Antiparasitic agents related to the milbemycins and avermectins

16 Pages·2013·1.89 MB·English

US005981500A
Ulllted States Patent [19] [11] Patent Number: 5,981,500
Bishop et al. [45] Date of Patent: Nov. 9, 1999
[54] ANTIPARASITIC AGENTS RELATED TO 5,055,596 10/1991 Baker et al. .......................... .. 549/268
THE MILBEMYCINS ANI) AVERMECTINS 5,169,839 12/1992 Linn et al. .............................. .. 514/30
[75] Inventors: Bernard Frank Bishop; Michael FOREIGN PATENT DOCUMENTS
Stephen Pacey, both of Sandwich,
United Kingdom; David Austen Perry, 379341 5/1990 European Pat Off- -
Grown, Conn 353959 7/1990 European Pat. Off. .
411897 2/1991 European Pat. Off. .
[73] Assignee: P?zer Inc., New York, NY. 428285 5/1991 European Pat O?- -
428286 5/1991 European Pat. Off. .
_ 480693 4/1992 European Pat. Off..
[21] App1'N°"08/853’686 506331 9/1992 European Pat. 011..
[22] Filed; May 9, 1997 519731 12/1992 European Pat. Off. .
535734 12/1992 European Pat. Off. .
Related US. Application Data
[63] Continuation of application No. 08/491,935, ?led as appli- P r imary Examiner —E11i Peselev
cation No. PCT/EP94/00095, Jan. 12, 1994, abandoned. Attorney, Agent, or Firm—Peter C. Richardson; Paul H.
[51] Int. Cl? .......................... .. A61K 31/70; c0711 17/08 Gmsburg; Gezma Holmlst
[52] US. Cl. ............................... .. 514/30; 514/27; 514/28; [57] ABSTRACT
536/71
[58] Field of Search .............................. .. 536/71; 514/30, A5-oximino-25-substimted avermec?n B1 or B2 K1090596
514/27, 28 charide of formula I herein has use in the treatment of
parasitic infections in animals and humans. The compounds
[56] References Cited may be prepared from corresponding 5-keto substituted
derivatives.
U.S. PATENT DOCUMENTS
5,055,454 10/1991 Blizzard et al. ........................ .. 514/30 11 Claims, No Drawings
5,981,500
1
ANTIPARASITIC AGENTS RELATED TO
THE MILBEMYCINS AND AVERMECTINS
This is a continuation of application Ser. No. 08/491,935,
?led on Jun. 22, 1995, noW abandoned, Which is a 371 of
PCT/EP 94/00095 ?led Jan. 12, 1994.
This invention relates to neW antiparasitic agents, related
to the milbemycins and avermectins and to processes for
their preparation and compositions thereof. 10
The avermectins are a group of broad spectrum antipara
sitic agents referred to previously as the C-076 compounds.
They are produced by fermenting a strain of the micro
organism Streptomyces avermitilis under aerobic conditions
15
in an aqueous nutrient medium containing inorganic salts
and assimilable sources of carbon and nitrogen. The isola o CH2R3
tion and the chemical structure of the eight individual H
N
components Which make up the C-076 complex is described |
in detail in British Patent Speci?cation 1573955 . 20 OR4
The C-076 complex comprises eight distinct but closely
related compounds described as C-076 A1 a, A1b, A2a, A2b, Wherein the broken line at the 22-23 position represents an
B1a, B1b, B2a and B2b. The “a” series of compounds refers optional bond and either this bond is present and R1 is absent
to the natural avermectins Wherein the 25-substituent is or this bond is absent and R1 is H, OH, oxo or oximino
25
(S)-sec-butyl and the “b” series to those Wherein the optionally substituted by a C1—C8 alkyl group, R2 is a C1—C8
25-substituent is isopropyl. The designations “A” and “B” alkyl, C2—C8 alkenyl or C3—C8 cycloalkyl group, or a 3- to
6- membered heterocyclic ring containing a sulphur or
refer to avermectins Wherein the 5 -substituent is methoxy or
oxygen atom, said ring being saturated or fully or partially
hydroxy, respectively, and the numeral “1” refers to aver
unsaturated and optionally substituted by one or more C1—C4
mectins Wherein a double bond is present at the 22-23 30
alkyl groups or halogen atoms,
position, and numeral “2” to avermectins lacking the 22-23
R3 is H or OH, and
double bond and having a hydrogen at the 22-position and
R4 is H or a group capable of being hydrolysed in vivo to
hydroxy at the 23 position.
yield a compound in Which R4 is H,
In our European Patent Applications 0214731, 0284176,
R5 is OH, optionally substituted With a group capable of
0317148, 0308145, 0340832, 0335541 and 0350187 there being hydrolysed in vivo to yield a compound in Which R5
are described preparations of compounds related to the
is OH.
avermectins but having a group at the 25-position other than and R6 is H or C1—C4 alkyl, or R6 is H and R5 is amino,
the isopropyl or (S)-sec-butyl groups found in the original optionally substituted With at least one group selected from
avermectin compounds disclosed in British Patent Speci? 40 C1—C8 alkyl and acyl (Which may be alkanoyl) groups.
cation 1573955. Such compounds may be prepared by Unless the context otherWise requires, all alkyl and alk
fermentation of particular strains of Streptomyces avermitilis enyl substituents having 3 or more carbon atoms may be
in the presence of organic acids or derivatives thereof. straight or branched-chain. The term “aryl” includes phenyl
Production of such avermectins is described in Journal of Which may be substituted by at least one C1—C6 alkyl,
45
Antibiotics (1991), 44, No. 3, pp 357—365. hydroxy, C1—C6 alkoxy, halo, nitro or CF3 group. In the
The milbemycins form another group of related mac present invention the term “alkyl” is intended to indicate
rolides Which are distinguished from the avermectins in those alkyl groups of from 1 to 8 carbon atoms such as
lacking a sugar residue attached at the C-13 position. methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, and the
Examples of such compounds are described in UK patent like, either straight or branched chain. The term “alkanoyl”
1390336, and European patent publications 170006, is intended to include those alkanoyl groups of from 1 to 8
254583, 334484 and 410615. In addition to these fermen carbon atoms such as formyl, acetyl, propionyl, buytryl,
tation products a large number of publications describe pentanoyl, hexanoyl, and the like.
compounds derived semisynthetically from these fermenta The term “carbamoyl” is intended to indicate the group
tion products many of Which possess useful antiparasitic 55 —CONR7R8 Where R7 and R8 are the same or different and
activities. Some of this chemistry is revieWed in Macrolia'e are H, alkyl, aryl, heteroaryl or from a 4—8 membered ring
Antibiotics, Omura 8., Ed., Academic press, NeW York containing 1 or more O, N, or S atoms.
