DESC:FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
THE PATENT RULES, 2003

COMPLTE SPECIFICATION
(See section 10; rule 13)

NOVEL CRYSTALLINE FORM OF INDIGO CARMINE

SKVEN TECHNOLOGIES PRIVATE LIMITED
A company incorporated under the laws of India having their office at
Shop No. 17, Shanti Kunj CHS, Sector-4, Kharghar, Navi Mumbai, Raigarh, Maharashtra 410210, India.

The following specification particularly describes the invention and the manner in which it is to be performed.

TITLE OF THE INVENTION
Novel crystalline form of indigo carmine.

FIELD OF THE INVENTION
The present invention relates to a novel crystalline form of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine] characterized by a powder x-ray diffraction comprising diffraction peaks at 2? values of 5.06±0.2, 10.13±0.2, 18.3±0.2, 22.20±0.2, and further characterized by having two or more additional diffraction peaks at 2? values selected from 19.46±0.2, 25.98±0.2, 29.88±0.2, 30.59±0.2; designated as Form A and also covers its process of preparation.

BACKGROUND OF THE INVENTION
Indigo carmine is also known as Indigotindisulfonate sodium, indigotine or FD&C Blue #2, it is an organic salt derived from indigo by aromatic sulfonation & soluble in water. It is chemically designated as disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate. The chemical structure of Indigo carmine is as follows:

Indigotindisulfonate sodium, sold under the brand name Bludigo, is used as a diagnostic dye during surgical procedures. Indigo carmine is originally employed as a kidney function test, the main application of Indigo Carmine at present is localizing ureteral orifices during cystoscopy and ureteral catheterization.

U.S. Patent No. 10,927,258 B2 relates to an improved process for the preparation of Indigotindisulfonate sodium having purity greater than 99.0% and total impurities less than 1.0%. It also relates to novel crystalline Form I of Indigotindisulfonate sodium and process for its preparation.

U.S. Patent No.10,738,192 B2 relates to a novel process for the preparation of Indigo carmine having high purity 99.90%, the process comprising treating an indole compound with peroxide in the presence of a catalyst in an alcoholic solvent; further reacting the Indigo with sulfuric acid and adding an organic solvent then washing or purifying in an ester solvent to produce an indigodisulfonic acid. It further discloses a crystalline Form I of Indigotindisulfonate sodium wherein X-ray powder diffraction (XRPD) pattern having peaks at 2 theta values 5.8, 11.67, 15.37, 17.62, 20.31 and 26.54±0.2°.

However, the present invention surprisingly found the novel crystalline form of disodium[2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine] characterized by a powder x-ray diffraction comprising diffraction peaks at 2? values of 5.06±0.2, 10.13±0.2, 18.3±0.2, 22.20±0.2, and further characterized by having two or more additional diffraction peaks at 2? values selected from 19.46±0.2, 25.98±0.2, 29.88±0.2, 30.59±0.2; designated as Form A.

SUMMARY OF THE INVENTION
The present invention relates to a novel crystalline form of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine] characterized by a powder x-ray diffraction comprising diffraction peaks at 2? values of 5.06±0.2, 10.13±0.2, 18.3±0.2, 22.20±0.2, and further characterized by having two or more additional diffraction peaks at 2? values selected from 19.46±0.2, 25.98±0.2, 29.88±0.2, 30.59±0.2; designated as Form A and also covers its process of preparation.

More precisely, the main object the present invention relates to a process for preparing crystalline Form A of indigotindisulfonate sodium, said process comprising:
i. treating a 2-nitrobenzaldehyde of compound formula (2) with base to give a indigo of compound formula (3)
ii) reacting the indigo of compound formula (3) with sulfuric acid and adding an organic solvent then isolated in an ester solvent to obtain an crude indigodisulfonic acid of compound formula (4)
iii) purifying the crude indigodisulfonic acid of compound formula (4) by suspending in methanol and filtering the suspension and washing with methanol to get pure indigodisulfonic acid of compound formula (4)
iv) pure indigodisulfonic acid of compound formula (4) reacting with sodium hydroxide to get crude indigotindisulfonate sodium compound of formula (1)
v) purifying the crude indigotindisulfonate sodium compound of formula (1), by
a) dissolving the compound of formula (1) in dimethylsulfoxide (DMSO) and filter the compound through micron filter to remove the undissolved matters
b) precipitated the compound by addition of acetone
c) cooling the suspension to 10 - 15°C and
d) filtering the suspension and washing with acetone to get pure crystalline Form A of indigotindisulfonate sodium compound of formula (1).

