HK1109391B - Adduct of fluorescent dye and tumor avid tetrapyrrole - Google Patents

Description

adduct of fluorescent dye and tumor avid tetrapyrrole

background

Detection of early neoplastic changes is important from a result standpoint because once invasive cancer and cancer metastasis occur, treatment becomes difficult. Currently, the removal of biopsies followed by histological examination is considered the "gold standard" for the diagnosis of changes in early neoplasia and cancer. Sometimes cytology, i.e. examination by surface or fecal cell analysis, is performed rather than by excisional biopsy. These techniques are powerful diagnostic tools because they provide high resolution spatial and morphological information about the cellular and subcellular structure of the tissue. The contrast and specificity of histopathological examination can be improved by using staining and treatment. However, both diagnostic procedures require physical removal of the sample followed by treatment of the tissue in the laboratory. These procedures require relatively high costs because of the handling of the sample for transport and important diagnostic information is not immediately available.

Fluorescence technology has the potential advantage of performing in vivo diagnosis of tissue, without the need to excise the sample and perform processing, and in recent years, fluorescence spectroscopy has been developed for the diagnosis of cancer. Infrared imaging (IRI) using spectroscopic agents has several advantages over in vitro and other in vivo techniques in that the technique is non-invasive and, under the appropriate conditions, can achieve deep penetration and obtain quantitative results, and can achieve a more comprehensive examination of the organ of interest than excisional biopsy or cytology. In addition, during the examination of the fluorescent material, both a complete profile of the absorption and information on the required retention and elimination of the spectroscopic agent can be obtained in one experimental animal, thus reducing the number of experimental animals required for preclinical testing.

The requirements for an ideal spectroscopic agent required for infrared imaging techniques are as follows: i) it is preferably localized in tumor cells; ii) has a higher fluorescence utilization rate; iii) does not produce phototoxicity or other side effects in the patient; iv) ease of synthesis; v) should be chemically pure; and vi) have long-wave emission so that deep-lying tumours can be detected.

Porphyrins, including chlorins, bacteriochlorins and other porphyrin-based derivatives, including analogs and derivatives thereof, have recently found preferred use as photodynamic compounds for the diagnosis and treatment of diseases, particularly certain cancers and other hyperproliferative diseases, such as macular degeneration. These compounds are also useful in the treatment of psoriasis and papillomatosis.

Such derivatives include dimers as well as trimers of these compounds. Derivatives that may be used also include cyclic variants of these compounds; provided that the sixteen pendant 4 nitrogen heterocycles remain intact in the center of these compounds. Thus, chlorophyllins, purpurins, and pheophorbides and their derivatives are included within the scope of "porphyrins, chlorins, and bacteriochlorins and their derivatives and analogs". Such derivatives include modifications of the substituents on these cyclic structures.

There are a number of publications on this subject, for example, "Use of the Chlorophyl Derivative pure Therapy-18, for Synthesis of Sensitizers for Use of the same Photodynemic Therapy", Lee et al, J.chem.Soc, 1993, (19) 2369-77; "Synthesis of New Bacteriochlorins And bed them Activity", Pandey et al, Biology And Med. chem. letters, 1992; "Photosensitzing Properties of Bacteriochlorophyllin a and bacteriochlorophylin a, TwoDerivatives of Bacteriochlorophyll a", Beems et al, Photochemistry and Photobiology, 1987, v.46, 639-; "photovoltaic therapy. II. Cu of animal Tumors With hematophagin and Light", Dougherty et al, Journal of the National Cancer Institute, July 1975, v.55, 115-; "Photodynamic therapy of C3H mole gamma with chemophorphyrin di-esters as sensors", Evansen et al, Br.J. cancer, 1987, 55, 483-; "bottom Effects in tetrapyrazole Subunit reaction and Picacol-Picacolone reactions: VIC-dihydroxyychlorins and VIC-Dihydroxybacteriochlorins″Pandey et al.，Tetrahedron Letters，1992，v.33，7815-7818；″Photodynamic Sensitizers from Chlorophyll：Purpurin-18and Chlorin P₆", Hoober et al, 1988, v.48, 579-; "Structure/Activity Relationships were amplified to Photopeptides and bacteriophages", Paney et al, Bioorganic and medicinal Chemistry Letters, 1992, v2, 491-; "Photodynamics therapeutics", Pandey et al, Proceedings Society of Photo-optical instrumentation Engineers (SPIE), 1989, v 1065, 164-; and "FastAtom Bombardment Mass Spectral Analyses of Photofrinand itsSynthetic antibodies ", Paney et al, Biomedical and Environmental Mass Spectrometry, 1990, v.19, 405-" 414. These articles are incorporated herein by reference for background.

