Preparation process of indocyanine green

Abstract

The present invention relates to a process for the preparation of indocyanine green (ICG, 1H-benz[e]indole,2-[7-[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-benz[e]indol-2-ylidene]-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulfobutyl)hydroxide, inner salt, sodium salt, CAS RN 3599-32-4) of formula (I) having a total impurity content of less than 0.5% and a single impurity of less than 0.10% (purity measured by a new analytical method HPLC at a wavelength of 254 nm) and a related composition containing stable NaI, which is water soluble and has a NaI content of less than 2.5%, also on an industrial scale. [Formula 1] JPEG2024510708000021.jpg5789

Description

[Technical Field]

[0001] The present invention relates to a process for preparing indocyanine green (ICG, 1H-benz[e]indole, 2-[7-[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-benz[e]indol-2-ylidene]-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulfobutyl)hydroxide, inner salt, sodium salt, CAS RN 3599-32-4) of formula (I) having a total impurity content of 0.5% or less and a single impurity content of 0.10% or less (purity measured by a new analytical method, HPLC, at a wavelength of 254 nm), as well as a related composition containing stable NaI that is water soluble and has a NaI content of 2.5% or less, even on an industrial scale. [ka] [Background technology]

[0002] Indocyanine green is a fluorescent dye used in medicine as a contrast agent in cardiac, circulatory, hepatic, and ophthalmological conditions (e.g., for photometric diagnosis of liver function and for fluorescence angiography). It is administered intravenously and is excreted by the body with a half-life of approximately 3–4 minutes, depending on liver function. The sodium salt of indocyanine green is usually available in powder form and is soluble in various solvents. To ensure better solubility, 5% sodium iodide is usually added (<5%, depending on the batch). Sterile, lyophilized water-indocyanine green solutions are approved as diagnostic agents for intravenous use in many European countries and the United States.

[0003] Various processes are known for the preparation of indocyanine green, including patents US 2,895,955 (filed in 1959) and US 10,287,436 (filed in 2016), and the recent US patent US2019 / 0337896.

[0004] The following synthesis schemes also report the above two synthetic approaches known in the literature and reported in the references mentioned above: [ka]

[0005] In US 2,895,955, the compound of formula (I) is synthesized by isolating intermediate (VI).

[0006] In US 10,287,436, the compound of formula (I) is synthesized by pre-forming and isolating intermediate (VII).

[0007] In US2019 / 0337896, the compound of formula (I) is again synthesized via isolation of intermediate (VI).

[0008] Patent application US2019 / 0337896 also described the preparation of an amorphous form of the compound of formula (I) having a purity of greater than 99%. The application also describes an HPLC method used to measure such purity.

[0009] Applicant prepared the compound of formula (I) according to the instructions present in said patent application, but discovered that the product obtained by reproducing the examples of US2019 / 0337896 was not characterized as being 99% or greater pure as claimed in said patent application, when analyzed by the HPLC analytical method used in the present invention, as detailed below. Summary of the Invention

[0010] The object of the present invention is to provide a new process for the preparation of compound of formula (I) which is characterized by a total impurity content of 0.5% or less and a single impurity content of 0.1% or less, and which can provide a final product which is also industrially scalable.

[0011] The process of the present invention also makes it possible to obtain compositions comprising the compound of formula (I) and containing a lower NaI content (≦2.5%) than those currently on the market, which are nevertheless soluble in water up to 5 mg / ml and stable, i.e., protected from light and oxygen, under storage conditions typically used for currently commercially available products.

[0012] In fact, currently available products have a higher NaI content (less than 5% according to the United States Pharmacopoeia USP).

[0013] The level of impurities of the compound of formula (I) of the present invention is determined using two new HPLC methods characterized by the use of analytical wavelengths different from those previously applied, for example, in patent application US2019 / 0337896.

[0014] In particular, by using a new HPLC analytical method with an analytical wavelength of 254 nm instead of 205 nm, the compound of formula (I) synthesized using the examples of US2019 / 0337896 was characterized by a much lower purity, i.e., 93%.

