(12) United States Patent
`US 6,818,633 132
`(10) Patent N0.:
`Balzarini et al.
`(45) Date of Patent:
`Nov. 16, 2004
`
`US006818633B2
`
`(54) ANTIVIRAL COMPOUNDS AND METHODS
`FOR SYNTHESIS AND THERAPY
`
`(75)
`
`Inventors: Jan M. R. Balzarini, Heverlee (BE);
`Erik D. A. De Clercq, Lovenjoel (BE);
`Antonin Holy, Horni Pocernice (CZ)
`
`(73) Assignees: Institute of Organic Chemistry and
`Biochemistry Academy of Sciences of
`the Czech Republic (CZ); Rega
`Stichting v.z.W. (BE)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 10/187,166
`
`(22)
`
`Filed:
`
`Jun. 28, 2002
`
`(65)
`
`Prior Publication Data
`US 2003/0109499 A1 Jun. 12, 2003
`
`(60)
`
`Related US. Application Data
`Provisional application No. 60/302,212, filed on Jun. 29,
`2001.
`
`Int. Cl.7 ...................... A61K 31/662; C07F 9/6512
`(51)
`(52) US. Cl.
`............................ 514/86; 514/88; 544/243
`(58) Field of Search ............................ 544/243; 514/86,
`514/88
`
`(56)
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4/1987 Holy et al.
`4,659,825 A
`2/1988 De Clercq et al.
`4,724,233 A
`2/1989 Holy et al.
`4,808,716 A
`8/1992 Holy et al.
`5,142,051 A
`5/1993 Kim et al.
`5,208,221 A
`4/1994 Yu et al.
`5,302,585 A
`10/1994 Jindrich et al.
`5,352,786 A
`10/1994 Harnden et al.
`5,356,886 A
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`EP
`W0
`W0
`W0
`
`0 269 947 A1
`0 369 409 A1
`0 398 231 A2
`0 434 450 A2
`0 454 427 A1
`0 468 119 A1
`0481 214 A1
`0 618 214 A1
`0 630 381 B1
`WO 94/03467
`WO 95/07920
`WO 96/33200
`
`6/1988
`5/1990
`11/1990
`6/1991
`10/1991
`1/1992
`4/1992
`10/1994
`12/1994
`2/1994
`3/1995
`10/1996
`
`Wormstadt et al., “Synthesis of Acyclic Nucleoside Phos-
`phonates as Antiviral Compounds”, J. Heterocyclic Chem.,
`2000, 37:1187—11191.
`Balzarini et al., “Antiretrovirus Activity of a Novel Class of
`Acyclic Pyrimidine Nucleoside Phosphonates”, Antimicro-
`bial Agents & Chemotherapy, Jul. 2002, 46(7):2185—2193.
`Cihlar et al., “Transport of 9—(2—Phosphonomethoxyethyl)
`Adenine across Plasma Membrane of HeLa S3 Cells .
`.
`. ”
`
`Antimicrob. Agents & Chemo., Jan. 1995, 39(1):117—124.
`Daluge et al., “A Novel Carbocyclic Nucleoside Analogue
`with Potent, Selective Anti—HIV Activity”, Abstracts of the
`34th ICAAC, Oct. 1994, 1592U89.
`Eger et al., “Synthesis of New Acyclic Pyrimidine Nucleo-
`side Analogs as Potential Antiviral Drugs”, J. Med. Chem.,
`1994, 37:3057—3061.
`Franchetti et al., “Acyclic Nucleotides Related to Clitocine:
`Synthesis and Anti—HIV Activity”, Nucleosides & Nucle-
`otides, 1995, 14(3—5):607—610.
`Holy et al., “Synthesis of Quaternary 1—[2—(Phosphono-
`methoxy)ethyl]
`Derivatives
`of
`2,4—Diaminopyrimi-
`dine .
`.
`. ”, Coll. Czech. Chem. Comm., 1999, 64(2):242—
`256.
`
`“6—[2—(Phosphonomethoxy)Alkoxy]Pyrim-
`al.,
`Holy et
`idines with Antiviral Activity”,
`J. Med. Chem., 2002,
`45:1918—1929.
`
`Holy et al., “Structure—Antiviral Activity Relationship in the
`Series of Pyrimidine & Purine N—[2—(2—Phospohnometh-
`oxy)Ethyl] .
`.
`. ”, J. Med. Chem. 1999, 42:2064—2086.
`Holy et al., “Phosphonylmethyl Ethers of Nucleosides &
`Their Acyclic Analogues”, ACS Symp. Ser.,
`1989,
`401:57—71.
