Patent Application
20220008537
The invention is directed to photosensitizing compounds useful for photodynamic therapy.
Photodynamic therapy (PDT) is an approach to cancer treatment aimed at minimizing the severe side effects associated with traditional chemotherapeutic agents. In PDT, excitation of a photosensitizer (PS) with low energy light triggers the production of highly localized reactive oxygen species (ROS) in diseased tissues with minimal involvement of surrounding cells. In the most common PDT mechanism, singlet oxygen (1O2) is generated by Type 2 energy transfer between the triplet excited state (3PS*) of the PS and ground state triplet oxygen (3O2). The triplet state can also react with 3O2 by Type I electron transfer to yield superoxide anion radicals (O2.-). Spontaneous dismutation of O2.- generates H2O2, which gives rise to hydroxyl radicals (.OH) by a Fenton reaction. With respective diffusion distances of 50-100 nm and 0.8-6.0 nm, the short-lived and highly reactive 1O2 and .OH formed upon dye excitation cause extensive oxidative damage to DNA and other cellular macromolecules in their vicinity.
The commonly used PDT agents porfimer sodium, talaporfin, and verteporfin directly sensitize cleavage of genomic DNA when irradiated in tissue culture and/or in circulating cells. While their absorption bands are compatible with visible light sources that emit at wavelengths ≤689 nm, alternative photosensitizers that possess near-infrared maxima extending from ˜700 nm to 900 nm are desired. This is due to enhanced penetration of incident irradiation afforded by minimal absorption of light in this range by molecules in the body. The light depth attained at 835 nm through biological tissue is approximately twice that at 630 nm, the wavelength used to activate porfimer sodium.
Although infrared light penetrates tissues deeply, red-shifting the λmax of a chromophore reduces triplet state energy, placing limits on near-IR ROS production. Type 2 singlet oxygen is generated only when a PS has a triplet state energy equivalent to or higher than the excitation energy of 1O2 (95 kJmol-1, ˜1270 nm). When taking into consideration the minimal energy gap between the first excited 1PS* and 3PS* states of PDT agents (≤63 kJmol-1), this translates into a ˜810 nm upper absorption limit for 1O2 production. In order for a photosensitizer to form Type 1 superoxide, the oxidation potential of its triplet state should be higher than the oxidation potential of ground state triplet oxygen (E° (3O2/O2.-)=0.16 V at pH 7.0), but excited state oxidation potentials decrease as a function of decreasing triplet state energy. As a result, there are relatively few examples of DNA photocleaving agents that are effective ROS generators in the near-infrared range. Using an anthracenyl-bis(pyridyl)Fe(III) catecholate complex to sensitize hydroxyl radical production, Chakravarty and co-workers cleaved plasmid DNA in high yield under 785 nm illumination. Until the present report, this was the longest wavelength ever to have been used to trigger DNA cleavage upon direct, single photon chromophore sensitization.
Near-infrared cyanine dyes are currently being developed as PDT agents and are used in clinical settings as fluorescent probes in the diagnosis and imaging of cancer. DNA interactions are facilitated by the cyanines' two flanking heteroaromatic nitrogen rings, which share a positive charge that is delocalized through a central polymethine bridge. DNA cleavage by hydroxyl radicals and singlet oxygen has been reported, with excitation wavelengths in the visible range up to 700 nm. A number of cyanine dyes, particularly those with 2-quinoline ring systems, avidly interact with the DNA minor groove as monomers, dimers, and higher order aggregates.
There remains a need for improved methods of photodynamic therapy. There remains a need for improved compounds for use in photodynamic therapy. There remains a need for photodynamic compounds capable of undergoing single photon excitation at longer wavelengths.
Disclosed herein are compounds for use in photodynamic therapy. The compounds are defined by the chemical formula:
wherein A1, A2, R, and X are defined herein.
The details of one or more embodiments are set forth in the descriptions below. Other features, objects, and advantages will be apparent from the description and from the claims.
Before the present methods and systems are disclosed and described, it is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes¬ from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
“Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. “Exemplary” means “an example of” and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes.
Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
The term “alkyl” as used herein is a branched or unbranched hydrocarbon group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, and the like. The alkyl group can also be substituted or unsubstituted. Unless stated otherwise, the term “alkyl” contemplates both substituted and unsubstituted alkyl groups. The alkyl group can be substituted with one or more groups including, but not limited to, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, phosphine or thiol. An alkyl group which contains no double or triple carbon-carbon bonds is designated a saturated alkyl group, whereas an alkyl group having one or more such bonds is designated an unsaturated alkyl group. Unsaturated alkyl groups having a double bond can be designated alkenyl groups, and unsaturated alkyl groups having a triple bond can be designated alkynyl groups. Unless specified to the contrary, the term alkyl embraces both saturated and unsaturated groups.
The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term “heterocycloalkyl” is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, selenium or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. Unless stated otherwise, the terms “cycloalkyl” and “heterocycloalkyl” contemplate both substituted and unsubstituted cyloalkyl and heterocycloalkyl groups. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol. A cycloalkyl group which contains no double or triple carbon-carbon bonds is designated a saturated cycloalkyl group, whereas an cycloalkyl group having one or more such bonds (yet is still not aromatic) is designated an unsaturated cycloalkyl group. Unless specified to the contrary, the term cycloalkyl embraces both saturated and unsaturated, non-aromatic, ring systems.
The term “aryl” as used herein is an aromatic ring composed of carbon atoms. Examples of aryl groups include, but are not limited to, phenyl and naphthyl, etc. The term “heteroaryl” is an aryl group as defined above where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, selenium or phosphorus. The aryl group and heteroaryl group can be substituted or unsubstituted. Unless stated otherwise, the terms “aryl” and “heteroaryl” contemplate both substituted and unsubstituted aryl and heteroaryl groups. The aryl group and heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, or thiol.
Exemplary heteroaryl and heterocyclyl rings include: benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyL cirrnolinyl, decahydroquinolinyl, 2H,6H˜1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, and xanthenyl.
The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl or cycloalkyl group bonded through an ether linkage; that is, an “alkoxy” group can be defined as—OA1 where A1 is alkyl as defined above. “Alkoxy” also includes polymers of alkoxy groups as just described; that is, an alkoxy can be a polyether such as —OA1—OA2 or —OA1-(OA2)a-OA3, where “a” is an integer of from 1 to 200 and A1, A2, and A3 are alkyl groups.
The terms “cycloalkoxy,” “heterocycloalkoxy,” “cycloalkoxy,” “aryloxy,” and “heteroaryloxy” have the aforementioned meanings for alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl, further providing said group is connected via an oxygen atom.
As used herein, the term “null,” when referring to a possible identity of a chemical moiety, indicates that the group is absent, and the two adjacent groups are directly bonded to one another. By way of example, for a genus of compounds having the formula CH3—X—CH3, if X is null, then the resulting compound has the formula CH3—CH3.
As used herein, two atoms connected via the symbol may be connected via a single or double bond.
As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. Unless specifically stated, a substituent that is said to be “substituted” is meant that the substituent can be substituted with one or more of the following: alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, phosphine, or thiol. In a specific example, groups that are said to be substituted are substituted with a protic group, which is a group that can be protonated or deprotonated, depending on the pH.
Unless specified otherwise, the term “patient” refers to any mammalian animal, including but not limited to, humans.
Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesirable toxicological effects. Examples of such salts are acid addition salts formed with inorganic acids, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, and nitric acids and the like; salts formed with organic acids such as acetic, oxalic, tartaric, succinic, maleic, fumaric, gluconic, citric, malic, methanesulfonic, p-toluenesulfonic, napthalenesulfonic, and polygalacturonic acids, and the like; salts formed from elemental anions such as chloride, bromide, and iodide; salts formed from metal hydroxides, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, lithium hydroxide, and magnesium hydroxide; salts formed from metal carbonates, for example, sodium carbonate, potassium carbonate, calcium carbonate, and magnesium carbonate; salts formed from metal bicarbonates, for example, sodium bicarbonate and potassium bicarbonate; salts formed from metal sulfates, for example, sodium sulfate and potassium sulfate; and salts formed from metal nitrates, for example, sodium nitrate and potassium nitrate. Pharmaceutically acceptable and non-pharmaceutically acceptable salts may be prepared using procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid comprising a physiologically acceptable anion. Alkali metal (for example, sodium, potassium, or lithium) or alkaline earth metal (for example, calcium) salts of carboxylic acids can also be made.
The compounds disclosed herein are broadly useful as photosensitizers, for instance as photosensitizers for use in photodynamic therapy. In some embodiments, the compounds can have an absorption maxima of at least 700 nm, at least 725 nm, at least 750 nm, at least 775 nm, at least 800 nm, at least 810 nm, at least 820 nm, at least 830 nm, at least 840 nm, at least 850 nm, at least 860 nm, at least 870 nm, at least 880 nm, at least 890 nm, at least 1,000 nm, at least 1,010 nm, at least 1,020 nm, at least 1,030 nm, at least 1,040 nm, at least 1,050 nm, at least 1,060 nm, at least 1,070 nm, at least 1,080 nm, at least 1,090 nm, or at least 1,100 nm. In some embodiments, the compounds disclosed herein can have an absorption maxima between 600-900 nm, between 650-900 nm, between 700-900 nm, between 725-900 nm, between 750-900 nm, between 775-900 nm, between 800-900 nm, between 825-900 nm, between 825-875 nm, between 850-900 nm, between 700-850 nm, between 750-850 nm, or between 800-850 nm. In other instances, the compounds disclosed herein can have an absorption maxima between 1,000-1,200 nm, between 1,000-1,150 nm, between 1,000-1,100 nm, between 1,000-1,050, between 1,025-1,075 nm, or between 1,050-1,100.
In some embodiments, the compounds disclosed herein do not strongly fluoresce when irradiated at the wavelengths described above. For instance, the compounds disclosed herein can have a fluorescence, as measured by quantum yield relative to quinine sulfate in sulfuric acid solution, no greater than 0.02 Φ, no greater than 0.01 Φ, no greater than 0.005 Φ, no greater than 0.001 Φ, no greater than 0.0005 Φ, no greater than 0.0001 Φ, no greater than 0.00005 Φ, or no greater than 0.00001 Φ.
In some embodiments, the compounds for use in photodynamic therapy can have the formula:
wherein A1 and A2 are individually selected from a heteroaryl ring system. Exemplary heteroaryl ring systems include pyridine, and benzofused analogs thereof, e.g., quinolines, isoquinolines, 1H-perimidines, acridines, phenantridines, and the like. In other embodiments, the heteroaryl ring system can include pyrrole, benzofused pyrroles, furans, benzofused furans, thiophenes, and benzothiophenes. In yet further embodiments, the heteroaryl ring system can include more than one heteroatoms. Such systems include phthalazines, cinnolines, napththryridines, purines, pteridines, quinazolines, quinoxalines, pyrimidines, indazoles, and the like;
R is in each case independently selected from F, Cl, Br, I, NO2, CN, Ra, ORa, N(Ra)2, SO2Ra, SO2N(Ra)2, C(O)Ra; C(O)ORa, OC(O)Ra; C(O)N(Ra)2, N(Ra)C(O)Ra, OC(O)N(Ra)2, N(Ra)C(O)N(Ra)2, wherein Ra is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl. When R is a substituted C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl group, said R groups may be substituted by one or more mitochondria targeting groups, e.g., phosphines, dequaliniums, rhodamines and mitochondrial penetrating peptides.
X is a pharmaceutically acceptable anion.
n can be any interger, for instance, n can be an integer from 1-5. In certain preferred embodiments, n is 1, 2 or 3.
In some embodiments, the compounds can include one or more chelating ligands. As used herein, a chelating ligand is a moiety having at least 2 chelating heteroatoms (e.g., oxygen, nitrogen, sulfur), disposed from one another by 2 or 3 atoms. Such systems bind metals such as copper and iron by forming five and six member rings that include the chelating heteroatoms and the metal. Exemplary systems include 1,2 diols, 1,3 diols, ethylene glycol ethers, propylene glycol ethers, 1,2 dicarbonyls, 1,3 dicarbonyl, 1,2 ethylene diamines, 1,3 propylene diamines, a-hydroxy carbonyls, β-hydroxy carbonyls, α-amino carbonyls, and β-amino carbonyls. In some instances, the chelating ligand can be a crown ether, thia-crown ether, or aza-crown ether, for instance [9]-crown-3; [12]-crown-4, [15]-crown-5; and [18]-crown-6. In this nomenclature, [X] refers to the total number of atoms in the ring system, and the subsequent number refers to the total number of heteroatoms. In some instances, the crown ether will contain only a single type of heteroatom, while in other cases a mixed crown ethers, e.g., those including both oxygen and nitrogen in the ring system, can be employed.
In some embodiments, A1 can be a heterocycle having the formula represented by A1a, A1b, A1c, A1d, A1e, A1f or A1g:
wherein Ra1 is C1-8alkyl, C1-8alkylaryl, C1-8alkoxy, C1-8alkoxyaryl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8alkyl-C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Ra2, is F, Cl, Br, I, NO2, CN, Ra2′, ORa2′, N(Ra2′)2, SO2Ra2′, SO2N(R)2, C(O)Ra2′; C(O)ORa2′, OC(O)Ra2′; C(O)N(Ra2′)2, N(Ra2′)C(O)Ra2′, OC(O)N(Ra2′)2, N(Ra2′)C(O)N(Ra2′)2, wherein Ra2′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Ra3 is F, Cl, Br, I, NO2, CN, Ra3′, ORa3′, N(Ra3′)2, SO2Ra3′, SO2N(Ra3′)2, C(O)Ra3′; C(O)ORa3′, OC(O)Ra3′; C(O)N(Ra3′)2, N(Ra3′)C(O)Ra3′, OC(O)N(Ra3′)2, N(Ra3′)C(O)N(Ra3′)2, wherein Ra3′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Ra4 is F, Cl, Br, I, NO2, CN, Ra4′, ORa4′, N(Ra4′)2, SO2N(Ra4′)2, C(O)Ra4′; C(O)ORa4′, OC(O)Ra4′; C(O)N(Ra4′)2, N(Ra4′)C(O)Ra4′, OC(O)N(Ra4′)2, N(Ra4′)C(O)N(Ra4′)2, wherein Ra4′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Ra5 is F, Cl, Br, I, NO2, CN, Ra5′, ORa5′, N(Ra5′)2, SO2Ra5′, SO2N(Ra5′)2, C(O)Ra5′; C(O)ORa5′, OC(O)Ra5′; C(O)N(Ra5′)2, N(Ra5′)C(O)Ra5′, OC(O)N(Ra5′)2, N(Ra5′)C(O)N(Ra5′)2, wherein Ra5′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Ra6 is F, Cl, Br, I, NO2, CN, Ra6′, ORa6′, N(Ra6′)2, SO2Ra6′, SO2N(Ra6′)2, C(O)Ra6′; C(O)ORa6′, OC(O)Ra6′; C(O)N(Ra6′)2, N(Ra6′)C(O)Ra6′, OC(O)N(Ra6′)2, N(Ra6′)C(O)N(Ra6′)2, wherein Ra6′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Ra7 is F, Cl, Br, I, NO2, CN, Ra7′, N(Ra7′)2, SO2Ra7′, SO2N(Ra7′)2, C(O)Ra7′; C(O)ORa7′, OC(O)Ra7′; C(O)N(Ra7′)2, N(Ra7′)C(O)Ra7′, OC(O)N(Ra7′)2, N(Ra7′)C(O)N(Ra7′)2, wherein Ra7′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl
Ra8 is F, Cl, Br, I, NO2, CN, Ra8′, ORa8′, N(Ra8′)2, SO2N(Ra8′)2, C(O)Ra8′; C(O)ORa8′, OC(O)Ra8′; C(O)N(Ra8)2, N(Ra8′)C(O)Ra8′, OC(O)N(Ra8′)2, N(Ra8′)C(O)N(Ra8′)2, wherein Ra8′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl.
