The su bject of our invention is process for the preparation of the compounds of general formula I,

I

where R represents a straight- or branched-chain Ci_ ₄ alkyl group.

M isoprostol (la) (R=methyl), a synthetic, modified PGEl derivative, falling under general formula I, is a racemic compound, a mixture of 4 stereoisomers. The following structural formula of the racemic compound indicates relative stereochemistry.

8(R), 11 (R), 12(R), 16(R) Therapeutic use of misoprostol is to reduce the risk to develop gastric and duodenal ulcer induced by nonsteroidal anti-inflammatories (NSAIDs). (http://www.drugs.com/pro/misoprostol.html, download 18 Feb. 2016.). Owing to this protective effect, it is also applied together with nonsteroidal anti- inflammatories, in combination preparations (http://www.drugs.com/cdi/diclofenac- misoprostol.html, down-load 18 Feb.2016.). Misoprostol in itself is also capable to induce labor (https://www.ferring.com/en/media/press-releases/2013/misode l· 17oct 13/, download 18 Feb. 2016.).

For the preparation of misoprostol several methods are known. In the process described in patent specification CA 1040197 A misoprostol was synthetized in a two- component cuprate coupling.

The unprotected or THP-group-protected (THP = tetrahydropyranyl-) cyclopentenone ester was reacted with a so-called„Lower Order" cuprate reagent (Figure 1.).

= THP

Figure 1.

The cuprate reagent was prepared from TBDMS-octynol (TBDMS=tert-butyldimethylsilyl-) (Figure

The triple bond was reduced with catecholborane or with diisobutylaluminum hydride, substituent incorporating the boron or the aluminum atom was then exchanged for iodine.

| ₂ , NaOH

TBDMS-octynol vinyl boron or

vinyl aluminum derivative

iodo vinyl derivative cuprate reagent

Figure 2. The vinyl lithium component -obtained from the iodo compound- was reacted with the solution of pentynyl-copper in hexamethylphosphoramide (HMPA) at -60°C to result the copper compound suitable for the cuprate coupling.

The advantage of the method is that the cuprate coupling was successfully applied for the preparation of misoprostol, however, the synthesis also has several disadvantages:

• reduction of the octynol with catecholborane or with diisobutylaluminum hydride proceeds in very poor yield

• during the reaction, beside the expected trans-olefin, the product with cis geometry is also formed

• preparation of the cuprate reagent requires cooling to -60°C and the use of carcinogenic HMPA.

The method described in patent specification US 4904820 is a developed version of the cuprate coupling. The TES-protected (TES = triethylsilyl-) cyclopentenone derivative was reacted with a „Higher Order" cuprate reagent (X= CN, SCN, OS0 ₂ CF ₃ , S-phenyl). The synthesis of the cuprate reagent was significantly simplified (Figure 3.).

TES-cyclopentenone cuprate reagent misoprostol

R = Me, Bu

Figure 3.

To obtain the cuprate reagent copper(l)cyanide was treated with Me- or Bu-lithium. Reaction of the thus obtained copper compound with another alkyllithium gave the dialkyl cuprate, which on reaction with vinyl stannane resulted the vinyl cuprate required for the coupling (Figure 4.).

R ¹ = Me, Bu, thiophenyl dialkyl cuprate

R ² = Me or Bu dialkyl cuprate vinyl stannane vinyl cuprate

Figure 4.

Advantages of the method:

• preparation of the vinyl stannane is more simple than that of the iodo vinyl derivative

• reaction of the„Higher-Order" cuprate with vinyl stannane does not require deep-freezing. Disadvantages of the method:

• it uses poisonous CuCN reagent

• the vinyl stannane is prepared by reduction of TMS-octynol (TMS = trimethylsilyl-) with tributyltinhydride. During the reduction 15% of cis-isomer impurity is formed (Figure 5.).

radical initiator

TMS-octynol vinyl stannane vinyl stannane impurity

85% 15%

Figure 5.

Patent specification US 5055604 describes further development, the reduction of TMS-octynol was namely carried out with zirconocene chloride hydride (Figure 6.).

TMS-octynol vinyl zirconium derivative

Figure 6.

