Halogenated compounds for photodynamic therapy

Abstract

Halo-organic heterocyclic compounds are described, in which at least two halogen atoms are bound to a nitrogen-containing heterocyclic terminal moiety of the compound, with at least one of such halogen atoms being iodine or bromine. Also described are polymethine dyes based on these heterocyclic compounds, and dendrimeric compounds and conjugates of such polymethine dyes. The polymethine dyes are characterized by enhanced properties, e.g., brightness, photostability, sensitivity and/or selective affinity that make them useful to target cancer cells, pathogenic microorganisms, and/or other biological materials, in applications such as photodynamic therapy, photodynamic antimicrobial chemotherapy (PACT), cancer treatment, selective removal or attachment of biological materials, antimicrobial coating materials, and other diagnostic, theranostic, spectrum shifting, deposition/growth, and analytic applications.

Claims

1. A method of generating reactive oxygen species (ROS) at a biological locus, comprising: (a) introducing to said locus a composition comprising a dye comprising at least one of moieties Het and Het.sup.+, or a conjugate thereof, wherein Het and Het.sup.+have the formulae: ##STR00152## wherein: ring A is an aromatic or heterocyclic ring having at least two halo substituents thereon, wherein at least one of said halo substituents is iodine or bromine; each of X.sup.1, X.sup.2, X.sup.3, and X.sup.4 is independently selected from the group consisting of N, .sup.+NR.sup.N3, and CR.sup.X, R.sup.N1, R.sup.N2 and R.sup.N3 may be independently selected from the substituents R.sup.N; R.sup.N is selected from H, L-S.sub.c, L-R.sup.R, L-R.sup., aliphatic groups, alicyclic groups, alkylaryl groups, and aromatic groups, wherein each aliphatic residue may incorporate up to ten heteroatoms selected from N, O, and S, and can be substituted one or more times by F, Cl, Br, I, hydroxy, alkoxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, alkyl-amino, dialkylamino or trialkylammonium; R.sup.X is H, F, Cl, Br, I, L-S.sub.c, L-R.sup.R, L-R.sup.; aliphatic, alicyclic, or aromatic group; amino or substituted amino; sulfo, trifluoromethyl, hydroxy, alkoxy, carboxy, phosphate, phosphonate, or sulfate; or adjacent R.sup.X substituents, taken in combination, form a fused aromatic or heterocyclic ring that is itself optionally further substituted by H, F, Cl, Br, I, L-S.sub.c, L-R.sup.R, L-R.sup.; aliphatic, alicyclic, or aromatic group; amino or substituted amino; sulfo, trifluoromethyl, hydroxy, alkoxy, carboxy, phosphate, phosphonate, or sulfate; each R.sup.C1 and R.sup.C2 is independently R.sup.C, or adjacent substituents (R.sup.C1, R.sup.C2) form a cyclic or heterocyclic system that further may be substituted or fused; R.sup.C is H, L-S.sub.c, L-R.sup.R, L-R.sup., aliphatic, alicyclic, aromatic, heteroatom-substituted aliphatic, polyether, alkyl-aryl, aryl-alkyl, F, Cl, Br, I, NH.sub.2, COOH, CN, azido, OH, NO.sub.2, SO.sub.3H, SO.sub.2NHR.sup.m, SO.sub.2NHN(R.sup.m).sub.2, sulfone, C.sub.6H.sub.4SO.sub.3H, C.sub.6H.sub.4PO.sub.3H, CH.sub.2C.sub.6H.sub.4SO.sub.3H, CH.sub.2C.sub.6H.sub.4PO.sub.3H, pyridylium, pyrylium, PO(OH).sub.2, OPO(OH).sub.2, PO(OH)(OR.sup.m), OP(OH)(OR.sup.m), PO(OR.sup.m).sub.2, (CH.sub.2CH.sub.2O).sub.nPO(OR.sup.m).sub.2, (OCH.sub.2CH.sub.2).sub.nPO(OR.sup.m).sub.2, wherein n=1-30, CONH.sub.2, CON(R.sup.m).sub.2, CONHN(R.sup.m).sub.2, COONHS or COOR.sup.m, wherein R.sup.m is selected from a group consisting of H, L-S.sub.c, L-R.sup.R, L-R.sup., aliphatic substituents, and aromatic substituents, and each aliphatic residue may incorporate up to ten heteroatoms selected from N, O, S, and can be substituted by F, Cl, Br, I, hydroxy, alkoxy, carboxy, sulfo, phosphate, amino, sulfate, phosphonate, cyano, nitro, azido, alkyl-amino, dialkyl-amino or trialkylammonium; R.sup.R is a reactive group; R.sup. is an ionic or hydrophilic group; L is a single covalent bond or is a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-20 non-hydrogen atoms selected from the group consisting of C, N, P, O and S, arranged so that the linkage contains any combination of ether, thioether, amine, ester, amide bonds; single, double, triple or aromatic carbon-carbon bonds; or carbon-sulfur bonds, carbon-nitrogen bonds, phosphorus-sulfur, nitrogen-nitrogen, nitrogen-oxygen or nitrogen-platinum bonds, or aromatic or heteroaromatic bonds; S.sub.c is a moiety that is conjugated to ring A through linker, L, and is selected from the group consisting of alkanes, alkenes, alkynes, polyethers, polyamines, cycloparaffins, cycloolefins, cyclynes, heteroalicyclic groups, aromatic groups, heteroaromatic groups, organic dyes, polymethines, dendrimers, nanoparticles, biological molecules, and biological compounds, and wherein the alkanes, alkenes, alkynes, polyethers, polyamines, cycloparaffins, cycloolefins, cyclynes, heteroalicyclic groups, aromatic groups, heteroaromatic groups, organic dyes, polymethines, and dendrimers are optionally substituted with one or more substituents selected from the group consisting of F, Cl, Br, I, carboxylic acid, sulfonic acid, CN, nitro, hydroxy, phosphate, phosphonate, sulfate, cyano, azido, amine, alkyl, alkoxy, trialkylammonium and aryl; and Y is O, S, Se, Te, C(R.sup.C1)(R.sup.C2), or NR.sup.N2; and (b) transmitting radiation to said composition at said locus that is effective to cause the composition to generate ROS at said locus.

2. The method of claim 1, further comprising introducing exogenous oxygen or ozone to said locus to increase available oxygen at said locus for generating ROS at said locus.

3. The method of claim 1, wherein said composition is introduced to said locus on a carrier.

4. The method according to claim 3, wherein the carrier comprises at least one carrier species selected from the group consisting of bacteriophages and antibodies.

5. The method of claim 1, wherein the dye in said composition is substantially completely photobleached by said radiation transmitted to said locus.

6. The method of claim 5, wherein the radiation transmitted to said locus is limited to the amount required for the dye in said composition to be substantially completely photobleached by said radiation transmitted to said locus.

7. The method of claim 1, wherein the biological locus comprises a corporeal locus of a human or animal subject.

8. The method of claim 1, wherein the composition comprises a self-limiting ROS generator photosensitizer dye or dye conjugate, the biological locus comprises a human body, and the radiation transmitted to said locus comprises low intensity activating spectrum light that is exposed over large areas of the body to deactivate residual photosensitizer dye or dye conjugate without inducing sufficient reactivity to damage the body.

9. The method of claim 8, wherein the transmitted radiation is progressively increased in intensity to remove sensitivity of the dye of the dye conjugate.

10. The method of claim 8, wherein the transmitted radiation is changed in intensity and/or spectrum during the transmission thereof to the locus.

11. The method of claim 1, wherein the composition comprises a self-limiting ROS generator photosensitizer dye or dye conjugate, the biological locus comprises a human body, and the radiation transmitted to said locus comprises light that is effective to activate the photosensitizer dye or dye conjugate to treat the body for cancer, pathogen, or other biomaterial in tissue, blood or bone of the body.

12. The method of claim 1, wherein the radiation is transmitted to said locus over a period of time in a range of from 4 to 5000 hours.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic representation of dye molecules bound to a carrier.

(2) FIG. 2 is a schematic representation of sensitizer molecules bound to a carrier by linker-spacer groups with reactive group(s).

(3) FIG. 3 is a schematic representation of conjugates of sensitizer molecules with quencher, reporter dye and carrier components.

(4) FIG. 4 is a graph of absorption spectra of iodinated cyanines I-52 and I-72 as compared to ICG in phosphate buffer (pH 7.4).

(5) FIG. 5 is a graph of relative decrease of the long-wavelength absorption band (photostability) of iodinated cyanine I-72 as compared to ICG in water upon irradiation with a 200 mW 660-nm diode laser.

(6) FIG. 6 is a graph of relative decrease of the emission intensity (photostability) of iodinated cyanine I-72 as compared to ICG in water upon irradiation with a 200 mW 660-nm diode laser.

(7) FIG. 7 is a graph of phototoxicity of sensitizers estimated via hemolysis level (0.1 M, 250 W IR lamp, 60 min exposure time).

(8) FIG. 8 is a graph of phototoxicity of sensitizers estimated via hemolysis level (0.5 M, 250 W IR lamp, 30 min exposure time).

(9) FIG. 9 is a graph of emission spectra of the 250 W IR lamp, which was used for irradiation.

(10) FIG. 10 is a graph of hemolysis level as a function of exposure time for 1 M dye concentrations at irradiation with constant laser power (786 nm laser, 68 mW).

(11) FIG. 11 is a graph of hemolysis level as a function of exposure time for 5 M dye concentrations at irradiation with constant laser power (786 nm laser, 68 mW).

DETAILED DESCRIPTION

(12) As used herein, the following abbreviations shall have the following definitions.

(13) TABLE-US-00001 Abbreviation Definition A Absorbance (Optical density) Alk Alkyl group arom Aromatic BSA Bovine serum albumin Bu Butyl d Doublet signal cm Centimeter (10.sup.2 meter) DCC Dicyclohexylcarbodiimide DMF Dimethylformamide DMSO Dimethyl sulfoxide D/P Dye-to-protein ratio Et Ethyl FDA US Food and Drug Administration g Grams h Hours HITC 1,1,3,3,3,3-hexamethylindotricarbocyanine (CAS 19764-96-6) .sup.1H-NMR Proton nuclear magnetic resonance (hydrogen-1 nuclear magnetic resonance) Hz Hertz ICG Indocyanine green (CAS 58984-23-9) IgG Immunoglobulin G IR Infrared NIR Near-infrared L Liters LED Light emitting diode m Milli (10.sup.3) m Multiplet signal in .sup.1H-NMR max Maximum M Molar mg Milligram (10.sup.3 gram) Me Methyl MHz Megahertz (10.sup.6 Hertz) mol Mole nm Nanometer (10.sup.9 meter) NHS N-hydroxysuccinimide NIR Near infrared PACT Photodynamic antimicrobial chemotherapy PB Phosphate buffer PDT Photodynamic therapy ppm Parts per million Py Pyridine q Quartet signal in .sup.1H-NMR ROS Reactive oxygen species (in this disclosure we include oxygen and non-oxygen containing reactive species induced by photosensitizers) s Singlet signal in .sup.1H-NMR SOG Singlet oxygen generation or singlet oxygen generator or sensitizer t Triplet signal in .sup.1H-NMR TMS Tetramethylsilane TSTU N,N,N,N-tetramethyl(succinimido)uronium tetra- fluoroborate W Watt Wavelength .sub.max (abs) Wavelength of absorption maximum .sub.max (em) Wavelength for emission (fluorescence) maximum Extinction coefficient Micro (10.sup.6) .sub.F Fluorescence (luminescence) quantum yield .sub. Quantum yield of singlet oxygen generation

(14) As used herein, the following terms shall have the following meanings, unless the context shall otherwise expressly require.

(15) Effects

(16) Photodynamic means generating ROS or localized toxicity from the absorption of light.

(17) Sonodynamic means generating ultrasound from the absorption of modulated light, or generation of light or photoreactivity using ultrasound.

(18) Parameters

(19) Extinction coefficient (E) is a wavelength-dependent measure of the absorbing power of a luminophore.

(20) Excitation spectrum is the dependence of emission intensity on the excitation wavelength (.sub.Ex), measured at a single constant emission wavelength.

(21) Emission spectrum is the wavelength distribution of the emission, measured at excitation with a single constant excitation wavelength.

(22) Stokes' shift (.sub.St) is the difference in wavelengths between the maximum of the emission spectrum and the maximum of the absorption spectrum.

(23) Luminescence lifetime () is the average time that a luminophore spends in the excited state prior to returning to the ground state.

(24) Fluorescence quantum yield (.sub.F) is the ratio of the number of photons emitted to the number of photons absorbed by a luminophore.

(25) Brightness is an extinction coefficient multiplied by a fluorescence quantum yield.

(26) Singlet oxygen generation quantum yield (.sub.A) is the ratio of the number of generated singlet oxygen molecules to the number of photons absorbed by a sensitizing dye.

(27) Clearing rate is an efficiency of compound removal from the body or some portion of the body.

(28) Compounds by Application

(29) Dye is a compound absorbing light in the ultraviolet (UV), visible, near-infrared (NIR, near-IR), or infrared (IR) spectral range.

(30) Chromophore is a part of a molecule responsible for the light absorption.

(31) Fluorophore is a molecule or a part of a molecule responsible for the fluorescence (luminescence) of the dye molecule.

(32) Reporter is a molecule or a part of a molecule that provides a signal, which is of sufficient character to be detected.

