EA039850B1 - Synthesis of omecamtiv mecarbil - Google Patents

Abstract

The invention relates to piperazine methyl carboxylate(PMEC) phosphate hydrate and a process for preparing the same, comprising (a) admixing piperazine and methyl chloroformate to form PMEC; and (b) admixing the PMEC and 0.5 molar equivalents of phosphoric acid in an aqueous solution to form PMEC phosphate hydrate. The PMEC phosphate hydrate is less hygroscopic, and it can be stored in air-sealed aluminum bags to avoid contact with moisture. The invention also discloses a process for synthesizing methyl 4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate(PIPN) using the above mentioned piperazine methyl carboxylate phosphate hydrate.

Description

The present invention relates to piperazine methylcarboxylate ⁿ phosphate hydrate (PMEC) and a process for its preparation, which comprises (a) mixing piperazine and methyl chloroformate to form PMEC; and (b) mixing PMEC and 0.5 molar equivalent of phosphoric acid in aqueous solution to form PMEC phosphate hydrate. Phosphate hydrate PMEC is less hygroscopic and can be stored in airtight aluminum bags to avoid contact with moisture. The present invention also discloses a method for the synthesis

methyl 4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate o*hume (PIPN) using the above piperazine methylcarboxylate phosphate hydrate.

Field of invention

Provided are piperazine methylcarboxylate phosphate (PMEC) phosphate hydrate, a method for synthesizing piperazine methylcarboxylate phosphate hydrate, and a method for synthesizing methyl 4-(2-fluoro-3-nitrobenzyl)piperazine-1carboxylate (PIPN).

Prerequisites for the invention

The cardiac sarcomere is the basic unit of muscle contraction in the heart. The cardiac sarcomere is a highly ordered cytoskeletal structure composed of myosin, cardiac muscle actin, and a set of regulatory proteins. The discovery and development of small molecular weight cardiac myosin activators will lead to promising treatments for acute and chronic heart failure, dilated cardiomyopathy (DCM), and conditions associated with left and/or right ventricular systolic dysfunction or systolic reserve. Cardiac myosin is a cytoskeletal motor protein in the cardiac muscle cell. It is directly responsible for the conversion of chemical energy into mechanical force, which causes the contraction of the heart muscle.

Existing positive inotropic agents, such as beta-adrenergic receptor agonists or inhibitors of phosphodiesterase activity, increase intracellular calcium concentration, thereby increasing cardiac sarcomere contractility. However, increasing calcium levels provides an increase in the rate of contraction of the heart muscle and a reduction in systolic ejection time, which has been associated with potentially life-threatening side effects. In contrast, cardiac myosin activators work through a mechanism that directly stimulates cardiac myosin motor protein activity without increasing intracellular calcium concentration. They speed up the rate-limiting step of the myosin enzymatic cycle and shift it in favor of a force-producing state. Instead of increasing the heart's rate of contraction, this mechanism increases the systolic ejection time, resulting in increased cardiac contractility and cardiac output in a potentially more oxygen-efficient manner.

US Pat. No. 7,507,735, incorporated herein by reference, discloses a genus of compounds comprising omecamtiva mecarbil (AMG 423, CK-1827452) having the structure:

Omecamtiva mecarbil is a first-in-class direct activator of cardiac muscle myosin, the motor protein that causes the heart to contract. It is being evaluated as a potential treatment for heart failure in both intravenous and oral formulations with the goal of establishing a new spectrum of agents for patient care in both hospital and outpatient settings.

There is an ongoing need for an industrial process for the manufacture of omecamtiv mecarbil that addresses API-specific issues, including Good Manufacturing Practices (GMP) requirements and regulatory approvals (eg, US Food and Drug Administration and EMA).

Brief description of the invention

Provided herein is a phosphate salt of piperazine methyl carboxylate (PMEC), for example a salt that is PMEC phosphate hydrate. PMEC is alternatively referred to as methylpiperazine-1-carboxylate.

Also provided herein are methods for synthesizing the phosphate salt of PMEC, comprising a) mixing piperazine and methyl chloroformate to form PMEC; (b) mixing PMEC and 0.5 molar equivalent of phosphoric acid to form PMEC phosphate; and (c) optionally filtering the PMEC phosphate from the mixture from step (b). In some cases, step (a) is carried out in an aqueous solution, which provides phosphate hydrate PMEC at a ratio of PMEC to water of approximately 2:1. In various cases, stage (a) is carried out at a temperature of from 20 to 55°C for 1-12 hours. In some cases, the PMEC formed in stage (a) is isolated as a solution in methylene chloride, dichloroethane, 2-methyltetrahydrofuran, or mixtures thereof. . More specifically, isolation can be carried out by (i) washing the resulting PMEC from step (a) with an organic solvent; (ii) adjusting the pH to 8-14 by adding a base to form a basic aqueous solution; and (iii) extracting the PMEC from the basic aqueous solution from step (ii) with methylene chloride, dichloroethane, 2-methyltetrahydrofuran, or mixtures thereof.

