United States Patent [191
`Taylor
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`US005344932A
`[11] Patent Number:
`[45) Date of Patent:
`
`5,344,932
`Sep.6, 1994
`
`[54] N-<PYRROL0(2,3-D)PYRIMIDIN-3-
`YLACYL)-GLUTAMIC ACID DERIVATIVES
`
`[56]
`
`[75]
`
`Inventor: Edward C. Taylor, Princeton, N.J.
`
`[73) Assignee: Trustees of Princeton University,
`Princeton, N.J.
`
`[21] Appl. No.: 674,541
`
`[22] Filed:
`
`Mar.22, 1991
`
`Related U.S. Application Data
`[63) Continuation ofSer. No. 448,742, Dec. 11, 1989, aban(cid:173)
`doned, and Ser. No. 479,655, Feb. 8, 1990, abandoned.
`
`Int. Cl.s .................. C07D 487/04; A61K 31!505
`[51]
`[52] U.S. CI •.................................................... 544/280
`[58) Field of Search ......................... 544/280; 514/258
`
`References Cited
`U.S. PATENT DOCUMENTS
`4,889,859 12/1989 Taylor et al ........................ 514/258
`4,996,206 2/1991 Taylor et al ........................ 514/258
`4,997,838 3/1991 Akimoto et al ..................... 514/258
`
`FOREIGN PATENT DOCUMENTS
`334636 9/1989 European Pat. Off ..
`Primary Examiner..:_ Emily Bernhard
`Attorney, Agent, or Firm-Mathews, Woodbridge &
`Collins
`[57]
`ABSTRACI'
`N-(Acyl)glutamic acid derivatives in which the acyl
`group is substituted with 4-hydroxypyrrolo[2,3-d)(cid:173)
`pyrimidin-3-yl group are antineoplastic agents. A typi(cid:173)
`cal embodiment is N-[4-(2-{ 4-hydroxy-6-aminopyrrolo(cid:173)
`[2,3-d]pyrimidin-3-yl}ethyl)benzoyl]-L-g1utamic acid.
`
`7 Claims, No Drawings
`
`NEPTUNE GENERICS 1003- 00001
`APOTEX 1003-0001
`
`

`
`1
`
`5,344,932
`
`N-<PYRROL0(2,3·D)PYRIMIDIN-3-YLACYL)·
`GLUTAMIC ACID DERIVATIVES
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`This is a continuation of Ser. No. 07/448,742 filed
`Dec. 11, 1989 and Ser. No. 07/479,655 ftled Feb. 8, 1990
`both now abandoned.
`The present invention pertains to glutamic acid deriv- 10
`atives having the formula:
`
`2
`processes for the preparation of these compounds and
`their salts, to chemical intermediates useful in prepara(cid:173)
`tion of these compounds, to a method of combatting
`neoplastic growth in a mammal, and to pharmaceutical
`5 compositions containing these compounds or their salts.
`A ftrst group of useful chemical intermediates, which
`can be converted directly to the desired final com(cid:173)
`pounds of Formula I through removal of protecting
`groups, are compounds of the formula:
`
`II
`
`20
`
`2S
`
`in which:
`Rl is -OH or -NH2;
`R 2 is hydrogen or a pharmaceutically acceptable
`cation:
`R3 is 1,4-phenylene or 1,3-phenylene unsubstituted or
`substituted with chloro, fluoro, methyl, methoxy,
`or trifluoromethyl; thienediyl or furanediyl each
`unsubstituted or substituted with chloro, fluoro,
`methyl, methoxy, or trifluoromethyl; cyclohex- 30
`anediyl; or alkanediyl;
`R4 is hydrogen, methyl, or hydroxymethyl;
`R5 is hydrogen, alkyl of 1 to 6 carbon atoms, or
`amino; and
`the configuration about the carbon atom designated * is 35
`s.
`The compounds of this invention are herein described
`as embodying the pyrrolo[2,3-d]pyrimidine heterocy(cid:173)
`clic ring system which ring system is numbered as fol(cid:173)
`lows:
`
`It will be appreciated that the pyrrolo[2,3-d]pyrimi(cid:173)
`dines as depicted by Formula I are the tautomeric
`equivalent of the corresponding 5-H-6-oxo or 5-H-6- so
`imino structures. Unless otherwise indicated, for sim(cid:173)
`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 60
`catalyses the methylation of deoxyuridylic acid to deox(cid:173)
`ythymidylic acid utilizing N5,Nl0.methylidenetetrahy(cid:173)
`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
`
`in which:
`R3 is as defmed above·
`R2' is hydrogen or a c~boxy protecting group;
`R 4' is hydrogen, methyl, hydroxymethyl, or hydroxy(cid:173)
`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(cid:173)
`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(cid:173)
`tions or formulating pharmaceutical compositions.
