Mixture of 3 isomers of a phthalocyanine derivative

Abstract

The present invention describes an industrial process for obtaining a mixture of only three positional isomers of [1,8(11 ),15(18),22(25)-tetrakis-(3-N,N,N- trimethylammoniumphenoxy)] Zinc phthalocyaninate tetrachloride, said mixture at a degree of purity suitable for use in the pharmaceutical field, in particular in the PDT treatment of both tumors and microbial infections.

Description

[0001] PATENT APPLICATION FOR INDUSTRIAL INVENTION TITLED:

[0002] MIXTURE OF 3 ISOMERS OF A PHTHALOCYANINE DERIVATIVE

[0003] FIELD OF THE INVENTION

[0004] The present invention refers to the field of Zn-phthalocyanine derivatives, their preparation and medical use thereof.

[0005] STATE OF THE ART

[0006] It is known that molecules containing the phthalocyanine chromofluorophore macrocycle produce reactive oxygen species, such as radicals or singlet oxygen, by interaction with visible light.

[0007] Due to these properties, phthalocyanine compounds have long been used in photodynamic therapy (hereinafter referred to as “PDT”) both for therapeutic treatment and diagnostic purposes.

[0008] WO201 1 / 012698 describes a process for preparing a mixture of the 4 isomers (Fig. 1 ) of the compound [1 ,8(1 1 ),15(18),22(25)-tetrakis-(3-N,N,N- trimethylammoniumphenoxy)] Zinc phthalocyaninate tetrachloride, said mixture wherein the isomer B (the one having symmetry class C4h) is present in amounts lower than or equal to 1 % by weight. The aforementioned mixture is obtained through a process that comprises a chromatographic separation step whose stationary phase consists of irregularly shaped silica gel particles with size of 20 - 75 pm and pores of 60 Angstroms, the X+Y mobile phase wherein X consists of a 94 / 5 / 1 DCM / THF / MeOH mixture and Y consists of n-hexane or n-heptane, the elution is carried out by eluting initially with X / Y 3 / 1 (until the isomers C and D begin to elute) and then with X / Y 4 / 1 until complete elution of the isomer A. The aforementioned mixture has proven to be particularly suitable for use in photodynamic therapy and related pharmaceutical formulations.

[0009] On an industrial scale, the process described in WO201 1 / 012698 involves important issues of both environmental nature and safety of the operators involved in such a process since the solvents used are toxic and low boiling, thus producing emissions into the atmosphere to which operators are exposed.

[0010] The aim of the present invention is to provide an alternative mixture of isomers of the compound [1 ,8(1 1 ),15(18),22(25)-tetrakis-(3-N,N,N- trimethylammoniumphenoxy)] Zinc phthalocyaninate tetrachloride, said mixture being therapeutically equivalent and sufficiently pure to be suitable for medical application. The aim of the present invention is also to provide an industrial process for preparing the aforementioned mixture which is industrially sustainable both for environmental and operator safety issues.

[0011] DEFINITIONS AND ABBREVIATIONS

[0012] DCM: dichloromethane

[0013] DMF: dimethylformamide

[0014] GMP: Good Manufacturing Practices

[0015] Mel: methyl iodide

[0016] MeOH: methanol

[0017] Me-THF: methyltetrahydrofuran

[0018] PDT: photodynamic therapy

[0019] PhMe: toluene

[0020] TBME: tert-butyl methyl ether

[0021] THF: tetrahydrofuran

[0022] SUMMARY OF THE INVENTION

[0023] The present invention addresses the aforementioned issues by means of a mixture of positional isomers of the compound [1 ,8(1 1 ), 15(18), 22(25)-tetrakis-(3-N,N,N- trimethylammoniumphenoxy)] Zinc phthalocyaninate tetrachloride (RLP068), said mixture consisting only of isomers A (D2h symmetry), C (Cssymmetry) and D (C2v symmetry) wherein the isomer B (the one with C4h symmetry) is absent or present in undetectable amounts (Figs. 7-8).

