FI100402B - Process for the preparation of therapeutically useful macrolide compounds - Google Patents

Description

100402

A process for the preparation of therapeutically useful macrolide compounds

Divided separated from patent application 864150 5

The invention relates to the preparation of novel macrolide derivatives of formula R2. Λ 10 CH3 ^ 3 c „rL ίΓ (i> 15 H 1 I H S * S V / °

\ QH H

20 0 ή r ch,

H * 4 J

OR

wherein R 1 is methyl, ethyl or isopropyl, R 2 is hydrogen or the group -OR 5, and R 3 is hydrogen, or R 2 and R 3 together with the 25 carbon atoms to which they are attached form a C = O group,. wherein OR 5 is -OH, -OCOR 6, wherein R 6 is C 1-6 alkyl which may be substituted by halogen, C 1-4 alkoxy or phenoxy; -OCOOR 6 *, wherein R 6a is C 1-6 alkyl; -OR 7, wherein R 7 is C 1-4 alkyl which may be substituted by halogen, or C 3-5 alkenyl; or -OCCO 2 R 9, wherein R 9 is hydrogen or C 1-6 alkyl; and OR 4 is -OH or -OCOR 4 *, wherein R 4 * is C 1-4 alkyl, which may be substituted by halogen or phenoxy; except for those compounds of formula 1 wherein R 2 is -OH and OR 4 is -OH.

• · 35 These compounds have an antibiotic effect.

100402 2

F1 patent publication 90 663 corresponding to FI-application 872517 (published on 7 December 1987) discloses compounds of formula I in which R2 and R3 together form an oxo group. These compounds are disclosed as intermediates in that publication.

GB Patent 2,166,436 (cont. FI 87,366) describes the production of antibiotics Antibiotics S541, which can be isolated from fermentation products of the species Streptomyces. These compounds obtained by natural fermentation correspond to compounds of formula I in which R 2 is OH, R 3 is H and R 4 is H or CH 3. It has now been found that the novel compounds of the formula I form an additional group of compounds having antibiotic activity, which are generally clearly more active than said known compounds and which can be prepared by chemically modifying the Antibiotics S541 compounds.

Compounds of formula (I) having an acidic group may form salts with suitable bases. Examples of such salts are alkali metal salts such as sodium and potassium salts.

· Significant compounds of formula (I) are those in which R1 represents a methyl, ethyl or isopropyl group:; R2 is an ethoxy group, OR4 is a hydroxy group or a substituted hydroxy group, and R3 represents a hydrogen atom, and salts thereof.

• · · ·

Also significant compounds of formula (I) are those in which R1 represents a methyl, ethyl or isopropyl group, R2 and R3 together with the carbon atom to which they are attached form a group> C = O and OR4 is a hydroxy group or substituted hydroxy group, and salts thereof.

Further important compounds of formula (I) are those in which R1 represents a methyl, ethyl or isopropyl group, R2 and R3 each represent hydrogen and OR4, ··. represents a hydroxyl or substituted hydroxyl group, and salts thereof.

il 100402 3

In the compounds of formula (I), the group R 1 is preferably an isopropyl group.

The group R 2 is preferably a hydrogen atom or a hydroxy group or a group -OR 7 in which R 7 is C 1-4 alkyl or allyl, or R 2 and R 3 together with the carbon atom to which they are attached form a> C = O group. In particular, R 2 is preferably a hydrogen atom or an ethoxy group, or R 2 and R 3 together with the carbon atom to which they are attached form a> C = O group.

The group -OR4 in the compounds of formula (I) is primarily a hydroxy, methoxy, acetoxy or methyloxycarbonyloxy group.

Significant active compounds of the formula I are those compounds of the formula (I) in which R1 is a methyl, ethyl or in particular isopropyl group, R2 is a hydrogen atom, or an ethoxy group and R3 is a hydrogen atom or R2 and R3 together with the carbon atom to which they are attached, form a> C = O group, and OR4 is a hydroxy, acetoxy or methoxycarbonyloxy group.

Particularly significant compounds of formula I are those compounds of formula (I) wherein R 1 is an isopropyl group, R 2 is a hydrogen atom, R 3 is a hydrogen atom and: I 'OR 4 is a hydroxyl group; : R1 is an isopropyl group, R2 and R3 together with the carbon atom to which they are attached are> C = O and -OR4 is a hydroxyl group; and R1 is an isopropyl group, R2 is an ethoxy group, R3 is a hydrogen atom and -OR4 is a hydroxyl group.

The compounds of the formula I and their salts can be prepared by the preparation of compounds of the formula (I) in which one or both of the groups R2 and • · • *: * OR4 is an acylated hydroxy group, the corresponding compound in which one or both of R2 and OR4 is a hydroxyl group is reacted with an acylating reagent or a reactive derivative thereof, using R6COOH of the formula R6COOH as the acylating agent. , R6aCO2OH, HOOCCOOR9 or R4aCOOH or a reactive derivative thereof, or a2) for the preparation of compounds of formula (I) wherein R2 is an etherified hydroxy group, the corresponding compound wherein R2 is a hydroxyl group is etherified with a compound of formula R7- Y, wherein R 7 is as defined above and Y is a leaving group, or (b) for the preparation of compounds of formula (I) wherein R 2 and R 3 are hydrogen, the corresponding y hdiste of formula

L

CH.

CH, X J

15 ^ y CH3 N ». 4H \ H | R1 (III> 20 \ O / 0: 1 \ x

•; oiS H

: 25 0 Pf 'CH

. *. * OR

Wherein R 1 is as defined above, -OR 4 is a substituted hydroxyl group as defined above, and L is a group R 1 OCSO, wherein R 11 is C 1-4 alkyl (aryl), such as p-tolyl chlorothionoformate, is reduced with a homolytically suitable reducing reagent, or (t) 100402 5 (c) for the preparation of compounds of formula (I) in which R2 and R3 together form an oxo group, the corresponding compound in which R2 and OH are oxidized, wherein OR4 is a protected group, in the preparation of compounds wherein OR4 is a hydroxyl group, or (d) for the preparation of compounds of formula (I) wherein R2 or OR4 is a hydroxyl group, the corresponding compound in which R2 or OR4 is a protected hydroxyl group is deprotected.

