WO2010072672A1

WO2010072672A1 - New crystal polymorphs of ceftobiprole

Info

Publication number: WO2010072672A1
Application number: PCT/EP2009/067514
Authority: WO
Inventors: Paolo Ghetti; Paul Hebeisen; Markus Heubes; Giovanni Pozzi; Michael Schleimer
Original assignee: Basilea Pharmaceutica Ag
Priority date: 2008-12-24
Filing date: 2009-12-18
Publication date: 2010-07-01
Also published as: TW201028427A

Abstract

A compound of formula (I) in a crystalline form, which has a X-ray diffraction pattern comprising, when measured using Cu- Kα₁ radiation, a diffraction line, having a relative intensity (rl) of 100 percent at about 11.6, 12.9, 14.7, 22.0, 23.1 or 24.5, given in degrees 2 Theta [2θ]. Five corresponding polymorphic crystal forms (Polymorph A; B; C; D or E) have been characterized by means of their XRPD and have been isolated.

Description

New crystal polymorphs of Ceftobiprole

The present invention relates to the compound of formula (I)

which is also known under the name "Ceftobiprole" (CAS-No. 09467-52-7) and is a broad spectrum antibiotic described e.g. in EP-A-O 849 269 and US-A-5,981,519. A derivative of Ceftobiprole having improved solubility in aqueous media is Ceftobiprole Medocaril having the following formula

which has been described e.g. in WO-A 99/65920. One way for preparing Ceftobiprole Medocaril is described e.g. in WO-A 01/90111 and includes the reaction of Ceftobiprole with carbonic acid 5-methyl-2-oxo-[l,3]dioxol-4-ylmethyl ester 4-nitro-phenyl ester. The compound of formula (I), Ceftobiprole, is thus not only important as an antibiotic by itself but also as a potential intermediate for the manufacture of derived antibiotics like e.g. Ceftobiprole Medocaril.

A manufacturing process for the compound of formula (I) is described in EP-A-O 849 269. In this manufacturing process (6R,7R)-7-[(Z)-2-(5-amino-[l,2,4]thiadiazol-3-yl)-2-trityl- oxyimino-acetylamino]-8-oxo-3-[(E)-(R)-2-oxo-[l,3']bipyrrol-idinyl-3-ylidenemethyl]-5- thia-l-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid is initially prepared and then converted to the compound of formula (I) as specifically described in Example 11.1 of EP- A-O 849 269. It has turned out, however, that the product obtainable by this process is not viable for industrial practice. In particular, it was found that the stability of the compound of formula (I) is much too low for practical use because almost half of the compound of formula (I) being originally present in a sample manufactured according to EP-A-O 849 269 decomposes already within four weeks. In addition to this unacceptable shelf life, the purity (i.e. the initial percentage in compound of formula (I)) of the material manufactured according to EP-A-O 849 269) was found to be only about 85% maximum, which is not satisfactory, either.

It was therefore necessary to improve the initial purity of the material obtained according to EP-A-O 849 269 as well as its long-time stability drastically in order to render the compound of formula (I) applicable at all in everyday medicinal practice. Conventional methods for an appropriate improvement would have included extensive purification of the initial Ceftobiprole substance and the use of appropriate stabilizer compounds in order to obtain a sufficient shelf life of the initial material.

It is known that beta-lactam antibiotics are compounds sensitive to hydrolytic degradation and not easy to crystallize. (Y. Ikeda et al, Chem. Pharm. Bull. 56(10) 1406-1411(2008). Moreover it is known, that only certain polymorphs of beta-lactam antibiotics display sufficient stability which is attributed to the binding of water molecules in the crystal lattice (Y. Takeuchi et al, Chem. Pharm. Bull 41(11) 1998-2002 (1993)).

Surprisingly, it has been found, however, that a specific polymorph of the compound of formula (I), hereinafter referred to as Polymorph D, shows such strongly improved purity and stability against decomposition that it provides a simple solution to the aforementioned problem.