Also provided herein are methods for the synthesis of methyl 4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate (PIPN) comprising (a) mixing 2-fluoro-3-nitrotoluene, sodium bromate, and sodium bisulfite in isopropyl acetate, and water to form 1-(bromomethyl)-2-fluoro-3-nitrobenzene (FNB); (b) optionally washing the FNB with an aqueous sodium thiosulfate solution, an aqueous sodium chloride solution, or both; and (c) mixing FNB, a trialky base

- 1 039850 lamin, and piperazine methyl carboxylate (PMEC) phosphate, such as PMEC phosphate hydrate, to form PIPN. In some cases, the FNB is washed with an aqueous sodium thiosulfate solution and an aqueous sodium chloride solution. Alternatively, PIPN can be prepared by (a) mixing 2-fluoro-3-nitrotoluene, benzoyl peroxide, N-bromosuccinimide and acetic acid at 70-95° C. to form 1-(bromomethyl)-2-fluoro-3-nitrobenzene (FNB); (b) optionally extracting the FNB with toluene, washing the FNB with an aqueous stock solution, or both; (c) mixing FNB, a trialkylamine base, and piperazine methylcarboxylate (PMEC) phosphate, such as PMEC phosphate hydrate, to form PIPN. In some cases, FNB is extracted with toluene and washed with aqueous sodium hydroxide. In any process for preparing PIPN, PIPN may be formed as the hydrochloride salt. In any process for the production of PIPN, a PMEC phosphate, such as PMEC phosphate hydrate, can be prepared as disclosed herein. In any process for preparing PIPN, the trialkylamine base includes diisopropylethylamine or triethylamine. In any process for preparing PIPN, prior to mixing FNB, trialkylamine base, and PMEC, the process may also include adding diethyl phosphite and trialkylamine and mixing the resulting mixture at a temperature of 30-65°C.

Brief description of the figures

In FIG. 1 is an isothermal dynamic vapor sorption (DVS) plot for three methylpiperazine-1-carboxylate (PMEC) salt forms, the phosphate hydrate salt form, the hemisulfate salt form, and the acetate salt form. The onset of weight gain at DVS for each salt was measured as indicated at 35% relative humidity (RH) for hemisulfate; 50% RH for acetate and 65% RH for phosphate hydrate. Phosphate hydrate is called phosphate or hemiphosphate and hemisulfate is called sulfate or hemisulfate in the figure.

In FIG. 2 shows the differential scanning calorimetry spectrum of phosphate hydrate PMEC.

In FIG. 3 shows the X-ray powder diffraction pattern for PMEC phosphate hydrate (square) and PMEC slurry (circle).

Detailed description

Mecarbil omecamtiv dihydrochloride hydrate is used in an oral formulation to treat heart failure. Specific conditions include, without limitation, acute (or decompensated) congestive heart failure and chronic congestive heart failure; in particular, diseases associated with systolic dysfunction of the heart.

A previous method for the manufacture of mecarbil omecamtiva dihydrochloride hydrate is disclosed in WO 2014/152270. The GMP process flow disclosed in this document differs from the previous synthesis flow in a number of ways. The GMP sequence has been increased from two to six stages. This longer GMP sequence provides alternative preparation sequences that include no solvents during preparation that are difficult to remove (e.g., N-methylpyrrolidone, NMP), elimination of the use of evaporation crystallization, and separation of intermediates to eliminate complex solvent substitutions.

A previous process for the manufacture of mecarbil omecamtiva dihydrochloride hydrate is depicted in Scheme 1 and discussed in detail in WO 2014/152270. This method involves non-GMP preparation of raw materials for regulatory APIs, piperazine nitro-HCl (PIPN) and phenyl carbamate-HCl (PCAR), from commercially available raw materials FN-toluene (FNT) and 5-amino-2-methylpyridine (APYR), respectively. An isolated GMP intermediate, piperazinaniline (PIPA), is prepared from PIPN by hydrogenation and subsequent coupling with PCAR to form omecamtiv mecarbil. Mecarbil omecamtiva dihydrochloride hydrate is prepared from the corresponding free base by telescoping (i.e. no isolation of omecamtiva mecarbil free base) and isolation as dihydrochloride hydrate by filtration after wet milling. All source materials for the API are marked in boxes.

