CA2369886A1 - Macrolide antiinfective agents - Google Patents

Abstract

Compounds of formula (1), (2) or (3) or the 10,11-anhydro forms thereof, wherein Ra is H or OH; Rb is H or halogen; Rc is H or a protecting group; Rd is methyl; unsubstituted alkyl(3-10C); substituted alkyl(1-10C); substituted or unsubstituted alkenyl(2-10C) or substituted or unsubstituted alkynyl(2- 10C); substituted or unsubstituted aryl(4-14C); substituted or unsubstituted arylalkyl(5-20C); substituted or unsubstituted arylalkenyl(5-20C); substitut ed or unsubstituted arylalkynyl(5-20C); substituted or unsubstituted amidoarylalkyl(5-20C); substituted or unsubstituted amidoarylalkenyl(5-20C); or substituted or unsubstituted amidoarylalkynyl(5-20C); Re is H or a protecting group or is mono- or disubstituted amino carbonyl; Rf is H; substituted or unsubstituted alkyl(1-10C), substituted or unsubstituted alkenyl(1-10C); substituted or unsubstituted alkynyl(1-10C); substituted or unsubstituted aryl(4-14C); substituted or unsubstituted arylalkyl(5-20C); or - ORf may be replaced by -H; one of Z and Y is H and the other is OH or protected OH, or is amino, mono- or dialkyl-amino, protected amino, or an aminoheterocycle or Z and Y together are =O, =NOH or a derivatized oxime; including any pharmaceutically acceptable salts thereof and any stereoisomer ic forms and mixtures of stereoisomeric forms thereof, are antimicrobial agents .

Description

J'~-~ -- ---t 3:35 PM FR MO-FOM SRN DIEG0858 720 51.25 TO 854A#t99999t#9~~"n ~
"°
06-06-2001 , ~ US 00000991 ~

MACROLIDE A,N~mYFEC'IfYE AGENTS
Technical icld The invention is dizectod to antibacterial compounds that expand the repertoire of eiytlwomycin-Like antibiotics. More particularly, the invention concerns macrolide antibiotics con~tainin an a g rythronolide nucleus modified at least at the substituent at C-13.
i Back, and Art 1'he increasing nunnber of microbial strains that have acqerired resistance to the currently available known anfs'biotic compounds is recognized as a dangerous threat to public health. As the use of such compounds has p i liferated, so too has the need for eatpanding the options available to treat ~a wide variety of microbial based conditions. The need for a larger choice of antimicrobial compounds extends ~eyond treatment of human infection and to a need to preserve food and other perishable commodities. New antibiotics can also be essential for resistant plants and animals as well as to provide resistance to materials that otherwise are subject to microbially caused corrosion.
Thus, there is a clear need for an expanded armament of compounds which can provide a multifaceted defense against unwanted microbial activity.
WO 98109978 published 12 March 1998 aid incorporated herein by reference 2Q discloses modified forms of erythxomycin which lack a cladinose residue at the 3 position and which are derivatized ><n various ways in positions 9-12 of the macrolide ring" Similarly, U.S.
Patent No. 5,750,5I0, issued 12 May 1998 aErd incorporated herein by reference, discloses i modified erythromycin derivatives. ' Empfangsteit 7.Juni 0:37 AMENDED SHEET

"~~"' ,s:,so ran rr~ I'IV-f Vfl JHI~I L~GVVO1.~P ILYJ J1LJ 1V UJ-tVfIJJvJVVIIVIV~-~~-~~ ~ -~~~~
06-06-2001 j US 00000991 E

' '~
I
The naturally occurring erythromycirj s have the structure ~vthramycin $' $"

B ' ~i -CH3 C -OH -H
D -H -H
wherein R' can be H or OH and lZ" can be H ox GFi3.
All of the compounds disclosed in the above-referenced patent documents contain a~n ethyl group ax pasitivn 13 of the macrolide nii~g. The present inventors have found that alterations in the substituent at position 13 results in a largo number of compounds with excelle~ntt antibacterial activity. .
Disclosure of the yention , The invention is directed to erythronolide derivatives that contain modifications firm l0 the native structure. All of the compounds of the invention are modified at least at position 13.
AMENDED SHEET
EmpfanBsZeit 7.Juni 0:37 3 : 36 PM FR MO-FOM SHN U 1 t~,ud~tt ~ce~ m c~ ~ V OJ~IYJtISJvJJJV tIJVVW ~ ~
m 06-06-2001 ' US 00000991:
i 300622003340 ' 3.
Thus, in one aspect, the invention is directed to compounds of the formula or.
or the 10,11-anhydro fornas thereof; , wherein Rn is H or OH, preferably OH;
Rb is H or halogen;
Rr is H or a protecting group;
Ra is methyl; unsubstituted alkyl (3-10C); substituted alkyl (1-lOC);
substituted or unsubstitutcd alkenyl (2-IflC); substituted ar unsubstituted alkynyl (2-l OC);
substituted or unsubstituted aryl (4-14C); substituted or unsubstituted arylalkyl (5-20C);
substituted or EmDfan6steit 7.Juni 0:37 AMENDED SHEET

' ."~-' wi:v30 fl'1 f rt 1'IV-rVnl amv mcuwu,~u mu ..rw,.r ..,.
...,.~~..,.r......_.....,.-._--_ . .._ 06-06-2001 , US 00000991 ~

unsubsfiituted arylalkenyI (5-ZOC); substituted or unsubstituted arylalkynyl (5-20C);
.
substituted or unsubstituted amidoarylalkyl (5-20C); substituted or unsubstituted amidoaryialkenyl (5-24C); or substituted or unsubstitutcd amidoarylalkynyl (5-20C);
R~ is H or a protecting group or is mono- or disubstituted amino carbonyl;
i S Rr is H; substituted or unsubstituted alkyl ( I-l OC); substituted or unsubstituted !.
atkenyl (1-I OC); substituted or unsubstituted all<ynyl (1-lOC); substituted or unsubstituted aryl (4-14C); substituted or unsubstituted arylalkyl (5-20C); or ORr may be replaced by H;
one of Z and Y is H and the other is OH or protected OH, or is amino, mono- or dialkyl-amino, pmtectcd amino, or an aminoheterocycle or Z and Y togethex are ~, =NOH or aj derivatizcd oxime;
i including any pharmaceutically acceptable salts thereof and any stereoisomenic forms and mixtures of stereoisomeric forms thereof In another aspect, the invention is directed to pharmaceutical or preservative compositions containing the compounds offormulas (1)-(3) and to methodsto treat infectious diseases by administering these compounds or to preserve materials by providing them.
The compounds of the invention have antibiotic acfiivity, but preferably are useful as semi-synthetic intermediates for forming 10, i 1 I aahydro forms of the compounds that arc further converted to compounds having an erythronolide nucleus and having a ring between the C10 and CI 1 positions of the eryrhronolide nucleus as descn'bed in PCT
Publication No.
WO 00/63224 which claims priority to U.S. provisional patent application Serial Al'os.
60II40,I75 filed I8 3une 1999 and 60/172,159 filed 17 December 1999 and U.S.
utilitypatent application Serial No. 091550,045 filed 14 April 2000 entitled "Macmiide ,A,ntiinfectives", which are incorporated by reference.
AsBrief Descrintioa o_f the Drawines Figure 1 shows a schematic of the synthesis of the compounds of the invention.
Figure 2 shows the post PKS biosynthesis of mycins. This pathway is employed in the present invention, as shown in Figure 1.
Figure 3 shows the syntlxesis of compounds of formula (3) wherein Rr is methyl.
Figure 4 shows the synthesis of compounds of formula (1) and their con~esponding 10,11-anhydro forms.
AMENDED SHEET
Empfangsteit 7.Juni 0:37 _. .._._1 ,j:,,ip rn rK rW-rUl'I 5H(V iJltUUi3Jti 1610 51G' lu t5J410R~~~~bpy .._._ .. ._..
06-Ofi-2001 ~ US 00000991 E

Figure 5 shows the synthesis of compounds of formula (3) wherein QRf is replaced by H.
Figure 6 illustrates the conversion of 15-azidoerythmmycin A into 15-amidoerythmmycins.
Modes of Carrvint Out the invention The compounds of the invention are conveniently synthesized by combining synthetic chemical techniques with,microbiological processes involving genetically engineered microorganisms. Briefly, in a preferred mode of carrying out the invention, a microbial host, preferably a host which does not itself pmduce a macrolide antibiotic, is provided with a recombinant expression system for the production of modified 6-deoxyerythronolide B
(6-dE~), which expression system in some instances will have been altered by a disruption in the catalytic domain of the ketvsyathase moiety in the first module. For substituents is which Rd is methyl, host cells arc used which do not have a disrupted domain of the ketosynthase moiety. This alteration in the d-dEB pol3tketide synthase (PKS) results in the inability of this PKS to utilize its native starter unit, and thus;permits inclusion of a synthetic diketide thiocster far its initial condensation pmduct in the sequence of reactions leading to modified 6-dTrB without competition from the diketide; that would otherwise, natively, have been produced. Thus, the recombinant host can be provided a synthetic diketide thioester for incorporation into the resulting polyketide. The incorporation of this diketide into the resulting polyketide results in a polyketide with a substituent at position 13 that may be selected as desired. Preferred methods for preparing the synthetic polyketide thioesters are set forti~ in PCT Publicafion No. WQ 00/44717 which claims priority to copending application 1J'.S. Serial No. 60/117,384 filed 27 January 1999 and 09/492,733 filed on 27 January 2000, which are incorporated herein by reference. , Recombinant forms of the 6-dEB PKS containing inactivated ketosynthase (KS) domains in the first module {KS1) and appropriate organisms modified to contain an expression system far this PKS are described~in PCT applications WO 97!02358, published 28 3anuary 1997 and WO 99/03986, published 28 January 1999, incorporated herein by reference.
Additional manipulations which provide alternative substituents on the macrolide ring are disclosed in PCT Publication No. WO 98/49315 which claims priority to U.S.
Sezial No.
AMENDED SHEET
EmvfangsZeit l.Juni 0:31 r.... v... . .~..~ ~ V ~ vl 1 I 11 l~~ 1\ I IV-f V11 JI'11'1 L~ G~:1VGJ0 ( LYJ
J 1 GJ 1 V OJ~~i1J1~1.7J.7.7CJhh~~~u"~ .- . r.
06-06-2001 i US 000009915 E
~oos2~oo3340 09!073,538 filed 6 May 1998, PCT Publication No. WO 00163361 which claims priority to U.S. Serial No. 601129,731 filed 16 April 1999, and PCT Publication No. WO

which claims priority to U.S. Serial No. 091x29,349 filed 28 October 1999 and are r irrcarporated herein in their entirety by reference.
The polyketide resulting from expres lion of the modified PKS is then isolated and purified, if desired, from the recombinantly ~odificd organism and fed to Saccharopolyspora erythraea, which contains the functionality for postpolyketide modifications, including glycosylation. Other modifications include hydroxylation at positions 6 and/or 12. The resultin modified g erythromycin is then isolated and chemically modified to obtain the compounds of the invention. Synthetic methods for providing these modifications are described in WO 98109978 and U.S. Patent No. 5,750,510, referenced hereinabove.
The general method for synthesizing compounds of the invention is shown in Figure I.
The resulting antiinfective compound is active in vitro and in vivo for activity against a panel of representative microorganisms. The compounds of the invention thus exhibit a sufficient diversity in specificity to cover the' spectrum of antibiotic activities desired, For use in treating infectious disease, the compounds of the invention are formulated into suitable compositions which will include typical excipients, pharmaceutically acceptable counterions if the compound is a salt, fiuthez~ additives as desired, such as antioxidants, buff=ers, and the lOce, and administered to aninnals or humans. The types of formulations that are appropriate for these compounds are similar to those for the macrolide antibiotics in general. Formulations may be foand, for example, in min en's Pharmaceutical Sciences Mack Publishing Co., latest edition. The compounds can be administered by any desired mute, including injection, oral administration, tisnsdermal administration, transmucosal adminustration, or any combination. The compounds of the invention can also be administered with additional active iugredieitts if desired.
The compounds of the invention are of fornaulas (1~(3) as set forth above, as well as any stereoisomeric forms of these compounds as shown. The particular stereoisomers depicted are those resulting from the preferred method of synthesis set forth above and exemplified herein; however, by modifying the expression system for the PK.S, or by altering r the chirality of the diketide, or by synthetic chemical conversion, other stereoisomers may also be prepared. Additional chiral centers ti2ay be present in the substituents, such as Rd and AMENDED SHEET
Emafanasteit l.Juni 0:31 ... ...... i ;a:;~ r rr~ rK r~u-rur~ 5HN uttuu~5~s rye 5m5 i a ~s54ea~a~aetts~~,--~. .. . .
Ofi-06-2001 i US 000009915 .i i i Rt: The stereoisomers may be administered as mixtures, or individual stereoisomers may be separated and utilized as is known in the art.
The properties of the compounds of formulas (1 ~(3) are defined by the substituents Ra-R~, Y and Z. Preferred embodiments of these substituents are set forth hereinbeiow. They contain moieties which are defined as follows: ' "Halogen" includes ffuoro, chloro, b imo and iodo, aad most preferably fluoro.
"Alkyl" refers to a saturated straight-chain, branched chain or cyclic hydrocarbyl moiety containing a specified number of carbons and that may contain one or more suitable heteroatoms; similarly, alkenyl aad aIkynyl refer to straight or branched chain or cyclic hydrocarbon substituents containing one or more double bonds or one or more triple bonds, respectively, and containing one or more suitable heteroatoms.
"Aryl" refers to an aromatic substituerit that may contain one or more suitable i heteroatoms such as phenyl, naphthyl, quiaolyl, or phenanthryl.
"ArylaIkyl," "atylalkenyl," or "arylalkynyl" refer to substituents wherein an aryl group is linked to the substituted moiety through an alkyl, alkenyl or alkynyl linkage, respectively.
Again, the number of carbons in the arylalkyl; arylalkenyl or arylalkynyl groups will be specified.
"Amidoarylalkyl," "amidoarylalkenyl,i or "anudoarylalkynyl" refer to substituents wherein an aryl group is linked to the substituted moiety through an amido and an alkyl, alkenyl or alkynyl linkage, respectively. Again, the number of carbons in the amidoarylalkyl, amidaarylalkenyl or amidoarylalkynyl groups~will be speeded.
Thus, included among the defined substituents herein are "hcteroalkyl,"
"heteroalkenyl," "heteroalkynyl;' "heteroatyl," "heteroarylallCyl," and the like. Suitable heteroatoms include N, O, and S.
All of the foregoing substituents may be unsubstituted ox may be further substituted.
Typical substituents include R, -OR, -SR, -NR,a, -COR, -COOR, -CONR~, -QOCR, NRCOit, -OCONRz, -CN, -CF3, -NOi, -SOR, -S02R, Halogen, wherein each R is independently H or is alkyl, allcenyl, allcynyl, aryl, arylalkyl, or the Iietero forms of these as definal above. In addition -al l, alken 1 and ky y alkynyl may be substituted by aryl or heteroaryl, which may, themselves, be further substituted. Aryl and lieteroaryl may also be substituted by alkyl, allcenyl or alkynyl, or by additional aryl or hetGroaryl moieties.
EmpfangsZeit 7.Jvni 0:37 AMENDED SHEET

... ... . .... _ ~ ~ : ~~ t..~.l t-K 1''IV-hUrl SHIV U 1 tlaUttSii ~Gl~ 51 G5 I U t35410R~~~~ldfi'J~ .. ._.. , .
06-06-2001 , s US 00000991 E
30U6zz003340 .g-"A derivatized oxime" is of the formula N-O-R, wherein R is other than H and is otherwise defined as above.
A "protecting group" far a hydroxy includes aryl groups, silyl groups, and tire like.
Suitable protecting groups are described by Cneene, T.W., et al., in Protecting Grouvs in Organic S~~nthesis, 2"d Ed., John Wiley & Sons, Inc. (1991), incorporated herein byreference.
The invention includes more preferred embodiments of the compound defined above.
Ra is preferably butyl, pentyl, methoxyethoxyinethyl, isobutyl, methylcyclo>~exyl, phenyl, benzyl, ethylphenyl, 3-{benzyIoxy)propyl, z-(pyrimidin-2-ylthio)ethyl, propyl, tluoroethyl, chloroethyl, vinyl, 3-buteriyl, ar azidaethyl and more preferably propyl, fluoroethyl, chloroethyl, vinyl, 3-butenyl, or azidoethyl. PCT Publication No. WO 00/44717 which . claims priority to U.S_ Serial No. 40/117,384 filed 27 January 1999 and U.S.
Serial No. 09/492,733 filed 27 January 2000 all of v~hich are incorporated herein by reference describe various oligoketide thioesters, preferably diketide thio~esters, that can be incorporated at the C-13 position. Such diketide thioesters! as described therein are incorporated into the compounds of the invention and thus determine preferred Ra groups at the C-13 position.
In another preferred embodiment, Rr is H or lower C1-C3 alkyl, and more preferably methyl. Rf is also preferably arylalkenyI or arylalkynyl such as 3-arylprop-2-enyl or 3-arylprop-2-ynyl. Preferably the aryl group in the preferred arylalkenyl or arylalkynyl embodiments are 3-quinolyl, 4-quinolyl, 5-quinolyl, phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-methoxyphenyl, 6-quinolyl, 6-quinoxalyl, ~6~ amino-3-quinolyl, or 4~isoquinolyl.
Synthesis of the Invention Compounds A.s dcscn'bed above, the antibiotic starting matezia,is for any further chemical synthesis to obtain the compounds of the invention are prepared, preferably, by feeding a suitable diketide to a microorganism modified to contain an expression system for the 6~EB PKS
containing a KS 1 knockout, or by a host cell that provides a methyl at the 13-position, followed by feeding the resulting polyketide to a recombinant strain of Saccharopolyspora erythraea that has been altered to eliminate pioduetion of 6-dEB. A strain can be prepared that is able to hydroxylate either both the 6- and 12-positions or the 12-position only. In this case, -ORf is replaced by -bI: Alternatively, a atrain can be prepared that hydroxylates only the 6-position. The recombinant S erythraea atrain, K40-57, is obtained by transforming an S. erythraea slain that produces high levels of erythromycin A with a plasnud comprising a AMENDED SHEET
Emafangsteit 7.Juni 0:37 " ! ;~ : ;~d NI'1 h-ht MU-f-OM SRN D 1 EG0858 720 S 1 25 TO 8540it9999et#9~"""
" ' "
06-06-2001 I US 00000991 ~
r i9_ mutated eryAl sequence encoding an inactivated KS I domain. By homologous recombination, the resulting transformants no~cv are unable to produce b-dEB
as a competitor to the fed polykebde and, instead, hydroxylate the 6-position and 12 position and glycosylate the 3-position and 5-position of the modified polyketide that has been made in Streptomyces or other polyketide-producing transformant If a macrolide having only the 12-position, and not the 6-position hydroxylated is desired (ORr is replaced by H), an S
erythraea strain is constructed by disrupting the tryF hydroxylase gene in strain K40-6?.
Alternatively, the eryK
gene can be disabled, wherein embodiments of compo~mds (1)-(3) wherein R" is H
ntay readily be produced.
14 The glycosylation reactions far the production of the erythromycins result in the diglycosylated forms analogous to the naturally occumng erythromycins. If the compounds r of formula (3) are to be prepared from the initial product, the hydroxyl gmup of the cladinose ring (attached to position 3) may then need to~be protected for subsequent modification of the macrolide substituents.
The modified erythromycins of the invention, in addition to modification at C-13, xnay contain an -OH group at position 6, unless ORf is replaced by H as desedbed above. To construct the compounds of formulas (1), (2) and (3) where position b is ORr, the compound.
of formula (3) is provided with protecting groups which form one embodiment of R~ and Rt.
Such otection is effected usin suitable mtec Pr g p ~ ting reagents such as acetic at~ydride, benzoic anhydride, benzochloro formate, hexamethyldisilazane, or a trialkylsilyl chloride in an aprotic solvent. Aprotic solvents include, for example, dichloromethane, chloroform, tetrahydrofl~ran, N methyl pymolidone, dimethyl sulfoxide (DMSO), dimethyl formamide (pMF) and the like. Mixtures may also be used. Protection of both sugar hydroxyls in formula (3) may be done simultaneously or sequentially. .
In addition to protecting the 2' and 4" hydroxyl groups of the two glycose residues, the keto ou at ~ ' gr p position 9 of the macrolide ring:must also be protected. Typically, this is effected by converting the keto group to a derxvatized oxime. Particularly preferred embodiments for R in the formula NOR include unsubstituted or substituted alkyl (I -12C), substituted or unsubstitutcd aryl (6-lOC), alkyl (1-t2C), substituted or unsubstituted heteroaryl (6-lOC), alkyl (1-I2C), and heteraa'lkyl (such as substitucuts ofthe formula. ' CR'20R wherein each R', in addition to being independently embodied as R as set forth Emafan8sieit l.Juni 0:37 AMENDED SHEET