(1984) and by Davies, H. G., Green, R. H. in Natural The above structural formula is shoWn Without a de?ni
product Reports (1986), 3, 87—121 and in Chem. Soc. Rev., tive stereochemistry. HoWever, during the course of the
60
1991, 2, 271—339. synthetic procedures used to prepare such compounds, the
It has been found that certain compounds synthetically products of such procedures can be a mixture of stereoiso
derivable from knoWn avermectins and avermectin deriva mers. In particular, the stereoisomers at the 4“-, 4‘-, 5, 13
tives possess unexpected bene?cial biological properties. and 23-positions may be oriented either ot- or [3-representing
According to one aspect of the invention there are 65 such groups being beloW or above the general plane of the
provided compounds of formula (I): molecule, respectively. In each of such case both the ot- and
5,981,500
3
[3-con?gurations are intended to be included Within the
ambit of this invention. In certain cases the term “epi” is (III)
used to distinguish the stereoisomer being of opposite con
?guration to the natural compound at one speci?c asym
metrical carbon atom.
Groups hydrolysable in vivo to yield corresponding com
pounds in Which the group is replaced by H are Well knoWn
10
in the pharmaceutical art in general and a Wide variety of
such groups are suitable for use in the compounds of this
invention. Examples of such groups are C2—C8 alkanoyl,
aroyl, carbamoyl, C1—C8 alkoxycarbonyl groups, and dicar
boxylic acid and amino acid residues. Particular groups are 15
identi?ed in the Examples beloW. Preferred compounds are
o CH2R3
those in Which R2 is a straight or branched C1—C8 alkyl or
cycloalkyl group, such as cyclohexyl, isopropyl or sec-butyl,
R3 is H, and the optional bond at the 22-23 position is
present or this optional bond is absent and R1 is H or OH.
and (ii) alloWing the compound of formula (III) to react With
a compound of formula R“—O—NH2 Where R4 is as
The oxime monosaccharides in Which R3 and R4 are H are de?ned above and Where R5 is ot-oleandrosyl oxy, hydrol
particularly preferred. ysing the compound obtained to yield a compound of
25 formula (I), and (iii) if necessary replacing group R4 When
the latter is H With said group capable of being hydrolysed
Individual compounds Within the invention are described in vivo to yield a compound in Which R4 is H,
in the Examples given beloW.
if necessary, the process further comprising one or more of
the folloWing steps before or after steps (i), (ii) and (iii),
The most preferred compound is 5-oximino-22,23 (iv) substituting group R5 When the latter is OH With said
dihydro-25-cyclohexyl avermectin B1 monosaccharide. group capable of being hydrolysed in vivo to yield a
compound in Which R5 is OH,
(v) oxidising group R1 When the latter is OH to oxo,
According to another aspect of the invention, there is
35
(vi) reacting the compound obtained from step (v) With
provided a process for preparing such a compound Which
hydroxylamine optionally substituted by a C1—C8 alkyl
comprises the steps (1) of oxidising a compound of formula
group to yield a compound in Which R1 is optionally
(II):
substituted oxo,
(vii) hydrogenating the compound to reduce a double bond
(II)
at the 22-23 position to a single bond,
(viii) oxidising a compound in Which R3 is H to a compound
in Which R3 is OH,
(ix) oxidising a compound in Which R5 is OH and R6 is H
45
to a compound in Which R5 is oxo and R6 is absent, and
either:
(x) reducing the compound obtained from to produce a
compound in Which R5 is an epi-OH group, or
(xi) reacting the compound obtained from With a Grig
nard reagent to yield a compound in Which R5 is OH and R6
is alkyl, or
(xii) subjecting the compound obtained from to reduc
tive amination to yield a compound in Which R5 is an amino
55
or alkylamino group, and if necessary acylating the com
pound obtained, any free OH groups being protected if
necessary during any of the above steps.
Preparation of compounds according to the invention is
discussed, by Way of illustration, beloW.
60
Wherein the broken line, R1, R2, R3 and R6 are as de?ned The compounds of the present invention may be prepared
starting from the compounds of formula (iv), Which may
above and R5 is as de?ned above or R5 is ot-oleandrosyl oxy
themselves be prepared as described in the above-mentioned
and R6 is H to yield a compound of formula (III):
patent publications.
5,981,500
(H)
OCH3
HO 4,.
OCH3
CH3 0 o 4,
CH3
CH3 0
CH3
Compound IIa Double bond present, R1 absent.
Compound IIb Double bond absent, R1 = H.
Compound IIc Double bond absent, R1 = OH.
30
The semisynthetic modi?cations required to provide the of the silyl compound With anhydrous pyridine-hydrogen
compounds given by formula I may require sequential ?uoride in tetrahydrofuran. The reaction is complete in from
reactions at the positions —4‘, 4“, 1, 4a, 13, 22, 23, 25 and 5 3 to 24 hours at from 0 to 25° C. Alternatively, the silyl
and the exact order in Which these transformations are group may be removed by stirring the silylated compound in
performed may vary. In addition, during the oxidation and 35 methanol catalyzed by an acid preferably a sulfonic acid
certain substitution reactions described beloW, it is necessary monohydrate such as p-toluenesulfonic acid monohydrate.
to protect the 5-hydroxy group to avoid substitution or The reaction is complete in about 1 to 12 hours at from 0 to
oxidation at that position. With this position protected, the 50° C. Compounds having a 23-hydroxyl group (or a pro
reactions may be carried out at the 4“-, or 4‘-positions tected derivative) may be converted to either the correspond
Without affecting the remainder of the molecule. Subsequent 40 ing 22,23-dihydro compound or alternatively to the corre
to any of the above-described reactions, the protecting group sponding compound having a double bond at the 22-23
may be removed and the unprotected product isolated. For position using methods described in US. Pat. No. 4,328,
compounds of this invention, conversion of the 5-hydroxy 335. The latter compounds may also be hydrogenated to the
group to a ketone or ketoxime is preferably performed after 22,23-dihydro compounds using Wilkinson’s catalyst under
substitution at the 4‘, 23 and 4a positions. The protecting 45 conditions described in US. Pat. No. 4,199,569.
group employed at the 5-position is ideally one Which may The preparation of the compounds of the invention can be
be readily synthesized, Will not be affected by the reactions accomplished by ?rst converting the aforementioned disac
at the 4“- and 4‘-positions and may be removed Without charides IIa, b and c into their corresponding monosaccha
affecting any other functionality of the molecule. One pre rides by hydrolysis. An alternative method of preparing the
ferred type of protecting group of the avermectin type of monosaccharides comprises a direct fermentation procedure
molecule is the tri-substituted silyl group, preferably the starting from a corresponding aglycone as described in
tri-loWeralkyl silyl group. One especially preferred example European Patent Application 463677. The 5-hydroxy posi
is the tert.-butyldimethylsilyl group. The reaction preparing tion is then protected to avoid substitution at this position so
the protected compound is carried out by reacting the that reaction may be carried out at the 4‘, or 23 positions. The
hydroxy compound With the appropriately substituted 55 4‘-hydroxy is more reactive than the 23-hydroxy, hence
silylhalide, preferably the silylchloride in an aprotic solvent additional suitable protection at C-4‘ alloWs selective reac
such as methylene chloride, benzene, toluene, ethyl acetate, tion at C-23.
