.
`
`United States Patent [19]
`Taylor
`
`US005344932A
`
`[11] Patent Number:
`[45] Date of Patent:
`
`5,344,932
`Sep. 6, 1994
`
`[54] N-(PYRROLO(2,3-D)PYR?VIIDIN-3-
`
`[56]
`
`References Cited
`
`-
`_
`[75] Invent“ Edward C‘ Taylor’ Pnnceton’ NJ‘
`
`[73] Assignee: Trustees of Princeton University,
`Princeton, N_J_
`
`[21] APPl' No‘: 674,541
`.
`[22] F11ed:
`
`Mar. 22, 1991
`
`[63]
`
`Related U's' Apphcanon Data
`Continuation of Ser. No. 448,742, Dec. 11, 1989, aban-
`doned, and s61‘. N0. 479,655, Feb. 8, 1990, abandoned.
`
`[51] Int. (:1.5 ................ .. C07D 487/04; A61K 31/505
`[52] US. Cl. .................................................. .. 544/280
`[58] Field of Search ....................... .. 544/280; 514/258
`
`4,889,859 12/1989 Taylor et a1. ..................... .. 514/258
`4,996,206 2/1991 Taylor et a1. ..... ..
`514/258
`4,997,838 3/1991 Akimoto et a1. .................. .. 514/258
`
`FOREIGN PATENT DOCUMENTS
`334636 9/1989 European Pat. Off. .
`Primary ExamineF-Emily Bernhard
`Attorney, Agent, or Firm—-Mathews, Woodbridge &
`Collins
`
`ABSTRACT
`[57]
`N-(Acyl)glutamic acid derivatives in which the acyl
`group is substituted with 4-hydroxypyrrolo[2,3-d]
`pyfimidin-B-yl grgup are antineoplastic agents_ A typi
`cal embodiment is N-[4-(2-{4-hydroxy-6-aminopyrrolo
`[2,3-dlpyrimidin-3-y1}ethy1)benz0y1]-L-glutamic acid.
`
`7 Claims, No Drawings
`
`Wockhardt Exhibit 1003 - 1
`
`

`
`1
`
`N-(PYRROLO(2,3-D)PYRIMIDIN-3-YLACYL)
`GLUTAMIC ACID DERIVATIVES
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`This is a continuation of Ser. No. 07/448,742 ?led
`Dec. 11, 1989 and Ser. No. 07/479,655 filed Feb. 8, 1990
`both now abandoned.
`The present invention pertains to glutamic acid deriv
`atives having the formula:
`
`5,344,932
`2
`processes for the preparation of these compounds and
`their salts, to chemical intermediates useful in prepara
`tion of these compounds, to a method of combatting
`neoplastic growth in a mammal, and to pharmaceutical
`compositions containing these compounds or their salts.
`A ?rst group of useful chemical intermediates, which
`can be converted directly to the desired ?nal com
`pounds of Formula I through removal of protecting
`groups, are compounds of the formula:
`
`5
`
`10
`
`15
`
`20
`
`25
`
`35
`
`R2 is hydrogen or a pharmaceutically acceptable
`cation:
`R3 is 1,4-phenylene or 1,3-phenylene unsubstituted or
`substituted with chloro, ?uoro, methyl, methoxy,
`or tri?uoromethyl; thienediyl or furanediyl each
`unsubstituted or substituted with chloro, ?uoro,
`methyl, methoxy, or tri?uoromethyl; cyclohex
`anediyl; or alkanediyl;
`R4 is hydrogen, methyl, or hydroxymethyl;
`R5 is hydrogen, alkyl of l to 6 carbon atoms, or
`amino; and
`the con?guration about the carbon atom designated * is
`S.
`The compounds of this invention are herein described
`as embodying the pyrrolo[2,3-d]pyrimidine heterocy
`clic ring system which ring system is numbered as fol
`lows:
`
`4
`C
`5 4 \C
`II
`
`c3
`II
`
`45
`
`It will be appreciated that the pyrrolo[2,3-d]pyrimi
`dines as depicted by Formula I are the tautomeric
`equivalent of the corresponding 5-H-6-oxo or 5-H-6
`imino structures. Unless otherwise indicated, for sim
`plicity’s sake the compounds are depicted herein and
`named using the 6-hydroxy and 6-amino convention, it
`being understood the 5-H-6-oxo and 5-H-6-imino struc
`tures are fully equivalent.
`The compounds of Formula I have an inhibitory
`effect on one or more enzymes which utilize folic acid,
`and in particular metabolic derivatives of folic acid, as a
`substrate. The compounds appear to be particularly
`active as inhibitors of thymidylate synthetase, which
`catalyses the methylation of deoxyuridylic acid to deox
`ythymidylic acid utilizing N5,Nlo-methylidenetetrahy
`drofolate as a coenzyme. The compounds thus can be
`used, alone or in combination, to inhibit the growth of
`those neoplasms which otherwise depend upon the
`65
`inhibited enzyme.
