KR100204739B1 - Process and antiparasitic intermediate for doramectin - Google Patents

Abstract

The present invention relates to a novel intermediate of formula (IV) and a process for the preparation thereof for the preparation of a compound called doramectin:

Formula IV

In the above formula,

R ¹ is (C ₂ -C ₅ ) alkanoyl or aryloxyacetyl.

Intermediates produced by the present invention also have utility as antiparasitic agents. The process of the present invention continues sparging the reaction inert gas during the pyrolysis step, with the result that the overall yield in the conversion is significantly improved.

Description

Insecticidal Intermediates of Doramectin and Methods of Making Doramectin

Doramectin is an extensive parasitic and parasitic remedy belonging to a group of secondary metabolites known as avermectin. Doramectin is a streptomycin, such as ATCC 31267, 31271 or 31272, under aerobic conditions in an aqueous nutrient medium containing inorganic salts and carbon and nitrogen assimilation sources, as described in US Pat. No. 5,089,480, which is incorporated herein by reference. It can be prepared by fermenting an avermectin producing strain of a microorganism called Streptomyces avermitilis . Another avermectin producing strain is Streptomyces avermitilis ATCC 53568, described in Duton et al., Journal of Antibiotics, 44, 357-65 (1991). Streptomyces armitillis ATCC 31267, 31271, 31272 and 53568 are currently deposited under the Budapest Treaty at the American Type Culture Collection, Rockville Parkron Drive 12301, 20852, Maryland, USA.

Various byproducts can be isolated during the fermentation process to prepare the compounds of formula (I) described above. The main by-product which can be isolated during the fermentation process of the microorganisms defined above has the structure of formula II above. Isolation of the compound of formula (II) from the fermentation broth of Streptomyces avermitilis ATCC 53568 is described in Duton et al., Supra. Compounds of formula (II) are active and parasitic control agents disclosed and claimed in US Pat. No. 5,089,480. However, doramectin is preferred. The process of the invention uses the by-product of formula (II) to convert it to the more valuable antiparasitic doramectin of formula (I).

The conversion made throughout the process is the removal of the C-23 hydroxyl group and leaving the olefin in the C-22 to C-23 position of the molecule. Low yields have been reported with the same conversion to slightly different avermectins. For example, when there is a C-25 cyclohexyl group instead of a secondary butyl group, the conversion takes place in five steps with a total yield of about 3.6% (Mrozik et al., Tetrhedron Letters, 1982, 23, 2377-78). ]). Given the low yields of the conventional methods, it is preferable to use a method in which the starting material is converted to doramectin more effectively.

Therefore, it is an object of the present invention to convert compounds of formula (II) to doramectin in high total yield. It is also an object of the present invention to provide intermediates useful for the process.

Summary of the Invention

The present invention provides novel intermediates of formula III:

Wherein R ¹ is (C ₂ -C ₅ ) alkanoyl or aryloxyacetyl and R ² is hydrogen or aryloxythiocarbonyl.

The present invention provides novel intermediates of formula IV:

Wherein R ¹ is (C ₂ -C ₅ ) alkanoyl or aryloxyacetyl.

Particularly preferred compounds of the invention are those wherein R ¹ is (C ₂ -C ₅ ) alkanoyl, phenoxyacetyl or (C ₁ -C ₄ ) alkylphenoxyacetyl and R ² is hydrogen or (C ₁ -C ₄ ) alkylphenoxy Citicarbonyl. A compound of Formula III. Particularly preferred compounds belonging to this group are compounds of formula III, wherein R ¹ is acetyl or phenoxyacetyl and R ² is hydrogen or p-tolyloxythiocarbonyl.

Also particularly preferred compounds of the present invention are compounds of formula IV, wherein R ¹ is (C ₂ -C ₅ ) alkanoyl, phenoxyacetyl or (C ₁ -C ₄ ) alkylphenoxyacetyl. Particularly preferred compounds belonging to this group are compounds of formula IV, wherein R ¹ is acetyl or phenoxyacetyl.

