Pyrrolobenzodiazepine conjugates for the treatment of cancer

Abstract

A compound useful as a biologically active compound is disclosed. The compound has the following structure (I): [Formula 1] JPEG2024536224000141.jpg53124(I) or a stereoisomer, tautomer or salt thereof. 1 , R 2 , R 3 , R 4 , R 5 , L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , M 1 , M 2 (wherein q, w, m and n are as defined herein.) The compounds of structure (I) find utility in many applications, including use as therapeutic agents for a variety of treatment methods.

Description

[Technical field]

[0001] SUMMARY OF THE DISCLOSURE Embodiments of the present disclosure are generally directed to biologically active polymeric compounds having alkylating agents and methods for their preparation. [Background technology]

[0002] Targeted drug conjugates deliver drugs to target cells, unlike chemotherapy, with little or no off-target activity.Typically, targeted drug conjugates contain a targeting molecule linked to a biologically active payload or drug.The conjugates combine the inherent targeting ability with the therapeutic efficacy of biologically active drugs, allowing the drug to be delivered only to the intended target and minimizing potential side effects. Antibody-drug conjugates (ADCs) are a class of targeted drug conjugates that are of particular interest, for example, in cancer treatment. ADCs combine the targeting characteristics of monoclonal antibodies with the cancer-killing potential of cytotoxic agents, resulting in a therapy with several advantages over other chemotherapeutic agents. However, the complexity of ADC construction, specifically the challenges associated with the chemical linker between the antibody and the drug, make the development of new and effective therapeutic agents quite challenging. The first ADC was approved in 2001, but it took almost a decade for the next ADC to be approved. At present, only Adcetris®, Besponsa®, Enhertu®, Mylotarg®, Padcev®, Polivy®, and Kadcyla® are commercially available worldwide (Zevalin® is only approved in China). Thus, there exists a need in the art for the development of potent targeted drug conjugates with high therapeutic indices and methods for their preparation. The present disclosure addresses this need and achieves further related advantages. Summary of the Invention

[0003] In one embodiment, a compound having the following structure (I): [ka] (I) or a stereoisomer, tautomer or salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 , L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , M 1 , M 2 , q, w, m and n are as defined herein.) The compounds of structure (I) find utility in a number of applications, including use as therapeutic agents in a variety of treatment methods.

[0004] In another embodiment, there is provided a composition comprising a compound of structure (I) and a pharma- ceutically acceptable carrier.

[0005] In yet another embodiment, a method of treating a disease is provided comprising administering to a subject in need thereof a therapeutically effective amount of a compound of structure (I) or a composition comprising a compound of structure (I), wherein M1 or M2 each independently comprises a biologically active moiety effective to treat the disease.

[0006] In yet another embodiment, a compound having the following structure (II): [ka] (II) or a salt, tautomer or stereoisomer thereof, wherein R 1” , R 2” , L 1b , R 6 , R 7 , R 8 , R 9 , R 10, and R 11 are as defined herein).

[0007] In yet another embodiment, a compound having the following structure (III): [ka] (III) or a salt, tautomer or stereoisomer thereof, wherein R 1” , R 2” , L 1b , R 6 , R 7 , R 8 , R 9 , R 10 , and R 11 are as defined herein). These and other aspects of the present disclosure will become evident upon reference to the following detailed description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments of the present disclosure. However, it will be understood by those skilled in the art that the present disclosure may be practiced without these details. Unless the context requires otherwise, throughout this specification and claims, the word "comprise" and variations thereof, such as "comprises" and "comprising," are to be interpreted in their open, inclusive sense, i.e., "including but not limited to."

[0009] With reference to the entire specification, the references to "one embodiment" or "an embodiment" mean that the particular features, structures, or properties described in connection with those embodiments are included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout the specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or properties may be combined in any suitable manner in one or more embodiments. "Amino" refers to the group -NH2. "Carboxy" refers to the group -CO2H. "Cyano" refers to the group -CN. "Formyl" refers to the group -C(=O)H. "Hydroxy" or "hydroxyl" refers to the group --OH. "Imino" refers to the group =NH. "Nitro" refers to the -NO2 group. "Oxo" refers to the =O substituent. "Sulfhydryl" refers to the -SH group. "Thioxo" refers to the group ═S. "Alkyl" means an alkyl group containing no unsaturation and having 1 to 12 carbon atoms (C1-C 12 "alkyl") refers to a straight or branched hydrocarbon chain radical consisting only of carbon and hydrogen atoms having 1 to 8 carbon atoms (C1-C8 alkyl) or 1 to 6 carbon atoms (C1-C6 alkyl) attached to the rest of the molecule by a single bond, such as methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specifically stated herein, alkyl groups may be optionally substituted.

[0010] "Alkylene" or "alkylene chain" refers to a divalent straight or branched hydrocarbon chain, containing no unsaturation and having 1 to 12 carbon atoms, consisting only of carbon and hydrogen, linking the remainder of the molecule to a radical group, e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless specifically stated otherwise in this specification, alkylene can be optionally substituted.

[0011] "Alkenylene" or "alkenylene chain" refers to a divalent straight or branched hydrocarbon chain, consisting only of carbon and hydrogen, containing at least one carbon-carbon double bond and having 2-12 carbon atoms, linking the rest of the molecule to a radical group, e.g., ethenylene, propenylene, n-butenylene, etc. The alkenylene chain is attached to the rest of the molecule through a single bond and to the radical group through a double bond or a single bond. The points of attachment of the alkenylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless specifically stated otherwise in this specification, alkenylene may be substituted. "Alkynylene" or "alkynylene chain" refers to a divalent straight or branched hydrocarbon chain, consisting only of carbon and hydrogen, containing at least one carbon-carbon triple bond and having 2 to 12 carbon atoms, linking the rest of the molecule to a radical group, such as ethenylene, propenylene, n-butenylene, and the like. The alkynylene chain is attached to the rest of the molecule through a single bond and to the radical group through a double bond or a single bond. The points of attachment of the alkynylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in this specification, alkynylene is optionally substituted.

[0012] "Alkyl ether" refers to any alkyl group, as defined above, in which at least one carbon-carbon bond is replaced by a carbon-oxygen bond. The carbon-oxygen bond may be terminal (as in an alkoxy group) or the carbon-oxygen bond may be internal (i.e., COC). An alkyl ether contains at least one carbon-oxygen bond, but may contain more than one. For example, polyethylene glycol (PEG) is included within the meaning of alkyl ether. Unless specifically stated otherwise herein, alkyl ether groups may be substituted. For example, in some embodiments, an alkyl ether may be an alcohol or -OP(=R a )(R b )R c is replaced by R a , R b and R c are each as defined for compounds of structure (I). "Alkoxy" means a group of the formula -OR a R refers to the group a is an alkyl group, as defined above, containing 1 to 12 carbon atoms. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted. "Alkoxyalkyl ether" means a group of the formula -OR a R b R refers to the group a is an alkylene group, as defined above, containing 1 to 12 carbon atoms, and Rb is an alkyl ether group, as defined herein. Unless otherwise specifically stated herein, an alkoxyalkyl ether group may be optionally substituted, for example, by an alcohol or -OP(=Ra)(Rb)Rc, where Ra, Rb, and Rc are each as defined for the compounds of structure (I).

[0013] "Heteroalkyl" refers to an alkyl group, as defined above, that contains at least one heteroatom (e.g., N, O, P, or S) within or at the terminus of the alkyl group. In some embodiments, the heteroatom is present within the alkyl group (i.e., a heteroalkyl has at least one carbon-[heteroatom] x -carbon bond, x is 1, 2, or 3). In other embodiments, the heteroatom is at the end of the alkyl group, thus serving to attach the alkyl group to the remainder of the molecule (e.g., M1-HA), where M1 is part of the molecule, H is the heteroatom, and A is the alkyl group. Unless otherwise specifically stated herein, heteroalkyl groups may be substituted. Exemplary heteroalkyl groups include ethylene oxide (e.g., polyethylene oxide), which may include a phosphorus-oxygen bond, such as a phosphodiester bond. "Heteroalkoxy" means a heteroalkoxy group of the formula -OR a R refers to the group a is a heteroalkyl group, as defined above, containing 1 to 12 carbon atoms. Unless stated otherwise specifically in the specification, a heteroalkoxy group may be optionally substituted.

[0014] "Heteroalkylene" refers to an alkylene group, as defined above, containing at least one heteroatom (e.g., Si, N, O, P, or S) within the alkylene chain or at the terminus of the alkylene chain. In some embodiments, the heteroatom is within the alkylene chain (i.e., the heteroalkylene contains at least one carbon-[heteroatom]-carbon bond, and x is 1, 2, or 3). In other embodiments, the heteroatom is at the terminus of the alkylene and serves to attach the alkylene to the remainder of the molecule (e.g., M1-HA-M2, where M1 and M2 are part of the molecule, H is a heteroatom, and A is an alkylene). Unless otherwise specifically stated herein, heteroalkylene groups may be substituted. Exemplary heteroalkylene groups include ethylene oxide (e.g., polyethylene oxide), as well as the "C", "HEG", and "PEG 1K" linking groups shown below: [ka] Various embodiments of heteroalkylene linkers include multimers of the C linker, HEG linker, and / or PEG 1K linker described above. In some embodiments of the PEG 1K linker, n ranges from 19 to 25, e.g., n is 19, 20, 21, 22, 23, 24, or 25. Multimers can have, for example, the following structure:

[0015] [ka] (wherein x is 0 or an integer greater than 0, for example, x is in the range of 0 to 100 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10)).

[0016] "Linker" refers to a continuous chain of at least one atom, such as carbon, oxygen, nitrogen, sulfur, phosphorus, and combinations thereof, that connects one portion of a molecule to another portion of the same molecule, or to a different molecule, moiety, or solid support (e.g., a microparticle). The linker can be attached to the molecule through covalent bonds or other means, such as ionic or hydrogen bonding interactions. In some embodiments, the linker is a heteroatom linker (e.g., containing 1-10 Si, N, O, P, or S atoms), a heteroalkylene (e.g., containing 1-10 Si, N, O, P, or S atoms and an alkylene chain), or an alkylene linker (e.g., containing 1-12 carbon atoms). In some embodiments, the heteroalkylene linker has the following structure:

[0017] [ka] (In the formula, x 9 and x 10are each independently an integer greater than 0. In some embodiments, the heteroatom linker is -O-, -S-, or -OP(=O)O - In some embodiments, the heteroalkylene linker is -OP(=O)O - In some embodiments, the heteroalkylene linker comprises at least one S-S bond.

[0018] A "physiologically cleavable linker" refers to a molecular linking group that can be cleaved or separated in a defined manner to yield two or more separate molecules while present in the in vivo or in vitro environment of an organism or cellular system. In general, physiological conditions that include such cleavage or scission events can include temperatures ranging from about 20 to 40° C., atmospheric pressure of about 1 atm (101 kPa or 14.7 psi), pH of about 6 to 8, glucose concentration of about 1 to 20 mM, atmospheric oxygen concentration, and gravity of the Earth. In some embodiments, physiological conditions include enzymatic conditions (i.e., cleavage by an enzyme). Bond cleavage or scission can be homogeneous or heterogeneous. "Heteroalkenylene" is a heteroalkylene, as defined above, containing at least one carbon-carbon double bond. Unless stated otherwise specifically in the specification, a heteroalkenylene group may be optionally substituted. "Heteroalkynylene" is a heteroalkylene containing at least one carbon-carbon triple bond. Unless stated otherwise specifically in the specification, a heteroalkynylene group may be optionally substituted.

[0019] "Heteroatom" in reference to a "heteroatom linker" refers to a linker group consisting of one or more heteroatoms. Exemplary heteroatom linkers include linkers having a single atom selected from the group consisting of O, N, P, and S, and multiple heteroatoms, such as linkers having the formula -P(O-)(=O)O- or -OP(O-)(=O)O-, and polymers and combinations thereof. "Phosphate" means -OP(=O)(R a )R b Group(R ais OH, O- or OR c and R b OH, O-, OR c ), a thiophosphate group or a further phosphate group (R c is the counter ion (e.g., Na + (etc.)

[0020] "Phosphoalkyl" means -OP(=O)(R a )R b R refers to the group a is OH, O- or OR c and R b is -Oalkyl, and R c is the counter ion (e.g., Na + Unless specifically stated otherwise in the specification, a phosphoalkyl group may be optionally substituted. For example, in certain embodiments, the -Oalkyl portion in a phosphoalkyl group is hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether, thiophosphoalkylether, or -OP(=R a )(R b )R c and R a , R b and R c are each as defined for compounds of structure (I).

[0021] "Phosphoalkyl ether" means -OP(=O)(R a )R b R refers to the group a is OH, O- or OR c and R b is -O alkyl ether, R c is the counter ion (e.g., Na +Unless specifically stated otherwise herein, a phosphoalkyl ether group may be substituted. For example, in certain embodiments, the -O alkyl ether portion of a phosphoalkyl ether group is hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether, or -OP(=R a )(R b )R c and R a , R b and R c are each as defined for compounds of structure (I). "Thiophosphate" is -OP(=R a )(R b )R c Group(R a is O or S, R b OH, O-, S-, OR d or SR d and R c OH, SH, O-, S-, OR d , S.R. d , a phosphate group or a further thiophosphate group, R d is the counter ion (e.g., Na + etc.), where i) R a is S, and ii) R b is S- or SR d and iii) R c is SH, S- or SR d or iv) any combination of i), ii) and / or iii).

[0022] "Thiophosphoalkyl" means -OP(=R a )(R b )R c R refers to the group a is O or S, R b OH, O-, S-, OR d or SR d and R c is -Oalkyl, and Rd is the counter ion (e.g., Na + etc.), where i) R a is S, and ii) R b is S- or SR d or iii) R a is S and R b is S- or SR d Unless stated otherwise specifically in the specification, a thiophosphoalkyl group may be optionally substituted. For example, in certain embodiments, the -Oalkyl portion in a thiophosphoalkyl group is hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkylether, thiophosphoalkylether, or -OP(=R a )(R b )R c and R a , R b and R c are each as defined for compounds of structure (I).

[0023] "Thiophosphoalkyl ether" means -OP(=R a )(R b )R c R refers to the group a is O or S, R b OH, O-, S-, OR d or SR d and R c is -O alkyl ether, R d is the counter ion (e.g., Na + etc.), where i) R a is S, and ii) R b is S- or SR d or iii) R a is S and R b is S- or SR dUnless otherwise specifically stated herein, a thiophosphoalkyl ether group may be optionally substituted. For example, in certain embodiments, the -O alkyl ether portion of a thiophosphoalkyl group may be hydroxyl, amino, sulfhydryl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, thiophosphoalkyl ether, or -OP(=R a )(R b )R c and R a , R b and R c are each as defined for compounds of structure (I).

[0024] "Carbocyclic" refers to a stable 3- to 18-membered aromatic or non-aromatic ring containing 3 to 18 carbon atoms. Unless otherwise specifically stated herein, a carbocyclic ring can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems, and may be partially saturated or fully saturated. Non-aromatic carbocyclyl radicals include cycloalkyl, while aromatic carbocyclyl radicals include aryl. Unless otherwise specifically stated herein, a carbocyclic group may be optionally substituted. "Cycloalkyl" refers to a stable non-aromatic monocyclic or polycyclic carbocyclic ring, which may include saturated or unsaturated fused or bridged ring systems having 3 to 15 carbon atoms, preferably 3 to 10 carbon atoms, attached to the remainder of the molecule by a single bond. Monocyclic cycloalkyls include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyls include, for example, adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo-[2.2.1]heptanyl, and the like. Unless otherwise specifically stated herein, cycloalkyl groups may be substituted.

