FIELD OF THE INVENTION

The present invention relates to a new process for the synthesis of D-(-)-p- hydroxyphenylglycine ester (on ahead D-HPG ester) in crystal form and with an enantiomeric excess which is useful in the synthesis of β-lactam antibiotics.

BACKGROUND OF THE INVENTION

Hydroxyphenylglycine derivatives such as esters may be used in the synthesis of β-lactam antibiotics for example by enzymatic coupling of a D-(-)-p- hydroxyphenyglycine ester (D-HPG ester) to a suitable β-lactam nucleus.

D-(-)-p-hydroxyphenylglycine esters are optically active phenylglycine derivatives which may be used in the synthesis of β-lactam antibiotics, for example by enzymatic coupling of a D-(-)-p-hydroxyphenylglycine ester to a suitable β- lactam nucleus, e.g. 6-amino penicillanic acid (6-APA) and 7- aminodesacetoxycephalosporanic acid (7-ADCA) to produce amoxicillin and cefadroxil, respectively (see for example WO 96/02663, WO 97/04086, or Akema et al. Eur. J. Biochem. 269, p.2093-2100, 2002).

However it has been found that in the enzymatic synthesis of β-lactam antibiotics using the known D-HPG esters, the reaction times, until completion of the enzymatic reactions, are very long and furthermore, recovery of the β-lactam antibiotics, e.g. by filtration, is difficult.

Applicants have found that problems encountered in the recovery of β-lactam antibiotics by filtration are caused by small quantities of L-HPG ester during the enzymatic coupling (this substrate is recognized by the enzyme, producing diasteromeric dipeptide that under acidic conditions gives very unsoluble diketopiperazine) or free HPG ester at the end of the reaction, which remain in the final reaction mixture comprising the β-lactam antibiotic. In this context HPG ester remaining in the final reaction mixture comprising β-lactam antibiotics may be L- or D-isomer of the HPG ester. Therefore it has been shown that is desirable to operate the process for the enzymatic synthesis of β-lactam antibiotics by coupling a D-(-)-p- hydroxyphenylglycine ester (D-HPG ester) to a β-lactam nucleus under such conditions that a low concentration of HPG ester is remaining in the final reaction mixture. A low concentration of HPG ester may be defined as below 1 .0 wt%, preferably below 0.5 wt%, more preferably below 0.4 wt%, and more preferably below 0.3 wt% of HPG ester present in the reaction mixture.

These drawbacks have been overcome with the use of D-(-)-p- hydroxyphenylglycine ester in crystal form with an ee _D above 95%. In this sense it has been shown that D-(-)-p-hydroxyphenylglycine ester in crystal form with an ee _D above 95% allows the enzymatic reaction with a β-lactam nucleus to provide a β- lactam antibiotic to proceed until completion within a short time, while the final reaction mixture comprising the β-lactam antibiotic is easy to process. Preferably the D-(-)-p-hydroxyphenylglycine ester in crystal form has an ee _D above 97%, preferably above 98%, more preferably above 99%, more preferably above 99.5%, even more preferably above 99.7% and most preferably above 99.8%.

In several publications processes for the synthesis of HPG esters are disclosed. W098/13335 describes a process for the production of a methyl D-(-)-p- hydroxyphenylglycine ester (on ahead D-HPGM), which is synthesized starting from a racemic mixture of methyl p-hydroxyphenylglycine ester (HPGM) containing both the D- and L- isomer and wherein the racemic mixture and L-mandelic acid are treated with HCI to prepare the hydrochloric salt of D-HPGM. The crystals of D- HPGM as a HCI salt have an ee _D of 92.4%. In WO 98/13335 the D-HPGM crystals are not in the form of the free base.

WO 98/49133 discloses the synthesis of HPG esters with relative low yields (88.1 %) and does not disclose the synthesis of HPG esters wherein the D- enantiomer is produced with high ee _D .

Therefore there is still the need in the art to provide an alternative process for the synthesis of D-HPG esters in crystal form and enantiomeric excess which solves or at least mitigates these problems providing thus D-HPG esters with high yield and high ee _D . As above mentioned the obtained D-(-)-p-hydroxyphenylglycine esters in crystal form with an ee _D above 95% are useful in the synthesis of β-lactam antibiotics since they allow the enzymatic reaction with a β-lactam nucleus to provide a β-lactam antibiotic, to proceed until completion within a short time, while the final reaction mixtures are also easy to process.