It has furthermore been found in this context that the compound of formula (I) exists in several polymorphic forms, which have now been isolated and characterized by X-ray powder diffraction (XRPD) methods. Polymorphism is defined as the ability of a drug substance to exist as two or more crystalline phases that have different arrangements and/or conformations of the molecules in the crystal lattice. Amorphous solids, on the other side, consist of disordered arrangements of molecules and do not possess a distinguishable crystal lattice. Polymorphs can have different chemical and physical properties such as melting point, chemical reactivity, apparent solubility, dissolution rate, optical and electrical properties, vapour pressure, and density. These properties can have a direct impact on the process-ability of drug substances and the quality/ performance of drug products, such as bioavailability, dissolution and especially stability. Metastable pharmaceutical solid forms, in particular, frequently change crystalline structure over time in response to changes in environmental conditions, like temperature or humidity, and/or processing and the like. The definitive criterion for the existence of polymorphism is via demonstration of non-equivalent crystal structures, usually by comparison of their x-ray diffraction patterns. Microscopy, thermal analysis methodology, and solid state NMR are generally considered as sources of supporting information obtained by X-ray diffraction analysis.

Manufacturing Ceftobiprole in one of the newly discovered polymorphic forms A, B, C, D and E described below facilitates the manufacture of medicaments comprising Ceftobiprole as such as well as the manufacture of derivatives of Ceftobiprole like in particular Ceftobiprole Medocaril.

In particular, the newly found Polymorph D of Ceftobiprole as defined herein, provides an unexpectedly strong improvement of the Ceftobiprole substance in stability and initial purity in comparison to the known amorphous prior art forms of Ceftobiprole, like those obtained according to EP-A-O 849 269.

The new polymorphs have an X-ray diffraction pattern comprising, when measured using Cu-Kαi radiation, a strong diffraction line (peak), in particular a diffraction line having a relative intensity (rl) of 100 percent, at about 11.6 or 12.9 or 14.7 or 22.0 or 23.1 or 24.5, said values being given in degrees 2 Theta [2Θ]. The estimated variation of the 2 Theta measurement is < +/- 0.2 degree. More particularly, the present invention is directed to Ceftobiprole in one of the polymorphic crystal forms indicated hereinafter as form A; B; C; D or E, which have X-ray diffraction patterns, when measured using Cu-Kαi radiation, comprising the characteristic diffraction lines shown below for each of the polymorphic forms and given in degrees 2 Theta [2θ]:

The indicated diffraction lines have a relative intensity of at least 20% based on the strongest peak of the respective diffraction pattern. It will be acknowledged by those skilled in the art that the relative intensities of the diffraction peaks of a specific compound may vary considerably, depending e.g. on the particle size of the sample, technical and/or device-related parameters used for recording the diffraction diagram, etc. This can be seen for example when one compares the intensity of the peak at 8.4 degrees 2-Theta in diagrams shown in Figures 2 and 7. The diffraction angles of the peaks are generally less variable and show usually a variation of 0.2 degree 2 Theta maximum.

The X-ray diffraction patterns of the new polymorphs A; B; C; D and E, when measured using Cu- Ka i radiation, comprise in particular the following diffraction peaks having a relative intensity (rl) of 40% and more: Form B

2Θ RI

7.9 m

10.3 m

10.5 m

11.9 m

12.9 vs

14.4 m

15.3

16.4 m

17.0 m

18.8 m

20.0 m

21.8 m

22.0 vs

24.1 m

25.3 m

27.3 m

Form D Form E

2Θ rl 2Θ rl

8.4 S 4.6 S

9.9 m 8.0 m

16.9 m 11.5 m

24.0 m 12.3 m

24.5 VS 14.7 VS

26.7 m 18.3 m

18.7 m

20.5 m

22.9 m

23.3 m

24.7 m

26.0 S

In the Tables above the relative intensities of the diffraction lines (rl) are classified as follows:

Symbol rl [%]

VS 100

S <100 to 85 m <85 to 40

W <40 The X-ray diffraction patterns of the mentioned crystalline forms may also show further peaks, in particular peaks having a relative intensity of less than 40. One or more of the weaker diffraction lines may, dependent on the exact recording conditions, not be found in any recorded x-ray diagram, and it can also not be entirely excluded that one or more of these reflections of low intensity may be reflections caused by impurities contained in the measured samples.