The invention also relates to a process for the preparation of a compound of formula (I) comprising the successive steps of (a) to (d):

(a) reacting a compound of formula (II) with an acylating agent (R ⁴ is aryloxymethyl and X is Cl or Br) of formula (R ⁴ CO) ₂ O or R ⁴ COX, wherein R ¹ is aryloxyacetyl and R Producing a compound of Formula III wherein ² is H;

(b) wherein R ¹ is an aryloxy acetyl and the formula R ³ a compound of formula III R ² is H OC (= S) compound of X (R ³ is aryl and X is Cl or Br Im) to yield R ¹ Producing a compound of Formula III wherein this is aryloxyacetyl and R ² is aryloxythiocarbonyl;

(c) reacting a compound of Formula III wherein R ¹ is aryloxyacetyl and R ² is aryloxythiocarbonyl in the presence of calcium carbonate in a reaction inert solvent at about 150 to about 200 ° C. for about 2 to about 48 hours Reacting with continued sparging of the inert gas to produce a compound of formula IV wherein R ¹ is aryloxyacetyl; And

(d) reacting a compound of Formula IV wherein R ¹ is aryloxyacetyl with a base in an alcohol solvent to produce a compound of Formula I.

Formula I

Formula II

Formula III

Formula IV

Particularly preferred among the present methods is a method wherein the reaction inert gas is nitrogen. Particularly preferred among these particularly preferred methods are those wherein the base is NH ₃ , KOH, KCN, Na ₂ CO ₃ , NaHCO ₃ or NaOAc. More particularly preferred methods among the above particularly preferred methods are those using particularly preferred compounds of the present invention.

The invention also relates to a process for the preparation of a compound of formula (I) comprising the successive steps of (a) to (d):

(a) a compound of formula formula II (R ⁴ CO) ₂ O or R ⁴ COX the acylating agent is R ¹ by (and R ⁴ are (C ₁ -C ₄₎ alkyl, X is Cl or Br Im) and the reaction Producing a compound of Formula III wherein (C ₁ -C ₄ ) alkanoyl and R ² is H;

(b) a compound of Formula III wherein R ¹ is (C ₁ -C ₄ ) alkanoyl and R ² is H, a compound of Formula R ³ OC (= S) X wherein R ³ is aryl and X is Cl or Br Reacting with to generate a compound of formula III wherein R ¹ is (C ₁ -C ₄ ) alkanoyl and R ² is aryloxythiocarbonyl;

(c) a compound of Formula III wherein R ¹ is (C ₁ -C ₄ ) alkanoyl and R ² is aryloxythiocarbonyl in a reaction inert solvent at about 150 to about 200 ° C. for about 2 to about 48 hours Reacting with continued sparging of the reaction inert gas in the presence of calcium carbonate to produce a compound of formula IV wherein R ¹ is (C ₁ -C ₄ ) alkanoyl; And

(d) reacting a compound of formula IV wherein R ¹ is (C ₁ -C ₄ ) alkanoyl with lithium aluminum hydride, sodium cyanoborohydride or lithium triethylborohydride in a reaction inert solvent Producing a compound of.

Particularly preferred among the above methods is a method wherein the reaction inert gas is nitrogen. More particularly preferred among these particularly preferred methods are those using particularly preferred compounds of the invention.

The present invention also provides a method of treating a mammal comprising administering to a mammal with parasites an effective amount of a compound of Formula III or (IV) in a parasitic.

The present invention relates to a novel intermediate in the preparation of a compound of formula (I) called doramectin and to an improved process for preparing said intermediate:

The invention also relates to the use of said intermediate as an antiparasitic agent. The present invention also relates to an improved process for preparing doramectin from a compound of formula II.

Compounds of the present invention are readily prepared according to the methods of the present invention, which will be described in detail below.

Compounds of formula (I) of the present invention are useful parasitic and parasitic control agents described in US Pat. No. 5,089,480, incorporated herein by reference. Dorametic is reacted with a lithium aluminum hydride, sodium cyanoborohydride or lithium triethylborohydride of the above-described compound of formula IV of the present invention or by saponifying an ester depending on the type of ester to be removed. It can be prepared from a compound of.

