United States Patent [19]
`Sisti et al.
`
`[54] METHOD FOR DOCETAXEL SYNTHESIS
`
`[75] Inventors: Nicholas J. Sisti,Jeffersonvi1le;
`Charles S. Swindell, Merion, both of
`Pa.
`
`[73] Assignees: NaPro BioTherapeutics, Inc, Boulder,
`Colo.; Bryn Mawr College, Bryn
`Mawr, Pa.
`
`[21] Appl. No.: 616,467
`[22] Filed:
`Mar. 19, 1996
`
`Related US. Application Data
`
`[63] Continuation-impart of Ser. No. 609,083, Feb. 29, 1996.
`
`[51] Int. Cl.6 ................................................. .. C07D 305/14
`[52] US. Cl.
`549/510
`[58] Field of Search ............................................. .. 549/510
`
`[56]
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`6/1993 Denis et al. .......................... .. 549/510
`Re. 34,277
`4,814,470 3/1989 Colin et a1.
`549/510
`4,857,653
`8/1989 Colin et al.
`549/510
`4,924,011
`5/1990 Denis et al.
`549/510
`4,924,012
`5/1990 Colin et al. .
`.. 549/510
`4,960,790 10/1990 Stella et al. .
`.. 549/510
`5,015,744
`5/1991 Holton ...... ..
`549/510
`5,136,060
`8/1992 Holton ...... ..
`549/510
`5,399,726
`3/1995 Holtonetal. ..... ..
`5491510
`5,422,364
`6/1995 Nicolaou et al.
`.. 549/510
`5,466,834 11/1995 Holtonetal. ..... ..
`549/510
`5,475,011 12/1995 Ojima et al. .......... ..
`.. 549/510
`5,530,020
`6/1996 Gunawardana m1. ........... .1. 549/510
`
`FOREIGN PATENT DOCUMENTS
`
`0400971 5/1990 European Pat. O?'. .
`0528729A1 8/1992 European Pat. 01f. .
`2687150 2/1992 France .
`WO91/13066 9/1991 WIPO .
`
`OTHER PUBLICATIONS
`
`“Application of the Vicinal Oxyamination Reaction With
`Asymmetric Induction to the Hemisynthesis of Taxol and
`Analogues”, L. Mangatal et a1, Tetrahedron, vol. 45, No. 13,
`pp. 4177 to 4190, 1989.
`
`A
`US00568
`[11] Patent Number:
`[45] Date of Patent:
`
`5,688,977
`Nov. 18, 1997
`
`“Synthesis of Biologically Active Taxol Analogues with
`Modi?ed Phenylisoserine Side Chains”, Goerg et al., J. Med.
`Chem, 1992, 35, 4230-4237.
`“Biologically Active Taxol Analogues with Deleted A-Ring
`Side Chain Substituents and Variable C-2’ Con?gurations”,
`Swindell et al, Journal of Medicinal Chemistry, 1991, vol.
`34, No. 3, pp. 1176-1184.
`“New and E?icient Approaches to the Semisynthesis of
`Taxol and its C-l3 Side Chain Analogs by Means of
`B-Lactam Synthon Method”, Ojima et a1, Tetrahedron, vol.
`48, No. 34, pp. 6985-7012, 1992.
`'
`“Improved Protection and Esteri?cation of a Precursor of the
`Taxotere and Taxol Side Chains," Commercon et a1, Tetra
`hedron, vol. 33, No. 36, pp. 5185-5188, 1992.
`“Highly Sterocontrolled and E?icient Preparation of the
`Protected, Esten'?cation-Ready Docetaxel (Taxotere) Side
`Chain”, Kanazawa et al, J. Org. Chem, vol. 59, No. 6, pp.
`1238-1240, 1994.
`“Novel Biologically Active Taxol AnalogueszBaccatin 11I
`13-(N-(p-Chlorobenzoy1)-(2'R,3‘S)-3'-phenylisoserinate)
`and
`Baccatin
`111
`13-{N-Benzoyl-(2'R.
`3‘S)-3'-(p-chlorophenyl)isoserinate),” Georg et a1, Bioor
`ganic & Medicinal Chem. Letters, vol. 2, No. 4, pp.
`295-298, 1992.
`
`Primary Examiner—James H. Reamer
`Attorney Agent, or Firm—TiInothy J. Martin; Michael R.
