In recent years, novel synthesized sulfonamides have been introduced as antitumor, antiviral and anti-inflammatory agents.Sulfonamides are the first effective chemotherapeutic agents used for several years to cure or prevent systemic bacterial infections. In addition, this agents showed anti-carbonic anhydrase and cause cell cycle perturbation in the G5 cells/mL for 24 h, and then logarithmic concentrations  of each drug were prepared, added to the plates and incubated for 72 h. Cell survival was then determined using ELISA plate reader in 540 nm applying MTT assay.In this paper, the cytotoxic effects of 8 synthesized sulfonamides were investigated by MTT assay on HeLa, MDA-MD-468 and MCF-7 cancer cell lines. Human cancer cells were cultured and passaged in RPMI-1640 medium. Cells incubated in 96-well plates in a concentration of 1 × 10All tested sulfonamides showed cytotoxic effect on HeLa and MCF-7 cells in the concentration range of 100-1000 μm. These sulfonamides were cytotoxic against MDA-MB-468 cell line at a concentration of 10-100 μm and reduced the cell survival less than 50%.50’s were as following: MDA-MB-468 < 30 μm; MCF-7 < 128 μm and HeLa< 360 μm. In conclusion, some tested sulfonamides had good cytotoxic effects against breast cancer cells, MDA-MB-468 and further investigations are needed to confirm their effects against other cells.According to the results calculated IC Recently, sulfa drugs were introduced as protease inhibitors; therefore, they can be used as anticancer, anti-inflammatory and antiviral agents , loop diuretics (including furosemide), some COX-2 inhibitors . Melting points were determined with a Thomas–Hoover capillary apparatus. Infrared spectra were acquired using a Perkin Elmer Model 1420 spectrometer. A Bruker FT-500 MHz instrument  was used to acquire 6 were used as solvents. Coupling constant (J) values are estimated in hertz (Hz) and spin multiples are given as s (singlet), d (double), t (triplet), q (quartet), m (multiplet), and br (broad). Low-resolution mass spectra were acquired with a MAT CH5/DF (Finnigan) mass spectrometer that was coupled online to a Data General DS 50 data system. The mass spectral measurements were performed on a 6410 Agilent LCMS triple quadruple mass spectrometer (LCMS) with an electrospray ionization (ESI) interface. Microanalyses, determined for C and H, were within ± 0.4% of theoretical values.Chloroform-D and DMSO-DChemistrySulfonamides used in this work were synthesized according to General procedure for preparation of 4- acetamido -4- substituted- phenyl- benzenesulfonamide 115 mmol of 4-acetamidobenzene sulfonyl chloride was added to a solution of 4-substituted-aniline (15 mmol) and triethyl amine (5 mL) in chloroform (25 mL) and stirred at 25°C for 15 min. Then the mixture was heated gently up to 55-60°C. After 5 h, the organic solvent was evaporated and 100 mL cold water was added. The produced precipitate was filtered and washed with water and recrystallized in ethanol (yields: 65-87%).4-Acetamido-4-phenyl-benzenesulfonamide 1a-1) 3310, 3200 (NH), 1670 (C=O), 1320, 1150 (SO2); LC-MS (ESI +) m/z: 291.1 (M + 1) (100).Yield: 87%; white crystalline powder; mp = 203-204°C; IR (KBr): ν (cm4-Acetamido -4-fluorophenyl-benzenesulfonamide 1b-1) 3320, 3200 (NH), 1680 (C=O), 1320, 1140 (SO2); LC-MS (ESI+) m/z: 309.1 (M + 1) (100).Yield: 67%; white crystalline powder; mp = 188-190°C; IR (KBr): ν (cm4-Acetamido-4-methoxyphenyl benzenesulfonamide 1c-1) 3300, 3200 (NH), 1650 (C=O), 1300, 1120 (SO2); LC-MS (ESI+) m/z: 321.1 (M + 1) (100).Yield: 65%; cream crystalline powder; mp = 200-201°C; IR (KBr): ν (cm4-Acetamido-4-methylphenyl-benzenesulfonamide 1d-1) 3300, 3200 (NH), 1660 (C=O), 1300, 1100 (SO2); LC-MS (ESI+) m/z: 305.1 (M + 1) (100).Yield: 65%; cream crystalline powder; mp = 200-201°C; IR (KBr): ν .4-Amino-4-phenyl-benzenesulfonamide 2a-1) 3380, 3320 (NH2), 3200(NH), 1310, 1150 (SO2); LC-MS (ESI+) m/z: 249.1 (M + 1) (100)Yield: 82%; yellow crystalline powder; mp = 195-196°C; IR (KBr): ν (cm4-Amino-4-fluorophenyl-benzenesulfonamide 2b-1) 3400, 3320 (NH2), 3200 (NH), 1300, 1140 (SO2); LC-MS (ESI+) m/z: 267.1 (M + 1) (100).Yield: 81%; white crystalline powder; mp = 165-167°C; IR (KBr): ν (cm4-Amino-4-methoxyphenyl-benzenesulfonamide 2c-1) 3400, 3300, (NH2), 3200 (NH), 1340, 1100 (SO2); LC-MS (ESI+) m/z: 279.1 (M + 1) (100).Yield: 90%; cream crystalline powder; mp = 195-196°C; IR (KBr): ν (cm4-Amino-4-methylphenyl-benzenesulfonamide 2d-1) 3350, 3250, (NH2), 3200 (NH), 1310, 1100 (SO2); LC-MS (ESI+) m/z: 263.1 (M + 1) (100).Yield: 79%; yellow crystalline powder; mp = 190-191°C; IR (KBr): ν  in water (5 mL) was added and the mixture was stirred for 1 h. The precipitated product was filtered, washed and crystallized by ethanol (yields: 83-96%).4-Azido-4-phenyl-benzenesulfonamide 3a-1) 3200(NH), 2100 (N3) 1300, 1150 (SO2); 1HNMR (CDCl3): δ ppm 6.46 , 7.07-7.29 , 7.77  ;LC-MS(ESI+) m/z : 275.1 (M + 1) (100).Yield: 90%; white crystalline powder; mp = 95°C; IR (KBr): ν (cm4-Azido-4-fluorophenyl-benzenesulfonamide 3b-1) 3200(NH), 2050 (N3) 1300, 1120 (SO2); 1HNMR (CDCl3): δ ppm 6.64 , 7.00 , 7.06-7.10 , 7.73  ; LC-MS(ESI+) m/z: 293.1 (M + 1) (100).Yield: 96%; white crystalline powder; mp = 97°C; IR (KBr): ν (cm4-Azido-4-methoxyphenyl-benzenesulfonamide 3c-1) 3200(NH), 2030 (N3) 1300, 1120 (SO2); 1HNMR (CDCl3): δ ppm 3.81 ,6.23 , 6.82 , 7.01 , 7.08 , 7.71  ;LC-MS (ESI+) m/z: 305.1 (M + 1) (100).Yield: 83%; cream crystalline powder; mp = 74°C; IR (KBr): ν (cm4-Azido-4-methylphenyl-benzenesulfonamide 3d-1) 3180(NH), 2040 (N3) 1300, 1120 (SO2); 1HNMR (CDCl3): δ ppm 2.33 ,6.43 , 6.98 , 7.07 , 7.11 , 7.75  ; LC-MS(ESI+) m/z: 289.1 (M + 1) (100).Yield: 94%; white crystalline powder; mp = 79-80°C; IR (KBr): ν .4-Methyl sulfamido-4-phenyl-benzenesulfonamide 4a-1) 3200(NH), 1340, 1150 (SO2); 1HNMR (CDCl3): δ ppm 2.84 , 6.89-7.06  7.15 , 7.58 , 9.43 , 9.89  ; LC-MS (ESI+) m/z: 327.2 (M + 1) (100).Yield: 72%; white crystalline powder; mp = 160-161°C; IR (KBr): ν (cm4-Methyl sulfamido-4-fluorophenyl-benzenesulfonamide 4b-1) 3200(NH), 1340, 1100 (SO2); 1HNMR (CDCl3): δ ppm 2.83 , 6.72 , 6.94 ),7.14 , 7.51 , 9.43 , 9.91  ; LC-MS (ESI+) m/z: 345.2 (M + 1) (100).Yield: 70%; white crystalline powder; mp = 175-176°C; IR (KBr): ν (cm4-Methyl sulfamido-4-methoxyphenyl-benzenesulfonamide 4c-1) 3200(NH),1300, 1120 (SO2); 1HNMR (CDCl3): δ ppm 2.10 ,2.82 , 6.81 , 6.85 ,7.12 , 7.52 , 9.22 , 9.87  ; LC-MS (ESI+) m/z: 341.2 (M + 1) (100).Yield: 83%; cream crystalline powder; mp = 181-182°C; IR (KBr): ν (cm4-Methyl sulfamido-4-methylphenyl-benzenesulfonamide 4d-1) 3200(NH), 1340, 1100 (SO2); 1HNMR (CDCl3): δ ppm 2.78 , 3.63 , 6.60 , 6.91 ,7.17 , 7.54 , 8.98 , 9.88  ; LC-MS (ESI+) m/z: 357.2 (M + 1) (100).Yield: 70%; cream crystalline powder; mp = 193-194°C; IR (KBr): ν  and other serial dilution were prepared from this stock solution, using PBS.Biological studiesCell lines HeLa (Human cervix carcinoma), MCF-7 (Human Caucasian/ breast cancer cells), MDA-MB-468 (Human breast adenocarcinoma) cell lines were purchased from Pasture Institute of Iran in Tehran. They were grown in RPMI-1640 . Completed media was sterilized through 0.22µ m microbiological filters after preparation and kept at 4°C before using. MTT-based cytotoxicity assay5 cells/mL) were seeded in 96-well microplates and incubated for 24 h . Then, 20 µl of final concentration of each compound was added and incubated for another 72 h in the same condition. Doxorubicin was used as a positive control. Cell survival was determined as already mentioned -2, 5-diphenyl tetrazolium bromide (MTT) and compared with untreated controls . This asentioned .Statistical analysis-values for significance were determined using the two-tailed Student›s t-test. Significance was assumed at 5% level.All results are expressed as mean ± SD of at least three experiments. P2 = 0.9407, 0.8695 and 0.9907, respectively). Intraday and interday variations for all standard curves were acceptable (%CV<15). Taxol (2 mg/mL), a known cytotoxic drug, significantly inhibited the proliferation of all tested cell lines as a positive control to less than 20%. Drugs were considered cytotoxic where cell viability decreased to less than 50%.A good relationship between the absorbance and the number of cells was observed for HeLa, MDA-MB-468 and MCF-7 cell lines,  of compounds. Results are shown in All tested sulfonamides used in this research in the concentration range of 100-1000 µM on HeLa cell line showed cytotoxic effect and reduced the cell survival to < 50%. To determine the exact ICCytotoxic effect of sulfonamide derivatives against MDA-MB-468 cell50’s were determined as illustrated in In the case of MDA-MB-468 cells, all tested compounds in the concentration range of 10-100 µM were cytotoxic and ICCytotoxic effect of sulfonamide derivatives against MCF-7 cell50’s were determined as seen in Tested sulfonamides in the concentration rang of 100-1000 µM on MCF-7 cell line showed cytotoxic effect and IC50’s of synthetic compounds against tested cell lines are shown in All calculated ICSulfonamides are usually used in prevention and treatment of bacterial infections in human, but their importance in the field of cancer treatment is considered in recent years. Researchers show that these compounds have multi-effects, in addition to their anti-microbial properties .in-vivo and in-vitro that introduces them as a suitable candidate for preventing proliferation treatment of tumor cells in different kind of cancers (New sulfonamides and their related derivatives have shown remarkable anti-tumor activities  cancers .α (TACE); therefore, they are effective in pathophysiology of most of diseases such as arthritis, metastatic tumor and bacterial meningitis, simultaneously  and transformed enzyme TNF-aneously .E7070  is one oin-vitro cytotoxic effects.Previous studies have been revealed that E7070 had an acceptable anti-tumor effect in cell lines including LX-1 of lung cancer, HCT-116 from colorectal  and A54950 ≤ 30 μm and ≤ 100 μm, respectively. On the other hand, these compounds did not show significant cytotoxic effects against HeLa cells, from cervix cancer (145 μm ≤ IC50 ≤ 360 μm). In agreement with these results, other researchers have shown that sulfonamides and cyclooxygenase inhibiting compounds did not show significant cytotoxic effect against hepatocellular carcinoma, cervix and lung cancer, however, they were much more effective against cell from breast, colorectal and bladder cancers  point of view, different groups on the sulfonamides nucleus (R1 and R2 in