In some of the methods described above, it may be necessary to protect the hydroxyl group at the 5- or 23-position of the starting material before carrying out the described reaction. In such cases, it may then be necessary to deprotect the same hydroxyl group after the reaction to obtain the desired compound. In this case, the usual protection and deprotection methods can be used. Examples of protected hydroxyl groups are well known and are described, for example, in "Protective Gropus in Organic Synthesis", Theodora W. Greene. (Wiley-Inter-20 science New York 1981) and in "Protective Groups in Organic Chemistry", J.F.W. McOmie (Plenum Press, London, 1973). Examples of R2 and OR4-protected hydroxy groups include phenoxyacetoxy, silyloxyacetoxy, (e.g., trimethylsilyloxyacetoxy and t-butyldimethylsilyl) hydroxyacetoxy), and silyloxy such as trimethylsilyl, .alpha.-oxy and t-butyldimethyl Thus, for example, an acyl group, such as an acetyl group, may be removed by hydrolysis with a base, e.g. using sodium or potassium hydroxide in aqueous alcohol, or by hydrolysis with ha-30, e.g. using concentrated sulfuric acid in methanol.

Acetal groups such as tetrahydropyranyl can be removed, e.g. by using acid hydrolysis (using acid such as acetic or trifluoroacetic acid or dilute. · · Inorganic acid). Silyl groups can be removed using · '. Fluoride ions (e.g. obtained from tetraalkylammonium fluoride such as tetra-n-butylammonium fluoride), hydrogen fluoride in aqueous acetonitrile or an acid such as p-toluenesulfonic acid (e.g. methanol).

Arylmethyl groups can be removed by treatment with a Lewis-5 acid (e.g. boron trifluoride etherate) in the presence of a thiol (e.g. ethanethiol) in a suitable solvent such as dichloromethane, e.g. at room temperature. Selective deprotection of the 5,23-disilyl compound of the invention can be performed using tetra-10 n-butylammonium fluoride, while selective deprotection of the 5,23-diacetoxy compound can be performed using sodium hydroxide in aqueous methanol.

Compounds of formula I in which R 2 and / or -OR 4 is a substituted hydroxyl group can usually be prepared by reacting Antibiotics S541 compounds or their 5- or 23-O-monosubstituted derivatives with reagents to form a substituted hydroxyl group. In general, the 5-hydroxyl group 20 is more reactive than the 23-hydroxyl group. Deprotection of 5-position hydroxyl groups is easier than of 23-position hydroxyl groups. In general, 5-monosubstituted compounds can be prepared by administering 5,23-sub-. the unsubstituted Hydroxy Antibiotics S541 to react with a limited amount of reagent, under milder conditions, while the 5,23-disubstituted compounds are formed using a greater amount of reagent and under less mild conditions and / or using a catalyst. 23-Mono-substituted compounds can be prepared by first forming a 5.23-substituted compound and selectively removing the 5-position. 5,23-disubstituted compounds having different substituents at the 5- and 23-positions can be obtained by reacting a compound having a mono-substituent at either the 5- or 23-position with a reagent of 100402 7 which to form a differently substituted hydroxyl group at the second position.

For the preparation of compounds of formula (I) wherein one of R 2 and OR 4 is a substituted hydroxyl group and the other is a hydroxyl or substituted hydroxyl group as defined above, a compound of formula OR 5 3 10 ch 3 CH 3

\ J ^ CH

cHrK, t ^ h v <“) 15 H | S4 rtii "20 O-: H CH3.:.: Or4 (wherein R1 is as previously defined and one of -OR4 and -OR5 is a hydroxyl group and the other is a hydroxyl group or a substituted hydroxyl group) is reacted with a reagent which allows the hydroxyl group is converted to a substituted hydroxyl group, followed, if desired, by a selective deprotection of a compound of formula (I) wherein R 2 and -OR 4 are both substituted hydroxyl groups to give a compound of formula (I) a compound wherein OR4 is a hydroxyl group and R is a substituted hydroxyl group.

, ·; The reaction is usually acylation, formylation, ··. sulfonylation, etherification, silylation or acetal form ‘1’. 35 teaching.

100402 8 Thus, for example, acylation may be carried out using an acylating agent such as an acid of formula R6COOH, or a reactive derivative thereof such as an acid halide (e.g. acid chloride), anhydride or activated ester, or a reactive derivative of the carboxylic acid R6aOCOOH.

Acylations using acid halides and anhydrides can be performed, if desired, with an acid scavenger such as a tertiary amine (e.g., triethylamine, dimethylaniline, or pyridine), inorganic bases (e.g., calcium carbonate or sodium bicarbonate), and oxiranes (such as lower 1,2-oxides). e.g. ethylene oxide or propylene oxide), which binds the hydrogen halide released in the acylation reaction.

Acylations using highly electrophilic acid chlorides (e.g., oxalyl chloride, methoxyacetyl chloride, chloroacetyl chloride, or bromoacetyl chloride) primarily in the presence of an acid neutralizing agent, such as calcium carbonate, can be used to selectively substitute the 23-position, wherein R 2 is a group OR 5, wherein R 5 is a substituted hydroxyl group and OR 4 is a hydroxyl group.

Acylations using acids are preferably performed with a condensing agent, e.g., a carbodiimide such as N, N'-dicyclohexylcarbodiimide or N-ethyl-N * -dimethylaminopropylcarbodiimide; a carbonyl compound such as [Π carbonyldiimidazole; or in the presence of an isoxazolium salt, such as N-ethyl-5-phenylisoxazolium perchlorate.

The activated ester can be conveniently formed in situ using, for example, 1-hydroxybenzotriazole in the presence of the above condensing agent. Alternatively, the optionally activated ester may be formed in advance.

The acylation reaction may be carried out in an aqueous or non-aqueous reaction medium, suitably at a temperature in the range of -20 ° to + 100 ° C, e.g. -10 ° to 0 ° C! + 50 ° C.

100402 9

The formylation can be performed using an activated formic acid derivative, e.g. N-formylimidazole or formic anhydride under standard reaction conditions.

