February 2002
`
`Chem. Pharm. Bull. 50(2) 229—234 (2002)
`
`229
`
`Photochemical Behavior of Sitafloxacin, Fluoroquinolone Antibiotic, in an
`Aqueous Solution
`
`Tetsuya ARAKI,*,a Yukinori KAWAI,b Ikumi OHTA,a and Hiroaki KITAOKAb
`Chemical Technology Research Laboratories,a Drug Metabolism and Physicochemical Properties Research Laboratory,b
`Daiichi Pharmaceutical Co., Ltd., 16–13 Kita-Kasai 1-chome, Edogawa-ku, Tokyo 134–8630, Japan.
`Received September 11, 2001; accepted October 26, 2001
`
`Sitafloxacin (STFX) hydrate, an antimicrobial agent, is photo-labile in aqueous solutions. The photodegra-
`dation rates (k) in neutral solutions were higher than those observed in acidic and alkaline solutions and maxi-
`mum at the maximum absorption wavelength of STFX. The structures of photodegradation products were eluci-
`dated as 7-[7-amino-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid and 1-(1-
`amino-2-{[6-fluoro-1-(2-fluoro-1-cyclopropyl)-1,4-dihydro-4-oxo-3-quinolin-7-yl]-amino}ethyl)cyclopropanecar-
`baldehyde. This implies that dechlorination is the key step in the photodegradation of STFX. The effect of halide
`ions on the photodegradation of STFX was estimated by observing the increments in the photostability of STFX
`with the addition of chloride ions. In contrast, in the presence of bromide ions, instead of increased photostabil-
`ity of the STFX rate, a new photodegradation product in the presence of bromide ion was observed. The struc-
`ture of this new photodegradation product was an 8-bromo form of STFX, which was substituted for chlorine at
`the 8-position, so the dissociation of C–Cl bond at the 8-position of STFX was the rate-limiting step in the initial
`process of the photodegradation. STFX generated · C (carbon centered radical) and · OH (hydroxyl radical) in
`the process of photodegradation in a pH 4.0 buffer. On the contrary, STFX did not generate · C in the presence of
`chloride ion in a pH 4.0 buffer. The · C was generated and then degraded into the above degradation products by
`photoirradiation in the absence of chloride ion, but the · C immediately reacted with chloride when it was pre-
`sent. As a result, the C–Cl bond was recovered leading to a possible increase in the apparent photostability.
`
`Key words
`
`antimicrobial; fluoroquinolone; sitafloxacin; photodegradation
`
`Sitafloxacin hydrate (STFX hydrate: (2)-7-[(7S)-7-amino-
`5-azaspiro[2.4]heptan-5-yl]-8-chloro-6-fluoro-1-[(1R,2S)-2-
`fluoro-1-cyclopropyl]-1,4-dihydro-4-oxo-3-quinolinecar-
`boxylic acid sesquihydrate) is a fluoroquinolone and its
`chemical structure is shown in Fig. 1.1) Fluoroquinolones are
`important synthetic antimicrobial agents and they have been
`widely used as therapeutic agents for general bacterial infec-
`tious diseases. However, some fluoroquinolones are known to
`induce phototoxicity as a side effect.2) A large number of
`studies have been reported to elucidate the mechanism of
`phototoxicity of fluoroquinolones. We reported that carbon
`centered radical (· C), hydroxyl radical (· OH), and singlet
`oxygen (1O2) are generated during photodegradation of flu-
`oroquinolones in neutral aqueous solutions.3) The fluoro-
`quinolones substituted at the 8-position by fluorine generated
`a high degree of 1O2 and · OH against photoirradiation. Such
`reactive species were related to DNA damage.4) Some fluoro-
`quinolones are photo-labile compounds and the phototoxicity
`might arise from photodegradation products. This study was
`done based on such a background. This report describes the
`photochemical behavior of STFX in aqueous solution includ-
`ing the identification of major photodegradation products of
`STFX, the mechanistic consideration of photodegradation,
`and the effect of pH. Furthermore, the possibility improving
`the photostability of STFX was examined in the presence of
`chloride ion.
