United States Patent
`Taylor
`
`[19]
`
`US005344932A
`
`[11] Patent Number:
`
`5,344,932
`
`[45] Date of Patent:
`
`Sep. 6, 1994
`
`[54] N-(PYRROLO(2,3-D)PYRIMIDIN-3-
`YI.ACYL)-GLUTAMIC ACID DERIVATIVES
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`[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 of Scr. No. 448,742, Dec. t 1, 1989, aban-
`doned, and Scr. No. 479,655, Feb. 8, 1990, abandoned.
`
`Int. Cl.S .................. C07D 487/04; A61K 31/505
`[51]
`[52] U.S. O ..................................................... 544/280
`[58] Field of Search ......................... 544/280; 514/258
`
`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 ExaminerL-Emily Bernhard
`Attorney, Agent, or Firm--Mathews, Woodbridge &
`Collins
`
`[57]
`
`ABSTRACT
`
`N-(Acyl)glutamie acid derivatives in which the acyl
`group is substituted with 4-hydroxypyrrolo[2,3-d]-
`pyrimidin-3-yl group are antineoplastic agents. A typi-
`cal embodiment is N-[4-(2-(4-hydroxy-6-aminopyrrolo-
`[2,3 -d]pydmidirt-3 -yl}ethyt)benzoyl] -L-glutamie acid.
`
`7 Clnimg~ No Drawings
`
`Sandoz Inc.
`Exhibit 1034-0001
`
`

`
`N.(PYRROLO(2,3-D)PY1HMIDIN-3-YLACYL)-
`GLUTAMIC ACID DERIVATIVES
`
`CROSS-REFERENCE TO RELATED
`APPLICATIONS
`
`5
`
`This is a continuation of Ser. No. 07/448,742 i’fled
`Dec. 11, 1989 and Set. No. 07/479,655 filed Feb. 8, 1990
`both now abandoned.
`The present invention pertains to glutamic acid dedv- 10
`atives having the formula:
`
`15
`
`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 first group of useful chemical intermediates, which
`can be converted directly to the desired final com-
`pounds of Formula I through removal of protecting
`groups, are compounds of the formula:
`
`0
`II
`o
`o
`R6~ ~.C~ . II * II ~
`N ~ C-- CH2CH--Ra"CNHCHCH2CH2C-- ORz’
`I
`II
`I
`I .
`C
`_C-
`CH
`R4’
`C--ORz°
`Ra’’~ ~N/ ~N"/
`II
`O
`I
`H
`
`II
`
`in which:
`R1 is --OH or --NH2;
`R 2 is hydrogen or a pharmaceutically acceptable
`cation:
`R 3 is 1,4-phenylene or 1,3-phenylene unsubstituted or
`substituted with chloro, fluoro, methyl, methoxy,
`or tritluoromethyl; thienediyl or furanediyl each
`unsubstituted or substituted with chloro, fluoro,
`methyl, methoxy, or trifluoromethyl; cyclohex-
`anediyl; or alkanediyl;
`R4 is hydrogen, methyl, or hydroxymethyl;
`R 5 is hydrogen, alkyl of 1 to 6 carbon atoms, or
`amino; and
`the configuration 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:
`
`5 N~C~c
`
`C 3
`
`7 1
`
`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 folie acid,
`and in particular metabolic derivatives of folio acid, as a
`substrate. The compounds appear to be particularly
`active as inhlbitors of thymidylate synthetase, which
`catalyses the methylation of deoxyuridylic acid to deox-
`ythymidylic acid utilizing NS,Nl0-methylidenetetrahy-
`drofolate as a eoenzyme. The compounds thus can be
`used, alone or in combination, to inhibit the growth of
`those neoplasms which otherwise depend upon the
`inhibited enzyme.
`The invention also pertains to the pharmaceutically
`acceptable salts of the compounds of Formula I, to
`
`in which:
`20 R3 is as defined 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, amin,O, or amino carrying a
`25 protecting group; and
`
`30
`
`R6 is hydrogen or alkanoyoxy;
`at least one of R2’,R4’, and Ry being a earboxy 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
`35 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.
`40 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, alnmirlum,
`
`45 zinc, ammonium, trimethy]ammonium, tdethanolam-
`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
`5o 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, fluoro, methyl, me-
`thoxy, or tdfluoromethyl.