When R ¹ is (C ₂ -C ₅ ) alkanoyl, Formula IV wherein R ¹ is (C ₂ -C ₅ ) alkanoyl in a reaction inert solvent at about −100 ° C. to 0 ° C. for about 15 minutes to about 24 hours. The (C ₂ -C ₅ ) alkanoyloxy group is converted to a hydroxy group by reacting a compound of with lithium aluminum hydride, sodium cyanoborohydride or lithium triethylborohydride. Suitable inert solvents for the reaction depend on the type of reducing agent selected and are selected from, but are not limited to, solvents such as diethyl ether, dioxane, tetrahydrofuran, 2-methoxymethyl ether and 1,2-dimethoxyethane. Do not. Particularly preferred reducing agents for this reaction are lithium triethylborohydride and a particularly good solvent is tetrahydrofuran. The temperature is generally maintained between -78 and -70 ° C during the mixing of the reagent with the material and thereafter for a short time, typically for about 15 minutes to 1 hour. Immediately after mixing is complete, generally after 15 minutes to 1 hour, the temperature is slowly raised to ambient temperature. The reaction mixture is quenched and the resulting compound of formula (I) is isolated according to standard methods known to those skilled in the art.

When R ¹ is aryloxyacetyl, aryloxy is reacted with an alkali metal hydroxide by reacting a compound of formula IV wherein R ¹ is aryloxyacetyl for about 15 minutes to about 24 hours at about −75 ° C. to about 0 ° C. in an alcoholic solvent. The acetyloxy group is converted to a hydroxy group. Particularly preferred alkali metal hydroxides are potassium hydroxide and particularly preferred solvents are methanol. Generally the reaction is carried out at a temperature of −35 ° C. for about 1 hour, after which the compound of formula I is isolated according to standard methods known to those skilled in the art. Alternatively, the reaction may be carried out using ammonia instead of alkali metal hydroxides. When using ammonia, the reaction is generally carried out at about −35 to about 0 ° C. for about 1 hour to about 16 hours. The reaction is preferably carried out in methanol at −15 ° C. for about 5 to about 6 hours. The resulting compounds of formula (I) are isolated according to standard organic chemistry methods known to the person skilled in the art.

By reacting a compound of formula III of the present invention at about 150 to about 200 ° C. for about 2 to about 48 hours in a high boiling solvent such as, but not limited to, 2-methoxyethyl ether or 2-ethoxyethyl ether Compounds of formula IV are readily prepared according to the process of the invention. To obtain maximum yield, the reaction mixture is kept sparged with a reaction inert gas such as nitrogen or argon. Particular preference is given to carrying out the reaction with continuous sparging with nitrogen gas for 12 h in 2-methoxyethyl ether at 156 ° C. to 158 ° C. When the reaction is complete, the resulting compound of formula IV is isolated according to standard methods known to those skilled in the art.

Pyridine or 4-dimethylaminopyridine, and a proton cows for about 30 minutes to compound of formula III of the present invention R ² is hydrogen for about 12 hours in the presence of a scavenger such as the formula R ³ OC (= S) X (R 3 is the According to the method of the invention to prepare a compound of formula III wherein R ² is aryloxythiocarbonyl. Suitable reaction inert solvents for the reaction include ethyl acetate, 1,2-dimethoxyethane, 2-methoxymethyl ether, 2-methoxyethyl ether, aromatic solvents such as toluene, xylene and benzene, and chlorinated solvents such as : Chloroform and methylene chloride). Preferred solvents for this reaction are ethyl acetate or toluene. It is preferable to carry out the reaction for 2 to 4 hours. The reaction mixture is heated at 40 ° C. to the reflux temperature of the solvent selected for each reaction. The reaction mixture is quenched and the resulting compound of formula III, wherein R ² is aryloxythiocarbonyl, is isolated according to standard methods.