Penicillium purpurogenum G59. Upon treatment of the G59 spores with a high concentration of gentamicin in aqueous DMSO, a total of 181 mutants were obtained by single colony isolation. In contrast to the strain G59, the EtOAc extracts of nine mutant cultures showed inhibitory effects on K562 cells, indicating that the nine mutants had acquired capability to produce antitumor metabolites. This was evidenced by TLC and HPLC analysis of EtOAc extracts of G59 and the nine mutants. Further isolation and characterization demonstrated that four antitumor secondary metabolites, janthinone (1), fructigenine A (2), aspterric acid methyl ester (3) and citrinin (4), were newly produced by mutant 5-1-4 compared to the parent strain G59, and which were also not found in the secondary metabolites of other Penicillium purpurogenum strains. However, Compounds 1–4 inhibited the proliferation of K562 cells with inhibition rates of 34.6% (1), 60.8% (2), 31.7% (3) and 67.1% (4) at 100 μg/mL, respectively. The present study demonstrated the effectiveness of a simple, yet practical approach to activate the production of dormant fungal secondary metabolites by introducing acquired resistance to aminoglycoside antibiotics, which could be applied to the studies for eliciting dormant metabolic potential of fungi to obtain cryptic secondary metabolites.A new approach to activate silent gene clusters for dormant secondary metabolite production has been developed by introducing gentamicin-resistance to an originally inactive, marine-derived fungal strain  Natural products are important sources of new drugs and drug leads ,2,3,4. IHowever, the majority of cultured microbes often do not produce bioactive metabolites under the laboratory culture conditions and thus could not be used for producing strains of anticipated metabolites. A number of these strains were thus stocked or even destroyed. Although these strains indeed possess potential for the production of bioactive metabolites, the gene clusters remain inactive under general culture conditions ,27,28,29In contrast, the one-strain-many-compounds (OSMAC) strategy  has beenPenicillium purpurogenum G59 + in ESI-MS were identified as janthinone 20D −17.3 , giving pseudo-molecular ion peak at m/z 251 [M + H]+ in ESI-MS, could be identified as citrinin  or 10% sulfuric acid reagent. HPLC analysis was performed using an analytical Venusil MP C18 column  on a Waters HPLC system equipped with Waters 600 controller, Waters 600 pump, Waters 2414 refractive index detector, Waters 2996 photodiode array (PDA) detector and Waters Empower™ software. Each 10 μL of sample solutions (8.3 mg/mL) in MeOH was injected into the column and eluted with MeOH–H2O in linear gradient  as mobile phase . The acquired photodiode array (PDA) data were processed by Empower PDA software to obtain targeted HPLC data.TLC analysis was carried out on precoated silica gel GF254 plates using CHClSpore suspension of mutant 5-1-4 (2 mL) was inoculated into a 500 mL cone-shaped flask with 200 mL of liquid medium  and fermented at 28 °C for 14 days on a rotary shaker at 180 rpm. Sampling was performed for a 15 mL portion with a 24 h interval from the third day of fermentation. After centrifugation of the sampled broth at 3000 rpm for 10 min, followed by measurement of the pH value of supernatant and the relative percentage (%) of mycelial volume, the whole broth (15 mL) was extracted with 30 mL acetone by ultrasonication for 1.5 h. The obtained aqueous acetone solution was concentrated under reduced pressure to remove acetone and the remaining water layer was extracted three times with equal volumes of EtOAc to obtain the extracts for assay./mL, from which the antitumor secondary metabolites 1–4 were isolated.Based on the result of the time course experiment mentioned, spore suspension of mutant 5-1-4 (2 mL) was inoculated into each of 245 cone-shaped 500 mL flasks with 200 mL of liquid medium  and fermented at 28 °C for 12 days on a rotary shaker at 180 rpm. The whole broth (49 L) was filtrated to separate into a filtrate and a mycelial cake. The filtrate was extracted three times with equal volumes of EtOAc to give an extract (14.3 g). The mycelial cake was extracted with 3 L of acetone-water (2:1) by ultrasonication for 1.5 h and the aqueous acetone solution obtained by filtration was evaporated under reduced pressure to remove acetone. The water layer remained was extracted three times with equal volumes of EtOAc to give another extract (16.3 g). The combined EtOAc extracts (30.6 g) showed an inhibitory effect on K562 cells, with an IR% value of 42.8% at the 100 µg/mL). This extract was used as negative control in the experiments for tracing antitumor secondary metabolites newly produced by the mutant 5-1-4.G59 spore suspension (2 mL) was inoculated into each of two 500 mL cone-shaped flasks containing 200 mL of liquid medium with the same composition and fermented at 28 °C for 12 days under the same conditions. The fermented broth (400 mL) was extracted as described above for mutant 5-1-4 to obtain an EtOAc extract (200 mg), which did not show any inhibitory effect on K562 cells  by vacuum liquid chromatography (VLC) using a silica gel column (bed 6.2 × 10 cm) dry-packed with 150 g of silica gel H (100–200 meshes) through stepwise elution with bp 60–90 °C petroleum ether (P)-ethyl acetate (E) (100:0→0:100) and then dichloromethane (D)-methanol (M) (90:10→0:100). Six fractions, Fr-4 (1.6 g) eluted by PE (9:1→8:2), Fr-5 (3.7 g) eluted by PE (8:2), Fr-6 (2.0 g) eluted by PE (8:2→7:3), Fr-7 (4.6 g) eluted by PE (7:3→4:6), Fr-8 (2.5 g) eluted by PE (2:8) and Fr-12 (5.1 g) eluted by DM (9:1→8:2), showed inhibitory effects against K562 cells, with IR% values of 34.4, 31.4 36.5, 52.8, 43.0, and 49.4%, respectively, at 100 μg/mL.The EtOAc extract (30.6 g) of mutant 5-1-4 was separated into 15 fractions (Fr-4 (1.6 g) was subsequently subjected to Sephadex LH-20 column (bed 1.5 × 135 cm in DM 1:1) chromatography using DM (1:1) as eluting solvent to obtain seven fractions, Fr-4-1 to Fr-4-7, in the order of elution. Fr-4-3 (1.45 g) was further separated by VLC using a silica gel column (bed 2.5 × 6 cm) dry-packed with 12 g of silica gel H (200–300 meshes) by a stepwise elution with PE (100:0→0:100) and EM (100:0→0:100) to give seven fractions, Fr-4-3-1 to Fr-4-3-7. Among them, Fr-4-3-2 (420 mg) eluted by PE (99:1→97:3) gave yellow crude crystals at room temperature, which were filtered and then repeatedly recrystallized from dichloromethane–methanol (1:1) to obtain 1 (28 mg) as yellowish needles.Fr-5 (3.7 g) was further separated into 12 fractions, Fr-5-1 to Fr-5-12, by VLC on a silica gel column (bed 2 × 12 cm) dry-packed with 25 g of silica gel H (100–200 meshes) through stepwise elution with PE (100:0→0:100) and DM (90:10→0:100). Fr-5-2 (160 mg) eluted by PE (99:1→96:4) was separated by Sephadex LH-20 column (bed 1.5 × 135 cm in DM 1:1) chromatography eluted with DM (1:1) to obtain five fractions, Fr-5-2-1 to Fr-5-2-5, in the order of elution. Fr-5-2-1 (70 mg) was separated by semi-preparative HPLC on a reversed-phase Senshu Pak C18 column (8 × 250 mm) using 65% aqueous MeOH as eluting solvent  to obtain a fraction corresponding to the peak with a retention time (tR) 38 min. This fraction was recrystallized in dichloromethane to give 2 (30 mg) as colorless needles.Fr-6 (2.0 g) was also further separated by Sephadex LH-20 column (bed 1.5 × 135 cm in DM 1:1) chromatography using DM (1:1) as eluents to give seven fractions, Fr-6-1 to Fr-6-7, in the order of elution. Fr-6-4 (490 mg) was separated by preparative TLC on silica gel GF254 plates  using cyclohexane-acetone-ammonia water (30:10:1) as the developing solvents. The silica gel on the band with the Rf value of 0.6 under 254 nm UV light was collected to afford 3 (8 mg) as colorless oil from MeOH solution.Fr-8 (2.5 g) gave yellow crude needles at room temperature in MeOH. The crystals were filtered and repeatedly recrystallized from MeOH to obtain 4 (40 mg) as lemon-yellow needles.1): yellowish needles (CH2Cl2-MeOH), mp 197–198 °C. Positive ESI-MS m/z: 285 [M + H]+, 307 [M + Na]+. 1H NMR  δ: 12.06 , 7.65 , 7.43 , 7.22 , 6.65 , 6.53 , 3.95 , 2.34 . 13C NMR  δ: 180.4 (C-9), 169.6 (C-10), 161.4 (C-8), 155.9 (C-4a), 155.6 (C-4b), 149.3 (C-6), 134.7 (C-3), 133.5 (C-1), 122.4 (C-2), 119.4 (C-4), 117.5 (C-9a), 111.6 (C-7), 107.3 (C-5), 106.9 (C-8a), 53.1 (C-11), 22.6 (C-12). The above data for 1 are identical with those reported for janthinone 20D −175.4 . Positive ESI-MS m/z: 444 [M + H]+; negative ESI-MS m/z: 442 [M − H]−. 1H NMR  δ: 7.94 , 7.30–7.11 , 7.06 , 5.98 , 5.70 , 5.57, 5.06 , 5.04 , 4.16 , 3.73 , 3.48 , 2.74 , 2.59 , 2.50 , 2.20 , 1.06 , 0.91 . 13C NMR  δ: 170.1 (C-22), 168.1 (C-4), 164.8 (C-1), 143.3 (C-7a), 143.0 (C-20) , 135.3 (C-13), 131.7 (C-10a), 129.4 , 129.1 , 127.7 (C-16), 124.6 , 119.1 (C-9), 114.6 (C-21), 79.5 (C-5a), 60.9 (C-10b), 59.1 (C-11a), 56.0 (C-3), 40.4 (C-19), 37.0 (C-11), 36.0 (C-12), 23.6 (C-19b), 23.2 (C-19a), 22.4 (C-23). The above data for 2 are identical with those of fructigenine A 20D −59 , molecular formula C16H24O4. Positive ESI-MS m/z: 281 [M + H]+, 303 [M + Na]+. Positive HR-ESI-MS m/z: measured 281.1757 [M + H]+, calculated for C16H25O4 [M + H] 281.1753. 1H NMR  δ: 4.31 , 3.71 , 3.67 , 3.34 , 2.37–2.45 , 2.32  2.25–2.35 , 2.16 , 2.01 , 1.96–2.07 , 1.73 , 1.71 , 1.62–1.67 , 1.60 , 1.49 , 1.42 . 13C-NMR  δ: 176.2 (C-12), 136.6 (C-10), 125.2 (C-11), 84.9 (C-2), 79.8 (C-3), 76.5 (C-13), 56.7 (C-6), 54.3 (C-7), 52.4 (C-16), 36.6 (C-1), 35.2 (C-4), 35.0 (C-8), 33.1 (C-9), 24.7 (C-5), 23.4 (C-15), 21.0 (C-14). The above NMR data for 3 assigned by the analysis of DEPT, 1H-1H COSY, HMQC and HMBC spectra closely resembled those of aspterric acid 20D −17.3 . Positive ESI-MS m/z: 251 [M + H]+, 273 [M + Na]+, 289 [M + K]+. 1H NMR  δ: 15.91 , 15.13 , 8.25 , 4.79 , 3.00 , 2.02 , 1.35 , 1.23 . 13C NMR  δ: 183.7 (C-6), 177.1 (C-8), 174.5 (C-12), 162.9 (C-1), 139.1 (C-4a), 123.0 (C-5), 107.3 (C-8a), 100.2 (C-7), 81.6 (C-3), 34.5 (C-4), 18.5 (C-11), 18.2 (C-9), 9.4 (C-10). The above data are identical with those reported for citrinin [Citrinin (citrinin ,55.Penicillium purpurogenum G59, which is inactive, to produce antitumor secondary metabolites. Treatment of the G59 spores with a high concentration of gentamicin in aqueous DMSO afforded a total of 181 mutants, nine of which acquired metabolic capability to produce antitumor secondary metabolites as indicated by the antitumor effects of their extracts. This was evidenced also by the TLC and HPLC analysis of extracts of G59 and the nine mutants. Identification of four newly produced antitumor metabolites by mutant 5-1-4, further demonstrated the effectiveness of this approach. The present study provides a simple and practical approach without a complex gene manipulation process to activate dormant secondary metabolite production in fungi, which could be applied to other fungal metabolite studies.To summarize, a new approach to activate dormant secondary metabolite production has been developed by introducing gentamicin-resistance in


Systemic-onset JIA (so-JIA) is often persisting into adulthood and can be refractory to treatment including biologicals. We report on efficacy and safety of Tocilizumab in adults with persistent active so-JIA in a retrospective single center descriptive cohort study.Disease activity scores (DAS-CRP), ESR and CRP were measured just before and at 1- 6- and 12 months of Tocilizumab treatment in all adults with refractory so-JIA at our University Hospital. Adverse events were recorded.Eight patients  with a median age of 27,5yr and median disease duration of 16 yr were treated. All previously failed Methotrexate, other classical DMARD’s, 7/8 also failed on one or more biologicals . Median HAQ before starting Tocilizumab was 1.25 (0.75-2.13). Tocilizumab dose was 8mg/kg/month, except for 1 patient receiving 6mg/kg/2 weeks. The median DAS-CRP decreased from 5,10(1.6-5.3) (n=6) at start to 2,25(1.3-2.9) (n=6) after six months . At 12 months, median DAS-CRP was 1,9(1.5-6.1)(n=3). Median ESR/CRP decreased from 35(15-87)(n=6)/69,2(9.1-292.6)mg/l(n=8) at start to2(1-7)/1,2(0.6-1.4)mg/l(n=7) at six months . At 1 year median ESR/CRP were 3,5(1-13)(n=4)/0,6(0.6-23.1)mg/l(n=5). A decrease/complete stop of steroids was observed in 6 patients. Atypical rash was seen in one patient, EBV-induced MAS occurred in 1 patient after 7 yrs of Tocilizumab.Our report on young adults with persistingly active so-JIA treated with Tocilizumab shows a fast, steep and persistent decrease in disease activity up to at least 1 year, demonstrating the efficacy of IL6-receptor antagonism also in patients with long-standing refractory so-JIA.