`55 Alternatively, R3 can be a thienediyl or furanediyl
`group, that is, a thiopheue 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
`60 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 uusubstituted or substituted with ehloro,
`fluoro, methyl, methoxy, or trifluoromethyl. It will be
`appreciated that whereas in the abstract the thiene-3,5-
`65 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:
`
`Sandoz Inc.
`Exhibit 1034-0002
`
`

`
`4
`With respect to R5’, an amino group can be protected
`as an amide utilizing an aeyl group which is selectively
`removable under mild conditions, especially formyl, a
`lower alkanoyl group which is branched ct to the ear-
`5 bonyl group, particularly tertiary alkanoyl such as pi-
`valoyl, or a lower alkanoyl group which is substituted
`in the position c~ to the carbonyl group, as for example
`trifluoroacetyl.
`Preferred compounds of Formula I are those wherein
`10 R5 is amino or hydrogen. Within this class, R1 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 ftrst process through catalytic hydroge-
`nation of a compound of the formula:
`
`15
`
`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 eycloalkane group of 6 carbon atoms
`such as eyelohexane-l,3-diyl and eyelohexane-l,4-diyl.
`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-l,2-diyl, propane-2,3-diyl, butane-
`2,3-diyl, butane-l,3-diyl, and butane-2,4-diyl. It again
`will be appreciated that whereas in the abstract pro-
`pane-l,2-diyl is the equivalent of propane-2,3-diyl, and
`butane-l,3-diyl the equivalent of butane-2,4-diyl, the
`two terms are utiJized 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 final 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, "Fla. W. "Protective Groups in Organic Synthe-
`sis", Wiley, New York, 1981; "The Peptides", Vol. I,
`SehriSder and Lubke, Academic Press, London and
`New York, 1965; "Methoden der orgardschen Chemic",
`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
`suffieieutly mild conditions not to disrupt the desired
`structure of the molecule, especially a lower alkyl ester
`of 1 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, 1-methoxyethyl, and ethox-
`ymethyl, (ii) lower alkylthio, such as for example me-
`thylthiomethyl and 1-ethylthioethyl; (iii) halogen, such
`as 2,2,2-tdehloroethyl, 2-bromoethyl, and 2-iodoethox-
`yearbonyl; (iv) one or two phenyl groups each of which
`can be unsubstituted or mono-, di- or td-snbstituted
`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 phenaeyl. 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-
`teeted through the formation of aeetals and ketals, as for
`example through formation of the tetrahydropyr-2-
`yloxy (THP) derivative.
`
`5,344,932
`
`35
`
`30
`
`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 def’med 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 1 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
`40 oxide, rhodium oxide, and the foregoing on a support
`such as carbon or calcium oxide.
`When in Formula IIl, 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’
`45 are bound, R4’ in the hydrogenation product will be
`
`hydrogen. Absent any ehirality in R3 (or any protecting
`group encompassed by R2’, R4’ and/or Rs’), the hydro-
`genation product will be a single enantiomer having the
`S-configuration about the carbon atom designated *.
`50 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,
`55 a mixture of the R,S and S,S 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. A1-
`teruatively, the individual diastereomers can be sepa-
`60 rated chemically by forming salts with a chiral acid,
`such as the individual enantiomers of 10-camphorsul-
`foulc acid, campbofic acid, alphabromocamphoric acid,
`methoxyacetie acid, tartaric acid, diaeetyltartarie acid,
`malic acid, pyrrolidone-5-carboxylic acid, and the like,
`65 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%.
`
`Sandoz Inc.
`Exhibit 1034-0003
`
`

`
`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 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 described in U.S. 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:
`
`10
`
`15
`
`VII
`
`The product of Formula VII then can be hydroge-
`nated, hydrolysed to remove the Rz’ and R6 protecting
`groups, and, optionally with intermediate protection of
`any amino group encompassed by Ry, and coupled with
`a protected glutamic acid derivative in the manner de-
`scribed in U.S. Pat. No. 4,684,653 using conventional
`condensation teehuiques for forming peptide bonds
`such as DCC or diphenylchiorophosphonate, following
`which the protecting groups are removed.
