OA10325A

OA10325A - Crystalline N-acetyl neuraminic acid derivatives and processes for their preparation

Info

Publication number: OA10325A
Application number: OA60842A
Authority: OA
Inventors: Christopher Williamson; William James White; Vipulkumar Patel
Original assignee: Biota Scient Management
Priority date: 1993-12-17
Filing date: 1996-06-14
Publication date: 1997-10-07
Also published as: CZ169396A3; NZ277789A; CO4340619A1; EP0734382A1; SI0734382T1; PT734382E; BR1100548KB1; ATE199255T1; FI962464A0; GR3035681T3; HU221971B1; US6294572B1; EP0734382B1; IL112011A; HU9601642D0; NO962545L; AU689185B2; PE31095A1; AP9400705A0; CN1142820A

Description

1 0 î 0325

CRYSTALLINE N-ACETYL NEURAMINIC ACID DERIVATIVES

AND PROCESSES FOR THEIR PREPARATION

The présent invention relates to dérivatives of N-acetyl neuraminic acid and theiruse in medicine. More particulariy the invention is concemed with particuiarphysical forms of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-£)-glycero-£)-galacto-non-2-enopyranosonic acid (the 4-guanidino analogue of DANA; alsoknown as 5-(acetylamino)-2,6 anhydro-3,4,5-trideoxy-4-guanidino-£)-giycereo-£)-galacto-non-2-enonic acid), pharmaceutical formulations thereof and their use intherapy. PCT/AU91/ÛÛ161 (publication no.WO91/16320) describes a number of dérivativesof 5-acetamidino-2,3,5-trideoxy-]0-glycero-]D-galacto-non-2-enopyranosonic acid(2,3,-dideoxy-2,3-didehydro-N-acetyl-neuraminic acid; DANA) including the 4-guanidino analogue of DANA. The 4-guanidino analogue of DANA is prepared bythe reaction of the corresponding O-acyl protected 4-amino analogue of DANA byreaction with S-methylisourea followed by deprotection, purification bychromatography and freeze-drying.

The structure of the 4-guanidino analogue of DANA is shown below:

NH

4-guanidino analogue of DANA

We hâve now found thatthe compound of formula (I) can be obtained in crystallineform. 2 0Î0325

There is thus provided in a first aspect of the invention 5-acetamido-2,3,4,5-tetradeoxy-4-quanidino-p-qlycero-p-qalacto-non-2-enopyranosonic acid incrystalline form.

We hâve further found that the compound of formula (I) may be obtained bycrystallisation under certain conditions in the form of a crystalline hydrate(hereinafter Hydrate I). Hydrate I exists in the form of crystals having a îow aspectratio, for example, tabuiar crystals, which are favoured for pharmaceuticalformulation because of their physical properties, e.g. good fîow characteristics.The water content of Hydrate I is related to relative humidity (RH). Water uptake ofHydrate I varies from zéro at RH of 0% up to 10% at RH of 90 - 100%.

The compound of formula (I) may also be crystailised in the form of a dihydrate(hereinafter Hydrate II). Hydrate il exists in the form of crystals having a highaspect ratio, for example, needle-shaped crystals. The water content of thesecrystais remains substantially constant over a broad relative humidity range (RHabout 10 - 90%). The stable water content of Hydrate 11 represents an advantageof this crystalline form for use in pharmacy.

There is thus provided in a further aspect of the invention 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-giycero-£)-galacto-non-2-enopyranosonic acid in theform of crystals having a îow aspect ratio, such as tabuiar crystals.

In a further aspect there is provided 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-£5-gtycero-Ë-gatacto-non-2-enopyranosonic acid in the form of crystals having ahigh aspect ratio, such as needle-shaped crystals.

Whilst tabuiar crystals are regarded as typical of Hydrate I and needle-shapedcrystals are regarded as typical of Hydrate il, it wiîl be appreciated that thepossibiiity of either Hydrate I or Hydrate il existing in alternative crystal habitsunder certain circumstances cannot be excluded. It is to be understood that ailsuch alternative crystal habits are within the scope of the présent invention.

There is also provided 5~acetamido-2,3,4,5-tetradeoxy-4-guanidino-jD-glycero-J0-galacto-non-2-enopyranosonic acid in the form of crystais which hâve stable watercontent over a broad humidity range, for example RH 10 - 80%. 3 010325

Hydrate I (oses substantialiy ali of its water of crystallisation at about 80-90°C.Décomposition occurs at 299°C.

