(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2013/057196 Al 25 April 2013 (25.04.2013) P O P C T

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C07D 501/36 (2006.01) A61P 31/04 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 31/546 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (21) Number: International Application DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, PCT/EP2012/070664 HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (22) International Filing Date: KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, 18 October 2012 (18.10.2012) ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, (25) Filing Language: English RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, (26) Publication Language: English TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (30) Priority Data: 11185954.2 20 October 201 1 (20. 10.201 1) EP (84) Designated States (unless otherwise indicated, for every kind of regional protection available): ARIPO (BW, GH, (71) Applicant: DSM SINOCHEM PHARMACEUTICALS GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, NETHERLANDS B.V. [NL/NL]; P.O.Box 245, Alexan UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, der Fleminglaan 1, NL-2613 AX Delft (NL). TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (72) Inventors: MOODY, Harold, Monro; P.O.Box 4, NL- MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, 6100 AA Echt (NL). MAAS, Peter, Johannes, Domini- TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, cus; P.O. Box 4, NL-6100 AA Echt (NL). SCHMITGES, ML, MR, NE, SN, TD, TG). Thomas; P.O. Box 4, NL-6100 AA Echt (NL). Published: (74) Agent: VROOM, DE, Erik; DSM Intellectual Property, P.O. Box 130, NL-6100 AC Echt (NL). — with international search report (Art. 21(3))

© o (54) Title: PROCESS FOR THE PREPARATION OF CEFAMANDOLE NAFATE (57) Abstract: The present invention relates to a process for the preparation of cefamandole nafate from cefamandole and to the use thereof in the manufacture of a medicament for treatment of a bacterial disease. PROCESS FOR THE PREPARATION OF CEFAMANDOLE NAFATE

Field of the invention

The present invention relates to a process for the preparation of cefamandole nafate from O-formyl cefamandole.

Background of the invention

Cefamandole ((1); CAS 34444-01-4; (6R,7R)-7-[[(2R)-hydroxyphenyl- acetyl]amino]-3-[[(1 -methyl-1 /-/-tetrazol-5-yl)thio]methyl]-8-oxo-5-thia-1 - azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid is a second generation broad-spectrum semi-synthetic cephalosporin antibiotic, the preparation of which was first disclosed in

US 3,641 ,021 . Cefamandole nafate ((3); CAS 42540-40-9) is the sodium salt of the formate ester of cefamandole and is the clinically used form of cefamandole. Various approaches towards the preparation of cefamandole nafate have been reported. In a first approach, the compound can be prepared by chemical N-acylation of the appropriate β-lactam nucleus, 7-amino-3-(1 -methyl-1 H-tetrazol-5-ylthiomethyl)-3- cephem-4-carboxylic acid (7-TMCA) as for instance described in Kaiser et al. (J. Infect.

Dis. (1978) 137, 10-16). In a second and environmentally more benign approach, an enzymatically catalyzed kinetically controlled acylation of 7-TMCA has been proposed in more recent literature by, amongst others, Nierstrasz et al. (Biocatalysis and Biotransformation (1999) 17, 209-223), Terreni et al. (Appl. Microbiol. Biotechnol. (2007) 77, 579-587) and Serra et al. (Eur. J. Org. Chem. (2009), 1384-1389). Although direct kinetically controlled acylation of 7-TMCA with D,L-0-formyl methyl mandelate towards O-formyl cefamandole (2) has been suggested by Fuganti et al. (Biotechnol. Lett. (1992) 14, 543-546) using the enzyme penicillin G acylase from Escherichia coii immobilized on Eupergit C, the general opinion is that the active site of penicillin G acylase is only moderately suited to accommodate the D,L-0-formyl mandelate side chain. Unfortunately, an efficient approach leading from cefamandole (1) or O-formyl cefamandole (2) to cefamandole nafate (3), has not been reported yet.

Detailed description of the invention

The current most promising approach for the preparation of cefamandole nafate (3) is kinetically controlled acylation of 7-TMCA with an ester of mandelic acid, such as methyl mandelate, to give cefamandole (1) followed by conversion into O- formyl cefamandole (2) and subsequently into cefamandole nafate (3).