`Henson
`
`[57]
`
`0
`
`ABSTRACT
`
`A method of producing docetaxel comprises the esteri?ca
`tion of C7, C10 di-CBZ IO-deacetyl baccatin Ill and an
`N-CBZ.C2'-protected 3-phenyl isoserine side chain wherein
`C2’ is protected by a hydrogenatable benyl-type protecting
`group. The C7, C10 carbobenzyloxy groups are then
`replaced with hydrogen and the carbobenzyloxy group at the
`C3‘ nitrogen site is replaced with t-butoxycarbonyl. Finally,
`the resulting compound is deprotected at C2’ by replacing
`the benzyl-type protecting group with hydrogen to produce
`dooetaxel. The esteri?cation preferably employs an excess,
`such as six equivalents, of the side chain for each equivalent
`of the C7, C10 di-CBZ IO-deac'etyl baccatin III. Benzy
`loxymethyl is the preferred protecting group at C2‘.
`
`24 Claims, No Drawings
`
`MYLAN PHARMS. INC. EXHIBIT 1029 PAGE 1
`
`

`
`5,688,977
`
`1
`METHOD FOR DOCETAXEL SYNTHESIS
`
`RELATED APPLICATION
`
`This application is a continuation-in-part of our earlier
`application, Ser. No. 08/609,083, ?led Feb. 29, 1996 and
`entitled, Intermediate For Docitaxel Synthesis and Produc
`tion Method Therefor.
`
`FIELD OF THE INVENTION
`
`This invention generally relates to the synthesis of doc
`etaxel from precursor compounds. More particularly,
`though. this invention concerns the synthesis of docetaxel
`using a suitably protected 10-deacetyl baccatin HI backbone
`which is esteri?ed with a suitably protected side chain acid
`to produce an intermediate that may thereafter be
`deprotected, acylated and further deprotected to produce
`docetaxel.
`
`BACKGROUND OF THE INVENTION
`
`Various taxane compounds are known to exhibit anti
`tumor activity. As a result of this activity, taxaues have
`received increasing attention in the scienti?c and medical
`cormnunity. Primary among these is a compound known as
`“paclitaxel” which is also referred to in the literature as
`“taxol”. Paclitaxel has been approved for the chemothera
`peutic treatment of several di?erent varieties of tumors, and
`the clinical trials indicate that paclitaxel promises a broad
`range of potent anti-leukemic and tumor-inhibiting activity.
`Paclitaxel is a naturally occurring taxane diterpenoid
`which is found in several species of the yew (genus Taxus,
`family Taxaceae). Unfortunately, the concentration of this
`compound is very low. The species of evergreen are also
`slow growing. Even though the bark of the yew trees
`typically exhibits the highest concentration of paclitaxel, the
`production of one kilogram of paclitaxel requires approxi
`mately 16,000 pounds of bark. Thus, the long term prospects
`for the availability of paclitaxel through isolation are dis
`couraging.
`While the presence of paclitaxel in the yew tree is in
`extremely low concentrations, there are a variety of other
`taxane compounds, such as baccatin III, cephalomannine,
`10-deacetylbaccatin III, etc., which are also able to be
`extracted from the yew bark. Some of these other taxane
`compounds are more readily extracted in higher yields.
`Indeed, a relatively high concentration of
`10-deacetylbaccatin lII can be extracted from the leaves of
`the yew as a renewable resource.
`Among the various taxane compounds which have been
`found to exhibit anti-tumor activity is the compound known
`as “docetaxel”. This compound is also sold under the
`trademark TAXUI'ERE® by Rhone-Poulenc Sante. Doc
`etaxel has the formula as follows:
`
`2
`(t-BOC) group at the C3‘ nitrogen position of the isoserine
`side chain and a free hydroxy group at the C10 position.
`Several possible syntheses of docetaxel and related com
`pounds have been reported in the Journal of Organic Chem
`istry: 1986, 51, 46; 1990, 55, 1957; 1991, 56, 1681; 1991,
`56, 6939; 1992, 57 4320; 1992, 57 6387; and 1993, 58, 255.
`In order to successfully synthesize docetaxel, convenient
`access to a chiral, non-racemic side chain and an abundant
`natural source of a usable baccatin [[l backbone as well as
`an elfective means of joining the two are necessary.
`However, the esteri?cation of the side chain to the baccatin
`llI backbone is di?icult because of the hindered C13
`hydroxyl in the baccatin III backbone which is located
`within the concave region of the hemispherical taxane
`skeleton. This di?iculty of synthesis is present both for the
`synthesis of docetaxel as well as for the synthesis of pacli
`taxel.
`One technique for the semi-synthesis of paclitaxel is
`found in co-pending patent application Ser. No. 08/483,081.
`In this application, paclitaxel is synthesized from C7 TES
`protected baccatin llI with N-carbamate protected C2‘
`hydroxyl-benzyl protected (2R,3S)-3-phenyl isoserine
`A-ring side chain with a hydrogenable benzyl-type protect
`ing group, such as a benzyloxymethyl (BOM) protecting
`group at the C2’ location for the side chain. Following the
`condensation of the C7 TES protected baccatin III and the
`side chain, the compound may be suitably deprotected,
`acylated, and further deprotected to yield paclitaxel.