Hydrate 11 loses one mole of water of crystallisation at about 84-90°C and a furthermole of water of crystallisation by about 135-143°C.

In a yet further aspect the invention provides 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-£)-glycero-£)-galacto-non-2-enopyranosonic acid in the form of acrystalline hydrate which loses substantialiy ail its water of crystallisation at 80-90°C.

In a yet further aspect the invention provides 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-£-g!ycero-n-galacto-non-2-enopyranosonic acid in the form of acrystalline hydrate which loses one mole of water of crystallisation at 84-90°C anda further mole of water of crystallisation at 135-143°C.

In a preferred aspect the invention provides 5-acetamîdo-2,3,4,5-tetradeoxy-4-guanidino-n-glycero-n-galacto-non-2-enopyranosonic acid in the form of Hydrate las herein defined substantialiy free of Hydrate II as herein deftned.

In a further preferred aspect the invention provides 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-£>-gîycero-n-galacto-non-2-enopyranosonic acid in theform of Hydrate II as herein defined substantialiy free of Hydrate I as hereindefined.

By "substantialiy free" is meant containing less than 5% of the alternative hydrate,such as less than 2%, for example less than 1% of the alternative hydrate. 5-Acetamido-2,3,4,5-tetradeoxy-4-guanidino-n-glycero-S-galacto-non-2-enopyranos: nie acid may be prepared in crystalline form by crystallisation of thecompound from aqueous solution.

Each of Hydrate I and Hydrate II may be prepared substantialiy free from thealternative Hydrate by controlling the solution concentration and température atwhich crystallisation occurs. 4 010325

In general, 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-Ë-glycero-E-gaiacto-non-2-enopyranosonic acid in the form of Hydrate I may be obtained by crystallisationof the compound from aqueous solution at a température greater than about50°C, preferably 50-55°C.

In general, 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-Ë-glycero-n-galacto-non-2-enopyranosonic acid in the form of Hydrate U may be obtained by crystallisationof the compound from aqueous solution at a température below about 40°C,preferably about 20-30°C.

Crystallisation of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-n-glycero-D-galacto-non-2-enopyranosonic acid from aqueous solution at a température in the rangeof about 40-50°C typically results in a mixture of tabular and needle-shapedcrystals. Such mixtures are disfavoured for the préparation of pharmaceuticalformulations because of the differing physical praperties of Hydrate I and Hydrateil, in particular their flow praperties.

Seeding of an aqueous solution of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-^-galacto-non-2-enopyranosonic acid w'rth crystals of Hydrate i or HydrateIl may lead to crystallisation of the seeded Hydrate. Préparation of Hydrate I orHydrate II should therefore be conducted in the absence of seeds of the undesiredHydrate. Conversely, Hydrate I may be prepared by seeding an aqueous solutionof 5-acetamido-2,3,4)5-tetradeoxy-4-guanidino-2.-g!ycero-Ë-gaîacto-non-2-enopyranosonic acid with crystals of Hydrate I, and Hydrate II may be prepared byseeding an aqueous solution of 5-acetamido-2,3,4,5-tetradeoxy-4-guanîdino-£)-giycero-û-galacto-non-2-enopyranosonic acid with crystals of Hydrate II.

For the préparation of Hydrate il it is préférable to employ a relatively diluteaqueous solution, for exampie a solution of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-n-glycero-Ë-gaîacto-non-2-enopyranosonic acid in 15-30 volumes ofwater, for example 20 volumes of water. Hydrate I may convenientfy becrystallised from a relatively concentrated aqueous solution, for example a solutionof 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-£)-glycero-i3-galacto-non-2- enopyranosonic acid in 12 - 20 volumes of water, such as 12 - 15 volumes ofwater. 5 010325

We hâve found that Hydrate li may be converted into Hydrate I in aqueoussuspension or saturated solution. Such interconversion may be effected byprolonged ageing of an aqueous suspension or saturated solution of Hydrate If, forexample ageing for a period of days, for example more than 10 days, such asabout 15 days. Altematively, interconversion may be effected in the presence of abase, for example an organic base such as imidazole.

Recovery of either Hydrate I or Hydrate II ffom aqueous solution may be enhancedby the addition to the solution of a suitable counter-solvent. Suitable counter-solvents are water-miscible solvents in which the compound of formula (l) has poorsolubiiity. Conveniently the counter-solvent will be a ketone, such as acetone, oran alkanol such as propan-2-ol. A preferred counter-solvent is acetone.

