean, N = sample size, Injected tumors: p = 0.049 compared to 13h. Significance was determined by one-way analysis of variance followed by post hoc analysis of multiple comparisons using Tukey's multiple comparisons test.

Frequency of CD8 ⁺ T cells to CD3 ⁺ T cells

Tumor Group Inserted, right side Not inserted, left side 4 Average (%) 29.23 29 SEM (%) 1.32 0.2 N 3 2 Ac-11b Average (%) 29 28.55 SEM (%) 1.10 2.05 N 3 2 13h Average (%) 23.93 26.33 SEM (%) 1.43 2.04 N 3 3 Ac-11b + human IL-2 Average (%) 38.83 , , 35 SEM (%) 2.21 1.2 N 3 2 13h+ human IL- 2 Average (%) 41.45 , , 48.53 , , SEM (%) 2.75 3.93 N 2 3

SEM = standard error of the mean, N = sample size, Tumors injected:p<0.02 compared toAc-11b,p<0.02 compared to4,p<0.001 compared to13h, Uninjected tumors:p = 0.0085 compared toAc-11b,p = 0.0096 compared to4,p = 0.0025 compared to13h. Significance was determined by one-way analysis of variance followed by post hoc analysis of multiple comparisons using Tukey's multiple comparisons test.

Ly-6C ⁺ T cell frequency for CD8 ⁺ T cells

Tumor Group Introduced, right side Not inserted, left side 4 Average (%) 53.7 48 SEM (%) 2.06 3.3 N 3 2 Ac-11b Average (%) 45.77 35.25 SEM (%) 3.47 0.75 N 3 2 13h Average (%) 49.1 50.4 SEM (%) 6.03 5.56 N 3 3 Ac-11b + human IL-2 Average (%) 56.17 54.8 SEM (%) 2.03 2.8 N 3 2 13h+ human IL- 2 Average (%) 68.35 68.17 , , SEM (%) 3.75 1.57 N 2 3

SEM = standard error of the mean, N = sample size, Injected tumors: p = 0.024 compared to Ac -11b, Non-injected tumors: p = 0.0029 compared to Ac-11b , p = 0.039 compared to 4 , p = 0.042 compared to 13h . Significance was determined by one-way analysis of variance followed by post hoc analysis of multiple comparisons using Tukey's multiple comparisons test.

Frequency of CD4 ⁺ T cells to CD3 ⁺ T cells

Tumor Group Inserted, right side Not inserted, left side 4 Average (%) 69.1 69.25 SEM (%) 1.31 0.05 N 3 2 Ac-11b Average (%) 69.57 69.8 SEM (%) 1.17 2.3 N 3 2 13h Average (%) 73.23 70.27 SEM (%) 1.09 1.45 N 3 3 Ac-11b + human IL-2 Average (%) 58.9 , , 62.45 SEM (%) 2.16 1.15 N 3 2 13h+ human IL- 2 Average (%) 54.5 , , 48.5 , , , SEM (%) 3 3.27 N 2 3

SEM = standard error of the mean, N = sample size, Tumors injected:p<0.009 compared toAc-11b,p<0.02 compared to4,p<0.002 compared to13h, uninjected tumors:p = 0.0021 compared toAc-11b,p = 0.0025 compared to4,p = 0.0009 compared to13h, p = 0.022 compared toAc-11b + human IL-2. Significance was determined by one-way analysis of variance followed by multiple comparisons using post hoc analysis using Tukey's multiple comparisons test.

Example 16: Flow Cytometric Profiling of Peripheral Blood

The study was conducted on female BALB/C mice aged 9-11 weeks on the day of tumor inoculation. Mice were implanted with 5×10 ⁵ CT26 tumor cells in the right hind flank. When the injected tumors had grown to an average tumor volume of ~80 mm ³ , mice were randomized to treatment groups (day 0) and given either one intravenous dose on day 0 and one intravenous dose on day 6, 200 μl control buffer, one intravenous dose on Day 0 and one intravenous dose on Day 6, 200 µL per 60 µg 16, a single intratumoral injection of 50 µL 12c on Day 0, or a combination of one intravenous dose on Day 0 and one intravenous dose on Day 6, 200 µL per 60 µg 16 and single intratumoral injection of 50 μL 12c on Day 0. Hydrogels were administered as suspensions in PTP buffer. Mice were bled 4 days after randomization for in vitro stimulation and flow cytometry (FACS). Blood was stimulated with leukocyte activation cocktail using BD GolgiPlug™ (BD Biosciences) for 5 hours in a humidified CO ₂ incubator at 37°C, then processed for FACS. Cells were washed with FACS buffer, supernatants were removed, and cells were resuspended in FACS buffer with 1 μg/ml Fc-Block and incubated at 4°C for 10 min in the dark. Mixtures of antibodies to surface markers in FACS buffer were added to each sample and samples were incubated in the dark at 4°C for 30 minutes. Red blood cell lysis buffer (Bio-gems) was added and the cells were further incubated at 4°C for 10 minutes. Cells were washed twice with FACS buffer, then fixed and permeabilized for 30 min at room temperature with Fix/Perm buffer (eBioscience). Cells were washed twice with permeabilization buffer and stained with intracellular antibodies in permeabilization buffer for 60 min at room temperature. Cells were washed twice in FACS buffer and prepared in the presence of 123count Ebeads (eBioscience).