(33) Fluorescent (luminescent) reporter is a molecule or a part of a molecule that provides a fluorescence (luminescence) signal that is of sufficient character to be detected.

(34) Quencher of fluorescence (luminescence) is a molecule or a part of a molecule, the fluorescence (luminescence) of which is not strong enough to be measured and/or that reduces fluorescence (luminescence) quantum yield of a fluorophore. Quenchers can absorb light in certain spectral regions to reduce photosensitizer efficiency when excited in that spectral region. Quenchers can be used as reporters in photo-acoustic measurements.

(35) Sensitizer is a molecule or a part of a molecule that provides generation of reactive species or initiation of chemical reaction.

(36) Photosensitizer is a molecule or a part of a molecule that provides generation of reactive species or initiation of chemical reaction under photo-excitation.

(37) Environment sensitive molecule or compound means a molecule or compound, the spectral and/or photosensitizing characteristics of which are dependent on its microenvironment. The environment sensitive molecules include, but not limited to, pH-sensitive, polarity sensitive and potential sensitive molecules, and ion indicators.

(38) Compounds by Structure

(39) Terminus means a terminal heterocyclic end group particular in polymethine dyes.

(40) Nitrogen containing heterocyclic compounds (heterocycles) are any cyclic compounds containing at least one ring that contains at least one nitrogen atom in the ring. Preferably these are five- and six-membered rings. These compounds can be formed by fusion of a nitrogen containing cycle with another cycle or multiple cycles.

(41) Indolenines are any compounds or fragments containing indolenine (3H-indole) moiety. Examples of indolenines and related nitrogen containing heterocyclic fragments are benzo[cd]indole, 3H-pyrrolo[2,3-b]pyridine, benzoxazole, benzothiazole, benzoselenazole, benzotellurazole, and their derivatives as shown below:

(42) ##STR00027##
Although some indole derivatives such as benzo[cd]indole are not 3H-indoles, in this disclosure they are also referred to as indolenines.

(43) Halogenated (iodinated, brominated, chlorinated, and fluorinated) derivatives (compounds or moieties) are heterocycles that contain iodine, bromine, chlorine, and/or fluorine atom(s) in the aromatic or heterocyclic ring, such as the following illustrative examples:

(44) ##STR00028##

(45) Polymethines are compounds containing one or more methine groups (CH) bound together by alternating single and double bonds (A. D. Kachkovski, M. L. Dekhtyar, Dyes and Pigments, 22 (1993) 83-97; http://en.wiktionary.org/wiki/polymethine). Monomethine dyes are also considered polymethine dyes in this disclosure. Polymethines can be neutral, zwitterionic, positively or negatively charged, and can include a single or multiple charge (2, 1, +1, +2, etc.). One or more substituent(s) (R) can be introduced in any position of the polymethine chain. The possibility of introducing these substituents in any structures disclosed herein is implicit. These substituents can form one or more cyclic systems or branch polymethine chains.

(46) ##STR00029##

(47) Cyanines are polymethine dyes consisting of two terminal nitrogen centers, one of which is positively charged and is linked by a conjugated chain of an odd number of carbon atoms to the other nitrogen; as a result the positive charge is delocalized (A. P. Demchenko (ed.), Advanced Fluorescence Reporters in Chemistry and Biology I: Fundamentals and Molecular Design, Springer Ser. Fluoresc. (2010) 8: 149-186, DOI 10.1007/978-3-642-04702-25, # Springer-Verlag Berlin Heidelberg 2010; A. Mishra et al. Cyanines during the 1990s: A Review. Chem. Rev., 2000, 100, 1973-2011). One or both nitrogen atoms can be included in a cyclic system.

(48) ##STR00030##
wherein A and A are independently alicyclic or heterocyclic groups but in this disclosure at least one on these groups is a heterocycle.

(49) Stryryls belong to a subclass of polymethine dyes bearing a positive charge delocalized within the polymethine chain; however, contrary to cyanines which contain an odd number of methine groups, styryl dyes have an even number of these groups in the polymethine chain (I. A. Fedyunyayeva et al, Dyes and Pigments, 90 (2011), 201-210).

(50) ##STR00031##
wherein A is alicyclic or heterocyclic groups; R.sup.2 and R.sup.3 may form a cycle A.sup.1. In this disclosure group A and/or A.sup.1 is a heterocycle.

(51) Merocyanines are unsymmetrical polymethine dyes with neutral chromophore comprised of two terminal fragmentsnitrogen donor and oxygen or sulfur acceptor connected by an ethylene or polyvinylene (polymethine) chain (A. P. Demchenko (ed.), Advanced Fluorescence Reporters in Chemistry and Biology I: Fundamentals and Molecular Design, Springer Ser. Fluoresc. (2010) 8: 149-186, DOI 10.1007/978-3-642-04702-2_5, # Springer-Verlag Berlin Heidelberg 2010; Top Heterocycl Chem (2008) 14: 75-105, DOI 10.1007/7081_2007_110).

(52) ##STR00032##
wherein A and B are independently alicyclic or heterocyclic groups, but in this disclosure group A is a heterocycle.

(53) Squaraines (squaraine or squarylium dyes) are a subclass of polymethines containing a derivatized 3-oxo-1-cyclobutene-1-olate substructure (a squaric acid residue) inside polymethine chain and represented by the structure:

(54) ##STR00033##
wherein substituents Z.sup.1, Z.sup.2, W.sup.1, and W.sup.2 are as disclosed below.

(55) Croconates or croconium dyes are a subclass of polymethines containing a croconium or derivatized croconium substructure (a croconic acid residue) inside polymethine chain and represented by structure:

(56) ##STR00034##
wherein substituents Z.sup.1, Z.sup.2, W.sup.1, and W.sup.2 are as disclosed below.

(57) Dendrimers are repetitively branched molecules (D. Astruc et at. (2010). Chem. Rev. 110 (4): 1857-1959). In this disclosure dendrimers may consist of uniform or non-uniform (different) molecules. Dendrimers may be symmetric or non-symmetric. In this disclosure dendrimers also include dendron molecules.

(58) Groups and Substituents

(59) Hydrophilic group means any group, which increases solubility of a compound in aqueous media. These groups include, but are not limited to, sulfo, sulfonic, phosphate, phosphonate, phosphonic, carboxylate, boronic, ammonium, cyclic ammonium, hydroxy, alkoxy, ester, polyethylene glycol, polyester, glycoside, and saccharide groups.

(60) Reactive group means any group allowing covalent or noncovalent binding to other molecule or carrier. These groups include, but are not limited to, activated carboxylic esters, acyl azides, acyl halides, acyl halides, acyl nitriles, aldehydes, ketones, alkyl halides, alkyl sulfonates, anhydrides, aryl halides, aziridines, boronates, carboxylic acids, carbodiimides, diazoalkanes, epoxides, haloacetamides, halotriazines, imido esters, isocyanates, isothiocyanates, N-hydroxysuccinimide, maleimides, phosphoramidites, silyl halides, sulfonate esters, sulfonyl halides, biotin, avidin, and streptavidin. In particular, the following reactive functional groups, among others, are especially useful for the preparation of labeled molecules or substances, and are therefore suitable reactive functional groups for the purposes of the reporter compounds: a) N-hydroxysuccinimide esters, isothiocyanates, and sulfonylchlorides, which form stable covalent bonds with amines, including amines in proteins and amine-modified nucleic acids; b) Iodoacetamides and maleimides, which form covalent bonds with thiol-functions, as in proteins; c) Carboxyl functions and various derivatives, including N-hydroxybenzotriazole esters, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters, and acyl imidazoles; d) Alkylhalides, including iodoacetamides and chloroacetamides; e) Hydroxyl groups, which can be converted into esters, ethers, and aldehydes and iodoacetamides; f) Aldehydes and ketones and various derivatives, including hydrazones, oximes, and semicarbozones; g) Isocyanates, which may react with amines; h) Activated CC double-bond-containing groups, which may react in a Diels-Alder reaction to form stable ring systems under mild conditions; i) Thiol groups, which may form disulfide bonds and react with alkylhalides (such as iodoacetamide); j) Alkenes, which can undergo a Michael addition with thiols, e.g., maleimide reactions with thiols; k) Phosphoramidites, which can be used for direct labeling of nucleosides, nucleotides, and oligonucleotides, including primers on solid or semi-solid supports; l) Primary amines that may be coupled to variety of groups including carboxyl, aldehydes, ketones, and acid chlorides, among others; m) Boronic acid derivatives that may react with sugars; n) Pyrylium moieties react with primary amines; o) Haloplatinates form stable platinum complexes with amines, thiols and heterocycles; p) Aryl halides react with thiols and amines; q) Azide, alkyne or other groups, which can be used for click chemistry reactions; r) Biotin, avidin, and streptavidin providing strong non-covalent binding.

(61) Aliphatic groups include groups of organic compounds characterized by straight- or branched-chain arrangement of the constituent carbon atoms. Aliphatic hydrocarbons comprise three subgroups: (1) paraffins (alkanes), which are saturated and comparatively unreactive; (2) olefins (alkenes or alkadienes), which are unsaturated and quite reactive; and (3) acetylenes (alkynes), which contain a triple bond and are highly reactive. In complex structures, the chains may be branched or cross-linked and may contain one or more heteroatoms (such as polyethers and polyamines, among others).

(62) Alicyclic groups include hydrocarbon substituents that incorporate closed rings. Alicyclic substituents may include rings in boat conformations, chair conformations, or conformations resembling bird cages. Most alicyclic groups are derived from petroleum or coal tar, and many can be synthesized by various methods. Alicyclic groups may optionally include heteroalicyclic groups that include one or more heteroatoms, typically nitrogen, oxygen, or sulfur. These compounds have properties resembling those of aliphatics and should not be confused with aromatic compounds having the hexagonal benzene ring. Alicyclics may comprise three subgroups: (1) cycloparaffins (saturated), (2) cycloolefins (unsaturated with two or more double bonds), and (3) cycloacetylenes (cyclynes) with a triple bond. The best-known cycloparaffins (sometimes called naphthenes) are cyclopropane, cyclohexane, and cyclopentane; typical of the cycloolefins are cyclopentadiene and cyclooctatetraene. Most alicyclics are derived from petroleum or coal tar, and many can be synthesized by various methods.

(63) Aromatic groups may include groups of unsaturated cyclic hydrocarbons containing one or more rings. A typical aromatic group is benzene, which has a 6-carbon ring formally containing three double bonds in a delocalized ring system. Aromatic groups may be highly reactive and chemically versatile. Most aromatics are derived from petroleum and coal tar. Heterocyclic rings include closed-ring structures, usually of either 5 or 6 members, in which one or more of the atoms in the ring is an element other than carbon, e.g., sulfur, nitrogen, etc. Examples include pyridine, pyrrole, furan, thiophene, and purine. Some 5-membered heterocyclic compounds exhibit aromaticity, such as furans and thiophenes, among others, and are analogous to aromatic compounds in reactivity and properties.

(64) Any substituent of the compounds of the invention, including any aliphatic, alicyclic, or aromatic group, may be further substituted one or more times by any of a variety of substituents, including without limitation, F, Cl, Br, I, carboxylic acid, sulfonic acid, CN, nitro, hydroxy, phosphate, phosphonate, sulfate, cyano, azido, amine, alkyl, alkoxy, trialkylammonium or aryl. Aliphatic residues can incorporate up to six heteroatoms selected from N, O, and S. Alkyl substituents include hydrocarbon chains having 1-22 carbons, more typically having 1-6 carbons, sometimes called lower alkyl.

(65) Squaraine moiety is a 3-oxo-1-cyclobutene-1-olate substructure, where one or both oxygen atoms may be substituted with a heteroatom or a group of atoms (substituents Z.sup.1 and Z.sup.2):

(66) ##STR00035##

(67) If a dye described in this disclosure has a total positive or negative electronic charge, an appropriate counterion is implied but may not be shown. Counterion can be organic or inorganic. Organic counterion can be covalently attached to the dye.

(68) To simplify drawings of chemical structures, a methyl group may be shown in this disclosure as a single bond. Thus, the structures on the left column below represent the same compound as on the right column:

(69) ##STR00036##

(70) The present disclosure relates to: iodinated, brominated, and other halogenated nitrogen containing heterocyclic compounds; organic dyes, in particular polymethine dyes including cyanines and squaraines based on these heterocyclic compounds; dendrimeric compounds comprising these halogenated polymethine dyes; conjugates of these polymethine and dendrimeric dyes with other organic and inorganic compounds, nanoparticles, biological molecules, and/or biological compounds that ensure improved brightness and photostability and/or sensitivity and selective affinity to target cancer cells, pathogenic microorganisms, and/or other biological materials; reporting and sensitizing compositions based on these polymethine compounds, dendrimers and conjugates of these compounds, and methods to make and use these compounds and compositions.

(71) More specifically, the disclosure relates to classes of fluorinated, chlorinated, brominated, and/or iodinated compounds, wherein at least two halogen atoms are directly bound to a nitrogen-containing heterocyclic terminal moiety of the compound, and wherein at least one of such halogen atoms is iodine or bromine. The disclosure also relates to derivatives and conjugates of these halogenated compounds, as well as to applications of such compounds and conjugates. In some embodiments, the dye compound includes a second halogenated terminal moiety.

