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Title:
PREPARATION AND USES OF GUANIDINIUM-MODIFIED PORPHYRINS AND PHTHALOCYANINES
Document Type and Number:
WIPO Patent Application WO/2010/028780
Kind Code:
A2
Abstract:
The invention relates to porphyrins, porphyrazines and phthalocyanines carrying a guanidine substituent. These compounds are obtained by treating a porphyrin, porphyrazine or phthalocyanine, respectively, carrying an amino substituent with cyanamide or a carbodiimide in an ionic liquid at elevated temperature. Described are valuable diagnostic, photoelectronic, and therapeutic uses, e.g. as photosensitizing agents in photodynamic therapy, fluorescent and magnetic probes for diagnosis, or to transport other molecules through the plasma membranes of living cells. Furthermore, the present invention relates to a method of removing a metal from metalloporphyrins, metalloporphyrazines and metallophthalocyanines by heating the zinc(II)-containing compounds in an ionic liquid and a proton source at temperatures between 60°C and 200°C.

Inventors:
LUEDTKE, Nathan (Ottikerstrasse 31, 8006 Zurich, CH)
ALZEER, Jawad (Allmendstrasse 32, 8304 Wallisellen, CH)
VUMMIDI, Balayeshwanth R. (29 Gandhi Road, 1 Tiruvallur, 60200, IN)
Application Number:
EP2009/006417
Publication Date:
March 18, 2010
Filing Date:
September 04, 2009
Export Citation:
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Assignee:
UNIVERSITÄT ZÜRICH (Prorektorat MNW, Rämistrasse 71, 8006 Zurich, CH)
LUEDTKE, Nathan (Ottikerstrasse 31, 8006 Zurich, CH)
ALZEER, Jawad (Allmendstrasse 32, 8304 Wallisellen, CH)
VUMMIDI, Balayeshwanth R. (29 Gandhi Road, 1 Tiruvallur, 60200, IN)
International Classes:
C07D487/22; C07F1/08; C07F3/06; C07F3/08; C07F3/14; C07F5/00
Other References:
SIBRIAN-VAZQUEZ, MARTHA ET AL: "Mitochondria Targeting by Guanidine- and Biguanidine-Porphyrin Photosensitizers" BIOCONJUGATE CHEMISTRY, vol. 19, no. 3, 13 February 2008 (2008-02-13), pages 705-713, XP002562194
SYNYTSYA ALLA ET AL: "Biolocalisation and photochemical properties of two novel macrocyclic photosensitisers: a spectroscopic study" JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B BIOLOGY, vol. 74, no. 2-3, 27 May 2004 (2004-05-27), pages 73-84, XP002562195 ISSN: 1011-1344
KRÁL VLADIMÍR ET AL: "Anion-controlled assembly of porphyrin-bicyclic guanidine conjugates." ORGANIC LETTERS, vol. 4, no. 1, 10 January 2002 (2002-01-10), pages 51-54, XP002562196 ISSN: 1523-7060
KUBAT, PAVEL ¦ LANG, KAMIL ¦ KROL, VLADIMIR ¦ SCHMIDTCHEN, FRANZ P: "Photophysical properties of two novel tetraphenylporphyrins substituted by guanidiniocarbonyl and monocyclic guanidine groups" INTERNATIONAL JOURNAL OF PHOTOENERGY, vol. 3, no. 3, 2001, pages 147-151, XP002562197
SALEHI, ASAAD ET AL: "Synthesis and DNA-binding studies of new cationic guanidine and betaineamide tetraphenylporphyrins" TETRAHEDRON LETTERS, vol. 32, no. 29, 1991, pages 3453-3456, XP002562198
SIBRIAN-VAZQUEZ M ET AL: "Synthesis and properties of cell-targeted Zn(II)-phthalocyanine-pept ide conjugates" BIOCONJUGATE CHEMISTRY 200703 US, vol. 18, no. 2, March 2007 (2007-03), pages 410-420, XP002562199 ISSN: 1043-1802
Attorney, Agent or Firm:
KASCHE, André (Ritscher & Partner Ag, Resirain 1, 8125 Zollikerberg, CH)
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Claims:
Claims

1. A compound selected from the group consisting of porphyrins, porphyrazines and phthalocyanines carrying a guanidine substituent.

2. The compound of claim 1 carrying four guanidine substituents.

3. The compound of claim 1 or 2 which is a phthalocyanine.

4. The compound of claim 1 or 2 which is a porphyrin.

5. The compound of anyone of claims 1 to 4 further carrying a metal atom.

6. The compound of claim 5 wherein the metal atom is selected from Zn, Cu, Ni, Co, Fe, Sn, Al, Hg, Pt, Pd, Mn, Ti, Si, Ga, and Ge.

7. A method of synthesis of a compound of claim 1, wherein a porphyrin, porphyrazine or phthalocyanine carrying an amino substituent is treated with cyanamide or a carbodiimide in an ionic liquid at elevated temperature.

8. A compound of anyone of claims 1 to 6 for use as a diagnostic agent.

9. A compound of claim 8 for use as a fluorescent probe.

10. A compound of anyone of claims 1 to 6 for use in the treatment of cancer, infections, autoimmune diseases, cardiovascular diseases, inherited genetic disorders, skin disorders, or primary immunodeficiency disorders.

11. A compound of claim 10 for use in the treatment of cancer and viral infections.

12. Use of a compound of anyone of claims 1 to 6 as a photoelectronic, photovoltaic, or data storage device, i

13. A pharmaceutical composition comprising a compound of claim 1.

14. A method of removing a metal from a compound selected from the group consisting of metalloporphyrins, metalloporphyrazines and metallophthalocyanines, wherein the zinc-containing compound is heated in an ionic liquid and a proton source at temperatures between 60°C and 200°C.

Description:
Preparation and Uses of Guanidinium-Modified Porphyrins and Phthalocyanines

Field of the Invention

The present invention relates to porphyrins, porphyrazines and phthalocyanines carrying a guanidine substituent, to a method of synthesis and to uses of the mentioned compounds. Furthermore the invention relates to a method of removing the metal atom of metallo-porphyrins, metallo-porphyrazines and metallo-phthalocyanines.

Background of the Invention

Over 5 x 10 10 g of synthetic porphyrins, porphyrazines, and phthalocyanines (together referred to here as PPcs) are produced annually. Their remarkable photophysical properties and extreme chemical, thermal, and photo stability make PPcs and metallo- PPcs important components of synthetic catalysts, photovoltaic devices, chemical sensors and data storage devices. PPcs can be used as inert dyestuffs or singlet oxygen sensitizers in vivo like in tattoo inks or for photodynamic therapy. The notorious (in)solubility properties of PPcs can pose significant problems to the development of new PPc applications in medicine and photoelectronic devices. Previous efforts have been directed towards the synthesis of soluble PPcs containing sulfonate, carboxylate, ether, thioether, alkylpyridinium, and/or alkylammonium substitutents on the periphery of the PPcs framework (see e.g. US 3,409,633; US 2,604,476; The Porphyrin Handbook, Volume 15, Kadish, K. M., Smith, K. M., and Guilard R., Ed., Academic Press, 2003; Phthalocyanine Materials: Synthesis, Structure and Function; McKeown, N. B., Ed.; Cambridge University Press, 1998). Phthalocyanines are typically prepared by high-temperature cyclotetramerization of phthalic acid derivatives, while porphyrins are synthesized by heating pyrroles or isoindoles with aldehydes. Metals, metalloids, and metal ion salts are included in these reactions to act as catalysts, reducing agents, and to tune the photophysical and catalytic properties of the resulting products. The inclusion of strongly coordinating metals like Mn, Fe, Co, Ni, Cu, or Zn can dramatically improve reaction yields for synthesis of PPcs, but the removal of these metal ions from metallo-

CONFIRMATION COPY phthalocyanines is generally thought to be impossible without destruction of the macrocycle itself.