Etherification can be performed using formula R7Y

wherein Y7 is as previously defined, and Y represents a leaving group such as a chlorine, bromine or iodine atom or a hydrocarbylsulfonyloxy group such as mesyloxy or tosyloxy group, or a haloalkanoyl-10 hydroxy group such as dichloroacetoxy. The reaction may be carried out by forming a magnesium alcoholate using a Grignard reagent such as methylmagnesium halide, e.g. methylmagnesium iodide or using a trialkylsilylmethylmagnesium halide, e.g. trimethylsilylmethyl-magnesium chloride, followed by treatment with reagent R7Y.

Alternatively, the reaction may be carried out in the presence of a silver salt such as silver oxide, silver perchlorate, silver carbonate or silver salicylate or mixtures thereof, and this system may be particularly suitable for performing etherification using an alkyl halide (e.g. methyl iodide).

The etherification may conveniently be carried out in a solvent such as ether, e.g. diethyl ether.

[· [· Acetal formation can be performed by reaction with a cyclic or acyclic vinyl ether. This method is particularly useful in the preparation of tetrahydropyranyl ethers, using dihydropyran as the reagent, or in the preparation of 1-alkoxy-1'-alkyl ethers, such as 1-ethoxyalkyl ether, using alkyl vinyl ether as the reagent. The reaction is preferably carried out in the presence of a strong acid catalyst, for example an inorganic acid such as sulfuric acid or an organic sulfonic acid such as p-toluenesulfonic acid, in a hydroxyl-free, substantially anhydrous solvent.

Anhydrous solvents that can be used in the above reactions include ketones (e.g. acetone), amides (e.g. N, N-dimethylformamide, N, N-dimethylacetamide or hexamethylphosphoramide), ethers (e.g. cyclic ethers such as tetrahydrofuran or dioxane, and acyclic ethers such as dimethoxyethane or diethyl ether), nitriles (e.g. acetonitrile), hydrocarbons such as halogenated hydrocarbons (e.g. methylene chloride), and esters such as ethyl acetate, as well as mixtures of two or more such solvents.

The silylation can be performed by allowing the reaction to proceed with a silyl halide (e.g., chloride), preferably in the presence of a base such as imidazole triethylamine or pyridine, using a solvent such as dimethylformamide.

In many cases, for example when a 5,23-dihydroxy compound of formula (II) is used as a starting material, a mixture of final products is formed, e.g. a mixture of 5-monosubstituted and 5,23-disubstituted derivatives. However, these can be distinguished by conventional methods, e.g.

•:. by chromatography, for example by chromatography on silica or hplcm.

·. ' A compound of formula (I) wherein R 2 and R 3 each represent a hydrogen atom may be prepared by reacting a compound of formula (III) wherein R 1 is as previously defined and OR 4 is a substituted hydroxyl group and OR 5 is a hydroxyl group, substituting 23 hydroxy group .. followed by deprotection of the group OR4, if desired, with a compound in which OR4 is a hydroxyl group, and, if desired, subsequent substitution to give a compound of formula (I) in which OR4 is a substituted hydroxyl group.

Thus, the starting materials of formula (II) in which OR4 is a substituted hydroxyl group and OR5 is a hydroxyl group may be reacted with a reagent which replaces the hydroxyl group with a leaving group to give compounds of formula (III):

L

CH, CH3 Γ] '* N "3 i ° Xo4n / chi

J «I

3h \ 1 '\ R1 S, v / ° 15 il nvc "3

OR

Wherein R 1 is as defined above and -OR 4 is a substituted hydroxyl group as defined above and L is a group R 11 OCSO- which can be removed by homolytic reduction - wherein R 11 is (C 1-4 alkylH) aryl such as p-tolyl-4 ' .

The reaction may be carried out in the presence of a base, for example an amine such as pyridine, in a solvent such as a halogenated hydrocarbon, e.g. dichloromethane.

If desired, intermediates of formula (III) can be isolated.

• · • · ♦ * ... Intermediates of formula (III) can then be * · \. reducing, to give the desired compound of formula (I) using a reducing agent such as alkyltin hydride (e.g. tri-n-butyltin hydride) in the presence of a radical initiator such as peroxide, azobisisobutyronitrile or light.

The reaction may conveniently be carried out in a suitable solvent which may be selected from the group consisting of a ketone, e.g. acetone; ether, e.g. dioxane; a hydrocarbon, e.g. hexane or toluene; halogenated hydrocarbon, e.g. trichlorobenzene; or an ester, e.g. ethyl acetate. Combinations of such solvents may also be used either alone or with water.

The reaction can be carried out at a temperature between 0 and 200 ° C, preferably between 20 and 130 ° C.

A compound of formula (I) wherein R 2 and R 3 together with the carbon atom to which they are attached represent> C = O may be prepared by oxidizing a compound of formula (II) wherein OR 5 is a hydroxyl group and OR 4 is substituted a hydroxyl group followed by deprotection of the group OR4, if desired, with a compound in which OR4 is a hydroxy group, followed, if desired, by a substitution to give a compound of formula (I) wherein OR4 is a substituted hydroxyl group. The reaction can be carried out with an oxidizing agent which converts a secondary hydroxyl group into an oxo group to give a compound of formula (I).

Suitable oxidizing agents are quinones in the presence of water, e.g. 2,3-dichloro-5,6-dicyano-1,4-benzoquinone or 2,3,5,6-tetrachloro-1,4-benzoquinone? a chromium (VI) oxidizing agent, e.g. pyridinium dichromate or chromium trioxide in pyridine; manganese (IV oxidizing agent, e.g. manganese-30 dioxide in dichloromethane; N-halosuccinimide, * ... e.g. N-chlorosuccinimide or N-bromosuccinimide; • «* \. dialkyl sulfoxide, e.g. dimethyl sulfoxide, activating :: in the presence of a substance such as N, N'-dicyclohexylcarbodiimide or an acyl halide, e.g. oxalyl chloride, or a pyridine-sulfur trioxide complex.

11 100402 13

The reaction may conveniently be carried out in a suitable solvent which may be selected from the group consisting of a ketone, e.g. acetone; an ether, e.g. diethyl ether, dioxane or tetrahydrofuran? a hydrocarbon, e.g. hexa-5 ni; a halogenated hydrocarbon, e.g. chloroform or methylene chloride; or an ester, e.g. ethyl acetate or a substituted amide, e.g. dimethylformamide. Combinations of such solvents alone or with water may also be used. The choice of solvent depends on the oxidizing agent used to cause the conversion.