`The invention also pertains to the pharmaceutically
`acceptable salts of the compounds of Formula I, to
`
`55
`
`R3 is as de?ned above;
`R2’ is hydrogen or a carboxy protecting group;
`R4’ is hydrogen, methyl, hydroxymethyl, or hydroxy
`methyl carrying a hydroxy protecting group;
`R5’ is hydrogen, alkyl, amino, or amino carrying a
`protecting group; and
`R6 is hydrogen or alkanoyoxy;
`at least one of R2',R4', and R5’ being a carboxy pro
`tecting group, a hydroxy protecting group, or an
`amino protecting group, respectively.
`The compounds of Formula I can be employed in the
`form of the free dicarboxylic acid, in which case both
`R2 groups are hydrogen. Alternatively, the compounds
`often can be employed advantageously in the form of a
`pharmaceutically acceptable salt, in which case one or
`both R2 groups are a pharmaceutically acceptable cat
`ion. Such salt forms, including hydrates thereof, are
`often crystalline and advantageous for forming solu
`tions or formulating pharmaceutical compositions.
`Pharmaceutically acceptable salts with bases include
`those formed from the alkali metals, alkaline earth met
`als, non-toxic metals, ammonium, and mono-, di- and
`trisubstituted amines, such as for example the sodium,
`potassium, lithium, calcium, magnesium, aluminum,
`zinc, ammonium, trimethylammonium, triethanolam
`monium, pyridinium, and substituted pyridinium salts.
`The mono and disodium salts, particularly the disodium
`salt, are advantageous.
`The group R3 is a divalent group having at least two
`carbon atoms between the carbon atoms carrying the
`free valence bonds. R3 for example can be a 1,4-pheny
`lene or 1,3-phenylene ring which is unsubstituted or
`optionally substituted with chloro, ?uoro, methyl, me
`thoxy, or tri?uoromethyl.
`Alternatively, R3 can be a thienediyl or furanediyl
`group, that is, a thiophene or furane ring from which
`two hydrogen atoms have been removed from two ring
`carbon atoms, as for example the thiene-2,5-diyl, thiene
`3,5-diyl, thiene-2,4-diyl, and thiene-3,4-diyl ring systems
`and the the furane-2,5-diyl, furane-3,5-diyl, furane-2,4
`diyl, and furane-3,4»diyl ring systems, which ring sys
`tems can be unsubstituted or substituted with chloro,
`?uoro, methyl, methoxy, or tri?uoromethyl. It will be
`appreciated that whereas in the abstract the thiene-3,5
`diyl system is the equivalent of the thiene-2,4-diyl sys
`tem, the two terms are utilized herein to denote the two
`isomeric forms resulting from the orientation of the
`thiophene ring within the remainder of the molecule:
`
`Wockhardt Exhibit 1003 - 2
`
`

`
`5,344,932
`3
`e.g. the structure in which the depicted carboxy group
`adjacent to R3 is in the 2-position of the thiophene ring
`and that in which the depicted carboxy group adjacent
`to R3 is in the 3-position of the thiophene ring. The same
`conventionas apply to the furane ring.
`Alternatively, R3 can be a cyclohexanediyl group,
`namely a divalent cycloalkane group of 6 carbon atoms
`such as cyclohexane-l,3-diyl and cyclohexane-1,4-diy1.
`Alternatively, R3 can be a alkanediyl, namely a
`straight or branched divalent aliphatic group of from 2
`to 4 carbon atoms such as ethano, trimethylene, tetra
`methylene, propane-1,2-diyl, propane-2,3-diyl, butane
`2,3-diyl, butane-1,3-diy1, and butane-2,4-diyl. It again
`will be appreciated that whereas in the abstract pro
`pane-1,2-diyl is the equivalent of propane-2,3-diyl, and
`butane-1,3-diyl the equivalent of butane-2,4-diyl, the
`two terms are utilized herein to denote the two isomeric
`forms resulting from the orientation of an unsymmetri
`cal alkanediyl chain with respect to the remainder of the
`molecule.
`The protecting groups designated by R2’, R4’ and R5’
`and utilized herein denote groups which generally are
`not found in the ?nal therapeutic compounds but which
`are intentionally introduced at a stage of the synthesis in
`order to protect groups which otherwise might react in
`the course of chemical manipulations, thereafter being
`removed at a later stage of the synthesis. Since com—
`pounds bearing such protecting groups thus are of im
`portance primarily as chemical intermediates (although
`some derivatives also exhibit biological activity), their
`precise structure is not critical. Numerous reactions for
`the formation and removal of such protecting groups
`are described in a number of standard works including,
`for example, “Protective Groups in Organic Chemis
`try”, Plenum Press, London and New York, 1973;
`Greene, Th. W. “Protective Groups in Organic Synthe
`sis”, Wiley, New York, 1981; “The Peptides”, Vol. I,
`Schriider and Lubke, Academic Press, London and
`New York, 1965; “Methoden der organischen Chemie”,
`Houben-Weyl, 4th Edition, Vol.15/I, Georg Thieme
`Verlag, Stuttgart 1974, the disclosures of which are
`incorporated herein by reference.