Summary of the Invention

The present invention relates to porphyrins, porphyrazines and phthalocyanines carrying a guanidine substituent, in particular to porphyrins, porphyrazines and phthalocyanines carrying four guanidine substituents.

Furthermore the invention relates to a method of synthesis of porphyrins, porphyrazines and phthalocyanines carrying a guanidine substituent, wherein a porphyrin, porphyrazine or phthalocyanine, respectively, carrying an amino substituent is treated with cyanamide or a carbodiimide in an ionic liquid at elevated temperature.

The invention also relates to particular uses of the porphyrins, porphyrazines and phthalocyanines carrying a guanidine substituent.

Furthermore, the present invention relates to a method of removing a metal from metalloporphyrins, metal loporphyrazines and metallophthalocyanines by heating the zinc-containing compounds in an ionic liquid and a proton source at temperatures between 6O 0 C and 200 0 C.

Brief Description of the Figures

Figure 1. Phthalocyanines and porphyrins react with carbodiimides in an ionic liquid and a proton source at elevated temperatures to furnish metal-free guanidium-containing phthalocyanines and porphyrins. Metal ions or metalloids can subsequently be inserted into the metal free guanidium-containing phthalocyanines and porphyrins to furnish the corresponding guanidium-containing metal lo-phthalocyanines and metal lo-porphyrins. Figure 2. Metallo-porphyrins and metallo-phthalocyanines treated with an ionic liquid and a proton source at elevated temperatures furnish metal-free phthalocyanines and porphyrins. Metal ions or metalloids can subsequently be inserted into the metal free phthalocyanines and porphyrins to furnish the corresponding metallo-phthalocyanines and metal lo-porphyrins carrying a different metal.

Figure 3. Removal of zinc(II) from unsubstituted or from a nitro-, amino-, oxysulfonyl-, alkyl-, alkenyl-, alkynyl-, aryl-, heteroaryl-, halo-, alkoxy-, aryloxy-, alkylthio, arylthio-, guanidino-, or alkylamino-substituted phthalocyanine, porphyrin and porphyrazine by heating in an ionic liquid and proton source.

Figure 4. Metal lo-porphyrins and metallo-phthalocyanines react with carbodiimides in an ionic liquid at elevated temperatures to furnish metal-containing guanidino- phthalocyanines and guanidino-porphyrins without loss of the metal ion or metalloid.

Figure 5.

A) 4,4',4",4"'-Tetraamino zinc phthalocyanine reacts with 4 to 100 equivalents of cyanamide, diisopropylcarbodiimide, or dicyclohexylcarbodiimide in a pyridine- pyridinium hydrochloride mixture (0.01 to 100 molar ratio) at 60 - 120°C in the presence or absence of added catalyst to furnish the metal-free tetraguanidino-phthalocyanines 1 - 3. Variable metalloids or metal ions are inserted into the metal-free tetraguanidino- phthalocyanines in the presence of cesium acetate at 90°C followed by precipitation with trifluoroacetic acid (TFA) and water to furnish the corresponding metallo-tetraguanidino- phthalocyanines 7 -21.

B) 3,3',3",3"'-Tetraamino zinc phthalocyanine reacts with 50 equivalents of diisopropylcarbodiimide in a pyridine-pyridium hydrochloride mixture (2:1 molar ratio) at 120 0 C in the presence of 1 molar equivalent of 4-dimethylaminopyridine to furnish the metal-free tetraguanidino-phthalocyanine 4. Zinc is inserted into the metal-free tetraguanidino-phthalocyanines 4 using zinc chloride in the presence of sodium acetate and acetic acid (1: 2 molar ratio) at 100°C for one hour to furnish the corresponding zinc- tetraguanidino-phthalocyanine 22. C) 3,3',3",3'"-Tetraamino zinc phthalocyanine reacts with 50 equivalents of l-ethyl-3-(3- dimethylaminopropyl)carbodiimide in a pyridine-pyridinium hydrochloride mixture (2:1 molar ratio) at 120°C to furnish the metal-free tetraguanidino-phthalocyanine 5. Zinc is inserted into the metal-free tetraguanidino-phthalocyanine 5 in the presence of zinc chloride, sodium acetate, and acetic acid at 100°C for one hour to furnish the corresponding zinc- tetraguanidino-phthalocyanine 23.

D) Zinc (II) 5,10,15,20-Tetrakis(4-aminophenyl)-21H,23H-porphine reacts with 50 equivalents of diisopropylcarbodiimide in a pyridine-pyridinium hydrochloride mixture (2:1 molar ratio) at 120°C to furnish the metal-free tetraguanidino-phthalocyanine 6. Zinc is inserted into the metal-free tetraguanidino-phthalocyanine 6 using zinc chloride in the presence of sodium acetate and acetic acid (1 :2 molar ratio) at 100°C for one hour to furnish the corresponding zinc- tetraguanidino-phthalocyanine 24.

Figure 6. Partial 1 H NMR spectra of metal-free tetraguanidino-phthalocyanines 1 - 3 in αVDMSO.

Figure 7. Partial 1 H NMR spectra of tetraguanidino zinc phthalocyanines 7 - 9 in d 6 - DMSO.

Figure 8. Absorbance and fluorescence changes of compound 8 upon selective binding of pharmacologically important biomolecules.

A) Absorbance spectra of a 1 μM solution of compound 8 upon adding 0 - 1 equivalents of a 22-nucleotide DNA sequence derived from the c-Myc promoter: TGAGGGTGGGG AGGGTGGGGAA, where ε = the molecular extinction coefficient and λ = wavelength.

B) - C) Fluorescence intensities "I" (arbitrary units) of 10 nM solutions of compound 8 (Ex. 620 nm, Em. 705 nm) upon titration with increasing concentrations "C" (in nM) of the following: c-Myc DNA (filled squares), a 30-nucleotide DNA derived from the 3' human telomeric overhang "Htelo": GTTA(GGGTTA) 4 GG (filled triangles), or an unstructured mutant of this sequence "Htelo-Mut": GTTA(GAGTTA) 4 GG (filled circles), a tRNA mixture Sigma Type X-SA (filled diamonds), or double stranded DNA from calf thymus (open circles). D) Fluorescence intensity "I" (arbitrary units) of a 10 nM solution of a 5'-fluorescein end- labeled c-Myc DNA (Ex. 495 nm, Em. 520 nm) upon addition of increasing concentrations "C" (in nM) of compound 8 (square symbols) or zinc(II) 5,10,15,20- tetra(N-methyl-4-pyridyl)porphin (triangle symbols) in the presence (open symbols) or absence (filled symbols) of 220 μM of CT DNA (nucleotide concentration). All samples were prepared and analyzed in a "TKE" buffer containing 50 mM Tris-HCl (pH 7.4) 150 mM KCl, and 0.5 mM EDTA.