`
`Holy et al., “Structure—Activity Studies in the Series of
`Acyclic Nucleotide Analogues”, Kem.
`Ind.,
`1989,
`38(10):457—462.
`
`Primary Examiner—Richard L. Raymond
`(74) Attorney, Agent, or Firm—Max D. Hensley
`
`(57)
`
`ABSTRACT
`
`Novel compounds are provided having formula (I)
`
`(1)
`
`R2
`
`N/
`
`Ax IN
`
`R1
`
`R4
`
`X
`
`flyovHO)(Z)2
`
`R3
`
`OTHER PUBLICATIONS
`
`Where
`
`Holy et al., “Synthesis of Quaternary 1—[2—(Phospho-
`nomethoxy)Ethyl] Derivatives .
`.
`. ”, Coll. Czech. Chem.
`Comm., 1999, Abstract XP—002215043.
`Snoeck et al., “Antivaccinia Activities of Acyclic Nucleo-
`side Phosphonate Derivatives .
`.
`. ,”,Antimocrobial Agents
`& Chem., Nov. 2002, 46(11):3356—3361.
`
`R1, R2, R3, R4, Z, X and * are defined herein. Also
`provided are antiviral methods for use and processes
`for synthesis of the compounds of formula (I).
`
`39 Claims, N0 Drawings
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`US 6,818,633 B2
`
`(1)
`
`R2
`
`N/
`
`Ax IN
`
`R1
`
`R4
`
`X
`
`Eff/0VP(O)(Z)2
`
`R3
`
`Where
`
`R1 is H, amino or methylsulfanyl;
`R2 is H, methyl, halo, —N(R5)2, hydroxy, protected
`hydroxy or a group of the formula (Ia)
`
`(Ia)
`
`—X
`
`*
`\/l\o/\ P(O)(Z)2
`R3
`
`R3 is independently H, methyl, hydroxymethyl, halom-
`ethyl or protected hydroxymethyl;
`R4 is H or halo;
`X independently is oxygen, sulfur or a bond;
`Z independently is hydroxy, an ester or amide;
`R5 is independently H, C1—C8 alkyl or a protecting group;
`and
`
`* designates a chiral carbon atom; and
`salts and solvates thereof.
`
`The objects also are accomplished by a method for
`preparation of compounds of the formula (I)
`
`It is an object of this invention to provide compounds
`having antiviral activity, in particular against RNA or DNA
`viruses such as HIV, HBV or HSV.
`
`55
`
`It is an additional object to provide compounds useful in
`the preparation of ion exchange resins or chiral media.
`It is a further object to provide intermediates and methods
`for making such compounds.
`These and other objects Will be more fully understood by
`further reference to the disclosures herein.
`
`SUMMARY OF THE INVENTION
`
`60
`
`65
`
`In accordance With the invention, novel compounds are
`provided having formula (I)
`
`R2
`
`N/ IxR1
`
`N
`
`R4
`
`XH
`
`Where
`
`R1 and R5 are defined above;
`R2 is H, methyl, halo, —N(R5)2, hydroxy or protected
`hydroxy; and
`XisOorS;
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`(1)
`
`(11)
`
`1
`ANTIVIRAL COMPOUNDS AND METHODS
`FOR SYNTHESIS AND THERAPY
`
`CROSS REFERENCED TO RELATED
`APPLICATIONS
`
`This application is based upon US. Provisional Applica-
`tion Ser. No. 60/302,212 filed Jun. 29, 2001, and is incor-
`porated herein by reference.
`
`BACKGROUND OF THE INVENTION
`
`FIELD OF THE INVENTION
`
`Acyclic nucleotide analogues containing phosphonate
`groups are disclosed for example in US. Pat. Nos. 4,659,
`825, 4,808,716, 4,724,233, 5,142,051, 5,302,585, 5,208,221,
`5,352,786, 5,356,886, in EP publication numbers 269,947,
`481,214, 630,381, 369,409, 454,427, 468,119, 434,450, 618,
`214 and 398,231 and in WO 95/07920, WO 94/03467 and
`WO 96/33200. The teachings of these patents include com-
`pounds in Which a phosphonate group is linked to a defined
`purine or pyrimidine base, generally at the 1- or 9-position
`of the pyrimidine or purine bases, respectively, by way of a
`2-(methoxy)propyl group, a 2-(methoxy)ethyl group, a
`2-methoxy-3-hydroxypropyl group, or a 2-methoxy-3-
`fluoropropyl group, known respectively as PMP, PME,
`HPMP and FPMP purine or pyrimidine compounds. These
`compounds exhibit antiviral and cytostatic activity.