[0024] The aforementioned mixture has a degree of purity suitable for use as an active ingredient in pharmaceutical preparations and, furthermore, the total absence of the isomer B surprisingly does not change the therapeutic efficacy of the compound compared to what is reported in WO2011 / 012698; even, and surprisingly, the total absence of the isomer B already at the level of purification of the intermediate RLP064 ( / .e. [1 ,8(11 ),15(18),22(25)-tetrakis-(3-N,N-dimethylammoniumphenoxy)] Zinc phthalocyaninate) increases the solubility / stability of the mixture in solution, ultimately providing it with a purity more favourable to therapeutic use. Subject-matter of the present invention is also an industrial process for the preparation of the above mixture, said process comprising:

[0025] (Step 2) submitting a mixture comprising the 4 positional isomers of the compound [1 ,8(11 ),15(18),22(25)-tetrakis-(3-N,N-dimethylammoniumphenoxy)] Zinc phthalocyaninate (Fig. 3, RLP064, mixture of 4 isomers) to chromatographic separation on a stationary phase consisting of silica gel with spherical particles using a mobile phase consisting of PhMe / X / MeOH = 92-95 / 4-8 / 1 -2 v / v, wherein X is selected from the group consisting of THF and 2-Me-THF, to obtain a mixture comprising 3 positional isomers of the compound [1 ,8(1 1 ),15(18),22(25)-tetrakis-(3- N,N-dimethylammoniumphenoxy)] Zinc phthalocyaninate (Fig.6 RLP064, mixture of 3 isomers), wherein the isomer B is absent or present in undetectable amounts.

[0026] After a long investigation for mobile phases alternative to the one described in WO201 1 / 012698, the aforementioned mobile phase, in which DCM is replaced by PhMe, not only allows an industrial production of the mixture of the invention which is safe for the operators but also ensures high purity thereof. Especially the isomer B is better separated by the solvent system of the present invention, in which DCM is replaced by PhMe, and this chromatographic behaviour is unexpected since the polarity of the solvents is very different.

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028] The undesirable impurities that may be present in the mixture subject-matter of the invention are those that are formed during the synthesis of the amine intermediate (Fig. 3, RLP064). The purity of the mixture object of the invention and the quantification of the impurities is determined by HPLC analysis. Preferably, the undesirable impurities are present in total amounts lower than 5% w / w, more preferably lower than 3%, even more preferably lower than 2%.

[0029] The chromatographic separation according to the invention preferably employs a mobile phase consisting of PhMe / 2-Me-THF / MeOH = 93:6:1 v / v.

[0030] According to the present invention, the process, upstream to the chromatographic separation (Step 2), comprises:

[0031] Step 1 ) subjecting 3-[3-(Dimethylamino)phenoxy]phthalonitrile (Fig.2, M64) to a condensation reaction catalysed by bases (preferably DABCO) and reducing agents (preferably Hydroquinone), in the presence of zinc acetate and at high temperature (120-160°C, preferably 130-150°C) to obtain a mixture comprising the 4 positional isomers of the compound [1 ,8(1 1 ),15(18),22(25)-tetrakis-(3-N,N,N- dimethylammoniumphenoxy)] Zinc phthalocyaninate (Fig.2, RLP064, mixture of 4 isomers).

[0032] Step 1 ) preferably includes a work-up comprising cooling to 60-80°C, adding MeOH and calcined diatomaceous earth (preferably Celite® Hyflo® Super-Cel®) and subsequent filtration. The solid residue is then preferably contacted with PhMe to dissolve the organic products and remove celite.

