Patent Description:
Larotrectinib ((<NUM>)-N-[<NUM>-[(2R)-<NUM>-(<NUM>,<NUM>-Difluorophenyl)-<NUM>-pyrrolidinyl]pyrazole-[<NUM>,<NUM>-a]pyrimidine-<NUM>-yl]-<NUM>-hydroxy-<NUM>-pyrrolidinecarboxamide) of formula I:
<CHM>
is a drug (trade name Vitravki®), indicated in the treatment of solid tumors characterized by an NTRK gene fusion in adult and pediatric patients with locally advanced disease, metastatic disease or when other acceptable therapeutic options are present. Larotrectinib is marketed in capsules or oral solution, in the form of sulfate salt.

Any impurities deriving from the synthesis process or from degradative processes must be carefully identified and monitored in order to guarantee appropriate safety and quality standards, as required, for examples, by international guidelines such as ICH Q7, a guideline concerning good laboratory practices (GMP) which provides the parameters to manage an appropriate purity control system of the active ingredient and the ICH Q3 A, which indicates the limits below which impurities and residual solvents must be maintained.

By reproducing the synthesis of Larotrectinib described in <CIT> (example <NUM>, page <NUM>), the only example reported with quantities applicable on an industrial scale, the API is obtained in the form of sulphate salt with a purity close to the limits of the ICH guidelines and which is difficult to control.

In the process described in <CIT>, Larotrectinib sulfate is precipitated with the addition of an anti-solvent from an ethanol solution. This step on an industrial scale is very delicate and leads to the formation of a precipitate that is difficult to manage. Said precipitate has a rubbery consistency which leads to problems in the processing phase, especially during filtration, and to the absorption of a large amount of ethanol, which must be reduced to <NUM>% by weight with respect to the total mass in the subsequent stage of hot suspension in water and <NUM>-butanone.

It has now been found that it is possible to obtain Larotrectinib of high purity by replacing ethanol with dichloromethane in the conversion from hydrochloride salt to sulphate salt.

A previously unidentified impurity corresponding to ((<NUM>)-N-[<NUM>-[(2R)-<NUM>-(<NUM>,<NUM>-Difluorophenyl)-<NUM>-pyrrolidinyl]-pyrazole [<NUM>,<NUM>- a] pyrimidine-<NUM>-yl] -<NUM>-hydroxysulfonyloxy-<NUM>-pyrrolidinecarboxamide of formula II (S-Larotrectinib) was also isolated:
<CHM>.

In a first aspect, the invention therefore provides a process for preparing Larotrectinib sulfate containing less than <NUM>% by weight S-Larotrectinib, comprising the following steps:.

In a second aspect, an object of the invention relates to the compound ((<NUM>)-N-[<NUM>-[(2R)-<NUM>-(<NUM>,<NUM>-Difluorophenyl)-<NUM>-pyrrolidinyl]pyrazole[<NUM>,<NUM>-a]pyrimidine-<NUM>-yl]-<NUM>-hydroxysulfonyloxy-<NUM>-pyrrolidinecarboxamide of formula II (S-Larotrectinib):
<CHM>.

Another object of the invention is an analytical procedure for the determination and quantitation of S-Larotrectinib.

Larotrectinib chloride, synthesized according to known methods, is transformed into Larotrectinib sulfate by means of an unblocking reaction in the presence of dichloromethane, and of a base, selected from alkaline or alkaline earth hydroxides, in particular NaOH or KOH. Said reaction is carried out at room temperature. The formation of Larotrectinib sulfate can occur by dropping a <NUM>% by weight sulfuric acid aqueous solution into a solution of demineralized water and methylethyl ketone <NUM>:<NUM> at room temperature.

The Larotrectinib sulphate thus obtained is filtered and dried at room temperature, to give without further processing the desired product within specification to the content of residual solvents and impurities in accordance to the ICH guidelines indicated above.

Another object of the invention is a process to determine the presence of S-Larotrectinib in a sample of Larotrectinib sulfate, comprising an HPLC analysis, which uses S-Larotrectinib compound as a reference standard.

Another object of the invention is a process to determine the amount of S-Larotrectinib in a sample of Larotrectinib sulfate, comprising an HPLC analysis using S-Larotrectinib as a reference standard.

The HPLC method used to determine the presence of S-Larotrectinib comprises:.

