CN118239857A - Preparation method of iodixanol impurity I - Google Patents

Abstract

The invention discloses a preparation method of iodixanol impurity I, which comprises the steps of reacting a compound 3 with a compound 6 in a solvent system under an alkaline condition, and obtaining a target product compound 7, namely iodixanol impurity I through post-treatment. The purity of the iodixanol impurity I prepared by the method is up to more than 97%, and the iodixanol impurity I can be used as a reference substance for detecting related substances of iodixanol and used for quality control of iodixanol and related preparations thereof.

Description

Preparation method of iodixanol impurity I

Technical Field

The invention relates to the technical field of chemical pharmacy, in particular to a preparation method of iodixanol impurity I.

Background

Iodine contrast agents are commonly used for X-ray imaging examinations, and are relatively low in toxicity compared with other interventional drugs, so that the iodine contrast agents have basically occupied the fields of enhanced CT, angiography and the like. For the structural analysis of the product market, the most domestic iodine contrast agents are iodixanol, iohexol, ioversol, iopromide and iopamidol. Compared with other single products, iodixanol has the advantage of small side effect, so the market demand continuously rises, and the industry development prospect is good.

Iodixanol impurity I is a byproduct generated in the iodixanol synthesis process, and the quality limit of the impurity is clearly described in USP and EP pharmacopoeias, so that the importance of the impurity in iodixanol quality research can be seen.

Iodixanol impurity I,5- (N- (3, 5-bis ((2, 3-dihydroxypropyl) carbamoyl) -2,4, 6-triiodophenyl) acetamido) -2-hydroxypropyl) acetamido) -N1- (3- (2, 3-dihydroxypropoxy) -2-hydroxypropyl) -N3- (2, 3-dihydroxypropyl) -2,4, 6-triiodoisophthalamide.

The molecular formula: c ₃₈H₅₀I₆N₆O₁₇

Molecular weight: 1624.3

Structural formula:

at present, the impurity has no disclosed synthetic method, most of the impurities are used for quality research by purchasing a first-level reference substance, but the custom-made period is long, the price is high (10 mg and 17 ten thousand at present), and the custom-made failure rate is high due to the complex structure, so that the impurity is extremely difficult to purchase.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a preparation method of iodixanol impurity I.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

a method for preparing iodixanol impurity I, comprising the following steps:

in a solvent system, the compound 3 and the compound 6 react under alkaline conditions, and the target product compound 7, namely iodixanol impurity I, is obtained after post-treatment:

further, the solvent is selected from ethylene glycol monomethyl ether, and the base is selected from sodium hydroxide; the pH is controlled between 11 and 12 in the reaction process, the reaction temperature is 20 ℃, and the reaction time is 50 hours.

Further, the post-treatment is to adjust the pH value of the solution obtained by the reaction to be neutral by acid, decompress and desolventize the solution, dissolve the solution in water, pass through macroporous resin, elute and purify the solution by water, decompress and concentrate the solution to collect the solid, namely iodixanol impurity I.

Still further, the macroporous resin is XAD-18 macroporous resin; the concentration and collection stage is carried out under reduced pressure at 65 ℃.

Further, the compound 3 is prepared by the following steps:

1) Compound 1 reacts with compound 8 in tetrahydrofuran or chloroform system to produce compound 2;

2) In the presence of an acid binding agent, the compound 2 reacts with the compound 9 in a dioxane or ethylene glycol monomethyl ether system to generate a compound 3:

further, step 2) uses triethylamine as an acid-binding agent.

Further, the compound 3 is prepared by the following steps:

1) Dissolving the compound 1 in tetrahydrofuran or chloroform, and cooling to 0 ℃ for standby; dissolving the compound 8 in isopropanol or ethanol, and slowly and uniformly dripping the obtained solution into tetrahydrofuran or chloroform solution of the compound 1; after the dripping is finished, heating to 10 ℃, keeping the reaction for 2 hours, filtering the solution obtained by the reaction after the reaction is finished, leaching a filter cake by using a 50v/v% tetrahydrofuran water solution or a 50v/v% chloroform water solution at 10 ℃, and drying in vacuum at 50 ℃ to obtain a compound 2;

2) Adding the compound 2 into dioxane or ethylene glycol monomethyl ether, heating to 30 ℃, stirring and dissolving, dripping the compound 9, adding triethylamine as an acid binding agent after dripping, heating to 45 ℃, keeping the reaction for 4 hours, filtering the solution obtained after the reaction is finished, adding water into a filter cake, heating to 90 ℃ for dissolving, naturally cooling to 30 ℃, stirring and crystallizing for 12 hours, filtering, leaching the filter cake with water, and drying in vacuum at 65 ℃ to obtain the compound 3.

