Dihydroisoxazole compound for use in controlling sea lice

Abstract

The present invention provides a dihydroisoxazole of formula: or a salt thereof, for use in the control of sea lice in fish. ##STR00001##

Claims

1. A compound of the formula: ##STR00014## or a salt thereof.

2. A method of controlling sea lice in fish comprising administering to the fish a veterinary effective amount of a compound of the formula: ##STR00015## or a salt thereof.

3. The method according to claim 2 wherein the fish are salmonids.

4. The method according to claim 2, wherein the compound, or a salt thereof, is orally administered.

5. A medicated fish feed comprising a compound of the formula: ##STR00016## or a salt thereof.

Description

EXAMPLE 1

(1) ##STR00013##

N-[2-oxo-2-(prop-2-ynylamino)ethyl]-3-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazol-3-yl]-4,5,6,7-tetrahydro-2-benzothiophene-1-carboxamide

(2) Stir a mixture of 3-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazol-3-yl]-4,5,6,7-tetrahydro-2-benzothiophene-1-carboxylic acid (15.00 g, 30.08 mmol), 2-amino-N-prop-2-ynyl-acetamide (4.90 g, 33.09 mmol, HCl), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (13.72 g, 36.10 mmol) and N,N-diisopropylethylamine (11.66 g, 90.24 mmol) in dichloromethane (500 mL) at 25 C. for 12 hours. Use thin layer chromatography (petroleum ether:ethyl acetate=3:1, R.sub.f=0.30) to show the reaction progress. Dilute the mixture in water (500 mL) and extract with dichloromethane (500 mL3). Wash the organic layer with brine (500 mL), dry over sodium sulfate, filter and concentrate under reduced pressure to dryness. Purify the crude material by silica gel chromatography (petroleum ether:ethyl acetate=10:1 to 1:1) to afford the corresponding product with 70% ee, which is further purified by supercritical fluid chromatography (SFC) to afford N-[2-oxo-2-(prop-2-ynylamino)ethyl]-3-[(5S)-5-(3,4,5-trichlorophenyl)-5-(trifluoromethyl)-4H-isoxazol-3-yl]-4,5,6,7-tetrahydro-2-benzothiophene-1-carboxamide (6.00 g, 34% yield, 99.8% ee) as off-white solid. .sup.1H NMR (CDCl.sub.3): 7.62 (s, 2H), 7.05-6.90 (m, 2H), 4.18 (d, J=4.8 Hz, 2H), 4.10 (t, J=2.4 Hz, 2H), 4.06 (d, J=16.8 Hz, 1H), 3.68 (d, J=16.8 Hz, 1H), 3.10-2.95 (m, 2H), 2.90-2.80 (m, 2H), 2.24 (t, J=2.4 Hz, 1H), 1.90-1.70 (m, 4H). LC/MS (m/z): 592 (M+H).sup.+. SFC conditions as follows SFC analytical conditions: Instrument: Berger SFC Chiralcel OD-3 1504.6 mm I.D., 3 um Mobile phase: A: CO.sub.2 B: ethanol (0.05% DEA) Gradient: from 5% to 40% of B in 5.0 min and hold 40% for 2.5 min, then 5% of B for 2.5 min Flow rate: 2.5 mL/min Wavelength: 220 nm Column temperature: 35 C. Retention time: 6.12 min (S-isomer) SFC separation conditions: Instrument: Thar SFC 200; Column: AS 250 mm*50 mm, 10 um; Mobile phase: A: Supercritical CO2, B: EtOH, Base-EtOH, Begin B: 40%, End B: 40% Wavelength 220 nm Flow rate 200 ml/min Column Temp: 38 C.; Nozzle Pressure: 100 Bar; Nozzle Temp: 40 C.; Evaporator Temp: 40 C.; Trimmer Temp: 25 C.; Retention time: 5.66 min (R-isomer), 5.96 min (S-isomer)
Activity In Vitro Against Lepeophtheirus Salmonis at Copepodid Stage

(3) Sea lice copepodids are used to seed a 96-well plate containing the compound of Example 1 (test compound). The test compound is tested by serial dilution in order to determine its minimal effective dose (MED). Copepodids are left in contact with the test compound diluted in sea water for 1 hour. They are then incubated in untreated sea water for 48 h. Efficacy against sea lice is then confirmed if no copepodid moves over a period of 80 seconds.

(4) In this test, the compound of Example 1 showed 100% efficacy at a dose of as low as 0.5 ng/ml sea water.

(5) Activity In Vivo Against Lepeophtheirus Salmonis on Atlantic Salmon Using in-Feed Application

(6) 60 Salmon (Atlantic Salmon, S. salar) of average weight 306.5 g are treated with medicated pellets containing the compound of Example 1 (test compound) at a dose of 5 mg/kg/day during 7 consecutive days. The medicated pellets are prepared by dry coating commercially available fish feed pellets with pre-mix containing the test compound to reach a content of 0.5% (w/w) test compound in the fish feed, sealing with 1% fish oil and administering at a 1% feeding rate. The treatment is carried out in sea water. After treatment, the fish are challenged with copepodids (the MERL resistant strain IoA-01) on 35, 100 and 150 days post-treatment. During the study the water temperature varies between 7 and 10 C. Fish weight and sea louse numbers are assessed 42, 107 and 157 days post-treatment. Counts are conducted on 30 fish at each sampling occasion.

(7) Efficacy is determined by comparison with a negative control (placebo) group.

(8) Efficacy is calculated using the formula:
% Efficacy=100(100mean of treatment group/mean of control).

(9) All fish are anaesthetized with 2-phenoxy-ethanol and examined for louse settlement under a dissecting microscope. After counting, fish are returned to the tanks.

(10) In this test, the compound of Example 1 showed 100% efficacy on the challenge performed 35, 100 and 150 days post treatment.