Particularly preferred embodiments of the present invention are

• the polymorphic crystal form D of Ceftobiprole(Polymorph D); • its polymorphic crystal form A (Polymorph A) and

• its polymorphic crystal form E (Polymorph E).

Most preferred is Form D. Polymorph D is the most stable polymorph of Ceftobiprole found, and appears to be the form being thermodynamically stable under usual conditions. Polymorph D of Ceftobiprole is of much higher purity than the amorphous prior art form of said compound and has furthermore a significantly reduced tendency to decompose during storage, also at elevated temperatures and under conditions of increased relative humidity as shown in Example 8 of this application.

The polymorphs A, B, C, D and E according to the invention can be easily distinguished by their X-ray powder diffraction diagrams which differ significantly from one another.

Brief description of the drawings:

Figure 1 shows a XRPD spectrum (recorded with Cu-Kαi radiation) of the polymorph A of the Ceftobiprole.

Figure 2 shows a XRPD spectrum (recorded with Cu-Kαi radiation) of polymorph D of Ceftobiprole. Figure 3 shows a XRPD spectrum (recorded with Cu-Kαi radiation) of polymorph E of Ceftobiprole.

Figure 4 shows a XRPD spectrum (recorded with Cu-Kαi radiation) of amorphous Ceftobiprole as obtained according to EP-A 0 849 269.

Figure 5 shows the change of the purity of Ceftobiprole in form of its Polymorph D versus that of corresponding amorphous material over four weeks.

Figure 6 shows the change in the percentage of Polymorph D in Ceftobiprole versus that of corresponding amorphous material over four weeks.

Figure 7 shows the XRPD spectrum of the freshly prepared Ceftobirole Polymorph D Batch 07/R0004 referred to in Example 8 (t=0 weeks).

Figure 8 shows the XRPD spectrum of the Ceftobirole Polymorph D Batch 07/R0004 referred to in Example 8 after a 4 weeks' storage at a temperature of 40⁰C and a relative humidity (r.h.) of 75% (t=4 weeks).

The crystalline Ceftobiprole polymorphs of the present invention can be prepared as follows:

Polymorph A can e.g. be obtained from (6R,7R)-7-[(Z)-2-(5-amino-[l,2,4]thiadiazol-3-yl)- 2-trityloxyimino-acetylamino]-8-oxo-3-[(E)-(R)-2-oxo-[l,3']bipyrrolidinyl-3-ylidene- methyl]-5-thia-l-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid dihydrochloride

C(Ph)₃

HCI by reaction with 90% formic acid, isolation of the obtained solid and purification thereof by chromatography with MCl Gel using a gradient of water with increasing concentrations of acetonitirile, and isolating the polymorphs from the collected fractions of pure compound of formula (I) obtained by the chromatography, as described in more detail in Example 3 or in an analogous manner.

Polymorph D can e.g. be obtained by reaction of (6R,7R)-7-[(Z)-2-(5-amino-

[l,2,4]thiadiazol-3-yl)-2-trityloxyimino-acetylamino]-8-oxo-3-[(E)-(R)-2-oxo-

[ 1 ,3']bipyrrolidinyl-3-ylidenemethyl]-5-thia- 1 -aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid with trifluoroacetic acid and triethylsilane at temperatures below about 0⁰C, adding a mixture of water, ethylacetate, acetonitrile and further trifluoroacetic acid, transferring the separated aqueous phase to a MCl gel column, eluting the product with an about 9: 1 :0.4 mixture of water:acetonitile:trifluoroacetic acid, removing the acetonitrile under vacuo, precipitating the product by adjusting the pH to about 3 and isolating the obtained polymorph D of the compound of formula (I). A more detailed description is given in Example 6.