In another embodiment of the present invention relates to a pharmaceutical composition comprising novel crystalline Form A of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine].

BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Crystalline Form A of Indigo Carmine by Powder X-ray diffraction (PXRD).

DETAILED DESCRIPTION OF THE INVENTION
The detailed description of the present invention described hereinafter.

The present invention relates to a novel crystalline form of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine] characterized by a powder x-ray diffraction comprising diffraction peaks at 2? values of 5.06±0.2, 10.13±0.2, 18.3±0.2, 22.20±0.2, and further characterized by having two or more additional diffraction peaks at 2? values selected from 19.46±0.2, 25.98±0.2, 29.88±0.2, 30.59±0.2; designated as Form A and also covers its process of preparation.

More precisely, the main object the present invention relates to a process for preparing crystalline Form A of indigotindisulfonate sodium, said process comprising:
i. treating a 2-nitrobenzaldehyde of compound formula (2) with base to give a indigo of compound formula (3)
ii) reacting the indigo of compound formula (3) with sulfuric acid and adding an organic solvent then isolated in an ester solvent to obtain an crude indigodisulfonic acid of compound formula (4)
iii) purifying the crude indigodisulfonic acid of compound formula (4) by suspending in methanol and filtering the suspension and washing with methanol to get pure indigodisulfonic acid of compound formula (4)
iv) pure indigodisulfonic acid of compound formula (4) reacting with sodium hydroxide to get crude indigotindisulfonate sodium compound of formula (1)
v) purifying the crude indigotindisulfonate sodium compound of formula (1), by
a) dissolving the compound of formula (1) in dimethylsulfoxide (DMSO) and filter the compound through micron filter to remove the undissolved matters
b) precipitated the compound by addition of acetone
c) cooling the suspension to 10 - 15°C and
d) filtering the suspension and washing with acetone to get pure crystalline Form A of indigotindisulfonate sodium compound of formula (1).

Further aspect of the present invention includes method for characterizing novel crystalline Form A of indigo carmine, said method comprising:
i) grinding the sample of indigo carmine into mortar and pestle and packed in the polymethyl methacrylate holder.
ii) the sample of indigo carmine kept in the goniometer and data generated by Powder X-ray diffraction (PXRD) having peak positions ±0.2 2? values.

In another embodiment of the present invention relates to a pharmaceutical composition comprising novel crystalline form of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine].

The route of synthesis for preparing a novel crystalline form of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate
[Indigo carmine] is schematically represented as follows:

The process for preparing a novel crystalline form of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine] is described as follows:

Example—1: Stage-1:
Preparation of Indigo from 2-Nitobenzaldehyde
2-Nitrobenzaldehyde (100g), water (500ml) acetone (1.0L) is cooled to 0-5°C. To this solution was slowly added aqueous Sodium hydroxide at 0-5°C. Maintain the reaction mass at 25-35°C till reaction completion. The reaction mass was filtered under vacuum and the solid obtained was washed with 500mL of demineralised water (DM) water and 200mL of methanol. The wet cake was heated in a mixture of 700mL acetone and 300mL methanol for 4- 5hrs at 55-60 °C. Then the reaction mixture was cooled to 25-30 °C and filtered under vacuum. The obtained solid was filtered and washed with a mixture of acetone and methanol (1:1). The wet solid was heated in a mixture of 700mL of acetone and 300mL of methanol for 4-5 hrs. at 55-60°C. Then the reaction mixture was cooled to 25-30 °C and filtered the solid under vacuum. The wet solid so obtained was stirred in l.0L of methanol for l-2hrs at 25-30 °C and filtered under vacuum at 25-30 °C. Dry the solid for 8-10hrs at 60-65 °C. To the obtained Solid, 10 volumes of dimethyl formamide was added and stirred for hr. at 100-110°C. The mixture was then cooled to 50-55 °C, filtered and washed the solid with 2 volumes of methanol and 1 volume of acetone. The solid so obtained was dried under vacuum at 60-65 °C to get Indigo.
Yield: 40-50g

Example: 2: Stage-2:
Preparation of Indigotindisulfonate Sodium
Add concentrated Sulphuric acid (380 gm.) and heat to 95-105° C. Slowly add Indigo (100 gm.) and maintain at 95-105° C. for 2 hrs. After completion of reaction cool the reaction mass to room temperature and add Acetone (5 L). Further cool the reaction mass to 0-10°C and add Ethyl acetate (5 L). Stir and filter. Wash with Acetone: Ethyl acetate (1:1) to get Indigo disulfonic acid. Dry the material under vacuum at 55-60°C. Purify the material in methanol twice to get pure acid.