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Scheme 1:

For the synthesis disclosed herein, API starting materials were moved upstream in sequence to meet the selection and justification requirements for API starting materials for various regulatory agencies, such as EMA and FDA. Thus, the method disclosed herein comprises six steps compared to the two-step sequence disclosed in WO 2014/152270. This longer GMP sequence provides several advantages over the shorter sequence. Methylpiperazine-1-carboxylate phosphate, (PMEC), is used in place of PMEC free base in the formation of the piperazine nitro-HCl (PIPN) intermediate. The PMEC free base is an oil that contains varying levels of piperazine resulting in impurities (eg BISN in a PIPN product, see Scheme 3). In contrast, PMEC phosphate is a stable crystalline salt that has low and constant levels of piperazine. Thus, the use of PMEC hemiphosphate hemihydrate instead of PMEC free base provides a significant reduction in the formation of impurities. The method disclosed in this document also allows the termination of the formation of N-methylpyrrolidinone (NMP) in the production of PCAR, an advantage since NMP is difficult to remove and which has appeared in the list of REACH protocols in the EU (list regarding the safety of chemical materials). In addition, the method disclosed herein provides for changing the solvent in which PIPN is hydrogenated to form PIPA, since the use of isopropyl acetate in the previous method involved an evaporative crystallization step, which often resulted in material contamination and inconsistent results. The method disclosed herein provides for the displacement of a complex solvent change considering the very low solubility of omecamtiv mecarbil free base in isopropanol (~12 mg/mL) at 20° C. and the formation of an unstirred slurry when the solvent is changed from tetrahydrofuran (THF) to isopropanol.

The novel industrial process disclosed herein for the preparation of mecarbil omecamtiva dihydrochloride hydrate is shown in Scheme 2. It includes six GMP steps. Designated commercial raw materials for API are 2-fluoro-3-nitrotoluene (FNT), 5-amino-2-methylpyridine (APYR) and PMEC phosphate hydrate.

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Scheme 2:

(ENT) Commercially available

APISM

i. NBS, (BzO)z AcOH ii. NRO(OEC _g Toluene

Stage I GMP

NOj

FN-bromide (FNB)

ΜοΟ-^'Ν'^ι 1ΰ Η _Ζ Ο u TsROU

U^hyStallic Medium APISM Poyaufmfat PMEC Pol>Tidrate

LRg ₂ WE

II. HCI

Iridium AmpnOl (APYR)

Commercially available APISM

Not isolated Use as a solution

Stage 2 GMP

Pipsrazinnigro-NS!

' (PIPN)

Crystalline CMP Intermediate Yield 82%

D-0 DOPhenylchloroformate, p^sh-HO

Acetonitrile (PCAR)

Stage 3 GMP Crystalline GMP

intermediate connection

Yield 95%

Pi perazni game -HC1 (P1PNJ

i. Toluene / NaOH (vod, i i. H j / Pd- C / toluene / la "

I. /-Pr _? NEt/THF ACN ii. H ₂ O

Phenplcarbamate-HCl (PCAR) iii. Heptane

GMP Stage 4 _{Pi11era1inand11n (PIPA)} Intermediate CMP GMP _Cr „ _{nsI s} Yield 90%

Η Η

Mecarbil amecamtiva free base Yield 85%

2-RgON

HjOiHCI

GMP Stage in *2HC1»H ₂ O

Crystalline DS

Yield 95%

FN-toluene is a raw material that is made from toluene through a short synthesis sequence. Fractional distillation of the resulting isomer mixture provides the desired regioisomer, 2-fluoro-3-nitrotoluene, in acceptable purity, with no more than 0.5% of the GC peak area of any other isomers. 2-Fluoro-3-nitrotoluene (FNT) produced by this process is of reproducible quality and can be identified as a commercial starting material for API.

PIPN manufacturing. PMEC phosphate, for example PMEC phosphate hydrate, is an API starting material made in a single step from piperazine. The previous method for preparing PIPN used PMEC free base as a commercially available raw material, but it is an oil that contains varying amounts of piperazine. When stored at 25°C, piperazine levels up to 18% of the LC peak area were observed in the case of PMEC free base. As shown in Scheme 3, residual piperazine results in the formation of a BISN impurity in the PIPN product.

Scheme 3:

A stable crystalline PMEC salt with low and constant levels of piperazine has been considered as a commercial API starting material. A plurality of salts were thus tested to determine a suitable candidate. The PMEC phosphate, for example PMEC phosphate hydrate, was found to be less hygroscopic than the corresponding sulfate and acetate salts, as shown in FIG. 1. It can be stored in airtight aluminum bags to avoid contact with moisture.

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As an advantage, PMEC phosphate, for example PMEC phosphate hydrate, can be added directly to the reaction mixture to form PIPN. In contrast, the PMEC acetate should be converted to the PMEC free base before being added to the reaction mixture, taking into account the by-product formation from FN-bromide (FNB) and the acetate anion. PMEC phosphate, such as PMEC phosphate hydrate, contains low levels of piperazine (<0.4% of GC peak area) that do not increase on storage. PMEC phosphate, such as PMEC phosphate hydrate, has been successfully used to make PIPN. The batch of PIPN thus obtained (5 kg) contained less than 0.1% of the LC peak area of residual BISN.