The polymorphs B, C and E can be obtained as decribed in Examples 4, 5 or 7 of this application, respectively, or in an analogous manner.

Example 1 :

(6R,7R)-7-|YZ)-2-(5-Amino-|^" 1 ,2,41thiadiazol-3-yl)-2-trityloxyimino-acetylaminol -8-oxo- 3-rrEV(RV2-oxo-ri.3'lbipyrrolidinyl-3-ylidenemethyll-5-thia-l-aza-bicvclor4.2.01oct-2- ene-2-carboxylic acid dihydrochloride

To a suspension of (6R,7R)-3-[(E)-(R)-r-allyloxycarbonyl-2-oxo-[l,3']bipyrrolidinyl-3- ylidenemethyl]-7-[(Z)-2-(5-amino-[l,2,4]thiadiazol-3-yl)-2-trityloxyimino-acetylamino]-8- oxo-5-thia-l-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (4.12 g, 4.79 mmol) prepared e.g. according to EP-A-O 849 269, in 280 ml dichloromethane is added 1.87 ml (7.66 mmol) N,O-bis(trimethylsilyl)acetamide whereby an orange solution is formed which is subsequently treated with 84 mg (0.12 mmol) bis-(triphenylphosphine)-palladium(II)- dichloride, 5.48 ml (95.8 mmol) acetic acid and 11.7 ml (44.1 mmol) tributyltin hydride and stirred at room temperature for 40 min. After addition of a few drops of water, the suspension is poured on 1500 ml diethyl ether containing 12 ml of a solution of 6 M hydrogen chloride in diethyl ether. The suspension is stirred for 2 h and the product is collected by filtration.

Yield: 4.04 g (99%) beige solid IR(KBr) 1781, 1659 cm^{' 1}

MS(ISP) 777.4 (M+H)⁺.

Example 2:

(6R,7R)-7-[(Z)-2-(5-Amino-[l,2,41thiadiazol-3-yl)-2-trityloxyimino-acetylaminol-8-oxo- 3-[(E)-(R)-2-oxo-[l,3'lbipyrrolidinyl-3-ylidenemethyll-5-thia-l-aza-bicyclo[4.2.01oct-2- ene-2-carboxylic acid

13.2 g Palladium(II) acetate (0.059 mol), 46.2 g triphenylphosphine (0.17 mol) are dissolved under nitrogen in 4.2 L of tetrahydrofurane and 116 ml formic acid (3 mol) are added at room temperature. The resulting solution is cooled to 0-10°C and a solution of (6R,7R)-3-[(E)-(R)- 1 '-Allyloxycarbonyl-2-oxo-[ 1 ,3']bipyrrolidinyl-3-ylidenemethyl]-7-

[(Z)-2-(5-amino-[ 1 ,2,4]thiadiazol-3-yl)-2-trityloxyimino-acetylamino]-8-oxo-5-thia- 1 -aza- bicyclo[4.2.0]oct-2-ene-2-carboxylic acid (704 g, 0.73 Mol) prepared e.g. according to EP- A-O 849 269, in 6 L of tetrahydrofurane is added within 2-10 min. Then 400 ml of tributyltinhydride (1.51 mol) are added within 2-10 min at -5 to -10⁰C. The cooling is removed and the mixture is stirred for 15-30 min. The resulting precipitate is collected by filtration, washed with 3.5 L tetrahydrofurane and dried at room temperature and under a pressure of less than 10 bar over night.

Yield: 450 g Example 3 (Ceftobiprole-Polymorph A):

17.89 g of (6R,7R)-7-[(Z)-2-(5-amino-[ 1 ,2,4]thiadiazol-3-yl)-2-trityloxyimino- acetylamino]-8-oxo-3-[(E)-(R)-2-oxo-[ 1 ,3']bipyrrolidinyl-3-ylidenemethyl]-5-thia- 1 -aza- bicyclo[4.2.0]oct-2-ene-2-carboxylic acid dihydrochloride are added to 200 ml 90% formic acid and are stirred for 1 h. This suspension is added under stirring to 2 L ethyl acetate and is stirred for further 30 min. The solid material is isolated by filtration, washed with ethyl acetate and diethyl ether and is dried at room temperature in vacuo for 3 h. The beige solid is purified by chromatography with MCI Gel (Mitsubishi, CHP-20P) using a gradient of water with increasing concentrations of acetonitrile. All fractions containing the pure product are combined and acetonitrile is removed by evaporation. The precipitate is collected by filtration and is dried in vacuo.