Numerous patents have also been filed and issued worldwide in this field relating to these photodynamic compounds. For example, reference is made to the following U.S. patents which are incorporated by reference: 4,649,151, respectively; 4,866,168, respectively; 4,889,129, respectively; 4,932,934, respectively; 4,968,715, respectively; 5,002,962, respectively; 5,015,463, respectively; 5,028,621, respectively; 5,145,863, respectively; 5,198,460, respectively; 5,225,433, respectively; 5,314,905, respectively; 5,459,159, respectively; 5,498,710, and 5,591,847.

One of these compounds ""approval has been obtained in canada and japan in the united states. Other such compounds have also been approved with at least some limitations, such as BPD for the treatment of macular degeneration, others are in clinical trials, or are being considered for use in such trials.

As noted above, and as described and illustrated in the above-mentioned articles and patents incorporated herein by reference as background art, the terms "porphyrins, chlorins, and bacteriochlorins" as used herein are meant to include derivatives and analogs thereof.

Such compounds have been found to have significant properties, excluding the liver and spleen, of preferentially accumulating in tumors, rather than in most normal cells and organs. Moreover, many of these tumors can be killed because the compounds can be activated by light to be toxic to the tumor.

Such compounds are preferentially absorbed into cancer cells and destroy cancer cells upon their preferred wavelength absorbance Near Infrared (NIR) absorption sensitization. Furthermore, such compounds emit radiation at wavelengths longer than the preferred absorption wavelength, and such light can penetrate several centimeters deep into tissue. The photosensitizer concentration in the subcutaneous tissue can thus be detected and determined from the measurement of the scattered light propagation. It is therefore believed that scattered NIR light can be used to detect and image diseased subcutaneous tissue based on NIR absorbance, fluorescence, and fluorescence decay kinetics associated with PDT drugs and other fluorescent whitening agents. It has been shown that frequency domain photon migration technology (FDPM) together with image-intensified charge-coupled devices (CCD) can be used for in vivo detection of diseased tissue using fluorescent contrast agents. Porphyrin-based compounds as described above are very strongly fluorescent compounds and this property has therefore been exploited for studying their use as optical imaging agents. Unfortunately, these compounds do not exhibit a sufficient shift between absorption and emission ("Stoke's shift") suitable for this purpose, and thus such compounds do not provide an excellent means for detection, i.e., the fluorescence emission wavelength of such compounds is close to that of their preferred absorbance, causing detection interference.

One approach to modify porphyrin structures to emit at longer wavelengths has been studied, for example, as described in U.S. Pat. No. 6,103,751, "carotenes Analogs of Porphyrins, Chlorrins and Bacteriochlorins as thermal and Diagnostic Agents". Unfortunately, the effect of adding the bilirubin moiety to the porphyrin is small and thus its therapeutic effect for treatment is impractical, and it is clear that such structures cannot be modified to trade off improvement in emission wavelength without loss of important properties.

However, many compounds that fluoresce at detectable wavelengths are known compounds that have been studied and used to diagnose almost every type of cancer, particularly in the discovery of early neoplastic changes in the human body. However, such an approach has significant difficulties due to several factors including: lack of significant preferred tumor absorbance, toxicity, and lack of sufficient penetration, whether due to activation of the fluorogenic compound or emission that has sufficiently penetrated to be detected in the tumor or in vitro. In addition, such compounds, while perhaps having the potential to be detected, do not have the ability to destroy tumors and other proliferative tissues.