We hâve aiso found that addition *' an aqueous solution of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-]3-glycero-,ti-galacto-non-2-enopyranosonic acid to asimilar volume of a counter-solvent as previously defined results in précipitation ofHydrate II. For example, addition of a solution of 5-acetamido-2,3,4,5-tetradeoxy- 4- guanidino-£)-glycero-D-galacto-non-2-enopyranosonic acid in 12 - 15 volumes ofwater to 12 - 20 volumes of acetone gives crystals of Hydrate II.

The methods for the préparation of crystalline material, and in particular methodsfor the préparation of Hydrate l and Hydrate II, described herein constitute furtheraspects of the présent invention. 5- Acetamido-2,3,4,5-tetradeoxy-4-guanidino-£)-glycero-n-galacto-non-2-enopyranosonic acid in crystalline form may be used as an antiviral agent asdescribed in WO 91/16320, which is incorporated herein by référencé. 5-Acetamido-2,3,4,5-tetradeoxy-4-guanidino-Ë-glycero-n-galacto-non-2-enopyranosonic acid in crystalline form may be formulated as a pharmaceuticalcomposition for use as an antiviral agent as described in WO 91/16320.

Preferred pharmaceutical formulations of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-J2-9iycero-£-galacto-non-2-enopyranosonic acid include powderformulations and aqueous solutions or suspensions. Préparation of powder 6 010325 formulations requires micronisation of the drug substance. The good fiowproperties of Hydrate I render it particulariy suitabie for micronisation. Hydrate ilhas adéquate fiow properties and also a particulariy rapid dissolution rate in water.These properties render Hydrate 11 particular advantageous for the préparation of 5 aqueous solutions/suspensions.

Hydrates I and II hâve been subjected to X-ray powder diffraction studies.Diffraction traces were obtained using a Seimens D-500 diffractometer and CuKqradiation. X-Ray intensifies were measured at 0.02° incréments for 5 second 10 intervals using a scintillation counter, between values of 5 and 55° 26. The d-spacings and line intensifies obtained for Hydrate ! and Hydrate II are shown inTables l and il, respectively.

TABLE I ύίΑ) 10.06 30.25 6.77 69.81 6.63 89.61 6.35 12.69 6.05 54.56 5.38 25.11 5.05 98.58 4.61 12.58 4.42 100.00 4.31 8.28 4.17 11.67 3.98 75.00 3.90 52.61 3.77 20.33 3.69 36.17 3.48 26.53 3.41 53.25 3.37 17.61 3.16 18.39 3.02 31.08 ·· * 'J>\. L·» 010325 2.98 9.25 2.92 6.28 2.87 13.58 2.82 10.78 2.78 6.78 2.74 18.03 2.69 15.33 2.65 6.25 2.63 6.44 2.59 11.44 2.49 14.31 2.45 18.81 2.41 8.64 2.35 11.36 2.19 5.42 2.13 12.25 2.11 6.56 2.02 8.33 1.98 5.47 TABLE 11 diAl iœ 16.88 66.34 10.38 50.60 9.50 16.08 8.47 40.46 7.12 100.00 5.84 11.78 5.33 18.83 5.21 33.99 4.78 12.94 4.57 75.81 4.32 16.37 4.25 18.49 010325 8 4.14 43.26 3.96 10.33 3.76 22.11 3.64 25.16 3.57 37.04 3.52 15.69 3.40 16.85 3.34 21.20 3.17 13.52 3.13 17.04 3.06 7.48 2.94 10.19 2.92 8.45 2.86 9.17 2.76 9.56 2.72 9.22 2.67 6.81 2.64 8.06 2.60 5.46 2.58 6.52 2.51 5.31 2.49 6.66 2.45 5.55 2.43 5.89 2.39 15.93 2.38 10.38 2.31 8.40 2.22 5.94 2.16 5.36 2.11 6.28 2.03 7.24 1.91 6.57

The following examples illustrate the invention but are not intended as a limitationthereof. Ail températures are in °C. δ 010325