tetrahydrofuran, dimethylformamide and the like. In order to Alternatively, the compounds of the invention may be
minimise side reactions, there is included in the reaction prepared by carrying out the above synthetic manipulations
mixture a base to react With the acid released during the 60 on the disaccharides IIa, b or c and ?nally hydrolysing them
course of the reaction. Preferred bases are amines such as to the desired monosaccharides.
imidazole, pyridine, or triethylamine. The base is required in When desired, hydroxy groups may be acylated to give
amounts equimolar to the amount of hydrogen halide liber esters using reagent such as acid anhydrides or acid chlo
ated; however, generally several equivalents of the amine rides and amine bases according to general procedures
are employed. The reaction is stirred at from 0° C. to the 65 knoWn to those skilled in the art. Hydroxy groups may be
re?ux temperature of the reaction mixture and is complete in converted to oxo groups by oxidation With manganese
from 1/2 to 16 hours. The silyl group is removed by treatment dioxide or tetrapropylammonium perruthenate. The oxo
5,981,500
7 8
compound may be treated With hydroxylamine or an starch, lactose, talc, or magnesium stearate. A drench for
O-substituted analogue thereof to produce the corresponding mulation may be prepared by dispersing the active ingredi
oxime. ent in an aqueous solution together With dispersing or
Compounds in Which R3 is OH are prepared from suitably Wetting agents and injectable formulations may be prepared
5-protected derivatives by ?rstly hydroxylating the 4a in the form of a sterile solution or emulsion. Pour-on or
methyl group using a procedure described in the Japanese spot-on formulations may be prepared by dissolving the
Patent Laid-Open Patent Application No. 83-59988. active ingredient in an acceptable liquid carrier vehicle, such
Compounds in Which R5 is an amino group optionally as butyl digol, liquid paraffin or non-volatile ester With or
substituted by one or more alkyl or acyl groups, may be Without addition of a volatile component such as isopro
prepared by reduction amination of a corresponding com 10 panol. Alternatively, pour-on, spot-on or spray formulations
pound in Which R5 is oxo, for example by reaction With an can be prepared by encapsulation to leave a residue of active
ammonium salt or amine salt and sodium cyanoborohydride agent on the surface of the animal. These formulations Will
in knoWn manner. The amino group may then be further vary With regard to the Weight of active compound depend
substituted, by acetylation, for example using acetic anhy ing on the species of host animal to be treated, the severity
dride. 15 and type of infection and the body Weight of the host. The
Compounds in Which R5 is an epi-OH group may be made compounds may be administered continuously, particularly
by reduction of such an oxo compound. The oxo compound for prophylaxis, by knoWn methods. Generally for oral,
may be converted to a compound in Which R5 is OH and R6 parenteral and pour-on administration a dose of from about
is alkyl by reaction With a Grignard reagent in knoWn 0.001 to 10 mg per kg of animal body Weight given as a
manner. 20 single dose or in divided doses for a period of from 1 to 5
The compounds of the invention are effective in treating days Will be satisfactory but of course there can be instances
a variety of conditions caused by endoparasites including, in Where higher or loWer dosage ranges are indicated and such
particular, helminthiasis Which is most frequently caused by are Within the scope of this invention.
a group of parasitic Worms described as nematodes and As an alternative the compounds may be administered
Which can cause severe economic losses in sWine, sheep, 25 With the animal feedstuff and for this purpose a concentrated
horses and cattle as Well as affecting domestic animals and feed additive or premix may be prepared for mixing With the
poultry. The compounds are also effective against other normal animal feed.
nematodes Which affect various species of animals For use as an insecticide and for treating agricultural pests
including, for example:- Diro?laria in dogs and various the compounds are applied as sprays, dusts, pour-on
parasites Which can infest livestock, companion animals 30 formulations, emulsions and the like in accordance With
such as cats and dogs and also humans including gastrointes standard agricultural practice.
tinal parasites such as Ancylostoma, Necator, Ascaris, For human use the compounds are administered as a
Strongyloides, Trichinella, Capillaria, Toxocara, Toxascaris, pharmaceutically acceptable formulation in accordance With
Trichuris, Enterobius and parasites Which are found in the normal medical practice.
blood or other tissues and organs such as ?larial Worms and 35 The preparation of compounds according to the invention
the extra intestinal stages of Strongyloides, Toxocara and are illustrated by the folloWing Examples.
Trichinella.
The compounds are also of particular value in treating EXAMPLE 1
ectoparasite infections including particular arthropod ecto
22,23-Dihydroavermectin Bla monosaccharide
parasites of humans, animals and birds such as ticks, mites, 40
lice, ?eas, bloW?y, biting insects and migrating dipterous 22,23-Dihydroavermectin Bla (50 g) Was dissolved in a
larvae Which can affect cattle and horses. mixture of isopropanol (100 ml) and sulphuric acid (1 ml)
The compounds are also insecticides active against house and stirred at room temperature under a nitrogen atmosphere
hold pests such as the cockroach, clothes moth, carpet beetle for 48 hours. The reaction mixture Was poured onto crushed
and the house?y as Well as being useful against arthropod 45 ice and extracted With dichloromethane (2x200 ml). The
pests of stored grain and of agricultural plants such as spider combined extracts Were Washed With aqueous saturated
mites, aphids, caterpillars and against migratory orthopter sodium bicarbonate solution (100 ml), dried over anhydrous
ans such as locusts. We have discovered that compounds magnesium sulphate and concentrated under vacuum to give
Within the scope of this invention are both safe and have White crystals (14 g) Which Were removed by ?ltration. Mass
unexpectedly high potent systemic activity against ?eas and 50 and NMR spectra Were fully consistent With the proposed
other important arthropod parasites of cats and dogs. structure.
The compounds of formula (I) may be administered as a The Blb analogue Was obtained by an identical method
formulation appropriate to the speci?c use envisaged and to starting from 22,23-dihydroavermectin Blb.
the particular species of host animal being treated and the
parasite or insect involved. The compounds may be admin 55 EXAMPLE 2
istered by injection, either subcutaneously or
5 -Oxo -22,23-dihydro avermectin B 1a
intramuscularly, alternatively they may be administered
monosaccharide
orally in the form of a capsule, bolus, tablet, cheWable tablet
or liquid drench, or they may be administered as a topical 22,23-dihydroavermectin Bla monosaccharide (14 g) Was
formulation or as an implant. For topical application dip, 60 dissolved in diethyl ether (200 ml) and activated manganese
spray, poWder, dust, pour-on, spot-on, jetting ?uid, dioxide (14 g) added. The mixture Was stirred at room
shampoos, collar, tag or harness may be used. Such formu temperature for 4 hours, ?ltered and evaporated to dryness
lations are prepared in a conventional manner in accordance under vacuum to yield the title product (11.4 g) Whose NMR
With standard veterinary practice. Thus capsules, boluses or spectrum Was fully consistent With the proposed structure.
tablets may be prepared by mixing the active ingredient With 65 The Blb analogue Was obtained by an identical method
a suitable ?nely divided diluent or carrier, additionally starting from 22,23-dihydroavermectin Blb monosaccha
containing a disintegrating agent and/or binder such as ride.
