WO2012150866A1 - Phosphoribosyltransferase inhibitors and uses thereof - Google Patents

Phosphoribosyltransferase inhibitors and uses thereof Download PDF

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Publication number
WO2012150866A1
WO2012150866A1 PCT/NZ2012/000053 NZ2012000053W WO2012150866A1 WO 2012150866 A1 WO2012150866 A1 WO 2012150866A1 NZ 2012000053 W NZ2012000053 W NZ 2012000053W WO 2012150866 A1 WO2012150866 A1 WO 2012150866A1
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Prior art keywords
nmr
mhz
compound
methyl
hydroxy
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PCT/NZ2012/000053
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French (fr)
Inventor
Keith Clinch
Douglas Ronald Crump
Gary Brian Evans
Keith Zachary HAZLETON
Jennifer Mary Mason
Vern L. Schramm
Peter Charles Tyler
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Industrial Research Limited
Albert Einstein College Of Medicine Of Yeshiva University
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Publication of WO2012150866A1 publication Critical patent/WO2012150866A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates generally to compounds that are inhibitors of hypoxanthine and/or guanine purine phosphoribosyltransferases and to pharmaceutical compositions containing the compounds, processes for preparing the compounds, and methods of treating diseases or conditions in which it is desirable to inhibit hypoxanthine and/or guanine purine phosphoribosyltransferases, e.g.
  • hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferases of protozoan parasites including those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani.
  • the diseases or conditions include malaria.
  • BACKGROUND Malaria is a major global health concern. It is estimated that malaria is responsible for greater than 200 million clinical cases and about 800000 deaths each year. The disease is caused by a parasite of the genus Plasmodium, which is transmitted by infected Anopheles mosquitos. There are four parasites that cause human malaria - Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. Plasmodium falciparum is considered to be the deadliest.
  • the vaccines for malaria currently in clinical trials confer limited protection and there is a problem with the emergence of resistance to current therapies such as artemisinin and its derivatives.
  • Plasmodium falciparum lacks the ability to synthesise purines de novo (Reyes, P., Rathod, P.K., Sanchez, D.J., Mrema, J.E., Rieckmann, K.H., and Heidrich, H.G. Mol. Biochem. Parasitol., 1982, 5, 275-290).
  • the parasite lacks adenosine kinase or adenine phosphoribosyltransferase activity and relies on the conversion of hypoxanthine to inosine 5'-monophosphate by hypoxanthine-guanine-xanthine phosphoribosyltransferase (PflHGXPRT) as its source of purines (Cassera, M.B., Hazleton, K.Z., Riegelhaupt, P.M., Merino, E.F., Luo, M., Akabas, M.H., and Schramm, V.L. J. Biol. Chem., 2008, 283, 32889-32899).
  • PflHGXPRT hypoxanthine-guanine-xanthine phosphoribosyltransferase
  • PNP purine nucleoside phosphorylase
  • HsHGPRT human hypoxanthine-guanine phosphoribosyltransferase
  • Keough et al. demonstrated inhibition of PflHGXPRT by acyclic nucleoside phosphonates (D.T. Keough, D. Hockova, A. Holy, L M. J. Naesens, T. S. Skinner-Adams, J. de Jersey, L. W. Guddat, J. Med. Chem, 2009, 52, 4391-4399; D. Hockova, A. Holy, M. Masojidkova, D.T. Keough, J. de Jersey, L. W. Guddat, Bioorg. Med. Chem., 2009, 17, 6218-6232).
  • Some of the phosphonate compounds incorporating a guanine or hypoxanthine base and a phosphonate 'tail' attached to the base, have high nanomolar to micromolar inhibition constants ( ,) against both PflHGXPRT and human hypoxanthine-guanine phosphoribosyltransferase (HsHGPRT).
  • HsHGPRT human hypoxanthine-guanine phosphoribosyltransferase
  • Branched acyclic nucleoside phosphonates are weak inhibitors of PflHGXPRT and HsHGPRT. The best compound had Ki of 100 ⁇ 20 nM for P/HGXPRT and Ki of of 1.0 ⁇ 0.5 ⁇ for HsHGPRT - a moderate selectivity of 10-fold.
  • the acyclic nucleoside phosphonates are substrate analogues.
  • Transition state analogues are attractive as chemotherapeutics. They can bind more strongly to an enzyme than, for example, substrate analogues.
  • HGXPRTs have resisted transition state analysis because of kinetic commitment factors. Nevertheless, the applicants have proposed a transition state structure for PflHGXPRT (C. M. Li et al., Nat. Struct. Biol., 1999, 6, 582-587) and reported that a nucleoside analogue, Immucillin-H 5'-phosphate (ImmHP) is a 1 nM inhibitor of P/HGXPRT, but with no selectivity relative to HsHGPRT.
  • ImmHP Immucillin-H 5'-phosphate
  • hypoxanthine and/or guanine purine phosphonbosyltransferases such as hypoxanthine and/or guanine and/or xanthine purine phosphonbosyltransferases of protozoan parasites, or to at least provide a useful choice.
  • the present invention provides a compound of the formula (I):
  • A is CH, CR 2 or N
  • D is H, OH or NH 2 ;
  • R 2 is halogen, alkyl, aralkyl or aryl; and n is 1 ;
  • R 1 is a radical of formula (i) where G is O; X is an optionally substituted C 3 or C 5 alkylene group and R 1 is attached to a terminal carbon atom of X; or n is 1 ;
  • R 1 is a radical of formula (i) where G is absent; X is an optionally substituted C 3 or C 4 alkylene group and R 1 is attached to a terminal carbon atom of X; or n is 2;
  • R is a radical of formula (i) where G is O or is absent; X is an optionally substituted C 2 alkylene group and R 1 is attached to a terminal carbon atom of X;
  • ester prodrug form of the compound of formula (I) is one in which one or more hydrogens in the group R 1 is replaced with one or more lipophilic groups, e.g. one or more alkoxyalkyl groups. It is further preferred that the ester prodrug form of the compound of formula (I) is one in which R 1 is a radical of formula (ii):
  • Z is -(CH 2 ) m -0-(CH 2 )p-CH 3 ;
  • Y is H, alkyl or -(CH 2 ) m -0-(CH 2 ) p -CH 3 ;
  • G is O or G is absent
  • n 2 or 3
  • p is an integer from 2 to 21 ;
  • each m and each p is independently selected.
  • the compound of formula (I) is an ester prodrug compound of formula (la):
  • A is CH, CR 2 or N
  • D is H, OH or NH 2 ;
  • R 2 is halogen, alkyl, aralkyl or aryl; and n is 1 ;
  • R is a radical of formula (ii) where G is O; Z is -(CH 2 )m-0-(CH2)p-CH 3 ; Y is H, alkyl or -(CH 2 )m-0-(CH2)p-CH 3 ;
  • X is an optionally substituted C 3 or C 5 alkylene group and R 1 is attached to a terminal carbon atom of X; or n is 1 ;
  • R 1 is a radical of formula (ii) where G is absent; Z is -(CH2) m -0-(CH2)p-CH 3 ; Y is H, alkyl or -(CH 2 ) m -0-(CH2)p-CH 3 ;
  • X is an optionally substituted C 3 or C 4 alkylene group and R 1 is attached to a terminal carbon atom of X; or n is 2;
  • R 1 is
  • m is 2 or 3 and p is an integer from 2 to 21 and where, when Z is -(CH 2 ) m -0- (CH 2 ) P -CH 3 and Y is -(CH 2 ) m -0-(CH2) p -CH 3 , each m and each p is independently selected; or a tautomer thereof, or a pharmaceutically acceptable salt thereof.
  • G is absent.
  • the C 2 , C 3 , C 4 or C 5 alkylene group X in the above formula (I) or (la) may optionally be substituted with one or more hydroxy groups and/or one or two fluorine atoms.
  • G when G is absent, the one or two fluorine atoms are attached to the terminal carbon of X, to which the group R 1 is also attached.
  • A is CH or N, more preferably CH.
  • D is H or NH 2 , most preferably D is H.
  • A is CH and D is H. In other examples A is N and D is H. In still other examples, A is CH and D is NH 2 .
  • the compound of formula (I) is a compound of formula ( ⁇ ):
  • X is a C 3 or C 4 alkylene group which is substituted with one or more hydroxy groups and/or one or two fluorine atoms.
  • the compound of formula (la) is a compound of formula (la'):
  • the compound of formula (la) is a compound of formula
  • the compound of formula (la) is a compound of formula (la'"):
  • n is 3. In other examples m is 2.
  • p is an integer from 7 to 21 , e.g. an integer from 7 to 17. In some examples p is 17. In other examples p is 15. In still other examples p is 7.
  • R 1 is a radical of formula (ii) where Z is -(CH 2 )m-0-(CH 2 )p-CH3 and Y is alkyl, e.g. lower alkyl, e.g. ethyl. In other examples R 1 is a radical of formula (ii) where Z is -(CH2)m-0-(CH 2 )p-CH3 and Y is H.
  • R 1 is a radical of formula (ii) where Z and Y are each independently -(CH2) m -0-(CH 2 )p-CH 3 .
  • R 1 is a radical of formula (ii) and the C 2 , C 3 , C 4 or C 5 alkylene group X is substituted with one or more hydroxy groups.
  • R 1 is a radical of formula (ii) and the C 2 , C 3 , C 4 or C 5 alkylene group X is substituted with one or two fluorine atoms.
  • R 1 is a radical of formula (ii) and the C 2 , C 3 , C 4 or C 5 alkylene group X is substituted with one or two fluorine atoms and one or more hydroxy groups.
  • R 1 is a radical of formula (i) and the C 2 , C 3 , C 4 or C 5 alkylene group X is substituted with one or more hydroxy groups. In other examples, R 1 is a radical of formula (i) and the C 2 , C 3 , C 4 or C 5 alkylene group X is substituted with one or two fluorine atoms. In other examples, R 1 is a radical of formula (i) and the C 2 , C 3 , C 4 or C 5 alkylene group X is substituted with one or two fluorine atoms and one or more hydroxy groups.
  • X is selected from the group consisting of ethylene, 1 ,3-propylene, 1 ,4- butylene, 1 ,5-pentylene, 3-hydroxy-1 ,2-propylene, 2-hydroxy-1 ,3-propylene, 2- hydroxymethyl-1 ,3-propylene, 2,2-bis(hydroxymethyl)-1 ,3-propylene, 4-hydroxy-1 ,3- butylene, 1-fluoro-4-hydroxy-1 ,3-butylene, 1 ,1-difluoro-4-hydroxy-1 ,3-butylene (where the carbon atom to which R 1 is attached is designated the 1 -position).
  • the compound of formula (I) is selected from the group consisting of:
  • the invention provides a composition
  • a composition comprising a pharmaceutically effective amount of a compound of formula (I) or (la) and optionally a carrier.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I) or (la) and, optionally, a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutically acceptable carrier e.g. a second drug compound.
  • the other compound may be, for example, an anti-malarial compound such as quinine, quinidine, cinchoine, cinchonidine, chloroquine, amodiaquine, amodiaquine, proguanil, sulfadoxine, sulfamethoxypyridazine, mefloquine, atovaquone, primaquine, artemisinin, artemether, artesunate, dihydroartemisinin, arteether, halofantrine, doxycycline, clindamycin or (3R,4R)-1-[(9-deazaguanin-9-yl)methyl]-3-hydroxy-4-(hydroxymethyl)pyrrolidine.
  • an anti-malarial compound such as quinine, quinidine, cinchoine, cinchonidine, chloroquine, amodiaquine, amodiaquine, proguanil, sulfadoxine, sulfamethoxypyridazine,
  • the invention provides the use of a compound of formula (I) or (la) for inhibiting a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g.
  • HGXPRT of Plasmodium falciparum or the HGPRT of Plasmodium vivax
  • HGXPRT of Tritrichomonas foetus Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani.
  • the invention provides the use of a compound of formula (I) or (la) as a medicament.
  • the invention provides the use of a compound of formula (I) or (la) for treating or preventing a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g.
  • HGXPRT of Plasmodium falciparum or the HGPRT of Plasmodium vivax
  • HGXPRT of Tritrichomonas foetus Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani.
  • the invention provides the use of a compound of formula (I) or (la) for treating or preventing an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani.
  • the invention provides the use of a compound of formula (I) or (la) for treating or preventing malaria.
  • the invention provides the use of a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I) or (la) for treating or preventing a disease or disorder in which it is desirable to inhibit a a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g.
  • HGXPRT of Plasmodium falciparum or the HGPRT of Plasmodium vivax
  • HGXPRT of Tritrichomonas foetus Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani.
  • the invention provides the use of a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I) or (la) for treating or preventing an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani.
  • the invention provides the use of a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I) or (la) for treating or preventing malaria.
  • the invention provides a compound of formula (I) or (la) for use in the manufacture of a medicament.
  • the invention provides a pharmaceutical composition for treating or preventing a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g.
  • HGXPRT of Plasmodium falciparum or the HGPRT of Plasmodium vivax
  • HGXPRT of Tritrichomonas foetus Cryptosporidium parvum
  • Eimeria tenella or Toxoplasma gondii
  • GPRT of Giardia lamblia the HPRT of Trypanosoma cruzi
  • HGPRT or XPRT of Leishmania donovani comprising a compound of formula (I) or (la).
  • the invention provides a pharmaceutical composition for treating or preventing an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani, comprising a compound of formula (I) or (la).
  • the invention provides a pharmaceutical composition for treating or preventing malaria.
  • the invention provides the use of a compound of formula (I) or (la) in the manufacture of a medicament for the treatment or prevention of a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g.
  • HGXPRT of Plasmodium falciparum or the HGPRT of Plasmodium vivax
  • HGXPRT of Tritrichomonas foetus Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani.
  • the invention provides the use of a compound of formula (I) or (la) in the manufacture of a medicament for the treatment or prevention of an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani.
  • the invention provides the use of a compound of formula (I) or (la) in the manufacture of a medicament for the treatment or prevention of malaria.
  • the invention provides a method of treating or preventing a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g.
  • HGXPRT of Plasmodium falciparum or the HGPRT of Plasmodium vivax
  • the invention provides a method of treating or preventing an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani, comprising administering a pharmaceutically effective amount of a compound of formula (I) or (la) to a patient requiring treatment.
  • the invention provides a method of treating or preventing malaria, comprising administering a pharmaceutically effective amount of a compound of formula (I) or (la) to a patient requiring treatment.
  • the invention provides the use of a compound of formula (I) or (la) in combination with at least one other compound, e.g. a second drug compound, e.g. an anti-malarial such as quinine, quinidine, cinchoine, cinchonidine, chloroquine, amodiaquine, amodiaquine, proguanil, sulfadoxine, sulfamethoxypyridazine, mefloquine, atovaquone, primaquine, artemisinin, artemether, artesunate, dihydroartemisinin, arteether, halofantrine, doxycycline, clindamycin or (3R,4R)-1-[(9-deazaguanin-9- yl)methyl]-3-hydroxy-4-(hydroxymethyl)pyrrolidine, for treating or preventing a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribon-
  • HGXPRT of Plasmodium falciparum or the HGPRT of Plasmodium vivax
  • HGXPRT of Tritrichomonas foetus Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani.
  • the compounds of formula (I) or (la) can be covalently attached via a biologically cleavable linkage to antimalarials such as quinine, quinidine, cinchoine, cinchonidine, chloroquine, amodiaquine, amodiaquine, proguanil, sulfadoxine, sulfamethoxypyridazine, mefloquine, atovaquone, primaquine, artemisinin, artemether, artesunate, dihydroartemisinin, arteether, halofantrine, doxycycline, clindamycin or (3R,4R)-1-[(9- deazaguanin-9-yl)methyl]-3-hydroxy-4-(hydroxymethyl)pyrrolidine.
  • antimalarials such as quinine, quinidine, cinchoine, cinchonidine, chloroquine, amodiaquine, amodiaquine, proguanil, sulfadox
  • the invention provides a method of treating or preventing a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g.
  • HGXPRT of Plasmodium falciparum or the HGPRT of Plasmodium vivax
  • HGXPRT of Tritrichomonas foetus Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani
  • administering a pharmaceutically effective amount of a compound of formula (I) or (la) in combination with at least one other compound e.g.
  • a second drug compound e.g. an anti-malarial compound such as quinine, quinidine, cinchoine, cinchonidine, chloroquine, amodiaquine, amodiaquine, proguanil, sulfadoxine, sulfamethoxypyridazine, mefloquine, atovaquone, primaquine, artemisinin, artemether, artesunate, dihydroartemisinin, arteether, halofantrine, doxycycline, clindamycin or (3R,4R)-1-[(9-deazaguanin-9-yl)methyl]-3-hydroxy-4- (hydroxymethyl)pyrrolidine.
  • an anti-malarial compound such as quinine, quinidine, cinchoine, cinchonidine, chloroquine, amodiaquine, amodiaquine, proguanil, sulfadoxine, sulfamethoxypyridazine
  • the compound of formula (I) or (la) and the other compound may be administered separately, simultaneously or sequentially, or the compound of formula (I) or (la) can be covalently attached via a biologically cleavable linkage to the other compound.
  • the disease or disorder is an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani.
  • the disease or disorder is malaria.
  • the compound of formula (I) or (la) may be selected from compounds (a) to (bt) as defined above.
  • a compound of the invention includes a compound in any form, e.g. in free form or in the form of a salt or a solvate.
  • alkyl means any saturated hydrocarbon radical having up to 30 carbon atoms and includes any C C 2 5, C Cao, Ci-C 5 , C C 10 , or C ⁇ Ce alkyl group, and is intended to include straight- and branched-chain alkyl groups.
  • alkyl groups include: methyl group, ethyl group, n-propyl group, /so-propyl group, n-butyl group, / ' so-butyl group, sec-butyl group, f-butyl group, n-pentyl group, 1 ,1-dimethylpropyl group, 1 ,2- dimethylpropyl group, 2,2-dimethylpropyl group, 1 -ethylpropyl group, 2-ethylpropyl group, n-hexyl group, 1 ,2-dimethylbutyl group.
  • the term "lower alkyl” means a C ⁇ Ce alkyl group, where alkyl is as defined above.
  • alkyl group may optionally be substituted with one or more fluorine or chlorine substituents.
  • alkylene has corresponding a meaning to "alkyl” and is intended to include saturated straight and branched chain groups, preferably C 2 -C 5 alkylene groups, e.g. ethylene, 1 ,3-propylene, 1 ,5-butylene, 1 ,2-propylene, 2-methyl-1 ,3-propylene, 2,2-di-1 ,3- propylene, and 1 ,3-butylene.
  • a C 2 , C 3 , C 4 or C 5 alkylene group X in the above formula (I) or (la) can be attached to the amino nitrogen atom via any carbon of the alkylene group, except the carbon to which the phosphate or phosphonate group R 1 is attached.
  • the carbon to which the phosphate or phosphonate group R 1 is attached is the terminal carbon.
  • a C 3 alkylene group can be attached at the 2- or 3-position of the carbon chain (where the carbon to which phosphate or phosphonate group R 1 is attached is designated position 1), or a C 4 alkylene group can be attached the 2-, 3- or 4- position of the carbon chain (where the carbon to which phosphate or phosphonate group R 1 is attached is designated position 1).
  • position 1 the carbon to which phosphate or phosphonate group R 1 is attached
  • a C 4 alkylene group can be attached the 2-, 3- or 4- position of the carbon chain (where the carbon to which phosphate or phosphonate group R 1 is attached is designated position 1).
  • the scope of the invention is intended to include both straight and branched alkylene groups.
  • Any alkylene group may be optionally substituted with one or two fluorine and/or one or more hydroxy groups, e.g.
  • alkoxyalkyl means -ROR' where R' is alkyl as defined above and R is alkylene.
  • aryl means an aromatic radical having 4 to 18 carbon atoms and includes heteroaromatic radicals. Examples include monocyclic groups, as well as fused groups such as bicyclic groups and tricyclic groups. Some examples include phenyl group, indenyl group, 1-naphthyl group, 2-naphthyl group, azulenyl group, heptalenyl group, biphenyl group, indacenyl group, acenaphthyl group, fluorenyl group, phenalenyl group, phenanthrenyl group, anthracenyl group, cyclopentacyclooctenyl group, and benzocyclooctenyl group, pyridyl group, pyrrolyl group, pyridazinyl group, pyrimidinyl group, pyrazinyl group, triazolyl group (including a 1-/7-1 ,2,3-triazol-1-yl and a
  • aralkyi means an aryl group covalently linked to an alkylene group.
  • Any aryl or aralkyi group may optionally be substituted with one or more fluorine, chlorine or C C 3 alkyl substituents.
  • the symbol " ", as used in structural formulae shown herein, is intended to denote the point of attachment of a radical in a structural formula.
  • prodrug means a pharmacologically acceptable derivative of the compounds of formula (I) such that an in vivo biotransformation of the derivative gives the compound as defined in formula (I).
  • Prodrugs of compounds of formula (I) may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to give the parent compound. Typically, prodrugs of the compounds of formula (I) will be ester prodrug forms.
  • salts are intended to apply to non-toxic salts such as ammonium salts, metal salts, e.g. sodium salts, or salts of organic cations, or a mixture thereof.
  • protecting group means a group that selectively protects an organic functional group, temporarily masking the chemistry of that functional group and allowing other sites in the molecule to be manipulated without affecting the functional group. Suitable protecting groups are known to those skilled in the art and are described, for example, in Protective Groups in Organic Synthesis (3 rd Ed.), T. W. Greene and P. G. M. Wuts, John Wiley & Sons Inc (1999).
  • protecting groups include, but are not limited to: O-benzyl, O-benzhydryl, O-trityl, O-tert-butyldimethylsilyl, O-tert-butyldiphenylsilyl, 0-4- methylbenzyl, O-acetyl, O-chloroacetyl, O-methoxyacetyl, O-benzoyl, O-4-bromobenzoyl, O-4-methylbenzoyl, O-fluorenylmethoxycarbonyl, O-levulinoyl or O-tert-butyl.
  • patient includes human and non-human animals.
  • treatment include the alleviation of one or more symptoms, or improvement of a state associated with the disease or disorder, for example reduction in malaria parasitaemia.
  • preventing include the prevention of one or more symptoms or states associated with the disease or disorder, for example, prevention of malaria parasitaemia.
  • prevention include the prevention of one or more symptoms or states associated with the disease or disorder, for example, prevention of malaria parasitaemia.
  • the compounds of the invention can exist in different tautomeric forms. For example, it will be appreciated that the representation of a compound of formula (I) or (la) where D is a hydroxy group is of the enol-type tautomeric form of a corresponding amide, and this will largely exist in the amide form.
  • the 4- hydroxy group of the 4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl moiety in compounds of the invention is represented in the nomenclature as the enol-type tautomeric form but will largely exist in the corresponding amide tautomeric form.
  • the scope of the invention is intended to cover all tautomeric forms of the compounds of the invention. It will also be appreciated that the compounds of the invention can exist in the form of optical isomers, racemates and diastereomers. The scope of this invention is intended to cover all possible stereoisomeric forms of the compounds of formulae (I) and (la).
  • one or more of the carbon atoms of the optionally substituted X group in the formula (I) or (la) may be asymmetric carbons and may be in the R- or S-configuration.
  • the phosphorus of the R 1 (phosphate or phosphonate) group may also be a chiral centre.
  • the structures shown for the ester prodrug compounds of the invention in which the phosphate or phosphonate group has four different substituents attached to the phosphorus atom are intended to represent racemates. Compounds of these structures exist in two enantiomeric forms. The scope of this invention is intended to cover all possible forms, i.e. the racemates and the two enantiomers.
  • the compounds of the invention are inhibitors of hypoxanthine and/or guanine purine phosphoribosyltransferases, e.g hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferases of protozoan parasites, including those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani, and are useful as pharmaceuticals, particularly for the treatment or prevention of diseases or conditions in
  • the diseases or conditions include malaria.
  • the compounds of the invention are useful in both free base or acid form and in the form of salts and/or solvates.
  • the compounds of the invention are surprisingly potent inhibitors of Plasmodium falciparum HGXPRT.
  • compound (95) has a K, of 0.65 nM against Plasmodium falciparum HGXPRT.
  • selectivity that the compounds of the invention show against Plasmodium falciparum HGXPRT compared to human HGPRT.
  • the K, for compound (95) against Plasmodium falciparum HGXPRT is more than 500 times lower than the K, against human HGPRT. This level of potency and selectivity would not have been predicted from the prior art.
  • the compounds of the invention include prodrug forms.
  • prodrugs of the compounds of formula (I), e.g. compounds of formula (la) can have increased efficacy.
  • Preferred prodrug forms are ester prodrugs, where one or more hydrogens of the phosphate or phosphonate (R 1 ) groups of the compounds of formula (I) are replaced with suitable lipophilic groups, such as alkoxyalkyl groups.
  • suitable lipophilic groups such as alkoxyalkyl groups.
  • lipopholic groups allows for transfer of the compounds through cell membranes.
  • the prodrugs are active against cultured Plasmodium falciparum.
  • Table 2 shows the IC 50 values for prodrug compounds of the invention against Plasmodium falciparum strain 3D7, the chloroquine/mefloquine resistant strain Dd2, and the chloroquine/quinine resistant strain FVO.
  • the compounds of the invention may be administered to a patient by a variety of routes, including orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally or via an implanted reservoir.
  • routes including orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally or via an implanted reservoir.
  • injections may be given intravenously, intra-arterially, intramuscularly or subcutaneously.
  • the amount of a compound of the invention to be administered to a patient will vary widely according to the nature of the patient and the nature and extent of the disorder to be treated. Typically the dosage for an adult human will be in the range of about 0.01 Mg/kg to about 1 g/kg, preferably about 0.01 mg/kg to about 100 mg/kg.
  • the specific dosage required for any particular patient will depend upon a variety of factors, such as the patient's age, body weight, general health, gender and diet. Optimal doses will depend on other factors such as mode of administration and level of progression of the disease or disorder. Doses may be given once daily, or two or more doses may be required per day. For example, a dosage regime for a malaria patient might require one dose in the morning and one in the evening. Alternatively, a dosage regime for such a patient might require four hourly doses.
  • the compounds can be formulated into solid or liquid preparations, for example tablets, capsules, granules, powders, solutions, suspensions, syrups, elixirs and dispersions. Such preparations are well known in the art as are other oral dosage regimes not listed here.
  • compounds of the invention can be formulated into sterile solutions, emulsions and suspension.
  • Compounds of the invention may be mixed with suitable vehicle and then compressed into the desired shape and size.
  • the compounds may be tableted with conventional tablet bases such as lactose, sucrose and corn starch, together with a binder, a disintegration agent and a lubricant.
  • the binder may be, for example, corn starch or gelatin
  • the disintegrating agent may be potato starch or alginic acid
  • the lubricant may be magnesium stearate.
  • diluents such as lactose and dried cornstarch may be employed. Other components such as colourings, sweeteners or flavourings may be added. Tablets, capsules or powders for oral administration may contain up to about 99% of a compound of the invention.
  • a compound of the invention may be combined with a pharmaceutically acceptable carriers such as water, an organic solvent such as ethanol, or a mixture of both, and optionally other additives such as emulsifying agents, suspending agents, buffers, preservatives, and/or surfactants may be used. Colourings, sweeteners or flavourings may also be added.
  • the compounds may also be administered by injection in a pharmaceutically acceptable diluent such as water or saline.
  • a pharmaceutically acceptable diluent such as water or saline.
  • the diluent may comprise one or more other ingredients such as ethanol, propylene glycol, an oil or a pharmaceutically acceptable surfactant.
  • the compounds of the invention may also be administered topically.
  • Carriers for topical administration of the compounds include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the compounds may be present as ingredients in lotions or creams, for topical administration to skin or mucous membranes. Such creams may contain the active compounds suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the compounds of the invention may further be administered by means of sustained release systems.
  • they may be incorporated into a slowly dissolving tablet or capsule.
  • the compounds of the invention are synthetically accessible and may be prepared by a variety of different methods.
  • the following are representative non- limiting examples.
  • R c will be a methyl, ethyl or tert-butyl group.
  • the groups X and X' are optionally substituted alkylene groups, preferably optionally substituted C 2 - C 5 linear or branched alkylene groups, to which the amino-moiety is attached to a primary, secondary or tertiary carbon atom of the alkylene group, and the phosphonic acid or ester moiety, respectively, is attached to a terminal carbon atom of the alkylene group.
  • the optional substituents on the alkylene group X' may be (a) one or two fluoro-atom substituents, preferably on the carbon bearing the phosphonic acid or ester moiety, and/or (b) one or more hydroxyl group substituents, or the O-protected forms on an otherwise unsubstituted primary, secondary or tertiary carbon atom of the linker.
  • the alkylene group X in the product compound (D) will be the same as the alkylene group X' in the precursor compound (C) but with O-protecting groups removed.
  • a suitable O- protecting group is a benzyl ether group.
  • a free phosphonic acid of general formula (B) (where R° is H) can be used in the reductive amination with the aldehyde of general formula (A), to give an adduct of general formula (C) where R c is H, which can then be converted as above to give a compound of general formula (D).
  • Reductive amination is conveniently effected by use of a reducing agent selected from sodium borohydride, 2- picoline borane complex, sodium triacetoxyborohydride or sodium cyanoborohydride.
  • the protecting groups in an intermediate adduct of general formula (C) are then removed to give the free phosphonic acid (D) (or salt form thereof).
  • This deprotection can be by stepwise treatment with strong mineral acid, typically concentrated hydrochloric acid, then with aqueous hydrobromic acid, typically 48% HBr in water.
  • Benzyl and benzyloxymethyl protecting groups can be removed by hydrogenolysis over a palladium catalyst if desired.
  • a (2-(4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)ethylamino)alkylphosphon acid (H) of the invention can be obtained as shown in Scheme B by reductive amination of a dialkyi ester of the requisite dialkyi ⁇ -oxoalkylphosphonate (F) (where the group X" is as defined below) with a 9-(2-aminoethyl)-9-deazapurine of general formula (E) (where D is H or OR b and where R b is H or an O-protecting group) to give an adduct of general formula (G) (where X ' is an optionally substituted alkylene linker as defined in General Procedure 1 above) from which any protecting groups are then removed as required to give a compound of general formula (H) (where X is an optionally substituted alkylene linker as defined in General Procedure 1 above).
  • the group X" in a compound of formula (F) is an optionally substituted alkylene group, preferably an optionally substituted Ci - C 4 linear or branched alkylene group, to which the amino-moiety is attached to a primary, secondary or tertiary carbon atom of the alkylene group, and the phosphonic ester moiety is attached to a terminal carbon atom of the alkylene group.
  • the optional substituents on the alkylene group X" may be (a) one or two fluoro-atom substituents, preferably on the carbon bearing the phosphonic acid or ester moiety, and/or (b) one or more hydroxy-group substituents or their O-protected forms on an otherwise unsubstituted primary, secondary or tertiary carbon atom of the linker.
  • the requisite dialkyi ⁇ -oxoalkylphosphonate (F) will be a diethyl ester.
  • Suitable O-protecting groups (R b ) in the compound of general formula (I) include methyl, benzyl and tert-butyl.
  • Alternative protecting groups can be introduced with the substitution of benzyl alcohol with an alternative alcohol (e.g. methanol or tert-butanol) if desired.
  • Scheme D A bis(alkyloxyalkyl) aminoalkylphosphonate of general structure (N), where R e is an optionally substituted alkyl group, n is 2 or 3, and X ' is an optionally substituted alkylene group, where the optional substituents can be (i) one or two fluoro-atom substituents, and/or (ii) one or more hydroxy-group substituents or O-protected forms thereof, can be prepared as shown in Scheme D from a dialkyl aminoalkylphosphonates (G) by: (a) hydrolysis to a phosphonic acid (J); (b) N-protection to give a carbamate (K) (where R d is an optionally substituted alkyl or aralkyl group); (c) chlorination to give dichloride (L); (d) displacement of both chlorides with an alkoxyalkyl alcohol to give (M); and (e) final N- deprotection to give the required bis(alkyloxyalkyl)
  • the starting amine (G) is the diethyl ester
  • suitable reagents for the conversions are: (a) hydrolysis using aqueous hydrobromic acid heated under reflux; (b) N-protection with benzyl chloroformate, di-tert-butyl dicarbonate or 2,2,2- trichloroethoxycarbonyl chloride; (c) dichlorination with thionyl chloride or oxalyl chloride; (d) chloride displacement with the alkoxyalkyi alcohol in the presence of base such as pyridine; and (e) N-deprotection using catalytic hydrogenolysis (e.g., over a palladium catalyst), treatment with acid (e.g. trifluoroacetic acid) or zinc in acetic acid, as suits the N-protecting group chosen.
  • catalytic hydrogenolysis e.g., over a palladium catalyst
  • An alkyi alkoxyalkyi aminoalkylphosphonate of general structure (S), where R e , n and X' are as in General Procedure 3 above, can be prepared as shown in Scheme E from a dialkyl aminoalkylphosphonates (B) by: (a) partial hydrolysis to the monoalkyl ester (O); (b) N-protection to give a carbamate (P); (c) chlorination to give chloride (Q); (d) displacement of the chloride with an alkoxyalkyi alcohol to give (R); and (e) final N- deprotection to give the required alkyi alkyloxyalkyl aminoalkylphosphonate (S).
  • B dialkyl aminoalkylphosphonates
  • the starting amine (B) is a d Ci-Cs alkyi) ester
  • suitable reagents for the conversions are: (a) hydrolysis with aqueous hydrobromic acid heated under reflux; (b) N-protection with benzyl chloroformate, di-tert-butyl dicarbonate or 2,2,2- trichloroethoxycarbonyl chloride; (c) chlorination with thionyl chloride or oxalyl chloride; (d) displacement of chloride with the alkoxyalkyl alcohol in the presence of base such as pyridine; and (e) N-deprotection using catalytic hydrogenolysis (e.g. over a palladium catalyst), treatment with acid (e.g. trifluoroacetic acid) or zinc in acetic acid, as suits the N-protecting group chosen.
  • catalytic hydrogenolysis e.g. over a palladium catalyst
  • acid e.g. trifluoroacetic acid
  • An alkoxyalkyl benzyl diester of an aminoalkylphosphonic acid general structure (V), where R e , n and X ' are as defined in General Procedure 3 above, can be prepared as shown in Scheme F from a dichloride (L) (see General Procedure 3) by (a) partial displacement with an alkyloxyalkyl alcohol, then hydrolysis of the remaining chloride to give mono ester (T); (b) chlorination and displacement of the chloride with an benzyl alcohol to give (U); and (e) final N-deprotection to give the required alkyloxyalkyl benzyl aminoalkylphosphonate (V).
  • Suitable reagents for the conversions are: (a) the use of limited alkoxyalkyl alcohol in the presence of pyridine and subsequent hydrolysis with aqueous NaHC0 3 (b) chlorination with thionyl chloride or oxalyl chloride and treatment of the resulting chloride with benzyl alcohol in the presence of pyridine; and (c) N- deprotection using catalytic hydrogenolysis (e.g. over a palladium catalyst), treatment with acid (e.g. trifluoroacetic acid) or zinc in acetic acid, as suits the N-protecting group chosen.
  • catalytic hydrogenolysis e.g. over a palladium catalyst
  • acid e.g. trifluoroacetic acid
  • zinc in acetic acid e.g. trifluoroacetic acid
  • a ((7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-3-yl)methylamino)alkylphosphonic acid (Y) of the invention (where X is as in General Procedure 1 above) can be obtained by reductive amination of a dialkyi ester of a aminoalkylphosphonic acid (B) (where R c is an alkyl or aralkyl group and X ' is as defined in General Procedure 1 above) with the formyl-8-aza- 9-deazapurine (77) (See Example 15) to give an adduct of general formula (X), from which any protecting groups are then removed as required to give a compound (Y) of the invention (Scheme A).
  • R c will be a methyl, ethyl or tert-butyl group.
  • a ((2-amino-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methylamino)alkylphos acid (AA) of the invention (where X is as in General Procedure 1 above) can be obtained by reductive amination of an aminoalkylphosphonic acid (B) (where R c is H and X' is as defined in General Procedure 1 above) or a dialkyi ester of (B) (where R° is a an alkyl or aralkyl group and X ' is as defined in General Procedure 1 above) with the 7-formyl-9- deazaguanine derivative (109) (See Example 21) to give an adduct of general formula (Z), from which any protecting groups are then removed as required to give a compound (AA) of the invention (Scheme J).
  • R c will be a methyl, ethyl or tert-butyl group.
  • Phosphates of general structure (AB) can be obtained from an aminoalcohol by (a) N- protection, e.g. as a N-fluorenyylmethoxycarbonyl derivative,; (b) selective mono- phosphorylation of a primary alcohol group, e.g. by P(OMe) 3 and l 2 in pyridine; (c) demethylation of the phosphate esters with bromotrimethylsilane; and (d) N-deprotection.
  • N- protection e.g. as a N-fluorenyylmethoxycarbonyl derivative
  • Figure 1a shows a graph of parasite growth inhibition by (54);
  • Figure 1 b shows extracellular purine analysis of metabolic labeling for uninfected erythrocytes treated with 100 ⁇ (54) and metabolically labeled with [ 3 H]hypoxanthine;
  • Figure 1c shows extracellular purine analysis of metabolic labeling for uninfected erythrocytes treated with 100 ⁇ (54) and metabolically labeled with [ 3 H]inosine.
  • Figure 2 shows the IC 50 values of (61) conducted at varying exogenous hypoxanthine concentrations.
  • Figure 3a shows purine quantification by HPLC of uninfected RBCs;
  • Figure 3b shows purine quantification by HPLC of erythrocyte-free parasites;
  • Figure 3c shows purine quantification by HPLC of parasites isolated from infected erythrocytes;
  • Figure 3d shows purine quantification by HPLC of extracellular purines from the experiment of Figure 3c.
  • the time (Figure 3e) and concentration (Figure 3f) dependence of label uptake are measured by liquid scintillation counting of spent media.
  • Figure 4a shows that compound (65) inhibits the incorporation of hypoxanthine into the parasite.
  • Cells are metabolically labeled with [2,8- 3 H]hypoxanthine in the presence or absence of 10 ⁇ compound (65).
  • Figure 4b shows that compound (76) does not inhibit the incorporation of hypoxanthine into the parasite during a two hour incubation.
  • Cells are metabolically labeled with [2,8- 3 H]hypoxanthine in the presence or absence of 15 ⁇ compound (76).
  • Incorporation of label into purines is quantified by HPLC of extracts of parasites isolated from infected erythrocytes.
  • Chromatography solvents are distilled prior to use.
  • Anhydrous solvents are those commercially available. Organic solutions are dried over anhydrous MgS0 4 and evaporated under reduced pressure. Air sensitive reactions are performed under Ar.5
  • Analytical TLC is performed on Merck pre-coated silica gel 60 F254, detection by UV absorption and/or by heating after dipping in a solution of ( ⁇ 4) 6 ⁇ 7 ⁇ 24- ⁇ 2 0 (5 wt%) and Ce(S0 4 ) 2 -4 H 2 0 (0.1 wt%) in 5% aq. H 2 S0 4 . Flash column chromatography is performed on silica gel (40-63 pm) or on an automated system with continuous gradient facilility.
  • Diethyl 1-aminomethylphosphonate (3a), diethyl 2-aminoethylphosphonate (3b), diethyl 3-aminopropylphosphonate (3c), diethyl 4-aminobutylphosphonate (3d) and diethyl 5- aminopentylphosphonate (3e) [P Bako et a/., Tetrahedron Asymm., 10 (1999) 2373- 2380] are prepared by the method of Hirschmann et al., (J. Amer. Chem.
  • the dimethylamine (9) (548 mg,1 eq.) in tetrahydrofuran (25 mL) is treated with methyl iodide (1.2 mL, 10 eq.) at room temperature for 18 h and then the solution is evaporated under high vacuum to give an orange foam of the quaternary ammonium salt (10).
  • Nitromethane (3.34 mL, 30 eq.) is added to methanol (32 mL) and methanolic sodium methoxide (7 mL, 30%, 20 eq.) and the mixture is stirred under argon for 10 min and then the crude quaternary ammonium salt (10) is added. The mixture is stirred for 2 h and then the methanol is evaporated.
  • NaBH 4 (393 mg, 10 eq.) is added in small portions to 4-(benzyloxy)-7-(2-nitroethyl)-5H- pyrrolo[3,2-d]pyrimidine (11) (310 mg 1 eq.) in methanol (20 mL) with CoCI 2 .6H 2 0 (495 mg, 2 eq.) at 0 °C and then the mixture is stirred at 0 °C for 30 min. The product is evaporated onto silica gel and flash chromatography (9/1/0.1 and 8/2/0.2 v/v/v DCM/MeOH/conc.
  • Triethylphosphite (8.4 mL) and 2-(2-bromoethyl)-1 ,3-dioxolane (10 g) are heated at 160 °C for 18 h and then evaporated under high vacuum at 100 °C .
  • the residue is refluxed with 1 N HCI under argon for 15 min and cooled to room temperature.
  • the product is salted out with solid NaCI and extracted with DCM, washed .with saturated NaHC0 3 , brine, dried and concentrated. Flash chromatography (EtOAc-MeOH 95:5 v/v) gives diethyl (3-oxopropyl)phosphonate (13) (2.5 g).
  • NMR spectral data identical with literature (J.M Varlet et a/., Tetrahedron 37 (1981) 1377-1384).
  • the phosphonate (14) (50 mg) is heated at 60 °C with cone. HCI (0.5 mL) for 1 h and then evaporated under reduced pressure twice from water. The residue is then heated with 48% aq. HBr (1 mL) at 90 °C for 5 h and evaporated again twice from water. The product is chromatographed on reverse phase C 18 silica gel eluting with water to give ⁇ 3- [(2- ⁇ 4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl ⁇ ethyl)amino]propyl ⁇ phosphonic acid (15) (30mg).
  • Triethylphosphite (12 mL) and 2-(bromomethyl)-1 ,3-dioxolane (10 g) are heated at 160 °C for 18 h and then evaporated under high vacuum at 100 °C. The residue is heated under reflux with 1 N HCI under argon for 15 min and cooled to room temperature. The product is salted out with solid NaCI and extracted with DCM, washed with saturated NaHC0 3 , brine, dried and concentrated. Flash chromatography (EtOAc-MeOH 95:5 v/v) gives diethyl (2-oxoethyl)phosphonate (16) (1.6 g) [NMR spectral data identical with literature, Org. Synth., Coll. Vol.