[0025] "Aryl" refers to a ring system containing at least one carbocyclic aromatic ring. In some embodiments, an aryl contains 6 to 18 carbon atoms. The aryl ring may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems. Aryl includes, but is not limited to, aryl derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, and triphenylene. Unless otherwise specifically stated herein, an aryl group may be optionally substituted.

[0026] "Heterocyclic" refers to a stable 3-18 membered aromatic or non-aromatic ring containing 1-12 carbon atoms and 1-6 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless otherwise specifically stated herein, a heterocyclic ring may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems, the nitrogen, carbon or sulfur atoms in the heterocyclic ring may be oxidized, the nitrogen atoms may be quaternized, and the heterocyclic ring may be partially or fully saturated. Examples of aromatic heterocyclic rings are listed below in the definition of heteroaryl (i.e., heteroaryl is a subset of heterocyclic). Examples of non-aromatic heterocyclic rings include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, pyrazolopyrimidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless stated otherwise specifically in the specification, a heterocyclic group may be optionally substituted.

[0027] "Heteroaryl" refers to a 5-14 membered ring system containing 1-13 carbon atoms, 1-6 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, and at least one aromatic ring. For purposes of certain embodiments of the present disclosure, the heteroaryl radical can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems, and the nitrogen, carbon or sulfur atoms in the heteroaryl radical can be optionally oxidized and the nitrogen atom can be optionally quaternized.Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzthiazolyl, benzoindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzyl, Benzofuranonyl, benzothienyl (benzothiophenyl), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, benzoxazolinonyl, benzimidazolethionyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizyl nyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, pteridinonyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyridinonyl, pyrazinyl, pyrimi Dinyl, pyrimidinonyl, pyridazinyl, pyrrolyl, pyrido[2,3-d]pyrimidinonyl, quinazolinyl, quinazolinonyl, quinoxalinyl, quinoxalinonyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, thieno[3,2-d]pyrimidin-4-onyl, thieno[2,3-d]pyrimidin-4-onyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless stated otherwise specifically in the specification, heteroaryl groups may be optionally substituted.

[0028] The suffix "-ene" refers to a particular structural feature (e.g., alkyl, aryl, heteroalkyl, heteroaryl) that is attached to the rest of the molecule through a single bond and to the radical group through a single bond. In other words, the suffix "-ene" refers to a linker that has the structural feature of the moiety to which it is attached. The points of attachment of the "-ene" chain to the rest of the molecule and to the radical group can be through one atom or any two atoms within the chain. For example, heteroarylene refers to a linker that includes a heteroaryl moiety as defined herein.

[0029] "Fused" refers to a ring system containing at least two rings, which share at least one common ring atom, e.g., two common ring atoms. When the fused ring is a heterocyclyl or heteroaryl ring, the common ring atom can be carbon or nitrogen. Fused rings include bicyclic, tricyclic, tertracyclic, etc.

[0030] As used herein, the term "substituted" refers to any of the above groups (e.g., alkyl, alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, alkoxy, alkyl ether, phosphoalkyl, phosphoalkyl ether, thiophosphoalkyl, thiophosphoalkyl ether, carbocyclic, cycloalkyl, aryl, heterocyclic, and / or heteroaryl) in which at least one hydrogen atom (e.g., one, two, three, or all of the hydrogen atoms) has been replaced with a halogen atom, such as, but not limited to, F, Cl, Br, and I; The term "substituted" also refers to any of the above groups in which one or more hydrogen atoms are replaced by a bond to a non-hydrogen atom, such as an oxygen atom in groups such as hydroxyl, alkoxy, and ester groups; a sulfur atom in groups such as thiol, thioalkyl, sulfone, sulfonyl, and sulfoxide groups; a nitrogen atom in groups such as amine, amide, alkylamine, dialkylamine, arylamine, alkylarylamine, diarylamine, N-oxide, imide, and enamine; a silicon atom in groups such as trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also refers to any of the above groups in which one or more hydrogen atoms are replaced by a higher bond (e.g., a double bond or a triple bond) to a heteroatom, such as an oxygen in groups such as oxo, carbonyl, carboxyl, and ester groups, and a nitrogen in groups such as imine, oxime, hydrazone, and nitrile. For example, "substituted" includes any of the above groups in which one or more hydrogen atoms are replaced by a bond to a non-hydrogen atom, such as an oxygen in groups such as -NR g R h , -NR g C(=O)R h , -NR g C(=O)NR g R h , -NR g C(=O)OR h , -NR g SO2R h , -OC(=O)NR g R h , -OR g , -SR g , -SOR g , -SO2R g , -OSO2Rg , -SO2OR g , =NSO2R g and -SO2NR g R h "Substituted" also includes any of the above groups replaced by one or more hydrogen atoms, such as -C(=O)R. g , -C(=O)OR g , -C(=O)NR g R h , -CH2SO2R g , -CH2SO2NR g R h In the above, R g and R h are the same or different and are independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. "Substituted" further refers to any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, cyano, hydroxyl, imino, nitro, oxo, thioxo, halo, alkyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl group. Additionally, any of the above-mentioned substituents may also be substituted with one or more of the above-mentioned substituents.

[0031] "Conjugation" refers to the overlap of one p orbital with another p orbital across an intervening sigma bond. Conjugation can occur in cyclic or acyclic compounds. "Degree of conjugation" refers to the overlap of at least one p orbital with another p orbital across an intervening sigma bond. For example, 1,3-butadiene has one degree of conjugation, while benzene and other aromatic compounds usually have multiple degrees of conjugation. Fluorescent and chromogenic compounds usually contain at least one degree of conjugation.

[0032] "Fluorescent" refers to a molecule that can absorb light of a particular frequency and emit light of a different frequency. Fluorescence is well known to those of skill in the art. "Colored" refers to molecules that absorb light within the color spectrum (ie, red, yellow, blue, etc.).

[0033] The term "biomolecule" refers to any of a variety of biological substances, including nucleic acids, carbohydrates, amino acids, polypeptides, glycoproteins, hormones, aptamers, and mixtures thereof. More specifically, the term is intended to include, without limitation, RNA, DNA, oligonucleotides, modified or derivatized nucleotides, enzymes, receptors, prions, receptor ligands (including hormones), antibodies, antigens, and toxins, as well as bacteria, viruses, blood cells, and tissue cells. The visually detectable biomolecules of the present disclosure (e.g., compounds of structure (I) having a biomolecule linked thereto) are prepared by contacting a biomolecule with a compound as described above having a reactive group that allows for the binding of the biomolecule to the compound through any available atom or functional group, such as an amino, hydroxyl, carboxyl, or sulfhydryl group, on the biomolecule, as further described herein.

[0034] A "reactive group" is a moiety that can react with a second reactive group (e.g., a "complementary reactive group") to form one or more covalent bonds, for example, by substitution, oxidation, reduction, addition, or cycloaddition reactions. Exemplary reactive groups are provided in Table 1 and include, for example, nucleophiles, electrophiles, dienes, dienophiles, aldehydes, oximes, hydrazones, alkynes, amines, azides, acyl azides, acyl halides, nitriles, nitrones, sulfhydryls, disulfides, sulfonyl halides, isothiocyanates, imidoesters, activated esters, ketones, α,β-unsaturated carbonyls, alkenes, maleimides, α-haloimides, epoxides, aziridines, tetrazines, tetrazoles, phosphines, biotins, thiiranes, and the like.

[0035] "Bioconjugation" or "bioconjugate" and related variations refer to a chemical reaction strategy that forms a stable covalent bond between two molecules. The term "bioconjugation" is commonly used when one of the molecules is a biomolecule (e.g., an antibody), but can be used to describe forming a covalent bond with a non-biomolecule (e.g., a polymeric resin). The product or compound resulting from such a reaction strategy is a "conjugate," "bioconjugate," or grammatical equivalent.

[0036] The terms "visible" and "visually detectable" are used herein to refer to substances observable by visual inspection without prior illumination or chemical or enzymatic activation. Such visually detectable substances absorb and emit light in the spectral region ranging from about 300 to about 900 nm. Preferably, such substances are strongly colored and have a chromaticity of preferably at least about 40,000, more preferably at least about 50,000, even more preferably at least about 60,000, even more preferably at least about 70,000, and most preferably at least about 80,000 M. -1 cm -1The compounds of the present disclosure can be detected by naked eye observation or by using optical-based detection devices, including, but not limited to, absorption spectrophotometers, transmitted light microscopes, digital cameras, and scanners. Visually detectable substances are not limited to those that emit and / or absorb light in the visible spectrum. Substances that emit and / or absorb light in the ultraviolet (UV) region (about 10 nm to about 400 nm), infrared (IR) region (about 700 nm to about 1 mm), and substances that emit and / or absorb in other regions of the electromagnetic spectrum are also included within the scope of "visually detectable" substances.

[0037] For the purposes of the present disclosure, the term "photostable visible dye" refers to a chemical moiety that is visually detectable and does not significantly change or decompose when exposed to light, as defined herein above.Preferably, the photostable visible dye does not show significant decolorization or decomposition after exposure to light for at least 1 hour.More preferably, the visible dye is stable after exposure to light for at least 12 hours, even more preferably at least 24 hours, even more preferably at least 1 week, and most preferably at least 1 month.Non-limiting examples of photostable visible dyes suitable for use in the compounds and methods of the present disclosure include azo dyes, thioindigo dyes, quinacridone pigments, dioxazines, phthalocyanines, perinones, diketopyrrolopyrroles, quinophthalones, and triarylcarboniums.

[0038] As used herein, the term "perylene derivative" is intended to include any substituted perylene that is visually detectable. However, the term is not intended to include perylene itself. The terms "anthracene derivative", "naphthalene derivative", and "pyrene derivative" are used analogously. In some preferred embodiments, the derivative (e.g., a perylene, pyrene, anthracene, or naphthalene derivative) is an imide, bisimide, or hydrazamimide derivative of perylene, anthracene, naphthalene, or pyrene.

[0039] The visually detectable molecules of various embodiments of the present disclosure are useful in a wide variety of analytical applications, such as biochemical and biomedical applications, where it is necessary to determine the presence, location, or amount of a particular analyte (e.g., a biomolecule). Thus, in another aspect, the present disclosure provides a method for visually detecting a biomolecule, comprising: (a) providing a biological system with a visually detectable biomolecule comprising a compound of structure (I) linked to the biomolecule; and (b) detecting the biomolecule by its visual characteristics. For the purposes of this disclosure, the phrase "detecting a biomolecule by its visual characteristics" means that the biomolecule is observed by the naked eye, or with an optical-based detection device, including, but not limited to, an absorption spectrophotometer, a transmitted light microscope, a digital camera, and a scanner, without illumination or chemical or enzymatic activation. A densitometer can be used to quantify the amount of visually detectable biomolecule present. For example, the relative amounts of biomolecules in two samples can be determined by measuring the relative optical density. If the stoichiometry of dye molecules per biomolecule is known and the extinction coefficient of the dye molecule is known, the absolute concentration of the biomolecule can also be determined from the optical density measurement. As used herein, the term "biological system" is used to refer to any solution or mixture that contains one or more biomolecules in addition to visually detectable biomolecules. Non-limiting examples of such biological systems include cells, cell extracts, tissue samples, electrophoretic gels, assay mixtures, and hybridization reaction mixtures.

[0040] "Solid support" or "solid resin" refers to any solid substrate known in the art for stationary phase support of molecules, for example, "microparticle" refers to any of a number of small particles useful for binding to compounds of the present disclosure, including, but not limited to, glass beads, magnetic beads, polymeric beads, non-polymeric beads, etc. In certain embodiments, the microparticle comprises a polystyrene bead. In some embodiments, the solid support or solid resin is controlled pore glass or macroporous polystyrene. "Solid support moiety" refers to a functional group that remains attached to a molecule once the molecule is cleaved from the solid support. Solid support moieties are known in the art and can be readily derivatized based on the structure of the solid support and the group linking the molecule to the solid support.

[0041] A "targeting moiety" is a moiety that selectively binds to or associates with a particular target, such as an analyte molecule. By "selectively" binds or associates, it is meant that the targeting moiety preferentially associates with or binds to the desired target over other targets. In some embodiments, the compounds disclosed herein include a linking group to the targeting moiety for the purpose of selectively binding or associating the compound with the analyte (i.e., the target of the targeting moiety), thereby allowing for detection of the analyte. Exemplary targeting moieties include, but are not limited to, antibodies, antigens, nucleic acid sequences, enzymes, proteins, cell surface receptor agonists, and the like. In some embodiments, the targeting moiety is a moiety, such as an antibody, that selectively binds to or associates with a target feature on or in a cell, such as a target feature on the cell membrane or other cellular structure surface, thereby allowing for detection of the cell of interest. Small molecules that selectively bind to or associate with the desired analyte are also contemplated as targeting moieties in certain embodiments. Those skilled in the art will recognize other analytes and corresponding targeting moieties that are useful in various embodiments. "Base pairing moiety" refers to a heterocyclic moiety capable of hybridizing with a complementary heterocyclic moiety through hydrogen bonding (e.g., Watson-Crick base pairing). Base pairing moieties include natural and unnatural bases. Non-limiting examples of base pairing moieties are RNA and DNA bases such as adenosine, guanosine, thymidine, cytosine and uridine, and analogs thereof.

[0042] The embodiments of the present disclosure disclosed herein are also meant to encompass all compounds that are isotopically labeled by replacing one or more atoms with an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compounds include, respectively:2 H, 3 H, 11 C. 13 C. 14 C. 13 N, 15 N, 15 O. 17 O. 18 O. 31 P, 32 P, 35 S, 18 F, 36 Cl, 123 I and 125 Includes isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as I. Isotopically labeled compounds of structure (I) may generally be prepared by conventional techniques known to those of skill in the art, or by methods analogous to those described below and in the Examples below, substituting appropriate isotopically labeled reagents in place of the non-labeled reagents used previously. "Stable compound" and "stable structure" are intended to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.

[0043] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when said event or circumstance occurs as well as instances when it does not occur. For example, "optionally substituted alkyl" means that the alkyl group may be substituted or unsubstituted, and that the description includes both substituted and unsubstituted alkyl groups. "Salt" includes both acid addition salts and base addition salts.

[0044] "Acid addition salts" include salts of inorganic acids, such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and salts of acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, schizoic acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, mucous acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, and the like. It refers to salts formed with organic acids such as 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid.

[0045] "Base addition salt" refers to a salt prepared from the addition of an inorganic or organic base to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

[0046] Crystallization may produce solvates of the compounds described herein. The embodiments of the present disclosure include all solvates of the compounds described. As used herein, the term "solvate" refers to an aggregate that includes one or more molecules of the compounds of the present disclosure and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may be an organic solvent. Thus, the compounds of the present disclosure may exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates, etc., as well as corresponding solvate forms. The compounds of the present disclosure may be true solvates, while in other cases, the compounds of the present disclosure may simply retain incidental water or another solvent, or may be a mixture of water and some incidental solvent.

[0047] The disclosed compounds (e.g., compounds of structure I), or salts, tautomers, or solvates thereof, may contain one or more stereocenters and may therefore give rise to enantiomers, diastereomers, and other stereoisomers that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, or, in the case of amino acids, (D)- or (L)-. The disclosed embodiments are intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from suitable optically pure precursors, or resolution of the racemates (or racemates of salts or derivatives) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise specified, the compounds are intended to include both E and Z geometric isomers, as well as all tautomers are also intended to be included.