Yield: 7.56 g light yellow crystals IR(MIR: 1764, 1658 cm^{' 1}

MS(ISP): 535.1 (M+H)⁺. 1H-NMR(25O MHz): δ [ppm] in DMSO-d6:

4H, 3.60, d,J = 17 Hz 3.79, d,J = 17 Hz; 6H, 5.07, d,J = 8 Hz; 7H, 5.75, dd,J = 5 Hz, J = 8 Hz; NH, 9.46, d,J= 8 Hz; NH2, 8.05, s; OH, 12.0, s br; 3Η, 7.30, s; 4¹H, 5¹H, 4"H, 5"H, 1.92-2.16, 2.5-3.5, m; 1"H, 10.3 s br; 3"H, 4.66, m.

The XRPD diffraction lines (recorded with Cu-Kαi radiation) are listed in the following Table 1 :

Example 4 (Ceftobrprole-Polvmorph B):

1 g of (6R,7R)-7-[(Z)-2-(5-Amino-[ 1 ,2,4]thiadiazol-3-yl)-2-trityloxyimino-acetylamino]-8- oxo-3-[(E)-(R)-2-oxo-[l,3']bipyrrolidinyl-3-ylidenemethyl]-5-thia-l-aza-bicyclo[4.2.0]oct- 2-ene-2-carboxylic acid dihydrochloride are stirred for 1 h in 10 ml of 90% formic acid, are then poured onto 100 ml of ethyl acetate and stirred for another 1 h. The solid is collected by filtration and is added to 8 ml aqua dest. resulting in a homogenous suspension. Acetonitrile (few drops) is added until a orange solution is obtained. This solution is purified on 100 ml of MCI-gel (Mitsubishi, CHP-20P), eluted with water (10 min) and a gradient of acetonitrile to 90% water/10% acetonitrile (100 min). The fractions containing the pure product are concentrated in vacuo and the resulting solid is collected by filtration and dried under high vacuum.

Yield: 270 mg beige solid

IR(MIR), MS(ISP) and 1H-NMR(25O MHz) cf. Example 3

The XRPD diffraction lines (recorded with Cu-Kαi radiation) are listed in the following Table 2:

Example 5 (Ceftobrprole-Polvmorph C):

To a mixture of 0.6 ml triethylsilane and 10 ml of trifluoroacetic acid at 0°C is added 1.62 g of(6R,7R)-7-[(Z)-2-(5-amino-[l,2,4]thiadiazol-3-yl)-2-trityloxyimino-acetylamino] -8- oxo-3-[(E)-(R)-2-oxo-[l,3']bipyrrolidinyl-3-ylidenemethyl]-5-thia-l-aza-bicyclo[4.2.0]oct- 2-ene-2-carboxylic acid with stirring. The mixture is continued to stirr at 0°C for 30 min. Trifluoroacetic acid is evaporated under aspirator vacuum and the residue is dried under high vacuum. The residue is distributed between water and ethyl acetate and the aqueous phase is slightly concentrated. The product is precipitated by adjusting the pH to 3.00 by addition of 2N sodium hydroxide solution at 0°C. The suspension is stirred at 0°C for 3 h and the product is collected by filtration.

The solid is dissolved in 15 ml IN HCl and 1.5 ml acetonitrile. The solution is passed through a bed of 5 g of MCI-gel (Mitsubishi, CHP-20P) and the gel is washed with 120 ml of a 9: 1 mixture of 1 N HCL and acetonitrile. The filtrate is concentrated to remove acetonitrile and the pH is adjusted to 3.0 by addition of cone. NaOH. The resulting suspension is concentrated to a volume of approx. 40 ml and stirred at 0°C for 2 h. The solid is collected by filtration and dried to constant weight.