A process was developed for the preparation of PMEC phosphate hydrate which involved treating piperazine with methyl chloroformate followed by extracting PMEC as the free base in the organic layer after neutralization with aqueous sodium hydroxide as shown in Scheme 4. After changing the solvent from dichloromethane to tert-butyl methyl ether, the desired the salt is crystallized by adding phosphoric acid and filtered. Phosphate hydrate PMEC is isolated in 45-50% yield from piperazine and >99% GC peak area. Piperazine levels in PMEC phosphate hydrate samples were observed to be <0.4% of the GC peak area. The DSC spectrum and XRPD powder diffraction pattern for PMEC phosphate hydrate are shown in Figures 2 and 3, respectively. PMEC phosphate has a stoichiometry of approximately 2:1 PMEC:phosphate and is thus referred to herein interchangeably as PMEC phosphate, or PMEC hemiphosphate, the phosphate salt of PMEC. The PMEC phosphate hydrate can be formed as detailed herein, and such a hydrate has a stoichiometry of approximately 2:1:1 PMEC:phosphate:water and is referred to interchangeably as PMEC phosphate hydrate, PMEC hemiphosphate hemihydrate, or PMEC phosphate hydrate. It is understood that the ratio of PMEC, phosphate and water in the PMEC phosphate hydrate may be somewhat different from the 2:1:1 stoichiometric ratio indicated above, for example up to a ratio of 6:4:3 or the like. Elemental analysis and/or single crystal X-ray diffraction analysis can be carried out on material obtained by the methods disclosed herein. The ratio of PMEC, phosphate, and water in the isolated salt is constant, and determining the exact ratio of PMEC:phosphate:water does not adversely affect the suitability of the phosphate hydrate PMEC salt in this document for intended use as a starting material in the preparation of omecamtiv dihydrochloride hydrate. mecarbil.

Scheme 4:

(1.0 equiv.) Water

15-25°C

th. Extraction about

NH'HCI

Bis-PMEC od analysis, substances) CH ₂ C1 ₂ layer

ΗΝη

L.NH 2HCI

Piperazine salt

_PMEC yield ₍ _ _{20% output} analysis, substances ₎

water layer

sh.Regulation of pH aq. NaOH iv.NaCl

v. Extraction

CH ₂ C1 ₂

hn η I___.ΝΗ

Piperazine

water layer

O ^{1/2 n} 2° L^NH ₂ 1/2 HPO ₄ ² PMEC hemiphosphate Hemihydrate ^ν ·-Solvent change MTBE H3PO4 (0.50 equiv.) vii. MTVE

40-45°C

Layer CH ₂ C1 ₂

Yield 45-50% >99% GC peak area

A general synthetic method for making PMEC phosphate, such as PMEC phosphate hydrate, involves mixing piperazine and methyl chloroformate to form PMEC, adding 0.5 molar equivalent of phosphoric acid (e.g. in aqueous solution) to form the phosphate salt, and optionally filtering the salt. The reaction of piperazine and methyl chloroformate can be carried out at a temperature of 20–55°C for 1–12 h.

Specific extraction methods and post-reaction work-up procedures are shown in Scheme 4 for the purification of PMEC phosphate. However, other processing procedures may be applied. PMEC can be purified from bis-PMEC formed in the reaction mixture of piperazine and methyl chloroformate by extraction with an organic solvent such as methylene chloride, dichloroethane or 2-methyltetrahydrofuran, or a mixture thereof. In some embodiments, the implementation of the organic solvent provides methylene chloride. The unwanted bis-PMEC is separated in the organic solvent layer and the desired PMEC remains in the aqueous layer. PMEC can be further purified. For example, RMES in

- 5 039850 aqueous solution can be adjusted to a basic pH value (eg 8-14) by adding a basic aqueous solution and extracted with an organic solvent such as methylene chloride, dichloroethane or 2-methyltetrahydrofuran or a mixture thereof, where PMEC is in an organic solvent. In some cases, the organic solvent includes methylene chloride. PMEC in an organic solvent can be subjected to a solvent change from the extracting organic solvent to methyl tert-butyl ether (MTBE) and reaction with phosphoric acid to form a phosphate salt.