[0015] The process of the present invention can save a synthetic step, making the overall process cheaper, and avoids both the isolation of intermediate (VI) and the isolation of intermediate (VII), and therefore differs from the prior art described above.

[0016] It is further emphasized that high purity is obtained thanks to the use of a special mixture of solvents and the special conditions used during the recrystallization step, and that such purity is in fact achieved despite the avoidance of isolation of both (VI) and (VII).

[0017] The present invention comprises the following steps: (a) reacting a compound of formula (II) below, 1,1,2-trimethyl-1h-benzo[e]indole, with a compound of formula (III) below, 1,4-butane sultone, [ka] [ka] reacting in a suitable high boiling solvent selected from anisole or xylene in the complete absence of methane to provide 4-(1,1,2-trimethyl-1H-benzo[e]indol-3-yl)butane-1-sulfonate of formula (IV) according to known methods; [ka] (b) reacting the compound of formula (IV) above with a compound of formula (V) below, benzeneamine, N-[(2E,4E)-5-(phenylamino)-2,4-pentadien-1-ylidene]-, hydrochloride (1:1) (known as GAD), [ka] reacting in the presence of acetic anhydride, sodium acetate, using a dipolar aprotic solvent to provide the final compound of formula (I) above, without isolating the intermediate; The present invention provides a novel synthesis process, including:

[0018] Step a) is also well known from the aforementioned references. The reaction can be carried out at a temperature that depends on the high-boiling solvent used. US2019 / 0337896 describes the following aprotic solvents: hexane, cyclohexane, toluene, xylene, tetrahydrofuran, acetone, acetonitrile, 1,4-dioxane, diethyl ether, dichloromethane, ethyl acetate, N,N-dimethylformamide, methyl tert-butyl ether, xylene, and acetone.

[0019] Applicant used xylene as the solvent at a temperature of about 130°C. Anisole can also be used with good results in terms of reaction rate, with complete conversion occurring in 7-8 hours at 140-150°C, compared to the typical 24 hours required in xylene at 125-130°C. The intermediate compound of formula (IV) is isolated by precipitation from the reaction mixture with acetone and used as is in the wet state without recrystallization as described in US 2019 / 0337896.

[0020] The presence of methanol in this step was evaluated as a crucial parameter. In fact, methanol can be used as a solvent to wash the reactor, as it is excellent at dissolving all materials used in this step, especially when the process is carried out on an industrial scale. However, even traces of methanol remaining in the reactor will react with compound (II) to produce N-methyl-benzindole (impurity G), which will then produce the impurity "methyl-indocyanine" (impurity H) in the subsequent step.

[0021] In pilot system tests ("demo batch" tests), this impurity was indeed found and was detected by LC / MS studies (LC / MS ESI + ;[MH] + :631,39), it had a hypothetical structure similar to "methyl indocyanine" (impurity H). Such a structure was confirmed throughout the total synthesis, as reported in the experimental part below.

[0022] The resulting compound (IV) was analyzed by LC / MS, and the expected m / z (LC / MS ESI + ;[MH] + The presence of an impurity, methylbenzindole (impurity G), characterized by the structure (identified as 224), was revealed. In this case, the structure was also confirmed throughout the total synthesis.

[0023] The absence of methanol, even as a residual solvent, in the starting material ensures that a product is obtained with specifically less than 0.10% impurity H. The characterization of this impurity has been improved by the development of an HPLC method adapted for this purpose.

[0024] The process according to the invention is therefore characterized by the direct implementation of step b), i.e. "one step", without the isolation of any intermediates and without the need to purify either intermediate (VI) or intermediate (VII), as occurs in the syntheses known in the prior art and described above.

