JP2008538771A - Use of compounds to improve telomerase processivity - Google Patents

Abstract

The present invention provides the use of acyclic nucleoside phosphonic acids and derivatives thereof for the preparation of a medicament that improves the processivity of telomerase to increase the number of mitotic cycles and mitotic cells. The present invention also provides a pharmaceutical composition comprising a compound of formula I, or a prodrug or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. The present invention also provides novel compounds of formula I as described herein, as well as prodrugs and pharmaceutically acceptable salts thereof.

Description

(Background of the Invention)
Human telomerase adds telomeric repeats to the 3 ′ end of the chromosome (Greider CW, Blackburn EH, Cell, 1985, 43, 405-13), thereby playing a role in compensating for the loss of telomeres associated with chromosome replication and cell division Large cellular ribonucleoprotein complex (Morin GB, Cell, 1989, 59, 521-9; Wenz C, et al., EMBO J, 2001; 20: 3526-34; and Collins K, Mitchell JR., Oncogene, 2002, 21, 564-79). Telomerase is upregulated in nearly 90% of all malignancies (Kim NW, et al., Science, 1994, 266, 2011-5; Shay JW, Bacchetti S, Eur J Cancer, 1997, 33, 787-91. And Hiyama E, Hiyama K, Oncogene, 2002, 21, 643-9). Thus, telomerase appears to be very promising not only as a tumor-specific marker but also as a target for anti-cancer treatment (Lichsteiner SP, et al., Ann NY Acad Sci, 1999, 886, 1- 11).

  The identification of the hTERT component of telomerase as a functional catalytic reverse transcriptase has led to established HIV reverse transcriptase inhibitors (eg, chain-terminated 3′-azido-3′-deoxythymidine (AZT) and 2 Research to inhibit telomerase using ', 3'-dideoxycytidine (ddC)) has been promoted (Non-patent document 1; Non-patent document 2; and Non-patent document 3).

These parent nucleosides are converted in vivo in the form of a triphosphate, which inhibits reverse transcriptase by acting as a competitive substrate for the enzyme and newly terminates DNA synthesis. AZT-TP inhibits the activity of telomerase to about 50% at 30 μmol / L. Other nucleotide inhibitors (both purine and pyrimidine derivatives) have been evaluated for telomerase, and effective IC ₅₀ values are in the micromolar range. In a more recent study, Fletcher reports the use of deazadeoxypurine as an inhibitor of telomerase, one compound (6-thio-7-deaza-2′-deoxyguanosine 5′-phosphate). Indicates an IC ₅₀ value of ₆₀ nmol / L. A series of other nucleotide analogs (ddGTP, ddATP, ddTTP, d4TTP, deaza dGTP, deaza dATP, thio dGTP, CBV-TP, araGTP and FaraTTP) have been previously reported to inhibit telomerase activity (Morin GB , Cell, 1989, 59, 521-9; Strahl C, Blackburn EH., Mol Cell Biol, 1996, 16, 53-56; Pai RB, et al., Cancer Res, 1998, 58, 1909-13; , Et al., Bioorg Chem, 2001, 29, 36-55; Tendian SW, Parker WB., Mol Pharmacol, 2000, 57, 695-9; Ahl C, Blackburn EH., Nucleic Acids Res, 1994, 22, 893-900; Fletcher ™, et al., Biochemistry, 1996, 35, 15611-7; and Raymond E, et al. 7, 583-91).

ANP has excellent antiviral activity against a wide range of DNA viruses and retroviruses, as well as significant antiproliferative capacity. (S) -HPMPC (cidofovir, CDV, Visitide (registered trademark)), (R) -PMPA (tenofovir, TDV, Viaread (registered trademark)), PMEA (adefovir, ADV, Hepsera (registered trademark)), Already approved for the treatment of cytomegalovirus retinopathy, HIV infection and chronic hepatitis B in AIDS patients, respectively. In cells, ANPs are activated by their conversion to diphosphate (an active metabolic antagonist), which inhibits viral replication and terminates the nascent DNA strand (Non-patent Document 4). ).
Nakamura ™, et al. , Science, 1997, 277, 955-9. Blackburn EH, Annu Rev Biochem, 1992, 61, 113-29 Cech TR, et al. , Biochemistry (Moscow), 1997, 62, 1202-5. Holy A, Curr Pharm Des, 2003, 9, 2567-92

  The present invention relates to the preparation of a medicament for improving telomerase processivity in the following acyclic nucleoside purines of formula I:

（式中；
ＸはＯＨ又は−Ｎ（Ｒ^１）_２であり、ここで、
Ｒ^１は独立して、
Ｈ；
Ｃ_２−Ｃ_１５アルキル、Ｃ_３−Ｃ_１５アルケニル、Ｃ_６−Ｃ_１５アリールアルケニル、Ｃ_３−Ｃ_１５アルキニル、Ｃ_７−Ｃ_１５アリールアルキニル、Ｃ_１−Ｃ_６−アルキルアミノ−Ｃ_１−Ｃ_６アルキル、Ｃ_５−Ｃ_１５アラルキル、Ｃ_６−Ｃ_１５ヘテロアルキル若しくはＣ_３−Ｃ_６ヘテロシクロアルキル（但し、ＮＨに隣接していないアルキル成分におけるメチレンは−Ｏ−によって置換されている）；又は
Ｃ_１−Ｃ_１５アルキル、Ｃ_２−Ｃ_１５アルケニル、Ｃ_６−Ｃ_１５アリールアルケニル、Ｃ_６−Ｃ_１５アリールアルキニル、Ｃ_２−Ｃ_１５アルキニル、Ｃ_１−Ｃ_６−アルキルアミノ−Ｃ_１−Ｃ_６アルキル、Ｃ_５−Ｃ_１５アラルキル、Ｃ_６−Ｃ_１５ヘテロアラルキル、Ｃ_４−Ｃ_６アリール、Ｃ_３−Ｃ_８シクロアルキル若しくはＣ_２−Ｃ_６ヘテロシクロアルキル
であるか；
或いは、必要に応じて、両方のＲ^１は一緒になって、１つ若しくは２つのＮヘテロ原子と、必要に応じて更なるＯ若しくはＳヘテロ原子とを含有する飽和若しくは不飽和Ｃ_２−Ｃ_５複素環を形成し；
ここで、前記Ｒ^１基の１つは、ハロ、−ＣＮ又は−Ｎ_３により置換されてもよく；
ＷはＨ又はＮＨ_２であり；
Ｙは独立して、ＯＨ、−ＯＲ^２、−ＯＣＨ（Ｒ^３）ＯＣ（Ｏ）Ｒ^２、モノリン酸、二リン酸、アミノ酸アミダート、ポリペプチドアミダート、−ＮＨＲ^２又は−Ｎ（Ｒ^２）_２であり；
Ｒ^２は独立して、非置換アルキル、アリール、アルケニル、アルキニル、アルカリール、アルキニルアリール若しくはアルケニルアリール；Ｈがハロ、カルボキシ、ヒドロキシル、シアノ、ニトロ、Ｎ−モリホリノ若しくはアミノによって置換されるアルキル、アリール、アルケニル、アルキニル、アルカリール、アルキニルアリール若しくはアルケニルアリール；又は−ＣＨ_２−成分がＮＨ、Ｓ若しくはＯによって置換されているアルキル、アルケニル、アルキニル、アルカリール、アルキニルアリール若しくはアルケニルアリールであり；
Ｚは、１つ以上のヒドロキシにより必要に応じて置換されるＣ_１−Ｃ_６アルキルであり；及び
Ｒ^３はＨ又はＲ^２である）；
或いはそのプロドラッグ、二リン酸、薬学的に許容される塩、又はその他のリン置換誘導体の使用を提供する。

(Where:
X is OH or —N (R ¹ ) ₂ , where
R ¹ is independently
H;
C ₂ -C _{15 alkyl,} C 3 _{-C 15 alkenyl,} C 6 _{-C 15 arylalkenyl,} C 3 _{-C 15 alkynyl,} C 7 _{-C 15 arylalkenyl,} C 1 -C ₆ - alkylamino _-C 1 -C ₆ alkyl, C ₅ -C ₁₅ aralkyl, C ₆ -C ₁₅ heteroalkyl or C ₃ -C ₆ heterocycloalkyl (provided that methylene in the alkyl moiety not adjacent to NH is replaced by —O—); or C ₁ -C _{15 alkyl,} C 2 _{-C 15 alkenyl,} C 6 _{-C 15 arylalkenyl,} C 6 _{-C 15 arylalkenyl,} C 2 _{-C 15 alkynyl,} C 1 -C ₆ - alkylamino -C ₁ - C ₆ alkyl, C ₅ -C ₁₅ aralkyl, C ₆ -C ₁₅ heteroaralkyl, C ₄ -C ₆ aryl, C ₃ -C ₈ or a cycloalkyl or _C 2 -C ₆ heterocycloalkyl;
Alternatively, if desired, both R ^{1 taken} together are saturated or unsaturated C ₂ -C containing one or two N heteroatoms and optionally further O or S heteroatoms. Forming a _5- heterocycle;
Wherein one of said R ¹ groups may be substituted by halo, —CN or —N ₃ ;
W is H or NH ₂ ;
Y is independently OH, —OR ² , —OCH (R ³ ) OC (O) R ² , monophosphate, diphosphate, amino acid amidate, polypeptide amidate, —NHR ² or —N (R ² ). ₂ ;
R ² is independently unsubstituted alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; H, alkyl, aryl substituted by halo, carboxy, hydroxyl, cyano, nitro, N-morpholino or amino , Alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; or alkyl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl in which the —CH ₂ — moiety is substituted by NH, S or O;
Z is C ₁ -C ₆ alkyl optionally substituted with one or more hydroxy; and R ³ is H or R ² );
Alternatively, the use of a prodrug, diphosphate, pharmaceutically acceptable salt, or other phosphorus-substituted derivative is provided.

  The present invention also provides a pharmaceutical composition comprising a compound of formula I, or a prodrug or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

  The present invention also provides a method for increasing the number of mitotic cycles in cells that continue to grow in tissue culture, comprising contacting the cells with a compound of formula I or a prodrug or salt thereof. To do.

  The present invention also provides a method for increasing mitotic cell division of non-dividing cells comprising contacting the cells with a compound of formula I or a prodrug or salt thereof in vitro or in vivo.

  The present invention also provides the use of a compound of formula I or a prodrug or pharmaceutically acceptable salt thereof for the preparation of a medicament that increases the number of mitotic cycles in continuously growing cells.

  The invention also provides the use of a compound of formula I or a prodrug or pharmaceutically acceptable salt thereof for the preparation of a medicament for increasing mitotic cell division of non-dividing cells.

  The present invention also provides novel compounds of formula I as described herein, as well as prodrugs and pharmaceutically acceptable salts thereof.