Further, the compound 6 is prepared by the following steps:

1) Reacting compound 4 with compound 10 to form compound 5;

2) In the presence of basic substances, compound 5 reacts with compound 11 in an N, N-dimethylformamide or an N, N-dimethylacetamide system, and then compound 12 is brominated and hydrolyzed to obtain compound 6:

Still further, the alkaline substance in step 2) is potassium carbonate or sodium carbonate.

Further, the compound 6 is prepared by the following steps:

1) Reflux-reacting the compound 4 and the compound 10 for 4 hours at the temperature of 81 ℃, and concentrating the mixture to dryness at the temperature of 60 ℃ after the reaction is finished to obtain a compound 5;

2) Stirring and dissolving the compound 5 and N, N-dimethylformamide or N, N-dimethylacetamide at 30 ℃, adding potassium carbonate or sodium carbonate, dropwise adding the compound 11 under stirring, keeping the temperature of 30 ℃ for 16 hours, then dropwise adding the compound 11, and keeping the temperature of 30 ℃ for 24 hours; after the reaction is finished, regulating the pH of the system to be neutral by using acid, concentrating to be dry at 80 ℃ under reduced pressure, adding methanol, dissolving in a reflux state, dripping into water at a constant speed, maintaining the temperature at 0-10 ℃ after dripping, stirring and crystallizing for 8 hours, filtering, leaching a filter cake by using water, adding water into the filter cake, cooling to 5 ℃, slowly dripping the aqueous solution of the compound 12 into the filter cake, continuously stirring and reacting at 5 ℃ after dripping for 2 hours, heating to 70 ℃ for reacting for 2 hours, regulating the pH to be neutral by using alkali, and concentrating to be dry at 65 ℃ under reduced pressure; adding water, stirring, passing through XAD-1600N macroporous resin, eluting with water, purifying, concentrating under reduced pressure at 65deg.C, and collecting to obtain compound 6.

Description of the compounds according to the invention:

compound 1:

5-acetamido-2,4,6-triiodoisophthaloyl dichloride (5-acetamido-2, 4,6-triiodoisophthaloyl dichloride); molecular weight 637.8;

Compound 2:

3-acetamido-5- ((2, 3-dihydroxypropyl) carbamoyl) -2,4,6-triiodobenzoyl chloride (3-acetamido-5- ((2, 3-dihydroxypropyl) carbamoyl) -2,4,6-triiodobenzoyl chloride); molecular weight 692.4;

compound 3:

5-acetamido-N1-(3-(2,3-dihydroxypropoxy)-2-hydroxypropyl)-N3-(2,3-dihydrox ypropyl)-2,4,6-triiodoisophthalamide(5- acetamido-N1- (3- (2, 3-dihydroxypropoxy) -2-hydroxypropyl) -N3- (2, 3-dihydroxypropyl) -2,4, 6-triiodoisophthalamide; molecular weight 821.1

Compound 4:

5-acetamido-N1, N3-bis (2, 3-dihydroxypropyl) -2,4,6-triiodoisophthalamide (5-acetamido-N1, N3-bis (2, 3-dihydroxypropyl) -2,4,6-triiodo isophthalamide); molecular weight 747.1;

compound 5:

5-acetamido-N1, N3-bis ((2, 2-dimethyl-1, 3-dioxolan-4-yl) methyl) -2,4,6-triiodoiso phthalamide (5-acetamido-N1, N3-bis ((2, 2-dimethyl-1, 3-dioxolan-4-yl) methyl) -2,4, 6-triiodoisophthalamide); molecular weight 827.2;

Compound 6:

5-(N-(3-bromo-2-hydroxypropyl)acetamido)-N1,N3-bis(2,3-dihydroxypropyl)-2,4,6-triiodoisophthalamide(5-(N-(3- Bromo-2-hydroxypropyl) acetamido) -N1, N3-bis (2, 3-dihydroxypropyl) -2,4, 6-triiodo m-benzenedicarboxamide; molecular weight 884.0;