In some specific embodiments, the piperazine is suspended in 4.0 volumes (V) of water at 20±5°C. Methyl chloroformate (1 equiv.) was added over >1 hour while maintaining the batch temperature at <20°C. The reaction mixture is stirred at 20±5° C. for >1 hour. One or more cycles of extraction with methylene chloride are carried out, with the methylene chloride layer discarded each time. The aqueous layer was treated with 10M aqueous NaOH (0.8 equiv.) to adjust the pH to 9.5-10.3. NaCl (1.47 equiv.) is added to the aqueous layer and washings are carried out with methylene chloride (2x4 V). The methylene chloride layers are combined and distilled to 2.5 V. Methyl butyl ether (MTBE) (8 V or 4.5 V) is added and the solution is concentrated to 2.5 V. MTBE (3.5 or 4.5 V) is added and concentrated to 2.5 V. MTBE (3.5 V) is added again and the mixture is finally filtered. The filtered solution is heated to 45±5°C (e.g. 40-50°C) and 85% phosphoric acid (0.5 equiv.) in MTBE (1.5 V or 3.5 V) is added over >3 h while maintaining the temperature of the party 45±5°C (for example, 40-50°C). The suspension is cooled to 20±5°C for 2 hours and shaken for 1 hour at 20±5°C. The suspension is filtered and the resulting precipitate is washed with MTBE (2 V) and dried (eg with nitrogen and vacuum for >24 h). The yield of phosphate hydrate PMEC is 48.5%, with 100% LC peak area, 64.6 wt.% analyte, 4.2 wt.% water content according to Karl Fischer titration, 0.44 wt.% residual MTBE and 0. 2% GC peak area of residual piperazine.

Procedure for making PIPN from FNT. FNT can be brominated to form FNB, which in turn can react with PMEC phosphate hydrate to form PIPN (see, for example, top of Scheme 2). FNT can be brominated to form FNB by reaction with NBS and benzoyl chloride in acetic acid at 70-95°C. The FNB may optionally be extracted with toluene and/or washed with an aqueous stock solution to remove impurities. Alternatively, FNT can be brominated to form FNB by reaction with sodium bromate and sodium bisulfite in isopropyl acetate and water. The FNB obtained by reaction with sodium bromate and sodium bisulfite may optionally be washed with an aqueous sodium thiosulfate solution and/or an aqueous sodium chloride solution to remove impurities. FNB, regardless of how it is derived from FNT, can optionally be treated with diethyl phosphite and a trialkylamine (eg, triethylamine or diisopropylethylamine) at 30-65° C. to reduce the level of unwanted dibromide impurities. FNB, regardless of how it is derived from FNT, can be mixed with a trialkylamine base (eg, triethylamine or diisopropylethylamine) and PMEC phosphate hydrate to form PIPN. PIPN can then be converted to the hydrochloride salt form by mixing with hydrochloric acid and can then be isolated.

In some specific embodiments, 2-fluoro-3-nitrotoluene (3.0 kg, 1 equiv.) is charged into the reactor, followed by benzoyl peroxide (0.03 equiv.) and N-bromosuccinimide (0.56 equiv.). Acetic acid (3 V) is loaded into the reactor and the batch is heated to 83°C. After 1.5 hours, a suspension of NBS (0.56 equiv.) in acetic acid (1 V) is loaded into the reactor. After a further 1.5 hours, a second suspension of NBS (0.56 equiv.) in acetic acid (1 V) is loaded into the reactor. After another 5 hours, a solution of H ₃ PO ₃ (0.1 equiv.) in acetic acid (0.1 V) is loaded into the reactor and the batch is shaken for 30 minutes, then cooled to 20°C. Water (5.5 V) and toluene (8 V) are charged to the reactor and the batch is vigorously shaken for 30 minutes. The shaking is then stopped and the layers are separated. The lower aqueous layer is discarded. A solution of NaOH (1.7 equiv.) in water (7 V) is loaded into the reactor while maintaining the batch temperature below 30°C. The batch is shaken vigorously for 30 minutes. The shaking is stopped and the layers are separated. The batch is filtered into a clean reactor and the layers are separated. The lower aqueous layer is discarded. Load into the reactor N,N-diisopropylethylamine (0.53 equiv.), and then methanol (0.23 V), and the party is heated to 40°C. A solution of diethyl phosphite (0.46 equiv.) in methanol (0.23 V) is loaded into the reactor and the batch is shaken for 3 hours. The batch is cooled to 20°C. In a solution of 1 equiv. 2-fluoro-3-nitrophenylmethyl bromide in toluene (9 V), obtained by radical bromination of 2-fluoro-3-nitrotoluene, add 2.3 equiv. diisopropylethylamine at 20°C. A solution of 1.05 equiv. is added dropwise to the stirred solution. PMEC phosphate hydrate in methanol (2.6 V). After stirring for >3 h, water (5 V) is added and the layers are separated. The organic phase is washed twice with saturated aqueous NH ₄ Cl (5 V), then once with saturated aqueous NaHCO ₃ (5 V). After final filtration, the toluene layer is diluted with isopropanol (9.7 V) and water (0.5 V). The solution is heated to

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55°C and add concentrated HCl (0.15 V) over 30 min. The solution is seeded with PIPNHCl (3 mol%) and maintained at 55°C for 15 minutes. Additional concentrated HCl (0.62 V) was added over 4 hours. The solution was kept at 55°C for 15 minutes and cooled to 20°C for >1 hour. The solution was stirred for 30 minutes and filtered. The crystals are washed twice with IPA (5.6 V). The precipitate is dried under vacuum and under nitrogen to give PIPN-HCl (yield 82%, 98.6 wt%, 99.6 LCAP).

