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WO1996011204A1 - NOVEL β-L-NUCLEOSIDES AND THEIR USE - Google Patents

NOVEL β-L-NUCLEOSIDES AND THEIR USE Download PDF

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Publication number
WO1996011204A1
WO1996011204A1 PCT/DE1995/001412 DE9501412W WO9611204A1 WO 1996011204 A1 WO1996011204 A1 WO 1996011204A1 DE 9501412 W DE9501412 W DE 9501412W WO 9611204 A1 WO9611204 A1 WO 9611204A1
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Prior art keywords
dideoxy
nucleosides
fluoro
hbv
chem
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PCT/DE1995/001412
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German (de)
French (fr)
Inventor
Eckart Matthes
Martin Von Janta-Lipinski
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Max-Delbrück-Centrum für Molekulare Medizin
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Priority claimed from DE19518216A external-priority patent/DE19518216A1/en
Application filed by Max-Delbrück-Centrum für Molekulare Medizin filed Critical Max-Delbrück-Centrum für Molekulare Medizin
Publication of WO1996011204A1 publication Critical patent/WO1996011204A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

  • the invention relates to new ⁇ -L nucleosides of the general formula
  • R 1 H, methyl, halogen, formyl, hydroxymethyl, ethyl, chloroethyl;
  • R 2 H, OH
  • R 4 OH, 0-acetyl, O-palmitoyl, alkoxy-carbonyl, phosphonate,
  • Mono-, di-, triphosphate, or another protective group which can be converted into the hydroxyl group in a subsequent reaction, and their use as pharmaceutical active substances or agents for the prophylaxis and / or treatment of infections, in particular those caused by hepatitis B. Virus (HBV) or HIV (human immunodeficiency virus) are caused.
  • HBV Virus
  • HIV human immunodeficiency virus
  • HBV is the trigger agent for hepatitis B, an infectious disease that affects approximately 200 million people worldwide and whose chronic form is associated with an increased risk of primary liver carcinoma, which leads to around one million new tumor diseases per year in China alone.
  • EP 0 330 992 Cyclopentane derivatives of purines and pyrimidines.
  • ß-L-nucleoside analogs have been prepared or purified in pure form, such as. B.: ß-L-dideoxycytidine (L-ddC) (M. Mansuri et al, Bioorg. Med. Chem Lett. 1991, 1, 65-68), ß-L-5-fluoride dideoxycytidine (L-FddC) and ß-L-5-fluoro-dideoxyuridine (L-FddU) (T.-S. Lin et al. J. Med. Chem. 1994, 37, 798-803), ß-L-3-thiacytidine (L- 3TC) (CN Chang et al, J. Biol.
  • L-ddC ß-L-dideoxycytidine
  • L-FddC ß-L-5-fluoride dideoxycytidine
  • L-FddU ß-L-5-fluoro-dideoxyuridine
  • L- 3TC
  • the invention is based on the object of developing new antivirally active ⁇ -L nucleosides which are active against hepatitis B and HIV infections and which, with good tolerability and low toxicity, are highly effective against these infections.
  • ⁇ -L nucleosides have the general formula
  • B guanine, 2-aminopurine
  • R 1 H, methyl, halogen, formyl, hydroxymethyl, ethyl, chloroethyl;
  • R 2 H, OH;
  • Mono-, di-, triphosphate, or another protective group which can be converted into the hydroxy group in a subsequent reaction, mean high antiviral activity.
  • 3'-Fluorine-modified compounds of the formula I are particularly effective, among them ⁇ -L-2 ', 3'-dideoxy-3'-fluorocytidine, ⁇ -L-2', 3'-dideoxy-3'-fluoro-5- methylcytidine, ß-L-2 ', 3'-dideoxy-3' -fluoro-5-chlorocytidine and ß-L-2 ', 3'-dideoxy-3' - fluororguanosine.
  • Ss-L-5-methylcytosine arabinoside is also highly effective.