`While, the existing techniques for synthesizing docetaxel
`certainly have merit, there is still a need for improved
`chemical processes which can produce this anti-cancer com
`pound. The present invention is directed to such a procedure
`utilizing the N-carbamate protected C2’ hydroxyl benzyl
`protected (2R,3S)-3-phenylisoserine A-ring side chain as
`described in my earlier co-pending application.
`
`10
`
`25
`
`30
`
`35
`
`SUMMARY OF THE INVENTION
`
`It is an object of the present invention to provide a new,
`useful and e?icient protocol for the attachment of a protected
`A-ring side chain to a protected baccatin III skeleton which
`may then be converted into docetaxel.
`It is still a further object of the present invention to
`provide a new and useful protocol for the semi-synthesis of
`docetaxel in an effort to produce a high yield of docetaxel in
`a cost e?icient manner.
`
`Yet another object of the present invention is to provide a
`method for the production of docetaxel which potentially
`can be scaled to commercial implementation.
`
`According to the present invention, then, a new and useful
`method for producing docetaxel is provided. According to
`the general method, C7, C10 di-CBZ IO-deacetyl baccatin
`5 III of the formula:
`
`Formula 1
`
`As may be seen in this formulation, docetaxel is similar-to
`paclitaxel except for the inclusion of the t-butoxycarbonyl
`
`is esteri?ed with an N—CBZ C2‘-protected
`3-pheny1isoserine side chain of the formula:
`
`65
`
`MYLAN PHARMS. INC. EXHIBIT 1029 PAGE 2
`
`

`
`5,688,977
`
`4
`the carbobenzyloxy group at the C3’ nitrogen site is subse
`quently replaced with t-BOC to produce the second inter
`mediate compound. The ?rst portion of this reaction is
`' accomplished by dissolving the ?rst intermediate compound
`in isopropanol/ethyl acetate in the presence of Pearlrnan’s
`5
`catalyst to form a ?rst mixture and then hydrogenating the
`?rst mixture for at least forty-eight hours. The next step in
`the replacement is accomplished by taking the amine up in
`tetrahydrofuran and then adding a tertiary amine base. The
`addition of the tertiary amine base is followed by the
`addition of di-tert-butyl dicarbonate in order to form C2‘
`OP1 docetaxel. This second mixture is stirred for about
`twenty-four hours and reduced in vacuum It is next redis
`solved in ethyl acetate, and washed with water and brine.
`This produces an organic phase which may be separated and
`chromotagraphed with ethyl acetatezhexane and/or recrys
`tallized to give the second intermediate compound in puri
`?ed form.
`The step of deprotecting the second intermediate com
`pound is then accomplished by dissolving the second inter
`mediate compound in isopropanol and ethyl acetate in the
`presence of Pearlman’s catalyst to form a second mixture.
`The second mixture is then hydrogenated for at least twenty
`four hours.
`These and other objects of the present invention will
`become more readily appreciated and understood from a
`consideration of the following detailed description of the
`exemplary embodiments.
`
`to form a ?rst intermediate compound of the formula:
`
`PhCIhOCO;
`
`0
`
`OCOgCHgPIl
`
`O
`
`Ph/\O)LNH O
`M :
`Ph
`:
`0_
`()pl
`
`"H
`
`I
`no 5
`PhCOg
`
`0
`
`E
`5
`OAO
`
`wherein P1 is a hydrogenable benzyl-type protecting group.
`Next, the C7 , C10 carbobenzyloxy in the ?rst intermediate
`compound is replaced with hydrogen and the carbobenzy
`loxy at the C3‘ nitrogen site is replaced with t-BOC to form
`a second intermediate compound of the formula:
`
`HO
`
`O
`
`wherein P1 is a hydrogenatable benzyl-type protecting
`group. Thereafter, the second intermediate compound is
`deprotected by replacing P1 with hydrogen thereby to pro
`duce docetaxel. It is preferred that the hydrogenatable ben
`zyl protecting group be selected from a group consisting of
`benzyloxymethyl and benzyl with benzyloxymethyl being
`the preferred protecting group.
`During esteri?cation, it is preferred that an excess
`N-CBZ C2'-protected 3-phenylisoserine be employed, and
`it is desirable that six equivalents of this side chain be used
`for each equivalent of a C7 , C10 di-CBZ IO-deacetyl
`baccatin HI during esteri?cation. This reaction is preferably
`performed by dissolving the two compounds in toluene to
`form a ?rst solution after which dimethylarnine pyridine
`(DMAP) and a dialkylcarbodiimide is added to the ?rst
`solution. The dialkylcarbodiimide is preferably in equal
`proportion to the amount of the side chain compound. This
`dialkylcarboiimide is selected from a group consisting of a
`I diisopropylcarbondiimide and dicyclohexylcarbondiirnide.
