Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D273/00—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D273/00—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00
  • C07D273/08—Heterocyclic compounds containing rings having nitrogen and oxygen atoms as the only ring hetero atoms, not provided for by groups C07D261/00 - C07D271/00 having two nitrogen atoms and more than one oxygen atom
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
  • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Definitions

• Neoplastic diseases characterized by the proliferation of cells not subject to the normal control of cell growth, are a major cause of death in humans and other mammals.
• Clinical experience in cancer chemotherapy has demonstrated that new and more effective drugs are desirable to treat these diseases.
• Such clinical experience has also demonstrated that drugs which disrupt the microtubule system of the cytoskeleton can be effective in inhibiting the proliferation of neoplastic cells.
• Cryptophycin compounds can now be prepared using a total synthetic process; see for example, Barrow, R.A. et al., J. Am . Chem . Soc . I l l , 2479 (1995).
• the present invention provides a process for preparing cryptophycin compounds, including cryptophycin 52, cryptophycin 55 and cryptophycin 55 glycinate, as well as a process for making cryptophycin compounds.
• the present invention provides a process for the preparation of a compound of the formula
• G is C1-C12 alkyl, C 2 -C ⁇ 2 alkenyl, C 2 -C ⁇ 2 alkynyl, or Ar;
• Ar is an aromatic or heteroaromatic group or a substituted aromatic or heteroaromatic group
• R 1 is halogen and R 2 is OH or glycinate ester; or R 1 and R 2 may be taken together to form an epoxide ring; or R 1 and R 2 may be taken together to form a bond;
• R 3 is C ⁇ -C 6 alkyl
• R 7 and R 8 are each independently hydrogen or C ⁇ -C 6 alkyl
• R 7 and R 8 taken together form a cyclopropyl or cyclobutyl ring
• R 9 is hydrogen, C ⁇ C 6 alkyl , C 2 -C 6 alkenyl , C 2 -C 6 alkynyl ,
• R 10 is hydrogen or C ⁇ -C 6 alkyl
• R 11 is hydrogen, C ⁇ -C 6 alkyl , phenyl or benzyl ;
• R , 1 1 4 is hydrogen or C_-C 6 alkyl ;
• Y is CH, 0, NR 12 , S, SO, S0 2 , wherein R 12 is H or C_-C 3 alkyl;
• R 6 is C ⁇ -C 6 alkyl, substituted (C ⁇ -C 6 ) alkyl, (C 3 -
• R 6a , R 615 , and R 6 ° independently are H, (C_-C 6 ) alkyl, halo NR 18 R 19 or OR 18 ;
• R 15 , R 16 , and R 17 independently are hydrogen, halo, (C_-
• R 18 and R 19 independently are hydrogen or C ⁇ -C 6 alkyl;
• R 23 is hydrogen or (C1-C 3 ) alkyl;
• Z is -(CH 2 ) n - or (C3-C5) cycloalkyl; n is 0, 1, or 2; and
• Z' is an aromatic or substituted aromatic group; or a pharmaceutically acceptable salt thereof
• R 3 is as defined above and M is hydrogen or a cation
• R is defined as above;
• R 2a is trityl or a suitable silyl protecting group, and R 3 is as defined above;
• G, R 3 and R 2a are as defined above and R a is hydrogen, allyl or C ⁇ -C 6 alkyl;
• pharmaceutically acceptable acid addition salt is intended to apply to any non-toxic organic or inorganic acid addition salt of the compounds of formula I or any of its intermediates.
• inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulphuric, and phosphoric acid and acid metal salts such as sodium monohydrogen orthophophate, and potassium hydrogen sulfate.
• organic acids which form suitable salts include the mono-, di- and tricaboxylic acids.
• Such acids are for example, acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicylic, 2-phenoxy-benzoic, and sulfonic acids such as p-toluenesulfonic acid, methane sulfonic acid and 2- hydroxyethane sulfonic acid.
• Such salts can exist in either hydrated or substantially anhydrous form.
• Suitable basic addition salts is intended to apply to any non-toxic organic or inorganic basic addition salts of the compounds of formula I or any of its intermediates.
• Illustrative bases which form suitable salts include alkali metal or alkaline-earth metal hydroxides such as sodium, potassium, calciu , magnesium or barium hydroxides; ammonia and aliphatic, cyclic or aromatic organic amines such as methylamine, dimethylamine, tri ethy1amine, diethylamine, triethylamine, isopropyldiethylamine, pyridine and picoline.
• C ⁇ -C 12 alkyl refers to a saturated straight or branched chain hydrocarbon group of from one to twelve carbon atoms. Included within the scope of this term are methyl, ethyl, n-propyl, isopropyl, n- butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, 2-methylbutyl, 3-methylbutyl, hexyl, heptyl, octyl, nonyl, decyl and the like.
• C_-C 6 alkyl refers to a saturated, unsaturated, straight or branched chain hydrocarbon radical of from one to six carbon atoms. Included within the scope of this term are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, 2-methylbutyl, 3- methylbutyl, hexyl and the like.
• C1-C12 alkyl and “C ⁇ -C 6 alkyl” is the terms “C_-C 3 alkyl” which refers to a saturated, unsaturated, straight or branched chain hydrocarbon radical of from one to three carbon atoms. Included within the scope of this term are methyl, ethyl, isopropyl, and the like.
• Substituted (C ⁇ -C 6 ) alkyl refers to a C_-C 6 alkyl group that may include up to three (3) substituents containing one or more heteroatoms. Examples of such substituents are OH, NH 2 , CONH 2 , C0 2 H, P0 3 H 2 and S0 2 R 21 wherein R 21 is hydrogen, C1-C3 alkyl or aryl.
• (C 3 -C 8 ) cycloalkyl refers to a saturated C 3 -C 8 cycloalkyl group. Included within this group are cyclopropyl, cyclobutyl, cyclohexyl, cyclooctyl, and the like.
• a "substituted (C 3 -C 8 ) cycloalkyl group” refers to a (C 3 -C 8 ) cycloalkyl group having up to three C_-C 3 alkyl, halo, or OR 21 substituents. The substituents may be attached at any available carbon atom. Cyclohexyl is an especially preferred cycloalkyl group.
• cycloalkyl where m is an integer one, two or three refers to a cyclopropyl, cyclobutyl or cyclopentyl ring attached to a methylidene, ethylidene or propylidene substituent.
• C 2 -C ⁇ 2 alkenyl refers to an unsaturated straight or branched chain hydrocarbon radical of from two to twelve carbon atoms and having from one to three triple bonds. Included within the scope of this term are ethenyl, propenyl, isopropenyl, n-butenyl, isobutenyl, pentenyl, 2- methylbutenyl, 3-methylbutenyl, hexenyl, octenyl, nonenyl, decenyl and the like. It is especially preferred that alkenyl have only one double bond.
• C 2 -C ⁇ 2 alkynyl refers to an unsaturated straight or branched chain hydrocarbon radical of from two to twelve carbon atoms and having from one to three triple bonds . Included within the scope of this term are ethynyl, propynyl, isopropynyl, 2-methypropynyl, hexynyl, decynyl, and the like. It is particularly preferred that alkynyl has only one triple bond.
• C_-C 6 alkoxy refers to a straight or branched alkoxy group containing from one to six carbon atoms, such as methoxy, ethoxy, n-propoxy, isopropoxy, n- butoxy, isobutoxy, pentoxy, 2-methylpentoxy, and the like.
• (C ⁇ -C 6 alkoxy) phenyl refers to a phenyl group substituted with a C ⁇ -C 6 alkoxy group at any available carbon on the phenyl ring.
• halo refers to chloro, bromo, fluoro, or iodo.
• aromatic group and “heteroaromatic group” refer to common aromatic rings having 4n + 2 pi electrons in a monocyclic or bicyclic conjugated system.
• aryl refers to an aromatic group, and the term
• aralkyl refers to an aryl (C ⁇ -C 6 -alkyl) group.
• aromatic groups are phenyl, benzyl and naphthyl .
• Heteroaromatic groups will contain one or more oxygen, nitrogen and/or sulfur atoms in the ring.
• heteroaromatic groups include furyl, pyrrolyl, thienyl, pyridyl and the like. When the aromatic or heteroaromatic groups are substituted, they may have from one to three independently selected C ⁇ -C 6 alkyl; Ci-Ce-alkoxy or halo, substituents.