A-steroid  scaffold starting from Grundmann’s ketone using two different pyridine annulation protocols. The biological evaluation of the pyridine and pyridinium products revealed that these compounds unexpectedly do not interfere with ergosterol and cholesterol biosynthesis. The pyridinium compound 6 showed significant antimicrobial and cytotoxic activities which are most likely due to its detergent-like structure.This paper describes a convenient approach to the 7-aza-des- In glucocorticoids, introduction of halogen substituents (mostly fluorine), additional double bonds, and oxygen-containing groups  resulted in significant enhancement of activity and selectivity [Steroids are of outstanding significance for human metabolism by acting as hormones  or as an essential part of cell membranes (cholesterol). In fungi and protozoa, ergosterol is an indispensible part of cell membranes, and inhibition of ergosterol biosynthesis is one of the central targets of antifungal and antiprotozoal chemotherapy. Highly active drugs have been designed in the past by introducing new structural motifs into the tetracyclic sterol scaffold by either semisynthesis starting from natural sterols, or by ectivity , whereasectivity , 3. Azasectivity , whereasectivity . Recentlectivity . 4-Azastectivity , and latectivity . Steroidectivity .1), covering the rings C+D as well as the aliphatic side chain of steroids, and containing amino (A) [B) [trans-dinitrile C shows moderate cytotoxicity, and its cis-isomer is inactive [A-analogue showed separation of androgenic and anabolic activities [In recent investigations we demonstrated that secosteroids, derived from Grundmann’s ketone (mino (A)  or amino (A) [B) , are potinactive  Fig. 1)1), covertivities .A-steroid in which ring B of the steroid backbone is replaced by a pyridine ring. The nitrogen atom is located at a position representing C-7 of steroids, and thus we expected that this azasteroid or its quaternary N-methyl derivative might interact with ergosterol biosynthesis in fungi or cholesterol biosynthesis in human cells by mimicking one of the numerous carbocationic HEIs [Here we report on the synthesis of a novel aza-des-nic HEIs , 13 of t1) [3), by instead using our improved workup procedure   was obta pentane ). For th1 was first converted to N,N-dimethylhydrazone 2 by condensation with N,N-dimethylhydrazine in ethanol, and significant conversion was only obtained upon microwave irradiation. In the next step, 2 was converted to its kinetic lithium enolate with lithium diisopropylamide (LDA), followed by trapping with 2-(2-bromoethyl)-1,3-dioxolane to give regioselectively the double-masked 1,5-dicarbonyl compound 3. The absolute configuration at the newly generated stereo-center at C-5 could not be determined by NMR, but was of minor interest, since it was to be destroyed in the next step anyway. Finally, refluxing intermediate 3 in glacial acetic acid under microwave irradiation produced the annulated pyridine 4 in a 29% yield. The mechanism of this cyclization comprises in situ opening of the acetal and extrusion of dimethylamine [4 in a 5.3% overall yield in three steps from Grundmann’s ketone (1). The structure of 4 was confirmed by the typical 1H- and 13C-NMR resonances of a 2,3-disubstituted pyridine ring and fitting MS data.In the style of Gladiali’s pyridoannulation protocol , ketone hylamine . This prO-allyl ether. Grundmann’s ketone (1) was easily converted into the oxime ether 5 by condensation with O-allylhydroxylamine. The thermolysis of 5 to yield the target pyridine 4 turned out to be the critical step in this approach. Heating neat 5 in a sealed glass tube at 180 °C, as described in [5 under air and pressure in a sealed tube led to the conversion to the annulated pyridine 4. Careful optimization of the reaction conditions led to the conclusion that high temperature (220 °C) combined with a short reaction time (5 min) gives the best yield (29%). With this protocol, pyridine 4 was obtained in a 23% overall yield in two steps from Grundmann’s ketone (1).Due to the poor overall yield of the approach described above, we also explored the pyridoannulation protocol of Koyama  involvinribed in , did not4 is not likely to be protonated to a significant extent under physiological conditions (pH 7.4). But a positive charge at the nitrogen would be a prerequisite for mimicking cationic HEIs in sterol biosynthesis. In order to introduce a positive charge into the molecule, we converted 4 into the N-methylpyridinium iodide 6 by reaction with methyl iodide.Due to its moderate basicity, pyridine 4 and 6 were tested in an agar diffusion assay against a panel of bacteria and fungi , whereas the pyridinium salt 6 was found to exhibit very strong cytotoxicity (IC50 = 0.8 μM).The cytotoxicity of the compounds was determined in a MTT assay on HL-60 cells (human leukemia cell line) using the method of Mosmann . The refYarrowia lipolytica[4 and 6 were devoid of effects on sterol biosynthesis in both assays. Analysis of the sterol patterns after appropriate times of incubation showed that the sterol patterns of the cell lines were unchanged.Finally, both compounds were subjected to the assays for the detection of enzyme inhibition in the post-squalene part of cholesterol biosynthesis (on the human cell line HL-60 ) and ergipolytica), which A-steroids, which were intended to inhibit enzymes in ergosterol or cholesterol biosynthesis. Surprisingly, pyridine 4 as well as its N-methylpyridinium analogue 6 did not interfere at all with sterol biosynthesis. In a test for antimicrobial activities, the pyridine derivative 4 was inactive. In contrast, the pyridinium salt 6 showed an activity pattern quite similar to the one of cetylpyridinium chloride, but its activity was significantly higher compared to this reference drug. This allows the conclusion that the antimicrobial activity, and probably also the significant cytotoxic activity against a human cancer cell line, of 6 are most likely due to its detergent-like structure. The sterol-like partial structure of 6 might support the incorporation of this compound into cell membranes and concomitant disturbance of membrane integrity, thus leading to the observed effects.In conclusion, we worked out a convenient approach to 7-aza-des-4 (300 eV); NMR: Jeol GSX 400 ; melting points: Büchi Melting Point B-540 (not corrected); flash column chromatography (FCC): silica gel 60 ; microwave-assisted reactions were performed in a CEM Discovery reactor .Mass spectra: Hewlett Packard 5989 A, EI at 70 eV, chemical ionisation (CI) with CH1) and 3.44 mL (45.4 mmol) of N,N-dimethylhydrazine in 30 mL EtOH was heated under microwave irradiation (150 W) for 4 h, then treated with 50 mL of hydrochloric acid (10%), and extracted with CH2Cl2 (3 × 50 mL). The combined organic layers were dried over MgSO4 and the solvent was evaporated. The residue was purified by FCC (isohexane/ethyl acetate 4:1) to give 1.62 g (47%) of 2 as a pale yellow oil. 1H-NMR (CDCl3): δ (ppm) = 2.36 , 2.32–2.20 , 2.15–0.97 , 0.96 , 0.89–0.84 . 13C-NMR (CDCl3): δ (ppm) = 170.57 (C-4), 54.96 (C-3a), 53.40 (C-1), 46.33 (C-7a), 44.93 (C-5), 39.84 (N(CH3)2), 38.39 (C-6), 36.39 (C-7), 33.38 (C-3), 32.83 (C-1’), 27.01 (C-5’), 26.39 (C-2), 23.79 (C-2’), 22.83 (7a-CH3), 21.78 (5’-CH3), 21.49 (C-6’), 21.06 (C-3’), 19.94 (C-4’), 18.71 (1’-CH3). CI-MS m/z (rel. int.): 307 , 278 (18). HR-MS (EI): calcd.: 306.3035, found: 306.3002.A solution of 3.00 g (11.3 mmol) Grundmann’s ketone  of dimethylhydrazone 2 in 30 mL anhydrous THF was added dropwise with stirring. After 2 h at 0 °C, the mixture was cooled to −78 °C, and 0.62 mL (5.28 mmol) 2-(2-bromoethyl)-1,3-dioxolane were added slowly. After stirring at −78 °C for 1 h, the mixture was allowed to warm up to room temp. and quenched with 40 mL of water. The mixture was extracted with diethyl ether (3 × 50 mL). The combined organic layers were dried over MgSO4 and the solvent was evaporated. The residue was purified by FCC (isohexane/ethyl acetate 7:3) to give 848 mg (39%) of 3 as a pale yellow oil. 1H-NMR (CDCl3): δ (ppm) = 4.83 , 3.99–3.91 , 3.88–3.79 , 2.50–2.43 2), 2.41 , 2.31–2.19 , 2.14–2.02 , 1.95–1.83 , 1.81–1.04 , 1.02 , 0.93 , 0.88–0.83 . 13C-NMR (CDCl3): δ (ppm) = 174.90 (C-4), 104.23 (C-3”), 64.87 , 64.84 , 58.79 (C-3a), 54.43 (C-1), 49.15 (C-7a), 48.42 (C-5), 39.84 (N(CH3)2), 39.38 (C-6), 36.57 (C-7), 35.44 (C-3), 34.17 (C-1’), 31.38 (C-5’), 28.27 (C-2), 27.96 (C-2’), 27.54 (C-2”), 26.01 (C-1”), 24.17 (7a-CH3), 22.78 (5’-CH3), 22.51 (C-6’), 22.01 (C-3’), 21.57 (C-4’), 19.02 (1’-CH3). CI-MS m/z (rel. int.): 407 . HR-MS (EI): calcd.: 406.3559, found: 406.3185.3.00 mL (5.28 mmol) of LDA solution (1.76 M in THF) was added to 20 mL of anhydrous THF under N3 in 5 mL glacial acetic acid was refluxed under microwave irradiation (300 W) for 3 h, then neutralized carefully with saturated Na2CO3 solution, and extracted with CH2Cl2 (3 × 50 mL). The combined organic layers were dried over MgSO4 and the solvent was evaporated. The residue was purified by FCC (isohexane/ethyl acetate 9:1) to give 182 mg (29%) of 4 as a light brown oil.A solution of 848 mg (2.08 mmol) of O-allyl oxime 5 was heated in a closed vessel without solvent under microwave irradiation  for 5 min. The product was purified by FCC (isohexane/ethyl acetate 9:1) to give 230 mg (29%) of 4 as a light brown oil.860 mg (2.69 mmol) of the 1H-NMR (CDCl3): δ (ppm) = 8.36 , 7.31 , 6.95 , 2.79 , 2.67–2.65 , 2.29–2.22 , 1.79–1.73 , 1.49–1.32 , 1.16–1.01 , 0.94 , 0.84 , 0.82 . 13C-NMR (CDCl3): δ (ppm) = 161.44 (C-9b), 147.26 (C-2), 135.48 (C-4), 131.67 (C-4a), 120.49 (C-3), 54.90 (C-9a), 53.27 (C-7), 42.52 (C-6a), 39.56 (C-4’), 35.71 (C-2’), 35.35 (C-8), 33.83 (C-1’), 31.73 (C-6) 28.88 (C-9), 28.06 (C-5’), 25.97 (C-5), 24.44 (C-3’), 23.40 (6a-CH3), 22.89 (C-6’), 22.64 (5’-CH3) 19.72 (1’-CH3). CI-MS m/z (rel. int.): 300 , 159 (12). HR-MS (EI): calcd.: 299.2613, found: 299.2608.1), 2.15 g (19.6 mmol) of O-allylhydroxylamine hydrochloride, 1.34 g (22.6 mmol) of sodium acetate, and 2.40 g (22.6 mmol) of Na2CO3 were dispersed in 25 mL ethanol. This mixture was refluxed for 6 h, stirred for another 12 h at ambient temp., and then evaporated to dryness. The residue was taken up in 60 mL CH2Cl2 and washed with 3 × 50 mL hydrochloric acid (1.5%). The organic layer was dried over MgSO4 and the solvent was evaporated. The residue was purified by FCC (isohexane/ethyl acetate 47:3) to give 2.93 g (80%) of 5 as a colourless oil. 1H-NMR (CDCl3): δ (ppm) = 6.02–5.95 , 5.29–5.15 , 4.52 , 2.30–2.25 , 2.06–1.99 , 1.88–1.82 , 1.62–1.46 , 1.33–1.24 , 1.12–1.09 , 0.96 , 0.89–0.85 . 