Compounds of formula III wherein R ² is hydrogen and R ¹ is (C ₂ -C ₅ ) alkanoyl or aryloxyacetyl are readily prepared according to the process of the invention. About -75 in a reaction inert solvent in the presence of a proton scavenger such as, but not limited to, pyridine, 4-dimethylaminopyridine, piperidine, pyrrolidine, triethylamine, morpholine or diisopropylethylamine The compound of formula II is reacted with an acylating agent at a temperature of from about 0 ° C. to about 0 ° C. for about 5 minutes to about 8 hours. Suitable reaction inert solvents for the reaction include aromatic solvents such as toluene, benzene or xylene or chlorinated solvents such as methylene chloride, chloroform or 1,2-dichloroethane. Suitable acylating agents are acid halides (usually acid chlorides) or acid anhydrides. When acid chloride is used as the acylating agent in the reaction, particularly preferred organic amines are pyridine. When acid anhydride is used as the acylating agent in the reaction, particularly preferred organic amines are triethylamine. Regardless of whether acid chloride or acid anhydride is used as the acylating agent, a generally preferred solvent for the reaction is a chlorinated solvent. Methylene chloride is particularly preferred. Upon completion of the reaction, the reaction mixture is quenched and the resulting compound of formula III, wherein R ² is hydrogen and R ¹ is (C ₂ -C ₅ ) alkanoyl or aryloxyacetyl, is isolated according to standard organic chemistry methods.

Compounds of formula (II) can be isolated via fermentation of an avermectin producing strain of Streptomyces avermitilis, such as ATCC 31267, 31271 or 31272, as described in US Pat. No. 5,089,480, which is incorporated herein by reference. have. Another method for obtaining the compound of formula II includes isolating from fermentation broth of Streptomyces avermitilis ATCC 53568 described in Durton et al., Journal of Antibiotics, 44, 357-65 (1991). .

The novel compounds (III) and (IV) of the present invention are useful as intermediates for the synthesis of compounds of formula I, ie doramectin, from compounds of formula II.

The novel compounds (III) and (IV) of the present invention are also useful as antiparasitic agents. The utility of using the compounds (III) and (IV) as an antiparasitic agent is useful for the activity of the compounds in the in vivo experiments of rodents described in Gration et al. Veterinary Parasitology, 42, 1992, 273-279. Is proved by

Compounds of formulas (III) and (IV) are highly active parasitic agents with particular utility as parasitic control agents, ectoparasitic control agents, insecticides and acaricides.

Compounds of formulas (III) and (IV) specifically include parasites, a disease most frequently caused by a group of parasites called nematodes that can infect livestock and poultry, as well as cause significant economic losses for pigs, sheep, horses, and cattle. It is effective in treating or preventing various conditions caused by internal parasites. Doramectin also contains Dirofilaria and Ancyolstoma, Necator, Ascaris, Strongyloides, Trikinella, and Capillaria in dogs. Various parasites such as Capillaria, Trichiris and Enterobius, and parasites found in the blood or other tissues and organs, such as filariasis, and parasites found in the intestine, such as strontyloids and trikinella It is also useful for the control of other nematodes that can infect a variety of animals, including.

Compounds of formulas III and IV are also ectoparasites, including arthropod portal parasites of animals and birds such as ticks, mites, teeth, fleas, flies, biting insects, and larvae of migratory twins that can infect cattle and horses. Infections can be treated and prevented.

The compounds of formulas III and IV are also useful for household grains such as cockroaches, moths, carpet beetles and houseflies, as well as control of insects and mobile locusts such as pests and locusts of stored cereals such as spider mites, aphids and caterpillars. It is useful for exterminating pests.