Brief description of antibodies used for FACS profiling

Markers Fluorophore Clone Isotype CD45 BV711 30-F11 Rat IgG2b, κ CD3 BUV395 17A2 Rat IgG2b, κ CD4 BUV737 GK1.5 Rat IgG2b, κ CD8 FITC 53-6.7 Rat IgG2a, k CD25 BV510 PC61 Rat IgG1, λ CD335 BV605 29A1.4 Rat IgG2a, k CD44 BV421 IM7 Rat IgG2b, κ Ly6C BV785 HK1.4 Rat IgG2c, k CTLA4 P.E. UC10-4B9 Armenian Hamster IgG FoxP3 PerCP-Cy 5.5 FJK-16S Rat IgG2a, k TNF-α APC MP6-XT22 Rat IgG1, k IFN-g PE-Cy7 XMG1.2 Rat IgG1, k GranzymeB PE-ef610 NGZB Rat IgG2a, k Life death efluo780 N.A. N.A.

After collection, FACS data were analyzed using FlowJo version 10.6.1. Compensation was digitally corrected using single antibody-stained beads, single antibody-stained cells, and fluorescence minus one (FMO) controls. CD8 ⁺ T cells were determined using the following gating strategy: FSC- A / SSC- A cells / FSC- H / FSC- A Singlets / LiveDead ^- / CD45 ⁺ /CD8 ⁺ . This gating scheme was used to simultaneously gate CD4+ and CD8+ T cells, and additional analyzes confirmed that these cells coexpress CD3 and are T cells.

Results:

Frequency of peripheral blood CD8 ⁺ T cells in CD45+ cells:

Group Buffer control 16 12c 12c+16 N 4 4 4 4 Average 4.77 8.51 3.52 13.45 S.E.M. 1.1 1.37 0.2691 1.664 P-value compared to control N.A. .051 .484 <.001 P-value compared to 16 .051 N.A. .014 .014 P-value compared to 12c .484 .014 N.A. <.001

According to this analysis, the combination of 12c + 16 showed a significantly higher frequency of CD8+ T cells in the blood within CD45+ cells (mean: 13.45%) compared to treatment with buffer control (mean: 4.77%) or treatment with 16 alone (mean : 8.51%) or 12c alone (average: 3.52%). Treatment 16 induced an approximately 1.78-fold increase in the percentage of CD8+ T cells in total CD45+ cells compared to control buffer treatment. Treatment with 12c+16 induced approximately 2.81-fold increase in the percentage of CD8+ T cells in total CD45+ cells compared to control buffer treatment. Treatment with 12c + 16 induced approximately 3.82-fold increase in the percentage of CD8+ T cells out of total CD45+ cells compared to treatment with 12c alone.

Example 17: In vivo PK study of plasma and concentrations of resiquimod and pharmacodynamic effects on peripheral blood mononuclear cell (PBMC) gene expression

The study was conducted on female BALB/C mice aged 6-8 weeks on the day of tumor inoculation. Mice were implanted with 5×10 ⁵ CT26 tumor cells in the right flank. When tumors had grown to a mean tumor volume of ~104 mm ³ , mice were randomly assigned to treatment groups (day 0). The day after randomization, animals received either a single intratumoral injection of 10 μg of resiquimod 4 (dissolved in 50 μl of 10 mM succinate, 90.0 mg/ml trehalose dihydrate, pH 5.0) or hydrogel 13i as a single intratumoral dose in the injection volume 50 µl. Hydrogels were introduced as suspensions in PTP buffer. At specific time points (0 hours, 6 hours, 22 hours and 72 hours after the start of treatment), 5 mice per group were sacrificed, and either plasma was prepared after blood collection or PBMC were isolated. Untreated tumor-bearing animals were sacrificed at time 0 hours to serve as untreated controls for assessing PBMC gene expression. The tumor was excised, weighed and quickly frozen. Plasma samples were further processed by solid phase extraction to determine the concentration of resiquimod by LC-MS/MS.