In some embodiments, A2 can be a heterocycle having the formula represented by A2a, A2b, A2c, A2d, A2e, A2f or A2g:
wherein Rb1 is C1-8alkyl, C1-8alkylaryl, C1-8alkoxy, C1-8alkoxyaryl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8alkyl-C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rb2 is F, Cl, Br, I, NO2, CN, Rb2′, ORb2′, N(Rb2′)2, SO2Rb2′, SO2N(Rb2′)2, C(O)Rb2′; C(O)ORb2′, OC(O)Rb2′; C(O)N(Rb2′)2, N(Rb2′)C(O)Rb2′, OC(O)N(Rb2′)2, N(Rb2′)C(O)N(Rb2′)2, wherein Rb2′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rb3 is F, Cl, Br, I, NO2, CN, Rb3′, ORb3′, N(Rb3′)2, SO2Rb3′, SO2N(Rb3′)2, C(O)Rb3′; C(O)ORb3′, OC(O)Rb3′; C(O)N(Rb3′)2, N(Rb3′)C(O)Rb3′, OC(O)N(Rb3′)2, N(Rb3′)C(O)N(Rb3′)2, wherein Rb3′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rb4 is F, Cl, Br, I, NO2, CN, Rb4′, N(Rb4′)2, SO2Rb4′, SO2N(Rb4′)2, C(O)Rb4′; C(O)ORb4′, OC(O)Rb4′; C(O)N(Rb4)2, N(Rb4′)C(O)Rb4′, OC(O)N(Rb4′)2, N(Rb4′)C(O)N(Rb4′)2, wherein Rb4′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rb5 is F, Cl, Br, I, NO2, CN, Rb5′, ORb5′, N(Rb5′)2, SO2Rb5′, SO2N(Rb5′)2, C(O)Rb5′; C(O)ORb5′, OC(O)Rb5′; C(O)N(Rb5′)2, N(Rb5′)C(O)Rb5′, OC(O)N(Rb5′)2, N(Rb5′)C(O)N(Rb5′)2, wherein Rb5′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rb6 is F, Cl, Br, I, NO2, CN, Rb6′, ORb6′, N(Rb6′)2, SO2Rb6′, SO2N(Rb6′)2, C(O)Rb6′; C(O)ORb6′, OC(O)Rb6′; C(O)N(Rb6′)2, N(Rb6′)C(O)Rb6′, OC(O)N(Rb6′)2, N(Rb6′)C(O)N(Rb6′)2, wherein Rb6′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rb7 is F, Cl, Br, I, NO2, CN, ORb7′, N(Rb7′)2, SO2Rb7′, SO2N(Rb7′)2, C(O)Rb7′; C(O)ORb7′, OC(O)Rb7′; C(O)N(Rb7′)2, N(Rb7′)C(O)Rb7′, OC(O)N(Rb7)2, N(Rb7′)C(O)N(Rb7′)2, wherein Rb7′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rb8 is F, Cl, Br, I, NO2, CN, Rb8′, N(Rb8′)2, SO2Rb8′, SO2N(Rb8′)2, C(O)Rb8′; C(O)ORb8′, OC(O)Rb8′; C(O)N(Rb8′)2, N(Rb8′)C(O)Rb8′, OC(O)N(Rb8′)2, N(Rb8′)C(O)N(Rb8′)2, wherein Rb8′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl.
wherein any two or more of Ra1, Ra2, Ra3, Ra4, Ra5, Ra6, Ra7, Ra8, R, Rb1, Rb2, Rb3, Rb3, Rb4, Rb5, Rb6, Rb7, and Rb8 may together form a ring.
In certain embodiments, Ra1 can be a C1-8alkyl group substituted with mitochondria target moiety, e.g., a triphenylphosphonium salt. For instance Ra1 can be CH2CH2—PPh3, CH2CH2CH2—PPh3, CH2CH2CH2CH2—PPh3, and the like.
In certain embodiments, when Ra1 is C1-8alkylaryl, it is preferred that it is benzyl, e.g., CH2-phenyl. The phenyl ring may be unsubstituted, or may be substituted one or more times by groups such as F, Cl, Br, I, NO2, CN, Raa, ORaa, N(Raa)2, SO2Raa, SO2N(Raa)2, C(O)Raa; C(O)ORaa, OC(O)Raa; C(O)N(Raa)2, N(Raa)C(O)Raa, OC(O)N(Raa)2, N(Raa)C(O)N(Raa)2, wherein Raa is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl. For instance, Rat may be a group having the formula:
wherein:
Raa1 is selected from hydrogen, F, Cl, Br, I, NO2, CN, COOH, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Raa2is selected from hydrogen, F, Cl, Br, I, NO2, CN, COOH, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Raa3 is selected from hydrogen, F, Cl, Br, I, NO2, CN, COOH, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Raa4 is selected from hydrogen, F, Cl, Br, I, NO2, CN, COOH, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Raa5 is selected from hydrogen, F, Cl, Br, I, NO2, CN, COOH, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl.
Preferably, one of Raa1, Raa2, Raa3, Raa4, Raa5 is COOH, and the remainder are hydrogen. Even more preferably, Raa3 is COOH and the other groups are hydrogen. In certain embodiments, when Ra1 is C1-8alkylaryl, it is preferred that it is benzyl, e.g., CH2-phenyl. However, in other embodiments Ra1 can be CH2CH2-phenyl, CH2CH2CH2-phenyl, CH2CH2CH2CH2-phenyl. The phenyl ring may be unsubstituted, or may be substituted one or more times by groups such as F, Cl, Br, I, NO2, CN, Raa, ORaa, N(Raa)2, SO2Raa, SO2N(Raa)2, C(O)Raa; C(O)ORaa, OC(O)Raa; C(O)N(Raa)2, N(Raa)C(O)Raa, OC(O)N(Raa)2, N(Raa)C(O)N(Raa)2, wherein Raa is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl.
For instance, Rb1 may be a group having the formula:
wherein:
Rbb1 is selected from hydrogen, F, Cl, Br, I, NO2, CN, COOH, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rbb2is selected from hydrogen, F, Cl, Br, I, NO2, CN, COOH, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rbb3 is selected from hydrogen, F, Cl, Br, I, NO2, CN, COOH, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rbb4 is selected from hydrogen, F, Cl, Br, I, NO2, CN, COOH, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rbb5 is selected from hydrogen, F, Cl, Br, I, NO2, CN, COOH, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl.
Preferably, one of Rbb1, Rbb2, Rbb3, Rbb4, Rbb5 is COOH, and the remainder are hydrogen. Even more preferably, Rbb3 is COOH and the other groups are hydrogen. In certain embodiments, when Ra1 is C1-8alkylaryl, it is preferred that it is benzyl, e.g., CH2-phenyl. However, in other embodiments Rb1 can be CH2CH2-phenyl, CH2CH2CH2-phenyl, CH2CH2CH2CH2-phenyl. The phenyl ring may be unsubstituted, or may be substituted one or more times by groups such as F, Cl, Br, I, NO2, CN, Rbb, ORbb, N(Rbb)2, SO2Rbb, SO2N(Rbb)2, C(O)Rbb; C(O)ORbb, OC(O)Rbb; C(O)N(Rbb)2, N(Rbb)C(O)Rbb, OC(O)N(Rbb)2, N(Rbb)C(O)N(Rbb)2, wherein Rbb is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl.