It is an object of the present invention to provide a method for the preparation of cefamandole nafate (3) from O-formyl cefamandole (2).

In a first aspect of the invention there is provided a method for the preparation of cefamandole nafate (3) comprising the steps of: (a) reacting cefamandole or a salt thereof and an impurity chosen from the list consisting of mandelic acid, methyl mandelate and O-acetyl mandelic acid or salts thereof with a formylating agent;

(b) contacting the mixture obtained in step (a) with a sodium source in an organic solvent,

characterized in that said impurity is present in an amount of from 0.5% to 25% by weight relative to the amount of O-formyl cefamandole or a salt thereof

formed in step (a).

As a result of the formylation reaction in step (a) the compounds present in step (b) are O-formyl cefamandole (2) or a salt thereof and an impurity chosen from the list consisting of mandelic acid, methyl mandelate, O-acetyl mandelic acid and O-formyl mandelic acid or salts thereof. The method remarkably enables production of highly pure cefamandole nafate (3) and is characterized in that any of the impurities mentioned above are removed to completion or near completion even when present in high amounts in the starting material, such as from 1% to 10%, from 2% to 20% or from 3% to 30%, all by weight relative to amount of said O-formyl cefamandole (2) or a salt thereof. Suitable organic solvents are esters such as acetates, formats and propionates and ketones. Preferred organic solvent are acetone, butyl acetate, butyl formate, ethyl acetate, ethyl formate, isobutyl acetate, isobutyl formate, isopropyl acetate, isopropyl formate but also mixtures of two or more of these and/or other solvents may be employed.

In one embodiment, suitable sodium sources are sodium halides or sodium salts of organic acids such as sodium acetate, sodium ethyl hexanoate or sodium formate or mixtures thereof. It is well known that mandelic acid is formed as a side product (usually in significant amounts) during the enzymatic synthesis of cefamandole (1) from 7-TMCA and an ester of mandelic acid. Like cefamandole (1), this mandelic acid is also formylated to give O-formyl mandelic acid. Surprisingly it is found that upon conversion of O-formyl cefamandole (2) to the sodium salt cefamandole nafate (3), only the latter crystallizes and can be isolated in high purity whereas the sodium salt of O-formyl mandelic acid remains dissolved in the mother liquor. This unexpected phenomenon results in a highly efficient purification step.

In another embodiment, O-formyl cefamandole (2) or a salt thereof is prepared by reacting cefamandole or a salt thereof with a formylating agent. In general, formylation of alcohols is well studied, as for instance in a review by G.A. Olah et al. (Chem. Rev. (1987) 87, 671-686) and can be carried out using a variety of reagents such as formic acid (M. Chakrabarty et al., Synth. Commun. (2000) 30:2, 187-200), formic anhydrides, formic esters, formic halides or N-formyl derivatives. Examples are N-formylbenzotriazole (A.R. Katritzky et al., Synthesis (1995), 503-505), ethyl formate (M.H. Habibi et al., Tetrahedron (2001 ) 57, 8333-8337), 2,2,2-trifluoroethyl formate

(D.R. Hill et al., Org. Lett. (2002), 111-1 13), acetic acid with ethyl formate (V. Mirkhani et al., Monatshefte fur Chemie (2004) 135, 1257-1263). However not much is known of formylation of the class of sensitive β-lactams. In US 4,877,783 formylation of a

β-lactam is reported using acetic formic anhydride, however the formylation concerns the carbon adjacent to the carbonyl of the β-lactam ring and the carboxyl group of the β-lactam is protected. If not for anything else this example stresses the ease of formation of unwanted side products in β-lactam formylation reactions.