There is therefore a need for a physiological compound which satisfies the following conditions:

1. preferably located in tumor tissue relative to normal tissue,

2. has the advantages of high fluorescent utilization rate,

3. should not be toxic, phototoxic, carcinogenic or teratogenic,

4. it should be convenient for the synthesis to be carried out,

5. should be of a chemical purity in that it is,

6. has a long wavelength absorption in the range of 600 to 800nm so that deep tumors can be detected,

7. will destroy the tumor located by activating it, and

8. have emission wavelengths sufficiently spaced (offset) from their preferred absorption wavelengths so as to prevent significant interference so that tumors can be readily detected by in vivo fluorescence spectroscopy.

Brief description of the invention

The invention includes compounds having the following properties: having a preferred localization in tumor tissue relative to normal tissue, having a preferred absorption of electromagnetic energy at a wavelength between about 660 and 900nm, and fluorescing at a wavelength shifted from the preferred absorption by at least +30nm and preferably at least +50 nm. The compound, when sensed at its preferred absorption wavelength, further preferentially destroys the tumor tissue in which the compound is absorbed. In a preferred embodiment of the invention, the compound is a conjugate of a tumor avid tetrapyrrole compound and a fluorescent dye, more preferably, the fluorescent dye is an indoleamine dye such as indocyanine green. The tumor avid tetrapyrrole compound is preferably a porphyrin derivative (collectively "porphyrin") selected from chlorins, bacteriochlorins, purpurins and derivatives thereof, and generally has the following general structure:

wherein:

R₁is substituted or unsubstituted-CH ═ CH₂-CHO, COOH, or

Wherein R is₉＝-OR₁₀Wherein R is₁₀Is lower alkyl of 1 to 8 carbon atoms, or- (CH)₂-O)_nCH₃；R₂，R_2a，R₃，R_3a，R₄，R₅，R_5a，R₇And R_7aIndependently hydrogen, lower alkyl, substituted lower alkyl, lower alkylene or substituted lower alkylene or 2R on adjacent carbon atoms₂，R_2a，R₃，R_3a，R₅，R_5a，R₇And R_7aThe groups may together form a covalent bond or 2R on the same carbon atom₂，R_2a，R₃，R_3a，R₅，R_5a，R₇And R_7aThe radicals may beA double bond forming a divalent pendant group; r₂And R₃May together form a 5-or 6-membered heterocyclic ring containing oxygen, nitrogen or sulfur; r₆is-CH₂-，-NR₁₁-, wherein R₁₁Is substituted or unsubstituted lower alkyl, or lower alkylene; or R₆Is a covalent bond; r₈Is- (CH)₂)₂CO₂R₁₂Wherein R is₁₂Is substituted or unsubstituted lower alkyl, lower alkylene or-NH₂。

Typically, at least R₁，R_2a，R₃，R_3a，R₄，R₅，R_5a，R₇，R_7a，R₈，R₉，R₁₀，R₁₁Or R₁₂One of which is substituted with a dye that fluoresces at a wavelength of from about 800 to about 900nm.

The fluorescent dye may be a non-toxic dye that causes the conjugate to preferentially emit (fluoresce) at wavelengths of 800 to about 900nm. Such dyes typically have at least 2 resonant ring structures, often chromophores, linked together by an intermediate resonant structure of conjugated double bonds, aromatic carbon rings, resonant heterocycles or combinations thereof.

Examples of such dyes include bis-indole dyes in which 2 indole or modified indole ring structures are linked together at their 32 and 21 carbon atoms respectively by the intermediate resonant structures described above. Such dyes are generally known as tricarbocyanine-type (tricarbocyanine) dyes. Such dyes typically have at least 1, and usually at least 2, hydrophilic substituents that render the dye water soluble. Such water solubility promotes the possibility of the structure entering the organism and its cellular structures and reducing toxicity, due to the reduced storage levels in adipose tissue and the rapid elimination from the system. The intermediate resonant structure typically contains a large number of double-bonded carbon atoms that are typically conjugated double bonds and may also contain unsaturated carbocyclic or heterocyclic rings. Such rings can bind to the porphyrin structure without significantly interfering with the resonance of the intermediate structure.