[0025] Step b) is carried out by condensing compounds of formula (IV) and (V) in the presence of a solvent (acetonitrile), acetic anhydride, and sodium acetate, as previously known. The reaction is carried out at a temperature of 40-50°C to form the crude compound of formula (I). Compounds of formula (V) and (IV) are dissolved in acetonitrile in the presence of sodium acetate (4 equivalents). Acetic anhydride (4 equivalents) is then added at a lower temperature than that disclosed in US2019 / 0337896 and the reaction is carried out at the same temperature for 1-3 hours. The use of acetonitrile in this "one-pot" step allows for the use of minimal amounts of acetic anhydride for complete reaction, thus avoiding the use of acetic anhydride as a reaction solvent. This makes its removal by distillation easier and therefore makes the use of a water / isopropanol mixture safer for isolating the crude form of compound of formula (I).

[0026] In fact, a subsequent workup was carried out using isopropanol to isolate the crude solid of the compound of formula (I).

[0027] The compound of formula (I) thus obtained in crude form is already characterized by a high HPLC purity level (>90%), with the only significant impurity present being impurity A. Compound (I) can be conveniently purified not by known methods, such as the methanol / isopropanol mixture of US 2019 / 0337896, or the acetone, isopropanol, or methanol used in other cited references, but rather, as surprisingly discovered by the applicant, by crystallization in isopropanol / HO. No references have previously mentioned the use of isopropanol / HO mixtures in the appropriate ratios selected from the following used in the present invention: isopropanol / water: 5.9 / 3.4, 7.4 / 3.4, or 9.9 / 3.4, expressed by volume in liters / kg for the crude compound of formula (I).

[0028] The use of isopropanol / H2O has the advantage of providing the compound of formula (I) in a purity of 99.5 or greater, despite the fact that intermediate (VI) is not isolated.

[0029] As mentioned above, the applicant prepared the compound of formula (I) described in application US2019 / 0337896, as reported in the experimental part, and verified that the maximum purity obtained was 93%, thus significantly lower than the claimed 99%. When analyzing the samples at 205 nm, as described in said application, such a low purity cannot be highlighted.

[0030] Known impurities that can be effectively quantified by this analytical method and reduced to less than 0.15% by the synthesis and purification process of the present invention are as follows: 1. Impurity A: N-phenylacetamide, 2. Impurity B: 4-(1,1-dimethyl-2-((1E,3E,5E)-6-(N-phenylacetamido)hexa-1,3,5-trienyl)-1H-benzo[e]indol-3-yl)butane-1-sulfonate, 3. Impurity C: 4-(1,1,2-trimethyl-1H-benzo[e]indol-3-yl)butane-1-sulfonate, 4. Impurity D: 1-(1,1-dimethyl-2-methylidene-1,2-dihydro-3H-benzo[e]indol-3-yl)ethan-1-one, 5. Impurity E: (naphthalen-2-yl)hydrazine, 6. Impurity F: N-acetyl-N'-(naphthalen-2-yl)acetohydrazide, 7. Impurity G: 1,1,2,3-tetramethyl-1H-benz[e]indol-3-ium, 8. Impurity H: 4-[(2Z)-1,1-dimethyl-2-[(2E,4E,6E)-7-(1,1,3-trimethylbenzo[e]indol-3-io-2-yl)hepta-2,4,6-trienylidene]benzo[e]indol-3-yl]butane-1-sulfonate (methyl-indocyanine), is.

[0031] Impurity G was only evaluated in the compound of formula (II) since it cannot be enriched and the final product is in fact impurity H.

[0032] The structures of these impurities are shown in the diagram below: [ka] [ka]

[0033] Impurities E and F in particular have warning structures due to their potential genotoxicity, and the process of the present invention is able to completely degrade them (≦0.05%).

[0034] The utility of the compound of formula (I) is rooted in its application in diagnosis, particularly ophthalmic angiography. The compound of formula (I) is sold in the solid state in the form of a sterile, lyophilized powder containing 25 mg or 50 mg of the compound of formula (I) in the presence of 5% or less sodium iodide. The amount that can be administered for ophthalmic angiography should not exceed 0.1-0.3 mg per kg of body weight as a bolus injection. A 25 mg dose of the compound is dissolved in 5 ml of water for injection, and a 50 mg dose of the compound is dissolved in 10 ml, so that 1 ml of reconstituted injection solution contains 5 mg of the compound of formula (I). The total daily dose for adults should be kept below 5 mg per kg of body weight.