13C-NMR (CDCl3): δ (ppm) = 160.63 (C-4), 134.72 (C-2”), 116.91 (C-3”), 74.19 (C-1”), 56.08 (C-3a), 52.68 (C-1), 46.59 (C-7a), 39.54 (C-5), 37.64 (C-6), 36.17 (C-7), 35.16 (C-3), 34.04 (C-1’), 28.03 (C-5’), 24.85 (C-2), 23.90 (C-2’), 23.13 (7a-CH3), 23.08 (5’-CH3), 22.65 (C-6’), 22.08 (C-3’), 19.65 (C-4’), 18.90 (1’-CH3). CI-MS m/z (rel. int.): 320 , 262 (50). HR-MS (EI): calcd.: 319.2875, found: 319.2867.3.03 g (11.5 mmol) of Grundmann’s ketone  of methyl iodide (caution! highly toxic agent!). The mixture was stirred in a well-ventilated hood for 16 h at ambient temp., then the precipitate was collected by filtration and recrystallized from ethanol to give 99 mg (53%) of 6 as white crystals. M.p. 218 °C. 1H-NMR (CDCl3): δ (ppm) = 9.43 , 8.17 , 7.83 , 4.56 , 3.21 , 2.89 , 2.48–2.38 , 2.08 , 2.00–1.90 , 1.67–1.35 , 1.23–1.10 , 0.99 , 0.86 . 13C-NMR (CDCl3): δ (ppm) = 157.70 (C-9b), 144.96 (C-2), 143.61 (C-4), 138.13 (C-4a), 123.65 (C-3), 51.10 (C-7), 48.58 (C-9a), 45.76 (1-CH3), 43.42 (C-6a), 38.35 (C-4’), 34.39 (C-2’), 33.34 (C-8), 31.91 (C-1’), 30.83 (C-6), 27.00 (C-5’), 26.87 (C-9), 25.07 (C-5), 24.16 (6a-CH3), 23.32 (C-3’), 21.77 (C-6’), 21.53 (5’-CH3), 18.81 (1’-CH3). CI-MS m/z (rel. int.): 314 , 300 (100). HR-MS (EI): calcd.: 313.2769, found: 313.2726.126 mg (0.42 mmol) of pyridine The assay was performed as described in detail previously  using thThe assay originally worked out by Mosmann  was perfThe qualitative assay was performed as described in detail previously  using HLYarrowia lipolytica as the test strain.The qualitative assay was performed as described in detail recently  using th


N-phenylpyrazolyl-N-glycinyl-hydrazone derivatives that were designed as novel prototypes of p38 mitogen-activated protein kinase (MAPK) inhibitors. All of the novel synthesized compounds described in this study were evaluated for their in vitro capacity to inhibit tumor necrosis factor α (TNF-α production in cultured macrophages) and in vitro MAPK p38α inhibition. The two most active anti-TNF-α derivatives, (E)-2-(3-tert-butyl-1-phenyl-1H-pyrazol-5-ylamino)-N’-((4-(2-morpholinoethoxy)naphthalen-1-yl)methylene)acetohydrazide (4a) and (E)-2-(3-tert-butyl-1-phenyl-1H-pyrazol-5-ylamino)-N’-(4-chlorobenzylidene)acetohydrazide (4f), were evaluated to determine their in vivo anti-hyperalgesic profiles in carrageenan-induced thermal hypernociception model in rats. Both compounds showed anti-inflammatory and antinociceptive properties comparable to SB-203580 used as a standard drug, by oral route at a dose of 100 µmol/kg. This bioprofile is correlated with the ability of NAH derivatives (4a) and (4f) suppressing TNF-α levels in vivo by 57.3 and 55.8%, respectively.Herein, we describe the synthesis and pharmacological evaluation of novel  For the proposed derivatives (4a–g), we investigated the replacement of the urea subunit of BIRB-796 (3) by a N-acylhydrazone unit N-phenyl-pyrazole nucleus through an NHCH2 spacer  N-phenyl-pyrazolyl-N-glycinyl hydrazone derivatives 4a–g  may exist as Z/E geometrical isomers about the C = N double bond and syn/anti amide conformers E) at the imine double bond -N’-(propan-2-ylidene)acetohydrazide (9), which was obtained by a reaction of the previously obtained hydrazide 8 with acetone , which showed a 90% yield. The alkylation of the monomethylamine derivative 12 with ethyl 2-bromoacetate in ethanol and sodium carbonate under reflux provided the corresponding ethyl ester 13 with a 60% yield. Hydrazinolysis of the ester 13 followed by condensation of the corresponding hydrazide 14 with benzaldehyde under acid catalysis resulted in the desired N-acylhydrazone derivative 15 with a 60% yield.To evaluate whether the amino spacer exerts some influence on the stabilization of the conformational isomers in solution, we inserted a methyl group into the amino spacer, as described in 1H-NMR spectrum of the N-methyl derivative 15 showed the same pattern of duplicity that was observed for the other synthesized N-acylhydrazones 4a-g. We were also able to observe conformational isomers of the amide unit of compound 15, suggesting that the amino spacer does not participate as a hydrogen bond donor in the stabilization of the conformational isomers in solution , 4a  and 4b  showed the most potent inhibitory effects. Compared with the unsubstituted phenyl ring compound 4g (cLogP = 5.3), the inhibitory potency increased when lipophilic groups [para-chloro for 4f (cLogP = 6.1), naphthyl for 4c (cLogP = 6.6), 4-hydroxynaphthyl for 4b (cLogP = 6.3) and 4-ethyl)morpholine for 4a (cLogP = 6.0) were added. These results indicate that the differences in hydrophobicity of the imine-attached framework play an important role for the in vitro anti-TNF-α activity of N-phenylpyrazolyl-N-glycinyl-hydrazone derivatives.We initially investigated the capacity of our N-acylhydrazone derivatives 4a–g were designed based on the p38α MAPK inhibitor BIRB-796 (3), they were all evaluated for their in vitro capacity to inhibit p38α MAPK activity Because the novel in vivo anti-inflammatory and antinociceptive profile of the NAH derivatives 4a, 4b, 4c and 4f, we employed the carrageenan-induced thermal hypernociception model p.o.) was used as a standard. in vitro  suppressed the elevation of tissue TNF-α level by 57.3 and 55.8%, respectively .in vivo profile of derivative 4a since both compounds, 4a and 4f, have the same theoretical lipophilicity, i.e. cLogP = 6.0 and 6.1, respectively. Considering that an adequate balance between the lipophilicity and aqueous solubility is essential for a good oral absorption of a drug candidate, we decided to determine experimentally the solubility of compounds 4a and 4f in buffer solutions of pH 6.4 and 7.4 , due to the partial ionization of its basic morpholine subunit. These solubility results allow us to rationalize that the improved in vivo activity of compound 4a is due to its better water solubility, which could favor its gastrointestinal absorption.About the best anti-hypernociceptive profile of the compound 4a in comparison to derivative 4f, we decided to investigate the molecular reasons associated with a probable distinction in the respective pharmacokinetic behaviors. The physicochemical property cLog P doesn’t seems to explain the better  and 7.4 . The derin vitro metabolic stability of derivatives 4a and 4f when placed in contact with preparations of liver and plasma of rats. The two NAH derivatives were resistant to oxidative microsomal metabolism, but the derivative 4a was about four times more resistant than derived 4f to plasma degradation, as described in in vivo pharmacological profile shown by the NAH derivative 4a when given orally.Moreover, we also evaluated the N-phenylpyrazolyl-N-glycinyl-hydrazone derivatives that were designed as novel prototypes of p38 MAPK inhibitors. All novel synthesized compounds described were evaluated for their in vitro capacity to inhibit TNF-α production in cultured macrophages and their in vitro p38α MAPK inhibition. The two most active anti-TNF-α derivatives were (E)-2-(3-tert-butyl-1-phenyl-1H-pyrazol-5-ylamino)-N’-((4-(2-morpholinoethoxy)naphthalen-1-yl)methylene)acetohydrazide (4a) and (E)-2-(3-tert-butyl-1-phenyl-1H-pyrazol-5-ylamino)-N’-(4-chlorobenzylidene)acetohydrazide (4f). These two compounds were evaluated for their in vivo anti-hypenociceptive profiles. Both compounds showed anti-inflammatory and anti-hypenociceptive properties that were comparable to SB-203580 (1), which was used as a standard.This study describes the synthesis and pharmacological evaluation of novel J) are given in Hz. Infrared (IR) spectra were obtained using a Nicolet Magna IR 760 spectrometer. Samples were examined as potassium bromide (KBr) disks. Elemental analyses were carried out on a Thermo Scientific Flash EA 1112 Series CHN-Analyzer. Melting points were determined using a Quimis instrument and are uncorrected and the compounds 4a-f had their melting points determined using a differential scanning calorimeter (Shimadzu DSC-60). Column chromatography purifications were performed using silica gel Merck 230–400 mesh. All described products showed 1H and 13C NMR spectra according to the assigned structures.Reactions were routinely monitored by thin-layer chromatography (TLC) in silica gel (F245 Merck plates) and the products visualized with ultraviolet lamp (254 and 365 nm). NMR spectra were recorded on a 200/50 MHz Bruker DPX-200, 250/62.5 MHz Bruker DPX-250, 400/100 MHz Varian 400-Mr, 300/75 MHz Varian Unity-300 spectrometer at room temperature. Peak positions are given in parts per million (δ) from tetramethylsilane as internal standard, and coupling constant values  or Kromasil 100-5C18 (4.6 mm × 250 mm) and a Shimadzu SPD-M20A detector at 254 nm wavelength. The solvent systems for HPLC purity analyses was acetonitrile:phosphate buffer solution pH7 = 70∶30. The isocratic HPLC mode was used, and the flow rate was 1.0 ml/min.1H NMR  δ = 7.59 ; 7.44 ; 7.26 ; 5.39 ; 5.19 ; 1.22 3). 13C NMR  δ = 160.7 (C3-pyrazole), 146.9 (C5-pyrazole), 139.6 (C1-phenyl), 128.9 (C3 and C5-phenyl), 125.5 (C4-phenyl), 122.4 (C2 and C6-phenyl), 87.0 (CH-pyrazole), 31.8 (C(CH3)3), 30.2 (3xCH3). IR (KBr): 3412, 3284, 3146, 2961, 1597, 1556, 1507, 1382, 1243, 988, 696 cm−1.A round-bottomed flask charged with phenylhydrazine , 4,4-dimethyl-3-oxo-pentanenitrile  and toluene (3 ml) was stirred and heated at reflux for 24 hours. The resulting mixture was concentrated on a rotary evaporator and the residue was purified by column chromatography on silica gel , to yield the title compound  as a white solid (mp: 50–52°C). 