(Detailed explanation)
The following abbreviations are used herein: ANP: acyclic nucleoside phosphonic acid; ANPpp: ANP diphosphate; TRAP: telomeric repeat amplification protocol; PMEG: 9- [2- (phosphonomethoxy) ethyl] guanine (R) -PMPG: (R) -9- [2- (phosphonomethoxy) propyl] guanine; (R) -HPPMG: (R) -9-[(3-hydroxy-2-phosphonomethoxy) propyl PMEDAP: 2,6-diamino-9- [2- (phosphonomethoxy) ethyl] purine; (S) -PMPG: (S) -9- [2- (phosphonomethoxy) propyl] guanine; S) -HPMPA: (S) -9-[(3-hydroxy-2-phosphonomethoxy) propyl] adenine; PMEO-DAPy: 2,4-diamino-6- [2- (e (R) -6-cyprPMPDAP: (R) -2-amino-6- (cyclopropylamino) -9- [2- (phosphonomethoxy) propyl] purine; (R) -PMPA: (R) -9- [2- (phosphonomethoxy) propyl] adenine; (R) -PMPDAP: (R) -2,6-diamino-9- [2- (phosphonomethoxy) propyl] purine; PMEA: 9- [2- (phosphonomethoxy) ethyl] adenine; PMEC: 1- [2- (phosphonomethoxy) ethyl] cytosine; PMET: 1- [2- (phosphonomethoxy) ethyl] thymine; (S)- PMPA: (S) -9- [2- (phosphonomethoxy) propyl] adenine; 6-Me ₂ PMEDAP: 2-amino-6- (dimethylamino) -9- [2- (phos Phonomethoxy) ethyl] purine; hTERT: human telomerase reverse transcriptase; AZT-TP: 3'-azido-3'-deoxythymidine 5'-triphosphate; ddC: 2 ', 3'-dideoxycytidine; d4TTP: 2' 3,3′-didehydro-2 ′, 3′-deoxythymidine 5′-phosphate; deaza dGTP: 7-deaza-2′-deoxyguanosine 5′-triphosphate; deaza dATP: 7-deaza-2′-deoxy Adenosine 5′-triphosphate; thio dGTP: 6-thio-2′-deoxyguanosine 5′-triphosphate; CBV-TP: 2 ′, 3′-didehydro-2 ′, 3′-dideoxyguanosine 5′- AraGTP: 9-β-D-arabinofuranosylguanine 5′-triphosphate; FaraTTP: 2′-fluoro-2′-deoxy β-D-arabinofuranosylthymine 5′-triphosphate CHAPS: 3 - [(3- cholamidopropyl) dimethyl ammonio] -1-propanesulfonic acid; and TS: telomerase substrate (non-telomeric oligonucleotide).

  Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings:

  As used herein, the term “prodrug” refers to a drug substance (as a result of a spontaneous chemical reaction, enzyme-catalyzed chemical reaction, photolysis, and / or metabolic chemical reaction when administered to a biological system. That is, it refers to any compound that produces an active ingredient). Thus, a prodrug is an analog or latent form modified by covalent attachment of a compound having therapeutic activity.

  A “prodrug component” refers to an unstable functional group that separates from an activity-inhibiting compound and becomes a cell systemically during metabolism by hydrolysis, enzymatic cleavage, or some other process (Bundgaad, Hans, “Design”). and Application of Prodrugs ", A Textbook of Drug Design and Development (1991), P. Krogsgaard-Larsen and H. Bundgad, H. Bundgad, H. Bundgad, Ed. Enzymes capable of enzyme activation by phosphonic acid compounds include, but are not limited to, amidase, esterase, microbial enzyme, phospholipase, cholinesterase and phosphatase. Prodrug ingredients can help to improve solubility, absorption and lipophilicity to optimize drug delivery, bioavailability and efficacy. Prodrug components may include active metabolites or the drug itself.

Exemplary prodrug components include acyloxymethyl esters —CH ₂ OC (═O) R ⁹ and acyloxymethyl carbonate —CH ₂ OC (═O) OR ⁹ that are susceptible to hydrolysis or unstable, where R ⁹ is C ₁ -C ₆ alkyl, C ₁ -C ₆ substituted alkyl, C ₆ -C ₂₀ aryl or C ₆ -C ₂₀ substituted aryl). Acyloxyalkyl esters were first used as a prodrug method for carboxylic acids by Farquar et al. And then applied to phosphoric and phosphonic acids (Farquahar, et al. (1983) J. Pharm. Sci, 72: 324; and U.S. Pat. Nos. 4,816,570, 4,968,788, 5,663,159 and 5,792,756). Subsequently, acyloxyalkyl esters were used to deliver phosphonic acid across cell membranes and to improve oral bioavailability. A close variant of the acyloxyalkyl ester, namely alkoxycarbonyloxyalkyl ester (carbonic acid), may also improve oral bioavailability as a prodrug component in the compounds of the combination of the present invention. Representative acyloxymethyl esters, pivaloyloxymethoxy _{(POM) -CH 2 OC (=} O) C (CH 3) 3. A representative acyloxymethyl carbonate prodrug component is pivaloyloxymethyl carbonate (POC) —CH ₂ OC (═O) OC (CH ₃ ) ₃ .

  Aryl esters of phosphate groups (especially phenyl esters) have been reported to improve oral bioavailability (De Lombert, et al. (1994) J. Med. Chem., 37: 498). ). Phenyl esters containing carboxylic acid esters in the ortho position of phosphoric acid have also been described (Khamnei and Torrance, (1996) J. Med. Chem., 39: 4109-4115). Benzyl esters have been reported to produce the parent phosphonic acid. If necessary, substituents at the ortho or para position may accelerate the hydrolysis. Benzyl analogs with acylated phenols or alkylated phenols may produce phenolic compounds through the action of enzymes (eg, esterases, oxidases, etc.), and then phenolic compounds are bound by benzylic C—O bonds. Cleavage occurs, producing phosphoric acid and quinone methide intermediates. For examples of this type of prodrug, see Mitchell, et al. (1992) J. Org. Chem. Soc. Perkin Trans. II, 2345; Glazier (WO 91/19721). Still other benzylic prodrugs containing carboxylic ester-containing groups attached to the methylene of benzyl have been described (Glazier, WO 91/19721). Thio-containing prodrugs have been reported to be useful for intracellular delivery of phosphonic acid drugs. These proesters contain an ethylthio group, in which case the thiol group is esterified with an acyl group or mixed with another thiol group to form a disulfide. Deesterification or reduction of the disulfide produces a free thiol intermediate which is then broken down into phosphoric acid and episulfide (Puech, et al. (1993) Antivirus Res., 22: 155-174; Benzaria, et al. (1996) J. Med. Chem. 39: 4958). Cyclic phosphonates have also been described as prodrugs of phosphorus-containing compounds (Erion, et al., US Pat. No. 6,312,661).

Examples of pharmaceutically acceptable salts include suitable bases such as alkali metals (eg, sodium), alkaline earths (eg, magnesium), ammonium and NX ₄ ⁺ , where X is C ₁ -C Salts derived from ₄ alkyl). Pharmaceutically acceptable salts of compounds having an amino group include organic carboxylic acids (eg acetic acid, benzoic acid, lactic acid, fumaric acid, tartaric acid, maleic acid, malonic acid, malic acid, isethionic acid, lactobionic acid and succinic acid. Acid); organic sulfonic acids (eg, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc.); and salts of inorganic acids (eg, hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid) It is. Pharmaceutically acceptable salts of compounds having a hydroxy group include suitable cations (eg, Na ⁺ and NX ₄ ⁺ , wherein X is independently selected from H or a C ₁ -C ₄ alkyl group. )) In combination with the anion of said compound. In therapeutic applications, the active ingredient salts are generally pharmaceutically acceptable. That is, the salt of the active ingredient is a salt derived from a pharmaceutically acceptable acid or base.

“Alkyl” is a branched or unbranched hydrocarbon containing normal, secondary or tertiary carbon atoms. Examples include methyl (Me, _-CH 3), ethyl _{(Et, -CH 2 CH 3)} , 1- propyl (n-Pr, _{n-propyl, -CH 2 CH 2 CH 3)} , 2- propyl (i -pr, _{i-propyl, -CH (CH 3) 2)} , 1- butyl (n-Bu, n- _{butyl, -CH 2 CH 2 CH 2 CH} 3), 2- methyl-1-propyl (i-Bu , i- _{butyl, -CH 2 CH (CH 3)} 2), 2- butyl (s-Bu, s- _{butyl, -CH (CH 3) CH 2} CH 3), 2- methyl-2-propyl (t- Bu, t-butyl, —C (CH ₃ ) ₃ ), 1-pentyl (n-pentyl, —CH ₂ CH ₂ CH ₂ CH ₂ CH ₃ ), 2-pentyl (—CH (CH ₃ ) CH ₂ CH ₂ CH _3), 3- pentyl _{(-CH (CH 2 CH 3)} 2), 2-methyl-2-butyl _{(-C (CH 3) 2 CH} 2 CH 3), 3- methyl-2-butyl _{(-CH (CH 3) CH (} CH 3) 2), 3- methyl-1-butyl _{(-CH 2 CH 2 CH (CH} 3) 2), 2- methyl-1-butyl _{(-CH 2 CH (CH 3)} CH 2 CH 3), 1- hexyl _{(-CH 2 CH 2 CH 2 CH} 2 CH ₂ CH _3), 2- hexyl _{(-CH (CH 3) CH 2} CH 2 CH 2 CH 3), 3- hexyl _{(-CH (CH 2 CH 3)} (CH 2 CH 2 CH 3)), 2- methyl 2-pentyl _{(-C (CH 3) 2 CH} 2 CH 2 CH 3), 3- methyl-2-pentyl _{(-CH (CH 3) CH (} CH 3) CH 2 CH 3), 4- methyl-2 - pentyl _{(-CH (CH 3) CH 2} CH (CH 3 _2), 3-methyl-3-pentyl _{(-C (CH 3) (CH} 2 CH 3) 2), 2- methyl-3-pentyl _{(-CH (CH 2 CH 3)} CH (CH 3) 2), 2,3-dimethyl-2-butyl (—C (CH ₃ ) ₂ CH (CH ₃ ) ₂ ), 3,3-dimethyl-2-butyl (—CH (CH ₃ ) C (CH ₃ ) ₃ ) .

“Alkenyl” is a branched or unbranched carbon containing a normal, secondary or tertiary carbon atom with at least one unsaturation (ie, a carbon-carbon sp ² double bond). Hydrogen. Examples include ethylene or vinyl (—CH═CH ₂ ), allyl (—CH ₂ CH═CH ₂ ), cyclopentenyl (—C ₅ H ₇ ), 5-hexenyl (—CH ₂ CH ₂ CH ₂ CH _2). CH = CH ₂₎ and 2,5-hexadienyl _{(-CH 2 CH = CHCH 2 CH} = CH 2) include, but are not limited to.

“Alkynyl” is a branched or unbranched hydrocarbon containing normal, secondary or tertiary carbon atoms with at least one unsaturation (ie, carbon-carbon sp triple bond). is there. Examples include, but are not limited to, acetylene (—C≡CH), propargyl (—CH ₂ C≡CH), and 2,5-hexadiynyl (—CH ₂ C≡CHCH ₂ C≡CH).

  “Aryl” means a monovalent aromatic hydrocarbon group having 6 to 20 carbon atoms, obtained by removing one hydrogen atom from one carbon atom of a parent aromatic ring system. To do. Representative aryl groups include, but are not limited to, groups derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like.

“Aralkyl” refers to an alkyl group in which one of the hydrogen atoms bonded to a carbon atom (generally a terminal or sp ³ carbon atom) is replaced by an aryl group. Representative arylalkyl groups include benzyl, 2-phenylethane-1-yl, naphthylmethyl, 2-naphthylethane-1-yl, naphthobenzyl, 2-naphthphenylethane-1-yl, and the like, It is not limited to these. An arylalkyl group contains 6 to 20 carbon atoms, for example, an alkyl component of an arylalkyl group (including an alkanyl, alkenyl or alkynyl group) contains 1 to 6 carbon atoms and an aryl component of 5 Contains from 14 to 14 carbon atoms.

“Arylalkenyl” refers to an alkenyl group in which one of the hydrogen atoms bonded to a carbon atom (generally a terminal or sp ³ carbon atom) is replaced by an aryl group.

“Arylalkynyl” refers to an alkynyl group in which one of the hydrogen atoms bonded to a carbon atom (generally the terminal or sp ³ carbon atom) is replaced by an aryl group.