`40 Pharmaceutically acceptable salts with bases include
`those formed from the alkali metals, alkaline earth met(cid:173)
`als, non-toxic metals, ammonium, and mono-, di- and
`trisubstituted amines, such as for example the sodium,
`potassium, lithium, calcium, magnesium, aluminum,
`45 zinc, ammonium, trimethylammonium, triethanolam(cid:173)
`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(cid:173)
`lene or 1,3-phenylene ring which is unsubstituted or
`optionally substituted with chloro, fluoro, methyl, me(cid:173)
`thoxy, or trifluoromethyl.
`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,
`fluoro, methyl, methoxy, or trifluoromethyl. 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(cid:173)
`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:
`
`55
`
`NEPTUNE GENERICS 1003-00002
`APOTEX 1 003 - 0002
`
`

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`5,344,932
`
`20
`
`4
`3
`With respect to R5', an amino group can be protected
`e.g. the structure in which the depicted carboxy group
`as an amide utilizing an acyl group which is selectively
`adjacent to R3 is in the 2-position of the thiophene ring
`removable under mild conditions, especially formyl, a
`and that in which the depicted carboxy group adjacent
`lower alkanoyl group which is branched a to the car-
`toR 3 is in the 3-position of the thiophene ring. The same
`5 bonyl group, particularly tertiary alkanoyl such as pi-
`conventionas apply to the furane ring.
`Alternatively, R3 can be a cyclohexanediyl group,
`valoyl, or a lower alkanoyl group which is substituted
`namely a divalent cycloalkane group of 6 carbon atoms
`in the position a to the carbonyl group, as for example
`such as cyclohexane-1,3-diyl and cyclohexane-1,4-diyl.
`trifluoroacetyl.
`~ternatively, R3 ~an be a. al~ediyl, namely a
`Preferred compounds of Formula I are those wherein
`stratght or branched divalent al1phat1c group of from 2 10 R s is amino or hydrogen. Within this class, R 1 prefera-
`to 4 carbon atoms such as ethano, trimethylene, tetra-
`bly is hydroxy, R3is 1,4-phenylene, and R4is hydrogen
`or hydroxymethyl. Also preferred within this class are
`methylene, propane-1,2-diyl, propane-2,3-diyl, butane-
`2,3-diyl, butane-1,3-diyl, and butane-2,4-diyl. It again
`the compounds in which R I is hydroxy, R 3 is thienediyl,
`will be appreciated that whereas in the abstract pro-
`and R4 is hydrogen or hydroxymethyl.
`pane-1,2-diyl is the equivalent of propane-2,3-diyl, and IS
`The compounds of this invention can be prepared
`butane-1,3-diyl the equivalent of butane-2,4-diyl, the
`two terms are utilized herein to denote the two isomeric
`according to a first process through catalytic hydroge-
`forms resulting from the orientation of an unsymmetri-
`nation of a compound of the formula:
`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 fmal therapeutic compounds but which
`are intentionally introduced at a stage of the synthesis in
`order to protect groups which otherwise might react in 25
`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-
`in which:
`Z 1 is hydrogen, or Z 1 taken together with R4' is a
`portance primarily as chemical intermediates (although
`carbon-carbon bond;
`some derivatives also exhibit biological activity), their 30
`R2' is hydrogen or a carboxy protecting group;
`precise structure is not critical. Numerous reactions for
`R3 and R6 are as defmed above;
`the formation and removal of such protecting groups
`R4', when taken independently of zl, is hydrogen,
`are described in a number of standard works including,
`methyl, hydroxymethyl, or hydroxymethyl substi-
`for example, "Protective Groups in Organic Chemis-
`tuted with a hydroxy protecting group; and
`try", Plenum Press, London and New York, 1973; 35
`Greene, Th. W. "Protective Groups in Organic Synthe-
`R5' is hydrogen, alkyl of 1 to 6 carbon atoms, amino,
`or an amino protecting group.
`sis", Wiley, New York, 1981; "The Peptides", Vol. I,
`Suitable hydrogenation catalysts include noble metals
`Schroder and Lubke, Academic Press, London and
`and noble metal oxides such as palladium or platinum
`New York, 1965; "MethodenderorganischenChemie",
`Houben-Weyl, 4th Edition, Vol.15/I, Georg Thieme 40 oxide, rhodium oxide, and the foregoing on a support
`Verlag, Stuttgart 1974, the disclosures of which are
`such as carbon or calcium oxide.