`This esteri?calion step is conducted at a ?rst temperature of
`about 60° to 80° C. for a ?rst interval of time, approximately
`one to ?ve hours after which the solution is allowed to cool
`to room temperature. Next, an equal volume of diethyl ether
`is added, and the resulting solution is cooled to a reduced
`second temperature of 0° C. or less for a second interval of
`about twenty-four hours.
`Moreover, it is preferred to dissolve the ?rst intermediate
`compound in a solution and elute this solution to purify the
`?rst intermediate compound before deprotecting at C7 and
`C10 and at the C3‘ nitrogen site. Here, also, the step of
`replacing the C7 and C10 carbobenzyloxy groups with
`hydrogen is conducted ?rst to produce an amine after which
`
`DETAILED DESCRIPTION OF THE
`EXEMPLARY EMBODIMENTS
`
`The present disclosure is broadly directed to a chemical
`process for the e?icient production of docetaxel as well as
`intermediates and precursors therefor. More specifically, the
`present invention discloses a new chemical compound in the
`form of C7, C10-di-CBZ IO-deacetylbaccatin III as a useful
`intermediate in the production of docetaxel. The C7 , C10
`di-CBZ lo-deacetylbaccatin III is esteri?ed with a
`3-phenylisoserine acid having a hydrogenable benzyl-type
`protecting group at C2‘ to the C13 hydroxyl of the baccatin
`III backbone. The general process described herein involves
`the production of the C7 , ClO-di-CBZ lO-deacetylbaccatin
`III backbone, the production of the suitably protected
`3-phenylisoserine acid having the hydrogenatable benzyl
`protecting group C2‘, the condensation of the two
`compounds, and the subsequent deprotection, acylation at
`the C3‘ nitrogen site to add the t-butoxycarbonyl group,
`followed by further deprotection to yield docetaxel.
`
`45
`
`A. Production of C7, C10 dicarbobenzyloxy 10
`deacetylbaccatin III
`
`C7, C10 di-CBZ lO-deacetylbaccatin III (Fonnula l) is
`produced by the following reaction:
`
`55
`
`HO
`
`Reaction I
`
`OH
`
`ll KW
`
`65
`
`HO
`PhC
`
`out" 2
`
`OAc
`
`MYLAN PHARMS. INC. EXHIBIT 1029 PAGE 3
`
`

`
`5
`—continued
`Reaction I
`
`PhCHzOCOz
`
`5,688,977
`
`6
`extracted with methylene chloride or ethyl acetate, as
`desired. The organic layer is separated, dried and reduced
`under vacuum to residue. The residue was then recrystal
`lized from ethyl acetate:hexane to result in N—CBZ
`3-phenylisoserine ethyl ester.
`This intermediate was next protected by the hydrogena
`table benzyl-type protecting group in several ways. For
`example. one route to the desired hydrogenatable benzyl
`type protected side chain is as follows:
`
`Reaction 111
`
`0
`
`OCO2CH2Ph 5
`
`10
`
`olllllll %
`
`Ph/\ 0/“\ gm
`Ph/\/ Cola‘ W
`I
`THF, —78 C.
`0H
`
`DOM-Cl
`
`'
`
`:
`
`20
`
`25
`
`0
`
`Ph/\ Oi 21H
`Ph/\/ COzEt
`630M
`
`Reaction ]]1
`
`Here, the hydrogeuable benzyl-type protecting group is
`benzyloxyrnethyl (BOM). To prepare this compound, the
`N—CBZ 3-phenylisoserine ethyl ester is dissolved in anhy
`drous THF under a nitrogen atmosphere and cooled to a
`reduced temperature such as -40° C. or —78° C., for
`example, in a dry ice/acetone bath followed by the dropwise
`addition of an alkyllithium agent such as n-butyl lithium,
`although it is desirable that the alkyllithium agent be a
`straight chain alkyl. In any event, the reaction is best done
`at a temperature no greater than 0° C. The resulting mixture
`is stirred for about ten minutes. Benzyloxymethyl chloride
`(BOM-Cl) is then added dropwise over an interval of about
`?ve minutes and the mixture stirred for approximately two
`to ?ve hours at the reduced temperature. Thereafter, the
`solution is warmed to 0° C. and quenched with water to
`eliminate excess n-butyl lithium. The resulting mixture is
`reduced under vacuum to residue, and this residue is there
`after taken up in ethyl acetate and washed with water and
`brine to remove unwanted salts. The organic layer may then
`be dried and reduced under vacuum and the residue recrys
`tallized from ethyl acetate:hexane or chromatographed with
`ethyl acetate:hexane to give the N—CBZ C2'-BOM
`3-phenylisoserine ethyl ester.