In one embodiment, the formylating agent is formic acid, a formic anhydride, a formic ester, a formic halide or an N-formyl derivative. Preferably, the formylating agent is a mixed anhydride such as acetic formic anhydride of formula CH3C(0)OC(0)H, a compound that can conveniently be prepared from acetic anhydride and formic acid. Alternatively, a sterically hindered derivative of acetic formic anhydride may be used, such as butyric formic anhydride of formula CH3CH2CHC(0)OC(0)H or 2-ethylbutyric formic anhydride of formula (C2H 5)2CH C(0)OC(0)H or isobutyric formic anhydride of formula (CH 3)2CHC(0)OC(0)H or 2-methylbutyric formic anhydride of formula

C H3C H2CH(CH 3)C(0)OC(0)H or propionic formic anhydride of formula

CH3CH2C(0)OC(0)l-l or mixtures thereof. These sterically hindered derivatives of acetic formic anhydride have the additional advantage of reduced formation of unwanted acylated products. For example, the presence of acetylated contaminations may lead to liberation of traces of acetate upon parenteral administration. Acetate is known (A.

Amore et al., J. Amer. Soc. Nephrol. (1997), 1431 ) for its negative effects in man leading to acetate intolerance, a condition associated with arterial hypertension, nausea, vomiting and headaches. For the purpose of the present invention it is also advantageous to use the mixed anhydride in the presence of formic acid as this suppresses the formation of unwanted acetylated products. For example, next to acetic formic anhydride, formic acid is preferably present in amounts of from 0.05 to 100 mol% based on the amount of acetic formic anhydride, more preferably of from 0.1 to 50 mol%, most preferably of from 0.2 to 10 mol%. Alternatively, the formation of unwanted acetylated products can be avoided by isolating the acetic formic anhydride by means of distillation prior to reaction with cefamandole (1). Distillation may be carried out at normal pressure or reduced pressure. Advantageously, acetic formic anhydride is prepared shortly prior to use as the compound is labile (degradation to form carbon monoxide). Thus, in certain instances it may be advisable to use the acetic formic anhydride within 24h after its preparation, preferably within 12h after its preparation, more preferably within 6h after its preparation and most preferably within 3h of its preparation. Also in view of the instability of acetic formic anhydride, this formylating agent is preferably added in excess compared to the amount of cefamandole (1). Preferably the molar ratio acetic formic anhydride/cefamandole (1) is between 100/1 and 100/99, more preferably between 50/1 and 100/95, most preferably between 10/1 and 10/9.

In another embodiment, the formylation is carried out in the presence of a base.

It has been found that improved conversions are obtainable, in particular when the formylation is carried out at lower temperatures such as from -78°C to 30°C when the base in question has a dissociation constant pKa (at 25°C) of from 3.0 to 11.0, preferably of from 6.0 to 9.0, more preferably of from 6.5 to 8.0. Examples are 1-amino-2-methoxyethane, allantoin, p-anisidine, benzylamine, 2-benzylpyridine, benzimidazole, n-butylamine, sec-butylamine, ie/f-butylamine, cyclohexylamine, Ν,Ν-diethylaniline, diethylmethylamine, dimethylamine, N,N,-dimethylaniline, 2.3-dimethylpyridine, 2,4-dimethylpyridine, 2,5-dimethylpyridine, 2,6-dimethylpyridine, 3.4-dimethylpyridine, 3,5-dimethylpyridine, ephedrine, ethylamine, 2-ethylbenzimidazole, N-ethylaniline, 1-ethylpiperidine, 2-ethylpyridine, heptylamine, hexadecylamine, hexylamine, imidazole, isoquinoline, melamine, methoxypyridine, methylamine, p-methylaniline, 1-methylimidazole, N-methylmorpholine, 1-methylpiperidine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, N-methylpyrrolidine, 2-methylquinoline, morfoline, pentylamine, p-phenetidine, phenethylamine, propylamine, pyridine, 2-pyridinamine, 2,5-pyridinediamine, theobromine, 2-thiazolamine, triethylamine, trimethylamine, 2,4,6-trimethylpyridine. Preferred examples of bases to be used in the method of the present invention are imidazole and derivatives thereof such as 1-methylimidazole, morpholine and derivatives thereof such as N-methylmorpholine, pyridine and derivatives thereof such as the mono-, di- and tri-methyl pyridines and triethylamine. In the context of the present invention the term dissociation constant pKa is defined as the negative logarithm of the acid dissociation constant Ka. Following its preparation as outlined according to any of the above embodiments, cefamandole nafate (3) may be isolated following known procedures. Hence the crystalline product may be separated from the mother liquor by centrifugation, decantation, filtration, sedimentation and the like. The solid particles so obtained may be dried, either at atmospheric pressure or under reduced pressure, at temperatures ranging from 0°C to 100°C, preferably ranging from 10°C to 80°C, more preferably ranging from 15°C to 60°C. Optionally, when this is required for specific purposes, the cefamandole nafate (3) thus obtained may be re-crystallized. The mother liquor remaining after the isolation procedure as described above may be used for recovering traces cefamandole nafate (3) or starting materials.