In other specific embodiments, 2-fluoro-3-nitrotoluene (3.0 kg, 1 equiv.) is charged to the reactor, followed by benzoyl peroxide (0.03 equiv.) and N-bromosuccinimide (NBS, 0.1 equiv.). Acetic acid (2 V) is loaded into the reactor and the mixture is heated to 83°C. The reaction mixture is shaken for 1.5 h and a suspension of NBS (0.4 equiv.) in acetic acid (0.9 V) is added. The reaction mixture was shaken for 1.5 h and a second suspension of NBS (0.4 equiv.) in acetic acid (0.9 V) was added. The reaction mixture was shaken for 1.5 h and a second suspension of NBS (0.8 equiv.) in acetic acid (1.6 V) was added. Acetic acid (1.0 equiv.) is added and the reaction mixture is shaken for 1.5 h, and a solution of phosphoric acid (H3PO3, 0.1 equiv.) in acetic acid (0.1 V) is charged into the reactor. The mixture is shaken for 60 min and cooled to 20°C. Water (5.5 V) and toluene (8 V) are added to the vial and the biphasic mixture is shaken vigorously for 30 minutes. The shaking is stopped and the layers are separated. The aqueous layer is discarded. Load a solution of sodium hydroxide (1.7 equiv.) in water (7 V) while maintaining the temperature below 30°C. The biphasic mixture was shaken vigorously for 30 minutes. The shaking is stopped and the layers are separated. The biphasic mixture is filtered and the layers are separated. The aqueous layer is discarded. The reaction mixture is transferred to a separate clean vessel, the original vessel is washed with toluene (1.2 V) and the wash volume is added to the reaction mixture. N,N-diisopropylethylamine (0.53 equiv.) and methanol (0.23 V) are loaded into the organic layer and the mixture is heated to 40°C. A solution of diethyl phosphite (0.46 equiv.) in methanol (0.23 V) is charged and the reaction mixture is shaken for 3 hours. The mixture is cooled to 20°C. To a solution of FNB in toluene, obtained by radical bromination of 2-fluoro-3-nitrotoluene (FNT), was added diisopropylethylamine (2.3 equiv.) and toluene (1 V). The FNB solution is added to a solution of methanol (1.8 V) and PMEC phosphate hydrate (1.05 equiv.). The original vessel, which contained the FNB solution, was washed with methanol (0.8 V) and the wash volume was added to the reaction mixture. The reaction mixture was shaken for 4 hours at 25°C and water (5 V) was added while maintaining the batch temperature below 30°C. The biphasic mixture is shaken for 30 minutes and the layers are separated. The organic phase is washed twice with 3 M aqueous ammonium chloride (5 V) and once with 1 M aqueous sodium bicarbonate (5 V). The reaction mixture is transferred to a separate clean vessel, the original vessel is washed with toluene (1 V) and the wash volume is added to the reaction mixture. After final filtration, isopropanol (9.7 V) and water (0.6 V) are added to the organic solution. The solution is heated to 55° C. and aqueous 32 wt% hydrochloric acid (0.25 equiv.) is added over 30 minutes. The solution is shaken at 55° C. for 15 minutes and seeded with a suspension of PIPN (hydrochloride salt, 0.045 equiv.) in isopropanol (0.2 V). The suspension is shaken at 55° C. for 30 minutes. Additional aqueous 32 wt% hydrochloric acid (1.0 equiv.) was added over 4 hours. The suspension was shaken at 55°C for 30 minutes and cooled to 20°C over 2 hours. The suspension was shaken for 30 minutes and filtered. The product precipitate is washed twice with isopropanol (5.6 V). The product precipitate was dried on a filter/dryer to form PIPN in 82% yield, with 98.6 wt% analyte and 99.6% LC peak area.

In some specific embodiments, 2-fluoro-3-nitrotoluene (5.1 g) is dissolved in isopropyl acetate (30 ml) and a solution of sodium bromate (14.9 g) in water (50 ml) is added. The mixture is cooled to 10°C. A solution of sodium bisulfite (10.3 g) in water (100 ml) was added over 20 minutes. The resulting mixture is heated to 80° C. for 3 hours. The reaction vessel is characterized by access to visible light. The contents are cooled to 20° C. and the phases are separated. The organic phase is then washed with 10% aqueous sodium thiosulfate and saturated aqueous sodium chloride. 1-(Bromomethyl)-2fluoro-3-nitrobenzene (FNB) is obtained with a yield of 74% of the analyte, with a yield of 11% of the analyte dibromide product.