2SO4, filtered, and concentrated. The brown residue was purified by silica gel chromatography hexane/ethyl acetate (gradient) to give the title compound  as a brown oil. 1H NMR  δ = 7.57-7.44 ; 7.31 ; 5.71 ; 5.37 ; 4.12 ; 3.81 ; 1.21-1.19 3 and CH3).To a solution of amine 5  in toluene (3.0 mL) and trietylamine (0.1 mL), was added ethyl 2-bromoacetate . The resulting mixture was stirred and heated at reflux for 4 hours. The residue was partitioned between water and ethyl acetate. The combined organic phases were dried over Na1H NMR  δ = 9.17 ; 7.58 ; 7.47 ; 7.31 ; 5.56 ; 5.35 ; 4.25 ; 3.57 ; 1,37 3). 13C NMR  δ = 168.9 (CO), 160.9 (C3-pyrazole), 148.1 (C5-pyrazole), 139,2 (C1-phenyl), 129.1 (C3 and C5-phenyl), 126.1 (C4-phenyl), 123.2 (C2 and C6-phenyl), 84,4 (CH-pyrazole), 47.5 (CH2), 31.2 (C(CH3)3), 30.2 (CH3). IR (NaCl) 3319, 2960, 1669, 1596, 1567, 1520, 1373, 1247, 991, 764 cm−1.A round-bottomed flask charged with 600 mg (2 mmol) of ester (7), hydrazine hydrate 100% (20 eq) and ethanol (5 mL) was stirred and heated at reflux for 2 hours. To the resulting mixture was added water and the aqueous phase was extracted with ethyl acetate to give the title compound  as a yellow oil. In a round flask containing hydrazide 8 (1.6 mmol) in ethanol (10 mL), was added aldehyde  and catalytic concentrated hydrochloric acid. The mixture was stirred for about 2 hours at room temperature. At the end of the reaction the volume of ethanol was reduced, saturated solution of sodium bicarbonate and ice were added to the reaction. The precipitate formed was filtered, or the mixture was extracted with dichloromethane.(Yield = 75%) (mp. 180°C).1H NMR  δ = 11.37 and 11.28 ; 8.91 and 8.72 ; 8.72 and 8.53 ; 8.26 ; 7.77 ; 7.40-7.60 ; 7.33 ; 7.04 ; 5.64 and 5.35 ; 5,45 ; 4.33 ; 4.24 and 3.78 ; 3.56 ; 2.88 ; 2.56 ; 1.23 3) [13C NMR  δ = 170.6 and 166.1 (C = O); 161.0 (C3-pyrazol); 155.7 (C4-naphthyl); 148.2 (C5-pyrazol); 147.7 and 144.3 (N = CH); 139.2 (C1-phenyl); 131.0 (C9-naphthyl); 129.2 (ArCH); 127.8 (ArCH); 126.2 (ArCH); 125.7 (ArCH); 125.2 (ArCH); 124.6 (Ar CH); 124.0 (ArCH); 123.1 (C1-nafhtyl); 122.3 (ArCH); 121.7 (C10-naphthyl); 105.2 (ArCH); 84.5 (CH-pyrazole); 66.4 (OCH2-ethoxyl); 66.2 (CH2-morfolyn); 56.9 (CH2-ethoxyl); 53.6 (CH2-morpholine); 48.0 and 46.5 (CH2); 31.9 (C(CH3)3); 30.2 (3xCH3) [−1. % purity >98% by HPLC. Anal. Calcd for C32H38N6O3H2O: C 67.11; H 7.04; N 14.67. Found: C 67.16; H 7.05; N 14.74. (CH3)3) . 13C NMR (3xCH3) . IR (KBr(Yield = 70%) (mp. 270°C).1H NMR  δ = 11.39 and 11.28 ; 10.78 ; 8.93 and 8.72 ; 8.73 and 8.52 ; 8.25 ; 7.74-7.36 ; 5.74 and 5.42 ; 5.49 and 5.46 ; 4.25 and 3.80 ; 1.23 3) [13C NMR  δ = 171.1 and 166.0 (CO); 161.6 (C3-pyrazole); 156.1 (C-OH); 148.8 (N = CH); 145.2 (C5-pyrazole); 139.8 (C1-phenyl); 132.04 (C9-naphthyl); 130.4 (C2-naphthyl); 129.8 (C3 and C5-phenyl); 128.2 (C5-naphthyl); 126.7 (C4 phenyl); 125.6 (C1-naphthyl); 125.3 (C7-naphthyl); 124.4 (C2 and C6-phenyl); 123.8 (C10-naphthyl); 123.3 (C6-naphthyl); 120.7 (C8-naphthyl); 108.6 (C3-naphthyl); 85.0 (CH-pyrazole); 47.0 (CH2); 32.5 (C(CH3)3); 30.8 (CH3)3) [−1. % purity >98% by HPLC. Anal. Calcd for C26H27N5O2: C 70.73; H 6.16; N 15.86. Found: C 70.25; H 6.20; N 15.52. (CH3)3) . 13C NMR (CH3)3) . IR (KBr(Yield = 90%) (mp. 140°C).1H NMR  δ = 11.60 and 11.53 ; 8.90 and 8.69 ; 8.84 and 8.64 ; 8.03-7.88 ; 7.63-7.49 ; 5.78 and 5.45 ; 5.50 ; 4.30 e 3.84 ; 1.23 3) [13C NMR  δ = 171.5 and 167.1 (CO); 161.6 (C3-pyrazole); 148.7 (C5-pyrazol); 147.6 and 144.1 (HC = N); 139.8 (C1-phenyl); 134.1 (C1-naphthyl); 131.0 (C10-naphthyl); 130.6 (C4-naphthyl); 129.8 (C3 e C5-phenyl); 129.4 (C5-naphthyl); 128.5 (C4-phenyl); 127.9 (C2-naphthyl); 126.8 (C7-naphthyl); 126.1 (C2 and C6-phenyl); 124.8 (C6-naphthyl); 124.2 (C9-naphthyl); 123.7 (C8-naphthyl); 85.1 (CH-pyrazole); 46.6 and 47.0 (CH2); 32.49 (C(CH3)3); 30.75 ((CH3)3) [−1. Anal. Calcd for C26H27N5O: C 73.39; H 6.40; N 16.46. Found: C 73.07; H 6.41; N 16.17. (CH3)3) . 13C NMR((CH3)3) . IR (KBr(Yield = 60%) (mp. 158°C).1H NMR  δ = 11.71 and 11.56 ; 8.10 and 7.90 ; 7.65-7.49 ; 7.30 ; 7.00 ; 5.47 and 5.40 ; 4.14-3.60 ; 3.57 ; 2.69 ; 2.49 ; 1.23 3) [13C NMR  δ = 171.3 and 166.7 (C = O); 161.6 (C3-pyrazole); 160.6 (C4-phenyl); 148.7 and 147.5 (N = CH); 144.3 (C5-pyrazole); 139.8 (C1-phenylpyrazol); 129.8 (C3 and C5-phenylpyrazole); 129.0 (C2 and C6-phenylpirazole); 127.3 (C1-phenyl); 126.7 (C4-phenylpyrazole); 123.6 (C2 and C6-phenyl); 115.4 (C3 and C5-phenyl); 85.0 (CH-pyrazole); 66.7 (CH2-morfolyn); 66.0 (OCH2-ethoxyl); 57.5 (CH2-ethoxyl); 54.2 (CH2-morfolyn); 46.8 (CH2); 32.5 (C(CH3)3); 30.8 (3xCH3) [−1. Anal. Calcd for C28H36N6O3: C 66.64; H 7.19; N 16.65. Found: C 66.10; H 7.13; N 16.51. (CH3)3) . 13C NMR (3xCH3) . IR (KBr(Yield = 70%) (mp. 280°C).1H NMR  δ = 11.36 and 11.23 ; 9.93 ; 8.14 and 7.91 ; 7.64-7.46 ; 7.36 ; 6.83 ; 5.70 and 5.37 ; 5.47 and 5.41 ; 4.16 and 3.74 ; 1.23 3) [13C NMR  δ = 171.4 and 166.6 (CO); 161.6 (C3-pyrazole); 159.9 (C4-OH); 148.7 and 144.7 (HC = N); 147.9 (C5-pyrazole); 139.8 (C1-phenyl); 129.8 (C2 and C6-phenol); 129.3 (C3 and C5-phenyl); 126.7 (C4-phenyl); 125.7 (C1-phenyl); 123.7 (C2 and C6-phenyl); 116.2 (C3 and C5-phenol); 85.1 and 85.0 (CH-pyrazole); 48.5 and 46.8 (CH2); 31.2 (C(CH3)3); 30.8 ((CH3)3) [−1. Anal. Calcd for C22H24N5O2: C 67,50; H 6,44; N 17,89. Found: C 67,27; H 6,33; N 17,88. (CH3)3) . 13C NMR((CH3)3) . IR  (mp. 193°C).1H NMR  δ = 11.62 and 11.60 ; 8.27 and 8.01 ; 7.75-7.30 ; 5.53 and 5.46 ; 4.21 and 3.78 ; 1.23 3) [13C NMR  δ = 171.6 (CO); 161.50 (C3-pyrazole); 148.9 (C5-pyrazole); 143.1 (HC = N); 139.1 (C1-phenylpyrazole); 134.9 (C4-phenyl); 133.5 (C1-phenyl); 129.8 (2xCH-Ar); 129.4 (2xCH-Ar); 129.2 (2xCH-Ar); 127.0 (CH-Ar); 123.9 (2xCH-Ar); 85.3 (CH-pyrazole); 46.7 (CH2); 32.5 (C(CH3)3); 30.7 ((CH3)3). IR (KBr): 3374, 2950, 1687, 1593, 1511, 1397, 1284, 984, 757 cm−1. Anal. Calcd for C22H24ClN5O: C 64.46; H 5.90; N 17.09. Found: C 64.04; H 5.81; N 16.68. (CH3)3) . 13C NMR.1H NMR  δ = 11.49 and 11.38 ; 8.27 and 8.02 ; 7.60-7.30 ; 5.49 and 5.43 ; 4.20 and 3.77 ; 1.23 3) [13C NMR  δ = 172.9 and 166.3 (CO); 160.9 (C3-pyrazole); 148.0 (C5-pyrazole); 147.0 and 143.8 (CH = N); 139.2 (C1-phenylpyrazole); 133.9 (C1-phenyl); 129.8 (CH-Ar); 129.2 (2xCH-Ar); 128.7 (2xCH-Ar); 126.8 (2xCH-Ar); 126.0 (CH-Ar); 123.0 (2xCH-Ar); 84.4 (CH-pyrazole); 47.9 and 46.2 (CH2); 31.9 (C(CH3)3); 30.1 ((CH3)3) [−1. % purity >98% by HPLC. Anal. Calcd for C22H25N5O: C 70.38; H 6.71; N 18.65. Found: C 70.26; H 6.65; N 18.42. (CH3)3) . 13C NMR((CH3)3) . IR (KBr2CO3 (1.5 mmol). The mixture was stirred and maintained at 40°C. The reaction was monitored by TLC. After the reaction was complete, the acetone was evaporated on a rotatory evaporator, ethanol was added and then ice. The solid was filtered and washed with water to give compound 4h . 1H NMR  δ = 8.01 ; 7.78 ; 7.59 ; 7.51-7.44 ; 7.32 ; 5.47 ; 5.39 ; 4.34 ; 3.34 ; 1.22 3).Iodomethane (1.5mmol) was added dropwise to the magnetically stirred solution of 8g  in acetone 50 mL and Ktert-butyl-1-phenyl-1H-pyrazol-5-ylamino)acetohydrazide (8) in 60 mL of acetone was refluxed for 3 hours. Then half of the volume was evaporated on a rotatory evaporator and the residue was cooled in the refrigerator for 3 hours. The crystals which formed were filtered, dried and recrystallized from acetone . 1H NMR  δ = 10.33 and 10.04 ; 7.60-7.46 ; 7.32 ; 5.38 and 5.41 ; 5.59 and 5.29 ; 4,03 and 3.74 ; 1.93, 1.86 and 1.83 ; 1.23 3) [A solution of 1 mmol of 2-(3- (CH3)3) .N-(3-tert-butyl-1-phenyl-1H-pyrazol-5-yl)acetamide (10) was obtained as a powder  after concentration on a rotary evaporator. 1H NMR  δ = 9.83 ; 7.46-7.38 ; 6.29 ; 1.96 ; 1.28 3). 13C NMR  δ = 168.8 (CO); 160.7 (C3-pyrazole); 138.9 (C1-phenyl); 136.0 (C5-pyrazole); 129.0 (C3 and C5-phenyl); 126.9 (C4-phenyl); 123.3 (C2 and C6-phenyl); 99.0 (C4-pyrazole); 32.0 (C(CH3)3); 30.1((CH3)3); 22.8 (COCH3). IR (KBr): 3255, 3218, 3058, 2946, 1671, 1554, 1501, 1373, 1399, 1277, 1235, 767, 687 cm−1.A solution of 0.3g (3.66 mmol) of sodium acetate anhydrous in 1.0 mL (17.5 mmol) of glacial acetic acid was prepared. 0.85g of amine 5 (3.95 mmol) was slowly added, and then 1.0 ml (10.6 mmol) of acetic anhydride was also added. The reaction mixture was stirred for about 3 h, at room temperature, when the end of reaction was observed by TLC. Water was added and the mixture was extracted with ethyl acetate. The 4Cl and ethyl acetate. The organic layer was separated and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with water, dried over Na2SO4, and concentrated to give the N-(3-tert-butyl-1-phenyl-1H-pyrazol-5-yl)-N-methylacetamide (11) as a solid in : mp: 108–110°C. 1H NMR  δ = 7.56-7.39 ; 6.48 ; 3.01 (NCH3COCH3); 1.67 (NCH3COCH3); 1.30 3). 13C NMR  δ = 169.7 (CO); 161.5 (C3-pyrazol); 141.6 (C5-pyrazole); 138.3 (C1-phenyl); 129.4 (C3 and C5-phenyl); 127.5 (C4-phenyl); 122.8 (C2 and C6-phenyl); 101.0 (C4-pyrazole); 35.8 (NCH3COCH3); 35.8((C(CH3)3); 30.0 ((CH3)3); 21.4 (NCH3COCH3). IR (KBr): 2969, 2867, 1677, 1594, 1566, 1502, 1366, 1334, 758, 688, 655 cm−1.Sodium hydride  was added to the magnetically stirred acetamide 10  in anhydrous THF (5 mL), and iodomethane  was added dropwise to the mixture, which was maintained below 5°C for 0.5 h, and stirred at room temperature. The reaction was monitored by TLC. After the reaction was complete, the reaction mixture was partitioned between saturated aqueous NHN-(3-tert-butyl-1-phenyl-1H-pyrazol-5-yl)-N-methylacetamide (11) (1 mmol) in ethylene glycol (0.75 ml). The reaction mixture was heated to reflux and the reaction was monitored by TLC. When the reaction was completed, saturated sodium bicarbonate solution was added and the reaction mixture was partitioned between water and ethyl acetate. The combined organic layers were washed with water, dried over Na2SO4, and concentrated to give 3-tert-butyl-N-methyl-1-phenyl-1H-pyrazol-5-amine (12) as oil . 1H NMR  δ = 7.55-7.23 ; 5.42 ; 5.33 (NH); 2.65 ; 1.24 3). 