In a second aspect of the invention, the cefamandole nafate obtained by the processes of the first aspect, is used for the manufacture of a medicament with antibacterial properties for the treatment of a bacterial disease. The medicament thus obtained has the advantage of being produced in high purity and with low environmental burden as compared to prior art approaches. As a result of the low levels of contamination, notably the sodium salt O-acetyl cefamandole, the cefamandole nafate obtained by the process of the first aspect is particularly suitable for use in the manufacture of a medicament for treatment of a bacterial disease in persons having acetate intolerance. MATERIALS AND METHODS

Preparation of immobilized penicillin acylase The production, isolation and purification of wild type and mutant penicillin G acylases may be carried out as described in WO 1996/005318 and WO 2003/055998. Alternatively, genes encoding mutant penicillin G acylases may be obtained by gene synthesis. Production of the mutant penicillin G acylase was achieved by cloning the genes encoding mutant penicillin G acylases into an appropriate expression vector, transforming a suitable host such as Escherichia coii with said vector and culturing the transformed host under conditions suitable for the production of the mutant penicillin G acylases and recovery and purification of the mutants was carried out as described in WO 2010/072765. Penicillin G acylase AA (the Escherichia coii wild type penicillin G acylase with mutations B:F24A and B:V148L) and penicillin G acylase mutant 1 (the Escherichia coii wild type penicillin G acylase with mutations V 11A, A:S3L, A:V192E,

B:F24A, B:V148L and B:F460L) as disclosed in Example 1 of WO 2010/072765 were immobilized according to the method disclosed in EP 839192 and EP 222462.

Analytical HPLC The reactions were followed by quantitative HPLC analysis. Samples were diluted (typically -50 mg sample was added up to 50 mL with buffer) in phosphate buffer pH

2.7 (50 mL 1 M H3P04 + 40 mL 1 M NaOH per liter MilliQ water).

Instrument: HPLC Hewlett Packard 1100, detection at 220 nm Column: Intersil ODS-3, 5µ ι 4.6x1 50mm C/N 5020-01731 Flow: 1 mL/min, stop time 37 min Method: Eluens A : Methanol 99.9%

Eluens B: Acetontirile

Eluens C: KH2P0 4 (2.72 g) and 1 M H3P0 4 (6 mL), to 1 L with MilliQ water Time (min) Eluens A (%) Eluens B (%) Eluens C (%) 0 0.5 98.5 2.5 3 96 11 25 74 15 40 59 25 60 39

28 80 19

32 80 19 32.1 0.5 98.5

Response factors and retention times:

Measurement of pH values

The pH values referred to in the present invention were measured as follows. The measurement is performed using 718 STAT Titrino from Metrohm. The pH electrode is from Metrohm, series number 6.0234.1 10. It contains 3M KCI. The pH meter calibration is performed at 20°C at pH 4 and pH 7 using standard solutions from

Merck, using the calibration program present in the instrument. EXAMPLES

Example 1 Enzymatic preparation of cefamandole (1) The enzymatic reaction was carried out with R-methyl mandelate (20.9 g ; 294 mmol), 7-TMCA (58.4 g ; 165 mmol) and water (671 g) at 2°C and pH 8.5. The reaction was started by adding immobilized penicillin G acylase mutant 1 (82.4 g , see Materials and Methods). At a conversion of 87% the reaction was stopped by removing the enzyme by filtration. The mother liquor of the enzymatic reaction was extracted at room temperature twice with isopropyl acetate (850 g each time) to remove the remaining methyl mandelate. The organic layer was evaporated under vacuum resulting in methyl mandelate oil. Remaining solvents were removed from the aqueous layer by evaporation. The pH was slowly reduced by adding 25% H2S0 4 solution. At pH values of 6.0 and 4.3 the solution was seeded with 0.1 g 7-TMCA and the resulting suspension was filtered at pH 4.7. The suspension was filtered and the organic and aqueous phases were separated. The water phase was extracted twice with ethyl acetate (210 g). The organic phases were combined and ethyl acetate was evaporated to completion (40°C). The remaining solid was analyzed by means of HPLC (see Materials and Methods):