`Another route to production of N—CBZ C2'-OBOM
`3-phenylisoserine ethyl ester is accomplished by dissolving
`the compound N—CBZ (2R,3S)-3-phenylisoserine ethyl
`ester in anhydrous methylene chloride. Thereafter, a tertiary
`amine base such as diisopropylethylamine is added along
`with BOM-Cl and the mix is re?uxed for twenty-four hours.
`While this reaction route will produce N—CBZ 2'-BOM—3
`phenylisoserine ethyl ester, the reaction proceeds much
`more slowly than the route discussed above. However, it
`may be preferred because of higher yield. Here, the com
`pound is not puri?ed, but rather is carried on to subsequent
`processing steps in nude form.
`In either instance, the resulting N—CBZ C2'-OBOM
`(2R3S)-3-phenylisoserine ethyl ester, either in the puri?ed
`form of the ?rst route or in the crude form from the second
`
`Reaction I
`Here, IO-deacetylbaccatin III is dissolved in anhydrous
`THF (tetrahydrofuran) and is cooled under a nitrogen atmo
`sphere to a temperature of less than —20° C. but preferably
`—78° C. n-butyl lithium (1.6M in hexane) is added dropwise
`and the solution is stirred at the reduced temperature for
`approximately ?ve minutes. At least 1.5 equivalents of
`n-butyl lithium are needed to get signi?cant product yield,
`however 2 equivalents are preferable. Benzyl chloroforrnate
`is then added dropwise (again, at least 1.5 equivalents of the
`benzyl chloroformate are needed for signi?cant yield, but 2
`equivalents are preferred) and the mixture is stirred over a
`period of one hour during which time it is allowed to warm
`to a temperature of no more than 0° C. The mixture is then
`quenched with cold saturated ammonium chloride to elimi
`nate any excess n-butyl lithium and acetyl chloride, and the
`mixture is reduced under vacuum. The residue is taken up in
`ethyl acetate and washed once with water and then with
`brine to remove unwanted salts. The organic layer may then
`be dried and reduced under vacuum, and the residue recrys
`tallized or column chromotagraphed with ethyl acetate!
`hexane to yield C7, C10 di-CBZ IO-deacetylbaccatin HI as
`a white solid in greater than 80% overall yield.
`
`35
`
`B. Production of the 3-Phenylisoserine Side Chain
`The production of the (2R,3S) N—CBZ C2‘ O-protected
`3-phenylisoserine ethyl ester side chain, where the C2‘
`hydroxy group is protected by a hydrogenatable benzyl-type
`protecting grouptForrnula 2) can be accomplished from the
`starting compound (2R, 3S) 3-phenylisoserine ethyl ester
`according to the following two reactions. The ?rst reaction
`1s:
`
`Reaction II
`
`NH;
`CBZ-Cl
`:
`Ph/\/ Coz'Et NaZCO3
`:
`Et 0: H O
`OH
`2
`2
`
`0
`/\ J‘L
`Ph
`0
`gm
`'
`Ph/\/ COgBt
`on
`
`45
`
`50
`
`55
`
`Reaction II
`Here, (2R, 3S) 3-phenylisoserine ethyl ester was alterna
`tively dissolved in either equal parts diethyl ether2water or
`equal parts methyl t-butyl etherzwater and the solution was
`cooled to 0° C. The sodium carbonate was then added to the
`solution and benzylchloroformate was added dropwise over
`an interval of about ?ve minutes and the resulting mixture
`stirred at 0° C. for approximately one hour. After the one
`hour stirring, the solution was then poured into water and
`
`65
`
`MYLAN PHARMS. INC. EXHIBIT 1029 PAGE 4
`
`

`
`7
`route, may simply be converted to the corresponding acid by
`the reaction:
`
`5,688,977
`
`8
`-continued
`Reaction V
`
`Reaction IV
`
`0
`
`P1]/\ CAL Em
`:
`LiOH
`ph/\/C0J'Et '?rorrzrrzo >
`630M
`
`0
`
`Ph/\ O/IL 13H
`Ph/\/ C0211
`630M
`
`Reaction IV
`
`10
`
`20
`
`0
`
`Ph/\ Oi 2TH
`Ph/\/ COzEt
`bBn
`
`Reaction V
`Here, the CH2 protected (2R,3S)~3-phenylisoserine ethyl
`ester is dissolved in anhydrous THF under a nitrogen atmo
`sphere and cooled to a reduced temperature such as —40° C.