Purification of Indigotindisulfonate Sodium: To 100g Acid added methanol (1.0L) and stir at 25-35°C for 1hr. Filter the mass and wash with methanol. Dry the material under vacuum at 50-55°C to get pure acid.
Yield: 90-100g
The obtained pure Indigo disulfonic acid compound (100 gm.) was dissolved in water (400 ml, 4 vol) and adjusted pH 9 to12 by using sodium hydroxide solution( in water. Add Methanol (500 ml, 5 vol) and stir. Cool the reaction mass to 5-10°C and maintain for 1hr. Filter and wash with methanol: water mixture to get Indigotindisulfonate Sodium, which is dried under vacuum at 55-60°C for 8hrs.
Yield: 50-60g.

Example-3: Stage-3
Preparation of Form A of Indigotindisulfonate Sodium
Indigotindisulfonate Sodium (10g) is added to dimethylsulfoxide (DMSO) (25V) and stir at 25-30°c. Filter the reaction mass to remove any undissolved solids. Take the filtrate and Add Acetone (55V) at 25-30°C. Maintain for 1hr at 25-30°C. Cool the mass to 10-15°C and maintain for 1hr. Filter the reaction mass and wash with acetone (5V) .slurry wash the material in acetone. Dry the material under vacuum at 60-65°C for 15hrs to get Form A of Indigotindisulfonate Sodium.
Characterization of crystalline Form A of Indigo Carmine by Powder X-ray diffraction (PXRD)
Sample Preparation Method:
The method for characterizing novel crystalline Form A of indigo carmine, said method comprising:
i) grinding the sample of indigo carmine into mortar and pestle and packed in the polymethyl methacrylate holder
ii) the sample of indigo carmine kept in the goniometer and data generated by Powder X-ray diffraction (PXRD) diffractogram having peak positions ±0.2 2? values.

The XRPD data of the novel crystalline form of Indigo Carmine obtained is presented in the Table 1.

Sr. 2? value (º) d value (Aº) Relative intensity %
1 5.06 17.44 100
2 10.13 8.73 36.7
3 12.14 7.28 2.1
4 15.18 5.83 4.6
5 16.50 5.37 0.9
6 18.30 4.84 64.2
7 19.46 4.56 23.9
8 20.25 4.38 6.8
9 22.20 4.00 47.9
10 24.42 3.64 1.3
11 24.99 3.56 0.7
12 25.98 3.43 15.4
13 26.60 3.35 15.3
14 28.88 3.09 1.7
15 29.88 2.99 12
16 30.59 2.92 14.2
17 31.30 2.86 5.1
18 32.97 2.71 3.4
19 34.18 2.62 3.8
20 34.84 2.57 0.8
21 36.61 2.45 8.7
22 37.34 2.41 3.5
23 38.09 2.36 2.2
24 38.68 2.33 2.5
25 39.66 2.27 1.5

Table 1 XRPD data of the novel crystalline form of Indigo Carmine
WE CLAIM,
1. A novel crystalline form of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine] characterized by a powder x-ray diffraction comprising diffraction peaks at 2? values of 5.06±0.2, 10.13±0.2, 18.3±0.2, 22.20±0.2, and further characterized by having two or more additional diffraction peaks at 2? values selected from 19.46±0.2, 25.98±0.2, 29.88±0.2, 30.59±0.2; designated as Form A.

2. A novel crystalline form of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate of claim 1, having an powder x-ray diffraction pattern in accordance with Figure 1.