.... v.uu n m n w n . . v.. ........ ... ......._ . ~._ -__- . _ ._ _ 06-06-2001 f US 00000991 30o6Z2003340 above, rnay, together with the other, form s cycloalkyl ring (3-12C)). A
pref~rod derivatizcd i crime is of the formula NOR wherein R is isopropoxycyclohexyI.
With the 9-kcto group and the 2' and 4, " hydroxyls protected, it is then possible to alkylate the fi-hydroxy group in the precursor to the compound of formula (3) by reaction with S an alkylating agent in the presence of base. ~ilkylating agents include alkyl halides and sulfonates. For example, the alkylating agents may include methyl tosylate, 2-fluoroethyl I
bromide, cinnamyl bromide, crotonyl bromide , allyl bromide, propargyl bromide, and the like. The alkylation is coimducted in the presence of base, such as potassium hydroxide, sodium hydride, potassium isopropoxide, potassium t-butoxide, and an aprotic solvcat.
Especially preferred for R.f are methyl; alIyl and ethyl.
Once the alkylation of the 6-hydroxxliis completed, the sugar residues and the macrolidc ring may be dcpmtectcd. Dcprotection of the glycoside moieties is conducted as described by Green, T.W., et al., in Protective Groups in Organic S n~, fnfra.
Similar conditions result in converting the derivati2ed crime to NOH. Zf formation of the 1S undcrivatized crime is rat concumxent with deprotection, the conversion to the crime is conducted separately.
The crime can then be removed and converted to a keto group by standard methods known in the art. Deoximating agents include inorganic sulfur oxide compounds such as sodium hydrogen sulfite, 'sodium pyrosulfate,~ sodium thiosulfxte, and the fke. In this case, I
profit solvents arc used, such as water, methanol, ethanol, isopropanol, trimethyl silanol and mixtures of these. In general, the deoximation reaction is conducted in the presence of an organic acid. ~ .
At this point in the process, or later, after the compound of formula (3) has been converted to the compounds of formulas (1) or (2} as further described below, the group introduced at the 6 hydroxyl can further be manipulated. Conveniently, the initial substitution may provide a 6-O-allyl -- i.e., O' CHzCH=CH2 -- which can further be derivatized by reduction to give the 6-0 propyl compound, or be treated with osmium tetroxide to provide the 2;3-dihydroxypropyl compound, which can further be esterified at tech oxygen atom. The O-allyl derivative can also be oxidized with m-chloroperoxxbenzoic acid in am aprotic solvent to provide the epoxy compound which can be opened with amines or N-containing heteroaryl compounds to provide compounds with N-containing sid0-chains, or can be oxidized vender blacker conditions to provide time substituent O-CH2-C(O)-CH3, or Emafangszeit 7.Juni 0:37 AMENDED SHEET

_. ..... 1 ;S : ;,i~ rn rtC hIU-rur~ 5f-IfV ~ 1 tCaUli5li r~l~ 51 G5 I U
ti54l~ii~~~~idii~' ...... ..
06-06-2001 ; US 000009915 -~11-can be ozonized to provide the aldehyde. The aldehyde can then be converted to the oxime or reacted with a suitable amine and reduced in the presence of a borohydride reducing agent to i provide an amine. The oxime can also be corivertod to a nitrite by reaction with a dehydration agent in an aprotic solvent. The O-allyl derivative can also be reacted with an aryl halide under Heck conditions (Pd(II) or Pd(O), phosphine and amine or inorganic base}
tv provide a 3-aryl prop-2-enyl derivative. This derivative can then be reduced with hydxogen and palladium on carbon to provide a 3-arylpropyl derivative. If the initial su6stituent Rf is a 2-propyae, similar reactions can be employod to provide alterations in the side-chain, including arylation. ' In order to convert the compound of formula (3) iato the compound of formula (1), by first removing the cladinose moiety, the compound of formula (3) is treated with mild aqueous acid or with a deglycosylating enzyzr'te. Suitable acids include hydrochloric, sulfuric, chloroacetic, trifluoroacetic and the like, in ttie presence of alcohol.
Reaction times are typically 0.5-24 hours at a temperature of -10:35°C. During this reaction, the 2' group of the I 5 remaining sugar is protected as set forth above and deprotected subsequent to the decladinizing reaction. The resulting hydroxyl group at the 3-position of the macmlide ring is then oxidized to the ketone using a modified Swern oxidation procedure. In this procedure, an oxidizing agent such as N-chlorosuccinimide-dimethyl sulfide or a carbodiamide-dimethylsulfoxide is used: Typically, a compound of formula (3) is added to pre-fonxted N-chlorosuceinimide and'dimethyl sulfide complex in a chlorinated solvent such as methylene chloride at -I O-25°C. After being stilted for 0.5-4 hours, a tertiary amine such as triethylamine is added to produce the corresponding ketone and the 2' protecting group is then removed. .
In order to halogenate the macrolide at position 2 (converting Rb is H to halogens the i compound of formula (1) is treated with a base and an electrophilic halogenating reagent such as pyridinium perbromide or N fluorobetazene sulfonic acid. The position 2 can be halogenated at any time after the 3 keto compound is prepared.
The appropriate substitucnt such as viayl, ethenyl, butenyl or azida at the C-position can be further maruipulatod. Por example, are amidoacetate salt of the compound of the invention can be derivatized using an arylacetyl chloride to yield az~
arylamino alkyl group on the C-I3 position. Preferably the C13 derivatives of an azido group take place before the AMENDED SHEET
EmufangsZeit 7.Juni 0:37 J.06=06-2001 t 3 : 39 PM FR MO-FOM SAN D I EG0858 720 5 125 TO 8540#99990#9 US
00000991 ~

-;12 -kctolide is formed. Derivations of an ethenyligroup can take place either before or after the ketolide is formed.
In order to obtain the compounds of formula (Z), the compound resulting from the deglycosylation reaction of formula ( 1 ) is treated with a dehydrating agent such as carbonyl diimidazole and base.
In order to prepare compounds of forri~ulas (1 )-(3) wherein one of Z and Y is H and i the other OH or protected OH or is an amino derivative as described above, either the i carbonyl or, oximo or derivatized oxime is reduced using a suitable reducing agent, such as i sodium horohydzide, Raney nickel/H2 or reductive aniiaation with the use of sodium IO cyanoborohydride and an amine. Substitutedjamines can also be obtained by alkylation.
Novel methods of synthesis of the compounds of the invention are also providod.
Exemplary Embodiments The compounds of formulas (1), (2) and (3) are defined by their various substituents.
Table 1 illustrates compounds within the scope of the present invention which are:
of formula (I) wherein R, is H or OHRb is H, Cl, or F, and R~ is H;
of formula (2) wherein R, is H or OH~and R~ is H; and of formula (3) wherein R" is H or OH; R~ is H, and R~ is H, or a radical a, b, c, oar d:
Vin).
v F F
F
-~N ~ (c): ~ ~..~ ~ (d).
Y
v Table 1 Rd . Rf Y Z

-CH3 -CHICHI =O

-~CH=CH2 -CHZCH=CH-~ ~ -_O

-CHZCH2CH3-CHZCti2NHCH3 =NOH

-CHa -CH2CHOHCH~ ' =NOCHzCH3 -CH(CH3}2 -CHz~ ~ H OH

-CHs -CH2-CH=CHZ . =O

-CH3 -CHa-CH=CH-(3-qeiinolyl) -CH3 -CHZ-CHa-CHZ-(3-QuitiOlyl) =O

Emafangsteit l.Juni 0:37 AMENDED SHEET

J"" "" ""'"1 3:40 PM FR MO-FOM SRN DIEGOB58 720 5125 TO 8540#99990#°--"- - '-06-06-2001 i US 00000991 ~

3'00622003340 ' -~ 13 -Table 1 Rd fRf Y Z

-CH9 =CHI-CH~H-(2-methi',yl-6-quinolyl) =O

-CH, -CHz-CH=CH-(5-isoquinalyl) =O

-CHs -CHrCH=CH-(3-bronio-6-quinofyl) =O
~

-CH3 -CHrC=CH-(6-meiho~2-naphthyl) =O

-CH3 -CHTC ~C-(2-phenylethenyt) =O

-CH$ -CH~-C eC-(3-quinolyl) O

-CH3 -CHrC eC-naphthyt =O
;

-CHI -CHI-C=C-(6-methyl.2-naphthyl) =O

-CH3 -CHrC~Cr(3-(2-furanyl)-6-quinolyl) =O

-CH=CHZ -CH3 , =O

-CHzOH -CHZ-C=CH-(4-fluorophenyl) =O
' -CHZOH -CHI-C=CH-(3-quinolyl) =O

-CH20H -CH2-C=CH-(6-quinolyl) -CH20CH9 -CHz-C=CH-(3-pyridylt) =O

-CHZCHzGH3-CHI-C=CH-(3-quinoljrl) =O

-CH=CHZCH3-CHI-C=CH-(6-chloro~3-qulnolyl) -_O

-CH2CH2CH3-CHrC=CH-(4-quinolyl) =O

-CHzCHzCH~-CHrC~H-(6-chtoro&quinolyt) =O

-CH2CH2CH3-CHI-C=CH-(8-hycbroXyr,3-quinolyl) =O

-CHZCHZCHa-CHz-C=CH-(fi-methoxy =O
3-quinolyl) -CHzCHZCH3-CHx-CCH-(G-aminocarbonyl-3-quinolyl) =O

-CH2CHZCH~-CHZ-C=CH-(3-(2-thiophenyl)-6-quinolyl) =Q

-CHxCHZCH~-CH2-C=CH-(6-hyclrouy =p 2-naphthyl) -CHZCHzCHa-CH~-C ~-(3-quinolyl) _O

-CHzCHZCH3-CHrC~-(6-chlora-2;
naphthyl) -CHZCHzCH~-CHZ-C=C-(6-quinoly!) =O

-CHzCH2CH3-CH2CH2NHGHZCHr(2-chlorophenyl) -_O

-CHS -CHZCH=NH2 ' -CH3 OR, replaced by H -NHs H

-CH3 -CH3 ~ -NHi H

-CH3 OR, replaced ~~ H
by H ' -CH3 . ~~ ~ H

-CH3 ' ~~, ~ H

AMENDED SHEET
Empfansszeit 7.Juni 0:37 .. .._.. ~ ;,f : 410 t'rl 1-K I'IU-1-UI''I 5f-lfV 1J 1 tlaUtiStf f~19 51 ~5 I
U t354bii~J'JJIJix~.,"",' ,-' . '.
06-06-2001 ~ , ~ US 000009915 I
304622003340 ' -14_ Table Rd Rf Y Z

-CH3 -CH2CHCIChl3 ~ H
v -CH3 ~ "; H

-CH3 j H --CHs -CHs ~ -H~., H

-CHZCH2CH3ORr replaced by H ~ H _,~NH

-CHzCHiCH'~~ -NHZ H

-CHzCHZChf-CHCH(OCH~)CH~ ~ .~~", H

-CH2CH2CH$-CNa H _,, v -CH2CH2CH3-CH2CH2CH3 : ~"~ H

-CH2CHZCH3-CHZCHBrCH3 . H

-CHs -CHZCHOHCHs ~ =NOCHCH$

-CH2CHzCHs-CHiCH2CH3 ~ -NHz H

x ' les i The following examples are intended to illustrate but not to limit the invention.
Compound cumbers and designations fare found in TIlustrative Scheme 1 In these examples,, in the first general 'step of the method, a 6-deoxyerythronolide B
(6-dEB) derivative compound is prepared by ferrreentation of a recombinant Streptomyces host cell.
The fermentation to produce 15-methyl-ti-deoxyerythronolide B and 14,15-dehydro-d-deoxyerythronolide B requires a synthetic diketide intermediate to be fed to the fermenting 1 D cells. The preparation of these s~thetic diketides is described in Example 1. These synthetic diketides are substrates for a 6-deoxyerythronolide B synthase (DEBS) that is unable to act on its natural substrate (propionyl CoA) due to a ,mutation in the ketosynthase domain of module 1 of DEBS. This recombipant DEBS is provided by plasmid pJRJ2 in Streptorriyce~r coelicolor CH999_ S. coelicolor CH999 is described in U.S. Patent No.
5,672,491, incorporated herein by reference. A derivative of S. coelicolor CH999, S.
coelicolor K39-02, AMENDED SHEET
Emvfangszeit 7.Juni 0:31 .

J"" "'' '""'l 3:40 PM FR MO-FOM SRN DIEG0858 720 5125 TO 8540#99990#9'"""" "
'"
06-06-2001 ; US 00000991 ~
300622003340 , that has been genetically modified to include aptpA gene, is described in U.S.
patent application Serial No. 09!181,833, incorporated herein by reference can also be employed for this purpose.
Plasmid 3RJ2 encodes the p cryAr, eryATX, and eryABI genes; the eryAl gene contained in the plasmid contains the KS 1 null mutation. The KS 1 null mutation prevents formation of the 6-deoxyerythronolide B produced by the wild-type gene unless exogenous substrate is provided. Plasmid pJRJ2 and a process.for using the plasmid to prepare novel 13-substituted erythromycins are descn'bed in PCT Publication Nos. WO 99!03986 and WO
97102358 and in PCT Publication No. WO 97/02358 which claims priority to U.S. patent application Serial Nos. 081675,817, filed 5 July 1996; 08/896,323, filed 17 July 1997; and 091311,756, bled 14 May 1999, each of which is incorporated herein by reference. The exogenous substrates provided can be prepared by the methods and~include the compounds described in PCT
Publication No. WO 00!44717 which claims priority to U.S. patent application Serial No.
09/492,733, both filed 27 Jan. 2000, by inventors G. Ashley et al., and both of which claim priority to U.S. patent application Serial No. 60!117,384, filed 27 Jan.1999, each of which is incorporated herein by reference. PK"S genes wither than the ery genes can also be employed;
suitable genes include the KS 1 null mutation containing oleaadoIide and megalomicin PKS
genes described in PCT Publication No. WO 01/27284 which claims priority to U.S. patent application Serial Nos. 60/158,305, filed 8 Oc~~t.1999 and 091428,517, filed 28 Oct.1999, and PCT Publication No. WO ~ 00/26349, filed 22 Oct.1999, each of which is incorporated herein by reference. .
The fermentation to produce x4-nor-6deoxyeTythronolida B does not require diketide feeding, bocause the desired compound is produced by the recombinant host cell Streptomyces coelicolor CH9991pQ~7. Plasatid pCK? is described in U.S. Patent No.
5,672,491 and comprises the DEBS genes. A~derivative of plasmid pCK?, pROS011-26, can also be used. The host cell comprising pI~OS01 I-26 and a recombinant ptpA
gene is S coelicolor 27-261pKOS01 I-26. These hosti cells produce both b-deoxyerftt~onolide B and I4-nor-6-deoxyerythronolide, due to the incorporation of propionyl CoA and acetyl CoA, both of wb,ich serve as substrates for DEBS.
The fermentation of Streptomyces coelicolor CH999lpJRJ2 and S. coeltcolor C~999IpCK7 is described in Example 2_ The isolation of the 6-deoxyerythranolide products resulting from this fermentation can be achieved by separation,.
AMENDED SHEET
Emafaossteit 7.Juni~ 0:37 J"" '" """ 1 :,i : 4 1 rri rK MV-r VI''I 5HN 1J 1 t(~Ul35ti lib 51 ~5 TO
854f~ti99990fi~9"'""" " "-06-06-2001 ~ US 000009915 300b22003340 -;lb-The isolated products are then added to the fermentation broth of Saccharopodyspora eryrhraea strains to make,other useful intermediate compounds of the invention. The S. erythraea strains catalyze the biosynthesis and attachment of sugar residues to the 3 and 5 positions of the b-dEB derivative compouads~ These strains also comprise a functional eryK
gene product and so hydroxylate the 6-dEB derivative compounds at the 12 position. The strains differ in regard to whether a functional eryF gene product is produced. If so, then the compounds produced are hydroxylated at the 6 position as well. If not, then a dcoxyerythromycin A derivative is produced ~ These S erythraea fermentations are described in Example 3, together with the isolation of the erythromycin A derivative compounds from the fermentation broth. ' The isolated products are then used ash intermediates in the chemical synthesis of other intermediate compounds of the inve~ntaon. Por erythromycin A derivative intermediates that comprise a b-hydroxyl, Eacamples 4-6 descn-be the process for alkylatiung the compounds to make the 6-O-alkyl intermediates of the invention and Example 11 describes the process for allylation to make 6-O-allyl intermediates which can be further derivatized as shown is Example 15 upon protection of the 2' u~d 4" Hydroxyl groups and protection of the 9-position as shown in Example 14. , The schematic for these reactions is shown in Figure 3.
i Examples ?-9 describe the conversions of the above-described compounds of formula (3) to compounds of formula {1), and corresponding compounds that are the 10,11-anhydro fozxns. 'This is shown schematically in Figure 4.
Example 10 also sets forth the process for making the 10,1 I-anhydro compounds of formula (3), but wherein ORf is replaced by H. The reaction scheme far these conversions is shown in Figure 5.
The compoc~nds in Example 11 can be converted to compounds of formula (1) or (2) as shown in Examples 12 and 13, respecdvcly.
Example 16 illustrates the halogenation of the 2 position.
Example 17 illustrates the conversion ~of 15-azidoerythromycin A into 15-anudoerythromycins, as shown in Figure 6.
AMENDED SHEET
Emufangszeit 7.Juni 0:37 J"" '"" '""" 1 3 : 41 PM FR MO-FOM SRN D I EG0858 720 5125 TO
8540#99990#9"'""" " "' 06-06-2001 ' US 00000991 ~