  • the compounds of the invention are prepared by processes known per se by condensation of the sugar part and heterocycle or by modification of the L-ribosyl radical.
  • L-Ribose acetylated and condensed with the heterocyclic base.
  • the resulting L-ribonucleoside is deoxygenated and then modified in the 3'-position, for example fluorinated.
  • the starting material L-ribose can be obtained in a simple manner by epimerization of L-arabinose, as a result of which the preparation of the compounds according to the invention is also economically viable.
  • HBV hepatitis B virus
  • L-FMetCdR is able to completely suppress the synthesis of HBV, the concentration of the inhibitor which reduces the amount of HBV-DNA released into the medium by 50% , is less than 0.2 ⁇ M.
  • a 50% inhibition of the proliferation of HepG2 2.2.15 cells (CD s -,) is only achieved at concentrations greater than 400 ⁇ M.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to novel β-L-pyrimidine and β-L-purine nucleosides and their use as pharmaceutical active substances and agents for the prophylaxis and/or treatment of infections caused particularly by the hepatitis-B virus (HBV) and the AIDS virus (HIV). Fields of application of the invention are medicine and the pharmaceutical industry.

Description

Neue β-L-Nucleoside und ihre VerwendungNew β-L nucleosides and their use
Beschreibungdescription
Die Erfindung betrifft neue ß-L-Nucleoside der allgemeinen FormelThe invention relates to new β-L nucleosides of the general formula
Figure imgf000003_0001
Figure imgf000003_0001
worinwherein
B = R Guanin, 2-Aminopurin;B = R guanine, 2-aminopurine;
Figure imgf000003_0002
R1 = H, Methyl, Halogen, Formyl, Hydroxymethyl, Ethyl, Chlor- ethyl;
Figure imgf000003_0002
R 1 = H, methyl, halogen, formyl, hydroxymethyl, ethyl, chloroethyl;
R2 = H, OH;R 2 = H, OH;
R3 = F, OH; wenn R2=H, dann R3=F, wenn R2=0H, dann R3=OHR 3 = F, OH; if R 2 = H, then R 3 = F, if R 2 = 0H, then R 3 = OH
R4 = OH, 0-Acetyl, O-Palmitoyl, Alkoxy-Carbonyl, Phosphonat,R 4 = OH, 0-acetyl, O-palmitoyl, alkoxy-carbonyl, phosphonate,
Mono-, Di-, Triphosphat, bzw. eine andere Schutzgruppe, die in einerFolgereaktion in die Hydroxygruppe umgewandelt werden kann, bedeuten, und ihre Verwendung als pharmazeutische Wirkstoffe bzw. Mittel zur Prophylaxe und/oder Behandlung von Infektionen, die insbesondere durch das Hepatitis B-Virus (HBV) bzw. das HIV (human immunodeficiency virus)verursacht sind. Anwendungsgebiete der Erfindung sind die Medizin und die pharmazeutische Industrie.Mono-, di-, triphosphate, or another protective group, which can be converted into the hydroxyl group in a subsequent reaction, and their use as pharmaceutical active substances or agents for the prophylaxis and / or treatment of infections, in particular those caused by hepatitis B. Virus (HBV) or HIV (human immunodeficiency virus) are caused. Areas of application of the invention are medicine and the pharmaceutical industry.
Das HBV ist das auslösende Agens für die Hepatitis B, einer Infektionskrankheit, von der weltweit etwa 200 Millionen Menschen betroffen sind und deren chronische Form mit einem erhöhten Risiko für ein primäres Leber-Carcinom verbunden ist, welches allein in China zu etwa einer Million Tumorneu¬ erkrankungen pro Jahr führt.HBV is the trigger agent for hepatitis B, an infectious disease that affects approximately 200 million people worldwide and whose chronic form is associated with an increased risk of primary liver carcinoma, which leads to around one million new tumor diseases per year in China alone.