From the resulting vinyl zirconium derivative the„Higher Order" cuprate reagent was prepared and reacted with TES-cyclopentenone. After acidic hydrolysis of the TES-group, misoprostol was obtained.

Advantage of the method:

• the vinyl zirconium derivative is not contaminated with the cis isomer. Disadvantage of the method :

• zirconocene chloride hydride is an expensive reagent.

In the method described in patent specification US 5684177 the vinyl cuprate reagent for the conjugated addition reaction was obtained by first reacting the alkyllithium with copper halide, then treating the obtained„Lower Order" d ialkyl cuprate with vinyl stannane (Figure 7.).

THF, 0°C

CuX + 2 R-Li R ₂ CuLi + LiX copper halide alkyllithium dialkyl cuprate

X = l, Br; = Ci_ ₆ alkyl group

+ BusSnR

dialkyl cuprate vinyl stannane vinyl cuprate

R = CL_6 alkyl group

Figu re 7.

Studying the process it was found that using more than 2 equivalents of alkyllithiu m for the preparation of the dialkyl cuprate, the reaction of the„Lower Order" cuprate with the vinyl stannane may be performed at 0-(-)-30°C, and cooling to -(78)°C to obtain the vinyl cuprate is not necessary.

If the amou nt of the alkyllithium is 2 equivalents or less, the vinyl cuprate is not formed.

If the amount of the alkyllithium is too high, undesired by-products are arising.

As described in the claims of the patent, the ratio of the alkyllithium is 2.05-4 mols for 1 mol of copper halide.

Favorable molar ratios are: alkyllithium: copper halide = 2.1 - 2.25: 1.

The effect of the addition order was also investigated. The orders copper halide - alkyllithium - vinyl stannane or vinyl stannane - copper halide - alkyllithium were equally applica ble to obtain the vinyl cuprate reagent.

Advantages of the method :

• by using more than 2 equivalents of alkyllithium the vinyl cuprate reagent may be prepared at 0-(-)-30°C and deep-freezing to (-78)°C is not necessary.

• the use of the poisonous CuCN is avoided. Disadvantages of the method : • the alkyllithium excess causes the formation of undesired by-products (e.g. instead of 1,4- addition 1,2-addition takes place)

• because of the formation of by-products the yield is lower.

In the method described in patent specification EP 0943607 the unprotected octynol was reacted with tributyltin hydride.

The unprotected cis- and trans-HO-vinyl stannane isomers may namely be separated by column chromatogra phy, thus the cuprate reagent used in the coupling reaction will not contain cis-isomer contamination (Figure 8.).

octynol trans-HO-vinyl stannane cis-HO-vinyl stannane

85% 15%

Figure 8.

The trans-HO-vinyl stannane was then transformed into the required vinyl cuprate reagent in the presence of copper salt (CuY), alkyllithium (RLi), Lewis acid (G) and lithium salt (Z) of various molar ratios (Figure 9.).

Addition order of the reagents was also varied and investigated when preparing the vinyl cuprate.

CN, SCN, R = alkyl G = Lewis acid Z vinyl stannane OS0 ₂ CF ₃ , BF ₃ .etherate,

TMSCI

Figure 9.

Molar ratios of the reagents

CuY R ⁴ Li G Z M

G-LiZ.Li(Y)Cu- 1 2 1

The yield of the cuprate coupling reaction was the highest if the CuY: RLi ratio was 1: 3 or 1: 4. Additives (Lewis acids, lithium salts) did not improve the yield of the cuprate coupling. The addition order of the reagents did not really influence the reaction. Advantages of the method:

• simple method to remove the cis stannane impurity

• the cuprate reagent is prepared at 0-(-)-40°C, deep-freezing is not needed. Disadvantage of the method:

• use of the poisonous copper cyanide.

Patent specification WO 2016005943 Al describes the preparation of prostaglandins by two- component coupling. According to the method the protected cyclopentenone is reacted with the vinyl boron derivative in the presence of rhodium-containing [RhCI(l,5-cyclooctadiene)] ₂ catalyst (Figure 10.).

Figure 10.