(72) The compounds and compositions of the present disclosure reflect the discovery that introduction of heavy atoms such as iodine and bromine in polymethine dyes surprisingly and unexpectedly increase sensitizing efficiency, photostability, and fluorescence quantum yields of the dye, in contradiction to conventional wisdom in the art, according to which sensitizing efficiency is increased at the cost of decreased fluorescence quantum yields. The dyes of the present disclosure thereby provide a unique, unexpected, and highly useful matrix of properties affording new functional advantages for reporting, diagnostics, and/or photodynamic therapies, among other applications.

(73) The multiply halogenated polymethine dyes of the disclosure can be used to form dendrimers, thereby enabling increased reactive oxygen species (ROS) generation per molecule, all other factors being equal, in relation to a single isolated polymethine dye.

(74) It will be recognized that quantum yield achievable by multiply halogenated polymethine dyes species of the present disclosure will vary among different compounds and classes of such compositions, and that the particular multiply halogenated polymethine dye desirably employed to achieve a predetermined quantum yield in a specific application can be readily determined within the skill in the art based on the disclosure herein.

(75) The disclosure also contemplates the provision and use of multiply halogenated polymethine dyes species wherein at least two halogen atoms are directly bound to a nitrogen-containing heterocyclic terminal moiety of the compound, and wherein the halogen atoms include two or more fluorine or chlorine atoms, optionally wherein two or more fluorine or chlorine atoms are directly bound to nitrogen-containing heterocyclic terminal moieties at two terminal regions of the molecule. In still other embodiments, such fluorinated or chlorine analogs may be conjugated with multiply iodinated or brominated molecules of the disclosure to form protected dendrimers.

(76) The disclosure thus broadly contemplates fluorinated, chlorinated, brominated, and iodinated nitrogen containing heterocyclic compounds, and in particular indolenines, benzoxazoles, benzothiazoles, benzoselenazoles, and benzotellurazoles having the formula:

(77) ##STR00037##
wherein:
X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are independently selected from the group consisting of N, .sup.+NR.sup.N3, and CR.sup.X with the proviso that at least two of the substituents X.sup.1, X.sup.2, X.sup.3 or X.sup.4 contain halogen atoms directly attached to aromatic or heterocyclic ring A wherein at least one of these halogen atoms is iodine or bromine. Fluorine and/or chlorine in these compounds can be introduced in the ring A to obtain additional identifiable reporters with high fluorescence quantum yield and photostability;
Y is O, S, Se, Te, C(R.sup.C1)(R.sup.C2), NR.sup.N2; and
other substituents are as disclosed herein.

(78) The disclosure further contemplates methods to synthesize the heterocycles and polymethine dyes of the disclosure, including methods for synthesizing the polymethine dyes wherein at least one of the heterocycle compounds of the disclosure is utilized as a starting or intermediate material or precursor.

(79) In another aspect, the disclosure relates to polymethine dyes and compositions of matter comprising the polymethine dyes containing at least one of the heterocyclic moieties Het and Het.sup.+:

(80) ##STR00038##
wherein at least one of the heterocyclic moieties contains at least two substituents attached to the aromatic or heteroaromatic ring, selected from iodine, bromine, chlorine and fluorine atoms, wherein at least one of these halogen atoms is iodine or bromine.

(81) Polymethine compounds and compositions of the disclosure are used for reporting and sensitizing applications, among others, and include compounds and compositions that absorb light in the greater-visible light range, e.g., in the red and NIR spectral range, to effect generation of ROS, as well as compounds and compositions that are effective to generate ROS under ultrasonic exposure. The compounds and compositions can be fluorescent or non-fluorescent, reactive or non-reactive, and/or hydrophilic or hydrophobic.

(82) The most significant and unpredicted features of the halogenated heterocyclic dyes of the present disclosure are increased brightness (identified as the extinction coefficient multiplied by the fluorescence quantum yield); and photostability simultaneously with increased sensitizing efficiency as compared to the dyes without these halogen atoms.

(83) One class of preferred dyes of the present disclosure includes iodinated and brominated indolenine-based pentamethine and heptamethine cyanines that absorb and emit in the red and near-infrared spectral region, where biological tissues demonstrate minimal auto-absorbance and auto-fluorescence.

(84) Heterocyclic compounds of the present disclosure include the following indolenines:

(85) ##STR00039## ##STR00040##
and the following polymethines:

(86) ##STR00041## ##STR00042##

(87) Molecular structures of the present disclosure comprise two or more halogen atoms, including at least one of either iodine and/or bromine, in the dye molecule that are effective to increase not only sensitizing efficiency but also fluorescence quantum yields. At least one of the termini of the molecular structure contains two or more halogen atoms, at least one of which is iodine or bromine. More than one terminus may contain such halogen atoms, and such molecular structures can be conjugated to form larger molecular structures characterized by high fluorescence brightness and photosensitizer ROS generation efficiency.

(88) Although cyanines and other polymethines have been previously utilized as fluorescent dyes, the conventional wisdom in the art has been that introduction of heavy atoms such as iodine and bromine in dye molecules will decrease fluorescence quantum yield, as well as generally the photostability of the dye. In consequence of such conventional wisdom, relating to the heavy atom effect of iodine and/or bromine halogen atoms as being correlative to expected low performance, the art has avoided use of multiple heavy halogen atoms in photosensitive dyes. The present disclosure therefore reflects a radical departure from prior practice in the provision of polymethine dyes with one or multiple heavy halogen atoms on one or more heterocyclic terminal groups, as a new class of fluorophores that in contradiction to accepted heavy-atom effect expectations exhibits high reactive oxygen species (ROS) generation efficiency photosensitizing characteristics with high fluorescence quantum yield.

(89) The disclosure contemplates conjugates of the halogenated heterocycle compounds herein disclosed as including two or more iodine and/or bromine atoms on heterocyclic terminal group(s), as well as ionic pairs collocated on carriers, and dendrimeric dyes comprising these halogenated dyes.

(90) Molecular structures of the present disclosure can be used as precursors to synthesize iodinated, brominated, and other halogenated dyes, in particular polymethines. The high ROS generation efficiency and high fluorescence quantum yield of the halogenated polymethine dyes of the disclosure, and their superior brightness and photostability, enable such improved reporting and sensitizing properties to be exploited in a wide variety of applications, including research, analysis, diagnostics, photodynamic therapies, theranostics, and photoinitiated deposition or growth of materials.

(91) Further improvement of the halogenated dyes of the disclosure can be achieved by conjugation of these dye molecules to a dendrimeric structure. The resulting dendrimer can be formed by the same dye molecules described herein, or combinations of different dye molecules with the dye molecules described herein. Any appropriate linker can be used to conjugate the dye molecules to a dendrimeric structure. The dendrimers can be hydrophilic, hydrophobic, reactive, or non-reactive. Dye molecules in a dendrimer may play a role of a reporter, sensitizer, or both. Dendrimeric structures comprised of dyes of the present disclosure have been found to improve light sensitivity, brightness, SOG and ROS efficiency.

(92) Thus, the disclosure contemplates iodinated, brominated, and other halogenated compounds having utility as photosensitive dyes, their conjugates, and methods of synthesizing and using these compounds and conjugates as reporters, sensitizing agents for photodynamic and sonodynamic therapy, photodynamic antimicrobial chemotherapy (PACT), cancer treatment, and antimicrobial coating materials. The utility of such halogenated compounds and their conjugates in such applications may be particularly advantageous as a result of enhancement of one or more of the following characteristics: (i) fluorescence quantum yield, (ii) sensitizing (photodynamic) efficiency, which can be estimated via the SOG or ROS generation effectiveness or performance to destroy certain cells, (iii) the sensitivity to excitation light, which can be estimated via the extinction coefficient at the excitation wavelength, (iv) spectral range causing depth of treatment, (v) dark toxicity, and (vi) photostability, as compared with compounds and conjugates other than those of the present disclosure.

(93) Compositions of the present disclosure include dyes useful as reporters for diagnosis or analysis in vitro or in vivo. The disclosure contemplates determining the presence and/or concentration of dyes described herein by various techniques, e.g., photoluminescent excitation or detection, or light absorption induced ultrasonic generation. Photodynamically active compounds of the present disclosure also can be remotely detected, thereby providing an intrinsic mechanism for reporting. High and low photodynamic activity molecules may be conjugated or otherwise linked to optimize chemical activity reporting functions.

(94) Compounds of the present disclosure include polymethine dyes such as cyanines, styryls, merocyanines, squaraines, and croconates, among others, as well as nitrogen-containing heterocyclic compounds (heterocycles) that can be used to synthesize such polymethines. All of these compounds (dyes and heterocycles) contain at least two halogen atoms in the heterocyclic compound or heterocyclic terminus, but at least one of these halogens is iodine or bromine.

(95) Nitrogen-containing heterocyclic compounds useful as precursors to synthesize polymethines include halogenated indolenines, benzo[cd]indole, 3H-pyrrolo[2,3-b]pyridine, benzoxazole, benzothiazole, benzoselenazole, benzotellurazole, and their derivatives. As previously indicated, the halogenated dyes themselves or in combination with other dyes can be bound together via linkers to form dendrimeric structures.

(96) Specific embodiments of indolenines and other nitrogen-containing heterocyclic compounds are set out below.

(97) ##STR00043## ##STR00044## ##STR00045##

(98) Specific embodiments of polymethines are set out below.

(99) ##STR00046## ##STR00047## ##STR00048## ##STR00049##

(100) Specific embodiments of dendrimers are set out below.

(101) ##STR00050## ##STR00051## ##STR00052## ##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057## ##STR00058## ##STR00059## ##STR00060##
where R.sup.D contains a dye;

(102) ##STR00061##
where R.sup.D contains a dye:

(103) ##STR00062## ##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067##

(104) ##STR00068##
where: R.sup.d is:

(105) ##STR00069##
where: R.sup.d is:

(106) ##STR00070##
where:
each R.sup.c1 and R.sup.c2 is independently selected from alkyl, (CH.sub.2).sub.nCOOH, (CH.sub.2).sub.nSO.sub.3H,

(107) ##STR00071##
(CH.sub.2).sub.nPO(OEt).sub.2, (CH.sub.2).sub.nPO(OH)(OEt), or (CH.sub.2).sub.nPO(OH).sub.2, or contains a dye;
each R.sup.N is independently selected from alkyl, (CH.sub.2).sub.kCOOH, (CH.sub.2).sub.kSO.sub.3H,

(108) ##STR00072##
(CH.sub.2).sub.kPO(OEt).sub.2, (CH.sub.2).sub.kPO(OH)(OEt), or (CH.sub.2).sub.kPO(OH).sub.2, or contains a dye;
each R.sup.h is independently selected from hydrogen, alkyl, aryl, or adjacent substituents R.sup.h form a cycle; and
k=1-20; m=1-20; n=1-20; p=1-20.

(109) Halogenated dendrimeric dyes of the present disclosure have substantially improved light sensitivity (extinction coefficients at the excitation wavelength), brightness (extinction coefficients multiplied by the fluorescence quantum yield), photostability, and sensitizing efficiency (minimal molar concentration threshold mediating a sensitizing effect).

(110) Two or more polymethine dyes of the present disclosure can be bound together to form a larger molecular conjugate with greater overall fluorescence and/or greater ROS efficiency, as shown in FIGS. 1 and 2. Such conjugates can be formed as of multiples of the same polymethine dye, different polymethine dyes, or at least one polymethine dye with other photosensitizer, reporting, or photosonic dyes (see FIG. 3).

(111) Spectral characteristics of selected polymethine dyes of the disclosure are set out in Table 1 in Example 38. Representative spectra are shown in FIG. 4, and photostability data are shown in FIGS. 5 and 6. These data demonstrate that the introduction of halogen atoms in accordance with the present disclosure increases both fluorescence quantum yield and photostability of the dyes.

(112) The efficacy of halogenated compounds of the present disclosure has been demonstrated by both photodynamic and dark toxicity testing using erythrocytes as model cells.

(113) The obtained data are shown in Table 2 (Example 42), FIGS. 7, 8, and FIGS. 10, 11 where the hemolysis percentage indicates the dark toxicity level and phototoxicity effectiveness. The hemolysis level was dependent on both sensitizing efficiency and cell uptake. The majority of compounds in Table 2 and FIGS. 7, 8, and FIGS. 10, 11 have generally similar hydrophobicity/hydrophilicity characteristics and therefore approximately equal uptake, as confirmed by fluorescence microscopy. The best sensitizers exhibit the lowest dark toxicity and highest phototoxicity at minimal concentrations. As can be seen from the data, the halogenated cyanines of the present disclosure exhibited sensitizing effectiveness that was substantially higher than non-halogenated cyanines such as ICG and HITC.

(114) Direct chemical singlet oxygen generation (SOG) and ROS quantum yield were measured using 1,3-diphenylisobenzofuran (DPBF) as the single oxygen sensitive dye, according to the procedure of W. Spiller et al., J. Porphyrins Phthalocyanines, 2 (1998) 1-5-158; H. Mojzisova et al. Photochem. Photobiol. Sci., 8 (2009) 778-787. The obtained data (Table 2 in Example 42 hereof) evidenced that the introduction of iodine and bromine atoms increased the SOG and ROS quantum yield.

(115) The dark cytotoxicity of almost all of the halogenated cyanines was found to be as low as that of non-halogenated cyanines of similar structure.

(116) The present disclosure in other aspects contemplates the use of oxic agents and conditions, e.g., ozone, oxygen-carrying fluorocarbons, carbogen, high pressure oxygen, and increased oxygen, to improve photodynamic therapy in conjunction with the use of sensitizing and reporting halogenated compounds and compositions of the present disclosure. Therapeutic approaches in this respect may include blood oxygen enhancement using pressure and/or high oxygen concentration chambers, the use of oxygen on protein as a delivery vehicle for increasing localized oxygen, etc.