  As used herein, “heterocycle” or “heterocyclo” includes, for example, Paquette, Leo A. et al. The Principles of Modern Heterocyclic Chemistry (WA Benjamin, New York, 1968) (especially Chapter 1, 3, 4, 6, 7 and 9); The Chemistry of He Hes. , New York, 1950 to present) (in particular, Volumes 13, 14, 16, 19 and 28); Am. Chem. Soc. (1960), 82: 5566, including but not limited to these heterocycles. In one specific embodiment of the invention, a “heterocycle” has one or more (eg, 1, 2, 3 or 4) carbon atoms heteroatoms (eg, O, N or Included are “carbocycles” as defined herein, which are substituted by S).

  Examples of heterocycles include, for example, pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl), thiazolyl, tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, Thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl Nil, azosinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H, 6H-1,5,2-dithiazinyl, thienyl, Anthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxatinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolidinyl, phthalazinyl, naphthyridinyl Quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochrominyl, chrominyl, imidazolinyl Pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindo Linyl, quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxyindolyl, benzoxazolinyl, isatinoyl and bis-tetrahydrofuranyl:

が含まれるが、これらに限定されない。

Is included, but is not limited thereto.

  The term “treatment” or “treating” includes, only when associated with a disease or condition, preventing the onset of the disease or condition, inhibiting the disease or condition, eliminating the disease or condition, and It includes alleviating one or more symptoms of a disease or condition.

  The stereochemical definitions and conventions used herein are generally described in S.H. P. Parker, Ed. McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E .; and Wilen, S .; , Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc. , New York. Many organic compounds exist in a form having optical activity. That is, many organic compounds have the ability to rotate the plane of plane-polarized light. In describing compounds with optical activity, the D and L or R and S prefixes are used to indicate the absolute configuration of the molecule around its chiral center. The d and l or (+) and (-) prefixes are used to indicate the sign of rotation of plane polarized light by the compound, where (-) or l means that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. In a particular chemical structure, these stereoisomers are identical except that they are mirror images of one another. Certain stereoisomers are also sometimes referred to as enantiomers, and mixtures of such isomers are often referred to as enantiomeric mixtures. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate and may occur when no stereoselection or stereospecificity is present in a chemical reaction or process. The terms “racemic mixture” and “racemate” refer to an equimolar amount of a mixture of two enantiomeric species that do not have optical activity.

  A variety of protecting groups are available, well known and used, and used as needed to prevent side reactions with protecting groups in synthetic procedures, ie, routes or methods for preparing compounds. Is done. In many cases, decisions regarding the protecting group, the timing to protect, and the nature of the chemical protecting group PG are determined by the chemical nature of the reaction being protected (eg, acidic, basic, oxidation, reduction or other conditions), and Depending on the intended purpose of the synthesis. When a compound is substituted with more than one PG, these PG groups need not be the same and are generally not the same. Generally, PG is used to protect functional groups (eg, carboxyl, hydroxyl, thio or amino groups), thereby preventing side reactions or otherwise promoting synthesis efficiency. . The order of deprotection to produce a free deprotecting group depends on the intended purpose of the synthesis and the reaction conditions spoken, and may be performed in any order determined by one skilled in the art. Protecting groups and the corresponding chemical cleavage reactions are numerous, and are described in Protective Groups in Organic Synthesis, Theodora W. et al. Green (John Wiley & Sons, Inc., New York, 1991, ISBN 0-471-62301-6) ("Green"). Kocienski, Philip J. et al. Also see Protective Groups (Georg Thime Verlag Stuttgart, New York, 1994).

Examples of suitable amino acids where the residue in Formula I may be represented by Y include: glycine; aminopolycarboxylic acids (eg, aspartic acid, β-hydroxyaspartic acid, glutamic acid, β-hydroxyglutamic acid) , Β-methylaspartic acid, β-methylglutamic acid, β, β-dimethylaspartic acid, γ-hydroxyglutamic acid, β, γ-dihydroxyglutamic acid, β-phenylglutamic acid, γ-methyleneglutamic acid, 3-aminoadipic acid, 2- Aminopimelic acid, 2-aminosuberic acid and 2-aminosebacic acid); amino acid amides (eg glutamine and asparagine); polyaminomonocarboxylic acids or polybasic monocarboxylic acids (eg arginine, lysine, β-aminoalanine, γ) -Aminobutyrin, ol Tin, citrulline, homoarginine, homocitrulline, hydroxylysine, allohydroxylysine and diaminobutyric acid); other basic amino acid residues (eg histidine); diaminodicarboxylic acids (eg α, α′-diaminosuccinic acid, α , Α′-diaminoglutaric acid, α, α′-diaminoadipic acid, α, α′-diaminopimelic acid, α, α′-diamino-β-hydroxypimelic acid, α, α′-diaminosuberic acid, α, α'-diaminoazelineic acid and α, α'-diaminosebacic acid); imino acids (eg proline, hydroxyproline, allohydroxyproline, γ-methylproline, pipecolic acid, 5-hydroxypipecolic acid and azetidine-2- acid); monoalkyl or dialkyl _(generally, branched or normal) amino of C 1 -C ₈ Acids (eg, alanine, valine, leucine, allylglycine, butyrin, norvaline, norleucine, heptillin, α-methylserine, α-amino-α-methyl-γ-hydroxyvaleric acid, α-amino-α-methyl-δ-hydroxy) Valeric acid, α-amino-α-methyl-ε-hydroxycaproic acid, isovaline, α-aminoglutamic acid, α-aminoisobutyric acid, α-aminodiethylacetic acid, α-aminodiisopropylacetic acid, α-aminodi-n-propylacetic acid, α-aminodiisobutylacetic acid, α-aminodi-n-butylacetic acid, α-aminoethylisopropylacetic acid, α-amino-n-propylacetic acid, α-aminodiisoamylacetic acid, α-methylaspartic acid, α-methylglutamic acid, 1 -Aminocyclopropane-1-carboxylic acid, isoleucine, alloisoleucine, tert-leucine, β-methyltryptophan and α-amino-β-ethyl-β-phenylpropionic acid); β-phenylserinyl; aliphatic α-amino-β-hydroxy acids (eg serine, β-hydroxyleucine, β-hydroxynorleucine, β-hydroxynorvaline and α-amino-β-hydroxystearic acid); α-aminohydroxy acid, γ-hydroxy acid, δ-hydroxy acid or ε-hydroxy acid (eg, homoserine residue, δ-hydroxynorvaline residue, γ-hydroxynorvaline residue and ε-hydroxynorleucine residue); canabin and canalin; γ-hydroxyornithine; 2-hexosamic acid (eg D-glucosamic acid or D-galactosamic acid) ); Α-amino-β-thiols (eg penicillamine, β-thiolno) Other sulfur-containing amino acid residues (including cysteine); homocystin, β-phenylmethionine, methionine, S-allyl-L-cysteine sulfoxide, 2-thiol histidine, cystathionine, and cysteine or Thiol esters of homocysteine; phenylalanine, tryptophan and ring-substituted α-amino acids (eg, phenyl amino acids or cyclohexyl amino acids, α-aminophenylacetic acid, α-aminocyclohexyl acetic acid, and α-amino-β-cyclohexylpropionic acid); aryl, Phenylalanine analogs and phenylalanine derivatives (eg, tyrosine, methylthio), including lower alkyl, hydroxy, guanidino, oxyalkyl ether, nitro, sulfur or halo-substituted phenyl. Syn, and o-chlorophenylalanine, p-chlorophenylalanine, 3,4-dichlorophenylalanine, o-methylphenylalanine, m-methylphenylalanine, p-methylphenylalanine, 2,4,6-trimethylphenylalanine, 2-ethoxy-5- Nitrophenylalanine, 2-hydroxy-5-nitrophenylalanine and p-nitrophenylalanine); furylalanine, thienylalanine, pyridylalanine, pyrimidinylalanine, purinylalanine or naphthylalanine; and tryptophan analogs and tryptophan derivatives (kynurenine, 3-hydroxykynurenine) , 2-hydroxytryptophan and 4-carboxytryptophan); α-amino substituted amino acids (sarcosine (N-methyl group) Syn), N-benzylglycine, N-methylalanine, N-benzylalanine, N-methylphenylalanine, N-benzylphenylalanine, N-methylvaline and N-benzylvaline); and α-hydroxy amino acids and substituted α-hydroxy Amino acids (including serine, threonine, allothreonine, phosphoserine and phosphothreonine).

  A polypeptide is a polymer of amino acids in which the carboxyl group of one amino acid monomer is bonded to the amino or imino group of the next amino acid monomer by an amide bond. Polypeptides include dipeptides, low molecular weight polypeptides (about 1500-5000 MW) and proteins. The protein optionally contains 3, 5, 10, 50, 75, 100 or more residues, preferably substantially human, animal, plant or microbial protein. Has sequence homology. Proteins include enzymes (hydrogen peroxidase), as well as immunogens (eg, KLH), or any type of antibody or protein that is desired to enhance the immune response. The nature and nature of a polypeptide can vary widely.

  Various peptide-degrading enzymes for cleaving polypeptide conjugates are well known and include in particular carboxypeptidases. Carboxypeptidases digest polypeptides by removing the C-terminal residue and are often specific for a particular C-terminal sequence. Such enzymes and their substrate requirements are generally well known. For example, a dipeptide (having a specific residue pair and a free carboxyl terminus) is covalently bonded to the phosphorus atom or carbon atom of the compounds described herein via its α-amino group. This peptide can be cleaved by an appropriate peptidolytic enzyme, which leaves the carboxyl of the proximal amino acid residue and cleaves the phosphonoamidate bond by autocatalysis.

  Suitable dipeptidyl groups (indicated by their single letter symbols) include AA, AR, AN, AD, AC, AE, AQ, AG, AH, AI, AL, AK, AM, AF, AP, AS, AT , AW, AY, AV, RA, RR, RN, RD, RC, RE, RQ, RG, RH, RI, RL, RK, RM, RF, RP, RS, RT, RW, RY, RV, NA, NR , NN, ND, NC, NE, NQ, NG, NH, NI, NL, NK, NM, NF, NP, NS, NT, NW, NY, NV, DA, DR, DN, DD, DC, DE, DQ , DG, DH, DI, DL, DK, DM, DF, DP, DS, DT, DW, DY, DV, CA, CR, CN, CD, CC, CE, CQ, CG, CH, CI, CL, CK , CM, CF, CP, CS, CT, CW, CY, CV, A, ER, EN, ED, EC, EE, EQ, EG, EH, EI, EL, EK, EM, EF, EP, ES, ET, EW, EY, EV, QA, QR, QN, QD, QC, QE, QQ, QG, QH, QI, QL, QK, QM, QF, QP, QS, QT, QW, QY, QV, GA, GR, GN, GD, GC, GE, GQ, GG, GH, GI, GL, GK, GM, GF, GP, GS, GT, GW, GY, GV, HA, HR, HN, HD, HC, HE, HQ, HG, HH, HI, HL, HK, HM, HF, HP, HS, HT, HW, HY, HV, IA, IR, IN, ID, IC, IE, IQ, IG, IH, II, IL, IK, IM, IF, IP, IS, IT, IW, IY, IV, LA, LR, LN, LD, LC, LE, LQ, LG, LH, LI, LL, K, LM, LF, LP, LS, LT, LW, LY, LV, KA, KR, KN, KD, KC, KE, KQ, KG, KH, KI, KL, KK, KM, KF, KP, KS, KT, KW, KY, KV, MA, MR, MN, MD, MC, ME, MQ, MG, MH, MI, ML, MK, MM, MF, MP, MS, MT, MW, MY, MV, FA, FR, FN, FD, FC, FE, FQ, FG, FH, FI, FL, FK, FM, FF, FP, FS, FT, FW, FY, FV, PA, PR, PN, PD, PC, PE, PQ, PG, PH, PI, PL, PK, PM, PF, PP, PS, PT, PW, PY, PV, SA, SR, SN, SD, SC, SE, SQ, SG, SH, SI, SL, SK, SM, SF, SP, SS, ST, SW, SY, SV, TA, TR, T N, TD, TC, TE, TQ, TG, TH, TI, TL, TK, TM, TF, TP, TS, TT, TW, TY, TV, WA, WR, WN, WD, WC, WE, WQ, WG, WH, WI, WL, WK, WM, WF, WP, WS, WT, WW, WY, WV, YA, YR, YN, YD, YC, YE, YQ, YG, YH, YI, YL, YK, YM, YF, YP, YS, YT, YW, YY, YV, VA, VR, VN, VD, VC, VE, VQ, VG, VH, VI, VL, VK, VM, VF, VP, VS, VT, VW, VY and VV are included. Tripeptide residues are also useful as protecting groups.