`
`Experimental
`Materials STFX was synthesized at Daiichi Pharmaceutical Co., Ltd.,
`Tokyo, Japan.
`Structural Elucidation of Photodegradation Products Ninety-four
`milligrams of STFX hydrate was dissolved in 1 l of purified water and irradi-
`ated with fluorescent lamps (Biophotochamber LX-2100 (TAITEC Co.,
`
`Tokyo)) at approximately 10000 lux for ca. 350000 lux · h. After irradiation,
`the solution was loaded onto a preparative HPLC system and fractionated.
`Preparative HPLC was carried out on a gradient system consisting of an
`LC 10AD pump (Shimadzu Co., Kyoto) equipped with a SPD-6A UV/vis
`detector (Shimadzu Co.). HPLC conditions were as follows: column,
`TSKgel ODS-80TM (21.5 mm i.d.3300 mm, Tosoh Co., Tokyo); eluent,
`0.05 M phosphate buffer (pH 2.4) : acetonitrile582 : 18 (from 0 to 65 min) to
`50 : 50 (from 65 to 100 min); flow rate, 5.0 ml/min (from 0 to 65 min) to
`7.0 ml/min (from 65 to 100 min); injection, 2 ml; detection, 278 nm; column
`temperature, 40 °C.
`The collected fractions were concentrated and lyophilized. Then the sam-
`ple was redissolved in water and loaded onto a desalting preparative HPLC.
`HPLC conditions were as follows: column, TSKgel ODS-80TM (21.5 mm
`i.d.3300 mm, Tosoh Co.); eluent, 0.1% acetic acid and acetonitrile530 : 70;
`flow rate, 7.0 ml/min; injection, 2 ml; detection, 278 nm; column tempera-
`ture, 40 °C. Isolated photodegradation products were elucidated by using a
`1H-NMR (JNM-GSX500 FT-NMR spectrometer (500 MHz), JEOL Co.,
`Tokyo) and electron impact (EI), chemical ionization (CI), fast atom bom-
`bardment (FAB)-MS (JMS-HX110 mass spectrometer, JEOL Co.) spectra.
`Kinetics of Photodegradation STFX was dissolved in Britton–Robin-
`son buffer solutions of pH 2.0—11.0 and 0.1 mol/l sodium hydroxide (pH
`13.0) (STFX, about 50mg/ml). Each sample solution (2 ml) was poured into
`a glass container (internal diameter: ca. 20 mm), which was covered with
`clear polyvinylidene chloride film. Then, an exposure test was performed at
`25 °C by irradiation with a D65 fluorescent lamp; this is an artificial daylight
`fluorescent lamp that emits visible and ultraviolet lights. The illumination of
`the D65 fluorescent lamp was adjusted to 4000 lux; at this illumination the
`near ultraviolet energy was about 120mW/cm2. At appropriate time inter-
`vals, the sample solution was withdrawn, mixed with the HPLC mobile
`phase, and the resulting solution (ca. 5mg/ml) was assayed by HPLC.
`
`Fig. 1. Chemical Structure of STFX Hydrate
`
`* To whom correspondence should be addressed.
`
`e-mail: araki45s@daiichipharm.co.jp
`
`© 2002 Pharmaceutical Society of Japan
`
`MYLAN Ex. 1007, Page 1
`
`

`
`230
`
`Vol. 50, No. 2
`
`Wavelength Dependency of Photodegradation Three milliliters of
`aqueous solution of STFX (50 mM, pH 7.4 phosphate buffer) in a quartz
`glass cell was irradiated for 0—10 J/cm2 at various wavelengths (699, 649,
`599, 499, 446, 419, 392, 365, 338, 311, 284, 257, 230 nm) by a grating
`monochrometer apparatus, CRM-FA equipped with a Xe lamp (Japan Spec-
`troscopic Co., Tokyo).