Figure 9. Cellular uptake of compound 8 by living cells demonstrated with fluorescence microscopy (Ex. 620 nm, Em. 700 nm). Shown are: A) HeLa, B) MCF7, C) SK-Mel-28 cells treated with 3 μM of compound 8 in PBS for 2 hours. Also shown are: D) SK-Mel- 28 cells treated with 10 μM of compound 8 in RPMl medium with 10% FCS, 2 mM L-glutamine, penicillin-streptomycin and 1 mM sodium pyruvate for 2 hours. Following imaging, the viability of the cells was confirmed using a resazurin cytotoxicity assay; no detectable decrease in respiration was observed as compared to untreated cells maintained in PBS or media.

Figure 10. Microscopy images of fixed SK-Mel-28 stained with 3 μM of compound 8 and 180 μM of Hoechst 33342. A) Fluorescence emitted from compound 8 (Ex. 620 nm, Em. 700 nm). B) White light absorbance of treated cells. C) Fluorescence emitted from Hoechst 33342 (Ex. 360 nm, Em. 470 nm). D) Composite image of A) - C).

Figure 11. Microscopy images of fixed SK-Mel-28 stained with 3 μM of compound 2 (Ex. 620 nm, Em. 700 nm). The white light absorbance and composite image of absorbance and fluorescence are also shown.

Figure 12. Photodynamic cytotoxicity. A standard viability assay was used to quantify % metabolic activity "Metab." of 10'0OO B16-F10 cells exposed to variable concentrations of guanidino-PPcs as compared to untreated cells. Cells were either maintained in the dark (-hv), or selected samples were exposed to 300 mW/cm 2 of red laser light (660 nm) for 30 seconds (+hv). All cells were then maintained in the dark for an additional 3 hours at 37°C with 5% CO 2 , and at 10% relative humidity. 100 μl of fresh media, containing 10% (v/v) of a 860 μM solution of resazurin was added to each sample and incubated an additional 3 hours. The metabolic activity in each sample was then quantified using a SpectraMax M5 fluorescence plate reader using excitation at 560 nm, emission at 590 nm, and a 570 nm cutoff filter to determine cell viability. The C 50 values are the concentration of each compound (with or without exposure to the laser) needed to decrease the metabolic activity of the sample by 50%.

Detailed Description of the Invention

The present invention provides porphyrin, porphyrazine, and phthalocyanine derivatives (PPcs) containing 1 - 4 guanidium groups and a variable metal ion or H 2 in the central cavity "M" having the following general structures:

Wherein the group "X" is independently a substituted or unsubstituted nitrogen or carbon atom. Wherein the group "R" is independently a substituted or unsubstituted alkyl, aryl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, amino alkyl, or amino aryl group. Wherein a divalent metal atom "M" is selected from the group consisting of Mn, Fe, Co, Ni, Cu, Zn, Pt, Pd, Pb, Sn, Mg, Ba, Ca, Ti, Hg, and Zn. Wherein a trivalent mono- substituted metal atom "M" is selected from the group consisting of AlCl, GeCl, InCl, Al(O-alkyl), Al(O-aryl), Al(O-arylalkyl) Ga(O-alkyl), Ga(O-aryl), Ga(O-arylalkyl), Al(OH), Hg(OH), and Ga(OH). Wherein a tetravalent di-substituted metal atom "M" is selected from the group consisting Of SiF 2 , TiCl 2 , GeC12 Si(O-alkyl) 2 , Si(O-aryl) 2 , Si(O- arylalkyl) 2 , Ge(O-alkyl) 2 , Ge(O-aryl) 2 , Ge(O-arylalkyl) 2 , Ge(OH) 2 , Hg(OH) 2 , V=O, Mn=O, and Ti=O.

The present invention is directed to the synthesis and uses of PPcs containing substituted guanidines. Central to this invention is a new synthetic process for removing the strongly coordinating metal ion, zinc (II), from metal loporphyrins, metallophthalocyanines, and metalloporphyrazines by heating these materials in an ionic liquid and a proton source. This method gives broad access to metal-free PPcs from starting materials that have been synthesized in the presence of zinc metal and/or zinc ions that serve as templates, reducing agents, and/or catalysts. Due to the extraordinary solubility properties of PPcs in ionic liquids, this invention is suitable for making combinatorial libraries with three variables: 1. scaffold selection; 2. metalloid/metal ion selection; and 3. guanidium group selection.

In one embodiment, PPcs carrying a guanidinium substituent are synthesized by reacting amino-containing PPcs with a carbodiimide and an acid in an ionic liquid at elevated temperature. Under these conditions, demetallation of starting materials and/or products provides metal-free PPcs carrying a guanidinium group (Figure 1). In another embodiment, metal ions are removed from metallo-PPcs by an ionic liquid and acid before, during, or after modification of PPcs (Figures 2 and 3). The physical properties of resulting metal-free PPcs and guanidino-PPcs are readily tuned by inserting variable metal ions or metalloids "M" to furnish the corresponding metallo-PPcs and metallo- guanidino-PPcs. In another embodiment, metal ions can remain associated with PPcs during and after the guanidinylation reaction to furnish the metal-containing guanidino- PPcs in one step (Figure 4).

Metal-free and metallo-guanidino-PPcs can be produced in large (multi-kiloton) scale as the method of synthesis requires only a small number of high yielding steps, utilizes inexpensive starting materials, and purification does not require chromatography. In another embodiment, this invention relates to PPcs carrying a guanidinium substituent to be used as fluorescent probes, photolelectronic components, and drugs. These PPcs carrying a guanidinium substituent demonstrate a wide range of useful photophysical, biophysical, and photoelectronic properties including water solubility, structure-selective DNA/RNA binding, singlet oxygen formation, fluorescence staining of cells, and photodynamic toxicity in cancer cells. These properties make PPcs carrying a guanidinium substituent useful in the treatment of cancer, infections (e.g. viral infections), autoimmune diseases (e.g. rheumatoid arthritis and multiple sclerosis), cardiovascular diseases (e.g. atherosclerosis), inherited genetic disorders (e.g. cystic fibrosis), skin disorders (e.g. psoriasis), or primary immunodeficiency disorders (e.g. Boder-Sedgwick syndrome). Furthermore, the compounds of the invention are useful for in vitro and in vivo diagnostic methods as fluorescent probes, magnetic probes, and chemical sensors. Further uses are as solid-state materials, e.g. as photoelectronic, photovoltaic, and data storage devices.