The reaction can be carried out at a temperature of -80 ° C to + 50 ° C.

Intermediates of Antibiotics S541 of formula (II) wherein OR5 is a hydroxy group and OR4 is a hydroxy or methoxy group can also be obtained using the fermentation and isolation methods described above, as described, for example, in GB Patent No. 2,166,436. Intermediates of formula (II) can be prepared from these compounds using the methods described above for the preparation of compounds of formula (I).

The compounds of the formula I have an antibiotic effect, e.g. an anthelmintic effect, e.g. an anthelmintic effect, and in particular an effect against endogenous and external parasites.

The compounds of formula I are therefore useful in the treatment of animals and humans with infections caused by endocrine and / or ectoparasites. Exterminators and parasites infect humans and various animals and .. 30 are particularly common in farm animals such as pigs, sheep, cattle, goats and poultry (e.g.

• · * ·), chickens and turkeys), horses, rabbits, game:: poultry, cage birds, and domestic animals such as dogs, cats, guinea pigs, jumping rats and hamsters.

35 Parasitic infection of livestock, which causes anemia, malnutrition, 100402 14 black and weight loss, is the main cause of economic losses throughout the globe.

Examples of endogenous genera that infect such animals and / or humans are Ancylostoma, Asca-5 ridia, Ascaris, Aspicularis, Brugia, Bunostomum, Capillary, Chabertia, Cooperia, Dictyocaulus, Dirofilaria, Dracunculus, Enterobius, Haemonchus Loa, Necator, Nematodirus, Nematospiroides (Heligomoroides), Nippostron-gylus, Desophagostomum, Onchocerca, Ostertagia, Oxyuris, 10 Parascaris, Strongylus, Syphacia, Toxascaris, Toxocara,

Trichonema, Trichostrongylus, Trichinella, Trichuris, Tri-odontophorus, Uncinaria and Wuchareria.

Examples of ectoparasites that infect animals and / or humans include arthropod ectoparasites such as stinging insects, meat flies, fleas, lice, mites, sucking insects, mites and other dicotyledonous cancers.

Examples of ectopic genera that infect animals and / or humans include Ambylomma, Boop-20 hilus, Chorioptes, Culliphore, Demodex, Damalinia, Derma- *; Tobia, Gastrophilus, Haematobia, Haematopinus, Haemophysalis, Hyaloma, Hypoderma, Ixodes, Linognathus, Lucilia,

Melophagus, Oestrus, Otobius, Otodectes, Psorergates, Pooroptes, Rhipicephalus, Sarcoptes, Stomoxys and Tapanus.

: 25 The compounds of formula I have been found to be effective against a variety of endocrine and extracellular drugs both in vitro and in vivo. The antibiotic activity of the compounds of the formula I can be demonstrated, for example, as their activity against free-living nematodes, e.g. the species .. Caenorhabiditis elegans. In particular, it has been found that the compounds are effective against parasitic worms such as Haemonchus contortus, Ostertagia circum-cincta, Trichostrongylus colubiformis, Dictyocaulus vivi-paris, Cooperia onchophera, Ostertagia ostertagi, Nematos-35 piroostius and Duboides dubiusides dubius parasitic 100402 against mites such as Sarcoptes and Psoroptes.

Parasitic runs vary depending on the host and mainly the site of infection. Thus, for example, Haemonchus 5 contortus, Ostertagia circumcincta and Trichostrongylus colubiformis usually infect the sheep and are located mainly in the stomach and small intestine, while Dic-tyocaulus viviparus, Cooperia oncophora and Ostertagia os-tertagi 10 stomach.

In addition, the compounds of the formula I have use as antifungal agents, for example against strains of Candida species, such as Candida albicans and Candida glabrata, and against yeasts, such as Saccharomyces carlsbergensis.

The compounds of formula I have been tested in an in vitro experiment with Caenorhab-ditis elegans. The susceptibility of the free live nematode C. elegans to S541 analogs and Ivermecin was used as one of the primary screens to determine anti-parasitic activity. The nematode C. elegans was maintained at room temperature with minimal agar medium supplemented with lysine and DAP cultured with E. coli as a food source. The nematodes were harvested and suspended in M9 buffer, and 200 25 μΐ containing 100-200 nematodes were dispensed into microtiter tubes. Solutions or suspensions of the compounds in propylene glycol and serial dilutions, 400 pg / ml>, were prepared in 20% propylene glycol. 10 μΐ of the resulting solutions were mixed with 200 30 μl of the resulting nematode suspension and kept for 20 • · • 1 'hours at room temperature. Results were expressed as the lethal minimum concentration mlc that caused> 98% death or paralysis of nematodes. Ivermectin was used as a control compound in each experiment. The results 35 are shown in Table 1.

100402 16

table 1

C. elegans test results for compounds of formula I (R1 = isopropyl) 5 4 r3 r2 R_ mlc (pg / ml) 20 h H, OH H 0.03 - 0.1 (Factor A, known) 10 H, 0CH3 H 0.005 H, OCH2CH3 H 0.005 h, och2ch2ch3 H 0.01 H, OCH2CH2CH2I H 0.002 H, OCH2CH = CH2 H 0.007 15 H, OCH2CH3 C (0) CH3 0.08 H, 0C (0) CH3 H <0.005 H, 0C ( 0) CH 2 Cl H 0.06 H, oc (o) ch 2 ch 3 H 0.01 h, oc (o) ch 2 ch 2 ch 3 H 0.01 20 H, 0C (O) CH (CH 3) CH 2 CH 3 H 0.01: ·; H, 0C (0) CH (CH2) 2 H 0.06 H, 0C (0) CH2OC6H5 H 0.03 H, 0C (0) CH2OCH3 H 0.005 H, 0C (0) C (0) 0CH3 H 0.005 25 H , 0C (O) OCH3 H 0.015 ·; H, 0C (0) CH3 C (0) CH3 0.03 * ·:: H, 0C (0) CH2CH2CH3 C (0) CH3 0.06 = 0 H 0.005 = 0 C (0) CH3 0.03 30 = 0 C (0) CH2C1 0.06 • · «· * · · * 1 ·« • · • · 100402 17

Table 1 (continued) R3, R2 R1 R4 mlc (pg / ml) 20 h 5 H, 0C (0) CH3 CH3 H 0.002 = 0 CH3 H 0.005 H, 0C (0) CH3 CH2CH3 H 0.001 H, 0C (0) CH3 CH2CH3 C (0) CH3 0.08 = 0 CH2CH3 H 0.0005 10 = 0 CH2CH3 C (0) CH3 0.005

The results show that the new compounds of formula I are generally clearly more active than the known compound Factor A.