[0033] The process according to the present invention, downstream to the chromatographic separation (Step 2), preferably comprises:

[0034] Step 3) contacting the mixture of three isomers obtained from Step 2 with Mel, preferably 10-20 eq, preferably at 15-25°C for 14-18 h, to obtain a mixture of 3 positional isomers of the compound [1 ,8(11 ),15(18),22(25)-tetrakis-(3-N,N,N- trimethylammoniumphenoxy)] Zinc phthalocyaninate tetraiodide, wherein the isomer B is absent or present in undetectable amounts.

[0035] Step 3) preferably includes a work-up, wherein MeOH and TBME are added to obtain the precipitation of the iodinated compound.

[0036] The process according to the present invention, subsequently to methyl iodination (Step 3), preferably comprises:

[0037] Step 4) contacting the mixture obtained from Step 3 with an anionic exchange resin, preferably with a Styrene-divinylbenzene matrix functionalized with trimethylammonium chloride, such as Amberlite Marathon 4200 (Cl), to obtain the mixture of the invention.

[0038] Step 4) preferably includes dissolving the mixture obtained from Step 3) in a mixture of H2O and MeOH. Preferably, after the anion exchange the mixture of the invention is precipitated by addition of TBME.

[0039] The mixture of three isomers of the compound [1 ,8(11 ),15(18),22(25)-tetrakis-(3- N,N,N-trimethylammoniumphenoxy)] Zinc phthalocyaninate tetrachloride obtained according to the present invention is useful in PDT treatment, both of tumors and microbial infections.

[0040] The present invention can be better understood in light of the following examples. BRIEF DESCRIPTION OF THE FIGURES

[0041] Fig. 1 shows the four positional isomers of the compound [1 ,8(1 1 ),15(18), 22(25)- tetrakis-(3-N,N,N-trimethylammoniumphenoxy)] Zinc phthalocyaninate tetrachloride.

[0042] Fig. 2 shows the industrial process according to the present invention.

[0043] Fig. 3 shows an HPLC plot of the crude compound RLP064.

[0044] Figs. 4-5 show a TLC of a chromatographic separation according to the present invention.

[0045] Fig. 6 shows a HPLC of the RLP064 product obtained after chromatographic separation according to the present invention, in which it can be noted that the isomer B results in amounts lower than the LoD.

[0046] Fig. 7 shows an HPLC (method RD108) of the product RLP068CI (A) 4 isomers obtained without chromatographic purification; (B) 4 isomers with isomer B < 1 % obtained according to the state-of-the-art process of WO201 1 / 012698; (C) 3 isomers obtained following chromatographic purification according to the present invention in which it can be noted that isomer B results in amounts lower than the LoD.

[0047] Fig. 8 shows an HPLC (method RD131 ) of the product RLP068CI (A) 4 isomers obtained without chromatographic purification; (B) 4 isomers with isomer B < 1 % obtained as per the state-of-the-art process of WO2011 / 012698; (C) 3 isomers obtained following chromatographic purification according to the present invention in which it can be noted that isomer B results in amounts lower than the LoD.

[0048] EXPERIMENTAL PART

[0049] MATERIALS AND ANALYTICAL METHODS

[0050] HPLC METHOD FOR RLP064

[0051] Mobile phase

[0052] CH3(CH2)4-SO3Na in phosphate buffer:MeOH:THF (20:48:32, v / v / v)

[0053] Preparation of RLP064 sample solution

[0054] Stock solution: 1 .0 mg / mL in DMF.

[0055] Sample solution: 250 pL of Stock solution + 750 pL of MeOH.

[0056] HPLC Column: Phenomenex Luna C18 150*4.6 mm (5 pm)

[0057] Ion pair solution: 60mM CH3(CH2)4-SO3Na in 10 mM phosphate buffer, pH 3

[0058] Eluent A: ion pair / MeOH / THF 20 / 48 / 32 v / v / v

[0059] Elution: isocratic with 50 min total time

[0060] Flow rate: 1 .0 mL / min

[0061] Temperature: 25°C ± 1 °C

[0062] Detection: 695nm, 20nm, REF. 500nm, 50nm

[0063] 254nm, 20nm, REF. 500nm, 50nm (optional)

[0064] Vinj: 20 pL

[0065] Retention times

[0066] RLP064 Isomer A (D2h)-. 25.5 min.