  • the diethyl phosphonate (17) (52 mg) is heated at 60 °C with cone. HCI (0.5 mL) for 1 h, evaporated under reduced pressure twice from water and the heated with 48% HBr (1 mL) at 90 °C for 5 h and evaporated again twice from water.
  • the product is chromatographed on reverse phase Ci 8 silica gel eluting with water to give ⁇ 2-[(2- ⁇ 4- hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl ⁇ ethyl)amino]ethyl ⁇ phosphonic acid (18) (39 mg).
  • Methanesuifonyl chloride (0.72 mL, 2 eq.) is added and the mixture stirred at room temperature for 1 h, diluted with DCM and the organic extract washed with satd. NaHC0 3i brine, dried and evaporated to give crude mesylate (28). Displacement of the mesylate is effected by heating a solution of crude (28) (1.93 g) in DMF (20 mL) with NaN 3 (0.9 g) at 80 °C for 7 h.
  • Ethanolamine 120 ⁇ _, 5 eq. is added to acetyl chloride (142 ⁇ _, 5 eq.) in methanol (8 mL) and a few microdrops of acetyl chloride added to adjust the pH to 5.
  • methanol 8 mL
  • 4-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine-7-carbaldehyde (4) 100 mg, 1 eq.
  • Picoline borane 64 mg, 1.5 eq.
  • the mixture stirred at 40 °C for 4 h.
  • the diethyl phosphonate (38) (10 mg) is heated at 60 °C with cone. HCI (0.5 mL) for 1 h, then evaporated twice from water and heated with 48% HBr (0.5 mL) at 90 °C for 5 h and evaporated twice from water.
  • the product is chromatographed on reverse phase C 18 silica gel eluting with water to give ⁇ 3-[( ⁇ 4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl ⁇ methyl) (methyl)amino]propyl ⁇ phosphonic acid (39) (9 mg).
  • Methyl 2- ⁇ [(3- ⁇ [(benzyloxy)carbonyl] amino ⁇ propyl)(phenoxy) phosphoryl] aminojpropanoate (42) (332 mg) in ethanol (5 mL) with TFA (5 drops) is hydrogenated at atmospheric pressure in the presence of 10% Pd/C (250 mg) for 1.5 h, then filtered and concentrated.
  • the crude product (43) (190 mg) is directly subjected to reductive amination with 4-benzyloxy-5H-pyrrolo[3.2-c/]pyrimidine-7-carbaldehyde (4) (60 mg, 0.5 eq.) and picoline borane (49 mg, 1 eq.) in MeOH (12 mL) for 2 h.
  • 3-Aminopropylphosphonic acid HBr salt (40) (290 mg, 1 eq.) dissolved in H 2 0 (2 mL) and MeOH (4 mL) is treated with EtN 3 (760 ⁇ , 4 eq.) and di-tert-butyl dicarbonate (288 mg, 1 eq.). After 30 min, the solution is evaporated and subjected to reverse phase chromatography on C 18 silica gel eluting with H 2 0 then 20% and 40% v/v MeOH in H 2 0 to give (3- ⁇ [(tert-butoxy)carbonyl]amino ⁇ propyl)phosphonic acid (46) (252 mg).
  • the protected amine (46) (220 mg) is dried by evaporation 2x with dry pyridine and dissolved in dry pyridine (5 mL). To this solution is added alanine methyl ester hydrochloride (236 mg, 2.6 eq.) in dry pyridine (1 mL) followed by Et 3 N (244 ⁇ , 2.6 eq.) and the mixture is stirred at 60 °C for 5 min.
  • Boc derivative (47) 50 mg is stirred with 80% TFA (0.5 mL) for 5 min, evaporated 2x with dioxane and subjected to flash chromatography (DCM and 20% v/v 6N NH 3 in MeOH) gives methyl 2- ⁇ [(3-aminopropyl)[(1-methoxy-1-oxopropan-2-yl)amino] phosphoryl]amino ⁇ propanoate (48) (58 mg).
  • H NMR 500 MHz, MeOD
  • ⁇ 3.98-3.92 m, 2H), 3.72 (s, 6H), 3.04-3.01 (m, 2H), 2.01-1.76 (m, 4H), 1.40-1.37 (m, 6H).
  • the protected amine (51) is hydrogenolysed with 10% Pd/C (500 mg) in ethanol for 1.5 h and evaporated. Flash chromatography (DCM/MeOH/conc. NH 3 9/9/0.1 v/v/v) gives ⁇ [(3- aminopropyl)( ⁇ [(2,2-dimethylpropanoyl)oxy]methoxy ⁇ )phosphoryl]oxy ⁇ methyl 2,2- dimethylpropanoate (52) (356 mg).
  • H NMR 500 MHz, CDCI 3 ) ⁇ 5.69, 5.66 (2s, 4H), 2.74-2.78 (m, 2H), 1.95-1.65 (m, 4H), 1.24(s, 18H).
  • the adduct (53) (65 mg) is hydrogenolysed with 10% Pd/C (50 mg) in ethanol (5 mL) for 4 h, filtered and concentrated. Flash chromatography (DCM/MeOH/conc. NH 3 9/1/0.1 then 8/2/0.2 v/v/v) gives [( ⁇ [(2,2-dimethylpropanoyl)oxy]methoxy ⁇ ( ⁇ 3-[( ⁇ 4-hydroxy-5H- pyrrolo [3,2-d]pyrimidin-7-yl ⁇ methyl)amino]propyl ⁇ )phosphoryl)oxy]methyl 2,2- dimethylpropanoate (54) (27 mg). The product is evaporated 2x from HOAc to form the acetic acid salt.
  • Benzyl N-(3- ⁇ ethoxy[3-(octadecyloxy)propoxy]phosphoryl ⁇ propyl)carbamate (58) (170 mg) in ethanol (15 mL) and TFA (0.06 mL) is hydrogenolysed in the presence of 10% Pd/C (150 mg) at atmospherice pressure for 2 h. Filtration and evaporation gives the TFA salt of ethyl 3-(octadecyloxy)propyl (3-aminopropyl)phosphonate (59) (111 mg).
  • Benzyl N-(3- ⁇ ethoxy[3-(hexadecyloxy)propoxy]phosphoryl ⁇ propyl)carbamate (62) (110 mg) in ethanol (10 mL) and TFA (0.04 ml.) is hydrogenolysed in the presence of 10% Pd/C (100 mg) at atmospherice pressure for 2 h. Filtration and evaporation gives the TFA salt of ethyl 3-(hexadecyloxy)propyl (3-aminopropyl)phosphonate (63) (85 mg).
  • Ethyl 3-(hexadecyloxy)propyl [3-( ⁇ [4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7- yl]methyl ⁇ amino)propyl]phosphonate (64) (20 mg) is hydrogenolysed with 10% Pd/C (20 mg) in ethanol (5 mL) containing TFA (0.06 mL) for 2 h. Filtration and evaporation gives ethyl 3-(hexadecyloxy) propyl ⁇ 3-[( ⁇ 4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl ⁇ methyl)amino]propyl ⁇ phosphonate (65) (18 mg).
  • Benzyl 3-(octadecyloxy)propyl (3-aminopropyl)phosphonate (70) (112 mg, 1.2 eq.) is heated at 70 °C in ethanol (7 mL) with 4-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine-7- carbaldehyde (4) (44 mg, 1 eq.) for 18 h and then cooled to room temperature and treated with NaBH 4 (20 mg). The product is evaporated onto silica gel and flash chromatographed (9/1/0.1 v/v/v DCM/MeOH/conc.
  • Benzyl 3-(octadecyloxy)propyl [3-( ⁇ [4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7- yl]methyl ⁇ amino)propyl]phosphonate (71) (44 mg) is hydrogenolysed with 10% Pd/C (50 mg) in ethanol (5 mL) containing TFA (0.04 mL).
  • Benzyl N-(3- ⁇ bis[3-(octadecyloxy)propoxy]phosphoryl ⁇ propyl)carbamate (73) (400 mg) is dissolved in methanol (20 mL) containing TFA (0.1 mL) and 10% Pd/C (400 mg) at 40 °C and hydrogenolysed at atmospheric pressure. The solution is filtered, the residue extracted with DCM and the combibed organic phase evaporated to give bis[3- (octadecyloxy)propyl] (3-aminopropyl)phosphonate (74) (200 mg).
  • 1 H NMR 500 MHz, CDCI 3 ) ⁇ .
  • Dess-Martin periodinane (0.390 g, 0.921 mmol) is added to a mixture of triethylamine (0.47 mL, 3.35 mmol) and alcohol (87) (0.214 g, 0.837 mmol) in THF (6 mL) and the mixture is stirred at room temperature for 40 min.
  • EtOAc 50 mL
  • sat.aq. NaHC0 3 3 mL
  • water 3 mL
  • the organic layer is separated and washed with brine then dried and the solvent evaporated to leave a solid which is dissolved in a 1 :1 v/v mixture of MeOH/CHCI 3 , silica gel added and the solvent evaporated.
  • n-Butyllithium (1.5M, 17.05 mL, 25.6 mmol) is added dropwise to a solution of di- tert butyl methylphosphonate (97) (5.33 g, 25.6 mmol) in THF (25 mL) keeping the temperature below -70 °C throughout the addition.
  • a mixture of diisopropyl azodicarboxylate (DIAD, 1.78 mL, 9.13 mmol) and diphenylphosphoryl azide (DPPA ,1.97 mL, 9.13 mmol) in dry toluene (10 mL) is added dropwise to a solution of di-terf-butyl [(3R)-4-(benzyloxy)-3-hydroxybutyl]phosphonate (98) (2 g, 5.37 mmol) and triphenyl phosphine (2.39 g, 9.13 mmol) in toluene (20 mL) at 0 °C.
  • the % d.e. of compound (99) is determined in two ways, as follows.
  • the (S, R) Mosher amide (Ward, D.E; Rhee, C.K, Tetrahedron Lett, 1991 , 32, 7165 for general method) is prepared by dissolution of di-tert-butyl [(3S)-3-amino-4-(benzyloxy)butyl]phosphonate (99) (6.3 mg, 0.017 mmol) in a mixture of CDCI 3 and Et 3 N (3 eq.) and a solution of (S)- MTPACI (1.2 eq.) in CDCI 3 (prepared from (R)-MTPA, 99% e.e.) is added to give a total volume of 0.6 ml_.
  • CFCI 3 ⁇ 0 ⁇ -69.3 (s, 97%), -69.4 (s, 3%).
  • the % d.e. is 94%.
  • the average % d.e. from these two assessments is 95%.
  • Dibenzyl diisopropylphosphoramidite (1.067 mL, 3.24 mmol) is added to te/ -butyl (4S)-4- (hydroxymethyl)-2,2-dimethyl-1 ,3-oxazolidine-3-carboxylate (0.5 g, 2.162 mmol) (106) [prepared from D-serine as described for the enantiomer in Organic Syntheses, Coll. Vol. 10, p. 320 (2004) and Vol. 77, p. 64 (2000)] and 1H-tetrazole (0.454 g, 6.49 mmol) in CH3CN (10 mL) and the mixture stirred for 1 h.
  • [(2fi)-2-Amino-3-hydroxypropoxy]phosphonic acid (108) (25 mg, 0.146 mmol) is suspended in methanol (50 mL) and brought to pH7 with Et 3 N.
  • 4-Benzyloxy-5 - - pyrrolo[3,2-c]pyrimidine-7-carbaldehyde (4) (22 mg, 0.088 mmol) and then picoline borane complex (20.3 mg, 0.19 mmol) are added and the suspension stirred at 50 °C for 60 h. The solution is evaporated onto silica gel.
  • MCPBA m- chloroperbenzoic acid
  • 4-Benzyloxy-5H-pyrrolo[3,2-d]pyrimidine-7- carbaldehyde (4) (93 mg, 0.26 mmol) and sodium cyanoborohydride (7.8 mg, 0.48 mmol) are added and the suspension stirred at 40 °C for 16 h and then at room temperature for 48 h.
  • Dibenzyl diisopropyl phoshoramidite (3.64 mL, 10.83 mmol) is added to an ice cold solution of terf-butyl /V-[5-(hydroxymethyl)-2,2-dimethyl-1 ,3-dioxan-5-yl]carbamate (147) [Ooi, H.; Ishibashi, N.; Iwabuchi, Y.; Ishihara, J. Hatakeyama, S., J. Org. Chem. 2004, 69, 7765-7768.] (1.35g, 5.42 mmol) and 1H-tetrazole (1.52 g, 21.7 mmol) in CH 2 CI 2 (50 mL).
  • [2-( ⁇ [4-(Benzyloxy)-5H-pyrrolo[3,2-c ]pyrimidin-7-yl]methyl ⁇ amino)-3-hydroxy-2- (hydroxymethyl)propoxy]phosphonic acid (150) is prepared in the same manner as compound (133) in Scheme 28 from compound [2-amino-3-hydroxy-2- (hydroxymethyl)propoxy]phosphonic acid (149) (55 mg, 0.18 mmol) and 4-benzyloxy-5 - - pyrrolo[3,2-c ]pyrimidine-7-carbaldehyde (4) to give [2-( ⁇ [4-(benzyloxy)-5H-pyrrolo[3,2- o pyrimidin-7-yl]methyl ⁇ amino)-3-hydroxy-2-(hydroxymethyl)propoxy]phosphonic acid (150) (7.0 mg, 0.015 mmol, 9%).
  • the crude product is purified by flash chromatography (8:2 to 1 :1 v/v petroleum ether-EtOAc) to give the 5-[(benzyloxy)methyl]- 4-(terf-butoxy)-5H-pyrrolo[3,2-c/]pyrimidine (153) as a pale yellow solid (4.00 g, 43%).
  • /V-Bromosuccinimide (2.29 g, 12.9 mmol) is added portionwise to a solution of 5- [(benzyloxy)methyl]-4-(terf-butoxy)-5H-pyrrolo[3,2-d]pyrimidine (153) (4.00 g, 12.9 mmol) in DCM (85.0 mL, 1321 mmol) at 0 °C over 1 h.
  • Di-ierf-butyl dicarbonate (346 mg, 1.59 mmol) is added to a solution of compound (156) (450 mg, 0.611 mmol) in CHCI 3 (20.0 mL) at ambient temperature under.
  • Et 3 N (86.0 pL, 611 ⁇ ) is added and the solution is stirred at ambient temperature for 18 h. The reaction mixture is concentrated under reduced pressure.
  • Tetrabutylammonium fluoride (1 M THF, 0.597 ml, 597 ⁇ ) is added to a solution of compound (157) (250 mg, 2.99 mmol) in THF (5.0 mL) at ambient temperature and the reaction mixture is stirred for 18 h. Further tetrabutylammonium fluoride (1 M THF, 0.597 mL, 5.97 mmol) is added and the reaction mixture is stirred for a further 36 h. The solvents are concentrated under reduced pressure and the residue is dissolved in CHCI 3 (50.0 mL) and washed with H 2 0 (2 *20.0 mL). The organic phase is dried, filtered and the solvent is concentrated under reduced pressure.
  • N,N-diethyl-1 ,5-dihydrobenzo[e][1 ,3,2]dioxaphosphepin-3-amine (130 ⁇ , 601 ⁇ ) is added to a solution of compound (158) (180 mg, 301 mol)and tetrazole (105 mg, 1.50 mmol) in MeCN (10.0 mL) and the solution is stirred at ambient temperature for 90 minutes. The solvent is concentrated under reduced pressure and the residue is dissolved in DCM (5.0 mL). The solution is cooled to 0 °C and m-chloroperoxybenzoic acid (259 mg, 902 pmol) is added.
  • Example 32 Inhibition of Plasmodium falciparum HGXPRT, Human HGPRT and Plasmodium vivax HGPRT Activity
  • PrHGXPRT activity is measured using spectrophotometric assays observing the conversion of xanthine and 5-phospho-a-D-ribose-1 -pyrophosphate to xanthosine-5'- monophosphate and inorganic pyrophosphate (PP,) at 247 nm ( ⁇ 257 - 6.8 mM "1 cm “1 ) or the conversion of guanine and PRPP to guanosine-5'-monophosphate and PP, ( ⁇ 257 - 5.8 mM "1 cm “1 ) on a Varian Cary 100 spectrophotometer (Palo Alto, CA) at 37 °C.
  • Compounds (7c) and (95) are competitive inhibitors of PflHGXPRT with R values of 10.6 and 0.65 n , respectively.
  • compounds (7c) and (95) display K, values of 4940 nM and 385 nM for human HGPRT.
  • compounds (7c) and (95) are, respectively, 466- and 592-fold more selective for the parasitic enzyme than the human enzyme.
  • These specific free phosphonate inhibitors show no activity against cultured parasites, consistent with their lack of membrane permeability, however other phosphonates are known to be permeable (Sheng et al., Bioorganic & Medicinal Chemistry Letters, 19 (2009) 3453-3457).
  • prodrug forms of these compounds show activity against the cultured parasites.
  • Example 33 Prodrug Compounds of the Invention Inhibit Proliferation Plasmodium falciparum in Culture
  • Bis-pivalate prodrug (54) is activated in infected erythrocytes - UPLC/MS/MS data from cell extracts of infected erythrocytes treated with compound (54). Values are arbitrary units and cannot be compared across columns.
  • falciparum strain 3D7 and compounds (61), (65), (72) are found to inhibit parasite growth in vitro with IC 50 values of 2.5 ⁇ 0.2 ⁇ , 1.9 ⁇ 0.1 ⁇ , and 7.0 ⁇ 0.1 ⁇ , respectively.
  • Compounds (61) and (65) show similar IC 50 values when compared with chloroquine/mefloquine-resistant strain Dd2 (3.0 ⁇ 0.1 ⁇ and 2.3 ⁇ 0.1 ⁇ ) or chloroquine/quinine resistant strain FVO (2.9 ⁇ 0.1 ⁇ and 3.1 ⁇ 0.1 ⁇ ).
  • Erythrocytes, infected erythrocytes and erythrocyte free parasites in the trophozoite stage are treated with 100 ⁇ (54), 10 ⁇ (61) and (65) or 15 ⁇ (76) for one hour at 37 °C.
  • Cells are then labeled with 1 ⁇ [2,8- 3 H]hypoxanthine (30 Ci/mmol, Moravek) or 1 ⁇ [2,8- 3 H]inosine (50 Ci/mmol, Moravek) for 1 hour at 37 °C.
  • Parasites from infected erythrocytes are isolated by lysis with 0.045% saponin either before addition of radiolabel or after incubation with radiolabel.
  • Proteins and nucleic acids are removed by perchloric acid treatment of supernatants and cell pellets (J. Biol. Chem., 2008, 283, 32889-32899). All samples are analyzed by HPLC with a modification in the previously described solvent gradient (J. Biol. Chem., 2008, 283, 32889-32899). Briefly, the mobile phases are 8 mM tetrabutylammonium bisulfate (Fluka) and 100 mM KH 2 P0 4 with the pH adjusted to 6.0 with KOH in water (solution A) or 30% acetonitrile (solution B). The HPLC gradient is from 0% to 10% solution B in 4 min and maintained for 2 min, 10% to 20% solution B in 1 min, 20% to 40% solution B in 10 min, 40% to 100% solution B in 3 min and maintained for 4 min.
  • Fluka tetrabutylammonium bisulfate
  • solution B acetonitrile
  • Table 4 Metabolic labeling summary showing the percentage change in incorporation of radiolabel from [2,8- 3 H 2 ]hypoxanthine into the nucleotide pool of erythrocytes, infected erythrocytes and free parasites on treatment with compound (61)
  • Inhibition of hypoxanthine uptake by (61) or (65) is stronger in infected erythrocytes than in isolated parasites, indicating that the erythrocytes play a role in the activation of the prodrug compound of the invention and/or transportation into the parasite.
  • Samples are placed in 96 deep-well plates, treated with 0.5 M HCI0 4 at 1 :7 (v/v, sample/HCI0 4 ), incubated for 20 min at 4 °C and neutralized with 5 M KOH at 10:1 (v/v, HCIO 4 /KOH) for 20 min at 4 °C. Plates are centrifuged (10 min at 4,000 rpm, 4 °C) and supernatants are filtered through a Multiscreen ® Filter Plate with Ultracel ® -10 Membrane (Millipore). Metabolite and inhibitor levels are analyzed by UPLC/MS/MS using a Xevo TQD mass spectrometer (Waters).
  • the separation of inosine, hypoxanthine, and inhibitors is achieved with an Acquity HSS T3 column (2.1 x 100 mm, 1.8 ⁇ , Waters) at 60 °C.
  • the eluent system is composed of 5 mM ammonium formate in water (A) and 5 mM ammonium formate in methanol (B) with a gradient of 98% eluent A to 30% eluent B from 0.1 to 1 min, 70% eluent A to 80% eluent B from 1 to 1.5 min and back to 98% eluent A from 1.5 to 3 min at a flow rate of 0.6 ml min "1 .
  • Detection is performed in ESI positive-ion mode using multiple-reaction monitoring (MRM) mode.
  • MRM multiple-reaction monitoring
  • ESI-MS/MS analysis the following ion transitions, cone voltage (CV) and collision energy (CE) are used: inosine m/z 269.1 >137.1 (CV: 14 V, CE: 12 eV), hypoxanthine m/z 137.1 >110.0 (CV: 42 V, CE: 18 eV), compound (7c) m/z 286.92 >139.74 (CV: 22 V, CE: 14 eV), compound (54) m/z 515.05 >205, (CV: 34 V, CE: 28 eV).
  • the ESI capillary voltage is 0.3 kV
  • source temperature is set at 150 °C and desolvation temperature at 450 °C.
  • Data acquisition and analysis are carried out by MassLynx V4.1 and QuanLynx software.
  • the invention relates to compounds that are inhibitors of hypoxanthine and/or guanine purine phosphoribosyltransferases, e.g hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferases of protozoan parasites, including those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani.
  • the compounds are therefore indicated for the treatment or prevention of diseases in which the inhibition of such purine phospho

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Abstract

The invention relates to compounds of formula (I) that are inhibitors of hypoxanthine and/or guanine purine phosphoribosyltransferases and to pharmaceutical compositions containing the compounds, processes for preparing the compounds, and methods of treating diseases or conditions in which it is desirable to inhibit hypoxanthine and/or guanine purine phosphoribosyltransferases. Such diseases include malaria.

Description

PHOSPHOR1BOSYLTRANSFERASE INHIBITORS AND USES THEREOF
STATEMENT OF GOVERNMENT SUPPORT
This invention is made with government support under grant number AI049512 awarded by the National Institutes of Health. The government has certain rights in the invention.
TECHNICAL FIELD This invention relates generally to compounds that are inhibitors of hypoxanthine and/or guanine purine phosphoribosyltransferases and to pharmaceutical compositions containing the compounds, processes for preparing the compounds, and methods of treating diseases or conditions in which it is desirable to inhibit hypoxanthine and/or guanine purine phosphoribosyltransferases, e.g. hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferases of protozoan parasites, including those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani. The diseases or conditions include malaria.
BACKGROUND Malaria is a major global health concern. It is estimated that malaria is responsible for greater than 200 million clinical cases and about 800000 deaths each year. The disease is caused by a parasite of the genus Plasmodium, which is transmitted by infected Anopheles mosquitos. There are four parasites that cause human malaria - Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae. Plasmodium falciparum is considered to be the deadliest. The vaccines for malaria currently in clinical trials confer limited protection and there is a problem with the emergence of resistance to current therapies such as artemisinin and its derivatives.
Plasmodium falciparum lacks the ability to synthesise purines de novo (Reyes, P., Rathod, P.K., Sanchez, D.J., Mrema, J.E., Rieckmann, K.H., and Heidrich, H.G. Mol. Biochem. Parasitol., 1982, 5, 275-290). In addition, the parasite lacks adenosine kinase or adenine phosphoribosyltransferase activity and relies on the conversion of hypoxanthine to inosine 5'-monophosphate by hypoxanthine-guanine-xanthine phosphoribosyltransferase (PflHGXPRT) as its source of purines (Cassera, M.B., Hazleton, K.Z., Riegelhaupt, P.M., Merino, E.F., Luo, M., Akabas, M.H., and Schramm, V.L. J. Biol. Chem., 2008, 283, 32889-32899). Hypoxanthine depletion through xanthine oxidase-mediated degradation or purine nucleoside phosphorylase (PNP) inhibition has been demonstrated to kill parasites in cell culture (Erman, P.A., Human, L. Adv. Exp. Med. Biol., 1991 , 309A, 165-168 and Kicska, G.A., Tyler, P.C., Evans, G.B., Furneaux, R.H., Schramm, V.L., and Kim, K. J. Biol. Chem., 2002, 277, 3226-3231.) This makes PflHGXPRT an attractive target for new chemotherapies.
Inhibition of human hypoxanthine-guanine phosphoribosyltransferase (HsHGPRT) by an inhibitor of PflHGXRPT is best avoided since genetic defects causing loss of human HGPRT activity leads to gouty arthritis. Furthermore, complete loss of activity is the cause of Lesch-Nyhan syndrome, a debilitating neurological disorder (Lesch, M. & Nyhan, W. L, Am. J. Med., 1964, 36, 561 ; Seegmiller, J. E., et al., Science, 1967, 155, , 1682. A selective inhibitor would avoid this potential problem and would also avoid increases in serum hypoxanthine levels by maintaining host HGPRT activity.
Keough et al. demonstrated inhibition of PflHGXPRT by acyclic nucleoside phosphonates (D.T. Keough, D. Hockova, A. Holy, L M. J. Naesens, T. S. Skinner-Adams, J. de Jersey, L. W. Guddat, J. Med. Chem, 2009, 52, 4391-4399; D. Hockova, A. Holy, M. Masojidkova, D.T. Keough, J. de Jersey, L. W. Guddat, Bioorg. Med. Chem., 2009, 17, 6218-6232). Some of the phosphonate compounds, incorporating a guanine or hypoxanthine base and a phosphonate 'tail' attached to the base, have high nanomolar to micromolar inhibition constants ( ,) against both PflHGXPRT and human hypoxanthine-guanine phosphoribosyltransferase (HsHGPRT). Branched acyclic nucleoside phosphonates are weak inhibitors of PflHGXPRT and HsHGPRT. The best compound had Ki of 100 ± 20 nM for P/HGXPRT and Ki of of 1.0 ± 0.5 μΜ for HsHGPRT - a moderate selectivity of 10-fold. The acyclic nucleoside phosphonates are substrate analogues.
Transition state analogues are attractive as chemotherapeutics. They can bind more strongly to an enzyme than, for example, substrate analogues. However, HGXPRTs have resisted transition state analysis because of kinetic commitment factors. Nevertheless, the applicants have proposed a transition state structure for PflHGXPRT (C. M. Li et al., Nat. Struct. Biol., 1999, 6, 582-587) and reported that a nucleoside analogue, Immucillin-H 5'-phosphate (ImmHP) is a 1 nM inhibitor of P/HGXPRT, but with no selectivity relative to HsHGPRT.
There remains an ongoing need to develop new antimalarial compounds against novel targets such as HGXPRT. It is therefore an object of the present invention to provide compounds that are inhibitors of hypoxanthine and/or guanine purine phosphonbosyltransferases, such as hypoxanthine and/or guanine and/or xanthine purine phosphonbosyltransferases of protozoan parasites, or to at least provide a useful choice.
SUMMARY OF INVENTION
In a first aspect, the present invention provides a compound of the formula (I):
Figure imgf000005_0001
wherein:
A is CH, CR2 or N;
D is H, OH or NH2;
R2 is halogen, alkyl, aralkyl or aryl; and n is 1 ; R1 is a radical of formula (i) where G is O; X is an optionally substituted C3 or C5 alkylene group and R1 is attached to a terminal carbon atom of X; or n is 1 ; R1 is a radical of formula (i) where G is absent; X is an optionally substituted C3 or C4 alkylene group and R1 is attached to a terminal carbon atom of X; or n is 2; R is a radical of formula (i) where G is O or is absent; X is an optionally substituted C2 alkylene group and R1 is attached to a terminal carbon atom of X;
Figure imgf000006_0001
(i) or an ester prodrug form thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof.
" _ '
Preferably the ester prodrug form of the compound of formula (I) is one in which one or more hydrogens in the group R1 is replaced with one or more lipophilic groups, e.g. one or more alkoxyalkyl groups. It is further preferred that the ester prodrug form of the compound of formula (I) is one in which R1 is a radical of formula (ii):
Figure imgf000006_0002
(ii)
wherein:
Z is -(CH2)m-0-(CH2)p-CH3;
Y is H, alkyl or -(CH2)m-0-(CH2)p-CH3;
G is O or G is absent;
m is 2 or 3; and
p is an integer from 2 to 21 ;
and where, when Z is -(CH2)m-0-(CH2)p-CH3 and Y is -(CH2)m-0-(CH2)p-CH3, each m and each p is independently selected.
Preferably the compound of formula (I) is an ester prodrug compound of formula (la):
Figure imgf000006_0003
wherein:
A is CH, CR2 or N;
D is H, OH or NH2;
R2 is halogen, alkyl, aralkyl or aryl; and n is 1 ; R is a radical of formula (ii) where G is O; Z is -(CH2)m-0-(CH2)p-CH3; Y is H, alkyl or -(CH2)m-0-(CH2)p-CH3; X is an optionally substituted C3 or C5 alkylene group and R1 is attached to a terminal carbon atom of X; or n is 1 ; R1 is a radical of formula (ii) where G is absent; Z is -(CH2)m-0-(CH2)p-CH3; Y is H, alkyl or -(CH2)m-0-(CH2)p-CH3; X is an optionally substituted C3 or C4 alkylene group and R1 is attached to a terminal carbon atom of X; or n is 2; R1 is a radical of formula (ii) where G is O or is absent; Z is -(CH2)m-0-(CH2)p-CH3; Y is H, alkyl or -(CH2)m-0-(CH2)p-CH3; X is an optionally substituted C2 alkylene group and R1 is attached to a terminal carbon atom of X;
Figure imgf000007_0001
(ii) where m is 2 or 3 and p is an integer from 2 to 21 and where, when Z is -(CH2)m-0- (CH2)P-CH3 and Y is -(CH2)m-0-(CH2)p-CH3, each m and each p is independently selected; or a tautomer thereof, or a pharmaceutically acceptable salt thereof. Preferably, G is absent.
The C2, C3, C4 or C5 alkylene group X in the above formula (I) or (la) may optionally be substituted with one or more hydroxy groups and/or one or two fluorine atoms. Preferably, when G is absent, the one or two fluorine atoms are attached to the terminal carbon of X, to which the group R1 is also attached. It is preferred that A is CH or N, more preferably CH.
It is also preferred that D is H or NH2, most preferably D is H.
In some examples, A is CH and D is H. In other examples A is N and D is H. In still other examples, A is CH and D is NH2.
Preferably the compound of formula (I) is a compound of formula (Γ):
Figure imgf000008_0001
where X is a C3 or C4 alkylene group which is substituted with one or more hydroxy groups and/or one or two fluorine atoms.
Preferably the compound of formula (la) is a compound of formula (la'):
Figure imgf000008_0002
(la')
where Y and Z are as defined above for formula (la);
or a tautomer thereof, or a pharmaceutically acceptable salt thereof.
Alternatively it is preferred that the compound of formula (la) is a compound of formula
Figure imgf000008_0003
(la")
where Y and Z are as defined above for formula (la);
or a tautomer thereof, or a pharmaceutically acceptable salt thereof.
Alternatively it is preferred that the compound of formula (la) is a compound of formula (la'"):
Figure imgf000009_0001
(la"') where Y and Z are as defined above for formula (la);
or a tautomer thereof, or a pharmaceutically acceptable salt thereof.
In some examples m is 3. In other examples m is 2.
In some examples p is an integer from 7 to 21 , e.g. an integer from 7 to 17. In some examples p is 17. In other examples p is 15. In still other examples p is 7.
In some examples m is 3 and p is an integer from 7 to 21 , e.g. an integer from 7 to 17, e.g. p is 17 or p is 15 or p is 7. In some examples, R1 is a radical of formula (ii) where Z is -(CH2)m-0-(CH2)p-CH3 and Y is alkyl, e.g. lower alkyl, e.g. ethyl. In other examples R1 is a radical of formula (ii) where Z is -(CH2)m-0-(CH2)p-CH3 and Y is H. In still other examples R1 is a radical of formula (ii) where Z and Y are each independently -(CH2)m-0-(CH2)p-CH3. In some examples, R1 is a radical of formula (ii) and the C2, C3, C4 or C5 alkylene group X is substituted with one or more hydroxy groups. In other examples, R1 is a radical of formula (ii) and the C2, C3, C4 or C5 alkylene group X is substituted with one or two fluorine atoms. In other examples, R1 is a radical of formula (ii) and the C2, C3, C4 or C5 alkylene group X is substituted with one or two fluorine atoms and one or more hydroxy groups.
In some examples, R1 is a radical of formula (i) and the C2, C3, C4 or C5 alkylene group X is substituted with one or more hydroxy groups. In other examples, R1 is a radical of formula (i) and the C2, C3, C4 or C5 alkylene group X is substituted with one or two fluorine atoms. In other examples, R1 is a radical of formula (i) and the C2, C3, C4 or C5 alkylene group X is substituted with one or two fluorine atoms and one or more hydroxy groups. Preferably X is selected from the group consisting of ethylene, 1 ,3-propylene, 1 ,4- butylene, 1 ,5-pentylene, 3-hydroxy-1 ,2-propylene, 2-hydroxy-1 ,3-propylene, 2- hydroxymethyl-1 ,3-propylene, 2,2-bis(hydroxymethyl)-1 ,3-propylene, 4-hydroxy-1 ,3- butylene, 1-fluoro-4-hydroxy-1 ,3-butylene, 1 ,1-difluoro-4-hydroxy-1 ,3-butylene (where the carbon atom to which R1 is attached is designated the 1 -position).
Preferably the compound of formula (I) is selected from the group consisting of:
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
10
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001

Figure imgf000019_0001
or a tautomer thereof, or a pharmaceutically acceptable salt thereof.
In another aspect the invention provides a composition comprising a pharmaceutically effective amount of a compound of formula (I) or (la) and optionally a carrier.
In another aspect the invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I) or (la) and, optionally, a pharmaceutically acceptable carrier, diluent or excipient. In another aspect the invention provides a compound of formula (I) or (la) in combination with at least one other compound, e.g. a second drug compound. The other compound may be, for example, an anti-malarial compound such as quinine, quinidine, cinchoine, cinchonidine, chloroquine, amodiaquine, amodiaquine, proguanil, sulfadoxine, sulfamethoxypyridazine, mefloquine, atovaquone, primaquine, artemisinin, artemether, artesunate, dihydroartemisinin, arteether, halofantrine, doxycycline, clindamycin or (3R,4R)-1-[(9-deazaguanin-9-yl)methyl]-3-hydroxy-4-(hydroxymethyl)pyrrolidine.
In another aspect the invention provides the use of a compound of formula (I) or (la) for inhibiting a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g. those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani.
In another aspect the invention provides the use of a compound of formula (I) or (la) as a medicament.
In another aspect the invention provides the use of a compound of formula (I) or (la) for treating or preventing a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g. those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani.
In another aspect the invention provides the use of a compound of formula (I) or (la) for treating or preventing an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani. Preferably, the invention provides the use of a compound of formula (I) or (la) for treating or preventing malaria.
In another aspect the invention provides the use of a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I) or (la) for treating or preventing a disease or disorder in which it is desirable to inhibit a a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g. those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani. In another aspect the invention provides the use of a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I) or (la) for treating or preventing an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani. Preferably, the invention provides the use of a pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I) or (la) for treating or preventing malaria.
In another aspect the invention provides a compound of formula (I) or (la) for use in the manufacture of a medicament.
In another aspect the invention provides a pharmaceutical composition for treating or preventing a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g. those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani, comprising a compound of formula (I) or (la).
In another aspect the invention provides a pharmaceutical composition for treating or preventing an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani, comprising a compound of formula (I) or (la). Preferably, the invention provides a pharmaceutical composition for treating or preventing malaria. In another aspect the invention provides the use of a compound of formula (I) or (la) in the manufacture of a medicament for the treatment or prevention of a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g. those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani.
In another aspect the invention provides the use of a compound of formula (I) or (la) in the manufacture of a medicament for the treatment or prevention of an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani. Preferably, the invention provides the use of a compound of formula (I) or (la) in the manufacture of a medicament for the treatment or prevention of malaria.
In another aspect the invention provides a method of treating or preventing a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g. those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani, comprising administering a pharmaceutically effective amount of a compound of formula (I) or (la) to a patient requiring treatment.
In another aspect the invention provides a method of treating or preventing an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani, comprising administering a pharmaceutically effective amount of a compound of formula (I) or (la) to a patient requiring treatment. Preferably, the invention provides a method of treating or preventing malaria, comprising administering a pharmaceutically effective amount of a compound of formula (I) or (la) to a patient requiring treatment.
In another aspect the invention provides the use of a compound of formula (I) or (la) in combination with at least one other compound, e.g. a second drug compound, e.g. an anti-malarial such as quinine, quinidine, cinchoine, cinchonidine, chloroquine, amodiaquine, amodiaquine, proguanil, sulfadoxine, sulfamethoxypyridazine, mefloquine, atovaquone, primaquine, artemisinin, artemether, artesunate, dihydroartemisinin, arteether, halofantrine, doxycycline, clindamycin or (3R,4R)-1-[(9-deazaguanin-9- yl)methyl]-3-hydroxy-4-(hydroxymethyl)pyrrolidine, for treating or preventing a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g. those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani.
The compounds of formula (I) or (la) can be covalently attached via a biologically cleavable linkage to antimalarials such as quinine, quinidine, cinchoine, cinchonidine, chloroquine, amodiaquine, amodiaquine, proguanil, sulfadoxine, sulfamethoxypyridazine, mefloquine, atovaquone, primaquine, artemisinin, artemether, artesunate, dihydroartemisinin, arteether, halofantrine, doxycycline, clindamycin or (3R,4R)-1-[(9- deazaguanin-9-yl)methyl]-3-hydroxy-4-(hydroxymethyl)pyrrolidine.
In another aspect the invention provides a method of treating or preventing a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or a guanine purine phosphoribosyltransferase, e.g a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, e.g. those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani, comprising administering a pharmaceutically effective amount of a compound of formula (I) or (la) in combination with at least one other compound, e.g. a second drug compound, e.g. an anti-malarial compound such as quinine, quinidine, cinchoine, cinchonidine, chloroquine, amodiaquine, amodiaquine, proguanil, sulfadoxine, sulfamethoxypyridazine, mefloquine, atovaquone, primaquine, artemisinin, artemether, artesunate, dihydroartemisinin, arteether, halofantrine, doxycycline, clindamycin or (3R,4R)-1-[(9-deazaguanin-9-yl)methyl]-3-hydroxy-4- (hydroxymethyl)pyrrolidine. The compound of formula (I) or (la) and the other compound may be administered separately, simultaneously or sequentially, or the compound of formula (I) or (la) can be covalently attached via a biologically cleavable linkage to the other compound. Preferably the disease or disorder is an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani. Most preferably the disease or disorder is malaria.
The compound of formula (I) or (la) may be selected from compounds (a) to (bt) as defined above.
Compounds of formulae (I) or (la) are hereinafter described as "compounds of the invention". A compound of the invention includes a compound in any form, e.g. in free form or in the form of a salt or a solvate.
DETAILED DESCRIPTION Definitions
The term "alkyl" means any saturated hydrocarbon radical having up to 30 carbon atoms and includes any C C25, C Cao, Ci-C 5, C C10, or C^Ce alkyl group, and is intended to include straight- and branched-chain alkyl groups. Examples of alkyl groups include: methyl group, ethyl group, n-propyl group, /so-propyl group, n-butyl group, /'so-butyl group, sec-butyl group, f-butyl group, n-pentyl group, 1 ,1-dimethylpropyl group, 1 ,2- dimethylpropyl group, 2,2-dimethylpropyl group, 1 -ethylpropyl group, 2-ethylpropyl group, n-hexyl group, 1 ,2-dimethylbutyl group. The term "lower alkyl" means a C^Ce alkyl group, where alkyl is as defined above.
Any alkyl group may optionally be substituted with one or more fluorine or chlorine substituents. The term "alkylene" has corresponding a meaning to "alkyl" and is intended to include saturated straight and branched chain groups, preferably C2-C5 alkylene groups, e.g. ethylene, 1 ,3-propylene, 1 ,5-butylene, 1 ,2-propylene, 2-methyl-1 ,3-propylene, 2,2-di-1 ,3- propylene, and 1 ,3-butylene. It will be appreciated that a C2, C3, C4 or C5 alkylene group X in the above formula (I) or (la) can be attached to the amino nitrogen atom via any carbon of the alkylene group, except the carbon to which the phosphate or phosphonate group R1 is attached. The carbon to which the phosphate or phosphonate group R1 is attached is the terminal carbon. For example, a C3 alkylene group can be attached at the 2- or 3-position of the carbon chain (where the carbon to which phosphate or phosphonate group R1 is attached is designated position 1), or a C4 alkylene group can be attached the 2-, 3- or 4- position of the carbon chain (where the carbon to which phosphate or phosphonate group R1 is attached is designated position 1). This gives rise to straight and branched- chain C3 and C4 alkylene groups. The scope of the invention is intended to include both straight and branched alkylene groups. Any alkylene group may be optionally substituted with one or two fluorine and/or one or more hydroxy groups, e.g. 3-hydroxy-1 ,2-propylene, 2-hydroxy-1 ,3-propylene, 2- hydroxymethyl-1 ,3-propylene, 2,2-bis(hydroxymethyl)-1 ,3-propylene, 4-hydroxy-1 ,3- butylene, 1-fluoro-4-hydroxy-1 ,3-butylene and 1 ,1-difluoro-4-hydroxy-1 ,3-butylene. The term "alkoxyalkyl" means -ROR' where R' is alkyl as defined above and R is alkylene.