`HPLC conditions were as follows: column, TSKgel ODS-80TM (4.6 mm
`i.d.3150 mm, Tosoh Co.); eluent, 0.05 M phosphate buffer (pH 2.4) : acetoni-
`trile580 : 20 (from 0 to 10 min), 36 : 64 (from 10 to 20 min), 80 : 20 (from
`20 to 40 min); flow rate, 1.0 ml/min; injection, 20ml; detection, 290 nm; col-
`umn temperature, 40 °C.
`Liquid Chromatography (LC)-MS Analysis of a Photodegradation
`Product of STFX in the Presence of Bromide Ions STFX was dissolved
`in 0.05 M citrate buffer (pH 4.0) containing 0.2 M of NaBr, and the resulting
`solution (about 2 mg/ml) was exposed to light from a D65 fluorescent lamp,
`which had an overall illumination of ca. 368000 lux · h. After the reaction,
`the sample solution was mixed with the HPLC mobile phase and the result-
`ing solution (ca. 50mg/ml) was subjected to LC-MS, the conditions of
`which were as follows: column, YMC AM-302, 4.6 mm i.d.3150 mm; elu-
`ent, trifluoroacetic acid (TFA) aqueous solution (pH 2.4)–acetonitrile
`(73 : 27, v:v); flow rate, 0.2 ml/min; detection, UV 294 nm; column tempera-
`ture, 40 °C. Electrospray ionization (ESI)-MS conditions of which were as
`follows: ion mode, positive; spray kilovolts, 4.5 kV; heated capillary, 225 °C;
`sheath gas (N2), 80 psi; auxiliary gas (N2), 10 units.
`Effect of Chloride Ion on the Photodegradation Kinetics of STFX
`STFX was dissolved in 0.05 M citrate buffer (pH 4.0) containing 0.2 M NaCl
`or 0.2 M of NaBr (STFX, about 50mg/ml). Each solution (2 ml) was poured
`into a glass container (internal diameter: ca. 20 mm), which was covered
`with clear polyvinylidene chloride film. Then, an exposure test was per-
`formed at 25 °C by irradiating these sample solutions with Light-Tron LT-
`120. The illumination of the D65 fluorescent lamp was adjusted to 4000 lux;
`at this illumination the near ultraviolet energy was about 120mW/cm2. At
`appropriate time intervals, the sample solution was withdrawn, mixed with
`the HPLC mobile phase, and the resulting solution (ca. 5mg/ml) was as-
`sayed by HPLC. The HPLC conditions were as follows: column, TSKgel
`ODS-80Ts (4.6 mm i.d.3250 mm, Tosoh Co.,); eluent, 0.05M phosphate
`buffer (pH 2.4)–acetonitrile (70 : 30, v/v) containing 0.02 M ammonium ac-
`etate and 5 mM sodium 1-nonanesulfonate; flow rate, 0.8 ml/min; injection,
`50ml; detection, UV 294 nm; column temperature, 40 °C.
`Determination of Acid Dissociation Constants (pKa) of STFX The
`acid dissociation constants of STFX were determined by the potentiometric
`titration method. The potentiometric titrator GT-05 (Mitsubishi Kagaku Co.,
`Tokyo) was used. A 100 ml solution of STFX in 2 mol/l HCl was titrated
`with 0.1 mol/l KOH at 25 °C. The pKa values were calculated with the com-
`puter program, PKAS.5)
`Detection of Free Radical and Active Oxygen during Photodegrada-
`tion by ESR Spin-Trapping ESR measurements were recorded on a
`JEOL JES-FE2XG spectrometer (JEOL) with 100 kHz field modulation op-
`erating at 9.42 GHz and at room temperature. The following instrumental
`parameters were employed: modulation amplitude, 0.063 mT; microwave
`power, 8.0 mW; scan time, 2 min.
`STFX solution (2.0 mg/ml pH 4, 50 mM citrate buffer solution) containing
`DMPO (ca. 1.80 M: 200ml) in the presence or absence of 0.2 M NaCl was ir-
`radiated with D65 fluorescent lamps at approximately 4000 lux for 20 min.