Example 1

Préparation of Hydrate l A mixture of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-Ëi-glycero-Ê-galacto-non-2-enopyranosonic acid (5.0g) and water (60mî) was heated at 100° to give a clearsolution. The solution was cooled during 30 min to 55° and maintained at between55° and 50° during 4h, to give a crystalline suspension. Acetone (80ml) wasadded during 90 min, the température being maintained between 48 and 55°.The résultant slurry was stirred 1 h, the température being allowed to fall to ca.20°, and the suspension was allowed to stand 17h at ambient température. Theproduct was collected by vacuum filtration and the filter bed was washed with 4:1acetone/water (2 x 10ml) then with acetone (10ml). The product was air dried atambient température and humidity to give Hydrate I (tabuler crystals) (4.5g). PMR (D2O) 2.04 (3H,s), 3.67 (2H,m), 4.23 (1H,m), 4.42 (2H,m), 5.63 (1H, d, J, 2.5Hz)

IR (Nujol) 3248, 3338, 3253; NH, OH

1692, 1666,1646, 1619,1575; CO (CH3CONH, CO2), CN

Water content 8.4% w/w; calculated for C12H20N4O7.1.7H2O

Exgmple.2

P-rgpgration Pf-Hydrate I A mixture of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-n-glycero-2-galacto-non-2-enopyranosonic acid (15.0g) and water (180ml) was heated at 100° to give aclear solution. The solution was darified by vacuum filtration through a filter paperthen cooled to ca. 55° and maintained at between 55° and 50° during 4h, allowingcrystallisation to become established. Acetone (210ml) was added with stirring,during 2h, the température being maintained at 48-55°. The résultant suspensionwas stirred and cooled to 30°and was then allowed to stand at ambienttempérature 17h. The soiid was filtered and the product was washed with 4:1acetone/water (2x30ml) and then acetone (30ml). The soiid was air dried atambient température and humidity to give Hydrate I (tabular crystals) (12.0g).

Characterisation as above. 10 010325

Préparation of Hydrate II A mixture of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-£)-glycero-£)-galacto-non-2-enopyranosonic acid (10.0g) and water (100ml) was heàted to 95°. Therésultant solution was clarified by vacuum filtration. The solution was then cooledto 30° and acetone (250ml) was added during 5 min stirring. The résultant thîckwhite suspension was allowed to stand ai ambient température 20 h. The solidwas collected by vacuum filtration and was washed with 4:1 acetone/water(2x20ml) then with acetone (20ml). The solid was dried in a vacuum oven at 35°for 24h and then equilibrated with atmospheric moisture at ambient températureand humidity to give Hydrate II.(needie-shaped crystals) (8.16g).

Characterisation as above.

Water content 10.6% w/w; calculated for C12H20N4O7.2H2O 9.8% w/w.

Example 4

Préparation of Hydrate II A mixture of 5-acetamido-2,3,4I5-tetradeoxy-4-guanidino-J0-glycero-D-gaiacto-non-2-enopyranosonic acid (10.0g) and water (400ml) was heated to 20° for 2h.The résultant solution was clarified by vacuum filtration. Acetone (110ml) wasadded and solid began to crystallise. The résultant suspension was stirred for2.5h at 20°. The solid was collected by vacuum filtration and was washed with 4:1v/v acetone/water (2x20m!) then with acetone (20ml). The solid was dried in avacuum oven at 30° and then equilibrated with atmospheric moisture at ambienttempérature and humidity to give Hydrate II (needie-shaped crystals) (7.7g)

Characterisation as above.

Water content 11.1% w/w calculated for C12H20N4O7.2H2O 9.8 w/w.

Example 5

Erepargtion-aiüydiaîË-ü A mixture of 5-acetamido-2,314,5-tetradeoxy-4-guanidino-iQ-giycero-S-galacto-non-2-eno-pyranosonic acid (50g) and water (1150ml) was heated to 75°. Therésultant solution was clarified by vacuum filtration, washing through with water 11 010325 (100ml). The solution was then cooled to 7° over 1h and acetone (500ml) wasadded. The résultant solution was stirred slowly for 0.5h, during which time solidbegan to crystaliise. Acetone (750ml) was then added to the suspension over 2h,maintaining the température at 5 - 10°. The solid was collected by vacuumfiltration and washed with 4 : 1 v/v acetone/water (2 x 100ml) then with acetone(100ml). The solid was air-dried at ambient température and humidity to giveHydrate H (needle-shaped crystals) (46.0g).

Water content 9.8% w/w; calculated for C12H20N4O7.2H2O 9.8% w/w. XRD: consistent with Hydrate II (>99%).