APYR manufacturing. 5-Amino-2-methylpyridine (APYR) is commercially available as a raw material, however it contains varying amounts of the hydrochloride salt (3-5% by weight) and is provided as a dark brown or black material. In addition, it may contain a variety of potentially genotoxic impurities, as depicted in Scheme 5. Therefore, for the use of APYR as a commercial starting material for high and consistent purity APIs, a purification protocol for APYR or a synthesis method for producing APYR is desirable.

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Scheme 5:

Nitropyridine (NPYR) Mutagenic in silico

Fe/HCi

Aminopyridine (APYR) Commercially available

Through these intermediate connections:

HOHN

Me

Hydrox lamp Mutagepny in silico

Intermediate azo compound Mutagenic in silico

Hydrazipe intermediate Mutagenic in silico

A method is provided for purifying APYR by washing an isopropyl acetate solution of APYR containing not more than 10% by weight of the corresponding hydrochloride salt with an aqueous solution of sodium hydroxide and then mixing the organic phase with activated charcoal. APYR can be crystallized from isopropyl acetate and heptane, optionally after azeotropic drying of the organic phase and final filtration. The method for purifying APYR is shown in Scheme 6. Purification of APYR involves converting the hydrochloride salt of APYR to the free base of APYR and simultaneously removing inorganic material by washing with basic aqueous sodium hydroxide of the isopropyl acetate solution of APYR. After treatment with activated charcoal (eg, mixing with activated charcoal and filtering the slurry or recirculating the isopropyl acetate solution through activated charcoal capsules), the APYR-containing solution is azeotropically dried and finally filtered. The clear isopropyl acetate solution is concentrated and the APYR is crystallized with the addition of heptane. APYR is isolated with >99% LC peak area and >99 wt% analyte.

Scheme 6:

,Me i . Water NaOH IPAc ii .Active treatment with coal

H ₂ N'

APYR ~3-5 wt. % Hydrochloride Dark

111. Azeotropic drying

IV. Final filtration .Me o ₂ n'

NPYR

Up to 5% hydrochloride

Dark color

vi. Heptane

v. Concentrated seed at 50°C

High purity

Yield 85% >99% LC peak area >99 wt. %

IPAc solution

Clear solution

V. Heptane

Crystallization from IPAc/heptane

Water NaOHIPAc

-Processing asset, coal, Me o ₂ n ^ ^

IPAc reaction mixture iii. IPAc/H^Pd-C

IV. Filtration with .Me catalyst

Crystallization from IPAc/heptane

H ₂ N Amnopyridine IPAc solution ^ν Concentrated seeding at 50C

>99% peak area LC >99wt. %

Yield 90%

In some specific embodiments, a solution of crude 5-amino-2-methylpyridine (APYR) in isopropyl acetate (IPAc) (15 volumes) is washed with 1N hydrochloric acid. NaOH aqueous solution (1.0 V) and circulated through activated charcoal capsules until the color of the solution (COS) in the production control corresponds to the specified one (COS <20). The solution is azeotropically dried by concentration to about 6 volumes and isopropyl acetate (8 volumes) is added. The mixture is finally filtered into a separate vessel. The original vessel is washed with isopropyl acetate (1.0 volume) and the volume of washing is added to the reaction mixture. The solution is concentrated, for example by distillation under reduced pressure, and the product is crystallized from isopropyl acetate and heptane (1:4, 10 V). In some cases, the solution is concentrated to 3 volumes at 60° C. and seeded with purified APYR (1 mole %). The suspension is shaken for 30 minutes, cooled to 20° C. over 3 hours and shaken for 1 hour. Heptane (8 volumes) is added over 3 hours to complete the crystallization of the material. The suspension is shaken for 1 hour, filtered and the product precipitate is washed with heptane (2 x 3 volumes). Purified APYR is isolated by filtration, dried and yielded in 90% with >99% LC peak area.

APYR from NPYR. In some cases, APYR is synthesized from NPYR, as shown in Scheme 6. Hydrogenation of NPYR is carried out in the presence of a palladium catalyst to form crude APYR, which can be crystallized from isopropyl acetate and heptane. Hydrogenation of NPYR to form crude APYR is carried out after washing with a basic aqueous solution and treatment with activated charcoal. Treatment with activated charcoal involves mixing with activated charcoal and filtering the slurry or recirculating the isopropyl acetate solution through activated charcoal capsules. The APYR solution is azeotropically dried and finally filtered. APYR crystallizes from

- 8 039850 isopropyl acetate and heptane. In some cases, the NPYR is purified prior to hydrogenation by washing with isopropyl acetate and aqueous sodium hydroxide and subjecting to activated carbon treatment (mixing with activated carbon, then filtering off the activated carbon).