(117) Halogenated compounds and compositions of the disclosure are useful as reporters and/or sensitizers and can be used to destroy cells and tissues following activation by light, including but not limited to applications such as photodynamic therapy, photodynamic antimicrobial chemotherapy (PACT), cancer treatment, targeted vascular disruption, lipid targeting on vascular walls, cosmetic applications, antimicrobial coating materials and sonodynamic therapy or diagnostic, analytical reporting and/or imaging. In general, the dyes and their compositions are administered in effective amount to a human or animal patient in whom it is desired to destroy certain cells or tissues or for diagnostic applications.

(118) In one aspect, the disclosure relates to a method of generating reactive oxygen species (ROS) at a biological locus, comprising introducing a composition of the disclosure to such locus, and transmitting radiation to the composition at the locus that is effective to cause the composition to generate ROS at the locus. The composition includes a dye of the disclosure, and/or a conjugate thereof, that is effective under irradiation conditions to generate ROS at the locus, e.g., a corporeal site of a human or animal subject.

(119) The method may further comprise introducing exogenous oxygen or ozone to the locus to increase available oxygen at the locus for generating ROS at the locus. The composition may be introduced to the locus on a carrier, e.g., at least one carrier species selected from the group consisting of bacteriophages and antibodies.

(120) The method is desirably carried out so that the dye in the composition is substantially completely photobleached by the radiation transmitted to the locus. Correspondingly, the radiation transmitted to the locus is desirably limited to the amount required for the dye in the composition to be substantially completely photobleached by the radiation transmitted to the locus.

(121) As discussed hereinabove, in vivo usage of photosensitizers for phototherapy targeting specific loci in the body entails many competing considerations deriving from the variable characteristics of the photosensitizer, including its photo-oxidation sensitivity, its concentration, its selectivity, its photostability/photolability properties, as well as associated variable properties of carriers that are used for delivery of the photosensitizer agent to the targeted corporeal locus, and the variables associated with the light source, such as intensity/flux characteristics, spectral characteristics of emitted radiation, permissible placement of the light source in relation to the body, etc., and the bodily variables themselves, e.g., size, shape and location of a tumoral mass or infected site and the characteristics of its proximate corporeal environment and constituent fluid, bone and tissue components.

(122) In the context of this plethora of variables, it is necessary to effect the therapeutic intervention on the target locus of the body undergoing treatment, while functionally minimizing adverse effect on corporeal regions outside the target locus. In some instances, the imperfect targeting of photosensitizer carriers, e.g., antibodies, proteins, etc. that invariably occurs in the therapeutic intervention makes it necessary to deactivate the photosensitizer in a selective, or non-selective, manner in order to limit damage to non-targeted corporeal regions. In these circumstances, photosensitizer dyes and dye conjugates of the present disclosure are particularly advantageous, exhibiting photostability characteristics that enable rapid photobleaching and photooxidation of the photosensitizer to be achieved with moderated ROS generation, e.g., in high light dose regions near the light source when deep tissue activation is being conducted. The photosensitizer dyes and dye conjugates of the present disclosure thereby dramatically broaden the range and types of approaches that can be utilized for effective phototherapy.

(123) In one embodiment, the disclosure contemplates a treatment methodology utilizing self-limiting ROS generator photosensitizers for photodynamic therapy where low intensity activating spectrum light is used over large areas of the body to slowly deactivate residual photosensitizer without inducing sufficient reactivity to do damage. As these concentrations should be low, a slowly increasing ramp of intensity can be used to remove sensitivity. The intensity and time matrix should be sufficient so as to leave the patient with unintended minimal photosensitizer activation risk from reasonable sun or other light source exposure (localized areas or full body, such as a tanning bed, but much higher intensity light at just the red to infra-red spectrum). This method can be accomplished by (1) using low intensity spectrum light for a long period of time sufficient for practical photosensitizer deactivation, (2) slowly ramping the activating light intensity upward over time to speed the deactivation process, (3) slowly changing the spectrum of the light from green or yellow light to red and near-infrared light, or (4) by changing both intensity and the spectrum over time.

(124) In another technique, the disclosure relates to a treatment methodology utilizing self-limiting ROS generator photosensitizers for photodynamic therapy where activating spectrum light is used to activate photosensitizer over large areas of the body, wherever the targeted photosensitizer has concentrated, but avoiding an intensity that would induce significant damage to internal organs in which the photosensitizer-carrier conjugates may accumulate without being directly targeted (e.g., liver, kidney, spleen, lungs, etc., depending on the carrier characteristics). The procedures described in the preceding paragraph can be used to deactivate non-targeted shallow tissue prior to high intensity exposure to treat broadly distributed targeted carriers with photosensitizer for cancer, pathogens, or other biomaterials (e.g., arterial plaque, fat, circulating cancer or pathogens) deep in tissue, blood, or in the bone.

(125) Light may also be used over long periods of time, e.g., in a range of from 4 to 5000 hours to activate the photosensitizer deep in tissue along with multiple oral, topically absorbed, or injected photosensitizer. Long term light applications can be accomplished with wearable light units or multiple long term light applications to activate the dye or dye conjugate in the administered composition.

(126) The disclosure in another aspect relates to mediating deactivation of a photosensitizer by high frequency modulation of a light source. Such high frequency modulation enables high rate in vivo deactivation of photosensitizer to be achieved. In specific embodiments, frequency modulation of light in a range of from 100 kHz to 100 THz is carried out to photodegrade the photosensitizer.

(127) In this frequency range of from 100 kHz to 100 THz, at the moderately high light intensities typically used in photodynamic therapy with photosensitizers, especially when activating the photosensitizer by light transmitted through a large volume of tissue, the photosensitizer exhibits a lower efficiency ROS generation and yet still exhibits high absorption and normal photodegradation.

(128) As a result, the photosensitizer present than non-targeted tissue can be degraded without causing as much damage to such tissue, in relation to photosensitizer exposure to corresponding light lacking such frequency modulation. This reduced damage result is markedly less pronounced at frequency below 100 kHz.

(129) The magnitude of the frequency modulated reduced damage effect and the optimal modulation frequency difference varies among photosensitizers and among different background environments, e.g., environments with different oxygen (O.sub.2) concentration, but the effect is almost always significant.

(130) Accordingly, the disclosure contemplates a method of treating a subject, e.g., a human or other animal subject, to whom photosensitizer has been administered resulting in presence of photosensitizer in a bodily region of the subject that is non-targeted for phototherapy, comprising transmitting to the non-targeted bodily region deactivatingly effective light that is frequency modulated in a range of from 100 kHz to 100 THz, to deactivate the photosensitizer present in such region. Such transmission of deactivatingly effective light is advantageously conducted contemporaneously with photodynamic therapy treatment of said subject, e.g., so that the PDT treatment of the subject in a targeted bodily region is carried out before, during, or after the deactivation of photosensitizer in the non-targeted bodily region of such subject, as part of a continuous or near-continuous therapeutic intervention.

(131) The frequency modulated light can additionally be ramped in wavelength, from shorter wavelength to longer wavelength, or intensity of the light can be slowly increased while modulating light had high-frequency, in order to confine photosensitizer degradation (and accompanying inefficient generation of ROS or other reactive species) to shallow bodily regions near the light source utilized for phototherapy, prior to carrying out continuous light transmission or low frequency pulsing of the transmitted light to reach a deeper targeted cancer or pathogen.

(132) The foregoing techniques for deactivating photosensitizers in non-targeted bodily regions are applicable to almost all photosensitizers and to all light source types that can be modulated at high frequencies, including light emitting diode (LED) and laser sources, and light sources that can be modulated with chopper devices. Such modulation frequency-controlled deactivation of photosensitizer with reduced cellular/tissue damage, in relation to damage occurring in the absence of such controlled deactivation, works particularly well with photosensitizer dyes and dye conjugates of the present disclosure.

(133) The synthesis of the cyanines, squaraines, other polymethines of the present disclosure, and their precursors, can be carried out with the use of techniques such as those disclosed in Heterocyclic Polymethine Dyes: Synthesis, Properties and Applications, Ser.: Topics in Heterocyclic Chemistry, Vol. 14, L. Strekowski (Ed.) 2008, Springer-Verlag, Berlin, Heidelberg, and A. Mishra et al., Cyanines during the 1990s: a review, Chem. Rev., 2000, V. 100, 1973-2011, and/or as otherwise described herein, including procedures that have been newly developed or improved to achieve better isolation yields. The synthesis of representative dyes, precursors, and conjugates is described in illustrative Examples hereafter. The dyes of the present disclosure can be fine-tuned to a desirable wavelength and made compatible with available light sources by appropriate modification of their chromophore system and substituents. These dyes can also be fine-tuned in respect of hydrophobic-hydrophilic properties, reactive groups, and other functionalities. Further improvements can be achieved by synthesizing dendrimeric dyes, as carried out in accordance with the present disclosure, utilizing generalized synthetic methods known in the art.

(134) The features and advantages of the present disclosure, and compounds, compositions, and methods thereof, are more fully shown by the following non-limiting examples, wherein all parts and percentages are by weight, unless otherwise expressly stated.

EXAMPLES

1. Synthesis of Intermediates

Example 1

Synthesis of 3,5-diiodophenylhydrazine

(135) ##STR00073##

(136) 3,5-Diiodoaniline was obtained by the procedure of M. Brub (M. Brub, et al. Synthesis of Simplified Hybrid Inhibitors of Type 1 17-Hydroxysteroid Dehydrogenase via the Cross-Metathesis and the Sonogashira Coupling Reactions, Organic Lett. 6 (2004) 3127-3130). 5 g (14.5 mmol) of 3,5-diiodoaniline were stirred with the solution of 5 mL of concentrated hydrochloric acid and 5 mL of water. The mixture was cooled to about 10 C. and 5.5 mL of 20% aqueous solution of NaNO.sub.2 was added dropwise with continuous stirring. The suspension was allowed to stir for another 40 minutes. Next, the cooled solution of 10.8 g (47.9 mmol) of SnCl.sub.2.2H.sub.2O in 11 mL of concentrated HCl was added dropwise at 10 C. to the suspension of diazocompound. The reaction mixture was kept 10 C. for one hour and at 5 C. overnight. The obtained precipitate of a double salt with tin chloride was filtered off and washed with water. The residue was resuspended in water and added concentrated aqueous solution of NaOH to alkaline medium. The 3,5-diiodophenylhydrazine was extracted with ether, the organic layer was washed in turn with aqueous NaOH, Na.sub.2S.sub.2O.sub.3 and water, and dried with CaCl.sub.2. Ether was evaporated to give 4.2 g of 3,5-diiodophenylhydrazine. .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 7.10 (2H, d, 1.2 Hz, arom.), 6.92 (1H, d, 1.2 Hz, arom.), 5.51 (1H, s, NH), 4.12 (2H, broad s, NH.sub.2).

Example 2

Synthesis of 4,6-diiodo-2,3,3-trimethyl-3H-indole

(137) ##STR00074##

(138) 2 g of 3,5-diiodophenylhydrazine (5.5 mmol), and 1 mL (9.3 mmol) of 3-methyl-2-butanone were refluxed in 15 mL of acetic acid for 20 hours. The acetic acid was evaporated and the residue was dissolved in ether. Insoluble precipitate was filtered off, and the etheric solution was washed with aqueous solutions of NaHCO.sub.3, followed by Na.sub.2S.sub.2O.sub.3 and water. The organic layer was dried with CaCl.sub.2 and ether was removed under reduced pressure by a rotary evaporator to give 1.4 g of 4,6-diiodo-2,3,3-trimethyl-3H-indole. .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 7.92 (1H, d, 1.1 Hz, arom.), 7.79 (1H, d, 1.1 Hz, arom.), 2.22 (3H, s, 2-CH.sub.3), 1.32 (6H, s, C(CH.sub.3).sub.2).

Example 3

Synthesis of 4,6-diiodo-1,2,3,3-tetramethyl-3H-indolium iodide

(139) ##STR00075##

(140) 1.05 g (2.5 mmol) of 4,5,6-triiodo-2,3,3-trimethyl-3H-indole and 2 mL of iodomethane were heated at 45 C. for 6 hours in a sealed tube. The formed precipitate was filtered, washed with acetone. Yield: 900 mg. .sup.1H-NMR (300 MHz, DMSO-d.sub.6), , ppm: 8.37 (2H, d, 5.4 Hz, arom.), 3.92 (3H, s, NCH.sub.3), 2.79 (3H, s, CH.sub.3), 1.61 (6H, s, C(CH.sub.3).sub.2).

Example 4

Synthesis of 3,4,5-triiodophenylhydrazine

(141) ##STR00076##

(142) 3,4,5-Triiodoaniline was obtained by the procedure of L. Kalb (L. Kalb et al., ber substituierte Indol-2-carbonsaure-8-propionsuren and einige jodierte Bensolderivate, Chem. Ber. 59 (1926) 1860-1870). 4 g (8.5 mmol) of 3,4,5-triiodoaniline were stirred with the solution of 2.3 mL of concentrated hydrochloric acid and 2.3 mL of water. The mixture was cooled to about 10 C. and the equivalent quantity of 2.5 M solution of NaNO.sub.2 was added dropwise with intensive stiffing. The reaction mixture was continued stirring for 30 min at 10 C. Next, the cooled solution of 5.75 g (25.5 mmol) of SnCl.sub.2.2H.sub.2O in 6 mL of concentrated HCl was added dropwise at 10 C. to the suspension of diazocompound. The reaction mixture was kept at 10 C. for one hour and at 5 C. overnight. The obtained precipitate was filtered off and washed with water to yield 4.7 g of 3,4,5-triiodophenylhydrazine, as a double salt with tin chloride, which was used for following step without additional treatment or purification. .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 10.14 (2H, broad s, NH.sub.2), 8.46 (1H, s, NH), 7.5 (2H., s, arom.).