The compounds of the formulas (III) and (IV) are administered in combination suitable for a particular use to a particular species of host animal and associated parasite or insect to be treated. When used as parasite remedies, the compounds can be administered orally in the form of capsules, pills, tablets or preferably liquid medicaments, on the one hand, can be injected, sprayed or administered as an insert. The combination is prepared by conventional methods according to standard veterinary practice. Thus, capsules, tablets or tablets are prepared by mixing the active ingredient with a suitable finely divided diluent or carrier further comprising a disintegrant and / or binder (eg, starch, lactose, talc, magnesium stearate, etc.). The medicament is prepared by dispersing the active ingredient in an aqueous solution containing a dispersing or wetting agent and the like, and injectables are sterile solutions which may contain sufficient amounts of salts or glucose to make the solution isotonic with the blood. It may be prepared in the form of. The combinations will vary depending on the species of the host animal to be treated, the severity and type of infection, and the weight of the active ingredient depending on the weight of the host. In general, in the case of oral administration or injection, it is satisfactory to administer a single dose or divided doses of about 0.001 to 10 mg / kg of animal body weight per day, but may indicate higher or lower doses. And they are within the scope of the present invention. In the case of oral administration or injection, it is more preferred to administer about 0.02 mg to 2 mg per kg body weight of a single dose or divided doses for 1 to 5 days.

Alternatively, the compound may be administered to animal feed, for which a concentrated feed additive or premix may be prepared and mixed with conventional animal feed.

For use in the control of pesticides and crop pests, the compounds may be administered in the form of sprays, dusts, emulsions and the like according to standard agricultural practice.

As used herein and in the appended claims, the reaction inert solvent refers to any solvent that does not react with the starting materials, reagents, intermediates or products in a way that negatively affects the yield of the desired product. Reaction inert gas refers to any gas that does not react with the starting materials, reagents, intermediates or products in a way that negatively affects the yield of the desired product.

Aryl as used herein and in the appended claims refers to phenyl or phenyl optionally substituted with ₁ to 3 (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkoxy or halo.

As used herein and in the appended claims, aryloxy refers to phenoxy or phenoxy optionally substituted with ₁ to 3 (C ₁ -C ₄ ) alkyl, (C ₁ -C ₄ ) alkoxy or halo.

The invention will be illustrated by the following examples. However, it should be understood that the present invention is not limited to the specific details of the following examples.

Example 1

R ^One Is phenoxyacetyl and R ² Is a compound of Formula III

A solution of 20 g of the compound of formula II in 400 ml methylene chloride and 10 ml anhydrous pyridine was cooled to -78 ° C under nitrogen and 15.1 ml phenoxyacetyl chloride was added dropwise over 10 minutes. The reaction mixture was maintained at −70 to −78 ° C. for 1.5 hours and the reaction was stopped using 250 ml of saturated aqueous sodium bicarbonate solution. The mixture was stirred for 1.5 h, the organic layer was separated, reextracted with 250 mL of saturated sodium bicarbonate solution and dried over anhydrous MgSO ₄ . The solvent was removed in vacuo to give 25.7 g of 97% purity of the title compound as a gray solid (96% yield). HPLC retention time was 7.9 minutes (Ultrasphere ODS 5μ, 25 cm × 4.6 mm (Beckman); Methanol: Acetonitrile: Water = 559: 383: 58; 1.5 mL / min; UV 245 nm) ).

Example 2

R ^One Is phenoxyacetyl and R ² Is a p-tolyloxythiocarbonyl

A solution of 10 g of the title compound of Example 1 in 100 ml toluene and 30 ml anhydrous pyridine was heated to 100 ° C. under nitrogen and 7.7 ml O-p-tolylchlorothionoformate was added dropwise over 5 minutes. The mixture was kept at 100 ± 5 ° C. for 2 hours to complete the reaction, cooled to about 50 ° C. and the solvent removed in vacuo to give a brown residue. It was dissolved in 120 ml of toluene, the solution was extracted with 120 ml of water and extracted twice with 100 ml of saturated sodium bicarbonate solution. The separated organic phase was concentrated to about 80 mL, filtered and chromatographed using a Prep-500 system (Waters associates) equipped with two silica gel cartridges. Eluent was 75:25 hexanes: ethyl acetate. Fractions containing the product were combined and the solvent removed in vacuo to yield 9.8 g of a product of purity 95.7% as a light tan solid (yield 83.2%). HPLC retention time was 15.2 min (Ultrasphere ODS 5μ, 25 cm × 4.6 mm (Beckman); Methanol: Acetonitrile: Water = 559: 383: 58; 1.5 mL / min; UV 245 nm) ).