Eine wirksame und verträgliche antivirale Therapie fehlt bisher. Der Einsatz von Adeninarabinosidmonophosphat und Acyclovir blieb auf wenige klinische Studien begrenzt, bedingt durch die z.Z. erheblichen Nebenwirkungen und die nur teilweisen und vorübergehenden Behandlungserfolge (Alexander et al. British Medical Journal 292, 915 (1986)). Einzig mit Interferon wurde in letzter Zeit in etwa 50% der behandelten Fälle ein längerdauernder Behandlungserfolg erzielt. Als ähnlich unbefriedigend muß die Therapie von HIV- Infektionen (AIDS) angesehen werden, das als Spätfolge einer Infektion von T-4-Lymphozyten mit dem HIV zum Zusammenbruch der immunologischen Abwehr führt. Die bisherige antivirale Therapie mit Azidothy idin und in letzter Zeit mit dem besser verträglichen Didesoxyinosin haben den tödlichen Ausgang des Immunschwächesyndroms zwar verzögern, jedoch nicht verhindern können.An effective and tolerable antiviral therapy has so far been lacking. The use of adenine arabinoside monophosphate and acyclovir was limited to a few clinical studies, due to the currently considerable side effects and the only partial and temporary treatment success (Alexander et al. British Medical Journal 292, 915 (1986)). Only with interferon has a long-term success in treatment been achieved in about 50% of the treated cases. The treatment of HIV infections (AIDS) must be regarded as similarly unsatisfactory, as the late consequence of infection of T-4 lymphocytes with HIV leads to the breakdown of the immunological defense. The previous antiviral therapy with azidothy idin and, more recently, with the better tolerated dideoxyinosin have delayed the fatal outcome of the immune deficiency syndrome, but have not been able to prevent it.
Neue potentiell wirksame Mittel sind eine Reihe von Nucleosidanaloga, die aus folgenden Schriften bekannt sind:New potentially effective agents are a number of nucleoside analogues which are known from the following publications:
1. EP 0 277 151 und EP 0 254 268 - 3'-Fluornucleoside von Adenin, Guanin, Cytosin und Thymin.1. EP 0 277 151 and EP 0 254 268 - 3'-fluoronucleosides of adenine, guanine, cytosine and thymine.
2. WO 89/01776 - 2'-Fluorarabinofuranosyl-5-ethyluracil.2. WO 89/01776 - 2'-fluoroarabinofuranosyl-5-ethyluracil.
3. EP 0 302 760 - 2 ' ,3'-Didesoxynucleoside verschiedener Purinderivate.3. EP 0 302 760-2 ', 3'-dideoxynucleosides of various purine derivatives.
4. EP 0 322 384 und EP 0 409 227 - Zuckermodifizierte Purin- und Pyrimidinnucleoside.4. EP 0 322 384 and EP 0 409 227 - sugar modified purine and pyrimidine nucleosides.
5. EP 0 330 992 - Cyclopentanderivate von Purinen und Pyrimidinen.5. EP 0 330 992 - Cyclopentane derivatives of purines and pyrimidines.
6. EP 0 434 450, EP 0 349 242, US 4 999 428 und WO 91/00282 - Carbozyclische Nucleoside von Purinderivaten.6. EP 0 434 450, EP 0 349 242, US 4 999 428 and WO 91/00282 - carbocyclic nucleosides of purine derivatives.
7. EP 0433 898 - Oxetan-Derivate von Purinen und Pyrimidinen.7. EP 0433 898 - oxetane derivatives of purines and pyrimidines.