3. A process for preparing crystalline Form A of indigotindisulfonate sodium, said process comprising:
i. treating a 2-nitrobenzaldehyde of compound formula (2) with base to give a indigo of compound formula (3)
ii) reacting the indigo of compound formula (3) with sulfuric acid and adding an organic solvent then isolated in an ester solvent to obtain an crude indigodisulfonic acid of compound formula (4)
iii) purifying the crude indigodisulfonic acid of compound formula (4) by suspending in methanol and filtering the suspension and washing with methanol to get pure indigodisulfonic acid of compound formula (4)
iv) pure indigodisulfonic acid of compound formula (4) reacting with sodium hydroxide to get crude indigotindisulfonate sodium compound of formula (1)
v) purifying the crude indigotindisulfonate sodium compound of formula (1), by
a) dissolving the compound of formula (1) in dimethylsulfoxide (DMSO) and filter the compound through micron filter to remove the undissolved matters
b) precipitated the compound by addition of acetone
c) cooling the suspension to 10 - 15°C and
d) filtering the suspension and washing with acetone to get pure crystalline Form A of indigotindisulfonate sodium compound of formula (1).
4. The process of preparing Form A of indigotindisulfonate sodium compound of formula (1), wherein said purification of crude indigotindisulfonate sodium compound of formula(1) performed by
a) dissolving the compound of formula (1) in dimethylsulfoxide (DMSO) and filter the compound through micron filter to remove the undissolved matters
b) precipitated the compound by addition of acetone
c) cooling the suspension to 10 - 15°C and
d) filtering the suspension and washing with acetone to get pure crystalline Form A of indigotindisulfonate sodium compound of formula (1).

5. The novel crystalline form of Indigo carmine as claimed in claim 1, wherein the crystalline form is used to prepare a pharmaceutical composition comprising novel crystalline Form A of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine].

Dated this 16th June 2024

Agent for the applicant,
Vijaykumar Shivpuje, IN/PA 1096,
Sri Kripa, Akshay Nagar,
Old Ausa Road, Latur, 413531,
Maharashtra, India.

NOVEL CRYSTALLINE FORM OF INDIGO CARMINE

ABSTRACT
The present invention relates to a novel crystalline form of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine] characterized by a powder x-ray diffraction comprising diffraction peaks at 2? values of 5.06±0.2, 10.13±0.2, 18.3±0.2, 22.20±0.2, and further characterized by having two or more additional diffraction peaks at 2? values selected from 19.46±0.2, 25.98±0.2, 29.88±0.2, 30.59±0.2; designated as Form A and also covers its process of preparation.

,CLAIMS:WE CLAIM,
1. A novel crystalline form of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine] characterized by a powder x-ray diffraction comprising diffraction peaks at 2? values of 5.06±0.2, 10.13±0.2, 18.3±0.2, 22.20±0.2, and further characterized by having two or more additional diffraction peaks at 2? values selected from 19.46±0.2, 25.98±0.2, 29.88±0.2, 30.59±0.2; designated as Form A.

2. A novel crystalline form of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate of claim 1, having an powder x-ray diffraction pattern in accordance with Figure 1.

3. A process for preparing crystalline Form A of indigotindisulfonate sodium, said process comprising:
i. treating a 2-nitrobenzaldehyde of compound formula (2) with base to give a indigo of compound formula (3)
ii) reacting the indigo of compound formula (3) with sulfuric acid and adding an organic solvent then isolated in an ester solvent to obtain an crude indigodisulfonic acid of compound formula (4)
iii) purifying the crude indigodisulfonic acid of compound formula (4) by suspending in methanol and filtering the suspension and washing with methanol to get pure indigodisulfonic acid of compound formula (4)
iv) pure indigodisulfonic acid of compound formula (4) reacting with sodium hydroxide to get crude indigotindisulfonate sodium compound of formula (1)
v) purifying the crude indigotindisulfonate sodium compound of formula (1), by
a) dissolving the compound of formula (1) in dimethylsulfoxide (DMSO) and filter the compound through micron filter to remove the undissolved matters
b) precipitated the compound by addition of acetone
c) cooling the suspension to 10 - 15°C and
d) filtering the suspension and washing with acetone to get pure crystalline Form A of indigotindisulfonate sodium compound of formula (1).
4. The process of preparing Form A of indigotindisulfonate sodium compound of formula (1), wherein said purification of crude indigotindisulfonate sodium compound of formula(1) performed by
a) dissolving the compound of formula (1) in dimethylsulfoxide (DMSO) and filter the compound through micron filter to remove the undissolved matters
b) precipitated the compound by addition of acetone
c) cooling the suspension to 10 - 15°C and
d) filtering the suspension and washing with acetone to get pure crystalline Form A of indigotindisulfonate sodium compound of formula (1).

5. The novel crystalline form of Indigo carmine as claimed in claim 1, wherein the crystalline form is used to prepare a pharmaceutical composition comprising novel crystalline Form A of disodium [2(2')E]-3,3'-dioxo-1,1',3,3'-tetrahydro[2,2'-biindolylidene]-5,5'-disulfonate [Indigo carmine].