`
`Daluge et al. (34th Interscience Conference on Antimi-
`crobial Agents and Chemotherapy, Oct. 4—7, 1994) discloses
`carbovir derivatives in Which the 6 position of the purine is
`substituted With cyclopropylamino, N-cyclopropyl-N-
`methylamino or N-aziridinyl.
`Cihlar et al., “Antimicrobial Agents and Chemotherapy”
`39(1):117—124 (1995) disclose NG-aminohexyl-PMEDAP.
`Holy et al., “ACS Symp. Ser.” 401:57—71 (1989) and
`Holy, “Kem.
`Ind.” 38(10):457—462 (1989) describe the
`antiviral activity of certain NG-substituted nucleotide ana-
`logues.
`Additional phosphonate-substituted pyrimidine analogues
`are disclosed by Holy et al., “Collect. Czech. Chem. Com-
`mun.” 64:242—256 (1999), Eger et al., “J. Med. Chem.”
`37:3057—3061 (1994), Wormstadt et al., “J. Heterocyclic
`Chem.” 37:1187—1191 (2000), and Franchetti et al.,
`“Nucleosides & Nucleotides” 14(3—5): 607—610(1995). The
`latter three publications have a phosphonate-containing
`side-chain linked via a 6-N substituent of 2,4-disubstituted
`pyrimidine.
`
`OBJECTS OF THE INVENTION
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`R
`
`4
`
`x K
`
`R3
`
`fiovP(O)(Z)2
`
`R2
`
`N/ l
`AR1
`
`N
`
`50
`
`Where
`
`R1, R2, R3, R4, X, Z, R5 and * are defined above;
`comprising reacting a compound of formula (II)
`
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`

`US 6,818,633 B2
`
`4
`R5 independently is H, C1—C8 alkyl or a protecting group;
`X is oxygen or sulfur;
`Z independently is hydroxy, an ester or amide; and
`* designates a chiral carbon atom;
`
`comprising reacting compound (IVa)
`
`(IVa)
`
`Cl
`
`N/
`
`P 0 Z
`O
`*
`k l
`\N X/Y V (
`)(
`)2
`
`R3
`
`HZN
`
`With N(R5)2 in anhydrous solvent, alkali hydroxide or alkali
`carbonate in aqueous solution and Z is optionally converted
`to the compound of formula (V) wherein 1 or 2 Z groups are
`hydroxy.
`In another embodiment, a method is provided for the
`preparation of compounds of formula (VI)
`
`(V1)
`
`NH2
`
`N/
`
`Ax IN
`
`R1
`
`0
`
`flyovP(O)(Z)2
`
`R3
`
`Where
`
`R1 is H, amino or methylsulfanyl;
`R3 is H, methyl, hydroxymethyl, halomethyl or protected
`hydroxymethyl;
`Z independently is hydroxy, an ester or amide; and
`* designates a chiral carbon atom;
`comprising reacting a compound of formula (VII)
`
`NH2
`
`N/ IAR1
`
`N
`
`C1
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`With a compound of the formula (III)
`
`3
`
`*
`
`(III)
`
`Y— CHZCH(R3)—O—CH2P(O)(Z)2
`
`Where
`
`Z is an ester or an amide;
`* designates a chiral carbon atom;
`R3 is H, methyl, halomethyl or protected hydroxymethyl;
`and
`
`Y is a leaving group
`in dipolar aprotic solvent in the presence of a base to obtain
`a compound of formula (I) Where Z is ester or amide; (b) one
`or both Z groups optionally are converted to produce the
`compound of formula (I) Where at least one Z is hydroxy.
`In another embodiment of this invention, a method is
`provided for the preparation of compounds of formula (I)
`Where
`
`R1 is H, amino or methylsulfanyl;
`R2 is —N(R5)2
`R3 is independently H, methyl, hydroxymethyl, halom-
`ethyl or protected hydroxymethyl;
`R4 is H or halo
`X is oxygen or sulfur;
`Z independently is hydroxy, an ester or amide;
`R5 is independently H, C1—C8 alkyl or a protecting group;
`and
`
`* designates a chiral carbon atom
`comprising reacting a compound (IV)
`
`(1V)
`
`X K
`
`R3
`
`rovP(O)(Z)2
`
`OH
`
`N / lAR1
`
`N
`
`Where
`
`R3 is H, methyl, halomethyl or protected hydroxymethyl;
`X is O or S; and
`Z is amide or ester;
`With N(R5)2. One or both Z groups optionally are converted
`to the compound of formula (I) Where at least one Z is
`hydroxy.