  The dipeptide or tripeptide species can be selected based on known transport properties and / or peptidase sensitivity that may affect transport to the intestinal mucosa or other cell types. Dipeptides and tripeptides lacking the α-amino group are transport substrates for peptide transport factors present in the brush border membrane of intestinal mucosal cells (Bai, JP, (1992) Pharm Res. 9: 969-978). Thus, transport component peptides can be used to improve the bioavailability of the compounds of the present invention. Dipeptides or tripeptides having one or more amino acids in the D conformation are also compatible with peptide transport and can be utilized in the compounds of the present invention. D conformational amino acids can be used to reduce the sensitivity of dipeptides or tripeptides to hydrolysis by proteases common in brush border membranes (eg, aminopeptidase N). In addition, dipeptides or tripeptides are alternatively selected based on their relative resistance to hydrolysis by proteases present in the intestinal lumen. For example, a tripeptide or polypeptide lacking asp and / or glu is a poor substrate for aminopeptidase A and is an amino acid residue on the N-terminal side of a hydrophobic amino acid (leu, tyr, phe, val, trp) Dipeptides or tripeptides lacking are a poor substrate for endopeptidase, and peptides lacking the pro residue second from the end of the free carboxyl terminus are poor substrates for carboxypeptidase P. Similar considerations can also be applied to the selection of peptides that are relatively resistant or relatively sensitive to hydrolysis by cytoplasm, kidney, liver, serum or other peptidases. Such polypeptide amidates that are not cleaved well are immunogens or are useful for conjugation to proteins to prepare immunogens.

(Specific Embodiment of the Present Invention)
The specific values listed for groups, substituents and ranges, and exemplary embodiments of the invention described herein are for illustrative purposes only; other defined values, or It does not exclude other values within the defined range.

  A specific acyclic nucleoside purine is a compound of formula I:

（式中；
ｎは２又は３であり；
Ｘは−Ｎ（Ｒ^１）_２であり、この場合、
Ｒ^１は独立して、
Ｈ；
Ｃ_２−Ｃ_１５アルキル、Ｃ_３−Ｃ_１５アルケニル、Ｃ_６−Ｃ_１５アリールアルケニル、Ｃ_３−Ｃ_１５アルキニル、Ｃ_７−Ｃ_１５アリールアルキニル、Ｃ_１−Ｃ_６−アルキルアミノ−Ｃ_１−Ｃ_６アルキル、Ｃ_５−Ｃ_１５アラルキル、Ｃ_６−Ｃ_１５ヘテロアルキル若しくはＣ_３−Ｃ_６ヘテロシクロアルキル（但し、ＮＨに隣接していないアルキル成分におけるメチレンは−Ｏ−によって置換されている）；又は
Ｃ_１−Ｃ_１５アルキル、Ｃ_２−Ｃ_１５アルケニル、Ｃ_６−Ｃ_１５アリールアルケニル、Ｃ_６−Ｃ_１５アリールアルキニル、Ｃ_２−Ｃ_１５アルキニル、Ｃ_１−Ｃ_６−アルキルアミノ−Ｃ_１−Ｃ_６アルキル、Ｃ_５−Ｃ_１５アラルキル、Ｃ_６−Ｃ_１５ヘテロアラルキル、Ｃ_４−Ｃ_６アリール、Ｃ_２−Ｃ_６ヘテロシクロアルキル
であるか；
或いは、必要に応じて、両方のＲ^１は一緒になって、１つ若しくは２つのＮヘテロ原子と、必要に応じて更なるＯヘテロ原子若しくはＳヘテロ原子とを含有する飽和若しくは不飽和Ｃ_２−Ｃ_５複素環を形成し；
この場合、前記Ｒ^１基の１つは、ハロ、−ＣＮ又は−Ｎ_３により置換されてもよいが、何れか１つ又は２つのＲ^１基はＨではなく；
Ｙは独立して、ＯＨ、−ＯＲ^２、−ＯＣＨ（Ｒ^３）ＯＣ（Ｏ）Ｒ^２、モノリン酸、二リン酸、アミノ酸アミダート、ポリペプチドアミダート、−ＮＨＲ^２又は−Ｎ（Ｒ^２）_２であり；
Ｒ^２は独立して、非置換アルキル、アリール、アルケニル、アルキニル、アルカリール、アルキニルアリール若しくはアルケニルアリール；Ｈがハロ、カルボキシ、ヒドロキシル、シアノ、ニトロ、Ｎ−モリホリノ若しくはアミノによって置換されるアルキル、アリール、アルケニル、アルキニル、アルカリール、アルキニルアリール若しくはアルケニルアリール；又は−ＣＨ_２−成分がＮＨ、Ｓ若しくはＯによって置換されているアルキル、アルケニル、アルキニル、アルカリール、アルキニルアリール若しくはアルケニルアリールであり；
Ｒ^３はＨ又はＲ^２である）；
或いはそのプロドラッグ、二リン酸又はその他のリン置換誘導体である。

(Where:
n is 2 or 3;
X is —N (R ¹ ) ₂ , where
R ¹ is independently
H;
C ₂ -C _{15 alkyl,} C 3 _{-C 15 alkenyl,} C 6 _{-C 15 arylalkenyl,} C 3 _{-C 15 alkynyl,} C 7 _{-C 15 arylalkenyl,} C 1 -C ₆ - alkylamino _-C 1 -C ₆ alkyl, C ₅ -C ₁₅ aralkyl, C ₆ -C ₁₅ heteroalkyl or C ₃ -C ₆ heterocycloalkyl (provided that methylene in the alkyl moiety not adjacent to NH is replaced by —O—); or C ₁ -C _{15 alkyl,} C 2 _{-C 15 alkenyl,} C 6 _{-C 15 arylalkenyl,} C 6 _{-C 15 arylalkenyl,} C 2 _{-C 15 alkynyl,} C 1 -C ₆ - alkylamino -C ₁ - C ₆ alkyl, C ₅ -C ₁₅ aralkyl, C ₆ -C ₁₅ heteroaralkyl, C ₄ -C ₆ aryl, C ₂ -C ₆ either heterocycloalkyl;
Alternatively, if desired, both R ^{1 taken} together are saturated or unsaturated C ₂ containing one or two N heteroatoms and optionally further O or S heteroatoms. -C ₅ heterocyclic ring is formed;
In this case, one of said R ¹ groups may be substituted by halo, —CN or —N _3, but either one or two R ¹ groups are not H;
Y is independently OH, —OR ² , —OCH (R ³ ) OC (O) R ² , monophosphate, diphosphate, amino acid amidate, polypeptide amidate, —NHR ² or —N (R ² ). ₂ ;
R ² is independently unsubstituted alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; H, alkyl, aryl substituted by halo, carboxy, hydroxyl, cyano, nitro, N-morpholino or amino , Alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; or alkyl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl in which the —CH ₂ — moiety is substituted by NH, S or O;
R ³ is H or R ² );
Alternatively, it is a prodrug, diphosphate or other phosphorus-substituted derivative.

  Another specific acyclic nucleoside purine of formula I is a compound of the following formula:

或いはそのプロドラッグ又は薬学的に許容される塩である。

Alternatively, it is a prodrug or a pharmaceutically acceptable salt thereof.

  Another specific acyclic nucleoside purine is a compound of the following chemical formula:

又はそのプロドラッグである。

Or a prodrug thereof.

A specific value for each R ¹ is independently H, C ₁ -C ₁₅ alkyl, C ₂ -C ₁₅ alkenyl, C ₂ -C ₁₅ alkynyl or C ₃ -C ₈ cycloalkyl, or as required Where appropriate, both R ¹ together form a saturated or unsaturated C ₂ -C ₅ heterocycle containing one or two N heteroatoms and optionally further O or S heteroatoms. Form.

A specific value for each R ¹ is independently H, methyl or cyclopropyl.

A specific value for each R ¹ is independently H.

A specific value for each R ¹ is independently methyl.

A specific value for ^one R ¹ is H, and a specific value for the other R ¹ is cyclopropyl.

  A specific value of W is H.

A specific value for W is NH _2.

  A specific value of X is OH.

A specific value for Z is —CH ₂ —CH ₂ —, —CH ₂ —CH (CH ₃ ) — or —CH ₂ —CH (CH ₂ OH) —.

  A specific value of n is 2.

  A specific value of n is 3.

A specific value for Z is the S-enantiomer of —CH ₂ —CH (CH ₃ ) — or —CH ₂ —CH (CH ₂ OH) —.

A specific compound of formula I is 6-Me ₂ PMEDAPpp or a pharmaceutically acceptable salt or prodrug thereof.

  A specific compound of formula I is (S) -PMPApp or a pharmaceutically acceptable salt or prodrug thereof.

  A specific compound of formula I is (S) -PMPA or a pharmaceutically acceptable salt or prodrug thereof.

A specific compound of formula I is 6-Me ₂ PMEDAPpp or a prodrug thereof.

  A specific compound of formula I is (S) -PMPApp or a prodrug thereof.

  A specific compound of formula I is (S) -PMPA or a prodrug thereof.

  The compounds of the present invention may have a chiral center (eg, a chiral carbon atom or a phosphorus atom). Accordingly, the compounds of the present invention include racemic mixtures of all stereoisomers (including enantiomers, diastereomers and atropisomers). In addition, the compounds of the present invention include enriched or resolved optical isomers at any asymmetric chiral atom or at all asymmetric chiral atoms. In other words, the chiral centers that are apparent from the description are provided as chiral isomers or racemic mixtures. Both racemic and diastereomeric mixtures, as well as isolated or synthesized individual optical isomers substantially free of their enantiomeric or diastereomeric partners are all within the scope of the present invention. Racemic mixtures can be prepared according to known techniques, for example by separating diastereomeric salts formed with optically active auxiliary components (eg acids or bases), followed by conversion to optically active substances, etc. Separated into optically pure isomers. In most cases, the desired optical isomer is synthesized by a stereospecific reaction initiated using the appropriate stereoisomer of the desired starting material.

  The compounds of the invention can also exist as tautomers in some cases. Although only one delocalized resonance structure may be shown, all such forms are contemplated within the scope of the present invention. For example, ene-amine tautomers can exist for purine, pyrimidine, imidazole, guanidine, amidine, and tetrazole, and all their possible tautomeric forms are in accordance with the present invention. Within the scope of

The present invention also provides salts of compounds of formula (I), in particular pharmaceutically acceptable non-toxic salts containing, for example, Na ⁺ , Li ⁺ , K ⁺ , Ca ⁺² and Mg ⁺² . Such salts include a combination of an appropriate cation (eg, alkali metal ions and alkaline earth metal ions, or ammonium ions or quaternary amino ions) and an acid anion component (generally a carboxylic acid). Derived salts can be included. When a water-soluble salt is desired, a monovalent salt is preferred.