-.17 -Example 1 P~e~aratian of Diketide Thioesters The processes used to prepare the N-acetylcysteaminethiotsters (NAcS) used to feed . the recombinant Str~eptomyces host cells to make the 15-methyl and 14,15-dehydr~o-6-I
deoxyerythronolide B intermediate compounds are described in this Example The synthesis i protocols d~cn'bed below are also described in PCT Publication No. W0 00/44717 which claims priority to U.S. provisional patent application Serial No. 501117,384, filed 27 Jan.
1999 and U.S. utility patent application Serial No. 091492,733, filed 27 Jan.
2000, both of Which are incorporated herein by reference. , Thus, (2S,3R)-2-methyl-3-hydroxyhe~anoate NAcS (Preparation E), which is used to prepare the 15-methyl-6-deoxyerythronalide B intermediate, is prepared from reacting (4S)-N-[(2S,3R)-Z-methyl-3 hydraxyhexauoyl]-4-benzyl 2-oxa2olidinone (Preparation D) with N-acetylcysteamine (Preparation B). N acetylcysteamine is, in taro, prepared from N,S-diacetylcysteamine (Preparation A). (4S)-N-[(2S,3R~Z-methyl-3 hydroxyhexanoyl]-benzyl-2-oxazolidinone (Preparation D) is prepared from (4S~N-Propionyl-4-benzyl-Z-oxazolidinone (Prapionyl-NOx; Preparation In similar fashion,. (2S,3R}-2-methyl-3-hydroxy-4-pentenoate NAcS (Prcgaration G), which isused to prepare the 14,15-dchydro-6; deoxyerythtonolide B
intermediate, is prepared from reacting (4S) N [(2S,3R~2-methyl-3-hydroxy-4-pentenoyl]-4-benzyl-2-oxazolidinone (Preparation F) with N-acetylcysteamine (preparation B). (4S)-N [(2S,3R~2-methyl-3-hydroxy~4-pentenoyl]-4-beazyl-Z-oxazoiidinone (Preparation F) is prepared from (4S~N
Propionyl-4-benzyl-2-oxazolidinone (Propionyl-NOx; Preparation C).
A. 1~T.S-Diacetvlcvsteamine: G~sfeamine hydrochloride (50.0 g) is added to a 1 L
3-neck round bottom flask fitted with a magnetic stir bar, 2 addition funnels, and a pH
electrode. Water (300 mL) is added, and the stirred solution is cooled on ice.
The pH is adjusted to 8.0 by addition of 8 N KOH. Acetic anhydride {125 mL) is placed in one addition funnel, and 8N KO~I (350 mL) is placed in tl ie other addition ftmnel. The acetic anhydride is added dropwise to the cysteamine solution, with 8 N KOH being added sv as to keep the reaction pH at 8 +i- l . After addition of acetic anhydride is complete, the pH was adjusted to 7.0 using 1 N HCl and the mixture is allowed;to stir for 75 min. on ice. Solid NaCI is added to saxuration, and'the solution is extracted 4 tinnes using 400 mL portions of CHiCl2. The organic extracts are combined, diied over lVlgSOa, filtered, and concentrated under reduced AMENDED SHEET
EmPfanasZeit 7.Ju~i 0:37 JI06-06-2001 ~ 3 ~ 42 PM FR MO-FOM SRN D I EG0858 720 5 I 25 TO 8548#99990#9 3oosz2oo~3ao -iI8 :.
pressure to yield 68.9 g (97% yield) of a pale yellow oil, which crystallizes upon standing at 4°C. . i B. ~j-,~9;ce3ylc3rsteamine: N,S-diacetylcysteamine (42.64 g3 is placed in a 2 L
round bottom Mask fitted with a magnetic s 'tuier, and dissolved in 1400 mL of water. The flask is purged with N2, and the nuxture is chilled in an ice bath. Potassium hydroxide (49.42 g) is added, and the mixture is stirred for 2 hr! on ice under inert atmosphere. The pH is adjusted to 7 using 6 N HCI, and solid NaCI '~s added to saturation. The mixture is extracted 7 times with 500 mL pvrtians of GHZCIZ. Thi organic extracts are combined, dried over MgSOa, filtered, and concentrated under reduced pressure to yield 30.2 g (96%
y~eld~ of product. This material is distilled immediately prior to use, by 138-140°C/7 mmHg.
C. (4S)-N-Pro~ionvl-4-benzvl-2-oxazolidinone (Prooionvl-NOx): A dry, 1 L
three-necked round bottomed flask equipped with a 500 mL addition funnel and a stir bar was charged with 20 g of (4S)-4-benzyl-2-oxazolidinone, capped with septa and flushed with nitrogen. Anhydrous THF (300 mL) was add~~ed by cannula and the resulting solution was cooled with a -78°C bath of dry i~sopropani 1. The addition futmel was charged with 78 mL of n-butyllithivart (1.6 M in hexane) by eannula, which was added in a stow stream to the reaction. Distilled propionyl chloride (bp 77 ~79°C), 8.0 mL, was added rapidly via syringe.
The reaction was allowed to stir for 30 min. is the dry icelisopropanol bath.
The reaction was removed from the cold bath, allowed to warm to >0°C, and quenched with 50 mL of saturated aqueous I~iIi~Cl. The mixture was concentrated to a slurry on a rotary evaporator. The slurry was extracted three times with 250 rnL
portions of ethyl ether. The organic extracts were combined and washed with 50 mL each of saturated aqueous NaHC03 and brine, dried with MgSp4, filtered, and concentrated to give a yellow oil. The material crystallized upon sitting. The crystals were triturated once with cold (-20°C) hexanes to give 2l.Q~g (80% yield) ofwhite crystalline material, m.p. 41-43°C.
a , APCI-MS: m/z = 234 (Ngi+),178,117. 1H-NMR (360 MHz, CDC13): 87.2-7.4 (SH,m); 4.67 (IH,m,H4); ,4.14-4.22 (2H,m,HS); 3.30 (lH,dd,J=3,13 Hz,benzylic);
2.89-3.03 (2H,m,H2'; 2.77 (lH,dd,J=9,13,benzylic); 1.20 (3H,t,J=7 Hz,H2~.
D. (4S1-N f(2S 381-2 methyl-3 hvdroxvhexanovll 4 benzYl 2-oxazolidinone: A
dry, 2 i, three-necked round bottomed flask equipped with a 500 mI, addition funnel, a low-temperature thermometer,'. and a stir bar was charged with 19.84 g of N
propionyl-oxazolidinone, capped with septa and flushed~with nitrogen. Anhydrous dichloromethane Empfao~szeit 7.Juni 0:31 AMENDED SHEET

w w ~i ~i:4C h~t~l rK I~IU-rui~i 5HN UltUUtiSa 720 5125 TO 8540tt99990i~9""""
06-06-2001 ~ : US 00000991:

-~19-(i00 mL) was added by cannula, and the resulting solution was cooled to -65°C in a bath of dry icelisopropanol. The addition funnel was charged by cannula with 100 mL of dibutylboron triflate (1.0 M in dichlomtnethaiie), which was added in a slow stream to the reaction. Triethylamine (15.6 mf.) was added dropwise by syringe, keeping the reaction temperature below -If°C. The reaction was then transferred to an ice bath and allowed to stir i at 0°C for 30 min. A.fteic that period, the reac i on was placed. back into the dry icelisopropanol bath and altowed to cool to -65°C. Butyraldehyde (8.6 mL) was added rapidly by syringe, aad the reaction was allowed to stir for 30 mia.
The reaction was transferred to an icelbath and the addition funnel was charged with I O I00 mL of a I M aqueous phosphate solution; pH ?.0 (the phosphate solution is comprised of equal molar amounts of mono- and dibasic potassiuan phosphate). The phosphate solution was added as quickly as possible while keeping the reaction temperature below 10°G. The addition funnel was then charged with 300 mL methanol which was added as quickly as possible while keeping the reaction temperature below 10°C. Finally, the addition funnel was charged with 300 mL of 2:1 methano1:30% hydrogea peroxide. This was added dropwise to ensure that the temperature was kept below 10°C. The reaction was stirred for one hx. after completion of addition. The solvent was then removed on a rotary evaporator until a slurry remained. The slurry was extracted 4 times with 500 rnL portions of ethyl ether. The combined organic extracts were washed with;250 mL each of saturated aqueous sodium bicarbonate and brine. The extract was then dried with MgS04, filtered, and concentrated to give a slightly yellow oil. , T'he nnaterial was then chromatographed on SiOz using 2:1 hexanes:ethyl acetate (product Rf= 0.4) resulting in 2z.0 g (8S% yield) of title compound as a colorless oil. i APCI-MS: mile 306 (MH+); !~ ~ (360 MHz, CDC13): 87.2-7.4 (SH,rn, Phenyl);
4.71 (lH,tn,H4); 4.17-4.25 (2H,m,HS); 3.96 (~IH,m,H3~; 3.77 (lH,dq,J~.S,? Hz, H2'; 3.26 (iH,dd,J=4,13 Hzbenzylic); 2.79 (lH,dd,r 9;13 Hz,benzylic);1.5-1.6 (2H,m,H4';1.3-1.5 (2H,m,HS~j;1.27 (3H,d,J=7 Hz,2: Me); 0.94 (3H,t,J=7 Hz,H6'~. .
E. (2S.3R)-2-nnethvl-3-hydrox hexanoate Iwacetvlcvsteamir~e thioeater: N
acetylcysteamine was distilled at 130°Cl7 mm Hg to give a colorless liquid at room temperature. A dry, 1 ~ t~_necked round bottomed flask equipped with a 500 mh addition funnel and a star bar was capped with septa and flushed with nitrogen. The flask was then charged with 10.7 mL of N-acetylcysteamine by syringe aad with 400 mL of anhydrous THF
AMENDED SHEET
EmpfangsZeit 7.Ju~i 0:37 J"" '"" ~"" l 3 : 42 PM FR MO-FOM SRN D I EG0858 720 5125 TO 8540t399990tt9--"-- - ' 06-06-2001 , ~ US 000009915 t 300622003340 ' by cannula. The mixture was cooled with a lv~fIeOHlice bath. Butyllithium (64 mL of 1.6 M
in hexanes) was added dtopwise by syringe, resulting in formation of a white precipitate.
After stirring for 30 min., arimethylaluminum~ (51 mL of 2.0 M in hexanes) was added dropwise by syringe. The reaction became clear after addition of trimethylaluminum and was allowed to stir an additional 30 min. During this period, 20.5 g (0.068 mot) of (4S~N-[(2S,3R}-2-methyl-3-hydroxylhexanoylJ-4-beilzyl-2-oxazolidinone was put under a blanket of nitrogen and dissvived in 100 mL of anhydrous THF; this solution was then transferred in a .
slow stream by camiula into the reaction. The resulting reaction mixture turned a yellow-I
green color and was allowed to stir for 1 hr. The reaction was finished whey the starting material could no longer be seen by thin-layer chromatographic analysis (ca. 1 hr.).
The reaction was treated with enough saturated oxalic acid to give a neutral reaction with pH paper (approximately 90 mL). The solvents were then removed on a rotary evaporator to give a white slurry. The slurry Gras extracted six times with 250 mL portions of ethyl ether. The organic extracts were combined and washed with brine, dried with MgS04, filtered, and concentrated to give a slightly yellow oil. The thioester product was purified by flash chromatography on SiOZ using 1:1 hexai~es:EtOAc until the elution of 4-benzyl-2-i oxazolidinone. At that point, the solvent system was switched to 100% EtOAc to give pure fractions of dl'ketide thioester. The product fractions were combined and concentrated to give 14.9 g {89% yield) of title compound. Tbis compound is referred to as the propyl diketide thioester in Example 2.
i APCI MS: m/z 248 (N1~I+);1H-NMR (360 Nffiz, CDCi~): a5.8 (br s,lH); 3.94 (dt,ll~, 3.46 (m,2~, 3.03 (dt,2I~, 2.71 (dq,l!1~, 1.97 (s,3Ii}, 1.50 (m,2I~,1.37 (m,2I~,1.21 (d,3H}, 0.94 (t,3HJ. ' t F. (4S)-N f (ZS.3Rl-2-methyl-3-hvdroxv-4-pentenoxl,)-4-Yl 2-oxazolidinone:
t A dry, 2 L three-necked round bottomed flask equipped with a 500 mL addition funnel, a low-temperature thermometer, and a stir bar v~ras charged with 20.0 g of propionyl oxazolidinone A, capped with septa and flushed with nitrogen. Anhydrous dichloromethane (100 ml) was added and the resulting solution was cooled to -15°C in a bath of methanollice.
Dibutylboron triflate (100 mL of i.0 M in dichlorometi~ane) was added in a slow stream via the addition funnel at such a rate as to keep the reaction temperature below 3°C.
Diisopropylethylamine (17.9 mL) was added dropwise by syringe, again keeping the internal temperature below 3°C. The reaction was then cooled to -65°C
using a dry ice/isopropanol AMENDED SHEET
Empfangszeit 7.Juni 0:37 .. ~ : 4:3 h'I") rht nlu-rlJ~'~ ShIIV L 1 CtWIts~ts r eYJ ~ 1 e~ i v o:W
r~rro~~~ri"oo_-,»,~, , 06-06-2001 , ~ US 00000991 ~

-! Z1-bath. Acrolein was added over S min. by syringe. The reaction was allowod to stir for 30 min. after completion of addition.
The reaction was then transferred to an ice bath and the addition funnel was charged with I20 mL (0.1 mol) of a 1 M aqueous phosphate solution, pH 7.0 (the phosphate solution is comprised of equal molar amounts of mono- and dibasic phosphate). The phosphate solution was added as quickly as possible while keeping the reaction temperature belaw 10°C.
The addition funnel was then charged with 400 mL ofmethanol that wen added as quickly as . i possible while keeping tha reaction temporatye below 10°C. Finally, the addition funnel was charged with 400 mL of 2,:1 methano1:30% hydrogen peroxide by initial drapwise addition to i keep the temperature below 10°C. The reaction was stirred for one hour.
The solvent was removed using a rotary evaporator, leaving a slurry. The slurry was extracted 4 tunes with 500 mL poxtions of ethyl ether. The organic extracts were combined and washed with 250 mL each of saturated sodium bicarbonate andjbrine, then dried with MgS04, fltered, and concentrated to give a slightly yellow oil. Trituration with hexane induced crystallization.
RecrystalIization from ether by addition of he~cane resulted in 13.67 g (55%
yield) of product.
IH NMR (360 MHz, CDC13): a7.2-?4 (m,51~; 5.86 (ddd,II~, 5.35 (dt,lI~, 5.22 (dt,lI~, 4.71 {m,lT~, 4.51 (m,II~, 4.21 (rn,2H), 3.89 (dq,lH), 3.26 (dd,lI~, 2.80 (dd,lH), 1.25 (d,3IT).
;
G. (2S.3R) 2-methyl-3-hy~,y-4! ventenoate N acet~rlc3rsteamine thioester: N
acetylcysteamine was distilled at 130°CJ7 mtri Hg to give a colorless liquid at room temperature. A dry, 1 L thrx-necked round bottomed flask equipped with a 500 mL addition funnel and a stir bar was capped with septa anld flushed with nitrogen. The flask was then charged with 7.5 mL of N-aeetyleystearnine by syringe and with 500 mL of anhydrous THF
by cannula. The reaction was then cooled with a MaOH/ice bath. 8utyllithium (44 mL of 1.6 M in hexane) was added dropwise by sy~ge. A white precipitate formed as the n BnLi was added. After stirring for 30 min., 35.5 mL (0.071 moi) of trim;ethylaluminum (2.0 M in I
hexane) were added drop-wise by syringe. The reaction became clear after addition of trimethylaluminum and was allowed to stir an'jadditional 30 min. (4S)-N
[(25,31't)-2-methyl-3-hydroxy 4-pentenoylJ-øbenzyl-2-oxazolidinone front Preparation F (13.6 g) yeas put under a'blanket of nitrogen, dissolved in 50 mL of anhydrous TTY, and this solution was then transferred in a slow stream by cannnla into the reaction. The resulting reaction mixture fumed a yellow-green color and was allowed to stir for 1 hr. The reaction was judged to be Emvfanssieit 7.Juni 0:31 AMENDED SHEET