Compound 7:

6-(N-(3-(N-(3,5-bis((2,3-dihydroxypropyl)carbamoyl)-2,4,6-triiodophenyl)aceta mido)-2-hydroxypropyl)acetamido)-N1-(3-(2,3-dihydroxypropoxy)-2-hydroxypropyl)-N3-(2,3-dihydroxypropyl)-2,4,6-triiodoisophthalamide(5-(N-(3-(N-(3,5- Bis ((2, 3-dihydroxypropyl) carbamoyl) -2,4, 6-triiodophenyl) acetamido) -2-hydroxypropyl) acetamido) -N1- (3- (2, 3-dihydroxypropoxy) -2-hydroxypropyl) -N3- (2, 3-dihydroxypropyl) -2,4, 6-triiodoisophthalamide; molecular weight 1624.3;

Compound 8:

3-Amino-1,2-propanediol (3-Amino-1, 2-propanediol); molecular weight 91.1;

Compound 9:

3- (3-amino-2-hydroxypropoxy) propane-1,2-diol (3- (3-amino-2-hydroxypropoxy) propane-1, 2-diol); molecular weight 165.2;

Compound 10:

2,2-dimethoxypropane (2, 2-dimethoxypropane); molecular weight 104.1;

compound 11:

3-bromoprop-1-ene (3-bromoprop-1-ene); molecular weight 121.0;

Compound 12:

bromine (Bromine); molecular weight 159.8;

Compound 13:

sodium hydroxide; molecular weight 40.0.

The invention has the beneficial effects that:

The purity of the iodixanol impurity I prepared by the method is up to more than 97%, the impurity is overlapped with a main peak in the iodixanol and is not easy to detect, and the iodixanol impurity I can be used as a reference substance for detecting related substances of the iodixanol and is applied to quality control of the iodixanol and related preparations thereof. The prior art has no related preparation method of the impurity, the invention fills the blank of the preparation method of the impurity, and overcomes the difficulty of synthesizing the impurity.

Drawings

FIG. 1 is a mass spectrum of compound 3 prepared in example 1.

FIG. 2 is a mass spectrum of compound 6 prepared in example 2.

FIG. 3 is a chart showing the infrared absorption spectrum of iodixanol impurity I, compound 7 prepared in example 3.

FIG. 4 is a graph showing the ultraviolet absorption spectrum of iodixanol impurity I, which is compound 7 prepared in example 3.

FIG. 5 is a ¹ H-NMR spectrum of compound 7 prepared in example 3, i.e., iodixanol impurity I.

FIG. 6 is a ¹³ C-NMR spectrum of iodixanol impurity I as compound 7 prepared in example 3.

FIG. 7 is a DEPT135℃spectrum of compound 7, iodixanol impurity I, prepared in example 3.

Fig. 8 is an HMBC spectrum of iodixanol impurity I, compound 7 prepared in example 3.

FIG. 9 is a HSQC spectrum of iodixanol impurity I, compound 7 prepared in example 3.

FIG. 10 is a ¹H-¹ H COSY spectrum of the iodixanol impurity I as compound 7 prepared in example 3.

FIG. 11 is a mass spectrum of compound 7 prepared in example 3, i.e., iodixanol impurity I +ESI.

FIG. 12 is a-ESI mass spectrum of compound 7, i.e., iodixanol impurity I, prepared in example 3.

FIG. 13 is an HPLC chart of iodixanol impurity I as compound 7 prepared in example 3.

Detailed Description

The invention will be further explained with reference to examples and figures. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.