Example 5

Synthesis of 4,5,6-triiodo-2,3,3-trimethyl-3H-indole

(143) ##STR00077##

(144) 2 g of 3,4,5-triiodophenylhydrazine, and 0.86 g (10 mmol) of 3-methyl-2-butanone were refluxed in 15 mL of acetic acid for 16 hours. The acetic acid was evaporated, the residue was dissolved in chloroform, washed with aqueous solutions of NaHCO.sub.3, followed by Na.sub.2S.sub.2O.sub.3 and water. The organic layer was dried and chloroform was removed under reduced pressure by a rotary evaporator. The residue was column purified (Silica gel 60, 0-2% methanol-chloroform) to give 0.6 g of 4,5,6-triiodo-2,3,3-trimethyl-3H-indole. .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 8.04 (1H, s, arom.), 2.19 (3H, s, 2-CH.sub.3), 1.30 (6H, s, C(CH.sub.3).sub.2).

Example 6

Synthesis of 4,5,6-triiodo-1,2,3,3-tetramethyl-3H-indolium iodide

(145) ##STR00078##

(146) 0.6 g (1.1 mmol) of 4,5,6-triiodo-2,3,3-trimethyl-3H-indole and 0.78 g (5.5 mmol) of iodomethane were heated at 30-40 C. for 20 hours in a sealed tube. The formed precipitate was filtered, and washed with small amounts of benzene and acetone to yield 350 mg of 4,5,6-triiodo-1,2,3,3-tetramethyl-3H-indolium iodide. .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 8.53 (1H, s, arom.), 3.93 (3H, s, NCH.sub.3), 2.77 (3H, s, CH.sub.3), 1.60 (6H, s, C(CH.sub.3).sub.2).

Example 7

Synthesis of 3-[3-(5-carboxypentyl)-4,5,6-triiodo-2,3-dimethyl-3H-1-indoliumyl]-1-propanesulfonate

(147) ##STR00079##

(148) A mixture of 3,4,5-triiodophenylhydrazine 2 g (4 mmol) and 1 g (5 mmol) of 7-methyl-8-oxononanoic acid was refluxed in 15 mL of acetic acid for 16 hours. The acetic acid was evaporated, and the residue was dissolved in CHCl.sub.3, and washed by aqueous NaHCO.sub.3 and water. The organic layer was dried with CaCl.sub.2 and solvent was removed under reduced pressure by a rotary evaporator. The residue was column purified (Silica gel 60, 0-4% methanol-chloroform) to give 0.6 g of 6-(4,5,6-triiodo-2,3-dimethyl-3H-3-indolyl)hexanoic acid. .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 8.02 (1H, s, arom.), 2.16 (3H, s, 2-CH.sub.3), 2.07 (2H, t, CH.sub.2COOH), 1.65-1.05 (6H, m, CH.sub.2), 1.26 (3H, s, CH.sub.3), 0.58-0.20 (2H, m, CH.sub.2).

(149) 600 mg (0.94 mmol) of 6-(4,5,6-triiodo-2,3-dimethyl-3H-3-indolyl)hexanoic acid was heated with 600 mg (4.7 mmol) of 1,3-propane sultone at 100 C. for 40 minutes. The raw product was column purified (Silica gel 60, 0-40% methanol-chloroform) to give 300 mg of 3-[3-(5-carboxypentyl)-4,5,6-triiodo-2,3-dimethyl-3H-1-indoliumyl]-1-propanesulfonate: 7.39 (1H, s, arom.), 4.31 (2H, m, NCH.sub.2), 3.27 (3H, s, CH.sub.3), 2.34-2.02 (2H, m, CH.sub.2), 2.19 (2H, t, CH.sub.2CO), 1.98-1.65 (4H, m, CH.sub.2), 1.31 (3H, s, CH.sub.3), 1.43-1.00 (4H, m, (CH.sub.2).sub.3), 0.62-0.35 (2H, m, CH.sub.2).

Example 8

Synthesis of 2,3,3-trimethyl-1-(3-triethylammoniopropyl)-3H-indolium bromide chloride (1)

(150) ##STR00080##

(151) Freshly distilled 2,3,3-trimethylindolenine (0.53 g, 3.3 mmol) and N-(3-bromopropyl)triethylammonium chloride (1.01 g, 3.9 mmol) were mixed in the thick-wall tube and placed under an argon atmosphere. The mixture was then heated at 140 C. for 1.5 h, giving a deep red viscous product, which solidified to a glass on cooling. It was ground to a powder under acetone; product was collected by filtration, triturated 3 times with 10 mL portions of acetone and dried under vacuum to give 0.45 g (36%) of 2,3,3-trimethyl-1-(3-triethylammoniopropyl)-3H-indolium bromide chloride as a white powder. .sub.H (200 MHz, DMSO-d.sub.6) 8.10 (1H, d, 6.7 Hz, arom.), 7.87 (1H, d, 5.8 Hz, arom.), 7.75-7.58 (2H, m, arom.), 4.56 (2H, t, 7.3 Hz, N.sup.+CH.sub.2), 3.27 (6H, q, 6.0 Hz, N.sup.+(CH.sub.2).sub.3), 2.91 (3H, s, CH.sub.3), 2.34-2.10 (2H, m, N.sup.+CH.sub.2), 1.56 (6H, s, (CH.sub.3).sub.2) 1.36-1.05 (11H, m, CH.sub.2, (CH.sub.3).sub.3).

Example 9

Synthesis of 3-[3-(5-carboxypentyl)-5-iodo-2,3-dimethyl-3H-1-indoliumyl]-1-propanesulfonate (2)

(152) ##STR00081##

(153) A mixture of 13.4 g (0.12 mol) of potassium tert-butoxide and 100 g of tert-butanol was stirred and heated until the tert-butoxide totally dissolved. The solution was cooled to about 50 C. and 17 g (0.12 mmol) of ethyl 2-methylacetoacetate was added dropwise. Ethyl-6-bromohexanoate (30 g, 0.13 mmol) was then added dropwise and the reaction mixture was stirred and refluxed for 5 hours. The mixture was filtered and the solvent was removed under reduced pressure. The residue was partitioned between 1 M HCl and chloroform. The organic layer was dried over magnesium sulfate and purified on silica gel using 1:10 ethyl acetate/hexane as the eluent to yield 25 g (75%) of diethyl 2-acetyl-2-methyloctanedioate (3) as yellow liquid.

(154) The compound 3 was dissolved in 160 mL of methanol. A solution of 5.4 g NaOH in 54 mL of water was added. The mixture was heated at 50 C. overnight. Then the solution was reduced to about 50 mL, acidified to pH 1 and extracted with ethyl acetate. The organic phase was collected, dried over MgSO.sub.4 and evaporated to yield 7.35 g of 7-methyl-8-oxononanonic acid (4).

(155) The 7-methyl-8-oxononanonic acid (4) 2 g (10.74 mmol) was refluxed in 15 mL of acetic acid with 2 g (8.55 mmol) of 4-iodophenylhydrazine for 20 hours. The acetic acid was evaporated and the product was purified on Silica gel 60 (0-15% methanol in chloroform) to yield 0.96 g (29%) of 6-(5-iodo-2,3-dimethyl-3H-3-indolyl)hexanoic acid (5) as an orange solid. .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 11.95 (1H, COOH, broad s), 7.76 (1H, arom., d, 1.4 Hz), 7.62 (1H, arom., dd, 8.1 Hz, 1.5 Hz), 7.23 (1H, arom., d, 8.1 Hz), 2.15 (3H, 2-CH.sub.3, s), 2.06 (2H, CH.sub.2, t, 7.2 Hz), 1.99-1.60 (2H, CH.sub.2, m), 1.42-1.24 (2H, CH.sub.2, m), 1.22 (3H, 3-CH.sub.3, s), 1.18-0.96 (2H, CH.sub.2, m), 0.72-0.24 (2H, CH.sub.2, m).

(156) 500 mg (1.30 mmol) of 6-(5-iodo-2,3-dimethyl-3H-3-indolyl)hexanoic acid was heated with 500 mg (4.09 mmol) of propane sultone at 100 C. for 30 minutes to generate the 450 mg of the final product 3-[3-(5-carboxypentyl)-5-iodo-2,3-dimethyl-3H-1-indoliumyl]-1-propanesulfonate (2).

Example 10

Synthesis of 3,4,5-tribromophenylhydrazine

(157) ##STR00082##

(158) 3,4,5-Tribromoaniline was obtained by the procedure of L. F. Tietze and T. Eicher (L. F. Tietze, T. Eicher. Reaktionen und Synthesen im organisch-chemischen Praktikum und Forschungslaboratorium. (1991) Georg Thieme Verlag, Stuttgart, New York). 7.8 g (24 mmol) of 3,4,5-tribromoaniline were stirred with a solution of 6.5 mL of concentrated hydrochloric acid and 6.5 mL of water. The mixture was cooled to about 10 C. and at this temperature the solution of 2.76 g of NaNO.sub.2 in 11 mL of water was added for 20 min with intensive stiffing. The reaction mixture was stirred for 30 min at 10 C. Then the cooled solution of 16.2 g of SnCl.sub.2.2H.sub.2O in 17 mL of concentrated HCl was added dropwise at 10 C. to the suspension of diazocompound. The reaction mixture was kept at 10 C. for one hour and at 5 C. overnight. The obtained precipitate was filtered off and washed with water. The residue was resuspended in 50 mL of water and concentrated aqueous solution of NaOH was added to get alkaline medium. The 3,4,5-tribromophenylhydrazine was extracted with ether. Organic layer was dried with MgSO.sub.4 and ether was evaporated. Yield: 5.5 g (67%). .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 7.09 (2H, s, arom.), 7.34 (1H, s, NH), 4.19 (2H, d, NH.sub.2).

Example 11

Synthesis of 4,5,6-tribromo-2,3,3-trimethyl-3H-indole

(159) ##STR00083##

(160) 1.73 g of 3,4,5-tribromophenylhydrazine and 2 mL (18.5 mmol) of 3-methyl-2-butanone were refluxed for an hour. Next, 15 mL of concentrated HCl were added and refluxed for another 3.5 h. The reaction mixture was neutralized with concentrated aqueous solution of sodium carbonate, and the product was extracted with benzene. The organic layer was dried with MgSO.sub.4 and benzene was removed under reduced pressure by a rotary evaporator to yield 1.12 g of 4,5,6-tribromo-2,3,3-trimethyl-3H-indole. .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 7.16 (1H, s, arom.), 2.22 (3H, s, CH.sub.3), 1.35 (6H, s, C(CH.sub.3).sub.2).

Example 12

Synthesis of 4,5,6-tribromo-1,2,3,3-tetramethyl-3H-indolium iodide

(161) ##STR00084##

(162) 1.12 g (2.8 mmol) of 4,5,6-tribromo-2,3,3-trimethyl-3H-indole and 2 mL (32 mmol) of iodomethane were heated in a sealed tube at 30-35 C. for 24 hours. The formed precipitate was filtered, washed with small amounts of benzene and ether to yield 700 mg (47%) of 4,5,6-tribromo-1,2,3,3-tetramethyl-3H-indolium iodide. .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 8.50 (1H, s, arom.), 3.99 (3H, s, NCH), 2.77 (3H, s, CH.sub.3), 1.60 (6H, s, C(CH.sub.3).sub.2).

2. Synthesis of Polymethine Dyes

Example 13

Synthesis of 4,5,6-triiodo-1,3,3-trimethyl-2-[7-(4,5,6-triiodo-1,3,3-trimethyl-2,3-dihydro-1H-2-indolyliden)-1,3,5-heptatrienyl]-3H-indolium iodide (I-76)

(163) ##STR00085##

(164) A mixture of 150 mg (0.22 mmol) of 4,5,6-triiodo-1,2,3,3-tetramethyl-3H-indolium iodide and 35 mg (0.12 mmol) of N-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride (glutacondianil hydrochloride) was refluxed in 2 mL of pyridine-acetic anhydride (1:1, v/v) mixture for 15 min. The dye was precipitated with ether, filtered off and washed with ether. The product was purified by a column chromatography (Silica gel 60, 0-4% methanol-chloroform) to give 20 mg of the 4,5,6-triiodo-1,3,3-trimethyl-2-[7-(4,5,6-triiodo-1,3,3-trimethyl-2,3-dihydro-1H-2-indolyliden)-1,3,5-heptatrienyl]-3H-indolium iodide (I-76). .sup.1H-NMR (200 MHz, CDCl.sub.3), , ppm: 8.03-7.74 (3H, m, CH), 7.63 (2H, s, arom.), 6.75 (2H, m, CH), 6.37 (2H, m, CH), 3.65 (3H, s, NCH.sub.3), 3.59 (3H, s, NCH.sub.3), 1.65 (12H, s, (CH.sub.3).sub.2). .sub.max (abs) 754 nm (methanol).