J"" '"'" ""1 3:43 PM FR MO-FOM SRN DIEG0858 720 5125 TO 8540#99990#9--"- - -' 06-06-2001 . ; US 000009915 (00622003340 _;
finished when starting material could no longer be seen by thin-layer chromatography (ea. 30 min.).
Enough saturated oxalic acid was adds to give a neutral reaction with pH paper (approximately b0 mL). The solvents were then removed by rotary evaporator to give a white slurry. The slurry was extt'aeted six times with 250 mL portions of ethyl ether. The organic extracts were combined, washed with brine, dried with MgS04, filtered, and concentrated to give a slightly yellow oil.. The thioestes was then purified by flash chromatography on SiOZ.
The column was run with 1:1 hexanes:ethyl acetate until the elution of oxazolidinone. At that point, the eluent was switched to 100% ethyl acetate to give pure fractions of product. ?he fractions wero combined and concentrated to give 7.7 g (71 % yield) of Title compound product. This product is referred to as the vinyl diketide thioester in Example 2.
1H-NMR (360 MHz, CDCl3): 85.82 (ddd,lH), 5.78 (br s, lf~, 5.32 (dt,lH?, 5.21 (dt,lH), 4.47 tm,lI~, 3.45 (m,2I~, 3.04 (m,2I~, 2.81 (dq,lFi), 1.96 (s,3H), 1.22 (d,3~.
. atn a I
' Preparation of lrrvthronolides A. ~ 5-methyl-6-deoxvervthronolide B (Compound P, R, H, Rd~ro 1 Py )~
Streptomyces coelicolor CI3999/pJRl2 is described in PCT Publication No. WO
97/02358 which claims priority to U.S. patent application Serial Nos.
08/896,323, filed 17 fuly 1997, and 081675,817, filed 5 ~ulx 1996,1 each of which is incorporated herein by reference. Plasmid p11ZT2 encodes a mutated~forrn ofAEBS in which the ketosynthase domain of module I (KS 1 ) has been inactivated via mutagenesis (KS I
°). S. coelicolor strains comprising this plasmid that are fed (2S, 3R)'2-meth 1-3-h o exanoate-N
i y Y~ ~
acetylcysteamine (Preparation E, propyl diketide) of Example 1 produce 15-methyl-5-i deoxyerythronolide B.
A 1 mI, vial of the CH9991pJRJ2 wor~la'ng cell bank is~thawed and the contents of the vial are added to 50 mL of Inoculum Medium 1 in a 250 mL baffled flask. The flask is placed in art incubatorlshaker maintained at 30t1°C and 1755 RPM for 48~10 hours. The I
50 mL culture is then added to a 2.8 L baffled flask containing 500. mi, of lnoculum Medium 1. This flask is incubated'in an incubator/shaker at 3011°C and 175~25 RPM for 48~10 hours. The 500.mL culture is divided equallyi among ten 2.8 L baffled flasks each containing 540 rrd:, of Inoculmn Medium 1. All flasks are then incubated as described previously.
AMENDED SHEET
Emvfan8szeit 7.Juni 0:37 J"" "~ ~"" 1 3 : 44 PM FR MO-FOM SAN D I EG0858 720 51 25 TO
8540ii99990tt9"""" '" ", 06-06-2001 , US 00000991 E

-'23 A 150 L fertnenter is prepared by sten'lizing ~I00 L of Production Medium 1 at 121°C
for 45 minutes. After incubation, all 10 flasks are combined in a 5 L sterile inoculation bottle i and aseptically added to a. I50 L fermenter. The fcrmenter is controlled at 30°C, pH 6.5 by addition of 2.5 N HIS04 and 2.5 N NaOH, 'dissolved oxygen >_ 80% air saturation by ag'station i rate (500-700 RPM), air flow rate (10-50 LPM), andlor back pressure control (0.1-0.4 bar).
Foam is controlled by the~intcrmittent atiditioin of a 50% solution of Antifoam B.
At 2415 hours (2S, 3R)-2-methyl-3-hydroxyhexanoyl-N aceiylcysteaminc (prapyl diketide, Preparation E in Example 1) is add i to a final concentration of 1 g/L. Propyl diketide is prepared by solubilizing in dimethyl sulfoxido at a ratio of 1:4 (diketide~to DMSO) i and then filter sterili2ed (0.2 ~tm, nylon filter). Production of 15-methyl-G-deoxyerythronolide B (15-methyl-6dEB) ceases on day 7 and the fermenter is harvested. The a fermentation broth is centrifuged at 20,500 g ~ n an Algha Laval AS-26 centrifuge. The product is predominantly in the centrate; the centrifuged cell mass is discarded.
This process has also been completed~in a 1000 L fermenter (700 L working volume).
The inoculum process is identical to the above process except that the 150 L
fermenter is charged with Inoculum Medium 1 and the 1000 L fermenter is charged with Production Mediuan 1. The fermenter is controlled at 30°C, pH 6.5 by addition of 2.5-5 N HzS04 aad 2.5-5 N NaOH, dissolved oxygen Z70% au saturation by agitation rate (140-205 RPNn, air flow rate (100-Z00 LP11~, andlor back press i 'e control (0.2-0.5 bar). Foam is controlled by the addition of a 50% solution of Antifoam B as needed. At 24~5 hours racemic 2-methyl-3-hydroxyhexanoyl-N propionylcysteamine (300 grams) is added to the 1000 L
fermenter. The femnenter is harvested at 4.6 days by centrifugation as desen'bed above.
Media used in this process include the following:
Inoculum Medium 1 Component ' C
oncentradon KN03 _ i 2 g/L

'Yeast extract ; 2p H case SF ; 20 Feso4-7H~o . as NaCl I2.5% stock ; ~ ~, M 04 I2.5% stock ' MaS04-HZO 0.5% stock ~ 1 ~

ZnSOa-7H20 1.0% stock ; 1 mLJL

CaCIZ-2H20 2.0% stock ~ 1 ~

AMENDED SHEET
Emafanssteit 7.Juni 0:37 v 06-06-2001 3 ~ 44 PM FR MO-FOM SRN D I EGOB58 72A 5125 TO B540tt9999ett~w "' i 300b22003340 ' ~-24-Sterilized by autoclaving for 60 minutes at r21 °C.
Post-sterile additions: I
1) 1 mL/L of 50 mglml Thiostrcpton in 100~o DMSO, sterile filtered.
2) 1 mT-!L 100% Antifoatn B silicon emulsion (J.T. Baker), autoclaved.
3) 40 mL of 500 g/L glucose, sterile filtered.
Production Medium 1 Com onent i Corn Starch I 45 Corn st li uor ~ 10 Dried, inactivated brewers ; 10 east CaC03 1 Sterilixed in fermenter for 45 minutes at 121°C.
Post-sterile additions for J'roduction Mcdiuui 1 I
1) 1 mIJf, of SO mg/mI Thiostrept~on in 100% DMSO, sterile filtered.
2) 1 mIJL of I00% Antifoam B (J.T. Baker), autoclaved.
After centrifugation, the centrate is filtered. The filtrate (approximately 700 L) are passed through an Amicon Moduline column (20 x 350 cm) containing 20 L of I-B'20 resin (Mitsubishi). The flow rate during loading i5 4 Lminute with a presswre drop below 8 psi.
After loading the resin is iwashed with 20 L of watcr and then 40 L of 30%
methanol. 15-tnethyl-6dEB is eluted using 100% methanol: Four 12 L fractions were collected with i fractions 2, 3 and 4 containing all of the detectable 15-methltl~dEB. The 15-methyl-6dEB
product pool is diluted with 36.7 L of water giving 7S L of a clear solution.
This solution is loaded dir~tly onto a 5 L Amicon Vantage Column containing HP20SS resin (Mitsubislui}.
Column loading is carried out at I LJminute. The column is eluted with 20 L of 65%
methanol, 20 L of 70% uiethanol, 20 L of $0% methanol, and finally 20 L of 100% methanol A total of I6 x 5 L fractious were collected. The 80% fractions along with the last 70%
fraction were combined (25 L) and evaporated to dryness. The resulting residue is dissolved in 1 L of 100% methanol, filtered, evaporated; and dried iri a vacuum oven at 40oC. This process resulted in 33 g of a solid product containing 93% 15 methyl-6dEB.
B. ~4.I 5-dehvdro-6-deoxvervthronolide B (Compound P --H Rd-alIvl):
S. coelicolor strains comprising this plasmid that are fed (2S,3Rr2-methyl-3-hydroxy-4-pentenoate NAc Cysteamine thioester (Preparation G) of Example 1 produce 14,15-Empfangsieit 7.Juni 0:3~AMENDEDSHEET

x.06-06-2001 3 ~ 44 PM I-K InU-1-UhI 'JHIV L I Cl7VCI~CS f GYJ J 1 GJ 1 V
OJ'nYJfTi7JJJCJfIJO:JHeJ n . wr r dehydro-6-deoxyerythroztolide B when prepared in accordance with the process described in Preparation A about to produce 15-methyl-6-deoxyerythronolide B.
C. 14-nor-6-iieoxvtrYthronolide B tCom~ P Ra,~I;I R.~amethvll;
i Similarly, 14-not 6-denxyerythmnolide B is produced u~g S. coelicolor 't CH999/pCK7 host, without using a diketide ithioester., when prepared in accordance with the process descn'bed in Example 2A.
xam le 3 . l~~r~aration of Enr~yg IO The 6-dE8 dezivative compounds produced in Example 2, Preparations ArC are converted to erythromycin derivatives using a recombinant strain of Saccharopolyspora erythraea. For production of erythromycins having both the 6 and 12 hydroxyl groups, the S. erythraea strain used was K40-67 or K39-14V. This strain was created by transforming sn S. erythraea strain capable of producing highs levels of arythromyein A with a pVVHIYI3-derived plasmid comprising a mutated eryAl sequence encoding an inactivated KS
1 domain.
By homologous recombination, the resulting transformants were rendered incapable of producing 6-dcoxyerythrnnolide B. Thus the~dE$ analog fed is not subject to competition for hydroxylation at the b position. For production of erythromycin derivatives having only the 12-hydroxyl group, the S.. erythraea strain used was K39.07. This strain was constructed i from strain K40-67 by disruption of the eryF hydroxylase gene; this destroys ability to hydroxylate the analog at the 6-position. Both strains were fermented under substantially similar conditions, as described below.
15-sncthyl-e~cin A: 15-methyl-orytbromycin A is produced according to tha following protocol: A I mL vial of the K39-~4V working cell bank is thawed and the contents of the vial arc added to 50 mL of Inoculum Medium 2 in a 250 mL
baffled flask. The flask is placed in an incubator/shaker maintains at 3411°C and 175125 RPM for 48110 hours. The 50 nnh culture is then added to a 2.'8 L baffled flask containing 504 mL of xnoculuna Medium 2. The flask is incubated hi an incubator/s~haker at 34~1 °C and 175125 RPM for 48110 hours. The 500 mL culture is divided equally among ten 2.$ L
bafllcd flasks i each containing 500 mL of Iraoculum Medium~2. All flasks are then incubated as described previously.
AMENDED SHEET
Empfaogsreit 7.Ju~oi 0:37 Un, ,~,~ ~.,...~. J,aJ ry ry ..v ~ v.. .rv", .,.... ~ ....... ,__ ___._ _ __ 06-06-2001 , US 000009915 i I
A 150 L fermenter is prepared by steFilizing 100 L of Production Mcdiutn 2 at 12I °C
for 45 minutes. After incubation, all 10 flask's are combined in a 5 L sterile inoculation bottle and aseptically added tv a I 50 L fermenter. The fermenter is controlled at 34°C, pH 7.0 by addition of 2.5 N H2SOa and 2.5 N NaOH, dissolved oxygen >_ 80% air saturation by agitation rate (500-700 RPM), air flow rate (15-50 LPlvn, andlor back pressure control (O.I-0.4 bar).
Foam is controlled by the addition of a 50% solution of Antifoam B.
At 24~5 hours a 58-60 mZJhour 15%idextrin (w/v) feed is initiated. The dextrin solution is continuously mixed during the fc I period. At 24~S hours 25 grams of 15-methyl-bdEB (Preparation A in Example 2) are added to the fermenter. The 15-methyl-6dEB is prepa3red by solubilizing 25 grams of 15-met$yl-6dFB in 400-600 mL of I00%
ethanol and filtering (0.2 Etm, nylon filter). Conversion of 15 methyl-6dEB to 15-methyl-erythromycin A
ceases after 60110 hours and the fermenter is harvested. The fermentation broth is centrifuged at 20,500 g in an Alpha Laval AS-26 ccntrifiige. The product is predominantly in the centrate;
the centrifuged cell mass is discarded. , ' Media used in this process include the following:
Inoculum Medium 2 Component ~ g/1, Corn Starch i 16.0 Corn dextrin ~ 10.0 Sa Meal Flour ' 15.0 CaCO~ ' 4.0 Corn st li uor i 5,0 So Bean Oil i 6.0 NaCI 2.5 ~)aSOa j Z.O

Sterilized by autoclaving for 60 minutes at 121°C.
Post-sterile addition:
1 mL/L 100% Antifoam B (J.?. Baker), autoclaved.
Production Medium 2 ' Com onent Corn Starch , - ~ 17.5 Com dextrin T a 3 I6.0 So Meal Flog 16.5 CaC03 ' ' 4.0 Corn ste li uor . g.0 So Bean OiI . 3.0 AMENDED SHEET
EmvfanasZeit l.Juni 0:37 X06-06-2001 3:45 PM FR MO-FOM SRN DIEG0858 720 5125 TO 8540~99990tt969"' ._ z~ _ i NaCI ~ 3.5 2Sd4 1.~
Sterilized in fermenter for 45 minutes at 121 °C.
Centrifuged fermentation broth (127 L) containing 34 g of the target molecule is passed through 18.3 L of HP20 sorbant packed into an Amicon P350 Moduline 2 chromatography column. At 4 Llmin loading, backpressure is found to be less than 5 psi.
Following loading, the resin is washed with 20 L deionized water and then 40 L
of 30%
methanol. I S-Methyl-Erythromycin A is eluted using 54 L of 100% methanol. The product pool is evaporated using a Buchi mtary evaporator (R-152). The solids were dissolved in a nunimal amount of 100% methanol, filtered and the filtrate evaporated to dryness. This resulted in 123 g of material containigg 30% i I5-Methyl-Faythromycin A by weight. 80 grams . of the 30% material is extracted twice with 1;L of 40°C acetone. The acetone extract is i filtered, and the filtrate is dried on the inside surface of a 20 L rotary evaporation flask. The r solids were extracted with 9:1 hexane to acetone three times at 40°C.
The organic extracts were pooled and evaporated to dryness ,giving 32 g of solids enriched (68%) in I5-Methyl Erythromycin A. The product pool from the acetonelhexane extraction is dissolved in 1 L of methanol to which an equal amount of water is added. The methanol solution is loaded onto a Hp20SS chromato~raphy~calumn (Kontes) pr~feviously washed arid equilibrated with 50%
methanol. Column dimensions were'4.8 x 115 em. Column loading with respect to I
Methyl-Erythromycin A is I 1 g/L. The colump is washed with 50% (0.8 L) and 50% (8 L) methanol in water. Elution of the target molecule is carried out using 70°!0 (8L), 80% (16 L) and 85% (8 L) methanol iit water. 1 L fractions were collected. Fractions 11-29 were combined, evaporated and dried in a vacuum oven giving 23 g of product with 93°!o purity.
This material served as starting material for the chemical derivatization procedures descn'bed in the following.examples. The following compounds are also produced by this methodology: 14-norerythromycin A (Re=Met; 14,15-dehydro-erythromycin A
(Ra=allyl);
la-nor-6-deoxy-czythromycin A;14,15-dehyclro-6-deoxy erythromycin A; and t5 methyl-6-deoxy erytluomycin A. When used to make 3descladinose-3-oxo-derivatives, the erythromycin A derivatives were not separated, from the orythrvmxein C
derivatives; instead, mixtures of the erytbmmycin A and erythromycin C compounds were used as starting materials for chemical derivatization.
These products were extracted and puri'~~fied as follows:
Emvfan8steit 7.Juni 0:3AMENDEDSHEET

~~06-06-2001 3' 45 PM FK ~tU-t-Ut~ 5Hrv m Cuvo~~ r c~ ,.~. ~~ ~ ., ~~..~.,~.,.,.,~".....,~.. . . __ i US 00000991 ~
300622003340 ~ ' .
=28-In general, fermentation troths are brought to phi 8.0 by addition of NaOH and ethanol is added (0.1 l:ll: broth). The broth is clarified by centrifugation and loaded onto an XAD-16 resin (Rohm and Haas) column (1 kg XAD/1 g erythromycin analogs) at a flow, rate of 2-4 mLlcmz-min. The loaded resin is washod with 2 column volumes of 20%
(vlv) ethanol in water and the erythromycin analogs are eluted from the resin with acetone and collected in l l2 column volume fractions. The fractions containing erythromycin analogs are identified by thin-layer chromatography (ethyl acetate:hexanes I:1) and HPLC/MS.
The acetone fractions containing erythromycin analogs are pooled and the volatiles are removed under reduced pressure. The resulting aqueous mixture is extracted with ethyl acetate. The ethyl acetate extract is washed with saturated Nal~sC03 and brine solutions, dried over sodium or magnesium sulfate, ~tltered, and concentrated to dryness under reduced pressure. Crude material is dissolved in dich~loromethane and loaded onto a pad of silica gel and washed with dichloromethane:methanol (96:4 vlv) until the eluent is no longer yellow.
The desired material is with dichloromethaneunethanolariethylamine (94:4:2 v/v) and IS collected in fractions. Fractions containing ei y0uomycin are identified bythin-layer chromatography, collected and concentrated under xeduced pressure. This material is recrystallized from dichloromethanelhexanes~
This general procedure is illustrated a's follows:
(i) 14-nor ~rt~c~nvcins: 1 liter of ethanol was added to each of 10 liters of fermentation broth. The broth was centrifu ed and the su 8 : pernatant was passed through 0.6 liters of XA.D (column dimensions 17 cm x 6~ 5 cm) at a flow rate of 100 mLniin. ARer I
loading, the column was washed with 1.5 liters of 20% (vlv) ethanol in water.
The desired I
material was then eluted with acetone. The fiactions containing this material were concentrated under reduced pressure until thej volatiles were removed and the aqueous remainder was extracted with ethyl acetate. The ethyl acetate layers were washed with .
saturated sodium bicarbonate solution, brine, dried with mtagaesium sulfate and concentrated under reduced pressure to give the crude extract.
Crude material (0.6 g) was dissolved in dichloromethane and gravity filtered through a 3 cm pad of silica gel in a'6 cm diameter fritEed funnel. The material was eluted with 400 mL
of dichloromethane fnilovved by 400 mL
dichloro~methane:methanolariethylatxtine (90,10:2 v/v) and collected in 40 mL fractions. Fractions containing eryth~coutycin were identified by thin-layer claromato h ~ ether:methanol.
~P Y ( -NH~OH 90:8:2 v/v, Rf.- 0,35 and AMENDED SHEET
Emvfa~sszeit l.Juni 0;37 ~ 06-06-2001 3 ~ 46 PM FR MO-FOM SRN U 1 E~UtsSts red ~ t ~c~ 1 V
0.7~fYJN.7JJJ'UffJO-~~~~ r . ..,..