`With respect to R2’, a carboxy group can be protected
`as an ester group which is selectively removable under
`sufficiently mild conditions not to disrupt the desired
`45
`structure of the molecule, especially a lower alkyl ester
`of l to 12 carbon atoms such as methyl or ethyl and
`particularly one which is branched at the 1-position
`such as t.-butyl; and such lower alkyl ester substituted in
`the 1- or 2-position with (i) lower alkoxy, such as for
`example, methoxymethyl, l-methoxyethyl, and ethox
`ymethyl, (ii) lower alkylthio, such as for example me
`thylthiomethyl and l-ethylthioethyl; (iii) halogen, such
`as 2,2,2-trichloroethyl, 2-bromoethyl, and Z-iodoethox
`ycarbonyl; (iv) one or two phenyl groups each of which
`can be unsubstituted or mono-, di- or tri-substituted
`with, for example lower alkyl such as tert.-butyl, lower
`alkoxy such as methoxy, hydroxy, halo such as chloro,
`and nitro, such as for example, benzyl, 4-nitrobenzyl,
`diphenylmethyl, di-(4-methoxyphenyl)methyl; or (v)
`aroyl, such as phenacyl. A carboxy group also can be
`protected in the form of an organic silyl group such as
`trimethylsilylethyl or tri-lower alkylsilyl, as for exam
`ple trimethylsilyloxycarbonyl.
`With respect to R4’, a hydroxy group can be pro
`tected through the formation of acetals and ketals, as for
`example through formation of the tetrahydropyr-2
`yloxy (THP) derivative.
`
`20
`
`25
`
`30
`
`35
`
`50
`
`55
`
`65
`
`4
`With respect to R5’, an amino group can be protected
`as an amide utilizing an acyl group which is selectively
`removable under mild conditions, especially formyl, a
`lower alkanoyl group which is branched a to the car
`bonyl group, particularly tertiary alkanoyl such as pi
`valoyl, or a lower alkanoyl group which is substituted
`in the position a to the carbonyl group, as for example
`tri?uoroacetyl.
`Preferred compounds of Formula I are those wherein
`R5 is amino or hydrogen. Within this class, Rl prefera
`bly is hydroxy, R3 is 1,4-phenylene, and R4 is hydrogen
`or hydroxymethyl. Also preferred within this class are
`the compounds in which R1 is hydroxy, R3 is thienediyl,
`and R4 is hydrogen or hydroxymethyl.
`The compounds of this invention can be prepared
`according to a ?rst process through catalytic hydroge
`nation of a compound of the formula:
`
`10
`
`15
`
`III
`
`ll
`c
`
`21
`
`l
`H
`
`in which:
`Z1 is hydrogen, or Z1 taken together with R4' is a
`carbon-carbon bond;
`R2’ is hydrogen or a carboxy protecting group;
`R3 and R6 are as de?ned above;
`R4’, when taken independently of Z1, is hydrogen,
`methyl, hydroxymethyl, or hydroxymethyl substi
`tuted with a hydroxy protecting group; and
`R5’ is hydrogen, alkyl of l to 6 carbon atoms, amino,
`or an amino protecting group.
`Suitable hydrogenation catalysts include noble metals
`and noble metal oxides such as palladium or platinum
`oxide, rhodium oxide, and the foregoing on a support
`such as carbon or calcium oxide.
`When in Formula III, Z1 taken together with R4’ is a
`carbon-carbon bond, that is, when a triple bond is pres
`ent between the two carbon atoms to which Z1 and R4’
`are bound, R4’ in the hydrogenation product will be
`hydrogen. Absent any chirality in R3 (or any protecting
`group encompassed by R2’, R4’ and/ or R5'), the hydro
`genation product will be a single enantiomer having the
`S-con?guration about the carbon atom designated *.
`This also is true when Z1 and R4’ are each hydrogen,
`that is, when a double bond is present between the two
`carbon atoms to which Z1 and R4’ are bound.
`When, on the other hand, R4’ is other than hydrogen,
`a mixture of the R,S and 8,3 diastereomers is obtained.
`The diastereomeric mixture can be used therapeutically
`as such (after removal of the protecting groups) or can
`be separated mechanically as by chromatography. Al
`ternatively, the individual diastereomers can be sepa
`rated chemically by forming salts with a chiral acid,
`such as the individual enantiomers of lO-camphorsul
`fonic acid, campboric acid, alphabromocamphoric acid,
`methoxyacetic acid, tartaric acid, diacetyltartaric acid,
`malic acid, pyrrolidone-S-carboxylic acid, and the like,
`and then freeing one or both of the individual diastere
`omeric bases, optionally repeating the process, so as
`obtain either or both substantially free of the other; i.e.,
`in a form having an optical purity of >95%.
`
`Wockhardt Exhibit 1003 - 3
`
`

`
`5,344,932
`5
`The protecting groups encompassed by R2’, R4’, R5’,
`and/or R6 can be removed following hydrogenation
`through acidic or basic hydrolysis, as for example with
`hydrogen chloride to cleave an R4’ protecting group or
`with sodium hydroxide to cleave RT or R5’ protecting
`groups, thereby yielding the compounds of Formula I.
`Methods of removing the various protective groups are
`described in the standard references noted above and
`incorporated herein by reference.
`Compounds of Formula III can be prepared utilizing
`procedures analogous to those described in US. Pat.