The term "aryl" means an aromatic radical having 4 to 18 carbon atoms and includes heteroaromatic radicals. Examples include monocyclic groups, as well as fused groups such as bicyclic groups and tricyclic groups. Some examples include phenyl group, indenyl group, 1-naphthyl group, 2-naphthyl group, azulenyl group, heptalenyl group, biphenyl group, indacenyl group, acenaphthyl group, fluorenyl group, phenalenyl group, phenanthrenyl group, anthracenyl group, cyclopentacyclooctenyl group, and benzocyclooctenyl group, pyridyl group, pyrrolyl group, pyridazinyl group, pyrimidinyl group, pyrazinyl group, triazolyl group (including a 1-/7-1 ,2,3-triazol-1-yl and a 1-H-1 ,2,3- triazol-4-yl group), tetrazolyl group, benzotriazolyl group, pyrazolyl group, imidazolyl group, benzimidazolyl group, indolyl group, isoindolyl group, indolizinyl group, purinyl group, indazolyl group, furyl group, pyranyl group, benzofuryl group, isobenzofuryl group, thienyl group, thiazolyl group, isothiazolyl group, benzothiazolyl group, oxazolyl group, and isoxazolyl group.
The term "aralkyi" means an aryl group covalently linked to an alkylene group.
Any aryl or aralkyi group may optionally be substituted with one or more fluorine, chlorine or C C3 alkyl substituents. The symbol " ", as used in structural formulae shown herein, is intended to denote the point of attachment of a radical in a structural formula.
The term "prodrug" as used herein means a pharmacologically acceptable derivative of the compounds of formula (I) such that an in vivo biotransformation of the derivative gives the compound as defined in formula (I). Prodrugs of compounds of formula (I) may be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved in vivo to give the parent compound. Typically, prodrugs of the compounds of formula (I) will be ester prodrug forms.
The term "pharmaceutically acceptable salts" is intended to apply to non-toxic salts such as ammonium salts, metal salts, e.g. sodium salts, or salts of organic cations, or a mixture thereof. The term "protecting group" means a group that selectively protects an organic functional group, temporarily masking the chemistry of that functional group and allowing other sites in the molecule to be manipulated without affecting the functional group. Suitable protecting groups are known to those skilled in the art and are described, for example, in Protective Groups in Organic Synthesis (3rd Ed.), T. W. Greene and P. G. M. Wuts, John Wiley & Sons Inc (1999). Examples of protecting groups include, but are not limited to: O-benzyl, O-benzhydryl, O-trityl, O-tert-butyldimethylsilyl, O-tert-butyldiphenylsilyl, 0-4- methylbenzyl, O-acetyl, O-chloroacetyl, O-methoxyacetyl, O-benzoyl, O-4-bromobenzoyl, O-4-methylbenzoyl, O-fluorenylmethoxycarbonyl, O-levulinoyl or O-tert-butyl. The term "patient" includes human and non-human animals.
The terms "treatment", "treating" and the like include the alleviation of one or more symptoms, or improvement of a state associated with the disease or disorder, for example reduction in malaria parasitaemia.
The terms "preventing", "prevention" and the like include the prevention of one or more symptoms or states associated with the disease or disorder, for example, prevention of malaria parasitaemia. Those skilled in the art will understand that the compounds of the invention can exist in different tautomeric forms. For example, it will be appreciated that the representation of a compound of formula (I) or (la) where D is a hydroxy group is of the enol-type tautomeric form of a corresponding amide, and this will largely exist in the amide form. The 4- hydroxy group of the 4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl moiety in compounds of the invention is represented in the nomenclature as the enol-type tautomeric form but will largely exist in the corresponding amide tautomeric form. The scope of the invention is intended to cover all tautomeric forms of the compounds of the invention. It will also be appreciated that the compounds of the invention can exist in the form of optical isomers, racemates and diastereomers. The scope of this invention is intended to cover all possible stereoisomeric forms of the compounds of formulae (I) and (la). For example, one or more of the carbon atoms of the optionally substituted X group in the formula (I) or (la) may be asymmetric carbons and may be in the R- or S-configuration. Furthermore, the phosphorus of the R1 (phosphate or phosphonate) group may also be a chiral centre. The structures shown for the ester prodrug compounds of the invention in which the phosphate or phosphonate group has four different substituents attached to the phosphorus atom are intended to represent racemates. Compounds of these structures exist in two enantiomeric forms. The scope of this invention is intended to cover all possible forms, i.e. the racemates and the two enantiomers.
The Compounds of the Invention
The compounds of the invention, particularly those exemplified, are inhibitors of hypoxanthine and/or guanine purine phosphoribosyltransferases, e.g hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferases of protozoan parasites, including those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani, and are useful as pharmaceuticals, particularly for the treatment or prevention of diseases or conditions in which it is desirable to inhibit hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferases. The diseases or conditions include malaria. The compounds of the invention are useful in both free base or acid form and in the form of salts and/or solvates. The compounds of the invention are surprisingly potent inhibitors of Plasmodium falciparum HGXPRT. For example, compound (95) has a K, of 0.65 nM against Plasmodium falciparum HGXPRT. Also surprising is the selectivity that the compounds of the invention show against Plasmodium falciparum HGXPRT compared to human HGPRT. For example, the K, for compound (95) against Plasmodium falciparum HGXPRT is more than 500 times lower than the K, against human HGPRT. This level of potency and selectivity would not have been predicted from the prior art.
The compounds of the invention include prodrug forms. Advantageously, prodrugs of the compounds of formula (I), e.g. compounds of formula (la), can have increased efficacy. Preferred prodrug forms are ester prodrugs, where one or more hydrogens of the phosphate or phosphonate (R1) groups of the compounds of formula (I) are replaced with suitable lipophilic groups, such as alkoxyalkyl groups. The use of lipopholic groups allows for transfer of the compounds through cell membranes. Without wishing to be bound by theory, it is proposed that the prodrug forms are transported into and activated inside the Plasmodium parasite, via cleavage by phospholipase C (S. A. Lauer, S. Chatterjee, K. Haldar, Molecular and Biochemical Parasitology, (2001), 115, 275-281). The prodrugs are active against cultured Plasmodium falciparum. For example, Table 2 shows the IC50 values for prodrug compounds of the invention against Plasmodium falciparum strain 3D7, the chloroquine/mefloquine resistant strain Dd2, and the chloroquine/quinine resistant strain FVO.
The compounds of the invention may be administered to a patient by a variety of routes, including orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally or via an implanted reservoir. For parenteral administration, injections may be given intravenously, intra-arterially, intramuscularly or subcutaneously.
The amount of a compound of the invention to be administered to a patient will vary widely according to the nature of the patient and the nature and extent of the disorder to be treated. Typically the dosage for an adult human will be in the range of about 0.01 Mg/kg to about 1 g/kg, preferably about 0.01 mg/kg to about 100 mg/kg. The specific dosage required for any particular patient will depend upon a variety of factors, such as the patient's age, body weight, general health, gender and diet. Optimal doses will depend on other factors such as mode of administration and level of progression of the disease or disorder. Doses may be given once daily, or two or more doses may be required per day. For example, a dosage regime for a malaria patient might require one dose in the morning and one in the evening. Alternatively, a dosage regime for such a patient might require four hourly doses.
For oral administration the compounds can be formulated into solid or liquid preparations, for example tablets, capsules, granules, powders, solutions, suspensions, syrups, elixirs and dispersions. Such preparations are well known in the art as are other oral dosage regimes not listed here.
For parenteral administration, compounds of the invention can be formulated into sterile solutions, emulsions and suspension.
Compounds of the invention may be mixed with suitable vehicle and then compressed into the desired shape and size. The compounds may be tableted with conventional tablet bases such as lactose, sucrose and corn starch, together with a binder, a disintegration agent and a lubricant. The binder may be, for example, corn starch or gelatin, the disintegrating agent may be potato starch or alginic acid, and the lubricant may be magnesium stearate. For oral administration in the form of capsules, diluents such as lactose and dried cornstarch may be employed. Other components such as colourings, sweeteners or flavourings may be added. Tablets, capsules or powders for oral administration may contain up to about 99% of a compound of the invention.
When liquid preparations are required for oral use, a compound of the invention may be combined with a pharmaceutically acceptable carriers such as water, an organic solvent such as ethanol, or a mixture of both, and optionally other additives such as emulsifying agents, suspending agents, buffers, preservatives, and/or surfactants may be used. Colourings, sweeteners or flavourings may also be added.
The compounds may also be administered by injection in a pharmaceutically acceptable diluent such as water or saline. The diluent may comprise one or more other ingredients such as ethanol, propylene glycol, an oil or a pharmaceutically acceptable surfactant.
The compounds of the invention may also be administered topically. Carriers for topical administration of the compounds include mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. The compounds may be present as ingredients in lotions or creams, for topical administration to skin or mucous membranes. Such creams may contain the active compounds suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
The compounds of the invention may further be administered by means of sustained release systems. For example, they may be incorporated into a slowly dissolving tablet or capsule.
Synthesis of the Compounds of the Invention Advantageously, the compounds of the invention are synthetically accessible and may be prepared by a variety of different methods. The following are representative non- limiting examples.
General Procedure 1
Synthesis of ((4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl)methylamino)alkylphosphonic acids (C), Scheme A.
Figure imgf000030_0001
Scheme A
A ((4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methylamino)alkylphosphonic acid (C) of the invention (where D = H or OH and X is as defined below) can be obtained by reductive amination of a dialkyl ester of a aminoalkylphosphonic acid (B) (where Rc is an alkyl or aralkyi group and X' is as defined below) with a 9-formyl-9-deazapurine of general formula (A) (where D' = H or ORbl; Ra is H or an N-protecting group; and Rb is H or an O-protecting group) to give an adduct of general formula (C), from which any protecting groups are then removed as required to give a compound (D) of the invention (Scheme A). Conveniently Rc will be a methyl, ethyl or tert-butyl group. The groups X and X' are optionally substituted alkylene groups, preferably optionally substituted C2 - C5 linear or branched alkylene groups, to which the amino-moiety is attached to a primary, secondary or tertiary carbon atom of the alkylene group, and the phosphonic acid or ester moiety, respectively, is attached to a terminal carbon atom of the alkylene group. The optional substituents on the alkylene group X' may be (a) one or two fluoro-atom substituents, preferably on the carbon bearing the phosphonic acid or ester moiety, and/or (b) one or more hydroxyl group substituents, or the O-protected forms on an otherwise unsubstituted primary, secondary or tertiary carbon atom of the linker. The alkylene group X in the product compound (D) will be the same as the alkylene group X' in the precursor compound (C) but with O-protecting groups removed. A suitable O- protecting group is a benzyl ether group.
Alternatively, a free phosphonic acid of general formula (B) (where R° is H) can be used in the reductive amination with the aldehyde of general formula (A), to give an adduct of general formula (C) where Rc is H, which can then be converted as above to give a compound of general formula (D).
Suitable aldehydes of general formula (A) can be prepared as detailed in WO 2009/082247 and WO2008/030119, where suitable combinations of substituents are Ra = H and Rb = Bn; Ra = Rb = H; or Ra = BnCH2 and Rb = Bu1. Reductive amination is conveniently effected by use of a reducing agent selected from sodium borohydride, 2- picoline borane complex, sodium triacetoxyborohydride or sodium cyanoborohydride. The protecting groups in an intermediate adduct of general formula (C) are then removed to give the free phosphonic acid (D) (or salt form thereof). This deprotection can be by stepwise treatment with strong mineral acid, typically concentrated hydrochloric acid, then with aqueous hydrobromic acid, typically 48% HBr in water. Benzyl and benzyloxymethyl protecting groups can be removed by hydrogenolysis over a palladium catalyst if desired.
General Procedure 2
Synthesis of a (2-(4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl)ethylamino)alkylphosphonic acid, Scheme B.
Figure imgf000032_0001
A (2-(4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)ethylamino)alkylphosphon acid (H) of the invention can be obtained as shown in Scheme B by reductive amination of a dialkyi ester of the requisite dialkyi ω-oxoalkylphosphonate (F) (where the group X" is as defined below) with a 9-(2-aminoethyl)-9-deazapurine of general formula (E) (where D is H or ORb and where Rb is H or an O-protecting group) to give an adduct of general formula (G) (where X' is an optionally substituted alkylene linker as defined in General Procedure 1 above) from which any protecting groups are then removed as required to give a compound of general formula (H) (where X is an optionally substituted alkylene linker as defined in General Procedure 1 above). The group X" in a compound of formula (F) is an optionally substituted alkylene group, preferably an optionally substituted Ci - C4 linear or branched alkylene group, to which the amino-moiety is attached to a primary, secondary or tertiary carbon atom of the alkylene group, and the phosphonic ester moiety is attached to a terminal carbon atom of the alkylene group. The optional substituents on the alkylene group X" may be (a) one or two fluoro-atom substituents, preferably on the carbon bearing the phosphonic acid or ester moiety, and/or (b) one or more hydroxy-group substituents or their O-protected forms on an otherwise unsubstituted primary, secondary or tertiary carbon atom of the linker. Conveniently the requisite dialkyi ω-oxoalkylphosphonate (F) will be a diethyl ester.
Figure imgf000032_0002
A compound of general formula (E) can be prepared from a protected 9- deazahypoxanthine of general formula (I) (where D = H or ORb) (Scheme C) by subjecting the compound of general formula (I) sequentially to (i) a Mannich reaction with dimethylamine and formaldehyde, (ii) N-quaternisation; (iii) displacement of the quaternary ammonium leaving group by a nitromethane anion; and (iv) reduction of the resulting nitro group in the product to a primary amine (Scheme C). Convenient reagents and reaction conditions for this conversion can be found in Example 2 (below) for the synthesis of compound (12). Suitable O-protecting groups (Rb) in the compound of general formula (I) include methyl, benzyl and tert-butyl. A compound of general formula (I) where R = Bn, D = H; and Rb = Bn and D = OBn, can be prepared by the methods described in Kamath et a/., Org. Proc. Devel., 13 (2009) 928-932 and Evans, et a/., J. Org. Chem., 66 (2001) 5723-5730, respectively. Alternative protecting groups can be introduced with the substitution of benzyl alcohol with an alternative alcohol (e.g. methanol or tert-butanol) if desired. A compound of general formula (I) where Rb = Me, D = H; can be prepared by the methods described in Sakamoto et a/., Chem. Pharm. Bull., 1993, 41 ,81 - 86.
General Procedure 3
Synthesis of a bis(alkoxyalkyl) ester of an aminoalkylphosphonic acid, Scheme D.
Figure imgf000033_0001
(N)
Scheme D A bis(alkyloxyalkyl) aminoalkylphosphonate of general structure (N), where Re is an optionally substituted alkyl group, n is 2 or 3, and X' is an optionally substituted alkylene group, where the optional substituents can be (i) one or two fluoro-atom substituents, and/or (ii) one or more hydroxy-group substituents or O-protected forms thereof, can be prepared as shown in Scheme D from a dialkyl aminoalkylphosphonates (G) by: (a) hydrolysis to a phosphonic acid (J); (b) N-protection to give a carbamate (K) (where Rd is an optionally substituted alkyl or aralkyl group); (c) chlorination to give dichloride (L); (d) displacement of both chlorides with an alkoxyalkyl alcohol to give (M); and (e) final N- deprotection to give the required bis(alkyloxyalkyl) aminoalkylphosphonate (N). Conveniently, the starting amine (G) is the diethyl ester, and suitable reagents for the conversions are: (a) hydrolysis using aqueous hydrobromic acid heated under reflux; (b) N-protection with benzyl chloroformate, di-tert-butyl dicarbonate or 2,2,2- trichloroethoxycarbonyl chloride; (c) dichlorination with thionyl chloride or oxalyl chloride; (d) chloride displacement with the alkoxyalkyi alcohol in the presence of base such as pyridine; and (e) N-deprotection using catalytic hydrogenolysis (e.g., over a palladium catalyst), treatment with acid (e.g. trifluoroacetic acid) or zinc in acetic acid, as suits the N-protecting group chosen.
General Procedure 4
Synthesis of an alkyi alkoxyalkyi diester of an aminoalkylphosphonic acid, Scheme E.
Figure imgf000034_0001
(R) (S)
Scheme E
An alkyi alkoxyalkyi aminoalkylphosphonate of general structure (S), where Re, n and X' are as in General Procedure 3 above, can be prepared as shown in Scheme E from a dialkyl aminoalkylphosphonates (B) by: (a) partial hydrolysis to the monoalkyl ester (O); (b) N-protection to give a carbamate (P); (c) chlorination to give chloride (Q); (d) displacement of the chloride with an alkoxyalkyi alcohol to give (R); and (e) final N- deprotection to give the required alkyi alkyloxyalkyl aminoalkylphosphonate (S). Conveniently, the starting amine (B) is a d Ci-Cs alkyi) ester, and suitable reagents for the conversions are: (a) hydrolysis with aqueous hydrobromic acid heated under reflux; (b) N-protection with benzyl chloroformate, di-tert-butyl dicarbonate or 2,2,2- trichloroethoxycarbonyl chloride; (c) chlorination with thionyl chloride or oxalyl chloride; (d) displacement of chloride with the alkoxyalkyl alcohol in the presence of base such as pyridine; and (e) N-deprotection using catalytic hydrogenolysis (e.g. over a palladium catalyst), treatment with acid (e.g. trifluoroacetic acid) or zinc in acetic acid, as suits the N-protecting group chosen.
General Procedure 5
Synthesis of an alkoxyalkyl benzyl diester of an aminoa!kylphosphonic acid, Scheme F.
Figure imgf000035_0001
Scheme F
An alkoxyalkyl benzyl diester of an aminoalkylphosphonic acid general structure (V), where Re, n and X' are as defined in General Procedure 3 above, can be prepared as shown in Scheme F from a dichloride (L) (see General Procedure 3) by (a) partial displacement with an alkyloxyalkyl alcohol, then hydrolysis of the remaining chloride to give mono ester (T); (b) chlorination and displacement of the chloride with an benzyl alcohol to give (U); and (e) final N-deprotection to give the required alkyloxyalkyl benzyl aminoalkylphosphonate (V). Suitable reagents for the conversions are: (a) the use of limited alkoxyalkyl alcohol in the presence of pyridine and subsequent hydrolysis with aqueous NaHC03 (b) chlorination with thionyl chloride or oxalyl chloride and treatment of the resulting chloride with benzyl alcohol in the presence of pyridine; and (c) N- deprotection using catalytic hydrogenolysis (e.g. over a palladium catalyst), treatment with acid (e.g. trifluoroacetic acid) or zinc in acetic acid, as suits the N-protecting group chosen.
General Procedure 6
Synthesis of prodrugs of ((4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl)methylamino)alkylphosphonic acids (C), Scheme G
Figure imgf000036_0001
Scheme G
A diester prodrug of general formula (X) of a ((4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl)methylamino)alkylphosphonic acid (C) of the invention, where D = H, OH or NH2 can be prepared as shown in Scheme G by reductive amination of a aminoalkylphosphonic acid diester of general formula (N) or (S) (see General Procedures 3 and 4) with a 9- formyl-9-deazapurine of general structure (A) to give the adduct (U), from which any protecting groups on the deazapurine moiety are then removed as required to give (V).
General Procedure 7
Synthesis of a monoester prodrug of ((4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl)methylamino)alkylphosphonic acids (C), Scheme H.
Figure imgf000036_0002
Scheme H
A alkoxyalkyl monoester prodrug of general formula (W) of a ((4-hydroxy-5H-pyrrolo[3,2- d]pyrimidin-7-yl)methylamino)alkylphosphonic acid (C) of the invention, where D = H, OH or NH2 can be prepared as shown in Scheme H by reductive amination of a aminoalkylphosphonic acid diester of general formula (V) (see General Procedures 5) with a 9-formyl-9-deazapurine of general structure (A) to give the adduct (U), from which any protecting groups on the deazapurine moiety are then removed as required to give (W).
General Procedure 8
Synthesis of ((7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-3- yl)methylamino)alkylphosphonic acid, Scheme I.
Figure imgf000037_0001
Scheme I
A ((7-hydroxy-1H-pyrazolo[4,3-d]pyrimidin-3-yl)methylamino)alkylphosphonic acid (Y) of the invention (where X is as in General Procedure 1 above) can be obtained by reductive amination of a dialkyi ester of a aminoalkylphosphonic acid (B) (where Rc is an alkyl or aralkyl group and X' is as defined in General Procedure 1 above) with the formyl-8-aza- 9-deazapurine (77) (See Example 15) to give an adduct of general formula (X), from which any protecting groups are then removed as required to give a compound (Y) of the invention (Scheme A). Conveniently Rc will be a methyl, ethyl or tert-butyl group.
General Procedure 9
Synthesis of ((2-amino-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl)methy!amino)alkylphosphonic acids (C), Scheme J.
Figure imgf000038_0001
Scheme J
A ((2-amino-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methylamino)alkylphos acid (AA) of the invention (where X is as in General Procedure 1 above) can be obtained by reductive amination of an aminoalkylphosphonic acid (B) (where Rc is H and X' is as defined in General Procedure 1 above) or a dialkyi ester of (B) (where R° is a an alkyl or aralkyl group and X' is as defined in General Procedure 1 above) with the 7-formyl-9- deazaguanine derivative (109) (See Example 21) to give an adduct of general formula (Z), from which any protecting groups are then removed as required to give a compound (AA) of the invention (Scheme J). Conveniently Rc will be a methyl, ethyl or tert-butyl group.
General Procedure 10
Synthesis of ((4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methylamino)alkyl phosphate (AD), Scheme K.
Figure imgf000038_0002
Scheme K A ((4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methylamino)alkyl phosphate (AD) of the invention (where D = H or OH; and X is as In General Procedure 1) can be obtained by reductive amination of an aminoalkyi phosphate (AB) with a 9-formyl-9-deazapurine of general formula (A) (where D' = H or ORb; Ra is H or an N-protecting group; and Rb is H or an O-protecting group) to give an adduct of general formula (AC), from which any protecting groups are then removed as required to give a compound (AD) of the invention (Scheme K). Phosphates of general structure (AB) can be obtained from an aminoalcohol by (a) N- protection, e.g. as a N-fluorenyylmethoxycarbonyl derivative,; (b) selective mono- phosphorylation of a primary alcohol group, e.g. by P(OMe)3 and l2 in pyridine; (c) demethylation of the phosphate esters with bromotrimethylsilane; and (d) N-deprotection. DESCRIPTION OF THE FIGURES
Figure 1a shows a graph of parasite growth inhibition by (54); Figure 1 b shows extracellular purine analysis of metabolic labeling for uninfected erythrocytes treated with 100 μΜ (54) and metabolically labeled with [3H]hypoxanthine; Figure 1c shows extracellular purine analysis of metabolic labeling for uninfected erythrocytes treated with 100 μΜ (54) and metabolically labeled with [3H]inosine.
Figure 2 shows the IC50 values of (61) conducted at varying exogenous hypoxanthine concentrations. Figure 3a shows purine quantification by HPLC of uninfected RBCs; Figure 3b shows purine quantification by HPLC of erythrocyte-free parasites; Figure 3c shows purine quantification by HPLC of parasites isolated from infected erythrocytes; Figure 3d shows purine quantification by HPLC of extracellular purines from the experiment of Figure 3c. The time (Figure 3e) and concentration (Figure 3f) dependence of label uptake are measured by liquid scintillation counting of spent media.
Figure 4a shows that compound (65) inhibits the incorporation of hypoxanthine into the parasite. Cells are metabolically labeled with [2,8-3H]hypoxanthine in the presence or absence of 10 μΜ compound (65). Figure 4b shows that compound (76) does not inhibit the incorporation of hypoxanthine into the parasite during a two hour incubation. Cells are metabolically labeled with [2,8-3H]hypoxanthine in the presence or absence of 15 μΜ compound (76). Incorporation of label into purines is quantified by HPLC of extracts of parasites isolated from infected erythrocytes.
ABBREVIATIONS
c 0
NMR Nuclear magnetic resonance
TLC Thin layer chromatography
DCM Dichloromethane
Ac Acetyl
0 DMF N,N-Dimethylformamide
ESI Electrospray ionization
HSQC Heteronuclear single quantum correlation
HRMS High resolution mass spectrum
MeOH Methanol
5 MS Mass spectrum
TFA Trifluoroacetic acid
THF Tetrahydrofuran
UV Ultraviolet
DMSO Dimethylsulfoxide
0 DQF-COSY Double quantum filtered correlation spectroscopy
HMBC Heteronuclear multiple bond coherence
DEPT Distortionless enhancement by polarisation transfer
Q-TOF Quadrupole time of flight
TFA Trifluoroacetic acid
5 MCPBA m-Chloroperoxybenzoic acid
EXAMPLES
The following examples further illustrate the invention. It is to be appreciated that the0 invention is not limited to the examples.
General Methods
Chromatography solvents are distilled prior to use. Anhydrous solvents are those commercially available. Organic solutions are dried over anhydrous MgS04 and evaporated under reduced pressure. Air sensitive reactions are performed under Ar.5 Analytical TLC is performed on Merck pre-coated silica gel 60 F254, detection by UV absorption and/or by heating after dipping in a solution of (ΝΗ4)6Μο7θ24- Η20 (5 wt%) and Ce(S04)2 -4 H20 (0.1 wt%) in 5% aq. H2S04. Flash column chromatography is performed on silica gel (40-63 pm) or on an automated system with continuous gradient facilility. 13C-, 31 P- and 19F-NMR spectra are 1H decoupled, chemical shifts are in ppm and coupling constants in Hz if not already stated. 1H NMR in CDCI3 (internal TMS, δ 0), CD3OD (internal TMS, δ 0), DMSOcfe (internal TMS, δ 0) or D20 (internal HOD) 13C NMR in CDCI3 (centre line of CDCI3), DMSOcfe (centre line of DMSOcfe) or D20, 3 P NMR in CDCI3 or D20 (external H3P04, δ 0), 19F NMR in CDCI3 or D20 (external CHF3, δ 0). Assignments of 1H and 13C resonances are based on 2D (1H-1H DQF-COSY, 1H-13C HSQC, HMBC) and DEPT experiments. High resolution positive and negative electrospray mass spectra (ESI-HRMS) are recorded on a Q-TOF Premier tandem mass spectrometer. Melting points are uncorrected. Microanalyses are performed by the Campbell Microanalytical Laboratory, University of Otago, New Zealand.
Example 1 u-((4-Hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl)methylamino)alkylphosphonic acids (7a) - (7e), Scheme 1.
Figure imgf000041_0001
Scheme 1 Example 1.1
Preparation of diethyl ω-aminoalkylphosphonates (3a) - (3e)
Diethyl 1-aminomethylphosphonate (3a), diethyl 2-aminoethylphosphonate (3b), diethyl 3-aminopropylphosphonate (3c), diethyl 4-aminobutylphosphonate (3d) and diethyl 5- aminopentylphosphonate (3e) [P Bako et a/., Tetrahedron Asymm., 10 (1999) 2373- 2380] are prepared by the method of Hirschmann et al., (J. Amer. Chem. Soc, 119 (1997) 8177-8190) via reaction of commercially available ω-bromoalkyl phthalamides (1) with triethyl phosphite to yield phosphonates (2) followed by treatment with hydrazine hydrate (Scheme 1). Reductive aminations of amines (3a) to (3e) with 4-(benzyloxy)-5H-pyrrolo[3,2-d] pyrimidine-7-carbaldehyde (4), prepared by the method detailed in WO2009/082247, provide compounds (7a) - (7e), respectively, as follows:
Example 1.2
{[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]methyl}phosphonic acid (7a).
Diethyl aminomethylphosphonate (3a) (98.5 mg, 1.5 eq.) and the aldehyde (4) (100 mg, 1 eq.) are heated to 60 °C in EtOH (6 mL) for 30 min until dissolved. The solution is then cooled to room temperature, NaBH4 (29.5 mg, 2eq) is added and the mixture is stirred for 30 min. The mixture is evaporated onto silica gel and flash chromatography ( 9/1/0.1 v/v/v DCM/MeOH/conc. NH3), gives diethyl [({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin- 7-yl]methyl}amino)methyl]phosphonate (5a, 76 mg). This product is heated with cone. HCI (0.5 mL) at 60 °C for 1 h and the mixture is evaporated in vacuo twice from water and purified by flash chromatography (9/1/0.1 and 8/2/0.2 v/v/v DCM/MeOH/conc. NH3) to give diethyl {[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]methyl}phosphonate (6a, 45 mg, 36%). 1H NMR (500 MHz, MeOD) δ 7.93 (s, 1 H), 7.51 (s, 1 H), 4.20-4.14 (m, 4H), 3.26-3.35 (m, 4H), 3.34 (m, 6H). 13C NMR (125MHz, MeOD) 6155.8, 145.0,143.3, 129.8, 119.5, 111.8, 64.4, 44.5, 44.4, 43.5, 42.3, 16.7. 31P NMR (202 MHz, MeOD) δ 22.5. ESI-HRMS for C12H19N404NaP [M+Na]+ calcd 337.1042; found 337.1039. The diethyl phosphonate (6a, 43mg) is heated with 48% HBr for 5 h at 90 °C and the solution is evaporated in vacuo twice from water. Reverse phase (C18) chromatography eluting with water gives title compound (7a, 40 mg) as a white water insoluble solid. 1H NMR (500 MHz, DMSO-cfe) δ 8.75 (s, 1 H), 7.73 (s, 1H), 3.39 (s, 2H), 3.36 (s, 2H). 13C (125 MHz, DMSO-d6) δ 152.3, 144.0, 137.2, 130.7, 117.7, 104.3, 42.2, 41.0. 31P (202 MHz, DMSO-cf6) δ 11.7. ESI-HRMS for C8H12N404P [M+H]+ calcd 259.0596; found 259.0599. Example 1.3
{2-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]ethyl}phosphonic acid (7b).
Using the procedure for the preparation of compound (6a), compound (3b) is converted to diethyl {2-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]ethyl}phosphonate (6b). 1H NMR (500 MHz, DMSO-d6) δ 7.77 (s,1 H), 7.27 (s, 1H), 3.95 (m, 4H), 3.76 (s, 2H), 2.72 (m, 2H), 2.50 (s, 2H),1.92 (m, 2H), 1.19 (m, 6H). 13C NMR (125MHz, MeOD) 6153.6, 143.0, 141.3, 125.8, 117.6, 115.3, 60.8, 42.1 , 26.0, 24.9, 16.2. 31P NMR (202 MHz, DMSO-d6 ) δ30.3. ESI-HRMS for C13H21N404 aP [M+Na]+ calcd 351.1198; found 351. 194. Using the procedure for the preparation of compound (7a), compound (6b) is converted to title compound (7b). 1H NMR (500 MHz, D20) δ 8.69 (s, 1 H), 7.69 (s,1H), 4.34 (s,2H), 3.23-3.19 (m, 2H), 2.02-1.95 (m, 2H). 13C NMR (125MHz, D20) δ 153.2, 144.5, 134.8, 132.3, 118.2, 103.6, 42.0, 40.2, 25.0, 23.9. 31P NMR (202 MHz, D20) δ 20.6. ESI-HRMS forC9H12N404P [M-H]- calcd 271.0596; found 271.0594. Example 1.4
{3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propyl}phosphonic acid (7c).
Using the procedure for the preparation of compound (6a), compound (3c) is converted to diethyl {3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- l}methyl)amino]propyl}phosphonate (6c). H NMR (500 MHz, D20) δ 8.79 (s,1 H), 7.70 (s,1 H), 4.29 (s, 2H), 3.98-3.92 (m, 4H), 3.07-3.04 (m,2H), 1.86-183 (m, 2H), 1.16-1.10 (m,6H). 13C NMR (125MHz, D20) δ 152.7, 144.8, 133.0, 132.6, 118.2, 103.3, 63.6, 46.7, 40.4, 21.8, 20.7, 18.9, 15.6. 31P NMR (202 MHz, D20) δ 33.4. ESI-HRMS for C14H24N404P [M+H]+ calcd 343.1535; found 343.1528. Using the procedure for the preparation of compound (7a), compound (6c) is converted to title compound (7c). 1H NMR (500 MHz, D20) δ 8.68 (s, 1 H), 7.69 (s, 1H), 4.30 (s, 2H), 3.07-3.01 (m, 2H), 1.88-1.80 (m, 2H), 1.74-1.68 (m 2H). 13C NMR (125MHz, D20) δ 152.5, 144.9, 132.8, 131.8, 118.3, 103.0, 47.1 , 40.5, 24.1 , 23.0, 19.4. 31P NMR (202 MHz, D20) δ 29.2. ESI-HRMS for C10H16N 404P [M+H]+ calcd 287.0909; found 287.0904.
Example 1.5 {4-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]butyl}phosphonic acid (7d).
Using the procedure for the preparation of compound (6a), compound (3d) is converted to first to diethyl [4-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino) butyl]phosphonate (5d). 1H NMR (500 MHz, CDCI3) δ 8.46 (s, 1H), 7.72 (s, 1H), 7.44- 7.27 (bm, 5H), 5.53 (s, 2H), 4.19 (s, 2H), 4.03-3.98 (m, 4H), 2.80-2.77 (m, 2H), 1.72-1.51 (bm, 6H), 1.30-1.23 (m, 6H). 13C NMR (125MHz, CDCI3) δ 149.7, 139.4, 130.5, 129.3, 128.5, 128.4, 128.2, 128.1 , 127.7, 115.0, 108.5, 67.7, 61.6, 47.1 , 41.5, 27.8, 27.7, 25.6, 24.5, 20.9, 16.4. 31P NMR (202 MHz, CDCI3) δ 31.6. ESI-HRMS for C22H32N4O4P [M+H]+ calcd 447.2161 ; found 447.2153. This is then converted to diethyl {4-[({4-hydroxy-5H- pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]butyl}phosphonate (6d). H NMR (500 MHz, DMSO-d6) δ 8.82 (s, 1 H), 7.78 (s, 1H), 4.36 (s, 2H), 4.01-4.07 (m, 4H), 3.10-3.08 (m, 2H), 1.91-1.85 (m, 2H), 1.81-1.75 (m, 2H), 1.65-1.54 (m, 2H), 1.26-1.20 (m, 6H).
Using the procedure for the preparation of compound (7a), compound (6d) is converted to title compound (7d). 1H NMR (500 MHz, D20) δ 8.93 (s, 1 H), 7.75 (s, 1 H), 4.31 (s, 2H), 3.02-3.06 (m, 2H), 1.75-1.67 (m, 4H), 1.55-1.51 (m, 2H). 13C NMR (125 MHz, D20) δ 152.5, 144.9, 132.8, 131.7, 118.3, 103.0, 46.6, 40.4, 26. 3, 26.2, 25.0, 19.3. 31P NMR (202 MHz, D20)6 31.2. ESI-HRMS for C11H18N404P [M+H]+ calcd 301.1066; found 301.1064.
Example 1.6 {5-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]pentyl}phosphonic acid (7e).
Using the procedure for the preparation of compound (6a), compound (3e) is converted to first to diethyl [5-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino) pentyljphosphonate (5e). 1H NMR (500 MHz, CDCI3) δ 8.53 (s, 1H), 7.33 (s, 1 H), 7.47- 7.28 (bm, 5H), 5.57(s, 2H), 4.09-4.00 (m, 4H), 4.01 (s, 1 H), 1/70-1.51 (bm, 6H), 1.39- 1.35(m, 2H), 1.32-1.27 (m, 6H). 13C NMR (125MHz, CDCI3) δ 155.4, 149.5, 149.1 , 136.3, 128.5, 128.4, 128.3, 128.0, 127.7, 115.3, 113.8, 67.8, 61.4, 48.8, 43.2, 29.0, 28.2, 26.0, 25.0, 22.2, 16.4. 31 P NMR (202 MHz, CDCI3) δ 32.2. ESI-HRMS for C23H34 404P [M+H]+ calcd 461.2318; found 461.2311. This is then converted to diethyl {5-[({4-hydroxy-5H- pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]pentyl}phosphonate (6e). 1H NMR (300 MHz, D20) δ 8.53 (s, 1 H), 7.68 (s,1 H), 4.36 (s, 2H), 4.11-3.97(m, 4H), 3.09-3,01 (m, 2H), 1.90- 1.40 (bm, 8H), 1.28-1.22 (m, 6H).
Using the procedure for the preparation of compound (7a), compound (6e) is converted to title compound (7e).1H NMR (500 MHz, D20) δ 8.93 (s, 1 H), 7.76 (s, 1 H), 4.31 (s, 2H), 3.01-3.05 (m, 2H), 1.71-1.58 (bm, 4H), 1.49-1.42 (m, 2H), 1.38-1.32 (m, 2H). 3C NMR (125MHz, D20) δ 152.4, 144.9, 132.8, 131.7, 118.3, 103.1 , 46.9, 40.3, 26.6, 26.5, 26.3, 25.1 , 21.4. 31 P NMR (202 MHz, D20) δ 32.3. ESI-HRMS for C12H2oN404P [M+H]+ calcd 315.1222; found 315.1221.
Example 2
{3-[(2-{4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}ethyl)amino]propyl}phosphonic acid (15), Scheme 2
Figure imgf000045_0001
Scheme 2
4-(Benzyloxy)-5H-pyrrolo[3,2-d]pyrimidine (8) (178 mg,1 eq.), prepared by the method detailed in WO2009/082247, in dioxane (1 mL) and water (1 mL) with NaOAc (64.6 mg, 1 eq.), Me2NH.HCI (64.6 mg,1 eq.) and HCHO (65.5 μΙ_, 1.1 eq., 37% aq) is heated at 95 °C for 4 h and then evaporated under reduced pressure. The residue is evaporated twice from MeOH-NH3 onto silica gel. Flash chromatography (DCM/MeOH-NH3 9:1 v/v) gives {[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}dimethylamine (9) (163 mg). H NMR (500 MHz, MeOD) δ 8.42 (s, 1 H), 7.54 (s, 1 H), 7.53-7.31 (m, 5H), 5.68 (s, 2H), 3.75 (s, 2H), 2.28 (s, 6H). 13C NMR (125 MHz, MeOD) δ 157.2, 150.3, 137.8, 131.8, 129.4, 116.4, 112.1 , 69.1 , 52.4, 44.8. ESI-HRMS for C15H19N40 [M+H]+ calcd 283.1559; found 283.1560.
The dimethylamine (9) (548 mg,1 eq.) in tetrahydrofuran (25 mL) is treated with methyl iodide (1.2 mL, 10 eq.) at room temperature for 18 h and then the solution is evaporated under high vacuum to give an orange foam of the quaternary ammonium salt (10). Nitromethane (3.34 mL, 30 eq.) is added to methanol (32 mL) and methanolic sodium methoxide (7 mL, 30%, 20 eq.) and the mixture is stirred under argon for 10 min and then the crude quaternary ammonium salt (10) is added. The mixture is stirred for 2 h and then the methanol is evaporated. The residue is diluted with water, the pH adjusted to 3 with 1 N HCI and extracted 3x with ethyl acetate. The organic extract is washed with brine, dried and evaporated. Flash chromatography (20-50% v/v EtOAc in hexanes) gives 4-(benzyloxy)-7-(2-nitroethyl)-5H-pyrrolo[3,2-d]pyrimidine ( 1) (310 mg, 53%).
1H NMR (500 MHz, DMSO-d6) δ 8.44 (s, 1 H), 7.54 (d, 1 H), 7.42-7.33 (m, 5H), 5.61 (s, 2H), 4.93 (m, 2H), 3.37 (m, 2H). 13C NMR (125MHz, DMSO-cfe) δ 154.9, 148.7, 136.6, 128.8, 128.4, 114.4, 109.7, 75.0, 67.0, 22.1. ESI-HRMS for C^H^OsNa [M+Na]+ calcd 321.0964; found 321.0970.
NaBH4 (393 mg, 10 eq.) is added in small portions to 4-(benzyloxy)-7-(2-nitroethyl)-5H- pyrrolo[3,2-d]pyrimidine (11) (310 mg 1 eq.) in methanol (20 mL) with CoCI2.6H20 (495 mg, 2 eq.) at 0 °C and then the mixture is stirred at 0 °C for 30 min. The product is evaporated onto silica gel and flash chromatography (9/1/0.1 and 8/2/0.2 v/v/v DCM/MeOH/conc. NH3> gives 2-[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl] ethan-1- amine (12) (195 mg, 70%). 1H NMR (500 MHz, MeOD) δ 8.4 (s, 1 H), 7.36 (s, 1 H), 7.53- 7.30 (m, 5H), 5.62 (s, 2H), 2.98-2.92 (m, 4H).13C NMR (125MHz, MeOD) δ 157.1 , 149.8, 137.9, 129,3,129.6,128.3, 116.8, 114.2, 69.1 , 43.0, 28.1. ESI-HRMS for C15H17N40 [M+H]+ calcd 269.1402; found 269.1404.
Triethylphosphite (8.4 mL) and 2-(2-bromoethyl)-1 ,3-dioxolane (10 g) are heated at 160 °C for 18 h and then evaporated under high vacuum at 100 °C . The residue is refluxed with 1 N HCI under argon for 15 min and cooled to room temperature. The product is salted out with solid NaCI and extracted with DCM, washed .with saturated NaHC03, brine, dried and concentrated. Flash chromatography (EtOAc-MeOH 95:5 v/v) gives diethyl (3-oxopropyl)phosphonate (13) (2.5 g). NMR spectral data identical with literature (J.M Varlet et a/., Tetrahedron 37 (1981) 1377-1384).
The amine (12) (60 mg, 1.5 eq.) and diethyl (3-oxopropyl)phosphonate (13) (36 mg, 1 eq.) in ethanol (2 mL) are heated at 70 °C for 30 min and cooled to room temperature. NaBH4 (15 mg, 2 eq.) is added and the mixture stirred for 30 min. Evaporation onto silica gel and flash chromatography (9/1/0.1 and 8/2/0.2 v/v/v DCM/MeOH/conc. NH^ gives diethyl [3-({2-[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7- yl]ethyl}amino)propyl]phosphonate (14, 50 mg). 1H NMR (500 MHz, CDCI3) δ 8.53 (s, 1 H), 7.26 (s, 1H), 7.49-7.19 (m, 5H), 5.57 (s, 2H), 4.09-4.01 (m, 4H), 2.98 (s, 4H), 2.70 (m, 2H), 1.78-1.71 (m, 4H), 1.31-1.26 (m, 6H). 13C NMR (125 MHz, CDCI3) δ 155.3, 149.3, 136.3, 128.5, 126.8, 115.4, 114.8, 67.9, 61.5, 49.7, 49.6, 24.5, 24.0, 22.8, 22.7, 16.4. 31P NMR (202 MHz, CDCI3) δ 32.1. ESI-HRMS for C22H32N404P [M+H]+ calcd 447.2161 ; found 447.2160.