[0035] For injectable formulations, doses of up to 40 mg of a compound of formula (I) can be used in 2 ml of sterile water. Immediately following the injection of the compound of formula (I), a 5 ml bolus of saline should be administered.

[0036] The experimental part also describes the preparation of a lyophilized formulation of the compound of formula (I) obtained according to the process of the present invention, containing NaI, which can be used to prepare bottles for diagnostic use.

[0037] [Experimental part] [ Analytical methods used to determine the purity of the compound of formula (I) ] HPLC column: ODS Hypersil 4.6x250mm 5μm Column temperature: 40℃ Detector: UV254nm Step A: Ammonium formate 4.09 g / L, pH = 5.0 (using formic acid) Step B: Acetonitrile ·Mixed phase: 70:30 A:B ·Flow rate: 1.5ml / min ·Injection volume: 10μL ·Analysis time: 30 minutes Gradient [Table 1] ·White: mixed phase

[0038] [ Sample preparation (Method 1) ] Dissolve 40 mg in a 50 ml flask and bring to the correct volume with mixing phases. Sonicate for 5 minutes to ensure complete solubilization. Inject immediately as sample is not stable for more than 30 minutes .

[0039] [ Sample preparation (Method 2) ] Dissolve 40 mg in a 50 ml flask and make up to the correct volume with methanol. Sonicate for 5 minutes to ensure complete solubilization. Immediately inject. Inject immediately as sample is not stable for more than 30 minutes .

[0040] The use of methanol can better stabilize the compound of formula (I), [MH] + :752.5, i.e., the formation of a degradation impurity having a "-1" with respect to the product can be prevented. Inject every 1 to 10 minutes.

[0041] The assessment of potentially genotoxic impurities is obtained by injecting larger volumes of solution and quantification is performed as a limit assay against standards.

[0042] [ Analytical Methods Used to Determine the Purity of Compounds of Formula (I) (2) ] HPLC column: Polaris3 C18-A 150x4.6mm Column temperature: 20℃ Detector: UV254nm Step A: 2.3 g of ammonium acetate in 1000 ml, adjusted to pH 6.8 ± 0.05 with dilute acetic acid or ammonia. Step B: Acetonitrile Diluent: Methanol ·Flow rate: 1.5ml / min ·Injection volume: 10μL ·Analysis time: 34 minutes Autosampler temperature: 5℃ Gradient [Table 2] Sample solution: 1.5mg / ml in methanol. Inject immediately after preparing the solution.

[0043] This method is particularly suitable for assessing the presence of impurity H, although it is also capable of separating all other impurities mentioned.

[0044] The assessment of potentially genotoxic impurities is obtained by injecting larger volumes of solution and quantification is performed as a limit assay against standards.

[0045] This "Analysis Method 2" for ICG is compatible with LC / MS, so it can be used with a UPLC / MS (ESI) equipped with the same column. + ) and used directly in ESI + A detector (Waters SQD with cone voltage: 20 volts) is also used to analyze the reaction mixture and products.

[0046] This method was used to identify impurities G and H.

[0047] [Example 1] [ Preparation of 4-(1,1,2-trimethyl-1H-benzo[e]indolyl-3-yl)butane-1-sulfonate of formula (IV) ] [ka] 31.1 g of the compound of formula (II) (0.15 mol, 1 equivalent, commercially available) and 40.5 g of the compound of formula (III) (0.30 mol, 2 equivalents, commercially available) in 93 ml of xylene are charged into a 2 L reactor under a nitrogen stream. The suspension is stirred and heated to a temperature of approximately 130 °C for 24 hours. The suspension is cooled and acetone (200 ml) is added. The resulting solid is then filtered and dried in vacuo. 48.5 g of the desired compound is thus obtained, which corresponds to a yield of 94.5% (HPLC purity: 97-98%).

[0048] Alternatively, anisole can be used in place of xylene as a solvent in the same amount. Following the same protocol, the reaction is carried out at 140 °C, and the conversion to the product is complete in 6-8 hours. The yields and amounts obtained are similar to those obtained in xylene.