The term "phthalocyanine, porphyrin and porphyrazine" (PPcs) refers to a family of closely related compounds all having a common core-structure containing four pyrrole units (in bold):

The four pyrrole units can be linked through either a nitrogen atom "X" (to furnish phthalocyanines and porphyrazines), or through a substituted or unsubstituted carbon atom "C" (to furnish porphyrins). Substituents "R" include, but are not limited to, alkyl, aryl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, amino alkyl, or amino aryl groups. The central groups "M" include, but are not limited to hydrogen, metalloids and metal ions. The terms "guanidine," "substituted guanidines," and "substituted guanidinium" refer to a carbon atom bound directly to three substituted or unsubstituted nitrogen atoms with the general structures:

wherein each of Ri, R 2, R 3 , R 4, R 5 , and R 6 is, independently, a hydrogen, alkyl group, aryl group, alkenyl group, alkynyl group, arylalkyl group, arylalkenyl group, arylalkynyl group, amino alkyl, amino aryl group, or heteroaryl group (including phthalocyanine, porphyrin, and porphyrazine). The term"guanidine group"or"guanidine moiety"as used herein is defined as a group or moiety derived from either unsubstituted guanidine or from an alkylguanidine, arylguanidine, arylalkyl, alkenyl, or heteroarylguanidine.

The term "ionic liquid" herein refers to ionic liquids, ionic liquid composites, and mixtures (or cocktails) of ionic liquids. The ionic liquid can comprise an anionic ionic component (such as, but not limited to chloride, bromide, iodide, acetate, BF 4 " , P F 6 " , phosphate, phosphonate, sulfate, sulfonate) and cationic ionic component (such as but not limited to ammonium, bipyridinium, imidazolium, phosphonium, pyrazolium, pyridinium, pyrrolidinium, quinolinium, sulfonium). As used herein, the term "ionic liquid" refers to a mixture of a salt (which can be solid at room temperature) with a proton donor (which can be a liquid or a solid).Upon mixing, these components turn into a liquid at about 60 - 100 0 C, or less, and the mixture behaves like an ionic liquid.

The term "alkyl" used herein refers to a monovalent straight or branched chain radical from one to ten carbon atoms, including, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and the like. Alkyl also represents cyclic radicals, including, but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The term "aryl" when used alone refers to an aromatic radical whether or not fused. Preferred aryl groups include phenyl, pyridyl, naphthyl, biphenyl and the like. The term "aryl" also refers to heteroaryl groups including, but not limited to, furanyl, pyrrolyl, thienyl, pyrazolyl, thiazolyl, oxazolyl, pyridyl, pyrimidinyl, indolyl, and the like. The term "aryl" encompasses substituted aryl groups and herein refers to an aryl group substituted with one, two or three substituents chosen from halogen, cyano, amino, guanidino, nitro, Cl-ClO alkyl, Cl-ClO-alkoxy, trifluoromethyl, alkoxycarbonyl, and the like. Examples of such groups are 4- chlorophenyl, 2-methylphenyl, and 3-ethoxyphenyl.

The term "alkenyl" as used herein refers to a straight or branched chain group of from two to ten carbon atoms containing a carbon-carbon double bond, including, but not limited to allyl, vinyl, and the like.

The term "akynyl" as used herein refers to a straight or branched chain group of from two to ten carbon atoms containing a carbon-carbon triple bond.

The term "arylalkyl" as used herein refers to an alkyl group which is substituted with one or more aryl groups. A typical arylalkyl group is the benzyl group.

The term "arylalkenyl" as used herein refers to an alkenyl group which is substituted with one or more aryl groups.

The term "arylalkynyl" as used herein refers to an alkynyl group which is substituted with an aryl group.

The term "amino alkyl" as used herein refers to a Ci-Ci 0 alkyl, alkenyl, or alkynyl group connected to a charged or uncharged nitrogen-containing functional groups such as ammonium, guanidinium, pyridinium, and the like. The term "amino aryl" group as used herein refers to an aryl group substituted with nitrogen-containing functional groups such as ammonium, guanidinium, pyridinium, and the like.

Embodiments of the present invention are directed to methods for synthesizing and uses of guanidine-containing phthalocyanines, porphyrins, and porphyrazines.

In one embodiment, the transport of other molecules through the plasma membranes of living cells relates to conjugates of the molecules presented herein and methods of using such conjugates in treating patients. Such treatments include modalities where delivery of nucleic acids, nucleosides, proteins, peptides, amino acid residues, lipids, carbohydrates, synthetic organic compounds, metals, vitamins, small molecules, dyes, isotopes, antibodies, toxins, ligands or any other compound that may need transport into a cell is required.

The present invention also provides suitable topical, oral, and parenteral pharmaceutical formulations for use in the treatment of various illnesses. The compounds of the present invention may be administered orally as tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, capsules, syrups or elixirs.

The present invention also provides suitable activation of such formulations by light energy (also known as photodynamic therapy) to treat various illnesses such as cancer, infections (e.g. viral infections), autoimmune diseases (e.g. rheumatoid arthritis and multiple sclerosis), cardiovascular diseases (e.g. atherosclerosis), inherited genetic disorders (e.g. cystic fibrosis), skin disorders (e.g. psoriasis), or primary immune- deficiency disorders (e.g. Boder-Sedgwick syndrome).

The present invention also provides suitable topical, oral, and parenteral pharmaceutical formulations for use in diagnostic studies in vivo and in vitro. These include fluorescent, magnetic, and photovoltaic probes and devices. The composition for oral use may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to produce pharmaceutically elegant and palatable preparations. The tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, (1) inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents, such as corn starch or alginic acid; (3) binding agents, such as starch, gelatin or acacia; and (4) lubricating agents, such as magnesium stearate, stearic acid or talc. These tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Coating may also be performed using techniques described in the art to form osmotic therapeutic tablets for control release.

The compounds furnished by the invention (including analogues, derivatives, or salts thereof) can be administered, for in vivo application, parenterally by injection or by gradual perfusion over time independently or together. Administration may be intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. For in vitro studies the agents may be added or dissolved in an appropriate biologically acceptable buffer and added to a cell or tissue.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. The following non-limiting examples are intended to illustrate embodiments of the invention.

In one embodiment of the invention PPcs carrying a guanidine substituent can be synthesized by reacting amine-containing PPcs with a carbodiimide in the presence a quinolinium, pyridium or imidazolium ionic liquid (Figure 1). Carbodiimides and the ionic liquid can be added to the reaction mixture, or, alternatively, generated in situ. In one example, a mono- or di-substituted urea and a sulfonyl chloride are reacted in pyridine at 60 - 200 0 C to generate a carbodiimide and pyridinium hydrochloride immediately before the guanidinylation reaction begins. In another example, a carbodiimide is added to amine-containing PPcs in the presence an ionic liquid and acid.

Upon heating metal-containing PPcs in an ionic liquid, metal ions ("M") can be removed from guanidino-PPcs and PPcs to furnish metal-free guanidino-PPcs and metal free PPcs (Figures 1 and 2). In one example, the ionic liquid is composed of a nitrogenous base and acid. In a preferred example, the nitrogenous base contains an imidazole, quinoline, or pyridine moiety. In a highly preferred example, a mixture of pyridine-pyridinium hydrochloride mixture (2:1 molar ratio) at 120 0 C is used to remove Zn(II) from porphyrins, phthalocyanines, and porphyrazines (Figure 3).