`or —78° C. for example in a dry ice/acetone bath followed by
`the dropwise addition of an alkyllithium agent such as
`n-butyl lithiurm although it is desirable that the alkyllithium
`agent be a straight chain alkyl. The resulting mixture is
`stirred for about ten minutes. Benzyl bromide (BnBr) is then
`added dropwise over an interval of about ?ve minutes and
`the mixture stirred for approximately two to ?ve hours at the
`reduced temperature. Thereafter, the solution is warmed to
`0° C. and quenched with water to destroy excess n-butyl
`lithium. The resulting mixture is reduced under vacuum to
`residue, and this residue is thereafter taken up in ethyl
`acetate and washed with water to remove any lithium
`bromide salt; it is then further washed with brine. The
`organic layer may the be dried and reduced under vacuum
`and the residue recrystallized from ethyl acetatezhexane or
`chromatographed with ethyl acetatezhexane to give
`N—CBZ 2‘-benzyl 3-phenylisoserine ethyl ester.
`Alternatively, the N——CBZ 2'-benzyl 3-phenylisosen'ne
`ethyl ester may be obtained according to the reaction:
`
`Reaction VI
`
`0
`l
`I,h/\ 0* gm
`:
`Ph/\/ CO’E‘
`on
`
`N DMF
`BnB_r 9
`
`25
`
`35
`
`Here, the protected ethyl ester is dissolved in ethanol]
`water (ratio 8:1). Lithium hydroxide (or other suitable alkali
`hydroxide) is added to the solution and the resulting mixture
`stirred for approximately three hours in order to saponify the
`compound. The mixture is then acidi?ed (1N hydrochloric
`acid) and extracted with ethyl acetate. The resulting organic
`layer is separated, dried and reduced under vacuum The
`residue acid is then isolated for use without further puri?
`cation. This produces the desired N—CBZ C2‘-OBOM
`(2R3S)-3-phenylisoserine.
`Where the N——CBZ C2'-OBOM 3-phenylisoserine ethyl
`ester is carried forward in the crude form and is converted
`into N—CBZ C2'-OBOM (2R,3S)-3-phenylisoserine, it is
`necessary for further puri?cation of the end product. This
`puri?cation is accomplished by dissolving the product in
`toluene followed by the dropwise addition of one equivalent
`of dicyclohexylamine and the resulting solution is stirred for
`one-half hour. This mixture is then concentrated in vacuo,
`and the resulting residue is recrystallized from ethyl
`acetatezhexane to give the dicyclohexylarnine salt of the
`N——CBZ C2'-0BOM (2R,3S)-3-phenylisoserine. The puri
`?ed N—CBZ C2'-OBOM (2R,3S)-3-phenylisoserine may
`then be liberated by dissolving this dicyclohexylamine salt
`in methylene chloride or another halogenated solvent fol
`lowed by washing the methylene chloride with several
`portions of 1N HCl. The organic layer is then washed with
`several portions of water to remove dicyclohexylamine
`hydrochloride. Next, it is washed with one portion of
`saturated brine and reduced in vacuo to give the desired acid.
`Benzyl itself is another example of a hydrogenatable
`benzyl-type protecting group that may be used instead of
`BOM. N—CBZ 2'-benzyl 3-phenylisoserine ethyl ester was
`produced as above with the substitution of benzyl bromide
`for BOM-Cl according to the reaction:
`
`Reaction V
`
`0
`
`Ph/\ OJL 2m
`2
`BnBr THF
`ph/\/ CO’E‘ _11-BuLi >
`on
`
`0
`
`Ph/\ OJL 12m
`Ph/\/ CD213t
`bBn
`
`Reaction VI
`
`Here, to a stirred solution of NaH in anhydrous DMF
`under nitrogen is added N—CBZ-3-phenylisoserine ethyl
`ester dissolved in DMF over ?ve minutes. The mixture is
`then stirred at 0° C. for one half hour. Then benzyl bromide
`(1.1 equivalents) is added dropwise over ?ve minutes and
`the reaction is stirred for two hours. The mixture is then
`quenched with water to destroy excess sodium hydride.
`Thereafter, either diethyl ether or methyl t-butyl ether is
`added. The organic layer is then washed with four por?ons
`of water to remove DMF and sodium bromide. Next, it is
`washed with brine and then dried and reduced under vacuum
`to produce N—CBZ C2’—benzyl 3-phenylisoserine ethyl
`ester may then be readily converted into N—CBZ C2'
`benzyl 3-phenylisoserine by the process of Reaction IV
`
`50
`
`55
`
`65
`
`MYLAN PHARMS. INC. EXHIBIT 1029 PAGE 5
`
`

`
`9
`above with the understanding that, in this case, benzyl is the
`C2' protecting group instead of benzyloxymethyl (BOM).
`
`10
`result in the desired C3’ NCBZ C2‘-OBOM-C7, C10-di
`CBZ IO-deacetyl baccatin III of the formula:
`
`5,688,977
`
`C. Esteri?cation of the Protected Baccatin III with
`the Side Chain '
`
`Esteri?cation of the C7 , C10 di-CBZ IO-deacetylbaccatin
`[[I with the N-CBZ C2'-protected 3-phenylisoserine side
`chain (where the C2‘ hydroxyl is protected by any hydro- ‘
`genatable benzyl-type group) is accomplished as follows.