DESCRIPTION OF THE INVENTION

Thus in one aspect the present invention refers to a process for the production of a D-(-)-p-hydroxyphenylglycine ester in crystal form with an ee _D above 95%. Said ee _D is preferably an ee _D above 97%, preferably above 98%, more preferably above 99%, more preferably above 99.5%, even more preferably above 99.7% and most preferably above 99.8%.

According to the present invention D-HPG ester in crystal form includes crystals of HPG ester (also referred to as HPG ester crystals: as used herein, the terms crystal of HPG ester and HPG ester crystals are intended to have the same meaning and will be used interchangeably). A single crystal of HPG ester may be a crystal of HPG ester in the D-form, a crystal of HPG ester in the L-form or a crystal in the D/L form (mixed crystals). The skilled man will appreciate that the ee _D of D- HPG ester in crystal form refers to the ee _D of the joint HPG ester crystals. The ee _D , i.e. the enantiomeric excess of the D-form, can suitably be determined using HPLC.

The process for the synthesis of D-(-)-p-hydroxyphenylglycine ester in crystal form of the invention, hereinafter also referred to as the process of the invention, preferably comprises (i) preparing a solution comprising dissolved D-HPG ester, and

(ii) crystallizing D-HPG ester from said solution to form a suspension comprising HPG ester crystals.

The ee _D of the D-HPG ester in the solution is preferably above 95%, preferably above 97%, more preferably above 98%, more preferably above 99%, more preferably above 99.5%. Preferably, the crystallization is effected under such conditions that the ee _D of the D-HPG ester crystals in the suspension is above 95%, preferably above 97%, more preferably above 98%, more preferably above 99%, more preferably above 99.5%. In a preferred embodiment, the ee _D of the HPG ester in the solution and the ee _D of the HPG ester crystals are above 95%, preferably above 97%, more preferably above 98%, more preferably above 99%, more preferably above 99.5%.

Preferably, during crystallizing HPG ester from the solution containing dissolved D-HPG ester to obtain the suspension comprising HPG ester crystals the suspension is stirred.

Crystallizing HPG ester in the process of the invention may be carried out at any suitable temperature. Preferably, said temperature is below 30 'C, more preferably below 25 °C, but most preferably below 20 ^. Crystallizing HPG ester from the solution containing dissolved D-HPG ester to obtain the suspension comprising HPG ester crystals with an ee _D above 95%, preferably above 97%, more preferably above 98%, more preferably above 99%, more preferably above 99.5% may preferably be carried out at temperature of between -5 and 30 'C, more preferably of between 0 and 25^ and most preferably of between 2 and 20 ^.

Crystallizing HPG ester from the solution containing dissolved D-HPG ester to obtain the suspension comprising HPG ester crystals may be performed in any suitable way. Preferably, said crystallizing is performed by the addition of water to the solution comprising dissolved D-HPG ester. Preferably, water is added to the solution comprising dissolved D-HPG ester and at the same time a base is added. A suitable base that may be added in crystallizing HPG ester to obtain the suspension comprising HPG ester crystals is for instance an aqueous solution comprising ammonia or sodium hydroxide (NaOH). Preferably, an aqueous solution comprising 1 to 25% w/w NaOH, preferably 2 to 15% w/w NaOH, more preferably 5 to 10% w/w NaOH is used as a base.

Crystallizing HPG ester in the process of the invention may be performed at any suitable pH. Preferably the pH is maintained between 3 and 10, more preferably between 4 and 9, most preferably between 5 and 8.

Surprisingly, it has been found that by using an aqueous solution of NaOH as a base during said crystallizing at the preferred pH values, the D-HPG ester crystals are formed such that the crystals are similar in size which results in improved separating and drying in the process of the invention, and that the ee _D of the D-HPG ester crystals is maintained above 95%, preferably above 97%, more preferably above 98%, more preferably above 99%, more preferably above 99.5%.

In an embodiment of the process of the invention the HPG ester crystals are separated from the suspension comprising HPG ester crystals, wherein the ee _D of the HPG ester crystals during said separating is maintained above 95%, preferably above 97%, more preferably above 98%, more preferably above 99%, more preferably above 99.5%. Separating the HPG ester crystals from the suspension may be performed in any suitable way, for example by filtration or centrifugation. Preferably, said separating is carried out by centrifugation.

Separating HPG ester crystals from the suspension comprising HPG ester crystals may be performed at any suitable temperature. Preferably said separating is carried out at a temperature of below 30 more preferably below 25 ^q C, but most preferably below 20 °C. Separating HPG ester crystals from the suspension comprising HPG ester crystals may preferably be performed at a temperature of between -5 and 30 °C, more preferably of between 0 and 25^ and most preferably of between 2 and 20 ^< €.