[0067] RLP064 Isomers C+D (Cs+C2v) 28.2 min.

[0068] RLP064 Isomer B (C4h) 31 .1 min.

[0069] HPLC METHOD RD108 FOR RLP068CI

[0070] With the method RD108 it is possible to identify the positional isomers and carry out specific quantification. With the same method RD108 it is possible to check whether the residual isomer B is within specification (<1 % a / a) through a limit test at the specification value. The method RD108 has not been validated in terms of LoD and LoQ on the isomer B specifications.

[0071] Mobile phase

[0072] Eluent A: 20mM KI with AcOH (0.1% v / v) in H2O milliQ

[0073] Eluent B: MeOH

[0074] Eluent C: MeOH I H2O milliQ 9 / 1

[0075] Preparation of RLP068 sample solution

[0076] Stock solution: 2.0 mg / mL in MeOH.

[0077] Sample solution: 125 pL of Stock solution + 875 pL of H2O milliQ.

[0078] HPLC

[0079] Column: Varian Polaris C18-Acid 150*4.6 mm (5pm)

[0080] Elution: gradient

[0081] Flow rate: 1 .0 mL / min

[0082] Temperature: 20°C ± 1 °C

[0083] Detection: 690 nm, 20 nm, REF. 500 nm, 50 nm

[0084] Vinj: 10pL

[0085] Retention times (Fig. 7)

[0086] RLP068 Isomer A (D2h)-. 10.1 min.

[0087] RLP068 Isomer C (Cs) 12.9 min.

[0088] RLP068 Isomer D (C2v) 13.9 min.

[0089] RLP064 Isomer B (C4h) 15.0 min.

[0090] HPLC METHOD RD131 FOR RLP068CI

[0091] With the method RD131 it was possible to determine a LoD and LoQ for the isomer B corresponding to 0.03% a / a and 0.1 % a / a, respectively.

[0092] Mobile phase

[0093] Eluent A: CH3(CH2)e-SO3Na in phosphate buffer (1.01 1 g CH3(CH2)e-SO3Na, 1.15 g H3PO4 85% brought to a volume of 1 L with H2O)

[0094] Eluent B: MeOH / MeCN 3 / 2 v / v

[0095] Preparation of RLP068 sample solution

[0096] Stock solution: 2.0 mg / mL in MeOH.

[0097] Sample solution: 125 pL of Stock solution + 875pL of H2O milliQ.

[0098] HPLC

[0099] Column: Agilent Polaris C18-Acid 150*4.6 mm (5pm)

[0100] Elution: gradient Flow rate: 1 .0 mL / min

[0101] Temperature: 40°C ± 1 °C

[0102] Detection: 695 nm, 20 nm, REF. 500 nm, 50 nm

[0103] Vinj: 5pL

[0104] Retention times (Fig. 8):

[0105] RLP068 Isomer A (D2h)-. 10.7 min.

[0106] RLP064 Isomer B (C4h) 1 1 .6 min.

[0107] RLP068 Isomer C (Cs) 1 1 .9 min.

[0108] RLP068 Isomer D (C2v) 12.7 min.

[0109] EXAMPLE 1 : STEP 1 - Synthesis of RLP064

[0110] 3-[3-(Dimethylamino)phenoxy]phthalonitrile (M64) (1.0 eq), hydroquinone (0.1 eq), diazabicyclooctane (3.0 eq), Zn(ll) acetate (0.5 eq) is added portionwise to DMF (1 .7 vol) in a 30L reactor at 80 °C. The addition system is rinsed with DMF (0.5 vol). The reaction mixture is heated to a temperature of 140 °C over 40 minutes; after 4 hours at 140 °C the M64 starting material tends to disappear and an in-process control is carried out to verify the end of the reaction.