[0048] "Stereoisomers" refers to compounds composed of the same atoms joined by the same bonds but having different three-dimensional structures and which cannot be interconverted. The present disclosure contemplates various stereoisomers and mixtures thereof, and includes "enantiomers," which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another. "Tautomer" refers to a proton transfer from one atom of a molecule to another atom of the same molecule. The present disclosure includes tautomers of any of the above compounds. Various tautomers of the compounds can be easily derived by one skilled in the art. The chemical naming protocols and structure diagrams used herein are a modification of the IUPAC nomenclature system using the ACD / Nomenclature version 9.07 software program and / or the ChemDraw Ultra version 11.0 software naming program (CambridgeSoft). Common names familiar to those skilled in the art are also used.

[0049] compound As noted above, in one embodiment of the disclosure, compounds are provided that are useful as covalent linkers between biologically active moieties, such as alkylating agents, and targeting moieties. In other embodiments, compounds are provided that are useful as synthetic intermediates for the preparation of compounds that include one or more biologically active moieties. Thus, in some embodiments, M 1 and M 2 is, independently at each occurrence, a moiety that comprises an alkylating agent or a fluorescent dye, provided that each occurrence of M 1 Or M 2 At least one of M is not a fluorescent dye. 1 Or M 2 is an alkylating agent (e.g., pyrrolobenzodiazepine (PBD) etc.).

[0050] A polymer backbone and then a biologically active moiety M attached to any targeting moiety. 1 and M 2 the number of biologically active moieties, the spacing between adjacent biologically active moieties on the polymer backbone (e.g., the number of biologically active moieties M 1 and M. 2Numerous advantages are provided by the embodiments disclosed herein, including the ability to control the spacing between the polymer backbone and the biologically active moiety (e.g., how far apart or close each of the linkers are) and the distance between the polymer backbone and the biologically active moiety (e.g., the length of the linker away from the polymer backbone). This allows for the construction of compounds that contain biologically active moieties that promote alkylation of guanine (G) in DNA such that the biologically active moieties attached to the polymer backbone are positioned at low energy positions in the minor groove of DNA. Compounds disclosed in this disclosure have multiple alkylating agents as biologically active moieties, allowing for the formation of interstrand and / or intrastrand DNA crosslinks, resulting in greater stabilization of DNA.

[0051] The biologically active moiety can be attached to the polymer backbone via a physiologically cleavable or non-cleavable linker. The procedures described in this disclosure provide the ability to selectively introduce a physiologically cleavable linker and / or a non-cleavable linker. This allows the synthesis of compounds with both physiologically cleavable and non-cleavable linkers with one or more biologically active moieties. In this regard, the biologically active moieties can be sequentially cleaved in response to physiological conditions. Furthermore, compounds can be synthesized with both biologically active moieties and fluorescent moieties attached by physiologically cleavable and / or non-cleavable linkers.

[0052] Some embodiments of the present disclosure provide combinations of therapeutic agents, targeting moieties and dye moieties (e.g., chromophores or fluorophores) that can be used for simultaneous targeting, treatment and detection. The ease of coupling the polymer-drug constructs to targeting agents such as antibodies, antibody fragments, proteins or other agents of clinical interest makes them useful for a wide range of applications of interest (e.g., surface chemistry, assay development, etc.). Thus, in some embodiments, M is a chromophore or fluorophore (e.g., FITC, 5-FAM, 6-FAM, etc.).

[0053] The compounds of certain embodiments also achieve other desirable properties, including enhanced permeability and retention. In addition to achieving the necessary solubility, the chemical characteristics of the compound embodiments can be adjusted to modulate the ability of the compounds to penetrate diseased cells / tissues and to be retained within them. These characteristics allow for effective delivery of bioactive agents through enhanced permeability, and increased efficacy through enhanced retention. Thus, it is understood that any embodiment of the compounds of structure (I), (II), or (III) above may be independently combined with other embodiments to form embodiments of the present disclosure not specifically described above. It is understood that in this description, combinations of substituents and / or variables of the depicted formulae are permissible if such contributions result in stable compounds. Thus, in one embodiment, a compound having the following structure (I): [ka] (I) or a stereoisomer, pharmaceutical salt, or tautomer thereof. (In the formula, M 1 is, independently at each occurrence, a moiety that contains a pyrrolobenzodiazepine, a minor groove binder, or a fluorescent dye that is not present, except that each occurrence of M 1 provided that at least one of the following is a pyrrolobenzodiazepine: M 2 is, independently at each occurrence, a moiety that comprises a pyrrolobenzodiazepine, a minor groove binder, or a fluorescent dye; L 1a is, independently at each occurrence, a heteroarylene linker; L 1b is, independently for each occurrence, H when M is absent; or 1 is an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker when is a pyrrolobenzodiazepine or a fluorescent dye; L 2 , L3 , L 5 , L 6 and L 7 is independently at each occurrence an optional alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene; L 4 is independently at each occurrence an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene linker; R 1 and R 2 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a )(R b )R c , Q or a protected form thereof, L′, or a minor groove binder; R 3 is independently at each occurrence H, alkyl or alkoxy; R 4 is independently O-, S-, OR d , or S.R. d and R 5 is independently at each occurrence oxo, thioxo, or absent; R a is O or S, R b OH, SH, O-, S-, OR d or SR d and R c OH, SH, O-, S-, OR d , O.L.', S.R. d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counterion, Q is, independently at each occurrence, a moiety that contains a reactive group, or a protected form thereof, capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q'; L' is, independently at each occurrence, a linker that includes a covalent bond to Q, a linker that includes a covalent bond to a targeting moiety, a linker that includes a covalent bond to an analyte molecule, a linker that includes a covalent bond to a solid support, a linker that includes a covalent bond to a solid support moiety, a linker that includes a covalent bond to a nucleoside, or a linker that includes a covalent bond to an additional compound of structure (I); m, for each occurrence, is an integer greater than or equal to zero, n is an integer equal to or greater than 1, q and w are, independently at each occurrence, 0 or 1, provided that at least one of q or w is 1 in one occurrence.

[0054] Various linkers and substituents in the compounds of structure (I) (e.g., M 1 , M 2 , Q, R 1 , R 2 , R 3 , R c , L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 and L 7) may be substituted with another substituent. For example, in some embodiments, the optional substituents are selected to optimize the water solubility or other properties of the compounds of structure (I). In certain embodiments, each chromophore, alkyl, alkoxy, alkyl ether, heteroarylene, heteroalkyl, alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, alkoxyalkyl ether, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, and thiophosphoalkyl ether in the compounds of structure (I) may be substituted with another substituent selected from the group consisting of hydroxyl, alkoxy, alkyl ether, alkoxyalkyl ether, sulfhydryl, amino, alkylamino, carboxyl, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, and thiophosphoalkyl ether. In certain embodiments, the optional substituent is -OP(=R a )(R b )R c where R a , R b and R c is as defined for compounds of structure (I).

[0055] In some embodiments, the occurring L 1a At least one of L is an optionally substituted 5- to 9-membered heteroarylene linker. 1a is a substituted 5-membered heteroarylene linker. In certain embodiments, L 1a is a substituted 6-membered heteroarylene linker. In certain embodiments, L 1a is a substituted 7-membered heteroarylene linker. In certain embodiments, L 1a is a substituted 8-membered heteroarylene linker. In certain embodiments, L 1a is a substituted 9-membered heteroarylene linker. In some related embodiments, L 1a is substituted with oxo, alkyl (e.g., methyl, ethyl, etc.), or combinations thereof. In certain embodiments, L 1aEach occurrence of L is unsubstituted. 1a is, independently at each occurrence, a pyrimidine. 1a is, independently at each occurrence, cytosine or thymine. 1a are independently selected from cytosine and thymine at each occurrence such that the compound comprises a sequence of cytosine and thymine bases capable of forming a triplex with the target DNA sequence. 1a has the following structure:

[0056] [ka] has.

[0057] In some embodiments, L 1b (L is L is) is an alkylene linker. In certain embodiments, L 1b The alkylene linker has an odd number of carbon atoms. 1b The alkylene linker in L is a C alkyl linker. 1b The alkylene linker in L is a C alkyl linker. 1b The alkylene linker of L has an even number of carbon atoms. 1b The alkylene linker in L is a C2 alkyl linker. 1b The alkylene linker in L is a C4 alkyl linker. 1bis independently at each occurrence optionally alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, alkyleneheteroarylenealkylene, alkyleneheterocyclylenealkylene, alkylenecarbocyclylenealkylene, heteroalkyleneheteroarylenealkylene, heteroalkyleneheterocyclylenealkylene, heteroalkylenecarbocyclylenealkylene, heteroalkyleneheteroaryleneheteroalkylene, heteroalkyleneheterocyclyleneheteroalkylene, heteroalkylenecarbocyclyleneheteroalkylene , alkyleneheteroaryleneheteroalkylene, alkyleneheterocyclyleneheteroalkylene, alkylenecarbocyclyleneheteroalkylene, heteroarylene, heterocyclylene, carbocyclylene, alkyleneheteroarylene, alkyleneheterocyclylene, heteroarylenealkylene, alkylenecarbocyclylene, carbocyclylenealkylene, heteroalkyleneheteroarylene, heteroalkyleneheterocyclylene, heteroaryleneheteroalkylene, heteroalkylenecarbocyclylene, carbocyclyleneheteroalkylene, or a heteroatom linker. 1b is an optionally substituted heteroalkenylene linker.

[0058] In some embodiments, the occurring L 1b At least one of L is substituted. 1b is replaced at each occurrence. In some more specific embodiments, L 1b is substituted with oxo.

[0059] Linker L 1a , L 1b , and L 7 is the M to the remainder of the compound 1 and M 2 For example, in some embodiments, a synthetic precursor of a compound of structure (I) is prepared, and M 1 and M 2The moiety is attached to the synthetic precursor using any number of coupling methods known in the art. 1b Or L 7 For at least one of the occurrences of L, the functional group comprises an alkene, an ester, an amide, a thioester, a disulfide, a carbocyclic group, a heterocyclic group, or a heteroaryl group. 1b Or L 7 For at least one of, the functional group comprises an alkene, an ester, an amide, a thioester, a thiourea, a disulfide, a carbocyclic group, a heterocyclic group, or a heteroaryl group. In other embodiments, the functional group comprises an amide or a thiourea.

[0060] In a more specific embodiment, the occurring L 7 At least one of the following structures: [ka] Contains one of the following:

[0061] In some embodiments, L 1b has the following structure: [ka] Contains one of the following: In some embodiments, the occurring L 2 In some more specific embodiments, at least one of L 2 In some specific embodiments, L is not present in each occurrence. 2 At least one of the occurrences of L is heteroalkylene. 2 At least one of the occurrences of L 2 At least one of the following structures:

[0062] [ka] has. (In the formula, x9 and x 10 are each independently an integer greater than 0)

[0063] In some embodiments, x 9 is 1, 2, 3, or 4. In certain embodiments, x 10 is 2, 3, 4, or 5. In some specific embodiments, x 9 is 1 or 2, and x 10 is 2, 3, or 4. In certain specific embodiments, L 2 Each occurrence of is heteroalkylene. In some more specific embodiments, L 2 Each occurrence of L includes oxygen. In certain more specific embodiments, L 2 For each occurrence, the following structure:

[0064] [ka] has. (In the formula, x 9 and x 10 are each independently an integer greater than 0)

[0065] In some embodiments, x 9 is 1, 2, 3, or 4. In certain embodiments, x 10 is 2, 3, 4, or 5. In more specific embodiments, x 9 is 1 or 2, and x 10 is 2, 3, or 4. In certain other embodiments, the L 2 At least one of the following structures:

[0066] [ka] Includes. (In the formula, x 9 and x 10 are each independently an integer greater than 0)

[0067] In certain embodiments, L 2 In some specific embodiments, the occurring L 2 At least one of the occurring L comprises an amide bond, an ester bond, a phosphodiester bond, a disulfide bond, a double bond, a triple bond, an ether bond, a hydrazone, an amino acid sequence comprising one or more amino acid residues, a ketone, a diol, a cyano, a nitro, or a combination thereof. 2 At least one of the occurring Ls comprises an amino acid sequence recognized by a sortase enzyme or a cysteine ​​protease. In certain embodiments, the amino acid sequence is Leu-Pro-X-Thr-Gly, where X is any amino acid residue. In more specific embodiments, the occurring Ls 2 At least one of the following structures:

[0068] [ka] Contains one of the following:

[0069] In certain embodiments, L 2 Each occurrence of L includes an amide bond, an ester bond, a phosphodiester bond, a disulfide bond, a double bond, a triple bond, an ether bond, a hydrazone, an amino acid sequence, a ketone, a diol, a cyano, a nitro, or a combination thereof. 2 For each occurrence, the following structure:

[0070] [ka] Contains one of the following:

[0071] In some more specific embodiments, the occurring L 2 In certain specific embodiments, at least one of the occurring L 2 At least one of the occurring L2 At least one of the following structures:

[0072] [ka] Contains one of the following:

[0073] In some embodiments, L 2 Each occurrence of L contains one or more amino acid residues. 2 Each occurrence of L comprises one or more amino acid residues selected from the group consisting of alanine, valine, and combinations thereof. 2 For each occurrence, the following structure: [ka] Contains one of the following:

[0074] In a more specific embodiment, the occurring L 2 At least one of the following structures: [ka] has one of the following:

[0075] In some specific embodiments, L 2 For each occurrence, the following structure: [ka] has one of the following: In some embodiments, the occurring L 3 At least one of L is an alkylene linker. 3 is an alkylene linker at each occurrence. In certain embodiments, the alkylene linker is a methylene linker.

[0076] In some embodiments, the occurring L 4 In some embodiments, at least one of the occurrences of L 5 Or L6 At least one of the occurrences of L is heteroalkylene. 5 Or L 6 At least one of L comprises an alkylene oxide. 5 Or L 6 The alkylene oxide in is ethylene oxide. In some more specific embodiments, the ethylene oxide is polyethylene oxide. In certain embodiments, the alkylene oxide in 5 Or L 6 At least one of L is an alkylene linker (e.g., methylene). 5 Or L 6 Each occurrence of L is an alkylene linker (e.g., methylene). 5 At least one of L is a heteroalkylene linker. 5 Each occurrence of L is a heteroalkylene linker. 5 At least one of the occurrences of L 5 At least one of L is an alkylene linker (e.g., methylene). 5 Each occurrence of L is an alkylene linker (e.g., methylene). 5 In some more specific embodiments, at least one of L 5 does not exist for every occurrence.

[0077] In certain embodiments, the occurring L 6 At least one of L is a heteroalkylene linker. 6 Each occurrence of L is a heteroalkylene linker. 6At least one of the L's comprises an alkylene oxide. In some of the foregoing embodiments, the alkylene oxide is ethylene oxide, e.g., polyethylene oxide. In certain embodiments, the L 6 At least one of L is an alkylene linker (e.g., methylene). 6 Each occurrence of L is an alkylene linker (e.g., methylene). 6 In some more specific embodiments, at least one of L 6 does not exist for every occurrence.