Example 6

Précipitation of Hydrate II A mixture of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-£)-glycero-D-galacto-non-2-eno-pyranosonic acid (50g) and water (600ml) was heated to 100°. Therésultant clear hot solution was added over 8 minutes to acetone (700ml) stirredrapidly at ambient température, causing the température of the mixture to rise from20° to 56° and précipitation of solid. The résultant suspension was allowed to coolto 20° with stirring, then the solid was cotlected by vacuum filtration and washedwith 4 : 1 acetone/water (2 x 100ml) then with acetone (100mi). The solid was air-dried at ambient température and humidity to give Hydrate II (needle-shapedcrystals) (47.9g).

Water content 10.0 w/w; calculated for C12H20N4O7.2H2O 9.8% w/w. XRD: consistent with Hydrate II (>99%).

Example 7

Crystallisation of Hydrate l from Water A mixture of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-£-glycero-n-galacto-non-2-eno-pyranosonic acid (35.7g) and water (350ml) was heated at 95° to give aclear solution. This was adjusted to pH7.0 (from pH6.4) with aqueous acetic acid(100μΙ, 10%v/v). The resulting solution was allowed to cool to ambienttempérature with stirring, to give a crystalline suspension. The product was 12 010325 collected by vacuum filtration, then vacuum-dried at ambient température to giveHydrate I (tabular crystals) (28.9g).

IR consistent with Hydrate I

Example 3

Préparation of Hydrate II by Seeding A mixture of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-gaiacto-non-2-eno-pyranosonic acid (30.0g) and water (540ml) was heated at 75° to give asolution. The résultant solution was clarified by vacuum filtration, washing throughwith water (54ml). The solution was heated to 100°, cooled to 40°, then seededwith Hydrate II (0.3g). Crystallisation occurred as the température was furtherreduced to ambient température. The résultant slurry was stirred for 1h at ambienttempérature, cooled to 5°, then acetone (600ml) was added over 1.5h. The solidwas collected by vacuum filtration and washed with 4:1v/v acetone/water (2x60ml)then with acetone (60ml). The solid was air-dried at ambient température andhumidity to give Hydrate II (needle-shaped crystals) (26.5g). XRD 90-95% Hydrate II (5-10% Hydrate I)

Examgls 9

Interconversion of Hydrate II to Hydrate I by Ageina A mixture of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-D-galacto-non-2-eno-pyranosonic acid (10.0g) and water (200ml) was heated at 100° to give asolution. The résultant solution was rapidly cooled to 30°, seeded with Hydrate II(0.05g) then left unstirred ovemight Light-microscopy showed exclusively thecharaderistic needles of Hydrate II. The suspension was aged unstirred atambient température for 11 days (when light-microscopy showed the presence ofsome crystals of Hydrate I), then stirred for 3 days. Acetone (200ml) was added,and the slurry was stirred for 1h. The solid was collected by vacuum filtration andwashed with 4:1v/v acetone/water (2 x 20ml) then with acetone (20ml). The solidwas air-dried at ambient température and humidity to give Hydrate I (tabularcrystals) (9.0g). XRD: Consistent with Hydrate I >99%) 13 010325

Examgte._10

Interconversion of Hydrate II to Hydrate I usina Base A suspension of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-£-giycero-p-galacto-non-2-enopyranosonic acid (5.0g, Hydrate II) in water (30mi), containing imidazole(2.96g) was stirred and heated at 30° for 40h. The remaining solid was coliectedby vacuum filtration and washed with water (2 x 1ml, 2 x 5ml) then air-dried atambient température and humidity to give Hydrate I (tabular crystals) (3.98g).

IR consistent with Hydrate I

Example 11

Préparation of Hydrate I by Seeding A mixture of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-iQ-glycero-D-galacto-non-2-eno-pyranosonic acid (5.0g) and water (60ml) was heated at 100° to give asolution. The résultant solution was ciarified by vacuum filtration. The résultantsolution was cooled to about 50°, seeded with Hydrate l, left unstirred at 50-55° for1 h, then stirred for 1 h at 50-55°. Acetone (70ml) was added whilst a températureof 48 - 55° was maintained. The slurry was stirred for 1h at 50 - 55°, then agedunstirred ovemight at ambient température. The solid was coliected by vacuumfiltration and washed with 4:1v/v acetone/water (2 x 10ml) then with acetone (2 x10ml). The solid was vacuum-dried, then allowed to re-equilibrate at ambienttempérature and humidity to give Hydrate l (tabular crystals) (3.8g). XRD: Consistant with Hydrate i (>99%)