`No. 4,818,819, utilizing however the corresponding
`halogenated pyrrolo[2,3-d]pyrimidine. Thus a pyr
`rolo[2,3-d]pyrimidine of the formula:
`
`15
`
`0
`II
`
`c-x
`ll
`cu
`
`H
`
`20
`
`I
`H
`
`The product of Formula VII then can be hydroge
`nated, hydrolysed to remove the R2’ and R6 protecting
`groups, and, optionally with intermediate protection of
`any amino group encompassed by R5’, and coupled with
`a protected glutamic acid derivative in the manner de
`scribed in US Pat. No. 4,684,653 using conventional
`condensation techniques for forming peptide bonds
`such as DCC or diphenylchlorophosphonate, following
`which the protecting groups are removed.
`In a further variant, compounds of Formula III can
`be prepared utilizing the procedures described in US.
`Pat. No. 4,818,819. Thus a compound of the formula:
`
`in which X is bromo or iodo, R5’, and R6 are as herein
`de?ned, is allowed to react with an unsaturated com
`pound of the formula:
`
`25
`
`6 R\ O
`
`C
`II
`
`21
`
`VIII
`
`30
`
`l
`H
`
`in which 2‘, R4’, R5’, and R6 are as herein de?ned, is
`allowed to react with a compound of the formula:
`
`in which Z1, R3 and R4’ are as herein de?ned, and R7
`35
`
`X—-R3--R7
`
`is
`
`1x
`
`in which X, R3, and R7 are as herein de?ned, in the
`presence of a palladium/trisubstituted phosphine cata
`lyst of the type described in US. Pat. No. 4,818,819.
`This variant of the process is particularly suitable for,
`but is not limited to, preparation of those compounds in
`which R4 is hydroxymethyl, in which case R4’ in For
`mula VI is a protected hydroxymethyl group, as for
`example tetrahydropyran-2-yloxymethyl.
`Compounds of Formula VIII also can be obtained by
`the methods of US. Pat. No. 4,818,819 by treating a
`compound of Formula IV with an unsaturated com
`pound of the formula:
`
`45
`
`in which R2’ is as herein defined, in the presence of a
`palladium/trisubstituted phosphine catalyst of the type
`described in US. Pat. No. 4,818,819, the disclosure of
`which is incorporated herein by reference.
`When R7 is —CON'HCH(COOR2')CH2CH2COOR2',
`the product of this coupling reaction is hydrogenated,
`and any protecting group removed, as described above.
`Alternatively, a compound of Formula IV is allowed
`to react with a compound of the formula:
`
`in which Z1, R2’, R3, and R4’ are as herein de?ned in
`the presence of a palladium/trisubstituted phosphine
`catalyst of the type described in US. Pat. No. 4,818,819
`to yield an intermediate of the formula:
`
`65
`
`in which R4" is methyl, a protected hydroxymethyl,
`or a trisubstituted silyl group in the presence of a pal
`ladium/trisubstituted phosphine catalyst of the type
`discussed above. This procedure is particularly suitable
`for, but is not limited to, preparation of those com
`pounds in which R4 is hydroxymethyl.
`Although not always the case, the compounds of
`Formula IV in which R6 is hydrogen can tend to be
`somewhat insoluble in solvents suitable for the reaction
`described in US. Pat. No. 4,818,819. In such instances,
`the compounds of Formula IV in which R6 is hydrogen
`can be ?rst treated with with sodium hydride and a
`suitable alkyl alkanoate (such as chloromethyl pivalate)
`to introduce an alkanoyloxy group in the 5-position and
`increase solubility.
`A useful subclass of compounds useful both as inter
`mediates and for their effect on enzymes are derivatives
`
`Wockhardt Exhibit 1003 - 4
`
`

`
`5,344,932
`8
`7
`3-[2-(thien-3-yl)ethyl]—4-hydroxypyrrolo[2,3
`dine,
`of Formula XI and XII lacking the glutamic acid side
`d]pyrimidine,
`3-[2-(thien-3-yl)ethyl]-4-hydroxy-6
`chain:
`methylpyrrolo[2,3-d]pyrimidine, 3-[2-(fur-2-yl)ethyl]-4
`hydroxy-6-aminopyrrolo[2,3-d]pyrimidine, 3-[2-(fur-2
`yl)ethy1]-4-hydroxypyrrolo[2,3-d]pyrimidine, 3-[2-(fur
`2-yl)ethyl]—4-hydroxy-6-methylpyrrolo[2,3-d]pyrimi
`dine,
`3-[2-(fur-3-y1)ethyl]—4-hydroxy-6-aminopyr
`rolo[2,3-d]pyrimidine,
`3-[2-(fur-3-yl)ethyl]—4-hydrox
`ypyrrolo[2,3-d]pyrimidine, and 3-[2-(fur—3-yl)ethyl]-4
`hydroxy-6-methylpyrrolo[2,3-d]pyrimidine.