Metal salts are generally prepared by reacting a metal hydroxide with a compound of the present invention. Examples of metal salts prepared by this method include salts containing Li ⁺ , Na ⁺ and K ⁺ . Less soluble metal salts can be precipitated from a more soluble salt solution by the addition of a suitable metal compound.

In addition, salts of certain organic and inorganic acids (eg, HCl, HBr, H ₂ SO ₄ , H ₃ PO ₄ or organic sulfonic acids) to basic centers (generally amines) or acidic groups Can be formed from acid addition. Finally, the compositions herein include the compound in its non-ionized form as well as the zwitterionic form, and the compound can be combined with a stoichiometric amount of water, such as a hydrate. Must be understood to include in combination.

  The scope of the present invention also includes salts formed with one or more amino acids. Any of the above amino acids are suitable (especially natural amino acids present as protein components). However, amino acids generally have side chains with basic or acidic groups (eg lysine, arginine or glutamic acid) or those with side groups with neutral groups (eg glycine, serine, Threonine, alanine, isoleucine or leucine).

  The compounds of the present invention can be formulated with conventional carriers and excipients selected according to conventional methods. Tablets contain excipients, glidants, fillers, binders and the like. Aqueous formulations are prepared in sterile form and are generally isotonic when intended for delivery by other than oral administration. All formulations optionally contain various excipients, such as those shown in Handbook of Pharmaceutical Excipients (1986). Excipients include ascorbic acid and other antioxidants, chelating agents (eg EDTA), carbohydrates (eg dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose), stearic acid and the like. The pH of the formulation ranges from about 3 to about 11, but is usually about 7-10.

  While it is possible for the active ingredient to be administered alone, it may be preferable to provide the active ingredient as a pharmaceutical formulation. The formulations of the present invention (both those used for animals and those used for humans) are at least one active ingredient as defined above, and one or more acceptable for it. It is included with the carrier and optionally other therapeutic ingredients. The carrier must be “acceptable” in the sense of being compatible with the other active ingredients of the formulation and physiologically innocuous to the recipient.

  Formulations include those suitable for the aforementioned route of administration. The formulations can conveniently be presented in unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. Various techniques and formulations are generally found in Remington's Pharmaceutical Sciences (Mack Publishing Co. [Easton, PA, USA)]. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, formulations are made by bringing the active ingredient uniformly and thoroughly together with the liquid phase carrier or finely divided solid phase carrier or both, and then, if necessary, shaping the product. Prepared.

  Formulations of the present invention suitable for oral administration are as individual units (eg, capsules, cachets or tablets) containing a predetermined amount of active ingredient, as powders or granules, or as aqueous liquids or It can be provided as a solution or suspension in a non-aqueous liquid, or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can also be administered as a bolus, electuary or paste.

  A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets contain the active ingredient in free-flowing form (eg powders or granules), optionally mixed with binders, lubricants, inert diluents, preservatives, surfactants or dispersants. It can be prepared by compression in a suitable apparatus. Molded tablets can be made by molding in a suitable apparatus a mixture of the powdered active ingredient moistened with an inert liquid diluent. These tablets can be coated or scored as needed and are optionally formulated to provide slow or controlled release of the active ingredient from the tablet.

  For administration to the eyes or other external tissues (eg mouth and skin), the formulation preferably contains the active ingredient in an amount (for example 0.075% w / w to 20% w / w). Is an active ingredient in a range between 0.1% and 20% in increments of 0.1% w / w, for example, an active ingredient at 0.6% w / w, 0.7% w / w, etc. Preferably, in an amount of 0.2% w / w to 15% w / w, most preferably in an amount of 0.5% w / w to 10% w / w Applied as an ointment or cream. When formulated in an ointment, the active ingredient can be used with either a paraffinic ointment base or a water-miscible ointment base. Or an active ingredient can be mix | blended with a cream with an oil-in-water type cream base.

  If desired, the aqueous phase of the cream base may be, for example, at least 30% w / w polyhydric alcohol (ie, an alcohol having two or more hydroxyl groups, such as propylene glycol, butane-1,3-diol. , Mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400)) and mixtures thereof. Topical formulations may desirably include compounds that enhance the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

  The oily phase of the emulsions of the present invention can be composed of known ingredients in a known manner. While the oil phase may simply contain an emulsifier (which is otherwise known as emulgent), the oil phase desirably includes fat or oil, or both fat and oil A mixture of at least one emulsifier with Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier that acts as a stabilizer. It is also preferred to include both oil and fat. In summary, an emulsifier with or without a stabilizer constitutes a so-called emulsifying wax, and the wax combined with oil and fat forms the oily dispersed phase of the cream formulation. Consists of an ointment base for emulsification.

  Emulgent and emulsion stabilizers suitable for use in the formulations of the present invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and lauryl sulfate. Contains sodium.

  The selection of a suitable oil or fat for the formulation is based on achieving the desired cosmetic properties. The cream should preferably be a product that has a suitable consistency to avoid leakage from tubes or other containers, is non-sticky, non-staining and drops with water. Linear or branched monobasic or dibasic alkyl esters such as diisoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acid, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, palmitic The acid 2-ethylhexyl or a mixture of branched chain esters known as Crodamol CAP, etc. can be used, the last three being the preferred esters. These can be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white petrolatum and / or liquid paraffin or other mineral oils can be used.

  A pharmaceutical formulation according to the invention comprises one or more compounds together with one or more pharmaceutically acceptable carriers or replication agents and optionally other therapeutic agents. The pharmaceutical formulation containing the active ingredient can be in any form suitable for the intended method of administration. For example, when used for oral use, tablets, troches, medicinal candy, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard capsules or soft capsules, syrups or elixirs Can be prepared. Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions are also palatable preparations. One or more agents may be included to provide a product, including sweeteners, flavoring agents, colorants and preservatives. Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets may be acceptable. Such excipients include, for example, inert diluents such as calcium carbonate or sodium carbonate, lactose, lactose monohydrate, croscarmellose sodium, povidone, calcium phosphate or sodium phosphate; granulating agents and disintegration Agents, such as corn starch or alginic acid; binders such as cellulose, microcrystalline cellulose, starch, gelatin or gum arabic; and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or coated by known techniques (including microencapsulation) to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period of time Can do. For example, a sustained release material (eg, glyceryl monostearate or glyceryl distearate) can be used alone or with a wax.

  Formulations for oral use are also as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent (eg calcium phosphate or kaolin) or the active ingredient is water or an oil vehicle (eg peanut oil, liquid paraffin) Or as a soft gelatin capsule mixed with olive oil).

  The aqueous suspensions of the present invention contain the active ingredients in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and gum arabic, and the like, as well as dispersing or wetting agents such as natural Phosphatides (eg, lecithin), condensation products of alkylene oxide and fatty acids (eg, polyoxyethylene stearate), condensation products of ethylene oxide and long-chain aliphatic alcohols (eg, heptadecaethyleneoxysetanol), ethylene oxide And a condensation product of fatty acid and a partial ester derived from hexitol anhydride (for example, polyoxyethylene sorbitan monooleate) and the like. Aqueous suspensions may also include one or more preservatives (eg, ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate), one or more colorants, one or more flavoring agents, and 1 One or more sweetening agents (eg, sucrose or saccharin) can be included.

  Oily suspensions may be formulated by suspending the active ingredient in vegetable oil (peanut oil, olive oil, sesame oil or coconut oil) or in mineral oil (eg, liquid paraffin). Oral suspensions can contain a thickening agent (eg, beeswax, hard paraffin or cetyl alcohol). Sweetening agents such as those set forth above and flavoring agents may be added to provide a palatable oral preparation. These compositions can be preserved by the addition of an antioxidant (eg, ascorbic acid).

  Dispersible powders and granules of the invention suitable for preparing an aqueous suspension by the addition of water comprise an active ingredient, a dispersing or wetting agent, a suspending agent, and one or more Provided in admixture with preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by the dispersing agents, wetting agents and suspending agents disclosed above. Additional excipients such as sweetening, flavoring and coloring agents can also be present.

  The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oily phase can be a vegetable oil (eg, olive oil or peanut oil) or a mineral oil (eg, liquid paraffin) or a mixture of these. Suitable emulsifiers include naturally occurring gums (eg, gum arabic and tragacanth), esters or partial esters derived from naturally occurring phosphatides (eg, soy lecithin), fatty acids and hexitol anhydrides (eg, sorbitan monooles). Art), as well as condensation products of such partial esters and ethylene oxide (eg, polyoxyethylene sorbitan monooleate). The emulsion can also contain sweetening and flavoring agents. Syrups and elixirs can be formulated with sweetening agents, for example glycerol, sorbitol or sucrose. Such formulations can also contain a demulsifier, a preservative, a flavoring agent or a colorant.

  The pharmaceutical compositions of the invention may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. Such suspensions may be formulated according to known techniques using such suitable dispersing or wetting agents and suspending agents described above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic diluent or solvent acceptable for parenteral administration (eg, a solution in 1,3-butanediol). Alternatively, it can be prepared as a lyophilized powder. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids (eg oleic acid) can likewise be used in the preparation of injectables.

  The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a sustained release formulation intended for oral administration to humans is formulated with a suitable and convenient amount of carrier material that can vary from about 5% to about 95% (weight: weight) of the total composition. From about 1 mg to 1000 mg of active material. The pharmaceutical composition can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion should contain about 3 μg to 500 μg of active ingredient per milliliter of solution so that a suitable volume of infusion can be performed at a rate of about 30 mL / hr. Can do.

  Formulations suitable for administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient. The active ingredient is preferably in such a formulation at a concentration of 0.5% to 20%, conveniently at a concentration of 0.5% to 10%, specifically about 1.5% w / w. Present at a concentration of.

  Formulations suitable for topical administration in the mouth include lozenges containing the active ingredient in a flavored base (usually sucrose or gum arabic or tragacanth); the active ingredient in an inert base (eg, , Gelatin and glycerin, or sucrose and gum arabic); and mouthwashes containing the active ingredients in a suitable liquid phase carrier.

  Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

  Formulations suitable for intrapulmonary or nasal administration are administered by rapid inhalation through the nasal passage or by inhalation through the mouth to reach the alveolar sac, e.g. 0.1 micron to 500 microns Have a particle size in the range (including in the range between 0.1 microns and 500 microns, including particle sizes in increments of 0.5 microns, 1 micron, 30 microns, 35 microns, etc.). Suitable formulations include aqueous or oily solutions of the active ingredients. Formulations suitable for aerosol or dry powder administration can be prepared according to conventional methods, and with other therapeutic agents (eg, compounds previously used in the treatment of inflammation as described below) Can be delivered.

  Formulations suitable for vaginal administration are as vaginal suppositories, tampons, creams, gels, pastes, foams or spray formulations containing carriers in addition to the active ingredient which are known to be suitable in the art. Can be provided.

  Formulations suitable for parenteral administration include aqueous and non-aqueous, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that render the formulation isotonic with the blood of the intended recipient. Aqueous sterile injectable solutions; and aqueous and non-aqueous sterile suspensions that may contain suspending and thickening agents.

  Such formulations are provided in unit dose containers or multi-dose containers (eg, sealed ampoules and vials) and require only the addition of a sterile liquid phase carrier (eg, water for injection) immediately prior to use. And can be stored in a freeze-dried (freeze-dried) state. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Preferred unit dosage formulations are unit dosage formulations containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate divided amount thereof, of the active ingredient.