Example 2 Formylation of cefamandole (1) to O-formyl cefamandole (2) Preparation mixed anhydride

Acetic anhydride (10.0 mL; 105.8 mmol) and formic acid (4 ml_; 106.0 mmol) were mixed and stirred at 55°C for 2h. Formylation 2.6 g of cefamandole obtained in Example 1 was mixed with isopropyl acetate (2.6 g) and mixed anhydride (6 g) after which imidazole (0.04 g) was added. The solution was stirred at room temperature for 8h and analyzed by HPLC. The conversion to formyl cefamandole was >97%. Next, isopropyl acetate (15 mL) and water (5 mL) were added and the pH was adjusted to 2 with 25% H2S0 4. The organic phase was separated and washed twice washed with water (5 mL, adjusted to pH 2). Yield on 7-TMCA after work-up: 95%.

Example 3 Formylation of cefamandole (1) to O-formyl cefamandole (2) using catalytic amounts of imidazole Preparation mixed anhydride Acetic anhydride (14.12 g ; 13.0 mL; 138.3 mmol) and formic acid (6.38 g ; 5.2 mL; 138.6 mmol) were mixed and stirred at 55°C for 2.5h. Formylation The mixed anhydride solution was added to a cefamandole mixture from an enzymatic reaction comprising cefamandole (5.290 g), methyl mandelate (0.124 g), mandelic acid

(0.166 g) and isopropyl acetate (16.92 g). After that, imidazole was added as solution in mixed anhydride (10.3 mg imidazole in 500 µ \- mixed anhydride). Samples were taken over time and analyzed by means of HPLC. After 20h, 95 mg imidazole was added. Isopropyl acetate (56 g) and water (50 g) were added, the pH of the aqueous phase was adjusted to 1.5 using 25% H2S0 4 and the phases were separated. The organic phase was washed with aqueous HCI (pH 1.8, 2 x 20 mL). Approximately 2.2 mol% of cefamandole remained in the combined aqueous phases.

Example 4 Cefamandole nafate

The organic phase obtained in Example 2 was evaporated to dryness at 37°C using a rotavap. Isopropanol (15 mL) and acetone (15 mL) were added and the resulting mixture was titrated at room temperature to pH 5.6 with a 10 % sod i u m

2-ethylhexanoate solution in acetone (718 STAT Titrino by Methrohm filled with 25%

NH3). The resulting crystals were isolated by filtration in a yield of 82.6%. Apart from the title product, HPLC analysis indicated the presence of cefamandole and acetyl cefamandole but not of any mandelic acid derivatives.

Example 5 Cefamandole nafate

The organic phase obtained in Example 3 was evaporated to 23.4 g at 38°C using a rotavap. Acetone (21 g) was added and the resulting mixture was titrated at room temperature to pH 5.66 with a 10% sodium 2-ethylhexanoate solution in acetone (55 mL). The resulting crystals were isolated by filtration to give, after drying overnight δ under vacuum at 30°C, 2.36 g of white solid. H NMR (700 MHz, DMSO-d6, CD3CN) 9.25 (d, 1H, J = 8.2 Hz), 8.27 (s, 1H), 7.5 (m, 2H), 7.4 (m, 3H), 6.1 1 (s, 1H), 5.55 (dd,

1H, i = 4.7 Hz, J2 = 8.4 Hz), 4.85 (d, 1H, J = 4.8 Hz), 4.43 (d, 1H, J = 12.5 Hz), 4.19 (d, 1H, J = 12.5 Hz), 3.88 (s, 3H), 3.50 (d, 1H, J = 17.6 Hz), 3.32 (d, 1H, J = 17.6 Hz). HPLC analysis showed the following composition: O-Formyl cefamandole (96.19 area%), cefamandole ( 1 .85 area%), acetyl cefamandole ( 1 .96 area%). CLAIMS