[0078] In certain embodiments, the occurring L 5 Or L 6 In a more specific embodiment, at least one of L 5 Or L 6 Each occurrence of L contains a phosphodiester moiety. 2 , L 3 , L 4 Or L 6 is independently at each occurrence C1-C6 alkylene, C2-C6 alkenylene, or C2-C6 alkynylene.

[0079] In some embodiments, the occurring L 5 At least one of L is heteroalkylene. 5 At each occurrence, is heteroalkylene, for example, the following structures: [ka] embedded image is a heteroalkylene, including one of the following:

[0080] In some embodiments, the occurring L 6 At least one of L is heteroalkylene. 6 At each occurrence, is heteroalkylene, for example, the following structures: [ka] Contains one of the following:

[0081] In some of the foregoing embodiments, heteroalkylene (e.g., L 3 , L 4 , L 5 Or L 6 ) has the following structure: [ka] where z is an integer ranging from 19 to 30. In some embodiments, z is in the range of 19 to 28. In certain embodiments, the average z is 23. In some embodiments, the average z is 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28.

[0082] In certain of the foregoing embodiments, the targeting moiety is an antibody or a cell surface receptor antagonist. In other more specific embodiments of any of the foregoing compounds of structure (I), R 1 or R 2 has the following structure: [ka] has one of the following:

[0083] In other more specific embodiments of any of the foregoing compounds of structure (I), R 1 or R 2 has the following structure: [ka] has one of the following:

[0084] In various other embodiments, R 1 and R 2 are each independently OH or -OP(=R a )(Rb )R c In some different embodiments, R 1 or R 2 is OH or -OP(=R a )(R b )R c and R 1 or R 2 The other is a linker that comprises a covalent bond to Q or Q.

[0085] In yet another embodiment of any of the foregoing compounds of structure (I), R 1 and R 2 are each independently -OP(=R a )(R b )R c In some of these embodiments, R c is OL'. In other embodiments, R 1 and R 2 are each independently -OP(=R a )(R b ) OL', where L' is an alkylene or heteroalkylene linker to Q, a targeting moiety, an analyte (e.g., an analyte molecule), a solid support, a solid support moiety, a nucleoside, or an additional compound of structure (I). The linker L' can be any linker suitable for attaching Q, a targeting moiety, an analyte (e.g., an analyte molecule), a solid support, a solid support residue, a nucleoside, or an additional compound of structure (I) to the compound of structure (I). Advantageously, certain embodiments include the use of an L' moiety that is selected to increase or optimize the aqueous solubility of the compound. In certain embodiments, L' is a heteroalkylene moiety. In some other certain embodiments, L' includes an alkylene oxide or a phosphodiester moiety, or a combination thereof.

[0086] In some embodiments, L' is a heteroalkylene linker to Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside, or an additional compound of structure (I). In some more specific embodiments, L' comprises an alkylene oxide or a phosphodiester moiety, or a combination thereof. In certain embodiments, L' has the following structure:

[0087] [ka] has. (In the formula, m″ and n″ are independently an integer from 1 to 10; R e is H, an electron pair or a counterion, L” is R e or Q is a direct bond or linkage to a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside, or a further compound of structure (I).

[0088] Certain embodiments of the compounds of structure (I) can be prepared following solid phase synthesis methods similar to those known in the art for the preparation of oligonucleotides. Thus, in some embodiments, L' is a linkage to a solid support or a solid support residue or a nucleoside. Solid supports containing activated deoxythymidine (dT) groups are readily available and, in some embodiments, can be used as starting materials for the preparation of compounds of structure (I). Thus, in some embodiments, R 1 or R 2 has the following structure:

[0089] [ka] has.

[0090] Those skilled in the art will appreciate that the dT group above is included only for ease of synthesis and economic efficiency, and is not required. Other solid supports can be used, different nucleosides or solid support moieties can be present on L', or the nucleosides or solid support moieties can be removed or modified after synthesis.

[0091] In some embodiments, the targeting moiety is an antibody, a cell surface receptor antagonist, or a cell surface receptor antagonist. In some embodiments, the targeting moiety is a monoclonal antibody. In some more specific embodiments, the monoclonal antibody is selected from the group consisting of abciximab, adalimumab, alemtuzumab, alirocumab, avibactam, basiliximab, benralizumab, bezlotoxumab, blinatumomab, brodalumab, burosumab, canakinumab, caplacizumab, certolizumab pegol, daclizumab, denosumab, dupilumab, eculizumab, emicizumab, erenumab, evolocumab, fremanezumab, galcanezumab, golimumab, guselkumab, ibalizumab, idarucizumab. , infliximab, itolizumab, ixekizumab, lanadelumab, loxivetomab, mepolizumab, natalizumab, obiltoxaximab, ocrelizumab, omalizumab, palivizumab, ranibizumab, raxibacumab, reslizumab, ramucirumab (Rmab), rovelizumab, ruplizumab, sarilumab, secukinumab, tildrakizumab, thiomab, tocilizumab, ustekinumab, vedolizumab, abrilumab, actoxumab, aducanumab, afacevicumab, afelimomab, anif Lorumab, Anrukinzumab (IMA-638), Acelizumab, Atollimumab, Bapineuzumab, BCD-100, Bertilimumab, Besilesomab, Biciromab, Bimagrumab, Bimekizumab, Virutamimab, Bleselumab, Brosozumab, Bococizumab, Brazikumab, Briakinumab, Brolucizumab, Carlumab, Carotuximab, Cedelizumab, Clazakizumab, Clenoliximab, Concizumab, Cosfrobiximab, CR6261, Crenezumab, Crizanlizumab, Clotedumab, De Patuxizumab, mafodotin, dellotuximab biotin, desamizumab, dilidabumab, domagrozumab, dusigitumab, ecromeximab, edovacomab, efalizumab, efungumab, eldelumab, elezanumab, enokizumab, eptinezumab, elizumab, etrolizumab, evinacumab, exbivirumab, fanolesomab, faralimoumab, faricimab, fasinumab, felvizumab, fezakinumab, framvotumab, fretikumab, flotetuzumab, fontolizumab,Foravirumab, Flobocimab, Flanumab, Gantenerumab, Gavilimomab, Gevokizumab, Gimsilumab, Gomiliximab, Goslanemab, Ianalumab, Inclacumab, Inolimomab, Iomab-B, Keliximab, Lampalizumab, Landgrozumab, Ralcaviximab, Lebrikizumab, Lenvervimab, Lerdelimumab, Letolizumab, Ribivirumab, Ligelizumab , roderucizumab, lurizumab pegol, marstacimab, mavrilimumab, metelimumab, mirikizumab, motavizumab, muromonab CD3, nebacumab, nemolizumab, NEOD001, nirsevimab, odulimomab, orendalizumab, olokizumab, OMS721, opicinumab, olticumab, otelixizumab, otilimab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, panobac Mab, pascolizumab, pateclizumab, PDR001, perakizumab, pexelizumab, placumab, prozalizumab, ponezumab, polgabiximab, prasinezumab, priliximab, PRO140, kiruzumab, rafivirumab, ralpancizumab, ranevetomab, ravagalimab, ravulizumab, refanezumab, regavirumab, relatorimab, rinukumab, risankizumab, loredumab, romosozumab , Rontalizumab, SA237, Satralizumab, Sevirumab, SHP647, Sifalimumab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Sonepcizumab, Spartalizumab, Stamulumab, Sulesomab, Sputavumab, Stimulimab, Suvisumab, Subratoxumab, Tadocizumab, Talizumab, Tamtubetomab, Tanezumab, Tefibazumab, Terimomab-alitoxumab (Telimomab aritox), Teneliximab, Teplizumab, Teprotumumab, Tezepelumab, Tiburizumab, Toralizumab, Tralokinumab, Trevoglumab, Tubilumab, Ulocuplumab, Urtoxazumab, Balisakumab, Bepalimomab, Vesencumab, Visilizumab, Bovalilizumab, Zolimomab aritox, Trastuzumab, Gemtuzumab, Brentuximab, Borsetuzumab, Lorvotuzumab, Cantuzumab, Bivatuzumab,or inotuzumab, or vadastuximab.

[0092] In some embodiments, the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof. In some embodiments, the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion. In some embodiments, the targeting moiety is an antibody, or a cell surface receptor antagonist. In some further embodiments, the solid support is a polymeric bead or a non-polymeric bead. In some embodiments, n is an integer from 1 to 100. In some more specific embodiments, n is an integer from 1 to 10. In some embodiments, m is an integer from 7 to 12. In certain more specific embodiments, m is an integer from 3 to 6.

[0093] In some embodiments, the R 3 At least one of R is H. 4 Each occurrence of R is oxo. 5 are independently expressed as OH, O - OR d In still other embodiments of any of the compounds of structure (I), R 5 are independently expressed as OH, O - OR d "OR d " and "SR d " is O bound to a cation - and S - For example, the disodium salt of a phosphate group is: [ka] (In the formula, R d is sodium (Na + ) is) It can be expressed as:

[0094] In other embodiments of any of the compounds of structure (I), each occurrence of R 4At least one of R is oxo. In other embodiments of any of the compounds of structure (I), R 4 Each occurrence of is oxo.

[0095] In some embodiments, the compound has the following structure (Ia): [ka] (Ia) has.

[0096] In a more specific embodiment, the compound has the following structure (Ib): [ka] (Ib) has. (In the formula, L 1b is, independently at each occurrence, a linker which may be alkylene or heteroalkylene.

[0097] In some embodiments, the compound has the following structure (Ic): [ka] has. (wherein z is an integer of 1 to 100).

[0098] In some embodiments, M 1 has the following structure: [ka] has one of the following: (In the formula, R 6 is independently at each occurrence H, CH=CHCONH2, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 7 , R8 , and R 9 are independently H, OH, and OR for each occurrence. f , S.H., S.R. f , NH2, NHR f , N.R. f R g , alkyl, alkoxy, alkyl ether, or heteroalkyl; R 10 is independently at each occurrence a nitrogen protecting group or H; R 11 is independently at each occurrence an oxygen protecting group, an alkyl, or H; R f and R g is independently at each occurrence alkyl, heterocyclyl, or aryl.

[0099] In some embodiments, M 2 , R 1 or R 2 In some more specific embodiments, the minor groove binder has the following structure: [ka] has one of the following: (In the formula, One substitutable position of the minor groove binder is covalently attached to the remainder of the compound via an optional linker.

[0100] In certain embodiments, the compound has the following structure (Id) or (Ie): [ka] has one of the following: (In the formula, R 6 is, independently at each occurrence, alkyl; R 7 and R 9 For each occurrence, H, R 8 are ORed independently for each occurrence. f and R 10is, independently at each occurrence, a nitrogen protecting group; R 11 is, independently at each occurrence, an oxygen protecting group; R f is an alkyl group)

[0101] In some embodiments, the occurring L 3 At least one of L is an alkylene linker. 3 is an alkylene linker at each occurrence. In certain embodiments, the alkylene linker is a methylene linker. In some embodiments, the occurring L 2 In a more specific embodiment, at least one of L 2 does not exist for every occurrence.

[0102] In yet other embodiments, Q, independently at each occurrence, is a moiety that includes a reactive group capable of forming a covalent bond with an analyte molecule or a solid support. In other embodiments, Q, independently at each occurrence, is a moiety that includes a reactive group capable of forming a covalent bond with a complementary reactive group, Q'. For example, in some embodiments, Q' is present in an additional compound of structure (I) (e.g., R 1 or R 2 Positions (Q, Q'), Q and Q' contain complementary reactive groups, such that reaction of a compound of structure (I) with an additional compound of structure (I) results in a covalent dimerization of the compound of structure (I). Multimeric compounds of structure (I) can also be prepared in an analogous manner and are included within the scope of embodiments of the present disclosure. The type of Q group and its attachability to the remainder of the compound of structure (I) are not limited, provided that Q contains a moiety with appropriate reactivity to form the desired bond. In certain embodiments, Q is a moiety (e.g., an amine, azide, or alkyne) that is not susceptible to hydrolysis under aqueous conditions, yet is sufficiently reactive to form a bond with a corresponding group on the analyte molecule or solid support.

[0103] Certain embodiments of the compounds of structure (I) include Q groups commonly used in the field of bioconjugation. For example, in some embodiments, Q includes a nucleophilic reactive group, an electrophilic reactive group, or a cycloaddition reactive group. In some more specific embodiments, Q includes a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functionality. In some embodiments, the activated ester is an N-succinimide ester, an imido ester, or a polyflourophenyl ester. In other embodiments, the alkyne is an alkyl azide or an acyl azide.

[0104] Q groups can be conveniently provided in protected forms that enhance storage stability or other desired properties, and the protecting groups can then be removed at the appropriate time for conjugation, for example, with a targeting moiety or analyte. Thus, Q groups include "protected forms" of reactive groups, including any of the reactive groups listed above and in Table 1 below. A "protected form" of Q refers to a moiety that has a lower reactivity under a given reaction condition relative to Q, but can be converted to Q under conditions that preferably do not decompose or react with other moieties of the compound of structure (I). Those skilled in the art can derive an appropriate protected form of Q based on the particular Q and the desired end-use and storage conditions. For example, if Q is -SH, the protected form of Q includes a disulfide that can be reduced using common known techniques and reagents to reveal an -SH moiety. Exemplary Q moieties are provided in Table I below.

[0105] [Table 1-1] [Table 1-2] [Table 1-3]

[0106] It should be noted that in some embodiments, when Q is -SH, the -SH moiety tends to form a disulfide bond with another sulfhydryl group, for example, of another compound of structure (I). Thus, some embodiments include a compound of structure (I) in the form of a disulfide dimer, where the disulfide bond is derived from the Q group that is -SH.

[0107] Similarly, compounds of structure (I) are included within the scope of certain embodiments, where R 1 and R 2 One or both of R 1 and R 2 One or both of -OP(=R a )(R b )R c and R c is OL', where L' is a linker comprising a covalent bond to a further compound of structure (I). Such compounds can be, for example, those having an "M 1 " and / or "M 2 " moiety (i.e., n=10) and suitable for reaction with a complementary Q' group on a second compound of structure (I). In this manner, any number of "M" moieties, e.g., 100 or more, can be prepared. 1 " and / or "M 2 Compounds of structure (I) having a "" moiety can be prepared without the need to sequentially couple each monomer. An exemplary embodiment of such a compound of structure (I) is the following structure (I'):

[0108] [ka] (I') has. (In the formula, R 1 , R 2 , R 3, R 4 , R 5 , L 1a , L 1b , L 2 , L 3 , L 4 , L 5 , L 6 , L 7 , M 1 , q, m, w, and n are, independently at each occurrence, as defined for compounds of structure (I); L″ is a linker that contains a functional group resulting from reaction of a Q moiety with the corresponding Q′ moiety; α is an integer greater than 1, for example, 1 to 100, or 1 to 10. Compounds of structure (I') can be derived by one of skill in the art, for example, by dimerizing or polymerizing compounds of structure (I) provided herein.

[0109] In other embodiments, the Q moiety is conveniently hidden (e.g., protected) as a disulfide moiety, which can be subsequently reduced to provide an activated Q moiety for conjugation to a desired analyte molecule or targeting moiety. For example, the Q moiety can have the following structure:

[0110] [ka] where R is an optionally substituted alkyl group. For example, in some embodiments, Q can be masked as a disulfide having the following structure: [ka] The disulfide moiety is provided as having the following structure: (wherein n is an integer of 1 to 10).