`incorporated herein by reference.
`When in Formula III zl taken together with R4' is a
`With respect to R2': a c:rrboxy ~oup can be protected
`carbon-carbon bond, th~t is, when a triple bond is pres-
`as an ester group which 1s selectively removable under
`ent between the two carbon atoms to which zl and R4'
`sufficiently mild conditions not. to disrupt the desired 45 are bound, R 4' in the hydrogenation product will be
`hydrogen. Absent any chirality in R 3 (or any protecting
`structure of the molecule, espec1ally a lower alkyl ester
`of 1_ to 12 carbon at~ms. such as methyl or ethyl_ ~d group encompassed by R2', R4' and/or R5'), the hydro-
`particularly one which IS branched at the l-pos1t1~n
`genation product will be a single enantiomer having the
`S-conflguration about the carbon atom designated *.
`such as t.-butyl;_ ~d su~h lo~er alkyl ester substituted m
`z1
`the 1- or 2-pos1t10n With (1) lower alkoxy, such as for 50 Thi al
`· tru
`h
`d R4'
`h h d
`s_. so IS
`e w en ~ are eac Y rogen,
`example, methoxymethyl, 1-methoxyethyl, and ethox-
`that IS, when a double bond 1s present between the two
`th 1 (" ·) 1
`alk lthi
`h
`£
`yme 7 • 11 ower
`1
`0~ sue as .. ~r examp e me-
`carbon atoms to which zl and R4' are bound.
`Y
`Wh
`th
`th h d R4' ·
`th
`th h d
`thylthiomethyl and 1-ethylthioethyl; (m) halogen, such
`. en, on e 0 er an •
`. IS 0 er ~ Y r~gen,
`as 2,2,2-trichloroethyl, 2-bromoethyl, and 2-iodoethox-
`ycarbonyl; (iv) one or two phenyl groups each of which 55 a IDIXt.ure of the ~S ~d S,S diastereomers 1s ob~ed.
`can be unsubstituted or mono- di- or tri-substituted
`The d1astereomenc miXture can be used therapeutically
`with, for example lower alkyl su~h as tert.-butyl, lower
`as such (after remova! of the protecting groups) or can
`alkoxy such as methoxy, hydroxy, halo such as chloro,
`be se~arated me~h~~ally ~ by chromatography. Al-
`temat1vely,. the md!Vldual_ d1astereo~ers can. be sel?a-
`and nitro, such as for example, benzyl, 4-nitrobenzyl,
`diphenylmethyl di-(4-methoxyphenyl)methyl· or (v) 60 rated chexmcally by fonnmg salts With a chiral ac1d,
`aroyl, such as ~henacyl. A carboxy group als~ can be
`such as the individual enantiomers of 10-camphorsul-
`protected in the form of an organic silyl group such as
`fonic acid, camphoric acid, alphabromocamphoric acid,
`trimethylsilylethyl or tri-lower alkylsilyl, as for exam-
`methoxyacetic acid, tartaric acid, diacetyltartaric acid,
`pie trimethylsilyloxycarbonyl.
`malic acid, pyrrolidone-5-carboxylic acid, and the like,
`With respect to R4', a hydroxy group can be pro- 65 and then freeing one or both of the individual diastere-
`omeric bases, optionally repeating the process, so as
`tected through the formation of acetals and ketals, as for
`example through formation of the tetrahydropyr-2-
`obtain either or both substantially free of the other; i.e.,
`yloxy (fHP) derivative.
`in a form having an optical purity of >95%.
`
`III
`
`NEPTUNE GENERICS 1003-00003
`APOTEX 1 003 - 0003
`
`

`
`5,344,932
`
`6
`
`VII
`
`5
`The protecting groups encompassed by R2', R4', RS',
`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 R2' or R5' protecting 5
`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 descnbed in U.S. Pat.
`No. 4,818,819, utilizing however the corresponding
`halogenated pyrrolo£2,3-d]pyrim.idine. Thus a pyr(cid:173)
`rolo[2,3-d]pyrimidine of the formula:
`
`10
`
`The product of Formula VII then can be hydroge-
`nated, hydrolysed to remove the R 2' and R 6 protecting
`groups, and, optionally with intermediate protection of
`any amino group encompassed by RS', and coupled with
`a protected glutamic acid derivative in the manner de-
`15 scribed in U.S. Pat. No. 4,684,653 using conventional
`condensation techniques for fonning peptide bonds
`such as DCC or diphenylchlorophosphonate, following
`which the protecting groups are removed.
`In a further variant, compounds of Formula III can
`20 be prepared utilizing the procedures described in U.S.