[0111] The reaction mixture is cooled to 70 °C and the mixture is added to methanol (8 vol) in a 150 L stir tank causing the crude product to precipitate; the suspension in the stir tank is stirred overnight at room temperature. Hyflo Super Cel (0.5 wt) is added to facilitate the filtration step.

[0112] The suspension is filtered using a 40 cm filter. At the end of the filtration step, the solid on the filter is rinsed with methanol, and is dried under a nitrogen flow overnight, with stirring to facilitate removal of the solvent.

[0113] The crude product is dissolved in PhMe and Hyflo is removed by filtration. The filtrate is evaporated to dryness; the crude product obtained is divided into 2 or 3 portions. Fig. 3 shows an HPLC plot of the crude RLP064 obtained according to the present example. EXAMPLE 2: STEP 2 - Chromatographic separation

[0114] Each chromatographic column is prepared using PhMe and 21 -22 kg of 20-70 mesh spherical silica gel.

[0115] The crude product obtained from Example 1 , divided into 2 or 3 portions, is dissolved in PhMe and loaded onto the column. Elution is performed with a solvent mixture of PhMe : 2-methyl-THF : methanol 93:6:1. For the purification of 1 kg of crude oil obtained from Example 1 , 14-15 L of mobile phase per kg of stationary phase were used.

[0116] The colourless initial fraction and the blue isomer B eluate are collected and discarded. Only the green eluate is fractionated, and a process control is performed for each fraction. (Figs. 4-5)

[0117] The eluted fractions containing the green eluate are combined and concentrated to obtain the purified product, a solvated mixture of three isomers of RLP064 with a residue of 2-methyl-THF and MeOH solvents (about 10%). Fig. 6 shows an HPLC plot of the purified product mixture of three isomers of RLP064, where the absence of the isomer B can be noted.

[0118] Optionally, to effectively remove residual solvents, the purified solvated portions (1 kg) are dissolved in DCM (9-10 L), and heptane (60 L) is added to obtain the precipitation of the product RLP064 as a mixture of 3 isomers (Fig. 5).

[0119] The suspension is stirred and filtered through a 40 cm diameter Buckner filter.

[0120] RLP064 purified mixture of 3 isomers is dried, first under an anhydrous nitrogen flow and subsequently under reduced pressure, until dry.

[0121] At the end of the drying phase, the in-process control (barrier phase) is carried out to evaluate the quality of the intermediate RLP064 mixture of three isomers.

[0122] EXAMPLE 3: STEP 3 - Methylation with Mel

[0123] RLP064 (1 .0 eq), as obtained from Example 2, is added to NMP (8 vol) at 20 °C in a 30L reactor. Mel (10.0 eq) is added to the deep green solution. After 4.5 hours another portion of Mel (4.0 eq) is added.

[0124] The reaction mixture is stirred for 16.5 hours at 20 °C. Methanol (5.4 vol) is added to the reaction mixture. The reaction mixture is transferred to a 150 L stir tank and the vessel is rinsed with methanol (11 vol). TBME (63 vol) is added. After 2 hours, the product is filtered (filtration time 4.5 hours using a 40 cm shaker filter). The filter cake is rinsed with TBME (30 vol, 1 hour) and the crude intermediate RLP068 / I is dried under a nitrogen flow.

[0125] EXAMPLE 4: STEP 4 - Ion exchange

[0126] An ion exchange column is prepared in a 50 liter filter. Amberlite Marathon 4200 Cl is loaded into the filter using purified water. The column is rinsed with a mixture of water / methanol (1 :1 and 1 :2) and with methanol.

