SALT OF NAPHTHYRIDINE CARBOXYLIC ACID DERIVATIVE

Abstract

7-(3-Aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate and hydrates thereof, processes for their preparation, pharmaceutical compositions comprising them, and their use in antibacterial therapy.

Claims

1. 7-(3-Aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate.

2. 7-(3-Aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate.nH.sub.2O, wherein n is in the range of from 1 to 4.

3. A compound according to claim 2 wherein n is 1.5.

4. A compound according to claim 2 having peaks at 2θ=8.0, 12.2 and 14.7° in its X-ray diffraction pattern.

5. A compound according to claim 2 having an X-ray diffraction pattern substantially as shown in FIG. 7.

6. A compound according to claim 2 where n is 3.

7. A compound according to claim 2 having peaks at 2θ=7.7, and 11.8° in its X-ray diffraction pattern.

8. A compound according to claim 2 having an X-ray diffraction pattern substantially as shown in FIG. 6.

9. A compound according to claim 2 which has a moisture content of from 4 to 6%.

10. A compound according to claim 2 which has a moisture content of from 9 to 11%.

11. A pharmaceutical composition comprising a compound according to claim 1, together with a pharmaceutically acceptable carrier or excipient.

12. A compound according to claim 1, for use as a pharmaceutical.

13. A method of treating bacterial infections in humans and animals which comprises administering a therapeutically effective amount of a compound according to claim 1.

14. (canceled)

15. A process for the preparation of a compound according to claim 1, which comprises reacting 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid with methanesulfonic acid and crystallizing the resulting compound from solution, and where desired or necessary, adjusting the hydration of the compound.

16. A process for the preparation of a compound according to claim 2, comprising exposing 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate anhydrate or a solvate thereof to a relative humidity of at least 75%.

17. A process according to claim 16, wherein the solvate is a solvate with one or more organic solvents selected from C.sub.1-C.sub.4 haloalkanes and C.sub.1-C.sub.8 alcohols.

18. A solvate of 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate with one or more organic solvents.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0034] The following examples and figures illustrate the invention but are not intended to limit the scope in any way.

[0035] FIG. 1 shows the moisture sorption profile of methanesulfonate anhydrate of Example 1 at 25° C. at several relative humidities.

[0036] FIG. 2 shows the isothermal moisture sorption profile of methanesulfonate anhydrate of Example 1 at 25° C.

[0037] FIG. 3 shows the equilibrium moisture content of the methanesulfonate n=3 hydrate of Example 2 at a relative humidity of 23 to 75%.

[0038] FIG. 4 shows the equilibrium moisture content of the methanesulfonate n=1.5 hydrate of Example 3 at a relative humidity of 23 to 75%.

[0039] FIG. 5 shows the powder X-ray diffraction pattern of the methanesulfonate anhydrate of Example 1.

[0040] FIG. 6 shows the powder X-ray diffraction pattern of the methanesulfonate n=3 hydrate of Example 2. The characteristic peaks are 2θ=7.7, 11.8°. The exact position of peaks can vary slightly depending on the experimental conditions.

[0041] FIG. 7 shows the powder X-ray diffraction pattern of the methanesulfonate n=1.5 hydrate of Example 3. The characteristic peaks are 2θ=8.0, 12.2, 14.7°. The exact position of peaks can vary slightly depending on the experimental conditions.

[0042] FIG. 8 shows the variation in moisture content with elapsed time of the methanesulfonate anhydrate of Example 1, taken after 0, 5, 10, 20, 30, and 60 minutes, respectively, from the initial point of passing humidified nitrogen gas through;

[0043] FIG. 9 shows the Differential Scanning Calorimetry on the methanesulfonate anhydrate of Example 1 and the methanesulfonate n=3 hydrate of Example 2.

[0044] FIG. 10 shows the results of thermogravimetric analysis on the methanesulfonate n=3 hydrate of Example 2.

[0045] FIG. 11 shows the change in X-ray diffraction pattern with elapsed time of the methanesulfonate solvate (ethanol content 0.11%) of Example 4, from initial point of passing the humidified nitrogen gas having a relative humidity of 93% through.

[0046] FIG. 12 shows the change in X-ray diffraction pattern with elapsed time of the methanesulfonate solvate (ethanol content 1.9%) of Example 5, from the initial point of standing the sample under a relative humidity of 93%.

[0047] FIG. 13 shows the change in X-ray diffraction pattern of the methanesulfonate solvate (ethanol content 0.12%) of Example 5 under various relative humidities, that is, relative humidity of 93% (1), relative humidity of 52% (2) and relative humidity of 11% (3), respectively.

BEST MODE FOR CARRYING OUT THE INVENTION

[0048] The present inventors have performed several experiments in order to identify the moisture content and physicochemical property of the methanesulfonate anhydrate and each hydrate, and the results are described in connection with the drawings in the following.

[0049] FIG. 1 shows the moisture sorption velocity profile of 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1, 4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate anhydrate at several relative humidities. Over the whole range of relative humidity tested, the initial moisture adsorption proceeds rapidly at each relative humidity. In most cases equilibrium is achieved within 2 hours. FIG. 2 shows the isothermal moisture sorption profile of the methanesulfonate anhydrate according to the change in relative humidity at 25° C. The weight increment (%) of Y-axis represents the equilibrium moisture content, from which it can be recognized that the equilibrium moisture content depends on the relative humidity. FIG. 3 shows the equilibrium moisture content of the n=3 hydrate (which is obtained by recrystallization from a solvent mixture of ethanol and water) after it is allowed to stand for 2 weeks under relative humidities in the range of 23 to 75%. The result shows that the n=3 hydrate is more stable than the anhydrate since it maintains a moisture content of around 10% under the relative humidities tested. FIG. 4 shows the isothermal moisture adsorption profile of the n=1.5 hydrate. Here, it maintains a moisture content of around 5% under the relative humidity in the range of 23 to 64%. Thus, it is also identified as a stable hydrate.

[0050] It has been identified that the physical properties of the hydrate are very different from those of the anhydrate.

[0051] For example, by comparing the powder X-ray diffraction patterns of the anhydrate in FIG. 5, the n=3 hydrate in FIG. 6, and the n=1.5 hydrate in FIG. 7, it can be seen that their crystal forms are different from each other. In addition, the thermal analysis using Differential Scanning Calorimetry (DSC) shows that the endothermic peak produced by the vaporization of the water molecules contained in the n=3 hydrate begins at around 50° C. and the exothermic peak by thermal decomposition is observed at around 185 to 220° C., whereas the anhydrate shows only an exothermic peak at around 185 to 220° C. due to the thermal decomposition without any endothermic peak (see, FIG. 9). At the same time, the thermogravimetric analysis shows a weight decrement at the temperature range of endothermic peak, the extent of which corresponds to the moisture content quantified by Karl-Fisher method (Mettler Toledo DL37KF Coulometer) (see, FIG. 10). Therefore, it is verified that the endothermic peak shown in the DSC analysis is due to the evaporation of a water molecule.

[0052] The present inventors also compared the chemical stability under heating of the hydrates with that of the anhydrate in order to identify the influence of hydration on the chemical stability. In this test, the anhydrate and hydrate were each kept at 70° C. for 4 weeks, and the extent of decomposition is analyzed by liquid chromatography. No difference in the extent of decomposition was noticed between the hydrates and the anhydrate, and thus confirming that the hydrate has the same chemical stability as the anhydrate.

[0053] The methanesulfonate anhydrate or a solvate thereof may be converted into a hydrate under appropriate conditions as described above.

[0054] This process can be monitored by the change in the X-ray diffraction pattern of the compound and the decrease in the amount of organic solvent in the compound. Such changes being caused by the water molecules newly intercalated into the crystal structure.

[0055] As can be seen from FIG. 11, the X-ray diffraction peaks based on the solvate disappear with the passing of humidified nitrogen gas to leave the peaks based on the hydrate. This shows that all the solvates is converted into hydrates. The residual solvent is decreased to the amount of less than the quantitative limit simultaneously with the change of X-ray diffraction. FIG. 12 shows that the X-ray diffraction peaks based on the solvate disappear when the solvate is allowed to stand under a relative humidity of 93%. However, there is no change in the X-ray diffraction pattern when the solvate is allowed to stand under a relative humidity of 11% or 52% (see FIG. 13). Therefore, it is recognized that the change shown in FIG. 12 occurs not by the spontaneous evaporation of the residual solvent but by the substitution of the organic solvents in the crystal by water molecules.

[0056] In preparing the hydrate according to the processes described above, the respective hydrates having a different hydration number can be obtained by changing conditions such as humidity, time, temperature, etc. or by changing the recrystallization condition. Such conditions should be adjusted according to whether the starting material is the anhydrate or a solvate, and depending on the nature of the solvate.

[0057] The present invention will be more specifically explained by the following examples and experimental examples. However, it should be understood that the examples are intended to illustrate but not in any manner limit the scope of the present invention.

Example 1: Synthesis of 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate anhydrate

[0058] 7-(3-Aminomethyl-4-methyloxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid (3.89 g, 10 mmol) was suspended in a mixture of dichloromethane and ethanol (110 ml, 8:2 v/v). Methanesulfonic acid (0.94 g, 9.8 mmol) was added dropwise and the resulting solution was stirred for 1 hour at 0° C. The resulting solid was filtered, washed with ethanol then dried to give the title compound (4.55).

[0059] m.p.: 195° C. (dec.)

[0060] .sup.1H NMR (DMSO-d.sub.6) δ (ppm): 8.57 (1H, s), 8.02 (1H, d), 7.98 (3H, br), 4.58 (2H, br), 4.39 (1H, m), 3.91 (3H, s), 3.85 (1H, m), 3.71 (1H, m), 3.42 (1H, m), 3.20˜3.10 (2H, m), 1.20˜1.10 (4H, m)

Example 2: Synthesis of 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate n=3 hydrate

[0061] A sonicator filled with water was adjusted to 40° C., sealed with a lid and a nitrogen inlet and outlet connected. When the pressure of the dried nitrogen introduced through the inlet was 20 psi the relative humidity of the nitrogen exiting through the outlet was more than 93%. The anhydrate of Example 1 having a moisture content of 2.5% (1.0 g) was introduced into a fritted filter and the humidified nitrogen produced as described above passed through the filter. Samples were taken after 0, 5, 10, 20, 30, and 60 minutes and the moisture content measured. From the results shown in FIG. 8 it can be seen that a moisture content of about 10% is maintained when the humidifying procedure is carried out over about 30 minutes. The X-ray diffraction pattern of the humidified sample was identical to that of the n=3 hydrate obtained by recrystallization.

Example 3: Synthesis of 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate n=1.5 hydrate

[0062] The title compound was prepared by the following routes:

[0063] Route A

[0064] The anhydrate of Example 1 (1.0 g) was dissolved in a mixture of water and acetone (17 ml, 10:7 v/v). The solvent was slowly evaporated in darkness leaving the title compound as a solid (0.8 g).

[0065] Route B

[0066] The anhydrate of Example 1 (5.0 g) was added to water (10 ml) and the mixture was heated to 45° C. to aid dissolution. Ethanol (20 ml) was added and the resulting solution stirred then allowed to stand. The resulting solid was filtered and dried under a flow of nitrogen to give the title compound (2.6 g).

Example 4: Synthesis of the hydrate from 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate solvate using a humidified nitrogen gas

[0067] A sonicator filled with water was adjusted to 40° C. and was sealed with a lid. Then, a nitrogen inlet and outlet were connected to the vessel. When the pressure of the dried nitrogen introduced through the nitrogen inlet was adjusted to about 20 psi, the relative humidity of the humidified nitrogen gas exiting through the outlet was more than 93%. The solvate (1 g, ethanol 0.11%) of the anhydrate of Example 1 was introduced into a fritted filter and the humidified nitrogen gas prepared as described above was passed through the filter. Samples were taken after 40 minutes, 3.5 and 6 hours, respectively. The change in the amount of residual organic solvent and X-ray diffraction pattern with the lapse of time were examined. After 3.5 hours, it was identified that the product contained the organic solvent in an amount of less than 50 ppm and that the peaks based on the solvate disappeared, whilst the peaks based on the mixture of n=3 hydrate and n=1.5 hydrate appeared.