The compounds of formula I may be formulated for administration by any suitable route for use as a veterinary or human medicament. Such combinations may be provided for use in a conventional manner with the aid of one or more suitable carriers or excipients. The combinations are specifically formulated for parenteral (including intramammary) administration, for oral, anal, topical or implant use. Suitable methods and substances for formulating compounds of formula I for use as medicaments in animals and humans include those methods which are · * ··. ·. ·. described in GB Patent No. 2,166,436 for Antibiotics S541.

The compounds of formula I may be administered in combination with other pharmaceutically active ingredients.

• · ♦ * Suitable size days for compounds used in both animal and human medicine are in the range of 1 to 200 pg per kilogram of body weight, preferably 10 to 1000 pg / kg, more preferably 100 to 500 pg / kg, and these can be • · 35 should be given in divided doses, eg 1-4 times a day.

100402 18

The following examples illustrate the invention. In the examples of the compound of formula (II) wherein R 1 is isopropyl and OR 4 and OR 5 are hydroxyl groups, the term "Factor A" is used. All temperatures are in degrees Celsius.

Example 1 5-Phenoxyacetoxy and 5,23-diphenoxyacetoxy

Factor A

Factor A (2.0 g) in dichloromethane (25 ml) and pyridine (0.35 ml) were treated at 0 ° C with a solution of phenoxyacetyl chloride (0.5 ml) in dichloromethane. After 18 hours at 3 ° C, the solution was treated with pyridine (1.0 mL) and phenoxyacetyl chloride (1.0 mL) in dichloromethane (5 mL). The solution was stirred at 150-5 ° C for 30 minutes before being poured into ice water. Ether (100 ml) was added and the mixture was stirred for 20 minutes. The aqueous layer was extracted with ether. The ether layers were combined, washed successively with water and brine, dried and evaporated to dryness. The residue was purified by chromatography on silica using dichloromethane / acetone (40: 1) to give the title compounds as a mixture (1.8 g, monoacyl: diacyl = 6: 1) which was separated from the reverse phase preparative using hplc. to give 5-phenoxyacetoxy 25 Factor A, δ (CDCl 3) 6.8-7.4 (m; 5H) and 4.66 (s; 2H), · m / z 746, 728, 710, 594 and 576 and 5,23-diphenoxyacetoxy

Factor A, δ (CDCl 3) 6.8-7.4 (m, 10H), 4.60 (s, 2H) and 4.70 (s, 2H).

Example 2 5-Phenoxyacetoxy-23- (4-methylphenoxythionocarbonyloxy) • · ·

v 'Factor A

5-Phenoxyacetoxy Factor A (747 mg) was treated with dichloromethane (10 mL) at 0 ° C under nitrogen with pyridine (0.81 mL) and then 4-methylphenyl chloro-100402 19 thionoformate ( 0.75 g) in dichloromethane (2 ml). The dark solution was stirred for 15 minutes at 0 ° C and then for 22 hours without cooling. The mixture was poured into cold water and brine, and extracted with ether. The ether layers of Compound 5 were washed with water and brine, dried and evaporated to dryness. The residue was purified by chromatography on a silica column using reverse phase preparative hplc to give the title compound (430 mg), δ (CDCl 3) 3.34 (m; 1H), 3.58 (m; 10 1H), 3 .97 (d10; 1H), 4.72 (s, 2H), 5.4 (m, 1H), 5.59 (d6; 1H) and 6.9-7.4 (m, 9H), m / z 728, 616, 576, 466, 464, 448, 354, 297, 247, 219 and 151.

Example 3

5-tert-butyldimethylsilyloxyacetoxy Factor A

Factor A (2.144 g) in dry ether (25 ml) and pyridine (2.5 ml) at 0 ° C under nitrogen was treated by dropwise addition of t-butyldimethylsilyloxyacetyl chloride (1.2 g) in ether (10 ml). mL).

The mixture was stirred for 90 minutes at 0 ° C before being treated dropwise with more acid chloride (1.10 g) in ether (10 ml). The mixture was stirred at 0 ° C for 60 minutes and poured into cold water and ether. The aqueous layer was washed with ether. The combined organic layers were washed with water and brine, dried and evaporated to dryness. The residue was purified by chromatography on silica using dichloromethane-acetone (25: 1) as eluent to give the title compound, δ (CDCl 3) 0.09 (s, 6H), 0.78 (d6, 3H). 0.90 (s, 30H), 0.93 (d6, 3H), 0.97 (d6, 3H), 1.03 (d6, 3H, 1.51 (s, f * · 3H), 1, 59 (s, 3H), 1.74 (s, 3H), 3.32 (m, 1H), 3.52 (d10, T 1H), 3.64 (m, 1H), 3.74 (d10) , 1H), 3.82 (m, 1H), 4.32 (s, · · · 2H) and 5.57 (d5, 1H), m / z 784, 766, 748, 595, 577, 484, 466, 354, 314, 297, 265, 247, 237, 219 and 151.