In one embodiment, zinc metal and/or zinc ions are included as catalysts and templates for the synthesis of metallo-PPcs, and subsequently removed by action of pyridinium hydrochloride at 20 - 200 0 C to furnish the metal-free PPcs and guanidino-PPcs.

In another embodiment, the resulting metal-free porphyrins and phthalocyanines are used as starting materials for insertion of a metal ion or metalloid to furnish PPcs and guanidino-PPcs with highly variable physical and biological properties.

In another embodiment, metal ion and metalloid-containing guanidino-PPcs can be prepared by reacting an amine-containing metallophthalocyanine or metalloporphyrin with a carbodiimide in the presence of an ionic liquid and proton source. In one example, the metal ions remain associated with the PPcs during the reaction to furnish the metal- containing guanidino-PPcs in one step (Figure 4). In a preferred example, the metal ions "M" are cobalt(II), palladium (II), platinum (II), copper (II), nickel (II), Mn(II), or Fe(III).

In one preferred embodiment, the general procedure for making guanidinium-containing porphyrins, porphyrazines, and phthalocyanines is the following: amine-containing PPcs are treated with 4 - 400 equivalents of a carbodiimide or cyanamide in a mixture of pyridinium hydrochloride in pyridine (0 — 100%) in the presence or absence of a catalyst at 60 - 200°C.

Example 1 : The metal-free guanidino-phthalocyanine derivative 1 was synthesized by reacting 4,4',4",4'"-tetraamino zinc phthalocyanine with 80 equivalents of cyanamide in a pyridine-pyridinium hydrochloride mixture (2:1 molar ratio) at 120°C (Figure 5A). By including 1 equivalent of dimethylaminopyridine (DMAP) as a catalyst the reaction reached completion after 48 hours. Purification was accomplished by precipitation of the desired product upon addition of 10% trifluoroacetic acid / water to the reaction mixture to furnish 1 as a blue-green precipitate in a 71% yield. High resolution ESI MS data: compound 1 [M+H] + calcd for C 36 H 31 N 20 , 743.3041; found 743.3037. A more detailed description is given below as Example 13.

Example 2: The metal-free guanidino-phthalocyanine derivative 2 was synthesized by reacting 4,4',4",4"'-tetraamino zinc phthalocyanine with 50 equivalents of diisopropyl- carbodiimide in pyridine-pyridinium hydrochloride (2:1 molar ratio) at 120°C for 18 hours (Figure 5A). Purification was accomplished by precipitation of the desired product upon addition of 10% trifluoroacetic acid / water to the reaction mixture to furnish 2 as a blue-green precipitate in an 83% yield. High resolution ESI MS data for compound 2 [M+H] + calcd for C 60 H 79 N 20 , 1079.6797; found 1079.6798. A more detailed description is given below as Example 10. Example 3: The metal-free guanidino-phthalocyanine derivative 3 was synthesized by reacting 4,4 l ,4",4'"-tetraamino zinc phthalocyanine with 50 equivalents of dicyclohexyl- carbodiimide in a pyridine-pyridinium hydrochloride mixture (2:1 molar ratio) at 120°C for 24 hours (Figure 5A). Purification was accomplished by precipitation of the desired product upon addition of 10% trifluoroacetic acid / water to the reaction mixture to furnish compound 3 as a blue-green precipitate in a 93 % yield. High resolution ESI MS data for compound 3 [M+H] + calcd for C 84 H 1n N 20 , 1399.9301; found 1399.9303. A more detailed description is given below as Example 12.

Metal-free and metallo 3,3',3",3'"- and 4,4',4",4"'-tetraamino phthalocyanines or mixed isomers thereof can be guanidinylated using this method.

Example 4: The metal-free guanidino-phthalocyanine derivative 4 was synthesized by reacting 3,3',3",3"'-tetraamino zinc phthalocyanine with 50 equivalents of diisopropyl- carbodiimide in a pyridine-pyridinium hydrochloride mixture (2:1 molar ratio) at 120°C for 48 hours (Figure 5B). Purification was accomplished by precipitation of the desired product upon addition of 10% trifluoroacetic acid / water to the reaction mixture to furnish 4 as a blue-green precipitate in a 63% yield. MALDI TOF MS data for compound 4: [M+H] + calcd for C 60 H 79 N 20 , 1079.7; found 1079.6.

In one preferred embodiment, mono-, di- and 1,2,3-tri-substituted guanidines can be synthesized using this invention.

Example 5: The metal-free guanidino-phthalocyanine derivative 5 was synthesized by reacting 3,3',3",3"'-tetraamino zinc phthalocyanine with 50 equivalents of l-ethyl-3-(3- dimethylaminopropyl)carbodiimide in a pyridine-pyridinium hydrochloride mixture (2:1 molar ratio) at 120 0 C for 48 hours (Figure 5C). Purification was accomplished by reversed-phase chromatography using a C- 18 column and an acetonitrile/water gradient to furnish 5 as a blue-green solid in a 32% yield. MALDI TOF MS data for compound 5: [M+H] + calcd for C 64 H 9I N 24 ; 1 195.8; found 1 195.7. In another preferred embodiment, metal-free guanidium-substituted porphyrins can be synthesized using this invention.

Example 6: 5,10,15,20-Tetrakis(4-aminophenyl)-21H,23H zinc porphine reacts with 50 equivalents of diisopropylcarbodiimide in a pyridine-pyridinium hydrochloride mixture (2:1 molar ratio) at 120°C for 24 hours (Figure 5D). Purification of the metal-free guanidino-porphine 6 was accomplished by precipitation upon addition of 10% trifluoroacetic acid / water to the reaction mixture to furnish the desired product as a green solid in a 82% yield. MALDI TOF MS data for compound 6: [M+H] + calcd for C 72 H 9 ]Ni 6 , 1179.8; found 1180.1.

In another preferred embodiment, metal ions and metalloids can be inserted into the metal free guanidino-PPcs to furnish the corresponding metallo- and metalloid-guanidino-PPcs.

Example 7: Zn(II) was reacted with the metal-free guanidino-phthalocyanines 1 - 3 in the presence of cesium acetate / acetic acid (1 : 4 molar ratio) at 90°C to for 2 hours to furnish the corresponding Zn-containing guanidino-phthalocyanines 7 - 9 in quantitative yields (Figure 4A). High resolution ESI MS data: compound 7: [M+H] + calcd for C 36 H 29 N 20 Zn, 805.2175; found 805.2177; compound 8: [M+H] + calcd for C 60 H 77 N 20 Zn, 1141.5932; found 1141.5923; compound 9: [M+H] + calcd for C 84 Hi 09 N 20 Zn, 1461.8436; found 1461.8423. A more detailed description is given below as Example 11.

Example 8: The metal-free guanidino-phthalocyanine 2 was reacted with different metals, metalloids, or metal ion salts including those of Cu, Ni, Co, Fe, Ru, In, Sn, Al, Hg, Pd, Mn, Si, Ga, and Ge in a cesium acetate / acetic acid mixture (0.1 - 100 molar ratio) at 20 - 200°C to furnish the corresponding metallo-guanidino-phthalocyanines 10 - 21 (Figure 5A).