  In addition to the ingredients specifically mentioned above, the formulations of the present invention can include other agents that are conventional in the art in view of the type of formulation in question, eg, oral It should be understood that formulations suitable for administration can include flavoring agents.

  The invention further provides an animal composition comprising at least one active ingredient as defined above together with an animal carrier therefor.

  An animal carrier is a substance useful for the purpose of administering the composition and is otherwise solid or acceptable in the veterinary field and compatible with the active ingredient, It can be a liquid material or a gaseous material. Such veterinary compositions can be administered by oral administration, parenteral administration or any other desired route.

  The compound should also be formulated to provide controlled release of the active ingredient to allow for less frequent dosing or to improve the pharmacokinetic or toxicity profile of the active ingredient. Can do. Accordingly, the present invention also provides a composition comprising one or more compounds formulated for sustained or controlled release.

  An effective dose of the active ingredient is delivered at least depending on the nature of the condition being treated, toxicity, whether the compound is being used prophylactically (lower doses), or is used against existing inflammation. Depending on the method and pharmaceutical formulation, it is determined by the clinician using conventional dose escalation studies. An effective dose of the active ingredient can be expected to be from about 0.0001 mg / kg body weight / day to about 100 mg / kg body weight / day. Generally from about 0.01 mg / kg body weight / day to about 10 mg / kg body weight / day. More generally from about 0.01 mg / kg body weight / day to about 5 mg / kg body weight / day. More generally from about 0.05 mg / kg body weight / day to about 0.5 mg / kg body weight / day. For example, a daily candidate dose for an adult weighing approximately 70 kg is in the range of 1 mg to 1000 mg, preferably between 5 mg and 500 mg, and can take single or multiple dose forms.

  One or more compounds (hereinafter referred to as active ingredients) can be administered by any route appropriate to the condition being treated. Suitable routes include oral, rectal, nasal, topical (including intraoral and sublingual), vaginal and parenteral (subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) ) Etc. are included. It will be appreciated that the preferred route may vary with for example the condition of the recipient. An advantage of the compounds of the present invention is that the compounds of the present invention have bioavailability by oral administration and can be taken by oral administration.

  The active ingredients of the present invention can also be used in combination with other active ingredients. Such combinations are generally selected based on the condition being treated, the cross-reactivity of the components, and the drug properties of the combination.

  It is also possible to combine any compound with one or more other active ingredients in a unitary dosage form for simultaneous or sequential administration to a patient. Such mixed therapy can be administered as simultaneous therapy or sequential therapy. When given sequentially, the combination may be given in two or more doses.

  Mixed therapy can result in a “synergistic” or “synergistic” effect, ie an effect that is achieved when the active ingredients used together are greater than the sum of the effects resulting from the separate use of the compound. it can. Synergistic effects when the active ingredients are (1) formulated simultaneously and administered or delivered simultaneously in the combined formulation; (2) when delivered alternately or in parallel as separate formulations; Or (3) can be achieved by some other therapy. When delivered in alternation therapy, a synergistic effect can be achieved when the compound is administered or delivered sequentially, eg, in separate tablets, pills or capsules, or by different injections in separate syringes. . In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, ie, sequentially, whereas in mixed therapy, two or more active ingredients are administered. Effective dosages of are administered together.

The scope of the present invention also includes in vivo metabolites of the compounds described herein. Such products may arise, for example, from the oxidation, reduction, hydrolysis, amidation and esterification of administered compounds, mainly by enzymatic processes. Accordingly, the present invention includes a compound produced by a process consisting of contacting a compound of the present invention with a mammal for a period of time sufficient to produce its metabolite. Such products are typically prepared by preparing radiolabeled (eg C ¹⁴ or H ³ ) labeled compounds, doses detectable radiolabeled compounds (eg about 0.5 mg / kg Parenteral administration to animals (eg, rats, mice, guinea pigs, monkeys) or humans at a dose greater than, giving sufficient time for metabolism to occur (generally about 30 seconds to 30 hours), And its conversion products are identified by isolation from urine, blood or other biological samples. Such products are easily isolated because they are labeled (other products are isolated by the use of antibodies that can bind to epitotes remaining in the metabolite). The structure of the metabolite is determined in a conventional manner, for example by MS analysis or NMR analysis. In general, analysis of metabolites is done in the same way as conventional drug metabolism studies well known to those skilled in the art. Unless the conversion product is found separately in vivo, the conversion product is useful in diagnostic tests for therapeutic use of the compound, even if the conversion product has no anti-inflammatory activity of its own.

  The invention also relates to methods of making the novel compounds herein. The compounds of the present invention are prepared by any applicable technique of organic synthesis. Many such techniques are well known in the art. However, many of the known techniques are described in Compendium of Organic Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T. Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and Shuyen Harrison, 1974; Vol. 3, Louis S. et al. Hegedus and Leroy Wade, 1977; Vol. 4, Leroy G. Wade, jr. , 1980; Vol. 5, Leroy G. Wade, Jr. , 1984; and Vol. 6, Michael B. Smith; and March, J. et al. , Advanced Organic Chemistry, Third Edition (John Wiley & Sons, New York, 1985), Comprehensive Organic Synthesis. Selectivity, Strategie & Efficiency in Modern Organic Chemistry. In 9 Volumes, Barry M. et al. Trost, Editor-in-Chief) (Pergamon Press, New York, 1993 printing).

  In general, reaction conditions (eg, temperature, reaction time, solvent and processing procedures, etc.) are those conditions that are common in the art for the particular reaction being performed. The cited references, as well as the references therein, contain a detailed description of such conditions. In general, the temperature is from −100 ° C. to 200 ° C., the solvent is aprotic or protic, and the reaction time is from 10 seconds to 10 days. Treatment generally consists of deactivating any unwanted reagents, followed by partitioning (extraction) in a water / organic layer system and separating the product containing layers.

  Resolution of a racemic mixture using a method such as formation of a diastereomer using an optically active resolving agent, and only one stereoisomer (eg, enantiomer) substantially free of that stereoisomer. (Streochemistry of Carbon Compounds (1962), EL Leliel, McGraw Hill; Lochmuller, CH (1975), J. Chromatogr., 113: (3) 283-302). Racemic mixtures of chiral compounds can be separated and isolated by any suitable method, such as (1) the formation of ionic diastereomeric salts with chiral compounds and fractional crystallization or other Separation by methods, (2) formation of diastereomeric compounds with chiral derivatization reagents, separation of diastereomers, and conversion to pure stereoisomers, and (3) substantial under direct chiral conditions Separation of purely pure or concentrated stereoisomers is included.

  In method (1), asymmetric compounds having acidic functional groups (for example, carboxylic acids and sulfonic acids) and enantiomers are pure chiral bases (for example, brucine, quinine, ephedrine, strychnine and α-methyl-β-phenylethylamine ( Diastereomeric salts can be formed by reacting amphetamines) and the like). Diastereomeric salts may be derived for separation by fractional crystallization or ionic chromatography. In order to separate optical isomers of amino compounds, a diastereomeric salt can be formed by adding a chiral carboxylic acid or sulfonic acid (for example, camphorsulfonic acid, tartaric acid, mandelic acid or lactic acid).

  Alternatively, in Method (2), the substrate to be resolved is reacted with one enantiomer of the chiral compound to form a diastereomeric pair (Eliel, E. and Wilen, S. (1994), Strokechemistry of Organic Compounds, John). Wiley & Sons, Inc., p.322). Diastereomeric compounds are obtained by reacting an asymmetric compound with an enantiomerically pure chiral derivative reagent (eg, a menthyl derivative), then separating the diastereomers and performing hydrolysis to concentrate the enantiomers. It can be formed by producing xanthene. Methods for measuring optical purity include chiral esters of racemic mixtures such as menthyl esters (eg (-) menthyl chloroformate in the presence of base), or Mosher esters (α-methoxy-α- (trifluoro). Methyl) phenyl acetate) (Jacob III. (1982) J. Org. Chem. 47: 4165), which involves analyzing the NMR spectrum for the presence of two atropisomeric diastereomers. Stable diastereomers of atropisomeric compounds can be separated and isolated by normal and reverse phase chromatography according to the method for separating atropisomeric naphthyl-isoquinoline compounds (Hoye, T., WO 96/15111). .

  In method (3), a racemic mixture of two enantiomers can be separated by chromatography using a chiral stationary phase (Chiral Liquid Chromatography (1989) WJ Lough, Ed., Chapman and Hall, New York. Okamoto (1990) J. of Chromatogr. 513: 375-378). Enriched or purified enantiomers can be distinguished by methods used to identify other chiral molecules having asymmetric carbon atoms (eg, optical rotatory and circular dichroic methods).

  The invention will now be illustrated by the following non-limiting examples.

Example 1
Were evaluated Materials and Methods The following compounds (Figure 1) :( S) -HPMPApp, ( R) -HPMPGpp, PMEApp, PMECpp, PMEGpp, PMETpp, PMEDAPpp, PMEO-DAPypp, 6-Me 2 -PMEDAPpp, (R) -6-cyprPMPDAPpp, (R) -PMPDAPpp, (R) -PMPApp, (S) -PMPApp, (R) -PMPGpp and (S) -PMPGpp. (S) -HPMPA and (R) -HPPMG diphosphate were prepared according to Otmar M, Masojidkova M, Votruba I, Holy A. , Collect Czech Chem Commun, 2001, 66, 500-6. Other ANP diphosphates were synthesized by a modified morpholid method. In a typical experiment, a mixture of ANP (free acid, 1 mmol), N, N′-dicyclohexylcarbodiimide (1.3 g) and morpholine (2 mL) in 80% aqueous tert-butanol (20 mL) was stirred for 6-8 hours. At reflux and evaporated in vacuo. The residue in water (100 mL) was filtered through Celite®, the filtrate was extracted with ether (3 × 50 mL), and the aqueous phase was concentrated in vacuo. The residue was transferred to a 100 mL flask, evaporated, co-distilled with ethanol (2 × 20 mL) and dried over phosphorus pentoxide at 15 Pa overnight. Add dry dimethyl sulfoxide solution (1 mol / L, 2.5 mL) of bis (tributylammonium) monophosphate or tris (tributylammonium) diphosphate and stir the mixture at ambient temperature for 4-6 days in a sealed flask. did. The reaction mixture was then acidified to pH 3 with 6M HCl and an appropriate amount of activated carbon was added. After the pelleted activated carbon was thoroughly washed with water for HPLC, the desalted nucleotide was eluted with 5% NH ₄ OH / 50% methanol. The eluate was evaporated at 30 ° C., dissolved in 0.05 M triethylammonium bicarbonate, and POROS® 50HQ anion in a linear gradient of triethylammonium bicarbonate (0.05-0.4 mol / L). Purification by chromatography on an exchanger (Applied Biosystems [Foster City, Calif., USA]). The peak corresponding to ANPpp (triethylammonium salt) was collected, evaporated in vacuo at room temperature, and then converted to ANPpp sodium salt with DOWEX ^™ 50X8 (Na ⁺ ) (SERVA Electrophoresis GmbH [Heidelberg, Germany]).

All other chemicals and materials were commercial products: eg activated carbon, N, N′-dicyclohexylcarbodiimide, morpholine, tert-butanol, Celite®, dimethyl sulfoxide, ddGTP, streptomycin, penicillin G , CHAPS, β-mercaptoethanol, RNase A, proteinase K, PBS and RPMI 1640 medium (Sigma-Aldrich [St. Louis, MO]), fetal calf serum (PAA Laboratories GmbH [Austria Passing]), vitamin B ₁₂ (Leciva aa) S [Prague, Czech Republic)), Pefablock-SC, Protector RNase inhibitor (Roche Diagnostics GmbH, Germany Man) ^{Im]), [γ- 32 P]} ATP (MP Biomedicals GmbH [ German]), T4 polynucleotide kinase buffer and T4 polynucleotide kinase (TaKaRa Bio, Inc. [Shiga Japan]), HEPES, deoxynucleotide triphosphates (DNTP), Taq polymerase reaction buffer, Taq DNA polymerase (Promega [Madison, Wis., USA]), TS, ACX, NT, TSNT primers (Invitrogen Ltd. [Paisley, UK]).