`In another embodiment, a method is provided for prepa-
`ration of compounds of formula (V)
`
`(v11)
`
`(VIII)
`
`N(Rs)2
`
`N/
`
`P 0 Z
`O
`*
`A l
`\N X/Y V ( X )2
`
`R3
`
`HZN
`
`55
`
`(V)
`
`Where
`
`R1 is H, amino or methylsulfanyl
`With a compound of the formula (VIII)
`
`*
`
`HOCHZCH(R3)OCH2P(O)(Z)2
`
`60
`
`65
`
`Where
`
`R3 is H, methyl, hydroxymethyl, halomethyl or protected
`hydroxymethyl;
`
`Where Z is amide or ester in the presence of a base.
`Optionally one or both Z groups are converted to produce a
`hydroxy.
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`

`5
`In another embodiment of this invention, a method is
`provided for the preparation of compounds of formula (XIII)
`
`6
`R2 is H, chloro or amino; and
`R3 is H, methyl, halomethyl or protected hydroxymethyl;
`and
`
`US 6,818,633 B2
`
`(XIII)
`
`5
`
`(b) reacting compound (XI) With a compound of the
`formula (XII)
`
`Y—CH2P(O)(OZ)2
`
`Y is a leaving group;
`
`(XII)
`
`Where
`Z is amide or ester in the presence of a base in dimeth-
`
`ylformamide or tetrahydrofurane to produce a com-
`pound of formula (XIII); and
`
`(c) optionally hydrolyzing Z group in compound (XIII) to
`produce a compound of formula (VI) Where 1 or 2 Z
`groups are hydroxy and X is oxygen atom.
`In another embodiment of this invention, a method is
`giggled for the preparation of compounds of formula (I)
`
`R1 is H, am1no or methylsulfanyl;
`R3 is H, methyl, hydroxymethyl, halomethyl or protected
`hydroxymethyl;
`R4 is halo;
`X is oxygen;
`
`10
`
`15
`
`20
`
`25
`
`Z independently is hydroxy, an ester or amide; and
`*
`.
`.
`.
`designates a chiral carbom atom,
`comprising (a) reacting a compound of the formula (VI)
`30 Where
`R1 is H, amino or methylsulfanyl;
`R3 is H, methyl, hydroxymethyl, halomethyl or protected
`hydroxymethyl;
`Z independently is an ester; and
`* designates a chiral carbon atom;
`With elemental halogen in an inert solvent to produce a
`d f f
`1
`I
`compoun o
`ormu a ( ).
`Optionally one or both Z groups are converted to hydroxy.
`Further objects of this invention are accomplished by a
`method comprising administering a therapeutically effective
`amount of a compound of formula (I) to a patient in need of
`such treatment.
`
`40
`
`35
`
`R2
`
`N / I
`MAN
`
`0
`RyovP(O)(Z)2
`
`R3
`
`Where
`R1 is H, amino or methylsulfanyl;
`* is a chiral carbon atom;
`R2 is H, chloro, hydroxy or amino;
`R3 is H, methyl, halomethyl or hydroxymethyl; and
`Z is amide or ester;
`comprising (a) reacting a compound of the formula (IX)
`
`R2
`
`/
`N
`A
`R1
`
`N
`
`I
`
`C1
`
`Where
`
`R1 is H, amino or methylsulfanyl;
`R2 is H, chloro or amino;
`With a compound 0f the formula (X)
`
`*
`
`R3
`>
`Rs
`
`\
`
`OH
`
`Where
`
`(IX)
`
`(X)
`
`45
`
`R3 is H, methyl, hydroxymethyl, halomethyl or protected
`DETAILED DESCRIPTION OF THE
`hydroxymethyl;
`INVENTION
`* is a chiral carbon atom;
`As used herein, and unless modified by the immediate
`R6 is hydroxy or protected hydroxy;
`context, alkyl means branched, normal or cyclic saturated
`or R3 and R6 are joined by a cyclic acetal or ketal
`hydrocarbons and includes methyl, ethyl, propyl,
`protecting group;
`in the presence of a base Without solvent or in the presence 50 CyC10pr0py1, cyclobutyl, isopropyl, n-; sec-, iso- and tert-
`of an aprotic solvent to produce a compound of formula (XI)
`butyl; pentyl,
`isopentyl, 1-methylbutyl, 1-ethylpropyl,
`neopentyl, and t-pentyl.
`Halo typically means chloro, but includes bromo, fiuoro,
`or iodo.