The invention also includes a method of detecting a tumor by injecting into an organism, administering a compound of the invention for a sufficient time to preferentially absorb in tumor tissue, sensing the absorbed compound at its preferred absorption wavelength and detecting the location of emission from the preferentially absorbed compound to locate tumor tissue, and a method of treating tumor tissue by injecting into an organism, administering a compound of the invention for a sufficient time to preferentially absorb into tumor tissue, and sensing the absorbed compound at its preferred absorption wavelength to destroy tumor tissue. It is to be understood that destruction of tumor tissue according to the present invention may be accomplished as part of the detection method.

Drawings

The nature and mode of operation of the present invention are more fully described in the following detailed description of the invention with reference to the accompanying drawings, in which:

FIG. 1 is a graph of the UV-visible spectrum of conjugate 5;

FIG. 2 is an in vitro fluorescence spectrum of conjugate 5 when excited at 660 nm;

FIG. 3 is a graph of the relative uptake of the combination 5 by the tumor and skin at 24 hours post-injection, shown by relative fluorescence;

FIG. 4 shows a graph of the relative absorption of conjugate 5 3-4 days after injection;

figure 5 shows relative to indocuzanine alone? Analog, tumor uptake of conjugate 5;

figure 6 shows the in vivo fluorescence of conjugate 5 at various injected concentrations, 24 hours after injection;

FIG. 7 shows the efficacy of photodynamic therapy using various concentrations of conjugate 5 for treatment of tumors;

FIG. 8 shows photosensitizer localization of conjugate 5 in mitochondria relative to known mitochondrial probes; and

figure 9 shows the in vivo efficacy of conjugate 5 at various doses 24 hours after injection on transplanted RIF tumors.

Detailed Description

By "preferred electromagnetic energy absorption at a wavelength between about 660 and 900 nm" is meant that there is a peak absorbance between 660 and 900nm in the UV band from about 300 to 900nm that is at least twice, and usually at least 3 times, the absorbance of the other peaks in that band. By "fluoresce at a wavelength shifted from the preferred absorption by at least +30nm and preferably at least +50 nm" is meant that the emission (fluorescence) wavelength resulting from excitation of the preferred absorption wavelength is shifted upwards by at least 30 and preferably at least 50nm from the peak absorbance wavelength. Although not essential according to the invention, the compound, when sensed at its preferred absorption wavelength, further preferably destroys the tumor tissue in which the compound is absorbed. It is believed that this occurs due to the localized formation of singlet oxygen in the cancer tissue that preferentially absorbs the compound.

As previously mentioned, in a preferred embodiment of the invention, the compound is a conjugate of a tumor avid tetrapyrrole compound and a fluorescent dye. Such dyes include, inter alia, bis-indole, tricarbocyanine type dyes, such as indolylamine dyes, which have a preferred absorbance in or near the UV wavelength range from about 300 to 900nm and an emission from about 600 to about 900nm. An example of such a dye is indocyanine green. Other suitable bis-indole dyes have the general structure:

wherein R is_1d，R_2d，R_3dAnd R_4dIs hydrogen, sulfonyl, amino, carboxyl, hydroxyl or alkyl; provided that R is_1dAnd R_3dAnd R_2dAnd R_4dMay together form a cycloalkenyl, aromatic or heterocyclic structure; r_5dAnd R_6dIndependently hydrogen, alkyl or substituted alkyl, wherein the substituents are carboxy, sulfonyl, hydroxy, amido, amino, alkyl ester or halogen or a combination thereofAn acid salt; and R is_7dIs a conjugated double-bonded carbon chain, or a resonating ring selected from the group consisting of aryl, unsaturated cycloalkyl, and resonating unsaturated heterocycle, the resonating ring being substituted with halogen, amino, or carboxyl, and n is an integer from 0 to 3. In preferred dyes of the above structure, R_7dIs that

Wherein X is halogen.