Advantages of the method:

• no reaction under deep-freezing is required

• no poisonous organic copper or tin compound or expensive and chemically sensitive organic zirconium compound is used

• no poisonous cyanide ligand is used.

Disadvantages of the method:

• the coupling has to be carried out in microwave reactor which hinders scale-up

• under thermic conditions the coupling reaction requires several days to take place. The su bject of our invention is preparation of the compound of general formula I

I where R stands for straight- or branched-chain Ci_ ₄ alkyl group by cuprate coupling of the vinyl cuprate of general formula I I

prepared by reacting the vinyl stanna ne of the general formula II I with copper halide CuX and alk llithium R ^x Li

II I II

wherein:

R ² stands for H or an alcohol-protecting group which may contain silicium atom, as for instance trimethylsilyl, triethylsilyl, iert.-butyldimethylsilyl group, or a cyclic or open-chain alkyl group containing oxygen atom, as for instance tetra hydropyranyl, methoxymethyl or ethoxymethyl group; X means I, Br, CN, SCN, OS0 ₂ CF ₃

R ¹ represents C ₆ alkyl group

n >2, if R ² is not hydrogen atom, n>3, if R ² is hydrogen atom; and the protected enone of the general formula IV

where R ³ represents TH P- or tria Ikylsilyl- group and the meaning of R is as defined a bove takes part in the cu prate reaction characterized by that

a.) the excess of the alkyllithium, which is applied as compared to the Cu(l)iodide

in the case of R ² ≠ H in 2-2.4 molar ratio,

in the case of R ² = H in 3-3.4 molar ratio,

is decomposed before the cou pling reaction of I I and IV, b.) protecting groups of the resulting compound of general formula V

V

where the meanings of R, R ² and R ³ are as defined above, are removed, the obtained compound of the general formula I is purified by chromatography.

To prepare misoprostol, compound I according to the invention where R stands for methyl group, the reagent needed for the cuprate coupling is prepared by the reaction of vinyl stannane (85:15 ratio mixture of the trans and cis isomers) with copper iodide and methyllithium.

Patent specification US 5684177 studies in detail the molar ratios of alkyllithium compared to copper(l)halide in the cuprate reagent preparation. As given in the claims, the amount of the alkyllithium is 2.05-4 mol for 1 mol of copper(l)halide. Favorable alkyllithium - copper(l)halide ratios are 2.1-2.25: 1.

According to our experiments, to obtain the vinyl cuprate reagent in acceptable yield, the MeLi / Cul molar ratio should be higher than 2. In our case (R ² ≠ H) a 2.4-fold excess proved to be the most favorable.

The excess of the methyllithium, however, causes by-products formation, which decreases the yield and renders purification of the product more difficult.

The novelty of our invention is that after the formation of the cuprate reagent, but before the cuprate coupling, the methyllithium excess is decomposed in„one-pot" method.

In our process, on the effect of the methyllithium excess, the vinyl cuprate reagent is formed in sufficiently high conversion and since the excess of the methyllithium is decomposed after the cuprate reagent formation, the amount of the impurities coming from the coupling reaction is significantly diminished.

Decomposition of the methyllithium excess may be effected with any kind of compound which in non-aqueous medium reacts with the methyllithium, but neither itself, nor its derivative given with methyllithium reacts with the starting materials or the product of the cuprate conjugated addition.

Reagents suitable to decompose the methyllithium excess are ketones, esters and halogenated silylating agents.

Most suitable reagents to decompose the excess of methyllithium are small molar weight ketones, esters or halogenated silylating agents, as their excess and the compounds arising from them in the methyllithium reaction are easily removed from the reaction mixture, for instance by evaporation or by chromatographic purification.

The most suitable reagents to decompose the excess of methyllithium are acetone, ethyl acetate or trimethylsilyl chloride.

After the acidic decomposition the reaction of methyllithium with acetone results tertiary-butanol, that with ethyl acetate gives acetone or acetone and tertiary-butanol, with trimethylsilyl chloride results tetramethylsilane. Each of these compounds has low boiling point and may be removed from the reaction mixture by simple evaporation.

Following decomposition of the methyllithium excess, the vinyl cuprate reagent is reacted in one-pot reaction with the TMS-enone at (-)-55°C, in tetrahydrofuran.

