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Sitafloxacin 1.5Hydrate (DU-6859a), a new-generation, broad-spectrum and oral fluoroquinolone class of antibiotic, is highly active against many Gram-positive, Gram-negative and anaerobic clinical isolates, including strains resistant to other fluoroquinolones, was recently approved in Japan for the treatment of respiratory and urinary tract infections. In terms of clinical efficacy, oral sitafloxacin was noninferior to oral levofloxacin in the treatment of community-acquired pneumonia or an infectious exacerbation of chronic respiratory tract disease, noninferior to oral tosufloxacin in the treatment of community-acquired pneumonia, and noninferior to oral levofloxacin in the treatment of complicated urinary tract infections, according to the results of randomized, double-blind, multicentre, noninferiority trials. Noncomparative studies demonstrated the efficacy of oral sitafloxacin in otorhinolaryngological infections, urethritis in men, C. trachomatis-associated cervicitis in women and odontogenic infections.
Physicochemical Properties
| Molecular Formula | C19H18CLF2N3O3.3/2H2O | |
| Molecular Weight | 436.84 | |
| Exact Mass | 409.1 | |
| CAS # | 163253-35-8 | |
| Related CAS # | Sitafloxacin;127254-12-0;Sitafloxacin hydrochloride;346607-37-2;Sitafloxacin monohydrate;163253-37-0; 163253-35-8 | |
| PubChem CID | 461399 | |
| Appearance | White to off-white solid powder | |
| Boiling Point | 629.2ºC at 760mmHg | |
| Melting Point | 145-147°C (dec.) | |
| Flash Point | 334.3ºC | |
| Vapour Pressure | 1.06E-16mmHg at 25°C | |
| LogP | 3.403 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 8 | |
| Rotatable Bond Count | 3 | |
| Heavy Atom Count | 28 | |
| Complexity | 761 | |
| Defined Atom Stereocenter Count | 3 | |
| SMILES | ClC1=C2C(C(C(C(=O)O[H])=C([H])N2[C@]2([H])C([H])([H])[C@]2([H])F)=O)=C([H])C(=C1N1C([H])([H])[C@]([H])(C2(C1([H])[H])C([H])([H])C2([H])[H])N([H])[H])F.O([H])[H] |
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| InChi Key | MPORYQCGWFQFLA-ONPDANIMSA-N | |
| InChi Code | InChI=1S/2C19H18ClF2N3O3.3H2O/c2*20-14-15-8(17(26)9(18(27)28)5-25(15)12-4-10(12)21)3-11(22)16(14)24-6-13(23)19(7-24)1-2-19;;;/h2*3,5,10,12-13H,1-2,4,6-7,23H2,(H,27,28);3*1H2/t2*10-,12+,13+;;;/m00.../s1 | |
| Chemical Name | 3-Quinolinecarboxylic acid, 7-((7S)-7-amino-5-azaspiro(24)hept-5-yl)-8-chloro-6-fluoro-1-((1R,2S)-2-fluorocyclopropyl)-1,4-dihydro-4-oxo-, hydrate (2:3) | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | DNA gyrase |
| ln Vitro |
Sitafloxacin (DU6859a) monohydrate exhibits antibacterial activity against wild-type ATCC 49619, gyrA mutant SP39, and parC mutant 1026523 streptococcus pneumoniae stran, with MIC values of 0.03, 0.12, and 0.06 mg/L, respectively[1]. For quinolone-susceptible strains of Streptococcus pneumoniae, sitafloxacin (DU6859a) monohydrate has antibacterial activity with MIC values of 0.03 mg/L for the EG 00218 strain and 0.03 mg/L for the EG 00093 strain, respectively[1]. DNA gyrase and topoisomerase IV (TopoIV) are inhibited by sitafloxacin (DU6859a) monohydrate, with >IC50s of 4.38 and 3.12 mg/L, respectively[1]. DC-159a and sitafloxacin exhibited potent antibacterial activities, low frequencies of mutant selection, low MPCs and narrow mutant selection windows against both quinolone-susceptible strains and first-step parC mutants of S. pneumoniae, compared with gatifloxacin, moxifloxacin and other quinolones tested. DC-159a and sitafloxacin showed relatively low MIC ratios against single gyrA or parC mutants relative to the wild-type strain and low IC(50) ratios against DNA gyrase and topoisomerase IV. Conclusions: DC-159a and sitafloxacin demonstrated a more balanced dual-targeting activity than gatifloxacin, moxifloxacin and other quinolones tested. In addition, DC-159a and sitafloxacin have a lower propensity for selecting first- and second-step resistant mutants [1]. |
| ln Vivo |