`Pat. No. 4,818,819. Thus a compound of the formula:
`
`IV
`
`in which X is bromo or iodo, R s·, and R 6 are as herein 25
`defmed, is allowed to react with an unsaturated com(cid:173)
`pound of the formula:
`
`VIII
`
`is
`
`v 30
`
`in which zt, R4', RS', and R6 are as herein defmed, is
`allowed to react with a compound of the formula:
`
`in which zl, R3 and R4' are as herein defmed, and R7 35
`
`IX
`
`or
`
`in which X, R3, and R7 are as herein defmed, in the
`presence of a palladium/trisubstituted phosphine cata(cid:173)
`lyst of the type described in U.S. Pat. No. 4,818,819.
`40 This variant of the process is particularly suitable for,
`but is not limited to, preparation of those compounds in
`which R4 is hydroxymethy1, in which case R4' in For(cid:173)
`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 U.S. Pat. No. 4,818,819 by treating a
`compound of Formula IV with an unsaturated com(cid:173)
`pound of the formula:
`
`45
`
`in which R 2' is as herein defmed, in the presence of a
`palladium/trisubstituted phosphine catalyst of the type 50
`described in U.S. Pat. No. 4,818,819, the disclosure of
`which is incorporated herein by reference.
`When R7 is -CONHCH(COOR2)CH2CH2COOR2',
`the product of this coupling reaction is hydrogenated,
`and any protecting group removed, as described above. 55
`Alternatively, a compound of Formula IV is allowed
`to react with a compound of the formula:
`
`Vl6Q
`
`in which zt, R2', R3, and R4' are as herein defined in 65
`the presence of a palladium/trisubstituted phosphine
`catalyst of the type described in U.S. Pat. No. 4,818,819
`to yield an intermediate of the formula:
`
`X
`
`in which R4'' is methyl, a protected hydroxymethyl,
`or a trisubstituted silyl group in the presence of a pal(cid:173)
`ladium/trisubstituted phosphine catalyst of the type
`discussed above. This procedure is particularly suitable
`for, but is not limited to, preparation of those com(cid:173)
`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 U.S. Pat. No. 4,818,819. In such instances,
`the compounds of Formula IV in which R6is hydrogen
`can be first 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(cid:173)
`mediates and for their effect on enzymes are derivatives
`
`NEPTUNE GENERICS 1003-00004
`APOTEX 1 003 - 0004
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`

`
`7
`of Formula XI and XII lacking the glutamic acid side(cid:173)
`chain:
`
`5,344,932
`
`XI
`
`and
`
`8
`3-[2-(thien-3-yl)ethyl]-4-hydroxypyrrolo[2,3-
`dine,
`d]pyrimidine,
`3-[2-(thien-3-yl)ethyl]-4-hydroxy-6-
`methylpyrrolo[2,3-d]pyrimidine, 3-[2-(fur-2-yl)ethyl]-4-
`hydroxy-6-aminopyrrolo[2,3-d]pyrimidine, 3-[2-(fur-2-
`5 yl)ethyl]-4-hydroxypyrrolo[2,3-d]pyrimidine, 3-[2-(fur-
`2-yl)ethyl]-4-hydroxy-6-methylpyrrolo[2,3-d]pyrimi(cid:173)
`dine,
`3-[2-(fur-3-yl)ethyl]-4-hydroxy-6-aminopyr(cid:173)
`rolo[2,3-d]pyrimidine,
`3-[2-(fur-3-yl)ethyl]-4-hydrox(cid:173)
`ypyrrolo[2,3-d]pyrimidine, and 3-[2-(fur-3-yl)ethyl]-4-
`10 hydroxy-6-methylpyrrolo[2,3-d]pyrimidine.
`As discussed above, the compounds of this invention
`can be prepared utilizing the palladium catalyzed cou(cid:173)
`pling of various unsaturated compounds described in
`U.S. Pat. No. 4,818,819 and the glutamic acid coupling
`15 reactions described in U.S. Pat. No. 4,684,653, substitut(cid:173)
`ing the appropriate pyrrolo[2,3-d]pyrimidine for the
`pyrido[2,3-d]pyrimidine therein disclosed. The pyr(cid:173)
`rolo[2,3-d]pyrimidine intermediates of Formula IV
`above can be obtained by treating a compound of the
`20 formula:
`
`XIII
`
`in which:
`Rl is -OH or -NH2;
`R4 is hydrogen, methyl, or hydroxymethyl;
`R5 is hydrogen, alkyl of I to 6 carbon atoms, or 25
`amino·
`R8 is h~drogen, chloro, fluoro, methyl, methoxy,
`trifluoromethyl, or carboxy; and
`Y is -S- or -0-; and
`the pharmaceutically acceptable salts thereof.