`In a further variant, compounds of Formula III can
`be prepared uftlizing the procedures described in U.S.
`Pat. No. 4,818,819. Thus a compound of the formula:
`
`o
`
`VIII
`
`C--C-----C--H
`I II IIR
`C C CH
`RS,-~ ~N~" ~N/
`I
`H
`
`in which Z1, R4’, Ry, and R6 are as herein defined, is
`allowed to react with a compound of the formula:
`
`o
`
`RtxN/C"c c--x
`II II
`
`IV
`
`20
`
`I
`H
`
`in which X is bromo or iodo, Ry, and R6 are as herein 25
`defined, is allowed to react with an unsaturated com-
`pound of the formula:
`
`ZI
`
`HC--C--R3~R7
`
`v
`
`30
`
`in which Z1, R3 and R4’ are as herein det’med, and R7
`
`35
`
`X--R3--R7
`
`is
`
`IX
`
`o
`--C--OR2’,
`
`o
`
`o
`
`--CNHCHCH2CH2C--OR2’
`
`~C~OR2’
`
`O
`
`in which X, R3, and R7 are as herein defined, in the
`presence of a palladium/tfisubstituted phosphine cata-
`lyst of the type described in U.S. 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 g4 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 U.S. 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 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 1V is allowed
`to react with a compound of the formula:
`
`Z1 ,
`
`HC-----C--R3--COOR2’
`
`in which Z1, 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:
`
`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 U.S. Pat. No. 4,818,819. In such instances,
`the compounds of Formula IV in which R6 is 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-
`mediates and for their effect on enzymes are derivatives
`
`Sandoz Inc.
`Exhibit 1034-0004
`
`

`
`7
`of Formula XI and XII lacking the glutamic acid side-
`chain:
`
`5,344,932
`
`XI
`
`XlI
`
`and
`
`dine, 3-[2-(thien-3-yl)ethyl]-4-hydroxypyrrolo [2,3-
`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-amlnopyrrolo[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-
`dine, 3-[2-(fur-3-yl)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-
`I0 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 eompotmds 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-
`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
`20 formula:
`
`o
`
`xIII
`
`Rr’~N.--* C~.C~ CH
`
`H
`
`in which:
`Rt is --OH or --NH2;
`R* is hydrogen, methyl, or hydroxymethyl;
`R 5 is hydrogen, alkyl of 1 to 6 carbon atoms, or 25
`amltaO;
`Ks is hydrogen, chloro, fluoro, methyl, methoxy,
`trifluoromethyl, or carboxy; and
`Y is --S-- or ---O--; and
`the pharmaceutically acceptable salts thereof.
`Compounds of Formulas XI and XII are obtained by
`allowing a compound of Formula V~I to react with a
`compound of the formula:
`
`30
`
`in which R5’ and R6 are as herein def’med 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-
`35 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 R! is --NH2 is de-
`40 sired, a compound in which R1 is --OH can be treated
`with 1,2,4-triazole and (4-chiorophenyl)dichlorophos-
`phate and the product of this reaction then treated with
`concentrated arnmoula.
`AS noted, the compounds of this invention have an
`45 effect on one or more enzymes which utilize folio 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]benzoyl)-L-
`glutamic acid demonstrates potent inhibitory effects
`50 against growth of human T-cell derived lyphoblastic
`leukemia cells (CCRF-CEM), exhibiting an IC50 of
`0.004/~/ml. Cytotoxicity is not reversed by addition of
`purines such as hypoxanthine or by addition of
`arnlnoimidazoleearboxamide but is reversed by addition
`55 of thymidine, indicating specific 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 chodoearcinoma, leukemia, adenocar-
`60 cinoma of the female breast, epidermid cancers of the
`head and neck, squamous or small-cell lung cancer, and
`various lymphosareomas. The compounds can also be
`used to treat mycosis fungoides and psoriasis.