`As discussed above, the compounds of this invention
`can be prepared utilizing the palladium catalyzed cou
`pling of various unsaturated compounds described in
`US. Pat. No. 4,818,819 and the glutamic acid coupling
`reactions described in US. Pat. No. 4,684,653, substitut
`ing the appropriate pyrrolo[2,3-d]pyrimidine for the
`pyrido[2,3-d]pyrimidine therein disclosed. The pyr
`rolo[2,3-d]pyrimidine intermediates of Formula IV
`above can be obtained by treating a compound of the
`formula:
`
`15
`
`R1 is —OH or —-NH;;
`R4 is hydrogen, methyl, or hydroxymethyl;
`R5 is hydrogen, alkyl of 1 to 6 carbon atoms, or
`amino;
`R8 is hydrogen, chloro, fluoro, methyl, methoxy,
`tri?uoromethyl, or carboxy; and
`Y is ——S— or —-O—; and
`the pharmaceutically acceptable salts thereof.
`30
`Compounds of Formulas XI and XII are obtained by
`allowing a compound of Formula VII to react with a
`compound of the formula:
`
`25
`
`R8
`
`01'
`
`Y
`
`in which X, Y, and R8 are as herein de?ned, by the
`methods of US. Pat. No. 4,818,819, namely in the pres
`ence of a palladium/tn'substituted phosphine catalyst,
`with the resulting coupled product being hydrogenated
`and hydrolysed to remove the R2' protecting group.
`Typical compounds of Formulas XI and XII are 3-(2
`phenylethyl)-4-hydroxy-é-aminopyrrolo[2,3-d]pyrimi
`dine,
`3-[2-(3-?uorophenyl)ethyl]—4-hydroxy-6
`aminopyrrolo[2,3-d]pyrimidine, 3-[2-(4-fluorophenyl)e
`thyl]-4-hydroxy-6-aminopyrrolo[2,3-d]pyrimidine, 3-[2
`(4-carboxyphenyl)ethylH-hydroxy-6-aminopyr
`rolo[2,3-d]-pyrimidine, 3-[2-(4-methoxyphenyl)ethyl]—4
`hydroxy-6-aminopyrrolo[2,3-d]pyrimidine,
`3-[2-(4
`methylphenyl)ethyl]-4-hydroxy-6-aminopyrrolo[2,3
`d]pyrimidine, 3-(Z-phenylethyl)-4-hydroxypyrrolo[2,3
`d]pyrimidine,
`3-(2—phenylethyl)—4-hydroxy-6-methyl
`pyrrolo[2,3-d]pyrimidine,
`3-(2-phenyl-3-hydroxy
`propyl)-4-hydroxy-6-aminopyrrolo[2,3-d]pyrimidine,
`3-[2-(thien-2-yl)ethy1]-4-hydroxy-6-aminopyrrolo[2,3
`d]pyrimidine,
`3-[2-(thien-2-yl)ethyl]—4-hydroxypyr
`rolo[2,3-d]pyrimidine,
`3-[2
`-(thien-2-yl)ethyl]-4
`hydroxy-6-methylpyrrolo[2,3-d]-pyrimidine, 3-[2-(thi
`en-3-yl)ethyl]-4-hydroxy-6-aminopyrrolo[2,3-d]pyrimi-'
`
`35
`
`45
`
`50
`
`55
`
`60
`
`65
`
`XIII
`
`in which R5’ and R6 are as herein de?ned with N
`iodosuccinimide to yield the corresponding 2,3-dii
`odopyrrolo[2,3-d]pyrimidine which then is treated with
`zinc and acetic acid to remove selectively the iodine
`atom in the 2-position, yielding the corresponding 3
`iodopyrrolo[2,3-d]pyrimidine of Formula IV.
`According to the foregoing processes, compounds of
`Formula II in which R1 is —OH are obtained. When a
`compound of Formula I in which R1 is —-NH2 is de
`sired, a compound in which R1 is —OH can be treated
`with 1,2,4-triazo1e and (4-chlorophenyl)dichlorophos
`phate and the product of this reaction then treated with
`concentrated ammonia.
`As noted, the compounds of this invention have an
`effect on one or more enzymes which utilize folic acid,
`and in particular metabolic derivatives of folic acid, as a
`substrate.
`For
`example,
`N-(4-[2-(4-hydroxy-6
`aminopyrrolo[2,3-d]pyrimidin-3-yl)ethyl]benzoy1)-L
`glutamic acid demonstrates potent inhibitory effects
`against growth of human T-cell derived lyphoblastic
`leukemia cells (CCRF-CEM), exhibiting an IC50 of
`0.004 u/ml. Cytotoxicity is not reversed by addition of
`purines such as hypoxanthine or by addition of
`aminoimidazolecarboxamide but is reversed by addition
`of thymidine, indicating speci?c inhibition in the
`tymidylate cycle and not in de novo purine synthesis.
`The compounds can be used, under the supervision of
`qualified professionals, to inhibit the growth of neo
`plasms including choriocarcinoma, leukemia, adenocar
`cinoma of the female breast, epidermid cancers of the
`head and neck, squamous or small-cell lung cancer, and
`various lymphosarcomas. The compounds can also be
`used to treat mycosis fungoides and psoriasis.