Example 3

5,4-O-diphenoxyacetyl-doramethin (R ^One Is a phenoxyacetyl)

4.5 g of calcium carbonate was added to a solution of 8.5 g of the title compound of Example 2 in 90 mL of 2-methoxyethyl ether. The mixture was heated to 156-158 ° C. with stirring while continuing sparging with a nitrogen stream. After 24 hours at 156-158 ° C., the mixture was analyzed and found to be 6.68 g of product (yield 90%). The mixture was filtered and the solvent was evaporated in vacuo to afford the product as a yellow gum. HPLC retention time was 10.4 minutes (Ultrasphere ODS 5μ, 25 cm × 4.6 mm (Beckman); Methanol: Acetonitrile: Water = 559: 383: 58; 1.5 mL / min; UV 245 nm) ).

Example 4

5,4-O-diphenoxyacetyl-doramethin (R ^One Is a phenoxyacetyl)

500 mg of calcium carbonate was added to a solution of 1 g of the title compound of Example 2 in 20 mL of 2-ethoxyethyl ether. The mixture was heated to 185 ° C. with stirring while continuing sparging with a nitrogen stream. After 5 hours at 185 ° C., the mixture was cooled to room temperature and filtered. The solution was analyzed by HPLC (Ultrasphere ODS 5μ, 25 cm × 4.6 mm (Beckman); methanol: acetonitrile: water = 559: 383: 58; 1.5 mL / min; UV 245 nm) It was found that 796 mg of product (yield 91%) was present.

Example 5

Doramectin (Compound of Formula I)

A solution of 500 mg of the title compound of Example 3 in 1 ml 2-methoxyethyl ether and 3.5 ml methanol was cooled to −35 ° C. and 2.1 ml 2M methanolic KOH solution was added dropwise over 1 minute. The mixture was kept at −35 ° C. with stirring for 1 hour to complete the reaction and 252 mg of glacial acetic acid dissolved in 0.5 ml of methanol was added. The cooling is stopped and dropwise addition of 2.2 ml of water for 1 hour initiates crystallization. After stirring for 1 hour at ambient temperature, the crystals were collected by filtration, washed twice with 0.5 ml of 7: 3 methanol: water and dried in a vacuum oven. Recrystallization from methanol / water gave 272 mg of product of purity 93.8% (yield 70.6%). HPLC retention time was 6.5 minutes (Ultrasphere ODS 5μ, 25 cm × 4.6 mm (Beck-man); Methanol: Acetonitrile: Water = 860: 51: 89; 1.5 mL / min; UV) 245 nm).

Example 6

Doramectin (Compound of Formula I)

A solution of 6.6 g of the title compound of Example 3 in 75 ml of methanol was cooled to -15 [deg.] C. and 75 ml of saturated ammonia solution in methanol was added thereto. The mixture was kept at −15 ° C. for 5.5 hours to complete the reaction and sparged with nitrogen for 30 minutes. The volatiles were removed in vacuo, the resulting pale yellow oil was dissolved in 45 ml of methanol and the solution was clarified by filtration. 5 ml of water is added dropwise to the solution to initiate crystallization. After the mixture was stirred at ambient temperature for 2 hours, 6.3 mL of water was added over 2.5 hours. After further stirring for 1.5 h, the solids were collected by filtration and washed twice with 2.5 ml of methanol: water (7: 3). After drying for 24 hours at ambient temperature in a vacuum oven, a product (yield 64%) of 3.75 g of purity 87.7% was obtained.