In certain embodiments, Rb1 can be a C1-8alkyl group substituted with mitochondria target moiety, e.g., a triphenylphosphonium salt. For instance Rb1 can be CH2CH2—PPh3, CH2CH2CH2—PPh3, CH2CH2CH2CH2—PPh3, and the like.
In certain embodiments, n is 1, i.e., a compound having the formula:
wherein A1, A2, and X have the meanings given above,
Rc1 is selected from F, Cl, Br, I, NO2, CN, N(Rc1′)2, SO2Rc1′, SO2N(Rc1′)2, C(O)Rc1′; C(O)ORc1′, OC(O)Rc1′; C(O)N(Rc1′)2, N(Rc1′)C(O)Rc1′, OC(O)N(Rc1′)2, N(Rc1′)C(O)N(Rc1′)2, wherein Rc1′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rc2 is selected from F, Cl, Br, I, NO2, CN, Rc2′, OR2′, N(Rc2′)2, SO2Rc2′, SO2N(Rc2′)2, C(O)Rc2′; C(O)ORc2′, OC(O)Rc2′; C(O)N(Rc2′)2, N(Rc2′)C(O)Rc2′, OC(O)N(Rc2′)2, N(Rc2′)C(O)N(Rc2′)2, wherein Rc2′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rcm is selected from F, Cl, Br, I, NO2, CN, Rcm′, ORcm′, N(Rcm′)2, SO2Rcm′, SO2N(Rcm′)2, C(O)Rcm′; C(O)ORcm′, OC(O)Rcm′; C(O)N(Rcm′)2, N(Rcm′)C(O)Rcm′, OC(O)N(Rcm′)2, N(Rcm′)C(O)N(Rcm′)2, wherein Rcm′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
wherein any two or more of A1, Rc1, Rc2, Rcm, and A2 may together form a ring.
Preferred Rcm groups include hydrogen, F, Cl, Br, optionally substituted phenyl and heteroaryl rings, and C1-8alkyl-COOH groups, e.g. CH2—COOH, CH2CH2—COOH, CH2CH2CH2—COOH, CH2CH2CH2CH2—COOH.
In certain embodiments, n is 2, i.e., a compound having the formula:
wherein A1, A2, and X have the meanings given above;
Rc1 is selected from F, Cl, Br, I, NO2, CN, Rc1′, ORc1′, N(Rc1′)2, SO2Rc1′, SO2N(Rc1′)2, C(O)Rc1′; C(O)ORc1′, OC(O)Rc1′; C(O)N(Rc1′)2, N(Rc1′)C(O)Rc1′, OC(O)N(Rc1′)2, N(Rc1′)C(O)N(Rc1′)2, wherein Rc1′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rc2 is selected from F, Cl, Br, I, NO2, CN, Rc2′, ORc2′, N(Rc2′)2, SO2Rc2′, SO2N(Rc2′)2, C(O)Rc2′; C(O)ORc2′, OC(O)Rc2′; C(O)N(Rc2′)2, N(Rc2′)C(O)Rc2′, OC(O)N(Rc2)2, N(Rc2′)C(O)N(Rc2′)2, wherein Rc2′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rc3 is selected from F, Cl, Br, I, NO2, CN, Rc3′, ORc3′, N(Rc3′)2, SO2Rc3′, SO2N(Rc3′)2, C(O)Rc3′; C(O)ORc3′, OC(O)Rc3′; C(O)N(Rc3′)2, N(Rc3′)C(O)Rc3′, OC(O)N(Rc3′)2, N(Rc3′)C(O)N(Rc3′)2, wherein Rc3′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
wherein Rc4 is selected from F, Cl, Br, I, NO2, CN, Rc4′, ORc4′, N(Rc4′)2, SO2Rc4′, SO2N(Rc4′)2, C(O)Rc4′; C(O)ORc4′, OC(O)Rc4′; C(O)N(Rc4′)2, N(Rc4′)C(O)Rc4′, OC(O)N(Rc4′)2, N(Rc4′)C(O)N(Rc4′)2, wherein Rc4′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rcm is selected from F, Cl, Br, I, NO2, CN, Rcm′, ORcm′, N(Rcm′)2, SO2Rcm′, SO2N(Rcm′)2, C(O)Rcm′; C(O)ORcm′, OC(O)Rcm′; C(O)N(Rcm′)2, N(Rcm′)C(O)Rcm′, OC(O)N(Rcm′)2, N(Rcm′)C(O)N(Rcm′)2, wherein Rcm′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl.
wherein any two or more of A1, Rc1, Rc2, Rc3, Rc4, Rcm, and A2 may together form a ring. Preferred Rcm groups include hydrogen, F, Cl, Br, optionally substituted phenyl and heteroaryl rings, and C1-8alkyl-COOH groups, e.g. CH2—COOH, CH2CH2—COOH, CH2CH2CH2—COOH, CH2CH2CH2CH2—COOH.
In certain embodiments, n is 3, i.e., a compound having the formula:
wherein A1, A2, and X have the meanings given above;
wherein Rc1 is selected from F, Cl, Br, I, NO2, CN, Rc1′, ORc1′, N(Rc1′)2, SO2Rc1′, SO2N(Rc1′)2, C(O)Rc1′; C(O)ORc1′, OC(O)Rc1′; C(O)N(Rc1′)2, N(Rc1′)C(O)Rc1′, OC(O)N(Rc1′)2, N(Rc1′)C(O)N(Rc1′)2, wherein Rc1′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rc2 is selected from F, Cl, Br, I, NO2, CN, Rc2′, ORc2′, N(Rc2′)2, SO2Rc2′, SO2N(Rc2′)2, C(O)Rc2′; C(O)ORc2′, OC(O)Rc2′; C(O)N(Rc2′)2, N(Rc2′)C(O)Rc2′, OC(O)N(Rc2)2, N(Rc2′)C(O)N(Rc2′)2, wherein Rc2′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rc3 is selected from F, Cl, Br, I, NO2, CN, Rc3′, ORc3′, N(Rc3′)2, SO2Rc3′, SO2N(Rc3′)2, C(O)Rc3′; C(O)ORc3′, OC(O)Rc3′; C(O)N(Rc3′)2, N(Rc3′)C(O)Rc3′, OC(O)N(Rc3′)2, N(Rc3′)C(O)N(Rc3′)2, wherein Rc3′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rc4 is selected from F, Cl, Br, I, NO2, CN, Rc4′, ORc4′, N(Rc4′)2, SO2Rc4′, SO2N(Rc4′)2, C(O)Rc4′; C(O)ORc4′, OC(O)Rc4′; C(O)N(Rc4′)2, N(Rc4′)C(O)Rc4′, OC(O)N(Rc4′)2, N(Rc4′)C(O)N(Rc4′)2, wherein Rc4′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
wherein Rc5 is selected from F, Cl, Br, I, NO2, CN, Rc5′, ORc5′, N(Rc5′)2, SO2Rc5′, SO2N(Rc5′)2, C(O)Rc5′; C(O)ORc5′, OC(O)Rc5′; C(O)N(Rc5′)2, N(Rc5′)C(O)Rc5′, OC(O)N(Rc5′)2, N(Rc5′)C(O)N(Rc5′)2, wherein Rc5′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
wherein Rc6 is selected from selected from F, Cl, Br, I, NO2, CN, Rc6′, ORc6′, N(Rc6′)2, SO2Rc6′, SO2N(Rc6′)2, C(O)Rc6′; C(O)ORc6′, OC(O)Rc6′; C(O)N(Rc6′)2, N(Rc6′)C(O)Rc6′, OC(O)N(Rc6′)2, N(Rc6′)C(O)N(Rc6′)2, wherein Rc6′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl;
Rcm is selected from F, Cl, Br, I, NO2, CN, Rcm′, ORcm′, N(Rcm′)2, SO2Rcm′, SO2N(Rcm′)2, C(O)Rcm′; C(O)Rcm′, OC(O)Rcm′; C(O)N(Rcm′)2, N(Rcm′)C(O)Rcm′, OC(O)N(Rcm′)2, N(Rcm′)C(O)N(Rcm′)2, wherein Rcm′ is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl, wherein any two or more of A1, Rc1, Rc2, Rc3, Rc4, Rc5, Rc6, Rcm, and A2may together form a ring. Preferred Rcm groups include hydrogen, F, Cl, Br, optionally substituted phenyl and heteroaryl rings, and C1-8alkyl-COOH groups, e.g. CH2—COOH, CH2CH2—COOH, CH2CH2CH2—COOH, CH2CH2CH2CH2—COOH.