• the aromatic groups may be further substituted with trifluoromethyl, COOR 57 (wherein R 57 is hydrogen or C_-C 6 alkyl), P0 3 H, S0 3 H, S0 2 R 57 , N(R 59 ) (R 60 ) (wherein R 59 is hydrogen or C ⁇ -C 6 alkyl and R 60 is hydrogen, C ⁇ -C 6 alkyl, BOC or FMOC) , -CN, -N0 2 , -OR 57 , -CH 2 OC(0) (CH 2 ) m' NH 2 (wherein m' is an integer 1 to 6) or -CH 2 -0-Si (R 57 ) (R 58 ) (R 59 ) (wherein R 58 is hydrogen or C_-C 6 alkyl) .
• substituents for the aromatic groups include methyl, halo, N(R 59 ) (R 60 ), and -OR 57 .
• the substituents may be attached at any available carbon
• heterocyclic or substituted heterocyclic groups include
• R is hydrogen or C ⁇ -C 6 alkyl.
• O-aryl refers to an aryloxy or an aryl group bonded to an oxy moiety.
• TBS tert- butyldimethylsilyl
• N-hydroxysuccinimide As used herein, the term “NHS” refers to N- hydroxysuccinimide represented by the formula
• Ph refers to a phenyl moiety
• base labile amino protecting group refers to common amino protecting groups which are known to be base labile. The artisan can consult common works such as Greene, T.W. "Protecting Groups in Organic Synthesis", Wiley (New York, 1981) . See particularly Chapter 7 of Greene. 7An especially preferred base labile amino protecting group is fluorenylmethoxycarbonyl (Fmoc)
• suitable activatable carboxy protecting group refers to carboxy protecting groups containing activatable ester substituents and are known by one of ordinary skill in the art and disclosed by Greene, T.W., supra.
• Suitable carboxy protecting groups are those which are activatable ester substituents including N-hydroxy- succinimide, N-hydroxysulfosuccinimide and salts thereof, 2- nitrophenyl, 4-nitrophenyl, 2, 4-dichlorophenyl, and the like.
• An especially preferred activatable carboxy protecting group is N-hydroxy-succinimide (NHS) .
• cryptophycin compound refers to a compound of formula (II) and to cryptophycins known in the art.
• Crystalptophycin 52 represents the compound of the formula:
• step 1 the a-cylacetate of formula (2) is cyclized with a suitable cyclizing agent to form a lactone of formula (3) .
• a suitable cyclizing agent is any agent capable of converting the acylacetate of formula (2) to the lactone of formula (3) .
• an acylacetate of formula (2) is added to a solution of a suitable base, such as potassium t- butoxide, lithium dialkylamides, for example, lithium diisopropylamide, sodium hydride and the like. Most preferred is potassium t-butoxide.
• the suitable base is dissolved in suitable organic solvent, for example, alcoholic solvents, such as methanol, ethanol, 2-propanol, or mixtures thereof; tetrahydrofuran, and the like. Most preferred are alcoholic solvents, such as 2-propanol.
• the amount of suitable base to be dissolved ranges from about 1.0 molar equivalents to about 2.0 molar equivalents as compared to the acylacetate of formula (2) .
• the amount of suitable base ranges from about 1.3 to about 1.7 molar equivalents. Most preferably, the amount of suitable base ranges from about 1.4 to about 1.6 molar equivalents.
• the basic solution is set to a temperature ranging from about -30°C to about 30°C, preferably under an inert atmosphere, such as nitrogen, in preparation for the reaction with the desired acylacetate of formula (2) . Most preferably, the solution is cooled to about 0°C.
• the acylacetate of formula (2) is added to the basic solution at a rate so as to maintain the temperature " at or below +10°C.
• the acylacetate of formula (II) is added so as to maintain the temperature between -5°C and +7°C.
• the acylacetate of formula (II) is added so as to maintain the temperature between 0°C and +5°C.
• the acylacetate basic solution is then reacted with a suitable aldehyde or ketone of formula (2b')/ which corresponds to the compound of formula (2b) wherein R 2b is hydrogen.
• the amount of aldehyde or ketone of formula (2b 1 ) to be added ranges from about 1.0 molar equivalents to about 3.0 molar equivalents as compared to the acylacetate of formula (2) .
• the amount of suitable base ranges from about 1.1 to about 2.2 molar equivalents. Most preferably, the amount of suitable base ranges from about
• the aldehyde or ketone of formula (2b') is reacted with the acylacetate solution at a temperature ranging from about 0°C to about 50°C. Most preferably, the reaction is carried out at room temperature.
• the resulting mixture is then acidified with a suitable acid, such as hydrochloric acid.
• a suitable acid such as hydrochloric acid.
• the acidified mixture is then isolated and purified according to methods appreciated by one of ordinary skill- in the art, such as extraction, evaporation, filtration and recrystallization to provide the lactone of formula (3) .
• acylacetates of formula (2) are known or readily prepared by one of ordinary skill in the art. Examples include ethyl 2-methylacetoacetate, ethyl 2-n- hexylacetoacetate, ethyl 2-ethylacetoacetate, ethyl 2-n- propylacetoacetate, ethyl 2-isopropylacetoacetate, and the like.
• the preferred aldehydes or ketones of formula (2b') include paraformaldehyde, acetaldehyde, acetone, and the like.
• step 2 the lactone of formula (3) is contacted with a stereoselective reducing agent to provide the stereoselectively reduced compound of formula (4) .
• the stereoselective reducing agent used in Scheme A, step 2 may be either chemical, or preferably biological.
• the preferred agents are microorganisms which contain reducing enzymes, more preferred microorganisms of genus Mortierella .
• Particular preference is given to the species: Mortierella isabellina , Mortierella alpina , Morti erella pusilla , Mortierella nana , Mortierella vinacea , and Mortierella ovata .
• the microorganism is Mortierella isabellina ATCC 42613.
• Suitable biological agents for this process include the genera: Pi chia , Saccharomyces , Candida , Kl uyveromyces, Zygosaccharomyces , Pi chia , Aureobasidium, Torulopsis , Trigonopsis , Kl oeckeva , Hanseniaspora , Schi zosaccharomyces , Cryptococcus , Rhodotorula , Geotrichum, Rhizopus and Cumminghamella .
• Torulopsis ethanoli tol erans ATCC 46859 Torul opsis ethanoli tolerans ATCC 46859, Torul opsi s ptarmiganii ATCC 26902, Torulopsis sonorensis ATCC 56511, Trigonopsis variabilis ATCC 10679, Torulopsis enokii ATCC 20432, Candida boidinii ATCC 18810, Candida blankii ATCC 18735,
• Cryptococcus laurentii ATCC 42922 Hansenula polymorpha ATCC 34438, Rhodotorula mucilaginosa A35210, Kl uyveromyces marxianus ATCC 8554, Saccharomyces bayanus ATCC 76516, Sporobolomyces salmonicolor ATCC 26697, Cryptococcus laurentii ATCC 36833, Arthroascus javanensi s NRRL Y1493, Hyphopicia burtonii NRRL Y1988, Saccharomycopsis capsulearis NRRL Y50, Yarrowia lipolytica NRRL YB423-3, Guill ermondella selenospora NRRL Y1357, Saccharomycopsis fibuligera NRRL Y3, Lipomyces tetrasporus NRRL 7074, Pachysol en tannophil us NRRL 2460, Geotrichum candidum
• NRRL 2458 Mortierella hyalina NRRL 6427, Mortierella pulchella ATCC 18078, Mortierella bisporalis NRRL 2493, Mortierella scleroti ella ATCC 18732, Mortierella minutissima ATCC 16268, Mortierella spinosa ATCC 16272 Peni cilli um glabrum ATCC 11080, Emericella quadrilineata ATCC 12067, Syncephalastrum racemosum ATCC 20471, Geotrichum sp. ATCC 32345, Aspergill us niveus ATCC 20922, Aspergill us niger ATCC 64958,
• a suitable microorganism such as Morti erella isabellina ATCC 42613 may be used in free state as wet cells, freeze-dried cells or heat-dried cells. Immobilized cells on support by physical adsorption or entrapment can also be used. Appropriate media for growing microorganisms for this process typically include necessary carbon sources, nitrogen sources, and trace elements. Inducers may also be added. As used herein, the term "inducer" refers to any compounds having keto or aldehyde groups, such as paraformaldehyde and the like.