`
`(X1)
`
`A
`R1
`
`N
`
`I
`
`O
`
`*
`
`OH
`
`R3
`
`Where
`R1 is H, amino or methylsulfanyl;
`* is a chiral carbon atom;
`
`55
`
`60
`
`R1 typically is H or amino, but also can be methylsulfanyl
`(i.e. methylthio).
`R2 generally is hydroxy or —N(R5)2 Where R5 indepen-
`dently is H or C1—C8 alkyl.
`R3 typically is H or methyl, but may be hydroxymethyl
`(typically (S) configuration substantially free of the (R)
`enantiomer) or halomethyl, and, if methyl or halomethyl,
`preferably is in the (2R) configuration substantially free of
`the (2S) configuration. Halomethyl generally is fiuorom-
`65 ethyl.
`R5 generally is H, but also can be lower (C1—C3) alkyl
`(one or both instances).
`
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`

`US 6,818,633 B2
`
`7
`As is further described infra, Z is suitably any ester or
`amide heretofore known for use with nucleotide phospho-
`nates. When Z is an ester,
`it has the structure OR7. R7
`ordinarily is H (i.e., Z is hydroxy) in compounds having
`antiviral activity per se, although other R7 ester groups
`described below are suitable for use as protecting groups or
`as pro-functionalities for prodrug embodiments.
`X preferably is O.
`Z is an ester or amide when it is desired to protect the
`compounds of this invention against undesired reactions or
`when the object is to provide an in vivo prodrug of the
`compound. Otherwise, Z is OH.
`The esters or amides are useful as protected intermediates
`in the synthesis of compounds of this invention where
`Z=OH. In this embodiment, the selection of ester or amide
`may not be important, depending upon the nature of the
`reaction involved. All that is needed is that the Z substituent
`not be removed until the step in synthesis at which this is
`desired, and if this is not apparent on theoretical grounds it
`can be readily determined by rudimentary experiments. For
`example, esters in particular are used to protect the phos-
`phonate hydroxy groups against alkylation.
`When Z serves as a prodrug functionality, the ester or
`amide is removed in vivo from the phosphonate. Suitable
`prodrug esters or amidates optionally are selected based on
`the substrate specificity of esterases and/or carboxypepti-
`dases expected to be found within cells where precursor
`hydrolysis is desired. To the extent that the specificity of
`these enzymes is unknown, one will screen a plurality of
`nucleotide analogues of this invention until
`the desired
`substrate specificity is found. This will be apparent from the
`appearance of free phosphonate or antiviral activity. One
`generally selects compounds that are (i) not hydrolyzed or
`hydrolyzed comparatively slowly in the upper gut, (ii) gut
`and cell permeable and (iii) are hydrolyzed in the cell
`cytoplasm and/or systemic circulation. Screens with cells
`from particular tissues are used to identify precursors that
`are released in organs susceptible to a target viral or micro-
`bial
`infection, e.g.
`in the case of liver, precursor drugs
`capable of hydrolysis in the liver. Other infections, e.g.
`CMV or HIV, optionally are treated with a precursor that is
`hydrolyzed at substantially the same rate and to substantially
`the same degree in all tissues. Assays known in the art are
`suitable for these purposes,
`including intestinal
`lumen
`stability, cell permeation,
`liver homogenate stability and
`plasma stability assays. These assays are used to determine
`the bioavailability characteristics of the precursors.
`Typical examples of ester and amide substituents group Z
`are found in WO95/07920, WO98/04569 and EP 481214
`A1. Any ester or amide genus or species described in these
`publications (and in the preference order set forth in such
`publications) can be used as group Z herein.
`Usually, both Z are hydroxyl or both are ester and/or
`amide,
`i.e,
`typically 2 or no Z groups are hydroxy. In
`general, when neither Z is OH then one Z is amide and one
`is ester. Amides with naturally occurring amino acids and a
`esters with phenyl are preferred. The free carboxyl(s) of
`amino acid Z groups generally are esterified with C1—C8
`alkyl.
`In general, Z is hydroxy in compounds to be used directly
`for antiviral purposes, i.e. such compounds are employed
`without any requirement for hydrolysis in vivo of the ester
`or amide.
`
`Protecting groups for hydroxyl include acetals, ketals or
`C1—C8 alkyl. A typical protecting group for amino is trityl.
`Other conventional protecting groups are known (Greene et
`al., “Protecting Groups in Organic Synthesis, 2”d Ed. 1991,
`pp. 10—142 and 309—405).