`The compounds can be administered orally but pref-
`65 erably are administered parenterally, alone or in combi-
`nation with other therapeutic agents including other
`anti-neoplastic agents, steroids, etc., to a mammal suf-
`feting from neoplasm and in need of treatment. Paten-
`
`in which X, Y, and R8 are as herein defined, by the
`methods of U.S. Pat. No. 4,818,819, namely in the pres-
`ence of a palladium/trisubstituted 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-6-amlnopyrrolo[2,3-d]pyrimi-
`dine, 3-[2-(3-fluorophenyl) 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)ethyl]-4-hydroxy-6-amlnopyr-
`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-arninopyrrolo[2,3-
`d]pyrimidine, 3-(2-phenylethyl)-4-hydroxypyrrolo[2,3o
`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)ethyl]-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-
`
`Sandoz Inc.
`Exhibit 1034-0005
`
`

`
`5,344,932
`
`10
`of water, and cooled. The solid is collected through
`fdtration and dried under vacuum over phosphorus
`pentoxide to yield 3-iodo-4-hydroxy-6-
`pivaloyiaminopyrrolo[2,3-d]pydmidine which can be
`5 purified further by chromatography over silica eluting
`with 2.5% methanol in methylene chloride, mp >240*
`C. 1NMR (d6-DMSO)81.20(s, 9I-I), 7.12 (d, J= 1.8 Hz, 1
`H), 10.82 (s, 1H), 11.79 (s, 1H), 11.89 (s, 1H). Anal. Calc.
`for CllH13N402I: C, 36.69; H 3.64; N, 15.56; I, 35.24.
`10 Found: C, 36.91; H, 3.58; N, 15.65; I, 35.56.
`In a similar fashion from 2,3-diiodo-4-hydroxy-6-
`methyipyrrolo [2,3 -d]pyrimidine and 2,3-diiodo-4-
`hydroxypyrrolo[2,3-d]pyrimidine, there are respec-
`tively obtained 3-iodo-4-hydroxy-6-methylpyrrolo[2,3-
`15 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)~7.20 (d, J=2.2 Hz, 1H),
`7.82 (d, J=2.8 Hz, 1H), 11.85 (d, J=l.1 Hz, lid), 12.17
`(s, IH).
`
`9
`teral routes of admini.~tration 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 rag/day for
`5-10 day.s or single daily administration of 250-500 rag,
`repeated periodically; e.g. every 14 days. While having
`a low toxicity as compared to other antimetabolltes 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 mgiml 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 "hr" denotes
`a broad peak.
`
`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]pyrimidine and 8.4 g (0.07 tool) 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
`methanol. Addition of 10% aqueous ammonia yields 4~2
`g (89%) of 4-hydroxy-6-pivaloylaminopyrrolo[2,3-
`d]pyfimidine which can be further purified by chroma-
`tography through silica gel, duting with 8% methanol
`in methylene chloride, mp 295° C. 1NMR (d6-
`DMSO)/~I.20(s, 9H), 6.37 (d, J=3.4 Hz, lI-I), 6.92 (d,
`J=3.4 Hz, 1H), 10.78 (s, 1H), 11.56 (s, 1H), 11.82 (s,
`1H). Anal. Cale. for CllH14N402: (2, 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 retool) of 4-hydroxy-6-
`pivaloylaminopyrrolo[2,3-d]pyrimidine in 200 mL of
`dimethylformamide are added 9.9 g (44 retool) 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 lrdtration and dried under vacuum over
`phosphorus pentoxide to yield 2,3-diiodo-4-hydroxy-6-
`pivaloylaminopyrrolo[2,3-d]pyrimidine which can be
`purified further by chromatography over silica eluting
`with 2.5% methanol in methylene chloride, mp > 290*
`C. INMR (d6-DMSO)~l.18(s, 9H), 10.85 (s, 1H), 11.85
`(s, 1H), 12.42 (s, liT). Anal. Cale. for CI1H12N40212: 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)~7.79 (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
`glacial acetic acid and 25 mL of water are added 1.3 g
`(20 retool) of zinc powder. The mixture is stirred at
`ambient temperature for 18 hours, diluted with 500 mL
`
`EXAMPLE 2
`
`Dimethyl
`N-[4-(4-hydroxy-6-pivaloyl aminopyrrolo[2,3-d]pyrimi-
`din-3-ylethynyl)benzoyl]-L-glutamate
`
`25
`
`To a mixture of 3.6 g (10 retool) of well-dried 3-iodo-
`4-hydroxy-6-pivaloy~amluopyrrolo[2,3-d]pyfimidine in
`40 mL of dimethylformamide are added 4.0 g (13.19
`retool) of dimethyl N-(4-ethynylbenzoyl)-L-glutamate,
`30 0.38 g of copper (I) iodide, 3 mL of tdethylamine, 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 f’titration, air dried, and then refluxed in
`35 200 mL of methanol. The mixture is cooled and the
`solid collected by f’titration, 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 first
`40 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 purified further by recrystallization from 50%
`methanol in methylene chloride, mp 280*-285* C.