`Compounds of Formulas XI and XII are obtained by
`allowing a compound of Formula VII to react with a
`compound of the formula:
`
`30
`
`or
`
`xfJ-R8
`
`y
`
`in which R5' and R6 are as herein defmed with N(cid:173)
`iodosuccinimide to yield the corresponding 2,3-dii(cid:173)
`odopyrrolo[2,3-d]pyrimidine which then is treated with
`zinc and acetic acid to remove selectively the iodine
`35 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 Rl is -OHare obtained. When a
`compound of Formula I in which R 1 is -NH2 is de-
`40 sired, a compound in which R 1 is -OH can be treated
`with 1,2,4-triazole and (4-chlorophenyl)dichlorophos(cid:173)
`phate and the product of this reaction then treated with
`concentrated ammonia.
`As noted, the compounds of this invention have an
`45 effect on one or more enzymes which utilize folic acid,
`in which X, Y, and R 8 are as herein defined, by the
`and in particular metabolic derivatives of folic acid, as a
`substrate.
`For
`example, N-(4-[2-(4-hydroxy-6-
`methods of U.S. Pat. No. 4,818,819, namely in the pres-
`amino~yrrol?[2,3-d]pyrimidin-3-yl)et~yl]~~nzoyl)-L-
`ence of a palladium/trisubstituted phosphine catalyst,
`glu~c acid demonstrates potent ~b1tory effec~s
`with the resulting coupled product being hydrogenated
`and hydrolysed to remove the R2' protecting group. 50 agamst. growth of human T-cell d~n.v~d lyphoblasttc
`leukerma cells (CC~-~EM), exhibltmg an ~<?so of
`Typical compounds of Formulas XI and XII are 3-(2-
`phenylethyl)-4-hydroxy-6-aminopyrrolo[2,3-d]pyrimi-
`0.~ ~/mi. CytotoXIcity 1s n~t reversed by ad?~t1on of
`dine,
`3-[2-(3-fluorophenyl)ethyl]-4-hydroxy-6-
`purmes such as hypoxanthine or by addition of
`aminopyrrolo[2,3-d]pyrimidine, 3-[2-(4-fluorophenyl)e-
`aminoimidazolecarboxamide but is reversed by addition
`thyl]-4-hydroxy-6-aminopyrrolo[2,3-d]pyrimidine, 3-[2- 55 of thymidine, indicating specific inhibition in the
`(4-carboxyphenyl)ethyl]-4-hydroxy-6-aminopyr-
`tymidylate cycle and not in de novo purine synthesis.
`rolo[2,3-d]-pyrimidine, 3-[2-(4-methoxyphenyl)ethyl]-4-
`The compounds can be used, under the supervision of
`hydroxy-6-aminopyrrolo[2,3-d]pyrimidine,
`3-[2-(4-
`qualified professionals, to inhibit the growth of neo-
`methylphenyl)ethyl]-4-hydroxy-6-aminopyrrolo[2,3-
`plasms including choriocarcinoma, leukemia, adenocar-
`d]pyrimidine, 3-(2-phenylethyl)-4-hydroxypyrrolo[2,3- 60 cinoma of the female breast, epidermid cancers of the
`head and neck, squamous or small-celllung cancer, and
`d]pyrimidine,
`3-(2-phenylethyl)-4-hydroxy-6-methyl-
`pyrrolo[2,3-d]pyrimidine,
`3-(2-phenyl-3-hydroxy-
`various lymphosarcomas. The compounds can also be
`propyl)-4-hydroxy-6-aminopyrrolo[2,3-d]pyrimidine,
`used to treat mycosis fungoides and psoriasis.