The phosphonate (14) (50 mg) is heated at 60 °C with cone. HCI (0.5 mL) for 1 h and then evaporated under reduced pressure twice from water. The residue is then heated with 48% aq. HBr (1 mL) at 90 °C for 5 h and evaporated again twice from water. The product is chromatographed on reverse phase C18 silica gel eluting with water to give {3- [(2-{4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}ethyl)amino]propyl}phosphonic acid (15) (30mg). 1H NMR (500 MHz, D20) δ 8.76 (s, 1H), 7.49 (s, 1 H), 3.26-3.17 (m, 2H), 3.07- 2.98 (m, 4H), 1.88-1.79 (m, 2H), 1.74-1.67 (m, 2H). 3C NMR (125MHz , D20) δ 152.9, 144.0, 132.4, 130.2, 117.9, 108.2, 47.9, 47.2, 24.3, 23.2, 20.3, 19.5. 31P NMR (202 MHz, D20) δ 28.3. ESI-HRMS for CnH18N404P [M+H]+ calcd 301.1066; found 301.1062.
Example 3
{2-[(2-{4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}ethyl)amino]ethyl}phosphonic acid (18), Scheme 3
Figure imgf000048_0001
Figure imgf000048_0002
Scheme 3
Triethylphosphite (12 mL) and 2-(bromomethyl)-1 ,3-dioxolane (10 g) are heated at 160 °C for 18 h and then evaporated under high vacuum at 100 °C. The residue is heated under reflux with 1 N HCI under argon for 15 min and cooled to room temperature. The product is salted out with solid NaCI and extracted with DCM, washed with saturated NaHC03, brine, dried and concentrated. Flash chromatography (EtOAc-MeOH 95:5 v/v) gives diethyl (2-oxoethyl)phosphonate (16) (1.6 g) [NMR spectral data identical with literature, Org. Synth., Coll. Vol. 6, 358]. The amine (12) (67 mg, 1.5 eq) and diethyl (2-oxoethyl)phosphonate (16) (40 mg, 1 eq.) in ethanol (2 mL) are heated at 70 °C for 30 min and cooled to room temperature. NaBH4 (17 mg, 2 eq.) is added and the mixture stirred for 30 min. The mixture is evaporated onto silica gel and subjected to flash chromatography (9/1/0.1 and 8/2/0.2 v/v/v DCM/MeOH/conc.NH3) to give diethyl [2-({2-[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7- yl]ethyl}amino)ethyl]phosphonate (17) (30 mg).
1H NMR (500 MHz, CDCI3) δ 8.48 (s, 1 H), 7.48-7.27 (m, 5H), 7.19 (s, 1 H), 5.55 (s, 2H), 4.07-4.01 (m, 4H), 3.04-2.95 (m, 6H), 2.08-2.02 (m, 2H), 1.28-1.25 (m, 6H). 13C NMR (125 MHz, CDCI3) 155.3, 149.1 , 148.8, 136.3, 128.4, 127.4, 115.4, 113.7, 67.9, 61.8, 49.1 , 42.8, 26.1 , 25.0, 24.0, 16.4. 3 P NMR (202 MHz, CDCI3) 29.2. ESI-HRMS for C21 H30N4O4P [M+H]+ calcd 433.2005; found 433.1999.
The diethyl phosphonate (17) (52 mg) is heated at 60 °C with cone. HCI (0.5 mL) for 1 h, evaporated under reduced pressure twice from water and the heated with 48% HBr (1 mL) at 90 °C for 5 h and evaporated again twice from water. The product is chromatographed on reverse phase Ci8 silica gel eluting with water to give {2-[(2-{4- hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}ethyl)amino]ethyl}phosphonic acid (18) (39 mg).
1H NMR (500M Hz, D20) δ 8.78 (s, 1 H), 7.50 (s, 1 H), 3.29-3.24 (m, 2H), 3.22-3.18 (m, 2H), 3.05-3.02 (m, 2H), 2.04-1.97 (m, 2H). 13C NMR (125MHz , D20) δ 152.8, 144.0, 132.2, 130.3, 1 17.9, 108.0, 47.0, 42.9, 24.9, 23.8, 20.3. 31 P NMR (202 MHz, D20) δ 21.3. ESI-HRMS for C10H16N4O4P [M+H]+ calcd 287.0909; found 287.0912.
Example 4
{2-Hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propyl} phosphonic acid (22), Scheme 4
Figure imgf000049_0001
Scheme 4
Diethyl (3-azido-2-hydroxypropyl)phosphonate (19) (620 mg) [prepared according to M. Ganesan and K.M. Muraleedharan, Nucleosides, Nucleotides, and Nucleic Acids, 29 (2010) 91-96] is reduced with 10% palladium on charcoal (250 mg) in ethanol (10 mL) under hydrogen at 1 atm. for 1 h, filtered and concentrated. Flash chromatography (9/1/0.1 and 8/2/0.2 v/v/v DCM/MeOH/conc. NH3> gives diethyl (3-amino-2- hydroxypropyl)phosphonate (20) (388 mg). 1H NMR (500 MHz, CDCI3) δ 4.18-4.09 (m, 4H), 3,98-3.92 (m, 1 H), 2.88-2.68 (m, 2H), 2.15-1.95 (bm, 3H),1.98-1.89 (m, 2H), 1.35- 1.33 (m, 6H). 13C NMR (125 MHz, CDCI3) δ 67.4, 61.9, 48.1 , 31.6, 31.0, 16.4. 31P NMR (202 MHZ,CDCl3)6 30.0. ESI-HRMS for C7H19N04P [M+H]+ calcd 212.1052; found 212.1049.
Diethyl 3-amino-2-hydroxypropylphosphonate (20) (95 mg,1.5 eq.) and 4-benzyloxy-5H- pyrrolo[3,2-d]pyrimidine-7-carbaldehyde (4) (80 mg, 1 eq.) in ethanol (3 mL) are heated at 70 °C for 10 min and then picoline borane (64 mg, 2 eq.) is added and the mixture stirred" for 3.5 h. The product is evaporated onto silica gel and subjected to flash chromatography (9/1/0.1 and 8/2/0.2 v/v/v DCM/MeOH/conc. NH3) to give diethyl [3-({[4- (benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)-2-hydroxypropyl]phosphonate (21) (20 mg). H NMR (500 MHz,CDCI3) δ 8.53 (s, 1 H), 7.49-7.35 (m, 5H), 7.26 (s, 1 H), 5.58 (s, 2H), 4.16-3.96 (m, 6H), 2.83-2.64 (m, 2H), 2.02-1.87 (m, 2H), 1.34-1.30(m, 6H). 13C NMR (125 MHz, CDCI3)6 155.4, 149.6, 149.0, 136.2, 128.5, 127.3, 115.4, 115.1 , 67.9, 65.0, 61.8, 55.1, 43.0, 32.1 , 31.0, 16.4. 31P NMR (202 MHz, CDCI3) δ.29.9. ESI-HRMS for C2iH3oN405P [M+H]+ calcd 449.1954; found 449.1951.
Diethyl [3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)-2- hydroxypropyl]phosphonate (21) (20 mg) is heated with cone. HCI (0.5 mL) at 70 °C for 30 min and then evaporated under reduced pressure twice from water. The product is heated with 48% HBr (1 mL) at 90 °C for 5 h and then evaporated under reduced pressure twice from water. Reverse phase chromatography on Ci8 silica gel eluting with water gives {2-hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]propyl} phosphonic acid (22) (7 mg).
1H NMR (500 MHz, D20) δ 8.89 (s, 1H), 7.44 (s, 1H), 4.38 (s, 2H), 4.24-4.10 (m, 2H), 3.27-2.99 (m, 2H), 2.04-1.97 (m, 2H). 3C NMR (125 MHz, D20 ) δ 152.7, 144.9, 132.9, 132.4, 118.2, 103.3, 62.3, 52.0, 40.5, 33.2, 32.1. 31P NMR (202 MHz, D20) δ 25.0. ESI- HRMS for CioH16N405P [M+H]+ calcd 303.0858; found 303.0862.
Example 5
(3-hydroxy-2-{[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino] methyl}propyl)phosphonic acid (32), Scheme 5
Figure imgf000051_0001
Scheme 5
2- [(Benzyloxy)methyl]propane-1 ,3-diol (23) (1.04 g,1 eq.) [J. Lee er a/., European J. Med. Chem., 44 (2009) 239-250] in dry THF (3 mL) is added to NaH (60%, 127 mg, 5.3 eq.) in dry THF (12 mL) with ice cooling. The mixture is stirred at room temperature for 30 min and then 'BuMezSiCI (800 mg, 5.3 eq.) in dry THF (2 mL) is added. After 2 h, water (6 mL) is added and the product extracted with EtOAc, washed with brine, dried and evaporated. Flash chromatography (hexanes-EtOAc 9:1 v/v) gives 2-[(benzyloxy)methyl]-
3- [(tert-butyldimethylsilyl)oxy]propan-1-ol (24) (1.25 g). 1H NMR (500 MHz, CDCI3) δ 7.37-7.30 (m, 5H), 4.51 (s, 2H), 3.80-3.71 (m, 4H), 3.64-3.53 (m, 2H), 2.07-2.01 (m, 1 H),
0.86 (s, 9H), 0.06 (s, 6H). 13C NMR (125MHz,CDCI3) δ 138.0, 128.4, 127.7, 73.2, 70.0, 64.5, 63.4, 43.0, 25.9, 18.2, 0.0. ESI-HRMS for C17H3o03NaSi [M+Na]+ calcd 333. 826; found 333.1858.
To (2-[(benzyloxy)methyl]-3-[(tert-butyldimethylsilyl)oxy]propan-1-ol 24) (1.25 g, 1 eq.) in dry DCM (2 mL) and dry pyridine (0.35 mL) under argon is added CBr4 (2 g, 1.5 eq.) followed by the dropwise addition of triphenylphosphine (1.07 g, 1.02 eq.) in dry DCM (2 mL). After 2.5 h, ice-cold hexanes (25 mL) are added and the precipitate filtered off. Flash chromatography (hexanes and then hexane with 5% v/v EtOAc) of the concentrated filtrate gives [3-(benzyloxy)-2-(bromomethyl)propoxy](tert- butyl)dimethylsilane (25) (788 mg).
1H NMR (500 MHz, CDCI3) δ 7.32-7.21 (m, 5H), 4.45 (s, 2H), 3.67-3.59 (m, 2H), 3.55- 3.41 (m, 2H), 2.16-2.09 (m, 1 H), 0.83 (s, 9H), 0.00 (s, 6H). 13C NMR (125 MHz,CDCI3) δ 138.3, 128.4, 127.6, 73.4, 69.1 , 61.7, 43.8, 33.0, 25.9, 18.2, 0. ESI-HRMS for C17H2902NaSi79Br [M+Na]+ calcd 395.1018; found 395.1020.
[3-(Benzyloxy)-2-(bromomethyl)propoxy](tert-butyl)dimethylsilane (25) (2.46 g, 1 eq.) and triethylphosphite (3.5 mL, 3 eq.) are heated at 175 °C for 18 h. Concentration of the product under high vacuum at 100 °C and flash chromatography (hexanes-EtOAc 20%- 100% v/v) of the residue gives diethyl {2-[(benzyloxy)methyl]-3-[(tert- butyldimethylsilyl)oxy] propyljphosphonate (26) (2.07g). 1H NMR (500 MHz, CDCI3) δ 7.29-7.22 (m, 5H), 4.45 (s, 2H), 4.04-4.03 (4H), 3.66-3.62 (m,2H), 3.52-3.50 (m,2H), 2.20-2.12 (m,1 H), 1.87-1.72 (m, 2H), 1.28-1.24 (m, 6H), 0.84 (s, 9H), 0.0 (s, 6H). 13C NMR (125 MHz,CDCI3) δ 138.6, 128.3, 127.5, 73.0, 70.2, 63.0, 61.4, 36.6, 25.9, 24.3, 23.1 , 18.3, 16.4, 0.0. 31P NMR (202 MHZ, CDCI3) δ.32.0. ESI-HRMS for C21H3905NaSiP [M+Na]+ calcd 453.2202; found 453.2205.
Diethyl {2-[(benzyloxy)methyl]-3-[(tert-butyldimethylsilyl)oxy] propyljphosphonate (26) (1.87 g) in MeOH (10 mL) and cone. HCI (10 mL) is stirred at room temperature for 1 h. Evaporation at reduced pressure gives crude diethyl {2-[(benzyloxy)methyl]-3- hydroxypropyljphosphonate (27) (1.45 g) which is dissolved in DCM (30 mL) and triethylamine (1.9 mL, 3 eq.) and cooled to 0 °C. Methanesuifonyl chloride (0.72 mL, 2 eq.) is added and the mixture stirred at room temperature for 1 h, diluted with DCM and the organic extract washed with satd. NaHC03i brine, dried and evaporated to give crude mesylate (28). Displacement of the mesylate is effected by heating a solution of crude (28) (1.93 g) in DMF (20 mL) with NaN3 (0.9 g) at 80 °C for 7 h. Addition of water and extraction with diethyl ether followed by flash chromatography (50%-100% v/v EtOAc in hexanes) gives diethyl [2-(azidomethyl)-3-(benzyloxy)propyl]phosphonate (29) (1.06 g) . H NMR (500 MHz, CDCI3) δ 7.38-7.27 (m, 5H), 4.53 (s, 2H), 4.14-4.03 (m, 4H), 3.58- 3.47 (m, 4H), 2.34-2.25 (m, 1H), 1.88-1.76 (m, 2H), 1.35-1.27 (m, 6H). 3C NMR (125 MHz, CDCI3) δ 138.0, 129.6, 128.3, 73.2, 70.2, 61.6, 52.6, 34.4, 25.4, 24.3, 16.4. ESI- HRMS for C15H24N304NaP [M+Na]+ calcd 364.1402; found 364 1393.
Diethyl [2-(azidomethyl)-3-(benzyloxy)propyl]phosphonate (29) (1.06 g) in MeOH (10 mL) with 20% Pd(OH)2 /C (0.5 g) is hydrogenated at 1 atm for 18 h. Filtration and flash chromatography (9/1/0.1 and 8/2/0.2 v/v DCM/MeOH/conc. NH3) gives diethyl [2- (aminomethyl)-3-hydroxypropyl]phosphonate (30) (300 mg). 1H NMR (500 MHz, CDCI3) δ 4.22-4.05 (m, 4H), 3.83-3.71 (m, 2H), 3.02-2.81 (m, 2H), 2.25-2.15 (m, 1 H), 2.10-1.98 (m, 1 H), 1.86-1.71 (m, 2H), 1.38-1.31 (m, 6H). 13C NMR (125 MHz, CDCI3) δ·67.4, 61.7, 46.2, 37.1 , 26.2, 25.1 , 16.5. 31P NMR (202 MHz, CDCI3) δ.31.8. ESI-HRMS for C8H21N04P [M+H]+ calcd 226.1208; found 226.1206.
Diethyl [2-(aminomethyl)-3-hydroxypropyl]phosphonate (30) (100 mg, 1.5 eq.) and 4- benzyloxy-5H-pyrrolo[3,2-d]pyrimidine-7-carbaldehyde (4) (80 mg, 1 eq.) are heated together in ethanol (3 mL) at 70 °C for 10 min and then picoline borane (64 mg, 2 eq.) is added and heated at 70 °C for 2 h. Evaporation and flash chromatography (9/1/0.1v/v/v DCM/MeOH/conc. NH3) gives diethyl {2-[({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7- yl]methyl}amino)methyl]-3-hydroxypropyl}phosphonate (31) (99 mg).
1H NMR (500 MHz, CDCI3) δ 8.55 (s, 1 H), 7.49-7.33 (m, 5H), 7.26 (s, 1 H), 5.58 (s, 2H), 4.13-3.99 (m, 6H), 3.81-3.63 (m, 2H), 3.01-2.81 (m, 2H), 2.26-2.19 (m, 1H), 1.75-1.63 (m, 2H), 1.35-1.24 (m, 6H). 3C NMR (125MHz, CDCI3) δ 155.3, 149.8, 149.1 , 136.2, 128.5, 127.3, 115.4, 114.3, 67.8, 61.7, 53.7, 43.3, 34.6, 26.6, 25.5, 16.4. 31P NMR (202 MHz, CDCI3) δ 31.3. ESI-HRMS for C22H32N405P [M+H]+ calcd 463.2110; found 463.2103.
Diethyl {2-[({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)methyl]-3- hydroxypropyljphosphonate (31) (45 mg) is heated at 70 °C in cone. HCI (0.5 mL) for 1 h and evaporated twice from water. The crude product is the heated with HBr (48%, 1 mL) at 90 °C for 3 h and then evaporated twice from water. Chromatography on reverse phase C18 silica gel eluting with water gives (3-hydroxy-2-{[({4-hydroxy-5H-pyrrolo[3,2- d]pyrimidin-7-yl}methyl)amino]methyl}propyl)phosphonic acid (32) (23 mg).1H NMR (500 MHz, D20) δ 8.19 (s, 1 H), 7.58 (s, 1 H), 4.30-4.21 (m, 2H), 3.56-3.37 (m, 2H), 3.15-3.04 (m, 2H), 2.19-2.11 (m, 1 H), 1.68-1.46 (m, 2H). 13C NMR (125 MHz, D20) δ 152.6, 144.9, 132.8, 118.4, 103.1 , 63.8, 49.8, 40.9, 33.0, 27.3, 26.2. 31P NMR (202 MHz, D20) δ.27.5. ESI-HRMS for C11H18N405P [M+H]+ calcd 317.1015; found 317.1007.
Example 6
{2-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]ethoxy}ph
acid), Scheme 6.
Figure imgf000054_0001
Figure imgf000054_0002
Scheme 6
Ethanolamine (120 μΙ_, 5 eq.) is added to acetyl chloride (142 μΙ_, 5 eq.) in methanol (8 mL) and a few microdrops of acetyl chloride added to adjust the pH to 5. To this solution is added 4-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine-7-carbaldehyde (4) (100 mg, 1 eq.) and the mixture is stirred at 40 °C for 30 min. Picoline borane (64 mg, 1.5 eq.) is added and the mixture stirred at 40 °C for 4 h. The product is evaporated onto silica gel and subjected to flash chromatography (9/1/0.1 v/v/v DCM/MeOH/conc. NH3) to give2-({[4- (benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)ethan-1-ol (33) (47 mg). 1H NMR (500 MHz, MeOD) δ 8.35 (s, 1 H), 7.51 (s, 1 H), 7.43-7.23 (m, 5H), 5.53 (s, 2H), 4.04 (s, 2H), 3.63 (m, 2H), 2.80 (m, 2H). 13C NMR (125 MHz, MeOD) δ 157.2, 150.5, 149.6, 137.0, 131.4, 129.5, 116.8, 111.8, 69.2, 60.2, 51.0, 42.9. ESI-HRMS for C16H19N402 [M+H]+ calcd 299.1508; found 299.1514.
To 2-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)ethan-1-ol (33) (47 mg, 1 eq.) in MeOH (5 mL) and Et3N (22.5 μί) is added di-tert-butyl dicarbonate (35 mg, 1 eq.) and the mixture is stirred for 30 min. Evaporation and flash chromatography (EtOAc) gives tert-butyl N-{[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}-N-(2- hydroxyethyl)carbamate (34) (32 mg).
To this carbamate (34) and tetrazole (28 mg, 5 eq.) in DCM (2 mL) is added dibenzyl diisopropylphosphoramidite (52.5 μί, 1.5 eq.) and the mixture is stirred under argon for 30 min. tert-Butyl hydroperoxide (0.08 mL) is then added followed by Na2S03 (10% aq., 0.08 mL) and the product is extracted with DCM, washed with brine, dried and evaporated. Flash chromatography (50%, 70%, then 100% v/v EtOAc in hexanes) gives tert-butyl N-{[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}-N-(2- [bis(benzyloxy)phosphoryl]oxy} ethyl)carbamate (35) (32 mg). 1H NMR (500 MHz, CDCI3) δ 8.52 (s, 1 H), 7.46-7.26 (m, 6H), 5.56 (s, 2H), 5.03-4.97 (m, 4H), 4.60 (s, 2H), 4.14-4.08 (bm, 2H), 3.60 (bs, 2H), 1.42 (s, 9H). 13C NMR (125 MHz, CDCI 3) δ 155.5, 155.3, 149.9, 136.3, 135.9, 128.4, 127.9, 114.9, 114.4, 113.9, 80.0, 69.3, 67.8, 65.7, 47.3, 41.9, 41.1, 28.4. 31P NMR (202 MHz, CDCI3 ) δ -1.0. ESI-HRMS for C35H40N4O7P [M+H]+ calcd 659.2635; found 659.2640. The carbamate (35) (23 mg) is hydrogenated at atmospheric pressure with 10% Pd/C (20 mg) in ethanol (5 mL) for 2 h, filtered and evaporated. The crude reduced product is hydrolysed with 80% aqueous TFA (5 mL) for 2 h and then evaporated twice from dioxane followed by evaporation from 1 N HCI to give ({2-[({4-hydroxy-5H-pyrrolo[3,2- ]pyrimidin-7-yl}methyl)amino]ethoxy}phosphonic acid (36) (14 mg). 1H NMR (500 MHz, D20) δ 8.05 (s, 1H), 7.71 (s, 1H), 4.42 (s, 2H), 4.05-4.02 (m, 2H), 3.32-3.30 (m, 2H). 3C NMR (125 MHz, D20 ) δ 153.2,144.4,134.7, 132.3,118.2,103.6,60.5, 46.9, 40.1. 31P NMR (202 MHz, D20) δ 2.3. ESI-HRMS for C9H14N405P [M+H]+ calcd 289.0702; found 289.0699.
Example 7 {3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)
(methyl)amino]propyl}phosphonic acid (39), Scheme 7
Figure imgf000055_0001
Scheme 7
To diethyl [3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl} amino) propyl]phosphonate (5c) (39 mg, 1 eq.) in methanol (5 mL) is added acetyl chloride (3 μί, 0.3 eq.), parafomaldehyde (14 mg, 5 eq.) and NaCNBH3 (20 mg, 3 eq.). The mixture is heated at 50 °C for 2 h and then evaporated onto silica gel. Flash chromatography (9/1/0.1 v/v/v DCM/MeOH/conc. NH3) gives diethyl [3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d] pyrimidin-7-yl]methyl}(methyl)amino)propyl]phosphonate (38) (10 mg). 1H NMR (500 MHz, CDCI3) δ 8.53 (s, 1H), 7.66-7.32 (m, 5H), 7.27 (s, 1H), 5.59 (s, 2H), 4.28 (s, 2H), 4.10-4.02 (m, 4H), 2.93 (m, 2H), 2.56 (s, 3H), 2.01-1.98 (m, 2H), 1.84-1.77 (m, 2H), 1.33- 1.26 (m, 6H). 13C NMR (125 MHz, CDCI3) δ 155.7, 150.2, 149.4, 136.1 , 131.2, 128.4, 115.3, 107.2, 68.0, 62.0, 56.1 , 50.1 , 40.5, 23.4, 22.3, 18.9, 16.4. 31P NMR (202 MHz, CDCI3) δ.31.7. ESI-HRMS for C22H32N4OP [M+H]+ calcd 447.2161 ; found 447.2157.
The diethyl phosphonate (38) (10 mg) is heated at 60 °C with cone. HCI (0.5 mL) for 1 h, then evaporated twice from water and heated with 48% HBr (0.5 mL) at 90 °C for 5 h and evaporated twice from water. The product is chromatographed on reverse phase C18 silica gel eluting with water to give {3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl} methyl) (methyl)amino]propyl}phosphonic acid (39) (9 mg). H NMR (500 MHz, D20) δ 8.8 (s, 1 H), 7.78 (s, 1 H), 4.50-4.34 (m, 2H), 3.30-3.09 (m, 2H), 2.76 (s, 3H), 1.98-1.86 (m, 2H), 1.76-1.69 (m, 2H). 13C NMR (125 MHz, D20) δ 152.9, 145.0, 134.0, 133.5, 118.5, 101.7, 55.5, 49.6, 38.9, 24.0, 23.0, 17.8. 31P NMR (202 MHz, D20) δ 28.7. ESI- HRMS for C11H18N404P [M+H]+ calcd 301.1066; found 301.1066. Example 8
Methyl 2-[({3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propyl} (phenoxy)phosphoryl)amino]propanoate (45), Scheme 8
Figure imgf000056_0001
Scheme 8
Diethyl (3-aminopropyl)phosphonate (3c) (2 g) is heated at 80 °C under reflux with aq. HBr (48%, 10 mL) for 18 h and then evaporated 2x from water. Reverse phase C 8-Silica chromatography eluting with water gives 3-aminopropylphosphonic acid (40) (2.28 g) as the HBr salt. This is dissolved in MeOH (30 ml_) and water (15 mL), then triethylamine (7 ml_, 5 eq.) and, slowly, benzylchloroformate (2.5 mL, 1 eq.) are added. The mixture is stirred at room temperature for 3 h and evaporated. Reverse phase C18-Silica chromatography eluting with 20 then 40% v/v MeOH in H20 gives (3- {[(benzyloxy)carbonyl]amino} propyl)phosphonic acid (41) as the triethylamine salt (2.7 g). 1H NMR (500 MHz, D20) δ 7.42-7.37 (m, 5H), 5.09 (s, 2H), 3.21-3.15 (m, 8H), 1.68- 1.65 (m, 2H), 1.57-1.51 (m, 2H), 1.26-1.24 (m, 9H). 13C NMR (125 MHz, D20) δ.128.8, 127.7, 67.2, 46.7, 41.5, 24.9, 23.2 ,8.3. 31P NMR (202 MHz,D20) δ.28.1 , 26.2. ESI- HRMS for CnHi6N05PNa [M+Na]+ calcd 296.0664; found 296.0669.
(3-{[(Benzyloxy)carbonyl]amino}propyl)phosphonic acid (41) as the triethylamine salt (820 mg, 1eq) in dry CH3CN (10 mL) is heated at 70 °C with SOCI2 (1 mL) for 2 h and then evaporated 2x from toluene. To the crude phosphonyl dichloride in dry toluene (10 mL) is added tetrazole (20 mg) at 0 °C and then a solution of phenol (110 mg, 0.5eq) and triethylamine (165 μΙ, 0.5eq) in toluene (10 mL) at 0 °C. The mixture is stirred at room temperature under Ar for 2 h and then alanine methyl ester hydrochloride (328 mg, 1eq) in pyridine (1 mL) followed by triethylamine (0.33 mL) is added and the reaction stirred at room temperature for 18 h. The solution is partitioned between EtOAc and saturated aqueous ammonium chloride, the aqueous layer is re-extracted with EtOAc, and the combined organic phase dried and concentrated. Flash chromatography (1 :1 v/v hexanes/EtOAc then EtOAc) gives methyl 2-{[(3-{[(benzyloxy)carbonyl]amino} propyl)(phenoxy)phosphoryl]amino}propanoate (42) (120 mg). 1H NMR (500 MHz, CDCI3) δ 7.36-7.10 (m, 10H), 5.28-5.07 (m, 2H), 4.15-3.95 (m, 1 H), 3.65 (s, 3H), 3.31- 3.30 (m, 2H), 1.95-1.85 (m, 4H), 1.28-1.21 (2d, 3H). 13C NMR (125 MHz, CDCI3) δ 174.6, 174.2, 156.6, 150.4, 136.6, 129.6, 128.5, 125.7, 125.0, 120.5, 66.6, 52.4, 49.5, 41.0, 26.0, 25.0, 23.0, 21.3. 31P NMR (202 MHz, CDCI3) δ 32.4, 31.7. ESI-HRMS for C21H27N206P [M+H]+ calcd 435.1685; found 435.1685.
Methyl 2-{[(3-{[(benzyloxy)carbonyl] amino}propyl)(phenoxy) phosphoryl] aminojpropanoate (42) (332 mg) in ethanol (5 mL) with TFA (5 drops) is hydrogenated at atmospheric pressure in the presence of 10% Pd/C (250 mg) for 1.5 h, then filtered and concentrated. The crude product (43) (190 mg) is directly subjected to reductive amination with 4-benzyloxy-5H-pyrrolo[3.2-c/]pyrimidine-7-carbaldehyde (4) (60 mg, 0.5 eq.) and picoline borane (49 mg, 1 eq.) in MeOH (12 mL) for 2 h. The mixture is concentrated and flash chromatographed (DCM then DCM/MeOH/HOAc 9/1/0.1 v/v/v) gives methyl 2-({[3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino) propyl] (phenoxy) phosphoryl}amino)propanoate (44) (27 mg). H NMR (500 MHz,CDCI3) δ 8.44 (m, 1 H), 7.94 (m, 1 H), 7.47-7.04 (m, 10H), 5.51 (s, 2H), 4.56-4.52 (m, 2H), 3.52-3.58 (2s, 3H), 3.24-3.13 (m, 1 H), 2.43-2.29 (m, 2H), 2.03 (s, 3H), 1.91-1.76 (m, 2H), 1.26-1.15 (2d, 3H). 13C NMR (125 MHz, CDCI3) δ 174.6, 155.9, 150.0, 148.9, 135.9, 133.6, 129.6, 128.5, 128.2, 124.8, 120.8, 120.4, 115.3, 103.2, 68.0, 52.3, 51.1 , 49.4, 46.5, 25.6, 24.5, 20.8, 17.6. 31P NMR (202 MHz, CDCI3) δ 31.4, 31.0. ESI-HRMS for C^HasNsOsP [M+H]+ calcd 538.2219; found 538.2220.
Methyl 2-({[3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)propyl] (phenoxy)phosphoryl}amino)propanoate (44) (27 mg) in ethanol (5ml)is hydrogenated at atmospheric pressure in the presence of 10% Pd/C (25 mg) and TFA (2 drops) for 2 h. Concentration gives methyl 2-[({3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl) amino]propyl}(phenoxy)phosphoryl)amino]propanoate (45) (23 mg). 1H NMR (500 MHz, D20) δ 8.05 (m, 1H), 7.75 (m, 1 H), 7.35-6.84 (m, 5H), 4.75-4.51 (2H), 3.83-3.77 (m, 1H), 3.60-3.50 (2s, 3H), 3.39-3.28 (m, 2H), 2.22-2.21 (m, 2H), 2.02-1.94 (2H), 1.18-1.14 (2d, 3H). 13C NMR (125 MHz, D20) δ 163.1 , 143.3, 132.0, 129.9, 125.8, 120.5, 117.9, 103.9, 52.8, 50.9, 49.5, 47.2, 24.8, 23.7, 19.5, 19.0, 17.5. 31P NMR (202 MHz, D20) δ 33.0, 32.3. ESI-HRMS for C2oH27N505P [M+H]+ calcd 448.1750; found 448.1754.
Example 9
Methyl 2-[({3-[({4-hydroxy-5H^yrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propyl}[(1- methoxy-1-oxopropan-2-yl)amino]phosphoryl)amino]propanoate (50), Scheme 9
Figure imgf000058_0001
3-Aminopropylphosphonic acid HBr salt (40) (290 mg, 1 eq.) dissolved in H20 (2 mL) and MeOH (4 mL) is treated with EtN3 (760 μΙ, 4 eq.) and di-tert-butyl dicarbonate (288 mg, 1 eq.). After 30 min, the solution is evaporated and subjected to reverse phase chromatography on C18 silica gel eluting with H20 then 20% and 40% v/v MeOH in H20 to give (3-{[(tert-butoxy)carbonyl]amino}propyl)phosphonic acid (46) (252 mg).
The protected amine (46) (220 mg) is dried by evaporation 2x with dry pyridine and dissolved in dry pyridine (5 mL). To this solution is added alanine methyl ester hydrochloride (236 mg, 2.6 eq.) in dry pyridine (1 mL) followed by Et3N (244 μί, 2.6 eq.) and the mixture is stirred at 60 °C for 5 min. 1 ,2-Di(pyridin-2-yl)disulfane (500 mg, 3.5 eq.) and triphenylphosphine (560 mg, 3.5 eq.) are dissolved in dry pyridine (2 mL) and this solution is added to the reaction and the mixture is stirred for 18 h under argon. The solvent is evaporated and the residue partitioned between EtOAc and saturated aqueous sodium bicarbonate. The organic layer is dried and evaporated. Flash chromatography (2%, 5% then 10% v/v MeOH in EtOAc) gives methyl 2-{[(3-{[(tert-butoxy)carbonyl] aminojpropyl) [(1-methoxy-1-oxopropan-2-yl)amino]phosphoryl]amino}propanoate (47) (80 mg). H NMR (500 MHz, CDCI3) δ 4.05-3.95 (m, 2H), 3.76 (s, 6H), 3.30-3.10 (m, 2H), 1.85-1.70 (m, 4H), 1.44 (s, 9H), 1.44-1.37 (m, 6H). 13C NMR (125 MHz, CDCI3) δ 175.2, 156.2, 52.3, 48.7, 48.4, 40.7, 28.4, 26.8, 25.9, 23.6, 21.4, 21.3. 3 P NMR (202 MHz, CDCI3) δ 29.0. ESI-HRMS for C16H32N307P Na [M+Na]+ calcd 432.1876; found 432.1872. The Boc derivative (47) ( 50 mg) is stirred with 80% TFA (0.5 mL) for 5 min, evaporated 2x with dioxane and subjected to flash chromatography (DCM and 20% v/v 6N NH3 in MeOH) gives methyl 2-{[(3-aminopropyl)[(1-methoxy-1-oxopropan-2-yl)amino] phosphoryl]amino}propanoate (48) (58 mg). H NMR (500 MHz, MeOD) δ 3.98-3.92 (m, 2H), 3.72 (s, 6H), 3.04-3.01 (m, 2H), 2.01-1.76 (m, 4H), 1.40-1.37 (m, 6H). 13C NMR (125 MHz, MeOD) δ 176.5, 52.7, 50.0, 41.2, 27.5, 26.5, 22.1 , 20.0. 31P NMR (202 MHz, MeOD ) δ 30.7. ESI-HRMS for CnHzsN^P [M+H]+ calcd 310. 532; found 3 0.1526.
To a mixture of the amine (48) (58 mg, 1.2 eq.) and the aldehyde (4) (40 mg, 1 eq.) in methanol (7 mL) is added methanolic HCI to adjust the pH to 5 and the mixture is stirred at 60 °C for 1 h. Picoline borane (33.4 mg, 2 eq.) is then added and the mixture is stirred for 18 h at room temperature. The solution is evaporated and flash chromatography (DCM then DCM/MeOH/conc.NH3 v/v/v 9/1/0.1) gives methyl 2-({[3-({[4-(benzyloxy)-5H- pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)propyl][(1-methoxy-1-oxopropan-2-yl)amino] phosphoryl}amino)propanoate (49) (20 mg). 1H NMR (500 MHz, CDCI3) δ 8.54 (s, 1 H), 7.48-7.33 (m, 5H), 7.26 (s, 1 H), 5.58 (s, 2H), 4.02-3.96 (m, 3H), 3.70(s,3H), 3.68 (s, 3H), 2.82-2.78 (m, 2H), 1.90-1.75 (m, 4H), 1.31-1.36 (m, 6H). 13C NMR (125 MHz, CDCI3) δ.175.2, 155.4, 149.7, 149.1 , 136.3, 128.4, 127.9, 115.3, 113.8, 67.8, 52.2, 48.7, 48.3, 42.6, 27.5, 26,6, 22.1 , 21.3. 31P NMR (202 MHz,CDCI3) δ 29.8. ESI-HRMS for C25H36N606P [M+H]+ calcd 547.2434; found 547.2427.
Hydrogenolysis of the dezahypoxanthine derivative (49) (20 mg) in ethanol (5 mL) with 10% Pd/C (20 mg) for 1 h, followed by filtration and evaporation gives methyl 2-[({3-[({4- hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propyl}[(1-methoxy-1-oxopropan-2- yl)amino]phosphoryl)amino]propanoate (50) (10 mg). 1H NMR (500 MHz, MeOD) δ 7.92 (s, 1 H), 7.50 (s, 1 H), 4.12 (m, 2H), 3.98-3.91 (m, 2H), 3.72-3.66 (2s, 6H), 2.94-2.91 (m, 2H), 1.93-1.75(m, 4H), 1.38-1.32 (m, 6H). 13C NMR (125 MHz, MeOD) δ.176.5, 155.9, 145.0, 143.3, 129.5, 119.5, 112.2, 52.7, 50.0, 49.8, 42.5, 27.9, 27.0, 22.1 , 21.1 , 21.0. 31P NMR (202 MHz, MeOD) δ 31.5. ES-HRMS for CieH3oNeOeP [M+H]+ calcd 457.1964; found 457.1964.
Example 10
[({[(2,2-Dimethylpropanoyl)oxy]methoxy}({3-[({4-hydroxy-5H-pyrrolo[3,2- d]pyrimidin-7-yl}methyl)amino]propyl})phosphoryl)oxy]methyl 2,2- dimethylpropanoate (54), Scheme 10
Figure imgf000060_0001
Scheme 10 (3-{[(Benzyloxy)carbonyl]amino}propyl)phosphonic acid (41) (1.7 g), chloromethyl pivalate (2.6 mL, 4 eq.) and Et3N (3.2 mL, 5 eq.) are heated at 60 °C in DMF (20 mL) for 4 h and then evaporated. Flash chromatography (hexanes/EtOAc 3:2 v/v) gives {[(3- {[(benzyloxy) . ca.rbonyl]amino}propyl)({[(2,2- dimethylpropanoyl)oxy]methoxy})phosphoryl]oxy}methyl 2,2-dimethylpropanoate (51) (600 mg). 1H NMR (500 MHz, CDCI3) δ 7.41-7.32 (m, 5H), 5.68-5.59 (m, 4H), 5.2 (s, 2H), 3.32-3.30 (m, 2H), 1.92-1.73 (m, 4H), 1.22 (s, 18H). 13C NMR (125 MHz, CDCI3) δ.176.9, 128.5, 128.1 , 81.4, 67.0, 38.7, 26.8, 24.3, 23.1 , 22.6. 31P NMR (202 MHZ, CDCI3) δ.32.4. ESI-HRMS for C23H36N09P23Na [M+Na]+ calcd 524.2025; found 524.2021. The protected amine (51) is hydrogenolysed with 10% Pd/C (500 mg) in ethanol for 1.5 h and evaporated. Flash chromatography (DCM/MeOH/conc. NH3 9/9/0.1 v/v/v) gives {[(3- aminopropyl)({[(2,2-dimethylpropanoyl)oxy]methoxy})phosphoryl]oxy}methyl 2,2- dimethylpropanoate (52) (356 mg). H NMR (500 MHz, CDCI3) δ 5.69, 5.66 (2s, 4H), 2.74-2.78 (m, 2H), 1.95-1.65 (m, 4H), 1.24(s, 18H). 13C NMR (125 MHz, CDCI3) δ.176.9, 81.4, 42.2, 27.6, 26.9, 25.9, 24.4, 23.3. 3 P NMR (202 MHz, CDCI3) δ.33.2. ESI-HRMS for C15H31N07P [M+Na]+ calcd 368.1838; found 368.1838.
The amine (52) (300 mg, 1.2 eq.), aldehyde (4) (170 mg, 1 eq.) and picoline borane (145 mg, 2 eq.) in ethanol (30 mL) are heated at 50 °C for 1 h and then evaporated. Flash chromatography (DCM/MeOH/NH3 9/1/0.1 v/v/v) gives ({[3-({[4-(benzyloxy)-5H- pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)propyl]({[(2,2-dimethylpropanoyl)
oxy]methoxy})phosphoryl}oxy)methyl 2,2-dimethylpropanoate (53) (188 mg). 1H NMR (500 MHz, CDCI3) δ 8.56 (s, 1 H), 7.51-7.26 (m, 6H), 5.65-5.62 (2s, 4H), 5.58 (s, 2H), 4.00 (s, 2H), 2.77-2.72 (m, 2H), 1.97-1.67 (m, 4H), 1.24 (s, 9H). 13C NMR (125 MHz, CDCI3) δ 176.9, 155.3, 149.5,149.2,136.3, 128.5, 126.9, 115.4, 81.4, 67.9, 49.2, 43.3, 38.7, 26.9, 24.9, 23.7, 22.4. 31P NMR (202 MHz, CDCI3) δ 33.3. ESI-HRMS for C29H42N408P [M+Na]+ calcd 605.2740; found 605.273 .
The adduct (53) (65 mg) is hydrogenolysed with 10% Pd/C (50 mg) in ethanol (5 mL) for 4 h, filtered and concentrated. Flash chromatography (DCM/MeOH/conc. NH3 9/1/0.1 then 8/2/0.2 v/v/v) gives [({[(2,2-dimethylpropanoyl)oxy]methoxy}({3-[({4-hydroxy-5H- pyrrolo [3,2-d]pyrimidin-7-yl}methyl)amino]propyl})phosphoryl)oxy]methyl 2,2- dimethylpropanoate (54) (27 mg). The product is evaporated 2x from HOAc to form the acetic acid salt. 1H NMR (500 MHz, MeOD) δ 7.95 (s, 1 H), 7.56 (s, 1 H), 5.72-5.62 (m, 4H), 4.26-4.32 (m, 2H), 3.06-3.09 (m, 2H), 2.06-1.96 (m, 4H), 1.93 (s, 3H), 1.22 (s, 18H). 13C NMR (125 MHz, MeOD) δ. 184.0,179.2,178.2, 156.0, 145.1, 144.1 , 131.3, 119.6, 107.9 83.1 ,79.0, 42.1 , 39.8, 27.2, 24.9, 23.7, 22.8, 20.8. 31P NMR (202 MHz, MeOD) δ 31.2. ESI-HRMS for C22H36N4O8P [M+Na]+ calcd 515.2271 ; found 515.2270.