5,981,500
9 10
EXAMPLE 3 compound (3.22 g) as a yelloW foam. This Was dissolved in
anhydrous pyridine (60 ml) and hydroxylamine hydrochlo
5 -Oximino-22,23 -dihydro avermectin B 1 a ride (3.22 g) Was added. After stirring for 15 hours at room
monosaccharide temperature a further aliquot of hydroxylamine hydrochlo
ride (3.22 g) Was added and the solution heated to 50° C.
5-Oxo-22,23-dihydroavermectin B1 a monosaccharide (1
until no starting material remained. The solution Was poured
g) Was dissolved in dry pyridine (25 ml) and hydroxylamine
into Water (50 ml) and extracted With diethyl ether (3x50
hydrochloride (1 g) added. The stirred reaction mixture Was
ml). The organic layer Was Washed With Water, saturated
heated under re?ux for 4 hours and after cooling poured onto
sodium chloride solution, dried over anhydrous sodium
crushed ice and extracted With dichloromethane (2><50ml).
10 sulphate and evaporated to dryness under vacuum. The
The combined extracts Were dried over anhydrous magne
crude product Was chromatographed on silica (25 g) eluting
sium sulphate and evaporated under vacuum to give a crude
With dichloromethane: ethyl acetate 4:1 and ?nally puri?ed
gum (1.1 g). This material Was puri?ed using high pressure
by high pressure liquid chromatography using a Dynamax
liquid chromatography on a Dynamax (trade mark) column
(trade mark) column (41.4><250 mm, 8 pm ODS-silica,
(41.4><250 mm, 8 pm, ODS-silica, Rainin) eluting With
15 Rainin) eluting With methanol:Water 9: 1 at 65 ml per minute.
methanol - Water 83:17 at 42 ml per minute. Appropriate
Appropriate fractions Were combined and evaporated under
fractions Were combined and evaporated to dryness to yield
vacuum to give the title compound (1.53 g). Mass and NMR
the title product as a White solid, melting point 180°—190° C.
spectra Were fully consistent With the proposed structure.
Mass and NMR spectra Were fully consistent With the
proposed structure. EXAMPLE 6
5-Oximino-22,23-dihydroavermectin B1b Was prepared by 20
25-Cyclohexylavermectin B2 monosaccharide
an identical method starting from 5-oxo-22,23
dihydroavermectin B1b monosaccharide. 25-Cyclohexylavermectin B2 (10 g) Was suspended in
isopropanol (100 ml) and a solution of sulphuric acid (2 ml)
EXAMPLE 4 in isopropanol (100 ml) Was added. After stirring at room
25 temperature for 24 hours the clear solution Was poured into
22,23-Dihydro-25-cyclohexylavermectin B1 ice (600 g) and extracted With dichloromethane (2x100 ml).
monosaccharide The organic layer Was dried over anhydrous sodium sulphate
and evaporated to dryness. The residue Was dissolved in
25-Cyclohexylavermectin B1 (9.9 g) Was dissolved in
tetrachloromethane and the solution stored at 4° C. The
toluene (1 liter) and Wilkinson’s catalyst (tris
30 crystals Which separated sloWly Were periodically removed
(triphenylphosphine)rhodium(I) chloride) (9.25 g) Was
by ?ltration and found to be the pure title compound. Mass
added. The solution Was hydrogenated on a large Parr (trade
and NMR spectra Were fully consistent With the proposed
mark) shaker at room temperature at 50 psi hydrogen
structure.
pressure. After 3 hours the reaction vessel Was depressurised
and alloWed to stand for 12 hours before addition of a further 35 EXAMPLE 7
portion of catalyst (5 g) and hydrogenated as before for a
5 -Oximino -25 -cyclohexylavermectin B2
further 2 hours after Which no starting material remained.
monosaccharide
The solution Was ?ltered, evaporated to dryness under
vacuum and the residue chromatographed on silica eluting Using the procedures of Examples 2 and 3,
With dichloromethane then dichloromethane:methanol 9:1. 25-cyclohexylavermectin B2 monosaccharide Was con
40
The crude product Was then chromatographed again on silica verted to the title compound. Mass and NMR spectra Were
(200 g) eluting With dichloromethane:methanol 19:1 to give fully consistent With the proposed structure.
after evaporation of the solvent under vacuum impure 22,23 EXAMPLE 8
dihydro-25-cyclohexylavermectin B1 as a broWn foam (10
25-Cyclohexylavermectin B1 monosaccharide
g). This material Was dissolved in a mixture of isopropanol
45
(200 ml) and sulphuric acid (2 ml) and the broWn solution 25-Cyclohexylavermectin B1 (20 g) Was dissolved in
Was stirred at room temperature for 15 hours then poured tetrahydrofuran (250 ml) and a mixture of tetrahydrofuran
into a mixture of ice and Water (500 ml) and extracted With (250 ml), Water (10 ml) and sulphuric acid (10 ml) Was
dichloromethane (3><200ml). The organic layer Was Washed added. The mixture Was stirred at room temperature for 15
With saturated aqueous potassium hydrogen carbonate solu hours then poured into a mixture of ice (500 g) and Water (1
tion (100 ml), Water (2x50 ml) dried over anhydrous mag l) and extracted With dichloromethane (2x500 ml). The
nesium sulphate and evaporated under vacuum to give a organic layer Was Washed With saturated aqueous sodium
crude gum Which Was chromatographed on silica (100 g) chloride solution, dried over anhydrous sodium sulphate and
eluting With dichloromethane then dichloromethane:ethyl evaporated under vacuum to give a foam. This Was chro
acetate 2:1 to give the title compound (8.2 g). Mass and matographed on silica (150 g) eluting With ethyl acetate -
55
NMR spectra Were fully consistent With the proposed struc dichloromethane 1:1 to give a crude product (13.3 g). Final
ture. puri?cation Was achieved by reverse phase hplc using a
Dynamax (trade mark) column (41.4><250mm, 8 pm ODS
EXAMPLE 5 silica, Rainin) eluting With methanol-Water 4:1 at 70 ml per
minute to give the pure title compound. Mass and NMR
5-Oximino 22,23-dihydro-25-cyclohexyavermectin 60
spectra Were fully consistent With the proposed structure.
B1 monosaccharide
EXAMPLE 9
22,23-Dihydro-25-cyclohexylavermectin B1 monosac
charide (8.2 g) Was oxidiZed to the 5-oxo derivative using 5 -Oximino -25 -cyclohexylavermectin B 1
manganese dioxide in anhydrous diethyl ether according to 65 monosaccharide
the procedure of Example 2. The crude product Was puri?ed Using the procedures of Examples 2 and 3,
by chromatography on silica (50 g) to give the 5-oxo 25-cyclohexylavermectin B1 monosaccharide Was con
5,981,500
11 12
verted to the title compound. Mass and NMR spectra Were Were dried over anhydrous magnesium sulphate and con
fully consistent With the proposed structure. centrated under vacuum. The resulting crude product (25
mg) Was puri?ed using high pressure liquid chromatography
EXAMPLE 10 on an Ultrasphere (trade mark) column (24><250 mm, 5
microns, ODS-silica, Beckman) eluting With methanol:
5 -O-t-Butyldimethylsilyl-25-cyclohexyl-22,23
Water 85:15 at 20 ml per minute. Appropriate fractions Were
dihydroavermectin B1 monosaccharide
combined, to give the title product. Mass and NMR spectra
Were fully consistent With the proposed structure.