• Carbon sources include sugars such as maltose, lactose, dextrose, glucose, fructose, glycerol, sorbitol, sucrose, starch, mannitol, propylene glycol, and the like; organic acids such as sodium acetate, sodium citrate, and the like; amino acids such as sodium glutamate and the like; alcohols such as ethanol, propanol, and the like.
• Nitrogen sources include N-Z amine A, corn steep liquor, soy bean meal, beef extracts, yeast extracts, molasses, baker's yeast, tryptone, nutrisoy, peptone, yeastamine, sodium nitrate, ammoonium sulfate, and the like.
• Trace elements include phosphates, magnesium, manganese, calcium, cobalt, nickel, iron, sodium, and potassium salts.
• appropriate media may include more than one carbon or nitrogen source and may include a mixture of several.
• the pH of the medium should be adjusted to 4.5 to 6.5, preferably 5.5.
• the pH may be maintained between about 4.0 and 6.0, preferably at 5.5 during the fermentation and 4.5 during the bioreduction.
• the temperature of the reaction mixture should be maintained to ensure that there is sufficient energy available for the process.
• the temperature is a measure of the heat energy available for the transformation process.
• a suitable temperature of reaction ranges from about 20°C to 35°C.
• a preferred temperature range is from about 25°C to about 30°C.
• the agitation and aeration of the reaction mixture affects the amount of oxygen available during the fermentation and bioreduction stages of the process. During both stages the agitation range from 150 to 450 rpm is preferable, with 150 to 275 rpm being most preferred. Aeration of about 0.5 to 3.5 standard cubic feet per minute (scfm) is preferable, with 0.5 to 1.0 scfm being most preferred.
• reaction time for the reduction of Scheme A, step 2 ranges from about 24 to 96 hours, preferably 24 to 48 hours, measured from the time of initially treating the substrate (3) with the microorganism to provide the lactone of formula (4) .
• step 3 the lactone of formula (4) is reacted with a hydroxy protecting agent to yield the protected lactone of formula (5) .
• a suitable hydroxy protecting agent includes compounds of the formula R 2a -LG where R 2a is trityl or a silyl protecting group, preferably tri (d-C 6 alkyl) silyl, and LG is a suitable leaving group, such as a halogen or a sulfonate, such as trifluoromethanesulfonate .
• Specific examples of hydroxy protecting agents include t- butyldimethysilyl chloride, t-butyldimethylsilyl trifluoromethane sulfonate, chlorotrimethylsilane and the like.
• the lactone of formula (4) is contacted with a suitable base, most preferably imidazole, in a suitable organic solvent such as CH 3 CN.
• a suitable hydroxy protecting agent such as t-butyldimethysilyl chloride, is then added to the solution, optionally with a suitable coupling catalyst such as dimethylaminopyridine.
• the mixture is then stirred at a temperature of from about 0°C to about 60°C, preferably room temperature, for a period of time ranging from about 2 to 24 hours.
• the protected alcohol of formula (5) can be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation.
• the product can be purified by chromatography and recrystallization.
• step 4 the protected alcohol of formula (5) is reacted with a reducing agent followed by an olefinating agent to provide the olefin of formula (6).
• a suitable reducing agent includes alkylated aluminum hydrides and other reagents that would convert the protected lactone of formula (5) into a lactol and/or open chained hydroxyaldehyde intermediate.
• alkylated aluminum hydrides and other reagents that would convert the protected lactone of formula (5) into a lactol and/or open chained hydroxyaldehyde intermediate.
• Examples include diisobutylaluminum hydride, bis (dialkylamino) aluminum hydride, either preformed or generated in si tu from alkali- aluminum compounds such as LiAlH 4 , NaAlH 4 , NaH 2 Al (C_-C 6 alkyl) 2 , NaH 2 Al (OCH 2 CH 2 OMe) 2 LiHAl(OtBu) 2 and the like, in combination with dialkyl or cyclic amines such as dimethylamine, diethylamine, dipropylamine, morpholine, piperidine and the like.
• a suitable olefinating agent includes aryl Wittig reagents, aryl Horner-Emmons Wadsworth reagents and other reagents that are known by one of ordinary skill in the art to convert aldehydes to olefins in either a one-step or stepwise fashion. Examples include benzyldiphenylphosphine oxide (BDPPO) , triphenyl benzyl phosphonium chloride and the like.
• BDPPO benzyldiphenylphosphine oxide
• triphenyl benzyl phosphonium chloride triphenyl benzyl phosphonium chloride
• the protected lactone of formula (5) is reacted with a suitable reducing agent such as DIBAL or DIBAH under an inert atmosphere, for a period ranging from about 0.5 to 12 hours.
• a suitable reducing agent such as DIBAL or DIBAH
• the reaction is carried out in the presence of a suitable organic solvent, such as methylene chloride or hexane while the temperature is maintained below -10°C to form portion A.
• a suitable olefinating agent such as BDPPO or triphenyl benzyl phosphonium chloride is contacted with a suitable base, such as sodium bis (trimethylsilyl) amide or potassium tert-butoxide in the presence of a suitable organic solvent such as tetrahydrofuran (THF) or methylene chloride.
• a suitable organic solvent such as tetrahydrofuran (THF) or methylene chloride.
• THF tetrahydrofuran
• the solution may be stirred at room temperature for a period of time ranging from about 10 minutes to 2 hours.
• the resulting reddish solution is then contacted with portion A and stirred for 1 to 36 hours at a temperature ranging from about 0°C to about 70°C.
• the olefin- of formula (6) may be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation. The product can be purified by chromatography and recrystallization.
• step 5 the olefin of formula (6) is oxidized with an oxidizing agent to provide the aldehyde of formula (7) .
• An oxidizing agent is a reagent capable of converting the hydroxy moiety on the olefin of formula (6) to aldehyde moiety of formula (7) .
• Suitable oxidizing agents include oxalyl chloride/DMSO, TEMPO/NaOCl, P 2 0 5 /DMSO, (COCl) 2 /DMS0, NBS/TEMPO, and the like.
• anhydrous dimethylsulfoxide is added to oxalyl chloride in a suitable organic solvent, such as methylene chloride over a period of time ranging from about 1 to about 30 minutes at a temperature ranging from about -30°C to about -78°C, preferably about -60°C.
• a suitable organic solvent such as methylene chloride
• a suitable base such as triethylamine is added and the reaction is allowed to warm to room temperature.
• the aldehyde of formula (7) may be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation.
• the product can be purified by chromatography and recrystallization.
• step 6 the aldehyde of formula (7) " is reacted with an alkyl ester forming agent to form the ester of formula (ID) .
• An alkyl ester forming agent is any agent capable of converting the aldehyde moiety of the compound formula (7) to the alkyl ester moiety of the compound of formula (ID), while inert to the other substituents on the molecules.
• the aldehyde of formula (7) may be converted to the ester of formula (ID) by means of a Horner- Emmons reaction.
• Suitable examples of alkyl ester forming agents include trimethyl phosphonoacetate, (CH 3 0) 2 POCH 2 CH 3 and the like.
• the aldehyde of formula (7) is contacted with an alkyl ester forming agent, such as trimethylphosphonoacetate, and tetramethylguanidine in a suitable organic solvent, such as tetrahydrofuran at ambient temperature and stirred for a period ranging from about 1 to about 24 hours.
• an alkyl ester forming agent such as trimethylphosphonoacetate, and tetramethylguanidine
• a suitable organic solvent such as tetrahydrofuran
• the ester of formula (ID) may be isolated by techniques well known in the art, such as extraction, evaporation, and precipitation.
• the product can be purified by techniques well known in the art, such as chromatography.
• step 7 the ester of formula (ID) is reacted with a hydrolyzing agent to -provide the acid of formula (IE) .
• a hydrolyzing agent is any agent that is capable of converting the ester moiety of the compound of formula (IA) to the acid moiety of the compound of formula (IB), while inert to the other substituents on the molecules.
• suitable hydrolyzing agents include inorganic bases such as sodium hydroxide and potassium hydroxide, with potassium hydroxide being preferred.
• the ester of formula (IA) is contacted with a suitable hydrolyzing agent, such as 2N KOH in a suitable organic solvent, such as 1,4-dioxane at ambient temperature.