The reaction mixture obtained after decomposition and work-up contains the protected TMS- misoprostol crude product.

Removal of the protecting groups in methanol with pyridinium tosylate gives the crude misoprostol.

Misoprostol is an oil, to meet the quality required by USP and PhEur specifications it has to be purified by column chromatography.

For column chromatographic purification, gravity chromatography was chosen.

Gravity chromatography is more advantageous than high-pressure preparative or medium-pressure „flash" chromatography, since

· it is cost-saving and easy to realize industrially

• it does not require expensive pressure-proof equipment • the silica gel used for the stationary phase is cheaper than those used in the medium- and high-pressure chromatographic systems

• on the column used in the gravity chromatography purification is performed in one run which shortens the production time.

In the purification process as stationary phase we applied the most widely used and most economical irregular Kieselgel Si 60 (0.063-0.200 mm) (maker: Merck), the significantly more expensive spherical YMC S75, YMC S150 (maker: YMC Co. Ltd.), Chromatorex MB 70-40/75, Chromatorex MB70-75/200 (maker: Fuji Silysia Chen. Ltd) and the irregular Sepra Silica 50 (Fenomenex Ltd) silica gels.

As for eluent we used multicomponent mixtures. As for polar component of the multicomponent system we applied ketone-, ether-, ester- and alcohol-type solvents, while for apolar component we used hydrocarbon, aromatic hydrocarbon, halogenated hydrocarbon and ether-type solvents.

Thus, stepwise gradient mixtures of solvents

hexane: ethyl acetate,

toluene: ethyl acetate, toluene: tetrahydrofuran

dichloromethane: acetone, dichloromethane: methyl ethyl ketone, dichloromethane: tetrahydrofuran

diisopropyl ether: acetone, diisopropyl ether: methyl ethyl ketone, diisopropyl ether: isopropanol

diisopropyl ether: acetone: methanol

were applied.

During our chromatographic experiments we found that the best purification may be reached by using solvent mixtures which contain alcohol. However, the amount of one impurity, 8-iso- misoprostol, significantly increased, instead of decreasing, during the chromatographic purification, rendering the use of this method questionable.

Solution to the problem was brought by our innovative finding of adding 0.1-0.01%, preferably 0.05% of acetic acid or formic acid to the eluent of the chromatography. Acidity of the eluent blocked the basic sites of the silica gel of amphoteric character hindering thus the degradation of the chemically sensitive misoprostol into 8-iso-misoprostol which takes place on the effect of base.

The favorable effect of the acid does not appear below an acid content of 0.01%, while an acid content higher than 0.1% may cause the formation of misoprostol-A and 8-iso-misoprostol impurities.

To hinder the formation of 8-iso-misoprostol impurity, both acetic and formic acid are suitable, however, because of easier removal the use formic acid is more advantageous. Misoprostol product meeting the quality according to the present specification was obtained in best yield by using YMC S75 silica gel and as eluent, diisopropyl ether: isopropanol gradient mixtures, containing 0.05% of formic acid.

Applying the much cheaper Kieselgel Si 60 (0.063-0.200 mm) silica gel the amount of the impurities eluting before misoprostol decreased to the value allowed by specification, but the amount of the related impurities eluting at T>1 remained higher than the allowed 0.10 mass%. If, however, the concentrated main fraction of the purification chromatography was clarified by filtration through active carbon, we found in surprise that the hardly removable related impurities more polar than misoprostol, bonded on the surface of the active carbon in such an extent that their amount decreased to below the allowed limit of 0.10 mass%.

Thus, misoprostol of appropriate quality may be prepared not only by chromatography on the expensive spherical YMC S75 silica gel using diisopropyl ether: isopropanol 0.05% formic acid mixtures as eluent, but also by chromatography on the much cheaper irregular Kieselgel Si 60 (0.063- 0.200 mm) silica gel using diisopropyl ether: isopropanol 0.05% formic acid or diisopropyl ether: acetone: methanol 0.05% formic acid mixtures as eluent, followed by filtration on active carbon.