`The resultant solution was transferred to capillary tubes (Drummond MI-
`CROCAPS: 50ml), sealed with TERUMOSEAL (Terumo, Tokyo), and mea-
`sured by ESR.
`
`Results and Discussion
`Structural Elucidation of Photodegradation Products
`of STFX STFX was decomposed rapidly in aqueous solu-
`tion by photoirradiation. Figure 2 shows the HPLC chro-
`matogram of STFX aqueous solution after being photoirradi-
`ated by fluorescent lamps for 350000 lux · h. Two major pho-
`todegradation products eluted around 22 and 24 min were ob-
`served in the chromatogram and designated P-1 and P-2, re-
`spectively. These products were isolated and identified by 1H-
`NMR and MS spectra. The product P-1 was identified as 7-
`[7-amino-5-azaspiro[2.4]heptan-5-yl]-6-fluoro-1,4-dihydro-
`4-oxo-3-quinolinecarboxylic acid from the spectrum analy-
`ses: field desorption (FD)-MS and FAB-MS m/z: 318
`
`Fig. 2. High Performance Liquid Chromatogram of STFX Aqueous Solu-
`tion Photoirradiated with Fluorescent Lamps for 350000 lux · h
`
`Fig. 3. Chemical Structures of Photodegradation Products
`
`(M1H)1, 1H-NMR (CD3COOD): 0.92, 1.19 (2H, m), 1.05
`(2H, m), 3.37 (1H, d), 3.67 (1H, d), 4.12 (2H, m), 4.24 (1H,
`m), 6.80 (1H, d), 7.88 (1H, d), 8.74 (1H, s). The results
`showed that a chlorine at the 8-position and a cyclopropyl
`group at 1-position were eliminated. The product P-2 was
`identified as 1-(1-amino-2-{[6-fluoro-1-(2-fluoro-1-cyclo-
`propyl)-1,4-dihydro-4-oxo-3-quinolin-7-yl]-amino}ethyl)-
`cyclopropanecarbaldehyde: CI-MS m/z: 391 (M1H)1, FAB-
`MS m/z: 391 (M1H)1, 1H-NMR (D2O1TFA): 0.14—0.33
`(4H, m), 0.53, 0.58 (1H, m), 0.70 (1H, m), 2.14 (1H, m),
`2.67 (1H, m), 2.77 (2H, m), 4.01, 4.14 (1H, m), 6.07 (1H, s),
`7.24 (1H, s), 7.84 (1H, s). The P-2 product had the structure
`of STFX oxidized at the 7-position and with a chlorine elimi-
`nated at the 8-position. These structures of P-1 and P-2 are
`shown in Fig. 3. Both compounds contained a dissociated
`C–Cl bond at the 8-position.
`Effect of pH on the Photodegradation In the case of
`ionizable compounds, the effect of pH on the photostability
`is significant.6) Figure 4 shows the first-order plots for the
`photodegradation reactions conducted at several pHs. The
`photodegradation of STFX followed an apparent first-order
`rate equation in the pH range of 2.0 to 12.9. The first-order
`plots exhibited good linear relationships with correlation co-
`efficients of 0.99 or higher at all pHs examined. The apparent
`first-order rate constants (k) were obtained from the slopes of
`the plots (Table 1). The log k–pH profile for the photodegra-
`dation of STFX is shown in Fig. 5. The photodegradation
`rates in neutral solution were higher than those observed in
`acidic and alkaline solutions. STFX is an amphoteric com-
`pound with pKa values of 5.7 for the carboxylic group and
`9.0 for the amine determined by potentiometric titration. This
`acid dissociation is shown in Chart 1. The isoelectric point of
`
`MYLAN Ex. 1007, Page 2
`
`

`
`February 2002
`
`231
`
`Fig. 4. Kinetics of STFX Photodegradation in Aqueous Solution at Sev-
`eral pHs
`s, pH 2.0; h, pH 4.0; n, pH 6.0; ,, pH 8.0; d, pH 10.0.
`
`Fig. 5.