22,23-dihydro-25-cyclohexyl-avermectin B1 monosac
charide (Example 4) (12.1 g) and imidaZole (7.2 g) Were
EXAMPLE 14
dissolved in dry dimethylformamide (10 ml). To this solu 10
tion at room temperature Was added t-butyldimethylsilyl 4‘-O-Acetyl-25-cyclohexyl-22,23-dihydroavermectin
chloride (7.9 g). After 18 hours the mixture Was poured into B1 monosaccharide
ice-Water (200 ml) acidi?ed to pH2 With 2N HCl and
5-O-t-Butyldimethylsilyl-25-cyclohexyl-22,23
extracted With diethyl ether (2x80 ml). The combined
extracts Were Washed With saturated aqueous potassium 15 dihydroavermect in B1 monosaccharide (Example 10) (216
mg) Was dissolved in dichloromethane (25 ml) containing
bicarbonate solution (50 ml) and Water (50 ml), dried over
pyridine (400 mg). Acetic anhydride (255 mg) in dichlo
anhydrous sodium sulphate and evaporated under vacuum to
give a crude product (14.9 g). This material Was further romethane (5 ml) Was added sloWly to this solution at room
puri?ed by chromatography on silica (Kieselgel 60, temperature and the reaction mixture Was alloWed to stand
230—240 mesh, Merck) (300 g) eluting With dichlo 20 for 72 hr. The solution Was poured into Water (20 ml), the
organic layer Was Washed With aqueous citric acid solution
romethane - ethyl acetate 9:1. Appropriate fractions Were
combined and evaporated to dryness to give the title product (20%, 2x10 ml), saturated aqueous potassium bicarbonate
solution (2x10 ml), dried over anhydrous sodium sulphate
(8.35 g). The NMR spectrum Was fully consistent With the
proposed structure. and evaporated to dryness under vacuum. This crude product
25 Was chromatographed on silica (25 g) eluting With dichlo
EXAMPLE 11 romethane - ethyl acetate 9:1. Appropriate fractions Were
combined and evaporated under vacuum to give 4‘-O-acetyl
5-Oxo-avermectin B1a 5-O-t-butyldimethylsilyl-25-cyclohexyl-22,23
dihydroavermectin B1 monosaccharide Which Was dissolved
Avermectin B1a (2.4 g) Was dissolved in diethyl ether (50
30 in methanol (20 ml) containing p-toluenesulphonic acid (230
ml) and activated manganese dioxide (2.0 g) added. The
mg). After stirring at room temperature for 1 hr, saturated
mixture Was stirred at room temperature for 18 hours,
aqueous potassium bicarbonate solution (5 ml) Was added
?ltered and evaporated to dryness under vacuum to yield the
and the product extracted With diethyl ether (2x10 ml). The
title product Whose NMR spectrum Was fully consistent With
combined organic extracts Were dried over anhydrous
the proposed structure.
35 sodium sulphate and evaporated to dryness under vacuum to
give the title product as a White poWder (139 mg) Whose
EXAMPLE 12
NMR spectra Were fully consistent With the proposed struc
ture. This material Was used in the folloWing example
5 -Oximino-avermectin B 1a
Without further puri?cation.
5-Oxo-avermectin B1a (800 mg) (Example 11) Was dis
40
solved in pyridine (10 ml) and hydroxylamine hydrochloride EXAMPLE 15
(800 mg) added. After stirring at room temperature for 1
4-O-Acetyl-5-oximino-25-cyclohexyl-22,23
hour the mixture Was poured into an ice (50 g) and Water(50
dihydroavermectin B1 monosaccharide
ml) mixture, acidi?ed to pH 4 With concentrated hydrochlo
ric acid and extracted With dichloromethane (3x30 ml). The 45 4‘-O-Acetyl-25-cyclohexyl-22,23-dihydroavermectin B1
combined extracts Were Washed With Water (20 ml), dried monosaccharide (139 mg) (Example 14) Was oxidised to the
over anhydrous sodium sulphate and evaporated to dryness 5-oxo-derivative using activated manganese dioxide (140
under reduced pressure to yield a crude material (1 g). This mg) in diethyl ether (20 ml) according to the method
material Was chromatographed on silica (Kieselgel 60, described in example 2. To a solution of this 4‘-O-acetyl-5
230—400 mesh, Merck) (10 g) eluting With dichloromethane: oxo-25-cyclohexyl-22,23-dihydroavermectin B1 monosac
ethyl acetate 2:1 and ?nally puri?ed by high pressure liquid
charide in a mixture of methanol - dioxan 1:1 (20 ml) Was
chromatography using a Dynamax (trade mark) column added a solution of hydroxylamine hydrochloride (176 mg)
(41.4><250 mm, 8 pm ODS-silica, Rainin) eluting With dissolved in Water (5 ml). This mixture Was stirred at 40° C.
methanol:Water 85:15 at 70 ml per minute. Appropriate for 3 hr before quenching With the addition of solid potas
fractions Were combined and evaporated under vacuum to 55 sium bicarbonate (200 mg) and diethyl ether (50 ml). The
give the title compound (290 mg). Mass and NMR spectra organic extract Was Washed With saturated aqueous sodium
Were fully consistent With the proposed structure. chloride solution (10 ml), dried over anhydrous sodium
sulphate and evaporated to dryness under vacuum to give the
EXAMPLE 13 crude product. This material Was then puri?ed by chroma
60 tography on silica (Kieselgel 60, 230—400 mesh, Merck)
5-Oximino-avermectin B1a monosaccharide
(log) eluting With dichloromethane - ethyl acetate 4:1.
5-oximino-avermectin B1a (50 mg) (Example 12 ) Was Appropriate fractions Were combined and evaporated to give
dissolved in a mixture of isopropanol (1 ml) and sulphuric the desired compound Which Was further puri?ed using high
acid (10 pl) and stirred at room temperature under a nitrogen pressure liquid chromatography on a Ultrasphere (trade
atmosphere for 48 hours. A saturated aqueous solution of 65 mark) column (10x250 mm, 5 pm, ODS-silica, Beckman)
sodium bicarbonate (1 ml) Was then added and the product eluting With acetonitrile:methanol:Water 71:14:15 at 5 ml
extracted With ethylacetate (2><5 ml). The combined extracts per minute. Appropriate fractions Were combined and
5,981,500
13 14
evaporated to dryness to yield the title product as a White ganese dioxide according to the procedure described in
solid (46 mg). Mass and NMR spectra Were fully consisted Example 2. The NMR spectrum of the product (160 mg) Was
With the proposed structure. fully consistent With the proposed structure.