Sitafloxacin monohydrate (DU6859a; 12.5–100 mg/kg; ir; daily for 4 weeks; female BALB/c mice) possesses antibacterial properties. M. There was no sign of footpad swelling and ulcerans cells could be separated from the infected footpads[2]. Efficacy of a new fluoroquinolone, sitafloxacin (DU-6859a), against Mycobacterium ulcerans was evaluated in vivo using the mouse footpad system. The growth of M. ulcerans in mouse footpads was completely inhibited when mice were fed with sitafloxacin at a dose of 25 mg/kg body weight per day; on the other hand similar effects were observed with ofloxacin at a dose of 100 mg/kg body weight per day. In the presence of rifampin, the above dose of sitafloxacin could be reduced by 75% to achieve total inhibition, while, under similar circumstances, the dose of ofloxacin could be reduced by only 50%. Either used singly or in combination with rifampin, the effects of sitafloxacin were bactericidal. The results suggest that sitafloxacin should be evaluated as a chemotherapeutic agent against M. ulcerans infection [2]. |
| Enzyme Assay |
Determination of inhibitory activities against DNA gyrase and topoisomerase IV from S. pneumoniae [1] DNA gyrase supercoiling and TopoIV decatenation assays were carried out as described previously22,23 with minor modifications. For the DNA gyrase supercoiling assay, the reaction mixture contained 40 mM Tris–HCl (pH 7.5), 30 mM KCl, 5 mM MgCl2, 1 mM spermidine, 4 mM ATP, 1 mM dithiothreitol (DTT) and 20 µg of BSA per mL. One unit of each purified GyrA and GyrB was incubated with 0.1 µg of relaxed pBR322 plasmid DNA for 60 min at 37°C. For the TopoIV decatenation assay, the reaction mixture contained 40 mM Tris–HCl (pH 7.5), 20 mM KCl, 5 mM MgCl2, 0.5 mM ATP, 1 mM DTT and 50 µg of BSA per mL. The decatenation activity was performed using 1 U of each purified ParC and ParE as well as 0.4 µg of catenated kinetoplast DNA (k-DNA) for 60 min at 37°C. |
| Cell Assay |
Bacterial strains and antimicrobial susceptibility testing [1] Two quinolone-susceptible strains of S. pneumoniae (EG00093 and EG00218), which did not harbour mutations in the QRDRs of the gyrA, gyrB, parC and parE genes, and two first-step parC mutants (60 and 1026523) were used for mutant selection experiments. In 2002, EG00093 and EG00218 were selected as representative strains of quinolone-susceptible S. pneumoniae from a surveillance collection in Japan. Strain 60 harbouring a single QRDR mutation in parC (Ser-79→Tyr) and isolate SP39 with a gyrA mutation (Ser-81→Phe) are both mutants of ATCC 49619. Strain 1026523 is a clinical isolate harbouring a combination of mutations within the QRDRs of parC (Ser-79→Phe) and parE (Ile-460→Val), which was obtained from the GLOBAL surveillance study in 2003 (Focus Bio-Inova Inc., Herndon, VA, USA). The determination of MICs was performed at least in duplicate, according to a standard agar dilution method.20 Determination of mutant selection frequency and MPC [1] The determination of mutant selection frequency and MPC experiments were performed concurrently. The method for measuring the mutation frequency and MPC was a modification of that described previously.21 Each isolate was grown for 12 h on heart infusion agar containing 5% sheep blood at 35°C. Several colonies were then suspended in sterile PBS at a turbidity equivalent to that of a 0.5 McFarland standard (1 × 108 cfu/mL). The suspension (20 mL) was divided equally into four flasks each containing 500 mL of fresh BHIY broth (total volume of 2 L) and incubated for an additional 6 h without shaking. Cultures were then concentrated 10- to 30-fold by centrifugation (5000 g for 30 min at 20°C) to yield a concentration of 1010–1011 cfu/mL. Aliquots of 100 µL of the bacterial suspension (containing 109–1010 cfu) were spread onto BHI agar plates containing 10% horse-defibrinated blood with multiples of the MICs for each quinolone. The plates containing quinolones were incubated at 35°C for 72 h, and the antibiotic-free plates were incubated at 35°C for 16–24 h. The frequency of mutant selection was calculated as the ratio of the number of resistant colonies at 72 h to the number of cfu plated. The MPC of each quinolone was determined as the lowest concentration that prevented the growth of resistant colonies