Specific examples of the dye used in the present invention are as follows: indocyanine green (bis-indole, i.e., tricarbocyanine-type dye); indocyanine green 820nm analogue CAS172616-80-7 (R)_7dIs thatFast green FCF (FD)&C green 3, triphenylmethane dyes); suzulene blue (triphenylmethane dye) and methylene blue (thiazine dye).

The tumor avid tetrapyrrole compound is preferably a porphyrin derivative (including porphyrin related compounds, whether or not actually derived from a porphyrin), generally selected from the group consisting of chlorins, bacteriochlorins and bacteriopurpurins. Preferred porphyrin derivatives generally have the following general structure:

wherein:

R₁is substituted or unsubstituted-CH ═ CH₂-CHO, COOH, or

Wherein R is₉＝-OR₁₀Wherein R is₁₀Is lower alkyl of 1 to 8 carbon atoms, or- (CH)₂-O)nCH₃；R₂，R_2a，R₃，R_3a，R₄，R₅，R_5a，R₇And R_7aIndependently hydrogen, lower alkyl, substituted lower alkyl, lower alkylene or substituted lower alkylene or 2R on adjacent carbon atoms₂，R_2a，R₃，R_3a，R₅，R_5a，R₇And R_7aThe groups may together form a covalent bond or 2R on the same carbon atom₂，R_2a，R₃，R_3a，R₅，R_5a，R₇And R_7aGroups may form double bonds of divalent side groups; r₂And R₃May together form a 5-or 6-membered heterocyclic ring containing oxygen, nitrogen or sulfur; r₆Is (-CH)₂-)，-NR₁₁-, wherein R₁₁Is substituted or unsubstituted lower alkyl, or lower alkylene; or R₆Is a covalent bond; r₈Is- (CH)₂)₂CO₂R₁₂Wherein R is₁₂Is hydrogen or a substituted or unsubstituted, lower alkyl, lower alkylene alkali or alkaline earth metal ion, or a dye moiety having a preferred absorbance in or near the UV wavelength range from about 300 to 900nm and an emission from about 600 to about 900nm, or R₈Is- (CH)₂)₂COR_12aWherein R is_12ais-NR₂R_2aWherein R is₂And R_2aAs previously mentioned, and may also include dye moieties having preferred absorbance in or near the UV wavelength range from about 300 to 900nm, and having emission from about 600 to about 900nm.

Typically, at least R₁，R_2a，R₃，R_3a，R₄，R₅，R_5a，R₇，R_7a，R₈，R₉，R₁₀，R₁₁Or R₁₂One of which is substituted with a dye moiety that fluoresces at a wavelength of from about 800 to about 900nm. When R is₁₂Is hydrogen, -NH₂or-NHR₁₃Wherein R is₁₃Is 1 to 6With lower alkyl groups of carbon atoms, such substitution typically occurs at R₈To (3).

The invention also includes a method of detecting a tumor by injecting into an organism, administering a compound of the invention for a sufficient time to preferentially absorb in tumor tissue, sensing the absorbed compound at its preferred absorption wavelength and detecting the location of emission from the preferentially absorbed compound to localize tumor tissue, and a method of treating tumor tissue by injecting into an organism, administering a compound of the invention for a sufficient time to preferentially absorb into tumor tissue, and sensing the absorbed compound at its preferred absorption wavelength to destroy tumor tissue. It will be appreciated that destruction of tumor tissue according to the invention may be achieved as an adjunct to the detection method.

The compounds of the present invention can be readily prepared from substantially all porphyrins including purpurins, chlorins and bacteriochlorins, as discussed in the background above; provided that such compounds have a free carboxyl group or a free carboxylate group, (commonly referred to as a "carboxyl functionality") suitable for binding to the appropriate dye structure as described above. Most porphyrins discussed in the background of the invention have such groups. In turn, it is desirable that the dye have or be modified to have a reactive amine site that is not critical to the fluorescence properties, so that the dye can react with the carboxyl functionality at the free amine to form the porphyrin conjugates of the present invention. Such dyes may also or optionally have reactive acid sites, for example, in the form of sulfonic or carboxylic acid moieties, which can react with basic substituents on the porphyrin structure.