«« 100402 20

Example 4 5-1ert-Butyldimethylsilyloxy

Factor A

Factor A (250 mg) and imidazole (163 mg) in dry dimethylformamide (10 ml) were treated with t-butyldimethylsilyl chloride (197 mg). The solution was stirred for 2 hours and poured into cold water. The mixture was extracted thoroughly with ether and the combined ether extracts dried and evaporated to dryness. The residue was purified by chromatography on silica using dichloromethane: -acetone (10: 1) as eluent to give the title compound (235 mg), δ (CDCl 3) 0.13 (s, 6H), 0.80 (d 6, 3H), O , 92 (s, 9H), 0.96 (d6, 3H), 1.00 (d6, 3H), 1.03 (d6, 3H), 1.53 (s, 3H), 1.60 (s , 3H), 1.80 (s, 3H), 3.37 (m, 1H), 3.56 (d10, 1H), 3.64 (m, 1H), 3.75 (d10, 1H) and 4 , 43 (d5, 1H), m / z 726, 708, 691, 651, 633, 466, 448, 354, 314, 297, 265, 247, 219 and 151.

Example 5 5-Acetoxy and 5,23-diacetoxy

Factor A

· Factor A (3.0 g) was treated in pyridine (20 ml) at -5 ° C with acetic anhydride (8 ml) and the resulting solution was left at 3 ° C for 20 hours. Benzene (100 mL) was added and the solution was concentrated in vacuo. Remaining. the residual oil was chromatographed on silica using dichloromethane-acetone (40: 1) as eluent to give Factor A 5-acetate (2.06 g) with 5,23-diacetate (10%). Compounds were isolated using 30 reverse phase preparative hplc to give 5- • ** acetoxy-Factor A (79% yield), Tl. (EtOH) 244.5 nm • · · (E \ 462), δ (CDCl 3) 2.14 (s, 3H), m / z 654, 594 and 576 and 5,23-diacetoxy Factor A (yield 6 .5%), δ (CDCl 3) 2.01 (s, 3H) and 2.13 (s, 3H), m / z 696 and 636.

100402 21

Example 6 5-Acetoxy-23-keto Factor Ä

A solution of oxalyl chloride (1.96 mL) in dry dichloromethane (25 mL) at -70 ° C under nitrogen was treated by dropwise addition of a solution of dimethyl sulfoxide (3.19 mL) in dry dichloromethane (15 mL) and then dropwise a solution of 5-acetoxy-Factor A (4.91 g) in dry dichloromethane (30 ml). The resulting solution was stirred at -70 ° C for 1.5 h before being treated by dropwise addition of a solution of triethylamine (12.6 mL) in dry dichloromethane (40 mL). The reaction mixture was stirred for 1.25 hours without cooling and poured into a mixture of cold water and ether (1: 1). The aqueous layer was extracted with ether. The combined organic layers were washed with water, brine, dried and evaporated to dryness. The residual foam was chromatographed on silica using dichloromethane / acetone (50: 1) to give the title compound (3.4 g), δ (CDCl 3) 3.33 (m, 1H), 3.49 (m, 1H) , 3.70 (d10, 1H) and 5.52 (d5, 1H), m / z 652, 634, 609, 591, 7: 574, 482, 263, 235 and 151.

Example 7 23-keto

25 Factor A

7! The compound of Example 6 (276 mg) was treated with methanol (5 ml) at 0 ° C by dropwise addition of a solution of 2-norm. sodium hydroxide (0.42 ml) in methanol (1.0 ml). The solution was left at 5 ° C for 5 hours before being poured into cold water. The mixture was extracted with ether and ethyl acetate. The combined organic layers were washed • ♦ ♦ ’· /. brine, dried and evaporated to dryness to leave a solid which was purified by preparative TLC using 35 dichloromethane: acetone (10: 1) as solvent to give 100402 22 title compound (140 mg), 6 (CDCl 3). 3.28 (m, 1H), 3.48 (m, 1H), 3.70 (d10, 1H) and 4.28 (tr7, 1H), m / z 592, 549, 482, 370, 265, 235 and 151.

Example 8 5-Phenoxyacetoxy-23-deoxy

Factor A

The compound of Example 2 (350 mg) and azobisisobutyronitrile (25 mg) in dry toluene (20 min) protected under nitrogen at 120 ° C were treated by the dropwise addition of a solution of tri-n-butyltin hydride (0.5 ml). in dry toluene (10 mL). The solution was boiled for 90 minutes, cooled and evaporated to dryness. The residue was chromatographed on silica using dichloromethane: acetone (40: 1) as solvent to give the title compound (280 mg), δ (CDCl 3) 3.32 (m, 1H), 3.42 (d 10, 1H), 3, 57 (m, 1H), 4.71 (s, 2H), 5.59 (d6, 1H) and 6.8-7.4 (m, 5H), m / z 730, 712, 578, 560, 468 , 450, 356, 314, 299, 249, 248, 221 and 151.

Example 9 20 23-deoxy

Factor A

The compound of Example 8 (240 mg) was added to a saturated solution of ammonia in methanol (10 ml) at -5 ° C.

· '. The solution was stirred at 0-10 ° C for 2 hours before being evaporated to dryness. The residue was chromatographed on silica using dichloromethane: acetone (20: 1) to give the title compound (180 mg), δ (CDCl 3) 3.27 (m, 1H), 3.42 ( d6, 1H), 3.54 (m, 1H) and 4.29 (t6, 1H), m / z 596, 578, 560, 468, 450, 356, 314, 299, 249, 248, 221 and 30151. .

• ·

II

100402 23 in excess of oxalyl chloride (1.0 mL). After 4-5 minutes the mixture was treated with water (15 ml) and after 5 minutes 2-mol. HCl (10 mL) and ethyl acetate (70 mL). The organic phase was separated, washed with water and brine, and then treated with decolorizing charcoal. After 5-10 minutes, the organic solution was filtered through phase separating paper and the solvent was evaporated from the filtrate. The residue dissolved in ether (50 ml) was clarified by filtration through 10 "Hyflo" and removal of the solvent gave a pale yellow foam. Trituration of this material with diisopropyl ether (ca. 7 mL) gave the title compound as a crystalline solid (750 mg) after washing the solid with diisopropyl ether and n-pentane. Abak «. (CHBr3) 3360-3600 (OH), 1805 (acid monomer) and 1720 cm-1 (acid dimer and esters); Δ (CDCl 3) 4.30 (d, 5Hz, 1H) and 5.14 (m, 1H).