In some embodiments, Rcm is selected from F, Cl, Br, I, aryl, C1-8heteroaryl, and chelating ligands. For instance, Rcm can be an aryl having the formula:
or a C1-8heteroaryl having the formula:
wherein Rd is in each case independently selected from F, Cl, Br, I, NO2, CN, Rd1′, N(Rd1′)2, SO2Rd1′, SO2N(Rd1′)2, C(O)Rd1′; C(O)ORd1′, OC(O)Rd1′; C(O)N(Rd1′)2, N(Rd1′)C(O)Rd1′, OC(O)N(Rd1′)2, N(Rd1′)C(O)N(Rd1′)2, wherein Rd1′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl. In a preferred embodiment, Rd can be a carboxylic acid, (meth)acrylate ester, azide, or aldehyde. Such groups can facilitate conjugation of the compound to a biopolymer such as a protein (including antibody), oligosaccharide, or other agent of interest.
Rd1 is selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl; wherein any two of more of Rd and Rd1 can together form a ring.
In certain case, Rcm is a moiety having the formula:
wherein X1 is F, Cl, Br, I, NO2, CN, Rx1′, ORx1′, N(Rx1′)2, SO2Rx1′, SO2N(Rx1′)2, C(O)Rx1′; C(O)ORx1′, OC(O)Rx1′; C(O)N(Rx1′)2, N(Rx1′)C(O)Rx1′, OC(O)N(Rx1′)2, N(Rx1′)C(O)N(Rx1′)2, wherein Rx1′ is in each case independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl. In some instances X1 can include a chelating ligand as defined above. For instance, X1 can include an ethylene glycol group of formula —(OCH2CH2)—ORL, wherein n is an integer greater or equal to 1, for instance, 1, 2, 3, 4, 5, or 6; and RL is hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl. Preferably, RL is hydrogen or methyl. In other instances, X1 can include a crown ether as defined above.
In some embodiments, any of RCM, X1, Rd and/or Rd1 can include a chelating ligand as defined above. For instance, Rd and/or Rd1 can include an ethylene glycol group of formula —(OCH2CH2)n—ORL, wherein n is an integer greater or equal to 1, for instance, 1, 2, 3, 4, 5, or 6; and RL is hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl. Preferably, RL is hydrogen or methyl. In other instances, RCM, X1, Rd and/or Rd1 can include a crown ether as defined above. In some embodiments, any of RCM, X1, Rd and/or Rd1 can include a mitochondria targeting ligand. For instance, a group having the formula:
wherein T1, T2, and T3 are independently selected from null, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloakyl, C1-8heterocyclyl, an ethylene glycol group of formula —(OCH2CH2)n— wherein n is an integer greater than 1; and Q can be:
wherein m is independently selected from 5-20, n is independently selected from 0-7, and Rq is in each case independently selected from C1-8alkyl, and NH2,
or
wherein R1, R2, R3 and R4 are independently selected from benzyl, 4-hydroxylbenzyl, 4-hydroxy-2,6-dimethylbenzyl, 3-guanidinyl-propyl, and 4-aminobutyl. In certain embodiments, R1 and R3 are independently selected from benzyl, 4-hydroxylbenzyl, 4-hydroxy-2,6-dimethylbenzyl, and R2 and R4 are independently selected from 3-guanidinyl-propyl, and 4-aminobutyl. In other embodiments, R2 and R4 are independently selected from benzyl, 4-hydroxylbenzyl, 4-hydroxy-2,6-dimethylbenzyl, and R1 and R3 are independently selected from 3-guanidinyl-propyl, and 4-aminobutyl.
In some embodiments, the compound can have the formula:
wherein A1, Rcm, Rc1, Rc2, Rcm, Rc5, Rc6, A2 and X have the aforementioned meanings, and Z is selected from null, C(Rz4)2, O, S, SO, SO2, or NRz4, wherein Rz4 is independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl. In some cases Rz4 can be a chelating ligand as described above. Preferred Z groups are those that form six-membered rings, e.g., CH2, O, NRz4, S, SO, and SO2.
Other ring systems can also be formed. For instance, Rc1 and Rc2 can together form a ring, e.g., an aryl ring; Rc2 and Rc3 can together form a ring, e.g., an aryl ring; Rc1 and Rcm can together form a ring, e.g., an aryl ring; Rc5 and Rc6 can together form a ring, e.g., an aryl ring; Rc4 and Rc5 can together form a ring, e.g., an aryl ring; or Rcm and Rc4 can together form a ring, e.g., an aryl ring.
In some embodiments, it is preferred that A1 and/or A2 include fused ring systems. For instance, Ra1 and Ra2 can together form an aryl or heteroaryl ring; Ra2 and Ra3 can together form an aryl or heteroaryl ring; Ra3 and Ra4 can together form an aryl or heteroaryl ring; Ra4 and Ra5 can together form an aryl or heteroaryl ring; Ra5 and Ra6 can together form an aryl or heteroaryl ring; Ra6 and Ra7 can together form an aryl or heteroaryl ring; Ra1 and Ra6 can together form an aryl or heteroaryl ring; or Ra1 and Ra7 can together form an aryl or heteroaryl ring. In certain embodiments, Rb1 and Rb2 can together form an aryl or heteroaryl ring; Rb2 and Rb3 can together form an aryl or heteroaryl ring; Rb3 and Rb4 can together form an aryl or heteroaryl ring; Rb4 and Rb5 can together form an aryl or heteroaryl ring; Rb5 and Rb6 can together form an aryl or heteroaryl ring; Rb6 and Rb7 can together form an aryl or heteroaryl ring; Rb1 and Rb6 can together form an aryl or heteroaryl ring; or Rb1 and Rb7 can together form an aryl or heteroaryl ring. The rings formed by the combination of these radicals may themselves be substituted one or more times by F, Cl, Br, I, NO2, CN, COOH, COOC1-8alkyl, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl. For instance, A1 can have the formula:
wherein Ra8 and Ra9 are independently selected from F, Cl, Br, I, NO2, CN, Ra, ORa, N(Ra)2, SO2Ra, SO2N(Ra)2, C(O)Ra; C(O)ORa, OC(O)Ra; C(O)N(Ra)2, N(Ra)C(O)Ra, OC(O)N(Ra)2, N(Ra)C(O)N(Ra)2, wherein Ra is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8 heterocyclyl.
In certain embodiments, it can be preferred that Ra7 is H, F, Cl, Br, or I. In certain sub-embodiments, each of Ra5, Ra6, Ra8, and Ra9 can be hydrogen, which in others, Ra3 and Ra4 together form a ring, e.g., an aryl ring; Ra4 and Ra5 together form a ring, e.g. an aryl ring; Ra1 and Rc1 together form a ring; Ra5 and Rc2 together form a ring, e.g., an aryl ring.