Many of the conjugates of the present invention have the general structure:

wherein R is₁₃Is hydrogen or methyl; r₈is-COR₁₇Wherein R is₁₇is-OH, -OR_n-NHR_nWherein R is_nIs lower alkyl of 1 to 8 carbon atoms, or R₁₇Is a dye moiety as previously described; r₁₄，R₁₅And R₁₆Independently hydrogen, methyl or ethyl; r₁And R₂Independently is-R₉，-OR₉，-C(R₁₂)(O)，-C(R₁₂)₂OR₉，-CH＝CHR₉Or is- (CH)₂)R₁₀；R₃is-R₉，-OR₉，-C(R₁₂)(O)，-C(R₁₂)₂OR₉，-CH＝CHR₉Or- (CH)₂)R₁₀Or together with R₃Is ═ O; r_2ais-R₉，-OR₉，-C(R₁₂)(O)，-C(R₁₂)₂OR₉，-CH＝CHR₉Or- (CH)₂)R₁₄Or together with R_3aIs a chemical bond; r_3ais-R₉，-OR₉，-C(R₁₂)(O)，-C(R₁₂)₂OR₉，-CH＝CHR₉Or- (CH)₂)R₁₀Or together with R_2aIs a chemical bond or together with R₃Is ═ O; r₄Is R₉OR-OR₉；R₉Each occurrence is independently hydrogen or a lower alkyl group of from 1 to 10 carbon atoms as previously described; r₁₀Is an amino acid residue; r₁₁is-R₉，-R₁₀or-C (O) NHR₉；R_4aAnd R_4bEach occurrence is independently hydrogen or lower alkyl of 1 to 4 carbon atoms or together may be-C (R)₉)₂C(Y)-，-C(O)O(O)C-，-C(NR₉) O (O) C-, or-C (O) N (R)₁₁) -c (O) -, and Y is ═ O, ═ S, or 2H-; provided that the compound comprises at least one dye moiety as described previously.

Preferred compounds of the present invention may be represented by the following general formula:

wherein the substituents are as described above.

Examples showing schematic diagrams for the preparation of preferred compounds according to the invention using HPPH and indocyanine green 820nm analogues (1) as described before, wherein the substituents on the carbon atoms a-d, f-g and m-o are usually hydrogen and possibly also lower alkyl, are as follows:

specific preferred compounds of the invention are:

the generally preferred compounds of the invention can be simply represented as follows:

photosensitizer: porphyrins, chlorins, bacteriochlorins, phthalocyanines, porphyrins in a broad sense.

R ═ alkyl, sulfonic acids or carboxyl groups containing carbon chains which alter the carbon unit.

R₂A wide variety of aromatic systems with and without fluorinated substituents.

Other preferred photosensitizer compounds of the invention may be represented as follows:

R＝COOH

R₁＝CONH-(CH₂)_n-photosensitizers

R＝R₁＝CONH-(CH₂)_n-photosensitizers

Photosensitizer: porphyrins, chlorins, bacteriochlorins, phthalocyanines, porphyrins in general

R₂Halogen ═ halogen

R₃Alkyl, sulfonic acids or carboxyl groups containing carbon chains of modified carbon units

R＝CONH(CH₂)_nNH-Folic acid

As shown in fig. 1, the UV-visible absorption spectrum of combination 5 shows characteristic absorption bands at 408, 660 and 830nm corresponding to the 3- (1' -hexyloxyethyl) derivative of pyropheophorbide-a (HPPH)4 and the modified long wavelength absorbing dye 3, respectively, as well as fig. 2 showing broad emission bands at 665, 710 and 860nm, indicating that the combination containing 2 chromophores (HPPH and dye) behaves similarly to a single molecule.