Example 11
Ethyl 3-(octadecyloxy)propyl {3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]propyl}phosphonate (61), Scheme 11
BnOCOCI, Et3N
Figure imgf000062_0001
Figure imgf000062_0002
(58) R1= CO
(ii) H2, Pd/C, TFA (60) R2= Bn
(59) R1=H (61) R2=H
Scheme 11
Diethyl 3-aminopropylphosphonate (3c) as the HBr salt (5.63 g) is heated at 90 °C under reflux with aq. HBr (48%, 34 mL) for 18 h and then evaporated 2x from water. Flash column chromatography (dioxane/water/Et3N 6:4:0.3 v/v/v) gives ethyl hydrogen 3- aminopropylphosphonate (55) (5.69 g) as the triethylammonium salt. To this aminophosphonate salt (55) (5.69 g) dissolved in MeOH (80 mL) and water (40 mL) is added triethylamine (15 mL, 5 eq.) and then slowly benzylchloroformate (6 mL, 1 eq.). The mixture is stirred at room temperature for 3 h and evaporated. Reverse phase C18Silica chromatography eluting with H20, then 20 and 40% v/v MeOH in H20 gives (3- {[(benzyloxy)carbonyl]amino}propyl)(ethoxy)phosphinic acid (56) as the triethylammoinium salt (2.33 g). 1H NMR (500 MHz, D20) δ 7.44-7.35 (m, 5H), 5.10 (s, 2H), 3.84-3.88 (m, 2H), 3.21-3.15 (m, 8H), 1.70-1.66m (m, 2H), 1.60-1.53 (m, 2H), 1.26- 1.24(m, 9H). 13C NMR (125 MHz, D20) δ 128.8, 127.9, 127.5, 66.8, 60.6, 46.7, 41.3, 23.9, 23.3, 22.8, 15.9, 8.3. ESI-HRMS for C13H2oN05PNa [M+Na]+ calcd 324.0977; found 324.0977. (3-{[(Benzyloxy)carbonyl]amino}propyl)(ethoxy)phosphinic acid (56) as the triethylammoinium salt (0.4 g) is twice evaporated from MeCN then dissolved in dry DCM (5 mL) and dry DMF (5 μί) and added to oxalyl chloride (445 μΙ, 5 eq.) in dry DCM (5 mL) under Ar, stirred at room temperature for 2 h and then evaporated twice from dry DCM. The resulting crude phosphonyl chloride (57) in dry DCM (5 mL) is added to a solution of 3-octadecyloxypropan-1-ol (426 mg, 1.3 eq.) and dry pyridine (1 mL) in dry DCM (3 mL). The mixture is stirred at room temperature for 3 h and then treated with saturated sodium bicarbonate solution, extracted with DCM, dried and evaporated. Flash chromatography (hexanes-EtOAc,10%-100%, followed by 5% v/v MeOH in EtOAc) gives benzyl N-(3-{ethoxy[3-(octadecyloxy)propoxy]phosphoryl}propyl)carbamate (58) (175 mg). 1H NMR (500 MHz, CDCI3) δ 7.37- 7.25 (m, 5H), 5.09 (s, 2H), 4.15-4.05 (m, 2H) 3.53.3.43 (m, 2H), 3.41-3.37 (m, 2H), 3.30-3.26 (m, 2H)i 1.94-1.90 (m, 2H), 1.88-1.73 (m, 2H), 1.53-1.59 (m, 2H), 1.37-1.25 (m, 33H), 0.89-0.84 (m, 3H). 13C NMR (125 MHz, CDCI3) δ.128.5, 128.1 , 71.0, 66.6, 62.9, 61.7, 31.9, 30.9, 29.7, 29.5, 29.3, 26.2, 22.7, 16.5, 14.1. ESI-HRMS for C34H62N06PNa [M+Na]+ calcd 634.4212; found 634.4210.
Benzyl N-(3-{ethoxy[3-(octadecyloxy)propoxy]phosphoryl}propyl)carbamate (58) (170 mg) in ethanol (15 mL) and TFA (0.06 mL) is hydrogenolysed in the presence of 10% Pd/C (150 mg) at atmospherice pressure for 2 h. Filtration and evaporation gives the TFA salt of ethyl 3-(octadecyloxy)propyl (3-aminopropyl)phosphonate (59) (111 mg). 1H NMR (500 MHz, CDCI3) δ 4.17-4.08 (m, 2H), 3.50-3.47 (m, 2H), 3.40-3.37 (m, 2H), 3.15- 3.07 (m, 2H), 2.81-2.17 (m, 2H), 2.08-1.89 (m, 4H), 1.55-1.53 (m, 2H), 1.34-1.25 (m, 33H), 0.89-0.84 (m, 3H). 13C NMR (125 MHz, CDCI3) δ.71.0, 66.3,63.4,62.9,41.0, 31.9, 29.5, 26.2, 22.7,16.6,13.7. ESI-HRMS for CzsH^ C^P [M+H]+ calcd 478.4025; found 478.4019. Ethyl 3-(octadecyloxy) propyl (3-aminopropyl)phosphonate (59) as the TFA salt (100 mg) is heated at 70 °C in ethanol (5 mL) with 4-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine-7- carbaldehyde (4) (30 mg) for 18 h and then cooled to room temperature and treated with NaBH4 (12 mg). The product is evaporated onto silica gel and flash chromatographed (9/1/0.1 v/v/v DCM/MeOH/conc. NH3) to give ethyl 3-(octadecyloxy)propyl [3-({[4- (benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)propyl]phosphonate (60)
(21 mg). 1H NMR (500 MHz, CDCI3) δ 8.49 (s, H), 7.66 (s, 1 H), 7.49-7.29 (m, 5H), 5.55 (s, 2H), 4.33 (s, 2H), 4.08-3.95 (m, 4H), 3.48-3.42 (m, 2H), 3.39-3.34 (m, 2H), 3.14- 3.07(m, 2H), 2.01-1.95 (m, 2H), 1.89-1.86 (m, 2H), 1.84-1.73 (m, 2H), 1.55-1.49 (m, 2H), 1.33-1.21 (m, 33H), 0.94-0.86 (m, 3H). 13C NMR (125 MHz, CDCI3) δ.162.0,155.6, 150.1, 148.6, 136.2, 130.9, 128.6, 128.3, 115.1 , 105.7, 71.2, 67.8, 66.4, 63.4, 62.3, 47.5, 45.6, 31.9, 30.8, 29.6, 26.1 , 23.2, 22.7, 22.0, 19.4, 16.3, 14.1. 31P NMR (202 MHz, CDCI3 ) δ 30.6. ESI-HRMS for C40H68N4O5P [M+H]+ calcd 715.4927; found 715.4930.
Ethyl 3-(octadecyloxy)propyl [3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7- yl]methyl}amino)propyl]phosphonate (60) (20 mg) is hydrogenolysed with hydrogen at 1 atm. over 10% Pd/C (20 mg) in ethanol (5 mL) containing TFA (0.06 mL) for 2 h. Filtration and evaporation gives ethyl 3-(octadecyloxy)propyl {3-[({4-hydroxy-5H- pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propyl}phosphonate (61) (16 mg). 1H NMR (500 MHz, MeOD) δ 7.99 (s, 1 H), 7.58 (s, 1 H), 4.36 (s, 2H), 4.17-4.07 (m, 4H), 3.54-3.52 (m, 2H), 3.44-3.40 (m, 2H), 3.18-3.15 (m, 2H), 2.04-1.98 (m, 4H), 1.55-1.52 (m, 2H), 1.44-1.24 (m, 33H), 0.94-0.88 (m, 3H). 13C NMR (125 MHz, MeOD) δ 155.7,145.0,143.7, 130.8, 120.0, 108.0, 72.4, 67.5, 64.7, 63.7, 47.9, 41.2, 33.1 , 31.8, 30.7, 30.5, 27.3, 23.6, 22.4, 20.6, 16.7, 14.4. 31P NMR (202 MHz, MeOD) δ 31.2. ESI-HRMS for C33H62N405P [M+H]+ calcd 625.4458; found 625.4456.
Example 12 Ethyl 3-(hexadecyloxy)propyl {3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]propyl}phosphonate (65), Scheme 12
Figure imgf000064_0001
Scheme 12 (3-{[(Benzyloxy)carbonyl]amino}propyl)(ethoxy)phosphinic acid (56) (0.52 g) is evaporated twice with MeCN then dissolved in dry DCM (5 mL) and dry DMF (5 pL). This solution is then added to oxalyl chloride (574 μΙ, 5 eq.) in dry DCM (5 mL) under Ar, stirred at room temperature for 2 h and then evaporated twice from dry DCM. The resulting crude phosphonyl chloride (57) in dry DCM (5 mL) is added to a solution of 3- hexadecyloxypropan-1-ol (504 mg, 1.3 eq.) and dry pyridine (1 mL) in dry DCM (3 mL). The mixture is stirred at room temperature for 3 h and then treated with saturated sodium bicarbonate solution, extracted with DCM, dried and evaporated. Flash chromatography (hexanes- EtOAc, followed by 5% v/v MeOH in EtOAc) gives benzyl N-(3-{ethoxy[3- (hexadecyloxy)propoxy]phosphoryl}propyl)carbamate (62) (110 mg). 1H NMR (500 MHz, CDCI3) δ 7.36-7.26 (m, 5H), 5.09 (s, 2H), 4.12-4.08 (m, 2H), 3.49-3.47 (m, 2H), 3.40-3.37 (m, 2H), 3.32-3.25 (m, 2H), 1.92-1.89 (m, 2H), 1.79-1.75 (m, 2H), 1.58-1.54 (m, 2H), 1.32-1.25 (m, 30H), 0.89-0.87(m, 3H). 3C NMR (125MHz, CDCI3) δ 156.4,136.6,128.5, 128.1 , 71.2, 66.6, 62.9, 61.7, 31.9, 30.9, 29.7, 29.5, 29.3, 26.2, 22.7, 16.4, 14.1. 31P NMR (202 MHz, CDCI3 ) δ 31.7. ESI-HRMS for C32H58N06PNa [M+Na]+ calcd 606.3899; found 686.3904.
Benzyl N-(3-{ethoxy[3-(hexadecyloxy)propoxy]phosphoryl}propyl)carbamate (62) (110 mg) in ethanol (10 mL) and TFA (0.04 ml.) is hydrogenolysed in the presence of 10% Pd/C (100 mg) at atmospherice pressure for 2 h. Filtration and evaporation gives the TFA salt of ethyl 3-(hexadecyloxy)propyl (3-aminopropyl)phosphonate (63) (85 mg). 1H NMR (500 MHz, CDCI3) δ 8.35-8.20(m,2H),4.18-4.09 (m, 2H), 3.49-3.42 (m, 2H), 3.40- 3.37 (m, 2H), 3.12-3.05 (m, 2H), 2.08-1.85 (m, 4H), 1.55-1.53 (m, 2H), 1.33-1.23 (m, 30H), 0.89-0.86 (m, 3H). 13C NMR (125 MHz, CDCI3) δ 71.2, 66.3, 63.5, 62.5, 31.9, 29.5, 26.2, 22.7, 16.2, 14.1. 31P NMR (202 MHz, CDCI3) δ 31.1. ESI-HRMS for C24H53N04P [M+H]+ calcd 450.3712; found 450.3723. Ethyl 3-(hexadecyloxy)propyl (3-aminopropyl)phosphonate (63) as the TFA salt (85 mg) is heated at 70 °C in ethanol (5 mL) with 4-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine-7- carbaldehyde (4) (35 mg) for 18 h and then cooled to room temperature and treated with NaBH4 (12 mg). The product is evaporated onto silica gel and flash chromatographed (9/1/0.1 v/v/v DCM/MeOH/conc. NH3) to give (ethyl 3-(hexadecyloxy)propyl [3-({[4- (benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)propyl]phosphonate (64) (23 mg). 1H NMR (500 MHz, CDCI3) δ 8.50 (s, 1 H), 7.69 (s, 1 H), 7.49-7.29 (m, 5H), 5.57 (s, 2H), 4.33 (s, 2H), 4.08-3.95 (m, 4H), 3.46-3.42 (m, 2H), 3.39-3.34 (m, 2H), 3.10-3.05 (m, 2H), 2.04-1.95 (m, 2H), 1.89-1.86 (m, 2H), 1.84-1.73 (m, 2H), 1.55-1.49 (m, 2H), 1.35- 1.28 (29H), 0.92-0.86 (m, 3H). 13C NMR (125 MHz,CDCI3) 6155.6, 150.1 , 148.6, 136.3, 130.9, 128.5, 128.1 , 115.1 , 105.8, 71.2, 67.8, 66.4, 63.4, 62.3, 47.5, 41.2, 31.9, 30.8, 29.6, 26.1 , 23.2, 22.7, 22.0, 19.4, 16.3, 14.1. 31P NMR (202 MHz, CDCI3) δ 30.6. ESI- HRMS for C38H64N405P [M+H]+ calcd 687.4614; found 687.4620.
Ethyl 3-(hexadecyloxy)propyl [3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7- yl]methyl}amino)propyl]phosphonate (64) (20 mg) is hydrogenolysed with 10% Pd/C (20 mg) in ethanol (5 mL) containing TFA (0.06 mL) for 2 h. Filtration and evaporation gives ethyl 3-(hexadecyloxy) propyl {3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]propyl}phosphonate (65) (18 mg). 1H NMR (500 MHz, MeOD) δ 7.99(s, 1 H), 7.58 (s, 1 H), 4.36 (s, 2H), 4.17-4.07 (m, 4H), 3.54-3.52 (m, 2H), 3.44-3.40 (m,2H), 3.18-3.15 (m,2H), 2.04-1.98 (m,4H), 1.55-1.52 (m,2H), 1.44-1.24 (m, 29H), 0.94-0.88 (m, 3H), 0.88 (m, 3H). 3C NMR (125 MHz, MeOD) δ 155.8,144.8,143.4, 130.6, 119.8, 107.8, 72.1 , 67.5, 64.7, 63.7, 47.9, 41.2, 33.1 , 31.8, 30.7, 30.5, 27.3, 23.6, 22.4, 20.6, 16.7, 14.4. 31P NMR (202 MHz, MeOD ) δ 31.2. ESI-HRMS for C3iH58 405P [M+H]+ calcd 597.4145; found 597.4152 .
Example 13 {3-[({4-Hydroxy-5H^yrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propyl}[3- (octadecyloxy)propoxy]phosphinic acid (72), Scheme 13
Figure imgf000066_0001
40 (66) (iii) H2, Pd/C, TFA
Figure imgf000066_0002
(70) (71) R2
H2, Pd/C, TFA, EtOH
(72) R2 =
Scheme 13
The HBr salt of 3-aminopropylphosphonic acid (40) (5 g, 1 eq.) in 1 M NaOH (aq) (50 mL) at 0 °C is treated with aliquots of 2,2,2-trichloroethoxycarbonyl chloride (6.2 mL, 2 eq.) and 1 M NaOH (5.7 mL, 2.2 eq.) over 3 h. The resulting solution is extracted with EtOAc and the aqueous phase acidified to pH 1 with HCI and then re-extracted with EtOAc, which is dried and evaporated to yield (3-{[(2,2,2-trichloroethoxy) carbonyl] amino} propyl) phosphonic acid (66) (4 g) 1H NMR (500 MHz, CDCI3) δ 7.40- 7.10(m,2H), 4.73 (s, 2H), 3.40-3.23 (bs, 2H), 2.04-1.85 (bs, 4H). 13C NMR (125 MHz, CDCI3) δ 155.3, 95.6, 74.5, 41.5, 24.2, 22.9. 31P NMR (202 MHz, CDCI3) δ 32.5. ESI- HRMS for C6H12N05 35CI3P [M+H]+ calcd 313.9519; found 313.9524.
(3-{[(2,2,2-Trichloroethoxy)carbonyl]amino}propyl)phosphonic acid (66) (1.33 g, 1 eq.) in MeCN (10 mL) is heated with thionyl chloride (0.9 mL, 3 eq.) at 70 °C for 2 h under Ar. The solution is evaporated twice from dry MeCN under high vacuum and dissolved in dry pyridine (5 mL). A solution of 3-octadecyloxypropan-1-ol (964 mg, 0.7 eq.) in dry pyridine (5 mL) is added dropwise and the mixture is stirred under Ar for 3 h. Saturated aq. NaHC03 is added and the product is stirred vigorously for 1 h and then extracted with DCM, which is washed with brine, dried and evaporated. Flash chromatography (EtOAc followed by 8/2/0.2 v/v/v DCM/MeOH/conc. NH3) gives first 2,2,2-trichloroethyl N-(3- {bis[3-(octadecyloxy)propoxy]phosphoryl}propyl)carbamate (68) (321 mg). 1H NMR (500 MHz, CDCI3) δ 4.72 (s, 2H), 4.17-4.07 (m, 4H), 3.50-3.48 (m, 4H), 3.42-3.38 (m, 4H), 3.34-3.31 (m, 2H), 1.94-1.91 (m, 2H), 1.65-1.52 (m,6H), 1.35-1.25 (m, 60H), 0.84-0.92 (m, 6H). 13C NMR (125 MHz, CDCI3) δ 74.5, 71.3, 66.6, 63.1 , 41.3, 31.9, 30.1 , 29.5, 26.2, 22.7, 14.1. 31P NMR (202 MHz, CDCI3) δ 31.6. ESI-HRMS for C48H96N07 35CI3P [M+H]+ calcd 934.5990; found 934.5992.
Next to elute is [3-(octadecyloxy)propoxy](3-{[(2,2,2-trichloroethoxy)carbonyl] amino}propyl)phosphinic acid (67) (302 mg). 1H NMR (500 MHz, CDCI3) δ 4.76-4.72 (m, 2H), 4.14-4.10 (m, 2H), 3.55-3.48 (m, 2H), 3.44-3.40 (m, 2H), 3.38-3.32 (m, 2H), 2.07- 1.82 (m, 4H), 1.56-1.52 (m, 2H), 1.38-1.30 (m, 30H), 0.89-0.83 (m, 3H). 13C NMR (125 MHz, CDCI3) δ 129.4, 95.3, 74.5, 67.1 , 66.5, 62.8, 41.5, 31.9, 30.9, 29.3, 26.2, 22.7, 14.1. 31P NMR (202 MHz, CDCI3) δ 26.4. ESI-HRMS for C27H54N06 35CI3P [M+H]+ calcd 624.2754; found 624.2764.
[3-(octadecyloxy)propoxy](3-{[(2,2,2-trichloroethoxy)carbonyl]amino}propyl)phosphinic acid (67) (300 mg, 1 eq.) in dry DCM (5 mL) and dry DMF (5 μί) is treated with oxalyl chloride (214 L, 5 eq.) for 2.5 h and then evaporated twice from dry DCM and dissolved in dry pyridine (2 mL). Dry benzyl alcohol (150 μί, 3 eq.) in dry pyridine (1 mL) is then added and the mixture stirred for 2 h. The product is extracted with DCM, washed with brine, dried and evaporated. Flash chromatography (hexanes, then 20 and 50% v/v EtOAc in hexanes) gives 2,2,2-trichloroethyl N-{3-[(benzyloxy)[3-(octadecyloxy)propoxy] phosphoryl]propyl}carbamate (69) (272 mg). 1H NMR (500 MHz, CDCI3) δ 7.47-7.35 (m, 5H), 5.12-5.03 (m, 2H), 4.71 (s, 2H), 4.14-4.01 (m, 2H), 3.46-3.44 (m, 2H), 3.38-3.36 (m, 2H), 3.30-3,27(m, 2H), 1.93-1.74 (m, 6H), 1.31-1.25 (m, 30H), 0.94-0.87 (m, 3H). 13C NMR (125 MHz, CDCI3) δ 154.7, 136.5,128.6, 128.0, 74.5, 71.2, 67.3, 63.1 , 41.2, 31.9, 30.9, 29.6, 26.2, 22.8, 14.1. 31P NMR (202 MHz, CDCI3) δ 32.0. ESI-HRMS for C34H6oN06 35CI3P [M+H]+ calcd 714.3224; found 714.3220.
2,2,2-Trichloroethyl 3-(benzyloxy(3-(octadecyloxy)propoxy)phosphoryl)propylcarbamate (69) (270 mg) is stirred with activated zinc dust (300 mg) and acetic acid (3 ml_) for 2 h. The product is diluted with chloroform, filtered and washed with water, dried and evaporated to yield benzyl 3-(octadecyloxy)propyl (3-aminopropyl)phosphonate (70) (266 mg). 1H NMR (500 MHz, CDCI3) δ 7.47-7.30 (m, 5H), 5.12-5.09 (m, 2H), 4.08-3.98 (m, 2H), 4.42-3.37 (m, 2H), 3.35-3.32 (m, 2H), 3.09-3.07 (m, 2H), 2.03-1.81 (m, 6H), 1.53-1.49 (m, 2H), 1.31-1.25 (m, 30H), 0.89-0.86 (m, 3H). 13C NMR (125 MHz, CDCI3) δ 176.6, 136.2, 128.6, 127.9, 71.2, 67.6, 66.5, 63.4, 40.0, 31.9, 30.8, 29.7, 26.1 , 22.2, 14.1. 31P NMR (202 MHz, CDCI3) δ 32.0. ESI-HRMS for C31H59N04 P [M+H]+ calcd 540.4182; found 540.4191.
Benzyl 3-(octadecyloxy)propyl (3-aminopropyl)phosphonate (70) (112 mg, 1.2 eq.) is heated at 70 °C in ethanol (7 mL) with 4-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine-7- carbaldehyde (4) (44 mg, 1 eq.) for 18 h and then cooled to room temperature and treated with NaBH4 (20 mg). The product is evaporated onto silica gel and flash chromatographed (9/1/0.1 v/v/v DCM/MeOH/conc. NH3) to give benzyl 3- (octadecyloxy)propyl [3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7- yl]methyl}amino)propyl]phosphonate (71) (62 mg). 1H NMR (500 MHz, CDCI3) δ 8.50(s,1 H), 7.60(s,1 H), 7.47-7.26(m,10H), 5.56(s,2H), 5.02-4.95(m,2H), 4.12(s,2H), 4.09- 3.94(m,2H), 3.41-3.39(m,2H), 3,37-3.34(m,2H), 2.86-2.82(m,2H), 1.96-1.75(m,6H), 1.51- 1.47(m,2H), 1.31-1.13(m,30H), 0.92-0.86(m,3H). 3C NMR (125 MHz, CDCI3) δ 155.4, 149.8, 149.1 , 136.0, 128.6, 128.4, 128.3, 115.1 , 73.3, 67.5, 67.4, 66.5, 63.2, 53.4, 48.4, 42.1 , 31.9, 30.8, 29.6, 26.2, 23.8, 22.7, 21.2, 14.2. 31P NMR (202 MHz, CDCI3) δ 32.2. ESI-HRMS for C45H7oN405 P [M+H]+ calcd 777.5084; found 777.5077.
Benzyl 3-(octadecyloxy)propyl [3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7- yl]methyl}amino)propyl]phosphonate (71) (44 mg) is hydrogenolysed with 10% Pd/C (50 mg) in ethanol (5 mL) containing TFA (0.04 mL). Dilution with chloroform(5ml) and filtration followed by extraction of the catalyst with chloroform-methanol (1 :1 ,10ml) gives after evaporation of the solvents {3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]propyl}[3-(octadecyloxy)propoxy]phosphinic acid (72) (20 mg). 1H NMR (500 MHz, CDCI3 -MeOD[1 :1]) δ 8.97 (s, 1 H), 7.85 (s, 1 H), 4.48 (s, 2H), 4.14-4.07 (m, 2H), 3.55-3.52 (m, 2H), 3.45-3.42 (m, 2H), 3.27-3.23(m, 2H), 2.11-2.02(m,2H) 1.98- 1.88(m,4H), 1.60-1.54(m,2H), 1.35-1.25 (m,30H), 0.91-0.87 (m, 3H). 13C NMR (125 MHz, CDCI3 -MeOD[1 :1]) δ 152.9, 146.1 , 133.3,, 119.9, 105.0,72.4, 67.9, 64.0, 42.0, 33.1 , 32.0,30.7, 27.3, 24.8, 23.8, 20.9, 14.9. 31P NMR (202 MHz, CDCI3 -MeOD[1 :1]) δ 30.0. ESI-HRMS for C45H7oN405 P [M+H]+ calcd 777.5084; found 777.5077.
Example 14
Bis[3-(octadecyloxy)propyl] {3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]propyl}phosphonate (76), Scheme 14.
Figure imgf000069_0001
(73) R1 = C02Bn
H2, Pd/C, TFA,
(74) R = H MeOH
Figure imgf000069_0002
Scheme 14
(3-{[(Benzyloxy)carbonyl]amino}propyl)phosphonic acid (41) (0.49 g) in dry DCM (10 mL) and dry DMF (10μί) is treated with oxalyl chloride (0.6 mL) and stirred for 3 h. The solvent is evaporated and then the product is evaporated twice from toluene and dissolved in dry pyridine (2 mL). 3-Octadecyloxypropan-1-ol (1.28 g) in dry pyridine (3 mL) is added and the mixture stirred for 2 h, diluted with DCM and then washed with brine and dried. Flash chromatography (petroleum ether, then 20 and 50% v/v EtOAc in petroleum ether) gives benzyl N-(3-{bis[3-
(octadecyloxy)propoxy]phosphoryl}propyl)carbamate (73) (406 mg). H NMR (500 MHz, CDCI3) δ 7.39-29 (m, 5H), 5.09 (s, 2H), 4.14-4.06 (m, 4H), 3.49-3.46 (m, 4H), 3.42-2.38 (m, 4H), 3.33-3.26(m, 2H), 1.93-1.54 (m, 12H), 1.30-1.24 (m, 60H), 0.93-0.84 (m, 6H). 13C NMR (125MHz, CDCI3) δ 156.4, 136.5, 128.5, 71.2, 69.0, 66.6, 60.4, 41.2, 31.9, 31.0, 29.6, 26.2, 23.2, 22.6, 21.0, 14.2. 31P NMR (202 MHz, CDCI3) δ 31.7. ESI-HRMS for C53H10oN07 P 23Na [M+Na]+ calcd 916.7135; found 916.7131.
Benzyl N-(3-{bis[3-(octadecyloxy)propoxy]phosphoryl}propyl)carbamate (73) (400 mg) is dissolved in methanol (20 mL) containing TFA (0.1 mL) and 10% Pd/C (400 mg) at 40 °C and hydrogenolysed at atmospheric pressure. The solution is filtered, the residue extracted with DCM and the combibed organic phase evaporated to give bis[3- (octadecyloxy)propyl] (3-aminopropyl)phosphonate (74) (200 mg). 1H NMR (500 MHz, CDCI3) δ. 4.15-4.08 (m, 2H), 3.50-3.45 (m, 4H), 3.42-3.38 (m, 4H), 2.05-1.85 (m, 8H), 1.60-1.50 (m, 4H), 1.35-1.24 (m, 60H), 0.92-0.85 (m, 6H). 13C NMR (125 MHz, CDCI3) δ .71.2, 66.4, 63.6, 39.9, 31.9, 30.8, 29.6, 26.2, 22.7, 14.1. 31P NMR (202 MHz, CDCI3) δ 31.2. ESI-HRMS for C45Hg5N05 P [M+H]+ calcd 760.6948; found 760.6945.
Bis[3-(octadecyloxy)propyl] (3-aminopropyl)phosphonate (74) (200 mg) and 4- benzyloxy-5H-pyrrolo[3,2-d]pyrimidine-7-carbaldehyde (4) (56 mg) in methanol (8 mL) are stirred at 60 °C for 18 h and then cooled to 30 °C. NaBH4 (20 mg) is added and the mixture stirred for 30 min and evaporated. Flash chromatography (eluting with EtOAc and then 5% v/v MeOH in EtOAc) gives bis[3-(octadecyloxy)propyl] [3-({[4-(benzyloxy)- 5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)propyl]phosphonate (75) (24 mg). 1H NMR (500 MHz, CDCI3) δ 8.50 (s, 1 H), 7.64 (s, 1 H), 7.47-7.35 (m, 5H), 5.58 (s, 2H), 4.34 (s, 2H), 4.12-4.01 (m, 4H), 3.47-3.42 (m, 4H), 3.40-3.34 (m, 4H), 3.12-3.09 (m, 2H), 2.06- 1.83 (m, 8H), 1.53-1.49 (m, 4H), 1.38-1.25 (m, 60H), 0.94-0.86 (m, 6H). 13C NMR (125 MHz, CDCI3) δ 162.2, 155.6, 150.1 , 148.7, 136.2, 130.8, 128.5, 128.4, 115.1 , 105.9, 71.2, 68.9, 66.4, 63.5, 47.4, 41.1 , 31.9, 30.7, 29.6, 29.3, 26.1 , 22.9, 21.8, 19.4 ,14.1. 31P NMR (202 MHz, CDCI3) δ 30.9. ESI-HRMS for
Figure imgf000070_0001
P [M+H]+ calcd 997.7850; found 997.7860.
Bis[3-(octadecyloxy)propyl] [3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7- yl]methyl}amino)propyl]phosphonate (75) (40 mg) in ethanol (5 mL) with TFA (0.06 mL) is hydrogenolysed over 10% Pd/C (30 mg) at atmospheric pressure. Filtration of the solvent and extraction of the residue with DCM gives bis[3-(octadecyloxy)propyl] {3-[({4- hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propyl}phosphonate (76) (32 mg) . 1H NMR (500 MHz, CDCI3) δ 7.95 (s, 1 H), 7.32 (s, 1 H), 4.32-4.23 (m, 2H), 4.23-4.02 (m, 4H), 3.42-3.40 (m, 4H), 3.38-3.36 (m, 4H), 3.36-3.30 (m, 2H), 2.25-1.85 (m, 8H), 1.60- 1.45 (m, 4H), 1.35-1.25 (m, 60H), 0.90-0.86 (m, 6H). 13C NMR (125 MHz, CDCI3) δ 162.3, 153.7, 142.8, 130.6, 117.8, 105.6, 71.2, 66.4, 63.6, 47.9, 41.4, 31.9, 30.7, 29.6, 26.2, 22.5, 19.5, 14.1. 31P NMR (202 MHz, CDCI3) δ 30.9. ESI-HRMS for C52H10oN406 P [M+H]+ calcd 907.7381 ; found 907.7385. Example 15
Ammonium 3-((7-hydroxy-1 H-pyrazolo[4,3-d]pyrimidin-3- yl)methylamino)propylphosphonate (79), Scheme 15.
Figure imgf000071_0001
Scheme 15
Anhydrous HCI in MeOH (1.0 M, 0.11 mL, 0.11 mmol) is added dropwise to a solution of the diethyl phosphonate (3c) (100 mg, 0.51 mmol) in anhydrous MeOH (2 mL) followed by the addition of the aldehyde (77) (90 mg, 0.34 mmol) and 2-picoline borane complex (50 mg, 0.44 mmol). The resulting suspension is left to stir for 2 h and the then homogeneous solution absorbed onto silica gel and the resulting residue subjected to flash chromatography (EtOAc then 5 to 10% v/v MeOH in CHCI3) to afford diethyl 3-((7- methoxy-2-(tetrahydro-2H-pyran-2-yl)-2H-pyrazolo[4,3-d]pyrimidin-3- yl)methylamino)propylphosphonate (78) (123 mg, 82 % yield) as a white solid, which is committed to the next step without further characterisation or purification.
Concentrated HCI (1.5 mL, 18 mmol) is added to a solution of compound (78) (90 mg, 0.204 mmol) in MeOH (3 mL, 0.204 mmol) and the resulting mixture heated to reflux for 6 h. The reaction mixture is concentrated in vacuo and partitioned between chloroform and water, the water layer is extracted with additional chloroform, the organic layers are combined, dried and concentrated in vacuo. The residue is dissolved in aqueous hydrobromic acid (2 mL, 17.68 mmol) and heated for a further 6 h at 80 °C. The reaction mixture is concentrated in vacuo and the resulting residue co-distilled with water (2 x 20 mL). Flash chromatography (1 :1 v/v dioxane/conc. NH4OH) afforded the ammonium 3- ((7-hydroxy-1 H-pyrazolo[4,3-d]pyrimidin-3-yl)methylamino)propylphosphonate (79) (35 mg, 60%) as a foam. 1H NMR (500 MHz, D20): S = 7.98 (s, 1 H), 4.47 (s, 2H), 3.16 (t, J = 6.9 Hz, 2H), 1.91 (septet, J = 7.0 Hz, 2H), 1.56 (m, 2H). 13C NMR (125 MHz, D20): δ = 155.2, 148.4, 137.5, 135.6, 128.6, 66.6, 48.7, 48.6 (d, J = 13.4 Hz), 40.8, 26.1 (d, J = 132 Hz), 25.6, 20.7(d, J = 3.8 Hz). 31P (202 MHz, D20): δ = 21.9. Example 16
{(±)-2(f?/S)-3-Hydroxy-2-[({4-hydroxy-5 -pyrrolo[3,2-dlpyrimidin-7- yl}methyl)amino]propoxy}phosphonic acid; triethylamine [(±)-(84)], Scheme 16.
Figure imgf000072_0001
Scheme 16
Iodine (1.94 g, 7.66 mmol) is added in one portion to a solution of trimethyl phosphite (0.943 mL, 7.98 mmol) in CH2CI2 (25 ml.) at 0 °C, which is then warmed to room temperature and stirred for 5 min to give a colourless solution (J.K. Stowell and T.S. Widlanski, Tetrahedron Lett, 1995, 36, 1825). A portion (6.7 mL, 1.3 eq) of this is added dropwise to a solution of 9H-fluoren-9-ylmethyl /V-(1 ,3-dihydroxypropan-2-yl)carbamate (80) (J. Y. Choi and R.F. Borch, Org. Lett, 2007, 9, 215) (0.5 g, 1.596 mmol) in a mixture of CH2CI2 (6 mL) and pyridine (3 mL) at 0 °C and stirred for 1 h. The mixture is warmed to room temperature and stirred for 30 min then the solvent is evaporated and the residue subjected to flash chromatography (EtOAc-hexanes, 9:1 v/v then EtOAc) to give first unreacted (80) (190 mg) then 9H-fluoren-9-ylmethyl /V-{(±)-2(ft/S)-1- [(dimethoxyphosphoryl)oxy]-3-hydroxypropan-2-yl}carbamate [(±)-(81)] (0.24 g, 36%) as a colourless gum. 1H NMR (500 MHz, CD3OD) 7.76 (d, J = 7.5, 2H), 7.63 (d, J = 7.3, 2H), 7.37 (t, J = 7.4, 2H), 7.29 (t, J = 7.4, 2H), 4.40-4.33 (m, 2H), 4.19-4.13 (m, 2H), 4.08 (m, 1 H), 3.87 (m, 1 H), 3.74 (d, J = 11.1 , 3H), 3.72 (d, J = 11.2, 3H), 3.65-3.56 (m, 2H). 13C NMR (125.7 MHz, CD3OD) 158.4 (C), 145.3 (d, J = 4.8, C), 142.6 (C), 128.7 (CH), 128.1 (CH), 126.1 (CH), 120.9 (CH), 67.74 (CH2), 67.70 (CH2), 61.5 (CH2), 55.3 (d, J = 6.1 , CH3), 54.2 (d, J = 7.0, CH), 48.4 (CH). Referenced to the centre line of CD3OD at 49.0 ppm. 31P (121.5 MHz, CD3OD) 2.2 (s). ESI-HRMS for C20H24NNaO7P [M+Na]+ calcd 444.1188; found 444.1179. Compound (±)-(81) (0.4 g, 0.949 mmol) is dissolved in CH2CI2 (10 mL) and bromotrimethylsilane (1.232 mL, 9.49 mmol) added. The clear solution is left at room temperature for 16 h then the solvent is evaporated. 10% aq. Et3N (2 mL) is added and the solution evaporated again. The residue is dissolved in DMF (4 mL) and piperidine (0.8 mL) added. After 30 min at room temperature the solvent is evaporated. The residue is dissolved in H20 (10 mL) and washed with EtOAc (10 mL). After evaporating the aqueous solution, the residue is repeatedly evaporated from 1 :1 v/v MeOH-Et3N (6 mL) to exchange most of the piperidine with Et3N. The residue is chromatographed on silica gel (1 ,4-dioxane-H20-Et3N, 60:40:1 v/v/v) then on RP-18 silica gel (H20) to give ([(±)- 2(f?/S)-2-amino-3-hydroxypropoxy)phosphonic acid; triethylamine [(±)-(82)] (160 mg, 0.428 mmol, - 80-85% pure) which is suspended in a mixture of MeOH (15 mL) and Et3N (0.040 mL, 0.286 mmol). 4-Benzyloxy-5 - -pyrrolo[3,2-c]pyrimidine-7-carbaldehyde (4) (0.072 g, 0.286 mmol) and sodium cyanoborohydride (0.023 g, 0.371 mmol) are added and the mixture heated at 50 °C for 16 h. The solvent is evaporated and the residue flash chromatographed (THF-H20-Et3N, 85:15:1 then 80:20:1 v/v/v) then on RP-18 silica gel (H20 then MeOH-THF-Et3N, 70:30:1 v/v/v) to give [(±)-2-(f?/S)-2-({[4-(benzyloxy)-5H- pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)-3-hydroxypropoxy]phosphonic acid; triethylamine [(±)-(83)] as a colourless amorphous solid (71 mg, 49%). 1H NMR (500 MHz, CD3OD) 8.48 (s, 1 H), 7.85 (s, 1 H), 7.52 (d, J = 7.2, 2H), 7.39-7.30 (m, 3H), 5.63 (s, 2H), 4.44 (s, 2H), 4.18 (ddd, J = 12.4, 8.8, 3.4, 1 H), 4.04 (ddd, J = 12.4, 9.7, 5.6 1 H), 3.85 (dd, J = 12.0, 5.1 , 1H), 3.79 (dd, J = 12.0, 6.1 , 1 H), 3.31 (m, residual CD3OD + 1 H), 3.07 (q, J = 7.3, 5.2H), 1.26 (t, J = 7.3, 8H). Approximately 0.9 eq of Et3N present. 13C NMR (125.7 MHz, CD3OD) 157.1 (C), 150.8 (CH), 149.6 (C), 137.7 (C), 133.3 (CH), 129.5 (CH), 129.4 (CH), 129.3 (CH), 116.5 (C), 107.9 (C), 69.1 (CH2), 62.6 (d, J = 3.8, CH2), 60.7 (d, J = 3.6, CH), 59.9 (CH2), 47.1 (CH2), 40.2 (CH2), 9.1 (CH3). Referenced to the centre line of CD3OD at δ 40.0. 31P (202.3 MHz CD3OD), 3.2 (s). ESI-HRMS for C17H2o 406P [M-Et3N-H]' calcd 407.1120; found 407.1128.
Compound [(±)-(83)] (0.07 g, 0.137 mmol) is dissolved in a 1 :1 mixture of MeOH-H20 (10 mL), 20% Pd(OH)2/C (40 mg) added and the mixture stirred under a hydrogen atmosphere at ambient pressure and temperature for 4 h. After filtering through Celite the solvent is evaporated to give {(±)-2(f?/S)-3-hydroxy-2-[({4-hydroxy-5H-pyrrolo[3,2- d]pyrimidin-7-yl}methyl)amino]propoxy}phosphonic acid; triethylamine [(±)-(84)] as a colourless solid (54 mg, 94%). 1H NMR (500 MHz, D20) 8.14 (s, 1 H), 7.82 (s, 1 H), 4.59 (s, 2H), 4.23 (ddd, J = 12.4, 6.7, 3.5, 1 H), 4.11 (ddd, J = 12.4, 7.5, 5.5, 1 H), 4.02 (dd, J = 12.6, 5.0, 1 H), 3.96 (dd, J = 12.6, 5.9, 1 H), 3.60 (m, 1 H), 3.27 (q, J = 7.4, 2.6H), 1.35 (t, J = 7.4, 3.9H). Referenced to HOD at 4.79 ppm. Approximately 0.4 eq. of Et3N present. 13C NMR (75.5 MHz, D20-CD3OD, 3:1) 156.0 (C), 144.5 (C), 143.8 (CH), 131.7 (CH), 118.6 (C), 107.8 (C), 61.6 (CH2), 60.2 (CH), 59.1 (CH2), 47.6 (CH2), 39.9 (CH2), 9.2 (CH3). Referenced to the centre line of CD3OD at δ 49.0. 31 P NMR (121.5 MHz, D20) 3.0 (s). ESI-HRMS for C10H14N4O6P [M-Et3N-H]" calcd 317.0651 ; found 317.0644. Example 17
{(±)-2( ?/S)-[({2-Chloro^-hydroxy-5H-pyrrolo[3,2-cqpyrimidin-7-yl}methyl)ami hydroxypropoxyjphosphonic acid; trrethylamrne [(±)-(90)], Scheme 17.
Figure imgf000074_0001
(±)-(89) (±)-(90)
Scheme 17 2,4-Dichloro-5H-pyrrolo[3,2-d]pyrimidine (85) (N.S. Girgis, H.B. Cottam, S.B. Larson and R.K. Robins, J. Het. Chem. 1987, 24, 821) (0.5 g, 2.66 mmol) and sublimed potassium ferf-butoxide (1.492 g, 13.30 mmol) are stirred in THF (25 mL) at 40 °C for 16 h. The solvent is evaporated, H20 (10 mL) added and the mixture extracted with EtOAc (50 mL), washed with brine, then dried and the solvent evaporated to yield a cream coloured solid. Flash chromatography (EtOAc-Hexanes, 3:7 v/v) gives 4-(terf-butoxy)-2-chloro-5H- pyrrolo[3,2-c]pyrimidine (86) (0.33 g, 55%) as a colourless solid. 1H NMR (500 MHz, CDCI3) 8.58 (bs, exchanged to D20, 1 H), 7.37 (t, J = 3.1 , after D20 became d, J = 3.1 , 1 H), 6.56 (dd, J = 3.1 , 2.2, after D20 became d, J = 3.1 , 1 H), 1.70 (s, 9H). 13C NMR (125.7 MHz, CD3OD) 157.3 (C), 152.4 (C), 150.4 (C), 132.0 (CH), 116.3 (C), 102.0 (CH), 85.0 (C), 28.7 (CH3). Referenced to the centre line of CD3OD at δ 49.0. ESI-HRMS for C10H13 (35)CIN3O [M+H]+ calcd 226.0747; found 226.0752.