[0127] The intermediate RLP068 / I, as obtained from Example 3, is dissolved in a H2O / MeOH mixture and the solution is loaded onto the ion exchange column and eluted with methanol. Only the blue-green eluent is collected. The eluent-containing product is divided into portions, if needed. TBME is added to each portion of eluent in a 150 L stir tank; the suspension obtained is stirred. The product is filtered using a 40 L shaken filter. The product is washed with TBME. An ongoing check is performed to evaluate residual iodide in the product. The RLP068 / CI product is dissolved in water and methanol, in-line filtered and combined in a 150 L stir tank. TBME is added to the solution causing the product to precipitate. The product is filtered and washed with TBME. The product is dried under a nitrogen flow (alternately wet and dry) until TBME reaches a content lower than or equal to 2.5%. The product is divided into portions of about 1 kg which are dissolved in water and methanol and precipitated in a 150L stir tank using TBME. All portions were filtered on the same 40 liter filter. The combined portions on the filter are washed twice in suspension with TBME to ensure homogeneous mixing.

[0128] The product is dried at room temperature under a nitrogen flow (alternating dry nitrogen and wet nitrogen) to reduce TBME and methanol content below specifications. This drying method was selected among many others tested because it is the one that has proven most effective in minimizing the formation of demethylated impurities.

[0129] At the end of the solvent elimination phase, release controls are performed to evaluate the quality of RLP068 / CI and verify that the amount of demethylated impurities remains as low as possible (Figs. 7C, 8C).

[0130] EXAMPLE 5 - Testing of alternative eluents to DCM / THF / MeOH for Step 2 As an alternative to the DCM / THF / MeOH / hexane mobile phase employed in WO201 1 / 012698, and to increase the chromatography efficiency, various solvent mixtures were tested by TLC (see Table 2). The most selective solvent mixture found is a mixture of PhMe - (methyl)THF - methanol. Especially the isomer B is better separated by the current solvent system. Furthermore, DCM is replaced by

[0131] PhMe. This behaviour is quite surprising since the polarity of both solvents is very different.

[0132] Table 1 - Separations using different TLC eluents

[0133] Solvent mix A = DCM:THF:MeOH 94:5:1

[0134] EXAMPLE 6 - STEP 2 chromatographic separation on 4 g With TLC separation, the mixture of PhMe / 2-methyl THF / methanol showed the best results. To verify these results, a column was run using 100 g of spherical silica and 4.0 g of crude product (from Step 1 , after quench with methanol). A mixture of PhMe / 2-methylTHF / methanol of 93: 6: 1 was used as the eluent. After 180 ml of elution, 20 fractions of 50 ml were collected, and the isomer B was well separated (fractions 4-7) from the other isomers (fractions from 12 to 16) (see Fig. 3). The isolation yield was of 30 g (75%) and therefore better than that described in the state of the art. Environmentally critical solvents (DCM, THF) have also been replaced by less critical solvents (PhMe, 2-methyl-THF).

[0135] EXAMPLE 7 - STEP 2 chromatographic separation on 25 g

[0136] Since the PhMe / 2-methyl THF / methanol solvent system showed good separation, it was reproduced on a larger scale. 25 g of a crude reaction product according to Example 1 was dissolved in PhMe (100 ml) and purified by chromatography using 25 wt spherical silica (625 g) and PhMe / 2-methyl THF / methanol (93: 6: 1 , total 8.0 L) (see Fig. 4). After evaporation of fractions 15 to 22, 18.07 g of product were obtained (68% yield) with an HPLC purity of 98.4%. To obtain a crystalline product, the product of fractions 15 to 22 was dissolved in MeTHF (3 vol) and this solution was added to methanol (10 vol). 6 vol of solvent were evaporated at 35 °C and methanol (6 vol) was added. Another 4 vol of solvent were evaporated. After filtration and vacuum drying, 16.3 g of a dark green solid with a 98.3% HPLC purity by area was obtained (see Figure 6 for details).