[0111] In some other embodiments, R 1 or R 2 One of the groups is OH or -OP(=R a )(R b )R c and R 1 or R2 the other is a linker comprising a covalent bond to the analyte molecule or a linker comprising a covalent bond to the solid support. For example, in some embodiments, the analyte molecule is a nucleic acid, an amino acid, or a polymer thereof. In other embodiments, the analyte molecule is an enzyme, a receptor, a receptor ligand, an antibody, a glycoprotein, an aptamer, or a prion. In some embodiments, the targeting moiety is an antibody, or a cell surface receptor antagonist. In yet different embodiments, the solid support is a polymeric bead or a non-polymeric bead.

[0112] The fluorescence intensity or efficacy for DNA alkylation can also be adjusted by selecting different values ​​of n. In certain embodiments, n is an integer between 1 and 100. In other embodiments, n is an integer between 1 and 10. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, n is 7. In some embodiments, n is 8. In some embodiments, n is 9. In some embodiments, n is 10.

[0113] The fluorescence or effectiveness for DNA alkylation can also be adjusted by selecting the value of m. The value of m includes adjacent M 1 Or M 2 In certain embodiments, m is an integer between 1 and 100. In other embodiments, m is an integer between 7 and 12. In some embodiments, m is an integer between 20 and 26. In some embodiments, m is an integer between 3 and 6. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. In some embodiments, m is 7. In some embodiments, m is 8. In some embodiments, m is 9. In some embodiments, m is 10. In some embodiments, m is 11.

[0114] In certain embodiments, the occurring M 1 Or M 2 At least one of the occurring M is a nitrogen mustard, a nitrosourea, a tetrazine, an aziridine, cisplatin or a cisplatin derivative, or a non-classical alkylating agent. 1 Or M 2 At least one of the following structures:

[0115] [ka] has. (In the formula, R 4 " is alkoxy, haloalkyl, alkyl, optionally substituted aryl or optionally substituted aralkyl, R 6 is independently at each occurrence H, CH=CHCONH2, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 7 , R 8 , and R 9 are independently H, OH, and OR for each occurrence. f , S.H., S.R. f , NH2, NHR f , N.R. f R g , alkyl, alkoxy, alkyl ether, or heteroalkyl; R 10 is independently at each occurrence a nitrogen protecting group or H; R 11 is independently at each occurrence an oxygen protecting group, an alkyl, or H; R f and R g is independently at each occurrence alkyl, heterocyclyl, or aryl. In certain embodiments, the occurring M 1 Or M 2 At least one of the following structures:

[0116] [ka] has one of the following:

[0117] M 1 and M 2 is selected based on the desired alkylation properties. 1 and M 2 is the same for each occurrence; however, M 1 and M 2 Each occurrence of M 1 and M 2 In certain embodiments, M 1 and M 2 It is important to note that compounds in which each occurrence of M is not the same are included. For example, in some embodiments, 1 and M 2 are not the same, and different M 1 and M 2 The portion is selected. 1 and M 2 The moiety can be appropriately selected by one of skill in the art based on the desired end use. 1 and M 2 Moieties include pyrrolobenzodiazepines (PBDs).

[0118] Furthermore, in some embodiments, the alkylating agent is protected by a protecting group such as an allyloxycarbonyl group (-Alloc) and a tert-butyldimethylsilyl ether (-TBS) to survive the DNA synthesis cycle. The Alloc protecting group is easily cleaved with a palladium catalyst, e.g., Pd(PPh3)4, using PhSiH3 to give the corresponding amine. The TBS protecting group is cleaved with a fluoride source, such as tetra-n-butylammonium fluoride (TBAF), to give the corresponding alcohol. Once deprotection is complete, the PBD moiety is activated with alkylation capability. In this regard, the PBD moiety is protected with a protecting group until the DNA synthesis cycle is completed. Deprotection then allows the conversion of the protected PBD moiety, which is inactive in alkylation, to a deprotected PBD moiety, which is active in alkylation.

[0119] In some embodiments, the occurring M 1 and M 2At least one of the moieties is an alkylating agent, an antimetabolite, a microtubule inhibitor, a topoisomerase inhibitor, or a cytotoxic antibiotic. In more specific embodiments, each occurrence of M is an alkylating agent, an antimetabolite, a microtubule inhibitor, a topoisomerase inhibitor, or a cytotoxic antibiotic. In some embodiments, at least one occurrence of M is an alkylating agent, an antimetabolite, a microtubule inhibitor, or a topoisomerase inhibitor. In more specific embodiments, each occurrence of M is an alkylating agent, an antimetabolite, a microtubule inhibitor, or a topoisomerase inhibitor. In certain embodiments, at least one occurrence of M is a nitrogen mustard, a nitrosourea, a tetrazine, an aziridine, cisplatin or a cisplatin derivative, or a non-classical alkylating agent. In more specific embodiments, at least one of the occurring M's is mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mytomycin, diaziquone (AZQ), cisplatin, carboplatin, oxaliplatin, procarbazine, or hexamethylmelamine. In some embodiments, at least one of the occurring M's is an antifolate, a fluoropyrimidine, a deoxynucleoside analog, or a thiopurine. In certain embodiments, at least one of the M's occurring is methotrexate, pemetrexed, fluorouracil, capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, thioguanine, and mercaptopurine. In some specific embodiments, at least one of the M's occurring is an auristatin, a vinca alkaloid, or a taxane. In certain specific embodiments, at least one of the M's occurring is auristatin F, auristatin E, vincristine, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, etoposide, or teniposide.In some more specific embodiments, at least one of the M's occurring is irinotecan, SN38, topotecan, camptothecin, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, or aclarubicin. In certain more specific embodiments, at least one of the M's occurring is an anthracycline or bleomycin. In some embodiments, at least one of the M's occurring is doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, or mitoxantrone. In some embodiments, at least one occurrence of M is auristatin F, monomethylauristatin F, monomethylauristatin E, paciltaxol, SN-38, calicheamicin, anthramycin, aveimicin, ticamycin, DC-81, mazethramycin, neothramycin A, neothramycin B, polothramycin, prothracarcin, sivanomycin, sibiromycin, tomamycin, mertansine, emtansine, irinotecan, camptothecin, topotecan, siratecan, cositecan, exatecan, lutotecan, gimatecan, belotecan, and rubitecan. In some embodiments, each occurrence of M is auristatin F, monomethylauristatin F, monomethylauristatin E, paciltaxol, SN-38, calicheamicin, anthramycin, aveimicin, ticamycin, DC-81, mazethramycin, neothramycin A, neothramycin B, polothramycin, prothracarcin, sivanomycin, sibiromycin, tomamycin, mertansine, emtansine, irinotecan, camptothecin, topotecan, siratecan, cositecan, exatecan, lutotecan, gimatecan, belotecan, and rubitecan.

[0120] M 1 Or M 2 are M 1 Or M 2 The bond to the remainder of the molecule can be at any position (i.e., atom) above. 1 Or M 2To the remainder of the molecule, one will recognize a means for attaching M 1 Or M 2 can be attached to the remainder of the molecule through the nitrogen of the diazepine, the oxygen of the diazepine or phenyl ring, or a carbon of the pyrrolidine ring.

[0121] In some specific embodiments, the compound is a compound selected from Tables 2A and 2B. The compounds of Tables 2A and 2B are prepared according to the procedures described in the Examples. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12]

[0122] In some embodiments, M 1 and M 2 is, independently at each occurrence, a fluorescent moiety or a chromogenic moiety. Any fluorescent and / or chromogenic moiety may be used, such as those known in the art and commonly used in colorimetric, UV and / or fluorescent assays. M is useful in various embodiments of the present disclosure. 1 and M 2Exemplary moieties include, but are not limited to, xanthene derivatives (e.g., fluorescein, rhodamine, Oregon Green, eosin, or Texas Red); cyanine derivatives (e.g., cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, or merocyanine); squaraine derivatives and ring-substituted squaraines (including Seta, SeTau, and Squara dyes); naphthalene derivatives (e.g., dansyl and polodan derivatives); coumarin derivatives; oxadiazole derivatives (e.g., pyridyloxazole, nitrobenzoxadiazo anthracene derivatives (e.g., anthraquinones, including DRAQ5, DRAQ7, and CyTRAK Orange); pyrene derivatives such as Cascade Blue; oxazine derivatives (e.g., Nile Red, Nile Blue, Cresyl Violet, Oxazine 170); acridine derivatives (e.g., Proflavine, Acridine Orange, Acridine Yellow); arylmethine derivatives: auramine, crystal violet, malachite green; and tetrapyrrole derivatives (e.g., porphine, phthalocyanine, or bilirubin). Other exemplary M 1 and M 2 Moieties include cyanine dyes, xanthate dyes (eg, Hex, Vic, Nedd, Joe, or Tet); Yakima Yellow, Redmond Red; Tamura; Texas Red, and Alexa Fluor® dyes.

[0123] In still other embodiments of any of the above, M 1 and M 2 independently at each occurrence, includes three or more aryl or heteroaryl rings, or combinations thereof, such as four or more aryl or heteroaryl rings, or combinations thereof, or even five or more aryl or heteroaryl rings, or combinations thereof. 1 and M 2 independently at each occurrence, contains six aryl or heteroaryl rings, or a combination thereof. In further embodiments, the rings are fused. For example, in some embodiments, M 1 and M2 may, independently at each occurrence, include three or more fused rings, four or more fused rings, five or more fused rings, or even six or more fused rings. In certain embodiments, M 1 and M 2 is, independently at each occurrence, a dimethylaminostilbene, quinacridone, fluorophenyl-dimethyl-BODIPY, his-fluorophenyl-BODIPY, acridine, terylene, sexiphenyl, porphyrin, benzopyrene, (fluorophenyl-dimethyl-difluorobora-diaza-indacene)phenyl, (bis-fluorophenyl-difluorobora-diaza-indacene)phenyl, quaterphenyl, bi-benzothiazole, ter-benzothiazole, bi-naphthyl, bi-anthracyl, squaraine, squarylium, 9,10-ethynylanthracene, or ter-naphthyl moiety. 1 and M 2 The fluorescent dye of is, independently at each occurrence, p-terphenyl, perylene, azobenzene, phenazine, phenanthroline, acridine, thioxanthrene, chrysene, rubrene, coronene, cyanine, perylene imide, or perylene amide, or a derivative thereof. 1 and M 2 The fluorescent dye of each occurrence is, independently, a coumarin dye, a resorufin dye, a dipyrrometheneboron difluoride dye, a ruthenium bipyridyl dye, an energy transfer dye, a thiazole orange dye, a polymethine, or an N-aryl-1,8-naphthalimide dye. 1 and M 2 is, independently at each occurrence, pyrene, perylene, perylene monoimide, or 6-FAM, or a derivative thereof. 1 and M 2 Each occurrence of the fluorescent dye has the following structure:

[0124] [ka] has one of the following:

[0125] M containing a carboxylic acid group 1 and M 2 The portion is in the anionic form (CO2 - ), but one of skill in the art will understand that this will vary depending on pH, and that the protonated form (i.e., -COH) is included in various embodiments.

[0126] In some specific embodiments, the compound is a compound selected from Tables 3A and 3B. The compounds in Tables 3A and 3B were prepared according to the procedures described in the Examples. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 4-9] [Table 4-10] [Table 4-11] [Table 4-12]

Table 4-13

Table 4-14

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

[0127] As used in Tables 3A-3B, and throughout this application, M has the definition provided for compounds of structure (I) unless otherwise indicated. In some embodiments, M is F, F', or F", where F, F', and F'' are, respectively, the following structures: [ka] It refers to a fluorescein moiety having the formula:

[0128] Interaction with DNA One embodiment provides a compound according to any one of the embodiments disclosed herein (e.g., a compound of structure (I), (Ia), (Ib), (Ic), (Id), or (Ie)) and a pharma- ceutically acceptable carrier. In some embodiments, the compounds disclosed in the present disclosure are represented by the formula: 11 A carbon capable of forming a covalent bond between the C2NH2 group of the guanine base, N 10 -C 11 Position of PBD part (M 1 Or M 2 ) containing an electrophilic imine moiety on the alkylating agent.

[0129] [ka]

[0130] As a result, the PBD moieties of the compounds in this disclosure can alkylate guanine bases of DNA to form inter- and intrastrand DNA crosslinks. Compounds with two PBD moieties interact with DNA to form the following adducts:

[0131] [ka]

[0132] [ka] In some embodiments, the compounds disclosed herein have two or more PBD moieties as biologically active moieties.For example, the compounds disclosed herein may contain four PBD moieties.In this regard, the compounds can form both interstrand and intrastrand crosslinks with DNA, as shown below.

[0133] [ka]

[0134] Dimers of alkylating agents such as PBD dimers do not allow for the formation of both interstrand and intrastrand crosslinks, since PBD dimers or compounds with two PBD moieties have only two alkylation sites. The combination of the compounds disclosed in the present disclosure with both interstrand and intrastrand crosslinks constitutes an absolute block to DNA strand separation, thus disrupting essential DNA metabolic processes such as replication and transcription. This leads to the arrest of cell division and ultimately cell death. In this regard, compounds disclosed herein with at least three PBD moieties attached to the polymer backbone are highly effective ADCs. Furthermore, triplex formation can be minimized, since alkylating agents such as the PBD moieties on the polymer backbone of the compounds disclosed herein form interstrand, intrastrand, or both interstrand and intrastrand crosslinks with guanine bases (G) of DNA. In some embodiments, the number of PBD moieties attached to the polymer backbone can be controlled to maximize the efficacy of the compound, depending on the number of interstrand and / or intrastrand crosslinks that are preferred for the treatment of some solid tumors. As noted above, depending on the number of other bases present between the two guanines in the DNA strand, the spacing between the alkylating agents can be controlled to position the imine moiety of the alkylating agent closer to the guanine base of the DNA.

[0135] Pharmaceutical Compositions One embodiment provides a composition comprising a compound according to any one of the embodiments disclosed herein (eg, a compound of structure (I)) and a pharma- ceutically acceptable carrier. Other embodiments are directed to pharmaceutical compositions. The pharmaceutical compositions include any one (or more) of the compounds of structure (I) and a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical compositions are formulated for oral administration. In other embodiments, the pharmaceutical compositions are formulated for injection. In further embodiments, the pharmaceutical compositions include a compound of structure (I) and an additional therapeutic agent (e.g., an anti-cancer agent). Non-limiting examples of such therapeutic agents are described herein below. Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ocular, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. Further, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injection, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injection.

[0136] In certain embodiments, the compound of structure (I) is administered locally rather than systemically, for example, by injecting the compound directly into an organ, often in a depot preparation or sustained release formulation. In specific embodiments, a long-acting formulation is administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. In yet other embodiments, the drug is delivered in a targeted drug delivery system, for example, in a liposome coated with an organ-specific antibody. In such embodiments, the liposome is targeted to the organ and taken up selectively by the organ. In yet other embodiments, the compound of structure (I) is provided in the form of a fast release formulation, a sustained release formulation, or an intermediate release formulation. In yet other embodiments, the compound of structure (I) is administered locally.

[0137] The compounds of structure (I) are effective over a wide dosage range. For example, in the treatment of adult humans, dosages of 0.01-1000 mg per day, 0.5-100 mg per day, 1-50 mg per day, and 5-40 mg per day are examples of dosages that may be used in certain embodiments. An exemplary dosage is 10-30 mg per day. The exact dosage will depend on the route of administration, the form in which the compound is administered, the subject being treated, the weight of the subject being treated, and the preference and experience of the attending physician. In some embodiments, the compound of structure (I) is administered in a single dose. Usually, such administration will be by injection, for example intravenous injection, to rapidly introduce the drug. However, other routes may be used when appropriate. A single dose of the compound of structure (I) may also be used to treat acute conditions.