Compound (86) (0.330 g, 1.462 mmol) is dissolved in a mixture of 1 ,4-dioxane (6 mL) and H20 (3 mL) then potassium carbonate (0.525 g, 3.80 mmol) and formaldehyde solution (37% aq., 1.53 mL, 19.10 mmol) are added and the mixture heated at 80 °C for 19 h. The mixture is cooled, extracted with EtOAc (50 mL), washed with brine, dried then the solvent evaporated to a gum which is dissolved in 7M NH3-MeOH (10 mL) and left at room temperature for 30 min After evaporation of the solvent the residue is flash chromatographed (EtOAc-hexanes, 7:3 then EtOAc) to give [4-(tert-butoxy)-2-chloro-5/-/- pyrrolo[3,2-d]pyrimidin-7-yl]methanol (87) (0.214 g, 57%) as a colourless solid. 1H NMR (500 MHz, CD3OD) 7.51 (s, 1 H), 4.75 (s, 2H), 1.72 (s, 9H). 13C NMR (125.7 MHz, CD3OD) 157.3 (C), 150.5(C x 2), 131.0 (CH), 116.8 (C), 116.6 (C), 85.0 (C), 55.2 (CH2), 28.8 (CH3). Referenced to the centre line of CD3OD at 49.0 ppm. ESI-HRMS for CnH ^CINaNaOz IM+Na calcd 278.0672; found 278.0671. Dess-Martin periodinane (0.390 g, 0.921 mmol) is added to a mixture of triethylamine (0.47 mL, 3.35 mmol) and alcohol (87) (0.214 g, 0.837 mmol) in THF (6 mL) and the mixture is stirred at room temperature for 40 min. EtOAc (50 mL), sat.aq. NaHC03 (3 mL) and water (3 mL) are added successively and the mixture filtered through Celite. The organic layer is separated and washed with brine then dried and the solvent evaporated to leave a solid which is dissolved in a 1 :1 v/v mixture of MeOH/CHCI3, silica gel added and the solvent evaporated. The residue is flash chromatographed (EtOAc-hexanes,4:6 then 1 :1 v/v) to give 4-(ferf-butoxy)-2-chloro-5H-pyrrolo[3,2-ci]pyrimidine-7-carbaldehyde (88) as a colourless solid which is triturated with a little EtOAc and dried (0.179 g, 84%). A sample is recrystallized from EtOAc. 1H NMR (500 MHz, DMSO d6) 13.08 (bs, 1 H), 10.06 (s, 1 H), 8.41 (s, 1H), 1.70 (s, 9H). 13C NMR (125.7 MHz, DMSO d6) 183.8 (CH), 156.2 (C), 151.0 (C), 149.6 (C), 137.7 (CH), 116.8 (C), 115.9 (C), 84.5 (C), 28.2 (CH3). Referenced to the centre line of DMSOcfe at δ 39.7. ESI-HRMS for CnH^CINaNaOz [M+Na]+ calcd 276.0516; found 276.0511.
A mixture of (±)-(82) (0.19 g, 0.509 mmol), Et3N (0.048 mL, 0.339 mmol), the aldehyde (88) (0.086 g, 0.339 mmol) and 2-picoline borane complex (0.047 g, 0.441 mmol) are stirred together in MeOH (15 mL) at 50 °C for 16 h then the solvent evaporated and the residue chromatographed on silica gel (1 ,4-dioxane-H20-1% aq. Et3N, 75:25:1 v/v/v). The crude product is further chromatographed on silica gel (THF-H20-Et3N, 85:15:1 then 80:20:1 v/v/v) to give [(±)-2(f?/S)-2-({[4-(tert-butoxy)-2-chloro-5H-pyrrolo[3,2-d]pyrimidin- 7-yl]methyl}amino)-3-hydroxypropoxy]phosphonic acid; triethylamine [(±)-(89)] as a colourless amorphous solid (105 mg, 51%). 1H NMR (500 MHz, CD3OD) 7.80 (s, 1 H), 4.32 (d, J = 13.9, 1 H), 4.29 (d, J = 13.9, 1 H), 4.13 (ddd, J = 12.4, 9.2, 3.6, 1 H), 4.00 (ddd, J = 12.4, 10.3, 5.6, 1 H), 3.82 (dd, J = 11.9, 5.2, 1 H), 3.77 (dd, J = 11.9, 5.9, 1 H), 3.16 (m, 1 H), 2.89 (q, J = 7.3, 12H), 1.72 (s, 9H), 1.19 (t, J = 7.3, 18H). Approximately 2 eq. of Et3N present. 13C NMR (125.7 MHz, CD3OD) 157.4 (C), 151.2 (C), 150.9 (C), 133.2 (CH), 116.5 (C), 109.4 (C), 85.2 (C), 62.8 (d, J = 4.5, CH2), 60.9 (d, J = 3.5, CH), 60.5 (CH2), 47.0 (CH2), 40.3 (CH2), 28.7 (CH3), 9.9 (CH3). Referenced to the centre line of CD3OD at δ 49.0. 31P NMR (202.3 MHz, CD3OD) 3.0 (s). ESI-HRMS for C14H21 (35)CIN406P [M-Et3N-H]"calcd 407.0887; found 407.0895. Compound (±)-(89) (0.095 g, 0.155 mmol) is dissolved in 80% aq. TFA (2 ml.) and left at room temperature. After 15 min the solvent is evaporated to leave a colourless solid which is chromatographed on silica gel (1 ,4-dioxane-H20-Et3N 75:25:1 v/v/v) to give {(±)- 2(f?/S)-[({2-Chloro-4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3- hydroxypropoxyjphosphonic acid; triethylamine [(±)-(90)] as a colourless gum (84 mg, 97%). 1H NMR (500 MHz, CD3OD) 7.53 (s, 1 H), 4.36 (s, 2H), 4.18 (ddd, J = 12.4, 8.7, 3.4, 1 H), 4.05 (ddd, J = 12.4, 9.7, 5.4, 1 H), 3.86 (dd, J = 12.0, 5.2, 1 H), 3.81 (dd, J = 12.0, 6.0, 1 H), 3.31 (residual CD3OD + 1 H), 3.08 (q, J = 7.3, 10.8H), 1.26 (t, J = 7.3, 16.2H). Approximately 1.8 eq. of Et3N present. 13C NMR (125.7 MHz, CD3OD) 164.7 (C), 152.5 (C), 147.2 (C), 129.5 (CH), 119.8 (C), 106.7 (C), 62.2 (CH2), 60.8 (CH), 59.9 (CH2), 47.1 (CH2), 40.7 (CH2), 9.5 (CH3). Referenced to the centre line of CD3OD at 49.0 ppm. 31 P NMR (202.3 MHz, CD3OD), 4.1 (s). ESI-HRMS for C10H13 (35)CIN4O6P [M-Et3N- H]"calcd 351.0261 ; found 351.0264.
Example 18
First route to [(3S)-4-hydroxy-3-[({4-hydroxy-5W-pyrrolo[3,2-d|pyrimidin yl}methyl)amino]butyl]phosphonic acid hydrobromide (95), Scheme 18
Figure imgf000077_0001
Scheme 18
Compound (91) (F.W. Foss, Jr., A.H. Snyder, M.D. Davis, M. Rouse, M.D. Okusa, K.R. Lynch and T.L. Macdonald, Bioorg. Med. Chem. Lett, 2007, 15, 663) (0.460 g, 1.27 mmol) and 10% Pd-C (50 mg) are stirred in EtOH (20 mL) under a hydrogen atmosphere at ambient pressure and temperature for 6 h. The mixture is filtered through Celite and the solvent evaporated. The residue is dissolved in a mixture of EtOH (3 mL) and 37% aq. HCI (2 mL) and left at room temperature for 1.5 h then the solvent is evaporated to give diethyl [(3S)-3-amino-4-hydroxybutyl]phosphonate hydrochloride (92) as a colourless gum (330 mg, 100%). [a] 22 +5.3 (c 0.57 MeOH). 1H NMR (500 MHz, D20)
4.28-4.17 (m, 4H), 3.91 (dd, = 12.5, 3.7, 1 H), 3.74 (dd, J = 12.5, 6.4, 1 H), 3.48 (m, 1 H), 2.14-1.93 (m, 4H), 1.40 (t, J = 7.1 , 6H). Referenced to HOD at 4.79 ppm. 13C NMR (125.7 MHz, D20), 64.3 (d, J = 6.6, CH2), 60.7 (CH2), 53.5 (d, J = 18.2, CH), 22.3 (d, J = 4.1 , CH2), 21.0 (d, J = 141.2, CH2), 16.2 (d, J = 5.7, CH3). Referenced to internal CH3CN at δ 1.47. 31P NMR (202.4 MHz, D20), 33.3 (s). ESI-HRMS for C8H21N04P [M-HCI+H]+ calcd 226.1208; found 226.1213.
A mixture of (92) (55 g, 0.591 mmol), triethylamine (0.030 mL, 0.211 mmol), 4-(tert- butoxy)-2-chloro-5H-pyrrolo[3,2-c ]pyrimidine-7-carbaldehyde (88) (0.1 g, 0.394 mmol) and 2-picoline borane complex (0.055 g, 0.512 mmol) are stirred together in MeOH (4 mL) at room temperature for 16 h. The solvent is evaporated and the residue chromatographed on silica gel (CHCI3-MeOH-7M NH3 in MeOH, 9.6:0.2:0.2 v/v/v) to give intermediate diethyl [(3S)-3-({[4-(terf-butoxy)-2-chloro-5H-pyrrolo[3,2-d]pyrimidin-7- yl]methyl}amino)-4-hydroxybutyl]phosphonate as a colourless gum (108 mg) which is dissolved in 1 :1 mixture of 37% aq. HCI-THF (3 mL) and left at room temperature for 10 min. The solvent is evaporated and the residue dissolved in EtOH and neutralized with Amberlyst A21 resin. After filtering off the resin, the filtrate is evaporated and the residue is chromatographed on silica gel (CHCI3-MeOH-7M NH3 in MeOH, 7.0:2.75:0.25 v/v/v) to give diethyl [(3S)-3-[({2-chloro-4-hydroxy-5H-pyrrolo[3,2-cf]pyrimidin-7-yl}methyl)amino]- 4-hydroxybutyl]phosphonate (93) (0.070 g, 44%) as a colourless amorphous solid, [a]^2 +10.3 (c 0.555, MeOH) 1H NMR (500 MHz, CD3OD) 7.37 (s, 1 H), 4.28 (d. J = 13.7, 1 H), 4.23 (d, J = 13.7, 1 H), 4.13-4.05 (m, 4H), 3.93 (dd, J = 12.3, 3.6, 1 H), 3.72 (dd, J = 12.3, 4.6, 1H), 3.22 (m, 1H), 2.04-1.90 (m, 4H), 1.31 (dt, J = 7.0, 0.5, 6H). 3C NMR (125.7 MHz, CD3OD) 162.5 (C), 149.7 (C), 146.4 (C), 129.3 (CH), 119.4 (C), 107.7 (C), 63.5 (d, J = 6.4, CH2), 60.1 (CH2), 59.7 (d, J = 17.5, CH), 40.4 (CH2), 22.8 (d, J = 3.7, CH2), 22.2 (d, J = 142.3, CH2), 16.7 (d, J = 5.8, CH3). Referenced to the centre line of CD3OD at δ 49.0. 31P NMR (202.3 MHz, CD3OD), 31.7 (s). ESI-HRMS for C15H25(35)CIN405P [M+H]+ calcd 407.1251 ; found 407.1250.
Compound (93) (0.060 g, 0.147 mmol) is dissolved in EtOH (5 ml_), 10% Pd-C (40 mg) added and the mixture stirred under a hydrogen atmosphere at ambient pressure and temperature for 3 h. The mixture is filtered through Celite and the solvent evaporated to give diethyl [(3S)-4-hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-cf]pyrimidin-7- yl}methyl)amino]butyl]phosphonate hydrochloride (94) (0.055 g, 91%) as a colourless solid, [a] ,2 +12.2 (c 0.54, MeOH). 1H NMR (500 MHz, CD3OD) 7.97 (s, 1H), 7.67 (bs, 1 H), 4.47 (d, J = 13.9, 1H), 4.43 (d, J = 13.9, 1 H), 4.15-4.06 (m, 4H), 4.00 (dd, J = 12.4, 2.9, 1 H), 3.76 (dd, J = 12.4, 4.1 , 1 H), 3.35 (m, partially hidden by residual CD3OD, 1 H), 2.11-1.88 (m, 4H), 1.32 (t, J = 7.1 , 6H). 13C NMR (125.7 MHz, CD3OD) 155.7 (C), 145.2 (C), 143.8 (CH), 130.8 (CH), 119.6 (C), 108.0 (C), 63.6 (d, J = 4.3, CH2), 59.6 (d, J = 17.7, CH), 59.0 (CH2), 39.7 (CH2), 22.3 (d, J = 3.7, CH2), 22.2 (d, J = 142.7, CH2), 16.7 (d, J = 5.8, CH3). Referenced to the centre line of CD3OD at δ 49.0. 31 P NMR (202.3 MHz, CD3OD) 31.2 (s). ESI-HRMS for C15H26N405P [M-HCI+H]+ calcd 373.1641 ; found 373.1642.
Compound (94) (0.052 g, 0.127 mmol) is heated at 80 °C in hydrobromic acid (48% aq., 1.002 ml_, 12.72 mmol) for 4 h. The solvent is evaporated and the residue eluted from a column of RP 18 silica gel (H20) to afford [(3S)-4-hydroxy-3-[({4-hydroxy-5/-/-pyrrolo[3,2- d]pyrimidin-7-yl}methyl)amino]butyl]phosphonic acid hydrobromide (95) as a colourless glass which slowly crystallized, [a]23 +9.3 (c 0.355, H20). H NMR (500 MHz, D20 +
DCI): δ 8.93 (s, 1 H), 7.84 (s, 1 H), 4.51 (d, J = 14.7 Hz, 1 H), 4.48 (d, J = 14.7 Hz, 1 H), 3.97 (dd, J = 13.1 , 2.7 Hz, 1 H), 3.81 (dd, J = 13.1 , 4.7 Hz, 1 H), 3.46 (m, 1 H), 2.08-1.77 (m, 4H). Referenced to HOD at 4.79 ppm. 13C NMR (125.7 MHz, D20 +DCI) 153.4 (C), 145.2 (CH), 133.8 (C) 133.7 (CH), 118.8 (C), 104.0 (C), 59.8 (d, J = 17.1 Hz, CH), 58.5 (CH2), 38.9 (CH2), 23.3 (d, J = 137.2 Hz, CH2), 21.5 (d, J = 3.2 Hz, CH2). Referenced to internal CH3CN at 1.47 ppm. 31P NMR (202.3 MHz, D20 + DCI) 28.5 (s). ESI-HRMS for CHH16N4O5P [M-HBr-H]" calcd 315.0858; found 315.0855.
Example 19
Second route to [(3S)-4-hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-ci]pyrimidin-7- yl}methyl)amino]butyl]phosphonic acid hydrobromide (95), Scheme 19.
Figure imgf000079_0001
Scheme 19
In a modification of a known method (Mark, V; Van Wazer, J.R, J. Org. Chem. 1964, 29, 1006), sodium hydride (60% in oil, 2.039 g, 51.0 mmol) is added in one portion to a solution of di-tert-butyl phosphonate (96) (6.6 g, 34.0 mmol) in CH3CN (30 mL) at room temperature and the mixture stirred 30 min. lodomethane (4.25 mL, 68.0 mmol) is added in 0.5 mL portions controlling the resulting exotherm with an ice-bath so that the temperature did not go above 50 °C. After the temperature has dropped back to room temperature, H20 (1 mL) is added and the solvent evaporated. The residue is suspended in DCM and filtered through Celite then the solvent evaporated. The residue is flash chromatographed (EtOAc-hexanes, 4:6 then 6:4 v/v) to give di-terf-butyl methylphosphonate (97) (5.4 g, 76%) as a colourless oil. The 1H and 3 P NMR are in agreement with that reported in the literature except for the sign in the 31P N R which is +ve and opposite to that reported.
13C NMR (500 MHz, CDCI3) δ 81.4 (d, J = 89.6 Hz, C), 30.4 (d, J = 3.7 Hz, CH3), 17.0 (d, J = 148.5 Hz, CH3). Referenced to the centre line of CDCI3 at 77.0 ppm. ESI-HRMS for C9H21Na03P [M+Na]+ calcd 231.1126; found 231. 132. n-Butyllithium (1.5M, 17.05 mL, 25.6 mmol) is added dropwise to a solution of di- tert butyl methylphosphonate (97) (5.33 g, 25.6 mmol) in THF (25 mL) keeping the temperature below -70 °C throughout the addition. After 15 min, boron trifluoride diethyl etherate (3.35 mL, 26.4 mmol) is added dropwise to the dark coloured mixture and after 10 min (2R)-2-[(benzyloxy)methyl]oxirane (1.4 g, 8.53 mmol) [Lindberg, J; Svensson, S.C.T; Pahlsson, P; Konradsson, Tetrahedron, 2002, 58, 5109] in THF (0.5 mL) added dropwise. After 30 min, sat. aq. NaHC03 (5 mL) is added then triethylamine (23.97 mL, 171 mmol) and the mixture warmed to room temperature. The solvent is evaporated and the residue dissolved in Et20 (150 mL) and washed with sat. aq. NaHC03, dried and the solvent evaporated. The residue is flash chromatographed (CHCI3-MeOH, 99:1 v/v) to give di-terf-butyl [(3R)-4-(benzyloxy)-3-hydroxybutyl]phosphonate (98) as a yellow oil
(2.53 g, 80%). [ct] *0 + 6.3 (c, 0.82, CHCI3). 1H NMR (500 MHz, CDCI3) δ 7.36-7.27 (m, 5H), 4.55 (s, 2H), 3.85 (m, 1 H), 3.48 (dd, J = 9.5 Hz, 4.1 , 1 H), 3.40 (dd, J = 9.5, 7.0 Hz, 1 H), 3.07 (d, J = 3.0, exchanged to D20, 1 H), 1.86-1.67 (m, 4H), 1.49 (s, 18H). 3C NMR (125.7 MHz, CDCI3) 138.0 (C), 128.4 (CH), 127.7 (CH), 81.6 (d, J = 8.5 Hz, C), 74.1 (CH2), 73.3 (CH2), 70.4 (d, J = 13.7 Hz, CH), 30.4 (CH3), 27.3 (d, J = 5.1 Hz, CH2), 26.5 (d, J = 146.5 Hz, CH2). Referenced to the centre line of CDCI3 at δ 77.0. 31P NMR (202.4 MHz, CDCI3) 24.4 (s). ESI-HRMS for Ci9H33Na05P [M+Na]+ calcd 395.1963, found 395.1969.
A mixture of diisopropyl azodicarboxylate (DIAD, 1.78 mL, 9.13 mmol) and diphenylphosphoryl azide (DPPA ,1.97 mL, 9.13 mmol) in dry toluene (10 mL) is added dropwise to a solution of di-terf-butyl [(3R)-4-(benzyloxy)-3-hydroxybutyl]phosphonate (98) (2 g, 5.37 mmol) and triphenyl phosphine (2.39 g, 9.13 mmol) in toluene (20 mL) at 0 °C. After 10 mins, the mixture is allowed to warm to room temperature and then stirred overnight, filtered through Celite and the solvent evaporated. The residue is flash chromatographed (EtOAc-hexanes, 4:6 v/v) to give the crude azide as a yellow oil (1.6 g) which is dissolved in dry Et20 (30 mL), cooled in an ice-bath and LiAIH4 (1 M in THF, 8.06 mL, 8.06 mmol) added dropwise. The mixture is warmed to room temperature and stirred for 30 min then cooled in an ice-bath and H20 (0.3 mL), 15% aq. NaOH (0.3 mL) then H20 (0.9 mL) added successively. After diluting with EtOAc, the mixture is filtered through Celite and the solvent evaporated. The residue is flash chromatographed (CHCI3-7M NH3 in MeOH, 98:2 then 96:4 v/v) to give di-terf-butyl [(3S)-3-amino-4-
(benzyloxy)butyl]phosphonate (99) as a colourless oil (0.688 g, 35%). [a]^0 + 1.2 (c,
1.09, CHCI3). 1H NMR δ (500 MHz, CDCI3) 7.36-7.26 (m, 5H), 4.54 (d, J = 12.1 Hz, 1 H), 4.51 (d, J = 12.1 Hz, 1 H), 3.45 (dd, J = 9.1 , 4.0 Hz, 1 H), 3.27 (dd, J = 9.1 , 7.3 Hz, 1 H), 3.01 (m, 1 H), 1.8-1.49 (m, 6H, after D20 exchange, became 4H), 1.49 (d, J = 1.5 Hz, 18H). 13C NMR (125.7 MHz, CDCI3) δ 138.2 (C), 128.3 (CH), 127.6 (CH), 81.4 (d, J = 8.6, C), 75.3 (CH2), 73.2 (CH2), 51.4 (d, J = 16.2 Hz, CH), 30.4 (d, J = 3.5 Hz, CH3), 28.1 (d, J = 5.5 Hz, CH2), 26.9 (d, J = 146.5 Hz, CH2). Referenced to the centre line of CDCI3 at δ 77.0. 31P NMR (202.4 MHz, CDCI3) 23.7 (s). ESI-HRMS for C19H35N04P [M+H]+ calcd 372.2304; found 372.2311.
The % d.e. of compound (99) is determined in two ways, as follows. The (S, R) Mosher amide (Ward, D.E; Rhee, C.K, Tetrahedron Lett, 1991 , 32, 7165 for general method) is prepared by dissolution of di-tert-butyl [(3S)-3-amino-4-(benzyloxy)butyl]phosphonate (99) (6.3 mg, 0.017 mmol) in a mixture of CDCI3 and Et3N (3 eq.) and a solution of (S)- MTPACI (1.2 eq.) in CDCI3 (prepared from (R)-MTPA, 99% e.e.) is added to give a total volume of 0.6 ml_. The mixture is spiked with CFCI3 and left for 30 min. 19F NMR (470.5 MHz, CDCI3, ref. CFCI3 δ 0): δ -69.3 (s, 2%), -69.4 (s, 98%). The % d.e. is 96%. The (S, S) Mosher amide. (Ward, D.E; Rhee, C.K, Tetrahedron. Lett, 1991, 32, 7165 for general method) is prepared by dissolution of di-tert-butyl [(3S)-3-amino-4- (benzyloxy)butyl]phosphonate (99) (5.0 mg, 0.019 mmol) is dissolved in a mixture of CDCI3 and Et3N (3.6 eq.) and a solution of (ft)-MTPACI (1.2 eq.) in CDCI3 (prepared from (S)-MTPA, 99% e.e.) added to give a total volume of 0.6 mL The mixture is spiked with CFCI3 and left for 30 min. 19F NMR (470.5 MHz, CDCI3, ref. CFCI3 δ 0): δ -69.3 (s, 97%), -69.4 (s, 3%). The % d.e. is 94%. The average % d.e. from these two assessments is 95%.
Di-terf-butyl [(3S)-3-amino-4-(benzyloxy)butyl]phosphonate (99) (0.15 g, 0.404 mmol), 2- picoline borane complex (0.043 g, 0.404 mmol), 4-benzyloxy-5H-pyrrolo[3,2- d]pyrimidine-7-carbaldehyde (4) (0.087 g, 0.311 mmol) and Et3N (0.043 mL, 0.311 mmol) are stirred in MeOH (15 mL) at 40 °C overnight to give a clear colourless solution. The solvent is evaporated and the residue is chromatographed (0 → 10% continuous gradient of 0.5% v/v Et3N/MeOH in EtOAc) to give di-tert-butyl [(3S)-4-(benzyloxy)-3-({[4- (benzyloxy)-5 -/-pyrrolo[3,2-cf]pyrimidin-7-yl]methyl}amino)butyl]phosphonate (100) as a colourless gum (0.135 g, 71%). [a] 0 -1.0 (c, 0.6, CHCI3). 1H NMR (500 MHz, CDCI3): δ
9.13 (bs, exchanged D20, 1 H), 8.53 (s, 1 H), 7.48-7.46 (m, 2H), 7.40-7.23 (m, 9H), 5.57 (s, 2H), 4.51 (d, J = 11.9, 1 H), 4.48 (d, J =11.9, 1 H), 4.05 (s, 2H), 3.53 (dd, J = 9.5, 4.6, 1 H), 3.46 (dd, J = 9.5, 6.0, 1 H), 2.94-2.89 (m, 1 H), 2.21 (bs, exchanged D20, 1 H), 1.84- 1.76 (m, 2H), 1.72-1.63 (m, 2H), 1.443, 1.442 (2s, 18H). 13C NMR (125.7 MHz, CDCI3) 155.3 (C), 149.6 (CH), 149.2 (C), 138.3 (C), 136.4 (C), 128.6 (CH), 128.4 (CH), 128.3 (CH), 127.6 (CH), 127.5 (CH), 127.1 (CH), 115.4 (C) 115.3 (C), 81.5 (d, J = 3.0, C), 81.4 (d, J = 2.6, C), 73.2 (CH2), 71.7 (CH2), 67.7 (CH2), 56.8 (d, J = 16.5, CH), 40.9 (CH2), 30.40 (CH3), 30.37 (CH3), 26.4 (d, J = 146.2, 2H), 25.2 (CH2). Referenced to the centre line of CDCI3 at δ 77.0. 31 P NMR (202.4 MHz, CDCI3) δ 24.1 (s). ESI-HRMS for C33H46N405P [M+H]+ calcd 609.3206; found 609.3203.
Di-terf-butyl [(3S)-4-(benzyloxy)-3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7- yl]methyl}amino)butyl]phosphonate (100) (0.120 g, 0.197 mmol) is heated at 80 °C in 48% aq. HBr (3 mL) for 16 h. After cooling, the solution is washed with CHCI3 (2x) then the aqueous phase evaporated and the residue chromatographed on RP 18 silica gel (H20) to give [(3S)-4-hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]butyl]phosphonic acid hydrobromide (95) as a colourless glass, [a]
+9.8 (C2.03, H20). ESI-HRMS for C11H18N405P [M+H]+ calcd 317.1015; found 317.1013. The 1H, 3C and 31P NMR of compound (95) made by this route are the same as those reported for compound (95) prepared in Example 18.
Example 20
[(3A?)-4-Hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-d|pyrimiclin-7- yl}methyl)amino]butyl]phosphonic acid hydrobromide (105), Scheme 20
Figure imgf000082_0001
Scheme 20 In the same way as that described for the synthesis of enantiomer (92) (Scheme 18), compound (101) (F.W. Foss, Jr., A.H. Snyder, M.D. Davis, M. Rouse, M.D. Okusa, K.R. Lynch and T.L. Macdonald, Bioorg. Med. Chem. Lett, 2007, 15, 663) (0.35 g, 0.96 mmol) is treated with 10% Pd/C (50 mg) in EtOH (10 mL) under a hydrogen atmosphere at ambient pressure and temperature for 6 h, then with a mixture of EtOH (3 mL) and 37% aq. HCI (2 mL) to give diethyl [(3R)-3-amino-4-hydroxybutyl]phosphonate hydrochloride
(102) (0.25 g, 99%). [a] 22 -5.1 (c 0.63, MeOH). ESI-HRMS for C8H21N04P [M-HCI+H]+ calcd 226.1208; found 226.1205. The 1H, 13C and 31P NMR spectra are identical to those of the enantiomer (92). In the same way as that described for the synthesis of enantiomer (93) (Scheme 18), compound (102) (0.083 g, 0.317 mmol) is treated with triethylamine (0.030 mL, 0.211 mmol), 4-(te t-butoxy)-2-chloro-5/- -pyrrolo[3,2-d]pyrimidine-7-carbaldehyde (88) (0.054 g, 0.21 mmol) and 2-picoline borane complex (0.029 g, 0.275 mmol) to give after treatment with a 1 :1 mixture of 37% aq. HCI-MeOH, diethyl [(3R)-3-[({2-chloro-4-hydroxy- 5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-4-hydroxybutyl]phosphonate (103) (0.039 g, 45%). [a]22 -10.6 (c 0.455, MeOH). ESI-HRMS for C15H25 (35)CIN405P [M+H]+ calcd
407.1251 ; found 407.1250. The 1H, 13C and 31P NMR spectra are identical to those of the enantiomer (93).
In the same way as that described for the synthesis of enantiomer (94) (Scheme 18), compound (103) (0.065 g, 0.160 mmol) is stirred under a hydrogen atmosphere in the presence of 10% Pd/C (40 mg) to give [(3f?)-4-hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2- d]pyrimidin-7-yl}methyl)amino]butyl]phosphonate hydrochloride (104) (0.063 g, 96%).
[a]25 -12.4 (c 0.37, MeOH). ESI-HRMS for C15H26N405P [M-HCI+H]+ calcd 373.1641 ; found 373.1647. The H, 13C and 31P NMR spectra are identical to those of the enantiomer (94).
In the same way as that described for the synthesis of enantiomer (95) (Scheme 18), compound (104) (0.058 g, 0.142 mmol) is treated with 48% aq. hydrobromic acid (1.12 mL. 14.2 mmol) to give [(3R)-4-hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]butyl]phosphonic acid hydrobromide (105) (0.04 g, 71%). [a] 2 -9.9 (c 0.365, H20). ESI-HRMS for CnH16N405P [M-HBr-H]" calcd 315.0858; found 315.0857. The 1H, 13C and 31P NMR spectra are identical to those of the enantiomer (95). Example 21
[(2 ?)-2-[({2-Amino-4-hydroxy-5H-pyrrolo[3,2-c lpyrimidin-7-yl}methyl)am
hydroxypropoxy]phosphonic acid hydrochloride (111), Scheme 21.
Figure imgf000084_0001
Scheme 21
Dibenzyl diisopropylphosphoramidite (1.067 mL, 3.24 mmol) is added to te/ -butyl (4S)-4- (hydroxymethyl)-2,2-dimethyl-1 ,3-oxazolidine-3-carboxylate (0.5 g, 2.162 mmol) (106) [prepared from D-serine as described for the enantiomer in Organic Syntheses, Coll. Vol. 10, p. 320 (2004) and Vol. 77, p. 64 (2000)] and 1H-tetrazole (0.454 g, 6.49 mmol) in CH3CN (10 mL) and the mixture stirred for 1 h. The solvent is evaporated and the residue flash chromatographed (EtOAc-hexanes, 1 :9 v/v) to give tert-butyl (4R)-4- ({[bis(benzyloxy)phosphanyl]oxy}methyl)-2,2-dimethyl-1 ,3-oxazolidine-3-carboxylate as a colourless oil. This is dissolved in CH2CI2 (10 mL) cooled in ice-water and MCPBA (m- chloroperoxybenzoic acid) (0.995 g, 4.32 mmol) added and stirred for 30 min. The mixture is diluted with CH2CI2 (50 mL) and washed with sat. aq. Na2S03, sat. aq. NaHC03 (3x) then brine, dried and the solvent evaporated. The residue is flash chromatographed (DCM-hexanes-EtOAc, 4:3:1 v/v/v) to give terf-butyl (4R)-4- ({[bis(benzyloxy)phosphoryl]oxy}methyl)-2,2-dimethyl-1 ,3-oxazolidine-3-carboxylate
(107) as a colourless oil (0.816 g, 77%). [a]^2 +21.5 (c 0.57, CHCI3). 1H NMR (500 MHz, CDCI3) 7.34 (s, 10H), 5.08-5.00 (m, 4H), 4.22-4.16 (m, 0.5H), 4.13-4.06 (m, 1 H), 3.97- 3.83 (m, 3H), 3.77 (q, J = 8.6, 0.5H), 1.52-1.41 (m, 15H). 13C NMR (125.7 MHz) 152.1 , 151.4 (C), 135.7 (C), 128.6 (CH), 127.9 (CH), 94.1 , 93.6 (C), 80.6, 80.3 (C), 69.4 (CH2j, 65.7, 65.3 (CH2), 64.8, 64.6 (CH2), 56.6, 56.5, 56.32, 56.26 (CH), 28.3 (CH3), 27.4, 24.3 (CH3), 26.6, 23.0 (CH3). Referenced to the centre line of CDCI3 at δ 77.0. 31 P NMR (202.3 MHz, CDCI3) -1.0 (s), -1.1 (s). ESI-HRMS for C25H34NNa07P [M+Na]+ calcd 514.1971 ; found 514.1968. The NMR spectra indicated (107) is a ~1 :1 mixture of conformational isomers.
Compound (107) (0.77 g, 1.567 mmol) and 10% Pd-C (100 mg) are stirred in EtOH (15 mL) under a hydrogen atmosphere at ambient temperature and pressure for 16 h. The mixture is filtered through cellulose paper and the solvent is evaporated to give {[(4f?)-3- [(fert-butoxy)carbonyl]-2,2-dimethyl-1 ,3-oxazolidin-4-yl]methoxy}phosphonic acid as a colourless gum (480 mg). This is dissolved in 80% aq. TFA (10 mL) and left at room temperature for 2 h. The solvent is evaporated and the residue dissolved in H20 (10 mL) and washed with CH2CI2 (2 x10 mL) then evaporated. The residue is dissolved in H20 and chromatographed on RP 18 silica gel (H20) to give [(2f?)-2-amino-3- hydroxypropoxyjphosphonic acid (108) as a colourless gum which solidified (0.26 g,
97%). [a] 22 0 (c 0.565, H20). No measurable rotation observed. 1H NMR (500 MHz, D20) 4.19-4.12 (m, 1H), 4.11-4.03 (m, 1 H), 3.90 (dd, J = 12.3, 4.7, 1H), 3.81 (dd, J = 12.3, 6.7, 1 H), 3.63 (m, 1 H). Referenced to HOD at δ 4.79. 13C NMR (125.7 MHz, D20) 62.9 (d, J = 3.2, CH2), 59.1 (CH2), 53.5 (d, J = 7.4, CH). Referenced to internal CH3CN at δ 1.47. 31P NMR (202.3 MHz, D20) 0.0 (s). ESI-HRMS for C3H9N05P [M-H]" calcd 170.0218; found 170.0211. The 9F NMR spectrum showed a very weak resonance at δ -75.5 and fluorine micro analysis indicated there to be < 0.7% F present. A mixture of (108) (0.15 g, 0.877 mmol), triethylamine (0.123 mL, 0.877 mmol), {2-[(£)- [(dimethylamino)methylidene]amino]-7-formyl-4-oxo-3/- ,4 V,5 - -pyrrolo[3,2-d]pyrimidin-3- yljmethyl 2,2-dimethylpropanoate (109) (T. Semeraro, A. Lossani, M. Botta, C. Ghiron, R. Alvarez, F. Manetti, C. Mugnaini, S. Valensin, F. Focher and F Corelli, J. Med. Chem. 2006, 49, 6037) (0.122 g, 0.351 mmol) and 2-picoline borane complex (0.049 g, 0.456 mmol) are stirred together in MeOH (15 mL) at 50 °C for 17 h then the solvent is evaporated and the residue flash chromatographed (3% aq. Et3N in 1 ,4-dioxane-H20, 8:2 v/v) to give [(2f?)-2-[({2-[(£)-[(dimethylamino)methylidene]amino]-3-{[(2,2- dimethylpropanoyl)oxy]methyl}-4-oxo-3H,4H5H-pyrrolo[3,2-c pyrimidin-7- yl}methyl)amino]-3-hydroxypropoxy]phosphonic acid; triethylamine (110) as a yellow solid (0.144 g, 68.0%). [a]^2 -15.1 (c 0.51 , MeOH). 1H NMR (500 MHz, CD3OD, 4 mg in 0.6 mL) 8.70 (s, 1 H), 7.47 (s, 1 H), 6.31 (s, 2H), 4.38 (d, J = 13.6, 1 H), 4.32 (d. J = 13.6, 1 H), 4.25 (ddd, J = 12.2, 8.6, 3.2, 1 H), 4.03 (ddd, J = 12.2, 10.1 , 5.1 , 1 H), 3.89 (dd, J = 12.0, 5.1 , 1 H), 3.84 (dd, J = 12.0, 6.7, 1 H), 3.34 (m, 1 H), 3.22 (s, 3H), 3.08 (s, 3H), 3.02 (q, J = 7.3, 5.4H), 1.24 (t, J = 7.3, 8H), 1.15 (s, 9H). Approximately 0.9 eq. of Et3N present. 1H NMR (500 MHz, CD3OD, 25 mg in 0.6 mL) 8.69 (s, 1 H), 7.47 (s, 1 H), 6.27 (d, J =9.0, 1 H), 6.22 (d, J = 9.1 , 1H), 4.37 (d, J = 13.6, 1 H), 4.32 (d. J = 13.6, partly overlapped with ddd at 4.28, 1 H), 4.28 (ddd, J = 12.3, 9.0, 3.0, 1 H, partly overlapped with d at 4.32), 4.08 (ddd, J = 12.6, 10.6, 5.3, 1 H), 3.96-3.86 (m, 2H), 3.38 (m, 1 H), 3.23 (s, 3H), 3.09 (s, 3H), 3.05 (q, J =7.3, 5.4H), 1.25 (t, J = 7.3, 8H), 1.13 (s, 9H). 13C NMR (125.7 MHz, CD3OD, 25 mg in 0.6 mL) 179.0 (C), 159.0 (CH), 156.2 (C), 155.6 (C), 144.9 (C), 130.5 (CH), 115.1 (C), 108.4 (C), 67.1 (CH2), 62.1 (d, J = 4.7, CH2), 61.4 (d, J = 2.9, CH), 60.3 (CH2), 46.9 (CH2), 41.3 (CH3), 39.8 (C), 35.3 (CH3), 27.4 (CH3), 9.9 (CH3). Referenced to the centre line of CD3OD at δ 49.0. 31P NMR (202.3 MHz, CD3OD) 3.4 (s). ESI-HRMS for C19H30N6O8P [M-Et3N-H]" calcd 501.1863; found 501.1857. The 1H and 3C NMR spectra displayed significant concentration effects.
Compound (110) (0.138 g, 0.229 mmol) is heated at 100 °C in 6M aq. HCI (6 mL) for 1.5 h. The solvent is evaporated and the residue flash chromatographed (1 ,4-dioxane-H20- Et3N, 75:25:3 then 70:30:3 v/v/v) to give the triethylammonium form of the product as a light brown solid. This is dissolved in 5% HCI (10 mL), the solvent evaporated and the residue dissolved in hot H20 and loaded on to an Amberlyst A21 resin column. The column is eluted first with H20 then the product is eluted off with 1 M HCI. The residue after evaporation of the solvent is chromatographed on RP 18 silica gel (H20) to give [(2f?)-2-[({2-amino-4-hydroxy-5/- -pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3- hydroxypropoxy]phosphonic acid hydrochloride (111) as a colourless solid after freeze drying (56 mg, 66%). [a] +4.9 (c 0.45, 1.5M HCI). 1H NMR (500 MHz, D20 + drop of DCI) 7.62 (s, 1 H), 4.49 (d, J = 14.2, 1 H), 4.43 (d, J = 14.2. 1 H), 4.31 (ddd, J = 12.1 , 6.1 , 3.6, 1 H), 4.19 (ddd, J = 12.1 , 6.0, 6.0, 1 H), 3.94 (dd, J = 12.5, 4.9, 1 H), 3.88 (dd, J = 12.5, 6.3, 1 H), 3.65 (m, 1 H). Referenced to HOD at 4.79 ppm. 13C NMR (125.7 MHz, D20 + drop of DCI) 154.2 (C), 151.2 (C), 133.1 (C), 132.3 (CH), 112.4 (C), 101.5 (C), 61.7 (d, J = 3.6, CH2), 59.2 (d, J = 6.8, CH), 58.5 (CH2), 39.3 (CH2). Referenced to internal CH3CN at 1.47 ppm. 31P NMR (202.3 MHz, D20 + DCI) -0.2 (s). ESI-HRMS for C10H15N5O6P [M-HCI-H]- calcd 332.0760; found 332.0755. Found 10.1% CI, CioH 6N506P»HCI required 9.6% CI indicating the product is a mono hydrochloride salt. Example 22
[(2S)-2-[({2-Amino-4-hydroxy-5W^yrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3- hydroxypropoxy]phosphonic acid hydrochloride (114), Scheme 22.
Figure imgf000087_0001
[(2S)-2-Amino-3-hydroxypropoxy]phosphonic acid (112) (0.15 g, 0.877 mmol), prepared in the same way as described for the enantiomer (108) in Scheme 21 , from L-serine derived terf-butyl (4R)-4-(hydroxymethyl)-2,2-dimethyl-1 ,3-oxazolidine-3-carboxylate [Organic Syntheses, Coll. Vol. 10, p. 320 (2004) and Vol. 77, p. 64 (2000)] is treated with Et3N (0.123 mL, 0.877 mmol), {2-[(£)-[(dimethylamino)methylidene]amino]-7-formyl-4- oxo-3W,4H,5 - -pyrrolo[3,2-d]pyrimidin-3-yl}methyl 2,2-dimethylpropanoate (109) (T. Semeraro, A. Lossani, M. Botta, C. Ghiron, R. Alvarez, F. Manetti, C. Mugnaini, S. Valensin, F. Focher and F Corelli, J. Med. Chem. 2006, 49, 6037) (0.122 g, 0.351 mmol) and 2-picoline borane complex (0.049 g, 0.456 mmol) in the same way as that described for the preparation of the enantiomer (110) (Scheme 21 ) to give [(2S)-2-[({2-[(E)- [(dimethylamino)methylidene]amino]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-4-oxo- 3H,4/-/,5H-pyrrolo[3,2-cf]pyrimidin-7-yl}methyl)amino]-3-hydroxypropoxy]phosphonic acid; triethylamine (113) as a yellow amorphous solid (0.168 g, 79%). [a] 2,2 +14.7 (c, 0.51 ,
MeOH). ESI-HRMS for CieH3oNe08P [M-Et3N-H]" calcd 501.1863; found 501.1858. The 1H, 13C and 31P NMR spectra are identical to those of the enantiomer (110).