13C NMR  δ = 161.1 (C3-pyrazole); 150.4 (C5-pyrazole); 139.6 (C1-phenyl); 129.1 (C3 and C5-phenyl); 125.8 (C4-phenyl); 122.8 (C2 and C6-phenyl); 84.2 (C4-pyrazole); 32.1 (NCH3); 32.0((C(CH3)3); 30.2 ((CH3)3). IR (NaCl) 3370, 2958, 1594, 1570, 1520, 1498, 1375, 1244, 760, 697 cm−1.Concentrated HCl (0.25 ml) was added to a stirred solution of tert-butyl-N-methyl-1-phenyl-1H-pyrazol-5-amine (12)  in EtOH (30 ml). The reaction mixture was stirred at room temperature for 30 min before Na2CO3  was added and refluxed overnight. After cooling to room temperature, the solution was concentrated by evaporation and the residue was partitioned between water and ethyl acetate. The combined organic phases were washed with water and saturated NaCl solution, dried over Na2SO4, filtered, and concentrated. The brown residue was purified by silica gel chromatography hexane/ethyl acetate (gradient) to give the title compound  as a brown oil. 1H NMR  δ = 7.69 ; 7.44 ; 5.86 ; 3.68 ; 3.48 ; 2.64 ; 1.34 3); 0.95 . 13C NMR  δ = 169.4 (CO); 160.5 (C3-pyrazol); 150.8 (C5-pyrazol); 140.0 (C1-phenyl); 128.9 (C3 and C5-phenyl); 126.2 (C4-phenyl); 122.6 (C2 and C6-phenyl); 91.6 (C4-pyrazole); 65.9 (CH2 ester); 60.0 (CH2 spacer); 41.1 (NCH3); 31.9((C(CH3)3); 30.0 ((CH3)3); 14.0 (CH3 ester). IR (NaCl): 2964, 2870, 1749, 1595, 1556, 1502, 1454, 1373, 1198, 1137, 1030, 765, 693 cm−1.Ethyl 2-bromoacetate  was added slowly at room temperature to a solution of 3-1H NMR  δ = 9.00 ; 7.71 ; 7.38 ; 7.24 ; 5.82 ; 4.18 ; 2.56 (NCH3); 1.19 3). 13C NMR  δ = 167.7 (CO), 160.5 (C3-pyrazole), 151.5 (C5-pyrazol), 140.0 (C1-phenyl), 128.9 (C3 and C5-phenyl), 126.1 (C4-phenyl), 122.7 (C2 and C6-phenyl), 91.7 (CH-pyrazole), 56.4 (CH2), 44.0 (NCH3), 32.0 (C(CH3)3), 30.2 (CH3). IR (NaCl): 3315, 2960, 2862, 1673, 1598, 1502, 1553, 1371, 1271, 1116, 1018, 767 cm-1.A round-bottomed flask charged with 472.5 mg (1.5 mmol) of ester (13), hydrazine hydrate 100% (20 eq) and ethanol (5 mL) was stirred and heated at reflux for 2 hours. To the resulting mixture was added water and the aqueous phase was extracted with dichloromethane to give the title compound  as a yellow oil after solvent concentration. 2SO4, filtered, and concentrated. The white solid obtained was washed with n-hexane and filtered under vacuum to give the title compound as a white solid : mp: 156–158°C. 1H NMR  δ = 11.34 ; 8.35 and 8.18 ; 7.89-7.20 ; 5.93 and 5.87 ; 4.06 and 3.61 ; 2.72 and 2.65 ;1.23 3). 13C NMR  δ = 170.1 and 165.0 (CO); 160.5 (C3-pyrazole); 151.4 (C5-pyrazole); 151.7 and 146.8 (N = CH); 143.1 (C1-phenylpyrazole); 140.2 and 140.0 (C1-phenyl); 134.1 and 134.0 (C4-phenyl); 130.1 (C4-phenylpyrazole); 128.8 and 128.7 (C3 and C5-phenylpyrazole); 127.0 and 126.7 (C3 and C5-phenyl); 126.2 and 126.0 (C2 and C6-phenylpyrazole); 122.9 and 122.6 (C2 and C6-phenyl); 91.9 and 91.3 (CH-pyrazole); 56.6 and 54.8 (CH2); 41.5; 32.0 (C(CH3)3); 30.1 ((CH3)3). IR (KBr): 3196, 3080, 2957, 2857, 1682, 1592, 1500, 1402, 1314, 1267, 759, 693 cm−1.In a round flask containing hydrazide 14 (0.93 mmol) in ethanol (5 ml), was added benzaldehyde  and catalytic concentrated hydrochloric acid. The mixture was stirred for about 2 hours at room temperature. At the end of the reaction the volume of ethanol was reduced, saturated solution of sodium bicarbonate and ice were added to the reaction and the mixture was extracted with dichloromethane. The combined organic phases were dried over NaAnimals were obtained from the LASSBio breeding unit . All animals were kept under standardized conditions, maintained in a 12-h light/dark cycle with water and food ad libitum until use. Animal experiments were performed according to the “Principles of Laboratory Animal Care and Use in Research” , based on international guidelines for the care and use of laboratory animals. All experiments were previously approved by the local ethics committee.2 atmophere. Then, macrophages were incubated with the vehicle or compounds and 1 hour later stimulated with LPS (100 ng/mL) for 24 hour when the supernatants were collected to evaluate TNF-α production by ELISA kit (BD Bioscience).BALBc mice were stimulated with thyoglicollate 3%  and 3 days later the peritoneal cavity were washed with RPMI 1640 and the peritoneal macrophages were plated onto 96-wells plate (30.000 cells/well) for 1 hour at 37°C in an humidified 5% COThe peritoneal macrophage were obtained and plated as described above. The cells were incubated with the vehicle or compounds for 20 hours when was added 20 µl of MTT -2,5-diphenyltetrazolium bromide) solution (5 mg/mL), followed by 4 hour of incubation. Then, the culture mediums were collected and the precipitates were solubilized in 200 µl of DMSO. The optical density was measured at 490 nm.Wistar rats of both sexes (150–200g) were used. The compounds were administered orally (100 µmol.5mL-1.kg-1) as a suspension in 5% arabic gum in saline (vehicle). Control animals received an equal volume of vehicle. One hour later, the animals were injected with either 0.1 ml of 1% carrageenan solution in saline or sterile saline (NaCl 0.9%), into the subplantar surface of one of the hind paws. The thermal hypernociception was determined using the modified hot-plate test. Rats are placed individually on a hot plate with the temperature adjusted to 51°C. The latency of the withdrawal response of the left hind paw is determined at 0, 30, 60, 120, 180, and 240 min post-challenge. The time of maximum permanence permitted on the hot surface is 20 s. Hypernociception to heat is defined as a decrease in withdrawal latency and calculated as follows: Δ paw withdrawal latency (s) =  – .The paw was homogenized 4 h after intraplantar injection of carrageenan, and the level of TNF-α in the supernatant was determined by ELISA.The solubility was evaluated after twenty-four hour agitation of 1 mg of test compound in 1 mL aqueous buffer (pH 6.4 and pH 7.4) at 37 °C followed by centrifugation and filtration for HPLC-UV analysis.2 (1.3 mM), NADP+ (0.4 mM), glucose-6-phosphate (3.5 mM), 0.5 U/mL glucose-6-phosphate dehydrogenase in a phosphate buffer  containing EDTA (1.5 mM) and the test compounds were added at final concentration of 50 µM with 0.25 mL of final volume. After the pre-warming of the mixture at 37 °C, the microsomal proteins were added to give a final protein concentration of 1 mg/mL. At the end of the incubation time the reaction was stopped by the addition of 375 µL of MeOH and 375 µL of CH3CN. The experiments were performed in duplicate. The samples were centrifuged and filtered for HPLC-UV analysis.The incubation was conducted at 37°C for 60 min. The experiments contains MgCl3CN. The experiments were performed in duplicate. The samples were centrifuged and filtered for HPLC-UV analysis.The rat plasma was obtained from blood by centrifugation and diluted in phosphate buffer (pH 7.4). The test compounds were added at final concentration of 50 µM with 0.25 mL of final volume and incubated at 37°C for 60 min under agitation. At the end of the incubation time the reaction was stopped by the addition of 375 µL of MeOH and 375 µL of CH4OH solution. The detection was carried out at 285 nm and 330 nm wavelength for compound 4f and 4a, respectively.The organic fraction was analyzed with the Shimadzu Prominence HPLC system  consisting of a vacuum degasser (DGU-20A5), a binary pump (LC-20AD), a autosampler (SIL-20A), UV/VIS Photodiode Array Detector (SPD-M20A) and fitted with a guard column (CLC G-ODS) and a Shimadzu  column (250 mm×4.6 mm i.d.) running at room temperature. Isocratic elution was performed with acetonitrile-water (40∶60 v/v), at a flow rate set at 1 mL/min. The mobile phase pH was adjusted to 8.0 with NHpost hoc test) for carrageenan-induced thermal hyperalgesia and Student’s t test or for the others experiments. In all cases p<0.05 was considered significant . When appropriate, the IC50 values (i.e. the concentration able to inhibit 50% of the maximum effect observed) were determined by non-linear regression using GraphPad Prism software v. 5.0.Data obtained from experiments were expressed as mean ± S.E.M., compared with vehicle control groups and statistically analyzed by the ANOVA one-way .(TIF)Click here for additional data file.Figure S213C NMR spectrum of 4a .(TIF)Click here for additional data file.Figure S31H NMR spectrum of 4b .(TIF)Click here for additional data file.Figure S413C NMR spectrum of 4b .(TIF)Click here for additional data file.Figure S51H NMR spectrum of 4c .(TIF)Click here for additional data file.Figure S613C NMR spectrum of 4c .(TIF)Click here for additional data file.Figure S71H NMR spectrum of 4d .(TIF)Click here for additional data file.Figure S813C NMR spectrum of 4d .(TIF)Click here for additional data file.Figure S91H NMR spectrum of 4e .(TIF)Click here for additional data file.Figure S1013C NMR spectrum of 4e .(TIF)Click here for additional data file.Figure S111H NMR spectrum of 4f .(TIF)Click here for additional data file.Figure S121H NMR spectrum of 4 g .(TIF)Click here for additional data file.Figure S131H NMR spectrum of 4 g .(TIF)Click here for additional data file.Figure S146, 300 MHz). Irradiation at 11.48 ppm.1D NOESY spectrum of 4 g (DMSO-(TIF)Click here for additional data file.Figure S156, 300 MHz). Irradiation at 8.26 ppm.1D NOESY spectrum of 4 g (DMSO-d(TIF)Click here for additional data file.Figure S1613C NMR spectrum of 4 g .(TIF)Click here for additional data file.Figure S171H NMR spectrum of 9 .(TIF)Click here for additional data file.Figure S181H NMR spectrum of 9 .(TIF)Click here for additional data file.Figure S191H NMR spectrum of 4 h .(TIF)Click here for additional data file.Figure S201H NMR spectrum of 15 .(TIF)Click here for additional data file.Figure S2113C NMR spectrum of 15 .(TIF)Click here for additional data file.Figure S22Chromatogram of compound 4 g obtained from reversed-phase HPLC studies.(TIF)Click here for additional data file.Figure S23E)-N’-benzylidene-2-(3-tert-butyl-1-phenyl-1H-pyrazol-5-ylamino)acetohydrazide (4 g) in DMSO-d6 (300 MHz). Irradiation at 11.48 ppm (A) and 8.26 ppm (B).1D NOESY spectrum of ((TIF)Click here for additional data file.Table S1p38α MAPK inhibitory activity of compounds (4a–g) at 10 µM.(DOC)Click here for additional data file.