`3-[2-(thien-2-yl)ethyl]-4-hydroxy-6-aminopyrrolo[2,3-
`The compounds can be administered orally but pref-
`d]pyrimidine,
`3-[2-(thien-2-yl)ethyl]-4-hydroxypyr- 65 erably are administered parenterally, alone or in combi-
`3-[2
`-(thien-2-yl)ethyl]-4-
`nation with other therapeutic agents including other
`rolo[2,3-d]pyrimidine,
`hydroxy-6-methylpyrrolo[2,3-d]-pyrimidine, 3-[2-(thi-
`anti-neoplastic agents, steroids, etc., to a mammal suf-
`en-3-yl)ethyl]-4-hydroxy-6-aminopyrrolo[2,3-d]pyrimi-
`fering from neoplasm and in need of treatment. Paren-
`
`NEPTUNE GENERICS 1003-00005
`APOTEX 1 003 - 0005
`
`

`
`5,344,932
`
`9
`10
`of water, and cooled. The solid is collected through
`teral routes of administration include intramuscular,
`filtration and dried under vacuum over phosphorus
`intrathecal, intravenous and intra-arterial. Dosage regi-
`pentoxide
`to
`yield
`3-iodo-4-hydroxy-6-
`mens must be titrated to the particular neoplasm, the
`condition of the patient, and the response but generally
`pivaloylaminopyrrolo[2,3-d]pyrimidine which can be
`doses will be from about 10 to about 100 mg/day for 5 purified further by chromatography over silica eluting
`5-10 days or single daily administration of 250-500 mg,
`with 2.5% methanol in methylene chloride. mp >240°
`repeated periodically; e.g. every 14 days. While having
`C. 'NMR (d6-DMS0)81.20(s, 9H), 7.12 (d, 1= 1.8 Hz, I
`a low toxicity as compared to other antimetabolites now
`H), 10.82 (s, I H), 11.79 (s, 1H), 11.89 (s, 1H). Anal. Calc.
`for c11H 13N4o2I: c, 36.69; H 3.64; N, 15.56; I, 35.24.
`in use, a toxic response often can be eliminated by either
`or both of reducing the daily dosage or administering 10 Found: C, 36.91; H, 3.58; N, 15.65; I, 35.56.
`the compound on alternative days or at longer intervals
`In a similar fashion from 2,3-diiodo-4-hydroxy-6-
`such as every three days. Oral dosage forms include
`methy1pyrrolo[2,3-d]pyrimidine
`and
`2,3-diiodo-4-
`tablets and capsules containing from 1-10 mg of drug
`hydroxypyrrolo[2,3-d]pyrimidine,
`there are respec-
`per unit dosage. Isotonic saline solutions containing
`tively obtained 3-iodo-4-hydroxy-6-methylpyrrolo[2,3-
`20-100 mg/ml can be used for parenteral administra- 15 d]pyrimidine m.p 245° c., and 3-iodo-4-hydroxypyr-
`tion.
`rolo[2,3-d]pyrimidine, mp >245° C. (compound loses
`The following examples will serve to further illus-
`iodine). INMR (d6-DMS0)87.20 (d, 1 =2.2 Hz, 1H),
`trate the invention. In the NMR data, "s': denotes sin-
`7.82 (d, 1=2.8 Hz, 1H), 11.85 (d, 1=1.1 Hz, 1H), 12.17
`s 1
`glet, "d" denotes doublet, "t" denotes tnplet, "q" de-
`notes quartet, "m" denotes multiplet, and "br" denotes 20 ( ' H).
`a broad peak.
`
`25
`
`EXAMPLE 2
`Dimethyl
`N-[4-(4-hydroxy-6-pivaloylaminopyrrolo[2,3-d]pyrimi(cid:173)
`din-3-ylethynyl)benzoyl]-L-glutamate
`
`EXAMPLE 1
`3-Iodo-4-hydroxy-6-Pivaloylaminopyrrolo[2,3-
`d]pyrimidine
`To a mixture of 3.6 g (10 mmol) of well-dried 3-iodo-
`A mixture of 3.0 g (0.02 mole) of 4-hydroxy-6-
`4-hydroxy-6-pivaloylaminopyrrolo[2,3-d]pyrimidine in
`aminopyrrolo[2,3-d]pyrimidine and 8.4 g (0.07 mol) of
`40 mL of ~imethylformamide are added 4.0 g (13.19
`pivaloyl chloride in 40 mL of pyridine is stirred for 30
`minutes at from so• to 90• c., the mixture then evapo-
`mmol) of dtmethyl ~-(~ethynylbenzo!'l)-L-gl~tamate,
`rated to dryness, and the residue dissolved in 30 mL of 30 0.38 g of coppe~ (I) ~odtde, 3 mL o~ triethyl~e, an?
`L? g o~ te~rakis-(tnph~nylphosphine)palladtum. This
`methanol. Addition of 10% aqueous ammonia yields 4.2
`rmxture 1s stirred a~ ambxent temperatures for two h~~s
`(89%) of 4-hydroxy-6-pivaloylaminopyrrolo[2,3-
`g
`and then poured ~to 5~ m~ of water. The solid :s
`d]pyrimidine which can be further purified by chroma-
`tography through silica gel, eluting with 8% methanol
`collected by flltration, arr dn~d, an~ then refluxed m
`in methylene chloride. mp 295° c. INMR (d6- 35 200 mL of methanol. The rmxture 1S cooled and the
`DMS0)81.20(s, 9H), 6.37 (d, 1=3.4 Hz, III), 6.92 (d,
`solid collected by flltration, dissolved in two liters of
`1=3.4 Hz, 1H), 10.78 (s, 1H), I1.56 (s, 1H), I1.82 (s,
`10% methanol in methylene chloride, and chromato-
`1H). Anal. Calc. for C11H 14N40 2: c, 56.40; H, 6.02; N,
`graphed over silica. Initial black bands are rechromato-
`23.92. Found: C, 56.16; H, 6.01; N, 23.67.