Example 14

Synthesis of 2-[7-(4,6-diiodo-1,3,3-trimethyl-2,3-dihydro-1H-2-indolyliden)-1,3,5-heptatrienyl]-4,6-diiodo-1,3,3-trimethyl-3H-indolium iodide (I-71)

(165) ##STR00086##

(166) 110 mg (0.20 mmol) of 4,6-diiodo-1,2,3,3-tetramethyl-3H-indolium iodide and 28.5 mg (0.10 mmol) of N-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride were dissolved in 2 mL of acetic anhydride at 100-110 C., following which 2 mL of pyridine were added and the reaction mixture was stirred for 10 min at this temperature. The dye was precipitated with ether, filtered off and washed with ether. The raw product was column purified on Silica gel 60 using 5-10% methanol-chloroform as eluent. Yield: 20 mg. .sup.1H-NMR (400 MHz, DMSO-d.sub.6), , ppm: 7.95 (2H, s, arom.), 7.91 (2H, t, 13.1 Hz, CH), 7.81 (2H, s, arom.), 7.77 (1H, t, 13.0 Hz, CH), 6.59 (2H, t, 12.6 Hz, CH), 6.34 (2H, d, 14 Hz, CH), 3.53 (6H, s, NCH.sub.3), 1.72 (12H, s, (CH.sub.3).sub.2). .sub.max (abs) 745 nm (methanol), .sub.max (fluor) 773 nm (methanol), QY (fluor) 0.46.

Example 15

Synthesis of 4,5,6-tribromo-1,3,3-trimethyl-2-[7-(4,5,6-tribromo-1,3,3-trimethyl-2,3-dihydro-1H-2-indolyliden)-1,3,5-heptatrienyl]-3H-indolium iodide (I-88)

(167) ##STR00087##

(168) A mixture of 91 mg (0.17 mmol) of 4,5,6-tribromo-1,2,3,3-tetramethyl-3H-indolium iodide and 24 mg (0.085 mmol) of N-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride (glutacondianil hydrochloride) was refluxed in 4 mL of pyridine-acetic anhydride (1:1, v/v) mixture for 15 min. The dye was precipitated with ether, filtered off and washed with ether. The product was purified by a column chromatography (Silica gel 60, chloroform) to give 14 mg of the 4,5,6-tribromo-1,3,3-trimethyl-2-[7-(4,5,6-tribromo-1,3,3-trimethyl-2,3-dihydro-1H-2-indolyliden)-1,3,5-heptatrienyl]-3H-indolium iodide (I-88). .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 8.06-7.72 (3H, m, CH), 7.93 (2H, s, arom.), 6.23 (2H, t, 12.6 Hz, CH), 6.35 (2H, d, 13.6 Hz, CH), 3.55 (6H, s, NCH.sub.3), 1.76 (12H, s, CH.sub.3). .sub.max (abs) 748 nm (s=240,000) (methanol), .sub.max (fluor) 778 nm (methanol), QY (fluor) 0.50 (methanol).

Example 16

Synthesis of 2-[6-anilino-1,3,5-hexatrienyl]-4,6-diiodo-1,3,3-trimethyl-3H-indolium iodide

(169) ##STR00088##

(170) 100 mg (0.18 mmol) of 4,6-diiodo-1,2,3,3-tetramethyl-3H-indolium iodide and 51 mg (0.18 mmol) of N-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride were stirred in 2 mL of acetic anhydride at 110-120 C. for 15 min. After cooling the product was precipitated with ether, filtered off and washed with ether. Yield: 67 mg (53%). .sub.max (abs) 483 nm (methanol). This product was used for following step without additional purification.

Example 17

Synthesis of 1-(5-carboxypentyl)-2-[7-(4,6-diiodo-1,3,3-trimethyl-3H-2-indoliumyl)-2,4,6-heptatrienylidene]-3-methyl-3-(4-sulfobutyl)-5-indolinesulfonate (I-72)

(171) ##STR00089##

(172) 67 mg (0.095 mmol) of 2-[6-anilino-1,3,5-hexatrienyl]-4,6-diiodo-1,3,3-trimethyl-3H-indolium iodide and 45 mg (0.095 mmol) of 4-[1-(5-carboxypentyl)-2,3-dimethyl-5-sulfo-3H-3-indoliumyl]-1-butanesulfonate were dissolved in 2 mL of DMF-acetic anhydride mixture (1:1, v/v). Three drops of triethylamine were added, and the solution was stirred for 15 min at 115 C. After cooling the product was precipitated with ethyl acetate, filtered off, washed with ether and column purified on Lichroprep RP-18 using 0-35% acetonitrile-water as eluent. Yield: 26 mg. .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 8.05 (1H, t, 12.8 Hz, CH), 7.81 (1H, s, arom.), 7.80-7.59 (5H, m, arom. and CH), 7.45 (1H, d, 8.4 Hz, arom.), 6.72-6.42 (3H, m, CH), 6.11 (1H, d, 13.0 Hz, CH), 4.19 (2H, m, NCH.sub.2), 3.42 (3H, s, NCH.sub.3), 2.20 (6H, t, 6.9 Hz, CH.sub.2COOH and (CH.sub.2).sub.2), 1.77-1.48 (2H, m, CH.sub.2), 1.71 (6H, s, (CH.sub.3).sub.2), 1.63 (3H, s, CH.sub.3), 1.48-1.30 (6H, m, (CH.sub.2).sub.3), 0.92-0.41 (2H, m, CH.sub.2). .sub.max (abs) 750 nm (methanol), .sub.max (fluor) 780 nm (methanol), QY (fluor) 0.41 (methanol), .sub.max (abs) 750 nm (s=270,000) (water), .sub.max (fluor) 777 nm (water), QY (fluor) 0.21 (water).

Example 18

Synthesis of 1-(5-carboxypentyl)-2-[7-(4,6-diiodo-1,3,3-trimethyl-3H-2-indoliumyl)-2,4,6-heptatrienylidene]-3,3-dimethyl-5-indolinesulfonate (I-73)

(173) ##STR00090##

(174) 70 mg (010 mmol) of 2-[6-anilino-1,3,5-hexatrienyl]-4,6-diiodo-1,3,3-trimethyl-3H-indolium iodide and 35 mg (0.10 mmol) of 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-5-indoliumsulfonate were dissolved in 2 mL of DMF-acetic anhydride mixture (1:1, v/v). Three drops of triethylamine were added, and the solution was stirred for 20 min at 110 C. After cooling the product was precipitated with ether, filtered off, washed with ether and column purified on Silica gel 60 using 15-55% methanol-chloroform as eluent. Yield: 28 mg (33%). .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 8.03 (1H, t, 12.3 Hz, CH), 7.82 (1H, s, arom.), 7.80-7.59 (5H, m, arom. and CH), 7.47 (1H, d, 8.4 Hz, arom.), 6.72-6.41 (3H, m, CH), 6.11 (1H, d, 13.2 Hz, CH), 4.19 (2H, m, NCH.sub.2), 3.42 (3H, s, NCH.sub.3), 2.20 (2H, t, 6.4 Hz, CH.sub.2COOH), 1.80-1.29 (6H, m, (CH.sub.2).sub.3), 1.70 (6H, s, (CH.sub.3).sub.2), 1.66 (6H, s, (CH.sub.3).sub.2). .sub.max (abs) 748 nm (methanol), .sub.max (fluor) 779 nm (methanol), QY (fluor) 0.41 (methanol).

Example 19

Synthesis of 3-(5-carboxypentyl)-2-[7-(4,6-diiodo-1,3,3-trimethyl-2,3-dihydro-1H-2-indolyliden)-1,3,5-heptatrienyl]-1,1-dimethyl-6-sulfo-8-(1H-benzo[e]indolium)sulfo-nate (I-74)

(175) ##STR00091##

(176) 70 mg (0.10 mmol) of 2-[6-anilino-1,3,5-hexatrienyl]-4,6-diiodo-1,3,3-trimethyl-3H-indolium iodide and 52 mg (0.11 mmol) of 3-(5-carboxypentyl)-1,1,2-trimethyl-6-sulfo-8-(1H-benzo[e]indolium)sulfonate were dissolved in 2 mL of DMF-acetic anhydride mixture (1:1, v/v). Three drops of triethylamine were added, and the solution was stirred for 10 min at 110-115 C. After cooling to room temperature the product was precipitated with ethyl acetate, filtered off, washed with ethyl acetate and ether and column purified on on Lichroprep RP-18 using 0-53% methanol-water as eluent. Yield: 10 mg (10%). .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 9.35 (1H, d, 6.2 Hz, CH), 9.08 (1H, d, 9.8 Hz, arom.), 8.47-7.55 (7H m, arom. and CH), 6.82-6.46 (3H, m, CH), 6.05 (1H, d, 12.8 Hz, CH), 4.37 (2H, m, NCH.sub.2), 3.40 (3H, s, NCH.sub.3), 2.21 (2H, t, 6.2 Hz, CH.sub.2COOH), 1.91 (6H, s, (CH.sub.3).sub.2), 1.71 (6H, s, (CH.sub.3).sub.2), 1.66-1.29 (6H, m, (CH.sub.2).sub.3). .sub.max (abs) 760 nm (methanol), .sub.max (fluor) 793 nm (methanol), QY (fluor) 0.22 (methanol).

Example 20

Synthesis of 3-(5-carboxypentyl)-2-7-[3-(5-carboxypentyl)-4,6-diiodo-1,3-dimethyl-2,3-dihydro-1H-2-indolyliden]-1,3,5-heptatrienyl-4,6-diiodo-1,3-dimethyl-3H-indolium iodide (I-79)

(177) ##STR00092##

(178) 130.6 mg (0.20 mmol) of 3-(5-carboxypentyl)-4,6-diiodo-1,2,3-trimethyl-3H-indolium iodide and 28.5 mg (0.10 mmol) of N-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride were stirred in 2 mL of pyridine-acetic anhydride (1:1, v/v) mixture at 115-120 C. for 12 min. After cooling to room temperature the dye was precipitated with 25 mL of ether, filtered off and washed with ether. The raw product was column purified on Silica gel 60 using 0-7% methanol-chloroform as eluent. Yield: 24.5 mg (10%). .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 7.95 (2H, s, arom.), 7.91-7.33 (3H, m, CH), 7.79 (2H, s, arom.), 6.59 (2H, t, 12.4 Hz, CH), 6.37 (2H, d, 13.5 Hz, CH), 3.56 (6H, s, NCH.sub.3), 2.05 (4H, m, CH.sub.2), 1.70 (6H, s, CH.sub.3), 1.45-0.99 (12H, m, (CH.sub.2).sub.2), 0.90-0.30 (4H, m, CH.sub.2). .sub.max (abs) 763 nm (chloroform).

Example 21

Synthesis of NHS ester of dye I-72

(179) ##STR00093##

(180) 50 mg (52 mol) of dye I-72 and 31 mg (104 mol) of 0-(N-succinimidyl)-N,N,N,N-tetramethyluronium tetrafluoroborate (TSTU) were dissolved in 2 mL of DMF, 36 L (207 mol) of N,N-diisopropyl ethyl amine (DIPEA) were added and the solution was stirred at room temperature for 1 hour. The NHS ester was precipitated with ether, filtered off, and washed with ether (310 mL). Pure NHS ester was obtained by column chromatography (Lichroprep RP-18, 10-31% acetonitrile-water gradient). Yield: 22 mg (40%). .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 8.06 (1H, t, 12.5 Hz, CH), 7.82 (1H, s, arom.), 7.80-7.62 (5H, m, arom. and CH), 7.45 (1H, d, 8.4 Hz, arom.), 6.72-6.42 (3H, m, CH), 6.12 (1H, d, 13.0 Hz, CH), 4.19 (2H, m, NCH.sub.2), 3.61 (2H, m, CH.sub.2CH.sub.3 (DIPEA)), 3.42 (3H, s, NCH.sub.3), 3.14 (2H, m, CH i-Pr (DIPEA)), 2.81 (4H, s, CH.sub.2NHS), 2.69 (2H, t, CH.sub.2CO), 2.20 (4H, m, CH.sub.2), 1.84-1.58 (2H, m, CH.sub.2), 1.70 (6H, s, CH.sub.3), 1.64 (3H, s, CH.sub.3), 1.58-1.34 (6H, m, (CH.sub.2).sub.3), 1.34-1.16 (15H, CH.sub.3 (DIPEA)), 0.90-0.35 (2H, m, CH.sub.2).

Example 22

Synthesis of NHS ester of dye I-79 (3-[5-(2,5-dioxotetrahydro-1H-1-pyrrolyloxycarbonyl)pentyl]-2-7-{3-[5-(2,5-dioxotetrahydro-1H-1-pyrrolyloxycarbonyl)pen-tyl]-4,6-diiodo-1,3-dimethyl-2,3-dihydro-1H-2-indolyliden}-1,3,5-heptatrienyl)-4,6-diiodo-1,3-dimethyl-3H-indolium iodide)

(181) ##STR00094##

(182) 40 mg (32 mol) of dye I-79 and 38.5 mg (128 mol) of O(N-succinimidyl)-N,N,N,N-tetra-methyluronium tetrafluoroborate (TSTU) were dissolved in 2 mL of DMF, 23 L (132 mol) of N,N-diisopropyl ethyl amine (DIPEA) were added and the solution was stirred at room temperature for 1 hour. The reaction was monitored by TLC (Sorbfil, methanol/chloroform azeotrope). The product was precipitated with ether, filtered off, washed with ether, and purified by column chromatography (Silica gel 60, 0-5% methanol-chloroform gradient). Yield: 18 mg (39.2%). .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 8.06-7.65 (3H, m, CH), 7.95 (2H, s, arom.), 7.80 (2H, s, arom.), 6.59 (2H, t, 12.6 Hz, CH), 6.38 (2H, d, 13.5 Hz, CH), 3.55 (6H, s, NCH.sub.3), 2.78 (8H, s, CH.sub.2NHS), 2.23-2.10 (4H, m, CH.sub.2CO), 1.71 (6H, s, CH.sub.3), 1.59-1.00 (12H, m, (CH.sub.2).sub.2), 0.94-0.30 (4H, m, CH.sub.2).