Excised tumor samples (weighing from 150 to 300 mg) were thawed and homogenized in the presence of 1 ml of saturated KOH in ethanol/water (9/1 v/v) using a FastPrep-24 5G homogenizer (MP Biomedicals, Eschwege) using slightly modified manufacturer's protocol (cooling with dry ice, 2 times 40 seconds at a speed of 6 m/s). The resulting cell lysate was further incubated at 37°C for 15 hours. After incubation, the dissolved samples were vortexed and diluted 1:10,000 in plasma. These samples were processed as described above and submitted for LC-MS analysis to determine the concentration of resiquimod. The amount of resiquimod in the tumor sample was calculated using the dilution factor and the determined tumor weight.

To isolate PBMCs, approximately 600 μl of whole blood was collected by cardiac puncture. Collected whole blood from each individual mouse was diluted 1:1 in prewarmed PBS supplemented with 2% fetal bovine serum (FBS). An equal volume of Histopaque-1083 was then added to a new sterile 15 mL conical tube where the diluted whole blood was applied to Histopaque-1083. The mixture was then centrifuged at 400 g for 30 minutes. The top layer of plasma was removed and the white translucent intermediate layer (mononuclear cells) was carefully transferred to a new sterile centrifuge tube. The mononuclear cells were then washed with PBS supplemented with 2% FBS and then centrifuged at 250 g for 10 minutes. Cells were then lysed with 2 ml ammonium chloride potassium (ACK) lysis buffer (Gibco) for 5 min at room temperature to remove red blood cells, following the manufacturer's instructions. Cells were then washed twice with PBS supplemented with 2% FBS and centrifuged at 250 g for 10 minutes. The supernatant was then removed, and the PBMC cell pellet was lysed in RLT buffer (Qiagen) and stored at −80°C before proceeding with RNA extraction and isolation.

Lysates from untreated control samples and 6-h treated samples were thawed and RNA isolated using the RNeasy Mini Kit (QIAGEN) according to the manufacturer's recommendations. After the first column washing step, DNA was digested on-column using an RNase-free DNase kit (TIANGEN) according to the manufacturer's recommendations. RNA was eluted with RNase-free water. RNA concentration was measured using NanoDrop (ThermoFisher) and then adjusted to 200 ng/ml with RNase-free water. RNA quality was assessed using a NanoDrop instrument (ThermoFisher). The concentrations of all RNA samples were >100 ng/μl, and the _A260 / _A280 ratio was confirmed to be close to or greater than 2, making it suitable for downstream qPCR analysis. 2 μg of RNA was reverse transcribed into cDNA using the RT2 First Strand Kit (QIAGEN). Reverse transcription was performed using random primers, 10 mM dNTP mixture, and RNase inhibitor (TIANGEN). Reverse transcription was performed with the following heat treatment steps: 25°C for 10 minutes, 37°C for 120 minutes, 55°C for 5 minutes. 200 ng of cDNA was used for qPCR using the RT² SYBR Green ROX qPCR Master mix (2X) kit (QIAGEN) according to the manufacturer's recommendations. The probe sets used for qPCR reactions were as follows:

Gene designation Catalog no. Il1a PPM03010F Ccl3 PPM02949F Il1b PPM03109F Cxcl2 PPM02969F Ccl2 PPM03151G Ccl4 PPM02948F Il10 PPM03017C Ifna4 PPM03549E Cxcl1 PPM03058C Cxcl10 PPM02978E Tnf PPM03113G B2m PPM03562A Ubc PPM03450A Gapdh PPM02946E

Cycle threshold (CT) values were collected using the ABI-7900H 384-well real-time qPCR system platform (Applied Biosystems). B2M, Ubb, and GAPDH were used as control housekeeping genes. Data are presented as mean 2^ - ΔΔCT for each treatment. The values of 2^ - ΔΔCT were calculated using the following formula:

2^- ΔΔCT = 2^-(ΔCT(treated)- ΔCT(untreated))

ΔCT(Treatment) = CT(Treatment)-CT(HomeTreatAverage), where CT(Treatment) = CT of the gene of interest in triplicate samples in the treatment group, and CT(HomeTreatment) = overall mean CT of the B2M, UBB housekeeping genes and GAPDH from the same sample in triplicate in one experimental group.