Example 9: Zn(II) was reacted with the metal-free guanidino-phthalocyanines 4 — 6 in sodium acetate / acetic acid (1 : 2 molar ratio) at 120°C to furnish the corresponding Zn- containing guanidino-phthalocyanines 22 - 24 in quantitative yields (Figure 5 B - D). Guanidino-phthalocyanines exhibit excellent solubility properties allowing their full characterization. The metal-free compounds (1 — 3) and corresponding zinc-containing guanidino-phthalocyanines (7 - 9) are freely soluble in DMSO allowing their characterization by NMR (Figures 6 and 7). The solubility properties of guanidino- phthalocyanines depend upon the identity of the centrally-coordinated metalloid/metal ion as well as the substituents and the counterions associated with the guanidinium groups.

The zinc-containing guanidino-phthalocyanines 7 - 9 exhibit better water solubility than the corresponding metal-free compounds 1 - 3. The chloride salts of compounds 2 and 8 are soluble in water, while the trifluoroacetate salts of 2 and 8 are insoluble in water, but soluble in DMSO, alcohol, and chlorinated solvents. The diisopropyl-substituted guanidinines 2 and 8 exhibit better water solubility than the mono-substituted guanidines 1 and 7 or the dicyclohexyl-substituted guanidines 3 and 9.

Particular combinations of guanidine substituents and metal ions generate guanidino- phthalocyanines that exhibit structure-selective binding interactions with DNA, RNA and proteins demonstrating their potential as agents to treat and diagnose diseases.

G-quadruplex-containing DNA and RNA structures are important anti-cancer and antiviral targets. Direct spectroscopic methods can be used to characterize the G-quadruplex binding affinity, specificity, and stoichiometry of guanidino-phthalocyanines. Guanidino- phthalocyanines exhibit high affinity and high specificity for G-quadruplex RNA and DNA and therefore are a source of promising anti-cancer and anti-viral agents (Figure 8).

The absorbance spectra of 1 μM solutions of compound 8 were monitored upon titration with 0 - 1 equivalents of a pre-folded 22-mer quadruplex DNA derived from the c-Myc promoter (Figure 8A), or the fluorescence intensities of 10 nM solutions of compound 8 (Ex. 620 nm, Em. 705 nm) were monitored upon titration with five different nucleic acids (Figure 8B-C). Highly selective c-Myc promoter binding is apparent by comparing binding isotherms for five different nucleic acids on a Logio nucleotide scale (Figure 8C). The extraordinarily high c-Myc G-quadruplex affinity and specificity of compound 8 can be confirmed by monitoring the fluorescence quenching of a 5'-fluorescein-labeled c-Myc DNA upon titration of compound 8 in the presence or absence of a 1, 000-fold nucleotide excess of CT DNA. (Figure 8D). As a control, the known cationic porphyrin zinc (II) 5,10,15,20-tetra(N-methyl-4-pyridyl) porphyrin (Zn-TMPyP4) was also evaluated. Consistent with its promiscuous binding of nucleic acids, a 10-fold loss in the apparent binding affinity of Zn-TMPyP4 was observed in the presence of competitor CT DNA (Figure 8D), while a high apparent affinity (Kj ≤ 2 nM) between compound 8 and c-Myc was measured even in the presence of a 1, 000-fold excess of CT DNA (Figure 8D).

Particular guanidine substituents and metal ions generate guanidino-phthalocyanines with highly variable photophysical and photoelectronic properties. The fluorescence emission from guanidino-phthalocyanines increases by over 100-fold upon binding G-quadruplex RNA and DNA. The quantum yield of compound 2 is approximately 0.2 +/- 0.1 when bound to biological macromolecules, while the quantum yield of the Zn (II) guanidino- phthalocyanine 8 is approximately 0.05 +/- 0.03 when bound to biological macromolecules. The quantum yield of Co (II) guanidino-phthalocyanine 12 is approximately 0 when bound to biological macromolecules.

Particular guanidine substituents and metal ions generate guanidino-phthalocyanines with valuable cellular uptake and localization properties. Sensitive detection of guanidino- phthalocyanine fluorescence is accomplished using standard fluorescence imaging systems. Internalization of guanidino-phthalocyanines is observed in living cells including HeLa, MCF7, and SK-Mel-28 (Figure 9). Due to their "pro-fluorescent" behavior guanidino-phthalocyanines can simply be added, incubated, and imaged, and no washing of the cells is required. Depending on the cell type, application method, and incubation conditions, compound 8 exhibits variable cellular localization (Figure 9 - 10). The metal-free guanidino-phthalocyanine 2 exhibits very distinct nuclear localization when applied to fixed cells (Figure 11). Particular guanidine substituents and metal ions generate guanidino-phthalocyanines with variable quantum yields for forming singlet oxygen upon photoexcitation in living cells. Compound 8 exhibits very large molecular extinction coefficients (ε) = 50O00 - 130'0OO Cm -1 M "1 over a wavelength range that overlaps with red lasers commonly used for photodynamic therapies (630 - 680 nm, Figure 1 A).

In a preferred embodiment, photoexcitation of guanidino-phthalocyanines is utilized for photodynamic therapy. The metabolic activities of cells treated with laser light (660 nm) and/or guanidino-phthalocyanines were measured (Figure 12). When maintained in the dark, little, if any, decrease in cellular respiration of SK-Mel-28, MCF7, macrophage, or B16-F10 cells exposed to 0 — 20 μM of guanidino-phthalocyanines containing hydrogen (2), Zn (8), Cu (11), Co (12), Sn (16), or Pd (18) for 24 hours was observed. Upon photoexcitation of the treated cells, a dramatic increase in cell death was observed for Pd(II)-containing guanidino-phthalocyanines resulting in 10 — 1000-fold lower EC50 values (Figure 12).

Preferably, guanidino-phthalocyanines containing aluminum (III), gallium (III), germanium (II), palladium (II), platinum (II), tin (II), or tin (IV) are utilized in photodynamic therapy. These metal ions and metalloids can adopt octahedral, square- pyramidal, or square-planar coordination spheres having one or two axial ligands.

The following non-limiting examples are intended to illustrate further some specific preparation methods and products of the invention.

Example 10:

4,4',4",4"'-Tetraamino zinc phthalocyanine (35 mg, 0.055 mmol), pyridine (2 mL), pyridinium-HCl (1 g), and diisopropylcarbodiimide (400 μL, 2.6 mmol, 47 equiv) were stirred under N 2 at 120°C for 17 h. The reaction was removed from the heat and 10 mL of H 2 O was used to transfer the hot mixture into a polypropylene centrifuge tube. Trifluoroacetic acid (TFA, 1.2 mL) was added, mixed, and the resulting precipitate was collected by centrifugation at 6'500 r.p.m. The supernatant was removed and mixed with TFA (1 mL) and centrifuged. The combined precipitate was sonicated with 0.1 N NaCl (7 mL) and then mixed with TFA (1.2 mL) and the colorless supernatant was carefully removed by glass pipette. The resulting precipitate was sonicated in water (7 mL) and then mixed with TFA (1.2 mL) and the precipitate collected as before. This procedure was repeated twice, and the precipitate was re-dissolved in TFA (2 mL) and then mixed with water (7 mL) and centrifuged. The resulting precipitate was collected by centrifugation, dissolved in a 1 :3 mixture of acetonitrile and water containing 0.1% TFA (4 mL), and lyophilized to yield 70 mg (83%). MALDI TOF MS (m/z): [M+H] + calcd for C 48 H 36 Ni 2 O 12 Zn, 1079.6; found 1079.9. UV- Vis (DMSO) λmax (nm) and ε (cm^M- 1 ): 347 (5.4 x 10 4 ), 682 (8.49 x 10 4 ), and 710 (9.24 x 10 4 ).