8. EP 0 442 757 - 3'-Fluornucleoside .8. EP 0 442 757 - 3'-fluoronucleosides.
Alle hier beschriebenen Nucleoside liegen in D-Form vor. L-Nucleoside, die Enantiomeren der natürlich vorkommenden D- Nucleoside, galten lange Zeit als enzymatisch nicht metabolisierbar und damit in biologischen Systemen als unwirksam. Mit diesem Dogma wurde 1992 durch die Befunde von Spadari et al gebrochen, die gezeigt haben, daß ß-L-Thymidin zwar von der zellulären TdR-Kinase nicht umgesetzt wird, aber ein Substrat des entsprechenden Enzyms des Herpes simplex Virus 1 ist (Spadari et al, J. Med. Chem. 1992, 35, 4214- 4220). In der Folgezeit sind eine Reihe von ß-L- Nucleosidanaloga in reiner Form hergestellt bzw. gereinigt worden, wie z. B. : ß-L-Didesoxycytidin (L-ddC) (M. Mansuri et al, Bioorg. Med. Chem Lett. 1991, 1, 65-68), ß-L-5-Fluor- didesoxycytidin (L-FddC) und ß-L-5-Fluor-didesoxyuridin (L- FddU) (T.-S. Lin et al. J. Med. Chem. 1994, 37, 798-803), ß-L- 3-Thiacytidin (L-3TC) (C. N. Chang et al, J. Biol. Chem. 11992, 267, 22414-22420) und ß-L-5-Fluorthiacytidin (L-FTC) (P. A. Furman et al, Antimicrob. Agents Chemother. 1992, 36, 2686-2692). Diese Verbindungen sind bezüglich ihrer antiviralen Wirksamkeit gegenüber der HBV- bzw. der HIV- Replikation sowie ihrer antiproliferativen Toxizität mit den entsprechenden Enantiomeren verglichen worden. Weitere Synthesen von L-Nucleosiden sind beschrieben inAll of the nucleosides described here are in D form. L-nucleosides, the enantiomers of the naturally occurring D-nucleosides, were long considered to be non-enzymatically metabolizable and thus in biological systems ineffective. This dogma was broken in 1992 by the findings of Spadari et al, who showed that ß-L-thymidine is not converted by the cellular TdR kinase, but is a substrate of the corresponding enzyme of the herpes simplex virus 1 (Spadari et al, J. Med. Chem. 1992, 35, 4214-4220). Subsequently, a number of ß-L-nucleoside analogs have been prepared or purified in pure form, such as. B.: ß-L-dideoxycytidine (L-ddC) (M. Mansuri et al, Bioorg. Med. Chem Lett. 1991, 1, 65-68), ß-L-5-fluoride dideoxycytidine (L-FddC) and ß-L-5-fluoro-dideoxyuridine (L-FddU) (T.-S. Lin et al. J. Med. Chem. 1994, 37, 798-803), ß-L-3-thiacytidine (L- 3TC) (CN Chang et al, J. Biol. Chem. 11992, 267, 22414-22420) and β-L-5-fluorothiacytidine (L-FTC) (PA Furman et al, Antimicrob. Agents Chemother. 1992, 36, 2686-2692). These compounds have been compared with the corresponding enantiomers with regard to their antiviral activity against HBV or HIV replication and their antiproliferative toxicity. Further syntheses of L-nucleosides are described in
- A. Holy, Collect. Czech. Chem. Commun 1972, 37, 4072-4087- A. Holy, Collect. Czech. Chem. Commun 1972, 37, 4072-4087
- M. J. Robins et al, J. Org. Chem 1970, 35, 636-639- M. J. Robins et al, J. Org. Chem 1970, 35, 636-639
- Y. Abe et al, Chem. Pharm Bull 1980, 28, 1324-1326.- Y. Abe et al, Chem. Pharm Bull 1980, 28, 1324-1326.
Es sind jedoch keine Verbindungen bekannt, die an der 3'- Position des Zuckerestes mit Fluor modifiziert sind bzw. die einen L-Arabinofuranosylrest enthalten.However, no compounds are known which are modified with fluorine at the 3'-position of the sugar residue or which contain an L-arabinofuranosyl residue.