• a suitable hydrolyzing agent such as 2N KOH
• a suitable organic solvent such as 1,4-dioxane
• the solution is then heated to reflux for a period of time ranging from about 1 to about 6 hours.
• the reaction is then quenched with a suitable acid, such as 2N HCl.
• the acid of formula (IB) is isolated by techniques well known in the art, such as extraction, evaporation, and precipitation.
• the product can be purified by techniques well known in the art, such as chromatography.
• Scheme B illustrates a general synthetic procedure for preparing a cryptophycin compound of formula (II) .
• R p is hydrogen or a suitable activatable carboxy protecting group
• R pl is hydrogen or C ⁇ -C 6 alkyl
• R 81 is C ⁇ -C 6 alkyl, C 3 -C 8 cycloalkyl, phenyl or benzyl
• R 82 is a base labile protecting group
• Hal is halogen, preferably chloro, bromo or iodo
• q is an integer 1 or 2.
• step 1 a compound of formula (IE) is optionally treated with a carboxy activating agent to provide the activatable ester of formula (8) .
• a compound of formula (IE) is reacted with a suitable coupling agent, such as a carbodiimide, for example, l-ethyl-3- (3-dimethylaminopropyl) carbodiimide, and a suitable carboxy activating agent, such as N- hydroxysuccinimide, in a suitable organic solvent, such as dry dimethylformamide.
• a suitable coupling agent such as a carbodiimide, for example, l-ethyl-3- (3-dimethylaminopropyl) carbodiimide
• a suitable carboxy activating agent such as N- hydroxysuccinimide
• the activatable ester of formula (8) is isolated by techniques well known in the art, such as extraction, evaporation, and precipitation.
• the product can be purified by techniques well known in the art, such as chromatography.
• step 2 an activatable ester of formula (8) is epoxidized with an epoxidizing agent to form an epoxide of formula (9) .
• the compound of activatable ester of formula (8) may be epoxidized non-selectively using a suitable epoxidizing agent.
• An "epoxidizing agent” is an agent capable of converting the activatable ester of formula (8) into the epoxide of compound (9) .
• Suitable epoxidizing agents include potassium peroxomonosulfate (oxone) in combination with acetone, m-CPBA, methyltrioxorhenium (VII) , trifluoroper-acetic acid, and magnesium monoperoxyphthalate, with Oxone in combination with acetone, or m-CPBA being preferred.
• Possible solvents for the epoxidation activatable ester of formula (8) include acetone, DMF, glyme, dioxane, CH 3 CN, alcohols, THF, EtOAc, halohydrocarbons, chlorobenzene, dichloromethane and toluene.
• the reaction optionally takes place in the presence of a suitable base such as NaHC0 3 . Reaction temperatures may range from about -30°C to about 50°C with about -10°C to about 25°C being preferred.
• the ⁇ -epoxide of formula (9) may be isolated and purified according to techniques and procedures well known in the art such as column chromatography.
• Either the ⁇ - and ⁇ - epoxides of formula (9) may be further separated by HPLC. It is preferred that the ⁇ -epoxide of formula (9), is separated from the ⁇ -epoxide of formula (9a), ' and further used in the remaining steps of the process of this invention to form a the ⁇ -epoxy form of a compound of formula (I) .
• the epoxidizing reaction of Scheme B, step 1 can also be used with the ⁇ -epoxide of formula (9a) or with a mixture of the two epoxides.
• the compound of formula (I) wherein R a is H may be epoxidized directly using m-CPBA.
• the m-CPBA epoxidation may be carried out on a compound of formula (I) to give a 1.2:1 b/a diastereomeric mixture of epoxides.
• the individual ⁇ - and ⁇ -diastereomers may be separated by HPLC, as described above. This direct epoxidation is illustrated in Scheme Bl. SCHEME Bl
• a compound of formula (9e) may be prepared by deesterifying a compound (9d) according to Scheme B2.
• R a is C ⁇ -C 6 alkyl whereas all of the remaining substituents are as previously defined.
• the alkyl ester of formula (9d) is deesterified with a suitable deesterifying agent to form the acid of formula (9e).
• suitable deesterifying agent encompasses any suitable means or conditions for removing the ester moiety of R a while inert to the epoxide.
• a suitable base such as potassium hydroxide
• a suitable solvent such as tetrahydrofuran.
• the biphasic mixture is then allowed to stir at a temperature ranging from about 20°C to about 80°C, preferably 40°C and 65°C, for a period of from about 6 to 24 hours.
• the aqueous layer is washed with an appropriate acid, such as IN hydrochloric acid, followed by brine.
• the mixture is dried, filtered and concentrated to provide the acid of (9e) .
• a compound of formula (I) or formula (8) may also be stereoselectively epoxidized to form either the compound of formula (9) or (9a) using a chiral ketone with Oxone in the presence of a suitable base such as NaHC0 3 using procedures analogous to those disclosed by Tu, Y. et al, J. Am. Chem. Soc . 118, 9806 (1996); Wang, Z-X et al . J. Org. Chem. 62, 2328 (1997); Wang, Z-X et al., J. Am. Chem . Soc . 119, 11224 (1997) .
• Preferred compounds of formula (8) for this reaction include those compounds where G is phenyl, R 3 is methyl, and R is NHS (N-hydroxysuccinimide) .
• the term "chiral ketone” refers to a ketone containing the following general features:
• the ketone has a fused ring and a quaternary center adjacent to a carbonyl group; and 3) one face of the ketone is sterically blocked.
• One especially preferred chiral ketone is of the structure:
• This preferred chiral ketone can be prepared from D-fructose by ketalization and oxidation under routine conditions.
• the ketalization can be completed using acetone, HCIO 4 , and the process is conducted at about 0° C.
• the oxidation can be completed using pyridinium chlorochromate at room temperature.
• the asymmetric epoxidation can be carried out at a pH within the range of from about 7.0 to about 11.5 during the reaction.
• Suitable solvents useful for the epoxidation step include H 2 0, DMF, glyme, dioxane, CH 3 CN, alcohols, THF, EtOAc, halohydrocarbons, chloro- benzene, and toluene, with a CH 3 CN/H 2 0 solvent combination being preferred. Reaction temperatures may range from about -20°C to about 25°C with about -10°C to about 10°C being preferred.
• the individual isomers, (9) or (9a) can be isolated from the crude mixture of isomers and purified by techniques well known in the art such as extraction, evaporation, chromatography and recr-ystallization.
• a preferred stereoselective epoxidation utilizes the chiral ketone of structure (9f) to provide a mixture of epoxides in the crude product in the ratio of about ⁇ : ⁇ 1:5.
• the ⁇ -epoxide of formula (9) is generally preferred and is used throughout the process of this invention.
• step 3 the epoxide of formula (9) is coupled to the amino acid of formula
• R 6 and R 14 are as defined above and R pl is hydrogen or C ⁇ -C 6 alkyl to yield a Fragment A-B compound of formula (10) .
• amino acids of formula (9g) are commercially available or are readily prepared by methods known in the art.
• Particularly preferred amino acids of formula (9g) include those where R 6 is a group of formula ( IA) and R 6a is methoxy, R 6b is chloro and R 6c is H; R 14 is hydrogen; and R pl is hydrogen; said amino acids being disclosed by PCT Intnl. Publ. No. WO 97/07798, published March 6, 1997, PCT Intnl. Publ. No. WO 96/40184, published December 19, 1996; Barrow, R.A. et al. J. Am. Chem. Soc . I ll , 2479 (1995).
• the epoxide of formula (9) where R p is NHS, is coupled to the amino acid of formula (9g) according to coupling procedures which are inert to the epoxide functionality.
• the epoxide of formula (9) is contacted with from about 1.5 to 3.5 equivalents of amino acid (9g), where R pl and R 14 are both hydrogen, and a suitable silylating agent in the presence of a suitable organic solvent.
• Suitable organic solvents include DMF, glyme, dioxane, CH 3 CN, THF, EtOAc, and halohydrocarbons, such as methylene chloride.
• the reaction is carried out at a temperature ranging from about -30°C to about 75°C, with a temperature ranging from about 20°C to about 60°C being preferred.
• the fragment A-B compound of formula (10) may be isolated and purified according to techniques and procedures well known in the art such as extraction, evaporation, chromatography and recrystallization.
• silylating agent is selected from any reagent capable of attaching a silyl group to a target substituent.