The Larotrectinib sulfate sample is dissolved in a water / acetonitrile mixture in a variable ratio comprised between <NUM>: <NUM> and <NUM>: <NUM>, advantageously in a ratio <NUM>:<NUM>, so as to obtain a solution containing Larotrectinib sulfate at a concentration of <NUM>-<NUM>/ml, advantageously <NUM>/ml. The solution obtained in step a) is injected into a reverse-phase chromatographic column (octadecylsilane), preferably Waters X-Bridge C18 150x4. <NUM>, <NUM>.

The sample is eluted in the column using a gradient of eluents A and B in different percentages, in a time comprised between <NUM>-<NUM> minutes, advantageously <NUM>-<NUM> minutes.

Eluent A consists of a solution of <NUM> / L ammonium formate at pH <NUM> with formic acid, while eluent B is acetonitrile. The ratio between eluent A and B changes during the analysis, in particular between <NUM> and <NUM> minute the eluting mixture contains <NUM>% phase A and <NUM>% phase B, at <NUM> minutes <NUM>% phase A and <NUM> % phase B, between <NUM> and <NUM> minutes <NUM>% phase A and <NUM>% phase B, between <NUM> and <NUM> minutes <NUM>% phase A and <NUM>% phase B, between <NUM> and <NUM> minutes <NUM>% phase A and <NUM>% phase B. The UV detector operates at a wavelength comprised between <NUM> and <NUM>, preferably <NUM>.

S-Larotrectinib is characterized by a relative retention time (RRT) comprised between <NUM>-<NUM>, more particularly <NUM>, in relation to Larotrectinib.

S-Larotrectinib impurity was isolated and characterized by HPLC, H-NMR and GC-MS.

UHPLC Thermo Fisher Vanquish system or equivalent equipped with: Autosampler with peltier system, UV Detector - Visible, Chromeleon data recording software or equivalent, column oven.

Gradient elution as shown in the scheme below:.

As proof of the formation of the S-Larotrectinib impurity, Larotrectinib sulfate was synthesized following the method described in <CIT> (example <NUM>, page <NUM>). The salification reaction with H<NUM>SO<NUM> in EtOH, the solvent that solvates the crystal, necessarily causes a subsequent stress of the product which is refluxed in a mixture of water and methylethylketone.

The combination of time and temperature used to reach the specification for residual solvents in the active ingredient, required by the ICH Q3 A guideline (ethanol <<NUM> ppm), causes an increase in the S-Larotrectinib impurity.

Below is the table of monitoring data of the percentage of ethanol residual in the crystal and the amount of impurity formed over time during the stripping procedure of the crystal in H<NUM>O: methylethylketone <NUM>:<NUM>.

(R)-<NUM>-(<NUM>- (<NUM>,<NUM>-difluorophenyl) pyrrolidin-<NUM>-yl) pyrazole [<NUM>,<NUM>-a] pyrimidine-<NUM>-amine (LARO <NUM>) (<NUM>, <NUM> mol) and dichloromethane (<NUM>) are sequentially loaded into a reactor. The solution is left under stirring at <NUM> for about <NUM> minutes.

A solution of phenylchloroformate (<NUM>, <NUM> mol) in dichloromethane (<NUM>) is dropped, maintaining a temperature below <NUM> and left under stirring at <NUM> for about an hour.

<NUM>-(R)-hydroxy pyrrolidine HCl (<NUM>, <NUM> mol) is loaded into the reactor and triethylamine (<NUM>, <NUM> mol) is subsequently dropped, maintaining a temperature below <NUM>. It is left under stirring at <NUM> for about <NUM> hours.

A solution of citric acid (<NUM>, <NUM> mol) and demineralized water (<NUM>) is prepared in another reactor and the previously prepared solution is loaded by dripping. The reaction is left under stirring for <NUM>-<NUM> minutes at room temperature, the phases are allowed to decant and the organic phase is separated from the aqueous one. The previously obtained organic phase is loaded into the flask. A solution of <NUM>% HCl (<NUM>, <NUM> mol) in demineralized water (<NUM>) is prepared in another reactor and cooled to a temperature between <NUM> and <NUM>. Half of the <NUM>% HCl solution maintained at room temperature is added to the organic phase. The mixture is left under stirring for <NUM>-<NUM> minutes, the phases are left to decant and the organic phase is separated from the aqueous one.