Der Erfindung liegt die Aufgabe zugrunde, neue antiviral wirksame ß-L-Nucleoside zu entwickeln, die gegen Hepatitis B- und HIV-Infektionen wirksam sind und die bei guter Verträglichkeit und geringer Toxizität eine hohe Wirksamkeit gegen diese Infektionen aufweisen.The invention is based on the object of developing new antivirally active β-L nucleosides which are active against hepatitis B and HIV infections and which, with good tolerability and low toxicity, are highly effective against these infections.
Überraschenderweise zeigen ß-L-Nucleoside der allgemeinen FormelSurprisingly, β-L nucleosides have the general formula
Figure imgf000005_0001
worin
Figure imgf000005_0001
wherein
B = Guanin, 2-Aminopurin;
Figure imgf000006_0001
B = guanine, 2-aminopurine;
Figure imgf000006_0001
R1 = H, Methyl, Halogen, Formyl, Hydroxymethyl, Ethyl, Chlor- ethyl; R2 = H, OH;R 1 = H, methyl, halogen, formyl, hydroxymethyl, ethyl, chloroethyl; R 2 = H, OH;
R3 = F, OH; wenn R2=H, dann R3=F, wenn R2=0H, dann R3=0H R* = OH, O-Acetyl , O-Palmitoyl, Alkoxy-Carbonyl, Phosphonat,R 3 = F, OH; if R 2 = H, then R 3 = F, if R 2 = 0H, then R 3 = 0H R * = OH, O-acetyl, O-palmitoyl, alkoxy-carbonyl, phosphonate,
Mono-, Di-, Triphosphat, bzw. eine andere Schutzgruppe, die in eine Folgereaktion in die Hydroxygruppe umgewandelt werden kann, bedeuten ,eine hohe antivirale Wirksamkeit.Mono-, di-, triphosphate, or another protective group, which can be converted into the hydroxy group in a subsequent reaction, mean high antiviral activity.
Besonders wirksam sind 3'-fluormodifizierte Verbindungen der Formel I, unter ihnen ß-L-2' ,3'-Didesoxy-3'-fluorcytidin, ß-L- 2 ' ,3'-Didesoxy-3'-fluor-5-methylcytidin, ß-L-2' ,3'-Didesoxy- 3 ' -fluor-5-chlorcytidin und ß-L-2 ' , 3 ' -Didesoxy-3 '- fluorguanosin. Auch ß-L-5-Methylcytosinarabinosid zeigt eine hohe Wirksamkeit.3'-Fluorine-modified compounds of the formula I are particularly effective, among them β-L-2 ', 3'-dideoxy-3'-fluorocytidine, β-L-2', 3'-dideoxy-3'-fluoro-5- methylcytidine, ß-L-2 ', 3'-dideoxy-3' -fluoro-5-chlorocytidine and ß-L-2 ', 3'-dideoxy-3' - fluororguanosine. Ss-L-5-methylcytosine arabinoside is also highly effective.
Die Herstellung der erfindungsgemäßen Verbindungen erfolgt nach an sich bekannten Verfahren durch Kondensation von Zuckerteil und Heterocyclus bzw. durch Abwandlung des L- Ribosylrestes.The compounds of the invention are prepared by processes known per se by condensation of the sugar part and heterocycle or by modification of the L-ribosyl radical.
So wird z. B. L-Ribose acetyliert und mit der heterocyclischen Base kondensiert. Das entstandene L-Ribonukleosid wird deoxygeniert und danach in 3'-Position modifiziert, bei¬ spielsweise fluoriert. Das Ausgangsmaterial L-Ribose kann auf einfache Weise durch Epimerisierung von L-Arabinose gewonnen werden, wodurch die Herstellung der erfindungsgemäßen Verbindungen auch ökonomisch tragfähig ist. So z. B. L-Ribose acetylated and condensed with the heterocyclic base. The resulting L-ribonucleoside is deoxygenated and then modified in the 3'-position, for example fluorinated. The starting material L-ribose can be obtained in a simple manner by epimerization of L-arabinose, as a result of which the preparation of the compounds according to the invention is also economically viable.