Example 11 Factor A

A solution of 23-hemioxalate Factor A (500 mg) in dichloromethane (30 mL) was stirred vigorously with aqueous sodium hydroxide (1.54 g in 30 mL H 2 O) for 2 h at 21 ° C. The organic phase was collected and the aqueous phase was washed with a small volume of dichloromethane. The organic phases were combined, washed with 2 mol. HCl, then dried (Na 2 SO 4) and the solvent evaporated to give the title compound (430 mg) as a foam. The compound was shown to be Factor A by comparison with hplc to an authentic 30 samples of Factor A, ϊ Example 12: *: * Factor A, 5-hemisuccinate

A solution of Factor A (306 mg) and succinic anhydride (60 mg) in dry pyridine (0.5 mL) was stirred at 22 ° C for 24 h. The mixture was diluted with ether-100402 24 blades and the organic phase was then washed with 2-norm. with hydrochloric acid and brine. Evaporation of the dried ether layer to dryness gave a foam from which the title compound was isolated by reverse phase preparative HPLC. The product in ether was precipitated as a white amorphous solid (86 mg) by the addition of n-pentane; [α] D 20 + 125 ° (c 0.44, CHCl 3) ABake. ethanol 245 nm (eBak ..

30,700), 7 \, naks. (CHBr 3) 3490 (OH) 3,300-2,200 (CO 2 H), 1730 (acid carbonyl) and 1710 cm -1 (ester); Δ (CDCl 3) 2.74 (s, 10 4H), m / z = 712 (M +).

Example 13

Factor A, 5-chloroacetate

A solution of Factor Aita (123 mg) and pyridine (0.1 ml) in dry dichloromethane (5 ml) was stirred at 0 ° C and chloroacetyl chloride (0.3 ml of 1 M dichloromethane solution) was added. . The resulting solution was stirred at 0 ° C for 15 minutes, then more chloroacetyl chloride (0.1 mL of a 1 M solution) was added, and stirring was continued for another 15 minutes. The solution was diluted with dichloromethane (50 mL), then washed with 2-norm.

; hydrochloric acid (2 x 20 ml), and saturated sodium hydrogen carbonate (20 ml), and dried (magnesium sulphate), and evaporated to dryness. The residue (140 mg) was purified by chromatography on Kieselgel 60 (15 g).

J 25 Elution of the column with petroleum ether: ethyl acetate (4: 1) gave the title compound (90 mg) as a colorless foam, [α] D + 143 ° (c, 1.11, CHCl 3), λ Bake (ethanol). 245.5 nm (δ 30,400), Λ Bake. (CHBr 3) 3500 (OH), 1760 (chloroacetate) and 1710 cm -1 (ester), δ (CDCl 3) 5.5-5.6 (m, 2H), 4.18 (s, 2H), about 3.81 (m, 1H), 3.74 (d, J 10 Hz, 1H) and 0.80 (d, J 7 Hz, 3H), m / z = 688 (M +, 35 Cl).

The compounds of Examples 15, 16, 17 and 18 were prepared in a similar manner.

• 1 100402 25

Example 14

Factor A, 5-chloroacetate, 23-ketone

A solution of Factor A, 5-chloroacetate (276 mg) and pyridinium dichromate (602 mg) in dry N, N-5 dimethylformamide (10 ml) was stirred at 20 ° C for 48 hours and then poured into a mixture of ethyl acetate (50 mL) and 2-norm. hydrochloric acid (25 ml). The organic phase was washed with 2-norm. hydrochloric acid and saturated sodium bicarbonate solution, and dried (magnesium sulfate), and evaporated to dryness. The residue was purified by chromatography on Kieselgel 60 (25 g). Elution of the column with petroleum ether: ethyl acetate (4: 1) gave the title compound (85 mg) as a colorless foam, [α] D + 120 ° (c 1.05, CHCl 3), Λ Bak ,.

15 (ethanol) 245 nm (ε 26,500), λ. Baks. (CHBr 3) 3480 (OH), 1760 (chloroacetate) and 1715 cm -1 (ester and ketone), δ (CDCl 3) 5.5-5.7 (m, 2H), 4.17 (s, 2H), 3 , 72 (d, J 10 Hz, 1H), 2.50 (s, 2H) and 0.85 (d, J 7 Hz, 3H), m / z = 686 (M +, 3SCl).

Example 15 Factor A, 5-bromoacetate (447 mg) [α] D 20 + 132 ° (c 0.77, CHCl 3), ABakB. (ethanol) 245.5 nm (ε 31,500), λ BakB.

(CHBr 3) 3495 (OH), 1730 (bromoacetate) and 1712 cm -1 (ester), δ (CDCl 3) 5.5-5.6 (m, 2H), 3.97 (d, J 12 Hz, 1H), 3.90 (d, J 12 Hz, 1H), 3.81 (m, 1H), 3.75 (d, J 10 Hz, 12 1H), and 0.81 (d, J 7 Hz, 3H), Factor A (613 g) and bromoacetyl bromide (0.13 ml).

• »

Example 16 23-Deoxy Factor A, 5-methyl carbonate (57 mg) [α] 30 + 152 ° (c 0.6, CHCl 3), Λ BakB. (ethanol) 244.5 nm 30 (δ 28,200), A Bak. (CHBr3) 3530 and 3460 (OH), 1740 (carbonate) and 1707 cm-1 (ester), δ (CDCl3) δ , 55 (s, 1H), 3.82: T (S, 3H), 3.42 (d, J 10 Hz, 1H) and 0.69 (d, J 4 Hz, 3H), 23-deoxy from Factor A (90 mg) and methyl chloroformate (0.3 ml of 1 M dichloromethane solution).

• I

100402 26

Example 17

Factor A, 5-methyl carbonate, 23-ketone (830 mg) [α] D 0 + 132 ° (c 0.82, CHCl 3), δ (ethanol) 245 nm (δ 29,800), from Factor A, 23-ketone (916 mg) 3a of methyl 5-chloroformate (0.23 ml).

Example 18

Factor A, 5-benzyl carbonate, 23-ketone (57 mg) [α] D + 99 ° (c 0.4, CHCl 3), Bake. (ethanol) 245.5 nm (δ 29,600), λ. 7.5 - 7.2 (m, 5H), 5.57 (s, 1H), 5.21 (s, 2H), 3.70 (d, J 10 Hz, 1H), 2.49 (s, 2H); ) and 0.86 (d, J 6 Hz, 3H), Factor A, from 23-ketone (92 mg) and benzyl chloroformate (0.05 mL).

