ANTIBIOTIC COMPOUNDS

FIELD OF THE INVENTION

The present invention relates to the new crystalline solid form Xl of Tigecycline and a process of preparing the same. Form Xl of Tigecycline is particularly suitable for the isolation of Tigecycline in the last step of the synthesis of Tigecycline. Further the present invention relates to a process of preparing amorphous Tigecycline by spray drying form Xl or another crystalline form of Tigecycline.

BACKGROUND OF THE INVENTION

Tigecycline, (4S,4aS,5aR,12aS)- 4,7-Bis(dimethylamino)-9-[[2-[(1 ,1 - dimethylethyl)amino]acetyl]amino]-1 ,4, 4a, 5, 5a, 6,1 1 ,12a-octahydro-3,10,12,12a-tetrahydroxy- 1 ,1 1 -dioxo-2-naphthacene carboxamide (Fig. A), is a 9-t-butylglycylamido derivative of minocycline (Merck Index 14 ^th Edition, monograph number 9432, CAS Registry Number 220620-09-7). Compared to other tetracycline antibiotics Tigecycline is more active against tetracycline-resistant strains and also more tolerable. Tigecycline possesses activity against bacterial isolates containing the two major determinants responsible for tetracycline- resistance: ribosomal protection and active efflux of the drug out of the bacterial cell. Further Tigecycline has broad spectrum activity, as it is active against gram-positive pathogens (e.g. methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci), gram- negative pathogens (e.g. Acinetobacter baumannii, Stenotrophomonas maltophilia) and anaerobic pathogens. It is used for the treatment of complicated skin and skin structure infections and intra-abdominal infections [PJ. Petersen et al., Antimicrob. Agents and Chemoth. 43:738-744 (1999); R. Patel et al., Diagnostic Microbiology and Infectious Disease 38:177-179 (2000); H.W. Boucher et al., Antimicrob. Agents and Chemoth. 44:2225-2229 (2000); DJ. Biedenbach et al., Diagnostic Microbiology and Infectious Disease 40:173-177 (2001 ); PJ. Petersen et al., Antimicrob. Agents and Chemoth. 46:2595-2601 (2002); D. Milatovic et al., Antimicrob. Agents and Chemoth. 47:400-404 (2003); T. Hirata et al., Antimicrob. Agents and Chemoth. 48:2179-2184 (2004); G.A. Pankey Journal of Antimicrobial Chemotherapy 56:470-480 (2005); R. Harris et al., P&T 31 :18-59 (2006)].

Figure A: Chemical structure of Tigecycline

At the moment Tigecycline is only available as injectable antibiotic, as its oral bioavailability is very limited. The orange lyophilized powder or cake is available in 5 ml vials containing 50 mg of the amorphous agent [R. Harris et al., P&T 31 :18-59 (2006)].

Patent application WO 2006/128150 discloses crystalline forms I, II, III, IV and V of Tigecycline and methods of their preparation. In WO 2007/127292 two crystalline forms but also an amorphous form of Tigecycline are disclosed as well as processes for their preparations. Nevertheless, there remains a need for alternative crystalline forms of Tigecycline, which have properties suitable for pharmaceutical processing on a commercial scale.

SUMMARY OF THE INVENTION

The present invention relates to novel form Xl of Tigecycline, a n-heptane solvate, characterized by an X-ray powder diffraction pattern with peaks at 3.9, 7.9, 8.8, 10.4, 10.9, 12.6, 13.5, 13.9, 15.8, 16.4, 17.8, 19.6, 20.3, 21.1 and 23.4 degrees 2-theta.

The present invention also provides a process of preparing form Xl of Tigecycline comprising the steps of: a) dissolving Tigecycline in methylene chloride b) crystallizing Tigecycline form Xl by the addition of n-heptane c) stirring the solution at room temperature or below to effect complete crystallization d) optionally isolating crystalline form Xl of Tigecycline

In another embodiment, the present invention also provides a simple, cost-effective process of preparing amorphous Tigecycline by spray drying form Xl or another crystalline form of Tigecycline. The experimental conditions of this process are easy to operate and suitable for large-scale production.

Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the description and the following specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the description and from reading the other parts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : X-ray powder diffraction pattern of form Xl of Tigecycline

Figure 2: Infrared spectrum of form Xl of Tigecycline

Figure 3: Differential scanning calorimetric curve of form Xl of Tigecycline

Figure 4: Thermogravimetric analysis curve of form Xl of Tigecycline

Figure 5: X-ray powder diffraction pattern of spray dried amorphous Tigecycline

Figure 6: X-ray powder diffraction pattern of spray dried amorphous Tigecycline

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term "room temperature" indicates that the applied temperature is not critical and that no exact temperature value has to be kept. Usually, "room temperature" is understood to mean temperatures of about 15 ⁹ C to about 25 °C (see e.g. EU Pharmacopoeia 5.0, page 6).