EXAMPLE 16
EXAMPLE 20
4‘-oxo -5 -O-Butyldimethylsilyl-25 -cyclohexyl-22,23
4‘-epi-5-Oximino-25-cyclohexyl-22,23
dihydroavermectin B 1 monosaccharide
dihydroavermectin B1 monosaccharide
5-O-Butyldimethylsilyl-25-cyclohexyl-22,23
dihydroavermectin B1 monosaccharide (Example 10) (1.4 4‘-epi-5-Oxo-25-cyclohexyl-22,23-dihydroavermectin
g) Was dissolved in dichloromethane (300 ml) at room 10 B1 monosaccharide (160 mg) Was treated With hydroxy
temperature together With N-methyl morpholine-N-oxide lamine hydrochloride (150 mg) in a methanol-dioxan-Water
(3.14 g) and tetra-n-propylammonium perruthenate (233 mixture 1:1:0.5 (25 ml) and the product extracted, according
mg). PoWdered molecular sieve, 4A(187 mg) Was added and to the procedure described in Example 15. Puri?cation Was
the mixture stirred. After 1 hour aqueous sodium sulphite achieved using high pressure liquid chromatography on a
solution, (50 ml, 5%) Was added and the separated organic 15 Ultrasphere (trade mark) column (10x250 mm, 5p, ODS
phase Washed With a second portion of aqueous sodium silica, Beckmann) eluting With acetonitrile-methanol-Water,
sulphite (50ml, 5%), Water (2><50ml), saturated aqueous 61:14:25 at 4 ml per minute. Appropriate fractions Were
sodium chloride solution (2x50ml) and dried over anhy combined and evaporated under vacuum to give the title
drous sodium sulphate. Evaporation of the solvent under product as a solid (25 mg). Mass and NMR spectra Were
vacuum folloWed by chromatography on silica (Kieselgel 20 fully consistent With the proposed structure.
60, 230—400 mesh, Merck) eluting With dichloromethane
ethyl acetate 9:1 and combination and evaporation of appro EXAMPLE 21
priate fractions, gave the title Droduct as a solid (857 mg).
4a-Hydroxy-25-cyclohexyl-22,23
The NMR spectra Were fully consistent With the proposed
structure. 25 dihydroavermectin B1 monosaccharide
EXAMPLE 17 To a solution of 25-cyclohexyl-22,23-dihydroavermectin
B1 monosaccharide (Example 4) (5g) in dichloromethane
4‘-epi-5-O-t-Butyldimethylsilyl-25-cyclohexyvl-22,
(150 ml) Was added a solution of selenium dioxide (370 mg)
23-dihydroavermectin B1 monosaccharide
and t-butylhydroperoxide (70%, 3.7 ml) in dichloromethane
4‘-oxo-5-O-t-Butyldimethylsilyl-25-cyclohexyl-22,23 30
(50 ml) and the Whole stirred at room temperature for 48
dihydroavermectin B1 monosaccharide (200 mg) Was dis hours. A further solution containing selenium dioxide (370
solved in methanol (10 ml) at 0° C. and sodium borohydride mg) and t-butylhydroperoxide (70%, 3.7 ml) in dichlo
(20 mg) added portionWise With stirring. After 15 minutes romethane (50 ml) Was added and stirring continued for 24
the mixture Was poured into Water, extracted With diethyl hours. After addition of Water (20 ml) the reaction mixture
ether (2><30ml) and the combined organic extract Washed Was extracted With dichloromethane (2><25 ml) and the
With Water (20 ml), saturated aqueous sodium chloride combined extracts Washed With 10% aqueous sodium bicar
solution (20 ml) and dried over anhydrous sodium sulphate. bonate solution (20 ml), and Water (20 ml), dried over
Evaporation of the solvent under vacuum afforded the title anhydrous sodium sulphate and evaporated under vacuum to
Droduct as a White solid, 141 mg. Mass and NMR spectra yield the crude product as a yelloW foam, 4.5 g. This
40
Were fully consistent With the proposed structure. material Was further puri?ed by chromatography over silica
(Kieselgel 60, 230—400 mesh, Merck) (120 g) eluting With
EXAMPLE 18
dichloromethane-methanol 97:3, taking 200 ml fractions.
4‘-epi-25-Cyclohexyl-22,23-dihydroavermectin B1
Material eluting in fractions 21—24 Was combined and
monosaccharide
evaporated under vacuum to yield the title comDound as a
45
4‘-epi-5-O-t-Butyldimethylsilyl-22,23 White poWder. Mass and NMR spectra Were fully consistent
dihydroavermectin B1 monosaccharide 141 mg, Was dis With the proposed structure.
solved in methanol (20 ml) containing p-toluenesulphonic
acid (200 mg). After 18 hours the reaction mixture Was EXAMPLE 22
basi?ed by adding saturated aqueous potassium bicarbonate
4a-t-Butyldimethylsilyloxy-25-cyclohexyl-22,23
solution (20 ml) and extracted With diethyl ether (2><50ml).
dihydroavermectin B1 monosaccharide
The combined extracts Were Washed With Water (20 ml),
saturated aqueous sodium chloride solution (20 ml), dried 4a-Hydroxy-25-cyclohexyl-22,23-dihydroavermectin B1
over anhydrous sodium sulphate and evaporated under monosaccharide (1.26 g), triethylamine (0.2 ml) and
vacuum to yield the crude product. Chromatography of this 55 4-dimethylaminopyridine (26 mg) Were dissolved by stirring
material on silica (Kieselgel 60, 230—400 mesh, Merck) (2g) in dry dichloromethane (120 ml) and t-butyldimethylsilyl
eluting With dichloromethane-ethyl acetate 4:1 and combi chloride (0.32 g) added portionWise. The Whole Was stirred
nation and evaporation under vacuum of appropriate under a nitrogen atmosphere for 18 hours after Which further
fractions, afforded the title product (100 mg). Mass and quantities of triethylamine (0.4 ml),
NMR spectra Were fully consistent With the proposed struc 4-dimethylaminopyridine (252 mg) and t-butyldimethylsilyl
60
ture. chloride (0.64 g) Were added and stirring continued for 3
EXAMPLE 19 hours. An aqueous solution of sodium bicarbonate (10%,
100 ml) Was added to the reaction and the organic layer
4‘-epi-5-Oxo-25-cyclohexyl-22,23
separated, Washed With Water (100 ml), dried over anhy
dihydroavermectin B1 monosaccharide
65 drous sodium sulphate and evaporated under vacuum to give
4‘-epi-25-Cyclohexyl-22,23-dihydroavermectin B1 the crude product. This material Was puri?ed by chroma
monosaccharide (196 mg) Was oxidised With activated man tography on silica (Kieselgel 60, 230—400 mesh, Merck)
5,981,500
15 16
(120 g) eluting With dichloromethane-methanol, 98:2 col (Example 25) Was dissolved in methanol (20 ml) containing
lecting 130 ml fractions. Material eluting in fractions 12—14 p-toluenesulphonic acid (36 mg) and stirred at ambient
Were combined and evaporated under vacuum to yield the temperature for 2 hours. After this time solid potassium
title compound as a White solid, (1.1 g). Mass and NMR bicarbonate (50 mg) Was added and the mixture diluted With
spectra Were fully consistent With the proposed structure. Water (20 ml) and extracted With diethyl ether (2><20 ml).