Example 7

R ^One Is acetyl and R ² Is a compound of Formula III

To a solution of 20 g of the compound of formula II in 200 ml of methylene chloride under nitrogen was added 28.6 g of triethylamine and 1.06 g of 4-dimethylaminopyridine. The solution was cooled to −5 ° C. to 0 ° C. and 14.5 g acetic anhydride was added dropwise over 5 minutes. The mixture was held at 0 ° C. for 20 minutes to complete the reaction and quenched with 200 mL saturated sodium bicarbonate solution at 0 ° C. The organic layer was reextracted twice with saturated sodium bicarbonate solution, dried (MgSO ₄ ) and the solvent removed in vacuo to yield 22.2 g of 95% purity product as a white solid (yield 96.7%). HPLC retention time was 6.6 minutes (Ultrasphere ODS 5μ, 25 cm × 4.6 mm (Beckman); Methanol: Acetonitrile: Water = 902: 36: 62; 1.0 mL / min; UV 245 nm) ).

Example 8

R ^One Is acetyl and R ² Is a compound of Formula III

To a slurry of 5 g of a compound of formula II in 100 ml of toluene under nitrogen was added 7.24 g of triethylamine and 266 mg of 4-dimethylaminopyridine. The mixture was cooled to -15 ° C and 3.58 g acetic anhydride was added dropwise over 5 minutes. The mixture was stirred at −10 ° C. to −15 ° C. for 30 minutes to complete the reaction, quenched with 50 ml of saturated aqueous sodium bicarbonate solution and stirred at ambient temperature for 1 hour. The organic layer was reextracted twice with 50 ml of saturated aqueous sodium bicarbonate solution and dried by partial distillation of the solvent using a Dean-Stark apparatus. The solution was analyzed by HPLC (Ultrasphere ODS 5μ, 25 cm × 4.6 mm (Beckman); methanol: acetonitrile: water = 902: 36: 62; 1.0 mL / min; UV 245 nm) It was found that 5.27 g of product (yield 96.5%) was contained.

Example 9

R ^One Is acetyl and R ² Is a p-tolyloxythiocarbonyl

To a solution of 20 g of the title compound of Example 7 in 200 ml of ethyl acetate under nitrogen was added 60 ml of anhydrous pyridine and 22.2 g of Op-tolylchlorothionoformate resulting in a yellow precipitate. The mixture was heated to reflux with stirring for 2 hours to complete the reaction and to cool to room temperature. The volatiles were removed in vacuo and the residue was partitioned between 150 ml of ethyl acetate and 150 ml of water and the organic layer was extracted twice with saturated aqueous sodium bicarbonate solution and once with water. The ethyl acetate layer was dried (MgSO ₄ ) and the solvent removed in vacuo. The residue was dissolved in 25 ml of ethyl acetate and 5 ml of methylene chloride and the solution was clarified by filtration and chromatographed using a Prep-500 system (Waters Associates) equipped with two silica gel cartridges. Eluent was hexane: ethyl acetate: methylene chloride of 70: 25: 5. Fractions containing the product were combined and the solvent removed in vacuo to yield 19 g (82.6% yield) of the product as a pale yellow solid. HPLC retention time was 17.1 minutes (Ultrasphere ODS 5μ, 25 cm × 4.6 mm (Beckman); Methanol: Acetonitrile: Water = 902: 36: 62; 1.0 mL / min; UV 245 nm) ).

Example 10

5,4-O-diacetyl-doramectin (R ^One Is acetyl)

A suspension of 9.5 g of calcium carbonate was heated to 156-158 ° C. while continuing sparging with a stream of nitrogen. 18.7 g of the title compound of Example 9 was added to the mixture, heated, stirred and continued sparging with nitrogen for 25 hours. After cooling to 50 ° C., the mixture was filtered and the solvent removed in vacuo. The residue was dissolved in 55 ml of methylene chloride, 25 ml of ethyl acetate and 70 ml of hexane were added, the solution was filtered and the Prep-500 system (Waters Associates) equipped with two silica gel cartridges Chromatography. Eluent was hexane: ethyl acetate: methylene chloride of 70: 25: 5. Fractions containing the product were combined and the solvent removed in vacuo to yield 13.7 g of a product of purity 87% as a yellow solid (yield 74.8%). HPLC retention time was 11.1 min (Ultrasphere ODS 5μ, 25 cm × 4.6 mm (Beckman); Methanol: Acetonitrile: Water = 902: 36: 62; 1.0 mL / min; UV 245 nm) ).