• silylating agents are employed. See for example, Calvin, E.W., “Silicon Reagents in Organic Synthesis", Academic Press, (London,
• silyl agents include any reagent with a trialkylsilyl group such as trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethylthexylsilyl, and t- butyldimethylsilyl, any reagent with an alkylarylsilyl group such as tribenzylsilyl, diphenylmethylsilyl, t- butylmethoxyphenylsilyl and tri-p-xylylsilyl, and any reagent with a triarylsilyl group such as triphenylsilyl .
• a trialkylsilyl group such as trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethylthexylsilyl, and
• the preferred silylating agent is a trimethyl silylating agent.
• Typical trimethyl silylating agents include N,0- Bis (trimethyl silyl) acetamide, allyltrimethylsilane, N,0- Bis (trimethylsilyl) - carbamate N,N- Bis (trimethylsilyl) methylamine, Bis (trimethylsilyl) sulfate, N, O-Bis (trimethylsilyl) trifluoroacetamide, N,N- Bis (trimethylsilyl) urea, (ethylthio) trimethylsilane, ethyl trimethyl silylacetate, hexamethyldisilane, hexamethyldisilazane, hexamethyldisiloxane, hexamethyldisilthiane, (isopropenyloxy) trimethyl silane, 1- methoxy-2-methyl-l-trimethyl-siloxy-propene, (methylthio) trimethly
• silylating agents include "tri-lower alkyl silyl” agents, the term of which contemplates triisopropylsilyl, trimethylsilyl and triethylsilyl, trimethylsilyl halides, silylated ureas such as bis (trimethylsilyl) urea (BSU) and silylated amides such as N,0-bis (trimethylsilyl) acetamide (BSA).
• BSU bis (trimethylsilyl) urea
• silylated amides such as N,0-bis (trimethylsilyl) acetamide (BSA).
• BSA bis N,0- trimethyl silyl acetamide
• the desired ⁇ -epoxide (9c) may be coupled with (9g) , when R pl is hydrogen, using a suitable coupling agent, preferably diphenylphosphinic chloride, and a silyl agent to give fragment A-B (10) .
• suitable coupling agents are well known in the art, as described by Greene, T.W. "Protecting Groups in Organic Synthesis", Wiley (New York, 1981) and include N, 0- diphenylphosphinic chloride, diphenyl chlorophosphate, DCC, EDCI, chloroformates, and 2- chloro-4, 6-dimethoxy-l, 3, 5-triazine.
• Diphenylphosphinic chloride is a preferred coupling agent.
• step 4 the fragment A-B compound of formula (10) is deprotected with a suitable alkoxy deprotecting agent to form a compound of formula (11) .
• a suitable alkoxy deprotecting agent is one that removes the hydroxy protecting group signified by the R 2a substituent while inert to the epoxide moiety of the fragment A-B compound of formula (10) .
• Preferred deprotecting agents include basic fluoride sources such as tetrabutylammoniu fluoride, pyridinium fluoride, triethylammonium fluoride, cesium fluoride, and the like, with tetrabutylammonium fluoride being preferred.
• the deprotection reaction takes place in the presence of a suitable organic solvent such as tetrahydrofuran, optionally in the presence of a suitable base, such as sodium bicarbonate (NaHC0 3 ) .
• the reaction takes place in the range of from about 0°C to about 80°C with from about 20°C to about 70°C being preferred.
• the reaction is run for a period of time ranging from about 3 to 24 hours.
• Crude product (11) may be used without further purification.
• the compound of formula (11) may be isolated and purified according to procedures well known well known in the art such as extraction, evaporation, chromatography and recrystallization.
• R pl for the compound of formula (11) is hydrogen
• the R pl moiety is actually the cationic salt of deprotecting agent, for example, cesium, tetrabutylammonium, and the like.
• step 5 the compound of formula (11) is contacted with a thioester forming agent to provide the ester of formula (12) .
• thioester forming agent encompasses any suitable means or conditions for forming the thioester moiety of formula (12). Included within this definition are the conditions set forth and/or analogously described in Ono, N. et al . , Bull . Chem. Soc . Jpn . 51 (8), 2401 (1978); Ho, Tse-Lok, Synth . Comm. 9(4), 267-270 (1979); Narasaka, K. et al., J. Am. Chem. Soc . 106 (10), 2954-2960 (1984); L.G. Wade, Jr. et al . , Tetrahedron Lett . 731-732 (1978); Mora, N. et al., Tetrahedron Lett . 34 (15), 2461-2464 (1993); and Dossena, A. et al . J. Chem . Soc . Perkin Trans . I, 2737 (1981) .
• the compound of formula (11) may be treated with a sterically hindered alkyl halide, such as tert-butylbromide, and a solvent of the formula (R 81 ) (Me) SO, wherein R 81 is C ⁇ -C 6 alkyl, C 3 -C 8 cycloalkyl, phenyl or benzyl, in the presence of a suitable base, such as sodium bicarbonate (NaHC0 3 ) .
• a preferred solvent for reaction is dimethylsulfoxide (DMSO) .
• DMSO dimethylsulfoxide
• Both the sterically hindered alkyl halide and the suitable base are added in a molar excess of about 7.0 to 12.0 in comparison to the compound of formula (11) .
• the reaction takes place in the range of from about 0°C to about 60°C with from about 10°C to about 30°C being preferred.
• the reaction is run for a period of time ranging from about 1 to 24 hours.
• Crude product (12) may be used without further purification.
• the ester of formula (12) may be isolated and purified according to procedures well known well known in the art such as extraction, evaporation, chromatography and recrystallization.
• the compound of formula (11) must first be carboxy-deprotected.
• Carboxy-deprotections under basic conditions are known by those of ordinary skill in the art.
• the compound of formula (11) may be treated with a suitable base, such as lithium hydroxide (LiOH) for a period of time sufficient to remove the carboxy protecting group, for example from about 1 to 24 hours.
• a suitable base such as lithium hydroxide (LiOH)
• step 6 the ester of formula (12) is coupled with a Fragment CD carboxylic acid of formula
• R 82 is a base labile protecting group; to provide the compound of formula (13) .
• the carboxylic acid of formula (12a) is dissolved in a suitable organic solvent, such as DMF, glyme, dioxane, THF, CH 3 CN, EtOAc, and halohydrocarbons, with dichloromethane being preferred.
• a suitable organic solvent such as DMF, glyme, dioxane, THF, CH 3 CN, EtOAc, and halohydrocarbons, with dichloromethane being preferred.
• a coupling reagents include DCC, EDCI, and similar reagents, such as DMAP which activate carboxylic acids towards esterification with alcohols.
• This solution may then be optionally treated with a suitable base such as solid sodium bicarbonate and then contacted with an ester of formula (12) .
• the concentration of (12a) after these additions should range from about 0.1 M to about 2.0 M.
• the reaction takes place in the range of from about -30°C to about 60°C with from about 10°C to about 30°C being preferred.
• the reaction is run for a period of time ranging from about 0.5 to 12 hours.
• the final concentration of Crude product (13) may be used without further purification.
• the compound of formula (13) may be isolated and purified according to procedures well known well known in the art such as extraction, evaporation, chromatography and recrystallization.
• step 7 the compound of formula (13) is oxidized with a suitable oxidizing agent to provide the sulfone or sulfoxide of formula (14) .
• a suitable oxidizing agent is an agent capable of converting the sulfide of formula (13) into the sulfone of formula (14), while inert to the epoxide moiety of the molecule.
• Suitable oxidizing agents include potassium peroxomonosulfate (Oxone), m-CPBA, methyltrioxorhenium (VII) , and magnesium monoperoxyphthalate, with Oxone being preferred.
• the sulfide of formula (13) is treated with a suitable base, such as sodium bicarbonate followed by a suitable oxidizing agent, such as Oxone.
• a suitable solvent such as acetone, DMF, glyme, dioxane, CH 3 CN, alcohols, THF, EtOAc, halohydro-carbons, chlorobenzene, and toluene, with acetone being preferred.
• the reaction is carried out at temperatures of from about -30°C to about 50°C with from about -10°C to about 10°C being preferred.
• the reaction requires from about 15 minutes to about 5 hours.
• Crude sulfone or sulfoxide (14) may be used without further purification.
• the sulfone or sulfoxide of formula (14) may be isolated and purified according to procedures well known well known in the art such as extraction, evaporation, chromatography and recrystallization.