Yield: 0.76 g off-white solid

IR(MIR), MS(ISP) and 1H-NMR(25O MHz) cf. Example 3

The XRPD diffraction lines (recorded with Cu-Kαi radiation) are listed in the following Table 3:

Example 6 (Ceftobiprole-Polymorph D):

1.6 g of (6R,7R)-7-[(Z)-2-(5-amino-[l,2,4]thiadiazol-3-yl)-2-trityloxyimino-acetylamino] - 8-oxo-3-[(E)-(R)-2-oxo-[ 1 ,3']bipyrrolidinyl-3-ylidenemethyl]-5-thia- 1 -aza- bicyclo[4.2.0]oct-2-ene-2-carboxylic acid are added to 5 ml trifluoroacetic acid at -10°C with stirring. Triethylsilane (0.3 ml) are added at once and the mixture is stirred at 0°C for 30 min. Approx. 4 g ice, 25 ml ethyl acetate and 25 ml of a 9: 1 :0.4 mixture of water:acetonitrile:trifluoroacetic acid are added. The clear phases are separated. The aqueous phase is transferred onto a column containing 17 g of MCI-gel (Mitsubishi, CHP- 20P) conditioned with a 9:1 mixture of water and acetonitrile. From this bed the product is eluted with a 9:1 :0.4 mixture of water: acetonitrile :trifluoroacetic acid. The product is contained in 4 fractions of approx. 12 ml each. Acetonitrile is removed under aspirator vacuum and the pH is adjusted to 3.00 by addition of cone. NaOH. The resulting precipitate is stirred at 0°C for 4 h and collected by filtration.

Yield: 0.65 g off-white crystalline solid

IR(MIR), MS(ISP) and 1H-NMR(25O MHz) cf. Example 3

The XRPD diffraction lines (recorded with Cu-Kαi radiation) are listed in the following Table 4:

Example 7 (Ceftobiprole-Polymorph E):

To a mixture of 820 ml trifluoroacetic acid and 72 ml of triethylsilane in 2.24 L of methylene chloride are added portion wise within 10-20 min 380 g of (6R,7R)-7-[(Z)-2- (5-amino-[ 1 ,2,4]thiadiazol-3-yl)-2-trityloxyimino-acetylamino] -8-oxo-3-[(E)-(R)-2-oxo- [ 1 ,3']bipyrrolidinyl-3-ylidenemethyl]-5-thia- 1 -aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid at -20°C. The resulting suspension is stirred for another 15 min at -15°C. From the biphasic solution the lower part is separated and the upper part (methylene chloride) is extracted with a mixture of 290 ml trifluoroacetic acid and 290 ml water. The lower phase and the extract are combined and diluted with 290 ml acetonitrile in 2.9 L of aqua dest.

The combined solution is filtrated over 130 g Dicalite Speedex and washed twice with 270 ml of a solution of 1.2:3:24 trifluoroacetic acid:acetonitrile:water. The combined filtrates are chromatographed over 4 kg MCI-gel (Mitsubishi, CHP-20P), eluted with 20 L of a solution of 1.2:3:24 trifluoroacetic acid:acetonitrile:water.

The main fraction containing the product is evaporated at 40-45 °C at 100 to 40 mbar to a volume of 6 to 8 L. The resulting light yellow solution is diluted with 1.9 L aqua dest. Under cooling to 0°C the pH is adjusted to 3.6 to 3.8 using 1.1 L 28% sodium hydroxide solution and at > pH 3 diluted (0.2 M) sodium hydroxide solution. After further stirring at 0-5°C for 3-4 h, the suspension is filtrated, washed with 1.9 L of cold aqua dest (2°C) and 1.9 L of cold acetone (2°C) and the resulting crystals are dried for 14.5 h at 40°C at < lbar.