In the same way as that described for the preparation of the enantiomer (111) (Scheme 21 ), compound (113) (0.14 g, 0.232 mmol) is treated with 6M aq. HCI and the product purified on an Amberlyst A21 ion exchange column to give [(2S)-2-[({2-amino-4-hydroxy- 5/- -pyrrolo[3,2-c/]pyrimidin-7-yl}methyl)amino]-3-hydroxypropoxy]phosphonic acid hydrochloride (114) as a colourless amorphous solid (0.059 g, 69%). [a]22. -4.6 (c 0.42,
1.5M HCI). ESI-HRMS for C10H15N5O6P [M-HCI-H]" calcd 332.0760; found 332.0756. The 1H, 13C and 31P NMR spectra are identical to those of the enantiomer (111).
Example 23
[(1 RIS, 3S)-1 -Fluoro-4-hydroxy-3-[({4-hydroxy-5W-pyrrolo[3,2-cGpyrimidin-7- yl}methyl)amino]butyl]phosphonic acid; triethylamine (118) as an -1 :1 mixture of diastereomers, Scheme 23.
Figure imgf000088_0001
Scheme 23 terf-Butyl (4S)-4-[(E)-2-(diethoxyphosphoryl)-2-fluoroethenyl]-2,2-dimethyl-1 ,3- oxazolidine-3-carboxylate (115) (F.W. Foss, Jr., A.H. Snyder, M.D. Davis, M. Rouse, M.D. Okusa, K.R. Lynch and T.L. Macdonald, Bioorg. Med. Chem. Lett., 2007, 15, 663) (0.222 g, 0.582 mmol) and 10% Pd/C (50 mg) are stirred in EtOH (10 mL) under a hydrogen atmosphere at ambient temperature and pressure for 5.5 h. The mixture is filtered through Celite and the solvent evaporated to a colourless gum (225 mg) which is dissolved in a mixture of EtOH (3 mL) and 37% aq. HCI (1 mL) and left at room temperature for 1.5 h. The solvent is evaporated to give diethyl [(1ft/S, 3S)-3-amino-1- fluoro-4-hydroxybutyl]phosphonate hydrochloride (116) as an ~1 :1 mixture of diastereomers as a dark yellow gum. 1H NMR (500 MHz, D20), 5.38 (m, 0.5H), 5.28 (m, 0.5H), 4.38-4.30 (m, 4H), 3.96 (t, J = 3.4, 0.5H), 3.94 (t, J = 3.4, 0.5H), 3.81-3.70 (m, 2H), 2.46-2.24 (m, 2H), 1.43 (t, J = 7.1 , 6H). Referenced to HOD at δ 4.79. 13C NMR (125.7 MHz, D20) 86.9 (dd, J = 175.6, 173.7, CHF), 85.9 (dd, J = 177.2, 173.3, CHF), 66.0 (d, J = 7.0, CH2), 65.8 (d, J = 6.7, CH2), 61.6 (CH2), 61.0 (CH2), 51.3 (d, J = 16.0, CH), 50.6 (d, J = 14.7 CH), 29.9 (d, J = 19.8, CH2), 29.8 (d, J = 19.2, CH2), 16.3 (d, J = 4.9, CH3). Referenced to internal CH3CN at δ 1.47. 31P NMR (202. MHz, D20), 18.0 (d, J = 76.0), 17.8 (d, J = 76.0). 19F NMR (470.5 MHz, D20) -209.7 (d, J = 76.0). ESI-HRMS for C8H20FNO4P [M-HCI+H]+ calcd 244.1114; found 244. 111. The NMR spectra indicated (116) is a ~1 :1 mixture of diastereomers.
Compound (116) (0.157 g, 0.561 mmol), 4-benzyloxy-5/-/-pyrrolo[3,2-d]pyrimidine-7- carbaldehyde (4) (0.113 g, 0.401 mmol), Et3N (0.034 mL, 0.241 mmol) and 2-picoline borane complex (0.056 g, 0.521 mmol) are stirred together in MeOH (15 mL) at 50 °C for 24 h. The solvent is evaporated and the residue flash chromatographed (CHCI3-7M NH3 in MeOH, 99:1 then 99:2 v/v) to give diethyl [(1ft/S, 3S)-3-({[4-(benzyloxy)-5H- pyrrolo[3,2-cdpyrimidin-7-yl]methyl}amino)-1 -fluoro-4-hydroxybutyl]phosphonate (117) as an -1 :1 mixture of diastereomers as a colourless gum. 1H NMR (500 MHz, CD3OD) 8.42 (s, 1 H), 7.52-7.49 (m, 3H), 7.39-7.29 (m, 3H), 5.61 (s, 2H), 5.13 (ddt, J = 46.4, 4.8, 2.4, 0.5H), 5.07 (ddt, J = 46.7, 6.9, 3.4, 0.5H), 4.23-4.12 (m, 4H), 4.08 (d, J = 13.5, 0.5H), 4.01 (s, 1 H), 3.98 (d, J = 13.5, 0.5H), 3.72 (dt, J = 11.5, 4.7, 1 H), 3.56 (dt, J = 10.7, 5.5, 1 H), 2.97 (pent, J = 5.6, 0.5H), 2.91 (sext, J = 4.6, 0.5H), 2.14-1.81 (m, 2H), 1.36-1.30 (m, 6H). 3C NMR (125.7 MHz, CD3OD) 157.0 (C), 150.1 (CH), 150.0 (CH), 149.6 (C), 137.9 (C), 130.3 (CH), 129.5 (CH), 129.4 (CH), 129.2 (CH), 116.7 (C), 115.2 (C), 114.8 (C), 87.8 (dd, J = 177.5, 171.9 CHF), 87.5 (dd, J = 177.5, 171.9 CHF), 69.0 (CH2), 64.8 (t, J = 8.4, CH2), 64.5 (t, J = 7.5, CH2), 64.1 (CH2), 63.7 (CH2), 56.4 (d, J = 13.6, CH), 55.7 (d, J = 13.9, CH), 41.3 (CH2), 41.2 (CH2), 33.8 (d, J = 20.1 , CH2), 32.7 (d, J = 19.7, CH2), 16.7 (CH3). Referenced to the centre line of CD3OD at δ 49.0. 31P NMR (202.3 MHz, CD3OD) 19.0 (d, J = 77.6), 18.5 (d, J = 77.6). 19F NMR (470.5 MHz, CD3OD) - 209.9 (d, J = 77.7), -210.7 (d, J = 77.4). ESI-HRMS for C22H31FN405P [M+H]+ calcd 481.2016; found 481.2014. The NMR spectra indicated (117) is a -1 :1 mixture of diastereomers.
Compound (117) (0.1 g, 0.208 mmol) and 10% Pd/C (30 mg) are stirred in EtOH (6 mL) under a hydrogen atmosphere at ambient temperature and pressure for 3 h. The mixture is filtered and the solvent evaporated. The residue is dissolved hydrogen bromide (48% aq. 1.639 mL, 20.81 mmol) and heated to 80 °C for 6 h. The solvent is evaporated several times from H20 then the residue dissolved in hot water and chromatographed on RP 18 silica gel (H20) to give the sparingly soluble product hydrobromide salt. It is dissolved in 5% aq. Et3N and the solvent evaporated then the residue flash chromatographed (1 ,4-dioxane-H20-Et3N, 80:20:3 then 70:30:3 v/v/v) to give [(1R/S, 3S)- 1 -fluoro-4-hydroxy-3-[({4-hydroxy-5 - -pyrrolo[3,2-c/]pyrimidin-7- yl}methyl)amino]butyl]phosphonic acid; triethylamine (118) as an ~1 :1 mixture of diastereomers as a colourless amorphous solid. 1H NMR (500 MHz, D20) 7.97 (s, 1 H), 7.69 (s, 1 H), 4.79 (HOD + 0.5H), 4.69 (bs, 0.5H), 4.46 (dd, J = 13.8, 1.9, 1 H), 4.36 (dd, J = 13.8, 2.7, 1 H), 4.08 (dd, J = 12.8, 3.6, 1 H), 3.91 (dt, J = 12.8, 5.0, 1 H), 3.66-3.58 (m, 1 H), 3.22 (q, J = 7.4, 6H), 2.44-2.24 (m, 2H), 1.30 (t, J = 7.4, 9H). Referenced to HOD at 4.79 ppm. Approximately 1 eq. of Et3N present. 13C NMR (125.7 MHz, D20) 155.5 (C), 143.8 (C), 143.3 (CH), 131.3 (CH), 131.2 (CH), 118.0 (C), 107.0 (C), 106.9 (C), 89.7 (bm, CHF), 60.4 (CH2), 59.5 (CH2), 57.6 (CH), 56.1 (CH), 47.3 (CH2), 38.7 (CH2), 38.5 (CH2), 31.1 (d, J = 20.9, CH2), 30.6 (d, J = 19.7, CH2), 8.9 (CH3). Referenced to internal CH3CN at δ 1.47. 31 P NMR (202.3 MHz, CD3OD + drop Et3N) 11.0 (d, J = 61.6), 10.9 (d, J = 63.1). 19F NMR (470.5 MHz, D20) -197.9 (d, J = 63.4), -203.9 (d, J = 62.1). ESI- HRMS for C11H15FN405P [M-Et3N-H] calcd. 333.0764; found 333.0754. The NMR spectra indicated compound (118) is an ~1 :1 mixture of diastereomers. Example 24
[(3S)-1 ,1-Difluoro-4-hydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]butyl]phosphonic acid; triethylamine (122), Scheme 24.
Figure imgf000090_0001
(121) (122)
Scheme 24 terf-Butyl (4S)-4-[2-(diethoxyphosphoryl)-2,2-difluoroethyl]-2,2-dimethyl-1 ,3-oxazolidine- 3-carboxylate (119) (A. Otaka, K. Miyoshi, T.R.Burke, Jr.; P.P. Roller, H. Kubota, H. Tamamura, N. Fujii, Tetrahedron Lett. 1995, 36, 927; D.B. Berkowitz, M.-J. Eggen, Q. Shen, R.K. Shoemaker, J. Org. Chem. 1996, 61, 4666) (0.345 g, 0.860 mmol) is dissolved in a mixture of EtOH (3 mL) and 37% aq. HCI (1 mL) and left to stand at room temperature for 1.5 h. The solvent is evaporated to give diethyl [(3S)-3-amino-1 ,1- difluoro-4-hydroxybutyl]phosphonate hydrochloride (120) as a yellow gum (0.256 g, 100%). 1H NMR (500 MHz, CDCI3) 8.24 (bs, 3H), 4.60 (bs, 1 H), 4.35-4.28 (m, 4H), 4.02 (d, J = 9.1 , 1 H), 3.92-3.81 (m, 2H), 2.91-2.74 (m, 1 H), 2.63-2.47 (m, 1 H), 1.39 (dt, J = 7.0, 1.0, 6H). 13C NMR (125.7 MHz, CDCI3) 119.3 (dt, J = 261.3, 216.5, CF2), 65.5 (d, J = 4.5, CH2), 61.5 (CH2), 48.2 (CH), 33.5 (m, CH2), 16.3 (d, J = 5.0, CH3). Referenced to the centre line of CDCI3 at δ 77.0. 3 P NMR (202.3 MHz, CDCI3), 5.4 (t, J = 104.9). 19F NMR (470.5 MHz, CDCI3) -107.7 (dd, J = 301.1 , 102.5), -111.2 (dd, J = 301.3, 105.9). ESI- HRMS for C8H19F2N04P [M-HCI+H]+ calcd 262.1020; found 262.1017.
Compound (120) (0.140 g, 0.470 mmol), 4-benzyloxy-5H-pyrrolo[3,2-d]pyrimidine-7- carbaldehyde (4) (0.095 g, 0.336 mmol), Et3N (0.042 mL, 0.302 mmol) and 2-picoline borane complex (0.047 g, 0.437 mmol) are stirred in MeOH (15 mL) at 50 °C for 24 h. The solvent is evaporated and the residue flash chromatographed (CHCI3-7M NH3 in MeOH, 99:1 then 98:2 v/v) to give diethyl [(3S)-3-({[4-(benzyloxy)-5H-pyrrolo[3,2- d]pyrimidin-7-yl]methyl}amino)-1 ,1-difluoro-4-hydroxybutyl]phosphonate (121) as a colourless gum (0.088 g, 53%). [a]^2 +7.5 (c 0.475, MeOH). 1H NMR (500 MHz, CD3OD)
8.42 (s, 1 H), 7.53 (s, 1 H), 7.51 (d, J = 7.3, 2H), 7.38-7.30 (m, 3H), 5.60 (s, 2H), 4.29-4.22 (m, 4H), 4.04 (d, J = 13.6, 1 H), 3.99 (d, J = 13.6, 1 H), 3.73 (dd, J = 11.3, 4.3 1 H), 3.53 (dd, J = 11.3, 6.2, 1 H), 3.18 (m, 1 H), 2.34-2.24 (m, 2H), 1.34 (t, J = 7.0, 6H). 3C NMR (125.7 MHz, CD3OD) 157.0 (C), 150.1 (CH), 149.5 (C), 137.8 (C), 130.4 (CH), 129.5 (CH), 129.4 (CH), 129.2 (CH), 122.0 (dt, J = 259.1 , 218.5, CF2), 116.7 (C), 114.5 (C), 69.0 (CH2), 66.2 (d, J = 6.7, CH2), 64.2 (CH2), 53.6 (CH), 41.1 (CH2), 36.3 (dt, J = 19.8, 14.3, CH2), 16.7 (CH3). Referenced to the centre line of CD3OD at δ 49.0. 31P NMR (202.4 MHz, CD3OD) 6.7 (t, J = 109.9). 19F NMR (470.5 MHz, CD3OD) -110.3 (dd, J = 299.3, 109.9, -111.6 (dd, J = 299.3, 109.9). ESI-HRMS C22H3oF2N405P [M+H]+ calcd 499.1922; found 499.1927.
Compound (121) (0.088 g, 0.177 mmol) and 10% Pd/C (30 mg) are stirred in EtOH (5 mL) under a hydrogen atmosphere at ambient temperature and pressure for 3 h. The mixture is filtered through Celite and the solvent evaporated. The residue is heated at 80 °C in hydrobromic acid (48% aq., 1.39 mL, 17.65 mmol) for 10 h. The solvent is evaporated and the residue dissolved in 5% aq. Et3N, evaporated, dissolved in H20 and loaded on to a column of Dowex 50WX8 (H+) resin (4 cm x 1 cm) and eluted with water (50 mL, discarded) then 5% aq. Et3N (250 mL). After leaving the column standing in 5% aq. Et3N overnight it is eluted with more 5% aq. Et3N (100 mL). The combined basic fractions are evaporated and the residue flash chromatographed (1 ,4-dioxane-H20-Et3N, 85:15:3 then 80:20:3 v/v/v)to give [(3S)-1 ,1-Difluoro-4-hydroxy-3-[({4-hydroxy-5H- pyrrolo[3,2- /]pyrimidin-7-yl}methyl)amino]butyl]phosphonic acid; triethylamine (122) as a cream coloured solid (0.058 g, 73%). [a]22 +6.7 (c 0.55, 3% aq. Et3N). 1H NMR (500
MHz, D20) 7.94 (s, 1 H), 7.68 (s, 1 H), 4.46 (d, J = 13.8, 1 H), 4.32 (d, J = 13.8, 1 H), 4.10 (dd, J = 12.8, 3.8, 1 H), 3.91 (dd, J = 12.8, 5.0, 1H), 3.72 (m, 1 H), 3.22 (q, J = 7.3, 6.5H), 2.62 (m, 1 H), 2.43 (m, 1 H), 1.3 (t, J = 7.3, 9.75H). Referenced to HOD at δ 4.79. Approximately 1.1 eq. of Et3N present. 13C NMR (125.7 MHz, D20) 155.4 (C), 143.6 (C), 143.2 (CH), 131.3 (CH), 123.0 (dt, J = 260.5, 179.1 , CF2), 117.9 (C), 106.9 (C), 60.4 (CH2), 54.6 (CH), 47.3 (CH2), 38.4 (CH2), 34.9 (dt, J = 23.1 , 13.8, CH2), 8.9 (CH3). Referenced to internal CH3CN at δ 1.47. 31 P NMR ( 202.4 MHz, D20) 4.6 (t, J = 83.1). 19F NMR (470.5 MHz, D20) -104.1 (dd, J = 286.6, 83.5, -110.6 (dd, J = 286.4, 82.8). ESI- HRMS for CiiHi4F2N405P [M-Et3N-H]-calcd 351.0670; found 351.0671. Example 25
[(3S)-3-[({2-Amino-4-hydroxy-5H^yrrolo[3,2-c0pyrimidin-7-yl}methyl)amino]-1,1- difluoro-4-hydroxybutyl]phosphonic acid; triethylamine (124), Scheme 25.
Figure imgf000092_0001
Scheme 25 [(3S)-3-Amino-1 ,1-difluoro-4-hydroxybutyl]phosphonate hydrochloride (120) (Scheme 24) (0.135 g, 0.454 mmol), {2-[(£)-[(dimethylamino)methylidene]amino]-7-formyl-4-oxo- 3H,4H,5H-pyrrolo[3,2-oilpyrimidin-3-yl}methyl 2,2-dimethylpropanoate (109) (T. Semeraro, A. Lossani, M. Botta, C. Ghiron, R. Alvarez, F. Manetti, C. Mugnaini, S. Valensin, F. Focher and F Corelli, J. Med. Chem., 2006, 49, 6037) (0.1 13 g, 0.324 mmol), Et3N (0.041 ml_, 0.292 mmol) and 2-picoline borane complex (0.045 g, 0.421 mmol) are stirred in MeOH (5 mL) at 50 °C for 1 h then at room temperature for 16 h. The solvent is evaporated and the residue flash chromatographed (CHCI3-7M NH3 in MeOH, 99:1 then 98:2 v/v) to give (123) as a yellow amorphous solid together with a mixture of (123) + (109). The latter is chromatographed twice more to give a total amount of [7- ({[(2S)-4-(diethoxyphosphoryl)-4,4-difluoro-1-hydroxybutan-2-yl]amino}methyl)-2-[(£)-
[(dimethylamino)methylidene]amino]-4-oxo-3H,4H,5H-pyrrolo[3,2-c]pyrimidin-3-yl]methyl 2,2-dimethylpropanoate (123), 0.109 g, 57%. [a]2 +5.4 (c 1.44, MeOH). 1H NMR (500 MHz, CD3OD) 8.58 (s, 1 H), 7.25 (s, 1 H), 6.31 (s, 2H), 4.31-4.24 (m, 4H), 3.90 (d, J = 13.2, 1 H), 3.83 (d, J = 13.2, 1 H), 3.73 (dd, J = 11.2, 4.6, 1 H), 3.52 (dd, J = 11.2, 6.7, 1 H), 3.22 (m, 1 H), 3.18 (s, 3H), 3.06 (s, 3H), 2.37-2.19 (m, 2H), 1.37 (t, J = 7.0, 6H), 1.15 (s, 9H). 13C NMR (125.7 MHz, CD3OD) 179.0 (C), 158.7 (CH), 156.5 (C), 155.3 (C), 144.9 (C), 128.6 (CH), 121.1 (dt, J = 259.0, 218.3, CF2), 115.3 (C), 114.7 (C), 67.0 (CH2), 66.3 (d, J = 6.9, CH2), 64.6 (CH2), 53.8 (CH), 41.7 (CH2), 41.1 (CH3), 39.8 (C), 36.2 (dt, J = 20.0, 14.1 , CH2), 35.2 (CH3), 27.4 (CH3), 16.7 (d, J = 5.0, CH3). Referenced to the centre line of CD3OD at 49.0 ppm. 31P NMR (202.4 MHz, CD3OD) 6.6 (t, J = 109.6). 19F NMR (470.5 MHz, CD3OD) -110.3 (dd, J = 298.9, 109.4), -111.4 (dd, J = 298.9. 109.8). ESI- HRMS for C24H4oF2N607P [M+H]+ calcd 593.2664; found 593.2667.
Compound (123) (0.1 g, 0.169 mmol) is heated at 80 °C in 48% aq. hydrobromic acid for 16 h then evaporated to the sparingly soluble hydrobromide salt form of (124). It is dissolved in 5% aq. Et3N, evaporated and the residue dissolved in H20 and loaded on to a column of Dowex 50WX8 (H+) resin (4 cm x 1 cm) and eluted with water (50 mL, discarded) then 5% aq. Et3N (250 mL). After leaving the column standing in 5% aq. Et3N overnight it is eluted with more 5% aq. Et3N (100 mL). The combined basic fractions are evaporated and the residue flash chromatographed (1 ,4-dioxane-H20-Et3N, 80:20:3 then 70:30:3 v/v/v) to give a yellow glassy solid which is dissolved in 3% aq. Et3N and chromatographed on RP 18 silica gel (3% aq. Et3N) to give [(3S)-3-[({2-amino-4-hydroxy- 5/- -pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-1 , 1 -difluoro-4-hydroxybutyl]phosphonic acid; triethylamine (124) as a cream coloured amorphous solid (0.051 g, 65%). [a]21 +7.7 (c 0.62, 3% aq. Et3N). 1H NMR (500 MHz, D20) 7.58 (s, 1 H), 4.43 (d, J = 13.7, 1 H), 4.32 (d, J = 13.7, 1 H), 4.08 (dd, J = 12.8, 4.0, 1 H), 3.92 (dd, J = 12.8, 6.0, 1 H), 3.75 (m, 1 H), 3.27 (q, J = 7.3, 7.3H), 2.65 (m, 1 H), 2.44 (m, 1 H), 1.35 (t, J = 7.3, 10.9H). Referenced to HOD at δ 4.79. Approximately 1.2 eq. of Et3N present. 13C NMR (125.7 MHz, D20) 158.0 (C), 152.6 (C), 141.8 (C), 130.6 (CH), 122.8 (dt, J = 260.6, 180.2, CF2), 112.9 (C), 103.1 (C), 60.4 (CH2), 54.6 (CH), 47.2 (CH2), 38.3 (CH2), 34.4 (dt, J = 23.9, 14.5, CH2), 8.8 (CH3). 31 P NMR (202.4 MHz, D20) 4.8 (t, J = 83.7). 19F NMR (470.5 MHz, D20) -103.8 (dd, J = 287.8, 84.2, -110.6 (dd, J = 287.6, 83.4). ESI-HRMS for CnH15F2N505P [M-Et3N- H]" calcd 366.0779; found 366.0785.
Example 26
[(2/?)-3-Hydroxy-2-[({4-hydroxy-5H-pyrrolo[3,2-t/Jpyrimidin-7- l}methyl)amino]propoxy]phosphonic acid; triethylamine (126), Scheme 26.
Figure imgf000094_0001
Et3N, MeOH
Scheme 26
[(2fi)-2-Amino-3-hydroxypropoxy]phosphonic acid (108) (25 mg, 0.146 mmol) is suspended in methanol (50 mL) and brought to pH7 with Et3N. 4-Benzyloxy-5 - - pyrrolo[3,2-c]pyrimidine-7-carbaldehyde (4) (22 mg, 0.088 mmol) and then picoline borane complex (20.3 mg, 0.19 mmol) are added and the suspension stirred at 50 °C for 60 h. The solution is evaporated onto silica gel. Flash chromatography (1 ,4-dioxane- H20-Et3N, 30:10:0.4 v/v/v then 20:10:0.3 v/v/v) gives [(2R)-3-hydroxy-2-[({4-hydroxy-5H- pyrrolo[3,2-c/]pyrimidin-7-yl}methyl)amino]propoxy]phosphonic acid; triethylamine (125) (18 mg, 40%). 1H NMR (500 MHz, CD3OD) 8.39 (s, 1 H), 7.75 (s, 1 H), 7.43 (m, 2H), 7.28 (m, 3H), 5.55 (s, 2H), 4.37 (m, 2H), 4.09 (m, 1 H), 3.95 (m, 1 H), 3.76 (dd, J = 12.1 , 5.0 Hz, 1 H), 3.69 (dd, J = 12.1 , 6.2 Hz, 1 H), 3.23 (m, 1 H), 3.01 (q, J = 7.1 Hz, 7H), 1.18 (t, J = 7.1 Hz, 11 H). Approximately 1.1 eq. of Et3N present. 13C NMR (125.7 MHz, CD3OD, centre line 49.0 ppm) 157.3, 150.8, 149.6, 137.8, 133.1 , 129.7, 116.8, 108.2, 69.4, 62.7 (d, JC,P = 5 Hz), 60.7 (d, Jc,p = 5 Hz), 60.0, 47.6, 40.4, 9.4. 31 P NMR (202.4 MHz, CD3OD) δ 2.7 ESI-HRMS for C17H2oN406P [M-H]" calcd. 407.1120, found 407.1129.
A solution of compound (125) (14 mg, 0.028 mmol) in methanol (5 mL) and water (5 mL) is stirred with Pearlmann's catalyst (2 mg) under a balloon of hydrogen for 16 h. The solution is filtered and the solvent evaporated to give [(2R)-3-hydroxy-2-[({4-hydroxy-5 - - pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propoxy]phosphonic acid; triethylamine (126) (4 mg, 46%). 1 H NMR (500 MHz, D20-CD3OD, 2: 1 ) δ 7.91 (s, 1 H), 7.61 (s, 1 H), 4.39 (m, 2H), 4.08 (m, 1 H), 3.96 (m, 1 H), 3.81 (dd, J = 12.1 , 5.5 Hz, 1 H), 3.74 (dd, J = 12.1 , 6.1 , Hz, 1 H), 3.41 (m, 1 H), 3.1 1 (q, J = 7.1 Hz, 4H), 1.14 (t, J = 7.1 Hz, 1 1 H). Approximately 0.7 eq. of Et3N present. 13C NMR (125.7 MHz, D20-CD3OD, 2: 1 centre line of CD3OD 49.0 ppm) 6 155.0, 143.6, 142.8, 130.8, 1 17.7, 106.0, 60.9 (d, JC,P = 5 Hz), 58.3 (d, JC,P = 7 Hz), 57.6, 46.6, 38.7, 8.1 . 31 P NMR (202.4 MHz, D20-CD3OD, 2: 1 ) δ 1 .0. ESI-HRMS for C10H14N4O6P [M-H]- calcd. 317.0651 , found 317.0644.
Example 27 [(2S)-3-Hydroxy-2-[({4-hydroxy-5H-pyrrolo[3,2-dlpyrimidin-7- l}methyl)amino]propoxy]phosphonic acid; triethylamine (128), Scheme 27.
Figure imgf000095_0001
Et3N, MeOH
Scheme 27
Compound [(2S)-3-hydroxy-2-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- l}methyl)amino]propoxy]phosphonic acid; triethylamine (128) is prepared, in the same manner as that of the enantiomer [(2R)-3-hydroxy-2-[({4-hydroxy-5H-pyrrolo[3,2- cf]pyrimidin-7-l}methyl)amino]propoxy]phosphonic acid; triethylamine (126) in Scheme 26 from [(2S)-2-Amino-3-hydroxypropoxy]phosphonic acid (112) via intermediate (127)and has the same 1H, 13C and 31P NMR as the enantiomer (126). ESI-HRMS for Ci0H15N4NaO6P [M+Na]+ calcd. 341 .0627, found 341 .0626.
Example 28
[(2f?,3S)-3,4-Dihydroxy-2-[({4-hydroxy-5W-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]butoxy]phosphonic acid; ammonia (134), Scheme 28.
Figure imgf000096_0001
Scheme 28
A solution of (2R)-2-amino-2-[(4S)-2,2-dimethyl-1 ,3-dioxolan-4-yl]ethan-1-ol (129) [S. Sagawa, H. Abe, Y. Hase and T. Inaba, J. Org. Chem., 1999, 64, 4962 - 4965] (1.14g, 4.02 mmol) and di-tert-butyl dicarbonate (0.878g, 4.02 mmol) in methanol (20 mL) is stirred at room temperature whilst triethylamine (0.54 mL, 3.82 mmol) is added. After 16h the solution is partitioned between water and EtOAc. The organic phase is dried and concentrated under reduced pressure to give tert-butyl A/-[(1R)-1-[(4S)-2,2-dimethyl-1 ,3- dioxolan-4-yl]-2-hydroxyethyl]carbamate (130) (1.05g, 100%). A solution of compound (130) (0.525g, 2.01 mmol) and 1H-tetrazole (422 mg, 6.0 mmol) in acetonitrile (20 mL) is stirred at room temperature and o-xylylene-A/,/V-diethylphosphoramidite (498 μί, 2.3 mmol) and is added. After 30 min the solution is cooled in an ice-water bath and m- chloroperbenzoic acid (MCPBA) (585 mg, 2.6 mmol) is added. The solution is warmed to room temperature and stirred for 1 h, quenched with sodium thiosulphate solution and portioned between water and EtOAc. The organic phase is washed with 10% aqueous NaHC03 and saturated aqueous NaCI, dried and the solvent evaporated. Flash chromatography of the residue (EtOAc-hexanes, 25:75) terf-butyl A/-[(1f?)-1-[(4S)-2,2- dimethyl-1 ,3- dioxolan-4-yl]-2-[(3-oxo-3,5-dihydro-1 H- 2,4,3A5-benzodioxaphosphepin-3- yl)oxy]ethyl]carbamate (131) as a waxy solid (0.66g, 74.1 %). 1H NMR (500 MHz, CDCI3) δ 7.37 (m, 2H), 7.28 (m, 2H), 5.21 (m, 4H), 4.88 (bs, 1 H), 4.32 (m, 1 H), 4.20 (m, 2H), 4.05 (dd, J = 8.2, 6.9 Hz, 1 H), 4.00 (m, 1 H), 3.72 (t, J = 7.5 Hz, 1 H), 1.44 (s, 12H), 1.33 (s, 3H). 13C NMR (125.7 MHz, CDCI3, centre line δ 77.0) δ 135.3, 129.2, 128.9, 109.6, 79.9, 73.7, 68.5 (d, Jc,p = 6 Hz), 67.3 (d, JC,P = 4 Hz), 66.0, 50.7, 28.2, 26.2, 24.9. 31P NMR (202.4 MHz, CDCI3) δ 0.0 ESI-HRMS for C2oH3oNNa08P [M+Na]+ calcd. 466.1607, found 466.1598.
A solution of terf-butyl A/-[(1f?)-1-[(4S)-2,2-dimethyl-1 ,3- dioxolan-4-yl]-2-[(3-oxo-3,5- dihydro-1 - - 2,4,3A5-benzodioxaphosphepin-3- yl)oxy]ethyl]carbamate (131) (200 mg, 0.45 mmol) in methanol (20 mL) is stirred with Pd on C (5%, 20 mg) under a balloon of hydrogen. After 3 h the catalyst is removed by filtration and the solvent evaporated. The residue is stirred in 90% aqueous TFA for 30 min and then concentrated to dryness. The residue is dissolved in water and several drops of pyridine added. Lyophilization gives [(2f?,3S)-2-amino-3,4-dihydroxybutoxy]phosphonic acid containing 1eq of pyridinium trifluoroacetate (132)(200 mg, >100%). 1H NMR (500 MHz, D20, HOD 4.69 ppm) δ 8.75 (d, J = 5.9 Hz, 2H), 8.61 (d, J = 7.8 Hz, 1 H), 8.06 (d, J = 7.0 Hz, 2H), 4.10 (m, 1 H), 4.03 (m, 1 H), 3.91 (m, 1 H), 3.75 (dd, J = 12.3, 3.6 Hz, 1 H), 3.67(dd, J = 12.3, 12.3 Hz, 1 H), 3.56 (m, 1H). 13C NMR (125.7 MHz, D20) δ 162.9 (d, JC,F = 36 Hz) 147.3, 141.1 , 127.5, 116.4 (q, JC,F = 290 Hz), 68.0, 62.7, 62.5 (d, JC,P = 4.5 Hz), 53.4 (d, JC,P = 8.3 Hz). 31P NMR (202.4 MHz, D20) δ 0.0. ESI-HRMS for C4HnN06P [M-HJ calcd. 200.0324, found 200.0321.
[(2 ,3S)-2-Amino-3,4-dihydroxybutoxy]phosphonic acid containing 1eq of pyridinium trifluoroacetate (132) (120 mg, 0.32 mmol) is suspended in methanol (10 mL) and brought to pH 6-7 by the addition of pyridine. 4-Benzyloxy-5H-pyrrolo[3,2-d]pyrimidine-7- carbaldehyde (4) (93 mg, 0.26 mmol) and sodium cyanoborohydride (7.8 mg, 0.48 mmol) are added and the suspension stirred at 40 °C for 16 h and then at room temperature for 48 h. The solution is concentrated onto silica gel and then flash chromatographed on silica (MeCN-H20, 4:1 v/v), Further purification by flash chromatography (CH2CI2-MeOH- 28% aq. NH4OH, 25:25:10 v/v/v then 50:80:20 v/v/v) gives [(2R,3S)-2-({[4-(benzyloxy)- 5/- -pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)-3,4-dihydroxybutoxy]phosphonic acid; ammonia (133) (23 mg, 16%). 1H NMR (500 MHz, CD3OD- D20, 1 :1) δ 8.28 (s, 1 H), 7.77 (s, 1 H), 7.33 (m, 2H), 7.22 (m, 3H), 5.39 (s, 2H), 4.84 (d, J = 14.1 Hz, 1 H), 4.36 (d, J = 14.1 Hz, 1 H), 4.15 (m, 1H), 3.87 (m, 2H), 3.58 (dd, J = 12.2, 3.1 Hz, 1 H), 3.48 (dd, J = 12.2, 4.5 Hz, 1 H), 3.23 (s, 1 H). 13C NMR (125.7 MHz, CD3OD-D20, 1 :1 , CD3OD centre line 48.0 ppm) δ 155.8, 149.5, 147.9, 135.8, 132.7, 128.5, 128.3, 127.9, 115.3, 105.3, 68.4, 68.3, 62.8, 59.7 (d, Jc,p = 4.5 Hz), 59.4 (d, JC,P = 4.6 Hz), 39.5. 31 P NMR (202.4
MHz, CD3OD-D20, 1 :1) δ 2.4. ESI-HRMSMS C18H24N407P [M+H]+ calcd. 439.1383, found 439.1375. A solution of [(2 3S)-2-({[4-(benzyloxy)-5H-pyrrolo[3,2-<^pyrimidin-7-yl]methyl}amino)- 3,4-dihydroxybutoxy]phosphonic acid; ammonia (133) (20 mg, 0.046 mmol) in methanol (5 mL), water (5 mL) and NH4OH (0.05 mL) is stirred with Pd on C (2 mg) under a balloon of hydrogen for 16 h. The solution is filtered and the solvent evaporated to give [(2R,3S)-3,4-dihydroxy-2-[({4-hydroxy-5H-pyrrolo[3,2-c]pyrimidin-7- yl}methyl)amino]butoxy]phosphonic acid; ammonia (134) (11 mg, 70%). H NMR (500 MHz, D20-CD3OD, 1 :3) δ 7.90 (s, 1 H), 7.61 (s, 1 H), 4.44 (d, J = 14.0 Hz, 1 H), 4.34 (d, J = 14.0 Hz, 1 H), 4.16 (m, 1 H), 3.91 (m, 2H), 3.63 (dd, J = 2.6, 12.2 Hz, 1 H), 3.52 (dd, J = 4.5, 12.2 Hz, 1 H), 3.30 (s, 1 H). 13C NMR (125.7 MHz, CD3OD- D20, 1 :3, CD3OD centre line 48.0 ppm) δ 155.0, 149.5, 142.9, 130.8, 117.6, 106.2, 68.2, 62.6, 59.5, 59.1 , 39.3.
31P NMR (202.4 MHz, D20-CD3OD, 1 :3) δ 3.7. ESI-HRMS for C11H16N407P [M-H]" calcd. 347.0757, found 347.0756.
Example 29
[(2S,3R)-2,4-Dihydroxy-3-[({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7- yl}methyl)amino]butoxy]phosphonic acid (146) Scheme 29.
Figure imgf000099_0001
NHCbz NH3 NH2
TFA, THF BnQ H2, Pd black °
— ► HO-P-0
86% OH OH OH
36% from (141)
(143) (144)
Figure imgf000099_0002
Scheme 29
A solution of compound (129) (2.0g, 12.4 mmol) and imidazole (3.38 g, 49.6 mmol) in DMF (50 mL) is cooled in an ice-water bath whilst ferf-butylchlorodiphenylsilane(11.5 mL, 43.4 mmol) is added from a syringe. The mixture is stirred at room temperature for 2 h and then diluted with water and extracted three times with EtOAc. The combined extracts are washed with saturated aqueous NaCI, dried and concentrated under reduced pressure. The residue is flash chromatographed EtOAc-CH2CI2-Et3N, 10:90:0.1 then stepwise to 40:60:0.1) to give silylated hydroxylamine (135) (3.9 g, 79%). This material is dissolved in THF-water (2:1 , 90 mL) and benzylchloroformate (1.53 mL, 10.7 mmol) and NaHC03 (2.46g, 29.3 mmol) are added. After stirring for 48 h the mixture is partitioned between EtOAc and water. The organic phase is washed with saturated aqueous NaCI, dried and concentrated under reduced pressure. The residue is flash chromatographed (EtOAc-hexanes, 1 :9 v/v then stepwise to 2:8 v/v) to give benzyl Λ/-[(1 R)-2-[(tert- butyldiphenylsilyl)oxy]-1-[(4S)-2,2-dimethyl-1 ,3-dioxolan-4-yl]ethyl]carbamate (136)(4.0 g, 77%). 1H NMR (500 MHz, CDCI3) δ 7.65 (m, 4H), 7.37 (m, 11 H), 5. 1 (d, J = 12.3 Hz, 1 H), 5.05 (d, J = 12.3 Hz, 1 H), 4.95 (bd, J = 9.4 Hz, 1 H), 4.44 (m, 1 H), 4.02 (m, 1 H), 3.91 (m, 1 H), 3.70 (m, 3H), 1.38 (s, 3H), 1.34 (s, 3H), 1.05 (s, 9H). 3C NMR (125 MHz, CDCI3, centre line δ 77.0) δ 156.4, 136.5, 135.6, 133.2, 129.8, 128.5, 128.1 , 127.7, 109.2, 73.9, 66.9, 66.2, 63.8, 52.7, 26.8, 26.3, 25.1 , 19.2. ESI-HRMS for C3iH39N05Si a [M+Na]+ calcd. 556.2496, found 556.2486.
A solution of benzyl /V-[( R)-2-[(ieri-butyldiphenylsilyl)oxy]-1-[(4S)-2,2-dimethyl-1 ,3- dioxolan-4-yl]ethyl]carbamate (136) (0.75g, 1.41 mmol) in AcOH- THF-water (3:1 :1 , 35 mL) is heated at 50 °C for 5 h. The solvents are removed under reduced pressure. The residue is dissolved in, and evaporated from, ethanol and toluene to give benzyl N- [(2 3S)-1-[(iert-butyldiphenylsilyl)oxy]-3,4-dihydroxybutan-2-yl]carbamate (137) (0.71 g, >100%) which is used without further purification. A portion of compound (137) (0.33g, 0.67 mmol) and pyridine (0.16 mL, 2.0 mmol) are dissolved in CH2CI2 (10 mL) and cooled in a dry ice-acetone bath then benzoyl chloride (0.135 mL, 1.15 mmol) is added. The solution is stirred for 30 min and then quenched with water, brought to room temperature and washed with water, 10% aqueous NaHC03 and saturated aqueous NaCI, dried and concentrated under reduced pressure. The resulting (2S,3R)-3- {[(benzyloxy)carbonyl]amino}-4-[(ferf-butyldiphenylsilyl)oxy]-2-hydroxybutyl benzoate (138) is taken up in acetone (16 mL) and 2,2-dimethoxypropane (4 mL), with a trace of toluenesulfonic acid, and stirred at 50 °C for 3 h. The solution is cooled and concentrated under reduced pressure. The residue is taken up in EtOAc, washed with 10% aqueous NaHC03, dried and concentrated under reduced pressure. The resulting benzyl (4R,5S)- 5-[(benzoyloxy)methyl]-4-{[(ie^-butyldiphenylsilyl)oxy]methyl}-2,2-dimethyl-1 ,3- oxazolidine-3-carboxylate (139) is dissolved in methanol (20 mL) and basified with sodium methoxide. After 1 h saturated aqueous ammonium chloride (0.5 mL) is added, the mixture is concentrated under reduced pressure, taken up in EtOAc, washed with water, dried (MgS04) and evaporated to dryness. Flash chromatography (EtOAc- hexanes 20:80 v/v, then stepwise to 25:75 v/v) gives benzyl (4f?,5S)-4-{[(rerf- butyldiphenylsilyl)oxy]methyl}-5-(hydroxymethyl)-2,2-dimethyl-1 ,3-oxazolidine-3- carboxylate (140) (0.16 g, 43%). 1H NMR (500 MHz, CDCI3) δ 7.60 (m, 4H), 7.47-7.12 (m, 1 1 H), 5.20-4.84 (m, 2H), 4.36 (m, 1 H), 4.05-3.60 (m, 5H), 2.10 (m, 1 H), 1.67 (s, 3H), 1.52 (s, 3H), 1.05 (s, 9H). 13C NMR (125.7 MHz, CDCI3 centre line δ 77.0, signals split due to hindered rotation) δ 152.4, 136.3, 135.7, 133.1 , 129.8, 128.4, 128.0, 127.8, 95.4, (78.5, 77.6), 66.9, (63.5, 61.7), (59.8, 58.9), 28.1 , 26.9, 26.1 , 19.2. ESI-HRMS
C31H39N05SiNa [M+Na]+ calcd. 556.2495, found 556.2495.