[0049] [Example 2] [ Preparation of Compounds of Formula (I) (One-Pot Synthesis) ] [ka] 20.0 g of the compound of formula (V) (0.07 mol, 1 equivalent, commercially available), 48.5 g of the compound of formula (IV) (prepared as described in Example 1, 0.14 mol, 2 equivalents), 23 g of sodium acetate (0.28 mol, 4 equivalents), and 180 ml of acetonitrile are charged into a 1 L reactor under a nitrogen stream. The suspension is stirred at 20-25°C, and 28.8 g of acetic anhydride (0.28 mol, 4 equivalents) is added dropwise after 5-10 minutes. The suspension is heated to a temperature of 45-50°C, and stirring is continued for approximately 2 hours. The reaction mixture is concentrated in vacuo while maintaining the temperature at 40-50°C. Then, atmospheric pressure is restored, 100 ml of isopropanol is added, and the reaction mixture is again concentrated in vacuo while maintaining the temperature at 40-50°C.

[0050] Water (180 ml) and isopropanol (320 ml) are then added, and the product is dissolved at 50-55°C. Isopropanol (100 ml) is then added, and the mixture is gradually cooled to 20-25°C. The mixture is filtered and washed with isopropanol. The desired wet compound is thus obtained (yield by weight loss: 46.3 g, 85.1% based on compound (V), HPLC purity 80-85%).

[0051] [Example 3] [ Purification of Compound of Formula (I) (without NaI) ] Charge 49 g of wet indocyanine green (prepared as described in Example 2, equivalent to 24.4 g dry) with isopropanol, 130 ml of isopropanol, and 77 ml of water into a 1-L flask. Heat to 50-55°C and stir until completely dissolved. Adjust the pH of the solution to 7.5-8.5 with 5% NaOH and cool to 40-45°C.

[0052] Isopropanol (48 ml) is added while maintaining the temperature at 40-45°C, then cooled gradually to 20-25°C and stirring continued for 1.5 hours before filtering and washing with isopropanol (2 x 48 ml). The powder is dried in vacuum at 60° C. for 40 hours. Yield: 20g (82.9%) HPLC purity: 99.5%; Impurity A: 0.40% Sodium iodide content: 0%.

[0053] [Example 4] [ Preparation of compounds of formula (I) according to the process described in US2019 / 0337896 ] The compound of formula (I) was prepared according to the process described in patent application US2019 / 0337896, starting from intermediates (II), (III), and (V) prepared as described in the previous examples, and the desired compound of formula (I) was obtained according to the synthetic procedures described in Examples 1, 2, 5, and 6 of US2019 / 0337896. The weight yield starting from the compound of formula (II) was 60%, while the process of the present invention was 86%.

[0054] The resulting product was then analyzed using the method of the present invention by preparing a solution of the sample using both Method 1 and Method 2. The purity was 93.27% as determined by HPLC. Impurity A: 0.68% Impurity C: 1.12% Impurities D, E, F: Unquantifiable Maximum unknown impurity: 2.42% (rt: 0.93) As reported in the aforementioned patent, when the purity is verified at a wavelength of 205 nm, the purity is 100%, which obviously does not correspond to reality.

[0055] [Example 5] [ Preparation of Compound of Formula (I) Using NaI (I Crystallization) ] 93.6 g of crude compound of formula (I) (46.3 g of theoretical dry compound, prepared as described in Example 2) and sodium iodide (1.39 g; 3% w / w) are suspended in 250 ml of isopropanol and 148 ml of water. The suspension is heated to a temperature of 55-60°C and stirred until completely dissolved. The pH is adjusted to 7.5-8.5 using 2.5% w / w sodium hydroxide solution. The solution is cooled to a temperature of 45-50°C, and after 15-30 minutes, 93 ml of isopropanol is added. The mixture is slowly cooled to a temperature of 20-25°C and stirring is continued for 30 minutes. The suspension is then brought to 35-40°C and stirred for approximately 1 hour, after which it is cooled again to 20-25°C after approximately 2 hours, and finally filtered at 20-30°C and washed with isopropanol.