`graphed and the combined colorless bands from the frrst
`To a mixture of 4.7 g (20 mmol) of 4-hydroxy-6- 40 and second runs are evaporated to give 3.5 g of di-
`pivaloylaminopyrrolo[2,3-d]pyrimidine in 200 mL of methyl N-[4-(4-hydroxy-6-pivaloylaminopyrrolo[2,3-
`d]pyrimidin-3-ylethynyl)benzoyl]-L-glutamate which
`dimethylformamide are added 9.9 g (44 mmol) of N-
`iodosuccinamide. The mixture is stirred at ambient tern-
`can be purified further by recrystallization from 50%
`perature in the dark for 18 hours. Most of the dimethyl-
`methanol in methylene chloride. mp 280• -285° C.
`formamide is removed by evaporation and the residual 45 1NMR (d6-DMS0)81.21 (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
`slurry poured into 300 mL of water. The resulting solid
`is collected by flltration and dried under vacuum over
`(m, III), 7.43 (s, 1H), 7.53 (d, 1=8.4 Hz, 2 H), 7.87 (d,
`phosphorus pentoxide to yield 2,3-diiodo-4-hydroxy-6-
`J=8.4 Hz, 2 H), 8.82 (d, 1=7.4 Hz, 1 H), 10.95 (s, 1H),
`pivaloylaminopyrrolo[2,3-d]pyrimidine which can be
`11.95 (s, 1H). Anal. Calc. for C21H29Ns07: C, 60.56; H
`purified further by chromatography over silica eluting 50 5.46; N, 13.08. Found: C, 60.55; H, 5.46; N, 12.89.
`In a similar fashion by substituting an equivalent
`with 2.5% methanol in methylene chloride. mp >290°
`C. INMR (d6-DMS0)81.18(s, 9H), 10.85 (s, 1H), 11.85
`amount of dimethyl N-(pent-4-ynoyl)-L-glutamate, di-
`(s, III), 12.42 (s, 1H). Anal. Calc. for C11H12N402h: C,
`methyl N-(hept-6-enoyl)-L-g1utamate, and dimethyl
`27.18; H 2.49; N, 11.53; I, 52.22. Found: C, 27.51; H,
`N-(hex-5-ynoyl)-L-glutamate
`for
`dimethyl N-(4-
`2.51; N, 11.27;I, 52.02.
`55 ethynylbenzoyl)glutamate in the foregoing procedure,
`In a similar fashion but starting with 4-hydroxy-6-
`there are obtained dimethyl N-[5-(4-hydroxy-6-
`methylpyrrolo[2,3-d]pyrimidine and 4-hydroxypyr-
`pivaloylaminopyrrolo[2,3-d]pyrimidin-3-yl)pent-4-
`rolo[2,3-d]pyrimidine (7-deazahypoxanthine) there are
`ynoyl]-L-glutamate,
`dimethyl N-[7-(4-hydroxy-6-
`respectively obtained 2,3-diiodo-4-hydroxy-6-methyl-
`pivaloylaminopyrrolo[2,3-d]-pyrimidin-3-yl)hept-6-
`pyrrolo[2,3-d]pyrimidine, mp 233° C., and 2,3-diiodo-4- 60 enoyl]-L-glutamate, and dimethyl N-[6-(4-hydroxy-6-
`pivaloylaminopyrrolo[2,3-d]pyrimidin-3-yl)hex-5-
`hydroxypyrrolo[2,3-d]pyrimidine, mp >205° C. (com-
`pound loses iodine). 'NMR (d6-DMS0)87.79 (s, 1H),
`ynoyl]-L-glutamate.
`11.93 (s, lH), 12.74 (s, III).
`Dimethyl N-(hex-5-ynoyl)-L-glutamate can be ob-
`To a mixture of 4.86 g of 2,3-diiodo-4-hydroxy-6-
`tained in the manner described generally in U.S. Pat.