dichloramethane:methanol 95:5 v/v, Rf~ Q)~and concentrated under reduced pressure. This material was recrystallized from dichloromethane/hcxanes.
i (ii) 1 S-methyl-ervri~romvr 'ns: 8 liters of ethanol was added to approximately 80 liters of fermentation broth. The broth was centrifuged and the supernatant was passod through 2.5 liters of SAD at a flow rate of 230 mlJmin. After loading the column was washed with 1 Liter of water and 5 liters of 20% (vlv) ethanol in water. The desired material was theft eluted with acetone. The fiactions containing this material were concentrated under reduced pressure until the volatiles were rea loved and the aqueous remainder was extracted with ethyl acebte. The ethyl acetate layers were washed with saturatod sodium bicarbonate solution, brine, dried with magnesium sulfate and concentrated under reduced pressure to give the crude extract Exude material (8.3 g) was dissolved in dichloromethane and gravity filtered through a 3 cm pad of silica gel in a 9 cm diameter ftitt~ed funnel. The material was eluted with 200 mL
of dichloromethane followed by 600 mL of dichloromethane: methanol (96:4 v/v) followed by 900 ml. dichloromethane:methanolariethylamine (89:9:2 v/v) and collected in 40 mL
fractions. Fractions containing erythromycini were identified by thin-layer chromatography (ether:methanol:NH40H 90:8:2 v/v, R f ~ 0.4 ;and dichloromethane:methanol 95:5, R,f ~ 0.05) and concentrated under reduced pressure. This material was re-subjected to the above procedure before it was sui#able for recrystalloiZation.
(iii) 14 nor-6-deoxv exvthsom~; 1 liter of ethanol was added to each of 210 liter fermenting. The broths were centrifuged and the supernatants were combined for a total of approximately 22 liters. The combined broths were then passed through 1 liter of XE~,D
(column dimensions 23.5 cm x 6.5 cm (i.d.) a't a flow rate of I70 mL/min.
After loading the column was washed with 2 liars of 20% (vlvj ethanol in water. The desired material was then eluted with acetone. The fractions containing this material were concentrated under reduced presswc until thc;volatiles were removed and the aqueous ranainder was extracted with ethyl acetate. The ethyl acetate layers were washed with saturated sodium bicarbonate soIutio brine dried with 'magnesium sulfate and concentrated under reduced pressure to give the crude extract_ ' ' (iv) 15-methyl-6-dcoxv-a ~rnmvr~ine~ 1 liter of ethanol was added to each of 3 ferrnentors containing 10 liters of broth. The broths were centrifuged and the supernatant was passed over I.25 liters of ~A,D (column dime~isions 40 cm x 6.5 cm) at a flow rate of 130 ' AMENDED SHEET
Emufaasszeit 7.Juni 0:37 ~ 06-06-2001 3 ~ 46 PM FR MO-FOM SHN 1J 1 ttauti't~ r ~e~ ~ i e:.r ~ ~
o.r..,~".r,~....,~.,,.,~,. ._.. . . _ .

~. 30 _ mIJmin. The column was then washed with 3 liters of 20% (v/v) ethaaol in water. T'he desired material was they eluted with acctoi je. The fractions containing this material we1'e concentrated under reduced pressure until the volatiles were removed and the aqueous remainder was extracted with ethyl acetate. iThe ethyl acetate layers were washed with saturated sodium bicarbonate solution, brine dried with magnesium sulfate and concezttratcd under reduced pressure to give the crude extiact.
Crude material (2.8 g) was dissolved) in dichlorotnethane and gravity filtered through a 3 cm pad of silica gel in a 6 cm diameter frilled funnel. The material was eluted with 400.mL
of dichloromethane:methanol (96:4 v/v) followed by 400 mL
dichloromethane :methanolariethylatnine (8 x:9:2 v/v) and collected in 40 mL
fractions.
Fractions eoatainmg eryrhromycm were identified by thin-layer chromatography (ether:methaaol:NH40H.90:8:2 v/v and dieh~oromethane~ethanol 95:5) and concentrated under reduced pressure. This material required further purification by silica gel chromatography.
IS
RQ=OH. R, Mc. R~Me ,RS=H, Rg=H Z ~'~
A. I4-N~ 'rthrom~vcin A g_Oxizne: A soh~tion of 14-noceryt~omycin A (4.621 g, 80% pure), hydroxytamine (0.5 ml of 50%j aqueous solution) and acetic acid (0.2 ml) in isopropanol (2 mi) was kept at 50°C for Z2 hours. It was extracted with chloroform/ethanol (3I2), washed with sodium bicarbonate, brine, and dried over MgS04. Filtration and evaporation in vacuo yielded a crude product~(0.65 g) as a white solid which was used directly for next transforaration.
B. 14-Noretvtin~omvcin A-9-f0-.~~ -isooropox~cvclohe;ryl)lox~: To a solution of above crude I4-noreythiomycin p~, goxime (O.fS g) and 1,1-diisopropoxy cyelohexanone (0.95 ml) in mcthylene chloride (2 ml) was added pyridiruiump-toluenesnlfonate (PATS) 0.333 y ~ ( ) ( g) in meth lone chloride 2 ml . After~stirring overnight, the mixture was extracted a (chloroform/ethanol 3:2), washed (NaF3C03 HzO, brine), and dried (lvlgsp4).
After. filtration and evaporation in vacuo, ahe crude product was repeatedly driven with toluene and isopropanol to yield 0.74 g of product, which was used directly for next reaction.
AMENDED SHEET
Emafan~sieit 7.Juni 0:37 ~.~~ ~~ ~~ IIU ~UI~I ~JH~ LICUVCS.7O IGrJ J1GJ I V VJ'lV.Iv.IVJW JVff.~VvJrtV
n ..r v..
06-06-2001 , i US 00000991;

C. ~ 4"-bis-0-trimethylsilvl-14-norervthromycin A-9-f O-f 1 ~ycvclohexy oxime: To a solutia i of 14-aore~hmm in A 9 Yc -gyp-(1_ isopropoxycyclohexyl)]oxime (0.74 g) is methylene chloride (6 ml) was added a solution of tnimethylsilyl imidazole (0.33 ml) and trimethylsilyl chloride {0.18 ml) in methylene chloride (2 mI) at 0°C. After 5 minute stirring, ethyl acetate was added, washed (NaHC03-HzO, brine), and dried (MgS04). Flash chromatography on silica gel (10:1 hexanes:acetone, l triethylamine) afforded port product as a white solid (0.50 g). Mass spectrometry reveals (M+H+] ~ 1020.
D. 6-O-Methyl-2' 4"- a il 1-14-no in A- - O- 1-isonropoxycvclohexyl)loXime: A solution of 2',4"-bis-Qtrimethylsilyl-!4-norerytluomycin A
9-[Q(1-isopropoxycyclohexyl)]oxime (0.3 g0.29. mmol) in 1:1 mcthylsulfoxide/tetrahydrofiuan (I?NISp!>"T~ (1.4 ml) was treated with 0.3 ml of a 2114 solution of methyl bmmide in ether arnd cooled to 10°C. A mixture of 1 M solution of potassium tent-butoxide in THF (0.6 ml ) and DMSO (0.6 mI) was added over 6 hours using a syringe pump. The reaction was then diluted) with ethyl acetate, washed with saturated NaHC03, brine, and dried over MgS04. Filttation and evaporation in vacuo yielded a crude product (0.29 g) as a white solid. Mass speetiometry reveals [M+I~j = I 034.

E. 6-D-Meth ~ 1-I4- orcr hxom~ ' in A -oxime: A mixture of 6-O-methyl-2',4"-bis-O-trimethylsilyl-14-norerythromycin A 9 ~(O-(1-isopropoxycyclohexyl)]oxime (0.29 g), acetic acid (3.6 ml), acctonitrile (6 ml) and water (3 ml) was stirred at ambient temperature for 4.5 hours. The mixture was driven to dryness using toluene to give a crude product as white solid (0.24 g), which was used directly for next step without further purification.
F. b-Q Mcthlrl- 4-note m tin ~ A mixture of 6-Q-methyl-14.
norerythromycin A 9-oxime (0.24 g), sodium~hydrosulfite (0.45 g, 85% pure), water (3 mI), ethanol (3 ml) and formic acid (0.07 ml) was kept at 85°C for 8 hours.
The reaction was brought to pH 8 with 1 N NaOH and extracted with ethyl acetate. The organic extract was washed with brim dried over MgSOa, filtered, and concentrated to yield a made preduct as a white solid (0.2 g). Mass Spectrometry revesis [M+H~'J = 735.
AMENDED SHEET
Emafangszeit l.Juni 0:37 L.... ...... _......, u,,,m v m ~ m my m,.. r.... ... ...... .__ __._ _ _.
06-06-2001 ~ US 00000991 ~' le S a ' of eth 1- 41 -de i. orm w ere ~~.-.Rs~
A. 14 15-deh er throw in A, 9-0 ' a A suspension of 14,15-dehydroersrth ' myein A (L984 g, 47% purity,1.2 mmol) in mL of 2-propanol was treated with 1.97 mL ~ f 50% aqueous hydroxylamine and stirred until dissolved. Acetic acid (0.62 mL} was added~and the mixture was stirred for 25 hours at 50°C.
Upon cooling to ambient temperature, saturated NaHC03 was added and the mixture was concentrated en vacuo to, remove isopropano'l. The resulting aqueous mixture was extracted I
three times with 250-mI, portions of CHC13. i The organic extracts were combined, washed with saturated NaHC03, water, and brine, then dried over MgSO4, filtered, and concentrated to yield 0.92 g of product.
B. I4 1 -do ' m in A 9- 1-iso o loh 1 oxime The oxime from (A) (0.92 gJ was dissolved in 6.2 mL of CH~C1Z and treated with 1,1 diisopropoxycyclohexane (1.Z3 g) and pyridinium p-toluenesulfonate (0.464 gar) for 15 hours at ambient temperature. The mixture was diluted with 160 mI, of CH2Clz, then washed sequentially with saturated NaHC03, water, and brine. The organic phase was dried with MgS04, filtered, and evaporated to yield a brown syrup. Chromatography on silica gel (gradient from toluene to~l:l toluene/acetone~+ 1°/a Et~ 3tielded 0.99$
g ofproduct.
C. ~',4"-bis(O-trimeth silv1~,14! 15-dehydr~, hromvcin A 9 (,O (I
iso 0 ox c rcIohex 1 oxime A solution of 14,15-dehydroeryttrCOmjre~ A 9_[~(1-isopropoxycyclohexyl)Ioxime (998 mg, 9.96) in 1 i.25 mL of CHZCIZ was cooled on iCe under inert atmosphere arid treated with a solution of chlorotrimethylsilane (0.24~mL) and 1-trimcthylsilylimidazole (0.44 mL ).
After 30 minutes, the reaction was diluted with 250. mI. of ethyl acetate and washed sequentially with saturated NaHC03, water, aad brine. The organic phase was dried with MgS04, filtered, aad evaporated to yield 1.002 g ofproduct.
D. ,~' 4"-bis O- eth 1 ' -6-O-meth 1- 4 15-deh oe m cin A 9- O-1-iso ro oxycvclohcx~_1 oxiime I
A solution of2',4"-bis-O-trimothylsilyl-14,15-dehydroerythromycin A 9-(O-(1-isopropoxycyclohexyl)]oxime ( 1.00 g, 20.7 mmol) in 9.69 mL of 1:1 t tetrahydrofuranlmethylsulfoxide was cooled to 10°C and treated with 0.97 mL of 2.0 M
AMENDED SHEET
Empfangsteit 7.Juni 0:37 L,06-06-2001 x:41 r'I'1 1'tC wV-rWn Jnlv u~wlvu.mr ,~c, ...~.. ...
....._.._____ ___ U S 00000991:
. a ~oo6z2oo3340 methyl bromide in ether under inert atmosphere. A mixture of methylsulfaxide (1.94 rnL}
and 1.0 M potassium tern-butoxide in tetrah idrofutan (1.94 mL) was added slowly. The reactioa was monitored by thin-layer chromatography (silica gel, 10:1 toluenelacetone), and was j udged complete after addition of 1 _6 molar equivalents of base. The reaction was diluted with 200 mL of ethyl acetate and 70 inL of satiuated NaHC03. The mixture was transferred to a separatory funnel, diluted with 850 mL of ethyl acetate and 280 nzL of saturated NaHCO3, then washed soquentially with water and brine. The organic phase was dried with MgSO4, f Itered thmugh Celitc, aid evaporated to yield 21.2 g of cmde 6-O-methyl 2',4"-bis-4-tcirnethylsilyl-14,15-dehydroezythromy~cin A 9-[O-(1- .
IO isopmpoxycyclohexyl))oxime. This was carded on without furtherpurifeation.
E. 6-O-me , v - 4 -dehvdroenit~mm~n a g~xitn~e ., A solution of 6-O-methyl-2',4" bis-p-trimethylsilyl-14,15-dehydroerythromycin [O-{1-isopropoxycyclohexyl)joxime (1.0 g) in 9.8 mL of 2:1 acetonitrilelwater was treated with 5.3 mL of acetic acid, and stirred for 8 Hours at ambient temperature.
The mixture was I
concentrated en vacuo, then repeatedlit concentxated after addition of tolu~e to yield 0.797 g of crude 6-O-methyl-14,15-dehydroerytiuomycin A 9-oxirne.
.
F. 6-O-methyl-14.I5-dehyd-rocr rfthro~mvcin A
A solution of 6-O-methyl-14,15-dehydroerythromycin A 9-oxime {0.797 g) and sodium hydrosuIfite (85%, 1.02 g~ in 7.5 mL'of 1:1 ethanol/water was placed under inert atmosphere. Formic acid (0.186 mL) was added dt~opwise, and the mixture was stirred at 80°C for 3 hours. After cooling to ambient temperature, the reaction wa,s adjusted to p~I 10 with 6 N NaOI~ and extracted three times with I50-mL portions of ethyl acetate. The organic extracts wear combined and washed sequentially with saturated NaHC03, water, acrd brine.
The organic phase was dried with MgS04, filtered, and evaporated to yield 0.68 g of 6-0-meth I-14 15-deh Y ~ Yaroe~rthrOmycin A suitable for furrhez conversion.
Exainple_6, Synthesis of 6-O-methyt_ I 5-methylervt_h~mycin A i a Formula t,3~ w a R~ OH.
R~~roDVL R~Me A. t5-Meth r~"erythromvcin A 9-Ozime: A suspension of IS-methyierythromycin A (20.0 g, 85% puzity, 22.6 mmol) in 40 m1~ of 2-pmpanol was treatod with 20.5 mL of 50"/0 ueous h ' aQ ydroxylaniine and stirred until dissolved. Acetic acid (6.41, mL) was ceded ~d the AMENDED SHEET
EmafanasZeit I.Juni 0:37 ~ 06-06-2001 3 ~ 48 PM FR MO-FOM SRN D I EG0858 720 51 25 TO
854ett~~~~eu~b'~~~ r .ara , , 30o62Z003340 mixture was stirred for 15 hours at SQ°C. Upon cooling to ambient temperature, saturated NaHC03 was added and,the mixture was concentrated en vacuo to remove isopropanol. The resulting aqueous mixture was extracted threie times with 250-mL portions of CHCI3. The organic extracts were combined, washed with saturated NaHCOa, water, and brine, then dried over MgSOo, filtered, arid concentrated to yield 20.5 g of crude product.
Analysis by LC/MS
revealed a 94:6 mixture of E and Z oximes, [M+N]+ = 764, B. 15-Methvlervthromyc'~A 9-jOJl-isoptopol,;)r~yclohexvl)loxime: The cmde oxime from above (20.5 g) was dissolved in 55 mL of CHZC12 and treated with 1,1-diisopropoxycyclohexane (27.3 mL) and pyridinium p-toluenesulfonate (9.8 gm) for 15 hours at ambient temperature. The mixtnre was diluted with 160 mL of CH2C11, then washed sequentially with saturated NaHC03, water, and brume. The organic phase was dried with MgS04, fxltcred, and ev ~ orated to eld a brown s ap Yi ~ yrup. Chromatography on silica gel (gradient from 2:I to 3:2 hexanes/acetone + 1% Et3l~ yielded 18.0 g ofproduct C. 2'. 4"-bis-~-trimethvlsitvi-1 S ii,pthvirr~rt",r~r"".~.:~, a o_rn_n _ isoflropoxvcvclohexvl)loicime: A solution of 15-Methyter~rthromycin A g-~O-(1-isopropoxycyclohexyl)Joacime (9.00 g, 9.96 trimol) in 25 mL of CHzCIz was cooled an ice under inert atmosphere and treated with a solution of chlorotrimethylsilatte (1.89 mL) aad 1-trimethyls~ylimidazoIc (3.65 mL ) in 8 mL of C1i2C1Z. After 30 minutes, the reaction was diluted with 250 mL of ethyl acetate and washed sequentially with saturated NaI~C03, water, and brine. The organic phase was dried with iVIgSO4, filtered, and evaporated.
The crude product was purified by silica gel chromatography (gradient from hexanes to 10:1 hexaneslacetone + 1 % Et3I~, yielding 7.8 g of product.
D. 6-O-Mcth 1-2' 4"-bis-O- eth lsil - -meth m i 9- O-iso ~ c ohe oxi e: A solution of 2',4' =bis-O-trimethylsilyl-15-,ZS methylerythramycin A 9-[O-(1-isopropoxycyclohexyl)Joxime (21.7 g, 20.7 mmol) in 41.4 mi, of tetrahydrofuran wa$ cooled to I 0°C and treafed with 41.4 mL of methylsulfoxide and 20.?
mL of 2.0 M methyl bromide in ether under inert atmosphere. A mixture of methylsulfoxide (41.4 mL) and 1.0 M potassium tent butoxide in tetrahydrofuran (41.4 mL) was added at a rate of ca 20 mL pes hour.' The reaction was ryonitored by thin-layer chromatography (silica gel, 1 O:I tolucnetacetone),, and was judged complete after addition of 1.6 molar equivalents of base. The reaction was diluted with 200 mL of ethyl acetate and 70 mL of saturated Na1-TCO3. The mixture was transferred to a separatory funnel, diluted with 850 mL of ethyl AMENDED SHEET
Em~faneszeit 7.Juni 0:37 ~ 06-06-2001 3: 48 PM FR MO-FOM SRN D I EG0858 720 5125 TO
8540tt99~5ertab;~~'~
. US 000009915 acetate and 280 mL of saturated NaHC03, then washed sequentially with water and br~me.
The organic phase was dried with MgS04, filtered through Celite, and evaporated to yield 21.2 g of crude 6-O-methyl-2',4"-bis-O-trimethylsilyI-I S-methylerythromycin A
9-[0-(I-isopropoxycyclohexyl)]oarime. This was cairied on without further purif ration.
E. 6-0-Methvl-I5-methylerythnomvcin A 9-oxime: A solution of 6-0-methyl-2',4"-bis-O-trimeth lsil 1-15-meth 1 y y y erythromycin A 9-[O-(1-isopropoxycyclohexyl)Joxime (21.2 g) in 110 mL of acetonitzile was treated with 55 mI. of water and 67 mL
of acetic said, and stirred for 8 hours at,ambient tempcratuTe. The mixture was concentrated en vacuo, then repeatedly concentrated after addition of toluene to yield 14.7 g of 5-O-methyl-1S-methylerythromycin A 9-oxime.
F. b-O-~ethyl-15-methy a omycin A: A solution of 6-O-methyl-15-methylerythromycin A 9-oxime (19.7 g) and sodium hydrosulfite (85%, 23.1 g~ in 280 mL of I :1 ethanol/water was placed under inert atm' sphere. Formic acid (3.75 rn~,) was added dropwise, acrd the mixtwe was stirred at 80°C for 4.5 hours. After cooling to ambient temperature, the reaction was treated with saturated NaHC03 and extracted three tirries with 400-mL portions of ethyl acetate. The orgaruic extracts were combined atld washod sequentially with saturated NaHC03, water, and brine. The organic phase was dried with MgS 04, filtered, and evaporated to yield 15.1! g of 6-O-methyl-1 S-methylerythmmycin A, suitable for further coavcrsion.
Exam a 7 Svnthcsis of 5-O-!2'-A,ectvldesosaminYl)~b 1 I-anh~eaxy~ -O methltl 14 ore onolide A fo o:f Formula =OH =Me Rp ~Ac i A.. S-O-Desosaminyl-6-O-methy -I4-norervthronolide A: A mixture of 6-Q
methyl-I 4-norerythromyein A (77 rng), 0.073 ~ml of 12 N HCl and water (2 ml) was stirred at ambient temperature for 3~hours. The mixture was brought to pH 8 with 8 N KOH, and r extracted with ethyl acetate. The organic extract was washed with brine, dried with Mgs04, filtered, and evaporated. The residue was chromatographed on silica gel (3:IJhexanes:aCetone, l % triethylamine) to gibe pure product as a white solid (42 mg). Mass spectrometry reveals [M+H~') = 576.
B. 5-O-f2'-Acetvldesosaminyll-6-O-methvt-14-norervthronolide A: A mixtuxe of 5-p desosamin 1-6-O-meth 1-14-pore Y y rythronolide A (73 mg), potassium carbonate(20 mg), , -AMENDED SHEET
Empfangszeit 7.Juni 0.3.