The inventors of the present invention have identified a novel solvate of Tigecycline. The novel crystalline form is a n-heptane solvate and may be characterized e.g. by a typical X-ray powder diffraction pattern, infrared spectrum, a characteristic differential scanning calorimetric (DSC) curve or by a characteristic thermogravimetric analysis (TGA) curve. Each of these characteristics on its own is sufficient to unambiguously define and identify the new pseudo polymorph but they also may be combined with each other.

The present invention relates to a novel form Xl of Tigecycline. Form Xl of Tigecycline is a n- heptane solvate, hereinafter also referred to as "form Xl" characterized by a X-ray powder diffraction pattern with peaks as shown in Table 1 at 3.9, 7.9, 8.8, 10.4, 10.9, 12.6, 13.5, 13.9, 15.8, 16.4, 17.8, 19.6, 20.3, 21.1 and 23.4 degrees 2-theta.

A characteristic X-ray powder diffraction pattern of form Xl of Tigecycline is shown in figure 1 and some characteristic peaks are listed in table 1.

Accordingly, in a preferred embodiment, the present invention relates to a novel form Xl of Tigecycline characterized by a X-ray powder diffraction pattern substantially in accordance with table 1 and figure 1 .

The powder pattern of Form Xl clearly can be distinguished from these of forms I, II, III, IV and V disclosed in patent application WO 2006/128150. For example, Form Xl of Tigecycline possesses an intensive peak at 3.9 degrees 2-theta. This peak does not appear in any of the powder patterns of forms I to V from patent WO 2006/128150. Furthermore the whole powder pattern of form Xl differs clearly from these of forms I, II, III, IV and V from WO 2006/128150.

In addition the powder pattern of form Xl also can be distinguished from these of forms I and Il from patent application WO 2007/127292. Form I of WO 2007/127292, for example, does not display peaks at 3.9, 7.9, 8.8, 10.4 and 10.8 degrees 2-theta, whereas form Xl of the present invention does. On the other hand form I of WO 2007/127292 shows a characteristic peak at 1 1.4 degrees 2-theta, which is missing in the powder pattern of form Xl.

Furthermore form Xl of the present invention also shows a different powder pattern compared to the powder pattern of form Il in WO 2007/127292, which does not display peaks at 3.9, 7.9, 8.8, 10.4 and 10.8 degrees 2-theta either. On the other hand form Il of WO 2007/127292 shows characteristic peaks, for example, at 4.8, 6.4 and 6.8 degrees 2-theta, which are missing in the powder pattern of form Xl.

Thus form Xl of the present invention can be seen as a novel crystalline form of Tigecycline.

Form Xl of Tigecycline may be also characterized by a typical infrared spectrum as shown in figure 2. Accordingly, in a further preferred embodiment, the present invention relates to form Xl of Tigecycline characterized by an infrared spectrum substantially in accordance with figure 2. Characteristic bands are present at 2957, 2927, 2788, 1675, 1612, 1518, 1281 , 1056 and 871 cm ¹ .

In addition, form Xl of Tigecycline shows a characteristic DSC curve at a heating rate of 10 ^< O/min. It can be seen in figure 3 that the DSC curve of form Xl displays a broad endothermic peak, which is due to desolvation.

The TGA curve shown in figure 4 shows a total weight loss of about 13.8 %, which is due to the desolvation process and shows good correspondence with GC-analysis.

Table 1 : X-Ray Powder Diffraction (XRPD) pattern of form Xl of Tigecycline

In one embodiment, the present invention provides a process of preparing form Xl of Tigecycline, comprising steps of: a) dissolving Tigecycline in methylene chloride b) crystallizing Tigecycline form Xl by the addition of n-heptane c) stirring the solution at room temperature or below to effect complete crystallization d) optionally isolating crystalline form Xl of Tigecycline

For preparing form Xl of Tigecycline according to the above process, any other form of Tigecycline may be used, e.g. the amorphous form, crystalline form I or Il disclosed in WO 2007/127292 or crystalline form I to V disclosed in WO 2006/128150. In addition, also forms of low crystallinity or mixtures of two or more different forms of Tigecycline may be used. The crystallization step b) of the above process may be facilitated by adding seed crystals of form Xl of Tigecycline. The process represents a practical purification method for Tigecycline, because most of the impurities of Tigecycline are soluble in methylene chloride and remain in solution.

Accordingly, form Xl of Tigecycline is also a particularly suitable form for the isolation of Tigecycline in the last step of the synthesis of Tigecycline. If, for example, 9-

chloroacetaminominocycline is reacted with tert.-butylamine in dimethylacetamide, Tigecycline can be obtained after a simple extractive work up in high yield and in high purity without an additional purification step. Analysis (area%) by HPLC shows a purity of the product of greater than 98.1 % with a C ₄ -epimer content of less than 1.4%.

For the preparation of form Xl of Tigecycline room temperature or an elevated temperature may be applied but usually it will be in the range of 10 °C to 40 °C. However, it is crucial that solvent and temperature are chosen such that the used form of Tigecycline crystallizes out of the solution, remains in the condition of a suspension and does not dissolve again. It is well within the general knowledge of a person skilled in the art to determine temperature accordingly.