`45 1NMR (d6-DMSO)~I.21 (s, 9H), 1.96-2.15 (m, 2H), 2.44
`(t, J=7.5 Hz, 2H) 3.56 (s, 3I-I), 3.62 (s, 3I-I), 4.40-4.45
`(m, 1H), 7.43 (s, 1H), 7.53 (d, J=8.4 az, 2 H), 7.87 (d,
`J=8.4 Hz, 2 H), 8.82 (d, J=7.4 Hz, 1 H), 10.95 (s, 1H),
`11.95 (s, lift). Anal. Calc. for C27H29NsOT: C, 60.56; H
`50 5.46; N, 13.08. Found: (2, 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-
`55 ethynylbenzoyl)glutamate in the foregoing procedure,
`there are obtained dimethyl N-[5-(4-hydroxy-6-
`pivaloylamln opyrrolo [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-
`60 enoyl]-L-glutamate, and dimethyl N-[6-(4-hydroxy-6-
`pivaloyJaminopyrrolo [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 U.S. Pat.
`65 No. 4,882,334 issued Nov. 21, 1989, the disclosure of
`which is incorporated herein by reference, by allowing
`hex-5-ynoic acid chloride (obtained by treating hex-5-
`ynoic acid with thionyl chloride) to react with dimethyl
`
`Sandoz Inc.
`Exhibit 1034-0006
`
`

`
`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
`
`EXAMPLE 3
`
`Diethyl
`N-J4-( 1-hydroxy-3-(4-hydroxy-06-amitxopyrrolo[2,3-
`d]pyrimidin-3-yl)prop-2-yl)benzoyl]glutamate
`
`10
`
`A mixture of 14.6 g of 3-iodo-4-hydroxy-6-
`pivaloylaralnopyrrolo[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 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-
`lene chloride. This filtrate is concentrated and chro-
`matographed on silica gel eluting with 20:1 ethyl aceta-
`te:hexane mixture to give 3-(3-tetrahydropyr-2-yloxy- 25
`prop-l-yn-l-yl)-4-hydroxy-6-pivaloylaminopyrrolo[2,3-
`d]pyrimidine which is further purified by recrystalliza-
`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 qninoline is stirred under 1 atmosphere hydro-
`gen pressure for 40 rain. 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 ohio-
`fide to remove catalyst and the filtrate then concen-
`trated to give an oil which upon adding ether yields 40
`3-(3-tetrahydropyr-2-yloxyprop- l-ca- l-yl)-4-hydroxy-
`6-pivaloylamlnopyrrolo[2,3-d]pyrimidine which can be
`further purified through column chromatography elut-
`ing with ethyl acetate and recrystallization using ethyl
`acetate. 45
`A mixture containing 3.48 g of 3-(3-tetrahydropyr-2-
`yloxyprop- 1 -en- 1 -yl)-4-hydroxy-6-pivaloylamir~opyr-
`rolo[2,3-d]pyrimidine, 3.12g (1.2 equiv.) of diethyl N-(4-
`iodobenzoyl)glutamate, 546 mg (20%) of tris-(2-methy!-
`phenyl)phosphine, 201 mg (10%) of palladium acetate 50
`and 85.5 mg (5%) of cuprous iodide in 15 ml of triethyl-
`amine and 240 ml of aeetonitrile is heated at reflux
`under nitrogen. After 12 hours., 1.17 g of diethyl N-(4-
`iodobenzoyl)glutamate are added and the reaction mix-
`tare is heated at reflux under nitrogen for an additional
`12 hours. The reaction mixture then is concentrated
`under reduced pressure and the residue chromato-
`graphed on silica gel, during with 20:1 ethyl acetate:-
`hexane. (Any recovered starting material can be recy- 60
`cled through the foregoing procedure.) The concen-
`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
`dro