~u06-06-2001. 3~49 PM FR MO-FUh1 bHN mtuub~ts r~r~ ,m~~ ~.. ~~-.~.,...-...-.~.,~~.."_ _ _ US 00000991 ~

-3b-acetic anhydride (1411,1) and acetone (I ml) was shard at ambient temperature for 18 boars.
Ethyl acetate was added, washed with wateriaad brine, dried over MgSO4, fittcred, and evaporated. The residue; was chromatograplied on silica gel (3:llhexanes:acetone, l%
triethylamine) to yield the pare product (71 x~ng~ as a white solid. Mass spectrometry reveals s [M+xr~ = 61 s.
C. 5-O-f2 =AoeriIdesosaminyl)-3-deoxy 3-oxo-6-O-meth 1-14 noretvthronolide ._ A (Formula (1 ) R~~H -Me, Rr=Me ~=H ~Acl: A solution of 5-D-(2'-acetyidcsosaminyl~6-t~rncthyl-14-norerythronolide A (99 mg) and 1-(3-dimethylatninopropyl)-3-ethylcarbodiidmide,' (EDC) hydrochloride (206 mph in dichloromethane (2 ml} was treated with DMSO (O.Z1 ml} and cooled to 5°C. A solution of i pyridinium trifluoroacetate (208 mg) in dichloromethane (2 ml) was added viu a syringe pump in 4 hours. Ethyl acetate was then added, washed with saturated NaHC03, water, brine, and dried over M,gS04, filtered, and evaporated. The residue was chromatographed on silica gel (3:1/hexaries:acetone, '1% triethyIamine) to yield the pure product (94 mg) as a whito i 5 solid. Mass spectrometry reveals [M+I~ = 616.
D. 5- 2' meth ~~1-14-nor'eiythronolide A: To a solution of 5-0-(2'-acetyldesosaminyl)-3-deoxy-3-oxo-6-D loath 1-I4-note Y rythronolidc A (93 mg) m dry pyridine (I ml) was added methanesulfonyl chloride (0.057 ml) at 5°C. A#ler 3 hours at 5°C, the reaction was warmed to ambient temperature and kept for~an additional 15 hours. The mixture was diluted with ethyl acetate, washed with saturated NaHC03(Zx}, water (~x}, b~nine, and dried over MgS04, filtered, and evaporated. The residue was chromatographed on silica gel (2:1/hexanes:acetone, 1 triethylamine) to yield the port product (72 trig) as a white solid. Mass spectrometry reveals [M+H~] = 695. , ' E. 5-O-t2'- ce , desosamimvl>-10.11-anhyd~o-3-deoxv-3-oxo-6 D methyl 14-z~or~rvthronolide A: A solution of 5-O (2'-acetyldesosaminyl)-3-deoxy-3-oxo-11-O
methanesulfonyl-b-t~methyl-14-norerythronolidc A (73 mg) in acetone {l m1) was treated with diazabic loundecene 32 Yo , { ~I) at ambient temperature for 18 hours. The mixture was diluted with ethyl acetate, washed with saturated NapiC03, water, brine, and dried over MgS04, filtered, and evaporated. The residue;. was chromatographed on silica gel (2:1/hexanes:acetone, 1 % triethylamine) to yield the pure product (50 mg) as a white solid.
Mass spectrometry reveals [M+1-1~ = 5gg. ' 3C NM~ (~C13, 100 MHz): 8 207.02, 204.50, EmPfangsZeit 7.Juni 0;31 AMENDED SHEET

J,OG-06-2001 1 3 : 49 PM FR MO-FOM SRN D I EG0858 720 5125 TO 8540ii99990#9""' ' ' ' 300b22003340 169.b3,168.72,142.52,139.40,101.87, 80.6 i, 80.02, 77,14, ?2.66, 71.48, 69.09, 63.56, 51.35, 50.56, 47.12, 40.61, 39.73, 37.36, 30.36, 21.32, 21.06, 20.96, 20.67,18.45,14.34, 13.89, 13.55,13.45.
$&-Benzovl) A. ~'-O-Ben~ovl-6-O-methyl-14.15-dehyrthlom~c'n A
A solution of G-O-methyl-14,15-deh~ecythromycin A (668 m~, benzoic anhydride (385 rnp~, and triethylamine (0.25 mL) in 3.6 .ImI. of CHZCIz was stirred for 2 days. After I
addition of saturated NaHCQ3, the mixture was extracted three times with CH2Cl2. The i organic extracts were combined and evaporated to dryness, and the product was purified by silica chromatography (90:9:1 toluene/acetone/Et~ to give 477 mg of product;
LC-MS
shows [M+Fi]+ = 850.6.
B. ~'~-Henzoyl-6-O-methyl-4":11 bis(O-methanesulfonvl)-1415 dehvdroervthromvcin A , A solution of2'-O-benzoyl-6-O-methyl-14,15-dehydrocxythromycin A (549 mg) and methanesulfonyl chloride (0.50 mL) in 2.39 ~rtiL of pyridine was stirred for 24 hours, then diluted with CI3zC12 and saturated NaHC03. The mixture was extracted three times with CHZClz. The organic extracts were combined and evaporated to dryness, and the product was purified by silica chmmato~raphy (90:9:1 toluenelacetoneJEt~ to give 530 mg of product;
LC-MS shows [M+ITJ* =1006.5.
C. __2'-O-l3enzovl-6-O-methyl,4"-0-methanesulfon7rl-1011-anhvdro-I41~
dehvdroervthromvein A ~ ' I
A mixture of 2'-O ; benzoyl-b-Q-methyl-4",11-bis(O-methaaesulfonyl) 14,15-dehydroerythromycin A (59 mg) and diazabicyclotmdecene. (0.018 mL) in 0.195 mL
of acetone was stirred for 24~hours, then dried ini vacuo. The pmduct was purifxad by silica ~ P Y~
chromato a h 90:9:1 toluene/acetonelEt~ to give 50 mg of product; LC-MS shows .jM+Hj* = 910.5. , AMENDED SHEET
~Empfansszeit 7.Juni 0:37 a : 4~ ri~i rK nu-ruri bNIV U 1 thuliJti (C1~ ~ 1 C' I a C'41~~i~~~~IOFIW "' 06-06-2001 ~ US 00000991 300622003340 ~ , I
D. 2'-O-Benzoyl-6-O-methyl-3-descladinosyl-l0~ydro-14,1 S-dehvdroervthiomv~cin A
A mixture of 2'-O-benzoyl-f>--O-methyl-4"-O-methu~esulfonyl-10, I 1-anhydro-I4, I S
dehydroerythromycin A (337 mg),1.5 mL of acetonitrile, and G.9 mL of 3 N HC!
was stirred for 22 hours. The acetonitrile was removed iri vacuo, the pH of the aqueous residue was adjusted to 12 by addition of NaOH, and the product was extracted using 4 portions of CHZCIz. The combined extracts were dried aid evaporated. The product was purified by i silica chromatography (gradient from 96:4 C~i2Cl2lMeOH to 95:4:1 CHZCl2/McOI3lEt3N) to give 197 mg, [M+H)+ = 674.4. .
IO E. 2_'-O-Benzovl-6-O-methyl-3-descladinosyl-3-oxo-10 lI-aa~hydro 14,~~5 dehydroeivrhromvcin A ~ ~ , , A suspension of 2'-O-be~zoyl-6-O-methyl-3-desoladinosyl-10,11-anhydro-I4,15-dehydroerythromycin A (22G mg) and the Dess-Martin periodiuaue (427 mg) in 14.6 mL of CHiCI2 ( 14.6 mL) was stirred for 1 hour. The mixture was diluted with C1i2C1Z
aad saturated NaHCO3. The product was extracted using 3',,po~t~ions of CHzCIZ, and the extracts were combined, driod, and evaporated. Silica gel chromatography (90:9:1 tolueaelacetone/Et~
yielded the product, 168 mg. [M+I~~* = 672.4. i3C-NMR (CDC13,100 IvlHz): S
206.78, 203 (br), 158.19, 165.08,141.36, 139.58, 132.74, .131.51,130.46, 129.79, 128.25, 120.18, 102.09, 80.79, 80.40, 78.70, 72.52, 71.91, 69.19, 63.76, 51.10, 50.54, 47.08, 40.73, 39.87, 37.77, 31.23, 22.13, 20.98,18.52,14.28,14.15,13.5. .
Ex~mle 9 ~ Ac A. 6O- methyl-3-descladinosvl-I S-methyler~~
A mixture of 6-O-meth 1-15-meth 1 Y Y ~Y~'omycin A (15.1 g) and 280 mL of 0.5 N
HCl was stirred at ambient temperature for 3 fours. The pH was adjusted to 9 by addition of 6 N NaOH, and the resulting precipitate was collected by vacuum filtration, washed with .
water, and dried. The filtrate was extracted thiee times with 400-mL portions of ethyl acetate.
The orga'rtic extracts were combined, washed s equentially with saturated NaHC03, water, and brine, then dried over MgSOd, filtered, and evaporated to provide. further product. .The AMENDED SHEET
Emvfangszeit 7.Juni 0:31 J.06-06-2001 1 3 : 50 PM FR MO-FOM SRN D I EG0858 720 5125 TO
8540tt99999#39~~"~ ~ '~

300622003340 , ' -'39 -combined crude products were chromatographed on silica gel to yield 9.35 g of pure 6-O- .
methyl-3-descladinosyl-I S-methylerythromycin A. ES-LClMS shows [M+HJ+ = 605.
B. ~'-O-Acetyl-6-O-meth~rl-3-descladino~y -15-methyl-~e ~rcin A
A solution of acetic anhydride (2.92 m~ L) in 35 mL of ethyl acetate was added dropwise to a solution of 6-O-methyl-3-descli~adinosyl-15-methylerythromycin A
(9.35 g) in 40 tnL of ethyl acetate. 'fhe mixture was st'~ed for 30 minutes after completion of addition, then concentrated. Chromatography on silicai gel (2:1 hexanes/acetone) gave 8.35 g of 2'-0-acetyl-6-O-methyl-3-descladinosyl-15 methylerythromycin A. ES-LCIMS shows [M+FI]' 647. ' C. 2'-O-Acctyl-6-O-methyl-3-descladinosvl-3-oxo-15-methvlcrvthmmvcin A
A solution of 2'-O=acetyl-6-O-methyl ~3-descladinosyl-15-methylerytbromycita A
(8.3 g) and I-ethyl-3-(dimcthylamino 1 carbo,~diimide b~ drochloride 16.51 PmPY ) ~ Y ( g) in 64 nlL, of dichloromethane and 15.47 mL of rnethylsulfoxide was placed under inert atmosphere and cooled on ice. A solution ofpyridinium trifluoroacetate (I6.63 g) in 64 mL of dichloromethane was added at a rate such that addition would be complete in 4 hours, and the reaction was monitored by thin-layer chromatography. Complete reaction was observed after addition of 73% of the solution, and so the reaction was then quenched by addition of b00 mL
of ethyl acetate and 200 mL of saturated NaHC03. The organic layer was collected and washed sequentially with saturated NaHC03, iwater, and brine, then dried over MgS04, filtered, and evaporated to yield 8.4 g of crude product. Chromatography on silica gel (3: I
hexaneslacetone) gave 6.75 g of 2'-O-ace~rl-6-p-methyl-3-descladinosyl-3-oxo-methylerythromycin A. ES-Y.CIMS shows [M+H]' = 645.
b. 2'-O Acetyl-6-O-methyl-3-descla inocvl oxo-l I-d methanesulfonvl 15 methvlervthrom cin A ;
. Methanesulfonylchloride (5.68 mL) was added dropwise to a solution of 2'-O-acetyl-6-O-methyl-3-descladinosyl-3-oxo-15-methylerythromycin A (6.73 g) in 35 rnL of pyridine at 0°C. The mixture was brought to ambient teriiperature and quenched by addition of 700 mL
of ethyl acetate and 200 niL of saturated NaHCO3. The organuc layer was collected and washed sequentially with 'saturated NaIiC03,'water, and brine, then dried over MgS04, 3a filtered, and eva orated to P yield 8.2 g of crude product. Chromatography on silica. gel (5:2 hexaaeslacetone gave 5_04 g of 2'-O-acetyl-6-O-methyl-3-descladinosyl-3-oxo-l 1-O-methanesulfonyl-IS-methylerythromycin A. ES-LCIMS shows [M+H~' _ X23.
AMENDED SHEET
EmPfansszeit 7.Juni 0;31 J.06-06-2001 I 3 : 50 PM FR MO-FOM SRN D I EG085B 720 51 25 TO
B540t399990tt9~~"~ ~ ' ' -40_ E. _2'-O-Acetyl-6-O-methyl-3-descladinosyt-3-oxo-10,11-~hydro-15 methvlervthromvcin A
l,&Diazabicyclo(5.4.0]under 7-ere (5.22 raL) was added dropwise to a solution of 2'-O-acetyl-6-O-methyl-3-descladinosyl-3-oxo-11-O-mcthanesulfonyl-15-methyierythromycin A (5_03 g) in 23 mL of ace~tane. The solution~was concentrated after 4.5 hours, and the residue was chromatographed on silica gel (S:I2 hexanes/acetone) to give 3.?2 g of 2'-O-acetyl-d-O-methyl-3-descladinosyl-3-oxo-10,1 I-anhydro-IS-methylerythromycin A. ES-LC/MS shows (M+H)' = 627.
! ~ Facatiiple 10 Synthesis of 5-O-(Z'-acetvldesosaminvl)-10 11-anhydro-3 6-dideoxv 3-0~ xo 15 meth le nolide A o a ~ dro fo - ro 1 OR re 1 ed by H,1t~=H, R~ Acl To a solution of 6-deoxy 15-methyl ezythromycin C (220 mg, 0.307 mmol) in .
dichloromethane {5 mL) were given potassium carbonate (50 mph and acetic anhydride (100 L, 0.9 mmol), and the reaction was stirred at room temperature for 16 hours.
The solution was filtered, sodium hydroxide (1N, 25 mL) and brine (25 mL) added and the aqueous Iayer was extracted with ethyl acetate 6 times. Thel combined organic layers were dried with sodium sulfate, filtered, and the solvent removed in vacuo. The crude product the 2' 24 acetylated form of the starting material was carried on to the next step.
The chide product;was dissolved in pyridine (5 tnL) and mesyl chloride (70 L, 0.9 mmoi) was added. The reaction was stirred at 20°C for 2' days, poured on sodium hydroxide (1 N, 2S mL) and brine (25 mL) and the ueous 1 aq ~ ayer was extracted with ethyl acetaxe 6 times. Tlae combined organic Layers were dried with sodium sulfate, filtered, and the solvent removed in vaeuo. The residue was purifaed liy chromatography on silica ,gel (toluenelacetone = 3:1,1% ammonium hydroatide) to yield 11,4"-dimesylated form (190 mg, 68% over two steps). ' The 11, 4"-dimesylated form (190 mg; 0.21 mmol) was dissolved in acetone {7 mL) and DBU (63 L, 0.42 mmol) was added, and the reaction was stirred at room temperature . over night. The muixture was poured an sodium hydroxide (1 N, 2~ ~,) ~d brine (25 mL) . and the aqueous layer was; extracted with ethyl acetate 6 times. The combined organic layers were dried with sodium sulfate, filtered, and the solvent removed in vacuo.
The crude .
AMENDED SHEET
Emvfangszeit 7.Ju~i 0;37 ' """ ;5:~1~ t'1'1 f K I'IV-1'VI'1 JHIV LlGl7VO.J0 ~ LY., .J~c_.J ~ v ..J-.un....,....v."....v~~ ~ . ,_ 06-06-2001 ; US 000009915 i -41 -product, tire 10,11-d~hydro form of G-deoxy li5-methyl erythromycin was carried on to the.
next step. ' To the crude product from the above step was added hydrochloric acid (30 ntL, 3 I~
and ethanol (2 mL) and the mixture was stirred vigorously for 6 hours. Sodium hydroxide (S
mL,10 N) was added and 'the aqueous layer v ias extracted with ethyl acetate b times. The combined organic layers were dried with sodium sulfate, filtered, and the solvent removed in vacuo_ The crude product; the anhydro form of formula (1) (but with aH at position 3) where 1~~OH, Rd=prapyl, ORf is replaced by H, Rb~R~~H, was carried on to the next step.
To the crude product from the above step in dichloromethane (5 mL) was added acetic I O anhydride (50 L, 0.45 mmol) and potassium carbonate (100 mg) and the mixture was stored vigorously for 9 hours. The reaction was filtered, sodium hydroxide (20 mL, l I~ and brine (ZS mL) were added and the aqueous layer was extracted with ethyl acetate 6 times. The combined organic layers were dried with sodium sulfate, filtered, and the solvent removed in vacuo. The residue was ptuified by chromatography on silica gel (toluene/acetone 3 3:1, 1%
ammonium hydroxide) to yield the 2' acctylatcd form of the starting material (110 mg, $9%
over three steps).
The product of the, above step (110 mg, 0.184 mmol) was dissolved in dichlommethanc (10 mL) and Dess-Martin reagent (220 mg, 0.53 mmol) was added.
The reaction was stirred at room temperature for 45 min. The reaction was quenched with Sodium hydroxide (20 mL,11~ and brine (25, mL) and the aqueous layer was extracted with ethyl acetate 6 times. The~combined organic layers were dried with sodium sulfate, filtered, and the solvent removed in vacuo. The residue was purified by chromatography on silica gel (tolueneJacetone; gradient ,= 6:1-3:1, 1 % ammonium hydroxide) to yield the compound of formula (1), anhydro form, where R,~H, R~ropyl, OR,f is replaced by H, Rb H, R~Ac (94 m,g, 86%).
Exaranle 11 I. Cvmnound of Formula ~): Rg=OH, R~~ropyl R~allvl i Step I . AIlyIation of Tntermediate Antibiotic at : OH: A solution of 2',4"-bis-Q
trimethylsilyl-i5 methylerythromycin A 9- O- 1-iso ro ox clohex 1 oxime formula I C P P YcY Y )1 ( {R, is OH, Rd is propyl, protected at 2' and 4" yrith. trimethylsiIyl and at C9~ by the .
isoproxycyclohexyl oxime)) (7.8 g, ?.44 mmol) it 30 mL of tetrahydro~uran was cooled on AMENDED SHEET
Empfangsteit 7.Juni 0:37 J"" "- ---1 3:51 PM FR MO-FOM SRN DIEG0858 720 5125 TO 8540#99990#9"-"- ' "' 06-06-2001 . ! US 00000991 E