In some specific embodiments, a solution of 2-methyl-5-nitropyridine (NPYR) in isopropyl acetate (15 V) is washed with 1 N. an aqueous solution of NaOH (2 V) and water (2 V). Optionally, the solution is circulated through the activated charcoal capsules until the color of the solution (COS) in the production control is correct (COS<20). Carry out hydrogenation NPYR at 4.5 bar of hydrogen, for example, at 70 psi. inch/50-60°C (for example, 55°C) in the presence of 5% Pd/C (on activated carbon sold by BASF Escat™ 1421, loading 1.5 wt%) for about 1 hour. The reaction mixture is filtered and Dry azeotropically by concentrating to about 7 V, add 8 V of isopropyl acetate and finally filter. The solution is concentrated to 3 V under reduced pressure at 60°C. The product is crystallized from isopropyl acetate and heptane (1:4), optionally by seeding pure APYR (1 mole %) and/or optionally by cooling to 20°C. The product is optionally filtered and washed with heptane (2x3 V). APYR is isolated in 75% yield with >99% LC peak area.

A number of methods disclosed in this document include steps indicated as optional. In some cases, an optional step is omitted. In other cases, an optional step is carried out.

Claims (17)

CLAIM 1. Piperazine methylcarboxylate phosphate hydrate O BUT MeO^N^ k^NH (PMEC). 2. Process for the synthesis of piperazine methylcarboxylate phosphate hydrate O BUT MeO^N^ (PMEC), including: (a) mixing piperazine and methyl chloroformate to form PMEC; (b) mixing PMEC and 0.5 molar equivalent of phosphoric acid in aqueous solution to form PMEC phosphate hydrate. 3. The method of claim 2, further comprising filtering off the phosphate hydrate PMEC from the mixture from step (b). 4. The method according to claim 2 or 3, further comprising isolating the PMEC formed in step (a) as a solution in methylene chloride, dichloroethane, 2-methyltetrahydrofuran, or mixtures thereof. 5. The method according to claim 4, where the selection is carried out by: (i) washing the resulting PMEC from step (a) with an organic solvent; (ii) adjusting the pH to 8-14 by adding a base to form a basic aqueous solution; and (iii) extracting the PMEC from the basic aqueous solution from step (ii) with methylene chloride, dichloroethane, 2-methyltetrahydrofuran, or mixtures thereof. 6. The method according to claim 2 or 3, wherein step (a) is carried out in an aqueous solution. 7. The method according to any one of paragraphs. 2-6, where stage (a) is carried out at a temperature of 20-55°C for 1-12 hours. 8. Method for the synthesis of methyl 4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate (PIPN), including: (a) mixing 2-fluoro-3-nitrotoluene, sodium bromate and sodium bisulfite in isopropyl acetate and water to form 1-(bromomethyl)-2-fluoro-3-nitrobenzene (FNB); and - 9 039850 (b) mixing FNB, trialkylamine base and piperazine methyl carboxylate phosphate hydrate MeO'^N'X (PMEC) to form PIPN. 9. The method of claim 8, further comprising, prior to step (b), washing the FNB with an aqueous sodium thiosulfate solution, an aqueous sodium chloride solution, or both. 10. The method of claim 9, wherein the FNB is washed with an aqueous sodium thiosulfate solution and an aqueous sodium chloride solution. 11. Method for the synthesis of methyl 4-(2-fluoro-3-nitrobenzyl)piperazine-1-carboxylate (PIPN), including: (a) mixing 2-fluoro-3-nitrotoluene, benzoyl peroxide, N-bromosuccinimide and acetic acid at a temperature of 70-95° C. to form 1-(bromomethyl)-2-fluoro-3-nitrobenzene (FNB); (b) mixing FNB, trialkylamine base and piperazine methyl carboxylate phosphate hydrate (PMEC) to form PIPN. 12. The method of claim 11, further comprising, prior to step (b), extracting the FNB with toluene, washing the FNB with an aqueous stock solution, or both. 13. The method of claim 12, wherein the FNB is extracted with toluene and washed with aqueous sodium hydroxide. 14. The method according to any one of paragraphs. 8-13, further comprising the formation of PIPN as a hydrochloride salt by mixing PIPN with an aqueous solution of hydrochloric acid. 15. The method according to any one of paragraphs. 8-14, where PMEC phosphate hydrate is obtained using the method according to any one of paragraphs. 2-7. 16. The method according to any one of paragraphs. 8-15, wherein the trialkylamine base is diisopropylethylamine or triethylamine. 17. The method according to any one of paragraphs. 8-16, where before mixing the FNB, trialkylamine base, and PMEC phosphate hydrate, the method further comprises adding diethyl phosphite and trialkylamine and stirring the resulting mixture at a temperature of 30-65°C.