`The preferred hydrogenatable benzyl group shown below is
`BOM (benzyloxymethyl).
`
`Reaction VII
`
`0
`
`P11/\ 01 NH
`5
`COZH
`Ph/\:/
`Pl\/ 0 \/ 0
`
`PhCHzOCOg
`
`0
`
`15
`
`.
`
`D. Deprotection and Treatment with Di-tert-Butyl
`dicarbonate and Deprotection to Form Docetaxel
`
`Diffs”
`
`2°
`
`The following reaction removes the CBZ protecting
`groups at C7 and C10 and the C3‘ nitrogen side chain site.
`(Again for clarity, BOM is used here as an example of a C2‘
`hydrogenatable benzyl-type protecting group):
`
`Reaction VIII
`
`PhCHZOCOZ
`
`O
`
`QCOzCHzPh
`
`| | H
`
`-
`-
`H0 5
`PhCO;
`
`O
`
`:
`2
`E
`OAe
`
`H:
`’s
`Pear
`catalyst
`
`HO
`
`O
`
`0
`P11/\ OJ‘L NH O
`M ~
`'
`t
`Ph
`:
`0
`m\/ 0V
`O
`
`Reaction VII
`
`45
`
`50
`
`55
`
`Reaction VIII
`
`Here, the C7, C10 di-CBZ lO-deacetylbaccatin III (1
`equivalent) of Formula 1 and the acid side chain (6
`equivalents) of Formula 2 are dissolved in toluene. To this
`mixture, 4-dimethylamino pyridine (DMAP) (one
`equivalent) and diisopropylcarbodiirnide (6 equivalents) are
`added, and the resulting mixture heated at about 60° to 80°
`C. for one to ?ve hours. It should also be noted, however,
`that other dialkylcarbodiirnides may be substituted for the
`diisopropylcarbodiimide, with one example being dicyclo-
`hexylcarbodiimide.
`The solution is then allowed to cool to room temperature,
`The coupled product of Formula 3 is dissolved in
`and next an equal volume of diethyl ether is added. The
`isopropanol/ethyl acetate to which Pearlrnan’s catalyst is
`resulting solution is cooled to 0° C. and held at this tem-
`added. The resulting mixture is hydrogenated at l atmo
`perature for twenty-four hours. This step crystallizes most of
`the urea impurity. After the twenty-four hour interval 60 sphere of hydrogen for at least twenty-four hours.
`elapses, the solution is ?ltered and the residue rinsed with
`Thereafter, the mixture is ?ltered through diatomaceous
`either ethyl ether or methyl t-butyl ether. The combined
`earth and reduced under vacuum to residue to result in the
`organics are then washed with hydrochloric acid (5%),
`amine shown which is used without further puri?cation.
`water, and ?nally brine. The organic phase is separated,
`dried, and reduced under vacuum. The resulting residue is 65
`then dissolved in ethyl aoetatezhexane and eluted over a
`silica gel plug. The eluent is then reduced under vacuum to
`
`Next, the t-BOC group can be attached at the N-C3' side
`chain site according to the following reaction:
`
`MYLAN PHARMS. INC. EXHIBIT 1029 PAGE 6
`
`

`
`11
`
`Reaction IX
`
`HO
`
`O
`
`5 688,977
`
`12
`-continued
`Reaction X
`
`NHgO
`
`Paco;
`
`Reaction X
`
`i
`M3110
`
`_
`Ph\/ 0 V b
`
`5
`E
`(3A6
`
`P1160:
`
`Reaction 1X
`
`Here, the puri?ed C2'-OBOM docetaxel is dissolved in
`isopropanol and Pearlmann’s catalyst is added. The mixture
`is then hydrogenated at either 1 Atm of hydrogen or at 40 psi
`hydrogen for at least twenty-four hours. The mixture is then
`?ltered through diatomaceous earth and reduced under
`vacuum to get crude docetaxel. Where the C2’ side chain site
`has been protected with O-Bn, conversion to crude doc
`etaxel may be accomplished according to the literature
`procedure (Kanazawa, A., Denis J. N. and Green, A. E. J.
`Org. Chem. 1994, 59, 1238).