ΔCT (untreated) = CT (raw -CT (untreated home), where CT (raw) = average of the CTs of untreated triplicates at the same time point as a CT (treated) comparator, and CT (untreated home) = Cumulative average CT of housekeeping genes B2M, UBB, and GAPDH of raw triplicates.

For each gene, 3 technical replicates were analyzed for each biological replicate. Uncertain CT values of the technical replicate were recorded as zero ΔCT values. A total of 4-5 biological replicates were evaluated.

Results:

Concentration of resiquimod in plasma samples

Time watch) 0 6 22 72 Group Resiquimod (pg/ml) 4 Average 210,000 360 22 14 SD 49,000 190 7 NC N 5 5 5 5 CV% 23 52 33 NC 13i Average 280 230 200 180 SD 130 49 46 31 N 5 5 5 5 CV% 47 22 23 17

SD = standard deviation, CV% = coefficient of variation, N = sample size, NC = not calculated, ND = not determined. 4/5 samples <LLOQ

Resiquimod content in tumors after complete release from the hydrogel:

Time watch) Group 0 h 72 h 13i 8.2 ± 2.3 µg (n = 5) 8.2 ± 1.5 µg (n = 5)

PBMC Gene Expression (6 hours after treatment):

Gene 4 13i Multiple change (4/13i) Il1a Average (2^ΔΔCT) 0.41 0.23 1.78 SEM(2^ΔΔCT) 0.26 0.11 N 4 5 Ccl3 Average (2^ΔΔCT) 1.43 0.87 1.64 SEM(2^ΔΔCT) 0.09 0.18 N 4 5 Il1b Average (2^ΔΔCT) 3.71 0.47 7.92 SEM(2^ΔΔCT) 1.12 0.27 N 4 5 Cxcl2 Average (2^ΔΔCT) 35.37 5.50 6.43 SEM(2^ΔΔCT) 17.41 5.21 N 4 5 Ccl2 Average (2^ΔΔCT) 6.75 2.39 2.82 SEM(2^ΔΔCT) 2.90 0.57 N 4 5 Ccl4 Average (2^ΔΔCT) 1.31 0.75 1.74 SEM(2^ΔΔCT) 0.35 0.27 N 4 5 Il10 Average (2^ΔΔCT) 2.77 1.21 2.29 SEM(2^ΔΔCT) 0.51 0.24 N 4 5 Ifna4 Average (2^ΔΔCT) 2.97 0.15 20.48 SEM(2^ΔΔCT) 2.35 0.04 N 4 5 Cxcl1 Average (2^ΔΔCT) 7.41 0.64 11.54 SEM(2^ΔΔCT) 6.41 0.39 N 4 5 Cxcl10 Average (2^ΔΔCT) 45.38 2.12 21.41 SEM(2^ΔΔCT) 34.14 0.86 N 4 5 Tnf Average (2^ΔΔCT) 4.64 1.04 4.45 SEM(2^ΔΔCT) 1.91 0.47 N 4 5

SEM = standard error of the mean, N = sample size, two-tailed p<0.05 vs. 4 . Significance was determined using an unpaired nonparametric Student's t test.

Abbreviations

AcOH Acetic acid AUC Area under the curve DCM dichloromethane DIPEA N,N-diisopropylethylamine DMAP 4-(dimethylamino)pyridine EDC N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride eq. equivalents EtOH ethanol Fmoc fluorenylmethyloxycarbonyl HOBt 1-hydroxybenzotriazole HOSu N-hydroxysuccinimide HPLC High performance liquid chromatography IV intravenously LC-MS Liquid chromatography in tandem with mass spectrometry LPLC low pressure liquid chromatography MeCN acetonitrile MeOH methanol NHS N-hydroxysuccinimide NMP N-Methyl-2-pyrrolidone PBST Phosphate buffered saline with Tween 20 P.E. polyethylene P.E.G. poly(ethylene glycol) P.K. pharmacokinetics/s PMM poly(methyl methacrylate) PTP 5 mM phosphate, 90 g/l trehalose dihydrate, 0.2% Pluronic F-68, pH 7.4 PyBOP benzotriazol-1-yl-oxytripyrrolidinephosphonium hexafluorophosphate RP-HPLC reverse phase high performance liquid chromatography RP-LPLC reverse phase low pressure liquid chromatography r.t. Room temperature S.C. Subcutaneously T.F.A. Trifluoroacetic acid THF Tetrahydrofuran TMEDA N,N,N′,N′-tetramethylethylenediamine Tween 20 polyethylene glycol sorbitan monolaurate UHPLC ultra-high performance liquid chromatography UPLC ultra high performance liquid chromatography UPLC-MS ultra-high performance liquid chromatography in tandem with mass spectrometry