`
`k–pH Profile for the Photodegradation of STFX
`
`Table 1. Apparent First-Order Rate Constants (k) for the Photodegradation
`of STFX at Several pHs
`
`Chart 1
`
`pH
`
`1.00
`2.00
`4.00
`6.00
`7.00
`8.00
`10.0
`12.3
`
`k (h21)
`
`0.750
`0.399
`0.599
`0.947
`1.238
`1.423
`0.870
`0.240
`
`the zwitter ion at pH 7.4. STFX was the most sensitive to
`photodegradation in zwitter ionic form at slightly basic pHs.
`STFX was stable in the acidic region, where the carboxyl
`group was not ionized and the basic nitrogen was completely
`protonated.
`Wavelength Dependence of Photodegradation Gener-
`ally, so-called photo-labile compounds decompose under
`light of specific wavelengths and thereby contribute to photo-
`degradation of the compounds.7) Figure 6a shows the pho-
`todegradated ratio of STFX after exposure to light of various
`wavelengths at the same energy (0.5 J/cm2); it clearly shows
`that STFX was degraded wavelength dependently. The degra-
`dation rate and UV spectrum of STFX (Fig. 6b) correlated
`well. That is, STFX was the most labile at 284 nm, which
`was the maximum absorption wavelength.
`Mechanistic Consideration of Photodegradation of
`STFX In some fluoroquinolones the structures of the pho-
`todegradation products have been already identified, such as
`levofloxacin (LVFX)8) and orbifloxacin (ORFX).9) These
`products are analogues altered at the piperazine moiety or
`having the fluorine eliminated at the 8-position of fluoro-
`
`Fig. 6. Photodegradated Ratio of STFX in pH 7.4 Buffered Solution under
`Irradiation for 0.5 J/cm2 at Various Wavelengths (a) and UV Spectrum of
`STFX in pH 7.4 Buffered Solution (b)
`
`quinolones. STFX was photodegradated in aqueous solution
`and gave two major products. The structures of the photo-
`degradation products had in common the chlorine eliminated
`at the 8-position of STFX and in being subsequently de-
`graded at the 1-position or 7-position of STFX. In general,
`mechanisms by which photodegradation of the compounds
`
`MYLAN Ex. 1007, Page 3
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`

`
`232
`
`Vol. 50, No. 2
`
`Chart 2
`
`Table 2. Apparent First-Order Rate Constants (k) for the Photodegradation
`of STFX in pH 4.0 Citrate Buffer in the Presence of Chloride Ion, Bromide
`Ion, and in the Absence of Halide Ion (Control)
`
`Without halide ion (control)
`With 0.2 M NaCl
`With 0.2 M NaBr
`
`a) k of control as 1.00.
`
`k (h21)
`
`0.733
`0.040
`0.739
`
`Ratioa)
`
`1.00
`18.4
`0.99
`
`Fig. 7. Photodegradation Kinetics of STFX in pH 4.0 Citrate Buffer in the
`Presence of Chloride Ion, Bromide Ion, and in the Absence of Halide Ion
`(Control)
`d, 0.2 M NaCl; s, 0.2 M NaBr; j, control.
`
`may occur are classified as follows. The Type I mechanism is
`a free radical chain process and is generally termed autoxida-
`tion. The Type II mechanism involves the excited 1O2 and is
`termed oxygenation. It is well known that heterocyclic com-
`pounds such as pyrrole and furan undergo photooxidation by
`a Type II mechanism. We demonstrated that STFX and the
`other fluoroquinolones generated 1O2 in response to photo-
`irradiation.3) We concluded from these that 1O2 production by
`irradiated STFX was responsible for photodegradation.
`STFX reacts with 1O2 to form a dioxetan intermediate, and
`then undergoes hydrolysis to afford the photodegradation
`products P-1 and P-2, as presented in Chart 2 according to
`the Type II mechanism. However, details of the mechanism
`of the photochemical reaction of STFX are not as clear.