[0138] In some embodiments, the compound of structure (I) is administered in multiple doses. In some embodiments, administration is about once, twice, three times, four times, five times, six times, or more times per day. In other embodiments, administration is about once a month, once every two weeks, once a week, or once every other day. In another embodiment, the compound of structure (I) and another agent are administered together about once a day to about six times a day. In another embodiment, administration of the compound of structure (I) and the agent continues for less than about 7 days. In yet another embodiment, administration continues for more than about 6, 10, 14, 28 days, 2 months, 6 months, or 1 year. In some cases, continuous dosing is achieved and maintained for as long as necessary. Administration of the compound of structure (I) may continue for as long as necessary. In some embodiments, the compound of structure (I) is administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In some embodiments, the compound of structure (I) is administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, the compound of structure (I) is administered continuously for an extended period of time, e.g., for treatment of long duration. In some embodiments, the compound of structure (I) is administered at a dosage.It is known in the art that due to the subject-to-subject variability of the pharmacokinetics of the compound, individualization of the administration regimen is necessary for optimal treatment.The administration for the compound of the present disclosure can be found by routine experimentation in light of the present disclosure.

[0139] In some embodiments, the compound of structure (I) is formulated into a pharmaceutical composition.In a specific embodiment, the pharmaceutical composition is formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliary agents, which facilitate the processing of active compound into medicament-usable preparations.The appropriate formulation depends on the route of administration selected. Any pharma- ceutical acceptable techniques, carriers, and excipients may be used to formulate the pharmaceutical compositions described herein, as appropriate: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

[0140] Provided herein is a pharmaceutical composition comprising a compound of structure (I) and a pharma- ceutical acceptable diluent, excipient or carrier.In certain embodiments, the described compounds are administered as pharmaceutical compositions in which the compound of structure (I) is mixed with other active ingredients, as in combination therapy.All combinations of active agents described in the combination therapy section below and throughout this disclosure are encompassed herein.In specific embodiments, the pharmaceutical composition comprises one or more compounds of structure (I). Pharmaceutical composition, as used herein, refers to a mixture of a compound of structure (I) with other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients. In certain embodiments, the pharmaceutical composition facilitates administration of the compound to an organism. In some embodiments of practicing the methods of treatment or methods of use provided herein, a therapeutically effective amount of a compound of structure (I) provided herein is administered in a pharmaceutical composition to a mammal having the disease, disorder, or medical condition to be treated. In a specific embodiment, the mammal is a human. In certain embodiments, the therapeutically effective amount will vary depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, and other factors. The compound of structure (I) is used as a component of a mixture, alone or in combination with one or more therapeutic agents.

[0141] In one embodiment, one or more compounds of structure (I) are formulated in an aqueous solution. In a specific embodiment, the aqueous solution is selected from physiologically compatible buffers, such as, by way of example only, Hanks' solution, Ringer's solution, or saline buffer. In other embodiments, one or more compounds of structure (I) are formulated for transmucosal administration. In a specific embodiment, the transmucosal formulation includes a penetrant appropriate for the barrier to be permeated. In yet other embodiments, when the compounds described herein are formulated for other parenteral injections, suitable formulations include aqueous or non-aqueous solutions. In a specific embodiment, such solutions include physiologically compatible buffers and / or excipients. In another embodiment, the compounds described herein are formulated for oral administration.The compounds described herein are formulated by combining the active compound with, for example, a pharma- ceutically acceptable carrier or excipient.In various embodiments, the compounds described herein are formulated in oral dosage forms, including, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions, and the like.

[0142] In certain embodiments, pharmaceutical preparations for oral use are obtained by mixing one or more solid excipients with one or more compounds described herein, optionally grinding the resulting mixture, and then processing the mixture of granules to obtain tablet or dragee cores, after adding suitable auxiliary agents, in particular.Suitable excipients are sugars, including lactose, sucrose, mannitol or sorbitol, cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, or fillers, such as polyvinylpyrrolidone (PVP or povidone) or others, such as calcium phosphate.In specific embodiments, disintegrants may be added.Disintegrants include, by way of example only, cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar or alginic acid or its salts, such as sodium alginate.

[0143] In one embodiment, dosage forms such as dragee cores and tablets are provided with one or more suitable coatings. In a specific embodiment, concentrated sugar solutions are used to coat the dosage forms. The sugar solutions may contain additional components such as, by way of example only, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. For identification purposes, dyes and / or pigments may be added to the coating. Furthermore, dyes and / or pigments may be utilized to characterize different combinations of active compound doses.

[0144] In certain embodiments, a therapeutically effective amount of at least one of the compounds described herein is formulated into other oral dosage forms. Oral dosage forms include push-fit capsules made of gelatin and soft sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. In a specific embodiment, the push-fit capsules contain the active ingredient in a mixture with one or more fillers. The fillers include, by way of example only, binders such as lactose, starch, and / or lubricants such as talc or magnesium stearate, and may also include stabilizers. In other embodiments, the soft capsules contain one or more active compounds dissolved or suspended in a suitable liquid. Suitable liquids include, by way of example only, one or more fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added.

[0145] In other embodiments, a therapeutically effective amount of at least one of the compounds described herein is formulated for buccal or sublingual administration. Formulations suitable for buccal or sublingual administration include, by way of example only, tablets, lozenges, or gels. In yet other embodiments, the compounds described herein are formulated for parental administration, including formulations suitable for bolus injection or continuous infusion. In specific embodiments, the injectable formulations are provided in unit dosage form (e.g., in ampoules) or in multi-dose containers. Preservatives may be added to the injectable formulations. In still other embodiments, the pharmaceutical compositions are formulated in a form suitable for parenteral injection as sterile suspensions, solutions, or emulsions in oily or aqueous vehicles. Parenteral injection formulations may contain formulating agents such as suspending, stabilizing, and / or dispersing agents. In specific embodiments, pharmaceutical formulations for parenteral administration include aqueous solutions of the active compound in water-soluble form. In additional embodiments, suspensions of the active compound (e.g., compounds of structure (I)) are prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles for use in the pharmaceutical compositions described herein include, by way of example only, fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. In certain embodiments, aqueous injection suspensions contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. The suspension may also contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions. Alternatively, in other embodiments, the active ingredient is in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0146] In yet another embodiment, the compound of structure (I) is administered topically. The compounds described herein are formulated into various compositions that can be administered topically, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. Such pharmaceutical compositions may contain solubilizers, stabilizers, tonicity enhancers, buffers and preservatives. In yet other embodiments, the compounds of structure (I) are formulated for transdermal administration. In specific embodiments, transdermal formulations can be lipophilic emulsions or buffered aqueous solutions dissolved and / or dispersed in polymers or adhesives using transdermal delivery devices and transdermal delivery patches. In various embodiments, such patches are constructed for continuous, pulsatile, or on-demand delivery of pharmaceutical agents. In additional embodiments, transdermal delivery of the compounds of structure (I) is achieved by iontophoretic patches and the like. In certain embodiments, transdermal patches provide controlled delivery of the compounds of structure (I). In specific embodiments, the rate of absorption is slowed by using rate-controlling membranes or by trapping the compound within a polymer matrix or gel. In alternative embodiments, absorption enhancers are used to increase absorption. Absorption enhancers or carriers include absorbable pharma- ceutically acceptable solvents that aid in passage through the skin. For example, in one embodiment, the transdermal device is in the form of a bandage that includes a backing member, a reservoir containing the compound (which may include a carrier), optionally a rate-controlling barrier for delivering the compound to the host's skin at a controlled and predetermined rate over an extended period of time, and a means for securing the device to the skin.

[0147] In other embodiments, the compound of structure (I) is formulated to be administered by inhalation.Various forms suitable for administration by inhalation include, but are not limited to, aerosols, mist or powders.Any pharmaceutical composition of the compound of structure (I) is conveniently delivered in the form of an aerosol spray supply from a pressurized pack or nebulizer with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).In a specific embodiment, the dosage unit of the pressurized aerosol is determined by providing a valve to deliver a metered amount.In certain embodiments, capsules or cartridges, such as gelatin, by way of example only, are formulated for use in an inhaler or insufflator, containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0148] In yet another embodiment, the compounds of structure (I) are formulated into rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories or retention enemas containing conventional suppository bases such as cocoa butter or other glycerides, and synthetic polymers such as polyvinylpyrrolidone, PEG, etc. Suppository forms of the composition contain a low melting wax, such as, but not limited to, a mixture of fatty acid glycerides, which may be combined with melted cocoa butter. In certain embodiments, the pharmaceutical composition is formulated in any conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries, which facilitate the processing of the active compound into a medicament that can be used pharmaceutically.The appropriate formulation depends on the route of administration selected.Any pharmaceutically acceptable technique, carrier and excipient may be used, if appropriate.The pharmaceutical composition containing the compound of structure (I) is prepared in a conventional manner, such as, by way of example only, conventional mixing, dissolving, granulating, dragee making, wet milling, emulsifying, encapsulating, entrapping or compressing processes.

[0149] The pharmaceutical compositions comprise at least one pharma- ceutically acceptable carrier, diluent or excipient, and at least one compound of structure (I) as described herein as an active ingredient. The active ingredient is in free acid or free base form, or in pharma- ceutically acceptable salt form. Additionally, the methods and pharmaceutical compositions described herein include the use of N-oxides, crystalline forms (also known as polymorphs), and active metabolites of these compounds having the same type of activity. All tautomers of the compounds described herein are included within the scope of the compounds provided herein. Additionally, the compounds described herein include unsolvated forms and solvated forms with pharma- ceutically acceptable solvents such as water, ethanol, and the like. Solvated forms of the compounds provided herein are also considered to be disclosed herein. Additionally, the pharmaceutical compositions may include other medicinal or pharmaceutical agents, carriers, preservatives, stabilizing agents, wetting or emulsifying agents, solution promoters, adjuvants such as salts for regulating osmotic pressure, buffering agents, and / or therapeutically valuable substances.

[0150] Methods for preparing compositions containing the compounds described herein include formulating the compounds with one or more inert pharma- ceutically acceptable excipients or carriers to form solids, semi-solids, or liquids. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include solutions in which the compounds are dissolved, emulsions containing the compounds, or solutions containing liposomes, micelles, or nanoparticles containing the compounds disclosed herein. Semi-solid compositions include, but are not limited to, gels, suspensions, and creams. The forms of the pharmaceutical compositions described herein include liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to use, or emulsions. These compositions may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like. In some embodiments, the pharmaceutical composition comprising at least one compound of structure (I) is illustratively in the form of a liquid in which the drug is in solution, in suspension, or both. Typically, when the composition is administered as a solution or suspension, a first portion of the drug is in solution, and a second portion of the drug is in particulate form in suspension in a liquid matrix. In some embodiments, the liquid composition comprises a gel formulation. In other embodiments, the liquid composition is aqueous.

[0151] In certain embodiments, useful aqueous suspensions contain one or more polymers as suspending agents. Useful polymers include water-soluble polymers such as cellulose polymers, e.g., hydroxypropylmethylcellulose, and water-insoluble polymers such as cross-linked carboxyl-containing polymers. Certain pharmaceutical compositions described herein include mucoadhesive polymers, e.g., selected from carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran. Useful pharmaceutical compositions may also include solubilizing agents to aid in the dissolution of the compounds of structure (I). The term "solubilizing agent" generally includes agents that result in the formation of a micellar or true solution of the drug. Certain acceptable non-ionic surfactants, such as polysorbate 80, are useful as solubilizing agents, as are ophthalmically acceptable glycols, polyglycols, such as polyethylene glycol 400, and glycol ethers.

[0152] In addition, useful pharmaceutical compositions may include one or more pH adjusting or buffering agents, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane, and buffers such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition in an acceptable range.

[0153] Further useful compositions may also contain one or more salts in an amount necessary to bring the osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, with suitable salts including sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate. Other useful pharmaceutical compositions may include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal, stabilized chlorine dioxide, and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0154] Still other useful compositions contain one or more surfactants to enhance physical stability or for other purposes. Suitable non-ionic surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, such as polyoxyethylene (60) hydrogenated castor oil, and polyoxyethylene alkyl ethers and alkyl phenyl ethers, such as octoxynol 10, octoxynol 40. Still other useful compositions optionally contain one or more antioxidants to enhance chemical stability. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite. In certain embodiments, aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multi-dose reclosable containers are used, in which case it is customary to include a preservative in the composition. In alternative embodiments, other delivery systems for hydrophobic pharmaceutical compounds are used. Liposomes and emulsions are examples of delivery vehicles or carriers useful herein. In certain embodiments, organic solvents such as N-methylpyrrolidone are also used. In additional embodiments, the compounds described herein are delivered using sustained release systems, such as semi-transparent matrices of solid hydrophobic polymers containing therapeutic agents. A variety of sustained release materials are useful herein. In some embodiments, sustained release capsules release compounds for several weeks up to 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, additional strategies for protein stabilization are used.

[0155] In certain embodiments, the formulations described herein include one or more antioxidants, metal chelators, thiol-containing compounds, and / or other general stabilizing agents. Examples of such stabilizers include, but are not limited to, (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc, or (n) combinations thereof.

[0156] In some embodiments, the concentration of the one or more compounds provided in the pharmaceutical composition is 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.1%. %, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v or v / v.

[0157] In some embodiments, the concentration of one or more compounds is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%,4.75%,4.50%,4.25%,4%,3.75%,3.50%,3.25%,3%,2.75%,2.50%,2.25%,2%,1.75%,1.50%,125%,1%,0.5%,0.4%,0.3%,0.2%,0.1%,0.09%,0.08%,0.07%,0.06%,0.05%,0.04%,0.03%,0.02%,0 greater than 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v or v / v.

[0158] In some embodiments, the concentration of the one or more compounds is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24%, from about 0.08% to about 100%, from about 0.09% to about 120%, from about 0.10% to about 130%, from about 0.11% to about 140%, from about 0.12% to about 150%, from about 0.13% to about 160%, from about 0.14% to about 170%, from about 0.15% to about 180%, from about 0.16% to about 190%, from about 0.18% to about 200%, from about 0.19% to about 210%, from about 0.19% to about 220%, from about 0.19% to about 230%, from about 0.19% to about 240%, from about 0.19% to about 250%, from about 0.19% to about 260%, from about 0.19% to about 250%, from about 0.19% to about 220%, from about 0.19% to about 230%, from about 0.19% to about 240%, from about 0.19% to about 25 ... about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12%, about 1% to about 10% w / w, w / v or v / v. In some embodiments, the concentration of the one or more compounds is within the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w / w, w / v or v / v.

[0159] In some embodiments, the amount of one or more compounds is 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15g, 0.1g, 0.09g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g, 0.009g, 0.008g, 0.007g, 0.006g, 0.005g, 0.004g, 0.003g, 0.002g, 0.001g, 0.0009g, 0.0008g, 0.0007g, 0.0006g, 0.0005g, 0.0004g, 0.0003g, 0.0002g, or 0.0001g.