[0056] The material was dried in vacuum at 50-80°C for 8-48 hours to give 35.89g of the desired product in 77.5% yield (based on the respective crude dried product charged). HPLC purity: 99.6%; Impurity A: 0.28% Iodide (potentiometric titration with a silver electrode): 1%.

[0057] If the known impurities are greater than 0.15% and the unknown impurities are greater than 0.1%, a second crystallization can be performed using less sodium iodide, which is essential to keep the amount of sodium iodide in the final product below 2.5% (Example 6).

[0058] [Example 6] [ Preparation of Compound of Formula (I) Using NaI (II Crystallization) ] A 1-liter reactor is charged with the wet compound of formula (I) obtained from the first crystallization (prepared as described in Example 5) (63.7%, equivalent to 35.9 g of the corresponding dry product on a weight loss basis), sodium iodide (0.54 g; 1.5% w / w), isopropanol (194 ml), and water (115 ml). After heating to 55-60°C and complete dissolution, the solution is filtered on cardboard and the filter is washed with water (7 ml) and then isopropanol (18 ml). The filtrate is brought to 55-60°C and, if necessary, the pH is adjusted to 7.5-8.5 with diluted NaOH. The filtrate is then cooled to 45-50°C, and after approximately 30 minutes, isopropanol (54 ml) is added.

[0059] The mixture is slowly cooled to 20-25°C, heated again to 35-40°C for about 1 hour, and then returned to 20-25°C after about 1 hour, with stirring continued for 30 minutes. The suspension is filtered and washed with isopropanol to obtain a wet product, which is dried in a vacuum at 50-80°C for 24-48 hours. Yield: 26.9g (75%). The purity of the product thus obtained is greater than 99.5% as determined using the HPLC method of the present invention. HPLC purity: 99.92% Impurities A, B, C, D, E and F: Not quantifiable (HPLC). Maximum unknown impurity: 0.084% (HPLC; rrt: 0.45). Sodium iodide (potentiometric titration per USP monograph): 0.9%. Residual isopropanol: 1597 ppm.

[0060] [Example 7] [ Preparation of a lyophilized formulation of the compound of formula (I) obtained according to the following process of the invention using NaI ] Indocyanine green (125 mg) obtained as described in Example 6 (containing ≦2.5% NaI) is dissolved in water (25 ml) and sonicated for 1 minute (5 mg / ml).

[0061] The above solution is filled into five different amber glass vials for use (approximately 5 ml of solution per vial). The vials are lyophilized using the following lyophilization conditions: Freeze dryer: Edwards MINIFAST 680 Temperature: -40°C at the start of freeze-drying; +5°C at the end of freeze-drying Pressure (vacuum): 6.6 at the start of freeze-drying * 10 2 mbar, at the end of freeze-drying 4.6 * 10 -2 mbar Freeze-drying time: 72 hours Amber glass vials were flushed with nitrogen and sealed upon completion of distillation.

[0062] [Example 8] [ Preparation of a lyophilized formulation of the compound of formula (I) obtained according to the following method of the invention without the use of NaI ] A lyophilized formulation is prepared as described in Example 7, but using the compound of formula (I) prepared as described in Example 3. The compound of formula (I) obtained without sodium iodide (sodium iodide = 0% by potentiometric titration with a silver electrode) is not soluble in water at 20-25°C at clinically useful concentrations of 5 mg / ml or 2.5 mg / ml when used directly as a dry, isolated powder after crystallization.

[0063] Surprisingly, the same lyophilized powder is soluble at concentrations of 2.5 mg / ml, 5 mg / ml, and even 10 mg / ml.

[0064] Solubility was assessed by filtering all the resulting solutions through a syringe filter with 0.45 μm holes, and it was observed that the filtration was fluid and no residue remained on the filter or in the vial from which the solution was removed.