A method for the preparation of cefamandole nafate comprising the steps of: (a) reacting cefamandole or a salt thereof and an impurity chosen from the list consisting of mandelic acid, methyl mandelate and O-acetyl mandelic acid or salts thereof with a formylating agent;

(b) contacting the mixture obtained in step (a) with a sodium source in an organic solvent, characterized in that said impurity is present in an amount of from 0.5% to 25% by weight relative to the amount of O-formyl cefamandole or a salt thereof formed in step (a).

Method according to claim 1 wherein said sodium source is sodium ethyl hexanoate.

Method according to claim 1 wherein said organic solvent is acetone, butyl acetate, butyl formate, ethyl acetate, ethyl formate, isobutyl acetate, isobutyl formate, isopropyl acetate, isopropyl formate or mixtures thereof.

Method according to claim 5 wherein said formylating agent is acetic formic anhydride of formula CH3C(0)OC(0)H or butyric formic anhydride of formula

CH3CH2CHC(0)OC(0)H or 2-ethylbutyric formic anhydride of formula

(C2H5)2CHC(0)OC(0)H or isobutyric formic anhydride of formula

(CH 3)2CHC(0)OC(0)H or 2-methylbutyric formic anhydride of formula

CH3CH2CH(CH3)C(0)OC(0)H or propionic formic anhydride of formula

CH3CH2C(0)OC(0)H or mixtures thereof.

Method according to claim 4 wherein formic acid is present.

Method according to claim 4 wherein said formylating agent is isolated by means of distillation prior to reaction with said cefamandole or a salt thereof.

Method according to any one of claims 1 to 6 further comprising isolation of cefamandole nafate. Use of cefamandole nafate obtained according to claim 7 in the manufacture of a medicament for treatment of a bacterial disease.

Use according to claim 8 wherein said bacterial disease occurs in a person having acetate intolerance. A . CLASSIFICATION O F SUBJECT MATTER INV. C07D501/36 A61K31/546 A61P31/04 ADD.

According to International Patent Classification (IPC) or to both national classification and IPC

B . FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) C07D

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)

EPO-Internal , CHEM ABS Data

C . DOCUMENTS CONSIDERED TO B E RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

X, P CN 102 276 629 A (PHARMACEUTICAL CO. , 1-3 , 7 LTD. ) 14 December 2011 (2011-12-14) Scheme page 5 . ; paragraph [0008] - paragraph [0018]

X US 3 928 592 A (GREENE JAMES M ET AL) 1-3 ,7-9 23 December 1975 (1975-12-23) exampl es 1-3

X CN 101 880 290 A (HAINAN NEW ZH0NGZHENG 1-3 , 7 PHARMACEUTICAL CO LTD) 10 November 2010 (2010-11-10) paragraph [0023] - paragraph [0025]

X DE 26 14 669 Al ( LI LLY CO ELI ) 1-3 , 7 2 1 October 1976 (1976-10-21) page 9 - page 10; exampl e A

-/--

X| Further documents are listed in the continuation of Box C . See patent family annex.

* Special categories of cited documents : "T" later document published after the international filing date or priority date and not in conflict with the application but cited to understand "A" document defining the general state of the art which is not considered the principle or theory underlying the invention to be of particular relevance "E" earlier application or patent but published o n or after the international "X" document of particular relevance; the claimed invention cannot be filing date considered novel or cannot be considered to involve an inventive "L" documentwhich may throw doubts on priority claim(s) orwhich is step when the document is taken alone cited to establish the publication date of another citation or other "Y" document of particular relevance; the claimed invention cannot be special reason (as specified) considered to involve an inventive step when the document is "O" document referring to an oral disclosure, use, exhibition or other combined with one o r more other such documents, such combination means being obvious to a person skilled in the art "P" document published prior to the international filing date but later than the priority date claimed "&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

17 January 2013 24/01/2013

Name and mailing address of the ISA/ Authorized officer European Patent Office, P.B. 5818 Patentlaan 2 NL - 2280 HV Rijswijk Tel. (+31-70) 340-2040, Fax: (+31-70) 340-3016 Von Daacke, Axel C(Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.