After separating the HPG ester crystals from the suspension comprising HPG ester crystals, the separated HPG ester crystals may be dried, wherein the ee _D of the HPG ester crystals during said drying is maintained above 95%, preferably above 97%, more preferably above 98%, more preferably above 99%, more preferably above 99.5%.

Drying HPG ester crystals in the process of the invention may be carried out at any suitable temperature. Preferably, the crystals are dried at a temperature of below 90 'Ό. Preferably, the crystals may be dried at a temperature of between 30 and ΘΟ 'Ό, more preferably of between 40 and 85 'Ό.

Surprisingly, it has been found that working under the preferred conditions according to the process of the invention, the ee _D of the D-HPG ester in crystal form is maintained above 95%, preferably above 97%, more preferably above 98%, more preferably above 99%, more preferably above 99.5%. Preferably, the ee _D of the HPG ester crystals obtained during said crystallizing, and/or said separating and/or said drying in the process according to the invention is maintained above 97%, more preferably above 98%, more preferably above 99%, more preferably above 99.5%, even more preferably above 99.7% and most preferably above 99.8%.

The solution comprising dissolved D-HPG ester may be prepared according to any suitable method, for example starting from D-(-)-p-hydroxyphenylglycine (on ahead D-HPG). Preparing a solution comprising dissolved D-HPG ester starting from D-HPG is preferably carried out by reacting D-HPG with an alcohol in the presence of sulfuric acid. The alcohol used may be any suitable alcohol. Preferably, the alcohol is methanol, ethanol, n-propanol or glycol. More preferably, the alcohol is methanol.

The D-HPG used in reacting D-HPG with an alcohol to prepare D-HPG ester with an ee _D above 95%, preferably has an ee _D of above 95%. More preferably, the ee _D of said D-HPG is above 97%, more preferably above 98%, more preferably above 99%, more preferably above 99.5%, more preferably above 99.7% and most preferably above 99.8%.

Reacting D-HPG in the process of the invention may be carried out at any suitable temperature resulting in D-HPG ester with an ee _D of above 95%. The temperature at which said reacting is carried out in the process according to the invention, is preferably below Ι ΟΟ 'Ό. Preferably, the temperature at which D-HPG may be reacted with an alcohol is below 90 °C, more preferably below 85 'Ό. The temperature at which the HPG may be reacted with an alcohol may range between 50 and 100°C, more preferably between 55 and 90 °C and most preferably between 60 and 85 °C.

Any β-lactam antibiotic wherein D-HPG is present as a side chain may be synthesised with the D-HPG ester in crystal form prepared according to the process of the present invention. The D-HPG ester in crystal form may be used for example in the synthesis of the β-lactam antibiotics amoxicillin, cefadroxil and cefprozil by enzymatic coupling of a D-HPG ester to 6-APA, 7-ADCA and 7-amino-3-[(Z)-1 - propenyl]-3-(desacetoxymethyl) cephalosporanic acid, respectively. Processes thereof are well known in the state of the art.

Thus, preferably the D-(-)-p-hydroxyphenylglycine ester in crystal form obtained by the process of the invention is a methyl, ethyl, n-propyl or 2-hydroxy- ethyl ester. More preferably the D-HPG ester is D-(-)-p-hydroxyphenylglycine methyl ester (D-HPGM). The D-HPG ester in crystal form may be obtained in any suitable form, for example in the form of its free base or in the form of a salt, such as a HCI salt, sulphuric acid salt or a formic acid salt of D-HPG ester. Preferably the D-HPG ester in crystal form is obtained in the form of its free base. In this sense it was surprisingly found that crystals of D-HPG esters as their free base can be used directly, i.e. without prior dissolving the D-HPG ester crystals, in the synthesis of β- lactam antibiotics.

The foregoing is illustrative of the present invention. This invention however is not limited to the following precise embodiments described herein, but encompasses all equivalent modifications within the scope of the claims which follow.

EXAMPLES

Example 1 : Synthesis of D-HPGM in crystal form i) Synthesis o. D-HPGM from D-HPG

A reactor was loaded with 7 moles of methanol (0.1 % v/v H ₂ 0), 1 .2 moles of sulphuric acid (H ₂ S04 98%) and 1 mol of HPG (ee >99.8%) (step a). The reaction mixture was heated until reflux to a temperature of about 74°C and the reaction mixture was stirred during 2 hours (step b). Subsequently, water which was formed during the reaction under b) and the remaining methanol were distilled off under vacuum in batch at <-0.94 bar and T=75 ⁵ C (step c). Thereafter, nitrogen was introduced into the reactor till atmospheric pressure was reached and subsequently 7 moles of methanol (0.1 % v/v H ₂ 0) were added to the reaction mixture (step d). Steps b, c and d were repeated twice, with the exception that in the last step 2,5 moles of methanol 0.1 % v/v water (230 g/IJ were added instead of 7 moles. The resulting reaction mixture is called "final D-HPGM solution".