[0160] In some embodiments, the amount of one or more compounds is 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, 0.0025g, 0.003g, 0.004 ... g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007g, 0.00 75g, 0.008g, 0.0085g, 0.009g, 0.0095g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 0.055g, 0.06g, 0.065g, 0.07g, 0.075g, 0.08g, 0 .085g, 0.09g, 0.095g, 0.1g, 0.15g, 0.2g, 0.25g, 0.3g, 0.35g, 0.4g, 0.45g, 0.5 g, 0.55g, 0.6g, 0.65g, 0.7g, 0.75g, 0.8g, 0.85g, 0.9g, 0.95g, 1g, 1.5g, 2g, 2.5, 3g, 3.5, 4g, 4.5g, 5g, 5.5g, 6g, 6.5g, 7g, 7.5g, 8g, 8.5g, 9g, 9.5g, or more than 10g. In some embodiments, the amount of one or more compounds ranges from 0.0001-10 g, 0.0005-9 g, 0.001-8 g, 0.005-7 g, 0.01-6 g, 0.05-5 g, 0.1-4 g, 0.5-4 g, or 1-3 g.

[0161] Treatment Certain compounds of the present disclosure are useful for treating diseases (i.e., compounds of structure (I)). The compounds disclosed herein provide a targeted approach to drug delivery strategies. Thus, in one embodiment, a method of treating a disease (or a symptom thereof) is provided, comprising administering a therapeutically effective amount of a compound of structure (I) to a mammal (e.g., a human) in need thereof.

[0162] For example, in certain embodiments, the disclosure provides methods of treating solid tumors, multiple myeloma, glioma, clear cell renal cell carcinoma, prostate cancer, ovarian cancer, non-small cell lung cancer, GI malignancies, acute lymphoblastic leukemia, acute myeloid leukemia, renal cell carcinoma, colorectal cancer, epithelial cancer, pancreatic and gastric cancer, renal cell carcinoma, non-Hodgkin's lymphoma, metastatic renal cell carcinoma, malignant mesothelioma, adenocarcinoma of the pancreas, ovary and / or lung, B-cell malignancies, breast cancer, melanoma, recurrent multiple myeloma, small cell lung cancer, CD22 positive B-cell malignancies, Hodgkin's lymphoma / anaplastic large cell lymphoma, or HER2 positive breast cancer.

[0163] In some of the foregoing embodiments, the disease is cancer. For example, in certain embodiments, the cancer is breast cancer, non-Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, gastric cancer, renal cell carcinoma, solid tumors, ovarian cancer, prostate cancer, colorectal cancer, pancreatic cancer, small cell lung cancer, diffuse large B-cell lymphoma, neoplasms, urothelial carcinoma, ALL, CLL, glioblastoma, Hodgkin's lymphoma, lymphoma, mesothelioma, non-small cell lung cancer, recurrent head and neck cancer, or a combination thereof.

[0164] Certain embodiments also relate to a method of treating a hyperproliferative disorder in a mammal (e.g., a human), comprising administering to the mammal a therapeutically effective amount of a compound of structure (I), or a pharma- ceutically acceptable salt, ester, prodrug, solvate, hydrate, or derivative thereof.In some embodiments, the method is directed to treating acute myeloid leukemia, adolescent cancer, adrenocortical carcinoma of childhood, AIDS-related cancers (e.g., lymphoma and Kaposi's sarcoma), anal cancer, appendix cancer, astrocytoma, atypical teratoid, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, atypical teratoid, embryonal tumor, germ cell tumor, primary lymphoma, cervical cancer, childhood cancer, chordoma, cardiac tumor, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic bile duct carcinoma in situ (DCIS), embryonal tumors, CNS cancer, endometrial cancer, ependymoma, esophageal cancer, nasal neuroblastoma, Ewing's sarcoma, extracranial germ cell tumors, extragonadal germ cell tumors, eye cancer, fibrous histiocytoma of bone, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, gestational trophoblastic tumors, hairy cell leukemia, head and neck cancer, cardiac cancer, liver cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, laryngeal cancer, lip and oral cancer Cavity cancer, liver cancer, lobular carcinoma in situ (LCIS), lung cancer, lymphoma, metastatic squamous neck cancer of unknown primary, midline canal carcinoma, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, multiple myeloma, Merkel cell carcinoma, malignant mesothelioma, malignant fibrous histiocytoma and osteosarcoma of bone, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer (NSCLC), oral cancer, lip and oral cavity cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, papillomatosis, paraneoplastic syndrome, For the treatment of cancers such as ganglionoma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach (gastric) cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, T-cell lymphoma, testicular cancer, laryngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, chorioepithelial tumor, rare cancers of childhood, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer or virus-induced cancer.In some embodiments, the methods relate to the treatment of non-cancerous hyperproliferative disorders such as benign hyperplasia of the skin (eg, psoriasis), restenosis or the prostate (eg, benign prostatic hyperplasia (BPH)).

[0165] Certain specific embodiments provide a method of treating lung cancer, comprising administering to a subject in need thereof an effective amount of any of the compounds of structure (I) above (or a pharmaceutical composition comprising same). In certain embodiments, the lung cancer is non-small cell lung cancer (NSCLC), such as adenocarcinoma, squamous cell lung carcinoma, or large cell lung carcinoma. In other embodiments, the lung cancer is small cell lung carcinoma. Other lung cancers treatable by the disclosed compounds include, but are not limited to, adenoma, carcinoid tumor, and undifferentiated carcinoma.

[0166] Thus, in some embodiments of structure (I), A is an antibody or cell surface receptor antagonist, such as an epidermal growth factor receptor (EGFR) inhibitor, a hepatocyte growth factor receptor (HGFR) inhibitor, an insulin-like growth factor receptor (IGFR) inhibitor, a folate or a MET inhibitor, such as those related to the specific antibody trastuzumab. In a further embodiment, the method further comprises inducing apoptosis. In some embodiments, the method of treatment includes treating a tumor having tumor cells with a tumor cell receptor. In some embodiments, the tumor cells have receptors in the range of 1,000-100,000, 1,000-50,000, 1,000-25,000, 1,000-10,000 receptors per cell. For example, in some embodiments, the tumor cells have about 1,000, about 10,000 receptors, or less than 100,000 receptors per cell. Additional therapeutic agents that can be combined with the compounds of the present disclosure can be found in "The Pharmacological Basis of Therapeutics," 10th Edition, by Goodman and Gilman, edited by Hardman, Limbird and Gilman, or the Physician's Desk Reference, both of which are incorporated herein by reference in their entireties.

[0167] The compounds of structure (I) described herein can be used in combination with the agents disclosed herein or other suitable agents depending on the condition being treated. Thus, in some embodiments, one or more compounds of the present disclosure will be co-administered with other agents as described above. When used in combination therapy, the compounds described herein are administered simultaneously or separately with the second agent. This combined administration can include co-administration of the two agents in the same dosage form, co-administration in separate dosage forms, and separate administration. That is, the compounds described herein and any of the agents described above can be formulated together in the same dosage form and administered at the same time. Alternatively, the compounds of the present disclosure and any of the agents described above can be administered at the same time, with both agents being in separate formulations. In another alternative, any of the agents described above can be administered immediately after administration of the compounds of the present disclosure, or vice versa. In some embodiments of the separate administration protocol, the compounds of the present disclosure and any of the agents described above are administered minutes apart, or hours apart, or days apart.

[0168] In some embodiments, the method further comprises administering an additional therapeutic agent selected from the group consisting of an anti-tumor agent, an enediyne anti-tumor antibiotic, a maytansinoid, a topoisomerase inhibitor, a kinase inhibitor, an anthracycline, and an EGFR inhibitor or alkylating agent, and combinations thereof. In some more specific embodiments, the method further comprises administering an additional therapeutic agent selected from the group consisting of an anti-tumor agent, an enediyne anti-tumor antibiotic, a maytansinoid, a topoisomerase inhibitor, a kinase inhibitor, an anthracycline, and an EGFR inhibitor or alkylating agent, and combinations thereof. In certain embodiments, the additional therapeutic agent comprises auristatin F, monomethylauristatin F, monomethylauristatin E, paciltaxol, SN-38, calicheamicin, anthramycin, aveimicin, ticamycin, DC-81, mazethramycin, neothramycin A, neothramycin B, polothramycin, prothracarcin, sivanomycin, sibiromycin, tomamycin, mertansine, emtansine, irinotecan, camptothecin, topotecan, ciratecan, cositecan, exatecan, lutotecan, gimatecan, belotecan, and rubitecan.

[0169] In some embodiments, the pharmaceutical composition comprises a compound of any of the above structures and a pharma- ceutically acceptable carrier, diluent, or excipient. In some embodiments, a method for treating a disease or disorder comprises administering a therapeutically effective amount of a compound of any of the above structures or a pharmaceutical composition thereof to a subject in need thereof. In certain embodiments, the disease or disorder is cancer. In some more specific embodiments, the cancer is breast cancer, gastric cancer, lung cancer, ovarian cancer, lymphoma, and bladder cancer. The examples and preparations provided below further describe and illustrate the compounds of the present disclosure and the methods of preparing such compounds. It should be understood that the scope of the present disclosure is in no way limited by the scope of the following examples and preparations. In the following examples and throughout the specification and claims, molecules and moieties that have a single stereocenter are present as racemic mixtures unless otherwise specified. Molecules and moieties that contain two or more stereocenters are present as racemic mixtures of diastereomers unless otherwise specified. Single enantiomers / diastereomers can be obtained by methods known to those skilled in the art.

[0170] As described in detail above, the compounds of structure (I), (Ia), (Ib), (Ic), (Id) and (Ie) can be prepared by oligomerization using well-known phosphoramidite chemistry. Applicants have discovered intermediate compounds useful for the synthesis of compounds of structure (II) and compounds of structure (III). Thus, embodiments of the present disclosure provide compounds of structure (II) or (III) as follows:

[0171] [ka] The present invention provides a compound having one of the following:

[0172] In some embodiments, R 1” is H, a protecting group, or an activated phosphorus moiety. For example, in some specific embodiments, R 1” is a dimethoxytrityl group (TMD). The TMD protecting group can be cleaved using a fluoride source such as tetra-n-butylammonium fluoride (TBAF) to yield a hydroxyl group. In some embodiments, R 2” is H or has the following phosphoramidite structure: [ka] The above phosphoramidite moieties can be introduced by reacting the free hydroxyl group (unprotected) of structure (II) or (III) with 3-((chloro(diisopropylamino)phosphanyl)oxy)propanenitrile under basic conditions, as described in the following section.

[0173] In some embodiments, R 6 is H, CH=CHCONH, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, cycloalkyl, heterocyclyl, aryl, or heteroaryl. For example, in some specific embodiments, R 6 is an alkyl group such as a methyl group (-CH3). In some embodiments, R 7 , R 8 , and R 9 are independently H, OH, and OR f , S.H., S.R. f , NH2, NHR f , N.R. f R g , alkyl, alkoxy, alkyl ether, or heteroalkyl. For example, in some specific embodiments, R 7 and R 9 are independently H. R 8 is an alkoxy group. For example, R 8 is a methoxy group (-OCH3).

[0174] In some embodiments, R 10 is a nitrogen protecting group or H. For example, in some specific embodiments, R 10 is allyloxycarbonyl (Alloc). The Alloc protecting group can be readily cleaved using Pd metal-based conditions to give the corresponding amine. In some embodiments, other nitrogen protecting groups can be used instead. For example, R 10 Nitrogen protecting groups include benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc), or tert-butyloxycarbonyl (Boc).

[0175] In some embodiments, R 11 is an oxygen protecting group, alkyl, or H. For example, in some specific embodiments, R 11 is a tert-butyldimethylsilyl ether (TBS) group. In some embodiments, other silyl ethers containing oxygen protecting groups can be used instead. For example, R 11Oxygen protecting groups include trimethylsilyl ether (TMS), triethylsilyl ether (TES), tert-butyldiphenylsilyl ether (TBDPS), or triisopropylsilyl ether (TIPS). The silyl ether protecting groups can be cleaved using a fluoride source such as tetra-n-butylammonium fluoride (TBAF) to provide the hydroxyl group.

[0176] In some embodiments, L 1b is a linker that may be an alkylene or a heteroalkylene. For example, in some specific embodiments, L 1b In other embodiments, L comprises an alkyl chain having an odd number of carbon atoms, such as C3, C5, or C7. 1b includes alkyl chains having an even number of carbon atoms, such as C2, C4, or C6.

[0177] In some embodiments, the compound has the following structure (IIa) or (IIIa): [ka] or stereoisomers. (In the formula, R 1” is H, a protecting group, or an activated phosphorus moiety, R 2” is H or has the structure:

[0178] [ka] having R 6 is methyl, R 10 is a nitrogen protecting group or H, R 11 is an oxygen protecting group or H.

[0179] In some specific embodiments, compound (II), (IIa), (III), or (IIIa) is selected from Table 4 or Table 5, respectively.

[0180] [Table 6-1] [Table 6-2] [Table 6-3]

[0181] [Table 7-1] [Table 7-2] [Table 7-3]

[0182] [Table 8] The following examples are offered by way of illustration and not by way of limitation.

[0183] A typical DNA synthesis cycle [ka]

[0184] Oligomerization is usually initiated by removing a protecting group (e.g., dimethoxytrityl group, DMTr) to reveal a free -OH (hydroxyl) group (step 1, detritylation). In a subsequent coupling step, a phosphoramidite monomer is introduced which reacts with the free OH group to form a new covalent bond with phosphorus with concomitant loss of a diisopropylamine group (step 2, coupling). The resulting phosphite triester is oxidized (e.g., with I2 and pyridine) to a more stable phosphate ester (step 3, oxidation), and in a capping step the remaining free OH group is rendered unreactive (step 4, capping). The new product, the phosphate oligomer, contains a DMTr-protected OH group and can be deprotected to restart the synthesis cycle so that another phosphoramidite monomer can be added to the oligomer.

[0185] Customization is performed in step 2 by the selection of phosphoramidite monomers. The nature of L (i.e., linker group) and M (i.e., chemotherapeutic agent) in the above scheme are selected to synthesize the desired compounds of structures (I), (Ia), (Ib), (Ic), (Id), and (Ie). M may be absent to incorporate desired spacing between M moieties. One skilled in the art can select multiple monomer types and vary the linker groups simultaneously to arrive at compounds of the present disclosure containing multiple therapeutic agents and / or other moieties (e.g., fluorophores or chromophores).

[0186] General reaction scheme 1 (phosphoramidites) [ka]

[0187] Reaction Scheme I illustrates a method for the preparation of phosphoramidite intermediates useful for the preparation of compounds of structures (I), (Ia), (Ib), (Ic), (Id), and (Ie). Referring to Reaction Scheme I, G 1represents the desired alkylating agent moiety containing a hydroxyl functionality (e.g., an alkylating agent moiety such as a pyrrolobenzodiazepine). Step 1 of Reaction Scheme I begins with alkylating the oxygen of the hydroxyl group with an alkyl halide such as 1,5-diiodopentane, as shown using known reagents under basic conditions (e.g., K2CO3 in acetone). The resulting ether is then coupled with a TMD-protected thymidine to provide the reaction product of step 2. The resulting adduct is then reacted with 3-((chloro(diisopropylamino)phosphaneyl)oxy)propanenitrile (or other suitable reagent) to provide the desired compound of structure (II) or (III) shown above.

[0188] The resulting compound of structure (II) or (III) can then be used to synthesize the desired compounds of structure (I), (Ia), (Ib), (Ic), (Id), and (Ie) by reaction under well-known (automated) DNA synthesis conditions. In some specific embodiments, the following reaction scheme can be used to synthesize compound III-5 shown in Table 5.