Example 1: compound 3: synthesis of 5-acetamido-N1- (3- (2, 3-dihydroxypropoxy) -2-hydroxypropyl) -N3- (2, 3-dihydroxypropyl) -2,4, 6-triiodo-isophthalamide

350Ml of tetrahydrofuran or chloroform (tetrahydrofuran is adopted in the embodiment) and 5-acetamido-2, 4, 6-triiodo isophthaloyl dichloride (compound 1, 200g,314 mmol) are put into a 500ml four-port reaction bottle, stirred and dissolved at room temperature, and then cooled to 0 ℃ for later use; 3-amino-1, 2-propanediol (compound 8, 57.2g, 6278 mmol) was weighed, dissolved in 100ml of isopropanol or ethanol (isopropanol was used in this example), stirred and mixed uniformly, and transferred to a constant pressure dropping funnel, and an isopropanol or ethanol solution of 3-amino-1, 2-propanediol (isopropanol solution of 3-amino-1, 2-propanediol was slowly and uniformly dropped into a tetrahydrofuran or chloroform solution of compound 1 (tetrahydrofuran solution of compound 1 was used in this example) for no less than 6 hours; after the completion of the dropping, the temperature was raised to 10℃and the reaction was kept for 2 hours, then the mixture was filtered with a Buchner funnel, and the cake was rinsed with 100ml of a 50v/v% aqueous tetrahydrofuran solution or a 50v/v% aqueous chloroform solution at 10℃in this example (50 v/v% aqueous tetrahydrofuran solution was used), and then dried in a vacuum oven at 50℃to give 195g of 3-acetamido-5- ((2, 3-dihydroxypropyl) carbamoyl) -2,4, 6-triiodobenzoyl chloride (Compound 2).

3-Acetamido-5- ((2, 3-dihydroxypropyl) carbamoyl) -2,4, 6-triiodobenzoyl chloride (compound 2, 195g,282 mmol) was put into a 1000ml four-port reaction flask, 680ml dioxane or ethylene glycol monomethyl ether (dioxane is adopted in this example) was added, the temperature was raised to 30 ℃ and stirred to dissolve, 3- (3-amino-2-hydroxypropoxy) propane-1, 2-diol (compound 9, 48.9g, 256 mmol) was added dropwise, about 1 hour was consumed, 5g triethylamine was added as an acid-binding agent after the dropwise addition, the temperature was raised to 45 ℃ and the reaction was maintained for 4 hours, filtering with a Buchner funnel after the reaction, putting the filter cake into a 2000ml four-port reaction bottle, adding 1200ml deionized water, heating to 90 ℃ for dissolution, naturally cooling to 30 ℃, stirring for crystallization for 12 hours, filtering with the Buchner funnel, leaching the filter cake with 100ml deionized water, and drying in a vacuum drying oven at 65 ℃ to obtain 192g of 5-acetamido-N1- (3- (2, 3-dihydroxypropoxy) -2-hydroxypropyl) -N3- (2, 3-dihydroxypropyl) -2,4, 6-triiodo-isophthalamide (compound 3), wherein the mass spectrum of the compound 3 is shown in figure 1.

Example 2: compound 6: synthesis of 5- (N- (3-bromo-2-hydroxypropyl) acetamido) -N1, N3-bis (2, 3-dihydroxypropyl) -2,4, 6-triiodo-isophthalamide

5-Acetamido-N1, N3-bis (2, 3-dihydroxypropyl) -2,4, 6-triiodo-isophthalamide (compound 4, 200g,268 mmol) was charged into a 500ml four-port reaction flask, 2-dimethoxypropane (compound 10, 200g,1921 mmol) was continuously charged, reflux reaction was carried out at a temperature of 81℃for 4 hours, and after completion of the reaction, the reaction was concentrated to dryness under reduced pressure at 60℃to obtain 221.4-acetamido-N1, N3-bis ((2, 2-dimethyl-1, 3-dioxolan-4-yl) methyl) -2,4, 6-triiodo-isophthalamide (compound 5) 221.4g.