In certain cases, Ra1 is a C1-4alkyl group, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl group. In other cases, Ra1 is a benzyl group. In yet further embodiments, Ra1 can form a ring with Ra2, Ra1 and Ra2 can together form an aryl ring; Ra2 and Ra1 together form a ring, e.g., an aryl ring.
In certain embodiments, A2 has the formula:
wherein Rb8 and Rb9 are independently selected from F, Cl, Br, I, NO2, CN, Rb, ORb, N(Rb)2, SO2Rb, SO2N(Rb)2, C(O)Rb; C(O)ORb, OC(O)Rb; C(O)N(Rb)2, N(Rb)C(O)Rb, OC(O)N(Rb)2, N(Rb)C(O)N(Ra)2, wherein Rb is in each case independently selected from hydrogen, C1-8alkyl, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl.
In certain embodiments, Rb7 is H, F, Cl, Br, or I. In certain sub-embodiments, each of Rb5, Rb6, Rb8, and Rb9 can be hydrogen, which in others, Rb3 and Rb4 together form a ring, e.g., an aryl ring; Rb4 and Rb5 together form a ring, e.g., an aryl ring; Rb1 and Rc5 together form a ring; Rb5 and Rc4 together form a ring, e.g., an aryl ring.
It can be preferred that Rb1 is a C1-4alkyl, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl group. In some instances, Rb1 is a benzyl group.
In some embodiments, the compounds disclosed herein may be conjugated to one or more targeting ligands to increase selectivity for target tissues. In some instances, the targeting ligand can be an antibody, for instance a monoclonal antibody, a peptide-fragment, or a small molecule. In some instances, the targeting ligand can be selective for one or more tumor or cancer types.
In some embodiments, the compounds can have the formula:
wherein Ra1 and Rb1 are independently selected from C1-8alkyl and C1-8alkylaryl, optionally substituted with one or more groups like COOH, PPh3, OH, F, Cl, and Br; Z is selected from null, C(Rz4)2, O, S, SO, SO2, or NRz4, wherein Rz4 is independently selected from hydrogen, C1-8alkyl, C1-8alkoxy, C2-8alkenyl, C2-8alkynyl, aryl, C1-8heteroaryl, C3-8cycloalkyl, or C1-8heterocyclyl, and Rcm is selected from F, Cl, Br, I, C1-8alkyl, aryl, C1-8heteroaryl. In preferred embodiments, Ra1 and Rb1 are (CH2)nPPh3 (CH2)nPh-4-COOH and (CH2)nPh, wherein n is 1, 2, 3, 4, or 5, and Ph-4-COOH refers to a 4-yl-benzoic acid group. In preferred embodiments, Z is CH2, and Rcm is selected from H, Cl, Br, aryl, heteroaryl, (CH2)nPPh3, and (CH2)nCOOH, wherein n is 1, 2, 3, 4, or 5. In certain embodiments, Rcm can be an aryl or heteroaryl group further substituted with (CH2)nPPh3 or (CH2)nCOOH.
Exemplary compounds include:
Compounds may be designated 1-A, 1-B, 1-C, 1-D, etc. . . , to reflect the substituent pattern and parent methine dye structure.
In certain instances, compounds having the following structures can be used:
The skilled person will appreciate that while the above compounds are depicted as iodide and chloride salts, other pharmaceutically acceptable anions may also be employed.
Also disclosed are compounds having the formula:
wherein Rcm and X are as defined above. In certain preferred embodiments, Rcm is H, Cl, or Br.
as well as the meso fluorine, bromine and iodine compound at the RCM position; the compound in which RCM is hydrogen has been published, however, it has not been disclosed or suggested as a compound useful for photodynamic therapy (i.e., photoinitiated cleavage of DNA);
while published, these compounds have not been explored for use in photodynamic therapy (i.e., photoinitiated cleavage of DNA).
The compounds disclosed herein may be formulated in a wide variety of pharmaceutical compositions for administration to a patient. Such compositions include, but are not limited to, unit dosage forms including tablets, capsules (filled with powders, pellets, beads, mini-tablets, pills, micro-pellets, small tablet units, multiple unit pellet systems (MUPS), disintegrating tablets, dispersible tablets, granules, and microspheres, multiparticulates), sachets (filled with powders, pellets, beads, mini-tablets, pills, micro-pellets, small tablet units, MUPS, disintegrating tablets, dispersible tablets, granules, and microspheres, multiparticulates), powders for reconstitution, transdermal patches and sprinkles, however, other dosage forms such as controlled release formulations, lyophilized formulations, modified release formulations, delayed release formulations, extended release formulations, pulsatile release formulations, dual release formulations and the like. Liquid or semisolid dosage form (liquids, suspensions, solutions, dispersions, ointments, creams, emulsions, microemulsions, sprays, patches, spot-on), injection preparations, parenteral, topical, inhalations, buccal, nasal etc. may also be envisaged under the ambit of the invention.
Suitable excipients may be used for formulating the dosage forms according to the present invention such as, but not limited to, surface stabilizers or surfactants, viscosity modifying agents, polymers including extended release polymers, stabilizers, disintegrants or super disintegrants, diluents, plasticizers, binders, glidants, lubricants, sweeteners, flavoring agents, anti-caking agents, opacifiers, anti-microbial agents, antifoaming agents, emulsifiers, buffering agents, coloring agents, carriers, fillers, anti-adherents, solvents, taste-masking agents, preservatives, antioxidants, texture enhancers, channeling agents, coating agents or combinations thereof.
The compounds disclosed herein may be administered by a number of different routes. For instance, the compounds may be administered orally, topically, transdermally, intravenously, subcutaneously, by inhalation, or by intracerebroventricular delivery.
In some embodiments, the compounds disclosed herein may be formulated as nanoparticles. The nanoparticles may have an average particle size from 1-1,000 nm, preferably 10-500 nm, and even more preferably from 10-200 nm.
The compounds may be administered to a patient systemically, e.g., by oral or intravenous administration, topically, i.e., by application of a cream, lotion or the like, or locally, e.g., by direct perfusion of a composition containing the compound to a target tissue. Once administered, the compounds may be activated for DNA cleavage by targeted application of light at the relevant wavelength.
After administration of the compounds, the patient may be exposed to irradiation directed at the site of interest, e.g., a tumor. In some embodiments, the patient is irradiated with a laser having a wavelength greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm, greater than 1000 nm, greater than 1025 nm, greater than 1050 nm, greater than 1075 nm, greater than 1100 nm, greater than 1125 nm, greater than 1150 nm, greater than 1125 nm, or greater than 1200 nm. In certain instances, because the disclosed compounds can preferentially accumulate in tumor cells and growths, a delay between administration and irradiation can be used to allow the compounds to be cleared from healthy tissues. For instance, the irradiation can take place 6 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 30 hours, 36 hours, or 48 hours after administration of the compounds.
The following examples are for the purpose of illustration of the invention only and are not intended to limit the scope of the present invention in any manner whatsoever.
Deionized distilled water was used for buffer and DNA sample preparation. PUC19 plasmid DNA was cloned in XL-1 blue E. coli competent cells (Stratagene) according to standard laboratory protocols and was purified using a QIAfilter Plasmid Mega Kit (Qiagen™, Cat. No. 12263) by following the manufacturer's instructions. All reagents were of the highest purity available. Sonicated calf thymus (CT) DNA was obtained from Invitrogen (Cat. No. 15633-019; 10 mg/mL, average size≤2000 bp). Sodium phosphate monobasic and sodium phosphate dibasic came from Thermo Fisher Scientific. Deuterium oxide (99.9%) was supplied by Cambridge Isotope Laboratories. All other chemicals, including sodium azide (≥99.99%), sodium benzoate (99%), and dimethyl sulfoxide (DMSO, ≥99.99%) were from Sigma-Aldrich.