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`8
`Utilities
`
`The compounds of the invention are useful for the treat-
`ment of viruses, or as intermediates in the preparation of
`such compounds. Exemplary viral infections to be treated or
`to be tested for susceptibility to compounds of this invention
`include infections caused by DNA or RNA viruses such as
`herpesviruses (CMV, HSV 1, HSV 2, EBV, varicella zoster
`virus [VZV], bovid herpesvirus type 1, equid herpesvirus
`type 1, HHV—6, papillomaviruses (HPV types 1—55 includ-
`ing carcinogenic HPV), fiaviviruses (including yellow fever
`virus, African swine fever virus and Japanese encephalitis
`virus), togaviruses (including Venezuelan equine encepha-
`lomyelitis virus), influenza viruses (types A—C), retroviruses
`(HIV-1, HIV-2, HTLV-I, HTLV-II, SIV, FeLV, FIV,
`MoMSV), adenoviruses (types 1—8), poxviruses (vaccinia
`virus), enteroviruses (poliovirus types 1—3, Coxsackie, hepa-
`titis A virus, and ECHO virus), gastroenteritis viruses
`(Norwalk viruses,
`rotaviruses), hantaviruses (Hantaan
`virus), polyomavirus, papovaviruses, rhinoviruses, parain-
`fiuenza virus types 1—4, rabies virus, respiratory synctial
`virus (RSV), hepatitis viruses A, B, C and E, and the like.
`Preferred compounds of this invention for the treatment of
`herpes viruses, hepadna viruses and HIV are those in which
`R1=NH2, R2=NH2 or OH, X=O and R3=H or methyl. Other
`antiviral activities of compounds of this invention are deter-
`mined by routine assay of antiviral activity using enzyme
`inhibition assays, tissue culture assays, animal model assays
`and the like as will be understood by those skilled in the art.
`The novel compounds of this invention also are useful per
`se or as intermediates in the preparation of polymers having
`a wide variety of diagnostic,
`therapeutic and industrial
`utilities.
`
`The compounds of this invention are suitable as interme-
`diates to prepare affinity absorption media bearing substitu-
`ent groups having properties useful for absorbing com-
`pounds from impure mixtures. These are prepared and used
`in the same fashion as other ion exchange media containing
`the same substituents, e.g. phosphonate or amino. For
`example, the phosphonate group of the compounds herein
`are covalently bound to insoluble matrix and free R1 amino
`substituents on the heterocyclic base serve as ion exchange
`sites. Alternatively, the heterocyclic base amino group is
`linked to the matrix and the free phosphonate group is then
`useful
`in the chromatographic absorption of positively
`charged molecules. Other immobilized embodiments of the
`compounds herein are useful in purifying proteins, e.g.,
`enzymes to which the compounds of this invention may
`bind, e.g. transport proteins (see Cihlar, supra).
`Suitable methods of incorporation of the compounds of
`this invention into insoluble matrices such as polymeric
`resins will be readily apparent to the skilled artisan. The
`compounds herein can be immobilized by covalently
`crosslinking the pyrimidine amino or hydroxy groups to an
`insoluble matrix. Similarly, compounds of this invention are
`incorporated into insoluble resins by binding the hydroxy of
`the phosphonate group or a hydroxymethyl R3 group to the
`matrix or resin using covalent
`linking agents heretofore
`known. Suitable linking methods are described in Cihlar
`(supra).
`The compounds of this invention also are useful as
`cross-linkers or spacers in preparing affinity absorption
`matrices (as opposed to functioning as affinity moieties per
`se as noted in the preceding paragraphs). The compounds
`herein contain a multiplicity of functional groups that are
`suitable as sites for cross-linking desired substances. It is
`conventional to link affinity reagents such as hormones,
`peptides, antibodies, enzymes, drugs, and the like to
`insoluble substrates. These insolubilized reagents are
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`US 6,818,633 B2
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`9
`employed in known fashion to absorb substances from
`manufactured preparations, diagnostic samples and other
`impure mixtures. Similarly, immobilized enzymes are used
`to perform catalytic conversions with facile separation of
`enzyme from product.
`In some embodiments, it is not necessary that the com-
`pounds of this invention be cross-linked to insoluble mate-
`rials. For example, they can be used to link analytes to
`detectable groups in preparing soluble diagnostic reagents.
`Methods for cross-linking using the substituent groups
`found in the compounds of this invention are well known in
`the art. For example, the phosphonic acid is used to form
`esters with alcohols or amides with amines. Similarly, the
`amino, halo, hydroxy and other reactive sites found on the
`pyrimidine are suitable. Of course, protection of reactive
`groups will be used where necessary while assembling the
`cross-linked reagent. In general, the inventive compounds
`are used by linking them through phosphonic acid to the
`hydroxy or amino groups of the linking partner, and
`covalently bonded to the other binding partner through
`another substituent of the compound of this invention. For
`example a first binding partner such as a steroid hormone is
`esterified to the phosphonic acid of this invention and then
`this conjugate is cross-linked through R3 hydroxymethyl to
`cyanogen bromide activated Sepharose, whereby the immo-
`bilized steroid is obtained. Other chemistries for conjugation
`are well known. See,
`for example Maggio, “Enzyme-
`Immunoassay” (CRC, 1988, pp 71—135) and references
`cited therein.