[0189] [ka]

[0190] Compounds III-1 to III-4 and III-6 shown in Table 5 can be similarly synthesized according to the above reaction scheme. The enantiomers or diastereomers can be synthesized in the same manner, except starting from the other enantiomer or diastereomer of the starting material.

[0191] The above reaction scheme describing the synthesis of protected PBD thymidine-based phosphoramidites is based on the use of thymidine as the diol scaffold. Other diols, such as solketal, can be used to generate other protected PBD phosphoramidites, and the following reaction schemes can be used to synthesize compounds IV-1 to IV-4 shown in Table 6.

[0192] [ka]

[0193] The protected PBD is added to a dry round bottom flask under an inert gas blanket containing a magnetic stir bar, followed by the addition of the solvent and base. The resulting solution is stirred and then methyl 4-iodobutyrate is added to the reaction flask. Workup affords the resulting crude ether product, which is purified by column chromatography. The ether product is treated with 0.4M NaOH in methanol and water to obtain the sodium salt.

[0194] The sodium salt (1.5 eq.) is added to a dry round-bottom flask under an inert gas blanket containing a magnetic stir bar, followed by the addition of DMF. The sodium salt is allowed to completely dissolve at room temperature under inert gas. DIPEA (3.3 eq.) is then added to the mixture, followed by HATU (1.2 eq.). 6,7-dihydroxy-4-oxaheptylamine (1.0 eq.) is added to a dry round-bottom flask under an inert gas blanket containing a magnetic stir bar, followed by the addition of DMF, and allowed to completely dissolve at room temperature. The sodium salt reaction mixture is then added to the solution containing 6,7-dihydroxy-4-oxaheptylamine; the resulting mixture is mixed at room temperature under inert gas. Upon completion of the reaction, the solvent is removed by rotary evaporation under vacuum (10 mbar) and heating (55° C.). The concentrated residue is placed under full vacuum at room temperature for several hours to give the crude diol.

[0195] The crude diol (1.0 equiv.) is added to a dry round-bottom flask containing a magnetic stir bar under an inert gas blanket, followed by anhydrous pyridine. The reaction flask is then transferred to an ice-water bath (0° C.) and allowed to cool with mixing until uniform (approximately 10 min). 4,4'-dimethoxytrityl chloride (1.5 equiv.) is then added to the cooled mixture with continuous mixing under inert gas. The reaction mixture is allowed to warm to room temperature and then sampled for TLC analysis. Once the reaction is confirmed to be complete, any remaining unreacted 4,4'-dimethoxytrityl chloride is quenched by adding methanol to the reaction mixture (1.0 equiv.). The solvent is removed by rotary evaporation under vacuum (10 mbar) and heating (55° C.). The concentrated residue is then suspended in toluene and the toluene is removed by rotary evaporation under vacuum (10 mbar) and heating (55° C.); repeat twice. The crude product is dissolved in dichloromethane, washed with sodium bicarbonate (saturated aqueous solution), and separated; this process is repeated once. The separated organic phase is washed with sodium chloride (saturated aqueous solution), and separated. The separated organic phase is dried over anhydrous sodium sulfate, and the sodium sulfate is filtered off. The solvent is removed by rotary evaporation, followed by silica gel flash chromatography to obtain the DMT-protected PBD, which is dried under vacuum for at least 24 hours, dissolved in dichloromethane under an inert gas blanket, containing a magnetic stir bar, followed by addition of DIPEA, followed by addition of Cl-Phos. The reaction is mixed for approximately 15 minutes, and then sampled for TLC analysis (TLC showed the reaction was complete). Once the reaction is complete, the reaction mixture is washed by adding directly to sodium bicarbonate (saturated aqueous solution), the organic phase is separated, and this is repeated once. The organic phases are combined, dried over anhydrous sodium sulfate, and then the sodium sulfate is filtered off. The product containing organic phase is sampled for TLC and LC-UV / MS analysis. The dichloromethane is then removed by rotary evaporation and proceeded to purification without crude weight. This crude material is then combined with crude material from a small scale pilot reaction.The combined crude material is purified by silica gel solid phase extraction, dichloromethane / methanol / triethylamine mobile phase, and product-containing fractions are pooled (determined by TLC) and sampled for TLC and LC-UV / MS analysis. The mobile phase is removed by rotary evaporation, then placed on a vacuum line for at least 24 hours to yield the DMT-protected PBD phosphoramidite as shown above.

[0196] The carbon chain length between the PBD moiety of the DMT-protected PBD phosphoramidite and the phosphoramidite / DMT group can be adjusted by using different halide methyl esters and / or diols. The enantiomers or diastereomers can be synthesized in the same way, except starting from the other enantiomer or diastereomer of the starting material. EXAMPLES

[0197] General method Mass spectral analysis was performed on a Waters / Micromass Quattro micro MS / System MS (MS mode only) using MassLynx 4.1 acquisition software. The mobile phase used for LC / MS was 100 mM 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), 8.6 mM triethylamine (TEA), pH 8. Phosphoramidites and precursor molecules were also analyzed using an acetonitrile / water mobile phase gradient held at 45° C. on a 2.1 mm x 50 mm Acquity BEH-C. 18 The analysis is performed using a Waters Acquity UHPLC system equipped with a 1000 .mu.m column. The molecular weights of the monomer intermediates are obtained on a Waters / Micromass Quattro micro MS / system MS (MS mode only) using tropylium cation injection enhanced ionization. Excitation and emission profile experiments are recorded on a Cary Eclipse spectrophotometer. All reactions are carried out in oven-dried glassware under a nitrogen atmosphere unless otherwise noted. Commercially available DNA synthesis reagents are purchased from Glen Research (Sterling, VA). Anhydrous pyridine, toluene, dichloromethane, diisopropylethylamine, triethylamine, acetic acid, pyridine and THF are purchased from Aldrich. All other chemicals are purchased from Aldrich or TCI and used as is without further purification.

[0198] Example 1 Synthesis of Compound I-1 Preparation of stock solutions Borate buffer prepared at 250 mM, pH 10 Fluorescein-NHS solution prepared at 350 mM (300 mg in 1.35 mL DMSO:acetonitrile 25:75)

[0199] solid phase synthesis Compound I-1 is prepared on a DNA synthesizer via a solid support using standard DNA synthesis techniques (i.e., DMT-protected 2-cyanoethyl phosphoramidite). The polymer is removed from the solid support using ammonium hydroxide and lyophilized to a paste. A 250 mg aliquot is reconstituted in water. A small aliquot is removed and serial dilutions are prepared in 100 mM NaCO3, pH 9 to determine the concentration (A263ε=10,000). The final stock concentration is found to be 14.5 mM. Dye Coupling Reaction In a 50 mL centrifuge tube equipped with a magnetic stir bar, add water (1.110 μL), borate buffer (1.800 μL), compound I-1 polymer solution (466 μL), acetonitrile (137.5 μL), triethylamine (313 μL), and fluorescein-NHS solution (675 μL). Wrap the tube in aluminum foil and stir the mixture at room temperature overnight. Size Exclusion Filtration Add 1 mL of water to an Amicon Ultra-15 centrifugal filter (Millipore UFC900324, molecular weight cutoff = 3000). Add the crude reaction mixture (4.5 mL) from the dye coupling reaction to the filtration apparatus. Rinse the reaction vessel twice with 4 mL of 100 mM NaOH and transfer the rinse to the filtration apparatus. Centrifuge the filtration apparatus at maximum speed (3220 g, swinging bucket, 30 min). Remove the filtrate and treat the retentate with an additional 10 mL of 100 mM NaOH. Centrifuge the filtration apparatus as before. Remove the filtrate again and add a third 10 mL aliquot of 100 mM NaOH to the retentate. Centrifuge the apparatus as before and remove the filtrate. Add a fourth 10 mL aliquot of 100 mM NaOH to the retentate and centrifuge as before. Remove the filtrate and add 10 mL of water to the filtration apparatus. Centrifuge the mixture as before. The retentate is removed and the filter vessel is washed with water and rinse is added to final volume (3.5 mL). The desired product is confirmed by LC-MS and the concentration is determined using absorbance.

[0200] Example 2 Activation of Compound I-1 and Antibody Conjugates [ka]

[0201] Maleimide-functionalized compound I-1 is prepared according to the method described in Example 1. In parallel, UCHT-1 antibody is treated with bis-maleimidoethane ("BMOE") to reduce disulfide bonds. The reduced antibody is reacted with compound I-1 at a polymer to antibody molar ratio of 5:1. This reaction results in a final product with a polymer to antibody ratio of 1:1, as detected by size exclusion chromatography. In some embodiments, anti-CD33, anti-CD70, or anti-CD123 can be used with bismaleimidoethane ("BMOE") to reduce disulfide bonds.

[0202] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the Application Data Sheets, including U.S. Provisional Patent Application No. 63 / 250,931, filed September 30, 2021, are incorporated by reference herein in their entirety to the extent not inconsistent herewith. If necessary, aspects of the embodiments can be modified to employ concepts from the various patents, applications and publications to provide still further embodiments.

[0203] From the foregoing it will be appreciated that, although specific embodiments of the present disclosure have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, the present disclosure is not to be limited except as by the appended claims.

Claims

1. The following structure (I): 【Chemical 1】 (I) or a stereoisomer, pharmaceutical salt or tautomer thereof. (In the formula, M 1 is independently at each occurrence absent, a moiety comprising a pyrrolobenzodiazepine, a minor groove binder, or a fluorescent dye, provided that each occurrence of M 1 is a pyrrolobenzodiazepine; M 2 is independently at each occurrence a moiety comprising a pyrrolobenzodiazepine, a minor groove binder, or a fluorescent dye; L 1a is, independently at each occurrence, a heteroarylene linker; L 1b is independently calculated for each occurrence, M 1 is absent, then H, or M 1 is a pyrrolobenzodiazepine or a fluorescent dye, the linker is an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or a combination of one or more thereof; L 2 , L 3 , L 5 , L 6 and L 7 is independently at each occurrence an optional linker consisting of alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, heteroalkynylene, or a combination of one or more thereof; L 4 is independently at each occurrence a linker that comprises an alkylene, alkenylene, alkynylene, heteroalkylene, heteroalkenylene, or heteroalkynylene; R 1 and R 2 are each independently H, OH, SH, alkyl, alkoxy, alkyl ether, heteroalkyl, -OP(=R a ) (R b ) R c , Q, or a protected form thereof, L′, a minor groove binder, or a combination of one or more thereof; R 3 is independently at each occurrence H, alkyl, or alkoxy; R 4 is independently calculated for each occurrence, - , S - , OR d , or SR d and R 5 is independently at each occurrence oxo, thioxo, or absent; R a is O or S; R b OH SH O - , S - , OR d or SR d and R c OH SH O - , S - , OR d ,OL',SR d , alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkyl ether, alkoxyalkyl ether, phosphate, thiophosphate, phosphoalkyl, thiophosphoalkyl, phosphoalkyl ether, or thiophosphoalkyl ether; R d is the counterion, Q is, independently at each occurrence, a moiety that includes a reactive group, or a protected form thereof, capable of forming a covalent bond with an analyte molecule, a targeting moiety, a solid support, or a complementary reactive group Q'; L′ is, independently at each occurrence, a linker that includes a covalent bond to Q, a linker that includes a covalent bond to a targeting moiety, a linker that includes a covalent bond to an analyte molecule, a linker that includes a covalent bond to a solid support, a linker that includes a covalent bond to a solid support residue, a linker that includes a covalent bond to a nucleoside, or a linker that includes a covalent bond to an additional compound of structure (I); m, for each occurrence, is an integer greater than or equal to zero; n is an integer of 1 or more, q and w are independently 0 or 1 in each occurrence, provided that at least one of q or w is 1 in one occurrence.

2. The following structure (Ia): 【Chemistry 2】 (Ia) 2. The compound of claim 1 having the formula:

3. Appearing L 2 3. The compound according to claim 1 or 2, wherein at least one of: is absent.

4. Appearing L 4 The compound of claim 1 or 2, wherein at least one of comprises an alkylene oxide.

5. Appearing L 5 or L 6 The compound of claim 1 or 2, wherein at least one of comprises an alkylene oxide.

6. The following structure (Ib): 【Chemistry 3】 3. The compound of claim 1 or 2, having the formula: (In the formula, L 1b is, independently at each occurrence, a linker which may be alkylene or heteroalkylene.

7. The following structure (Ic): 【Chemistry 4】 3. The compound of claim 1 or 2, having the formula: (wherein z is an integer from 1 to 100)

8. M 1 but has the following structure: 【Chemistry 5】 3. The compound of claim 1 or 2, having one of the following: (In the formula, R 6 is independently at each occurrence H, CH═CHCONH2, alkyl, alkoxy, heteroalkyl, heteroalkoxy, alkylether, alkoxyalkylether, cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 7 , R 8 , and R 9 is independently H, OH, OR for each occurrence. f , S.H., S.R. f , N.H. 2 , N.H.R. f , N.R. f R g , alkyl, alkoxy, alkyl ether, or heteroalkyl; R 10 is independently at each occurrence a nitrogen protecting group or H; R 11 is independently at each occurrence an oxygen protecting group, alkyl, or H; R f and R g is independently at each occurrence alkyl, heterocyclyl, or aryl.

9. The following structures (Id) or (Ie): 【Chemistry 6】 3. The compound of claim 1 or 2, having one of the following: (In the formula, R 6 is, independently at each occurrence, alkyl; R 7 and R 9 is, for each occurrence, H, and R 8 are independently ORed for each occurrence. f and R 10 is independently at each occurrence a nitrogen protecting group; R 11 is independently at each occurrence an oxygen protecting group; R f is alkyl)

10. 3. The compound of claim 1 or 2, wherein L' is a heteroalkylene-containing linker to Q, a targeting moiety, an analyte molecule, a solid support, a solid support residue, a nucleoside, or an additional compound of structure (I).

11. 3. The compound of claim 1 or 2, wherein the targeting moiety is an antibody, a cell surface receptor antagonist, or a cell surface receptor antagonist.

12. R 1 or R 2 but has the following structure: 【Chemistry 7】 3. The compound of claim 1 or 2, having one of the following:

13. R 1 or R 2 but has the following structure: 【Chemistry 8】 3. The compound of claim 1 or 2, having the formula:

14. 3. The compound of claim 1 or 2, wherein Q comprises a sulfhydryl, disulfide, activated ester, isothiocyanate, azide, alkyne, alkene, diene, dienophile, acid halide, sulfonyl halide, phosphine, α-haloamide, biotin, amino, or maleimide functionality.

15. Appearing M 1 or M 2 3. The compound of claim 1 or 2, wherein at least one of is a nitrogen mustard, a nitrosourea, a tetrazine, an aziridine, cisplatin or a cisplatin derivative, or a non-classical alkylating agent.

16. Appearing M 1 or M 2 3. The compound of claim 1, wherein at least one of the following is mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan, N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mitomycin, diaziquone (AZQ), cisplatin, carboplatin, oxaliplatin, procarbazine, or hexamethylmelamine.

17. Appearing M 1 or M 2 at least one of which has the following structure: 【Chemistry 9】 3. The compound of claim 1 or 2, having one of the following:

18. Appearing L 7 at least one of which has the following structure: 【Chemistry 10】 3. The compound of claim 1 or 2, comprising one of:

19. 10. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier, diluent, or excipient.

20. 20. A method for treating a disease or disorder, comprising administering a therapeutically effective amount of a compound of claim 1 or 2, or a pharmaceutical composition of claim 19, to a subject in need thereof.

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