Tumor uptake of conjugate 5 was determined by in vivo reflectance spectroscopy. For these experiments, C3H mice with RIF tumors were injected with 5.0 μmole/kg of conjugate 5 and in vivo absorption spectra were taken at different time intervals. As can be seen from fig. 3, conjugate 5 showed more significant absorption in the tumor than the skin 24 hours after injection. At 3-4 days post injection (fig. 4), the conjugate was cleared from the skin without significantly reducing the intratumoral concentration. In contrast, the indole cyanine green analogue (1), here ICG, produced a higher level of uptake in the skin than the tumor (tumor bearing C3H mice) alone at the same dose (5.0 μmole/kg) and the ICG analogue alone showed a significantly lower level of tumor uptake compared to HPPH-ICG conjugate 5 (figure 5). ICG dye was rapidly cleared from the tumor and skin 4-5 hours after injection (1). These results clearly show that in conjugate 5, HPPH not only acts as a medium to deliver a dye with the desired photophysical properties to the tumor, but also acts as a medium to hold the dye on the tumor surface by altering the overall photodynamic properties of the binding molecule compared to the dye or photosensitizer alone.

The in vivo fluorescence spectra of conjugate 5 were determined by in vivo fluorescence spectroscopy at different concentrations (10, 5.0 and 2.5. mu. mole/kg). The results are summarized in fig. 6. In a typical experiment, conjugate 5 was injected into each mouse (a group of three mice with RIF tumors) at doses of 10, 5.0 and 2.5 μmoles/kg. 24 hours after injection, the absorption peak at 660nm was excited and the longest wavelength emission (from band 830-890 nm) was recorded. At different concentrations, the resulting fluorescence has equal intensity, probably due to saturation effects.

To measure photosensitizing efficiency, RIF tumors were implanted subcutaneously in the axilla of 5-7 week old female C3H mice. When the tumor grows to 4 to 5mm³Conjugate 5(0.5, 1.0, 1.5 and 2.5 μmole/kg) was injected at different doses in size. 24 hours after injection, the injection was carried out at 665nm wavelength (in vivo absorption band of HPPH), 135J/cm²The tumors were treated with energy light and the mice were observed daily. From the results summarized in fig. 7, it can be seen that at a dose of 2.5 μmole/kg (tumor imaging dose), conjugate 5 resulted in 100% tumor cure rate. At lower doses, limited photosensitizing efficiency was observed.

Typically, porphyrin-based compounds exhibit different localization profiles, depending at least in part on structure, lipophilicity, and charge. Localization in lysosomes and mitochondria has been reported to be predominant; however, photosensitizers that localize primarily in the mitochondria are generally found to be more effective. Thus, the site of localization of HPPH-ICG conjugate 5 (2.5. mu. mole/kg) after 24 hours incubation was comparable to known mitochondrial probesLocalization of green (400nM) in RIF tumor cells (a well-known tumor cell line). The structure shown in fig. 8 clearly shows that conjugate 5 is localized in mitochondria, a more sensitive site for cellular damage in photodynamic therapy (PDT). A in FIG. 8 shows Compound 5Localization in mitochondria. In FIG. 8B shows the localization of the known mitochondrial probe and C shows the overlap of A and B.

These results show that tumor-avid porphyrin based photosensitizers, which may not be tumor specific but which show strong emission in the IR spectral region, can be used as a vehicle for dye delivery to tumors. In particular, HPPH bound to the isocyanine derivative is specifically localized within the tumor and can be detected by fluorescence, while maintaining the property of destroying the tumor after exposure to light. This result is predictive of the performance of other porphyrin-based photosensitizers in combination with other dyes with similar absorption and emission properties. The method thus provides a means of generating multiple conjugates in which the photosensitizer moiety can be made available by a range of long wavelength tumor-avid photosensitizers, such as purpurinimides and bacteriochlorins that exhibit long wavelength absorption in the 700-800nm range. These other conjugates provide the ability to excite molecules at longer wavelengths (700-800nm, instead of 660nm for HPPH) and detect emissions beyond 860nm, compared to Compound 5. This is a unique advantage provided by the conjugates of the present invention. In addition, the compounds of the present invention have the advantage of treating larger tumors by implanting fewer fibers to deliver light of the appropriate wavelength.

The tumor affinity optical imaging agents developed according to the present invention are themselves very advanced, and the dual functional properties of the compounds of the present invention provide for the first time a good opportunity for diagnosis followed by targeted photodynamic therapy, thereby combining 2 modalities into a single low cost "examination and treatment" approach.