15 Example 19

(a) 5-Acetoxy-23-n-butoxy Factor A

Silver carbonate (1 g) was added to a solution of 5-acetoxy Factor A (325 mg) in dry ether, followed by iodobutane (0.5 ml) and silver perchlonate 20 (550 mg). The mixture was stirred at room temperature for 20 hours with the addition of collidine (0.5 ml). After stirring for a further 20 minutes, the mixture was filtered and the filtrate was washed successively with 2-norm. hydrochloric acid, saturated aqueous sodium hydrogencarbonate solution and water. The dried organic phase was evaporated to near dryness and the oil was purified by chromatography on Merck Kieselgel 60, 230-400 mesh (100 ml). Elution of the column with hexane: ethyl acetate (3: 1) gave the title compound as a colorless foam (276 mg, 78%), [α] 22 + 160 ° (c 0.94, 30 CHCl 3) λ makB. (ethanol) 245 nm (eBak. 28300); Δ (CDCl 3) 3.16 • · i * · (m, 1H), 3.43 (m, 1H), 3.59 (m, 1H).

• · · V (b) 5-Acetoxy-23-ethoxy Factor A was prepared in a similar manner from 5-acetoxy Factor A and ethyl iodide. The title compound was obtained as a colorless foam; 35 [α] 22 + 1670 (c 1.02, CHCl 3), Λ Bake. (ethanol) 245 nm

II

100402 27 (G 28070), δ (CDCl 3) 3.25 (m, 1H), 3.46 (m, 1H), 3.64 (m, 1H).

Example 20 23-Ethoxy

5 Factor A

A solution of 5-acetoxy-23-ethoxy Factor Aita (806 mg) in methanol (18 mL) was cooled in an ice bath, 1-norm. aqueous sodium hydroxide solution (1.3 ml) and a pale yellow solution were stirred in an ice bath for 1.25 hours. The solution was diluted with ethyl acetate (80 mL), then washed successively with 1-norm. hydrochloric acid, water and brine. The dried organic phase was evaporated to dryness and the residual resin was purified by Chromatography on Merck Kieselgel 60 silica, 230-1500 mesh (200 ml). Elution of the column with 15% ethyl acetate in dichloromethane gave the title compound as a colorless foam (623 mg), [α] D + 178 ° (c 1.13, CHCl 3) λ »<1 .. (ethanol) 244 nm (€ ac. 29.400 ); δ (CDCl 3) 4.29 (t, 7, 1H), 3.65 (m, 1H), 3.47 (m, 1H), 3.26 (m, 2H), 1.15 (t, 7 , 3H).

The compound of Example 21 was prepared in a similar manner:

Example 21 23-n-Butoxy Factor Fence (61%) was obtained as a colorless foam; [α] 11 + 161 ° (c 1.47, CHCl 3), Λ. »« κβ. (ethanol) 244 nm (6 33100); δ (CDCl 3) 4.30 (t, 7, 1H), 3.60 (m, 1H), 3.43 • · (m, 1H), 3.17 (m, 1H) 5-acetoxy-23-n ~ butoxy-Factor A.

• ♦ - «« • · ♦ · · • 1 • ·

Claims (4)

A process for the preparation of therapeutically useful macrolide compounds of formula 5. (2) / 3 CH, CH 3 Wherein O is methyl, ethyl or isopropyl, R2 is hydrogen or a group -OR5, and R3 is hydrogen, or R2 and R3 form together-; with the carbon atom to which they are attached a C = O group, wherein OR5 is -OH, -OCOR6, where R6 is C1-4 alkyl, which may be: substituted by halogen, C1-4 alkoxy or phenoxy; : -OCOOR 6a, where R 6a is C 1-4 alkyl; -OR7, where R7 is C1-4 alkyl, which may be substituted by halogen or C3-5 alkenyl; **** or -COCOCO2 R9, where R9 is hydrogen or C1-4 alkyl; and OR 4 is • 4 -OH or -OCOR 4a, where R 4a is C 1-4 alkyl which may be substituted by halogen or phenoxy; in addition to such compounds of formula (I) wherein R 2 is -OH and OR 4 is -OH, characterized in that ·. (ai) for the preparation of such compounds of formula (I), wherein one or each of the groups R2 and OR4 is an acylated hydroxy group, a corresponding compound is reacted where one or each of the groups R2 and OR4 is reacted Is a hydroxyl group, with an acylation reagent or a reactive derivative thereof, wherein as the acylation medek is used an acid of the formula R6COOH, R6aC020H, HOOCCOOR9 or R4aCOOH or reactive derivative thereof, or (a2) (I), where R 2 is an etherified hydroxy group, a corresponding compound is etherified, wherein R 2 is a hydroxyl group, with a compound of formula R 7 -Y, where R 7 is the same as above and Y is a leaving group, or (b) for the preparation of such compounds of formula (I), wherein R 2 and R 3 are hydrogen, homologically a compound of the formula L CH 1 CH 3 / J. \ L 3. H \ i H I R (III) 20 \ \ / ° H Wherein R 1 is the same as above, -OR 4 is a previously defined substituted hydroxyl group and L is a group R 11OCSO, where R 11 is C 1-6 alkyl (aryl), such as p-tolyl chloroformate, with .. Or (c) for the preparation of such compounds of formula (I). wherein R 2 and R 3 together form an oxo group, a corresponding compound is oxidized, wherein R 2 is -OH, wherein: OR 4 is a protected group in the preparation of compounds wherein OR 4 is a hydroxyl group, or II 100402 (D) for the preparation of compounds of formula (I) wherein R 2 or OR 4 is a hydroxyl group, the protecting group is removed from a corresponding compound wherein R 2 or OR 4 is a protected hydroxyl group. wherein a R 2 is hydrogen or a hydroxy group or a group of the formula -OR 7, wherein R 7 is C 1-4 alkyl or allyl, or R 2 and R 3 form together with the carbon atom to form a compound of formula (I) 10 they are bound, a C = O group .. · · · · · · · · · · · · · · · · · · · ·