5-Acetamido-N1, N3-bis ((2, 2-dimethyl-1, 3-dioxolan-4-yl) methyl) -2,4, 6-triiodoisophthalamide (compound 5, 221g,267 mmol) was put into a 500ml single-port bottle, 300ml of N, N-Dimethylformamide (DMF) or N, N-Dimethylacetamide (DMAC) (DMF was used in this example) was added, and stirred and dissolved at 30 ℃, potassium carbonate (55.4 g,400.5 mmol) or sodium carbonate (potassium carbonate was used in this example) was added, 3-bromoprop-1-ene (compound 11, 26.7g,221 mmol) was added dropwise thereto, and after the reaction was continued at 30℃for 16 hours, 3-bromoprop-1-ene (compound 11,8.9g,74 mmol) was added dropwise thereto, and the reaction was continued at 30℃for 24 hours; after the reaction is finished, regulating the pH of the system to be neutral by glacial acetic acid, concentrating to be dry at 80 ℃, adding 150ml of methanol, dissolving in a reflux state, dripping into 1500ml of deionized water at a constant speed, maintaining the temperature at 0-10 ℃ after dripping, stirring and crystallizing for 8 hours at 10 ℃ in the embodiment, filtering by using a Buchner funnel, leaching filter cakes by using 100ml of deionized water, putting all filter cakes back into a 5000ml four-port reaction bottle, adding 2700ml of deionized water, cooling to 5 ℃, dissolving bromine (compound 12, 50g,313 mmol) into 1500ml of deionized water, slowly dripping into a reaction solution at 5 ℃ for not less than 4 hours, continuously stirring and reacting for 2 hours at 5 ℃, heating to 70 ℃ for reacting for 2 hours, regulating the pH to be neutral by using liquid alkali (concentration of 30 wt%) and concentrating to be dry at 65 ℃ under reduced pressure; the material was further stirred with water and placed in a 5L volume macroporous resin column, resin model XAD-1600N, eluting with deionized water, concentrating under reduced pressure at 65deg.C, collecting to give 117g of 5- (N- (3-bromo-2-hydroxypropyl) -acetamido) -N1, N3-bis (2, 3-dihydroxypropyl) -2,4, 6-triiodo-isophthalamide (compound 6), mass spectrum of compound 6 is shown in FIG. 2.

Example 3: synthesis of iodixanol impurity I

300Ml of ethylene glycol monomethyl ether was charged into a 500ml four-port reaction flask, 5-acetamido-N1- (3- (2, 3-dihydroxypropoxy) -2-hydroxypropyl) -N3- (2, 3-dihydroxypropyl) -2,4, 6-triiodoisophthalamide (compound 3, 50g,61 mmol), 5- (N- (3-bromo-2-hydroxypropyl) acetamido) -N1, N3-bis (2, 3-dihydroxypropyl) -2,4, 6-triiodoisophthalamide (compound 6, 54g,61 mmol), sodium hydroxide (compound 13,4.1g,103 mmol) were weighed and charged into the reaction flask together, the temperature was controlled at 20 ℃, the pH was controlled at 11-12, and the reaction was maintained for 50 hours, during which time the required liquid alkali to maintain pH stability was 30wt% concentration, and the required hydrochloric acid was 36.5wt% concentration; after the reaction, the pH of the reaction mixture was adjusted to be neutral by hydrochloric acid (concentration: 36.5 wt%), the reaction mixture was placed in a rotary evaporator at 70 ℃ and the solvent was removed under reduced pressure, the reaction mixture was redissolved in 150g of deionized water, the reaction mixture was placed in a 1.4L volume macroporous resin column, the resin model was XAD-18, the reaction mixture was eluted and purified by deionized water, the reaction mixture was concentrated and collected under reduced pressure at 65 ℃ to scrape out a solid, and 5- (N- (3, 5-bis ((2, 3-dihydroxypropyl) carbamoyl) -2,4, 6-triiodophenyl) acetamido) -2-hydroxypropyl) acetamido) -N1- (3- (2, 3-dihydroxypropoxy) -2-hydroxypropyl) -N3- (2, 3-dihydroxypropyl) -2,4, 6-triiodoisophthalamide (compound 7) was 46g, i.e., iodixanol impurity I was obtained, the purity was up to 97.374% (FIG. 13) by HPLC analysis, and the structure of compound 7 was confirmed to be shown in FIG. 3-FIG. 12.

Claims (10)

1. The preparation method of iodixanol impurity I is characterized by comprising the following steps:

in a solvent system, the compound 3 and the compound 6 react under alkaline conditions, and the target product compound 7, namely iodixanol impurity I, is obtained after post-treatment:

2. The method for preparing iodixanol impurity I according to claim 1, characterized in that the solvent is selected from ethylene glycol monomethyl ether and the base is selected from sodium hydroxide; the pH is controlled between 11 and 12 in the reaction process, the reaction temperature is 20 ℃, and the reaction time is 50 hours.