Claims

010325 14 CLAIMS 1. 5-Acetamido-2,3,4,5-tetradeoxy-4-guaniclino-D-g!ycero-Drga!acto-non-2-enopyranosonic acid in crystalline form.
2. 5-Acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-glycero-Q-galacto-non-2-enopyranosonic acid in the form of crystals having a low aspect ratio.
3. The crystalline form as claimed in Claim 2 wherein the crystals aretabular.
4. The crystalline form as claimed in any one of Claims 1 to 3 whereinsubstantially ail water of crystallisation is lost at about 80 to 9Q°C.
5. The crystalline form as ciaimed in any one of Claims 1 to 4 for which theX-ray data are as shown in Table J.
6. 5-Acetamido-2,3,4,5-tetradeoxy-4-guanidino-Q-glycero-D-galacto-non-2-enopyranosonic acid in the form of crystals having a high aspect ratio.
7. The crystalline form as claimed in Claim 6 wherein the crystals areneedle-shaped.
8. The crystalline form as claimed in any one of Claims 1, 6 or 7 whereinwater content is stable over a broad range of relative humidity.
9. The crystalline form as claimed in any one of Claims 1, 6 to 8 whereinone mole of water of crystallisation is lost at about 84-90°C and a furthermole of water of crystallisation is lost by about 135-143°C.
10. The crystalline form as claimed in any one of Claims 1, 6 to 9 for whichthe X-ray data are as shown in Table 11.
11. The crystalline form claimed in any one of Claims 2 to 5 substantially freeof the crystalline form claimed in any one of Claims 6 to 10. 15 010325
12. The crystalline form claîmed in any one of Claims 6 to 10 substantiallyfree of the crystalline form ciaimed in any one of Claims 2 to 5. 5
13. A method for the préparation of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-B-glycero-n-galacto-non-2-enopyranosonic acid in crystallineform, which method comprises crystallisation of 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-û-glycero-n-gaIacto-non-2-enopyranosonic acidfrom aqueous solution. 10
14. A method as ciaimed in Claim 13 for the préparation of the crystallineform as ciaimed in any one of Claims 2 to 5.
15. 15 A method as ciaimed in Claim 14 wherein the température of theaqueous solution is greaterthan about 50°C.
16. A method as ciaimed in Claim 15 wherein the température of theaqueous solution is in the range 50 to 55°C. 20
17. A method as ciaimed in any one of Claims 14 to 16 wherein the aqueoussolution is seeded with crystals of the crystalline form as ciaimed in anyone of Claims 2 to 5.
18. 25 A method as ciaimed in Claim 13 for the préparation of the crystallineform as ciaimed in any one of Claims 6 to 10.
19. A method as ciaimed in Claim 18 wherein the température of theaqueous solution is less than about 40°C. 30
20 A method as ciaimed in Claim 19 wherein the température of theaqueous solution is in the range 20 to 30°C.
- 21. A method as ciaimed in any one of Claims 18 to 20 wherein the aqueoussolution is seeded with crystals of the crystalline form as ciaimed in any 35 one of Claims 6 to 10. 010325 16
22. A method as claimed in any one of Claims 13 to 21 comprising additionof a counter soivent to the aqueous solution.
23. A method as claimed in Ciaim 22 wherein the counter solvent is a ketoneor an alkanol.
24. A method as claimed in Ciaim 23 wherein the counter solvent is acetone.
25. A method for the préparation of the crystalline form as claimed in àny oneof Claims 2 to 5, which method comprises interconversion of thecrystalline form as claimed in any one of Claims 6 to 10.
26. The method as claimed in Ciaim 25 wherein interconversion is effectedby ageing of the aqueous solution.
27. The method as claimed in Ciaim 25 wherein the interconversion iseffected by addition of a base to the aqueous solution.
28. A method for the préparation of the crystalline form as claimed in any oneof Claims 6 to 10, which process comprises addition of an aqueoussolution of 5-acetamido-2,3t4,5-tetradeoxy-4-guanidino-D-giycero-D-galacto-non-2-enopyranosonic acid to a similar volume of a countersolvent.
29. The method as claimed in Ciaim 28 wherein the counter solvent isacetone.
30. A pharmaceutical formulation comprising 5-acetamido-2,3,4,5-tetradeoxy-4-guanidino-D-giycero-D-galacto-non-2-enopyranosonic acid incrystalline form and a pharmaceutically acceptable carrier therefor.
31. A pharmaceutical formulation as claimed in Ciaim 30 in the form of apowder. 17 010325
32. A pharmaceutical formulation as claimed in Claim 30 in the form of anaqueous solution or suspension.