`Effect of Halide Ion on the Photodegradation ORFX,
`a fluoroquinolone having a fluorine at the 8-position, was
`converted into a photoproduct substituted by chlorine at the
`8-position in an aqueous solution containing chloride ions.10)
`Since STFX has a chloride at the 8-position, if photodegra-
`dation of STFX were done in aqueous solution containing
`chloride ion, its apparent stability might be improved. There-
`fore, we demonstrated the photodegradation studies of STFX
`at several pHs in the presence and absence of chloride ion.
`Figure 7 shows the first-order plots for the photodegradation
`of STFX in pH 4.0 citrate buffer solutions containing chlo-
`ride ion, bromide ion, or without halogen ion (control). The
`photodegradation of STFX followed an apparent first-order
`rate equation in each buffer. The first-order plots exhibited
`good linear relationships with correlation coefficients of 0.99
`
`Fig. 8. High Performance Liquid Chromatogram of STFX Photoirradiated
`in pH 4.0 Citrate Buffer Containing Bromide Ion with a D65 Fluorescent
`Lamp for 0.5 h
`
`or higher. The apparent first-order rate constants (k) were ob-
`tained from the slopes of the plots. Table 2 shows the k for
`the photodegradation of STFX in the pH 4.0 citrate buffer so-
`lutions containing chloride ion, bromide ion or without halo-
`gen ion (control). The photostability of STFX was improved
`approximately 18 fold in the presence of chloride ion, while
`it was not affected in the presence of bromide ion.
`LC-MS Analysis of Photodegradation Product in the
`Presence of Bromide Ion Figure 8 shows a high-perfor-
`mance liquid chromatogram of STFX in a pH 4.0 buffer con-
`taining 0.2 M NaBr following irradiation with a D65 fluores-
`cent lamp. Even though the photostability was not affected in
`the presence of bromide ion, an unknown photodegradation
`product was newly observed. Quasi-molecular ions at
`m/z5454 and m/z5456 were present in the LC-ESI mass
`spectrum. Therefore, the structure of the newly observed
`photodegradation product of STFX in the presence of bro-
`mide is the 8-bromo form of STFX, which is substituted for
`chlorine as shown in Chart 3.
`Kinetics of the Photodegradation in the Presence of
`Chloride Ion at Several pHs Figure 9 shows the first-
`order plots for the photodegradation of STFX in 0.1 M
`
`MYLAN Ex. 1007, Page 4
`
`

`
`February 2002
`
`233
`
`Chart 3
`
`Table 3. Apparent First-Order Rate Constants (k) for the Photodegradation
`of STFX in the Presence and in the Absence of Chloride Ion in Aqueous So-
`lution at Several pHs
`
`k (h21)
`
`Without Cl2 (A) With 0.2 M NaCl (B)
`
`0.1 M HClO4
`pH 4.0 citrate buffer
`pH 7.0 phosphate buffer
`0.1 M NaOH
`
`0.750
`0.733
`1.238
`0.240
`
`0.025
`0.040
`0.264
`0.209
`
`Ratio
`A/B
`
`30.6
`18.4
`4.7
`1.1
`
`Fig. 10. Apparent First-Order Rate Constants (k) of the Photodegradation
`of STFX in the Presence or Absence of Chloride Ions in Aqueous Solutions
`at Several pHs
`s, control (without chloride ion); d, with 0.2 M NaCl.
`
`Fig. 9. Photodegradation Kinetics of STFX in the Presence or Absence of
`Chloride Ions in Aqueous Solutions at Several Different pHs
`a) 0.1 M HCl, b) phosphate buffer (pH 7.0), c) 0.1 M NaOH.
`s, control (without chloride ion); h, with 0.2 M NaCl.
`
`HClO4, pH 7.0 phosphate buffer, and 0.1 M NaOH in the
`presence and absence of chloride ion. Table 3 and Fig. 10
`show k for the photodegradation of STFX in the presence
`and absence of chloride ion in the above solutions. The pho-
`tostability of STFX was improved approximately 31 fold in
`the presence of chloride ion in 0.1 M HClO4. Conversely, it
`was not affected in the presence of chloride ion in 0.1 M
`NaOH. Furthermore, the photodegradation rate in a neutral
`solution was almost equal to that in an alkaline solution in
`the presence of chloride ion.