EXAMPLE 23 The combined extracts Were Washed With Water (20 ml),
saturated aqueous sodium chloride solution (10 ml), dried
4a-t-Butyldimethylsilyloxy-5-oxo-25-cyclohexyl-22,
over anhydrous sodium sulphate and evaporated under
23-dihydroavermectin B1 monosaccharide
vacuum to yield a crude product. This material Was chro
4a-t-Butyldimethylsilyloxy-25-cyclohexyl-22,23
10 matographed on silica (Kieselgel 60, 230—400 mesh, Merck)
dihydroavermectin B1 monosaccharide (1.1 g) Was oxidised (10 g) eluting With dichloromethane-ethyl acetate 4:1.
With activated manganese dioxide (1 g) in ether (10 ml)
Appropriate fractions Were combined and evaporated under
according to the method described in Example 2 to give the
vacuum to give the product Which Was further puri?ed by
title compound (0.9 g) Whose NMR spectrum Was fully
preparative high pressure liquid chromatography on an
consistent With the proposed structure.
15 Ultrasphere (trade mark) column (10><250 mm, 5 pm, ODS
EXAMPLE 24 silica, Beckmann) eluting With methanol-Water 85:15 at 5 ml
4a-Hydroxy-5-oximino-25-cyclohexyl-22,23 per minute. Appropriate fractions Were combined and
dihydroavermectin B1 monosaccharide evaporated under vacuum to yield the title product as a White
poWder. Mass and NMR spectra Were fully consistent With
4a-t-Butyldimethylsilyloxy-5-oxo-25-cyclohexyl-22,23
20 the proposed structure.
dihydroavermectin B1 monosaccharide (0.9 g) Was dis
solved in a methanol-dioxan (1:1 mixture , 36 ml) and EXAMPLE 27
hydroxylamine hydrochloride (0.9 g) dissolved in Water (18
4‘-Methyl-5 -oxo-25 -cyclohexyl-22,23
ml) added. This reaction mixture Was stirred and heated at
dihydroavermectin B 1 monosaccharide
40° C. for 1 hour then concentrated to approximately 20 ml
25
by evaporation under vacuum and the product extracted With 4‘-Methyl-25-cyclohexyl-22,23-dihydroavermectin B1
diethyl ether (2x50 ml). The combined extracts Were Washed monosaccharide (255 mg) Was oxidised With activated man
With aqueous sodium bicarbonate solution (10%, 30 ml), ganese dioxide (250 mg) in diethyl ether (50 ml) according
Water (50 ml), dried over anhydrous sodium sulphate and to the procedure described in Example 2 to give the title
evaporated under vacuum to yield the crude product. This compound (208 mg) Whose NMR spectrum Was fully con
30
material Was chromatographed on silica (Kieselgel, 60, sistent With the proposed structure.
230—400 mesh, Merck) (50 g) eluting With dichloromethane
methanol gradient 99:1 to 96:4 collecting 50 ml fractions EXAMPLE 28
over 1.5 hours. Fractions 50—54 Were combined and evapo 4-Methyl-5 -oximino-25 -cyclohexyl-22,23
rated under vacuum to give material Which Was further
35 dihydroavermectin B 1 monosaccharide
puri?ed by high pressure liquid chromatography on an
Dynamax (trade mark) column (24><250 mm, 5 pm, ODS To a solution of 4‘-methyl-5-oxo-25-cyclohexyl-22,23
silica, Rainin) eluting With methanol-Water 80:20 at 20 ml dihydroavermectin B1 monosaccharide (208 mg) in a mix
per minute. Appropriate fractions Were combined and ture of methanol:dioxan 1:1 (40 ml) Was added a solution of
hydroxylamine hydrochloride (416 mg) dissolved in Water
evaporated under vacuum to yield the title compound as a
White poWder. Mass and NMR spectra Were fully consistent 40 (10 ml). This mixture Was stirred at room temperature and a
With the proposed structure. further amount of hydroxylamine hydrochloride (208 mg)
Was added after 3 hr and stirring continued at 50° C. for 5
EXAMPLE 25
hr before quenching With the addition of saturated aqueous
4‘-Methyl-5-O-t-butyldimethylsilyl-25-cyclohexyl potassium bicarbonate solution (20 ml) and diethyl ether (50
22,23-dihydroavermectin B1 monosaccharide 45 ml). The organic extract Was Washed sequentially With Water
4‘-Oxo-5-O-t-butyldimethylsilyl-25-cyclohexyl-22,23 (20 ml) and saturated aqueous sodium chloride solution (20
dihydroavermectin B1-monosaccharide (Example 16) (174 ml), dried over anhydrous sodium sulphate and evaporated
to dryness under vacuum to give the crude product. This
mg) Was dissolved in dry ether (15ml) and cooled to 0° C.
material Was then chromatographed on silica (Kieselgel 60,
under a nitrogen atmosphere. To this solution Was added a
230—400 mesh, Merck) (10 g) eluting With dichloromethane:
solution of methyl magnesium bromide (115 pl, 3M in
ethyl acetate 4:1. Appropriate fractions Were combined and
diethyl ether) dropWise, and stirring continued for a further
evaporated to give the desired compound Which Was further
1 hour. After quenching, by adding an aqueous solution of
puri?ed using high pressure liquid chromatography on a
ammonium chloride, (10 ml, 10%), the organic layer Was
Dynamax (trade mark) column (10><250mm, 5 pm, Ultras
separated, Washed With Water (2x10 ml), dried over anhy
55 phere (trade mark) ODS-silica, Beckman) eluting With
drous sodium sulphate and evaporated under vacuum to give
acetonitrile:methanol:Water 63:12:25 at 5 ml per minute.
the crude product. This material Was chromatographed on
silica (Kieselgel 60, 230—400 mesh, Merck) (20 g) eluting Appropriate fractions Were combined and evaporated to
dryness to yield the title product as a White solid (63 mg).
With dichloromethane-ethyl acetate 9:1. Combining appro
Mass and NMR spectra Were fully consisted With the
priate fractions, and evaporation under vacuum afforded the
60 proposed structure.
product Which Was used Without further puri?cation.