ice and treated with 30 mL of methylsulfoxid~ and 2.58 mL of freshly distilled allyl bromide under inert atmosphere. A mixture of methylsulfoxide (29.8 mL) and 1.0 M
potassium tert-butoxide in tetrahydrofuran (29.8 mL) was added at a rate of 1.33 molar equivalents of base per hour. The reaction was monitored by think layer chromatography (silica gel,10:1 toluenelacetone), and was~judged complete after addition of 3.6 molar equivalents of base.
The reaction was diluted with 700 mL of ethyl acetate and washed sequentially with saturated NaHC03, water, and brine. The organic phase was dried with MgS04, filtered, and evaporated to yield 8.08 g; of crude 6-O-allyl-2',4' ~bis-O-trimethylsilyl-1 S-methylerythromycin A 9-[O-(1-isopropoxycyclohexyl)Joxime. This was carried on without further purification.
Step 2: A solution of 6-O-allyi-2',4"~iis-O-trimethylsilyl-15-methylerythromycin A 9-[O-(1-isopropoxycycloheXyl)joxime {8.08 g) in 42 mL of acetonitrile was treated with 21 mL
of water and 24 mL of acetic acid, and stined jfor 18 hours at ambient temperature. The mixture was concentrated after addition of 2 propanol, then repeatedly after addition of toluene to yield 7.7 g of crude product. Chromatography on silica gel (gradient ii'om 2:1 to 1: x hexanes/acetone + 1 % Et3I~ gave 3.75 g of 6-O-allyl-15-methylerythroznyoin A 9-oxime.
Step 3: A solution of b-O-allyt-15-methylerythromycin A 9-oxime (3.75 g) and sodium hydrosulfite (85%, 5.37 g) in 65 rnL o'f 1:1 ethanollwater was placad under inert atmosphere. Formic acid (0.845 mL) was added dropwise, and the mixture was stiaed at r 80°C for 3.5 hours. After cooling to ambient temperature, the reaction was adjusted to pH 1 p with 6 N NaOH and extracted three times with 150-mL portions of ethyl acetate.
The organic extracts were combined and washed sequentially with saturated NaHC03, water, and brine.
The organic phase was dried with MgSOs, filtered, and evaporated to yield 3.42 g~of 6-O-allyl-1 S-methylerythrotxlycin A suitable for ~u~cther conversion.
ZS
II. Compound of Formula l3) ' R~=O~ Me R~allvl Step 1: Alllrlation'of Intermediate Antibiotic at 5-OH: A solution of 2',4"-bis-O-trimtethylsilyl-I4-norerythromycin A 9-[O-(1-~sopropoxycyclohexyl)]oxime, Formula ()7, (Ra is OH, Rd is methyl, protected at 2' and 4" with trimethylsilyI and at C9=O by the isoproxycyclohexyl oxime) (202 mg) in tetrahydrofuran (0.4 mL), DMSO (0.4 mL), and ether (0.04 mL) was cooled to 10°C and treated with 0.035 mL of freshly distilled allyl bromide r under inert atmosphere. A mixture of methylsWfoxide (0.4 mL) and 1.0 M
potassiuru iert-' AMENDED SHEET
Emvfanssteit 7.Joni 0:31 J"~~ -- ---t 3:51 PM FR MO-FOM SRN DIEG0858 720 5125 TO 8540#99990#9"'""'" "
"' 06=06-2001 , US 00000991 E

butoxide in tetrahydrofuran {0.4 mL) was add! at a rate 0.22 mLlhour. The reaction was monitored by thin-layer chromatography (silica gel, 5:1 toluenelacetone. The reaction was i diluted with ethyl acetate and washed scquent~ally with saturated NaHC03, water, and brine.
The organic phase was dried with MgS04, filtered, and evaporated to yield 222 mg of crude fi-0-allyl-2',4°-bis-0-trimethylsilyl-14-norerythromycin A 9-(O-(1-isopropoxycyclohexyl)]oxime. This was carried on without further purification.
a Step 2: A solution of 6-0-allyl-2',4"-firs-O-trimcthylsilyl-14-norerythmmycin A 9-[O-(1-isopropoxycyclohexyl)]oxime (222 mg) in~4 mL'of acetonitrile was treated with 2 mL of water and 2.4 mL of acetic acid, and stirred f i r I8 hours at ambient temperature. Tha mixture was concentrated after addition of 2-propanol; then repeatedly after addition of toluene to yield 220 mg of crude 6-0-allyl-14-norerythromycin A 9-oxime.
Step 3: A solution of 6-O-allyl-14-noierythromycin A 9-oxime (220 mg) and sodium hydrosulfite (8S%, 322 mg) in 4 mL of 1:1 ethanol/water was placed under inert atmosphere.
Formic acid (0.050 mL) was added dropwise,j and the mixture was stirred at 80°C for 15 hours. After cooling to ambient temperature, jthe reaction was adjusted to pH
10 with 6 N
NaOII and extracted three times with 1 SO-mL portions of ethyl acetate. The organic extr8cts were combined and washed sequentially with] saturated NaHC03, water, and brine. The organic phase was dried with MgS04, filtered, and evaporated to yield 15d mg of 6-O-allyl-14-norerythromycin A suitable for fiuther eoaversion.
Other embodiments: In a similar manner, compounds of formula (3) wherein Y aad Z
are, together, =O, Ra is OH, Rf is allyl, is prepared from an intermediate where R~ is butyl, benzyl, vinyl, or 3-hydroXybutyl.
Ex ~ 12 ' Conversion;to Formula (1) Step 1: A mixture of the compound prepared in Example 11, II (77 mg, crude), 0.073 mI of l2 N HCl and water (2 mi) was stirred at ambient temperature for 3 hours. The mixture was brought to pH 8 with :8 N KOTd, and extracted with ethyl acetate. The organic extract was washed with brine, deed with MgS04, filtered, and evaporated. The residue was chromatographed on silica gel (3:1/hexanes:acetone, I% triethyIamine) to give pure product as a white solid (42 mg).
AMENDED SHEET
Fmofan~s~ait 7..luni 0:37 _ __ -___ _ _. . .. , ,. . , . ..... ,r..., ~yVVJU IGVJ J1GJ IV
CY~4YJH~~~~~j~"'-~~».. .. ....
06-06-2001 ; US 00000991 300622003340 , , Step 2: To protect rthe 2' OH, a mixture the above compound (73 mg), potassium i carbonate(20 mg), acetic anhydride (14w1) and acetone (1 ml) was stirred at ambient temperature for I 8 hours. Ethyl acetate was added, washed with water and brine, dried over MgSO~, filtered, and evaporated. The residuelwas chromatographed on silica gel (3:Ilhexanes:acetone,1 % ~iethylamine) to yield the pure product (7I mg) as a white solid.
Step 3: A solution of the compound resulting from step 2 (99 mg) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiid~ide(EDC) hydrochloride (206 mg) in dichloromcthane (2 ml) was treated with AMSO (0.21 ml) and cooled to 5°C. A solution of pyridinium trifluoroacetate (208 mg) in dichloromethane (2 ml) was added via a syringe pump in 4 hours. Ethyl acetate was then added, washed with saturated NaHC03, water, brine, and dried over MgSOa, filtered, and evaporated. Tlie residue was chmmatographed on silica gel (3:llhexanes:acetone, I% triethylanzinc) to yield the pure compound of formula (1) (94 r mg, R~ is OH, R~ is acetate, Re is CHs, and Rfis allyl).
Step 4: To deprotect 2' OH, a solutioti of the compound resulting 6om step 3 (94 mg) in 5 mL methanol was stirred at room Eemperature for 24 hours. The solvent was removed in vacuo to give~the desired compound of formula (1) (R' is OH, R~ is H, Ita is CH3, and Rf is allyl).
Other embodiments: In a similar ma inner, compounds of formula (1 ) wherein RQ
is OH, R~ is H, R~ is allyl, and Rd is propyl, butxl, benzyl, vinyl, or 3-hyd~roxybutyl is prepared.
Prepaxatxon of Compounds of Formula l2) The compound of formula (3), prepared as the 6-aliyl derivative in Example 11, is protected at the 2' position, treated with acid and dehydrated, then deprotected to obtain the compound of formula (2), as shown in Figure 1, wherein R, is OH, R~ is H, and Rf is allyl.
Similarly, compounds of'~ormula (1) wherein Rd is propyl, butyl, benzyl, vinyl, or 3-h drox a 1 are Y Yb tY , prepared as descn~bed above using as starting material the compounds of formula (n wherein Rd is as set forth above.
Emufansszeit 7.Juni 0:37 AMENDED SHEET

J um uu ~rm.~ 1 J ~ ,.rte r m r r~ ~~m-r vrm Jr»v L 1 CqVi3~t5 fGVJ ~ J. C~ 1 V tf~41011~~~~19ii~'~..r.""
06-06-2001 . ; US 000009915 i x le 1 Conversion of ~ at Position 9 to NOH
According to the procedure of Example 6A, the carbonyl at position 9 of eryrhromycins are converted to the corresponding oximes.
S
Fxaniple 15 Conversions at -OR
A. Aryl --° Propyl: A solution of any of the compounds prepared above (0.2 i mmol) in ethanol is flushed with nitrogen and~IO% palladium on carbon (20 mg) added. The mixture is then flushed with hydrogen and theireaction mixture stimd overnight under positive hydrogen pressure. The reaction mixture is filtered and concentrated in vacuo to give .
a glass. Chromatography on silica gel (95:5:0( 5 dichloromethane-methanol-ammonia) gives the propyl compounds as white solids.
B. A.llvl --y -CH3CH0: Ozone is passed through a -?8°C solution is dichloromethane (I00 mL) of any of the compounds resulting above (4.0 mmol) for 45 minutes. The reaction mixture is then flushed with nitrogen for 10 minutes.
Dimethyl sulfide (1.46 mL, 20 mmol) is ad~.ed at -78°C and the reaction mixture stirred for 30 minutes at 0°C.
The reaction mixture is concentrated in vacuo to give a white foam which is used without further purification by heating a solution of the compound in THF (40 mL, 4.0 mmol) and triphenylphosphine (2.62 g, 10.0 mmoi) at 55~ C for 2.5 hours. The reaction mixture is concentrated in vacuo to give a white foam. Chromatography on silica gel (1:1 acetone-hexane, then ?5:25:0.5 acetone-hexane-tniethylamane) gives !he desired compound as a white solid. , ;
C. AIlyl ~ -CH,~CH-NOH: To a solution in methanol (5 mL) of the compound prepared in B wherein Rf is -CHzCHO, (0.08 ~mmol) is added triethylamine (31 p.L, 0.225 mmol) and hydroxylamine hydrochloride (?.7, mg, 0.1 I2 mmol) and the reaction mixtwe stirred for 6 hours at ambient temperature. The xeaction mixture is taken up in ethyl acetate , and washed with aqueous. 5% sodium bicarbonate and brine, dried over sodium sulfate, and concentrated in vacuo to give a clear glass. Chromatography on silica gel (g5:5 :0.5 dichloronaethane-methanol-ammonia) gives the compound as a white solid.
D. -__CH~CH NOH i -CHzCN: ~'o a solution under nitrogen of the compound prepared in C {0.267 mmol) in T'1~ {5 mL) is added diisopropylcarbodiimide (83 uL, 0.534 AMENDED SHEET
Emofanosteit 7.J.uni 0:31 ""' "'" '""~1 3:53 FM FR MO-FOM SRN DIEG0858 720 5125 TO 8540#99990#~'-' 06-06-2001 ' ~ US 00000991 ~
~30062Z003340 , tnmol) and CuCI (2.7 mg, 0.027 mmol) and the reaction mixture is stirred overnight at ambient tomperature. The reaction mixture is taken up in ethyl acetate and washed witth aqueous 5% sodium bicailionate and brine, dried over sodium sulfate, and concentratod in ' I
vacuo to give a clear glass: Chromatography on silica gel (95:5:0.5 dichloromethane-methanol-ammonia) gives the desired compound as a white solid.
E. -CH~CHO --f -CH~CH21~'i~: To a solution in methanol (10 mL) of the compound prepared in B (0.276 mmol) is added ammonium acetate (212 mg, 2.76 mmol) and I
the mixture is cooled to 0°C. Sodium cyanoborohydride (34 mg, 0.553 mmol) is added and the reaction mixtuie stirred for 30 hours at 0°C. The reaction mixture is taken up in ethyl ' ~ I
acetate and washed with aqueous 5% sodium carbonate, aqueous 2%
tris(hydroxymethyl)aminomethane, and brines dried over sodium sulfate, filtered, and I
concentrated in vacuo. Chromatography on silica gel (90:10:0.5 dichloromethane-methanol i ammonia) gives the desired compound as a white solid.
p'. -CH"~,CHO'-~ -CH~~~iCH~-Phenol: To a 0°C solution in methanol (10 mL of the co ) mpound prepared in B (0.200 mmol) is added acetic acid (114 ~tL, 2.00 ramol) and benzylamine (2I 8 ~r,Y., 2.00 moron and the mixture is stirred for IO
minutes. Sodium cyanobomhydride (24.8 mg, 0.400 mmol) is added and the reaction mixture stirred for lb hours. Additional sodium cyanoborohydride x(24.8 mg, 0.400 mmol) is then added and stirring continued for 5 hours. The reaction mixture is taken up in ethyl acetate and washed with aqueous 5% sodium.carbonate, aqueous~2% tris(hydroxymethyl)aminolmethane, and brine, dried over sodium sulfate, filtered, and concentrated in vacuo.
Chromatographx on silica gel (95:5:0.5 dichloromethane-methanol-ammonia) followed by a second chromatography (50:50:0.5 acetone-hexanes !triethylamiae) gives the desired compound as a white foam. ~ ~ ' i G. -CH~CHO ~ -CH_CHZNHC1:I2CHa Phenyl: To a 0°C solution in methanol (10 mT.) of the compound prepared in B (0.200 mmol) is added acetic acid (114 p,L,, 2.00 nlmol) and phenethylamine (218 ~,, 2.00 minol) and the mixture stirred for 10 minutes.
Sodium cyanoborohydride (24.8 mg, 0.400 ~mol) is added and the reaction mixture stirred . for 16 hours. The reaction mixhire is taken up in ethyl acetate and washed with aqueous 5%
sodium carbonate, aqueous 2% tris(hydroxymethyl)aminornethaue, and brine, dried over sodium sulfate, filtered, and concentrated in vacuo. Chromatography on silica gel (90:10:0.5 dicbloxoznethane-methanol-ammonia) gives the desired compound.
Emvfa~ssteit 7.Juni 0:37 AMENDED SHEET