`The compounds can be administered orally but pref
`erably are administered parenterally, alone or in combi
`nation with other therapeutic agents including other
`anti-neoplastic agents, steroids, etc., to a mammal suf
`fering from neoplasm and in need of treatment. Paren
`
`Wockhardt Exhibit 1003 - 5
`
`

`
`5,344,932
`teral routes of administration include intramuscular,
`intrathecal, intravenous and intra-arterial. Dosage regi
`mens must be titrated to the particular neoplasm, the
`condition of the patient, and the response but generally
`doses will be from about 10 to about 100 mg/day for
`5-10 days or single daily administration of 250-500 mg,
`repeated periodically; e.g. every 14 days. While having
`a low toxicity as compared to other antimetabolites now
`in use, a toxic response often can be eliminated by either
`or both of reducing the daily dosage or administering
`the compound on alternative days or at longer intervals
`such as every three days. Oral dosage forms include
`tablets and capsules containing from 1-10 mg of drug
`per unit dosage. Isotonic saline solutions containing
`20-100 mg/ml can be used for parenteral administra
`tion.
`The following examples will serve to further illus
`trate the invention. In the NMR data, “s” denotes sin
`glet, “d” denotes doublet, “t” denotes triplet, “q” de
`notes quartet, “m” denotes multiplet, and “br” denotes
`a broad peak.
`
`10
`
`20
`
`10
`of water, and cooled. The solid is collected through
`?ltration and dried under vacuum over phosphorus
`pentoxide
`to
`yield
`3-iodo-4-hydroxy-6
`pivaloylaminopyrrolo[2,3-d]pyrimidine which can be
`puri?ed further by chromatography over silica eluting
`with 2.5% methanol in methylene chloride. mp >240°
`C. 1NMR (d6-DMSO)81.20(s, 9H), 7.12 (d, J = 1.8 Hz, 1
`H), 10.82 (s, 1H), 11.79 (s, 1H), 11.89 (s, 1H). Anal. Calc.
`fOI' C11H13N4O2I: C, 36.69; H 3.64; N, 15.56; I, 35.24.
`Found: C, 36.91; H, 3.58; N, 15.65; I, 35.56.
`In a similar fashion from 2,3-diiodo-4-hydroxy-6
`methylpyrrolo[2,3-d]pyrimidine
`and
`2,3-diiodo-4~
`hydroxypyrrolo[2,3-d]py1imidine, there are respec
`tively obtained 3-iodo-4-hydroxy-6-methylpyrrolo[2,3
`d]pyrimidine m.p 245° C., and 3-iodo-4-hydroxypyr
`rolo[2,3-d]pyrimidine, mp >245° C. (compound loses
`iodine). 1NMR (d6-DMSO)87.20 (d, J :22 Hz, 1H),
`7.82 (d, J=2.8 Hz, 1H), 11.85 (d, J=1.1 Hz, 1H), 12.17
`(s, 1H).
`
`25
`
`35
`
`EXAMPLE 1
`3-Iodo-4-hydroxy-6-Pivaloylaminopyrrolo[2,3
`d]pyrimidine
`A mixture of 3.0 g (0.02 mole) of 4-hydroxy-6
`aminopyrrolo[2,3-d]pyiimidine and 8.4 g (0.07 mol) of
`pivaloyl chloride in 40 mL of pyridine is stirred for 30
`minutes at from 80° to 90° C., the mixture then evapo
`rated to dryness, and the residue dissolved in 30 mL of
`30
`methanol. Addition of 10% aqueous ammonia yields 4.2
`g (89%) of 4-hydroxy-6-pivaloylaminopyrrolo[2,3
`d]pyrimidine which can be further puri?ed by chroma
`tography through silica gel, eluting with 8% methanol
`in methylene chloride. mp 295° C. 1NMR (d6
`DMSO)81.20(s, 9H) , 6.37 (d, J=3.4 Hz, 1H), 6.92 (d,
`J =3.4 Hz, 1H), 10.78 (s, 1H), 11.56 (s, 1H), 11.82 (s,
`1H). Anal. Calc. for C11H14N4O2: C, 56.40; H, 6.02; N,
`23.92. Found: C, 56.16; H, 6.01; N, 23.67.
`To a mixture of 4.7 g (20 mmol) of 4-hydroxy-6
`40
`pivaloylaminopyrrolo[2,3-d]pyrimidine in 200 mL of
`dimethylformamide are added 9.9 g (44 mmol) of N
`iodosuccinamide. The mixture is stirred at ambient tem
`perature in the dark for 18 hours. Most of the dimethyl
`formamide is removed by evaporation and the residual
`slurry poured into 300 mL of water. The resulting solid
`is collected by ?ltration and dried under vacuum over
`phosphorus pentoxide to yield 2,3-diiodo-4-hydroxy-6
`pivaloylaminopyrrolo[2,3-d]pyrimidine which can be
`puri?ed further by chromatography over silica eluting
`with 2.5% methanol in methylene chloride. mp >290°
`C. 1NMR (d6-DMSO)81.18(s, 9H), 10.85 (s, 1H), 11.85
`(s, 1H), 12.42 (s, 1H). Anal. Calc. for C11H12N402I2: C,
`27.18; H 2.49; N, 11.53; I, 52.22. Found: C, 27.51; H,
`2.51; N, 11.27;I, 52.02.