Example 11

5,4-O-diacetyl-doramectin (R ^One Is acetyl)

250 mg of calcium carbonate was added to a solution of 504 mg of the title compound of Example 9 in 25 ml of 2-ethoxyethyl ether. The mixture was heated to 183-185 ° C. with good stirring while continuing sparging with a nitrogen stream. After 1.5 hours at 183-185 ° C., the mixture was analyzed to show that 364.4 mg of product (yield 84.7%) was present.

Example 12

Doramectin (Compound of Formula I)

To a solution of 7.3 g of the title compound of Example 10 in 145 ml anhydrous tetrahydrofuran cooled to −72 ° C. under nitrogen, 59.5 ml of 1.0 M lithium triethylborohydride in tetrahydrofuran solution was added for 45 minutes. Dropwise over. The reaction mixture was stirred for 1 hour at -72 to -70 ° C and slowly warmed to room temperature to complete the reaction. The mixture was quenched with 75 ml of water and 75 ml of methylene chloride and stirred for 1 hour. The layers were separated, the aqueous layer was extracted with 75 ml of methylene chloride, and the combined organic layers were washed twice with 75 ml of saturated aqueous sodium bicarbonate solution. The solvent was evaporated in vacuo, the residue was dissolved in 54 ml of methanol and the solution was clarified by filtration. 6.9 mL of water was added dropwise to the solution over 20 minutes to initiate crystallization. The mixture was stirred at ambient temperature for 15 minutes and 6.2 mL of water was added dropwise over 30 minutes. The mixture was stirred for 18 hours, the solids were collected by filtration, washed twice with 2 ml of 75:25 methanol: water and dried in a vacuum oven to yield 4.27 g of a product of purity 89.5% (yield 57.3%). It was.

Example 13

Effect of Nitrogen Spazing on Thermal Elimination Reactions

2.5 g of calcium carbonate was added to a solution of 5.3 g of the title compound of Example 2 in 55 mL of 2-methoxyethyl ether. The mixture was heated to 157 ° C. with good stirring while the nitrogen stream was still sparged through the gas dispersion tube. After 30 hours at 154 to 157 ° C., the mixture was cooled to room temperature, filtered and analyzed by HPLC (Ultrasphere ODS 5μ, 25 cm × 4.6 mm (Beckman); methanol: aceto Nitrile: water = 559: 383: 58; 1.5 ml / min; UV 245 nm). As a result, it was found that 3.93 g of 5,4-O-diphenoxyacetyl-doramethin (yield 84.8%) was present.

The examples were repeated under the same conditions using the same amount of the title compound of Example 2 but without nitrogen sparging. Since the product yield in this case was 47.5%, it was found to be advantageous to nitrogen sparging.

Claims (6)

A compound of formula IV: Formula IV In the above formula, R ¹ is (C ₂ -C ₅ ) alkanoyl or aryloxyacetyl. The method of claim 1, R ¹ is (C ₂ -C ₅ ) alkanoyl, phenoxyacetyl or (C ₁ -C ₄ ) alkylphenoxyacetyl. The method of claim 2, R ¹ is CH ₃ C (═O)- The method of claim 2, R ¹ is C ₆ H ₅ OCH ₂ C (═O)-. A compound of formula (IV) comprising reacting a compound of formula (III) in the presence of calcium carbonate with continuing sparging of the reaction inert gas at about 150 to about 200 ° C. for about 2 to about 48 hours in a reaction inert solvent Manufacturing method: Formula IV Formula III In the above formula, R ¹ is (C ₂ -C ₅ ) alkanoyl or aryloxyacetyl, and R ² is aryloxythiocarbonyl. The method of claim 5, The reaction inert gas is nitrogen.