Original text: "Expression of other proteins was detected using anti-PorA P1.7 mAb MN14C11.6 [65], anti-PorB P3.15 mAb 2-1-P15 [65], anti-RmpM mAb 173,G-1 [66] and anti-fHbp variant 1 mAb JAR4 [67]"Corrected text: "Expression of other proteins was detected using anti-PorA P1.7 mAb MN14C11.6 [65], anti-PorB P3.15 mAb 2-1-P15 [65], anti-RmpM mAb 173,G-1 [66]  and anti-fHbp variant 1 mAb JAR4 [67]"


The one-pot cyclization of 5-hexynoic acid to produce 3-alkoxy-2-cyclohexenones proceeds in good yields (58–90%). 3-Hexynoic acid was converted to its acyl chloride with the aid of oxalyl chloride and was cyclized to 3-chloro-2-cyclohexenone upon addition of indium(III) chloride. Subsequent addition of alcohol nucleophiles led to the desired 3-alkoxy-2-cyclohexenones. This result is consistent with those reported by Smit whose acyclic cases led exclusively to acylation at the unsubstituted end of terminal alkynes . Additio3), see 1H NMR analysis of aliquots from the reaction mixture indicated that 3 was completely converted to its acid chloride 2 within 2.5 hours. Indium(III) chloride was added directly to the flask upon formation of acid chloride 2. Significantly, omitting nitromethane as a solvent did not hinder the reaction, indicating that the chloride ion concentration was sufficient to promote the cyclization event. In addition to simplifying the overall process, the yield was also improved from 71% to 81% for the ethyl case. Consistent by-products under these conditions were dialkyl oxalates formed from esterification of unreacted oxalyl chloride with excess alcohol nucleophiles, and thus a basic hydrolysis step was added to our work-up procedure. Secondary alcohols required heating to 90 °C following addition of the alcohol nucleophile to promote product formation. IR and NMR spectral data can be found in Recently we have developed one-pot conditions for this reaction starting from 5-hexynoic acid (4) [tert-butanol   from rea-butanol . Enone 4 omitted .4 and the apparent steric influence of the alcohol nucleophiles, as observed in the yield data in 3 was converted to the acid chloride 2 by treatment of oxalyl chloride [2 to 3-chloro-2-cyclohexenone (4) and was followed by quenching with alcohol nucleophiles, yielding 3-alkoxy-2-cyclohexenones 1.Based on the isolation of chloride . In situRelated examples by Forsyth  and ClauBuilding on the work of Tedder  and SmitGeneral methods: All experiments were carried out under an atmosphere of dry nitrogen in oven-dried glassware. Solvents, starting materials and reagents were purchased (Sigma-Aldrich and Fisher Scientific) and used without further purification. 1H and 13C NMR spectra were obtained on a Varian Gemini 2000 (300 MHz) spectrometer with CDCl3 as solvent. Infrared spectra were obtained on a Thermo Nicolet 380 FT-IR spectrometer with Smart IR ATR (Si or ZnSe). Mass spectra were taken with a Varian 3800 GC/Saturn 2000 MS (ion trap).3-Methoxy-2-cyclohexenone (1b): 5-Hexynoic acid (3)  was combined with oxalyl chloride  in anhydrous dichloromethane (1.5 mL) in a nitrogen-purged 25 mL round-bottom flask and stirred for 2.5 hours. Indium(III) chloride  was added and the mixture was stirred for 3 hours. Methanol  was then added and the mixture was stirred for 15 minutes. Next, a saturated NaHCO3 solution (6 mL) and THF (3 mL) were added and stirred for 15 minutes, followed by addition of NaOH  and 5 minutes of stirring. The mixture was extracted with dichloromethane (3 × 10 mL) and the combined organic layers were dried over CaCl2 and concentrated in vacuo. Solvent exchange with CHCl3 (2 × 5 mL) gave the product  as an oil. 1H NMR  δ 5.36 , 3.68 , 2.40 , 2.34 , 1.97 ; 13C NMR  δ 195.1, 174.3, 97.6, 50.9, 31.9, 24.0, 16.4; IR  2946, 1645, 1603, 1378, 1225, 1186, 1135, 1003, 826, 757 cm−1; EIMS (m/z): 127.3-Isopropoxy-2-cyclohexenone (1d): 5-Hexynoic acid  and oxalyl chloride  were combined with anhydrous dichloromethane (1.5 mL) in a nitrogen-purged 25 mL round-bottom flask and stirred for 2.5 hours. Indium(III) chloride  was added and the mixture was stirred for 3 hours. 2-propanol  was then added and the mixture was stirred for 15 minutes at 90 °C. The reaction mixture was cooled to room temperature and a saturated NaHCO3 solution (6 mL) and THF (3 mL) were added and stirred for 15 minutes, followed by addition of NaOH  and 15 more minutes of stirring. The mixture was extracted with DCM (2 × 15 mL) and the combined organic layers were dried over CaCl2 and concentrated in vacuo. Solvent exchange with CHCl3 (3 × 5 mL) gave the pure product  as an oil. 1H NMR  δ 5.33 , 4.41 , 2.35 , 2.32 , 1.95 , 1.27 ; 13C NMR (CDCl3) δ 195.4, 172.4, 98.3, 66.2, 31.9, 24.8, 16.6; IR : 2980, 1645, 1594, 1379, 1219, 1183, 1105, 940, 824, 752 cm−1; EIMS (m/z): 155.3-Ethoxy-2-cyclohexenone (1a): 1H NMR  δ 5.34 , 3.89 , 2.39 , 2.34 , 1.97 , 1.35 .3-Propoxy-2-cyclohexenone (1c): 1H NMR  δ 5.33 , 3.76 , 2.39 , 2.32 , 1.96 , 1.73 , 0.96 .3-Butoxy-2-cyclohexenone (1e): 1H NMR  δ 5.33 , 3.81 , 2.38 , 2.32 , 1.96 , 1.69 , 1.41 , 0.93 .3-(sec-Butoxy)-2-cyclohexenone (1f): 1H NMR  δ 5.32 , 4.19 , 2.36 , 2.33 , 1.96 , 1.61 , 1.24 , 0.90 .3-Phenoxy-2-cyclohexenone (1h): 1H NMR  δ 7.37 , 7.22 , 7.02 , 5.10 , 2.64 , 2.36 , 2.06 .3-Benzyloxy-2-cyclohexenone (1i): 1H NMR  δ 7.37 , 5.48 , 4.89 , 2.47 , 2.37 , 2.00 .3-Allyloxy-2-cyclohexenone (1j): 1H NMR  δ 5.95 , 5.38 , 5.35 , 5.29 , 4.36 , 2.42 , 2.33 , 1.97 .3-Chloro-2-cyclohexenone (4): 1H NMR  δ 6.21 , 2.68 , 2.39 , 2.08 .File 11a–f, 1h–j and 4.IR and NMR data for compounds


Roseobacter clade bacterium Rugeria pomeroyi were collected by use of a closed-loop stripping headspace apparatus (CLSA) and analysed by GC–MS. Several lactones were found for which structural proposals were derived from their mass spectra and unambiguously verified by the synthesis of reference compounds. An enantioselective synthesis of two exemplary lactones was performed to establish the enantiomeric compositions of the natural products by enantioselective GC–MS analyses. The lactones were subjected to biotests to investigate their activity against several bacteria, fungi, and algae. A specific algicidal activity was observed that may be important in the interaction between the bacteria and their algal hosts in fading algal blooms.Volatiles released by the marine  Roseobacter clade form one of the most abundant lineages of marine bacteria that occur globally in marine ecosystems from polar to tropical regions , 111 (16), 95 (18), 69 (96), 55 (100), 53 (19), 43 (21), 41 (92), 40 (20); IR (ATR) : 2985 (w), 2933 (w), 2847 (w), 1731 (m), 1677 (m), 1450 (w), 1376 (w), 1344 (w), 1284 (m), 1242 (w), 1202 (m), 1181 (m), 1152 (s), 1110 (s), 1036 (s), 990 (m), 924 (m), 834 (w), 761 (w), 696 (w), 553 (w) cm−1; UV–vis λmax (log ε): 240 (2.22) nm.Pent-1-en-3-yl methacrylate (16): Yield: 3.0 g . TLC (hexane/ethyl acetate 10:1): Rf 0.29; GC (BPX-5): I = 988; 1H NMR  δ 6.11 , 5.81 , 5.54–5.52 , 5.25–5.19 , 5.17–5.13 , 1.93 , 1.69 , 0.90 ; 13C NMR  δ 166.7 (CO), 136.6 (Cq), 136.3 (CH), 125.1 (CH2), 116.5 (CH2), 76.0 (CH), 27.2 (CH2), 18.3 (CH3), 9.3 (CH3); MS  m/z (%): 154 (<1) [M+], 125 (8), 111 (7), 109 (6), 69 (100), 67 (14), 41 (44), 39 (22); IR (ATR) : 3087 (w), 2696 (m), 2931 (m), 2879 (w), 1721 (s), 1640 (w), 1456 (w), 1381 (w), 1294 (w), 1261 (w), 1164 (m), 1078 (w), 1059 (w), 990 (w), 930 (m), 810 (w), 657 (w) cm−1; UV–vis: λmax (log ε): 228 (2.83) nm.General procedure for the ring-closing metathesis to butenolides: Grubbs catalyst of the second generation (0.05 equiv) was added to a solution of the ester 15 or 16 (1.0 equiv) in dry dichloromethane (0.05 M). The mixture was stirred under reflux for 5 d. The solvent was removed under reduced pressure and the residue was purified by column chromatography on silica gel (pentane/diethyl ether 3:1) to give the butenolides as colourless oils.2-Methylpent-2-en-4-olide (11): Yield: 0.48 g . TLC (hexane/ethyl acetate 3:1): Rf 0.27; GC (BPX-5): I = 1017; 1H NMR  δ 7.02 , 5.01–4.94 , 1.90 , 1.39 ; 13C NMR  δ 173.9 (CO), 149.6 (CH), 129.3 (Cq), 77.1 (CH), 18.7 (CH3), 10.2 (CH3); MS  m/z [%]: 112 (28), 98 (16), 84 (4), 69 (42), 52 (24), 41 (78), 39 (100); IR (ATR) : 3382 , 3084 (w), 2985 (w), 2933 (w), 1743 (s), 1659 (w), 1448 (w), 1376 (w), 1342 (w), 1321 (w), 1209 (w), 1188 (w), 1103 (m), 1082 (s), 1048 (m), 1028 (m), 997 (s), 929 (m), 866 (m), 763 (m), 610 (m), 574 (w), 540 (m) cm−1; UV–vis λmax (log ε): 228 (2.70), 221 (2.03) nm. NMR spectroscopic data are in agreement with previously published data , 99 (6), 70 (34), 55 (100), 42 (72), 39 (69); IR (ATR) : 2977 (w), 2935 (w), 2878 (w), 1762 (s), 1454 (w), 1387 (w), 1349 (w), 1293 (w), 1177 (s), 1121 (m), 1069 (m), 1042 (s), 994 (w), 949 (s), 922 (w), 872 (w), 775 (w), 704 (w), 625 (m), 569 (w) cm−1; UV–vis λmax (log ε): 239 (1.22), 232 (0.69) nm. NMR spectroscopic data are in agreement with previously published data , 127 (1), 99 (49), 84 (24), 71 (23), 69 (29), 56 (30), 55 (69), 41 (100), 39 (92); IR (ATR) : 3082 (w), 2973 (w), 2934 (w), 2883 (w), 1746 (s), 1659 (w), 1460 (w), 1383 (w), 1344 (w), 1281 (w), 1209 (w), 1087 (s), 1046 (w), 1023 (m), 1009 (m), 959 (m), 892 (m), 857 (m), 786 (m), 762 (w), 612 (w), 555 (w) cm−1; UV–vis: λmax (log ε): 228 (1.44), 222 (0.90) nm.General procedure for the cis/trans isomerisation: To a solution of the cis-substituted lactone 7 or 9 (1.0 equiv) in t-BuOH (0.2 M), KOt-Bu (2.0 equiv) was added. The mixture was stirred under reflux for 24 h and then quenched by the addition of an equal volume of HCl (0.5 M). The mixture was extracted three times with diethyl ether. The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. Column chromatography of the residue on silica gel (pentane/diethyl ether 3:1) yielded mixtures of the cis- and trans-configured lactones as colourless oils, which were inseparable by chromatographic means.trans-2-Methylpentan-4-olide (8): Yield: 0.30 g , TLC (hexane/ethyl acetate 2:1): Rf 0.48; GC (BPX-5): GC: I = 1006; 1H NMR  δ 4.63 , 2.74–2.62 , 2.08–1.96 , 1.33 , 1.24 ; 13C NMR  δ 179.9 (CO), 74.5 (CH), 36.9 (CH2), 33.9 (CH), 20.9 (CH3), 15.6 (CH3); MS  m/z (%): 114 (<1) [M+], 99 (8), 70 (38), 55 (100), 42 (80), 39 (75); IR (ATR) : 2977 (w), 2936 (w), 2879 (w), 1763 (s), 1455 (w), 1386 (w), 1349 (w), 1178 (s), 1122 (m), 1068 (w), 1042 (m), 996 (w), 950 (s), 922 (w), 872 (w), 774 (w), 704 (w), 625 (w), 570 (w) cm−1; UV–vis λmax (log ε): 239 (1.20), 232 (0.53) nm. NMR spectroscopic data are in agreement with previously published data, apart from the chemical shifts of the 13C NMR signals for the methyl groups, which were previously reported at 20.7 and 26.3 ppm , 99 (54), 84 (25), 71 (27), 69 (31), 55 (57), 41 (100), 39 (87); IR (ATR) : 2971 (w), 2983 (w), 2881 (w), 1762 (s), 1457 (w), 1378 (w), 1354 (w), 1291 (w), 1177 (s), 1134 (w), 1053 (w), 1024 (w), 996 (m), 960 (m), 929 (m), 868 (w), 755 (w), 733 (w), 583 (w) cm−1; UV–vis λmax (log ε): 238 (1.30), 233 (0.92) nm.Enantioselective synthesis of -7 and -8. Treatment of -18  in dry CH2Cl2 (1 mL) for 1 h at room temperature was followed by acidic work-up with 2 N HCl (5 mL) and extraction with diethyl ether (3 × 5 mL). The combined organic layers were dried with MgSO4 and concentrated. The crude product was purified by chromatography over silica gel (hexane/EtOAc 3:1). Due to its volatility and the low amounts of material used for the synthesis, the solvent was not completely removed. The product (+)-11 was identical to racemic 11 by GC–MS analysis , [11 (6 mg) was dissolved in methanol (1 mL) and a small amount of Pd(OH)2  was added. The catalytic hydrogenation was carried out in a H2 atmosphere (1 bar) at 20 °C for 24 h. The reaction mixture was filtered through silica gel and concentrated to yield -7. The product was used in the next step without purification. Treatment of -7 with potassium tert-butoxide (5 mg) in tert-butanol (1 mL) under reflux for 6 h gave a mixture of -7 and -8, which was used for enantioselective GC analyses.lsewhere ) with Et CHCl3), ). CompouAgar diffusion assay for antimicrobial activity. The substances were dissolved in MeOH at a concentration of 2 mg/mL. Twenty-five microlitres of the solutions  was pipetted onto a sterile filter disk , which was placed onto an appropriate agar growth medium for the respective test organism and subsequently sprayed with a suspension of the test organism. The bacteria Escherichia coli and Bacillus megaterium were grown on Luria-Bertani (LB) medium , the fungus Microbotryum violaceum and the alga Chlorella fusca were grown on MPY medium , and B. cinerea was grown on biomalt agar  [−1 agar) . Referen