EXAMPLE 26 EXAMPLE 29
4‘-Methyl-25-cyclohexyl-22,.23-dihvdroavermectin 5 -O-t-Butyldimethylsilyloximino-22,23-dihyro-25 -
B1 monosaccharide cyclohexylavermectin B1 monosaccharide
65
4‘-Methyl-5-O-t-butyldimethylsilyl-25-cyclohexyl-22, Method 1. 5-Oximino-22,23-dihydro-25
23-dihydroavermectin B1 monosaccharide (74 mg) cyclohexylavermectin B1 monosaccharide (5.93 g)
5,981,500
17 18
(Example 5), t-butyldimethylsilyl chloride (2.11 g) and ODS-silica, Rainin) eluting With methanol-Water 85: 15 at 20
imidaZole (1.9 g) Were dissolved in dichloromethane (20 ml) ml per minute. Appropriate fractions Were combined and
and the mixture stirred at room temperature for 1 hr. Further evaporated under vacuum to give the product Which Was
amounts of t-butyldimethylsilyl chloride (2.11 g) and imi further puri?ed by high pressure liquid chromatography on
daZole (1.9 g) Were then added and the reaction mixture an Dynamax (trade mark) column (24x250mm, 5 pm, ODS
stirred at 40° C. for another 0.5 hr. The mixture Was Washed silica, Rainin) eluting With methanol-Water 82:18 at 20 ml
With Water (2><20 ml), saturated aqueous potassium bicar per minute. Appropriate fractions Were combined and
bonate solution (10 ml), dried over anhydrous sodium sul evaporated under vacuum to yield the title compound as a
phate and evaporated to dryness under vacuum to give the White poWder (50 mg). Mass and NMR spectra Were fully
crude product. Puri?cation Was achieved by chromatogra 10 consistent With the proposed structure.
phy on silica (Kieselgel 60, 230—400 mesh, Merck) (250 g)
eluting With dichloromethane: ethyl acetate 9:1. Appropriate EXAMPLE 31
fractions Were combined and evaporated to give the title
compound as a White poWder (4.0 g). 5 -Oximino -25 -cyclohexylavermectin B2
Method 2. 22,23-Dihydro-25-cyclohexylavermectin B1 15 25-Cyclohexylavermectin B2 (50 g) Was oxidised to its
monosaccharide (Example 4) Was oxidiZed to the 5-oxo 5 -oxo-derivative With activated manganese dioxide (2><50 g)
derivative using manganese dioxide in anhydrous diethyl in diethyl ether (500 ml) according to the method described
ether according to a procedure of Example 2. The 5-oxo in Example 2. Further reaction With hydroxylamine hydro
22,23-dihydro-25-cyclohexylavermectin B1 monosaccha chloride (53 g) in an aqueous methanol-dioxan-Water mix
ride (5.0 g) Was dissolved in dichloromethane (200 ml) and 20 ture (1:1:1) (900 ml) Was carried out according to the
O-(t-butyldimethylsilyl)-hydroxylamine (2.5 g) and glacial
procedure described in Example 28. The title product Was
acetic acid (10 ml) Were added. The reaction mixture Was isolated from the reaction by pouring into Water (500 ml)
stirred at room temperature for 18 hrs and then Washed With and extracting With diethyl ether (3x500 ml). The combined
Water (50 ml), saturated aqueous potassium bicarbonate organic extracts Were dried over anhydrous magnesium
solution (50 ml), dried over anhydrous sodium sulphate and 25 sulphate and evaporated to dryness under vacuum to give the
evaporated to dryness under vacuum to give the crude product (53 g) Which Was used directly in the next example
product. Final puri?cation Was achieved by chromatography Without further puri?cation.
over silica (Kieselgel 60, 230—400 mesh, Merck) (250 g)
eluting With a gradient of hexane:diethyl ether 2:1 changing EXAMPLE 32
to 1:1. Appropriate fractions Were combined and evaporated 30
to give the title compound as a White poWder (3.9 g). 5 -Oximino-23-oxo -25 -cyclohexylavermectin B2
The NMR spectrum of the products from the tWo methods monosaccharide
Was fully consistent With the proposed structure.
A mixture of 5-Oximino-25-cyclohexylavermectin B2
(500 mg) and pyridinium dichromate (1.87 g) in dimethyl
EXAMPLE 30 35
formamide (25 ml) Was stirred at room temperature under a
4‘-Acetylamino -4‘-deoxy-5 -oximino -22,23 -dihydro nitrogen atmosphere for 18 hrs. The reaction mixture Was
then poured into an ice (25 g) and (50 ml) mixture and
25 -cyclohexylavermectin B 1 monosaccharide
extracted With diethyl ether (2><50ml). The combined ether
To a solution of 5-O-t-Butyldimethylsilyloximino-22,23 extract Was Washed With 2N hydrochloric acid (20 ml),
40
dihydro-25-cyclohexylavermectin B1 monosaccharide Water (50 ml), dried over anhydrous magnesium sulphate
(620 mg) (Example 29) in dichloromethane (40 ml) Were and evaporated to dryness under vacuum to give a residue
added tetrapropylammonium perruthenate (100 mg) and (350 mg) Which Was dissolved in isopropanol (25 ml)
N-methylmorpholine N-oxine (600 mg). The reaction mix containing 1% sulphuric acid v/v. After stirring this reaction
ture Was stirred at room temperature for 1 hour and then 45 mixture for 18 hrs under a nitrogen atmosphere it Was
applied to the top of a silica chromatography column poured into an ice-Water mixture (50 ml) and the product
(Kieselgel 60, 230—400 mesh, Merck) (30 g). FolloWing extracted With dichloromethane (2><30 ml). The combined
elution With dichloromethane, appropriate fractions Were organic extracts Were dried over anhydrous magnesium
combined and evaporated to give the 4‘-oxo-derivative sulphate and evaporated to dryness under vacuum to give the
Which Was used directly in the next step by dissolution in crude product (300 mg) Which Was puri?ed by high pressure
methanol (10 ml), adding ammonium acetate (1.0 g) fol liquid chromatography on a Zorbax column (212x250 mm,
loWed by sodium cyanoborohydride portionWise until TLC 8 pm, ODS-silica, Rainin) eluting With methanol-Water
indicated complete reduction. The solvent Was then removed 78:22 at 9 ml per minute. Appropriate fractions Were com
by evaporation under reduced pressure, the residue taken up bined and evaporated under vacuum to yield the title com
in dichloromethane (10 ml) and triethylamine (5001) and 55 pound as a White poWder. Mass and NMR spectra Were fully
acetic anhydride (2001) Were added. After 1 hr TLC indi consistent With the proposed structure.
cated that the reaction Was complete. The mixture Was
EXAMPLE 33
evaporated under reduced pressure, the residue taken up in
methanol (10 ml) and solid p-toluenesulphonic acid added
5-Oximino-23-methoximino-25
until the solution pH Was 3.0. After 1 hr TLC indicated that 60 cyclohexylavermectin B2 monosaccharide
deprotection Was complete and the reaction mixture Was
poured into a mixture of aqueous sodium bicarbonate solu To a solution of 5-oximino-23-oxo-25
tion and ether 1:1 (20 ml). The organic layer Was separated, cyclohexylavermectin B2 monosaccharide (300 mg)
dried over anhydrous sodium sulphate and evaporated to (Example 32) in dioxan (150 ml) containing sodium acetate
dryness under vacuum to give the crude product. Puri?ca 65 (320 mg) and methoxylamine hydrochloride (370 mg) Was
tion Was achieved by high pressure liquid chromatography added glacial acetic acid (10 ml). The reaction Was stirred at
on an Dynamax (trade mark) column (24x250 mm, 5 pm, room temperature for 18 hrs then poured into Water (200 ml)