Yield: 172 g crystals

IR(MIR), MS(ISP) and 1H-NMR(25O MHz) cf. Example 3

The XRPD diffraction lines (recorded with Cu-Kαi radiation) are listed in the following Table 5:

Example 8

The stability of a Ceftobiprole batch having crystal form D obtained according to Example

6 (batch 07R0004) and of a amorphous batch Ceftobiprole obtained according

EP-A 0 849 269 (batch 966/15) are compared after storage at 40°C/75% r.h. for 0, 1, 2, and

4 weeks. The Purity/ Assay are monitored by HPLC and the crystallinity is investigated by

XRPD.

HPLC measurements:

Equipment:

HPLC system: Quaternary pump module (Waters Alliance 2695) DAD detector (Waters Alliance 2996) Auto sampler (Waters Alliance 2695) Column oven (Waters Alliance 2695) Empower-control and integration software

Mettler Toledo MT5. balance capable of weighing the required amount of compounds in a tolerance of 0.5% and a ratio of reading error/tolerance of 1/5

HPLC Method Details:

Flow: 1.2 mL/min

Mobile phase A: 3/97/0.1 , ACN/water/TFA (v/v/v) Mobile phase B: 1000/1 , ACN/TFA(v/v) Column: Prontosil 120-3 -C 18 (75 X 4.6 mm), 3μm particle size Column temp: 3O⁰C Autosampler: 5°C

Injection: 10μL

Detection: UV or DAD λ=229nm

Gradient program for this method

Time (min) %A %B

0.0 100 0

1.0 100 0

10.0 88 0

25.0 82 18

42.0 60 40

46.0 30 70

48.0 5 95

50.0 5 95

50.4 100 0

55.0 100 0

Materials:

Acetonitrile: Merck lichrosolv gradient grade

Water: Merck lichrosolv gradient grade.

Sample solvent:

Water: add 400 mL of M.P.A. to a 500 mL graduated cylinder. Make to volume with acetonitrile. Add 15 mL Trifluoroacetic acid. Samples:

Preparation of mobile phases:

Mobile phase A

Add 30 mL acetonitrile to a IL graduated cylinder. Make to volume with water. Add ImL trifluoroacetic acid.

Mobile phase B

Add 1 mL acetonitrile to ImL trifluoroacetic acid. Calculation of the Assay of a Ceftobiprole sample, i.e. the analytic content of the sample in Ceftobiprole:

Assay(sample) =

* _Assaysm * _{1 O}o Area_STD mg_sample

Results:

The crystalline form Polymorph D of Ceftobiprole is significantly more stable than its amorphous form of (cf. Table 6, Table 7 and particularly Table 8).

The purity of the crystalline Ceftobiprole-Polymorph D remains constant over the storage while the purity of the corresponding amorphous compound decreases by 25%. "Purity" as used herein means %area of the Ceftobiprole peak in the HPLC diagram.

The Assay (as defined above) of the crystalline Ceftobiprole-Polymorph D decreases by about 3% only over the four weeks of storage while the Assay of the amorphous sample of Ceftobiprole decreases by almost 40%.

Table 6: Ceftobiprole batch 07R0004(crystalline) Stability

Table 7: Ceftobiprole batch 966/15 (amorphous) Stability

Table 8: Overview, relative percent change

calculated as Purity(time point) - Purity(t=O) [^b]calculated as Assay(time point) - Assay(t=0)

Figure 5 and Figure 6 are a graphical representation of the data listed in Table 8.

As seen in the XRPDs (cf. Table 9 below), the Ceftobiprole Polymorph does not change crystal form during the test. The XRPD pattern of the Ceftobiprole batch 07R0004 corresponds to the crystal form D.

Table 9: Comparison of the 2-Theta peak intensities of Ceftobiprole batch 07R0004, after storage of t=0 weeks and t=4 weeks

The NMR spectrum of Ceftobiprole-batch 966/15 shows impurity signals after four weeks storage due to the amount of degradation.