Gatifloxacin is an antibacterial agent belonging to the fourth-generation fluoroquinolone family. Four piperazine-linked fluoroquinolone dimers of Gatifloxacin were observed during the laboratory process for Gatifloxacin and they were identified. The present work describes the origin, synthesis, characterization, and control of these dimers along with the synthesis of Despropylene Gatifloxacin (metabolite). Gatifloxacin 1 is marketed by Bristol-Myers Squibb in the brand name of Tequin® for the treatment of respiratory tract infections. It is available as aqueous solutions for intravenous therapy. The ophthalmic solution is marketed by Allergan in the brand name of Zymer®.Gatifloxacin The presence of impurities in an Active Pharmaceutical Ingredient (API) will influence the quality and safety of the drug product. In the regulatory guidelines of the International Conference on Harmonization (ICH), it is recommended that impurities more than 0.1% .1 in the laboratory, we observed four unknown impurities along with the known impurities . In comparison with Gatifloxacin 1, twice the number of quinoline moiety protons in 1H NMR were observed.Compound mpound 6 . The mas9 originated from the condensation of the piperazine moiety of Gatifloxacin 1 with the carboxylic acid of 8-methoxyquinolinecarboxylic acid 7 during the hydrolysis of Gatifloxacin borondifluoride chelate 4.Gatifloxacin dimer-2 8 was independently prepared from compound 7. Reaction of 7 with thionyl chloride in dichloromethane to produce compound 8, which was further reacted with Gatifloxacin 1 in dichloromethane in presence of diisopropylethylamine, produced compound 9  impurity was formed by the self-condensation of Gatifloxacin 1 during the hydrolysis of Gatifloxacin borondifluoride chelate 4.The Gatifloxacin dimer-3 11 was independently prepared from compound 1. Reaction of 1 with thionyl chloride in dichloromethane to produce compound 10, which was further reacted with compound 1 in dichloromethane in presence of diisopropylethylamine, produced compound 11  & 3.75 (2H) ppm and also NH2 protons at δ 7.89 (3H) ppm.Compound pound 14 . The mas16 originated by the condensation of ethylenediamine, present as an impurity in the raw material, and 2-methylpiperazine with two molecules of compound 3.Gatifloxacin dimer-4 16 was independently prepared from compound 14 from the reaction with 3 in DMSO to produce compound 15, which was further reacted with triethylamine in methanol to produce Gatifloxacin dimer-4 16 (+] and its sodium adduct at m/z 633.1 amu [(M+Na)+]. In comparison with compound 14, we observed twice the number of quinoline moiety protons in the 1H NMR.Compound mer-4 16 . The mas6, 9, 11, 16 are novel and process-related compounds. These impurities were eliminated during the purification of Gatifloxacin 1. Compounds 14 & 16 originated due to the presence of the ethylenediamine impurity found in the raw material 2-methylpiperazine, and thus ethylenediamine is controlled within a specified limit (0.1%) in the specifications of the 2-methylpiperazine raw material.Gatifloxacin dimers 1–4, 1H NMR and 13C NMR spectral data were performed on the Bruker-Avance 300-MHz spectrometer in DMSO-d6. The chemical shift values were reported on the δ scale in parts per million (ppm), downfield from tetramethylsilane (TMS) as an internal standard. IR spectra were recorded in the solid state as KBr pellets using a Perkin-Elmer FT-IR spectrophotometer. The mass spectrum was recorded using a Perkin-Elmer PE SCIEX-API 2000, equipped with ESI source used online with a HPLC system after the ultraviolet (UV) detector.Solvents and reagents were obtained from the commercial sources and used without purification.3  in DMSO (275 mL), 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxo-quinoline-3-carboxylic acid 1  was added at room temperature. The reaction mass was heated to 80–85 °C and stirred for 24 h at 80–85 °C. The reaction mass was cooled to room temperature and added triethylamine . Further, the reaction mass was heated and stirred for another 10 h at 80–85 °C. The reaction mass was cooled to 5–10 °C, filtered the product, and dried to obtain compound 6, which was purified by crystallizing from methanol to yield a white solid 6 ; HPLC Purity: 82.19%; IR : 3442 , 3082 , 2942, 2867 , 1727, 1619 , 1444 , 777, 739 (Aromatic CH out-of-plane bend); 1H-NMR : 0.8–1.07 , 1.092 , 1.132 , 3.74–3.84 , 3.1 , 3.3–3.46 , 7.66–7.693 , 8.49–8.55 , MS m/z: 651.2271 [(M+H)+].To a suspension of 1-cyclopropyl-6,7-difluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid borondifluoride chelate 7  in dichloromethane (100 mL), thionyl chloride  was added at room temperature. The reaction mass was heated to 35–40 °C and stirred for 2 h to complete the formation of 1-cyclopropyl-6,7-difluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carbonyl chloride 8. The reaction mass was concentrated under reduced pressure to remove excess thionyl chloride and dissolved the residue in dichloromethane (100 mL). In a separate flask, 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxo-quinoline-3-carboxylic acid 1  was dissolved in dichloromethane (750 mL) and added N,N-diisopropylethylamine  at room temperature. Thereafter, the above prepared acid chloride 8 solution was added at room temperature. After 1 h stirring at 20–25 °C, the reaction mass was washed with DM water (250 mL). DM water (250 mL) was added to the organic layer and adjusted the pH to 5.5 with 10% aqueous hydrochloric acid. The organic layer was taken and concentrated at >40 °C under vacuum to obtain a residue, which was triturated with ethyl acetate to obtain a white solid 9. ; HPLC Purity: 91.71%; IR : 3452 , 3050 , 2950, 2850 , 1731,1621,1599 , 1469 , 783, 760 (Aromatic CH out-of-plane bend); 1H-NMR : 1.03–1.14 , 1.34 , 1.35 , 3.73 , 3.42 , 3.44–3.49 , 4.05–4.08 , 7.75–7.87 , 8.19–8.71 ; 13C-NMR (DMSO-D6): 8.9, 15.36, 16.21, 37.01, 40.6, 42.5, 45.06, 50.61, 54.73, 55.24, 63.14, 63.67, 106.47, 106.94, 117.12, 121.15, 123.68, 132.65, 134.18, 139.8, 140.69, 146.58, 148.83, 149.05, 150.62, 154.28, 156.77, 164.66, 165.25, 170.84, 176.3. MS m/z: 653.2220 [(M+H)+].To a suspension of 1-cyclopropyl-6,7-difluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 1  in dichloromethane (100 mL), thionyl chloride  was added at room temperature. The reaction mass was heated to 35–40 °C and stirred for 2 h to complete the formation of 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarbonyl chloride 10. The reaction mass was concentrated under reduced pressure to remove excess thionyl chloride and dissolved the residue in dichloromethane (100mL). In a separate flask, 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic acid 1  was dissolved in dichloromethane (750 mL), N,N-diisopropylethylamine  was added and cooled the reaction mass to 0–5 °C. Thereafter, the above prepared acid chloride 10 solution was added at 0–5 °C. After 1 h stirring at 20–25 °C, the reaction mass was washed with DM water (250 mL). DM water (250 mL) was added to the organic layer and adjusted pH to 7.0 with 10% aqueous hydrochloric acid. The organic layer was separated, washed with DM water (250 mL), and concentrated at 35–40 °C under reduced pressure to obtain a residue, which was triturated with hexanes and further purified by using column chromatography to obtain a white solid 11. ; HPLC Purity: 99.42%; IR : 3425 , 3084 , 2978, 2847 , 1725, 1620, 1589 , 1448 , 734, 806 (Aromatic CH out-of-plane bend); 1H-NMR : 0.91–1.1 , 1.1–1.33 , 1.9 , 3.74 , 2.7–3.35 , 3.76 , 3.97 , 7.59–7.63 , 7.76–7.8 , 8.1 , 8.71 ; 13C-NMR (DMSO-D6): 8.9, 19.08, 21.29, 40.67, 45.93, 48.6, 50.73, 57.87, 62.53, 63.68, 106.72, 121.17, 122.65, 133.57, 134.21, 137.87, 139.84, 145.86, 146.15, 150.65, 153.86, 154.29, 156.32, 156.78, 165.12, 165.68, 172.21, 176.31; MS m/z: 733.3148 [(M+H)+]To a suspension of 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-methyl-1-piperazin-yl)-4-oxo-3-quinolinecarboxylic acid 13 was cooled to 5–10 °C, filtered, and suspended in methanol (232 mL) at 25–30 °C. Triethylamine  was added to the above at 25–30 °C and heated the slurry mass to 60–65 °C for 16 h. Cooled the slurry mass to 25–30 °C, filtered, and dried to obtain a white solid 14. ; HPLC Purity: 99.64%; IR : 3332, 3083 , 2967 , 1620 , 1466 , 770.8, 747 (Aromatic CH out-of-plane bend); 1H-NMR : 1.04–1.17 , 3.12 , 3.75 , 4.18 , 7.74–7.79 , 7.89 , 8.68 ; 13C-NMR (DMSO-D6): 9.07, 44.62, 61.39, 105.93, 106.84, 107, 115.28, 133.37, 137.23, 149.17, 149.95, 151.61, 165.95, 176; MS m/z: 336.1362 [(M+H)+]To a suspension of 1-cyclopropyl-6,7-difluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid borondifluoride chelate 3  in acetonitrile (200 mL), ethylenediamine 12  was added at room temperature. The reaction mass was stirred for 16 h at 25–30 °C. The resulting product 14  in DMSO (40 mL), 1-cyclo propyl-6,7-difluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid borondifluoride chelate 3  was added. The reaction mass was heated to 70–75 °C and maintained at the same temperature for 16 h. Thereafter, the resulting slurry mass was cooled to 25–30 °C and filtered the product 14. This wet material was suspended in the mixture of methanol (70 mL) and sodium hydroxide . The reaction mass was heated to 40–45 °C and stirred for 4 h at 40–45 °C. The reaction mass was cooled to 25–30 °C. DM water (200 mL) was added to the slurry mass and adjusted pH to 7.0 with 10% aqueous hydrochloric acid at 25–30 °C, the resulting product was filtered and dried to obtain a white solid 16 ; HPLC Purity: 99.45%; IR : 3366 , 3077 , 2964 , 1725, 1620 , 1465 , 771, 749 (Aromatic CH out-of-plane bend); 1H-NMR : 1.02–1.13 , 3.68 , 3.76 , 4.14 , 7.67–7.72 , 8.64 ; MS m/z: 611.1953 [(M+H)+].To a suspension of 7-[(2-aminoethyl)amino]-1-cyclopropyl-6-fluoro-8-methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 1H-NMR spectra , 13C-NMR spectra , HRMS & elemental analyses , IR spectra , HPLC purity spectra , and the impurity mixture chromatogram of dimers are available in the online version : http://dx.doi.org/10.3797/scipharm.1212-21.Supporting information containing