`
`Pharmaceutical Formulations
`
`The compounds of this invention and their physiologi-
`cally acceptable salts and solvates (hereafter collectively
`referred to as the active ingredients) are formulated for
`administration by any route appropriate to the condition to
`be treated. The compounds and formulations preferably will
`be sterile.
`
`The active ingredients are placed into pharmaceutical
`formulations. The formulations, both for veterinary and for
`human use, comprise at least one active ingredient, as above
`defined, together with one or more acceptable carriers there-
`for and optionally other therapeutic ingredients. The carrier
`(s) must be “acceptable” in the sense of being compatible
`with the other ingredients of the formulation and not del-
`eterious to the recipient.
`The formulations conveniently are presented in unit dos-
`age form and may be prepared by any of the methods well
`known in the art of pharmacy. In general the formulations
`are prepared by uniformly and intimately bringing into
`association the active ingredient with liquid carriers or finely
`divided solid carriers or both, and then, if necessary, shaping
`the product.
`Formulations of the present invention suitable for oral
`administration may be presented as discrete units such as
`capsules, cachets or tablets each containing a predetermined
`amount of the active ingredient; as a powder or granules; as
`solution or a suspension in an aqueous liquid or a non-
`aqueous liquid; or as an oil-in-water liquid emulsion or a
`water-in-oil liquid emulsion. The active ingredient may also
`be presented as a bolus, electuary or paste.
`For external infections of the eye or other external tissues
`e.g. mouth and skin, the formulations are preferably applied
`as a topical ointment or cream containing the active
`ingredient(s) in an amount of, for example, 0.075 to 20%
`w/w (including active ingredient(s) in a range between 0.1%
`and 20% in increments of 0.1% w/w such as 0.6% w/w,
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`10
`
`0.7% w/w, etc), typically 0.2 to 15% w/w and most typically
`0.5 to 10% w/w. When formulated in an ointment, the active
`ingredients may be employed with either a paraffinic or a
`water-miscible ointment base. Alternatively,
`the active
`ingredients may be formulated in a cream with an oil-in-
`water cream base.
`
`the aqueous phase of the cream base may
`If desired,
`include, for example, at least 30% w/w of a polyhydric
`alcohol, i.e. an alcohol having two or more hydroxy groups
`such as propylene glycol, butane 1,3-diol, mannitol, sorbitol,
`glycerol and polyethylene glycol (including PEG 400) and
`mixtures thereof. The topical formulations may desirably
`include a compound which enhances absorption or penetra-
`tion of the active ingredient
`through the skin or other
`affected areas. Examples of such dermal penetration enhanc-
`ers include dimethyl sulphoxide and related analogues.
`The oily phase of the emulsions of this invention may be
`constituted from known ingredients in a known manner. This
`phase may comprise an emulsifier alone, or a mixture of at
`least one emulsifier with a fat or an oil or with both a fat and
`
`an oil. Preferably, a hydrophilic emulsifier is included
`together with a lipophilic emulsifier which acts as a stabi-
`lizer. It is also preferred to include both an oil and a fat.
`Emulsion stabilizers suitable for use in the formulation of
`
`the present invention include Tween® 60, Span® 80, ceto-
`stearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl
`mono-stearate and sodium lauryl sulfate. Suitable oils or fats
`include straight or branched chain, mono- or dibasic alkyl
`esters such as di-isoadipate,
`isocetyl stearate, propylene
`glycol diester of coconut fatty acids, isopropyl myristate,
`decyl oleate,
`isopropyl palmitate, butyl stearate or
`2-ethylhexyl palmitate. These may be used alone or in
`combination depending on the properties required.
`Alternatively, high melting point lipids such as white soft
`paraffin and/or liquid paraffin or other mineral oils can be
`used.
`
`Formulations suitable for topical administration to the eye
`also include eye drops wherein the active ingredient
`is
`dissolved or suspended in a suitable carrier, especially an
`aqueous solvent for the active ingredient. The active ingre-
`dient
`is typically is present
`in such formulations in a
`concentration of 0.01 to 20% by weight.