• step 8 the sulfone or sulfoxide of formula (14) is deprotected with a suitable deprotecting agent to provide the amine of formula (14a).
• a suitable deprotecting agent is an agent capable of removing the base labile substituent R 8Z on the compound of formula (14) while inert to the epoxide moiety of the molecule.
• Suitable deprotecting agents include bases such as secondary and tertiary amines and inorganic bases, for example, piperidine, morpholine, dicyclohexylamine, p- dimethylaminopyridine, diisopropylethylamine, and the like, with piperidine being preferred.
• the reaction is carried out in a suitable solvent such as DMF, glyme, dioxane, CH 3 CN, alcohols, THF, EtOAc, halohydrocarbons, chlorobenzene, or toluene.
• the reaction is carried out at a temperature ranging from about 0°C to about 120 °C. Generally, the reaction requires from about 1 to 72 hours.
• the compound of formula (IIB) may be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization. Alternatively, the compound of formula (14a) is isolated and may be further cyclized with a cyclizing agent to provide a compound of formula (IIB) .
• the compound of formula (14) undergoes spontaneous cyclization.
• some particular compounds of formula (14) may require an additional cyclization step.
• the sulfide of formula (13) although much less -active than its oxidized counterpart, upon removal of the base-labile protecting group may be cyclized with a second suitable cyclizing agent, such as 2-hydroxypyridine to form a compound of formula (IIB) .
• a second suitable cyclizing agent such as 2-hydroxypyridine
• the sulfide of formula (13) or alternatively a selected compound of formula (14a) , is heated in a suitable solvent, such as DMF at about 60 °C for several days in the presence of piperidine and 2- hydroxypyridine.
• the compound of formula (IIB) is isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
• step 9 the epoxide of formula (IIB) is optionally treated with a halohydrin forming reagent to produce the halohydrin of formula (IIC), where Hal is halogen, preferably chlorine.
• a "halohydrin forming reagent” is any agent capable of coverting the epoxide moiety of compound (IIB) to the halohydrin moiety of compound (IIC) .
• Suitable halohydrin forming reactions are disclosed in PCT Intnl. Publ. No. WO 96/40184, published December 19, 1996 and PCT Intnl. Publ. No. WO 98/09988, published March 12, 1998.
• the epoxide of formula (IIB) is treated with a suitable halo-acid, such as hydrochloric acid in a suitable organic solvent or solvent mixture, such as dimethoxy- ethane/water .
• the mixture is then stirred at a temperature ranging from about 10°C to about 50°C for a period of time ranging from about 6.to 36 hours.
• the mixture is then neutralized with a suitable base or buffer, such as potassium carbonate.
• a suitable base or buffer such as potassium carbonate.
• the halohydrin of formula (IIC) is isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
• step 10 the halohydrin of formula
• a "glycinating agent” is any agent capable of converting the halohydrin of formula (IIC) into the glycinate ester of formula (IID) .
• Suitable glycinating reactions are disclosed in PCT Intnl. Publ. No. WO 98/08505, published March 5, 1998.
• the halohydrin of formula (IIC) is coupled with N- ( ert-butoxycarbonyl) glycine (Boc-Gly) under coupling conditions well known in the art.
• the halohydrin of formula (IIC) is contacted with Boc-Gly, dimethylaminopyridine (DMAP) and 1,3- dicyclohexylcarbodiimide (DCC) .
• the resulting mixture is stirred at a temperature ranging from 10°C to 50°C for a period of time ranging from 0.5 to 24 hours.
• the glycinate ester of formula (IID) is isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
• a synthetic scheme for making the Fragment CD carboxylic acids of formula (12a) is set forth in Scheme C.
• the reagents and starting material are readily available to one of ordinary skill in the art. In Scheme C, all substituents, unless otherwise indicated, are as previously defined.
• step 1 the Boc-protected amine of formula (15) is deprotected to provide the deprotected amine of formula (16) .
• the deprotection reaction involves the removal of an amino protecting group by techniques and procedures well known and appreciated by one of ordinary skill in the art.
• the selection, use, and removal of protecting groups are set forth by Greene, T.W. "Protecting Groups in Organic Synthesis", Wiley (New York, 1981) .
• the Boc-protected amine of formula (15) is dissolved in a suitable acid, such as trifluoroacetic acid or hydrochloric acid.
• the reaction is carried out at a temperature ranging from about 0°C to about 60 °C.
• the reaction requires from about 1 to 24 hours.
• the deprotected amine of formula (16) may be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
• Boc-protected amine of formula (15) is described in Barrow, R.A. et al . J. Am. Chem . Soc . I ll , 2479 (1995); PCT Intnl. Publ. No. WO 96/40184, published December 19, 1996; and PCT Intnl. Publ. No. WO 97/07798, published March 6, 1997.
• step 2 the deprotected amine of formula (16) is amino-protected with a base-labile amino protecting group to provide the carboxylic acid of formula (12a) .
• the protection of an amino group with a base-labile amino protecting group involves the addition of a base-labile amino protecting group by techniques and procedures well known and appreciated by one of ordinary skill in the art.
• the selection, use, and removal of base- labile amino protecting groups are set forth by Greene, T.W. "Protecting Groups in Organic Synthesis", Wiley (New York, 1981) .
• a preferred base-labile amino protecting group is Fmoc.
• a suitable solvent such as dioxane
• a suitable base such as sodium bicarbonate
• a compound of the formula R 82 -C1 or R 82 -ONHS such as Fmoc-Cl or Fmoc-ONHS succinimide.
• the mixture may be optionally diluted with a small amount of water and stirred for a period of time ranging from 12 to 48 hours at a temperature ranging from about 0°C to about 60°C.
• the mixture may be quenched with a suitable acid, such as hydrochloric acid.
• the carboxylic acid of formula (12a) may be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
• Scheme D illustrates a general synthetic procedure for preparing a cryptophycin compound of formula (II) .
• all substituents unless otherwise indicated, are as peviously defined.
• the substituent "Hal” stands for halogen.
• step 1 a compound of formula (IB) is coupled with a Fragment B amino acid of formula (9g) to provide an alkoxy-protected Fragment AB compound of formula (17) according to the procedure set forth in Scheme B, step 3.
• step 2 an alkoxy-protected Fragment AB compound of formula (17) is alkoxy-deprotected according to the procedure set forth in Scheme B, step 4 to provide a Fragment AB compound of formula (18) .
• the alkoxy-protected Fragment AB compound of formula (17) is deprotected according to techniques and procedures well known to one of ordinary skill in the art. Since the alkoxy-protected Fragment AB compound of formula (17) does not possess an epoxide group as does the corresponding analog in Scheme B, the deprotecting reaction conditions are not required to be as sensitive.
• an alkoxy- protected Fragment AB compound of formula (17) may be deprotected according to the procedure set forth in Barrow, R.A. et al, J. Am. Chem. Soc . I ll , 2479 (1995), which includes 50% aqueous HF in a CH 3 CN solution.
• step 3 a Fragment AB compound of formula (18) is coupled with a Fragment CD carboxylic acid of the formula
• R 7 , R 8 , R 9 , R 10 , R 11 , R 50 and Y are as defined above and Pg is a suitable amino protecting group, according to the procedure set forth in Scheme B, step 6 to provide a Fragment ABCD compound of formula (19).
• Suitable amino protecting groups are well known by one of ordinary skill in the art and are disclosed in Greene, "Protective Groups in Organic Chemistry", John Wiley & sons, New York (1981), the disclosure of which is hereby incorporated by reference.
• a particularly preferred amino protecting group is t-Boc.
• step 4 a Fragment ABCD compound of formula (19) is deprotected with a suitable second deprotecting agent to provide the deprotected Fragment ABCD compound of formula (20) .
• a suitable "second deprotecting agent” is any agent or combination of agents which are effective in removing both the "Pg" amino protecting group and the "R P1 " carboxy protecting group, either sequentially or concomitantly. Since the Fragment ABCD compound of formula (19) does not possess an epoxide group as does the sulfoxide or sulfone of formula (14) in Scheme B, step 8, the deprotecting reaction conditions are not required to be as sensitive. For example, a Fragment ABCD compound of formula (19) may be deprotected according to the procedure set forth in Barrow, R.A. et al, J. Am . Chem. Soc . I ll , 2479 (1995) . The deprotected Fragment ABCD compound of formula (20) may be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
• step 5 the deprotected ABCD compound of formula (20) is cyclized with a second suitable cyclizing agent according to Barrow, R.A. et al, J. Am. Chem. Soc . I ll , 2479 (1995) to form the cyclic alkene of formula (IIA) .