Claims (25)

1. A water-insoluble controlled-release pattern recognition receptor agonist (PRRA) or a pharmaceutically acceptable salt thereof, wherein said water-insoluble controlled-release PRRA releases one or more PRRAs and at least 25% of the amount of PRRA remains locally in such tissue 3 days after interstitial administration, wherein the water-insoluble PRRA contains a carrier fragment Z to which one or more -L ² -L ¹ -D fragments are conjugated, where each -L ² - individually represents a C _1-20 alkyl chain, which is optionally interrupted by one or more groups independently selected from -O-, -T- and -C(O)N(R ^y1 )-, and where C ₁ The _-20 alkyl chain is optionally substituted with one or more groups independently selected from -OH, -T and -C(O)N(R ^y6 R ^y6a ), where -R ^y1 , -R ^y6 , -R ^y6a are independently selected from the group, consisting of H and C _1-4 alkyl, and where T is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C _3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8 -30-membered carbopolycyclyl and 8-30-membered heteropolycyclyl; each -L ^1- individually represents a linker moiety to which -D is reversibly and covalently conjugated, selected from the group consisting of formulas (X -1), (X-2), (X-3), (X-4) , (X-5), (X-6), (X-7), (X-8), (X-9), (X-10), (X-11) and (X-12) Where the dotted line marked with an asterisk indicates the addition of an amine functional group -D to the nitrogen atom, the unmarked dotted line shows the attachment to -L ² -, -R ¹ is selected from the group consisting of - H, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl, -R ² , and -R ^2a are independently selected from the group consisting of - H, halogen, C _1-10 alkyl, C _2-10 alkenyl and C _2-10 alkynyl, n is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25, m is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25, o represents an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, p is an integer selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and q is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25; each -D represents resiquimod; And Z is a water-insoluble PEG-based hydrogel. 2. The water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof according to claim 1, wherein interstitial administration is intratumoral administration. 3. A water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the antitumor administration is to a solid tumor or lymphoma. 4. The water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof according to claim 3, wherein the solid tumor or lymphoma is selected from the group consisting of lip and oral cavity cancer, oral cavity cancer, liver/hepatocellular cancer, primary liver cancer, lung cancer, lymphoma, malignant mesothelioma, malignant thymoma, skin cancer, intraocular melanoma, metastatic squamous cell carcinoma of the neck with primary latency, multiple endocrine neoplasia syndrome in children, mycosis fungoides, cancer of the paranasal sinuses and nasal cavity, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer , ovarian cancer, pancreatic cancer, parathyroid cancer, pheochromoblastoma, pituitary tumor, adrenocortical carcinoma, AIDS-related malignancies, anal cancer, bile duct cancer, bladder cancer, brain and nervous system cancer, breast cancer, adenoma bronchus/carcinoid, carcinoid tumor of the gastrointestinal tract, carcinoma, colorectal cancer, endometrial cancer, esophageal cancer, extracranial germ cell tumor, extragonadal gonocytoma, extrahepatic bile duct cancer, gallbladder cancer, gastric cancer (stomach), gestational trophoblastic tumor, head cancer and neck, hypopharyngeal cancer, islet cell carcinoma (endocrine pancreas), kidney/renal cell carcinoma, laryngeal cancer, pleuropulmonary blastoma, prostate cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, salivary gland cancer, sarcoma , Sezary syndrome, small bowel cancer, genitourinary cancer, malignant thymoma, thyroid cancer, Wilms tumor and cholangiocarcinoma. 5. A water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein at least 30% of the total amount of administered PRRA remains in such tissue after 3 days. 6. A water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein at least 25% of the total amount of administered PRRA remains in such tissue after 7 days. 7. A water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein at least 30% of the total amount of administered PRRA remains in such tissue after 7 days. 8. A water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein at least 25% of the total amount of administered PRRA remains in such tissue after 10 days. 9. The water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the PRRA is released from the water-insoluble controlled-release PRRA with a half-life under physiological conditions of at least 3 days. 10. Water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the PRRA is released from the water-insoluble controlled-release PRRA with a half-life under physiological conditions of at least 10 days. 11. A pharmaceutical composition comprising one or more water-insoluble controlled-release PRRA or a pharmaceutically acceptable salt thereof according to claim 1 or 2 and at least one excipient.