`Formulations suitable for nasal administration wherein
`
`the carrier is a solid include a coarse powder having a
`particle size for example in the range 20 to 500 microns
`(including particle sizes in a range between 20 and 500
`microns in increments of 5 microns such as 30 microns, 35
`microns, etc), which is administered by rapid inhalation
`through the nasal passage from a container of the powder.
`Suitable formulations wherein the carrier is a liquid, for
`administration as for example a nasal spray or as nasal
`drops, include aqueous or oily solutions of the active ingre-
`dient. Formulations suitable for aerosol administration may
`be prepared according to conventional methods and may be
`delivered with other therapeutic agents such as pentamidine
`for treatment of pneumocystis pneumonia.
`Formulations suitable for vaginal administration may be
`presented as pessaries, tampons, creams, gels, pastes, foams
`or spray formulations containing in addition to the active
`ingredient such carriers as are known in the art
`to be
`appropriate.
`Formulations suitable for parenteral administration
`include aqueous and non-aqueous sterile injection solutions
`which may contain anti-oxidants, buffers, bacteriostats and
`solutes which render the formulation isotonic with the blood
`
`of the intended recipient; and aqueous and non-aqueous
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`US 6,818,633 B2
`
`11
`sterile suspensions which may include suspending agents
`and thickening agents. The formulations may be presented in
`unit-dose or multi-dose containers,
`for example sealed
`ampoules and vials, and may be stored in a freeze-dried
`(lyophilized) condition requiring only the addition of the
`sterile liquid carrier, for example water for injections, imme-
`diately prior to use. Extemporaneous injection solutions and
`suspensions may be prepared from sterile powders, granules
`and tablets of the kind previously described. Preferred unit
`dosage formulations are those containing a daily dose or unit
`daily sub-dose, as herein above recited, or an appropriate
`fraction thereof, of an active ingredient.
`The present invention further provides veterinary compo-
`sitions comprising at least one active ingredient as above
`defined together with a veterinary carrier therefor. Veteri-
`nary carriers are materials for administering the composition
`and may be solid, liquid or gaseous materials which are
`otherwise inert or acceptable in the veterinary art and are
`compatible with the active ingredient. These veterinary
`compositions may be administered orally, parenterally or by
`any other desired route.
`Compounds herein optionally are used in controlled
`release pharmaceutical formulations containing as active
`ingredient one or more active compounds in which the
`release of the active ingredient is controlled and regulated to
`allow less frequent dosing or to improve the pharmacoki-
`netic or toxicity profile of a given compound. In general, the
`compounds are administered from controlled release sys-
`tems such as the implant of WO 92/14450 or US. Pat. No.
`5,098,443, or the matrices of US. Pat. No. 4,740,365 or US.
`Pat. No. 5,141,752. Many others are known and are suitable
`for use herein.
`
`Therapeutic Administration
`
`Suitable routes for administration include oral, rectal,
`nasal,
`topical (including ocular, buccal and sublingual),
`vaginal and parenteral
`(including subcutaneous,
`intramuscular,
`intravitreous,
`intravenous,
`intradermal,
`intrathecal and epidural). The preferred route of administra-
`tion will depend upon the condition of the patient,
`the
`toxicity of the compound and the site of infection, among
`other considerations known to the clinician.
`
`For each of the above-indicated therapeutic indications
`the amount required of an active ingredient (as above
`defined) will depend upon a number of factors including the
`severity of the condition to be treated, the infectious agent,
`whether the use is prophylactic or to treat an acute infection,
`the site of infection or pathology and other factors ultimately
`at the discretion of the attending physician or veterinarian.
`In general, however, a suitable dose for consideration by the
`clinician will be in the range of analogous methoxyphos-
`phonates (see supra),
`taking into account differences in
`potency in in vitro testing, generally 0.1 to 250 mg per
`kilogram bodyweight of recipient per dose (including active
`ingredient(s) in a range between 0.1 mg and 400 mg/Kg/
`dose in increments of 0.5 mg/Kg/dose such as 2.5 mg/Kg/
`dose, 3.0 mg/Kg/dose, 3.5 mg/Kg/dose, etc), typically in the
`range 0.5 to 50 mg per kilogram body weight per dose and
`most usually in the range 1 to 300 mg per kilogram body
`weight per dose.
`The desired dose is administered at appropriate intervals
`in unit dosage forms, usually with a relatively higher induc-
`tion dose and lower, less frequent maintenance doses. The
`compounds also are used prophylactically, for example, by
`administration on about from 1 to 7 days before viral
`infection. HPV tumors or growths and herpes lesions often
`
`10
`
`15
`
`20
`
`25
`
`