[0137] EXAMPLE 8 - STEP 2 chromatographic separation on 100 g

[0138] Starting from 100 g of M64, 107 g of crude RLP064 (101 % yield) were obtained with an HPLC purity of 98.6%. This crude was purified by chromatography on a column (diameter: 12 cm, height: 40 cm) packed with 2.10 kg of spherical silica (21 times by weight with respect to the crude to be purified) and PhMe. The crude was dissolved in PhMe, loaded onto the column, and eluted with a solvent mixture of PhMe / Me-THF / MeOH (93: 6: 1 ).

[0139] The following fractions were collected:

[0140] Head fraction: 5 L (colourless eluent)

[0141] Fraction 1 10 L Fraction 2 5 L

[0142] Fraction 3 2 L

[0143] Fraction 4 -13 1 L (total: 32 L)

[0144] The fractions from 3 to 7 / 8 / 9 were combined and analysed:

[0145] Fractions 3 to 9 were combined and evaporated. After evaporation, 76.3 g of product were obtained (72% yield) as MeOH / Me-THF solvate complex. After precipitation from DCM / heptane, 68.3 g of RLP064 were obtained (64% yield).

[0146] 0.5 g of the product RLP064 were methylated using 10 eq of Mel and the isomeric ratio of the resulting RLP068 / I was analysed by HPLC to be 2.6: n.d.: 54.7: 42.8 (A: B: C: D).

[0147] EXAMPLE 9 - STEP 2 chromatographic separation on GMP production scale GMP production began with 2.81 kg of M64 and delivered 2.86 kg of crude RLP064 (96% yield) with an HPLC purity of 98.7% a / a. The product RLP064 was separated into 3 portions of 0.95 kg each. These portions were purified separately using a chromatographic column of suitable size and materials suited for the purpose.

[0148] PORTION 1

[0149] Portion 1 was purified using 21 kg of silica and a total of 300L of eluent (PhMe : Me- THF : MeOH = 93 : 6 : 1 ). About 80L of eluate as head fraction and 22 fractions of 10L were collected.

[0150] The undesirable isomer B was collected in fractions 1 to 7 including an impurity visible only under UV. The desired isomers C + D eluted in fractions 13 to 20 and the isomer A eluted in fractions 16 to 20. Based on TLC, fraction 16 was the most concentrated. Several other impurities were also eluted from fraction 18 onwards. For a pooling test on a ml-scale, fractions 13 to 16 / 17 / 18 were analysed by HPLC. Fractions 13 to 18 were combined and evaporated. 597 g of products were obtained (60% yield for phase 1 + 2). The purity by HPLC is 99.3% of the area for isomer A + C + D.

[0151] 23 g of the product isolated after chromatography were dissolved in DCM (210 ml) and the product was precipitated with heptane (1242 ml). After vacuum drying at 40°C, 20.78 g were obtained (90% yield). This material was methylated using 10 eq plus 4 eq of Mel. The purity of the isolated RLP068 / I showed an isomeric ratio of 0.7: n.d. : 56.8: 42.5 (A: B: C: D).

[0152] PORTION 2

[0153] 0.95 kg of crude were purified using 22 kg of silica and a total of 300 L of eluent (PhMe: Me-THF: MeOH = 93: 6: 1 ). Approximately 150L of eluate as head fraction and 15 fractions of 10L were collected.

[0154] The undesirable isomer B was removed with the head fraction. The desired isomers C + D eluted in fractions 6 to 13, and the isomer A was eluted in fractions 10 to 13. Based on TLC, fractions 8 and 9 were the most concentrated fractions. Many other impurities were also eluted from fraction 1 1 onwards. For a pooling test on a ml- scale, fractions 6 to 12 were analysed in different combinations by HPLC from which fractions 8 to 1 1 were selected and pooled and evaporated. 546 g of product were thus obtained (55% yield for Steps 1 + 2).

[0155] PORTION 3:

[0156] 0.95 kg of crude were purified using 22 kg of silica and a total 320 L of eluent (PhMe: Me-THF: MeOH = 93: 6: 1 ). About 200L of eluate as head fraction and 12 fractions of 10L were collected.