Example 23

Synthesis of 4-2-[6-anilino-1,3,5-hexatrienyl]-3,3-dimethyl-5-sulfo-3H-1-indoliumyl-1-butanesulfonate

(183) ##STR00095##

(184) A mixture of 187 mg (0.5 mmol) of 4-(2,3,3-trimethyl-5-sulfo-3H-1-indoliumyl)-1-butanesulfonate, 152 mg (0.53 mmol) of N-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride, 2 mL of acetic anhydride, and 1 mL of acetic acid was stirred at 120-125 C. for 45 min. After cooling the product was precipitated with ethyl acetate, filtered off and washed with ethyl acetate and ether. Yield: 210 mg. This product was used for following step without additional purification.

Example 24

Synthesis of 4-(2-7-[3-(5-carboxypentyl)-4,6-diiodo-1-methyl-3H-2-indoliumyl]-2,4,6-heptatrienylidene-3,3-dimethyl-5-sulfo-2,3-dihydro-1H-1-indolyl)-1-butanesulfonate (I-83)

(185) ##STR00096##

(186) 53 mg (0.10 mmol) of 4-2-[6-anilino-1,3,5-hexatrienyl]-3,3-dimethyl-5-sulfo-3H-1-indoliumyl-1-butanesulfonate were dissolved in a mixture of 1.5 mL of (1:1, v/v) acetic acid-acetic anhydride under gently warming. To the obtained solution 69 mg (0.11 mmol) of 3-(5-carboxypentyl)-4,6-diiodo-1,2,3-trimethyl-3H-indolium iodide, dissolved in 2 mL of DMF, were added followed by three drops of triethylamine. The mixture was allowed to stir for 10 min at 110 C. After cooling to room temperature the product was precipitated with 25 mL of ethyl acetate, filtered off, washed with ethyl acetate and ether. Purification by column chromatography on Lichroprep RP-18 using gradient 0-35% acetonitrile-water as eluent gave 9 mg (9%) of I-83. .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 8.04 (1H, t, 12.5 Hz, CH), 7.90-7.44 (7H, m, arom. and CH), 6.90-6.40 (3H, m, CH), 6.10 (1H, d, 13.0 Hz, CH), 4.21 (2H, m, NCH.sub.2), 3.40 (3H, s, NCH.sub.3), 2.72 (2H, t, CH.sub.2SO.sub.3H), 2.07 (2H, t, CH.sub.2COOH), 1.90-1.47 (2H, m, CH.sub.2), 1.75 (3H, s, CH.sub.3), 1.67 (6H, s, CH.sub.3), 1.43-1.00 (8H, m, CH.sub.2), 0.90-0.32 (2H, m, CH.sub.2).

Example 25

Synthesis of 4-[2-[6-anilino-1,3,5-hexatrienyl]-3-methyl-5-sulfo-3-(4-sulfobutyl)-3H-1-indoliumyl]-1-butanesulfonate

(187) ##STR00097##

(188) 300 mg (0.55 mmol) of 2,3-dimethyl-5-sulfo-1,3-di(4-sulfobutyl)-3H-indolium and 190 mg (0.66 mmol) of N-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride were heated in 6 mL acetic anhydride-acetic acid (1:1, v/v) mixture for 30 min at 110 C. After cooling the product was precipitated with ethyl acetate, filtered off and washed with ether to give 240 mg (56%) of 4-[2-[6-anilino-1,3,5-hexatrienyl]-3-methyl-5-sulfo-3-(4-sulfobutyl)-3H-1-indoliumyl]-1-butanesulfonate. .sub.max (abs) 505 nm (methanol). This product was used for following step without additional purification.

Example 26

Synthesis of 4-[2-7-[3-(5-carboxypentyl)-4,5,6-triiodo-3-methyl-1-(3-sulfopro-pyl)-2,3-dihydro-1H-2-indolyliden]-1,3,5-heptatrienyl-3-methyl-5-sulfo-3-(4-sulfobutyl)-3H-1-indoliumyl]-1-butanesulfonate (I-84)

(189) ##STR00098##

(190) A mixture of 200 mg (0.26 mmol) of 4-[2-[6-anilino-1,3,5-hexatrienyl]-3-methyl-5-sulfo-3-(4-sulfobutyl)-3H-1-indoliumyl]-1-butanesulfonate, 197 mg (0.26 mmol) of 3-[3-(5-carboxypentyl)-4,5,6-triiodo-2,3-dimethyl-3H-1-indoliumyl]-1-propanesulfonate, 2 mL of acetic anhydride, 2 mL of acetic acid and 3 mL of pyridine were stirred for 20 min at 110 C. After cooling the product was precipitated with ethyl acetate, filtered off, washed with ether and column purified on Lichroprep RP-18 using 0-15% acetonitrile-water as eluent. Yield: 50 mg (10%). .sub.max (abs) 758 nm (=270,000) (water), .sub.max (fluor) 787 nm (water), QY (fluor) 0.20 (water).

Example 27

Synthesis of 3-(3-(5-carboxypentyl)-2-7-[3-(5-carboxypentyl)-4,5,6-triiodo-3-methyl-1-(3-sulfopropyl)-2,3-dihydro-1H-2-indolyliden]-1,3,5-heptatrienyl-4,5,6-triiodo-3-methyl-3H-1-indoliumyl)-1-propanesulfonate (I-86)

(191) ##STR00099##

(192) 150 mg (0.20 mmol) of 3-[3-(5-carboxypentyl)-4,5,6-triiodo-2,3-dimethyl-3H-1-indoliumyl]-1-propanesulfonate and 28.5 mg (0.1 mmol) of N-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride (glutacondianil hydrochloride) were refluxed in a mixture of 1 mL of acetic anhydride, 1 mL of acetic acid and 2 mL of pyridine for 15 min at 110 C. The dye was precipitated with ether, filtered off and washed with ether. The product was purified by column chromatography (Silica gel 60, 0-10% methanol-chloroform) to give 22 mg (8%) of I-86. .sup.1H-NMR (200 MHz, CDCl.sub.3), , ppm: 8.12 (2H, s, arom.), 7.94 (2H, t, CH), 7.71 (1H, t, CH), 6.58 (2H, t, CH), 6.37 (2H, d, CH), 4.20 (4H, m, NCH.sub.2), 2.15 (4H, m, 4H, CH.sub.2COOH), 1.95-1.80 (4H, m, (CH.sub.2).sub.2), 1.48-0.98 (16H, s, (CH.sub.3).sub.2), 0.55-0.30 (4H, m, CH.sub.2). .sub.max (abs) 765 nm (methanol), .sub.max (fluor) 795 nm (methanol), QY (fluor) 0.44 (methanol), .sub.max (abs) 721 nm (=210,000) (water), .sub.max (fluor) 786 nm (water), QY (fluor) 0.006 (water).

Example 28

Synthesis of NHS Ester of Dye I-84

(193) ##STR00100##

(194) A mixture of 8 mg (6 mol) of 4-[2-7-[3-(5-carboxypentyl)-4,5,6-triiodo-3-methyl-1-(3-sulfopropyl)-2,3-dihydro-1H-2-indolyliden]-1,3,5-heptatrienyl-3-methyl-5-sulfo-3-(4-sulfo-butyl)-3H-1-indoliumyl]-1-butanesulfonate (I-84) and 3.6 mg (12 mol) of TSTU were dissolved in 0.5 mL of DMF, 10 g L of N,N-diisopropyl ethyl amine (DIPEA) were added and the solution was stirred at room temperature for an hour. The product was precipitated with ether, filtered off, washed with ether and column purified on Lichroprep RP-18 using 0-22% acetonitrile-water as eluent. Yield: 2 mg.

Example 29

Synthesis of NHS Ester of Dye I-86

(195) ##STR00101##

(196) 14 mg (8.8 mol) of 3-(3-(5-carboxypentyl)-2-7-[3-(5-carboxypentyl)-4,5,6-triiodo-3-methyl-1-(3-sulfopropyl)-2,3-dihydro-1H-2-indolyliden]-1,3,5-heptatrienyl-4,5,6-triiodo-3-methyl-3H-1-indoliumyl)-1-propanesulfonate (I-86) and 10.6 mg (35.2 mol) of TSTU were dissolved in 0.6 mL of DMF, 9 L of N,N-diisopropyl ethyl amine (DIPEA) were added and the solution was stirred for 5 min at room temperature. The product was precipitated with ether, filtered off, washed with ether and column purified on Silica gel 60, 0-60% methanol-chloroform as eluent. Yield: 6 mg.

Example 30

Synthesis of 1-[5-(2-2-[2-(2-carboxyethoxy)ethoxy]ethoxyethylcarbamoyl)-pentyl]-2-[7-(4,6-diiodo-1,3,3-trimethyl-3H-2-indoliumyl)-2,4,6-heptatrienylidene]-3-methyl-3-(4-sulfobutyl)-5-indolinesulfonate (I-89)

(197) ##STR00102##

(198) 58 mg (55 mol) of NHS ester of dye I-72 and 61 mg (220 mol) of tert-butyl 3-2-[2-(2-aminoethoxy)ethoxy]ethoxypropanoate were dissolved in 2 mL of DMF, 35 L of N,N-diisopropyl ethyl amine (DIPEA) were added and the solution was stirred for an hour at room temperature. The reaction was monitored by TLC (Silica gel 60 RP-18, acetonitrile/water (1:1, v/v)). The product was precipitated with ether, filtered off, washed with ether and dried in a vacuum desiccator. The residue was hydrolyzed in 400 pt of formic acid for 5 hours at room temperature. After completion of hydrolysis (TLC on Silica gel 60 RP-18, acetonitrile/water (1:1, v/v)) the dye was precipitated with ether, filtered off, washed with ether and column purified on Lichroprep RP-18, 15-35% acetonitrile-water as eluent. Yield: 28 mg (43%). .sup.1H-NMR (200 MHz, DMSO-d.sub.6), , ppm: 8.06 (1H, t, CH), 7.94-7.61 (6H, m, arom.), 7.45 (1H, d, 8.6 Hz, CH), 6.73-6.55 (3H, m, CH), 6.11 (1H, d, 13.1 Hz, CH), 4.31-4.07 (2H, m, NCH.sub.2), 3.58 (2H, t, NHCH.sub.2CH.sub.2), 3.48 (8H, s, CH.sub.2CH.sub.2OCH.sub.2CHD, 3.42 (3H, s, NCH), 2.43 (2H, t, 6.2 Hz, CH.sub.2COOH), 2.28-2.13 (m, 2H, CH.sub.2), 2.05 (2H, t, 6.8 Hz, CH.sub.2), 1.78-1.59 (2H, m, CH.sub.2), 1.71 (6H, s, CH.sub.3), 1.63 (3H, s, CH.sub.3), 1.59- 1.31 (8H, m, CH.sub.2), 0.94-0.41 (2H, m, CH.sub.2).

Example 31

Synthesis of NHS Ester of Dye I-89

(199) ##STR00103##

(200) 10 L of DIPEA were added to a mixture of 17 mg (0.0146 mmol) of dye I-89 and 8.8 mg (0.029 mmol) of TSTU in 1 mL of dry DMF, stirred at room temperature for 40 min and then 10 L of DIPEA were added and stirred for 30 min. The reaction was monitored using TLC (RP-18, AcCN-water, 1:1). The product was precipitated with diethyl ether (25 mL), filtered, washed with diethyl ether (210 mL), and dried using a vacuum dissicator. The resulted NHS ester was column purified (RP-18, 30-35% AcCN in water). Yield: 7.8 mg (35%).

Example 32

Synthesis of Dye I-90: Binding of Proline Decamer to Cyanine I-72 NHS Ester

(201) ##STR00104##

(202) A solution of 30 mg (0.0246 mmol) of dye I-72 NHS ester in 1 mL DMF was mixed with a solution of 36 mg (0.0251 mmol) of proline decamer in 0.5 mL DMF. Next, 8.5 mL (0.0492 mmol) of DIPEA were added to the above solution and stirred at room temperature for 20 h. The product was precipitated with diethyl ether, filtered, washed with diethyl ether, dried using a vacuum dissicator, and column purified (RP-18, 27-33% AcCN in water). Yield: 16.7 mg (41%).

Example 33

Synthesis of NHS Ester of Dye I-90

(203) ##STR00105##

(204) 3.5 L of DIPEA were added to a mixture of 10 mg (0.00517 mmol) of dye I-90 and 3 mg (0.01 mmol) of TSTU in 1 mL of dry DMF, stirred at room temperature for 40 min and then 3 L of DIPEA were added and stirred for 30 min. The product was precipitated with diethyl ether (25 mL), filtered, washed with diethyl ether (210 mL), and dried using a vacuum dissicator for 12 h. The resultant NHS ester was column purified (RP-18, 33-35% AcCN in water). Yield: 4.6 mg (45%).