• the deprotected ABCD compound of formula (20) may be cyclized with a suitable cyclizing agent according to Scheme B, step 8.
• the cyclic alkene of formula (IIA) may be isolated and purified by techniques well known in the art, such as extraction, evaporation, chromatography and recrystallization.
• step 6 the cyclic alkene of formula (IIA) is epoxidized according to the procedures set forth in Scheme B, step 2 or Scheme Bl to provide the epoxide of formula (IIB) .
• step 7 the epoxide of formula (IIB) is treated with a halohydrin forming reagent according to
• the cyclic alkene of formula (IIA) is contacted sequentially with an epoxidizing agent and a trialkylsilyl chloride according to PCT Intnl. Publ. No. WO 98/09988, published March 12, 1998 to provide the halohydrin of formula (IIC) where "Hal" is chloro.
• step 8 the halohydrin of formula (IIC) is reacted with a glycinating agent according to
• salts of the compounds of formulae (I) or (II) may be formed using standard techniques.
• the free base may be dissolved in aqueous or aqueous-alcohol solution or other suitable solvent containing the appropriate acid and the salt isolated by evaporating the solution.
• the free base may be reacted in an organic solvent containing the appropriate acid and the salt isolated by evaporating the solution.
• the free base may be reacted in an organic solvent in which case the salt separates directly or can be obtained by concentration of the solution or in a solvent such as water which is then removed in vacuo or by freeze-drying, or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin.
• one aspect of the invention represents a convergent synthesis to produce a cryptophycin compound of formula (II)
• alternate sequences of couplings may be utilized.
• Fragment A may be first coupled to Fragment B to form Fragment AB and Fragment C to Fragment D to form Fragment CD.
• Fragment AB may then be coupled to Fragment CD to form Fragment ABCD.
• G is phenyl, p-fluorophenyl, or p-chlorophenyl;
• R 1 is chloro and R 2 is OH;
• R 1 is chloro and R 2 is glycinate ester
• R 3 is methyl;
• R 6 is a group of formula (IA) wherein R 6a is chloro, R 6b is methoxy and R 6c is hydrogen;
• one of R 7 or R 8 is hydrogen while the other is methyl;
• R 7 and R 8 are both methyl;
• R 9 is hydrogen and R 10 is C ⁇ -C 6 methyl ;
• R 11 is hydrogen;
• R 14 is hydrogen ;
• R 50 i s ( 0) ;
• the crude product was purified by column chromatography (Biotage-Si ⁇ 2 : gradient elution; 10%- 75% EtOAC: Hexanes) to provide Fmoc amine as a pale yellow solid (850mg, 37%).
• Product was contaminated with amino acid, which was removed by dissolving the product in EtOAc and stirring with IN HCl aq for several hours. Organics were dried and concentrated to give product (85:15 product: amino acid) .
• reaction mixture was then filtered through a small pad of celite and the filtrate was washed with 5 % NaHC ⁇ 3 , brine and dried over Na 2 S ⁇ 4 .
• the solvent was removed in vacuo and the residue was flash chromatographed on Si ⁇ 2 (15 % EtOAc/hexane) to give the title compound as a clear oil.
• IR (cm -1 ) 2961, 2933, 1742, 1715, 1497, 1366, 1249, 1170,
• the resulting slurry was treated with paraformaldehyde (6.00 g, 200 mmol) , in one portion, the ice bath was removed, and the suspension was stirred at room temperature for 90 min. The resulting cloudy yellow mixture was evaporated and the residue partitioned between ice water and TBME. The layers were separated and the aqueous layer was diluted with tetrahydrofuran (150 mL) , cooled to 0°C, and acidified with HCl ⁇ cone , 10 mL, 120 mmol) .
• the culture Mortierella isabellina ATCC 42613 as a frozen vegetative mycelia, was thawed and used to inoculate 10 mL of Difco YM Broth ( 42 g/L) with 0.3% Difco YM Agar in a 50 ml Erlenmeyer flask. The flask was incubated at 26°C for 48 hours on a shaker orbiting in a two inch circle at 250 rpm. Cells were harvested by centrifugation at 1,700 x g for 10 minutes. These cells were suspended in 100 mM potassium phosphate buffer pH 6.0 with 4 % glucose to obtain a 5 mL volume.
• TLC system consisted of Whatman silica gel 60 F-254 run in ethyl acetate / hexane (8/2, v/v) and detection with UV and 5% aqueous potassium permanganate.
• the ethyl acetate extract was concentrated to dryness by vacuum.
• Dried extracts for chiral analysis were reconstituted with methylene chloride.
• Derivatization of 2- methyl-3-hydroxy-valerolactone was conducted with trifluoroacetic anhydride.
• the diastereomeric and enantiomeric purity of the (S,S) isomer of 2-methyl-3- hydroxy-valerolactone in this example were determined to be 97% and 96% respectively by utilizing gas chromatography analysis. This analysis was conducted under the following conditions :
• a -70°C frozen vegetative mycelia preparation of Morti erella isabellina ATCC 42613 was thawed and used to inoculate the vegetative medium consisting of Difco YM Broth (42 g/L) with 0.2% YM agar.
• Difco YM Broth 42 g/L
• YM agar 0.2% YM agar.
• One ml of culture stock was used to inoculate 50 L of medium in a 250 mL Erlenmeyer flask. This inoculated medium was incubated at 26°C for 48 hours on a shaker orbiting in a two inch circle at 250 rpm. This growth (8 mL) was used to inoculate fermentation medium (200mL/flask) of the same composition in 1 liter Erlenmeyer flasks.
• the fermentation stage was incubated at 26°C for 48 hours on a shaker orbiting in a two inch circle at 250 rpm. Cells were harvested by centrifugation at 17,700x g for 15 minutes. The cells were placed in a 2 liter bioreactor and suspended in 150 mM citrate phosphate buffer pH 4.5 for a final volume of 2L. Dextrose was added for final concentration of 1% (w/v) . The hydride form of 2-methyl-3-keto- ⁇ -valerolactone was added for a final concentration at 23.4 mmoles/L. The pH was adjusted to pH 4.5 with 6N HCl and maintained during the bioconversion at pH 4.5 by addition of 8N NHOH and 3N HCl.
• the dissolved oxygen level was controlled at 30% by the addition of sterile air at a flow rate from 0.5 liters per minute (1pm) and an agitation rate from 500 to 950 rpm.
• the temperature was maintained at 26°C during the bioconversion.
• Progress of the bioconversion was detected on high pressure liquid chromatography (HPLC) by monitoring the disappearance of the substrate.
• HPLC high pressure liquid chromatography
• the HPLC system utilized was an isocratic system at 1.0 mL min "1 on a Waters RCM 8x10 RadialPak containing a NovaPak C18 column cartridge with a NovaPak C18 guard column with a detection at 254 nm.
• the solvent system is comprised of 25 mM ammonium phosphate buffer, adjusted to pH 3.5 with acetic acid/ acetonitrile (9/1, v/v) . Retention time of substrate is 5.5 minutes. The hydroxylactone could not be detected under these conditions. After 23 hours, the cells were removed by centrifugation at 30,100 x g. The supernatant then was saturated with sodium chloride ( ⁇ 20g/L) and then extracted 3 times with equal volume of acetonitrile. The aqueous layer was discarded. Acetonitrile layers were combined and concentrated to dryness by vacuum. Determination of the enantiomeric purity of the 2-methyl-3- hydroxy-valerolactone was conducted as follows. When the
• Carrier gas He at 1.5 mL/min
• Mortierella isabellxna ATCC 42613 100 L Scale Bioconversion of 2-methyl-3-keto- ⁇ -valerolactone, hydride or potassium salt form
• Inoculum for the tank fermentation and bioconversion were prepared in two stages.