`Fluoroquinolones generated · C and · OH in the process of
`photodegradation in pH 7.0 buffer by means of the 5,5-di-
`methylpyrroline-1-oxide (DMPO) spin trapping method.3)
`The 8-fluorine-substituted fluoroquinolones such as lome-
`floxacin and sparfloxacin produced · C; however, STFX did
`not produce the · C in pH 7.0 buffer. Figure 11a shows the
`ESR spectrum of photoirradiated STFX in pH 4.0, and Fig.
`11b shows the ESR spectrum of photoirradiated STFX in pH
`
`Fig. 11. ESR Spectra of Photoirradiated STFX Solution in the Presence of
`DMPO
`a) STFX in pH 4.0 citrate buffer in the absence of chloride ion was irradiated with
`D65 lamps (approximately 4000 lux) for 20 min. b) STFX in pH 4.0 citrate buffer in the
`presence of 0.2 M NaCl was irradiated with D65 lamps (approximately 4000 lux) for
`20 min.
`
`4.0 in the presence of 0.2 M NaCl. Figure 11a shows that
`STFX generated · C and · OH in the process of photodegrada-
`tion in pH 4.0 buffer. Therefore, the homolytic bond scission
`of the C–Cl took place in the absence of chloride ion in pH
`4.0 buffer solution. On the contrary, STFX did not generate
`the · C in the presence of 0.2 M NaCl in pH 4.0 buffer (Fig.
`11b). These results suggest that the chloride ion competes
`with DMPO in reacting with the generated · C.
`The Mechanism of the Increments in Photostability in
`the Presence of Chloride Ion The photostability of STFX
`
`MYLAN Ex. 1007, Page 5
`
`

`
`234
`
`Vol. 50, No. 2
`
`Chart 4
`
`increased in the presence of chloride ions. In their absence,
`· C was generated at pH 4.0 by homolytic bond scission of
`the C–Cl bond; however, · C was not observed in the pres-
`ence of chloride ions. These results might arise because the
`dechlorination reaction, the initial process of photodegrada-
`tion, was suppressed in the presence of chloride ions. The · C
`was generated and degraded into P-1 and P-2 by photoirradi-
`ation in the absence of chloride ions; but, in the presence of
`these ions, · C immediately reacted with them. As a result,
`the C–Cl bond was recovered, and the apparent photostability
`was increased. Furthermore, the photodegradation rate of
`STFX was not affected by the presence of bromide ions. A
`new major photodegradation product was the 8-bromo form
`of STFX, which was not observed in the absence of bromide
`ions, indicating that the dissociation of the C–Cl bond at the
`8-position of STFX is the rate-limiting step in the initial
`process of photodegradation of STFX. Postulated mecha-
`nisms for the photodegradation of STFX solution in the pres-
`ence of chloride ions and bromide ions are summarized in
`Chart 4.
`No effects on the photostability of STFX in the presence
`of chloride ions were observed in alkaline solutions, so the
`dissociation of the C–Cl bond of STFX is not the rate-limit-
`ing step in these solutions. The carboxyl group of STFX
`completely dissociates (anionic form) in alkaline solutions.
`The photodegradation reactions of STFX in acidic and neu-
`
`tral solutions were different from that in alkaline solutions,
`because the conjugated system of the quinolone ring in car-
`boxylic acid might be quite different from that in carboxylate
`anions. However, the mechanisms of this process are uncer-
`tain.
`In conclusion, the photodegradation behavior of STFX
`was influenced by the addition of chloride ions. The photo-
`stability of STFX was increased in the presence of chloride
`ions in acidic and neutral solutions.
`
`References
`1) Sato K., Hoshino K., Tanaka M., Hayakawa I., Osada Y., Antimicrob.
`Agents Chemother., 36, 1491—1498 (1992).
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`MYLAN Ex. 1007, Page 6