/ ^' / ^' ) Neutralisation

The "final D-HPGM solution" was introduced into a second reactor, wherein said solution was neutralised by adding water and NaOH 8,5% (w/v) to maintain the pH between 6 and 7. When all the "final D-HPGM solution" was loaded, the pH was adjusted to 7.5, while stirring. During the neutralisation step, D-HPGM crystallizes to obtain a suspension comprising D-HPGM crystals. The temperature during neutralisation was maintained below 20 'Ό.

/ ^' / ^' / ^' ) Centrifugation and drying

The D-HPGM crystals that were formed during neutralisation under ii) were separated from the suspension comprising D-HPGM crystals by centrifugation at a temperature of below 20 ⁵ C. The product was washed with 300 g of water. The D-

HPGM crystals separated by centrifugation were dried at 70 ⁵ C under vacuum. iv) Determination enantiomeric excess (ee) of D-HPGM in crystal form

The ee _D of D-HPGM was determined on HPLC, with a HPLC column Crownpack CR(+) (Daicel) 150 mm x 4 mm I.D., 5 μηι.. The eluent was HCI0 ₄ pH

1 ,2: Methanol 90:10 (v/v), at flow velocity of 1 ,0 ml/min and the column temperature was maintained at 30°C. Standard solutions of racemic HPG and HPGM were prepared by dissolving 50 mg of D,L-HPG in 0.1 N HCL and 50 mg of D,L-HPGM in 0,1 N HCI. Solutions of the HPGM crystals obtained with the process under steps i- iii were prepared in a similar way as the solutions of standards. Solutions of standards and samples were filtered through 0,22 μηι filters prior to injection on HPLC. D-HPG, L-HPG, D-HPGM and L-HPGM were determined at 220 nm.

The enantiomeric excess was calculated by the expression:

A _D - A _L

Enantiomeric excess (HPLC, % area) = x 100

A _D + A _L

A _D is the average area of the D(-)HPGM peak in the sample. A _L is the average area of the L(+)HPGM peak in the sample.

The ee of the D-HPGM (= ee _D ) prepared according to the process described above under steps i) to iii) was between 99.7 and 99.9%.

Example 2: Influence of neutralisation temperature on ee _D of D-HPGM crystals

All steps for the synthesis of D-HPGM in crystal form were similar to those as described in Example 1 , except that the neutralisation step ii) was carried out at 35 °C. The ee _D of D-HPGM thus obtained was determined by HPLC and was 97.4%.

Examples 1 and 2 show that an increase in the temperature at which the neutralisation step ii) is performed from 20 to 35^ results in a decrease of the ee _D of D-HPGM.

Example 3: Enzymatic synthesis of cefadroxil with D-HPGM In crystal form with different ee _D values

/ ^' Enzyme and Immobilisation

The penicillin acylase as used herein was a Pen-G acylase mutant Phe-24- Ala as described in WO 98/20120. The enzyme was immobilised as described in EP

222 462, with gelatin and chitosan being used as gelling agent and glutaraldehyde as crosslinker. ii) S\nthesis of cefadroxil with D-HPGM in e stal fonn with an ee _n of 92. 5% 95% 97.5% and 99.9 %

8.0 9 7-ADCA (37.1 mmol) and 6.9 9 HPGM with an ee _D of 99.9 % (37.9 mmol) were suspended in 48.5 ml_ water, the mixture was cooled to 12 ^< Ό and the pH increased to 7.3 with ammonia. At t = 0 min., 6,50 g immobilized Pen-G acylase mutant Phe-24-Ala was added. The reaction was carried out at constant temperature (12 ^< Ό) and constant pH (titration with formic acid at pH = 7.3). The time until the concentration of HPGM had reached a value of below or equal to 0.5 wt. % and the time until the concentration of HPGM had reached a value of below or equal to 0.3 wt. % were determined.

This procedure was also carried out using HPGM with an ee _D of 97.5 %, 95.0 %, and 92.5 %. The results are summarized in Table 1 .

Table 1 . The reaction time until completion of the reaction at different HPGM concentrations

Time until [HPGM] <= 0.5% Time until [HPGM] <=0.3% ee _D (%)

(min.) (min.)

99.9 135 150

97.5 155 >1000

95 300

92.5 >1000