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Preparation of Milbe oxime

Sep 30，2020

Background and overview^[1][2]

Milbemycin Oxime (Milbemycin Oxime) is a macrolide antibody internal and external parasite drug, and is an oxime derivative of Milbemycin A3 and A4. In 1997, my country's Taiwan region launched the Milbex oxime produced by Taiwan Novartis Co., Ltd. as an internal medicine to prevent heartworm and intestinal parasites in dogs and cats. This is the first application of Milbex oxime in China. Milbex oxime has a broad-spectrum antiparasitic effect, and has a good repellent effect on internal and external parasites, especially nematodes and arthropods.

Preparation ^[1]

The preparation method of milbe oxime, its steps are as follows:

1. Weigh 130Kg of dried milbe mycelium, the calibrated content of milbemycin A3 and A4 are 0.55% and 2.57%, respectively, and add it to 1500L and stir to extract To the tank, add 750L of a mixed solvent of n-heptane and acetone with a volume ratio of 6:1, stir and extract at 30 rpm for 2 hours, filter, and extract the filter cake twice under the above conditions, and combine the filtrate. The filtrate was pumped into a concentration kettle, and concentrated under reduced pressure at 55°C to about 300L to obtain concentrated solution A.

2. Transfer the concentrated solution A of the previous step to a 1000L extraction kettle, add 300L of 85% ethanol aqueous solution, stir for 15min at 25rpm, let stand for 30min, and separate, the upper n-heptane phase is extracted once more with 300L of 85% ethanol solution, and the extracts are combined , 55°C under reduced pressure and concentrated to 150L to obtain concentrated liquid B.

3. Transfer the concentrated liquid B from the previous step to a 500L extraction kettle, adjust the pH to 9.5 with ammonia, add 150L of a 95:5 volume ratio of n-heptane and acetone mixed solvent, stir at 30rpm for 15min, stand for 30min, and separate. Extract the lower aqueous phase again, combine the extracts, transfer them to a 500L stirring tank, add 15Kg of anhydrous magnesium sulfate, stir for 10min, filter, and dry the filtrate under reduced pressure at 55℃ to obtain 14.9Kg of milbemycin extract The calibrated contents of milbemycin A3 and A4 were 4.03% and 20.12%, respectively. The extraction yields of milbemycin A3 and A4 were 84.0% and 89.7%, respectively.

4. Dissolve the above milbemycin extract with 60L dichloromethane and transfer it to a 100L reactor, add 52g Tempo (tetramethylpiperidine nitrogen oxide), 1350g ferric chloride, 578g sodium nitrite, stir at 35℃ Reaction for 2h, sampling and testing, after the completion of the reaction, filtration, the filtrate was concentrated and dried under reduced pressure at 35°C to obtain milbetone.

5. Dissolve the milbedone with 300L of 65% ethanol aqueous solution, transfer to a 1000L extraction kettle, add 300L of n-heptane and acetone solution with a volume ratio of 95:5, stir for 15min, stand for 60min, separate, and extract the lower ethanol solution again Once, combine the n-heptane phases, concentrate and dry under reduced pressure at 45°C to obtain a purified product of milbeketone.

6. The purified product of milbetone was dissolved in methanol and dioxane, and hydroxylamine hydrochloride solution was added dropwise, and reacted at 30°C for 16 hours. The reaction system was concentrated, extracted with a dichloromethane-water system, and the dichloromethane phase was dried and concentrated Obtain the crude milbe oxime; crystallize the crude milbe oxime with a mixed solvent of chloroform and n-heptane, dissolve the crystals in ethanol, add dropwise to water with stirring to crystallize, filter and dry to obtain 2.85Kg of the finished milbe oxime. The yield was 70.3%.

Assay ^[2]

An LC-MS/MS method for the determination of milbex oxime in animal plasma. The plasma sample is processed as follows: 1mL plasma sample is added with 4mL acetonitrile and 0.3g sodium chloride, vortexed on a micro mixer for 1 min, and the temperature is 3500r/ Centrifuge for 5 minutes for 5 minutes, take the supernatant in a sharp-bottomed glass centrifuge tube, and blow dry under a nitrogen stream at 50℃. Add 3mL methanol-5mmol/L ammonium acetate to the residue in a ratio of 1:9, vortex to mix, and apply to a C18 solid phase extraction column Purify; sequentially activate the extraction cartridge with 3mL methanol and 3mL water, add the sample solution, rinse with 2mL water, and then elute with 3mL10:90 volume ratio methanol-5mmol/L ammonium acetate and 3mL methanol, collect the eluent , Dried under a nitrogen stream at 50°C, the residue was dissolved in 1mL mobile phase, passed through a 0.22μm microporous nylon membrane, 5μL was injected, and LC-MS/MS analysis was carried out using liquid chromatography-mass spectrometry under the following chromatographic conditions:

The ionization method is electrospray condition, nitrogen spray pressure is 49psi, spray voltage is 4800V, argon pressure is 20psi, temperature is 300℃, SourceCID-12V. The m/z detected by Milbe oxime is 536. Full scan mode detects fragment ions with m/z of 536; mobile phase composition: acetonitrile: 0.5 mmol/L ammonium acetate = 85: 15, flow rate 0.25 mL/min, column temperature 20°C.