The last step of the purification process of misoprostol is filtration through silica gel, in order to remove the contaminations arising from the solvents used during the purifications.

Filtration through silica gel was performed on Kieselgel Si 60 (0.063-0.200 mm) silica gel, using stepwise gradient mixtures of distilled dichloromethane: acetone 0.05% formic acid and methyl tert.- butyl ether: acetone and 0.05% formic acid solvents.

The fractions containing the product were united, washed with sodium hydrogen carbonate solution and with water, clarified, dried, filtered and evaporated.

Applying the above method according to the invention misoprostol product of appropriate quality meeting the present specification may be prepared.

Here below we demonstrate the characteristics of some of the stationary phases applied in the above purification process:

Fuji Silysia Chemical Ltd.

CHROMATOREX MB 70-40/75

Item Unit Specifications Lot Data

Surface Area m ² /g 450 - 550 483

Pore Volume mi/g 0.70 - 0.90 0.85

Bu!k Density g/ml 0,40 - 0.60 0.53 pH - 6.0 - 8.0 7.5

Loss on Drying % 3.0 - 7.0 5.6

ParticleSize Distribution

on 75 μιη % 10,0 % max. 1.4

38 - 75 pm % 80.0 % min. 94.0

thru 38 pm % 10.0 % max. 4.6 CHROMATOREX MB 70-75/200

YMC Co. , Ltd.

Item Result

Average Particle Size ^* D50 (μιη) 79

Uniformity Coefficient : D4O D90 1.51

Average Pore Siase (nm) 6.5

Specific Surface Area (m ^z /g) 761

Pore Volume (mL g) 1,23

Volatile Matter (%) 0.3 pH 5.9 EXAMPLES Example 1.

(+)-5-oxo-3-[(trimethylsilyl)oxyl-l-cyclopenten-l-heptanoic acid methyl ester

HO-enone TMS-enone

Mr: 240.30 Mr: 312.48 1955g of 3-hydroxy-5-oxo-l-cyclopenten-l-heptanoic acid methyl ester (HO-enone) is dissolved in tetrahydrofuran (20 kg) under inert atmosphere. To the solution 1.7L of triethylamine and then 1.14L of trimethylsilyl chloride are added. After reaching the desired conversion the excess of the trimethylsilyl chloride is decomposed with methanol, the reaction mixture is filtered, the precipitate is washed with tetrahydrofuran, to the liquid filtrate triethylamine is added and the mixture is concentrated with evaporation.

The silylated product (TMS-enone) is transferred into the next step without further purification.

Example 2.

(+Hlla.l3El-16-methyl-9-oxo-11.16-bisf(trimethvlsilvlloxv l-13-en-prostanoic acid methyl ester

Preparation of the cuprate reagent vinyl stannane vinyl cuprate

C24H ₅₂ OSiSn C ₁₃ H ₂₈ CuOSi+Li

Mr: 503.46 Mr: 298.93

Decomposition of the methyllithium excess

O ni i OH

Me-Li A — J< ^→ <

methyllithium acetone iert.-butanol

Coupling (conjugated addition)

TMS-enone TMS-misoprostol

C16H28O4S1 C28H54O5S12

Mr: 312.48 Mr: 526.92

Preparation of the cuprate reagent

8.4kg of tributyl[l(f)-4-methyl-4-[trimethylsilyl)oxy]l-octen-l-yl]-s tannane (vinyl stannane) is dissolved in tetrahydrofuran in an inert atmosphere. To the solution 2.72kg of copper(l)iodide is added. The reaction mixture is agitated at room temperature for 30 minutes, then cooled to (-)-35°C and methyllithium solution equivalent to 34.5 mol of methyllithium is added and the reaction mixture is agitated at (-)-20— (-)-25°C. If the conversion after 30 minutes of stirring is not sufficient, a further amount of methyllithium solution equivalent to 0.7 mol of methyllithium is added and stirring at (-)-20— (-)25°C is continued for another 30 minutes.