Claims

Claims:

1. A compound of formula (I)

(Ceftobiprole) in a crystalline form, which has a X-ray diffraction pattern comprising, when measured using Cu-Kαi radiation, a diffr _CX_Daction line, having the relative intensity (rl) of 100 percent at about 11.6 or 12.9 or 14.7 or 22.0 or 23.1 or 24.5, given in degrees 2 Theta [2Θ].

2. A compound of formula (I) according to claim 1 in one of the polymorphic crystal forms indicated hereinafter as form A; B; C; D or E, which have X-ray diffraction patterns, when measured using Cu- Kαi radiation, comprising the diffraction peaks indicated hereinafter for each of the polymorphic forms and given in degrees 2 Theta [2Θ], the indicated diffraction peaks having a relative intensity of 20% and more based on the strongest peak of the respective diffraction pattern:

3. A compound of formula (I) according to claim 2 which have X-ray diffraction patterns, when measured using Cu-Kαi radiation, comprising the diffraction lines indicated hereinafter for each of the polymorphic forms and given in degrees 2 Theta [2Θ], the indicated diffraction peaks having a relative intensity of 40% and more based on the strongest peak of the respective diffraction pattern:

wherein the relative intensities of the diffraction lines (rl) are classified as follows:

Symbol rl [%] vs 100 s <100 to 85 m <85 to 40 w <40

4. A compound of formula (I) according to any one of claims 2 to 3 in the one of the polymorphic crystal forms A, D or E.

5. A compound of formula (I) according to any one of claims 2 to 4 in the polymorphic crystal forms D and E.

6. A compound of formula (I) according to any one of claims 2 to 5 in the polymorphic crystal form D.

7. A compound according to any one of claims 2 to 6 having a x-ray powder diffraction diagram comprising the following diffraction lines (recorded with Cu- Ka i radiation):

8. A compound of formula (I) according to any one of claims 1 to 4 in the polymorphic crystal form A.

9. A compound according to any one of claims 1 to 4 having a x-ray powder diffraction diagram comprising the following diffraction lines (recorded with Cu- Ka i radiation):

10. A compound according to anyone of claims 1 to 4 in the polymorphic crystal form E.

11. A compound according to any one of claims 1 to 4 or 10 having a x-ray powder diffraction diagram comprising the following diffraction lines (recorded with Cu-Kαi radiation):

12. A process for the manufacture of a crystalline polymorph of the compound of formula

(I)

(Ceftobiprole) by • reacting(6R,7R)-7-[(Z)-2-(5-amino-[l,2,4]thiadiazol-3-yl)-2-trityloxyimino- acetylamino]-8-oxo-3-[(E)-(R)-2-oxo-[ 1 ,3']bipyrrolidinyl-3-ylidenemethyl]-5-thia- 1 - aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid with trifluoroacetic acid and triethylsilane at temperatures below about 0⁰C; adding a mixture of water, ethylacetate, acetonitrile and further trifluoroacetic acid, separating the aqueous phase from the obtained mixture and transferring the separated aqueous phase to a MCl gel column; eluting the product with an about 9:1 :0.4 mixture of water: acetonitile trifluoroacetic acid; removing the acetonitrile under vacuo; precipitating the product of the Ceftobiprole polymorph by adjusting the pH to about 3 and isolating the polymorph.

13. A process for the manufacture of a crystalline polymorph of the compound of formula (I)

(Ceftobiprole) by • reacting (6R,7R)-7-[(Z)-2-(5-amino-[ 1 ,2,4]thiadiazol-3-yl)-2-trityloxyimino- acetylamino]-8-oxo-3-[(E)-(R)-2-oxo-[ 1 ,3']bipyrrolidinyl-3-ylidenemethyl]-5-thia- 1 - aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid dihydrochloride of formula

C(Ph)₃

with a 90 percent formic acid,

• isolating the obtained solid and

• purifying it by chromatography with MCl Gel using a gradient of water with increasing concentrations of acetonitirile, and

• isolating the polymorph from the collected fractions of pure compound of Ceftobiprole obtained by the chromatography;

• removing acetonitrile by evaporation and e precipitate is isolating the obtained precipitate of the Ceftobiprole polymorph.