Synthetic Procedures
4-methylquinolinium iodide (I): Quinolinium salt 1 was obtained by the reaction of 4-methylquinoline (1 equiv) with iodomethane (4 equiv) in anhydrous acetonitrile refluxed at 90° C. for 72 h. Thin layer chromatography (TLC) was used to monitor the progress of the reaction eluting with a 4:1 mixture of DCM:hexanes. Upon completion, the reaction mixture was allowed to cool to room temperature and diethyl ether was added to precipitate the iodide salt. The solid was collected by vacuum filtration and washed with diethyl ether (3×25 mL). The salt was used without further purification in subsequent reactions.
General Procedure for the Synthesis of Polymethine Precursors
Polymethine precursor 3 was purchased from Sigma Aldrich and used as-is. Mucochloric acid (1 equiv) was dissolved in ethanol. A solution of aniline (2 equiv) was added dropwise over 10 min, and the resulting mixture was stirred and heated to 40° C. until the evolution of CO2 (g) was observed to cease. The mixture was then cooled in an ice bath and diethyl ether was slowly added to induce precipitation of the reaction product. The resulting solid was collected by vacuum filtration, washed with diethyl ether (3×25 mL), and used without any additional purification.
Salt 1 (2 equiv) and the corresponding polymethine precursors 2 or 3 (1 equiv) were dissolved in acetic anhydride. Trimethylamine (TEA) (0.1 mL) was added and the reaction mixture was stirred and heated to 75° C. Reaction progress was monitored by UV-visible spectrophotometry. Upon completion of the reaction, diethyl ether was added to precipitate the final dyes 4 and 5, which were collected by vacuum filtration. The dyes were purified by recrystallization from methanol/diethyl ether.
1-methyl-4-((1E,3E)-5-((Z)-1 -methylquinolin-4(1H)-ylidene)penta-1,3-dien-1-yl)quinolin-1-ium iodide (4): MP 239° C. (Dec); 1H NMR (400 MHz, DMSO-d6) δ ppm 3.97 (s, 6 H) 6.65 (t, J=12.25 Hz, 1 H) 6.99 (d, J=13.39 Hz, 2 H) 7.32 (d, J=7.33 Hz, 2 H) 7.59 (t, J=7.07 Hz, 2 H) 7.78-7.89 (m, 4 H) 7.95 (t, J=13.01 Hz, 2 H) 8.09 (d, J=7.07 Hz, 2 H) 8.42 (d, J=8.34 Hz, 2 H); 13C NMR (100 MHz, DMSO-d6) δ ppm 41.47, 107.80, 110.48, 117.22, 118.93, 124.18, 124.74, 125.90, 128.62, 132.46, 138.86, 141.36, 145.80, 146.89; HRMS (TOF MS ESI+): calc'd for C25H23N2+: m/z 351.1856 ([M]+), Found: m/z 351.0218 [M]+
4-((1E,3Z)-3-chloro-5-((Z)-1-methylquinolin-4(1H)-ylidene)penta-1,3-dien-1-yl)-1-methylquinolin-1-ium iodide (5): MP>260° C.; 1H NMR (400 MHz, DMSO-d6) δ ppm 4.05 (s, 6 H) 6.94 (d, J=13.14 Hz, 2 H) 7.41 (d, J=7.33 Hz, 2 H) 7.67 (t, J=5.81 Hz, 2 H) 7.86-7.97 (m, 4 H) 8.14 (d, J=12.88 Hz, 2 H) 8.30 (d, J=7.07 Hz, 2 H) 8.41 (d, J=8.34 Hz, 2 H); HRMS (TOF MS ESI+): calc'd for C25H22ClN2+: m/z 385.1466 ([M]+), Found: m/z 385.1964 [M]+.
Cyanine dyes 4 and 5 were stored in a −4° C. freezer as 2.5 mM stock solutions in DMSO.
Compound 6 (X═Br) has also been prepared.
A PerkinElmer Lambda 35 spectrophotometer, a Shimadzu UV-2401PC spectrophotometer and a PerkinElmer LS-55 fluorescence spectrometer were respectively used to record UV-visible absorption spectra and fluorescence emission spectra. Circular dichroism (CD) and induced circular dichroism (ICD) spectra were acquired with a Jasco J-810 or a Jasco J-1500 CD spectropolarimeter. At wavelengths from 190 nm to 1000 nm, the Jasco J-1500 CD spectropolarimeter was fitted with a Jasco EXPM-531 NIR extender to enhance the sensitivity of the CD signal in this range.
The absorbance of cyanine dyes was measured at 22° C. with a UV-visible spectrophotometer. Cuvettes contained 10 μM of dye in DMSO or 10 μM of dye in 10 mM of sodium phosphate pH 7.0 buffer without and with 150 μM bp CT DNA. Absorption spectra were recorded at 5 min time intervals from 0 min up to 25 or 30 min
In DNA titration experiments, small volumes of an aqueous solution of 15,111 μM bp CT DNA were sequentially added to samples containing 20 μM of cyanine dye in 10 mM sodium phosphate buffer pH 7.0 (500 μL initial volume). All absorption spectra were corrected for sample dilution. Final concentrations of CT DNA in each sample ranged from 0 μM bp up to 2684 μM bp.
Individual DNA cleavage reactions containing 5 μM to 50 μM concentrations of cyanine dye, 38 μM bp of pUC19 plasmid, and 10 mM of sodium phosphate pH 7.0 were prepared in a total volume of 40 μL. In order maintain reaction temperature at 10° C., 22° C., or 37° C., the samples were placed in a thermometer-fitted metal block that was either heated, kept at room temperature, or immersed in an ice bath. While monitoring temperature with the thermometer, the samples were either kept in the dark or were irradiated at time interval ranging from 5 min to 120 min using a light emitting diode (LED) laser (LaserLands) with a peak emission wavelength of either 532 nm (100 mW), 808 nm (300 mW), or 830 nm (300 mW). At the end of the irradiation time interval, a total of 3 μL of electrophoresis loading buffer containing 15.0% (w/v) ficoll and 0.025% (w/v) bromophenol blue) was added to each reaction and 20 μL of the resulting solution was added to one of the wells of 1.5% agarose gel stained with ethidium bromide (0.5 μg/mL, final concentration). Completely loaded gels were then electrophoresed for ˜60 min at 105 V in a Bio-Rad Laboratories gel box using 1×tris-acetate-EDTA (TAE) containing 0.5 μg/mL ethidium bromide as the running buffer. Electrophoresed gels were visualized at 302 nm with a VWR Scientific LM-20E transilluminator and then photographed with a UVP PhotoDoc-It™ Imaging System. For quantitating the gels, ImageQuant version 5.2 software was employed. The DNA photocleavage yields were then calculated using the formula:
Percent Photocleavage=[(Linear+Nicked DNA)/(Linear+Nicked+Supercoiled DNA)]×100.
Individual samples for CD analysis consisted of 10 mM sodium phosphate buffer pH 7.0 with 10 μM of dye and 120 μM of CT-DNA present alone and in combination (total volume of 3000 μL). Spectra were collected from 900 to 200 nm in 3 mL (1.0 cm) quartz cuvettes (Starna) using the following instrument settings: scan speed, 100 nm/min; response time, 2 s; bandwidth, 0.5 nm; sensitivity, 100 millidegrees. Final spectra were averaged over 12 acquisitions.