`
`25
`
`30
`
`Accordingly, the present invention has been described
`with some degree of particularity directed to the exemplary
`_
`_
`_
`embodiment of the present invention. It should be
`Ben’ th? amme ‘5 takcn up In anhydrous THF an? a
`appreciated, though. that the present invention is de?ned by
`tcmary amme base
`be addefi to 30.36161?“ the macho“,
`the following claims construed in light of the prior art so that
`f°!1°Wed_ by file addmou of dl'tcl't'butyldlcarbonate- The
`mm 15 surfed for Fwenty'fQ‘" how's’ and then reduceid 35 modi?cations or changes may be made to the exemplary
`under vacuum and Tedlssolvsd 1" ethyl acetate- The Orgamc
`embodiment of the present invention without departing from
`phase was then washed with water and brine. The resulting
`the inven?vc conccpts contained harem
`organic phase was then separated, dried, and reduced under
`vacuum to get crude C2'-OBOM docetaxel. It is necessary at
`this “38¢ of Processing to Purify the crude C2"OBOM 4o
`docetaxel. This can be accomplished by column chromatog-
`raphy and/or recrystalization from ethyl/acetatezhexane.
`Preferably both column chromatography with ethyl!
`acetatezhexane to produce an eluent that is reduced in
`.
`_
`_
`vacuum to form a residue followed by recrystahzation of the 45
`residue from ethyl acetatezhexane is employed to yield
`C2'-OBOM docetaxel in a substantially pure form.
`
`W6 claim;
`LAmethod of producing docetaxel. comprising the steps
`of;
`
`t- m f
`1b
`C1 di_CBZ1 _de
`t -f -
`(a) cs
`Hg C7’ 0
`0 “my accam O
`the formula
`
`phc?zocoz
`
`0
`
`ocozc?zyh
`
`The benzyloxymethyl protecting group is removed as 50
`follows:
`
`ReactionX
`
`0
`
`55
`
`65
`
`HO
`
`:
`HO 5
`PhCOz
`
`g
`E
`CA4:
`
`.- H
`
`0
`
`with an N—-CBZ C2'-protected 3-phenyl isoserine side
`chain of the formula
`
`'opl
`
`MYLAN PHARMS. INC. EXHIBIT 1029 PAGE 7
`
`

`
`5,688,977
`
`13
`to form a ?rst intermediate compound of the formula
`
`PhCHzOCOZ
`
`O
`
`,
`
`OCOzCHzPh
`
`Ph/\ 0
`
`O
`|
`
`2TH O
`lib/Ki 0\
`2)?
`1
`
`I I H
`o
`
`=
`H0 5
`P1100;
`
`g
`a
`OAc
`
`wherein P1 is a hydrogenatable benzyl-type protecting
`group;
`(b) substituting hydrogen for the C7, C10 carbobenzyloxy
`groups and substituting t-butoxycarbonyl for the car
`bobenzyloxy group at the C3’ nitrogen site to form a
`second intermediate compound of the formula:
`
`HO
`
`O
`
`6P1
`
`Paco
`2
`
`wherein P1 is a hydrogenatable benzyl-type protecting
`group; and
`(c) deprotecting the second intermediate compound by
`substituting hydrogen for P1 to produce docetaxel.
`2. The method of producing docetaxel according to claim
`1 wherein P1 is selected from a group consisting of benzy
`loxymethyl and benzyl.
`3. The method of producing docetaxel according to claim
`1 wherein six equivalents of the N—CBZ C2'-protected
`3-phenylisoserine side chain are used for each equivalent of
`C7, C10 di-CBZ IO-deacetyl baccatin ]]1 during the esteri
`fying step.
`4. The method of producing docetaxel according to claim
`1 wherein the N—CBZ C2'-protected 3-phenylisoserine side
`chain and the C7, C10 di-CBZ IO-deacetyl baccatin I[[ are
`dissolved in toluene to form a ?rst solution during the
`esterifying step after which DMAP and a dialkylcarbodiim
`ide is added to the ?rst solution to produce a second solution
`containing the ?rst intermediate compound.
`5. The method of producing docetaxel according to claim
`4 wherein the dialkylcarbodiirnide is in equal proportion to
`the N—CBZ C2'-protected 3-phenylisoserine.
`6. The method of producing docetaxel according to claim
`4 wherein the dialkylcarbodiimide is selected from a group
`consisting of diisopropylcarbodiimide and dicyclohexylcar
`bodiimide.
`7. The method of producing docetaxel according to claim
`4 wherein the esterifying step is conducted at a ?rst tem
`perature of 60°-80° C. for a ?rst interval.
`8. The method of producing docetaxel according to claim
`7 wherein diethyl ether is added to the second solution to
`produce a third solution that is then cooled to a reduced
`temperature su?icient to crystalline urea therefrom.
`9. The method of producing docetaxel according to claim
`1 wherein the esterifying step is conducted at a ?rst tem
`perature of 60°—80° C. from a ?rst interval.
`10. The method of producing docetaxel according to
`claim 1 wherein the ?rst intermediate compound is dissolved
`
`45
`
`55
`
`65
`
`15
`
`25
`
`30
`
`35
`
`14
`in a solution and is column chromatographed to purify the
`?rst intermediate compound prior to replacing the C7, C10
`and N-C3' carbobenzyloxy groups to form the