w" ,,:~,, rm rK r~u-r~r~ SHrv ~ttuua~a rib 515 rU 8540~99990a~'US~OOOOO9915 06-06-2001 ' 300622003340 f i H. -G'~H~S~ O i -CH~CH~NHyC42C,.~~1(.~'H~ Phenyl: To a 0°C solution in i methanol (10 mL) of the compound prepared i~ B (0.200 mmol) is added L-phenylalanine methyl ester hydrochloride (129 mg, 0.604 znrirol) and the mixture stirred for 10 minutes.
Sodium cyanoborohydride 924.8 mg, 0.400 minol) is added and the reaction mixture stin~ed for 22 hours. The ruction mixture is taken up in ethyl acetate and washed with aqueous 5%
sodium carbonate, aqueous 2% tcis(hydroxymethyl)aminomethane, and brine, dried over sodium sulfate, filtered, arid concentrated in vacuo. Chromatography on silica gel (95:5:0.5 dichloromethane-methanol-ammonia) gives the desired compound.
i r. -CHaCHO -: -C 2CHZNHCHZ-(4 p~ri~l): The desired compound is prepared according to the inethod in G, except substituting 4-aminomethylpyridiune for phenethylamine. ;
J. -CH2C~NHg -~ -CH,2CH~NH~ 'nolxl~: To a solution of the compound prepared in E (0.15 mmol) in methanol (2 mT.) is added 4-quinolinecarboxaldehyde (23 mg, O.I S mmol), acetic acid (8.6 ~cL, 0.15 mmol), and sodium a cyanoborohydride (9.4 mg, 0.15 mmol) and the reaction mixture is stirred for i5 hours. The reaction mixtuxe is taken up in ethyl acetate and washed with aqueous 5%
sodium carbonate,, aqueous 2% tris(hydroxymethyl)aminomcthane, and brine, dried over sodium sulfate, filtered, I
and concentrated in vacua. Chromatographyion silica gel (95;10:0.5 dichloromethane-methanol-ammonia) gives the desired compound.
ZO K. All~rl -~ -CHaCH=CH-Phenyl: To a solution under nitrogen of the 2' protected compound prepared in Example 10 (1.00 miriol), palladium(II)acetate (22 mg, 0.100 nrmol), and triphenylphosphine (52 mg, 0.200 mmol) in acetonitrile (5 mL) was added iodobenzene (220 uL, 2.00 mmol) and triethylamine (280 ~L, 2.00 mmol) and the mixture is cooled to -78°C, degassed, and sealed. The reaction mixture is then warmed to 60°C for 0.5 hours and stirred at 80°C far 12 hours, taken up in ethyl acetate and washed twice with aqueous 5%
sodium bicarbonate, once with aqueous 2% tiffs(hydroxymethyl}aminomethane, and once with brine, dried over sodium sulfate, filtered, and concentrated in vacuo.
Chromatographx on silica gel (95:5:0.5 dichlommethano-methanol-ammonia) gives the desired compound.
Deproteetion is accomplished by heating in methanol Other embodiments of formulas (1)-(.3} where Rb is H, Its is H, R,, is OH, Y
and Z are together -0 and Rd is propyl, butyl, bent 1, vinyl, or 3 h Y , - ydroxybutyl are those wherein Rris:
-CHzCHzCHz phenyl;
AMENDED SHEET
Emvfansszeit 7.Juni 0:37 J'"' -- 3:54 PM FR MO-FOM SRN DIEG085B
---t 720 5125 TO 8540~19~~~dtt9''""~
06-06-2001 i US 00000991 ~

l 3oos22oo~ao , i .

-CH2CH~CH (4-methoxyphenyl);

-CHZCH~CH-(4-cb~lorophenyl);

-CHZCH~CH-(3-quinolyl);
, -~z~z~zOH; , -CHzC(O)OH;

-CHZCHzNHCH3; ~ I

-CHzCHzNHCH20H; ' _~z~zN(Cg3~y -CHyCH2( 1 3riOrph011Ilyj);

1 ~ -CH2C(O)NH2;

-CHzNHC(O)NHz;

-CH~VHC(O)CH3; . ;

-CHIF;

-CHzCHzOCH3;

-CHz~3; ~ .

-~Z~=C~(CH3)2i .

-CHzCHzCH(CH3)CH3;

-CH2CHZOCHZC~izOCH3;

-CHZSCH3; ~ i -cyclopropyl;

-CHzOCHs;

-CHzCHzF; ~ i _CH2-cyclopropyl; .

-CH2CH2CH0; ;

-C(O)CHzCHZCH3;

-CHZ-(4-~itrophenyl);

-CHz-(4-chiorophenyl);

-CHz-(4-methoxyphenyl);

-CHZ-(4-c o hen 1 Y~ p Y )~

-CHZCH=CHC(O)OCH3;
i -CH2CH=CHC(O)OCHzCH3;

-CHZCH=CHCH3; .

AMENDED SHEET
EmPfangszeit 7.Juni 0:37 .. v' m-.~. .-~.v ~ a . ,-,~ n v' ' m ' m ' w' Jim a a LuVU.~O i LYJ J 1 GJ I
V OJai'UH055JU' ~1;'~~'~aw ~~~ w<
06-06-2001 ; ~ US 00000991 ~

I

-CH2CHaCHCHzCH3; ;
-CHzCH~HCH2CHaCFi3; i -CHiCH=CHSOz-phcayl;
-CHiC ~Si(CH3~
-CHIC ~CH2CH2CH2CHZCHZCH3; I
-CH2C ~CH3;
-~2-(2-P~dyl)~
-CHr{3-PYridYI)i -CHz-(4-PYndY1)~
-CHz-{4-quinolyl);
I I
-CH2N0z;
-CH2C{O)OCH3; ; .
-CHZC(U)-phenyl; ~ .
-CH2C(O)CHiCH3;
-CH3C1; , ;
-cHZs(o)2 phe~yi;
-cHZcH~z~r ~ -;
-CHZCH=CFi-(4-quinolyl); j -CHaCHzCHZ-{4-quinolyl);
-CHzCH=CH-(5-quinolyl);
-CH2CHZCH2-(5-quinolyl);
i -CH2GH~H-(4-tienzoxazolyl); or -CH2CH=CH-(7-benzimidazolyl).
Any of the foregoing compounds can~be converted to the correspoztding derivatives wherein Y and Z are together NOH in the manner descn'bed in Example 34 above.
i am le 16 Fluorinatioa of C2 Position Synthesis of 2'-O benzovl-6-O-provar~~rl-3-descladinosStl-3-oxo 10 i 1 anhydro i fluoro-15-meths tomycin A
34 A solution of 2'-O-benzoyl -6-O-propargyl-3-descladinosyl-3-oxo-10,11-anhydro-! 5-methyl-erythromycin A in tet<ahydrofuran, under inert atmosphere is cooled to -7$°C and treated with 1.0 M potassium tern-butoxide iit tetrahydz~ofuran. The mixture is stirred for 5 ' . i ' AMENDED SHEET
Empfangsteit l.Juni 0:31 .

J"~'~ "~~ ~~""W ~J'~f rll 1I< IIV-rVl1 Jf71'1 L1L17VIJJU 1LCJ JiLJ W
OJ'lCJff.JJJJYJr1.70JY1YJ f .J'I
06-06-2001 ~ US 00000991 E
i i ' - 50 -minutes, and a solution of N-ffuorobenzenesulfonimide in tetrahydrofuran is added in three portions over 2 hours. After addition, the reaction is allowed to warm to ambient temperature and kept for an additional 5 hours. Aqueous KiC03 is added, and the mixture is extracted With CHZC12. The organic extracts are combined, drib over MgS04, filtered, and evaporated.

Chromatography on silica gel gives the prod ~ t.
c ! of berivatization ~of C-13 Position . .~
Starting Material. 15-Aminoerythromyeiti A diacetate salt apHOAc A solution of 15-azidoerythromycin A (7.75 g, 10 mmol) in 50 mL of methanol is treated with acetic acid (2.0 mL) and 10% palladium on carbon (0.1 g) and stirred under 1 i atm of hydrogen gas until, thin-layer chromatographic analysis reveals complete reduction of the starting material. The suspension is littered through Celite to remove the catalyst, then evaporated to dryness to yield the product, which is used as a starting material for the following derivatizations.
A. Synthesis of 15-lquinol-4-ylacetatnido~er~rthromycm A
~Me2 A solution of 15-aminoerythromycin A diacetate salt (1.0 g) in 10 mL of dichloromethaxxe is treated sequentially with qninol-4-ylacetyl chloride (350 mg) and AMENDED SHEET
Emvfangszeit 7.Juni 0:31 06-06-2001 , ~ . _ .. _ _ _ _ , ~ -US~ ~~0000991 i 300622003340 ~' i . _51 _ triethylamine (0.5 mL) at 0°C. . After 3 hours, tie reaction is diluted with dichloromethane and washed three times with saturated aqueous NaHC03. The organic phase is dried ovar MgS04, filtered, and evaporated to yield the ciude groduct. Purification by silica gel chmmatographyyields the.pure product. !
;
B. Synthesis of 1 S-(3dquinol-4.. llarovio,~amidol~pr~yein A
i H: H
\.
"' ~ ~OH "" M
\ N~,,. ~~, ~ H~7~T~~~a2 ~O
H . y, O~ ~ ~ Me O fiI..OH
A solution of 15-aminoerythromycin A diacetate salt (1.0 g) in 10 mL of dichloroFnGthane is treated sequentially with 3-(quinol-4-yl)propionyl chloride (400 mph and triethylamine (0.5 mL) at 0°C. After 3 hours, ;the reaction is diluted with dachloromethane and washed three times with saturated aqueous NaHC03. The organic phase is dried over MgS04, filtered, and evaporated to yield the crude product. Purification by silica. gel chromatography yields the pure product. i i C. S~mthesis of 15-rsgquinol-4-vlacetaniidol~~,~.A
.;
~I H
OH "~ e2 V,, ~4, ., HO
i A solution of 15-amino erythromycin A diacetate salt (1.0 g) in 10 mL of dichlommethane is treated s equentially with isoquinol-4-ylacetyl chloride (350 mg) and tricthylanunc (0.5 mL) at!0°C. After 3 hours ~ the reaction is diluted with dichlorrnnethane and washed three times. with saturated aqueous NaHC03. The organic phase is dried over i ' AMENDED SHEET
Emvfangszeit 7.Juni 0:37 J' -- ---1 3:55 PM FR MO-FOM SRN D1EG0858 728 5125 TO 854C~~t9S5~4~tt9''~""
06=06-2001 ~ US 00000991 E

-5,2-MgS4a~ fiitcred, and evaporated to yield tire ar~udc product. PuriEcation by silica gel chromatography yields the;pure product.
D. ~ynthcsis of 15-(3-(isoquinol-4-yl)nrooio~lamidolen~thromvcin A
.
A solution of 15-aminoersrthromycin A diacetate salt (1.0 g) in 10 mL of dichioromethane is treated sequentially with 3~ (isoqwinol-4-yl)propionyl chloride (400 mg) and triethylamine {0.5 mL) at 0°C. After 3 hoe~rs, the reaction is diluted with dichlorornethaae and washed three times withlsaturated aqueous NaHCOa. The organic phase TO is dried over MgSOa, filtered, and evaporated xo yield the crude product.
Purification by silica gel chromatography;yields the pure product.
j E. Synthesis of 15-((quinol-5-ylamino)acetamido?erythsom~rcin A
p , N I ~. N
i A solution of 15-amino erythromycin A diacetate salt (1.0 g) in 10 mL of dichloromethane is treated sequentially with (quinol-5 ylamino)acetic acid (0.30 g}, ' dicyclahexyicarbodiimide:(0.4 g}, l~ydroxybenzotriazole (0.25 g}, and triethylamine (0.5 mL) at 0°C. After 3 hours, the reaction is diluted with dichlommethane and washed three times with saturated a evus NaHC . The or qu 03 , ganic phase is dried over MgSOa, filtered, and evaporated to yield the. crude produet_ Purification by silica gel chromatography yields the pure product. ~ j ' Fmofanx~~ait 7.,Ivni 0;37 AMENDED SHEET

06-06-2001 ; ~ US 00000991 ~

i F. Svnthesis of 15-(fauinol-6-vlamino)acetamido)ervthromvcin A
I
N~
A solution of 15-aiminoerythromycin ~r diacetate salt (1.0 g) in 10 mL of dichloromethane is treated sequentially with (quinol-6-ylamino)acetic acid (0.30 g), i dicyclohexylcarbodiinude (0.4 g), I-hydroxybenzotriazole (0.25 g), and triethylamine (0.5 mL) at 4°C. After 3 hours, the reactuon is diluted with dichloromethane and washed three times with saturated aqueous NaHC03. The organic phase is dried over MgS04, filtered, and I 4 evaporated to yield the crude product. Purification by silica gel chromatography yields the pure product. ' I
G.
N~ j I S A solution of I S-atninoerythromycin A diacetate salt (I .0 g) in 10 mI, of dichloromethane is treated sequentially with quinoline,-q..methoxycarbonyl chloride (400 tng) and triethylamine (0.5 znL) at 0°C. After 3 hours, the reaction is diluted with dichloromethane and washed three times with saturated aqueous NaHC03. The organic phase is dried over MgSp4, filtered, and, evaporated to yield the crude product.
Purification by 20 silica gel chromatography yields the pure product.
Emvfangszeit 7.Jiuni 0:31 AMENDED SHEET

Claims (26)

Claims

1. A compound of the formula or the 10,11-anhydro forms thereof wherein R a is H or OH;
R d is H or halogen;
R c is H or a protecting group;

R d is methyl; unsubstituted alkyl (3-10C); substituted alkyl (1-10C);
substituted or unsubstituted alkenyl (2-10C); substituted or unsubstituted alkynyl (2-10C);
substituted or unsubstituted aryl (4-14C); substituted or unsubstituted arylalkyl (5-20C);
substituted or unsubstituted arylalkenyl (5-20C); substituted or unsubstituted arylalkynyl (5-20C);
substituted or unsubstituted amidoarylalkyl (5-20C) substituted or unsubstituted amidoarylalkenyl (5-20C); or substituted or unsubstituted amidoarylalkynyl (5-20C);
R ~ is H or a protecting group or is mono- or disubstituted amino carbonyl;
R f is H; substituted or unsubstituted alkyl (1-10C); substituted or unsubstituted alkenyl (1-10C); substituted or unsubstituted alkynyl (1-10C); substituted or unsubstituted aryl (4-14C); substituted or unsubstituted arylalkyl (5-20C); or -OR f may be replaced by -H
provided that when OR f is replaced by H, then R d is not methyl;
one of Z and Y is H and the other is OH or protected OH, or is amino, mono- or dialkyl-amino, protected amino, or as aminoheterocycle or Z and Y together are =O, NOH or a derivatized oxime;
including any pharmaceutically acceptable salts thereof and any stereoisomeric forms and mixtures of stereoisomeric forms thereof.

2. The compound of claim 1 wherein R d is methyl, propyl or vinyl.

3. The compound of claim 1 wherein R f is arylalkenyl or arylalkynyl.

4. The compound of claim 3 wherein R f is 3-aryl prop-2-enyl or 3-aryl prop-2-ynyl.

5. The compound of claim 4 wherein said aryl is 3-quinolyl, 4-quinolyl or 5-quinolyl, phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-methoxyphenyl, 6-quinolyl, 6-quinoxalyl, 6-amino-3-quinolyl, or 4-isoquinolyl.

6. The compound of claim 1 wherein R f is H or C1-C3 alkyl.

7. The compound of claim 6 wherein R f is methyl.

8. The compound of claim 1 wherein R b is fluoro.

9. A pharmaceutical composition comprising the compound of claim 1 in admixture with a pharmaceutically acceptable excipient.

10. A method to control infection din a subject which method comprises administering to a subject in need of such control an effective amount of the compound of claim 1 or a pharmaceutical composition thereof.

11. A method to preserve material from microbial decay which method comprises providing said material with as effective amount of the compound of claim 1.

12. The compound as in claim 1 wherein R a is hydroxyl;
R b is H;
R c is H or a protecting group;
R d is methyl, propyl, vinyl, fluoroethyl or azidoethyl;
R e is H or a protecting group;
R f is methyl, allyl or propargyl; and Z and Y together are =O, NOH or a derivatized oxime.

13. The compound as in claim 1 of formula wherein R a is hydroxyl R b is H;

R c is H or a protecting group;
R d is propyl;
R f is allyl; and Z and Y together are =O.

14. The compound as in claim 1 of formula wherein R a is hydroxyl;
R b is fluoro R c is H or H or a protecting group;
R d is propyl;
R f is allyl; and Z and Y together are =O.

15. The compound as in claim 1 of formula wherein R a is hydroxyl;

R b is H;
R e is H or a protecting group;
R d is propyl;
R f is 3-arylprop-2-enyl; and Z and Y together are =O.

16. The compound as in claim 17 wherein the aryl is selected from the group consisting of 3-quinolyl, 6-quinolyl and 6 quinoxalyl.

17. The compound as in claim 1 of formula wherein R a is hydroxyl;
R b is fluoro;
R c is H or a protecting group;
R d is propyl;
R r is 3-arylprop-2-enyl; and Z and Y together ,are =O.

18. The compound as in claim 17 wherein the aryl is selected from the group consisting of 3-quinolyl, 6-quinolyl and 6-quinoxalyl.

19. The compound as in claim 1 of formula wherein R a is hydroxyl;
R b is H;
R c is H or a protecting group;
R d is fluoroethyl;
R f is allyl; and Z and Y together are =O.

20. The compound as in claim 1 of formula wherein R a is hydroxyl;
R b is fluoro;
R c is H or a protecting group;
R d is fluoroethyl;
R f is allyl; and Z and Y together are =O.

21. The compound as in claim 1 of formula wherein R a is hydroxyl;
R b is H;
R c is H or a protecting group;
R d is fluoroethyl;
R f is 3-arylprop-2-enyl; and Z and Y together are =O.

22. The compound as in claim 21, wherein the aryl is selected from the group consisting of 3-quinolyl, 6-quinolyl and 6 quinoxalyl.

23. The compound as is claim 1 of formula wherein R a is hydroxyl;
R b is fluoro;
R c is H or a protecting group;

R d is fluoroethyl;
R f is 3-arylprop-2-enyl; and Z and Y together are =O
.

24. The compound as in claim 23 wherein the aryl is selected from the group consisting of 3-quinolyl, 6-quinolyl and 6-quinoxalyl.

25. The compound as in claim 1 of formula wherein R a is hydroxyl;
R b is H;
R c is H;
R d is propyl;
R e is H;
R f is H; and Z and Y together are =O.

26. The compound as in claim 1 of formula wherein R a is hydroxyl;
R b is H;
R c is H;
R d is fluoroethyl;
R e is H;
R f is H; and Z and Y together are =O.