Die Erfindung soll nachfolgend durch Ausführungsbeispiele näher erläutert werden.The invention will be explained in more detail below by means of exemplary embodiments.
Ausführungsbeispiele:EXAMPLES
1. Synthese von ß-L-2' ,3'-Didesoxy-3'-fluor-5-methylcytidin1. Synthesis of ß-L-2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine
Eine Lösung von l-(5-0-Acetyl-2,3-didesoxy-3-fluor-ß-L- ribofuranosyl)thymin (788 mg, 2,8 mmol, 1,2,4-Triazol (400 mg, 5,6 mmol) und 4-Chlorphenyldichlorphosphat (0,67 ml, 4,2 mmol) in Pyridin (25 ml) verbleibt für fünf Tage bei Raumtemperatur. Anschließend wird dem dunkelbraunen Reaktionsgemisch konzentrierte Ammoniaklösung (40 ml) hinzugefügt [ (W.L.J.Sung, J. Chem. Soc. Chem. Commun. 1089 (1981)]. Nach 10 Stunden wird das Lösungsmittel im Vakuum entfernt. Der verbleibende Rückstand wird in 50 ml Wasser gelöst und an Dowex WX 8 (H+- Form, 50 ml) mit Wasser (1000 ml) und 5%iger Ammoniaklösung (300 ml) als Elutionsmittel säulenchromatographisch gereinigt. Aus dem ammoniakalkalischen Eluat wird die Titelverbindung als Roh-produkt erhalten. Eine säulenchromatographische Trennung des Rohmaterials an Kieselgel 60 (0,063-0,2 mm) (Merck), mit Chloroform (15% Methanol) liefert ß-L-2' ,3'-Didesoxy-3'-fluor- 5-methylcytidin, das aus Methanol mit wenig HCL als Hydrochlorid erhalten wird (314 mg, 41% Ausbeute). MS: m/z 243 (M+-HCL); UV (H20, pH=7): MX278 nm( 7430).A solution of 1- (5-0-acetyl-2,3-dideoxy-3-fluoro-ß-L-ribofuranosyl) thymine (788 mg, 2.8 mmol, 1,2,4-triazole (400 mg, 5 , 6 mmol) and 4-chlorophenyl dichlorophosphate (0.67 ml, 4.2 mmol) in pyridine (25 ml) remain at room temperature for five days, after which concentrated ammonia solution (40 ml) is added to the dark brown reaction mixture [(WLJSung, J. Chem. Soc. Chem. Commun. 1089 (1981)]. After 10 hours the solvent is removed in vacuo. The remaining residue is dissolved in 50 ml of water and on Dowex WX 8 (H + form, 50 ml) with water ( 1000 ml) and 5% ammonia solution (300 ml) as the eluent are purified by column chromatography. The title compound is obtained as a crude product from the ammonia-alkaline eluate. with chloroform (15% methanol) gives ß-L-2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine, which is obtained from methanol with a little HCL as the hydrochloride (314 mg, 41% yield). MS: m / z 243 (M + -HCL); UV (H 2 0, pH = 7): MX 278 nm (7430).