[0157] The undesirable isomer B was removed with the head fraction. The desired isomers C + D eluted in fractions 3 to 11 , and the isomer A eluted in fractions 8 to 12. Fractions 6 to 8 were the most concentrated fractions. Several other impurities were also eluted from fraction 1 1 onwards. For a pooling test on a ml-scale, fractions 3 to 1 1 were analysed in different combinations by HPLC. Fractions 4 to 1 1 were evaporated to obtain 678 g of product (68%) with an HPLC purity of 98.76 area%. The products obtained from the three separations were combined and dissolved in DCM (16.1 L) and the solution was added to heptane (100 L) to precipitate the product. After 55 minutes, the product was filtered and washed with heptane (1 1 L) (filtering time 3.5 hours plus 1 hour washing). The filter cake was pre-dried under a nitrogen flow overnight. After drying on the rotary evaporator at 40 °C for 48 hours, 1616 g of product were obtained (54% yield) (Figs. 7C, 8C).

Claims

CLAIMS1. A mixture of positional isomers of the compound [1 ,8(11 ),15(18),22(25)-tetrakis-(3-N,N,N-trimethylammoniumphenoxy)] Zinc phthalocyaninate tetrachloride consisting of the isomers A (D2h symmetry), C (Cssymmetry) and D (C2v symmetry)2. The mixture according to the preceding claim, wherein undesirable impurities are present in total amounts lower than 5% w / w, and wherein the isomer B (C4h symmetry) is absent or present in undetectable amounts.

3. The mixture according to any one of the preceding claims for use in the photodynamic treatment of tumors and / or microbial infections.

4. An industrial process for preparing the mixture according to any one of claims 1 -3, said process comprising:(Step 2) subjecting a mixture comprising the 4 positional isomers of the compound [1 ,8(1 1 ),15(18),22(25)-tetrakis-(3-N,N-dimethylammoniumphenoxy)] Zinc phthalocyaninate to chromatographic separation on a stationary phase consisting of silica gel with spherical particles using a mobile phase consisting of PhMe / X / MeOH = 92-95 / 4-8 / 1 -2 v / v, wherein X is selected from the group consisting of THF and 2-Me-THF, to obtain a mixture comprising 3 positional isomers of the compound [1 ,8(1 1 ),15(18),22(25)-tetrakis-(3-N,N-dimethylammoniophenoxy)] Zinc phthalocyaninate, wherein the isomer B is absent or present in undetectable amounts.

5. The process according to the preceding claim, wherein the chromatographic separation employs a mobile phase consisting of PhMe / 2-Me- THF / MeOH = 93:6:1 v / v.

6. The process according to any one of claims 4-5 comprising, upstream of the chromatographic separation (Step 2)(Step 1 ) subjecting 3-[3-(Dimethylamino)phenoxy]phthalonitrile to a condensation reaction catalysed by bases and reducing agents, in the presence of zinc acetate and at a temperature of 120-160°C, to obtain a mixture comprising the4 positional isomers of the compound [1 ,8(1 1 ),15(18),22(25)-tetrakis-(3-N,N,N- dimethylammoniophenoxy)] Zinc phthalocyaninate.

7. The process according to any one of claims 4-5 comprising, downstream to the chromatographic separation (Step 2): (Step 3) contacting the mixture of three isomers obtained from Step 2 with Mel, to obtain a mixture of 3 positional isomers of the compound [1 ,8(1 1 ),15(18), 22(25)- tetrakis-(3-N,N,N-trimethylammoniophenoxy)] Zinc phthalocyaninate tetraiodide, wherein the isomer B is absent or present in undetectable amounts.

8. The process according to the preceding claim comprising, subsequent to the methyl-iodination (Step 3):(Step 4) contacting the mixture obtained from Step 3 with an anion exchange resin, to obtain the mixture according to any one of claims 1 -3.

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