`In a similar fashion but starting with 4-hydroxy-6
`methylpyrrolo[2,3-d]pyrimidine and 4-hydroxypyr
`rolo[2,3-d]pyrimidine (7-deazahypoxanthine) there are
`respectively obtained 2,3-diiodo-4-hydroxy-6-methyl
`pyrrolo[2,3-d]pyrimidine, mp 233° C., and 2,3-diiodo-4
`hydroxypyrrolo[2,3-d]pyrimidine, mp >205° C. (com
`pound loses iodine). 1NMR (d6-DMSO)67.79 (s, 1H),
`11.93 (s, 1H), 12.74 (s, 1H).
`To a mixture of 4.86 g of 2,3-diiodo—4~hydroxy-6
`pivaloylaminopyrrolo[2,3-d]pyrimidine in 100 mL of
`65
`glacial acetic acid and 25 mL of water are added 1.3 g
`(20 mmol) of zinc powder. The mixture is stirred at
`ambient temperature for 18 hours, diluted with 500 mL
`
`60
`
`45
`
`50
`
`55
`
`EXAMPLE 2
`Dimethyl
`N-[4-(4-hydroxy-6-pivaloylaminopyrrolo[2,3-d]pyrimi
`din-3-ylethynyl)benzoyl]-L-glutamate
`To a mixture of 3.6 g (10 mmol) of well-dried 3-iodo
`4-hydroxy-6-pivaloy1arninopyrrolo[2,3-d]pyrimidine in
`40 mL of dimethylformamide are added 4.0 g (13.19
`mmol) of dimethyl N-(4-ethynylbenzoyl)-L-glutamate,
`0.38 g of copper (I) iodide, 3 mL of triethylamine, and
`1.0 g of tetrakis-(triphenylphosphine)palladium. This
`mixture is stirred at ambient temperatures for two hours
`and then poured into 500 mL of water. The solid is
`collected by ?ltration, air dried, and then re?uxed in
`200 mL of methanol. The mixture is cooled and the
`solid collected by ?ltration, dissolved in two liters of
`10% methanol in methylene chloride, and chromato
`graphed over silica. Initial black bands are rechromato
`graphed and the combined colorless bands from the ?rst
`and second runs are evaporated to give 3.5 g of di
`methyl N-[4-(4-hydroxy-6-pivaloylaminopyrrolo[2,3
`d]pyrimidin-3-ylethynyl)benzoyl]-L-glutamate which
`can be puri?ed further by recrystallization from 50%
`methanol in methylene chloride. mp 280°-285° C.
`lNMR(d,;-DMSO)'61.21 (s, 9H), 1.96-2.15,(m, 2H), 2.44
`(t, J =7.5 Hz, 2H) 3.56 (s, 3H), 3.62 (s, 3H), 4.40-4.45
`(m, 1H), 7.43 (s, 1H), 7.53 (d, J :84 Hz, 2 H), 7.87 (d,
`J :84 Hz, 2 H), 8.82 (d, J=7.4 Hz, 1 H), 10.95 (s, 1H),
`11.95 (s, 1H). Anal. Calc. for C27H29N5O7: C, 60.56; H
`5.46; N, 13.08. Found: C, 60.55; H, 5.46; N, 12.89.
`In a similar fashion by substituting an equivalent
`amount of dimethyl N-(pent-4-ynoyl)-L-glutamate, di
`methyl N-(hept-6-enoyl)-L-glutamate, and dimethyl
`N-(hex-5-ynoyl)-L-glutamate for dimethyl N-(4
`ethynylbenzoyl)glutamate in the foregoing procedure,
`there are obtained dimethyl N-[5-(4-hydroxy-6
`pivaloylaminopyrrolo[2,3-d]pyrimidin-3-yl)pent-4
`ynoyl]-L-glutamate,
`dimethyl
`N-[7-(4-hydroxy-6
`pivaloylaminopyrrolo[2,3-d]-pyrimidin-3-yl)hept-6
`enoyl]-L-glutamate, and dimethyl N-[6-(4-hydroxy-6
`pivaloylaminopyrrolo[2,3-d]pyrimidin-3-yl)hex-5
`ynoyl]-L-glutamate.
`Dimethyl N-(hex-5-ynoyl)-L-glutamate can be ob
`tained in the manner described generally in US. Pat.
`No. 4,882,334 issued Nov. 21, 1989, the disclosure of
`which is incorporated herein by reference, by allowing
`hex-S-ynoic acid chloride (obtained by treating hex-5
`ynoic acid with thionyl chloride) to react with dimethyl
`
`Wockhardt Exhibit 1003 - 6
`
`

`
`11
`L-glutamate in the presence of an acid acceptor such as
`triethylamine. Hex-S-ynoic acid in turn can be prepared,
`for example, by alkaline hydrolysis of 5-cyanopent~l
`yne.