[0065] While the molecule is likely soluble itself, for kinetic reasons it will not dissolve on a reasonable timescale, whereas freeze-dried powders have a large relative surface area in contact with water and therefore tend to dissolve completely and immediately, even without the need for sonication.

[0066] [Example 9] [ Stability Test ] The stability of the powder of compound of formula (I) obtained by the method described according to Example 6 was evaluated in the presence or absence of oxygen (i.e., air or nitrogen) without protection from light (i.e., packaged in a polyethylene bag or aluminium and polyethylene double bag according to the method described in the applicant's international patent application WO2013168186) and compared to the stability of a commercial product containing a higher amount of NaI (3.6% vs. 1.4%).

[0067] The powders produced according to the described method are significantly more stable, despite the low content of sodium iodide and the absence of nitrogen, and are generally characterized by a higher purity even at T 0 (zero).

[0068] The results are shown in Table 1. [Table 3]

[0069] [Example 10] [ (A) Preparation of 4-(1,1,2-trimethyl-1H-benzo[e]indolyl-3-yl)butane-1-sulfonate of formula (IV) with formation of impurity G in the presence of methanol ] The preparation was carried out as described in Example 1, but in the presence of 3.3% v / v methanol. LC / MS analysis of this reaction revealed [M] + It is emphasized that there is 0.55% of impurity G characterized by: 224.18.

[0070] [ (B) Confirmation of the structure of impurity G ] To confirm the structure of the previously hypothesized impurity G in Example 10A based on LC / MS analysis, impurity G (methyl benzindole) was synthesized by reacting the starting benzindole with methyl iodide according to a process known from the literature (Ind. Chem. Res, 2012, 51, 3630-3638). The product thus obtained had the expected mass spectrum, i.e., [M]+ :224, which coeluted with a peak of equal weight identified as Intermediate 1 in Example 10A, confirming its identity. H-NMR: Conforms to the structure.

[0071] [ (C) Preparation of Impurity H (Methylindocyanine) ] Impurity B (US 2895955, prepared according to the procedure reported in Example 3; 20.0 g), iodomethylbenzindole (14.24 g), sodium acetate (37.7 g), glacial acetic acid (23.6 ml), and acetonitrile (240 ml) are charged in the given order into a 500 ml flask. The suspension is heated to 45 / 50°C and stirring is continued for 6 hours. The reaction mass is cooled to 20 / 25°C, and the reaction is stirred at 20 / 25°C for another 40 hours. Glacial acetic acid (1 ml) is added, and the reaction mixture is heated to 45 / 50°C and stirring is continued for 5 hours. The reaction mixture is cooled to 20 / 25°C, and stirring is continued for another 64 hours. A portion of the solvent (approximately 130 ml) is distilled in vacuo at 30 / 50°C. Isopropanol (40 g) is added, and the solvent is distilled in vacuo at 30 / 50°C. Water (100 ml) and isopropanol (122 g) are added to the reaction residue. The suspension is heated to 50 / 55°C without complete dissolution being observed. The suspension is maintained at 50 / 55°C for 30 minutes. Isopropanol (40 ml) is added while maintaining the suspension at 50 / 55°C. After about 2 hours, it is cooled to 30 / 35°C and the suspension is stirred for 1 hour. The reaction mass is further heated to 40 / 45°C and the suspension is stirred for another 30 minutes. The reaction mass is cooled to 20 / 25°C after about 1 hour and stirred for 1 hour. The solid is filtered through a Buchner funnel and washed with isopropanol (2 x 20.0 g). The crude solid is dried at 55°C for 16 hours.

[0072] First purification: The crude solid (23.2 g), water (74.4 g) and isopropanol (98.4 g) are charged in the given order to a 500 ml flask. The suspension is heated to 60 / 65° C. without any observed dissolution. The suspension is stirred at 60 / 65° C. for 1 hour. Isopropanol (36.4 g) is added and stirred at 60 / 65° C. for 1 hour. The reaction mass is cooled to 20 / 25° C. and stirred for 5 hours. The solid is filtered on a Buchner funnel and washed with isopropanol (25 ml). The solid is dried in vacuo at 55° C. for 16 hours.