The following examples illustrate preferred methods of synthesizing the compounds of the present invention.

Synthesis of ICG analogue 3:

commercially available dye 1(60mg) and 4-aminothiophenol 2(60mg) were dissolved in dry DMF and stirred overnight. After removal of the solvent, silica column chromatography with MeOH/CH₂Cl₂(1: 3) the residue was purified as the eluting solvent and intermediate 3 was obtained in about 60% yield. UV-vis: 830nm (in methanol))(ε＝207,000)。¹H NMR(CHCl₃) Delta (ppm)9.0(d, 2H 5H-a), 8.2(d, 2H, H-b), 8.0(t, 4H, H-C), 7.62(d, 4H, H-d), 7.48(2d overlapped into triplet, 2H, H-e), 7.12(d, 2H, H-f), 6.70(d, 2H, H-g), 6.35(d, 2H, H-H), 4.30(t, 4H, H-i), 2.95(t, 4H, H-j), 2.80(m, 4H, H-k), 2.00(m, 10H, 4H of H-l), 6H of m, n, o, 1.90(s, 12H, H-p), 1.30(s, H-q) 3 (C)₅₂H₅₆N₃NaO₆S₃) MS analysis of (1): 937, found: 938

Synthesis of HPPH-ICG conjugate 5:

hexanylether derivative of pheophorbide (HPPH)4(100mg) and DCCI (110mg) were dissolved in DMF (1 ml). After stirring for 10 min, a solution of 3(60mg) DMF (2ml) and DMAP (10mg) was added. After stirring the reaction mixture for 24 hours, it was diluted with dichloromethane (100ml) washed with water (2X 100 ml). The organic phase was dried over anhydrous sodium sulfate. The residue obtained after removal of the solvent from the filtrate was chromatographed using MeOH/CH₂Cl₂(1: 3) as the elution solvent, and the desired conjugate 5 was obtained in about 65% yield. UV-vis in H₂In O: 848nm (∈ 975,47), 664nm (∈ 53,800), 413nm (∈ 101' 456). UV-vis in MeOH; 833nm (∈ 207,455), 660nm (∈ 53,856), 408nm (∈ -95,222).¹HNMR(CHCL₃) δ (ppm): 9.47(s, 1H of meso-H, in HPPH moieties), 8.46(s, 1H, meso-H in HPPH moieties), 8.35(br-s, 3H, 1H of meso-H, in HPPH, 2H of H-a), 7.50(m, 5H, 1H of H-b, 4H of H-c), 7.30(m, 3H, 1H of H-b, 2H of H-e), 7.20(s, 2H, H-f), 7.05(s, 4H, H-d), 6.85(s, 2H, H-g), 6.61(s, 2H, H-H), 5.70(br, 3H, H-3)¹1H of (1H), H-17 1H, H-18 1H), 4.54 (br-doublet, 1H, H-13)²)，4.22(br，2H，H-i)，3.66(br，2H，H-i)，3.52(br，1H，H-13²) 3.20(br, 9H, 5H of HPPH moiety: 7-CH₃3H, 3 of¹-OCH ₂(CH₂)₄CH₃2H of H-j), 3.03(m, 4H, H-k), 2.90(s, 1H, -CONH-)，2.72(br，7H，8-CH₂CH₃2H, 17-CH of₂CH₂2H, 2-CH of CO-₃3H) 2.55(br, 5H, 17-CH)₂CH₂2H, 12-CH of CO-₃3H) of (1.88 (br, 3H, 3-CHCH)₃) 1.72-0.72 (multiplicities, 36 protons, 22H of dye moiety: 12H for H-p, 4H for H-l, 6H for H-m, n, o; 14H of HPPH moiety: 18-CH ₃3H, 8-CH of₂CH₃3H, 3 of¹-OCH ₂(CH₂)₄CH₃8H) of (3), 0.62(m, 3H, 3)¹-OCH₂(CH₂)₄CH₃. Conjugate 5 (C)₉₁H₁₀₂N₇NaO₉S₃) The MS of (1): 1555.7, found: 1556.7.

Claims (1)

1. A compound represented by the formula: