OA11161A

OA11161A - Salt of naphthyridine carboxylic acid derivative

Info

Publication number: OA11161A
Application number: OA9900213A
Authority: OA
Inventors: Jay Hyok Chang; Jong Ryoo Choi; Hoon Choi; Ae Ri Kim; Jin Hwa Lee; Tae Hee Lee; Do Hyun Nam; Ki Sook Park
Original assignee: Lg Chemical Ltd
Priority date: 1997-03-21
Filing date: 1999-09-20
Publication date: 2003-04-25
Also published as: CA2283671C; ES2198697T3; CZ328299A3; DK1179533T3; HU227536B1; JP4781614B2; HU225412B1; NO312515B1; NO994595L; SA98181055B1; DZ2448A1; HUP0000728A2; ATE239724T1; SK284107B6; PE67999A1; IL131812A0; IL131812A; BG103750A; WO1998042705A1; BR9808290A

Description

1

SALT QF NAPHTHYRIDINE CARBOXYLÏC ACID DERIVATIVE

TECHNICAL FIELD

The présent invention relates to a sait and associated hydrates ofracemic 7-(3-aminomethyl-4-methoxyiminopyrrolidin-l-yl)-l-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-l,8-naphthyridine-3-carboxylic acid, processes fortheir préparation, pharmaceutical compositions comprising them, and theiruse in antibacterial therapy.

BACKGROUND ART EP 688772 (coiresponding to Korean Patent Laid open PublicationNo 96-874) discloses novel quinoline(naphthyridine)carboxylic aciddérivatives, including anhydrous 7-(3-aminomethyl-4-methoxyiminopyrro-lidin-1 -yl)-1 -cyclopropyl-6-fluoro-4-oxo- 1,4-dihydro- l,8-naphthyridine-3-car-boxylic acid of formula I, having antibacterial activity. o o

OH

DISCLOSURE OF INVENTION

According to the invention there is provided 7-(3-Aminomethyl- 011161 4-metlioxyiminopyrrolidin-1 -yl)-1 -cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naph- thyridine-3-carboxylic acid methanesulfonate. 7-(3-Aminomethyl-4-methoxyiminopyrrolidin-1 -yl)-1 -cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulf- 5 onate (hereinafter referred to as 'the methanesulfonate') may be obtainedas an anhydrate or a hydrate i.e. methanesulfonate.nHjO.

Hydrates of the methanesulfonate wherein n is in the range 1 to4 are preferred. Particular hydrates of the methanesulfonate which may be mentioned are those in which n is 1, 1.5, 2, 2.5, 3, 3.5 and 4.lu Particulary preferred compounds are those in which n is 1.5 or 3, with n=1.5 being most preferred.

The moisture content of the methanesulfonate hydrates varies withthe hydration number (n) of the hydrated molécule. The methane-sulfonate has a molecular weight of 485.5 thus the calculated moisture 15 content of hydrates where n is 1, 1.5, 2, 2.5, 3, 3.5 and 4 is 3.6%,5.0%, 6.9%, 8.5%, 10.0%, 11.5% and 12.9% respectively. However,the actual moisture content of the methanesulfonate hydrates may differfrom the calculated value depending on various factors includingrecrystallization conditions and drying conditions. The obseived 2e moisture content for the methanesulfonate hydrates where n is 1, 1.5, 2,2.5, 3, 3.5 and 4 is shown in Table 1: 3 0111 61

Table 1.

Hydration Number (n) Moisture Content (% w/w) 1 2—4 1.5 4-6 2 6-8 2.5 8-9 3 9-11 3.5 11 - 12 4 12 - 13 _

It is possible to mix methanesulfonate hydrates having differentmoisture contents together to give materials having intermediate moisturecontents.

Preferred methanesulfonate hydrates hâve a moisture content offrom 4 to 6% or from 9 to 11%, especially a moisture content of from4 to 6%.

The methanesulfonate has been observed to exist as a stablehydrate over a range of hydration numbers (n). Stability of thehydrate refers to its résistance to loss or gain of water moléculescontained in the compound. The methanesulfonate hydrates maintain aconstant moisture content over an extended relative humidity range.The n=3 hydrate has a constant moisture content at a relative humidityof from at least 23 to 75%, and the n=1.5 hydrate has a constantmoisture content at a relative humidity of from 23 to 64% (see Figures3 and 4). In contrast, moisture absorption by the anhydrate varies greatly with relative humidity. 4

Both the methanesulfonate anhydrate and n=3 hydrate undergotransition to the n=1.5 hydrate in aqueous suspension indicating that tlielatter is thermodynamically more stable. The n=1.5 hydrate is asesquihydrate at 11 to 64% of relative humidity. Above 75% relativehumidity, it takes up water over 10% and its X-ray diffraction patternchanges. The hydrate (another form of n=3 having different physio-chemical properties from the n=3 hydrate of Example 2) obtained fromn=1.5 hydrate at 93% relative humidity is not stable at lower relativehumidity, it converts back to n=1.5 hydrate at a relative humidity below75%.

Since the moisture content of the anhydrate changes readilydepending on the environment e.g. relative humidity, formulationadditives etc, it may require careful handling during storage orformulation, with operations such as quantifying procedures beingperformed in a dry room. The hydrates do not change in moisturecontent as easily and hence products which are stable to prolongedstorage and formulation may be obtained. The hydrate can be tabletted without the addition of a binder since the water contained inthe compound itself acts a binder, whereas it may not be possible totablet the anhydrate at a similar pressure.

The présent invention also provides a process for the préparationof 7-(3-aminomethyl-4-methoxyiminopyrrolidin-l-yl)-l-cyclopropyl-6-fluoro- 4-oxo-l,4-dihydro-l,8-naphthyridine-3-carboxylic acid methanesulfonate andhydrates thereof which comprises reacting 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1 -y 1)-1 -cy clopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid with methanesulfonic acid and crystallizingthe resulting methanesulfonate from solution, and where desired or 01 Π 61 necessary adjusting the hydration of the compound.

The methanesulfonate and its hydrates may be prepared by theaddition of methanesulfonic acid to the free base which may be preparedas described in EP 688772. Preferably, 0.95 to 1.5 molar équivalents 5 of methanesulfonic acid is added to the free base, or 1 molar équivalentof methanesulfonic acid dissolved in a suitable solvent is added to thefree base. Suitable solvents for the préparation of the methanesulfonateand its hydrates include any solvent in which the methanesulfonate issubstantially insoluble, suitable solvents include C1-C4 haloalkanes, Ci-Cs 1ui alcohols and water, or mixtures thereof. Dichloromethane, chloroform,1,2-dichloroethane, methanol, éthanol, propanol and water, or mixturesthereof, are preferred solvents. If necessary, the free base may beheated in the solvent to facilitate solution before methanesulfonic acid isadded, altematively the methanesulfonic acid may be added to a 15 suspension, or partial suspension, of the free base in the solvent.Foliowing addition of the methanesulfonic acid the reaction mixture ispreferably allowed to stand or is stirred for 1 to 24 hours at atempérature of from about -10 to 40 °C- The resulting methanesulfonateis obtained as a solid which can be isolated by filtration or by removal 2U of the solvent under reduced pressure.

Different hydrates may be obtained by altering the recrystallizationconditions used in the préparation of the methanesulfonate, suchconditions may be ascertained by conventional methods known to thoseskilled in the art.

The présent invention also provides a process for the préparationof a hydrate of 7-(3-aminomethyl-4-methoxyiminopyrrolidin-l-yl)-l-cyclopropyl-6-fluoro-4-oxo-l,4-dihydro-l,8-naphthyridine-3-carboxylic acid 6 om 61 methanesulfonate comprising exposing the methanesulfonate anhydrate ora solvaté thereof to a high relative humidity.

The methanesulfonate anhydrate or solvaté thereof is preferablyexposed to a relative humidity of at least 75%. j The methanesulfonate anhydrate or solvaté thereof may be exposed to high relative humidity by passing humidified nitrogen gasthrough the methanesulfonate anhydrate or solvaté thereof or by standingthe methanesulfonate anhydrate or solvatés thereof under a high relativehumidity. 1b The humidified nitrogen gas used in this process, for example nitrogen gas having a humidity of at least 75%, may be made byconventional methods. In this process it is désirable to maintain thetempérature in the range above which moisture condensation could occur.

Also, particularly in large scale production, it is préférable to stir the 1b sample thoroughly while the humidified nitrogen gas is passed through.When the hydrate is prepared by standing the methanesulfonate anhydrateor solvaté thereof under a high relative humidity, for example a relativehumidity of at least 75%, it is préférable to spread the sample as thinlyas possible in order to raise the conversion efficiency.

The solvatés of methanesulfonate anhydrate which may be used in the process according to this aspect of the présent invention include solvatés with one or more organic solvents. Preferred solvents include C1-C4 haloalkanes and Ci-Cs alcohols, for example those selected from the group consisting of éthanol, dichloromethane, isopropanol and 25 2-methyl-2-propanol.

Z 011161

Solvatés of the methanesulfonate anhydrate are novel. Thusaccording to a further aspect of the invention there is provided a solvatéof 7-(3-aminomethyl-4-methoxyiminopyrrolidin-l-yl)-l-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid methanesulfonate b with one or more organic solvents.

The solvatés of the methanesulfonate are prepared byrecrystallization and controlled by the condition of recrystallizing System.

The methanesulfonate and its hydrates exhibit the same potentantibacterial activity as the corresponding free base disclosed in EP688772. The methanesulfonate and its hydrates also exhibit désirablephysicochemical properties including improved solubïlity and constantmoisture content regardless of the ambient relative humidity whencompared to the free base and other salts thereof. The methanesulfonateand its hydrates thus exhibit greater ease of handling, quality control and Ί7 formulation than the free base and other salts thereof.

As mentioned above the methanesulfonate and its hydrates exhibitantibacterial activity. The methanesulfonate and its hydrates may beformulated for administration in any convenient way for use in human orveterinary medicine, according to techniques and procedures per seknown in the art with reference to other antibiotics, and the inventiontherefore includes within its scope a pharmaceutical compositioncomprising 7-(3-aminomethyl-4-methoxyiminopyrrolidin-1 -yl)-1 -cyclopropyl- 6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylie acid methanesulf-onate or a hydrate thereof together with a pharmaceutically acceptablecarrier or excipient.

Compositions comprising the methanesulfonate or hydrate thereof 8 Γ·. Λ ή χ <1 u i ι ι 6 1 as active ingrédient may be formulated for administration by any suitable route, such as oral, parentéral or topical application. The compositions may be in the form of tablets, capsules, powders, granules, lozenges, creams or liquid préparations, such as oral or stérile parentéral solutions 5 or suspensions. Tablets and capsules for oral administration may be inunit dose présentation form and may contain conventional excipients suchas binding agents, for example, hydroxypropyl methyl cellulose, hydroxypropyl celullose, syrup acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrollidone; fillers, for example microcrystalline cellulose, 10 lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine;tabletting lubricants, for example magnésium stéarate, talc, polyethyleneglycol or silica; disintegrants, for example sodium starch glycolate, crosslinked polyvinyl pyrollidone or potato starch; or acceptable wetting agentssuch as sodium lauryl sulfate. The tablets may be coated according to 15 methods well known in normal pharmaceutical practice. Oral liquidpréparations may be in the form of, for example, aqueous or oilysuspensions, solutions, émulsions, syrups or élixirs, or may be presentedas a dry product for reconstitution with water or other suitable vehiclebefore use. Such liquid préparations may contain conventional additives 25 such as suspending agents, for example sorbitol, methyl cellulose, glucosesyrup, gelatin, hydroxyethyl cellulose, caboxymethyl cellulose, aluminiumstéarate gel or hydrogenated edible fats; emulsifying agents, for examplelecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which mayinclude edible oils), for example almond oil, oily esters, glycérine,propylene glycol, or ethyl alcohol; preservatives, for example methyl orpropyl /?-hydroxybenzoate or sorbic acid; and, if desired conventionalflavouring or coloring agents. Suppositories will contain conventionalsuppository base, e.g. cocoa-butter or other glyceride.

For parentéral administration, fluid unit dosage forms are prepared or,161 utilising the compound and a stérile vehicle, water being preferred.The methanesulfonate or hydrate thereof, can be either suspended ordissolved in the vehicle, depending on the vehicle and concentrationused. In preparing solutions the methanesulfonate or hydrate thereofcan be dissolved in water for injection and filter sterilized before fillinninto a suitable vial or ampoule and seahng. Advantageously, agents such as local anaesthetic, preservative and buffering agents can bedissolved in the vehicle. To enhance the stability, the compositioncan be lyophilised and the dry lyophilised powder sealed in a vial, anaccompanying vial of water for injection may be supplied to reconstitutethe powder prior to use. Parentéral suspensions are prepared insubstantially the same manner except that the methanesulfonate or hydratethereof is suspended in the vehicle instead of being dissolved andsterilization cannot be accomplished by filtration. The methansulfonateor hydrate thereof can be sterilized by exposure to ethylene oxide beforesuspending in the stérile vehicle. Advantageously, a surfactant orwetting agent is included in the composition to facilitate uniformdistribution of the methanesulfonate or hydrate thereof.

The methanesulfonate or hydrate thereof may also be formulatedas an intramammary composition for veterinary use.

The composition may contain from 0.1% to 100% by weight,preferably from 10 to 99.5% by weight, more preferably from 50 to99.5% by weight of the active ingrédient measured as the free base,depending on the method of administration. Where the compositionscomprise dosage units, each unit will preferably contain from 50-1500rng of the active ingrédient measured as the free base. The dosage asemployed for adult human treatment will preferably range from 100 mgto 12 g per day for an average adult patient (body weight 70 kg), for 10 011161 instance 1500 mg per day, depending on the route and frequency ofadministration. Such dosages correspond to approximately 1.5 to 170mg/kg per day. Suitably the dosage is from 1 to 6 g per day.

The daily dosage is suitably given by administering the activeingrédient once or several rimes in a 24-hour period. e.g. up to 400 mgmaybe adminstered once a day, in practice, the dosage and frequency ofadministration which will be most suitable for an individual parient willvary with the âge, weight and response of the patients, and there will beoccasions when the physician will choose a higher or lower dosage anda different frequency of administration. Such dosage regimens are withinthe scope of this invention.

The présent invention also includes a method of treating bacterialinfections in hum ans and animais which method comprises administeringa therapeutically effective amount of 7-(3-aminomethyl-4-methoxyimino-pyrrolidin-l-yl)-l-cyclopropyl-6-fluoro-4-oxo-l,4-dihydro-l,S-naphthyridine-3-carboxylic acid methanesulfonate or a hydrate thereof.

In a further aspect, the présent invention also provides the use of 7-(3-aminomethyl-4-methoxyiminopy rrolidin-1 -yl)-1 -cyclopropyl-6-fluoro-4-oxo-l,4-dihydro-l,8-naphthyridine-3-carboxylic acid methanesulfonate or ahydrate thereof for the manufacture of a médicament for treating bacterialinfection.

The methanesulfonate and its hydrates are active against a broadrange of Gram-positive and Gram-negarive bacteria, and may be used totreat a wide range of bacterial infections including those inimmunocompromised patients. 11 011161

Amongst many other uses, the methanesulfonate and its hydratesare of value in the treatment of skin, soft tissue, respiratory tract andurinary tract infections, and sexually transmitted diseases in humans.The methanesulfonate and its hydrates may also be used in tire treatmentof bacterial infections in animais, such as mastitis in cattle.

BRIEF DESCRIPTION OF DRAWINGS

The following examples and figures illustrate the invention but arenot intended to limit the scope in any way.

Figure 1 shows the moisture sorption profile of methanesulfonateanhydrate of Example 1 at 25 °C at several relative humidifies.

Figure 2 shows the isothermal moisture sorption profile ofmethanesulfonate anhydrate of Example 1 at 25 °C.

Figure 3 shows the equilibrium moisture content of themethanesulfonate n=3 hydrate of Example 2 at a relative humidity of 23to 75%.

Figure 4 shows the equilibrium moisture content of themethanesulfonate n=1.5 hydrate of Example 3 at a relative humidity of23 to 75%.

Figure 5 shows the powder X-ray diffraction pattern of themethanesulfonate anhydrate of Example 1.

Figure 6 shows the powder X-ray diffraction pattern of the 12 011161 methanesulfonate n=3 hydrate of Example 2. The characteristic peaksare 2 <9 = 7.7, 11.8 ° . The exact position of peaks can vary slightlydepending on the experimental conditions.

Figure 7 shows the powder X-ray diffraction pattern of themethanesulfonate n=1.5 hydrate of Example 3. The characteristic peaksare 2 0 = 8.0, 12.2, 14.7 " . The exact position of peaks can varyslightly depending on the experimental conditions.

Figure 8 shows the variation in moisture content with elapsedtime of the methanesulfonate anhydrate of Example 1, taken after 0, 5,10, 20, 30, and 60 minutes, respectively, from the initial point ofpassing humidifïed nitrogen gas through;

Figure 9 shows the Differential Scanning Calorimetry on themethanesulfonate anhydrate of Example 1 and the methanesulfonate n-3hydrate of Example 2.

Figure 10 shows the results of thermogravimetric analysis on themethanesulfonate n=3 hydrate of Example 2.

Figure 11 shows the change in X-ray diffraction pattern withelapsed time of the methanesulfonate solvaté (éthanol content 0.11%) ofExample 4, from initial point of passing the humidifïed nitrogen gashaving a relative humidity of 93% through.

Figure 12 shows , the change in X-ray diffraction pattern withelapsed time of the methanesulfonate solvaté (éthanol content 1.9%) ofExample 5, from the initial point of standing the sample under a relativehumidity of 93%. 13 011161

Figure 13 shows the change in X-ray diffraction pattern of themethanesulfonate solvaté (éthanol content 0.12%) of Example 5 undervarious relative humidities, that îs, relative humidity of 93% (1), relativehumidity of 52% (2) and relative humidity of 11% (3), respectively.

BEST MODE FOR CARRYING OUT THE INVENTION 5 The présent inventors hâve performed several experiments in order to identify the moisture content and physicochemical property of themethanesulfonate anhydrate and each hydrate, and the results aredescribed in connection with the drawings in the following.

Figure 1 shows the moisture sorption velocity profile of 7-(3-% aminomethyl-4-methoxyiminopyrrolidin-1 -yl)-1 -cyclopropy l-6-fluoro-4-oxo-1, 4-dihydro-l,8-naphthyridine-3-carboxylic acid methanesulfonate anhydrateat several relative humidities. Over the whole range of relativehumidity tested, the initial moisture adsorption proceeds rapidly at eachrelative humidity. In most cases equilibrium is achieved within 2 13 hours. Figure 2 shows the isothermal moisture sorption profile of themethanesulfonate anhydrate according to the change in relative humidityat 25 ’C. The weight incrément (%) of Y-axis represents the equilibriummoisture content, from which it can be recognized that the equilibriummoisture content dépends on the relative humidity. Figure 3 showsthe equilibrium moisture content of the n=3 hydrate (which is obtainedby recrystallization from a solvent mixture of éthanol and water) after itis allowed to stand for 2 weeks under relative humidities in the range of23 to 75%. The resuit shows that the n=3 hydrate is more stable thanthe anhydrate since it maintains a moisture content of around 10% under 14 011161 the relative humidities tested. Figure 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. S It has been identified that the physical properties of the hydrate are very different from those of the anhydrate.

For example, by comparing the powder X-ray diffraction patternsof the anhydrate in Figure 5, the n=3 hydrate in Figure 6, and the n=1.5hydrate in Figure 7, it can be seen that their crystal forms aie different 1 from each other. In addition, the thermal analysis using Differential

Scanning Calorimetry (DSC) shows that the endothermie peak producedby the vaporization of the water molécules contained in the n=3 hydratebegins at around 50°C and the exothermic peak by thermal décomposition is observed at around 185 to 220 °C, whereas the 4anhydrate shows only an exothermic peak at around 185 to 220 °C due tothe thermal décomposition without any endothermie peak (see, Figure 9).At the same time, the thermogravimetric analysis shows a weightdécrément at the température range of endothermie peak, the extent ofwhich corresponds to the moisture content quantified by Karl-Fishermethod (Mettler Toledo DL37KF Coulometer)(see, Figure 10).Therefore, it is verified that the endothermie peak shown in the DSCanalysis is due to the évaporation of a water molécule.

The présent inventors also compared the Chemical stability underheating of the hydrates with that of the anhydrate in order to identifythe influence of hydration on the Chemical stability. In this test, theanhydrate and hydrate were each kept at 70 °C for 4 weeks. and theextent of décomposition is analyzed by liquid chromatography. No 15 011161 différence in the extent of décomposition was noticed between thehydrates and the anhydrate, and thus confirming that the hydrate has thesame Chemical stability as the anhydrate.

The methanesulfonate anhydrate or a solvaté thereof may beconverted into a hydrate under appropriate conditions as described above.This process can be monitored by the change m the X-ray diffractionpattern of the compound and the decrease in the amount of organicsolvent in the compound. Such changes being caused by the water molécules newly intercalated into the crystal structure.

As can be seen from Figure 11, the X-ray diffraction peaks based'on the solvaté disappear with the passing of humidified nitrogen gas toleave the peaks based on the hydrate. This shows that ail the solvatésis converted into hydrates. The residual solvent is decreased to theamount of less than the quantitative limit simultaneously with the changeof X-ray diffraction. Figure 12 shows that the X-ray diffraction peaksbased on the solvaté disappear when the solvaté is allowed to standunder a relative humidity of 93%. However, there is no change inthe X-ray diffraction pattern when the solvaté is allowed to stand undera relative humidity of 11% or 52% (see Figure 13). Therefore, it is recognized that the change shown in Figure 12 occurs not by thespontaneous évaporation of the residual solvent but by the substitution ofthe organic solvents in the crystal by water molécules.

In preparing tlie hydrate according to the processes describedabove. the respective hydrates havrng a different hydration number canbe obtained by changing conditions such as humidity, time, température,etc. or by changing the recrystallization condition. Such conditions should be adjusted according to whether tire starting material is the 16 011161 anhydrate or a solvaté, and depending on the nature of the solvaté.

The présent invention will be more specifîcally explained by thefollowing examples and experimental examples. However, it should beunderstood that the examples are mtended to illustrate but not in anymanner limit the scope of the présent invention.

Example 1: Synthesis of 7-(3-aminomethyl-4-methoxyiminopyrrol-idin-1 -yl)-1 -cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbo xylic acid methanesulfonate anhydrate 7-(3 -Aminomethy 1-4-methyloxyiminopyrrolidin-1 -y 1)-1 -cyclopropy 1-6-t fluoro-4-oxo-l,4-dihydro-l,8-naphthyridme-3-carboxylic acid (3.89g, 10 mmol) was suspended in a mixture of dichloromethane and éthanol (110mf, 8:2 v/v). Methanesulfonic acid (0.94g, 9.8mmol) was addeddropwise and the resulting solution was stirred for 1 hour at 0°C.The resulting solid was filtered, washed with éthanol then dried to give 1_ the title compound (4.55g). m.p. : 195 °C (dec.) 'H NMR(DMSO-dô) ô (ppm) 8.57(lH,s), 8.02(lH,d), 7.98(3H,br), 4.58(2H,br), 4.39(lH,m), 3.91(3H,s), 3.85(lH,m), 3.71(lH,m),3.42(lH,m), 3.20~3.10(2H,m), 1.20 ~~ 1.10(4H,m)

Example 2: Synthesis of 7-(3-aminomethyl-4-methoxyiminopyrrol- idin-1 -yl)-1 -cy clopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carbo xylic acid methanesulfonate n=3 hydrate A sonicator filled with water was adjusted to 40 °C, sealed with alid and a nitrogen inlet and outlet connected. When the pressure of 17 011161 the dried nitrogen introduced through the inlet was 20psi the relativehumidity of the nitrogen exiting through the outlet was more than 93%.The anhydrate of Example 1 having a moisture content of 2.5% (1.0g)was introduced into a fritted filter and the humidified nitrogen producedas described above passed through the filter. Samples were taken after0, 5, 10, 20, 30, and 60 minutes and the moisture content measured.From the results shown in Figure 8 it can be seen that a moisturecontent of about 10% is maintained when the humidifying procedure iscarried out over about 30 minutes. The X-ray diffraction pattern of the 11, humidified sample was identical to that of the n=3 hydrate obtained byrecrystallization.

Example 3: Synthesis of 7-(3-aminomethyl-4-methoxyiminopyrrol- idin-l-yl)-l-cyclopropyl-6-fluoro-4-oxo-l,4-dihydro-l,8-naphthyridine-3-carbo xylic acid methanesulfonate n=1.5 hydrate % The title compound was prepared by the following routes:

Route A

The anhydrate of Example 1 (1.0g) was dissolved in a mixtureof water and acetone (17m£, 10:7 v/v). The solvent was slowlyevaporated in darkness leaving the title compound as a solid (0.8g).

Route B

The anhydrate of Example 1 (5.0g) was added to water (lOmf)and the mixture was heated to 45 °C to aid dissolution. Ethanol (20mf)was added and the resulting solution stirred then allowed to stand.The resulting solid was filtered and dried under a flow of nitrogen to 18 011161 give the title compound (2.6g).

Example 4: Synthesis of the hydrate from 7-(3-aminomethyl-4-m ethoxyiminopyrrolidin-1 -yl)-1 -cyclopropvl-6-fluoro-4-oxo-1,4-dihydro- 1,8-naphthyridine-3-carboxylic acid methanesulfonate solvaté using a humid-ified nitrogen gas A sonicator filled with water was adjusted to 40 °C and wassealed with a lid. Then, a nitrogen inlet and outlet were connected tothe vessel. When the pressure of the dried nitrogen introduced throughthe nitrogen inlet was adjusted to about 20psi, the relative humidity ofthe humidified nitrogen gas exiting through the outlet was more than93%. The solvaté (lg, éthanol 0.11%) of the anhydrate of Example 1was introduced into a fritted filter and the humidified nitrogen gasprepared as described above was passed through the filter. Sampleswere taken after 40 minutes, 3.5 and 6 hours, respectively. The changein the amount of residual organic solvent and X-ray diffraction patternwith the lapse of time were examined. After 3.5 hours, it wasidentified that the product contained the organic solvent in an amount ofless than 50ppm and that the peaks based on the solvaté disappeared,whilst the peaks based on the mixture of n=3 hydrate and n=1.5 hydrateappeared.

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

Saturated aqueous potassium nitrate solution was placed in adesiccator, and accordingly the relative humidity inside the desiccator was 19 011161 controlled to 93%. For tests under relative humidity of 11% or 52%, desiccators containing saturated aqueous solutions of lithium chloride and magnésium nitrate, respectively, were prepared. Into tire desiccator having a relative humidity of 93% was introduced a solvate(1.9% 5 éthanol) of the anhydrate of Example 1, and into each of the desiccatorshaving a relative humidity of 93%, 52% or 11% was introduced asolvaté (0.12% éthanol) of the anhydrate of Example 1. The solvatéswere stored so as not to directly contact the aforementioned saitsolutions. After a certain period of time has passed, samples were 1 taken and subjected to gas chromatography in order to analyze theresidual solvent. As a resuit, it was identified that solvatés stored for4 weeks under a relative humidity of 93% contained the organic solventin an amount of less than 50ppm. Also, it was identified by X-raydiffraction pattern that peaks based on the solvatés disappeared after 4weeks. To the contrary, in the case where the samples were storedunder a relative humidity of 52% or 11%, the amount of residualorganic solvent and X-ray diffraction pattern after 4 weeks were identicalwith those at the beginning.

Example 6: Synthesis of n=3 hydrates from 7-(3-amino-methyl- 2 : 4-methoxyiminopyrrolidm-1 -yl)-1 -cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8 -naphthyridine-3-carboxylic acid methanesulfonate solvatés

Dried nitrogen gas and humidified nitrogen gas having a relativehumidity of 78 to 84% were passed over 24 hours, respectively, through10g of four different solvatés each of which had a different kind andamount of organic solvent from the others. The amount of residualorganic solvent was measured and the change in X-ray diffraction patternwas analvzed, the results of which are shown in Table 2. The X-raydiffraction analysis shows that the samples through which dried nitrogen 20 01 11 61 gas was passed remained as the original solvatés, while the samplesthrough which humidified nitrogen gas was passed had the same X-raydiffraction pattern and crystallinity as those of the n=3 hydrate obtainedby recrystallization.

The results from this Example suggests that water moléculescontained in the humidified nitrogen gas replace the organic solvents inthe solvaté. This suggestion is also supported by the change in X-raydiffraction pattern influenced by a relative humidity.

Table 2.

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

Example 7; Synthesis of the ethanolate containing éthanol 0.11%

The anhydrate of Example 1 (5.0g) was added to a solventmixture of éthanol (25ml) and water (25ml) and the mixture was heatedto 50°C to facilitate dissolution. Then, the solution was cooled slowlyto -3°C and allowed to stand at that température for about 3 hours. The 21 01 1161 resulting solid was filtered and washed with a solvent mixture of éthanoland water (16.5ml, éthanol : water = 20 : 8, v/v) to give the titlecompound quantitatively.

Test Example 1: Moisture sorption of the anhydrate of Example 1

The moisture sorption velocity and the equilibrium moisturecontent of the anhydrate of Example 1 was determined by means of anautomatic moisture sorption analyzer (MB 300G Gravimétrie SorptionAnalyzer). This instrument produces a spécifie relative humidity at aspécifie température and continuously records the weight change of asample due to adsorption or desorption of moisture as measured by amicro balance inside the instrument. The anhydrate of Example 1 (16mg) was loaded onto the micro balance and the moisture contained in thesample removed under a stream of dried nitrogen at 50°C. A weightchange of less than 5/zg per 5 minutes was the criterion for complétédryness. Thereafter, the inner température was adjusted to 25 °C andthe sample tested at 5% intervals whilst varying the humidity from 0 to95%. The sample was considered to hâve reached equilibrium whenthe weight change was less than 5/zg per 5 minutes. Figure 1 showsthe moisture adsorption velocity, that is the time required for the sampleto reach equilibrium at each relative humidity. As can be seen initialmoisture adsorption proceeded rapidly at each relative humidity tested, inmost cases equilibrium was reached within 2 hours. Figure 2 showsthe weight incrément at each relative humidity, i.e. the equilibriummoisture content. It is clear from Figure 2 that the equilibriummoisture content of the anhydrate is dépendent on the relative humidity.

Test Example 2: Thermal analysis of the anhydrate of Example 1 and n-3 hydrate of Example 2 22 011161

For the Differential Scanning Calorimetry, METTLER TOLEDODSC821e and METTLER TOLEDO STARe System were used. Thesample (3.7mg) was weighed into the aluminum pan which was thenpress sealed with an aluminum lid. Three tiny needle holes were madeon the lid and the sample tested by heating from normal température to250°C at a rate of 10°C/min. As can be seen from Figure 9, theendothermie peak due to the vaporization of the water moléculescontained in the n=3 hydrate begins at around 50 °C and the exothermicpeak due to the thermal décomposition is observed at around 180 to 220°C. In contrast, the anhydrate showed only an exothermic peak due tothe thermal décomposition at around 185 to 220 °C without anyendothermie peak.

In the thermogravimetric analysis, SEIKO TG/DTA220 was used.The sample (3.8mg) was weighed into an aluminum pan and was heatedfrom normal température to 250°C at a rate of 10°C/min according tothe température raising program. As can be seen from Figure 10,weight décrément was observed at the température range of endothermiepeak, the extent of which corresponds to the moisture content determinedby Karl-Fisher method (Mettler Toledo DL37KF Coulometer).

Test Example 3: Equilibrium moisture content détermination of hydrates

Six saturated aqueous sait solutions were introduced into eachdesiccator to control the inner relative humidity to a spécifie value asshown in Table 3. Then, equilibrium moisture contents of n=3 hydrate and n=1.5 hydrate of Examples 2 and 3, respectively, weredetermined at several relative humidities. 23 011161

Table 3. Saturated sait solutions inside the desiccator

Sait Solution Relative Humidity (%) at 25 “C Potassium Acetate --- 23

Magnésium Chloride JJ Potassium Carbonate 43 Magnésium Nitrate 52 Sodium Nitrite 64 Sodium Chloride 75

The sample (lOOmg) was spread on a pre-weighed Pétri dish andthe total weight was accurately measured, then three of the sample wereplaced in each desiccator of Table 3. The desiccators were allowed tostand at normal température for 7 days and then the sample was takento be weighed. After 13 days, one of the three samples inside eachdesiccator was taken and the moisture content of each was measured bythe thermogravimetric analysis described in Test Example 2. Equilibriummoisture content at each relative humidity is represented in Figure 3(n=3 hydrate) and Figure 4 (n=1.5 hydrate). Figure 3 shows thatmoisture content of the n=3 hydrate is maintained around 10% for thewhole relative humidity range tested; Figure 4 shows that the moisturecontent of the n=1.5 hydrate is maintained around 5% at the relativehumidity of 23 to 64%. Both hydrates are stable since they maintaina constant equilibrium moisture content regardless of the relativehumidity change. 24 011161

Test Example 4: X-ray diffraction analysis

The anhydrate of Example 1, n=3 hydrate of Example 2 andn=1.5 hydrate of Example 3 (50mg of each) were thinly spread on thesample holder, X-ray diffraction analysis (35kV x 20mA RigakuGergeflex D/max-III C) were performed under the conditions listedbelow. - scan speed (2 Q ) 57min - sampling time : 0.03 sec - scan mode : continuons - 2 θ / Θ reflection

If. - Cu-target (Ni filter)

Results of X-ray diffraction analyses on the anhydrate, n=3hydrate, and the n=1.5 hydrate are shown in Figure 5, 6, and 7. Thediffraction patterns illustrate the différence in crystal form of these 3compounds. 1-. According to a further aspect of the invention we provide 7-(3-ammomethyl-4-methoxyiminopynOlidin-1 -yl)-1 -cyclopropyl-6-fluoro-4-oxo-l,4-dihydro-l,8-naphthyridine-3-carboxylic acid methanesulfonate havingan X-ray diffraction pattern substantially as shown in Figure 5, 6 or 7.

We also provide 7-(3-aminomethyl-4-methoxyiminopyrrolidin- l-yl)-l-cyclopropyl-6-fluoro-4-oxo-l,4-dihidro-l,8-naphthyridine-3-carboxyIicacid 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 -cyclopropy l-6-fluoro-4-oxo-1,4-dihidro-1,8-naphthyridine-3-carboxylic acid methanesulfonate hydrate having peaks at 2 25 011161 θ = 7.7 and 11.8° in its X-ray diffraction pattern.

The change of crystallinity during the conversion from the solvatéto the hydrate in Examples 4 and 5 was identified by X-ray diffractionanalysis under the same conditions as mentioned above (see, Figure 11to 13). Figure 11 shows tire X-ray diffraction pattern of the solvaté ischanged into that of the n=3 hydrate (see, Example 4); Figure 12represents the change in X-ray diffraction pattern of the solvatécontaining 1.9% of éthanol before and after storage of one week, twoweeks, three weeks and four weeks at 93% of relative humidity; andFigure 13 represents the change in X-ray diffraction pattern of thesolvaté containing 0.12% of éthanol after storage of four weeks at 93%,52% and 11% of relative humidity, respectively (see, Example 5).

Test Example 5: Chemical stability

The Chemical stability of the n=3 hydrate of Example 2 and then=1.5 hydrate of Example 3 and the anhydrate of Example 1 werecompared at elevated température in order to détermine the effect onChemical stability of the extent of hydration.

The anhydrate and each of the hydrates were introduced into aglass vial and maintained at 70 °C. The extent of décomposition withelapsed time was analyzed by liquid chromatography. The resultsobtained are shown in Table 4. 26 011161

Table 4. Thermal stability with elapsed time (at 70°C, Unit: %)

Time(week) S am p l Initia] 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

As can be seen from Table 4, the n=3 hydrate and the n=1.5hydrate both show the same degree of Chemical stability as theanhydrate.

Test Example 6: In vitro antibacterial activity

In order to détermine whether 7-(3-aminomethyl-4-methyloxyimino-pyrrolidin-l-yl)-l-cyclopropyI-6-fluoro-4-oxo-l,4-dihydro-l,8-naphthyridine-3-carboxylic acid methanesulfonate has the same antibacterial activity asthe free base, in vitro antibacterial activity of the methanesulfonate wasmeasured using agar medium dilution method. The results are shown 16 in Tables 5. The minimum inhibitory concentration (MIC, ^g/m£) was simply calculated in the ratio of weight without considering themolecular weight, and ciprofloxacin was chosen as the control. 27 011161

Table 5. In vitro Antibacterial activity (Minimum Inhibitory Concentration: MIC, /zg/mi)

Test Strains Methanesulfonic acid sait 1 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 887E 0.016 0.13 Staphylococcus aureus epidermidis 178 4 128 Staphylococcus aureus faecalis 29212 0.13 0.5 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 ΊΈΜ3 3455E 0.25 0.5 Escherichia coli ΊΈΜ5 3739E 0.13 0.13 Escherichia coli TEM9 2639E 0.031 0.016 i 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 aerosenes 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 1 0.031 J.. 0,031 28 011161

Test Example 7: Water solubility of the anhydrate of Example 1

The water solubility of the free base and various salts of7-(3-aminomethyl-4-methoxyiminopyrrolidin-l-yl)-l-Cyclopropyl-6-fluoro-4-oxo-l,4-dihydro-l,8-naphthyridine-3-carboxylic acid, including the methane-sulfonate of Example 1, was measured at 25 °C. The results areshown in Table 6.

Table 6. Water Solubility (at 25 °C)

Sample Solubility in water(mg/ml) Free form 0.007 Tartrate 6.7 Sulfurate 11.4 p-Toluenesulfonate 7.5 Methanesulfonate >30

As can be seen, the methanesulfonate shows increased watersolubility compared to that of the tartrate, the sulfurate, and thep-toluenesulfonate and the free base.

Claims

29 011161 WHAT IS CLAIMED IS : 1. 7-(3-Aminomethyl-4-methoxyimmopyrrolidin- 1-yl)-1 -cyclopropvl- 6-fluoro-4-oxo-l,4-dihydro-l,8-naphthyridine-3-carboxylic acid methanesulf-onate.
2. 7-(3-Aminomethyl-4-methoxyiminopyrrolidin-l-yl)-l-cyclopropyl-6- -, fluoro-4-oxo-l,4-dihydro-l,8-naphthyridine-3-carboxylic acid methanesulfo-nate.nHzO, 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 2 Q = 8.0, 12.2and 14.7° in its X-ray diffraction pattern. ηj
5. A compound according to claim 2 having an X-ray diffraction pattern substantially as shown in Figure 7.
6. A compound according to claim 2 wherein n is 3.
7. A compound according to claim 2 having peaks at 2 Θ = 7.7 and11.8° in its X-ray diffraction pattern.
, 8. A compound according to claim 2 having an X-ray diffraction pattern substantially as shown in Figure 6.
9. A compound according to claim 2 which has a moisture contentof from 4 to 6%.
10. A compound according to claim 2 which has a moisture content 30 011161 of from 9 to 11%.
11. A pharmaceutical composition comprising a compound accordingto any one of the preceding daims, together with a pharmaceuticallyacceptable carrier or excipient.
12. A compound according to any one of daims 1 to 10, for use asa pharmaceutical.
13. A method of treating bacterial infections in humans and animaiswhich comprises administering a therapeutically effective amount of acompound according to any one of daims 1 to 10.
14. The use of a compound according to any one of daims 1 to 10 1. for the manufacture of a médicament for treating bacterial infection.
15. A process for the préparation of a compound according to anyone of daims 1 to 10, which comprises reacting 7-(3-aminomethyI- 4-methoxyiminopyrrolidin-1 -yl)-1 -cy clopropyl-6-fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid with methanesulfonic acid and 15 crystallizing the resulting compound from solution, and where desired or necessary, adjusting the hydration of the compound.
16. A process for the préparation of a compound according to anyone of daims 2 to 10, comprising exposing 7-(3-aminomethyl-4-m ethoxy iminopyrrolidin-1 -yl)-1 -cyclopropy l-6-fluoro-4-oxo-1,4-dihydro-1,8- L naphthyridine-3-carboxylic acid methanesulfonate anhydrate or a solvaté thereof to a relative humidity of at least 75%.
17. A process according to daim 16, wherein the solvaté is a solvaté 31 01 1 161 with one or more organic solvents selected from Ci-C4 haloalkanes andCi-C8 alcohols.
18. A solvaté of 7-(3-aminomethyl-4-methoxyiminopyrrolidin-l-yl)-l-cyclopropyl-6-fluoro-4-oxo-l,4-dihydro-l,8-naphthyridine-3-carboxylic acidmethanesulfonate with one or more organic solvents.