Example 5: Synthesis of the hydrate from 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate solvate using a high relative humidity

[0068] Saturated aqueous potassium nitrate solution was placed in a desiccator, and accordingly the relative humidity inside the desiccator was controlled to 93%. For tests under relative humidity of 11% or 52%, desiccators containing saturated aqueous solutions of lithium chloride and magnesium nitrate, respectively, were prepared. Into the desiccator having a relative humidity of 93% was introduced a solvate (1.9% ethanol) of the anhydrate of Example 1, and into each of the desiccators having a relative humidity of 93%, 52% or 11% was introduced a solvate (0.12% ethanol) of the anhydrate of Example 1. The solvates were stored so as not to directly contact the aforementioned salt solutions. After a certain period of time has passed, samples were taken and subjected to gas chromatography in order to analyze the residual solvent. As a result, it was identified that solvates stored for 4 weeks under a relative humidity of 93% contained the organic solvent in an amount of less than 50 ppm. Also, it was identified by X-ray diffraction pattern that peaks based on the solvates disappeared after 4 weeks. To the contrary, in the case where the samples were stored under a relative humidity of 52% or 11%, the amount of residual organic solvent and X-ray diffraction pattern after 4 weeks were identical with those at the beginning.

Example 6: Synthesis of n=3 hydrates from 7-(3-amino-methyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate solvates

[0069] Dried nitrogen gas and humidified nitrogen gas having a relative humidity of 78 to 84% were passed over 24 hours, respectively, through 10 g of four different solvates each of which had a different kind and amount of organic solvent from the others. The amount of residual organic solvent was measured and the change in X-ray diffraction pattern was analyzed, the results of which are shown in Table 2. The X-ray diffraction analysis shows that the samples through which dried nitrogen gas was passed remained as the original solvates, while the samples through which humidified nitrogen gas was passed had the same X-ray diffraction pattern and crystallinity as those of the n=3 hydrate obtained by recrystallization.

[0070] The results from this Example suggests that water molecules contained in the humidified nitrogen gas replace the organic solvents in the solvate. This suggestion is also supported by the change in X-ray diffraction pattern influenced by a relative humidity.

TABLE-US-00002 TABLE 2 The kind and amount of the residual organic solvent The kind and amount of the after humidified residual organic solvent after nitrogen gas (78~84% dried nitrogen gas has passed RH) has passed for 24 Sample No. for 24 hours hours 1 Methylene chloride 1.14%, 0.08% Ethanol 3.73% <50 ppm 2 Isopropanol 0.45% 0.06% 3 2-Isopropanol 0.24% 0.04% 4 2-Methyl-2-propanol 0.07% 0.01% Ethanol 0.06% <50 ppm

Example 7: Synthesis of the Ethanolate Containing Ethanol 0.11%

[0071] The anhydrate of Example 1 (5.0 g) was added to a solvent mixture of ethanol (25 ml) and water (25 ml) and the mixture was heated to 50° C. to facilitate dissolution. Then, the solution was cooled slowly to −3° C. and allowed to stand at that temperature for about 3 hours. The resulting solid was filtered and washed with a solvent mixture of ethanol and water (16.5 ml, ethanol:water=20:8, v/v) to give the title compound quantitatively.

Test Example 1: Moisture Sorption of the Anhydrate of Example 1

[0072] The moisture sorption velocity and the equilibrium moisture content of the anhydrate of Example 1 was determined by means of an automatic moisture sorption analyzer (MB 300G Gravimetric Sorption Analyzer). This instrument produces a specific relative humidity at a specific temperature and continuously records the weight change of a sample due to adsorption or desorption of moisture as measured by a micro balance inside the instrument. The anhydrate of Example 1 (16 mg) was loaded onto the micro balance and the moisture contained in the sample removed under a stream of dried nitrogen at 50° C. A weight change of less than 5 μg per 5 minutes was the criterion for complete dryness. Thereafter, the inner temperature was adjusted to 25° C. and the sample tested at 5% intervals whilst varying the humidity from 0 to 95%. The sample was considered to have reached equilibrium when the weight change was less than 5 μg per 5 minutes. FIG. 1 shows the moisture adsorption velocity, that is the time required for the sample to reach equilibrium at each relative humidity. As can be seen initial moisture adsorption proceeded rapidly at each relative humidity tested, in most cases equilibrium was reached within 2 hours. FIG. 2 shows the weight increment at each relative humidity, i.e. the equilibrium moisture content. It is clear from FIG. 2 that the equilibrium moisture content of the anhydrate is dependent on the relative humidity.

Test Example 2: Thermal Analysis of the Anhydrate of Example 1 and n=Hydrate of Example 2

[0073] For the Differential Scanning Calorimetry, METTLER TOLEDO DSC821e and METTLER TOLEDO STARe System were used. The sample (3.7 mg) was weighed into the aluminum pan which was then press sealed with an aluminum lid. Three tiny needle holes were made on the lid and the sample tested by heating from normal temperature to 250° C. at a rate of 10° C./min. As can be seen from FIG. 9, the endothermic peak due to the vaporization of the water molecules contained in the n=3 hydrate begins at around 50° C. and the exothermic peak due to the thermal decomposition is observed at around 180 to 220° C. In contrast, the anhydrate showed only an exothermic peak due to the thermal decomposition at around 185 to 220° C. without any endothermic peak.

[0074] In the thermogravimetric analysis, SEIKO TG/DTA220 was used. The sample (3.8 mg) was weighed into an aluminum pan and was heated from normal temperature to 250° C. at a rate of 10° C./min according to the temperature raising program. As can be seen from FIG. 10, weight decrement was observed at the temperature range of endothermic peak, the extent of which corresponds to the moisture content determined by Karl-Fisher method (Mettler Toledo DL37KF Coulometer).

Test Example 3: Equilibrium Moisture Content Determination of Hydrates

[0075] Six saturated aqueous salt solutions were introduced into each desiccator to control the inner relative humidity to a specific value as shown in Table 3. Then, equilibrium moisture contents of n=3 hydrate and n=1.5 hydrate of Examples 2 and 3, respectively, were determined at several relative humidities.

TABLE-US-00003 TABLE 3 Saturated salt solutions inside the desiccator Salt Solution Relative Humidity (%) at 25° C. Potassium Acetate 23 Magnesium Chloride 33 Potassium Carbonate 43 Magnesium Nitrate 52 Sodium Nitrite 64 Sodium Chloride 75

[0076] The sample (100 mg) was spread on a pre-weighed Petri dish and the total weight was accurately measured, then three of the sample were placed in each desiccator of Table 3. The desiccators were allowed to stand at normal temperature for 7 days and then the sample was taken to be weighed. After 13 days, one of the three samples inside each desiccator was taken and the moisture content of each was measured by the thermogravimetric analysis described in Test Example 2. Equilibrium moisture content at each relative humidity is represented in FIG. 3 (n=3 hydrate) and FIG. 4 (n=1.5 hydrate). FIG. 3 shows that moisture content of the n=3 hydrate is maintained around 10% for the whole relative humidity range tested; FIG. 4 shows that the moisture content of the n=1.5 hydrate is maintained around 5% at the relative humidity of 23 to 64%. Both hydrates are stable since they maintain a constant equilibrium moisture content regardless of the relative humidity change.

Test Example 4: X-Ray Diffraction Analysis

[0077] The anhydrate of Example 1, n=3 hydrate of Example 2 and n=1.5 hydrate of Example 3 (50 mg of each) were thinly spread on the sample holder, X-ray diffraction analysis (35 kV×20 mA Rigaku Gergeflex D/max-III C) were performed under the conditions listed below. [0078] scan speed (2θ) 5°/min [0079] sampling time: 0.03 sec [0080] scan mode: continuous [0081] 2θ/θ reflection [0082] Cu-target (Ni filter)

[0083] Results of X-ray diffraction analyses on the anhydrate, n=3 hydrate, and the n=1.5 hydrate are shown in FIGS. 5, 6, and 7. The diffraction patterns illustrate the difference in crystal form of these 3 compounds.

[0084] According to a further aspect of the invention we provide 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate having an X-ray diffraction pattern substantially as shown in FIG. 5, 6 or 7.

[0085] We also provide 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate hydrate having peaks at 2θ=8.0, 12.2 and 14.7° in its X-ray diffraction pattern; and 7-(3-aminomethyl-4-methoxy-iminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate hydrate having peaks at 2 θ=7.7 and 11.8° in its X-ray diffraction pattern.