2. Bestimmung der antiviralen Aktivität von ß-L-2' ,3'-Didesoxy- 3'-fluor-5-methylcytidin (L-FMetCdR)2. Determination of the antiviral activity of β-L-2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine (L-FMetCdR)
Menschliche Hepatoblastomzellen, die mit dem Hepatitis B Virus (HBV) transfiziert wurden (HepG2 2.2.15 Zellen) und permanent das Virus produzieren [(Seils et al., Proc. Natl. Acad. Sei. USA 84, 1005 (1987)] wurden in RPMI 1640 Medium inkubiert, dem 2mM Glutamin und 10% fetales Kälberserum zugesetzt wurde. Nach 5-tägiger Inkubation wurde das Medium erneuert und das L- FMetCdR den Ansätzen in verschiedenen Konzentrationen zugesetzt. Alle zwei Tage wurde das Medium gewechselt und dabei auch die Hemmstofflösung ersetzt.Human hepatoblastoma cells infected with the hepatitis B virus (HBV) were transfected (HepG2 2.2.15 cells) and permanently produce the virus [(Seils et al., Proc. Natl. Acad. Sci. USA 84, 1005 (1987)] were incubated in RPMI 1640 medium, the 2 mM glutamine and 10% fetal calf serum was added. After 5 days of incubation, the medium was renewed and the L-FMetCdR was added to the batches in various concentrations. The medium was changed every two days and the inhibitor solution was also replaced.
Nach 8-tägiger Inkubation der Zellen mit L-FMetCdR wurde das Medium zentrifugiert und die Viren aus dem Überstand mit 10% Polyethylenglykol gefällt, die HBV-DNA daraus gereinigt und mittels Dot-Blot-Analyse quantifiziert [(E.Matthes et al. Antimicrob. Agents Chemother. 34, 1986 (1990)]. L-FMetCdR ist in der Lage, die Synthese von HBV vollständig zu unterdrücken. Die Konzentration des Hemmstoffes, die die von den Zellen ins Medium abgegebene Menge an HBV-DNA um 50% reduziert, ist kleiner als 0,2 μM. Eine 50%ige Hemmung der Proliferation der HepG2 2.2.15 Zellen (CDs-,) wird erst bei Konzentrationen größer als 400 μM erreicht. After the cells had been incubated for 8 days with L-FMetCdR, the medium was centrifuged and the viruses were precipitated from the supernatant with 10% polyethylene glycol, the HBV-DNA was purified therefrom and quantified by means of dot blot analysis [(E. Matthes et al. Antimicrob Agents Chemother. 34, 1986 (1990)] L-FMetCdR is able to completely suppress the synthesis of HBV, the concentration of the inhibitor which reduces the amount of HBV-DNA released into the medium by 50% , is less than 0.2 μM. A 50% inhibition of the proliferation of HepG2 2.2.15 cells (CD s -,) is only achieved at concentrations greater than 400 μM.

Claims

Patentansprüche claims
1. Neue ß-L-Nucleoside der allgemeinen Formel1. New β-L nucleosides of the general formula
Figure imgf000009_0001
worin
Figure imgf000009_0001
wherein
B = " ,, GGUueanin, 2-Aminopurin;B = " ,, GGUueanin, 2-aminopurine;
Figure imgf000009_0002
Figure imgf000009_0002
R1 = H, Methyl , Halogen, Formyl, Hydroxymethyl, Ethyl, Chlor- ethyl; R2 = H, OH;R 1 = H, methyl, halogen, formyl, hydroxymethyl, ethyl, chloroethyl; R 2 = H, OH;
R3 = F, OH; wenn R2=H, dann R3=F, wenn R2=0H, dann R3=OH R4 = OH, O-Acetyl, O-Palmitoyl, Alkoxy-Carbonyl, Phosphonat,R 3 = F, OH; if R 2 = H, then R 3 = F, if R 2 = 0H, then R 3 = OH R 4 = OH, O-acetyl, O-palmitoyl, alkoxy-carbonyl, phosphonate,
Mono-, Di-, Triphosphat, bzw. eine andere Schutzgruppe, die in einer Folgereaktion in die Hydroxygruppe umgewandelt werden kann, bedeuten.Mono-, di-, triphosphate or another protective group which can be converted into the hydroxy group in a subsequent reaction.