Example 34

(205) Synthesis of Positively Charged Cyanine Dye I-91

(206) ##STR00106##

3. Synthesis of Conjugates

Example 35

General Protein Labeling Procedure

(207) Amino-modified molecule or particle (protein, antibody, phage, etc.) labeling reactions were carried out using 50 mM bicarbonate buffer (pH 9.0). A stock solution of 0.5 mg of dye in 50 L of anhydrous DMF was prepared. Amino-modified compound (1.5 mg in case of protein or antibody such as BSA or IgG) was dissolved in 0.5 mL of a 50 mM bicarbonate buffer pH 9.0. To obtain different dye-to-protein (D/P) ratio a series of these protein solutions were prepared. Then, various amounts of the dye stock solution (e.g. 3, 12, 25 uL) were added to the above protein solution(s) in order to obtain different dye-to-protein (D/P) ratios and the mixture was allowed to stir for 2 h at room temperature. Unconjugated dye was separated from the labeled proteins using gel permeation chromatography with Sephadex G25 for BSA conjugates or Sephadex G50 for IgG (0.5 cm20 cm column) and a 67 mM phosphate buffer solution of pH 7.4 as the eluent. The first colored fraction containing the dye-protein conjugate is isolated, while the blue or bluish-green band with a much higher retention time (free label) was discarded. A series of labeling reactions as described above were set up to obtain different dye-to-protein ratios.

Example 36

Binding of Sensitizer Molecule to Carrier Via Rigid Polyproline Linker

(208) This example demonstrates one of the possible ways to synthesize a conjugate of a sensitizer molecules bound to an amino-modified carrier with a polyproline linker.

(209) ##STR00107##

Example 37

Covalent Conjugate of Counterion with Heptamethine Cyanine

(210) ##STR00108##

4. Spectral and Photophysical Characteristics

Example 38

Spectral Properties of the Invented Dyes

(211) This example shows the spectral properties of the representative dyes (Table 1).

(212) Absorption spectra were measured at RT on a Perking Elmer Lambda 35 UV/Vis spectrophotometer. For measurement of the extinction coefficients, each dye (7-10 mg) was dissolved in 50 mL of PB (pH 7.4). The stock solution was diluted (1:2000) and the absorbance was measured in a 5-cm standard quartz cell. All dye concentrations were in the range of 1.1 to 2.010.sup.7 M. The extinction coefficients were calculated according to Lambert-Beer's law.

(213) Fluorescence spectra for the dyes were determined at RT on a Varian Cary Eclipse spectrofluorometer in a standard 1-cm quartz cell. The spectra were corrected. All concentrations of the fluorophores were chosen to be between 0.2 and 1.0 M.

(214) For the determination of the quantum yields (QY), the integrated relative intensities of the dyes or dye-protein conjugates, were measured against HITC as the reference. All absorbances at excitation wavelength (.sub.exc) were in the range of 0.04-0.08 (when measured in a 1-cm cell). The fluorescence spectra of the solutions were measured and the absolute QYs were determined relative to HITC in methanol (QY 28%) by known methodology (Lakowicz J. R. Principles of Fluorescence Spectroscopy, 4th ed. Springer, New York, 2006).

(215) The QY of each sample was independently measured 3-4 times and the average value was calculated.

(216) Results are shown in Table 1 below.

(217) TABLE-US-00002 TABLE 1 Spectral properties of representative dyes of this disclosure compared to prototypes (ICG and HITC) Absorption Extinction Emission Fluorescence max. Coefficient max. quantum yield Sample Media [nm] [M.sup.1cm.sup.1] [nm] [%] 09 ICG Water Methanol 780 784 114,000 200,000 810 818 16 0 HITC Methanol 740 768 28 I-25 Methanol Water Chloroform 753 750 771 784 779 801 30 I-51 Methanol Chloroform 758 783 788 809 I-52 Methanol Phosphate buffer (pH 7.4) Conjugates with IgG (antibody) 763 760 763-767 240,000 791 788 788 I-55 Phosphate buffer (pH 7.4) Chloroform Methanol 764 787 767 235,000 794 819 799 I-56 Methanol 784 806 16 I-61 Methanol 792 812 14 I-68 Methanol 794 826 6 I-71 Methanol 745 773 46 I-72 Water Methanol 750 750 270,000 777 780 21 41 0 I-73 Methanol 748 779 41 I-74 Methanol 760 793 22 I-76 Methanol 755 785 43 I-79 Chloroform 763 I-83 Methanol Water Conjugates with IgG 750 748 755 779 776 776 43 22 I-84 Water (PB, pH 7.4) Conjugates with IgG 758 762 787 787 20 I-86 Methanol Water BSA conjugate 765 760 778 210,000 100,000 795 796 44 5-7 (subject to D/P) I-88 Methanol 748 240,000 778 50 I-89 Water 750 270,000 777 21.5 I-90 Water Methanol 751 751 270,000 778 780 27 47 0 I-91 Methanol 766 795 26 I-92 Methanol 764 794 39 I-93 Methanol Water 755 783 783 n.f. 47 I-94 Methanol Water 759 695, 756 788 782 38 1.8 I-96 Methanol 787 808 21

Example 39

Photostability of Halogenated Dyes

(218) Photostability of the water soluble halogenated dyes was measured in water. Solutions with absorbance (optical density) in the range between 0.15 and 0.20 (measured in standard 1-cm quartz cells) at the excitation (laser) wavelength were prepared. The laser was placed on the top of a cuvette containing the dye solution with a magnetic stirrer, and the solution was irradiated with continuous stiffing. The absorption and emission spectra of the solutions were measured before irradiation and during light exposure. Relative photostabilities were calculated as the ratio between (i) the measured absorbances at the long-wavelength maximum before and after exposure (A/A.sub.0) and (ii) relative fluorescence intensities before and after exposure (I/I.sub.0), and corresponding plots were generated. The plots showing A/A.sub.0 and I/I.sub.0 vs. the exposure time are shown in FIGS. 5 and 6, respectively.

5. Biological Testing

Example 40

Determination of the Dye-to-Protein Ratios

(219) The molar dye-to-protein ratio (degree of labeling) for each purified dye conjugate was calculated as the molarity of dye divided by the molarity of protein. The dye concentration of the conjugate was determined according to Lambert-Beer's Law from the absorbance of the dye at the absorption maximum. The protein concentration was assessed by measurement of the absorption of the protein at 278 nm. The dye-to-protein ratios (D/P) were calculated using the following formula (with the assumption that the extinction coefficients for the free and conjugated dyes are approximately equal):

(220) $D/P=\frac{A_{\mathrm{conj}\left(\max \right)}{\mathrm{.Math.}}_p}{\left(A_{\mathrm{conj}\left(278\right)}-{\mathrm{xA}}_{\mathrm{conj}\left(\max \right)}\right){\mathrm{.Math.}}_{\mathrm{dye}}},$
where .sub.conj (max), A.sub.conj(278) are the absorbances (optical densities) at the long-wavelength absorption maxima and at 278 nm of the dye-protein conjugate respectively; .sub.dye is the extinction coefficient of the dye at .sub.max, .sub.p is the extinction coefficient of the protein at 278 nm, for antibody (IgG): .sub.p=201,700 M.sup.1 cm.sup.1. The factor x (x=A.sub.dye(278)/A.sub.dye(max)) in the denominator accounts for dye absorption at 278 nm (A.sub.dye(278)) which is a percent of the absorption of the dye at its maximum absorption (A.sub.dye(max)).

Example 41

Preparation of Bovine Erythrocytes

(221) Bovine erythrocytes were isolated from blood plasma stored with sodium citrate. 1 mL of bovine blood was treated at centrifuge (3,000 rpm) for 10 min to precipitate erythrocytes. The supernatant was decanted, 2 mL of physiological solution were added, treated and erythrocytes were precipitated with centrifuge. The washing procedure was repeated for 3 times and the precipitated erythrocytes were collected.

Example 42

Testing of Phototoxicity Under Exposure with Glow Lamp and Dark Toxicity of Photosensitizing Compositions Using Bovine Erythrocytes

(222) For dye incubation approximately 72510.sup.6 erythrocytes were placed in a 1 mL physiological solution, containing a known dye concentration. Cells were dye-incubated for 45 minutes at 37 C. in the dark. Then the examined suspensions of cells (erythrocytes) were placed at a distance of 30-cm from a 250 W glow red-lamp equipped with a water-based heat filter and irradiated with occasional stirring. The emission spectrum of the lamp is shown in FIG. 9. After irradiation, cells were stored at 4 C. for 15 hours. Then the whole suspension was centrifuged and the absorbance of supernatant was measured at 542 nm. The hemolysis level (percentage of hemolyzed cells) was determined as the ratio of absorbances of the supernatant and a solution of 100% hemolysed cells. The 100% hemolysis was taken as the value obtained when the given number of erythrocytes were suspended in distilled water.

(223) Representative data are shown in Table 2 below.

(224) TABLE-US-00003 TABLE 2 Hemolysis level of bovine erythrocytes in dark and under light exposure with glow lamp Exposure Concentration Time Hemolysis Composition [M] Excitation [min] [%] ICG 10 5 1 Dark Dark Glow Lamp Glow Lamp Glow Lamp 120 60 120 1.7 1.6 65 16 4 HITC 10 5 1 Dark Dark Glow Lamp Glow Lamp Glow Lamp Glow Lamp 120 60 120 60 6.7 2.0 74 65 5.4 4.4 I-25 50 0.5 0.1 Dark Glow Lamp Glow Lamp 30 60 51 83 39 I-71 50 5 0.5 0.1 Dark Dark Glow Lamp Glow Lamp 30 60 30 2 75 21 I-76 50 5 0.5 0.1 Dark Dark Glow Lamp Glow Lamp 30 60 12 2 46 43 0 I-79 50 1 0.5 Dark Glow Lamp Glow Lamp 30 60 4 80 75 I-86 10 0.5 0.1 Dark Glow Lamp Glow Lamp 30 60 3 74 70

Example 43

Testing of Phototoxicity Under Irradiation with Laser and Dark Toxicity of Photosensitizing Compositions Using Bovine Erythrocytes

(225) For dye incubation, approximately 72510.sup.6 erythrocytes were placed in a 1 mL physiological solution, containing a known dye concentration. Cells were incubated for 45 minutes at 37 C. in the dark. Then the examined suspensions of cells (erythrocytes) were irradiated with diode laser (786 nm, 68 mW) with occasional stirring. After irradiation, cells were stored at 4 C. for 15 hours. Then erythrocytes were precipitated using centrifugation and the absorbance of supernatant was measured at 542 nm. The hemolysis level was determined as the ratio of absorbances of the investigated supernatant and a solution of 100% hemolysed cells. The 100% hemolysis was taken as the value obtained when the given number of erythrocytes were suspended in distilled water. Representative data are shown in FIG. 9.

6. The Singlet Oxygen Generation Efficiency

Example 44

Determination of the Singlet Oxygen Quantum Yield ()

(226) The modified relative photochemical method of Spiller W. et al. (J. Porphyrins Phthalocyanines, 1998, V. 2, 145-158) utilizing 1,3-diphenylisobenzofuran (DPBF) as a chemical scavenger was used to determine the photosensitiser's singlet oxygen quantum yields (.sub.) in ethanol solutions. ICG (.sub.=12%) was used as a reference compound (J. A. Cardillo, et al. Br. J. Ophthalmol., 2008, V. 92, 276-280).

(227) Ethanolic solutions containing DPBF (10 M) and sensitizers with absorbance (optical density) in a range between 0.09 and 0.10 at 650 nm (measured in a standard 1-cm quartz cell) were prepared. Samples were saturated with oxygen by bubbling. Next, 3 mL of the solution were placed in a 1-cm cell equipped with a stirring bar and a 10 mW, 650 nm diode laser was hermetically attached to the top of cuvette so as to avoid solvent evaporation and to provide a light beam direction from top to bottom of the cuvette. The solution in the cuvette was irradiated with the diode laser under continuous stirring for a certain time. DPBF consumption was determined by monitoring DPBF bleaching over irradiation time using absorption spectroscopy at 411 nm (extinction coefficient 22,000 M.sup.1 cm.sup.1). At least six absorption spectra were obtained for each solution at different irradiation times until the DPBF absorbance reduced at least to the 10% of the initial absorbance.

(228) The decrease of DPBF absorbance at 411 nm versus irradiation time was plotted for each photosensitizer and the DPBF degradation rate constant (K) characterizing the reaction rate of singlet oxygen with DPBF in presence of a sensitizer was calculated.

(229) The .sub. values were calculated according to the following formula:
.sub.A=.sub.Ce6K/K.sub.Ce6A.sub.Ce6/A,
where .sub. and .sub.Ce6 are the singlet oxygen quantum yield of a SOG and the reference (ICG);
K and K.sub.Ce6 are the DPBF degradation rate constant of a SOG and ICG, respectively, and
A and A.sub.Ce6 are the absorbances (optical densities) of SOG of the dye sample and of ICG at the excitation wavelength (650 nm).

(230) Representative .sub.A data are shown in Table 3 below.

(231) TABLE-US-00004 TABLE 3 Singlet oxygen generation quantum yield (.sub.) measured in ethanol Singlet oxygen generation quantum Composition yield .sub. [%] ICG 12 HITC 5 I-25 20 I-71 19 I-72 12 I-76 50 I-86 13 I-89 13 0 I-88 11 I-90 11