[0073] Second purification: The first dried purified product (16.9 g), water (68.3 g), and isopropanol (111.9 g) are charged in the specified order to a 250 ml flask and the suspension is heated to 60 / 65° C. The suspension is stirred at 60 / 65° C. for 4 hours. The suspension is cooled to 20 / 25° C. and stirred at 20 / 25° C. for 2 hours. The solid is filtered on a Buchner funnel and washed with isopropanol (20 ml). The solid is dried in vacuo at 50° C. for 20 hours.

[0074] Third purification: The second purified product (14.2 g) and isopropanol (60 g) are charged in the specified order into a 250 ml flask. The suspension is heated to 70 / 80°C (solvent reflux) and maintained for 1 hour. The reaction mass is cooled to 20 / 25°C and stirred for 5 hours. The solid is filtered through a Buchner funnel and washed with isopropanol (2 x 15 g). The solid is dried in a stove at 50°C for 6 hours, then at 60°C for another 8 hours. 1.57g dry solid, (HPLC) Purity: 93.07%, [MH] + :631.39(ESI + ).

[0075] Impurity H prepared in this way coelutes with the 8 m / z impurity seen in the "demo batch" mentioned in the description part.

Claims

1. 1. A method for measuring the purity of indocyanine green of formula (I), 1H-benz[e]indolium, 2-[7-[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-benz[e]indol-2-ylidene]-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulfobutyl)hydroxide, inner salt, sodium salt, comprising: 【Chemistry 1】 below: Column HPLC: Polaris3 C18-A 150 x 4.6 mm Column temperature: 20°C Detector: UV 254 nm Step A: 2.3 g of ammonium acetate in 1000 ml, adjusted to pH 6.8 ± 0.05 with dilute acetic acid or ammonia Step B: Acetonitrile Diluent: Methanol ・Flow rate: 1.5ml / min ・Injection volume: 10μL ・Analysis time: 34 minutes Autosampler temperature: 5°C Gradient Table 1 A purity measurement method comprising:

2. A composition comprising indocyanine green of formula (I), 1H-benz[e]indolium, 2-[7-[1,3-dihydro-1,1-dimethyl-3-(4-sulfobutyl)-2H-benz[e]indol-2-ylidene]-1,3,5-heptatrienyl]-1,1-dimethyl-3-(4-sulfobutyl)hydroxide, inner salt, sodium salt, having a total impurity content of 0.5% or less, a single impurity content of 0.10% or less, and a NaI content of 2.5% or less, as measured by the HPLC method of claim 1.

3. 10. A process for preparing the compound of formula (I) according to claim 2, comprising the steps of: a) reacting the compound 1,1,2-trimethyl-1h-benzo[e]indole of formula (II) with 1,4-butanesultone of formula (III), 【Chemistry 2】 【Transformation 3】 reacting in a high boiling solvent selected from anisole or xylene to give 4-(1,1,2-trimethyl-1H-benzo[e]indol-3-yl)butane-1-sulfonate of formula (IV) according to known methods; 【Chemistry 4】 b) reacting a compound of formula (IV) with a compound of formula (V), N-phenyl-N-((1E,3E,5E)-5-(phenylammonium)penta-1,3-dienyl hydrochloride; 【Transformation 5】 reacting in the presence of acetic anhydride, sodium acetate, using a dipolar aprotic solvent to give the final compound of formula (I) without isolating the intermediate; The process includes:

4. 4. The process according to claim 3, wherein the dipolar aprotic solvent in step b) is acetonitrile, and acetic anhydride and sodium acetate are equal to 4 equivalents relative to compound (IV).

5. 5. The process according to claim 3 or 4, wherein the compound of formula (I) in crude form obtained after step b) is purified by crystallization from an isopropanol / water mixture selected from 5.9 / 3.4 or 7.4 / 3.4 or 9.9 / 3.4, expressed in liters of volume per kg of crude compound of formula (I).

6. A composition comprising indocyanine green of formula (I) according to claim 2, obtainable by any one of claims 3 to 5.

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