Coupling (conjugated addition)

The reaction mixture is cooled to (-)-60°C and in order to decompose the methyllithium excess, acetone is added. At (-)-55°C the tetrahydrofuran solution of the TMS-enone derivative is added to the reaction mixture. After 30-40 minutes of agitation the reaction mixture is decomposed by adding it to ammonium chloride - ammonium hydroxyde solution (77kg of water, 9.2g of ammonium hydroxide solution, 25.3kg of ammonium chloride).

The decomposed reaction mixture is extracted with methyl ieri.-butyl ether, the organic phase is washed with sodium hydrogen sulfate in sodium chloride solution, and then with saturated sodium chloride solution, then dried over sodium sulfate.

The evaporated reaction mixture is transferred into the next reaction step without further purification.

Example 3.

Misoprostol

(+)-(lla,13E)-ll,16-dihydroxy-16-methyl-9-oxo-13-en-prostano ic acid methyl ester

TMS-misoprostol misoprostol

Mr: 526.92 Mr: 382.53 The TMS-misoprostol (8.11 mol) prepared in the coupling reaction according to Example 2. is dissolved in methanol (17 kg) at room temperature, 205g of pyridinium tosylate is added to it and the mixture is agitated until the desired conversion is reached. The mixture is then let onto sodium chloride solution. The product is extracted with methyl ieri.-butyl ether, the united organic phase is washed with sodium chloride solution, dried over sodium sulfate, filtered and the filtrate solution is evaporated.

Example 4. Misoprostol

The crude misoprostol concentrate (8.11 mol) prepared according to Example 3. is dissolved in diisopropyl ether and purified by chromatography on a column made of 100kg of silica gel (Kieselgel Si 60 (0.063-0.200 mm)) using as eluent stepwise gradient mixtures of diisopropyl ether: acetone: methanol and 0.05% formic acid, wherein

diisopropyl ether: acetone: methanol=100:5:2, 100:10:2, 100:0.5:5, 100:0.5:7.5.

The united main fraction is concentrated. To the diisopropyl ether solution of the main fraction hexane is added until it undergoes opaque, then it is filtered through a 1.6kg active carbon bed using hexane: acetone=5:l and hexane: acetone=l:l solvent mixtures. The filtrate containing the product is concentrated, during concentration the solvent is changed to toluene.

The pre-purified misoprostol concentrate is purified by chromatography using a column of 20kg of silica gel (Kieselgel Si 60 (0.063-0.200 mm) and eluent mixtures made of dichloromethane: acetone=10:l, 0.05 % formic acid, dichloromethane: acetone =7:1, 0.05 % formic acid, methyl ieri.- butyl ether: acetone=2:l, 0.05% formic acid.

The aim of the filtration chromatography is to remove the contaminations arising from the solvents used during the purification, therefore to this chromatography distilled solvents are used.

The main fraction of the chromatography is neutralized with sodium hydrogen carbonate solution, washed to neutral with water, dried over sodium sulfate which contains active carbon, filtered, evaporated and made solvent-free.

Yield: 1.65kg, 53% (calculated on HO-enone), colorless oil.

Example 5.

Misoprostol

The crude misoprostol concentrate (1.3 mol) prepared of from 1.3 mol of enone according to Example 3., is dissolved in methyl ieri.-butyl ether and purified by chromatography using a column made of 22.5kg of silica gel (YMC S75 ) and eluent mixtures made of diisopropyl ether: isopropanol= 15:1, 0.05 % formic acid and diisopropyl ether: isopropanol =10:1, 0.05 % formic acid.

The united main fraction is washed with sodium hydrogen carbonate solution and then with sodium chloride solution, the neutralized solution is evaporated. The concentrate obtained after evaporation is dissolved in dichloromethane and purified by chromatography using silica gel column (Kieselgel Si 60 (0.063-0.200 mm) and eluent mixtures made of dichloromethane: acetone=10:l, 0.05 % formic acid, dichloromethane: acetone =7:1, 0.05 % formic acid and methyl ieri.-butyl ether: acetone=2:l, 0.05% formic acid. To the filtration chromatography distilled solvents are used.

The united main fraction is washed to neutral with sodium hydrogen carbonate solution and then with water, dried over sodium sulfate which contains active carbon, filtered and evaporated to solvent-free.

Yield: 275g, 55% (calculated to HO-enone), colorless oil.