2. ß-L-2' ,3'-Didesoxy-3'-fluorcytidin2. β-L-2 ', 3'-dideoxy-3'-fluorocytidine
3. ß-L-2' ,3'-Didesoxy-3'-fluor-5-methylcytidin3. β-L-2 ', 3'-dideoxy-3'-fluoro-5-methylcytidine
4. ß-L-2' ,3'-Didesoxy-3'-fluor-5-chlorcytidin4. ß-L-2 ', 3'-dideoxy-3'-fluoro-5-chlorocytidine
5. ß-L-2' ,3'-Didesoxy-3'-fluorguanosin5. β-L-2 ', 3'-dideoxy-3'-fluoroguanosine
6. ß-L-5-Methylcytosinarabinosid 6. β-L-5-methylcytosine arabinoside
PCT/DE1995/001412 1994-10-07 1995-10-05 NOVEL β-L-NUCLEOSIDES AND THEIR USE WO1996011204A1 (en)

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US7795238B2 (en) 1998-08-10 2010-09-14 Idenix Pharmaceuticals, Inc. β-L-2′-deoxy-nucleosides for the treatment of hepatitis B
US6569837B1 (en) 1998-08-10 2003-05-27 Idenix Pharmaceuticals Inc. β-L-2′-deoxy pyrimidine nucleosides for the treatment of hepatitis B
US6946450B2 (en) 1998-08-10 2005-09-20 Idenix Pharmaceuticals, Inc. β-L-2′-deoxy-nucleosides for the treatment of hepatitis B
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US7304043B2 (en) 1998-08-10 2007-12-04 Idenix Pharmaceuticals, Inc. β-L-2′-deoxy-nucleosides for the treatment of hepatitis B
US6566344B1 (en) 1998-08-10 2003-05-20 Idenix Pharmaceuticals, Inc. β-L-2′-deoxy-nucleosides for the treatment of hepatitis B
EP2415776A1 (en) * 1998-08-10 2012-02-08 IDENIX Pharmaceuticals, Inc. Beta-L-2'-Deoxy-Nucleosides for the Treatment of Hepatitis B
US6596700B2 (en) 2000-05-26 2003-07-22 Idenix Pharmaceuticals Inc. Methods of treating hepatitis delta virus infection with β-L-2'-deoxy-nucleosides
US6787526B1 (en) 2000-05-26 2004-09-07 Idenix Pharmaceuticals, Inc. Methods of treating hepatitis delta virus infection with β-L-2′-deoxy-nucleosides
US6875751B2 (en) 2000-06-15 2005-04-05 Idenix Pharmaceuticals, Inc. 3′-prodrugs of 2′-deoxy-β-L-nucleosides
US7585851B2 (en) 2000-06-15 2009-09-08 Idenix Pharmaceuticals, Inc. 3′-prodrugs of 2′-deoxy-β-L-nucleosides
US7323451B2 (en) 2002-08-06 2008-01-29 Idenix Pharmaceuticals, Inc. Crystalline and amorphous forms of beta-L-2′-deoxythymidine
US7589079B2 (en) 2002-08-06 2009-09-15 Novartis Ag Crystalline and amorphous forms of beta-L-2′-deoxythymidine
US7858594B2 (en) 2002-08-06 2010-12-28 Novartis Pharma Ag Crystalline and amorphous forms of beta-L-2′-deoxythymidine
US7928086B2 (en) 2002-09-13 2011-04-19 Novartis Ag β-L-2′-deoxynucleosides for the treatment of resistant HBV strains and combination therapies
US8158606B2 (en) 2002-09-13 2012-04-17 Novartis, Ag β-L-2′-deoxynucleosides for the treatment of resistant HBV strains and combination therapies
US7186700B2 (en) 2002-09-13 2007-03-06 Idenix Pharmaceuticals, Inc. β-L-2′-deoxynucleosides for the treatment of resistant HBV strains and combination therapies
US7582748B2 (en) 2003-03-20 2009-09-01 Microbiologica Quimica E Farmaceutical Ltd. Methods of manufacture of 2′-deoxy-β-L-nucleosides
US7595390B2 (en) 2003-04-28 2009-09-29 Novartis Ag Industrially scalable nucleoside synthesis

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