when more than 1010 bacteria were spread on agar plates and incubated for 72 h at 35°C. PCR amplification of QRDRs and DNA sequence analysis [1] The presence of mutations in the QRDRs of the gyrA, gyrB, parC and parE genes was investigated by PCR. The primer sequences used to amplify the gyrA QRDR were as follows: SPGA3, 5′-GTCAATCTGACAAAGGAGATGAAGG-3′ (position 25 to 49) and SPGA6, 5′-CAATCTCTGTACGAGAACGTAGGAC-3′ (position 715 to 739). For the amplification of the gyrB QRDR, SPGB3: 5′-TTACCAATCGCCTCTTCAGTGAAGC-3′ (position 1070 to 1094) and SPGB4: 5′-CTTCCAACCTTGACACCATAGATTGG-3′ (position 1621 to 1646) were used; for the amplification of the parC QRDR, SPPC1: 5′-GGCTTTGTATCTTATGTCTAACATTC-3′ (position −14 to 27) and SPPC6: 5′-AAACTGCAGCATCTATGACCTCAGC-3′ (position 548 to 573) and for the amplification of the parE QRDR, SPPE3: 5′-AGTTGTGGATGGAATAGTGGCT-3′ (position 1061 to 1084) and SPPE4: 5′-GGACATCTTGTAAGAGGTGGGAG-3′ (position 1615 to 1638). Amplification was performed in a total volume of 50 µL containing 1 µL of template DNA, 4 µL of each deoxynucleoside triphosphate (2.5 mM each), 5 µL of 10× Ex Taq DNA polymerase buffer, 1 µL of each primer (10 pmol/µL) and 0.25 µL of Ex Taq DNA polymerase (5 U/μL). Amplification conditions were 94°C for 2 min, 35 cycles of 98°C for 20 s, 62°C for 2 min and 72°C for 3 min, with a final extension of 3 min at 68°C. DNA sequencing of PCR products was carried out with an ABI PRISM 3100 Avant Genetic Analyzer in accordance with the manufacturer's instructions. |
| Animal Protocol |
Animal/Disease Models: BALB/c female mice[2] Doses: 12.5, 25, 50 and 100 mg/kg Route of Administration: po (oral gavage); daily, for 4 weeks Experimental Results: Inhibits the growth of Mycobacterium ulcerans and the M. ulcerans cells. |
| References |
[1]. Dual-targeting properties of the 3-aminopyrrolidyl quinolones, DC-159a and sitafloxacin, against DNA gyrase and topoisomerase IV: contribution to reducing in vitro emergence of quinolone-resistant Streptococcus pneumoniae. J Antimicrob Chemother. 2008 Jul;62(1):98-104. [2]. Activities of sitafloxacin (DU-6859a), either singly or in combination with rifampin, against Mycobacterium ulcerans infection in mice. J Chemother. 2003 Feb;15(1):47-52. [3]. Drugs Today (Barc).2008 Jul;44(7):489-501;[4]. Drugs.2011 Apr 16;71(6):731-44. |
| Additional Infomation |
Sitafloxacin is a member of quinolines, a quinolone antibiotic and a fluoroquinolone antibiotic. Objectives: DC-159a (a novel quinolone) and sitafloxacin (DU-6859a) are structurally related quinolones, bearing a 3-aminopyrrolidyl substitution. We investigated the relationship between the target preferences of these 3-aminopyrrolidyl quinolones, in vitro potencies and emergence of quinolone-resistant mutants in Streptococcus pneumoniae, compared with other quinolones. Methods: MICs, resistance frequencies and mutant prevention concentrations (MPCs) were determined using quinolone-susceptible strains and first-step parC mutant strains of S. pneumoniae. Target preferences were tested by the following two methods: antibacterial activities against gyrA or parC mutants and in vitro enzyme assays for the determination of 50% inhibition (IC(50)) values. [1] In conclusion, the balanced dual-targeting activity of DC-159a and sitafloxacin against DNA gyrase and TopoIV might provide a greater potential to minimize the development of quinolone resistance. DC-159a and sitafloxacin could be potential therapeutic options for pneumococcal pneumonia caused by quinolone-susceptible and less quinolone-susceptible isolates. [1] |
Solubility Data
| Solubility (In Vitro) |
DMSO : ~15 mg/mL (~34.33 mM) H2O : ~1 mg/mL (~2.29 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.76 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (4.76 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.08 mg/mL (4.76 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.08 mg/mL (4.76 mM) (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2892 mL | 11.4458 mL | 22.8917 mL | |
| 5 mM | 0.4578 mL | 2.2892 mL | 4.5783 mL | |
| 10 mM | 0.2289 mL | 1.1446 mL | 2.2892 mL |