`pivaloylaminopyrrolo[2,3-d]pyrimidine in 100 mL of 65 No. 4,882,334 issued Nov. 21, 1989, the disclosure of
`glacial acetic acid and 25 mL of water are added 1.3 g
`which is incorporated herein by reference, by allowing
`(20 mmol) of zinc powder. The mixture is stirred at
`hex-5-ynoic acid chloride (obtained by treating hex-5-
`ambient temperature for 18 hours, diluted with 500 mL
`ynoic acid with thionyl chloride) to react with dimethyl
`
`NEPTUNE GENERICS 1003-00006
`APOTEX 1 003 - 0006
`
`

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`5
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`10
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`55
`
`EXAMPLE3
`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(cid:173)
`dium chloride, 2.36 g (20%) of triphenyl phosphine, 428
`mg (5%) of cuprous iodide, 45 ml oftriethyl amine and 15
`700 ml of acetonitrile is heated at reflux under nitrogen
`for 12 hours. There then are added to the hot reaction
`mixture 3.2 g of 2-(2-propynyloxy)-tetrahydropyran
`and reflux is continued for an additional 12 hours. After
`heating for a total of 24 hours under reflux, the solvent 20
`is removed under reduced pressure, and the residue
`filtered through silica gel using 2% methanol in methy(cid:173)
`lene chloride. This ftltrate is concentrated and chro(cid:173)
`matographed on silica gel eluting with 20: 1 ethyl aceta(cid:173)
`te:hexane mixture to give 3-(3-tetrahydropyr-2-yloxy- 25
`prop-1-yn-1-yl)-4-hydroxy-6-pivaloylaminopyrrolo[2,3-
`d]pyrimidine which is further purified by recrystalliza(cid:173)
`tion with ethyl acetate.
`A mixture of 2 g of 3-(3-tetrahydropyr-2-yloxyprop- 30
`1-yn-1-yl)-4-hydroxy-6-pivaloylaminopyrrolo[2,3-
`d]pyrimidine, 40 ml of methanol, 20 ml of chloroform,
`40 mg of 5% palladium on barium sulfate, and 40 mg of
`synthetic quinoline is stirred under 1 atmosphere hydro(cid:173)
`gen pressure for 40 min. The solvent then is removed by 35
`evaporation and the residue diluted with methylene
`chloride. The methylene chloride solution is filtered
`through silica gel with 2% methanol in methylene chlo(cid:173)
`ride to remove catalyst and the filtrate then concen(cid:173)
`trated to give an oil which upon adding ether yields 40
`3-(3-tetrahydropyr-2-yloxyprop-1-en-1-yl)-4-hydroxy-
`6-pivaloy1aminopyrrolo[2,3-d]pyrimidine which can be
`further purified through column chromatography elut(cid:173)
`ing with ethyl acetate and recrystallization using ethyl
`acetate.
`A mixture containing 3.48 g of 3-(3-tetrahydropyr-2-
`yloxyprop-1-en-1-yl)-4-hydroxy-6-pivaloylaminopyr(cid:173)
`rolo[2,3-d]pyrimidine, 3.12g (1.2 equiv.) of diethyl N-(4-
`iodobenzoyl)glutamate, 546 mg (20%) of tris-(2-methyl(cid:173)
`phenyl)phosphine, 201 mg (10%) of palladium acetate 50
`and 85.5 mg (5%) of cuprous iodide in 15 ml of triethyl(cid:173)
`amine and 240 ml of acetonitrile is heated at reflux
`under nitrogen. After 12 hours., 1.17 g of diethyl N-(4-
`iodobenzoyl)glutamate are added and the reaction mix-
`ture is heated at reflux under nitrogen for an additional
`12 hours. The reaction mixture then is concentrated
`under reduced pressure and the residue chromato(cid:173)
`graphed on silica gel, eluting with 20: 1 ethyl acetate:(cid:173)
`hexane. (Any recovered starting material can be recy- 60
`cled through the foregoing procedure.) The concen(cid:173)
`trated material is dissolved in 1:5 ethyl acetate:ether and
`this solution is refrigerated for 15 hours. The solid
`which forms is collected by filtration, washed with cold
`ethyl acetate and dried to yield diethyl N-[4-(1-(tetrahy- 65
`dropyr-2-yloxy)-3-(4-hydroxy-6
`-pivaloylaminopyr-
`rolo[2,3-d]pyrimidin-3-yl)prop-2-en-2yl)benzoyl]gluta(cid:173)
`mate.
`
`45
`
`11
`L-glutamate in the presence of an acid acceptor such as
`triethylamine. Hex-5-ynoic acid in turn can be prepared,
`for example, by alkaline hydrolysis of 5-cyanopent-1-
`yne.
`
`5,344,932
`
`12
`EXA