Human acute promyelocytic leukemia HL-60 cells (ATCC CCL 240), 10% (v / v) heat-inactivated fetal calf serum, antibiotics (200 μg / mL streptomycin and 200 units / mL penicillin G), with 10mM of β- mercaptoethanol and vitamin _{B 12} is supplemented with RPMI-1640 medium and incubated at 37 ° C. in a humidified atmosphere containing 5% of CO _2. Cells harvested in log phase growth were pelleted, washed with PBS and frozen at -70 ° C.

Extracts having telomerase activity were obtained from Kim NW, et al. , Science, 1994, 266, 2011-5, and prepared and analyzed with some modifications. That is, cells were thawed, 0.5% CHAPS, 10 mM of HEPES-NaOH (pH7.5), the 1mM of MgCl _2, 1MM EGTA, of 5 mM beta-mercaptoethanol, the 2 mM Pefabloc-SC, 10% of the Incubate 30 minutes on ice with CHAPS lysis buffer containing glycerol and 1 μL (40 U) Protector RNase inhibitor, and lyse 1 million cells using 200 μL CHAPS lysis buffer. In order to ensure lysis of HL-60 cells, these cells were subjected to two freeze / thaw cycles during the incubation period. This further procedure did not affect telomerase activity in HL-60 cells. Cell debris was pelleted at 16000 xg for 20 minutes at 4 ° C. The supernatant was removed, aliquoted, frozen with dry ice and stored at -70 ° C. The protein concentration of the supernatant was determined by the Bradford test.

Telomerase activity was determined by Kim NW, Wu F., with the modifications described below. , Nucleic Acids Res, 1997, 25, 2595-7. In this study, the sensitivity of the TRAP test was increased by extending the incubation time from 10 minutes to 15 minutes and increasing the number of PCR cycles from 27 to 33. The dependence of the amount of amplified telomerase product on the number of PCR cycles was linear in the range of 30-35 cycles (no data). An aliquot of 800 pmol TS substrate primer (5′-AATCCGTCGAGCAGAGTT-3 ′) (SEQ ID NO: 1) was added to 60 μCi [γ- ³² P] ATP (60 mCi / mL, 7000 Ci / mmol), T4 polynucleotide kinase buffer and 40 U. Labeled in a 100 μL reaction mixture containing a T4 polynucleotide kinase. After incubation at 37 ° C. for 30 minutes and then at 85 ° C. for 2 minutes, unincorporated excess [γ- ³² P] ATP is reacted with a MicroSpin ^™ G-25 column (Amersham Biosciences [Piscataway, NJ, USA]) Removed from the mixture. 40 μl of TRAP reaction solution was mixed with Taq polymerase reaction buffer (50 mM KCl, 10 mM Tris-HCl (pH 9.0 at 25 ° C.), 1.5 mM MgCl ₂ , 0.1% Triton X (registered trademark) -100, dNTP ( 30, 60 and 125 μmol / L), 18 pmol of end-labeled TS substrate primer, containing appropriate amounts of tested AMPpp, ANPp and ANP, respectively, the reaction was initiated by adding cell extract (0.15 μg protein). Each TRAP reaction mixture is placed in a thermocycler block previously heated to 30 ° C., incubated at 30 ° C. for 15 minutes, and then heated at 95 ° C. for 2 minutes (in one cycle) to stop the telomerase reaction. 6 pmol ACX reverse primer (5′-G _{GCGG [CTTACC] 3 CTAACC-3} ') ( SEQ ID NO: 2), NT internal control primers 3 pmol, after adding a mixture of 10μL containing TSNT internal control primers, and 1.25U of Taq DNA polymerase 0.01amol The reaction solution was subjected to 33 cycles of 94 ° C. for 20 seconds, 52 ° C. for 30 seconds, and 72 ° C. for 20 seconds.

  Several inactivation experiments were performed to ensure that the measured telomerase activity was truly dependent on telomerase itself. An aliquot of cell lysate was incubated with RNase A (50 μg / mL) at 37 ° C. for 30 minutes. Proteinase K and heat-inactivated cell extracts were incubated with proteinase K (50 μg / mL) for 30 minutes at 37 ° C. before testing 3 μL of extract by TRAP test. Each was prepared by heating at 75 ° C. for 10 minutes. The HL-60 cell extract showed telomerase activity, and a characteristic primer extension binding pattern was seen in the autoradiograph. We considered that this sample was positive for telomerase activity when the signal disappeared after RNase A treatment and no signal was detected in the lysis buffer alone (negative control). By treatment with RNase A and proteinase K, the ladder band of the PCR product was eliminated, and both protein dependency and RNA dependency of enzyme activity were confirmed.

Amplified telomerase products were analyzed on denaturing 15% polyacrylamide / 7M urea sequencing gel using Tris-borate-EDTA for 2 hours at 1,900V. The dried gel was exposed to a PhosphorImager storage screen and the amount of reaction product was evaluated using the ImageQuant ^™ software (Molecular Dynamics [Sunnyvale, Calif., USA]) of a TYPHON ^™ 9410 image analyzer. To compare relative telomerase activity in the presence of inhibitors, the telomerase ladder TRAP test signal was normalized to the signal of the corresponding internal standard after background removal. The signal intensity of the bands of ANPpp, ANPp and ANP treated samples was expressed as a percentage of the signal intensity detected in the control. These relative intensities have been calculated using ImageQuant software. All results were expressed as the mean ± SD of 4 independent measurements.

Results and Discussion Table 1 shows the approximate IC ₅₀ values of the PME derivatives. Table 1 shows IC ₅₀ values according to the following magnitude of telomerase inhibitory capacity: PMEGpp>PMEDAPpp>PMEO-DAPyppp>PMEApp> PMECpp ≧ PMETpp> 6-Me ₂ PMEDAPPpp.

^ａ 値は４つの独立した測定の平均±ＳＤである；^ｂ 測定されず；^ｃ 阻害なし；^ｄ プロセッシビティーの増強。

^a value is the mean ± SD of 4 independent measurements; ^b not measured; ^c no inhibition; ^d enhanced processivity.

The guanine derivative PMEGpp is the most potent telomerase inhibitor of all the acyclic nucleotide analogs tested, with an IC ₅₀ of 12.7 ± 0.5 μmol / L at 125 μM dNTPs (Table 1). , FIG. 2). This inhibitory potency against telomerase is the inhibitory potency of ddGTP, known to be one of the most effective nucleotide analog telomerase inhibitors (IC ₅₀ is 8.1 ± 0.4 μmol / L with 125 μM dNTP) Comparable with. Depending on the concentration of dNTP, PMEGpp inhibits telomerase activity by 50% only when present in the dGTP concentration range of 0.07 to 0.11. PMEG monophosphate and PMEG itself show no effect on telomerase activity up to a concentration of 300 μM PMEG and / or PMEGp, respectively, with 125 μM dNTPs.

PMEDAPpp, which selectively inhibits DNA polymerase δ (Holy A., Curr Pharm Des, 2003, 9, 2567-92) and exerts a significant cytostatic effect, is 76 ± 13.5 μmol / L (125 μM dNTP). Inhibits the activity of telomerase with an IC ₅₀ of Surprisingly, 6-Me ₂ PMEDAPpp, an N ⁶ -dimethyl derivative, increases the processivity of the enzyme (FIG. 3A). The non-phosphorylated form 6-Me ₂ PMEDAP has no effect on the telomerase ladder pattern.

  PMETApp and PMECpp, which are pyrimidine derivatives of PMEApp, that inhibit retroviral reverse transcriptase (Holly A., Curr Pharm Des, 2003, 9, 2567-92) do not show significant inhibition of telomerase. Inhibition is observed with PMEO-DAPypp; this open ANP is considered to be a PMEDApp analog (FIG. 1).

  The ability of PMEGpp and PMEDAPpp to inhibit telomerase is consistent with the parent compounds PMEG and PMEDAP inducing apoptosis, excellent cytostatic activity, and anticancer activity (Holy A., Curr Pharm Des, 2003). , 9, 2567-92).

Estimated IC ₅₀ values of PMP derivatives diphosphate are shown in Table 2 in order of potency as telomerase inhibitors shown below: (R) -PMPGpp> (S) -PMPGpp> (R) -6-cyprPMPDAPpp> (R) -PMPApp> (R) -PMPDAPpp ≧ (S) -PMPApp.

^ａ 値は４つの独立した測定の平均±ＳＤである；^ｂ 測定されず；^ｃ プロセッシビティーの増強。

^a value is the mean ± SD of 4 independent measurements; ^b not measured; ^c enhancement of processivity.

The most potent inhibitor of PMP-type analogs is (R) -PMPGpp, a guanine derivative, which has enzymatic activity at an IC ₅₀ concentration of 1/5 to 1/8 compared to the natural substrate dGTP. Inhibits. (S) -PMPGpp has significantly less inhibitory activity than its (R) -enantiomer, and its IC ₅₀ is almost 5 times greater than (R) -PMPGpp. This suggests that the absolute configuration plays an important role in the inhibition of telomerase, allowing the enzyme to distinguish between (R) -enantiomer and (S) -enantiomer. Similarly, (S) -PMPApp increases enzyme processivity (FIG. 3B), while (R) -PMPApp (125 μM dNTPs with an IC ₅₀ of 224 ± 30 μmol / L) has significant telomerase activity. No inhibition is seen, which is very efficient as a retroviral reverse transcriptase chain termination inhibitor (Holy A., Curr Pharm Des, 2003, 9, 2567-92). These results are reported in Pai RB, et al., Who reported on the discrimination of the D- and L-enantiomers of FaraTTP. , (Cancer Res, 1998, 58, 1909-13) and reported on the difference in inhibition efficiency in the D- and L-enantiomer pairs of CBV-TP in Tendian SW, Parker WB. , (Mol Pharmacol, 2000, 57, 659-9). Structure activity relationship studies also show the enantioselectivity of some of these ANP-type inhibitors of human telomerase. In general, the (R) -enantiomer of a PMP derivative has better affinity for the enzyme than the (S) -enantiomer.

The approximate IC ₅₀ values for the diphosphate of the purine HPMP derivative are shown in Table 3. Similar to the PME system and the PMP system, (S) -HPMPApp, which is an adenine derivative, has a smaller inhibitory activity than (R) -HPMPPGpp, which is a guanine derivative.

^ａ 値は４つの独立した測定の平均±ＳＤである；^ｂ 測定されず。

^a value is the mean ± SD of 4 independent measurements; ^b not measured.

The previously reported inhibitory effect of ddGTP on telomerase activity (see Pai RB, et al., Cancer Res, 1998, 58, 1909-13) is the most efficient ANP-based compound, PMEGpp and (R)- Comparable to the inhibitory ability of PMPGpp. (R) -HPMGPGpp, PMEDAPpp, (S) -PMPGpp, (R) -6-cyprPMPDAPPpp, PMEO-DAPyppp and (S) -HPMPApp are shown as moderate inhibitors, IC ₅₀ values are natural substrates Comparable to the concentration of. Although the active site of telomerase has already been shown to be related to that of other reverse transcriptases (Nugent CI, Lundbald V., Genes Dev, 1998, 12, 1073-85), retroviral reverse transcription The nucleotide analogs (R) -PMPApp and PMEAPp (Holy A., Curr Pharm Des, 2003, 9, 2567-92)) known to be chain termination inhibitors of the enzyme do not inhibit telomerase activity. .