In another embodiment the present invention relates to a method of preparing amorphous Tigecycline by spray drying form Xl or another crystalline form of Tigecycline. It has been surprisingly found that spray drying yields amorphous Tigecycline in high purity without a significant increase of the C ₄ -epimer content although Tetracycline epimerization is known to be temperature dependent (P. H. Yuen, T.D. Sokoloski, J. Pharm. Sci. 1977, (66), 1646 - 1650). As lyophilization is a cost-intensive process of preparing amorphous Tigecycline the present invention provides a cost effective alternative process of preparing amorphous Tigecycline comprising the steps of: a) dissolving or slurrying Tigecycline in a suitable solvent b) spray drying the solution or suspension

Suitable solvents may be polar solvents like water, alcohols (e.g. methanol) and ketones (e.g. acetone). Furthermore methylene chloride is suitable too.

The invention is further described by reference to the following examples. These examples are provided for illustration purposes only and are not intended to be limiting the present invention in any way.

EXAMPLES

Powder diffractogram of form Xl was collected on a Unisantis XMD 300 X-ray powder diffractometer with a position sensitive detector in parallel beam optics using the following acquisition conditions: tube anode: Cu, 40 kV, 0.8 mA; 3 - 43 degrees theta/2-theta; simultaneous detection of regions of 10 degrees per step with detector resolution 1024, counting time 300 seconds per step. A typical precision of the 2-theta values is in the range ± 0.2 degrees 2-theta. Thus a diffraction peak that appears at 5.0 degrees 2-theta can appear

between 4.8 and 5.2 degrees 2-theta on most X-ray diffractometers under standard conditions.

Infrared spectra were collected on a diamond ATR cell with a Bruker Tensor 27 FTIR spectrometer with 4 cm ^"1 resolution. A typical precision of the wavenumber values is in the range ± 2 cm ^"1 . Thus an infrared peak that appears at 1716 cm ^"1 can appear between 1714 and 1718 cm ^"1 on most infrared spectrometers under standard conditions.

Differential scanning calorimetry (DSC) was performed on a Netzsch DSC 204 instrument. Samples were heated in 25 μl Al-Pans with loose lids from room temperature to 250 °C at a rate of 10 'O/min. Nitrogen (purge rate 20 ml/min) was used as purge gas.

Thermogravimetric analysis (TGA) was performed on a Netzsch STA 409 PC/PG instrument. Samples were heated in an AI ₂ O ₃ crucible from room temperature to 300 °C at a rate of 10 ^< O/min. Nitrogen (purge rate 50 ml/min) was used as purge gas.

Spray drying was performed on a Bϋchi mini spray dryer 190.

Example 1 : Preparation of Tigecycline Form Xl

25 ml N,N-dimethylacetamide and 10.0 g 9-chloroacetaminominocycline x 1.5 TBA (t- butylamine) were put into a three-necked- flask at room temperature. 9.05 g t-butylamine and 3.0 g sodium iodide were added to the suspension and the reaction mixture was stirred for 2 hours at 50 °C. Then the reaction mixture was transferred into a Schmizo reactor and diluted with 250 ml methylene chloride and 250 ml water. The pH was adjusted to 8.29 (± 0.1 ) by dropwise adding concentrated hydrochloric acid. After stirring the mixture for 5 to 10 minutes the layers were separated. Before the aqueous layer was washed two times with 250 ml methylene chloride, the pH was adjusted to 8.0 ± 0.1 with 0.1 N sodium hydroxide. The united organic layers were washed three times with 250 ml water and again the pH was adjusted to 8.0 by dropwise adding either 0.1 N hydrochloric acid or 0.1 N sodium hydroxide before washing. Then the organic layer was filtered through a fluted filter and evaporated at 40 °C on the rotavapor. The liquid residue was diluted with 100 ml of methylene chloride and again transferred into a Schmizo reactor. Tigecycline was crystallized within a few minutes by the addition of 120 ml n-heptane. The suspension was stirred for 3 hours at room temperature and over night at 5 °C. The solid was filtered off, washed with a cold methylene chloride/ n-heptane (3:7) mixture and dried under vacuum over night to obtain 8.2 g (92 % of

yield) of crystalline form Xl of Tigecycline. (HPLC area % 98.8 with a C4-epimer content of 0.6%; GC: 13.21 % n-heptane)

Example 2: Preparation of amorphous Tigecycline

883 mg crystalline Tigecycline Form Xl were dissolved in 53 ml water at room temperature. The solution was subjected to spray drying in the following setting of spray dryer:

Spray drying resulted in amorphous Tigecycline with an X-ray powder pattern in accordance with figure 5.

Example 3: Preparation of amorphous Tigecycline

500 mg crystalline Tigecycline Form Xl were dissolved in 75 ml of a methanol/methylene chloride mixture (1 1 :4) at room temperature. The solution is subjected to spray drying in the following setting of spray dryer:

Spray drying resulted in amorphous Tigecycline with an X-ray powder pattern in accordance with figure 6.