Example 11 :

4,4',4",4'"-Tetra-diisopropylguanidino-phthalocyanine (135 mg, 0.088 mmol), acetic acid (3 mL), sodium acetate (1 g), and ZnCl 2 (150 mg, 1.1 mmol, 12.5 equiv) were stirred under N 2 at 110°C for 30 min. The reaction was removed from the heat and 7 mL of H 2 O was used to transfer the hot mixture into a polypropylene centrifuge tube. TFA (2 mL) was added, mixed, and the resulting precipitate was collected by centrifugation at 6'500 r.p.m. The precipitate was sonicated with 0.1 N NaCl (7 mL) and then mixed with TFA (0.6 mL) and the colorless supernatant was carefully removed by glass pipette. The resulting precipitate was sonicated in water (7 mL) and then mixed with TFA (0.5 mL) and the precipitate collected as before. The precipitate was re-dissolved in TFA (1.5 mL) and then mixed with water (5 mL) and centrifuged. The resulting precipitate was collected by centrifugation, dissolved in a 1 :3 mixture of acetonitrile and water containing 0.1% TFA (4 mL), and lyophilized to yield 110 mg (79%). UV- Vis (DMSO) λmax (nm) and ε (CnT 1 M- 1 ): UV- Vis (DMSO) λmax (nm) and ε (CnT 1 M- 1 ): 360 (1.0 x 10 5 ), 620 (3.3 x 10 4 ), and 690 (1.4 x 10 5 ).

Example 12

4,4',4",4"'-Tetraamino zinc phthalocyanine (35 mg, 0.055 mmol), pyridine (2 mL), pyridinium-HCl (1 g), and dicyclohexylcarbodiimide (227 mg, 1.1 mmoles, 20 equiv) were stirred under N 2 at 120°C for 17 h. The reaction was removed from the heat and 5 mL OfH 2 O was used to transfer the hot mixture into a polypropylene centrifuge tube, the resulting precipitate was collected by centrifugation at 6'500 r.p.m. and washed repeatedly with hot water (3 mL) and centrifuged. The dark green solid was dissolved in acetonitrile (10 mL) and filtered. The filtrate was evaporated and passed over C- 18 reverse phase column (3:5, acetonitrile:water, containing 0.1% TFA), evaporation of the solvent yielded 75 mg (73.5%). MALDI TOF MS (m/z): [M+H] + calcd for C 84 H 110 N 20 , 1399.92; found 1399.9. UV-Vis (DMSO) λmax (nm) and ε (CnT 1 M "1 ): 347 (5.2 x 10 4 ), 682 (8.56 x 10 4 ), and 710 (9.69 x 10 4 ). Example 13:

4,4',4",4'"-Tetraamino zinc phthalocyanine (35 mg, 0.055 mmol), pyridine (2 mL), pyridinium-HCl (1 g) and dimethylaminopyridine (8.06 mg, 0.06 mmol) were stirred under N 2 at 120°C and cyanamide (115 mg, 2.75 mmol, 50 equiv) was added in three portions over 2 days. The reaction was removed from the heat and 7 mL of acetic acid was used to transfer the hot mixture into a polypropylene centrifuge tube, the resulting precipitate was collected by centrifugation at 6'500 r.p.m. and washed repeatedly with acetic acid, ethyl acetate, NaHCO 3 solution, and water. The dark green solid was re- dissolved in trifluoroacetic acid (TFA, 2 mL) and then mixed with water (5 mL) and centrifuged. The resulting precipitate was collected by centrifugation, dissolved in a 1 :3 mixture of acetonitrile and water containing 0.1% TFA (4 mL), and lyophilized to yield 46 mg (70%). MALDI TOF MS (Wz): [M+H] + calcd for C 36 H 30 N 20 , 743.30; found 743.4. UV-Vis (DMSO) λmax (nm) and ε (011 "1 M "1 ): 342 (6.0 x 10 4 ), 677 (8.9 x 10 4 ), and 704 (1.0 x l O 5 ).

Example 14:

pyπdιne-HCI 120 °C

3,3',3",3'"-Tetranitro zinc phthalocyanine (35 mg, 0.046 mmol), pyridine (2 mL), and pyridinium-HCl (1 g) were stirred under N 2 at 110 0 C for 17 h. The reaction was removed from the heat and 5 mL of H 2 O was used to transfer the hot mixture into a polypropylene centrifuge tube, and the resulting precipitate was collected by centrifugation at 6'500 r.p.m. The dark green precipitate was washed repeatedly with H 2 O and methanol, and dried under high vacuum to yield 28 mg (87.2%). MALDI TOF MS (jnlz): [M+H] + calcd for C 32 H 14 N 12 O 8 , 695.1; found 695.2.

Example 15:

4,4',4",4'"-Tetranitro zinc phthalocyanine (35 mg, 0.046 mmol), pyridine (2 mL), and pyridinium-HCl (1 g) were stirred under N 2 at 110 0 C for 17 h. The reaction was removed from the heat and 5 mL OfH 2 O was used to transfer the hot mixture into a polypropylene centrifuge tube, and the resulting precipitate was collected by centrifugation at 6'500 r.p.m. The dark green precipitate was washed repeatedly with H 2 O and methanol, and dried under high vacuum to yield 26 mg (81%). MALDI TOF MS (mlz): [M+H] + calcd for C 32 H 14 Ni 2 O 8 , 695.1; found 695.2.

Example 16:

pyπdιne-HCI 120 °C

3,3',3",3"'-Tetraamino zinc phthalocyanine (35 mg, 0.055 mmol), pyridine (2 mL), and pyridinium-HCl (1 g) were stirred under N 2 at 110 0 C for 17 h. The reaction was removed from the heat and 10 mL OfH 2 O was used to transfer the hot mixture into a polypropylene centrifuge tube, and the resulting precipitate was collected by centrifugation at 6'500 r.p.m. The dark green precipitate was washed repeatedly with H 2 O, methanol, and ethyl acetate, and dried under high vacuum to yield 24.5 mg (77%).

Example 17:

pyridine-HCI 120°C

4,4',4",4"'-Tetraamino zinc phthalocyanine (35 mg, 0.055 mmol), pyridine (2 mL), and pyridinium-HCl (1 g) were stirred under N 2 at 110 0 C for 17 h. The reaction was removed from the heat and 10 mL of H 2 O was used to transfer the hot mixture into a polypropylene centrifuge tube, and the resulting precipitate was collected by centrifugation at 6'500 r.p.m. The dark green precipitate was washed repeatedly with H 2 O, methanol and ethyl acetate, and dried under high vacuum to yield 30.2 mg (95%). MALDI TOF MS (mlz): [M] + calcd for C 32 H 22 N 12 , 574.2; found 574.2.