3. The method for preparing iodixanol impurity I according to claim 1, wherein the post-treatment is to adjust the pH of the solution obtained by the reaction to neutral with acid, dissolve the solution in water after decompression and desolventization, pass through macroporous resin, elute and purify with water, and decompress and concentrate and collect the phase, thus obtaining the solid, i.e. iodixanol impurity I.

4. A process for the preparation of iodixanol impurity I according to claim 3, characterized in that said macroporous resin is XAD-18 macroporous resin; the concentration and collection stage is carried out under reduced pressure at 65 ℃.

5. The method for preparing iodixanol impurity I according to claim 1, wherein the compound 3 is prepared by the following steps:

1) Compound 1 reacts with compound 8 in tetrahydrofuran or chloroform system to produce compound 2;

2) In the presence of an acid binding agent, the compound 2 reacts with the compound 9 in a dioxane or ethylene glycol monomethyl ether system to generate a compound 3:

6. The method for preparing iodixanol impurity I according to claim 5, wherein step 2) uses triethylamine as an acid-binding agent.

7. The method for preparing iodixanol impurity I according to claim 6, wherein the compound 3 is prepared by the following steps:

1) Dissolving the compound 1 in tetrahydrofuran or chloroform, and cooling to 0 ℃ for standby; dissolving the compound 8 in isopropanol or ethanol, and slowly and uniformly dripping the obtained solution into tetrahydrofuran or chloroform solution of the compound 1; after the dripping is finished, heating to 10 ℃, keeping the reaction for 2 hours, filtering the solution obtained by the reaction after the reaction is finished, leaching a filter cake by using a 50v/v% tetrahydrofuran water solution or a 50v/v% chloroform water solution at 10 ℃, and drying in vacuum at 50 ℃ to obtain a compound 2;

2) Adding the compound 2 into dioxane or ethylene glycol monomethyl ether, heating to 30 ℃, stirring and dissolving, dripping the compound 9, adding triethylamine as an acid binding agent after dripping, heating to 45 ℃, keeping the reaction for 4 hours, filtering the solution obtained after the reaction is finished, adding water into a filter cake, heating to 90 ℃ for dissolving, naturally cooling to 30 ℃, stirring and crystallizing for 12 hours, filtering, leaching the filter cake with water, and drying in vacuum at 65 ℃ to obtain the compound 3.

8. The method for preparing iodixanol impurity I according to claim 1, wherein the compound 6 is prepared by the following steps:

1) Reacting compound 4 with compound 10 to form compound 5;

2) In the presence of basic substances, compound 5 reacts with compound 11 in an N, N-dimethylformamide or an N, N-dimethylacetamide system, and then compound 12 is brominated and hydrolyzed to obtain compound 6:

9. the method for preparing iodixanol impurity I according to claim 8, wherein the alkaline substance in step 2) is potassium carbonate or sodium carbonate.

10. The method for preparing iodixanol impurity I according to claim 9, characterized in that the compound 6 is prepared by the following steps:

1) Reflux-reacting the compound 4 and the compound 10 for 4 hours at the temperature of 81 ℃, and concentrating the mixture to dryness at the temperature of 60 ℃ after the reaction is finished to obtain a compound 5;

2) Stirring and dissolving the compound 5 and N, N-dimethylformamide or N, N-dimethylacetamide at 30 ℃, adding potassium carbonate or sodium carbonate, dropwise adding the compound 11 under stirring, keeping the temperature of 30 ℃ for 16 hours, then dropwise adding the compound 11, and keeping the temperature of 30 ℃ for 24 hours; after the reaction is finished, regulating the pH of the system to be neutral by using acid, concentrating to be dry at 80 ℃ under reduced pressure, adding methanol, dissolving in a reflux state, dripping into water at a constant speed, maintaining the temperature at 0-10 ℃ after dripping, stirring and crystallizing for 8 hours, filtering, leaching a filter cake by using water, adding water into the filter cake, cooling to 5 ℃, slowly dripping the aqueous solution of the compound 12 into the filter cake, continuously stirring and reacting at 5 ℃ after dripping for 2 hours, heating to 70 ℃ for reacting for 2 hours, regulating the pH to be neutral by using alkali, and concentrating to be dry at 65 ℃ under reduced pressure; adding water, stirring, passing through XAD-1600N macroporous resin, eluting with water, purifying, concentrating under reduced pressure at 65deg.C, and collecting to obtain compound 6.