Extended, near-infrared circular dichroism spectra were recorded from 1000 nm to 600 nm for samples containing 10 mM sodium phosphate buffer pH 7.0, 25 μM of dye, and 990 μM bp of CT DNA (3,000 μL total volume). The scan speed was set at 100 nm/min, the response time was 2 s, and the bandwidth and sensitivity were 0.5 nm and 200 millidegrees, respectively. Final spectra were averaged over 12 acquisitions.
Solutions containing 10 mM sodium phosphate buffer pH 7.0 and 10 μM of cyanine dye in the absence and presence of either 100 μM bp or 990 μM bp CT DNA were transferred to 3.0 mL Starna quartz cuvettes (3,000 μL, total volume). The samples were excited at 550 nm and 800 nm and emission spectra were respectively recorded from 555 nm to 900 nm and from 805 nm to 900 nm (22° C.).
In an argon-purged glove box, 40 μL photocleavage reactions containing 10 mM sodium phosphate buffer pH 7.0, 20 μM of cyanine dye, and 38 μM bp of pUC19 plasmid were prepared from solutions bubbled with argon and then irradiated at 830 nm for 30 min The procedure was repeated using aerated solutions in a glove box purged with air.
A second set of reactions containing 10 mM of sodium phosphate buffer pH 7.0, 38 μM bp pUC19 plasmid DNA and 20 μM of dye were prepared in the presence and absence of either 100 mM of the singlet oxygen scavenger sodium azide, 100 mM of the hydroxyl radical scavenger sodium benzoate, or 84% D2O (v/v). The reactions were aerobically irradiated on the bench top for 30 min (830 nm).
After the irradiation, the above DNA reactions were electrophoresed on 1.5% non-denaturing agarose gels, visualized, and quantitated as just described. The percent change in DNA photocleavage was then calculated using the formula, where additive stands for either argon, sodium azide, sodium benzoate, or D2O:
Percent Change in Cleavage=[(% Total of Linear and Nicked DNAwith additive−% Total of Linear and Nicked DNAwithout additive)/(% Total of Linear and Nicked DNAwithout additive)]×100.
1O2 & •OH
1O2 > •OH
1O2
Reactions consisting of 38 μM bp of pUC19 plasmid DNA equilibrated with 20 μM of 5 or 6, with and without 100 mM of scavenger or 84% D2O (v/v) were irradiated for 30 mM with a 830 nm, 300 mW LED laser (10 mM sodium phosphate buffer pH 7.0;
Solutions containing 10 mM sodium phosphate buffer pH 7.0, and 3 μM of hydroxyphenyl fluorescein (HPF) in the presence and absence of 20 μM of 5 were prepared. In a parallel reaction, a total 100 mM of sodium benzoate was used as a hydroxyl radical scavenging reagent. Samples were then kept in the dark or irradiated with an 830 nm LED laser (2.8 W/cm2) for 30 mM To generate hydroxyl radicals, aqueous solutions containing 10 μM H2O2, 10 μM ammonium iron(II) sulfate, 3 μM HPF, and 10 mM sodium phosphate buffer pH 7.0 in the presence and absence of 100 mM of sodium benzoate were equilibrated in the dark for a few minutes (22° C).5 Fluorescence emission spectra were immediately recorded using a PerkinElmer LS55 spectrofluorometer (□ex=490 nm).
Reactions containing 10 mM sodium phosphate buffer pH 7.0 and 0.75 μM of Singlet Oxygen Sensor Green® (SOSG) in the presence and absence of 20 μM of 5 were prepared. Samples were then kept in the dark or irradiated with an 830 nm LED laser (2.8 W/cm2) for 30 mM As a positive control for hydroxyl radical detection, an aqueous solution containing 10 μM H2O2, 10 μM ammonium iron(II) sulfate, 750 nM of SOSG, and 10 mM sodium phosphate buffer pH 7.0 was equilibrated in the dark for a few minutes (22° C).5 To generate singlet oxygen, solutions containing 1 μM of methylene blue and 10 mM sodium phosphate buffer pH 7.0 were kept in the dark or irradiated with a 638 nm LED laser (2.8 W/cm2, LaserLand) for 2 s. Fluorescence emission spectra were immediately recorded with a PerkinElmer LS55 spectrofluorometer (□ex=480 nm).
ES2 human clear cell ovarian carcinoma cell line was obtained from ATCC (Manassas, Va.). All cancer cells were cultured in DMEM medium (Sigma, St. Louis, Mo.) with 10% fetal bovine serum (VWR, Visalia, Calif.) and 1.2 mL/100 mL penicillinstreptomycin (Sigma, St. Louis, Mo.). All cells were grown in a humidified atmosphere of 5% CO2 (v/v) in air at 37° C.6
Cellular Uptake and Fluorescence Imaging
ES2 cells were plated in 96-well plates at a density of 10×103 cells/well and cultured for 24 h. After that cells were incubated with the dye 5 (10 μg/mL) dissolved in DMEM (10% fetal bovine serum) for 24 h. To visualize the subcellular distribution of the dye, nuclei of ES2 cells were stained with Hoechst 33342. Before imaging, cells were washed with DPBS. Images were collected with an BZ-X710 Keyence microscope using DAPI filter and Cy® 7 filter cubes.7
ROS Measurements
Intracellular ROS levels were evaluated with the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay according the previously described procedure.8 Briefly, ES2 cells were seeded in 96-well plates at a density of 10×103 cells/well and cultured for 24 h. Subsequently, cells were incubated with the dye 5 dissolved in cell culture medium (1.0 μg/mL) for 24 h. Then, the cells were rinsed with DPBS and 100 μL of 10 μM DCFH-DA was added under dark conditions and incubated for 30 mM prior to light treatment. The test samples were exposed to a 808 nm laser diode light for 5 mM (0.3 W/cm2). Non-treated cells, cells incubated with the same concentration of the dye 5 under dark conditions, and cells exposed to a 808 nm laser diode for 5 mM were used as controls. Fluorescence was measured using a multiwell plate reader with 485 nm excitation and 528 nm emission filters.
Evaluation of Phototherapeutic Effect
ES2 cells were plated in 96-well plates at a density of 10×103 cells/well and cultured for 24 h. After that cells were incubated in the dark with the dye 5 (10 μg/mL) dissolved in DMEM (10% fetal bovine serum) for 24 h. The dye-containing medium was then removed and the cells were rinsed with warm DPBS before fresh medium was added. Subsequently, cells were exposed to a 808 nm laser diode light for 10 min (0.3 W/cm2). After treatment, cells were cultured for 24 h in growth medium prior to viability measurements with Calcein AM as previously described.9 Non-treated cells, cells incubated with the same concentration of the dye 5 under dark conditions, and cells exposed to a 808 nm laser diode for 5 min were used as controls.
The following compounds were also evaluated:
ES2 cells were plated at 10 k cells/well and left overnight. The solubilized dye was then put into the wells and left for 24 hours. The wells were then washed with DPBS and appropriate wells were exposed to the laser (5 min/well). The wells were then left in fresh media overnight. CalceinAM was then incubated was allowed to incubate for 1 hour and fluorescence was recorded on a plate reader (Synergy HT, BioTek Instruments, Winooski, Vt.) using 485 nm excitation and 528 nm emission filters.
ES2 cells were plated in a 96 well plate. 0.5 mg/ml of the dyes were placed in the wells for 24 hours.
ES2 cells were plated at 10 k cells/well and left overnight. The solubilized dye was then put into the wells and left for 24 hours. The plate was then washed with DPBS and H2DCFDA was added to each well. After 40 min, they wells were lasered and read at Read at Ex/Em: ˜492−495/517−527 nm.
The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than in the examples, or where otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches
This application claims the benefit of U.S. Provisional Application 62/769,301, filed Nov. 19, 2018, the contents of which are hereby incorporated in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/062210 | 11/19/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62769301 | Nov 2018 | US |