Example 18:

pyridine-HC1 120 0 C

Zinc phthalocyanine (35 mg, 0.06 mmol), pyridine (2 mL), and pyridinium-HCl (1 g) were stirred under N 2 at 11O 0 C for 17 h. The reaction was removed from the heat and 10 mL ofH 2 O was used to transfer the hot mixture into a polypropylene centrifuge tube, and the resulting precipitate was collected by centrifugation at 6'500 r.p.m. The green bluish precipitate was washed repeatedly with H 2 O, methanol, and ethyl acetate, and dried under high vacuum to yield 21 mg (65.7%).

5,10,15,20-Tetrakis(4-aminophenyl)-21H,23H zinc poφhine (9 mg, 0.0122 mmoles), pyridine (1 mL), and pyridinium-HCl (0.5 g) were stirred under N 2 at 110°C for 2 h. The reaction was removed from the heat and 5 mL OfH 2 O was used to transfer the hot mixture into a polypropylene centrifuge tube, and the resulting precipitate was collected by centrifugation at 6'500 r.p.m. The brown precipitate was washed repeatedly with saturated solution OfNaHCO 3 , H 2 O, and dried under high vacuum to yield 7.4 mg (90%). MALDI TOF MS (Wz): [M+H] + calcd for C 44 H 34 N 8 , 674.29; found 679.20.

Example 20:

In another example, 4,4',4",4'"-tetra-diisopropylguanidine phthalocyanine (1 mg, 0.63 μmoles), cobalt (II) sulfate (5 mg), and 100 μL of a 1 M solution OfCs 2 CO 3 in acetic acid were heated at 90 0 C on shaker for 30 min, removed from the heat and 800 μL of H 2 O added, mixed, and centrifuged at 20'0OO g for 3 min, the supernatant was removed into clean tube and 170 μL of 30% TFA in water (v/v ratio) was added, mixed, centrifuged as before and the supernantant discarded. The precipitate was dissolved into 500 μL of 0.1 M KCl using sonication for 3 min and 300 μL of 5% TFA in water (v/v ratio) added, mixed, and centrifuged as before and the supernantant discarded. The precipitate was washed twice with 300 μL of 5% TFA in water (v/v ratio), and dried under high vaccum to furnish approximately 1 mg (95%) of a blue-green solid. MALDI TOF MS (m/z): [M+H] + calcd for C 60 H 77 CoN 20 , 1136.6; found 1136.5.

Example 21 :

chloπde

In another example, 4,4',4",4'"-tetra-diisopropylguanidine phthalocyanine (1 mg, 0.63 μmoles), copper (II) chloride (5 mg), and 100 μL of a 1 M solution Of Cs 2 CO 3 in acetic acid were heated at 90 0 C on shaker for 30 min, removed from the heat and 800 μL of H 2 O added, mixed, and centrifuged at 20'0OO g for 3 min, the supernatant was removed into clean tube and 170 μL of 30% TFA in water (v/v ratio) was added, mixed, centrifuged as before and the supernantant discarded. The precipitate was dissolved into 500 μL of 0.1 M KCl using sonication for 3 min and 300 μL of 5% TFA in water (v/v ratio) added, mixed, and centrifuged as before and the supernantant discarded. The precipitate was washed twice with 300 μL of 5% TFA in water (v/v ratio), and dried under high vaccum to furnish approximately 1 mg (95%) of a blue-green solid. MALDI TOF MS (m/z): [M+H] + calcd for C 60 H 77 CuN 20 , 1140.6; found 1140.5. Example 22:

In another example, 4,4',4",4'"-tetra-diisopropylguanidine phthalocyanine (1 mg, 0.63 μmoles), palladium (II) acetate (5 mg), and 100 μL of a 1 M solution Of Cs 2 CO 3 in acetic acid were heated at 90 0 C on shaker for 30 min, removed from the heat and 800 μL of H 2 O added, mixed, and centrifuged at 20'0OO g for 3 min, the supernatant was removed into clean tube and 170 μL of 30% TFA in water (v/v ratio) was added, mixed, centrifuged as before and the supernantant discarded. The precipitate was dissolved into 500 μL of 0.1 M KCl using sonication for 3 min and 300 μL of 5% TFA in water (v/v ratio) added, mixed, and centrifuged as before and the supernantant discarded. The precipitate was washed twice with 300 μL of 5% TFA in water (v/v ratio), and dried under high vaccum to furnish approximately 1 mg (95%) of a blue-green solid. MALDI TOF MS (mlz): [M+H] + calcd for C 60 H 77 PdN 20 , 1183.6; found 1183.6.

Example 23:

In another example, 4,4',4",4'"-tetra-diisopropylguanidine phthalocyanine (1 mg, 0.63 μmoles), tin (II) chloride (5 mg), and 100 μL of a 1 M solution Of Cs 2 CO 3 in acetic acid were heated at 90 0 C on shaker for 30 min, removed from the heat and 800 μL of H 2 O added, mixed, and centrifuged at 20'0OO g for 3 min, the supernatant was removed into clean tube and 170 μL of 30% TFA in water (v/v ratio) was added, mixed, centrifuged as before and the supernantant discarded. The precipitate was dissolved into 500 μL of 0.1 M KCl using sonication for 3 min and 300 μL of 5% TFA in water (v/v ratio) added, mixed, and centrifuged as before and the supernantant discarded. The precipitate was washed twice with 300 μL of 5% TFA in water (v/v ratio), and dried under high vaccum to furnish approximately 1 mg (95%) of a blue-green solid. MALDI TOF MS (m/z): [M+H] + calcd for C 60 H 77 SnN 20 , 1197.6; found 1197.6.

The inserted metal ions or metalloids can adopt octahedral, square-pyramidal, or square- planar coordination spheres with one or two axial ligands.

Example 24:

In another example, 4,4',4",4'"-tetra-diisopropylguanidine phthalocyanine (1 mg, 0.63 μmoles), mercury (II) chloride (5 mg), and 100 μL of a 1 M solution Of Cs 2 CO 3 in acetic acid were heated at 90 0 C on shaker for 30 min, removed from the heat and 800 μL of H 2 O added, mixed, and centrifuged at 20'0OO g for 3 min, the supernatant was removed into clean tube and 170 μL of 30% TFA in water (v/v ratio) was added, mixed, centrifuged as before and the supernantant discarded. The precipitate was dissolved into 500 μL of 0.1 M KCl using sonication for 3 min and 300 μL of 5% TFA in water (v/v ratio) added, mixed, and centrifuged as before and the supernantant discarded. The precipitate was washed twice with 300 μL of 5% TFA in water (v/v ratio), and dried under high vaccum to furnish approximately 1 mg (95%) of a blue-green solid. MALDI TOF MS (m/z): [M+H] + calcd for C 60 H 79 HgN 20 O 2 , 1313.6; found 1313.6.