`
`5,344,932
`
`12
`EXAMPLE 4
`Dimethyl
`N-[5-(4-hydroxy-6-pivaloylaminopyrrolo [2,3-d]pyrimi
`din-3-yl}ethynyl)thien-2-ylcarbonyl]-L-glutamate
`A mixture of 2.0 g of 3-iodo-4-hydroxy-6
`pivaloylaminopyrrolo[2,3-d]pyrimidine, 1.2 g. of trime
`thylsilylacetylene, 0.1 g of palladium chloride, 0.23 g of
`triphenylphosphine, 0.06 g of cuprous iodide, and 2.6 g
`of triethylamine in 100 mL of acetonitrile is heated in a
`sealed tube for 1.5 hours at 50° C. and then at re?ux for
`3 hours. The solvent is removed under reduced pressure
`and the residue triturated with 1:1 ethyl acetatezhexanes
`and ?ltered. The solid thus collected is dissolved in
`methylene chloride and this solution is passed through a
`pad of silica gel eluting with 1% methanol on methy
`lene chloride. The eluate is concentrated to yield 3
`trimethylsilylethynyl-4-hydroxy-6-pivaloylaminopyr
`rolo[2,3-d1pyrimidine.
`To a solution of 1.5 g of 3-trimethylsilylethynyl-4
`hydroxy-6-pivaloylaminopyrrolo[2,3-d]pyrimidine in
`100 mL of anhydrous tetrahydrofuran cooled to 0° C.
`are added under nitrogen 4.75 mL of 1M tetrabutylam
`monium ?uoride in anhydrous tetrahydrofuran. After 5
`minutes, the reaction mixture is allowed to attain room
`temperature and is then stirred for 2 hours. The solvent
`is removed under reduced pressure and the residue
`puri?ed by chromatography over silica gel to yield
`3-ethynyl-4-hydroxy-6-pivaloylaminopyrrolo[2,3
`dlpyrimidine.
`A mixture of 1.70 g. of 3-ethynyl-4-hydroxy-6
`pivaloylaminopyrrolo[2,3-d]pyrimidine, 2.30 g. of di
`methyl
`N-(S-bromothien-2-ylcarbonyl)-L-glutamate
`(prepared as described in U.S. Pat. No. 4,882,333 issued
`Nov. 21, 1989, the disclosure of which is incorporated
`herein by reference), 44 mg. of palladium chloride, 130
`mg. of triphenylphosphine, 25 mg. of cuprous iodide,
`and 1.13 mL. of triethylamine in 30 mL. of acetonitrile
`is heated at re?ux for 3 hours and then cooled to ambi
`ent temperature. The solvent is removed under reduced
`pressure and the residue column chromatographed
`(Waters 500) eluting with 1:19 methanolzmethylene
`chloride to
`yield dimethyl N-[5-(4~hydroxy-6
`pivaloylaminopyrrolo[2,3-d]pyrimidin-3-yl)ethynyl)thi
`en-2-ylcarbonyl]~L-glutamate.
`By substituting equivalent amounts of diethyl N-(4
`bromothien-Z-ylcarbony)-L-glutamate, and diethyl N
`(5-bromothien-3-ylcarbony)-L-glutamate in the forego
`ing procedure, there are respectively obtained diethyl
`N-[4-(4-hyclroxy-6-pivaloylaminopyrrolo[2,3-d]pyrimi
`din-3-ylethynyl)thien-2-ylcarbonyl]-L-glutamate
`and
`diethyl
`N-[5-(4-hydroxy-?-pivaloylaminopyrrolo[2,3
`d]pyrimidin3-ylethynyl)thien-3-ylcarbonyl]-L-gluta
`mate.
`N-[4-(4-hydroxy-6-pivaloylaminopyr
`Diethyl
`rolo[2,3-d]pyrimidin-3-ylethynyl)fur-2-ylcarbonyl]-L
`glutamate
`and
`diethyl
`N-[5-(4-hydroxy-6
`pivaloylaminopyrrolo[2,3-d]pyrimidin-3-ylethynyl)fur
`3-ylcarbonyl]-L-glutamate can be similarly obtained
`from diethyl N(4-bromofur-2-ylcarbony)-L-glutamate
`and diethyl N-(5-bromofur-3-ylcarbony)-L-glutamate,
`respectively.
`Similarly from dimethyl N-(2-?uoro-4-iodobenzoyl)
`L-glutamate and dimethyl N-(3-fluoro-4-iodobenzoy1)
`L-glutamate (prepared as described in U.S. Pat. No.
`4,889,859 issued Dec. 26, 1989, the disclosure of which
`is incorporated herein by reference), there are respec
`
`10
`
`15
`
`20
`
`30
`
`EXAMPLE 3
`Diethyl
`N-[4-{1-hydroxy-3-(4-hydroxy-06-aminopyrrolo[2,3
`d]pyrimidin-3-yl)prop-2-yl}benzoyl] glutamate
`A mixture of 14.6 g of 3-iodo-4-hydroxy-6
`pivaloylaminopyrrolo[2,3-d]pyrimidine, 7.6 g of 2-(2
`propynyloxy)-tetrahydropyran, 798 mg (10%) of palla
`dium chloride, 2.36 g (20%) of triphenyl phosphine, 428
`mg (5%) of cuprous iodide, 45 ml of triethyl amine and
`700 ml of acetonitrile is heated at re?ux under nitrogen
`for 12 hours. There then are added to the hot reaction
`mixture 3.2 g of 2-(2-propynyloxy)-tetrahydropyran
`and re?ux is continued for an additional