The other half of the <NUM>% HCl solution at room temperature is added to the organic phase. The mixture is left under stirring for <NUM>-<NUM> minutes, the phases are left to decant and the organic phase is separated from the aqueous one.

The combined aqueous phases are loaded into the reactor and dichloromethane (<NUM>) is added. The mixture is left under stirring for <NUM>-<NUM> minutes, the phases are left to decant and the organic phase is separated from the aqueous one.

The washing step of the aqueous phase with dichloromethane is repeated for another <NUM> times.

A mixture composed of NaOH aq <NUM>% (<NUM>, <NUM> mol) and dichloromethane (<NUM>) is prepared in another reactor and this mixture is dropped into the aqueous phase previously cooled to <NUM> and <NUM>, maintaining the temperature below <NUM>.

The mixture is left under stirring for about <NUM> minutes at <NUM>, the phases are decanted and the organic phase is separated from the aqueous one. The organic phase is distilled under reduced pressure to a small volume.

The absolute ethanol (<NUM>) is loaded onto residue and the organic phase is distilled under reduced pressure to small volumes. A solution of <NUM>% HCl in ethanol (<NUM>, <NUM> mol) is slowly added to the reactor while maintaining the temperature at <NUM>. The mixture is left under stirring at <NUM> for about <NUM> hours. <NUM> of methyl-t-butyl ether (MTBE) are slowly added while maintaining the temperature at <NUM>. The mixture is left stirred at <NUM> for about an hour. It is filtered on buchner washing with a mixture of absolute ethanol (<NUM>) and MTBE (<NUM>). The solid thus obtained is dried under vacuum at <NUM> for at least <NUM> hours. <NUM> of pink-orange solid product (<NUM>%) are obtained.

Larotrectinib chloride (<NUM>, <NUM> mol), dichloromethane (<NUM><NUM>) and demineralized water (<NUM>) are sequentially loaded into a reactor. The suspension is stirred for <NUM> minutes at room temperature and then an aqueous solution of <NUM>% NaOH (<NUM>, <NUM> mol) is dropped. It is left under stirring for <NUM> minutes at room temperature. The aqueous phase is separated from the organic one and the organic phase is distilled at reduced pressure. Methylethylketone (<NUM>) is loaded into the reactor and distilled under reduced pressure to a small volume. Demineralized water (<NUM>) is added and the mixture is heated to <NUM>. <NUM>% H<NUM>SO<NUM> is dropped for about <NUM> minutes maintaining the temperature comprised between <NUM> and <NUM>. Methylethylketone (<NUM>) is charged while maintaining the temperature below <NUM>. The mixture is stirred for <NUM> minutes at <NUM>. The mass is cooled to room temperature, methylethylketone (<NUM>) is added and stirred at room temperature for <NUM> minutes.

The suspension is cooled to a temperature between <NUM> and <NUM> and is left stirred for at least one hour. It is filtered by washing the solid with methylethylketone (<NUM>) and dried under vacuum at <NUM> for at least <NUM> hours. <NUM> of light pink solid (<NUM>%) are obtained.

In order to demonstrate the advantage in terms of purity on the final active ingredient, a preparation batch of Larotrectinib chloride (Example <NUM> stage a) was divided before the hydrochloride salt formation stage into three batches of equal weight.

The first batch was transformed into Larotrectinib chloride following the procedure reported in example <NUM>, stage a), and subsequently the hydrochloride was transformed into Larotrectinib sulfate (batch: <NUM>/<NUM>-<NUM>), following the reaction reported in example <NUM>, stage b).

The second batch was transformed directly into Larotrectinib sulfate (batch: <NUM>/<NUM>-<NUM>), following the procedure described in <CIT>.

The third batch was transformed into Larotrectinib chloride following the procedure reported in example <NUM>, stage a), and subsequently the hydrochloride was transformed into Larotrectinib sulfate (batch: <NUM>/<NUM>), following the reaction reported in example <NUM>, stage b).

Claim 1:
A process for preparing Larotrectinib sulfate containing less than <NUM>% by weight S-Larotrectinib, comprising:
(a) the unblocking reaction of Larotrectinib hydrochloride in dichloromethane, in the presence of bases selected from an alkaline or alkaline earth hydroxide;
(b) the treatment of Larotrectinib obtained in step a) with sulfuric acid in methyl ethyl ketone, and water;
(c) the filtration of Larotrectinib sulfate.