The data in Tables 1, 2 and 3 show that the IC ₅₀ values for inhibition of telomerase activity by all tested guanine derivatives [PMEGpp, (R) -PMPGpp, (S) -PMPGpp and (R) -HPMGPGpp] It shows that it is 1/2 to 1/10 of the dGTP concentration in the test. Thus, the affinity of these nucleotide analogs for telomerase is significantly greater than the affinity of the natural substrate dGTP (FIG. 4A). As with human telomere sequences, adenine ANPpp and / or 2,6-diaminopurine ANPpp are not very efficient inhibitors (FIG. 4B).

Interestingly, the two compounds from the tested strains are very different from the other ANPpp molecules: (S) -PMPApp and 6-Me ₂ PMEDAPpp do not inhibit telomerase, but 125 μM dNTP concentration Increases telomerase processivity (FIG. 3). We can infer that both (S) -PMPApp and 6-Me ₂ PMEDAPpp may interfere with the telomerase reaction cycle in some way, similar to dGTP. In the two subunits of the telomerase complex (see Wenz C et al., EMBO J, 2001, 20, 3526-34), separation of the telomerase RNA template and the DNA strand is facilitated and repeated addition It is estimated that processivity is increased (Hammond PW, Cech TR., Biochemistry, 1998, 37, 5167-72; and Hardy CD, et al., J Biol Chem, 2001, 276, 4863-71).

According to the “anchor site model”, the RNA template of one subunit is thought to be used primarily for substrate binding, while the other template is considered to be replicated upon the addition of telomeric repeats. (Kelleher C, et al., Trends Biochem Sci, 2002, 27, 572-9). It is believed that so-called DNA anchor sites, which differ from the catalytic site, are subject to the action of both (S) -PMPApp and / or 6-Me ₂ PMEDAPpp, thereby promoting processivity during the synthesis of telomeric repeats.

Example 2
Using a procedure similar to the procedure described above, (S) -PMPA reduces telomere length in vivo in CCRF-CEM cells when fed to growth medium for 5 weeks at concentrations of 10 and 20 μM. It was found to increase over about 1 kb (see FIG. 5). This data is consistent with the fact that (S) -PMPA diphosphate did not inhibit telomerase activity and was shown to increase telomerase processivity in vitro at 125 μM dNTPs.

Example 3
Using a procedure similar to the procedure described above, (S) -PMPA is also extracted from CCRF-CEM T lymphoblastoid cell extract (human acute lymphoblastic leukemia, ATCC CCL 119), human cervical cancer HeLa. S3 (ATCC CCL 2.2) cell extract, murine lymphocytic leukemia L1210 cells, and T cell spontaneous leukemia / lymphoma (inbred Sprague-Dawley rat strain; Otova B, et al., Folia Biol (Praha)) , 2002, 48, 213-26) have been shown to improve telomerase processivity.

  All references and patent literature mentioned in this specification are hereby expressly incorporated by reference in their entirety. Specifically cited portions or pages of the above cited references are specifically incorporated by reference. The present invention has been described in sufficient detail to enable those skilled in the art to make and use the subject matter of the following claims. It will be apparent that the methods and compositions of the following claims may be subject to specific modifications within the scope and spirit of the present invention.

FIG. 1 illustrates the purine and pyrimidine structures of PME, PMEO, PMP and HPMP. FIG. 2 illustrates inhibition of HL-60 telomerase by PMEGpp as measured in Example 1. FIG. 3 illustrates the enhancement of telomerase processivity by 6-Me ₂ PMEDAPpp (A) and (S) -PMPApp as measured in Example 1. FIG. 4A shows inhibition of HL-60 telomerase by PMEGpp (1), (R) -PMPGpp (2), (R) -HPMGPGpp (3), and (S) -PMPGpp (4) which are guanine derivatives. Illustratively, FIG. 4B shows adenine and 2,6-diaminopurine derivatives PMEDAPpp (5), (S) -HPMPApp (6), PMEO-DAPypp (7) and (R) -6-cyprPMPDAPpp ( The inhibition by 8) is illustrated. Enzyme activity was measured in HL-60 cell extracts in the presence of 125 μmol / L dNTPs. FIG. 5 illustrates the change in telomere length in the cell line CCRF-CEM after treatment with 10 μM and 20 μM (S) -PMPA (see Example 2).

Claims (23)

In the manufacture of a medicament for improving telomerase processivity, the following acyclic nucleoside purines of formula I:

(Where:
X is OH or —N (R ¹ ) ₂ , where
R ¹ is independently
H;
C ₂ -C _{15 alkyl,} C 3 _{-C 15 alkenyl,} C 6 _{-C 15 arylalkenyl,} C 3 _{-C 15 alkynyl,} C 7 _{-C 15 arylalkenyl,} C 1 -C ₆ - alkylamino _-C 1 -C ₆ alkyl, C ₅ -C ₁₅ aralkyl, C ₆ -C ₁₅ heteroalkyl or C ₃ -C ₆ heterocycloalkyl (provided that methylene in the alkyl moiety not adjacent to NH is replaced by —O—); or C ₁ -C _{15 alkyl,} C 2 _{-C 15 alkenyl,} C 6 _{-C 15 arylalkenyl,} C 6 _{-C 15 arylalkenyl,} C 2 _{-C 15 alkynyl,} C 1 -C ₆ - alkylamino -C ₁ - C ₆ alkyl, C ₅ -C ₁₅ aralkyl, C ₆ -C ₁₅ heteroaralkyl, C ₄ -C ₆ aryl, C ₃ -C ₈ or a cycloalkyl or _C 2 -C ₆ heterocycloalkyl;
Alternatively, if desired, both R ^{1 taken} together are saturated or unsaturated C ₂ containing one or two N heteroatoms and optionally further O or S heteroatoms. -C ₅ heterocyclic ring is formed;
In this case, one of said R ¹ groups may be substituted by halo, —CN or —N ₃ ;
W is H or NH ₂ ;
Y is independently OH, —OR ² , —OCH (R ³ ) OC (O) R ² , monophosphate, diphosphate, amino acid amidate, polypeptide amidate, —NHR ² or —N (R ² ). ₂ ;
R ² is independently unsubstituted alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; alkyl in which H is substituted by halo, carboxy, hydroxyl, cyano, nitro, N-morpholino or amino; Aryl, alkenyl, alkynyl, alkynyl, alkynylaryl or alkenylaryl; or alkyl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl in which the —CH ₂ — moiety is substituted by NH, S or O;
Z is C ₁ -C ₆ alkyl optionally substituted with one or more hydroxy; and R ³ is H or R ² );
Alternatively, the use of prodrugs, diphosphates, pharmaceutically acceptable salts, or other phosphorus-substituted derivatives. In the manufacture of a medicament that improves telomerase processivity or inhibits an enzyme that causes neoplasia in an animal, said acyclic nucleoside purine is a compound of formula I:

(Where:
n is 2 or 3;
X is —N (R ¹ ) ₂ , where
R ¹ is independently
H;
C ₂ -C _{15 alkyl,} C 3 _{-C 15 alkenyl,} C 6 _{-C 15 arylalkenyl,} C 3 _{-C 15 alkynyl,} C 7 _{-C 15 arylalkenyl,} C 1 -C ₆ - alkylamino _-C 1 -C ₆ alkyl, C ₅ -C ₁₅ aralkyl, C ₆ -C ₁₅ heteroalkyl or C ₃ -C ₆ heterocycloalkyl (provided that methylene in the alkyl moiety not adjacent to NH is replaced by —O—); or C ₁ -C _{15 alkyl,} C 2 _{-C 15 alkenyl,} C 6 _{-C 15 arylalkenyl,} C 6 _{-C 15 arylalkenyl,} C 2 _{-C 15 alkynyl,} C 1 -C ₆ - alkylamino -C ₁ - C ₆ alkyl, C ₅ -C ₁₅ aralkyl, C ₆ -C ₁₅ heteroaralkyl, C ₄ -C ₆ aryl, C ₂ -C ₆ or a heterocycloalkyl;
Alternatively, if desired, both R ^{1 taken} together are saturated or unsaturated C ₂ containing one or two N heteroatoms and optionally further O or S heteroatoms. -C ₅ heterocyclic ring is formed;
Wherein one of the R ¹ groups may be substituted by halo, —CN or —N _3, but any one or two R ¹ groups are not H;
Y is independently OH, —OR ² , —OCH (R ³ ) OC (O) R ² , monophosphate, diphosphate, amino acid amidate, polypeptide amidate, —NHR ² or —N (R ² ). ₂ ;
R ² is independently unsubstituted alkyl, aryl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl; alkyl in which H is substituted by halo, carboxy, hydroxyl, cyano, nitro, N-morpholino or amino; Aryl, alkenyl, alkynyl, alkynyl, alkynylaryl or alkenylaryl; or alkyl, alkenyl, alkynyl, alkaryl, alkynylaryl or alkenylaryl in which the —CH ₂ — moiety is substituted by NH, S or O;
R ³ is H or R ² );
Alternatively, the use according to claim 1, which is a prodrug, diphosphate or other phosphorus substituted derivative thereof. Said acyclic nucleoside purine of formula I is a compound of the following formula:

Alternatively, the use according to claim 1, which is a prodrug or a pharmaceutically acceptable salt thereof. The acyclic nucleoside purine is a compound of the following chemical formula:

Or use according to claim 2, which is a prodrug thereof. Each R ¹ is independently H, C ₁ -C ₁₅ alkyl, C ₂ -C ₁₅ alkenyl, C ₂ -C ₁₅ alkynyl or C ₃ -C ₈ cycloalkyl; or optionally both R ¹ together form a saturated or unsaturated C ₂ -C ₅ heterocycle containing one or two N heteroatoms and optionally further O or S heteroatoms. The use according to any one of claims 1 to 4. Use according to any one of claims 1 to 4, wherein each R ¹ is independently H, methyl or cyclopropyl. Use according to any one of claims 1 to 4, wherein each R ¹ is independently H. Use according to any one of claims 1 to 4, wherein each R ¹ is independently methyl. Use according to any one of claims 1 to 4, wherein one R ¹ is H and the other R ¹ is cyclopropyl. 4. Use according to claim 1 or 3, wherein W is H. W is NH _2, Use according to claim 1 or 3. Z is _{-CH 2 -CH 2 -, - CH} 2 -CH (CH 3) - or _{-CH 2 -CH (CH 2 OH)} - is a use according to claim 1, 3 or 10. Use according to claim 2 or 4, wherein n is 2. Use according to claim 2 or 4, wherein n is 3. Use according to claim 1, 3 or 10, wherein the compound of formula I is the S-enantiomer. The compounds of Formula I is _6-Me 2 PMEDAPpp or a prodrug thereof, Use according to claim 1. The use according to claim 1, wherein the compound of formula I is (S) -PMPApp or a prodrug thereof. The use according to claim 1, wherein the compound of formula I is (S) -PMPA or a prodrug thereof. A pharmaceutical composition comprising the compound according to any one of claims 1 to 18 and a pharmaceutically acceptable excipient. 19. A method for increasing the number of mitotic cycles of cells that continue to grow in tissue culture, comprising contacting the cells with a compound according to any one of claims 1-18. A method of increasing mitotic cell division of a non-dividing cell, comprising contacting the cell with a compound according to any one of claims 1-18 in vitro or in vivo. Use of a compound according to any one of claims 1 to 18 for the preparation of a medicament for increasing the number of mitotic cycles of continuously proliferating cells. Use of a compound according to any one of claims 1 to 18 for the preparation of a medicament for increasing mitotic cell division of non-dividing cells.

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