EP 0 156 771 A2 (BIOCHEMIE GMBH [AT]) 1,2,7 2 October 1985 (1985-10-02) example 4

DE 27 30 579 A l (PIERREL SPA) 1,7 12 January 1978 (1978-01-12) example 5 Patent document Publication Patent family Publication cited in search report date member(s) date

CN 102276629 14-12-2011 NONE

US 3928592 23-12-1975 A R 203895 Al 31-10- 1975 AT 356264 B 25- 04- 1980 AU 7833975 A 19- 08- 1976 BE 825653 Al 18- 08- 1975 CA 1043261 Al 28-11- 1978 CH 620929 A5 31-12- 1980 CS 189689 B2 30-04- 1979 DD 117807 A5 05- 02- 1976 DE 2506622 Al 28-08- 1975 DK 65575 A 20- 10- 1975 ES 434976 Al 16- 12- 1976 FR 2261772 Al 19- 09- 1975 GB 1493676 A 30- 11- 1977 HU 174129 B 28- 11- 1979 I E 40671 Bl 18-07- 1979 I L 46650 A 31- 10- 1977 P 50126817 A 06- 10- 1975 P 61019605 B 17- 05- 1986 NL 7502045 A 26- 08- 1975 PH 12058 A 18- 10- 1978 PL 109311 Bl 31-05- 1980 SE 406153 B 29- 01- 1979 SE 7501985 A 25-08- 1975 SU 659094 A3 25-04- 1979 US 3928592 A 23-12- 1975 YU 41875 A 31-05- 1982 ZA 7500975 A 29-09- 1976

CN 101880290 10-11-2010 NONE

DE 2614669 Al 21-10-1976 A R 210880 Al 30-09-1977 AT 343806 B 26-06-1978 AU 500013 B2 10-05-1979 AU 1143576 A 01-09-1977 BE 840179 Al 30-09-1976 CA 1045114 Al 26-12-1978 CH 621554 A5 13-02-1981 CS 191970 B2 31-07-1979 DD 125950 A5 01-06-1977 DE 2614669 Al 21-10-1976 DK 150676 A 12-10-1976 ES 446907 Al 16-09-1977 FI 760788 A 12-10-1976 FR 2306698 Al 05-11-1976 GB 1546898 A 31-05-1979 GR 59912 Al 20-03-1978 HU 175592 B 28-09-1980 I L 49100 A 12-03-1979 P 51123812 A 28-10-1976 P 61010474 B 29-03-1986 LU 74740 Al 11-11-1976 MX 3687 E 29-04-1981 NL 7603660 A 13-10-1976 NO 761017 A 12-10-1976 OA 5290 A 28-02-1981 PL 104803 Bl 29-09-1979

page 1 of 2 Patent document Publication Patent family Publication cited in search report date member(s) date

PT 64917 A 01-04-1976 RO 68501 Al 30- 04-1981 SE 435290 B 17-09-1984 SE 7602565 A 12-10-1976 SU 668608 A3 15-06-1979 US 4006138 A 01-02-1977 YU 79276 A 31- 10-1982 ZA 7601103 A 26-10-1977

EP 0156771 A2 02-10-1985 AU 587974 B2 07-09 1989 AU 3599489 A 21-09 1989 AU 4234085 A 01- 11 1985 CA 1277667 C2 11-12 1990 EP 0156771 A2 02- 10 1985 ES 8706154 Al 16-08 1987 ES 8706696 Al 16-09 1987 ES 8706697 Al 16-09 1987 FI 854577 A 20-11 1985 HU T39746 A 29-10 1986 I L 74751 A 29-04 1990 NZ 211594 A 26-02 1990 PH 21445 A 20-10 1987 0 8504404 Al 10-10 1985

DE 2730579 Al 12-01-1978 DE 2730579 Al 12-01-1978 FR 2357567 Al 03-02-1978 P 53023991 A 06-03-1978 NL 7707678 A 12-01-1978

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