[0086] The change of crystallinity during the conversion from the solvate to the hydrate in Examples 4 and 5 was identified by X-ray diffraction analysis under the same conditions as mentioned above (see, FIG. 11 to 13). FIG. 11 shows the X-ray diffraction pattern of the solvate is changed into that of the n=3 hydrate (see, Example 4); FIG. 12 represents the change in X-ray diffraction pattern of the solvate containing 1.9% of ethanol before and after storage of one week, two weeks, three weeks and four weeks at 93% of relative humidity; and FIG. 13 represents the change in X-ray diffraction pattern of the solvate containing 0.12% of ethanol after storage of four weeks at 93%, 52% and 11% of relative humidity, respectively (see, Example 5).

Test Example 5: Chemical Stability

[0087] The chemical stability of the n=3 hydrate of Example 2 and the n=1.5 hydrate of Example 3 and the anhydrate of Example 1 were compared at elevated temperature in order to determine the effect on chemical stability of the extent of hydration.

[0088] The anhydrate and each of the hydrates were introduced into a glass vial and maintained at 70° C. The extent of decomposition with elapsed time was analyzed by liquid chromatography. The results obtained are shown in Table 4.

TABLE-US-00004 TABLE 4 Thermal stability with elapsed time (at 70° C., Unit: %) Time (week) Sample Initial 1 2 3 4 Anhydrate 100 99.8 98.6 97.7 96.7 n = 3 hydrate 100 102.4 100.7 99.2 99.2 n = 1.5 hydrate 100 97.3 95.8 97.2 96.2

[0089] As can be seen from Table 4, the n=3 hydrate and the n=1.5 hydrate both show the same degree of chemical stability as the anhydrate.

Test Example 6: In Vitro Antibacterial Activity

[0090] In order to determine whether 7-(3-aminomethyl-4-methyloxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate has the same antibacterial activity as the free base, in vitro antibacterial activity of the methanesulfonate was measured using agar medium dilution method. The results are shown in Tables 5. The minimum inhibitory concentration (MIC, μg/ml) was simply calculated in the ratio of weight without considering the molecular weight, and ciprofloxacin was chosen as the control

TABLE-US-00005 TABLE 5 In vitro Antibacterial activity (Minimum Inhibitory Concentration: MIC, μg/ml) Methanesulfonic Test Strains acid salt Ciprofloxacin Staphylococcus aureus 6538p 0.016 0.13 Staphylococcus aureus giorgio 0.016 0.13 Staphylococcus aureus 77 0.031 0.25 Staphylococcus aureus 241 4 128 Staphylococcus aureus epidermidis 0.016 0.13 887E Staphylococcus aureus epidermidis 4 128 178 Staphylococcus aureus faecalis 0.13 0.5 29212 Bacillus subtilis 6633 0.016 0.031 Micrococcus luteus 9431 0.13 2 Escherichia coli 10536 0.008 <0.008 Escherichia coli 3190Y 0.008 <0.008 Escherichia coli 851E 0.016 <0.008 Escherichia coli TEM3 3455E 0.25 0.5 Escherichia coli TEM5 3739E 0.13 0.13 Escherichia coli TEM9 2639E 0.031 0.016 Pseudomonas aeruginosa 1912E 0.25 0.13 Pseudomonas aeruginosa 10145 0.5 0.5 Acinetobacter calcoaceticus 15473 0.031 0.25 Citrobacter diversus 2046E 0.031 0.016 Enterobacter cloacae 1194E 0.031 0.016 Enterobacter cloacae P99 0.016 <0.008 Klebsiella aerogenes 1976E 0.13 0.13 Klebsiella aerogenes 1082E 0.031 0.016 Proteus vulgaris 6059 0.25 0.031 Seratia marsecence 1826E 0.13 0.063 Salmonella thypimurium 14028 0.031 0.031

Test Example 7: Water Solubility of the Anhydrate of Example 1

[0091] The water solubility of the free base and various salts of 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid, including the methanesulfonate of Example 1, was measured at 25° C. The results are shown in Table 6.

TABLE-US-00006 TABLE 6 Water Solubility (at 25° C.) Solubility in water Sample (mg/ml) Free form 0.007 Tartrate 6.7 Sulfurate 11.4 p-Toluenesulfonate 7.5 Methanesulfonate >30

[0092] As can be seen, the methanesulfonate shows increased water solubility compared to that of the tartrate, the sulfurate, and the p-toluenesulfonate and the free base.