A solution of benzyl (4f?,5S)-4-{[(fe/ -butyldiphenylsilyl)oxy]methyl}-5-(hydroxymethyl)- 2,2-dimethyl-1 ,3-oxazolidine-3-carboxylate (140) (160 mg, 0.30 mmol) and 1 H-tetrazol (42 mg, 0.60 mmol) in MeCN (9 mL) is cooled in an ice-H20 bath and dibenzyl diisopropylphosphoramidite (0.168 mL, 0.51 mmol) is added. The mixture is briefly warmed to room temperature, then cooled again in the ice-water bath whilst MCPBA (138 mg, 0.60 mmol) is added. The solution is warmed to room temperature and partitioned between EtOAc and 10% aqueous NaHC03. The organic phase is concentrated under reduced pressure and the residue flash chromatographed (EtOAc- hexanes, 25:75) to give crude benzyl (4f?,5S)-5-({[bis(benzyloxy)phosphoryl]oxy}methyl)- 4-{[(ierf-butyldiphenylsilyl)oxy]methyl}-2,2-dimethyl-1 ,3-oxazolidine-3-carboxylate (141) (200 mg, 0.25 mmol, 83%). A portion of this is further purified by flash chromatography (EtOAc-CH2CI2, 4:96) to give clean benzyl (4 ,5S)-5- ({[bis(benzyloxy)phosphoryl]oxy}methyl)-4-{[(ferf-butyldiphenylsilyl)oxy]methyl}-2,2- dimethyl-1 ,3-oxazolidine-3-carboxylate (141). 1H NMR (500 MHz, CDCI3) δ 7.58 (bs, 4H), 7.30(m, 20H), 7.11 (bs, 1 H), 5.20-4.82 (bm, 2H), 5.02(m, 4H), 4.46(m, 1 H), 4.11 (m, 1 H), 4.05(m, 1 H), 3.85(bs, 1 H), 3.71 (bs, 2H), 1.61(bs, 3H), 1.50(bs, 3H), 1.09(s, 9H). 13C NMR (125.7 MHz, CDCI3, centre line δ 77.0, signals split due to hindered rotation) δ 152.2, 135.4, 129.9, 128.4, 128.1 , 128.0, 127.8, (95.7, 95.1), (76.4, 75.6), 69.4, 67.7, (67.1 , 66.8), (63.0, 61.5), (60.2, 59.2), 28.3, 26.9, 26.5, 14.1. 31P NMR (202,4 MHz, CDCI3) δ -1.0 ESI-HRMS for C45H52N08pSi a [M+Na]+ calcd. 816.3098, found 816.3095. Benzyl (4f?,5S)-5-({[bis(benzyloxy)phosphoryl]oxy}methyl)-4-{[(rerf- butyldiphenylsilyl)oxy]methyl}-2,2-dimethyl-1 ,3-oxazolidine-3-carboxylate (141) (200 mg, 0.25 mmol) is dissolved in THF (5 mL) and TBAF (1 M, 0.44 mL, 0.44 mmol) is added. After a few minutes the solution is filtered through a pad of silica gel and concentrated under reduced pressure to give crude benzyl (4ft,5S)-5-({[bis(benzyloxy)phosphoryl]oxy} methyl)-4-(hydroxymethyl)-2,2-dimethyl-1 ,3-oxazolidine-3-carboxylate (142). The residue is stirred in THF-TFA-H20(1:1 :1) for 4 h, concentrated under reduced pressure and flash chromatographed (EtOAc) to give benzyl A/-[(2R,3S)-4- {[bis(benzyloxy)phosphoryl]oxy}-1 ,3-dihydroxybutan-2-yl]carbamate (143) ( 46 mg, mmol, 36%). 1H NMR (500 MHz, CDCI3) δ 7.34 (m, 15H), 5.48 (m, 1 H), 5.06 (m, 6H), 4.08 (m, 1 H), 3.99 (m, 2H), 3.79 (m, 1 H), 3.71 (m, 2H), 2.10 (bs, 2H). 13C NMR (125.7 MHz, CDCI3, centre line δ 77.0) δ 156.7, 136.3, 135.4, 133.1 , 128.8, 128.7, 128.2, 128.1 , ,
128.1 , 71.7, 70.2, 69.9, 67.1 , 64.2, 52.6. 31P NMR (202.4 MHz, CDCI3) δ 0.5 ESI-HRMS for C26H3oN08PNa [M+Na]+ calcd. 538.1067, found 538.1605.
A solution of benzyl A/-[(2R,3S)-4-{[bis(benzyloxy)phosphoryl]oxy}-1 ,3-dihydroxybutan-2- yl]carbamate (143) (100 mg, 0.19 mmol) in methanol (10 mL) is stirred with Pd black (5 mg) under a balloon of hydrogen. After 3 h the catalyst is removed by filtration, 7M NH3 in MeOH (1 drop) added, and the solution concentrated to dryness to give [(2S,3R)-3- amino-2,4-dihydroxybutoxy]phosphonic acid; ammonia (144) (33 mg, 86%). 1H NMR (500 MHz, D20, HOD 4.70 ppm) δ 4.00 (m, 3H), 3.85 (dd, J = 12.4, 4.2 Hz, 1 H), 3.74 (dd, J = 12.4, 7.1 Hz, 1 H), 3.49 (m, 1 H), 3.31 (s, 1 H). 13C NMR (125.7 MHz, D20) δ 66.9 (d, JC.P = 8 Hz), 65.1 (d, JC,P = 6 Hz), 59.0, 54.6. 31P NMR (202.4 MHz, D20) δ 4.8. ESI- HRMS for C4H12NNa06P [M+Na]+ calcd. 224.0300, found 224.0299.
[(2S,3f?)-3-Amino-2,4-dihydroxybutoxy]phosphonic acid; ammonia (144) (15 mg, 0.075 mmol) is suspended in methanol and brought to pH 6 with triethylamine. 4-benzyloxy-5/7- pyrrolo[3,2-c |pyrimidine-7-carbaldehyde (4) (20 mg, 0.060 mmol) and sodium cyanoborohydride (7.0 mg, 0.1 1 mmol) are added and the mixture stirred at 50 °C for 48 h. The solution is concentrated onto silica gel and the residue flash chromatographed (/- PrOH-H20, 8:2 v/v) to give crude give [(2S,3R)-3-({[4-(benzyloxy)-5H-pyrrolo[3,2- c|pyrimidin-7-yl]methyl}amino)-2,4-dihydroxybutoxy]phosphonic acid (145) which is dissolved in H20, and chromatographed on a Strata-X cartridge (Phenomenex) eluting with H20-methanol-28% aq. NH OH (5:4: 1 v/v/v) to give [(2S,3R)-3-({[4-(benzyloxy)-5H- pyrrolo[3,2-c]pyrimidin-7-yl]methyl}amino)-2,4-dihydroxybutoxy]phosphonic acid (145) (12 mg, 38%). 1H NMR (500 MHz, D20-MeOD, 9:1 , HOD 4.70 ppm) δ 8.38 (s, 2H) 8.34 (s, 1 H), 7.81 (s, 1 H), 7.46 (m, 2H), 7.37 (m, 3H), 5.50 (s, 2H), 4.54 (d, J = 14.1 Hz, 1 H), 4.48 (d, J = 14.1 Hz, 1 H), 4.02-3.92 (m, 3H), 3.86 (m, 2H), 3.40 (m, 1 H). 13C NMR (125.7 MHz, D20-MeOD, 9: 1) δ 170.6 155.8, 149.6, 147.6, 135.9, 132.7, 128.7, 128.5, 127.9, 115.3, 104.6, 68.5, 67.1 , 65.6, 59.9, 56.8, 39.0. 31P NMR (202.4 MHz, D20) δ 1.3. ESI-HRMS for C18H24N407P [M+H]+ calcd. 439.1383, found 439.1378.
A solution [(2S,3R)-3-({[4-(benzyloxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl]methyl}amino)-2,4- dihydroxybutoxy]phosphonic acid (145) ( mg, 0.022 mmol) in 20% aqueous methanol (7mL) is stirred with Pd black (1 mg) under a balloon of hydrogen. After 14 h the catalyst is removed by filtration and the residue after evaporation purified by flash chromatography (/-PrOH-H20, 7:3 v/v) to give [(2S,3ft)-2,4-dihydroxy-3-[({4-hydroxy-5H- pyrrolo[3,2-c(]pyrimidin-7-yl}methyl)amino]butoxy]phosphonic acid (146)(3.2 mg, 42%) 1H NMR (500 MHz, D20, HOD 4.70 ppm) δ 8.05 (s, 1 H), 7.73 (s, 1 H), 4.56 (d, J = 14.1 Hz, 1 H), 4.49 (d, J = 14.1 Hz, 1 H), 4.00(m, 3H), 3.90 (m, 2H), 3.48 (m, 1 H). 13C NMR (125.7 MHz, D20) δ 155.2, 150.6, 143.1 , 130.9, 117.7, 106.2, 67.3 (d, Jc,p = 6 Hz), 65.4, 60.0, 56.8, 39.0. 31P NMR (202.4 MHz, D20) δ 2.9. ESI-HRMS for C11H17N4Na07P [M+Na]+ calcd. 371.0733, found 371.0733.
Example 30
{3-Hydroxy-2-[({4-hydroxy-5H^yrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-2- (hydroxymethyl)propoxyjphosphonic acid (151), Scheme 30.
Figure imgf000103_0001
9% (150) (151)
Scheme 30
Dibenzyl diisopropyl phoshoramidite (3.64 mL, 10.83 mmol) is added to an ice cold solution of terf-butyl /V-[5-(hydroxymethyl)-2,2-dimethyl-1 ,3-dioxan-5-yl]carbamate (147) [Ooi, H.; Ishibashi, N.; Iwabuchi, Y.; Ishihara, J. Hatakeyama, S., J. Org. Chem. 2004, 69, 7765-7768.] (1.35g, 5.42 mmol) and 1H-tetrazole (1.52 g, 21.7 mmol) in CH2CI2 (50 mL). The solution is stirred at room temperature for 1 h and then cooled to -30 °C. A solution of MCPBA (4.67g, 16.3 mmol) in CH2CI2 (25 mL) is added and the solution bought slowly to room temperature. The solution is partitioned between CH2CI2 and 0% aqueous sodium bicarbonate. The organic phase is dried and concentrated under reduced pressure. The residue is flash chromatographed (CH2CI2-MeOH, 9:1) to give crude terf-butyl A/-[5-(hydroxymethyl)-2,2-dimethyl-1 ,3-dioxan-5-yl]carbamate (148) (2.81 g, 99%). A portion of compound (148) (0.2 g) is further flash chromatographed (EtOAc- hexanes) and the purified product is then dissolved in methanol (40 mL) and stirred with palladium on charcoal (5%, 22 mg) under a balloon of hydrogen. After 2 h the catalyst is removed by filtration. The solution is concentrated to dryness and the residue dissolved in 90% aqueous TFA. After 30 min the solution is concentrated under reduced pressure and the residue dissolved in H20. Lyophilization gives [2-amino-3-hydroxy-2- (hydroxymethyl)propoxy]phosphonic acid (149) (90 mg, 0.29 mmol, 76%). 1H NMR (500 MHz, D20, HOD 4.70 ppm) δ 3.99 (s, 2H), 3.75 (s, 4H). 13C NMR (125.7 MHz, D20, referenced to internal acetone at δ 30.3) δ 62.8 (d, Jc,p = 4.6 Hz), 60.8 (d, Jc,p = 8.1 Hz), 59.3. 31P NMR (202.4, MHz, D20) δ 0.0. ESI-HRMS C4H13N06P [M+H]+calcd. 202.0481 , found 202.0484.
[2-({[4-(Benzyloxy)-5H-pyrrolo[3,2-c ]pyrimidin-7-yl]methyl}amino)-3-hydroxy-2- (hydroxymethyl)propoxy]phosphonic acid (150) is prepared in the same manner as compound (133) in Scheme 28 from compound [2-amino-3-hydroxy-2- (hydroxymethyl)propoxy]phosphonic acid (149) (55 mg, 0.18 mmol) and 4-benzyloxy-5 - - pyrrolo[3,2-c ]pyrimidine-7-carbaldehyde (4) to give [2-({[4-(benzyloxy)-5H-pyrrolo[3,2- o pyrimidin-7-yl]methyl}amino)-3-hydroxy-2-(hydroxymethyl)propoxy]phosphonic acid (150) (7.0 mg, 0.015 mmol, 9%). 1H NMR (500 MHz, D20, HOD 4.70 ppm) δ 8.35 (s, 1 H), 7.79 (s, 1 H), 7.46 (m, 2H), 7.41 (m, 3H), 5.45 (s, 2H), 4.47 (s, 2H), 4.09 (m, 2H), 3.86 (m, 4H). 13C NMR (125.7 MHz, D20) δ 155.9, 149.3, 146.6, 135.6, 133.0, 128.8, 128.6, 127.8, 115.2, 104.6, 68.6, 65.7(d, JC,P = 7 Hz), 60.9, 57.9, 35.4. 31P NMR (202.4 MHz, D20) δ 1.2. ESI-HRMSMS Ci8H24N407P [M+H]+ calcd. 439.1383, found 439.1385.
[2-({[4-(Benzyloxy)-5/- -pyrrolo[3,2-cf]pyrimidin-7-yl]methyl}amino)-3-hydroxy-2- (hydroxymethyl)propoxy]phosphonic acid (150) is treated in the same manner as for the conversion of compound (125) to compound (126) in Scheme 26 to give {3-hydroxy-2- [({4-hydroxy-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-2- (hydroxymethyl)propoxy}phosphonic acid (151) (6.0 mg, 0.014 mmol) and crystallised from MeCN-H20. (2.0 mg, 41%) 1H NMR (500 MHz, D20, HOD 4.70 ppm) δ 7.98 (s, 1 H), 7.61 (s, 1 H), 4.22 (s, 2H), 3.90 (d, J = 6.4 Hz, 2H,), 3.77 (m, 4H). 13C NMR (125.7 MHz, D20) δ 155.6, 143.1 , 142.7, 130.0, 117.4, 109.8, 63.7, 61.5, 59.0, 35.0. 31P NMR (202.4 MHz, D20) δ 4.5. ESI-HRMS CnH18N407P [M+H]+ calcd. 349.0913, found 349.0911.
Example 31
2-({[Bis(aminooxy)phosphoryl]oxy}methyl)-2-{[({4-hydroxy-5W-pyrrolo[3,2- d|pyrimidin-7-yl}methyl)amino]methyl}propane-1,3-diol (161), Scheme 31.
Figure imgf000105_0001
(152) (153) XX == HH (155)
NBS
(154) X = Br
Figure imgf000105_0002
MCPBA
Figure imgf000105_0003
Scheme 31
Sodium hydride (60% dispersion in mineral oil, 2.38 g, 59.4 mmol) is added to a suspension of 4-chloro-5 - -pyrrolo[3,2-d]pyrimidine (152) (4.56 g, 29.7 mmol) in THF (70.0 mL, 854 mmol) at 0 °C and the mixture is stirred at 0 °C for 20 min. Benzyl chloromethyl ether (8.95 mL, 38.6 mmol) is added dropwise and the mixture is warmed to ambient temperature and stirred for 1 h.
t-Butanol (20.0 mL) and DMF (20.0 mL) are added followed by NaH (60% dispersion in mineral oil, 2.38 g, 59.4 mmol) and the reaction mixture is stirred at ambient temperature for 18 h. The reaction mixture is quenched with water (50.0 mL) and the THF is removed under reduced pressure. Water (150 mL) and EtOAc (100 mL) are added and the biphasic mixture is filtered through Celite, separated and the organic phase is dried, and concentrated under reduced pressure. The crude product is purified by flash chromatography (8:2 to 1 :1 v/v petroleum ether-EtOAc) to give the 5-[(benzyloxy)methyl]- 4-(terf-butoxy)-5H-pyrrolo[3,2-c/]pyrimidine (153) as a pale yellow solid (4.00 g, 43%). /V-Bromosuccinimide (2.29 g, 12.9 mmol) is added portionwise to a solution of 5- [(benzyloxy)methyl]-4-(terf-butoxy)-5H-pyrrolo[3,2-d]pyrimidine (153) (4.00 g, 12.9 mmol) in DCM (85.0 mL, 1321 mmol) at 0 °C over 1 h. The reaction mixture is then concentrated under reduced pressure and the crude material purified by flash chromatography (9:1 to 5:4 v/v petroleum ether-EtOAc) to give 5-[(benzyloxy)methyl]-7- bromo-4-(terf-butoxy)-5H-pyrrolo[3,2-d]pyrimidine (154) as a clear oil (3.20 g, 64%) n-Butyllithium (1.6M hexanes, 1.48 mL, 1.92 mmol) is added to a solution of 5- [(benzyloxy)methyl]-7-bromo-4-(fe Tf-butoxy)-5 -pyrrolo[3,2-c]pyrimidine (154) (750 mg, 1.92 mmol) in THF (2.0 mL) at -78 °C. The solution is stirred for 10 min at -78 °C. DMF (0.74 mL, 9.61 mmol) is added and the mixture is stirred at -78 °C for 1 h. The reaction mixture is quenched with water (10.0 mL) whilst at -78 °C and then warmed to ambient temperature. The reaction is concentrated under reduced pressure and diethyl ether (20.0 mL) and H20 (10.0 mL) are added. The phases are separated and the aqueous phase is extracted with diethyl ether (20.0 mL). The organic phase is dried, filtered and the solvent concentrated under reduced pressure. The residue is purified by flash chromatography (9:1 to 5:4 v/v petroleum ether-EtOAc) to give 5-(benzyloxymethyl)-4- tert-butoxy-5H-pyrrolo[3,2-d]pyrimidine-7-carbaldehyde (155) as an orange solid (330 mg, 51%). A solution of (5-((tert-butyldiphenylsilyloxy)methyl)-2,2-dimethyl-1 ,3-dioxan-5- yl)methanamine (575 mg, 1.39 mmol) and 5-[(benzyloxy)methyl]-4-(terf-butoxy)-5 -/- pyrrolo[3,2-d]pyrimidine-7-carbaldehyde (155) (262 mg, 0.772 mmol) in anhydrous 1 ,2- dichloroethane (5.0 mL), is stirred at ambient temperature for 18 hours.
Sodium triacetoxyborohydride (213 mg, 1.00 mmol) is added and the reaction mixture is stirred for a further 6 hours. The reaction mixture is diluted with DCM (10.0 mL) and washed with saturated NaHC03 (10.0 mL). The organic phase is dried, filtered and the solvent concentrated under reduced pressure to give ({5-[(benzyloxy)methyl]-4-(terf- butoxy)-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)[(5-{[(fert-butyldiphenylsilyl)oxy]methyl}- 2,2-dimethyl-1 ,3-dioxan-5-yl)methyl]amine (156) as an orange oil (450 mg, 79%).
Di-ierf-butyl dicarbonate (346 mg, 1.59 mmol) is added to a solution of compound (156) (450 mg, 0.611 mmol) in CHCI3 (20.0 mL) at ambient temperature under. Et3N (86.0 pL, 611 μιηοΙ) is added and the solution is stirred at ambient temperature for 18 h. The reaction mixture is concentrated under reduced pressure. The residue is purified by flash chromatography (7:3 v/v petroleum ether-EtOAc) to give tert-butyl /-({5- [(benzyloxy)methyl]-4-(ierf-butoxy)-5 -/-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)-/\/-[(5-{[(tert- butyldiphenylsilyl)oxy]methyl}-2,2-dimethyl-1 ,3-dioxan-5-yl)methyl]carbamate (157) as a clear oil (495 mg, 97%)
Tetrabutylammonium fluoride (1 M THF, 0.597 ml, 597 μηηοΙ) is added to a solution of compound (157) (250 mg, 2.99 mmol) in THF (5.0 mL) at ambient temperature and the reaction mixture is stirred for 18 h. Further tetrabutylammonium fluoride (1 M THF, 0.597 mL, 5.97 mmol) is added and the reaction mixture is stirred for a further 36 h. The solvents are concentrated under reduced pressure and the residue is dissolved in CHCI3 (50.0 mL) and washed with H20 (2 *20.0 mL). The organic phase is dried, filtered and the solvent is concentrated under reduced pressure. The residue is purified by flash chromatography (40% v/v EtOAc in CHCI3) to give terf-butyl N-({5-[(benzyloxy)methyl]-4- (ieri-butoxy)-5H-pyrrolo[3,2-cf]pyrimidin-7-yl}methyl)-/\/-{[5-(hydroxymethyl)-2,2-dimethyl- 1 ,3-dioxan-5-yl]methyl}carbamate (158) as a clear oil (130 mg, 73%).
N,N-diethyl-1 ,5-dihydrobenzo[e][1 ,3,2]dioxaphosphepin-3-amine (130 μΐ, 601 μηηοΙ) is added to a solution of compound (158) (180 mg, 301 mol)and tetrazole (105 mg, 1.50 mmol) in MeCN (10.0 mL) and the solution is stirred at ambient temperature for 90 minutes. The solvent is concentrated under reduced pressure and the residue is dissolved in DCM (5.0 mL). The solution is cooled to 0 °C and m-chloroperoxybenzoic acid (259 mg, 902 pmol) is added. The reaction mixture is stirred for 30 minutes at 0 °C then washed with NaHC03 and NaHS03, dried and then concentrated under reduced pressure. The residue is purified by flash chromatography (50-100% v/v EtOAc in petroleum ether) to give compound terf-butyl A/-({5-[(benzyloxy)methyl]-4-(te/i-butoxy)- 5H-pyrrolo[3,2-cf]pyrimidin-7-yl}methyl)-/V- [(2,2-dimethyl-5-{[(3-oxo-3,5-dihydro-1H- 2,4,3A5-benzodioxaphosphepin-3-yl)oxy]methyl}- 1 ,3-dioxan-5-yl)methyl]carbamate (159) as a clear oil (110 mg, 47%), which is contained an impurity that resisted separation.
Compound (159) (110 mg, 141 μητιοΙ) is dissolved in EtOH (10.0 mL) containing a few drops of cone. aq. NH3. 10% Palladium on carbon (1.499 mg, 14.09 μιηοΙ) is added and the mixture is treated with H2 (hydrogen balloon) over the weekend.
The reaction had not reached completion, so the reaction mixture is filtered and concentrated under reduced pressure and the residue redissolved in EtOH (10.0 mL) containing a few drops of 28%. aq. NH3. 10% Palladium on carbon (15.0 mg, 0.1419 mmol) is added and the mixture is treated with H2 (hydrogen balloon) at ambient temperature for 18 h. The reaction mixture is filtered through Celite and the residue washed with EtOH. The solvent is concentrated under reduced pressure and the crude product is purified by flash chromatography (DCM-MeOH- 28% aq. NH3, 8:8:2 v/v/v) to give the product te/†-butyl /V-{[5-({[bis(aminooxy)phosphoryl]oxy}methyl)-2,2-dimethyl- 1 ,3-dioxan-5-yl]methyl}-/V-{[4-(te i-butoxy)-5H-pyrrolo[3,2-c]pyrimidin-7- yl]methyl}carbamate (160) as a clear oil (22 mg, 26%).
Compound (160) (20 mg, 33.7 μιηοΙ) is dissolved in 80% TFA in water (1.0 mL) and stirred at ambient temperature for 3 h. Dioxane (2.0 mL) is added and the solvent is concentrated under reduced pressure and the residue is azeotroped with MeOH (*3). The crude material is purified by flash chromatography (6:3:1 v/v/v MeCN-water-NH3 to give 2-({[Bis(aminooxy)phosphoryl]oxy}methyl)-2-{[({4-hydroxy-5H-pyrrolo[3,2- c ]pyrimidin-7-yl}methyl)amino]methyl}propane-1 ,3-diol (161) as a white solid (9 mg, 67%). 1H NMR (500 MHz, D20) δ 8.02 (s, 1 H), 7.69 (s, 1 H), 4.37 (s, 1 H), 3.79 (d, 2H), 3.52 (s, 4H), 3.14 (s, 1 H).13C NMR (125MHz, DMSO-of6) δ 155.2, 143.9, 142.9, 131.0, 1 17.7, 105.9, 63.3 (d), 62.1 , 48.7, 43.3 (d), 41.4. 31P NMR (202 MHz, D20) δ 5.30.
Example 32: Inhibition of Plasmodium falciparum HGXPRT, Human HGPRT and Plasmodium vivax HGPRT Activity
PrHGXPRT activity is measured using spectrophotometric assays observing the conversion of xanthine and 5-phospho-a-D-ribose-1 -pyrophosphate to xanthosine-5'- monophosphate and inorganic pyrophosphate (PP,) at 247 nm (ε257 - 6.8 mM"1cm"1) or the conversion of guanine and PRPP to guanosine-5'-monophosphate and PP, (ε257 - 5.8 mM"1cm"1) on a Varian Cary 100 spectrophotometer (Palo Alto, CA) at 37 °C. All assays are conducted in 10 mM potassium phosphate, pH 7.6, 10 mM MgCI2, 1 mM PRPP, 150 μΜ xanthine or 50 μΜ guanine, 0.5 mM DTT with varying amounts of inhibitor and concentrations of P/HGXPRT between 2 and 10 nM or human HGPRT of 1 nM or FVHGPRT of 5 nM. As the dissociation constants of many of the inhibitors are near or below that of the enzyme concentration used, the data are fit to the Morrison equation for tight-binding inhibitors (Morrison, J. F. Kinetics of the reversible inhibition of enzyme- catalysed reactions by tight-binding inhibitors. Biochim Biophys Acta 185, 269-286, (1969). Table 1 : Inhibition Data for Certain Compounds of the Invention
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
(126)
(134)
(146)
(151)
(161)
Compounds (7c) and (95) are competitive inhibitors of PflHGXPRT with R values of 10.6 and 0.65 n , respectively. When tested against human HGPRT, compounds (7c) and (95) display K, values of 4940 nM and 385 nM for human HGPRT. Advantageously, compounds (7c) and (95) are, respectively, 466- and 592-fold more selective for the parasitic enzyme than the human enzyme. These specific free phosphonate inhibitors show no activity against cultured parasites, consistent with their lack of membrane permeability, however other phosphonates are known to be permeable (Sheng et al., Bioorganic & Medicinal Chemistry Letters, 19 (2009) 3453-3457). As described below, prodrug forms of these compounds show activity against the cultured parasites. Example 33: Prodrug Compounds of the Invention Inhibit Proliferation Plasmodium falciparum in Culture
Compound (54), the bis-pivalate of (7c), inhibits the growth of cultured parasites with an IC50 of 45 ± 6 μΜ (Figure 1 a). Metabolic labeling of erythrocytes with [3H]hypoxanthine in the presence of 100 μΜ (54) (Figure 1 b) reveals incorporation of radiolabel into extracellular inosine and other intermediates and labeling with [3H]inosine (Figure 1 c) shows inhibition of inosine conversion to hypoxanthine. UPLC/MS/MS analysis of infected erythrocytes treated with 100 and 200 μΜ of (54) for 30 minutes confirms that (54) is processed to (7c) in infected erythrocytes, causing an increase in inosine concentration (Table 1 ). Hypoxanthine is not found in treated or control samples, suggesting that human HGPRT activity is unaffected. The accumulation of extracellular inosine from labeled erythrocytes indicates that (54) is permeable, but is converted to (7c) before crossing the parasite membranes. At higher concentrations, (54) also crosses the parasite membranes and inhibits PflHGXPRT activity.
Table 2: Bis-pivalate prodrug (54) is activated in infected erythrocytes - UPLC/MS/MS data from cell extracts of infected erythrocytes treated with compound (54). Values are arbitrary units and cannot be compared across columns.
Figure imgf000113_0001
ND=Not detected
Example 34: Plasmodium falciparum Cell Culture Inhibition Assay
Activity of the compounds is tested as previously described (M. B. Cassera et al. Erythrocytic adenosine monophosphate as an alternative purine source in Plasmodium falciparum, (J Biol Chem 283, 32889-32899, (2008)). Compounds (61) and (65) are dissolved in sterile water by sonication in a warm water bath; compound (72) is dissolved in DMSO; compound (76) is dissolved in sterile water, added to malaria culture media and concentrated by lyphophilization to achieve the desired concentration. Compounds (61), (65), (72) and (76) are assayed to determine the IC50 values against P. falciparum strain 3D7 and compounds (61), (65), (72) are found to inhibit parasite growth in vitro with IC50 values of 2.5 ± 0.2 μΜ, 1.9 ± 0.1 μΜ, and 7.0 ± 0.1 μΜ, respectively. Compounds (61) and (65) show similar IC50 values when compared with chloroquine/mefloquine-resistant strain Dd2 (3.0 ± 0.1 μΜ and 2.3 ± 0.1 μΜ) or chloroquine/quinine resistant strain FVO (2.9 ± 0.1 μΜ and 3.1 ± 0.1 μΜ). Inhibition of P/HGXPRT cannot be rescued by exogenous hypoxanthine (Kicska, G.A., Tyler, P.C., Evans, G.B., Furneaux, R.H., Schramm, V.L., and Kim, K. J. Biol. Chem., 2002, 277, 3226-3231). The IC50 assays are conducted at a range of exogenous hypoxanthine concentrations from 0 to 185 μΜ. The IC50 values show no significant variation with increasing concentrations of exogenous hypoxanthine (all IC50 values between 2.2 and 2.4 μΜ from 3 to 185 μΜ hypoxanthine) (Figure 2). This indicates that the site of action is inhibition of P/HGXPRT. Compound (76) does not inhibit parasite growth in vitro, at concentrations up to 15 μΜ.
Table 3: ICS0 Data for Certain Prodrug Compounds of the Invention
Figure imgf000114_0001
Figure imgf000114_0002
Example 34: Metabolic Labeling Studies
Erythrocytes, infected erythrocytes and erythrocyte free parasites in the trophozoite stage are treated with 100 μΜ (54), 10 μΜ (61) and (65) or 15 μΜ (76) for one hour at 37 °C. Cells are then labeled with 1 μΜ [2,8-3H]hypoxanthine (30 Ci/mmol, Moravek) or 1 μΜ [2,8-3H]inosine (50 Ci/mmol, Moravek) for 1 hour at 37 °C. Parasites from infected erythrocytes are isolated by lysis with 0.045% saponin either before addition of radiolabel or after incubation with radiolabel. Proteins and nucleic acids are removed by perchloric acid treatment of supernatants and cell pellets (J. Biol. Chem., 2008, 283, 32889-32899). All samples are analyzed by HPLC with a modification in the previously described solvent gradient (J. Biol. Chem., 2008, 283, 32889-32899). Briefly, the mobile phases are 8 mM tetrabutylammonium bisulfate (Fluka) and 100 mM KH2P04 with the pH adjusted to 6.0 with KOH in water (solution A) or 30% acetonitrile (solution B). The HPLC gradient is from 0% to 10% solution B in 4 min and maintained for 2 min, 10% to 20% solution B in 1 min, 20% to 40% solution B in 10 min, 40% to 100% solution B in 3 min and maintained for 4 min.
Incorporation of radiolabeled hypoxanthine by uninfected erythrocytes is unchanged by incubation with 10 μΜ of (61) (Figure 4a and Table 4). The label is incorporated into the erythrocyte IMP and GMP pools with smaller amounts appearing in GDP and GTP. The lack of incorporation into the adenine nucleotides is expected since human erythrocytes lack adenylosuccinate synthase activity. (Figure 4b and Table 4).
Table 4: Metabolic labeling summary showing the percentage change in incorporation of radiolabel from [2,8-3H2]hypoxanthine into the nucleotide pool of erythrocytes, infected erythrocytes and free parasites on treatment with compound (61)
Figure imgf000115_0001
Adenosine/AMP -2 ± 1 -37 ± 31 -91 ± 3
ADP -9 ± 4 -41 ± 20 -79 ± 11
ATP -3 ± 3 -42 ± 22 -68 ± 22
Compounds (61) and (65) are shown to decrease incorporation of radiolabeled hypoxanthine into the parasite nucelotide pool by 85% (Figures 3c and 4a) and result in the accumulation of extracellular hypoxanthine (Figure 3d). Inhibition of uptake of the radiolabeled hypoxanthine is similar for (61) or (65) during incubation of parasites with radiolabeled hypoxanthine or in preincubation experiments where excess compound of the invention is removed prior to the addition of the radiolabel. Inhibition of hypoxanthine uptake by (61) or (65) is stronger in infected erythrocytes than in isolated parasites, indicating that the erythrocytes play a role in the activation of the prodrug compound of the invention and/or transportation into the parasite.
In time- and concentration-dependent inhibition experiments, maximum inhibition of hypoxanthine uptake by (61) is complete after thirty minutes of treatment and increases linearly with concentrations of (61) between 0.5 and 5 μΜ (Figures 3e and 3f).
Example 36: UPLC/MS/MS
Samples are placed in 96 deep-well plates, treated with 0.5 M HCI04 at 1 :7 (v/v, sample/HCI04), incubated for 20 min at 4 °C and neutralized with 5 M KOH at 10:1 (v/v, HCIO4/KOH) for 20 min at 4 °C. Plates are centrifuged (10 min at 4,000 rpm, 4 °C) and supernatants are filtered through a Multiscreen® Filter Plate with Ultracel®-10 Membrane (Millipore). Metabolite and inhibitor levels are analyzed by UPLC/MS/MS using a Xevo TQD mass spectrometer (Waters). The separation of inosine, hypoxanthine, and inhibitors is achieved with an Acquity HSS T3 column (2.1 x 100 mm, 1.8 μπι, Waters) at 60 °C. The eluent system is composed of 5 mM ammonium formate in water (A) and 5 mM ammonium formate in methanol (B) with a gradient of 98% eluent A to 30% eluent B from 0.1 to 1 min, 70% eluent A to 80% eluent B from 1 to 1.5 min and back to 98% eluent A from 1.5 to 3 min at a flow rate of 0.6 ml min"1. Detection is performed in ESI positive-ion mode using multiple-reaction monitoring (MRM) mode. For ESI-MS/MS analysis, the following ion transitions, cone voltage (CV) and collision energy (CE) are used: inosine m/z 269.1 >137.1 (CV: 14 V, CE: 12 eV), hypoxanthine m/z 137.1 >110.0 (CV: 42 V, CE: 18 eV), compound (7c) m/z 286.92 >139.74 (CV: 22 V, CE: 14 eV), compound (54) m/z 515.05 >205, (CV: 34 V, CE: 28 eV). The ESI capillary voltage is 0.3 kV, source temperature is set at 150 °C and desolvation temperature at 450 °C. Data acquisition and analysis are carried out by MassLynx V4.1 and QuanLynx software.
Although the invention has been described by way of example, it should be appreciated the variations or modifications may be made without departing from the scope of the invention. Furthermore, when known equivalents exist to specific features, such equivalents are incorporated as if specifically referred to in the specification.
INDUSTRIAL APPLICABILITY
The invention relates to compounds that are inhibitors of hypoxanthine and/or guanine purine phosphoribosyltransferases, e.g hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferases of protozoan parasites, including those of Leishmania, Plasmodium, Toxoplasma or Trypanosoma species, preferably the HGXPRT of Plasmodium falciparum, or the HGPRT of Plasmodium vivax, or the HGXPRT of Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella or Toxoplasma gondii, or the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, or the HGPRT or XPRT of Leishmania donovani. The compounds are therefore indicated for the treatment or prevention of diseases in which the inhibition of such purine phosphoribosyltransferases is desirable, e.g. malaria.

Claims

A compound of the formula (I):
Figure imgf000118_0001
A is CH, CR2 or N;
D is H, OH or NH2;
R2 is halogen, alkyl, aralkyl or aryl; and n is 1 ; R1 is a radical of formula (i) where G is O; X is an optionally substituted C3 or C5 alkylene group and R1 is attached to a terminal carbon atom of X; or n is 1 ; R1 is a radical of formula (i) where G is absent; X is an optionally substituted C3 or C4 alkylene group and R1 is attached to a terminal carbon atom of X; or n is 2; R1 is a radical of formula (i) where G is O or is absent; X is an optionally substituted C2 alkylene group and R1 is attached to a terminal carbon atom of X;
Figure imgf000118_0002
or an ester prodrug form thereof, or a tautomer thereof, or a pharmaceutically acceptable salt thereof.
2. A compound as claimed in claim 1 which is an ester prodrug compound of formula (la):
Figure imgf000119_0001
wherein:
A is CH, CR2 or N;
D is H, OH or NH2;
R2 is halogen, alkyl, aralkyl or aryl; and n is 1 ; R1 is a radical of formula (ii) where G is O; Z is -(CH2)m-0-(CH2)p-CH3; Y is H, alkyl or -(CH2)m-0-(CH2)p-CH3; X is an optionally substituted C3 or C5 alkylene group and R1 is attached to a terminal carbon atom of X; or n is 1 ; R1 is a radical of formula (ii) where G is absent; Z is -(CH2)m-0-(CH2)p-CH3; Y is H, alkyl or -(CH2)m-0-(CH2)p-CH3; X is an optionally substituted C3 or C4 alkylene group and R1 is attached to a terminal carbon atom of X; or n is 2; R1 is a radical of formula (ii) where G is O or is absent; Z is -(CH2)m-0- (CH2)P-CH3; Y is H, alkyl or -(CH2)m-0-(CH2)p-CH3; X is an optionally substituted C2 alkylene group and R1 is attached to a terminal carbon atom of X;
Figure imgf000119_0002
where m is 2 or 3 and p is an integer from 2 to 21 and where, when Z is -(CH2)m- 0-(CH2)p-CH3 and Y is -(CH2)m-0-(CH2)p-CH3, each m and each p is independently selected; or a tautomer thereof, or a pharmaceutically acceptable salt thereof.
3. A compound as claimed in claim 1 or claim 2 wherein the C2, C3, C4 or C5 alkylene group X in the formula (I) or (la) is substituted with one or more hydroxy groups.
4. A compound as claimed in any one of claims 1 to 3 wherein the C2, C3, C4 or C5 alkylene group X in the formula (I) or (la) is substituted with one or two fluorine atoms.
5. A compound as claimed in any one of claims 1 to 4 wherein A is CH or N.
6. A compound as claimed in any one of claims 1 to 5 wherein D is H or NH2.
7. A compound as claimed in any one of claims 2 to 6 wherein p is an integer from 7 to 17.
8. A compound as claimed in any one of claims 2 to 7 wherein R1 is a radical of formula (ii) where Z is -(CH2)m-0-(CH2)p-CH3 and Y is alkyl.
9. A compound as claimed in any one of claims 2 to 7 wherein R1 is a radical of formula (ii) where Z is -(CH2)m-0-(CH2)p-CH3 and Y is H.
10. A compound as claimed in any one of claims 1 to 9 wherein X is selected from the group consisting of ethylene, 1 ,3-propylene, 1 ,4-butylene, 1 ,5-pentylene, 3- hydroxy- ,2-propylene, 2-hydroxy-1 ,3-propylene, 2-hydroxymethyl- ,3-propylene, 2,2-bis(hydroxymethyl)-1 ,3-propylene, 4-hydroxy-1 ,3-butylene, 1-fluoro-4- hydroxy-1 ,3-butylene, 1 ,1-difluoro-4-hydroxy-1 ,3-butylene.
11. A compound as claimed in claim 1 , selected from the group consisting of:
Figure imgf000120_0001
Figure imgf000121_0001
Figure imgf000122_0001
120
Figure imgf000123_0001
121
Figure imgf000124_0001
ı22
Figure imgf000125_0001
Figure imgf000126_0001
124
Figure imgf000127_0001
Figure imgf000128_0001
Figure imgf000129_0001
or a tautomer thereof, or a pharmaceutically acceptable salt thereof.
12. 4-(te/t-Butoxy)-2-chloro-5 - -pyrrolo[3,2-d]pyrimidine, a compound of formula:
Figure imgf000130_0001
A pharmaceutical composition comprising a pharmaceutically effective amount of a compound as claimed in any one of claims 1 to 1 1 and, optionally, a pharmaceutically acceptable carrier, diluent or excipient.
A method of treating or preventing a disease or disorder in which it is desirable to inhibit a hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase of a protozoan parasite, comprising administering a pharmaceutically effective amount of a compound as claimed in any one of claims 1 to 11 to a patient requiring treatment.
A method as claimed in claim 14 wherein the hypoxanthine and/or guanine and/or xanthine purine phosphoribosyltransferase is selected from the group consisting of: the HGXPRT of Plasmodium falciparum, the HGPRT of Plasmodium vivax, the HGXPRT of Tritrichomonas foetus, the HGXPRT of Cryptosporidium parvum, the HGXPRT of Eimeria tenella the HGXPRT of Toxoplasma gondii, the GPRT of Giardia lamblia, the HPRT of Trypanosoma cruzi, and the HGPRT or XPRT of Leishmania donovani, comprising administering a pharmaceutically effective amount of a compound as claimed in any one of claims 1 to 11 to a patient requiring treatment.
A method of treating or preventing an infection caused by Plasmodium falciparum, Plasmodium vivax, Tritrichomonas foetus, Cryptosporidium parvum, Eimeria tenella, Toxoplasma gondii, Giardia lamblia, Trypanosoma cruzi or Leishmania donovani, comprising administering a pharmaceutically effective amount of a compound as claimed in any one of claims 1 to 1 1 to a patient requiring treatment.
A method of treating or preventing malaria, comprising administering pharmaceutically effective amount of a compound as claimed in any one claims 1 to 1 to a patient requiring treatment.
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CN103073582A (en) * 2013-02-05 2013-05-01 中国科学院上海有机化学研究所 Synthesis method of monoalkyl hydrocarbyl phosphonate
JP2016531119A (en) * 2013-07-31 2016-10-06 メルク パテント ゲゼルシャフト ミット ベシュレンクテル ハフツングＭｅｒｃｋ Ｐａｔｅｎｔ Ｇｅｓｅｌｌｓｃｈａｆｔ ｍｉｔ ｂｅｓｃｈｒａｅｎｋｔｅｒ Ｈａｆｔｕｎｇ Pyridine, pyrimidine and pyrazine and their use as inhibitors of BTK
US10793572B2 (en) 2014-09-29 2020-10-06 The Provost Fellows Foundation Scholars And The Other Members Of Board Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth Substituted pyrimidine derivatives useful in the treatment of autoimmune diseases
US11229652B2 (en) 2014-09-29 2022-01-25 The Provost, Fellows, Foundation Scholars, And The Other Members Of Board, Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Treatments for autoimmune disease
US11518765B2 (en) 2014-09-29 2022-12-06 The Provost, The Fellows, Foundation Scholars, And The Other Members Of Board, Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Substituted pyrimidine derivatives useful in the treatment of autoimmune diseases
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