• a -70°C frozen vegetative mycelia preparation of Morti erella isabellina ATCC 42613 was thawed and used to inoculate first stage vegetative medium consisting 2.6% dextrose, 1.6% yeast extract, and 0.1%
• Bacto Agar One ml of culture stock was used to inoculate 50 mL of medium in a 250 mL Erlenmeyer flask. This inoculated medium was incubated at 26°C for 48 hours on a shaker orbiting in a two inch circle at 250 rpm. This growth (lOmL) was used to inoculate a second stage vegetative medium (400mL) of the same composition in 2 liter Erlenmeyer flasks. This second stage was incubated at 26°C for 48 hours on a shaker orbiting in a two inch circle at 250 rpm.
• Two liters of the second stage were used to inoculate a 150 liter fermentor containing 100 liters of medium of same composition without the agar. 7 ⁇ mmonium hydroxide and sulfuric acid were used to maintain the pH between 5.0-6.0. The culture was allowed to grow for 24 hours in the fermentor maintaining the temperature at 26°C. The dissolved oxygen level was controlled at 30% by the addition of sterile air at a flow rate from 0.5 to 3.5 scfm and an agitation rate from 150 to 450 rpm.. At 24 hours the pH of the fermentation was adjusted to 4.5 with 30% sulfuric acid.
• the substrate 2-methyl-3-keto-d- valerolactone, hydride or potassium salt form was added for a final concentration of 23.4 mmoles/ L.
• the rate of the bioconversion was monitored on high pressure liquid chromatography (HPLC) .
• HPLC high pressure liquid chromatography
• the HPLC system utilized was an isocratic system at 1.0 ml min "1 on a Waters RCM 8x10 RadialPak containing a NovaPak C18 column cartridge with a NovaPak C18 guard column with a detection at 254 nm.
• the solvent system is comprised of 25 mM ammonium phosphate buffer, adjusted to pH 3.5 with acetic acid/ acetonitrile (95/5, v/v). Retention time of substrate is 5.5 minutes.
• Inoculum for the tank fermentation and bioconversion were prepared in three stages.
• a -70°C frozen vegetative mycelia preparation of Mortierella isabellina ATCC 42613 was thawed and used to inoculate first stage vegetative medium consisting of 2.6% dextrose, 1.6% yeast extract, and 0.1% Bacto Agar.
• One ml of culture stock was used to inoculate 50 mL of medium in a 250 mL Erlenmeyer flask. This inoculated medium was incubated at 26°C for 48 hours on a shaker orbiting in a two inch circle at 250 rpm.
• This growth was used to inoculate a second stage vegetative medium (400 mL) of the same composition in 2 liter Erlenmeyer flasks. This second stage was incubated at 26°C for 48 hours on a shaker orbiting in a two inch circle at 250 rpm. Two liters of the second stage were used to inoculate a 150 liter fermentor containing 100 liters of medium of same composition without the agar. Ammonium hydroxide and sulfuric acid were used to maintain the pH between 5.0-6.0. The culture was allowed to grow for 24 hours in the fermentor maintaining the temperature at 26°C.
• the dissolved oxygen level was maintained at 30% by first controlling the air flow rate between 0.5 to 3.5 scfm, then by controlling the agitation rate from 150 to 450 rpm using a PID controller. Twenty liters of this tank were used to inoculate a 1300 liter fermentor containing 1000 liters of medium consisting of 3.5% dextrose and 1.6% yeast extract. Ammonium hydroxide and sulfuric acid were used to maintain the pH between 5.0-6.0. The culture was allowed to grow until glucose depletion in the fermentor maintaining the temperature at 26°C. The dissolved oxygen level was controlled at 30% by controlling the air flow and agitation under a PID controller.
• the pH of the fermentation was adjusted to 4.5 with 30% sulfuric acid and the substrate 2-methyl-3-keto- ⁇ -valerolactone was added for a final concentration of 23.4 mmoles/ L.
• a glucose feed was started at the delivery rate of 200 grams of glucose/ hour.
• Three additional shots of substrate at the same concentration were added to the bioconversion tank for a total addition of 93.6 moles.
• the rate of the bioconversion was monitored on high pressure liquid chromatography (HPLC) .
• HPLC high pressure liquid chromatography
• the HPLC system utilized was an isocratic system at 1.0 ml min "1 on a Phenomenex Luna C18 (2), 5 m (250 x 4.6 mm) with a guard column (30 x 4.6 mm) of the same resin using a detection at 254 nm.
• the solvent system is comprised of 25 mM ammonium phosphate buffer, adjusted to pH 3.5 with acetic acid/ acetonitrile (95/5, v/v). Retention time of substrate is 5.5 minutes.
• the cells were removed by filtration of broth through a 6" single plate filter. Sodium chloride was added to the filtrate (20%, w/v) . This solution was extracted 3 times with equal volume of acetonitrile .
• reaction was then stirred for 1.25 hours at 50°C and monitored by TLC (1:1 EtOAc/heptane) .
• the reaction mixture was quenched by addition of solid sodium sulfate decahydrate (20. Og) in several portions, resulting in a milky white suspension which was mixed with hexane (600.0ml) and filtered.
• the filtering cake was washed with hexane (50.0ml) and the combined filtrate was washed twice with 750 L of 10% citric acid solution.
• the organic layer was then dried over MgS ⁇ 4, filtered and concentrated under reduced pressure to afford 51.0 g of an oil (27) with an E:Z ratio of 29:1 as determined by 1 H NMR.
• Example 7 To a solution of the product of Example 7 (29, 22.05g, 58.83mmoles) in 1,4-dioxane (118.0ml) at room temperature was added 2 N KOH (118.0ml, 235.3mmoles) . The solution was then heated at reflux for 2.5 hours when TLC (1:1 EtOAc/heptane) indicated no starting material was present. The reaction mixture was then allowed to warm up to temperature and quenched with 2N HCl (160.0ml, 308.3mmoles) .
• Acetone (lOmL) was added to a solution of the active ester of Example 9 (2.90 g, 6.35 mmol) in dichloromethane (20 mL) and the solution cooled to 0°C.
• the resulting solution was added to the reaction mixture and stirred at 0°C for 7h (tlc- 50% conversion).
• Example 18A To a solution of silyl ether of Example 17 (160mg, 0.272mmols) in dry DMF (3.5mL) was added sodium bicarbonate (228mg, 2.72mmols) followed by solid tetrabutylammonium fluoride-hydrate (TBAF) (358mg, 1.36mmols). The mixture was heated at 60°C for 17h and then further TBAF (358mg, 1.36mmols) and heated for 9h and finally a solution of IM TBAF in THF (360uL, 1.36mmols) added turning the reaction a brown colour. The mixture was heated for 20 mins and then the reaction quenched in water (lOOmL) and extracted with EtOAc (3x50mL) . Combined, dried (Na 2 S0 4 ) organics were concentrated in vacuo to give a brown oily gum (248mg) . Crude carboxylate salt was used in the next step without further purification.
• Example 18A To a solution of
• step (a) 122 mg, 0.141 mmol
• a 4.0 M solution of hydrogen chloride in 1,4-dioxane 178 ml, 0.707 mmol
• the clear, colorless reaction mixture was concentrated in vacuo to provide 120 mg (99%, corrected for 7 wt% dioxane) of the title compound as a white foam: 500
• the aqueous layer was extracted with 2 L of heptane.
• the combined organic layers were extracted successively with 0.2 N HCl solution (3 L) , deionized water (3 L) , and brine (3 L) .
• the organic layer was dried (sodium sulfate) and concentrated in vacuo to give 2984 g of compound (48) as an oil.
• EXAMPLE 25A Alternate Preparation of [5S- (2E,5R* , 6S* ,7E) ] -3-chloro-N- [5- [ [ (1 ,1-dimethylethyl) dimethylsilyl] oxy] -6-methyl-l-oxo-8- phenyl-2 , 7-octadienyl ] -O-methyl-2 , 2 , 2-trichloroethyl ester D-T ⁇ rosine (52) .
• the Boc-amine (57), as prepared by Example 30 (109 mg, 0.154 mmol), was dissolved in trifluoroacetic acid (5 mL, 5 mM) and stirred at room temperature for 2 h. The reaction was concentrated in vacuo and dried under high vacuum to give the trifluoroacetate salt of amine (57) as a light brown foam.
• the crude amine salt (max. 0.154 mmol) was dissolved in dry DMF (31 mL) and diisopropylethylamme (80 ⁇ L, 0.462 mmol) , followed by addition of pentafluorophenyl diphenyl- phosphinate (77 mg,0.2 mmol).

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